1           General

1.1           Synthesis and characterization of iodixanol.

1. Priebe et al

Acta Radiologica Supplement, 399, 21-31 (1995)         

 

Iodixanol (Visipaque) is a new nonionic roentgen contrast medium intended for general use. Visipaque is a pharmaceutical formulation of iodixanol which is isotonic and isoosmotic with blood. Two synthetic routes from 5-nitro-isophthalic acid to iodixanol are described. The chemical structure is confirmed by spectroscopical data (¹H-NMR, ¹³C-NMR, electrospray-MS, UV, IR and Raman). Chromatographic characteristics are related to the isomerism of iodixanol.

 

1.2           Physical properties of iodixanol.

Eivindvik, K. and Sjøgren, E.

Acta Radiologica Supplement, 399, 32-38 (1995)

 

Iodixanol, the radiopaque in Visipaque, is a new nonionic, dimeric roentgen contrast medium for intravascular use. Compared to aqueous solutions of nonionic monomers, which have a higher osmolality than blood, aqueous solution of iodixanol have a lower osmolality due to the dimeric structure of the molecule. As a consequence of this advantageous property, solutions of all clinical concentrations of iodixanol can be made isotonic by the addition of salts of the key electrolytes sodium and calcium to the formulation. The viscosity of all iodixanol (Visipaque) solutions is less than or equal to that of iohexol (Nycodenz). Iodixanol itself is an amorphous and hygroscopic solid which is freely soluble in water. Partition coefficients show that iodixanol is even more hydrophilic than the nonionic monomers such as iohexol. The high hydrophilicity and the good aqueous solubility of iodixanol are due to the hydroxyl group in the dimer linkage and the hydrophilic amide side chains of the molecule.

 

1.3           Visipaque is isotonic to human and rat blood plasma.

Karlsson, J.O.G., Gregersen, M. and Refsum, H.

Acta Radiologica Supplement, 399, 39-42 (1995)

 

Even at its highest concentration, 320 mgI/ml, Visipaque - based on the nonionic dimer iodixanol - is isoosmotic to blood plasma, whereas Omnipaque (300 mgI/ml) - based on the nonionic monomer iohexol - has an osmolality of about twice that of the plasma. However, the fact that a solution is isoosmotic to plasma does not necessarily mean that it is isotonic to plasma. An isoosmotic solution can still cause a net movement of water over the plasma membranes of, for example, erythrocytes and endothelial cells.

 

Determination of the tonicity of Visipaque 320 mgI/ml and comparison with that of           Omnipaque 300 mgI/ml and hypertonic NaCl have been performed. No change in the water content of human erythrocytes was seen after mixing whole blood 10:1 with either Visipaque 320 mgI/ml or 155 mM NaCl, whereas a significant decrease in water content occurred with Omnipque 300 mgI/ml and 620 mM NaCl. No difference in the water content of rat erythrocytes was evident after mixing whole blood with Visipaque 320 mgI/ml or isotonic NaCl. However, as with human erythrocytes, a significant decrease in water content occurred after rat blood was mixed with Omnipaque 300 mgI/ml.

 

In conclusion, Visipaque 320 mgI/ml does not cause any net movement of water over the human or rat erythrocyte plasma membrane, i.e., Visipaque is isotonic to human and rat blood plasma.

 

1.4           Formulation, stability and compatibility of iodixanol.

Aars, E.V. and Eivindvik, K.

Acta Radiologica Supplement, 399, 50-58 (1995)

 

Iodixanol (Visipaque) is an isotonic, electrolyte-balanced roentgen contrast medium for intravascular use. The patented and well-proven formulation and the rationale for it are described, and the efficacy and safety documented. The stability of iodixanol is well within the specifications under all relevant conditions, both in glass and polypropylene bottles; the product has a recommended shelf-life of at least 36 months when stored at room temperature and protected from light. Heating to body temperature before use is acceptable and recommendable, and storage at 37⁰C for 1 month does not jeopardize product quality. Iodixanol has no apparent immediate in vitro incompatibility reactions with drugs used in connection with roentgen contrast examination.

1.5           Preclinical pharmacokinetics and general toxicology of iodixanol.

Heglund, F. et al

Acta Radiologica Supplement, 399, 69-82  (1995)

 

To document the safety of iodixanol and to assess its pharmacokinetic properties, extensive tests have been performed. Iodixanol was rapidly excreted, mainly via the     kidney, with a plasma half-life in rats and monkeys of 25 and 76 min. respectively. The pharmacokinetic data was consistent with an extracellular distribution of iodixanol. During the 24 hours post-dosing, the urinary excretion was from 72 to 100% in rats and 78% in monkeys. Biliary excretion was 1.5% during the first 4 hours in rats. Fecal excretion was about 7% in rats and 0.8% in monkeys over the first 24 hours after injection. Approximately 0.5 and 1% of the dose was found in the kidneys of rats and monkeys, respectively, 24 hours after dosing. Acute toxicity of iodixanol in rats was low, with an LD₅₀ greater than 21gI/kg. In mice the LD₅₀ was 21gI/kg and the approximated median lethal dose (ALD₅₀) was found to range from 15 to 21 gI/kg. A single dose of 1 and 3 gI/kg was well tolerated in monkeys. As for other roentgen contrast media, a reversible, dose-related, vacuolation of the proximal tubules in the kidneys was seen in the acute and subacute studies in rats and monkeys. No relationship was seen between the vacuolisation and kidney function. Local tolerance studies demonstrated a low irritation potential for iodixanol when injected by a variety of intravascular and extravascular routes.

 

The reproductive capacity of male and female rats was unaffected by iodixanol when administered daily at doses up to 2 gI/kg/day. No teratogenic potential in rats and rabbits of iodixanol was observed. Further, no toxic effects on pups were seen when rats were dosed during the lactation period. Each of 4 standard genotoxicity tests was negative. No antigenic potential of iodixanol was observed when assessed by the passive cutaneous anaphylaxis test and the active systemic anaphylaxis test in guinea pigs.

 

The intravascular tolerability of iodixanol is high and, therefore, iodixanol should be considered as a safe roentgen contrast medium for intravascular use.

 

1.6           Iodixanol: A nonionic iso-osmotic centrifugation medium for the formation of self generated gradients.

Ford, T., Graham, J. and Rickwood, D.

Anal. Biochem., 220, 360-366 (1994)

 

The physical and biological properties of iodixanol, a new nonionic density gradient medium, are described in this paper. It is effectively a dimer of Nycodenz and it exhibits two significant advantages over previous iodinated density gradient media- its  aqueous solutions are iso-osmotic up to a density of 1.32 g/ml and it is capable of forming self-generating gradients in 1 to 3 h. It has a very low toxicity towards biological material and enzyme assays can be carried out in its presence.

 

1.7           Spectrophotometric determination of iodixanol in subcellular fractions of mammalian cells.

Schroder, M., Schafer, R. and Friedl, P.

Anal. Biochem., 244, 174-176 (1997)

 

Nonionic iodinated density gradient media are widely used for the separation of cells and organelles. The most suitable of them is iodixanol. It displays no cytotoxicity and a number of marker enzymes for cellular organelles can be assayed in the presence of iodixanol. In contrast to other nonionic iodinated density gradient media, such as metrizamide or Nycodenz, iodixanol readily forms self-generated gradients, thus obviating the preparation of gradients using a gradient marker.

 

A subcellular fractionation of cellular organelles is evaluated by the distribution of marker enzymes in the gradient and the density profile of the gradient. The density profile of a gradient is commonly analyzed by measuring the refractive index of the gradient fractions. Cellular material present in subcellular fractions interferes with the determination of the refractive index, thus necessitating a mock fractionation to determine the density profile of the gradient.

 

Here we report the direct and specific determination of iodixanol as an example for a nonionic density gradient medium in subcellular fractions. No mock runs are necessary to determine the density profile of the gradient when this method is used. This assay should be more convenient for many laboratories because a refractometer is not required.

 

1.8           Determination of particle sedimentation rate by ultrasonic interferometry: role of particle size, density and volume fraction

Razavian, S.M., Wenby, R.B., Fisher, T.C. and Meiselman, H.J.

Biorheology, 34(4/5), 349-362 (1997)

 

The sedimentation rate (SR) of non-aggregated spherical particles in suspension was determined using an ultrasonic interferometry technique (Echo-Cell); this method is based on A-mode echography and measures the rate of formation of a sediment on a solid plate during settling. The particle accumulation rate, which is related to SR, is obtained from the interference of two waves reflected by two interfaces: one between the plate and the sediment and the other between the sediment and the suspension. Studies were carried out at 25°C using latex spheres of different diameters (7 to 20 mm) and densities (1.062 to 1.190 g/cm³) suspended in distilled water at various volume fractions (1% to 5%). As anticipated by the Stokes model, linear relations were found between SR and both particle density and the square of particle radius. Experimental SR values decreased with increasing suspension particle concentration; these concentration effects were in good agreement with those predicted by the Steinour model. Our results thus serve to validate the theoretical aspects of the Echo-Cell method and suggest its usefulness as a tool for studies of RBC interaction and RBC aggregation.

 

1.9           Iodixanol is readily eliminated by hemodialysis

Berg, K.J., Rolfsen, B. and Stake, G.

Acta Radiologica, 39, 372-374 (1998)

 

The dialyzability of the high-molecular X-ray contrast medium iodixanol was examined in an in vitro hemodialysis model using two different hollow fiber membranes: one high flux (polysulfone) membrane and one intermediate-flux (cellulose triacetate) membrane. Blood flow was 200 ml/min and membrane area 1.3 m². The dialyzer clearance of iodixanol dissolved in a mixture of leucocyte-filtered SAG-M blood and compatible citrate plasma was 143.2 ± 3.6 ml/min for the polysulfone membrane and 113.0 ± 3.6 ml/min for the triacetate membrane. Iodixanol is readily dialyzed through commercial high-flux membranes.

 

1.10           Stability of the X-ray contrast agent iodixanol - 3,3’5,5’-tetrakis(2,3dihydroxy-propylcarbamoyl)-2,2’,4,4’,6,6’-hexaiodo-N,N’-(2-hydroxypropane-1,3-diyl)-diacetanilide towards acid, base, oxygen, heat and light.

Priebe, H. et al.

1. Clin. Pharm. and Therapeut., 24, 227-235 (1999)

 

Background: During the production of the X-ray contrast agent iodixanol the drug substance may be exposed to acid, base, air, heat and daylight, conditions that may cause decomposition products.

 

Objectives: To investigate the chemical stability of iodixanol under accelerating conditions.

 

Method: Chemometrical stability studies were undertaken to investigate the effect of acid and base on the contrast agent’s stability.

 

Results: Cleavage of the central bridge in iodixanol occurred under ultraviolet irradiation via a Norrish Type-II reaction. Basic conditions (pH 14) combined with heat (60°C) initiated a cyclization reaction. Less than 1% iodixanol decomposed in solution heated to 140°C for 2 days or under both basic conditions (pH 11, 20°C, 5 days) and acidic conditions (pH 0-4, 80°C, 5 days) or under oxygen atmosphere (100°C, 3 days).

 

Conclusion: Even under highly acidic and basic conditions, iodixanol is stable.

2           Macromolecules

 

2.1           A new method for the rapid separation of plasma lipoproteins

Graham, J.M., Higgins, J.A. and Gillot, T.

Atherosclerosis, 115 (Suppl), S123 (1995)

 

Variations in the relative amounts of plasma lipoprotein classes and /or molecular changes in their lipid components and apoproteins, are important factors in the development of atherosclerosis. A new equilibrium centrifugation method, which avoids high salt gradients, provides high resolution and concentration of lipoproteins for electrophoretic, compositional and structural analysis. The method uses a new non-ionic medium, iodixanol, available as a 60% (w/v) solution called OptiPrep (Axis-Shield PoC, Oslo). Plasma is adjusted to 12.5% iodixanol; overlaid with 0.1-0.2x its volume of saline and centrifuged at approx. 350,000g for 2.5-3 h in a vertical or near-vertical rotor. Using 3.9 ml tubes, unloaded dense end first; soluble proteins are contained in the first 1.0 ml, HDL in the 1.2-2.4 ml fraction; LDL in the 2.6-3.4 ml fraction and VLDL in the 3.6-3.9 ml fraction. There is little overlap between the three classes. Fractions can be analysed directly, by gel electrophoresis and for lipid content. The method may resolve different sub-classes of these lipoproteins: this is currently being investigated. Because of the concentration effect achieved by the gradient, Lp(a), which is recovered in a single 0.25 ml fraction, can be easily demonstrated on gels when levels in the whole plasma are below the limits of detection. The technique thus provides a means of fractionating and concentrating all plasma lipoproteins in a single step; it should prove to be a valuable diagnostic and analytical tool in studies of lipoprotein metabolism.

 

2.2           A novel method for the rapid separation of plasma lipoproteins using self-generated gradients of iodixanol.

Graham, J. et al

Atherosclerosis, 124, 125-135 (1996)

 

We describe a new method for the rapid fractionation of plasma lipoproteins, which makes use of a new

is simple: plasma or serum is mixed with iodixanol followed by centrifugation in a vertical or near vertical rotor. Separation of VLDL, LDL and HDL can be achieved in 3 h and the lipoprotein fractions are comparable in density and composition with those prepared using conventional salt based gradients. Each class of lipoprotein can be removed in a single fraction, or a profile of lipoprotein distribution can be obtained using a gradient fractionation. Because the medium is inert, fractions from the gradient can be analyzed by agarose gel electrophoresis or assayed for lipid content or apolipoprotein composition by SDS-PAGE without removing the iodixanol. Small differences in electrophoretic mobility of HDL and LDL across several gradient fractions suggest that subfractionation of these classes may occur. The new method is simple, rapid and versatile with potential application for preparation of lipoproteins and for analysis of lipoprotein profiles in the research and clinical laboratory.

 

2.3           Isolation of plasmid DNA.

Rickwood, D.and Patel, N.

Mol. Biol. Cell, 7, 162a (1996)

 

Centrifugation of DNA in CsCl-ethidium bromide gradients remains a widely used technique for the isolation of high purity plasmid DNA. There are two major problems with separations using this technique. The first is that because ethidium bromide intercalates into the DNA helix, it is a powerful mutagen, leading to dangers in working with it and difficulties in disposing of the ethidium bromide after use. The other problem is that ethanol precipitation of the plasmid can cause the precipitation of CsCl thus complicating recovery of the DNA. We have devised a new method for the purification of plasmid DNA using self-generating OptiPrep gradients containing a fluorescent dye, DAPI, to mark the position of the DNA in the gradient. Crude plasmid DNA is prepared using the standard alkaline lysis method and then purified by centrifugation in a gradient of 28% OptiPrep containing 0.005% DAPI at 150,000g for 40 hours at 5°C. The native DNA is separated from the denatured chromosomal DNA, RNA and proteins. As judged by electrophoresis, the plasmid DNA obtained after centrifugation is free of contaminating chromosomal DNA and RNA and can be ethanol precipitated directly from the gradient solution.

 

2.4           Rate zonal sedimentation of proteins in one hour or less.

Basi, N.S. and Rebois, V.

Anal. Biochem., 251, 103-109 (1997)

 

Rate zonal sedimentation gives information about the shape and size of proteins, and is useful for investigating protein-protein interactions. However, rate zonal sedimentation experiments typically last approximately 1 day. In contrast, this report describes a rate zonal sedimentation method requiring 1 h or less. This was accomplished by centrifuging small density gradients (200 ul) prepared with sucrose or OptiPrep in a fixed-angle rotor at high relative centrifugal force. By using small gradient volumes, the sample dilution that occurs with larger gradients and with many chromatographic techniques was also avoided. For a variety of proteins, plots of s_20,w  versus distance sedimented during centrifugation in a TLA 120.2 rotor were linear. As a practical application, sedimentation of the heterotrimeric stimulatory G protein and its dissociated a-subunits were determined. The results were similar to those obtained in an SW 50.1 rotor and agreed with previously published values. Long periods of centrifugation might preclude the study of some unstable proteins or the investigation of protein-protein interactions whose affinities are to low to survive the lengthy centrifugations required to carry out traditional rate zonal sedimentation experiments. A rate zonal sedimentation technique that rivals many chromatographic methods in celerity will help to circumvent these problems.

 

2.5           Effect of dietary fish oil or sunflower oil on plasma lipoproteins and hepatic gene expression in the hamster

Bennett, A.J., Kendrick, J.S., Anderton, K.L., Higgins, J.A. and White, D.A.

Atherosclerosis, 130 (Suppl. 1), S24 (1997)

 

An increased dietary consumption of fish oils reduces the occurrence of coronary artery disease. Although this probably involves several mechanisms, one effect of increased consumption of fish oil is a fall in plasma triacylglycerol (TAG) in humans and experimental animals. This has led to the idea that fish oil fatty acids lower VLDL secretion by the liver and hence reduce the risk of atherosclerosis. To investigate this, we have supplemented the diets of hamsters with 10% fish-oil or 10% sunflower oil and examined blood lipoproteins, VLDL secretion and the mRNA levels of genes involved in apolipoprotein B metabolism. Hamsters were fed 1.5 ml of either fish oil (Maxepa) or sunflower oil by daily gavage. Blood samples were taken at weekly intervals. The lipids were analysed and the lipoprotein classes separated on iodixanol gradients and by agarose electrophoresis. In fish oil fed animals, plasma TAG fell by 25% over a three-week period. There was no significant change in the TAG levels of either chow fed animals or sunflower oil fed animals or in the cholesterol levels of the three groups of animals. Plasma VLDL were considerably reduced and there was a small increase in LDL and HDL in the fish-oil fed animals. IN the sunflower oil fed and chow fed animals, there was no significant change in the plasma lipoproteins. Consistent with these results from in vivo experiments, secretion of VLDL by hepatocytes isolated from fish-oil hamsters were considerably reduced compared with hepatocytes from chow-fed and sunflower-oil fed animals. The fall in plasma VLDL in fish-oil fed hamsters was not accompanied by a fall in LDL. To determine whether this is due decreased LDL uptake we determined the mRNA levels for the LDL receptor (LDL-R) in liver from hamsters fed fish-oils, sunflower oil or chow for three weeks using the mRNA protection assay. The mRNA for apo-B, HMG-CoA reductase and microsomal triacylglycerol transfer protein (MTP) were also measured in the same liver samples to examine possible effects on other candidate proteins involved in apo-B metabolism 9Table 1). Dietary fish-oils reduced the levels of mRNA for the LDL-R by approximately 60% while dietary sunflower oil had no significant effect, mRNA for HMG-CoA reductase was reduced 60% by feeding fish-oils and 20% by feeding sunflower oils. These results suggest that there may be a complex metabolic relationship between apo-B synthesis and uptake by the liver which is perturbed by feeding fish-oils.

 

2.6           Dietary fish oils modify the assembly of VLDL and expression of the LDL receptor in rabbit liver

Wilkinson J., Higgins, J.A., Fitzsimmons, C. and Bowyer, D.E.

Arterioscler. Thromb. Vasc. Biol., 18: 1490-1497 (1998)

 

Supplementation of the diet of rabbits with fish oil or sunflower oil resulted in significant changes in the lipoproteins and lipids in serum. Compared with chow-fed rabbits, dietary fish oils decreased very low-density lipoprotein (VLDL), increased low-density lipoprotein (LDL), and shifted the peak of the LDL to denser fractions, whereas sunflower oil increased high density lipoprotein and shifted LDL to the lighter fractions. The amount of LDL receptors in fish oil-fed rabbit liver decreased by >70% while there was only a small fall in these levels in sunflower oil-fed rabbit liver. The concentrations of apolipoprotein (apo) B in the subcellular organelles of the secretory compartment (rough and smooth endoplasmic reticula and Golgi fractions) were also changed by dietary lipids. In both sunflower oil- and fish oil-fed liver, apo B was increased in the lumen of the rough endoplasmic reticulum compared with fractions from chow-fed rabbit liver. The apo B in the trans-Golgi lumen from fish oil-fed livers was reduced and occurred in particles of r~1.21 g/mL. In contrast, apo B in the trans-Golgi lumen from livers of sunflower oil-fed rabbits was increased and occurred in particles of r<l .21 g/mL. These results suggests that feeding of fish oils causes an interruption in the intracellular transfer of apo B and hence assembly of VLDL. This leads to an enrichment of the rough endoplasmic reticulum membranes with cholesterol, thus down-regulating the expression of the LDL receptor.

 

2.7           Carotenoid composition and antioxidant potential in subfractions of human low-density lipoprotein.

Lowe, G.M. et al

Ann. Clin. Biochem., 36, 323-332 (1999)

 

Carotenoids and vitamin E are transported in human plasma complexed with lipoproteins. The bulk of them are associated with low-density lipoprotein (LDL) where they may act as antioxidants and thus delay the onset of atherosclerosis. In this study we have used a simple, rapid ultracentrifugation technique in which plasma lipoproteins are fractionated in self-generated gradients of iodixanol, a non-ionic iodinated density gradient medium. The carotenoid content and composition of a number of LDL subfractions was determined by reversed-phase HPLC. Lycopene, b-carotene and b-cryptoxanthin were mainly located in the larger, less-dense, LDL particles whereas lutein and zeaxanthin were found preferentially in the smaller, more-dense, LDL particles. When the antioxidant content of these fractions was expressed per mg of LDL protein, a different distribution was found with significantly lower concentrations of carotenoid and vitamin E associated with the smaller protein-rich, fractions of LDL. Strong positive correlations were found for total carotenoid and vitamin E concentrations with the lag-time of Cu²⁺-mediated oxidation of LDL subfractions. The more dense LDL subfractions, which had lower levels of these antioxidants, were more readily oxidised, highlighting their possible role in atherosclerotic events.

 

 

2.8           Blood appearance, metabolic transformation and plasma transport proteins of ¹⁴C-   astaxanthin in Atlantic salmon (Salmo salar L.)

Aas, G.H., Bjerkeng, B., Storebakken, T. And Ruyter, B.

Fish Physiol. Biochem., 21, 325-334 (1999)

 

The time of appearance in blood, and transport of astaxanthin, and catabolic transformation of astaxanthin to idoxanthin were investigated in Atlantic salmon (Salmo salar) that had been force-fed a single dose ¹⁴C-astaxanthin. In addition to the LPs, a major protein, associated with radiolabeled astaxanthin was detected. The maximum level of radiolabeled carotenoids in blood was attained 30 h after administration of ¹⁴C-astaxanthin. Radioactive idoxanthin (combined 3’4’-cis and 3’4’-trans glycolic isomers of idoxanthin) appeared after 6 h and a stable level was obtained after 18 h. LPDP and LP, separated by ultracentrifugation, contained on average 89 and 11% of the total radioactivity in plasma, respectively. During the 168 h experiment, maximum radioactivity in LP appeared after 22h. Separation of plasma by ultracentrifugation on a discontinuous NaCl/KBr-gradient and an iodixanol-gradient confirmed that most of the radiolabel carotenoids were present in the HDPF that did not contain LPs (58%), whereas HDL and LDL contained 36 and 6% of the radioactivity, respectively. Of the recovered radioactivity, astaxanthin in the HDPF comprised 82%, idoxanthin 5% and unidentified compounds 12%, whereas HDL contained 78% astaxanthin, 22% idoxanthin and no unidentified compounds. Protein from the fractions with high density and high radioactivity (iodixanol-gradient) were separated by PAGE under non-denaturing conditions and showed a radioactive band with parallel migration length to BSA and salmon albumin. These results show that astaxanthin is rapidly converted to idoxanthin and that the majority of astaxanthin in the plasma is associated with a protein other than LPs, presumably albumin. The identity of this protein requires verification.

 

2.9           In healthy adults, postprandial lipaemia results in triglyceride enrichment of very low-density lipoprotein, enhanced oxidative stress and deterioration in endothelial function

Anderson, R.A. et al

Atherosclerosis Suppl 154(2), 434 (1999)

 

There is a significant relationship between postprandial lipaemia (PPL) and coronary artery disease in patients with both normal and abnormal carbohydrate metabolism. PPL has recently been shown to cause endothelial dysfu8nction in normal individuals: attenuated by antioxidants. We therefore studied the effects of PPL on endothelial function (EF), free radicals (FR) and triglyceride (TG) metabolism in healthy subjects. Methods: Twelve subjects with no history of vascular disease or diabetes were studied, (five male, seven female; mean age 43 years). After a 12-h overnight fast, EF, lipid profiles and venous FR levels were measured in the fasting and peak postprandial phase (at 4 h) subsequent to ingestion of a standard fat meal. Flow-mediated dilatation (FMD) was assessed, as a measure of EF in the brachial artery, and expressed as the percentage change in brachial artery diameter. Lipoprotein subfractions were assessed using a density gradient medium, iodixanol. Lipid-derived FR levels were measured in venous blood by electron paramagnetic resonance spectroscopy; results expressed in arbitrary units. Results: There was an increase in venous FR postprandially (mean ± SD), 2.4 ± 0.1 to 3.3 ± 0.2 (P < 0.05), and a decrease in FMD, 6.26 ± 1.3% to 4.8 ± 1.3% (P < 0.05). TG levels (in mmol/l) were also significantly elevated at 4 h., 1.3 ± 0.5 to 2.2 ± 0.7 (P < 0.05). There were no changes in TG content of high- and low-density lipoprotein cholesterol but there was an increase in TG content of very low-density lipoprotein (VLDL), 54 ± 6.5% to 59.3 ± 5.7%. Conclusion: We show for the first time that PPL in healthy individuals is associated with increased lipid-derived free radicals and deterioration of endothelial function. This was associated with relative TG-enrichment of VLDL particles.

 

2.10           Ciprofibrate reduces the postprandial generation of triglyceride-rich lipoproteins and attenuates the associated endothelial dysfunction and oxidative stress in non-insulin dependent diabetes mellitus

Evans, L.M. et al

Atherosclerosis Suppl 154(2), 434 (1999)

 

Introduction: Triglyceride-rich lipoproteins (TGRL) may be involved in athero­genesis by mechanisms including endothelial dysfunction and enhanced oxida­tive stress. Non-insulin-dependent diabetes mellitus (NIDDM) results in excess vascular disease and exaggerated excursions of postprandial TGRL. We studied the effect of fibrate therapy on the relationship between postprandial lipaemia (PPL), endothelial function (EF) and oxidative stress in NIDDM. Methods: Twenty NIDDM patients were studied following an overnight fast and 4 h after a fatty meal. Lipoproteins were separated using an iodixanol density gradient medium. Free radicals (FR) were directly measured using electron paramag­netic resonance spectroscopy. EF was assessed by measuring flow-mediated brachial artery dilatation (FMD). Subjects were randomised in a double-blind manner to 3 months of placebo (P) or ciprofibrate (C) (100 mg OD). Results (mean ± SD): Seventeen subjects completed the study. At base line, both groups exhibited similar changes in FMD [(% change) 3.8 ± 1.8% to 1.8 ± 1.3% (C) vs. 3.3 ± 1.7 to 1.7 ± 1.1% (P)]. Increase in FR [(arbitrary units) 2.9 ± 1.3 (C) vs. 3.1 ± 1.5 (P)]. Rise in plasma TG [(mmol/l) 2.8 ± 2.1 to 6.7 ± 6 (C) vs. 2.8 ± 1.7 to 7 ± 7.3 (P)] and TG enrichment of very low-density lipoprotein (VLDL) [(% TG content) 59.6 ± 4.6% to 73.4 ± 6.9% (C) vs. 61.2 ± 5.9% to 76.1 ± 9.8% (P)]. Following treatment fasting and PP, FMD improved in the treatment group with reductions in fasting and PP TG, VLDL-TG content and FR. FMD, 4.8 ± 1.1 to 3.4 ± 1.1% (C) vs. 3.4 ± 1.2 to 1.8 ± 1.1% (P) (P <0.05). TG, 1.5 ± 0.8 to 2.8 ± 1.3 mmol/l (C) vs. 3.1 ± 2.1 to 6.6 ±4.1(P) mmol/l (P< 0.05). VLDL-TG (%), 50.1 ± 6.2 to 59.5 ± 4.3% (C) vs. 60.6 ± 3.9 to 72.9 ± 6.9% (P) (P<0.05). FR, 0.3 ± 0.6 (C) vs. 1.1 ± 0.9 (P) (P<0.05). Conclusions: This study demonstrates that ciprofibrate may reduce the risk of vascular disease in NIDDM by mechanisms involving improved EF, attenuated PPL, enhanced catabolism of TGRL and reduced FR release.

 

2.11           Consequences of interaction of a lipophilic endotoxin antagonist with plasma lipoproteins

Rose, J.R. et al

Antimicrobial Agents and Chemotherapy, 44(3), 504-510 (2000)

 

E5531, a novel synthetic lipid A analogue, antagonizes the toxic effects of lipopolysaccharide, making it a potential intravenously administered therapeutic agent for the treatment of sepsis. This report describes the distribution of E5531 in human blood and its activity when it is associated with different lipoprotein subclasses.  After in vitro incubation of  (¹⁴C) E5531 with blood, the great majority (>92%) of material was found in the plasma fraction.  Analysis by size-exclusion and affinity chromatography and density gradient centrifugation indicates that (¹⁴C)E5531 binds to lipoproteins, primarily high-density lipoproteins (HDLs), with distribution into low-density lipoproteins (LDLs) and very low density lipoproteins (VLDLs) being dependent on the plasma LDL or VLDL cholesterol concentration.  Similar results were also seen in a limited study of (¹⁴C)E5531 administration to human volunteers.  The potency of E5531 in freshly drawn human blood directly correlates to increasing LDL cholesterol levels.  Finally, preincubation of E5531 with plasma or purified lipoproteins indicated that binding to HDL resulted in a time-dependent loss of drug activity.  This loss in activity was not observed with drug binding to LDLs or to VLDLs or chylomicrons.  Taken together, these results indicate that E5531 binds to plasma lipoproteins, making its long-term antagonistic potency dependent on the plasma lipoprotein composition.

 

 

2.12           Ciprofibrate therapy improves endothelial function and reduces postprandial lipemia and oxidative stress in type 2 diabetes mellitus

Evans, M. et al

Circulation, 101, 1773-1779 (2000)

 

Background-Exaggerated postprandial lipemia (PPL) is a factor in atherogenesis, involving endothelial dysfunction and enhanced oxidative stress.  We examined the effect of ciprofibrate therapy on these parameters in type 2 diabetes mellitus.

 

Methods and Results-Twenty patients entered a 3-month, double blind, placebo-controlled study.  Each subject was studied fasting and after a fatty meal, at baseline, and after 3 months of treatment.  Glucose and lipid profiles were measured over an 8-hour postprandial period.  Endothelial function (flow-mediated endothelium-dependent vasodilatation [FMDI) and oxidative stress (electron paramagnetic resonance spectroscopy) were measured after fasting and 4 hours postprandially.  At baseline, both groups exhibited similar PPL and deterioration in endothelial function.  After ciprofibrate, fasting and postprandial FMD values were significantly higher (from 3.8±1.8% and 1.8 ±1.3% to 4.8 ± 1.1% and 3.4 ± 1.1%; P<0.05). This was mirrored by a fall in fasting and postprandial triglycerides (3.1±2.1 and 6.6±4.1 mmol/L to 1.5±0.8 and 2.8±1.3 mmol/L, P<0.05). Fasting and postprandial HDL cholesterol was also elevated (0.9±0.1 and 0.8±0.1 mmol/L and 1.2±0.2 and 1.2±0.1 mmol/L, P<0.05). There were no changes in total or LDL cholesterol.  Fasting and postprandial triglyceride enrichment of all lipoproteins was attenuated, with cholesterol depletion of VLDL and enrichment of HDL.  There were similar postprandial increases in oxidative stress in both groups at baseline, which was significantly attenuated by ciprofibrate (0.3±0.6 versus 1.5± 1.1 U, P<0.05).

 

Conclusions-This study demonstrates that fibrate therapy improves fasting and postprandial endothelial function in type 2 diabetes.  Attenuation of PPL and the associated oxidative stress, with increased HDL cholesterol levels, may be important.

 

2.13           Collagen-bound von Willebrand factor has reduced affinity for factor VIII

Bendetowicz, A.V., Wise, R.J. and Gilbert, G.E.

J. Biol. Chem., 274(18), 12300-12307 (2000)

 

von Willebrand factor (vWf) is a multimeric adhesive glycoprotein that serves as a carrier for factor VIII in plasma.  Although each vWf subunit displays a high affinity binding site for factor VIII in vitro, in plasma, only 2% of the vWf sites for factor VIII are occupied.  We investigated whether interaction of plasma proteins with vWf or adhesion of vWf to collagen may alter the affinity or availability of factor VIII-binding sites on vWf. When vWf was immobilized on agarose-linked monoclonal antibody, factor VIII bound to vWf with high affinity, and neither the affinity nor binding site availability was influenced by the presence of 50% plasma.  Therefore, plasma proteins do not alter the affinity or availability of factor VIII-binding sites.  In contrast, when vWf was immobilized on agarose-linked collagen, its affinity for factor VIII was reduced 4-fold, with K, increasing from 0.9 to 3.8 nm.  However, one factor VIII-binding site remained available on each vWf subunit.  A comparable reduction in affinity for factor VIII was observed when vWf was a constituent of the subendothelial cell matrix and when it was bound to purified type VI collagen.  In parallel with the decreased affinity for factor VIII, collagen-bound vWf displayed a 6-fold lower affinity for monoclonal antibody W5-6A, with an epitope composed of residues 78-96 within the factor VIII-binding motif of vWf.  We conclude that Collagen induces a conformational change within the factor VIII-binding motif of vWf that lowers the affinity for factor VIII.

 

2.14           Plasma appearance and distribution of astaxanthin E/Z and R/S isomers in plasma lipoproteins of men after single dose administration of astaxanthin

Osterlie, M., Bjerkeng, B. And Liaaen-Jensen, S.

1. Nutr. Biochem., 11, 482-490 (2000)

 

Appearance, pharmacokinetics, and distribution of astaxanthin E/Z and R/S isomers in plasma and lipoprotein fractions were studied in 3 middle-aged male volunteers (37-43 years) after ingestion of a single meal containing a 100 mg dose of astaxanthin. The astaxanthin source consisted of 74% all-E-, 9% 9Z-, 17% 13Z-astaxanthin (3R, 3’R, 3R, 3’S: meso-, and 3S, 3’S-astaxanthin in a 1:2:1 ratio). The plasma astaxanthin concentration – time curves were measured during 72 hr. Maximum levels of astaxanthin (1.3 ± 0.1 mg/L) were reached 6.7 ± 1.2 hr after administration, and the plasma astaxanthin elimination half-life was 21 ± 11 hr. 13Z-astaxanthin accumulated selectively, whereas the 3 and 3’R/S astaxanthin distribution was similar to that of the experimental meal. Astaxanthin was present mainly in very low-density lipoproteins containing chylomicrons (VLDL/CM); 36-64% of total astaxanthin), whereas low-density lipoprotein (LDL) and high-density lipoprotein (HDL) contained 29% and 24% of total astaxanthin, respectively. The astaxanthin isomer distribution in plasma, VLDL/CM, LDL and HDL was not affected by time. The results indicate that a selective process increases the relative proportion of astaxanthin Z-isomers compared to the all-E-astaxanthin during blood uptake and that astaxanthin E/Z isomers have similar pharmacokinetics.

 

2.15           Fractionation and characterization of oligomeric, protofibrillar and fibrillar forms of b-amyloid peptide

Ward, R.V. et al

Biochem. J., 348, 137-144 (2000)

 

The b-amyloid (Ab) peptide, a major component of senile plaques in Alzheimer disease brain, has been shown previously to undergo a process of polymerisation to produce neurotoxic forms of amyloid. Recent literature has attempted to define precisely the form of Ab responsible for its neurodegenerative properties. In the present study we describe a novel density-gradient centrifugation method for the isolation and characterization of structurally distinct polymerized forms of Ab peptide. Fractions containing protofibrils, fibrils, sheet structures and low molecular mass oligomers were prepared. The fractionated forms of Ab were characterized structurally by transmission electron microscopy. The effects on cell viability of these fractions were determined in the B12 neuronal cell line and hippocampal neurons. Marked effects on cell viability in the cells were found to correspond to the presence of protofibrillar and fibrillary structures, but not to monomeric peptide or sheet-like structures of polymerized Ab. Biological Activity correlated with a positive reaction in an immunoassay that specifically detects protofibrillar and fibrillary Ab; those fractions that were immunoassay negative had no effect on cell viability. These data suggest that the effect of Ab on cell viability is not confined to a single conformational form but that both fibrillary and protofibrillar species have the potential to be active in this assay.

 

2.16           Capillary isotachophoretic analysis of serum lipoprotein using a carrier ampholyte as spacer ion

Inano, K., Tezuka, S., Miida, T. and Okada, M.

Ann. Clin. Biochem., 37, 708-716 (2000)

 

We have developed a novel analytical method for serum lipoproteins using a commercially available capillary electrophoresis apparatus, BioFocus 3000 (Bio-Rad Laboratories Co Ltd, USA). The analytical principle is isotachophoresis (ITP), using a carrier ampholyte, BioLyte 7/9, as a spacer ion. The method allows a much higher resolution of lipoproteins than of amino acid mixtures. Serum lipoproteins are normally separated into 13-15 peaks, including some shoulder peaks. The reproducibility of repeated analysis within a day was relatively good with the coefficient of variation within the range 0.9-1.1%. VLDL, LDL and HDL prepared by discontinuous density ultracentrifugation could be further separated by capillary ITP. This high-resolving ability of our method enabled detection of small amounts of abnormal lipoprotein species. For example, small dense LDL, which is thought to be an atherogenic lipoprotein, could be detected within the LDL group peak. Moreover, an abnormal HDL, apolipoprotein E-rich HDL, was also detected by a single analysis. These findings suggest that our capillary ITP method is a useful means for detailed analysis of lipoproteins and thus for clinical diagnosis of hyperlipoproteinaemic subjects.

 

2.17           A randomized trial of the effects of garlic oil upon coronary heart disease risk factors in trained male runners

Zhang, X-H., Lowe, D., Giles, P., Fell, S., Board, A. R., Baughan, J. A., Connock, M. J. and Maslin, D. J.

Blood Coagulat. Fibrinolysis, 11, 67-74 (2000)

 

Most trials of bulb garlic and garlic powder tablets indicate reduced coronary heart disease (CHD) risk in elevated-risk subjects. Most persons taking garlic supplements lack overt risk of Cl-ED. However, no trials have tested steam-distilled garlic oil (GO) capsules with healthy subjects. The objectives of the present randomized, double-blind, placebo-controlled study were to determine whether GO capsules reduce CHD risk in trained male runners. Twenty-seven volunteers (mean age, 28.8 years) completed the study. Each took 12.3 mg/day GO (or placebo) capsules for 16 weeks. Main outcome measures were 95% confidence intervals (CIs) between GO and placebo groups for differences in changes of blood pressure (BP), plasma lipids, total antioxidant status (TAS), low-density lipoprotein (LDL) composition and blood clotting factors. Principal results as mean differences (95% CI) between GO and placebo are: pulse, 2.9 beats/mm (-0.8 to 6.7), P = 0.12; systolic BP, -4.5 mmHg (-10.8 to 1.9), P = 0.16; plasma total cholesterol, 0.01 mmol/l (-0.34 to 0.37), P = 0.95; plasma triglycerides, -0.20 mmol/l (-0.43 to 0.03), P = 0.09; plasma TAS, 45 pmol Trolox equivalent/l (-35 to 124), P = 0.26; LDL density, 0.0019 g/ml (-0.0005 to -0.0043), P = 0.12; LDL triglycerides/protein, -0.078 mg/mg (-0.149 to -0.007), P = 0.03; LDL cholesterol/protein, 0.24 mg/mg (-0.69 to 0.22), P = 0.3; LDL TAS/triglycerides, 29 nmol/mg (11 to 68), P = 0.15; prothrombin time, 0.99s (-0.36 to 2.35), P = 0.14; partial thromboplastin time, 3.0s (-1.0 to 7.1), P = 0.13. Results were null statistically. Trends with GO were mostly towards lower CHD risk, and a larger study (-150 subjects) is required to test their validity.

 

2.18           Putative fusogenic activity of NSF is restricted to a lipid mixture whose coalescence is also triggered by other factors

Brügger, B. et al

EMBO J., 19(6), 1272-1278 (2000)

 

It has recently been reported that N-ethylmaleimide-sensitive fusion ATPase (NSF) can fuse protein-free liposomes containing substantial amounts of 1,2-dioleoyl-phosphatidylserine (DOPS) and 1,2-dioleoyl-phosphatidyl-ethanolamine (DOPE) (Otter-Nilsson et al., 1999).  The authors impart physiological significance to this observation and propose to re-conceptualize the general role of NSF in fusion processes.  We can confirm that isolated NSF can fuse liposomes of the specified composition.  However, this activity of NSF is resistant to inactivation of- N-ethylmaleimide and does not depend on the presence of a-SNAP (soluble NSF-attachment protein). Moreover, under the same conditions, either a-SNAP, other proteins apparently unrelated to vesicular transport (glyceraldehyde-3-phosphate dehydrogenase or lactic dehydrogenase or even 3 mM magnesium ions can also cause lipid mixing.  In contrast, neither NSF nor the other proteins nor magnesium had any significant fusogenic activity with liposomes composed of a biologically occurring mixture of lipids.  A straightforward explanation is that the lipid composition chosen as optimal for NSF favors non-specific fusion because it is physically unstable when formed into liposomes.  A variety of minor perturbations could then trigger coalescence.

 

 

2.19           The relationships between post-prandial lipaemia, endothelial function and oxidative stress in healthy individuals and patients with type 2 diabetes

Anderson, R.A. et al

Atherosclerosis, 154, 475-483 (2001)

 

Post-prandial lipaemia (PPL) is a factor in atherosclerosis and results in reversible endothelial dysfunction in healthy individuals. Oxidative stress and triglyceride (TG)-rich lipoproteins have been implicated. Type 2 diabetes (NIDDM) results in exaggerated PPL. We attempted to delineate the mechanisms of PPL induced, endothelial dysfunction (EF) and oxidative stress in 12 NIDDM and 12 matched healthy subjects. Subjects underwent a fat tolerance test, with endothelial function assessed by flow-mediated vasodilatation and oxidative stress measured by venous lipid-derived free radicals ex vivo and lipid peroxidation products over the postprandial phase. Fasting TG, post-prandial hypertriglyceridaemia and the TG enrichment of all lipoproteins was significantly greater in NIDDM. Post-prandial endothelial function inversely correlated with fasting HDL-C (r = -0.84, P= 0.001) in both the control and NIDDM groups. The deterioration in EF in the NIDDM group also correlated with TG enrichment of VLDL and LDL. PPL in both groups also resulted in increased oxidative stress. The increment in free radicals correlated with TG enrichment of VLDL in both groups and was, therefore, greater in NIDDM. Thus, PPL – with the production of TG-enrichment of VLDL – results in endothelial dysfunction by an oxidative stress mechanism in both groups. The magnitude is greater in NIDDM. Fasting HDL-C appears to contribute to the protection of the endothelium against this phenomenon. Hence, exaggerated PPL associated with reduced HDL-C may be important in the pathogenesis of vascular disease, particularly in NIDDM

 

2.20           Rapid identification of LDL subclass phenotypes by iodixanol gradient centrifugation

Davis, I.G. and Griffin, B.A.

Atherosclerosis, 159, 247-252 (2001) 

A rapid and simple method was developed to identify LDL subclass phenotypes for the classification of an atherogenic lipoprotein phenotype. Plasma (3 ml adjusted to 12% iodixanol) was pre-stained (Coomassie Blue R250), under-layered beneath 9% iodixanol and subject to ultracentrifugation (3 h, 65,000 rpm 16°C, (341,000 g)) in a near vertical rotor (Beckman NVT65). A digital photograph of separated LDL bands was downloaded onto a PC and analysed using Total Lab ID gel-scan software (Pharmacia, UK). LDL subclass phenotypes were characterised by Rf values, density intervals and by cross-reference to LDL subclass profiles obtained by our established salt density gradient technique. LDL profiles corresponded to a predominance of light LDL-I, LDL-II or small dense LDL-III. This new method provides a more rapid separation of LDL subclasses than existing salt gradients, with multiple runs and larger rotors (Beckman NVT 65.2 or VTi 65.2) increasing throughput to 48 samples in 24 h.

 

2.21           Direct evidence for a two-step assembly of ApoB48-containing lipoproteins in the lumen of the smooth endoplasmic reticulum of rabbit enterocytes

Cartwright, I.J. and Higgins, J.A.

1. Biol. Chem, 276(51), 48048-48057(2001)

 

The aim of this study was to investigate the types and characteristics of chylomicrons precursor in the lumen of the secretory compartment of rabbit enterocytes. Luminal contents were separated into density subfractions in two continuous self-generating gradients of different density profiles. In enterocytes from rabbits fed a low far diet, newly synthesized and Immunodetectable apoB48 was only in the subfraction of density similar to high density lipoprotein (dense particles); the luminal triacylglycerol (TAG) content was low and only in the subfraction of density similar to that of chylomicrons/very low density lipoproteins (light particles). After feeding fat, newly synthesized and immunodetectable apoB48 was in both dense (phospholipid-rich) and light (TAG-rich) particles. Luminal TAG mass and synthesis increased after fat feeding and was only in light particles. Pulse-chase experiments showed that the luminal-radiolabeled apoB48 lost from the dense particles was recovered in the light particles and the secreted chylomicrons. All of the light particle lipids (mass and newly synthesized) co-immunoprecipitated with apoB48. However, in the dense particles, there was a preferential co-precipitation of the preexisting rather than newly synthesized phospholipid. Assembly of apoB48-containing TAG-enriched lipoproteins is therefore a two-step process. The first step produces dense apoB48 phospholipid-rich particles, which accumulate in the smooth endoplasmic reticulum lumen. In the second step, these dense particles rapidly acquire the bulk of the TAG and additional phospholipid in a single and rapid step.

 

2.22           Superior role of apolipoprotein B48 over apolipoprotein B100 in chylomicron assembly and fat absorption: an investigation of apobec-1 knock-out and wild-type mice.

Kendrick, J.S., Chan, L., and Higgins, J.A.

Biochem. J., 356, 821-827 (2001)

 

Editing of apolipoprotein (apo)-B100 mRNA to yield apo-B48 is a specific and developmentally regulated step in enterocytes of mammals. However, the functional significance of this step is not known. Since mice containing only apo-B100 have not been documented to exhibit any difference in intestinal fat absorption from wild-type mice, the evolutionary advantage of apoB mRNA editing has been questioned. In the present study, we have compared fat absorption and chylomicron assembly in apobec-1 knock-out (KO) or wild-type (WT) mice subjected to different dietary manipulations: low-fat chow, a fat-enriched “Western” diet and overnight fasting. Experiments in vivo and in vitro revealed differences in the ability of KO and WT enterocytes to assemble and secrete chylomicrons under different dietary conditions. After overnight fasting, chylomicrons secretion is reduced considerably in KO compared with WT enterocytes. This is not due to reduced synthesis of apo-B or triacylglycerol (TAG), but appears to be a result of impaired assembly of chylomicrons, so that triacylglycerol accumulates in the enterocytes. After feeding with fat, secretion of chylomicrons enriched in pre-existing TAG is stimulated in KO compared with WT mice. IN the present study, we have documented for the first time that apo-B100 is considerably less efficient that apo-B48 in exerting its role in the early stage of chylomicron assembly, which is rate-limiting in the fat-fed state. Apo-B mRNA editing may result in more efficient fat absorption, specifically under conditions of food shortage or low-fat content, and thus provide an evolutionary advantage.

 

2.23           Vesicle permeabilization by protofibrillar a-synuclein: Implications for the pathogenesis and treatment of Parkinson’s disease

Volles, M.J. et al

Biochemistry, 40, 7812-7819 (2001)

 

Fibrillar alpha-synuclein is a component of the Lewy body, the characteristic neuronal inclusion of the Parkinson's disease (PD) brain. Both alpha-synuclein mutations linked to autosomal dominant early-onset forms of PD promote the in vitro conversion of the natively unfolded protein into ordered prefibrillar oligomers, suggesting that these protofibrils, rather than the fibril itself, may induce cell death. We report here that protofibrils differ markedly from fibrils with respect to their interactions with synthetic membranes. Protofibrillar alpha-synuclein, in contrast to the monomeric and the fibrillar forms, binds synthetic vesicles very tightly via a beta-sheet-rich structure and transiently permeabilizes these vesicles. The destruction of vesicular membranes by protofibrillar alpha-synuclein was directly observed by atomic force microscopy. The possibility that the toxicity of alpha-synuclein fibrillization may derive from an oligomeric intermediate, rather than the fibril, has implications regarding the design of therapeutics for PD.

 

2.24           A rapid single-step centrifugation method for determination of HDL, LDL, and VLDL cholesterol, and TG, and identification of predominant LDL subclass

Sawle, A., Higgins, M.K., Olivant, M.P., and Higgins, J.A.

1. Lipid Res., 43, 335-343 (2002)

 

Determination of the circulating levels of plasma lipoprotein HDL, LDL, and VLDL is critical in the assessment of risk of coronary heart disease. More recently it has become apparent that the LDL subclass pattern is a further important diagnostic parameter. The reference method for separation of plasma lipoproteins is ultracentrifugation. However, current methods often involve prolonged centrifugation steps and use high salt concentrations, which can modify the lipoproteins structure and must be removed before further analysis. To overcome these problems we have now investigated the use of rapid self-generating gradients of iodixanol for separation and analysis of plasma lipoproteins. A protocol is presented in which HDL, LDL, and VLDL, characterized by electron microscopy and agarose gel electrophoresis, separate in three bands in a 2.5 h centrifugation step. Recoveries of cholesterol and TG from the gradient were close to 100%. The distribution profiles of cholesterol and TG in the gradient were used to calculate the concentrations of individual lipoprotein classes. The values correlated with those obtained using commercial kits for HDL and LDL cholesterol. The position of the LDL peak in the gradient and its shape varied between plasma samples and was indicative of the density of the predominant LDL class. The novel protocol offers a rapid, reproducible and accurate single-step centrifugation method for the determination of HDL, LDL, and VLDL, cholesterol, and TG, and identification of LDL subclass pattern.

 

2.25           Persistent and transient replication of full-length hepatitis C virus genomes in cell culture

Pietschmann, T. et al

1. Virol., 76, 4008-4021 (2002)

 

The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the en­velope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these full-length HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgl compart­ments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were de­tectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are Important for virus particle assembly and/or release.

 

2.26           Protein interactions with myocilin

Wentz-Hunter, K., Ueda, J. and Yue, B.Y.J.T.

Invest. Ophtalmol. Vis. Sci., 43(1), 176-182 (2002)

PURPOSE. To identify factors that interact in vivo with myocilin,a glaucoma gene product.

METHODS. The yeast two-hybrid system with myocilin as the baitand a human skeletal muscle cDNA library as the prey was usedto identify potential factors that interact with myocilin. Interactionswere also examined in bovine trabecular meshwork (TM) cellsthrough a mammalian two-hybrid system. Biochemical coimmunoprecipitationfrom both human TM cell lysate and in vitro translated proteinswas also used to confirm results obtained from yeast analysis.

RESULTS. Twenty positive clones isolated through yeast two-hybridscreening were deemed potential myocilin partners. Sequenceanalysis determined that two of them encoded for myocilin fromamino acids 64 to 268. Myocilin was also found to interact witha component of the myosin motor protein, myosin regulatory lightchain (RLC). The myocilin–myocilin and myocilin–RLCinteractions revealed by the yeast system were further confirmedand demonstrated in cultured TM cells, by means of a mammaliantwo-hybrid system, and through biochemical coimmunoprecipitation,subcellular fractionation, immunofluorescence, and immunogolddouble labeling.

CONCLUSIONS. These results indicate that myocilin can form homomultimers in vivo, independent of the olfactomedin-like domain. Further analysis established that the leucine zipper motif of myocilin may be necessary for the myocilin–RLC interaction. The interaction of myocilin with RLC, a component of the myosin motor protein complex, implies a role for myocilin in the actomyosin system, linking in turn this novel protein to functional status of the TM.

 

2.27           Evidence for an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial dysfunction and oxidative stress generation

Ceriello, A.C. et al

Circulation, 106, 1211-1218 (2002)

 

Background— Postprandial hypertriglyceridemia and hyperglycemiaare considered risk factors for cardiovascular disease. Evidencesuggests that postprandial hypertriglyceridemia and hyperglycemiainduce endothelial dysfunction through oxidative stress; however,the distinct role of these two factors is a matter of debate.

Methods and Results— Thirty type 2 diabetic patients and20 normal subjects ate 3 different meals: a high-fat meal; 75g glucose alone; and high-fat meal plus glucose. Glycemia, triglyceridemia,nitrotyrosine, and endothelial function were assayed duringthe tests. Subsequently, diabetics took 40 mg/d simvastatinor placebo for 12 weeks. The 3 tests were performed again atbaseline, between 3 to 6 days after the start, and at the endof each study. High-fat load and glucose alone produced a decreaseof endothelial function and an increase of nitrotyrosine innormal and diabetic subjects. These effects were more pronouncedwhen high fat and glucose were combined. Short-term simvastatintreatment had no effect on lipid parameters but reduced theeffect on endothelial function and nitrotyrosine observed duringeach different test. Long-term simvastatin treatment was accompaniedby a lower increase in postprandial triglycerides, which wasfollowed by smaller variations of endothelial function and nitrotyrosineduring the tests.

Conclusions— This study shows an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial function, suggesting oxidative stress as common mediator of such effect. Simvastatin shows a beneficial effect on oxidative stress and endothelial dysfunction, which may be ascribed to a direct effect as well as the lipid-lowering action of the drug.

 

2.28           Characterization of HCV RNA particles from the serum of a patient with common variable immunodeficiency on isotonic iodixanol (OptiPrep) gradients. Association with apolipoprotein-B100

Nielsen, S. et al

1. Hepatol., 36, Suppl. 1, 87 (2002)

 

Objective: To investigate the association of HCV RNA and lipoprotein in the serum of an antibody negative patient with common variable immunodeficiency.

Methods: The serum collected six weeks post-orthotopic liver transplant from a patient with common variable immunodeficiency and chronic HCV infection was fractionated by sequential density centrifugation on sodium bromide and by isopycnic centrifugation on isotonic iodixanol (Optiprep).

Results: Whilst sequential density centrifugation yielded HCV RNA fractions of high (> 1.2 g/ml), intermediate (1.063-1.21 g/ml) and low (< 1.063 g/ml) density of approximately equal titre, on iodixanol  gradients all the RNA was recovered at the top of the gradient with a density below 1.13 g/ml. Following precipitation with anti-apoB100 or manganese chloride and heparin the majority of the HCV RNA was removed from the serum leaving only a minor peak with a density of 1.13 g/ml on iodixanol gradients. Treatment of sera with sodium desoxycholate released further particles with a density of 1.13 g/ml in iodixanol gradients, suggesting that this is the density of the virus particle stripped of serum lipoproteins. Treatment with 0.18% NP40 produced a single peak of HCV RNA in a fraction with a density of 1.23 g/ml and a sedimentation coefficient of 150S in iodixanol gradients, taken to be naked viral cores.

Conclusions: Fractionation of HCV RNA on iodixanol in isotonic conditions indicates that essentially all RNA is present in lower density fractions and is associated with low density or very low density lipoproteins.

 

2.29           Characterization of the structural proteins of HCV isolated from human liver

Nielsen, S. et al

1. Hepatol., 36, Suppl. 1, 87 (2002)

 

Objective: To determine the molecular weights of the structural protein of HCV recovered from infected human liver.

Methods: Macerates of a six week post-orthotopic liver transplant from a patient with common variable immunodeficiency and chronic HCV infection were shown to contain 9 LogIU of HCV RNA/g. Macerates were analyzed by isopycnic centrifugation on iodixanol density gradients. HCV RNA was precipitated from crude macerates with manganese chloride and heparin and the solubilised precipitate was analysed by SDS-PAGE and western blotting with monoclonal antibodies to the viral structural proteins.

Results: Following fractionation of the liver macerate on iodixanol density gradients all the HCV RNA was recovered in fractions of density of 1.13 g/ml and below suggesting that the RNA is associated with host lipoprotein. In line with this, manganese/heparin treatment precipitated essentially all of the HCV RNA. SDS-polyacrylamide gel electrophoresis of the proteins present in manganese/heparin precipitates and western blotting revealed a single band of core protein of molecular weight 21 kDa and an E1 band of 31 kDa which migrated in approximately the same position in the gel as the corresponding proteins transiently expressed from a recombinant vaccinia virus system. A single E2 band, however, migrated with a molecular weight of 62 kDa some 8 kDa smaller than the equivalent band expressed from recombinant vaccinia virus.

Conclusions: The molecular weights of the HCV structural proteins recovered from beta-lipoprotein associated virions suggest that processing of the virus polyprotein in the liver may differ from that recombinant vaccinia virus expression system.

 

2.30           Characterization of cytoplasmic a-synuclein aggregates

Lee, H-J. and Lee, S-J.

1. Biol. Chem., 277, 48976-48983 (2002)

 

The a-synuclein fibrillation process has been associated with the pathogenesis of several neuro-degenerative diseases. Here, we have characterized the cytoplasmic a-synuclein aggregates using a fractionation procedure with which different aggregate species can be separated. Overexpression of a-synuclein in cells produce two distinct types of aggregates: large juxtanuclear inclusion bodies and small punctate aggregates scattered throughout the cytoplasm. Biochemical fractionation results in an inclusion-enriched fraction and two small aggregate fractions. Electron microscopy and thioflavin S reactivity of the fractions show that the juxtanuclear inclusion bodies are filled with amyloidlike a-synuclein fibrils, whereas both the small aggregate fractions contain non-fibrillar spherical aggregates with distinct size distributions. These aggregates appear sequentially, with the smallest population appearing the earliest and the fibrillar inclusions the latest. Based on the structural and kinetic properties, we suggest that the small spherical aggregates are the cellular equivalents of the protofibrils. The proteins that co-exist in the Lewy bodies, such as proteasome subunit, ubiquitin, and hsp70 chaperone, are present in the fibrillar inclusions but absent in the protofibrils, suggesting that these proteins may not be directly involved in the early aggregation stage. As predicted in the aggresome model, disruption of microtubules with nocodazole

reduced the number of inclusions and increased the size of the protofibrils. Despite the increased size, the

protofibrils remained non-fibrillar, suggesting that the deposition of the protofibrils in the juxtanuclear region is important in fibril formation. This study provides evidence that the cellular fibrillation also involves nonfibrillar intermediate species, and the microtubule-dependent inclusion-forming process is required for the protofibril-to-fibril conversion in cells.

 

2.31           Properties of the chaperonin comples from the halophilic archaeon Haloferax volcanii

Large, A.T., Kovacs, E. and Lund, P.A.

FEBS Lett., 532, 309-312 (2002)

 

The halophilic archaeon Haloferax volcanii has three genes encoding type II chaperonins, named cct1, cct2 and cct3. We show here that the three CCT proteins are all expressed but not to the same level. All three proteins are further induced on heat shock. The CCT proteins were purified by ammonium sulphate precipitation, sucrose gradient centrifugation and hydrophobic interaction chromatography. This procedure yields a high molecular mass complex (or complexes). The complex has ATPase activity, which is magnesium dependent, low salt-sensitive and stable to at least 75°C. Activity requires high levels of

potassium ions and was reduced in the presence of an increasing concentration of sodium ions.

 

2.32           Methods for measuring lipid metabolism in vivo

Patterson, B. W.

Curr., Opin., Clin., Nutr., Metab. Care, 5, 475-479 (2002)

 

Purpose of review

This review discusses diverse methods that have been used in several recent papers for the qualitative and quantitative analysis of lipids, studies of lipid oxidation, lipoprotein fractionation, and studies of lipid metabolism and metabolic kinetics using tracers. Papers for this review were selected on the basis of their

timeliness, novelty, and/or their potential impact on diverse fields of lipid metabolism.

Recent findings

Many methods used for studies of lipid metabolism employ advanced chromatographic and mass spectrometric techniques to characterize lipids. In particular, the use of stable isotopically labeled tracers has become increasingly important to study metabolic kinetics.

Summary

Such developments in methodology will continue to advance studies of lipid metabolism in many areas of clinical interest, including heart disease, obesity, and diabetes.

 

2.33           Functional reconstitution of purified metabotrophic glutamate receptor expressed in the fly eye

Eroglu, C., Croner, P., Panneels, V., Beaufils, P. and Sinning, I.

EMBO Reports, 3(5), 491-496 (2002)

 

G-protein-coupled receptors (GPCRs) form one of the largest superfamilies of membrane proteins. Obtaining high yields of GPCRs remains one of the major factors limiting a detailed understanding of their structure and function. Photoreceptor cells (PRCs) contain extensive stacks of specialized membranes where high levels of rhodopsins are naturally present, which makes them ideal for the overexpression of GPCRs. We have generated transgenic flies expressing a number of GPCRs in the PRCs. Drosophila melanogaster metabotropic glutamate receptor (DmGluRA) expressed by this novel strategy was purified to homogeneity, giving at least 3-fold higher yields than conventional baculovirus expression systems due to the higher membrane content of the PRCs. Pure DmGluRA was then reconstituted into liposomes of varying composition. Interestingly, glutamate binding was strictly dependent on the presence of ergosterol.

 

2.34           Vesicular restriction of synaptobrevin suggests a role for calcium in membrane fusion

Hu, K. et al

Nature, 415, 646-650 (2002)

 

Release of neurotransmitter occurs when synaptic vesicles fuse with the plasma membrane. This neuronal exocytosis is triggered by calcium and requires three SNARE (soluble-N-ethylmaleimide-sensitive factor attachment protein receptors) proteins: synaptobrevin (also known as VAMP) on the synaptic vesicle, and syntaxin and SNAP-25 on the plasma membrane. Neuronal SNARE proteins form a parallel four-helix bundle that is thought to drive the fusion of opposing membranes. As formation of this SNARE complex in solution does not require calcium, it is not clear what function calcium has in triggering SNARE-mediated membrane fusion. We now demonstrate that whereas syntaxin and SNAP-25 in target membranes are freely available for SNARE complex formation, availability of synaptobrevin on synaptic vesicles is very limited. Calcium at micromolar concentrations triggers SNARE complex formation and fusion between synaptic vesicles and reconstituted target membranes. Although calcium does promote interaction of SNARE proteins between opposing membranes, it does not act by releasing synaptobrevin from synaptic vesicle restriction. Rather, our data suggest a mechanism in which calcium-triggered membrane apposition enables syntaxin and SNAP-25 to engage synaptobrevin, leading to membrane fusion.

 

2.35           Massive and Selective Delivery of Lipid-Coated Cationic Lipoplexes of Oligonucleotides Targeted in Vivo to Hepatic Endothelial Cells

Bartsch, M., Weeke-Klimp, A.H., Meijer, D.K.F., Scherphof, G.L. and Kamps, J.A.A.M.

Pharmaceutical Res.,19(5), 676-680 (2002)

 

Purpose. Previously we reported on massive uptake of liposomes surface-modified with negatively charged aconitylated albumin (Aco-HSA) by liver sinusoidal endothelial cells (EC) in vivo. In the present work we applied this principle for the in vivo delivery of antisense oligonucleotides (ODN) to these cells.

Methods. Anti ICAM-1 ODN was complexed with the cationic lipid DOTAP and the complex was coated by an excess of neutral lipids including a lipid-anchored poly(ethylene glycol). Aco-HSA was coupled to the coated cationic lipoplexes (CCLs). Plasma disappearance, organ and intrahepatic distribution of Aco-HSA modified CCLs were determined in rats, using [³H]-cholesteryl oleyl ether and ³²P-labeled ODN as markers.

Results. The Aco-HSA coupled CCLs were <160 nm="" in="" size,="" contained="" 1.03="" ±="" 0.35="" nmol="" odn="" and="" 54="" ±="" 18=""> g Aco-HSA per mol total lipid. These CCLs were rapidly eliminated from plasma, about 60% the injected dose of ³H- or ³²P-label being recovered in the liver after 30 min. Within the liver, the EC accounted for two thirds of total liver uptake. Control non-targeted CCLs were eliminated very slowly: after 30 min still >90% of the particles was in the blood.

Conclusions. Our results demonstrate efficient targeting of antisense ODN to EC in vivo, employing plasma-stable coated cationic lipoplexes, surface modified with negatively charged albumin. 40% of the injected ODN was delivered to the target cells within 30 min.

 

2.36           Antioxidant properties of aged garlic extract: an in vitro study incorporating human low density lipoprotein

Dillon, S.A., Burmi, R.S., Lowe, G.M., Billington, D. and Rahman, K.

Life Sciences, 72, 1538-1594 (2003)

 

Oxidation of low-density lipoprotein (LDL) has been recognized as playing an important role in the development and progression of atherosclerotic heart disease. Human LDL was isolated and challenged with a range of oxidants either in the presence or absence of AGE or its diethyl ether extract. Oxidative modification of the LDL fraction using CuSO₄, 5-lipoxygenase and xanthine/xanthine oxidase was monitored by both the appearance of thiobarbituric-acid substances (TBA-RS) and an increase in electrophoretic mobility.This study indicates that AGE is an effective antioxidant as it scavenged superoxide ions and reduced lipid peroxide formation in cell free assays. Superoxide production was completely inhibited in the presence of a 10% (v/v) aqueous preparation of AGE and reduced by 34% in the presence of a 10% (v/v) diethyl ether extract of AGE. The presence of 10% (v/v) diethyl ether extract of AGE significantly reduced Cu²⁺ and 15-lipoxygenase-mediated lipid peroxidation of isolated LDL by 81% and 37%, respectively. In addition, it was found that AGE also had the capacity to chelate copper ions. In contrast, the diethyl ether extract of AGE displayed no copper binding capacity, but demonstrated distinct antioxidant properties. These results support the view that AGE inhibits the in vitro oxidation of isolated LDL by scavenging superoxide and inhibiting the formation of lipid peroxides. AGE was also shown to reduce LDL oxidation by the chelation of Cu²⁺. Thus, AGE may have a role to play in preventing the development and progression of atherosclerotic disease.

 

2.37           Separation of bovine plasma lipoproteins by a rapid ultracentrifugation method

Gardner, R.S., Ogden, N.H., Cripps, P.J. and Billington, D.

1. Comp. Path., 128, 15-23 (2003)

 

The recently described method of centrifugation with iodixanol for the rapid separation of human plasma lipoproteins was adapted to separate bovine plasma lipoproteins. Density gradients were generated by mixing plasma with iodixanol 12% (w/v), followed by centrifugation at 350000g and 16 degrees C for 3h 10min in a vertical rotor. Gradients were unloaded dense-end first into 10 fractions. Human very low density lipoprotein (VLDL; density <1.011g/ml), low density lipoprotein (LDL; density = 1.016-1.039 g/ml) and high density lipoprotein (HDL; density = 1.039-1.090 g/ml) were resolved well at densities considerably lower than those traditionally reported in salt gradients. In gradients generated from 12% iodixanol, bovine LDL and HDL exhibited even lower densities (1.016-1.028 and 1.016-1.048g/ml, respectively) with all lipoproteins occurring at the lower density region of the gradient. In contrast, density gradients generated from layers of equal volumes of 6% and 12% iodixanol readily separated bovine HDL from VLDL, whilst LDL still overlapped with HDL. The latter accounts for >80% of all bovine lipoproteins and exists as two populations, namely light and heavy HDL. Gradients generated from two layers of iodixanol recovered bovine HDL in five fractions. The hypercholesterolaemia associated with lactation resulted in a modest shift in the profile of HDL cholesterol towards lipoprotein particles of lower density (light HDL). Significant between-farm differences were also detected in the density profiles of bovine plasma cholesterol. This new method is suitable for use in research and diagnosis in relation to lipoprotein metabolism disorders in cows.

 

2.38           Characterization of the genome and structural proteins of b-lipoprotein associated HCV extracted from infected human liver

Nielsen, S., Bassendine, M.F., Burt, A. And Toms, G.L.

GUT, Abstract from the British association for the study of liver meeting 2002, abstract 94 (2003)

 

Serum and liver macerates from a patient with common variable immunodeficiency and chronic HCV infection six weeks post-transplant were analysed by isopycnic centrifugation on isotonic iodixanol (optiprep) density gradients. All of the HCV RNA fractionated at the top of the gradient, in fractions of density of < 1.13 g/ml and was precipitable with manganese chloride and heparin (Mn/Hep) indicating that it is all associated with host beta-lipoprotein. Treatment with desoxycholate released putative hepatitis C virions with a density of 1.13 g/ml and treatment with 0.18% NP40 released putative virus cores with a density of 1.21 g/ml and a sedimentation coefficient of 150S. Northern blotting of Mn/Hep precipitates revealed a single band of HCV RNA of 9.4 kb corresponding in size to the full HCV genome. SDS-polyacrylamide gel electrophoresis and western blotting with monoclonal antibodies revealed a single 20 kDa band of core protein and a single 31 kDa E1 band reduced to 20 kDa after deglycosylation with endoglycosidase F. Both core and E1 bands co-migrated with corresponding bands derived from a recombinant vaccinia virus system expressing the HCV structural protein genes. Anti-E2 Mabs blotted a single 62 kDa band from Mn/Hep precipitates, approximately 7 kDa smaller than the anti-E2 staining band in the recombinant vaccinia virus system. Following deglycosylation, E2 glycoprotein ran with an apparent molecular weight of 36 kDa, co-migrating with E2, which forms a minor band in the deglycosylated vaccinia virus recombinant. No band equivalent to the major 40 kDa E2/P7 band in the deglycosylated vaccinia virus system was observed in the Mn/Hep precipitates, indicating that E2/P7 is not a structural protein. Under reducing conditions both E1 and E2 ran as monomers, suggesting that the two glycoproteins are not disulphide linked in the virion.

 

2.39           A novel role for CD36 in VLDL-enhanced platelet activation

Englyst, N.A., Taube, J.M., Aitman, T.J., Baglin, T.P. and Byrne C.D.

Diabetes, 52, 1248-1255 (2003)

 

Type 2 diabetes is characterized by increased plasma triglyceride levels and a fourfold increase in ischemic heart disease, but the mechanism is unclear. CD36 is a receptor/transporter that binds fatty acids of lipoproteins. CD36 deficiency has been linked with insulin resistance. There is strong evidence of in vivo interaction between platelets and atherogenic lipoproteins suggesting that atherogenic triglyceride-rich lipoproteins, such as VLDL, that are increased in diabetic dyslipidemia are important in this process. This study demonstrates that VLDL binds to the platelet receptor CD36, enhances platelet thromboxane A2 production, and causes increased collagen-mediated platelet aggregation. VLDL enhanced collagen-induced platelet aggregation by 1) shortening the time taken for aggregation to begin (lag time) to 70% of control (P = 0.001); 2) increasing maximum aggregation to 170% of control (P = 0.008); and 3) increasing thromboxane production to 3,318% of control (P = 0.004), where control represents platelets stimulated with collagen (100%). A monoclonal antibody against CD36 attenuated VLDL-enhanced collagen-induced platelet aggregation by 1) inhibiting binding of VLDL to platelets by 75% (P = 0.041); 2) lengthening lag time to 190% (P < 0.001); and 3) decreasing thromboxane production to 8% of control (P < 0.001). In support of this finding, platelets from Cd36-deficient rats showed no increase in aggregation, thromboxane production, and VLDL binding in contrast to platelets from rats expressing CD36. These data suggest that platelet Cd36 has a key role in VLDL-induced collagen-mediated platelet aggregation, possibly contributing to atherothrombosis associated with increased VLDL levels.

 

2.40           Rapid separation of LDL subclasses by iodixanol gradient ultracentrifugation

Davies, I.G., Graham, J.M. and Griffin, B.A.

Clin. Chem., 49(11), 1865-1872 (2003)

 

Background: A predominance of small, dense LDL (sdLDL) confersin excess of a threefold increase in coronary heart disease(CHD) risk. The conventional method for the detection of sdLDL,salt density gradient ultracentrifugation (DGUC) has been supersededby more rapid techniques. This report presents novel methodologyfor the separation of sdLDL by a combination of iodixanol densitygradient centrifugation and digital photography.

Methods: LDL subclasses were separated in 3 h from prestainedplasma on a self-forming density gradient of iodixanol. LDLsubclass profiles were generated by digital photography andgel-scan software. Plasma samples from 106 normo- and dyslipidemicindividuals were used to optimize the gradient for the resolutionof LDL heterogeneity. A subgroup of 47 LDL profiles were thencompared with LDL subclasses separated by salt DGUC.

Results: The peak density of the predominant LDL band correlated significantly with the relative abundance (as a percentage) of sdLDL as resolved by salt DGUC (P <0.001). As shown previously, LDL isolated at a lighter density in iodixanol compared with salt gradients. A predominance of sdLDL corresponded to a peak density on iodixanol of 1.028 kg/L. This density and the area under the LDL profile lying above this density were sensitive and specific markers for the prediction of a predominance of sdLDL (P <0.001) and showed predictable associations with plasma triglycerides (r = 0.59; P <0.001) and HDL (r = -0.4; P <0.001).

Conclusions: This simple method for the detection of sdLDL candifferentiate a predominance of sdLDL, is highly reproducible,and can be used preparatively to isolate sdLDL.

 

2.41           Protein-protein, protein-RNA and protein-lipid interactions of signal-recognition particle components in the hyperthermoacidophilic archeon Arcidianus ambivalens

Moll, R.G.

Biochem. J., 374, 247-254 (2003)

 

The signal-recognition particle (SRP) of one of the most acidophilic and hyperthermophilic archaeal cells, Acidianus ambivalens, and its putative receptor component, FtsY (prokaryotic SRP receptor), were investigated in detail. A. ambivalens Ffh (fifty-four-homologous protein) was shown to be a soluble protein with strong affinity to membranes. In its membrane-residing form, Ffh was extracted from plasma membranes with chaotropic agents like urea, but not with agents diminishing electrostatic interactions. Using unilamellar tetraether phospholipid vesicles, both Ffh and FtsY associate independently from each other in the absence of other factors, suggesting an equilibrium of soluble and membrane-bound protein forms under in vivo conditions. The Ffh protein precipitated from cytosolic cell supernatants with anti-Ffh antibodies, together with an 7 S-alike SRP–RNA, suggesting a stable core ribonucleoprotein composed of both components under native conditions. The SRP RNA of A. ambivalens depicted a size of about 309 nucleotides like the SRP RNA of the related organism Sulfolobus acidocaldarius. A stable heterodimeric complex composed of Ffh and FtsY was absent in cytosolic super-natants, indicating a transiently formed complex during archaeal SRP targeting. The FtsY protein precipitated in cytosolic super-natants with anti-FtsY antisera as a homomeric protein lacking accessory protein components. However, under in vitro conditions, recombinantly generated Ffh and FtsY associate in a nucleotide-independent manner, supporting a structural receptor model with two interacting apoproteins.

 

2.42           The effect of CTAB concentration in cationic PLG microparticles on DNA adsorption and in vivo performance

Singh, M. et al

Pharmaceut, Res., 20(2), 247-251 (2003)

 

Purpose. Cationic PLG microparticles with adsorbed DNA have previously been shown to efficiently target antigen presenting cells in vivo for generating higher immune responses in comparison to naked DNA. In this study we tried to establish the role of surfactant (CTAB) concentration on the physical behavior of these formulations.

Methods. Cationic PLG microparticle formulations with adsorbed DNA were prepared using a solvent evaporation technique. Formulations with varying CTAB concentrations and a fixed DNA load were prepared. The loading efficiency and 24 h DNA release was evaluated for each formulation. Select formulations were tested in vivo.

Results. Higher CTAB concentration correlated with higher DNA binding efficiency on the microparticles and lower in vitro release rates. Surprisingly though, the in vivo performance of formulations with varying CTAB concentration was comparable to one another.

Conclusions. Cationic PLG microparticles with adsorbed DNA, as described here, offer a robust way of enhancing in vivo responses to plasmid DNA.

 

2.43           Trans Unsaturated Fatty Acids Are Less Oxidizable than Cis Unsaturated Fatty Acids and Protect Endogenous Lipids from Oxidation in Lipoproteins and Lipid Bilayers

Sargis, R.M. and Subbaiah, P.V.

Biochemistry, 42, 11533-11543 (2003)

 

Epidemiological data suggest that dietary trans unsaturated fatty acids increase the risk of heart disease; however, the underlying mechanisms are unclear. In this study, we investigated one possible mechanism, namely, their effect on LDL oxidation. Supplementation of LDL with 10% 16:1 trans-cholesteryl ester (CE) inhibited the oxidation compared to that with 16:1 cis-CE. Total replacement of core lipids with 18:2 trans,trans-CE decreased the rate of LDL oxidation by 19% compared to replacement with 18:2 cis,cis-CE. When the surface phosphoglycerides were replaced with either 16:0-18:2 cis,cis-phosphatidylcholine (PC) or 16:0-18:2 trans,trans-PC, the latter was found to inhibit the rate and increase the lag time of oxidation to a greater extent than the former. To confirm these findings, we studied the oxidation of PC liposomes by assessing the formation of conjugated dienes or the degradation of a fluorescently labeled PC. By both methods, the 16:0-18:2 trans,trans-PC exhibited greater resistance to oxidation than the 16:0-18:2 cis,cis-PC. Eliminating the fluidity differences did not completely eliminate the differences in oxidation rates, suggesting that the trans double bond is inherently resistant to oxidation. The composition of the conjugated hydroperoxy products formed after oxidation differed markedly for the two 18:2 isomers. Supplementation of 16:0-18:2 cis,cis-PC liposomes with 20 mol % di16:1 trans-PC retarded oxidation rates to a greater extent than supplementation with di16:1 cis-PC. These studies show that dietary trans unsaturated fatty acids decrease the rate of lipid peroxidation, an effect that may mitigate the atherogenic effect of these fatty acids.

 

2.44           Copper-mediated LDL oxidation by homocysteine and related compounds depends largely on copper ligation

Nakano, E., Williamson, M.P., Williams, N.H. and Powers, H.J.

Biochim. Biophys. Acta, 1688, 33-42 (2004)

 

Oxidation of low-density lipoprotein (LDL) is thought to be a major factor in the pathophysiology of atherosclerosis. Elevated plasma homocysteine is an accepted risk factor for atherosclerosis, and may act through LDL oxidation, although this is controversial. In this study, homocysteine at physiological concentrations is shown to act as a pro-oxidant for three stages of copper-mediated LDL oxidation (initiation, conjugated diene formation and aldehyde formation), whereas at high concentration, it acts as an antioxidant. The affinity for copper of homocysteine and related copper ligands homocysteine, cystathionine and djenkolate was measured, showing that at high concentrations (100 M) under our assay conditions, they bind essentially all of the copper present. This is used to rationalise the behaviour of these ligands, which stimulate LDL oxidation at low concentration but generally inhibit it at high concentration. Albumin strongly reduced the effect of homocystine on lag time for LDL oxidation, suggesting that the effects of homocystine are due to copper binding. In contrast, copper binding does not fully explain the pro-oxidant behaviour of low concentrations of homocysteine towards LDL, which appears in part at least to be due to stimulation of free radical production. The likely role of homocysteine in LDL oxidation in vivo is discussed in the light of these results.

 

2.45           Effect of postprandial hypertriglyceridemia and hyperglycemia on circulating adhesion molecules and oxidative stress generation and the possible role of  simvastatin treatment

Ceriello, A. et al

Diabetes, 53, 701-710 (2004)

 

Adhesion molecules, particularly intracellular adhesion molecule(ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin,have been associated with cardiovascular disease. Elevated levelsof these molecules have been reported in diabetic patients.Postprandial hypertriglyceridemia and hyperglycemia are consideredrisk factors for cardiovascular disease, and evidence suggeststhat postprandial hypertriglyceridemia and hyperglycemia mayinduce an increase in circulating adhesion molecules. However,the distinct role of these two factors is a matter of debate.Thirty type 2 diabetic patients and 20 normal subjects ate threedifferent meals: a high-fat meal, 75 g of glucose alone, anda high-fat meal plus glucose. Glycemia, triglyceridemia, plasmanitrotyrosine, ICAM-1, VCAM-1, and E-selectin were assayed duringthe tests. Subsequently, diabetic subjects took simvastatin40 mg/day or placebo for 12 weeks. The three tests were performedagain at baseline, between 3 and 6 days after starting the study,and at the end of each study. High-fat load and glucose aloneproduced an increase of nitrotyrosine, ICAM-1, VCAM-1, and E-selectinplasma levels in normal and diabetic subjects. These effectswere more pronounced when high fat and glucose were combined.Short-term simvastatin treatment had no effect on lipid parameters,but reduced the effect on adhesion molecules and nitrotyrosine,which was observed during every different test. Long-term simvastatintreatment was accompanied by a lower increase in postprandialtriglycerides, which was followed by smaller variations in ICAM-1,VCAM-1, E-selectin, and nitrotyrosine during the tests. Thisstudy shows an independent and cumulative effect of postprandialhypertriglyceridemia and hyperglycemia on ICAM-1, VCAM-1, andE-selectin plasma levels, suggesting oxidative stress as a commonmediator of such effects. Simvastatin shows a beneficial effecton oxidative stress and the plasma levels of adhesion molecules,which may be ascribed to a direct effect in addition to thelipid-lowering action of the drug.

 

2.46           Effects of Rosiglitazone on endothelial function in men with coronary artery disease without diabetes mellitus

Sidhu, J.S., Cowan, D. and Kaski, J.C.

Am. J. Cardiol., 94, 151-156 (2004)

 

Recent data have shown that peroxisome proliferator-activated receptor- agonists may exert protective effects on the vascular endothelium by amelioration of insulin resistance and through direct anti-inflammatory effects. In this study we assessed the effect of rosiglitazone on biochemical and biophysical indexes of endothelial function in male, nondiabetic patients with coronary artery disease. Consecutive male subjects (n = 71) with clinically stable, angiographically documented coronary artery disease and without diabetes mellitus were investigated. Patients were randomized in a double-blind manner to placebo or rosiglitazone for a total of 24 weeks. Flow-mediated dilation (FMD) of the brachial artery, C-reactive protein, von Willebrand factor, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 levels, and parameters of glucose and lipid metabolism were measured at baseline and after 12 and 24 weeks of treatment. Rosiglitazone treatment significantly reduced C-reactive protein (median 0.56 mg/L [interquartile range 0.33 to 1.02] to 0.33 mg/L [interquartile range 0.26 to 0.40], p <0.01), von Willebrand factor (139 ± 47 to 132 ± 44 IU/dl, P = 0.02), insulin resistance index (p = 0.05), and mean low-density lipoprotein (LDL) density (p <0.001) compared with placebo. However, no significant differences were seen between the rosiglitazone and placebo groups with regard to brachial artery FMD, intercellular adhesion molecule-1, or vascular cell adhesion molecule-1 levels. Rosiglitazone treatment significantly increased LDL (2.62 ± 0.72 to 2.95 ± 0.84 mmol/L, P = 0.03) and triglyceride (1.23 ± 0.63 to 1.56 ± 0.98 mmol/L, P = 0.04) levels. Thus, rosiglitazone reduced markers of inflammation and endothelial activation, but this did not translate into an improvement in FMD. Increased LDL and triglyceride levels may have played a role.

 

2.47           Plasma appearance of unesterified astaxanthin geometrical E/Z and optical R/S isomers in men given single doses of a micture of optical 3 and 3’R/S isomers of astaxanthin fatty acyl diesters

Coral-Hinostroza, G.N., Ytrestøyl, T., Ruyter, B. and Bjerkeng, B.

Comp. Biochem. Biophys.Part C, 139, 99-110 (2004)

 

Appearance, pharmacokinetics and distribution of astaxanthin all-E-, 9Z- and 13Z-geometrical and (3R,3′R)-, (3R,3′S)- and (3S,3′S)-optical isomers in plasma fractions were studied in three middle-aged male volunteers (41–50 years) after ingestion of a single meal containing first a 10-mg dose equivalent of astaxanthin from astaxanthin diesters, followed by a dose of 100 mg astaxanthin equivalents after 4 weeks. Direct resolution of geometrical isomers and optical isomers of astaxanthin dicamphanates by HPLC after saponification showed that the astaxanthin consisted of 95.2% all-E-, 1.2% 9Z- and 3.6% 13Z-astaxanthin, of (3R,3′R)-, (3R,3′S; meso)- and (3S,3′S)-astaxanthin in a 31:49:20 ratio. The plasma astaxanthin concentration–time curves were measured during 76 h. Astaxanthin esters were not detected in plasma. Maximum levels of astaxanthin (C_max=0.28±0.1 mg/l) were reached 11.5 h after administration and the plasma astaxanthin elimination half-life was 52±40 h. The C_max at the low dose was 0.08 mg/l and showed that, the dose response was non-linear. The (3R,3′R)-astaxanthin optical isomer accumulated selectively in plasma compared to the (3R,3′S)- and (3S,3′S)-isomers, and comprised 54% of total astaxanthin in the blood and only 31% of total astaxanthin in the administered dose. The astaxanthin Z-isomers were absorbed selectively into plasma and comprised 32% of total astaxanthin 6–7.5 h postprandially. The proportion of all-E-astaxanthin was significantly higher in the very low density lipoproteins and chylomicrons (VLDL/CM) plasma lipoprotein fraction than in the high density lipoproteins (HDL) and low denisty lipoproteins (LDL) fractions (P<0.05). The results indicate that a selective process increase the relative proportion of astaxanthin Z-isomers compared to the all-E-astaxanthin before uptake in blood and that the astaxanthin esters are hydrolyzed selectively during absorption.

 

2.48           Prevention of Alzheimer’s disease-associated Ab aggregation by rationally designed nonpeptidic b-sheet logands

Rzepecki, P. et al

1. Biol. Chem., 279(46), 47497-47505 (2004)

 

A new concept is introduced for the rational design of -sheet ligands, which prevent protein aggregation. Oligomeric acylated aminopyrazoles with a donor-acceptor-donor (DAD) hydrogen bond pattern complementary to that of a -sheet efficiently block the solvent-exposed -sheet portions in A -(1–40) and thereby prevent formation of insoluble protein aggregates. Density gradient centrifugation revealed that in the initial phase, the size of A aggregates was efficiently kept between the trimeric and 15-meric state, whereas after 5 days an additional high molecular weight fraction appeared. With fluorescence correlation spectroscopy (FCS) exactly those two, i.e. a dimeric aminopyrazole with an oxalyl spacer and a trimeric head-to-tail connected aminopyrazole, of nine similar aminopyrazole ligands were identified as efficient aggregation retardants whose minimum energy conformations showed a perfect complementarity to a -sheet. The concentration dependence of the inhibitory effect of a trimeric aminopyrazole derivative allowed an estimation of the dissociation constant in the range of 10^–5 M. Finally, electrospray ionization mass spectrometry (ESI-MS) was used to determine the aggregation kinetics of A -(1–40) in the absence and in the presence of the ligands. From the comparable decrease in A monomer concentration, we conclude that these -sheet ligands do not prevent the initial oligomerization of monomeric A but rather block further aggregation of spontaneously formed small oligomers. Together with the results from density gradient centrifugation and fluorescence correlation spectroscopy it is now possible to restrict the approximate size of soluble A aggregates formed in the presence of both inhibitors from 3- to 15-mers.

 

2.49           A common mechanism for the regulation of vesicular SNAREs on phospholipid membranes

Hu, K., Rickman, C., Carroll, J. and Davletov, B.

Biochem. J., 377, 781-795 (2004)

 

The SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) family of proteins is essential for membrane fusion in intracellular traffic in eukaryotic organisms. v-SNAREs (vesicular SNAREs) must engage target SNAREs in the opposing membrane to form the fusogenic SNARE complex. Temporal and spatial control of membrane fusion is important for many aspects of cell physiology and may involve the regulation of the SNAREs resident on intracellular membranes. Here we show that the v-SNARE synaptobrevin 2, also known as VAMP (vesicle-associated membrane protein) 2, is restricted from forming the SNARE complex in chromaffin granules from adrenal medullae to the same degree as in brain-purified synaptic vesicles. Our analysis indicates that the previously reported synaptophysin–synaptobrevin interaction is not likely to be involved in regulation of the v-SNARE. Indeed, the restriction can be reproduced for two distinct v-SNARE homologues, synaptobrevin 2 and cellubrevin/VAMP3, by reconstituting them in pure liposomal membranes. Overall, our data uncover a common mechanism for the control of SNARE engagement where intact phospholipid membranes rather than proteins down-regulate vesicular SNAREs in different cellular organelles.

 

2.50           Hyperlipidemic subjects have reduced uptake of newly absorbed vitamin E into their plasma lipoproteins, erythrocytes, platelets, and lymphocytes, as studied by deuterium-labeled a-tocopherol biokinetics

Hall, W.L., Jeanes, Y.M. and Lodge, J.K.

1. Nutr., 135, 58-63 (2005)

 

Vitamin E homeostasis in hyperlipidemia is poorly understood. The biokinetics of deuterated -tocopherol ( -T) in blood components was investigated in normolipidemic (N; total cholesterol < 5.5 mmol/L and triglycerides < 1.5 mmol/L, n = 9), hypercholesterolemic (HC; total cholesterol > 6.5 mmol/L and triglycerides < 1.5 mmol/L, n = 10), and combined hypercholesterolemic and hypertriglyceridemic (HCT; total cholesterol > 6.5 mmol/L and triglycerides > 2.5 mmol/L, n = 6) subjects. Subjects ingested 150 mg hexadeuterated RRR- -tocopheryl acetate, and blood was collected up to 48 h after ingestion. Labeled -T was measured in plasma, lipoproteins, erythrocytes, platelets, and lymphocytes by liquid chromatography/mass spectroscopy. In plasma, HC had an earlier time of maximum concentration (6 h) compared with N and HCT (12 h) (P < 0.05). HCT had a lower uptake of labeled -T (P < 0.005) and a longer half-life (P < 0.05). In chylomicrons, the maximum labeled -T concentration was higher in HC compared with N and HCT (P < 0.00005); however, HCT had a lower uptake of labeled -T in LDL. In all groups, the lowest density LDL subfraction contained more labeled -T than denser subfractions (P < 0.05). In platelets, lymphocytes, and erythrocytes, the areas under the labeled -T concentration vs. time curves were in the order N > HC > HCT. In lymphocytes, differences in labeled -T were found at 6 and 48 h (P < 0.05). These data demonstrate that there are differences in the uptake of newly absorbed -T into blood components in hyperlipidemia. Because these blood components are functionally affected by vitamin E, reduced uptake of -T may be relevant to the pathogenesis of atherosclerosis.

  

2.51           Cell surface heparan sulfate proteoglycans contribute to intracellular lipid accumulation in adipocytes

Wilsie, L.C., Chanchani, S., Navaratna, D. and Orlando, R.A.

Lipids in Health and Disease, 4(2), 1-15 (2005)

 

Background

Transport of fatty acids within the cytosol of adipocytes and their subsequent assimilation into lipid droplets has been thoroughly investigated; however, the mechanism by which fatty acids are transported across the plasma membrane from the extracellular environment remains unclear. Since triacylglycerol-rich lipoproteins represent an abundant source of fatty acids for adipocyte utilization, we have investigated the expression levels of cell surface lipoprotein receptors and their functional contributions toward intracellular lipid accumulation; these include very low density lipoprotein receptor (VLDL-R), low density lipoprotein receptor-related protein (LRP), and heparan sulfate proteoglycans (HSPG).

Results

We found that expression of these three lipoprotein receptors increased 5-fold, 2-fold, and 2.5-fold, respectively, during adipocyte differentiation. The major proteoglycans expressed by mature adipocytes are of high molecular weight (>500 kD) and contain both heparan and chondroitin sulfate moieties. Using ligand binding antagonists, we observed that HSPG, rather than VLDL-R or LRP, play a primary role in the uptake of DiI-lableled apoE-VLDL by mature adipocytes. In addition, inhibitors of HSPG maturation resulted in a significant reduction (>85%) in intracellular lipid accumulation.

Conclusions

These results suggest that cell surface HSPG is required for fatty acid transport across the plasma membrane of adipocytes.

 

2.52           Optimized targeting of polyethylene glycol-stabilized anti-intercellular adhesion molecule 1 oligonucleotide/lipid particles to liver sinusoidal endothelial cells

Bartsch, M. et al

Mol. Pharmacol., 67(3), 883-890 (2005)

 

We prepared polyethylene glycol (PEG)-stabilized antisense oligonucleotide (ODN)/lipid particles from a lipid mixture including the positively charged amphiphile 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and anti-intercellular adhesion molecule 1 (ICAM-1) antisense ODN by an extrusion method in the presence of 40% ethanol. These particles were targeted to scavenger receptors on liver endothelial cells by means of covalently coupled polyanionized albumin. Two types of such targeted particles were prepared, one with the albumin coupled to a maleimide group attached to the particle's lipid bilayer and the other with the protein coupled to a maleimide group attached at the distal end of added bilayer-anchored PEG chains. Upon intravenous injection, the ODN particles with bilayer-coupled albumin were cleared from the blood circulation at the same low rate as untargeted particles (<5% in 30 min). By contrast, the distal-end coupled particles were very rapidly cleared from the blood and preferentially taken up by the endothelial cells of the hepatic sinusoid (55% of injected dose after 30 min). Despite this substantial endothelial targeting, no consistent inhibition of ICAM-1 expression could be demonstrated in this cell type, either in vivo or in vitro. However, in J774 cells that also express scavenger receptors and ICAM-1, significant down-regulation of ICAM-1 mRNA was achieved with distal-end targeted lipid particles, as determined with real-time RT-PCR. It is concluded that massive delivery of ODN to cell types that express scavenger receptors can be achieved if lipid particles are provided with negatively charged albumin distally attached to bilayer anchored PEG chains.

 

2.53           Downstream effects on human low density lipoprotein of homoocysteine exported from endothelial cells in an in vitro system

Nakano, E. et al

1. Lipid Res., 46, 484-493 (2005)

 

A model system is presented using human umbilical vein endothelial cells (HUVECs) to investigate the role of homocysteine (Hcy) in atherosclerosis. HUVECs are shown to export Hcy at a rate determined by the flux through the methionine/Hcy pathway. Additional methionine increases intracellular methionine, decreases intracellular folate, and increases Hcy export, whereas additional folate inhibits export. An inverse relationship exists between intracellular folate and Hcy export. Hcy export may be regulated by intracellular S-adenosyl methionine rather than by Hcy. Human LDLs exposedto HUVECs exporting Hcy undergo time-related lipid oxidation,a process inhibited by the thiol trap dithionitrobenzoate. Thisis likely to be related to the generation of hydroxyl radicals,which we show are associated with Hcy export. Although Hcy isthe major oxidant, cysteine also contributes, as shown by theeffect of glutamate. Finally, the LDL oxidized in this systemshowed a time-dependent increase in uptake by human macrophages,implying an upregulation of the scavenger receptor.

These results suggest that continuous export of Hcy from endothelialcells contributes to the generation of extracellular hydroxylradicals, with associated oxidative modification of LDL andincorporation into macrophages, a key step in atherosclerosis.Factors that regulate intracellular Hcy metabolism modulatethese effects.

 

2.54           Evidence for the presence of three distinct binding sites for the thioflavin T class of Alzheimer’s disease PET imaging agents on b-amyloid peptide fibrils

Lockhart, A. et al

1. Biol. Chem., 280(9), 7677-7684 (2005)

 

Imaging the progression of Alzheimer's disease would greatly facilitate the discovery of therapeutics, and a wide range of ligands are currently under development for the detection of -amyloid peptide (A )-containing plaques by using positron emission tomography. Here we report an in-depth characterization of the binding of seven previously described ligands to in vitro generated A -(1–40) polymers. All of the compounds were derived from the benzothiazole compound thioflavin T and include 2-[4'-(methylamino)phenyl]benzothiazole and 2-(4'-dimethylamino-)phenyl-imidazo[1,2-a]-pyridine derivatives, 2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole and 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and a benzofuran compound (5-bromo-2-(4-dimethylaminophenyl)benzofuran). By using a range of fluorescent and radioligand binding assays, we find that these compounds display a more complex binding pattern than described previously and are consistent with three classes of binding sites on the A fibrils. All of the compounds bound with very high affinity (low nM K_d) to a low capacity site (BS3) (1 ligand-binding site per 300 A -(1–40) monomers) consistent with the previously recognized binding site for these compounds on the fibrils. However, the compounds also bound with high affinity (K_d 100 nM) to either one of two additional binding sites on the A -(1–40) polymer. The properties of these sites, BS1 and BS2, suggest they are adjacent or partially overlapping and have a higher capacity than BS3, occurring every 35 or every 4 monomers of A -(1–40)-peptide, respectively. Compounds appear to display selectivity for BS2 based on the presence of a halogen substitution (2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole, 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and 5-bromo-2-(4-dimethylaminophenyl)benzofuran) on their aromatic ring system. The presence of additional ligand-binding sites presents potential new targets for ligand development and may allow a more complete modeling of the current positron emission tomography data.

 

2.55           Distinct signaling particles containing ERK/MEK and B-Raf in PC12 cells

MacCormick, M. et al

Biochem. J., 387, 155-164 (2005)

 

Although several multiprotein complexes containing MAPKs (mitogen-activated protein kinases) have been identified using overexpression of kinases and scaffold proteins, the components of the complexes and their physical properties at endogenous expression levels have not been defined. We characterized a large protein complex containing a nerve-growth-factor-activated ERK (extracellular-signal-regulated kinase) and MEK (MAPK/ERK kinase) in rat pheochromocytoma (PC12) cells. This protein complex fractionated into a high-speed pellet and was resistant to non-ionic detergent treatments that solubilized membranes. Disruption of protein–protein interactions by treatment with high salt was required to facilitate immunoprecipitation of active ERK1 and co-precipitation of MEK1. Microtubule fragments were also present in the detergent-resistant high-speed pellet, and some kinases were bound to them, especially ERK1b (an alternatively spliced isoform of ERK1), which showed a strong preference for binding microtubules. The large protein complex containing ERK1 and MEK1 was resolved by velocity sedimentation from fragments of microtubules; however, it did not contain other scaffolding components known to bind ERK and MEK. B-Raf was also present in a distinct detergent-resistant, microtubule-independent protein complex slightly larger than that containing ERK and MEK. We conclude that there are two independent nerve growth factor-regulated ‘signalling particles’ with an estimated size of 60–75 S, one containing ERK1 and MEK1 and the other containing B-Raf. These signalling particles may have a role in the temporal and spatial regulation of kinase activity inside cells.

 

2.56           A Dictyostelium homologue of WASP is required for polarized F-actin assembly during chemotaxis

Myers, S.A., Han, J.W., Lee, Y., Firtel, R.A. and Chung, C.Y.

Mol. Biol. Cell, 16, 2191-2206 (2005)

 

The actin cytoskeleton controls the overall structure of cells and is highly polarized in chemotaxing cells, with F-actin assembled predominantly in the anterior leading edge and to a lesser degree in the cell's posterior. Wiskott-Aldrich syndrome protein (WASP) has emerged as a central player in controlling actin polymerization. We have investigated WASP function and its regulation in chemotaxing Dictyostelium cells and demonstrated the specific and essential role of WASP in organizing polarized F-actin assembly in chemotaxing cells. Cells expressing very low levels of WASP show reduced F-actin levels and significant defects in polarized F-actin assembly, resulting in an inability to establish axial polarity during chemotaxis. GFP-WASP preferentially localizes at the leading edge and uropod of chemotaxing cells and the B domain of WASP is required for the localization of WASP. We demonstrated that the B domain binds to PI(4,5)P₂ and PI(3,4,5)P₃ with similar affinities. The interaction between the B domain and PI(3,4,5)P₃ plays an important role for the localization of WASP to the leading edge in chemotaxing cells. Our results suggest that the spatial and temporal control of WASP localization and activation is essential for the regulation of directional motility.

 

2.57           Monomeric and dimeric states exhibited by the kinesin-related motor protein KIF1A

Rashid, D.J., Bononi, J., Tripet, B.P., Hodges, R.S. and Pierce, D.W.

J.Peptide Res., 65, 538-549 (2005)

 

KIF1A, a kinesin-related motor protein that transports pre-synaptic vesicles in neurons, was originally presumed to translocate along microtubules (MT) as a monomer. Protein structure predictions from its amino acid sequence failed to identify the long coiled-coil domains typical of kinesins, which led researchers to believe it does not oligomerize into the canonical kinesin dimer. However, mounting evidence using recombinant chimeric protein indicates that KIF1A, like conventional kinesin, requires dimerization for fast, unidirectional processive movement along MTs. Because these studies are somewhat indirect, we wished to test the oligomerization state of native KIF1A, and to compare that to full-length recombinant protein. We have performed hydrodynamic analyses to determine the molecular weights of the respective complexes. Our results indicate that most native KIF1A is soluble and indeed monomeric, but recombinant KIF1A is a dimer. MT-binding studies also showed that native KIF1A did not bind to MTs in either the presence of AMP-PNP, apyrase, or adenosine triphosphate (ATP), but recombinant KIF1A bound to MTs most stably in the presence of ATP, indicating very different motor functional states. To further characterize KIF1A's dimerization potential, we prepared peptides corresponding to the neck domains of MmKIF1A and CeUnc104, and by circular dichroism spectroscopy compared these peptides for their ability to form coiled-coils. Interestingly, both MmKIF1A and CeUnc104 neck peptides formed homodimeric coiled-coils, with the MmKIF1A neck coiled-coil exhibiting the greater stability. Collectively, from our data and from previous studies, we predict that native KIF1A can exist as both an inactive monomer and an active homodimer formed in part through its neck coiled-coil domain.

 

2.58           Effect of Atorvastatin and Irbesartan, alone and in combination, on postprandial endothelial dysfunction, oxidative stress, and inflammation in type 2 diabetic patients

Ceriello, A. et al

Circulation, 111, 2518-2524 (2005)

 

Background— Postprandial hypertriglyceridemia and hyperglycemiaare considered risk factors for cardiovascular disease. Evidencesuggests that postprandial hypertriglyceridemia and hyperglycemiainduce endothelial dysfunction and inflammation through oxidativestress. Statins and angiotensin type 1 receptor blockers havebeen shown to reduce oxidative stress and inflammation, improvingendothelial function.

Methods and Results— Twenty type 2 diabetic patients ate3 different test meals: a high-fat meal, 75 g glucose alone,and a high-fat meal plus glucose. Glycemia, triglyceridemia,endothelial function, nitrotyrosine, C-reactive protein, intercellularadhesion molecule-1, and interleukin-6 were assayed during thetests. Subsequently, diabetics took atorvastatin 40 mg/d, irbesartan300 mg/d, both, or placebo for 1 week. The 3 tests were performedagain between 5 and 7 days after the start of each treatment.High-fat load and glucose alone produced a decrease in endothelialfunction and increases in nitrotyrosine, C-reactive protein,intercellular adhesion molecule-1, and interleukin-6. Theseeffects were more pronounced when high-fat load and glucosewere combined. Short-term atorvastatin and irbesartan treatmentssignificantly counterbalanced these phenomena, and their combinationwas more effective than either therapy alone.

Conclusions— This study confirms an independent and cumulativeeffect of postprandial hypertriglyceridemia and hyperglycemiaon endothelial function and inflammation, suggesting oxidativestress as a common mediator of such an effect. Short-term treatmentwith atorvastatin and irbesartan may counterbalance this phenomenon;the combination of the 2 compounds is most effective.

 

2.59           Distribution of brevetoxin (PbTx-3) in mouse plasma: association with high-density lipoprotein

Woofter, R.T., Spiess, P.C. and Ramsdell. J.S.

Environ. Health Perspect., 113(11), 1491-1496 (2005)

 

We investigated the brevetoxin congener PbTx-3 to determine its distribution among carrier proteins, including albumin and blood lipoproteins. Using a radiolabeled brevetoxin tracer (PbTx-3), we found that 39% of the radiolabel remained associated with components in mouse plasma after > 15 kDa cutoff dialysis. Of this portion, only 6.8% was bound to serum albumin. We also examined the binding of brevetoxin to various lipoprotein fractions. Plasma, either spiked with PbTx-3 or from mice treated for 30 min with PbTx-3, was fractionated into different-sized lipoproteins by iodixanol gradient ultracentrifugation. Each fraction was then characterized and quantified by agarose gel electrophoresis and brevetoxin radioimmunoassay, respectively. In both the in vitro and in vivo experiments, the majority of brevetoxin immunoreactivity was restricted to only those gradient fractions that contained high-density lipoproteins (HDLs). Independent confirmation of brevetoxin binding to HDLs was provided by high molecular weight (100 kDa cutoff) dialysis of [3H]PbTx-3 from lipoprotein fractions as well as a scintillation proximity assay using [3H]PbTx-3 and purified human HDLs. This information on the association of brevetoxins with HDLs provides a new foundation for understanding the process by which the toxin is delivered to and removed from tissues and may permit more effective therapeutic measures to treat intoxication from brevetoxins and the related ciguatoxins.

 

2.60           Phosphorylation-induced autoinhibition regulates the cytoskeletal protein lethal (2) giant larvae

Betschinger, J., Eisenhaber, F. and Knoblich, J.A.

Current Biol., 15(3), 276-282 (2005)

 

During asymmetric cell division, cell fate determinants localize asymmetrically and segregate into one of the two daughter cells. In Drosophila neuroblasts, the asymmetric localization of cell fate determinants to the basal cell cortex requires aPKC. aPKC localizes to the apical cell cortex and phosphorylates the cytoskeletal protein Lethal (2) giant larvae (Lgl). Upon phosphorylation, Lgl dissociates from the cytoskeleton and becomes inactive. Here, we show that phosphorylation regulates Lgl by allowing an autoinhibitory interaction of the N terminus with the C terminus of the protein. We demonstrate that interaction with the cytoskeleton is mediated by a C-terminal domain while the N terminus is not required. Instead, the N terminus can bind to the C terminus and can compete for binding to the cytoskeleton. Interaction between the N- and C-terminal domains requires phosphorylation of Lgl by aPKC. Our results suggest that unphosphorylated, active Lgl exists in an open conformation that interacts with the cytoskeleton while phosphorylation changes the protein to an autoinhibited state.

 

2.61           E-Cadherin Tethered to Micropatterned Supported Lipid Bilayers as a Model for Cell Adhesion

Perez, T.D., Nelson, W.J., Boxer, S.G. and Kam, L.

Langmuir, 21, 11963-11968 (2005)

 

Cell−cell adhesion is a dynamic process requiring recruitment, binding, and reorganization of signaling proteins in the plane of the plasma membrane. Here, we describe a new system for investigating how this lateral mobility influences cadherin-based cell signaling. This model is based on tethering of a GPI-modified E-cadherin protein (hEFG) to a supported lipid bilayer. In this report, membrane microfluidics and micropatterning techniques are used to adopt this tethered protein system for studies with the anchorage-dependent cells. As directly formed from proteoliposomes, hEFG exhibits a diffusion coefficient of 0.6 ± 0.3 μm²/s and mobile fraction of 30−60%. Lateral structuring of the supported lipid bilayer is used to isolate mobile proteins from this mixed mobile/immobile population, and should be widely applicable to other proteins. MCF-7 cells seeded onto hEFG-containing bilayers recognize and cluster this protein, but do not exhibit cell spreading required for survival. By micropatterning small anchors into the supported lipid bilayer, we have achieved cell spreading across the bilayer surface and concurrent interaction with mobile hEFG protein. Together, these techniques will allow more detailed analysis of the cellular dynamics involved in cadherin-dependent adhesion events.

 

2.62           A discoidal lipoprotein from the coelomic fluid of the polychaete Nereis virens

Schenk, S., Harris, J.R. and Hoeger, U.

Comp. Biochem. Physiol., Part B, 143, 236-243 (2006)

 

A discoidal lipoprotein was isolated from the coelomic fluid of the polychaete, Nereis virens, by density gradient centrifugation. The lipoprotein was present in both sexes and moved as a uniform band in an agarose gel. The average diameter of the lipoprotein particles determined by electron microscopy was 42 nm with a thickness of 10 nm. SDS electrophoresis showed two apoprotein subunits with molecular masses of 247 and 85 kDa, respectively. In lectin blots, both apoproteins were reactive with Concanavalin A indicating the presence of N-glycans. The small subunit was also reactive with peanut lectin, indicating additional O-glycosylation. The total lipid content was 48% and consisted mainly of phospholipids and some diglycerides as judged by thin layer chromatography. The estimated native molecular mass of N. virens lipoprotein ( 675 kDa) lies in the range of vertebrate high-density lipoprotein and insect lipophorins. The size of the apoproteins is similar to those found in insects, while the composition of the lipid fraction is more similar to that of crustacean lipoproteins.

 

2.63           Blood lipid concentrations and lipoprotein patterns in captive and wild American black bears (Ursus americanus)

Frank, N., Elliott, S.B., Allin, S.B. and Ramsay, E.C.

Am. J. Vet. Res., 67(2), 335-341 (2006)

 

Objective-To compare blood lipid concentrations and lipoprotein patterns for captive and wild American black bears (Ursus americanus). Animals-7 captive and 9 wild adult (>/= 4 years old) black bears. Procedure-Blood was collected from 2 groups of captive black bears (groups A and B) and 1 group of wild black bears (group C). Blood triglyceride (TG) and cholesterol concentrations were compared among groups. Plasma lipoproteins were isolated by use of a self-generating gradient of iodixanol, and lipoprotein patterns were compared between groups A and B. Results-Captive bears (mean +/- SD, 187.8 +/- 44.4 kg) weighed significantly more than wild bears (mean, 104.8 +/- 41.4 kg), but mean body weight did not differ between groups A and B. Mean blood TG concentrations for groups B (216.8 +/- 16.0 mg/dL) and C (190.7 +/- 34.0 mg/dL) were significantly higher than that of group A (103.9 +/- 25.3 mg/dL). Mean blood cholesterol concentration was also significantly higher for group B (227.8 +/- 8.2 mg/dL) than for groups A (171.7 +/- 35.5 mg/dL) or C (190.8 +/- 26.8 mg/dL). Mean very-low-density lipoprotein TG and low-density lipoprotein cholesterol concentrations were 2- and 3-fold higher, respectively, for group B, compared with concentrations for group A. Conclusions and Clinical Relevance-Blood lipid concentrations vary significantly among populations of black bears. Plasma lipoprotein patterns of captive bears differed significantly between colonies and may have reflected differences in diet or management practices.

 

2.64           Prolonged deterioration of endothelial dysfunction in response to postprandial lipaemia is attenuated by vitamin C in type 2 diabetes

Anderson, R.A. et al

Diabetic Med., 23, 258-264 (2006)

 

Background  Endothelial dysfunction (ED) has been described in Type 2 diabetes (T2DM). We have described previously a diminution of flow-mediated arterial dilatation and, by implication, further ED in T2DM in response to postprandial lipaemia (PPL) at 4 h. This is possibly mediated by oxidative stress/alteration of the nitric oxide (NO) pathway. T2DM subjects tend to exhibit both exaggerated and prolonged PPL. We therefore studied the relationship of PPL to the duration of ED in T2DM subjects and oxidative stress with or without the antioxidant, vitamin C.

Methods  Twenty subjects with T2DM with moderate glycaemic control (mean HbA_1c 8.4%) were studied. After an overnight fast, all subjects consumed a standard fat meal. Endothelial function (EF), lipid profiles, and venous free radicals were measured in the fasting, peak lipaemic phase (4 h) and postprandially to 8 h. The study was repeated in a double-blinded manner with placebo, vitamin C (1 g) therapy for 2 days prior to re-testing and with the fat meal. Oxidative stress was assessed by lipid-derived free radicals in plasma, ex vivo by electron paramagnetic resonance spectroscopy (EPR) and by markers of lipid peroxidation (TBARS). Endothelial function was assessed by flow-mediated vasodilatation (FMD) of the brachial artery.

Results  There was a significant decrease in endothelial function in response to PPL from baseline (B) 1.3 ± 1.3% to 4 h 0.22 ± 1.1% (P < 0.05) and 8 h 0.7 ± 0.9% (P < 0.05) (mean ± sem). The endothelial dysfunction seen was attenuated at each time point with vitamin C. Baseline EF with vitamin C changed from (fasting) 3.8 ± 0.9–2.8 ± 0.8 (at 4 h) and 2.9 ± 1.3 (at 8 h) in response to PPL. Vitamin C attenuated postprandial (PP) oxidative stress significantly only at the 4-h time point [301.1 ± 118 (B) to 224.7 ± 72 P < 0.05] and not at 8 h 301.1 ± 118 (B) to 260 ± 183 (P = NS). There were no changes with placebo treatment in any variable. PPL was associated with a PP rise in TG levels (in mmol/l) from (B) 1.8 ± 1 to 2.7 ± 1 at 4 h and 1.95 ± 1.2 at 8 h (P = 0.0002 and 0.33, respectively).

Conclusion  PPL is associated with prolonged endothelial dysfunction for at least 8 h after a fatty meal. Vitamin C treatment improves endothelial dysfunction at all time points and attenuates PPL-induced oxidative stress. This highlights the importance of low-fat meals in T2DM and suggests a role for vitamin C therapy to improve endothelial function during meal ingestion.

 

2.65           Soy-isoflavone-enriched foods and markers of lipid and glucose metabolism in postmenopausal women: interactions with genotype and equol production

Hall, W.L. et al

Am. J. Clin. Nutr., 83, 592-600 (2006)

 

Background: The hypocholesterolemic effects of soy foods arewell established, and it has been suggested that isoflavonesare responsible for this effect. However, beneficial effectsof isolated isoflavones on lipid biomarkers of cardiovasculardisease risk have not yet been shown.

Objective: The objective was to investigate the effects of isolated soy isoflavones on metabolic biomarkers of cardiovascular disease risk, including plasma total, HDL, and LDL cholesterol; triacylglycerols; lipoprotein(a); the percentage of small dense LDL; glucose; nonesterified fatty acids; insulin; and the homeostasis model assessment of insulin resistance. Differences with respect to single nucleotide polymorphisms in selected genes [ie, estrogen receptor (XbaI and PvuII), estrogen receptor ß (AluI), and estrogen receptor ß(cx) (Tsp509I), endothelial nitric oxide synthase (Glu298Asp), apolipoprotein E (Apo E2, E3, and E4), cholesteryl ester transfer protein (TaqIB), andleptin receptor (Gln223Arg)] and with respect to equol productionwere investigated.

Design: Healthy postmenopausal women (n = 117) participatedin a randomized, double-blind, placebo-controlled, crossoverdietary intervention trial. Isoflavone-enriched (genistein-to-daidzeinratio of 2:1; 50 mg/d) or placebo cereal bars were consumedfor 8 wk, with a wash-out period of 8 wk before the crossover.

Results: Isoflavones did not have a significant beneficial effect on plasma concentrations of lipids, glucose, or insulin. A significant difference between the responses of HDL cholesterol to isoflavones and to placebo was found with estrogen receptor ß(cx) Tsp509I genotype AA, but not GG or GA.

Conclusions: Isoflavone supplementation, when provided in theform and dose used in this study, had no effect on lipid orother metabolic biomarkers of cardiovascular disease risk inpostmenopausal women but may increase HDL cholesterol in anestrogen receptor ß gene–polymorphic subgroup.

 

2.66           Inducible expression of Tau repeat domain in cell models of tauopathy

Khlistunova, I. et al

1. Biol. Chem., 281(2), 1205-1214 (2006)

 

We generated several cell models of tauopathy in order to study the mechanisms of neurodegeneration in diseases involving abnormal changes of tau protein. N2a neuroblastoma cell lines were created that inducibly express different variants of the repeat domain of tau (tau_RD) when exposed to doxycycline (Tet-On system). The following three constructs were chosen: (i) the repeat domain of tau that coincides with the core of Alzheimer paired helical filaments; (ii) the repeat domain with the deletion mutation K280 known from frontotemporal dementia and highly prone to spontaneous aggregation; and (iii) the repeat domain with K280 and two proline point mutations that inhibit aggregation. The comparison of wild-type, pro-aggregation, and anti-aggregation mutants shows the following. (a) Aggregation of tau_RD is toxic to cells. (b) The degree of aggregation and toxicity depends on the propensity for -structure. (c) Soluble mutants of tau_RD that cannot aggregate are not toxic. (d) Aggregation is preceded by fragmentation. (e) Fragmentation of tau_RD in cells is initially due to a thrombin-like protease activity. (f) Phosphorylation of tau_RD (at KXGS motifs) precedes aggregation but is not correlated with the degree of aggregation. (g) Aggregates of tau_RD disappear when the expression is silenced, showing that aggregation is reversible. (h) Aggregation can be prevented by drugs and even pre-formed aggregates can be dissolved again by drugs. Thus, the cell models open up new insights into the relationship between the structure, expression, phosphorylation, aggregation, and toxicity of tau_RD that can be used to test current hypotheses on tauopathy and to develop drugs that prevent the aggregation and degeneration of cells.

 

2.67           Effects of dairy products naturally enriched with cis-9, trans-11 conjugated linoleic acid on the blood lipid profile in healthy middle-aged men

Tricon, S. et al

Am. J. Clin. Nutr., 83, 744-753 (2006)

 

Background: Interest in the development of dairy products naturally enriched in conjugated linoleic acid (CLA) exists. However, feeding regimens that enhance the CLA content of milk also increase concentrations of trans-18:1 fatty acids. The implications forhuman health are not yet known.

Objective: This study investigated the effects of consuming dairy products naturally enriched in cis-9,trans-11 CLA (and trans-11 18:1) on the blood lipid profile, the atherogenicityof LDL, and markers of inflammation and insulin resistance inhealthy middle-aged men.

Design: Healthy middle-aged men (n = 32) consumed ultra-heat-treated milk, butter, and cheese that provided 0.151 g/d (control) or 1.421 g/d (modified) cis-9,trans-11 CLA for 6 wk. This was followedby a 7-wk washout and a crossover to the other treatment.

Results: Consumption of dairy products enriched with cis-9,trans-11 CLA and trans-11 18:1 did not significantly affect body weight, inflammatory markers, insulin, glucose, triacylglycerols, or total, LDL, and HDL cholesterol but resulted in a small increase in the ratio of LDL to HDL cholesterol. The modified dairy products changed LDL fatty acid composition but had no significant effect on LDL particle size or the susceptibility of LDL to oxidation. Overall, increased consumption of full-fat dairy products and naturally derived trans fatty acids did not cause significantchanges in cardiovascular disease risk variables, as may beexpected on the basis of current health recommendations.

Conclusion: Dairy products naturally enriched with cis-9,trans-11 CLA and trans-11 18:1 do not appear to have a significant effecton the blood lipid profile.

 

2.68           Influence of an algal triacylglycerol containing docosahexaenoic acid (22: 6n-3) and docosapentaenoic acid (22: 5n-6) on cardiovascular risk factors in healthy men and women

Sanders, T.A.B., Gleason, K., Griffin, B. and Miller, G.J.

Br. J. Nutrition, 95, 525-531 (2006)

 

The intake of long-chain n-3 PUFA, including DHA (22: 6n-3), is associated with a reduced risk of CVD. Schizochytrium sp. are an important primary source of DHA in the marine food chain but they also provide substantial quantities of the n-6 PUFA docosapentaenoic acid (22: 5n-6; DPA). The effect of this oil on cardiovascular risk factors was evaluated using a double-blind randomised placebo-controlled parallel-design trial in thirty-nine men and forty women. Subjects received 4 g oil/d for 4 weeks; the active treatment provided 1[middle dot]5 g DHA and 0[middle dot]6 g DPA. Active treatment increased plasma concentrations of arachidonic acid, adrenic acid, DPA and DHA by 21, 11, 11 and 88 mg/l respectively and the proportions of DPA and DHA in erythrocyte phospholipids by 78 and 27 % respectively. Serum total, LDL- and HDL-cholesterol increased by 0[middle dot]33 mmol/l (7[middle dot]3 %), 0[middle dot]26 mmol/l (10[middle dot]4 %) and 0[middle dot]14 mmol/l (9[middle dot]0 %) compared with placebo (all P<=0[middle dot]001). Factor VII (FVII) coagulant activity increased by 12 % following active treatment (P=0[middle dot]006). There were no significant differences between treatments in LDL size, blood pressure, plasma glucose, serum C-reactive protein, plasma FVII antigen, FVII activated, fibrinogen, von Willebrand factor, tocopherol or carotenoid concentrations, plasminogen activator inhibitor-1, creatine kinase or troponin-I activities, haematology or liver function tests or self-reported adverse effects. Overall, the oil was well tolerated and did not adversely affect cardiovascular risk.

 

2.69           Metformin prevents alcohol-induced liver injury in the mouse: critical role of plasminogen activator inhibitor-1

Bergheim, I. et al

Gastroenterology, 130, 2099-2112 (2006)

 

Background & Aims: The biguanide drug metformin has recently been found to improve steatosis and liver damage in animal models and in humans with nonalcoholic steatohepatitis. Methods: The aim of the present study was to determine whether metformin also prevents steatosis and liver damage in mouse models of acute and chronic alcohol exposure. Results: Acute ethanol exposure caused a >20-fold increase in hepatic lipids, peaking 12 hours after administration. Metformin treatment significantly blunted the ethanol effect by >60%. Although metformin is a known inducer of AMP kinase (AMPK) activity, the hepatoprotective property of metformin did not correlate with activation of AMPK or of AMPK-dependent pathways. Instead, the protective effects of metformin correlated with complete prevention of the upregulation of plasminogen activator inhibitor (PAI)-1 caused by ethanol. Indeed, a similar protective effect against acute alcohol-induced lipid accumulation was observed in PAI-1^−/− mice. Hepatic fat accumulation caused by chronic enteral ethanol feeding was also prevented by metformin or by knocking out PAI-1. Under these conditions, necroinflammatory changes caused by ethanol were also significantly attenuated. Conclusions: Taken together, these findings suggest a novel mechanism of action for metformin and identify a new role of PAI-1 in hepatic injury caused by ethanol.

 

2.70           Historical milestones in measurement of HDL-cholesterol: Impact on clinical and laboratory practice 

Langlois, M.R. and Blaton, V.H.

Clin. Chem. Acta, 369, 168-178 (2006)

 

High-density lipoprotein cholesterol (HDL-C) comprises a family of particles with differing physicochemical characteristics. Continuing progress in improving HDL-C analysis has originated from two separate fields—one clinical, reflecting increased attention to HDL-C in estimating risk for coronary heart disease (CHD), and the other analytical, reflecting increased emphasis on finding more reliable and cost-effective HDL-C assays. Epidemiologic and prospective studies established the inverse association of HDL-C with CHD risk, a relationship that is consistent with protective mechanisms demonstrated in basic research and animal studies. Atheroprotective and less atheroprotective HDL subpopulations have been described. Guidelines on primary and secondary CHD prevention, which increased the workload in clinical laboratories, have led to a revolution in HDL-C assay technology. Many analytical techniques including ultracentrifugation, electrophoresis, chromatography, and polyanion precipitation methods have been developed to separate and quantify HDL-C and HDL subclasses. More recently developed homogeneous assays enable direct measurement of HDL-C on an automated analyzer, without the need for manual pretreatment to separate non-HDL. Although homogeneous assays show improved accuracy and precision in normal serum, discrepant results exist in samples with atypical lipoprotein characteristics. Hypertriglyceridemia and monoclonal paraproteins are important interfering factors. A novel approach is nuclear magnetic resonance spectroscopy that allows rapid and reliable analysis of lipoprotein subclasses, which may improve the identification of individuals at increased CHD risk. Apolipoprotein A-I, the major protein of HDL, has been proposed as an alternative cardioprotective marker avoiding the analytical limitations of HDL-C.

 

2.71           Syndecan-1 mediates internalization of apoE-VLDL through a low density lipoprotein receptor-related protein (LRP)-independent, non-clathrin-mediated pathway

Wilsie, L.C., Gonzales, M. and Orlando, R.A.

Lipids in Health and Disease, 5(23), 1-14 (2006)

 

Background

Triacylglyerol-rich very low density lipoprotein (VLDL) particles are the primary carriers of fatty acids in the circulation and as such serve as a rich energy source for peripheral tissues. Receptor-mediated uptake of these particles is dependent upon prior association with apolipoprotein E (apoE-VLDL) and is brought about by cell surface heparan sulfate proteoglycans (HSPG) in some cell types and by the low density lipoprotein receptor-related protein (LRP) in others. Although LRP's role in apoE-VLDL uptake has been well studied, the identity of the HSPG family member that mediates apoE-VLDL uptake has not been established. We investigated if syndecan-1 (Syn-1), a transmembrane cell surface HSPG, is able to mediate the internalization of apoE-VLDL and examined the relationship between Syn-1 and LRP toward apoE-VLDL uptake. For this study, we used a human fibroblast cell line (GM00701) that expresses large amounts of LRP, but possesses no LDL receptor activity to eliminate its contributions toward apoE-VLDL uptake.

Results

Although LRP in these cells is fully active as established by substantial α₂macroglobulin binding and internalization, uptake of apoE-VLDL is absent. Expression of human Syn-1 cDNA restored apoE-VLDL binding and uptake by these cells. Competition for this uptake with an LRP ligand-binding antagonist had little or no effect, whereas co-incubation with heparin abolished apoE-VLDL internalization. Depleting Syn-1 expressing cells of K⁺, to block clathrin-mediated endocytosis, showed no inhibition of Syn-1 internalization of apoE-VLDL. By contrast, treatment of cells with nystatin to inhibit lipid raft function, prevented the uptake of apoE-VLDL by Syn-1.

Conclusion

These data demonstrate that Syn-1 is able to mediate apoE-VLDL uptake in human fibroblasts with little or no contribution from LRP and that the endocytic path taken by Syn-1 is clathrin-independent and relies upon lipid raft function. These data are consistent with previous studies demonstrating Syn-1 association with lipid raft domains.

 

2.72           Effects of altering the ratio of dietary n–6 to n–3 fatty acids on insulin sensitivity, lipoprotein size, and postprandial lipemia in men and postmenopausal women aged 45–70 y: the OPTILIP Study

Griffin, M.D. et al

Am. J. Clin. Nutr., 84, 1290-1298 (2006)

 

Background: Insulin resistance is associated with elevated plasmatriacylglycerol, low HDL concentrations, elevated postprandiallipemia, and a predominance of small, dense LDLs (sdLDLs). Ithas been hypothesized that the dietary ratio of n–6 ton–3 (n–6:n–3) polyunsaturated fatty acids(PUFAs) may have favorable effects on these risk factors byincreasing insulin sensitivity.

Objective: The objective was to measure changes in insulin sensitivity,lipoprotein size, and postprandial lipemia after a 6-mo alterationin n–6:n–3.

Design: In a randomized, parallel design in 258 subjects aged45–70 y, we compared 4 diets providing 6% of energy asPUFAs with an n–6:n–3 between 5:1 and 3:1 with acontrol diet that had an n–6:n–3 of 10:1. The dietswere enriched in -linolenic acid, eicosapentaenoic acid (EPA)and docosahexaenoic acid (DHA), or both. Insulin sensitivitywas assessed with the homeostatic model assessment of insulinresistance and the revised quantitative insulin sensitivitytest.

Results: Dietary intervention did not influence insulin sensitivityor postprandial lipase activities. Fasting and postprandialtriacylglycerol concentrations were lower, and the proportionof sdLDLs decreased (by 12.7%; 95% CI: –22.9%, 2.4%),with an n–6:n–3 of 3:1, which was achieved by theaddition of long-chain n–3 PUFAs (EPA and DHA).

Conclusions: Decreasing the n–6:n–3 does not influence insulin sensitivity or lipase activities in older subjects. The reduction in plasma triacylglycerol after an increased intake of n–3 long-chain PUFAs results in favorable changes in LDL size.

 

2.73           Prolonged deterioration of endothelial dysfunction in response to postprandial lipaemia is attenuated by vitamin C in Type 2 diabetes

Anderson, R.A., Evans, L.M., Ellis, G.R., Khan, N., Morrist, K., Jackson, S.K., Rees, A., Lewis, M.J. and Frenneaux, M.P.

Diabetic Med., 2383), 258-264 (2006)

Background  Endothelial dysfunction (ED) has been described in Type 2 diabetes (T2DM). We have described previously a diminution of flow-mediated arterial dilatation and, by implication, further ED in T2DM in response to postprandial lipaemia (PPL) at 4 h. This is possibly mediated by oxidative stress/alteration of the nitric oxide (NO) pathway. T2DM subjects tend to exhibit both exaggerated and prolonged PPL. We therefore studied the relationship of PPL to the duration of ED in T2DM subjects and oxidative stress with or without the antioxidant, vitamin C.

Methods  Twenty subjects with T2DM with moderate glycaemic control (mean HbA_1c 8.4%) were studied. After an overnight fast, all subjects consumed a standard fat meal. Endothelial function (EF), lipid profiles, and venous free radicals were measured in the fasting, peak lipaemic phase (4 h) and postprandially to 8 h. The study was repeated in a double-blinded manner with placebo, vitamin C (1 g) therapy for 2 days prior to re-testing and with the fat meal. Oxidative stress was assessed by lipid-derived free radicals in plasma, ex vivo by electron paramagnetic resonance spectroscopy (EPR) and by markers of lipid peroxidation (TBARS). Endothelial function was assessed by flow-mediated vasodilatation (FMD) of the brachial artery.

Results  There was a significant decrease in endothelial function in response to PPL from baseline (B) 1.3 ± 1.3% to 4 h 0.22 ± 1.1% (P < 0.05) and 8 h 0.7 ± 0.9% (P < 0.05) (mean ± sem). The endothelial dysfunction seen was attenuated at each time point with vitamin C. Baseline EF with vitamin C changed from (fasting) 3.8 ± 0.9–2.8 ± 0.8 (at 4 h) and 2.9 ± 1.3 (at 8 h) in response to PPL. Vitamin C attenuated postprandial (PP) oxidative stress significantly only at the 4-h time point [301.1 ± 118 (B) to 224.7 ± 72 P < 0.05] and not at 8 h 301.1 ± 118 (B) to 260 ± 183 (P = NS). There were no changes with placebo treatment in any variable. PPL was associated with a PP rise in TG levels (in mmol/l) from (B) 1.8 ± 1 to 2.7 ± 1 at 4 h and 1.95 ± 1.2 at 8 h (P = 0.0002 and 0.33, respectively).

Conclusion  PPL is associated with prolonged endothelial dysfunction for at least 8 h after a fatty meal. Vitamin C treatment improves endothelial dysfunction at all time points and attenuates PPL-induced oxidative stress. This highlights the importance of low-fat meals in T2DM and suggests a role for vitamin C therapy to improve endothelial function during meal ingestion.

 

2.74           Novel Porphyrin Conjugates with a Potent Photodynamic Antitumor Effect: Differential Efficacy of Mono- and Bis-β-cyclodextrin Derivatives

Kralova, J., Synytsya, A., Pouckova, P., Koc, M., Dvorak, M. and Kral, V.

Photochem. Photobiol., 82(2), 432-438 (2006)

 

In the present study we investigated the photosensitizing properties of two novel mono- and bis-cyclodextrin tetrakis (pentafluorophenyl) porphyrin derivatives in several tumor cell lines and in BALB/c mice bearing subcutaneously transplanted syngeneic mouse mammary carcinoma 4T1. Both studied sensitizers were localized mainly in lysosomes and were found to induce cell death by triggering apoptosis in human leukemic cells HL-60. In 4T1 and other cell lines both apoptotic and necrotic modes of cell death occurred depending on drug and light doses. Mono-cyclodextrin porphyrin derivative P(β-CD)l exhibited stronger in vitro phototoxic effect than bis-cyclodextrin derivative P(β-CD)2. However, in vivo P(β-CD)2 displayed faster tumor uptake with maximal accumulation 6 h after application, leading to complete and prolonged elimination of subcutaneous tumors within 3 days after irradiation (100 J cm^-2). In contrast, P(β-CD)1 uptake was slower (48 h) and the reduction of tumor mass was only transient, reaching the maximum at the 12 h interval when a favorable tumor-to-skin ratio appeared. Thus, P(β-CD)2 represents a new photosensitizing drug displaying fast and selective tumor uptake, strong antitumor activity and fast elimination from the body.

 

2.75           Gasoline Exhaust Emissions Induce Vascular Remodeling Pathways Involved in Atherosclerosis

Lund, A.K. et al

Toxicol. Sci., 95(2), 485-494 (2007)

 

Epidemiological evidence indicates that environmental air pollutants are positively associated with the development of chronic vascular disease; however, the mechanisms involved have not been fully elucidated. In the present study we examined molecular pathways associated with chronic vascular disease in atherosclerosis-prone apolipoprotein E–deficient (ApoE^–/–) mice, including markers of vascular remodeling and oxidative stress, in response to exposure to the ubiquitous environmental pollutant, gasoline engine emissions. ApoE^–/– mice, on a high-fat diet, were exposed by inhalation to either filtered air; 8, 40, or 60 µg/m³ particulate matter whole exhaust; or filtered exhaust with gases matching the 60-µg/m³ concentration, for 7 weeks. Aortas and plasma were collected and assayed for changes in histochemical markers, real-time reverse transcriptase–polymerase chain reaction, and indicators of oxidative damage. Inhalational exposure to gasoline engine emissions resulted in increased aortic mRNA expression of matrix metalloproteinase-3 (MMP-3), MMP-7, and MMP-9, tissue inhibitor of metalloproteinases-2, endothelin-1 and heme oxygenase-1 in ApoE^–/– mice; increased aortic MMP-9 protein levels were confirmed through immunohistochemistry. Elevated reactive oxygen species were also observed in arteries from exposed animals, despite absence of plasma markers. Similar findings were also observed in the aortas of ApoE^–/– mice exposed to particle-filteredatmosphere, implicating the gaseous components of the wholeexhaust in mediating the expression of markers associated withthe vasculopathy. These findings demonstrate that exposure togasoline engine emissions results in the transcriptional upregulationof factors associated with vascular remodeling, as well as increasedmarkers of vascular oxidative stress, which may contribute tothe progression of atherosclerosis and reduced stability ofvulnerable plaques.

 

2.76           The AAA+ protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization

He, J. et al

1. Cell Biol., 176(2), 141-146 (2007)

 

Many copies of mammalian mitochondrial DNA contain a short triple-stranded region, or displacement loop (D-loop), in the major noncoding region. In the 35 years since their discovery, no function has been assigned to mitochondrial D-loops. We purified mitochondrial nucleoprotein complexes from rat liver and identified a previously uncharacterized protein, ATAD3p. Localization studies suggested that human ATAD3 is a component of many, but not all, mitochondrial nucleoids. Gene silencing of ATAD3 by RNA interference altered the structure of mitochondrial nucleoids and led to the dissociation of mitochondrial DNA fragments held together by protein, specifically, ones containing the D-loop region. In vitro, a recombinant fragment of ATAD3p bound to supercoiled DNA molecules that contained a synthetic D-loop, with a marked preference over partially relaxed molecules with a D-loop or supercoiled DNA circles. These results suggest that mitochondrial D-loops serve to recruit ATAD3p for the purpose of forming or segregating mitochondrial nucleoids.

 

2.77           Functional refolding of a recombinant C-type lectin-like domain containing intramolecular disulfide bonds 

Vohra, R., Murphy, J.E., Walker, J.H., Homer-Vanniasinkam, S. and Ponnambalam, S.

Protein Expression & Purification, 52(2), 415-421 (2007)

 

The lectin-like oxidized low-density lipoprotein scavenger receptor (LOX-1) is a pro-inflammatory marker and Type II membrane protein expressed on vascular cells and tissues. The LOX-1 extracellular domain mediates recognition of oxidized low-density lipoprotein (oxLDL) particles that are implicated in the development of atherosclerotic plaques. To study the molecular basis for LOX-1-mediated ligand recognition, we have expressed, purified and refolded a recombinant LOX-1 protein and assayed for its biological activity using a novel fluorescence-based assay to monitor binding to lipid particles. Overexpression of a hexahistidine-tagged cysteine-rich LOX-1 extracellular domain in bacteria leads to the formation of aggregates that accumulated in bacterial inclusion bodies. The hexahistidine-tagged LOX-1 molecule was purified by affinity chromatography from solubilized inclusion bodies. A sequential dialysis procedure was used to refold the purified but inactive and denatured LOX-1 protein into a functionally active form that mediated recognition of oxLDL particles. This approach allowed slow LOX-1 refolding and assembly of correct intrachain disulfide bonds. Circular dichroism analysis of the refolded LOX-1 molecule demonstrated a folded state with substantial α-helical content. Using immobilized recombinant, refolded LOX-1 we demonstrated a 70-fold preferential recognition for oxLDL over native LDL particles. Thus, a protein domain containing intrachain disulfide bonds can be reconstituted into a functionally active state using a relatively simple dialysis-based technique.

 

2.78           Simultaneous Control of Hyperglycemia and Oxidative Stress Normalizes Endothelial Function in Type 1 Diabetes

Ceriello, A., Kumar, S., Piconi, L., Esposito, K. and Guigliano, D.

Diabetes Care, 30(3), 649-654 (2007)

 

OBJECTIVE—Previous studies have shown that in type 1 diabetesendothelial dysfunction persists even when glycemia is normalized.Moreover, oxidative stress has recently been demonstrated tobe the mediator of hyperglycemia-induced endothelial dysfunction.

RESEARCH DESIGN AND METHODS—Thirty-six type 1 diabeticpatients and 12 control subjects were enrolled. The diabeticpatients were divided into three groups. The first group wastreated for 24 h with insulin, achieving a near-normalizationof glycemia. After 12 h of this treatment, vitamin C was addedfor the remaining 12 h. The second group was treated for 24h with vitamin C. After 12 h of this treatment, insulin wasstarted, with achievement of near-normalization of glycemiafor the remaining 12 h. The third group was treated for 24 hwith both vitamin C and insulin, achieving near-normalizationof glycemia.

RESULTS—Neither normalization of glycemia nor vitaminC treatment alone was able to normalize endothelial dysfunctionor oxidative stress. However, a combination of insulin and vitaminC normalized endothelial dysfunction and decreased oxidativestress to normal levels.

CONCLUSIONS—This study suggests that long-lasting hyperglycemia in type 1 diabetic patients induces permanent alterations in endothelial cells, which may contribute to endothelial dysfunction by increased oxidative stress even when hyperglycemia is normalized.

 

2.79           Secretion of the glucose-regulated selenoprotein SEPS1 from hepatoma cells 

Gao, Y. et al

Biochem. Biophys. Res. Comm., 356, 636-641 (2007)

 

SEPS1 (also called selenoprotein S, SelS, Tanis or VIMP) is a selenoprotein, localized predominantly in the ER membrane and also on the cell surface. In this report, we demonstrate that SEPS1 protein is also secreted from hepatoma cells but not from five other types of cells examined. The secretion can be abolished by the ER-Golgi transport inhibitor Brefeldin A and by the protein synthesis inhibitor cycloheximide. Using a sandwich ELISA, SEPS1 was detected in the sera of 65 out of 209 human subjects (31.1%, average = 15.7 ± 1.1 ng/mL). Fractionation of human serum indicated that SEPS1 was associated with LDL and possibly with VLDL. The function of plasma SEPS1 is unclear but may be related to lipoprotein metabolism.

 

2.80           Differences in cell morphology, lipid and apo B secretory capacity in caco-2 cells following long term treatment with saturated and monounsaturated fatty acids 

Bateman, P.A., Jackson, K.G., Maitin, V., Yaqoob, P. and Williams, C.M.

Biochim. Biophys. Acta, 1771, 475-485 (2007)

 

The suitability of the caco-2 cell line as a model for studying the long term impact of dietary fatty acids on intestinal lipid handling and chylomicron production was examined. Chronic supplementation of caco-2 cells with palmitic acid (PA) resulted in a lower triacylglycerol secretion than oleic acid (OA). This was coupled with a detrimental effect of PA, but not OA, on transepithelial electrical resistance (TER) measurements, suggesting a loss of structural integrity across the cell monolayer. Addition of OA reversed the adverse effects of PA and stearic acid on TER and increased the ability of cells to synthesise and accumulate lipid, but did not normalise the secretion of lipids by caco-2 cells. Increasing amounts of OA and decreasing amounts of PA in the incubation media markedly improved the ability of cells to synthesise apolipoprotein B and secrete lipids. Real time RT-PCR revealed a down regulation of genes involved in lipoprotein synthesis following PA than OA. Electron microscopy showed adverse effects of PA on cellular morphology consistent with immature enterocytes such as stunted microvilli and poor tight junction formation. In conclusion, previously reported differences in lipoprotein secretion by caco-2 cells supplemented with saturated fatty acids (SFA) and OA may partly reflect early cytotoxic effects of SFA on cellular integrity and function.

 

2.81           Large-scale preparation of human low- and high-density lipoproteins by density gradient centrifugation using iodixanol

Billington, D., Maxwell, E., Graham, J.M. and Newland, P.

Anal. Biochem., 367(1), 137-139 (2007)

 

No abstract available

 

2.82           Telmisartan Shows an Equivalent Effect of Vitamin C in Further Improving Endothelial Dysfunction After Glycemia Normalization in Type 1 Diabetes

Ceriello, A., Piconi, L., Esposito, K. and Gugliano, D.

Diabetes Care, 30(7), 1694-1698 (2007)

 

OBJECTIVE— Long-lasting hyperglycemia in type 1 diabeticpatients induces permanent alterations of endothelial functionby increased oxidative stress, even when glycemia is normalized.

RESEARCH DESIGN AND METHODS— In this study, 36 type 1diabetic patients and 12 control subjects were enrolled. Thediabetic patients were divided into three groups. The firstgroup was treated for 24 h with insulin, achieving a near normalizationof glycemia. After 12 h of this treatment, vitamin C was addedfor the remaining 12 h. The second group was treated for 24h with vitamin C. After 12 h of this treatment, insulin wasstarted, achieving a near normalization of glycemia for theremaining 12 h. The third group was treated for 24 h with bothvitamin C and insulin, achieving near normalization of glycemia.The same protocols were performed after 1 month of telmisartanor placebo.

RESULTS— Neither normalization of glycemia nor vitaminC treatment alone was able to normalize endothelial dysfunctionor oxidative stress. Combining insulin and vitamin C normalizedendothelial dysfunction and decreased oxidative stress to normallevels. Telmisartan significantly improved basal endothelialfunction and decreased nitrotyrosine plasma levels. In patientstreated with telmisartan, a near normalization of both flow-mediatedvasodilation and oxidative stress was achieved when glycemiawas normalized, whereas adding vitamin C infusion did not showfurther effect on endothelial function or nitrotyrosine plasmalevels.

CONCLUSIONS— These data indicate that combining the normalizationof glycemia with an antioxidant can normalize endothelial functionin type 1 diabetic patients and that telmisartan works as anantioxidant like vitamin C.

 

2.83           Separation of the principal HDL subclasses by iodixanol gradient ultracentrifugation

Harman, N.L., Davies, I.G. and Griffin, B.A.

Atherosclerosis, 194(1), 283-284 (2007)

 

Introduction: HDL is the smallest and most dense lipoprotein, its main function is in reverse cholesterol transport and as such it can be described as an anti-atherogenic lipoprotein. HDL exists as a number of subclasses differing in size and density; it has been proposed that these subclasses show a variable relationship with CVD risk. The use of HDL subclasses as a CVD risk marker is limited due to the expensive and time consuming nature of current techniques. The aim of the current study was to develop a rapid and cost effective method for the measurement of the principal plasma HDL subclasses.

Methods: HDL subclasses were separated by iodixanol gradient ultracentrifugation (I_xDGUC) from pre-stained plasma, in a run time of 2 h 30 min. A digital image of the separated HDL bands was downloaded and analysed by TotalLab 1D gel scan software to generate HDL subclass profiles. These profiles were validated by co-isolation of the HDL fraction on gradient gel electrophoresis (GGE). HDL was further fractionated and characterised by the distribution of particle size and apo A-1.

Results: Delineation between HDL₂ and HDL₃ was at a density cut-off of 1.059 g/L. There was a significant correlation between HDL subclass for both GGE and I_xDGUC methods as measured by area under the curve [HDL₃ r = 0.77; P < 0.01]. HDL₃% correlated inversely with total HDL-cholesterol [r = −0.64; P < 0.01)].

Conclusion: I_xDGUC provides a rapid, high throughput and cost effective method for the separation of HDL₂ and HDL₃ subclasses.

 

2.84           Characterization of hepatitis C virus associated with very low density lipoprotein (VLDL) in infected human serum and liver

Nielsen, S., Bassendine, M., Neely, D., Ibrahim, S. and Toms, G.

Atherosclerosis, 194(1), 284 (2007)

 

Hepatitis C virus (HCV) particles in serum are complexed with host very low density lipoprotein (VLDL) in lipo-viro particles. These structures are fragile and heterogeneous in density and size but, hitherto, their titres have been too low for detailed structural studies. Our group has previously described a unique liver transplant patient with an unusually high titre of HCV, sufficient for comprehensive biochemical and biophysical characterization.

In order to characterize native HCV lipo-viro particles in this material, serum and liver macerate was first fractionated on iodixanol density gradients. HCV containing fractions with densities below 1.10 g/ml were further characterized by gel filtration, which separates VLDL, LDL and HDL according to size. HCV RNA in serum was found in particles, which co-eluted with large VLDL particles. These are the putative lipo-viro particles and support our earlier findings that HCV circulates in blood in association with VLDL. HCV in liver was found to co-elute with endoplasmic reticulum membranes. However, particles of a similar size to HCV lipo-viro particles in serum contained a high ratio of positive to negative strand HCV RNA suggesting that these fractions are enriched in HCV virus particles associated with VLDL.

 

2.85           Endurance swimming activates trout lipoprotein lipase: plasma lipids as a fuel for muscle

Magnoni, L. and Weber, J-M.

1. Exp. Biol., 210, 4016-4023 (2007)

 

Fish endurance swimming is primarily powered by lipids supplied to red muscle by the circulation, but the mechanism of delivery remains unknown. By analogy to mammals, previous studies have focused on non-esterified fatty acids (NEFA bound to albumin), but lipoproteins have not been considered as an energy shuttle to working muscles. The effects of exercise on fish lipoprotein lipase (LPL) have never been investigated. We hypothesized that LPL and circulating lipoproteins would be modified by prolonged swimming. Because LPL is naturally bound to the endothelium, we have used heparin to release the enzyme in the circulation and to characterize reserve capacity for lipoprotein catabolism. The effects of exercise (4 days at 1.5 body lengths s^–1 in a swim tunnel) were measured for red muscle LPL, post-heparin plasma LPL, and lipoprotein concentration/composition. Red muscle LPL activity increased from 18±5 (rest) to 49± 9 nmol fatty acids min^–1 g^–1 (swimming). In resting fish, heparin administration caused a 27-fold increase in plasma LPL activity that reached a maximum of 1.32± 0.67 µmol fatty acids min^–1 ml^–1 plasma. This heparin-induced response of plasma LPL was not different between resting controls and exercised fish. Heparin or prolonged swimming had no effect on the concentration/composition of lipoproteins that contain 92% of the energy in total plasma lipids. We conclude that (1) red muscle LPL is strongly activated by endurance swimming, (2) rainbow trout have a high reserve capacity for hydrolyzing lipoproteins, and (3) future studies should aim to measure lipoprotein flux because their concentration does not reflect changes in flux. These novel characteristics of fish LPL imply that lipoproteins are used as a metabolic shuttle between fat reserves and working muscles, a strategy exploiting an abundant source of energy in rainbow trout.

 

2.86           Targeting of stabilized plasmid lipid particles to hepatocytes in vivo by means of coupled lactoferrin

Weeke-Klimp, A.H. et al

1. Drug Targeting, 15(9), 585-594 (2007)

 

For non-viral gene delivery we prepared stabilized plasmid lipid particles (SPLPs), to which lactoferrin (LF) was coupled as a hepatocyte specific targeting ligand. LF-SPLPs and untargeted SPLPs labeled with [³H]cholesteryloleyl-ether were injected into rats. About 87% of the LF-SPLPs were eliminated from the blood within 5 min, while 80% of untargeted SPLPs were still circulating after 2 h. Fifty-two percent of the LF-SPLPs were taken up by hepatocytes, while non-parenchymal liver cells accounted for 16% of the uptake. Despite the efficient targeting of LF-SPLPs to hepatocytes and their capacity to transfect HepG2 and COS-7 cells in vitro, expression of a reporter gene was not detected in vivo. Overall, covalent coupling of LF to SPLPs leads to massive delivery in hepatocytes after systemic administration. However, these LF-SPLPs are not able to transfect these cells in vivo.

 

2.87           Lipoprotein-Heparan Sulfate Interactions in the Hh Pathway

Eugster, C., Panakova, D., Mahmoud, A. and Eaton, S.

Developmental Cell, 13(1), 57-71 (2007)

 

The Drosophila lipoprotein particle, Lipophorin, bears lipid-linked morphogens on its surface and is required for long-range signaling activity of Wingless and Hedgehog. Heparan sulfate proteoglycans are also critical for trafficking and signaling of these morphogens. Here we show that Lipophorin interacts with the heparan sulfate moieties of the glypicans Dally and Dally-like. Membrane-associated glypicans can recruit Lipophorin to disc tissue, and remain associated with these particles after they are released from the membrane by cleavage of their gpi anchors. The released form of Dally colocalizes with Patched, Hedgehog, and Lipophorin in endosomes and increases Hedgehog signaling efficiency without affecting its distribution. These data suggest that heparan sulfate proteoglycans may influence lipid-linked morphogen signaling, at least in part, by binding to Lipophorin. They further suggest that the complement of proteins present on lipoprotein particles can regulate the activity of morphogens.

 

2.88           Binding of liver derived, low density hepatitis C virus to human hepatoma cells

Martin, C., Nielsen, S.U., Ibrahim, S., Bassendine, M.F. and Toms, G.L.

1. Med. Virol., 80, 816-823 (2008)

 

HCV recovered from low density fractions of infected blood is associated with lipid and host apo-lipoproteins in lipo-viro-particles (LVP). It has been proposed that these particles are capable of binding and entering hepatocytes by viral glycoprotein independent mechanisms utilizing uptake pathways of normal host lipoproteins after binding to cell surface glycosaminoglycans (GAG), the low density lipoprotein receptor (LDL-r) or scavenger receptor B1 (SR-B1). In this study binding to human hepatoma cells of HCV low density RNA containing particles, semi-purified from macerates of infected human liver, is compared with that of normal host low density lipoprotein (LDL). Binding of both LDL and HCV low density RNA containing particles paralleled LDL-r but not SR-B1 expression on the recipient cells. Binding of both particle types was sensitive to suramin at 0°C but less so at 37°C suggesting that they both bind initially to GAG but, at 37°C, are internalized or transferred to a suramin resistant receptor. Suramin resistant uptake of both particles was blocked in the presence of excess LDL or oxidized LDL. However, whilst LDL uptake was blocked by anti-apoB-100, HCV low density RNA uptake was enhanced by anti-apoB100 and further enhanced by a cocktail of anti-apo-B100 and anti-apoE. Pre-incubation of HCV low density RNA containing particles with antibodies to the E2 glycoprotein had little or no effect on uptake. These data indicate that whilst liver derived HCV RNA containing particles are taken up by HepG2 cells by a virus glycoprotein independent mechanism, the mechanism differs from that of LDL uptake.

 

2.89           TRANSPORT OF GHRELIN AND OBESTATIN IN PLASMA

Holmes, E., Davies, I., Lowe, G. and Ranganath, L.

Atherosclerosis Supplements, 9(1), 26-27 (2008)

 

Introduction: Since it’s discovery in 1999, ghrelin has emerged as a key player in central appetite regulation, and also has cardiovascular actions. Roles for ghrelin in the development of atherosclerosis have also been described. Ghrelin is secreted in an active, acylated form, which is rapidly des-acylated in plasma. Obestatin, a peptide produced from the cleavage of pre-proghrelin, is also emerging as a metabolic regulator. While it has been reported that ghrelin can bind to HDL, knowledge about ghrelin and

obestatin transport and metabolism is limited.

Aims: To investigate the transport of ghrelin and obestatin by lipoproteins.

Methods: Ghrelin and obestatin were measured by EIA in plasma fractions generated using an iodixanol gradient.

Results: Acylated ghrelin (AG) bound to all lipoproteins and was present as a plasma protein (VLDL 12%, LDL 33%, HDL 23%, protein 32% of total AG). In contrast, unacylated ghrelin (UAG) was bound more

specifically to HDL (LDL 5%, HDL 43%, protein 51%). Obestatin did not bind to lipoproteins.

Discussion: AG binding to lipoproteins, may be due to a non-specific, hydrophobic interaction between the acyl group and the phospholipid membrane. The interaction between UAG and HDL may indicate a more

specific interaction; possibly due to des-acylation of ghrelin by a HDL bound enzyme. The implications of these various forms of Ghrelin in the context of obesity and cardiovascular disease is unknown.

 

2.90           Lipoprotein separation in a novel iodixanol density gradient, for composition, density, and phenotype analysis

Yee, M.S. et al

1. Lipid Res., 49, 1364-1371 (2008)

 

Separation of lipoproteins by traditional sequential salt density floatation is a prolonged process ( 72 h) with variable recovery, whereas iodixanol-based, self-generating density gradients provide a rapid ( 4 h) alternative. A novel, three-layered iodixanol gradient was evaluated for its ability to separate lipoprotein fractions in 63 subjects with varying degrees of dyslipidemia. Lipoprotein cholesterol, triglycerides, and apolipoproteins were measured in 21 successive iodixanol density fractions. Iodixanol fractionation was compared with sequential floatation ultracentrifugation. Iodixanol gradient formation showed a coefficient of variation of 0.29% and total lipid recovery from the gradient of 95.4% for cholesterol and 84.7% for triglyceride. Recoveries for VLDL-, LDL-, and HDL-cholesterol, triglycerides, and apolipoproteins were approximately 10% higher with iodixanol compared with sequential floatation. The iodixanol gradient effectively discriminated classic lipoproteins and their subfractions, and there was evidence for improved resolution of lipoproteins with the iodixanol gradient. LDL particles subfractionated by the gradient showed good correlation between density and particle size with small, dense LDL (<25.5 nm) separated in fractions with density >1.028 g/dl. The new iodixanol density gradient enabled rapid separation with improved resolution and recovery of all lipoproteins and their subfractions, providing important information with regard to LDL phenotype from a single centrifugation step with minimal in-vitro modification of lipoproteins.

 

2.91           Phenolsulfonphthalein, but Not Phenolphthalein, Inhibits Amyloid Fibril Formation: Implications for the Modulation of Amyloid Self-Assembly

Levy, M., Porat, Y., Bacharach, E., Shalev, D.E. and Gazit, E.

Biochemistry, 47, 5896-5904 (2008)

 

The study of the mechanism of amyloid fibril formation and its inhibition is of key medical importance due to the lack of amyloid assembly inhibitors that are approved for clinical use. We have previously demonstrated the potent inhibitory potential of phenolsulfonphthalein, a nontoxic compound that was approved for diagnostic use in human subjects, on aggregation of islet amyloid polypeptide (IAPP) that is associated with type 2 diabetes. Here, we extend our studies on the mechanism of action of phenolsulfonphthalein by comparing its antiamyloidogenic effect to a very similar compound that is also approved for human use, phenolphthalein. While these compounds have very similar primary chemical structures, they significantly differ in their three-dimensional conformation. Our results clearly demonstrated that these two compounds had completely different inhibitory potencies: While phenolsulfonphthalein was a very potent inhibitor of amyloid fibril formation by IAPP, phenolphthalein did not show significant antiamyloidogenic activity. This behavior was observed with a short amyloid fragment of IAPP and also with the full-length polypeptide. The NMR spectrum of IAPP₂₀₋₂₉ in the presence of phenolsulfonphthalein showed chemical shift deviations that were different from the unbound or phenolphthalein-bound peptide. Differential activity was also observed in the inhibition of insulin amyloid formation by these two compounds, and density-gradient experiments clearly demonstrated the different inhibitory effect of the two compounds on the formation of prefibrillar assemblies. Taken together, our studies suggest that the three-dimensional arrangement of the polyphenol phenolsulfonphthalein has a central role in its amyloid formation inhibition activity.

 

2.92           Increased dietary cholesterol does not increase plasma low density lipoprotein when accompanied by an energy-restricted diet and weight loss

Harman, N.L., Leeds, A.R. and Griffin, B.A.

Eur. J. Nutr., 47, 287-293 (2008)

 

Background  Diets enriched with dietary cholesterol, frequently from eggs, have been shown to produce a small but variable increase in plasma low density lipoprotein (LDL) cholesterol. There is evidence to suggest that energy-restricted diets, that may contain a relatively high proportion of fat and cholesterol, can attenuate the cholesterol-raising effect of dietary cholesterol on plasma LDL.

Aim of the study  To determine the combined effects of increased dietary cholesterol and weight loss produced by energy restriction on plasma LDL cholesterol and lipoproteins.

Methods  A randomized, controlled, parallel study was performed in two groups of free-living volunteers on an energy-restricted diet for 12 weeks, one group was instructed to consume two eggs a day (n = 24), the other, to exclude eggs (n = 21). Dietary advice on energy restriction was based on the British Heart Foundation guidelines on how to lose weight for men and women.

Results  Energy intake fell by 25 and 29% in the egg-fed and non-egg-fed groups, resulting in a moderate weight loss of 3.4 kg (P < 0.05) and 4.4 kg (P < 0.05), respectively. The daily intake of dietary cholesterol increased significantly in the egg-fed group from 278 to 582 mg after 6 weeks. The concentration of plasma LDL cholesterol decreased in the non-egg-fed groups after 6 weeks (P < 0.01) and in the egg-fed and non-egg-fed at 12 weeks relative to baseline. There were no other significant changes in plasma lipoproteins or LDL particle size.

Conclusions  An increased intake of dietary cholesterol from two eggs a day, does not increase total plasma or LDL cholesterol when accompanied by moderate weight loss. These findings suggest that cholesterol-rich foods should not be excluded from dietary advice to lose weight on account of an unfavorable influence on plasma LDL cholesterol.

 

2.93           The Potential for β-Structure in the Repeat Domain of Tau Protein Determines Aggregation, Synaptic Decay, Neuronal Loss, and Coassembly with Endogenous Tau in Inducible Mouse Models of Tauopathy

Mocanu, M-M. et al

1. Neurosci., 28(3), 737-748 (2008)

 

We describe two new transgenic mouse lines for studying pathological changes of Tau protein related to Alzheimer's disease. They are based on the regulatable expression of the four-repeat domain of human Tau carrying the FTDP17 (frontotemporal dementia and parkinsonism linked to chromosome 17) mutation K280 (Tau_RD/ K280), or the K280 plus two proline mutations in the hexapeptide motifs (Tau_RD/ K280/I277P/I308P). The K280 mutation accelerates aggregation ("proaggregation mutant"), whereas the proline mutations inhibit Tau aggregation in vitro and in cell models ("antiaggregation mutant"). The inducible transgene expression was driven by the forebrain-specific CaMKII (calcium/calmodulin-dependent protein kinase II ) promoter. The proaggregation mutant leads to Tau aggregates and tangles as early as 2–3 months after gene expression, even at low expression (70% of endogenous mouse Tau). The antiaggregation mutant does not aggregate even after 22 months of gene expression. Both mutants show missorting of Tau in the somatodendritic compartment and hyperphosphorylation in the repeat domain [KXGS motifs, targets of the kinase MARK (microtubule affinity regulating kinase)]. This indicates that these changes are related to Tau expression rather than aggregation. The proaggregation mutant causes astrogliosis, loss of synapses and neurons from 5 months of gene expression onward, arguing that Tau toxicity is related to aggregation. Remarkably, the human proaggregation mutant Tau_RD coaggregates with mouse Tau, coupled with missorting and hyperphosphorylation at multiple sites. When expression of proaggregation Tau_RD is switched off, soluble and aggregated exogenous Tau_RD disappears within 1.5 months. However, tangles of mouse Tau, hyperphosphorylation, and missorting remain, suggesting an extended lifetime of aggregated wild-type Tau once a pathological conformation and aggregation is induced by a proaggregation Tau species.  

 

2.94           mRNA Translation Regulation by the Gly-Ala Repeat of Epstein-Barr Virus Nuclear Antigen 1

Apcher, S., Komarova, A., Daskalogianni, C., Yin, Y., Malbert-Colas, L. And Fåhraes, R.

1. Virol., 83(3), 1289-1298 (2009)

 

The glycine-alanine repeat (GAr) sequence of the Epstein-Barr virus-encoded EBNA-1 prevents presentation of antigenic peptides to major histocompatibility complex class I molecules. This has been attributed to its capacity to suppress mRNA translation in cis. However, the underlying mechanism of this function remains largely unknown. Here, we have further investigated the effect of the GAr as a regulator of mRNA translation. Introduction of silent mutations in each codon of a 30-amino-acid GAr sequence does not significantly affect the translation-inhibitory capacity, whereas minimal alterations in the amino acid composition have strong effects, which underscores the observation that the amino acid sequence and not the mRNA sequence mediates GAr-dependent translation suppression. The capacity of the GAr to repress translation is dose and position dependent and leads to a relative accumulation of preinitiation complexes on the mRNA. Taken together with the surprising observation that fusion of the 5' untranslated region (UTR) of the c-myc mRNA to the 5' UTR of GAr-carrying mRNAs specifically inactivates the effect of the GAr, these results indicate that the GAr targets components of the translation initiation process. We propose a model in which the nascent GAr peptide delays the assembly of the initiation complex on its own mRNA.

 

 

2.95           Flow-mediated vasodilatation: variation and interrelationships with plasma lipids and lipoproteins

Rasmussen, J.G., Eschen, R.B., Aardestrup, I.V., Dethlefsen, C., Griffin, B.A. and Schmidt, E.B.

Scand. J. Clin. Lab. Invest., 69(1), 156-160 (2009)

 

Objective . Endothelial dysfunction is a critical, prerequisite step in atherosclerosis, and may be evaluated by flow-mediated vasodilatation (FMD). The objective of this study was to examine interrelationships between FMD and plasma lipids and lipoproteins, and to determine the between-operator and within-subject variability associated with this technique. Material and methods. FMD, plasma lipids and lipoproteins, including small dense LDL (sdLDL), were measured twice in 40 healthy volunteers, 4 weeks apart. Interrelationships between mean FMD responses and plasma lipids and lipoproteins were examined by correlation analysis. FMD measurements were taken by two independent operators, allowing determination of between-operator variability. Within-subject variability was determined by obtaining two measurements, 4 weeks apart, in every subject, and carried out by the same operator. Results. FMD was inversely related to plasma triglycerides (r = -0.47, p = 0.002), total cholesterol/HDL cholesterol (r = -0.35, p = 0.03) and apolipoprotein B (r = -0.36, p = 0.02), but not to other plasma lipids and lipoproteins. When measuring variation in FMD, the following results were found: Between operators (SD = 4.0 FMD%) and within subjects (SD = 2.9 FMD%). Conclusions. The associations between FMD, plasma triglycerides and apoB provide evidence supporting a role for triglyceride-rich lipoproteins in endothelial dysfunction.

 

2.96           Phosphotyrosine-dependent in vitro reconstitution of recombinant LAT-nucleated multiprotein signalling complexes on liposomes

Sangani, D., Venien-Bryan, C. and Harder, T.

Mol. Med. Biol., 26(2), 159-170 (2009)

 

Numerous cell surface receptors propagate activation signals to the interior of the cell via tyrosine phosphorylation of transmembrane proteins. This leads to the phosphotyrosine (PiY)-mediated recruitment of cytoplasmic signalling protein complexes which catalyze crucial biochemical signalling reactions. Here we describe the first in vitro reconstitution of such PiY-nucleated protein complexes on an artificial lipid membrane. A tyrosine phosphorylated recombinant variant of the transmembrane adaptor protein Linker for Activation of T cells (PiYLAT) was anchored in liposomes. These PiYLAT proteoliposomes specifically recruited cooperative high avidity signalling protein complexes from Jurkat cytosol. Nucleation of signalling protein assemblies readily occurred on PiYLAT liposomes composed of phosphatidylserine, but not on PiYLAT liposomes composed of phosphatidylcholine. Purified recombinant grb2 alone did not stably associate with tyrosine phosphorylated LAT proteoliposomes. However, when grb2 was presented to the PiYLAT proteoliposomes in the context of Jurkat cytosol it was incorporated into multiprotein signalling complexes. Together the data suggest that these reconstituted high-avidity signalling protein complexes represent a cooperative protein network. This novel in vitro approach offers a novel technology permitting biochemical, structural, and pharmacological analyses of plasma membrane receptor signalling complexes.

 

2.97           The scavenger receptor CD36 plays a role in cytokine-induced macrophage fusion

Helming, L.,Winter, J. and Gordon, S.

1. Cell Sci., 122, 453-459 (2009)

 

Multinucleated giant cells, characteristic of granulomatous infections, originate from the fusion of macrophages. Using an antibody screening strategy we found that the scavenger receptor CD36 participates in macrophage fusion induced by the cytokines IL-4 and GM-CSF. Our results demonstrate that exposure of phosphatidylserine on the cell surface and lipid recognition by CD36 are required for cytokine-induced fusion of macrophages. We also show that CD36 acts in a heterotypic manner during giant-cell formation and that the formation of osteoclasts is independent of CD36. The discovery of molecules involved in the formation of multinucleated giant cells will enable us to determine their functional significance. Furthermore, our results suggest that lipid capture by cell surface receptors may be a general feature of cell fusion.

 

2.98           Loss of Modifier of Cell Adhesion Reveals a Pathway Leading to Axonal Degeneration

Chen, Q., Peto, C.A., Shelton, G.D., Mizisin, A., Sawchenko, P.E. and Schubert, D.

1. Neurosci., 29(1), 118-130 (2009)

 

Axonal dysfunction is the major phenotypic change in many neurodegenerative diseases, but the processes underlying this impairment are not clear. Modifier of cell adhesion (MOCA) is a presenilin binding protein that functions as a guanine nucleotide exchange factor for Rac1. The loss of MOCA in mice leads to axonal degeneration and causes sensorimotor impairments by decreasing cofilin phosphorylation and altering its upstream signaling partners LIM kinase and p21-activated kinase, an enzyme directly downstream of Rac1. The dystrophic axons found in MOCA-deficient mice are associated with abnormal aggregates of neurofilament protein, the disorganization of the axonal cytoskeleton, and the accumulation of autophagic vacuoles and polyubiquitinated proteins. Furthermore, MOCA deficiency causes an alteration in the actin cytoskeleton and the formation of cofilin-containing rod-like structures. The dystrophic axons show functional abnormalities, including impaired axonal transport. These findings demonstrate that MOCA is required for maintaining the functional integrity of axons and define a model for the steps leading to axonal degeneration.

 

2.99           DDX3 DEAD-Box RNA Helicase Inhibits Hepatitis B Virus Reverse Transcription by Incorporation into Nucleocapsids

Wang, H., Kim, S.. and Ryu, W-S.

1. Virol., 83(11), 5815-5824 (2009)

 

Viruses utilize host factors in many steps of their life cycles. Yet, little is known about host factors that contribute to the life cycle of hepatitis B virus (HBV), which replicates its genome by reverse transcription. To identify host factors that contribute to viral reverse transcription, we sought to identify cellular proteins that interact with HBV polymerase (Pol) by using affinity purification coupled with mass spectrometry. One of the HBV Pol-interacting host factors identified was DDX3 DEAD-box RNA helicase, which unwinds RNA in an ATPase-dependent manner. Recently, it was shown that DDX3 is essential for both human immunodeficiency virus and hepatitis C virus infection. In contrast, we found that the ectopic expression of DDX3 led to significantly reduced viral DNA synthesis. The DDX3-mediated inhibition of viral DNA synthesis did not affect RNA encapsidation, a step prior to reverse transcription, and indicated that DDX3 inhibits HBV reverse transcription. Mutational analysis revealed that mutant DDX3 with an inactive ATPase motif, but not that with an inactive RNA helicase motif, failed to inhibit viral DNA synthesis. Our interpretation is that DDX3 inhibits viral DNA synthesis at a step following ATP hydrolysis but prior to RNA unwinding. Finally, OptiPrep density gradient analysis revealed that DDX3 was incorporated into nucleocapsids, suggesting that DDX3 inhibits viral reverse transcription following nucleocapsid assembly. Thus, DDX3 represents a novel host restriction factor that limits HBV infection.

 

2.100           Photoinitiated Destruction of Composite Porphyrin−Protein Polymersomes

Robbins, G.P., Jimbo, M., Swift, J., Therien, M.J., Hammer, D.A. and Dmochoxski, I.J.

1. Am. Chem. Soc., 131, 3872-3874 (2009)

 

Bilayer vesicles assembled from amphiphilic diblock copolymers (polymersomes) adopt asymmetric structures when loaded with moderate concentrations (≥1.5 mg/mL) of horse spleen ferritin (HSF) or its iron-free variant (HSAF). Incorporation of both ferritin and a zinc porphyrin dimer (PZn₂) generates photoresponsive vesicles: irradiation with focused light of near-UV to near-IR wavelengths induces polymersome deformation and destruction on the minute time scale. To investigate this phenomenon, polymersomes were loaded with dye-labeled ferritin and PZn₂. Confocal microscopy identified BODIPY-FL-labeled ferritin at the membrane, whereas Cy3-labeled ferritin was found both at the membrane and throughout the aqueous core. Fluorescence recovery after photobleaching (FRAP) experiments confirmed that Cy3- and BODIPY-FL-labeled ferritin and PZn₂ exhibited slow diffusion at the membrane, consistent with membrane association. Furthermore, micropipette aspiration experiments revealed increased elastic moduli and altered bending rigidity in vesicles incorporating HSAF. Finally, a small molecule (biocytin) was encapsulated within the ferritin−PZn₂ vesicles and released upon exposure to light. These data indicate synergy between ferritin, whose membrane association lowers the barrier to deformation, and PZn₂, which embeds in the membrane, harvests light energy and produces local heating that may lead to membrane budding. This appears to be a general protein−polymer membrane phenomenon, as replacement of ferritin with bovine serum albumin or equine skeletal myoglobin resulted in vesicles with similar asymmetric morphology and photosensitivity.

 

2.101           Four Conserved Cysteine Residues of the Hepatitis B Virus Polymerase Are Critical for RNA Pregenome Encapsidation

Kim, S., Lee, J. and Ryu, W-S.

1. Virol., 83(16), 8032-8040 (2009)

 

Hepadnaviruses replicate via reverse transcription of an RNA template, the pregenomic RNA (pgRNA). Although hepadnaviral polymerase (Pol) and retroviral reverse transcriptase are distantly related, some of their features are distinct. In particular, Pol contains two additional N-terminal subdomains, the terminal protein and spacer subdomains. Since much of the spacer subdomain can be deleted without detrimental effects to hepatitis B virus (HBV) replication, this subdomain was previously thought to serve only as a spacer that links the terminal protein and reverse transcriptase subdomains. Unexpectedly, we found that the C terminus of the spacer subdomain is indispensable for the encapsidation of pgRNA. Alanine-scanning mutagenesis revealed that four conserved cysteine residues, three at the C terminus of the spacer subdomain and one at the N terminus of the reverse transcriptase subdomain, are critical for encapsidation. The inability of the mutant Pol proteins to incorporate into nucleocapsid particles, together with other evidence, argued that the four conserved cysteine residues are critical for RNA binding. One implication is that these four cysteine residues might form a putative zinc finger motif. Based on these findings, we speculate that the RNA binding activity of HBV Pol may be mediated by this newly identified putative zinc finger motif.

 

2.102           Effect of hyperthermia on plasma lipids and gene expression in Atlantic Cod. (Gadus Morhua I.)

Aursnes, I.A.S., Gjoen, T. and Rishovd, A-L.

Toxico. Lett., 189S, S57 (2009)

 

Purpose: Wild fish occupy their preferred temperature range by vertical movements in the water column. During fish farming, this migration may be restricted leading to thermal stress. We have investigated stress responses in Atlantic Cod (Gadus Morhua) exposed to elevated water temperature. The main goal was to identify stable housekeeping genes that can be used for internal normalization for quantitative real-time PCR analyses across treatment groups in experiments. The software used was Normfinder and REST. We also obtained blood samples and analyzed for differences in plasma lipid concentrations.

Methods: Wild Atlantic cod (Gadus Morhua) weighing 200–500 g was divided into two groups (5 fish in each tan were one served as control (kept at 12 °C) and one group in which the water temperature was increased from 12 °C to 17 °C. Tissue samples and blood samples were withdrawn from all individuals. Blood was analyzed by separation on a continuous gradient using iodixanol and divided into fractions which was analyzed for triglycerides, cholesterol and level of oxidation. The tissue samples were prepared for RNA extraction and RT-qPCR analyses.

Results: Plasma lipid concentrations (total cholesterol and triglycerides), and oxidation levels in the hyperthermally stressed fish were significantly increased compared to the control group. Across both groups and all tissues Normfinder ranked the gene analyzed in this order, from most stable to least stable EF1α > 18s > ubiquitin > β-actin > β-2-microglobulin > tubulin α > ARP > G6PDH. In liver tissue Normfinder identified ubiquitin as the most stable gene. REST analysis of relative expression revealed that in liver HSP90, IL-1β and TNFα was significantly upregulated after hyperthermia.

 

2.103           Comparability of methods for LDL subfraction determination: A systematic review

Chung, M., Lichtenstein, A.H., Ip, S., Lau, J. and Balk, E.M.

Atherosclerosis, 205, 342-348 (2009)

 

Identifying and aggressively treating individuals at elevated risk of developing cardiovascular disease (CVD) is critical to optimizing health outcomes. The CVD risk factors defined by the National Cholesterol Education Program do not fully predict individuals at high risk of developing CVD. Validation of potential methodologies against a reference method is essential to the adoption of a potential new risk factor to improve risk prediction. Low-density lipoprotein (LDL) subfraction has been advanced as a potential additional CVD risk factor. Currently, there is no reference method for determining LDL subfractions or standardizing the different methods used to measure LDL subfractions. We conducted a systematic review to identify reports comparing two or more methods of measuring LDL subfractions. Nine articles were identified that separated and quantified LDL subfractions by at least two methods. Comparative data were available for nuclear magnetic resonance vs. gel electrophoresis (GE), LipoPrint^® vs. other GE methods, ultracentrifugation vs. GE, and high performance gel filtration chromatography vs. GE. We found a wide range of agreement (from 7 to 94% concordance for classifying LDL patterns) among methods for LDL subfraction determinations. Different criteria and definitions were used among the articles to classify individuals with respect to CVD risk. No study used CVD or other clinical outcomes as an outcome measure. In summary, the currently available literature does not provide adequate data about comparability in terms of test performance to choose one or another method to serve as a standard nor are data on comparability in terms of predicting CVD outcomes.

 

2.104           Changes in lipoprotein profile and urinary albumin excretion in familial LCAT deficiency with lipid lowering therapy

Yee, M.S., Pavitt, D.V., Richmond, W., Cook, H.T., McLean, A.G., Valabhji, J. and Elkeles, R.S.

Atherosclerosis, 205, 528-532 (2009)

 

Familial lecithin:cholesterol acyltransferase deficiency (FLD) is a monogenic autosomal recessive condition, affecting cholesterol esterification and leads to progressive renal impairment and end-stage renal failure, probably due to the abnormal lipoprotein (X) (Lp(X)).

We report a case of FLD, whom we treated with a combination of nicotinic acid 1.5 g nocte and fenofibrate M/R 160 mg od and report changes in lipid profile and Lp(X), after six weeks and serum creatinine and urine albumin/creatinine ratio after 12 months. We assessed the cardiovascular risk using electron beam computed tomography.

At baseline total cholesterol was 6.61 mmol/L; HDL cholesterol 0.57 mmol/L; Lp(X) cholesterol 3.24 mmol/L; triglyceride 4.13 mmol/L; apolipoprotein A1 46 mg/dL; and apolipoprotein B 53 mg/dL. After six weeks of treatment his total cholesterol was 4.16; HDL cholesterol 0.52; Lp(X) cholesterol 1.73 mmol/L; triglyceride 1.80 mmol/L; apolipoprotein A1 36 mg/dL; and apolipoprotein B 50 mg/dL. Baseline serum creatinine was 106 μmol/L and urine albumin/creatinine ratio was 127.3 mg/mmol and after 12 months was 101 μmol/L and 31.5 mg/mmol respectively. His coronary artery calcification score was zero.

We have shown, we believe for the first time, that combination lipid modifying therapy in FLD leads to a reduction in Lp(X) concentration and an associated reduction in urine albumin excretion at 12 months.

 

2.105           Long-Term Glycemic Control Influences the Long-Lasting Effect of Hyperglycemia on Endothelial Function in Type 1 Diabetes

Ceriello, A., Esposito, K., Ihnat, M., Thorpe, J. and Giugliano, D.

1. Clin. Endocrinol. Metab., 94(8), 2751-2756 (2009)

 

Objective: The objective of the study was to investigate theeffect of different periods of hyperglycemia on the reversalof endothelial dysfunction by glucose normalization and antioxidanttherapy.

Research Design and Methods: Ten healthy subjects and threesubgroups of 10 type 1 diabetic subjects were enrolled as follows:1) patients within 1 month of diagnosis; 2) patients between4.5 and 5.2 yr from diagnosis and with glycosylated hemoglobinlevels 7% or greater since diagnosis; 3) patients between 4.8and 5.4 yr from diagnosis and with glycosylated hemoglobin levelsgreater than 7% since diagnosis. Each patient participated inthree experiments: 1) 24-h insulin treatment, achieving a nearnormalization of glycemia, together with the addition of theantioxidant vitamin C during the last 12 h; 2) 24-h vitaminC treatment with insulin treatment for the last 12 h; and 3)treatment with both vitamin C and insulin for 24 h.

Results: Endothelial function, as measured by flow-mediatedvasodilation of the brachial artery and levels of nitrotyrosine,an oxidative stress marker, were normalized by each treatmentin subgroups 1 and 2. In the third subgroup, neither glucosenormalization nor vitamin C treatment alone was able to normalizeendothelial dysfunction or oxidative stress. Combining insulinand vitamin C, however, normalized endothelial dysfunction andnitrotyrosine.

Conclusions: This study suggests that long-lasting hyperglycemiain type 1 diabetic patients induces long-term alterations inendothelial cells, which may contribute to endothelial dysfunctionand is interrupted only by both glucose and oxidative stressnormalization.

 

2.106           The accessory subunit of mitochondrial DNA polymerase determines the DNA content of mitochondrial nucleoids in human cultured cells

Re, M.D., Sembongi, H., He, J., Reyes, J.H., Yasukawa, T., Martinsson, P., Bailey, L.J., Goffart, S., Boyd-Kirkup, J.D., Wong, T.S., Fersht, A.R., Spelbrink, J.N. and Holt, I.J.

Nucleic Acids Res., 37(17), 5701-5713 (2009)

 

The accessory subunit of mitochondrial DNA polymerase , POLGβ, functions as a processivity factor in vitro. Here we show POLGβ has additional roles in mitochondrial DNA metabolism. Mitochondrial DNA is arranged in nucleoprotein complexes, or nucleoids, which often contain multiple copies of the mitochondrial genome. Gene-silencing of POLGβ increased nucleoid numbers, whereas over-expression of POLGβ reduced the number and increased the size of mitochondrial nucleoids. Both increased and decreased expression of POLGβ altered nucleoid structure and precipitated a marked decrease in 7S DNA molecules, which form short displacement-loops on mitochondrial DNA. Recombinant POLGβ preferentially bound to plasmids with a short displacement-loop, in contrast to POLG . These findings support the view that the mitochondrial D-loop acts as a protein recruitment centre, and suggest POLGβ is a key factor in the organization of mitochondrial DNA in multigenomic nucleoprotein complexes.

 

2.107           Ultrastructures and strain comparison of under-glycosylated scrapie prion fibrils

Sim, V.L. and Caughey, B.

Neurobiology of Aging, 30, 2031-2042 (2009)

 

Prions, composed primarily of misfolded, often fibrillar, polymers of prion protein, have poorly understood structures. Heavy surface glycosylation may obscure visualization of their fibrillar cores, so we purified severely under-glycosylated prion protein fibrils from scrapie-infected transgenic mice expressing anchorless prion protein. Using electron and atomic force microscopy, we obtained dimensions and morphological information about prion protein core protofilaments which variably intertwined to form scrapie fibrils. Occasional isolated protofilaments were observed, suggesting that the lateral association of protofilaments is neither essential nor invariant in prion protein polymerization. Strain comparisons suggested basic structural differences; ME7 and 22L fibrils contained thinner protofilaments, 22L fibrils preferred left-handed twists, and 22L fibril periodicities averaged 106 nm per half-turn, compared with 64 and 66 nm for RML and ME7 fibrils, respectively. The strains displayed overlapping fibril morphologies, providing evidence that prion fibril morphology is influenced, but not dictated, by strain-dependent differences in protofilament structure. These measurements of the amyloid core of scrapie fibrils should aid development of models of prion structure and strain determination.

 

2.108           Phytosterol-Enriched Yogurt Increases LDL Affinity and Reduces CD36 Expression in Polygenic Hypercholesterolemia

Ruiu, G., Pinach, S., Veglia, F., Gambino, R., Marena, S., Uberti, B.., Alemanno, N., Burt, D., Pagano, G. and Cassader, M.

Lipids, 44, 153-160 (2009)

 

Dietary enrichment with phytosterols (plant sterols similar to cholesterol) is able to reduce plasma cholesterol levels due to reduced intestinal absorption. The aim of this study was to investigate the effect of phytosterol-enriched yogurt consumption on the major serum lipid parameters, low density lipoprotein (LDL) receptor activity, LDL-receptor affinity, and CD36 expression in hypercholesterolemic subjects. Fifteen patients affected by polygenic hypercholesterolemia were evaluated in a single-blind randomized crossover study after a 4 weeks treatment with a phytosterol-enriched yogurt containing 1.6 g esterefied phytosterols (equivalent to 1.0 g free phytosterol). Lipid parameters were compared with a phytosterol-free placebo-controlled diet. The effect of the two treatments on each variable, measured as percentage change, was compared by paired samples t test and covariance analysis. The treatment induced a modest but significant decrease in LDL-cholesterol levels (4.3%, P = 0.03) and a significant increase in high density lipoprotein (HDL) 3-cholesterol (17.1%, P = 0.01). Phytosterol consumption had no effect on LDL-receptor activity whereas patient LDL-receptor affinity significantly increased (9.7%, P = 0.01) and CD36 expression showed a marked significant decrease (18.2%, P = 0.01) in the phytosterol-enriched yoghurt patients. Our data show that the oral administration of a phytosterol-enriched yogurt has modest but significant effects on commonly measured lipid parameters. The improvement of LDL-receptor affinity and the reduction in CD36 expression may reflect an important antiatherogenic effect.

 

2.109           Circulating ghrelin exists in both lipoprotein bound and free forms

Holmes, E., Davies, I., Lowe, G. and Ranganath, L.R.

Ann. Clin. Biochem., 46, 514-516 (2009)

 

Introduction: Ghrelin is a gastric peptide that has been implicated in thedevelopment of obesity and cardiovascular disease. It has beenreported that ghrelin binds to lipoproteins, although the differentbinding patterns of acylated ghrelin (AG) and unacylated ghrelin(UAG) are still to be determined.

Methods: Lipoprotein fractions were generated using a self-generating iodixanol gradient. AG and UAG were measured using specificenzyme immunoassays.

Results: AG bound to all lipoproteins in approximately equal concentrations(VLDL 26%, LDL 22%, HDL 23%) and was present as a plasma protein(27%). UAG bound more specifically to HDL (49%) and was presentas a plasma protein (48%).

Conclusions: The different binding patterns of AG and UAG may have significant implications for their biological effects, including roles in energy metabolism, the development of obesity and potentially in the modulation of cardiovascular disease.

 

2.110           Impact of Saturated, Polyunsaturated and Monounsaturated Fatty Acid-Rich Micelles on Lipoprotein Synthesis and Secretion in Caco-2

Jackson, K.G., Bateman, P.A., Yaqoob, P. and Williams, C.M.

Lipids, 44, 1081-1089 (2009)

 

Meal fatty acids have been shown to modulate the size and composition of triacylglycerol (TAG)-rich lipoproteins influencing the magnitude and duration of the postprandial plasma TAG response. As a result there is considerable interest in the origin of these meal fatty-acid induced differences in particle composition. Caco-2 cells were incubated over 4 days with fatty acid mixtures resembling the composition of saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acid (PUFA)-rich meals fed in a previous postprandial study to determine their impact on lipoprotein synthesis and secretion. The MUFA- and PUFA-rich mixtures supported greater intracellular TAG, but not cholesterol accumulation compared with the SFA-rich mixture (P < 0.001). The MUFA-rich mixture promoted significantly greater TAG and cholesterol secretion than the other mixtures and significantly more apolipoprotein B-100 secretion than the PUFA-rich mixture (P < 0.05). Electron microscopy revealed the SFA-rich mixture had led to unfavourable effects on cellular morphology, compared with the unsaturated fatty acid-rich mixtures. Our findings suggest the MUFA-rich mixture, may support the formation of a greater number of TAG-rich lipoproteins, which is consistent with indirect observations from our human study. Our electron micrographs are suggestive that some endocytotic uptake of MUFA-rich taurocholate micelles may promote greater lipoprotein synthesis and secretion in Caco-2 cells.

 

2.111           Incorporation of Eukaryotic Translation Initiation Factor eIF4E into Viral Nucleocapsids via Interaction with Hepatitis B Virus Polymerase

Kim, S., Wang, H. and Ryu, W-S.

1. Virol., 84(1), 52-58 (2010)

 

The DNA genome of hepatitis B virus (HBV) replicates via reverse transcription within capsids following the encapsidation of an RNA template, the pregenomic RNA (pgRNA). We previously demonstrated that the 5' cap proximity of the stem-loop structure ( or epsilon), an encapsidation signal, is critically important for the encapsidation of the pgRNA (J. K. Jeong, G. S. Yoon, and W. S. Ryu, J. Virol. 74:5502-5508, 2000). Therefore, we speculated that the viral polymerase (Pol), while bound to the 5' stem-loop structure, could recognize the cap via its interaction with eIF4E, a eukaryotic translation initiation factor. Our data showed the direct interaction between HBV Pol and eIF4E, as measured by coimmunoprecipitation. Further, we demonstrated that eIF4E interacts with the Pol- ribonucleoprotein complex (RNP) rather than Pol alone, resulting in eIF4E-Pol- RNP complex formation. In addition, we asked whether eIF4E remains engaged to the Pol- RNP complex during nucleocapsid assembly. Density gradient analysis revealed that eIF4E indeed was incorporated into nucleocapsids. It is of great importance to uncover whether the incorporated eIF4E contributes to viral reverse transcription or other steps in the HBV life cycle.

 

2.112           Unpacking a gel-forming mucin: a view of MUC5B organization after granular release

Kesimer, M., Makhov, A.M., Griffith, J.D., Verdugo, P. and Sheehan, J.K.

Am. J. Physiol. Lung Cell Mol. Physiol., 298, L15-L22 (2010)

 

Gel-forming mucins are the largest complex glycoprotein macromolecules in the body. They form the matrix of gels protecting all the surface epithelia and are secreted as disulfide-bonded polymeric structures. The mechanisms by which they are formed and organized within cells and thereafter released to form mucus gels are not understood. In particular, the initial rate of expansion of the mucins after release from their secretory granules is very rapid (seconds), but no clear mechanism for how it is achieved has emerged. Our major interest is in lung mucins, but most particularly in MUC5B, which is the major gel-forming mucin in mucus, and which provides its major protective matrix. In this study, using OptiPrep density gradient ultracentrifugation, we have isolated a small amount of a stable form of the recently secreted and expanding MUC5B mucin, which accounts for less than 2% of the total mucin present. It has an average mass of 150 x 10⁶ Da and size Rg of 150 nm in radius of gyration. In transmission electron microscopy, this compact mucin has maintained a circular structure that is characterized by flexible chains connected around protein-rich nodes as determined by their ability to bind colloidal gold. The appearance indicates that the assembled mucins in a single granular form are organized around a number of nodes, each attached to four to eight subunits. The organization of the mucins in this manner is consistent with efficient packing of a number of large heavily glycosylated monomers while still permitting their rapid unfolding and hydration. For the first time, this provides some insight into how the carbohydrate regions might be organized around the NH₂- and COOH-terminal globular protein domains within the granule and also explains how the mucin can expand so rapidly upon its release.  

 

2.113           Distribution of Chlorophyll- and Bacteriochlorophyll-derived Photosensitizers in Human Blood Plasma

Dandler, J., Wilhelm, B. and Scheer, H.

Photochem. and Photobiol., 86, 182-193 (2010)

 

Chlorophyll a and, in particular, bacteriochlorophyll a derivatives are promising candidates for photosensitizers in photodynamic therapy. The distribution of 21 (bacterio)chlorophyll derivatives among human blood plasma fractions was studied by iodixanol gradient ultracentrifugation and in situ absorption spectroscopy. Modifications of the natural pigments involved the central metal (Mg²⁺, Zn²⁺, Pd²⁺, none), the isocyclic ring (closed, open and taurinated), substituents at C-3 (vinyl, acetyl, 1-hydroxyethyl) and C-17³ (phytyl ester, free acid). Cellular blood components bound only a small fraction of the pigments. Distribution among low-density lipoproteins (LDL), high-density lipoproteins (HDL) and high-density proteins (HDP) of the plasma was influenced as follows: (1) application in Cremophor^® EL slightly altered pigment distribution by lipoprotein modification, (2) only very polar pigments with multiple hydrophilic substituents showed substantial HDP binding, (3) the presence of the esterifying alcohol at C-17³ caused enrichment in LDL, this was more pronounced with bacteriochlorophylls than with chlorophylls, (4) substituents at C-3 had only little influence on the distribution, (5) Zn²⁺-complexes were enriched in HDL compared to Mg²⁺ and Pd²⁺ complexes, indicating specific binding of the former. Equilibration of pigments among the different fractions was largely complete within 3 h.

 

2.114           Acrolein consumption induces systemic dyslipidemia and lipoprotein modification

Conklin, D.J., Barski, O.A., Lesgards, J-F., Juvan, P., rezen, T., Rozman, D., Prough, R.A., Vladykovskaya, E., Liu, S., Srivastava, S. and Bhatnagar, A.

Tox. Appl. Pharmacol., 243(1), 1.12 (2010)

 

Aldehydes such as acrolein are ubiquitous pollutants present in automobile exhaust, cigarette, wood, and coal smoke. Such aldehydes are also constituents of several food substances and are present in drinking water, irrigation canals, and effluents from manufacturing plants. Oral intake represents the most significant source of exposure to acrolein and related aldehydes. To study the effects of short-term oral exposure to acrolein on lipoprotein levels and metabolism, adult mice were gavage-fed 0.1 to 5 mg acrolein/kg bwt and changes in plasma lipoproteins were assessed. Changes in hepatic gene expression related to lipid metabolism and cytokines were examined by qRT-PCR analysis. Acrolein feeding did not affect body weight, blood urea nitrogen, plasma creatinine, electrolytes, cytokines or liver enzymes, but increased plasma cholesterol and triglycerides. Similar results were obtained with apoE-null mice. Plasma lipoproteins from acrolein-fed mice showed altered electrophoretic mobility on agarose gels. Chromatographic analysis revealed elevated VLDL cholesterol, phospholipids, and triglycerides levels with little change in LDL or HDL. NMR analysis indicated shifts from small to large VLDL and from large to medium-small LDL with no change in the size of HDL particles. Increased plasma VLDL was associated with a significant decrease in post-heparin plasma hepatic lipase activity and a decrease in hepatic expression of hepatic lipase. These observations suggest that oral exposure to acrolein could induce or exacerbate systemic dyslipidemia and thereby contribute to cardiovascular disease risk.

 

2.115           Comparative proteomic profiling of plasma very-low-density and low-density lipoproteins

Sun, H-Y., Chen, S-F., Lai, M-D., Chang, T-T., Chen, T-L., Li, P-Y., Shieh, D-B. and Young, K-C.

Clin. Chim. Acta., 411, 336-344 (2010)

 

Background

Low-density lipoprotein (LDL) is a natural metabolite of very-low-density lipoprotein (VLDL) in the circulation. Systematic investigation of total protein components and dynamics might provide insights into this normal metabolic process.

Methods

VLDL and LDL were purified from normolipidemia pooled plasma by gradient ultracentrifugation with either ionic or non-ionic media. The protein contents were compared by liquid chromatography tandem mass analyses based on isobaric tag for relative and absolute quantitation and two-dimensional gel electrophoresis.

Results

Our comparative lipoproteomes revealed 21 associated proteins. Combined with Western blot analysis, and on the basis of the differential expression levels we classified them into 3 groups: (i) VLDL > LDL [apolipoprotein (apo) A-IV, apo(a), apoCs, apoE, apoJ and serum amyloid A-4]; (ii) VLDL < LDL [albumin, α-1-antitrypsin, apoD, apoF, apoM, and paraoxonase-1]; and (iii) VLDL = LDL [apoA-I, apoA-II, apoB-100, apoL-I and prenylcysteine oxidase-1]. The apoA-I level positively correlated with PCYOX1 but negatively with apoM in VLDL and LDL. Furthermore, the two-dimensional maps displayed 5 apoA-I isoforms in which phosphorylation at Ser55, Ser166, Thr185, Thr221 and Ser252 residues were identified.

Conclusions

This study revealed the VLDL- and LDL lipoproteomes and the full-spectrum protein changes during physiological VLDL-to-LDL transition. It provides a valuable dataset VLDL and LDL proteomes potentially applied to the development of diagnostics.

 

2.116           Isolation and Characterization of Cytoplasmic Cofilin-Actin Rods

Minamide, L.S., Maiti, S., Boyle, J.A., Davis, R.C., Coppinger, J.A., Bao, Y., Huang, T.Y., yates, J., Bokoch, G.M. and Bamburg, J.R.

1. Biol. Chem., 285(8), 5450-5460 (2010)

 

Cofilin-actin bundles (rods), which form in axons and dendrites of stressed neurons, lead to synaptic dysfunction and may mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-depolymerizing factor (ADF)/cofilin fluorescent proteins by differential and equilibrium sedimentation on OptiPrep gradients and then from neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated rods are stable in dithiothreitol, EGTA, Ca²⁺, and ATP. Cofilin-GFP-containing rods are stable in 500 mm NaCl, whereas rods formed from endogenous proteins are significantly less stable in high salt. Proteomic analysis of rods formed from endogenous proteins identified other potential components whose presence in rods was examined by immunofluorescence staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation; furthermore, the rapid assembly of rods in vitro from these purified proteins at physiological concentration shows that they are the only proteins necessary for rod formation. Cytoplasmic rod formation is inhibited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultrastructural tomography ranged from 22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated F-actin.

 

2.117           Increased Glycation and Oxidative Damage to Apolipoprotein B100 of LDL Cholesterol in Patients With Type 2 Diabetes and Effect of Metformin

Rabbani, N., Chittari, M.V., Bopdmer, C.W., Zehnder, D., Ceriello, A. and Thornalley, P.J.

Diabetes, 59, 1038-1045 (2010)

 

OBJECTIVE The aim of this study was to investigate whether apolipoprotein B100 of LDL suffers increased damage by glycation, oxidation, and nitration in patients with type 2 diabetes, including patients receiving metformin therapy.

RESEARCH DESIGN AND METHODS For this study, 32 type 2 diabetic patients and 21 healthy control subjects were recruited; 13 diabetic patients were receiving metformin therapy (median dose: 1.50 g/day). LDL was isolated from venous plasma by ultracentrifugation, delipidated, digested, and analyzed for protein glycation, oxidation, and nitration adducts by stable isotopic dilution analysis tandem mass spectrometry.

RESULTS Advanced glycation end product (AGE) content of apolipoprotein B100 of LDL from type 2 diabetic patients was higher than from healthy subjects: arginine-derived AGE, 15.8 vs. 5.3 mol% (P < 0.001); and lysine-derived AGE, 2.5 vs. 1.5 mol% (P < 0.05). Oxidative damage, mainly methionine sulfoxide residues, was also increased: 2.5 vs. 1.1 molar equivalents (P < 0.001). 3-Nitrotyrosine content was decreased: 0.04 vs. 0.12 mol% (P < 0.05). In diabetic patients receiving metformin therapy, arginine-derived AGE and methionine sulfoxide were lower than in patients not receiving metformin: 19.3 vs. 8.9 mol% (P < 0.01) and 2.9 vs. 1.9 mol% (P < 0.05), respectively; 3-nitrotyrosine content was higher: 0.10 vs. 0.03 mol% (P < 0.05). Fructosyl-lysine residue content correlated positively with fasting plasma glucose. Arginine-derived AGE residue contents were intercorrelated and also correlated positively with methionine sulfoxide.

CONCLUSIONS Patients with type 2 diabetes had increased arginine-derived AGEs and oxidative damage in apolipoprotein B100 of LDL. This was lower in patients receiving metformin therapy, which may contribute to decreased oxidative damage, atherogenicity, and cardiovascular disease.

 

2.118           The Physical Relationship between Infectivity and Prion Protein Aggregates Is Strain-Dependent

Tixador, P., Herzog, L., Reine, F., Jaumain, E., chapuis, J., Le Dur, A., Laude, H. and Beringue, V.

PloSPathogens, 6(4), e1000859 (2010)

 

Prions are unconventional infectious agents thought to be primarily composed of PrP^Sc, a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP^C). They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrP^Sc conformation could encode this ‘strain’ diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrP^Sc aggregation state, and how this varies according to the strain. We applied a sedimentation velocity technique to a panel of natural, biologically cloned strains obtained by propagation of classical and atypical sheep scrapie and BSE infectious sources in transgenic mice expressing ovine PrP. Detergent-solubilized, infected brain homogenates were used as starting material. Solubilization conditions were optimized to separate PrP^Sc aggregates from PrP^C. The distribution of PrP^Sc and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrP^Sc peak was observed in the middle part of the gradient. This population approximately corresponds to multimers of 12–30 PrP molecules, if constituted of PrP only. For two strains, infectivity peaked in a markedly different region of the gradient. This most infectious component sedimented very slowly, suggesting small size oligomers and/or low density PrP^Sc aggregates. Extending this study to hamster prions passaged in hamster PrP transgenic mice revealed that the highly infectious, slowly sedimenting particles could be a feature of strains able to induce a rapidly lethal disease. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics.

Prions are unconventional transmissible agents causing fatal neurodegenerative diseases in human and animals. They are thought to be formed from polymers of abnormal conformations of the host-encoded prion protein (PrP), but little is known about the physical organization of the infectious particles and any relationship between packing order and infectivity. As an additional layer of complexity, different PrP conformational variants associated with distinct biological phenotypes, or ‘strains’, can propagate in the same host. We subjected PrP polymers from eight different ovine and hamster prion strains to sedimentation velocity centrifugation, which allows separation of macromolecular complexes according to their size, density or shape. We showed that, whereas the PrP sedimentation profiles share common features, the infectivity profiles exhibit striking differences amongst the strains. For four of them, the infectious component was predominantly associated with slowly sedimenting particles, suggestive of small size oligomers and/or low density PrP aggregates. Such particles appeared to be a feature of strains able to induce a rapidly lethal disease in the recipient host. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics.

 

2.119           The Conserved Bardet-Biedl Syndrome Proteins Assemble a Coat that Traffics Membrane Proteins to Cilia

Jin, H., White, S.R., Shida, T., Schulz, S., Aguiar, M., Gygi, S.P., Bazan, J.F. and Nachury, M.V.

Cell, 141, 1208-1219 (2010)

 

The BBSome is a complex of Bardet-Biedl Syndrome (BBS) proteins that shares common structural elements with COPI, COPII, and clathrin coats. Here, we show that the BBSome constitutes a coat complex that sorts membrane proteins to primary cilia. The BBSome is the major effector of the Arf-like GTPase Arl6/BBS3, and the BBSome and GTP-bound Arl6 colocalize at ciliary punctae in an interdependent manner. Strikingly, Arl6^GTP-mediated recruitment of the BBSome to synthetic liposomes produces distinct patches of polymerized coat apposed onto the lipid bilayer. Finally, the ciliary targeting signal of somatostatin receptor 3 needs to be directly recognized by the BBSome in order to mediate targeting of membrane proteins to cilia. Thus, we propose that trafficking of BBSome cargoes to cilia entails the coupling of BBSome coat polymerization to the recognition of sorting signals by the BBSome.

 

2.120           Calcium-dependent Regulation of SNARE-mediated Membrane Fusion by Calmodulin

Di Giovanni, J., Iborra, C., Maulet, Y., Leveque, C., El Far, O. and Seagar, M.

1. Biol. Chem., 285(31), 23665-23675 (2010)

 

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca²⁺ sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca²⁺-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca²⁺/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K_D = 500 nm) and syntaxin 1 (K_D = 2 μm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca²⁺ sensors act antagonistically in SNARE-mediated fusion.

 

2.121           Cigarette smoke and human plasma lycopene depletion

Graham, D.L., Carail, M., Caris-Veyrat, C. and Lowe, G.M.

Food and Chem. Tox., 48, 2413-2420 (2010)

 

It is known that smokers have a higher risk of developing cardiovascular disease and lung cancer. Plasma carotenoid concentrations in smokers are generally lower than in non-smokers and this may be due to modifications in diet or a direct or indirect action of cigarette smoke on carotenoids in the plasma. Recently it was reported that reactive nitrogen species derived from cigarette smoke could diffuse across the lung alveolar cell wall into the plasma. Such species may modify circulating low density lipoprotein (LDL) and in the process reduce circulating carotenoid concentrations. In an effort to address this rational we have treated lycopene solutions, human plasma and isolated LDL with cigarette smoke and monitored all-(E)-lycopene, 5(Z)-lycopene and β-carotene depletion. In plasma, the depletion of all-(E)-lycopene (15.0 ± 11.0%, n = 10) was greater than 5(Z)-lycopene (10.4 ± 9.6%) or β-carotene (12.4 ± 10.5%). In LDL, both all-(E)- and 5(Z)-lycopene were more susceptible than β-carotene (20.8 ± 11.8%, 15.4 ± 11.5% and 11.5 ± 12.5%, n = 3 respectively). The effects have been compared with Sin-1 reactions and isomerization of all-(E) lycopene is common to both treatments. The results clearly indicate that low plasma lycopene may be a direct consequence of smoke inhalation.

 

2.122           Lipoprotein Particles Cross the Blood–Brain Barrier in Drosophila

Brankatschk, M. and Eaton, S.

1. Neurosci., 30(31), 10441-10447 (2010)

 

The blood–brain barrier (BBB) regulates passage of nutrients and signaling molecules from the circulation into the brain. Whether lipoproteins cross the BBB in vivo has been controversial, and no clear requirement for circulating lipoproteins in brain development has been shown. We address these issues in Drosophila, which has an functionally conserved BBB, and lipoproteins that resemble those of vertebrates. We show that the Drosophila lipoprotein lipophorin exists in two isoforms. Both isoforms cross the BBB, but accumulate on distinct subsets of cells within the brain. In addition to acting as a lipid carrier, lipophorin carries both sterol-linked and GPI-linked proteins into the circulation and transports them across the BBB. Finally, lipophorin promotes neuroblast proliferation by a mechanism that does not depend on delivery of dietary lipids. Transport of lipophorin and its cargo across the BBB represents a novel mechanism by which peripherally synthesized proteins might enter the brain and influence its development. Furthermore, lipid-linkage may be an efficient method to transport therapeutic molecules across the BBB.

 

2.123           Tunable Leuko-polymersomes That Adhere Specifically to Inflammatory Markers

Robbins, G.P., Saunders, R.L., Haun, J.B., Rawson, J., Therien, M.J. and Hammer, D.A.

Langmuir, 26(17), 14089-14096 (2010)

 

The polymersome, a fully synthetic cell mimetic, is a tunable platform for drug delivery vehicles to detect and treat disease (theranostics). Here, we design a leuko-polymersome, a polymersome with the adhesive properties of leukocytes, which can effectively bind to inflammatory sites under flow. We hypothesize that optimal leukocyte adhesion can be recreated with ligands that mimic receptors of the two major leukocyte molecular adhesion pathways, the selectins and the integrins. Polymersomes functionalized with sialyl Lewis X and an antibody against ICAM-1 adhere avidly and selectively to surfaces coated with inflammatory adhesion molecules P-selectin and ICAM-1 under flow. We find that maximal adhesion occurs at intermediate densities of both sialyl Lewis X and anti-ICAM-1, owing to synergistic binding effects between the two ligands. Leuko-polymersomes bearing these two receptor mimetics adhere under physiological shear rates to inflamed endothelium in an in vitro flow chamber at a rate 7.5 times higher than those to uninflamed endothelium. This work clearly demonstrates that polymersomes bearing only a single ligand bind less avidly and with lower selectivity, thus suggesting proper mimicry of leukocyte adhesion requires contributions from both pathways. This work establishes a basis for the design of polymersomes for targeted drug delivery in inflammation.

 

2.124           Engineering Therapeutic Nanocarriers with Optimal Adhesion for Targeting 

Haun, J.B., Robbins, G.P. and Hammer, D.A.

1. Adhesion, 86, 131-159 (2010)

 

There is considerable interest in developing therapeutic delivery carriers that can be targeted via receptor-ligand interactions to sites within the blood stream. The adhesion of carriers is determined by the combined effects of transport phenomena, hydrodynamic force, and the dynamics of multivalent receptor/ligand bonding. Optimizing the adhesion of carriers requires developing relationships between these factors and carrier properties such as size and receptor coating density. Recently, we developed canonical relationships for the binding of antibody-conjugated 200 nm particles to surfaces coated with a vascular adhesion molecule, intercellular adhesion molecule-1. Here we extend our previous studies of adhesion to particles of different size, including 40 nm and 1  m particles. Particle binding is assessed under fluid flow in a parallel plate flow chamber while varying particle receptor density, substrate ligand density, and flow rate. Using a stochastic simulation and transport-reaction model we then extract multivalent kinetic rate constants for particle attachment and detachment from the binding data. We demonstrate that particles go though a maximum in binding with particle size. For small particles, increasing size increases receptor-ligand encounter rates; for larger particles, fluid shear force begins to dominate, leading to higher forces and decreased adhesion. Our methods provide a means for optimizing particle size and receptor density for the selective binding of particles to vascular endothelium under flow.

 

2.125           Lack of effect of cold water prawns on plasma cholesterol and lipoproteins in normo-lipidaemic men

Isherwood, C., Wong, M., Jones, W.S., Davies, I.G. and Griffin, B.A.

Cell. Mol. Biol., 56(1), 52-58 (2010)

 

OBJECTIVE: Dietary guidelines for the prevention of coronary heart disease (CHD) have restricted the intake of foods rich in dietary cholesterol, on the grounds that the dietary cholesterol will increase blood cholesterol. In the case of shellfish, this recommendation may limit the intake of a valuable dietary source of long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA). The objective of this study was to undertake a dietary intervention to determine the effects of cold water prawns on plasma lipids and lipoproteins.

METHODS: 23 healthy male subjects were randomised to receive either 225 g of cold water prawns or an equivalent weight of fish ('crab') sticks as a control for 12 weeks in a cross-over design. Blood samples were taken at the beginning and end of each intervention for the determination of plasma lipids and lipoproteins by routine enzymatic assays and iodixanol density gradient centrifugation respectively.

RESULTS: The diets were well matched for the intake of total energy and macronutrients, and body weight remained stable throughout the study. The prawn intervention increased the intake of dietary cholesterol to 750 mg/d against 200 mg/d on the control. The intake of LC n-3 PUFA from prawns was estimated to be between 0.5-0.7 g/d. The consumption of prawns produced no significant effects on the concentration of plasma total or LDL cholesterol, triacylglycerol, HDL cholesterol or apolipoproteins A-I and B relative to the control, or within each intervention group over time. There was also no significant effect on LDL density (particle size) relative to the control, or any difference between and within treatments in total plasma lipoprotein profiles by density gradient centrifugation.

CONCLUSION: These findings provide evidence to suggest that the consumption of cold water prawns, at least in healthy, male subjects, should not be restricted on the grounds of this seafood producing an adverse effect on plasma LDL cholesterol.

 

2.126           A Generalized System for Photoresponsive Membrane Rupture in Polymersomes

Kamat, N.P., Robbins, G.P., Rawson, J., Therien, M.J., Dmochowski, I.J. and Hammer, D.A.

Adv. Funct. Mater., 20, 2588-2596 (2010)

 

Polymersomes are vesicles whose membranes comprise self-assembled block copolymers. It has recently been shown that co-encapsulating conjugated multiporphyrin dyes in a polymersome membrane with ferritin protein in the aqueous lumen confers photolability to the polymersome. In the present study, the photolability is shown to be extendable to vesicles containing dextran, an inert and inexpensive polysaccharide, as the luminal solute. How structural features of the polymersome/porphyrin/dextran composite affect its photoresponse is explored. Increasing dextran molecular weight, decreasing block copolymer molecular weight, and altering fluorophore-membrane interactions results in increasing the photoresponsiveness of the polymersomes. Amphiphilic interactions of the luminal encapsulant with the membrane coupled with localized heat production in the hydrophobic bilayer likely cause differential thermal expansion in the membrane and the subsequent membrane rupture. This study suggests a general approach to impart photoresponsiveness to any biomimetic vesicle system without chemical modification, as well as a simple, bio-inert method for constructing photosensitive carriers for controlled release of encapsulants.

 

2.127           Lipid accumulation and metabolism in polychaete spermatogenesis: Role of the large discoidal lipoprotein

Schenk, S. and Hoeger, U.

Mol. Reprod. Dev., 77, 710-719 (2010)

 

In most oviparous animals, lipoprotein-mediated lipid transport plays an important role in the nutrient supply for the oocyte. In male gametes, lipids are used as energy substrates in spermatozoa but nothing is yet known about their origin and metabolism throughout spermatogenesis. The lipid profiles analyzed from different stages of male germ cell development in the marine annelid Nereis virens were found to undergo a dramatic change from primary triacylglycerides at the beginning of germ cell development to cholesterol and phospholipids at the end of development as demonstrated by HPLC with evaporative light scattering detection and mass spectrometry. The uptake of a large discoidal lipoprotein into the developing germ cells could be demonstrated by fluorescence labeling and electron microscopic techniques as well as by the presence of a lipoprotein receptor in the germ cells, thus establishing its role in lipid supply. The incorporated lipoprotein discs were found to be stored as intact complexes indicating that they are not readily degraded upon endocytotic uptake. The change in lipid composition during germ cell development reflects their metabolic activity, especially in spermatogonia. The high concentration of lipids maintained by spermatogonia during the early phase of gametogenesis seems to be required for the later rapid processes of meiosis and spermatocyte differentiation. At times when peak demand of lipids arises for membrane synthesis and increased metabolism, this may be met more efficiently by a rapid on-site mobilization of lipids instead of an external supply.

 

2.128           Functional roles of VASP phosphorylation in the regulation of chemotaxis and osmotic stress response

Lin, W-H., Nelson, S.E., Hollingsworth, R.J. and Chung, C.Y.

Cytoskeleton, 67, 259-271 (2010)

 

Vasodilator-stimulated phosphoprotein (VASP) plays crucial roles in controlling F-actin-driven processes and growing evidence indicates that VASP function is modulated by phosphorylation at multiple sites. However, the complexity of mammalian system prevents the clear understanding of the role of VASP phosphorylation. In this study, we took advantage of Dictyostelium which possesses only one member of the Ena/VASP family to investigate the functional roles of VASP phosphorylation. Our results demonstrated that hyperosmotic stress and cAMP stimulation cause VASP phosphorylation. VASP phosphorylation plays a negative role for the early steps of filopodia/microspikes formation. VASP phosphorylation appears to modulate VASP localization at the membrane cortex and its interactions with WASP and WIPa. Analysis of chemotaxis of cells expressing VASP mutants showed that VASP phosphorylation is required for the establishment of cell polarity under a cAMP gradient.

 

2.129           Photochemistry of Bacteriochlorophylls in Human Blood Plasma: 1. Pigment Stability and Light-induced Modifications of Lipoproteins

Dandler, J., Wilhelm, B. and Scheer, H.

Photochem. Photobiol., 86, 331-341 (2010)

 

Transmetalated derivatives of bacteriochlorophyll are promising sensitizers in photodynamic therapy. Protocols using short delay times between injection and irradiation cause interest in the photochemistry of these pigments in the blood. Using near-infrared irradiation where these pigments absorb strongly, we have studied the photochemistry of Zn- and Pd-bacteriopheophorbide (WST09), and of the highly polar taurinated Pd-derivative, WST11, in isolated fractions of human blood plasma. The stability of all pigments is increased in blood plasma, compared with monomeric solutions. Pd-bacteriopheophorbide is much more stable than the other two derivatives. It also has a higher capacity for inducing reactive oxygen species, yet the consumption of oxygen is comparable. There is furthermore evidence for photobleaching under anoxic conditions. The generation of hydroperoxides (ROOH) is faster with Pd- than with Zn-complexes; the formation of endoperoxides (ROOR′), measured as thiobarbituric acid reactive substances, is comparable with the two central metals. Formation of both ROOH and ROOR′ is increased in low-density lipoproteins (LDL) compared with high-density lipoproteins (HDL), which is probably related to the higher concentration of target molecules in the former. In HDL, extensive cross-linking is induced among the apolipoproteins; judged from the electrophoretic mobility of LDL and HDL particles, there is also a gross structural change. Photosensitized cross-linking is much less pronounced with high-density proteins.

 

2.130           Effect of changing the amount and type of fat and carbohydrate on insulin sensitivity and cardiovascular risk: the RISCK (Reading, Imperial, Surrey, Cambridge, and Kings) trial

Jebb, S.A., Lovegrove, J.A., Griffin, B.A., Frost, G.S., Moore, C.S., Chatfield, M.D., Bluck, L.J., Williams, C.M. and Sanders, T.A.B.

Am. J. Clin. Nutr., 92, 748-758 (2010)

 

Background: Insulin sensitivity (Si) is improved by weight lossand exercise, but the effects of the replacement of saturatedfatty acids (SFAs) with monounsaturated fatty acids (MUFAs)or carbohydrates of high glycemic index (HGI) or low glycemicindex (LGI) are uncertain.

Objective: We conducted a dietary intervention trial to studythese effects in participants at risk of developing metabolicsyndrome.

Design: We conducted a 5-center, parallel design, randomizedcontrolled trial [RISCK (Reading, Imperial, Surrey, Cambridge,and Kings)]. The primary and secondary outcomes were changesin Si (measured by using an intravenous glucose tolerance test)and cardiovascular risk factors. Measurements were made after4 wk of a high-SFA and HGI (HS/HGI) diet and after a 24-wk interventionwith HS/HGI (reference), high-MUFA and HGI (HM/HGI), HM andLGI (HM/LGI), low-fat and HGI (LF/HGI), and LF and LGI (LF/LGI)diets.

Results: We analyzed data for 548 of 720 participants who were randomly assigned to treatment. The median Si was 2.7 x 10^–4 mL · µU^–1 · min^–1 (interquartile range: 2.0, 4.2 x 10^–4 mL · µU^–1 · min^–1), and unadjusted mean percentage changes (95% CIs) after 24 wk treatment (P = 0.13) were as follows: for the HS/HGIgroup, –4% (–12.7%, 5.3%); for the HM/HGI group,2.1% (–5.8%, 10.7%); for the HM/LGI group, –3.5%(–10.6%, 4.3%); for the LF/HGI group, –8.6% (–15.4%,–1.1%); and for the LF/LGI group, 9.9% (2.4%, 18.0%).Total cholesterol (TC), LDL cholesterol, and apolipoproteinB concentrations decreased with SFA reduction. Decreases inTC and LDL-cholesterol concentrations were greater with LGI.Fat reduction lowered HDL cholesterol and apolipoprotein A1and B concentrations.

Conclusions: This study did not support the hypothesis thatisoenergetic replacement of SFAs with MUFAs or carbohydrateshas a favorable effect on Si. Lowering GI enhanced reductionsin TC and LDL-cholesterol concentrations in subjects, with tentativeevidence of improvements in Si in the LF-treatment group. Thistrial was registered at clinicaltrials.gov as ISRCTN29111298.

 

2.131           Effects of antioxidants on postprandial oxidative stress and endothelial dysfunction in subjects with impaired glucose tolerance and Type 2 diabetes

Neri, S., Calvagno, S., Mauceri, B., Misseri, M., Tsami, A., Vecchio, C., Mastrosimone, G., Di Pino, A., Maiorca, D., Judica, A., Romano, G., Rizzotto, A. and Signorelli, S.S.

Eur. J. Nutr., 49, 409-416 (2010)

 

Aim  

To compare changes in the oxidation–reduction balance and endothelial function before and after meal in patients with type 2 diabetes or impaired glucose tolerance and determine the effects of standard antioxidant supplementation.

Methods  

Forty diabetics and 40 subjects with impaired glucose tolerance were compared with a control group. We assessed before and after a test meal (homogenized milkshake containing 80 g of saturated fat, amounting to 1,480 kcal), some reactive oxygen species, inflammation markers and flow-mediated vascular dilatation. These parameters were then reassessed after standard antioxidant treatment.

Results  

After the meal, diabetics, subjects with impaired glucose tolerance and controls had higher levels of oxidant compounds compared to fasting levels. In subjects with diabetes and impaired glucose tolerance (IGT), Vascular Adhesion Molecule-1 and CRP were higher after the meal—diabetic subjects exhibited lower fasting flow-mediated dilatation, which deteriorated significantly after the meal. Antioxidant administration significantly improved the parameters investigated in all subjects.

Conclusions  

In diabetic subjects, altered glycaemia and lipaemia are closely correlated with markers of systemic oxidative stress. Our results show that the abnormal changes in oxidative-reductive balance parameters are paralleled by similar changes in markers of endothelial dysfunction and inflammation at 4 h after ingestion of a fatty meal. Supplementation with a pool of antioxidants can reduce oxidative stress and inflammation in healthy subjects and, more importantly, in IGT patients. This previous aspect suggests that the timing of antioxidant supplementation has an important role in endothelium protection in healthy and pre-diabetic subjects, and along with prompt antioxidant treatment before irreversible endothelial damage has occurred, may have an important protective role in subjects with IGT—patients who require administration of adequate dietary antioxidants.

 

2.132           Role of the Highly Conserved Middle Region of Prion Protein (PrP) in PrP−Lipid Interaction

Wang, F., Yin, S., Wang, X., Zha, L., Sy, M-S. and Ma, J.

Biochemistry, 49, 8169-8176 (2010)

 

Converting normal prion protein (PrP^C) to the pathogenic PrP^Sc isoform is central to prion disease. We previously showed that, in the presence of lipids, recombinant mouse PrP (rPrP) can be converted into the highly infectious conformation, suggesting a crucial role of lipid−rPrP interaction in PrP conversion. To understand the mechanism of lipid−rPrP interaction, we analyzed the ability of various rPrP mutants to bind anionic lipids and to gain lipid-induced proteinase K (PK) resistance. We found that the N-terminal positively charged region contributes to electrostatic rPrP−lipid binding but does not affect lipid-induced PK resistance. In contrast, the highly conserved middle region of PrP, consisting of a positively charged region and a hydrophobic domain, is essential for lipid-induced rPrP conversion. The hydrophobic domain deletion mutant significantly weakened the hydrophobic rPrP−lipid interaction and abolished the lipid-induced C-terminal PK resistance. The rPrP mutant without positive charges in the middle region reduced the amount of the lipid-induced PK-resistant rPrP form. Consistent with a critical role of the middle region in lipid-induced rPrP conversion, both disease-associated P105L and P102L mutations, localized between lysine residues in the positively charged region, significantly affected lipid-induced rPrP conversion. The hydrophobic domain-localized 129 polymorphism altered the strength of hydrophobic rPrP−lipid interaction. Collectively, our results suggest that the interaction between the middle region of PrP and lipids is essential for the formation of the PK-resistant conformation. Moreover, the influence of disease-associated PrP mutations and the 129 polymorphism on PrP−lipid interaction supports the relevance of PrP−lipid interaction to the pathogenesis of prion disease.

 

2.133           Oral Treatment with the d-Enantiomeric Peptide D3 Improves the Pathology and Behavior of Alzheimer’s Disease Transgenic Mice

Funke, S.A., van Groen, T., Kadish, I., Bartnik, D., Nage-Steger, L., Brener, O., Sehl, T., Batra-Safferling, R., Moriscot, C., Schoehn, G., Horn, A.H.C., Müller-Schiffmann, A., Korth, C., Sticht, H. and Willbold, D.

ACS Chem. Neurosci., 1(9), 639-648 (2010)

 

Several lines of evidence suggest that the amyloid-β-peptide (Aβ) plays a central role in the pathogenesis of Alzheimer’s disease (AD). Not only Aβ fibrils but also small soluble Aβ oligomers in particular are suspected to be the major toxic species responsible for disease development and progression. The present study reports on in vitro and in vivo properties of the Aβ targeting d-enantiomeric amino acid peptide D3. We show that next to plaque load and inflammation reduction, oral application of the peptide improved the cognitive performance of AD transgenic mice. In addition, we provide in vitro data elucidating the potential mechanism underlying the observed in vivo activity of D3. These data suggest that D3 precipitates toxic Aβ species and converts them into nonamyloidogenic, nonfibrillar, and nontoxic aggregates without increasing the concentration of monomeric Aβ. Thus, D3 exerts an interesting and novel mechanism of action that abolishes toxic Aβ oligomers and thereby supports their decisive role in AD development and progression.

 

2.134           Phosphorylation of Aquaporin-2 Regulates Its Water Permeability

Eto, K., Noda, Y., Horikawa, S., Uchida, S. and Sasaki, S.

1. Biol. Chem., 285(52), 40777-40784 (2010)

 

Vasopressin-regulated water reabsorption through the water channel aquaporin-2 (AQP2) in renal collecting ducts maintains body water homeostasis. Vasopressin activates PKA, which phosphorylates AQP2, and this phosphorylation event is required to increase the water permeability and water reabsorption of the collecting duct cells. It has been established that the phosphorylation of AQP2 induces its apical membrane insertion, rendering the cell water-permeable. However, whether this phosphorylation regulates the water permeability of this channel still remains unclear. To clarify the role of AQP2 phosphorylation in water permeability, we expressed recombinant human AQP2 in Escherichia coli, purified it, and reconstituted it into proteoliposomes. AQP2 proteins not reconstituted into liposomes were removed by fractionating on density step gradients. AQP2-reconstituted liposomes were then extruded through polycarbonate filters to obtain unilamellar vesicles. PKA phosphorylation significantly increased the osmotic water permeability of AQP2-reconstituted liposomes. We then examined the roles of AQP2 phosphorylation at Ser-256 and Ser-261 in the regulation of water permeability using phosphorylation mutants reconstituted into proteoliposomes. The water permeability of the non-phosphorylation-mimicking mutant S256A-AQP2 and non-phosphorylated WT-AQP2 was similar, and that of the phosphorylation-mimicking mutant S256D-AQP2 and phosphorylated WT-AQP2 was similar. The water permeability of S261A-AQP2 and S261D-AQP2 was similar to that of non-phosphorylated WT-AQP2. This study shows that PKA phosphorylation of AQP2 at Ser-256 enhances its water permeability.

 

2.135           The fibrate drug gemfibrozil disrupts lipoprotein metabolism in rainbow trout

Prindiville, J.S., Mennigen, J.A., Zamora, J.M., Moon, T.W. and Weber, J-M.

Tox. Appl. Pharmacol., 251, 201-208 (2011)

 

Gemfibrozil (GEM) is a fibrate drug consistently found in effluents from sewage treatment plants. This study characterizes the pharmacological effects of GEM on the plasma lipoproteins of rainbow trout (Oncorhynchus mykiss). Our goals were to quantify the impact of the drug on: 1) lipid constituents of lipoproteins (phospholipids (PL), triacylglycerol (TAG), and cholesterol), 2) lipoprotein classes (high, low and very low density lipoproteins), and 3) fatty acid composition of lipoproteins. Potential mechanisms of GEM action were investigated by measuring lipoprotein lipase activity (LPL) and the hepatic gene expression of LPL and of the peroxisome proliferator-activated receptor (PPAR) α, β, and γ isoforms. GEM treatment resulted in decreased plasma lipoprotein levels (− 29%) and a reduced size of all lipoprotein classes (lower PL:TAG ratios). However, the increase in HDL-cholesterol elicited by GEM in humans failed to be observed in trout. Therefore, HDL-cholesterol cannot be used to assess the impact of the drug on fish. GEM also modified lipoprotein composition by reducing the abundance of long-chain n−3 fatty acids, thereby potentially reducing the nutritional quality of exposed fish. The relative gene expression of LPL was increased, but the activity of the enzyme was not, and we found no evidence for the activation of PPAR pathways. The depressing effects of GEM on fish lipoproteins demonstrated here may be a concern in view of the widespread presence of fibrates in aquatic environments. Work is needed to test whether exposure to environmental concentrations of these drugs jeopardizes the capacity of fish for reproduction, temperature acclimation or migratory behaviors.

 

2.136           Small dense LDL particles - a predictor of coronary artery disease evaluated by invasive and CT-based techniques: a case-control study

Toft-Petersen, A.P., Tilsted, H.H., aarøe, J., Rasmussen, K., Christensen, T., Griffin, B.A., Aardestrup, I.V., Andreasen, A. and Schmidt, E.B.

Lipids in Health and Disease, 10, 21-27 (2011)

 

Background

Coronary angiography is the current standard method to evaluate coronary atherosclerosis in patients with suspected angina pectoris, but non-invasive CT scanning of the coronaries are increasingly used for the same purpose.

Low-density lipoprotein (LDL) cholesterol and other lipid and lipoprotein variables are major risk factors for coronary artery disease. Small dense LDL particles may be of particular importance, but clinical studies evaluating their predictive value for coronary atherosclerosis are few.

Methods

We performed a study of 194 consecutive patients with chest pain, a priori considered of low to intermediate risk for significant coronary stenosis (>50% lumen obstruction) who were referred for elective coronary angiography. Plasma lipids and lipoproteins were measured including the subtype pattern of LDL particles, and all patients were examined by coronary CT scanning before coronary angiography.

Results

The proportion of small dense LDL was a strong univariate predictor of significant coronary artery stenosis evaluated by both methods. After adjustment for age, gender, smoking, and waist circumference only results obtained by traditional coronary angiography remained statistically significant.

Conclusion

Small dense LDL particles may add to risk stratification of patients with suspected angina pectoris.

 

2.137           LIPOPROTEIN SECRETION PROFILES AND VLDL PRODUCTION IN HEPATOCYTE CELL LINES

Jammart, B., Zoulin, F.and Durantel, D.

1. Hepatol., 54, S318 (2011)

 

Background and Aims: Hepatitis C virus (HCV) is highly associated to apolipoprotein-B containing lipoproteins (LDL and VLDL) in infected patient sera, as most viral RNA is co-immunoprecipitated

with anti-ApoB antibodies, but this association is barely seen in vitro (e.g. Huh7.5 cells infected with the HCV JFH-1 strain). Recent data suggested that these cells may be deficient for mature VLDL

production. Thus, our aim was to: i. characterize lipoprotein secretion in Huh7.5 cells; ii. compare this secretion to natural VLDL production by primary human hepatocytes (PHH); iii. find other hepatocyte cell lines competent for VLDL production.

Methods: Cell culture supernatants were harvested, concentrated using an Amicon centrifugal filter unit with a cut-off of 100 kDa (Millipore™) and ultracentrifuged over an iodixanolsucrose gradient. Density distributions of apolipoproteins were determined using ELISA or western blot. The production of mature

VLDL was further assessed by co-immunoprecipitation of different apolipoproteins.

Results: We found that Huh7.5 cells secrete a large amount of ApoB as compared to PHH. However, ApoB was detected at a density corresponding to LDL or IDL (sup. than 1.01 g/mL), but not VLDL, and was not associated with ApoE, as neither co-segregation nor co-immunoprecipitation were observed. Importantly, HCV infection increased ApoB secretion but did not affect the density of secreted particles. In contrast, secretion of ApoB/ApoE-containing lipoproteins with a density and composition comparable to that of PHH was observed in differentiated HepaRG cells, although the amount of secreted particles was lower. Finally, the hepatoblastoma cell line HepG2 was also able to secrete very-low-density particles

containing ApoB and ApoE upon treatment with oleic acid (to stimulate lipoprotein production) and MEK/ERK inhibitors (to reduce the over-activation of MEK1 kinase in these cells).

Conclusion: We have characterized lipoprotein secretion profiles in 3 different cell lines (Huh7.5, HepG2 and HepaRG) as well as in PHH. Huh7.5 cell line, which is commonly used to study HCV replication, does not seem to produce mature VLDL and is therefore a poor model to study HCV particle secretion and its association to lipoproteins as observed in vivo. The other hepatocyte cell lines may

therefore be more relevant study models of HCV morphogenesis.

 

2.138           The putative diabetic plasma marker, soluble CD36, is non-cleaved, non-soluble and entirely associated with microparticles

Alkhatatbeh, M.J., Mhaidat, N.M., Enjeti, A.K., Lincz, L.F. and Thorne, R.F.

1. Thrombosis and Haemostasis, 9, 844-851 (2011)

 

Background:CD36 is a widely expressed cell surface receptor that binds  lipoproteins, and its function has been implicated in many complications of the metabolic syndrome. A cell-free form of CD36, soluble CD36 (sCD36), has been reported in human plasma, found to be elevated in obesity and diabetes, and claimed as a marker of insulin resistance. Objective:To determine the  nature of sCD36; in particular, whether sCD36 is truly soluble or, as hypothesized, is found as a component of circulating microparticles (MPs). Methods:Lipoproteins were fractionated by density gradient  centrifugation, and plasma MPs were isolated by ultracentrifugation, size exclusion, and immunoprecipitation with CD36 detected by immunoblotting. MPs from plasma and activated platelets were analyzed by multicolor flow cytometry, with a DyLight-488 anti-CD36 conjugate in combination with antibodies against different cellular markers. Results:Cell-free plasma CD36 was not  observed associated with lipoproteins and was not a proteolytic fragment; rather, it was associated with the plasma MP fraction, suggesting that sCD36 in the plasma of normal subjects is a product of circulating MPs. Cytometric and immunoblotting analyses of plasma from normal donors showed that these MPs were derived mainly from platelets. Analysis of in vitro activated platelets also showed that CD36 to be secreted in the form of MPs. Conclusions:sCD36  is not a proteolytic product, but rather is associated with a specific subset of circulating MPs that can readily be analysed. This finding will enable more specific investigations into the cellular source of the increased levels of plasma CD36 found in subjects with diabetes.

 

2.139           Cell stress is related to re-localization of Argonaute 2 and to decreased RNA interference in human cells

Detzer, A., Engel, C., Wûnsche, W. and Sczakiel, G.

Nucelic Acids Res., 39(7), 2727-2741 (2011)

 

Various kinds of stress on human cells induce the formation of endogenous stress granules (SGs). Human Argonaute 2 (hAgo2), the catalytic core component of the RNA-induced silencing complex (RISC), can be recruited to SGs as well as P-bodies (PBs) indicating that the dynamic intracellular distribution of hAgo2 in SGs, in PBs or at other sub-cellular sites could be related to the efficiency of the RNA interference (RNAi) machinery. Here, we studied the influence of heat shock, sodium arsenite (NaAsO₂), cycloheximide (CHX) and Lipofectamine^TM 2000-mediated transfection of phosphorothioate (PS)-modified oligonucleotides (ON) on the intracellular localization of hAgo2 and the efficiency of RNAi.

Fluorescence microscopy and sedimentation analysis of cell fractions indicate stress-induced accumulation of hAgo2 in SGs and the loss of distinctly composed complexes containing hAgo2 or their sub-cellular context. Transfection of cells with PS-ON induces cell stress that is phenotypically similar to the established inducers heat shock and NaAsO₂. The intracellular re-distribution of hAgo2 is related to its increased metabolic stability and to decreased RNAi directed by microRNA or by short interfering RNA. Here, we propose a functional model of the relationship between cell stress, translocation of hAgo2 to SGs providing a depot function, and loss of RNAi activity.

 

2.140           Forming giant vesicles with controlled membrane composition, asymmetry, and contents

Richmond, D.L., Schmidt, E.M., Martens, S., Stachowiak, J.C., Liska, N. and Fletcher, D.A.

PNAS, 108(23), 9431-9436 (2011)

 

Growing knowledge of the key molecular components involved in biological processes such as endocytosis, exocytosis, and motility has enabled direct testing of proposed mechanistic models by reconstitution. However, current techniques for building increasingly complex cellular structures and functions from purified components are limited in their ability to create conditions that emulate the physical and biochemical constraints of real cells. Here we present an integrated method for forming giant unilamellar vesicles with simultaneous control over (i) lipid composition and asymmetry, (ii) oriented membrane protein incorporation, and (iii) internal contents. As an application of this method, we constructed a synthetic system in which membrane proteins were delivered to the outside of giant vesicles, mimicking aspects of exocytosis. Using confocal fluorescence microscopy, we visualized small encapsulated vesicles docking and mixing membrane components with the giant vesicle membrane, resulting in exposure of previously encapsulated membrane proteins to the external environment. This method for creating giant vesicles can be used to test models of biological processes that depend on confined volume and complex membrane composition, and it may be useful in constructing functional systems for therapeutic and biomaterials applications.

 

2.141           Actin and myosin contribute to mammalian mitochondrial DNA maintenance

Reyes, A. et al

Nucleic Acid Res., 39(12), 5098-5108 (2011)

 

Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and β-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of β-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some β-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance.

 

2.142           Macromolecular organization of ATP synthase and complex I in whole mitochondria

Davies, K.M., Strauss, M., Daum, B., Kief, J.H., Osiewacz, H.D., Rycovska, A., Zickermann, V and Kühlbrandt, W.

PNAS, 108(34), 14121-14126 (2011)

 

We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F₁ heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.

 

2.143           Spongiform Encephalopathy in Transgenic Mice Expressing a Point Mutation in the β2–α2 Loop of the Prion Protein

Sigurdson, C., Joshi-Barr, S., Bett, C., Winson, O., Manco, G., Schwarz, P., Rülicke, T., Nilsson, K.P.R., Margalith, I., Raeber, A., Peretz, D., Hornemann, S., Wüthrick, K. and Aguzzi, A.

1. Neurosci., 31(39), 13840-13847 (2011)

 

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases attributed to misfolding of the cellular prion protein, PrPC, into a β-sheet-rich, aggregated isoform, PrPSc. We previously found that expression of mouse PrP with the two amino acid substitutions S170N and N174T, which result in high structural order of the β2–α2 loop in the NMR structure at pH 4.5 and 20°C, caused transmissible de novo prion disease in transgenic mice. Here we report that expression of mouse PrP with the single-residue substitution D167S, which also results in a structurally well ordered β2–α2 loop at 20°C, elicits spontaneous PrP aggregation in vivo. Transgenic mice expressing PrPD167S developed a progressive encephalopathy characterized by abundant PrP plaque formation, spongiform change, and gliosis. These results add to the evidence that the β2–α2 loop has an important role in intermolecular interactions, including that it may be a key determinant of prion protein aggregation.

 

2.144           Fish Oil Supplementation During Late Pregnancy Does Not Influence Plasma Lipids or Lipoprotein Levels in Young Adult Offspring

Rytter, D., Schmidt, E.B., Bech, B.H., Christensen, J.H., henriksen, T.B. and Olsen, S.F.

Lipids, 46, 1091-1099 (2011)

 

Nutritional influences on cardiovascular disease operate throughout life. Studies in both experimental animals and humans have suggested that changes in the peri- and early post-natal nutrition can affect the development of the various components of the metabolic syndrome in adult life. This has lead to the hypothesis that n-3 fatty acid supplementation in pregnancy may have a beneficial effect on lipid profile in the offspring. The aim of the present study was to investigate the effect of supplementation with n-3 fatty acids during the third trimester of pregnancy on lipids and lipoproteins in the 19-year-old offspring. The study was based on the follow-up of a randomized controlled trial from 1990 where 533 pregnant women were randomized to fish oil (n = 266), olive oil (n = 136) or no oil (n = 131). In 2009, the offspring were invited to a physical examination including blood sampling. A total of 243 of the offspring participated. Lipid values did not differ between the fish oil and olive oil groups. The relative adjusted difference (95% confidence intervals) in lipid concentrations was −3% (−11; 7) for LDL cholesterol, 3% (−3; 10) for HDL cholesterol, −1% (−6; 5) for total cholesterol,−4% (−16; 10) for TAG concentrations, 2%(−2; 7) for apolipoprotein A1, −1% (−9; 7) for apolipoprotein B and 3% (−7; 15) in relative abundance of small dense LDL. In conclusion, there was no effect of fish oil supplementation during the third trimester of pregnancy on offspring plasma lipids and lipoproteins in adolescence.

 

2.145           An albumin-associated PLA2-like activity inactivates surfactant phosphatidylcholine secreted from fetal type II pneumocytes

Damas, J.E. and Cake, M.H.

Am. J. Physiol. Lung Cell. Mol. Physiol., 301(6), L966-L974 (2011)

 

Type II pneumocytes are responsible for the synthesis and secretion of pulmonary surfactant, which reduces surface tension in lung alveoli, thus decreasing their tendency to collapse during expiration. For this effect to be sustained, the integrity of the surface-active components of surfactant must be maintained. This study has shown that, when cultured type II pneumocytes are exposed to lipoprotein-free serum (LFS), the level of lyso-phosphatidylcholine (lyso-PC) in the secreted surfactant phospholipids is markedly elevated with a concomitant decline in the level of phosphatidylcholine (PC). This effect is the result of hydrolysis of surfactant PC by a phospholipase A₂ (PLA₂)-like activity present within serum. Anion-exchange chromatography, gel filtration chromatography and preparative electrophoresis of human LFS have shown that this PLA₂-like activity coelutes with albumin and is biochemically distinct from the secretory form of PLA₂. Furthermore, specific inhibitors of PLA₂ such as p-bromophenacyl bromide, aristolochic acid, and palmitoyl trifluoromethyl ketone do not inhibit this activity of serum. Commercially purified human serum albumin fraction V and recombinant human serum albumin (rHSA) are almost as effective as LFS in enhancing the level of lyso-PC in the media. The latter finding implies that rHSA directly generates lyso-PC from secreted PC and suggests that this PLA₂-like activity may be an intrinsic attribute of albumin.

 

2.146           BAP31 and BiP are essential for dislocation of SV40 from the endoplasmic reticulum to the cytosol

Geiger, R., Andritschke, D., Friebe, S., Herzog, F., Luisoni, S., heger, T. and Helenius, A.

Nature Cell Biol., 13(11), 1305-1314 (2011)

 

Non-enveloped viruses such as SV40 are transported from the extracellular space into the host cell nucleus through a pathway involving endocytosis, trafficking to the endoplasmic reticulum (ER) lumen, transport across the ER membrane to the cytoplasm, and subsequent nuclear import. Helenius and colleagues provide insight into how SV40 escapes from the ER by showing that viral proteins interact with components of the host ER-associated degradation machinery (ERAD). These interactions are crucial for translocation of SV40 into the cytoplasm and infectivity.

 

2.147           Does lycopene offer human LDL any protection against myeloperoxidase activity?

Chew, P.Y., Riley, L., Graham, D.L., Rahman, K. and Lowe, G.M.

Mol. Cell. Biochem., 361(1-2), 181-187 (2012)

 

Lycopene is a lipophilic antioxidant that is largely transported in human blood by Low Density Lipoproteins (LDL). One of the early events in the aetiology of atherosclerosis is thought to be the oxidation of LDL. Myeloperoxidase an enzyme secreted by neutrophils and macrophages is thought to oxidise human LDL particles. In this study, isolated human LDL was challenged with myeloperoxidase or copper, and the LDL was screened for lipoperoxidation and oxidation of apolipoprotein B100, depletion of lycopene and oxidation of cholesterol. Myeloperoxidase induced oxidation of LDL through direct interaction with apolipoprotein B100. No lipoperoxidation was observed following myeloperoxidase treatment; however, 7-ketocholesterol was detected indicating the products of myeloperoxidase interact with the surface of the LDL particles. Lycopene does react with the products of myeloperoxidase in solvent, but played no role in protecting against enzyme derived oxidation of human LDL.

 

2.148           Shedding of syndecan-1 from human hepatocytes alters very low density lipoprotein clearance

Deng, Y., Foley, E.M., Gonzales, J.C., Gordts, P.L., Li, Y. and Esko, J.D.

Hepatology, 55(1), 277-286 (2012)

 

We recently showed that the heparan sulfate proteoglycan syndecan-1 mediates hepatic clearance of triglyceride-rich lipoproteins in mice based on systemic deletion of syndecan-1 and hepatocyte-specific inactivation of sulfotransferases involved in heparan sulfate biosynthesis. Here, we show that syndecan-1 expressed on primary human hepatocytes and Hep3B human hepatoma cells can mediate binding and uptake of very low density lipoprotein (VLDL). Syndecan-1 also undergoes spontaneous shedding from primary human and murine hepatocytes and Hep3B cells. In human cells, phorbol myristic acid induces syndecan-1 shedding, resulting in accumulation of syndecan-1 ectodomains in the medium. Shedding occurs through a protein kinase C–dependent activation of ADAM17 (a disintegrin and metalloproteinase 17). Phorbol myristic acid stimulation significantly decreases DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate)-VLDL binding to cells, and shed syndecan-1 ectodomains bind to VLDL. Although mouse hepatocytes appear resistant to induced shedding in vitro, injection of lipopolysaccharide into mice results in loss of hepatic syndecan-1, accumulation of ectodomains in the plasma, impaired VLDL catabolism, and hypertriglyceridemia. Conclusion: These findings suggest that syndecan-1 mediates hepatic VLDL turnover in humans as well as in mice and that shedding might contribute to hypertriglyceridemia in patients with sepsis.

 

2.149           Human telomerase acts as a hTR-independent reverse transcriptase in mitochondria

Sharma, N.K., Reyes, A., Green, P., Caron, M.J., Bonini, M.G., Gordon, D.M., Holt, I.J. and Hertzog Santos, J.

Nucleic Acids Res., 40(2), 712-725 (2012)

 

Human telomerase reverse transcriptase (hTERT) is localized to mitochondria, as well as the nucleus, but details about its biology and function in the organelle remain largely unknown. Here we show, using multiple approaches, that mammalian TERT is mitochondrial, co-purifying with mitochondrial nucleoids and tRNAs. We demonstrate the canonical nuclear RNA [human telomerase RNA (hTR)] is not present in human mitochondria and not required for the mitochondrial effects of telomerase, which nevertheless rely on reverse transcriptase (RT) activity. Using RNA immunoprecipitations from whole cell and in organello, we show that hTERT binds various mitochondrial RNAs, suggesting that RT activity in the organelle is reconstituted with mitochondrial RNAs. In support of this conclusion, TERT drives first strand cDNA synthesis in vitro in the absence of hTR. Finally, we demonstrate that absence of hTERT specifically in mitochondria with maintenance of its nuclear function negatively impacts the organelle. Our data indicate that mitochondrial hTERT works as a hTR-independent reverse transcriptase, and highlight that nuclear and mitochondrial telomerases have different cellular functions. The implications of these findings to both the mitochondrial and telomerase fields are discussed.

 

2.150           Large Aggregates Are the Major Soluble Aβ Species in AD Brain Fractionated with Density Gradient Ultracentrifugation

Sehlin, D., Englund, H., Simu, B., Karlsson, M., Ingelsson, M., Nikolajeff, f., Lannfelt, L. and Pettersson, F.E.

PloS One, 7(2), e32014 (2012)

 

Soluble amyloid-β (Aβ) aggregates of various sizes, ranging from dimers to large protofibrils, have been associated with neurotoxicity and synaptic dysfunction in Alzheimer's Disease (AD). To investigate the properties of biologically relevant Aβ species, brain extracts from amyloid β protein precursor (AβPP) transgenic mice and AD patients as well as synthetic Aβ preparations were separated by size under native conditions with density gradient ultracentrifugation. The fractionated samples were then analyzed with atomic force microscopy (AFM), ELISA, and MTT cell viability assay. Based on AFM appearance and immunoreactivity to our protofibril selective antibody mAb158, synthetic Aβ42 was divided in four fractions, with large aggregates in fraction 1 and the smallest species in fraction 4. Synthetic Aβ aggregates from fractions 2 and 3 proved to be most toxic in an MTT assay. In AβPP transgenic mouse brain, the most abundant soluble Aβ species were found in fraction 2 and consisted mainly of Aβ40. Also in AD brains, Aβ was mainly found in fraction 2 but primarily as Aβ42. All biologically derived Aβ from fraction 2 was immunologically discriminated from smaller species with mAb158. Thus, the predominant species of biologically derived soluble Aβ, natively separated by density gradient ultracentrifugation, were found to match the size of the neurotoxic, 80–500 kDa synthetic Aβ protofibrils and were equally detected with mAb158.

 

2.151           The small G protein Arl1 directs the trans-Golgi–specific targeting of the Arf1 exchange factors BIG1 and BIG2

Christis, C. and Munro, S.

1. Cell. Biol., 196(3), 327-335 (2012)

 

The small G protein Arf1 regulates Golgi traffic and is activated by two related types of guanine nucleotide exchange factor (GEF). GBF1 acts at the cis-Golgi, whereas BIG1 and its close paralog BIG2 act at the trans-Golgi. Peripheral membrane proteins such as these GEFs are often recruited to membranes by small G proteins, but the basis for specific recruitment of Arf GEFs, and hence Arfs, to Golgi membranes is not understood. In this paper, we report a liposome-based affinity purification method to identify effectors for small G proteins of the Arf family. We validate this with the Drosophila melanogaster Arf1 orthologue (Arf79F) and the related class II Arf (Arf102F), which showed a similar pattern of effector binding. Applying the method to the Arf-like G protein Arl1, we found that it binds directly to Sec71, the Drosophila ortholog of BIG1 and BIG2, via an N-terminal region. We show that in mammalian cells, Arl1 is necessary for Golgi recruitment of BIG1 and BIG2 but not GBF1. Thus, Arl1 acts to direct a trans-Golgi–specific Arf1 GEF, and hence active Arf1, to the trans side of the Golgi.

 

2.152           Crystal structure and biochemical analyses reveal Beclin 1 as a novel membrane binding protein

Huang, W., Choi, W., Hu, W., Mi, N., Guo, Q., Ma, M., Liu, M., Tian, Y., Lu, P., Wang, F-L., Deng, H., Liu, L., Gao, N., Yu, L. and Shi, Y.

Cell Res., 22, 473-489 (2012)

 

The Beclin 1 gene is a haplo-insufficient tumor suppressor and plays an essential role in autophagy. However, the molecular mechanism by which Beclin 1 functions remains largely unknown. Here we report the crystal structure of the evolutionarily conserved domain (ECD) of Beclin 1 at 1.6 Å resolution. Beclin 1 ECD exhibits a previously unreported fold, with three structural repeats arranged symmetrically around a central axis. Beclin 1 ECD defines a novel class of membrane-binding domain, with a strong preference for lipid membrane enriched with cardiolipin. The tip of a surface loop in Beclin 1 ECD, comprising three aromatic amino acids, acts as a hydrophobic finger to associate with lipid membrane, consequently resulting in the deformation of membrane and liposomes. Mutation of these aromatic residues rendered Beclin 1 unable to stably associate with lipid membrane in vitro and unable to fully rescue autophagy in Beclin 1-knockdown cells in vivo. These observations form an important framework for deciphering the biological functions of Beclin 1.

 

2.153           Density Gradient Multilayer Polymerization for Creating Complex Tissue

Karpiak, J.V., Ner, Y. and Almutairi, A.

Adv. Mater., 24(11), 1466-1470 (2012)

 

An adaptable density gradient multilayer polymerization (DGMP) method facilitates simple fabrication of complex multicompartment scaffolds with structurally continuous interfaces. Solvent density liquid-liquid phase segregation compartmentalizes varied mechanical and chemical cues independently. Bulk photopolymerization produces stratified three-dimensional and two-dimensional matrices. Cells attach to patterned adhesion peptides on biomimetic 2D substrates.

 

2.154           Distribution of perfluorooctanesulfonate and perfluorooctanoate into human plasma lipoprotein fractions

Butenhoff, J.L., Pieterman, E., Ehresman, D.J., Gorman, G.S., Olsen, G.W., Chang, S-C. and Princen, H.M.G.

Toxicology Letters, 210, 360-365 (2012)

 

Some cross-sectional epidemiological studies have reported positive associations of serum concentrations of non-high density lipoprotein cholesterol with serum perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA). However, the strength of the reported associations is inconsistent for exposure–response across three orders of magnitude of serum PFOS and/or PFOA concentrations. These positive associations are unexpected based on toxicological/mechanistic studies, suggesting that the associations may have a biological, rather than a causal, basis. This study tested the hypothesis that PFOS and PFOA distribute into serum lipoprotein fractions such that increases in serum lipoproteins would result in corresponding increases in serum concentrations of PFOS and PFOA. Based on observed binding of PFOS and PFOA to isolated β-lipoproteins in physiological saline (96% and 40% bound, respectively) in preliminary experiments using ultrafiltration and LC–MS/MS methods, binding to human donor plasma lipoprotein fractions was investigated by two density gradient methods. The majority of PFOS and PFOA recovered masses were found in lipoprotein-depleted plasma. Plasma density gradient fractionation data suggested that maximally 9% of PFOS distributes to lipoprotein-containing fractions, yet only 1% or less of PFOA is so distributed. These data do not support a strong role for plasma lipoprotein fractions in explaining the inconsistent dose–response associations reported in cross-sectional epidemiological studies.

 

2.155           Fusion of single proteoliposomes with planar, cushioned bilayers in microfluidic flow cells

Karatekin, E. and Ropthman, J.E.

Nature Protocols, 7(5), 903-920 (2012)

 

Many biological processes rely on membrane fusion, and therefore assays to study its mechanisms are necessary. Here we report an assay with sensitivity to single-vesicle, and even to single-molecule events using fluorescently labeled vesicle-associated v-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) liposomes and target-membrane-associated t-SNARE–reconstituted planar, supported bilayers (t-SBLs). Docking and fusion events can be detected using conventional far-field epifluorescence or total internal reflection fluorescence microscopy. In this assay, fusion is dependent on SNAP-25, one of the t-SNARE subunits that is required for fusion in vivo. The success of the assay is due to the use of: (i) bilayers covered with a thin layer of poly(ethylene glycol) (PEG) to control bilayer-bilayer and bilayer-substrate interactions, and (ii) microfluidic flow channels that present many advantages, such as the removal of nonspecifically bound liposomes by flow. The protocol takes 6–8 d to complete. Analysis can take up to 2 weeks.

 

2.156           P52—Distribution of perfluorooctanesulfonate and perfluorooctanoate into human plasma lipoprotein fractions over a wide range of concentrations

Butenhoff, J.L., Pieterman, E.J., Ehresman, D.J., Olsen, G.W., Chang, S-C. and Princen, H.M.G.

Reprod. Toxicol., 33, 1-29, abstract P-52 (2012)

 

Certain observational epidemiological studies have been characterized as finding modest positive associations between serum concentrations of non-high density lipoprotein cholesterol (non-HDL-C) and serum concentrations of PFOS and/or PFOA. However, collectively, these primarily cross-sectional investigations of occupational, community-exposed, and general populations are remarkably inconsistent for any dose–response relationship across a range of PFOS and/or PFOA concentrations. These PFOS and PFOA concentrations span three orders of magnitude between the least and highest exposed populations. Contrary to the epidemiological positive associations, toxicological studies of PFOS and PFOA in laboratory animals, including cynomologus monkeys, have observed either no change in serum lipids or decreases in serum cholesterol and/or triglycerides. These conflicting observations suggest that the reported epidemiologic associations of serum PFOS and PFOA with serum cholesterol may have biological, but not causal, significance. A possible non-causal hypothesis for the reported epidemiological associations between serum PFOS and/or PFOA and serum cholesterol is that PFOS and/or PFOA have affinity for and distribute into serum lipoprotein fractions. Thus, it can be postulated that, as serum lipoprotein concentration increases, distribution of PFOS and PFOA into serum lipoprotein fractions and total serum PFOS and/or PFOA concentrations would increase correspondingly. The study reported herein was undertaken to test the latter hypothesis through investigation of the effect of plasma concentration of PFOS and PFOA on the proportion of plasma PFOS and PFOA bound to the human plasma lipoprotein fractions VLDL, LDL, and HDL at background general population serum PFOS and PFOA concentrations and in serum spiked with either approximately 0.19 or 19 μM PFOS or PFOA. Plasma from a senior investigator was obtained and used to represent general population background concentrations of PFOS and PFOA, as well as to prepare approximately 0.19 and 19 μM concentrations of each fluorochemical. All experiments were performed in triplicate. Fractionation of 300 μL plasma into 25 fractions was accomplished by density gradient ultracentrifugation using both Redgrave and iodixanol density gradients. For each density gradient, fractions were pooled into very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL), and lipoprotein depleted plasma (LPDP) fractions as well as intermediate fractions based on determination of cholesterol concentrations in each of 25 fractions. Both density gradients were adjusted to give clean separations between the lipoprotein fractions and between HDL and albumin contained in LPDP. PFOS and PFOA concentrations in pooled fractions were determined by LC–MS/MS with lower limits of quantitation (LLOQ) ranging from 10 to 25 pg/mL. Total recovered mass of PFOS and PFOA based on duplicate determinations of initial concentrations ranged from 71 to 84% and 95 to 109% for PFOS and from 82 to 102% and 96 to 110% for PFOA for Redgrave and iodixanol density gradients, respectively. The majority of PFOS and PFOA recovered mass was found in LPDP (52–60% and 87–104% of PFOS mass and 81–98% and 94–109% of PFOA mass for Redgrave and iodixanol density gradients, respectively). Percent of mass recovered from fractions 1 to 19 (lipoprotein-containing fractions) ranged from 17 to 24% and <5 to 9% for PFOS and from <4 to <8% and <1 to <4% for PFOA for the Redgrave and iodixanol density gradients, respectively. PFOS concentrations in LDL and HDL were clearly higher than in VLDL and intermediate pooled lipoprotein fractions. For both PFOS and PFOA, there was minimal difference between concentrations tested and the resulting proportion distributed to the various pooled fractions. The results using the iodixanol method may be more representative physiologically, as iodixanol is a water soluble, non-ionic fluid. The iodixanol data suggest that maximally 9% of PFOS may be distributed to lipoprotein-containing fractions in plasma, yet only 1% or less of PFOA is so distributed. Taken together, these data do not support a strong role for plasma lipoprotein fractions in explaining the inconsistent dose–response associations reported in observational epidemiological studies.

 

2.157           Identification and Characterization of an Aβ Oligomer Precipitating Peptide That May Be Useful to Explore Gene Therapeutic Approaches to Alzheimer Disease

Funke, S.A:, Liu, H., Sehl, T., Bartnik, D., Brener, O., Nagel-Steger, L., Wiesehan, K. and Wilbold, D.

Rejuvenation Res., 15(2), 144-147 (2012)

 

A key feature of Alzheimer disease (AD) is the pathologic self-association of the amyloid-β (Aβ) peptide, leading to the formation of diffusible toxic Aβ oligomers and extracellular amyloid plaques. Next to extracellular Aβ, intraneuronal Aβ has important pathological functions in AD. Agents that specifically interfere with the oligomerization processes either outside or inside of neurons are highly desired for the elucidation of the pathologic mechanisms of AD and might even pave the way for new AD gene therapeutic approaches. Here, we characterize the Aβ binding peptide L3 and its influence on Aβ oligomerization in vitro. Preliminary studies in cell culture demonstrate that stably expressed L3 reduces cell toxicity of externally added Aβ in neuroblastoma cells.

 

2.158           A Dual Role for UVRAG in Maintaining Chromosomal Stability Independent of Autophagy

Zhao, Z., Oh, S., Li, D., Ni, D., Pirooz, S.D., Lee, J-H., yang, S., Lee, J-Y., Ghozalli, I., Costanzo, V., Stark, J.M. and Liang, C.

Developmental Cell, 22(5), 1-16 (2012)

 

Autophagy defects have recently been associated with chromosomal instability, a hallmark of human cancer. However, the functional specificity and mechanism of action of autophagy-related factors in genome stability remain elusive. Here we report that UVRAG, an autophagic tumor suppressor, plays a dual role in chromosomal stability, surprisingly independent of autophagy. We establish that UVRAG promotes DNA double-strand-break repair by directly binding and activating DNA-PK in nonhomologous end joining. Disruption of UVRAG increases genetic instability and sensitivity of cells to irradiation. Furthermore, UVRAG was also found to be localized at centrosomes and physically associated with CEP63, an integral component of centrosomes. Disruption of the association of UVRAG with centrosomes causes centrosome instability and aneuploidy. UVRAG thus represents an autophagy-related molecular factor that also has a convergent role in patrolling both the structural integrity and proper segregation of chromosomes, which may confer autophagy-independent tumor suppressor activity.

 

2.159           Differential Effects of Grape (Vitis vinifera) Skin Polyphenolics on Human Platelet Aggregation and Low-Density Lipoprotein Oxidation

Shanmuganayagam, D., Beahm, M.R., Kuhns, M.A., Krueger, C.G., Reed, J.D. and Folts, J.D.

1. Agric. Food Chem., 60(23), 5787-5794 (2012)

 

Antioxidant and antiplatelet properties of grape products are thought to be responsible for observed antiatherosclerotic effects. Diverse classes of phenolics are derived from the seed and skin (GSK) of grapes. The relative contributions of the classes of phenolics to observed properties of grape products are unknown. In this paper, GSK fractions were used to examine effects on platelet aggregation, low-density lipoprotein (LDL) oxidation in vitro, and relative binding of phenolics to LDL. GSK was separated into six fractions (fractions 1–6), and primary phenolics were characterized using high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Fractions 4, 5, and 6, enriched in polygalloyl polyflavan-3-ols (PGPFs) with 3–6, 4–8, and 6–15 degrees of polymerization, respectively, inhibited platelet aggregation. Fractions 1–3, containing various amounts of oligosaccharides, hydroxycinnamic acids, anthocyanins, flavanols, and low molecular weight PGPFs, significantly increased platelet aggregation. Fractions 4–6 were most effective in binding LDL and inhibiting LDL oxidation. Fractions 5 and 6 exhibited the greatest inhibition of platelet aggregation and LDL oxidation, suggesting that polymeric PGPFs are responsible for the beneficial effects of grape products. Conversely, phenolics in fractions 1–3 may reduce the net biological potency of the grape products and have undesirable effects on cardiovascular disease risk factors.

 

2.160           , 58-63 (2012)small mitochondrial ribosomal subunit

He, J., Cooper, H.M., Reyes, A., Di Re, M., Kazak, L., Wood, S.R., Mao, C.C., Fearnley, I.M., Walker, J.E. and Holt, I.J.

Nucleic Acids Res., 40(13), 6097-6108 (2012)

 

The bacterial homologue of C4orf14, YqeH, has been linked to assembly of the small ribosomal subunit. Here, recombinant C4orf14 isolated from human cells, co-purified with the small, 28S subunit of the mitochondrial ribosome and the endogenous protein co-fractionated with the 28S subunit in sucrose gradients. Gene silencing of C4orf14 specifically affected components of the small subunit, leading to decreased protein synthesis in the organelle. The GTPase of C4orf14 was critical to its interaction with the 28S subunit, as was GTP. Therefore, we propose that C4orf14, with bound GTP, binds to components of the 28S subunit facilitating its assembly, and GTP hydrolysis acts as the release mechanism. C4orf14 was also found to be associated with human mitochondrial nucleoids, and C4orf14 gene silencing caused mitochondrial DNA depletion. In vitro C4orf14 is capable of binding to DNA. The association of C4orf14 with mitochondrial translation factors and the mitochondrial nucleoid suggests that the 28S subunit is assembled at the mitochondrial nucleoid, enabling the direct transfer of messenger RNA from the nucleoid to the ribosome in the organelle.

 

2.161           Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis

He, J., Cooper, H.M., Reyes, A., Di Re, M., Sembongi, H., Litwin, T.R., Gao, J., Neuman, K.C., Fearnley, I.M., Spinazzola, A., Walker, J.E. and Holt, I.J.

Nucleic Acids Res., 40(13), 6109-6121 (2012)

 

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

 

2.162           Benzophenones and xanthones from Garcinia cantleyana var. cantleyana and their inhibitory activities on human low-density lipoprotein oxidation and platelet aggregation

Jantan, I. and Saputri, C.

Phytochemistry, 80, 58-63 (2012)

 

Three benzophenones, 2,6,3′,5′-tetrahydroxybenzophenone (1), 3,4,5,3′,5′-pentahydroxybenzophenone (3) and 3,5,3′,5′-tetrahydroxy-4-methoxybenzophenone (4), as well as a xanthone, 1,3,6-trihydroxy-5-methoxy-7-(3′-methyl-2′-oxo-but-3′-enyl)xanthone (9), were isolated from the twigs of Garcinia cantleyana var. cantleyana. Eight known compounds, 3,4,5,3′-tetrahydroxy benzophenone (2), 1,3,5-trihydroxyxanthone (5), 1,3,8-trihydroxyxanthone (6), 2,4,7-trihydroxyxanthone (7), 1,3,5,7-tetrahydroxyxanthone (8), quercetin, glutin-5-en-3β-ol and friedelin were also isolated. The structures of the compounds were elucidated by spectroscopic methods. The compounds were investigated for their ability to inhibit low-density lipoprotein (LDL) oxidation and platelet aggregation in human whole blood in vitro. Most of the compounds showed strong antioxidant activity with compound 8 showing the highest inhibition with an IC₅₀ value of 0.5 μM, comparable to that of probucol. Among the compounds tested, only compound 4 exhibited strong inhibitory activity against platelet aggregation induced by arachidonic acid (AA), adenosine diphosphate (ADP) and collagen. Compounds 3, 5 and 8 showed selective inhibitory activity on platelet aggregation induced by ADP.

 

2.163           Purified and synthetic Alzheimer’s amyloid beta (Aβ) prions

Stöhr, J., Watts, J.C., Mensinger, Z.L., Oehler, A., Grillo, S.K., DeArmond, S.J., Prusiner, S.B. and Giles, K.

PNAS, 109(27), 11025-11030 (2012)

 

The aggregation and deposition of amyloid-β (Aβ) peptides are believed to be central events in the pathogenesis of Alzheimer’s disease (AD). Inoculation of brain homogenates containing Aβ aggregates into susceptible transgenic mice accelerated Aβ deposition, suggesting that Aβ aggregates are capable of self-propagation and hence might be prions. Recently, we demonstrated that Aβ deposition can be monitored in live mice using bioluminescence imaging (BLI). Here, we use BLI to probe the ability of Aβ aggregates to self-propagate following inoculation into bigenic mice. We report compelling evidence that Aβ aggregates are prions by demonstrating widespread cerebral β-amyloidosis induced by inoculation of either purified Aβ aggregates derived from brain or aggregates composed of synthetic Aβ. Although synthetic Aβ aggregates were sufficient to induce Aβ deposition in vivo, they exhibited lower specific biological activity compared with brain-derived Aβ aggregates. Our results create an experimental paradigm that should lead to identification of self-propagating Aβ conformations, which could represent novel targets for interrupting the spread of Aβ deposition in AD patients.

 

2.164           Feedback Regulation of Transcriptional Termination by the Mammalian Circadian Clock PERIOD Complex

Padmanabhan, K. eet al

Science, 337, 599-602 (2012)

 

Eukaryotic circadian clocks are built on transcriptional feedback loops. In mammals, the PERIOD (PER) and CRYPTOCHROME (CRY) proteins accumulate, form a large nuclear complex (PER complex), and repress their own transcription. We found that mouse PER complexes included RNA helicases DDX5 and DHX9, active RNA polymerase II large subunit, Per and Cry pre-mRNAs, and SETX, a helicase that promotes transcriptional termination. During circadian negative feedback, RNA polymerase II accumulated near termination sites on Per and Cry genes but not on control genes. Recruitment of PER complexes to the elongating polymerase at Per and Cry termination sites inhibited SETX action, impeding RNA polymerase II release and thereby repressing transcriptional reinitiation. Circadian clock negative feedback thus includes direct control of transcriptional termination.

 

2.165           The dual PH domain protein Opy1 functions as a sensor and modulator of PtdIns(4,5)P2 synthesis

Ling, Y., Stefan, C.J., MacGurn, J.A., Audhya, A. and Emr, S.D.

EMBO J., 31(13), 2882-2894 (2012)

 

Phosphatidylinositol-4,5-bisphosphate, PtdIns(4,5)P₂, is an essential signalling lipid that regulates key processes such as endocytosis, exocytosis, actin cytoskeletal organization and calcium signalling. Maintaining proper levels of PtdIns(4,5)P₂ at the plasma membrane (PM) is crucial for cell survival and growth. We show that the conserved PtdIns(4)P 5-kinase, Mss4, forms dynamic, oligomeric structures at the PM that we term PIK patches. The dynamic assembly and disassembly of Mss4 PIK patches may provide a mechanism to precisely modulate Mss4 kinase activity, as needed, for localized regulation of PtdIns(4,5)P₂ synthesis. Furthermore, we identify a tandem PH domain-containing protein, Opy1, as a novel Mss4-interacting protein that partially colocalizes with PIK patches. Based upon genetic, cell biological, and biochemical data, we propose that Opy1 functions as a coincidence detector of the Mss4 PtdIns(4)P 5-kinase and PtdIns(4,5)P₂ and serves as a negative regulator of PtdIns(4,5)P₂ synthesis at the PM. Our results also suggest that additional conserved tandem PH domain-containing proteins may play important roles in regulating phosphoinositide signalling.

 

2.166           Lipoproteins in Drosophila melanogaster—Assembly, Function, and Influence on Tissue Lipid Composition

Palm, W., Sampaio, J.L., Brankatschk, M., Carvalho, M., Mahmoud, A., Shevchenko, A. and Eaton, S.

PloS One, 8(7), e1002828 (2012)

 

Interorgan lipid transport occurs via lipoproteins, and altered lipoprotein levels correlate with metabolic disease. However, precisely how lipoproteins affect tissue lipid composition has not been comprehensively analyzed. Here, we identify the major lipoproteins of Drosophila melanogaster and use genetics and mass spectrometry to study their assembly, interorgan trafficking, and influence on tissue lipids. The apoB-family lipoprotein Lipophorin (Lpp) is the major hemolymph lipid carrier. It is produced as a phospholipid-rich particle by the fat body, and its secretion requires Microsomal Triglyceride Transfer Protein (MTP). Lpp acquires sterols and most diacylglycerol (DAG) at the gut via Lipid Transfer Particle (LTP), another fat body-derived apoB-family lipoprotein. The gut, like the fat body, is a lipogenic organ, incorporating both de novo–synthesized and dietary fatty acids into DAG for export. We identify distinct requirements for LTP and Lpp-dependent lipid mobilization in contributing to the neutral and polar lipid composition of the brain and wing imaginal disc. These studies define major routes of interorgan lipid transport in Drosophila and uncover surprising tissue-specific differences in lipoprotein lipid utilization.

 

 

2.167           Functions of Nonmuscle Myosin II in Assembly of the Cellular Contractile System

Shutova, M., Yang, C., Vasiliev, J.M. and Svitkina, T.

PloS One, 7(7), e40814 (2012)

 

The contractile system of nonmuscle cells consists of interconnected actomyosin networks and bundles anchored to focal adhesions. The initiation of the contractile system assembly is poorly understood structurally and mechanistically, whereas system’s maturation heavily depends on nonmuscle myosin II (NMII). Using platinum replica electron microscopy in combination with fluorescence microscopy, we characterized the structural mechanisms of the contractile system assembly and roles of NMII at early stages of this process. We show that inhibition of NMII by a specific inhibitor, blebbistatin, in addition to known effects, such as disassembly of stress fibers and mature focal adhesions, also causes transformation of lamellipodia into unattached ruffles, loss of immature focal complexes, loss of cytoskeleton-associated NMII filaments and peripheral accumulation of activated, but unpolymerized NMII. After blebbistatin washout, assembly of the contractile system begins with quick and coordinated recovery of lamellipodia and focal complexes that occurs before reappearance of NMII bipolar filaments. The initial formation of focal complexes and subsequent assembly of NMII filaments preferentially occurred in association with filopodial bundles and concave actin bundles formed by filopodial roots at the lamellipodial base. Over time, accumulating NMII filaments help to transform the precursor structures, focal complexes and associated thin bundles, into stress fibers and mature focal adhesions. However, semi-sarcomeric organization of stress fibers develops at much slower rate. Together, our data suggest that activation of NMII motor activity by light chain phosphorylation occurs at the cell edge and is uncoupled from NMII assembly into bipolar filaments. We propose that activated, but unpolymerized NMII initiates focal complexes, thus providing traction for lamellipodial protrusion. Subsequently, the mechanical resistance of focal complexes activates a load-dependent mechanism of NMII polymerization in association with attached bundles, leading to assembly of stress fibers and maturation of focal adhesions.

 

2.168           Structural and genetic basis for development of broadly neutralizing influenza antibodies

Lingwood, D., McTamney, P., Yassine, H.M., Whittle, J.R., Guo, X., Boyington, J.C., Wei, C-J. and Nabel, G.J.

Nature, 487, 566-570 (2012)

 

Influenza viruses take a yearly toll on human life despite efforts to contain them with seasonal vaccines. These viruses evade human immunity through the evolution of variants that resist neutralization. The identification of antibodies that recognize invariant structures on the influenza haemagglutinin (HA) protein have invigorated efforts to develop universal influenza vaccines. Specifically, antibodies to the highly conserved stem region of HA neutralize diverse viral subtypes. These antibodies largely derive from a specific antibody gene, heavy-chain variable region IGHV1-69, after limited affinity maturation from their germline ancestors^{1, 2}, but how HA stimulates naive B cells to mature and induce protective immunity is unknown. To address this question, we analysed the structural and genetic basis for their engagement and maturation into broadly neutralizing antibodies. Here we show that the germline-encoded precursors of these antibodies act as functional B-cell antigen receptors (BCRs) that initiate subsequent affinity maturation. Neither the germline precursor of a prototypic antibody, CR6261 (ref. 3), nor those of two other natural human IGHV1-69 antibodies, bound HA as soluble immunoglobulin-G (IgG). However, all three IGHV1-69 precursors engaged HA when the antibody was expressed as cell surface IgM. HA triggered BCR-associated tyrosine kinase signalling by germline transmembrane IgM. Recognition and virus neutralization was dependent solely on the heavy chain, and affinity maturation of CR6261 required only seven amino acids in the complementarity-determining region (CDR) H1 and framework region 3 (FR3) to restore full activity. These findings provide insight into the initial events that lead to the generation of broadly neutralizing antibodies to influenza, informing the rational design of vaccines to elicit such antibodies and providing a model relevant to other infectious diseases, including human immunodeficiency virus/AIDS. The data further suggest that selected immunoglobulin genes recognize specific protein structural ‘patterns’ that provide a substrate for further affinity maturation.

 

2.169           Small dense LDL: An emerging risk factor for cardiovascular disease

Hirayama, S. and Miida, T.

Clinica Chemica Acta, 414, 215-224 (2012)

 

Although low-density lipoprotein cholesterol (LDL-C) is a strong risk factor for coronary artery disease (CAD), LDL-C levels are not always elevated in CAD patients. LDL consists of several subclasses with distinct sizes, densities, and physicochemical compositions. Thus, LDL subclasses can be separated by various laboratory procedures. Among them, ultracentrifugation and electrophoresis have been used most frequently for determining LDL subclasses. Accumulating evidence has shown that a predominance of small dense LDL (sd-LDL) is closely associated with CAD. Moreover, sd-LDL-cholesterol (sd-LDL-C) concentrations are elevated in groups at a high risk for CAD, such as patients with type 2 diabetes and metabolic syndrome. Therefore, sd-LDL concentration is recognized as a surrogate marker for CAD. However, some studies failed to show therapeutic modulation of sd-LDL, likely because separating methods and sd-LDL particle definitions have not yet been standardized. Recently, a detergent-based homogenous assay for sd-LDL-C has been developed. This method does not require any pretreatment, and the measured values are highly reproducible with an automated analyzer. These features are suitable for large-scale clinical studies. This homogeneous assay is a useful tool for clarifying whether sd-LDL-C is a superior marker to LDL-C, and whether sd-LDL-C lipid-lowering therapies decrease the incidence of CAD.

 

2.170           Inhibitory Activities of Compounds from the Twigs of Garcinia hombroniana Pierre on Human Low-density Lipoprotein (LDL) Oxidation and Platelet Aggregation

Saputri, F.C. and Jantan, I.

Phytother. Res., 26(12), 1845-1850 (2012)

 

The methanol extract of the twigs of Garcinia hombroniana, which showed strong LDL antioxidation and antiplatelet aggregation activities, was subjected to column chromatography to obtain 3,5,3′,5′-tetrahydroxy-4-methoxybenzophenone, 1,7-dihydroxyxanthone and eight triterpenoids, garcihombronane B, D, E and F, friedelin, glutin-5-en-3β-ol, stigmasterol and lupeol. The structures of the compounds were elucidated by spectroscopic methods. The compounds were evaluated for their ability to inhibit copper-mediated LDL oxidation and arachidonic acid (AA)-, adenosine diphosphate (ADP)-, collagen-induced platelet aggregation in vitro. Among the compounds tested, 3,5,3′,5′-tetrahydroxy-4-methoxybenzophenone and 1,7-dihydroxyxanthone showed strong inhibitory activity on LDL oxidation with half-maximal inhibitory concentration (IC₅₀) values of 6.6 and 1.7 µm, respectively. 3,5,3′,5′-Tetrahydroxy-4-methoxybenzophenone exhibited strong activity on AA-, ADP- and collagen-induced platelet aggregation with IC₅₀ values of 53.6, 125.7 and 178.6 µm, respectively, while 1,7 dihydroxyxanthone showed significant and selective inhibitory activity against ADP-induced aggregation with IC₅₀ value of 5.7 µm. Of the triterpenoids tested, garcihombronane B showed moderate activity against LDL oxidation and garcihombronane D and F showed selective inhibition on ADP-induced platelet aggregation.

2.171           Why working with porcine circulating serum amyloid A is a pig of a job

Soler, L., Molenaar, A., Merola, N., Eckersall, P.D., Butierrez, A., Ceron, J.J.., Mulero, V. and Niewold, T.A:

J. Theoretical Biol., 317, 119-125 (2012)

 

Serum amyloid A (SAA) is a major acute phase protein in most species, and is widely employed as a health marker. Systemic SAA isoforms (SAA1, and SAA2) are apolipoproteins synthesized by the liver which associate with high density lipoproteins (HDL). Local SAA (SAA3) isoforms are synthesized in other tissues and are present in colostrums, mastitic milk and mammary dry secretions. Of systemic SAA the bulk is monomeric and bound to HDL, and a small proportion is found in serum in a multimeric form with a buried HDL binding site. In most species, systemic SAA could easily be studied by purifying it from serum of diseased individuals by hydrophobic interaction chromatography methods. For years, we were not able to isolate systemic pig SAA using the latter methods, and found that the bulk of pig SAA did not reside in the HDL-rich serum fractions but in the soluble protein fraction mainly as a multimeric protein.

Based on these surprising results, we analysed in silico the theoretical properties and predicted the secondary structure of pig SAA by using the published pig primary SAA amino acid sequence. Results of the analysis confirmed that systemic pig SAA had the highest homology with local SAA3 which in other species is the isoform associated with non-hepatic production in tissues such as mammary gland and intestinal epithelium. Furthermore, the primary sequence of the pig SAA N-terminal HDL binding site did differ considerably from SAA1/2. Secondary structure analysis of the predicted alpha–helical structure of this HDL binding site showed a considerable reduction in hydrophobicity compared to SAA1/2. Based on these results, it is argued that systemic acute phase SAA in the pig has the structural properties of locally produced SAA (SAA3). It is proposed that in pig SAA multimers the charged N-terminal sequence is buried, which would explain their different properties.

It is concluded that pig systemic SAA is unique compared to other species, which raises questions about the proposed importance of acute phase SAA in HDL metabolism during inflammation in this species.

 

2.172           Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication

Baird, N.L., York, J. and Nunberg, J.H.

J. Virol., 86(20), 11301-11310 (2012)

 

Arenaviruses are responsible for acute hemorrhagic fevers with high mortality and pose significant threats to public health and biodefense. These enveloped negative-sense RNA viruses replicate in the cell cytoplasm and express four proteins. To better understand how these proteins insinuate themselves into cellular processes to orchestrate productive viral replication, we have identified and characterized novel cytosolic structures involved in arenavirus replication and transcription. In cells infected with the nonpathogenic Tacaribe virus or the attenuated Candid#1 strain of Junín virus, we find that newly synthesized viral RNAs localize to cytosolic puncta containing the nucleoprotein (N) of the virus. Density gradient centrifugation studies reveal that these replication-transcription complexes (RTCs) are associated with cellular membranes and contain full-length genomic- and antigenomic-sense RNAs. Viral mRNAs segregate at a higher buoyant density and are likewise scant in immunopurified RTCs, consistent with their translation on bulk cellular ribosomes. In addition, confocal microscopy analysis reveals that RTCs contain the lipid phosphatidylinositol-4-phosphate and proteins involved in cellular mRNA metabolism, including the large and small ribosomal subunit proteins L10a and S6, the stress granule protein G3BP1, and a subset of translation initiation factors. Elucidating the structure and function of RTCs will enhance our understanding of virus-cell interactions that promote arenavirus replication and mitigate against host cell immunity. This knowledge may lead to novel intervention strategies to limit viral virulence and pathogenesis.

 

2.173           Alternative translation initiation augments the human mitochondrial proteome

Kazak, L., Reyes, A., Duncan, A.L., Rorbach, J., Wood, S.R., Brea-Calvo, g., Gammage, P.A., Robinson, A.J., Minczuk, M. and Holt, I.J.

Nucleic Acids Res., 41(4), 2354-2369 (2013)

 

Alternative translation initiation (ATI) is a mechanism of producing multiple proteins from a single transcript, which in some cases regulates trafficking of proteins to different cellular compartments, including mitochondria. Application of a genome-wide computational screen predicts a cryptic mitochondrial targeting signal for 126 proteins in mouse and man that is revealed when an AUG codon located downstream from the canonical initiator methionine codon is used as a translation start site, which we term downstream ATI (dATI). Experimental evidence in support of dATI is provided by immunoblotting of endogenous truncated proteins enriched in mitochondrial cell fractions or of co-localization with mitochondria using immunocytochemistry. More detailed cellular localization studies establish mitochondrial targeting of a member of the cytosolic poly(A) binding protein family, PABPC5, and of the RNA/DNA helicase PIF1α. The mitochondrial isoform of PABPC5 co-immunoprecipitates with the mitochondrial poly(A) polymerase, and is markedly reduced in abundance when mitochondrial DNA and RNA are depleted, suggesting it plays a role in RNA metabolism in the organelle. Like PABPC5 and PIF1α, most of the candidates identified by the screen are not currently annotated as mitochondrial proteins, and so dATI expands the human mitochondrial proteome.

 

2.174           FisB mediates membrane fission during sporulation in Bacillus subtilis

Doan, T.., Coleman, J., Marquis, K.A. et al

Gens Dev., 27(3), 322-334 (2013)

 

How bacteria catalyze membrane fission during growth and differentiation is an outstanding question in prokaryotic cell biology. Here, we describe a protein (FisB, for fission protein B) that mediates membrane fission during the morphological process of spore formation in Bacillus subtilis. Sporulating cells divide asymmetrically, generating a large mother cell and smaller forespore. After division, the mother cell membranes migrate around the forespore in a phagocytic-like process called engulfment. Membrane fission releases the forespore into the mother cell cytoplasm. Cells lacking FisB are severely and specifically impaired in the fission reaction. Moreover, GFP-FisB forms dynamic foci that become immobilized at the site of fission. Purified FisB catalyzes lipid mixing in vitro and is only required in one of the fusing membranes, suggesting that FisB–lipid interactions drive membrane remodeling. Consistent with this idea, the extracytoplasmic domain of FisB binds with remarkable specificity to cardiolipin, a lipid enriched in the engulfing membranes and regions of negative curvature. We propose that membrane topology at the final stage of engulfment and FisB–cardiolipin interactions ensure that the mother cell membranes are severed at the right time and place. The unique properties of FisB set it apart from the known fission machineries in eukaryotes, suggesting that it represents a new class of fission proteins.

 

2.175           Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains

Volmer, R., van der Ploeg, K. and Ron, D.

PNAS, 110(12), 4628-4633 (2013)

 

Endoplasmic reticulum (ER) stress sensors use a related luminal domain to monitor the unfolded protein load and convey the signal to downstream effectors, signaling an unfolded protein response (UPR) that maintains compartment-specific protein folding homeostasis. Surprisingly, perturbation of cellular lipid composition also activates the UPR, with important consequences in obesity and diabetes. However, it is unclear if direct sensing of the lipid perturbation contributes to UPR activation. We found that mutant mammalian ER stress sensors, IRE1α and PERK, lacking their luminal unfolded protein stress-sensing domain, nonetheless retained responsiveness to increased lipid saturation. Lipid saturation-mediated activation in cells required an ER-spanning transmembrane domain and was positively regulated in vitro by acyl-chain saturation in reconstituted liposomes. These observations suggest that direct sensing of the lipid composition of the ER membrane contributes to the UPR.

 

2.176           Low Density Lipoprotein Binds to Proprotein Convertase Subtilisin/Kexin Type-9 (PCSK9) in Human Plasma and Inhibits PCSK9-mediated Low Density Lipoprotein Receptor Degradation

Kosenko, T., Golder, M., Leblond, G., Weng, W. and Lagace, T.A.

1. Biol. Chem., 288(12), 8279-8288 (2013)

 

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a secreted protein that binds to the epidermal growth factor-like-A domain of the low density lipoprotein receptor (LDLR) and mediates LDLR degradation in liver. Gain-of-function mutations in PCSK9 are associated with autosomal dominant hypercholesterolemia in humans. Size-exclusion chromatography of human plasma has shown PCSK9 to be partly associated with undefined high molecular weight complexes within the LDL size range. We used density gradient centrifugation to isolate LDL in plasma pooled from 5 normolipidemic subjects and report that >40% of total PCSK9 was associated with LDL. Binding of fluorophore-labeled recombinant PCSK9 to isolated LDL in vitro was saturable with a K_D ∼ 325 nm. This interaction was competed >95% by excess unlabeled PCSK9, and competition binding curves were consistent with a one-site binding model. An N-terminal region of the PCSK9 prodomain (amino acids 31–52) was required for binding to LDL in vitro. LDL dose-dependently inhibited binding and degradation of cell surface LDLRs by exogenous PCSK9 in HuH7 cells. LDL also inhibited PCSK9 binding to mutant LDLRs defective at binding LDL. These data suggest that association of PCSK9 with LDL particles in plasma lowers the ability of PCSK9 to bind to cell surface LDLRs, thereby blunting PCSK9-mediated LDLR degradation.

 

2.177           Secretion and Signaling Activities of Lipoprotein-Associated Hedgehog and Non-Sterol-Modified Hedgehog in Flies and Mammals

Palm, W., Swierczynska, M.M., Kumari, V., Ehrhart-Bornstein, M., Bornstein, S.R. and Eaton, S.

PloS Biology, 11(3), e1001505 (2013)

 

Hedgehog (Hh) proteins control animal development and tissue homeostasis. They activate gene expression by regulating processing, stability, and activation of Gli/Cubitus interruptus (Ci) transcription factors. Hh proteins are secreted and spread through tissue, despite becoming covalently linked to sterol during processing. Multiple mechanisms have been proposed to release Hh proteins in distinct forms; in Drosophila, lipoproteins facilitate long-range Hh mobilization but also contain lipids that repress the pathway. Here, we show that mammalian lipoproteins have conserved roles in Sonic Hedgehog (Shh) release and pathway repression. We demonstrate that lipoprotein-associated forms of Hh and Shh specifically block lipoprotein-mediated pathway inhibition. We also identify a second conserved release form that is not sterol-modified and can be released independently of lipoproteins (Hh-N*/Shh-N*). Lipoprotein-associated Hh/Shh and Hh-N*/Shh-N* have complementary and synergistic functions. In Drosophila wing imaginal discs, lipoprotein-associated Hh increases the amount of full-length Ci, but is insufficient for target gene activation. However, small amounts of non-sterol-modified Hh synergize with lipoprotein-associated Hh to fully activate the pathway and allow target gene expression. The existence of Hh secretion forms with distinct signaling activities suggests a novel mechanism for generating a diversity of Hh responses

 

2.178           Very-Low-Density Lipoprotein (VLDL)-Producing and Hepatitis C Virus-Replicating HepG2 Cells Secrete No More Lipoviroparticles than VLDL-Deficient Huh7.5 Cells

Jammart, B., Michelet, M., Pecheur, E-I., parent, R., bartosch, B., Zoulim, F. and Durantel, D.

1. Virol., 87(9), 5065-5080 (2013)

 

In the plasma samples of hepatitis C virus (HCV)-infected patients, lipoviroparticles (LVPs), defined as (very-) low-density viral particles immunoprecipitated with anti-β-lipoproteins antibodies are observed. This HCV-lipoprotein association has major implications with respect to our understanding of HCV assembly, secretion, and entry. However, cell culture-grown HCV (HCVcc) virions produced in Huh7 cells, which are deficient for very-low-density lipoprotein (VLDL) secretion, are only associated with and dependent on apolipoprotein E (apoE), not apolipoprotein B (apoB), for assembly and infectivity. In contrast to Huh7, HepG2 cells can be stimulated to produce VLDL by both oleic acid treatment and inhibition of the MEK/extracellular signal-regulated kinase (ERK) pathway but are not permissive for persistent HCV replication. Here, we developed a new HCV cell culture model to study the interaction between HCV and lipoproteins, based on engineered HepG2 cells stably replicating a blasticidin-tagged HCV JFH1 strain (JB). Control Huh7.5-JB as well as HepG2-JB cell lines persistently replicated viral RNA and expressed viral proteins with a subcellular colocalization of double-stranded RNA (dsRNA), core, gpE2, and NS5A compatible with virion assembly. The intracellular RNA replication level was increased in HepG2-JB cells upon dimethyl sulfoxide (DMSO) treatment, MEK/ERK inhibition, and NS5A overexpression to a level similar to that observed in Huh7.5-JB cells. Both cell culture systems produced infectious virions, which were surprisingly biophysically and biochemically similar. They floated at similar densities on gradients, contained mainly apoE but not apoB, and were not neutralized by anti-apoB antibodies. This suggests that there is no correlation between the ability of cells to simultaneously replicate HCV as well as secrete VLDL and their capacity to produce LVPs.

 

2.179           Production, Purification and Characterization of Recombinant, Full-Length Human Claudin-1

Bonander, N., Jamshad, M., Oberthür, D., Clare, M., Barwell, J., Hu, K., Farquhar, M.J., Stamataki, Z., Harris, H.J., Dierks, K., daffron, T.R., Betzel, C., McKeating, J.A. and Bill, R.M.

PloS One, 8(5), e64517 (2013)

 

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1:2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.

 

2.180           A Cryptic Targeting Signal Creates a Mitochondrial FEN1 Isoform with Tailed R-Loop Binding Properties

Kazak, L., Reyes, A., He, J., Wood, S.R., Brea-Calvo, G., Holen, T.T. and Holt, I.J.

PloS One, 8(5), e62340 (2013)

 

A growing number of DNA transacting proteins is found in the nucleus and in mitochondria, including the DNA repair and replication protein Flap endonuclease 1, FEN1. Here we show a truncated FEN1 isoform is generated by alternative translation initiation, exposing a mitochondrial targeting signal. The shortened form of FEN1, which we term FENMIT, localizes to mitochondria, based on import into isolated organelles, immunocytochemistry and subcellular fractionation. In vitro FENMIT binds to flap structures containing a 5′ RNA flap, and prefers such substrates to single-stranded RNA. FENMIT can also bind to R-loops, and to a lesser extent to D-loops. Exposing human cells to ethidium bromide results in the generation of RNA/DNA hybrids near the origin of mitochondrial DNA replication. FENMIT is recruited to the DNA under these conditions, and is released by RNase treatment. Moreover, high levels of recombinant FENMIT expression inhibit mtDNA replication, following ethidium bromide treatment. These findings suggest FENMIT interacts with RNA/DNA hybrids in mitochondrial DNA, such as those found at the origin of replication.

 

2.181           Separation of the principal HDL subclasses by iodixanol ultracentrifugation

Harman, N.L., Griffin, B.A. and Davies, I.G.

1. Lipid Res., 54, 2273-2281 (2013)

 

HDL subclasses detection, in cardiovascular risk, has been limited due to the time-consuming nature of current techniques. We have developed a time-saving and reliable separation of the principal HDL subclasses employing iodixanol density gradient ultracentrifugation (IxDGUC) combined with digital photography. HDL subclasses were separated in 2.5 h from prestained plasma on a three-step iodixanol gradient. HDL subclass profiles were generated by digital photography and gel scan software. Plasma samples (n = 46) were used to optimize the gradient for the resolution of HDL heterogeneity and to compare profiles generated by IxDGUC with gradient gel electrophoresis (GGE); further characterization from participants (n = 548) with a range of lipid profiles was also performed. HDL subclass profiles generated by IxDGUC were comparable to those separated by GGE as indicated by a significant association between areas under the curve for both HDL₂ and HDL₃ (HDL₂, r = 0.896, P < 0.01; HDL₃, r = 0.894, P < 0.01). The method was highly reproducible, with intra- and interassay coefficient of variation percentage < 5 for percentage area under the curve HDL₂ and HDL₃, and < 1% for peak Rf and peak density. The method provides time-saving and cost-effective detection and preparation of the principal HDL subclasses.

 

2.182           Galectin-3 mediates oligomerization of secreted hensin using its carbohydrate-recognition domain

Vijayakumar, S., Peng, H. and Schwartz, G.J.

Am. J: Physiol. Renal Physiol., 305, F90-F99 (2013)

 

A multidomain, multifunctional 230-kDa extracellular matrix (ECM) protein, hensin, regulates the adaptation of rabbit kidney to metabolic acidosis by remodeling collecting duct intercalated cells. Conditional deletion of hensin in intercalated cells of the mouse kidney leads to distal renal tubular acidosis and to a significant reduction in the number of cells expressing the basolateral chloride-bicarbonate exchanger kAE1, a characteristic marker of α-intercalated cells. Although hensin is secreted as a monomer, its polymerization and ECM assembly are essential for its role in the adaptation of the kidney to metabolic acidosis. Galectin-3, a unique lectin with specific affinity for β-galactoside glycoconjugates, directly interacts with hensin. Acidotic rabbits had a significant increase in the number of cells expressing galectin-3 in the collecting duct and exhibited colocalization of galectin-3 with hensin in the ECM of microdissected tubules. In this study, we confirmed the increased expression of galectin-3 in acidotic rabbit kidneys by real-time RT-PCR. Galectin-3 interacted with hensin in vitro via its carbohydrate-binding COOH-terminal domain, and the interaction was competitively inhibited by lactose, removal of the COOH-terminal domain of galectin-3, and deglycosylation of hensin. Galectin-9, a lectin with two carbohydrate-recognition domains, is also present in the rabbit kidney; galectin-9 partially oligomerized hensin in vitro. Our results demonstrate that galectin-3 plays a critical role in hensin ECM assembly by oligomerizing secreted monomeric hensin. Both the NH₂-terminal and COOH-terminal domains are required for this function. We suggest that in the case of galectin-3-null mice galectin-9 may partially substitute for the function of galectin-3.

 

2.183           Serum Proprotein Convertase Subtilisin/Kexin Type 9 and Cell Surface Low-Density Lipoprotein Receptor: Evidence for a Reciprocal Regulation

Tavori, H., Fan, D., Blakemore, J.L., Yancey, P.G., Ding, L., MacRae, F.-L. and Fazio, S.

Circulation, 127, 2403-2413 (2013)

 

Background—Proprotein convertase subtilisin/kexin type 9 (PCSK9) modulates low-density lipoprotein (LDL) receptor (LDLR) degradation, thus influencing serum cholesterol levels. However, dysfunctional LDLR causes hypercholesterolemia without affecting PCSK9 clearance from the circulation.

Methods and Results—To study the reciprocal effects of PCSK9 and LDLR and the resultant effects on serum cholesterol, we produced transgenic mice expressing human (h) PCSK9. Although hPCSK9 was expressed mainly in the kidney, LDLR degradation was more evident in the liver. Adrenal LDLR levels were not affected, likely because of the impaired PCSK9 retention in this tissue. In addition, hPCSK9 expression increased hepatic secretion of apolipoprotein B–containing lipoproteins in an LDLR-independent fashion. Expression of hPCSK9 raised serum murine PCSK9 levels by 4.3-fold in wild-type mice and not at all in LDLR^−/− mice, in which murine PCSK9 levels were already 10-fold higher than in wild-type mice. In addition, LDLR^+/− mice had a 2.7-fold elevation in murine PCSK9 levels and no elevation in cholesterol levels. Conversely, acute expression of human LDLR in transgenic mice caused a 70% decrease in serum murine PCSK9 levels. Turnover studies using physiological levels of hPCSK9 showed rapid clearance in wild-type mice (half-life, 5.2 minutes), faster clearance in human LDLR transgenics (2.9 minutes), and much slower clearance in LDLR^−/− recipients (50.5 minutes). Supportive results were obtained with an in vitro system. Finally, up to 30% of serum hPCSK9 was associated with LDL regardless of LDLR expression.

Conclusions—Our results support a scenario in which LDLR represents the main route of elimination of PCSK9 and a reciprocal regulation between these 2 proteins controls serum PCSK9 levels, hepatic LDLR expression, and serum LDL levels.

 

2.184           Very low-density lipoprotein/lipo-viro particles reverse lipoprotein lipase-mediated inhibition of hepatitis C virus infection via apolipoprotein C-III

Sun, H-Y., Lin, C-C., Lee, J-C., Wang, S-W., Cheng, P-N., Wu, I-C., Chang, T-T., Lai, M-D., Shieh, D.B. and Young, K-C.

Gut, 62(8), 1193-1203 (2013)

 

Objective Circulating hepatitis C virus (HCV) virions are associated with triglyceride-rich lipoproteins, including very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), designated as lipo-viro-particles (LVPs). Previous studies showed that lipoprotein lipase (LPL), a key enzyme for hydrolysing the triglyceride in VLDL to finally become LDL, may suppress HCV infection. This investigation considers the regulation of LPL by lipoproteins and LVPs, and their roles in the LPL-mediated anti-HCV function.

Design The lipoproteins were fractionated from normolipidemic blood samples using iodixanol gradients. Subsequent immunoglobulin-affinity purification from the canonical VLDL and LDL yielded the corresponding VLDL-LVP and LDL-LVP. Apolipoprotein (apo) Cs, LPL activity and HCV infection were quantified.

Results A higher triglyceride/cholesterol ratio of LDL was found more in HCV-infected donors than in healthy volunteers, and the triglyceride/cholesterol ratio of LDL-LVP was much increased, suggesting that the LPL hydrolysis of triglyceride may be impaired. VLDL, VLDL-LVP, LDL-LVP, but not LDL, suppressed LPL lipolytic activity, which was restored by antibodies that recognised apoC-III/-IV and correlated with the steadily abundant apoC-III/-IV quantities in those particles. In a cell-based system, treatment with VLDL and LVPs reversed the LPL-mediated inhibition of HCV infection in apoC-III/-IV-dependent manners. A multivariate logistic regression revealed that plasma HCV viral loads correlated negatively with LPL lipolytic activity, but positively with the apoC-III content of VLDL. Additionally, apoC-III in VLDL was associated with a higher proportion of HCV-RNA than was IgG.

Conclusion This study reveals that LPL is an anti-HCV factor, and that apoC-III in VLDL and LVPs reduces the LPL-mediated inhibition of HCV infection.

 

 

2.185           Quaternary Structure of Pathological Prion Protein as a Determining Factor of Strain-Specific Prion Replication Dynamics

Laferriere, F., Tixador, P., Moudjou, M., Chapuis, J., Sibille, P., Herzog, L., Reine, F., Jaumain, E., Laude, H., Rezaei, H. and Beringue, V.

PloS Pathogens, 9(10), e1003702 (2013)

 

Prions are proteinaceous infectious agents responsible for fatal neurodegenerative diseases in animals and humans. They are essentially composed of PrP^Sc, an aggregated, misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP^C). Stable variations in PrP^Sc conformation are assumed to encode the phenotypically tangible prion strains diversity. However the direct contribution of PrP^Sc quaternary structure to the strain biological information remains mostly unknown. Applying a sedimentation velocity fractionation technique to a panel of ovine prion strains, classified as fast and slow according to their incubation time in ovine PrP transgenic mice, has previously led to the observation that the relationship between prion infectivity and PrP^Sc quaternary structure was not univocal. For the fast strains specifically, infectivity sedimented slowly and segregated from the bulk of proteinase-K resistant PrP^Sc. To carefully separate the respective contributions of size and density to this hydrodynamic behavior, we performed sedimentation at the equilibrium and varied the solubilization conditions. The density profile of prion infectivity and proteinase-K resistant PrP^Sc tended to overlap whatever the strain, fast or slow, leaving only size as the main responsible factor for the specific velocity properties of the fast strain most infectious component. We further show that this velocity-isolable population of discrete assemblies perfectly resists limited proteolysis and that its templating activity, as assessed by protein misfolding cyclic amplification outcompetes by several orders of magnitude that of the bulk of larger size PrP^Sc aggregates. Together, the tight correlation between small size, conversion efficiency and duration of disease establishes PrP^Sc quaternary structure as a determining factor of prion replication dynamics. For certain strains, a subset of PrP assemblies appears to be the best template for prion replication. This has important implications for fundamental studies on prions.

 

2.186           Mitochondrial Ribosomal RNA (rRNA) Methyltransferase Family Members Are Positioned to Modify Nascent rRNA in Foci near the Mitochondrial DNA Nucleoid

Lee, K-W., Okot-Kotber, C., LaComb, J.F. and Bogenhagen, D.F.

1. Biol. Chem., 288(43), 31386-31399 (2013)

 

We have identified RNMTL1, MRM1, and MRM2 (FtsJ2) as members of the RNA methyltransferase family that may be responsible for the three known 2′-O-ribose modifications of the 16 S rRNA core of the large mitochondrial ribosome subunit. These proteins are confined to foci located in the vicinity of mtDNA nucleoids. They show distinct patterns of association with mtDNA nucleoids and/or mitochondrial ribosomes in cell fractionation studies. We focused on the role of the least studied protein in this set, RNMTL1, to show that this protein interacts with the large ribosomal subunit as well as with a series of non-ribosomal proteins that may be involved in coupling of the rate of rRNA transcription and ribosome assembly in mitochondria. siRNA-directed silencing of RNMTL1 resulted in a significant inhibition of translation on mitochondrial ribosomes. Our results are consistent with a role for RNMTL1 in methylation of G¹³⁷⁰ of human 16 S rRNA.

 

2.187           Infrared Microspectroscopy Detects Protein Misfolding Cyclic Amplification (PMCA)-induced Conformational Alterations in Hamster Scrapie Progeny Seeds

Daus, M.L., Wagenführ, K., Thomzig, A., Boerner, S., Hermann, P., Hermelink, A., Beekes, M. and Lasch, P.

1. Biol. Chem., 288(49), 35068-35080 (2013)

 

The self-replicative conformation of misfolded prion proteins (PrP) is considered a major determinant for the seeding activity, infectiousness, and strain characteristics of prions in different host species. Prion-associated seeding activity, which converts cellular prion protein (PrP^C) into Proteinase K-resistant, infectious PrP particles (PrP^TSE), can be monitored in vitro by protein misfolding cyclic amplification (PMCA). Thus, PMCA has been established as a valuable analytical tool in prion research. Currently, however, it is under discussion whether prion strain characteristics are preserved during PMCA when parent seeds are amplified in PrP^C substrate from the identical host species. Here, we report on the comparative structural analysis of parent and progeny (PMCA-derived) PrP seeds by an improved approach of sensitive infrared microspectroscopy. Infrared microspectroscopy revealed that PMCA of native hamster 263K scrapie seeds in hamster PrP^C substrate caused conformational alterations in progeny seeds that were accompanied by an altered resistance to Proteinase K, higher sedimentation velocities in gradient ultracentrifugations, and a longer incubation time in animal bioassays. When these progeny seeds were propagated in hamsters, misfolded PrP from brain extracts of these animals showed mixed spectroscopic and biochemical properties from both parental and progeny seeds. Thus, strain modifications of 263K prions induced by PMCA seem to have been partially reversed when PMCA products were reinoculated into the original host species.

 

2.188           Loss of Plasma Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) After Lipoprotein Apheresis

Tavori, H., Giunzioni, I., MacRae, F.L. and Fazio, S.

Circ. Res., 113, 1290-1295 (2013)

 

Rationale: Lipoprotein apheresis (LA) reduces low-density lipoprotein (LDL) levels in patients with severe familial hypercholesterolemia (FH). We have recently reported that >30% of plasma proprotein convertase subtilisin/kexin 9 (PCSK9) is bound to LDL, thus we predicted that LA would also reduce plasma PCSK9 levels by removing LDL.

Objective: Pre- and post-apheresis plasma from 6 patients with familial hypercholesterolemia on 3 consecutive treatment cycles was used to determine changes in PCSK9 levels.

Methods and Results: LA drastically reduced plasma LDL (by 77±4%). Concomitantly, PCSK9 levels fell by 52±5%, strongly correlating with the LDL drop (P=0.0322; r²=0.26), but not with decreases in triglyceride (49±13%) or high-density lipoprotein levels (18±2%). Levels of albumin, creatinine, and CK-MB did not show significant changes after LA. Similar to LDL, PCSK9 levels returned to pretreatment values between cycles (2-week intervals). Fractionation of pre- and post-apheresis plasma showed that 81±11% of LDL-bound PCSK9 and 48±14% of apolipoprotein B–free PCSK9 were removed. Separation of whole plasma, purified LDL, or the apolipoprotein B–free fraction through a scaled-down, experimental dextran sulfate cellulose beads column produced similar results.

Conclusions: Our results show, for the first time, that modulation of LDL levels by LA directly affects plasma PCSK9 levels, and suggest that PCSK9 reduction is an additional benefit of LA. Because the loss of PCSK9 could contribute to the LDL-lowering effect of LA, then (1) anti-PCSK9 therapies may reduce frequency of LA in patients currently approved for therapy, and (2) LA and anti-PCSK9 therapies may be used synergistically to reduce treatment burden.

 

2.189           PrimPol, an Archaic Primase/Polymerase Operating in Human Cells

Garcia-Gomez, S., Reyes, A., Martinez-Jimenez, M.I., Chocron, E.S., Mouron, S., Terrados, G., Powell, C., Salido, E., Mendez, J., Holt, I.J. and Blanco, L.

Molecular  Cell, 52, 541-553 (2013)

 

We describe a second primase in human cells, PrimPol, which has the ability to start DNA chains with deoxynucleotides unlike regular primases, which use exclusively ribonucleotides. Moreover, PrimPol is also a DNA polymerase tailored to bypass the most common oxidative lesions in DNA, such as abasic sites and 8-oxoguanine. Subcellular fractionation and immunodetection studies indicated that PrimPol is present in both nuclear and mitochondrial DNA compartments. PrimPol activity is detectable in mitochondrial lysates from human and mouse cells but is absent from mitochondria derived from PRIMPOL knockout mice. PRIMPOL gene silencing or ablation in human and mouse cells impaired mitochondrial DNA replication. On the basis of the synergy observed with replicative DNA polymerases Polγ and Polε, PrimPol is proposed to facilitate replication fork progression by acting as a translesion DNA polymerase or as a specific DNA primase reinitiating downstream of lesions that block synthesis during both mitochondrial and nuclear DNA replication.

 

2.190           Interactome of Two Diverse RNA Granules Links mRNA Localization to Translational Repression in Neurons

Fritzsche, R. et al

Cell Reports, 5, 1749-1762 (2013)

 

Transport of RNAs to dendrites occurs in neuronal RNA granules, which allows local synthesis of specific proteins at active synapses on demand, thereby contributing to learning and memory. To gain insight into the machinery controlling dendritic mRNA localization and translation, we established a stringent protocol to biochemically purify RNA granules from rat brain. Here, we identified a specific set of interactors for two RNA-binding proteins that are known components of neuronal RNA granules, Barentsz and Staufen2. First, neuronal RNA granules are much more heterogeneous than previously anticipated, sharing only a third of the identified proteins. Second, dendritically localized mRNAs, e.g., Arc and CaMKIIα, associate selectively with distinct RNA granules. Third, our work identifies a series of factors with known roles in RNA localization, translational control, and RNA quality control that are likely to keep localized transcripts in a translationally repressed state, often in distinct types of RNPs.

 

2.191           Hepatic trans-Golgi action coordinated by the GTPase ARFRP1 is crucial for lipoprotein lipidation and assembly

Hesse, D., Radloff, K., Jaschke, A., Lagerpusch, M., Chung, B., Tailleux, A., Staels, B. and Schürmann, A.

1. Lipid Res., 55, 41-52 (2014)

 

The liver is a major organ in whole body lipid metabolism and malfunctioning can lead to various diseases including dyslipidemia, fatty liver disease, and type 2 diabetes. Triglycerides and cholesteryl esters are packed in the liver as very low density lipoproteins (VLDLs). Generation of these lipoproteins is initiated in the endoplasmic reticulum and further maturation likely occurs in the Golgi. ADP-ribosylation factor-related protein 1 (ARFRP1) is a small trans-Golgi-associated guanosine triphosphatase (GTPase) that regulates protein sorting and is required for chylomicron lipidation and assembly in the intestine. Here we show that the hepatocyte-specific deletion of Arfrp1 (Arfrp1^liv−/−) results in impaired VLDL lipidation leading to reduced plasma triglyceride levels in the fasted state as well as after inhibition of lipoprotein lipase activity by Triton WR-1339. In addition, the concentration of ApoC3 that comprises 40% of protein mass of secreted VLDLs is markedly reduced in the plasma of Arfrp1^liv−/− mice but accumulates in the liver accompanied by elevated triglycerides. Fractionation of Arfrp1^liv−/− liver homogenates reveals more ApoB48 and a lower concentration of triglycerides in the Golgi compartments than in the corresponding fractions from control livers. In conclusion, ARFRP1 and the Golgi apparatus play an important role in lipoprotein maturation in the liver by influencing lipidation and assembly of proteins to the lipid particles.

 

2.192           The 51 kDa FADS3 is Secreted in the ECM of Hepatocytes and Blood in Rat

Blanchard, H., Boulier-Monthean, N., Legrand, P. and Pedrono, F.

1. Cell. Biochem., 115(1), 199-207 (2014)

 

The fatty acid desaturase (Fads) cluster is composed of three genes encoding for the Δ5- and Δ6-desaturases and FADS3. The two former proteins are involved in the fatty acid biosynthesis; the latter one shares a high sequence identity but has still no attributed function. In a previous work performed in rat, we described three isoforms of FADS3 expressed in a tissue-dependent manner. In the present study, we demonstrated a specific subcellular targeting depending on the isoform. In cultured hepatocytes, which mainly expressed the 51 kDa protein, FADS3 was unexpectedly present in the cytosolic fraction, but was also secreted in the extracellular matrix on fibronectin-containing fibers. The secretion pathway was investigated and we determined the presence of exosome-like vesicles on the FADS3-stained fibers. In parallel, FADS3 was detected in blood of hepatic vessel, and particularly in serum. In conclusion, this study demonstrated a very specific intra- and extracellular location of FADS3 in comparison with the Δ5- and Δ6-desaturases, suggesting a unique function for this putative desaturase, even if no activity has been yet identified neither in the extracellular matrix of hepatocytes nor in serum.

 

2.193           Pathogenic Mutations within the Hydrophobic Domain of the Prion Protein Lead to the Formation of Protease-Sensitive Prion Species with Increased Lethality

Coleman, B.M., Harrison, C.F., Guo, B., Masters, C.L., Barnham, K.J., Lawson, V.A. and Hill, A.F.

1. Virol., 88(5), 2690-2703 (2014)

 

Prion diseases are a group of fatal and incurable neurodegenerative diseases affecting both humans and animals. The principal mechanism of these diseases involves the misfolding the host-encoded cellular prion protein, PrP^C, into the disease-associated isoform, PrP^Sc. Familial forms of human prion disease include those associated with the mutations G114V and A117V, which lie in the hydrophobic domain of PrP. Here we have studied the murine homologues (G113V and A116V) of these mutations using cell-based and animal models of prion infection. Under normal circumstances, the mutant forms of PrP^C share similar processing, cellular localization, and physicochemical properties with wild-type mouse PrP (MoPrP). However, upon exposure of susceptible cell lines expressing these mutants to infectious prions, very low levels of protease-resistant aggregated PrP^Sc are formed. Subsequent mouse bioassay revealed high levels of infectivity present in these cells. Thus, these mutations appear to limit the formation of aggregated PrP^Sc, giving rise to the accumulation of a relatively soluble, protease sensitive, prion species that is highly neurotoxic. Given that these mutations lie next to the glycine-rich region of PrP that can abrogate prion infection, these findings provide further support for small, protease-sensitive prion species having a significant role in the progression of prion disease and that the hydrophobic domain is an important determinant of PrP conversion.

 

2.194           Immobilization of Homogeneous Monomeric, Oligomeric and Fibrillar Aβ Species for Reliable SPR Measurements

Frenzel, D., Glück, J.M., Brener, O., Oesterhelt, F., Nagel-Steger, L. and Willbold, D.

PloS One, 9(3), e89490 (2014)

 

There is strong evidence that the amyloid-beta peptide (Aβ) plays a central role in the pathogenesis of Alzheimer's disease (AD). In this context, a detailed quantitative description of the interactions with different Aβ species is essential for characterization of physiological and artificial ligands. However, the high aggregation propensity of Aβ in concert with its susceptibility to structural changes due to even slight changes in solution conditions has impeded surface plasmon resonance (SPR) studies with homogeneous Aβ conformer species. Here, we have adapted the experimental procedures to state-of-the-art techniques and established novel approaches to reliably overcome the aforementioned challenges. We show that the application of density gradient centrifugation (DGC) for sample purification and the use of a single chain variable fragment (scFv) of a monoclonal antibody directed against the amino-terminus of Aβ allows reliable SPR measurements and quality control of the immobilized Aβ aggregate species at any step throughout the experiment.

 

2.195           Replication factors transiently associate with mtDNA at the mitochondrial inner membrane to facilitate replication

Rajala, N., Gerhold, J.M., Martinsson, P., Klymov, A. and Spelbrink, J.N.

Nucleic Acids Res., 42(2), 952-967 (2014)

 

Mitochondrial DNA (mtDNA) is organized in discrete protein–DNA complexes, nucleoids, that are usually considered to be mitochondrial-inner-membrane associated. Here we addressed the association of replication factors with nucleoids and show that endogenous mtDNA helicase Twinkle and single-stranded DNA-binding protein, mtSSB, co-localize only with a subset of nucleoids. Using nucleotide analogs to identify replicating mtDNA in situ, the fraction of label-positive nucleoids that is Twinkle/mtSSB positive, is highest with the shortest labeling-pulse. In addition, the recruitment of mtSSB is shown to be Twinkle dependent. These proteins thus transiently associate with mtDNA in an ordered manner to facilitate replication. To understand the nature of mtDNA replication complexes, we examined nucleoid protein membrane association and show that endogenous Twinkle is firmly membrane associated even in the absence of mtDNA, whereas mtSSB and other nucleoid-associated proteins are found in both membrane-bound and soluble fractions. Likewise, a substantial amount of mtDNA is found as soluble or loosely membrane bound. We show that, by manipulation of Twinkle levels, mtDNA membrane association is partially dependent on Twinkle. Our results thus show that Twinkle recruits or is assembled with mtDNA at the inner membrane to form a replication platform and amount to the first clear demonstration that nucleoids are dynamic both in composition and concurrent activity.

 

2.196           Initial Steps in RNA Processing and Ribosome Assembly Occur at Mitochondrial DNA Nucleoids

Borgenhagen, D.F., martin, D.W. and Koller, A.

Cell Metabolism, 19, 618-629 (2014)

 

Mammalian mitochondrial DNA (mtDNA) resides in compact nucleoids, where it is replicated and transcribed into long primary transcripts processed to generate rRNAs, tRNAs, and mRNAs encoding 13 proteins. This situation differs from bacteria and eukaryotic nucleoli, which have dedicated rRNA transcription units. The assembly of rRNAs into mitoribosomes has received little study. We show that mitochondrial RNA processing enzymes involved in tRNA excision, ribonuclease P (RNase P) and ELAC2, as well as a subset of nascent mitochondrial ribosomal proteins (MRPs) associate with nucleoids to initiate RNA processing and ribosome assembly. SILAC pulse-chase labeling experiments show that nascent MRPs recruited to the nucleoid fraction were highly labeled after the pulse in a transcription-dependent manner and decreased in labeling intensity during the chase. These results provide insight into the landscape of binding events required for mitochondrial ribosome assembly and firmly establish the mtDNA nucleoid as a control center for mitochondrial biogenesis.

 

2.197           Regulated Oligomerization Induces Uptake of a Membrane Protein into COPII Vesicles Independent of Its Cytosolic Tail

Springer, S., Malkus, P., Borchert, B., Wellbrock, U., Duden, R. and Schekman, R.

Traffic, 15, 531-545 (2014)

 

Export of transmembrane proteins from the endoplasmic reticulum (ER) is driven by directed incorporation into coat protein complex II (COPII)-coated vesicles. The sorting of some cargo proteins into COPII vesicles was shown to be mediated by specific interactions between transmembrane and COPII-coat-forming proteins. But even though some signals for ER exit have been identified on the cytosolic domains of membrane proteins, the general signaling and sorting mechanisms of ER export are still poorly understood. To investigate the role of cargo protein oligomer formation in the export process, we have created a transmembrane fusion protein that – owing to its FK506-binding protein domains – can be oligomerized in isolated membranes by addition of a small-molecule dimerizer. Packaging of the fusion protein into COPII vesicles is strongly enhanced in the presence of the dimerizer, demonstrating that the oligomeric state is an ER export signal for this membrane protein. Surprisingly, the cytosolic tail is not required for this oligomerization-dependent effect on protein sorting. Thus, an alternative mechanism, such as membrane bending, must account for ER export of the fusion protein.

 

 

 

2.198           The metabolic inter-relationships between changes in waist circumference, triglycerides, insulin sensitivity and small, dense low-density lipoprotein particles with acute weight loss in clinically obese children and adolescents

Hobkirk, J.P., King, R.E., davies, I., Harman, N., Gately, P., Pemberton, P., Smith, A., barth, J.H. and Carroll, S.

Pediatric Obesity, 9(3), 209-217 (2014)

 

Objective

Small, dense low-density lipoprotein (LDL) particles are highly atherogenic and strongly associated with obesity-related dyslipidemia. The metabolic inter-relationships between weight loss induced changes in waist circumference, triglycerides, insulin sensitivity and small-dense LDL particles in clinically obese children and adolescents have not been studied.

Methods

Seventy-five clinically obese boys and girls (standardized body mass index 3.07 ± 0.59, aged 8–18 years) were recruited. Anthropometric, body composition and cardiometabolic risk factors were measured pre- and post-weight loss.

Results

There were highly significant reductions in anthropometric, body composition and cardiometabolic risk factors. Triglyceride change was positively correlated with LDL peak particle density and percentage LDL pattern B changes (relative abundance of small, dense LDL particles). Multiple regression analyses showed that changes in triglyceride concentration accounted for between 24 and 18% of the variance in LDL peak particle density and percentage LDL pattern B change, respectively. Changes in waist circumference and insulin sensitivity did not predict these changes in LDL characteristics.

Conclusion

Acute and highly significant weight loss significantly decreased LDL peak particle density and percentage LDL pattern B. The change in triglycerides was a strong predictor of LDL peak particle density and percentage LDL pattern B change.

 

2.199           Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

Brindley, M.A., Plattet, P. and Plemper, R.K.

PNAS, 111(36), E3795-E3804 (2014)

 

Enveloped viruses such as HIV and members of the paramyxovirus family use metastable, proteinaceous fusion machineries to merge the viral envelope with cellular membranes for infection. A hallmark of the fusogenic glycoproteins of these pathogens is refolding into a thermodynamically highly stable fusion core structure composed of six antiparallel α-helices, and this structure is considered instrumental for pore opening and/or enlargement. Using a paramyxovirus fusion (F) protein, we tested this paradigm by engineering covalently restricted F proteins that are predicted to be unable to close the six-helix bundle core structure fully. Several candidate bonds formed efficiently, resulting in F trimers and higher-order complexes containing covalently linked dimers. The engineered F complexes were incorporated into recombinant virions efficiently and were capable of refolding into a postfusion conformation without temporary or permanent disruption of the disulfide bonds. They efficiently formed fusion pores based on virus replication and quantitative cell-to-cell and virus-to-cell fusion assays. Complementation of these F mutants with a monomeric, fusion-inactive F variant enriched the F oligomers for heterotrimers containing a single disulfide bond, without affecting fusion complementation profiles compared with standard F protein. Our demonstration that complete closure of the fusion core does not drive paramyxovirus entry may aid the design of strategies for inhibiting virus entry.

 

2.200           Virus-Inspired Membrane Encapsulation of DNA Nanostructures To Achieve In Vivo Stability

Perrault, S.D. and Shih, W.M.

ACSNano, 8(5), 5132-5140 (2014)

 

DNA nanotechnology enables engineering of molecular-scale devices with exquisite control over geometry and site-specific functionalization. This capability promises compelling advantages in advancing nanomedicine; nevertheless, instability in biological environments and innate immune activation remain as obstacles for in vivo application. Natural particle systems (i.e., viruses) have evolved mechanisms to maintain structural integrity and avoid immune recognition during infection, including encapsulation of their genome and protein capsid shell in a lipid envelope. Here we introduce virus-inspired enveloped DNA nanostructures as a design strategy for biomedical applications. Achieving a high yield of tightly wrapped unilamellar nanostructures, mimicking the morphology of enveloped virus particles, required precise control over the density of attached lipid conjugates and was achieved at 1 per 180 nm². Envelopment of DNA nanostructures in PEGylated lipid bilayers conferred protection against nuclease digestion. Immune activation was decreased 2 orders of magnitude below controls, and pharmacokinetic bioavailability improved by a factor of 17. By establishing a design strategy suitable for biomedical applications, we have provided a platform for the engineering of sophisticated, translation-ready DNA nanodevices.

 

2.201           Abstract 433: Examination of Factors Affecting the Association of PCSK9 With Low-Density Lipoprotein Particles in Human Plasma

Golder, M., Sarkar, S., Kosenko, T., McPherson, R. and Lagace, T.A.

Arterioscler. Thromb. Vasc. Biol., 34:A433 (2014)

 

Rationale: We have previously shown that a substantial proportion of plasma PCSK9 (30-40%) is associated with LDL particles in normolipidemic subjects. Cellular assays show that LDL-bound PCSK9 is less active for binding to cell surface LDLRs. Therefore, the ability of circulating PCSK9 to direct LDLR degradation in liver could be regulated by plasma LDL levels. In addition, LDL subspecies may have altered abilities in binding PCSK9. We have mapped the LDL binding region to a short stretch of amino acids (aa 31-52) in the PCSK9 prodomain. It is unknown whether a common loss-of-function PCSK9 mutation (R46L) within this region affects LDL binding.

Objective: To determine whether plasma PCSK9 distribution (LDL-bound versus unbound) is affected in hypercholesterolemic subjects. To further characterize the interaction of PCSK9 and LDL, we investigated the interaction of PCSK9 with two subspecies of LDL - large, buoyant LDL (LBLDL; d=1.019-1.044 g/ml) and small, dense LDL (SDLDL; d=1.044-1.063 g/ml). Additionally, we investigated the effect of the R46L PCSK9 mutation on the LDL binding affinity of PCSK9.

Methods and Results: We used flotation ultracentrifugation in Optiprep density gradients to fractionate human plasma samples followed by immunoprecipitation and western blot to quantify PCSK9 distribution in LDL and non-LDL fractions. In a pilot study, the proportion of total plasma PCSK9 in the LDL fraction was increased from 38±5% to 57±3% (N=6) in hypercholesterolemic subjects (LDL>4.9mM, TG<2.3mM) versus normal controls (LDL<3 mM, TG<2.3mM). Saturation binding assays showed that SDLDL bound PCSK9 with lower affinity (Kd = 361.9 nM) than LDLDL (Kd = 263.9 nM). Competition binding assays determined that recombinant purified PCSK9-R46L secreted from HEK293 cells did not bind to isolated LDL with significantly altered affinity compared to wild-type PCSK9.

Conclusion: Our preliminary results indicate that plasma PCSK9 distribution is altered in hypercholesterolemia, with an increased proportion of total PCSK9 bound to LDL particles. Our in vitro results suggest that circulating small, dense LDL may bind more poorly to PCSK9 than larger LDL subspecies.

 

2.202           Absence of an effect of vitamin E on protein and lipid radical formation during lipoperoxidation of LDL by lipoxygenase

Ganini, D. and Mason, R.P.

Free Radical Biology and Medicine, 76, 61-68 (2014)

 

Low-density lipoprotein (LDL) oxidation is the primary event in atherosclerosis, and LDL lipoperoxidation leads to modifications in apolipoprotein B-100 (apo B-100) and lipids. Intermediate species of lipoperoxidation are known to be able to generate amino acid-centered radicals. Thus, we hypothesized that lipoperoxidation intermediates induce protein-derived free radical formation during LDL oxidation. Using DMPO and immuno-spin trapping, we detected the formation of protein free radicals on LDL incubated with Cu²⁺ or the soybean lipoxidase (LPOx)/phospholipase A₂ (PLA₂). With low concentrations of DMPO (1 mM), Cu²⁺ dose-dependently induced oxidation of LDL and easily detected apo B-100 radicals. Protein radical formation in LDL incubated with Cu²⁺ showed maximum yields after 30 min. In contrast, the yields of apo B-100 radicals formed by LPOx/PLA₂ followed a typical enzyme-catalyzed kinetics that was unaffected by DMPO concentrations of up to 50 mM. Furthermore, when we analyzed the effect of antioxidants on protein radical formation during LDL oxidation, we found that ascorbate, urate, and Trolox dose-dependently reduced apo B-100 free radical formation in LDL exposed to Cu²⁺. In contrast, Trolox was the only antioxidant that even partially protected LDL from LPOx/PLA₂. We also examined the kinetics of lipid radical formation and protein radical formation induced by Cu²⁺ or LPOx/PLA₂ for LDL supplemented with α-tocopherol. In contrast to the potent antioxidant effect of α-tocopherol on the delay of LDL oxidation induced by Cu²⁺, when we used the oxidizing system LPOx/PLA₂, no significant protection was detected. The lack of protection of α-tocopherol on the apo B-100 and lipid free radical formation by LPOx may explain the failure of vitamin E as a cardiovascular protective agent for humans.

 

2.203           MPV17L2 is required for ribosome assembly in mitochondria

Rosa, I.D., Durigon, R., Pearce, S.F., Rorbach, J., Hirst, E.M.A., Vidoni, S., Reyes, A., Brea-Calvo, G., Minczuk, M., Woellhaf, M.W., Herrmann, J.M., Huynen, M.A., Holt, I.J. and Spinazzola, A.

Nucleic Acids Res., 42(13), 8500-8515 (2014)

 

MPV17 is a mitochondrial protein of unknown function, and mutations in MPV17 are associated with mitochondrial deoxyribonucleic acid (DNA) maintenance disorders. Here we investigated its most similar relative, MPV17L2, which is also annotated as a mitochondrial protein. Mitochondrial fractionation analyses demonstrate MPV17L2 is an integral inner membrane protein, like MPV17. However, unlike MPV17, MPV17L2 is dependent on mitochondrial DNA, as it is absent from ρ0 cells, and co-sediments on sucrose gradients with the large subunit of the mitochondrial ribosome and the monosome. Gene silencing of MPV17L2 results in marked decreases in the monosome and both subunits of the mitochondrial ribosome, leading to impaired protein synthesis in the mitochondria. Depletion of MPV17L2 also induces mitochondrial DNA aggregation. The DNA and ribosome phenotypes are linked, as in the absence of MPV17L2 proteins of the small subunit of the mitochondrial ribosome are trapped in the enlarged nucleoids, in contrast to a component of the large subunit. These findings suggest MPV17L2 contributes to the biogenesis of the mitochondrial ribosome, uniting the two subunits to create the translationally competent monosome, and provide evidence that assembly of the small subunit of the mitochondrial ribosome occurs at the nucleoid.

 

2.204           Transcriptomic characterization of short duration endoplasmic reticulum stress on cultured human proximal tubule cells

Zhang, Y., barati, M., Munoz, I., Li, M., Wilkey, D., Rouchka, E.and Merchant, M.

BMC Bioformatics, 15 (Suppl 10) P5 (2014)

 

Stress granules (SG) are formed as collections of protein and RNA (ribonucleoprotein structures) and continuously assembled/disassembled in response to stresses such as heat, osmotic, or oxidant stress; representing an attempt to survive the stress through salvage of important proteins and RNA. Recent research suggests diabetic nephropathy (DN) may change or alter SG biology and in conditions that model DN may involve the receptor for activated C-kinases (RACK1). The incorporation of RACK1 into stress granules may down-regulate programmed cell death and further may impart the ability to scaffold to and sequester key signaling proteins to affect cell survival or death. We hypothesized that the inappropriate or dysregulated scaffolding of proteins or RNA into stress granules may be of importance in the development of diabetic nephropathy. The goal of this study is to qualitatively and semi-quantitatively characterize the effects of cell culture conditions modeling diabetes and ER stress in conjunction with over-expression studies of SG stabilizing proteins on RNA transcripts.

 

2.205           A C-terminal Membrane Anchor Affects the Interactions of Prion Proteins with Lipid Membranes

Chu, N.K., Shabbir, W., Bove-Fwenderson, E., Araman, C., Lemmens-Gruber, R., harris, D.A. and Becker, C.F.W.

1. Biol. Chem., 289, 30144-30160 (2014)

 

Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrP^C into pathogenic PrP^Sc. Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23–231, FL_PrP), N-terminally truncated PrP (residues 90–231, T_PrP), and PrP missing its central hydrophobic region (Δ105–125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions.

 

2.206           RuvB-like ATPases Function in Chromatin Decondensation at the End of Mitosis

Magalska, A., Schellhaus, a.K., Moreno-Andres. D., Zanini, F., Schooley, A., Sachdev, R., Schwarz, H., Madlung, J. and Antonin, W.

Developmental Cell, 31(3), 305-318 (2014)

 

Chromatin undergoes extensive structural changes during the cell cycle. Upon mitotic entry, metazoan chromatin undergoes tremendous condensation, creating mitotic chromosomes with 50-fold greater compaction relative to interphase chromosomes. At the end of mitosis, chromosomes reestablish functional interphase chromatin competent for replication and transcription through a decondensation process that is cytologically well described. However, the underlying molecular events and factors remain unidentified. We describe a cell-free system that recapitulates chromatin decondensation based on purified mitotic chromatin and Xenopus egg extracts. Using biochemical fractionation, we identify RuvB-like ATPases as chromatin decondensation factors and demonstrate that their ATPase activity is essential for decondensation. Our results show that decompaction of metaphase chromosomes is not merely an inactivation of known chromatin condensation factors but rather an active process requiring specific molecular machinery. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its coordination with concomitant processes.

 

2.207           Amphipathic α-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles

Fukuhara, T., Wada, M., Nakamura, S., Ono, C., Shiokawa, M., yamamoto, S., Motomura, T., Okamoto, T., Okuzaki, D., Yamamoto, M., Saito, I., Wakita, T., Koike, K. and matsuura, Y.

PloS Pathogens, 10(12), e1004534 (2014)

 

Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly.

 

2.208           Cytotoxicity of Human Endogenous Retrovirus K–Specific T Cells toward Autologous Ovarian Cancer Cells

Rycaj, K., Plummer, J.B., Yin, B., Li, M., Garza, J., Radvanyi, L., Ramondetta, L.M., Lin, K., Johanning, G.L., Tang, D.G. and Wang-Johanning, F.

Clin. Cancer Res., 21(2), 471-483 (2015)

 

Purpose: To determine whether HERV-K envelope (ENV) protein could function as a tumor-associated antigen and elicit specific T-cell responses against autologous ovarian cancer cells.

Experimental Design: The expression of HERV-K transcripts and ENV protein, the presence of serum antibodies against HERV-K, reverse transcriptase (RT) activities, and cellular immune responses in primary ovarian cancer tissues and patient blood samples were analyzed and compared with samples from patients with benign ovarian diseases and normal female donors.

Results: Ovarian cancer cells in primary tumors and ascites expressed markers of cancer stem cells and markers of both mesenchymal and epithelial cells. Expression of HERV transcripts and HERV-K ENV protein and reverse transcriptase activities were higher in ovarian cancer compared with adjacent normal and benign tissues. The ovarian cancer patient plasma also had high reverse transcriptase activities and the ovarian cancer patient sera contained HERV-K immunoreactive antibodies. HERV-K–specific T cells generated from autologous dendritic cells pulsed with HERV-K ENV antigens exhibited phenotypes and functions consistent with a cellular immune response including T-cell proliferation, IFNγ production, and HERV-K–specific cytotoxic T lymphocyte (CTL) activity. Significantly higher CTL lysis of autologous tumor cells than of uninvolved normal cells was demonstrated in patients with ovarian cancer than patients with benign diseases and further enhanced lysis was observed if T regulatory cells were depleted.

Conclusion: Endogenous retroviral gene products in ovarian cancer may represent a potentially valuable new pool of tumor-associated antigens for targeting of therapeutic vaccines to ovarian cancer.

 

2.209           Atomic Structure of T6SS Reveals Interlaced Array Essential to Function

Clemens, D.L., Ge, P., Horwitz, M.A. and Zhou, Z.H.

Cell, 160, 940-951 (2015)

 

Type VI secretion systems (T6SSs) are newly identified contractile nanomachines that translocate effector proteins across bacterial membranes. The Francisella pathogenicity island, required for bacterial phagosome escape, intracellular replication, and virulence, was presumed to encode a T6SS-like apparatus. Here, we experimentally confirm the identity of this T6SS and, by cryo electron microscopy (cryoEM), show the structure of its post-contraction sheath at 3.7 Å resolution. We demonstrate the assembly of this T6SS by IglA/IglB and secretion of its putative effector proteins in response to environmental stimuli. The sheath has a quaternary structure with handedness opposite that of contracted sheath of T4 phage tail and is organized in an interlaced two-dimensional array by means of β sheet augmentation. By structure-based mutagenesis, we show that this interlacing is essential to secretion, phagosomal escape, and intracellular replication. Our atomic model of the T6SS will facilitate design of drugs targeting this highly prevalent secretion apparatus.

 

2.210           MinD-like ATPase FlhG effects location and number of bacterial flagella during C-ring assembly

Schuhmacher, J.S., Rossmann, F., Dempwollf, F., Knauer, C., Altegoer, f., Steinchen, W., Dörrich, A.K., Klingl, A., Stephen, M., Linne, U., Thormann, K.M. and Bange, G.

PNAS, 7(10), 3092-3097 (2015)

 

The number and location of flagella, bacterial organelles of locomotion, are species specific and appear in regular patterns that represent one of the earliest taxonomic criteria in microbiology. However, the mechanisms that reproducibly establish these patterns during each round of cell division are poorly understood. FlhG (previously YlxH) is a major determinant for a variety of flagellation patterns. Here, we show that FlhG is a structural homolog of the ATPase MinD, which serves in cell-division site determination. Like MinD, FlhG forms homodimers that are dependent on ATP and lipids. It interacts with a complex of the flagellar C-ring proteins FliM and FliY (also FliN) in the Gram-positive, peritrichous-flagellated Bacillus subtilis and the Gram-negative, polar-flagellated Shewanella putrefaciens. FlhG interacts with FliM/FliY in a nucleotide-independent manner and activates FliM/FliY to assemble with the C-ring protein FliG in vitro. FlhG-driven assembly of the FliM/FliY/FliG complex is strongly enhanced by ATP and lipids. The protein shows a highly dynamic subcellular distribution between cytoplasm and flagellar basal bodies, suggesting that FlhG effects flagellar location and number during assembly of the C-ring. We describe the molecular evolution of a MinD-like ATPase into a flagellation pattern effector and suggest that the underappreciated structural diversity of the C-ring proteins might contribute to the formation of different flagellation patterns.

 

2.211           Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2

Cortesio, C.L., Lewellyn, E.B.and Drubin, D.G.

Mol. Biol. Cell, 26, 1509-1522 (2015)

 

Clathrin-mediated endocytosis (CME) is facilitated by a precisely regulated burst of actin assembly. PtdIns(4,5)P2 is an important signaling lipid with conserved roles in CME and actin assembly regulation. Rhomboid family multipass transmembrane proteins regulate diverse cellular processes; however, rhomboid-mediated CME regulation has not been described. We report that yeast lacking the rhomboid protein Rbd2 exhibit accelerated endocytic-site dynamics and premature actin assembly during CME through a PtdIns(4,5)P2-dependent mechanism. Combined genetic and biochemical studies showed that the cytoplasmic tail of Rbd2 binds directly to PtdIns(4,5)P2 and is sufficient for Rbd2's role in actin regulation. Analysis of an Rbd2 mutant with diminished PtdIns(4,5)P2-binding capacity indicates that this interaction is necessary for the temporal regulation of actin assembly during CME. The cytoplasmic tail of Rbd2 appears to modulate PtdIns(4,5)P2 distribution on the cell cortex. The syndapin-like F-BAR protein Bzz1 functions in a pathway with Rbd2 to control the timing of type 1 myosin recruitment and actin polymerization onset during CME. This work reveals that the previously unstudied rhomboid protein Rbd2 functions in vivo at the nexus of three highly conserved processes: lipid regulation, endocytic regulation, and cytoskeletal function.

 

2.212           Prevalence of plasma small dense LDL is increased in obesity in a Thai population

Kulanuwar, S., Tungtrongchitr, R., Billington, D. and Davies, I.G.

Lipis in Health and Disease, 14:30 (2015)

 

Background

Plasma low density lipoprotein (LDL) particles vary in size, density, electrical charge and chemical composition. An increased presence of small dense LDL (sdLDL), along with raised triglyceride concentrations and decreased high density lipoprotein (HDL) cholesterol concentrations is commonly known as the atherogenic triad and has been observed in some cases of obesity, principally in Europe and America. This study examines the prevalence of sdLDL in the plasma of an obese (BMI ≥ 25 kg/m²) Thai population.

Methods

Plasma from fasted obese (n = 48) and non-obese (n = 16) Thai participants was subjected to density gradient ultracentrifugation in iodixanol to separate lipoproteins. Gradients were unloaded top-to-bottom into 20 fractions which were assayed for cholesterol, triglyceride, apo B and apo A-1 to identify lipoprotein types and subtypes.

Results

LDL cholesterol was subfractionated into LDL I + II (fractions 3–6, ρ = 1.021-1.033 g/ml) which was considered to represent large buoyant LDL (lbLDL), LDL III (fractions 7–9, ρ = 1.036-1.039 g/ml) which was considered to represent sdLDL, and, LDL IV (fractions 10–12, ρ = 1.044-1.051 g/ml) which was considered to represent very sdLDL. Concentrations of LDL III and IV were increased by 15-20% in obese participants whilst that of LDL I + II was concomitantly decreased by 10%. This was accompanied by a 50% increase in plasma triglyceride concentrations and 15% decrease in HDL cholesterol concentrations. Only 3/16 (19%) non-obese participants had a pattern B LDL cholesterol profile (peak density of >1.033 g/ml), whilst 28/48 (58%) obese participants were pattern B. When expressed as a fraction of the LDL concentration, total sdLDL (i.e. LDL III + IV) showed highly significant correlations to plasma triglyceride concentrations and the triglyceride/HDL cholesterol ratio.

Conclusions

The prevalence of sdLDL is increased in obesity in a Thai population such that they demonstrate a similar atherogenic triad to that previously observed in European and American populations.

 

2.213           Cry Protein Crystals: A Novel Platform for Protein Delivery

Nair, M.S., Lee, M.M., Bonnegarde-Bernard, A., Wallace, J.A., Dean, D.H., Ostrowski, M.C., Burry, R.W., Boyaka, P.N. and Chan, K.

PloS One, 10(6), e0127669 (2015)

 

Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer’s patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.

 

2.214           Effects of impaired membrane interactions on α-synuclein aggregation and neurotoxicity

Ysselstein, Joshi, M., Mishra, V., Grigg, A.M., Asiago, J.M., McCabe, G.P., Stanciu, L.A., Post, C.B. and Rochet, J-C.

Neurobiology of Disease, 79, 150-163 (2015)

 

The post-mortem brains of individuals with Parkinson's disease (PD) and other synucleinopathy disorders are characterized by the presence of aggregated forms of the presynaptic protein α-synuclein (aSyn). Understanding the molecular mechanism of aSyn aggregation is essential for the development of neuroprotective strategies to treat these diseases. In this study, we examined how interactions between aSyn and phospholipid vesicles influence the protein's aggregation and toxicity to dopaminergic neurons. Two-dimensional NMR data revealed that two familial aSyn mutants, A30P and G51D, populated an exposed, membrane-bound conformer in which the central hydrophobic region was dissociated from the bilayer to a greater extent than in the case of wild-type aSyn. A30P and G51D had a greater propensity to undergo membrane-induced aggregation and elicited greater toxicity to primary dopaminergic neurons compared to the wild-type protein. In contrast, the non-familial aSyn mutant A29E exhibited a weak propensity to aggregate in the presence of phospholipid vesicles or to elicit neurotoxicity, despite adopting a relatively exposed membrane-bound conformation. Our findings suggest that the aggregation of exposed, membrane-bound aSyn conformers plays a key role in the protein's neurotoxicity in PD and other synucleinopathy disorders.

 

2.215           A novel and rapid method for obtaining high titre intact prion strains from mammalian brain

Wenborn, A., terry, C., Gros, N., Joiner, S., D’Castro, L., Panico, S., Sells, J., Cronier, S., Linehan, J.M., Brandner, S., Saibil, H.R., Collinge, J. and Wadsworth, J.D.F.

Scientific Reports, 5:10062 (2015)

 

Mammalian prions exist as multiple strains which produce characteristic and highly reproducible phenotypes in defined hosts. How this strain diversity is encoded by a protein-only agent remains one of the most interesting and challenging questions in biology with wide relevance to understanding other diseases involving the aggregation or polymerisation of misfolded host proteins. Progress in understanding mammalian prion strains has however been severely limited by the complexity and variability of the methods used for their isolation from infected tissue and no high resolution structures have yet been reported. Using high-throughput cell-based prion bioassay to re-examine prion purification from first principles we now report the isolation of prion strains to exceptional levels of purity from small quantities of infected brain and demonstrate faithful retention of biological and biochemical strain properties. The method’s effectiveness and simplicity should facilitate its wide application and expedite structural studies of prions.

 

2.216           The Effect of n-3 Fatty Acids on Small Dense Low-Density Lipoproteins in Patients With End-Stage Renal Disease: A Randomized Placebo-Controlled Intervention Study

Sørensen, G.V.B., Svensson, M., Strandhave, C., Schmidt, E.B., Jørgensen, K.A. and Christensen, J.H.

1. Renal Nutrition, 25(4), 376-380 (2015)

 

Objective

Patients with end-stage renal disease (ESRD) have a high risk of cardiovascular disease. Small dense low-density lipoprotein (sdLDL) particles are particularly atherogenic. Marine n-3 polyunsaturated fatty acids (PUFA) may have a beneficial effect on numbers of sdLDL particles, and the aim of this study was to investigate the effect of n-3 PUFA on plasma levels of sdLDL in patients with ESRD.

Methods

ESRD patients with cardiovascular disease (n = 161) on chronic hemodialysis were randomized to treatment with 1.7 g of n-3 PUFA (n = 81) or 2 g of placebo (olive oil; n = 80) for 3 months. The study was double-blinded. Densities of LDL and percentages of sdLDL (sdLDL%) of total LDL were measured before and after intervention. On the basis of sdLDL%, patients were classified as having lipid pattern A, I (intermediate), or B defined by a successive increase in sdLDL concentration and decrease in lipid particle size.

Results

n-3 PUFAs significantly reduced triglycerides. However, LDL cholesterol remained unchanged. In the n-3 group, the LDL density did not change significantly during follow-up. Similarly, the LDL density remained unchanged in the placebo group. In the n-3 group, the sdLDL% was 34% at baseline and unchanged at follow-up. At baseline 71% had LDL pattern A, 9% had pattern I, and 20% had pattern B, and none of these patterns were significantly changed by n-3 PUFA supplementation.

Conclusion

Dietary supplementation with 1.7 g of n-3 PUFA had no effect on LDL density or sdLDL levels in patients with ESRD.

 

2.217           Co-option of Membrane Wounding Enables Virus Penetration into Cells

Luisoni, S., Suomalainen, M., Boucke, K., Grzybek, M., Coskun, U. and Greber, U.F.

Cell Host & Microbe, 18, 75-85 (201)

 

During cell entry, non-enveloped viruses undergo partial uncoating to expose membrane lytic proteins for gaining access to the cytoplasm. We report that adenovirus uses membrane piercing to induce and hijack cellular wound removal processes that facilitate further membrane disruption and infection. Incoming adenovirus stimulates calcium influx and lysosomal exocytosis, a membrane repair mechanism resulting in release of acid sphingomyelinase (ASMase) and degradation of sphingomyelin to ceramide lipids in the plasma membrane. Lysosomal exocytosis is triggered by small plasma membrane lesions induced by the viral membrane lytic protein-VI, which is exposed upon mechanical cues from virus receptors, followed by virus endocytosis into leaky endosomes. Chemical inhibition or RNA interference of ASMase slows virus endocytosis, inhibits virus escape to the cytosol, and reduces infection. Ceramide enhances binding of protein-VI to lipid membranes and protein-VI-induced membrane rupture. Thus, adenovirus uses a positive feedback loop between virus uncoating and lipid signaling for efficient membrane penetration.

 

2.218           Hybrid pulmonary surfactant-coated nanogels mediate efficient in vivo delivery of siRNA to murine alveolar macrophages

De Backer, L., Naessens, t., De Koker, S., Zagato, E., Demeester, J., Grooten, J., De Smedt, S.C. and Raemdonck, K.

1. Controlled Release, 217, 53-63 (2015)

 

The local delivery of small interfering RNA (siRNA) to the lungs may provide a therapeutic solution to a range of pulmonary disorders. Resident alveolar macrophages (rAM) in the bronchoalveolar lumen play a critical role in lung inflammatory responses and therefore constitute a particularly attractive target for siRNA therapeutics. However, achieving efficient gene silencing in the lung while avoiding pulmonary toxicity requires appropriate formulation of siRNA in functional nanocarriers. In this study, we evaluated pulmonary surfactant-coated dextran nanogels for the delivery of siRNA to rAM upon pharyngeal aspiration in BALB/c mice. Both the surfactant-coated and uncoated nanogels achieved high levels of siRNA uptake in rAM, yet only the surfactant-coated formulation could significantly reduce gene expression on the protein level. Surfactant-coated nanogels induced a profound downregulation of target mRNA levels, reaching 70% knockdown with ~ 1 mg kg^− 1 siRNA dose. In addition, only mild acute pro-inflammatory cytokine and chemokine responses were detected one day after nanoparticle aspiration, accompanied by a moderate neutrophil infiltration in the bronchoalveolar lumen. The latter could be substantially reduced by removal of excess surfactant from the formulation. Overall, our hybrid core-shell nanoparticles have demonstrated safe and effective siRNA delivery to rAM, providing a new therapeutic approach for treatment of inflammatory pathologies in the lung.

 

2.219           QIAD assay for quantitating a compound’s efficacy in elimination of toxic Aβ oligomers

Brener, O. et al

Scientific Reports, 5:13222 (2015)

 

Strong evidence exists for a central role of amyloid β-protein (Aβ) oligomers in the pathogenesis of Alzheimer’s disease. We have developed a fast, reliable and robust in vitro assay, termed QIAD, to quantify the effect of any compound on the Aβ aggregate size distribution. Applying QIAD, we studied the effect of homotaurine, scyllo-inositol, EGCG, the benzofuran derivative KMS88009, ZAβ3W, the D-enantiomeric peptide D3 and its tandem version D3D3 on Aβ aggregation. The predictive power of the assay for in vivo efficacy is demonstrated by comparing the oligomer elimination efficiency of D3 and D3D3 with their treatment effects in animal models of Alzheimer´s disease.

 

2.220           Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

Gilbert, G.E., Novakovic, V.A., Shi, J., Rasmussen, J. and Pipe, S.W.

Blood, 126(10), 1237-1244 (2015)

 

Thrombin-stimulated platelets expose very little phosphatidylserine (PS) but express binding sites for factor VIII (fVIII), casting doubt on the role of exposed PS as the determinant of binding sites. We previously reported that fVIII binding sites are increased three- to sixfold when soluble fibrin (SF) binds the α_IIbβ₃ integrin. This study focuses on the hypothesis that platelet-bound SF is the major source of fVIII binding sites. Less than 10% of fVIII was displaced from thrombin-stimulated platelets by lactadherin, a PS-binding protein, and an fVIII mutant defective in PS-dependent binding retained platelet affinity. Therefore, PS is not the determinant of most binding sites. FVIII bound immobilized SF and paralleled platelet binding in affinity, dependence on separation from von Willebrand factor, and mediation by the C2 domain. SF also enhanced activity of fVIII in the factor Xase complex by two- to fourfold. Monoclonal antibody (mAb) ESH8, against the fVIII C2 domain, inhibited binding of fVIII to SF and platelets but not to PS-containing vesicles. Similarly, mAb ESH4 against the C2 domain, inhibited >90% of platelet-dependent fVIII activity vs 35% of vesicle-supported activity. These results imply that platelet-bound SF is a component of functional fVIII binding sites.

 

2.221           Structure-based drug design identifies polythiophenes as antiprion compounds

Herrmann, U.S. et al

Science Translational Medicine, 7(299), 299ra123 (2015)

 

In a mouse model of prion disease, Herrmann et al. evaluated the therapeutic efficacy of luminescent conjugated polythiophenes (LCPs), which are molecules with a high affinity for ordered protein aggregates. Intracerebral administration of LCPs into prion-infected mice using osmotic pumps increased survival. Solid-state nuclear magnetic resonance and in silico binding studies of LCPs to simplified model fibrils allowed the authors to define structural rules, which they then used for the design of LCPs with superior prophylactic and therapeutic potency. The new work demonstrates the feasibility of rational drug design for developing therapeutics to treat prion diseases.

 

2.222           Nutritional values and bioactive components of under-utilised vegetables consumed by indigenous people in Malaysia

Wahab, N.A., Ahdan, R., Aufa, Z.A., Kong, K.W., Johar, M.H., Shariff, Z.M. and ismail, A.

1. Sci. Food Agric., 95(13), 2704-2711 (2015)

 

BACKGROUND

Diverse plants species in the forest remain under-utilised and they are mainly consumed only by local people. However, increasing issues in food security prompted the present study, which explores the nutritional and antioxidant aspects of Malaysian under-utilised vegetables. The studied vegetables were Paku Nyai (Stenochlaena palustris), Cemperai (Champereia manillana), Maman Pasir (Cleome viscose), Dudung (Erechtites valerianifolia) and Semambuk (Ardisia pendula).

RESULTS

Overall, these vegetables exhibited a low proximal content but they were high in vitamin C [7.07–1263 mg kg⁻¹ edible fresh sample (EFS)] and β-carotene content (18.4–43.9 mg kg⁻¹ kg⁻¹ EFS). Cemperai had the highest calcium content (565 mg kg⁻¹ EFS), whereas Semambuk had the highest total phenolic content [28.21 g gallic acid equivalents kg⁻¹ edible dried sample (EDS)] and antioxidant activity (86.1%) measured using β-carotene bleaching assay. Maman Pasir contained the highest total flavonoid content (39.99 g CE kg⁻¹ EDS) and 1,1-diphenyl-2-picryl hydrazyl radical scavenging activity (82.2%). The extracts of these vegetables had significantly prevented the oxidation of haemoglobin and low-density lipoprotein, which yielded a reduced production of malondialdehyde.

CONCLUSION

Semambuk and Maman Pasir are potent to be used as new food and functional food sources as they are rich in nutrients and antioxidants. © 2014 Society of Chemical Industry.

 

2.223           Human Mitochondrial DNA-Protein Complexes Attach to a Cholesterol-Rich Membrane Structure

Gerhold, J.M., Cansiz-Arda, S., Löhmus, M., Engberg, O., Reyes, A., van Rennes, H., Sanz, A., Holt, I.J., Cooper, H.M. and Spelbrink, J.N.

Scientific Reports, 5:15292 (2015)

 

The helicase Twinkle is indispensable for mtDNA replication in nucleoids. Previously, we showed that Twinkle is tightly membrane-associated even in the absence of mtDNA, which suggests that Twinkle is part of a membrane-attached replication platform. Here we show that this platform is a cholesterol-rich membrane structure. We fractionated mitochondrial membrane preparations on flotation gradients and show that membrane-associated nucleoids accumulate at the top of the gradient. This fraction was shown to be highly enriched in cholesterol, a lipid that is otherwise low abundant in mitochondria. In contrast, more common mitochondrial lipids, and abundant inner-membrane associated proteins concentrated in the bottom-half of these gradients. Gene silencing of ATAD3, a protein with proposed functions related to nucleoid and mitochondrial cholesterol homeostasis, modified the distribution of cholesterol and nucleoids in the gradient in an identical fashion. Both cholesterol and ATAD3 were previously shown to be enriched in ER-mitochondrial junctions, and we detect nucleoid components in biochemical isolates of these structures. Our data suggest an uncommon membrane composition that accommodates platforms for replicating mtDNA, and reconcile apparently disparate functions of ATAD3. We suggest that mtDNA replication platforms are organized in connection with ER-mitochondrial junctions, facilitated by a specialized membrane architecture involving mitochondrial cholesterol.

 

2.224           α-Tocopherol bioavailability is lower in adults with metabolic syndrome regardless of dairy fat co-ingestion: a randomized, double-blind, crossover trial

Mah, E., Sapper, T.., Chitchumroonchokchai, C., Failla, M.L., Schill, K.E., Clinton, S.K., Bobe, G., Traber, M.G. and Bruno, R.S.

Am. J. Clin. Nutr., 102(5), 1070-1080 (2015)

 

Background: Increasing dietary fat intake is expected to improve α-tocopherol bioavailability, which could be beneficial for improving α-tocopherol status, especially in cohorts at high cardiometabolic risk who fail to meet dietary α-tocopherol requirements.

Objective: Our objective was to assess dose-dependent effects of dairy fat and metabolic syndrome (MetS) health status on α-tocopherol pharmacokinetics in plasma and lipoproteins.

Design: A randomized, crossover, double-blind study was conducted in healthy and MetS adults (n = 10/group) who ingested encapsulated hexadeuterium-labeled (d₆)–RRR–α-tocopherol (15 mg) with 240 mL nonfat (0.2 g fat), reduced-fat (4.8 g fat), or whole (7.9 g fat) milk before blood collection at regular intervals for 72 h.

Results: Compared with healthy participants, those with MetS had lower (P < 0.05) baseline plasma α-tocopherol (μmol/mmol lipid) and greater oxidized low-density lipoprotein (LDL), interleukin (IL)–6, IL-10, and C-reactive protein. Regardless of health status, d₆–α-tocopherol bioavailability was unaffected by increasing amounts of dairy fat provided by milk beverages, but MetS participants had lower estimated d₆–α-tocopherol absorption (±SEM) than did healthy participants (26.1% ± 1.0% compared with 29.5% ± 1.1%). They also had lower plasma d₆–α-tocopherol AUC from 0 to 72 h, as well as maximal concentrations (C_max: 2.04 ± 0.14 compared with 2.73 ± 0.18 μmol/L) and slower rates of plasma disappearance but similar times to C_max. MetS participants had lower d₆–α-tocopherol AUC from t = 0–12 h (AUC_{0–t final}) in lipoprotein fractions [chylomicron, very-low-density lipoprotein (VLDL), LDL, high-density lipoprotein]. Percentages of d₆–α-tocopherol AUC_{0–t final} in both the chylomicron (r = −0.46 to −0.52) and VLDL (r = −0.49 to −0.68) fractions were inversely correlated with oxidized LDL, IL-10, IL-6, and C-reactive protein.

Conclusions: At dietary intakes equivalent to the Recommended Dietary Allowance, α-tocopherol bioavailability is unaffected by dairy fat quantity but is lower in MetS adults, potentially because of greater inflammation and oxidative stress that limits small intestinal α-tocopherol absorption and/or impairs hepatic α-tocopherol trafficking. These findings support higher dietary α-tocopherol requirements for MetS adults. This trial was registered at www.clinicaltrials.gov as NCT01787591.

 

2.225           Surface acoustic wave controlled integrated band-pass filter

Skowronek, V., Rambach, R.W. and Franke, T.

Microfluid. Nanofluid., 19(2), 335-341 (2015)

 

We introduce a microfluidic band-pass filter for particles that is fully integrated in a polydimethylsiloxane-based microchannel device. This acoustic filter allows a continuous and label-free separation of particles. To demonstrate the functionality, mixtures of particles with different sizes are exposed to propagating surface acoustic waves generated by two laterally displaced interdigitated transducers, one on each side of the microchannel. Dependent on the frequency used, a specific size or even a size range of particles can be extracted. We sort particles of sizes of ~1–10 µm and estimate the size resolution to be smaller than ∆r < 0.88 µm. We examine the performance of the device and achieve a throughput of ~10⁵ particles/s with an efficiency as high as 99 %.

 

2.226           Hitchhiking nanoparticles: Reversible coupling of lipid-based nanoparticles to cytotoxic T lymphocytes

Wayteck, L., Dewitte, H., De Backer, L., Breckpot, K., Demeester, J., De Smedt, S.C. and Raemdonck, K.

Biomaterials, 77, 143-154 (2016)

 

Following intravenous injection of anti-cancer nanomedicines, many barriers need to be overcome en route to the tumor. Cell-mediated delivery of nanoparticles (NPs) is promising in terms of overcoming several of these barriers based on the tumoritropic migratory properties of particular cell types. This guided transport aims to enhance the NP accumulation in the tumor and moreover enhance the infiltration of regions that are typically inaccessible for free NPs. Within this study, cytotoxic CD8⁺ T cells were selected as carriers based on both their ability to migrate to the tumor and their intrinsic cytolytic activity against tumor cells. Many anti-cancer nanomedicines require tumor cell internalization to mediate cytosolic drug delivery and enhance the anti-cancer effect. This proof-of-concept therefore reports on the reversible attachment of liposomes to the surface of cytotoxic T lymphocytes via a reduction sensitive coupling. The activation status of the T cells and the liposome composition are shown to strongly influence the loading efficiency. Loading the cells with liposomes does not compromise T cell functionalities like proliferation and cytolytic function. Additionally, the triggered liposome release is demonstrated upon the addition of glutathione. Based on this optimization using liposomes as model NPs, a small interfering RNA (siRNA)-loaded NP was developed that can be coupled to the surface of CD8⁺ T cells.

 

2.227           In vitro reconstitution of B cell receptor–antigen interactions to evaluate potential vaccine candidates

Weaver, G.C., Villar, R.F., Kanekiyo, M., Nabel, G.J., Mascola, J.R. and Lingwood, D.

Nature Protocols, 11(2), 193-213 (2016)

 

Predicting immune responses before vaccination is challenging because of the complexity of the governing parameters. Nevertheless, recent work has shown that B cell receptor (BCR)-antigen engagement in vitro can prove a powerful means of informing the design of antibody-based vaccines. We have developed this principle into a two-phased immunogen evaluation pipeline to rank-order vaccine candidates. In phase 1, recombinant antigens are screened for reactivity to the germline precursors that produce the antibody responses of interest. To both mimic the architecture of initial antigen engagement and facilitate rapid immunogen screening, these antibodies are expressed as membrane-anchored IgM (mIgM) in 293F indicator cells. In phase 2, the binding hits are multimerized by nanoparticle or proteoliposome display, and they are evaluated for BCR triggering in an engineered B cell line displaying the IgM sequences of interest. Key developments that complement existing methodology in this area include the following: (i) introduction of a high-throughput screening step before evaluation of more time-intensive BCR-triggering analyses; (ii) generalizable multivalent antigen-display platforms needed for BCR activation; and (iii) engineered use of a human B cell line that does not display endogenous antibody, but only ectopically expressed BCR sequences of interest. Through this pipeline, the capacity to initiate favorable antibody responses is evaluated. The entire protocol can be completed within 2.5 months.

 

2.228           Association of mitotane with chylomicrons and serum lipoproteins: practical implications for treatment of adrenocortical carcinoma

Kroiss, M., Plonne, D., Kendl, S., Schirmer, D., Ronchi, C.L., Schirbel, A., Zink, M., Lapa, C., Klinker, H., Fassnacht, M., Heinz, W and Sbiera, S.

Eur. J. Endocrinol., 174(3), 343-353 (2016)

 

Objective Oral mitotane (o,p′-DDD) is a cornerstone of medical treatment for adrenocortical carcinoma (ACC).

Aim Serum mitotane concentrations >14 mg/l are targeted for improved efficacy but not achieved in about half of patients. Here we aimed at a better understanding of intestinal absorption and lipoprotein association of mitotane and metabolites o,p′-dichlorodiphenylacetic acid (o,p′-DDA) and o,p′-dichlorodiphenyldichloroethane (o,p′-DDE).

Design Lipoproteins were isolated by ultracentrifugation from the chyle of a 29-year-old patient and serum from additional 14 ACC patients treated with mitotane. HPLC was applied for quantification of mitotane and metabolites. We assessed NCI–H295 cell viability, cortisol production, and expression of endoplasmic reticulum (ER) stress marker genes to study the functional consequences of mitotane binding to lipoproteins.

Results Chyle of the index patient contained 197 mg/ml mitotane, 53 mg/ml o,p′-DDA, and 51 mg/l o,p′-DDE. Of the total mitotane in serum, lipoprotein fractions contained 21.7±21.4% (VLDL), 1.9±0.8% (IDL), 8.9±5.5% (LDL1), 18.9±9.6% (LDL2), 10.1±4.0% (LDL3), and 26.3±13.0% (HDL2). Only 12.3±5.5% were in the lipoprotein-depleted fraction.

Discussion Mitotane content of lipoproteins directly correlated with their triglyceride and cholesterol content. O,p′-DDE was similarly distributed, but 87.9±4.2% of o,p′-DDA found in the HDL2 and lipoprotein-depleted fractions. Binding of mitotane to human lipoproteins blunted its anti-proliferative and anti-hormonal effects on NCI–H295 cells and reduced ER stress marker gene expression.

Conclusion Mitotane absorption involves chylomicron binding. High concentrations of o,p′-DDA and o,p′-DDE in chyle suggest intestinal mitotane metabolism. In serum, the majority of mitotane is bound to lipoproteins. In vitro, lipoprotein binding inhibits activity of mitotane suggesting that lipoprotein-free mitotane is the therapeutically active fraction.

 

 

2.229           Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

Rudolph, S., Klein, A.N., Tusche, M., Schlosser, C., Elfgen, A., Brener, O, Teunissen, C., Gremer, L., Funke, S.A., Kutzsche, J. and Willbold, D.

PloS One, 11(2), e0147470 (2016)

 

Alzheimer´s disease is the most prominent type of dementia and currently no causative treatment is available. According to recent studies, oligomeric species of the amyloid beta (Aβ) peptide appear to be the most toxic Aβ assemblies. Aβ monomers, however, may be not toxic per se and may even have a neuroprotective role. Here we describe a competitive mirror image phage display procedure that allowed us to identify preferentially Aβ_1–42 monomer binding and thereby stabilizing peptides, which destabilize and thereby eliminate toxic oligomer species. One of the peptides, called Mosd1 (monomer specific d-peptide 1), was characterized in more detail. Mosd1 abolished oligomers from a mixture of Aβ_1–42 species, reduced Aβ_1–42 toxicity in cell culture, and restored the physiological phenotype in neuronal cells stably transfected with the gene coding for human amyloid precursor protein.

 

2.230           Simvastatin Efficiently Lowers Small LDL-IgG Immune Complex Levels: A Therapeutic Quality beyond the Lipid-Lowering Effect

Hörl, G., Froehlich, H., Fersti, U., ledinski, G., Binder, J., Cvirn, G., Stojakovic, T., Trauner, M., Koidl, C., Tafeit, E., Amrein, K., Scharnagi, H., Jürgens, G. and Hallström, S.

PloS One, 11(2), e0148210 (2016)

 

We investigated a polyethylene glycol non-precipitable low-density lipoprotein (LDL) subfraction targeted by IgG and the influence of statin therapy on plasma levels of these small LDL-IgG-immune complexes (LDL-IgG-IC). LDL-subfractions were isolated from 6 atherosclerotic subjects and 3 healthy individuals utilizing iodixanol density gradient ultracentrifugation. Cholesterol, apoB and malondialdehyde (MDA) levels were determined in each fraction by enzymatic testing, dissociation-enhanced lanthanide fluorescence immunoassay and high-performance liquid chromatography, respectively. The levels of LDL-IgG-IC were quantified densitometrically following lipid electrophoresis, particle size distribution was assessed with dynamic light scattering and size exclusion chromatography. The influence of simvastatin (40 mg/day for three months) on small LDL-IgG-IC levels and their distribution among LDL-subfractions (salt gradient separation) were investigated in 11 patients with confirmed coronary artery disease (CAD). We demonstrate that the investigated LDL-IgG-IC are small particles present in atherosclerotic patients and healthy subjects. In vitro assembly of LDL-IgG-IC resulted in particle density shifts indicating a composition of one single molecule of IgG per LDL particle. Normalization on cholesterol levels revealed MDA values twice as high for LDL-subfractions rich in small LDL-IgG-IC if compared to dominant LDL-subfractions. Reactivity of affinity purified small LDL-IgG-IC to monoclonal antibody OB/04 indicates a high degree of modified apoB and oxidative modification. Simvastatin therapy studied in the CAD patients significantly lowered LDL levels and to an even higher extent, small LDL-IgG-IC levels without affecting their distribution. In conclusion simvastatin lowers levels of small LDL-IgG-IC more effectively than LDL-cholesterol and LDL-apoB levels in atherosclerotic patients. This antiatherogenic effect may additionally contribute to the known beneficial effects of this drug in the treatment of atherosclerosis.

 

2.231           Scalable Isolation of Mammalian Mitochondria for Nucleic Acid and Nucleoid Analysis

Lee, K-W. and Bogenhagen, D.F.

Methods in Mol. Biol., 1351, 67-79 (2016)

 

Isolation of mitochondria from cultured cells and animal tissues for analysis of nucleic acids and bona fide mitochondrial nucleic acid binding proteins and enzymes is complicated by contamination with cellular nucleic acids and their adherent proteins. Protocols presented here allow for quick isolation of mitochondria from a small number of cells and for preparation of highly purified mitochondria from a larger number of cells using nuclease treatment and high salt washing of mitochondria to reduce contamination. We further describe a method for the isolation of mitochondrial DNA–protein complexes known as nucleoids from these highly purified mitochondria using a combination of glycerol gradient sedimentation followed by isopycnic centrifugation in a non-ionic iodixanol gradient.

 

 

 

2.232           Immunogenicity of Leishmania-derived hepatitis B small surface antigen particles exposing highly conserved E2 epitope of hepatitis C virus

Czarnota, A., Tyborowska, J., Peszynska-Sularz, G., Growatzka, B., Bienkowska-Szewczyk, K. and Grzyb, K.

Microb. Cell Fact., 15:62 (2016)

 

Background

Hepatitis C virus (HCV) infection is a major health problem worldwide, affecting an estimated 2–3 % of human population. An HCV vaccine, however, remains unavailable. High viral diversity poses a challenge in developing a vaccine capable of eliciting a broad neutralizing antibody response against all HCV genotypes. The small surface antigen (sHBsAg) of hepatitis B virus (HBV) has the ability to form highly immunogenic subviral particles which are currently used as an efficient anti-HBV vaccine. It also represents an attractive antigen carrier for the delivery of foreign sequences. In the present study, we propose a bivalent vaccine candidate based on novel chimeric particles in which highly conserved epitope of HCV E2 glycoprotein (residues 412–425) was inserted into the hydrophilic loop of sHBsAg.

Results

The expression of chimeric protein was performed in an unconventional, Leishmania tarentolae expression system resulting in an assembly of particles which retained immunogenicity of both HCV epitope and sHBsAg protein. Direct transmission electron microscopy observation and immunogold staining confirmed the formation of spherical particles approximately 22 nm in diameter, and proper foreign epitope exposition. Furthermore, the sera of mice immunized with chimeric particles proved reactive not only to purified yeast-derived sHBsAg proteins but also HCV E2 412–425 synthetic peptide. Most importantly, they were also able to cross-react with E1E2 complexes from different HCV genotypes.

Conclusions

For the first time, we confirmed successful assembly of chimeric sHBsAg virus-like particles (VLPs) in the L. tarentolae expression system which has the potential to produce high-yields of properly N-glycosylated mammalian proteins. We also proved that chimeric Leishmania-derived VLPs are highly immunogenic and able to elicit cross-reactive antibody response against HCV. This approach may prove useful in the development of a bivalent prophylactic vaccine against HBV and HCV and opens up a new and low-cost opportunity for the production of chimeric sHBsAg VLPs requiring N-glycosylation process for their proper functionality and immunogenicity.

 

2.233           Hepatitis C Virus-Induced Degradation of Cell Death-Inducing DFFA-Like Effector B Leads to Hepatic Lipid Dysregulation

Lee, E.M., Alsagheir, A., Wu, X., Hammack, C., Mclaughlan, J., Watanabe, N., Wakita, T., Kneteman, N.M., Douglas, D.N. and Tang, H.

1. Virol., 90(8), 4174-4185 (2016)

 

Individuals chronically infected with hepatitis C virus (HCV) commonly exhibit hepatic intracellular lipid accumulation, termed steatosis. HCV infection perturbs host lipid metabolism through both cellular and virus-induced mechanisms, with the viral core protein playing an important role in steatosis development. We have recently identified a liver protein, the cell death-inducing DFFA-like effector B (CIDEB), as an HCV entry host dependence factor that is downregulated by HCV infection in a cell culture model. In this study, we investigated the biological significance and molecular mechanism of this downregulation. HCV infection in a mouse model downregulated CIDEB in the liver tissue, and knockout of the CIDEB gene in a hepatoma cell line results in multiple aspects of lipid dysregulation that can contribute to hepatic steatosis, including reduced triglyceride secretion, lower lipidation of very-low-density lipoproteins, and increased lipid droplet (LD) stability. The potential link between CIDEB downregulation and steatosis is further supported by the requirement of the HCV core and its LD localization for CIDEB downregulation, which utilize a proteolytic cleavage event that is independent of the cellular proteasomal degradation of CIDEB.

 

2.234           Increased Presence of Remnant Lipoprotein Cholesterol in The Hdl of Diabetic Subjects

Gonzalez, M., Heras, M., Rosales, R., Guardiola, M., Plana, N., Vallve, J.C., Masana, L. and Ribalta, J.

Ann. Clin. Lab. Sci., 46(2), 229 (2016)

 

The atherogenic dyslipidemia associated with type II diabetes (T2DM) is characterized by elevated fasting triglycerides (TG) and remnant lipoproteins (RL) as well as small and dense low-density lipoprotein (sdLDL) particles and low high-density lipoprotein cholesterol (HDLc) levels [1]. Epidemiological studies have demonstrated that both fasting and non-fasting hyperlipidemia are important risk factors for atherosclerosis and cardiovascular events [2]. In the non-fasting state, the lipid profile is characterized by the accumulation of RL, a heterogeneous group of catabolized hepatic and intestinal lipoproteins, which differ in size, density, electrophoretic mobility, composition, and receptor affinity [3]. RL are considered an independent risk factor in cardiovascular disease [4–6]. The analytical determination of RL is not straightforward. Measurements of circulating apolipoprotein (apo) B48 estimate postprandial remnant lipoproteins [7]. On the other hand, the remnant-like particle cholesterol (RLPc) fraction obtained by immunchromatography identifies a wider spectrum of lipoproteins, ranging from VLDL to HDL, that cannot effectively bind to antibodies against apoA1 and apoB100 [8]. The RLPs density range varies according to the population studied; for instance, in normolipidemic subjects, RLPc is normally undetectable in the IDL fraction [5]. However, in diabetic or dysbetalipoproteinemic patients, the IDL can be found in the RLPc fraction [9].

The aim of this study is two-fold: 1) To analyze which lipoproteins have RLPs and how this varies between normolipidemic and T2DM patients; 2) To test how this distribution varies postprandially in normolipidemic subjects with iodixanol gradient ultracentrifugation, a method that allows for more accurate subfraction separation permitting the quantification of up to 21 lipoprotein subclasses [10].

 

2.235           Optimization of the All-D Peptide D3 for Aβ Oligomer Elimination

Klein, A.-N., Ziehm, T., Tusche, M., Buitenhuis, J., bartnik, D., Boeddrich, A., Wiglenda, T., Wanker, E., Funke, S.A., Brener, O., Gremer, L., Kutzsche, J. and Willbold, D.

PloS One, 11(4), e0153035 (2016)

 

The aggregation of amyloid-β (Aβ) is postulated to be the crucial event in Alzheimer’s disease (AD). In particular, small neurotoxic Aβ oligomers are considered to be responsible for the development and progression of AD. Therefore, elimination of thesis oligomers represents a potential causal therapy of AD. Starting from the well-characterized d-enantiomeric peptide D3, we identified D3 derivatives that bind monomeric Aβ. The underlying hypothesis is that ligands bind monomeric Aβ and stabilize these species within the various equilibria with Aβ assemblies, leading ultimately to the elimination of Aβ oligomers. One of the hereby identified d-peptides, DB3, and a head-to-tail tandem of DB3, DB3DB3, were studied in detail. Both peptides were found to: (i) inhibit the formation of Thioflavin T-positive fibrils; (ii) bind to Aβ monomers with micromolar affinities; (iii) eliminate Aβ oligomers; (iv) reduce Aβ-induced cytotoxicity; and (v) disassemble preformed Aβ aggregates. The beneficial effects of DB3 were improved by DB3DB3, which showed highly enhanced efficacy. Our approach yielded Aβ monomer-stabilizing ligands that can be investigated as a suitable therapeutic strategy against AD.

 

2.236           A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion

Xu, W., Nathwani, B., Lin, C., Wang, J., karatekin, E., Pincet, F., Shih, W. and Rothman, J.E.

J. Am. Chem. Soc., 138(13), 4439-4447 (2016)

 

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes are the core molecular machinery of membrane fusion, a fundamental process that drives inter- and intracellular communication and trafficking. One of the questions that remains controversial has been whether and how SNAREs cooperate. Here we show the use of self-assembled DNA-nanostructure rings to template uniform-sized small unilamellar vesicles containing predetermined maximal number of externally facing SNAREs to study the membrane-fusion process. We also incorporated lipid-conjugated complementary ssDNA as tethers into vesicle and target membranes, which enabled bypass of the rate-limiting docking step of fusion reactions and allowed direct observation of individual membrane-fusion events at SNARE densities as low as one pair per vesicle. With this platform, we confirmed at the single event level that, after docking of the templated-SUVs to supported lipid bilayers (SBL), one to two pairs of SNAREs are sufficient to drive fast lipid mixing. Modularity and programmability of this platform makes it readily amenable to studying more complicated systems where auxiliary proteins are involved.

 

2.237           Ex vivo mammalian prions are formed of paired double helical prion protein fibrils

Terry, C., Wenborn, A., Gros, N., Sells, J., Joiner, S., Hosszu, L.P., tattum, M.H., Panico, S., Clare, D.K., Collinge, J., Saibil, H.R. and Wadsworth, J.D.F.

Open Biology, 6, 160035 (2016)

 

Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity and the three-dimensional structure of infectious prions has remained obscure. Recently, we developed novel methods to obtain exceptionally pure preparations of prions from mouse brain and showed that pathogenic PrP in these high-titre preparations is assembled into rod-like assemblies. Here, we have used precise cell culture-based prion infectivity assays to define the physical relationship between the PrP rods and prion infectivity and have used electron tomography to define their architecture. We show that infectious PrP rods isolated from multiple prion strains have a common hierarchical assembly comprising twisted pairs of short fibres with repeating substructure. The architecture of the PrP rods provides a new structural basis for understanding prion infectivity and can explain the inability to systematically generate high-titre synthetic prions from recombinant PrP.

 

2.238           Isolation of high density lipoproteins in ovine follicular and oviductal fluid

Bernecic, N.C., Gadella, B.M., de Graaf, S.P. and Leahy, T.

Animal Reproduction Science, 169, 121.122 (2016)

 

Capacitation is a key maturation process in spermatozoa that is vital for successful fertilisation. Following the initiation of this process, cholesterol is lost from the sperm plasma membrane via a series of regulated mechanisms which then permits appropriate sperm binding to the zona pellucida and the induction of the acrosome reaction. High density lipoproteins (HDL) have been shown to play a key role in cholesterol efflux in somatic cells but their physiological role in sperm cholesterol efflux is uncertain. The objective of this study was to isolate and identify HDL in ovine follicular and oviductal fluid for potential use in media that supports ram sperm capacitation in vitro.

Density gradients were generated by diluting Iodixanol (OptiPrep; Sigma Aldrich, Australia) to 20% (v/v) with follicular or oviductal fluid and then layering this mixture underneath equal volumes of 6% and 12.5% iodixanol in PBS (v/v). Samples were centrifuged (27,7320 × g at 16 °C for 5 h) and the resulting gradients were harvested into 10 fractions by aspiration. Proteins from each fraction were separated based on molecular weight using 1D SDS-PAGE. Liquid Chromatography Mass Spectrometry (LC–MS) was used to detect the presence of the major HDL apolipoprotein, apoA-I, in the relevant molecular weight band on the gels.

This proteomic approach confirmed the presence of apoA-I in fractions 7–10 of follicular fluid and fractions 8–10 of oviductal fluid. This indicates that HDLs in ovine follicular and oviductal fluid can be isolated using iodixanol and density ultracentrifugation. The establishment of this protocol in sheep provides an opportunity to investigate the function of HDLs as potential cholesterol acceptors from ram spermatozoa during in vitro capacitation, which will be the focus of future studies.

 

2.239           Self-assembly of size-controlled liposomes on DNA nanotemplates

Yang, Y., Wang, J., Shigematsu, H., Xu, W., Shih, W.M., Rothman, J.E. and Lin, C.

Nature Chem., 8(5), 476-483 (2016)

 

Artificial lipid-bilayer membranes are valuable tools for the study of membrane structure and dynamics. For applications such as the study of vesicular transport and drug delivery, there is a pressing need for artificial vesicles with controlled size. However, controlling vesicle size and shape with nanometre precision is challenging, and approaches to achieve this can be heavily affected by lipid composition. Here, we present a bio-inspired templating method to generate highly monodispersed sub-100-nm unilamellar vesicles, where liposome self-assembly was nucleated and confined inside rigid DNA nanotemplates. Using this method, we produce homogeneous liposomes with four distinct predefined sizes. We also show that the method can be used with a variety of lipid compositions and probe the mechanism of templated liposome formation by capturing key intermediates during membrane self-assembly. The DNA nanotemplating strategy represents a conceptually novel way to guide lipid bilayer formation and could be generalized to engineer complex membrane/protein structures with nanoscale precision.

 

2.240           PCSK9 Association With Lipoprotein(a)

Tavori, H., Christian, D., Minnier, J., Plubell, D., Shapiro, M.D., Yeang, C., Giunzioni, I., Croyal, M., Duell, P.B., Lambert, G., Tsimikas, S. and Fazio, S.

Circ. Res., 119, 29-35 (2016)

 

Rationale: Lipoprotein(a) [Lp(a)] is a highly atherogenic low-density lipoprotein–like particle characterized by the presence of apoprotein(a) [apo(a)] bound to apolipoprotein B. Proprotein convertase subtilisin/kexin type 9 (PCSK9) selectively binds low-density lipoprotein; we hypothesized that it can also be associated with Lp(a) in plasma.

Objective: Characterize the association of PCSK9 and Lp(a) in 39 subjects with high Lp(a) levels (range 39–320 mg/dL) and in transgenic mice expressing either human apo(a) only or human Lp(a) (via coexpression of human apo(a) and human apolipoprotein B).

Methods and Results: We show that PCSK9 is physically associated with Lp(a) in vivo using 3 different approaches: (1) analysis of Lp(a) fractions isolated by ultracentrifugation; (2) immunoprecipitation of plasma using antibodies to PCSK9 and immunodetection of apo(a); (3) ELISA quantification of Lp(a)-associated PCSK9. Plasma PCSK9 levels correlated with Lp(a) levels, but not with the number of kringle IV-2 repeats. PCSK9 did not bind to apo(a) only, and the association of PCSK9 with Lp(a) was not affected by the loss of the apo(a) region responsible for binding oxidized phospholipids. Preferential association of PCSK9 with Lp(a) versus low-density lipoprotein (1.7-fold increase) was seen in subjects with high Lp(a) and normal low-density lipoprotein. Finally, Lp(a)-associated PCSK9 levels directly correlated with plasma Lp(a) levels but not with total plasma PCSK9 levels.

Conclusions: Our results show, for the first time, that plasma PCSK9 is found in association with Lp(a) particles in humans with high Lp(a) levels and in mice carrying human Lp(a). Lp(a)-bound PCSK9 may be pursued as a biomarker for cardiovascular risk.

 

2.241           TREM2 Binds to Apolipoproteins, Including APOE and CLU/APOJ, and Thereby Facilitates Uptake of Amyloid-Beta by Microglia

Yeh, F.L., Wang, Y., Tom, I., Gonzales, L.C. and Sheng, M.

Neuron, 91, 328-340 (2016)

 

Genetic variants of TREM2, a protein expressed selectively by microglia in the brain, are associated with Alzheimer’s disease (AD). Starting from an unbiased protein microarray screen, we identified a set of lipoprotein particles (including LDL) and apolipoproteins (including CLU/APOJ and APOE) as ligands of TREM2. Binding of these ligands by TREM2 was abolished or reduced by disease-associated mutations. Overexpression of wild-type TREM2 was sufficient to enhance uptake of LDL, CLU, and APOE in heterologous cells, whereas TREM2 disease variants were impaired in this activity. Trem2 knockout microglia showed reduced internalization of LDL and CLU. β-amyloid (Aβ) binds to lipoproteins and this complex is efficiently taken up by microglia in a TREM2-dependent fashion. Uptake of Aβ-lipoprotein complexes was reduced in macrophages from human subjects carrying a TREM2 AD variant. These data link three genetic risk factors for AD and reveal a possible mechanism by which mutant TREM2 increases risk of AD.

 

2.242           Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

Fogeron, M-L. et al

1. Biomol. NMR, 65(2), 87-98 (2016)

 

We describe the expression of the hepatitis C virus nonstructural protein 4B (NS4B), which is an integral membrane protein, in a wheat germ cell-free system, the subsequent purification and characterization of NS4B and its insertion into proteoliposomes in amounts sufficient for multidimensional solid-state NMR spectroscopy. First spectra of the isotopically [²H,¹³C,¹⁵N]-labeled protein are shown to yield narrow ¹³C resonance lines and a proper, predominantly α-helical fold. Clean residue-selective leucine, isoleucine and threonine-labeling is demonstrated. These results evidence the suitability of the wheat germ-produced integral membrane protein NS4B for solid-state NMR. Still, the proton linewidth under fast magic angle spinning is broader than expected for a perfect sample and possible causes are discussed.

 

2.243           Eicosapentaenoic Acid Inhibits Oxidation of ApoB-containing Lipoprotein Particles of Different Size In Vitro When Administered Alone or in Combination With Atorvastatin Active Metabolit Compared With Other Triglyceride-lowering Agents

Mason, R.P., Sherratt, S.C.R. and Jacob, R.F.

1. Cardiovasc. Pharmacol., 68(1), 33-40 (2016)

 

Eicosapentaenoic acid (EPA) is a triglyceride-lowering agent that reduces circulating levels of the apolipoprotein B (apoB)-containing lipoprotein particles small dense low-density lipoprotein (sdLDL), very–low-density lipoprotein (VLDL), and oxidized low-density lipoprotein (LDL). These benefits may result from the direct antioxidant effects of EPA. To investigate this potential mechanism, these particles were isolated from human plasma, preincubated with EPA in the absence or presence of atorvastatin (active) metabolite, and subjected to copper-initiated oxidation. Lipid oxidation was measured as a function of thiobarbituric acid reactive substances formation. EPA inhibited sdLDL (IC50 ∼2.0 μM) and LDL oxidation (IC50 ∼2.5 μM) in a dose-dependent manner. Greater antioxidant potency was observed for EPA in VLDL. EPA inhibition was enhanced when combined with atorvastatin metabolite at low equimolar concentrations. Other triglyceride-lowering agents (fenofibrate, niacin, and gemfibrozil) and vitamin E did not significantly affect sdLDL, LDL, or VLDL oxidation compared with vehicle-treated controls. Docosahexaenoic acid was also found to inhibit oxidation in these particles but over a shorter time period than EPA. These data support recent clinical findings and suggest that EPA has direct antioxidant benefits in various apoB-containing subfractions that are more pronounced than those of other triglyceride-lowering agents and docosahexaenoic acid.

 

2.244           Increase of Positive Net Charge and Conformational Rigidity Enhances the Efficacy of d-Enantiomeric Peptides Designed to Eliminate Cytotoxic Aβ Species

Ziehm, T., Brener, O., van Groen, T., Kadish, I., Frenzel, D., Tusche, M., Kutzsche, J., Reiss, K., Gremer, L., Nagel-Steger, L. and Willbold, D.

ACS Chem. Neurosci., 7(8), 1088-1096 (2016)

 

Alzheimer’s disease (AD) is a neurodegenerative disorder and the most common type of dementia. Until now, there is no curative therapy available. Previously, we selected the amyloid-beta (Aβ) targeting peptide D3 consisting of 12 d-enantiomeric amino acid residues by mirror image phage display as a potential drug candidate for the treatment of AD. In the current approach, we investigated the optimization potential of linear D3 with free C-terminus (D3_COOH) by chemical modifications. First, the impact of the net charge was investigated and second, cyclization was introduced which is a well-known tool for the optimization of peptides for enhanced target affinity. Following this strategy, three D3 derivatives in addition to D3_COOH were designed: C-terminally amidated linear D3 (D3_CONH2), cyclic D3 (cD3), and cyclic D3 with an additional arginine residue (cD3r) to maintain the net charge of linear D3_CONH2. These four compounds were compared to each other according to their binding affinities to Aβ(1–42), their efficacy to eliminate cytotoxic oligomers, and consequently their potency to neutralize Aβ(1–42) oligomer induced neurotoxicity. D3_CONH2 and cD3r versions with equally increased net charge showed superior properties over D3_COOH and cD3, respectively. The cyclic versions showed superior properties compared to their linear version with equal net charge, suggesting cD3r to be the most efficient compound among these four. Indeed, treatment of the transgenic AD mouse model Tg-SwDI with cD3r significantly enhanced spatial memory and cognition of these animals as revealed by water maze performance. Therefore, charge increase and cyclization imply suitable modification steps for an optimization approach of the Aβ targeting compound D3.

 

2.245           14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells

Li, T. and Paudel, H.K.

PloS One, 11(8), e0160635 (2016)

 

Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer’s disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445–6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau

 

2.246           Hepatitis C virus (HCV) and atherosclerosis risk: A role for low-density immune complexes?

Bassendine, M., Nielsen, S. and Neely, D.

Atherosclerosis, 252, e206 (2016)

 

Objectives: HCV is associated with an increased risk of atherosclerosis, despite inducing a favourable lipid profile with accompanying low classical risk score. HCV lipo-viral particles and sub-viral particles are found in the low–density fraction (LDF) of blood (<1.08 g/ml) associated with apoB-containing lipoproteins. The aim of this study was to examine the proteome of LDF in chronic HCV, compared to non-HCV control.

Methods: Proteins in iodixanol fractions of blood were separated on SDS PAGE; 14 bands excised from the gel were analysed by MALDI TOF. Peptides generated by trypsin digestion were analysed by mass spectrometry. Protein bands were identified using the peptide mass fingerprint data and the Mascot search engine program, searched against NCBI protein.

Results: Several protein bands were detected in the LDF only in HCV patients. After identification of protein bands by mass spectrometry, lanes were scanned to produce an intensity profile. Peaks corresponding to proteins identified by mass spectrometry were labelled indicating that four proteins, IgM heavy chain, IgG3 heavy chain, IgG1 heavy chain and Kappa light chain were present only in LDF purified from HCV patients.

 

2.247           Mechanisms of selective delivery of xanthophylls to retinal pigment epithelial cells by human lipoproteins

Thomas, S.E. and Harrison, E.H.

1. Lipid Res., 57, 1865-1878 (2016)

 

The xanthophylls, lutein and zeaxanthin, are dietary carotenoids that selectively accumulate in the macula of the eye providing protection against age-related macular degeneration. To reach the macula, carotenoids cross the retinal pigment epithelium (RPE). Xanthophylls and β-carotene mostly associate with HDL and LDL, respectively. HDL binds to cells via a scavenger receptor class B1 (SR-B1)-dependent mechanism, while LDL binds via the LDL receptor. Using an in-vitro, human RPE cell model (ARPE-19), we studied the mechanisms of carotenoid uptake into the RPE by evaluating kinetics of cell uptake when delivered in serum or isolated LDL or HDL. For lutein and β-carotene, LDL delivery resulted in the highest rates and extents of uptake. In contrast, HDL was more effective in delivering zeaxanthin and meso-zeaxanthin leading to the highest rates and extents of uptake of all four carotenoids. Inhibitors of SR-B1 suppressed zeaxanthin delivery via HDL. Results show a selective HDL-mediated uptake of zeaxanthin and meso-zeaxanthin via SR-B1 and a LDL-mediated uptake of lutein. This demonstrates a plausible mechanism for the selective accumulation of zeaxanthin greater than lutein and xanthophylls over β-carotene in the retina. We found no evidence of xanthophyll metabolism to apocarotenoids or lutein conversion to meso-zeaxanthin.

 

2.248           Characterizing the Effect of Multivalent Conjugates Composed of Aβ-Specific Ligands and Metal Nanoparticles on Neurotoxic Fibrillar Aggregation

Streich, C., Akkari, L., Decker, C., Bormann., J., Rehbock, C., Müller-Sciffmann, A., Carlsson Niemayer, F., Nagel-Steger, L., Wilbold, d., Sacca, B., Korth, C., Schrader, T. and Barcikowski, S.

ACS Nano, 10(8), 7582-7597 (2016)

 

Therapeutically active small molecules represent promising nonimmunogenic alternatives to antibodies for specifically targeting disease-relevant receptors. However, a potential drawback compared to antibody–antigen interactions may be the lower affinity of small molecules toward receptors. Here, we overcome this low-affinity problem by coating the surface of nanoparticles (NPs) with multiple ligands. Specifically, we explored the use of gold and platinum nanoparticles to increase the binding affinity of Aβ-specific small molecules to inhibit Aβ peptide aggregation into fibrils in vitro. The interactions of bare NPs, free ligands, and NP-bound ligands with Aβ are comprehensively studied via physicochemical methods (spectroscopy, microscopy, immunologic tests) and cell assays. Reduction of thioflavin T fluorescence, as an indicator for β-sheet content, and inhibition of cellular Aβ excretion are even more effective with NP-bound ligands than with the free ligands. The results from this study may have implications in the development of therapeutics for treating Alzheimer’s disease.

 

2.249           Size Determination of a Liposomal Drug by Small-Angle X-ray Scattering Using Continuous Contrast Variation

Garcia-Diez, R., Gollwitzer, C., Krumrey, M. and Varga, Z.

Langmuir, 32(3), 772-778 (2016)

 

The continuously growing complexity of nanodrugs urges for complementary characterization techniques which can elude the current limitations. In this paper, the applicability of continuous contrast variation in small-angle X-ray scattering (SAXS) for the accurate size determination of a complex nanocarrier is demonstrated on the example of PEGylated liposomal doxorubicin (Caelyx). The mean size and average electron density of Caelyx was determined by SAXS using a gradient of aqueous iodixanol (Optiprep), an iso-osmolar suspending medium. The study is focused on the isoscattering point position and the analysis of the Guinier region of the scattering curves recorded at different solvent densities. An average diameter of (69 ± 5) nm and electron density of (346.2 ± 1.2) nm^–3 were determined for the liposomal formulation of doxorubicin. The response of the liposomal nanocarrier to increasing solvent osmolality and the structure of the liposome-encapsulated doxorubicin after the osmotic shrinkage of the liposome are evaluated with sucrose contrast variation in SAXS and wide-angle X-ray scattering (WAXS). In the case of using sucrose as contrast agent, a clear osmolality threshold at 670 mOsm kg^–1 was observed, above which the liposomal drug carriers start to shrink, though preserving the intraliposomal doxorubicin structure. The average size obtained by this technique is smaller than the value measured by dynamic light scattering (DLS), though this difference is expected due to the hydrodynamic size of the PEG moieties attached to the liposomal surface, which are not probed with solvent contrast variation in SAXS. The advantages and drawbacks of the proposed technique are discussed in comparison to DLS, the most frequently used sizing method in nanomedicine.

 

2.250           High-Affinity Binding of Monomeric but Not Oligomeric Amyloid-β to Ganglioside GM1 Containing Nanodiscs

Thomaier, M., Gremer, L., Dammers, C., Fabig, J., Neudecker, P. and Willbold, D.

Biochemistry, 55(48), 6662-6672 (2016)

 

The interaction of the amyloid-β protein (Aβ) with neuronal cell membranes plays a crucial role in Alzheimer’s disease. Aβ undergoes structural changes upon binding to ganglioside GM1 containing membranes leading to altered molecular characteristics of the protein. The physiological role of the Aβ interaction with the ganglioside GM1 is still unclear. In order to further elucidate the molecular requirements of Aβ membrane binding, we tested different nanodiscs varying in their lipid composition, regarding the charge of the headgroups as well as ganglioside GM1 concentration. Nanodiscs are excellent model membrane systems for studying protein membrane interactions, and we show here their suitability to investigate the membrane interaction of Aβ. In particular, we set out to investigate whether the binding activity of GM1 to Aβ is specific for the assembly state of Aβ and compared the binding affinities of monomeric with oligomeric Aβ. Using fluorescence titration experiments, we demonstrate high-affinity binding of Aβ(1–40) to GM1 containing nanodiscs, with dissociation constants, K_D, in the range from 25 to 41 nM, in a GM1 concentration-dependent manner. Biolayer interferometry experiments confirmed the high-affinity binding of monomeric Aβ(1–40) (K_D of 24 nM to 49 nM) as well as of Aβ(1–42) (K_D of 30 nM) to GM1 containing nanodiscs, and no binding to phospholipid containing nanodiscs. Interestingly, and in contrast to monomeric Aβ, neither oligomeric Aβ(1–40) nor oligomeric Aβ(1–42) binds to GM1 nanodiscs. To the best of our knowledge, this is the first report of a loss of function for monomeric Aβ upon aggregation.

 

2.251           Plasma transport of ergocalciferol and cholecalciferol and their 25-hydroxylated metabolites in dairy cows

Hymøller, L. and Jensen, S.K.

Domestic Animal Endocrinology, 59, 44-52 (2017)

 

In cattle, there are 2 significant forms of vitamin D: ergocalciferol (ERG) from fungi on roughage and cholecalciferol (CHO) from vitamin supplements or endogenous synthesis in the skin. The hypothesis of the present study is that vitamin D from the 3 sources is transported in different plasma fractions in the body. This is hypothesized to explain the lower efficiency of ERG compared to CHO in securing a sufficient plasma status of 25-hydroxyvitamin D and explain the inefficient excretion of dietary CHO into milk compared to endogenous CHO. Twenty vitamin D–depleted cows were assigned to 5 treatments: D2, housed indoor and fed 625-μg/d (25.000 IU) ERG; D3, housed indoor and fed 625-μg/d CHO; D2+D3, housed indoor and fed 625-μg/d ERG and 625-μg/d CHO; SUN, let out for daily pasture to facilitate CHO synthesis from sunlight; and D2+SUN, fed 625-μg/d ERG and let out for daily pasture. Blood samples were taken twice weekly and plasma fractionated by ultracentrifugation into 3 fractions: light lipoprotein (LLP), heavy lipoprotein (HLP), and protein and analyzed for content of ERG and CHO and their liver derived metabolites 25-hydroxyergocalciferol (25ERG) and 25-hydroxycholecalciferol (25CHO), respectively. Liver biopsies were taken on the last day of the study to asses gene expression related to vitamin D metabolism. During 4 wk of study, the vitamin D status in plasma increased to 19.3 to 22.8 ng/mL 25ERG in ERG-treated cows with the highest concentration in D2 (P ≤ 0.05) and to 25.0 to 33.4 ng/mL 25CHO in pasture or CHO-treated cows with the highest concentration in SUN (P ≤ 0.01). In plasma fractions, CHO was mainly found in the HLP fraction, whereas 25CHO was almost exclusively found in the protein fraction, probably due to its reported high binding affinity to vitamin D–binding protein. About 70% to 90% of 25ERG was found in the protein fraction and the remaining 25ERG was found in HLP, whereas ERG was found in both HLP and LLP fractions. In liver tissue, the expression of vitamin D-25-hydroxylase was lower in D2+D3 (P ≤ 0.05) and SUN (P ≤ 0.05) than that in the remaining groups, and the vitamin D receptor was expressed in the liver to a larger extent in D2+SUN than that in D2+D3 (P ≤ 0.05) and SUN (P ≤ 0.05). In conclusion, different plasma transport mechanisms may explain the lower physiological efficiency of ERG compared to CHO in securing the vitamin D status in plasma but do not explain the lower efficiency of synthetic CHO compared to endogenous CHO from sunlight or UV light in securing a high CHO content in milk.

 

2.252           Omega-3 fatty acid fish oil dietary supplements contain saturated fats and oxidized lipids that may interfere with their intended biological benefits

Preston Mason, R. and Sherratt, A.C.R.

Biochem. Biophys. Res. Comm., 483, 425-429 (2017)

 

Widely available fish oil dietary supplements (DS) may contain fats and oxidized lipids in addition to the beneficial omega-3 fatty acids (OM3FAs) for which they are purchased. Little is known about the potential biological effects of these oxidized lipids. The objective of this study was to assess the fatty acid content, oxidation products, and biological effects of leading fish oil DS available in the United States. Three top-selling fish oil DS in the US were included in this analysis. Fatty acid composition was measured using gas chromatography. Lipid oxidation (primary and secondary products) was measured by spectroscopy in both DS and a prescription OM3FA product. OM3FAs were also isolated and concentrated from DS and were tested for the ability to inhibit copper-induced oxidation of human small dense low-density lipoprotein particles (sdLDL) in vitro. Fish oil DS were found to contain more than 30 different fatty acids, including 10 to 14 different saturated species comprising up to 36% of the total fatty acid content. Levels of OM3FAs also varied widely among DS (33%–79%). Primary (peroxide), secondary (anisidine), and total oxidation products exceeded maximum levels established by international standards of quality in the DS but not the prescription OM3FA product. Oxidation of sdLDL was inhibited by >95% (P < 0.001) with non-oxidized forms of OM3FA but not with OM3FAs isolated from DS, which were a mixture of oxidized and non-oxidized OM3FAs. These data indicate that levels of saturated fat and oxidized OM3FAs found in common DS may interfere with their intended/potential biological benefits.

 

2.253           Controlling the gastrointestinal fate of nutraceutical and pharmaceutical-enriched lipid nanoparticles: From mixed micelles to chylomicrons

Yao, M., McClements, D.J., Zhao, F., Craig, R.W. and Xiao, H.

NanoImpact, 5(1), 13-21 (2017)

 

The oral bioavailability of lipophilic bioactive compounds such as many pharmaceuticals and nutraceuticals can be enhanced using triacylglycerol-based lipid nanoparticle delivery systems. These digestible lipid nanoparticles are dissembled in the gastrointestinal tract to form mixed micelles that solubilize and transport the lipophilic bioactives to the intestinal epithelium cells where they are absorbed. In these cells, the lipid digestion products and bioactive agents contained within the mixed micelles are then packaged into biological lipid protein nanoparticles (e.g., chylomicrons) that are secreted into the lymph. In this study, we examined the influence of fatty acid type (i.e., oleic acid, linoleic acid, and linolenic acid) on the properties of mixed micelles, cellular lipid droplets, and lipoprotein nanoparticles, and on the bioavailability of a highly lipophilic nutraceutical: 5-demethylnobiletin (5DN). There were distinct differences in the structural properties of lipoprotein nanoparticles formed depending on fatty acid unsaturation. Oleic acid (C_18:1) was most effective in enhancing intestinal uptake of 5DN and led to the formation of the largest chylomicrons. Linoleic acid (C_18:2) and linolenic acid (C_18:3) also promoted intestinal uptake of 5DN and formation of chylomicrons, but they were less efficient than oleic acid. The metabolism of 5DN within the intestinal epithelium cells was greatly reduced when 5DN was incorporated into chylomicrons, presumably because they were isolated from metabolic enzymes in the cytoplasm. These results have important implications for the rational design of lipid nanoparticle-based delivery systems for lipophilic nutraceuticals and pharmaceuticals by targeting them to the lymphatic circulation.

 

2.254           Anionic Phospholipids and the Albino3 Translocase Activate Signal Recognition Particle-Receptor Interaction during Light-harvesting Chlorophyll a/b-binding Protein Targeting

Chandrasekar, S. and Shan, S-o.

1. Biol. Chem., 292(1), 397-406 (2017)

 

The universally conserved signal recognition particle (SRP) co-translationally delivers newly synthesized membrane and secretory proteins to the target cellular membrane. The only exception is found in the chloroplast of green plants, where the chloroplast SRP (cpSRP) post-translationally targets light-harvesting chlorophyll a/b-binding proteins (LHCP) to the thylakoid membrane. The mechanism and regulation of this post-translational mode of targeting by cpSRP remain unclear. Using biochemical and biophysical methods, here we show that anionic phospholipids activate the cpSRP receptor cpFtsY to promote rapid and stable cpSRP54·cpFtsY complex assembly. Furthermore, the stromal domain of the Alb3 translocase binds with high affinity to and regulates GTP hydrolysis in the cpSRP54·cpFtsY complex, suggesting that cpFtsY is primarily responsible for initial recruitment of the targeting complex to Alb3. These results suggest a new model for the sequential recruitment, remodeling, and unloading of the targeting complex at membrane translocase sites in the post-translational cpSRP pathway.

 

2.255           Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly

Haddad, J.G., Rouille, Y., Hanoulle, X., Descamps, V., Hamze, M., Dabbousi, F., Baumert, T.F., Duverlie, G., Lavie, M. and Dubuisson, J.

1. Virol., 91(8), e-00048-17 (2017)

 

Hepatitis C virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis.

 

2.256           Lipid Membrane Encapsulation of a 3D DNA Nano Octahedron

Perrault, S.D. and Shih, W.M.

Methods in Mol. Biol., 1500, 165-184 (2017)

 

Structural DNA nanotechnology methods such as DNA origami allow for the synthesis of highly precise nanometer-scale materials (Rothemund, Nature 440:297–302, 2006; Douglas et al., Nature 459:414–418, 2009). These offer compelling advantages for biomedical applications. Such materials can suffer from structural instability in biological environments due to denaturation and nuclease digestion (Hahn et al., ACS Nano 2014; Perrault and Shih, ACS Nano 8:5132–5140, 2014). Encapsulation of DNA nanostructures in a lipid membrane compartmentalizes them from their environment and prevents denaturation and nuclease digestion (Perrault and Shih, ACS Nano 8:5132–5140, 2014). Here, we describe the encapsulation of a 50 nm DNA nanostructure having the geometry of a wireframe octahedron in a phospholipid membrane containing poly-(ethylene glycol), resulting in biocompatible DNA nanostructures.

 

2.257           The Survival of Motor Neuron Protein Acts as a Molecular Chaperone for mRNP Assembly

Donlin-Asp, P.G., Fallini, C., Campos, J., Chou, C.C., Merritt, C.C., Bassell, G.J. and Rossoll, W.

Cell Reports, 18, 1660-1673 (2017)

 

Spinal muscular atrophy (SMA) is a motor neuron disease caused by reduced levels of the survival of motor neuron (SMN) protein. SMN is part of a multiprotein complex that facilitates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). SMN has also been found to associate with mRNA-binding proteins, but the nature of this association was unknown. Here, we have employed a combination of biochemical and advanced imaging methods to demonstrate that SMN promotes the molecular interaction between IMP1 protein and the 3′ UTR zipcode region of β-actin mRNA, leading to assembly of messenger ribonucleoprotein (mRNP) complexes that associate with the cytoskeleton to facilitate trafficking. We have identified defects in mRNP assembly in cells and tissues from SMA disease models and patients that depend on the SMN Tudor domain and explain the observed deficiency in mRNA localization and local translation, providing insight into SMA pathogenesis as a ribonucleoprotein (RNP)-assembly disorder.

 

2.258           Protease resistance of infectious prions is suppressed by removal of a single atom in the cellular prion protein

Leske, H., Hornemann, U., Zhu, C. et al

PloS One, 12(2), e0170503 (2017)

 

Resistance to proteolytic digestion has long been considered a defining trait of prions in tissues of organisms suffering from transmissible spongiform encephalopathies. Detection of proteinase K-resistant prion protein (PrP^Sc) still represents the diagnostic gold standard for prion diseases in humans, sheep and cattle. However, it has become increasingly apparent that the accumulation of PrP^Sc does not always accompany prion infections: high titers of prion infectivity can be reached also in the absence of protease resistant PrP^Sc. Here, we describe a structural basis for the phenomenon of protease-sensitive prion infectivity. We studied the effect on proteinase K (PK) resistance of the amino acid substitution Y169F, which removes a single oxygen atom from the β2–α2 loop of the cellular prion protein (PrP^C). When infected with RML or the 263K strain of prions, transgenic mice lacking wild-type (wt) PrP^C but expressing MoPrP^169F generated prion infectivity at levels comparable to wt mice. The newly generated MoPrP^169F prions were biologically indistinguishable from those recovered from prion-infected wt mice, and elicited similar pathologies in vivo. Surprisingly, MoPrP^169F prions showed greatly reduced PK resistance and density gradient analyses showed a significant reduction in high-density aggregates. Passage of MoPrP^169F prions into mice expressing wt MoPrP led to full recovery of protease resistance, indicating that no strain shift had taken place. We conclude that a subtle structural variation in the β2–α2 loop of PrP^C affects the sensitivity of PrP^Sc to protease but does not impact prion replication and infectivity. With these findings a specific structural feature of PrP^C can be linked to a physicochemical property of the corresponding PrP^Sc.

 

2.259           CD63 Regulates Epstein-Barr Virus LMP1 Exosomal Packaging, Enhancement of Vesicle Production, and Noncanonical NF-κB Signaling

Hurwitz, S.N., Nkosi, D., Conlon, M.M., York, S.B., Liu, X., Tremblay, D.C. and Meckes, D.G.

1. Virol., 91(5), e02251-16 (2017)

 

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. Recently, LMP1 was shown to be copurified with CD63, a conserved tetraspanin protein enriched in late endosomal and lysosomal compartments. Here, we demonstrate the importance of CD63 presence for exosomal packaging of LMP1. Nanoparticle tracking analysis and gradient purification revealed an increase in extracellular vesicle secretion and exosomal proteins following LMP1 expression. Immunoisolation of CD63-positive exosomes exhibited accumulation of LMP1 in this vesicle population. Functionally, CRISPR/Cas9 knockout of CD63 resulted in a reduction of LMP1-induced particle secretion. Furthermore, LMP1 packaging was severely impaired in CD63 knockout cells, concomitant with a disruption in the perinuclear localization of LMP1. Importantly, LMP1 trafficking to lipid rafts and activation of NF-κB and PI3K/Akt pathways remained intact following CD63 knockout, while mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and noncanonical NF-κB activation were observed to be increased. These results suggest that CD63 is a critical player in LMP1 exosomal trafficking and LMP1-mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling.

 

2.260           Post-translational modifications in PrP expand the conformational diversity of prions in vivo

Aguilar-Calvo, P., Xiao, X., Bett, C., Erana, H., Soldau, K., Castilla, J., Nilsson, K.P.R., Surewicz, W.K. and Sigurdson, C.J.

Scientific Reports, 7:43295 (2017)

 

Misfolded prion protein aggregates (PrPSc) show remarkable structural diversity and are associated with highly variable disease phenotypes. Similarly, other proteins, including amyloid-β, tau, α-synuclein, and serum amyloid A, misfold into distinct conformers linked to different clinical diseases through poorly understood mechanisms. Here we use mice expressing glycophosphatidylinositol (GPI)-anchorless prion protein, PrPC, together with hydrogen-deuterium exchange coupled with mass spectrometry (HXMS) and a battery of biochemical and biophysical tools to investigate how post-translational modifications impact the aggregated prion protein properties and disease phenotype. Four GPI-anchorless prion strains caused a nearly identical clinical and pathological disease phenotype, yet maintained their structural diversity in the anchorless state. HXMS studies revealed that GPI-anchorless PrPSc is characterized by substantially higher protection against hydrogen/deuterium exchange in the C-terminal region near the N-glycan sites, suggesting this region had become more ordered in the anchorless state. For one strain, passage of GPI-anchorless prions into wild type mice led to the emergence of a novel strain with a unique biochemical and phenotypic signature. For the new strain, histidine hydrogen-deuterium mass spectrometry revealed altered packing arrangements of β-sheets that encompass residues 139 and 186 of PrPSc. These findings show how variation in post-translational modifications may explain the emergence of new protein conformations in vivo and also provide a basis for understanding how the misfolded protein structure impacts the disease.

 

2.261           Actin-Interacting Protein 1 Contributes to Intranuclear Rod Assembly in Dictyostelium discoideum

Ishikawa-Ankerhold, H.C., Daszkiewicz, W., Schleicher, M. and Müller-Taubenberger, A.

Scientific Reports, 7:40310 (2017)

 

Intranuclear rods are aggregates consisting of actin and cofilin that are formed in the nucleus in consequence of chemical or mechanical stress conditions. The formation of rods is implicated in a variety of pathological conditions, such as certain myopathies and some neurological disorders. It is still not well understood what exactly triggers the formation of intranuclear rods, whether other proteins are involved, and what the underlying mechanisms of rod assembly or disassembly are. In this study, Dictyostelium discoideum was used to examine appearance, stages of assembly, composition, stability, and dismantling of rods. Our data show that intranuclear rods, in addition to actin and cofilin, are composed of a distinct set of other proteins comprising actin-interacting protein 1 (Aip1), coronin (CorA), filactin (Fia), and the 34 kDa actin-bundling protein B (AbpB). A finely tuned spatio-temporal pattern of protein recruitment was found during formation of rods. Aip1 is important for the final state of rod compaction indicating that Aip1 plays a major role in shaping the intranuclear rods. In the absence of both Aip1 and CorA, rods are not formed in the nucleus, suggesting that a sufficient supply of monomeric actin is a prerequisite for rod formation.

 

2.262           Enhanced neuroinvasion by smaller, soluble prions

Bett, C., Lawrence, J., Kurt, T.D., Orru, C., Aguilar-Calvo, P., Kincaid, A.E., Surewicz, W.K., Caughey, B., Wu, C. and Sigurdson, C.J.

Acta Neuropathol.Commun., 5:32 (2017)

 

Infectious prion aggregates can propagate from extraneural sites into the brain with remarkable efficiency, likely transported via peripheral nerves. Yet not all prions spread into the brain, and the physical properties of a prion that is capable of transit within neurons remain unclear. We hypothesized that small, diffusible aggregates spread into the CNS via peripheral nerves. Here we used a structurally diverse panel of prion strains to analyze how the prion conformation impacts transit into the brain. Two prion strains form fibrils visible ultrastructurally in the brain in situ, whereas three strains form diffuse, subfibrillar prion deposits and no visible fibrils. The subfibrillar strains had significantly higher levels of soluble prion aggregates than the fibrillar strains. Primary neurons internalized both the subfibrillar and fibril-forming prion strains by macropinocytosis, and both strain types were transported from the axon terminal to the cell body in vitro. However in mice, only the predominantly soluble, subfibrillar prions, and not the fibrillar prions, were efficiently transported from the tongue to the brain. Sonicating a fibrillar prion strain increased the solubility and enabled prions to spread into the brain in mice, as evident by a 40% increase in the attack rate, indicating that an increase in smaller particles enhances prion neuroinvasion. Our data suggest that the small, highly soluble prion particles have a higher capacity for transport via nerves. These findings help explain how prions that predominantly assemble into subfibrillar states can more effectively traverse into and out of the CNS, and suggest that promoting fibril assembly may slow the neuron-to-neuron spread of protein aggregates.

 

 

 

2.263           Multiple pathogen biomarker detection using an encoded bead array in droplet PCR

Rajeswari, P.K.P., Soderberg, L.M., Yacoub, A., Leijon, M., Andersson Svahn, H. and Joensson, H.N.

1. Microbiol. Methods, 139, 22-28 (2017)

 

We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color-coded Luminex® beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads.

 

2.264           Covalently linked dengue virus envelope glycoprotein dimers reduce exposure of the immunodominant fusion loop epitope

Rouvinski, A. et al

Nature Communications, 8:15411 (2017)

 

A problem in the search for an efficient vaccine against dengue virus is the immunodominance of the fusion loop epitope (FLE), a segment of the envelope protein E that is buried at the interface of the E dimers coating mature viral particles. Anti-FLE antibodies are broadly cross-reactive but poorly neutralizing, displaying a strong infection enhancing potential. FLE exposure takes place via dynamic ‘breathing’ of E dimers at the virion surface. In contrast, antibodies targeting the E dimer epitope (EDE), readily exposed at the E dimer interface over the region of the conserved fusion loop, are very potent and broadly neutralizing. We here engineer E dimers locked by inter-subunit disulfide bonds, and show by X-ray crystallography and by binding to a panel of human antibodies that these engineered dimers do not expose the FLE, while retaining the EDE exposure. These locked dimers are strong immunogen candidates for a next-generation vaccine.

 

2.265           Phosphorylation of TXNIP by AKT Mediates Acute Influx of Glucose in Response to Insulin

Waldhart, A.N., Dykstra, H., Peck, A.S., Cantley, L.C., Graw, T.E. and Wu, N.

Cell Reports, 19, 2005-2013 (2017)

 

Growth factors, such as insulin, can induce both acute and long-term glucose uptake into cells. Apart from the rapid, insulin-induced fusion of glucose transporter (GLUT)4 storage vesicles with the cell surface that occurs in muscle and adipose tissues, the mechanism behind acute induction has been unclear in other systems. Thioredoxin interacting protein (TXNIP) has been shown to be a negative regulator of cellular glucose uptake. TXNIP is transcriptionally induced by glucose and reduces glucose influx by promoting GLUT1 endocytosis. Here, we report that TXNIP is a direct substrate of protein kinase B (AKT) and is responsible for mediating AKT-dependent acute glucose influx after growth factor stimulation. Furthermore, TXNIP functions as an adaptor for the basal endocytosis of GLUT4 in vivo, its absence allows excess glucose uptake in muscle and adipose tissues, causing hypoglycemia during fasting. Altogether, TXNIP serves as a key node of signal regulation and response for modulating glucose influx through GLUT1 and GLUT4.

 

2.266           Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles

Oliveira, C. et al

Hepatology, 65(6), 1851-1864 (2017)

 

The development of different cell culture models has greatly contributed to increased understanding of the hepatitis C virus (HCV) life cycle. However, it is still challenging to grow HCV clinical isolates in cell culture. If overcome, this would open new perspectives to study HCV biology, including drug-resistant variants emerging with new antiviral therapies. In this study we hypothesized that this hurdle could be due to the presence of inhibitory factors in patient serum. Combining polyethylene glycol precipitation, iodixanol gradient, and size-exclusion chromatography, we obtained from HCV-seronegative sera a purified fraction enriched in inhibitory factors. Mass spectrometric analysis identified apolipoprotein(a) (apo[a]) as a potential inhibitor of HCV entry. Apo(a) consists of 10 kringle IV domains (KIVs), one kringle V domain, and an inactive protease domain. The 10 KIVs are present in a single copy with the exception of KIV type 2 (KIV2), which is encoded in a variable number of tandemly repeated copies, giving rise to numerous apo(a) size isoforms. In addition, apo(a) covalently links to the apolipoprotein B component of a low-density lipoprotein through a disulfide bridge to form lipoprotein(a). Using a recombinant virus derived from the JFH1 strain, we confirmed that plasma-derived and recombinant lipoprotein(a) as well as purified recombinant apo(a) variants were able to specifically inhibit HCV by interacting with infectious particles. Our results also suggest that small isoforms are less inhibitory than the large ones. Finally, we observed that the lipoprotein moiety of HCV lipoviroparticles was essential for inhibition, whereas functional lysine-binding sites in KIV7, KIV8, and KIV10 were not required. Conclusions: Our results identify apo(a) as an additional component of the lipid metabolism modulating HCV infection.

 

2.267           Heterogeneous Defect Domains in Single-Crystalline Hexagonal WS2

Jeong, H.Y., Jin, Y., Yun, S.J., Zhao, J., Baik, J., Keum, D.H., Lee, H.S. and Lee, Y.H.

Advanced Materials, 29:160543 (2017)

 

Single-crystalline monolayer hexagonal WS₂ is segmented into alternating triangular domains: sulfur-vacancy (SV)-rich and tungsten-vacancy (WV)-rich domains. The WV-rich domain with deep-trap states reveals an electron-dedoping effect, and the electron mobility and photoluminescence are lower than those of the SV-rich domain with shallow-donor states by one order of magnitude. The vacancy-induced strain and doping effects are investigated via Raman and scanning photoelectron microscopy.

 

2.268           Placing and shaping liposomes with reconfigurable DNA nanocages

Zhang, Z., Yang, Y., Pincet, F., Llaguno, M.C. and Lin, C.

Nature Chem., 9(7), 653-659 (2017)

 

The diverse structure and regulated deformation of lipid bilayer membranes are among a cell's most fascinating features. Artificial membrane-bound vesicles, known as liposomes, are versatile tools for modelling biological membranes and delivering foreign objects to cells. To fully mimic the complexity of cell membranes and optimize the efficiency of delivery vesicles, controlling liposome shape (both statically and dynamically) is of utmost importance. Here we report the assembly, arrangement and remodelling of liposomes with designer geometry: all of which are exquisitely controlled by a set of modular, reconfigurable DNA nanocages. Tubular and toroid shapes, among others, are transcribed from DNA cages to liposomes with high fidelity, giving rise to membrane curvatures present in cells yet previously difficult to construct in vitro. Moreover, the conformational changes of DNA cages drive membrane fusion and bending with predictable outcomes, opening up opportunities for the systematic study of membrane mechanics.

 

2.269           Discrete cytosolic macromolecular BRAF complexes exhibit distinct activities and composition

Diedrich, B., Rigbolt, K.T.G., Röring, M., herr, R., Kaeser-Pebernard, S., Gretzmeier, C., Murphy, R.F., Brummer, T. and Dengjel, J.

EMBO J., 36(5), 646-663 (2017)

 

As a central element within the RAS/ERK pathway, the serine/threonine kinase BRAF plays a key role in development and homeostasis and represents the most frequently mutated kinase in tumors. Consequently, it has emerged as an important therapeutic target in various malignancies. Nevertheless, the BRAF activation cycle still raises many mechanistic questions as illustrated by the paradoxical action and side effects of RAF inhibitors. By applying SEC‐PCP‐SILAC, we analyzed protein–protein interactions of hyperactive BRAF^V600E and wild‐type BRAF (BRAF^WT). We identified two macromolecular, cytosolic BRAF complexes of distinct molecular composition and phosphorylation status. Hyperactive BRAF^V600E resides in large complexes of higher molecular mass and activity, while BRAF^WT is confined to smaller, slightly less active complexes. However, expression of oncogenic K‐Ras^G12V, either by itself or in combination with RAF dimer promoting inhibitors, induces the incorporation of BRAF^WT into large, active complexes, whereas pharmacological inhibition of BRAF^V600E has the opposite effect. Thus, the quaternary structure of BRAF complexes is shaped by its activation status, the conformation of its kinase domain, and clinically relevant inhibitors.

 

2.270           Impact of Lipid Phase on the Bioavailability of Vitamin E in Emulsion-Based Delivery Systems: Relative Importance of Bioaccessibility, Absorption, and Transformation

Yang, Y., Xiao, H. and McClements, D.J.

Agric. Food Chem., 65, 3946-3955 (2017)

 

A simulated gastrointestinal tract/Caco-2 cell culture model was used to investigate the effects of lipid phase type on vitamin E (VE) bioavailability. Oil-in-water emulsions fortified with α-tocopherol acetate were fabricated using a natural emulsifier (quillaja saponin) and long or medium chain triglycerides (LCTs or MCTs) as lipids. The impact of lipid type on VE bioaccessibility, absorption, and transformation was determined. VE bioaccessibility was greater for LCT (46%) than MCT (19%) due to greater solubilization in mixed micelles assembled from longer fatty acids. VE absorption by Caco-2 cells was similar for LCT (28%) and MCT (30%). The transformation of α-tocopherol acetate to α-tocopherol was higher for LCT (90%) than MCT (75%) due to differences in esterase accessibility to VE. Emulsion-based delivery systems formulated using LCT are therefore more suitable for encapsulating and delivering vitamin E than those formulated using MCT.

 

2.271           Microfluidic platform for efficient Nanodisc assembly, membrane protein incorporation, and purification

Wade, J.H., Jones, J.D., lenov, I., Riordan, C.M., Sligar, S.G. and Bailey, R.C.

Lab on a Chip, 17, 2951-2959 (2017)

 

The characterization of integral membrane proteins presents numerous analytical challenges on account of their poor activity under non-native conditions, limited solubility in aqueous solutions, and low expression in most cell culture systems. Nanodiscs are synthetic model membrane constructs that offer many advantages for studying membrane protein function by offering a native-like phospholipid bilayer environment. The successful incorporation of membrane proteins within Nanodiscs requires experimental optimization of conditions. Standard protocols for Nanodisc formation can require large amounts of time and input material, limiting the facile screening of formation conditions. Capitalizing on the miniaturization and efficient mass transport inherent to microfluidics, we have developed a microfluidic platform for efficient Nanodisc assembly and purification, and demonstrated the ability to incorporate functional membrane proteins into the resulting Nanodiscs. In addition to working with reduced sample volumes, this platform simplifies membrane protein incorporation from a multi-stage protocol requiring several hours or days into a single platform that outputs purified Nanodiscs in less than one hour. To demonstrate the utility of this platform, we incorporated Cytochrome P450 into Nanodiscs of variable size and lipid composition, and present spectroscopic evidence for the functional active site of the membrane protein. This platform is a promising new tool for membrane protein biology and biochemistry that enables tremendous versatility for optimizing the incorporation of membrane proteins using microfluidic gradients to screen across diverse formation conditions.

 

2.272           Reversible unfolding of infectious prion assemblies reveals the existence of an oligomeric elementary brick

Igel-Egalon, A., Moudjou, M., Martin, D., Busley, A., Knäpple, T., Herzog, L., Reine, F., Lepajova, N., Richard, C-A., Beringue, V. and Rezaei, H.

PloS Pathogens, 13(9), e1006557 (2017)

 

Mammalian prions, the pathogens that cause transmissible spongiform encephalopathies, propagate by self-perpetuating the structural information stored in the abnormally folded, aggregated conformer (PrP^Sc) of the host-encoded prion protein (PrP^C). To date, no structural model related to prion assembly organization satisfactorily describes how strain-specified structural information is encoded and by which mechanism this information is transferred to PrP^C. To achieve progress on this issue, we correlated the PrP^Sc quaternary structural transition from three distinct prion strains during unfolding and refolding with their templating activity. We reveal the existence of a mesoscopic organization in PrP^Sc through the packing of a highly stable oligomeric elementary subunit (suPrP), in which the strain structural determinant (SSD) is encoded. Once kinetically trapped, this elementary subunit reversibly loses all replicative information. We demonstrate that acquisition of the templating interface and infectivity requires structural rearrangement of suPrP, in concert with its condensation. The existence of such an elementary brick scales down the SSD support to a small oligomer and provide a basis of reflexion for prion templating process and propagation.

 

 

2.273           A human APOC3 missense variant and monoclonal antibody accelerate apoC-III clearance and lower triglyceride-rich lipoprotein levels

Khetarpal, S.A. et al

Nature Med., 23(9), 1086-1094 (2017)

 

Recent large-scale genetic sequencing efforts have identified rare coding variants in genes in the triglyceride-rich lipoprotein (TRL) clearance pathway that are protective against coronary heart disease (CHD), independently of LDL cholesterol (LDL-C) levels1. Insight into the mechanisms of protection of these variants may facilitate the development of new therapies for lowering TRL levels. The gene APOC3 encodes apoC-III, a critical inhibitor of triglyceride (TG) lipolysis and remnant TRL clearance2. Here we report a detailed interrogation of the mechanism of TRL lowering by the APOC3 Ala43Thr (A43T) variant, the only missense (rather than protein-truncating) variant in APOC3 reported to be TG lowering and protective against CHD3, 4, 5. We found that both human APOC3 A43T heterozygotes and mice expressing human APOC3 A43T display markedly reduced circulating apoC-III levels. In mice, this reduction is due to impaired binding of A43T apoC-III to lipoproteins and accelerated renal catabolism of free apoC-III. Moreover, the reduced content of apoC-III in TRLs resulted in accelerated clearance of circulating TRLs. On the basis of this protective mechanism, we developed a monoclonal antibody targeting lipoprotein-bound human apoC-III that promotes circulating apoC-III clearance in mice expressing human APOC3 and enhances TRL catabolism in vivo. These data reveal the molecular mechanism by which a missense variant in APOC3 causes reduced circulating TG levels and, hence, protects from CHD. This protective mechanism has the potential to be exploited as a new therapeutic approach to reduce apoC-III levels and circulating TRL burden.

 

2.274           Metalloprotease-mediated cleavage of PlexinD1 and its sequestration to actin rods in the motoneuron disease spinal muscular atrophy (SMA)

Rademacher, S., Verheijen, B.M., Hensel, N., Peters, M., Bora, G., Brandes, G., de Sa, R.V., Heidrich, N., Fischer, S., Brinkmann, H.,van  der Pol, W.L., Wirth, B., Pasterkamp, R.J. and Claus, P.

Hum. Mol. Genet., 26(20), 3946-3959 (2017)

 

Cytoskeletal rearrangement during axon growth is mediated by guidance receptors and their ligands which act either as repellent, attractant or both. Regulation of the actin cytoskeleton is disturbed in Spinal Muscular Atrophy (SMA), a devastating neurodegenerative disease affecting mainly motoneurons, but receptor-ligand interactions leading to the dysregulation causing SMA are poorly understood. In this study, we analysed the role of the guidance receptor PlexinD1 in SMA pathogenesis. We showed that PlexinD1 is cleaved by metalloproteases in SMA and that this cleavage switches its function from an attractant to repellent. Moreover, we found that the PlexinD1 cleavage product binds to actin rods, pathological aggregate-like structures which had so far been described for age-related neurodegenerative diseases. Our data suggest a novel disease mechanism for SMA involving formation of actin rods as a molecular sink for a cleaved PlexinD1 fragment leading to dysregulation of receptor signaling.

 

Conclusions Our findings highlight a new mechanism in lipid metabolism regulation and interaction of the lipid metabolism with the HCV life cycle, which may be important for viral pathogenesis and might also be explored for antiviral therapy.

 

2.275           Human Herpesvirus 8 Interleukin-6 Interacts with Calnexin Cycle Components and Promotes Protein Folding

Chen, D., Xiang, Q. and Nicholas, J.

1. Virol., 91(22), e00965-17 (2017)

 

Viral interleukin-6 (vIL-6) encoded by human herpesvirus 8 (HHV-8) is believed to contribute via mitogenic, survival, and angiogenic activities to HHV-8-associated Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease through autocrine or paracrine mechanisms during latency or productive replication. There is direct evidence that vIL-6 promotes latently infected PEL cell viability and proliferation and also viral productive replication in PEL and endothelial cells. These activities are mediated largely through endoplasmic reticulum (ER)-localized vIL-6, which can induce signal transduction via the gp130 signaling receptor, activating mitogen-activated protein kinase and signal transducer and activator of transcription signaling, and interactions of vIL-6 with the ER membrane protein vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2). The latter functional axis involves suppression of proapoptotic lysosomal protein cathepsin D by promotion of the ER-associated degradation of ER-transiting, preproteolytically processed procathepsin D. Other interactions of VKORC1v2 and activities of vIL-6 via the receptor have not been reported. We show here that both vIL-6 and VKORC1v2 interact with calnexin cycle proteins UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), which catalyzes monoglucosylation of N-glycans, and oppositely acting glucosidase II (GlucII), and that vIL-6 can promote protein folding. This activity was found to require VKORC1v2 and UGGT1, to involve vIL-6 associations with VKORC1v2, UGGT1, and GlucII, and to operate in the context of productively infected cells. These findings document new VKORC1v2-associated interactions and activities of vIL-6, revealing novel mechanisms of vIL-6 function within the ER compartment.

 

2.276           Optimization of d-Peptides for Aβ Monomer Binding Specificity Enhances Their Potential to Eliminate Toxic Aβ Oligomers

Klein, A.N., Ziehm, T., van Groen, T., Kadish, I., Elfgen, A., Tusche, M., Thomaier, M., Reiss, K., Brener, O., Gremer, l., Kutzsche, J. and Wilbold, D.

ACS Chem. Neurosci., 8, 1889-1900 (2017)

 

Amyloid-beta (Aβ) oligomers are thought to be causative for the development and progression of Alzheimer’s disease (AD). Starting from the Aβ oligomer eliminating d-enantiomeric peptide D3, we developed and applied a two-step procedure based on peptide microarrays to identify D3 derivatives with increased binding affinity and specificity for monomeric Aβ(1–42) to further enhance the Aβ oligomer elimination efficacy. Out of more than 1000 D3 derivatives, we selected seven novel d-peptides, named ANK1 to ANK7, and characterized them in more detail in vitro. All ANK peptides bound to monomeric Aβ(1–42), eliminated Aβ(1–42) oligomers, inhibited Aβ(1–42) fibril formation, and reduced Aβ(1–42)-induced cytotoxicity more efficiently than D3. Additionally, ANK6 completely inhibited the prion-like propagation of preformed Aβ(1–42) seeds and showed a nonsignificant tendency for improving memory performance of tg-APPSwDI mice after i.p. application for 4 weeks. This supports the hypothesis that stabilization of Aβ monomers and thereby induced elimination of Aβ oligomers is a suitable therapeutic strategy.

 

2.277           How We Make DNA Origami

Wagenbauer, K.F., Engelhardt, F.A.S., Stahl, E., Hechtl, V.K., Stömmer, P., Seebacher, F., Meregalli, L., Ketterer, P., Gerling, T. and Dietz, H.

ChemBioChem., 18(19), 1873-1885 (2017)

 

DNA origami has attracted substantial attention since its invention ten years ago, due to the seemingly infinite possibilities that it affords for creating customized nanoscale objects. Although the basic concept of DNA origami is easy to understand, using custom DNA origami in practical applications requires detailed know-how for designing and producing the particles with sufficient quality and for preparing them at appropriate concentrations with the necessary degree of purity in custom environments. Such know-how is not readily available for newcomers to the field, thus slowing down the rate at which new applications outside the field of DNA nanotechnology may emerge. To foster faster progress, we share in this article the experience in making and preparing DNA origami that we have accumulated over recent years. We discuss design solutions for creating advanced structural motifs including corners and various types of hinges that expand the design space for the more rigid multilayer DNA origami and provide guidelines for preventing undesired aggregation and on how to induce specific oligomerization of multiple DNA origami building blocks. In addition, we provide detailed protocols and discuss the expected results for five key methods that allow efficient and damage-free preparation of DNA origami. These methods are agarose-gel purification, filtration through molecular cut-off membranes, PEG precipitation, size-exclusion chromatography, and ultracentrifugation-based sedimentation. The guide for creating advanced design motifs and the detailed protocols with their experimental characterization that we describe here should lower the barrier for researchers to accomplish the full DNA origami production workflow.

 

2.278           The Aβ oligomer eliminating D-enantiomeric peptide RD2 improves cognition without changing plaque pathology

Van Groen, T., Schemmert, S., Brener, O., gremer, l., Ziehm, T., Tusche, m., nagel-Steger, L., Kadish, I., Schartmann, E., Elfgen, A., Jürgens, D., Willuweit, A., Kutzsche, J. and Willbold, D.

Scientific Reports, 7.16275 (2017)

 

While amyloid-β protein (Aβ) aggregation into insoluble plaques is one of the pathological hallmarks of Alzheimer’s disease (AD), soluble oligomeric Aβ has been hypothesized to be responsible for synapse damage, neurodegeneration, learning, and memory deficits in AD. Here, we investigate the in vitro and in vivo efficacy of the d-enantiomeric peptide RD2, a rationally designed derivative of the previously described lead compound D3, which has been developed to efficiently eliminate toxic Aβ42 oligomers as a promising treatment strategy for AD. Besides the detailed in vitro characterization of RD2, we also report the results of a treatment study of APP/PS1 mice with RD2. After 28 days of treatment we observed enhancement of cognition and learning behaviour. Analysis on brain plaque load did not reveal significant changes, but a significant reduction of insoluble Aβ42. Our findings demonstrate that RD2 was significantly more efficient in Aβ oligomer elimination in vitro compared to D3. Enhanced cognition without reduction of plaque pathology in parallel suggests that synaptic malfunction due to Aβ oligomers rather than plaque pathology is decisive for disease development and progression. Thus, Aβ oligomer elimination by RD2 treatment may be also beneficial for AD patients.

 

2.279           UNC-45a promotes myosin folding and stress fiber assembly

Lehtimäki, J.I., Fenix, A.M., Kotila, T.M., Balistreri, G., Paavolainen, L., varjosalo, M., Burnette, D.T. and Lappalainen, P.

J. Cell Biol., 216(12), 4053-4072 (2017)

 

Contractile actomyosin bundles, stress fibers, are crucial for adhesion, morphogenesis, and mechanosensing in nonmuscle cells. However, the mechanisms by which nonmuscle myosin II (NM-II) is recruited to those structures and assembled into functional bipolar filaments have remained elusive. We report that UNC-45a is a dynamic component of actin stress fibers and functions as a myosin chaperone in vivo. UNC-45a knockout cells display severe defects in stress fiber assembly and consequent abnormalities in cell morphogenesis, polarity, and migration. Experiments combining structured-illumination microscopy, gradient centrifugation, and proteasome inhibition approaches revealed that a large fraction of NM-II and myosin-1c molecules fail to fold in the absence of UNC-45a. The remaining properly folded NM-II molecules display defects in forming functional bipolar filaments. The C-terminal UNC-45/Cro1/She4p domain of UNC-45a is critical for NM-II folding, whereas the N-terminal tetratricopeptide repeat domain contributes to the assembly of functional stress fibers. Thus, UNC-45a promotes generation of contractile actomyosin bundles through synchronized NM-II folding and filament-assembly activities.

 

2.280           A stretch of residues within the protease-resistant core is not necessary for prion structure and infectivity

Munoz-Montesino, C., Sizun, C., Moudjou, M., Herzog, L., Reine, F., Igel-Egalon, A., Barbereau, C., Chapuis, J., Ciric, D., Laude, H., Beringue, V., Rezaei, H. and Dron, M.

Prion, 11(1), 25-30 (2017)

 

Mapping out regions of PrP influencing prion conversion remains a challenging issue complicated by the lack of prion structure. The portion of PrP associated with infectivity contains the α-helical domain of the correctly folded protein and turns into a β-sheet-rich insoluble core in prions. Deletions performed so far inside this segment essentially prevented the conversion. Recently we found that deletion of the last C-terminal residues of the helix H2 was fully compatible with prion conversion in the RK13-ovPrP cell culture model, using 3 different infecting strains. This was in agreement with preservation of the overall PrP^C structure even after removal of up to one-third of this helix. Prions with internal deletion were infectious for cells and mice expressing the wild-type PrP and they retained prion strain-specific characteristics. We thus identified a piece of the prion domain that is neither necessary for the conformational transition of PrP^C nor for the formation of a stable prion structure.

 

2.281           Circulating ApoJ is closely associated with insulin resistance in human subjects

Seo, J.A., Kang, M-C., Ciaraldi, T.P., Kim, S.S., Park, K.S., Choe, C., hwang, W.H., Lim, D.M., Farr, O., Mantzoros, C., Henry, R. and Kim, Y-B.

Metabolism, 78, 155-166 (2018)

 

Objective

Insulin resistance is a major risk factor for type 2 diabetes. ApolipoproteinJ (ApoJ) has been implicated in altered pathophysiologic states including cardiovascular and Alzheimer's disease. However, the function of ApoJ in regulation of glucose homeostasis remains unclear. This study sought to determine whether serum ApoJ levels are associated with insulin resistance in human subjects and if they change after interventions that improve insulin sensitivity.

Methods

Serum ApoJ levels and insulin resistance status were assessed in nondiabetic (ND) and type 2 diabetic (T2D) subjects. The impacts of rosiglitazone or metformin therapy on serum ApoJ levels and glucose disposal rate (GDR) during a hyperinsulinemic/euglycemic clamp were evaluated in a separate cohort of T2D subjects. Total ApoJ protein or that associated with the HDL and LDL fractions was measured by immunoblotting or ELISA.

Results

Fasting serum ApoJ levels were greatly elevated in T2D subjects (ND vs T2D; 100 ± 8.3 vs. 150.6 ± 8.5 AU, P < 0.0001). Circulating ApoJ levels strongly correlated with fasting glucose, fasting insulin, HOMA-IR, and BMI. ApoJ levels were significantly and independently associated with HOMA-IR, even after adjustment for age, sex, and BMI. Rosiglitazone treatment in T2D subjects resulted in a reduction in serum ApoJ levels (before vs. after treatment; 100 ± 13.9 vs. 77 ± 15.2 AU, P = 0.015), whereas metformin had no effect on ApoJ levels. The change in ApoJ levels during treatment was inversely associated with the change in GDR. Interestingly, ApoJ content in the LDL fraction was inversely associated with HOMA-IR.

Conclusion

Serum ApoJ levels are closely correlated with the magnitude of insulin resistance regardless of obesity, and decrease along with improvement of insulin resistance in response only to rosiglitazone in type 2 diabetes.

 

2.282           Eicosapentaenoic acid inhibits oxidation of high density lipoprotein particles in a manner distinct from docosahexaenoic acid

Sherratt, S.C.R. and mason, R.P.

Biochem. Biophys. Res. Comm., 496, 315-338 (2018)

 

The omega-3 fatty acid eicosapentaenoic acid (EPA) reduces oxidation of ApoB-containing particles in vitro and in patients with hypertriglyceridemia. EPA may produce these effects through a potent antioxidant mechanism, which may facilitate LDL clearance and slow plaque progression. We hypothesize that EPA antioxidant effects may extend to ApoA-containing particles like HDL, potentially preserving certain atheroprotective functions. HDL was isolated from human plasma and incubated at 37 °C in the absence (vehicle) or presence of EPA and/or DHA; 5.0 or 10.0 μM each. Samples were then subjected to copper-induced oxidation (10 μM). HDL oxidation was inhibited similarly by EPA and DHA up to 1 h. EPA (10 μM) maintained significant HDL oxidation inhibition of 89% (0.622 ± 0.066 μM MDA; p < .001) at 4 h, with continued inhibition of 64% at 14 h, vs. vehicle (5.65 ± 0.06 to 2.01 ± 0.10 μM MDA; p < .001). Conversely, DHA (10 μM) antioxidant benefit was lost by 4 h. At a lower concentration (5 μM), EPA antioxidant activity remained at 81% (5.53 ± 0.15 to 1.03 ± 0.10 μM MDA; p < .001) at 6 h, while DHA lost all antioxidant activity by 4 h. The antioxidant activity of EPA was preserved when combined with an equimolar concentration of DHA (5 μM each). EPA pretreatment prevented HDL oxidation in a dose-dependent manner that was preserved over time. These results suggest unique lipophilic and electron stabilization properties for EPA as compared to DHA with respect to inhibition of HDL oxidation. These antioxidant effects of EPA may enhance certain atheroprotective functions for HDL.

 

2.283           Update on the laboratory investigation of dyslipidemias

Ramasamy, I.

Clin. Chim. Acta, 479, 103-125 (2018)

 

The role of the clinical laboratory is evolving to provide more information to clinicians to assess cardiovascular disease (CVD) risk and target therapy more effectively. Current routine methods to measure LDL-cholesterol (LDL-C), the Friedewald calculation, ultracentrifugation, electrophoresis and homogeneous direct methods have established limitations. Studies suggest that LDL and HDL size or particle concentration are alternative methods to predict future CVD risk. At this time there is no consensus role for lipoprotein particle or subclasses in CVD risk assessment. LDL and HDL particle concentration are measured by several methods, namely gradient gel electrophoresis, ultracentrifugation-vertical auto profile, nuclear magnetic resonance and ion mobility. It has been suggested that HDL functional assays may be better predictors of CVD risk. To assess the issue of lipoprotein subclasses/particles and HDL function as potential CVD risk markers robust, simple, validated analytical methods are required. In patients with small dense LDL particles, even a perfect measure of LDL-C will not reflect LDL particle concentration. Non-HDL-C is an alternative measurement and includes VLDL and CM remnant cholesterol and LDL-C. However, apolipoprotein B measurement may more accurately reflect LDL particle numbers. Non-fasting lipid measurements have many practical advantages. Defining thresholds for treatment with new measurements of CVD risk remain a challenge. In families with genetic variants, ApoCIII and lipoprotein (a) may be additional risk factors. Recognition of familial causes of dyslipidemias and diagnosis in childhood will result in early treatment. This review discusses the limitations in current laboratory technologies to predict CVD risk and reviews the evidence for emergent approaches using newer biomarkers in clinical practice.

 

2.284           Isolation and Characterization of Endogenous RNPs from Brain Tissues

Schieweck, R., Ang, F.y., Fritzsche, R. and Kiebler, M.A.

Methods in Mol. Biol., 1649, 419-426 (2018)

 

Identification of physiological target RNAs and protein interactors bound to RNA-binding proteins is a key prerequisite to understand the underlying mechanisms of posttranscriptional expression control and RNA granule assembly. Here, we describe a multistep biochemical approach to isolate endogenous ribonucleoprotein particles from brain tissues by exploiting differential centrifugation and gradient fractionation followed by immunoprecipitation with monospecific, affinity-purified antibodies directed against selected RNA-binding proteins. This protocol results in highly enriched endogenous ribonucleoprotein particles that then can be analyzed by mass spectrometry (for proteins composition) and microarray or RNA sequencing technologies (for target mRNAs).

 

2.285           Functional innate immunity restricts Hepatitis C Virus infection in induced pluripotent stem cell–derived hepatocytes

Schöbel, A., Rösch, K. and Herker, E.

Scientific Reports, 8:3893 (2018)

 

Knowledge of activation and interplay between the hepatitis C virus (HCV) and the hosts’ innate immunity is essential to understanding the establishment of chronic HCV infection. Human hepatoma cell lines, widely used as HCV cell culture system, display numerous metabolic alterations and a defective innate immunity, hindering the detailed study of virus-host interactions. Here, we analysed the suitability of induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHLCs) as a physiologically relevant model to study HCV replication in vitro. Density gradients and triglyceride analysis revealed that iHLCs secreted very-low density lipoprotein (VLDL)-like lipoproteins, providing a putative platform for bona fide lipoviroparticles. iHLCs supported the full HCV life cycle, but in contrast to Huh7 and Huh7.5 cells, replication and viral RNA levels decreased continuously. Following HCV infection, interferon-stimulated gene (ISG)-expression significantly increased in iHLCs, whereas induction was almost absent in Huh7/7.5 cells. However, IFNα-stimulation equally induced ISGs in iHLCs and hepatoma cells. JAK-STAT pathway inhibition increased HCV replication in mature iHLCs, but not in Huh7 cells. Additionally, HCV replication levels where higher in STAT2-, but not STAT1-knockdown iHLCs. Our findings support iHLCs as a suitable model for HCV-host interaction regarding a functional innate immunity and lipoprotein synthesis.

 

2.286           Prion-like propagation of β-amyloid aggregates in the absence of APP overexpression

Ruiz-Riquelme, A., Lau, H.H., Stuart, E., Goczi, A.N., Wang, Z., Schmitt-Ulms, G. and Watts, J.C.

Acta Neuropathol. Comm., 6:26 (2018)

 

The amyloid cascade hypothesis posits that the initiating event in Alzheimer’s disease (AD) is the aggregation and deposition of the β-amyloid (Aβ) peptide, which is a proteolytic cleavage product of the amyloid precursor protein (APP). Mounting evidence suggests that the formation and spread of prion-like Aβ aggregates during AD may contribute to disease progression. Inoculation of transgenic mice that overexpress APP with pre-formed Aβ aggregates results in the prion-like induction of cerebral Aβ deposition. To determine whether Aβ deposition can also be induced when physiological APP levels are present in the brain, we inoculated App^NL-F mice, a knock-in model of AD that avoids potential artifacts associated with APP overexpression, with Aβ aggregates derived from the brains of AD patients or transgenic mice. In all cases, induced Aβ deposition was apparent in the corpus callosum, olfactory bulb, and meningeal blood vessels of inoculated mice at 130–150 days post-inoculation, whereas uninoculated and buffer-inoculated animals exhibited minimal or no Aβ deposits at these ages. Interestingly, despite being predominantly composed of protease-resistant Aβ42 aggregates, the induced parenchymal Aβ deposits were largely diffuse and were unreactive to an amyloid-binding dye. These results demonstrate that APP overexpression is not a prerequisite for the prion-like induction of cerebral Aβ deposition. Accordingly, spreading of Aβ deposition may contribute to disease progression in AD patients.

 

 

 

2.287           One‐Way Particle Transport Using Oscillatory Flow in Asymmetric Traps

Lee, J. and Burns, M.A.

Small, 14, 1702724 (2018)

 

One challenge of integrating of passive, microparticles manipulation techniques into multifunctional microfluidic devices is coupling the continuous‐flow format of most systems with the often batch‐type operation of particle separation systems. Here, a passive fluidic technique—one‐way particle transport—that can conduct microparticle operations in a closed fluidic circuit is presented. Exploiting pass/capture interactions between microparticles and asymmetric traps, this technique accomplishes a net displacement of particles in an oscillatory flow field. One‐way particle transport is achieved through four kinds of trap–particle interactions: mechanical capture of the particle, asymmetric interactions between the trap and the particle, physical collision of the particle with an obstacle, and lateral shift of the particle into a particle–trapping stream. The critical dimensions for those four conditions are found by numerically solving analytical mass balance equations formulated using the characteristics of the flow field in periodic obstacle arrays. Visual observation of experimental trap–particle dynamics in low Reynolds number flow (<0.01) confirms the validity of the theoretical predictions. This technique can transport hundreds of microparticles across trap rows in only a few fluid oscillations (<500 ms per oscillation) and separate particles by their size differences.

 

2.288           High-density lipoprotein suppresses tumor necrosis factor alpha production by mycobacteria-infected human macrophages

Inoue, M., Niki, M., Ozeki, Y., Nagi, S. et al

Scientific Reports, 8:6736 (2018)

 

Immune responses to parasitic pathogens are affected by the host physiological condition. High-density lipoprotein (HDL) and low-density lipoprotein (LDL) are transporters of lipids between the liver and peripheral tissues, and modulate pro-inflammatory immune responses. Pathogenic mycobacteria are parasitic intracellular bacteria that can survive within macrophages for a long period. Macrophage function is thus key for host defense against mycobacteria. These basic facts suggest possible effects of HDL and LDL on mycobacterial diseases, which have not been elucidated so far. In this study, we found that HDL and not LDL enhanced mycobacterial infections in human macrophages. Nevertheless, we observed that HDL remarkably suppressed production of tumor necrosis factor alpha (TNF-α) upon mycobacterial infections. TNF-α is a critical host-protective cytokine against mycobacterial diseases. We proved that toll-like receptor (TLR)-2 is responsible for TNF-α production by human macrophages infected with mycobacteria. Subsequent analysis showed that HDL downregulates TLR2 expression and suppresses its intracellular signaling pathways. This report demonstrates for the first time the substantial action of HDL in mycobacterial infections to human macrophages.

 

2.289           Eicosapentaenoic acid improves endothelial function and nitric oxide bioavailability in a manner that is enhanced in combination with a statin

Mason, R.P., Dawoud, H., Jacob, R.F., Sherratt, S.C.R. and Malinski, T.

Biomedicine Pharmacother., 103, 1231-1237 (2018)

 

The endothelium exerts many vasoprotective effects that are largely mediated by release of nitric oxide (NO). Endothelial dysfunction represents an early but reversible step in atherosclerosis and is characterized by a reduction in the bioavailability of NO. Previous studies have shown that eicosapentaenoic acid (EPA), an omega-3 fatty acid (O3FA), and statins individually improve endothelial cell function, but their effects in combination have not been tested. Through a series of in vitro experiments, this study evaluated the effects of a combined treatment of EPA and the active metabolite of atorvastatin (ATM) on endothelial cell function under conditions of oxidative stress. Specifically, the comparative and time-dependent effects of these agents on endothelial dysfunction were examined by measuring the levels of NO and peroxynitrite (ONOO⁻) released from human umbilical vein endothelial cells (HUVECs). The data suggest that combined treatment with EPA and ATM is beneficial to endothelial function and was unique to EPA and ATM since similar improvements could not be recapitulated by substituting another O3FA docosahexaenoic acid (DHA) or other TG-lowering agents such as fenofibrate, niacin, or gemfibrozil. Comparable beneficial effects were observed when HUVECs were pretreated with EPA and ATM before exposure to oxidative stress. Interestingly, the kinetics of EPA-based protection of endothelial function in response to oxidation were found to be significantly different than those of DHA. Lastly, the beneficial effects on endothelial function generated by combined treatment of EPA and ATM were reproduced when this study was expanded to an ex vivo model utilizing rat glomerular endothelial cells. Taken together, these findings suggest that a combined treatment of EPA and ATM can inhibit endothelial dysfunction that occurs in response to conditions such as hyperglycemia, oxidative stress, and dyslipidemia.

 

2.290           Favouring modulation of circulating lipoproteins and lipid loading capacity by direct antiviral agents grazoprevir/elbasvir or ledipasvir/sofosbuvir treatment against chronic HCV infection

Sun, H-Y., Cheng, P-N., Tseng, C-Y., Tsai, W-J., Chiu, Y-C. and Young, K-C.

Gut, 67, 1342-1350 (2018)

 

Objective Lipid homoeostasis is disturbed in patients with HCV infection. Direct-acting antiviral agent (DAA) treatment eradicates chronic HCV viraemia, but the dynamics of lipid components remain elusive. This study investigates the clinical manifestation and mechanistic relevance of plasma triglyceride (TG), cholesterol (Chol), lipoproteins and apolipoproteins (apos) after DAA treatment.

Design Twenty-four patients with chronic genotype 1 (GT1) HCV treated with elbasvir/grazoprevir or ledipasvir/sofosbuvir for 12 weeks, and followed-up thereafter, were recruited. Their TG, Chol, apoAI and apoB levels were quantified in plasma samples and individually fractionated lipoprotein of various classes. Liver fibrosis was evaluated using the FIB-4 Score. The TG and Chol loading capacities were calculated with normalisation to apoB, which represents per very low density lipoprotein (VLDL) and LDL particle unit

Results DAA treatment achieved a sustained virological response rate of 91.7% and reduced the FIB-4 Score. Relative to the baseline, the plasma TG level was reduced but the Chol level increased gradually. Plasma apoB levels and apoB/apoAI ratio were transiently downregulated as early as the first 4 weeks of treatment. The TG and Chol loading capacities in VLDL were elevated by ~20% during the period of DAA treatment and had steadily increased by 100% at follow-up. Furthermore, the TG-to-Chol ratio in VLDL was increased, while the ratio in LDL was reduced, indicating an efficient catabolism.

Conclusion The DAA treatment of patients with chronic hepatitis C might lead to efficient HCV eradication and hepatic improvement concomitantly evolving with favouring lipoprotein/apo metabolisms.

 

2.291           Dual role of the RNA helicase DDX5 in post-transcriptional regulation of myelin basic protein in oligodendrocytes

Hoch-Kraft, P., White, R., Tenzer, S., Krämer-Albers, E-M., Trotter, J. and Gonsior, C.

1. Cell Science, 131, jcs20470 (2018)

 

In the central nervous system, oligodendroglial expression of myelin basic protein (MBP) is crucial for the assembly and structure of the myelin sheath. MBP synthesis is tightly regulated in space and time, particularly at the post-transcriptional level. We have identified the DEAD-box RNA helicase DDX5 (also known as p68) in a complex with Mbp mRNA in oligodendroglial cells. Expression of DDX5 is highest in progenitor cells and immature oligodendrocytes, where it localizes to heterogeneous populations of cytoplasmic ribonucleoprotein (RNP) complexes associated with Mbp mRNA in the cell body and processes. Manipulation of the amount of DDX5 protein inversely affects the level of MBP. We present evidence that DDX5 is involved in post-transcriptional regulation of MBP protein synthesis, with implications for oligodendroglial development. In addition, knockdown of DDX5 results in an increased abundance of MBP isoforms containing exon 2 in immature oligodendrocytes, most likely by regulating alternative splicing of Mbp. Our findings contribute to the understanding of the complex nature of MBP post-transcriptional control in immature oligodendrocytes where DDX5 appears to affect the abundance of MBP proteins via distinct but converging mechanisms.

 

2.292           High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis

Ramakrishnan, S., Gohlke, A., Li, F., Coleman, J., Xu, W., Rothman, J.E. and Pincet, F.

Langmuir, 34(20), 5849-5859 (2018)

 

In vivo membrane fusion primarily occurs between highly curved vesicles and planar membranes. A better understanding of fusion entails an accurate in vitro reproduction of the process. To date, supported bilayers have been commonly used to mimic the planar membranes. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that induce membrane fusion usually have limited fluidity when embedded in supported bilayers. This alters the kinetics and prevents correct reconstitution of the overall fusion process. Also, observing content release across the membrane is hindered by the lack of a second aqueous compartment. Recently, a step toward resolving these issues was achieved by using membranes spread on holey substrates. The mobility of proteins was preserved but vesicles were prone to bind to the substrate when reaching the edge of the hole, preventing the observation of many fusion events over the suspended membrane. Building on this recent advance, we designed a method for the formation of pore-spanning lipid bilayers containing t-SNARE proteins on Si/SiO2 holey chips, allowing the observation of many individual vesicle fusion events by both lipid mixing and content release. With this setup, proteins embedded in the suspended membrane bounced back when they reached the edge of the hole which ensured vesicles did not bind to the substrate. We observed SNARE-dependent membrane fusion with the freestanding bilayer of about 500 vesicles. The time between vesicle docking and fusion is ∼1 s. We also present a new multimodal open-source software, Fusion Analyzer Software, which is required for fast data analysis.

 

2.293           Cryo-EM structure of human mitochondrial trifunctional protein

Liang, K., Li, N., Wang, X., Dai, J., Liu, P., Wang, C., Chen, X-W., Gao, N. and Xiao, J.

PNAS, 115(27), 7039-7044 (2018)

 

The mitochondrial trifunctional protein (TFP) catalyzes three reactions in the fatty acid β-oxidation process. Mutations in the two TFP subunits cause mitochondrial trifunctional protein deficiency and acute fatty liver of pregnancy that can lead to death. Here we report a 4.2-Å cryo-electron microscopy α2β2 tetrameric structure of the human TFP. The tetramer has a V-shaped architecture that displays a distinct assembly compared with the bacterial TFPs. A concave surface of the TFP tetramer interacts with the detergent molecules in the structure, suggesting that this region is involved in associating with the membrane. Deletion of a helical hairpin in TFPβ decreases its binding to the liposomes in vitro and reduces its membrane targeting in cells. Our results provide the structural basis for TFP function and have important implications for fatty acid oxidation related diseases.

 

2.294           Mitochondrial maintenance under oxidative stress depends on mitochondrially localised α-OGG1

Lia, D., Reyes, A., de Melo Campos, J.A.T., Piolot, T., Baijer, J., Radicella, J.P. and Campalans, A.

1. Cell Science, 131, jcs213538 (2018)

 

Accumulation of 8-oxoguanine (8-oxoG) in mitochondrial DNA and mitochondrial dysfunction have been observed in cells deficient for the DNA glycosylase OGG1 when exposed to oxidative stress. In human cells, up to eight mRNAs for OGG1 can be generated by alternative splicing and it is still unclear which of them codes for the protein that ensures the repair of 8-oxoG in mitochondria. Here, we show that the α-OGG1 isoform, considered up to now to be exclusively nuclear, has a functional mitochondrial-targeting sequence and is imported into mitochondria. We analyse the sub-mitochondrial localisation of α-OGG1 with unprecedented resolution and show that this DNA glycosylase is associated with DNA in mitochondrial nucleoids. We show that the presence of α-OGG1 inside mitochondria and its enzymatic activity are required to preserve the mitochondrial network in cells exposed to oxidative stress. Altogether, these results unveil a new role of α-OGG1 in the mitochondria and indicate that the same isoform ensures the repair of 8-oxoG in both nuclear and mitochondrial genomes. The activity of α-OGG1 in mitochondria is sufficient for the recovery of organelle function after oxidative stress.

 

2.295           Interfering surface and localized plasmon: Tuning the Wood anomaly for biosensing

Shaimanov, A.N., Orlikovsky, N.A., Khasbushev, E.M., Zverev, A.V., Pishmova, A.A., Sharonov, G.V., Yankovskii, G.M., Rodionov, L.A. and Baryshev, A.V.

Photonics and Nanostructures – Fundamentals and Applications, 32, 1-5 (2018)

 

We demonstrate spectra of slabs of plasmonic 1D nanostructures and show their changes when detecting specific biomolecular binding. The slabs were fabricated by electron-beam lithography, they had sub-millimeter dimensions and allowed us to detect a specific binding of low-density lipoproteins. Optical spectra of the slabs exhibiting a spectrally sharp resonant peak have been analyzed numerically to interpret their features and to define structural parameters governing the quality factor of the resonance. We show a comparison between the sensing performances of the different slabs under study, thus discussing their better designs and experimental geometries.

 

2.296           DNA-induced liquid phase condensation of cGAS activates innate immune signaling

Du, M. and Chen, Z.J.

Science, 361(6403), 704-709 (2018)

 

The binding of DNA to cyclic GMP–AMP synthase (cGAS) leads to the production of the secondary messenger cyclic GMP–AMP (cGAMP), which activates innate immune responses. We have shown that DNA binding to cGAS robustly induced the formation of liquidlike droplets in which cGAS was activated. The disordered and positively charged cGAS N terminus enhanced cGAS-DNA phase separation by increasing the valencies of DNA binding. Long DNA was more efficient in promoting cGAS liquid phase separation and cGAS enzyme activity than short DNA. Moreover, free zinc ions enhanced cGAS enzyme activity both in vitro and in cells by promoting cGAS-DNA phase separation. These results demonstrated that the DNA-induced phase transition of cGAS promotes cGAMP production and innate immune signaling.

 

2.297           Rbfox1 Mediates Cell-type-Specific Splicing in Cortical Interneurons

Warmsley, B., Jaglin, X.H., Favuzzi, E., Khodadadi-Jamayran, A., Rudy, B. and Fishell, G.

Neuron, 100, 846-859 (2018)

 

Cortical interneurons display a remarkable diversity in their morphology, physiological properties, and connectivity. Elucidating the molecular determinants underlying this heterogeneity is essential for understanding interneuron development and function. We discovered that alternative splicing differentially regulates the integration of somatostatin- and parvalbumin-expressing interneurons into nascent cortical circuits through the cell-type-specific tailoring of mRNAs. Specifically, we identified a role for the activity-dependent splicing regulator Rbfox1 in the development of cortical interneuron-subtype-specific efferent connectivity. Our work demonstrates that Rbfox1 mediates largely non-overlapping alternative splicing programs within two distinct but related classes of interneurons.

 

2.298           Ciprofloxacin impairs mitochondrial DNA replication initiation through inhibition of Topoisomerase 2 

Hangas, A., Aasumets, K., Kekäläinen, N.J., Paloheinä, M., Pohjoismäki, L., Gerhold, J.M. and Goffart, S.

Nucleic Acids Res., 46(18), 9625-9636 (2018)

 

Maintenance of topological homeostasis is vital for gene expression and genome replication in all organisms. Similar to other circular genomes, also mitochondrial DNA (mtDNA) is known to exist in various different topological forms, although their functional significance remains unknown. We report here that both known type II topoisomerases Top2α and Top2β are present in mammalian mitochondria, with especially Top2β regulating the supercoiling state of mtDNA. Loss of Top2β or its inhibition by ciprofloxacin results in accumulation of positively supercoiled mtDNA, followed by cessation of mitochondrial transcription and replication initiation, causing depletion of mtDNA copy number. These mitochondrial effects block both cell proliferation and differentiation, possibly explaining some of the side effects associated with fluoroquinolone antibiotics. Our results show for the first time the importance of topology for maintenance of mtDNA homeostasis and provide novel insight into the mitochondrial effects of fluoroquinolones.

 

2.299           Intermolecular crosslinking of abnormal prion protein is efficiently induced by a primuline-sensitized photoreaction

Teruya, K., Nishizawa, K., Oguma, A., Sakasegawa, Y. and Kitamoto, T.

BBA – General Subjects, 1863, 384-394 (2019)

 

In prion diseases, infectious pathogenic particles that are composed of abnormal prion proteins (PrP^Sc) accumulate in the brain. PrP^Sc is biochemically characterized by its protease-resistance core (PrP^res), but its structural features have not been fully elucidated. Here, we report that primuline, a fluorescent dye with photosensitization activity, dramatically enhances UV-irradiation-induced SDS-resistant PrP^Sc/res oligomer formation that can be detected by immunoblot analysis of prion-infected materials. This oligomer formation occurs specifically with PrP^Sc/res but not with normal prion protein, and it was demonstrated using purified PrP^Sc/res as well as unpurified materials. The oligomer formation proceeded in both primuline-dose- and UV irradiation time-dependent manners. Treatment with urea or formic acid did not break oligomers into monomers. Neither did the presence of aromatic amino acids modify oligomer formation. Analysis with a panel of anti-prion protein antibodies showed that the antibodies against the N-terminal region of PrP^res were less reactive in the dimer than the monomer. These findings suggest that the primuline-sensitized photoreaction enhances intermolecular crosslinking of PrP^Sc/res molecules at a hydrophobic area of the N-terminal region of PrP^res. In the screening of other compounds, photoreactive compounds such as luciferin exhibited a similar but lower activity with respect to oligomer formation than primuline. The enhanced photoreaction with these compounds will be useful for evaluating the structural features of PrP^Sc/res, especially the interactions between PrP^Sc/res molecules.

 

2.300           Cell-associated heparin-like molecules modulate the ability of LDL to regulate PCSK9 uptake

Galvan, A.M. and Chorba, J.S.

1. Lipid Res., 60, 71-84 (2019)

 

Proprotein convertase subtilisin/kexin type 9 (PCSK9) targets the LDL receptor (LDLR) for degradation, increasing plasma LDL and, consequently, cardiovascular risk. Uptake of secreted PCSK9 is required for its effect on the LDLR, and LDL itself inhibits this uptake, though how it does so remains unclear. In this study, we investigated the relationship between LDL, the PCSK9:LDLR interaction, and PCSK9 uptake. We show that LDL inhibits binding of PCSK9 to the LDLR in vitro more impressively than it inhibits PCSK9 uptake in cells. Furthermore, cell-surface heparin-like molecules (HLMs) can partly explain this difference, consistent with heparan sulfate proteoglycans (HSPGs) acting as coreceptors for PCSK9. We also show that HLMs can interact with either PCSK9 or LDL to modulate the inhibitory activity of LDL on PCSK9 uptake, with such inhibition rescued by competition with the entire PCSK9 prodomain, but not its truncated variants. Additionally, we show that the gain-of-function PCSK9 variant, S127R, located in the prodomain near the HSPG binding site, exhibits increased affinity for HLMs, potentially explaining its phenotype. Overall, our findings suggest a model where LDL acts as a negative regulator of PCSK9 function by decreasing its uptake via direct interactions with either the LDLR or HLMs.

 

2.301           Membrane trafficking of the bacterial adhesin GspB and the accessory Sec transport machinery

Spencer, C., Bensing, B.A., Mishra, N.N. and Sullam, P.M.

1. Biol. Chem., 294(5), 1502-1515 (2019)

 

The serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria are large, cell wall–anchored adhesins that mediate binding to many host cells and proteins and are associated with bacterial virulence. SRR glycoproteins are exported to the cell surface by the accessory Sec (aSec) system comprising SecA2, SecY2, and 3–5 additional proteins (Asp1 to Asp5) that are required for substrate export. These adhesins typically have a 90-amino acid-long signal peptide containing an elongated N-region and a hydrophobic core. Previous studies of GspB (the SRR adhesin of Streptococcus gordonii) have shown that a glycine-rich motif in its hydrophobic core is essential for selective, aSec-mediated transport. However, the role of this extended N-region in transport is poorly understood. Here, using protein–lipid co-flotation assays and site-directed mutagenesis, we report that the N-region of the GspB signal peptide interacts with anionic lipids through electrostatic forces and that this interaction is necessary for GspB preprotein trafficking to lipid membranes. Moreover, we observed that protein–lipid binding is required for engagement of GspB with SecA2 and for aSec-mediated transport. We further found that SecA2 and Asp1 to Asp3 also localize selectively to liposomes that contain anionic lipids. These findings suggest that the GspB signal peptide electrostatically binds anionic lipids at the cell membrane, where it encounters SecA2. After SecA2 engagement with the signal peptide, Asp1 to Asp3 promote SecA2 engagement with the mature domain, which activates GspB translocation.

2.302           Highly Reproducible Physiological Asymmetric Membrane with Freely Diffusing Embedded Proteins in a 3D‐Printed Microfluidic Setup

Heo, P., Ramakrishnan, S., Coleman, J., Rothman, J.E., Fleury, J-B. and Pincet, F.

Small, 15(21), 1900725 (2019)

 

Experimental setups to produce and to monitor model membranes have been successfully used for decades and brought invaluable insights into many areas of biology. However, they all have limitations that prevent the full in vitro mimicking and monitoring of most biological processes. Here, a suspended physiological bilayer‐forming chip is designed from 3D‐printing techniques. This chip can be simultaneously integrated to a confocal microscope and a path‐clamp amplifier. It is composed of poly(dimethylsiloxane) and consists of a ≈100 µm hole, where the horizontal planar bilayer is formed, connecting two open crossed‐channels, which allows for altering of each lipid monolayer separately. The bilayer, formed by the zipping of two lipid leaflets, is free‐standing, horizontal, stable, fluid, solvent‐free, and flat with the 14 types of physiologically relevant lipids, and the bilayer formation process is highly reproducible. Because of the two channels, asymmetric bilayers can be formed by making the two lipid leaflets of different composition. Furthermore, proteins, such as transmembrane, peripheral, and pore‐forming proteins, can be added to the bilayer in controlled orientation and keep their native mobility and activity. These features allow in vitro recapitulation of membrane process close to physiological conditions.

 

2.303           Aβ Oligomer Elimination Restores Cognition in Transgenic Alzheimer’s Mice with Full-blown Pathology

Schemmert, S., Schartmann, E., Zafiu, C., Kass, B., Hartwig, S., Lehr, S., Bannach, O., Langen, K-J., Shah, N.J., Kutzche, J., Willuweit, A. and Willbold, D.

Mol. Neurobiol., 56, 2211-2223 (2019)

 

Oligomers of the amyloid-β (Aβ) protein are suspected to be responsible for the development and progression of Alzheimer’s disease. Thus, the development of compounds that are able to eliminate already formed toxic Aβ oligomers is very desirable. Here, we describe the in vivo efficacy of the compound RD2, which was developed to directly and specifically eliminate toxic Aβ oligomers. In a truly therapeutic, rather than a preventive study, oral treatment with RD2 was able to reverse cognitive deficits and significantly reduce Aβ pathology in old-aged transgenic Alzheimer’s Disease mice with full-blown pathology and behavioral deficits. For the first time, we demonstrate the in vivo target engagement of RD2 by showing a significant reduction of Aβ oligomers in the brains of RD2-treated mice compared to placebo-treated mice. The correlation of Aβ elimination in vivo and the reversal of cognitive deficits in old-aged transgenic mice support the hypothesis that Aβ oligomers are relevant not only for disease development and progression, but also offer a promising target for the causal treatment of Alzheimer’s disease.

 

2.304           Cellular Trafficking of Amyloid Precursor Protein in Amyloidogenesis Physiological and Pathological Significance

Manucat-Tan, N., Saadipour, K., Wang, Y-J., Bobrovskaya, L. and Zhou, X-F.

Mol. Neurobiol., 56(2), 812-830 (2019)

 

The accumulation of excess intracellular or extracellular amyloid beta (Aβ) is one of the key pathological events in Alzheimer’s disease (AD). Aβ is generated from the cleavage of amyloid precursor protein (APP) by beta secretase-1 (BACE1) and gamma secretase (γ-secretase) within the cells. The endocytic trafficking of APP facilitates amyloidogenesis while at the cell surface, APP is predominantly processed in a non-amyloidogenic manner. Several adaptor proteins bind to both APP and BACE1, regulating their trafficking and recycling along the secretory and endocytic pathways. The phosphorylation of APP at Thr668 and BACE1 at Ser498, also influence their trafficking. Neurotrophins and proneurotrophins also influence APP trafficking through their receptors. In this review, we describe the molecular trafficking pathways of APP and BACE1 that lead to Aβ generation, the involvement of different signaling molecules or adaptor proteins regulating APP and BACE1 subcellular localization. We have also discussed how neurotrophins could modulate amyloidogenesis through their receptors.

 

2.305           New insights into the ORF2 capsid protein, a key player of the hepatitis E virus lifecycle

Ankavay, M., Montpellier, C., Sayed, I.M., Saliou, J-M., Wychowski, C., Saas, L., Duvet, S., Aliouat-Denis, C-M., Farhat, R., de Masson d’Autume, V., Meuleman, P.M Dubuisson, J. and Cocquerel, L.

Scientific Reports, 9:6243 (2019)

 

Hepatitis E Virus (HEV) genome encodes three proteins including the ORF2 capsid protein. Recently, we demonstrated that HEV produces three different forms of ORF2: (i) the ORF2i form (infectious ORF2) which is the component of infectious particles, (ii) the secreted ORF2g (glycosylated ORF2) and ORF2c (cleaved ORF2) forms that are not associated with infectious particles, but are the major antigens in HEV-infected patient sera. The ORF2 protein sequence contains three highly conserved potential N-glycosylation sites (N1, N2 and N3). The status and biological relevance of ORF2 N-glycosylation in HEV lifecycle remain to be elucidated. Here, we generated and extensively characterized a series of ORF2 mutants in which the three N-glycosylation sites were mutated individually or in combination. We demonstrated that the ORF2g/c protein is N-glycosylated on N1 and N3 sites but not on the N2 site. We showed that N-glycosylation of ORF2 protein does not play any role in replication and assembly of infectious HEV particles. We found that glycosylated ORF2g/c forms are very stable proteins which are targeted by patient antibodies. We also demonstrated that the ORF2i protein is translocated into the nucleus of infected cells. Hence, our study led to new insights into the molecular mechanisms of ORF2 expression.

 

2.306           The Legionella effector RavD binds phosphatidylinositol-3-phosphate and helps suppress endolysosomal maturation of the Legionella-containing vacuole

Pike, C.M., Boyer-Andersen, R., Kinch, L.N., Caplan, J.L. and Neunuebel, M.R.

J. Biol. Chem., 294(16), 6405-6415 (2019)

 

Upon phagocytosis into macrophages, the intracellular bacterial pathogen Legionella pneumophila secretes effector proteins that manipulate host cell components, enabling it to evade lysosomal degradation. However, the bacterial proteins involved in this evasion are incompletely characterized. Here we show that the L. pneumophila effector protein RavD targets host membrane compartments and contributes to the molecular mechanism the pathogen uses to prevent encounters with lysosomes. Protein–lipid binding assays revealed that RavD selectively binds phosphatidylinositol-3-phosphate (PI(3)P) in vitro. We further determined that a C-terminal RavD region mediates the interaction with PI(3)P and that this interaction requires Arg-292. In transiently transfected mammalian cells, mCherry-RavD colocalized with the early endosome marker EGFP-Rab5 as well as the PI(3)P biosensor EGFP-2×FYVE. However, treatment with the phosphoinositide 3-kinase inhibitor wortmannin did not disrupt localization of mCherry-RavD to endosomal compartments, suggesting that RavD's interaction with PI(3)P is not necessary to anchor RavD to endosomal membranes. Using superresolution and immunogold transmission EM, we observed that, upon translocation into macrophages, RavD was retained onto the Legionella-containing vacuole and was also present on small vesicles adjacent to the vacuole. We also report that despite no detectable effects on intracellular growth of L. pneumophila within macrophages or amebae, the lack of RavD significantly increased the number of vacuoles that accumulate the late endosome/lysosome marker LAMP-1 during macrophage infection. Together, our findings suggest that, although not required for intracellular replication of L. pneumophila, RavD is a part of the molecular mechanism that steers the Legionella-containing vacuole away from endolysosomal maturation pathways.

 

2.307           Oxidized LDL, homocysteine, homocysteine thiolactone and advanced glycation end products act as pro-oxidant metabolites inducing cytokine release, macrophage infiltration and pro-angiogenic effect in ARPE-19 cells

AnandBabu, K., Sen, P. and Angayarkanni, N.

PloS One, 14(5), e0216899 (2019)

 

Age-related Macular Degeneration (AMD) is one of the major vision-threatening diseases of the eye. Oxidative stress is one of the key factors in the onset and progression of AMD. In this study, metabolites associated with AMD pathology more so at the systemic level namely, oxidized LDL (oxLDL), homocysteine (Hcy), homocysteine thiolactone (HCTL), advanced glycation end product (AGE) were evaluated for their pro-oxidant nature in a localized ocular environment based on in vitro studies in human retinal pigment epithelial cells (ARPE-19 cells). Human ARPE-19 cells were treated with pro-oxidants 50 μg/mL oxLDL, 500 μM Hcy, 500 nM HCTL, 100 μg/mL AGE, 200 μM H₂O₂ and 200 μM H₂O₂ with and without pre-treatment of 5 mM N-acetyl cysteine (NAC). The cytokines IL-6, IL-8 and vascular endothelial growth factor (VEGF) secreted from ARPE-19 cells exposed to pro-oxidants were estimated by ELISA. In vitro angiogenesis assay was performed with conditioned media of the pro-oxidant treated ARPE-19 cells in Geltrex-Matrigel coated 96-well plate. The human acute monocytic leukemia cell line (THP-1) was differentiated into macrophages and its migration in response to conditioned media of ARPE-19 cells insulted with the pro-oxidants was studied by transwell migration assay. Western blot was performed to detect the protein expression of Bax, Bcl-2 and NF-κB to assess apoptotic changes. The compounds involved in the study showed a significant increase in reactive oxygen species (ROS) generation in ARPE-19 cells (oxLDL; Hcy; AGE: p < 0.001 and HCTL: p < 0.05). NAC pre-treatment significantly lowered the oxidative stress brought about by pro-oxidants as seen by lowered ROS and MDA levels in the cells. Treatment with pro-oxidants significantly increased the secretion of IL-6 (oxLDL: p < 0.05; Hcy, HCTL and AGE: p < 0.01) and IL-8 cytokines (oxLDL: p < 0.05; HCTL: p <. 001 and AGE: p < 0.01) in ARPE-19 cells. Serum samples of AMD patients (n = 23) revealed significantly higher IL-6 and IL-8 levels compared to control subjects (n = 23) (IL6: p < 0.01 and IL8: p < 0.05). The pro-oxidants also promoted VEGF secretion by ARPE-19 cells compared to untreated control (oxLDL: p < 0.001; Hcy: p < 0.01; HCTL and AGE: p < 0.05). In vitro angiogenesis assay showed that the conditioned media significantly increased the tube formation in RF/6A endothelial cells. Transwell migration assay revealed significant infiltration of macrophages in response to pro-oxidants. We further demonstrated that the pro-oxidants increased the Bax/Bcl-2 ratio and increased the NF-κB activation resulting in pro-apoptotic changes in ARPE-19 cells. Thus, oxLDL, Hcy, HCTL and AGE act as pro-oxidant metabolites in RPE that promote AMD through oxidative stress, inflammation, chemotaxis and neovascularization.

 

2.308           Chimaeric Rift Valley Fever Virus‐Like Particle Vaccine Candidate Production in Nicotiana benthamiana

Mbewana, S., Meyers, A.E. and Rybicki, E.P.

Biotech. J., 14(4), 1800238 (2019)

 

Rift Valley fever virus (RVFV) is an emerging mosquito‐borne virus and hemorrhagic fever agent, which causes abortion storms in farmed small ruminants and potentially causes miscarriages in humans. Although live‐attenuated vaccines are available for animals, they can only be used in endemic areas and there are currently no commercially available vaccines for humans. Here the authors describe the production of chimaeric RVFV virus‐like particles transiently expressed in Nicotiana benthamiana by Agrobacterium tumefaciens‐mediated gene transfer. The glycoprotein (Gn) gene is modified by removing its ectodomain (Gne) and fusing it to the transmembrane domain and cytosolic tail‐encoding region of avian influenza H5N1 hemagglutinin. This is expressed transiently in N. benthamiana with purified protein yields calculated to be ≈57 mg kg⁻¹ fresh weight. Transmission electron microscopy shows putative chimaeric RVFV Gne‐HA particles of 49–60 nm which are immunogenic, eliciting Gn‐specific antibody responses in vaccinated mice without the use of adjuvant. To our knowledge, this is the first demonstration of the synthesis of Gne‐HA chimaeric RVFV VLPs and the first demonstration of a detectable yield of RVFV Gn in plants.

 

2.309           The LipoGlo reporter system for sensitive and specific monitoring of atherogenic lipoproteins

Thierer, J.H., Ekker, S.C. and Farber, S.A.

Nature Communications, 10:3426 (2019)

 

Apolipoprotein-B (ApoB) is the structural component of atherogenic lipoproteins, lipid-rich particles that drive atherosclerosis by accumulating in the vascular wall. As atherosclerotic cardiovascular disease is the leading cause of death worldwide, there is an urgent need to develop new strategies to prevent lipoproteins from causing vascular damage. Here we report the LipoGlo system, which uses a luciferase enzyme (NanoLuc) fused to ApoB to monitor several key determinants of lipoprotein atherogenicity including particle abundance, size, and localization. Using LipoGlo, we comprehensively characterize the lipoprotein profile of individual larval zebrafish and collect images of atherogenic lipoprotein localization in an intact organism. We report multiple extravascular lipoprotein localization patterns, as well as identify Pla2g12b as a potent regulator of lipoprotein size. ApoB-fusion proteins thus represent a sensitive and specific approach to study atherogenic lipoproteins and their genetic and small molecule modifiers.

 

2.310           A programmable DNA-origami platform for studying lipid transfer between bilayers

Bian, X., Zhang, Z., Xiong, Q., De Camilli, P. and Lin, C.

Nature Chem. Biol., 15, 830-837 (2019)

 

Non-vesicular lipid transport between bilayers at membrane contact sites plays important physiological roles. Mechanistic insight into the action of lipid-transport proteins localized at these sites requires determination of the distance between bilayers at which this transport can occur. Here we developed DNA-origami nanostructures to organize size-defined liposomes at precise distances and used them to study lipid transfer by the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain of extended synaptotagmin 1 (E-Syt1). Pairs of DNA-ring-templated donor and acceptor liposomes were docked through DNA pillars, which determined their distance. The SMP domain was anchored to donor liposomes via an unstructured linker, and lipid transfer was assessed via a Förster resonance energy transfer (FRET)-based assay. We show that lipid transfer can occur over distances that exceed the length of an SMP dimer, which is compatible with the shuttle model of lipid transport. The DNA nanostructures developed here can also be adapted to study other processes occurring where two membranes are closely apposed to each other.

 

2.311           The role of surface chemistry in serum protein corona-mediated cellular delivery and gene silencing with lipid nanoparticles

Chen, D., Ganesh, S., Wang, W. and Amiiji, M.

Nanoscale, 11, 8760-8775 (2019)

 

Delivery of genetic medicines, such as small interfering RNA (siRNA), by lipid nanoparticles (LNPs) is a promising approach towards the treatment of diseases, such as solid tumors. However, in vitro and in vivo nanoparticle delivery efficiency is influenced by the formation of a protein corona in biological media. In this study, we have formulated four types of EnCore nanoparticles (F1 to F4) with a similar composition, but different polyethylene glycol (PEG) conjugated lipid chain lengths (carbon 14 vs. carbon 18) and molar ratios (6% vs. 3%). These LNPs showed dramatic differences in cellular delivery and transfection in hepatocellular carcinoma (HepG2) cells in the absence and presence of fetal bovine serum (FBS). The presence of proteins inhibited the cellular uptake of C18 (3%) nanoparticles, while it facilitated the cellular uptake of C14 nanoparticles. Among the adsorbed proteins from FBS, apolipoprotein E, but not apolipoprotein A1, affected the cellular uptake of the carbon 14 LNPs. Additionally, surface PEG was one of the determinants for the protein corona amount and composition. Finally, different serum to LNP volume ratios resulted in different protein enrichment patterns. Overall, the results showed a correlation between surface chemistry of LNPs and the protein corona composition suggesting a potential use for targeted delivery.

 

2.312           Early stage prion assembly involves two subpopulations with different quaternary structures and a secondary templating pathway

Igel-Egalon, A., Laferriere, F., Moudjou, M., Bohl, J., Mezache, M., Knäpple, T., Herzog, L., Reine, F., Jas-Duval, C., Doumic, M., Rezaei, h. and Beringue, V.

Communications Biol., 2:363 (2019)

 

The dynamics of aggregation and structural diversification of misfolded, host-encoded proteins in neurodegenerative diseases are poorly understood. In many of these disorders, including Alzheimer’s, Parkinson’s and prion diseases, the misfolded proteins are self-organized into conformationally distinct assemblies or strains. The existence of intrastrain structural heterogeneity is increasingly recognized. However, the underlying processes of emergence and coevolution of structurally distinct assemblies are not mechanistically understood. Here, we show that early prion replication generates two subsets of structurally different assemblies by two sequential processes of formation, regardless of the strain considered. The first process corresponds to a quaternary structural convergence, by reducing the parental strain polydispersity to generate small oligomers. The second process transforms these oligomers into larger ones, by a secondary autocatalytic templating pathway requiring the prion protein. This pathway provides mechanistic insights into prion structural diversification, a key determinant for prion adaptation and toxicity.

 

2.313           Mitochondrial Alkbh1 localizes to mtRNA granules and its knockdown induces the mitochondrial UPR in humans and C. elegans

Wagner, A., Hofmeister, O., Rolland, S.G., maiser, A., Aasumets, K., Schmitt, S., Schorpp, K., Feuchtinger, A., Hadian, K., Scheneider, S., Zischka, H., Leonhardt, H., Conradt, B., Gerhold, J.M. and Wolf, A.

1. Cell Sci., 132, jcs223891 (2019)

 

The Fe(II) and 2-oxoglutarate-dependent oxygenase Alkb homologue 1 (Alkbh1) has been shown to act on a wide range of substrates, like DNA, tRNA and histones. Thereby different enzymatic activities have been identified including, among others, demethylation of N³-methylcytosine (m³C) in RNA- and single-stranded DNA oligonucleotides, demethylation of N¹-methyladenosine (m¹A) in tRNA or formation of 5-formyl cytosine (f⁵C) in tRNA. In accordance with the different substrates, Alkbh1 has also been proposed to reside in distinct cellular compartments in human and mouse cells, including the nucleus, cytoplasm and mitochondria. Here, we describe further evidence for a role of human Alkbh1 in regulation of mitochondrial protein biogenesis, including visualizing localization of Alkbh1 into mitochondrial RNA granules with super-resolution 3D SIM microscopy. Electron microscopy and high-resolution respirometry analyses revealed an impact of Alkbh1 level on mitochondrial respiration, but not on mitochondrial structure. Downregulation of Alkbh1 impacts cell growth in HeLa cells and delays development in Caenorhabditis elegans, where the mitochondrial role of Alkbh1 seems to be conserved. Alkbh1 knockdown, but not Alkbh7 knockdown, triggers the mitochondrial unfolded protein response (UPR^mt) in C. elegans.

 

2.314           The role of apolipoprotein- and vitronectin-enriched protein corona on lipid nanoparticles for in vivo targeted delivery and transfection of oligonucleotides in murine tumor models

Chen, D., Parayath, N., Ganesh, S., Wnag, W. and Amiji, M.

Nanoscale, 11, 18806-18824 (2019)

 

The application of lipid-based nanoparticle (LNP) delivery systems remains a popular strategy for the systemic delivery of gene therapies to specific disease targets, including solid tumors. It is now well acknowledged that upon systemic administration, biomolecules from blood will adsorb onto nanoparticles’ surfaces, forming a “protein corona”, affording nanoparticles a “biological identity” on top of their “synthetic identity”. Detailed analysis of nanoparticle protein corona is gradually revealing the “missing link” between nanoparticle chemical properties and the biological identity. Nevertheless, the discovery of nanoparticle protein corona's impact on tumor delivery is limited. In this study, we demonstrate that protein corona can be manipulated by formulation composition and particle surface charge changes, and a single lipid switch could switch the nanoparticle protein corona profile. The protein corona composition differences had a profound impact on cell transfection, in vivo biodistribution as well as tumor-specific delivery efficiency. Nanoparticles with apolipoprotein-rich corona showed better delivery to hepatocellular carcinoma (HepG2) as compared to those with vitronectin-rich corona. In addition, we found that, the PEG conjugated lipid chain length and PEG amount in LNPs were key factors to consider in successful RNA interference therapy for solid tumors.

 

2.315           Elevated Lipoprotein(a) Levels Lower ABCA1 Cholesterol Efflux Capacity

Tavori, H., Fenton, A.M., Plubell, D.L., Rosario, S., Yerkes, E., Gasik, R., Miles, J., Bergstrom, P., Minnier, J., Fazio, S. and Pamir, N.

1. Clin. Endocrinol. Metab., 104(10), 4793-4803 (2019)

 

Context

Elevated serum lipoprotein(a) [Lp(a)] levels are associated with increased cardiovascular disease risk. ABCA1-mediated cholesterol efflux from macrophages may be an antiatherogenic process. Plasminogen (PLG) is a driver of ABCA1-mediated cholesterol efflux, and its action is inhibited by purified human Lp(a).

Objective

To determine the effects of Lp(a) in human serum on ABCA1 cholesterol efflux.

Methods

Cholesterol efflux capacity (CEC) was measured with two different cell-culture models using serum from 76 patients with either low (<50 mg/dL) or high (>50 mg/dL) Lp(a) levels.

Results

Using cAMP-stimulated J774 macrophages or baby hamster kidney fibroblasts overexpressing human ABCA1, we show that CEC was lower in patients with high Lp(a) levels compared with patients with low levels (−30.6%, P = 0.002 vs −24.1%, P < 0.001, respectively). Total-serum CEC negatively correlated with Lp(a) levels (r = −0.433, P = 0.0007 vs r = −0.505, P = 0.0011, respectively). These negative associations persisted after adjusting for serum cholesterol, age, sex, and statin use in a multiple linear regression model (adjusted R² = 0.413 or 0.405, respectively) and were strengthened when further adjusting for the interaction between Lp(a) and PLG levels (adjusted R² = 0.465 and 0.409, respectively). Total-serum and isolated Lp(a) from patients with high Lp(a) inhibited PLG-mediated ABCA1 cholesterol efflux.

Conclusion

Total-serum CEC is reduced in patients with high Lp(a) levels. This is in part due to the inhibition of PLG-mediated ABCA1 cholesterol efflux by Lp(a). Our findings suggest an atherogenic role for Lp(a) through its ability to inhibit CEC.

 

2.316           Critical Role of a Sheath Phosphorylation Site On the Assembly and Function of an Atypical Type VI Secretion System

Ziveri, J., Chhuon, C., Jamet, A., Rytter, h., Prigent, G., tros, F., Barel, M., Coureuil, M., Lays, C., Henry, T., Keep, N.H., Guerrera, I.C. and Charbit, A.

Mol. Cell. Proteomics, 18, 2418-2432 (2019)

 

The bacterial pathogen Francisella tularensis possesses a noncanonical type VI secretion system (T6SS) that is required for phagosomal escape in infected macrophages. KCl stimulation has been previously used to trigger assembly and secretion of the T6SS in culture. By differential proteomics, we found here that the amounts of the T6SS proteins remained unchanged upon KCl stimulation, suggesting involvement of post-translational modifications in T6SS assembly. A phosphoproteomic analysis indeed identified a unique phosphorylation site on IglB, a key component of the T6SS sheath. Substitutions of Y139 with alanine or phosphomimetics prevented T6SS formation and abolished phagosomal escape whereas substitution with phenylalanine delayed but did not abolish phagosomal escape in J774–1 macrophages. Altogether our data demonstrated that the Y139 site of IglB plays a critical role in T6SS biogenesis, suggesting that sheath phosphorylation could participate to T6SS dynamics.

 

2.317           Structural evidence for the critical role of the prion protein hydrophobic region in forming an infectious prion

Abskharon, R., Wang, F., Wohlkonig, A., Ruan, J., Soror, s., Giachin, G., Pardon, E., Zou, W., Legname, G., Ma, J. and Steyaert, J.

PloS Pathogens, 15(12), e1008139 (2019)

 

Prion or PrP^Sc is the proteinaceous infectious agent causing prion diseases in various mammalian species. Despite decades of research, the structural basis for PrP^Sc formation and prion infectivity remains elusive. To understand the role of the hydrophobic region in forming infectious prion at the molecular level, we report X-ray crystal structures of mouse (Mo) prion protein (PrP) (residues 89–230) in complex with a nanobody (Nb484). Using the recombinant prion propagation system, we show that the binding of Nb484 to the hydrophobic region of MoPrP efficiently inhibits the propagation of proteinase K resistant PrP^Sc and prion infectivity. In addition, when added to cultured mouse brain slices in high concentrations, Nb484 exhibits no neurotoxicity, which is drastically different from other neurotoxic anti-PrP antibodies, suggesting that the Nb484 can be a potential therapeutic agent against prion disease. In summary, our data provides the first structure-function evidence supporting a crucial role of the hydrophobic region of PrP in forming an infectious prion.

 

2.318           A transient amphipathic helix in the prodomain of PCSK9 facilitates binding to low-density lipoprotein particles

Sarkar, S.K., Foo, A.C.Y., Matyas, A., Asikhia, I., Kosenko, T., Goto, N.K., Vergara-jaque, A. and Lagace, T.A.

1. Biol. Chem., 295(8), 2285-2298 (2020)

 

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a ligand of low-density lipoprotein (LDL) receptor (LDLR) that promotes LDLR degradation in late endosomes/lysosomes. In human plasma, 30–40% of PCSK9 is bound to LDL particles; however, the physiological significance of this interaction remains unknown. LDL binding in vitro requires a disordered N-terminal region in PCSK9's prodomain. Here, we report that peptides corresponding to a predicted amphipathic α-helix in the prodomain N terminus adopt helical structure in a membrane-mimetic environment. This effect was greatly enhanced by an R46L substitution representing an atheroprotective PCSK9 loss-of-function mutation. A helix-disrupting proline substitution within the putative α-helical motif in full-length PCSK9 lowered LDL binding affinity >5-fold. Modeling studies suggested that the transient α-helix aligns multiple polar residues to interact with positively charged residues in the C-terminal domain. Gain-of-function PCSK9 mutations associated with familial hypercholesterolemia (FH) and clustered at the predicted interdomain interface (R469W, R496W, and F515L) inhibited LDL binding, which was completely abolished in the case of the R496W variant. These findings shed light on allosteric conformational changes in PCSK9 required for high-affinity binding to LDL particles. Moreover, the initial identification of FH-associated mutations that diminish PCSK9's ability to bind LDL reported here supports the notion that PCSK9-LDL association in the circulation inhibits PCSK9 activity.

 

2.319           Eicosapentaenoic acid (EPA) has optimal chain length and degree of unsaturation to inhibit oxidation of small dense LDL and membrane cholesterol domains as compared to related fatty acids in vitro

Sherratt, S.C.R., Juliano, R.A. and Mason, R.P.

BBA-Biomembranes, 1862, 183254 (2020)

 

Background

Oxidation of small dense low-density lipoprotein (sdLDL) and membranes is causally related to atherosclerosis. The omega-3 fatty acid (FA) eicosapentaenoic acid (EPA, 20:5, ω-3) significantly reduced oxidized LDL in patients with hypertriglyceridemia by unknown mechanisms. We compared EPA effects to related FAs of varying chain length and unsaturation on oxidation of sdLDL and model membranes, and on cholesterol crystal domains. We compared EPA to the FAs: stearic (SA, 18:0), oleic (OA, 18:1, ω-9), linoleic (LA, 18:2, ω-6), alpha-linolenic (ALA, 18:3, ω-3), eicosanoic (EA, 20:0), eicosatrienoic (ETE, 20:3, ω-3), arachidonic (AA, 20:4, ω-6), docosapentaenoic (DPA, 22:5, ω-3), and docosahexaenoic (DHA, 22:6, ω-3).

Methods

Human sdLDL or model membranes of cholesterol and 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine [18:2(cis)PC or DLPC] were preincubated with FAs followed by copper-induced oxidation. Malondialdehyde (MDA) or lipid hydroperoxides (LOOH) levels measured oxidation; small-angle X-ray diffraction assessed cholesterol domain formation.

Results

After 40 min, EPA reduced MDA levels 70% compared to vehicle (p < 0.001). Lesser inhibition was observed with DHA, DPA, ETE, and ALA (33%, 34%, 32%, and 16%, respectively; all p < 0.001 versus vehicle). Similar relative FA effects were observed in model membranes where EPA more substantially inhibited cholesterol crystal domain formation.

Conclusion

We observed relationships between hydrocarbon length and unsaturation with antioxidant activity and membrane cholesterol domain formation. EPA had the most favorable molecular structure, likely contributing to membrane stability, improved lipoprotein clearance, and reduced inflammation.

 

2.320           A muscle-specific calpain, CAPN3, forms a homotrimer

Hata, S., Doi, N., Shinkai-Ouchi, F. and Ono, Y.

BBA-Proteins and Proteomics, 1868, 140411 (2020)

 

Calpain-3 (CAPN3), a 94-kDa member of the calpain protease family, is abundant in skeletal muscle. Mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A, indicating that CAPN3 plays important roles in muscle physiology. CAPN3 has several unique features. A crystallographic study revealed that its C-terminal penta–EF-hand domains form a homodimer, suggesting that CAPN3 functions as a homodimeric protease. To analyze complex formation of CAPN3 in a more convenient manner, we performed blue native polyacrylamide gel electrophoresis and found that the observed molecular weight of native CAPN3, as well as recombinant CAPN3, was larger than 240 kDa. Further analysis by cross-linking and sequential immunoprecipitation revealed that CAPN3 in fact forms a homotrimer. Trimer formation was abolished by the deletion of the PEF domain, but not the CAPN3-specific insertion sequences NS, IS1, and IS2. The PEF domain alone formed a homodimer, as reported, but addition of the adjacent CBSW domain to its N-terminus reinforced the trimer-forming property. Collectively, these results suggest that CAPN3 forms a homotrimer in which the PEF domain's dimer-forming ability is influenced by other domains.

 

2.321           Sciadonic acid derived from pine nuts as a food component to reduce plasma triglycerides by inhibiting the rat hepatic Δ9-desaturase

Pedrono, F., Boulier-Monthean, N., Boissel, F., Ossemond, J., Viel, R., Fautrel, A., Marchix, J. and Dupont, D.

Scientific Reports, 10:6223 (2020)

 

Sciadonic acid (Scia) is a Δ5-olefinic fatty acid that is particularly abundant in edible pine seeds and that exhibits an unusual polymethylene-interrupted structure. Earlier studies suggested that Scia inhibited the in vitro expression and activity of the Stearoyl-CoA Desaturase 1 (SCD1), the hepatic Δ9-desaturase involved in the formation of mono-unsaturated fatty acids. To confirm this hypothesis, rats were given 10% Scia in diets balanced out with n-6 and n-3 fatty acids. In those animals receiving the Scia supplement, monoene synthesis in the liver was reduced, which was partly attributed to the inhibition of SCD1 expression. As a consequence, the presence of Scia induced a 50% decrease in triglycerides in blood plasma due to a reduced level of VLDL-secreted triglycerides from the liver. In non-fasting conditions, results showed that Scia-induced inhibition of SCD1 led to a decrease in the proportions of 16:1n-7 and 18:1n-7 in the liver without impacting on the level of 18:1n-9, suggesting that only triglycerides with neosynthesized monoenes are marked out for release. In conclusion, this in vivo study confirms that Scia highly inhibits SCD1 expression and activity. The work was performed on normo-triglyceride rats over six weeks, suggesting promising effects on hyper-triglyceridemic models.

 

2.322           Prion protein post-translational modifications modulate heparan sulfate binding and limit aggregate size in prion disease

Callender, J.A., Sevillano, A.M., Soldau, K., Kurt, T.D., Schumann, T., Pizzo, D.P., Altmeppen, H., Glatzel, M., Esko, J.D. and Sigurdson, C.J.

Neurobiol. of Disease, 142, 104955 (2020)

 

Many aggregation-prone proteins linked to neurodegenerative disease are post-translationally modified during their biogenesis. In vivo pathogenesis studies have suggested that the presence of post-translational modifications can shift the aggregate assembly pathway and profoundly alter the disease phenotype. In prion disease, the N-linked glycans and GPI-anchor on the prion protein (PrP) impair fibril assembly. However, the relevance of the two glycans to aggregate structure and disease progression remains unclear. Here we show that prion-infected knockin mice expressing an additional PrP glycan (tri-glycosylated PrP) develop new plaque-like deposits on neuronal cell membranes, along the subarachnoid space, and periventricularly, suggestive of high prion mobility and transit through the interstitial fluid. These plaque-like deposits were largely non-congophilic and composed of full length, uncleaved PrP, indicating retention of the glycophosphatidylinositol (GPI) anchor. Prion aggregates sedimented in low density fractions following ultracentrifugation, consistent with oligomers, and bound low levels of heparan sulfate (HS) similar to other predominantly GPI-anchored prions. Collectively, these results suggest that highly glycosylated PrP primarily converts as a GPI-anchored glycoform, with low involvement of HS co-factors, limiting PrP assembly mainly to oligomers. Since PrP^C is highly glycosylated, these findings may explain the high frequency of diffuse, synaptic, and plaque-like deposits in the brain as well as the rapid conversion commonly observed in human and animal prion disease.

 

2.323           Small sequence variations between two mammalian paralogs of the small GTPase SAR1 underlie functional differences in coat protein complex II assembly

Melville, D. B., Studer, S. and Schekman, R.

1. Biol. Chem., 295(25), 8401-8412 (2020)

 

Vesicles that are coated by coat protein complex II (COPII) are the primary mediators of vesicular traffic from the endoplasmic reticulum to the Golgi apparatus. Secretion-associated Ras-related GTPase 1 (SAR1) is a small GTPase that is part of COPII and, upon GTP binding, recruits the other COPII proteins to the endoplasmic reticulum membrane. Mammals have two SAR1 paralogs that genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion in the case of SAR1B. Here we identified two amino acid clusters that have conserved SAR1 paralog–specific sequences. We observed that one cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site for two COPII components, SEC31 homolog A COPII coat complex component (SEC31) and SEC23. We found that the latter cluster confers to SAR1B a binding preference for SEC23A that is stronger than that of SAR1A for SEC23A. Unlike SAR1B, SAR1A was prone to oligomerize on a membrane surface. SAR1B knockdown caused loss of lipoprotein secretion, overexpression of SAR1B but not of SAR1A could restore secretion, and a divergent cluster adjacent to the SEC31/SEC23-binding site was critical for this SAR1B function. These results highlight that small primary sequence differences between the two mammalian SAR1 paralogs lead to pronounced biochemical differences that significantly affect COPII assembly and identify a specific function for SAR1B in lipoprotein secretion, providing insights into the mechanisms of large cargo secretion that may be relevant for COPII-related diseases.

 

2.324           pH-responsive Frame-Guided Assembly with hydrophobicity controllable peptide as leading hydrophobic groups

Wang, C., Zhang, Y., Shao, Y., Tian, X., Piao, J., Dong, Y. and Liu, D.

Giant, 1, 100006 (2020)

 

Frame-Guided Assembly (FGA) strategy has been recently reported to prepare vesicles with customized shapes and sizes. However, the effects of the interaction between leading hydrophobic groups (LHGs) and amphiphiles on the thermodynamic and kinetic control of the FGA process haven't been fully understood. In this work, we employed the pH low-insertion peptide (pHLIP) as the LHGs because its interaction with lipids could be finely tuned by pH and investigated the mechanism of FGA in detail. Our study demonstrated the peptide frames could successfully guide the assembly of lipids to form hetero-liposomes below the pH transition point owing to the strong peptide and lipids interaction. The pH-dependent kinetic controlled FGA process was proved and factors affecting the FGA process were also investigated systematically. We believe this pH-responsive FGA strategy improved our knowledge on the mechanism of the FGA and provide inspiration in understanding the sophisticated assembly behavior in life.

 

2.325           Isolation of infectious, non-fibrillar and oligomeric prions from a genetic prion disease

Vanni, I., Pirisinu, L., Acevedo-Morantes, C., Kamali-Jamil, R., Rathod, V. et al

Brain, 143(5), 1512-1524 (2020)

 

Prions are transmissible agents causing lethal neurodegenerative diseases that are composed of aggregates of misfolded cellular prion protein (PrP^Sc). Despite non-fibrillar oligomers having been proposed as the most infectious prion particles, prions purified from diseased brains usually consist of large and fibrillar PrP^Sc aggregates, whose protease-resistant core (PrP^res) encompasses the whole C-terminus of PrP. In contrast, PrP^Sc from Gerstmann-Sträussler-Scheinker disease associated with alanine to valine substitution at position 117 (GSS-A117V) is characterized by a small protease-resistant core, which is devoid of the C-terminus. We thus aimed to investigate the role of this unusual PrP^Sc in terms of infectivity, strain characteristics, and structural features. We found, by titration in bank voles, that the infectivity of GSS-A117V is extremely high (10^9.3 ID₅₀ U/g) and is resistant to treatment with proteinase K (10^9.0 ID₅₀ U/g). We then purified the proteinase K-resistant GSS-A117V prions and determined the amount of infectivity and PrP^res in the different fractions, alongside the morphological characteristics of purified PrP^res aggregates by electron microscopy. Purified pellet fractions from GSS-A117V contained the expected N- and C-terminally cleaved 7 kDa PrP^res, although the yield of PrP^res was low. We found that this low yield depended on the low density/small size of GSS-A117V PrP^res, as it was mainly retained in the last supernatant fraction. All fractions were highly infectious, thus confirming the infectious nature of the 7 kDa PrP^res, with infectivity levels that directly correlated with the PrP^res amount detected. Finally, electron microscopy analysis of these fractions showed no presence of amyloid fibrils, but only very small and indistinct, non-fibrillar PrP^resparticles were detected and confirmed to contain PrP via immunogold labelling. Our study demonstrates that purified aggregates of 7 kDa PrP^res, spanning residues ∼90–150, are highly infectious oligomers that encode the biochemical and biological strain features of the original sample. Overall, the autocatalytic behaviour of the prion oligomers reveals their role in the propagation of neurodegeneration in patients with Gerstmann-Sträussler-Scheinker disease and implies that the C-terminus of PrP^Sc is dispensable for infectivity and strain features for this prion strain, uncovering the central PrP domain as the minimal molecular component able to encode infectious prions. These findings are consistent with the hypothesis that non-fibrillar prion particles are highly efficient propagators of disease and provide new molecular and morphological constraints on the structure of infectious prions.

 

2.326           Transbilayer Movement of Sphingomyelin Precedes Catastrophic Breakage of Enterobacteria-Containing Vacuoles

Ellison, C.J., Kukulski, W., Boyle, K.B., Munro, S. and Randow, F.

Current Biol., 30, 2974-2983 (2020)

 

Pathogenic bacteria enter the cytosol of host cells through uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the vacuolar membrane [1]. Bacterial invaders are sensed either directly, through cytosolic pattern-recognition receptors specific for bacterial ligands, or indirectly, through danger receptors that bind host molecules displayed in an abnormal context, for example, glycans on damaged BCVs [2, 3, 4]. In contrast to damage caused by Listeria monocytogenes, a Gram-positive bacterium, BCV rupture by Gram-negative pathogens such as Shigella flexneri or Salmonella Typhimurium remains incompletely understood [5, 6]. The latter may cause membrane damage directly, when inserting their Type Three Secretion needles into host membranes, or indirectly through translocated bacterial effector proteins [7, 8, 9]. Here, we report that sphingomyelin, an abundant lipid of the luminal leaflet of BCV membranes, and normally absent from the cytosol, becomes exposed to the cytosol as an early predictive marker of BCV rupture by Gram-negative bacteria. To monitor subcellular sphingomyelin distribution, we generated a live sphingomyelin reporter from Lysenin, a sphingomyelin-specific toxin from the earthworm Eisenia fetida [10, 11]. Using super resolution live imaging and correlative light and electron microscopy (CLEM), we discovered that BCV rupture proceeds through two distinct successive stages: first, sphingomyelin is gradually translocated into the cytosolic leaflet of the BCV, invariably followed by cytosolic exposure of glycans, which recruit galectin-8, indicating bacterial entry into the cytosol. Exposure of sphingomyelin on BCVs may therefore act as an early danger signal alerting the cell to imminent bacterial invasion.

 

2.327           Exploring the effects of carrier oil type on in vitro bioavailability of β-carotene: A cell culture study of carotenoid-enriched nanoemulsions

Xia, Z., Han, Y., Du, H., McClements, D.J., Tang, Z. and Xiao, H.

LWT-Food Sci. Technol., 134, 110224 (2020)

 

β-carotene was encapsulated in either olive oil or flaxseed oil-in-water nanoemulsions, and it's in vitro bioaccessibility and bioavailability were determined in a simulated gastrointestinal digestion model (mouth, stomach and small intestine) combined with a Caco-2 cell monolayer model. Nanoemulsions fabricated from both types of oils significantly increased the in vitro bioaccessibility and bioavailability of β-carotene. Olive oil, however, was digested more efficiently, which generated more free fatty acids capable of forming mixed micelles. Furthermore, the mixed micelles stimulated the formation of lipoprotein particles (chylomicrons and very low-density lipoproteins), which are the transcellular carriers of β-carotene in the intestinal epithelium, leading to an increase in bioavailability. Interestingly, olive oil led to the formation of larger chylomicrons than flaxseed oil, suggesting that fatty acid type impacts the nature of the lipoprotein particles formed after lipid digestion.

 

2.328           A seven-residue deletion in PrP leads to generation of a spontaneous prion formed from C-terminal C1 fragment of PrP

Munoz-Montesino, C., larkem, D., Barbereau, C., Igel-Egalon, A., Truchet, S., Jacquet, E., Nhiri, N., Moudjou, M., Sizun, C., Rezaei, H., Beringue, V.and Dron, M.

J. Biol. Chem., 295(41), 14025-14039 (2020)

 

Prions result from a drastic conformational change of the host-encoded cellular prion protein (PrP), leading to the formation of β-sheet–rich, insoluble, and protease-resistant self-replicating assemblies (PrPSc). The cellular and molecular mechanisms involved in spontaneous prion formation in sporadic and inherited human prion diseases or equivalent animal diseases are poorly understood, in part because cell models of spontaneously forming prions are currently lacking. Here, extending studies on the role of the H2 α-helix C terminus of PrP, we found that deletion of the highly conserved 190HTVTTTT196 segment of ovine PrP led to spontaneous prion formation in the RK13 rabbit kidney cell model. On long-term passage, the mutant cells stably produced proteinase K (PK)–resistant, insoluble, and aggregated assemblies that were infectious for naïve cells expressing either the mutant protein or other PrPs with slightly different deletions in the same area. The electrophoretic pattern of the PK-resistant core of the spontaneous prion (ΔSpont) contained mainly C-terminal polypeptides akin to C1, the cell-surface anchored C-terminal moiety of PrP generated by natural cellular processing. RK13 cells expressing solely the Δ190–196 C1 PrP construct, in the absence of the full-length protein, were susceptible to ΔSpont prions. ΔSpont infection induced the conversion of the mutated C1 into a PK-resistant and infectious form perpetuating the biochemical characteristics of ΔSpont prion. In conclusion, this work provides a unique cell-derived system generating spontaneous prions and provides evidence that the 113 C-terminal residues of PrP are sufficient for a self-propagating prion entity.

 

2.329           Overexpression of α-Synuclein by Oligodendrocytes in Transgenic Mice Does Not Recapitulate the Fibrillar Aggregation Seen in Multiple System Atrophy

Laferriere, F., He, X., Zinghirino, F., Doudnikoff, E., Faggiani, E., Meissner, W.G., Bezard, E., De Giorgi, F. and Ichas, F.

Cells, 9:2371 (2020)

 

The synucleinopathy underlying multiple system atrophy (MSA) is characterized by the presence of abundant amyloid inclusions containing fibrillar α-synuclein (α-syn) aggregates in the brains of the patients and is associated with an extensive neurodegeneration. In contrast to Parkinson’s disease (PD) where the pathological α-syn aggregates are almost exclusively neuronal, the α-syn inclusions in MSA are principally observed in oligodendrocytes (OLs) where they form glial cytoplasmic inclusions (GCIs). This is intriguing because differentiated OLs express low levels of α-syn, yet pathogenic amyloid α-syn seeds require significant amounts of α-syn monomers to feed their fibrillar growth and to eventually cause the buildup of cytopathological inclusions. One of the transgenic mouse models of this disease is based on the targeted overexpression of human α-syn in OLs using the PLP promoter. In these mice, the histopathological images showing a rapid emergence of S129-phosphorylated α-syn inside OLs are considered as equivalent to GCIs. Instead, we report here that they correspond to the accumulation of phosphorylated α-syn monomers/oligomers and not to the appearance of the distinctive fibrillar α-syn aggregates that are present in the brains of MSA or PD patients. In spite of a propensity to co-sediment with myelin sheath contaminants, the phosphorylated forms found in the brains of the transgenic animals are soluble (>80%). In clear contrast, the phosphorylated species present in the brains of MSA and PD patients are insoluble fibrils (>95%). Using primary cultures of OLs from PLP-αSyn mice we observed a variable association of S129-phosphorylated α-syn with the cytoplasmic compartment, the nucleus and with membrane domains suggesting that OLs functionally accommodate the phospho-α-syn deriving from experimental overexpression. Yet and while not taking place spontaneously, fibrillization can be seeded in these primary cultures by challenging the OLs with α-syn preformed fibrils (PFFs). This indicates that a targeted overexpression of α-syn does not model GCIs in mice but that it can provide a basis for seeding aggregation using PFFs. This approach could help establishing a link between α-syn aggregation and the development of a clinical phenotype in these transgenic animals.

 

2.330           Complex multicomponent patterns rendered on a 3D DNA-barrel pegboard

Wickham, S.F.J., Auer, A., Min, J., Ponnuswamy, N., Wooehrstein, J.B., Schueder, F. et al

Nature Communications, 11:5768 (2020)

 

DNA origami, in which a long scaffold strand is assembled with a many short staple strands into parallel arrays of double helices, has proven a powerful method for custom nanofabrication. However, currently the design and optimization of custom 3D DNA-origami shapes is a barrier to rapid application to new areas. Here we introduce a modular barrel architecture, and demonstrate hierarchical assembly of a 100 megadalton DNA-origami barrel of ~90 nm diameter and ~250 nm height, that provides a rhombic-lattice canvas of a thousand pixels each, with pitch of ~8 nm, on its inner and outer surfaces. Complex patterns rendered on these surfaces were resolved using up to twelve rounds of Exchange-PAINT super-resolution microscopy. We envision these structures as versatile nanoscale pegboards for applications requiring complex 3D arrangements of matter, which will serve to promote rapid uptake of this technology in diverse fields beyond specialist groups working in DNA nanotechnology.

 

2.331           RCC1L (WBSCR16) isoforms coordinate mitochondrial ribosome assembly through their interaction with GTPases

Reyes, A., Favia, P., Vidoni, S., Petruzzella, V. and Zeviani, M.

PloS Genetics, 16(7), e1008923 (2020)

 

Mitochondrial translation defects can be due to mutations affecting mitochondrial- or nuclear-encoded components. The number of known nuclear genes involved in mitochondrial translation has significantly increased in the past years. RCC1L (WBSCR16), a putative GDP/GTP exchange factor, has recently been described to interact with the mitochondrial large ribosomal subunit. In humans, three different RCC1L isoforms have been identified that originate from alternative splicing but share the same N-terminus, RCC1L^V1, RCC1L^V2 and RCC1L^V3. All three isoforms were exclusively localized to mitochondria, interacted with its inner membrane and could associate with homopolymeric oligos to different extent. Mitochondrial immunoprecipitation experiments showed that RCC1L^V1 and RCC1L^V3 associated with the mitochondrial large and small ribosomal subunit, respectively, while no significant association was observed for RCC1L^V2. Overexpression and silencing of RCC1L^V1 or RCC1L^V3 led to mitoribosome biogenesis defects that resulted in decreased translation. Indeed, significant changes in steady-state levels and distribution on isokinetic sucrose gradients were detected not only for mitoribosome proteins but also for GTPases, (GTPBP10, ERAL1 and C4orf14), and pseudouridylation proteins, (TRUB2, RPUSD3 and RPUSD4). All in all, our data suggest that RCC1L is essential for mitochondrial function and that the coordination of at least two isoforms is essential for proper ribosomal assembly.

 

2.332           Inactivation of the mitochondrial protease Afg3l2 results in severely diminished respiratory chain activity and widespread defects in mitochondrial gene expression

Pareek, G. and Pallanck, L.J.

PloS Genetics, 16(10), e1009118 (2020)

 

The m-AAA proteases play a critical role in the proteostasis of inner mitochondrial membrane proteins, and mutations in the genes encoding these proteases cause severe incurable neurological diseases. To further explore the biological role of the m-AAA proteases and the pathological consequences of their deficiency, we used a genetic approach in the fruit fly Drosophila melanogaster to inactivate the ATPase family gene 3-like 2 (AFG3L2) gene, which encodes a critical component of the m-AAA proteases. We found that null alleles of Drosophila AFG3L2 die early in development, but partial inactivation of AFG3L2 using RNAi allowed survival to the late pupal and adult stages of development. Flies with partial inactivation of AFG3L2 exhibited behavioral defects, neurodegeneration, accumulation of unfolded mitochondrial proteins, and diminished respiratory chain (RC) activity. Further work revealed that the reduced RC activity was primarily a consequence of severely diminished mitochondrial transcription and translation. These defects were accompanied by activation of the mitochondrial unfolded protein response (mito-UPR) and autophagy. Overexpression of mito-UPR components partially rescued the AFG3L2-deficient phenotypes, indicating that protein aggregation partly accounts for the defects of AFG3L2-deficient animals. Our work suggests that strategies designed to activate mitochondrial stress pathways and mitochondrial gene expression could be therapeutic in the diseases caused by mutations in AFG3L2.

 

2.333           Identification of distinct pathological signatures induced by patient-derived α-synuclein structures in nonhuman primates

Bourdenx, M., Nioche, A., Dovero, S., Arotcarena, M.L., Camus, S., Porras, G. et al

Sci. Adv., 6:eaa9165 (2020)

 

Dopaminergic neuronal cell death, associated with intracellular α-synuclein (α-syn)–rich protein aggregates [termed “Lewy bodies” (LBs)], is a well-established characteristic of Parkinson’s disease (PD). Much evidence, accumulated from multiple experimental models, has suggested that α-syn plays a role in PD pathogenesis, not only as a trigger of pathology but also as a mediator of disease progression through pathological spreading. Here, we have used a machine learning–based approach to identify unique signatures of neurodegeneration in monkeys induced by distinct α-syn pathogenic structures derived from patients with PD. Unexpectedly, our results show that, in nonhuman primates, a small amount of singular α-syn aggregates is as toxic as larger amyloid fibrils present in the LBs, thus reinforcing the need for preclinical research in this species. Furthermore, our results provide evidence supporting the true multifactorial nature of PD, as multiple causes can induce a similar outcome regarding dopaminergic neurodegeneration.

 

2.334           The Free Radical Scavenging and Anti-Isolated Human LDL Oxidation Activities of Pluchea indica (L.) Less. Tea Compared to Green Tea (Camellia sinensis)

Sirichaiwetchakoon, K., Lowe, G.M. and Eumkeb, G.

BioMed Res. Int., 2020:4183643 (2020)

 

Tea is one of the most popular beverages in the world. Camellia sinensis tea (CST) or green tea is widely regarded as a potent antioxidant. In Thailand, Pluchea indica (L.) Less. tea (PIT) has been commercially available as a health-promoting drink. This study focused on free radical scavenging activities of PIT, and its ability to protect isolated human low-density lipoproteins (LDL) from oxidation by chemical agents. A preliminary study to investigate the antioxidant nature of PIT was undertaken. These included common antioxidant assays involving 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis-(3- ethylbenzothiazoline)-6-sulfonic acid (ABTS), hypochlorous acid (HOCl), and its potential to scavenge peroxynitrite. In separated experiments, isolated human LDL was challenged with either 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), copper (Cu2+), or 3-Morpholinosydnonimine hydrochloride (SIN-1) to induce LDL oxidation. PIT exhibited antioxidant activity in all test systems and performed significantly better than CST in both DPPH (P < 0:05; IC50PIT = 245:85 ± 15:83 and CST = 315:41 ± 24:18 μg/ml) and peroxynitrite scavenging assays. PIT at 75 μg/ml almost fully prevented the peroxynitrite over a 5 h period. Moreover, it displayed similar properties to CST during the antioxidation of isolated human LDL using AAPH, Cu2+, SIN-1, and hypochlorous acid scavenging assays. However, it revealed a significantly lower ABTS scavenging activity than CST (P < 0:05; IC50PIT = 30:47 ± 2:20 and CST = 21:59 ± 0:67 μg/ml). The main constituents of the PIT were identified using LC-MS/MS. It contained 4-O-caffeoylquinic acid (4-CQ), 5-O-caffeoylquinic acid (5-CQ), 3,4-O-dicaffeoylquinic acid (3,4-CQ), 3,5-O-dicaffeoylquinic acid (3,5-CQ), and 4,5-O-dicaffeoylquinic acid (4,5-CQ). In conclusion, caffeoyl derivatives in PIT could play an important role in potent antioxidant properties. So, it may be further developed to be antioxidant beverages for preventing atherosclerosis and cardiovascular diseases associated with oxidative stress

 

2.335           Optimizing the method of plasma lipoprotein isolation for elucidating their differential association to proprotein convertase subtilisin/kexin 9 (PCSK9)

Canclini, L., Malvandi, A.M., Uboldi, P., Zampoleri, V., Bellosta, S., Bargetti, A., Grigore, l., Zambon, A. and Catapano, A.L.

Atherosclerosis, 315, e161 (2020)

 

Background and Aims: PCSK9 is a secretory protease that has emerged as a target to regulate lipoprotein metabolism. Antibodies blocking PCSK9 reduce LDL-C levels through the prevention of PCSK9 binding to the LDL receptor. The existence of PCSK9-lipoprotein complexes is reported, but data in literature are discordant. This project aims at studying the existence of this interaction and its possible biological consequences. To reach this goal, we aimed at setting the optimal condition in the lipoprotein isolation method that helps build up the capacity for future clinical studies.

Methods: Fresh sera were collected from 60 healthy individuals to isolate the lipoprotein fraction using different methods including precipitation with phosphotungstic acid, fast protein liquid chromatography (FPLC), ultracentrifugation using KBr or iodixanol gradient (IGr), as a nonionic hydrophilic compound that would not interfere with molecular associations. The resulting fractions were analyzed to detect PCSK9 with ELISA and LP(a), ApoB, ApoA1, and Cholesterol using clinical-grade turbidimetry assays.

Results: In the precipitation-mediated assay, around 60% of PCSK9 was found in ApoB fraction. In the FPLC, 10±0.5% of recovered PCSK9 was detected in the LDL fraction. No PCSK9 was detectable in lipoprotein fraction isolated by ultracentrifugation in the presence of KBr, whereas 23±8% of PCSK9 was found in LDL fraction isolated with the iodixanol gradient.

Conclusions: Based on our observations, it appears that the association of PCSK9 and LDL is sensitive to salt concentrations and changes with the isolation method. No binding to other lipoproteins was detected. More studies are requested to define the type of interaction.

 

2.336           Adiponectin and related C1q/TNF-related proteins bind selectively to anionic phospholipids and sphingolipids

Ye, J.J., Bian, X., Lim, J. and Medzhitov, R.

PNAS, 117(229), 17381-17388 (2020)

 

Adiponectin (Acrp30) is an adipokine associated with protection from cardiovascular disease, insulin resistance, and inflammation. Although its effects are conventionally attributed to binding Adipor1/2 and T-cadherin, its abundance in circulation, role in ceramide metabolism, and homology to C1q suggest an overlooked role as a lipid-binding protein, possibly generalizable to other C1q/TNF-related proteins (CTRPs) and C1q family members. To investigate this, adiponectin, representative family members, and variants were expressed in Expi293 cells and tested for binding to lipids in liposomes using density centrifugation. Binding to physiological lipids were also analyzed using gradient ultracentrifugation, liquid chromatography-mass spectrometry, and shotgun lipidomics. Interestingly, adiponectin selectively bound several anionic phospholipids and sphingolipids, including phosphatidylserine, ceramide-1-phosphate, glucosylceramide, and sulfatide, via the C1q domain in an oligomerization-dependent fashion. Binding to lipids was observed in liposomes, low-density lipoproteins, cell membranes, and plasma. Other CTRPs and C1q family members (Cbln1, CTRP1, CTRP5, and CTRP13) also bound similar lipids. These findings suggest that adiponectin and CTRPs function not only as hormones, but also as lipid opsonins, as may other C1q family proteins.

 

2.337           Medin aggregation causes cerebrovascular dysfunction in aging wild-type mice

Degenhardt, K., Wagner, J., Skodras, A., Candlish, M. et al

PNAS, 117(38), 23925-23931 (2020)

 

Medin is the most common amyloid known in humans, as it can be found in blood vessels of the upper body in virtually everybody over 50 years of age. However, it remains unknown whether deposition of Medin plays a causal role in age-related vascular dysfunction. We now report that aggregates of Medin also develop in the aorta and brain vasculature of wild-type mice in an age-dependent manner. Strikingly, genetic deficiency of the Medin precursor protein, MFG-E8, eliminates not only vascular aggregates but also prevents age-associated decline of cerebrovascular function in mice. Given the prevalence of Medin aggregates in the general population and its role in vascular dysfunction with aging, targeting Medin may become a novel approach to sustain healthy aging.

 

2.338           Range of SHH signaling in adrenal gland is limited by membrane contact to cells with primary cilia

Mateska, I., Nanda, K., Dye, N.A., Alexaki, V.I. and Eaton, S.

1. Cell Biol., 219(12), e201910087 (2020)

 

The signaling protein Sonic Hedgehog (SHH) is crucial for the development and function of many vertebrate tissues. It remains largely unclear, however, what defines the range and specificity of pathway activation. The adrenal gland represents a useful model to address this question, where the SHH pathway is activated in a very specific subset of cells lying near the SHH-producing cells, even though there is an abundance of lipoproteins that would allow SHH to travel and signal long-range. We determine that, whereas adrenal cells can secrete SHH on lipoproteins, this form of SHH is inactive due to the presence of cosecreted inhibitors, potentially explaining the absence of long-range signaling. Instead, we find that SHH-producing cells signal at short range via membrane-bound SHH, only to receiving cells with primary cilia. Finally, our data from NCI-H295R adrenocortical carcinoma cells suggest that adrenocortical tumors may evade these regulatory control mechanisms by acquiring the ability to activate SHH target genes in response to TGF-β.

 

2.339           EIF3H Orchestrates Hippo Pathway–Mediated Oncogenesis via Catalytic Control of YAP Stability

Zhou, Z., Zhou, H., Ponzoni, L., Luo, A., He, M., Huang, Y., Guan, K-L-. Bahar, I., Liu, Z. and Wan, Y.

Cancer Res., 80, 2550-2563 (2020)

 

EIF3H is presumed to be a critical translational initiation factor. Here, our unbiased screening for tumor invasion factors has identified an unexpected role for EIF3H as a deubiquitylating enzyme that dictates breast tumor invasion and metastasis by modulating the Hippo–YAP pathway. EIF3H catalyzed YAP for deubiquitylation, resulting in its stabilization. Structure-based molecular modeling and simulations coupled with biochemical characterization unveiled a unique catalytic mechanism for EIF3H in dissociating polyubiquitin chains from YAP through a catalytic triad consisting of Asp90, Asp91, and Gln121. Trp119 and Tyr 140 on EIF3H directly interacted with the N-terminal region of YAP1, facilitating complex formation of EIF3H and YAP1 for YAP1 deubiquitylation. Stabilization of YAP via elevated EIF3H promoted tumor invasion and metastasis. Interference of EIF3H-mediated YAP deubiquitylation blocked YAP-induced tumor progression and metastasis in breast cancer models. These findings point to a critical role for YAP regulation by EIF3H in tumor invasion and metastasis.

 

2.340           Association of circulating levels of total and protein-bound sphingosine 1-phosphate with osteoporotic fracture

Song, H.E., Lee, S.H., Kim, S.J., Kim, B-J., Yoo, H.J. and Koh, J-M.

1. Investig. Med., 68, 1295-1299 (2020)

 

The biological activity and effects of circulating sphingosine 1-phosphate (S1P) might be dependent on the carrier protein. Although S1P is known to be a biomarker for osteoporotic fracture (OF), its role according to its carrier protein (high-density lipoprotein (HDL), low-density lipoprotein (LDL), or albumin) has not yet been studied. We measured the protein-bound S1P levels and bone mineral density (BMD) in 58 postmenopausal women with OF and 58 age-matched and body mass index–matched postmenopausal women without OF. Albumin-bound S1P was the most abundant. Before adjustment, women with OF had higher total S1P (p=0.046) and albumin-bound S1P (p=0.026) levels than those without OF, but there was no difference in the levels of HDL-bound or LDL-bound S1P. After adjustment for confounders including BMD, women with OF had only higher levels of total S1P than those without OF (p=0.047). Before adjustment, the OR for OF was higher in subjects in the highest quartile for total S1P (OR 5.36, 95% CI 1.22 to 23.63) or albumin-bound S1P (OR 4.48, 95% CI 1.22 to 16.42). After adjustment for confounders including BMD, statistical significance persisted only for total S1P (OR 2.23, 95% CI 1.12 to 4.81). These findings suggest that the positive association of S1P with OF is mainly due to level of total plasma S1P and not due to the differing contributions from specific carrier protein-bound fractions.

 

2.341           Unveiling the pitfalls of the protein corona of polymeric drug nanocarriers

Berrecoso, G., Crecente-Campo, J. and Alonso, M.J.

Drug Delivery and Transl. Res., 10, 730-750 (2020)

 

The protein corona is a natural protein layer spontaneously formed around nanomaterials when exposed to biological media. This layer can alter the nanosystems’ biological performance, particularly their tissue accumulation, cellular uptake, clearance by the immune system, toxicity, and even the release profile of their payloads. Hence, the characterization of this protein layer has become a critical step when developing a new nanomedicine. The modification of the nanosystem fate by the protein corona, systematically ignored in the vast majority of the nanotechnology-based research, may have contributed to the low in vitro/in vivo correlation. Actually, the protein corona of polymeric nanosystems has been scarcely studied in the literature, and most studies have been focused instead on metallic nanoparticles and liposomes. In this review, we analyzed the influence of the physicochemical properties and composition of the polymeric nanosystems on the protein layer deposited around them. In addition, we present some recommendations on how to perform the protein corona studies of polymeric nanoparticles, which, hopefully, will contribute to obtain more reliable and reproducible data in the future.

 

2.342           High-Density Lipoproteins Are the Main Carriers of PCSK9 in the Circulation

S.A. Burnap and M. Mayr et. al.

1. Am. Coll. Cardial., 75(12), 2667-2676 (2020)

 

Proprotein convertase subtilisin/kexin type 9 (PCSK9), a secreted protein that regulates circulating low-density lipoprotein (LDL) through the hepatic LDL receptor degradation pathway, is the latest therapeutic target to further lower cholesterol in patients on maximal statin therapy (1). Circulating PCSK9 has been shown to bind to LDL and lipoprotein(a) (Lp[a]). The latter is an LDL particle carrying apolipoprotein(a) as an additional protein component (2,3). Furthermore, the multimeric state of PCSK9 is thought to be influenced by lipoproteins, including high-density lipoprotein (HDL), regulating the LDL receptor–degrading capabilities of PCSK9 (4). However, the presence of PCSK9 on other lipoprotein particles is less well established, in particular in humans.

The potential associations of PCSK9 with different human lipoproteins was first determined by immunocapture, as previously described (5). Alirocumab, a human monoclonal antibody to PCSK9, was coated as a capture antibody to measure plasma PCSK9 in 20 healthy volunteers. Antibodies specific for apolipoprotein(a), apolipoprotein B (ApoB), and apolipoprotein A1 were then used to interrogate PCSK9-lipoprotein associations with Lp(a), ApoB-containing lipoproteins, and HDL, respectively. Measurement of lipoprotein-associated PCSK9 suggested a predominant association of PCSK9 with HDL (Figure 1A). For validation, plasma was subject to an anti-ApoB immunoprecipitation (Sun Diagnostics, New Gloucester, Maine). Successful removal of ApoB-carrying lipoproteins, including Lp(a), as confirmed by targeted mass spectrometry, led to only a <20% removal of PCSK9 as measured by enzyme-linked immunosorbent assay (DY3888, R&D Systems, Minneapolis, Minnesota; data not shown). In contrast, HDL removal using an HDL immunodepletion column (Genway Biotech, San Diego, California) resulted in a >90% removal of PCSK9 from fasting plasma (data not shown).

 

2.343           Pharmacologic Suppression of B7-H4 Glycosylation Restores Antitumor Immunity in Immune-Cold Breast Cancers

Sosng, X., Zhou, Z., Li, H., Xue, Y., Lu, X., Baha, I., Keep, O., Hung, M-C., Kroemer, G. and Wan, Y.

CancerDiscov., 10(12), 1872-1893 (2020)

 

Despite widespread utilization of immunotherapy, treating immune-cold tumors has proved to be a challenge. Here, we report that expression of the immune checkpoint molecule B7-H4 is prevalent among immune-cold triple-negative breast cancers (TNBC), where its expression inversely correlates with that of PD-L1. Glycosylation of B7-H4 interferes with its interaction/ubiquitination by AMFR, resulting in B7-H4 stabilization. B7-H4 expression inhibits doxorubicin-induced cell death through the suppression of eIF2α phosphorylation required for calreticulin exposure vis-à-vis the cancer cells. NGI-1, which inhibits B7-H4 glycosylation causing its ubiquitination and subsequent degradation, improves the immunogenic properties of cancer cells treated with doxorubicin, enhancing their phagocytosis by dendritic cells and their capacity to elicit CD8⁺ IFNγ-producing T-cell responses. In preclinical models of TNBC, a triple combination of NGI-1, camsirubicin (a noncardiotoxic doxorubicin analogue) and PD-L1 blockade was effective in reducing tumor growth. Collectively, our findings uncover a strategy for targeting the immunosuppressive molecule B7-H4.

 

2.344           Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion

Matoba, K., Kotani, T., Tsutsumi, A., Tsuji, T., Mori, T., Noshiro, D., Sugita, Y., Nomura, n., Iwata, S., Ohsumi, Y., Fujimoto, T., Nakatogawa, H., Kikkawa, M. and Noda, N.N.

Nature Struct. Mol. Biol., 27, 1185-1193 (2020)

 

The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9’s ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.

 

2.345           A critical review: Recent advances in “digital” biomolecule detection with single copy sensitivity

Liu, H. and Lei, Y.

Biosensors and Bioelectronics, 177, 112901 (2021)

 

Detection of a single biomolecule, ranging from nucleic acids, proteins, viruses to bacteria, is of paramount importance in various fields including biology, environment, food and agriculture industry, public health, and medicine. With the understanding of the biological functions of these biomolecules (or bioparticles) and their impacts on public health, environmental pollution, and food safety, advanced detection techniques are unprecedentedly demanded for their early and/or sensitive detection. In this critical review, a series of elegant research about digital detection of biomolecules with potential single copy sensitivity is reviewed and summarized with the focus on the design principle and the innovation of how to accomplish the “digital” detection concept. Starting with a brief introduction on the importance of digital detection, recent advances in “digital” biomolecule detection with single copy sensitivity are grouped and discussed based on the difference of signal reporting systems, including surrogate signal development for “digital” detection, direct visualization for “digital” detection, and nucleic acid amplification enabled “digital” detection. Interdisciplinary combination and integration of different cutting-edge techniques are also discussed with details. The review is closed with the conclusion and future trends.

 

2.346           Expanding the codes: The development of density-encoded hydrogel microcarriers for suspension arrays

Hou, M., Shi, L., Zhou, Y., Wang, J., Jiang, J., Jiang, j. and He, J.

Biosensors and Bioelectronics, 181, 113133 (2021)

 

Although suspension array technology (SAT), which uses encoded microspheres, provides high-quality results with versatile applicability for information-intensive bioanalytic applications, current encoding strategies limit the number of codes that can be distinguished. In this paper, we introduce density-encoded hydrogel microcarriers (DMs), which employ the intrinsic density property of biomaterials as a high-capacity coding dimension. Two hydrogel monomers were employed at different ratios to synthesize microgels with distinctive densities. DMs not only can be simultaneously decoded and separated using density gradient centrifugation, but also are compatible with flow cytometry detection. The size and color of DMs have been used as extra coding parameters, to construct an 8 × 2 × 4 (density × size × color) three-dimensionally encoded hydrogel microcarrier library. With aptamer-functionalized DMs (ADMs), we developed a 4-plex protein quantification method for the label-free detection of plasma biomarkers with sub-nanomolar detection limits and good linearities. Moreover, ADMs can be used for label-free naked-eye detection of tumor-derived exosomes. We believe that the simplicity and functionality of DMs will advance the field of suspension arrays and inspire the development of DM-based diagnostic applications.

 

2.347           Lipoprotein compartmentalisation as a regulator of PCSK9 activity

Burnap, S.A. and Mayr, M.

1. Mol. Cell. Cardiol., 155, 21-24 (2021)

 

Lipoprotein compartmentalisation and PCSK9-mediated LDLR degradation. PCSK9 binds to the extracellular domain of the LDLR to promote its degradation through the lysosomal compartment. PCSK9 exists in a free and lipoprotein-bound form in the circulation. PCSK9 association with LDL is thought to sequester PCSK9 and inhibit PCSK9-mediated LDLR degradation. The functional consequence of the interaction between PCSK9 and HDL is currently unknown. PCSK9 probably shuttles between a lipoprotein-bound pool of PCSK9 and circulating free-PCSK9. This lipoprotein compartmentalisation may impact on its LDLR degrading activity.

 

 

2.348           A Comparative Analysis of the Lipoprotein(a) and Low-Density Lipoprotein Proteomic Profiles Combining Mass Spectrometry and Mendelian Randomization

Bourgeois, R., Girard, A., Perrot, N., Guertin, J., Mirchell, P.L., Couture, Cc. et al

CJC Open, 3, 450-459 (2021)

 

Background

Lipoprotein(a) (Lp[a]), which consists of a low-density lipoprotein (LDL) bound to apolipoprotein(a), is one of the strongest genetic risk factors for atherosclerotic cardiovascular diseases. Few studies have performed hypothesis-free direct comparisons of the Lp(a) and the LDL proteomes. Our objectives were to compare the Lp(a) and the LDL proteomic profiles and to evaluate the effect of lifelong exposure to elevated Lp(a) or LDL cholesterol levels on the plasma proteomic profile.

Methods

We performed a label-free analysis of the Lp(a) and LDL proteomic profiles of healthy volunteers in a discovery (n = 6) and a replication (n = 9) phase. We performed inverse variance weighted Mendelian randomization to document the effect of lifelong exposure to elevated Lp(a) or LDL cholesterol levels on the plasma proteomic profile of participants of the INTERVAL study.

Results

We identified 15 proteins that were more abundant on Lp(a) compared with LDL (serping1, pi16, itih1, itih2, itih3, pon1, podxl, cd44, cp, ptprg, vtn, pcsk9, igfals, vcam1, and ttr). We found no proteins that were more abundant on LDL compared with Lp(a). After correction for multiple testing, lifelong exposure to elevated LDL cholesterol levels was associated with the variation of 18 plasma proteins whereas Lp(a) did not appear to influence the plasma proteome.

Conclusions

Results of this study highlight marked differences in the proteome of Lp(a) and LDL as well as in the effect of lifelong exposure to elevated LDL cholesterol or Lp(a) on the plasma proteomic profile.

 

 

2.349           TFAM knockdown-triggered mtDNA-nucleoid aggregation and a decrease in mtDNA copy number induce the reorganization of nucleoid populations and mitochondria-associated ER-membrane contacts

Aasumets, K., Basikhina, Y., Pohjoismäki, J., Goffart, S. and Gerhold, J.

Biochem. Biophys. Reports, 28, 101142 (2021)

 

The correct organization of mitochondrial DNA (mtDNA) in nucleoids and the contacts of mitochondria with the ER play an important role in maintaining the mitochondrial genome distribution within the cell. Mitochondria-associated ER membranes (MAMs) consist of interacting proteins and lipids located in the outer mitochondrial membrane and ER membrane, forming a platform for the mitochondrial inner membrane-associated genome replication factory as well as connecting the nucleoids with the mitochondrial division machinery. We show here that knockdown of a core component of mitochondrial nucleoids, TFAM, causes changes in the mitochondrial nucleoid populations, which subsequently impact ER-mitochondria membrane contacts. Knockdown of TFAM causes a significant decrease in the copy number of mtDNA as well as aggregation of mtDNA nucleoids. At the same time, it causes significant upregulation of the replicative TWNK helicase in the membrane-associated nucleoid fraction. This is accompanied by a transient elevation of MAM proteins, indicating a rearrangement of the linkage between ER and mitochondria triggered by changes in mitochondrial nucleoids. Reciprocal knockdown of the mitochondrial replicative helicase TWNK causes a decrease in mtDNA copy number and modifies mtDNA membrane association, however, it does not cause nucleoid aggregation and considerable alterations of MAM proteins in the membrane-associated fraction. Our explanation is that the aggregation of mitochondrial nucleoids resulting from TFAM knockdown triggers a compensatory mechanism involving the reorganization of both mitochondrial nucleoids and MAM. These results could provide an important insight into pathological conditions associated with impaired nucleoid organization or defects of mtDNA distribution.

 

2.350           The role of the Cer1 transposon in horizontal transfer of transgenerational memory

Moore, R.S., Kaletsky, R.S., Lesnik, C., Parsons, L.R., Gital, Z. and Murphy, C.T.

Cell, 184, 4697-4712 (2021)

 

Animals face both external and internal dangers: pathogens threaten from the environment, and unstable genomic elements threaten from within. C. elegans protects itself from pathogens by “reading” bacterial small RNAs, using this information to both induce avoidance and transmit memories for four generations. Here, we found that memories can be transferred from either lysed animals or from conditioned media to naive animals via Cer1 retrotransposon-encoded virus-like particles. Moreover, Cer1 functions internally at the step of transmission of information from the germline to neurons and is required for learned avoidance. The presence of the Cer1 retrotransposon in wild C. elegans strains correlates with the ability to learn and inherit small-RNA-induced pathogen avoidance. Together, these results suggest that C. elegans has co-opted a potentially dangerous retrotransposon to instead protect itself and its progeny from a common pathogen through its inter-tissue signaling ability, hijacking this genomic element for its own adaptive immunity benefit.

 

2.351           An evaluation of different methods to study the association of proprotein convertase subtilisin-kexin type 9 to lipoproteins.

Canclini, L., Malvandi, A.M., Uboldi, P., Zampoleri, V., Baragetti, A., Grigore, L., and Catapano, A.L.

Atherosclertosis, 331, e139 (2021)

 

Background and Aims: Proprotein convertase subtilisin-kexin type 9 (PCSK9) enhances the degradation of the hepatic low-density lipoprotein (LDL) receptor, increasing LDL-cholesterol levels in plasma. PCSK9 has been reported to associate with lipoproteins (LPs) in plasma, but data in literature are discordant. We compared different methods for LPs isolation, aiming at finding the best one for studying this association.

Methods: Fresh serum was collected from healthy volunteers. LPs were isolated with different methods, including precipitation with phosphotungstic acid, fast protein liquid chromatography (FPLC), ultracentrifugation using both KBr and iodixanol gradient (IGr). The PCSK9 content of the LP fractions obtained was quantified with ELISA. Cholesterol, APOB and APOA1 were measured using clinical-grade reactives.

Results: In the precipitation-mediated assay, more than 80% of PCSK9 was found in the APOB precipitate. Negligible amount of PCSK9 was detected in the LPs isolated with the KBr ultracentrifugation. PCSK9 was found in the LDL fraction obtained with both IGr ultracentrifugation and FPLC. The percentage of association showed inter and intra individual variability, ranging from 1% to 30% of total recovered PCSK9.

Conclusions: Based on our observations, the PCSK9-LDL association exists and is sensitive to high salt concentrations. IGr ultracentrifugation and FPLC appear to be both suitable for further studies.

 

2.352           Breakfast partly restores the anti-inflammatory function of high-density lipoproteins from patients with type 2 diabetes mellitus

Lemmers, R.E.H., Martens, N.E.M.A., Maas, A.H., van Verk-van Zee, L.C., Leijten, F.P.J., Groot-van Ruijven, C.M., van Hoek, M., Lieverse, A.G., Sijbrands, E.J.G., Haak, H.R., Leenen, P.J.M., Verhoeven, A.J.M., Dik, W.A. and Mulder, M.T.

Atherosclerosis Plus, 44, 43-50 (2021)

 

Background and aims

High-density lipoproteins (HDL) of patients with type 2 diabetes mellitus (T2DM) have impaired anti-inflammatory activities. The anti-inflammatory activity of HDL has been determined ex vivo after isolation by different methods from blood mostly obtained after overnight fasting. We first determined the effect of the HDL isolation method, and subsequently the effect of food intake on the anti-inflammatory function of HDL from T2DM patients.

Methods

Blood was collected from healthy controls and T2DM patients after an overnight fast, and from T2DM patients 3 h after breakfast (n = 17 each). HDL was isolated by a two-step density gradient ultracentrifugation in iodixanol (HDL_DGUC2), by sequential salt density flotation (HDL_SEQ) or by PEG precipitation (HDL_PEG). The anti-inflammatory function of HDL was determined by the reduction of the TNFα-induced expression of VCAM-1 in human coronary artery endothelial cells (HCAEC) and retinal endothelial cells (REC).

Results

HDL isolated by the three different methods from healthy controls inhibited TNFα-induced VCAM-1 expression in HCAEC. With apoA-I at 0.7 μM, HDL_DGUC2 and HDL_SEQ were similarly effective (16% versus 14% reduction; n = 3; p > 0.05) but less effective than HDL_PEG (28%, p < 0.05). Since ultracentrifugation removes most of the unbound plasma proteins, we used HDL_DGUC2 for further experiments. With apoA-I at 3.2 μM, HDL from fasting healthy controls and T2DM patients reduced TNFα-induced VCAM-1 expression in HCAEC by 58 ± 13% and 51 ± 20%, respectively (p = 0.35), and in REC by 42 ± 13% and 25 ± 18%, respectively (p < 0.05). Compared to preprandial HDL, postprandial HDL from T2DM patients reduced VCAM-1 expression by 56 ± 16% (paired test: p < 0.001) in HCAEC and by 34 ± 13% (paired test: p < 0.05) in REC.

Conclusions

The ex vivo anti-inflammatory activity of HDL is affected by the HDL isolation method. Two-step ultracentrifugation in an iodixanol gradient is a suitable method for HDL isolation when testing HDL anti-inflammatory function. The anti-inflammatory activity of HDL from overnight fasted T2DM patients is significantly impaired in REC but not in HCAEC. The anti-inflammatory function of HDL is partly restored by food intake.

 

2.353           A hexokinase isoenzyme switch in human liver cancer cells promotes lipogenesis and enhances innate immunity

Perrin-Cocon , L., Vidalain, P-O., Jacquemin, C., Aublin-Gex, A., Olmstead, K., Panthu, B. et al

Communications Biol., 4:4217 (2021)

 

During the cancerous transformation of normal hepatocytes into hepatocellular carcinoma (HCC), the enzyme catalyzing the first rate-limiting step of glycolysis, namely the glucokinase (GCK), is replaced by the higher affinity isoenzyme, hexokinase 2 (HK2). Here, we show that in HCC tumors the highest expression level of HK2 is inversely correlated to GCK expression, and is associated to poor prognosis for patient survival. To further explore functional consequences of the GCK-to-HK2 isoenzyme switch occurring during carcinogenesis, HK2 was knocked-out in the HCC cell line Huh7 and replaced by GCK, to generate the Huh7-GCK⁺/HK2⁻ cell line. HK2 knockdown and GCK expression rewired central carbon metabolism, stimulated mitochondrial respiration and restored essential metabolic functions of normal hepatocytes such as lipogenesis, VLDL secretion, glycogen storage. It also reactivated innate immune responses and sensitivity to natural killer cells, showing that consequences of the HK switch extend beyond metabolic reprogramming.

 

 

2.354           Dual size-exclusion chromatography for efficient isolation of extracellular vesicles from bone marrow derived human plasma

Jung, J-H., Back, W., Yoon, J., Han, H., Kang, K-W., Choi, B., Jeong, H. et al

Scientific Reports, 11:217 (82021)

 

Isolation of pure extracellular vesicles (EVs), especially from blood, has been a major challenge in the field of EV research. The presence of lipoproteins and soluble proteins often hinders the isolation of high purity EVs upon utilization of conventional separation methods. To circumvent such problems, we designed a single-step dual size-exclusion chromatography (dSEC) column for effective isolation of highly pure EVs from bone marrow derived human plasma. With an aim to select appropriate column design parameters, we analyzed the physiochemical properties of the major substances in bone marrow derived plasma, which include EVs, lipoproteins, and soluble proteins. Based on these findings, we devised a novel dSEC column with two different types of porous beads sequentially stacked each other for efficient separation of EVs from other contaminants. The newly developed dSEC columns exhibited better performance in isolating highly pure EVs from AML plasma in comparison to conventional isolation methods.

 

2.355           FisB relies on homo-oligomerization and lipid binding to catalyze membrane fission in bacteria

Landajuela, A., Braun, M., Rodrigues, C.D.A., Martinez-Cavo, A., Doan, T., Horenkamp, F., Andronicos, A., Shteyn, V., Williams, N.D., Lin, C., Wingreen, N.S., Rudner, D.Z. and Karatekin, E.

PloS Biology, 19(6), e3001314 (2021)

 

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, fission protein B (FisB), is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here, we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of approximately 12 molecules that give way to an immobile cluster at the engulfment pole containing approximately 40 proteins at the time of membrane fission. Analysis of FisB mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Experiments using artificial membranes and filamentous cells suggest that FisB does not have an intrinsic ability to sense or induce membrane curvature but can bridge membranes. Finally, modeling suggests that homo-oligomerization and trans-interactions with membranes are sufficient to explain FisB accumulation at the membrane neck that connects the engulfment membrane to the rest of the mother cell membrane during late stages of engulfment. Together, our results show that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid binding, and the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains, negative-curvature lipids, or curvature sensing.

 

2.356           Turnover of Variant Surface Glycoprotein in Trypanosoma brucei Is a Bimodal Process

Garrison, P., Khan, U., Cipriano, M., Bush, P.J., McDonald, J., Sur, A., Myler, P.J., Smith, T.K., Hajduk, S.L. and bangs, J.D.

mBio, 12(4), e01725-21 (2021)

 

African trypanosomes utilize glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) to evade the host immune system. VSG turnover is thought to be mediated via cleavage of the GPI anchor by endogenous GPI-specific phospholipase C (GPI-PLC). However, GPI-PLC is topologically sequestered from VSG substrates in intact cells. Recently, A. J. Szempruch, S. E. Sykes, R. Kieft, L. Dennison, et al. (Cell 164:246–257, 2016, https://doi.org/10.1016/j.cell.2015.11.051) demonstrated the release of nanotubes that septate to form free VSG⁺ extracellular vesicles (EVs). Here, we evaluated the relative contributions of GPI hydrolysis and EV formation to VSG turnover in wild-type (WT) and GPI-PLC null cells. The turnover rate of VSG was consistent with prior measurements (half-life [t_1/2] of ∼26 h) but dropped significantly in the absence of GPI-PLC (t_1/2 of ∼36 h). Ectopic complementation restored normal turnover rates, confirming the role of GPI-PLC in turnover. However, physical characterization of shed VSG in WT cells indicated that at least 50% is released directly from cell membranes with intact GPI anchors. Shedding of EVs plays an insignificant role in total VSG turnover in both WT and null cells. In additional studies, GPI-PLC was found to have no role in biosynthetic and endocytic trafficking to the lysosome but did influence the rate of receptor-mediated endocytosis. These results indicate that VSG turnover is a bimodal process involving both direct shedding and GPI hydrolysis.

 

2.357           Long-term fasting improves lipoprotein-associated atherogenic risk in humans

Grundler, F., Plonne, D., Mesnage, R., Müller, D., Sirtori, C.R., Ruscica, M., de Toledo, F.W.

Eur. J. Nutr., 60, 4031-4044 (82021)

 

Purpose

Dyslipidemia is a major health concern associated with an increased risk of cardiovascular mortality. Long-term fasting (LF) has been shown to improve plasma lipid profile. We performed an in-depth investigation of lipoprotein composition.

Methods

This observational study included 40 volunteers (50% men, aged 32–65 years), who underwent a medically supervised fast of 14 days (250 kcal/day). Changes in lipid and lipoprotein levels, as well as in lipoprotein subclasses and particles, were measured by ultracentrifugation and nuclear magnetic resonance (NMR) at baseline, and after 7 and 14 fasting days.

Results

The largest changes were found after 14 fasting days. There were significant reductions in triglycerides (TG, − 0.35 ± 0.1 mmol/L), very low-density lipoprotein (VLDL)-TG (− 0.46 ± 0.08 mmol/L), VLDL-cholesterol (VLDL-C, − 0.16 ± 0.03 mmol/L) and low-density lipoprotein (LDL)-C (− 0.72 ± 0.14 mmol/L). Analysis of LDL subclasses showed a significant decrease in LDL1-C (− 0.16 ± 0.05 mmol/L), LDL2-C (− 0.30 ± 0.06 mmol/L) and LDL3-C (− 0.27 ± 0.05 mmol/L). NMR spectroscopy showed a significant reduction in large VLDL particles (− 5.18 ± 1.26 nmol/L), as well as large (− 244.13 ± 39.45 nmol/L) and small LDL particles (− 38.45 ± 44.04 nmol/L). A significant decrease in high-density lipoprotein (HDL)-C (− 0.16 ± 0.04 mmol/L) was observed. By contrast, the concentration in large HDL particles was significantly raised. Apolipoprotein A1 decreased significantly whereas apolipoprotein B, lipoprotein(a), fibrinogen and high-sensitivity C-reactive protein were unchanged.

Conclusion

Our results suggest that LF improves lipoprotein levels and lipoprotein subclasses and ameliorates the lipoprotein-associated atherogenic risk profile, suggesting a reduction in the cardiovascular risk linked to dyslipidemia.

 

2.358           Butyrylcholinesterase–Protein Interactions in Human Serum

Jasiecki, J., Szczoczarz, A., Cysewski, D., Lewandowaki, K., Skowron, P., Waleron, K. and Wasag, B.

Int. J. Mol. Sci., 22(19), 10662 (2021)

 

Measuring various biochemical and cellular components in the blood is a routine procedure in clinical practice. Human serum contains hundreds of diverse proteins secreted from all cells and tissues in healthy and diseased states. Moreover, some serum proteins have specific strong interactions with other blood components, but most interactions are probably weak and transient. One of the serum proteins is butyrylcholinesterase (BChE), an enzyme existing mainly as a glycosylated soluble tetramer that plays an important role in the metabolism of many drugs. Our results suggest that BChE interacts with plasma proteins and forms much larger complexes than predicted from the molecular weight of the BChE tetramer. To investigate and isolate such complexes, we developed a two-step strategy to find specific protein–protein interactions by combining native size-exclusion chromatography (SEC) with affinity chromatography with the resin that specifically binds BChE. Second, to confirm protein complexes′ specificity, we fractionated blood serum proteins by density gradient ultracentrifugation followed by co-immunoprecipitation with anti-BChE monoclonal antibodies. The proteins coisolated in complexes with BChE were identified by mass spectroscopy. These binding studies revealed that BChE interacts with a number of proteins in the human serum. Some of these interactions seem to be more stable than transient. BChE copurification with ApoA-I and the density of some fractions containing BChE corresponding to high-density lipoprotein cholesterol (HDL) during ultracentrifugation suggest its interactions with HDL. Moreover, we observed lower BChE plasma activity in individuals with severely reduced HDL levels (≤20 mg/dL). The presented two-step methodology for determination of the BChE interactions can facilitate further analysis of such complexes, especially from the brain tissue, where BChE could be involved in the pathogenesis and progression of AD.

 

2.359           Reconstitution of Functional Integrin αIIbβ3 and Its Activation in Plasma Membrane-Mimetic Lipid Environments

Janke, U., Mitlehner, A., Weide, A., Gutmann, T. and Delcea, M.

Membranes, 11, 499 (2021)

 

The study of the platelet receptor integrin αIIbβ3 in a membrane-mimetic environment without interfering signalling pathways is crucial to understand protein structure and dynamics. Our understanding of this receptor and its sequential activation steps has been tremendously progressing using structural and reconstitution approaches in model membranes, such as liposomes or supported-lipid bilayers. For most αIIbβ3 reconstitution approaches, saturated short-chain lipids have been used, which is not reflecting the native platelet cell membrane composition. We report here on the reconstitution of label-free full-length αIIbβ3 in liposomes containing cholesterol, sphingomyelin, and unsaturated phosphatidylcholine mimicking the plasma membrane that formed supported-lipid bilayers for quartz-crystal microbalance with dissipation (QCM-D) experiments. We demonstrate the relevance of the lipid environment and its resulting physicochemical properties on integrin reconstitution efficiency and its conformational dynamics. We present here an approach to investigate αIIbβ3 in a biomimetic membrane system as a useful platform do dissect disease-relevant integrin mutations and effects on ligand binding in a lipid-specific context, which might be applicable for drug screening.

 

2.360           Lipoprotein Proteomics and Aortic Valve Transcriptomics Identify Biological Pathways Linking Lipoprotein(a) Levels to Aortic Stenosis

Bourgeois, R., Bourgault, J., Despres, A-A., Perrot, N., Guertin, J., Girard, A. et al

Metabolites, 11, 459 (2021)

 

Lipoprotein(a) (Lp(a)) is one of the most important risk factors for the development of calcific aortic valve stenosis (CAVS). However, the mechanisms through which Lp(a) causes CAVS are currently unknown. Our objectives were to characterize the Lp(a) proteome and to identify proteins that may be differentially associated with Lp(a) in patients with versus without CAVS. Our second objective was to identify genes that may be differentially regulated by exposure to high versus low Lp(a) levels in explanted aortic valves from patients with CAVS. We isolated Lp(a) from the blood of 21 patients with CAVS and 22 volunteers and performed untargeted label-free analysis of the Lp(a) proteome. We also investigated the transcriptomic signature of calcified aortic valves from patients who underwent aortic valve replacement with high versus low Lp(a) levels (n = 118). Proteins involved in the protein activation cascade, platelet degranulation, leukocyte migration, and response to wounding may be associated with Lp(a) depending on CAVS status. The transcriptomic analysis identified genes involved in cardiac aging, chondrocyte development, and inflammation as potentially influenced by Lp(a). Our multi-omic analyses identified biological pathways through which Lp(a) may cause CAVS, as well as key molecular events that could be triggered by Lp(a) in CAVS development

 

2.361           The protease-sensitive N-terminal polybasic region of prion protein modulates its conversion to the pathogenic prion conformer

Zhang, X., Pan, Y-H., Chen, Y., Pan, C., Ma, J., Yan, C., Yu, G. and Ma, J.

1. Biol. Chem., 297(5), 101344 (2021)

 

Conversion of normal prion protein (PrP^C) to the pathogenic PrP^Sc conformer is central to prion diseases such as Creutzfeldt–Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrP^C-to-PrP^Sc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrP^Sc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrP^Sc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrP^Sc mutants were significantly different from each other and from that of WT recPrP^Sc. Together, our results support that the NPR greatly influences PrP^C-to-PrP^Sc conversion, but it is not essential for the generation of PrP^Sc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.

 

 

2.362           Mammalian BCAS3 and C16orf70 associate with the phagophore assembly site in response to selective and non-selective autophagy

Kojima, W., Yamano, K., Kosako, H., Imai, K., Kikuchi, R., Tanak, k. and Matsuda, N.

Autophagy, 17(8), 2011-2036 (2021)

 

Macroautophagy/autophagy is an intracellular degradation process that delivers cytosolic materials and/or damaged organelles to lysosomes. De novo synthesis of the autophagosome membrane occurs within a phosphatidylinositol-3-phosphate-rich region of the endoplasmic reticulum, and subsequent expansion is critical for cargo encapsulation. This process is complex, especially in mammals, with many regulatory factors. In this study, by utilizing PRKN (parkin RBR E3 ubiquitin protein ligase)-mediated mitochondria autophagy (mitophagy)-inducing conditions in conjunction with chemical crosslinking and mass spectrometry, we identified human BCAS3 (BCAS3 microtubule associated cell migration factor) and C16orf70 (chromosome 16 open reading frame 70) as novel proteins that associate with the autophagosome formation site during both non-selective and selective autophagy. We demonstrate that BCAS3 and C16orf70 form a complex and that their association with the phagophore assembly site requires both proteins. In silico structural modeling, mutational analyses in cells and in vitro phosphoinositide-binding assays indicate that the WD40 repeat domain in human BCAS3 directly binds phosphatidylinositol-3-phosphate. Furthermore, overexpression of the BCAS3-C16orf70 complex affects the recruitment of several core autophagy proteins to the phagophore assembly site. This study demonstrates regulatory roles for human BCAS3 and C16orf70 in autophagic activity.

 

2.363           Self-Assembling Nanoparticle Vaccines Displaying the Receptor Binding Domain of SARS-CoV-2 Elicit Robust Protective Immune Responses in Rhesus Monkeys

Li, H., Guo, L., Zheng, H., Li, J., Zhao, X., Li, J., Liang, Y. et al

Bioconjugate Chem., 32, 1034-1046 (2021)

 

SARS-CoV-2 caused the COVID-19 pandemic that lasted for more than a year. Globally, there is an urgent need to use safe and effective vaccines for immunization to achieve comprehensive protection against SARS-CoV-2 infection. Focusing on developing a rapid vaccine platform with significant immunogenicity as well as broad and high protection efficiency, we designed a SARS-CoV-2 spike protein receptor-binding domain (RBD) displayed on self-assembled ferritin nanoparticles. In a 293i cells eukaryotic expression system, this candidate vaccine was prepared and purified. After rhesus monkeys are immunized with 20 μg of RBD–ferritin nanoparticles three times, the vaccine can elicit specific humoral immunity and T cell immune response, and the neutralizing antibodies can cross-neutralize four SARS-CoV-2 strains from different sources. In the challenge protection test, after nasal infection with 2 × 10⁵ CCID50 SARS-CoV-2 virus, compared with unimmunized control animals, virus replication in the vaccine-immunized rhesus monkeys was significantly inhibited, and respiratory pathology observations also showed only slight pathological damage. These analyses will benefit the immunization program of the RBD–ferritin nanoparticle vaccine in the clinical trial design and the platform construction to present a specific antigen domain in the self-assembling nanoparticle in a short time to harvest stable, safe, and effective vaccine candidates for new SARS-CoV-2 isolates.

 

2.364           The Association of Proprotein Convertase Subtilisin/Kexin Type 9 to Plasma Low-Density Lipoproteins: An Evaluation of Different Methods

Canclini, L., Malvandi, A.M., Uboldi, P., Jabnati, N., Grigore, L., Zambon, A., Baragetti, A. and Catapano, A.L.

Metabolites, 11:861 (2021)

 

Background: Proprotein convertase subtilisin/kexin type-9 (PCSK9) is key regulator of low-density lipoprotein (LDL) metabolism. A significant proportion of PCSK9 is believed to be associated with LDL in plasma as it circulates, although this finding is still a matter of debate. The purpose of this study was to establish an experimental method to investigate the presence of such an interaction in the bloodstream. Methods: We compared a number of well-established methods for lipoprotein (LP) isolation to clarify whether PCSK9 associates differently to circulating lipoproteins, such as KBr gradient ultracentrifugation, physical precipitation of ApoB-LPs, fast protein liquid chromatography (FPLC) and iodixanol gradient ultracentrifugation. Results: Our data show heterogeneity in PCSK9 association to lipoproteins according to the method used. Two methods, iodixanol ultracentrifugation and column chromatography, which did not involve precipitation or high salt concentration, consistently showed an interaction of PCSK9 with a subfraction of LDL that appeared to be more buoyant and have a lower size than average LDL. The percent of PCSK9 association ranged from 2 to 30% and did not appear to correlate to plasma or LDL cholesterol levels. Conclusions: The association of PCSK9 to LDL appeared to be sensitive to high salt concentrations. FPLC and iodixanol gradient ultracentrifugation appeared to be the most suitable methods for the study of this association.

 

2.365           Sorting sub-150-nm liposomes of distinct sizes by DNA-brick-assisted centrifugation

Yang, Y., Wu, Z., Wang, l., Zhou, K., Xia, K., Xiong, Q., Liu, L., Zhang, Z., Chapman, E.R., Xiong, Y., Melia, T.J., Karatekin, E., Gu, H. and Lin, C.

Nature Chem., 13, 335-342 (2021)

 

In cells, myriad membrane-interacting proteins generate and maintain curved membrane domains with radii of curvature around or below 50 nm. To understand how such highly curved membranes modulate specific protein functions, and vice versa, it is imperative to use small liposomes with precisely defined attributes as model membranes. Here, we report a versatile and scalable sorting technique that uses cholesterol-modified DNA ‘nanobricks’ to differentiate hetero-sized liposomes by their buoyant densities. This method separates milligrams of liposomes, regardless of their origins and chemical compositions, into six to eight homogeneous populations with mean diameters of 30–130 nm. We show that these uniform, leak-resistant liposomes serve as ideal substrates to study, with an unprecedented resolution, how membrane curvature influences peripheral (ATG3) and integral (SNARE) membrane protein activities. Compared with conventional methods, our sorting technique represents a streamlined process to achieve superior liposome size uniformity, which benefits research in membrane biology and the development of liposomal drug-delivery systems.

 

2.366           Cryptic splicing events result in unexpected protein products from calpain-10 (CAPN10) cDNA

Ono, Y., Doi, N., Shindo, M., Panico, P. and Salazar, A.M.

BBA-Mol. Cell Res., 1869, 119188 (2022)

 

Calpain-10 (CAPN10) belongs to the calpain superfamily. Genetic polymorphisms of the CAPN10 gene are associated with susceptibility to develop type 2 diabetes mellitus. Although the role of CAPN10 in the pathophysiology of diabetes has been extensively investigated, its biochemical properties are largely unknown. In this report, we made the surprising discovery that CAPN10 cDNA transcripts are subject to cryptic splicing and unexpected protein products were expressed. The same set of splicing products was reproducibly detected in four types of cultured cells including the primary culture of mouse myoblast. At least, one of the products was identical to a natural splicing variant. Sequence analysis of the splicing potential of CAPN10 cDNA, together with mutagenesis studies, resulted in the identification of a powerful splicing acceptor site at the junction of the sequences encoded by exons 9 and 10. We successfully extended the analysis to create expression construct resistant to splicing for both human and mouse CAPN10. The construct allowed us to analyze two major CAPN10 isoforms and reveal their difference in substrate proteolysis and potential cell functions.

These results demonstrate that proteins produced from cDNA do not necessarily reflect the original nucleotide sequence. We provide insight into the property of recombinantly expressed CAPN10 proteins in cultured cells circumventing unexpected protein products.

 

2.367           ILF2 enhances the DNA cytosine deaminase activity of tumor mutator APOBEC3B in multiple myeloma cells

Kazuma, Y., Shirakawa, K., Tashiro, Y., Yamazaki, H., Nomura, R. et al

Scientific Reports, 12:2278 (2022)

 

DNA cytosine deaminase APOBEC3B (A3B) is an endogenous source of mutations in many human cancers, including multiple myeloma. A3B proteins form catalytically inactive high molecular mass (HMM) complexes in nuclei, however, the regulatory mechanisms of A3B deaminase activity in HMM complexes are still unclear. Here, we performed mass spectrometry analysis of A3B-interacting proteins from nuclear extracts of myeloma cell lines and identified 30 putative interacting proteins. These proteins are involved in RNA metabolism, including RNA binding, mRNA splicing, translation, and regulation of gene expression. Except for SAFB, these proteins interact with A3B in an RNA-dependent manner. Most of these interacting proteins are detected in A3B HMM complexes by density gradient sedimentation assays. We focused on two interacting proteins, ILF2 and SAFB. We found that overexpressed ILF2 enhanced the deaminase activity of A3B by 30%, while SAFB did not. Additionally, siRNA-mediated knockdown of ILF2 suppressed A3B deaminase activity by 30% in HEK293T cell lysates. Based on these findings, we conclude that ILF2 can interact with A3B and enhance its deaminase activity in HMM complexes.

2.368           Bay41-4109-induced aberrant polymers of hepatitis b capsid proteins are removed via STUB1-promoted p62-mediated macroautophagy

Lin, J., Yin, L., Xu, X-Z., Sun, H-C., Huang, Z-H., Ni, X-Y., Chen, Y. and Lin, X.

PloS Pathogens, 18(1), e1010204 (2022)

 

The hepatitis B virus (HBV) core protein (HBc) functions in multiple steps of the viral life cycle. Heteroaryldihydropyrimidine compounds (HAPs) such as Bay41-4109 are capsid protein allosteric modulators that accelerate HBc degradation and inhibit the virion secretion of HBV, specifically by misleading HBc assembly into aberrant non-capsid polymers. However, the subsequent cellular fates of these HAP-induced aberrant non-capsid polymers are not well understood. Here, we discovered that that the chaperone-binding E3 ubiquitin ligase protein STUB1 is required for the removal of Bay41-4109-induced aberrant non-capsid polymers from HepAD38 cells. Specifically, STUB1 recruits BAG3 to transport Bay41-4109-induced aberrant non-capsid polymers to the perinuclear region of cells, thereby initiating p62-mediated macroautophagy and lysosomal degradation. We also demonstrate that elevating the STUB1 level enhances the inhibitory effect of Bay41-4109 on the production of HBeAg and HBV virions in HepAD38 cells, in HBV-infected HepG2-NTCP cells, and in HBV transgenic mice. STUB1 overexpression also facilitates the inhibition of Bay41-4109 on the cccDNA formation in de novo infection of HBV. Understanding these molecular details paves the way for applying HAPs as a potentially curative regimen (or a component of a combination treatment) for eradicating HBV from hepatocytes of chronic infection patients.

 

2.369           Ice nucleation in a Gram-positive bacterium isolated from precipitation depends on a polyketide synthase and non-ribosomal peptide synthetase

Failor, K., Liu, H., Llontop, M.E.M., LeBlanc, S., Exkshtain-Levi, N., Sharma, P., Reed, A., Yang, S., Tian, L., lefevre, C.T., Menguy, N., Du, L., Monteil, C.L. and Vinatzer, B.

ISME J., 16, 890-897 (2022)

 

Earth’s radiation budget and frequency and intensity of precipitation are influenced by aerosols with ice nucleation activity (INA), i.e., particles that catalyze the formation of ice. Some bacteria, fungi, and pollen are among the most efficient ice nucleators, but the molecular basis of INA is poorly understood in most of them. Lysinibacillus parviboronicapiens (Lp) was previously identified as the first Gram-positive bacterium with INA. INA of Lp is associated with a secreted, nanometer-sized, non-proteinaceous macromolecule or particle. Here a combination of comparative genomics, transcriptomics, and a mutant screen showed that INA in Lp depends on a type I iterative polyketide synthase and a non-ribosomal peptide synthetase (PKS-NRPS). Differential filtration in combination with gradient ultracentrifugation revealed that the product of the PKS-NRPS is associated with secreted particles of a density typical of extracellular vesicles and electron microscopy showed that these particles consist in “pearl chain”-like structures not resembling any other known bacterial structures. These findings expand our knowledge of biological INA, may be a model for INA in other organisms for which the molecular basis of INA is unknown, and present another step towards unraveling the role of microbes in atmospheric processes.

 

2.370           Hepatic Reduction in Cholesterol 25-Hydroxylase Aggravates Diet-induced Steatosis

Dong, Z., He, F., Yan, X., Xing, Y., Lei, Y., Gao, J., He, M., Li, D., Bai, L., Yuan, Z. and Shyy, J.-Y.

Cell. Mol. Gastroenterol. Hepatol., 13, 1161-1179 (2022)

 

Background & Aims

Cholesterol 25-hydroxylase (Ch25h), converting cholesterol to 25-hydroxycholesterol (25-HC), is critical in modulating cellular lipid metabolism and anti-inflammatory and antiviral activities. However, its role in nonalcoholic fatty liver disease remains unclear.

Methods

Ch25h expression was detected in livers of ob/ob mice and E3 rats fed a high-fat diet (HFD). Gain- or loss-of-function of Ch25h was performed using Ch25h^+/+ (wild type [WT]) mice receiving AAV8-Ch25h or Ch25h knockout (Ch25h^-/-) mice. WT mice fed an HFD were administered with 25-HC. The Ch25h–LXRα–CYP axis was measured in primary hepatocytes isolated from WT and Ch25h^-/- mice.

Results

We found that Ch25h level was decreased in livers of ob/ob mice and E3 rats fed an HFD. Ch25h^-/- mice fed an HFD showed aggravated fatty liver and decreased level of cytochrome P450 7A1 (CYP7A1), in comparison with their WT littermates. RNA-seq analysis revealed that the differentially expressed genes in livers of HFD-fed Ch25h^-/- mice were involved in pathways of positive regulation of lipid metabolic process, steroid metabolic process, cholesterol metabolic process, and bile acid biosynthetic process. As gain-of-function experiments, WT mice receiving AAV8-Ch25h or 25-HC showed alleviated NAFLD, when compared with the control group receiving AAV8-control or vehicle control. Consistently, Ch25h overexpression significantly elevated the levels of primary and secondary bile acids and CYP7A1 but decreased those of small heterodimer partner and FGFR4.

Conclusions

Elevated levels of Ch25h and its enzymatic product 25-HC alleviate HFD-induced hepatic steatosis via regulating enterohepatic circulation of bile acids. The underlying mechanism involves 25-HC activation of CYP7A1 via liver X receptor. These data suggest that targeting Ch25h or 25-HC may have therapeutic advantages against nonalcoholic fatty liver disease.

 

2.371           Recombinant VLPs empower RBM peptides showing no immunogenicity in native SARS-COV-2 protein to elicit a robust neutralizing antibody response

Long, Q., Yang, Y., Yang, M., Bai, H., Sun, W., Yang, X., Huang, W., Li, D. and Ma, Y.

Nanomed.: Nanotechnol. Biol. Med., 41, 102527 (2022)

 

New SARS-COV-2 vaccine strategies are still urgently needed, especially for emerging virus mutations and variants. In this study, we focused on analyzing the antigenicity and vaccine potency of linear peptide epitopes located in receptor binding motif (RBM) of spike (S) protein. Nine 12 to 16-mer overlapping peptides (P1-P9) were synthesized chemically and coupled to carrier protein KLH for the immunization in mice. Four of identified peptides were further engineered to present on the surface of recombinant Hepatitis B core antigen (HBcAg) virus-like particles (VLPs) respectively. Antisera obtained from VLPs -immunized mice demonstrated strong reactivity and affinity to S1 protein or inactivated virus and neutralizing activity against virus infection in vitro. This study indicates that recombinant VLPs empower peptides which display underprivileged antigenicity in native protein to elicit high levels of neutralizing antibody, providing potential epitope candidates and an effective delivery strategy for the development of a multi-epitope vaccine.

 

2.372           Aβ oligomer concentration in mouse and human brain and its drug-induced reduction ex vivo

Kass, B., Schemmert, S., Zafiu, C., Kutzsche, j., Bujnicki, T. and Willbold, D.

Cell Report Med., 3, 100630 (2022)

 

The elimination of amyloid beta (Aβ) oligomers is a promising strategy for therapeutic drug development of Alzheimer’s disease (AD). AD mouse models that develop Aβ pathology have been used to demonstrate in vivo efficacy of compounds that later failed in clinical development. Here, we analyze the concentration and size distribution of Aβ oligomers in different transgenic mouse models of AD and in human brain samples by surface-based fluorescence intensity distribution analysis (sFIDA), a highly sensitive method for detecting and quantitating protein aggregates. We demonstrate dose- and time-dependent oligomer elimination by the compound RD2 in mouse and human AD brain homogenates as sources of native Aβ oligomers. Such ex vivo target engagement analyses with mouse- and human-brain-derived oligomers have the potential to enhance the translational value from pre-clinical proof-of-concept studies to clinical trials.

 

2.373           The Cell Adhesion Molecule L1 Interacts with Methyl CpG Binding Protein 2 via Its Intracellular Domain

Loeers, G., Kleene, R., Minguez, M.G. and Schachner, M.

Int. J. Mol. Sci., 23:3554 (2022)

 

Cell adhesion molecule L1 regulates multiple cell functions, and L1 deficiency is linked to several neural diseases. Recently, we have identified methyl CpG binding protein 2 (MeCP2) as a potential binding partner of the intracellular L1 domain. By ELISA we show here that L1’s intracellular domain binds directly to MeCP2 via the sequence motif KDET. Proximity ligation assay with cultured cerebellar and cortical neurons suggests a close association between L1 and MeCP2 in nuclei of neurons. Immunoprecipitation using MeCP2 antibodies and nuclear mouse brain extracts indicates that MeCP2 interacts with an L1 fragment of ~55 kDa (L1−55). Proximity ligation assay indicates that metalloproteases, β-site of amyloid precursor protein cleaving enzyme (BACE1) and ɣ-secretase, are involved in the generation of L1−55. Reduction in MeCP2 expression by siRNA decreases L1-dependent neurite outgrowth from cultured cortical neurons as well as the migration of L1-expressing HEK293 cells. Moreover, L1 siRNA, MeCP2 siRNA, or a cell-penetrating KDET-containing L1 peptide leads to reduced levels of myocyte enhancer factor 2C (Mef2c) mRNA and protein in cortical neurons, suggesting that the MeCP2/L1 interaction regulates Mef2c expression. Altogether, the present findings indicate that the interaction of the novel fragment L1−55 with MeCP2 affects L1-dependent functions, such as neurite outgrowth and neuronal migration.

3. Membranes and Cell Organelles

 

3.1           The preparation of subcellular organelles from mouse liver in self-generated gradients of iodixanol.

Graham, J., Ford, T. and Rickwood, D. Anal. Biochem., 220, 367-373 (1994)   This paper reports the use of a new density gradient compound, iodixanol, for the resolution of the major organelles from mouse liver. A major advantage of iodixanol over other iodinated density gradient media is its ready ability to form self-generated gradients. Gradient-forming conditions have been modulated to provide optimal recoveries of Golgi membranes, lysosomes, mitochondria and peroxisomes. The organelles were isolated in high yield (80-90% of gradient input) and high purity. Nycodenz and iodixanol were compared using preformed gradients. Iodixanol provided resolution superior to that of Nycodenz, notably of peroxisomes and mitochondria and the separation of lysosomes from endoplasmic reticulum. Because iodixanol does not interfere significantly with marker enzyme activities, gradient fractions can be analyzed without removal of the gradient media.  

3.2           A detergent-free method for purifying caveolae membrane from tissue culture cells.

Smart, E.J., Ying, Y-S, Mineo, C. and Anderson, R.G.W. Proc. Natl. Acad. Sci. USA, 92, 10104-10108 (1995)   Current methods for purifying caveolae from tissue culture cells take advantage of the Triton X-100 insolubility of this membrane domain. To circumvent the use of detergents, we have developed a method that depends upon the unique buoyant density of caveolae membrane. The caveolae fractions that we obtain are highly enriched in caveolin. As a consequence we are able to identify caveolae-associated proteins that had previously gone undetected. Moreover, resident caveolae proteins that are soluble in Triton X-100 are retained during the isolation.  

3.3           Acylation targets endothelial nitric‑oxide synthase to plasmalemmal caveolae.

Shaul, P.W. et al

1. Biol. Chem., 271(11), 6518‑6522 (1996)

  Endothelial nitric‑oxide synthase (eNOS) generates the key signaling molecule nitric oxide in response to intralumenal hormonal and mechanical stimuli. We designed studies to determine whether eNOS is localized to plasmalemmal microdomains implicated in signal transduction called caveolae. Using immunoblot analysis, eNOS protein was detected in caveolar membrane fractions isolated from endothelial cell plasma membranes by a newly developed detergent‑free method; eNOS protein was not found in noncaveolar plasma membrane. Similarly, NOS enzymatic activity was 9.4‑fold enriched in caveolar membrane versus whole plasma membrane, whereas it was undetectable in non‑caveolar plasma membrane. 51‑86% of total NOS activity in postnuclear supernatant was recovered in plasma membrane, and 57‑100% of activity in plasma membrane was recovered in caveolae. Immunoelectron microscopy showed that eNOS heavily decorated endothelial caveolae, whereas coated pits and smooth plasma membrane were devoid of gold particles. Furthermore, eNOS was targeted to caveolae in COS‑7 cells transfected with wild‑type eNOS cDNA. Studies with eNOS mutants revealed that both myristoylation and palmitoylation are required to target the enzyme to caveolae and that each acylation process enhances targeting by 10‑fold. Thus, acylation targets eNOS to plasmalemmal caveolae. Localization to this microdomain is likely to optimize eNOS activation and the extracellular release of nitric oxide.  

3.4           Clustered folate receptors deliver 5‑methyltetrahydrofolate to cytoplasm of MA104 cells

Smart, E.J., Mineo, C. and Anderson, R.G.W.

1. Cell Biol., 134(5), 1169‑1177 (1996)

  Previously, a high affinity, glycosylphosphatidylinositol‑anchored receptor for folate and a caveolae internalization cycle have been found necessary for potocytosis of 5‑methyltetrahydrofolate in MA104. We now show by cell fractionation that folate receptors also must be clustered in caveolae for potocytosis. An enriched fraction of caveolae from control cells retained 65‑70% of the [³H]folic acid bound to cells in culture. Exposure of cells to the cholesterol‑binding drug, filipin, which is known to uncluster receptors, shifted approximately 50% of the bound [³H]folic acid from the caveolae fraction to the noncaveolae membrane fraction and markedly inhibited internalization of [³H]folic acid. An mAb directed against the folate receptor also shifted approximately 50% of the caveolae associated [³H]folic acid to noncaveolae membrane, indicating the antibody perturbs the normal  receptor distribution. Concordantly, the mAb inhibited the delivery of  5‑methyl[³H]tetrahydrofolate to the cytoplasm. Receptor bound 5‑methyl [³H]tetrahydrofolate moved directly from caveolae to the cytoplasm and was not blocked by phenylarsine oxide, an inhibitor of receptor‑mediated endocytosis. These results suggest cell fractionation can be used to study the uptake of molecules by caveolae.  

3.5           Localization of epidermal growth factor‑stimulated Ras/Raf‑1 interaction to caveolae membrane.

Mineo, C., James, G.L., Smart, E.J. and Anderson, R.G.W.

1. Biol. Chem., 271(20), 11930‑11935 (1996)

  An essential step in the epidermal growth factor (EGF)‑dependent activation of MAP kinase is the recruitment of Raf‑1 to the plasma membrane. Here we present evidence that caveolae are the membrane site where Raf‑1 is recruited. Caveolae fractions prepared from normal Rat‑1 cells grown in the absence of serum were highly enriched in both EGF receptors and Ras. Thirty seconds after EGF was added to these cells Raf‑1 began to appear in caveolae but not in non‑caveolae membrane fractions. The maximum concentration was reached at 3 min followed by a decline over the next 60 min. During this time EGF receptors disappeared from the caveolae fraction while the concentration of Ras remained constant. The Raf‑1 in this fraction was able to phosphorylate MAP kinase, whereas cytoplasmic Raf‑1 in the same cell was inactive. Elevation of cellular cAMP blocked the recruitment of Raf‑1 to caveolae. Overexpression of Ha‑Ras^v12 caused the recruitment of Raf‑1 to caveolae independently of EGF stimulation, and this was blocked by the farnesyltransferase inhibitor BZA‑5B. Finally, prenylation appeared to be required for localization of Ras to caveolae.  

3.6           A role for caveolin in transport of cholesterol from endoplasmic reticulum to plasma membrane.

Smart, E.J., Ying, Ys., Donzell, W.C. and Anderson, R.G.W.

1. Biol. Chem., 271(46), 29427‑29435 (1996)

  Caveolin is a 22‑kDa membrane protein found associated with a coat material decorating the inner membrane surface of caveolae. A remarkable feature of this protein is its ability to migrate from caveolae directly to the endoplasmic reticulum (ER) when membrane cholesterol is oxidized. We now present evidence caveolin is involved in transporting newly synthesized cholesterol from the ER directly to caveolae. MA104 cells and normal human fibroblasts transported new cholesterol to caveolae with a half‑time of approximately 10 min. The cholesterol then rapidly flowed from caveolae to non‑caveolae membrane. Cholesterol moved out of caveolae even when the supply of fresh cholesterol from the ER was interrupted. Treatment of cells with 10 mg/ml progesterone blocked cholesterol movement from ER to caveolae. Simultaneously, caveolin accumulated in the lumen of the ER, suggesting  cholesterol transport is linked to caveolin movement. Caveolae fractions from  cells expressing caveolin were enriched in cholesterol 3‑4‑fold, while the same fractions from cells lacking caveolin were not enriched. Cholesterol transport to the cell surface was nearly 4 times more rapid in cells expressing caveolin than in matched cells lacking caveolin.  

3.7           Subcellular colocalization of the cellular and scrapie prion proteins in caveolae‑like membranous domains.

Vey, M. et al

Proc. Natl. Acad. Sci. USA, 93, 14945‑14949 (1996)   Results of transgenetic studies argue that the scrapie isoform of the prion protein (PrP^Sc) interacts with the substrate cellular PrP (PrP^C) during conversion into nascent PrP^Sc. While PrP^Sc appears to accumulate primarily in lysosomes, caveolae‑like domains (CLDs) have been suggested to be the site where PrP^C is converted into PrP^Sc. We report herein that CLDs isolated from scrapie‑infected neuroblastoma (ScN2a) cells contain PrP^C and PrP^Sc. After lysis of ScN2a cells in ice‑cold Triton X‑100, both PrP isoforms and an N‑terminally truncated form of PrP^C (PrP^C‑II) were found concentrated in detergent‑insoluble complexes resembling CLDs that were isolated by flotation in sucrose gradients. Similar results were obtained when CLDs were purified from plasma membranes by sonication and gradient centrifugation; with this procedure no detergents are used, which minimizes artifacts that might arise from redistribution of proteins among subcellular fractions. The caveolar markers ganglioside GM1 and H‑ras were found concentrated in the CLD fractions. When plasma membrane proteins were labeled with the impermeant reagent sulfo-N‑hydroxysuccinimide-biotin, both PrP^C and PrP^Sc were found biotinylated in CLD fractions. Similar results on the colocalization of PrP^C and PrP^Sc were obtained when CLDs were isolated from Syrian hamster brains. Our findings demonstrate that both PrP^C and PrP^Sc are present in CLDs and, thus, support the hypothesis that the PrP^Sc formation occurs within this subcellular compartment.  

3.8           Iodixanol (OptiPrep), an improved density gradient medium for the iso-osmotic isolation of rat liver peroxisomes.

Van Veldhoven, P.P., Baumgart, E. and Mannaerts, G.P. Anal. Biochem. 237, 17-23 (1996)                    The suitability of iodixanol, a nonionic iodinated compound with a molecular weight of 1550, for the isolation of peroxisomes from rat liver was investigated. Centrifugation of light mitochondrial fractions in 20 to 40% (w/v) iodixanol gradients, made iso-osmotic by the addition of sucrose, resulted in an excellent separation of peroxisomes from the remaining organelles, which were not able to enter the gradient. Peroxisomes banded around 30% (w/v) iodixanol (d-1.175) and, as revealed by marker enzyme analysis, were enriched 35- to 40-fold. Morphological examination of the peroxisomal fractions confirmed the near absence of other organelles and revealed structurally well-preserved peroxisomes. Free cores, also present in the starting fractions, migrated to higher densities and were trapped on a cushion. No interference of iodixanol with marker enzyme determinations was observed, except for the UV-metric determination of urate oxidase and for the analysis of protein.  

3.9           Potential chitinase activating factor from yeast cells of Candida albicans.

Jackson, D.J., Saunders, V.A. and Humphreys, A.M. Lett. Appl. Microbiol., 23, 159-162 (1996)   Microsomal chitinase from yeast and hyphal cells of Candida albicans was activated endogenously by incubation at 30⁰C and exogenously by trypsin. The putative activating factor of yeast cells was separated from chitinase activity by fractionation of lysed protoplasts on an Iodixanol density gradient. The vacuole fraction contained no significant chitinase activity, but was enriched in chitinase activating factor. Activity of microsomal chitinase increased upon incubation with this, but no other gradient factor. Results suggest that the regulatory system governing microsomal chitinase activity, like that governing chitin synthase, involves a >vacuolar= activating factor in Candida albicans.  

3.10           Localization of platelet-derived growth factor-stimulated phosphorylation cascade to caveolae

Liu, P., Ying, Y., Ko, Y.G  and  Anderson, R.G.W. J Biol. Chem., 271(17), 10299-10303 (1996)   Previously we showed that interleukin lb stimulates the conversion of sphingomyelin to ceramide in the caveolae fraction of normal human fibroblasts.  The ceramide, in turn, blocked platelet-derived growth factor (PDGF) stimulated DNA synthesis.  We now present evidence that the PDGF receptor initiates signal transduction from caveolae.  Cell fractionation and immunocytochemistry show caveolae to be the principal location of PDGF receptors at the cell surface.  Multiple caveolae proteins acquire phosphotyrosine when PDGF binds to its receptor, but the hormone appears to have little effect on the tyrosine phosphorylation of non-caveolae membrane proteins.  Five proteins known to interact with the phosphorylated receptor were found to be highly enriched in caveolae membrane.  PDGF caused the concentration of three of these proteins to significantly increase in the caveolae fraction.  Finally, PDGF stimulated the association of a 190-kDa phosphoprotein with the caveolae marker protein, caveolin.  Therefore, ceramide may modulate PDGF receptor function directly in caveolae.  

3.11           Tissue-specific distribution and subcellular distribution of phospholipase D in rat: evidence for distinct RhoA-  and ADP-ribosylation factor (ARF)-regulated isoenzymes.

Provost, J.J. et al.  Biochem. J., 319(2), 285-291 (1996)   Phospholipase D (PLD) is regulated by many factors including the small G-proteins, RhoA and ADP-ribosylation factor (ARF). The present study examined the distribution of RhoA- and ARF-responsive PLD in membranes, microsomes and cytosol of rat tissues and in rat liver subcellular fractions. PLD was present in all tissue fractions examined and was stimulated by guanosine 5'-[g -thio]triphosphate (GTP[S]), with the highest specific activities being in lung, kidney and spleen. When myristoylated recombinant ARF (mARF) was added with GTP[S], the PLD activity was stimulated further, but the addition of RhoA was without effect. However, in extracts from crude membranes both mARF and RhoA enhanced the stimulation by GTP[S], with high specific activities of PLD being observed in all tissues except muscle. The response to mARF was usually greater than to RhoA, and the responses were additive, except for liver, which showed synergism. When the PLD activity of subcellular fractions of liver was examined, GTP[S] caused increases in all fractions except microsomes and mitochondria, which exhibited low activity. All fractions except mitochondria showed responses to RhoA and mARF, with the response to RhoA being greater in plasma membranes and that to mARF being greater in Golgi and nuclei. Western blotting showed that RhoA was located mainly in the cytosol and plasma membranes, whereas ARF was principally in the cytosol. These findings demonstrate the widespread occurrence of significant activity of both Rho- and ARF-responsive forms of PLD in membranes from all tissues except muscle, and the presence of both forms in liver subcellular fractions except mitochondria. The large variations in the relative responses of PLD to Rho and ARF observed in different tissues and fractions support the existence of different isoforms of the enzyme.  

3.12           Transport of an external Lys-Asp-Glu-Leu (KDEL) protein from the plasma membrane to the endoplasmic reticulum: studies with cholera toxin in Vero cells

Majoul, I.V., Bastiaens, P.I.H. and Soling H-D.

1. Cell Biol., 133, 777-789 (1996)

  The A2 chain of cholera toxin (CTX) contains a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. We have, therefore, analyzed by immunofluorescence and by subcellular fractionation in Vero cells whether CTX can be used to demonstrate a retrograde transport of KDEL proteins from the Golgi to the ER. Immunofluorescence studies reveal that after a pulse treatment with CTX, the CTX-A and B subunits CTX-A and CTX-B) reach Golgi-like structures after 15-20 min (maximum after 30 min). Between 30 and 90 min, CTX-A (but not CTX-B) appear in the intermediate compartment and in the ER, whereas the CTX-B are translocated to the lysosomes. Subcellular fractionation studies confirm these results: after CTX uptake for 15 min, CTX-A is associated only with endosomal and Golgi compartments. After 30 min, a small amount of CTX-A appears in the ER in a trypsin-resistant form, and after 60 min, a significant amount  appears. CTX-A seems to be transported mainly in its oxidized form (CTX-A1-S-S-CTX-A2) from the Golgi to the ER, where it becomes slowly reduced to form free CTX A1 and CTX-A2, as indicated by experiments in which cells were homogenized 30 and 90 rain after the onset of CTX uptake in the presence of N-ethylmaleimide. Nocodazol applied after accumulation of CTX in the Golgi inhibits the appearance of CTX-A in the ER and delays the increase of 3', 5'cAMP, indicating the participation of microtubules in the retrograde Golgi-ER transport.  

3.13           Murine SR‑BI, a high density lipoprotein receptor that mediates selective lipid uptake, is N‑glycosylated and fatty acylated and colocalizes with plasma membrane caveolae.

Babitt, J. et al

1. Biol. Chem., 272(20), 13242‑13249 (1997)

  The class B, type I scavenger receptor, SR‑BI, was the first molecularly well defined cell surface high density lipoprotein (HDL) receptor to be described. It mediates transfer of lipid from HDL to cells via selective lipid uptake, a mechanism distinct from receptor‑mediated endocytosis via clathrin‑coated pits and vesicles. SR‑BI is expressed most abundantly in steroidogenic tissues (adrenal gland, ovary), where trophic hormones coordinately regulate its expression with steroidogenesis, and in the liver, where it may participate in reverse cholesterol transport. Here we have used immunochemical methods to study the structure and subcellular localization of murine SR‑BI (mSR‑BI) expressed either in transfected Chinese hamster ovary cells or in murine adrenocortical Y1‑BS1 cells. mSR‑BI, an approximately 82‑kDa glycoprotein, was initially synthesized with multiple high mannose N‑linked oligosaccharide chains, and some, but not all, of these were processed to complex forms during maturation of the protein in the Golgi apparatus. Metabolic labeling with [³H]palmitate and [³H]myristate demonstrated that mSR‑BI was fatty acylated, a property shared with CD36, another class B scavenger receptor, and other proteins that concentrate in specialized, cholesterol‑ and glycolipid‑rich plasma membrane microdomains called caveolae. OptiPrep density gradient fractionation of plasma membranes established that mSR‑BI copurified with caveolin‑1, a constituent of  caveolae; and immunofluorescence microscopy demonstrated that mSR‑BI colocalized with caveolin‑1 in punctate microdomains across the surface of cells and on the edges of cells. Thus, mSR‑BI colocalizes with caveolae, and this raises the possibility that the unique properties of these specialized cell surface domains may play a critical role in SR‑BI‑mediated transfer of lipids between lipoproteins and cells.  

3.14           Investigation of the role of lipids in the assembly of very low density lipoproteins in rabbit hepatocytes.

Cartwright, I.J. et al

1. Lipid Res., 38, 531-545 (1997)

  Our aims were (i) to determine which lipids co-localise with newly-synthesised apo-B in the lumen of the rough endoplasmic reticulum (RER), and thus may play a role in the stabilisation and/or translocation of this protein: and (ii) to determine the intracellular sites of assembly of lipids into VLDL. In order to do this, we have developed a new method for the separation of ER derived microsomes on self-generated gradients of iodixanol (OptiPrep). Rabbit liver microsomes were resolved into two broad peaks, the lighter peak contained smooth vesicles, and the heavier peak contained rough vesicles. Each peak was collected in a number of fractions. A single gradient thus separates the initial events in the secretion process (RER fractions), from later events (SER fractions). The microsomal fractions were separated into membranes and lumenal contents, and the mass of apo-B and VLDL lipids determined by ELISA or high performance thin layer chromatography, respectively. The biosynthetic relationships of apo-B and lipids were investigated, in timed or chase-experiments, by incubation of isolated rabbit hepatocytes with radiolabelled precursors of apo-B or lipids, followed by isolation, and analysis of the microsomal fractions. The results indicate that very small amounts of triacylglycerol, cholesterol and cholesterol ester co-localise with apo-B into the lumen of RER. The bulk of the VLDL lipids were in the lumen of the SER. However, some newly synthesised triacylglycerol, phospholipid, cholesterol and cholesterol ester were transferred to the lumen of the RER and were chased into the SER lumen. Double-labelling experiments, showed that cholesterol ester produced from newly synthesised cholesterol (labelled with [³H]-mevalonate and [¹⁴C]-oleate) was almost exclusively present in the RER, while cholesterol ester in the SER was labelled only with [¹⁴C]-oleate. Thus, distinct intracellular lipid-pools may be involved at different stages in the assembly of VLDL.  

3.15           Protein-disulfide isomerase-mediated reduction of the A subunit of cholera toxin in a human intestinal cell line.

Orlandi, P.A.

1. Biol. Chem., 272(7), 4591-4599 (1997)

  A key step in the action of cholera toxin (CT) is the reduction of its A subunit to the A₁ peptide. The latter is an ADP-ribosyltransferase, which activates the a-subunit of the stimulatory G protein of adenylyl cyclase. In this study, the enzymatic reduction of membrane-bound CT in CaCo-2 human intestinal epithelial cells was characterized. Whereas diphtheria toxin was found to be reduced by a cell surface population of protein-disulfide isomerase (PDI) and its cytotoxicity was inhibited by p- chloromercuri-benzenesulfonic acid, bacitracin, or anti-PDI antibodies, these inhibitors had no effect on CT reduction or activity in intact cells. In contrast, the reduction of CT in vitro by either postnuclear supernatants (PNS) or microsomal membranes in the presence of Triton X-100 was significantly inhibited by p-chloromercuribenzenesulfonic acid and bacitracin. Anti-PDI monoclonal antibodies likewise inhibited the in vitro reduction of CT and also were effective in depleting reductase activity from PNS. Since inhibition and depletion were not observed in the absence of detergent, these results suggested that the reductase activity was a soluble component localized to the lumen of microsomal vesicles and correlated with the presence of protein-disulfide isomerase. This was further confirmed by showing a corresponding depletion of reductase activity and PDI in alkali-treated microsomes. This activity was restored when purified bovine PDI was added back to alkali-treated microsomes in a redox buffer that reflected conditions found in the lumen of endoplasmic reticulum (ER). When the CT-related reductase activity was assayed in subcellular fractions of PNS-derived membranes isolated on a 9-30% iodixanol gradient, the activity, as measured by CT-A₁ peptide formation localized to those membrane fractions containing the majority of cellular PDI. Furthermore, the banding density corresponded to a region of the gradient containing ER-derived membranes. These results indicated that CT was a substrate for PDI-catalyzed reduction in intact cells and supported the hypothesis that CT reduction and activation occurs in the ER.  

3.16           The transmembrane domain of a carboxyl-terminal anchored protein determines localization to the endoplasmic reticulum.

Yang, M., Ellenberg, J., Bonifacino, J.S. and Weissman, A.M.

1. Biol.Chem., 272(3), 1970-1975 (1997)

  UBC6 is a C-terminal membrane-anchored (type IV) protein, native to Saccharomyces cerevisiae, where it is found in the endoplasmic reticulum. When expressed in mammalian cells, this novel ubiquitin-conjugating enzyme also localizes to the endoplasmic reticulum. UBC6 lacks a lumenal domain and contains no known endoplasmic reticulum retention signals. Analysis of chimeric proteins in which the cytosolic domain of UBC is linked to a heterologous transmembrane domain, or in which the UBC6 transmembrane domain is appended to an unrelated soluble protein, led to the determination that the transmembrane domain of UBC6 plays a dominant role in its compartmental localization. The basis for the transmembrane domain-mediated subcellular targeting of UBC6 was evaluated by lengthening the wild type UBC6 hydrophobic segment from 17 to 21 amino acids, which resulted in re-targeting to the Golgi complex. A further increase in length to 26 amino acids allowed this modified protein to traverse the secretory pathway and gain expression at the plasma membrane. These findings are consistent with models in which, in the absence of dominant cytosolic or lumenal targeting determinants, proteins may be sorted within the secretory pathway based on interactions between their transmembrane domains and the surrounding lipid bilayer.  

3.17           Tyrosine kinase receptors concentrated in caveolae-like domains from neuronal plasma membrane

Wu, C., Butz, S., Ying, Y-S and  Anderson, R.G.W.

1. Biol. Chem., 272(6), 3554-3559 (1997)

  Recent evidence suggests that tyrosine kinases are highly organized in caveolae of tissue culture cells.  We now report the isolation of a membrane domain from neuronal plasma membranes that has the biochemical characteristics of caveolae.  A low density membrane (LDM) fraction with the same density as caveolae was highly enriched in tyrosine kinases such as insulin receptors, neurotrophin receptors, Eph family receptors, and Fyn.  Grb2, Ras, heterotrimeric GTP-binding proteins, and Erk2 were also concentrated in the LDM.  Incubation of the LDM fraction at 37°C stimulated the phosphorylation on tyrosine of multiple, resident proteins, whereas the bulk membrane fraction was devoid of tyrosine kinase activity.  The LDM, which makes up ~5-10% of the plasma membrane protein, appears to be organized for signal transduction.  

3.18           Physical association with Ras enhances activation of membrane-bound Raf (RafCAAX)

Mineo, C., Anderson, R.G.W. and White, M.A.

1. Biol. Chem., 272(16), 10345-10348 (1997)

  The transforming activity of artificially membrane targeted Rafl suggests that Ras-mediated recruitment of Rafl to the plasma membrane is an important step in Rafl activation.  Cellular Ras is concentrated in the caveolae, a microdomain of the plasma membrane that is highly enriched in caveolin, glycosylphosphatidylinositol-anchored proteins, and signal transduction molecules.  Growth factor stimulation recruits Rafl to this membrane domain.  Whether Ras simply promotes Rafl association with caveolae membranes or also modulates subsequent activation events is presently unclear.  We have identified a ras variant, ras12V,37G, that does not interact with Raf1 but does interact with a mutant raf1, raf1(257L).  To examine the role of Ras in the activation of membrane-bound Raf1, raf1CAAX, and raf1(257L)CAAX membrane-targeted variants of Raf1 and raf1(257L), respectively, were expressed in fibroblasts with or without coexpression of ras12V,37G.  Cell fractionation localized both raf1CAAX and raf1(257L)CAAX to caveolae membranes independent of rasl2V,37G expression; however, coexpression of rasl2V,37G enhanced the activation of raf(257L)CAAX, but not raf1CAAX, as monitored by induction of cellular transformation, increased Raf kinase activity, and induction of activated MAP kinase.  These results suggest that the Ras/Raf1 interaction plays a role in Raf1 activation that is distinct from membrane recruitment.  

3.19           Organization of G proteins and adenylyl cyclase at the plasma membrane

Huang, C., Hepler, J.R., Chen, L.T., Gilman, A.G., Anderson, R.G.W. and Mumby, S.M. Mol. Biol. Cell, 8, 2365-2378 (1997)   There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane.  We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane.  These subtractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae.  Immunofluorescence experiments revealed a punctate pattern of G protein a and b subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane.  Partial coincidence of localization of G protein a subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence.  Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically.  Because regulated adenylyl cyclase activity is present in low-density subtractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system.  The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.  

3.20           Platelet-derived growth factor activates mitogen-activated protein kinase in isolated caveolae

Liu, P., Ying, Y-S., and  Anderson. R.G.W. Proc. Natl. Acad. Sci., USA, 94, 13666-13670 (1997)   The ability of a peptide hormone to affect many different intracellular targets is thought to be possible because of the modular organization of signal transducing molecules in the cell. Evidence for the presence of signaling modules in metazoan cells, however, is incomplete.  Herein we show, with morphology and cell fractionation, that all the components of a mitogen-activated protein kinase pathway are concentrated in caveolae of unstimulated human fibroblasts.  Addition of platelet-derived growth factor to either the intact cell or caveolae isolated from these cells stimulates tyrosine phosphorylation and activates mitogen-activated protein kinases in caveolae.  The molecular machinery for kinase activation, therefore, is preorganized at the cell surface of quiescent cells.  

3.21           Aminopeptidase I is targeted to the vacuole by a nonclassical vesicular mechanism

Scott, S.V., Baba, M., Ohsumi, Y. and Klionsky, D.J.

1. Cell Biol., 138(1), 37-44 (1997)

  The yeast vacuolar protein aminopeptidase I (API) is synthesized as a cytosolic precursor that is transported to the vacuole by a nonclassical targeting mechanism. Recent genetic studies indicate that the biosynthetic pathway that transports API uses many of the same molecular components as the degradative autophagy pathway. This overlap coupled with both in vitro and in vivo analysis of API import suggested that, like autophagy, API transport is vesicular. Subcellular fractionation experiments (OptiPrep) demonstrate that API precursor (prAPI) initially enters a nonvacuolar cytosolic compartment. In addition, subvacuolar vesicles containing prAPI were purified from a mutant strain defective in breakdown of autophagosomes, further indicating that prAPI enters the vacuole inside a vesicle. The purified subvacuolar vesicles do not appear to contain vacuolar marker proteins. Immunogold EM confirms that prAPI is localized in cytosolic and in subvacuolar vesicles in a mutant strain defective in autophagic body degradation. These data suggest that the cytosolic vesicles containing prAPI fuse with the vacuole to release a membrane-bounded intermediate compartment that is subsequently broken down, allowing API maturation.  

3.22           Bisecting GlcNac structures act as negative sorting signals for cell surface Glycoproteins in forskolin-treated rat hepatoma cells

Sultan, A.S. et al

1. Biol. Chem., 272(5), 2866-2872 (1997)

  The bisecting N-acetylglucosamine residue is formed by UDP-N-acetylglucosamine:b-D-mannoside-b-1,4-N-acetylglucosaminyltransferase III (GnT-III), a key branching enzyme for N-glycans.  We found that forskolin, an adenylyl cyclase activator, markedly enhanced GnT-III at the transcriptional level in various hepatoma cells and hepatocytes, resulting in an increase of bisecting GlcNAc residues in various glycoproteins, as judged from the lectin binding to erythroagglutinating phytohemagglutinin (E-PHA).  In whole cell lysates, the E-PRA binding was increased, and leukoagglutinating phytohemagglutinin (L-PHA) binding was decreased at 12 h after forskolin treatment, by time, both GnT-III activity and mRNA had reached the maximum levels.  In contrast, the binding capacity as to E-PHA, determined by fluorescence-activated cell sorting on the cell surface, was decreased, suggesting that bisecting GlcNAc structures in certain glycoproteins changed the expression levels of glycoproteins and decreased their sorting on the cell surface.  Fractionated organelles of M31 cells showed that the binding capacity as to E-PHA was mainly localized in Golgi membranes and lysosomes. This was also supported by a fluorescence microscopy. In order to determine whether or not the bisecting GlcNAc residue acts as a sorting signal for glycoproteins, N-oligosaccharide structures of lysosomal-associated membrane glycoprotein 1 and b-glucuronidase, g-glutamyltranspeptidase, and secretory glycoproteins such as ceruloplasmin and a-fetoprotein were measured by E-PHA and L-PHA blotting after immunoprecipitation. The expression levels of lysosomal membrane glycoprotein 1 and g-glutamyltranspeptidase on the cell surface were decreased at 12 h after forskolin treatment, indicating that the bisecting GlcNAc structure may act as a negative sorting signal for the cell surface glycoproteins and may alter the characteristics of hepatoma cells. This is the first report on glycoprotein sorting related to a specific structure of oligosaccharides, bisecting GlcNAc.  

3.23           Rapid plasma membrane anchoring of newly synthesized p59^fyn. Selective requirement for NH₂-terminal myristoylation and palmitoylation at cysteine-3.

van’t Hof, W. and Resh, M.D.

1. Cell Biol., 136(5), 1023-1035 (1997)

  Abstract.  The trafficking of Src family proteins after biosynthesis is poorly defined.  Here we studied the role of dual fatty acylation with myristate and palmitate in biosynthetic transport of p59^fyn.  Metabolic labeling of transfected COS or NIH 3T3 cells with [³⁵S]methionine followed by analysis of cytosolic and total membrane fractions showed that Fyn became membrane bound within 5 min after biosynthesis.  Newly synthesized Src, however, accumulated in the membranes between 20-60 min.  Northern blotting detected Fyn mRNA specifically in soluble polyribosomes and soluble Fyn protein was only detected shortly (1-2 min) after radiolabeling. Use of chimeric Fyn and Src constructs showed that rapid membrane targeting was mediated by the myristoylated NH₂-terminal sequence of Fyn and that a cysteine at position 3, but not 6, was essential. Examination of Ga₀-, Ga_s-, or GAP43-Fyn fusion constructs indicated that rapid membrane anchoring is exclusively conferred by the combination of N-myristoylation plus palmitoylation of cysteine-3.  Density gradient analysis colocalized newly synthesized Fyn with plasma membranes.  Interestingly, a 10-20-min lag phase was observed between plasma membrane binding and the acquisition of non-ionic detergent insolubility.  We propose a model in which synthesis and myristoylation of Fyn occurs on soluble ribosomes, followed by rapid palmitoylation and plasma membrane anchoring, and a slower partitioning into detergent-insoluble membrane subdomains.  These results serve to define a novel trafficking pathway for Src family proteins that are regulated by dual fatty acylation.  

3.24           Molecular cloning and expression of a chloride ion channel of cell nuclei

Valenzuela, S.M. et al

1. Biol. Chem., 272(19), 12575-12582 (1997)

  Ion channels are known to be present on the plasma membrane of virtually all cells and have been found on the membranes of various intracellular organelles.  However, until recently they were believed not to occur at the nuclear membrane.  In this study we describe the molecular cloning and characterization of a nuclear ion channel protein, designated nuclear chloride channel-27 (NCC27), from the human myelomonocytic cell line, U937.  NCC27 is a novel chloride ion channel protein that was found to localize principally to the cell nucleus. Its only known homologue is a bovine chloride ion channel protein (p64) believed to localize to internal organelles.  NCC27 therefore represents the first human member of a new class of organellar chloride ion channel proteins.  

3.25           Bradykinin sequesters B2 bradykinin receptors and the receptor-coupled Ga subunits Ga_q and Ga_i in caveolae in DDT₁ MF-2 smooth muscle cells

de Weerd, W.F.C. and Leeb-Lundberg, L.M.F.

1. Biol. Chem., 272(28), 17858-17866 (1997)

  In this report, we show that the vasoactive peptide agonist bradykinin (BK) when bound to B2 BK receptors on DDT₁MF-2 smooth muscle cells promotes the recruitment and sequestration of the occupied receptors and the receptor-coupled G-protein a subunits Ga_q and Ga_i in caveolae.  Association of ligand receptor complexes and Ga subunits with caveolae was indicated by their co-enrichment on density gradients with caveolin, a marker protein for caveolae.  Caveolin and Ga subunits were monitored by immunoblotting, whereas receptors were monitored as ligand receptor complexes formed by labeling receptors with the agonist BK or the antagonist NPC17731 prior to cell disruption and caveolae enrichment.  These complexes were detected with radioligand and by immunoblotting with BK antibodies.  A direct interaction of Ga subunits with caveolin was also indicated by their co-immunoprecipitation.  Immunoelectron microscopy revealed that the enriched caveolin, Ga subunits, and BK receptor complexes were present in structures of 0.1-0.2 mm.  At 4^oC, BK and NPC17731 receptor complexes were detected in caveolae, and both complexes were sensitive to acid washing prior to cell disruption and caveolae enrichment.  Elevation of the temperature to 37°C increased the amount of BK receptor complexes in caveolae with a maximal response at 10 min (continuous labeling) or 20 min (single-round labeling), and the complexes became acid-resistant.  These conditions also increased the amount of Ga_q and Ga_i. in caveolae with a maximal response at 5-10 min.  In contrast, the NPC17731 receptor complexes remained acid sensitive and dissociated at this temperature, and antagonists did not increase the amount of Ga subunits in caveolae.  These results show that some agonists that act through G-protein-coupled receptors promote the association of their receptors and receptor-coupled Ga subunits with caveolae.

3.26           Vacuoles induced by Helicobacter pylori toxin contain both late endosomal and lysosomal markers

Molinari, M.et al

1. Biol. Chem., 272(40), 25339-25344 (1997)

  Intoxication of mammalian cells with the vacuolating toxin (VacA) released by Helicobacter pylori causes the formation of large acidic vacuoles containing the vacuolar ATPase proton pump and Rab7, a late endosome marker.  Here, we describe a novel subcellular fractionation procedure, and we show that nanomolar concentrations of VacA induce a clear redistribution of lysosomal membrane glycoproteins among endocytic compartments.  This redistribution is an early event in the process of cellular intoxication by VacA and precedes the formation of macroscopic vacuoles.  The absence of the cation independent mannose 6-P receptor and the presence of Rab7 and of lysosomal membrane proteins in the newly formed compartment suggest that the vacuolating toxin induces the accumulation of a post-endosomal hybrid compartment presenting both late endosomal and lysosomal features.  

3.27           Inhibition of endosome function in CHO cells bearing a temperature-sensitive defect in the coatomer (COPI) component Î-COP

Daro, E., Sheff, D., Gomez, M., Kreis, T. and Mellman, I.

1. Cell Biol., 139(7), 1747-1759 (1997)

  Abstract.  Recent evidence has suggested that subunits of the coatomer protein (COPI) complexes are functionally associated with endosomes in mammalian cells.  We now provide genetic evidence that COPI plays a role in endocytosis in intact cells.  The 1D1F mutant CHO cell line bears a temperature-sensitive defect in the COPI subunit Î-COP.  In addition to exhibiting conditional defects in the secretary pathway, we find that the cells are also defective at mediating endosome-associated functions.  As found for cells microinjected with anti-COPI antibodies, 1D1F cells at the restrictive temperature could not be infected by vesicular stomatitis (VSV) or Semliki Forest virus (SFV) that require delivery to acidic endosomes to penetrate into the cytosol.  Although there was no temperature-sensitive defect in the internalization of receptor-bound transferrin (Tfn), Tfn recycling and accumulation of HRP were markedly inhibited at the restrictive temperature.  Sorting of receptor-bound markers such as EGF to lysosomes was also reduced, although delivery of fluid-phase markers was only partially inhibited.  In addition, lysosomes redistributed from their typical perinuclear location to the tips of the 1D1F cells.  Mutant phenotypes began to emerge within 2 h of temperature shift, the time required for the loss of detectable Î-COP, suggesting that the endocytic defects were not secondary to a block in the secretary pathway.  Importantly, the mutant phenotypes were also corrected by transfection of wild-type Î-COP cDNA demonstrating that they directly or indirectly reflected the Î-COP defect.  Taken together, the results suggest that Î-COP acts early in the endocytic pathway, most likely inhibiting the normal sorting and recycling functions of early endosomes.

3.28           Identification of caveolin and caveolin-related proteins in the brain

Cameron, P.L., Ruffin, J.W., Bollag, R., Rasmussen, H. and Cameron, R.S.

1. Neurosci., 17(24), 9520-9535 (1997)

  Caveolae are 50-100 nm, nonclathrin-coated, flask-shaped plasma membrane microdomains that have been identified in most mammalian cell types, except lymphocytes and neurons.  To date, multiple functions have been ascribed to caveolae, including the compartmentalization of lipid and protein components that function in transmembrane signaling events, biosynthetic transport functions, endocytosis, potocytosis, and transcytosis.  Caveolin, a 21-24 kDa integral membrane protein, is the principal structural component of caveolae.  We have initiated studies to examine the relationship of detergent-insoluble complexes identified in astrocytes to the caveolin-caveolae compartment detected in cells of peripheral tissues. Immunolocalization studies performed in astrocytes reveal caveolin immunoreactivity in regions that correlate well to the distribution of caveolae and caveolin determined in other cell types, and electron microscopic studies reveal multiple clusters of flask-shaped invaginations aligned along the plasma membrane. Immunoblot analyses demonstrate that detergent insoluble complexes isolated from astrocytes are composed of caveolin-1 a, an identification verified by Northern blot analyses and by the cloning of a CDNA using reverse transcriptase-PCR amplification from total astrocyte RNA.  Using a full-length caveolin-1 probe, Northern blot analyses suggest that the expression of caveolin-1 may be regulated during brain development. Immunoblot analyses of detergent-insoluble complexes isolated from cerebral cortex and cerebellum identify two immunoreactive polypeptides with apparent molecular weight and isoelectric points appropriate for caveolin.  The identification of caveolae microdomains and caveolin-1 in astrocytes and brain, as well as the apparent regulation of caveolin-1 expression during brain development, identifies a cell compartment not detected previously in brain.    

3.29           High-density-lipoprotein subfraction 3 interaction with glycosylphosphatidyl-inositol-anchored proteins

Nion, S. et al Biochem. J., 328, 415-423 (1997)   To elucidate further the binding of high-density-lipoprotein subfraction 3 (HDL₃) to cells, the involvement of glycosylphosphatidylinositol-anchored proteins (GPI-proteins) was studied. Treatment of cultured cells, such as fibroblasts or SK-MES-1 cells, with a phosphatidylinositol-specific phospholipase C(PI-PLC) significantly decreases specific HDL₃ binding. Moreover, PI-PLC treatment of cultured cells or cellular plasma membrane fractions results in releasing proteins. These proteins have a soluble form and can also bind HDL₃, as revealed by ligand blotting experiments with HDL₃. In order to obtain enriched GPI-proteins, we used a detergent-free purification method to prepare a caveolar membrane fraction. In the caveolar fraction, we obtained, by ligand blotting experiments, the enrichment of two HDL₃-binding proteins with molecular masses of 120 and 80 kDa. These proteins were also revealed in a plasma membrane preparation with two other proteins, with molecular masses of 150 and 104 kDa, and were sensitive to PI-PLC treatment. Electron microscopy also showed the binding of Au-labelled HDL₃ inside the vacuolar membrane invaginations. In SK-MES-1 cells, HDL₃ are internalized into a particular structure, resulting in the accumulation and concentration of such specific membrane domains. To sum up, a demonstration has been made of the implication of GPI-proteins as well as caveolae in the binding of HDL₃ to cells.  

3.30           Reduction of protein disulfide bonds in an oxidizing environment

Majoul, I., Ferrari, D. and Soling, H-D. FEBS Lett., 401, 104-108 (1997)   Following retrograde transport to the endoplasmic reticulum (ER) the A-subunit of cholera toxin (CTX-A) is partially cleaved into CTX-A1 and CTX-A2 by reduction of a disulfide bridge [Majoul et al (1996) J. Cell Biol. 133, 777-789], although the redox state in the ER favors disulfide formation. We show here that the disulfide bridge of CTX-A is cleaved in vitro already at GSH/GSSG ratios between 1 and 3. Protein disulfide isomerase (PDI) exerts only a minor accelerating effect. Various mixed disulfide intermediates (CTX-A1-S-S-CTX-A1; PDI-S-S-A2; PDI-S-S-A1) appear during CTX-A reduction. These results indicate that in the ER protein disulfide formation and protein disulfide reduction can take place simultaneously.  

3.31           Calbindin-D_28k in nerve cell nuclei

German, D.C., Ng, M.C., Liang, C.L., McMahon, A and Iacopino, A.M. Neurosci., 81(3), 735-743 (1997)   Calbindin-D_28k is a member of the large EF-hand family of calcium-binding proteins that is believed to function, in part, as a cytosolic calcium buffer. Recent studies have demonstrated that cells containing Calbindin-D_28k are protected from degeneration caused by conditions that elevate intracellular calcium concentration. Since its initial discovery in 1966, Calbindin-D_28k has been localized in the cytoplasm of many neuronal populations, but its nuclear localization has been uncertain. Using light and electron microscopic immunochemistry, and nuclear fractionation methods, we demonstrate localization of Calbindin-D_28k not only in the cytoplasm, but also in the nucleus of rodent midbrain dopaminergic neurons and cerebellar Purkinje cells. The Calbindin-D_28k immunoreactive staining intensity in the nucleus was routinely equal or greater than that in the cytoplasm. Since calcium signals are propagated to the nucleus, where they can regulate gene expression, the existence of nuclear Calbindin-D_28k has important implications for cellular functions.  

3.32           Membrane association of FtsY, the E. coli SRP receptor

DeLeeuw, et al FEBS Lett., 416, 225-229 (1997)   FtsY, the Escherichia coli homologue of the eukaryotic SRP receptor (SRa), is located both in the cytoplasm and in the inner membrane of E. coli. Similar to SRa, FtsY consists of the two major domains: a strongly acidic N-terminal domain (A) and a C-terminal GTP binding domain (NG) of which the crystal structure has recently been determined. The domains were expressed both in vivo and in vitro to examine their subcellular localization. The results suggest that both domains associated with the membrane but that the nature of the association differs.

3.33           Role for the target enzyme in deactivation of photoreceptor G protein in vivo

Tsang, S.H., et al. Science, 282(5386), 117-121(1998)   Heterotrimeric guanosine 5’-triphosphate (GTP)-binding proteins (G proteins) are deactivated by hydrolysis of the GTP that they bind when activated by transmembrane receptors. Transducin, the G protein that relays visual excitation from rhodopsin to the cyclic guanosine 3’, 5’-monophosphate phosphodiesterase (PDE) in retinal photoreceptors, must be deactivated for the light response to recover. A point mutation in the g subunit of PDE impaired transducin-PDE interactions and slowed the recovery rate of the flash response in transgenic mouse rods. These results indicate that the normal deactivation of transducin in vivo requires the G protein to interact with its target enzyme.  

3.34           Characterization of a cytosolic heat-shock protein-caveolin chaperone complex.

Uittenbogaard, A., Ying, Y.S. and Smart, E.J.

1. Biol. Chem., 273(11), 6525-6532, (1998)

  Caveolin is a 22-kDa protein that appears to play a critical role in regulating the cholesterol concentration of caveolae. Even though caveolin is thought to be a membrane protein, several reports suggest that this peculiar protein can traffic independently of membrane vesicles. We now present evidence that a cytosolic pool of caveolin is part of a heat-shock protein-immunophilin chaperone complex consisting of caveolin, heat-shock protein 56, cyclophilin 40, cyclophilin A, and cholesterol. Treatment of NIH 3T3 cells with 1 mm cyclosporin A or 100 nm rapamycin disrupted the putative transport complex and prevented rapid (10-20 min) transport of cholesterol to caveolae. The lymphoid cell line, L1210-JF, does not express caveolin, does not form an immunophilin-caveolin complex, and does not transport newly synthesized cholesterol to caveolae. Transfection of caveolin cDNA into L1210-JF cells allowed the assembly of a transport complex identical to that found in NIH 3T3 cells. In addition, newly synthesized cholesterol in transfected cells was rapidly (10-20 min) and specifically transported to caveolae. These data strongly suggest that a caveolin-chaperone complex is a mechanism by which newly synthesized cholesterol is transported from the endoplasmic reticulum through the cytoplasm to caveolae.  

3.35           Dissection of hepatic receptor-mediated endocytic pathways using self-generated gradients of iodixanol (OptiPrep).

Billington, D., Maltby, P.J. Jackson, A.P. and Graham, J.M. Anal. Biochem., 258, 251-258 (1998).        Iodixanol is a new, non-ionic, iodinated density gradient medium which has the advantage over other similar media in that it rapidly forms self-generated gradients in vertical or near-vertical rotors.  Endocytosis of ^99mTc-labelled neogalactosyl albumin (^99mTc-NGA), a synthetic ligand for the asialoglycoprotein receptor, was studied by administering the ligand as a short pulse to perfused rat livers operating under single pass conditions.  Intracellular processing was arrested at various times after the pulse and the resultant homogenate cleared of nuclei and heavy mitochondria by centrifugation at 3000g for 10 min.  After adjusting to 12.5% (w/v) iodixanol, the 3000g supernatants were centrifuged at 350,000g for 60 min to form the gradients in which early, clathrin-containing vesicles, low-density endosomes and lysosomes were well-resolved.  ^99mTc-NGA bound to the sinusoidal membrane could be partially resolved from clathrin-containing vesicles by inclusion of 1 mM CaCl₂ in the homogenisation and gradient buffers. Two populations of early clathrin-containing vesicles could be resolved by rate-zonal centrifugation in pre-formed iodixanol gradients.  Thus, iodixanol is an excellent density gradient medium for the rapid and efficient resolution of endosome compartments.  

3.36           Subcellular distribution and turnover of presenilins in transfected cells.

Zhang, J. et al

1. Biol. Chem., 273(20), 12436-12442 (1998)

  The mechanisms by which mutations in presenilin-1 (PS1) and presenilin-2 (PS2) result in the Alzheimer=s disease phenotype are unclear. Full-length PS1 and PS2 are each processed into stable proteolytic fragments after their biosynthesis in transfected cells. PS1 and PS2 have been localized by immunocytochemistry to the endoplasmic reticulum (ER) and Golgi compartments, but previous studies could not differentiate between the full-length presenilins and their fragments. Full-length PS1 and PS2 were principally distributed in ER fractions, whereas the N- and C-terminal fragments were localized predominantly to the Golgi fractions. In cells expressing the PS1 mutant lacking exon (DE9), we observed only full-length molecules that were present in the ER and Golgi fractions. The turnover rate was considerably slower for the DE9 holoprotein, apparently due to decreased degradation within the ER. Our results suggest that full-length presenilin proteins are primarily ER resident molecules and undergo endoproteolysis within the ER. The fragments are subsequently transported to the Golgi compartment, where their turnover rate is much slower than that of the full-length presenilin in the ER.  

3.37           Dietary fish oils modify the assembly of VLDL and expression of the LDL receptor in rabbit liver.

Wilkinson, J., Higgins, J.A., Fitzsimmons, C. and Bowyer, D.E. Arterioscler. Thromb. Vasc. Biol., 18, 1490-1497 (1998)   Supplementation of the diet of rabbits with fish oil or sunflower oil resulted in significant changes in the lipoproteins and lipids in serum. Compared with chow-fed rabbits, dietary fish oils decreased very low density lipoprotein (VLDL), increased low density lipoprotein (LDL), and shifted the peak of the LDL to denser fractions, whereas sunflower oil increased high density lipoprotein and shifted LDL to the lighter fractions. The amount of LDL receptors in fish oil-fed rabbit liver decreased by > 70% while there was only a small fall in these levels in sunflower oil-fed rabbit liver. The concentrations of apolipoprotein (apo) B in the subcellular organelles of the secretory compartment (rough and smooth endoplasmic reticula and Golgi fractions) were also changed by dietary lipids. In both sunflower oil- and fish oil-fed liver, apo B was increased in the lumen of the rough endoplasmic reticulum compared with fractions from chow-fed rabbit liver. The apo B in the trans-Golgi lumen from fish oil-fed livers was reduced and occurred in particles of d » 1.21 g/mL. In contrast, apo B in the trans-Golgi lumen from livers of sunflower oil-fed rabbits was increased and occurred in particles of d< 1.21 g/mL. These results suggests that feeding of fish oils causes an interruption in the intracellular transfer of apo B and hence assembly of VLDL. This leads to an enrichment of the rough endoplasmic reticulum membranes with cholesterol, thus down regulating the expression of the LDL receptor.  

3.38           Phosphatidylinositol 4-phosphate synthesis in immunoisolated caveolae-like vesicles and low buoyant non-caveolar membranes.

Waugh, M.G., Lawson, D., Tan, S.K. and Hsuan, J.J.

1. Biol. Chem., 273(27), 17115-17121 (1998)

  This study examined phosphatidylinositol 4-phosphate (PtdIns4P) synthesis in caveolae that have been suggested to be discrete signaling microdomains of the plasma membrane and are enriched in the marker protein caveolin. Caveolin-rich light membranes (CLMs) were isolated from A431 cells by detergent-free, discontinuous density-gradient centrifugation method. The CLM fraction was separated from the bulk of the cellular protein and was greatly enriched in PtdIns, PtdIns4P, and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) and an adenosine-sensitive type II PtdIns 4-kinase activity. Preparation of CLMs by an OptiPrep-based cell fractionation procedure confirmed the co-localization of PtdIns 4-kinase and caveolin. Electron microscopy confirmed that an anti-caveolin antiserum immunopurified vesicles from CLMs that were within the size range described for caveolae in other systems. Co-immunoprecipitated PtdIns 4-kinase activity could utilize endogenous PtdIns, present within the caveolae and CLMs. However, less than 1% of the total cellular PtdIns and PtdIns 4-kinase activity was present in caveolae-like vesicles, indicating that non-caveolar light membranes rafts are the main sites for cellular PtdIns4P production.  

3.39           Rapid enzyme-free preparation of starch-free nuclei from plants facilitates studies of chromatin structure.

Ford, T.C., Baldwin, J.P. and Lambert, S.J. Plant proteins in abiotic stress responses, Plant Protein Club, 1998 Annual Symposium, University of York, p24 (1998)   Several studies of the chromatin structure of normal or stressed plant cells require rapid purification of the cell nuclei under conditions which do not alter the structure of the active or inactive genes under investigation. We have developed a very simple, rapid and enzyme-free method for preparing wheatgerm nuclei by centrifugation on an iodixanol step between densities 1.168 and 1.234 g/ml with a minimal contribution of the iodixanol to solution colligative properties (osmotic pressure). Histone octamers, which are key molecular complexes in the regulation of transcription and which are acetylated in specific lysines in active genes have been purified rapidly from the pure nuclei.    

3.40           Insulin-induced protein tyrosine phosphorylation cascade and signalling molecules are localized in a caveolin-enriched cell membrane domain.

Smith, R.M., Harada, S., Smith, J.A., Zhang, S. and Jarett, L. Cell. Signalling, 10(5), 355-362 (1998)   The cellular localisation of time- and temperature-dependent ¹²⁵I-insulin binding, insulin-sensitive signalling proteins and the insulin-induced protein tyrosine phosphorylation cascade were assessed in subcellular fractions isolated on Iodixanol gradients from control and insulin-treated H35 hepatoma cells. Western blot analysis demonstrated that the concentrations of IRS-1, Shc, GRB-2, SOS, Syp, PI 3-kinase, MAP kinase and G_1a were at least 10-fold higher in cell surface-derived, caveolin-enriched fraction than in a cell surface-derived, caveolin-poor fraction (i.e., the plasma membranes). Insulin treatment caused a 15-fold increase in tyrosine phosphorylation of IRS-1 in the caveolin-enriched fraction in 5 min at 37°C compared with a 3-fold increase in plasma membranes and a 6-fold increase in the cytosol and endosomes. Insulin also increased tyrosine phosphorylation of both a 72-kDa protein and the 46-kDa Shc isoform only in the caveolin-enriched fraction. Insulin treatment did not change the concentrations of insulin receptors or Shc but increased IRS-1 in the caveolin-enriched fraction, possibly recruited from the cytosolic pool. Insulin also increased the concentrations of insulin receptors, IRS-1 and Shc in endosomes, suggesting insulin-induced internalization of the insulin receptors and proteins activated with them. Electron microscopic analysis, with the use of a combination of colloidal gold-labelled insulin to label the insulin receptor and immunolabelling to detect caveolin or IRS-1, demonstrated the co-localisation of insulin receptors in caveolin- and IRS-1 containing vesicular structures. Differences in the insulin-induced protein tyrosine phosphorylation and concentrations of these proximal signalling proteins in the caveolin-enriched fraction, plasma membranes, and cytosol suggest that insulin receptors in the caveolae play a major role in the initiating insulin’s signal transduction processes.  

3.41           Purification and characterization of autophagosomes from rat hepatocytes.

Strømhaug, P.E., Berg, T.O., and Seglen, P.O. Biochem. J., 335, 217-224 (1998)   To investigate the properties and intracellular origin of autophagosomes, a procedure for the purification and isolation of these organelles from rat liver has been developed. Isolated hepatocytes were incubated with vinblastine to induce autophagosome accumulation; the cells were then homogenized and treated with the cathepsin C substrate glycyl-1-phenylalanine 2-naphthylamide to cause osmotic disruption of the lysosomes. Nuclei were removed by differential centrifugation, and the postnuclear supernatant was fractionated on a discontinuous Nycodenz density gradient. The autophagosomes, recognized by their content of autophagocytosed lactate dehydrogenase (LDH), could be recovered in an intermediated-density fraction, free from cytosol and mitochondria. Finally, the autophagosomes were separated from the endoplasmic reticulum and other membraneous elements by centrifugation in a Percoll colloidal density gradient, followed by flotation in iodixanol to remove the Percoll particles. The final autophagosome preparation represented a 24-fold purification of autophagocytosed LDH relative to intact cells, with a 12% recovery. The purified autophagosomes contained sequestered cytoplasm with a normal ultrastructure, including mitochondria, peroxisomes and endoplasmic reticulum in the same proportions as in intact cells. However, immunoblotting indicated a relative absence of cytoskeletal elements (tubulin, actin and cytokeratin), which may evade autophagic sequestration. The autophagosomes showed no enrichment in protein markers typical of lysosomes (acid phosphatase, cathepsin B, lysosomal glycoprotein of 120 kDa), endosomes (early-endosome-associated proteins 1, cation-independent mannose 6-phosphate-regulated protein of 78 kDa, protein disulphide isomerase), suggesting that the sequestering membranes are not derived directly from any of these organelles, but rather represent unique organelles (phagophores).  

3.42           Presenilin 1 regulates the processing of b-amyloid precursor protein C-terminal fragments and the generation of amyloid b-protein in endoplasmic reticulum and Golgi.

Xia, W. et al Biochemistry, 37(47), 16465-71 (1998)   Progressive cerebral deposition of the amyloid b-protein (Ab) is believed to play a pivotal role in the pathogenesis of Alzheimer’s disease (AD). The highly amyloidogenic 42-residue form of Ab (Ab₄₂) is the first species to be deposited in both sporadic and familial AD. Mutations in two familial AD-linked genes, presenilins 1 (PS1) and 2 (PS2), selectively increase the production of Ab₄₂ in cultured cells and the brains of transgenic mice, and gene deletion of PS1 shows that it is required for normal gamma-secretase cleavage of the beta-amyloid precursor protein (APP) to generate Ab. To establish the subcellular localization of the PS1 regulation of APP processing to Ab, fibroblasts from PS1 wild-type (wt) or knockout (KO) embryos as well as Chinese hamster ovary (CHO) cells stably transfected with wt or mutant PS1 were subjected to subcellular fractionation on discontinuous Iodixanol gradients. APP C-terminal fragments (CTF) were markedly increased in both endoplasmic reticulum- (ER-) and Golgi-rich fractions of fibroblasts from KO mice; moreover, similar increases were documented directly in KO brain tissue. No change in the subcellular distribution of full-length APP was detectable in fibroblasts lacking PS1. In CHO cells, a small portion of APP, principally the N-glycosylated isoform, formed complexes with PS1 in both ER- and Golgi-rich fractions, as detected by coimmunoprecipitation. When the same fractions were analyzed by enzyme-linked immunosorbent assays for Ab_total and Ab₄₂, Ab₄₂ was the major Ab species in the ER fraction (Ab₄₂:Ab_total ratio 0.5-1.0), whereas absolute levels of both Ab₄₂ and Ab₄₀ were higher in the Golgi fraction and the Ab₄₂:Ab_total ratio was 0.05-0.16 there. Mutant PS1 significantly increased Ab₄₂ levels in the Golgi fraction. Our results indicate PS1 and APP can interact in the ER and Golgi, where PS1 is required for proper g-secretase processing of APP CTFs, and that PS1 mutations augment Ab₄₂ levels principally in Golgi-like vesicles.  

3.43           Role of plasmalemmal caveolae in signal transduction

Shaul, P.W. and Anderson, R.G.W. Am. J. Physiol., 275, 843-851 (1998)   Caveolae are specialized plasmalemmal microdomains originally studied in numerous cell types for their involvement in the transcytosis of macromolecules.  They are enriched in glycosphingolipids, cholesterol, sphingomyelin, and lipid-anchored membrane proteins, and they are characterized by a light buoyant density and resistance to solubilization by Triton X-100 at 4°C.  Once the identification of the marker protein caveolin made it possible to purify this specialized membrane domain, it was discovered that caveolae also contain a variety of signal transduction molecules.  This includes C protein-coupled receptors, G proteins and adenylyl cyclase, molecules involved in the regulation of intracellular calcium homeostasis, and their effectors including the endothelial isoform of nitric oxide synthase, multiple components of the tyrosine kinase-mitogen-activated protein kinase pathway, and numerous lipid signaling molecules.  More recent work has indicated that caveolae further serve to compartmentalize, modulate, and integrate signaling events at the cell surface.  This specialized plasmalemmal domain warrants direct consideration in future investigations of both normal and pathological signal transduction in pulmonary cell types.  

3.44           Targeting of protein Kinase Ca to caveolae

Mineo, C., Ying, Y-S, Chapline, C., Jaken, S. and Anderson, R.G.W.

1. Cell Biol., 141(3), 601-610 (1998)

  Previously, we showed caveolae contain a population of protein kinase Ca (PKCa) that appears to regulate membrane invagination. We now report that multiple PKC isoenzymes are enriched in caveolae of unstimulated fibroblasts. To understand the mechanism of PKC targeting, we prepared caveolae lacking PKCa and measured the interaction of recombinant PKCa with these membranes. PKCa bound with high affinity and specificity to caveolae membranes. Binding was calcium dependent, did not require addition of factors that activate the enzyme, and involved the regulatory domain of the molecule. A68-kD PKCa-binding protein identified as sdr (serum deprivation response) was isolated by interaction cloning and localized to caveolae. Antibodies against sdr inhibited PKCa binding. A 100-aminoacid sequence from the middle of sdr competitively blocked PKCa binding while flanking sequences were inactive. Caveolae appear to be a membrane site where PKC enzymes are organized to carry out essential regulatory functions as well as to modulate signal transduction at the cell surface.  

3.45           SR-BII, an isoform of the scavenger receptor BI containing an alternate cytoplasmic tail, mediates lipid transfer between high density lipoprotein and cells

Webb, N.R. et al

1. Biol. Chem., 273(24), 15241-15248 (1998)

  The scavenger receptor class B, type I (SR-BI), binds high density lipoprotein (HDL) and mediates selective uptake of cholesteryl ester from HDL and HDL-dependent cholesterol efflux from cells.  We recently identified a new MRNA variant that differs from the previously characterized form in that the encoded C-terminal cytoplasmic domain is almost completely different.  In the present study, we demonstrate that the mRNAs for mouse SR-El and SR-BII (previously termed SR-BI.2) are the alternatively spliced products of a single gene.  The translation products predicted from human, bovine, mouse, hamster, and rat cDNAs exhibit a high degree of sequence similarity within the SR-BII C-terminal domain (62-67% identity when compared with the human sequence), suggesting that this variant is biologically important.  SR-BII protein represents approximately 12% of the total immunodetectable SR-BI/II protein in mouse liver.  Subcellular fractionation of transfected Chinese hamster ovary cells showed that SR-BII, like SR-BI, is enriched in caveolae, indicating that the altered cytoplasmic tail does not affect targeting of the receptor.  SR-BII mediated both selective cellular uptake of cholesteryl ether from HDL as well as HDL-dependent cholesterol efflux from cells, although with approximately 4-fold lower efficiency than SR-BI. In vivo studies using adenoviral vectors showed that SR-BII was relatively less efficient than SR-BI in reducing plasma HDL cholesterol.  These studies show that SR-BII, an HDL receptor isoform containing a distinctly different cytoplasmic tail, mediates selective lipid transfer between HDL and cells, but with a lower efficiency than the previously characterized variant.  

3.46           Cholesterol depletion of caveolae causes hyperactivation of extracellular signal-related kinase (ERK)

Furuchi, T. and Anderson, R.G.W.

1. Biol. Chem., 273(33), 21009-21104 (1998)

  Previously we showed that activation of Erk in quiescent cells occurs in the caveolae fraction isolated from fibroblasts.  Since the structure and function of caveolae is sensitive to the amount of cholesterol in the membrane, it might be that a direct link exists between the concentration of membrane cholesterol and mitogen activated protein (MAP) kinase activation.  We acutely lowered the cholesterol level of the caveolae fraction by incubating Rat-1 cells in the presence of either cyclodextrin or progesterone.  Cholesterol-depleted caveolae had a reduced amount of several key protein components of the MAP kinase complex, including Ras, Grb2, Erk2, and Src.  Incubation of these cells in the presence of epidermal growth factor (EGF) caused a rapid loss of EGF receptor from the caveolae fraction, but the usual recruitment of c-Raf was markedly inhibited.  Despite the reduced amount of c-Raf and Erk2 in the cholesterol depleted caveolae fraction, EGF caused a hyperactivation of the remaining caveolae Erk isoenzymes.  This was followed by an increase in the amount of active Erk in the cytoplasm.  The increased amount of activated Erk produced under these conditions was linked to a 2-fold higher level of EGF-stimulated DNA synthesis.  Even cholesterol depletion by itself stimulated Erk activation and DNA synthesis.  These results suggest that the MAP kinase pathway can connect the cholesterol level of caveolae membrane to the control of cell division.  

3.47           Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells

Grindstaff, K.K., Yeaman, C., Anandasabapathy, N., Hsu, S.C., Rodriguez-Boulan,E., Scheller R.H. and Nelson, W.J. Cell, 93, 731-740 (1998)   In budding yeast, the Sec6/8p complex is essential for generating cell polarity by specifying vesicle delivery to the bud tip. We show that Sec6/8 homologs are components of a cytosolic, ~17S complex in nonpolarized MDCK epithelial cells. Upon initiation of calcium-dependent cell-cell adhesion, ~70% of Sec6/8 is rapidly (t_1/2 » 3-6 hr) recruited to sites of cell-cell contact. In streptolyslin-O-permeabilized MDCK cells, Sec6/8 antibodies inhibit delivery of LDL receptor to the basal-lateral membrane, but not p75^NTR to the apical membrane. These results indicate that lateral membrane recruitment of the Sec6/8 complex is a consequence of cell-cell adhesion and is essential for the biogenesis of epithelial cell surface polarity.  

3.48           ARNO is a guanine nucleotide exchange factor for ADP-ribosylation factor 6

Frank, S., Upender, S., Hansen, S.H. and Casanova, J.E.

1. Biol. Chem., 273(1), 23-27(1998)

  ADP-ribosylation factors (ARFS) constitute a family of small monomeric GTPases.  ARFs 1 and 3 function in the recruitment of coat proteins to membranes of the Golgi apparatus, whereas ARF6 is localized to the plasma membrane, where it appears to modulate both the assembly of the actin cytoskeleton and endocytosis.  Like other GTPases, ARF activation is facilitated by specific guanine nucleotide exchange factors (GEFs).  ARNO (ARF nucleotide-binding site opener) is a member of a growing family of ARF-GEFs that share a common, tripartite structure consisting of an N-terminal coiled-coil domain, a central domain with homology to the yeast protein Sec7p, and a C-terminal pleckstrin homology domain.  Recently, ARNO and its close homologue cytohesin-1 were found to catalyze in vitro nucleotide exchange on ARF1 and ARF3, respectively, raising the possibility that these GEFs function in the Golgi.  However, the actual function of these proteins may be determined in part by their ability to interact with specific ARFs and in part by their subcellular localization.  We report here that in vitro ARNO can stimulate nucleotide exchange on both ARF1 and ARF6. Furthermore, based on subcellular fractionation and immunolocalization experiments, we find that ARNO is localized to the plasma membrane in mammalian cells rather than the Golgi.  It is therefore likely that ARNO functions in plasma membrane events by modulating the activity of ARF6 in vivo. These findings are consistent with the previous observation that cytohesin-1 regulates the adhesiveness of aLb₂ integrins at the plasma membrane of lymphocytes.  

3.49           Retrograde transport of Golgi-localized proteins to the ER

Cole, N.B., Ellenberg, J., Song, J., DiEuliis, D. and Lippincott-Schwarz, J.

1. Cell Biol., 140(1), 1-15 (1998)

  Abstract.  The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment.  Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretary pathway are able to return to the ER folding environment during their lifetime.  Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins.  The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains.  At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules.  Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained.  This occurred over a time period of 15 min-2 h depending on the chimera, and did not require new protein synthesis.  Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex.  This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained.  The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.  

3.50           Isolation of functional Golgi-derived vesicles with a possible role in retrograde transport

Love, H.D., Lin, C.C., Short, C.S. and Ostermann, J.

1. Cell Biol., 140(3), 541-551 (1998)

  Secretory proteins enter the Golgi apparatus when transport vesicles fuse with the cis -side and exit in transport vesicles budding from the trans-side.  Resident Golgi enzymes that have been transported in the cis-to-trans direction with the secretary flow must be recycled constantly by retrograde transport in the opposite direction.  In this study, we describe the functional characterization of Golgi-derived transport vesicles that were isolated from tissue culture cells.  We found that under the steady-state conditions of a living cell, a fraction of resident Golgi enzymes was found in vesicles that could be separated from cistemal membranes.  These vesicles appeared to be depleted of secretary cargo.  They were capable of binding to and fusion with isolated Golgi membranes, and after fusion their enzymatic contents most efficiently processed cargo that had just entered the Golgi apparatus.  Those results indicate a possible role for these structures in recycling of Golgi enzymes in the Golgi stack.  

3.51           Caveolin-1 and –2 in the exocytic pathway of MDCK cells

Scheiffele, P. et al

1. Cell Biol., 140(4), 795-806 (1998)

   Abstract.  We have studied the biosynthesis and transport of the endogenous caveolins in MDCK cells.  We show that in addition to homooligomers of caveolin-1, heterooligomeric complexes of caveolin-1 and -2 are formed in the ER.  The oligomers become larger, increasingly detergent insoluble, and phosphorylated on caveolin-2 during transport to the cell surface.  In the TGN caveolin-l/-2 heterooligomers are sorted into basolateral vesicles, whereas larger caveolin-I homooligomers are targeted to the apical side.  Caveolin-1 is present on both the apical and basolateral plasma membrane, whereas caveolin-2 is enriched on the basolateral surface where caveolae are present.  This suggests that caveolin-1 and -2 heterooligomers are involved in caveolar biogenesis in the basolateral plasma membrane.  Anti-caveolin-1 antibodies inhibit the apical delivery of influenza virus hemagglutinin without affecting basolateral transport of vesicular stomatitis virus G protein.  Thus, we suggest that caveolin-1 homooligomers play a role in apical transport.  

3.52           Caveolae, plasma membrane microdomains for a-secretase-mediated processing of the amyloid precursor protein

Ikezu, T et al

1. Biol. Chem., 273(17), 10485-10495 (1998)

  Caveolae are plasma membrane invaginations where key signaling elements are concentrated.  In this report, both biochemical and histochemical analyses demonstrate that the amyloid precursor protein (APP), a source of Ab amyloid peptide, is enriched within caveolae. Caveolin-1, a principal component of caveolae, is physically associated with APP, and the cytoplasmic domain of APP directly participates in this binding. The characteristic C-terminal fragment that results from APP processing by a-secretase, an as yet unidentified enzyme that cleaves APP within the Ab amyloid sequence, was also localized within these caveolae-enriched fractions. Further analysis by cell surface biotinylation revealed that this cleavage event occurs at the cell surface. Importantly, a-secretase processing was significantly promoted by recombinant overexpression of caveolin in intact cells, resulting in increased secretion of the soluble extracellular domain of APP. Conversely, caveolin depletion using antisense oligonucletotides prevented this cleavage event. Our current results indicate that caveolae and caveolins may play a pivotal role in the a-secretase-mediated proteolysis of APP in vivo.  

3.53           Lipid domain structure of the plasma membrane revealed by patching of membrane components

Harder, T., Scheiffele, P., Verkade, P. and Simons K.

1. Cell Biol., 141(4), 929-942 (1998)

  Abstract.  Lateral assemblies of glycolipids and cholesterol, "rafts," have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion.  We studied the structure of raft domains in the plasma membrane of non-polarized cells.  Overexpressed plasma membrane markers were evenly distributed in the plasma membrane.  We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers.  For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin.  The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T-lymphoma cells.  Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains.  In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases.  This view is supported by the finding that cholesterol depletion abrogated segregation.  Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.  

3.54           Cholesterol depletion inhibits the generation of b-amyloid in hippocampal neurons

Simons, M., Keller, P., De Strooper, B., Beyreuther, K., Dotti, C.G. and Simons, K. Proc. Natl. Acad. Sci. USA, 95, 6460-6464 (1998)   The amyloid precursor protein (APP) plays a crucial role in the pathogenesis of Alzheimer's disease.  During intracellular transport APP undergoes a series of proteolytic cleavages that lead to the release either of an amyloidogenic fragment called b-amyloid (Ab) or of a nonamyloidogenic secreted form consisting of the ectodomain of APP (APP_sec). It is Ab that accumulates in the brain lesions that are thought to cause the disease.  By reducing the cellular cholesterol level of living hippocampal neurons by 70% with lovastatin and methyl-b-cyclodextrin, we show that the formation of Ab is completely inhibited while the generation of APP_sec is unperturbed.  This inhibition of Ab formation is accompanied by increased solubility in the detergent Triton X-100 and is fully reversible by the readdition of cholesterol to previously depleted cells.  Our results show that cholesterol is required for Ab formation to occur and imply a link between cholesterol, Ab, and Alzheimer's disease.  

3.55           CD14-dependent endotoxin internalization via a macropinocytic pathway

Poussin, C., Foti, M., Carpentier, J.L. and Pugin, J.

1. Biol. Chem., 273(32), 20285-20291 (1998)

  Gram-negative bacterial endotoxin (a lipopolysaccharide (LPS)) specifically binds to CD14, a glycosylphosphatidyl inositol (GPI)-anchored surface myeloid glycoprotein.  This interaction leads to cell activation, but it also promotes LPS internalization and detoxification.  In this work, we investigated the route of LPS and CD14 internalization and the relevance of CD14 GPI anchor in the endocytic pathway.  In promonocytic THP-1 ells transfected with a GPI or a chimeric integral form of CD14, we showed by differential buoyancy 'm sucrose density gradients that these two forms of CD14 were sorted to different plasma membrane subdomains.  However, both forms of CD14 associated preferentially with the same surface microfilament-enriched microvilli or ruffles.  Electron microscopic studies indicated that CD14 internalized via macropinocytosis, a process resembling that of phagocytosis, different from "classical" receptor-mediated endocytic pathways, such as clathrin-coated pits or caveolae.  With cell warming, the CD14-enriched ruffles fused and formed large vesicles. Later, these vacuoles made stacks and condensed into phago-lysosomes.  CD14 was specifically associated with all of these structures.  Radiolabeled LPS internalization paralleled CD14 internalization.  Confocal microscopic studies confirmed the co-localization of LPS and CD14 both at the cell surface and in endosomal compartments. The microfilament-disrupting, macropinocytosis blocking agent cytochalasin D inhibited LPS and CD14 internalization but did not prevent LPS-dependent activation, indicating that these two processes are dissociated.  

3.56           Isolation and characterization of rat liver amphisomes

Berg, T.O., Fengsrud, M., Stromhaug, P.E., Berg, T. and Seglen, P.O.

1. Biol. Chem., 273(34), 21883-21892 (1998)

  Amphisomes, the autophagic vacuoles (AVs) formed upon fusion between autophagosomes and endosomes, have so far only been characterized in indirect, functional terms.  To enable a physical distinction between autophagosomes and amphisomes, the latter were selectively density-shifted in sucrose gradients following fusion with AOM-gold-loaded endosomes (endosomes made dense by asialoorosomucoid-conjugated gold particles, endocytosed by isolated rat hepatocytes prior to subcellular fractionation).  Whereas amphisomes, by this criterion, accounted for only a minor fraction of the AVs in control hepatocytes, treatment of the cells with leupeptin (an inhibitor of lysosomal protein degradation) caused an accumulation of amphisomes to about one-half of the " population.  A quantitative electron microscopic study confirmed that leupeptin induced a severalfold increase in the number of hepatocytic amphisomes (recognized by their gold particle contents; otherwise, their ultrastructure was quite similar to antophagosomes).  Leupeptin caused, furthermore, a selective retention of endocytosed AOM-gold in the amphisomes at the expense of the lysosomes, consistent with an inhibition of amphisome-lysosome fusion.  The electron micrographs suggested that autophagosomes could undergo multiple independent fusions, with multivesicular (late) endosomes to form amphisomes and with small lysosomes to form large autolysosomes. A biochemical comparison between autophagosomes and amphisomes, purified by a novel procedure, showed that the amphisomes were enriched in early endosome markers (the asialoglycoprotein receptor and the early endosome-associated protein 1) as well as in a late endosome marker (the cation-independent mannose 6-phosphate receptor).  Amphisomes would thus seem to be capable of receiving inputs both from early and late endosomes.  

3.57           Apg14p and Apg6/Vps30p form a protein complex essential for autophagy in the yeast Saccharomyces cerevisiae

Kametaka, S., Okano, T., Ohsumi, M. and Ohsumi, Y.

1. Biol. Chem., 273(35), 22284-22291 (1998)

  Mutation in the Saccharomyces cerevisiae APG14 gene causes a defect in autophagy. Cloning and structural analysis of the APG14 gene revealed that APG14 encodes a novel hydrophilic protein with a predicted molecular mass of 40.5 kDa, and that Apgl4p has a coiled-coil motif at its N terminus region.  We found that overproduction of Apgl4p partially reversed the defect in autophagy induced by the apg6-1 mutation.  The apg6-1 mutant was found to be defective not only in autophagy but also in sorting of carboxypeptidase Y (CPY), a vacuolar-soluble hydrolase, to the vacuole.  However, overexpression of APG14 did not alter the CPY sorting defect of the apg6-l mutant, nor did the apgl4 null mutation affect the CPY sorting pathway.  Structural analysis of APG6 revealed that APG6 is identical to VPS30, which is involved in a retrieval step of the CPY receptor, Vpsl0p, to the late Golgi from the endosome (Seaman, M. N. J., Marcusson, E. G., Cereghino, J. L., and Emr, S. D. (1997) J.  Cell Biol 137, 79-92).  Subcellular fractionation indicated that Apgl4p and Apg6p peripherally associated with a membrane structure(s).  Apgl4p was co-immunoprecipitated with Apg6p, suggesting that they form a stable protein complex.  These results imply that Apg6/Vps30p has two distinct functions in the autophagic process and the vacuolar protein sorting pathway.  Apgl4p may be a component specifically required for the function of Apg6/Vps30p through the autophagic pathway.  

3.58           Annexin XIIIb associates with lipid microdomains to function in apical delivery

Lafont, F., Lecat, S., Verkade, P. and Simons K.

1. Cell Biol., 142(6), 1413-1427 (1998)

  A member of the annexin XIII sub-family, annexin XIIIb, has been implicated in the apical exocytosis of epithelial kidney cells.  Annexins are phospholipid-binding proteins that have been suggested to be involved in membrane trafficking events although their actual physiological function remains open. Unlike the other annexins, annexin XIIIs are myristoylated.  Here, we show by immunoelectron microscopy that annexin XIIIb is localized to the trans-Golgi network (TGN), vesicular carriers and the apical cell surface.  Polarized apical sorting involves clustering of apical proteins into dynamic sphingolipid-cholesterol rafts.  We now provide evidence for the raft association of annexin XIIIb. Using in vitro assays and either myristoylated or unmyristoylated recombinant annexin XIIIb, we demonstrate that annexin XIIIb in its native myristoylated form stimulates specifically apical transport whereas the unmyristoylated form inhibits this route.  Moreover, we show that formation of apical carriers from the TGN is inhibited by an anti-annexin XIIIb antibody whereas it is stimulated by myristoylated recombinant annexin XIIIb.  These results suggest that annexin XIIIb directly participates in apical delivery.  

3.59           The Escherichia coli SRP and SecB targeting pathways converge at the translocon

Valent, Q.A. et al The EMBO J., 17(9), 2504-2512 (1998)   Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane.  The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins.  SecB targets the pre-protein to SecA that mediates preprotein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins.  By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane.  The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP.  Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.  

3.60           Functions of lipid rafts in biological membranes

Brown, D.A. and London, E. Annu. Rev. Cell Dev. Biol., 14, 111-136 (1998)   Recent studies showing that detergent-resistant membrane fragments can be isolated from cells suggest that biological membranes are not always in a liquid-crystalline phase. Instead, sphingolipid and cholesterol-rich membranes such as plasma membranes appear to exist, at least partially, in the liquid-ordered phase or a phase with similar properties.  Sphingolipid and cholesterol-rich domains may exist as phase-separated “rafts” in the membrane.  We discuss the relationship between detergent-resistant membranes, rafts, caveolae, and low-density plasma membrane fragments.  We also discuss possible functions of lipid rafts in membranes.  Signal transduction through the high-affinity receptor for IgE on basophils, and possibly through related receptors on other hematopoietic cells, appears to be enhanced by association with rafts.  Raft association may also aid in signaling through proteins anchored by glycosylphosphatidylinositol, particularly in hematopoietic cells and neurons.  Rafts may also function in sorting and trafficking through the secretary and endocytic pathways.    

3.61           The calcium-sensing receptor is localized in caveolin-rich plasma membrane domains of bovine parathyroid cells

Kifor, O., Diaz, R., Butters, R., Kifor, I. And Brown, E.M.

1. Biol. Chem., 273(34), 21708-21713 (1998)

  Parathyroid cells have an intracellular machinery for parathyroid hormone (PTH) secretion that is inversely regulated by the extracellular calcium concentration (Ca²⁺_o).  The recently characterized Ca²⁺_o sensing receptor (CaR) is a G protein-coupled, seven-transmembrane receptor mediating the inhibitory effects of high Ca²⁺_o on PTH secretion.  The CaR's precise cell surface localization and the signal transduction pathway(s) mediating its inhibitory effects on PTH secretion have not been characterized fully.  Here, we demonstrate that the CaR resides within caveolin-rich membrane domains in bovine parathyroid cells.  Chief cells within bovine parathyroid glands exhibit a similar pattern of staining for caveolin-1 and for alkaline phosphatase, a glucosylphosphatidyl-inositol-anchored protein often enriched in caveolae.  Purified caveolin-enriched membrane fractions (CEMF) from bovine parathyroid cells are highly enriched in the CaR and alkaline phosphatase.  Other signaling proteins, including G_q/11, ENOS, and several protein kinase C isoforms (i.e. a, d, and z) are also present in CEMF.  Activation of the CaR by high Ca²⁺_o increases tyrosine phosphorylation of caveolin-1 in CEMF, suggesting that CaR-mediated signal transduction potentially involved in Ca²⁺_o regulated processes in parathyroid cells occur in caveolae-like domains.  

3.62           Palmitoylation of Neurofascin at a Site in the Membrane-Spanning Domain Highly Conserved Among the L1 Family of Cell Adhesion Molecules

Ren, Q. and Bennett, V.

1. Neurochem., 70(5), 1839-1849 (1998)

  This report presents the first evidence that a member of the L1 family of nervous system cell-adhesion molecules is covalently modified by thioesterification with palmitate, and identifies a highly conserved cysteine in the predicted membrane-spanning domain as the site of modification. Neurofascin is constitutively palmitoylated at cysteine-1213 at close to a 1:1 molar stoichiometry. Kinetics of palmitate incorporation into neurofascin expressed in resting neuroblastoma cells indicate that the palmitate modification has the same turnover rate as the polypeptide chain and does not affect the protein stability of neurofascin. Palmitoylation of neurofascin expressed in dorsal root ganglion neurons is not required for delivery of neurofascin to the plasma membrane or targeting to axons. Palmitoylation also has no effect on ankyrin-binding activity of neurofascin, on the oligomeric state of neurofascin in solution, or on cell-adhesion activity of neurofascin expressed in neuroblastoma cells. A significant difference between native and C1213L neurofascin is that these proteins were localized in distinct fractions within a low-density membrane population enriched in signaling molecules. These results indicate a palmitate-dependent targeting of neurofascin to a specialized membrane microdomain.  

3.63           The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions.

Sheff, D.R., Daro, E.A., Hull, M. and Mellmann, I.

1. Cell Biol., 145(1), 123-139 (1999)

  Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF₄ selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes.  

3.64           Opposing effects of reactive oxygen species and cholesterol on endothelial nitric oxide synthase and endothelial cell caveolae.

Peterson, T.E. et al. Circulation Res., 85, 29-37 (1999)   Synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is critical for normal vascular home-ostasis. eNOS function is rapidly regulated by agonists and blood flow and chronically by factors that regulate mRNA stability and gene transcription. Recently, localization of eNOS to specialized plasma membrane invagin-ations termed caveolae has been proposed to be required for maximal eNOS activity. Because caveolae are highly enriched in cholesterol, and hypercholesterolemia is associated with increased NO production, we first studied the effects of cholesterol loading on eNOS localization and NO production in cultured bovine aortic endothelial cells (BAECs). Caveolae-enriched fractions were prepared by OptiPrep gradient density centrifug-ation. Treatment of BAECs with 30 mg/mL cholesterol for 24 hours stimulated significant increases in total eNOS protein expression (1.50-fold), eNOS associated with caveolae-enriched membranes (2.23-fold), and calcium ionophore-stimulated NO production (1.56-fold). Because reactive oxygen species (ROS) contribute to endothelial dysfunction in hypercholesterolemia, we next studied the effects of ROS on eNOS localization and caveolae number. Treatment of BAECs for 24 hours with 1 mmol/L LY83583, a superoxide-generating naptho-quinolinedione, decreased caveolae number measured by electron microscopy and prevented the cholesterol-mediated increases in eNOS expression. In vitro exposure of caveolae-enriched membranes to ROS (xanthine + xanthine oxidase) dissociated caveolin more readily than eNOS from the membranes. These results show that cholesterol treatment increases eNOS expression, whereas ROS treatment decreases eNOS expression and the association of eNOS with caveolin in caveolae-enriched membranes. Our data suggest that oxidative stress modulates endothelial function by regulating caveolae formation, eNOS expression, and eNOS-caveolin interactions.  

3.65           Purification and characterization of rat hippocamal CA3-dendritic spines associated  with mossy fiber terminals

Kiebler, M.A., Lopez-Garcia, J.C. and Leopold, P.L. FEBS Lett., 445, 80-86 (1999)   We report a revised and improved isolation procedure for CA3-dendritic spines, most of them still in association with mossy fiber terminals resulting in a 7.5-fold enrichment over nuclei and a 29-fold enrichment over myelin. Additionally, red blood cells, medullated fibers, mitochondria and small synaptosomes were significantly depleted. We show by high resolution electron microscopy that this subcellular fraction contains numerous dendritic spines with a rich ultrastructure, e.g. an intact spine apparatus, membranous organelles, free and membrane-bound polyribosomes, endocytic structures and mitochondria. This improved experimental system will allow us to study aspects of post-synaptic functions at the biochemical and molecular level.  

3.66           Separation of the intracellular secretory compartment of rat liver and isolated rat hepatocytes in a single step using self-generating gradients of iodixanol

Plonne, D., Cartwright, I., Linss, W., Dargel, R., Graham, J.M. and Higgins, J.A.Anal. Biochem., 276(1), 88-96 (1999)   A novel method is described for the separation on a single gradient of the major intracellular organelles of the secretory pathway, the Golgi, the smooth endoplasmic reticulum (ER), and the rough (ER). Total microsomes were prepared from rat liver by differential centrifugation and resuspended in 20% iodixanol. The microsomal suspension was then layered between a 30% iodixanol cushion and a layer of 15% iodixanol and centrifuged in a vertical rotor for 2 h. The microsomes distributed in four visible bands. The gradients were collected by upward displacement and were characterized (i) by determination of UDP galactose-galactosyl-transferase (Golgi marker) NADPH-cytochrome c reductase (ER marker) and RNA (rough endoplasmic reticulum marker); (ii) by immunoblotting for TGN38 (trans-Golgi marker) and GS28 (cis-Golgi marker) and for protein disulfide isomerase (endoplasmic reticulum lumenal marker); (iii) by determination of the lipid composition; and (iv) by electron microscopy. The results suggest that the top band (density 1.045-1.090 g/ml), which contains 68% of the galactosyltransferase activity, consists of vesicles derived from the Golgi. The second broad band in the middle of the tube (density 1.130-1.160 g/ml), which contains 54% of the NADPH-cytochrome c reductase activity, consists mainly of vesicles derived from the smooth endoplasmic reticulum, overlapped at the top by a small band of Golgi-derived lamellae. The two bands at the bottom of the tube (density 1.130-1.160 and density 1.180-1.220 g/ml) appear to contain two subfractions of vesicles derived from the rough endoplasmic reticulum.  

3.67           Raft association of SNAP receptors acting in apical trafficking in Madin-Darby canine kidney cells

Lafont, F., Verkade, P., Galli, T., Wimmer, C., Louvard, D. and Simons, K. Proc. Natl. Acad. Sci. USA, 96, 3734-3738 (1999)   We have investigated the relationship between apical sorting mechanism using lipid rafts and the soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) machinery, which is involved in membrane docking and fusion. We first confirmed that anti-alpha-SNAP antibodies inhibit the apical pathway in Madin-Darby canine kidney (MDCK) cells; in addition, we report that a recombinant SNAP mutant inhibits this transport step. Based on t-SNARE overexpression experiments and the effect of botulinum neurotoxin E, syntaxin 3 and SNAP-23 have been implicated in apical membrane trafficking. Here, we show in permeabilized MDCK cells that antisyntaxin 3 and anti-SNAP-23 antibodies lower surface delivery of an apical reporter protein. Moreover, using a similar approach, we show that tetanus toxin-insensitive, vesicle-associated membrane protein (TI-VAMP; also called VAMP7), a recently described apical v-SNARE, is involved. Furthermore, we show the presence of syntaxin 3 and TI-VAMP in isolated apical carriers. Polarized apical sorting has been postulated to be mediated by the clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We provide evidence that syntaxin 3 and TI-VAMP are raft-associated. These data support a raft-based mechanism for the sorting of not only apically destined cargo but also of SNAREs having functions in apical membrane-docking and fusion events.  

3.68           Analysis of CD44-containing lipid rafts: Recruitment of Annexin II and stabilization by the actin cytoskeleton

Oliferenko, S., et al

1. Cell Biol., 146(4), 843-854 (1999)

  CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II-p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II-lipid raft complexes were stabilized by addition of GTPgS or phalloidin in a semipermeabilized and cholesterol depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton.  

3.69           Subcellular distribution of tuberin in cells derived from brain and liver

Yamamoto, Y. and Yeung, R.S. Proc. Amer. Assoc. Cancer Res., 40, #4515 (1999)   Mutations of TSC1 and TSC2 are responsible for the autosomal syndrome of tuberous sclerosis which predisposes carriers to the development of hamartomas and neoplasia. The TSC2 product, tuberin, possesses tumor suppressor activity and in vitro GAP activity towards Rap1 and Rab5. However, the physiological function of tuberin remains poorly understood. Primary sequence analysis suggested a putative transmembrane domain consistent with its abundance in the P100 post nuclear fraction and indirect immunofluorescence demonstrated co-localization with Rap1 and other Golgi markers. In light of the potential role of tuberin in the vesicular trafficking, we studied its subcellular distribution in cells of primary tissues using biochemical fractionation. Microsomal fractions of rat brain and liver were separated on a continuous Iodixanol gradient and analyzed for protein expression of tuberin, rabaptin-5, a TSC2 associated protein, EEA1, and endosomal protein, and TGN38, a trans-Golgi marker. The pattern of expression was similar between tuberin, rabaptin-5 and EEA1 but distinct from that of TGN38. Further, a substantial fraction of tuberin was found free in the cytosol and was re-distributed to the supernatant under conditions of high pH. These findings suggest that tuberin may shuttle between the cytosol and membranous organelles. Further, tuberin localization is not restricted to the Golgi but includes the endosomal compartment. This is consistent with a role of tuberin in vesicular transport.  

3.70           Spatial organization of EGF receptor transmodulation by PDGF

Liu, P. and Anderson, R.G.W. Biochem. Biophys. Res. Commun., 261, 695-700 (1999)   Even though the modulation of EGF receptors by PDGF is well documented, it is not known where on the cell surface cross-talk between the two receptor systems takes place.  The recent finding that both populations of receptors are concentrated in cell surface caveolae suggests that the confinement of the two' receptors to this space might facilitate their interaction.  Here we show that stimulation of PDGF receptors in caveolae with PDGF causes a subpopulation of EGF receptors in the same membrane fraction to become phosphorylated on tyrosine.  Coincident with tyrosine phosphorylation, the binding of EGF to its receptor markedly declines.  Loss of EGF binding is partially blocked by tyrosine kinase inhibitors.  Despite the close proximity of the two receptors in caveolae, we saw no evidence that EGF could stimulate PDGFR tyrosine phosphorylation.  These results suggest that these two receptor systems are highly organized in caveolae.  

3.71           Co-expression of scavenger receptor-BI and caveolin-1 is associated with enhanced selective cholesterol ester uptake in THP-1 macrophages

Matveev, S., van der Westhuyzen, D.R. and Smart, E.J.

1. Lipid Res., 40, 1647-1654 (1999)

  Scavenger receptor (SR)-BI mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters and the efflux of free cholesterol. In Chinese hamster ovary (CHO) cells, SR-BI is predominantly associated with caveolae which we have recently demonstrated are the initial loci for membrane transfer of HDL cholesteryl esters. Because cholesterol accumulation in macrophages is a critical event in atherogenesis, we investigated the expression of SR-BI and caveolin-1 in several macrophage cell lines. Human THP-1 monocytes were examined before and after differentiation to macrophages by treatment with 200 nM phorbol ester for 72 h. Undifferentiated THP-1 cells expressed caveolin-1 weakly whereas differentiation upregulated caveolin-1 expression greater than 50-fold. In contrast, both undifferentiated and differentiated THP-1 cells expressed similar levels of SR-BI. Differentiation of THP-1 cells increased the percent of membrane cholesterol associated with caveolae from 12% ± 1.9% to 38% ± 3.1%. The increase in caveolin-1 expression was associated with a 2- to 3-fold increase in selective cholesterol ether uptake from HDL. Two mouse macrophage cell lines, J774 and RAW, expressed levels of SR-BI similar to differentiated THP-1 cells but did not express detectable levels of caveolin-1. In comparison to differentiated THP-1 cells, RAW and J774 cells internalized 9- to 10-fold less cholesteryl ester. We conclude that differentiated THP-1 cells express both caveolin-1 and SR-BI and that their co-expression is associated with enhanced selective cholesteryl ester uptake.  

3.72           The class B, type I scavenger receptor promotes the selective uptake of high density  lipoprotein cholesterol ethers into caveolae

Graf, G.G., Connell, P.M., van der Westhuyzen, D.R. and Smart, E.J.

1. Biol. Chem., 274(17), 12043-12048 (1999)

  The uptake of cholesterol esters from high density lipoproteins (HDLS) is characterized by the initial movement of cholesterol esters into a reversible plasma membrane pool.  Cholesterol esters are subsequently internalized to a nonreversible pool.  Unlike the uptake of cholesterol from low density lipoproteins, cholesterol ester uptake from HDL does not involve the internalization and degradation of the particle and is therefore termed selective.  The class B, type I scavenger receptor (SR-BI) has been identified as an HDL receptor and shown to mediate selective cholesterol ester uptake. SR-BI is localized to cholesterol- and sphingomyelin- rich microdomains called caveolae.  Caveolae are directly involved in cholesterol trafficking.  Therefore, we tested the hypothesis that caveolae are acceptors for HDL-derived cholesterol ether (CE).  Our studies demonstrate that in Chinese hamster ovary cells expressing SR-BI, >80% of the plasma membrane associated CE is present in caveolae after 7.5 min of selective cholesterol ether uptake. We also show that excess, unlabeled HDL can extract the radiolabeled CE from caveolae, demonstrating that caveolae constitute a reversible  plasma membrane pool of CE.  Furthermore, 50% of the caveolae-associated CE can be chased into a nonreversible pool. We conclude that caveolae are acceptors for HDL-derived cholesterol ethers, and that caveolae constitute a reversible, plasma membrane pool of cholesterol ethers.  

3.73           Polarized distribution of endogenous Racl and RhoA at the cell surface

Michaely, P.A., Mineo, C. Ying, Y-S. and Anderson, R.G.W.

1. Biol. Chem., 274(30), 21430-21436 (1999)

  Racl and RhoA regulate membrane ruffling and stress fiber formation.  Both molecules appear to exert their control from the plasma membrane.  In fibroblasts stimulated with platelet-derived growth factor or lysophosphatidic acid, the reorganization of the cytoskeleton begins at specific sites on the cell surface.  We now report that endogenous Racl and RhoA also have a polarized distribution at the cell surface.  Cell fractionation and immunogold labeling show that in quiescent fibroblasts both of these molecules are concentrated in caveolae, which are plasma membrane domains that are associated with actin-rich regions of the cell.  Treatment of these cells with platelet-derived growth factor stimulated the recruitment of additional Racl and RhoA to caveolae fractions, while lysophosphatidic acid only caused the recruitment of RhoA.  We could reconstitute the recruitment of RhoA using either whole cell lysates or purified caveolae.  Surprisingly, pretreatment of the lysates with exoenzyme C3 shifted both resident and recruited RhoA from caveolae to noncaveolae membranes.  The shift in location was not caused by inactivation of the RhoA effector domain.  Moreover, chimeric proteins containing the C-terminal consensus site for Racl and RhoA prenylation were constitutively targeted to caveolae fractions.  These results suggest that the polarized distribution of Rho family proteins at the cell surface involves an initial targeting of the protein to caveolae and a mechanism for retaining it at this site.  

3.74           Regulated migration of epidermal growth factor receptor from caveolae

Mineo, C., Gill, G.N. and Anderson, R.G.W.

1. Biol. Chem., 274(43), 30636-30643 (1999)

  In quiescent fibroblasts, epidermal growth factor (EGF) receptors (EGFR) are initially concentrated in caveolae but rapidly move out of this membrane domain in response to EGF.  To better understand the dynamic localization of EGFR to caveolae, we have studied the behavior of wild-type and mutant receptors expressed in cells lacking endogenous EGFR.  All of the receptors we examined, including those missing the first 274 amino acids or most of the cytoplasmic tail, were constitutively concentrated in caveolae.  By contrast, migration from caveolae required EGF binding, an active receptor kinase domain, and at least one of the five tyrosine residues present in the regulatory domain of the receptor.  Movement appears to be modulated by Src kinase, is blocked by activators of protein kinase C, and occurs independently of internalization by clathrin coated pits.  Two mutant receptors previously shown to induce an oncogenic phenotype lack the ability to move from caveolae in response to EGF, suggesting that a prolonged residence in this domain may contribute to abnormal cell behavior.  

3.75           Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation

Blair, A., Shaul, P.W., Yuhanna, I.S., Conrad, P.A. and Smart, E.J.

1. Biol Chem., 274(45), 32512-32519 (1999)

  Hypercholesterolemia-induced vascular disease and atherosclerosis are characterized by a decrease in the bioavailability of endothelium-derived nitric oxide.  Endothelial nitric-oxide synthase (eNOS) associates with caveolae and is directly regulated by the caveolar protein, caveolin.  In the present study, we examined the effects of oxidized low density lipoprotein (oxLDL) on the subcellular location of eNOS, on eNOS activation, and on caveolar cholesterol in endothelial cells: We found that treatment with 10 mg/ml oxLDL for 60 min causes greater than 90% of eNOS and caveolin to leave caveolae.  Treatment with oxLDL also inhibited acetylcholine-induced activation of eNOS but not prostacyclin production. oxLDL did not affect total cellular eNOS abundance.  Oxidized LDL also did not affect the palmitoylation, myristoylation or phosphorylation of eNOS. Oxidized LDL, but not native LDL, or HDL depleted caveolae of cholesterol by serving as an acceptor for cholesterol.  Cyclodextrin also depleted caveolae of cholesterol and caused eNOS and caveolin to translocate from caveolae.  Furthermore, removal of oxLDL allowed eNOS and caveolin to return to caveolae.  We conclude that oxLDL-induced depletion of caveolar cholesterol causes eNOS to leave caveolae and inhibits acetylcholine-induced activation of the enzyme.  This process may be an important mechanism in the early pathogenesis of atherosclerosis.  

3.76           Subcellular location of enzyme involved in oxidation on n-alkane by Cladosporium resinae

Goswami, P. and Cooney, J.J. Appl. Microbiol. Biotechnol., 51(6), 860-864 (1999)   More than 70% of n-hexadecane-grown cells of Cladosporium resinae ATCC 22711 were converted to spheroplasts when they were treated with chinitase and lytic enzyme from Trichoderma harziamum. The light mitochondrial fraction, containing microbodies, mitochondria and vacuoles, was isolated from spheroplasts. Vacuoles in cells were demonstrated by the inability of acridine orange to stain organelles previously treated with 2.5 mM Bafilomycin A₁, a vacuolar ATPase inhibitor. Microbodies, mitochondria and vacuoles were separated from the light mitochondrial fraction by self-generated density gradient ultracentrifugation using iodixanol as gradient medium. NADH-dependent n-alkane monooxygenase activity and fatty alcohol oxidase activity were located in the cytoplasm and mitochondrial fractions respectively.  

3.77           Persistent membrane association of activated and depalmitoylated G protein a subunits

Huang, C., Duncan, J.A., Gilman, A.G. and Mumby, S.M. Proc. Natl. Acad. Sci. USA, 96, 412-417 (1999)   Heterotrimeric signal-transducing G proteins are organized at the inner surface of the plasma membrane, where they are positioned to interact with membrane-spanning receptors and appropriate effectors.  G proteins are activated when they bind GTP and inactivated when they hydrolyze the nucleotide to GDP.  However, the topological fate of activated G protein a subunits is disputed.  One model declares that depalmitoylation of a, which accompanies activation by a receptor, promotes release of the protein into the cytoplasm.  Our data suggest that activation of G protein a subunits causes them to concentrate in subdomains of the plasma membrane but not to be released from the membrane. Furthermore, a subunits remained bound to the membrane when they were activated with guanosine5'-(3-0-thio)triphosphate and depalmitoylated with an acyl protein thioesterase. Limitation of a subunits to the plasma membrane obviously restricts their mobility and may contribute to the efficiency and specificity of signaling.  

3.78           The yeast frataxin homologue mediates mitochondrial iron efflux

Radisky, D.C., Babcock, M.C. and Kaplan, J.

1. Biol. Chem.,274(8), 4497-4499 (1999)

  Mutations in the nuclear gene encoding the mitochondrial protein frataxin are responsible for the neurological disorder Friedreich ataxia (FA).  Yeast strains with a deletion in the frataxin homologue YFH1 accumulate excess iron in mitochondria and demonstrate mitochondrial damage.  We show that in the absence of YFH1, mitochondrial damage is proportional to the concentration and duration of exposure to extracellular iron, establishing mitochondrial iron accumulation as causal to mitochondrial damage.  Reintroduction of YFH1 results in the rapid export of accumulated mitochondrial iron into the cytosol as free, non-heme bound iron, demonstrating that mitochondrial iron in the yeast FA model can be made bioavailable.  These results demonstrate a mitochondrial iron cycle in which Yfhlp regulates mitochondrial iron efflux.  

3.79           Induction of caveolae in the apical plasma membrane of Madin-Darby canine kidney cells

Verkade, P., Harder, T., Lafont, F. And Simons, K.

1. Cell Biol., 148(4), 727-739 (1999)

  In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells.  We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane.  Whereas antibody cross-linking induced the formation of large clusters on the basolateral membrane, resembling those observed on the surface of fibroblasts (Harder, T, P. Scheiffele, P. Verkade, and K. Simons. 1998.  J Cell Biol. 929-942), only small (~100 nm) clusters formed on the apical plasma membrane.  Cross-linked apical raft proteins e.g., GPI-anchored placental alkaline phosphatase (PLAP), influenza hemagglutinin, and gpl14 coclustered and were internalized slowly (~10% after 60 min).  Endocytosis occurred through surface invaginations that corresponded in size to caveolae and were labeled with caveolin-1 antibodies.  Upon cholesterol depletion the internalization of PLAP was completely inhibited.  In contrast, when a non-raft protein, the mutant LDL receptor LDLR-CT22, was cross-linked, it was excluded from the clusters of raft proteins and was rapidly internalized via clathrin-coated pits. Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins.  

3.80           Regulation of b-amyloid secretion by FE65, an amyloid protein precursor-binding  protein

Sabo, S.L. et al

1. Biol. Chem., 274(12), 7952-7957 (1999)

  The principal component of Alzheimer's amyloid plaques, Ab, derives from proteolytic processing of the Alzheimer's amyloid protein precursor (APP).  FE65 is a brain-enriched protein that binds to APP.  Although several laboratories have characterized the APP-FE65 interaction in vitro, the possible relevance of this interaction to Alzheimer's disease has remained unclear.  We demonstrate here that APP and FE65 co-localize in the endoplasmic reticulum/Golgi and possibly in endosomes.  Moreover, FE65 increases translocation of APP to the cell surface, as well as both aAPP_s and Ab secretion.  The dramatic (4-fold) FE65-dependent increase in Ab secretion suggests that agents which inhibit the interaction of FE65 with APP might reduce Ab secretion in the brain and therefore be useful for preventing or slowing amyloid plaque formation.  

3.81           The mixed lineage kinase DLK utilizes MKK7 and not MKK4 as substrate

Merritt, S.E. et al

1. Biol. Chem., 274(15), 10195-10202 (1999)

  Mixed lineage kinases DLK (dual leucine zipper-bearing kinase) and MLK3 have been proposed to function as mitogen-activated protein kinase kinase kinases in pathways leading to stress-activated protein kinase/c-Jun NH₂-terminal kinase activation.  Differences in primary protein structure place these MLK (mixed lineage kinase) enzymes in separate subfamilies and suggest that they perform distinct functional roles.  Both DLK and MLK3 associated with, phosphorylated, and activated MKK7 in vitro. Unlike MLK3, however, DLK did not phosphorylate or activate recombinant MKK4 in vitro. In confirmatory experiments performed in vivo, DLK both associated with and activated MKK7. The relative localization of endogenous DLK, MLK3, MKK4, and MKK7 was determined in cells of the nervous system.  Distinct from MLK3, which was identified in non-neuronal cells, DLK and MKK7 were detected predominantly in neurons in sections of adult rat cortex by immunocytochemistry.  Subcellular fractionation experiments of cerebral cortex identified DLK and MKK7 in similar nuclear and extranuclear subcellular compartments.  Concordant with biochemical experiments, however, MKK4 occupied compartments distinct from that of DLK and MKK7.  That DLK and MKK7 occupied subcellular compartments distinct from MKK4 was confirmed by immunocytochemistry in primary neuronal culture.  The dissimilar cellular specificity of DLK and MLK3 and the specific substrate utilization and subcellular compartmentation of DLK suggest that specific mixed lineage kinases participate in unique signal transduction events.  

3.82           Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA

Meeusen, S. et al

1. Cell Biol., 145(2), 291-304 (1999)

  Maintenance of mitochondrial DNA (mtDNA) during cell division is required for progeny to be respiratory competent.  Maintenance involves the replication, repair, assembly, segregation, and partitioning of the mitochondrial nucleoid.  MGM101 has been identified as a gene essential for mtDNA maintenance in S. cerevisiae, but its role is unknown.  Using liquid chromatography coupled with tandem mass spectrometry, we identified Mgm101p as a component of highly enriched nucleoids, suggesting that it plays a nucleoid specific role in maintenance.  Subcellular fractionation, indirect immunofluorescence and GFP tagging show that Mgm101p is exclusively associated with the mitochondrial nucleoid structure in cells.  Furthermore, DNA affinity chromatography of nucleoid extracts indicates that Mgm101p binds to DNA, suggesting that its nucleoid localization is in part due to this activity.  Phenotypic analysis of cells containing a temperature sensitive mgm101 allele suggests that Mgml0lp is not involved in mtDNA packaging, segregation, partitioning or required for ongoing mtDNA replication.  We examined Mgml0lp's role in mtDNA repair.  As compared with wild-type cells, mgm101 cells were more sensitive to mtDNA damage induced by UV irradiation and were hypersensitive to mtDNA damage induced by gamma rays and H₂O₂ treatment.  Thus, we propose that Mgm101p performs an essential function in the repair of oxidatively damaged mtDNA that is required for the maintenance of the mitochondrial genome.  

3.83           Association of sterol- and glycosylphosphatidylinositol-linked proteins with Drosophilia raft lipid microdomains

Rietveld, A., Neutz, S., Simons, K. and Eaton, S.

1. Biol. Chem., 274(17), 12049-12054 (1999)

  In vertebrates, the formation of raft lipid microdomains plays an important part in both polarized protein sorting and signal transduction.  To establish a system in which raft-dependent processes could be studied genetically, we have analyzed the protein and lipid composition of these microdomains in Drosophila melanogaster. Using mass spectrometry, we identified the phospholipids, sphingolipids, and sterols present in Drosophila membranes.  Despite chemical differences between Drosophila and mammalian lipids, their structure suggests that the biophysical properties that allow raft formation have been preserved.  Consistent with this, we have identified a detergent-insoluble fraction of Drosophila membranes that, like mammalian rafts, is rich in sterol, sphingolipids, and glycosylphosphatidylinositol-linked proteins.  We show that the sterol-linked Hedgehog N-terminal fragment associates specifically with this detergent-insoluble membrane fraction.  Our findings demonstrate that raft formation is preserved across widely separated phyla in organisms with different lipid structures.  They further suggest sterol modification as a novel mechanism for targeting proteins to raft membranes and raise the possibility that signaling and polarized intracellular transport of Hedgehog are based on raft association.  

3.84           N-glycans mediate the apical sorting of a GPI-anchored, raft-associated protein in  Madin-Darby canine kidney cells

Benting, J.H., Rietveld, A.G. and Simons, K.

1. Cell Biol., 146(2), 313-320 (1999)

  Glycosyl-phosphatidylinositol (GPI)-anchored proteins are preferentially transported to the apical cell surface of polarized Madin-Darby canine kidney (MDCK) cells.  It has been assumed that the GPI anchor itself acts as an apical determinant by its interaction with sphingolipid-cholesterol rafts.  We modified the rat growth hormone (rGH), an unglycosylated, unpolarized secreted protein, into a GPI-anchored protein and analyzed its surface delivery in polarized MDCK cells.  The addition of a GPI anchor to rGH did not lead to an increase in apical delivery of the protein.  However, addition of N-glycans to GPI-anchored rGH resulted in predominant apical delivery, suggesting that N-glycans act as apical sorting signals on GPI-anchored proteins as they do on transmembrane and secretary proteins, In contrast to the GPI-anchored rGH, a transmembrane form of rGH which was not raft-associated accumulated intracellularly.  Addition of N-glycans to this chimeric protein prevented intracellular accumulation and led to apical delivery.  

3.85           Identification and characterization of polycystin-2, the PKD2 gene product

Cai, Y. et al

1. Biol. Chem., 274(40), 28557-28565 (1999)

  PKD2, the second gene for the autosomal dominant polycystic kidney disease (ADPKD), encodes a protein, polycystin-2, with predicted structural similarity to cation channel subunits.  However, the function of polycystin-2 remains unknown.  We used polyclonal antisera specific for the intracellular NH₂ and COOH termini to identify polycystin-2 as an ~110-kDa integral membrane glycoprotein.  Polycystin-2 from both native tissues and cells in culture is sensitive to Endo H suggesting the continued presence of high-mannose oligosaccharides typical of pre-middle Golgi proteins.  Immunofluorescent cell staining of polycystin-2 shows a pattern consistent with localization in the endoplasmic reticulum. This finding is confirmed by co-localization with protein-disulfide isomerase as determined by double indirect immunofluorescence and co-distribution with calnexin in subcellular fractionation studies.  Polycystin-2 translation products truncated at or after Gly⁸²¹ retain their exclusive endoplasmic reticulum localization while products truncated at or before Glu⁷⁸⁷ additionally traffic to the plasma membrane.  Truncation mutants that traffic to the plasma membrane acquire Endo H resistance and can be biotinylated on the cell surface in intact cells.  The 34-amino acid region GLU⁷⁸⁷-Ser⁸²⁰, containing two putative phosphorylation sites, is responsible for the exclusive endoplasmic reticulum localization of polycystin-2 and is the site of specific interaction with an as yet unidentified protein binding partner for polycystin-2.  The localization of full-length polycystin-2 to intracellular membranes raises the possibility that the PKD2 gene product is a subunit of intracellular channel complexes.  

3.86           NK lytic-associated molecule: A novel gene selectively expressed in cells with cytolytic function

Kozlowski, M., Schorey, J., Portis, T., Grigoriev, V. and Kornbluth, J.

1. Immunol., 163, 1775-1785 (1999)

  NK cells are most effective in killing a broad spectrum of primary tumor cells after stimulation with cytokines.  We have cloned a novel gene, designated NKLAM (for NK lytic-associated molecule), whose expression is associated with this cytokine-enhanced process.  NKLAM expression is up-regulated in NK cells by IL-2 and IFNb.  NKLAM is also selectively expressed by activated macrophages and CTL.  Treatment of NK cells and CTL with NKLAM antisense oligonucleotides specifically decreases their cytolytic activity, while having no effect on cell growth.  The NKLAM gene encodes a 62-kDa ring finger-containing protein that localizes to the cytoplasmic granules in NK cells.  Further study of this gene may add to our understanding of cytotoxic processes common to NK cells, CTL, and activated macrophages.  

3.87           Glycoprotein reglucosylation and nucleotide sugar utilization in the secretory pathway: identification of a nucleoside diphosphatase in the endoplasmic reticulum

Trombetta, E.S. and Helenius, A. The EMBO J., 18(12), 3282-3292 (1999)   UDP is generated in the lumen of the endoplasmic reticulum (ER) as a product of the UDP-glucose-dependent glycoprotein reglucosylation in the calnexin/calreticulin cycle.  We describe here the identification, purification and characterization of an ER enzyme that hydrolyzes UDP to UMP.  This nucleoside diphosphatase is a ubiquitously expressed, soluble 45 kDa glycoprotein devoid of transmembrane domains and KDEL-related ER localization sequences.  It requires divalent cations for activity and hydrolyzes UDP, GDP and IDP but not any other nucleoside di-, mono- or triphosphates, nor thiamine pyrophosphate.  By eliminating UDP, which is an inhibitory product of the UDP-Glc:glycoprotein glucosyltransferase, it is likely to promote reglucosylation reactions involved in glycoprotein folding and quality control in the ER.  

3.88           Characterization of the internalization pathways for the cystic fibrosis transmembrane conductance regulator

Bradbury, N.A. et al Am. J. Physiol., 276 (Lung Cell. Mol. Physiol., 20), L659-L668 (1999)   Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel give rise to the most common lethal genetic disease of Caucasian populations, CF. Although the function of CFTR is primarily related to the regulation of apical membrane chloride permeability, biochemical, immunocytochemical, and functional studies indicate that CFTR is also present in endosomal and trans Golgi compartments.  The molecular pathways by which CFTR is internalized into intracellular compartments are not fully understood. To define the pathways for CFTR internalization, we investigated the association of CFTR with two specialized domains of the plasma membrane, clathrin-coated pits and caveolae.  Internalization of CFTR was monitored after cell surface biotinylation and quantitation of cell surface CFTR levels after elution of cell lysates from a monomeric avidin column.  Cell surface levels of CFTR were determined after disruption of caveolae or clathrin-coated vesicle formation. Biochemical assays revealed that disrupting the formation of clathrin-coated vesicles inhibited the internalization of CFTR from the plasma membrane, resulting in a threefold increase in the steady-state levels of cell surface CFTR.  In contrast, the levels of cell surface CFTR after disruption of caveolae were not different from those in control cells.  In addition, although our studies show the presence of caveolin at the apical membrane domain of human airway epithelial cells, we were unable to detect CFTR in purified caveolae.  These results suggest that CFTR is constitutively internalized from the apical plasma membrane via clathrin-coated pits and that CFTR is excluded from caveolae.  

3.89           Cloning, expression, and cellular localization of a human prenylcysteine lyase

Tschantz, W.R., Zhang, L. and Casey, P.J

1. Biol. Chem., 274(50), 35802-35808 (1999)

  Prenylated proteins contain either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid covalently attached to cysteine residues at or near their C terminus.  These proteins constitute up to 2% of total cellular protein in eukaryotic cells.  The degradation of prenylated proteins raises a metabolic challenge to the cell, because the thioether bond of the modified cysteine is quite stable.  We recently identified and isolated an enzyme termed prenylcysteine lyase that cleaves the prenylcysteine to free cysteine and an isoprenoid product (Zhang, L., Tschantz, W. IL, and Casey, P. J. (1997) J.  Biol Chem. 272,23354-23359).  To facilitate the molecular characterization of this enzyme, its cloning was undertaken.  Overlapping cDNA clones encoding the complete coding sequence of this enzyme were obtained from a human cDNA library.  The open reading frame of the gene encoding prenylcysteine lyase is 1515 base pairs and has a nearly ubiquitous expression pattern with a message size of 6 kilobase pairs.  Recombinant prenylcysteine lyase was produced in a baculovirus-Sf9 expression system.  Analysis of both the recombinant and native enzyme revealed that the enzyme is glycosylated and contains a signal peptide that is cleaved during processing.  Additionally, the subcellular localization of this enzyme was determined to be lysosomal. These findings strengthen the notion that prenylcysteine lyase plays an important role in the final step in the degradation of prenylated proteins and will allow further physiological and biochemical characterization of this enzyme.  

3.90           The amyloid precursor protein interacts with G_o heterotrimeric protein within a cell compartment specialized in signal transduction

Brouillett, E. et al

1. Neurosci., 19(5), 1717-1727 (1999)

  The function of the b-amyloid protein precursor (bAPP), a transmembrane molecule involved in Alzheimer pathologies, is poorly understood.  We recently reported the presence of a fraction of bAPP in cholesterol and sphingoglycolipid-enriched microdomains (CSEM), a caveolae-like compartment specialized in signal transduction.  To investigate whether bAPP actually interferes with cell signaling, we reexamined the interaction between bAPP and G_o GTPase.  In strong contrast with results obtained with reconstituted phospholipid vesicles (Okamoto et al., 1995), we find that incubating total neuronal membranes with 22Cl1, an antibody that recognizes an N-terminal bAPP epitope, reduces high-affinity G_o GTPase activity.  This inhibition is specific of G_ao and is reproduced, in the absence of 22Cl1, by the addition of the bAPP C-terminal domain but not by two distinct mutated bAPP C-terminal domains that do not bind G_ao. This inhibition of G_ao GTPase activity by either 22Cl1 or wild-type bAPP cytoplasmic domain suggests that intracellular interactions between bAPP and G_ao could be regulated by extracellular signals.  To verify whether this interaction is preserved in CSEM, we first used biochemical, immunocytochemical, and ultrastructural techniques to unambiguously confirm the colocalization of G_ao and bAPP in CSEM.  We show that inhibition of basal G_ao GTPase activity also occurs within CSEM and correlates with the coimmunoprecipitation of G_ao and bAPP. The regulation of G_ao GTPase activity by bAPP in a compartment specialized in signaling may have important consequences for our understanding of the physiopathological functions of bAPP.  

3.91           ER/Golgi intermediates acquire Golgi enzymes by Bredelfin A – sensitive retrograde transport in vitro

Lin, C-C., Love, H.D., Gushue, J.N., Bergeron, J.J.M. and Osterman, J.

1. Cell. Biol., 147(7), 1457-1472 (1999)

  Secretary proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus.  Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates.  Secretary cargo was arrested at distinct steps of the secretary pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined.  Complementation yield increased after ER exit of secretary cargo and was optimal when transport was blocked at an ER/Golgi intermediate step.  The rapid drop of the complementation yield as secretary cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack.  Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA).  Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I-mediated step.  Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.        

3.92           Membrane raft microdomains mediate front-rear polarity in migrating cells

Manes, S. et al EMBO J., 18(22), 6211-6220 (1999)   The acquisition of spatial and functional asymmetry between the rear and the front of the cell is a necessary step for cell chemotaxis. Insulin-like growth factor-1 (IGF-I) stimulation of the human adenocarcinoma MCF-7 induces a polarized phenotype characterized by asymmetrical CCR5 chemokine receptor redistri-bution to the leading cell edge. CCR5 associates with membrane raft microdomains, and its polarization parallels redistribution of raft molecules, including the raft-associated ganglioside GM1, glycosylphosphat-idylinositol- anchored green fluorescent protein and ephrinBl, to the leading edge. The non-raft proteins  transferrin receptor and a mutant ephrinB1 are distributed homogeneously in migrating MCF-7 cells, supporting the raft localization requirement for polarization.  IGF-I stimulation  of cholesterol-depleted cells induces projection of multiple pseudopodia over the entire cell  periphery, indicating that raft disruption specifically affects the acquisition of cell polarity but not IGF-I-induced protrusion activity.  Cholesterol depletion inhibits MCF-7 chemotaxis, which is restored by replenishing cholesterol.  Our results indicate that initial segregation between raft and non-raft membrane proteins mediates the necessary, redistribution of specialized molecules for cell migration.  

3.93           Isolated rabbit enterocytes as a model cell system for investigations of chylomicron  assembly and secretion

Cartwright, I.J. and Higgins, J.A.

1. Lipid Res., 40, 1357-1365 (1999)

  A method is described for the isolation of viable enterocytes from rabbit small intestine. The procedure can also be used to isolate populations of epithelial cells from the crypt/villus gradient.  The isolated enterocytes synthesized and secreted apoB-48 and triacylglycerol in particles of the density of chylomicrons.  Secretion was stimulated by addition of bile salt/lipid micelles.  Pulse-chase experiments demonstrated that newly synthesized apoB-48 is degraded intracellularly and that degradation is inhibited by provision of lipid micelles, suggesting that regulation of chylomicron assembly and secretion is broadly similar to that of very low density lipoprotein assembly in hepatocytes.  This procedure for preparation of isolated enterocytes will provide a useful model system for investigation of the molecular details of chylomicron assembly.  

3.94           Caveolin-3 upregulation activates b-secretase-mediated cleavage of the amyloid  precursor protein in Alzheimer’s disease

Nisshiyama, K. et al

1. Neurosci., 19(15), 6538-6548 (1999)

  Here, we investigate the involvement of caveolins in the pathophysiology of Alzheimer's disease (AD). We show dramatic upregulation of caveolin-3 immunoreactivity in astroglial cells surrounding senile plaques in brain tissue sections from authentic AD patients and an established transgenic mouse model of AD.  In addition, we find that caveolin-3 physically interacts and biochemically colocalizes with amyloid precursor protein (APP) both in vivo and in vitro.  Interestingly, recombinant overexpression of caveolin-3 in cultured cells stimulated b-secretase-mediated processing of APP.  Immunoreactivities of APP and presenilins were concomitantly increased in caveolin-3-positive astrocytes.  Because the presenilins also form a physical complex with caveolin-3, caveolin-3 may provide a common platform for APP and the presenilins to associate in astrocytes.  In AD, augmented expression of caveolin-3 and presenilins in reactive astrocytes may alter APP processing, leading to the overproduction of its toxic amyloid metabolites.  

3.95           Immunoisolation of caveolae with high affinity antibody binding to the oligomeric caveolin cage

Oh, P. and Schnitzer, J.E.

1. Biol. Chem, 274 (33), 23144-23154 (1999)

  Defining the molecular composition of caveolae is es­sential in establishing their molecular architecture and functions. Here, we identify a high affinity monoclonal antibody that is specific for caveolin-h and rapidly binds caveolin oligomerized around intact caveolae. We use this antibody (i) to develop a new simplified method for rapidly isolating caveolae from cell and tissue homo­genates without using the silica-coating technology and (ii) to analyze various caveolae isolation techniques to understand how they work and why they yield different compositions. Caveolae are immunoisolated from rat lung plasma membrane fractions subjected to mechani­cal disruption. Sonication of plasma membranes, iso­lated with or without silica coating, releases caveolae along with other similarly buoyant microdomains and, therefore, requires immunoisolation to purify caveo­lae. Shearing of silica-coated plasma membranes pro­vides a homogeneous population of caveolae whose con­stituents (i) remain unchanged after immunoisolation, (ii) all fractionate bound to the immunobeads, and (iii) appear equivalent to caveolae immunoisolated after sonication. The caveolae immunoisolated from different low density fractions are quite similar in molecular composition. They contain a subset of key signaling molecules (i.e. G protein and endothelial nitric oxide synthase) and are markedly depleted in glycosylphos­phatidylinositol-anchored proteins, b-actin, and angio­tensin-converting enzyme. All caveolae isolated from the cell surface of lung microvascular endothelium in vivo appear to be coated with caveolin-la. Caveolin-1b and -2 can also exist in these same caveolae. The isola­tion and analytical procedures as well as the time-de­pendent dissociation of signaling molecules from caveo­lae contribute to key compositional differences reported in the literature for caveolae. This new, rapid, magnetic immunoisolation procedure provides a consistent prep­aration for use in the molecular analysis of caveolae.  

3.96           The growth-related, translationally controlled protein P23 has properties of a tubulin protein and associates transiently with microtubules during the cell cycle

Gachet, Y. et al

1. Cell Sci., 112, 1257-1271 (1999)

  The translationally controlled protein P23 was discovered by the early induction of its rate of synthesis after mitogenic stimulation of mouse fibroblasts. P23 is expressed in almost all mammalian tissues and it is highly conserved between animals, plants and yeast. Based on its amino acid sequence. P23 cannot be attributed to any known protein family, and its cellular function remains to be elucidated. Here, we present evidence that P23 has properties of a tubulin binding protein that associates with microtubules in a cell cycle-dependent manner. (1) P23 is a cytoplasmic protein that occurs in complexes of 100-150 kDa, and part of P23 can be immunoprecipitated from HeLa cell extracts with anti-tubulin antibodies. (2) In Immunolocalization experiments we find P23 associated with microtubules during G₁, S, G₂ and early M phase of the cell cycle. At metaphase, P23 is also bound to the mitotic spindle, and it is detached from the spindle during metaphase-anaphase transition. (3) A GST-P23 fusion protein interacts with a- and b-tubulin, and recombinant P23 binds to taxol-stabilised microtubules in vitro. The tubulin binding domain of P23 was identified by mutational analysis; it shows similarity to part of the tubulin binding domain of the microtubule-associated protein MAP-1B. (4) Overexpression of P23 results in cell growth retardation and its alterations of cell morphology. Moreover, elevation of P23 levels leads to microtubule rearrangements and to an increase in microtubule mass and stability.  

3.97           VIP36 localisation to the early secretory pathway

Fullekrug, J., Scheiffele, P. and Simons, K.

1. Cell Sci., 112, 2813-2821 (1999)

  VIP36, an integral membrane protein previously isolated from epithelial MDCK cells, is an intracellular lectin of the secretory pathway. Overexpressed VIP36 had been localized to the Golgi complex, plasma membrane and endocytic structures suggesting post-Golgi trafficking of this molecule (Fiedler et al, 1994). Here we provide evidence that endogenous VIP36 is localized to the Golgi apparatus and the early secretory pathway of MDCK and Vero cells and propose that retention is easily saturated. High resolution confocal microscopy, shows partial overlap of VIP36 with Golgi marker proteins. Punctate cytoplasmic structures colocalize with coatomer and ERGIC-53, labeling ER-Golgi intermediate membrane structures. Cycling of VIP36 is suggested by colocalization with anterograde cargo trapped in pre-Golgi structures and modification of its N-linked carbohydrate by glycosylation enzymes of medial Golgi cisternae. Furthermore, after brefeldin A treatment VIP36 is segregated from resident Golgi proteins and codistributes with ER-Golgi recycling proteins.  

3.98           Acyl and alkyl chain length of GPI-anchors is critical for raft association in vitro

Benting, J., Rietveld, A., Ansorge, I. and Simons, K FEBS Lett., 462, 47-50 (1999)   We determined the acyl and alkyl chain composition of GPI-anchors isolated from MDCK and Fischer rat thyroid (FRT) cells. Both cell lines synthesize GPI-anchors containing C16/C18 or C18/C18 saturated acyl and alkyl chains. The GPI-anchored placental alkaline phosphatase (PLAP) expressed in both cells is raft-associated and PLAP purified from FRT cells is raft-associated in vitro when reconstituted into liposomes containing raft lipids. In contrast, the GPI-anchored variant surface glycoprotein from Trypanosoma brucei, which contains C14 acyl and alkyl chains, shows no significant raft association after reconstitution in vitro. These data indicate that the acyl and alkyl chain composition of GPI-anchors determines raft association.  

3.99           EphrinB ligands recruit GRIP family PDZ adaptor proteins into raft membrane microdomains

Bruckner, K. et al Neuron, 22, 511-524 (1999)   Transmembrane ephrinB proteins have important functions during embryonic patterning as ligands for Eph receptor tyrosine kinases and presumably as signal-transducing receptor-like molecules. Consistent with “reverse” signaling, ephrinB1 is localized in sphingo-lipid/cholesterol-enriched raft microdomains, platforms for the localized concentration and activation of signaling molecules. Glutamate receptor-interacting protein (GRIP) and a highly related protein, which we have termed GRIP2, are recruited into these rafts through association with the C-terminal PDZ target site of ephrinB1. Stimulation of ephrinB1 with soluble EphB2 receptor ectodomain causes the formation of large raft patches that also contain GRIP proteins. Moreover, a GRIP-associated serine/threonine kinase activity is recruited into ephrinB1-GRIP complexes. Our findings suggest that GRIP proteins provide a scaffold for the assembly of a multiprotein signaling complex downstream of ephrinB ligands.  

3.100           In vivo imaging of tumors with protease-activated near-infrared fluorescent probes

Weissleder, R., Tung, C-H., Mahmood, U. and Bogdanov, A. Nature Biotech., 17, 375-378 (1999)   We have developed a method to image tumor-associated lysosomal protease activity in a xenograft mouse model In vivo using autoquenched near-infrared fluorescence (NIRF) probes. NIRF probes were bound to a long circulating graft copolymer consisting of poly-L-lysine and methoxypolyethylene glycol succinate. Following intravenous Injection, the NIRF probe carrier accumulated in solid tumors due to its long circulation time and leakage through tumor neovasculature. Intratumoral NIRF signal was generated by lysosomal proteases in tumor cells that cleave the macromolecule, thereby releasing previously quenched fluorochrome. In vivo imaging showed a 12-fold increase in NIRF signal, allowing the detection of tumors with submillimeter-sized diameters. This strategy can be used to detect such early stage tumors in vivo and to probe for specific enzyme activity.  

3.101           Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and g-secretase activity

Wolfe, M.S., Xia, W., Ostraszewski, B.L., Diehl, T.S., Kimberly, W.T and Selkoe, D.J. Nature 398, 513-517 (1999)   Accumulation of the amyloid-b protein (Ab) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimer’s disease. The final step in the generation of Ab from the b-amyloid precursor protein is an apparently intramem­branous proteolysis by the elusive g-secretase(s). The most common cause of familial Alzheimer’s disease is mutation of the genes encoding presenilins 1 and 2, which alters g-secretase activity to increase the production of the highly amyloidogenic Ab₄₂ isoform. Moreover, deletion of presenilin-1 in mice greatly reduces g-secretase activity, indicating that presenilin-1 medi­ates most of this proteolytic event. Here we report that mutation of either of two conserved transmembrane (TM) aspartate resi­dues in presenilin-1, Asp 257 (in TM6) and Asp 385 (in TM7), substantially reduces Ab production and increases the amounts of the carboxy-terminal fragments of b-amyloid precursor protein that are the substrates of g-secretase. We observed these effects in three different cell lines as well as in cell-free microsomes. Either of the Asp® Ala mutations also prevented the normal endo­proteolysis of presenilin-1 in the TM6®TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimer’s disease and which does not require this cleavage, the Asp 385®Ala mutation still inhibited g-secretase activity. Our results indicate that the two transmembrane aspartate residues are critical for both presenilin-I endoproteolysis and g-secretase activity, and suggest that pre­senilin 1 is either a unique diaspartyl cofactor for g-secretase or is itself g-secretase, an autoactivated intramembranous aspartyl protease.  

3.102           Evaluation of the affinity and turnover number of both hepatic mitochondrial and microsomal carnitine acyltransferases: relevance to intracellular partitioning of Acyl-CoAs

Abo-Hashema, K.A., Cake, M.H., Lukas, M.A. and Knudsen, J. Biochemistry, 38, 15840-15847 (1999)   Mitochondrial carnitine palmitoyltransferase I (CPT I) and microsomal carnitine acyltransferase I (CAT I) regulate the entry of fatty acyl moieties into their respective organelles. Thus, CPT I and CAT I occupy prominent positions in the pathways responsible for energy generation in mitochondria and the assembly of VLDL in the endoplasmic reticulum, respectively. Previous attempts to determine the intrinsic kinetic properties of CPT I and CAT I have been hampered by the occurrence of sigmoidal velocity curves. This was overcome, in this study, by the inclusion of recombinant acyl-CoA binding protein in the assay medium. For the first time, we have determined the concentrations of total functional enzyme (E(t)) by specific radiolabeling of the active site, the dissociation constants (K(d)) and the turnover numbers of CPT I and CAT I toward the CoA esters of oleic acid (C18:1) and docosahexaenoic acid (C22:6). The data show that carnitine inhibits CAT I at physiological concentrations which are not inhibitory to CPT I. Thus, carnitine concentration is likely to be a significant factor in determining the partitioning of acyl-CoAs between mitochondria and microsomes, a role which has not been previously recognized. Moreover, the finding that CAT I elicits a lower turnover toward the CoA ester of C22:6 (25 s(-)(1)) than toward that of C18:1 (111 s(-)(1)), while having similar K(d) values, suggests the use of this polyunsaturated fatty acid to inhibit VLDL biosynthesis.  

3.103           The protective role of high-density lipoproteins in atherosclerosis

Mingpeng, S. and Zongli, W. Exp. Gerontol., 34, 539-548 (1999)   Serum high-density lipoprotein level is known to be correlated inversely with the incidence and mortality rates of ischemic heart disease. Although some reports pointed out that in case of hyperalphalipoproteinemia, lesions in the coronary arteries were occasionally found, it is also noticed that in very rare condition, no atheromatous lesions found even in patients with hereditary alphalipoprotein deficiency (Funke et al., 1991). However, clinical surveys have confirmed that high high-density-lipoprotein cholesterol level is favorable in preventing the development of atheroclerotic lesion and high-density lipoprotein together with apolipoprotein AI are currently considered to be the most reliable parameters in predicting the development of atherosclerosis in hyperlipidemia.  

3.104           Membrane structure of caveolae and isolated caveolin-rich vesicles

Westermann, M., Leutbecher, H. and Meyer, H.W. Histochem. Cell Biol., 111, 71-81 (1999)   Caveolae are specialized invaginated domains of the plasma membrane. Using freeze-fracture electron microscopy, the shape of caveolae and the distribution of intramembrane particles (integral membrane proteins) were analyzed. The caveolar membrane is highly curved and forms flask-like invaginations with a diameter of 80-120 nm with an open porus of 30-50 nm in diameter. The fracture faces of caveolar membranes are nearly free of intramembrane particles. Protein particles in a circular arrangement surrounding the caveolar opening were found on plasma membrane fracture faces. For isolation of caveolin-enriched membrane vesicles, the method of Triton X-100 solubilization, as well as a detergent-free isolation method, was used. The caveolin-rich vesicles had an average size of between 100 and 200 nm. No striated coat could be detected on the surface of isolated caveolin-rich vesicles. Areas of clustered intramembrane particles were found frequently on membrane fracture faces of caveolin-rich vesicles. The shape of these membrane protein clusters is often ring-like with a diameter of 30-50 nm. Membrane openings were found to be present in the caveolin-rich membrane vesicles, mostly localized in the areas of the clustered membrane proteins. Immunogold labeling of caveolin showed that the protein is a component within the membrane protein clusters and is not randomly distributed on the membrane of caveolin-rich vesicles.            

3.105           Caveolin interacts with Trk A and p75^NTR and regulates neurotrophin signaling pathways

Bilderback, T.R., Gazula, V-R., Lisanti, M.P. and Dobrowsky, R.T.

1. Biol. Chem., 274(1), 257-263 (1999)

  Neurotrophins signal through Trk tyrosine kinase receptors and the low-affinity neurotrophin receptor p75^NTR. We have shown previously that activation of Trk A tyrosine kinase activity can inhibit p75^NTR-dependent sphingomyelin hydrolysis, that caveolae are a localized site for p75^NTR signaling, and that caveolin can directly interact with p75^NTR. The ability of caveolin to also interact with tyrosine kinase receptors and inhibit their activity led us to hypothesize that caveolin expression may modulate interactions between neurotrophin signaling pathways. PC12 cells were transfected with caveolin that was expressed efficiently and targeted to the appropriate membrane domains. Upon exposure to nerve growth factor (NGF), caveolin-PC12 cells were unable to develop extensive neuritic processes. Caveolin expression in PC12 cells was found to diminish the magnitude and duration of Trk A activation in vivo. This inhibition may be due to a direct interaction of caveolin with Trk A, because Trk A co-immunoprecipitated with caveolin from Cav-Trk A-PC12 cells, and a glutathione S-transferase-caveolin fusion protein bound to Trk A and inhibited NGF-induced autophosphorylation in vitro. Furthermore, the in vivo kinetics of the inhibition of Trk A tyrosine kinase activity by caveolin expression correlated with an increased ability of NGF to induce sphingomyelin hydrolysis through p75^NTR. In summary, our results suggest that the interaction of caveolin with neurotrophin receptors may have functional consequences in regulating signaling through p75^NTR and Trk A in neuronal and glial cell populations.  

3.106           Presence of cholyl- and chenodexoycholyl- coenzyme A thioesterase activity in human liver

Solaas, K., Sletta, R.J., Srreide, O. and Kase, B.F. Scand. J. Clin. Lab. Invest., 60, 91-102 (2000)   In human liver homogenate the formation of bile acid..CoA thioesters is localized both to the microsomal fraction catalysed by an ATP-dependent synthetase and to the peroxisomal fraction catalysed by the thiolase in the last step of the b-oxidative cleavage of the 5b-cholestanoyl side chain. The cytosolic bile acid-CoA:amino acid N-acyltransferase catalyse the conjugation of the CoA-activated bile acids with taurine or glycine prior to secretion into bile. The formation of bile acid-CoA esters is considered the rate-limiting step in bile acid amidation. So far, a bile acid-CoA cleaving activity has not been assessed in the research of bile acid aniidation in human liver. In this work, a bile acid-CoA cleaving activity has been demonstrated at a rate that may influence the concentration of bile acid-CoA thioesters, free bile acids and amidated bile acids within the hepatocyte. Recently, it was shown that free chenodeoxycholic acid, formed by the thioesterase, is the physiological ligand of the farnesoid X receptor.   A multiorganelle distribution of the bile acid-CoA hydrolytic activity was found. In the postnuclear fraction of human liver homogenate, apparent K_m and V_max for the cleavage of choloyl-CoA were 7.7 x 10^-5 mol/L and 3.6 nmol x mg^-1 x min^-1, respectively. The corresponding values for chenodeoxycholoyl-CoA cleavage were 7.1 x 10^-5 mol/L and 4.8 nmol x mg^-1 x min^-1. Hydrolytic activities were detected in the microsomal and the peroxisomal fractions where the bile acid-­CoA esters are formed as well as in cytosol housing the N-acyltransferase activity. Compared to the bile acid-CoA synthetase activities, the hydrolytic activities were considerably higher, both in the postnuclear fraction and in the microsomal fraction. The thioesterase activities were in the same range as detected for the N-­acyltransferase activities both in the postnuclear fraction and in the cytosolic fraction. The mere presence of thioesterase in microsomes, peroxisomes and cytosol seems counterproductive to bile acid amidation. The thioesterases may have an indirect regulatory function on the bile acid synthesis and are important for the regulation of bile acid synthesis by providing free chenodeoxycholic acid, the most potent activator of the farnesoid X receptor.  

3.107           Characterization of insulin-responsive GLUT4 storage vesicles isolated from 3T3-L1 adipocytes

Hashiramoto, M. and James, D.E. Mol. Cell Biol., 20(1), 416-427 (2000)   Insulin regulates glucose transport in muscle and adipose tissue by triggering translocation of a facilitative glucose transporter, GLUT4, from an intracellular compartment to the cell surface. It has previously been suggested that GLUT4 is segregated between endosomes, the trans-Golgi network (TGN), and a postendosomal storage compartment. The aim of the present study was to isolate the GLUT4 storage compartment in order to determine the relationship of this compartment to other organelles, its components, and its presence in different cell types. A crude intracellular membrane fraction was prepared from 3T3-L1 adipocytes and subjected to iodixanol equilibrium sedimentation analysis. Two distinct GLUT4-containing vesicle peaks were resolved by this procedure. The lighter of the two peaks (peak 2) was comprised of two overlapping peaks: peak 2b contained recycling endosomal markers such as the transferrin receptor (TfR), cellubrevin, and Rab4, and peak 2a was enriched in TGN markers (syntaxin 6, the cation-dependent mannose 6-phosphate receptor, sortilin, and sialyltransferase). Peak 1 contained a significant proportion of GLUT4 with a smaller but significant amount of cellubrevin and relatively little TfR. In agreement with these data, internalized transferrin (Tf) accumulated in peak 2 but not peak 1. There was a quantitatively greater loss of GLUT4 from peak 1 than from peak 2 in response to insulin stimulation. These data, combined with the observation that GLUT4 became more sensitive to ablation with Tf-horseradish peroxidase following insulin treatment, suggest that the vesicles enriched in peak 1 are highly insulin responsive. Iodixanol gradient analysis of membranes isolated from other cell types indicated that a substantial proportion of GLUT4 was targeted to peak 1 in skeletal muscle, whereas in CHO cells most of the GLUT4 was targeted to peak 2. These results indicate that in insulin-sensitive cells GLUT4 is targeted to a subpopulation of vesicles that appear, based on their protein composition, to be a derivative of the endosome. We suggest that the biogenesis of this compartment may mediate withdrawal of GLUT4 from the recycling system and provide the basis for the marked insulin responsiveness of GLUT4 that is unique to muscle and adipocytes.  

3.108           Activated cardiac adenosine A₁ receptors translocate out of caveolae

Lasley, R.D., Narayan, P., Uittenbogaard, A. and Smart, E.J.

1. Biol. Chem., 275(6), 4417-4421 (2000)

  The cardiac affects of the purine nucleoside, adenosine, are well known.  Adenosine increases coronary blood flow, exerts direct negative chronotropic and dromotropic effects, and exerts indirect anti-adrenergic effects.  These effects of adenosine are mediated via the activation of specific G protein-coupled receptors. There is increasing evidence that caveolae play a role in the compartmentalization of receptors and second messengers in the vicinity of the plasma membrane.  Several reports demonstrate that G protein-coupled receptors redistribute to caveolae in response to receptor occupation.  In this study, we tested the hypothesis that adenosine A₁ receptors would translocate to caveolae in the presence of agonists.  Surprisingly, in unstimulated rat cardiac ventricular myocytes, 67±5% of adenosine A₁ receptors were isolated with caveolae.  However, incubation with the adenosine A₁ receptor agonist 2-chlorocyclopentyladenosine induced the rapid translocation of the A₁ receptors from caveolae into non-caveolae plasma membrane, an effect that was blocked by the adenosine A₁ receptor antagonist, 8-cyclopentyl-1,3dipropylxanthine.  An adenosine A₁ receptor agonist did not alter the localization of A₁ receptors to caveolae. These data suggest that the translocation of A₁ receptors out of caveolae and away from compartmentalized signaling molecules may explain why activation of ventricular myocyte A₁ receptors are associated with few direct effects.  

3.109           High density lipoprotein prevents oxidized low density lipoprotein-induced inhibition of endothelial nitric-oxide synthase localization and activation in caveolae

Uittenbogaard, A., Shaul, P.W., Yuhanna, I.S., Blair, A. and Smart, E.J.

1. Biol. Chem., 275(15), 11278-11283 (2000)

  Oxidized LDL (oxLDL) depletes caveolae of cholesterol, resulting in the displacement of endothelial nitric-oxide synthase (eNOS) from caveolae and impaired eNOS activation.  In the present study, we determined if the class B scavenger receptors, CD36 and SR-BI, are involved in regulating nitric-oxide synthase localization and function.  We demonstrate that CD36 and SR-BI are expressed in endothelial cells, co-fractionate with caveolae, and co-immuno-precipitate with caveolin-1.  Co-incubation of cells with 10 ug/ml high density lipoprotein (HDL) prevented oxLDL induced translocation of eNOS from caveolae and restored acetylcholine-induced nitric-oxide synthase stimulation. Acetylcholine caused eNOS activation in cells incubated with 10 ug/ml oxLDL (10-15 thiobarbituric acid-reactive substances) and blocking antibodies to CD36, whereas cells treated with only oxLDL were unresponsive. Furthermore, CD36-blocking antibodies prevented oxLDL induced  redistribution of eNOS.  SR-BI-blocking antibodies were used to demonstrate that the effects of HDL are mediate by SR-BI. HDL binding to SR-BI maintained the concentration of caveola-associated cholesterol by promoting the uptake of cholesterol esters, thereby preventing oxLDL induced depletion of caveola cholesterol We conclude that CD36 mediates the effects of oxLDL on caveola composition and eNOS activation.  Furthermore, HDL prevents oxLDL from decreasing the capacity for eNOS activation by preserving the cholesterol concentration in caveolae and, thereby maintaining the subcellular location of eNOS.  

3.110           Gastropod mollusc aliphatic alcohol oxidase: subcellular localisation and properties

Grewal, N., Parveen, Z., Large, A., Perry, C. and Connock, M. Comp. Biochem. Biophys., 125, 543-554 (2000)   The digestive gland and other tissues of several species of terrestrial gastropod mollusc contain an aliphatic alcohol oxidase activity (EC1.1.3.13). The enzyme is FAD dependent, consumes oxygen and generates hydrogen peroxide and the corresponding aldehyde.  Saturated primary alcohols arc favoured as substrates with octanol preferred with an apparent K_m of 3-4 mM.  The activity is clearly distinguishable from previously reported molluscan aromatic alcohol oxidase (EC1.1.3.7) on the basis of FAD dependence, sensitivity to heat treatment and hi-h salt concentration and with regard to substrate preferences.  The aliphatic alcohol oxidase is membrane associated and most likely localised to the endoplasmic reticulum.  Extraction of membranes with 1% Igipal solubilises the enzyme in active form.  This enzyme is a further example of an oxidase apparently restricted to molluscs.  

3.111           CCC1 suppresses mitochondrial damage in the yeast model of Friedreich’s ataxia by limiting mitochondrial iron accumulation

Chen, O.S. and Kaplan J.

1. Biol. Chem., 275(11), 7626-7632 (2000)

  Deletion of YFH1 in Saccharomyces cerevisiae leads to a loss of respiratory competence due to excessive mitochondrial iron accumulation. A suppressor screen identified a gene, CCC1, that maintained respiratory function in a Dyfh1 yeast strain regardless of extracellular iron concentration. CCC1 expression prevented excessive mitochondrial iron accumulation by limiting mitochondrial iron uptake rather than by increasing mitochondrial iron egress. Expression of CCC1 did not result in sequestration of iron in membranous compartments or cellular iron export. CCC1 expression in wild type cells resulted in increased expression of the high affinity iron transport system composed of FET3 and FTR1, suggesting that intracellular irons is not sensed by the iron-dependent transcription factor Aft1p. Introduction of AFT1^up, a constitutive allele of the iron transcription factor, AFT1, that also leads to increased high affinity iron transport did not prevent Dyfh1 cells from becoming respiratory-incompetent. Although the mechanism by which CCC1 expression affects cytosolic iron is not known, the data suggest that excessive mitochondrial iron accumulation only occurs when cytosolic free iron levels are high.  

3.112           Subcellular localization of presenilins: association with a unique membrane pool in cultured cells

Kim, S.H., Lah, J.J., Thinakaran, G., Levey, A. and Sisodia S.S Neurobiol. Dis., 7(2), 99-117 (2000)   We have investigated the subcellular distribution of presinilin-1 (PS1) and presenilin-2 (PS2) in a variety of mammalian cell lines. In Iodixanol –based density gradients, PS1 derivatives show a biphasic distribution, cofractionating with membranes containing ER-resident proteins and an additional population of membranes with low buoyant density that do not contain markers of the Golgi complex, ERGIC, COP II vesicles, ER exit compartment, COP II receptor, SNARE, trans-Golgi network, caveolar membranes, or endocytic vesicles. Confocal immunofluorescence and immunoelectron microscopy studies fully supported the fractionation studies. These data suggest that PS1 fragments accumulate in a unique subcompartment(s) of the ER or ER to Golgi trafficking intermediates. Interestingly, the FAD-linked PS1 variants show a marked redistribution toward the heavier region of the gradient. Finally, and in contrast PS1 and PS2 fragments are detected preponderantly in more densely sedimenting membranes, suggesting that the subcellular compartments in which these molecules accumulate are distinct.  

3.113           Mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes

Sims, A.C., Ostermann, J. and Denison, M.R.

1. Virol., 74(12), 5647-5654 (2000)

  The coronavirus replicase gene (gene1) is translated into two co-amino-terminal polyproteins that are proteolytically processed to yield more than 15 mature proteins. Several gene1 proteins have been shown to localize at sites of viral RNA synthesis in the infected cell cytoplasm, notably on late endosomes at early times of infection. However, both immunofluorescence and electron microscopy studies have also detected gene 1 proteins at sites distinct from the putative sites of viral RNA synthesis or virus assembly. In this study, mouse hepatitis virus (MHV)-infected cells were fractionated and analyzed to determine if gene 1 proteins segregated to more than one membrane population. Following differential centrifugation of lysates of MHV-infected DBT cells, gene 1 proteins as well as the structural N and M proteins were detected almost exclusively in a high-speed small membrane pellet. Following fractionation of the small membrane pellet on an iodixanol density gradient, the gene 1 proteins p28 and helicase cofractionated with dense membranes (1.12 to 1.13 g/ml) that also contained peak concentrations of N. In contrast, p65 and p1a-22 were detected in a distinct population of less dense membranes (1.05 to 1.09 g/ml). Viral RNA was detected in membrane fractions containing helicase, p28, and N but not fractions containing p65 and p1a-22. LAMP-1, a marker for late endosomes and lysosomes, was detected in both membrane populations. These results demonstrate that multiple gene 1 proteins segregate into two biochemically distinct but tightly associated membrane populations and that only one of the populations appears to be a site for viral RNA synthesis. The results further suggest that p28 is a component of the viral replication complex whereas the gene 1 proteins p1a-22 and p65 may serve roles during infection that are distinct from viral RNA transcription or replication.  

3.114           Characterization of isolated acidocalcisomes of Trypanosoma cruzi

Scott, D.A. and Docampo, R.

1. Biol. Chem., 275(31), 24215-24221 (2000)

  The acidocalcisome is an acidic calcium store in trypanosomatids, with a vacuolar-type proton-translocating pyrophosphatase (V-H⁺-PPase) located in the membrane. In this paper, we describe a new method using iodixanol density gradients for purification of the acidocalcisomes from Trypanosoma cruzi epimastigotes. Pyrophosphatase assays indicated that the isolated organelle was at least 60-fold purified compared with the large organelle (10,000xg) fraction. Assays for other organelles generally indicated no enrichment in the acidocalcisome fraction; glycosomes were concentrated 5-fold. Vanadate-sensitive ATP-driven Ca²⁺ uptake (Ca²⁺-ATPase) activity was detectable in isolated acidocalcisome, but ionophore experiments indicated that it was not acidic. However, when pyrophosphate was added, the organelle acidified and rate of Ca²⁺ uptake increased. Use of the indicator Oxonol VI showed that PPase activity generated a membrane potential. Use of sulfate or nitrate in place of chloride in the assay buffer did not affect V-H⁺-ATPase activity, but there was less activity with gluconate. Organelle acidification was countered by the chloride/proton symport cycloprogidiosin. No vacuolar H⁺-ATPase activity was detectable in isolated acidocalcisomes. However, immunoblots showed the presence of at least a membrane-bound V-H⁺-ATPase subunit, while experiments employing permeabilized epimastigotes suggested that vacuolar H⁺-ATPase and V-H⁺-PPase activities are present in the same Ca²⁺-containing compartment.  

3.115           The existence of a lysosomal redox chain and the role of ubiquinone

Gille, L. and Nohl, H. Arch. Biochem. Biophys., 375(2), 347-354 (2000)   Several studies concerning the distribution of ubiquinone (UQ) in the cell report a preferential accumulation of this biogenic quinone in mitochondria, plasma membranes, Golgi vesicles, and lysosomes.  Except for mitochondria, no recent comprehensive experimental evidence exists on the particular function of UQ in these subcellular organelles.  The aim of a recent study was to elucidate whether UQ is an active part of an electron-transfer system in lysosomes.  In the present work, a lysosomal fraction was prepared from a light mitochondrial fraction of rat liver by isopycnic centrifugation.  The purity of our preparation was verified by estimation of the respective marker enzymes.  Analysis of lysosomes for putative redox carriers and redox processes in lysosomes was carried out by optical spectroscopy, HPLC, oxymetry, and ESR techniques.  UQ was detected in an amount of 2.2 nmol/mg of protein in lysosomes.  Furthermore, a b -type cytochrome and a flavin-adenine dinucleotide (FAD) were identified as other potential electron carriers.  Since NADH was reported to serve as a substrate of UQ redox chains in plasma membranes, we also tested this reductant in lysosomes.  Our experiments demonstrate a NADH-dependent reduction of UQ by two subsequent one-electron-transfer steps giving rise to the presence of ubisemiquinone and an increase of the ubiquinol pool in lysosomes. Lysosomal NADH oxidation was accompanied by an approximately equimolar oxygen consumption, suggesting that O₂ acts as a terminal acceptor of this redox chain.  DMPO/OH spin adducts were detected by ESR in NADH-supplemented lysosomes, suggesting a univalent reduction of oxygen.  The kinetic analysis of redox changes in lysosomes revealed that electron carriers operate in the sequence NADH > FAD > cytochrome b > ubiquinone > oxygen.  By using the basic spin label TEMPAMINE, we showed that the NADH-related redox chain in lysosomes supports proton accumulation in lysosomes.  In contrast to the hypothesis that UQ in lysosomes is simply a waste product of autophagy in the cell, we demonstrated that this lipophilic electron carrier is a native constituent of a lysosomal electron transport chain, which promotes proton translocation across the lysosomal membrane.  

3.116           Caveolar structure and protein sorting are maintained in NIH cells independent of glycosphingolipid depletion

Shu, L et al Arch. Biochem. Biophys., 373(1), 83-90 (2000)   Glycosphingolipids have been proposed to be critical components of cluster lipids within cell membranes that serve as rafts for the attachment and sorting of proteins to the cell membrane. Density gradient centrifugation was used to isolate and to ascertain the lipid composition of caveolin-enriched membranes. These membranes demonstrated a significant enrichment of sphingolipids and cholesterol containing up to 20% and 30%, respectively, of the cellular glucosylceramide and lactosylceramide. A specific inhibitor of glucosylceramide synthase, d-threo-1-phenyl-2-palmitoyl-3-pyrrolidino-propanol, was used to test the hypothesis that glycosphingolipids are required for the sorting of proteins to caveolae. When NIH 3T3 cells were depleted of their glucosylceramide based glycosphingolipid mass, the caveolar structure remained intact as determined by electron microscopy and confocal microscopy. The caveolar proteins caveolin and annexin II sorted normally to caveolae, as determined by immunoblotting and confocal microscopy. When the GPI-linked protein B61 was inducibly expressed in these cells, sorting to caveolar membranes occurred normally, even in the presence of glucosylceramide depletion. These observations suggest that protein sorting to caveolae in fibroblasts occurs independently of glycosphingolipid synthesis.  

3.117           Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast

Bagnat, M., Keranen, S., Shevchenko, A., Shevchenko, A. and Simons, K. Proc. Natl. Acad. Sci.,. USA, 97(7), 3254-3259 (2000)   Lipid rafts, formed by lateral association of sphingolipids and cholesterol, have been implicated in membrane traffic and cell signaling in mammalian cells. Sphingolipids also have been shown to play a role in protein sorting in yeast. Therefore, we wanted to investigate whether lipid rafts exist in yeast and whether these membrane microdomains have analogous function to their mammalian counterparts. We first developed a protocol for isolating detergent-insoluble glycolipid-enriched complexes (DIGs) from yeast cells. Sequencing of the major protein components of the isolated DIGs by mass spectrometry allowed us to identify, among others, Gas1p, Pma1p, and Nce2p. Using lipid biosynthetic mutants we could demonstrate that conditions that impair the synthesis of sphingolipids and ergosterol also disrupt raft association of Gas1p and Pma1p but not the secretion of acid phosphatase. That endoplasmic reticulum (ER)-to-Golgi transport of Gas1p is blocked in the sphingolipid mutant lcb1-100 raised the question of whether proteins associate with lipid rafts in the ER or later as shown in mammalian cells. Using the sec 18-1 mutant we found that DIGs are present already in the ER. Taken together, our results suggest that lipid rafts are involved in the biosynthetic delivery of proteins to the yeast plasma membrane.  

3.118           Dissecting the role of the Golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface

Heino, S. et al Proc. Natl. Acad. Sci., USA, 97(15), 8375-8380 (2000)   In this study, we compared the transport of newly synthesized cholesterol with that of influenza hemagglutinin (HA) from the endoplasmic reticulum to the plasma membrane. The arrival of cholesterol on the cell surface was monitored by cyclodextrin removal, and HA transport was monitored by surface trypsinization and endoglycosidase H digestion. We found that disassembly of the Golgi complex by brefeldin A treatment resulted in partial inhibition of cholesterol transport while completely blocking HA transport. Further, microtubule depolymerization by nocodazole inhibited cholesterol and HA transport to a similar extent. When the partitioning of cholesterol into lipid rafts was analyzed, we found that newly synthesized cholesterol began to associate with low-density detergent-resistant membranes rapidly after synthesis, before it was detectable on the cell surface, and its raft association increased further upon chasing. When cholesterol transport was blocked by using 15°C incubation, the association of newly synthesized cholesterol with low-density detergent-insoluble membranes was decreased and cholesterol accumulated in a fraction with intermediate density. Our results provide evidence for the partial contribution of the Golgi complex to the transport of newly synthesized cholesterol to the cell surface and suggest that detergent-resistant membranes are involved in the process.  

3.119           Palmitoylation of caveolin-1 is required for cholesterol binding, chaperone complex formation, and rapid transport of cholesterol to caveolae

Uittenbogaard, A. and Smart, E.J.

1. Biol. Chem., 275(33), 25595-25599 (2000)

  We previously demonstrated that a caveolin-chaperone complex transports newly synthesized cholesterol from the endoplasmic reticulum through the cytoplasm to caveolae. Caveolin-1 has a 33-amino acid hydrophobic domain and three sites of palmitoylation in proximity to the hydrophobic domain. In the present study, we hypothesized that palmitoylation of caveolin-1 is necessary for binding of cholesterol, formation of a caveolin-chaperone transport complex, and rapid direct transport of cholesterol to caveolae. To test this hypothesis, four caveolin-1 constructs were generated that substituted an alanine for a cysteine at position 133, 143, or 156 or all three sites (triple mutant). These mutated caveolins and wild type caveolin-1 were stably expressed in the lymphoid cell line, L1210-JF which does not express caveolin-1, does not form a caveolin-chaperone complex, and does not transport newly synthesized cholesterol to caveolae. All of the caveolins were expressed and the proteins localized to plasma membrane caveolae. Wild type caveolin-1 and mutant 133 assembled into complete transport complexes and rapidly (10-20 min) transported cholesterol to caveolae. Caveolin mutants 143 and 156 did not assemble into complete transport complexes, weakly associated with cholesterol, and transported small amounts of cholesterol to caveolae. The triple mutant did not assemble into complete transport complexes and did not associate with cholesterol. We conclude that palmitoylation of caveolin-1 at positions 143 and 156 is required for cholesterol binding and transport complex formation.  

3.120           Mechanism of residence of cytochrome b(5), a tail-anchored protein, in the endoplasmic reticulum

Pedrazzini, E., Villa, A., Longhi, R., Bulbarelli, A. and Borgese, N.

1. Cell Biol., 148(5), 899-913 (2000)

  Endoplasmic reticulum (ER) proteins maintain their residency by static retention, dynamic retrieval, or a combination of the two.  Tail-anchored proteins that contain a cytosolic domain associated with the lipid bilayer via a hydrophobic stretch close to the COOH terminus are sorted within the secretary pathway by largely unknown mechanisms.  Here, we have investigated the mode of insertion in the bilayer and the intracellular trafficking of cytochrome b(5) (b[5]), taken as a model for ER-resident tail-anchored proteins.  We first demonstrated that b(5) can acquire a transmembrane topology posttranslationally, and then used two tagged versions of b(5), N-glyc and O-glyc b(5), containing potential N- and O-glycosylation sites, respectively, at the COOH-terminal lumenal extremity, to discriminate between retention and retrieval mechanisms.  Whereas the N-linked oligosaccharide provided no evidence for retrieval from a downstream compartment, a more stringent assay based on carbohydrate acquisition by O-glyc b(5) showed that b(5) gains access to enzymes catalyzing the first steps of O-glycosylation.  These results suggest that b(5) slowly recycles between the ER and the cis-Golgi complex and that dynamic retrieval as well as retention are involved in sorting of tail-anchored proteins.  

3.121           The role of the COOH terminus of Sec2p in the transport of post-Golgi vesicles

Elkind., N.B., Walch-Solimena, C. and Novick, P.J.

1. Cell Biol., 149(1), 95-110 (2000)

  Sec2p is required for the polarized transport of secretory vesicles in S cerevisiae. The Sec2p NH₂ terminus encodes an exchange factor for the Rab protein Sec4p. Sec2p associates with vesicles and in Sec2p COOH-terminal mutants Sec4p and vesicles no longer accumulate at bud tips.  Thus, the Sec2p COOH terminus functions in targeting vesicles, however, the mechanism of function is unknown.  We found comparable exchange activity for truncated and full-length Sec2 proteins, implying that the COOH terminus does not alter the exchange rate.  Full-length Sec2-GFP, similar to Sec4p, concentrates at bud tips.  A COOH-terminal 58-amino acid domain is necessary but not sufficient for localization.  Sec2p localization depends on actin, Myo2p and Seclp, Sec6p, and Sec9p function.  Full-length, but not COOH-terminally truncated Sec2 proteins are enriched on membranes.  Membrane association of full-length Sec2p is reduced in sec6-4 and sec9-4 backgrounds at 37°C but unaffected at 25°C.  Taken together, these data correlate loss of localization of Sec2 proteins with reduced membrane association.  In addition, Sec2p membrane attachment is substantially Sec4p independent, supporting the notion that Sec2p interacts with membranes via an unidentified Sec2p receptor, which would increase the accessibility of Sec2p exchange activity for Sec4p.  

3.122           Desferrioxamine-mediated iron uptake in Saccharomyces cerevisiae. Evidence for two pathways of iron uptake

Yun, C-W. et al

1. Biol. Chem., 275(14), 10709-10715 (2000)

  In the yeast Saccharomyces cerevisiae, uptake of iron is largely regulated by the transcription factor Aftl. cDNA microarrays were used to identify new iron and AFT1-regulated genes.  Four homologous genes regulated as part of the AFT1-regulon (ARN1-4) were predicted to encode members of a subfamily of the major facilitator superfamily of transporters.  These genes were predicted to encode proteins with 14 membrane spanning domains and were from 26 to 53% identical at the amino acid level. ARN3 is identical to SIT1, which is reported to encode a ferrioxamine B permease.  Deletion of ARN3 did not prevent yeast from using ferrioxamine B as an iron source; however, deletion of ARN3 and FET3, a component of the high affinity ferrous iron transport system, did prevent uptake of ferrioxamine bound iron and growth on ferrioxamine as an iron source.  The siderophore-mediated transport system and the high affinity ferrous iron transport system were localized to separate cellular compartments.  Epitope-tagged Arn3p was expressed in intracellular vesicles that co-sediment with the endosomal protein Pepl2.  In contrast, Fet3p was expressed on the plasma membrane and was digested by extracellular proteases.  These data indicate that S. cerevisiae has two pathways for ferrrioxamine-mediated iron uptake, one occurring at the plasma membrane and the other occurring in an intracellular compartment.  

3.123           Assembly of Trp1 in a signaling complex associated with caveolin-scaffolding lipid raft  domains

Lockwich, T.P. et al

1. Biol. Chem., 275(16), 11934-11942 (2000)

  Trpl has been proposed as a component of the store-operated Ca²⁺ entry (SOC) channel.  However, neither the molecular mechanism of SOC nor the role of Trp in this process is yet understood.  We have examined possible molecular interactions involved in the regulation of SOC and Trpl and report here for the first time that Trpl is assembled in signaling complex associated with caveolin-scaffolding lipid raft domains.  Endogenous hTrpl and caveolin-1 were present in low density fractions of Triton X-100-extracted human submandibular gland cell membranes.  Depletion of plasma membrane cholesterol increased Triton X-100 solubility of Trpl and inhibited carbachol-stimulated Ca²⁺ signaling.  Importantly, thapsigargin stimulated Ca²⁺ influx, but not internal Ca²⁺ release, and inositol 1,4,5-triphosphate (IP₃) stimulated I_soc were also attenuated.  Furthermore, both anti-Trp1 and anti-caveolin-1 antibodies co-immunoprecipitated hTrpl, caveolin-1, Ga_q/11 and IP₃ receptor-type 3 (IP₃R3).  These results demonstrate that caveolar microdomains provide a scaffold for (i) assembly of key Ca²⁺ signaling proteins into a complex and (ii) coordination of the molecular interactions leading to the activation of SOC.  Importantly, we have shown that Trpl is also localized in this microdomain where it interacts with one or more components of this complex, including IP₃ R3.  This finding is potentially important in elucidating the physiological function of Trp.  

3.124           Enterotoxigenic Escherichia coli secretes active heat-labile enteroxin via outer membrane vesicles

Horstman, A.L. and Kuehn, M.J.

1. Biol. Chem., 275(17), 12489-12496 (2000)

  Escherichia coli and other Gram-negative bacteria produce outer membrane vesicles during normal growth.  Vesicles may contribute to bacterial pathogenicity by serving as vehicles for toxins to encounter host cells.  Enterotoxigenic E.coli (ETEC) vesicles were isolated from culture supernatants and purified on velocity gradients, thereby removing any soluble proteins and contaminants from the crude preparation.  Vesicle protein profiles were similar but not identical to outer membranes and differed between strains.  Most vesicle proteins were resistant to dissociation, suggesting they were integral or internal.  Thin layer chromatography revealed that major outer membrane lipid components are present in vesicles.  Cytoplasmic membranes and cytosol were absent in vesicles; however, alkaline phosphatase and AcrA, periplasmic residents, were localized to vesicles.  In addition, physiologically active heat-labile enterotoxin (LT) was associated with ETEC vesicles. LT activity correlated directly with the gradient peak of vesicles, suggesting specific association, but could be removed from vesicles under dissociating conditions. Further analysis revealed that LT is enriched in vesicles and is located both inside and on the exterior of vesicles. The distinct protein composition of ETEC vesicles and their ability to carry toxin may contribute to the pathogenicity of ETEC strains.  

3.125           Biosynthesis of a major lipofuscin fluorophore in mice and humans with ABCR–mediated retinal and macular degeneration

Mata, N.L., Weng, J. and Travis, G.H. Proc. Natl. Acad. Sci. USA, 97(13), 7154-7159 (2000)   Increased accumulation of lipofuscin in cells of the retinal pigment epithelium (RPE) is seen in several forms of macular degeneration, a common cause of blindness in humans. A major fluorophore of lipofuscin is the toxic bis-retinoid, N-retinylidene-N-retinylethanolamine (A2E).  Previously, we generated mice with a knockout mutation in the abcr gene.  This gene encodes rim protein (RmP), an ATP-binding cassette transporter in rod outer segments.  Mice lacking RmP accumulate A2E in RPE cells at a greatly increased rate over controls.  Here, we identify three precursors of A2E in ocular tissues from abcr/mice and humans with ABCR-mediated recessive macular degenerations.  Our results corroborate the scheme proposed by C. A. Parish, M. Hashimoto, K. Nakanishi, J. Dillon & J. Sparrow [Proc. Natl. Acad. Sci. USA (1998) 95,14609-14613], for the biosynthesis of A2E: (i) condensation of all-trans-retinaldehyde (all-trans-RAL) with phosphatidylethanolamine to form a Schiff base; (ii) condensation of the amine product with a second all-trans-RAL to form a bis-retinoid; (iii) oxidation to yield a pyridinium salt; and (iv) hydrolysis of the phosphate ester to yield A2E.  The latter two reactions probably occur within RPE phagolysosomes.  As predicted by this model, formation of A2E was completely inhibited when abcr / mice were raised in total darkness.  Also, once formed, A2E was not eliminated by the RPE.  These data suggest that humans with retinal or macular degeneration caused by loss of RmP function may slow progression of their disease by limiting exposure to light. The precursors of A2E identified in this study may represent pharmacological targets for the treatment of ABCR-mediated macular degeneration.  

3.126           Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb

Plant PJ. et al

1. Cell Biol., 149(7), 1473-1483 (2000).

  Nedd4 is a ubiquitin protein ligase (E3) containing a C2 domain, three or four WW domains, and a ubiquitin ligase HECT domain.  We have shown previously that the C2 domain of Nedd4 is responsible for its Ca2+-dependent targeting to the plasma membrane, particularly the apical region of epithelial MDCK cells.  To investigate this apical preference, we searched for Nedd4-C2 domain-interacting proteins that might be involved in targeting Nedd4 to the apical surface.  Using immobilized Nedd4-C2 domain to trap interacting proteins from MDCK cell lysate, we isolated, in the presence of Ca2+, a ~35-40-kD protein that we identified as annexin XIII using mass spectrometry.  Annexin XIII has two known isoforms, a and b, that are apically localized, although XIIIa is also found in the basolateral compartment.  In vitro binding and coprecipitation experiments showed that the Nedd4-C2 domain interacts with both annexin XIIIa and b in the presence of Ca2+, and the interaction is direct and optimal at 1 mM Ca2+. Immunofluorescence and immunogold electron microscopy revealed colocalization of Nedd4 and annexin XIIIb in apical carriers and at the apical plasma membrane.  Moreover, we show that Nedd4 associates with raft lipid microdomains in a Ca2+ -dependent manner, as determined by detergent extraction and floatation assays.  These results suggest that the apical membrane localization of Nedd4 is mediated by an association of its C2 domain with the apically targeted annexin XIIIb.  

3.127           Mutation of conserved aspartates affects maturation of both aspartate mutant and  endogenous presenilin 1 and presenilin 2 complexes

Yu, G. et al

1. Biol. Chem., 275(35), 27348-27353 (2000)

  Presenilin (PS1 and PS2) holoproteins are transiently incorporated into low molecular weight (MW complexes.  During subsequent incorporation into a higher MW complex, they undergo endoproteolysis to generate stable N- and C-terminal fragments.  Mutation of either of two conserved aspartate residues in transmembrane domains inhibits both presenilin-endoproteolysis and the proteolytic processing of b-amyloid precursor protein and Notch.  We show that although PS1/PS2 endoproteolysis is not required for inclusion into the higher MW N- and C-terminal fragment-containing complex, aspartate mutant holoprotein presenilins are not incorporated into the high MW complexes.  Aspartate mutant presenilin holoproteins also preclude entry of endogenous wild type PS1/PS2 into the high MW complexes but do not affect the incorporation of wild type holoproteins into lower MW holoprotein complexes.  These data suggest that the loss of function effects of the aspartate mutants result in altered PS complex maturation and argue that the functional presenilin moieties are contained in the high molecular weight complexes.  

3.128           ³¹P NMR spectroscopy of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major

Moreno, B. et al

1. Biol. Chem., 275(37), 28356-28362 (2000)

  High resolution ³¹P nuclear magnetic resonance spectra at 303.6 MHz (corresponding to a ¹H resonance frequency of 750 MHz) have been obtained of perchloric acid extracts of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, the causative agents of African sleeping sickness, Chagas’disease, and leishmaniasis.  Essentially complete assignments have been made based on chemical shifts and by direct addition of authentic reference compounds.  The results indicate the presence of high levels of short chain condensed polyphosphates: di-, tri-, tetra-, and pentapolyphosphate. ³¹P NMR spectra of purified T. brucei, T. cruzi and L. major acidocalcisomes, calcium and phosphorus storage organelles, indicate that polyphosphates are abundant in these organelles and have an average chain length of 3.11-3.39 phosphates.  In the context of the recent discovery of several pyrophosphate-utilizing enzymes in trypanosomatids, the presence of these inorganic polyphosphates implies a critical role for these molecules in these parasites and a potential new route to chemotherapy.  

3.129           Epidermal growth factor-mediated caveolin recruitment to early endosomes and MAPK activation

Pol, A., Lu, A., Pons, M., Peiro, S. and Enrich, C.

1. Biol. Chem., 275(39), 30566-30572 (2000)

 

The endocytic compartment of eukaryotic cells is a complex intracellular structure involved in sorting, processing, and degradation of a great variety of internalized molecules.  Recently, the uptake through caveolae has emerged as an alternative internalization pathway, which seems to be directly related with some signal transduction pathways.  However, the mechanisms, molecules, and structures regulating the transport of caveolin from the cell surface into the endocytic compartment are largely unknown.  In this study, normal quiescent fibroblasts (normal rat kidney (NRK)) were used to demonstrate that epidermal growth factor causes partial redistribution of caveolin from the cell surface into a cellubrevin early endocytic compartment.  Treatment of NRK cells with cytochalasin D or latrunculin A inhibit this pathway and the concomitant activation of Mek and mitotic-activated protein (MAP) kinase; however, if cells were pre-treated with filipin, cytochalasin D does not inhibit the phosphorylation of MAP kinase induced by epidermal growth factor.  From these results we conclude that in NRK cells the intact actin cytoskeleton is necessary for the EGF-mediated transport of caveolin from the cell surface into the early endocytic compartment and the activation of MAP kinase pathway.  

3.130           Dystrophin associates with a caveolae of rat cardiac myocytes

Doyle, D.D. et al Circulation Res., 87, 480-488 (2000)

 

The possibility of an interaction between the cytoskeletal protein dystrophin and cell surface caveolae in the mammalian myocardium was investigated by several techniques. Caveolin (cav)-3-enriched, detergent-insoluble membranes isolated from purified ventricular sarcolemma by density-gradient fractionation were found to contain dystrophin and dystroglycan.  Further purification of cav-3-containing membranes by immunoprecipitation using anti-cav-3-coated magnetic beads yielded dystrophin but not always dystroglycan.  Electron microscopic analysis of precipitated material revealed caveola-sized vesicular profiles that could be double-labeled with anti-dystrophin and anti-cav-3 antibodies.  In contrast, immunoprecipitation of membranes with anti-dystrophin-coated beads yielded both cav-3 and dystroglycan.  Electron microscopic analysis of this material showed heterogeneous membrane profiles, some of which could be decorated with anti-cav-3 antibodies.  To confirm that dystrophin and cav-3 were closely associated in cardiac myocytes, we verified that dystrophin was also present in immunoprecipitated cav-3-containing membranes from detergent extracts, as well as in sonicated extracts of purified ventricular myocytes.  Confocal immunofluorescence microscopy of ventricular and atrial cardiac myocytes showed that the cellular distributions of cav-3 and dystrophin partially overlapped.  Immuno-electron micrographs of thin sections of rat atrial myocytes revealed a fraction of dystrophin molecules that are in apparently close apposition to caveolae.  These results suggest that a subpopulation of dystrophin molecules interacts with cardiac myocyte caveolae in vivo and that some of the dystrophin is engaged in linking cav-3 with the dystroglycan complex.  

3.131           Chemical stimulation of synaptosomes modulates a-Ca²⁺/calmodulin-dependent protein kinase II mRNA association to polysomes

Bagni, C., Mannucci, L., Dotti, C.G. and Amaldi, F.

1. Neurosci., 20 RC76, 1-6 (2000)

 

The presence of specific mRNAs in dendrites and at synapses is well established, but a direct and reliable demonstration that they are associated with polysomes is still missing.  To address this point we analyzed the polysomal association of the mRNAs for the a-subunit of Ca²⁺ /calmodulin-dependent protein kinase II (a-CaMKII), for type 1 inositol 1,4,5-trisphosphate receptor (InsP3Rl) and for the activity-regulated cytoskeleton-associated protein (Arc) in a synaptosomal preparation devoid of contaminating material from neuronal and glial perikarya.  We show that a fraction of a-CaMKII, InsP3Rl, and Arc mRNAs present in synaptosomes is indeed associated with polysomes. Moreover, we show that polysomal association of a-CaMKII mRNA, but not InsP3Rl and Arc mRNAs, increases with depolarization of the synaptosomal membrane.  Finally, we show that the synthesis of a-CaMKII protein increases with stimulation.  Dendritic mRNA recruitment onto polysomes in response to synaptic stimulation might represent one of the mechanisms underlying the processes of learning and memory.  

3.132           Mutant presenilin 1 increases the levels of Alzheimer amyloid b-peptide Ab42 in late compartments of the constitutive secretory pathway

Petanceska, S., Seeger, M., Checler, F. and Gandy, S.

1. Neurochem., 74, 1878-1884 (2000)

  Mutations in the presenilin 1 (PS1) gene are associated with autosomal dominant, early-onset, familial Alzheimer's disease and result in increased release of the hyperaggregatable 42-amino acid form of the amyloid b-peptide (Ab42).  To determine which subcellular compartments are potential source(s) of released Ab42, we compared the levels and spatial segregation of intracellular Ab40 and Ab42 peptides between N2a neuroblastoma cells doubly transfected with the "Swedish" familial Alzheimer's disease-linked amyloid precursor protein variant and either wild-type PS1 (PS1^wt) or familial Alzheimer's disease-linked D9 mutant PS1 (PS1^D9).  As expected, PSl^D9-expressing cells had dramatically higher levels of intracellular Ab42 than did cells expressing PS1^wt.  However, the highest levels of Ab42 colocalized not with endoplasmic reticulum or Golgi markers but with rab8, a marker for trans-Golgi network (TGN)-to-plasma membrane (PM) transport vesicles.  We show that PS1 mutants are capable of causing accumulation of Ab42 in late compartments of the secretary pathway, generating there a readily releasable source of Ab42.  Our findings indicate that PS1 "bioactivity" localizes to the vicinity of the TGN and/or PM and reconcile the apparent discrepancy between the preponderant concentration of PS1 protein in proximal compartments of the secretary pathway and the recent findings that PS1 "bioactivity" can control g-secretase-like processing of another transmembrane substrate, Notch, at or near the PM.  

3.133           Golgi targeting of the GLUT1 glucose transporter in lactating mouse mammary gland

Nemeth, B.A., Tsang, S.W.Y., Geske, R.S. and Haney, P. Pediatr Res., 47, 444-450 (2000)   Lactose, the major carbohydrate of human milk, is synthesized in the Golgi from glucose and UDP-galactose.  The lactating mammary gland is unique in its requirement for the transport of glucose into Golgi.  Glucose transporter-1 (GLUT1) is the only isoform of the glucose transporter family expressed in mammary gland.  In most cells, GLUT1 is localized to the plasma membrane and is responsible for basal glucose uptake; in no other cell type is GLUT1 a Golgi resident.  To test the hypothesis that GLUT1 is targeted to Golgi during lactation, the amount and subcellular distribution of GLUT1 were examined in mouse mammary gland at different developmental stages.  Methods including immunohistochemistry, immunofluorescence, subcellular fractionation, density gradient centrifugation, and Western blotting yielded consistent results.  In virgins, GLUT1 expression was limited to plasma membrane of epithelial cells.  In late pregnant mice, GLUT1 expression was increased with targeting primarily to basolateral plasma membrane but also with some intracellular signal. During lactation, GLUT1 expression was further increased, and targeting to Golgi, demonstrated by colocalization with the 110-kD coatomer-associated protein b-COP, predominated.  Removal of pups 18 d after delivery resulted in retargeting of GLUT1 from Golgi to plasma membrane and a decline in total cellular GLUT1 within 3 h. In mice undergoing natural weaning, GLUT1 expression declined.  Changes in the amount and targeting of GLUT1 during mammary gland development are consistent with a key role for GLUT1 in supplying substrate for lactose synthesis and milk production.  

3.134           Membrane raft microdomains mediate lateral assemblies for HIV-1 infection

Manes, S. et al EMBO reports, 1(2), 190-196 (2000)   HIV-1 infection triggers lateral membrane diffusion following interaction of the viral envelope with cell surface receptors. We show that these membrane changes are necessary for infection, as initial gpl2O-CD4 engagement leads to redistribution and clustering of membrane microdomains, enabling subsequent interaction of this complex with HIV-1 co-receptors.  Disruption of cell membrane rafts by cholesterol depletion before viral exposure inhibits entry by both X4 and R5 strains of HIV-1, although viral replication in infected cells is unaffected by this treatment.  This inhibitory effect is fully reversed by cholesterol replenishment of the cell membrane. These results indicate a general mechanism for HIV-1 envelope glycoprotein-mediated fusion by reorganization of membrane microdomains in the target cell, and offer new strategies for preventing HIV-1 infection.  

3.135           Subcellular organization of bile acid amidation in human liver: a key in regulating the biosynthesis of bile salts

Solaas, K., Ulvestad, A., Soreide, O. and Kase, B.F.

1. Lipid Res., 41, 1154-1162 (2000)

 

To extend our knowledge of how the synthesis of free bile acids and bile salts is related within the hepatocytes, bile acid-CoA:amino acid N-acyltransferase and bile acid-CoA thioesterase activities were measured in subcellular fractions of human liver homogenates.  Some bile acids, both conjugated and unconjugated, have been reported to be natural ligands for the farnesoid X receptor (FXR), an orphan nuclear receptor.  The conversion of [¹⁴C]choloyl-CoA and [¹⁴C]chenodeoxycholoyl-CoA into the corresponding tauro- and glyco-bile acids or the free bile acids was measured after high-pressure liquid radiochromatography.  There was an enrichment of the N-acyltransferase in the cytosolic and the peroxisomal fraction.  Bile acid-CoA thioesterase activities were enriched in the cytosolic, peroxisomal, and mitochondrial fractions.  The highest amidation activities of both choloyl-CoA and chenodeoxycholoyl-CoA were found in the peroxisomal fraction (15-58 nmol/mg protein/min).  The K_m, was higher for glycine than taurine both in cytosol and the peroxisomal fractions These results show that the peroxisomal de novo synthesis of bile acids is rate limiting for peroxisomal amidation, and the microsomal bile acid-CoA synthetase is rate limiting for the cytosolic amidation.  The peroxisomal location may explain the predominance of glyco-bile acids in human bile.  Both a cytosolic and a peroxisomal bile acid-CoA thioesterase may, influence the intracellular levels of free and conjugated bile acids.  

3.136           Human bleomycin hydrolase regulates the secretion of amyloid precursor protein

Lefterov, I.M., Koldamova, R.P. and Lazo, J.S. FASEB J., 14, 1837-1847 (2000)   Human bleomycin hydrolase (hBH) is a neutral cysteine protease genetically associated with increased risk for Alzheimer disease.  We show here that ectopic expression of hBH in 293APPwt and CHOAPPsw cells altered the processing of amyloid precursor protein (APP) and increased significantly the release of its proteolytic fragment, b amyloid (Ab).  We also found that hBH interacted and colocalized with APP as determined by subcellular fractionation, in vitro binding assay, and confocal immunolocalization.  Metabolic labeling and pulse-chase experiments, showed that ectopic hBH expression increased secretion of soluble APPa/b products without changing the half-life of cellular APP. We also observed that this increased Ab secretion was independent of hBH isoforms.  Our findings suggest a regulatory role for hBH in APP processing pathways.    

3.137           Identification, purification and characterization of an acetoacetyl-CoA thiolase from rat liver peroxisomes

Antonenkov, V.D., Croes, K., Waelkens, E., Van Veldhoven, P.P. and Mannaerts, G.P. Eur. J. Biochem., 267, 2981-2990 (2000)   Acetoacetyl-CoA specific thiolases catalyse the cleavage of acetoacetyl-CoA into two molecules of acetyl-CoA and the synthesis (reverse reaction) of acetoacetyl-CoA. The formation of acetoacetyl-CoA is the first step in cholesterol and ketone body synthesis. In this report we describe the identification of a novel acetoacetyl-CoA thiolase and its purification from isolated rat liver peroxisomes by column chromatography. The enzyme, which is a homotetramer with a subunit molecular mass of 42 kDa, could be distinguished from the cytosolic and mitochondrial acetoacetyl-CoA thiolases by its chromatographic behaviour, kinetic characteristics and partial internal amino-acid sequences. The enzyme did not catalyse the cleavage of medium or long chain 3-oxoacyl-CoAs. The enzyme cross-reacted with polyclonal antibodies raised against cytosolic acetoacetyl-CoA thiolase. The latter property was exploited to confirm the peroxisomal localization of the novel thiolase in subcellular fractionation experiments. The peroxisomal acetoacetyl-CoA thiolase most probably catalyses the first reaction in peroxisomal cholesterol and dolichol synthesis. In addition, its presence in peroxisomes along with the other enzymes of the ketogenic pathway indicates that the ketogenic potential of peroxisomes needs to be re-evaluated.

 

3.138           Human oxytocin receptors in cholesterol-rich vs. cholesterol-poor microdomains of the plasma membrane

Gimpl, G. and Fahrenholz, F. Eur. J. Biochem., 267, 2483-2497 (2000)   We analyzed the properties of a G protein-coupled receptor localized in cholesterol-poor vs. cholesterol-rich microdomains of the plasma membrane.  For this purpose, the human oxytocin receptor, which is very sensitive against alterations of the membrane cholesterol level, was stably expressed in HEK293 cells, To calculate the total number of receptors independent of ligand binding studies, the oxytocin receptor was tagged with an enhanced green fluorescent protein (EGFP) which did not change the functional properties of the receptor.  Only 1% of the oxytocin receptors were present in cholesterol-rich detergent-insoluble domains.  In contrast, employing a detergent-free fractionation scheme that preserves the functional activity of the receptor, we detected 10-15% of the receptors in cholesterol-rich low-density membranes and therein the high-affinity state receptors were twofold enriched.  In cholesterol-poor vs. cholesterol-rich domains, high-affinity oxytocin receptors behaved similar with respect to their agonist binding kinetics and GTP sensitivity. However, high-affinity oxytocin receptors localized in cholesterol-rich low-density membranes showed a markedly enhanced (t, » threefold) stability at 37°C as compared with the oxytocin receptors localized in the cholesterol-poor high-density membranes.  Addition of cholesterol to the high-density membranes fully protected the oxytocin receptors against loss of function.  The importance of cholesterol to stabilize the oxytocin receptor was supported in experiments with solubilized receptors. Cholesterol markedly delayed the inactivation of oxytocin receptors solubilized with Chapso.  In conclusion, the data of this report suggest that functional properties of heptahelical receptor proteins could differ in dependence of their localization in different membrane microdomains.  

3.139           Involvement of gangliosides in GPI-anchored neuronal cell adhesion molecule TAG-1 signaling in lipid rafts

Kasahara, K. et al

1. Biol. Chem., 275(44), 34701-34709 (2000)

The association of ganglioside GD3 with TAG-1, a glycosylphosphatidylinositol (GPI)-anchored neuronal cell adhesion molecule, was examined by coimmunoprecipitation experiments. Previously, we have shown that the anti-ganglioside GD3 antibody (R24) immunoprecipitated the src-family kinase Lyn from the rat cerebellum, and R24 treatment of primary cerebellar cultures induced Lyn activation and rapid tyrosine phosphorylation of an 80-kDa protein (p80). We now report that R24 coimmunoprecipitates a 135-kDa protein (p135) from primary cerebellar cultures. Treatment with phosphatidylinositol-specific phospholipase C revealed that p135 was GPI-anchored to the membrane. It was identified as TAG-1 by sequential immunoprecipitation with an anti-TAG-1 antibody. Antibody-mediated crosslinking of TAG-1 induced Lyn activation and rapid tyrosine phosphorylation of p80. Selective inhibitor for src-family kinases reduced the tyrosine phosphorylation of p80. Sucrose density gradient analysis revealed that the TAG-1 and tyrosine-phosphorylated p80 in cerebellar cultures were present in the lipid raft fraction. These data show that TAG-1 transduces signals via Lyn to p80 in the lipid rafts of the cerebellum. Furthermore, degradation of cell-surface glycosphingolipids by endoglycoceramidase induced an alteration of TAG-1 distribution on an OptiPrep gradient and reduced the TAG-1 mediated Lyn activation and tyrosine phosphorylation of p80. These observations suggest that glycosphingolipids are involved in TAG-1 mediated signaling in lipid rafts.  

3.140           Presence of oxidized cholesterol in caveolae uncouples active platelet-derived growth factor receptors from tyrosine kinase substrates

Liu, P., Wang, P-y., Michaely, P., Zhu, M. and Anderson, R.G.W.

1. Biol. Chem., 275(41), 31648-31654 (2000)

  Platelet-derived growth factor receptor b (PDGFRb) in fibroblasts is concentrated in caveolae where it controls the tyrosine phosphorylation of multiple proteins. Caveolae are enriched in cholesterol and sphingolipids, but the role of these lipids in PDGFR signal transduction is unknown.  We report that introduction of cholest-4-en-3-one into caveolae membranes uncouples PDGFR autophosphorylation from tyrosine phosphorylation of neighboring proteins. Cholest-4-en-3-one appears to interfere with the normal interaction between PDGFR and its partners.  The results suggest that tightly packed caveolae lipids form a membrane platform that functions as a lipid scaffold for organizing the molecular interactions of multiple signaling pathways.  

3.141           Huntingtin expression stimulates endosomal-lysosomal activity, endosome tubulation and autophagy

Kegel, K.B. et al

1. Neurosci., 20(19), 7268-7278 (2000)

  An expansion of polyglutamines in the N terminus of huntingtin causes Huntington’s disease (HD) and results in the accrual of mutant protein in the nucleus and cytoplasm of affected neurons.  How mutant huntingtin causes neurons to die is unclear, but some recent observations suggest that an autophagic process may occur.  We showed previously that huntingtin markedly accumulates in endosomal-lysosomal organelles of affected HD neurons and, when exogenously expressed in clonal striatal neurons, huntingtin appears in cytoplasmic vacuoles causing cells to shrink.  Here we show that the huntingtin-enriched cytoplasmic vacuoles formed in vitro internalized the lysosomal enzyme cathepsin D in proportion to the polyglutamine-length in huntingtin.  Huntingtin-labeled vacuoles displayed the ultrastructural features of early and late autophagosomes (autolysosomes), had little or no overlap with ubiquitin, proteasome, and heat shock protein 70/heat shock cognate 70 immunoreactivities, and altered the arrangement of Golgi membranes mitochondria, and nuclear membranes.  Neurons with excess cytoplasmic huntingtin also exhibited increased tubulation of endosomal membranes.  Exogenously expressed human full-length wild-type and mutant huntingtin codistributed with endogenous mouse huntingtin in soluble and membrane fractions, whereas human N-terminal huntingtin products were found only in membrane fractions that contained lysosomal organelles.  We speculate that mutant huntingtin accumulation in HD activates the endosomal-lysosomal system, which contributes to huntingtin proteolysis and to an autophagic process of cell death.  

3.142           The human DIMINUTO/DWARF1 homolog seladin-1 confers resistance to  Alzheimer’s disease-associated neurodegeneration and oxidative stress

Greeve, I. et al

1. Neurosci., 20(19), 7345-7352 (2000)

  In Alzheimer's disease (AD) brains, selected populations of neurons degenerate heavily, whereas others are frequently spared from degeneration.  To address the cellular basis for this selective vulnerability of neurons in distinct brain regions, we compared gene expression between the severely affected inferior temporal lobes and the mostly unaffected fronto-parietal cortices by using an mRNA differential display.  We identified seladin-1, a novel gene, which was down regulated in large pyramidal neurons in vulnerable regions in AD but not control brains.  Seladin- 1 is a human homolog of the DIMINUTO/DWARF1 gene described in plants and Caenorhabditis elegans.  Its sequence shares similarities with flavin-adenin-dinucleotide (FAD)-dependent oxidoreductases.  In human control brain, seladin-1 was highly expressed in almost all neurons.  In PC12 cell clones that were selected for resistance against AD-associated amyloid-b peptide (Ab)-induced toxicity, both mRNA and protein levels of seladin-1 were approximately threefold higher as compared with the non-resistant wild-type cells.  Functional expression of seladin-1 in human neuroglioma H4 cells resulted in the inhibition of caspase 3 activation after either Ab-mediated toxicity or oxidative stress and protected the cells from apoptotic cell death.  In apoptotic cells, however, endogenous seladin-1 was cleaved to a 40 kDa derivative in a caspase-dependent manner.  These results establish that seladin-1 is an important factor for the protection of cells against Ab toxicity and oxidative stress, and they suggest that seladin-1 may be involved in the regulation of cell survival and death, Decreased expression of seladin-1 in specific neurons may be a cause for selective vulnerability in AD.  

3.143           Carboxyl-terminal fragments of Alzheimer b-amyloid precursor protein accumulate in restricted and unpredicted intracellular compartments in presenilin 1 deficient cells

Chen, F. et al

1. Biol. Chem., 275, 36794-36802 (2000)

  Absence of functional presenilin 1 (PS1) protein leads to loss of g-secretase cleavage of the amyloid precursor protein (bAPP), resulting in a dramatic reduction in amyloid b peptide (Ab) production and accumulation of a- or b-secretase-cleaved C-terminal fragments of bAPP (a- or b-CTFs). The major C-terminal fragment (CTF) in brain was identified as bAPP-CTF-(11-98), which is consistent with the observation that cultured neurons generate primarily Ab(11-40). In PS1^-/- murine neurons and fibroblasts expressing the loss-of-function PS1_D385A mutant, CTFs accumulated in the endoplasmic reticulum, Golgi, and lysosomes, but not late endosomes. There were some subtle differences in the subcellular distribution of CTFs in PS1^-/- neurons as compared to PS1_D385A mutant fibroblasts. However, there was no obvious redistribution of full-length bAPP or of markers of other organelles in either mutant. Blockade of endoplasmic reticulum-to-Golgi trafficking indicated that in PS1^-/- neurons (as in normal cells) trafficking of bAPP to the Golgi compartment is necessary before a-and b-secretase cleavage occurs. Thus, while we cannot exclude a specific role for PS1 in trafficking of CTFs, these data argue against a major role in general protein trafficking. These results are more compatible with a role for PS1 either as the actual g-secretase catalytic activity or in other functions indirectly related to g-secretase catalysis (e.g. an activator of g-secretase, a substrate-adaptor for g-secretase, or delivery of g-secretase to bAPP-containing compartments.  

3.144           Differential targeting of b-adrenergic receptor subtypes and adenyl cyclase to cardiomyocyte caveolae: A mechanism to functionally regulate the cAMP signaling  pathway

Rybin, V.O., Xu, X., Lisanti, M.P. and Steinberg, S.F.

1. Biol. Chem., 275(52), 41447-41457(2000)

  Differential modes for b₁ and b₂-adrenergic receptor (AR) regulation of adenylyl cyclase in cardiomyocytes is most consistent with spatial regulation in microdo­mains of the plasma membrane. This study examines whether caveolae represent specialized subdomains that concentrate and organize these moieties in car­diomyocytes. Caveolae from quiescent rat ventricular cardiomyocytes are highly enriched in b₂-ARs, Ga_i, protein kinase A, RIIa subunits, caveolin-3, and flotillins (caveolin functional homologues); b₁-ARs, m₂-muscarinic cholinergic receptors, Ga_s and cardiac types V/VI adenylyl cyclase distribute between caveolae and other cell fractions, whereas protein kinase A RIa subunits, G protein-coupled receptor kinase-2, and clathrin are largely excluded from caveolae. Cell surface b₂-ARs lo­calize to caveolae in cardiomyocytes and cardiac fibro­blasts (with markedly different b₂-AR expression levels), indicating that the fidelity of b₂-AR targeting to caveo­lae is maintained over a physiologic range of b₂-AR ex­pression. In cardiomyocytes, agonist stimulation leads to a marked decline in the abundance of b₂-ARs (but not b₁-ARs) in caveolae. Other studies show co-immunopre­cipitation of cardiomyocytes adenylyl cyclase V/VI and caveolin-3, suggesting their in vivo association. How­ever, caveolin is not required for adenylyl cyclase tar­geting to low density membranes, since adenylyl cyclase targets to low buoyant density membrane fractions of HEK cells that lack prototypical caveolins. Neverthe­less, cholesterol depletion with cyclodextrin augments agonist-stimulated cAMP accumulation, indicating that caveolae function as negative regulators of cAMP accu­mulation. The inhibitory interaction between caveolae and the cAMP signaling pathway as well as domain-specific differences in the stoichiometry of individual elements in the b-AR signaling cascade represent impor­tant modifiers of cAMP-dependent signaling in the heart.    

3.145           Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling  assemblies

Harder, T. and Kuhn, M.

1. Cell Biol., 151(2), 199-207 (2000)

  Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes.  Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction.  Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells.  We employed a novel method to immunoisolate plasma membrane subfragments that were highly concentrated in activated TCR-CD3 complexes and associated signaling proteins.  We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner.  In contrast, other raft-associated molecules, including protein tyrosine kinases Lek and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates.  Many downstream signaling proteins coisolated with the TCR/LAT-enriched plasma membrane fragments, suggesting that LAT/ TCR assemblies form a structural scaffold for TCR signal transduction proteins.  Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.  

3.146           Presenilin complexes with the C-terminal fragments of amyloid precursor protein at the sites of amyloid b-protein generation

Xia, W. et al Proc. Natl. Acad. Sci., 97(16), 9299-9304 (2000)   An unusual intramembranous cleavage of the b-amyloid precursor protein (APP) by g-secretase is the final step in the generation of amyloid b-peptide (Ab).  Two conserved aspartates in transmembrane (TM) domains 6 and 7 of presenilin (PS) 1 are required for Ab production by g-secretase.  Here we report that the APP C-terminal fragments, C83 and C99, which are the direct substrates of g-secretase, can be coimmunoprecipitated with both PS1 and PS2.  PS/C83 complexes were detected in cells expressing endogenous levels of PS.  The complexes accumulate when g-secretase is inactivated either pharmacologically or by mutating the PS aspartates.  PS1/ C83 and PS1/C99 complexes were detected in Golgi-rich and trans-Golgi network-rich vesicle fractions.  In contrast, complexes of PS1 with APP holoprotein, which is not the immediate substrate of g-secretase, occurred earlier in endoplasmic reticulum-rich vesicles.  The major portion of intracellular Ab at steady state was found in the same Golgi/trans-Golgi network-rich vesicles, and Ab levels in these fractions were markedly reduced when either PS1 TM aspartate was mutated to alanine.  Furthermore, de novo generation of Ab in a cell-free microsomal reaction occurred specifically in these same vesicle fractions and was markedly inhibited by mutating either TM aspartate.  Thus, PSs are complexed with the g-secretase substrates C83 and C99 in the subcellular locations where Ab is generated, indicating that PSs are directly involved in the pathogenically critical intramembranous proteolysis of APP.  

3.147           Long-term insulin treatment of 3T3-L1 adipocytes results in mis-targeting of GLUT4: implications for insulin-stimulated glucose transport

Maier, V.H. and Gould G.W. Diabetologia, 43(10), 1273-1281 (2000)   Aimslhypothesis.  Insulin stimulates glucose transport in adipose and muscle tissue by the translocation of a specialised pool of intracellular GLUT4-containing vesicles to the cell surface.  It is well established that defective insulin-stimulated GLUT4 translocation is associated with insulin resistance.  Long-term insulin treatment (500 nmol/l for 24 h) of 3T3-Ll adipocytes has previously been shown to decrease cellular GLUT4 content and reduce insulin-stimulated GLUT4 translocation.  Here, we test the hypothesis that the insulin resistance observed after long-term insulin treatment arises by the selective loss of GLUT4 from a specific intracellular compartment.   Methods  Using iodixanol gradient centrifugation we have separated intracellular GLUT4 containing membranes into two distinct populations corresponding to recycling endosomes and a distinct intracellular compartment, which probably represents GLUT4 storage vesicles (GSVs).   Results.  A short-term insulin stimulation reduced the content of GLUT4 in the GSV fraction (51 ± 3.5 %) with only a modest decrease from the endosomal fraction (23 ± 2.6 %).  Long-term insulin treatment decreased cellular GLUT4 content by about 40 % and diminished the ability of a short-term insulin challenge to promote GLUT4 translocation.  We further show that this depletion of cellular GLUT4 is selectively from the GSV fraction (68 ± 7 % decrease compared to untreated cells).   Conclusions/interpretation.  Such data argue that long-term insulin treatment results in the mistargeting of GLUT4 such that it no longer accesses the GSV compartment.  These data imply that defective targeting of GLUT4 away from the GSV compartment plays an important role in the aetiology of insulin resistance.  

3.148           Assembly of myelin by association of proteolipid protein with cholesterol and  galactosylceramide-rich membrane domains

Simons, M., Kramer, E-M., Thiele, C., Stoffel, W. And Trotter, J.

1. Cell Biol., 151(1), 143-153 (2000)

  Myelin is a specialized membrane enriched in glycosphingolipids and cholesterol that contains a limited spectrum of proteins. We investigated the assembly of myelin components by oligodendrocytes and analyzed the role of lipid-protein interactions in this process. Proteolipid protein (PLP), the major myelin protein, was recovered from cultured oligodendrocytes from a lower-density CHAPS-insoluble membrane fraction (CIMF) enriched in myelin lipids. PLP associated with the CIMF after leaving the endoplasmic reticulum but before exiting the Golgi apparatus, suggesting that myelin lipid and protein components assemble in the Golgi complex. The specific association of PLP with myelin lipids in CIMF was supported by the finding that it was efficiently cross-linked to photoactivable cholesterol, but not to phosphatidylcholine, which is underrepresented in both myelin and CIMF: Furthermore, depletion of cholesterol or inhibition of sphingolipid synthesis in oligodendrocytes abolished the association of PLP with CIMF. Thus PLP may be recruited to myelin rafts, represented by CIMF, via lipid-protein interactions. In contrast to oligodendrocytes, after transfection in BHK cells, PLP is absent from isolated CIMF, suggesting that PLP requires specific lipids for raft association. In mice deficient in the enzyme ceramide galactosyl transferase, which cannot synthesize the main myelin glycophingolipids, a large fraction of PLP no longer associates with rafts. Formation of a cholesterol- and galactosylceramide-rich membrane domain (myelin rafts) may be critical for the sorting of PLP and assembly of myelin in oligodendrocytes.  

3.149           Intracellular events in the assembly of chylomicrons in rabbit enterocytes

Cartwright, I.J., Plonne, D. and Higgins, J.A.

1. Lipid Res., 41, 1728-1739 (2000)

  The aim of this study was to determine the intracellular events in chylomicron assembly in adult villus enterocytes. We have used novel methods for separation of the intracellular components of the secretory compartment [ rough and smooth endoplasmic reticulum (RER and SER), respectively) and Golgi], and their membrane and luminal components, from villus enterocytes isolated from rabbit small intestine. The steady state composition of the components of the secretory compartment and the intracellular pools of newly synthesized. Apolipoprotein B-48 (apoB-48) and triacylglycerol (TAG) was determined. The observations indicate that the SER is the main site of the TAG synthesis and of chylomicron assembly. Newly synthesized apoB-48 and TAG accumulate in the SER membrane and are transferred into the lumen in a microsomal triglyceride transfer protein-dependent step. In enterocytes isolated from chow-fed rabbits, in which fat absorption is relatively slow, transfer of apoB-48 and TAG from the SER membrane into the lumen appears to be rate limiting. In enterocytes from fat-fed rabbits, TAG accumulates into the lumen of the SER, suggesting that movement out of the SER lumen becomes rate limiting, when chylomicron secretion is markedly stimulated. In these cells, the cytosolic TAG also increased to 450 mg/g enterocytes, compared with 12 mg/g enterocytes from chow-fed rabbits, indicating that transfer of TAG from the SER membrane into the secretory pathway can become saturated, so that newly synthesized TAG moves into the cytosol.  

3.150           Brain plasmin enhances APP a-cleavage and Ab degradation and is reduced in Alzheimer’s disease brains

Ledesma, M.D., Da Silva, J.S., Crassaerts, K., Delacourte, A., De Strooper, B and Dotti, C.G. EMBO Reports, 11(61), 530-535 (2000)   The proteolytic processing of amyloid precursor protein (APP) has been linked to sphingolipid-cholesterol microdomains (rafts). However, the raft proteases that may be involved in APP cleavage have not yet been identified. In this work we present evidence that the protease plasmin is restricted to rafts of cultured hippocampal neurons. We also show that plasmin increases the processing of human APP preferentially at the a-cleavage site, and efficiently degrades secreted amyloidogenic and non-amyloidogenic APP fragments. These results suggest that brain tissue from Alzheimer’s disease patients contains reduced levels of plasmin, implying that plasmin downregulation may cause amyloid plaque deposition accompanying sporadic Alzheimer’s disease.  

3.151           Cytosolic phospholipase A₂ regulates Golgi structure and modulates intracellular trafficking of membrane proteins

Choukroun, G.J. et al

1. Clin. Invest, 106, 983-993 (2000)

  The Golgi complex and the trans-Golgi network are critical cellular organelles involved in the endocytic and biosynthetic pathways of protein trafficking. Lipids have been implicated in the regulation of membrane protein trafficking vesicular fusion, and targeting. We have explored the role of cytosolic group IV phospholipase A₂ (cPLA₂) in membrane-protein trafficking in kidney epithelial cells. Adenoviral expression of cPLA₂ in LLC-PK₁ kidney epithelial cells prevents constitutive trafficking to the plasma membrane of an aquaporin 2-green fluorescent protein chimera, with retention of the protein in the rough endoplasmic reticulum. Plasma membrane Na⁺-K⁺-ATPase a-subunit localization is markedly reduced in cells expressing cPLA₂, whereas the trafficking of a Cl ^-/ HCO₃^- anion exchanger to the plasma membrane is not altered in these cells. Expression of cPLA₂ results in dispersion of giantin and b-COP from their normal, condensed Golgi localization, and in marked disruption of the Golgi cisternae. cPLA₂  is present in Golgi fractions from noninfected LLC-PK₁ cells and rat kidney cortex. The distribution of tubulin and actin was not altered by cPLA₂  indicating that the microtubule and actin cytoskeleton remain intact. Total cellular protein synthesis is unaffected by the increase in cPLA₂ activity. Thus cPLA₂ plays an important role in determining Golgi architecture and selective control of constitutive membrane-protein trafficking in renal epithelial cells.  

3.152           Tropism of human cytomegalovirus for endothelial cells is determined by a post-entry step dependent on efficient translocation to the nucleus

Sinzger, C. et al

1. Gen. Virol., 81, 3021-3035 (2000)

  Marked interstrain differences in the endothelial cell (EC) tropism of human cytomegalovirus (HCMV) isolates have been described. This study aimed to define the step during the replicative cycle of the HCMV that determines this phenotype. The infection efficiency of various HCMV strains in EC versus fibroblasts was quantified by immunodetection of immediate early (IE), early and late viral antigens. Adsorption and penetration were analyzed by radiolabeled virus binding assays and competitive HCMV-DNA-PCR. The translocation of penetrated viral DNA to the nucleus of infected cells was quantified by competitive HCMV-DNA-PCR in pure nuclear fractions. The intracytoplasmic translocation of capsids that had penetrated was followed by immunostaining of virus particles on a single particle level; this was correlated with the initiation of viral gene expression by simultaneous immunostaining of viral IE antigens. The infectivity of nonendotheliotropic HCMV strains in EC was found to be 100-1000-fold lower when compared to endotheliotropic strains. The manifestation of this phenotype at the level of IE gene expression indicated the importance of initial replication events. Surprisingly, no interstrain differences were detected during virus entry. However, dramatic interstrain differences were found regarding the nuclear translocation of penetrated viral DNA. With nonendotheliotropic strains, the content of viral DNA in the cell nucleus was 100-1000-fold lower in EC when compared to endotheliotropic strains, thereby reflecting the strain differences in IE gene expression. Simultaneous staining of viral particles and viral IE antigen revealed that interstrain differences in the transport of penetrated capsids towards the nucleus of endothelial cells determine the EC tropism of HCMV.  

3.153           B cell antigen receptor signaling occurs outside lipid rafts in immature B cells

Sproul, T.W., Malapati, S., Kim, J. and Pierce, S.

1. Immunol., 165, 6020-6023 (2000)

  B cell Ag receptor (BCR) signaling changes dramatically during B cell development, resulting in activation in mature B cells and apoptosis, receptor editing, or anergy in immature B cells. BCR signaling in mature B cells was shown to be initiated by the translocation of the BCR into cholesterol- and sphingolipid-enriched membrane microdomains that include the Src family kinase Lyn and exclude the phosphatase CD45. Subsequently the BCR is rapidly internalized into the cell. Here we show that the BCR in the immature B cell line, WEHI-231, does not translocate into lipid rafts following cross-linking nor is the BCR rapidly internalized. The immature BCR initiates signaling from outside lipid rafts as evidenced by the immediate induction of an array of phosphoproteins and subsequent apoptosis. The failure of the BCR in immature B cells to enter lipid rafts may contribute to the dramatic differences in the outcome of signaling in mature and immature B cells.  

3.154           Different properties of two isoforms of annexin XIII in MDCK cells

Lecat, S. et al

1. Cell Sci., 113, 2607-2618 (2000)

  Annexins form a family of proteins that are widely expressed and known to bind membranes in the presence of calcium. Two isoforms of the annexin XIII subfamily are expressed in epithelia. We previously reported that annexin XIIIb is apically localized in MDCK cells and that it is involved in raft-mediated delivery of apical proteins. We have now analyzed the properties of annexin XIIIa, which differs from annexin XIIIb by a deletion of 41 amino acids in the amino-terminal domain, and is distributed both apically and basolaterally. Annexin XIIIa binding to membranes is independent of calcium but requires its myristoyl amino-terminal modification, as observed with annexin XIIIb. Our biochemical and functional data show that annexin XIIIa behaves differently in the apical and in the basolateral compartments. Whereas annexin XIIIa apically can associate with rafts independently of calcium, the basolateral pool requires calcium for this. Annexin XIIIa, like annexin XIIIb, stimulates apical transport of influenza virus hemagglutinin but, in contrast, only annexin XIIIa inhibits basolateral transport of vesicular stomatitis virus G protein. Our results suggest that annexin XIIIa and XIIIb have specific roles in epithelial cells, and because of their structural similarities, these isoforms offer interesting tools for unravelling the functions of annexins.  

3.155           New and re-emerging diseases: A dedication to Norman D. Levine

Docampo, R. Parasitology Today, 16(8), 316, (2000)   No abstract  

3.156           Differential effects of acyl-CoA binding protein on enzymatic and non-enzymatic thioacylation of protein and peptide substrates

Dunphy, J.T. et al Biochem. Biophys. Acta, 1485, 185-198 (2000)   Both enzymatic and autocatalytic mechanisms have been proposed to account for protein thioacylation (commonly known as palmitoylation). Acyl-CoA binding proteins (ACBP) strongly suppress non-enzymatic thioacylation of cysteinyl-containing peptides by long-chain acyl-CoAs. At physiological concentrations of ACBP, acyl-CoAs, and membrane lipids, the rate of spontaneous acylation is expected to be too slow to contribute significantly to thioacylation of signaling proteins in mammalian cells (Leventis et al., Biochemistry 36 (1997) 5546-5553). Here we characterized the effects of ACBP on enzymatic thioacylation. A protein S-acyltransferase activity previously characterized using G-protein a-subunits as a substrate (Dunphy et al., J. Biol. Chem., 271 (1996) 7154-7159), was capable of thioacylating short lipid-modified cysteinyl-containing peptides.The minimum requirements for substrate recognition were a free cysteins thiol adjacent to a hydrophobic lipid anchor, either myristate or farnesyl isoprenoid. PAT activity displayed specificity for the acyl donor, efficiently utilizing long-chain acyl-CoAs, but not free fatty acid or S-palmitoyl-N-acetylcysteamine. ACBP only modestly inhibited enzymatic thioacylation of a myristoylated peptide or G-protein a-subunits under conditions where non-enzymatic thioacylation was reduced to background. Thus, protein S-acyltransferase remains active in the presence of physiological concentrations of ACBP and acyl CoA in vitro and is likely to represent the predominant mechanism of thioacylation in vivo.  

3.157           Cytosolic Hsp70s are involved in the transport of aminopeptidase 1 from the cytoplasm into the vacuole

Satyanarayana, C., Schroder-Kohne, S., Craig, E.A., Schu, P.V. and Horst, M. FEBS Lett., 470, 232-238 (2000)   Eukaryotic 70 kDa heat shock proteins (Hsp70s) are localized in various cellular compartments and exhibit functions such as protein translocation across membranes, protein folding and assembly. Here we demonstrate that the constitutively expressed members of the yeast cytoplasmic Ssa subfamily, Ssa1/2p, are involved in the transport of the vacuolar hydrolase aminopeptidase 1 from the cytoplasm into the vacuole. The Ssap family members displayed overlapping functions in the transport of aminopeptidase 1. In SSAI and SSAII deletion mutants the precursor of aminopeptidase 1 accumulated in a dodecameric complex that is packaged in prevacuolar transport vesicles. Ssa1/2p was prominently localized to the vacuolar membrane, consistent with the role we propose for Ssa proteins in the fusion of transport vesicles with the vacuolar membrane.  

3.158           Seasonal variation in mussel Mytilus edulis digestive gland cytochrome P4501A- and 2E-immunoidentified protein levels and DNA strand breaks (Comet assay)

Shaw, J.P., Large, A.T., Chipman, J.K., Livingstone, D.R. and Peters, L.D. Marine Environmental Res., 50, 405-409 (2000)   Mytilus edulis digestive gland microsomes were prepared from indigenous populations sampled from a clean reference site (Pot Quin) and an urban-industrial contaminated site (Blackpool) in the UK. Samples were collected in March/April, May, August and December 1998. Western blot analysis was performed using polyclonal antibodies to fish CYP1A and rat CYP2E using partially purified M. edulis CYP as a positive control, to aid identification. CYP1A- and CYP2E-immunopositive protein levels showed different site-specific seasonal variation with higher levels of CYP2E determined in May (P< 0.05). At both sites, lower levels of CYP1A-immunopositive protein but not CYP2E-immunopositive protein were observed in the samples collected in December (P < 0.05). This correlated with lower levels of nuclear DNA damage (Comet assay expressed as per cent tail DNA) observed in December compared to August (P < 0.05).  

3.159           Tyrosine mutants are capable of prodrug activation in transfected nonmelanotic cells

Simonova, M., Wall, A., Weissleder, R. and Bogdanov, A. Cancer Res., 60, 6656-6662 (2000)   Tyrosinase has been suggested as a prodrug-converting enzyme for the treatment of melanoma. We hypothesized that tyrosinase expression in transfected nonmelanotic cells can be used in a gene therapy paradigm of prodrug activation. To verify our hypothesis, we used the following tyrosinase variants: (a) a full-length human tyrosinase clone (T); (b) a mutant lacking the COOH-terminal cytoplasmic domain (TDC); (c) a mutant lacking the COOH-terminal transmembrane and cytoplasmic domains (TDTC); and (d) a fusion with the eight COOH-terminal amino acids of lysosome-associated membrane protein-1 (TL). Expression of mutant and wild-type tyrosinases was induced by transfection in nontumorigenic human cells of epithelial origin (293HEK, MCF-10A adenoma, and NHDF-Ad human dermal fibroblasts) as well as in tumour cells (9L gliocarcinoma, MCF7 adenocarcinoma and HT-1080 fibrosarcoma). When compared with the wild-type tyrosinase transfectants, truncated mutant expression resulted in higher mRNA levels that paralleled higher enzyme activity of the truncated mutants. Two model tyrosinase prodrugs, hydroxyphenyl-propanol (HPP) and N-acetyl-4-S-cysteaminylphenol (NAcSCAP) inhibited proliferation and caused cell death of transfected cells in a dose-dependent manner. Effects of prodrug treatment were compared for tumorigenic cells and their nontumorigenic counterparts. Two truncated mutants (TDC and TDTC) showed low endogenous cytotoxicity and efficiently suppressed proliferation and induced cytotoxicity in transfected tumor cells in the presence of NAcSCAP. Overall, these results indicate that the developed tyrosinase mutants hold promise as prodrug activation systems for tumoral gene therapy.  

3.160           GFRa –mediated localization of RET to lipid rafts is required for effective downstream signaling, differentiation, and neuronal survival

Tansey, M.G., Baloh, R.H., Milbrandt, J. and Johnson, Jr., E.M. Neuron, 25, 611-623 (2000)   The GDNF family ligands (GFLs: GDNF, neurturin, persephin, and artemin) signal through RET and a glycosyl-phosphatidylinositol (GPI)-anchored coreceptor (GFRa1-a4) that binds ligand with high affinity and provides specificity. The importance of the GPI anchor is not fully understood; however, GPI-linked proteins cluster into lipid rafts, structures that may represent highly specialized signaling organelles. Here, we report that GPI-anchored GFRa 1 recruits RET to lipid rafts after GDNF stimulation and results in RET/Src association. Disruption of RET localization using either transmembrane-anchored or soluble GFRa1 results in RET phosphorylation, but GDNF-induced intracellular signaling events are markedly attenuated as are neuronal differentiation and survival responses. Therefore, proper membrane localization of RET via interaction with a raft-localized, GPI-linked coreceptor is of fundamental importance in GFL signaling.  

3.161           Amyloid precursor proteins inhibit heme oxygenase activity and augment neurotoxicity in Alzheimer’s disease

Takahashi, M. et al Neuron, 28, 461-473 (2000)   Amyloid precursor protein (APP) generates the b-amyloid peptide, postulated to participate in the neurotoxicity of Alzheimer’s disease. We report that APP and APLP bind to heme oxygenase (HO), an enzyme whose product, bilirubin, is antioxidant and neuroprotective. The binding of APP inhibits HO activity, and APP with mutations linked to the familial Alzheimer’s disease (FAD) provides substantially greater inhibition of HO activity than wild-type APP. Cortical cultures from transgenic mice expressing Swedish mutant APP have greatly reduced bilirubin levels, establishing that mutant APP inhibits HO activity in vivo. Oxidative neurotoxicity is markedly greater in cerebral cortical cultures from APP Swedish mutant transgenic mice than wild-type cultures. These findings indicate that augmented neurotoxicity caused by APP-HO interactions may contribute to neuronal cell death in Alzheimer’s disease.  

3.162           Mutational and biochemical analysis of plasma membrane targeting mediated by the farnesylated, polybasic carboxy terminus of K-ras4B

Roy, M-O., Leventis, R. and Silvius, J.R. Biochemistry, 39, 8298-8307 (2000)   Mutational analysis and in vitro assays of membrane association have been combined to investigate the mechanism of plasma membrane targeting mediated by the farnesylated, polybasic carboxy-terminal sequence of K-ras4B in mammalian cells. Fluorescence-microscopic localization of chimeric proteins linking the enhanced green fluorescent protein (EGFP) to the K-ras4B carboxy-terminal sequence, or to variant forms of this sequence, reveals that the normal structure of this targeting motif can be greatly altered without compromising plasma membrane-targeting activity so long as an overall strongly polybasic/amphiphilic character is retained. An EGFP/K-ras4B(171-188) chimeric protein was readily abstracted from isolated cell membranes by negatively charged lipid vesicles, and this abstraction was markedly enhanced by the anionic lipid-binding agent neomycin. Our results strongly favor a mechanism in which at the plasma membrane the carboxy-terminal sequence of K-ras4B associates not with a classical specific proteinaceous receptor but rather with nonspecific but highly anionic 'sites' formed at least in part by the membrane lipid bilayer. Our findings also suggest that the recently demonstrated prenylation-dependent trafficking of immature forms of K-ras4B through the endoplasmic reticulum [Choy et al. (1999) Cell 98, 69-80], while required for maturation of the protein, beyond this stage may not be essential to allow the ultimate delivery of the mature protein to the plasma membrane.  

3.163           An ever-expanding story of cyst formation

Gallagher, A.R., Obermüller, N., Cedzich, A., Gretz, N. and Witzgall, R. Cell Tissue Res., 300, 361-371 (2000)   Autosomal-dominant polycystic kidney disease represents one of the most common monogenetic human disorders. The cloning of the PKD1 and PKD2 genes, which are mutated in far more than 90% of the patients affected by this disease, has generated high hopes for a quick understanding of the pathogenesis of cyst formation. However, these expectations have not yet been fulfilled, since the function of both polycystin-1 and polycystin-2, the two proteins encoded by PKD1 and PKD2, still remains a puzzle. In this review, we will highlight some of the characteristics of polycystic kidney disease, briefly touch on polycystin-1, and then go on to describe recent results of experiments with polycystin-2, since the latter is the major focus of our work. We will discuss new evidence which suggests that autosomal-dominant polycystic kidney disease actually behaves recessively on a cellular level. Finally, a model will be presented that tries to explain the available data.  

3.164           Ultrastructural characterization of the delimiting membranes of isolated autophagosomes and amphisomes by freeze-fracture electron microscopy

Fengsrud, M., Erichsen, E.S., Berg, T.O., Raiborg, C. And Seglen, P.O. Eur. J. Cell Biol., 79, 871-882 (2000)   The delimiting membranes of isolated autophagosomes from rat liver had extremely few transmembrane proteins, as indicated by the paucity of intramembrane particles in freeze-fracture images (about 20 particles/um², whereas isolated lysosomes had about 2000 particles/um²). The autophagosomes also appeared to lack peripheral surface membrane proteins, since attempts to surface-biotinylate intact autophagosomes only yielded biotinylation of proteins from contaminating damaged mitochondria. All the membrane layers of multilamellar autophagosomes were equally particle-poor; the same was true of the autophagosome-forming, sequestering membrane complexes (phagophores). Isolated amphisomes (vacuoles formed by fusion between autophagosomes and endosomes) had more intramembrane particles than the autophagosomes (about 90 particles/um²), and freeze-fracture images of these organelles frequently showed particle-rich endosomes fusing with particle-poor or particle-free autophagosomes. The appearance of multiple particle clusters suggested that a single autophagic vacuole could undergo multiple fusions with endosomes. Only the outermost membrane of bi-or multilammelar autophagic vacuoles appeared to engage in such fusions.          

3.165           The phagacytosis-associated respiratory burst in human monocytes is associated with increased uptake of glutathione

Seres, T., Knickelbein, R.G., Warshaw, J.B. and Johnston, Jr, R.B.

1. Immunol., 165, 3333-3340 (2000)

  During the phagocytic respiratory burst, oxygen is converted to potent cytotoxic oxidants. Monocytes and macrophages are potentially long-lived, and we have hypothesized that protective mechanisms against oxidant stress are varied and fully expressed in these cells. We report here that the respiratory burst in monocytes is accompanied by an increase in the uptake of [³⁵S]glutathione ([³⁵S]GSH) after 20–30 min to levels up to 10-fold greater than those at baseline. By 30 min, 49% of the cell-associated radioactivity was in the cytosol, 41% was in membrane, and 10% was associated with the nuclear fraction. GSH uptake was inhibited by catalase, which removes hydrogen peroxide (H₂O₂), and micromolar H₂O₂ stimulated GSH uptake effectively in monocytes and also lymphocytes. Oxidation of GSH to glutathione disulfide with H₂O₂ and glutathione peroxidase prevented uptake. Acivicin, which inhibits GSH breakdown by -glutamyl transpeptidase (GGT), had no effect on the enhanced uptake seen during the respiratory burst. Uptake of cysteine or cystine, possible products of GGT activity, stayed the same or decreased during the respiratory burst. These results suggest that a GGT-independent mechanism is responsible for the enhanced GSH uptake seen during the respiratory burst. We describe here a sodium-independent, methionine-inhibitable transport system with a K_m (8.5 µM) for GSH approximating the plasma GSH concentration. These results suggest that monocytes have a specific GSH transporter that is triggered by the release of H₂O₂ during the respiratory burst and that induces the uptake of GSH into the cell. Such a mechanism has the potential to protect the phagocyte against oxidant damage.  

3.166           Expression and localization of rab escort protein isoforms in parotid acinar cells from rat

Chan, D., Lin, J. and Raffaniello, R.D.

1. Cell. Physiol., 185(3), 339-347 (2000)

  Rab proteins are geranylgeranylated on their carboxyl terminal cysteine motifs by geranylgeranyltransferase II (GGTase). Rab escort protein (REP) is required to present Rab proteins to GGTase. REP may remain bound to newly isoprenylated Rab proteins and present them to their target membrane. Other studies have shown that Rab proteins cycle between the membrane and cytosolic compartments and that cytosolic Rab proteins are complexed with rab-GDI. In the present study, we examined the expression and localization of REP isoforms in parotid acinar cells. Although both REP isoforms, REP-1 and REP-2, were detected in parotid cytosol, REP-2 was the predominant isoform. Subcellular fractionation revealed that approximately 42% of cellular REP-2 is membrane-associated. REP-2 was partially removed from parotid membranes with 1 M NaCl or Na2CO3, indicating that REP-2 is a peripheral membrane protein. Membrane-associated REP-2 did not colocalize with Rab3D on secretory granule membranes. However, density gradient centrifugation revealed that membrane-associated REP-2 and Rab3D colocalize on low- and high-density membrane fractions in parotid acinar cells. Isoproterenol, an agent which induces amylase release from parotid glands, caused a shift in both REP-2 and Rab3D to less dense membrane fractions. When acinar cell cytosol was fractionated by gel filtration chromatography, Rab3D eluted exclusively with REP, not rab-GDI. In contrast, Rab1B and Rab5 eluted with both REP and Rab-GDI. Colocalization of Rab3D and REP-2 on acinar cell membranes suggests that REP-2 plays a role in delivering Rab3D to parotid membranes and may regulate guanine nucleotide binding to membrane-associated Rab3D. In addition, unlike other Rab proteins, cytosolic Rab3D appears to associate exclusively with REP, not rab-GDI in parotid acinar cells.  

3.167           Mutation of conserved aspartates affect maturation of presenilin 1 and presenilin 2 complexes

Yu, G., Chen, F., Nishimura, M., Steiner, H., Tandon, A., Kawarai, T., Arawaka, S., Supala, A., Song, Y-Q., Rogaeva, E., Holmes, E., Zhang, D.M., Milman, P., Fraser, P., Haass, C. and  St. George-Hyslop, P. Acta Neurol Scand., Suppl. 176, 6-11 (2000)   Presenilin (PS1 and PS2) holoproteins are transiently incorporated into low molecular weight (MW) complexes. During subsequent incorporation into a higher MW complex, they undergo endoproteolysis to generate stable N- and C-terminal fragments (NTF/CTF). Mutation of either of two conserved aspartate residues in transmembrane domains inhibits both presenilin-endoproteolysis and the proteolytic processing of APP and Notch. We show that aspartate-mutant holoprotein presenilins are not incorporated into the high molecular weight, NTF/CTF-containing complexes. Aspartate-mutant presenilin holoproteins also preclude entry of endogenous wild-type PS1/PS2 into the high molecular weight complexes, but do not affect the incorporation of wild-type holoproteins into lower molecular weight holoprotein complexes. These data suggest that the loss-of-function aspartate-mutants cause altered PS complex maturation, and argue that the functional presenilin moieties are contained in the high molecular weight presenilin NTF/CTF-containing complexes.  

3.168           Degradation of lipid vesicles in the yeast vacuole requires function of Cvt17, a  putative lipase

Teter, S.A. et al

1. Biol. Chem., 276(1), 2083-2087 (2001)

  The vacuole/lysosome serves an essential role in allowing cellular components to be degraded and recycled under starvation conditions.  Vacuolar hydrolases are key proteins in this process.  In Saccharomyces cerevisiae, some resident vacuolar hydrolases are delivered by the cytoplasm to vacuole targeting (Cvt) pathway, which shares mechanistic features with autophagy.  Autophagy is a degradative pathway that is used to degrade and recycle cellular components under starvation conditions.  Both the Cvt pathway and autophagy employ double-membrane cytosolic vesicles to deliver cargo to the vacuole.  As a result, these pathways share a common terminal step, degradation of subvacuolar vesicles.  We have identified a protein, Cvt17, which is essential for this membrane lytic event.  Cvt17 is a membrane glycoprotein that contains a motif conserved in esterases and lipases.  The active-site serine of this motif is required for sub-vacuolar vesicle lysis. This is the first characterization of a putative lipase implicated in vacuolar function in yeast.  

3.169           An ephrinA-dependent signaling pathway controls integrin function and is linked to the tyrosine phosphorylation of a 120 kDa protein

Huai, J.and Drescher, U.

1. Biol. Chem., 276(9), 6689-6694 (2001)

  The Eph family of receptor tyrosine kinases and their ligands, the ephrins, have been implicated in the development of the retinotectal projection.  Here, glycosylphoaphatidylinositol-anchored A-ephrins are not only expressed in the tectum, but also on retinal axons, raising the possibility that they function in this context as receptors.  We now show that activation of ephrinA2 or ephrinA5 by one of their receptors, ephA3, results in a b1-integrin dependent increased adhesion of ephrinA-expressing cells to laminin.  In the search for an ephrinA-dependent signaling pathway controlling integrin activation, we identified a 120 kDa raft membrane protein which is tyrosine phosphorylated specifically after ephrinA activation.  Tyrosine phosphorylation of this protein is not seen after stimulating ephrinA2-expressing cells with basic fibroblast growth factor, epidermal growth factor, insulin growth factor or fetal calf serum containing a large set of different growth factors.  The role of p120 as a mediator of an ephrinA - integrin coupling is supported by the finding that inhibiting tyrosine phosphorylation of pl20 correlates with an abolishment of the b1-dependent cell adhesion.  

3.170           The synaptic vesicle protein, cysteine-string, is associated with the plasma membrane in 3T3-L1 adipocytes and interacts with syntaxin 4

Chamberlain, L.H. et al

1. Cell Sci., 114(2), 445-455 (2001)

  Adipocytes and muscle cells play a major role in blood glucose homeostasis. This is dependent upon the expression of Glut4, an insulin-responsive facilitative glucose transporter. Glut4 is localized to specialized intracellular vesicles that fuse with the plasma membrane in response to insulin stimulation. The insulin-induced translocation of Glut4 to the cell surface is essential for the maintenance of optimal blood glucose levels, and defects in this system are associated with insulin resistance and type II diabetes. Therefore, a major focus of recent research has been to identify and characterize proteins that regulate Glut4 translocation. Cysteine-string protein (Csp) is a secretory vesicle protein that functions in presynaptic neurotransmission and also in regulated exocytosis from non-neuronal cells. We show that Csp1 is expressed in 3T3-L1 adipocytes and that cellular levels of this protein are increased following cell differentiation. Combined fractionation and immunofluorescence analyses reveal that Csp1 is not a component of intracellular Glut4-storage vesicles (GSVs), but is associated with the adipocytes plasma membrane. This association is stable, and not affected by either insulin stimulation or chemical depalmitoylation of Csp1. We also demonstrate that Csp1 interacts with the t-SNARE syntaxin 4. As syntaxin 4 is an important mediator of insulin-stimulated GSV fusion with the plasma membrane, this suggests that Csp1 may play a regulatory role in this process. Syntaxin 4 interacts specifically with Csp1, but not with Csp2. In contrast, syntaxin 1A binds to both Csp isoforms, and actually exhibits a higher affinity for the Csp2 protein. The results described raise a number of interesting questions concerning the intracellular targeting of Csp in different cell types, and suggest that the composition and synthesis of GSVs may be different from synaptic and other secretory vesicles. In addition, the interaction of Cps1 with syntaxin 4 suggests that this Csp isoform may play a role in insulin-stimulated fusion of GSVs with plasma membrane.  

3.171           Pro-caspase-8 is pre-dominantly localized in mitochondria and released into cytoplasm upon apoptotic stimulation

Qin, Z-H. et al

1. Biol. Chem., 276(11), 8079-8086 (2001)

  The recruitment and cleavage of pro-caspase-8 to produce the active form of caspase-8 is a critical biochemical event in death receptor-mediated apoptosis. However, the source of pro-caspase-8 available for activation by apoptotic triggers is unknown. In human fibroblasts and mouse clonal striatal cells, confocal microscopy revealed that pro-caspase-8 immunofluorescence was co-localized with cytochrome c in mitochondria and was also distributed diffusely in some nuclei. Biochemical analysis of subcellular fractions indicated that pro-caspase-8 was enriched in mitochondria and in the nucleus. Pro-caspase-8 was found in the intermembrane space, inner membrane and matrix of mitochondria after limited digestion of mitochondrial fractions and this distribution was confirmed by immunogold electron microscopy. Pro-caspase-8 and cytochrome c were released from isolated mitochondria that were treated with an inhibitor of the ADP/ATP carrier atractyloside (Atr), which opens the mitochondrial permeability transition pore. Release was blocked by the mitochondrial permeability transition pore inhibitor cyclosporin A (CsA). After clonal striatal cells were exposed for 6 hr to the apoptotic inducer, tumor necrosis factor-a (TNF-a), mitochondria immunoreactive for cytochrome c and pro-caspase-8 became clustered at perinuclear sites. Pro-caspase-8 and cytochrome c levels decreased in mitochondrial fractions and increased, along with pro-caspase-8a cleavage products, in the cytoplasm of the TNF-alpha treated striatal cells. CsA blocked the TNF-a-induced release of pro-caspase 8, but not cytochrome c. Internucleosomal DNA fragmentation started at 6 hr and peaked at 12 hr after TNF-a treatment. These results suggest that pro-caaspase-8 is predominately localized in mitochondria and is released upon apoptotic stimulation through a CsA-sensitive mechanism.  

3.172           Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth  factor receptor

Chin, L-S., Raynor, M.C., Wei, X., Chen, H-Q. and  Li, L.

1. Biol. Chem., 276(10), 7069-7078 (2001)

  Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a mammalian homologue of yeast vacuolar protein sorting (Vps) protein Vps27p, however the role of Hrs in lysosomal trafficking is unclear. Here, we report that Hrs interacts with sorting nexin 1 (SNX1), a recently identified mammalian homologue of yeast Vps5p that recognize the lysosomal targeting code of epidermal growth factor receptor (EGFR) and participates in lysosomal trafficking of the receptor. Biochemical analyses demonstrate that Hrs and SNX1 are ubiquitous proteins that exist in both cytosolic and membrane-associated pools, and that the association of Hrs and SNX occurs on cellular membranes but not in the cytosol. Furthermore, endogenous SNX1 and Hrs form a ~550-kDa complex that excludes EGFR. Immunofluorescence and subcellular fractionation studies show that Hrs and SNX1 colocalize on early endosomes. By using depletion analysis, we have mapped the binding domains of Hrs and SNX1 that mediate their association. Overexpression of Hrs or its SNX1-binding domain inhibits ligand-induced degradation of EGFR, but does not affect either constitutive or ligand-induced receptor-mediated endocytosis. These results suggest that Hrs may regulate lysosomal trafficking through its interaction with SNX1.  

3.173           Localization and insulin-regulated relocation of phosphoinositide 5-kinase PIKfyve in 3T3-L1 adipocytes

Shisheva, A., Rusin, B., Ikonomov, O.C., DeMarco, C. and Sbrissa, D.

1. Biol. Chem., 276(15), 11859-11869 (2001)

  The mammalian phosphoinositide kinase PIKfyve catalyzes the synthesis of phosphatidylinositol 5-P and phosphatidylinositol 3,5-P₂, thought essential in cellular functions, including membrane trafficking. To discern the intracellular loci of PIKfyve product’s formation, we have examined the localization of PIKfyve protein vs. enzymatic activity, and a possible acutely regulated redistribution in 3T3-L1 adipocytes. Subcellular fractions of resting cells that were positive for immunoreactive PIKfyve, such as cytosol (~76%), internal structures (LDM, composed of recycling endosomes, GLUT4 storage compartment, Golgi and cytoskeletal elements) (~20%), and plasma membrane (~4%), expressed enzymatically active PIKfyve. While presence of FYVE finger in PIKfyve predicts early endosome targeting, density gradient sedimentation, immunoadsorption and fluorescence microscopy analyses segregated the LDM-associated PIKfyve from the membranes of the recycling endosomes and GLUT4. PIKfyve fluorescence staining largely coincided with trans-Golgi network/multivesicular body markers, indicating PIKfyve’s role in the late endocytic/biosynthetic pathways. A subfraction of particulate PIKfyve resisted nonionic detergent treatment, implying association with cytoskeletal structures, previously found positive for key members of the insulin signaling cascade. Upon acute stimulation of 3T3-L1 adipocytes with insulin or pervanadate, a portion of the cytosolic PIKfyve was recruited onto LDM, which was coupled with a commensurate increase of PIKfyve lipid kinase activity and an electrophoretic mobility shift. We suggest the recruited PIKfyve specifies the site and timing of phosphoinositide signals that are relevant to the acute insulin action.  

3.174           Active (9.6S) and inactive (21S) oligomers of NHE3 in distinct microdomains of the  renal brush border

Biemesderfer, D., DeGray, B. and Aronson, P.S.

1. Biol. Chem., 276(13), 10161-10167 (2001)

  We have previously shown that Na⁺-H⁺ exchanger isoform NHE3 exists as both 9.6 S and 21 S (megalin-associated) oligomers in the renal brush border (Biesmesderfer et al., JBC 274, 17518, 1999). To characterize the oligomeric forms of the renal brush border Na⁺-H⁺ exchanger in more detail, we performed membrane fractionation studies. We found that similar amounts of NHE3 were present in microvilli and a non-microvillar membrane domain of high density (dense vesicles). Horseradish peroxidase-labeled endosomes were not prevalent in the dense membrane fraction. However, megalin, which localizes primarily to the intermicrovillar microdomain of the brush border, was enriched in the dense vesicles, implicating this microdomain as the likely source of these membranes. Immunolocalization of NHE3 confirmed that a major fraction of the transporter co-localized with megalin in the intermicrovillar region of the brush border. Immunoprecipitation studies demonstrated that in microvilli the majority of NHE3 was not bound to megalin, while in the dense vesicles most of the NHE3 co-precipitated with megalin. Moreover, sucrose velocity gradient centrifugation experiments revealed that most NHE3 in microvilli sedimented with an S-value of 9.6 while the S-value of NHE3 in dense vesicles was 21. Finally, we examined the functional state of NHE3 in both membrane fractions. As assayed by changes in acridine orange fluorescence, imposing an outwardly directed Na⁺ gradient caused generation of an inside-acid pH gradient in the microvilli, indicating Na⁺-H⁺ exchange activity, but not in the dense vesicles. Taken together, these data demonstrate that renal brush border NHE3 exists in two oligomeric states; a 9.6 S active form present in microvilli, and a 21 S, megalin-associated, inactive form in the intermicrovillar microdomain of the apical plasma membrane. Thus, regulation of renal brush border Na⁺-H⁺ exchange activity may be mediated by shifting the distribution between these forms of NHE3.  

3.175           Membrane lipid rafts are necessary for the maintenance of the a7 nicotinic  acetylcholine receptor in somatic spines of ciliary neurons

Bruses, J.L., Chauvet, N. and Rutishauser, U.

1. Neurosci., 21(2), 504-512 (2001)

  Calcium-permeable neurotransmitter receptors are concentrated into structurally and biochemically isolated cellular compartments to localize calcium-mediated events during neurotransmission.  The cytoplasmic membrane contains lipid microdomains called lipid rafts, which can gather into microscopically visible clusters, and thus the association of a particular protein with lipid rafts can result in its redistribution on the cell surface.  The present study asks whether lipid rafts participate in the formation and maintenance of the calcium-permeable a7-subunit nicotinic acetylcholine receptor (a7nAChR) clusters found in somatic spines of ciliary neurons.  Lipid rafts and a7nAChR become progressively colocalized within somatic spines during synaptogenesis.  To determine whether these rafts are required for the maintenance of a7nAChR aggregates, cholesterol was extracted from dissociated ciliary neurons by treatment with methyl-b-cyclodextrin.  This treatment caused the dispersion of lipid rafts and the redistribution of a7nAChR into small clusters over the cell surface, suggesting that the integrity of lipid rafts is required to maintain the receptor clustering.  However, lipid raft dispersion also caused the depolymerization of the F-actin cytoskeleton, which can also tether the receptor at specific sites.  To assess whether interaction between rafts and a7nAChR is independent of F-actin filaments, the lipid raft patches were stabilized with a combination of the cholera toxin B subunit (CTX), which specifically binds to the raft component ganglioside GM1, and an antibody against CTX.  The stabilized rafts were then treated with latrunculin-A to depolymerize F-actin.  Under these conditions, large patches of CTX persisted and were colocalized with a7nAChR, indicating that the aggregates of receptors can be maintained independently of the underlying F-actin cytoskeleton.  Moreover, it was found that the a7nAChR is resistant to detergent extraction at 4⁰C and floats with the caveolin-containing lipid-rich fraction during density gradient centrifugation, properties that are consistent with a direct association between the receptor and the membrane microdomains.  

3.176           Mutations in sialidosis impair sialidase binding to the lysosomal multienzyme complex

Lukong, K.E. et al

1. Biol. Chem., 276, 17286-17290 (2001)

  Sialidosis is an autosomal recessive disease caused by the genetic deficiency of lysosomal sialidase, which catalyzes the catabolism of sialoglycoconjugates.  The disease is associated with progressive impaired vision, macular cherry-red spots, and myoclonus (sialidosis type 1) or with skeletal dysplasia, Hurler-like phenotype, dysostosis multiplex, mental retardation, and hepatosplenomegaly (sialidosis type II).  We have analyzed the effect of missense mutations, G68V, Sl82G, G227R, F26OY, L270F, A298V, G328S and L363P, identified in the sialidosis type I and sialidosis type II patients on the activity, stability and intracellular distribution of the sialidase.  We found that 3 mutations, F260Y, L270F and A298V which are clustered in the same region on the surface of sialidase molecule dramatically reduce the enzyme activity and cause a rapid intralysosomal degradation of the expressed protein.  We suggested that this region might be involved in the sialidase binding with lysosomal cathepsin A and/or b-galactosidase in the multienzyme lysosomal complex required for the expression of sialidase activity.  Transgenic expression of mutants followed by density gradient centrifugation of cellular extracts confirmed this hypothesis and showed that sialidase deficiency in some sialidosis patients results from disruption of the lysosomal multienzyme complex.  

3.177           Mammalian sprouty-1 and –2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells

Impagnatiello, M-A. et al

1. Cell Biol., 152(5), 1087-1098 (2001)

  Growth factor-induced signaling by receptor tyrosine-kinases (RTKs) plays a central role in embryonic development and in pathogenesis and, hence, is tightly controlled by several regulatory proteins. Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered. Subse-quently, four mammalian Sprouty homologues (Spry 1-4) have been identified. Here, we report for funct-ional characterization of two of them, Spry-1 and –2, in endothelial cells. Overexpressed Spry-1 and -2 inhibit fibroblast growth factor- and vascular endothelial growth factor-induced proliferation and different-iation by repressing pathways leading to p42/44 mitogen activating protein (MAP) kinase activation. In contrast, although epidermal growth factor-induced proliferation of endothelial cells were also inhibited by Spry-1 and –2, activation of p42/44 MAP kinase was not affected. Biochemical and immunofluorescence analysis of endogenous and overexpressed Spry-1 and –2 reveal that both Spry-1 and –2 are anchored to membranes by palmitoylation and associate with caveolin-1 in perinuclear and vesicular structures. They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane. The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in an RTK-specific fashion.  

3.178           Cytosolic phospholipase A₂-a associates with plasma membrane, endoplasmic reticulum and nuclear membrane in glomerular epithelial cells

Liu, J., Takano, T., Papillon, J., Khadir, A. and Cybulsky, A.V. Biochem. J., 353, 79-90 (2001)   Eicosanoids mediate complement-dependent glolmerular epi­thelial injury in experimental membranous nephropathy. The release of arachidonic acid from phospholipids by cytosolic phospholipase A₂ (cPLA₂) is the rate-limiting step in eicosanoid synthesis. The present study examines the association of cPLA₂ with membranes of organelles. Glomerular epithelial cells were disrupted by homogenization in Ca²⁺ -free buffer; organelles were separated by gradient centrifugation. The distribution of cPLA₂, and organelles was analyzed by immunoblotting with antibodies against cPLA₂ and organelle markers, or by enzyme assay. In cells incubated with or without the Ca²⁺ ionophore ionomycin plus PMA, cPLA₂ co-localized with plasma mem­brane, endoplasmic reticulum and nuclei, but not with mito­chondria or Golgi. A greater amount of cPLA₂ was associa­ted with membranes in stimulated cells, but membrane-associated cPLA₂ was readily detectable under resting conditions. The pattern of association of cPLA₂ with membrane in cells treated with antibody and complement was similar to that in cells stimulated with ionomycin plus PMA; however, complement did not enhance the membrane association of cPLA₂ protein. To determine the functional role of membrane association of cPLA₂, phospholipids were labeled with [³H]arachidonic acid. Cells were then incubated with or without antibody and complement and were fractionated. Complement induced a loss of radio­activity from the plasma membrane, endoplasmic reticulum and nuclei, but not from the mitochondrial fraction. Thus the release of arachidonic acid by cPLA₂ is due to the hydrolysis of phospholipids at multiple subcellular membrane sites, including the endoplasmic reticulum, plasma membrane and nucleus.  

3.179           Raft-partitioning of the ubiquitin ligases CbI and Nedd4 upon IgE-triggered cell signaling

Lafont, F. and Simons, K. Proc. Natl. Acad. Sci., USA., 98; 3180-3184 (2001).   The high affinity receptor for IgE, FceRI on mast calls and basophils plays an essential role in immunological defense. Upon multivalent antigen binding,  FceRI becomes phoshorylated by the protein-­tyrosine kinase Lyn, as a result of receptor clustering in lipid rafts. FceRI has been shown to be ubiquitinated. Ubiquitination can lead to degradation by proteasomes, but it can also act as a sorting signal to internalize proteins destined to the endosomal/lysosomal pathway. We have analyzed whether FceRI ubiquitination takes place within rafts. We report biochemical and imaging evidence in rat basolukemia cells for the presence of ubiquitinated FceRI in clustered rafts upon receptor activation. Moreover, we demon­strated that the ubiquitin ligases CbI and Nedd4 co-localize with FceRI patches and showed that both ligases become assoc-iated with lipid rafts after activation of IgE signaling. Because CbI is known to interact with the FceRI signaling complex, ubiquitination is likely to be an important parameter regulating IgE-triggered signaling occurring in rafts.  

3.180           Cell-specific targeting of caveolin-1 to caveolae, secretory vesicles, cytoplasm or mitochondria

Li, W-P et al

1. Cell Sci., 114(7), 1397-1408 (2001)

  In commonly used tissue culture cells, caveolin-1 is embedded in caveolae membranes. It appears to reach this location after being cotranslationally inserted into ER membranes, processed in the Golgi and shipped to the cell surface. We now report that caveolae are not the preferred location for caveolin-1 in all cell types. Skeletal muscle cells and keratinocytes target caveolin-1 to the cytosol while in exocrine and endocrine cells it accumulates in the secretory pathway. We also found that airway epithelial cells accumulate cevaolin-1 in modified mitochondria. The cytosolic and the secreted forms appear to be incorporated into a soluble, lipid complex. We conclude that caveolin-1 can be targeted to a variety of intracellular destinations, which suggests a novel mechanism for the intracellular traffic of this protein.  

3.181           Glycolipid antigen processing for presentation by CD1d molecules

Prigozy, T.I. et al Science, 291, 664-667 (2001)   The requirement for processing glycolipid antigens in T cell recognition was examined with mouse CD1d-mediated responses to glycosphingolipids (GSLs). Although some disaccharide GSL antigens can be recognized without processing, the responses to three other antigens, including the disaccharide GSL Gal(a1®2)GalCer (Gal, galactose; GalCer, galactosylceramide), required removal of the terminal sugars to permit interaction with the T cell receptor. A lysosomal enzyme, a-galactosidase A, was responsible for the processing of Gal(a1®2)GalCer to generate the antigenic monosaccharide epitope. These data demonstrate a carbohydrate antigen processing system analogous to that used for peptides and an ability of T cells to recognize processed fragments of complex glycolipids.  

3.182           Segregation of heterotrimeric G proteins in cell surface microdomains

Oh, P. and Schnitzer, J.E. Mol. Biol. Cell, 12, 685-698 (2001)   Select lipid-anchored proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins and nonreceptor tyrosine kinase may preferentially partition into sphingomyelin-rich and cholesterol-rich plasmalemmal microdomains, thereby acquiring resistance to detergent extraction. Two such domains, caveolae and lipid rafts, are morphologically and biochemically distinct, contain many signaling molecules, and may function in compartmentalizing cell surface signaling. Subfractionation and confocal immunofluorescence microscopy reveal that, in lung tissue and in cultured endothelial and epithelial cells, heterotrimeric G proteins (G_i, G_q, G_s, and G_bg) target discrete cell surface microdomains. G_q specifically concentrates in caveolae, whereas G_i and G_s concentrate much more in lipid rafts marked by GPI-anchored proteins (5’ nucleotidase and folate receptor). G_q, apparently without G_bg subunits, stably associates with plasmalemmal and cytosolic caveolin. G_i and G_s interact with G_bg subunits but not caveolin.G_i and G_s unlike G_q, readily move out of caveolae. Thus, caveolin may function as a scaffold to trap, concentrate, and stabilize G_q preferentially within caveolae over lipid rafts. In N2a cells lacking caveolae and caveolin, G_q, G_i, and G_s all concentrate in lipid rafts as a complex with G_bg. Without effective physiological interaction with caveolin, G proteins tend by default to segregate in lipid rafts. The ramifications of the segregated microdomain distribution and the G_q-caveolin complex without G_bg for trafficking, signaling, and mechanotransduction are discussed.  

3.183           Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane

Lusa, S., et al

1. Cell Sci., 114(10), 1893-1900 (2001)

  In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signaling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [³H]cholesterol released from [³H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.  

3.184           The Leishmania ATP-binding cassette protein PGPA is an intracellular metal-thiol transporter ATPase

Legare, D., et al

1. Biol. Chem., 276(28), 26301-26307 (2001)

  The Leishmania ATP-binding cassette (ABC) transporter PGPA is involved in metal resistance (arsenicals and antimony), although the exact mechanism by which PGPA confers resistance to antimony, the first line drug against Leishmania, is unknown. The results of co-transfection experiments, transport assays, and the use of inhibitors suggest that PGPA recognizes metals conjugated to gluthathione or trypanothione, a glutathione-spermidine conjugate present in Leishmania. The HA epitope tag of the influenza hemagglutinin as well as the green fluorescent protein (GFP) were fused at the COOH-terminus of PGPA. Immunofluorescence, confocal and electron microscopy studies of the fully functional tagged molecules clearly indicated that PGPA is localized in membranes that are close to the flagellar pocket, the site of endocytosis and exocytosis in this parasite. Subcellular fractionation of Leishmania tarentolae PG-PAHA transfectants was performed to characterize further this ABC transporter. The basal PGPA ATPase activity was determined to be 115 nmoles/mg/min. Transport experiments using radioactive arsenite-glutathione conjugates clearly showed that PGPA recognizes and actively transport thiol-metal conjugates. Overall, the results are consistent with PGPA being an intracellular ABC transporter that confers arsenite and antimonite resistance by sequestration of the metal-thiol conjugates.  

3.185           Cvt9/Gsa9 functions in sequestering selective cytosolic cargo destined for the vacuole

Kim, J. et al

1. Cell Biol., 153(2), 381-396 (2001)

  Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.   We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.  

3.186           Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the a-secretase ADAM 10

Kojro, E., Gimpl, G., Lammich, S., Marz, W. and Fahrenholz, F. Proc. Natl. Acad. Sci., 98(10), 5815-5820 (2001)   Biochemical, epidemiological, and genetic findings demonstrate a link between cholesterol levels, processing of the amyloid precursor protein (APP), and Alzheimer’s disease. In the present report, we identify the a-secretase ADAM 10 (a disintegrin and metalloprotease) as a major target of the cholesterol effects on APP metabolism. Treatment of various peripheral and neutral cell lines with either the cholesterol-extracting agent methyl-b-cyclodextrin or the hydroxymethyl glutaryl-CoA reductase inhibitor lovastin resulted in a drastic increase of secreted a-secretase cleaved soluble APP. This strong stimulatory effect was in the range obtained with phorbol esters and was further increased in cells overexpressing ADAM 10. In cells overexpressing APP, the increase of a-secretase activity resulted in a decreased secretion of Ab peptides. Several mechanisms were elucidated as being the basis of enhanced a-secretase activity: increased membrane fluidity and impaired internalization of APP were responsible for the effect observed with methyl-b-cyclodextrin; treatment with lovastatin resulted in higher expression of the a-secretase ADAM 10. Our results demonstrate that cholesterol reduction promotes the nonamyloidogenic a-secretase pathway and the formation of neuroprotective a-secretase cleaved soluble APP by several mechanisms and suggest approaches to prevention of or therapy for Alzheimer’s disease.  

3.187           Neuregulin-1 proteins in rat brain and transfected cells are localized to lipid rafts

Frenzel, K.E. and Falls, D.L.

1. Neurochem., 77, 1-12 (2001)

  Neuregulin–1 proteins and their receptors, which are members of the ErbB subfamily of receptor tyrosine kinases, play essential roles in the development of the nervous system and heart. Most neuregulin-1 isoforms are synthesized as transmembrane proproteins that are proteolytically processed to yield an N-terminal fragment containing the bioactive EGF-like domain. In this study we investigated whether neuregulins are found in lipid rafts, membrane microdomains hypothesized to have important roles in signal transduction, protein trafficking, and proteolytic processing. We found that 45% of a 140-kDa neuregulin protein in rat brain synatosomal plasma membrane fractions was insoluble in 1% Triton X-100. Flotation gradient analysis demonstrated the presence of the brain 140-kDa neuregulin protein in low-density fractions enriched in PSD-95, a known lipid raft protein. In transfected cells expressing the neuregulin I-b1a or the III-b1a isoform, most of the neuregulin proprotein was insoluble in 1% Triton X-100, and neuregulin proproteins and C-terminal fragments were detected in lipid raft fractions. In contrast, the III-b1a N-terminal fragment was detected only in the detergent-soluble fraction. These results suggest that localization of neuregulins to lipid rafts may play a role in neuregulin signaling within the nervous system.  

3.188           Yeast Rab GTPase-activating protein Gyp1p localizes to the Golgi apparatus and is a negative regulator of Ypt1p

Du, L-L and Novick, P. Mol. Biol. Cell, 12, 1215-1226 (2001)   A family of related proteins in yeast Saccharomyces cerevisiae is known to have in vitro GTPase-activating protein activity on the Rab GTPases. However, their in vivo function remains obscure. One of them, Gyp1p, acts on Sec4p, Ypt1p, Ypt7p and Ypt51p in vitro. Here, we present data to reveal its in vivo substrate and the role that it plays in the function of the Rab GTPase. Red fluorescent protein-tagged Gyp1p is concentrated on cytoplasmic punctate structures that largely colocalize with a cis-Golgi marker. Subcellular fractionation of a yeast lysate confirmed that Gyp1p is peripherally associated with membranes and that it cofractionates with Golgi markers. This localization suggests that Gyp1p may only act on Rab GTPases on the Golgi. A gyp1D strain displays a growth defect on synthetic medium at 37°C. Overexpression of Ypt1p, but not other Rab GTPases, strongly inhibits the growth of gyp1D cells. Conversely, a partial loss-of-function allele of YPT1, ypt1-2, can suppress the growth defect of gyp1D cells. Furthermore, deletion of GYP1 can partially suppress growth defects associated with mutants in subunits of transport protein particle complex, a complex that catalyzes nucleotide exchange on Ypt1p. These results establish that Gyp1p functions on the Golgi as a negative regulator of Ypt1p.  

3.189           SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis

Lang, T. et al EMBO J., 20(9), 2202-2213 (2001)   During exocytosis, SNARE proteins of secretory vesicles interact with the corresponding SNARE proteins in the plasmalemma to initiate the fusion reaction. However, it is unknown whether SNAREs are uniformly distributed in the membrane or whether specialized fusion sites exist. Here we report that in the plasmalemma, syntaxins are concentrated in 200 nm large, cholesterol-dependent clusters at which secretory vesicles preferentially dock and fuse. The syntaxin clusters are distinct from cholesterol-dependent membrane rafts since they are Triton X-100 soluble and do not co-patch with raft markers. Synaptosomal-associated protein (SNAP)-25 is also clustered in spots, which partially overlap with syntaxin. Cholesterol depletion causes dispersion of these clusters, which is associated with a strong reduction in the rate of secretion, whereas the characteristics of individual exocytic events are unchanged. This suggests that high local concentrations of SNAREs are required for efficient fusion.  

3.190           WFS1 (Wolfram syndrome 1) gene product: predominant subcellular localization to endoplasmic reticulum in cultured cells and neuronal expression in rat brain

Takeda, K. et al Hum. Mol. Genet., 10(5), 477-484 (2001)   Wolfram (DIDMOAD) syndrome is an autosomal recessive neurodegenerative disorder accompanied by insulin-dependent diabetes mellitus and progressive optic atrophy. Recent positional cloning led to identification of the WFS1 (Wolfram syndrome 1) gene, a member of a novel gene family of unknown function. In this study, we generated a specific antibody against the C-terminus of the WFS1 protein and investigated its subcellular localization in cultured cells. We also studied its distribution in the rat brain. Biochemical studies indicated the WFS1 protein to be an integral, endoglycosidase H-sensitive membrane glycoprotein that localizes primarily in the endoplasmic reticulum (ER). Consistent with this, immunofluorescence cell staining of overexpressed WFS1 showed a characteristic reticular pattern over the cytoplasm and overlapped with the ER marker staining. No co-localization of WFS1 with mitochondria argues against an earlier clinical hypothesis that Wolfram syndrome is a mitochondria-mediated disorder. In the rat brain, at both the protein and mRNA level, WFS1 was found to be present predominantly in selected neurons in the hippocampus CA1, amygdaloid areas, olfactory tubercle and superficial layer of the allocortex. These expression sites, i.e. components of the limbic system or structures closely associated with this system, may be involved in the psychiatric, behavioral and emotional abnormalities characteristic of this syndrome. ER localization of WFS1 suggests that this protein plays an as yet undefined role in membrane trafficking, protein processing and/or regulation of ER calcium homeostasis. These studies represent a first step toward the characterization of WFS1 protein, which presumably functions to maintain certain populations of neuronal and endocrine cells.  

3.191           Regulation of tumor angiogenesis by oxygen-regulated protein 150, an inducible endoplasmic reticulum chaperone

Ozawa, K. et al Cancer Res., 61, 4206-4213 (2001)   Expression of angiogenic factors such as vascular endothelial growth factor (VEGF) under conditions of cell stress involves both transcriptional and translational events, as well as an important role for inducible endoplasmic reticulum (ER) chaperones. Coexpression of VEGF and 150-kDa oxygen-regulated protein (ORP), a novel ER chaperone, in human glioblastoma suggested a link between angiogenesis and ORP150. C6 glioma cells stably transfected with ORP150 antisense displayed selectively reduced ORP150 expression. Tumors raised after inoculation of immuno-compromised mice with ORP150 antisense C6 glioma transfect-ants demonstrated an initial phase of growth comparable to wild-type C6 glioma cells which was followed by marked regression within 8 days. Decreased density of platelet/endothelial cell adhesion molecule 1-positive structures within the tumor bed was consistent with reduced angiogenesis in C6 gliomas expressing ORP150 antisense, compared with tumors derived from C6 cells overexpressing ORP150 sense or vector controls. In vitro, inhibition of ORP150 expression decreased release of VEGF into culture supernatants; in ORP150 antisense transfectants, VEGF accumulated intracellularly within the ER. These findings demons-trate a critical role for the inducible ER chaperone ORP150 in tumor-mediated angiogenesis via processing of VEGF, and thus, highlight a new facet of mechanism amenable to therapeutic manipulation in tumors.  

3.192           Subcellular compartment and molecular subdomain of b-amyloid precursor protein relevant to the Ab42-promoting effects of Alzheimer mutant presenilin 2

Iwata, H., Tomita, T., Maruyama, H. And Iwatsubo, T.

1. Biol. Chem., 276(24), 21678-21685 (2001)

  Increased production of amyloidb peptides ending at position 42 (Ab42) is one of the pathogenic phenotypes caused by mutant forms of presenilins (PS) linked to familial Alzheimer’s disease. To identify the subcellular compartment(s) in which familial Alzheimer’s disease mutant PS2 (mt PS2) affects the g-cleavage of bAPP to increase Ab42, we co-expressed the C-terminal 99-amino acid fragment of bAPP (C100) tagged with sorting signals to the endoplasmic reticulum (C100/ER) or to the trans-Golgi network (C100/TGN) together with mt PS2 in N2a cells. C100/TGN co-transfected with mt PS2 increased levels or ratios of intracellular as well as secreted Ab42 at similar levels to those with C100 without signals (C100/WT), whereas C100/ER yielded a negligible level of Ab, which was not affected by co-transfection of mt PS2. To identify the molecular subdomain of bAPP required for the effects of mt PS2, we next co-expressed C100 variously truncated at the C-terminal cytoplasmic domain together with mt PS2. All types of C-terminally truncated C100 variants including that lacking the entire cytoplasmic domain yielded the secreted form of Ab at levels comparable with those from C100/WT, and co-transfection of mt PS2 increased the secretion of Ab42. These results suggest that (i) late intracellular compartments including TGN are the major sites in which Ab42 is produced and up-regulated by mt PS2 and that (ii) the anterior half of C100 lacking the entire cytoplasmic domain is sufficient for the overproduction of Ab42 caused by mt PS2.  

3.193           Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole

Hakansson, S., Charron, A.J. and Sibley, L.D. The EMBO J., 20(12), 3132-3144 (2001)   Rapid discharge of secretory organelles called rhoptries is tightly coupled with host cell entry by the protozoan parasite Toxoplasma gondii. Rhoptry contents were deposited in clusters of vesicles within the host cell cytosol and within the parasitophorous vacuole. To examine the fate of these rhoptry-derived secretory vesicles, we utilized cytohalasin D to prevent invasion, leading to accumulation of protein-rich vesicles in the host cell cytosol. These vesicles lack an internal parasite and are hence termed evacuoles. Like the mature parasite-containing vacuole, evacuoles became intimately associated with host cell mitochondria and endoplasmic reticulum, while remaining completely resistant to fusion with host cell endo-somes and lysosomes. In contrast, evacuoles were recruited to pre-existing, parasite-containing vacuoles and were capable of fusing and delivering their contents to these compartments. Our findings indicate that a two-step process involving direct rhoptry secretion into the host cell cytoplasm followed by incorporation into the vacuole generates the parasitophorous vacuole occupied by Toxoplasma. The characteristic properties of the mature vacuole are likely to be determined by this early delivery of rhoptry components.  

3.194           Rapid changes in polyphosphate content within acidocalcisomes in response to cell growth, differentiation and environmental stress in Trypanosoma cruzi

Ruiz, F.A., Rodrigues, C.O. and Docampo, R.

1. Biol. Chem., 276(28), 26114-26121 (2001)

  Inorganic polyphosphate (polyP) has been identified and measured in different stages of Trypanosoma cruzi. Millimolar levels (in terms of P_i residues) in chains of less than 50 residues long, and micromolar levels in chains of about 700-800 residues long, were found in different stages of T. cruzi. Analysis of purified T. cruzi acidocalcisomes indicated that polyPs were preferentially located in these organelles. This was confirmed by visualization of polyPs in the acidocalcisomes using 4,6-diamidino-2-phenylindole (DAPI). A rapid increase (within 2-4 h) in the levels of short and long-chain polyPs was detected during trypomastigote to amastigote differentiation and during the lag phase of growth of epimastigotes (within 12-24 h). Levels rapidly decreased after epimastigotes  resumed growth. Short– and long-chain polyP levels rapidly decreased upon exposure of epimastigotes to hypo-osmotic or alkaline stresses while levels increased after hyperosmotic stress. Ca²⁺ release from acidocalcisomes by a combination of ionophores (ionomycin, nigericin) was associated with the hydrolysis of short – and long-chain polyPs. In agreement with these results, acidocalcisomes were shown to contain polyphosphate kinase and exopolyphosphatase activities. Together, these results suggest a critical role for these organelles in the adaptation of the parasite to environmental changes.  

3.195           Apg2 is a novel protein required for the cytoplasm to vacuole targeting, autophagy, and pexophagy pathways

Wang, C-W., et al

1. Biol. Chem, 276(32), 30442-30451 (2001)

  To survive starvation conditions, eukaryotes have developed an evolutionarily conserved process, termed autophagy, by which the vacuole/lysosome mediates the turnover and recycling of non-essential intracellular material for re-use in critical biosynthetic reactions. Morphological and biochemical studies in Saccharomyces cerevisiae have elucidated the basic steps and mechanisms of the autophagy pathway. Although it is a degradative process, autophagy shows substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway that delivers resident hydrolases to the vacuole. Recent molecular genetics analyses of mutants defective in autophagy and the Cvt pathway, apg, aut and cvt, have begun to identify the protein machinery and provide a molecular resolution of the sequestration and import mechanism that are characteristic of these pathways. In this study, we have identified a novel protein, termed Apg2, required for both the Cvt and autophagy pathways as well as the specific degradation of peroxisomes. Apg2 is required for the formation and/or completion of cytosolic sequestering vesicles that are required for vacuolar import through both the Cvt pathway and autophagy. Biochemical studies revealed that Apg2 is a peripheral membrane protein. Apg2 localizes to the previously identified perivacuolar compartment that contains Apg9, the only characterized integral membrane protein that is required for autophagosome/Cvt vesicle formation.  

3.196           Apg2p functions in autophagosome formation on the perivacuolar structure

Shintani, T., Suzuki, K., Kamada, Y., Noda, T. and Ohsumi,Y.

1. Biol. Chem., 276(32), 30452-30460 (2001)

  Autophagy is a degradative process in which cytoplasmic components are non-selectively sequestered by double-membrane structures, termed autophagosomes, and transported to the vacuole. We have identified and characterized a novel protein Apg2 essential for autophagy in yeast. Biochemical and fluorescence microscopic analyses indicate that Apg2p functions at the step of autophagosome formation. Apg2p localizes to some membranous structure distinct from any known organelle. Using fluorescent protein-tagged Apg2p, we showed that Apg2p localizes to a dot structure close to the vacuole, where Apg8p also exists, but not on autophagosomes unlike Apg8p. This punctate localization of Apg2p depends on the function of Apg1p kinase, phosphatidylinositol-3 kinase complex and Apg9p. Apg2p^G83E, encoded by an apg2-2 allele, shows a severely reduced activity of autophagy and a dispersed localization in the cytoplasm. Overexpression of the mutant Apg2p lessens the defect in autophagy. These results suggest that the dot structure is physiologically important. Apg2p and Apg8p are independently recruited to the structure but coordinately function there to form the autophagosome.  

3.197           Cross-talk between caveolae and glycosylphosphatidylinositol-rich domains

Abrami, L., et al.

1. Biol. Chem., 276(33), 30729-30736 (2001)

  Most mammalian cells have in their plasma membrane at least two types of lipid microdomains, non-invaginated lipid rafts and caveolae. Glycosylphosphatidylinositol (GPI)-anchored proteins constitute a class of proteins that are enriched in rafts but not caveolae at steady state. We have analyzed what the effects of abolishing GPI-biosynthesis are on rafts, caveolae and cholesterol levels. GPI-deficient cells were obtained by screening for resistance to the pore-forming toxin aerolysin, which uses this class of proteins as receptors. Despite the absence of GPI-anchored proteins, mutant cells still contained lipid rafts indicating that GPI-anchored proteins are not crucial structural elements of these domains. Interestingly, the caveolae-specific membrane proteins, caveolin 1 and 2, were up regulated in GPI-deficient cells, in contrast to flotillin-1 and GM1, which were expressed at normal levels. Also the number of surface caveolae was increased. This effect was specific since recovery of GPI biosynthesis by gene recomplementation, restored caveolin expression and the number of surface caveolae to wild type levels. The inverse correlation between the expression of GPI-anchored proteins and caveolin-1 was confirmed by the observation that overexpression of caveolin-1 in wild type cells led to a decrease in the expression of GPI-anchored proteins. In cells lacking caveolae, the absence of GPI-anchored proteins caused an increase in cholesterol levels suggesting a possible role of GPI-anchored proteins in cholesterol homeostasis, which in some cells, such as Chinese hamster ovary cells, can be compensated by caveolin up regulation.  

3.198           Heterogeneous fatty acylation of Src family kinases with polyunsaturated fatty acids regulates raft localization and signal transduction

Liang, X et al

1. Biol. Chem., 276(33), 30987-30994 (2001)

  Fatty acylation of Src family kinases is essential for localization of the modified proteins to the plasma membrane and to plasma membrane rafts. It has been suggested that the presence of saturated fatty acyl chains on proteins is conducive for their insertion into the liquid ordered lipid domains present in rafts. The ability of unsaturated dietary fatty acids to be attached to Src family kinases has not been investigated. Here, we demonstrate that heterogeneous fatty acylation of Src family kinases occurs and that the nature of the attached fatty acid influences raft-mediated signal transduction. By using matrix-assisted-laser-desorption/ionization time-of-flight mass spectrometry, we show that in addition to 14:0 (myristate), 14:1 and 14:2 fatty acids can be attached to the N-terminal glycine of the Src family kinase Fyn when the growth media is supplemented with these dietary fatty acids. Moreover, we synthesized novel iodinated analogs of oleate and stearate, and showed that heterogeneous S-acylation can occur on cysteine residues within Fyn as well as Ga, GAP43 and Ras. Modification of Fyn with unsaturated or polyunsaturated fatty acids reduced its raft localization and resulted in decreased T cell signal transduction. These studies establish that heterogeneous fatty acylation is a widespread occurrence that serves to regulate signal transduction by membrane bound proteins.  

3.199           Lipoprotein lipase and leptin are accumulated in different secretory compartments in rat adipocytes

Roh, C., Roduit, R., Thorens, B., Fried, S. and Kandror, K.V.

1. Biol. Chem., 276(38), 35990-35994 (2001)

  Adipose cells produce and secrete several physiologically proteins, such as lipoprotein lipase (LPL), leptin, adipsin, Acrp30, etc. However, secretory pathways in adipocytes have not been characterized, and vesicular carriers responsible for accumulation and transport of secreted proteins have not been identified. We have compared the intracellular localization of two proteins secreted from adipose cells: leptin and LPL. Adipocytes accumulate large amounts of both proteins, suggesting that neither of them is targeted to the constitutive secretory pathway. By means of velocity centrifugation in sucrose gradients, equilibrium density centrifugation in iodixanol gradients and immunofluorescence confocal microscopy, we determined that LPL and leptin were localized in different membrane structures. LPL was found mainly in the endoplasmic reticulum with a small pool being present in low-density membrane vesicles that may represent a secretory compartment in adipose cells. Virtually all intracellular leptin was localized in these low-density secretory vesicles. Insulin-sensitive Glut4-vesicles did not contain either LPL or leptin. Thus, secretion from adipose cells is controlled both at the exit from the endoplasmic reticulum as well as at the level of “downstream” secretory vesicles.  

3.200           Activation of mitogen-activated protein kinase by membrane targeted Raf chimeras is independent of raft localization

Chen, X. and Resh, M.D.

1. Biol. Chem., 276(37), 34617-34623 (2001)

  Binding of proteins to the plasma membrane can be achieved with various membrane targeting motifs, including combinations of fatty acids, isoprenoids, and basic domains. In this study, we investigate whether attachment of different membrane targeting motifs influences the signaling capacity of membrane-bound signal transduction proteins by directing the proteins to different membrane microdomains. We used c-Raf-1 as a model for a signaling protein that is activated when membrane-bound. Three different membrane targeting motifs from K-Ras, Fyn and Src proteins were fused to the N- or C-terminus of Raf-1. The ability of the modified Rafs to initiate MAPK signaling was then investigated. All three modified Raf-1 constructs activated MAPK to nearly equivalent levels. The extent of localization of the Raf-1 constructs to membrane microdomains known as rafts did not correlate with the level of MAPK activation. Moreover, treatment of cells with the raft disrupting drug methyl-b-cyclodextrin (MbCD) caused activation of MAPK to levels equivalent to those achieved with membrane targeted Raf constructs. The use of pharmacological agents as well as dominant mutants revealed that MAPK activation by MbCD proceeds via a PI3K dependent mechanism that is Ras/Raf independent. We conclude that cholesterol depletion from the plasma membrane by MbCD constitutes an alternative pathway for activating MAPK.  

3.201           Biochemical and morphological analysis on the localization of Rac1 in neurons

Kumanogoh, H., Miyata, S., Sokawa, Y. and Maekawa, S. Neuroscience Res., 39, 189-196 (2001)   The acquisition of cell type-specific morphologies is a central feature of neuronal differentiation. Many extra- and intracellular signals are known to cause the morphological changes of neuronal cells through the reconstruction of the microfilaments underneath the cell membrane. The membrane microdomain called “raft” has been paid much attention, for this domain contains many signal-transducing molecules including trimeric G proteins and cytoskeletal proteins. The raft domain is recovered in a low-density fraction after the treatment of the membrane with the non-ionic detergent such as Triton X-100 and the enrichment of cholesterol and sphingolipids is ascribed to be responsible for the detergent insolubility. In contrast to the well-known localization of trimeric G proteins in raft, the localization of small G proteins in the raft is poorly characterized. Since Rho family small G proteins  (Rho, Rac, and Cdc42) regulate the microfilament system, we studied the localization of Rho family small G proteins in the raft of rat brain with western blotting. Specific localization of Rac1 was detected in the raft from 1-day-old and 8-week-old rat whole brain, and also in the raft prepared from the growth cone and synaptic plasma membrane fractions. Rho and Cdc42 were, in contrast, recovered in the Triton soluble fraction. Double immunostaining of cultured hippocampal neurons with antibodies to Rac1 and MAP-2, or Rac1 and tau, showed punctate distribution of Rac1 in axons as well as in dendrites.  

3.202           Serum-activated assembly and membrane translocation of an endogenous Rac1: effector complex

Hansen, M.D.H. and Nelson, W.J. Current Biology, 11, 356-360 (2001)   Rho family GTPases (Cdc42, Rac1, and RhoA) function downstream of Ras, and in a variety of cellular processes. Studies to examine these functions have not directly linked endogenous protein interactions with specific in vivo functions of Rho GTPases. Here, we show that endogenous Rac1 and two known binding partners, Rho GDP dissociation inhibitor (RhoGDI) and p21-activated kinase (PAK), fractionate as distinct cytosolic complexes. A Rac1: PAK complex is translocated from the cytosol to ruffling membranes upon cell activation by serum. Overexpression of dominant-negative (T17N) Rac1 does not affect the assembly or distribution of this Rac1: PAK complex. This is the first direct evidence of how a specific function of Rac1 is selected by the assembly and membrane translocation of a distinct Rac1:effector complex.  

3.203           Subcellular study of sphingoid base phosphorylation in rat tissues: evidence for multiple sphingosine kinases

Gijsbers, S., Van der Hoeven, G. And Van Veldhoven, P.P. Biochim. Biophys. Acta, 1532, 37-50 (2001)   The enzymatic phosphorylation of sphingoid bases was analysed in rat tissues, using D-erythro-[4,5-³H]sphinganine as substrate. After optimization of the assay, taking care to block sphingosine-phosphate lyase and sphingosine phosphatase, highest ATP-dependent kinase activities were present in testis, followed by kidney, and intestinal mucosa. Approximately two thirds of the kidney activity was membrane bound, the remaining being cytosolic. Classical cell fractionation studies of a particulate fraction from kidney homogenates by Percoll gradient and sucrose density gradient centrifugation revealed that kinase activities are associated with vesicles derived from the endoplasmic reticulum and the plasma membrane. Based on indirect data, such as the effect of detergents and divalent ions, the cytosolic and both membrane bound activities appear to reside in different proteins. N,N-dimethylsphingenine was inhibitory to all three different kinases, which were mainly active towards the D-erythro isomers of sphingenine and sphinganine.  

3.204           GPI-anchored proteins and glycoconjugates segregate into lipid rafts in Kinetoplastida

Denny, P.W., Field, M.C. and Smith, D.F. FEBS Lett., 491, 148-153 (2001)   The plasma membranes of the divergent eukaryotic parasites, Leishmania and Trypanosoma, are highly specialized, with a thick coat of glycoconjugates and glycoproteins playing a control role in virulence. Usually, the majority of these surface macromolecules are attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. In mammalian cells and yeast, many GPI-anchored molecules associate with sphingolipid and cholesterol-rich detergent-resistant membranes, known as lipid rafts. Here we show that GPI-anchored parasite macromolecules (but not the dual acylated Leishmania surface protein (hydrophilic acylated surface protein) or a subset of the GPI-anchored glycoinositol phopholipid glycolipids) are enriched in a sphingolipid/sterol-rich fraction resistant to cold detergent extraction. This observation is consistant with the presence of functional lipid rafts in these ancient, highly polarized organisms.  

3.205           Endoplasmic reticulum and cis-Golgi localization of human T-lymphotropic virus type 1 p12^I : association with calreticulin and calnexin

Ding, W. et al

1. Virol., 75(16), 7672-7682 (2001)

  Human T-lymphotropic virus type 1 (HTLV-1) is a complex retrovirus encoding regulatory and accessory genes in four open reading frames (ORF I to IV) of the pX region. We have demonstrated an important role of pX ORF I expression, which encodes p12^I, in establishment of HTLV-1 in a rabbit model and for optimal viral infectivity in quiescent primary lymphocytes. These data indicated that p12^I may enhance lymphocyte activation and thereby promote virus infection. To further define the role of p12^I in cell activation, we characterized the subcellular localization of p12^I transfected 293T cells and HeLa-Tat cells by multiple methods, including immunofluorescence confocal microscopy, and subcellular fractionation. Herein, we demonstrate that p12^I accumulates in the endoplasmic reticulum (ER) and cis-Golgi apparatus. The localization of p12^I was unchanged following treatments with both cycloheximide (blocking de novo protein synthesis) and brefeldin A (disrupting ER-to-Golgi protein transport), indicating that the protein is retained in the ER and cis-Golgi. Moreover, using coimmunoprecipitation assays, we identify the direct binding of p12^I with both calreticulin and calnexin, resident ER proteins which regulate calcium storage. Our results indicate that p12^I directly binds key regulatory proteins involved in calcium-mediated cell signalling and suggest a role of p12^I in the establishment of HTLV-1 by activation of host cells.  

3.206           Multimerization of human immunodeficiency virus type 1 Gag promotes its localization to barges, raft-like membrane microdomains

Lindwasser, O.W. and Resh, M.D.

1. Virol., 75(17), 7913-7924 (2001)

  The Gag polyprotein of human immunodeficiency virus type 1 (HIV-1) organizes the assembly of nascent virions at the plasma membrane of infected cells. Here we demonstrate that a population of Gag is present in distinct raft-like membrane microdomains that we have termed “barges”. Barges have a higher density than standard rafts, most likely due to the presence of oligomeric Gag-Gag assembly complexes. The regions of the Gag protein responsible for barge targeting were mapped by examining the flotation behavior of wild-type and mutant proteins on OptiPrep density gradients. N-myristoylation of Gag was necessary for association with barges. Removal of the NC and p6 domains shifted much of the Gag from barges into typical raft fractions. These data are consistent with a model in which multimerization of myristoylated Gag proteins drive association of Gag oligomers into raft-like barges. The functional significance of barge association was revealed by several lines of evidence. First, Gag isolated from virus-like particles was almost entirely localized in barges. Moreover, a comparison of wild-type Gag with Fyn(10)Gag, a chimeric protein containing the N-terminal sequence of Fyn, revealed that Fyn(10)Gag exhibited increased affinity for barges and a two- to fourfold increase in particle production. These results imply that association of Gag with raft-like barge membrane microdomains plays an important role in the HIV-1 assembly process.  

3.207           Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization

Gomez-Mouton, C. et al Proc. Natl. Acad, Sci., USA, 98(17), 9642-9647 (2001)   Redistribution of specialized molecules in migrating cells develops asymmetry between two opposite cell poles, the leading edge and the uropod. We show that acquisition of a motile phenotype in T lymphocytes results in the asymmetric redistribution of ganglioside GM3- and GM1-enriched raft domains to the leading edge and to the uropod, respectively. This segregation to each cell pole parallels the specific redistribution of membrane proteins associated to each raft subfraction. Our data suggest that raft partitioning is a major determinant for protein redistribution in polarized T cells, as ectopic expression of raft-associated proteins results in their asymmetric redistribution, whereas non-raft partitioned mutants of these proteins are distributed homogeneously in the polarized cell membrane. Both acquisition of a migratory phenotype and SDF-1 a-induced chemotaxis are cholesterol depletion-sensitive. Finally, GM3 and GM1 raft redistribution requires an intact actin cytoskeleton, but is insensitive to microtubule disruption. We propose that membrane protein segregation not only between raft and nonraft domains but also between distinct raft subdomains may be an organizational principle that mediates redistribution of specialized molecules needed for T cell migration.  

3.208           The Sec6/8 complex in mammalian cells: characterization of mammalian Sec3, subunit interactions, and expression of subunits in polarized cells

Matern, H.T., Yeaman, C., Nelson, W.J. and Scheller, R.H. Proc. Natl. Acad. Sci, USA, 98(17), 9648-9653 (2001)   The yeast exocyst complex (also called Sec6/8 complex in higher eukaryotes) is a multiprotein complex essential for targeting exocytic vesicles to specific docking sites on the plasma membrane. It is composed of eight proteins (Sec3, -5, -6, -8, -10, and -15, and Exo70 and -84), with molecular weights ranging from 70 to 144 kDa. Mammalian orthologues for seven of these proteins have been described and here we report the cloning and initial characterization of the remaining subunit, Sec3. Human Sec3 (hSec3) shares 17% sequence identity with yeast Sec3p, interacts in the two-hybrid system with other subunits of the complex (Sec5 and Sec8), and is expressed in almost all tissues tested. In yeast, Sec3p has been proposed to be a spatial landmark for polarized secretion (1), and its localization depends on its interaction with Rho1p (2). We demonstrate here that hSec3 lacks the potential Rho1-binding site and GFP-fusions of hSec3 are cytosolic. Green fluorescent protein (GFP)-fusion of nearly every subunit of the mammalian Sec6/8 complex were expressed in Madin-Darby canine kidney (MDCK) cells, but they failed to assemble into a complex with endogenous proteins and localized in the cytosol. Of the subunits tested, only GFP-Exo70 localized to lateral membrane sites of cell-cell contact when expressed in MDCK cells. Cells overexpressing GFP-Exo70 fail to form a tight monolayer, suggesting the Exo70 targeting interaction is critical for normal development of polarized epithelial cells.  

3.209           Nerve growth factor activates persistent Rap1 signaling in endosomes

Wu, C., Lai, C-F. and Mobley, W.C.

1. Neurosci., 21(15), 5406-5416 (2001)

  We investigated a role for endogenous Rap1, a small monomeric GTP-binding protein of the Ras family, in nerve growth factor (NGF) signaling in PC12 cells. Although both epidermal growth factor (EGF) and NGF caused transient activation of Ras, only NGF induced the activation of Rap1. Moreover, Rap1 activation was sustained for hours, an effect that matched the sustained activation of the mitogen-activated protein kinase (MAPK) pathway. To investigate the molecular basis for Rap1 activation, we examined complexes containing C3G, a guanine nucleotide exchange factor for Rap1, and CrkL, an adapter protein known to influence Rap1 signaling. NGF induced the formation of a long-lived complex containing C3G/CrkL/Shp2/Gab2/TrkA. Linking the complex to Rap1 activation, we coprecipitated activated TrkA and activated MAPK with activated Rap1 in NGF-treated cells. Confocal microscopy and subcellular fractionation showed that activated Rap1 and the other proteins of the signaling complex were present in endosomes. Pretreatment of PC12 cells with brefeldin A (BFA), which disrupts the Golgi and endosomal compartments, had little effect on Ras activation but strongly inhibited NGF-induced Rap1 activation and continuing MAPK activation. We propose that endosomes are a site from which NGF induces the prolonged activation of Rap1 and MAPK.  

3.210           The first proline of PALP motif at the C terminus of presenilins is obligatory for stabilization, complex formation, and g-secretase activities of presenilins

Tomita, T. et al

1. Biol. Chem., 276(35), 33273-33281 (2001)

  Mutations in presenilin (PS) genes cause early-onset familial Alzheimer’s disease by increasing production of the amyloidogenic form of amyloid b peptides ending at residue 42 (Ab42). PS is an evolutionarily conserved multipass transmembrane protein, and all known PS proteins contain a proline-alanine-leucine-proline (PALP) motif starting at proline (P) 414 (amino acid numbering based on human PS2) at the C terminus. Furthermore, missense mutations that replace the first proline of PALP with leucine (P414L) lead to a loss-of-function of PS in Drosophila melanogaster and Caenorhabditis elegans. To elucidate the roles of the PALP motif in PS structure and function, we analyzed neuro2a as well as PS1/2 null fibroblast cell lines transfected with human PS harboring mutations at the PALP motif. P414L mutation in PS2 (and its equivalent in PS1) abrogated stabilization, high molecular weight complex formation, and entry to Golgi/trans-Golgi network of PS proteins, resulting in failure of Ab42 overproduction on familial Alzheimer’s disease mutant basis as well as of site-3 cleavage of Notch. These data suggest that the first proline of the PALP motif plays a crucial role in the stabilization and formation of the high molecular weight complex of PS, the latter being the active form with intramembrane proteolytic activities.  

3.211           A role for smooth endoplasmic reticulum membrane cholesterol ester in determining the intracellular location and regulation of sterol-regulatory element-binding protein-2

Iddon, R. et al Biochem. J., 358, 415-422 (2001)   Cellular cholesterol homoeostasis is regulated through proteolysis of the membrane-bound precursor sterol-regulatory element-binding protein (SREBP) that releases the mature transcription factor form, which regulates gene expression. Our aim was to identify the nature and intracellular site of the putative sterol-regulatory pool which regulates SREBP proteolysis in hamster liver. Cholesterol metabolism was modulated by feeding hamsters control chow, or a cholesterol-enriched diet, or by treatment with simvastatin or with the oral acyl-CoA: cholesterol acyltransferase inhibitor C1-1011 plus cholesterol. The effects of the different treatments on SREBP activation were confirmed by determination of the mRNAs for the low-density lipoprotein receptor and hydroxymethylglutaryl-CoA (HMG-CoA) reductase and by measurement of HMG-CoA reductase activity. The endoplasmic reticulum was isolated from livers and separated into subfractions by centrifugation in self-generating iodixanol gradients. Immunodetectable SREBP-2 accumulated in the smooth endoplasmic reticulum of cholesterol-fed animals. Cholesterol ester levels of the smooth ER membrane (but not the cholesterol levels) increased after cholesterol feeding and fell after treatment with simvastatin or C1-1011. The results suggest that an increased cellular cholesterol load causes accumulation of SREBP-2 in the smooth endoplasmic reticulum and, therefore, that membrane cholesterol ester may be one signal allowing exit of the SREBP-2/SREBP-cleavage-regulating protein complex to the Golgi.  

3.212           Intracellular distribution of lysosomal sialidase is controlled by the internalisation signal in its cytoplasmic tail

Lukong, K.E. et al

1. Biol. Chem., 276(49), 46172-46181 (2001)

  Sialidase (neuraminidase), encoded by the neu-1 gene in the major histocompatibility complex locus catalyzes the intralysosomal degradation of sialylated glycoconjugates. Inherited deficiency of sialidase results in sialidosis or galactosialidosis, both severe metabolic disorders associated with lysosomal storage of oligosaccharides and glycopeptides. Sialidase also plays an important role in cellular signaling and is specifically required for the production of cytokine interleukin-4 by activated T lymphocytes. In these cells, neu-1-encoded sialidase activity is increased on the cell surface, suggesting that a specific mechanism regulates sorting of this enzyme to the plasma membrane. We investigated that mechanism by first showing that sialidase contains the internalization signal found in lysosomal membrane proteins targeted to endosomes via clathrin-coated pits. The signal consists of a C-terminal tetrapeptide ⁴¹²YGTL⁴¹⁵, with Tyr⁴¹² and Leu⁴¹⁵ essential for endocytosis of the enzyme. We further demonstrated that redistribution of sialidase from lysosomes to the cell surface of activated lymphocytes is accompanied by increased reactivity of the enzyme with antiphosphotyrosine antibodies. We speculate that phosphorylation of Tyr⁴¹² results in inhibition of sialidase internalization in activated lymphocytes.  

3.213           Involvement of lipid rafts in nephrin phosphorylation and organization of the glomerular slit diaphragm

Simons, M. et al Am. J. Pathol., 159(3), 1069-1077 (2001)   NPHS1 has recently been identified as the gene whose mutations cause congenital nephrotic syndrome of the Finnish type. The respective gene product nephrin is a transmembrane protein expressed in glomerular podocytes and primarily localized to the glomerular slit diaphragm. This interpodocyte junction functions in the glomerular filtration by restricting the passage of plasma proteins into the urinary space in a size-selective manner. The functional role of nephrin in this filtration process is so far not very well understood. In this study, we show that nephrin associates in an oligomerized form with signaling microdomains, also known as lipid rafts, and that these localize to the slit diaphragm. We also show that the nephrin-containing rafts can be immunoisolated with the 27A antibody recognizing a podocyte-specific 9-O-acetylated GD3 ganglioside. In a previous study it has been shown that the in vivo injection of this antibody leads to morphological changes of the filtration slits resembling foot process effacement. Here, we report that, in this model of foot process effacement, nephrin dislocates to the apical pole of the narrowed filtration slits and also that it is tyrosine phosphorylated. We suggest that lipid rafts are important in the spatial organization of the glomerular slit diaphragm under physiological and pathological conditions.  

3.214           Presenilin, notch, and the genesis and treatment of Alzheimer’s disease

Selkoe, D.J. Proc. Natl. Acad, Sci., 98(20), 111039-11041 (2001)   Elucidation of the proteolytic processing of the amyloid b-protein precursor (APP) has revealed that one of the two proteases (g-secretase) that cleave APP to release amyloid-b protein (Ab) is likely to be presenilin. Presenilin also mediates the g-secretase-like cleavage of Notch receptors to enable signaling by their cytoplasmic domains. Therefore, APP and Notch may be the first identified substrates of a unique intramembranous aspartyl protease that has presenilin as its active-site component. In view of the evidence for a central role of cerebral build-up of Ab in the pathogenesis of Alzheimer’s disease, this disorder appears to have risen in the human population as a late-life consequence of the conservation of a critical developmental pathway.  

3.215           Cellular membrane-binding ability of the C-terminal cytoplasmic domain of human immunodeficiency virus type 1 envelope transmembrane protein gp41

Chen, S.S-L., Lee, S-F. and Wang, C-T.

1. Virol., 75(20), 9925-9938 (2001)

  The amphipathic a-helices located in the cytoplasmic tail of the envelope (Env) transmembrane glycoprotein gp41 of human immunodeficiency virus type 1 have been implicated in membrane association and cytopathicity. Deletion of the last 12 amino acids in the C terminus of this domain severely impairs infectivity. However, the nature of the involvement of the cytoplasmic tail in Env-membrane interactions in cells and the molecular basis for the defect in infectivity of this mutant virus are still poorly understood. In this study we examined the interaction of the cytoplasmic tail with membranes in living mammalian cells by expressing a recombinant cytoplasmic tail fragment and an Escherichia coli b-galactosidase/cytoplasmic tail fusion protein, both of them lacking gp120, the gp41 ectodomain, and the transmembrane region. We found through cell fractionation, in vivo membrane flotation, and confocal immunofluorescence studies that the cytoplasmic tail contained determinants to be routed to a perinuclear membrane region in cells. Further mapping showed that each of the three lentivirus lytic peptide (LLP-1, LLP-2, and LLP-3) sequences conferred this cellular membrane-targeting ability. Deletion of the last 12 amino acids from the C terminus abolished the ability of the LLP-1 motif to bind to membranes. High salt extraction, in vitro transcription and translation, and posttranslational membrane binding analyses indicated that the b-galactosidase/LLP fusion proteins were inserted into membranes via the LLP sequences. Subcellular fractionation and confocal microscopy studies revealed that each of the LLP motifs, acting in a position-independent manner, targeted non-endoplasmic reticulum (ER)-associated-b-galactosidase and enhanced green fluorescence protein to the ER. Our study provides a basis for the involvement of the gp41 cytoplasmic tail during Env maturation and also supports the notion that the membrane apposition of the C-terminal cytoplasmic tail plays a crucial role in virus-host interaction.  

3.216           The polyphosphate bodies of Chlamydomas reinhardtii possess a proton pumping pyrophosphatase and are similar to acidocalcisomes

Ruiz, F.A., Marchesini, N., Seufferheld, M., Govindjee and Docampo, R.

1. Biol. Chem., 276/49, 46196-46203 (2001)

  Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and apicomplexan parasites. In this work, we describe organelles with properties similar to acidocalcisomes in the green alga Chlamydomonas reinhardtii. Nigericin and NH₄CL released ⁴⁵Ca²⁺ from preloaded permeabilized cells suggesting the incorporation of a significant amount of this cation into an acidic compartment. X-ray microanalysis of the electron-dense vacuoles or polyphosphate bodies of C. reinhardtii showed large amounts of phosphorus, magnesium, calcium, and zinc. Immunofluorescence microscopy, using antisera raised against a peptide sequence of the vacuolar-type proton pyrophosphatase (H⁺-PPase) of Arabidopsis thaliana which is conserved in the C. reinhardtii enzyme, indicated localization in the plasma membrane, in intracellular vacuoles and the contractile vacuole where it co-localized with the vacuolar proton ATPase (V-H⁺-ATPase). Purification of the electron-dense vacuoles using iodixanol density gradients indicated a preferential localization of the H⁺-PPase and the V-H⁺-ATPase activities in addition to high concentrations of PP_i and short and long chain polyphosphate, but lack of markers for mitochondria and chloroplasts. In isolated electron-dense vacuoles, PP_i-driven proton translocation was stimulated by potassium ions and inhibited by the PP_i analog aminomethylene-diphosphonate. Potassium fluoride, imidodiphosphate, N,N’-decyclohexylcarbodi-imide and N-ethylmaleimide also inhibited PP_i  hydrolysis in the isolated organelles in a dose-dependent manner. These results indicate that the electron-dense vacuoles of C.reinhardtii are very similar to acidocalcisomes with regard to their chemical composition and the presence of proton pumps. Polyphosphate was also localized to the contractile vacuole by 4’, 6-diamino-2-phenylindole staining, suggesting, with the immunochemical data, a link between these organelles and the acidocalcisomes.  

3.217           Characterization of a presenilin-mediated APP carboxyl terminal fragment g: Evidence for distinct mechanisms involved in “gamma-secretase processing of the APP and notch 1 transmembrane domains

Yu, C et al

1. Biol. Chem.,276(47), 43756-43760 (2001)

  A variety of investigations have led to the conclusion that presenilins (PS) play a critical role in intramembranous, g-secretase proteolysis of selected type I membrane proteins, including Notch1 and amyloid precursoe protein (APP). We now show that the generation of the S3/Notch intracellular domain and APP-carboxy-terminal fragment g (CTFg) derivatives are dependent on PS expression and inhibited by a highly selective and potent g-secretase inhibitor. Unexpectedly, the APP-CTFg derivative is generated by processing between Leu645 and Val646 (of APP₆₉₅), several amino acids carboxyl-terminal to the scissile bonds for production of amyloid b protein peptides. Although the relationship of APP-CTFg to the production of amyloid b protein peptides is not known, we conclude that in contrast to the highly selective PS-dependent processing of Notch, the PS-dependent g-secretase processing of APP is largely nonselective, and occurs at multiple sites within the APP transmembrane domain.  

3.218           Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent-resistant membranes

Falguieres, T. et al Mol. Biol. Cell, 12, 2453-2468 (2001)   In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100 resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to the trans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.  

3.219           C-terminal domain of the Epstein-Barr virus LMP2A membrane protein contains a clustering signal

Matskova, L., Ernberg, I., Pawson, T. and Winberg, G.

1. Virol., 75(22), 10941-10949 (2001)

  The latency-regulated transmembrane protein LMP2A interferes with signaling from the B-cell antigen receptor by recruiting the tyrosine kinases Lyn and Syk and by targeting them for degradation by binding the cellular E3 ubiquitin ligase AIP4. It has been hypothesized that this constitutive activity of LMP2A requires clustering in the membrane, but molecular evidence for this has been lacking. In the present study we show that LMP2A coclusters with chimeric rat CD2 transmembrane molecules carrying the 27-amino-acid (aa) intracellular C terminus of LMP2A and that this C-terminal domain fused to the glutathione-S-transferase protein associates with LMP2A in cell lysates. This molecular association requires neither the cysteine-rich region between aa 471 and 480 nor the terminal three aa 495 to 497. We also show that the juxtamembrane cysteine repeats in the LMP2A C terminus are the major targets for palmitoylation but that this acylation is not required for targeting of LMP2A to detergent-insoluble glycolipids-enriched membrane microdomains.  

3.220           Agonist-promoted trafficking of human bradykinin receptors: arrestin- and dynamin-independent sequestration of the B₂ receptor and bradykinin in HEK293 cells

Lamb, M.E., de Weerd, W.F.C. and Leeb-Lundberg, L.M.F. Biochem. J., 355, 741-750 (2001)   In this study, we analyzed the agonist trafficking of human B₂ (B₂R) and B₁ (B₁R) bradykinin (BK) receptors using wild-type and green fluorescent protein (GFP)-tagged receptors in HEKL293 cells. B₂R was sequestered to a major extent upon exposure to BK, as determined by the loss of cell-surface B₂R using radioligand binding and by imaging of B₂R-GFP using laser-scanning confocal fluorescence microscopy. Concurrent BK sequestration was revealed by the appearance of acid-resistant specific BK receptor binding. The same techniques showed that B₁R was sequestered to a considerably lesser extent upon binding of des-Arg¹⁰-kallidin. B₂R sequestration was rapid (half-life ~ 5 min) and reached a steady-state level that was significantly lower than that of BK sequestration. B₂R sequestration was minimally inhibited by K44A dynamin (22.4 ± 3.7%), and was insensitive to arrestin- (319-418), which are dominant-negative mutants of dynamin I and b-arrestin respectively. Furthermore, the B₂R-mediated sequestration of BK was completely insensitive to both mutants, as well as the association of BK with a caveolae-enriched fraction of the cells. On the other hand, agonist-promoted sequestration of the b₂-adrenergic receptor was dramatically inhibited by K44A dynamin (81.2 ± 16.3%) and by arrestin- (319-418) (36.9 ± 4.4%). Our results show that B₂R is sequestered to a significantly greater extent than is B₁R upon agonist treatment in HEK293 cells. Furthermore, B₂R appears to be recycled in the process of sequestering BK, and this process occurs in a dynamin- and b-arrestin-independent manner and, at least in part, involves caveolae.  

3.221           Subcellular site of superoxide dismutase expression differentially controls AP-1 activity and injury in mouse liver following ischemia/reperfusion

Zhou, W. et al Hepatology, 33, 902-914 (2001)   Acute damage following ischemia and reperfusion (I/R) in the liver is in part caused by the generation of reactive oxygen species, such as superoxides, during the reperfusion event. Gene therapy directed at attenuating mitochondrial superoxide production following warm I/R injury in the liver has demonstrated great promise in reducing acute hepatocellular damage. In the present study, we have compared the therapeutic effects of ectopic expression of mitochondrial (MnSOD) and cytoplasmic (Cu/ZnSOD) superoxide dismutase using recombinant adenoviral vectors for reducing I/R damage in the liver. Consistent with previous observations, recombinant adenoviral delivery of MnSOD to the liver significantly attenuated both acute liver damage and AP-1 activation following I/R injury to the livers of mice. However, ectopic expression of Cu/ZnSOD diminished neither I/R-induced elevations in serum alanine transaminase (ALT) nor AP-1 activation. Interestingly, baseline activation of AP-1 before I/R-induced injury was seen in livers infected with recombinant Ad.Cu/ZnSOD, but not Ad.MnSOD or Ad.LacZ, vectors. The level of Cu/ZnSOD-induced AP-1 activation was significantly reduced by ablation of Kupffer cells or by coexpression of catalase, suggesting that increased H₂O₂ production facilitated by Cu/ZnSOD in hepatocytes and/or Kupffer cells may be responsible for AP-1 activation. In vitro reconstitution studies using hepatocyte and macrophage cell lines demonstrated that Cu/ZnSOD overexpression induces AP-1 in both cell types, and that secretion of a Cu/ZnSOD-induced macrophage factor is capable of elevating AP-1 in hepatocytes. In summary, our findings demonstrate that subcellular sites of superoxide production in the liver can differentially affect the outcome.  

3.222           Autophagosome requires specific early Sec proteins for its formation and NSF/SNARE for vacuolar fusion

Ishihara, N. et al Mol. Biol. Cell, 12, 3690-3702 (2001)   Double membrane structure, autophagosomes, is formed de novo in the process of autophagy in the yeast Saccharomyces cerevisiae, and many Apg proteins participate in the process. To further understand autophagy, we analyzed the involvement of factors engaged in the secretory pathway. First, we showed that Sec18p (N-ethylmaleimide-sensitive fusion protein, NSF) and Vti1p (soluble N-ethylmaleimide-sensitive fusion protein attachment protein, SNARE), and soluble N-ethylmaleimide-sensitive fusion protein receptor are required for fusion of the autophagosomes to the vacuole but are not involved in autophagosome formation. Second, Sec12p was shown to be essential for autophagy but not for the cytoplasm to vacuole-targeting (Cvt) pathway, which shares mostly the same machinery with autophagy. Subcellular fractionation and electron microscopic analyses showed that Cvt vesicles, but not autophagosomes, can be formed in sec12 cells. Three other coatmer protein (COPII) mutants, sec16, sec23, and sec24, were also defective in autophagy. The blockage of autophagy in these mutants was not dependent on transport from endoplasmic reticulum-to-Golgi, because mutations in two other COPII genes, SEC13 and SEC31, did not affect autophagy. These results demonstrate the requirement for subgroup of COPII proteins in autophagy. This evidence demonstrating the involvement of Sec proteins in the mechanism of autophagosome formation is crucial for understanding membrane flow during the process.  

3.223           Yarrowia lipolytica Pex20p, Saccharomyces cerevisiae Pex18p/Pex 21p and mammalian Pex5pL fulfil a common function in the early steps of the peroxisomal PTS2 import pathway

Einwachter, H., Sowinski, S., Kunau, W-H. and Schliebs, W. EMBO Reports, 2(11), 1035-1039 (2001)   Import of peroxisomal matrix proteins is essential for peroxisome biogenesis. Genetic and biochemical studies using a variety of different model systems have led to the discovery of 23 PEX genes required for this process. Although it is generally believed that, in contrast to mitochondria and chloroplasts, translocation of proteins into peroxisomes involves a receptor cycle, there are reported differences of an evolutionary conservation of this cycle either with respect to the components or the steps involved in different organisms. We show here that the early steps of protein import into peroxisomes exhibit a greater similarity than was thought previously to be the case. Pex20p of Yarrowia lipolytica, Pex18p and Pex21p of Saccharomyces cerevisiae and mammalian Pex5pL fulfill a common function in the PTS2 pathway of their respective organisms. These non-orthologous proteins possess a conserved sequence region that most likely represents a common PTS2-receptor binding site and di-aromatic pentapeptide motifs that could be involved in binding of the putative docking proteins. We propose that no necessarily the same proteins but functional modules of them are conserved in the early steps of peroxisomal protein import.  

3.224           Sec6/8 complexes on trans-Golgi network and plasma membrane regulate stages of exocytosis in mammalian cells

Yeaman, C., Grindstaff, K.K., Wright, J.R. and Nelson, W.J.

1. Cell Biol., 155(12), 593-604 (2001)

  Sec6/8 complex regulates delivery of exocytic vesicles to plasma membrane docking sites, but how it is recruited to specific sites in the exocytic pathway is poorly understood. We identified a Sec6/8 complex on trans-Golgi network (TGN) and plasma membrane in normal rat kidney (NRK) cells that formed either fibroblast- (NRK-49F) or epithelial-like (NRK-52E) intercellular junctions. At both TGN and plasma membrane, Sec6/8 complex colocalizes with exocytic cargo protein, vesicular stomatitis virus G protein (VSVG)-tsO45. Newly synthesized Sec6/8 complex is simultaneously recruited from the cytosol to both sites. However, brefeldin A treatment inhibits recruitment to the plasma membrane and other treatments that block exocytosis (e.g., expression of kinase-inactive protein kinase D and low temperature incubation) cause accumulation of Sec6/8 on the TGN, indicating that steady-state distribution of Sec6/8 complex depends on continuous exocytic vesicle trafficking. Addition of antibodies specific for TGN- or plasma membrane-bound Sec6/8 complexes to semiintact NRK cells results in cargo accumulation in a perinuclear region or near the plasma membrane, respectively. These results indicate that Sec6/8 complex is required for several steps in exocytic transport of vesicles between TGN and plasma membrane.  

3.225           Open reading frame III of Borna disease virus encodes a nonglycosylated matrix protein

Kraus, I et al

1. Virol., 75(24), 12098-12104 (2001)

  The open reading frame III of Borna virus (BDV) codes for a protein with a mass of 16kDa, named p16 or BDV-M. p16 was described as an N-glycosylated protein in several previous publications and therefore was termed gp18, although the amino acid sequence of p16 does not contain any regular consensus sequence for N glycosylation. We examined glycosylation of p16 and studied its membrane topology using antisera raised against peptides, which comprise the N and the C termini. Neither an N- nor a C-terminal peptide is cleaved from p16 during maturation. Neither deglycosylation of p16 by endoglycosidases nor binding of lectin to p16 was detectable. Introduction of typical N-glycosylation sites at the proposed sites of p16 failed in carbohydrate attachment. Flotation experiments with membranes of BDV-infected cells on density gradients revealed that p16 is not an integral membrane protein, since it can be dissociated from membranes. Our experimental data strongly suggest that p16 is a typical nonglycosylated matrix protein associated at the inner surface of the viral membrane, as is true for homologous protein of other members of the Mononegavirales order.  

3.226           Association of Na⁺ -H⁺ exchanger isoform NHE3 and dipeptidyl peptidase IV in the renal proximal tubule

Girardi, A.C.C., Degray, B.C., Nagy, T., Biemesderfer, D. and Aronsen, P.

1. Biol. Chem., 276(49), 46671-46677 (2001)

  In an attempt to identify proteins that assemble with the apical membrane Na⁺ -H⁺ exchanger isoform NHE3, we generated monoclonal antibodies (mAbs) against affinity-purified NHE3 protein complexes isolated from solubilized renal microvillus membrane vesicles. Hybridomas were selected based on their ability to immunoprecipitate NHE3. We have characterized in detail one of the mAbs (1D11) that specifically co-precipitated NHE3 but not villin or NaPi-2. Western blot analyses of microvillus membranes and immunoelectron microscopy of kidney sections showed that mAb 1D11 recognizes a 110-kDa protein highly expressed on the apical membrane of proximal tubule cells. Immunoaffinity chromatography was used to isolate the antigen against which mAb 1D11 is directed. N-terminal sequencing of the purified protein identified it as dipeptidyl peptidase IV (DPPIV) (EC 3.4.14.15), which was confirmed by assays of DPPIV enzyme activity. We also evaluated the distribution of the NHE3-DPPIV complex in microdomains of rabbit renal brush border. In contrast to the previously described NHE3-megalin complex, which principally resides in a dense membrane population (coated pits) in which NHE3 is active, the NHE3-DPPIV complex was predominantly in the microvillar fraction in which NHE3 is active. Serial precipitation experiments confirmed that anti-megalin and anti-DPPIV antibodies co-precipitate different pools of NHE3. Taken together, these studies revealed an unexpected association of the brush border Na⁺ -H⁺ exchanger NHE3 with dipeptidyl peptidase IV in the proximal tubule. These findings raise the possibility that association with DP-PIV may affect NHE3 surface expression and/or activity.  

3.227           Evidence for coupling of membrane targeting and function of the signal recognition particle (SRP) receptor FtsY

Herskovits, A.A. et al EMBO Reports, 2(11), 1040-1046 (2001)    Recent studies have indicated that FtsY, the signal recognition particle receptor of Escherichia coli, plays a central role in membrane protein biogenesis. For proper function, FtsY must be targeted to the membrane, but its membrane-targeting pathway is unknown. We investigated the relationship between targeting and function of FtsY in vivo, by separating its catalytic domain (NG) from its putative targeting domain (A) by three means: expression of split ftsY, insertion of various spacers between A and NG, and separation of A and NG by the in vivo proteolysis. Proteolytic separation of A and NG does not abolish function, whereas separation by long linkers or expression of split ftsY is detrimental. We propose that proteolytic cleavage of FtsY occurs after completion of co-translational targeting and membrane assembly of NG. In contrast, separation by other means may interrupt proper synchronization of co-translational targeting and membrane assembly of NG. The co-translational interaction of FtsY with the membrane was confirmed by in vitro experiments.  

3.228           Direct evidence for a two-step assembly of apoB48-containing lipoproteins in the lumen of the smooth endoplasmic reticulum of rabbit enterocytes

Cartwright, I.J. and Higgins, J.A.

1. Biol. Chem., 276(51), 48048-48057 (2001)

  The aim of this study was to investigate the types and characteristics of chylomicrons precursor in the lumen of the secretory compartment of rabbit enterocytes. Luminal contents were separated into density subfractions in two continuous self-generating gradients of different density profiles. In enterocytes from rabbits fed a low fat diet, newly synthesized and immunodetectable apoB48 was only in the subfraction of density similar to high density lipoprotein (dense particles); the luminal triacylglycerol (TAG) content was low and only in the subfraction of density similar to that of chylomicrons/very low density lipoproteins (light particles). After feeding fat, newly synthesized and immunodetectable apoB48 was in both dense (phospholipid-rich) and light (TAG-rich) particles. Luminal TAG mass and synthesis increased after fat feeding and was only in light particles. Pulse-chase experiments showed that the luminal-radiolabeled apoB48 lost from the dense particles was recovered in the light particles and the secreted chylomicrons. All of the light particle lipids (mass and newly synthesized) co-immunoprecipitated with apoB48. However, in the dense particles, there was a preferential co-precipitation of the preexisting rather than newly synthesized phospholipid. Assembly of apoB48-containing TAG-enriched lipoproteins is therefore a two-step process. The first step produces dense apoB48 phospholipid-rich particles, which accumulate in the smooth endoplasmic reticulum lumen. In the second step, these dense particles rapidly acquire the bulk of the TAG and additional phospholipid in a single and rapid step.  

3.229           Quantitative and reproducible two-dimensional gel analysis using Phoretix 2D Full

Mahon, P. and Dupree, P. Electrophoresis, 22, 2075-2085 (2001)   Quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is used to determine changes in individual protein levels in complex protein mixtures. To provide reliable data, the software used for 2-D gel image analysis must provide a linear response over a wide dynamic range of data output. Here, we show that Phoretix 2D Full analysis of 2-D gels stained with colloidal Coomassie Brilliant Blue G-250 can provide a linear measure of changes in protein quantity. We show using a complex mixture of Arabidopsis thaliana proteins, that this is true for essentially all focused proteins, in a data output range greater than three orders of magnitude. An analysis of the factors that affect errors in the results demonstrated that reproducibility of the data is significantly improved by user seeding, whereas it is reduced by use of the background subfraction algorithms.

3.230           Cvt18/Gsa12 is required for cytoplasm-to-vacuole transport, pexophagy, and autophagy in Saccharomyces cerevisiae and Pichia pastoris

Guan, J. et al Mol. Biol. Cell, 12(12), 3821-3838 (2001)   Eukaryotic cells have the ability to degrade proteins and organelles by selective and nonselective modes of micro- and macroautophagy. In addition, there exist both constitutive and regulated forms of autophagy. For example, pexophagy is a selective process for the regulated degradation of peroxisomes by autophagy. Our studies have shown that the differing pathways of autophagy have many molecular events in common. In this article, we have identified a new member in the family of autophagy genes. GSA12 in Pichia pastoris and its Saccharomyces cerevisiae counterpart, CVT18, encode a soluble protein with two WD40 domains. We have shown that these proteins are required for pexophagy and autophagy in P. pastoris and the Cvt pathway, autophagy, and pexophagy in S. cerevisiae. In P. pastoris, Gsa12 appears to be required for an early event in pexophagy. That is, the involution of the vacuole or extension of vacuole arms to engulf the peroxisomes does not occur in the gsa12 mutant. Consistent with its role in vacuole engulfment, we have found that this cytosolic protein is also localized to the vacuole surface. Similarly, Cvt18 displays a subcellular localization that distinguishes it from the characterized proteins required for cytoplasm-to-vacuole delivery pathways.  

3.231           Plasma membrane proton ATPase Pma1p requires raft association for surface delivery in yeast

Bagnat, M., Chang, A. and Simons, K. Mol. Biol. Cell, 12(12), 4129-4138 (2001)   Correct sorting of proteins is essential to generate and maintain the identity and function of the different cellular compartments. In this study we demonstrate the role of lipid rafts in biosynthetic delivery of Pma1p, the major plasma membrane proton ATPase, to the cell surface. Disruption of rafts led to mistargeting of Pma1p to the vacuole. Conversely, Pma1-7, an ATPase mutant that is mistargeted to the vacuole, was shown to exhibit impaired raft association. One of the previously identified suppressors, multicopy AST1, not only restored surface delivery but also raft association of Pma1-7. Ast1p, which is a peripheral membrane protein, was found to directly interact with Pma1p inducing its clustering into a SDS/Triton X100-resistant oligomer. We suggest that clustering facilitates partition of Pma1p into rafts and transport to cell surface.  

3.232           Na⁺-H⁺ exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3

Li, X et al

1. Physiol., 537(2), 537-552 (2001)

  Rabbit ileal Na⁺-absorbing cell Na⁺-H⁺ exchanger 3 (NHE3) was shown to exist in three pools in the brush border (BB), including a population in lipid rafts. Approximately 50% of BB NHE3 was associated with Triton X-100-soluble fractions and the other ~50% with Triton X-100-insoluble fractions; ~33% of the detergent-insoluble NHE3 was present in cholesterol-enriched lipid microdomains (rafts).   The raft pool of NHE3 was involved in the stimulation of BB NHE3 activity with epidermal growth factor (EGF). Both EGF and clonidine treatments were associated with a rapid increase in the total amount of BB NHE3. This EGF- and clonidine-induced increase of BB NHE3 was associated with an increase in the raft pool of NHE3 and to a smaller extent with an increase in the total detergent-insoluble fraction, but there was no change in the detergent-soluble pool. In agreement with the rapid increase in the amount of NHE3 in the BB, EGF also caused a rapid stimulation of BB Na⁺-H⁺ exchange activity.   Disrupting rafts by removal of cholesterol with methyl-b-cyclodextrin (MbCD) or destabilizing the actin cytoskeleton with cytochalasin D decreased the amount of NHE3 in early endosomes isolated by OptiPrep gradient fractionation. Specifically, NHE3 was shown to associate with endosomal vesicles immunoisolated by anti-EEA1 (early endosomal autoantigen 1) antibody-coated magnetic beads and the endosome-associated NHE3 was decreased by cytochalasin D and MbCD treatment.   We conclude that (i) a pool of ileal BB NHE3 exists in lipid rafts; (ii) EGF and clonidine increase the amount of BB NHE3; (iii)lipid rafts and to a lesser extent, the cytoskeleton, but not the detergent-soluble NHE3 pool, are involved in the EGF- and clonidine-induced acute increase in amount of BB NHE3; (iv) lipid rafts and the actin cytoskeleton play important roles in the basal endocytosis of BB NHE3.  

3.233           YFH1-mediated iron homeostasis is independent of mitochondrial respiration

Chen, O.S. and Kaplan, J. FEBS Lett., 509, 131-134 (2001)   The human gene frataxin and its yeast homolog YFH1 affect mitochondrial function. Deficits in frataxin result in Friedreich ataxia, while deletion of YFH1 results in respiratory incompetence. We determined that as long as respiratory incompetent yeast express Yfh1p they do not accumulate excessive mitochondrial iron. Deletion of YFH1 in respiratory incompetent yeast results in mitochondrial iron accumulation, while the reintroduction of Yfh1p results in mitochondrial iron export. Further, overexpression of Yfh1p has no effect on oxygen consumption in wild-type yeast grown in either fermentative or respiratory carbon sources. We conclude that the effect of Yfh1p on mitochondrial iron metabolism is independent of respiratory activity.  

3.234           Subcellular localization of presenilin 2 endoproteolytic C-terminal fragments

Tekirian, T.L. et al Mol. Brain Res., 96, 14-20 (2001)   Mutations in the genes that encode the presenilin 1 and 2 (PS1 and PS2) proteins cause the majority of familial Alzheimer’s disease (FAD). Differential cleavage of the presenilins results in a generation of at least two C-terminal fragments (CTFs). An increase in the smaller of these two CTFs is one of the few changes in presenilin processing associated with FAD mutations in both PS1 and PS2. Interestingly, the phosphorylation of PS2 modulates the production of the smaller, caspase-derived PS2 CTF, which indicates that the generation of this fragment is a regulated physiologic event. To date, there is no data concerning the subcellular distribution of the caspase-derived PS2 CTF. Because this fragment is normally present at levels that are difficult to detect, we have used cell lines in which the production of wild-type or N141I mutant PS2 is controlled by a tetracycline-regulated promoter in order to assess the subcellular localization of the caspase CTF in relation to the larger, constitutive PS2 CTF and to PS2 holoprotein. We have found that when levels of PS2 are low, the constitutive CTF colocalizes with markers consistent with localization in the early Golgi-ER-Golgi intermediate compartment (ERGIC) while the caspase CTF colocalizes with markers for the endoplasmic reticulum (ER). Following induction of wild-type or mutant PS2, when the levels of PS2 are high, the primary localization of the constitutive CTF appears to shift from the early Golgi-ERGIC in addition to the ER. Interestingly, while the induction of wild-type PS2 resulted in the localization of the caspase CTF primarily in the ER, the induction of mutant PS2 resulted in the localization of the caspase CTF to both the ER and the early Golgi-ERGIC. In summary, these data suggest that the two presenilin 2 CTFs have different patterns of subcellular localization and that the N141I PS2 mutation alters the localization pattern of the PS2 caspase fragment.  

3.235           The role of caveolae and caveolin in vesicle-dependent and vesicle-independent trafficking

Matveev, S., Li, X., Everson, W. and Smart, E.J. Adv. Drug. Deliver. Rew., 49, 237-240 (2001)   Caveolae can mediate endocytosis, transcytosis, and potocytosis. Our understanding of these processes as well as the elucidation of the molecular machinery involved has greatly expanded. In addition, caveolin, a 22 kDa protein often associated with caveolae, can promote the trafficking of sterol through the cytoplasm independent of vesicles. Caveolin also influences the formation, morphology, and function of caveolae. The ability of caveolae and caveolin to mediate macromolecular transport directly impacts a variety of physiological and pathophysiological processes.  

3.236           Localization of p24 putative cargo receptors in the early secretory pathway depends on the biosynthetic activity of the cell

Kuiper, R.P. et al Biochem. J., 360, 421-429 (2001)   Members of the p24 family of putative cargo receptors (subdivided into p24-a, -b, -g and -d) are localized in the intermediate- and cis-Golgi compartment of the early secretory pathway, and are thought to play an important role in protein transport. In the present study, we wondered what effect increased biosynthetic cell activity with resulting high levels of protein transport would have on the subcellular localization of p24. We examined p24 localization in Xenopus intermediate pituitary melanotrope cells, which in black- and white-adapted animals are biosynthetically highly active and virtually inactive respectively. In addition, p24 localization was studied in Xenopus anterior pituitary cells whose activity is not changed during background adaptation. Using organelle fractionation, we found that in the inactive melanotropes and moderately active anterior pituitary cells of white-adapted animals, the p24-a, -b, -g and -d proteins are all located in the Golgi compartment. In the highly active melanotropes, but not in the anterior cells of black-adapted animals, the steady-state distribution of all four p24 members changed towards the intermediated compartment and subdomains of the endoplasmic reticulum (ER), most probably the ER exit sites. In the active melanotropes, the major cargo protein pro-opiomelanocortin was mostly localized to ER subdomains and partially co-localized with the p24 proteins. Furthermore, in the active cells, in vitro blocking of protein biosynthesis by cycloheximide or dispersion of the Golgi complex by brefeldin A led to a redistribution of the p24 proteins, indicating their involvement in ER-to-Golgi protein transport and extensive cycling in the early secretory pathway. We conclude that the subcellular localization of p24 proteins is dynamic and depends on the biosynthetic activity of the cell.  

3.237           Preptin derived from proinsulin-like growth factor II (proIGF-II) is secreted from pancreatic islet b-cells and enhances insulin secretion

Buchanan, C.M., Phillips, A.R. and Cooper, G.J.S. Biochem. J., 360, 431-439 (2001)   Pancreatic islet b-cells secrete the hormones insulin, amylin and pancreastatin. To search for further b-cell hormones, we purified peptides from secretory granules isolated from cultured murine bTC6-F7 b-cells. We identified a 34 amino-acid peptide (3948 Da), corresponding to Asp⁶⁹-Leu¹⁰² of the proinsulin-like growth factor II E-peptide, which we have termed “preptin”. Preptin is present in islet b-cells and undergoes glucose-mediated co-secretion with insulin. Synthetic preptin increase insulin secretion from glucose-stimulated bTC6-F7 cells in a concentration-dependent and saturable manner. Preptin infusion into the isolated, perfused rat pancreas increases the second phase of glucose-mediated insulin secretion by 30%, while antipreptin immunoglobulin infusion decreases the first and second phases of insulin secretion by 29 and 26% respectively. These findings suggest that preptin is a physiological amplifier of glucose-mediated insulin secretion.  

3.238           Ubiquitin sorts proteins into the intralumenal degradative compartment of the late-endosome/vacuole

Urbanowski, J.L. and Piper, R.C. Traffic, 2, 622-630 (2001)   Many studies have demonstrated a role for ubiquitin (Ub) in the down-regulation of cell surface proteins. In yeast, down-regulation is marked by the internalization of proteins, followed by their delivery to the lumen of the vacuole where both the cytosolic and lumenal domains are degraded. It is generally believed that the regulatory step of this process is internalization from the plasma membrane and that protein delivery to the lysosome or vacuole is by default. By separating the process of internalization from degradation, we demonstrate that incorporation of proteins into intralumenal vesicles represents a distinct sorting step along the endocytic pathway that is controlled by recognition of ubiquitin. We show that attachment of a single ubiquitin can serve as a specific sorting signal for the degradative pathway by redirecting recycling Golgi proteins and resident vacuolar proteins into intralumenal vesicles of the yeast vacuole. This pathway is independent of Ptdlns (3,5) P2 and does not rely on the specific composition of transmembrane domain segments. These data provide a physiological basis for how ubiquitination of cell surface proteins guides their degradation and removal from the recycling pathway.  

3.239           Membrane protein diffusion sets the speed of rod phototransduction

Calvert, P.D. et al Nature, 411, 90-94 (2001)   Retinal rods signal the activation of a single receptor molecule by a photon. To ensure efficient photon capture, rods maintain about 10⁹ copies of rhodopsin densely packed into membranous disks. But a high packing density of rhodopsin may impede other steps in phototransduction that take place on the disk membrane, by restricting the lateral movement of, and hence the rate of encounters between, the molecules involved. Although it has been suggested that lateral diffusion of proteins on the membrane sets the rate of onset of the photoresponse, it was later argued that the subsequent processing of the complexes was the main determinant of this rate. The effects of protein density on response shut-off have not been reported. Here we show that a roughly 50% reduction in protein crowding achieved by the hemizygous knockout of rhodopsin in transgenic mice accelerates the rising phases and recoveries of flash responses by about 1.7-fold in vivo. Thus, in rods the rates of both response onset and recovery are set by the diffusional encounter frequency between proteins on the disk membrane.  

3.240           Kinesin-mediated axonal transport of a membrane compartment containing b-secretase and presenilin-1 requires APP

Kamal, A. et al Nature, 414, 643-648 (2001)   Proteolytic processing of amyloid precursor protein (APP) generates amyloid-b peptide and has been implicated in the pathogenesis of Alzheimer's disease. However, the normal function of APP, whether this function is related to the proteolytic processing of APP, and where this processing takes place in neurons in vivo remain unknown. We have previously shown that the axonal transport of APP in neurons is mediated by the direct binding of APP to the kinesin light chain subunit of kinesin-I, a microtubule motor protein. Here we identify an axonal membrane compartment that contains APP, b-secretase and presenilin-1. The fast anterograde axonal transport of this compartment is mediated by APP and kinesin-I. Proteolytic processing of APP can occur in the compartment in vitro and in vivo in axons. This proteolysis generates amyloid-b and a carboxy-terminal fragment of APP, and liberates kinesin-I from the membrane. These results suggest that APP functions as a kinesin-I membrane receptor, mediating the axonal transport of b-secretase and presenilin-1, and that processing of APP to amyloid-b by secretases can occur in an axonal membrane compartment transported by kinesin-I.  

3.241           Acyl-coenzyme A: cholesterol acyltransferase modulates the generation of the amyloid b-peptide

Puglielli, L. et al Nature Cell Biol., 3, 905-912 (2001)   The pathogenic event common to all forms of Alzheimer’s disease is the abnormal accumulation of the amyloid b-peptide (Ab). Here we provide strong evidence that intracellular cholesterol compartmentation modulates the generation of Ab. Using genetic, biochemical and metabolic approaches, we found that cholesteryl-ester levels are directly correlated with Ab production. Acyl-coenzyme A: cholesterol acyltransferase (ACAT), the enzyme that catalyses the formation of cholesteryl esters, modulates the generation of Ab through the tight control of the equilibrium between free cholesterol and cholesteryl esters. We also show that pharmacological inhibitors of ACAT, developed for the treatment of atherosclerosis, are potent modulators of Ab generation, indicating their potential for use in the treatment of Alzheimer’s disease.  

3.242           Caspase-3 is localized to endothelial caveolar domains

Oxhorn, B.C., Wadia, R. and Buxton, I.L.O. Proc. West. Pharmacol. Soc., 44, 45-48 (2001)   No abstract available    

3.243           Vesicle Permeabilization by Protofibrillar -Synuclein: Implications for the Pathogenesis and Treatment of Parkinson's Disease

Volles, M.J. et al Biochemistry, 40(26), 7812-7819 (2001)   Fibrillar -synuclein is a component of the Lewy body, the characteristic neuronal inclusion of the Parkinson's disease (PD) brain. Both -synuclein mutations linked to autosomal dominant early-onset forms of PD promote the in vitro conversion of the natively unfolded protein into ordered prefibrillar oligomers, suggesting that these protofibrils, rather than the fibril itself, may induce cell death. We report here that protofibrils differ markedly from fibrils with respect to their interactions with synthetic membranes. Protofibrillar -synuclein, in contrast to the monomeric and the fibrillar forms, binds synthetic vesicles very tightly via a -sheet-rich structure and transiently permeabilizes these vesicles. The destruction of vesicular membranes by protofibrillar -synuclein was directly observed by atomic force microscopy. The possibility that the toxicity of -synuclein fibrillization may derive from an oligomeric intermediate, rather than the fibril, has implications regarding the design of therapeutics for PD.  

3.244           The clustered Fc receptor II is recruited to Lyn-containing membrane domains and undergoes phosphorylation in a cholesterol-dependent manner

Kwiatkowska, K. and Sobota, A. Eur. J. Immunol., 31(4), 989-998 (2001)   Phosphorylation of clustered Fc receptor II (Fc RII) by Src family tyrosine kinases is the earliest event in the receptor signaling cascade. However, the molecular mechanisms for the interaction between Fc RII and these kinases are not elucidated. To asses this problem we isolated high molecular weight complexes of cross-linked Fc RII from non-ionic detergent lysates of U937 monocytic cells. CD55, a glycosylphosphatidylinositol-anchored protein, a ganglioside GM1 and Lyn, a Src family tyrosine kinase, were also located in these complexes. Gradient centrifugation demonstrated that the complexes containing cross-linked Fc RII displayed a low buoyant density. The Fc RII present in the complexes underwent tyrosine phosphorylation. Cross-linked Fc RII and Lyn occupied common 100–200 nm detergent-resistant membrane fragments, as demonstrated by immunoprecipitation and microscopy studies. Pretreatment of the cells with β-cyclodextrin, a cholesterol acceptor, depleted membrane cholesterol and released CD55, GM1 and Lyn from the detergent-resistant complexes. In parallel, the association of Lyn with cross-linked Fc RII was disrupted and phosphorylation of the receptor inhibited. Reincorporation of cholesterol evoked the relocation of Lyn into the detergent-resistant membrane fraction and restored both Lyn association with cross-linked Fc RII and tyrosine phosphorylation of the receptor. Our data demonstrate that cholesterol-enriched membrane rafts can facilitate tyrosine phosphorylation of clustered Fc RII by Lyn kinase.  

3.245           The influence of CD40 on the association of the B cell antigen receptor with lipid rafts in mature and immature cells

Malapati, S. and Pierce, S.K. Eur. J. Immunol., 31(12), 3789-3797 (2001)   Cholesterol- and sphingolipid-rich membrane microdomains termed lipid rafts appear to play a central role in B cell activation. In mature B cells, signaling through the B cell antigen receptor(BCR) is initiated from within rafts and leads to activation. In immature B cells, the BCR is excluded from rafts and signaling leads to apoptosis. CD40, a member of the tumor necrosis receptor family, is expressed by B cells throughout development and has been shown to influence the results of the engagement of antigen by the BCR in both mature B and immature B cells. Here evidence is provided that CD40 is excluded from the lipid rafts of both mature and immature B cells and remains excluded from rafts even after cross-linking. Nevertheless, in mature B cells CD40 signaling influences the association of the BCR with rafts resulting in an increase in the amount of BCR that translocates into rafts following ligand binding and a subsequent acceleration of the movement of the BCR from rafts. In immature B cells, the cross-linked BCR remains excluded from rafts in the presence of CD40 signaling, conditions under which BCR-induced apoptosis is blocked. These results indicate that CD40 functions outside lipid rafts to influence raft-dependent events in mature B cells and raft-independent events in immature B cells.  

3.246           Mechanisms of Aβ Production and Aβ Degradation: Routes to the Treatment of Alzheimer's Disease

Selkoe, D.J., Xia, W., Kimberly, W.T., Vekrellis, K., Walsh, D., Esler, W.P. and Wolfe, M.S: Alzheimer’s Disease: Advances in Ethiology, Pathogenesis and Therapeutics, 421-432 (2001)   In this chapter, we summarize recent work from our laboratories on three interrelated aspects of the role of amyloid β-protein as the initiator of Alzheimer's disease: its production, degradation and aggregation. By analyzing each of these three steps in the economy of Beta-amyloid independently and then attempting to bring together the findings into a unified cycle, we hope to shed light, not only on multiple mechanisms by which Beta-amyloid can accumulate to induce neuronal dysfunction, but also on discrete points of therapeutic intervention.  

3.247           Convergence of multiple autophagy and cytoplasm to vacuole components to a perivacuolar membrane compartment prior to de novo vesicle formation

Kim, J., Huang, W-P., Stromhaug, P.E. and Klionsky, D.J.

1. Biol. Chem., 277(1), 763-773 (2002)

  Under starvation conditions, the majority of intracellular degradation occurs at the lysosome or vacuole by the autophagy pathway. The cytoplasmic substrates destined for degradation are packaged inside unique double-membrane transport vesicles called autophagosomes and targeted to the lysosome/vacuole for subsequent breakdown and recycling. Genetic analyses of yeast autophagy mutants, apg and aut, have begun to identify to molecular machinery as well as indicate a substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway. Transport vesicle formation is a key regulatory step of both pathways. In this study, we characterize the putative compartment from which both autophagosomes and the analogous Cvt vesicles may originate. Microscopy analyses identified a perivacuolar membrane as the resident compartment for both the Apg1-Cvt9 signaling complex, which mediates the switching between autophagic and Cvt transport, and the autophagy/Cvt-specific phosphatidylinositol 3-kinase complex. Further, the perivacuolar compartment designates the initial site of membrane binding by the Apg/Cvt vesicle component Aut7, the Cvt cargo receptor Cvt19, and the Apg conjugation machinery, which functions in the de novo formation of vesicles. Biochemical isolation of the vesicle component Aut7 and density gradient analyses recapitulate the microscopy findings while also supporting the paradigm that components required for vesicle formation and packaging concentrate at subdomains within the donor membrane compartment.  

3.248           Membrane-bound a-synuclein has a high aggregation propensity and the ability to seed the aggregation of cytosolic form

Lee, H-J., Choi, C. and Lee, S-J.

1. Biol. Chem., 277(1), 671-678 (2002)

  a-Synuclein exists as at least two structural isoforms: a helix-rich, membrane-bound form and a disordered, cytosolic form. Here, we investigated the role of membrane-bound a-synuclein in the aggregation process. In a cell-free system consisting of isolated brain fractions, spontaneous and progressive aggregation of a-synuclein was observed in membranes starting at day 1, whereas no aggregation was observed in the cytosolic fraction in a 3-day period. Addition of antioxidants reduced the aggregation in membrane fraction, implicating the role of oxidative modifications. When excess cytosolic a-synuclein was added to brain membranes, the rate of aggregation was increased while the lag-time was unaffected. Incorporation of cytosolic a-synuclein into membrane-associated aggregates was demonstrated by fractionation and coimmunoprecipitation experiments. In a previous study, we showed that mitochondrial inhibitors such as rotenone, induced a-synuclein aggregation in cells. In the present study using rotenone-treated cells, the earliest appearance of a-synuclein oligomeric species was observed in membranous compartments. Furthermore, a-synuclein-positive inclusions were co-stained with DiI, a membrane-partitioning  fluorescent dye, confirming the presence of lipid components in a-synuclein aggregates. These results suggest that membrane-bound a-synuclein can generate nuclei that seed the aggregation of the more abundant cytosolic form.  

3.249           Characterization of an acyl-CoA thioesterase that functions as a major regulator of peroxisomal lipid metabolism

Hunt, M.C., Solaas, K., Kase, B.F and Alexon, E.H.

1. Biol. Chem., 277(2), 1128-1138 (2002)

  Peroxisomes function in b-oxidation of very long- and long-chain fatty acids, dicarboxylic fatty acids, bile acid intermediates, prostaglandins, leukotrienes, thromboxanes, pristanic acid and xenobiotic carboxylic acids. These lipids are mainly chain-shortened for excretion as the carboxylic acids are transported to mitochondria for further metabolism. Several of these carboxylic acids are slowly oxidized and may therefore sequester coenzyme A (CoASH). To prevent CoASH sequestration and to facilitate excretion of chain-shortened carboxylic acids, acyl-CoA thioesterases, which catalyze the hydrolysis of acyl-CoA to the free acid and CoASH, may play important roles. We have here cloned and characterized a peroxisomal acyl-CoA thioesterase from mouse, named PTE-2 (peroxisomal acyl-CoA thioesterase 2. PTE-2 is ubiquitously expressed and induced at mRNA level by treatment with the peroxisome proliferator WY-14,643 and fasting. Induction seen by these treatments was dependent on the peroxisome proliferator-activated receptor a. Recombinant PTE-2 showed a broad chain-length specificity with acyl-CoAs from short- and medium-, to long-chain acyl-CoAs, and other substrates including trihydroxycoprostanoyl-CoA, hydroxymethylglutaryl-CoA and branched chain acyl-CoAs, all of which are present in peroxisomes. Highest activities were found with the CoA esters of primary bile acids choloyl-CoA and chenodeoxycholoyl-CoA as substrates. PTE-2 activity is inhibited by free CoASH, suggesting that intraperoxisomal free CoASH levels regulate the activity of this enzyme. The acyl-CoA specificity of recombinant PTE-2 closely resembles that of purified mouse liver peroxisomes, suggesting that PTE-2 is the major acyl-CoA thioesterase in peroxisomes. Addition of recombinant PTE-2 to incubations containing isolated mouse liver peroxisomes strongly inhibited bile acid-CoA: amino acid N-acyltransferase activity, suggesting that this thioesterase can interfere with CoASH-dependent pathways. We propose that PTE-2 functions as a key regulator of peroxisomal lipid metabolism.  

3.250           Dynamic association of human insulin receptor with lipid rafts in cells lacking caveolae

Vainio, S. et al EMBO Reports, 3(1), 1-6 (2002)   Cholesterol-sphingolipid rich plasma membrane domains, known as rafts, have emerged as important regulators of signal transduction. The adipocytes insulin receptor (IR) is localized to and signals via caveolae that are formed by polymerization of caveolins. Caveolin binds to IR and stimulates signaling. We report that, in liver-derived cells lacking caveolae, autophosphorylation of the endogenous IR is dependent on raft lipids, being compromised by acute cyclodextrin-mediated cholesterol depletion or by antibody clustering of glycosphingolipids. Moreover, we provide evidence that IR becomes recruited to detergent-resistant domains upon ligand binding and that clustering of GM2 ganglioside inhibits IR signaling apparently by excluding the ligand-bound IR from these domains. Our results indicate that, in cells derived from liver, an important insulin target tissue, caveolae are not required for insulin signaling. Rather, the dynamic recruitment of the ligand-bound IR into rafts may serve to regulate interactions in the initiation of the IR signaling cascade.  

3.251           Cholesteryl ester is transported from caveolae to internal membranes as part of a caveolin-annexin II lipid-protein

Uittenbogaard, A., Everson, W.V., Matveev, S.V. and Smart, E.J.

1. Biol. Chem., 277(7), 4925-4931 (2002)

  We previously demonstrated that in Chinese hamster ovary cells scavenger receptor, class B, type I-dependent selective cholesteryl ester uptake occurs in caveolae. In the present study we hypothesized that cholesteryl ester is transported from caveolae through the cytosol to an internal membrane by a caveolin chaperon complex similar to the one we originally described for the transport of newly synthesized cholesterol. To test this hypothesis we incubated Chinese hamster ovary cells expressing scavenger receptor, class B, type I with (³H)cholesteryl ester-labeled high density lipoprotein, subfractionated the cells and looked for a cytosolic pool of (³H)cholesteryl ester. The radiolabeled sterol initially appeared in the caveolae fraction, then in the cytosol, and finally in the internal membrane fraction. Caveolin IgG precipitated all of the (³H)cholesteryl ester associated with the cytosol. Co-immunoprecipitation studies demonstrated that in the presence of high density lipoprotein, but not low density lipoprotein or lipoprotein-deficient serum, caveolin IgG precipitated four proteins; annexin II, cyclophilin 40, caveolin, and cyclophilin A. Caveolin acylation-deficient mutants were used to demonstrate that acylation of cysteine 133 but not cysteine 143 or 156 is required for annexin II association with caveolin and the rapid transport of cholesteryl esters out of caveolae. We conclude that a caveolin-annexin II lipid-protein complex facilitates the rapid internalization of cholesteryl esters from caveolae.  

3.252           Alzheimer’s disease-related overexpression of the cation-dependent mannose-6-phosphate receptor increases Ab secretion: Role for altered lysosomal hydrolase distribution in b-amyloidogenesis.

Mathews, PM et al

1. Biol. Chem., 277(7), 5299-5307 (2002)

  Prominent endosomal and lysosomal changes are an invariant feature of neurons in sporadic Alzheimer’s disease (AD). These changes include increased levels of lysosomal hydrolases in early endosomes and increased expression of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is partially localized to early endosomes. To determine whether AD-associated redistribution of lysosomal hydrolases resulting from changes in CD-MPR expression affects amyloid precursor protein (APP) processing, we stably transfected APP-overexpressing murine L cells with human CD-MPR. As controls for these cells, we also expressed CD-MPR trafficking-mutants that either localize to the plasma membrane (CD-MPRpm) or to early endosomes (CD-MPRendo). Expression of CD-MPR resulted in a partial redistribution of a representative lysosomal hydrolase, cathepsin D, to early endosomal compartments. Turnover of APP and secretion of sAPPa and sAPPb were not altered by overexpression of any of the CD-MPR constructs. However, secretion of both human Ab40 and Ab42 into the growth media nearly tripled in CD-MPR and CD-MPRendo expressing cells when compared to parental or CD-MPRpm expressing cells. Comparable increases were confirmed for endogenous mouse Ab40 in L cells expressing these CD-MPR constructs but not overexpressing human APP. These data suggest that redistribution of lysosomal hydrolases to early endocytic compartments mediated by increased expression of the CD-MPR may represent a potentially pathogenic mechanism for accelerating Ab generation in sporadic AD, where the mechanism of amyloidogenesis is unknown.  

3.253           Paxillin associates with poly(A)-binding protein 1 at the dense endoplasmic reticulum and the leading edge of migrating cells

Woods, A.J. et al

1. Biol. Chem., 277(8), 6428-6437 (2002)

  Using mass spectrometry we have identified proteins which co-immunoprecipitate with paxillin, an adaptor protein implicated in the integrin-mediated signaling pathways of cell motility. A major component of paxillin immunoprecipitates was poly(A)-binding protein 1, a 70 kDa mRNA-binding protein. Poly(A)-binding protein 1 associated with both the a and b isoforms of paxillin, and this was unaffected by RNase treatment consistent with a protein-protein interaction. The NH₂-terminal region of paxillin (residues 54-313) associated directly with poly(A)-binding protein 1 in cell lysates, and with His-poly(A)-binding protein 1 immobilized in microtiter wells. Binding was specific, saturable and of high affinity (K_d of » 10nM). Cell fractionation studies showed that at steady state, the bulk of paxillin and poly(A)-binding protein 1 was present in the “dense” polyribosome-associated endoplasmic reticulum. However, inhibition of nuclear export with leptomycin B caused paxillin and poly(A)-binding protein1 to accumulate in the nucleus, indicating that they shuttle between the nuclear and cytoplasmic compartments. When cells migrate, poly(A)-binding protein 1 colocalized with paxillinb at the tips of lamellipodia. Our results suggest a new mechanism whereby a paxillin-poly(A)-binding protein 1 complex facilitates transport of mRNA from the nucleus to sites of protein synthesis at the endoplasmic reticulum and the leading lamella during cell migration.  

3.254           Huntingtin is present in the nucleus, interacts with the transcriptional corepressor C-terminal binding protein, and represses transcription

Kegel, K. et al

1. Biol. Chem., 277(9), 7466-7476 (2002)

  Huntingtin is a protein of unknown function that contains a polyglutamine tract, which is expanded in patients with Huntington’s disease (HD). We investigated the localization and a potential function for huntingtin in the nucleus. In human fibroblasts from normal and HD patients, huntingtin localized diffusely in the nucleus and in subnuclear compartments identified as speckles, promyelocytic leukemia protein bodies, and nucleoli. Huntingtin-positive nuclear bodies redistributed after treatment with sodium butyrate. By Western blot, purified nuclei had low levels of full-length huntingtin compared with the cytoplasm but contained high levels of N- and C-terminal huntingtin fragments, which tightly bound the nuclear matrix. Full-length huntingtin co-immunoprecipitated with the transcriptional corepressor C-terminal binding protein, and polyglutamine expansion in huntingtin reduced this interaction. Full-length wild-type and mutant huntingtin repressed transcription when targeted to DNA. Truncated N-terminal mutant huntingtin repressed transcription, whereas the corresponding wild-type fragment did not repress transcription. We speculate that wild-type huntingtin may function in the nuclei in the assembly of nuclear matrix-bound protein complexes involved with transcriptional repression and RNA processing. Proteolysis of mutant huntingtin may alter nuclear functions by disrupting protein complexes and inappropriately repressing transcription in HD.  

3.255           ORP2, a homolog of oxysterol binding protein, regulates cellular cholesterol metabolism

Laitinen, S. et al

1. Lipid Res., 43, 245-255 (2002)

  Oxysterol binding protein (OSBP) related proteins (ORPs) constitute a family that has at least 12 members in humans. In the present study we characterize one of the novel OSBP homologs, ORP2, which we show to be expressed ubiquitously in mammalian tissues. The ORP2 cDNA encodes a deduced 55 kDa protein that lacks a pleckstrin homology (PH) domain, a feature found in the other family members. Sucrose gradient centrifugation analysis of Chinese hamster ovary (CHO) cell post-nuclear supernatant demonstrated that ORP2 is distributed in soluble and membrane-bound fractions. Immunofluorescence microscopy of the endogenous and overexpressed ORP2 in CHO cells suggested that the membrane-bound fraction of the protein localizes to the Golgi apparatus. Stably transfected CHO cells that overexpress ORP2 showed an increase in [¹⁴C]cholesterol efflux to serum, apolipoprotein A-I (apoA-I), and phosphatidyl choline vesicles. The proportion of cellular [¹⁴C]cholesterol that is esterified and the ACAT activity measured as [¹⁴C]oleyl-CoA conversion into cholesteryl [¹⁴C]oleate by the cellular membranes, were markedly decreased in the ORP2 expressing cells. Transient high level overexpression of ORP2 interfered with the clearance of a secretory pathway protein marker from the Golgi complex. The results implicate ORP2 as a novel regulator of cellular sterol homeostasis and intracellular membrane trafficking.  

3.256           ARL2 and BART enter mitochondria and bind the adenine nucleotide transporter

Sharer, J.D., Shern, J.S., Van Valkenburg, H., Wallace, D.C., and Kahn, R.A. Mol. Biol. Cell, 13, 71-83 (2002)   The ADP-ribosylation factor-like 2 (ARL2) GTPase and its binding partner binder of ARL2 (BART) are ubiquitously expressed in rodent and human tissues and are most abundant in brain. Both ARL2 and BART are predominantly cytosolic, but a pool of each was found associated with mitochondria in a protease-resistant form. ARL2 was found to lack covalent N-myristoylation, present on all other members of the ARF family, thereby preserving the N-terminal amphipathic a-helix as a potential mitochondrial import sequence. An overlay assay was developed to identify binding partners for the BART-ARL2-GTP complex and revealed a specific interaction with a protein in bovine brain mitochondria. Purification and partial microsequencing identified the protein as an adenine nucleotide transporter (ANT). The overlay assay was performed on mitochondria isolated from five different tissues from either wild-type or transgenic mice deleted for ANT1. Results confirmed that ANT1 is the predominant binding partner for the BART-ARL2-GTP complex and that the structurally homologous ANT2 protein does not bind the complex. Cardiac and skeletal muscle mitochondria from ant1^-/ant1^- mice had increased levels of ARL2, relative to that seen in mitochondria from wild-type animals. We conclude that the amount of ARL2 in mitochondria is subject to regulation via an ANT1-sensitive pathway in muscle tissues.  

3.257           Acidocalcisomes are functionally linked to the contractile vacuole of Dictyostelium discoideum

Marchesini, N., Ruiz, F.A., Vieira, M. and Docampo, R.

1. Biol. Chem., 277(10), 8146-8153 (2002)

  The mass-dense granules of Dictyostelium discoideum were shown to contain large amounts of phosphorus, magnesium, and calcium, as determined by x-ray microanalysis, either in situ or when purified using iodixanol gradient centrifugation. The high phosphorus content was due to the presence of pyrophosphate and polyphosphate, which were also present in the contractile vacuoles. Both organelles also possessed a vacuolar H⁺ ATPase, an H⁺-pyrophosphatase, and a Ca²⁺-ATPase, as determined by biochemical methods or by immunofluorescence microscopy. The H⁺-pyrophosphatase activity of isolated mass-dense granules was stimulated by potassium ions and inhibited by the pyrophosphate analogs aminomethylene-diphosphonate and imidodiphosphate and by KF and N-ethylmaleimide in a dose-dependent manner. The mass-dense granules and the contractile vacuole appeared to contact each other when the cells were submitted to hyposmotic stress. Acetazolamide inhibited the carbonic anhydrase activity of the contractile vacuoles and prolonged their contraction cycle in a dose-dependent manner. Similar effects were observed with the anion exchanger inhibitor 4,4’-diisothiocyanatodihydrostilbene-2, 2’-disulfonic acid and the vacuolar H⁺-ATPase inhibitor bafilomycin A₁. Together, these results suggest that the mass-dense granules of D. discoideum are homologous to the acidocalcisomes described in protozoan parasites and are linked to the function of the contractile vacuole.  

3.258           Agonist-induced translocation of the kinin B₁ receptor to caveolae-related rafts

Sabourin, T., Bastien, L., Bachvarov, D.R., and Marceau, F. Mol. Pharmacol., 61(3), 546-553 (2002)   The kallikrein-kinin system, activated during inflammatory conditions and the regulation of specific cardiovascular and renal functions, includes two G protein coupled receptors for bradykinin (BK)-related peptides. The B₁ receptor (B₁R) subtype is not believed to undergo agonist-induced phosphorylation and endocytosis. A conjugate made of the rabbit B₁R fused with the yellow variant of green fluorescent protein (YFP) was expressed in mammalian cells. In COS-1 or human embryonic kidney (HEK) 293 cells, the construction exhibited a nanomolar affinity for the agonist radioligand [³H]Lys-des-Arg⁹-BK or the antagonist ligand [³H]Lys-[Leu⁸]des-Arg⁹-BK and a pharmacological profile virtually identical to that of wild-type B₁R. Lys-des-Arg⁹-BK stimulation of HEK 293 cells stably expressing B₁R-YFP but not stimulation of untransfected cells released [³H]arachidonate in a phospholipase A₂ assay. B₁R-YFP was visualized as a continuous labeling of the plasma membranes in stably transfected HEK 293 cells (confocal microscopy). Addition of Lys-des-Arg⁹-BK (1-100nM) rapidly concentrated the receptor-associated fluorescence into multiple aggregates that remained associated with the plasma membrane (no significant internalization) and colocalized with caveolin-1. This reaction was slowly reversible upon agonist washing at 37°C and prevented pretreatment with B₁R antagonist. b-Cyclodextrin treatment, which extracts cholesterol from membranes and disrupts caveolae-related rafts, prevented agonist-induced redistribution of B₁R-YFP but not the PLA₂ activation mediated by this receptor. The agonist radioligand copurified with caveolin-1 to a greater extent than the tritiated antagonist in buoyant fractions of HEK 293 cells treated with the ligands. Agonist-induced cellular translocation of the kinin B₁R to caveolae-related rafts without endocytosis is a novel  variation on the theme of G-protein-coupled receptor adaptation.  

3.259           Conversion of raft associated prion protein to the protease-resistant state requires insertion of PrP-res (PrP^Sc) into contiguous membranes

Baron, G.S., Wehrly, K., Dorward, D.W., Chesebro, B., and Caughey, B. EMBO J., 21(5), 1031-1040 (2002)   Prion protein (PrP) is usually attached to membranes by a glycosylphosphatidylinositol-anchor that associates with detergent-resistant membranes (DRMs), or rafts. To model the molecular processes that might occur during the initial infection of cells with exogenous transmissible spongiform encephalopathy (TSE) agents, we examined the effect of membrane association on the conversion of the normal protease-sensitive PrP isoform (PrP-sen) to the protease-resistant isoform (PrP-res). A cell-free conversion reaction approximating physiological conditions was used, which contained purified DRMs as a source of PrP-sen and brain microsomes from scrapie-infected mice as a source of PrP-res. Interestingly, DRM-associated PrP-sen was not converted to PrP-res until the PrP-sen was either released from DRMs by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), or the combined membrane fractions were treated with the membrane-fusing agent polyethylene glycol (PEG). PEG-assisted conversion was optimal at pH 6-7, and acid pretreating the DRMs was not sufficient to permit conversion without PI-PLC or PEG, arguing against late endosomes/lysosomes as primary compartments for PrP conversion. These observations raise the possibility that generation of new PrP-res during TSE infection requires (i) removal of PrP-sen from target cells; (ii) an exchange of membranes between cells; or (iii) insertion of incoming Prp-res into the raft domains of recipient cells.  

3.260           Heterologous desensitization of EGF receptors and PDGF receptors by sequestration in caveolae

Matveev, S.V. and Smart, E.J. Am. J. Physiol Cell Physiol, 282, C935-C946 (2002)   Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors have been reported to signal via caveolin-containing membranes called caveolae. In contrast, others report that EGF and PDGF receptors are exclusively associated with caveolin-devoid membranes called rafts. Our subcellular fractionation and coimmunoprecipitation studies demonstrate that, in the absence of ligand, EGF and PDGF receptors are associated with rafts. However, in the presence of ligand, EGF and PDGF receptors transiently associate with caveolae. Surprisingly, pretreatment of cells with EGF prevents PDGF-dependent phosphorylation of PDGF receptors and extracellular signal-regulated kinase (ERK) ½ kinase activation. Furthermore, cells pretreated with PDGF prevent EGF-dependent phosphorylation of EGF receptors and ERK1/2 kinase activation. Radioligand binding studies demonstrate that incubation of cells with EGF and PDGF causes both EGF and PDGF receptors to be reversibly sequestered from the extracellular space. Experiments with methyl-b-cyclodextrin, filipin, and antisense caveolin-1 demonstrate that sequestration of the receptors is dependent on cholesterol and caveolin-1. We conclude that ligand-induced stimulation of EGF or PDGF receptors can cause the heterologous desensitization of the other receptor by sequestration in cholesterol-rich, caveolin-containing membranes or caveolae.  

3.261           Podocyte slit-diaphragm protein nephrin is linked to the actin cytoskeleton

Yuan, H.Y., Takeuchi, E., and Salant, D.J. Am. J. Physiol., Renal Physiol., 282, F585-F591 (2002)   Nephrin is an Ig-like transmembrane protein. It is a major component of the podocytes slit diaphragm and is essential for maintaining normal glomular permeability. CD2-associated protein (CD2AP) is also necessary for normal glomerular permeability and is a putative nephrin adaptor molecule. Here, we document that nephrin and CD2AP are linked to the actin cytoskeleton. As detected by Western blot analysis, nephrin and CD2AP were both insoluble when cell membranes from normal rat glomeruli were extracted with 0.5% Triton X-100 (TX-100) at 4°C in the presence of divalent cations, but they were solubilized when the extraction included potassium iodide (KI) to depolymerize F-actin. In addition, a small fraction of the solubilized nephrin and CD2AP was recovered in the low-density fractions of OptiPrep flotation gradients, which indicates that a portion of nephrin, possibly associated with CD2AP, resides in a cholesterol- or sphingolipid-rich region of the plasma membrane. Immunofluorescent staining of unfixed sections of normal rat kidney for nephrin, CD2AP, and F-actin was unaltered by treatment with TX-100 but was greatly diminished by addition of KI. Nephrin staining was slightly reduced by cholesterol depletion with methyl-b-cyclodextrin in the presence of TX-100 but was nearly absent after addition of KI. These results document that nephrin anchors the slit diaphragm to the actin cytoskeleton, possibly by linkage to CD2AP, and that nephrin traverses a relatively cholesterol-poor region of the podocytes plasma membrane. In addition, a small pool of actin-associated nephrin and CD2AP resides in lipid rafts, possibly in the cholesterol-rich apical region of the podocytes-foot processes.  

3.262           Functional characterization of D³, D²-enoyl-CoA isomerase from rat liver

Zhang, D. et al

1. Biol. Chem., 277(11), 9127-9132 (2002)

  The degradation of unsaturated fatty acids by b-oxidation involves D³, D²-enoyl-CoA isomerase (enoyl-CoA isomerases) that catalyze 3-cis ® 2-trans and 3-trans ® 2-trans isomerizations of enoyl-CoAs and the 2,5 ® 3,5 isomerization of dienoyl-CoAs. An analysis of rat liver enoyl-CoA isomerases revealed the presence of a monofunctional enoyl-CoA isomerase (ECI) in addition to mitochondrial enoyl-CoA isomerase (MECI) in mitochondria, whereas peroxisomes contain ECI and multifunctional enzyme 1 (MFE1). Thus ECI, which previously had been described as peroxisomal enoyl-CoA isomerase, was found to be present in both peroxisomes and mitochondria. This enzyme seems to be identical with mitochondrial long-chain enoyl-CoA isomerase (Kilponen, J.M., Palosaari, P.M., and Hiltunen, J.K. 1990 Biochem. J., 269, 223-226). All three hepatic enoyl-CoA isomerases have broad chain length specificities but are distinguishable by their preferences for one of the three isomerization reactions. MECI is most active in catalyzing the 3-cis ® 2-trans isomerization; ECI has a preference for the 3-trans ® 2- trans isomerization, and MFEI is the optimal isomerase for the 2,5 ® 3,5 isomerization. A functional characterization based on substrate specificities and total enoyl-CoA isomerase activities in rat liver leads to the conclusion that the 3-cis ® 2-trans  and 2,5 ® 3,5 isomer-izations in mitochondria are catalyzed overwhelmingly by MECI, whereas ECI contributes significantly to the 3-trans ® 2-trans isomerization. In peroxisomes, ECI is predicted to be the dominant enzyme for the 3-cis ® 2-trans and 3-trans ® 2-trans isomerizations of long-chain intermediates, whereas MFE1 is the key enzyme in the 2,5 ® 3,5 isomerization.  

3.263           Trafficking and cell surface stability of the epithelial Na⁺ channel expressed in epithelial Madin-Darby canine kidney cells

Hanwell, D., Ishikawa, T., Saleki, R., and Rotin, D.

1. Biol. Chem., 277(12), 9772-9779 (2002)

  The apically located epithelial Na⁺ channel (abg-EnaC) plays a key role in the regulation of salt and fluid transport in the kidney and other epithelia, yet its mode of trafficking to the plasma membrane and its cell surface stability in mammalian cells are poorly understood. Because the expression of EnaC in native tissues/cells is very low, we generated epithelial Madin-Darby canine kidney (MDCK) cells stably expressing abg-EnaC, where each subunit is tagged differentially at the intracellular C terminus and the b-subunit is also Myc-tagged at the ectodomain (a_HAb_Myc,T7g_FLAG). EnaC expression in these cells was verified by immunoblotting with antibodies to the tags, and patch clamp analysis has confirmed that the tagged channel is functional. Moreover, using electron microscopy, we demonstrated apical, but not basal, membrane localization of EnaC in these cells. The glycosylation pattern of the intracellular pool of EnaC revealed peptide N-glycosidase F and endoglycosidase H sensitivity. Surprisingly, the cell surface pool of EnaC, analyzed by surface biotinylation, was also core glycosylated and lacked detectable endoglycosidase H-resistant channels. Extraction of the channel from cells in Triton X-100 demonstrated that both intracellular and cell surface pools of EnaC are largely soluble. Moreover, flotation assays to analyze the presence of EnaC in lipid rafts showed that both intracellular and cell surface pools of this channel are not associated with rafts. We have shown previously that the total cellular pool of EnaC is turned over rapidly (t_1/2 ~ 1-2 h). Using cycloheximide treatment and surface biotinylation we now demonstrate that the cell surface pool of EnaC has a similarly short half time (t_1/2 ~ 1 h), unlike the long half-life reported recently for the Xenopus A6 cells. Collectively, these results help elucidate key aspects of EnaC trafficking and turnover rates in mammalian kidney epithelial cells.  

3.264           Mannosomes: a molluscan intracellular tubular membrane system related to heavy metal stress

Knigge, T. et al Comp. Biochem. and Physiol. Part C, 131, 259-269 (2002)   Amongst animals, several hydrogen peroxide-generating oxidases are apparently restricted to molluscs. On of these, D-mannitol oxidase, is concentrated in the alimentary system where it is associated with its own subcellular membrane system of unique tubular morphology, most likely presenting a structural modification of the ER. These structures can be purified by subcellular fractionation and have been termed “mannosomes”. Little is known about the functions of mannitol oxidase or of mannosomes, but the previously reported molluscicide-induced increase in mannosomes implies their involvement in a general stress reaction. In this study, we examined the effects of heavy metal stress in the terrestrial gastropod Arion lusitanicus. The activity of mannitol oxidase and mannosome abundance were monitored, together with metal effects on heat-shock protein level, and these parameters were compared to heavy metal accumulation in the digestive gland. We found that mannitol oxidase is inhibited by heavy metals more than other oxidases. On the other hand, hsp70 levels and mannosomal protein were increased with enhanced heavy metal stress, the latter indicating a probably increase in the number of mannosome organelles. Thus, stress protein (hsp70) and mannosomal protein were positively correlated with heavy metal accumulation, whereas the enzyme activity showed a negative correlation with increasing heavy metal content of the slugs.  

3.265           A proton pumping pyrophosphatase in the Golgi apparatus and plasma membrane vesicles of Trypanosoma cruzi

Martinez, R. et al Mol. & Biochem. Parasitol,, 120, 205-213 (2002)   The proton pumping pyrophosphatase (H⁺ -PPase) is an enzyme that has been identified in membranes of plant vacuoles, in the Golgi complex of plants and Chlamydomonas reinhardii, and more recently in acidocalcisomes of different trypanosomatids and apicomplexan parasites. Immunofluorescence and immunoelectron microscopy studies using antibodies against the plant enzyme also suggested a plasma membrane localization in different stages of Trypanosoma cruzi. In this report we provide immunogold electron microscopy evidence of the presence of the H⁺ -PPase in the Golgi complex and plasma membrane of epimastogotes of T. cruzi. Pyrophosphate promoted acidification of plasma membrane vesicles as determined using acridine orange. This activity was stimulated by K⁺ ions, inhibited by the pyrophosphate analogs imidodiphosphate (IDP) and aminoethylenediphosphate (AMDP) by KF, NaF and DCCD, and it had different responses to ions and inhibitors as compared with the activity present in acidocalcisomes. Surface localization of the H⁺ -PPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against H⁺ -PPase. Taken together, these results are consistent with the presence of a functional H⁺ -PPase in the plasma membrane of these parasites.  

3.266           Alternative splicing unmasks dendritic and axonal targeting signals in metabotropic glutamate receptor 1

Francesconi, A. and Duvoisin, R.M.

1. Neurosci., 22(6), 2196-2205 (2002)

  Precise targeting of neurotransmitter receptors to different neuronal compartments is a fundamental step for the establishment and function of synaptic circuitry. Group I metabotropic glutamate receptors, mGluR1 and mGluR5, control glutamatergic neurotransmission by acting both postsynaptically and presynaptically. Four alternative sliced variants of the mGluR1 gene exist, which differ in their signaling properties and subcellular localization. The present study was undertaken to identify the molecular signals responsible for trafficking of these receptors to different neuronal compartments. Here we report that targeting of mGluR1 to dendrites and axons of transfected retina neurons is controlled by alternative splicing. We have identified in the tail of the receptor a tripeptide motif, which is necessary and sufficient to exclude the splice variant mGluR1b from distal dendrites and to drive it to the axon. This motif, which is present in all the mGluR1 receptors, is masked in mGluR1a by a dominant dendritic signal sequence harbored by the extended C-terminal tail of this splice variant. Furthermore, we show that the identified axonal and dendritic targeting signals are also necessary and sufficient to localize mGluR1b and mGluR1a to the apical and basolateral compartments of Madin-Darby canine kidney cells, respectively, consistent with the existence of common trafficking components for polarized targeting in epithelial cells and neurons.  

3.267           Segregation of Bad from lipid rafts is implicated in the induction of apoptosis

Ayllon, V., Fleischer, A., Cayla, X., Garcia, A. and Rebollo, A.

1. Immunol., 168, 3387-3393 (2002)

  Many molecules relocate subcellularly in cells undergoing apoptosis. Using coimmunoprecipitation experiments we demonstrate that Bad is not associated to 14-3-3 protein, suggesting a new mechanism for the control of the proapoptotic role of Bad. Here we show, by confocal microscopy and cellular fractionation, that Bad is attached to lipid rafts in IL-4-stimulated cells and thymocytes while associated with mitochondria in IL-4-deprived cells. Disruption of lipid rafts by methyl-b-cyclodextrin treatment induces segregation of Bad from rafts, which correlates with apoptosis. Our results suggest that the interaction of Bad with rafts is a dynamic process regulated by IL-4 and involved in the control of apoptosis.  

3.268           Expression of myosin VI within the early endocytic pathway in adult and developing proximal tubules

Biemesderfer, D., Mentone, S.A., Mooseker, M. And Hasson, T. Am. J. Physiol., Ren. Physiol., 282(5), F785-F794 (2002)   Myosin VI is a reverse-direction molecular motor implicated in membrane transport events. Because myosin VI is most highly expressed in the kidney, we investigated its renal localization by using high-resolution immunocytochemical and biochemical methods. Indirect immunofluorescence microscopy revealed myosin VI at the base of the brush border in proximal tubule cells. Horseradish peroxidase uptake studies, which labeled endosomes, and double staining for clathrin adapter protein-2 showed that myosin VI was closely associated with the intermicrovillar (IMV) coated-pit region of the brush border. Localization of myosin VI to the IMV region was confirmed at the electron microscopic level by colloidal gold labeling of ultrathin cryosections. In addition, antigen retrieval demonstrated a small but significant pool of myosin VI on the microvilli. To confirm the association of myosin VI with the IMV compartment, these membranes were separated from other membrane compartments by using 15-25% OptiPrep density gradients. Immunoblotting of the gradient fractions confirmed that myosin VI was enriched with markers for the IMV microdomain of the brush border, suggesting that myosin VI associates with proteins in this compartment. Finally, we examined the expression of myosin VI during nephron development. We found myosin VI present in a diffuse cytoplasmic pattern at stage II (S-shaped body phase) and that it was only redistributed fully to the brush border in the stage IV nephron. These studies support a model for myosin VI function in the endocytic process of the proximal tubule.  

3.269           Rafts promote assembly and atypical targeting of a nonenveloped virus, rotavirus, in Caco-2 cells

Sapin, C. et al

1. Virol., 76(9), 4591-4602 (2002)

  Rotavirus follows an atypical pathway to the apical membrane of intestinal cells that bypasses the Golgi. The involvement of rafts in this process was explored here. VP4 is the most peripheral protein of the triple-layered structure of this nonenveloped virus. High proportions of VP4 associated with rafts within the cell as early as 3 h postinfection. In the meantime a significant part of VP4 was targeted to the Triton X-100-resistant microdomains of the apical membrane, suggesting that this protein possesses an autonomous signal for its targeting. At a later stage the other structural rotavirus proteins were also found in rafts within the cells together with NSP4, a nonstructural protein required for the final stage of virus assembly. Rafts purified from infected cells were shown to contain infectious particles. Finally purified VP4 and mature virus were shown to interact with cholesterol- and sphingolipid-enriched model lipid membranes that changed their phase preference from inverted hexagonal to lamellar structures. Together these results indicate that a direct interaction of VP4 with rafts promotes assembly and atypical targeting of rotavirus in intestinal cells.  

3.270           GCAP1 and rescues rod photoreceptor response in GCAP1/GCAP2 knockout mice

Howes, K.A. et al The EMBO J., 21(7), 1545-1554 (2002)   Visual transduction in retinal photoreceptors operates through a dynamic interplay of two second messengers, Ca²⁺ and cGMP. Ca²⁺ regulates the activity of guanylate cyclase (GC) and the synthesis of cGMP by acting on a GC-activating protein (GCAP). While this action is critical for rapid termination of the light response, the GCAP responsible has not been identified. To test if GCAP1, one of two GCAPs present in mouse rods, supports the generation of normal flash responses, transgenic mice were generated that express only GCAP1 under the control of the endogenous promoter. Paired flash responses revealed a correlation between the degree of recovery of the rod a-wave and expression levels of GCAP1. In single cell recordings, the majority of the rods generated flash responses that were indistinguishable from wild type. These results demonstrate that GCAP1 at near normal levels supports the generation of wild-type flash responses in the absence of GCAP2.  

3.271           Vesicular restriction of synaptobrevin suggests a role for calcium in membrane fusion

Hu, K. et al Nature, 415, 646-650 (2002)   Release of neurotransmitter occurs when synaptic vesicles fuse with the plasma membrane. This neuronal exocytosis is triggered by calcium and requires three SNARE (soluble-N-ethylmaleimide-sensitive factor attachment protein receptors) proteins: synaptobrevin (also known as VAMP) on the synaptic vesicle, and syntaxin and SNAP-25 on the plasma membrane. Neuronal SNARE proteins form a parallel four-helix bundle that is thought to drive the fusion of opposing membranes. As formation of this SNARE complex in solution does not require calcium, it is not clear what function calcium has in triggering SNARE-mediated membrane fusion. We now demonstrate that whereas syntaxin and SNAP-25 in target membranes are freely available for SNARE complex formation, availability of synaptobrevin on synaptic vesicles is very limited. Calcium at micromolar concentrations triggers SNARE complex formation and fusion between synaptic vesicles and reconstituted target membranes. Although calcium does promote interaction of SNARE proteins between opposing membranes, it does not act by releasing synaptobrevin from synaptic vesicle restriction. Rather, our data suggest a mechanism in which calcium-triggered membrane apposition enables syntaxin and SNAP-25 to engage synaptobrevin, leading to membrane fusion.  

3.272           Naturally secreted oligomers of amyloid b protein potently inhibit hippocampal long-term potentiation in vivo

Walsh, D.M. et al Nature, 416, 535-539 (2002)   Although extensive data support a central pathogenic role for amyloid b protein (Ab) in Alzheimer's disease, the amyloid hypothesis remains controversial, in part because a specific neurotoxic species of Ab and the nature of its effects on synaptic function have not been defined in vivo. Here we report that natural oligomers of human Ab are formed soon after generation of the peptide within specific intracellular vesicles and are subsequently secreted from the cell. Cerebral microinjection of cell medium containing these oligomers and abundant Ab monomers but no amyloid fibrils markedly inhibited hippocampal long-term potentiation (LTP) in rats in vivo. Immunodepletion from the medium of all Ab species completely abrogated this effect. Pretreatment of the medium with insulin-degrading enzyme, which degrades Ab monomers but not oligomers, did not prevent the inhibition of LTP. Therefore, Ab oligomers, in the absence of monomers and amyloid fibrils, disrupted synaptic plasticity in vivo at concentrations found in human brain and cerebrospinal fluid. Finally, treatment of cells with g-secretase inhibitors prevented oligomer formation at doses that allowed appreciable monomer production, and such medium no longer disrupted LTP, indicating that synaptotoxic Ab oligomers can be targeted therapeutically.  

3.273           Polycystin-2 is an intracellular calcium release channel

Koulen, P. et al Nature Cell Biol., 4, 191-197 (2002)   Polycystin-2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease (ADPKD), is the prototypical member of a subfamily of the transient receptor potential (TRP) channel superfamily, which is expressed abundantly in the endoplasmic reticulum (ER) membrane. Here, we show by single channel studies that polycystin-2 behaves as a calcium-activated, high conductance ER channel that is permeable to divalent cations. Epithelial cells overexpressing polycystin-2 show markedly augmented intracellular calcium release signals that are lost after carboxy-terminal truncation or by the introduction of a disease-causing missense mutation. These data suggest that polycystin-2 functions as a calcium –activated intracellular calcium release channel in vivo and that polycystic kidney disease results from the loss of a regulated intracellular calcium release signaling mechanism.  

3.274           Specific SHP-2 partitioning in raft domains triggers integrin-mediated signaling via Rho activation

Lacalle, R.A. et al

1. Cell Biol., 157(2), 277-289 (2002)

  Cell signaling does not occur randomly over the cell surface, but is integrated within cholesterol-enriched membrane domains, termed rafts. By targeting SHP-2 to raft domains or to a non-raft plasma membrane fraction, we studied the functional role of rafts in signaling. Serum-depleted, nonattached cells expressing the raft SHP-2 form, but not non-raft SHP-2, display signaling events resembling those observed after fibronectin attachment, such as ß₁ integrin clustering, ³⁹⁷Y-FAK phosphorylation, and ERK activation, and also increases Rho-GTP levels. Expression of the dominant negative N19Rho abrogates raft-SHP-2–induced signaling, suggesting that Rho activation is a downstream event in SHP-2 signaling. Expression of a catalytic inactive SHP-2 mutant abrogates the adhesion-induced feedback inhibition of Rho activity, suggesting that SHP-2 contributes to adhesion-induced suppression of Rho activity. Because raft recruitment of SHP-2 occurs physiologically after cell attachment, these results provide a mechanism by which SHP-2 may influence cell adhesion and migration by spatially regulating Rho activity.  

3.275           Overexpression of the myelin proteolipid protein leads to accumulation of cholesterol and proteolipid protein in endosomes/lysosomes: implications for Pelizaeus-Merzbacher disease

Simons, M. et al

1. Cell Biol., 157(2), 327-336 (2002)

  Duplications and overexpression of the proteolipid protein (PLP) gene are known to cause the dysmyelinating disorder Pelizaeus-Merzbacher disease (PMD). To understand the cellular response to overexpressed PLP in PMD, we have overexpressed PLP in BHK cells and primary cultures of oligodendrocytes with the Semliki Forest virus expression system. Overexpressed PLP was routed to late endosomes/lysosomes and caused a sequestration of cholesterol in these compartments. Similar results were seen in transgenic mice overexpressing PLP. With time, the endosomal/lysosomal accumulation of cholesterol and PLP led to an increase in the amount of detergent-insoluble cellular cholesterol and PLP. In addition, two fluorescent sphingolipids, BODIPY–lactosylceramide and –galactosylceramide, which under normal conditions are sorted to the Golgi apparatus, were missorted to perinuclear structures. This was also the case for the lipid raft marker glucosylphosphatidylinositol–yellow fluorescence protein, which under normal steady-state conditions is localized on the plasma membrane and to the Golgi complex. Taken together, we show that overexpression of PLP leads to the formation of endosomal/lysosomal accumulations of cholesterol and PLP, accompanied by the mistrafficking of raft components. We propose that these accumulations perturb the process of myelination and impair the viability of oligodendrocytes.  

3.276           Platelet-derived growth factor mediates tyrosine phosphorylation of the cytoplasmic domain of the low density lipoprotein receptor-related protein in caveolae

Boucher, P. et al

1. Biol. Chem., 277(18), 15507-15513 (2002)

  The low density lipoprotein (LDL) receptor gene family represents a class of multifunctional, endocytic cell surface receptors. Recently, roles in cellular signaling have also emerged. For instance, the very low density lipoprotein receptor (VLDLR) and the apolipoprotein receptor-2 (apoER2) function in a developmental signaling pathway that regulates the lamination of cortical layers in the brain and involves the activation of tyrosine kinases. Furthermore, the cytoplasmic domain of the LDL receptor-related protein (LRP) was found to be a substrate for the non-receptor tyrosine kinase Src, but the physiological significance of this phosphorylation event remained unknown. Here we show that tyrosine phosphorylation of LRP occurs in caveolae and involves the platelet-derived growth factor (PDGF) receptor b and phosphoinositide 3-kinase. Receptor-associated protein, an antagonist of ligand binding to LRP, and apoE-enriched b-VLDL, a ligand for LRP, reduce PDGF-induced tyrosine phosphorylation of the LRP cytoplasmic domain. In the accompanying paper (Loukinova, E., Ranganathan, S., Kuznetsov, S., Gorlatova, N., Migliorini, M., Ulery, P. G., Mikhailenko, I., Lawrence, D. L., and Strickland, D. K. (2002) J. Biol. Chem. 277, 15499-15506) Loukinova et al. further demonstrate that one form of PDGF, PDGF-BB, binds specifically to LRP and that phosphorylation of LRP requires the activation of Src family kinases. Taken together, these findings provide a biochemical basis for a cellular signaling pathway that involves apoE and LRP.  

3.277           EphrinB phosphorylation and reverse signaling: regulation by Src kinases and PTP-BL phosphatase

Palmer, A. et al Mol. Cell, 9, 725-737 (2002)   Ephrins are cell surface-associated ligands for Eph receptors and are important regulators of morphogenic processes such as axon guidance and angiogenesis. Transmembrane ephrinB ligands act as “receptor-like” signaling molecules, in part mediated by tyrosine phosphorylation and by engagement with PDZ domain proteins. However, the underlying cell biology and signaling mechanisms are poorly understood. Here we show that Src family kinases (SFKs) are positive regulators of ephrinB phosphorylation and phosphotyrosine-mediated reverse signaling. EphB receptor engagement of ephrinB causes rapid recruitment of SFKs to ephrinB expression domains and transient SFK activation. With delayed kinetics, ephrinB ligands recruit the cytoplasmic PDZ domain containing protein tyrosine phosphatase PTP-BL and are dephosphorylated. Our data suggest the presence of a switch mechanism that allows a shift from phosphotyrosine/SFK-dependent signaling to PDZ-dependent signaling.  

3.278           Induction of protein aggregation in an early secretory compartment by elevation of expression level

Schroder, M., Schafer, R. and Friedl, P. Biotechnol. Bioeng., 78(2), 131-140 (2002)   A variety of valuable therapeutic proteins are expressed in mammalian cells. Currently, rate-limiting for secretion of recombinant glycoproteins are activities in the secretory pathway of eukaryotic cells, i.e., folding and glycosylation of the naked polypeptide chain. In this paper we provide evidence that elevation of expression level alone is sufficient to cause intracellular aggregation of a structurally relatively simple glycoprotein, antithrombin III (ATIII). Elevation of expression level by selection for increased drug resistance in Chinese hamster ovary cells stably expressing ATIII resulted in formation of disulfide-bonded aggregates of ATIII. Aggregated ATIII displayed incomplete sialylation and Endo H-sensitivity and located to the endoplasmic reticulum and the cis-Golgi compartment in subcellular fractionations. To explore possible causes for aggregation of ATIII at elevated expression levels we investigated the influence of the two major energy sources of cultured mammalian cells, D-glucose and L-glutamine, on the ATIII-yield. We found that utilization of D-glucose was not limiting for synthesis of ATIII at elevated expression levels. However, the amount of ATIII-synthesized per L-glutamine consumed did not seem to increase steadily with expression level for ATIII, indicating that secretion of ATIII may be limited by the capacity of the cell to utilize L-glutamine.  

3.279           Presenilin 1 is required for maturation and cell surface accumulation of nicastrin

Leem, J.Y. et al

1. Biol. Chem., 277(21), 19236-19240 (2002)

  Proteolytic processing of amyloid precursor protein generates b-amyloid (Ab) peptides that are deposited in senile plaques in brains of aged individuals and patients with Alzheimer's disease. Presenilins (PS1 and PS2) facilitate the final step in Ab production, the intramembranous g-secretase cleavage of amyloid precursor protein. Biochemical and pharmacological evidence support a catalytic or accessory role for PS1 in g-sec-retase cleavage, as well as a regulatory role in select membrane protein trafficking. In this report, we demon-strate that PS1 is required for maturation and cell surface accumulation of nicastrin, an integral component of the multimeric g-secretase complex. Using kinetic labeling studies we show that in PS1^-/-/PS2^-/- cells nicastrin fails to reach the medial Golgi compartment, and as a consequence, is incompletely glycosylated. Stable expression of human PS1 restores these deficiencies in PS1^-/- fibroblasts. Moreover, membrane fractionation studies show co-localization of PS1 fragments with mature nicastrin. These results indicate a novel chaperone-type role for PS1 and PS2 in facilitating nicastrin maturation and transport in the early biosynthetic compartments. Our findings are consistent with PS1 influencing g-secretase processing at multiple steps, including maturation and intracellular trafficking of substrates and component(s) of the g-secretase complex.  

3.280           Distribution of microsomal triglyceride transfer protein within sub-endoplasmic reticulum in human hepatoma cells

Higashi, Y. et al Biochim. Biophys. Acta, 1581, 127-136 (2002)   Very low-density lipoprotein (VLDL) particles are formed in the endoplasmic reticulum (ER) through the association of lipids with apolipoprotein B (apoB). Microsomal triglyceride transfer protein (MTP), which transfers lipid molecules to nascent apoB, is essential for VLDL formation in ER. However, little is known of the distribution and interaction of MTP with apoB within ER. In this study, distribution patterns of apoB and MTP large subunits (lMTP) within ER were examined. Microsomes prepared from HuH-7 cells, a human hepatoma cell line, were further fractionated into rough ER (RER)-enriched subfractions (ER-I fraction) and smooth ER (SER)-enriched subfractions (ER-II fraction) by iodixanol density gradient ultracentrifugation. ApoB was evenly distributed in the ER-I and the ER-II fractions, while 1.5 times more lMTP molecules were present in the ER-I fraction than in the ER-II fraction. lMTP and apoB were coprecipitated both in the ER-I and in the ER-II fractions by immunoprecipitation whenever anti-apoB or an anti-lMTP antibody was used. ApoB-containing lipoprotein particles showed a lower density in the ER-II fraction than those in the ER-I fraction. From these results, it is suggested that MTP can function in both rough and smooth regions of ER in human hepatoma cells.  

3.281           Molecular determinants of the sensory and motor neuron-derived factor insertion into plasma membrane

Cabedo, H., Luna, C., Fernandez, A.M., Gallar, J. and Ferrer-Montiel, A.

1. Biol. Chem., 277(22), 19905-19912 (2002)

  The sensory and motor neuron-derived factor (SMDF) is a type III neuregulin that regulates development and proliferation of Schwann cells. Although SMDF has been shown to be a type II protein, the molecular determinants of membrane biogenesis, insertion, and topology remain elusive. Here we used heterologous expression of a yellow fluorescent protein-SMDF fusion protein along with a stepwise deletion strategy to show that the apolar/uncharged segment (Ile⁷⁶-Val¹⁰⁰) acts as an internal, uncleaved membrane insertion signal that defines the topology of the protein. Unexpectedly, removal of the transmembrane segment (TM) did not eliminate completely membrane association of C-terminal fragments. TM-deleted fusion proteins, bearing the amino acid segment (Ser²⁸³-Glu²⁹⁶) located downstream to the epidermal growth factor-like motif, strongly interacted with plasma membrane fractions. However, synthetic peptides patterned after this segment did not insert into artificial lipid vesicles, suggesting that membrane interaction of the SMDF C terminus may be the result of a post-translational modification. Subcellular localization studies demonstrated that the 40-kDa form, but not the 83-kDa form, of SMDF was segregated into lipid rafts. Deletion of the N-terminal TM did not affect the interaction of the protein with these lipid microdomains. In contrast, association with membrane rafts was abolished completely by truncation of the protein C terminus. Collectively, these findings are consistent with a topological model for SMDF in which the protein associates with the plasma membrane through both the TM and the C-terminal end domains resembling the topology of other type III neuregulins. The TM defines its characteristic type II membrane topology, whereas the C terminus is a newly recognized anchoring motif that determines its compartmentalization into lipid rafts. The differential localization of the 40- and 83-kDa forms of the neuregulin into rafts and non-raft domains implies a central role in the protein biological activity.  

3.282           Amyloid-lowering isocoumarins are not direct inhibitors of g-secretase

Esler, W.P. et al Nature Cell Biol., 4, E110 (2002)   The last step in the production of the amyloid-bprotein (Ab), the major protein component of the cerebral plaques of Alzheimer's disease, is proteolysis within the transmembrane region of the Amyloid-b Precursor Protein (APP) by g-secretase. The Notch receptor (N) is processed in a similar manner as part of a signalling mechanism essential for metazoan development. Missense mutations in the polytopic presenilins, PS1 and PS2, cause Alzheimer's disease and alter the specificity of g-secretase to increase production of a much more aggregation-prone form of Ab. Knockout studies, site-directed mutagenesis, pharmacological profiling, affinity labelling and biochemical isolation all strongly support the hypothesis that g-secretase is a complex of integral membrane proteins, an aspartyl protease in which the active site resides between the two subunits of a processed form of PS. As a corollary, PS processes both APP and N.  

3.283           Identification, characterization, and localization of a novel kidney polycystin-1-polycystin-2 complex

Newby, L.J. et al

1. Biol. Chem., 277(23), 20763-20773 (2002)

  The functions of the two proteins defective in autosomal dominant polycystic kidney disease, polycystin-1 and polycystin-2, have not been fully clarified, but it has been hypothesized that they may heterodimerize to form a "polycystin complex" involved in cell adhesion. In this paper, we demonstrate for the first time the existence of a native polycystin complex in mouse kidney tubular cells transgenic for PKD1, non-transgenic kidney cells, and normal adult human kidney. Polycystin-1 is heavily N-glycosylated, and several glycosylated forms of polycystin-1 differing in their sensitivity to endoglycosidase H (Endo H) were found; in contrast, native polycystin-2 was fully Endo H-sensitive. Using highly specific antibodies to both proteins, we show that polycystin-2 associates selectively with two species of full-length polycystin-1, one Endo H-sensitive and the other Endo H-resistant; importantly, the latter could be further enriched in plasma membrane fractions and co-immunoprecipitated with polycystin-2. Finally, a subpopulation of this complex co-localized to the lateral cell borders of PKD1 transgenic kidney cells. These results demonstrate that polycystin-1 and polycystin-2 interact in vivo to form a stable heterodimeric complex and suggest that disruption of this complex is likely to be of primary relevance to the pathogenesis of cyst formation in autosomal dominant polycystic kidney disease.  

3.284           Ceramide biosynthesis is required for the formation of the oligomeric H⁺ -ATPase Pma1p in the yeast endoplasmic reticulum

Lee, M.C., Hamamoto, S. and Schekman, R.

1. Biol. Chem., 277(25), 23395-23401 (2002)

  The yeast plasma membrane H⁺-ATPase Pma1p is one of the most abundant proteins to traverse the secretory pathway. Newly synthesized Pma1p exits the endoplasmic reticulum (ER) via COPII-coated vesicles bound for the Golgi. Unlike most secreted proteins, efficient incorporation of Pma1p into COPII vesicles requires the Sec24p homolog Lst1p, suggesting a unique role for Lst1p in ER export. Vesicles formed with mixed Sec24p-Lst1p coats are larger than those with Sec24p alone. Here, we examined the relationship between Pma1p biosynthesis and the requirement for this novel coat subunit. We show that Pma1p forms a large oligomeric complex of >1 MDa in the ER, which is packaged into COPII vesicles. Furthermore, oligomerization of Pma1p is linked to membrane lipid composition; Pma1p is rendered monomeric in cells depleted of ceramide, suggesting that association with lipid rafts may influence oligomerization. Surprisingly, monomeric Pma1p present in ceramide-deficient membranes can be exported from the ER in COPII vesicles in a reaction that is stimulated by Lst1p. We suggest that Lst1p directly conveys Pma1p into a COPII vesicle and that the larger size of mixed Sec24pLst1p COPII vesicles is not essential to the packaging of large oligomeric complexes.  

3.285           The epithelial cell cytoskeleton and intracellular trafficking. I. Shiga toxin B-subunit system: retrograde transport, intracellular vectorization, and more

Ludger, J. Am. J. Physiol. Gastrointest Liver Physiol., 283, G1-G7 (2002)   Many intracellular transport routes are still little explored. This is particularly true for retrograde transport between the plasma membrane and the endoplasmic reticulum. Shiga toxin B subunit has become a powerful tool to study this pathway, and recent advances on the molecular mechanisms of transport in the retrograde route and on its physiological function(s) are summarized. Furthermore, it is discussed how the study of retrograde transport of Shiga toxin B subunit allows one to design new methods for the intracellular delivery of therapeutic compounds.  

3.286           Hypercholesterolemia promotes a CD36-dependent and endothelial nitric oxide synthase mediated vascular dysfunction

Kincer, J.F. et al

1. Biol. Chem., 277(26), 23525-23533 (2002)

  Numerous studies have implicated either the pres­ence or absence of CD36 in the development of hypertension. In addition, hypercholesterolemia is associated with the loss of nitric oxide-induced vasodilation and the subsequent increase in blood pressure. In the cur­rent study, we tested the hypothesis that diet-induced hypercholesterolemia promotes the disruption of ago­nist-stimulated nitric oxide generation and vasodilation in a CD36-dependent manner. To test this, C57BL/6, apoE null, CD36 null, and apoE/CD36 null mice were maintained on chow or high fat diets. In contrast to apoE null mice fed a chow diet, apoE null mice fed a high fat diet did not respond to acetylcholine with a decrease in blood pressure. Caveolae isolated from in vivo vessels did not contain endothelial nitric-oxide synthase and were depleted of cholesterol. Age-matched apoE/CD36 null mice fed a chow or high fat diet responded to ace­tylcholine with a decrease in blood pressure. The mech­anism underlying the vascular dysfunction was revers­ible because vessels isolated from apoE null high fat-fed mice regained responsiveness to acetylcholine wheri in­cubated with plasma obtained from chow-fed mice. Fur­ther analysis demonstrated that the plasma low density lipoprotein fraction was responsible for depleting caveolae of cholesterol, removing endothelial nitric-ox­ide synthase from caveolae, and preventing nitric oxide production. In addition, the pharmacological removal of caveola cholesterol with cyclodextrin mimicked the ef­fects caused by the low density lipoprotein fraction. We conclude that the ablation of CD36 prevented the negative impact of hypercholesterolemia on agonist-stimu­lated nitric oxide-mediated vasodilation in apoE null mice. These studies provide a direct link between CD36 and the early events that underlie hypercholesterol­emia-mediated hypertension and mechanistic linkages between CD36 function, nitric-oxide synthase activa­tion, caveolae integrity, and blood pressure regulation.  

3.287           Construction of a catalytically inactive cholesterol oxidase mutant: inverstigation of the interplay between active site-residues glutamate 361 and histidine 447.

Yin, Y., Liu, P., Anderson, R.G.W. and Sampson, N.S. Arch. Biochem. Biophys., 402, 235-242 (2002)   Cholesterol oxidase catalyzes the oxidation of cholesterol to cholest-5-en-3-one and its subsequent isomerization into cholest­-4-en-3-one. Two active-site residues, His447 and Glu361, are important for catalyzing the oxidation and isomerization reactions, respectively. Double-mutants were constructed to test the interplay between these residues in catalysis. We observed that the k_cat of oxidation for the H447Q/E361Q mutant was 3-fold less than that for H447Q and that the k_cat of oxidation for the H447E/E361Q mutant was 10-fold slower than that for H447E. Because both doubles-mutants do not have a carboxylate at position 361, they do not catalyze isomerization of the reaction intermediate cholest-5-en-3-one to cholest-4-en-3-one. These results suggest that Glu361 can compensate for the loss of histidine at position 447 by acting as a general base catalyst for oxidation of cholesterol. Importantly, the construction of the double-mutant H447E/E361Q yields an enzyme that is 31,000-fold slower than wild type in k_cat for oxidation. The H447E/E361Q mutant is folded like native enzyme and still associates with model membranes. Thus, this mutant may be used to study the effects of membrane binding in the absence of catalytic activity. It is demonstrated that in assays with caveolae membrane fractions, the wild-type enzyme uncouples platelet-derived growth factor receptor b (PDGFRb) autophosphorylation from tyrosine phosphorylation of neighboring proteins, and the H447E/E361Q mutant does not. Thus maintenance of membrane structure by cholesterol is important for PDGFRb-mediated signaling. The cholesterol oxidase mutant probe described will be generally useful for investigating the role of membrane structure in signal transduction pathways in addition to the PDGFRb-dependent pathway tested.  

3.288           Optimization of the workup procedure for the analysis of 8-oxo-7, 8-dihydro-2’-deoxyguanosine with electrochemical detection

Hofer, T. and Moller, L. Chem. Res. Toxicol., 15, 426-432 (2002)   The artifactual generation of the biomarker for oxidative stress, 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG), during the workup procedure for its analysis is a difficult problem to solve, and the responsible factors are unclear. Here, peroxide removal and other antioxidant procedures during workup were compared using a limited amount of rat liver (50 mg) as starting material, with subsequent hydrolysis of 50 mg of DNA. A cold (0°C) high salt GTC (4 M guanidine thiocyanate) non-phenol DNA extraction method was developed where DNA is quickly isolated. GSH (reduced glutathione) generated artifactual formation of 8-oxodG during the workup procedure, whereas H₂0₂ removal using catalase, Fe³⁺ removal and passivation using desferal, peroxide removal using glutathione peroxidase, ebselen and a peroxidase mimic lowered the 8-oxodG levels, all identifying peroxides as the responsible oxidants. Desferal was more protective when excluding Mg²⁺ and Ca²⁺ from buffers but was found to disturb the electrochemical detector when repeatedly injected five to six times, even at 100 mM. Addition of the OH· scavenger ethanol in all steps at 2% v/v had no protective effect. Zn²⁺ was found necessary for efficient DNA hydrolysis using nuclease P₁ which was poor below 37°C. Use of water substitutes was tested but inhibited DNA hydrolysis completely. H₂¹⁸O could, however, work for mass spectrometry methods. Long-term (38 days) storage of 0.5% v/v Triton X-100 generated more 8-oxodG than Tween 20 when Incubated with free dG. The cold GTC DNA extraction method was used for analysis of freshly isolated human lymphocytes/monocytes from 60 healthy men using catalase and TEMPO as antioxidants, giving a background level of 0.074 ± 0.027 8-oxodG/10⁵ dG (or 16 8-oxodG/10⁸ nucleotides or 1943 8-oxodG/nuclei) which is probably the lowest value obtained yet. No increase with age was seen. Oxidation of dG to 8-oxodG during workup was found to fit a mathematically defined curve, and a calculated background level of 0.047 8-oxodG/10⁵ dG was obtained. To obtain more reliable results it is recommended that control samples are included during the workup procedure, having an equal amount of cells (or DNA) as the exposed samples.  

3.289           Studying the behavior of mitochondria

Nunnari, J., Wong, E.D., Meesusen, S. and Wagner, J.A. Methods in Enzymol., 351, 381-393 (2002)   Yeast mitochondria form dynamic tubular structures that are distributed uniformly at the cell cortex and contain an average of 50 to 100 copies of the mitochondrial genome (mtDNA) packaged into higher order nucleoid structures. Saccharamyces cerevisiae is an ideal system for studying the mechanisms of both mitochondrial structure and mtDNA maintenance. Even after cells lose mtDNA, often as a secondary consequence of abnormal mitochondrial morphology, they can still be propagated and studied when a fermentable carbon source such as glucose is present. However, because of its role in various metabolic processes, the mitochondrial organelle, even in the absence of mtDNA, is an essential structure that cannot be created de novo. Therefore, a daughter cell survives only if a portion of the mitochondrion is inherited from the mother cell before cytokinesis.. Much insight into the mechanisms that govern the shape, distribution, and movement of mitochondria, as well as the behavior of mtDNA, has been gained through the use of genetic, cytological, and biochemical approaches. This chapter describes some of the cytological and biochemical techniques that have been developed and used in our laboratory and by others in the field to study the behavior of this complex organelle.  

3.290           Lipid microdomains are required sites for the selective endocytosis and nuclear translocation of IFN-g, its receptor chain IFN-g receptor-1, and phosphorylation and nuclear translocation of STAT1a

Subramanian, P.S. and Johnson, H.M.

1. Immunol, 169, 1959-1969 (2002)

  IFN-g contains a nuclear localization sequence that may play a role in the nuclear transport of activated STAT1a via a complex of IFN-g/IFN-g receptor (IFNGR)-1/STAT1a with the nuclear importer nucleoprotein interactor 1. In this study, we examine the mechanism of endocytosis of IFNGR-1 and the relationship of its nuclear translocation to that of STAT1a. In untreated WISH cells, both IFNGR-1 and IFNGR-2 were constitutively localized within caveolae-like microdomains isolated from plasma membrane. However, treatment of cells with IFN-g resulted in rapid migration of IFNGR-1, but not IFNGR-2, from these microdomains. Filipin pretreatment, which specifically inhibits endocytosis from caveolae-like microdomains, inhibited the nuclear translocation of IFN-g and IFNGR-1 as well as the tyrosine phosphorylation and nuclear translocation of STAT1a, but did not affect the binding of IFN-g to these cells. In the Jurkat T lymphocyte cell line, which does not express caveolin-1, nuclear translocation of IFNGR-1 and STAT1a were similarly inhibited by filipin pretreatment. Isolation of lipid microdomains from Jurkat cells showed that both IFNGR-1 and IFNGR-2 were associated with lipid microdomains only after stimulation with IFN-g, suggesting that the IFNGR subunits are recruited to lipid microdomains by IFN-g binding in lymphocytes (Jurkat) in contrast to their constitutive presence in epithelial (WISH) cells. In contrast, treatments that block clathrin-dependent endocytosis did not inhibit either activation or nuclear translocation of STAT1a or the nuclear translocation of IFN- or IFNGR-1. Thus, membrane lipid microdomains play an important role in IFN-g-initiated endocytic events involving IFNGR-1, and the nuclear translocation of IFN-g, IFNGR-1, and STAT1a  

3.291           Second cysteine-rich region of epidermal growth factor receptor contains targeting information for caveolae/rafts

Yamabhai, M. and Anderson, R.G.W.

1. Biol. Chem., 277(28), 24843-24846 (2002)

  Previous studies have shown that ~60% of the epidermal growth factor receptors (EGFRs) in quiescent fibroblasts are concentrated in the caveolae/raft fraction from purified plasma membranes. This high degree of localization suggests the EGFR contains targeting information for lipid domains. We have used mutagenesis to determine that the region of the receptor that controls targeting to caveolae/rafts resides in the juxtamembrane, second cysteine-rich region. A 60-amino acid-long sequence within this region that is continuous with the transmembrane domain was sufficient to target the transmembrane and cytoplasmic tails of both EGFR and the low density lipoprotein receptor to caveolae/rafts. Two N-linked sugars in this segment were not required for proper targeting, although unglycosylated wild-type receptors did not localize properly. We conclude that, in contrast to signals for coated pit localization that are in the cytoplasmic tail, the targeting information for caveolae/rafts is on the extracellular side of the EGFR very close to the membrane.    

3.292           Mature glycosylation and trafficking of nicastrin modulate its binding to presenilins

Yang, D-S, et al

1. Biol. Chem., 277(31), 28135-28142 (2002)

  Nicastrin is an integral component of the high molecular weight presenilin complexes that control proteolytic processing of the amyloid precursor protein and Notch. We report here that nicastrin is most probably a type 1 transmembrane glycoprotein that is expressed at moderate levels in the brain and in cultured neurons. Immunofluorescence studies demonstrate that nicastrin is localized in the endoplasmic reticulum, Golgi, and a discrete population of vesicles. Glycosidase analyses reveal that endogenous nicastrin undergoes a conventional, trafficking-dependent maturation process. However, when highly expressed in transfected cells, there is a disproportionate accumulation of the endo-b-N-acetylglucosaminidase H-sensitive, immature form, with no significant increase in the levels of the fully mature species. Immunoprecipitation revealed that presenilin-1 interacts preferentially with mature nicastrin, suggesting that correct trafficking and co-localization of the presenilin complex components are essential for activity. These findings demonstrate that trafficking and post-translational modifications of nicastrin are tightly regulated processes that accompany the assembly of the active presenilin complexes that execute -secretase cleavage. These results also underscore the caveat that simple overexpression of nicastrin in transfected cells may result in the accumulation of large amounts of the immature protein, which is apparently unable to assemble into the active complexes capable of processing amyloid precursor protein and Notch.  

3.293           Blocking of HIV-1 infection by targeting CD4 to nonraft membrane domains

del Real, G. et al

1. Exp. Med., 196(3), 293-301 (2002)

  Human immunodeficiency virus (HIV)-1 infection depends on multiple lateral interactions between the viral envelope and host cell receptors. Previous studies have suggested that these interactions are possible because HIV-1 receptors CD4, CXCR4, and CCR5 partition in cholesterol-enriched membrane raft domains. We generated CD4 partitioning mutants by substituting or deleting CD4 transmembrane and cytoplasmic domains and the CD4 ectodomain was unaltered. We report that all CD4 mutants that retain raft partitioning mediate HIV-1 entry and CD4-induced Lck activation independently of their transmembrane and cytoplasmic domains. Conversely, CD4 ectodomain targeting to a nonraft membrane fraction results in a CD4 receptor with severely diminished capacity to mediate Lck activation or HIV-1 entry, although this mutant binds gp120 as well as CD4wt. In addition, the nonraft CD4 mutant inhibits HIV-1 X4 and R5 entry in a CD4⁺ cell line. These results notonly indicate that HIV-1 exploits host membrane raft domainsas cell entry sites, but also suggest new strategies for preventingHIV-1 infection.  

3.294           Stomatin-related olfactory protein, SRO, spedifically expressed in the murine olfactory sensory neurons

Kobayakawa, K. et al

1. Neurosci., 22(14), 5931-5937 (2002)

  We identified a stomatin-related olfactory protein (SRO) that is specifically expressed in olfactory sensory neurons (OSNs). The mouse sro gene encodes a polypeptide of 287 amino acids with a calculated molecular weight of 32 kDa. SRO shares 82% sequence similarity with the murine stomatin, 78% with Caenorhabditis elegans MEC-2, and 77% with C. elegans UNC-1. Unlike other stomatin-family genes, the sro transcript was present only in OSNs of the main olfactory epithelium. No sro expression was seen in vomeronasal neurons. SRO was abundant in most apical dendrites of OSNs, including olfactory cilia. Immunoprecipitation revealed that SRO associates with adenylyl cyclase type III and caveolin-1 in the low-density membrane fraction of olfactory cilia. Furthermore, anti-SRO antibodies stimulated cAMP production in fractionated cilia membrane. SRO may play a crucial role in modulating odorant signals in the lipid rafts of olfactory cilia.  

3.295           Differential sorting and fate of endocytosedGPI-anchored proteins

Fivaz, M. et al EMBO J., (21/15), 3989-4000 (2002)   In this paper, we studied the fate of endocytosed glycosylphosphatidylinositol anchored proteins (GPI- APs) in mammalian cells, usingaerolysin, a bacterial toxin that binds to the GPI anchor, asa probe. We find that GPI-APs are transported down the endocyticpathway to reducing late endosomes in BHK cells, using biochemical,morphological and functional approaches. We also find that thistransport correlates with the association to raft-like membranesand thus that lipid rafts are present in late endosomes (inaddition to the Golgi and the plasma membrane). In marked contrast,endocytosed GPI-APs reach the recycling endosome in CHO cellsand this transport correlates with a decreased raft association.GPI-APs are, however, diverted from the recycling endosome androuted to late endosomes in CHO cells, when their raft associationis increased by clustering seven or less GPI-APs with an aerolysinmutant. We conclude that the different endocytic routes followedby GPI-APs in different cell types depend on the residence timeof GPI-APs in lipid rafts, and hence that raft partitioningregulates GPI-APs sorting in the endocytic pathway.  

3.296           Akt1 regulates a JNK scaffold during excitotoxic apoptosis

Kim, A.H. et al Neuron, 35, 697-709 (2002)   Cell survival is determined by a balance among signaling cascades, including those that recruit the Akt and JNK pathways. Here we describe a novel interaction between Akt1 and JNK interacting protein 1 (JIP1), a JNK pathway scaffold. Direct association between Akt1 and JIP1 was observed in primary neurons. Neuronal exposure to an excitotoxic stimulus decreased the Akt1-JIP1 interaction and concomitantly increased association between JIP1 and JNK. Akt1 interaction with JIP1 inhibited JIP1-mediated potentiation of JNK activity by decreasing JIP1 binding to specific JNK pathway kinases. Consistent with this view, neurons from Akt1-deficient mice exhibited higher susceptibility to kainate than wild-type littermates. Overexpression of Akt1 mutants that bind JIP1 reduced excitotoxic apoptosis. These results suggest that Akt1 binding to JIP1 acts as a regulatory gate preventing JNK activation, which is released under conditions of excitotoxic injury.  

3.297           Hsp90 interactions and acylation target the G protein Ga₁₂ but not Ga₁₃ to lipid rafts

Waheed, A.A. and Jones, T.L.Z.,

1. Biol. Chem., 277(36), 32409-32412 (2002)

  The heterotrimeric G proteins, G₁₂ and G₁₃, are closely related in their sequences, signaling partners, and cellular effects such as oncogenic transformation and cytoskeletal reorganization. Yet G₁₂ and G₁₃ can act through different pathways, bind different proteins, and show opposing actions on some effectors. We investigated the compartmentalization of G₁₂ and G₁₃ at the membrane because other G proteins reside in lipid rafts, membrane microdomains enriched in cholesterol and sphingolipids. Lipid rafts were isolated after cold, nonionic detergent extraction of cells and gradient centrifugation. Ga₁₂ was in the lipid raft fractions, whereas Ga₁₃ was not associated with lipid rafts. Mutation of Cys-11 on Ga₁₂, which prevents its palmitoylation, partially shifted Ga₁₂ from the lipid rafts. Geldanamycin treatment, which specifically inhibits Hsp90, caused a partial loss of wild-type Ga₁₂ and a complete loss of the Cys-11 mutant from the lipid rafts and the appearance of a higher molecular weight form of Ga₁₂ in the soluble fractions. These results indicate that acylation and Hsp90 interactions localized Ga₁₂ to lipid rafts. Hsp90 may act as both a scaffold and chaperone to maintain a functional Ga₁₂ only in discrete membrane domains and thereby explain some of the nonoverlapping functions of Ga₁₂ and Ga₁₃ and control of these potent cell regulators.  

3.298           Complex N-linked glycosylated nicastrin associates with active g-secretase and undergoes tight cellular regulation

Taylor Kimberly, W. et al

1. Biol. Chem., 277(38), 35113-35117 (2002)

  The intramembranous proteolysis of Notch and the amyloid precursor protein by g-secretase exemplifies an unusual and newly recognized mechanism of signal transduction in multicellular organisms. Here, we show that only a form of nicastrin (NCT) containing N-linked complex oligosaccharides is present in active g-secretase complexes. Overexpression of NCT does not generate more of this mature protein, a phenomenon analogous to the strictly regulated formation of mature presenilin heterodimers from immature holoprotein. The absence of presenilin severely limits the maturation of NCT, yet combined overexpression of both proteins does not increase respective mature types. Taken together, our findings describe unusual regulatory features of this key signaling protease: the association of NCT with g-secretase is tightly regulated via glycosylation; at least one other cofactor exists; the least abundant member of the complex becomes limiting; and the cofactor that serves this role may vary by cell type.  

3.299           FATS1 channels exogenous FA into 1,2,3-triacyl-sn-glycerol and down-regulates sphingomyelin and cholesterol metabolism in growing 293 cells

Hatch, G.M., Smith, A.J., Xu, F.Y., Hall, A.M. and Bernlohr, D.A.

1. Lipid. Res., 43, 1380-1389 (2002)

  Biosynthesis of lipids was investigated in growing 293 cells stably expressing fatty acid (FA) transport protein 1 (FATP1), a bifunctional polypeptide with FA transport as well as fatty acyl-CoA synthetase activity. In short-term (30 s) incubations, FA uptake was increased in FATP1 expressing cells (C8 cells) compared with the vector (as determined by BODIPY 3823 staining and radioactive FA uptake). In long-term (4 h) incubations, incorporation of [¹⁴C]acetate, [3H]oleic acid, or [¹⁴C]lignoceric acid into 1,2,3-triacyl-sn-glycerol (TG) was elevated in C8 cells compared with vector, whereas incorporation of radiolabel into glycerophospholipids was unaltered. The increase in TG biosynthesis correlated with an increase in 1,2-diacyl-sn-glycerol acyltransferase activity in C8 cells compared with vector. In contrast, incorporation of [¹⁴C]acetate into sphingomyelin (SM) and cholesterol, and [3H]oleic acid or [¹⁴C]lignoceric acid into SM was reduced due to a reduction in de novo biosynthesis of these lipids in C8 cells compared with vector. The results indicate that exogenously supplied FAs, and their subsequently produced acyl-CoAs, are preferentially channeled by an FATP1 linked mechanism into the TG biosynthetic pathway and that such internalized lipids down-regulate de novo SM and cholesterol metabolism in actively growing 293 cells.  

3.300           Inversin forms a complex with catenins and N-cadherin in polarized epithelial cells

Nürnberger, J., Bacallao, R.L. and Phillips, C.P. Mol. Biol. Cell, 13, 3096-3106 (2002)   Nephrogenesis starts with the reciprocal induction of two embryonically distinct analages, metanephric mesenchyme and ureteric bud. This complex process requires the refined and coordinated expression of numerous developmental genes, such as inv. Mice that are homozygous for a mutation in the inv gene (inv/inv) develop renal cysts resembling autosomal-recessive polycystic kidney disease. The gene locus containing inv has been proposed to serve as a common modifier for some human and rodent polycystic kidney disease phenotypes. We generated polyclonal antibodies to inversin to study its subcellular distribution, potential binding partners, and functional aspects in cultured murine proximal tubule cells. A 125-kDa inversin protein isoform was found at cell-cell junctions. Two inversin isoforms, 140- and 90-kDa, were identified in the nuclear and perinuclear compartments. Plasma membrane allocation of inversin is dependent upon cell-cell contacts and was redistributed when cell adhesion was disrupted after incubation of the cell monolayer with low-calcium/EGTA medium. We further show that the membrane-associated 125-kDa inversin forms a complex with N-cadherin and the catenins. The 90-kDa nuclear inversin complexes with b-catenin. These findings indicate that the inv gene product functions in several cellular compartments, including the nucleus and cell-cell adhesion sites.  

3.301           Differential localization of the vacuolar H⁺ pump with G subunit isoforms (G1 and G2) in mouse neurons

Murata, Y. et al

1. Biol. Chem., 277(39), 36296-36303 (2002)

  Vacuolar H⁺-ATPases (V-ATPases), a family of multimeric proton pumps, are involved in a wide variety of physiological processes. We have identified two mouse genes, Atp6g1 and Atp6g2, encoding the G1 and G2 isoforms of the V-ATPase G subunit, respectively. G1 was distributed ubiquitously in the tissues examined, whereas G2 was specifically distributed in central nervous system neurons. G1 was expressed at an early embryonic stage, whereas G2 transcription was significantly induced at 10.5 days postcoitus (embryonic day 10.5, i.e. 2 days before axon outgrowth). Both G1 and G2 were strongly expressed in cortical and hippocampal neurons, cerebellar granule cells, and Purkinje cells. Immunohistochemistry with isoform-specific antibodies revealed that G2 was localized in cell bodies, dendrites, and axons. In addition, electron microscopy and subcellular fractionation indicated that G2 was localized in synaptic vesicles, whereas G1 was not detectable. G1 and G2 exhibit 62% identity, and both isoforms were immunoprecipitated with the c and A subunits of V-ATPase. G2 could complement the yeast deletion mutant Dvma10, which lacks the G subunit. The V-ATPases containing the G1 and G2 isoforms, respectively, showed similar K_m(ATP) values and maximal velocity. These results indicate that G1 and G2 are bona fide subunits of V-ATPases and that the enzyme with the G2 isoform is involved in synaptic vesicle  acidification.  

3.302           Myristoylation as a target for inhibiting HIV assembly: Unsaturated fatty acids block viral budding

Lindwasser, O.W. and Resh, M.D. Proc. Natl. Acad. Sci.,99(20), 13037-13042 (2002)   Modification of HIV-1 Gag with myristic acid, a saturated 14-carbon fatty acid (14:0), is essential for HIV-1 assembly. We recently showed that exogenous treatment of cells with unsaturated 14-carbon fatty acids, 5-cis-tetradecenoic acid (14:1n-9) and 5-cis,8-cis-tetradecadienoic acid (14:2n-6), reduces the affinity of some myristoylated proteins for plasma membrane rafts, membrane subdomains that have been shown to be required for efficient assembly of HIV. Here we demonstrate that treatment of cells with 14:1n-9 and 14:2n-6 fatty acids reduced the affinity of Gag for rafts but not membranes in general. Furthermore, treatment of cells with 14-carbon unsaturated fatty acids inhibited Gag-driven particle assembly. These effects most likely reflect covalent modification of Gag with unsaturated fatty acids. Treatment with 14:1n-9 and 14:2n-6 fatty acids did not alter intracellular protein trafficking, nor did it reduce cell viability. These studies suggest a strategy to attack HIV assembly by selectively altering the patterns of fatty acid modification.  

3.303           Elevation of cytochrome P450-immunopositive protein and DNA damage in mussels (Mytilus edulis) transplanted to a contaminated site

Shaw, J.P., Large, A.T., Livingstone, D.R., Doyotte, A., Renger, J., Chipman, J.K. and Peters, L.D. Marine Environ. Res., 54, 505-509 (2002)   Mytilus edulis were collected from a reference site (Port Quin) and an urban/industrial contaminated site (New Brighton) in the UK during June 1999.Levels of PCBs (S7 congeners) and CB-138 were determined to be, respectively, 21 fold and 16 fold higher in the mussel digestive glands from New Brighton. Levels of CYP1A-immunopositive protein were 1.5 fold higher (P<0.05) at the polluted site but the levels of DNA strand breaks were 1.3 fold higher (P<0.05) at the reference site. Mussels from Port Quin were placed in cages at both sites and both transplanted and indigenous populations sampled in September (13 weeks). Mussels transplanted from the reference site to the industrial site, reported elevated levels of CYP1A-immunopositive protein (1.4 fold; P<0.05) and higher levels of DNA damage (1.2 fold; P<0.05) compared to caged populations at the reference site and a PCB loading similar to the populations from the polluted site.Moreover, transplanted mussels had DNA damage 1.8 fold greater (P<0.05) than indigenous mussels at the transplant site. These changes were small but significant when compared to the observed temporal changes in the indigenous populations.  

3.304           Rat liver acyl-CoA synthetase 4 is a peripheral-membrane protein located in two distinct subcellular organelles, peroxisomes and mitochondrial-associated membranes

Lewin T.M., Van Horn, C.G., Krisans, S.K. and Coelman, R.A. Arch. Biochem. Biophys., 404, 263-270 (2002)   Obesity and non-insulin-dependent diabetes favor storage of fatty acids in triacylglycerol over oxidation. Recently, individual acyl-CoA synthetase (ACS) isoforms have been implicated in the channeling of fatty acids either toward lipid synthesis or toward oxidation. Although ACS1 had been localized to three different subcellular regions in rat liver, endoplasmic reticulum, mitochondria, and peroxisomes, the study had used an antibody raised against the full-length ACS1 protein which cross-reacts with other isoforms, probably because all ACS family members contain highly conserved amino acid sequences. Therefore, we examined the subcellular location of ACS1, ACS4, and ACS5 in rat liver to determine which isoform was present in peroxisomes, whether the ACSs were intrinsic membrane proteins, and which ACS isoforms were up-regulated by PPARa ligands. Non-cross-reacting ACS1, ACS4, and ACS5 peptide antibodies showed that ACS4 was the only ACS isoform present in peroxisomes isolated from livers of gemfibrozil-treated rats. ACS4 was also present in fractions identified as mitochondria associated membrane (MAM). ACS1 was present in endoplasmic reticulum fractions and ACS5 was present in mitochondrial fractions. Incubation with troglitazone, a specific inhibitor of ACS4, decreased ACS activity in the MAM fractions 30–45% and in the peroxisomal fractions about 30%. Because the signal for ACS4 protein in peroxisomes was so strong compared to the MAM fraction, we examined ACS4 mRNA abundance in livers of rats treated with the PPARa agonist GW9578. Treatment with GW9578 increased ACS4 mRNA abundance 40% and ACS1 mRNA 25%. Although we had originally proposed thatACS4 is linked to triacylglycerol synthesis, it now appears that ACS4 may also be important in activating fatty acids destined for peroxisomal oxidation. We also determined that, unlike ACS1 and 5, ACS4 is not an intrinsic membrane protein. This suggests that ACS4 is probably targeted and linked to MAM and peroxisomes by interactions with other proteins.  

3.305           Multicompartmental distribution of tuberous sclerosis gene products, hamartin and tuberin

Yamamoto, Y., Jones, K.A., Mak, B.C., Muehlenbachs, A. and Yeung, R.S. Arch. Biochem. Biophys., 404, 210-217 (2002)   Mutations of the TSC1 and TSC2 genes give rise to the clinical disorder of tuberous sclerosis characterized by the development of hamartomas predominantly affecting the central nervous system, kidney, skin, lung, and heart. The function of the gene products, hamartin and tuberin, is not well understood but we have previously suggested a role in vesicular transport. To define the subcellular compartment(s) involved with these two proteins, biochemical characterization of hamartin and tuberin was performed in primary tissues and cell lines. Fractionation of cell lysates identifed both proteins in the cytosolic, microsomal, and cytoskel-etal compartments. In each of these fractions, hamartin and tuberin formed a stable complex in coimmuno-precipitation analyses. Further, they colocalized extensively in discrete, vesicular structures in the cyto-plasm. Within the microsomal compartment, hamartin and tuberin behaved as peripheral membrane proteins that associate with the cytosolic leaflet of membranous domains. Immunoisolation of tuberin-bound vesicles using magnetic beads showed an enrichment of rap1, rab5, and caveolin-1, all of which have been found in specialized lipid microdomains, caveolae. Our data suggest that hamartin and tuberin are multicompart-mental proteins that partially reside in caveolin-1-enriched structures and potentially affect their signaling.  

3.306           Sphingolipid and cholesterol dependence of alphavirus membrane fusion

Waarts, B-L., Bittman, R. and Wilschutt, J.

1. Biol. Chem., 277(41), 38141-38147 (2002)

  Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped viruses that infect their host cells by receptor-mediated endocytosis and subsequent fusion from within acidic endosomes. Fusion of the viral envelope requires the presence of both cholesterol and sphingolipids in the target membrane. This is suggestive of a possible involvement of sphingolipid-cholesterol microdomains, or "lipid rafts," in the membrane fusion and cell entry process of the virus. In this study, large unilamellar vesicles (LUVs) were prepared from synthetic sphingolipids and sterols that vary with respect to their capacity to promote microdomain formation, as assessed by gradient flotation analysis in the presence of Triton X-100. SFV and SIN fused with LUVs irrespective of the presence or absence of Triton X-100-insoluble microdomains. These results suggest that SFV and SIN do not require the presence of lipid rafts for fusion with target membranes. Furthermore, it is not necessary for sphingolipids to reside in a detergent-insoluble complex with cholesterol to promote SFV or SIN fusion.  

3.307           ER-X: A novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury

Toran-Allerand, C.D. et al

1. Neurosci., 22(19), 8391-8401 (2002)

  We showed previously in neocortical explants, derived from developing wild-type and estrogen receptor (ER)-a gene-disrupted (ERKO) mice, that both 17a- and 17b-estradiol elicit the rapid and sustained phosphorylation and activation of the mitogen-activated protein kinase (MAPK) isoforms, the extracellular signal-regulated kinases ERK1 and ERK2. We proposed that the ER mediating activation of the MAPK cascade, a signaling pathway important for cell division, neuronal differentiation, and neuronal survival in the developing brain, is neither ER-a nor ER-b but a novel, plasma membrane-associated, putative ER with unique properties. The data presented here provide further evidence that points strongly to the existence of a high-affinity, saturable, ³H-estradiol binding site (K_d, ~1.6 nM) in the plasma membrane. Unlike neocortical ER-a, which is intranuclear and developmentally regulated, and neocortical ER-b, which is intranuclear and expressed throughout life, this functional, plasma membrane-associated ER, which we have designated "ER-X," is enriched in caveolar-like microdomains (CLMs) of postnatal, but not adult, wild-type and ERKO neocortical and uterine plasma membranes. We show further that ER-X is functionally distinct from ER-a and ER-b, and that, like ER-a, it is re-expressed in the adult brain, after ischemic stroke injury. We also confirmed in a cell-free system that ER-a is an inhibitory regulator of ERK activation, as we showed previously in neocortical cultures. Association with CLM complexes positions ER-X uniquely to interact rapidly with kinases of the MAPK cascade and other signaling pathways, providing a novel mechanism for mediation of the influences of estrogen on neuronal differentiation, survival, and plasticity.  

3.308           A novel monoclonal antibody recognizes lysosome-like structures and reflects regional and age-related differences in the rat dentate gyrus

Maeda, S. Et al Neurosci. Lett., 330, 275-279 (2002)   The granule cells (GCs) of dentate gyrus exhibit regionally specific morphology, and continue to be born and to develop well into adult life. We used a novel monoclonal antibody, MAb2G7, elicited by immunization of a mouse with a microsome fraction of the hippocampus, to evaluate regional and age-related differences in GCs immunohistochemically. Weak cytoplasmic reactions were observed in many neurons, but intense MAb2G7-positive dots were observed only in GCs. Using electron microscopy, we observed that these dots were localized in the internal droplets of secondary lysosome-like structures in GCs. The MAb2G7-positive granules were quantitatively analyzed in young adult and middle-aged rats. Larger numbers of reactive granules were observed in the infrapyramidal blade (IPB) than in the suprapyramidal blade (SPB) and the numbers of positive granules were proportionally reduced in the two areas in middle-aged rats. The changes in the MAb2G7 immunoreactivity may reflect different activation or neurogeneration of GCs in the IPB versus the SPB, and in middle-aged versus young adult rats.  

3.309           Nuclear translocation of extracellular superoxide dismutase

Ookawara, T. et al Biochem. Biophys. Res. Comm., 296, 54-61 (2002)   Histochemical examination of mouse tissues showed nuclear staining of extracellular superoxide dismutase (EC-SOD), and the nuclear translocation of EC-SOD was also confirmed in cultured cells that had been transfected with its gene, as shown by immunohistochemistry and Western blot analysis. The EC-SOD which was secreted into the medium was incorporated into 3T3-L1 cells and a significant fraction of the material taken up was localized in the nucleus. Site-directed mutagenesis indicated that the heparin-binding domain of EC-SOD functions as the nuclear localization signal. These results suggest that the mechanism of the nuclear transport of EC-SOD involves a series of N-terminal signal peptide- and C-terminal heparin-binding domain-dependent processes of secretion, re-uptake and the subsequent nuclear translocation. The findings herein provide support for the view that the role of EC-SOD is to protect the genome DNA from damage by reactive oxygen species and/or the transcriptional regulation of redox-sensitive gene expression.  

3.310           Initial steps of Shigella infection depend on the cholesterol/shingolipid raft-mediated CD44-IpaB interaction

Lafont, F. Et al The EMBO J., 21(17), 4449-4457 (2002)   Shigellosis is an acute inflammatory bowel disease caused by the enteroinvasive bacterium Shigella. Upon host cell–Shigella interaction, major host cell signalling responses are activated. Deciphering the initial molecular events is crucial to understanding the infectious process. We identified a molecular complex involving proteins of both the host, CD44 the hyaluronan receptor, and Shigella, the invasin IpaB, which partitions during infection within specialized membrane microdomains enriched in cholesterol and sphingolipids, called rafts. We also document accumulation of cholesterol and raft-associated proteins at Shigella entry foci. Moreover, we report that Shigella entry is impaired after cholesterol depletion using methyl-ß-cyclodextrin. Finally, we find that Shigella is less invasive in sphingosid-based lipid-deficient cell lines, demonstrating the involvement of sphingolipids. Our results show that rafts are implicated in Shigella binding and entry, suggesting that raft-associatedmolecular machineries are engaged in mediating the cell signallingresponse required for the invasion process.  

3.311           Plasma membrane phospholipid scamblase 1 is enriched in lipid rafts and interacts with the epidermal growth factor receptor

Sun, J., Nanjundan, M., Pike, L.J., Wiedmer, T. and Sims, P.J. Biochemistry, 41, 6338-6345 (2002)   We have identified physical and functional interactions between the epidermal growth factor (EGF) receptor and phospholipid scramblase 1 (PLSCR1), an endofacial plasma membrane protein proposed to affect phospholipid organization. PLSCR1, a palmitoylated protein, was found to partition with the EGF receptor in membrane lipid rafts. Cell stimulation with EGF transiently elevated Tyr-phosphorylation of PLSCR1, peaking at 5 min. Although PLSCR1 is known substrate of c-Ab1 (Sun, J., et al. (2001) J. Biol. Chem. 276, 28984-28990), the Ab1 inhibitor STI571 did not substantially affect its EGF-dependent phosphorylation, suggesting PLSCR1 is a substrate of the EGF receptor kinase, or another EGF-activated kinase. Coinciding with phosphorylation, there was a transient increase in physical association of PLSCR1 with both the EGF receptor and the adapter protein Shc, as determined by immunoprecipitation and Western blotting. Confocal immunofluorescence analysis revealed that EGF initiates rapid internalization of both the EGF receptor and PLSCR1, with trafficking into both distinct and common endosomal pools. These data also suggested that whereas the EGF receptor is ultimately degraded, much of the endocytosed PLSCR1 is recycled to the cell surface with 3 h after EGF treatment. Consistent with this interpretation. Western blotting revealed neither ubiquitination nor proteolysis of PLSCR1 under these conditions, whereas the ubiquitination and degradation of the EGF receptor were rapidly confirmed. Finally, stimulation with EGF was also found to markedly increase the total cellular expression of PLSCR1, suggesting that in addition to its initial interactions with activated EGF receptor, PLSCR1 may also contribute to posttranscriptional effector pathway(s) mediating the cellular response to EGF.  

3.312           Truncated soluble Nogo receptor binds Nogo-66 and blocks inhibition of axon growth by myelin

Fournier, A.E., Gould, G.C., Liu, B.P. and Strittmatter, S.M.

1. Neurosci., 22(20), 8876-8883 (2002)

  CNS myelin contains axon outgrowth inhibitors, such as Nogo, that restrict regenerative growth after injury. An understanding of the mechanism of Nogo signaling through its receptor (NgR) is critical to developing strategies for overcoming Nogo-mediated inhibition. Here we analyze the function of NgR domains in outgrowth inhibition. Analysis of alkaline phosphatase (AP)-Nogo binding in COS-7 cells reveals that the leucine-rich repeat domain is necessary and sufficient for Nogo binding and NgR multimerization. Viral infection of embryonic day 7 chick retinal ganglion cells with mutated NgR demonstrates that the NgR C-terminal domain is required for inhibitory signaling but not ligand binding. The NgR glycosylphosphatidylinositol domain is not essential for inhibitory signaling but may facilitate Nogo responses. From this analysis, we have developed a soluble, truncated version of the Nogo receptor that antagonizes outgrowth inhibition on both myelin and Nogo substrates. These data suggest that NgR mediates a significant fraction of myelin inhibition of axon outgrowth.  

3.313           Cell surface polarization during yeast mating

Bagnat, M. and Simons, K. Proc. Natl. Acad. Sci., 99(22), 14283-14188 (2002)   Exposure to mating pheromone in haploid Saccharomyces cerevisiae cells results in the arrest of the cell cycle, expression of mating-specific genes, and polarized growth toward the mating partner. Proteins involved in signaling, polarization, cell adhesion, and fusion are localized to the tip of the mating cell (shmoo) where fusion will eventually occur. The mechanisms ensuring the correct targeting and retention of these proteins are poorly understood. Here we show that in pheromone-treated cells, a reorganization of the plasma membrane involving lipid rafts results in the retention of proteins at the tip of the mating projection, segregated from the rest of the membrane. Sphingolipid and ergosterol biosynthetic mutants fail to polarize proteins to the tip of the shmoo and are deficient in mating. Our results show that membrane microdomain clustering at the mating projection is involved in the generation and maintenance of polarity during mating.  

3.314           RNA incorporation is critical for retroviral particle integrity after cell membrane assembly of Gag complexes

Wang, S-W. and Aldovini, A.

1. Virol., 76(23), 11853-11865 (2002)

  The nucleocapsid (NC) domain of retroviruses plays a critical role in specific viral RNA packaging and virus assembly. RNA is thought to facilitate viral particle assembly, but the results described here with NC mutants indicate that it also plays a critical role in particle integrity. We investigated the assembly and integrity of particles produced by the human immunodeficiency virus type 1 M1-2/BR mutant virus, in which 10 of the 13 positive residues of NC have been replaced with alanines and incorporation of viral genomic RNA is virtually abolished. We found that the mutations in the basic residues of NC did not disrupt Gag assembly at the cell membrane. The mutant Gag protein can assemble efficiently at the cell membrane, and viral proteins are detected outside the cell as efficiently as they are for the wild type. However, only ~10% of the Gag molecules present in the supernatant of this mutant sediment at the correct density for a retro-viral particle. The reduction of positive charge in the NC basic domain of the M1-2/BR virus adversely affects both the specific and nonspecific RNA binding properties of NC, and thus the assembled Gag poly-protein does not bind significant amounts of viral or cellular RNA. We found a direct correlation between the % of Gag associated with sedimented particles and the amount of incorporated RNA. We con-clude that RNA binding by Gag, whether the RNA is viral or not, is critical to retroviral particle integrity after cell membrane assembly and is less important for Gag-Gag interactions during particle assembly and release.  

3.315           Proteomic analysis of a detergent-resistant membrane skeleton from neutrophil plasma membranes

Nebl, T. et al

1. Biol. Chem., 277(45), 43999-43409 (2002)

  Plasma membranes are organized into functional domains both by liquid-ordered packing into "lipid rafts," structures that resist Triton extraction, and by attachments to underlying cytoskeletal proteins in assemblies called "membrane skeletons." Although the actin cytoskeleton is implicated in many lipid raft-mediated signaling processes, little is known about the biochemical basis for actin involvement. We show here that a subset of plasma membrane skeleton proteins from bovine neutrophils co-isolates with cholesterol-rich, detergent-resistant membrane fragments (DRMs) that exhibit a relatively high buoyant density in sucrose (DRM-H; d ~1.16 g/ml). By using matrix-assisted laser desorption/ionization time of flight and tandem mass spectrometry, we identified 19 major DRM-H proteins. Membrane skeleton proteins include fodrin (nonerythroid spectrin), myosin-IIA, myosin-IG, a-actinin 1, a-actinin 4, vimentin, and the F-actin-binding protein, supervillin. Other DRM-H components include lipid raft-associated integral membrane proteins (stomatin, flotillin 1, and flotillin 2), extracellular surface-bound and glycophosphatidylinositol-anchored proteins (IgM, membrane-type 6 matrix metalloproteinase), and intracellular dually acylated signaling proteins (Lyn kinase, Ga_i-2). Consistent with cytoskeletal association, most DRM-H-associated flotillin 2, Lyn, and Ga_i-2 also resist extraction with 0.1 M octyl glucoside. Supervillin, myosin-IG, and myosin-IIA resist extraction with 0.1 M sodium carbonate, a treatment that removes all detectable actin, suggesting that these cytoskeletal proteins are proximal to the DRM-H bilayer. Binding of supervillin to the DRM-H fragments is confirmed by co-immunoaffinity purification. In spreading neutrophils, supervillin localizes with F-actin in cell extensions and in discrete basal puncta that partially overlap with Ga_i staining. We suggest that the DRM-H fraction represents a membrane skeleton-associated subset of leukocyte signaling domains.  

3.316           Presence of detergent-resistant microdomains in lysosomal membranes

Taute, A. et al Biochem. Biophys. Res. Comm., 298(3), 5-9 (2002)   We examined the association of acetyl-CoA:a -glucosaminide N-acetyltransferase, a lysosomal enzyme participating in the degradation of heparan sulfate with other components of the lysosomal membrane. We prepared lysosomal membranes from human placenta and treated them with zwitterionic and non-ionic detergents. Membrane proteins were solubilized either in the presence of CHAPS at room temperature or of Triton X-100 at 4 °C. The CHAPS-containing extract was subjected to gel filtration in a column with the nominal size exclusion of 0.6 MDa. Under these conditions the enzyme fractionated near the void volume. To examine the association of the enzyme with detergent-resistant lipid microdomains, the extract that had been prepared with Triton X-100 was subjected to flotation in a density gradient medium. After centrifug-ation, a major portion of the activity of the acetyltransferase was found at the top of the gradient along with the bulk of alkaline phosphatase. Alkaline phosphatase is a glycosylphosphatidylinositol-anchored protein; possibly a contaminant in the lysosomal fraction originating from the plasma membrane and adventitiously an internal control for the flotation in the gradient. In contrast, acetyltransferase is a genuine lysosomal protein that obligatorily spans the membrane since it transfers acetyl residues from acetyl-CoA in cytosol to glucosaminyl residues in heparan sulfate fragments in the lysosomal matrix. To our knowledge this is the first report on association of a lysosomal membrane protein with detergent-resistant membrane microdomains or rafts.  

3.317           A specific binding protein/receptor for 1a,25-dihydroxyvitamin D₃ is present in an intestinal caveolae membrane fraction

Norman, A.W., Olivera, C.J., Silva, F.R.M.B. and Bishop, J.E. Biochem. Biophys. Res. Comm., 298(3), 414-419 (2002)   The steroid hormone 1a,25-dihydroxyvitamin D₃ [1a,25(OH)₂D₃] produces biological responses by interaction with both a well-characterized nuclear receptor (VDR_nuc) to regulate gene transcription and with an as-yet uncharacterized membrane-associated protein/receptor (VDR_mem) to generate a variety of rapid, non-genotropic responses. We report for the first time that [³H]1a,25(OH)₂D₃ binds with high affinity to a chick duodenal caveolae-enriched membrane fraction (CMF) isolated without the use of detergents. Caveolae are plasma membrane invaginations implicated in signal transduction and molecular transport processes. Using the CMF fraction as a possible source of VDR_mem, we found that the in vitro binding of [³H]1a,25(OH)₂D₃ was ligand dependent and saturable; the K_D and B_max were 1.3±0.6 nM and 29±11 fmol 1,25(OH)₂D₃/mg protein (n=17), respectively. Immunoblot analysis of the CMF confirms the presence of caveolin-1, a marker protein for membranes with caveolae. Therefore, chick CMF may represent a good source for isolation and characterization of the putative VDR_mem for 1a,25(OH)₂D₃.  

3.318           Novel non-labile covalent binding of sulfamethoxazole reactive metabolites to cultured human lymphoid cells

Summan, M. and Cribb, A.E. Chemico-Biol. Interactions, 142, 155-173 (2002)   Sulfamethoxazole (SMX) causes rare hypersensitivity syndrome reactions characterized by fever and multi-organ toxicity. Covalent binding of SMX reactive metabolites to cellular proteins has been demonstrated but the link between cytotoxicity and targets of covalent binding has not been explored. We therefore invest-igated the relationship between covalent binding of the reactive SMX-hydroxylamine (SMX-HA) metabo-lite, and its cytotoxicity to a hystiocytic lymphoma (U937) cell line. Incubation of U937 cells with 0–1 mM SMX-HA for 3 h resulted in dose-dependent cytotoxicity, as assessed by tetrazolium dye conversion at 24 h. SMX-HA caused dose-dependent covalent binding to cellular proteins as assessed by immunoblotting with SMX antisera at 3 and 24 h. Covalent binding was predominantly to proteins of approximately 45, 59 and 75 kDa, but other targets were also observed. The relative extent of binding to proteins was significantly different from the relative cytotoxicity at 24 h. Further, cells surviving at 24 h also had extensive covalent binding. Covalent binding was observed under reducing (b-mercaptoethanol) and non-reducing conditions to plasma membrane and microsomal but not cytosolic proteins. This non-labile covalent binding has not been previously reported. These observations suggest that extensive covalent binding does not necessarily lead to cell death, allowing the accumulation of potentially immunogenic drug-protein conjugates. These observations in whole cells may be relevant to the immunopathogenesis of SMX hypersensitivity syndrome reactions.  

3.319           Molecular mechanism for orienting membrane and actin dynamics to nascent cell-cell contacts in epithelial cells

Hansen, M.D., Ehrlich, J.S. and Nelson, W.J.

1. Biol. Chem., 277(47), 45371-45376 (2002)

  The small GTPase Rac1 has been implicated in regulation of cell migration and cell-cell adhesion in epithelial cells. Little is known, however, about the spatial and temporal coordination of Rac1 activity required to balance these competing processes. We fractionated endogenous Rac1-containing protein complexes from membranes of Madin-Darby canine kidney cells and identified three major complexes comprising a Rac1·PAK (p21-activated kinase) complex, and 11 S and 16 S Rac1 complexes. Significantly, Rac1 shifts from the 11 S to a 16 S particle during initiation of cell-cell adhesion. This shift may reflect a diffusion trapping mechanism by which these Rac1 complexes are localized to cadherin-mediated cell-cell contacts through an interaction with annexin II.  

3.320           Regulated expression and intracellular localization of cystatin F in human U937 cells

Nathanson, C-M, Wasselius, J., Wallin, H. and Abrahamson, M. Eur. J. Biochem., 269, 5502-5511 (2002)   Cystatin F is a cysteine peptidase inhibitor recently discovered in haematopoietic cells by cDNA cloning. To further investigate the expression, distribution and properties of the native human inhibitor the promyeloid cell line U937 has been studied. The cells expressed relatively large quantities of cystatin F, which was found both secreted and intracellularly. The intracellular levels were unusually high for a secreted cystatin (»25% of the cystatin F in 2- or 4-day culture medium). By contrast, U937 cells contained only 3–4% of the related inhibitor, cystatin C. Cystatin F purified from lysates of U937 cells showed three major forms carrying two, one or no carbohydrate chains. Immunocytochemistry demonstrated a marked cytoplasmic cystatin F staining in a granular pattern. Double staining with a marker for endoplasmic reticulum revealed no colocal-ization for cystatin F. Analysis of the promoter region of the cystatin F gene (CST7) showed that it, like that of the cystatin C gene (CST3), is devoid of typical TATA- and CAAT-box elements. In contrast to the cyst-atin C promoter, it does not contain multiple Sp1 binding sites, but has a unique site for C/EBPa, possibly explaining the restricted expression of the cystatin F gene. Cells stimulated with all-trans retinoic acid to differentiate them towards a granulocytic pathway, showed a strong (»18-fold) down-regulation of intra-cellular cystatin F and almost abolished secreted levels of the inhibitor. Stimulation with tetradecanoyl phorbol acetate, causing monocytic differentiation, also resulted in down-regulation (two fold to threefold) of cystatin F expression, whereas the cystatin C expression was essentially unaltered in both experiments. The results suggest that cystatin F as an intracellular cysteine peptidase inhibitor with readily regulated expression, may be a candidate to control the cysteine peptidase activity known to be essential for antigen presentation in different blood cell lineages.  

3.321           The Ccz1-Mon1 protein complex is required for the late step of multiple vacuole delivery pathways

Wang, C-W, Stromhaug, P.E., Shima, J. and Klionsky, J.

1. Biol. Chem., 277(49), 47917-47927 (2002)

  Mon1 and Ccz1 were identified from a gene deletion library as mutants defective in the vacuolar import of aminopeptidase I (Ape1) via the cytoplasm to vacuole targeting (Cvt) pathway. The mon1D and ccz1D strains also displayed defects in autophagy and pexophagy, degradative pathways that share protein machinery and mechanistic features with the biosynthetic Cvt pathway. Further analyses indicated that Mon1, like Ccz1, was required in nearly all membrane-trafficking pathways where the vacuole represented the terminal acceptor compartment. Accordingly, both deletion strains had kinetic defects in the biosynthetic delivery of resident vacuolar hydrolases through the CPY, ALP, and MVB pathways. Biochemical and microscopy studies suggested that Mon1 and Ccz1 functioned after transport vesicle formation but before (or at) the fusion step with the vacuole. Thus, ccz1D and mon1D are the first mutants identified in screens for the Cvt and Apg pathways that accumulate precursor Ape1 within completed cytosolic vesicles. Subcellular fractionation and co-immunoprecipitation experiments confirm that Mon1 and Ccz1 physically interact as a stable protein complex termed the Ccz1-Mon1 complex. Microscopy of Ccz1 and Mon1 tagged with a fluorescent marker indicated that the Ccz1-Mon1 complex peripherally associated with a perivacuolar compartment and may attach to the vacuole membrane in agreement with their proposed function in fusion.  

3.322           Lipid rafts are enriched in arachidonic acid and plasmenylethanolamine and their composition is independent of caveolin-1 expression: a quantitative electrospray ionization/mass spectrometric analysis

Pike, L.J., Han, X., Chung, K-N and Gross, R.W. Biochemistry, 41, 2075-2088 (2002)   Lipid rafts are specialized cholesterol-enriched membrane domains that participate in cellular signaling processes. Caveolae are related domains that become invaginated due to the presence of the structural protein, caveolin-1. In this paper, we use electrospray ionization mass spectrometry (ESI/MS) to quantitatively compare the phospholipids present in plasma membranes and nondetergent lipid rafts from caveolin-1-expressing and nonexpressing cells. Lipid rafts are enriched in cholesterol and sphingomyelin as compared to the plasma membrane fraction. Expression of caveolin-1 increases the amount of cholesterol recovered in the lipid raft fraction but does not affect the relative proportions of the various phospholipid classes. Surprisingly, ESI/MS demonstrated that lipid rafts are enriched in plasmenylethanolamines, particularly those containing arachidonic acid. While the total content of anionic phospholipids was similar in plasma membranes and nondetergent lipid rafts, the latter were highly enriched in phosphatidylserine but relatively depleted in phosphatidylinositol. Detergent-resistant membranes made from the same cells showed a higher cholesterol content than nondetergent lipid rafts but were depleted in anionic phospholipids. In addition, these detergent-resistant membranes were not enriched in arachidonic acid-containing ethanolamine plasmalogens. These data provide insight into the structure of lipid rafts and identify potential new roles for these domains in signal transduction.  

3.323           Endoproteolysis of presenilin in vitro: inhibition by g-secretase inhibitors

Campbell, W.A., Iskandar, M-K., Reed, M.L.O. and Xia, W. Biochemistry, 41, 3372-3379 (2002)   The final proteolytic step to generate the amyloid beta-protein (Abeta) of Alzheimer's disease (AD) from beta-amyloid precursor protein (APP) is achieved by presenilin (PS)-dependent gamma-secretase cleavage. AD-causing mutations in PS1 and PS2 result in a selective and significant increase in production of the more amyloidogenic Abeta42 peptide. PS1 and PS2 undergo endoproteolysis by an unknown enzyme termed presenilinase to generate the functional complex of N- and C-terminal fragments (NTF/CTF). To investigate the endoproteolytic activity that generates active PS, we used a mammalian cell-free system that allows de novo human PS NTF and CTF generation. PS NTF and CTF generation in vitro was observed in endoplasmic reticulum (ER)-enriched fractions of membrane vesicles and to a lesser extent in Golgi/trans-Golgi-network (TGN)-enriched fractions. AD-causing mutations in PS1 and PS2 did not alter de novo generation of PS fragments. Removal of peripheral membrane-associated and cytosolic proteins did not prevent de novo generation of fragments, indicating that presenilinase activity corresponds to an integral membrane protein. Among several general inhibitors of different protease classes that blocked the presenilinase activity, pepstatin A was the most potent inhibitor. Screening available transition state analogue gamma-secretase inhibitors led to the identification of two compounds that were able to prevent the de novo generation of PS fragments, with an expected inhibition of Abeta generation. Our studies provide a biochemical approach to characterize and identify this elusive presenilinase.  

3.324           Annular a-synuclein protofibrils are produced when spherical protofibrils are incubated in solution or bound to brain-derived membranes

Ding, T.T., Lee, S-J., Rochet, J-C. and Lansbury, Jr., P.T. Biochemistry, 41, 10209-10217 (2002)   The Parkinson's disease substantia nigra is characterized by the loss of dopaminergic neurons and the presence of cytoplasmic fibrillar Lewy bodies in surviving neurons. The major fibrillar protein of Lewy bodies is alpha-synuclein. Two point mutations in the alpha-synuclein gene are associated with autosomal-dominant Parkinson's disease (FPD). Studies of the in vitro fibrillization behavior of the mutant proteins suggest that fibril precursors, or alpha-synuclein protofibrils, rather than the fibrils, may be pathogenic. Atomic force microscopy (AFM) revealed two distinct forms of protofibrillar alpha-synuclein: rapidly formed spherical protofibrils and annular protofibrils, which were produced on prolonged incubation of spheres. The spherical protofibrils bound to brain-derived membrane fractions much more tightly than did monomeric or fibrillar alpha-synuclein, and membrane-associated annular protofibrils were observed. The structural features of alpha-synuclein annular protofibrils are reminiscent of bacterial pore-forming toxins and are consistent with their porelike activity in vitro. Thus, abnormal membrane permeabilization may be a pathogenic mechanism in PD.  

3.325           Characterization of isolated acidocalcisomes from Toxoplasma gondii Tachyzoites reveals a novel pool of hydrolysable polyphosphate

Rodrigues, C.O., Ruiz, F.A., Rohloff, P., Dcott, D.A. and Moreno, S.N.J.

1. Biol. Chem., 277(50), 48650-48656 (2002)

  Toxoplasma gondii tachyzoites were fractionated by modification of an iodixanol density gradient method previously used for acidocalcisome isolation from Trypanosoma cruzi epimastigotes. Fractions were characterized using electron microscopy, x-ray microanalysis, and enzymatic markers, and it was demonstrated that the heaviest (pellet) fraction contains electron-dense vacuoles rich in phosphorus, calcium, and magnesium, as found before for acidocalcisomes. Staining with 4',6-diamidino-2-phenylindole (DAPI) indicated that poly- phosphate (polyP) was preferentially localized in this fraction together with pyrophosphate (PP_i). Using an enzyme-based method, millimolar levels (in terms of P_i residues) of polyP chains of less than 50 residues long and micromolar levels in polyP chains of about 700-800 residues long were found to be preferentially localized in this fraction. The fraction also contained the pyrophosphatase and polyphosphatase activities characteristic of acidocalcisomes. Western blot analysis using antibodies against proteins from micronemes, dense granules, rhoptries, and plasma membrane showed that the acidocalcisomal fraction was not contaminated by these other organelles. T. gondii polyP levels rapidly decreased upon exposure of the parasites to a calcium ionophore (ionomycin), to an inhibitor of the V-H⁺-ATPase (bafilomycin A₁), or to the alkalinizing agent NH₄Cl. These changes were in parallel to an increase in intracellular Ca²⁺ concentration, suggesting a close association between polyP hydrolysis and Ca²⁺ release from the acidocalcisome. These results provide a useful method for the isolation and characterization of acidocalcisomes, showing that they are distinct from other previously recognized organelles present in T. gondii, and provide evidence for the role of polyP metabolism in response to cellular stress.  

3.326           Formation of mutually exclusive Rab11 complexes with members of the family of Rab11-interacting proteins regulates Rab11 endocytic targeting and function

Meyers, J.M. and Prekeris, R.

1. Biol. Chem., 277(50), 49003-49010 (2002)

  Several Rabs, including Rab11, regulate the traffic and sorting of proteins in the endosomal pathway. Recently, six novel Rab11 family interacting proteins (FIPs) were identified. Although they share little overall sequence homology, all FIPs contain a conserved Rab11-binding domain. Here we investigate the role of FIPs as Rab11-targeting proteins and show that the Rab11-binding domain assumes an a-helical structure, with the conserved residues forming a hydrophobic Rab11-binding patch. This hydrophobic patch mediates the formation of mutually exclusive complexes between Rab11 and various members of FIP protein family. Furthermore, the formation of Rab11/FIP complexes regulates Rab11 localization by recruiting it to distinct endocytic compartments. Thus, we propose that Rab11/FIP complexes serve as targeting patches, regulating Rab11 localization and recruitment of additional cellular factors to different endocytic compartments.  

3.327           A specific structural requirement for ergosterol in long-chain fatty acid synthesis mutants important for maintaining raft domains in yeast

Eisenkolb, M., Zenzmaier, C., Leitner, E., and Schneiter, R. Mol. Biol. Cell, 13, 4414-4428 (2002)   Fungal sphingolipids contain ceramide with a very-long-chain fatty acid (C26). To investigate the physiological significance of the C26-substitution on this lipid, we performed a screen for mutants that are synthetically lethal with ELO3. Elo3p is a component of the ER-associated fatty acid elongase and is required for the final elongation cycle to produce C26 from C22/C24 fatty acids. elo3D mutant cells thus contain C22/C24- instead of the natural C26-substituted ceramide. We now report that under these conditions, an otherwise nonessential, but also fungal-specific, structural modification of the major sterol of yeast, ergosterol, becomes essential, because mutations in ELO3 are synthetically lethal with mutations in ERG6. Erg6p catalyzes the methylation of carbon atom 24 in the aliphatic side chain of sterol. The lethality of an elo3D erg6D double mutant is rescued by supplementation with ergosterol but not with cholesterol, indicating a vital structural requirement for the ergosterol-specific methyl group. To characterize this structural requirement in more detail, we generated a strain that is temperature sensitive for the function of Erg6p in an elo3D mutant background. Examination of raft association of the GPI-anchored Gas1p and plasma membrane ATPase, Pma1p, in the conditional elo3D erg6^ts double mutant, revealed a specific defect of the mutant to maintain raft association of preexisting Pma1p. Interestingly, in an elo3D mutant at 37°C, newly synthesized Pma1p failed to enter raft domains early in the biosynthetic pathway, and upon arrival at the plasma membrane was rerouted to the vacuole for degradation. These observations indicate that the C26 fatty acid substitution on lipids is important for establishing raft association of Pma1p and stabilizing the protein at the cell surface. Analysis of raft lipids in the conditional mutant strain revealed a selective enrichment of ergosterol in detergent-resistant membrane domains, indicating that specific structural determinants on both sterols and sphingolipids are required for their association into raft domains.  

3.328           Lipid-dependent subcellular relocalization of the acyl chain desaturase in yeast

Tatzer, V., Zellnig, G., Kohlwein, S.D. and Schneiter, R. Mol. Biol. Cell, 13, 4429-4442 (2002)   The degree of acyl chain desaturation of membrane lipids is a critical determinant of membrane fluidity. Temperature-sensitive mutants of the single essential acyl chain desaturase, Ole1p, of yeast have previously been isolated in screens for mitochondrial inheritance mutants (Stewart, L.C., and Yaffe, M.P. (1991). J. Cell Biol. 115, 1249-1257). We now report that the mutant desaturase relocalizes from its uniform ER distribution to a more punctuate localization at the cell periphery upon inactivation of the enzyme. This relocalization takes place within minutes at nonpermissive conditions, a time scale at which mitochondrial morphology and inheritance is not yet affected. Relocalization of the desaturase is fully reversible and does not affect the steady state localization of other ER resident proteins or the kinetic and fidelity of the secretory pathway, indicating a high degree of selectivity for the desaturase. Relocalization of the desaturase is energy independent but is lipid dependent because it is rescued by supplementation with unsaturated fatty acids. Relocalization of the desaturase is also observed in cells treated with inhibitors of the enzyme, indicating that it is independent of temperature-induced alterations of the enzyme. In the absence of desaturase function, lipid synthesis continues, resulting in the generation of lipids with saturated acyl chains. A model is discussed in which the accumulation of saturated lipids in a microdomain around the desaturase could induce the observed segregation and relocalization of the enzyme.  

3.329           The mechanism of g-secretase activities through high molecular weight complex formation pof presenilins is conserved in Drosophila melanogaster and mammals

Takasugi, N., Takahashi, Y., Morohashi, Y. and Tomita, T.

1. Biol. Chem., 277(51), 50198-50205 (2002)

  Mutations in presenilin 1 (PS1) and PS2 genes contribute to the pathogenesis of early onset familial Alzheimer's disease by increasing secretion of the pathologically relevant Ab42 polypeptides. PS genes are also implicated in Notch signaling through proteolytic processing of the Notch receptor in Caenorhabditis elegans, Drosophila melanogaster, and mammals. Here we show that Drosophila PS (Psn) protein undergoes endoproteolytic cleavage and forms a stable high molecular weight (HMW) complex in Drosophila S2 or mouse neuro2a (N2a) cells in a similar manner to mammalian PS. The loss-of-function recessive point mutations located in the C-terminal region of Psn, that cause an early pupal-lethal phenotype resembling Notch mutant in vivo, disrupted the HMW complex formation, and abolished g-secretase activities in cultured cells. The overexpression of Psn in mouse embryonic fibroblasts lacking PS1 and PS2 genes rescued the Notch processing. Moreover, disruption of the expression of Psn by double-stranded RNA-mediated interference completely abolished the g-secretase activity in S2 cells. Surprisingly, g-secretase activity dependent on wild-type Psn was associated with a drastic overproduction of Ab1-42 from human bAPP in N2a cells, but not in S2 cells. Our data suggest that the mechanism of g-secretase activities through formation of HMW PS complex, as well as its abolition by loss-of-function mutations located in the C terminus, are highly conserved features in Drosophila and mammals.  

3.330           Axonal transport of mitochondria to synapses depends on Milton, a novel Drosophilia protein

Stowers, R.S., Megeath, L.J., Gorska-Andrzejak, J., Meinertzhagen, I.A. and Schwartz, T.L. Neuron, 36, 1063-1077 (2002)   A protein required to localize mitochondria to Drosophila nerve terminals has been identified genetically. Photoreceptors mutant for milton show aberrant synaptic transmission despite normal phototransduction. Without Milton, synaptic terminals and axons lack mitochondria, although mitochondria are numerous in neuronal cell bodies. In contrast, synaptic vesicles continue to be transported to and concentrated at synapses. Milton protein is associated with mitochondria and is present primarily in axons and synapses. A likely explanation of the apparent trafficking defect is offered by the coimmunoprecipitation of Milton and kinesin heavy chain. Transfected into HEK293T cells, Milton induces a redistribution of mitochondria within the cell. We propose that Milton is a mitochondria-associated protein required for kinesin-mediated transport of mitochondria to nerve terminals.  

3.331           The organizing principle in the formation of the T cell receptor-CD3 complex

Call, M.E., Pyrdol, J., Wiedmann, M. and Wucherpfennig, K.W. Cell, 111, 967-979 (2002)   The T cell receptor (TCR) serves a critical function in the immune system and represents one of the most complex receptor structures. A striking feature is the presence of nine highly conserved, potentially charged residues in the transmembrane helices. Previous models have attempted to explain assembly based on pairwise interactions of these residues. Using a novel method for the isolation of intact radiolabeled protein complexes, we demonstrate that one basic and two acidic transmembrane residues are required for the assembly of each of the three signaling dimers with the TCR. This remarkable three-helix arrangement applies to all three assembly steps and represents the organizing principle for the formation of this intricate receptor structure.  

3.332           Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDA and phospholipase Cg1 are required for NF-kB activation and lipid raft recruitment of protein kinase Cq induced by T cell costimulation

Dienz, O. et al

1. Immunol., 169, 365-372 (2002)

  The NF-kB activation pathway induced by T cell costimulation uses various molecules including Vav1 and protein kinase C (PKC)q. Because Vav1 inducibly associates with further proteins including phospholipase C (PLC)g 1 and Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), we investigated their role for NF-kB activation in Jurkat leukemia T cell lines deficient for expression of these two proteins. Cells lacking SLP-76 or PLCg1 failed to activate NF-kB in response to T cell costimulation. In contrast, replenishment of SLP-76 or PLCg1 expression restored CD3/CD28-induced IkB kinase (IKK) activity as well as NF-kB DNA binding and transactivation. PKCq activated NF-kB in SLP-76- and PLCg1-deficient cells, showing that PKCq is acting further downstream. In contrast, Vav1-induced NF-kB activation was normal in SLP-76^- cells, but absent in PLCg1^- cells. CD3/CD28-stimulated recruitment of PKCq and IKKg to lipid rafts was lost in SLP-76- or PLCg1-negative cells, while translocation of Vav1 remained unaffected. Accordingly, recruitment of PKCq to the immunological synapse strictly relied on the presence of SLP-76 and PLCg1, but synapse translocation of Vav1 identified in this study was independent from both proteins. These results show the importance of SLP-76 and PLCg1 for NF-kB activation and raft translocation of PKCq andIKKg.  

3.333           Genetic analysis of iron citrate toxicity in yeast: implications for mammalian iron homeostasis

Chen, O.S., Hemenway, S. and Kaplan, J. Proc. Natl. Acad. Sci., USA, 99(26), 16922-16927 (2002)   Deletion of the yeast homologue of frataxin, YFH1, results in mitochondrial iron accumulation and respiratory deficiency (petite formation). We used a genetic screen to identify mutants that modify iron-associated defects in respiratory activity in Dyfh1 cells. A deletion in the peroxisomal citrate synthase CIT2 in Dyfh1 cells decreased the rate of petite formation. Conversely, overexpression of CIT2 in Dyfh1 cells increased the rate of respiratory loss. Citrate toxicity in Dyfh1 cells was dependent on iron but was independent of mitochondrial respiration. Citrate toxicity was not restricted to iron-laden mitochondria but also occurred when iron accumulated in cytosol because of impaired vacuolar iron storage. These results suggest that high levels of citrate may promote iron-mediated tissue damage.  

3.334           Regulated secretion and subcellular distribution of rabbit lacrimal gland alpha-fucosidases

Gierow, J., Andersson, S.V. and Sjögren, E.C. Invest. Ophthamol. Vis. Sci., 43, E-Abstract 3125 (2002)   Purpose: Our previous results have indicated the presence of two forms of alpha-fucosidase, one acidic and one neutral, that are expressed and secreted by rabbit lacrimal acinar cells in primary culture (IOVS 42: s259, 2001). The purpose of the present study was to further characterize the two forms of alpha-fucosidase regarding their subcellular distribution and regulated secretion Methods: Single acinar cells were isolated from female NZW rabbit lacrimal glands and cultured for 40 h on Matrigel, thus allowing the cells to re-organize into acinar-like structures. The cells were then rinsed and incubated at 37C for 1 h in the absence or presence of 0.1 mM carbachol. Media were collected and analyzed for alpha-fucosidase catalytic activity, at pH 4.0 and pH 7.0, using a 4-methyl umbelliferyl conjugate as substrate.Tissue and cultured cells were homogenized and fractionated by centrifugation yielding a soluble, a nuclear and a microsomal fraction. The membrane fractions were further fractionated by density gradient centrifugation on 10-30% Iodioxanol (Optiprep) gradients, and the resulting fractions were analyzed for fucosidase activity. Results: Secretion of the acidic and the neutral form were both stimulated 5-fold by carbachol. Subcellular fractionation of tissue resulted in 60% recovery of both the acidic and the neutral fucosidase activity in the soluble fraction and 15% in the nuclear (low-speed) pellet, whereas fractionation of cultured cells resulted in a reversed distribution, the difference probably a result of the harsher homogenization procedures used for tissue. Density gradient centrifugation of the membrane fractions revealed the presence of at least three fucosidase-containing membrane populations: One fraction of low density of unknown origin, one high-density, potentially lysosomal population and one intermediate fraction, possibly Golgi-related. Conclusion: No sigificant differences were observed between the distribution and secretion of the acidic and neutral forms of alpha-fucosidase, indicating that they are sequestered in the same endomembrane compartments and that they are under the same secretory control.  

3.335           Human B1 and B2 bradykinin receptors and their agonists target caveolae-related lipid rafts to different degrees in HEK293 cells

Lamb, M.E., Zhang, C., Shea, T., Kyle, D.J. and Leeb-Lundberg, L.M.F. Biochemistry, 41, 14340-14347 (2002)   To address the targeting of G protein-coupled receptors to caveolae-related lipid rafts (CLR), we studied the human B2 (B2R) and B1 (B1R) bradykinin receptor subtypes in HEK293 cells. CLR were enriched on the basis of their unique buoyant density and composition of cholesterol, caveolin-1, and flotillin-1 but not clathrin. CLR contained B2R and B1R as determined by both receptor immunoblotting and the increase in specific activity of receptor agonist binding to cells at both 4 and 37 degrees C when binding was followed by CLR enrichment. B2R was highly enriched in this fraction, whereas B1R was not enriched. Furthermore, acid washing of cells prior to cell disruption minimally affected the CLR-associated B2R agonist binding, whereas it dissociated a major portion of the CLR-associated B1R agonist binding. In addition, when agonist binding at 4 degrees C was followed by an increase in the temperature to 37 degrees C, B2R agonist binding in CLR transiently increased, and this increase was dependent on the C-terminal domain. On the other hand, B1R agonist binding remained unchanged and was independent of the C-terminal domain. Our results show that B2R is constitutively targeted to CLR in HEK293 cells and appears to shuttle the agonist through these domains, whereas B1R may be there by default.  

3.336           Hormone-induced subcellular redistribution of trimeric G proteins

Svobada, P. and Novotny, J. Cell. Mol. Life Sci., 59, 501-512 (2002)   Trimeric guanine nucleotide-binding proteins (G proteins) function as the key regulatory elements in a number of transmembrane signaling cascades where they convey information from agonist-activated receptors to effector molecules. The subcellular localization of G proteins is directly related to their funct-ional role, i.e., the dominant portion of the cellular pool of G proteins resides in the plasma membrane. An intimate association of G protein subunits with the plasma membrane has been well known for a long time. However, results of a number of independent studies published in the past decade have indicated clearly that exposure of intact target cells to agonists results in subcellular redistribution of the cognate G proteins from plasma membranes to the light-vesicular membrane fractions, in internalization from the cell surface into the cell interior and in transfer from the membrane to the soluble cell fraction (high-speed supernatant), i.e. solubilization. Solubilization of G protein  asubunits as a consequence of stimulation of G protein-coupled receptors (GPCRs) with agonists has also been observed in isolated membrane preparations. The membrane -cytosol shift of G proteins was detected even after direct activation of these proteins by non-hydrolyzable analogues of GTP or by cholera toxin-induced ADP-ribosylation. In addition, prolonged stimulation of GPCRs with agonists has been shown to lead to down-regulation of the relevant G proteins. Together, these data suggest that G proteins might potentially participate in a highly complex set of events, which are gene-rally termed desensitization of the hormone response. Internalization, subcellular redistribution, solubiliza-tion, and down-regulation of trimeric G proteins may thus provide an additional means (i.e., beside receptor -based mechanisms) to dampen the hormone or neurotransmitter response after sustained (long-term) exposure.  

3.337           Reversible depression of transcription during hibernation

van Breukelen, F. and Martin, S.L.

1. Comp. Physiol. B., 172, 355-361 (2002)

  Mammalian hibernators downregulate processes of energy production and consumption while maintaining cellular homeostasis. Energetic costs of transcription must be balanced with demands for gene products. Data from nuclear run-on assays indicate transcriptional initiation is reduced two fold in torpid golden-mantled ground squirrels (Spermophilus lateralis) as compared to euthermic animals between bouts of torpor. In addition, elongation rates across the temperature range experienced by hibernators indicate a virtual arrest of transcription at the low body temperatures of torpor. Finally, there is no seasonal compensation or species-specific adaptation for increased elongational capacity in the cold. Thus, it appears that hibernators are not specifically adapted to continue transcription during torpor. Taken together, these data indicate that transcription arrests during torpor because of a moderate depression of initiation and a more severe inhibition of elongation, largely due to temperature effects. Restoration of euthermic body temperatures during the interbout arousals reverses this transcriptional depression and permits gene expression.  

3.338           Myocilin is associated with mitochondria in human trabecular meshwork cells

Wentz-Hunter, K., Ueda, J., Shimuzi, N. and Yue, B.Y.J.T.

1. Cell. Physiol., 190, 46-53 (2002)

  The trabecular meshwork (TM) is a specialized tissue located at the chamber angle of the eye next to the cornea. This tissue is believed to be responsible for regulation of the aqueous humor outflow and control of the intraocular pressure (IOP). Alterations in functions of the TM may lead to IOP elevation and develop-ment of glaucoma, a major cause of blindness. The myocilin gene has recently been directly linked to open-angle glaucomas. The gene product was originally identified as a protein inducible in TM cells by treatment with glucocorticoids such as dexamethasone (DEX) and termed TIGR (TM inducible-glucocorticoid resp-onse). The exact nature and function of the myocilin protein so far still remain elusive. In this study, myocil-in was localized to the perinuclear region of both DEX-treated and control TM cells. Its distribution over-lapped considerably with that of mitochondria. Subcellular fractionation and Western blot analyses suggest-ed a rather extensive association of myocilin with mitochondria. The DEX-treated TM cells were found to undergo apoptosis, when exposed to anti-Fas antibody, to a signilicantly higher degree than the untreated control cells. It appears that the TM cell integrity remains intact after DEX treatment. However, the induc-ed myocilin or myocilin-mitochondria association seems to render the cells more susceptible to a second stress or challenge. This vulnerability may be the basis that ultimately leads to pathological consequences.  

3.339           RVS161p and sphingolipids are required for actin repolarization following salt stress

Balguerie, A., Bagnat, M., Bonneu, M., Aigle, M. and Breton, A.M. Eukaryotic Cell, 1, 1021-1031 (2002) In Saccharomyces cerevisiae, the actin cytoskeleton is depolarized by NaCl stress. In this study, the response was maximal after 30 mm, and then actin patches repolarized. Rvsl6lp was required for actin repolarization because the rvs161D mutant did not repolarize actin patches after growth in a salt medium. Mutations suppressing the rvsl6Th-related salt sensitivity all occurred in genes required for sphingolipid biosynthesis: FEN1, SUR4, SUR2, SUR!, and IPT1. These suppressors also suppressed act1-l-related salt sensitivity and the defect in actin repolarization of the rvs161D mutant, providing a link between sphingolipids and actin polarization. Indeed, deletion of the suppressor genes suppressed the rvs161D defect in actin repolarization in two ways: either actin was not depolarized at the wild-type level in a set of suppressor mutants, or actin was repolarized in the absence of Rvs16lp in the other suppressor mutants. Rvsl6lp was localized as cortical patches that concentrated at polarization sites, i.e., bud emergence and septa, and was found to be associated with lipid rafts. An important link between sphingolipids and actin polarization is that Rvsl61p was required for actin repolarization and was found to be located in lipid rafts.  

3.340           Huntingtin-associated protein 1 interacts with hepatocyte growth factor-regulated tyrosine kinase substrate and functions in endosomal trafficking

Li, Y., Chin, L-S., Levey, A.L. and Li, L.

1. Biol. Chem., 277(31), 28212-28221 (2002)

  Huntingtin-associated protein 1 (HAP1) is a novel protein of unknown function with a higher binding affinity for the mutant form of Huntington's disease protein huntingtin. Here we report that HAP1 interacts with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a mammalian homologue of yeast vacuolar protein sorting protein Vps27p involved in the endosome-to-lysosome trafficking. This novel interaction was identified in a yeast two-hybrid screen using full-length Hrs as bait, and confirmed by in vitro binding assays and co-immunoprecipitation experiments. Deletion analysis reveals that the association of HAP1 with Hrs is mediated via a coiled-coil interaction between the central coiled-coil domains of both proteins. Immunofluorescence and subcellular fractionation studies show that HAP1 co-localizes with Hrs on early endosomes. Like Hrs, overexpression of HAP1 causes the formation of enlarged early endosomes, and inhibits the degradation of internalized epidermal growth factor receptors. Whereas overexpression of HAP1 does not affect either constitutive or ligand-induced receptor-mediated endocytosis, it potently blocks the trafficking of endocytosed epidermal growth factor receptors from early endosomes to late endosomes. These findings implicate, for the first time, the involvement of HAP1 in the regulation of vesicular trafficking from early endosomes to the late endocytic compartments.  

3.341           The adenosine 2b receptor is required to the plasma membrane and associates with E3KARP and ezrin upon agonist stimulation

Sitaraman, S.V. et al

1. Biol. Chem., 277(36), 33188-33195 (2002)

  We have previously shown that adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5'-AMP. Acting through the adenosine A2b receptor (A2bR), the luminally derived adenosine induces vectorial chloride secretion and a polarized secretion of interleukin-6 to the intestinal lumen. Although some G protein-coupled receptors interact with anchoring or signaling molecules, not much is known in this critical area for the A2bR. We used the model intestinal epithelial cell line, T84, and Caco2-BBE cells stably transfected with GFP-A2b receptor to study the intestinal A2bR. The A2bR is present in both the apical and basolateral membranes of intestinal epithelia. Apical or basolateral stimulation of the A2bR induces recruitment of the receptor to the plasma membrane and caveolar fractions. The A2bR co-immunoprecipitates with E3KARP and ezrin upon agonist stimulation. Ezrin interacts with E3KARP and PKA and the interaction between ezrin and E3KARP is enhanced by agonist stimulation. Our data suggest that the A2bR is recruited to the plasma membrane upon apical or basolateral agonist stimulation and interacts with E3KARP and ezrin. We speculate that such an interaction may not only anchor the A2bR to the plasma membrane but may also function to stabilize the receptor in a signaling complex in the plasma membrane.  

3.342           Organization of the recveptor-kinase signaling array that regulates Escherichia coli chemotaxis

Levit, M.N., Grebe, T.W. and Stock, J.B.

1. Biol. Chem., 277(39), 36748-36754 (2002)

  Motor behavior in prokaryotes is regulated by a phosphorelay network involving a histidine protein kinase, CheA, whose activity is controlled by a family of Type I membrane receptors. In a typical Escherichia coli cell, several thousand receptors are organized together with CheA and an Src homology 3-like protein, CheW, into complexes that tend to be localized at the cell poles. We found that these complexes have at least 6 receptors per CheA. CheW is not required for CheA binding to receptors, but is essential for kinase activation. The kinase activity per mole of bound CheA is proportional to the total bound CheW. Similar results were obtained with the E. coli serine receptor, Tsr, and the Salmonella typhimurium aspartate receptor, Tar. In the case of Tsr, under conditions optimal for kinase activation, the ratio of subunits in complexes is ~6 Tsr:4 CheW:1 CheA. Our results indicate that information from numerous receptors is integrated to control the activity of a relatively small number of kinase molecules.  

3.343           A novel membrane protein, Ros3p, is required for phospholipid translocation across the plasma membrane in Saccharomyces cerevisiae

Kato, U. et al

1. Biol. Chem., 277(40), 37855-37862 (2002)

  Ro09-0198 (Ro) is a tetracyclic peptide antibiotic that binds specifically to phosphatidylethanolamine (PE) and causes cytolysis. To investigate the molecular basis of transbilayer movement of PE in biological membranes, we have isolated a series of budding yeast mutants that are hypersensitive to the Ro peptide. One of the most sensitive mutants, designated ros3 (Ro-sensitive 3), showed no significant change in the cellular phospholipid composition or in the sensitivity to amphotericin B, a sterol-binding polyene macrolide antibiotic. These results suggest that the mutation of ros3 affects the PE organization on the plasma membrane, rather than PE synthesis or overall organization of the membrane structures. By functional complementation screening, we identified the gene ROS3 affected in the mutant, and we showed that the hypersensitive phenotype was caused by the defective expression of the ROS3 gene product, Ros3p, an evolutionarily conserved protein with two putative transmembrane domains. Disruption of the ROS3 gene resulted in a marked decrease in the internalization of fluorescence-labeled analogs of PE and phosphatidylcholine, whereas the uptake of fluorescence-labeled phosphatidylserine and endocytic markers was not affected. Neither expression levels nor activities of ATP-binding cassette transporters of the ros3 cells differed from those of wild type cells, suggesting that Ros3p is not related to the multidrug resistance activities. Immunochemical analyses of the structure and subcellular localization showed that Ros3p was a glycosylated membrane protein localized in both the plasma membrane and the endoplasmic reticulum, and that a part of Ros3p was associated with the detergent-insoluble glycolipid-enriched complexes. These results indicate that Ros3p is a membrane glycoprotein that plays an important role in the phospholipid translocation across the plasma membrane.  

3.344           The biofunctional activity of ubiquinone in lysosomal membranes

Nohl, H. and Gille, L. Biogerontology, 3, 125-131 (2002)   Ubiquinone is inhomogenously distributed insubcellular biomembranes. Apart frommitochondria where ubiquinone was demonstratedto exert bioenergetic and pathophysiologicalfunctions, unusually high levels of ubiquinonewere also reported to exist in Golgi vesiclesand lysosomes. In lysosomes the interiordiffers from other organelles by the low pH-value, which is important not only to arrestproteins but also to ensure optimal activity ofhydrolytic enzymes. Since redox-cycling ofubiquinone is associated with the acceptanceand release of protons, we assumed thatubiquinone is a part of a redox chaincontributing to unilateral proton distribution.A similar function of ubiquinone was earliersuggested by Crane to operate in Golgivesicles. Support for the involvement ofubiquinone in a presumed couple ofredox-carriers came from our observation thatalmost 70% of total lysosomal ubiquinone wasin the divalently reduced state. Furtherreduction was seen in the presence of externalNADH. Analysis of the components involved inthe transfer of reducing equivalents fromcytosolic NADH to ubiquinone revealed theexistence of a FAD-containingNADH-dehydrogenase. The latter was found toreduce ubiquinone by means of a b-typecytochrome. Proton translocation into theinterior was linked to the activity of thenovel lysosomal redox chain. Oxygen was foundto be the terminal electron acceptor, therebyalso regulating acidification of the lysosomalmatrix. In contrast to mitochondrialrespiration, oxygen was only trivalently reduced,giving rise to the release ofHO -radicals. The role of this novelproton-pumping redox chain and the significanceof the associated ROS formation has to beelucidated.  

3.345           Palmitic is the main fatty acid carried by lipids of detergent-resistant membrane fractions from neural and non-neural cells

Pitto, M. et al Neurochem. Res., 27(7/8), 729-734 (2002)   Lipids extracted from detergent-resistant membrane fractions, thought to derive from membrane domains, were analyzed for fatty acid composition. The proportion of palmitic acid in fractions isolated from neurons (cerebellar granule cells) and from neural-like cell lines (neuroblastoma-glioma NG108-15) nearly doubled (reaching about 54% of total fatty acids) with respect to cell WCL, indicating their enrichment in palmitic acid-carrying lipids. The proportion of palmitic acid in detergent-resistant fractions obtained from caveolin-transfected NG108-15 cells was comparable with that obtained from caveolin-negative cells, ruling out a specific role of this protein in recruiting palmitoylated lipid species. The enrichment in palmitic acid was remarked also in membrane fractions isolated from non-neuronal cell lines (A431) using either detergents or detergent-free techniques. Lipid fractionation and mass spectrometry experiments show that palmitic acid–rich phosphatidylcholine species are responsible of the peculiar fatty acid composition of these fractions. All together these results suggest that the enrichment in palmitic acid–rich phosphatidylcholine species is a common feature of neural and non-neural cell lines and may play a major role in the biogenesis of membrane domains.    

3.346           Phosphorylation of FcgRIIA is required for the receptor-induced actin rearrangement and capping: the role of membrane rafts

Kwiatkowska, K., Frey, J. and Sobota, A.

1. Cell Sci., 116, 537-550 (2003)

  Activation of Fcg receptor II (FcgRII) induces rearrangement of the actin-based cytoskeleton that serves as a driving force for FcRII-mediated phagocytosis and FcgRII capping. To get insight into the signaling events that lead to the actin reorganization we investigated the role of raft-associated Src family tyrosine kinases in capping of FcgRII in U937 cells. After crosslinking, FcgRII was found to be recruited to detergent-resistant membrane domains (DRMs), rafts, where it coexisted with Lyn kinase and underwent tyrosine phosphorylation. Lyn was displaced from DRMs under the influence of DL-a-hydroxymyristic acid and 2-bromopalmitic acid, agents blocking N-terminal myristoylation and palmitoylation of proteins, respectively, and after disruption of DRM integrity by depletion of plasma membrane cholesterol with ß-cyclodextrin. Under these conditions, phosphorylation of the crosslinked FcgRII was diminished and assembly of FcgRII caps was blocked. The similar reduction of FcgRII cap formation correlated with inhibition of receptor phosphorylation was achieved with the use of PP1 and herbimycin A, specific inhibitors of Src family tyrosine kinases. Phosphorylation of FcgRIIA expressed in BHK cells, lacking endogenous FcgRs, was abolished by substitution of tyrosine 298 by phenylalanine in the ITAM of the receptor. The mutant receptor did not undergo translocation towards cap-like structures and failed to promote the receptor-mediated spreading of the cells, as compared to BHK cells transfected with the wild-type FcgRIIA. On the basis of these data, we suggest that tyrosine phosphorylation of activated FcgRIIA by raft-residing tyrosine kinases of the Src family triggers signaling pathways that control the rearrangement of the actin cytoskeleton required for FcgRII-mediated motility.  

3.347           Subcellular localization of host and viral proteins associated with tobamovirus RNA replication

Hagiwara, Y. et al EMBO J., 22(2), 344-353 (2003)   Arabidopsis TOM1 (AtTOM1) and TOM2A (AtTOM2A) are integral membrane proteins genetically identified to be necessary for efficient intracellular multiplication of tobamoviruses. AtTOM1 interacts with the helicase domain polypeptide of tobamovirus-encoded replication proteins and with AtTOM2A, suggest-ing that both AtTOM1 and AtTOM2A are integral components of the tobamovirus replication complex. We show here that AtTOM1 and AtTOM2A proteins tagged with green fluorescent protein (GFP) are targeted to the vacuolar membrane (tonoplast)-like structures in plant cells. In subcellular fractionation analyses, GFPAtTOM2A, AtTOM2A and its tobacco homolog NtTOM2A were predominantly fractionated to low-density tonoplast-rich fractions, whereas AtTOM1–GFP, AtTOM1 and its tobacco homolog NtTOM1 were distributed mainly into the tonoplast-rich fractions and partially into higher-buoyant-density fractions con-taining membranes from several other organelles. The tobamovirus-encoded replication proteins were co-fractionated with both NtTOM1 and viral RNA-dependent RNA polymerase activity. The replication proteins were also found in the fractions containing non-membrane-bound proteins, but neither NtTOM1 nor the polymerase activity was detected there. These observations suggest that the formation of tobamo-viral RNA replication complex occurs on TOM1-containing membranes and is facilitated by TOM2A.  

3.348           Amyloidgenic processing of the Alzheimer b-amyloid precursor protein depends on lipid rafts

Ehehalt, R.E., Keller, P., Haass, C., Thiele, C. and Simons, K

1. Cell Biol., 160(1), 113-123 (2003)

  Formation of senile plaques containing the ß-amyloid peptide (Aß) derived from the amyloid precursor protein (APP) is an invariant feature of Alzheimer's disease (AD). APP is cleaved either by ß-secretase or by a-secretase to initiate amyloidogenic (release of Aß) or nonamyloidogenic processing of APP, respectively. A key to understanding AD is to unravel how access of these enzymes to APP is regulated. Here, we demonstrate that lipid rafts are critically involved in regulating Aß generation. Reducing cholesterol levels in N2a cells decreased Aß production. APP and the ß-site APP cleavage enzyme (BACE1) could be induced to copatch at the plasma membrane upon cross-linking with antibodies and to segregate away from nonraft markers. Antibody cross-linking dramatically increased production of Aß in a cholesterol-dependent manner. Aß generation was dependent on endocytosis and was reduced after expression of the dynamin mutant K44A and the Rab5 GTPase-activating protein, RN-tre. This inhibition could be overcome by antibody cross-linking. These observations suggest the existence of two APP pools. Although APP inside raft clusters seems to be cleaved by ß-secretase, APP outside rafts undergoes cleavage by a-secretase. Thus, access of a- and ß-secretase to APP, and therefore Aß generation, may be determined by dynamic interactions of APP with lipid rafts.  

3.349           Independent segregation of human immunodeficiency virus type I Gag protein complexes and lipid rafts

Ding, L., Derdowsky, A., Wang, J-J- and Spearman, P.

1. Virol., 77(3), 1916-1926 (2003)

  Formation of human immunodeficiency virus type 1 (HIV-1) particles takes place at the plasma membrane of cells and is directed by the Pr55^Gag polyprotein. A functional assembly domain (the M domain) within the N-terminal portion of Pr55^Gag mediates the interaction of Gag with cellular membranes. However, the determinants that provide specificity for assembly on the plasma membrane, as opposed to intracellular membranes, have not been identified. Recently, it was reported that Pr55^Gag interacts with lipid raft microdomains of the plasma membrane. We sought to identify the domains within Pr55^Gag that contribute to lipid raft association of Gag. Here we demonstrate that the I domain is required for interaction with detergent-resistant membrane fractions (DRMs). Mutation of key I-domain residues or loss of myristylation abrogated the association of Gag with DRMs. Thus, the I domain and the M domain combine to mediate Gag-lipid raft interactions as defined by these biochemical criteria. However, Gag protein complexes defined by flotation studies were much denser than classical lipid rafts, failed to incorporate classical lipid raft marker proteins, and were not disrupted by cholesterol extraction. Large sheets of Gag protein were identified in DRM fractions upon examination by electron microscopy. These results indicate that HIV-1 Pr55^Gag forms detergent-resistant complexes at the cellular periphery that are distinct from lipid raft microdomains.  

3.350           A third human carnitine/organic cation transporter (OCTN3) as a candidate for the 5q31 Crohn’s disease locus (IBD5)

Lamhonwah, A-M., Skaug, J., Scherer, S. and Tein, I. Biochem. Biophys. Res. Comm., 301, 98-101 (2003)   Organic cation transporters function primarily in the elimination of cationic drugs in kidney, intestine, and liver [1, 2 and 3]. The murine organic cation/carnitine (Octn) transporter family, Octn1, Octn2, and Octn3 is clustered on mouse chromosome 11 (NCBI Accession No NW_00039). The human OCTN1 and OCTN2 orthologs map to the syntenic IBD5 locus at 5q31 [1], which has been shown to confer susceptibility to Crohn's disease [4]. We show that the human OCTN3 protein, whose corresponding gene is not yet cloned or annotated in the human reference DNA sequence, does indeed exist and is uniquely involved in carnitine-dependent transport in peroxisomes. Its functional properties and inferred chromosomal location implicate it for involvement in Crohn's disease.  

3.351           Regulation of vascular endothelial growth factor receptor-2 activity by caveolin-1 and plasma membrane cholesterol

Labrecque, L. et al Mol. Biol. Cell, 14, 334-347 (2003)   The stimulation of vascular endothelial growth factor receptor-2 (VEGFR-2) by tumor-derived VEGF represents a key event in the initiation of angiogenesis. In this work, we report that VEGFR-2 is localized in endothelial caveolae, associated with caveolin-1, and that this complex is rapidly dissociated upon stimulation with VEGF. The kinetics of caveolin-1 dissociation correlated with those of VEGF-dependent VEGFR-2 tyrosine phosphorylation, suggesting that caveolin-1 acts as a negative regulator of VEGF R-2 activity. Interestingly, we observed that in an overexpression system in which VEGFR-2 is constitutively active, caveolin-1 overexpression inhibits VEGFR-2 activity but allows VEGFR-2 to undergo VEGF-dependent activation, suggesting that caveolin-1 can confer ligand dependency to a receptor system. Removal of caveolin and VEGFR-2 from caveolae by cholesterol depletion resulted in an increase in both basal and VEGF-induced phosphorylation of VEGFR-2, but led to the inhibition of VEGF-induced ERK activation and endothelial cell migration, suggesting that localization of VEGFR-2 to these domains is crucial for VEGF-mediated signaling. Dissociation of the VEGFR-2/caveolin-1 complex by VEGF or cyclodextrin led to a PP2-sensitive phosphorylation of caveolin-1 on tyrosine 14, suggesting the participation of Src family kinases in this process. Overall, these results suggest that caveolin-1 plays multiple roles in the VEGF-induced signaling cascade.  

3.352           Regulation of integrin growth factor interactions in oligodendrocytes by lipid raft microdomains

Baron, W., Decker, L., Colognato, H. and ffrench-Constant, C. Current Biol., 13, 151-155 (2003)   Individual growth factors can regulate multiple aspects of behavior within a single cell during differentiation, with each signaling pathway controlled independently and also responsive to other receptors such as cell surface integrins. The mechanisms by which this is achieved remain poorly understood. Here we use myelin-forming oligodendrocytes and their precursors to examine the role of lipid rafts, cholesterol and sphingolipid-rich microdomains of the cell membrane implicated in cell signaling [1]. In these cells, the growth factor PDGF has sequential and independent roles in proliferation and survival [2and 3]. We show that the oligodendrocyte PDGFa receptor becomes sequestered in a raft compartment at the developmental stage when PDGF ceases to promote proliferation, but is now required for survival. We also show that laminin-2, which is expressed on axons in the CNS and which provides a target-dependent signal for oligodendrocyte survival by amplification of PDGFaR signaling [4] induces clustering of the laminin binding integrin a6b1 with the PDGFaR-containing lipid raft domains. This extracellular matrix-induced colocalization of integrin and growth factor receptor generates a signaling environment within the raft for survival-promoting PI3K/Akt activity. These results demonstrate novel signaling roles for lipid rafts that ensure the separation and amplification of growth factor signaling pathways during development.  

3.353           SR-BI does not require raft/caveola localization for cholesteryl ester selective uptake in the human adrenal cell line NCI-H295R

Briand, O. et al Biochim. Biophys. Acta, 163, 42-50 (2003)   Class B type I scavenger receptor (SR-BI) mediates the selective uptake of high-density lipoprotein (HDL)-derived cholesteryl esters (HDL-CE) in steroidogenic cells and hepatocytes. SR-BI is enriched in the caveolae of some cell types, genetically modified or not, and these domains have already been shown to constitute primary acceptors for HDL-CE. Nevertheless, the fate of caveola-free cell types has not yet been discussed. NCI-H295R, a human adrenal cell line, highly active in HDL-CE uptake via SR-BI, does not display any morphologically defined caveolae and expresses caveolin at a very low level. Using two different fractionation protocols, we have shown, in this cell type, that SR-BI is homogeneously distributed along the plasma membrane and consists principally of a non-raft membrane-associated pool. Raft destabilisation and caveolin-1 displacement from plasma membrane did not modify the SR-BI-mediated HDL-CE selective uptake. Moreover, the induction of SR-BI expression that is associated with increased CE selective uptake was not associated with any modification in caveolin-1 expression or any raft-targeting mechanism of SR-BI in NCI-H295R. In conclusion, we provide evidence that SR-BI does not require raft/caveola localisation to be implicated in CE selective uptake either in basal or in induced conditions.  

3.354           The uracil transporter Fur4p associates with lipid rafts

Hearn, J.D., Lester, R.L. and Dickson, R.C.

1. Biol. Chem., 278, 3679-3686 (2003)

  Sphingolipids are abundant components of eucaryotic membranes, where they perform essential functions. To uncover new roles for sphingolipids, we studied Saccharomyces cerevisiae lcb1–100 cells, which have a temperature-sensitive block in the first step in sphingolipid synthesis. We find that the level of all five species of the sphingoid long chain base intermediates is reduced 2–7-fold in cells grown at a permissive temperature, and the level of complex sphingolipids is reduced 50%. In addition, lcb1–100 cells make no detectable phosphorylated sphingoid bases. After transfer to a restrictive temperature (a heat shock), the level of the major sphingoid bases drops rather than transiently rising, as in wild type cells. These changes affect lcb1–100 cells in multiple ways. Basal uracil transport by Fur4p is reduced 25%, and when cells are heat-shocked, uracil transport activity falls rapidly and is not restored as it is in wild type cells. Restoration requires a functional secretory pathway and synthesis of complex sphingolipids, leading us to hypothesize that Fur4p associates with lipid rafts. The finding that Fur4p is insoluble in TritonX-100 at 4 °C and behaves like a raft-associated protein on a density gradient supports this hypothesis. Raft association may be essential for regulating breakdown of Fur4p in response to stresses and other factors that govern uracil transport activity. Our results show that long chain bases do not contribute to the inactivation of Fur4p transport activity after heat stress, but they are essential for some later, but unknown, process that leads to degradation of the protein. Further studies using lcb1–100 cells should reveal new roles of sphingolipids in nutrient uptake and other membrane-dependent processes.  

3.355           Transient mechanoactivation of neutral sphingomyelinase in caveolae to generate ceramide

Czarny, M., Liu, J., Oh, P. and Schnitzer, J.E.

1. Biol. Chem., 278, 4424-4430 (2003)

  The vascular endothelium acutely autoregulates blood flow in vivo in part through unknown mechano-sensing mechanisms. Here, we report the discovery of a new acute mechanotransduction pathway. Hemodynamic stressors from increased vascular flow and pressure in situ rapidly and transiently induce the activity of neutral sphingomyelinase but not that acid sphingomyelinase in a time- and flow rate-dependent manner, followed by the generation of ceramides. This acute mechanoactivation occurs directly at the luminal endothelial cell surface primarily in caveolae enriched in sphingomyelin and neutral sphingo-myelinase, but not acid sphingomyelinase. Scyphostatin, which specifically blocks neutral but not acid sphingomyelinase, inhibits mechano-induced neutral sphingomyelinase activity as well as downstream activation of extracellular signalregulated kinase 1 and 2 (ERK1 and ERK2) by increased flow in situ. We postulate a novel physiological function for neutral sphingomyelinase as a new mechanosensor initiating the ERK cascade and possibly other mechanotransduction pathways.  

3.356           Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process

Abrami, L., Liu, S., Cosson, P., Leppla, S.H. and van der Groot, F.G.

1. Cell Biol., 160(3), 321-328 (2003)

  The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of anthrax toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal anthrax toxin receptor.    

3.357           APH-1 I teracts with mature and immature forms of presenilins and nicastrin and may play a role in maturation of presenilin-nicastrin complexes

Gu, Y. et al

1. Biol. Chem., 278(9), 7374-7380 (2003)

  APH-1 and PEN-2 genes modulate the function of nicastrin and the presenilins in Caenorhabditis elegans. Preliminary studies in transfected mammalian cells overexpressing tagged APH-1 proteins suggest that this genetic interaction is mediated by a direct physical interaction. Using the APH-1 protein encoded on human chromosome 1 (APH-1₁L; also known as APH-1a) as an archetype, we report here that endogenous forms of APH-1 are predominantly expressed in intracellular membrane compartments, including the endoplasmic reticulum and cis-Golgi. APH-1 proteins directly interact with immature and mature forms of the presenilins and nicastrin within high molecular weight complexes that display g- and e-secretase activity. Indeed APH-1 proteins can bind to the nicastrin D312-369 loss of function mutant, which does not undergo glycosylation maturation and is not trafficking beyond the endoplasmic reticulum. The levels of expression of endogenous APH-1₁L can be suppressed by overexpression of any other members of the APH-1 family, suggesting that their abundance is coordinately regulated. Finally, although the absence of APH-1 destabilizes the presenilins, in contrast to nicastrin and PEN-2, APH-1 itself is only modestly destabilized in cells lacking functional expression of presenilin 1 or presenilin 2. Taken together, our data suggest that APH-1 proteins, and APH-1₁ in particular, may have a role in the initial assembly and maturation of presenilin·nicastrin complexes.  

3.358           Dynamin-like protein 1 is involved in peroxisomal fission

Koch, A. et al

1. Biol. Chem., 278(10), 8597-8605 (2003)

  The mammalian dynamin-like protein 1 (DLP1), a member of the dynamin family of large GTPases, possesses mechanochemical properties known to constrict and tubulate membranes. In this study, we have combined two experimental approaches, induction of peroxisome proliferation by Pex11pb and expression of dominant-negative mutants, to test whether DLP1 plays a role in peroxisomal growth and division. We were able to localize DLP1 in spots on tubular peroxisomes in HepG2 cells. In addition, immunoblot analysis revealed the presence of DLP1 in highly purified peroxisomal fractions from rat liver and an increase of DLP1 after treatment of rats with the peroxisome proliferator bezafibrate. Expression of a dominant negative DLP1 mutant deficient in GTP hydrolysis (K38A) either alone or in combination with Pex11pb caused the appearance of tubular peroxisomes but had no influence on their intracellular distribution. In co-expressing cells, the formation of tubulo-reticular networks of peroxisomes was promoted, and peroxisomal division was completely inhibited. These findings were confirmed by silencing of DLP1 using siRNA. We propose a direct role for the dynamin-like protein DLP1 in peroxisomal fission and in the maintenance of peroxisomal morphology in mammalian cells.  

3.359           Oligomeric and polymeric aggregates formed by proteins containing expanded polyglutamine

Iuchi, S., Hoffner, G., Verbeke, P., Djian, P. And Green, H. PNAS, 100(5), 2409-2414 (2003)   Neurological diseases resulting from proteins containing expanded polyglutamine (polyQ) are characteristic-ally associated with insoluble neuronal inclusions, usually intranuclear, and neuronal death. We describe here oligomeric and polymeric aggregates formed in cells by expanded polyQ. These aggregates are not dissocia-ted by concentrated formic acid, an extremely effective solvent for otherwise insoluble proteins. Perinuclear inclusions formed in cultured cells by expanded polyQ can be completely dissolved in concentrated formic acid, but a soluble protein oligomer containing the expanded polyQ and released by the formic acid is not dissociated to monomer. In Huntington's disease, a formic acid-resistant oligomer is present in cerebral cort-ex, but not in cerebellum. Cortical nuclei contain a polymeric aggregate of expanded polyQ that is insoluble in formic acid, does not enter polyacrylamide gels, but is retained on filters. This finding shows that the process of polymerization is more advanced in the cerebral cortex than in cultured cells. The resistance of oligomer and polymer to formic acid suggests the participation of covalent bonds in their stabilization.  

3.360           Influenza B virus BM2 protein is an oligomeric integral membrane protein expressed at the cell surface

Paterson, R.G., Takeda, M., Ohigashi, Y., Pinto, L.H. and Lamb, R.A. Virology, 306, 7-17 (2003)   The influenza B virus BM2 protein contains 109 amino acid residues and it is translated from a bicistronic mRNA in an open reading frame that is +2 nucleotides with respect to the matrix (M1) protein. The amino acid sequence of BM2 contains a hydrophobic region (residues 7–25) that could act as a transmembrane (TM) anchor. Analysis of properties of the BM2 protein, including detergent solubility, insolubility in alkali pH 11, flotation in membrane fractions, and epitope-tagging immunocytochemistry, indicates BM2 protein is the fourth integral membrane protein encoded by influenza B virus in addition to hemagglutinin (HA), neuraminidase (NA), and the NB glycoprotein. Biochemical analysis indicates that the BM2 protein adopts an N_outC_in orientation in membranes and fluorescence microscopy indicates BM2 is expressed at the cell surface. As the BM2 protein possesses only a single hydrophobic domain and lacks a cleavable signal sequ-ence, it is another example of a Type III integral membrane protein, in addition to M₂, NB, and CM2 prot-eins of influenza A, B, and C viruses, respectively. Chemical cross-linking studies indicate that the BM2 protein is oligomeric, most likely a tetramer. Comparison of the amino acid sequence of the TM domain of the BM2 protein with the sequence of the TM domain of the proton-selective ion channel M₂ protein of influenza A virus is intriguing as M₂ protein residues critical for ion selectivity/activation and channel gating (H³⁷ and W⁴¹, respectively) are found at the same relative position and spacing in the BM2 protein (H¹⁹ and W²³).  

3.361           Mammalian Ykt6 is a neuronal SNARE targeted to a specialized compartment by its profilin-like amino terminal domain

Hasegawa, H. et al Mol. Biol. Cell, 14, 698-720 (2003)   SNAREs are required for specific membrane fusion throughout the endomembrane system. Here we report the characterization of rat ykt6, a prenylated SNARE selectively expressed in brain neurons. Immunofluor-escence microscopy in neuronal and neuroendocrine cell lines revealed that membrane-associated ykt6 did not colocalize significantly with any conventional markers of endosomes, lysosomes, or the secretory path-way. However, ykt6-containing membranes displayed very minor overlaps with lysosomes and dense-core secretory granules and were similar to lysosomes in buoyant density. Thus, ykt6 appears to be specialized for the trafficking of a unique membrane compartment, perhaps related to lysosomes, involved in aspects of neuronal function. Targeting of this SNARE to the ykt6 compartment was mediated by its profilin-like amino-terminal domain, even in the absence of protein prenylation. Although several other R-SNAREs contain related amino-terminal domains, only the ykt6 version was able to confer the specialized localiz-ation. Rat ykt6, which contains an arginine in its SNARE motif zero-layer, was found to behave like other R-SNAREs in its SNARE assembly properties. Interestingly, cytosolic ykt6, constituting more than half of the total cellular pool, appeared to be conformationally inactive for SNARE complex assembly, perhaps indicative of a regulatory mechanism that prevents promiscuous and potentially deleterious SNARE interactions.  

3.362           Inhibitors of glycosphingolipid biosynthesis reduce transepithelial electrical resistance in MDCK I and FRT cells

Leung, L.W., Contreras, R.G., Flores-Maldomado, C., Cereijido, M. and Rodriguez-Boulan, E. Am. J. Physiol. Cell Physiol., 284, C1021-C1030 (2003)   Madin-Darby canine kidney (MDCK) I and Fisher rat thyroid (FRT) cells exhibit transepithelial electrical resistance (TER) values in excess of 5,000 W·cm². When these cells were incubated in the presence of various inhibitors of sphingolipid biosynthesis, a >5-fold reduction of TER was observed without changes in the gate function for uncharged solutes or the fence function for apically applied fluorescent lipids. The localization of ZO-1 and occludin was not altered between control and inhibitor-treated cells, indicating that the tight junction was still intact. Furthermore, the complexity of tight junction strands, analyzed by freeze-fracture microscopy, was not reduced. Once the inhibitor was removed and the cells were allowed to synthesize sphingolipids, a gradual recovery of the TER was observed. Interestingly, these inhibitors did not attenuate the TER of MDCK II cells, a cell line that typically exhibits values below 800 W·cm^2. These results suggest that glycosphingolipids play a role in regulating the electrical properties of epithelial cells.  

3.363           Regulatory volume decrease in Trypanosoma cruzi involves amoni acid efflux and changes in intracellular calcium

Rohloff, P., Rodrigues, C.O. and Dacompo, R. Mol. Biochem. Parasit., 126, 219-230 (2003)   A regulatory volume decrease (RVD) in response to hyposmotic stress has been characterized in different life-cycle stages of Trypanosoma cruzi. Hyposmotic stress initially caused swelling, but this was rapidly reversed by a compensatory volume reversal that was essentially complete by 5 min. Volume recovery was associated with an amino acid efflux that accounted for approximately 50% of the regulatory volume decrease in all three life-cycle stages. The amino acid efflux was selective for neutral and anionic amino acids, but excluded cationic amino acids. Acidocalcisomes contained an amino acid pool over four times more concentrated than whole-cell levels, but about 90% of this was composed of Arg and Lys, so involvement of this pool in amino acid efflux was ruled out. Hyposmotic stress induced a rise in intracellular calcium that was dependent on influx of calcium across the plasma membrane, since chelation of extracellular calcium abolished the response. Influx of calcium was confirmed by demonstration of manganese-mediated quenching of intracellular fura-2 fluorescence and partial inhibition of the rise in calcium by calcium channel blockers. Manipulation of intra- and extracellular calcium levels had minor effects on the initial rate of amino acid efflux and no effect on the rate of volume recovery.  

3.364           Caveolar compartmentation of caspase-3 in cardiac endothelial cells

Oxhorn, B.C. and Buxton, I.L.O. Cellular Signalling, 15, 489-496 (2003)   Endothelial cell apoptosis is intimately involved in the balance between blood vessel growth and regression and is promoted by numerous stimuli including angiostatin and endostatin, reactive oxygen species (ROS) released during inflammatory processes, and chronic use of drugs of abuse such as cocaine. Apoptosis is characterized by many biological signalling events, including the activation of caspases. Caveolar domains have been hypothesized to mediate apoptotic signalling. We have addressed this hypothesis in cardiac endothelial cells and here we show that caspase-3 proenzyme (32 kDa) and its activated counterpart (17 kDa) co-purify with low-density, caveolin-enriched microdomains and that caspase-3 can be localized with caveolae in intact cells using fluorescent microscopy. Disruption of caveolae results in temporal and spatial changes in enzyme activity. While caspase-3 has been associated with mitochondrial, cytosolic, and high-density regions, the co-purification of activated caspase-3 and caveolar domains reported here suggests the possibility that sarcolemmal caspase-3 may be targeted to plasma-membrane associated substrates.  

3.365           Rapid localization of Gag/GagPol Complexes to detergent-resistant membrane during the assembly of human immunodeficiency virus type 1

Halwani, R., Khorchid, A., Cen, S. and Kleiman, L

1. Virol., 77(7), 3973-3984 (2003)

  During human immunodeficiency virus type 1 (HIV-1) assembly in HIV-1-transfected COS7 cells, almost all steady-state Gag/Gag and Gag/GagPol complexes are membrane bound. However, exposure to 1% Triton X-100 gives results indicating that while all Gag/GagPol complexes remain associated with the detergent-resistant membrane (DRM), only 30% of Gag/Gag complexes are associated with the DRM. Analysis of the localization of newly synthesized Gag/Gag and Gag/GagPol to the membrane indicates that after a 10-min pulse with radioactive [³⁵S]Cys-[³⁵S]Met, all newly synthesized Gag/GagPol is found at the DRM. Only 30% of newly synthesized Gag/Gag moves to the membrane, and at 0 min of chase, only 38% of this membrane-bound Gag/Gag is associated with the DRM. During the first 30 min of chase, most membrane-bound Gag/Gag moves to the DRM, while between 30 and 60 min of chase, there is a significant decrease in membrane-bound Gag/Gag and Gag/GagPol. Since the localization of newly synthesized Gag/Gag to the DRM and the interaction of GagPol with Gag both depend upon Gag multimerization, the rapid localization of GagPol to the DRM probably reflects the interaction of all newly synthesized GagPol with the first newly synthesized polymeric Gag to associate with the DRM.  

3.366           Localization of presenilin-nicastrin complexes and g-secretase activity to the trans-Golgi network

Siman, R. and Velji, J.

1. Neurochem., 84, 1143-1153 (2003)

  Abundant biochemical and genetic evidence suggests that presenilins are catalytic components of g-secretase, the protease responsible for generating the Alzheimer amyloid ß-protein. However, the differential localization of presenilins to early secretory compartments and g-secretase substrates to late secretory compartments and the plasma membrane (the ‘spatial paradox’) argues against this view. We investigated this issue by studying the localization of nicastrin, another putative g-secretase component, and its association with presenilin-1 into proteolytically active complexes. Glycosidase digests revealed that nicastrin exists in multiple glycoforms and is terminally sialylated, a modification often associated with the trans -Golgi network. Trafficking of nicastrin to the trans -Golgi network was confirmed by density gradient fractionation and immunofluorescence microscopy. In presenilin-deficient cells, however, nicastrin trafficking and maturation were abnormal, as the protein was restricted to early secretory compartments and failed to be sialylated. Mature sialylated nicastrin in trans -Golgi network fractions was complexed quantitatively with N- and C-terminal fragments of presenilin-1, whereas immature nicastrin present in early secretory compartments was not. Additionally, trans -Golgi network fractions contained the g-secretase substrate ß-amyloid precursor protein C83 and were enriched in presenilin-dependent g-secretase proteolytic activity. The results resolve the apparent spatial paradox by demonstrating that presenilin–nicastrin complexes and presenilin-dependent g-secretase activity are co-localized to a late secretory compartment. The findings provide further evidence that presenilin-containing complexes are the g-secretase, and indicate that presenilins also regulate g-secretase assembly.  

3.367           Do lipid raft mediate virus assembly and pseudotyping?

Briggs, J.A., Wilk, T. and Fuller, S.D.

1. Gen. Virol., 84, 757-768 (2003)

  Co-infection of a host cell by two unrelated enveloped viruses can lead to the production of pseudotypes: virions containing the genome of one virus but the envelope proteins of both viruses. The selection of components during virus assembly must therefore be flexible enough to allow the incorporation of unrelated viral membrane proteins, yet specific enough to exclude the bulk of host proteins. This apparent contradiction has been termed the pseudotypic paradox. There is mounting evidence that lipid rafts play a role in the assembly pathway of non-icosahedral, enveloped viruses. Viral components are concentrated initially in localized regions of the plasma membrane via their interaction with lipid raft domains. Lateral interactions of viral structural proteins amplify the changes in local lipid composition which in turn enhance the concentration of viral proteins in the rafts. An affinity for lipid rafts may be the common feature of enveloped virus proteins that leads to the formation of pseudotypes.    

3.368           Mastoparan selectively activates phospholipase D2 in cell membranes

Chahdi, A., Choi, W.S., Kim, Y. M. and Beaven, M.A.

1. Biol. Chem., 278(14), 12039-12045 (2003)

  Both known isoforms of phospholipase (PL) D, PLD1 and PLD2, require phosphatidylinositol 4,5-bisphosphate for activity. However, PLD2 is fully active in the presence of this phospholipid, whereas PLD1 activation is dependent on additional factors such as ADP-ribosylation factor-1 (ARF-1) and protein kinase Ca. We find that mastoparan, an activator of G_i and mast cells, stimulates an intrinsic PLD activity, most likely PLD2, in fractions enriched in plasma membranes from rat basophilic leukemia 2H3 mast cells. Overexpression of PLD2, but not of PLD1, results in a large increase in the mastoparan-inducible PLD activity in membrane fractions, particularly those enriched in plasma membranes. As in previous studies, expressed PLD2 is localized primarily in the plasma membrane and PLD1 in granule membranes. Studies with pertussis toxin and other agents indicate that mastoparan stimulates PLD2 independently of G_i, ARF-1, protein kinase C, and calcium. Kinetic studies indicate that mastoparan interacts synergistically with phosphatidylinositol 4,5-bisphosphate and that oleate, itself a weak stimulant of PLD2 at low concentrations, is a competitive inhibitor of mastoparan stimulation of PLD2. Therefore, mastoparan may be useful for investigating the regulation of PLD2, particularly in view of the well studied molecular interactions of mastoparan with certain other strategic signaling  proteins.  

3.369           Human immunodeficiency virus type 1 assembly and lipid rafts: Pr55^gag associates with membrane domains

Holm, K., Weclewicz,K., Hewson, R. and Suomalainen, M.

1. Virol.,77, 4805-4817 (2003)

  The assembly and budding of human immunodeficiency virus type 1 (HIV-1) at the plasma membrane are directed by the viral core protein Pr55^gag. We have analyzed whether Pr55^gag has intrinsic affinity for sphingolipid- and cholesterol-enriched raft microdomains at the plasma membrane. Pr55^gag has previously been reported to associate with Triton X-100-resistant rafts, since both intracellular membranes and virus-like Pr55^gag particles (VLPs) yield buoyant Pr55^gag complexes upon Triton X-100 extraction at cold temperatures, a phenotype that is usually considered to indicate association of a protein with rafts. However, we show here that the buoyant density of Triton X-100-treated Pr55^gag complexes cannot be taken as a proof for raft association of Pr55^gag, since lipid analyses of Triton X-100-treated VLPs demonstrated that the detergent readily solubilizes the bulk of membrane lipids from Pr55^gag. However, Pr55^gag might nevertheless be a raft-associated protein, since confocal fluorescence microscopy indicated that coalescence of GM1-positive rafts at the cell surface led to copatching of membrane-bound Pr55^gag. Furthermore, extraction of intracellular membranes or VLPs with Brij98 yielded buoyant Pr55^gag complexes of low density. Lipid analyses of Brij98-treated VLPs suggested that a large fraction of the envelope cholesterol and phospholipids was resistant to Brij98. Collectively, these results suggest that Pr55^gag localizes to membrane microdomains that are largely resistant to Brij98 but sensitive to Triton X-100, and these membrane domains provide the platform for assembly and budding of Pr55^gag VLPs.  

3.370           Regulation of cytochrome c oxidase activity by c-Src in osteoclasts

Miyazaki, T., Neff, L., Tanaka, S., Horne, W.C. and Baron, R.

1. Cell Biol., 160(5), 709-718 (2003)

  The function of the nonreceptor tyrosine kinase c-Src as a plasma membrane–associated molecular effector of a variety of extracellular stimuli is well known. Here, we show that c-Src is also present within mitochondria, where it phosphorylates cytochrome c oxidase (Cox). Deleting the c-src gene reduces Cox activity, and this inhibitory effect is restored by expressing exogenous c-Src. Furthermore, reducing endogenous Src kinase activity down-regulates Cox activity, whereas activating Src has the opposite effect. Src-induced Cox activity is required for normal function of cells that require high levels of ATP, such as mitochondria-rich osteoclasts. The peptide hormone calcitonin, which inhibits osteoclast function, also down-regulates Cox activity. Increasing Src kinase activity prevented the inhibitory effect of calcitonin on Cox activity and osteoclast function. These results suggest that c-Src plays a previously unrecognized role in maintaining cellular energy stores by activating Cox in mitochondria.  

3.371           Peroxisomal membrane monocarboxylate transporters: evidence for a redox shuttle system

McClelland, G.B., Khanna, S., Gonzales, G.F., Butz, C.E. and Brooks, G.A. Biochem. Biophys. Res. Comm., 304, 130-135 (2003)   One of the many functions of liver peroxisomes is the b-oxidation of long-chain fatty acids. It is essential for the continuation of peroxisomal b-oxidation that a redox shuttle system exist across the peroxisomal membrane to reoxidize NADH. We propose that this redox shuttle system consists of a substrate cycle between lactate and pyruvate. Here we present evidence that purified peroxisomal membranes contain both monocarboxylate transporter 1 (MCT 1) and MCT 2 and that along with peroxisomal lactate dehydrogenase (pLDH) form a Peroxisomal Lactate Shuttle. Peroxisomal b-oxidation was greatly stimulated by the addition of pyruvate and this increase was partially inhibited by the addition of the MCT blocker a-cyano-4-hydroxycinnamate (CINN). We also found that peroxisomes generated lactate in the presence of pyruvate. Together these data provide compelling that the Peroxisome Lactate Shuttle helps maintain organelle redox and the proper functioning of peroxisomal b-oxidation.  

3.372           Modulation of Rho GTPase signaling regulates a switch between adigenesis and myogenesis

Sordella, R. et al Cell, 113, 147-158 82003)   Mature adipocytes and myocytes are derived from a common mesenchymal precursor. While IGF-1 promotes the differentiation of both cell types, the signaling pathways that specify the distinct cell fates are largely unknown. Here, we show that the Rho GTPase and its regulator, p190-B RhoGAP, are components of a critical switch in the adipogenesis-myogenesis ''decision.'' Cells derived from embryos lacking p190-B RhoGAP exhibit excessive Rho activity, are defective for adipogenesis, but undergo myogenesis in response to IGF-1 exposure. In vitro, activation of Rho-kinase by Rho inhibits adipogenesis and is required for myogenesis. The activation state of Rho following IGF-1 signaling is determined by the tyrosine-phosphorylation status of p190-B RhoGAP and its resulting subcellular relocalization. Moreover, adjusting Rho activity is sufficient to alter the differentiation program of adipocyte and myocyte precursors. Together, these results identify the Rho GTPase as an essential modulator of IGF-1 signals that direct the adipogenesis-myogenesis cell fate decision.  

3.373           The SNARE motif contributes to rbet1 intracellular targeting and dynamics independently of SNARE interactions

Joglekar, A.P., Xu, D., Rigotti, D.J., Fairman, R. and Hay, J.C.

1. Biol. Chem., 278(16), 14121-14133 (2003)

  The endoplasmic reticulum/Golgi SNARE rbet1 cycles between the endoplasmic reticulum and Golgi and is essential for cargo transport in the secretory pathway. Although the quaternary SNARE complex containing rbet1 is known to function in membrane fusion, the structural role of rbet1 is unclear. Furthermore, the structural determinants for rbet1 targeting and its cyclical itinerary have not been investigated. We utilized protein interaction assays to demonstrate that the rbet1 SNARE motif plays a structural role similar to the carboxyl-terminal helix of SNAP-25 in the synaptic SNARE complex and demonstrated the importance to SNARE complex assembly of a conserved salt bridge between rbet1 and sec22b. We also examined the potential role of the rbet1 SNARE motif and SNARE interactions in rbet1 localization and dynamics. We found that, in contrast to what has been observed for syntaxin 5, the rbet1 SNARE motif was essential for proper targeting. To test whether SNARE interactions were important for the targeting function of the SNARE motif, we used charge repulsion mutations at the conserved salt bridge position that rendered rbet1 defective for binary, ternary, and quaternary SNARE interactions. We found that heteromeric SNARE interactions are not required at any step in rbet1 targeting or dynamics. Furthermore, the heteromeric state of the SNARE motif does not influence its interaction with the COPI coat or efficient recruitment onto transport vesicles. We conclude that protein targeting is a completely independent function of the rbet1 SNARE motif, which is capable of distinct classes of protein interactions.    

3.374           Effect of glycosylphosphatidylinositol anchor-dependent and -independent prion protein association with model raft membranes on conversion to the protease-resistant isoform

Baron, G.S. and Caughey, B.

1. Biol. Chem., 278(17), 14883-14892 (2003)

  Prion protein (PrP) is usually bound to membranes by a glycosylphosphatidylinositol (GPI) anchor that associates with detergent-resistant membranes, or rafts. To examine the effect of membrane association on the interaction between the normal protease-sensitive PrP isoform (PrP-sen) and the protease-resistant isoform (PrP-res), a model system was employed using PrP-sen reconstituted into sphingolipid-cholesterol-rich raft-like liposomes (SCRLs). Both full-length (GPI⁺) and GPI anchor-deficient (GPI^-) PrP-sen produced in fibroblasts stably associated with SCRLs. The latter, alternative mode of membrane association was not detectably altered by glycosylation and was markedly reduced by deletion of residues 34-94. The SCRL-associated PrP molecules were not removed by treatments with either high salt or carbonate buffer. However, only GPI⁺ PrP-sen resisted extraction with cold Triton X-100. PrP-sen association with SCRLs was pH-independent. PrP-sen was also one of a small subset of phosphatidylinositol-specific phospholipase C (PI-PLC)-released proteins from fibroblast cells found to bind SCRLs. A cell-free conversion assay was used to measure the interaction of SCRL-bound PrP-sen with exogenous PrP-res as contained in microsomes. SCRL-bound GPI⁺ PrP-sen was not converted to PrP-res until PI-PLC was added to the reaction or the combined membrane fractions were treated with the membrane-fusing agent polyethylene glycol (PEG). In contrast, SCRL-bound GPI^- PrP-sen was converted to PrP-res without PI-PLC or PEG treatment. Thus, of the two forms of raft membrane association by PrP-sen, only the GPI anchor-directed form resists conversion induced by exogenous PrP-res.  

3.375           Plasma membrane rafts of rainbow trout are subject to thermal acclimation

Zehmer, J.K. and Hazel, J.R.

1. Exp. Biol., 206, 1657-1667 (2003)

  Rafts are cholesterol- and sphingolipid-enriched microdomains of the plasma membrane (PM) that organize many signal transduction pathways. Interactions between cholesterol and saturated lipids lead to patches of liquid-ordered membrane (rafts) phase-separating from the remaining PM. Phase behavior is temperature sensitive, and acute changes in temperature experienced by poikilotherms would be expected to perturb raft structure, necessitating an acclimatory response. Therefore, with thermal acclimation, we would expect compositional changes in the raft directed to offset this perturbation. Using differential and density gradient centrifugation, we separated PM from the livers of rainbow trout acclimated to 5°C and 20°C into raft-enriched (raft) and raft-depleted PM (RDPM). Compared with RDPM, the raft fractions were enriched in cholesterol, the ß₂-adrenergic receptor and adenylyl cyclase, which are commonly used markers for this microdomain. Furthermore, cholesterol was enriched in all fractions from warm-compared with cold-acclimated animals, but this increase was 3.4 times greater in raft than in PM. We developed a novel approach for measuring membrane molecular interaction strength (and thus the tendency to stabilize raft structure) based on the susceptibility of membranes to detergent. Specifically, studies with model vesicles demonstrated that the capacity of a membrane to accommodate detergent prior to solubilization (saturation point) was a good index of this property. The saturation point of the isolated membrane preparations was temperature sensitive and was significantly different in 5°C- and 20°C-acclimated RDPM when assayed at 5°C and 20°C, respectively. By contrast, this comparison in rafts was not significantly different, suggesting compensation of this property. These data suggest that compositional changes made in the PM during thermal acclimation act to offset thermal perturbation of the raft but not the RDPM structural integrity.    

3.376           Ligand-dependent recruitment of the ErbB4 signaling complex into neuronal lipid rafts

Li Ma et al

1. Neurosci., 23(8), 3164-3175 (2003)

  Neuregulin (NRG) regulates synapse formation and synaptic plasticity, but little is known about the regulation of NRG signaling at synapses. Here we show that the NRG receptor ErbB4 was localized in anatomically defined postsynaptic densities in the brain. In cultured cortical neurons, ErbB4 was recruited to the neuronal lipid raft fraction after stimulation by NRG. Along with ErbB4, adaptor proteins Grb2 and Shc were translocated to lipid rafts by NRG stimulation. In transfected human embryonic kidney 293 cells, the partitioning of ErbB4 into a detergent-insoluble fraction that includes lipid rafts was increased by PSD-95 (postsynaptic density-95), through interaction of the ErbB4 C terminus with the PDZ [PSD-95/Discs large/zona occludens-1] domains of PSD-95. Disruption of lipid rafts inhibited NRG-induced activation of Erk and prevented NRG-induced blockade of induction of long-term potentiation at hippocampal CA1 synapses. Thus, our results indicate that NRG stimulation causes translocation of ErbB4 into lipid rafts and that lipid rafts are necessary for signaling by ErbB4.  

3.377           Raft partitioning of the yeast uracil permease during trafficking along the endocytic pathway

Dupre, S. and Haguenauer-Tsapis, R. Traffic, 4, 83-96 (2003)   Lipid rafts, formed by the lateral association of sphingolipids and cholesterol in the external membrane leaflet, have been implicated in membrane traffic and cell signaling in mammalian cells. Yeast plasma membranes were also recently shown to contain lipid raft microdomains consisting of sphingolipids and ergosterol, and containing several plasma membrane proteins, including Gas1p, a GPI-anchored protein, and the [H⁺] ATPase Pma1p. In this study, we investigated whether lipid rafts were involved in the intracellular trafficking of a yeast transporter, uracil permease, which undergoes ubiquitin-dependent endocytosis. Regardless of its ubiquitination status, uracil permease was found to be associated with rafts in the plasma membrane. The expression of Fur4p in lcb1-100 cells, deficient in the first enzyme of sphingolipid synthesis, impaired the association of Fur4p with detergent-resistant fractions. When targeted to endocytic compartments, uracil permease appeared to be progressively transferred to detergent-soluble fractions, suggesting that the lipid environment might change between plasma membrane and endosomes. Consistent with this hypothesis, the wild-type form of the v-SNARE Snc1p, which is known to cycle between the plasma membrane and endosomal compartments, was recovered in both detergent-resistant and detergent-soluble fractions. In contrast, a variant Snc1p that accumulates at the plasma membrane was recovered exclusively in detergent-resistant fractions.  

3.378           The structure of murine outer segment disk membranes using atomic force microscopy

Saperstein, D.A. et al Invest. Ophthalmol Vis. Sci., 44, E-abstract 3175 (2003)   Purpose: Atomic force microscopy (AFM) is a powerful new tool to study biologic membranes. We used AFM to study the surface of murine rod outer segment disk membranes Methods: Dark adapted wildtype C57BL/6 mice were sacrificed and their retinas were removed. Osmotically intact rod outer segments (ROS) were isolated via centrifugation in an Optiprep gradient. The ROS were then burst using 2mM Tris-HCl, pH 7.4, at 0°C for 15 hr and isolated using centrifugation in an Optiprep gradient. The isolated disks were adsorbed to mica and scanned using a Nanoscope Multimode microscope (Digital Instruments) equipped with an infrared laser head, fluid cell, and oxide-sharpened silicon nitride cantilevers (OMCL-TR400PSA, Olympus) in aqueous fluid using the contact scanning mode. All procedures were carried out in complete darkness with the aid of night vision goggles. The disk integrity was verified by scanning and transmission electron microscopy. Results: The superstructure of the disk membrane was revealed. The cytoplasmic surface of the disks are textured and under high magnification consist of rows of rhodopsin pairs densely packed in paracrystalline arrays. The density of rhodopsin monomers averages 48,300 molecules per µm². The distance measured between rhodopsin molecules in the dimer was 3.8 nm (N=40). This measurement was consistent with measurements using the angularly averaged powder diffraction pattern. Conclusions:This study represents the first description of the higher orderstructure of rhodopsin molecules within the native disk membranes.The resolution is sufficient to visualize individual unstainedrhodpsin molecules. The dimeric nature of rhodopsin in the diskmembrane is clearly demonstrated supporting published pharmacologicaland biochemical analyses.

3.379           Amphipathic helix-dependent localization of NS5A mediates hepatitis C virus RNA replication

Elazar, M. et al

1. Virol., 77(10), 6055-6061 (2003)

  We identified an N-terminal amphipathic helix (AH) in one of hepatitis C virus (HCV)’s nonstructural proteins, NS5A. This AH is necessary and sufficient for membrane localization and is conserved across isolates. Genetically disrupting the AH impairs HCV replication. Moreover, an AH peptide-mimic inhibits the membrane association of NS5A in a dose-dependent manner. These results have exciting implications for the HCV life cycle and novel antiviral strategies.  

3.380           Role of caveolae in signal-tranducing function of cardiac Na⁺/K⁺ -ATPase

Liu, L. et al Am. J. Physiol. Cell Physiol., 284, C1550-C1560 (2003)   Ouabain binding to Na⁺/K⁺-ATPase activates Src/epidermal growth factor receptor (EGFR) to initiate multiple signal pathways that regulate growth. In cardiac myocytes and the intact heart, the early ouabain-induced pathways that cause rapid activations of ERK1/2 also regulate intracellular Ca²⁺ concentration ([Ca²⁺]_i) and contractility. The goal of this study was to explore the role of caveolae in these early signaling events. Subunits of Na⁺/K⁺-ATPase were detected by immunoblot analysis in caveolae isolated from cardiac myocytes, cardiac ventricles, kidney cell lines, and kidney outer medulla by established detergent-free procedures. Isolated rat cardiac caveolae contained Src, EGFR, ERK1/2, and 20-30% of cellular contents of a₁- and a₂-isoforms of Na⁺/K⁺-ATPase, along with nearly all of cellular caveolin-3. Immunofluorescence microscopy of adult cardiac myocytes showed the presence of caveolin-3 and a-isoforms in peripheral sarcolemma and T tubules and suggested their partial colocalization. Exposure of contracting isolated rat hearts to a positive inotropic dose of ouabain and analysis of isolated cardiac caveolae showed that ouabain caused 1) no change in total caveolar ERK1/2, but a two- to threefold increase in caveolar phosphorylated/activated ERK1/2; 2) no change in caveolar a₁-isoform and caveolin-3; and 3) 50-60% increases in caveolar Src and a₂-isoform. These findings, in conjunction with previous observations, show that components of the pathways that link Na⁺/K⁺-ATPase to ERK1/2 and [Ca²⁺]_i are organized within cardiac caveolae microdomains. They also suggest that ouabain-induced recruitments of Src and a₂-isoform to caveolae are involved in the manifestation of the positive inotropic effect of ouabain.  

3.381           Resistance of cell membranes to different antigens

Schuck, S., Honsho, M., Ekroos, K., Shevchenko, A. and Simons, K. Proc. Natl. Acad. Sci. USA, 100, 5795-5800 (2003)   Partial resistance of cell membranes to solubilization with mild detergents and the analysis of isolated detergent-resistant mem­branes (DRMs) have been used operationally to define membrane domains. Given the multitude of detergents used for this purpose. we sought to investigate whether extraction with different deter gents might reflect the same underlying principle of domain formation. We therefore compared the protein and lipid content of DRM5 prepared with a variety of detergents from two cell lines. We found that the detergents differ considerably in their ability to selectively solubilize membrane proteins and to enrich sphingo­lipids and cholesterol over glycerophospholipids as well as satu­rated over unsaturated phosphatidyicholine. In addition, we observed cell type-dependent variations of the molecular charac­teristics of DRMs and the effectiveness of particular detergents. These results make it unlikely that different detergents reflect the same aspects of membrane organization and underscore both the structural complexity of cell membranes and the need for more sophisticated analytical tools to understand their architecture.    

3.382           Nephrin and Neph1 co-localize at the podocyte foot process intercellular junction and form cis hetero-oligomers

Barletta, G-M., Kovari, I.A., Verma, R.K., Kerjaschki, D. and Holzmann, L.B.

1. Biol. Chem., 278, 19266-19271 (2003)

  Glomerular visceral epithelial cells (podocytes) ap­pear to play a central role in maintaining the selective filtration barrier of the renal glomerulus. While the im­munoglobulin superfamily member Nephrin was pro­posed to act as a cell adhesion molecule at the podocyte intercellular junction necessary for maintaining gb­merular perm selectivity, the Nephrin ligand has not been identified. The existence of a new subfamily of Nephrin-like molecules including Neph1 was recently described. Genetic deletion of Nephrin or Neph1 re­sulted in similar phenotypes of podocyte foot process effacement and proteinuria. The subcellular localiza­tion of Neph1 and the possibility that Nephrin and Neph1 interact was investigated. Polyclonal antiserum for Neph1 was raised and characterized. Neph1 mi­grated as a 90-kDa protein on SDS-PAGE under reduc­ing conditions. Neph1 was identified in a glomerular and podocyte-specific distribution in adult rat kidney. Like Nephrin and Podocin, Neph1 was enriched in Tri­ton X-100 detergent-resistant membrane fractions. Con­sistent with this observation, immunogold electron mi­croscopy demonstrated that Neph1 localized exclusively to lateral margins of podocyte foot processes at the in­sertion of the slit diaphragm. Neph1 and Nephrin par­ticipate in a direct cis-interaction involving their cyto­plasmic domains. In addition, interactions between the extracellular domain of Nephrin and itself and between the extracellular domain of Nephrin and that of Neph1 were detected. Neph1 did not interact via a homophilic interaction. These observations suggest that Nephrin and Neph1 form a hetero-oligomeric receptor complex in the plane of the membrane that might interact across the foot process intercellular junction through interac­tions between Nephrin with itself and Neph1.  

3.383           Distinct rates of palmitate turnover on membrane-bound cellular and oncogenic H-Ras

Baker, T.L., Zheng, H., Walker, J., Coloff, J.L. and Buss, J.E.

1. Biol. Chem., 278, 19292-19300 (2003)

  H-Ras displays dynamic cycles of GTP binding and palmitate turnover. GTP binding is clearly coupled to activation, but whether the palmitoylated COOH termi­nus participates in signaling, especially when con­strained by membrane tethering, is unknown. As a way to compare COO!! termini of membrane-bound, lipid-modified H-Ras, palmitate removal rates were measured for various forms of H-Ras in NIH 3T3 cells. Depalmito­ylation occurred slowly (t₂ ~2.4 h) in cellular (H-RasWT) or dominant negative (H-Rasl7N) forms and more rap­idly (t₂ ~1 h) in oncogenic H-Ras6lL or H-RasRl2,T59. Combining this data with GTP binding measurements, the palmitate half-life of H-Ras in the fully GTP-bound state was estimated to be less than 10 min. Slow palmi­tate removal from cellular H-Ras was not explained by sequestration in caveolae, as neither cellular nor onco­genic H-Ras showed alignment with caveolin by immu­nofluorescence. Conversely, although it had faster palmitate removal, oncogenic H-Ras was located in the same fractions as H-RasWT on four types of density gra­dients, and remained fully membrane-bound. Thus the different rates of deacylation occurred even though on­cogenic and cellular H-Has appeared to be in similar locations. Instead, these results suggest that acyiprotein thioesterases access oncogenic H-Ras more easily be­cause the conformation of its COOH terminus against the membrane is altered. This previously undetected difference could help produce distinctive effector inter­actions and signaling of oncogenic H-Ras.    

3.384           Palmitoylation regulates regulators of G-protein signaling (RGS) 16 function: I Mutation of amino-terminalcysteine residues on RGS16 prevents its targeting to lipid rafts and palmitoylation of an internal cysteine residue.

Hiol, A et al

1. Biol. Chem., 278, 19301-19308 (2003)

  Regulators of G-protein signaling (RGS) proteins down-regulate signaling by heterotrimeric G-proteins by accelerating GTP hydrolysis on the Ga subunits. Palmitoylation, the reversible addition of palmitate to cysteine residues, occurs on several RGS proteins and is critical for their activity. For RGS16, mutation of Cys-2 and Cys-12 blocks its incorporation of E³Hlpalmitate and ability to turn-off G_i and G_q signaling and significantly inhibited its GTPase activating protein activity toward a Ga subunit fused to the 5-hydroxytryptamine receptor 1A, but did not reduce its plasma membrane localization based on cell fractionation studies and immunoelectron microscopy. Palmitoylation can target proteins, includ­ing many signaling proteins, to membrane microdo­mains, called lipid rafts. A subpopulation of endogenous RGS16 in rat liver membranes and overexpressed RGS16 in COS cells, but not the non-palmitoylated cys­teine mutant of RGS16, localized to lipid rafts. However, disruption of lipid rafts by treatment with methyl-j3-cyclodextrin did not decrease the GTPase activating protein activity of RGS16. The lipid raft fractions were enriched in protein acyltransferase activity, and RGS16 incorporated E³Hlpalmitate into a peptide fragment con­taining Cys-98, a highly conserved cysteine within the RGS box. These results suggest that the amino-terminal palmitoylation of an RGS protein promotes its lipid raft targeting that allows palmitoylation of a poorly accessi­ble cysteine residue that we show in the accompanying article (Osterhout, J. L., Waheed, A. A., Hiol, A., Ward, R. J., Davey, P. C., Nini, L., Wang, J., Milligan, G., Jones, T. L. Z., and Druey, K. M. (2003) J. BioL Chem. 278,19309-19316) was critical for RGS16 and RGS4 GAP activity.  

3.385           Palmitoylation regulates regulators of G-protein signaling (RGS) 16 function: II Palmitoylation of a cysteine residue in the RGS box is critical for RGS16 GTPase accelerating activity and regulation of G_i-coupled signaling

Osterhout, J.L. et al.

1. Biol. Chem., 278, 19309-19316 (2003)

  Palmitoylation is a reversible post-translational mod­ification used by cells to regulate protein activity. The regulator of G-protein signaling (RGS) proteins RGS4 and RGS16 share conserved cysteine (Cys) residues that undergo palmitoylation. In the accompanying article (Hiol, A., Davey, P. C., Osterhout, J. L., Waheed, A. A., Fischer, E. H., Chen, C. K., Milligan, G., Druey, K. M., and Jones, T. L. Z. (2003) J. BioL Chem. 278, 19301-19308), we determined that mutation of NH₂-terminal cysteine res­idues in RGS16 (Cys-2 and Cys-12) reduced GTPase ac­celerating (GAP) activity toward a 5-hydroxytryptamine (5-HT_1A)/Ga_o1 receptor fusion protein in cell mem­branes. NH₂-terminal acylation also permitted palmito­ylation of a cysteine residue in the RGS box of RGS16 (Cys-98). Here we investigated the role of internal pal­mitoylation in RGS16 localization and GAP activity. Mutation of RGS16 Cys.98 or RGS4 Cys-95 to alanine re­duced GAP activity on the 5-HT_1A/Ga_o1 fusion protein and regulation of adenylyl cyclase inhibition. The C98A mutation had no effect on RGS16 localization or GAP activity toward purified G-protein a subunits. Enzymatic palmitoylation of RGS16 resulted in internal palmito­ylation on residue Cys-98. Palmitoylated RGS16 or RGS4 WT but not C98A or C95A preincubated with membranes expressing 5-HT_1A/Ga_o1 displayed increased GAP activ­ity over time. These results suggest that palmitoylation of a Cys residue in the RGS box is critical for RGS16 and RGS4 GAP activity and their ability to regulate G_i-cou­pled signaling in mammalian cells.    

3.386           Differential mobilization of newly synthesized cholesterol and biosynthetic sterol precursors from cells

Lusa, S., Heino, S. and Ikonen, E.

1. Biol. Chem., 278, 19844-19851 (2003)

  Previous work demonstrates that the biosynthetic precursor of cholesterol, desmosterol, is released from cells and that its efflux to high density lipoprotein or phosphatidylcholine vesicles is greater than that of newly synthesized cholesterol (Johnson, W. J., Fischer, H. T., Phillips, M. C., and Rothblat, G. H. (1995) J. BioL Chem. 270, 25037-25046). Here we report that the release of individual precursor sterols varies with the efflux of newly synthesized zymosterol being greater than that of lathosterol and both exceeding that of newly synthe­sized cholesterol when using either methyl-b-cyclodex-trin or complete serum as acceptors. The transfer of newly synthesized lathosterol to methyl-b-cyclodextrin was inhibited by actin polymerization but not by Golgi disassembly whereas that of newly synthesized choles­terol was inhibited by both conditions. Newly synthe­sized lathosterol associated with cellular detergent-re­sistant membranes more rapidly than newly synthesized cholesterol. Upon efflux to serum, newly synthesized cholesterol precursors associated with both high and low density lipoproteins. Stimulation of the formation of direct endoplasmic reticulum-plasma membrane contacts was accompanied by enhanced ef­flux of newly synthesized lathosterol but not of newly synthesized cholesterol to serum acceptors. The data indicate that the efflux of cholesterol precursors differs not only from that of cholesterol but also from each other, with the more polar zymosterol being more avidly effluxed. Moreover, the results suggest that the intracel­lular routing of cholesterol precursors differs from that of newly synthesized cholesterol and implicates a poten­tial role for the actin cytoskeleton and endoplasmic re­ticulum-plasma membrane contacts in the efflux of lathosterol.  

3.387           Presenilin 1 and Presenilin 2 have differential effects on the stability and maturation of Nicastrin in mammalian brain

Chen, F. et al.

1. Biol. Chem., 278, 19974-19979 (2003)

  The presenilins and nicastrin form high molecular mass, multimeric protein complexes involved in the in. tramembranous proteolysis of several proteins. Post-translational glycosylation and trafficking of nicastrin is necessary for the activity of these complexes. We re­port here that although there are differences in the post-translational processing of nicastrin in neurons and glia, both of the presenilins are required for the physi­ological post-translational modification and for the cor­rect subcellular distribution of nicastrin. Absence of presenilin 1 (PS1) is associated with dramatic reduc­tions in the level of mature glycosylated nicastrin and with redistribution of nicastrin away from the cell sur­face. In contrast, absence of presenilin 2 (PS2) is associ­ated with only modest reductions in the levels of imma­ture nicastrin. It is notable that these differential effects parallel the differential effects of null mutations in PS1 and PS2 on APP and Notch processing. Our data there. fore suggest that the differential interactions of PS1 and PS2 with nicastrin reflect different functions for the PS1 and PS2 complexes.    

3.388           Synergistic assembly of linker for activation of T cells signaling protein complexes in T cell plasma membrane domains

Hartgrove, L., Lin, J., Langen, H., Zech, T., Weiss, A. and Harder, T.

1. Biol. Chem., 278, 20389-20394 (2003)

  Transmembrane adaptor molecule LAT (linker for ac­tivation of T cells) forms a central scaffold for signaling protein complexes that accumulate in the vicinity of activated T cell antigen receptors (TCR). Here we used biochemical analysis of immunoisolated plasma mem­brane domains and fluorescence imaging of green fluo­rescence protein-tagged signaling proteins to investi­gate the contributions of different tyrosine.based signaling protein docking sites of LAT to the formation of LAT signaling protein assemblies in TCR membrane domains. We found that the phospholipase C g docking site of LAT and different Grb2/Gads docking sites func­tion in an interdependent fashion and synergize to ac­cumulate LAT, Grb2, and phospholipase C g in TCR sig­naling assemblies. Two-dimensional gels showed that Grb2 is a predominant cytoplasmic adaptor in the iso­lated LAT signaling complexes, whereas Gads, Crk-1, and Grap are present in lower amounts. Taken together our data suggest a synergistic assembly of multimolecu­lar TCR-LAT signal transduction complexes in T cell plasma membrane domains.  

3.389           A novel method of imaging lysosomes in living human mammary epithelial cells

Glunde, K., Guggino, S.E., Ichikawa, Y. and Bhujwalla, Z.M. Mol. Imaging, 2, 24-36 (2003)   Cancer cells Invade by secreting degradative enzymes which, under normal conditions, are sequestered in lysosomal vesicles. The ability to non-invasively label lysosomes and track lysosomal trafficking would be extremely useful to understand the mechanisms by which degradative enzymes are secreted in the presence of pathophysiological environments, such as hypoxia and acidic extracellular pH, which are frequently encountered in solid tumors. In this study, a novel method of introducing a fluorescent label into lysosomes of human mammary epithellal cells (HMECs) was evaluated. Highly glycosylated lysosomal membrane proteins were labeled with a newly synthesized compound, 5-dimethylamino-naphthalene-1-sulfonic acid 5-amino-3,4,6-trihydroxy-tetrahydro-pyran-2-ylmethyl ester (6-O-dansyl.G1cNH₂). The ability to optically image lysosomes using this new probe was validated by determining the colocalization of the fluorescence from the dansyl group with immunofluorescent staining of two well-established lysosomal marker proteins, LAMP-1 and LAMP-2. The location of the dansyl group in lysosomes was also verified by using an anti-dansyl antibody in Western blots of lysosomes isolated using isopycnic density gradient centrifugation. This novel method of labeling lysosomes biosynthetically was used to image lysosomes in living HMECs perfused In a microscopy-compatible cell perfusion system.  

3.390           Fyn binds to and phosphorylates the kidney slit diaphragm component Nephrin

Verma, R. et al.

1. Biol. Chem., 278, 20716-20723 (2003)

  Recent investigations have focused on characterizing the molecular components of the podocyte intercellular junction, because several of these components, includ­ing Nephrin, are functionally necessary for develop. ment of normal podocyte structure and filter integrity. Accumulating evidence suggests that the Nephrin-asso. ciated protein complex is a signaling nexus. As such, Nephrin-dependent signaling might be mediated in part through Nephrin phosphorylation. Described are bio­chemical and mouse genetics experiments demonstrat­ing that membrane-associated Nephrin is tyrosine-­phosphorylated by the Src family kinase Fyn. Nephrin fractionated in detergent-resistant glomerular mem­brane fractions with Fyn and Yes. Fyn directly bound Nephrin via its SH3 domain, and Fyn directly phospho­rylated Nephrin. Glomeruli in which Fyn, Yes, or Fyn and Yes were genetically deleted in mice were charac. terized to explore the relationship between these ki­nases and Nephrin. Fyn deletion resulted in coarsening of podocyte foot processes and marked attenuation of Nephrin phosphorylation in isolated glomerular deter­gent-resistant membrane fractions. Yes deletion had no identifiable effect on podocyte morphology but dramat­ically increased Nephrin phosphorylating activity. Sim­ilar to Fyn deletion, simultaneous deletion of Fyn and Yes reduced Nephrin phosphorylating activity. These results demonstrate that endogenous Fyn catalyzes Nephrin phosphorylation in podocyte detergent-resis­tant membrane fractions. Although Yes appears to effect the regulation of Nephrin phosphorylation, the mecha­nism by which this occurs requires investigation.  

3.391           Ultracentrifugation-based approaches to study regulation of Sec6/8 (exocyst) complex function during development of epithelial cell polarity

Yeaman, C. Methods 30, 198-206 (2003)   The Sec6/8 (exocyst) complex is an essential component of the exocytic apparatus and plays an evolutionarily conserved role in polarized membrane growth. During development of epithelial cell polarity, this cytosolic protein complex is recruited to plasma membrane sites of cell-cell contact, where it facilitates exocytosis to the lateral membrane domain. However, the identity of membrane binding sites for Sec6/8 complex, mechanisms regulating association of Sec6/8 complex with these sites, and the precise function of the complex in polarized trafficking are not known. Biochemical strategies involving differential, rate-zonal, and iso­pycnic density gradient ultracentrifugation are providing clues to these questions.  

3.392           Disruption of the epithelial apical-junction complex by Helicobacter pylori CagA

Amieva, M.R., Vogelmann, R., Covacci, A., Tompkins, L.S., Nelson, W.J. and Falkow, S. Science, 300, 1430-1434 (2003)   Helicobacter pylori translocates the protein CagA into gastric epithelial cells and has been linked to peptic ulcer disease and gastric carcinoma. We show that injected CagA associates with the epithelial tight-junction scaffolding protein ZO-1 and the transmembrane protein junctional adhesion molecule, causing an ectopic assembly of tight-junction components at sites of bacterial attachment, and al­tering the composition and function of the apical-junctional complex. Long-term CagA delivery to polarized epithelia caused a disruption of the epithelial barrier function and dysplastic alterations in epithelial cell morphology. CagA appears to target H. pylori to host cell intercellular junctions and to disrupt junction-mediated functions.  

3.393           Expression of the molecular chaperone Hsp70 in detergent-resistant microdomains correlates with its membrane delivery and release

Broquet, A.H., Thomas, G., Masliah, J., Trugnan, G. and Bachelet, M.

1. Biol. Chem., 278, 21601-21606 (2003)

  Accumulating evidence suggests that some heat shock proteins (Hsps), in particular the 72-kDa inducible Hsp70, associate to the cell membrane and might be secreted through an unknown mechanism to exert im­portant functions in the immune response and signal transduction. We speculated that specialized structures named lipid rafts, known as important platforms for the delivery of proteins to the cell membrane, might be in­volved in the unknown mechanism ensuring membrane association and secretion of Hsp70. Lipid rafts are sphingolipid-cholesterol-rich structures that have been mainly characterized in polarized epithelial cells and can be isolated as detergent-resistant microdomains (DRMs). Analysis of soluble and DRM fractions prepared from unstressed Caco-2 epithelial cells revealed that Hsp70, and to a lesser extent calnexin, were present in DRM fractions. Increased expression of Hsps, through heat shock or by using drugs acting on protein traffick­ing or intracellular calcium level, induced an efficient translocation to DRM. We also found that Hsp70 was released by epithelial Caco-2 cells, and this release dra­matically increased after heat shock. Drugs known to block the classical secretory pathway were unable to reduce Hsp70 release. By contrast, release of the protein was affected by the raft-disrupting drug methyl-b-cyclodextrin. Our data suggest that lipid rafts are part of a mechanism ensuring the correct functions of Hsps and provide a rational explanation for the observed mem­brane association and release of Hsp70.    

3.394           Organization of the G protein-coupled receptors rhodopsion and opsin in native membranes

Liang, Y., Fotiadis, D., Filipek, S., Saperstein, D.A., Palczewski, K. and Engel, A.

1. Biol. Chem., 278, 21655-21662 (2003)

  G protein-coupled receptors (GPCRs), which constitute the largest and structurally best conserved family of signaling molecules, are involved in virtually all physiological processes. Crystal structures are available only for the de­tergent-solubilized light receptor rhodopsin. In addition, this receptor is the only GPCR for which the presumed higher order oligomeric state in native membranes has been demonstrated (Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D. A., Engel, A., and Palczewski, K (2003) Na­ture 421, 127-128). Here, we have determined by atomic force microscopy the organization of rhodopsin in native membranes obtained from wild-type mouse photoreceptors and opsin isolated from photoreceptors of Rpe65-/- mu­tant mice, which do not produce the chromophore 11-cis-retinal. The higher order organization of rhodopsin was present irrespective of the support on which the mem­branes were adsorbed for imaging. Rhodopsin and opsin form structural dimers that are organized in paracrystal­line arrays. The intradimeric contact is likely to involve helices IV and V, whereas contacts mainly between helices I and II and the cytoplasmic loop connecting helices V and VI facilitate the formation of rhodopsin dimer rows. Con­tacts between rows are on the extracellular side and in­volve helix I. This is the first semi-empirical model of a higher order structure of a GPCR in native membranes, and it has profound implications for the understanding of how this receptor interacts with partner proteins.  

3.395           Novel localization of the DNA-PK complex in lipid rafts: a putative role in the signal transduction pathway of the ionizing radiation response

Lucero, H., Gae, D. and Taccioli, G.E.

1. Biol. Chem., 278, 22136-22143 (2003)

  Increased sensitivity to ionizing radiation (IR) has been shown to be due to defects in DNA double-strand break repair machinery. The major pathway in mamma­lian cells dedicated to the repair of DNA double-strand breaks is by the nonhomologous end-joining machinery. Six components function in this pathway, of which three (Ku70, Ku86, and DNA-PKcs) constitute a protein com­plex known as DNA-dependent protein kinase (DNA­PK). However, it is now recognized that the cellular radiation response is complex, and radiosensitivity may be also regulated at different levels in the radiation signal transduction pathway. In addition to DNA dam­age, exposure to IR triggers intracellular signaling cas­cades that overlap with pathways initiated by ligand engagement to a receptor. In this study, we provide evidence for the novel localization of the DNA-PK com­plex in lipid rafts. We also show this property is not a generalized characteristic of all DNA repair proteins. Furthermore, we have detected Ku86 in yeast lipid rafts. Our results suggest that the components of this complex might be recruited separately to the plasma membrane by tethering with raft-resident proteins. In addition, we found an irradiation-induced differential protein phos­phorylation pattern dependent upon DNA-PKcs in lipid rafts. Thus, we speculate that another role for the DNA­PKcs subunit and perhaps for the holoenzyme is in the signal transduction of IR response.  

3.396           Virus entry, assembly, budding and membrane rafts (Review article)

Chazal, N. and Gerlier, D. Microbiol. Mol. Biol. Rev., 67, 226-237 (2003)   Specific microdomains of the plasma membrane called rafts appear to be involved in many biological events such as bio­synthetic traffic, endocytic traffic, and the signal transduction pathway. Among pathogens, viruses, which are obligate intra­cellular parasites, are confronted with the plasma membrane during their life cycle. They have to enter their host cells by fusion, permeation, or endocytic vesicle discharge and to exit them by budding or membrane disruption. In this review, we focus on data supporting the involvement of membrane rafts in the virus replication cycle, their role as a viral entry site, a platform for the assembly of viral compo­nents, and a scaffold for the budding of virus from infected cells. The elucidation of these interactions requires a detailed understanding of raft structures and dynamics.    

3.397           GAP43 stimulates inositol trisphosphate-mediated calcium release in response to hypotonicity

Caprini, M. et al. The EMBO J., 22, 3004-3014 (2003)   The identification of osmo/mechanosensory proteins in mammalian sensory neurons is still elusive. We have used an expression cloning approach to screen a human dorsal root ganglion cDNA library to look for proteins that respond to hypotonicity by raising the intracellular Ca²⁺ concentration ([Ca²⁺]_i). We report the unexpected identification of GAP43 (also known as neuromodulin or B50), a membrane-anchored neuro­nal protein implicated in axonal growth and synaptic plasticity, as an osmosensory protein that augments [Ca²⁺]_i, in response to hypoton-icity. Palmitoylation of GAP43 plays an important role in the protein osmo­sensitivity. Depletion of intracellular stores or inhib­ition of phospholipase C (PLC) activity abrogates hypotonicity-evoked, GAP43-mediated [Ca²⁺]_i eleva­tions. Notably, hypotonicity promoted the selective association of GAP43 with the PLC-d₁ isoform, and a concomitant Increase in inositol-1,4,5-trisphospbate (IP₃) formation. Collectively, these findings Indicate that hypo-osmotic activation of GAP43 induces Ca²⁺ release from IP₃-sensitive intracellular stores. The osmosensitivity of GAP43 furnishes a mechanistic framework that links axon elongation with phospho­inositide metabolism, spontaneous triggering of cyto­solic Ca²⁺ transients and the regulation of actin dynamics and motility at the growth cone in response to temporal and local mechanical forces.  

3.398           Presenilin endoproteolysis mediated by an aspartyl protease activity pharmacologically distinct from g-secretase

Campbell, W.A., Reed, M.L.O., Strahle, J., Wolfe, M.S. and Xia, W.

1. Neurochem., 85, 1563-1574 (2003)

  Presenilin (PS)-dependent g-secretase cleavage is the final proteolytic step in generating amyloid b protein (Ab), a key peptide involved in the pathogenesis of Alzheimer’s disease. PS undergoes endoproteolysis by an unidentified ‘presenilin­ase’ to generate the functional N-terminal and C-terminal fragment heterodimers (NTF/CTF) that may harbor the g-secretase active site. To better understand the relationship between presenilinase and g-secretase, we characterized the biochemical properties of presenilinase and compared them with those of g-secretase. Similar to g-secretase, presenilin­ase was most active at acidic pH 6.3. Aspartyl protease inhibitor pepstatin A blocked presenilinase activity with an IC₅₀ of ~1 mM. Difluoroketone aspartyl protease transition state analogue MW167 was relatively selective for presenilinase (IC₅₀ < 1mM) over g-secretase (IC₅₀ - 16 mM). Importantly, removing the transition state mimicking moiety simultaneously abolished both presenilinase and g-secretase inhibition, sug­gesting that presenilinase, like g-secretase, is an aspartyl protease. Interestingly, several of the most potent g-secretase inhibitors (IC₅₀ = 0.3 or 20 mM) failed to block presenilinase activity. Although de novo generation of PS1 fragments coincided with production of Ab in vitro, blocking presenilinase activity without reducing pre-existing fragment levels permit­ted normal do novo generation of Ab and amyloid intracellular domain. Therefore, presenilinase has characteristics of an aspartyl protease, but this activity is distinct from g-secretase.  

3.399           NGF signaling in sensory neurons: evidence that early endosomes carry NGF retrograde signals

Delcroix, J-D., Valletta, J.S., Wu, C., Hunt, S.J., Kowal, A.S. and Mobley, W.C. Neuron, 39¸69-84 (2003)   Target-derived NGF promotes the phenotypic mainte­nance of mature dorsal root ganglion (DRG) nocicep­tive neurons. Here, we provide in vivo and in vitro evidence for the presence within DRG neurons of en­dosomes containing NGF, activated TrkA, and signal­ing proteins of the Rapl/Erkl/2, p38MAPK, and PI3K/Akt pathways. Signaling endosomes were shown to be retrogradely transported in the isolated sciatic nerve in vitro. NGF injection in the peripheral target of DRG neurons increased the retrograde transport of p-Erkl/2, p-p38, and pAkt in these membranes. Con­versely, NGF antibody injections decreased the retro­grade transport of p-Erkl/2 and p-p38. Our results are evidence that signaling endosomes, with the charac­teristics of early endosomes, convey NGF signals from the target of nociceptive neurons to their cell bodies.    

3.400           Cell surface expression of functional hepatitis C virus E1 and E2 glycoproteins

Drummer, H.E., Maerz, A. and Poumbourios, P. FEBS Lett., 546, 385-390 (2003)   Hepatitis C virus (HCV) glycoproteins El and E2 are believed to be retained in the endoplasmic reticulum (ER) or cis-Golgi compartment via retention signals located in their transmembrane domains. Here we describe the detection of El and E2 at the surface of transiently transfected HEK 293T and Huh7 cells. Surface-localized E1E2 heterodimers presented ex­clusively as non-covalently associated complexes. Surface-ex­pressed E2 contained trans-Golgi modified complex/hybrid type carbohydrate and migrated diffusely between 70 and 90 kDa while intracellular El and E2 existed as high mannose 35 kDa and 70 kDa precursors, respectively. In addition, sur­face-localized E1E2 heterodimers were incorporated into E1E2-pseudotyped HIV-l particles that were competent for entry into Huh7 cells. These studies suggest that functional HCV glyco­proteins are not retained exclusively in the ER and transit through the secretory pathway.  

3.401           Distinct endosomal compartments in early trafficking of low density lipoprotein-derived cholesterol

Sugii, S., Reid, P.C., Ohgami, N., Du, H. and Chang, T-Y.

1. Biol. Chem., 278, 27180-27189 (2003)

  We previously studied the early trafficking of low den­sity lipoprotein (LDL)-derived cholesterol in mutant Chi­nese hamster ovary cells defective in Niemann-Pick type Cl (NPC1) using cyclodextrin (CD) to monitor the arrival of cholesterol from the cell interior to the plasma mem­brane (PM) (Cruz, J. C., Sugii, S., Yu, C., and Chang, T.-Y. (2000) J. BioL Chem. 215,4013-4021). We found that newly hydrolyzed cholesterol derived from LDL first appears in certain CD-accessible pool(s), which we assumed to be the PM, before accumulating in the late endosome/lysosome, where NPC1 resides. To determine the identity of the early CD-accessible pool(s), in this study, we performed additional experiments, including the use of revised CD incubation protocols. We found that prolonged incuba­tion with CD (>30 min) caused cholesterol in internal membrane compartment(s) to redistribute to the PM, where it became accessible to CD. In contrast, a short incubation with CD (5-10 min) did not cause such an effect. We also show that one of the early compartments contains acid lipase (AL), the enzyme required for liber­ating cholesterol from cholesteryl ester in LDL Biochem­ical and microscopic evidence indicates that most of the AL is present in endocytic compartment(s) distinct from the late endosome/lysosome. Our results suggest that cho­lesterol is liberated from LDL cholesteryl ester in the hydrolytic compartment containing AL and then moves to the NPC1-containing late endosome/lysosome before reaching the PM or the endoplasmic reticulum.  

3.402           Syntaxin 6 regulates Glut4 trafficking in 3T3-L1 adipocytes

Perera, H.K.I., Clarke, M., Morris, N.J., Hong, W., Chamberlain, L.H. and Gould, G.W. Mol. Biol. Cell, 14, 2946-2958 (2003)   Insulin stimulates the movement of glucose transporter-4 (Glut4)-containing vesicles to the plasma membrane of adipose cells. We investigated the role of post-Golgi t-soluble N-ethylma­leimide-sensitive factor attachment protein receptors (SNAREs) in the trafficking of Glut4 in 3T3-L1 adipocytes. Greater than 85% of syntaxin 6 was found in Glut4-containing vesides, and this t-SNARE exhibited insulin-stimulated movement to the plasma membrane. In contrast, the colocalization of Glut4 with syntaxin 7, 8, or 12/13 was limited and these molecules did not translocate to the plasma membrane. We used adenovirus to overexpress the cytosolic domain of these syntaxins and studied their effects on GIut4 traffic. Overexpression of the cytosolic domain of syntaxin 6 did not affect insulin-stimulated glucose transport, but increased basal deGlc transport and cell surface Glut4 levels. Moreover, the syntaxin 6 cytosolic domain significantly reduced the rate of Glut4 re-internalization after insulin withdrawal and perturbed sub-endosomal Glut4 sorting; the corresponding domains of syntaxins 8 and 12 were without effect. Our data suggest that syntaxin 6 is involved in a membrane-trafficking step that sequesters Glut4 away from traffic destined for the plasma membrane. We speculate that this is at the level of traffic of Glut4 into its unique storage compartment and that syntaxin 16 may be involved.    

3.403           Bursts of high-frequency stimulation trigger rapid delivery of pre-existing a-CaMKII mRNA to synapses: a mechanism in dendritic protein synthesis during long-term potentiation in adult awake rats

Havik, B., Rokke, H., Bardsen, K., Davanger, S. and Bramham, C.R. Eur. J. Neurosci., 17, 2679-2689 (2003)   Messenger ribonucleic acid encoding the alpha-subunit of calcium/calmodulin-dependent protein kinase II (camkII) is abundantly and constitutively expressed in dendrites of pyramidal and granule cell neurons of the adult hippocampus. Recent evidence suggests that camkII messenger ribonucleic acid is stored in a translationally dormant state within ribonucleic acid storage granules. Delivery of camkII messenger ribonucleic acid from sites of storage to sites of translation may therefore be a key step in activity-driven dendritic protein synthesis and synaptic plasticity. Here we explored possible camkII trafficking in the context of long-term potentiation in the dentate gyrus of awake, adult rats. Long-term potentiation was induced by patterned high-frequency stimulation, synaptodendrosomes containing pinched-off dendritic spines were obtained from microdissected dentate gyrus, and messenger ribonucleic acid levels were determined by real-time polymerase chain reaction. High-frequency stimulation triggered a rapid 2.5-fold increase in camkII messenger ribonucleic acid levels in the synaptodendrosome fraction. This increase occurred in the absence of camkII upregulation in the homogenate fraction, indicating trafficking of pre-existing messenger ribonucleic acid to synaptodendrosomes. The elevation in camkII messenger ribonucleic acid was paralleled by an increase in protein expression specific to the synaptodendrosome fraction, and followed by depletion of camkII message. Activity-dependent regulation of camkII messenger ribonucleic acid and protein did not require N-methyl-D-aspartate receptor activation. In contrast, N-methyl-D-aspartate receptor activation was required for induction of the immediate early genes zif268 and activity-regulated cytoskeleton-associated protein in dentate gyrus homogenates. The results support a model in which locally stored camII messenger ribonucleic acid is rapidly transported to dendritic spines and translated during long-term potentiation in behaving rats.  

3.404           Ergosterol is required for targeting of tryptophan permease to the yeast plasma membrane

Umebayashi, K. and Nakano, A.

1. Cell Biol., 161, 1117-1131 (2003)

  It was known that the uptake of tryptophan is reduced in the yeast erg6 mutant, which is defective in a late step of ergosterol biosynthesis. Here, we show that this is because the high affinity tryptophan permease Tat2p is not targeted to the plasma membrane. In wild-type cells, the plasma membrane localization of Tat2p is regulated by the external tryptophan concentration. Tat2p is transported from the Golgi apparatus to the vacuole at high tryptophan, and to the plasma membrane at low tryptophan. However, in the erg6 mutant, Tat2p is missorted to the vacuole at low tryptophan. The plasma membrane targeting of Tat2p is dependent on detergent-insoluble membrane domains, suggesting that sterol affects the sorting through the organization of lipid rafts. The erg6 mutation also caused missorting to the multi­vesicular body pathway in late endosomes. Thus, sterol composition is crucial for protein sorting late in the secretory pathway. Tat2p is subject to polyubiquitination, which acts as a vacuolar-targeting signal, and the inhibition of this process suppresses the Tat2p sorting defects of the erg6 mutant. The sorting mechanisms of Tat2p that depend on both sterol and ubiquitin will be discussed.  

3.405           Pharmacological characterization and immunoaffinity purification of metabotropic glutamate receptor from Drosophila overexpressed in Sf9 cells

Panneels, V., Eroglu, C., Cronet, P. and Sinning, I. Prot. Expression Purification, 20, 275-282 (2003)   Metabotropic glutamate receptors (mGluRs) play important roles in the function and regulation of the central nervous system. Structural studies are necessary for the detailed understanding of their mechanisms of action. However, overexpression and puri­fication of functional receptors in quantities required for these studies proves to be a major challenge. In this study we report the overexpression of a Drosophila melanogaster mGluR (DmG1uRA) by using a baculovirus—insect cell expression system. Expression was tested in two different insect cell hosts (Sf9 and Hi5) and analyzed by performing expression kinetics. Pharmacological characterization of the recombinant receptor by radioactive glutamate binding assays showed a profile similar to group II mGluRs, as previously reported, when the receptor was expressed in mammalian systems. The B_max value reached 11 pmol receptor/mg Sf9-membrane protein. A monoclonal antibody against DmG1uRA was generated by genetic immunization and used to purify the receptor.  

3.406           Inhibition of GTP-dependent vesicle trafficking impairs internalization of palsmalemmal eNOS and cellular nitric oxide production

Chaterjee, S., Cao, S., Peterson, T.E., Simari, R.D. and Shah, V.

1. Cell Sci., 116, 3645-3655 (2003)

  The Ca²⁺ mobilizing peptide, bradykinin (BK), stimulates endothelial nitric oxide synthase (eNOS)-derived cellular nitric oxide (NO) production in association with altering the subcellular distribution of the enzyme. In the present study we examine the influence of cellular GTPases, particularly the large GTPase dynamin, on BK-mediated eNOS localization and cellular NO production. BK stimulation of ECV cells, which were stably transfected with eNOS-GFP (eNOS-GFP ECV304), increased NO production. This was associated with the mobilization of eNOS-GFP protein into Triton X-100-insoluble fractions of cell lysates, and an internalization of plasmalemmal eNOS-GFP in live and fixed ECV 304 cells. Incubation of digitonin-permeabilized ECV304 cells with the non-hydrolyzed GTP analog, GTPg-S, abrogated the BK-mediated internalization of eNOS-GFP as assessed by confocal microscopy. Conversely, inhibition of clathrin-­dependent endocytosis, via overexpression of AP 180 or pretreatment of cells with chlorpromazine, did not influence BK-mediated eNOS redistribution. Furthermore, specific inhibition of dynamin-2 GTPase function by overexpression of a dominant negative construct, K44A, prevented the BK-mediated enrichment of eNOS-GFP within low buoyant density, caveolin-enriched fractions of eNOS-GFP ECV304 cell lysates. Dynamin-2 K44A overexpression also markedly impaired BK-dependent, L-NAME-inhibited NO production as did incubation of permeabilized cells with GTP-g-s. These studies demonstrate that disruption of dynamin- and GTP­dependent, but clathrin-independent, vesicle trafficking pathways impairs BK-dependent cellular NO production, via inhibition of the internalization of eNOS-containing plasmalemmal vesicles.  

3.407           Identification of organelles in bacteria similar to acidocalsisomes of unicellular eukaryotes

Seufferheld, M. et al

1. Biol. Chem., 278(32), 29971-29978 (2003)

  Acidocalcisomes are acidic calcium storage compartments described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds. In this work, we report that the volutin granules of Agrobacterium tumefaciens possess properties similar to the acidocalcisomes. Transmission electron microscopy revealed that each intracellular granule was surrounded by a membrane. X-ray microanalysis of the volutin granules showed large amounts of phosphorus, magnesium, potassium, and calcium. Calcium in the volutin granules increased when the bacteria were incubated at high extracellular calcium concentration. Immunofluorescence and immunoelectron microscopy, using antisera raised against peptide sequences conserved in the A. tumefaciens proton pyrophosphatase, indicated localization in intracellular vacuoles. Purification of the volutin granules using iodixanol density gradients indicated a preferential localization of the pyrophosphatase activity in addition to high concentrations of phosphate, pyrophosphate, short- and long-chain polyphosphate, but lack of markers of the plasma membrane. The pyrophosphatase activity was potassium-insensitive and inhibited by the pyrophosphate analogs, amynomethylenediphosphonate and imidodiphosphate, by dicyclohexylcarbodiimide, and by the thiol reagent N-ethylmaleimide. Polyphosphate was also localized to the volutin granules by 4',6'-diamino-2-phenylindole staining. The organelles were acidic, as demonstrated by staining with LysoSensor blue DND-167, a dye especially used to detect very acidic compartments in cells, and cycloprodigiosin, a compound isolated from a marine bacterium that has been shown to uncouple proton pyrophosphatase activity acting as a chloride/proton symport. The results suggest that acidocalcisomes arose before the prokaryotic and eukaryotic lineages diverged.  

3.408           Association of the Golgi UDP-galactose transporter with UDP-galactose: ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum

Sprong, H. et al Mol. Biol. Cell, 14, 3482-3493 (2003)   UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose:ceramide galactosyltransferase. It is not known how UDP-galactose is transported from the cytosol into the endoplasmic reticulum. We transfected ceramide galactosyltransferase cDNA into CHOlec8 cells, which have a defective UGT and no endogenous ceramide galactosyltransferase. Cotransfection with the human UGT1 greatly stimulated synthesis of lactosylceramide in the Golgi and of galactosylceramide in the endoplasmic reticulum. UDP-galactose was directly imported into the endoplasmic reticulum because transfection with UGT significantly enhanced synthesis of galactosylceramide in endoplasmic reticulum membranes. Subcellular fractionation and double label immunofluorescence microscopy showed that a sizeable fraction of ectopically expressed UGT and ceramide galactosyltransferase resided in the endoplasmic reticulum of CHOlec8 cells. The same was observed when UGT was expressed in human intestinal cells that have an endogenous ceramide galactosyltransferase. In contrast, in CHOlec8 singly transfected with UGT 1, the transporter localized exclusively to the Golgi complex. UGT and ceramide galactosyltransferase were entirely detergent soluble and form a complex because they could be coimmunoprecipitated. We conclude that the ceramide galactosyltransferase ensures a supply of UDP-galactose in the endoplasmic reticulum lumen by retaining UGT in a molecular complex.  

3.409           Screening for nitric oxide-dependent protein-protein interactions

Matsumoto, A., Comatas, K.E., Liu, L. and Stamler, J.S. Science, 301, 657-661 (2003)   Because nitric oxide (NO) may be a ubiquitous regulator of cellular signaling, we have modified the yeast two-hybrid system to explore the possibility of NO-dependent protein-protein interactions.We screened for binding partners of procaspase-3, a protein implicated in apoptotic signaling pathways, and identified multiple NO-dependent interactions.Two such interactions, with acid sphingomyelinase and NO synthase, were shown to occur in mammalian cells dependent on endogenous NO.Nitrosylation may thus provide a broad-based mechanism for regulating interactions between proteins.If so, systematic proteomic analyses in which redox state and NO bioavailability are carefully controlled will reveal a large array of novel interactions.  

3.410           The yeast deubiquitinating enzyme Ubp16 is anchored to the outer mitochondrial membrane

Kinner, A. and Kölling, R. FEBS Lett., 549, 135-140 (2003)   We looked for membrane-associated Dubs (deubiquitinating enzymes) among the 16 yeast members of the ubiquitin-specific processing protease (Ubp) family to identify potential regulators of ubiquitin-dependent processes at membranes. For each of the Ubps examined, a certain fraction was found to be membrane associated. This fraction was only small for most Ubps but quite substantial for some Ubps. For Ubp4/Doa4 almost 40% of the protein was found in the membrane fraction suggesting that this protein performs a major function at membranes, probably at endosomes. Among the proteins tested, only one protein (Ubp16) was exclusively membrane associated. By cell fractionation and immunofluorescence experiments, we could show that Ubp16 is localized to mitochondria. Ubp16 contains an N-terminal hydrophobic domain that is similar to N-terminal sequences of other yeast outer mitochondrial membrane proteins. The presence of this putative signal sequence and the result of protease protection experiments suggest that Ubp16 is an integral membrane protein of the outer mitochondrial membrane with an N_in–C_out orientation. Phenotypic characterization of the ubp16 mutant and overexpression studies further suggest that Ubp16 is probably not important for the general functioning of mitochondria, but that it rather performs a more specialized function at mitochondria.  

3.411           Rotavirus infectious particles use lipid rafts during replication for transport to the cell surface in vitro and in vivo

Cuadras, M.A. and Greeberg, H.B. Virology, 313, 308-321 (2003)   The pathway by which rotavirus is released from the cell is poorly understood but recent work has shown that, prior to cell lysis, rotavirus is released almost exclusively from the apical surface of the infected cell. By virtue of their unique biochemical and physical properties, viruses have exploited lipid rafts for host cell entry and/or assembly. Here we characterized the association of rhesus rotavirus (RRV) with lipid rafts during the rotavirus replication cycle. We found that newly synthesized infectious virus associates with rafts in vitro and in vivo. RRV proteins cosegregated with rafts on density gradients. Viral infectivity and genomic dsRNA also cosegregated with the raft fractions. Confocal microscopic analysis of raft and RRV virion proteins demonstrated colocalization within the cell. In addition, cholesterol depletion interfered with the association of RRV particles with rafts and reduced the release of infectious particles from the cell. Furthermore, murine rotavirus associates with lipid rafts in intestinal epithelial cells during a natural infection in vivo. Our results confirm the association of rotavirus infectious particles with rafts during replication in vitro and in vivo and strongly support the conclusion that this virus uses these microdomains for transport to the cell surface during replication.  

3.412           Glutamate-binding affinity of Drosophila metabotropic glutamate receptor is modulated by association with lipid rafts

Eroglu, C., Brügger, B., Wieland, F. And Sinning, I. PNAS, 100(18), 10219-10224 (2003)   Metabotropic glutamate receptors (mGluRs) are responsible for the effects of glutamate in slow synaptic transmission, and are implicated in the regulation of many processes in the CNS. Recently, we have reported the expression and purification of a mGluR from Drosophila melanogaster (DmGluRA), a homologue of mammalian group II mGluRs. We have shown that ligand binding to reconstituted DmGluRA requires the presence of ergosterol in the liposomes [Eroglu, C., Cronet, P., Panneels, V., Beaufils, P. & Sinning, I. (2002) EMBO Rep. 3, 491-496]. Here we demonstrate that the receptor exists in different affinity states for glutamate, depending on the membrane composition. The receptor is in a high-affinity state when associated with sterol-rich lipid microdomains (rafts), and in a low-affinity state out of rafts. Enrichment of the membranes with cholesterol shifts the receptor into the high-affinity state, and induces its association with rafts. The receptor was crosslinked to photocholesterol. Our data suggest that sterol-rich lipid rafts act as positive allosteric regulators of DmGluRA.  

3.413           Mutational analysis of the cytoplasmic domain of b1,4-galactosyltransferase I: influence of phosphorylation on cell surface expression

Hathaway, H.J. et al

1. Cell Sci., 116, 4319-4330 (2003)

  b1,4-Galactosyltransferase I (GalT I) exists in two subcellular compartments where it performs two distinct functions. The majority of GalT I is localized in the Golgi complex where it participates in glycoprotein biosynthesis; however, a small portion of GalT I is expressed on the cell surface where it functions as a matrix receptor by binding terminal N-acetylglucosamine residues on extracellular glycoside ligands. The GalT I polypeptide occurs in two alternate forms that differ only in the length of their cytoplasmic domains. It is thought that the longer cytoplasmic domain is responsible for GalT I function as a cell surface receptor because of its ability to associate with the detergent-insoluble cytoskeleton. In this study, we demonstrate that the long GalT I cytoplasmic and transmembrane domains are capable of targeting a reporter protein to the plasma membrane, whereas the short cytoplasmic and transmembrane domains do not have this property. The surface-localized GalT I reporter protein partitions with the detergent-insoluble pool, a portion of which co-fractionates with caveolin-containing lipid rafts. Site-directed mutagenesis of the cytoplasmic domain identified a requirement for serine and threonine residues for cell surface expression and function. Replacing either the serine or threonine with aspartic acid reduces surface expression and function, whereas substitution with neutral alanine has no effect on surface expression or function. These results suggest that phosphorylation negatively regulates GalT I function as a surface receptor. Consistent with this, phosphorylation of the endogenous, full-length GalT I inhibits its stable expression on the cell surface. Thus, the 13 amino acid extension unique to the long GalT I isoform is required for GalT I expression on the cell surface, the function of which is regulated by phosphorylation.  

3.414           Abnormal cholesterol processing in Alzheimer’s disease patient’s fibroblasts

Dufour, D., Zhao, W-Q., Ravindranath, L. and Alkon, D.L. Neurobiol. Lipids., 1(7), 34-44 (2003)   Cholesterol has recently received attention as a potentially important factor in Alzheimer’s disease etiology. Caveolin, which binds cholesterol, plays a prominent role in cellular cholesterol transport. Here, we found a higher level of cholesterol and caveolin in the caveolae-enriched fractions prepared from Alzheimer's disease patients' (AD) fibroblasts compared with age and sex matched controls (AC). Furthermore, the cross-linking activation of the prion protein, which is known to link to signal transduction of caveolin, is altered in AD fibroblasts. Our results suggest a dysregulation of cholesterol processing in AD fibroblasts which may contribute to the pathogenesis of AD.      

3.415           Distribution of myocilin, a glaucoma gene product, in human corneal fibroblasts

Wentz-Hunter, K., Shen, X. and Yue, B.Y.J. Mol. Vision., 9, 308-314 (2003)   Purpose: Myocilin is a gene linked to open-angle glaucomas. In this study, the expression and distribution of myocilin in corneal fibroblasts with or without dexamethasone (DEX) treatment were investigated. Methods: Human corneal fibroblasts were treated with 100 nM DEX for 10-14 days. Immunofluorescence staining for myocilin was performed. Cell lysates and ultracentrifugation fractions were assessed by western blotting for distribution of myocilin and its possible association with various organelles. Staurosporine was used to induce apoptosis and apoptotic cells were detected using a monoclonal single stranded DNA antibody. Results: By immunofluorescence, myocilin protein was found to distribute throughout the cytoplasm of corneal fibroblasts including perinuclear regions. Myocilin distribution overlapped to varying degrees with that of the Golgi complex, endoplasmic reticulum, and mitochondria. Subsequent examination by subcellular fractionation however revealed that myocilin, while co-sedimenting with the Golgi complex, lysosomes, and endoplasmic reticulum, did not fractionate or associate with mitochondria. On western blots, protein bands at approximately 66, 57, and 55 kDa were detected and the intensity of the bands was not affected by DEX treatment in corneal fibroblasts. Apoptosis was induced by staurosporine to a similar extent in both DEX-treated and untreated corneal cultures. Conclusions: In corneal fibroblasts, myocilin expression is not enhanced by DEX treatment and the protein was not associated with mitochondria, in contrast to what were found in human trabecular meshwork (TM) cells. Such differences suggest that the expression and distribution of myocilin may be distinctive for TM cells and may explain why pathology with myocilin mutations is only evident in glaucoma even though myocilin is expressed ubiquitously in ocular and nonocular tissues.  

3.416           Proteomic characterisation of neuronal sphingolipid-cholesterol microdomains: role in plasminogen activation

Ledesma, M.D., Da Silva, J.S., Schevchenko, A., Wilm, M. And Dotti, C.G. Brain Res., 987, 107-116 (2003)   Sorting of certain membrane proteins requires a mechanism involving rafts, protein–lipid complexes enriched in glycosphingolipids and cholesterol. These microdomains remain at the plasma membrane of different cell types and play a role in signal transduction. Although recent reports have begun to describe molecules associated with rafts, their protein composition remains largely unknown, especially in neuronal cells. To address this question, we have purified detergent-insoluble raft fractions (DRMs) from primary cultures of hippocampal neurons. Bidimensional gel analysis and pharmacological raft lipid manipulation allowed the identification of neuronal raft proteins and their characterisation by MALDI-TOF analysis. Enolases were found among the proteins identified and functional studies demonstrate their participation in plasminogen binding. We also show the specific enrichment in rafts of several other plasminogen binding molecules and the exclusive activation of plasminogen to the protease plasmin in these microdomains. These observations suggest that neuronal rafts may play, in addition to intracellular signaling, a role in extracellular/membrane protein proteolysis.  

3.417           Characterization of the expression, intracellular localization, and replication complex association of the putative mouse hepatitis virus RNA-dependent RNA polymerase

Brockway, S.M., Clay, C.T., Lu, X.T. and Denison, M.R.

1. Virol., 77(19), 10515-10527 (2003)

  Mouse hepatitis virus (MHV) RNA synthesis is mediated by a viral RNA-dependent RNA polymerase (RdRp) on membrane-bound replication complexes in the host cell cytoplasm. However, it is not known how the putative MHV RdRp (Pol) is targeted to and retained on cellular membranes. In this report, we show that a 100-kDa protein was stably detected by an anti-Pol antiserum as a mature product throughout the virus life cycle. Gradient fractionation and biochemical extraction experiments demonstrated that Pol was not an integral membrane protein but was tightly associated with membranes and coimmunoprecipitated with the replicase proteins 3CLpro, p22, and p12. By immunofluorescence confocal microscopy, Pol colocalized with viral proteins at replication complexes, distinct from sites of virion assembly, over the entire course of infection. To determine if Pol associated with cellular membranes in the absence of other viral factors, the pol domain of gene1 was cloned and expressed in cells as a fusion with green fluorescentprotein, termed Gpol. In Gpol-expressing cells that were infectedwith MHV, but not in mock-infected cells, Gpol relocalized froma diffuse distribution in the cytoplasm to punctate foci thatcolocalized with markers for replication complexes. Expressionof Gpol deletion mutants established that the conserved enzymaticdomains of Pol were dispensable for replication complex association,but a 38-amino-acid domain in the RdRp unique region of Polwas required. This study demonstrates that viral or virus-inducedfactors are necessary for Pol to associate with membranes ofreplication complexes, and it identifies a defined region ofPol that may mediate its interactions with those factors.  

3.418           Endoplasmic reticulum export of glycosyltransferases depends on interaction of a cytoplasmic dibasic motif with Sar1

Giraudo, C.G. and Maccioni, J.F. Mol. Biol. Cell, 14, 3753-3766 (2003)   Membrane proteins exit the endoplasmic reticulum (ER) in COPII-transport vesicles. ER export is a selective process in which transport signals present in the cytoplasmic tail (CT) of cargo membrane proteins must be recognized by coatomer proteins for incorporation in COPII vesicles. Two classes of ER export signals have been described for type I membrane proteins, the diacidic and the dihydrophobic motifs. Both motifs participate in the Sar1-dependent binding of Sec23p–Sec24p complex to the CTs during early steps of cargo selection. However, information concerning the amino acids in the CTs that interact with Sar1 is lacking. Herein, we describe a third class of ER export motif, [RK](X)[RK], at the CT of Golgi resident glycosyltransferases that is required for these type II membrane proteins to exit the ER. The dibasic motif is located proximal to the transmembrane border, and experiments of cross-linking in microsomal membranes and of binding to immobilized peptides showed that it directly interacts with the COPII component Sar1. Sar1GTP-bound to immobilized peptides binds Sec23p. Collectively, the present data suggest that interaction of the dibasic motif with Sar1 participates in early steps of selection of Golgi resident glycosyltransferases for transport in COPII vesicles.  

3.419           Endostatin associates with lipid rafts and induces reorganisation of the actin cytoskeleton via down-regulation of RhoA activity

Wickström, S.A., Alitalo, K. and Keski-Oja, J.

1. Biol. Chem., 278(39), 37895-37901 (2003)

  Endostatin, the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Observations that endostatin inhibits endothelial cell migration and induces disassembly of the actin cytoskeleton provide putative cellular mechanisms for this effect. To understand the mechanisms of endostatin-induced intracellular signaling, we analyzed the association of recombinant endostatin with endothelial cell lipid rafts and the roles of its heparin- and integrin-binding properties in this interaction. We observed that a fraction of cell surface-bound endostatin partitioned in low density membrane raft fractions together with caveolin-1. Heparinase treatment of cells prevented the recruitment of endostatin to the lipid rafts but did not affect the association of endostatin with the non-raft fraction, whereas preincubation of endostatin with soluble _{5 1} integrin prevented the association of endostatin with the endothelial cell membrane. Endostatin treatment induced recruitment of _{5 1} integrin into the raft fraction via a heparan sulfate proteoglycan-dependent mechanism. Subsequently, through _{5 1} integrin, heparan sulfate, and lipid raft-mediated interactions, endostatin induced Src-dependent activation of p190RhoGAP with concomitant decrease in RhoA activity and disassembly of actin stress fibers and focal adhesions. These observations provide a cell biological mechanism, which plausibly explains the anti-angiogenic mechanisms of endostatin in vivo.  

3.420           Phagosomes are competent organelles for antigen cross-presentation

Houde, M. et al Nature, 425, 402-406 (2003)   The ability to process microbial antigens and present them at the surface of cells is an important aspect of our innate ability to clear infections. It is generally accepted that antigens in the cytoplasm are loaded in the endoplasmic reticulum and presented at the cell surface on major histocompatibility complex (MHC) class I molecules, whereas peptides present in endo/phagocytic compartments are presented on MHC class II molecules. Despite the apparent segregation of the class I and class II pathways, antigens from intracellular pathogens including mycobacteria, Escherichia coli, Salmonella typhimurium, Brucella abortus and Leishmania, have been shown to elicit an MHC class-I-dependent CD8⁺ T-cell response, a process referred to as cross-presentation. The cellular mechanisms allowing the cross-presentation pathway are poorly understood. Here we show that phagosomes display the elements and properties needed to be self-sufficient for the cross-presentation of exogenous antigens, a newly ascribed function linked to phagocytosis mediated by the endoplasmic reticulum.  

3.421           Compartmentalization of integrin a6b4 signaling in lipid rafts

Gagnoux-Palacios, L. et al

1. Cell Biol., 162(7), 1189-1196 (2003)

  Integrin 6ß4 signaling proceeds through Src family kinase (SFK)–mediated phosphorylation of the cytoplasmic tail of ß4, recruitment of Shc, and activation of Ras and phosphoinositide-3 kinase. Upon cessation of signaling, 6ß4 mediates assembly of hemidesmosomes. Here, we report that part of 6ß4 is incorporated in lipid rafts. Metabolic labeling in combination with mutagenesis indicates that one or more cysteine in the membrane-proximal segment of ß4 tail is palmitoylated. Mutation of these cysteines suppresses incorporation of 6ß4 in lipid rafts, but does not affect 6ß4-mediated adhesion or assembly of hemidesmosomes. The fraction of 6ß4 localized to rafts associates with a palmitoylated SFK, whereas the remainder does not. Ligation of palmitoylation-defective 6ß4 does not activate SFK signaling to extracellular signal–regulated kinase and fails to promote keratinocyte proliferation in response to EGF. Thus, compartmentalization in lipid rafts is necessary to couple the 6ß4 integrin to a palmitoylated SFK and promote EGF-dependent mitogenesis.  

3.422           Mislocalization of membrane proteins associated with multidrug resistance in cisplastin-resistant cancer cell lines

Liang, X-J., Shen, D-W., Garfield, S. And Gottesman, M.M. Cancer Res., 63, 5909-5916 (2003)   The accumulation of [¹⁴C]carboplatin and [³H]methotrexate is reduced in single-step KB epidermoid adenocarcinoma (KB-CP) cells, which are cross-resistant to carboplatin, methotrexate, and sodium arsenite. In these KB-CP cells, multidrug resistance is accompanied by mislocalization of multidrug resistance associated protein (MRP) 1 and other membrane proteins such as folate-binding protein. MRP1 was not decreased in amount in single-step variants but accumulates in a cytoplasmic fraction, and its apparent molecular weight was altered probably because of reduced glycosylation in resistant cells. This low-density compartment was partially labeled with antibodies to lectin-GSII (a Golgi marker) and Bip/GRP78 (an endoplasmic reticulum marker). Pulse-chase labeling of MRP1 with ³⁵S-methionine and ³⁵S-cysteine and pulse-chase biotinylation of cell surface MRP1 suggests that membrane protein mislocalization is caused mainly by a defect of plasma membrane protein recycling, manifested also as a defect in acidification of lysosomes. The reduced accumulation of cytotoxic compounds in the KB-CP cells is presumed to result from the failure of carrier proteins and/or transporters to localize to the plasma membrane.  

3.423           The major surface protease (MSP or GP63) of Leishmania sp.  Biosynthesis, regulation of expression and function

Yao, C., Donelson, J.E. and Wilson, M.E. Mol. Biol. Parasitol., 132, 1-16 (2003)   Leishmania sp. are digenetic protozoa that cause an estimated 1.5–2 million new cases of leishmaniasis per year worldwide. Among the molecular factors that contribute to Leishmania sp. virulence and pathogenesis is the major surface protease, alternately called MSP, GP63, leishmanolysin, EC3.4.24.36, and PSP, which is the most abundant surface protein of leishmania promastigotes. Recent studies using gene knockout, antisense RNA and overexpression mutants have demonstrated a role for MSP in resistance of promastigotes to complement-mediated lysis and either a direct or indirect role in receptor-mediated uptake of leishmania. The MSP gene clusters in different Leishmania sp. include multiple distinct MSPs that tend to fall into three classes, which can be distinguished by their sequences and by their differential expression in parasite life stages. Regulated expression of MSP class gene products during the parasite life cycle occurs at several levels involving both mRNA and protein metabolism. In this review we summarize advances in MSP research over the past decade, including organization of the gene families, crystal structure of the protein, regulation of mRNA and protein expression, biosynthesis and possible functions. The MSPs exquisitely demonstrate the multiple levels of post-transcriptional gene regulation that occur in Leishmania sp. and other trypanosomatid protozoa.  

3.424           APP processing is regulated by cytoplasmic phosphorylation

Lee, M-S. et al

1. Cell Biol., 163(1), 83-95 (2003)

  Amyloid-ß peptide (Aß) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers and the ß-secretase, BACE1. Western blot analysis reveals increased levels of T668-phosphorylated APP COOH-terminal fragments in hippocampal lysates from many AD but not control subjects. Importantly, P-APP cofractionates with endosome markers and BACE1 in an iodixanol gradient and displays extensive colocalization with BACE1 in rat primary cortical neurons. Furthermore, APP COOH-terminal fragments generated by BACE1 are preferentially phosphorylated on T668 verses those produced by -secretase. The production of Aß is significantly reduced when phosphorylation of T668 is either abolished by mutation or inhibited by T668 kinase inhibitors. Together, these results suggest that T668 phosphorylation may facilitate the BACE1 cleavage of APP to increase Aß generation.  

3.425           Functional g-secretase complex assembly in Golgi/trans-Golgi network: interactions among presenilin, nicastrin, Aph1, Pen-2, and g-secreatase substrates

Baulac, S. et al Neurobiology of Disease, 14, 194-204 (2003)   g-Secretase is a proteolytic complex whose substrates include Notch, -amyloid precursor protein (APP), and several other type I transmembrane proteins. Presenilin (PS) and nicastrin are known components of this high-molecular-weight complex, and recent genetic screens in invertebrates have identified two additional gene products, Aph1 and Pen-2, as key factors in -secretase activity. Here, we examined the interaction of the components of the -secretase complex in Chinese hamster ovary cells stably expressing human forms of APP, PS1, Aph1, and Pen-2. Subcellular fractionation of membrane vesicles and subsequent coimmunoprecipitation of individual -secretase components revealed that interactions among all proteins occurred in the Golgi/trans-Golgi network (TGN) compartments. Furthermore, incubation of the Golgi/TGN-enriched vesicles resulted in de novo generation of amyloid -protein and APP intracellular domain. Immunofluorescent staining of the individual -secretase components supported our biochemical evidence that the -secretase components assemble into the proteolytically active -secretase complex in the Golgi/TGN compartment.  

3.426           The conversion of apoB100 low density lipoprotein/high density lipoprotein particles to apoB100 very low density lipoproteins in response to oleic acid occurs in the endoplasmic reticulum and not in the Golgi in Mca RH7777 cells

Yamaguchi, J., Gamble, M.V., Conlon, D., Liang, J-S. and Ginsberg, H.N.

1. Biol. Chem., 278(43), 42643-42651 (2003)

  The site where bulk lipid is added to apoB100 low density lipoproteins (LDL)/high density lipoproteins (HDL) particles to form triglyceride-enriched very low density lipoproteins (VLDL) has not been identified definitively. We employed several strategies to address this question. First, McA RH7777 cells were pulse-labeled for 20 min with [³⁵S]methionine/cysteine and chased for 1 h (Chase I) to allow study of newly synthesized apoB100 LDL/HDL remaining in the endoplasmic reticulum (ER). After Chase I, cells were incubated for another hour (C2) with/without brefeldin A (BFA) and nocodazole (Noc) (to block ER to Golgi trafficking) and with/without oleic acid (OA). OA treatment alone during C2 increased VLDL secretion. This was prevented by the addition of BFA/Noc in C2. When C2 media were replaced by control media for another 1-h chase (C3), VLDL formed during OA treatment in C2 were secreted into C3 medium. Thus, OA-induced conversion of apoB100 LDL/HDL to VLDL during C2 occurred in the ER. Next, newly synthesized apoB100 lipoproteins were trapped in the Golgi by treatment with Noc and monensin during Chase I (C1), and C2 was carried out in the presence of BFA/Noc with/without OA and without monensin. Under these conditions, OA treatment during C2 did not stimulate VLDL secretion. The same pulse/chase protocols were followed by iodixanol subcellular fractionation, extraction of lipoproteins from ER and Golgi, and sucrose gradient separation of extracted lipoproteins. Cells treated with BFA/Noc and OA in C2 had VLDL in the ER. In the absence of OA, only LDL/HDL were present in the ER. The density of Golgi lipoproteins in these cells was not affected by OA. Similar results were obtained when ER were immuno-isolated with anti-calnexin antibodies. In conclusion, apoB100 bulk lipidation, resulting in conversion of LDL/HDL to VLDL, can occur in the ER, but not in the Golgi, in McA RH7777 cells.    

3.427           Pressure-induced differential regulation of the two tryptophan permeases Tat1 and Tat2 by ubiquitin ligase Rsp5 and its binding proteins, Bu11 and Bu12

Abe, F. and Iida, H. Mol. Cell. Biol., 23(21), 7566-7584 (2003)   Tryptophan uptake appears to be the Achilles' heel in yeast physiology, since under a variety of seemingly diverse toxic conditions, it becomes the limiting factor for cell growth. When growing cells of Saccharomyces cerevisiae are subjected to high hydrostatic pressure, tryptophan uptake is down-regulated, leading to cell cycle arrest in the G₁ phase. Here we present evidence that the two tryptophan permeases Tat1 and Tat2 are differentially regulated by Rsp5 ubiquitin ligase in response to high hydrostatic pressure. Analysis of high-pressure growth mutants revealed that the HPG1 gene was allelic to RSP5. The HPG1 mutation or the bul1 bul2 double mutation caused a marked increase in the steady-state level of Tat2 but not of Tat1, although both permeases were degraded at high pressure in an Rsp5-dependent manner. There were marked differences in subcellular localization. Tat1 localized predominantly in the plasma membrane, whereas Tat2 was abundant in the internal membranes. Moreover, Tat1 was associated with lipid rafts, whereas Tat2 localized in bulk lipids. Surprisingly, Tat2 became associated with lipid rafts upon the occurrence of a ubiquitination defect. These results suggest that ubiquitination is an important determinant of the localization and regulation of these tryptophan permeases. Determination of the activation volume ( V ) for Tat1- and Tat2-mediated tryptophan uptake (89.3 and 50.8 ml/mol, respectively) revealed that both permeases are highly sensitive to membrane perturbation and that Tat1 rather than Tat2 is likely to undergo a dramatic conformational change during tryptophan import. We suggest that hydrostatic pressure is a unique tool for elucidating the dynamics of integral membrane protein functions as well as for probing lipid microenvironments where they localize.  

3.428           The AP-1A and AP1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains

Fölsch, H., Pypaert, M., Maday, S., Pelletier, L. and Mellman, I.

1. Cell Biol., 163(2), 351-362 (2003)

  Most epithelial cells contain two AP-1 clathrin adaptor complexes. AP-1A is ubiquitously expressed and involved in transport between the TGN and endosomes. AP-1B is expressed only in epithelia and mediates the polarized targeting of membrane proteins to the basolateral surface. Both AP-1 complexes are heterotetramers and differ only in their 50-kD µ1A or µ1B subunits. Here, we show that AP-1A and AP-1B, together with their respective cargoes, define physically and functionally distinct membrane domains in the perinuclear region. Expression of AP-1B (but not AP-1A) enhanced the recruitment of at least two subunits of the exocyst complex (Sec8 and Exo70) required for basolateral transport. By immunofluorescence and cell fractionation, the exocyst subunits were found to selectively associate with AP-1B–containing membranes that were both distinct from AP-1A–positive TGN elements and more closely apposed to transferrin receptor–positive recycling endosomes. Thus, despite the similarity of the two AP-1 complexes, AP-1A and AP-1B exhibit great specificity for endosomal transport versus cell polarity.  

3.429           Epstein-Barr virus latent membrane protein 2A activates b-catenin signaling in epithelial cells

Morrison, J.A., Klingelhutz, A.J. and Raab-Traub, N.

1. Virol., 77(22), 12276-12284 (2003)

  The Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) functions to maintain latency in EBV-infected B lymphocytes. Although LMP2A is nonessential for the immortalization of B lymphocytes by EBV, its expression in B lymphocytes prevents viral reactivation by blocking B-cell receptor activation and signaling. LMP2A also provides an antiapoptotic signal in transgenic mice that express LMP2A in B lymphocytes. LMP2A activates phosphatidylinositol 3-kinase (PI3K) and the serine/threonine kinase Akt in lymphocytes and epithelial cells. Here we show that EBV LMP2A activates the PI3K and ß-catenin signaling pathways in telomerase-immortalized human foreskin keratinocytes (HFK). LMP2A activated Akt in a PI3K-dependent manner, and the downstream Akt targets glycogen synthase kinase 3ß (GSK3ß) and the Forkhead transcription factor FKHR were phosphorylated and inactivated in LMP2A-expressing HFK cells. GSK3ß is a negative regulator of the Wnt signaling pathway, and inactivation of GSK3ß by LMP2A signaling led to stabilization of ß-catenin, the central oncoprotein of Wnt signaling. In LMP2A-expressing cells, ß-catenin accumulated in the cytoplasm and translocated into the nucleus via a two-step mechanism. The cytoplasmic accumulation of ß-catenin downstream of LMP2A was independent of PI3K signaling, whereas its nuclear translocation was dependent on PI3K signaling. In the nucleus, ß-catenin activated a reporter responsive to T-cell factor, and this activation was augmented by LMP2A coexpression. The Wnt pathway is inappropriately activated in 90% of colon cancers and is dysregulated in several other cancers, and these data suggest that activation of this pathway by LMP2A may contribute to the generation of EBV-associated cancers.  

3.430           Function, expression and localization of annexin A7 in platelets and red blood cells: insights derived from an annexin A7 mutant mouse

Herr, C. et al BMC Biochem., 4(8), 1-11 (2003)   Annexin A7 is a Ca²⁺- and phospholipid-binding protein expressed as a 47 and 51 kDa isoform, which is thought to be involved in membrane fusion processes. Recently the 47 kDa isoform has been identified in erythrocytes where it was proposed to be a key component in the process of the Ca²⁺-dependent vesicle release, a process with which red blood cells might protect themselves against an attack by for example complement components. The role of annexin A7 in red blood cells was addressed in erythrocytes from anxA7^-/- mice. Interestingly, the Ca²⁺-mediated vesiculation process was not impaired. Also, the membrane organization appeared not to be disturbed as assessed using gradient fractionation studies. Instead, lack of annexin A7 led to an altered cell shape and increased osmotic resistance of red blood cells. Annexin A7 was also identified in platelets. In these cells its loss led to a slightly slower aggregation velocity which seems to be compensated by an increased number of platelets. The results appear to rule out an important role of annexin A7 in membrane fusion processes occurring in red blood cells. Instead the protein might be involved in the organization of the membrane cytoskeleton. Red blood cells may represent an appropriate model to study the role of annexin A7 in cellular processes.   We have demonstrated the presence of both annexin A7 isoforms in red blood cells and the presence of the small isoform in platelets. In both cell types the loss of annexin A7 impairs cellular functions. The defects observed are however not compatible with a crucial role for annexin A7 in membrane fusion processes in these cell types.  

3.431           Vesicular localization and activity-dependent trafficking of presynaptic choline transporters

Ferguson, S.M. et al

1. Neurosci., 23(30), 9697-9709 (2003)

  Presynaptic synthesis of acetylcholine (ACh) requires a steady supply of choline, acquired by a plasma membrane, hemicholinium-3-sensitive (HC-3) choline transporter (CHT). A significant fraction of synaptic choline is recovered from ACh hydrolyzed by acetylcholinesterase (AChE) after vesicular release. Although antecedent neuronal activity is known to dictate presynaptic CHT activity, the mechanisms supporting this regulation are unknown. We observe an exclusive localization of CHT to cholinergic neurons and demonstrate that the majority of CHTs reside on small vesicles within cholinergic presynaptic terminals in the rat and mouse brain. Furthermore, immunoisolation of presynaptic vesicles with multiple antibodies reveals that CHT-positive vesicles carry the vesicular acetylcholine transporter (VAChT) and synaptic vesicle markers such as synaptophysin and Rab3A and also contain acetylcholine. Depolarization of synaptosomes evokes a Ca²⁺-dependent botulinum neurotoxin C-sensitive increase in the V_max for HC-3-sensitive choline uptake that is accompanied by an increase in the density of CHTs in the synaptic plasma membrane. Our study leads to the novel hypothesis that CHTs reside on a subpopulation of synaptic vesicles in cholinergic terminals that can transit to the plasma membrane in response to neuronal activity to couple levels of choline re-uptake to the rate of ACh release.  

3.432           Visualization of protein compartmentation within the plasma membrane of living yeast cells

Malinska, K., Malinsky, J., Opekarova, M and Tanner, W. Mol. Biol. Cell., 14(11), 4427-4436 (2003)   Different distribution patterns of the arginine/H⁺ symporter Can1p, the H⁺ plasma membrane ATPase Pma1p, and the hexose transport facilitator Hxt1p within the plasma membrane of living Saccharomyces cerevisiae cells were visualized using fluorescence protein tagging of these proteins. Although Hxt1p-GFP was evenly distributed through the whole cell surface, Can1p-GFP and Pma1p-GFP were confined to characteristic subregions in the plasma membrane. Pma1p is a well-documented raft protein. Evidence is presented that Can1p, but not Hxt1p, is exclusively associated with lipid rafts, too. Double labeling experiments with Can1p-GFP– and Pma1p-RFP–containing cells demonstrate that these proteins occupy two different nonoverlapping membrane microdomains. The size of Can1p-rich (Pma1p-poor) areas was estimated to 300 nm. These domains were shown to be stable in growing cells for >30 min. To our knowledge, this is the first observation of a cell polarization-independent lateral compartmentation in the plasma membrane of a living cell.  

3.433           Inhibition of heme biosynthesis prevents transcription of iron uptake genes in yeast

Crisp, R.J. et al

1. Biol. Chem., 278(46), 45499-45506 (2003)

  Yeast are capable of modifying their metabolism in response to environmental changes. We investigated the activity of the oxygen-dependent high-affinity iron uptake system of Saccharomyces cerevisiae under conditions of heme depletion. We found that the absence of heme, due to a deletion in the gene that encodes -aminolevulinic acid synthase (HEM1), resulted in decreased transcription of genes belonging to both the iron and copper regulons, but not the zinc regulon. Decreased transcription of the iron regulon was not due to decreased expression of the iron sensitive transcriptional activator Aft1p. Expression of the constitutively active allele AFT1-1^up was unable to induce transcription of the high affinity iron uptake system in heme-depleted cells. We demonstrated that under heme-depleted conditions, Aft1p-GFP was able to cycle normally between the nucleus and cytosol in response to cytosolic iron. Despite the inability to induce transcription under low iron conditions, chromatin immunoprecipitation demonstrated that Aft1p binds to the FET3 promoter in the absence of heme. Finally, we provide evidence that under heme-depleted conditions, yeast are able to regulate mitochondrial iron uptake and do not accumulate pathologic iron concentrations, as is seen when iron-sulfur cluster synthesis is disrupted.  

3.434           Syndecan-1 transmembrane and extracellular domains have unique and distinct roles in cell spreading

McQuade, K.J. and Repraeger, A.C.

1. Biol. Chem., 278(47), 46607-46615 (2003)

  Raji cells expressing syndecan-1 (Raji-S1) adhere and spread when plated on heparan sulfate-binding extracellular matrix ligands or monoclonal antibody 281.2, an antibody directed against the syndecan-1 extracellular domain. Cells plated on monoclonal antibody 281.2 initially extend a broad lamellipodium, a response accompanied by membrane ruffling at the cell margin. Membrane ruffling then becomes polarized, leading to an elongated cell morphology. Previous work demonstrated that the syndecan-1 cytoplasmic domain is not required for these activities, suggesting important roles for the syndecan-1 transmembrane and/or extracellular domains in the assembly of a signaling complex necessary for spreading. Work described here demonstrates that truncation of the syndecan-1 extracellular domain does not affect the initial lamellipodial extension in the Raji-S1 cells but does inhibit the active membrane ruffling that is necessary for cell polarization. Replacement of the entire syndecan-1 transmembrane domain with leucine residues completely blocks the cell spreading. These data demonstrate that the syndecan-1 transmembrane and extracellular domains have important but distinct roles in Raji-S1 cell spreading; the extracellular domain mediates an interaction that is necessary for dynamic cytoskeletal rearrangements whereas an interaction of the transmembrane domain is required for the initial spreading response.  

3.435           Raft disorganization leads to reduced plasmin activity in Alzheimer’s disease brains

Ledesma, M.D. et al EMBO Reports, 4(12), 1190-1196 (2003)   The serine protease plasmin can efficiently degrade amyloid peptide in vitro, and is found at low levels in the hippocampus of patients with Alzheimer's disease (AD). The cause of such paucity remains unknown. We show here that the levels of total brain plasminogen and plasminogen-binding molecules are normal in these brain samples, yet plasminogen membrane binding is greatly reduced. Biochemical analysis reveals that the membranes of these brains have a mild, still significant, cholesterol reduction compared to age-matched controls, and anomalous raft microdomains. This was reflected by the loss of raft-enriched proteins, including plasminogen-binding and -activating molecules. Using hippocampal neurons in culture, we demonstrate that removal of a similar amount of membrane cholesterol is sufficient to induce raft disorganization, leading to reduced plasminogen membrane binding and low plasmin activity. These results suggest that brain raft alterations may contribute to AD by rendering the plasminogen system inefficient.  

3.436           Novel cadherin-related membrane proteins, alcadeins, enhance the X11-like protein-mediated stabilization of amyloid b-protein precursor metabolism

Araki, Y. et al

1. Biol.Chem., 278(49), 49448-49458 (2003)

  Previously we found that X11-like protein (X11L) associates with amyloid -protein precursor (APP). X11L stabilizes APP metabolism and suppresses the secretion of the amyloid -protein (A ) that are the pathogenic agents of Alzheimer's disease (AD). Here we found that Alcadein (Alc), a novel membrane protein family that contains cadherin motifs and originally reported as calsyntenins, also interacted with X11L. Alc was abundant in the brain and occurred in the same areas of the brain as X11L. X11L could simultaneously associate with APP and Alc, resulting in the formation of a tripartite complex in brain. The tripartite complex stabilized intracellular APP metabolism and enhanced the X11L-mediated suppression of A secretion that is due to the retardation of intracellular APP maturation. X11L and Alc also formed another complex with C99, a carboxyl-terminal fragment of APP cleaved at the -site (CTF ). The formation of the Alc·X11L·C99 complex inhibited the interaction of C99 with presenilin, which strongly suppressed the -cleavage of C99. In AD patient brains, Alc and APP were particularly colocalized in dystrophic neurites in senile plaques. Deficiencies in the X11L-mediated interaction between Alc and APP and/or CTF enhanced the production of A , which may be related to the development or progression of AD.    

3.437           RGS16 inhibits signaling through the Ga13-Rho

Johnson, E.N. et al Nature Cell Biol., 5(12), 1095-1103 (2003)   Ga13 stimulates the guanine nucleotide exchange factors (GEFs) for Rho, such as p115Rho-GEF¹. Activated Rho induces numerous cellular responses, including actin polymerization, serum response element (SRE)-dependent gene transcription and transformation². p115Rho-GEF contains a Regulator of G protein Signalling domain (RGS box) that confers GTPase activating protein (GAP) activity towards G 12 and G 13 (ref. 3). In contrast, classical RGS proteins (such as RGS16 and RGS4) exhibit RGS domain-dependent GAP activity on G i and G q, but not G 12 or G 13 (ref 4). Here, we show that RGS16 inhibits G 13-mediated, RhoA-dependent reversal of stellation and SRE activation. The RGS16 amino terminus binds G 13 directly, resulting in translocation of G 13 to detergent-resistant membranes (DRMs) and reduced p115Rho-GEF binding. RGS4 does not bind G 13 or attenuate G 13-dependent responses, and neither RGS16 nor RGS4 affects G 12-mediated signalling. These results elucidate a new mechanism whereby a classical RGS protein regulates G 13-mediated signal transduction independently of the RGS box.  

3.438           The lack of annexin A7 affects functions of primary astrocytes

Clemen, C.S., Herr, C., Hövelmeyer, N. and Noegel, A.A. Exp. Cell Res., 291, 406-414 (2003)   Annexin A7 is a Ca²⁺- and phospholipid-binding protein, which is thought to function in membrane organization and Ca²⁺-dependent signaling processes. It localizes to different cellular compartments and exists in a 47- and 51-kDa isoform with the large isoform being expressed in brain, skeletal, and heart muscle. In human temporal brain annexin A7 was found exclusively in astroglial cells. As astrocytes are thought to play key roles in several processes of the brain we focused on Ca²⁺-dependent signaling processes and astrocyte proliferation. Primary astrocytes from an anxA7^-/- mouse exhibited an increased velocity of mechanically induced astrocytic Ca²⁺ waves as compared to wild type. We also observed a remarkably increased proliferation rate in cultured mutant astrocytes. A search for annexin A7 binding partners with advanced biochemical methods confirmed sorcin as the major binding protein. However, in vivo GFP-tagged annexin A7 and sorcin appeared to redistribute mainly independently from each other in wild type and in mutant astrocytes. Our results favor an involvement of annexin A7 in Ca²⁺-dependent signaling or Ca²⁺ homeostasis in astrocytes.  

3.439           Subcellular localization and regulation of coenzyme A synthetase

Zhyvoloup, A. et al

1. Biol. Chem., 278(50), 50316-50321 (2003)

  CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme. Transient expression studies and confocal microscopy allowed us to demonstrate that full-length CoA synthase is associated with the mitochondria, whereas the removal of the N-terminal region relocates the enzyme to the cytosol. In addition, we showed that the N-terminal sequence of CoA synthase (amino acids 1–29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis, indicated that CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.  

3.440           Yeast homotypic vacuole fusion requires the Ccz1-Mon1 complex during the tethering/docking stage

Wang, C-W., Stromhaug, P.E., Kauffman, E.J., Weisman, L.S. and Klionsky, D.J.

1. Cell Biol., 163(5), 973-985 (2003)

  The function of the yeast lysosome/vacuole is critically linked with the morphology of the organelle. Accordingly, highly regulated processes control vacuolar fission and fusion events. Analysis of homotypic vacuole fusion demonstrated that vacuoles from strains defective in the CCZ1 and MON1 genes could not fuse. Morphological evidence suggested that these mutant vacuoles could not proceed to the tethering/docking stage. Ccz1 and Mon1 form a stable protein complex that binds the vacuole membrane. In the absence of the Ccz1–Mon1 complex, the integrity of vacuole SNARE pairing and the unpaired SNARE class C Vps/HOPS complex interaction were both impaired. The Ccz1–Mon1 complex colocalized with other fusion components on the vacuole as part of the cis-SNARE complex, and the association of the Ccz1–Mon1 complex with the vacuole appeared to be regulated by the class C Vps/HOPS complex proteins. Accordingly, we propose that the Ccz1–Mon1 complex is critical for the Ypt7-dependent tethering/docking stage leading to the formation of a trans-SNARE complex and subsequent vacuole fusion.  

3.441           Active PIKfyve associates with and promotes the membrane attachment of the late endosome-to-trans-Golgi network transport factor Rab9 effector p40

Ikonomov, O.C. et al

1. Biol. Chem., 278(51), 50863-50871 (2003)

  PIKfyve, a kinase that displays specificity for phosphatidylinositol (PtdIns), PtdIns 3-phosphate (3-P), and proteins, is important in multivesicular body/late endocytic function. Enzymatically inactive PIKfyve mutants elicit enormous dilation of late endocytic structures, suggesting a role for PIKfyve in endosome-to-trans-Golgi network (TGN) membrane retrieval. Here we report that p40, a Rab9 effector reported previously to bind Rab9-GTP and stimulate endosome-to-TGN transport, interacts with PIKfyve as determined by yeast two-hybrid assays, glutathione S-transferase (GST) pull-down assays, and co-immunoprecipitation in doubly transfected HEK293 cells. The interaction engages the PIKfyve chaperonin domain and four out of the six C-terminally positioned kelch repeats in p40. Differential centrifugation in a HEK293 cell line, stably expressing PIKfyve^WT, showed the membrane-associated immunoreactive p40 co-sedimenting with PIKfyve in the high speed pellet (HSP) fraction. Remarkably, similar analysis in a HEK293 cell line stably expressing dominant-negative kinase-deficient PIKfyve^K1831E demonstrated a marked depletion of p40 from the HSP fraction. GST-p40 failed to specifically associate with the PIKfyve lipid products PtdIns 5-P and PtdIns 3,5-P₂ in a liposome binding assay but was found to be an in vitro substrate of the PIKfyve serine kinase activity. A band with the p40 electrophoretic mobility was found to react with a phosphoserine-specific antibody mainly in the PIKfyve^WT-containing fractions obtained by density gradient sedimentation of total membranes from PIKfyve^WT-expressing HEK293 cells. Together these results identify the Rab9 effector p40 as a PIKfyve partner and suggest that p40-PIKfyve interaction and the subsequent PIKfyve-catalyzed p40 phosphorylation anchor p40 to discrete membranes facilitating late endosome-to-TGN transport.      

3.442           Generation of the b-amyloid peptide and the amyloid percursor protein C-terminal fragment g are potentiated by FE65L1

Chang, Y. et al

1. Biol. Chem., 278(51), 511000-51107 (2003)

  Members of the FE65 family of adaptor proteins, FE65, FE65L1, and FE65L2, bind the C-terminal region of the amyloid precursor protein (APP). Overexpression of FE65 and FE65L1 was previously reported to increase the levels of -secretase-derived APP (APPs ). Increased -amyloid (A ) generation was also observed in cells showing the FE65-dependent increase in APPs . To understand the mechanism for the observed increase in both A and APPs given that -secretase cleavage of a single APP molecule precludes A generation, we examined the effects of FE65L1 overexpression on APP C-terminal fragments (APP CTFs). Our data show that FE65L1 potentiates -secretase processing of APP CTFs, including the amyloidogenic CTF C99, accounting for the ability of FE65L1 to increase generation of APP C-terminal domain and A 40. The FE65L1 modulation of these processing events requires binding of FE65L1 to APP and APP CTFs and is not because of a direct effect on -secretase activity, because Notch intracellular domain generation is not altered by FE65L1. Furthermore, enhanced APP CTF processing can be detected in early endosome vesicles but not in endoplasmic reticulum or Golgi membranes, suggesting that the effects of FE65L1 occur at or near the plasma membrane. Finally, although FE65L1 increases APP C-terminal domain production, it does not mediate the APP-dependent transcriptional activation observed with FE65.  

3.443           Rat liver bile acid CoA:amino acid N-acyltransferase: expression, characterization, and peroxisomal localization

He, D., Barnes, S. and Falany, C.N.

1. Lipid Res., 44, 2242-2249 (2003)

  Bile acid CoA:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids taurine and glycine. Rat liver BAT (rBAT) cDNA was isolated from a rat liver ZAP cDNA library and expressed in Sf9 insect cells using a baculoviral vector. rBAT displayed 65% amino acid sequence homology with human BAT (hBAT) and 85% homology with mouse BAT (mBAT). Similar to hBAT, expressed rBAT was capable of forming both taurine and glycine conjugates with cholyl-CoA. mBAT, which is highly homologous to rBAT, forms only taurine conjugated bile acids (Falany, C. N., H. Fortinberry, E. H. Leiter, and S. Barnes. 1997. Cloning and expression of mouse liver bile acid CoA: Amino acid N-acyltransferase. J. Lipid Res. 38: 86–95).Immunoblot analysis of rat tissues detected rBAT only in ratliver cytosol following homogenization and ultracentrifugation.Subcellular localization of rBAT detected activity and immunoreactiveprotein in both cytosol and isolated peroxisomes. Rat bile acidCoA ligase (rBAL), the enzyme responsible for the formationof bile acid CoA esters, was detected only in rat liver microsomes.Treatment of rats with clofibrate, a known peroxisomal proliferator,significantly induced rBAT activity, message, and immunoreactiveprotein in rat liver. Peroxisomal membrane protein-70, a markerfor peroxisomes, was also induced by clofibrate, whereas rBALactivity and protein amount were not affected. In summary, rBAT is capable of forming both taurine and glycinebile acid conjugates and the enzyme is localized primarily inperoxisomes in rat liver.  

3.444           Activation of nuclear factor-kB p50 homodimer/Bcl-3 complexes in nasapharyngeal carcinoma

Thornburg, N.J., oathmanathan, R. and Raab-Traub, N. Cancer. Res., 63, 8293-8301 (2003)   EBV latent infection is associated with the development of lymphoid and epithelial malignancies such as nasopharyngeal carcinoma (NPC). The EBV latent membrane protein 1 (LMP1) acts as a constitutively active tumor necrosis factor receptor and activates cellular signaling pathways such as c-Jun-NH₂-terminal kinase, cdc42, Akt, and nuclear factor (NF)- B. In epithelial cells, two regions of LMP1 induce specific forms of NF- B. COOH-terminal activating region 2 only activates p52/p65 dimers, whereas COOH-terminal activating region 1 activates p50/p50, p50/p52, and p52/p65 dimers and also uniquely up-regulates the epidermal growth factor receptor (EGFR) at the mRNA level. Deregulation of specific NF- B members is associated with the development of many cancers. In this study, the status of NF- B activation was investigated in NPC to determine which NF- B dimers may contribute to the development of NPC. Electrophoretic mobility shift assay, immunoblot, ELISA, and immunohistochemistry data demonstrate that in NPC, NF- B p50 homodimers are specifically activated, and this activation is not dependent on LMP1 expression. Coimmunoprecipitation assays indicate that homodimers are bound to the transcriptional coactivator Bcl-3, and chromatin immunoprecipitation indicates that this complex is bound to NF- B consensus motifs within the egfr promoterin NPC. The discrete yet striking NF-B p50 activation in NPCsuggests that p50/p50 homodimers may be important factors inthe development of NPC and may contribute to oncogenesis throughtranscriptional up-regulation of target genes through theirinteraction with Bcl-3.  

3.445           Downregulation of caveolin-1 function by EGF leads to the loss of E-cadherin, increased transcriptional activity of  b-catenin, and enhanced tumor cell invasion

Lu, Z., Ghosh, S., Wang, Z. and Hunter, T. Cancer Cell, 4, 499-515 (2003)   EGF receptor (EGFR) overexpression correlates with metastasis in a variety of carcinomas, but the underlying mechanisms are poorly understood. We demonstrated that EGF disrupted cell-cell adhesion and caused epithelial-to-mesenchymal transition (EMT) in human tumor cells overexpressing EGFR, and also induced caveolin-dependent endocytosis of E-cadherin, a cell-cell adhesion protein. Chronic EGF treatment resulted in transcriptional downregulation of caveolin-1 and induction of the transcriptional repressor Snail, correlating with downregulation of E-cadherin expression. Caveolin-1 downregulation enhanced β-catenin-TCF/LEF-1 transcriptional activity in a GSK-3β-independent manner. Antisense RNA-mediated reduction of caveolin-1 expression in EGFR-overexpressing tumor cells recapitulated these EGF-induced effects and enhanced invasion into collagen gels. We propose that EGF-induced negative regulation of caveolin-1 plays a central role in the complex cellular changes leading to metastasis.  

3.446           Src signaling links mediators of inflammation to Cx43 gap junction channels in primary and transformed CFTR-expressing airway cells

Huang, S. et al Cell Comm. and Aadhesion, 10, 279-285 (2003)   Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with recurrent pulmonary infections and inflammation. We previously reported that tumor necrosis factor (TNF)-f decreases gap junction connectivity in cell lines derived from the airway epithelium of non-cystic fibrosis (non-CF) subjects, a mechanism that was defective in cells derived from CF patients, and identified the tyrosine kinase c-Src as a possible bridge between TNF-f and Cx43. To examine whether this modulation also takes place in primary epithelial cells, the functional expression of Cx43 was studied in non-CF and CF airway cells, obtained from surgical polypectomies and turbinectomies, which were grown either on culture dishes or permeable filters. Expression of Cx43 was detected by immunofluorescence on cells grown under both culture conditions. Non-CF and CF airway cells also showed intercellular diffusion of Lucifer Yellow. Dye coupling was rapidly abolished in non-CF cells in the presence of TNF-f, lipopolysaccharide and lysophosphatidic acid, and could be prevented by tyrphostin47, an inhibitor of Src tyrosine kinases. This down-regulation, however, was not detected in CF airway cells. These data indicate that CFTR dysfunction is associated with altered Src signaling, resulting in the persistence of gap junction connectivity in primary and transformed CF airway cells.  

3.447           Large clusters of a7-containing nicotinic acetylcholine receptors on chick spinal cord neurons

Roth, A.L. and Berg, D.K.

1. Comp. Neurol., 465, 195-204 (2003)

  Nicotinic acetylcholine receptors containing the 7 gene product are widely expressed in the nervous system and have high calcium permeabilities that allow them to influence numerous calcium-dependent processes. Though often found at presynaptic locations, where they enhance transmitter release, the receptors can also occupy postsynaptic sites. Highest levels have been reported for chick ciliary ganglion neurons, where the postsynaptic receptors are concentrated on somatic spines arranged in clumps and appear as large receptor clusters. We show here that subpopulations of chick spinal cord neurons also express high levels of 7-containing receptors and arrange them in large clusters. The populations include peripheral motoneurons, presumptive preganglionic neurons, neurons adjacent to the lateral motor column, and possible interneurons in the ventral horn. In many cases, the receptor clusters codistribute with filamentous actin, as do clusters on ciliary ganglion neurons, where the actin represents a somatic spine constituent. In other respects, the spinal cord clusters differ. Those on motoneurons codistribute with the actin-associated component drebrin, as do the clusters on ciliary ganglion neurons, but the clusters on preganglionic neurons do not. Preganglionic neurons do, however, stain for lipid raft components as found for ciliary ganglion neurons, where the rafts embed the receptor-enriched spines. The results demonstrate that CNS neurons can configure 7-containing nicotinic receptors into large clusters but also suggest that the clusters are not likely to reflect a common molecular substructure on all neurons.  

3.448           Lipid rafts: bringing order to chaos

Pike, L.J.

1. Lipid Res., 44, 655-667 (2003)

  Lipid rafts are subdomains of the plasma membrane that containhigh concentrations of cholesterol and glycosphingolipids. Theyexist as distinct liquid-ordered regions of the membrane thatare resistant to extraction with nonionic detergents. Raftsappear to be small in size, but may constitute a relativelylarge fraction of the plasma membrane. While rafts have a distinctiveprotein and lipid composition, all rafts do not appear to beidentical in terms of either the proteins or the lipids thatthey contain. A variety of proteins, especially those involvedin cell signaling, have been shown to partition into lipid rafts.As a result, lipid rafts are thought to be involved in the regulationof signal transduction. Experimental evidence suggests thatthere are probably several different mechanisms through whichrafts control cell signaling. For example, rafts may containincomplete signaling pathways that are activated when a receptoror other required molecule is recruited into the raft. Raftsmay also be important in limiting signaling, either by physicalsequestration of signaling components to block nonspecific interactions,or by suppressing the intrinsic activity of signaling proteinspresent within rafts. This review provides an overview of the physical characteristicsof lipid rafts and summarizes studies that have helped to elucidatethe role of lipid rafts in signaling via receptor tyrosine kinasesand G protein-coupled receptors.  

3.449           Role of nephrin in proteinuric renal diseases

Salant, D.J. and Topham, P.S. Immunopathol., 24, 423-439 (2003)   No abstract available  

3.450           Transport of plasma membrane-derived cholesterol and the function of Niemann-Pick C1 protein

Wiegand, V., Chang, T-Y., StraussIII, J.F., Fahrenholz, F. and Gimpl, G. FASEB J., 17(6), 782-784 (2003)   To visualize the intracellular transport of plasma membrane-derived cholesterol under physiological and pathophysiological conditions, a novel fluorescent cholesterol analog, 6-dansyl cholestanol (DChol), has been synthesized. We present several lines of evidence that DChol mimics cholesterol. The cholesterol probe could be efficiently incorporated into the plasma membrane via cyclodextrin-donor complexes. The itinerary of DChol from the plasma membrane to the cell was studied to determine its dependence on the function of Niemann-Pick C1 (NPC) protein. In all cells, DChol moved from the plasma membrane to the endoplasmic reticulum. Its further transport to the Golgi complex was observed but with marked differences among various cell lines. DChol was finally transported to small (~0.5 μm diameter) lipid droplets, a process that required functional acyl-CoA:cholesterol acyltransferase. In human NPC fibroblasts, NPC-like cells, or in cells mimicking the NPC phenotype, DChol was found in enlarged (>1 μm diameter) droplets. When the NPC-phenotype was corrected by transfection with NPC1, DChol was again found in small-sized droplets. Our data show that NPC1 has an essential role in the distribution of plasma membrane-derived cholesterol by maintaining the small size of cholesterol-containing lipid droplets in the cell.  

3.451           Insights into the Association of Fc RII and TCR with Detergent-Resistant Membrane Domains: Isolation of the Domains in Detergent-Free Density Gradients Facilitates Membrane Fragment Reconstitution

Korzeniowaski, M., Kwiatkowska, K. and Sobota, A. Biochemistry, 42, 5358-5367 (2003)   Plasma membrane rafts are routinely isolated as detergent-resistant membranes (DRMs) floating in detergent-free density gradients. Here we show that both the presence and exclusion of TX-100 during the density gradient fractionation have profound effects on the location of Fc RII and TCR in DRM fractions. The presence of TX-100 during fractionation promoted solubilization of non-cross-linked Fc RII when the receptor was insufficiently dissolved upon cell lysis. In the detergent-supplemented gradients, TX-100 micelles floated, further enhancing dissociation of Fc RII and TCR from DRMs and promoting a shift of the receptors toward higher-density fractions. Hence, fractionation of cell lysates over the detergent-containing gradients enables isolation of DRMs devoid of weakly associated proteins, like nonactivated Fc RII and TCR. On the other hand, in a detergent-free gradient, non-cross-linked Fc RII, fully soluble in 0.2% TX-100, was recovered in DRM fractions. Moreover, employment of the TX-100-free gradient for refractionation of intermediate-density fractions, derived from detergent-supplemented gradients and containing Fc RII and TCR, resulted in flotation of the receptors to buoyant fractions. An analysis of the TX-100 concentration revealed that after fractionation of 0.2% TX-100 cell lysates in the absence of detergent, the level of TX-100 in DRM fractions was reduced to 0.01%, below the critical micelle concentration. Therefore, fractionation of detergent cell lysates over detergent-free gradients can mimic conditions for a membrane reconstitution, evoking association of a distinct subset of membrane proteins, including Fc RII and TCR, with DRMs.  

3.452           Engineering of technetium-99m-binding artificial receptors for imaging gene expression

Simonova, M., Shtanko, O., Serrgeyev, N., Weissleder, R. and Bogdanov Jr, A.

1. Gene Med., 5(12), 1956-1066 (2003)

  Background Optimization of gene therapy protocols requires accurate and non-invasive quantification of vector delivery and gene expression. To facilitate non-invasive imaging of gene expression, we have genetically engineered ‘artificial receptors’, i.e. membrane proteins that bind 99mTc-oxotechnetate (99mTcOT) via transchelation from a complex with glucoheptonate. The latter is a component of a widely used clinical imaging kit. Methods The engineered marker proteins were designed as type I and II membrane proteins and consisted of (1) an 99mTcOT-binding domain, metallothionein (MT), and (2) a membrane-anchoring domain. Engineered constructs were used for transfection of COS-1 and 293 cells; the expression of mRNA was verified by RT-PCR. Results Immunofluorescent analysis, cell fractionation and immunoblotting revealed expression of marker proteins on plasma membrane. Transfection of cells resulted in strong positive staining of plasma membrane with anti-His-tag antibodies. Scintigraphic imaging in vitro confirmed the ability of transfected cells to bind 99mTcOT. The fraction of bound radioactivity reached a peak (3.53%) when 0.93 MBq 99mTcOT was added to transfected COS-1 cells. The experiment-to-control signal ratio was equal to 32 at the same added dose. Conclusions (1) Both types of engineered ‘artificial receptors’ were expressed on the surface of eukaryotic cells; (2) marker proteins were functional in binding 99mTcOT; and (3) type II membrane proteins were more efficient in binding 99mTcOT than type I proteins. We anticipate that the developed approach could be useful for ‘tagging’ transfected cells with 99mTcOT enabling imaging of tracking in vivo transduced cells or cell therapies.  

3.453           p53 is present in synapses where it mediates mitochondrial dysfunction and synaptic degeneration in response to DNA damage, and oxidative and excitotoxic insults

Gilman, C.P., Chan, S.L., Guo, Z., Zhu, X., Greig, N. and mattson, M:P. NeuroMol. Med., 3(3), 159-172 (2003)   A form of programmed cell-death called apoptosis occurs in neurons during development of the nervous system, and may also occur in a variety of neuropathological conditions. Here we present evidence obtained in studies of adult mice and neuronal cell cultures showing that p53 protein is present in synapses where its level and amount of phosphorylation are increased following exposure of the cells to the DNA-damaging agent etoposide. We also show that levels of active p53 increase in isolated cortical synaptosomes exposed to oxidative and excitotoxic insults. Increased levels of p53 also precede loss of synapsin I immunoreactive terminals in cultured hippocampal neurons exposed to etoposide. Synaptosomes from p53-deficient mice exhibit increased resistance to oxidative and excitotoxic insults as indicated by stabilization of mitochondrial membrane potential and decreased production of reactive oxygen species. Finally, we show that a synthetic inhibitor of p53 (PFT-α) protects synaptosomes from wild-type mice against oxidative and excitotoxic injuries, and preserves presynaptic terminals in cultured hippocampal neurons exposed to etoposide. Collectively, these findings provide the first evidence for a local transcription-independent action of p53 in synapses, and suggest that such a local action of p53 may contribute to the dysfunction and degeneration of synapses that occurs in various neurodegenerative disorders.  

3.454           Latent infection membrane protein transmembrane FWLY is critical for intermolecular interaction, raft localization, and signaling

Yasui, T., Luftig, M., Soni, V. and Kieff, E. PNAS, 101(1), 278-283 (2004)   Relatively little is known about the biochemical mechanisms through which the Epstein-Barr virus latent infection integral membrane protein 1 (LMP1) transmembrane domains cause constitutive LMP1 aggregation and continuous cytoplasmic C terminus-mediated signal transduction. We now evaluate the role of the three consecutive LMP1 hydrophobic transmembrane pairs, transmembrane domains (TM)1-2, TM3-4, and TM5-6, in intermolecular aggregation and NF- B activation. LMP1TM1-2 enabled 40% of wild-type LMP1 cytoplasmic domain-mediated NF- B activation, whereas TM3-4 or TM5-6 assayed in parallel had almost no effect independent of LMP1TM1-2. Alanine mutagenesis of conserved residues in LMP1TM1-2 identified FWLY_38-41 to be critical for LMP1TM1-2 intermolecular association with LMP1TM3-6. Further, in contrast to wild-type LMP1, LMP1 with FWLY_38-41 mutated to AALA_38-41 did not (i) significantly partition to lipid Rafts or Barges and effectively intermolecularly associate, (ii) enable cytoplasmic C terminus engagement of tumor necrosis factor receptor-associated factor 3, (iii) activate NF- B, and thereby (iv) induce tumor necrosis factor receptor-associated factor 1 expression. Other LMP1 intermolecular associations were observed that involved LMP1TM1-2/LMP1TM1-2 or LMP1TM3-4/LMP1TM3-6 interactions; these probably also contribute to LMP1 aggregation. Because FWLY_38-41 was essential for LMP1-mediated signal transduction, and LMP1 activation of NF- B is essential for proliferating B lymphocyte survival, inhibition of LMP1FWLY₄₁-mediated LMP1/LMP1 intermolecular interactions is an attractive therapeutic target.  

3.455           Distinct subcellular localizations of Nox1 and Nox4 in vascular smooth muscle cells

Hilenski, L.L., Clempus, R.E., Quinn, M.T., Lambeth, J.D. and Griendling, K.K. Arterioscler. Thromb. Vasc. Biol., 24, 1-8 (2004)   Objective--Reactive oxygen species (ROS) that act as signaling molecules in vascular smooth muscle cells (VSMC) and contribute to growth, hypertrophy, and migration in atherogenesis are produced by multi-subunit NAD(P)H oxidases. Nox1 and Nox4, two homologues to the phagocytic NAD(P)H subunit gp91^phox, both generate ROS in VSMC but differ in their response to growth factors. We hypothesize that the opposing functions of Nox1 and Nox4 are reflected in their differential subcellular locations. Methods and Results--We used immunofluorescence to visualize the NAD(P)H subunits Nox1, Nox4, and p22^phox in cultured rat and human VSMC. Optical sectioning using confocal microscopy showed that Nox1 is co-localized with caveolin in punctate patches on the surface and along the cellular margins, whereas Nox4 is co-localized with vinculin in focal adhesions. These immunocytochemical distributions are supported by membrane fractionation experiments. Interestingly, p22^phox, a membrane subunit that interacts with the Nox proteins, is found in surface labeling and in focal adhesions in patterns similar to Nox1 and Nox4, respectively. Conclusions--The differential roles of Nox1 and Nox4 in VSMC may be correlated with their differential compartmentalization in specific signaling domains in the membrane and focal adhesions.  

3.456           Akt2, phosphatidylinositol 3-kinase, and PTEN are in lipid rafts of intestinal cells: role in absorption and differentiation

Li, X., et al Gastroenterology, 126, 122-135 (2004)   Background & Aims: In intestinal Na absorptive cells, phosphatidylinositol 3-kinase (PI 3-K) is involved in rapid epidermal growth factor (EGF) stimulation of Na absorption by the brush border membrane (BBM) Na⁺/H⁺ exchanger NHE3. However, how NHE3 is regulated by the PI 3-K pathway and the role of Akt2 are poorly defined. Methods: The localization of Akt, PI 3-K, and NHE3 was determined by either immunocytochemistry and/or membrane fractionation using OptiPrep density gradient centrifugation. Results: In ileum, active total Akt was present most in the villi and basal layer of the crypts, and Akt2 was mostly in villi. In villus cells, PI 3-K and Akt2 were mostly at the apical surface at which they were present partially in lipid rafts (LR). EGF increased PI 3-K and active Akt2 in ileal BBM at the same time that it increased PI 3-K-dependent trafficking of NHE3 to BBM and stimulation of Na absorption. However, Akt2 was only active in the detergent soluble (DS) pool and not LR of ileal BBM, which correlated with the presence of PTEN in LR. In Caco-2 cells, while EGF stimulated BB NHE3, Akt2 was active in both LR and DS pools. This correlated with the lack of PTEN in the LR of Caco-2 membranes. Akt2 also correlated with epithelial cell differentiation. Akt2 amount and activity were greater in differentiated than undifferentiated Caco-2 cells. Conclusions: These results suggest that LR may play an important role in determining the function of PI 3-K/Akt2 signaling, including stimulation of intestinal Na absorption. These results also suggest that LR-associated Akt2 may be involved in enterocyte differentiation.  

3.457           Mutant presenilin (A260V) affects Rab8 in PC12D cell

Kametani, F., Usami, M., Tanaka, K., Kume, H. And Mori, H. Neurochem. Int., 44, 313-320 (2004)   Most familial early-onset Alzheimer's disease (FAD) is caused by mutations in the presenilin-1 (PS1) gene. A is derived from amyloid precursor protein (APP) and an increased concentration of A 42 is widely believed to be a pathological hallmark of abnormal PS function. Therefore, the interaction between PS1 and APP is a central theme in attempts to clarify the molecular mechanism of AD. To examine the effect of PS1 mutations on APP metabolism, we made PC12D cell lines that express human PS1 or mutant PS1 (A260V). In PC12D cells expressing the PS1A260V mutant, we found that Rab8, a GTPase involved in transport from the trans-Golgi network (TGN) to the plasma membrane (PM), was significantly reduced in PC12D cells expressing the A260V mutant and that APP C-terminal fragment (CTF), the direct precursor of A , accumulated in the heavy membrane fraction including membrane vesicles involved in TGN-to-PM transport. Furthermore, the total intracellular A production was reduced in these cells. Combined together, we have observed that PS1 mutation disturbs membrane vesicle transport, resulting in prolonged residence of APP CTF during TGN-to-PM transport pathway. Therefore, it is highly likely that reduction of A is closely related to the retention of APP CTF during TGN-to-PM transport.  

3.458           BACE1 suppression by RNA interferences in primary cortical neurons

Kao, S-C., Krichevsky, A.M., Kosik, K.S. and Tsai, L-H.

1. Biol. Chem., 279(3), 1942-1949 (2004)

  Extracellular deposition of amyloid- (A ) aggregates in the brain represents one of the histopathological hallmarks of Alzheimer's disease (AD). A peptides are generated from proteolysis of the amyloid precursor proteins (APPs) by - and -secretases. -Secretase (BACE1) is a type I integral membrane glycoprotein that can cleave APP first to generate C-terminal 99- or 89-amino acid membrane-bound fragments containing the N terminus of A peptides ( CTF). As BACE1 cleavage is an essential step for A generation, it is proposed as a key therapeutic target for treating AD. In this study, we show that small interfering RNA (siRNA) specifically targeted to BACE1 can suppress BACE1 (but not BACE2) protein expression in different cell systems. Furthermore, BACE1 siRNA reduced APP CTF and A production in primary cortical neurons derived from both wild-type and transgenic mice harboring the Swedish APP mutant. The subcellular distribution of APP and presenilin-1 did not appear to differ in BACE1 suppressed cells. Importantly, pretreating neurons with BACE1 siRNA reduced the neurotoxicity induced by H₂O₂ oxidative stress. Our results indicate that BACE1 siRNA specifically impacts on -cleavage of APP and may be a potential therapeutic approach for treating AD.  

3.459           Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles

Kesty, N.C. and Kuehn, M.J.

1. Biol. Chem., 279(3), 2069-2076 (2003)

  Gram-negative bacteria shed outer membrane vesicles composed of outer membrane and periplasmic components. Since vesicles from pathogenic bacteria contain virulence factors and have been shown to interact with eukaryotic cells, it has been proposed that vesicles behave as delivery vehicles. We wanted to determine whether heterologously expressed proteins would be incorporated into the membrane and lumen of vesicles and whether these altered vesicles would associate with host cells. Ail, an outer membrane adhesin/invasin from Yersinia enterocolitica, was detected in purified outer membrane and in vesicles from Escherichia coli strains DH5 , HB101, and MC4100 transformed with plasmid-encoded Ail. In vesicle-host cell co-incubation assays we found that vesicles containing Ail were internalized by eukaryotic cells, unlike vesicles without Ail. To determine whether lumenal vesicle contents could be modified and delivered to host cells, we used periplasmically expressed green fluorescent protein (GFP). GFP fused with the Tat signal sequence was secreted into the periplasm via the twin arginine transporter (Tat) in both the laboratory E. coli strain DH5 and the pathogenic enterotoxigenic E. coli ATCC strain 43886. Pronase-resistant fluorescence was detectable in vesicles from Tat-GFP-transformed strains, demonstrating that GFP was inside intact vesicles. Inclusion of GFP cargo increased vesicle density but did not result in morphological changes in vesicles. These studies are the first to demonstrate the incorporation of heterologously expressed outer membrane and periplasmic proteins into bacterial vesicles.  

3.460           AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture

Benaud, C.B. et al

1. Cell Biol., 164(1), 133-144 (2004)

  Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell–cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca²⁺-dependent cell–cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2–specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.    

3.461           Mechanism of recruiting Sec6/8 (exocyst) complex to the apical juntional complex during polarization of epithelial cells

Yeaman, C., Grindstaff, K.K. and Nelson, W.J.

1. Cell Sci., 117, 559-570 (2004)

  Sec6/8 (exocyst) complex regulates vesicle delivery and polarized membrane growth in a variety of cells, but mechanisms regulating Sec6/8 localization are unknown. In epithelial cells, Sec6/8 complex is recruited to cell-cell contacts with a mixture of junctional proteins, but then sorts out to the apex of the lateral membrane with components of tight junction and nectin complexes. Sec6/8 complex fractionates in a high molecular mass complex with tight junction proteins and a portion of E-cadherin, and co-immunoprecipitates with cell surface-labeled E-cadherin and nectin-2 . Recruitment of Sec6/8 complex to cell-cell contacts can be achieved in fibroblasts when E-cadherin and nectin-2 are co-expressed. These results support a model in which localized recruitment of Sec6/8 complex to the plasma membrane by specific cell-cell adhesion complexes defines a site for vesicle delivery and polarized membrane growth during development of epithelial cell polarity.  

3.462           Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control

Molinari, M. et al Mol. Cell, 13, 125-135 (2004)   Calreticulin and calnexin are homologous lectins that serve as molecular chaperones for glycoproteins in the endoplasmic reticulum of eukaryotic cells. Here we show that calreticulin depletion specifically accelerates the maturation of cellular and viral glycoproteins with a modest decrease in folding efficiency. Calnexin depletion prevents proper maturation of some proteins such as influenza hemagglutinin but does not interfere appreciably with the maturation of several others. A dramatic loss of stringency in the ER quality control with transport at the cell surface of misfolded glycoprotein conformers is only observed when substrate access to both calreticulin and calnexin is prevented. Although not fully interchangeable during assistance of glycoprotein folding, calreticulin and calnexin may work, independently, as efficient and crucial factors for retention in the ER of nonnative polypeptides.  

3.463           Smooth muscle raft-like membranes

Baron, C.B. and Coburn, R.F.

1. Lipid Res.,45, 41-53 (2004)

  We developed a method for extracting raft-like, liquid-orderedmembranes from the particulate fraction prepared from porcinetrachealis smooth muscle. This fraction, which contains mostof the plasma membrane in this tissue, was homogenized in thepresence of cold 0.5% Triton X-100. After centrifugation, membranescontaining high contents of sphingomyelin (SM) and cholesteroland low phosphatidylcholine (PC) contents remained in the pellet.Thirty-five millimolar octyl glucoside (OG) extracted 75% ofthese membranes from the Triton X-100-resistant pellet. Thesemembranes had low buoyant densities and accounted for 28% ofthe particulate fraction lipid. Their lipid composition, 22%SM, 60% cholesterol, 11% phosphatidylethanolamine, 8% PC, <1%phosphatidylinositol, and coisolation with 5'-nucleotidase andcaveolin-1 suggest that they are liquid-ordered membranes. Wecompared characteristics of OG and Triton X-100 extractionsof the particulate fraction. In contrast to Triton X-100 extractions,membranes released from the particulate fraction by OG weremainly collected in low buoyant fractions at densities rangingfrom 1.05 to 1.11 g/ml and had phospholipid and cholesterolcontents consistent with a mixture of liquid-ordered and liquid-disorderedmembranes.  

3.464           The EF-hand Ca²⁺-binding protein p22 plays a role in microtubule and endoplasmic reticulum organization and dynamics with distinct Ca²⁺-binding requirements

Andrade, j., Zhao, H., Titus, B., Pearce, T. and Barroso, M. Mol. Biol. Cell, 15, 481-496 (2004)   We have reported that p22, an N-myristoylated EF-hand Ca²⁺-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca²⁺, p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca²⁺-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based "bulk microinjection" assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca²⁺-binding p22 mutant, which is unable to undergo Ca²⁺-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca²⁺-independent manner and affects ER network assembly in a Ca²⁺-dependent manner.  

3.465           Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death

Muppidi, J. and Siegel, R.M. Nature Immunol., 5(2), 182-189 (2004)   Clonotypic elimination of activated T cells through Fas–Fas ligand (CD95–CD95L) interactions is one mechanism of peripheral self-tolerance. T cell receptor (TCR) stimuli trigger FasL synthesis but also sensitize activated T cells to Fas-mediated apoptosis through an unknown mechanism. Here we show that TCR restimulation of activated human CD4⁺ T cells resulted in Fas translocation into lipid raft microdomains before binding FasL, rendering these cells sensitive to apoptosis after stimulation with bivalent antibody or FasL. Disruption of lipid rafts reduced sensitivity to Fas-mediated apoptosis after TCR restimulation. Thus, the redistribution of Fas and other tumor necrosis factor family receptors into and out of lipid rafts may dynamically regulate the efficiency and outcomes of signaling by these receptors.  

3.466           A novel fluorescence resonance energy transfer assay demonstrates that the human immunodeficiency virus type I Pr55^Gag  I domain mediates Gag-Gag interactions

Derdowski, A., Ding, L. and Spearman, P. J.Virol., 78(3), 1230-1242 (2004)   Human immunodeficiency virus type 1 (HIV-1) assembly takes place at the plasma membrane of cells and is directed by the Pr55^Gag polyprotein (Gag). One of the essential steps in the assembly process is the multimerization of Gag. We have developed a novel fluorescence resonance energy transfer (FRET) assay for the detection of protein-protein interactions between Gag molecules. We demonstrate that Gag multimerization takes place primarily on cellular membranes, with the majority of these interactions occurring on the plasma membrane. However, distinct sites of Gag-Gag interaction are also present at punctate intracellular locations. The I domain is a functional assembly domain within the nucleocapsid region of Gag that affects particle density, the subcellular localization of Gag, and the formation of detergent-resistant Gag protein complexes. Results from this study provide evidence that the I domain mediates Gag-Gag interactions. Using Gag-fluorescent protein fusion constructs that were previously shown to define the minimal I domain within HIV-1 Pr55^Gag, we show by FRET techniquesthat protein-protein interactions are greatly diminished whenGag proteins lacking the I domain are expressed. Gag-Tsg101interactions are also seen in living cells and result in a shiftof Tsg101 to the plasma membrane. The results within this studyprovide direct evidence that the I domain mediates protein-proteininteractions between Gag molecules. Furthermore, this studyestablishes FRET as a powerful tool for the detection of protein-proteininteractions involved in retrovirus assembly.        

3.467           The K1 protein of Kaposi’s sarcoma-associated herpesvirus activates the Akt signaling pathway

Tomlinson, C.C. and Damania, B.

1. Virol., 78(4), 1918-1927 (2004)

  Kaposi's sarcoma-associated herpesvirus (KSHV) has been implicated in Kaposi's sarcoma, as well as in primary effusion lymphoma and multicentric Castleman's disease. The K1 protein of KSHV has been shown to induce cellular transformation and focus formation and to deregulate B-lymphocyte signaling pathways by functionally mimicking the activated B-cell receptor complex. Here we show that expression of K1 in B lymphocytes targets the phosphatidylinositol-3 kinase pathway, leading to the activation of the Akt kinase and the inhibition of the phosphatase PTEN. We also demonstrate that activation of Akt by the K1 protein leads to the phosphorylation and inhibition of members of the forkhead (FKHR) transcription factor family, which are key regulators of cell cycle progression and apoptosis. We demonstrate that K1 can inhibit apoptosis induced by the FKHR proteins and by stimulation of the Fas receptor. Our observations suggest that the K1 viral protein promotes cell survival pathways and may contribute to KSHV pathogenesis by preventing virally infected cells from undergoing apoptosis prematurely.  

3.468           Rap1 up-regulation and activation on plasma membrane regulates T cell adhesion

Bivona, T.G. et al

1. Cell Biol., 164(3), 461-470 (2004)

  Rap1 and Ras are closely related GTPases that share some effectors but have distinct functions. We studied the subcellular localization of Rap1 and its sites of activation in living cells. Both GFP-tagged Rap1 and endogenous Rap1 were localized to the plasma membrane (PM) and endosomes. The PM association of GFP-Rap1 was dependent on GTP binding, and GFP-Rap1 was rapidly up-regulated on this compartment in response to mitogens, a process blocked by inhibitors of endosome recycling. A novel fluorescent probe for GTP-bound Rap1 revealed that this GTPase was transiently activated only on the PM of both fibroblasts and T cells. Activation on the PM was blocked by inhibitors of endosome recycling. Moreover, inhibition of endosome recycling blocked the ability of Rap1 to promote integrin-mediated adhesion of T cells. Thus, unlike Ras, the membrane localizations of Rap1 are dynamically regulated, and the PM is the principle platform from which Rap1 signaling emanates. These observations may explain some of the biological differences between these GTPases.  

3.469           The Arf activator Gea2p and P-type ATPase Drs2p interact at the Golgi in Saccharomyces cerevisiae

Chantalai, S.C. et al

1. Cell Sci., 117, 711-722 (2004)

  Arf GTPases regulate both the morphological and protein sorting events that are essential for membrane trafficking. Guanine nucleotide exchange factors (GEFs) specific for Arf proteins determine when and where Arf GTPases will be activated in cells. The yeast Gea2p Arf GEF is a member of an evolutionarily conserved family of high molecular mass Arf GEFs that are peripherally associated with membranes. Nothing is known about how these proteins are localized to membranes, and few direct binding partners have been identified. In yeast, Gea2p has been implicated in trafficking through the Golgi apparatus and in maintaining Golgi structure. A major function of the Golgi apparatus is the packaging of cargo into secretory granules or vesicles. This process occurs through a series of membrane transformation events starting with fenestration of a saccular membrane, and subsequent remodeling of the fenestrated membrane into a mesh-like tubular network. Concentration of secretory cargo into nodes of the tubular network leads to enlargement of the nodes, which correspond to forming vesicles/granules, and thinning of the surrounding tubules. The tubules eventually break to release the secretory vesicles/granules into the cytoplasm. This process is highly conserved at the morphological level from yeast to mammalian cells. Drs2p, a multi-span transmembrane domain protein and putative aminophospholipid translocase, is required for the formation of a class of secretory granules/vesicles in yeast. Here we show that Drs2p interacts directly with Gea2p, both in vitro and in vivo. We mapped the domain of interaction of Drs2p to a 20-amino-acid region of the C-terminal cytoplasmic tail of the protein, adjacent to a region essential for Drs2p function. Mutations in Gea2p that abolish interaction with Drs2p are clustered in the C-terminal third of the Sec7 domain, and are important for Gea2p function. We characterize one such mutant that has a thermosensitive phenotype, and show that it has morphological defects along the secretory pathway in the formation of secretory granules/vesicles.    

3.470           The matrix protein of Marburg virus is transported to the plasma membrane along cellular membranes: exploiting the retrograde late endosomal pathway

Kolesnikova, L., Bamberg, S., Berghöfer, B. and Becker, S.

1. Virol., 78(5), 2382-2393 (2004)

  VP40, the matrix protein of Marburg virus, is a peripheral membrane protein that has been shown to associate with membranes of multivesicular bodies (MVBs) (L. Kolesnikova, H. Bugany, H.-D. Klenk, and S. Becker, J. Virol. 76:1825-1838, 2002). The present study revealed that VP40 is bound to cellular membranes rapidly after synthesis. Time course studies were performed to trace the distribution of VP40 during the course of expression. First, VP40 was homogenously distributed throughout the cytoplasm, although the majority of protein (70%) was already membrane associated. Next, VP40 accumulated in MVBs and in tubular protrusions emerging from MVBs. Finally, VP40 appeared in a patch-like pattern beneath the plasma membrane. These morphological results were supported by iodixanol density gradient analyses. The majority of VP40-positive membranes were first detected comigrating with small vesicles. VP40 was then shifted to fractions containing endosomal marker proteins, and later, to fractions containing plasma membrane marker proteins. Blocking of protein synthesis by use of cycloheximide at the time when VP40 was mainly associated with the small vesicles did not prevent the redistribution of VP40 to the late endosomes and further to the plasma membrane. The inhibition of intracellular vesicular trafficking by monensin significantly reduced the appearance of VP40 at the plasma membrane. In conclusion, we suggest that the transport of the Marburg virus matrix protein VP40 involves its accumulation in MVBs followed by the redistribution of VP40-enriched membrane clusters to the plasma membrane.  

3.471           Bicarbonate-responsive “soluble” adenylyl cyclase defines a nuclear camp microdomain

Zippin, J.H. et al

1. Cell Biol., 164(4), 527-534 (2004)

  Bicarbonate-responsive "soluble" adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation.  

3.472           Site of docking and fusion of insulin secretory granules in live MIN6 b cells analyzed by TAT-conjugated anti-syntaxin 1 antibody and total internal reflection fluorescence microscopy

Ohara-Imaizumi, M. et al

1. Biol. Chem., 279(9), 8403-8408 (2004)

  To determine the site of insulin exocytosis in the pancreatic cell plasma membrane, we analyzed the interaction between the docking/fusion of green fluorescent protein-tagged insulin granules and syntaxin 1 labeled by TAT-conjugated Cy3-labeled antibody (Ab) using total internal reflection fluorescence microscopy (TIRFM). Monoclonal Ab against syntaxin 1 was labeled with Cy3 then conjugated with the protein transduction domain of HIV-1 TAT. TAT-conjugated Cy3-labeled anti-syntaxin 1 Ab was transduced rapidly into the subplasmalemmal region in live MIN6 cells, which enabled us to observe the spatial organization and distribution of endogenous syntaxin 1. TIRFM imaging revealed that syntaxin 1 is distributed in numerous separate clusters in the intact plasma membrane, where insulin secretory granules were docked preferentially to the sites of syntaxin 1 clusters, colocalizing with synaptosomal-associated protein of 25 kDa (SNAP-25) clusters. TIRFM imaging analysis of the motion of single insulin granules demonstrated that the fusion of insulin secretory granules stimulated by 50 mM KCl occurred exclusively at the sites of the syntaxin 1 clusters. Cholesterol depletion by methyl- -cyclodextrin treatment, in which the syntaxin 1 clusters were disintegrated, decreased the number of docked insulin granules, and, eventually the number of fusion events was significantly reduced. Our results indicate that 1) insulin exocytosis occurs at the site of syntaxin 1 clusters; 2) syntaxin 1 clusters are essential for the docking and fusion of insulin granules in MIN6 cells; and 3) the sites of syntaxin 1 clusters are distinct from flotillin-1 lipid rafts.

     

3.473           Sphingolipid-cholesterol domains (lipid rafts) in normal human and dog thyroid follicular cells are not involved in thyrotropin receptor signaling

Costa, M.J. et al Endocrinology, 145(3), 1464-1472 (2004)   Partition of signaling molecules in sphingolipid-cholesterol-enriched membrane domains, among which are the caveolae, may contribute to signal transduction efficiency. In normal thyroid, nothing is known about a putative TSH/cAMP cascade compartmentation in caveolae or other sphingolipid-cholesterol-enriched membrane domains. In this study we show for the first time that caveolae are present in the apical membrane of dog and human thyrocytes: caveolin-1 mRNA presence is demonstrated by Northern blotting in primary cultures and that of the caveolin-1 protein by immunohistochemistry performed on human thyroid tissue. The TSH receptor located in the basal membrane can therefore not be located in caveolae. We demonstrate for the first time by biochemical methods the existence of sphingolipid-cholesterol-enriched domains in human and dog thyroid follicular cells that contain caveolin, flotillin-2, and the insulin receptor. We assessed a possible sphingolipid-cholesterol-enriched domains compartmentation of the TSH receptor and the - subunit of the heterotrimeric G_s and G_q proteins using two approaches: Western blotting on detergent-resistant membranes isolated from thyrocytes in primary cultures and the influence of 10 mM methyl-ß-cyclodextrin, a cholesterol chelator, on basal and stimulated cAMP accumulation in intact thyrocytes. The results from both types of experiments strongly suggest that the TSH/cAMP cascade in thyroid cells is not associated with sphingolipid-cholesterol-enriched membrane domains.  

3.474           Characterization of the Lassa virus matrix protein Z: electron microscopic study of virus-like particles and interaction with the nucleoprotein (NP)

Eichler, R. et al Virus Res., 100, 249-255 (2004)   Lassa virus is the causative agent of a hemorrhagic fever endemic in west Africa. The RNA genome of Lassa virus encodes the glycoprotein precursor GP-C, a nucleoprotein (NP), the viral polymerase L and a small protein Z (11 kDa). Here, we analyze the role of Z protein for virus maturation. We have recently shown that expression of Z protein in the absence of other viral proteins is sufficient for the release of enveloped Z-containing particles. In this study, we examined particles secreted into the supernatant of a stably Z protein-expressing CHO cell line by electron microscopy. The observed Z-induced virus-like particles did not significantly differ in their morphology and size from Lassa virus particles. Mutation of two proline-rich domains within Z which are known to drastically reduce the release of virus-like particles, had no effect on the cellular localization of the protein nor on its membrane-association. Furthermore, we present evidence that Z interacts with the NP. We assume that Z recruits NP to cellular membranes where virus assembly takes place. We conclude from our data that Lassa virus Z protein plays an essential role in Lassa virus maturation.  

3.475           Glycosylphosphatidylinositol-anchored proteins and actin cytoskeleton modulate chloride transport by channels formed by the Helicobacter pylori vacuolating cytotoxin VacA in HeLa cells

Gauthier, N.C. et al

1. Biol. Chem., 279(10), 9481-9489 (2004)

  The vacuolating cytotoxin VacA is an important virulence factor of Helicobacter pylori. Removing glycosylphosphatidylinositol-anchored proteins (GPI-Ps) from the cell surface by phosphatidylinositol-phospholipase C or disrupting the cell actin cytoskeleton by cytochalasin D reduced VacA-induced vacuolation of cells (Ricci V., Galmiche, A., Doye, A., Necchi, V., Solcia, E., and Boquet, P. (2000) Mol. Biol. Cell 11, 3897-3909). Using the fluorescent dye 6-methoxy-N-ethylquinolinium chloride, an indicator for cytosolic chloride, we have investigated the role of either GPI-Ps or actin cytoskeleton in the activity of the selective anionic channel formed by VacA at the plasma membrane level. Removal of GPI-Ps from HeLa cell surfaces did not impair VacA localization into lipid rafts but strongly reduced VacA channel-mediated cell influx and efflux of chloride. Disruption of the actin cytoskeleton of HeLa cells by cytochalasin D did not affect VacA localization in lipid rafts but blocked VacA cell internalization and inhibited cell vacuolation while increasing the overall chloride transport by the toxin channel at the cell surface. Specific enlargement of Rab7-positive compartments induced by VacA could be mimicked by the weak base chloroquine alone, and the vacuolating activities of either chloroquine alone or VacA were blocked with the same potency by the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid shown to inhibit VacA channel activity (Tombola, F., Oregna, F., Brutsche, S., Szabò, I., Del Giudice, G., Rappuoli, R., Montecucco, C., Papini, E., and Zoratti, M. (1999) FEBS Lett. 460, 221-225). We suggest that formation of functional VacA channels at the cell surface required GPI-Ps and that endocytosis of these channels by an actin-dependent process increases the chloride content of late endosomes that accumulate weak bases, provoking their enlargement by osmotic swelling.  

3.476           Human immunodeficiency virus type 1 Nef activates p21-activated kinase vis recruitment into lipid rafts

Krautkrämer, E., Giese, S.I., Gasteier, J.E., Muuranyi, W. and Fackler, O.T.

1. Virol., 78(8), 4085-4097 (2004)

  The Nef protein of human immunodeficiency virus type 1 is an important factor in AIDS pathogenesis. In addition to downregulating CD4 and major histocompatibility complex class I molecules from the cell surface, as well as increasing virion infectivity, Nef triggers activation of the T-cell receptor (TCR) cascade to facilitate virus spread. Signaling pathways that are induced by Nef have been identified; however, it is unclear how and in which subcellular compartment Nef triggers signaling. Nef recruits a multiprotein complex to activate the cellular Pak kinase that mediates downstream effector functions. Since a subpopulation of Nef is present in detergent-insoluble microdomains (lipid rafts) from where physiological TCR signaling is initiated, we tested whether lipid rafts are instrumental for Nef-mediated Pak activation. In flotation analysis, Nef-associated Pak activity exclusively fractionated with lipid rafts. Activation of Pak in the presence of Nef coincided with lipid raft recruitment of the kinase, which was otherwise excluded from detergent-insoluble microdomains. Experimental solubilization of lipid rafts interfered with the association of Pak activity with Nef. To analyze the importance of the raft localization for Nef function more rigorously, we generated a palmitoylated Nef (PalmNef). PalmNef was highly enriched in lipid rafts and associated with significantly higher levels of Pak activity than Nef. Notably, activation of Pak by its physiological activators, Cdc42 and Rac, also occurred in lipid rafts and required raft integrity. Together, these data suggest that Nef induces signal transduction via the recruitment of a signaling machinery including Pak into lipid rafts, thereby mimicking a physiological cellular mechanism to initiate the TCR cascade.  

3.477           Conserved “PAL” sequence in presenilins is essential for g-secretase activity, but not required for formation or stabilization of g-secretase complexes

Wang, J., Brunkan, A.L., Hecimovic, S., Walker, E. and Goate, A. Neurobiol.of Disease, 15, 654-666 (2004)   Generation of A from the -amyloid precursor protein (APP) requires a series of proteolytic processes, including an intramembranous cleavage catalyzed by an aspartyl protease, -secretase. Two aspartates in presenilins (PS) are required for -secretase activity (D257 and D385 of PS1), suggesting that PS may be part of this protease. Little is known concerning the importance of other sequences in PS for activity. We introduced point mutations (P433L, A434D, L435R) into a completely conserved region C-terminal to transmembrane domain eight of PS1. The P433L mutation abolished PS1 endoproteolysis as well as -secretase cleavage of APP and Notch in PS1/2 K/O cells. In HEK cells, expression of PS1/P433L reduced A production and caused accumulation of APP C-terminal stubs. When the P433L mutation was introduced into the non-cleavable exon 9 ( E9) variant of PS1, it abolished -secretase cleavage of APP and Notch. The P433L holoprotein is stable and incorporated into the high molecular weight -secretase complex, arguing that P433 is not necessary for formation or stabilization of the -secretase complex. Other non-conservative mutations in the invariant P₄₃₃A₄₃₄L₄₃₅ sequence also result in a phenotype that is indistinguishable from the aspartate mutants, suggesting a direct involvement of this sequence in -secretase activity.  

3.478           Secreted MMP9 promotes angiogenesis more efficiently than constitutive active MMP9 bound to the tumor cell surface

Mira, E. et al

1. Cell Sci., 117, 1847-1856 (2004)

  Association of matrix metalloprotease 9 (MMP9) to the cell membrane is considered important in tumor growth and angiogenesis. To dissect this regulatory mechanism, we generated raft and non-raft MMP9 chimeras to force membrane expression in the MCF-7 human breast carcinoma cell line. MMP9 targeting to non-raft cell surface domains rendered a constitutive active membrane MMP9 form, suggesting a contribution by the lipid environment in MMP activation. We generated human breast cancer xenograft models using MCF-7 cells overexpressing secreted and membrane-anchored MMP9. The non-raft MMP9 chimera was constitutively active at the cell membrane in xenografts, but this activation did not correlate with an increase in MMP9-induced angiogenesis. Capillary number and vessel perimeter were specifically increased only in tumors overexpressing wild-type MMP9 (the secreted form); this increase was inhibited when tumors were induced in doxycycline-treated mice. Xenografts from tumor cells overexpressing wild-type MMP9 showed increased vascular endothelial growth factor (VEGF)/VEGFR2 receptor association, which was also dependent on MMP9 activity. These observations indicate that membrane location can influence MMP9 activity in vitro and in vivo, and confirm the relevance of stromal-associated, but not tumor-bound MMP9 in mediating tumor-induced angiogenesis.  

3.479           Implication of ZW10 in membrane trafficking between the endoplasmic reticulum and Golgi

Hirose, H. et al EMBO J., 23, 1267-1278 (2004)   ZW10, a dynamitin-interacting protein associated with kinetochores, is known to participate directly in turning off of the spindle checkpoint. In the present study, we show that ZW10 is located in the endoplasmic reticulum as well as in the cytosol during interphase, and forms a subcomplex with RINT-1 (Rad50-interacting protein) and p31 in a large complex comprising syntaxin 18, an endoplasmic reticulum-localized t-SNARE implicated in membrane trafficking. Like conventional syntaxin-binding proteins, ZW10, RINT-1 and p31 dissociated from syntaxin 18 upon Mg²⁺-ATP treatment in the presence of NSF and -SNAP, whereas the subcomplex was not disassembled. Overexpression, microinjection and knockdown experiments revealed that ZW10 is involved in membrane trafficking between the endoplasmic reticulum and Golgi. The present results disclose an unexpected role for a spindle checkpoint protein, ZW10, during interphase.  

3.480           Appropriate NR1-NR1 disulfide-linked homodimer formation is requisite for efficient expression of functional, cell surface N-methyl-D-aspartate NR1/NR2 receptors

Papadakis, M., Hawkins, L.M. and Stephenson, F.A.

1. Biol. Chem., 279(15), 14703-14712 (2004)

  A c-Myc epitope-tagged N-methyl-D-aspartate receptor NR1-2a subunit was generated, NR1-2a_c-Myc, where the tag was inserted after amino acid 81. NR1-2a_c-Myc /NR2A receptors when expressed in mammalian cells are not trafficked to the cell surface nor do they yield cell cytotoxicity post-transfection. NR1-2a_c-Myc was, however, shown to assemble with NR2A subunits by immunoprecipitation and [³H]MK801 radioligand binding assays. Immunoblots of cells co-transfected with wild-type NR1-2a/NR2A subunits yielded two NR1-2a immunoreactive species with molecular masses of 115 and 226 kDa. Two-dimensional electrophoresis under non-reducing and reducing conditions revealed that the 226-kDa band contained disulfide-linked NR1-2a subunits. Only the 115-kDa NR1-2a species was detected for NR1-2a_c-Myc/NR2A. The c-Myc epitope is inserted adjacent to cysteine 79 of the NR1-2a subunit; therefore, it is possible that the tag may prevent the formation of NR1 disulfide bridges. A series of cysteine alanine NR1-2a mutants was generated, and the NR1-2a mutants were co-expressed with NR2A or NR2B subunits in mammalian cells and characterized with respect to cell surface expression, cell cytotoxicity post-transfection, co-association by immunoprecipitation, and immunoblotting following SDS-PAGE under both reducing and non-reducing conditions. When co-expressed with NR2A in mammalian cells, NR1-2a_C79A/NR2A displayed similar properties to NR1-2a_c-Myc/NR2A in that the 226-kDa NR1 immunoreactive species was not detectable, and trafficking to the cell surface was impaired compared with wild-type NR1/NR2 receptors. These results provide the first biochemical evidence for the formation of NR1-NR1 intersubunit disulfide-linked homodimers involving cysteine 79. They suggest that disulfide bridging and structural integrity within the NR1 N-terminal domain is requisite for cell surface N-methyl-D-aspartate receptor expression.  

3.481           Gene and protein characterization of the human glutathione S-transferase kappa and evidence for a peroxisomal localization

Morel, F. et al

1. Biol. Chem., 279(16), 16246-16253 (2004)

  Kappa class glutathione S-transferase (GST) cDNA sequences have been identified in rat, mouse, and human. In the present study, we determined the structure and chromosomal location of the human GST Kappa 1 (hGSTK1) gene, characterized the protein, and demonstrated its subcellular localization. The human gene spans 5 kb, has 8 exons, and maps onto chromosome 7q34. The 5'-flanking region lacks TATA or CCAAT boxes, but there is an initiator element overlapping the transcription start site. hGSTK1 amino acid sequence showed homology to bacterial 2-hydroxychromene-2-carboxylate isomerase, an enzyme involved in naphthalene degradation pathway. hGSTK1 mRNA was expressed in all of the organs examined. Subcellular fractionation of HepG2 cells showed that the protein was located in peroxisomes and mitochondria and was not detectable in cytoplasm. The peroxisomal localization was confirmed by transfection of HepG2 cells with a plasmid coding a green fluorescent protein fused inframe to the N terminus of hGSTK1. The C terminus of hGSTK1 was essential for localization of the protein to peroxisomes, and the C-terminal sequence Ala-Arg-Leu represents a peroxisome targeting signal. This is the first time that a human GST has been found in peroxisomes, suggesting a new function for this family of enzymes.  

3.482           Pen-2 is sequestered in the endoplamic reticulum and subjected to ubiquitylation and proteasome-mediated degradation in the absence of presenilin

Bergmann, A. et al

1. Biol. Chem., 279(16), 16744-16753 (2004)

  The -secretase complex catalyzes intramembrane proteolysis of a number of transmembrane proteins, including amyloid precursor protein, Notch, ErbB4, and E-cadherin. -Secretase is known to contain four major protein constituents: presenilin (PS), nicastrin, Aph-1, and Pen-2, all of which are integral membrane proteins. There is increasing evidence that the formation of the complex and the stability of the individual components are tightly controlled in the cell, assuring correct composition of functional complexes. In this report, we investigate the topology, localization, and mechanism for destabilization of Pen-2 in relation to PS function. We show that PS1 regulates the subcellular localization of Pen-2: in the absence of PS, Pen-2 is sequestered in the endoplasmic reticulum (ER) and not transported to post-ER compartments, where the mature -secretase complexes reside. PS deficiency also leads to destabilization of Pen-2, which is alleviated by proteasome inhibitors. In keeping with this, we show that Pen-2, which adopts a hairpin structure with the N and C termini facing the luminal space, is ubiquitylated prior to degradation and presumably retrotranslocated from the ER to the cytoplasm. Collectively, our data suggest that failure to become incorporated into the -secretase complex leads to degradation of Pen-2 through the ER-associated degradation-proteasome pathway.  

3.483           Seasonal varation in cytochrome P450 immunopositive protein levels, lipid peroxidation and genetic toxicity in digestive gland of the mussel Mytilus edulis

Shaw, J.P. et al Aquatic Tox., 67, 325-336 (2004)   The relationship between cytochrome P450 1A- and 2E-immunopositive proteins, lipid peroxidation and DNA strand breaks (SBs) was studied in Mytilus edulis digestive gland at different seasons and at different sites around the UK coast. Cytochrome P4501A (CYP1A)-immunopositive protein and DNA strand breaks were generally lowest in December but there was no correlation between PAH exposure (indicated by chemical measurement and CYP1A-immunopositive protein expression) and DNA strand breaks which was highest at the relatively non-polluted site (Port Quin). As with CYP1A, CYP2E1-immunopositive protein was maximal at most sites in May. Lipid peroxidation, in contrast, did not alter markedly throughout the year. In conclusion, DNA strand breakage was not correlated with any of the above parameters although it did correlate with "scope for growth" as did the inverse of PAH levels. The study highlights the need to establish the relative contribution of DNA damage and DNA repair processes to the production of DNA strand breaks and emphasises the need to consider seasonal variation in interpretation of biomarkers.      

3.484           The 2003 ASBMB-Avanti Award in Lipids Address: applications of novel synthetic lipids to biological problems

Bittmann, R. Chemistry & Lipids, 129, 111-131 (2004)   This paper is an overview of the 2003 Avanti Award in Lipids address that was presented by Robert Bittman at the American Society for Biochemistry and Molecular Biology (ASBMB) Annual Meeting held in San Diego, CA in conjunction with meetings of five other FASEB Societies, April 15, 2003. The theme of the lecture is: "How can the chemical synthesis of unnatural lipids provide insights into problems ranging from cell biology to biophysics?" The following examples are presented: (1) novel ceramide analogs as experimental anticancer agents, (2) photoactivatable sphingosine 1-phosphate analogs as probes of protein targets of this bioactive lipid, (3) a ¹³C-enriched cerebroside as a quantitative probe of glycosphingolipid (GSL) transbilayer distribution in bilayers with and without sphingomyelin, (4) cis and trans unsaturated sphingomyelin analogs as modulators of the existence of cholesterol-enriched microdomains (rafts) that may facilitate fusion of alphaviruses with target membranes, (5) ceramide as an indirect enhancer of the permeabilization of membranes induced by cholesterol-specific cytolysins, (6) fluorescent GSL analogs of widely disparate structure as probes of the molecular features responsible for the selective internalization of GSLs in caveolae of living mammalian cells, (7) enantiomeric lysophosphatidic acid (LPA) analogs as probes of receptor subtypes that mediate LPA signaling, and (8) phosphonocholine analogs of the antitumor ether lipid ET-18-OCH₃ as tools for discerning the primary targets that are critical for cytotoxic activity in tumor cells.  

3.485           CD44 interaction with ankyrin and IP₃ receptor in lipid rafts promotes hyaluronan-mediated Ca²⁺ signaling to nitric oxide production and endothelial cell adhesion and proliferation

Singleton, P.A. and Bourguignon, L.Y.W. Exp. Cell Res., 295, 102-118 (2004)   In this study, we have showed that aortic endothelial cells (GM7372A cell line) express CD44v10 [a hyaluronan (HA) receptor], which is significantly enriched in cholesterol-containing lipid rafts (characterized as caveolin-rich plasma membrane microdomains). HA binding to CD44v10 promotes recruitment of the cytoskeletal protein, ankyrin and inositol 1,4,5-triphosphate (IP₃) receptor into cholesterol-containing lipid rafts. The ankyrin repeat domain (ARD) of ankyrin is responsible for binding IP₃ receptor to CD44v10 at lipid rafts and subsequently triggering HA/CD44v10-mediated intracellular calcium (Ca²⁺) mobilization leading to a variety of endothelial cell functions such as nitric oxide (NO) production, cell adhesion and proliferation. Further analyses indicate (i) disruption of lipid rafts by depleting cholesterol from the membranes of GM7372A cells (using methyl- -cyclodextrin treatment) or (ii) interference of endogenous ankyrin binding to CD44 and IP₃ receptor using overexpression of ARD fragments (by transfecting cells with ARDcDNA) not only abolishes ankyrin/IP₃ receptor accumulation into CD44v10/cholesterol-containing lipid rafts, but also blocks HA-mediated Ca²⁺ signaling and endothelial cell functions. Taken together, our findings suggest that CD44v10 interaction with ankyrin and IP₃ receptor in cholesterol-containing lipid rafts plays an important role in regulating HA-mediated Ca²⁺ signaling and endothelial cell functions such as NO production, cell adhesion and proliferation.  

3.486           Heparan sulphate proteoglycans modulate fibroblast growth factor-2 binding through a lipid-raft-mediated mechanism

Chu, C.L., Buczek-Thomas, J.A. and Nugent, M.A. Biochem. J., 379, 331-341 (2004)   We investigated how lipid raft association of HSPG (heparan sulphate proteoglycans) modulates FGF-2 (fibroblast growth factor-2/basic fibroblast growth factor) interactions with vascular smooth-muscle cells. When lipid rafts were disrupted with sterol-binding agents, methyl-alpha-cyclodextrin and filipin, FGF-2 binding to HSPG was reduced 2-5-fold, yet the amount and turnover of cell-surface HSPG were unaffected. Approx. 50-65% of bound FGF-2 was in lipid raft-associated fractions based on insolubility in unlabelled Triton X-100 and flotation in OptiPrep density gradients, and this level was increased with higher FGF-2 concentrations. Less FGF-2 (50-90%) was associated in raft fractions when cholesterol was depleted or HSPG were degraded with heparinase III. To investigate how lipid raft-HSPG interactions altered binding, we compared the rates of FGF-2 dissociation with native, MbetaCD (methyl-beta-cyclodextrin)- and filipin-treated cells. We found that FGF-2 dissociation rates were increased when lipid rafts were disrupted. These results suggest that localization of HSPG within lipid rafts creates high local concentrations of binding sites such that dissociation of FGF-2 is hindered. The localization of FGF-2 and HSPG to lipid rafts also correlated with the activation of protein kinase Calpha. Thus raft association of HSPG might create growth factor traps resulting in increased binding and signal transduction to enhance cell sensitivity.  

3.487           Lipid rafts and integrin activation regulate oligodendrocyte survival

Decker, L. and ffrench-Constant, C.

1. Neurosci., 24(15) 3816-3825 (2004)

  Newly formed oligodendrocytes in the CNS derive survival cues from their target axons. These cues are provided in part by laminins expressed on the axon, which are recognized by 6 1 integrin on the oligdendrocyte and amplify platelet-derived growth factor (PDGF) signaling through the phosphatidylinositol 3'-kinase (PI3K) pathway. The 6 1 integrin is localized in oligodendrocyte lipid rafts. We show here using the sphingolipid synthesis inhibitor fumonisin-B1 to deplete rafts that this localization is important for normal survival signaling, because depletion increases oligodendrocyte apoptosis and inhibits PI3K signaling. We have shown previously that PDGF-mediated integrin activation is an important component of oligodendrocyte proliferation signaling, and here we present evidence that a similar mechanism operates in survival signaling. Integrin activation using manganese increases raft localization and rescues the effects of both raft depletion and PDGF removal on survival and PI3K signaling. Together, these results point to an essential role for rafts in oligodendrocyte survival signaling on the basis of the provision of a favorable environment for growth factor-mediated integrin activation.  

3.488           Regulated membrane trafficking of the insulin-responsive glucose transporter 4 in adipocytes

Watson, R.T., Kanzaki, M. and Pessin, J.E. Endocrine Reviews, 25, 177-204 (2004)   Since the discovery of insulin roughly 80 yr ago, much has been learned about how target cells receive, interpret, and respond to this peptide hormone. For example, we now know that insulin activates the tyrosine kinase activity of its cell surface receptor, thereby triggering intracellular signaling cascades that regulate many cellular processes. With respect to glucose homeostasis, these include the function of insulin to suppress hepatic glucose production and to increase glucose uptake in muscle and adipose tissues, the latter resulting from the translocation of the glucose transporter 4 (GLUT4) to the cell surface membrane. Although simple in broad outline, elucidating the molecular intricacies of these receptor-signaling pathways and membrane-trafficking processes continues to challenge the creative ingenuity of scientists, and many questions remain unresolved, or even perhaps unasked. The identification and functional characterization of specific molecules required for both insulin signaling and GLUT4 vesicle trafficking remain key issues in our pursuit of developing specific therapeutic agents to treat and/or prevent this debilitating disease process. To this end, the combined efforts of numerous research groups employing a range of experimental approaches has led to a clearer molecular picture of how insulin regulates the membrane trafficking of GLUT4.        

3.489           Activation of the tumor suppresor merlin modulates its interaction with lipid rafts

Stickney, J.T., Bacon, W.C., Rojas, M., Ratner, N. and Ip W. Cancer Res., 64, 2717-2724 (2004)   Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by bilateral schwannomas of the eighth cranial nerve. The NF2 tumor suppressor protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of membrane/F-actin linkers. Merlin resists solubilization by the detergent Triton X-100 (TX-100), a property commonly attributed to association with the cytoskeleton. Accordingly, NF2 patient mutations that encode merlins with enhanced TX-100 solubility have been explained previously in terms of loss of cytoskeletal attachment. However, here we present data to suggest that the detergent resistance of merlin is a result of its constitutive residence in lipid rafts. Furthermore, when cells are grown to high density, merlin shifts to a more buoyant lipid raft fraction in a density gradient. This shift is mimicked in subconfluent cells treated with cytochalasin D, suggesting that the shift results from merlin dissociation from the actin cytoskeleton, but not from lipid rafts. Intramolecular NH₂- and COOH-terminal binding, which occurs when merlin transitions to the growth-suppressive form, also brings about a similar change in buoyant density. Our results suggest that constitutive residence of merlin in lipid rafts is crucial for its function and that as merlin becomes growth suppressive in vivo, one significant molecular event may be the loss of interaction with the actin cytoskeleton. To our knowledge, merlin is the first tumor suppressor known to reside within lipid rafts, and the significance of this finding is underscored by known loss-of-function NF2 patient mutations that encode merlins with enhanced TX-100 solubility.  

3.490           Carbachol regulation of rabbit ileal brush border Na⁺-H⁺ exchanger 3 (NHE3) occurs through changes in NHE3 trafficking and complex formation and is Src dependent

Li, X. et al

1. Physiol., 3, 791-804 (2004)

  The epithelial brush border membrane (BBM) Na⁺-H⁺ exchanger 3 (NHE3) is the major transport protein responsible for ileal electroneutral Na⁺ absorption. We have previously shown that ileal BBM NHE3 activity is rapidly inhibited by carbachol, an agonist that mimics cholinergic activation in digestion. In this study, we investigated the mechanisms involved in this NHE3 inhibition. Carbachol decreased the amount of ileal Na⁺ absorptive cell BBM NHE3 within 10 min of exposure. Based on OptiPrep gradient centrifugation, carbachol increased the amount of NHE3 in early endosomes and decreased the amount of NHE3 in BBM, consistent with effects on NHE3 trafficking. The decrease in BBM NHE3 occurred in the detergent-soluble BBM fraction with no change in the amount of NHE3 in the BBM detergent-resistant membranes. The size of BBM NHE3 complexes increased in carbachol-exposed ileum, as studied with sucrose gradient centrifugation. The NHE3 complex size increased in the total BBM, but did not change in the detergent-soluble fraction. This suggests that carbachol treatment enhanced the association of proteins with NHE3 complexes specifically in the detergent-resistant fraction of ileal BBM. NHERF2, -actinin-4 and protein kinase C were among those NHE3-associated proteins because they were more efficiently coimmunoprecipitated from total BBM after carbachol treatment. Moreover, Src was involved in the carbachol-mediated inhibition since: (1) c-Src was rapidly activated in the detergent-resistant membranes by carbachol; and (2) carbachol inhibition of ileal Na⁺ absorption was completely abolished by  the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, the carbachol-induced increase in the size of NHE3-containing complexes was reversed by PP2. These data demonstrate that regulation of NHE3 activity by carbachol can be achieved at several interrelated levels: (1) the subcellular level, at which NHE3 is rapidly endocytosed from BBM to endocytic vesicles upon treatment with carbachol; (2) multiple BBM pools, in which carbachol selectively decreases the amount of NHE3 in the BBM detergent-soluble fraction but not the detergent-resistant membrane; and (3) the molecular level, at which NHE3 complex-associated proteins can be changed upon carbachol treatment, with carbachol leading to larger BBM NHE3 complexes and increased co-IP of NHERF2 with -actinin-4 and activated PKC. The study further describes NHE3 presence simultaneously in multiple dynamic BBM pools in which NHE3 distribution and associated proteins are altered as part of carbachol-induced and Src-mediated rapid signal transduction, which decreases the amount of BBM NHE3 and thus inhibits NHE3 activity.          

3.491           Ubiquitin-mediated targeting of a mutant plasma membrane ATPase, Pma1-7, to the endosomal/vacuolar system in yeast

Pizzirusso, M. and Chang, A. Mol. Biol. Cell, 15, 2401-2409 (2004)   Pma1-7 is a mutant plasma membrane ATPase that is impaired in targeting to the cell surface at 37°C and is delivered instead to the endosomal/vacuolar pathway for degradation. We have proposed that Pma1-7 is a substrate for a Golgibased quality control mechanism. By contrast with wild-type Pma1, Pma1-7 is ubiquitinated. Ubiquitination and endosomal targeting of Pma1-7 is dependent on the Rsp5-Bul1-Bul2 ubiquitin ligase protein complex but not the transmembrane ubiquitin ligase Tul1. Analysis of Pma1-7 ubiquitination in mutants blocked in protein transport at various steps of the secretory pathway suggests that ubiquitination occurs after ER exit but before endosomal entry. In the absence of ubiquitination in rsp5-1 cells, Pma1-7 is delivered to the cell surface and remains stable. Nevertheless, Pma1-7 remains impaired in association with detergent-insoluble glycolipid-enriched complexes in rsp5-1 cells, suggesting that ubiquitination is not the cause of Pma1-7 exclusion from rafts. In vps1 cells in which protein transport into the endosomal pathway is blocked, Pma1-7 is routed to the cell surface. On arrival at the plasma membrane in vps1 cells, Pma1-7 remains stable and its ubiquitination disappears, suggesting deubiquitination activity at the cell surface. We suggest that Pma1-7 sorting and fate are regulated by ubiquitination.  

3.492           Rotavirus RRV associates with lipid membrane microdomains during cell entry

Isa, P., Realpe, M., Romero, P., Lopez, S. and Arias, C.F. Virology, 322, 370-381 (2004)   Rotavirus cell entry is a multistep process, not completely understood, which requires at least four interactions between the virus and cell surface molecules. In this work, we investigated the role of the sphingolipid- and cholesterol-enriched lipid microdomains (rafts) in the entry of rotavirus strain RRV to MA104 cells. We found that ganglioside GM1, integrin subunits 2 and 3, and the heat shock cognate protein 70 (hsc70), all of which have been implicated as rotavirus receptors, are associated with TX-100 and Lubrol WX detergent-resistant membranes (DRMs). Integrin subunits 2 and 3 were found to be particularly enriched in DRMs resistant to lysis by Lubrol WX. When purified RRV particles were incubated with cells at 4 °C, about 10% of the total infectious virus was found associated with DRMs, and the DRM-associated virus increased to 37% in Lubrol-resistant membrane domains after 60-min incubation at 37 °C. The virus was excluded from DRMs if the cells were treated with methyl- -cyclodextrin (M CD). Immunoblot analysis of the viral proteins showed that the virus surface proteins became enriched in DRMs upon incubation at 37 °C, being almost exclusively localized in Lubrol-resistant DRMs after 60 min. These data suggest that detergent-resistant membrane domains play an important role in the cell entry of rotaviruses, which could provide a platform to facilitate the efficient interaction of the rotavirus receptors with the virus particle.  

3.493           Tula hantavirus L protein is a 250 kDa perinuclear membrane-associated protein

Kukkonen, S.K.J., Vaheri, A. And Plyusin, A.

1. Gen. Virol., 85, 1181-1189 (2004)

  The complete open reading frame of Tula hantavirus (TULV) L RNA was cloned in three parts. The middle third (nt 2191–4344) could be expressed in E. coli and was used to immunize rabbits. The resultant antiserum was then used to immunoblot concentrated TULV and infected Vero E6 cells. The L protein of a hantavirus was detected, for the first time, in infected cells and was found to be expressed as a single protein with an apparent molecular mass of 250 kDa in both virions and infected cells. Using the antiserum, the expression level of the L protein was followed and image analysis of immunoblots indicated that there were 10⁴ copies per cell at the peak level of expression. The antiserum was also used to detect the L protein in cell fractionation studies. In cells infected with TULV and cells expressing recombinant L, the protein pelleted with the microsomal membrane fraction. The membrane association was confirmed with membrane flotation assays. To visualize L protein localization in cells, a fusion protein of L and enhanced green fluorescent protein, L–EGFP, was expressed in Vero E6 cells with a plasmid-driven T7 expression system. L–EGFP localized in the perinuclear region where it had partial co-localization with the Golgi matrix protein GM130 and the TULV nucleocapsid protein.  

3.494           Cholesterol-independent interactions with CD47 enhance a_vb₃ activity

McDonald, J.F., Zheleznyak, A. and Frazier, W.A.

1. Biol. Chem., 279(17), 17301-17311 (2004)

  Expression in OV10 cells of either wild-type CD47 or its extracellular IgV domain linked to a glycosylphosphatidylinositol anchor-(IgV-GPI) enhanced ligand-induced _{v 3} activation as detected by the binding of LIBS1 and LIBS6 mAbs. The amplitude of LIBS binding was greater with both CD47 and IgV-GPI expression, indicating an increase in the population of "activable" integrin molecules. Expression of either CD47 species also increased _{v 3}-mediated adhesion to vitronectin, and to surfaces coated with the anti- ₃ antibody AP3, because of enhanced clustering of _{v 3} as confirmed by chemical cross-linking. Cholesterol depletion with methyl- -cyclodextrin did not prevent the increase in anti-LIBS binding, but reduced cell adhesion to vitronectin and AP3. However, cells expressing CD47 were partially insulated against this disruption, and IgV-GPI was even more effective. Both CD47 and IgV-GPI were found in cholesterol-rich rafts prepared in the absence of detergent, but only CD47 could recruit _{v 3} and its associated signaling molecules to these domains. Thus CD47- _{v 3} complexes in cholesterol-rich raft domains appear to engage in G_i-dependent signaling whereas CD47- _{v 3} interactions that lead to integrin clustering are also detergent resistant, but are insensitive to cholesterol depletion and do not require the transmembrane region of CD47.  

3.495           Brain-specific deletion of neuropathy target esterase/swisscheese results in neurodegeneration

Akassoglou, K. et al PNAS, 101(14), 5075-5080 (2004)   Neuropathy target esterase (NTE) is a neuronal membrane protein originally identified for its property to be modified by organo-phosphates (OPs), which in humans cause neuropathy characterized by axonal degeneration. Drosophila mutants for the homolog gene of NTE, swisscheese (sws), indicated a possible involvement of sws in the regulation of axon-glial cell interaction during glial wrapping. However, the role of NTE/sws in mammalian brain pathophysiology remains unknown. To investigate NTE function in vivo, we used the cre/loxP site-specific recombination strategy to generate mice with a specific deletion of NTE in neuronal tissues. Here we show that loss of NTE leads to prominent neuronal pathology in the hippocampus and thalamus and also defects in the cerebellum. Absence of NTE resulted in disruption of the endoplasmic reticulum, vacuolation of nerve cell bodies, and abnormal reticular aggregates. Thus, these results identify a physiological role for NTE in the nervous system and indicate that a loss-of-function mechanism may contribute to neurodegenerative diseases characterized by vacuolation and neuronal loss.  

3.496           The G protein-coupled receptor rhodosin in the native membrane

Fotiadis, D. et al FEBS Lett., 564, 281-288 (2004)   The higher-order structure of G protein-coupled receptors (GPCRs) in membranes may involve dimerization and formation of even larger oligomeric complexes. Here, we have investigated the organization of the prototypical GPCR rhodopsin in its native membrane by electron and atomic force microscopy (AFM). Disc membranes from mice were isolated and observed by AFM at room temperature. In all experimental conditions, rhodopsin forms structural dimers organized in paracrystalline arrays. A semi-empirical molecular model for the rhodopsin paracrystal is presented validating our previously reported results. Finally, we compare our model with other currently available models describing the supramolecular structure of GPCRs in the membrane.  

3.497           Copatching and lipid raft association of different viral glycoproteins expressed on the surfaces of Pseudorabies virus-infected cells

Favoreel, H.W., Mettenleiter, T.C. and Nauwynck, H.J.

1. Virol., 78(10), 5279-5287 (2004)

  Pseudorabies virus (PRV) is a swine alphaherpesvirus that is closely related to human herpes simplex virus (HSV). Both PRV and HSV express a variety of viral envelope glycoproteins in the plasma membranes of infected cells. Here we show that at least four major PRV glycoproteins (gB, gC, gD, and gE) in the plasma membrane of infected swine kidney cells and monocytes seem to be linked, since monospecific antibody-induced patching of any one of these proteins results in copatching of the others. Further, for all four PRV glycoproteins, monospecific antibody-induced patches were enriched in GM1, a typical marker of lipid raft microdomains, but were excluded for transferrin receptor, a nonraft marker, suggesting that these viral proteins may associate with lipid rafts. However, only gB and, to a lesser extent, gE were found in lipid raft fractions by using detergent floatation assays, indicating that gC and gD do not show strong lipid raft association. Addition of methyl-ß-cyclodextrin (MCD), a cholesterol-depleting agent that is commonly used to disrupt lipid rafts, only slightly reduced copatching efficiency between the different viral proteins, indicating that other factors, perhaps tegument-glycoprotein interactions, may be important for the observed copatching events. On the other hand, MCD strongly reduced polarization of the antibody-induced viral glycoprotein patches to a cap structure, a gE-dependent process that has been described for specific PRV- and HSV-infected cells. Therefore, we hypothesize that efficient gE-mediated capping of antibody-antigen patches may require the lipid raft-associated signal transduction machinery.  

3.498           Endogenous SHIP2 does not localize in lipid rafts in 3T3-L1 adipocytes

Jacobs, C., Onnockx, S., Vandenbroere, I. and Pirson, I. FEBS Lett., 565, 70-74 (2004)   SH2 domain containing inositol polyphosphate 5-phosphatase (SHIP2) dephosphorylates phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) into phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P₂). SHIP2 knock-out mice demonstrated that SHIP2 acts as a negative regulator of insulin cascade in vivo. Our two-hybrid study showed that SHIP2 interacts with c-Cbl associated protein (CAP) and c-Cbl, implicated in the insulin signaling. As some proteins implicated in insulin signaling, like insulin receptor, CAP, c-Cbl or TC10, were reported to localize in lipid rafts, we addressed the same question for SHIP2. SHIP2 was detected in the non-raft fraction in CHO-IR, C2C12 myotubes and 3T3-L1 adipocytes except when it is overexpressed in CHO-IR, where we detected SHIP2 in the raft fraction.  

3.499           Uptake and transsport of high-density lipoprotein (HDL) and HDL-associated a-tocopherol by an in vitro blood-brain barrier model

Balazs, Z. et al

1. Neurochem., 89, 939-950 (2004)

  The present study aimed to investigate pathways that contribute to uptake and transcytosis of high-density lipoproteins (HDLs) and HDL-associated -tocopherol ( TocH) across an in vitro model of the blood-brain barrier (BBB). In primary porcine brain capillary endothelial cells HDL-associated TocH was taken up in 10-fold excess of HDL holoparticles, indicating efficient selective uptake, a pathway mediated by scavenger receptor class B, type I (SR-BI). SR-BI was present in caveolae of brain capillary endothelial cells and expressed almost exclusively at the apical membrane. Disruption of caveolae with methyl- -cyclodextrin (CDX) resulted in (mis)sorting of SR-BI to the basolateral membrane. Immunohistochemistry of porcine brain cryosections revealed SR-BI expression on brain capillary endothelial cells and presumably astrocytic endfeet. HDL-associated [¹⁴C] TocH taken up by brain capillary endothelial cells was recovered in sucrose gradient fractions containing the majority of cellular caveolin-1, the major caveolae-associated protein. During mass transfer studies using TocH-enriched HDL, approximately 50% of cellular TocH was recovered with the bulk of cellular caveolin-1 and SR-BI. Efflux experiments revealed that a substantial amount of cell-associated [¹⁴C] TocH could be mobilized into the culture medium. In addition, apical-to-basolateral transport of HDL holoparticles and HDL-associated TocH was saturable. Results from the present study suggest that part of cerebral apolipoprotein A-I and TocH originates from plasma HDL transcytosed across the BBB and that caveolae-located SR-BI facilitates selective uptake of HDL-associated TocH at the BBB.    

3.500           Caveolar and lipid raft localization of the growth hormone receptor ans its signaling elements

Yang, N., Huang, Y., Jiang, J. And Frank, S.J.

1. Biol. Chem., 279(20)

  The growth hormone receptor (GHR) is a cell surface receptor that mediates the somatogenic and metabolic effects of the growth hormone (GH). GHR signaling is transduced via the receptor-associated cytoplasmic tyrosine kinase called Janus protein kinase 2 (JAK2). The major intracellular signaling systems activated by JAK2 in response to GH include the signal transducer and activator of transcription (STAT) 5 and extracellular signal-regulated kinase (ERK)-1 and -2 pathways. In this report, we investigate the role of cholesterol-rich plasma membrane microdomains (caveolae and lipid rafts) in GH signaling. By subcellular fractionation of the GH-responsive 3T3-F442A murine preadipocyte, we found dramatic enrichment (6.7-fold) of plasma membrane GHR in the caveolae membranes (CM). JAK2 was also represented in the CM fraction, but was less enriched (2.5-fold) than GHR. ERK1/2 and the important ERK pathway upstream small adaptor protein, Grb2 (growth factor receptor-bound protein 2), were also enriched in caveolae (2.3- and 8.3-fold, respectively), but STAT5 was barely detected in the same fraction. Correspondingly, GH-induced tyrosine-phosphorylated GHR, JAK2, and ERK1/2 were highly represented in the CM fraction, whereas tyrosine-phosphorylated STAT5 was enriched in the non-membranous fraction of the post-nuclear supernatant. Additionally, GH induced further accumulation of GHR, Grb2, and SHC proteins in the CM fraction. Interestingly, treatment of the cells with the caveolae-disrupting agent, methyl- -cyclodextrin (m CD), selectively inhibited GH-induced ERK1/2 activation but not STAT5 phosphorylation; repletion of cholesterol in m CD-treated cells restored GH-induced ERK activation. Comparison of 3T3-F442A cells with the GHR-expressing human IM-9 lymphoblasts revealed similar enrichment of GHR in the lipid raft fraction of IM-9 as in the CM fraction of 3T3-F442A, but there were dramatic differences in the ERKs and Grb2. The IM-9 cell, in which ERKs are not activated by GH, displayed no enrichment of ERKs and Grb2 in the lipid raft fraction. Our results suggest that localization of GHRs in the CM fraction of the plasma membrane plays important roles in signaling.  

3.501           A role for myosin-1A in the localization of a brush border disaccharidase

Tyska, M.J. and Mooseker, M.S.

1. Cell Biol., 165(3), 395-405 (2004)

  To gain insight regarding myosin-1A (M1A) function, we expressed a dominant negative fragment of this motor in the intestinal epithelial cell line, CACO-2_BBE. Sucrase isomaltase (SI), a transmembrane disaccharidase found in microvillar lipid rafts, was missing from the brush border (BB) in cells expressing this fragment. Density gradient centrifugation, affinity purification, and immunopurification of detergent-resistant membranes isolated from CACO-2_BBE cells and rat microvilli (MV) all indicate that M1A and SI reside on the same population of low density ( 1.12 g/ml) membranes. Chemical cross-linking of detergent-resistant membranes from rat MV indicates that SI and M1A may interact in a lipid raft complex. The functional significance of such a complex is highlighted by expression of the cytoplasmic domain of SI, which results in lower levels of M1A and a loss of SI from the BB. Together, these studies are the first to assign a specific role to M1A and suggest that this motor is involved in the retention of SI within the BB.  

3.502           Svp1p defines a family of phosphatidylinositol 3,5-bisphosphate effectors

Dove, S.K. et al EMBO J., 23, 1922-1933 (2004)   Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂), made by Fab1p, is essential for vesicle recycling from vacuole/lysosomal compartments and for protein sorting into multivesicular bodies. To isolate PtdIns(3,5)P₂ effectors, we identified Saccharomyces cerevisiae mutants that display fab1 -like vacuole enlargement, one of which lacked the SVP1/YFR021w/ATG18 gene. Expressed Svp1p displays PtdIns(3,5)P₂ binding of exquisite specificity, GFP-Svp1p localises to the vacuole membrane in a Fab1p-dependent manner, and svp1 cells fail to recycle a marker protein from the vacuole to the Golgi. Cells lacking Svp1p accumulate abnormally large amounts of PtdIns(3,5)P₂. These observations identify Svp1p as a PtdIns(3,5)P₂ effector required for PtdIns(3,5)P₂-dependent membrane recycling from the vacuole. Other Svp1p-related proteins, including human and Drosophila homologues, bind PtdIns(3,5)P₂ similarly. Svp1p and related proteins almost certainly fold as ß-propellers, and the PtdIns(3,5)P₂-binding site is on the ß-propeller. It is likely that many of the Svp1p-related proteins that are ubiquitous throughout the eukaryotes are PtdIns(3,5)P₂ effectors. Svp1p is not involved in the contributions of FAB1/PtdIns(3,5)P₂ to MVB sorting or to vacuole acidification and so additional PtdIns(3,5)P₂ effectors must exist.  

3.503           Differential regulation of cytosolic and peroximal bile acid amidation by PPARa activation favors the formation of unconjugated bile acids

Solaas, K. et al

1. Lipid. Res.,45, 1051-1060 (2004)

  In human liver, unconjugated bile acids can be formed by the action of bile acid-CoA thioesterases (BACTEs), whereas bile acid conjugation with taurine or glycine (amidation) is catalyzed by bile acid-CoA:amino acid N-acyltransferases (BACATs). Bothpathways exist in peroxisomes and cytosol. Bile acid amidationfacilitates biliary excretion, whereas the accumulation of unconjugatedbile acids may become hepatotoxic. We hypothesized that theformation of unconjugated and conjugated bile acids from theircommon substrate bile acid-CoA thioesters by BACTE and BACATis regulated via the peroxisome proliferator-activated receptor(PPAR). Livers from wild-type and PPAR-null mice either untreatedor treated with the PPAR activator WY-14,643 were analyzed forBACTE and BACAT expression. The total liver capacity of taurochenodeoxycholateand taurocholate formation was decreased in WY-14,643-treatedwild-type mice by 60% and 40%, respectively, but not in PPAR-nullmice. Suppression of the peroxisomal BACAT activity was responsiblefor the decrease in liver capacity, whereas cytosolic BACATactivity was essentially unchanged by the treatment. In bothcytosol and peroxisomes, the BACTE activities and protein levelswere upregulated 5- to 10-fold by the treatment. These effectscaused by WY-14,643 treatment were abolished in PPAR-null mice. The results from this study suggest that an increased formationof unconjugated bile acids occurs during PPAR activation.  

3.504           Passive and active inclusion of host proteins in human immunodeficiency virus type 1 Gag particles during budding at the plasma membrane

Hammerstedt, M. and Garoff, H.

1. Virol., 78(11), 5686-5697 (2004)

  Human immunodeficiency virus type 1 particles form by budding at the surface of most cell types. In this process, a piece of the plasma membrane is modified into an enveloped virus particle. The process is driven by the internal viral protein Pr55^gag. We have studied how host proteins in the membrane are dealt with by Pr55^gag during budding. Are they included in or excluded from the particle? The question was approached by measuring the relative concentrations of host and viral proteins in the envelope of Pr55^gag particles and in their donor membranes in the cell. We observed that the bulk of the host proteins, including actin and clathrin, were passively included into the virus-like Gag particles. This result suggests that budding by Pr55^gag proceeds without significant alteration of the original host protein composition at the cell membrane. Nevertheless, some proteins were concentrated in the particles, and a few were excluded. The concentrated proteins included cyclophilin A and Tsg-101. These were recruited to the plasma membrane by Pr55^gag. The membrane-bound cyclophilin A was concentrated into particles as efficiently as Pr55^gag, whereas Tsg-101 was concentrated more efficiently. The latter finding is consistent with a role for Tsg-101 in Gag particle release.  

3.505           Functions of pancreatic b cells and adipocytes in bombesin receptor subtype-3-deficient mice

Nakamichi, Y. et al Biochem. Biophys. Res. Comm., 318, 698-703 (2004)   We previously reported that mice lacking bombesin receptor subtype-3 (BRS-3) exhibit mild late-onset obesity and glucose intolerance [Nature 390 (1997) 160]. To examine the mechanism by which glucose intolerance is developed in these mice, we studied insulin release and proinsulin biosynthesis in isolated pancreatic islets and glucose uptake and facilitative glucose transporter (GLUT)-4 translocation in adipose tissues. Although islet insulin contents and the size and number of islets of Langerhans in BRS-3-deficient mice decreased, there was no difference in glucose-stimulated insulin release and proinsulin biosynthesis between BRS-3-deficient and wild-type control mice. In contrast, adipose tissues exhibited a marked difference: the uptake of [¹⁴C]2-deoxy- -glucose by adipocytes isolated from BRS-3-deficient mice was not stimulated by 10⁻⁷ M insulin addition, and membrane fractionation analysis showed that GLUT4 was barely detected in the fraction of plasma membrane in BRS-3-deficient mice in the presence of 10⁻⁷ M insulin. Quantitative reverse transcription-PCR (RT-PCR) showed that mRNA levels of GLUT4, insulin receptor, insulin receptor substrate (IRS)-1 and IRS-2, syntaxin 4, SNAP23, and VAMP-2 in adipose tissues of BRS-3-deficient mice were unchanged compared with those in wild-type control mice. We concluded that impaired glucose metabolism observed in BRS-3-deficient mice was mainly caused by impaired GLUT4 translocation in adipocytes.  

3.506           Analysis of lipid rafts in T cell

Thomas, S., Preda-Pais, A., Casares, S. and Brumeanu, T-D. Mol. Immunol., 41, 399-409 (2004)   The plasma membrane of T cells is made of a combination of glycosphingolipids and protein receptors organized in glycolipoprotein microdomains termed lipid rafts. The structural assembly of lipid rafts was investigated by various physical and biochemical assays. Depending on the differentiation status of T cells, the lipid rafts seclude various protein receptors involved in T cell signaling, cytoskeleton reorganization, membrane trafficking, and the entry of infectious organisms into the cells. This review article summarizes the most common methods, and their limits and advantages for analyzing the composition and assembly of lipid rafts with protein receptors into lipid rafts microdomains in plasma membrane of T cells. It also includes new methods such as ELISA/Polysorp and flow cytometry, and a combined sucrose gradient centrifugation–FPLC–Western blot strategy developed in our laboratory to study non-covalent interactions between the GM1 glycosphingolipid and protein receptors in plasma membrane of T cells.  

3.507           Functional domains in presenilin. The TYR-288 residue controls g-secretase activity and endoprotoeolysis

Laudon, H. et al

1. Biol. Chem., 279(23), 23925-23932 (2004)

  Processing of the Alzheimer amyloid precursor protein (APP) into the amyloid -protein and the APP intracellular domain is a proteolysis event mediated by the -secretase complex where presenilin (PS) proteins are key constituents. PS is subjected to an endoproteolytic cleavage, generating a stable heterodimer composed of an N-terminal and a C-terminal fragment. Here we aimed at further understanding the role of PS in endoproteolysis, in proteolytic processing of APP and Notch, and in assembly of the -secretase complex. By using a truncation protocol and alanine scanning, we identified Tyr-288 in the PS1 N-terminal fragment as critical for PS-dependent intramembrane proteolysis. Further mutagenesis of the 288 site identified mutants differentially affecting endoproteolysis and -secretase activity. The Y288F mutant was endoproteolyzed to the same extent as wild type PS but increased the amyloid -protein 42/40 ratio by 75%. In contrast, the Y288N mutant was also endoproteolytically processed but was inactive in reconstituting -secretase in PS null cells. The Y288D mutant was deficient in both endoproteolysis and -secretase activity. All three mutant PS1 molecules were incorporated into -secretase complexes and stabilized Pen-2 in PS null cells. Thus, mutations at Tyr-288 do not affect -secretase complex assembly but can differentially control endoproteolysis and -secretase activity.  

3.508           The T cell receptor g chain alternate reading frame protein (TARP), a prostate-specific protein localized in mitochondria

Maeda, H. et al

1. Biol. Chem., 279(23), 24561-24568 (2004)

  We previously showed that mRNA encoding TARP (T cell receptor chain alternate reading frame protein) is exclusively expressed in the prostate in males and is up-regulated by androgen in LNCaP cells, an androgen-sensitive prostate cancer cell line. We have now developed an anti-TARP monoclonal antibody named TP1, and show that TARP protein is up-regulated by androgen in both LNCaP and MDA-PCa-2b cells. We used TP1 to determine the subcellular localization of TARP by Western blotting following subcellular fractionation and immunocytochemistry. Both methods showed that TARP is localized in the mitochondria of LNCaP cells, MDA-PCa-2b cells, and PC-3 cells transfected with a TARP-expressing plasmid. We also transfected a plasmid encoding TARP fused to green fluorescent protein into LNCaP, MDA-Pca-2b, and PC-3 cells and confirmed its specific mitochondrial localization in living cells. Fractionation of mitochondria shows that TARP is located in the outer mitochondrial membrane. Immunohistochemistry using a human prostate cancer sample showed that TP1 reacted in a dot-like cytoplasmic pattern consistent with the presence of TARP in mitochondria. These data demonstrate that TARP is the first prostate-specific protein localizing in mitochondria and indicate that TARP, an androgen-regulated protein, may act on mitochondria to carry out its biological functions.  

3.509           Lipid raft polarization contributes to hyphal growth in Candida albicans

Martin, S.W. and Konopka, J.B. Eukaryotic cell, 3(3), 675-684 (2004)   The polarization of sterol- and sphingolipid-enriched domains (lipid rafts) has been linked to morphogenesis and cell movement in diverse cell types. In the yeast Saccharomyces cerevisiae, a dramatic polarization of sterol-rich domains to the shmoo tip was observed in pheromone-induced cells (M. Bagnat and K. Simons, Proc. Natl. Acad. Sci. USA 99:14183-14188, 2002). We therefore examined whether plasma membrane lipid polarization contributes to the ability of the fungal pathogen Candida albicans to grow in a highly polarized manner to form hyphae. Interestingly, staining with filipin revealed that membrane sterols were highly polarized to the leading edge of growth during all stages of hyphal growth. Budding and pseudohyphal cells did not display polarized staining. Filipin staining was also enriched at septation sites in hyphae, where colocalization with septin proteins was observed, suggesting a role for the septins in forming a boundary domain. Actin appeared to play a role in sterol polarization and hyphal morphogenesis in that both were disrupted by low concentrations of latrunculin A that did not prevent budding. Furthermore, blocking either sphingolipid biosynthesis with myriocin or sterol biosynthesis with ketoconazole resulted in a loss of ergosterol polarization and caused abnormal hyphal morphogenesis, suggesting that lipid rafts are involved. Since hyphal growth is required for the full virulence of C. albicans, these results suggest that membrane polarization may contribute to the pathogenesis of this organism.  

3.510           CD44 interaction with Na⁺-H⁺ exchanger (NHE1) creates acidic microenvironment leading to hyaluronidase-2 and cathepsin B activation and breast tumor cell invasion

Bourguignon, L.Y.W., Singleton, P.A., Diedrich, F., Stern, R. and Gilad, E.

1. Biol. Chem., 279(26), 26691-27007 (2004)

  We have explored CD44 (a hyaluronan (HA) receptor) interaction with a Na⁺-H⁺ exchanger (NHE1) and hyaluronidase-2 (Hyal-2) during HA-induced cellular signaling in human breast tumor cells (MDA-MB-231 cell line). Immunological analyses demonstrate that CD44s (standard form) and two signaling molecules (NHE1 and Hyal-2) are closely associated in a complex in MDA-MB-231 cells. These three proteins are also significantly enriched in cholesterol and ganglioside-containing lipid rafts, characterized as caveolin and flotillin-rich plasma membrane microdomains. The binding of HA to CD44 activates Na⁺-H⁺ exchange activity which, in turn, promotes intracellular acidification and creates an acidic extracellular matrix environment. This leads to Hyal-2-mediated HA catabolism, HA modification, and cysteine proteinase (cathepsin B) activation resulting in breast tumor cell invasion. In addition, we have observed the following: (i) HA/CD44-activated Rho kinase (ROK) mediates NHE1 phosphorylation and activity, and (ii) inhibition of ROK or NHE1 activity (by treating cells with a ROK inhibitor, Y27632, or NHE1 blocker, S-(N-ethyl-N-isopropyl) amiloride, respectively) blocks NHE1 phosphorylation/Na⁺-H⁺ exchange activity, reduces intracellular acidification, eliminates the acidic environment in the extracellular matrix, and suppresses breast tumor-specific behaviors (e.g. Hyal-2-mediated HA modification, cathepsin B activation, and tumor cell invasion). Finally, down-regulation of CD44 or Hyal-2 expression (by treating cells with CD44 or Hyal-2-specific small interfering RNAs) not only inhibits HA-mediated CD44 signaling (e.g. ROK-mediated Na⁺-H⁺ exchanger reaction and cellular pH changes) but also impairs oncogenic events (e.g. Hyal-2 activity, hyaluronan modification, cathepsin B activation, and tumor cell invasion). Taken together, our results suggest that CD44 interaction with a ROK-activated NHE1 (a Na⁺-H⁺ exchanger) in cholesterol/ganglioside-containing lipid rafts plays a pivotal role in promoting intracellular/extracellular acidification required for Hyal-2 and cysteine proteinase-mediated matrix degradation and breast cancer progression.  

3.511           Carboxyl ester lipase cofractionates with scavenger receptor BI in hepatocyte lipid rafts and enhances selective uptake and hydrolysis of cholesteryl esters from HDL₃

Camarota, L.M., Chapman, J.M., Hui, D.Y. and Howles, P.N.

1. Biol. Chem., 279(26), 27599-27606 (2004)

  Cholesteryl esters are selectively removed from high density lipoproteins by hepatocytes and steroidogenic cells through a process mediated by scavenger receptor BI. In the liver this cholesterol is secreted into bile, primarily as free cholesterol. Previous work showed that carboxyl ester lipase enhanced selective uptake of cholesteryl ether from high density lipoprotein by an unknown mechanism. Experiments were performed to determine whether carboxyl ester lipase plays a role in scavenger receptor BI-mediated selective uptake. When added to cultures of HepG2 cells, carboxyl ester lipase cofractionated with scavenger receptor BI and [³H]cholesteryl ether-labeled high density lipoprotein in lipid raft fractions of cell homogenates. Confocal microscopy of immunostained carboxyl ester lipase and scavenger receptor BI showed a close association of these proteins in HepG2 cells. The enzyme and receptor also cofractionated from homogenates of mouse liver using two different fractionation methods. Antibodies that block scavenger receptor BI function prevented carboxyl ester lipase stimulation of selective uptake in primary hepatocytes from carboxyl ester lipase knockout mice. Heparin blockage of cell-surface proteoglycans also prevented carboxyl ester lipase stimulation of cholesteryl ester uptake by HepG2 cells. Inhibition of carboxyl ester lipase activity in HepG2 cells reduced hydrolysis of high density lipoprotein-cholesteryl esters 40%. In vivo, hydrolysis was similarly reduced in lipid rafts from the livers of carboxyl ester lipase-null mice compared with control animals. Primary hepatocytes from these mice yielded similar results. The data suggest that carboxyl ester lipase plays a physiological role in hepatic selective uptake and metabolism of high density lipoprotein cholesteryl esters by direct and indirect interactions with the scavenger receptor BI pathway.  

3.512           PSPN/GFRa4 has a significantly weaker capacity thab GDNF/GFRa1 to recruit RET to rafts, but promotes neuronal survival and neurite outgrowth

Yang, J. et al FEBS Lett., 569, 267-271 (2004)   Previously, it was shown that the recruitment of RET into lipid rafts by glial cell line-derived neurotrophic factor (GDNF)/GFR 1 is crucial for efficient signal transduction. Here, we show that the mouse GFR 4 is a functional, N-glycosylated, glycosylphosphatidylinositol (GPI)-anchored protein, which mediates persephin (PSPN)-induced phosphorylation of RET, but has an almost undetectable capacity to recruit RET into the 0.1% Triton X-100 insoluble membrane fraction. In spite of this, PSPN/mGFR 4 promotes neurite outgrowth in PC6-3 cells and survival of cerebellar granule neurons. As we show that also human PSPN/GFR 4 is unable to recruit RET into lipid rafts, we propose that the mammalian GFR 4 in this respect differs from GFR 1.  

3.513           Sphingolipid C4 hydroxylation influences properties of yeast detergent-insoluble glycolipid-enriched membranes

Idkowiak-Baldys, J., Grilley, M.M. and Takemoto, J.Y. FEBS Lett., 569, 272-276 (2004)   Sphingoid base C4 hydroxylation is required for syringomycin E action on the yeast plasma membrane. Detergent-insoluble glycolipid-enriched membranes (DIGs) from a yeast strain lacking C4 hydroxylated sphingoid bases (sur2Δ) are composed of linear membrane fragments instead of vesicular structures observed for wild-type DIGs, though they have similar lipid compositions and amounts of DIG marker proteins. Light-scattering bands collected from sur2Δ after centrifugation of Triton X-100-treated cell lysates in continuous density gradients have lower buoyant densities than that of the wild-type. The results show that C4 hydroxylation influences the physical and structural properties of DIGs and suggest that syringomycin E interacts with lipid rafts.  

3.514           Membrane order conservation in raft and non-raft regions of hepatocyte plasma membranes from thermally acclimated rainbow trout

Zehmer, J.K. and Hazel, J.R. Biochim, Biophys. Acta., 1664, 108-116 (2004)   Homeoviscous adaptation (HVA), the thermal conservation of membrane fluidity/order at different body temperatures, has been observed to varying degrees in different membranes. However, HVA has not been studied in raft and non-raft regions of the plasma membrane (PM) separately. Rafts are ordered PM microdomains implicated in signal transduction, membrane traffic and cholesterol homeostasis. Using infrared spectroscopy, we measured order in raft-enriched PM (raft) and raft-depleted PM (RDPM) isolated from hepatocytes of rainbow trout (Oncorhynchus mykiss) acclimated to 5 and 20 °C. We found approximately 130% and 90% order compensation in raft and RDPM, respectively, suggesting their independent regulation. Raft was more ordered than RDPM in the warm-acclimated trout, a difference fully explained by a 58% enrichment of cholesterol, compared to RPDM. Unexpectedly, raft and RDPM from cold-acclimated trout did not differ in cholesterol content or order. Freezing the membrane samples during preparation had no effect on order. Treatment with cyclodextrin depleted cholesterol by 36%, 56%, and 55%, producing significant decreases in order in raft and RDPM from warm-acclimated trout and RDPM from cold-acclimated trout, respectively. However, a 69% depletion of cholesterol from raft from cold-acclimated trout had no significant effect on order. This result, and the lack of a difference in order between raft and RDPM, suggests that raft and non-raft PM in cold-acclimated trout are not spatially segregated by phase separation due to cholesterol.  

3.515           Cellular distribution of lysyl-tRNA synthetase and its interaction with Gag during human immunodeficiency virus type 2 assembly

Halwani, R. et al

1. Virol., 78(14), 7553-7564 (2004)

  Lysyl-tRNA synthetase (LysRS) is packaged into human immunodeficiency virus type 1 (HIV-1) via its interaction with Gag, and this enzyme facilitates the selective packaging of tRNA₃^Lys, the primer for initiating reverse transcription, into HIV-1. The Gag/LysRS interaction is detected at detergent-resistant membrane but not in membrane-free cell compartments that contain Gag and LysRS. LysRS is found (i) in the nucleus, (ii) in a cytoplasmic high-molecular-weight aminoacyl-tRNA synthetase complex (HMW aaRS complex), (iii) in mitochondria, and (iv) associated with plasma membrane. The cytoplasmic form of LysRS lacking the mitochondrial import signal was previously shown to be efficiently packaged into virions, and in this report we also show that LysRS compartments in nuclei, in the HMW aaRS complex, and at the membrane are also not required as a primary source for viral LysRS. Exogenous mutant LysRS species unable to either enter the nucleus or bind to the cell membrane are still incorporated into virions. Many HMW aaRS components are not packaged into the virion along with LysRS, and the interaction of LysRS with p38, a protein that binds tightly to LysRS in the HMW aaRS complex, is not required for the incorporation of LysRS into virions. These data indicate that newly synthesized LysRS may interact rapidly with Gag before the enzyme has the opportunity to move to the above-mentioned cellular compartments. In confirmation of this idea, we found that newly synthesized LysRS is associated with Gag after a 10-min pulse with [³⁵S]cysteine/methionine. This observationis also supported by previous work indicating that the incorporationof LysRS into HIV-1 is very sensitive to the inhibition of newsynthesis of LysRS.  

3.516           Microtubules regulate angiotensin II type 1 receptor and Rac1 localization in caveolae/lipid rafts

Zuo, L., Ushio-Fukai, M., Hilenski, L.L. and Alexander, R.W. Arterioscler. Thromb. Vasc. Biol., 24, 1223-1228 (2004)    Objective— Microtubules are important in signal transduction temporal–spatial organization. Full expression of angiotensin II (Ang II) signaling in vascular smooth muscle cells (VSMCs) is dependent on the reactive oxygen species (ROS) derived from nicotinamide-adenine dinucleotide phosphate (NAD(P)H) oxidase and the dynamic association of the Ang II type 1 receptor (AT₁R)with caveolae/lipid rafts. Translocation of the small GTPaseRac1 to the plasma membrane is an essential step for activationof NAD(P)H oxidase; however, its precise localization in theplasma membrane after agonist stimulation and how it is targetedare unknown. We hypothesized that microtubules are involvedin regulating multiphasic Ang II signaling events in VSMC. Methods and Results— We show that Ang II promotes Rac1 and AT₁R trafficking into caveolae/lipid rafts, which is blocked by disruption of microtubules with nocodazole. As a consequence, nocodazole significantly inhibits Ang II–stimulated H₂O₂production, its downstream ROS-dependent epidermal growth factorreceptor transactivation, Akt phosphorylation, and vascularhypertrophy without affecting Rac1 activation or ROS-independentextracellular signal-regulated kinase 1/2 phosphorylation. Conclusions— These results suggest that proper Rac1 and AT₁R trafficking into caveolae/lipid rafts requires the integrity of microtubules and provide insight into the essential role of microtubules for the spatial–temporal organization of ROS-dependent and caveolae/lipid rafts–dependent AT₁Rsignaling linked to vascular hypertrophy. The role of microtubules in angiotensin II (Ang II) signaling remains unknown. We demonstrate that Ang II promotes Rac1 and Ang II type 1 receptor trafficking into the caveolae/lipid rafts, which requires the integrity of microtubules. We also found that intact microtubules mediate Ang II–stimulated H₂O₂production, its downstream EGF-R transactivation, Akt phosphorylation,and vascular hypertrophy.  

3.517           Stoichiometry of the T-cell receptor-CD3 complex and key intermediates assembled in the endoplasmic reticulum

Call, M.E., Pyrdol, J. and Wucherpfennig, K.W. EMBO J., 22, 2348-2357 (2004)   The T-cell receptor (TCR)–CD3 complex is critical forT-cell development and function, and represents one of the mostcomplex transmembrane receptors. Models of different stoichiometryand valency have been proposed based on cellular experimentsand these have important implications for the mechanisms ofreceptor triggering. Since determination of receptor stoichiometryin T-cells is not possible due to the presence of previouslysynthesized, unlabeled receptor components with different half-lives,we examined the stoichiometry of the receptor assembled in endoplasmicreticulum (ER) microsomes of B-cell origin. The stoichiometricrelationship among all subunits was directly determined usingintact radiolabeled TCR–CD3 complexes that were isolatedwith a sequential, non-denaturing immunoprecipitation method,and identical results were obtained with two detergents belongingto different structural classes. The results firmly establishthat the ß TCR–CD3 complex assembled in theER is monovalent and composed of one copy of the TCRß,CD3, CD3 and – dimers.  

3.518           PKD1/PKCm promotes avb3 integrin recycling and delivery to nascent focal adhesions

Woods, A.J., White, D.P., Caswell, P.T. and Norman, J.C. EMBO J., 23, 2531-2543 (2004)   To identify kinases that regulate integrin recycling, we have immunoprecipitated vß3 integrin from NIH 3T3 fibroblasts in the presence and absence of primaquine (a drug that inhibits receptor recycling and leads to accumulation of integrins in endosomes) and screened for co-precipitating kinases. Primaquine strongly promoted association of vß3 integrin with PKD1, and fluorescence microscopy indicated that integrin and PKD1 associate at a vesicular compartment that is downstream of a Rab4-dependent transport step. PKD1 association was mediated by the C-terminal region of the ß3 integrin cytodomain, and mutants of ß3 that were unable to recruit PKD1 did not recycle in a PDGF-dependent fashion. Furthermore, suppression of endogenous PKD1 levels by RNAi, or overexpression of catalytically inactive PKD1 inhibited PDGF-dependent recycling of vß3 from early endosomes to the plasma membrane and blocked recruitment of vß3 to newly formed focal adhesions during cell spreading. These data indicate that PKD1 influences cell migration by directing vesicular transport of the vß3 integrin heterodimer.  

3.519           Human Doppel and prion protein share common membrane microdomains and internalization pathways

Massimino, M.L. et al Int. J. Biochem. and  Cell Biol., 36, 2016-2031 (2004)   Doppel is the first identified homologue of the prion protein (PrP^c) implicated in prion disease. Doppel is considered an N-truncated form of PrP^c, and shares with PrP^c several structural and biochemical features. When over expressed in the brain of some PrP knockout animals, it provokes cerebellar ataxia. As this phenotype is rescued by reintroducing the PrP gene, it has been suggested that Doppel and PrP^c have antagonistic functions and may compete for a common ligand. However, a direct interaction between the two proteins has recently been observed. To investigate whether the neuronal environment is suitable for such possibility, human Doppel and PrP^c were expressed separately, or together, in neuroblastoma cells, and then studied by biochemical and immunomicroscopic tools, as well as in intact cells expressing fluorescent fusion constructs. The results demonstrate that Doppel and PrP^c co-patch extensively at the plasma membrane, and get internalized together after ganglioside cross-linking by cholera toxin or addition of an antibody against only one of the proteins. These processes no longer occur if the integrity of rafts is disrupted. We also show that, whereas each protein expressed alone occupies Triton X-100-insoluble membrane microdomains, co-transfected Doppel and PrP^c redistribute together into a less ordered lipidic environment. All these features are consistent with interactions occurring between Doppel and PrP^c in our neuronal cell model.  

3.520           Synaptotagmins are trafficked to distinct subcellular domains including the postsynaptic compartment

Adolfsen, B., Sarawati, S., Yoshihara, M. and Littleton, J.T.

1. Cell Biol., 166(2), 249-260 (2004)

  The synaptotagmin family has been implicated in calcium-dependent neurotransmitter release, although Synaptotagmin 1 is the only isoform demonstrated to control synaptic vesicle fusion. Here, we report the characterization of the six remaining synaptotagmin isoforms encoded in the Drosophila genome, including homologues of mammalian Synaptotagmins 4, 7, 12, and 14. Like Synaptotagmin 1, Synaptotagmin 4 is ubiquitously present at synapses, but localizes to the postsynaptic compartment. The remaining isoforms were not found at synapses (Synaptotagmin 7), expressed at very low levels (Synaptotagmins 12 and 14), or in subsets of putative neurosecretory cells (Synaptotagmins and ß). Consistent with their distinct localizations, overexpression of Synaptotagmin 4 or 7 cannot functionally substitute for the loss of Synaptotagmin 1 in synaptic transmission. Our results indicate that synaptotagmins are differentially distributed to unique subcellular compartments. In addition, the identification of a postsynaptic synaptotagmin suggests calcium-dependent membrane-trafficking functions on both sides of the synapse.  

3.521           Drosophila Wnt-1 undergoes a hydrophobic modification and is targeted to lipid rafts, a process that requires porcupine

Zhai, L., Chaturvedi, D. and Cumberledge, S.

1. Biol. Chem., 279(32), 33220-33227 (2004)

  Wnt signaling pathways regulate many developmental responses; however, little is known about how Wnt ligands function on a biochemical level. Recent studies have shown that Wnt-3a is palmitoylated before secretion. Here we report that Drosophila Wnt-1 (Wingless) also undergoes a lipid modification. Lipidation occurs in the endoplasmic reticulum and is dependent on Porcupine, a putative O-acyltransferase. After modification, DWnt-1 partitions as a membrane-anchored protein and is sorted into lipid raft detergent-insoluble microdomains. Lipidation, raft targeting, and secretion can be blocked by the addition of 2-bromopalmitate, a competitive inhibitor of O-acyltransferase activity. Based on these results we propose a model whereby lipidation targets Wnt-1 to secretory vesicles that deliver the ligand to specialized microdomains at the cell surface where it can be packaged for secretion.    

3.522           Oligomerization of the sensory and motor neuron-derived factor prevents protein O-glycosylation

Cabedo, H., Carteron, C. and Ferrer-Montiel, A.

1. Biol. Chem., 279(32), 33623-33629 (2004)

  The sensory and motor neuron-derived factor (SMDF) is a neuregulin that promotes Schwann cell proliferation and differentiation. Hence, understanding axon myelination is important to unveil the mechanisms involved in SMDF biogenesis, membrane delivery, and compartmentalization. SMDF is a type II membrane protein expressed as two distinct polypeptides of 40 and 83 kDa. Whether the 83-kDa polypeptide results from posttranslational modifications of the protein monomers or protein dimerization remains unknown. Here we have addressed this question and shown that the 83-kDa polypeptide is an O-glycosylated form of the protein. Deletion of the N-terminal domain fully abrogates the SMDF O-glycosylation, indicating that incorporation of O-glycans occurs in the intracellular domain of the protein. Notably, O-glycosylated forms are excluded from partitioning into lipid raft microdomains. In addition, we found that heterologously expressed SMDF monomers interact in intact living cells as evidenced from fluorescence resonance energy transfer of cyan fluorescent protein/yellow fluorescent protein·SMDF fusion proteins. A stepwise deletion approach demonstrated that SMDF self-association is primarily determined by its transmembrane segment. Notably, biochemical analysis revealed that SMDF multimers are exclusively composed of the 40-kDa polypeptide. Collectively, these findings indicate that the 40-kDa form corresponds to unmodified SMDF, which may be present as multimers, whereas the 83-kDa polypeptide is a monomeric O-glycosylated form of the protein. Furthermore, our observations imply a role for oligomerization as a potential modulator of the distribution in membrane domains and O-glycosylation of the protein.  

3.523           Differential signaling pathways are activated in the Epstein-Barr virus-activated malignancies nasopharyngeal carcinoma and Hodgkin lymphoma

Morrison, J.A., Gulley, M.L., Pathmanathan, R. and Raab-Traub, N. Cancer. Res.,64, 5251-5260 (2004)   EBV is associated with the epithelial cancer, nasopharyngeal carcinoma (NPC), and the lymphoid malignancy, Hodgkin lymphoma (HL). The EBV latent membrane proteins 1 and 2A are expressed in these tumors. These proteins activate the phosphatidylinositol 3'-OH kinase (PI3K)/Akt pathway, which is commonly activated inappropriately in malignancy. In this study, the status of Akt activation and its targets, glycogen synthase kinase-3ß (GSK-3ß) and ß-catenin, was investigated in NPC and HL clinical specimens. In the majority of HL and NPC specimens, Akt was activated, indicating an important role for this kinase in the development and/or progression of these tumors. Akt phosphorylates and inactivates GSK-3ß, a negative regulator of the proto-oncoprotein ß-catenin that is aberrantly activated in many cancers. GSK-3ß was phosphorylated and inactivated with concomitant nuclear ß-catenin accumulation in the majority of NPC specimens. The malignant cells of the majority of HL cases, however, did not have inactivated GSK-3ß and lacked nuclear ß-catenin expression. These data indicate that this signaling arm of PI3K/Akt is universal and important in NPC pathogenesis but is apparently not affected in HL. These findings point to a divergence in pathways activated by EBV in different cellular contexts.  

3.524           Comparisons of physical separation methods of Kunjin virus-induced membranes

Kim, M., Mackenzie, J.M. and Westaway, E.G.

1. Virol. Meth., 120, 179-187 (2004)

  two sets of connected membranes induced in Kunjin virus-infected cells are characterized by the presence of NS3 helicase/protease in both, and by RNA-dependent RNA polymerase (RdRp) activity plus the associated double-stranded RNA (dsRNA) template in vesicle packets (VP), or by the absence of both the VP-specific markers in the convoluted membranes/paracrystalline arrays (CM/PC). Attempts were made to separate flavivirus-induced membranes by sedimentation or flotation analyses in density gradients of sucrose or iodixanol, respectively, after treatment of cell lysates by sonication, osmotic shock, or tryptic digestion. Only osmotic shock treatment provided suggestive evidence of separation. This was explored by flow cytometry analysis (FCA) of RdRp active membrane fractions from a sucrose gradient, using dual fluorescent labelling via antibodies to NS3 and dsRNA. FCA revealed the presence of a dual labelled membrane population indicative of VP, and in a faster sedimenting fraction a membrane population able to be labelled only in NS3, representative of CM/PC and associated (R)ER. It was postulated that osmotic shock ruptured the bounding membrane of the VP, releasing the enclosed small vesicles associated with the Kunjin virus replication complex characterized previously. Notably, the presence of the full spectrum of nonstructural proteins in some membrane fractions was not a reliable marker for RdRp activity. These experiments may provide the opportunity for isolation of relatively pure flavivirus replication complexes in their native membrane-associated state by fluorescence-activated cell sorting.  

3.525           Membrane dynamics in giant unilamminar vesicles (GUVS) of raft and non-raft fractions of brushborders membranes

Cheng, M. et al Biophysical Meeting 2004 abstract   Lipid rafts are discrete regions in the plasma membrane which are composed of lipids that exist in the liquid ordered state, and are believed to function as platforms for protein and lipid transport. These structures are reported to be present in numerous membrane systems, including brushborder membranes in kidney cells. This research aims to study the membrane dynamics in brushborder membranes and membrane fractions of rat renal proximal tubular cells. Fluorescence (Laurdan) provided direct visualization of GUVs formed through electroformation of raft and non-raft fractions of intact renal brushborder membranes. Flotation through a density gradient (Optiprep) separated the raft and non-raft fractions without the use of a detergent. Two-photon scanning microscopy of the GUVs formed from the raft fraction showed uniform fluorescence intensity images with some non-fluorescent domains of a few microns in size. Previous GUV studies of raft fractions obtained through detergent extraction yielded vesicles devoid of domains. Membrane fluidity measured as Laurdan Generalized Polarization (GP) function was monitored across a physiological temperature range (25°-42°C) in the GUVs. The GP in the raft fraction indicated a less fluid phase than in the non-raft fraction. This result is consistent with the compositional differences in these membrane fractions, particularly in terms of sphingomyelin and cholesterol content. Scanning fluctuation correlation spectroscopy performed on the intact brushborder membrane revealed the diffusion rate of NaPi-II cotransporter, a brushborder membrane specific protein, to be consistent with the mobility of a membrane associated protein.  

3.526           Phagosome microdomains define foci of specialized functions in innate immunity

Goyette, G. et al ICI/FOCIS 2004 meeting abstract no. 2246 (2004)   Macrophages are cells of the immune system specialized in the destruction of invading pathogens, and the elaboration of an efficient immune response. Phagocytosis, the process by which pathogens are internalized by host cells, leads to the formation of phagosomes at the cell surface by the direct recruitment of the endoplasmic reticulum. Phagosomes engage in a complex maturation process leading to the formation of phagolysosomes, allowing the killing and degradation of microorganisms. Unfortunately, several microorganisms have evolved strategies to alter phagolysosome biogenesis, a process still poorly understood. Proteomics analyses revealed that phagosomes are made of hundreds of proteins, highlighting the complexity of the molecular mechanisms involved in phagosome functions. We have shown recently that the phagosome membrane is not homogeneous but rather made of microdomains. The functions of these cholesterol-enriched microdomains on phagosomes are unknown. To understand these functions, we initiated the systematic characterization of these domains using a proteomics approach. Phagosome microdomains were isolated based on their insolubility in Triton X-100 and their flotation on Optiprep™ step gradients. MS/MS analyses of these microdomains led to the identification of 264 proteins indicating that functions such as acidification, cholesterol metabolism, signal transduction, and membrane fusion are likely to take place on phagosome membrane microdomains. Results also indicate that the intracellular pathogen Leishmania donovani survives in macrophages by using a surface glycolipid to disrupt the proper organization of phagosome lipid rafts, highlighting the potential involvement of these structures in our ability to fight infection.  

3.527           Linking receptor-mediated endocytosis and cell signaling

Zou, Z. et al

1. Biol. Chem., 279(33), 34302-34310 (2004)

  Megalin, a member of the low density lipoprotein receptor gene family, is required for efficient protein absorption in the proximal tubule. Recent studies have shown that the low density lipoprotein receptor-related protein, another member of this gene family, is proteolytically processed by -secretase implying a role for low density lipoprotein receptor-related protein in a Notchlike signaling pathway. This pathway has been shown to involve: 1) metalloprotease-mediated ectodomain shedding and -secretase-mediated intramembrane proteolysis of some receptors. Experiments were performed to determine whether megalin undergoes similar processing. By immunocytochemistry, immunoblotting, and a fluorogenic enzyme assay presenilin-1 (required for -secretase activity) and -secretase activity were found in the brush border of proximal kidney tubules where megalin is localized. Using a fluorogenic peptide containing an amyloid precursor protein -secretase cleavage site and Compound E, a specific -secretase inhibitor, we found high levels of -secretase activity in renal brush border membrane vesicles. Immunoblotting analysis of renal microsomes and opossum kidney proximal tubule (OKP) cells using antibodies directed to the cytosolic domain of megalin showed a 35–40-kDa, membrane-associated, carboxyl-terminal fragment of megalin (MCTF). When cells were incubated with 200 nM phorbol 12-myristate 13-acetate, the appearance of the MCTF increased 2.5-fold and was blocked by metalloprotease inhibitors. When the cells were incubated with -secretase inhibitor Compound E, it caused a 2-fold increase in MCTF. Finally, incubating the cells with 1 µM vitamin D-binding protein resulted in a 25% increase in the appearance of the MCTF. In summary, the MCTF is produced by protein kinase C regulated, metalloprotease-mediated ectodomain shedding and is the substrate for -secretase. We postulate that the enzymatic processing of megalin represents part of a novel ligand-dependent signaling pathway in the proximal tubule that links receptor-mediated endocytosis with cell signaling.  

3.528           Bph1p, the Saccharomyces cerevisiae homologue of CHS1/beige, functions in cell wall formation and protein sorting

Shiffert, S.L., Vaughn, M.B., Huynh, D., Kaplan, J. and McVey Ward, D. Traffic, 5, 700-710 (2004)   Mutations in the Chediak-Higashi syndrome gene (CHS1) and its murine homologue Beige result in the formation of enlarged lysosomes. BPH1 (Beige Protein Homologue 1) encodes the Saccharomyces cerevisiae homologue of CHS1/Beige. BPH1 is not essential and the encoded protein was found to be both cytosolic and peripherally bound to a membrane. Neither disruption nor overexpression of BPH1 affected vacuole morphology as assessed by fluorescence microscopy. The bph1 strain showed an impaired growth on defined synthetic media containing potassium acetate buffered below pH 4.25, increased sensitivity to calcofluor white, and increased agglutination in response to low pH. A library screen identified VPS9, FLO1, FLO9, BTS1 and OKP1 as high copy suppressors of the growth defect of bph1 on both low pH potassium acetate and calcofluor white. The bph1 strain demonstrated a mild defect in sorting vacuolar components, including increased secretion of carboxypeptidase Y and missorting of alkaline phosphatase. Overexpression of VPS9, BTS1 and OKP1 suppressed the carboxypeptidase Y secretion defect of bph1. Overexpression of BPH1 was found to suppress the calcofluor white sensitivity of a class E VPS deletion strain, vta1. Together, these data suggest that Bph1p associates with a membrane and is involved in protein sorting and cell wall formation.  

3.529           Nicalin and its binding partner Nomo are novel Nodal aignaling antagonists

Haffner, C. et al   Nodals are signaling factors of the transforming growth factor-ß (TGFß) superfamily with a key role in vertebrate development. They control a variety of cell fate decisions required for the establishment of the embryonic body plan. We have identified two highly conserved transmembrane proteins, Nicalin and Nomo (Nodal modulator, previously known as pM5), as novel antagonists of Nodal signaling. Nicalin is distantly related to Nicastrin, a component of the Alzheimer's disease-associated -secretase, and forms a complex with Nomo. Ectopic expression of both proteins in zebrafish embryos causes cyclopia, a phenotype that can arise from a defect in mesendoderm patterning mediated by the Nodal signaling pathway. Accordingly, downregulation of Nomo resulted in an increase in anterior axial mesendoderm and the development of an enlarged hatching gland. Inhibition of Nodal signaling by ectopic expression of Lefty was rescued by reducing Nomo levels. Furthermore, Nodal- as well as Activin-induced signaling was inhibited by Nicalin and Nomo in a cell-based reporter assay. Our data demonstrate that the Nicalin/Nomo complex antagonizes Nodal signaling during mesendodermal patterning in zebrafish.  

3.530           Cell surface ceramide generation precedes and controls FcgRII clustering and phosphorylation in rafts

Shakor, A.B.A., Kwiatkowska, K. and Sobota, A.

1. Biol. Chem., 279(35), 36778-36787 (2004)

  Despite the role of sphingolipid/cholesterol rafts as signaling platforms for Fc receptor II (Fc RII), the mechanism governing translocation of an activated receptor toward the rafts is unknown. We show that at the onset of Fc RII cross-linking acid sphingomyelinase is rapidly activated. This enzyme is extruded from intracellular compartments to the cell surface, and concomitantly, exofacially oriented ceramide is produced. Both non-raft and, to a lesser extent, raft sphingomyelin pools were hydrolyzed at the onset of Fc RII cross-linking. The time course of ceramide production preceded the recruitment of Fc RII to rafts and the receptor phosphorylation. Exogenous C₁₆-ceramide facilitated clustering of Fc RII and its association with rafts. In contrast, inhibition of acid sphingomyelinase diminished both the ceramide generation and clustering of cross-linked Fc RII. Under these conditions, tyrosine phosphorylation of Fc RII and receptor-accompanying proteins was also reduced. All the inhibitory effects were bypassed by treatment of cells with exogenous ceramide. These data provide evidence that the generation of cell surface ceramide is a prerequisite for fusion of cross-linked Fc RII and rafts, which triggers the receptor tyrosine phosphorylation and signaling.  

3.531           Neu4, a novel human lysosomal lumen sialidase, confers normal phenotype to sialidosis and galactosialidosis cells

Seyrantepe, V. et al

1. Biol. Chem., 279(35), 37021-37029 (2004)

  Three different mammalian sialidases have been described as follows: lysosomal (Neu1, gene NEU1), cytoplasmic (Neu2, gene NEU2), and plasma membrane (Neu3, gene NEU3). Because of mutations in the NEU1 gene, the inherited deficiency of Neu1 in humans causes the severe multisystemic neurodegenerative disorder sialidosis. Galactosialidosis, a clinically similar disorder, is caused by the secondary Neu1 deficiency because of genetic defects in cathepsin A that form a complex with Neu1 and activate it. In this study we describe a novel lysosomal lumen sialidase encoded by the NEU4 gene on human chromosome 2. We demonstrate that Neu4 is ubiquitously expressed in human tissues and has broad substrate specificity by being active against sialylated oligosaccharides, glycoproteins, and gangliosides. In contrast to Neu1, Neu4 is targeted to lysosomes by the mannose 6-phospate receptor and does not require association with other proteins for enzymatic activity. Expression of Neu4 in the cells of sialidosis and galactosialidosis patients results in clearance of storage materials from lysosomes suggesting that Neu4 may be useful for developing new therapies for these conditions.  

3.532           Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure during the Cvt pathway

Meiling-Wesse, K. et al

1. Biol. Chem., 279(36), 37741-37759 (2004)

  Atg21 and Atg18 are homologue yeast proteins. Whereas Atg18 is essential for the Cvt pathway and autophagy, a lack of Atg21 only blocks the Cvt pathway. Our proteinase protection experiments now demonstrate that growing atg21 cells fail to form proaminopeptidase I-containing Cvt vesicles. Quantitative measurement of autophagy in starving atg21 cells showed only 35% of the wild-type rate. This suggests that Atg21 plays a nonessential role in improving the fidelity of autophagy. The intracellular localization of Atg21 is unique among the Atg proteins. In cells containing multiple vacuoles, Atg21-yellow fluorescent protein clearly localizes to the vertices of the vacuole junctions. Cells with a single vacuole show most of the protein at few perivacuolar punctae. This distribution pattern is reminiscent to the Vps class C(HOPS) (homotypic fusion and vacuolar protein sorting) protein complex. In growing cells, Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure. Consistently, the covalent linkage of Atg8 to the lipid phosphatidylethanolamine is significantly retarded. Lipidated Atg8 is supposed to act during the elongation of autophagosome precursors. However, despite the reduced autophagic rate and the retardation of Atg8 lipidation, electron microscopy of starved atg21 ypt7 double mutant cells demonstrates the formation of normally sized autophagosomes with an average diameter of 450 nm.  

3.533           Interactions of EGFR and caveolin-1 in human glioblastoma cells: evidence that tyrosine phosphorylation regulates EGFR association with caveolae

Abulrob, A. et al Oncogene, 23, 6967-6979 (2004)   Epidermal growth factor receptor (EGFR) amplification and type III mutation (EGFRvIII), associated with constitutive tyrosine kinase activation and high malignancy, are commonly observed in glioblastoma tumors. The association of EGFR and EGFRvIII with caveolins was investigated in human glioblastoma cell lines, U87MG and U87MG-EGFRvIII. Caveolin-1 expression, determined by RT-PCR, real-time quantitative PCR and Western blot, was upregulated in glioblastoma cell lines (two-fold) and tumors (20-300-fold) compared to primary human astrocytes and nonmalignant brain tissue, respectively. U87MG-EGFRvIII expressed higher levels of caveolin-1 than U87MG. In contrast, the expression of caveolin-2 and -3 were downregulated in glioblastoma cells compared to astrocytes. A colocalization of EGFR, but not of EGFRvIII, with lipid rafts and caveolin-1 was observed by immunocytochemistry. Association of EGFR and EGFRvIII with caveolae, assessed in vitro by binding to caveolin scaffolding domain peptides and in vivo by immunocolocalization studies in cells and caveolae-enriched cellular fraction, was phosphorylation-dependent: ligand-induced phosphorylation of EGFR resulted in dissociation of EGFR from caveolae. In contrast, inhibition of the EGFRvIII constitutive tyrosine phosphorylation by AG1478 increased association of EGFRvIII with caveolin-1. AG1478 also increased caveolin-1 expression and reduced glioblastoma cell growth in a semi-solid agar. The evidence suggests that the phosphorylation-regulated sequestration of EGFR in caveolae may be involved in arresting constitutive or ligand-induced signaling through EGFR responsible for glial cell transformation.  

3.534           Isolated plant nuclei as mechanical and thermal sensors involved in calcium signaling

Xiong, T.C., Jauneau, A., Ranjeva, R. and Mazars, C. The Plant J., 40, 12-21 (2004)   Calcium signals in the nucleus elicit downstream effects that are distinct from those of cytosolic calcium signals. In the present work, we have evaluated the ability of plant nuclei to sense stimuli directly and to convert them into calcium changes. We show that individual mechanical stimulation of isolated nuclei elicits a single calcium transient at acidic pHs, whereas a series of stimulations leads to oscillations whose frequency reflects that of the stimuli. Conversely, at alkaline pHs, nuclei respond to temperature but not to stretch. The stretch- and the temperature-activated processes differ by their sensitivity to pharmacological drugs known to affect ion channel activities in animal cells. Our data demonstrate that isolated nuclei are able to gauge physical parameters of their environment. This might have a profound influence on the functioning of calcium-dependent processes known to control a large array of molecular events in the nucleus.  

3.535           CLIPR-59 is a lipid raft-associated protein containing a cytoskeleton-associated protein glycine-rich domain (CAP-Gly) that perturbs microtubule dynamics

Lallamand-Breitenbach, V. et al

1. Biol. Chem., 279(39), 41168-41178 (2004)

  We recently have identified a new cytoplasmic linker protein (CLIP), CLIPR-59, which is involved in the regulation of early endosome/trans-Golgi network dynamics. In contrast with CLIP-170, CLIPR-59 is not localized to microtubules at steady state but is associated with the trans-Golgi network and the plasma membrane. Here we show that the last 30 amino acids (C30) are sufficient for membrane targeting and that two cysteines in the C30 domain are palmitoylated. We demonstrate that CLIPR-59 is associated with lipid rafts via its C-terminal palmitoylated domain. In vitro experiments suggest that CLIPR-59 and its microtubule-binding domain alone have a better affinity for unpolymerized tubulin or small oligomers than for microtubules. In contrast with the CLIP-170 microtubule-binding domain, the CLIPR-59 microtubule-binding domain diminishes microtubule regrowth after nocodazole washout in vivo, showing that this domain can prevent microtubule polymerization. In contrast with the role of linker between membranes and microtubules that was proposed for CLIP function, CLIPR-59 thus may have an "anti-CLIP" function by preventing microtubule-raft interactions.  

3.536           Reorganization of lipid rafts during capacitation of human sperm

Cross, N.L. Biol. Reprod., 71, 1367-1373 (2004)   Ejaculated mammalian sperm must complete a final maturation, termed capacitation, before they can undergo acrosomal exocytosis and fertilize an egg. In human sperm, loss of sperm sterol is an obligatory, early event in capacitation. How sterol loss leads to acrosomal responsiveness is unknown. These experiments tested the hypothesis that loss of sperm sterol affects the organization of cold detergent-resistant membrane microdomains (lipid "rafts"). The GPI-linked protein CD59, the ganglioside GM1, and the protein flotillin-2 were used as markers for lipid rafts. In uncapacitated sperm, 51% of the CD59, 41% of the GM1, and 90% of the flotillin-2 were found in the raft fraction. During capacitation, sperm lost 67% of their 3ß-hydroxysterols, and the percentages of CD59 and GM1 in the raft fraction decreased to 34% and 31%, respectively. The distribution of flotillin-2 did not change. Preventing a net loss of sperm sterol prevented the loss of CD59 and GM1 from the raft fraction. Fluorescence microscopy showed CD59 and GM1 to be distributed over the entire sperm surface. Flotillin-2 was located mainly in the posterior head and midpiece. Patching using bivalent antibodies indicated that little of the GM1 and CD59 was stably associated in the same membrane rafts. Likewise, GM1 and flotillin-2 were not associated in the same membrane rafts. In summary, lipid rafts of heterogeneous composition were identified in human sperm and the two raft components, GM1 and CD59, showed a partial sterol loss-dependent shift to the nonraft domain during capacitation.  

3.537           An N-terminal amphipathic helix in hepatitis C virus (HCV) NS4B mediates membrane association, correct localization of replication complex proteins, and RNA replication

Elazar, M., Liu, P., Rice, C.M. and Glenn, J.S.

1. Virol., 78(20), 11393-11400 (2004)

  Like other positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its RNA in association with host cell cytoplasmic membranes. Because of its association with such membranes, NS4B, one of the virus's nonstructural proteins, may play an important role in this process, although the mechanistic details are not well understood. We identified a putative N-terminal amphipathic helix (AH) in NS4B that mediates membrane association. Introduction of site-directed mutations designed to disrupt the hydrophobic face of the AH abolishes the AH's ability to mediate membrane association. An AH in NS4B is conserved across HCV isolates. Completely disrupting the amphipathic nature of NS4B's N-terminal helix abolished HCV RNA replication, whereas partial disruption resulted in an intermediate level of replication. Finally, immunofluorescence studies revealed that HCV replication complex components were mislocalized in the AH-disrupted mutant. These results identify a key membrane-targeting domain which can form the basis for developing novel antiviral strategies.  

3.538           Functional similarity between the peroxisomal PTS2 receptor binding protein Pex18p and the N-terminal half of the PTS1 receptor Pex5p

Schäfer, A., Kerssen, D., Veenhuis, M., Kunau, W-H. And Schliebs, W. Mol. Cell Biol., 24(20), 8895-8906 (2004)   Within the extended receptor cycle of peroxisomal matrix import, the function of the import receptor Pex5p comprises cargo recognition and transport. While the C-terminal half (Pex5p-C) is responsible for PTS1 binding, the contribution of the N-terminal half of Pex5p (Pex5p-N) to the receptor cycle has been less clear. Here we demonstrate, using different techniques, that in Saccharomyces cerevisiae Pex5p-N alone facilitates the import of the major matrix protein Fox1p. This finding suggests that Pex5p-N is sufficient for receptor docking and cargo transport into peroxisomes. Moreover, we found that Pex5p-N can be functionally replaced by Pex18p, one of two auxiliary proteins of the PTS2 import pathway. A chimeric protein consisting of Pex18p (without its Pex7p binding site) fused to Pex5p-C is able to partially restore PTS1 protein import in a PEX5 deletion strain. On the basis of these results, we propose that the auxiliary proteins of the PTS2 import pathway fulfill roles similar to those of the N-terminal half of Pex5p in the PTS1 import pathway.  

3.539           BZLF1, an Epstein-Barr virus immediate-early protein, induces p65 nuclear translocation while inhibiting p65 transcriptional function

Morrison, T.E. and Kenncy, S.C. Virology, 328, 219-232 (2004)   We have previously demonstrated that the Epstein–Barr virus immediate–early BZLF1 protein interacts with, and is inhibited by, the NF-κB family member p65. However, the effects of BZLF1 on NF-κB activity have not been intensively studied. Here we show that BZLF1 inhibits p65-dependent gene expression. BZLF1 inhibited the ability of IL-1, as well as transfected p65, to activate the expression of two different NF-κB-responsive genes, ICAM-1 and IκB-α. BZLF1 also reduced the constitutive level of IκB-α protein in HeLa and A549 cells, and increased the amount of nuclear NF-κB to a similar extent as tumor necrosis factor-alpha (TNF-α) treatment. In spite of this BZLF1-associated increase in the nuclear form of NF-κB, BZLF1 did not induce binding of NF-κB to NF-κB responsive promoters (as determined by chromatin immunoprecipitation assay) in vivo, although TNF-α treatment induced NF-κB binding as expected. Overexpression of p65 dramatically inhibited the lytic replication cycle of EBV in 293-EBV cells, confirming that NF-κB also inhibits BZLF1 transcriptional function. Our results are consistent with a model in which BZLF1 inhibits the transcriptional function of p65, resulting in decreased transcription of IκB-α, decreased expression of IκB-α protein, and subsequent translocation of NF-κB to the nucleus. This nuclear translocation of NF-κB may promote viral latency by negatively regulating BZLF1 transcriptional activity. In situations where p65 activity is limiting in comparison to BZLF1, the ability of BZLF1 to inhibit p65 transcriptional function may protect the virus from the host immune system during the lytic form of infection.      

3.540           Targeting, import, and dimerization of a mammalian mitochondrial ATP binding cassette (ABC) transporter, ABCB10 (ABC-me)

Graf, S.A., Haigh, S.E., Corson, E.D. and Shirihai, O.S.

1. Biol. Chem., 279(41), 42954-42963 (2004)

  ATP binding cassette (ABC) transporters are a diverse superfamily of energy-dependent membrane translocases. Although responsible for the majority of transmembrane transport in bacteria, they are relatively uncommon in eukaryotic mitochondria. Organellar trafficking and import, in addition to quaternary structure assembly, of mitochondrial ABC transporters is poorly understood and may offer explanations for the paucity of their diversity. Here we examine these processes in ABCB10 (ABC-me), a mitochondrial inner membrane erythroid transporter involved in heme biosynthesis. We report that ABCB10 possesses an unusually long 105-amino acid mitochondrial targeting presequence (mTP). The central subdomain of the mTP (amino acids (aa) 36–70) is sufficient for mitochondrial import of enhanced green fluorescent protein. The N-terminal subdomain (aa 1–35) of the mTP, although not necessary for the trafficking of ABCB10 to mitochondria, participates in the proper import of the molecule into the inner membrane. We performed a series of amino acid mutations aimed at changing specific properties of the mTP. The mTP requires neither arginine residues nor predictable -helices for efficient mitochondrial targeting. Disruption of its hydrophobic character by the mutation L46Q/I47Q, however, greatly diminishes its efficacy. This mutation can be rescued by cryptic downstream (aa 106–715) mitochondrial targeting signals, highlighting the redundancy of this protein's targeting qualities. Mass spectrometry analysis of chemically cross-linked, immunoprecipitated ABCB10 indicates that ABCB10 embedded in the mitochondrial inner membrane homodimerizes and homo-oligomerizes. A deletion mutant of ABCB10 that lacks its mTP efficiently targets to the endoplasmic reticulum. Quaternary structure assembly of ABCB10 in the ER appears to be similar to that in the mitochondria.  

3.541           Differential compartmentalization of the calpain/calpastatin network with the endoplasmic reticulum and Golgi apparatus

Hood, J.L., Brooks, W.H. and Roszman, T.L.

1. Biol. Chem., 279(41), 43126-43135 (2004)

  Calpain, a calcium-activated cysteine protease, is involved in modulating a variety of cell activities such as shape change, mobility, and apoptosis. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to various sites in the cell. The activity of calpain is modulated by two regulatory proteins, calpastatin, the specific endogenous inhibitor of calpain, and the 28-kDa regulatory subunit. Using velocity gradient centrifugation, the results of this study confirm and greatly expand upon our previous finding that the calpain/calpastatin network is associated with the endoplasmic reticulum and Golgi apparatus in cells. Moreover, confocal microscopy demonstrates that calpain II colocalizes with specific proteins found in these organelles. Additional experiments reveal that hydrophobic rather than electrostatic interactions are responsible for the association of the calpain/calpastatin network with these organelles. Treatment of the organelles with Na₂CO₃ or deoxycholate reveal that calpain I, 78-kDa calpain II, and the regulatory subunit are "embedded" within the organelle membranes similar to integral membrane proteins. Proteinase K treatment of the organelles shows that calpain I and II, calpastatin, and the regulatory subunit localize to the cytosolic surface of the organelle membranes, and a subset of calpain II and the regulatory subunit are also found within the lumen of these organelles. These results provide a new and novel explanation for how the calpain/calpastatin network is organized in the cell.  

3.542           Hepatitis C virus core protein associates with detergent-resistant membranes distinct from classical plasma membrane rafts

Matto, M., Rice, C.M., Aroeti, B. and Glenn, J.S.

1. Virol., 78(21), 12047-12053 (2004)

  A subpopulation of hepatitis C virus (HCV) core protein in cells harboring full-length HCV replicons is biochemically associated with detergent-resistant membranes (DRMs) in a manner similar to that of markers of classical lipid rafts. Core protein does not, however, colocalize in immunofluorescence studies with classical plasma membrane raft markers, such as caveolin-1 and the B subunit of cholera toxin, suggesting that core protein is bound to cytoplasmic raft microdomains distinct from caveolin-based rafts. Furthermore, while both the structural core protein and the nonstructural protein NS5A associate with membranes, they do not colocalize in the DRMs. Finally, the ability of core protein to localize to the DRMs did not require other elements of the HCV polyprotein. These results may have broad implications for the HCV life cycle and suggest that the HCV core may be a valuable probe for host cell biology.  

3.543           Dynamic confinement of NK2 receptors in the plasma membrane

Cezanne, L. et al

1. Biol. Chem., 279(43), 45057-45067 (2004)

  A functional fluorescent neurokinin NK2 receptor, EGFP-NK2, was previously used to follow, by fluorescence resonance energy transfer measurements in living cells, the binding of its fluorescently labeled agonist, bodipy-neurokinin A (NKA). Local agonist application suggested that the activation and desensitization of the NK2 receptors were compartmentalized at the level of the plasma membrane. In this study, fluorescence recovery after photobleaching experiments are carried out at variable observation radius (vrFRAP) to probe EGFP-NK2 receptor mobility and confinement. Experiments are carried out at 20 °C to maintain the number of receptors constant at the cell surface during recordings. In the absence of agonist, 35% EGFP-NK2 receptors diffuse within domains of 420 ± 80 nm in radius with the remaining 65% of receptors able to diffuse with a long range lateral diffusion coefficient between the domains. When cells are incubated with a saturating concentration of NKA, 30% EGFP-NK2 receptors become immobilized in small domains characterized by a radius equal to 170 ± 50 nm. Biochemical experiments show that the confinement of EGFP-NK2 receptor is not due to its association with rafts at any given time. Colocalization of the receptor with -arrestin and transferrin supports that the small domains, containing 30% of activated EGFP-NK2, correspond to clathrin-coated pre-pits. The similar amount of confined EGFP-NK2 receptors found before and after activation (30–35%) is discussed in term of putative transient interactions of the receptors with preexisting scaffolds of signaling molecules.  

3.544           ATP-binding cassette (ABC) transporters mediate nonvesicular, raft-modulatd sterol movement from the plasma membrane to the endoplasmic reticulum

Li, Y and Priz, W.A.

1. Biol. Chem., 279(43), 45226-45234 (2004)

  Little is known about the mechanisms of intracellular sterol transport or how cells maintain the high sterol concentration of the plasma membrane (PM). Here we demonstrate that two inducible ATP-binding cassette (ABC) transporters (Aus1p and Pdr11p) mediate nonvesicular movement of PM sterol to the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. This transport facilitates exogenous sterol uptake, which we find requires steryl ester synthesis in the ER. Surprisingly, while expression of Aus1p and Pdr11p significantly increases sterol movement from PM to ER, it does not alter intracellular sterol distribution. Thus, ER sterol is likely rapidly returned to the PM when it is not esterified in the ER. We show that the propensity of PM sterols to be moved to the ER is largely determined by their affinity for sterol sphingolipid-enriched microdomains (rafts). Our findings suggest that raft association is a primary determinant of sterol accumulation in the PM and that Aus1p and Pdr11p facilitate sterol uptake by increasing the cycling of sterol between the PM and ER.  

3.545           Participation of G protein in natriuretic peptide hormone secretion from heart atria

Bensimon, M. et al Endocrinol., 145, 5313-5321 (2004)   The involvement of G proteins in the mechanism underlying the increased atrial natriuretic factor (ANF) secretion observed after atrial muscle stretch (stretch-secretion coupling) was assessed using a combined pharmacological, immunocytochemical, and tissue fractionation approach. It was found that G_i/o inhibition by pertussis toxin (PTX) abolished stretch-secretion coupling without affecting baseline secretion through a mechanism that is independent of G_q signaling agonists. Mastoparan-7, a G_i/o agonist, significantly increased ANF secretion even in the absence of muscle stretch through a PTX-sensitive mechanism. By confocal and electron immunocytochemistry, ANF and G_o partially colocalized, whereas ultracentrifugation analysis suggested the presence of two populations of granules, one of which was partially associated with G_o, as demonstrated by Western blotting. PTX did not affect basal or endothelin-1-stimulated ANF secretion, in line with the view that endothelin-1 signals mainly through G_q. It is concluded there are at least two types of regulated secretory processes in atrial cardiocytes: one is acutely responsive to muscle stretch and is PTX sensitive, and the other is G_qmediated and PTX insensitive and may be responsible for changes in secretion after chronic changes in the neuroendocrine environment.  

3.546           Real time analysis of intact organelle using surface plasmon resonance

Ferraci, G., Seagar, M., Joël, C., Miquelis, R. And Leveque, C. Anal. Biochem., 334, 367-375(2004)   Membrane proteins remain refractory to standard protein chip analysis. They are typically expressed at low densities in distinct subcellular compartments, their biological activity can depend on assembly into macromolecular complexes in a specific lipid environment. We report here a real-time, label-free method to analyze membrane proteins inserted in isolated native synaptic vesicles. Using surface plasmon resonance-based biomolecular interaction analysis (Biacore), organelle capture from minute quantities of 10,000g brain supernatant (1–10 μg) was monitored. Immunological and morphological characterization indicated that pure intact synaptic vesicles were immobilized on sensor chips. Vesicle chips were stable for days, allowing repetitive use with multiple analytes. This method provides an efficient way in which to characterize organelle membrane components in their native context. Organelle chips allow a broad range of measurements, including interactions of exogenous ligands with the organelle surface (kinetics, K_d), and protein profiling.  

3.547           The vitamin D receptor is present in caveolae-enriched plasma membranes and binds 1a,25(OH)₂-vitamin D₃ in vivo and in vitro

Huhtakangas, J.A., Olivera, C.J., Bishop, J.E., Zanello, L.P. and Norman, A.W. Mol. Endocrinol., 18(11), 2660-2671 (2004)   The steroid hormone 1 ,25(OH)₂-vitamin D₃ (1,25D) regulates gene transcription through a nuclear receptor [vitamin D receptor (VDR)] and initiation of rapid cellular responses through a putative plasma membrane-associated receptor (VDR_mem). This study characterized the VDR_mem present in a caveolae-enriched membrane fraction (CMF), a site of accumulation of signal transduction agents. Saturable and specific [³H]-1,25D binding in vitro was found in CMF of chick, rat, and mouse intestine; mouse lung and kidney; and human NB4 leukemia and rat ROS 17/2.8 osteoblast-like cells; in all cases the 1,25D K_D binding dissociation constant = 1–3 nM. Our data collectively support the classical VDR being the VDR_mem in caveolae: 1) VDR antibody immunoreactivity was detected in CMF of all tissues tested; 2) competitive binding of [³H]-1,25D by eight analogs of 1,25D was significantly correlated between nuclei and CMF (r² = 0.95) but not between vitamin D binding protein (has a different ligand binding specificity) and CMF; 3) confocal immunofluorescence microscopy of ROS 17/2.8 cells showed VDR in close association with the caveolae marker protein, caveolin-1, in the plasma membrane region; 4) in vivo 1,25D pretreatment reduced in vitro [³H]-1,25D binding by 30% in chick and rat intestinal CMF demonstrating in vivo occupancy of the CMF receptor by 1,25D; and 5) comparison of [³H]-1,25D binding in VDR KO and WT mouse kidney tissue showed 85% reduction in VDR KO CMF and 95% reduction in VDR KO nuclear fraction. This study supports the presence of VDR as the 1,25D-binding protein associated with plasma membrane caveolae.        

3.548           Both the sequence and length of the C terminus of PEN-2 are critical for intermolecular interactions and function of presenilin complexes

Hasegawa, H. et al

1. Biol. Chem., 279(45), 46455-46463 (2004)

  Presenilin 1 or presenilin 2, nicastrin, APH-1, and PEN-2 form high molecular weight complexes that play a pivotal role in the cleavage of various Type I transmembrane proteins, including the -amyloid precursor protein. The specific function of PEN-2 is unclear. To explore its function and intermolecular interactions, we conducted deletion and mutagenesis studies on a series of conserved residues at the C terminus of PEN-2. These studies suggest that: 1) both the presence and amino acid sequence of the conserved DYLSF domain at the C terminus of PEN-2 (residues 90–94) is critical for binding PEN-2 to other components in the presenilin complex and 2) the overall length of the exposed C terminus is critical for functional -secretase activity.  

3.549           The target cell plasma membrane is a critical interface for Salmonella cell entry effector-host interplay

Cain, R.J., Hayward, R.D. and Koronakis, V. Mol. Microbiol., 54(4), 887-904 (2004)   Salmonella species trigger host membrane ruffling to force their internalization into non-phagocytic intestinal epithelial cells. This requires bacterial effector protein delivery into the target cell via a type III secretion system. Six translocated effectors manipulate cellular actin dynamics, but how their direct and indirect activities are spatially and temporally co-ordinated to promote productive cytoskeletal rearrangements remains essentially unexplored. To gain further insight into this process, we applied mechanical cell fractionation and immunofluorescence microscopy to systematically investigate the subcellular localization of epitope-tagged effectors in transiently transfected and Salmonella-infected cultured cells. Although five effectors contain no apparent membrane-targeting domains, all six localized exclusively in the target cell plasma membrane fraction and correspondingly were visualized at the cell periphery, from where they induced distinct effects on the actin cytoskeleton. Unexpectedly, no translocated effector pool was detectable in the cell cytosol. Using parallel in vitro assays, we demonstrate that the prenylated cellular GTPase Cdc42 is necessary and sufficient for membrane association of the Salmonella GTP exchange factor and GTPase-activating protein mimics SopE and SptP, which have no intrinsic lipid affinity. The data show that the host plasma membrane is a critical interface for effector-target interaction, and establish versatile systems to further dissect effector interplay.  

3.550           Segregation of Nogo66 receptors into lipid rafts in rat brain and inhibition of Nogo66 signaling by cholesterol depletion

Yu, W., Guo, W. and Feng, L. FEBS Lett., 577, 87-92 (2004)   NogoA, a myelin-associated component, inhibits neurite outgrowth. Nogo66, a portion of NogoA, binds to Nogo66 receptor (NgR) and induces the inhibitory signaling. LINGO-1 and p75 neurotrophin receptor (p75), the low-affinity nerve growth factor receptor, are also required for NogoA signaling. However, signaling mechanisms downstream to Nogo receptor remain poorly understood. Here, we observed that NgR and p75 were colocalized in low-density membrane raft fractions derived from forebrains and cerebella as well as from cerebellar granule cells. NgR interacted with p75 in lipid rafts. In addition, disruption of lipid rafts by -methylcyclodextrin, a cholesterol-binding reagent, reduced the Nogo66 signaling. Our results suggest an important role of lipid rafts in facilitating the interaction between NgRs and provide insight into mechanisms underlying the inhibition of neurite outgrowth by NogoA.  

3.551           The behavior of peroxisomes in vitro: mammalian peroxisomes are osmotically sensitive particles

Antonenko, V.D., Sormunen, R.T. and Hiltunen, J.K. Am. J. Physiol., 287, C1623-C1635 (2004)   It has been known for a long time that mammalian peroxisomes are extremely fragile in vitro. Changes in the morphological appearance and leakage of proteins from purified particles demonstrate that peroxisomes are damaged during isolation. However, some properties of purified peroxisomes, e.g., the latency of catalase, imply that their membranes are not disrupted. In the current study, we tried to ascertain the mechanism of this unusual behavior of peroxisomes in vitro. Biochemical and morphological examination of isolated peroxisomes subjected to sonication or to freezing and thawing showed that the membrane of the particles seals after disruption, restoring permeability properties. Transient damage of the membrane leads to the formation of peroxisomal "ghosts" containing nucleoid but nearly devoid of matrix proteins. The rate of leakage of matrix proteins from broken particles depended inversely on their molecular size. The effect of polyethylene glycols on peroxisomal integrity indicated that these particles are osmotically sensitive. Peroxisomes suffered an osmotic lysis during isolation that was resistant to commonly used low-molecular-mass osmoprotectors, e.g., sucrose. Damage to peroxisomes was partially prevented by applying more "bulky" osmoprotectors, e.g., polyethylene glycol 1500. A method was developed for the isolation of highly purified and nearly intact peroxisomes from rat liver by using polyethylene glycol 1500 as an osmoprotector.  

3.552           A common mechanism for the regulation of vesicular SNAREs on phospholipid membranes

Hu, K., Rickman, C., Carroll. J. and Davletov, B. Biochem.J., 377, 781-765 (2004)   The SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) family of proteins is essential for membrane fusion in intracellular traffic in eukaryotic organisms. v-SNAREs (vesicular SNAREs) must engage target SNAREs in the opposing membrane to form the fusogenic SNARE complex. Temporal and spatial control of membrane fusion is important for many aspects of cell physiology and may involve the regulation of the SNAREs resident on intracellular membranes. Here we show that the v-SNARE synaptobrevin 2, also known as VAMP (vesicle-associated membrane protein) 2, is restricted from forming the SNARE complex in chromaffin granules from adrenal medullae to the same degree as in brain-purified synaptic vesicles. Our analysis indicates that the previously reported synaptophysin–synaptobrevin interaction is not likely to be involved in regulation of the v-SNARE. Indeed, the restriction can be reproduced for two distinct v-SNARE homologues, synaptobrevin 2 and cellubrevin/VAMP3, by reconstituting them in pure liposomal membranes. Overall, our data uncover a common mechanism for the control of SNARE engagement where intact phospholipid membranes rather than proteins down-regulate vesicular SNAREs in different cellular organelles.  

3.553           Membrane and raft association of reggie-1/flotillin-2: role of myristoylation, palmitoylation and oligomerization and induction of filopodia by overexpression

Neumann-Giesen, C. Et al Biochem. J., 378, 509-518 (2004)   The reggie protein family consists of two proteins, reggie-1 and -2, also called flotillins, which are highly ubiquitous and evolutionarily conserved. Both reggies have been shown to be associated with membrane rafts and are involved in various cellular processes such as T-cell activation, phagocytosis and insulin signalling. However, the exact molecular function of these proteins remains to be determined. In addition, the mechanism of membrane association of reggie-1, which does not contain any transmembrane domain, is not known. In this study, we have produced a fusion protein of reggie-1 with enhanced green fluorescent protein and generated targeted substitutions for the inactivation of putative palmitoylation and myristoylation sites. We were able to show that reggie-1 is myristoylated and multiply palmitoylated and that lipid modifications are necessary for membrane association of reggie-1. Overexpression of reggie-1 resulted in the induction of numerous filopodia-like protrusions in various cell lines, suggesting a role for reggie-1 as a signalling protein in actin-dependent processes.    

3.554           Different subcellular localization of sulphotransferase 2B1b in human placenta and prostate

He, D., Meloche, C.A., Dumas, N.A., Frost, A.R. and Falany, C.N. Biochem. J., 379, 533-540 (2004)   The human hydroxysteroid SULT (sulphotransferase) 2B1 subfamily consists of two isoforms, SULT2B1a and SULT2B1b. These two isoenzymes are transcribed from the same gene by alternative splicing of their first exons and share 94% amino acid sequence identity. The SULT2B1 isoforms are highly selective for the sulphation of 3b-hydroxysteroids. Immunoblot analysis of SULT2B1 expression in several human tissues indicates the presence of only SULT2B1b protein. Immunoreactive SULT2B1b protein was detected in human prostate, skin, placenta and lung tissue. SULT2B1b mRNA expression was detected in RNA isolated from term placenta, normal prostate, prostate carcinoma, benign prostate hyperplasia, LNCaP prostate cancer cells, breast cancer specimens and MCF-7 breast cancer cells. Immunohistochemical localization of SULT2B1b, in terms placental and prostate tissues, detected it in nuclei of placental syncytiotrophoblasts and cytoplasm of epithelial cells in prostate tissues. Immunoreactive and catalytically active SULT2B1b was identified in nuclei isolated from term human placenta. Also SULT2B1b was capable of translocating to nuclei in BeWo placental cells after stable transfection and differentiation. In contrast, immunohistochemical analysis of human prostate showed only cytosolic localization of SULT2B1b in the basal and luminal prostate epithelial cells. SULT2B1b was not detected in isolated nuclei from LNCaP prostate cancer cells but was present in the cytosolic fraction. Differential subcellular localization of SULT2B1b in prostate and placenta suggests that SULT2B1b may be differentially regulated and have different physiological functions in these two hormonally responsive human tissues.  

3.555           Recruitment of the cross-linked opsonic receptor CD32A (FcgRIIA) to high density detergent-resistant membrane domains in human neutrophils

Rollet-Labelle, E., Marois, S., Barbeau, K., Malawista, S.E. and Naccache, P.H. Biochem. J., 381, 919-928 (2004)   We have previously shown that CD32A (or FcgRIIA), one of the main opsonin receptors, was rapidly insolubilized and degraded in intact neutrophils after its cross-linking. In view of these experimental difficulties, the early signalling steps in response to CD32A activation were studied in purified plasma membranes of neutrophils. After CD32A cross-linking in these fractions, the tyrosine phosphorylation of two major substrates, the receptor itself and the tyrosine kinase Syk, was observed. Phosphorylation of these two proteins was observed only in the presence of orthovanadate, indicating the presence, in the membranes, of one or more tyrosine phosphatases that maintain CD32A dephosphorylation. The tyrosine phosphorylation of these two proteins was inhibited by the Src kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). The ligation of CD32A led to its recruitment to a previously uncharacterized subset of high-density flotillin-1-positive DRMs (detergent-resistant membranes). The changes in the solubility properties of CD32A were observed in the absence of added ATP; therefore, they were probably not secondary to the tyrosine phosphorylation of the receptor, rather they preceded it. Src kinases as well as Syk were constitutively present in DRMs of high and low density and no evident changes in their distribution were detected after cross-linking of CD32A. Pretreatment of plasma membranes with methyl-b-cyclodextrin did not inhibit the recruitment of CD32A to DRMs, although it led to the loss of the Src kinase Lyn from these fractions. In addition, methyl-b-cyclodextrin inhibited the tyrosine phosphorylation of CD32A and Syk induced by cross-linking of CD32A. This membrane model allowed us to observe a movement of CD32A from detergent-soluble regions of the membranes to DRMs, where it joined Src kinases and Syk and became tyrosine-phosphorylated.  

3.556           Structure and cholesterol domain dynamics of an enriched caveolae/raft isolate

Gallegos, A.M., McIntosh, A.L., Atshaves, B.P. and Schroeder, F. Biochem. J., 382, 451-461 (2004)   Despite the importance of cholesterol in the formation and function of caveolar microdomains in plasma membranes, almost nothing is known regarding the structural properties, cholesterol dynamics or intracellular factors affecting caveolar cholesterol dynamics. A non-detergent method was employed to isolate caveolae/raft domains from purified plasma membranes of murine fibroblasts. A series of fluorescent lipid probe molecules or a fluorescent cholesterol analogue, dehydroergosterol, were then incorporated into the caveolae/raft domains to show that: (i) fluorescence polarization of the multiple probe molecules {diphenylhexatriene analogues, DiI₁₈ (1,1´-dioctadecyl-3,3,3´,3´-tetramethylindocarbocyanine perchlorate), parinaric acids and NBD-stearic acid {12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadecanoic acid} indicated that acyl chains in caveolae/raft domains were significantly less ‘fluid’ (i.e. more rigid) and the transbilayer ‘fluidity gradient’ was 4.4-fold greater than in plasma membranes; (ii) although sterol was more ordered in caveolae/raft domains than plasma membranes, spontaneous sterol transfer from caveolae/raft domains was faster (initial rate, 32%; half-time, t_1/2, 57%) than from the plasma membrane; (iii) although kinetic analysis showed similar proportions of exchangeable and non-exchangeable sterol pools in caveolae/raft domains and plasma membranes, addition of SCP-2 (sterol carrier protein-2) 1.3-fold more selectively increased sterol transfer from caveolae/raft domains by decreasing the t_1/2 (50%) and increasing the initial rate (5-fold); (iv) SCP-2 was also 2-fold more selective in decreasing the amount of non-exchangeable sterol in caveolae/raft domains compared with plasma membranes, such that nearly 80% of caveolar/raft sterol became exchangeable. In summary, although caveolae/raft lipids were less fluid than those of plasma membranes, sterol domains in caveolae/rafts were more spontaneously exchangeable and more affected by SCP-2 than those of the bulk plasma membranes. Thus caveolae/raft domains isolated without the use of detergents display unique structure, cholesterol domain kinetics and responsiveness to SCP-2 as compared with the parent plasma membrane.  

3.557           Rafts can trigger contact-mediated secretion of bacterial effectors via a lipid-based mechanism

Van der Goot, F.G., Tran van Nhieu, G., Allaoui, A., Sansonetti, P and Lafont, F.

1. Biol. Chem., 279(46), 47792-47798 (2004)

  Infection by the Gram-negative bacterial pathogen Shigella flexneri depends on its ability to invade host cells. Bacterial engulfment requires a functional type III secretion system (TTSS) allowing the translocation into host cells of bacterial effectors that activate cell-signaling cascades. We demonstrated previously that specialized lipid membrane domains enriched in cholesterol and sphingolipids (rafts) are involved during early steps of invasion, namely in binding and host cell entry. In this study, we addressed the issue of contact-mediated secretion by the TTSS. We show that contact-mediated and TTSS-induced hemolysis depend on the presence of cholesterol on the host cell surface. We found that purified detergent resistant membranes were able to activate TTSS. Finally, we found that artificial liposomes, devoid of proteins, were able to activate the TTSS but only when their composition mimicked that of lipid rafts. Altogether, these data indicate that specific lipid packing can trigger contact-mediated secretion by S. flexneri.  

3.558           Rhodopsin signaling and organization in heterozygote rhodosin knockout mice

Linag, Y. et al

1. Biol. Chem., 279(46), 48189-48196 (2004)

  Rhodopsin (Rho) resides within internal membrane structures called disc membranes that are found in the rod outer segments (ROS) of photoreceptors in the retina. Rho expression is essential for formation of ROS, which are absent in knockout Rho-/- mice. ROS of mice heterozygous for the Rho gene deletion (Rho+/-) may have a lower Rho density than wild type (WT) membranes, or the ROS structure may be reduced in size due to lower Rho expression. Here, we present evidence that the smaller volume of ROS from heterozygous mice is most likely responsible for observed electrophysiological response differences. In Rho+/- mice as compared with age-matched WT mice, the length of ROS was shorter by 30–40%, and the average diameter of ROS was reduced by 20%, as demonstrated by transmission and scanning electron microscopy. Together, the reduction of the volume of ROS was 60% in Rho+/- mice. Rho content in the eyes was reduced by 43% and 11-cis-retinal content in the eye was reduced by 38%, as determined by UV-visible spectroscopy and retinoid analysis, respectively. Transmission electron microscopy of negatively stained disc membranes from Rho+/- mice indicated a typical morphology apart from the reduced size of disc diameter. Power spectra calculated from disc membrane regions on such electron micrographs displayed a diffuse ring at 4.5 nm^-1, indicating paracrystallinity of Rho. Atomic force microscopy of WT and Rho+/- disc membranes revealed, in both cases, Rho organized in paracrystalline and raftlike structures. From these data, we conclude that the differences in physiological responses measured in WT and Rho+/- mice are due to structural changes of the whole ROS and not due to a lower density of Rho.  

3.559           Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains

Bravo, A. et al Biochim. Biophys. Acta, 1667, 38-46 (2004)   Bacillus thuringiensis Cry1A toxins, in contrast to other pore-forming toxins, bind two putative receptor molecules, aminopeptidase N (APN) and cadherin-like proteins. Here we show that Cry1Ab toxin binding to these two receptors depends on the toxins' oligomeric structure. Toxin monomeric structure binds to Bt-R₁, a cadherin-like protein, that induces proteolytic processing and oligomerization of the toxin (Gómez, I., Sánchez, J., Miranda, R., Bravo A., Soberón, M., FEBS Lett. (2002) 513, 242–246), while the oligomeric structure binds APN, which drives the toxin into the detergent-resistant membrane (DRM) microdomains causing pore formation. Cleavage of APN by phospholipase C prevented the location of Cry1Ab oligomer and Bt-R₁ in the DRM microdomains and also attenuates toxin insertion into membranes despite the presence of Bt-R₁. Immunoprecipitation experiments demonstrated that initial Cry1Ab toxin binding to Bt-R₁ is followed by binding to APN. Also, immunoprecipitation of Cry1Ab toxin-binding proteins using pure oligomeric or monomeric structures showed that APN was more efficiently detected in samples immunoprecipitated with the oligomeric structure, while Bt-R₁ was preferentially detected in samples immunoprecipitated with the monomeric Cry1Ab. These data agrees with the 200-fold higher apparent affinity of the oligomer than that of the monomer to an APN enriched protein extract. Our data suggest that the two receptors interact sequentially with different structural species of the toxin leading to its efficient membrane insertion.  

3.560           Fas ligand is enriched in the caveolae membrane domains of thymic epithelial cells

Lalor, D., Liu, P. and Hayashi, J. Cellular Immunol., 230, 10-16 (2004)   Both Fas and Fas ligand (FasL) are expressed in the thymus. Although reports suggest that they are important throughout the thymocyte maturation process their precise role remains elusive. The present paper characterizes the expression of FasL in the thymus and in the TEA3A1 and BT1B functional thymic epithelial cell (TEC) lines. FasL expression by thymus fractions, TEA3A1, and BT1B cells was detected by Northern blot analysis. In TEA3A1 cells, we discovered that FasL protein expression was localized to caveolae membrane domains. This restricted subcellular localization of FasL, together with reports describing the localization of the major histocompatibility complex proteins, the T cell receptor and Fas to caveolae membrane domains, may provide a mechanism for the deletion of thymocytes during negative selection. Finally, using semi-quantitative RT-PCR we found that FasL expression by TECs is regulated by glucocorticoids.  

3.561           Partitioning of NAP_i cotransporter in cholesterol-, sphingonyelin-, and glycophingolipid-enriched membrane domains modulates NAP_i protein diffusion, clustering, and activity

Inoue, M. et al

1. Biol. Chem., 279(47), 49160-49171 (2004)

  In dietary potassium deficiency there is a decrease in the transport activity of the type IIa sodium/phosphate cotransporter protein (NaP_i) despite an increase in its apical membrane abundance. This novel posttranslational regulation of NaP_i activity is mediated by the increased glycosphingolipid content of the potassium-deficient apical membrane. However, the mechanisms by which these lipids modulate NaP_i activity have not been determined. We determined if in potassium deficiency NaP_i is increasingly partitioned in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched microdomains of the apical membrane and if the increased presence of NaP_i in these microdomains modulates its activity. By using a detergent-free density gradient flotation technique, we found that 80% of the apical membrane NaP_i partitions into the low density cholesterol-, sphingomyelin-, and GM1-enriched fractions characterized as "lipid raft" fractions. In potassium deficiency, a higher proportion of NaP_i was localized in the lipid raft fractions. By combining fluorescence correlation spectroscopy and photon counting histogram methods for control and potassium-deficient apical membranes reconstituted into giant unilamellar vesicles, we showed a 2-fold decrease in lateral diffusion of NaP_i protein and a greater than 2-fold increase in size of protein aggregates/clusters in potassium deficiency. Our results indicate that NaP_i protein is localized in membrane microdomains, that in potassium deficiency a larger proportion of NaP_i protein is present in these microdomains, and that NaP_i lateral diffusion is slowed down and NaP_i aggregation/clustering is increased in potassium deficiency, both of which could be associated with the decreased Na/P_i cotransport activity in potassium deficiency.  

3.562           Nef associates with p21-activated kinase 2 in a p21-GTPase-dependent dynamic activation complex within lipid rafts

Pulkkinen, K., Renkema, G.H., Kirchhoff, F. And Saksela, K.

1. Virol., 78(23), 12773-12780 (2004)

  We have previously reported that Nef specifically interacts with a small but highly active subpopulation of p21-activated kinase 2 (PAK2). Here we show that this is due to a transient association of Nef with a PAK2 activation complex within a detergent-insoluble membrane compartment containing the lipid raft marker GM1. The low abundance of this Nef-associated kinase (NAK) complex was found to be due to an autoregulatory mechanism. Although activation of PAK2 was required for assembly of the NAK complex, catalytic activity of PAK2 also promoted dissociation of this complex. Testing different constitutively active PAK2 mutants indicated that the conformation associated with p21-mediated activation rather than kinase activity per se was required for PAK2 to become NAK. Although association with PAK2 is one of the most conserved properties of Nef, we found that the ability to stimulate PAK2 activity differed markedly among divergent Nef alleles, suggesting that PAK2 association and activation are distinct functions of Nef. However, mutations introduced into the p21-binding domain of PAK2 revealed that p21-GTPases are involved in both of these Nef functions and, in addition to promoting PAK2 activation, also help to physically stabilize the NAK complex.    

3.563           Identification of Epstein-Barr virus RK-BARF0-interacting proteins and characterization of expression pattern

Thornburg, N.J., Kusano, S. and Raab-Traub, N.

1. Virol., 78(23), 12848-12856 (2004)

  The Epstein-Barr virus (EBV) BamHI A transcripts are a family of transcripts that are differentially spliced and can be detected in multiple EBV-associated malignancies. Several of the transcripts may encode proteins. One transcript of interest, RK-BARF0, is proposed to encode a 279-amino-acid protein with a possible endoplasmic reticulum-targeting sequence. In this study, the properties of RK-BARF0 were examined through identification of cellular-interacting proteins through yeast two-hybrid analysis and characterization of its expression in EBV-infected cells and tumors. In addition to the interaction previously identified with cellular Notch, it was determined that RK-BARF0 also bound cellular human I-mfa domain-containing protein (HIC), epithelin, and scramblase. An interaction between RK-BARF0 and Notch or epithelin induced proteasome-dependent degradation of Notch and epithelin but not of HIC or scramblase. Low levels of endogenous Notch expression in EBV-positive cell lines may correlate with RK-BARF0 expression. However, a screen of EBV-positive cell lines and tumors with an affinity-purified -RK-BARF0 antiserum did not consistently detect RK-BARF0. These data suggest that while RK-BARF0 may have important cellular functions during EBV infection, and while the phenotype of EBV-positive cells suggest its expression, RK-BARF0 levels may be too low to detect.  

3.564           A mammalian ortholog of Saccharomyces cerevisiae Vac14 that associates with and Up-regulates PIKfyve phosphoinositide 5-kinase activity

Sbrissa, D. et al Mol. Cell Biol., 24(23), 10437-10447 (2004)   Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P₂, produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14. PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated. A small-interfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P₂ production. Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions. Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity. Concordantly, intracellular PtdIns(3)P-to-PtdIns(3,5)P₂ conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14. These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P₂ synthesis and endomembrane homeostasis in mammalian cells.  

3.565           Localization of organelle proteins by isotope tagging (LOPIT)

Dunkley, T.P.J., Watson, R., Griffin, J.L., Dupree, P. and Liley, K.S. Mol. Cell. Proteomics, 3, 1128-1134 (2004)   We describe a proteomics method for determining the subcellular localization of membrane proteins. Organelles are partially separated using centrifugation through self-generating density gradients. Proteins from each organelle co-fractionate and therefore exhibit similar distributions in the gradient. Protein distributions can be determined through a series of pair-wise comparisons of gradient fractions, using cleavable ICAT to enable relative quantitation of protein levels by MS. The localization of novel proteins is determined using multivariate data analysis techniques to match their distributions to those of proteins that are known to reside in specific organelles. Using this approach, we have simultaneously demonstrated the localization of membrane proteins in both the endoplasmic reticulum and the Golgi apparatus in Arabidopsis. Localization of organelle proteins by isotope tagging is a new tool for high-throughput protein localization, which is applicable to a wide range of research areas such as the study of organelle function and protein trafficking.      

3.566           Modulation of cyclin D1 and early growth response factor-1 gene expression in interleukin-1b-treated rat smooth muscle cells by n-6 and n-3 polyunsaturated fatty acids

Bousserouel, S., Raymondjean, M., Brouillet, A., Bereziat, G. and Andreani, M. Eur. J. Biochem., 271, 4462-4473 (2004)   The proliferation of smooth muscle cells (SMC) is a key event in the development of atherosclerosis. In addition to growth factors or cytokines, we have shown previously that n-3 polyunsaturated fatty acids (PUFAs) act in opposition to n-6 PUFAs by modulating various steps of the inflammatory process. We have investigated the molecular mechanisms by which the incorporation of the n-6 PUFA, arachidonic acid, increases the proliferation of rat SMC treated with interleukin-1 , while the n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), elicit no mitogenic response. Incorporation of EPA or DHA into SMC, which are then activated by interleukin-1 to mimic inflammation, decreases promoter activity of the cyclin D1 gene and phosphorylation of the retinoblastoma protein. Together, our data demonstrate that n-3 effects are dependent on the Ras/Raf-1/extracellular signal regulated kinase (ERK)/mitogen-activated protein kinase pathway, and that down-regulation of the cyclin D1 promoter activity is mediated by the specific binding of the early growth response factor-1. Finally, we have shown that the incorporation of EPA and DHA also increased the concentration of caveolin-1 and caveolin-3 in caveolae, which correlated with n-3 PUFA inhibition of SMC proliferation through the mitogen-activated protein kinase pathway. We provide evidence indicating that, in contrast to n-6 PUFAs, n-3 PUFAs exert antiproliferative effects on SMC through the mitogen-activated protein kinase/ERK pathway.  

3.567           Distribution of Can1p into stable domains reflects lateral protein segregation within the plasma membrane of living S. cerevisiae cells

Malinska, K., Malinsky, J., Opekarova, M. And Tanner, W.

1. Cell Sci., 117, 6031-6041 (2004)

  Recently, lipid-raft-based subdomains within the plasma membrane of living Saccharomyces cerevisiae cells were visualized using green fluorescent protein fusions, and non-overlapping subdomains containing either Pma1p or Can1p were distinguished. In this study, the long-term stability of the subdomains was investigated. Experiments with latrunculin A and nocodazole ruled out the involvement of cytoskeletal components in the stabilization of the subdomains. Also a putative role of the cell wall was excluded, because protoplasting of the cells changed neither the pattern nor the stability of the subdomains. By contrast, the expected inner dynamics of the membrane subdomains was documented by FRAP experiments. Finally, two other proteins were localized within the frame of the Can1p/Pma1p plasma-membrane partition. We show that Fur4p (another H⁺ symporter) and Sur7p (a protein of unknown function) occupy the Can1p subdomain.  

3.568           The H⁺ -pyrophosphatase of Rhodospirillum rubrun is predominantly located in polyphosphate-rich acidocalcisomes

Seufferheld, M., Lea, C.R., Vieira, M., Oldfiled, E. and Docampo, R.

1. Biol. Chem., 279(49), 51193-51202 (2004)

  Acidocalcisomes are acidic, calcium storage compartments with a H⁺ pump located in their membrane that have been described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds, and have also been found in the bacterium Agrobacterium tumefaciens. In this work, we report that the H⁺-pyrophosphatase (H⁺-PPase) of Rhodospirillum rubrum, the first enzyme of this type that was identified and thought to be localized only to chromatophore membranes, is predominantly located in acidocalcisomes. The identification of the acidocalcisomes of R. rubrum was carried out by using transmission electron microscopy, x-ray microanalysis, and immunofluorescence microscopy. Purification of acidocalcisomes using iodixanol gradients indicated co-localization of the H⁺-PPase with pyrophosphate (PP_i) and short and long chain polyphosphates (polyPs) but a lack of markers of the plasma membrane. polyP was also localized to the acidocalcisomes by using 4',6'-diamino-2-phenylindole staining and identified by using ³¹P NMR and biochemical methods. Calcium in the acidocalcisomes increased when the bacteria were incubated at high extracellular calcium concentrations. The number of acidocalcisomes and chromatophore membranes as well as the amounts of PP_i and polyP increased when bacteria were grown in the light. Taken together, these results suggest that the H⁺-PPase of R. rubrum has two distinct roles depending on its location acting as an intracellular proton pump in acidocalcisomes but in PP_i synthesis in the chromatophore membranes.  

3.569           Bovine caveolin-2 cloning and effects of shear stress on its localization in bovine aortic endothelial cells

Boyd, N.L. et al Endothelium, 11, 189-198 (2004)   Caveolae are plasmalemmal domains enriched with cholesterol, caveolins, and signaling molecules. Normally, cells that express caveolin-1 also express caveolin-2, but this has not been demonstrated in bovine aortic endothelial cells (BAECs). Here, we show that BAECs express caveolin-2, which localizes in caveolae with caveolin-1. We have cloned the bovine caveolin-2 gene and after comparison with known protein sequences (human, murine, rat, and canine) have found divergent immunogenic regions (amino acid [aa] 21 to aa 50 and aa 79 to 88), which may explain the inability to detect caveolin-2 in different cell types. We developed a bovine caveolin-2–specific antibody to examine this protein's expression and localization in BAECs. We used differential gradient centrifugations and immunoprecipitation to show that bovine caveolin-2 and caveolin-1 form a hetero-oligomer in plasma membrane caveolae. Using immunocytochemistry we show that a pool of caveolin-2 also colocalizes with the cis-Golgi in static culture, but unlike caveolin-1, this Golgi associated pool is maintained after 1 day of shear exposure. Therefore, the interaction of caveolin-2 with caveolin-1 could play an important role in caveolae biogenesis and shear stimulated mechano-signal transduction.  

3.570           Plasmin deficiency in Alzheimer’s disease brains: causal or casual?

Dotti, C.G., Galvan, C. And Ledesma, M.D. Neurodegenerative Dis., 1, 205-212 (2004)   Substantial recent evidence suggests that defects in amyloid peptide degradation can be at the base of cases of sporadic Alzheimer's disease (AD). Among the discovered brain enzymes with the capacity to degrade amyloid peptide, the serine protease plasmin acquires special physiological relevance because of its low levels in areas of AD human brains with a high susceptibility to amyloid plaque accumulation. In this article we comment on a series of observations supporting the fact that plasmin paucity in the brain is not simply a secondary event in the disease but rather a primary defect in certain cases of sporadic AD. We also refer to recent data pointing to alterations in raft membrane domains and diminished membrane cholesterol as the underlying cause. Finally, we discuss the possibility that plasmin deficiency in the brain could lead to AD symptomatology because of amyloid aggregation and the triggering of cell death signaling cascades.  

3.571           Leaky b-oxidation of a trans –fatty acid

Yu, W. et al

1. Biol. Chem., 279(50), 52160-52167 (2004)

  The degradation of elaidic acid (9-trans-octadecenoic acid), oleic acid, and stearic acid by rat mitochondria was studied to determine whether the presence of a trans double bond in place of a cis double bond or no double bond affects -oxidation. Rat mitochondria from liver or heart effectively degraded the coenzyme A derivatives of all three fatty acids. However, with elaidoyl-CoA as a substrate, a major metabolite accumulated in the mitochondrial matrix. This metabolite was isolated and identified as 5-trans-tetradecenoyl-CoA. In contrast, little or none of the corresponding metabolites were detected with oleoyl-CoA or stearoyl-CoA as substrates. A kinetic study of long-chain acyl-CoA dehydrogenase (LCAD) and very long-chain acyl-CoA dehydrogenase revealed that 5-trans-tetradecenoyl-CoA is a poorer substrate of LCAD than is 5-cis-tetradecenoyl-CoA, while both unsaturated acyl-CoAs are poor substrates of very long-chain acyl-CoA dehydrogenase when compared with myristoyl-CoA. Tetradecenoic acid and tetradecenoylcarnitine were detected by gas chromatography/mass spectrometry and tandem mass spectrometry, respectively, when rat liver mitochondria were incubated with elaidoyl-CoA but not when oleoyl-CoA was the substrate. These observations support the conclusion that 5-trans-tetradecenoyl-CoA accumulates in the mitochondrial matrix, because it is less efficiently dehydrogenated by LCAD than is its cis isomer and that the accumulation of this -oxidation intermediate facilitates its hydrolysis and conversion to 5-trans-tetradecenoylcarnitine thereby permitting a partially degraded fatty acid to escape from mitochondria. Analysis of this compromised but functional process provides insight into the operation of -oxidation in intact mitochondria.  

3.572           Acidocalcisomes and the contractile vacuole complex are involved in osmoregulation in Trypanosoma cruzi

Rohloff, P., Montalvetti, A. and Docompo, R.

1. Biol. Chem., 279(50), 52270-52281 (2004)

  Trypanosoma cruzi, the etiologic agent of Chagas disease, resists extreme fluctuations in osmolarity during its life cycle. T. cruzi possesses a robust regulatory volume decrease mechanism that completely reverses cell swelling when submitted to hypo-osmotic stress. The efflux of amino acids and K⁺ release could account for only part for this volume reversal. In this work we demonstrate that swelling of acidocalcisomes mediated by an aquaporin and microtubule- and cyclic AMP-mediated fusion of acidocalcisomes to the contractile vacuole complex with translocation of this aquaporin and the resulting water movement are responsible for the volume reversal not accounted for by efflux of osmolytes. Contractile vacuole bladders were isolated by subcellular fractionation in iodixanol gradients, showed a high concentration of basic amino acids and inorganic phosphate, and were able to transport protons in the presence of ATP or pyrophosphate. Taken together, these results strongly support a role for acidocalcisomes and the contractile vacuole complex in osmoregulation and identify a functional role for aquaporin in protozoal osmoregulation.  

3.573           Intracellular processing and activation of membrane type 1 matrix metalloprotease depends on its partitioning into lipid domains

Mazzone, M. et al

1. Cell Sci., 117, 6275-6287 (2004)

  The integral membrane type 1 matrix metalloprotease (MT1-MMP) is a pivotal protease in a number of physiological and pathological processes and confers both non-tumorigenic and tumorigenic cell lines with a specific growth advantage in a three-dimensional matrix. Here we show that, in a melanoma cell line, the majority (80%) of MT1-MMP is sorted to detergent-resistant membrane fractions; however, it is only the detergent-soluble fraction (20%) of MT1-MMP that undergoes intracellular processing to the mature form. Also, this processed MT1-MMP is the sole form responsible for ECM degradation in vitro. Finally, furin-dependent processing of MT1-MMP is shown to occur intracellularly after exit from the Golgi apparatus and prior to its arrival at the plasma membrane. It is thus proposed that the association of MT1-MMP with different membrane subdomains might be crucial in the control of its different activities: for instance in cell migration and invasion and other less defined ones such as MT1-MMP-dependent signaling pathways.  

3.574           Role of myosin VIIa and Rab27a in the motility and localization of RPE nelanosomes

Gibbs, D. Et al

1. Cell Sci., 117, 6473-6483 (2004)

  Myosin VIIa functions in the outer retina, and loss of this function causes human blindness in Usher syndrome type 1B (USH1B). In mice with mutant Myo7a, melanosomes in the retinal pigmented epithelium (RPE) are distributed abnormally. In this investigation we detected many proteins in RPE cells that could potentially participate in melanosome transport, but of those tested, only myosin VIIa and Rab27a were found to be required for normal distribution. Two other expressed proteins, melanophilin and myosin Va, both of which are required for normal melanosome distribution in melanocytes, were not required in RPE, despite the association of myosin Va with the RPE melanosome fraction. Both myosin VIIa and myosin Va were immunodetected broadly in sections of the RPE, overlapping with a region of apical filamentous actin. Some 70-80% of the myosin VIIa in RPE cells was detected on melanosome membranes by both subcellular fractionation of RPE cells and quantitative immunoelectron microscopy, consistent with a role for myosin VIIa in melanosome motility. Time-lapse microscopy of melanosomes in primary cultures of mouse RPE cells demonstrated that the melanosomes move in a saltatory manner, interrupting slow movements with short bursts of rapid movement (>1 µm/second). In RPE cells from Myo7a-null mice, both the slow and rapid movements still occurred, except that more melanosomes underwent rapid movements, and each movement extended approximately five times longer (and further). Hence, our studies demonstrate the presence of many potential effectors of melanosome motility and localization in the RPE, with a specific requirement for Rab27a and myosin VIIa, which function by transporting and constraining melanosomes within a region of filamentous actin. The presence of two distinct melanosome velocities in both control and Myo7a-null RPE cells suggests the involvement of at least two motors other than myosin VIIa in melanosome motility, most probably, a microtubule motor and myosin Va.  

3.575           N-octyl-b-valienamine up-regulates activity of F213I mutant b-glucosidase in cultured cells: a potential chemical chaperone therapy for Gaucher disease

Lin, H. et al Biochim. Biophys. Acta, 1689, 219-228 (2004)   Gaucher disease (GD) is the most common form of sphingolipidosis and is caused by a defect of β-glucosidase (β-Glu). A carbohydrate mimic N-octyl-β-valienamine (NOV) is an inhibitor of β-Glu. When applied to cultured GD fibroblasts with F213I β-Glu mutation, NOV increased the protein level of the mutant enzyme and up-regulated cellular enzyme activity. The maximum effect of NOV was observed in F213I homozygous cells in which NOV treatment at 30 μM for 4 days caused a 6-fold increase in the enzyme activity, up to 80% of the activity in control cells. NOV was not effective in cells with other β-Glu mutations, N370S, L444P, 84CG and RecNciI. Immunofluorescence and cell fractionation showed localization of the F213I mutant enzyme in the lysosomes of NOV-treated cells. Consistent with this, NOV restored clearance of ¹⁴C-labeled glucosylceramide in F213I homozygous cells. F213I mutant β-Glu rapidly lost its activity at neutral pH in vitro and this pH-dependent loss of activity was attenuated by NOV. These results suggest that NOV works as a chemical chaperone to accelerate transport and maturation of F213I mutant β-Glu and may suggest a therapeutic value of this compound for GD.  

3.576           Overexpression of caveolin-1 increases plasma membrane fluidity and reduces P-glycoprotein function in Hs578T/Dox

Cai, C., Zhu, H. and Chen, J. Biochem. Biophys Res. Comm., 320, 868-874 (2004)   Cholesterol is a key lipid in mediating the enzyme activity or signaling pathway of many proteins on the plasma membrane in mammalian cells. In this report, we demonstrate for the first time that after overexpressing caveolin-1, the plasma membrane cholesterol level was decreased by about 12% and 30% for doxorubicin-sensitive and doxorubicin-resistant Hs578T breast cancer cells, respectively. However, the total cholesterol level in both cell lines was increased by about 10%. By measuring fluorescence and flow cytometry using the fluorescence dyes 1,6-diphenyl-1,3,5-hexatriene and Merocyanine 540, we found that overexpressing caveolin-1 resulted in a similar increase in membrane fluidity and loosening of lipid packing density as cholesterol depletion by 1 mM methyl-β-cyclodextrin (MβCD) or 2-hydroxypropyl-β-cyclodextrin (HβCD). Moreover, we found that the transport activity of P-gp was significantly inhibited by 1 mM MβCD or HβCD, which is also similar to the inhibitory effect of caveolin-1 overexpression. Our data demonstrate for the first time that the reduction of the plasma membrane cholesterol level induced by overexpressing caveolin-1 may indirectly inhibit P-gp transport activity by increasing plasma membrane fluidity.  

3.577           Depression of transcription and translation during daily torpor in the Djungarian hamster (Phodopus sungorus)

Diaz, M.B., Lange, M., Heldmaier, G. And Klingenspor, M.-

1. Comp. Physiol. B, 174, 495-502 (2004)

  During daily torpor, Djungarian hamsters reduce their metabolic rate by more than 70% below their resting metabolic rate for several hours per day. We investigated whether this depression of metabolism is associated with a reduction in transcription and translation. Liver tissue was sampled in defined metabolic states: during normometabolism, in the torpid state and after arousal from torpor. Nuclei were isolated from liver tissue and subjected to nuclear run-on assays at an assay temperature of 25 °C. We observed a ~40% decrease in transcriptional initiation in liver nuclei of hamsters which had attained minimal metabolic rate during torpor as compared to nuclei from normometabolic hamsters. During arousal from torpor, the transcriptional run-on activity recovered to the normometabolic level. Polysome profile analysis of liver tissue was used to determine the proportion of actively translating polysomes. Profiles of liver samples from torpid animals show a disaggregation of polysomes compared to profiles from normometabolic hamsters, which indicates that, in addition to transcription, protein synthesis decreases during torpor. These results indicate that during torpor a specific inhibition of the energetically costly processes of RNA and protein synthesis contributes to the overall metabolic depression.  

3.578           Tyrosine phosphatase activity in mitochondria: presence of Shp-2 phosphatase in mitochondria

Salvi, M. Et al Cell. Mol. Life Sci., 61, 2393-2404 (2004)   Tyrosine phosphorylation by unidentified enzymes has been observed in mitochondria, with recent evidence indicating that non-receptorial tyrosine kinases belonging to the Src family, which represent key players in several transduction pathways, are constitutively present in mitochondria. The extent of protein phosphorylation reflects a coordination balance between the activities of specific kinases and phophatases. The present study demonstrates that purified rat brain mitochondria possess endogenous tyrosine phosphatase activity. Mitochondrial phosphatases were found to be capable of dephosphorylating different exogenous substrates, including paranitrophenylphosphate, ³²P-poly(Glu-Tyr)_4:1 and ³²P-angiotensin. These activities are strongly inhibited by peroxovanadate, a well-known inhibitor of tyrosine phosphatases, but not by inhibitors of alkali or Ser/Thr phosphatases, and mainly take place in the intermembrane space and outer mitochondrial membrane. Using a combination of approaches, we identified the tyrosine phosphatase Shp-2 in mitochondria. Shp-2 plays a crucial role in a number of intracellular signalling cascades and is probably involved in several human diseases. It thus represents the first tyrosine phosphatase shown to be present in mitochondria.  

3.579           Paradoxical downregulation of the glucose oxidation pathway despite enhanced flux in severe heart failure

Lei, B. Et al

1. Mol. Cell. Cardiol., 36, 567-576 (2004)

  Free fatty acid (FFA) oxidation is depressed in severe heart failure due to reduced activity of mitochondrial fatty acid oxidation enzymes. It is unknown whether the concomitant enhancement in cardiac glucose use is a consequence of reduced FFA oxidation, or also due to potentiation of the carbohydrate oxidative pathway. FFA and glucose oxidation rates were measured in vivo in 9 normal dogs and 9 dogs with pacing-induced heart failure by infusing ³H-oleate and ¹⁴C-glucose. FFA oxidation was lower (39 ± 9 vs. 73 ± 5 nmol min^–1 g^–1), while glucose oxidation was higher (42 ± 8 vs. 17 ± 6 nmol min^–1 g^–1) in failing compared to normal hearts (P < 0.05). At the end of the in vivo experiment, clamp-frozen biopsies were harvested from the left ventricle. Messenger RNAs encoding for proteins involved in both glucose and fatty acid metabolism, and for citrate synthase, were significantly reduced. Protein expression of GLUT-1 and GLUT-4, and GLUT-4 translocation to the sarcolemma showed no significant differences between the two groups despite a significant reduction in mRNAs with heart failure. GAPDH mRNA, protein expression, and activity were all reduced. The E2 subunit of pyruvate dehydrogenase was decreased both at the mRNA and protein level, with no effect on either fractional or maximal activity. In conclusion, we found either no changes or moderate downregulation of key enzymes of the carbohydrate metabolism in failing hearts, which suggests that the increase in glucose oxidation in vivo was principally due to impaired FFA oxidation and that the maximal myocardial capacity to obtain energy from substrate is globally depressed.  

3.580           Acidocalsisomes of Phytomonas francai possess distinct morphological characteristics and contain iron

Miranda, K. et al Microsc. Microanal., 10, 647-655 (2004)   Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and apicomplexan parasites, and recently found in other unicellular eukaryotes. The aim of this study was to identify the presence of acidocalcisomes in the plant trypanosomatid Phytomonas francai. Electron-dense organelles of P. francai were shown to contain large amounts of oxygen, sodium, magnesium, phosphorus, potassium, calcium, iron, and zinc as determined by X-ray microanalysis, either in situ or when purified using iodixanol gradient centrifugation or by elemental mapping. The presence of iron is not common in other acidocalcisomes. In situ, but not when purified, these organelles showed an elongated shape differing from previously described acidocalcisomes. However, these organelles also possessed a vacuolar H+-pyrophosphatase (V-H+-PPase) as determined by biochemical methods and by immunofluorescence microscopy using antibodies against the enzyme. Together, these results suggest that the electron-dense organelles of P. francai are homologous to the acidocalcisomes described in other trypanosomatids, although with distinct morphology and elemental content.  

3.581           Mechanisms of neurotrophin receptor vesicular transport

Yano, H. and Chao, M.V.

1. Neurobiol., 58, 244-257 (2004)

  Accumulating evidence has indicated that neurotrophin receptor trafficking plays an important role in neurotrophin-mediated signaling in developing as well as mature neurons. However, little is known about the molecular mechanisms and the components of neurotrophin receptor vesicular transport. This article will describe how neurotrophin receptors, Trk and p75 neurotrophin receptor (p75^NTR), are intimately involved in the axonal transport process. In particular, the molecules that may direct Trk receptor trafficking in the axon will be discussed. Finally, potential mechanisms by which receptor-containing vesicles link to molecular cytoskeletal motors will be presented.  

3.582           Effect of Membrane Perturbants on the Activity and Phase Distribution of Inositol Phosphorylceramide Synthase; Development of a Novel Assay

Aeed, P.A., Sperry, A.E., Young, C.L., Nagiec, M.M. andf Elhammer, Å.P. Biochemistry, 43, 8483-8493 (2004)   The effect of 26 different membrane-perturbing agents on the activity and phase distribution of inositol phosphorylceramide synthase (IPC synthase) activity in crude Candida albicans membranes was investigated. The nonionic detergents Triton X-100, Nonidet P-40, Brij, Tween, and octylglucoside all inactivated the enzyme. However, at moderate concentrations, the activity of the Triton X-100- and octylglucoside-solubilized material could be partially restored by inclusion of 5 mM phosphatidylinositol (PI) in the solubilization buffer. The apparent molecular mass of IPC synthase activity solubilized in 2% Triton X-100 was between 1.5 × 10⁶ and 20 × 10⁶ Da, while under identical conditions, octylglucoside-solubilized activity remained associated with large presumably membrane-like structures. Increased detergent concentrations produced more drastic losses of enzymatic activity. The zwitterionic detergents Empigen BB, N-dodecyl-N,N-(dimethylammonio)butyrate (DDMAB), Zwittergent 3-10, and amidosulfobetaine (ASB)-16 all appeared capable of solubilizing IPC synthase. However, these agents also inactivated the enzyme essentially irreversibly. Solubilization with lysophospholipids again resulted in drastic losses of enzymatic activity that were not restored by the inclusion of PI. Lysophosphatidylinositol also appeared to compete, to some extent, with the donor substrate phosphatidylinositol. The sterol-containing agent digitonin completely inactivated IPC synthase. By contrast, sterol-based detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and taurodeoxycholate (tDOC) had little or no effect on the enzyme activity. The IPC synthase activity in C. albicans membranes remained largely intact and sedimentable at CHAPS concentrations (4%) where >90% of the phospholipids and 60% of the total proteins were extracted from the membranes. At 2.5% CHAPS, a concentration where approximately 50% of the protein and 80% of the phospholipids are solubilized, there was no detectable loss of enzyme activity, and it was found that the detergent-treated membranes had significantly improved properties compared to crude, untreated membranes as the source of IPC synthase activity. In contrast to assays utilizing intact membranes or Triton X-100 extracts, assays using CHAPS- or tDOC-washed membranes were found to be reproducible, completely dependent on added acceptor substrate (C₆-7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-ceramide), and >95% dependent on added donor substrate (PI). Product formation was linear with respect to both enzyme concentration and time, and transfer efficiency was improved more than 20-fold as compared to assays using crude membranes. Determination of kinetic parameters for the two IPC synthase substrates using CHAPS-washed membranes resulted in K_m values of 3.3 and 138.0 M for C₆-NBD-ceramide and PI, respectively. In addition, the donor substrate, PI, was found to be inhibitory at high concentrations with an apparent K_i of 588.2 M.  

3.583            

 

3.584           CD4-induced down-regulation of T cell adhesion to B cells is associated with localization of phosphatidyl inositol 3-kinase and LFA-1 in distinct membrane domains

Trucy, M., Barbat, C., Sorice, M., Fischer, A. and Mazerolles, F. Eur. J: Immunol., 34(8), 2168-2178 (2004)   We have previously shown that binding of anti-CD4 antibody inhibit LFA-1-dependent adhesion between CD4⁺ T cells and B cells in a p56^lck and a PI3-kinase-dependent manner. In this work, we investigated with two different T cell lines (Jurkat and A201) whether CD4 binding could alter interactions of the proteins putatively involved in this adhesion regulatory pathway. Anti-CD4 binding was shown to induce a transient association between PI3-kinase and LFA-1, which took place in different regions of the plasma membrane. It was detected in detergent soluble membrane but also in detergent insoluble membrane consisting in raft microdomains, composed of GM1 and/or GM3 gangliosides. These results show that anti-CD4 Ab could modify the interaction between LFA-1 and signaling molecules, such as PI3-kinase and induce, in part, their recruitment in raft domains. By using specific inhibitors, raft integrity and CD4 association with GM3 were found necessary for observing the CD4-dependent inhibition of LFA-1-mediated adhesion. These results strongly suggest that these molecular rearrangements in the membrane are necessary to induce down-regulation of LFA-1-mediated adhesion.  

3.585           Comparison of nerve terminal events in vivo effecting retrograde transport of vesicles containing neurotrophins or synaptic vesicle components

Weible II, M.W., Ozsarac, N., Grimes, M.L. and Hendry, I.A.

1. Neurosci. Res., 75(6), 771-781 (2004)

  Although vesicular retrograde transport of neurotrophins in vivo is well established, relatively little is known about the mechanisms that underlie vesicle endocytosis and formation before transport. We demonstrate that in vivo not all retrograde transport vesicles are alike, nor are they all formed using identical mechanisms. As characterized by density, there are at least two populations of vesicles present in the synaptic terminal that are retrogradely transported along the axon: those containing neurotrophins (NTs) and those resulting from synaptic vesicle recycling. Vesicles containing nerve growth factor (NGF), NT-3, or NT-4 had similar densities with peak values at about 1.05 g/ml. Synaptic-derived vesicles, labeled with anti-dopamine β-hydroxylase (DBH), had densities with peak values at about 1.16 g/ml. We assayed the effects of pharmacologic agents in vivo on retrograde transport from the anterior eye chamber to the superior cervical ganglion. Inhibitors of phosphatidylinositol-3-OH (PI-3) kinase and actin function blocked transport of both anti-DBH and NGF, demonstrating an essential role for these molecules in retrograde transport of both vesicle types. Dynamin, a key element in synaptic vesicle recycling, was axonally transported in retrograde and anterograde directions, and compounds able to interfere with dynamin function had a differential effect on retrograde transport of NTs and anti-DBH. Okadaic acid significantly decreased retrograde axonal transport of anti-DBH and increased NGF retrograde transport. We conclude that there are both different and common proteins involved in endocytosis and targeting of retrograde transport of these two populations of vesicles.

3.586           Distinct protective mechanisms of HO-1 and HO-2 against hydroperoxide-induced cytotoxicity

Kim, Y-S., Zhuang, H., Koehler, R.C. and Dore, S. Free Radical Biology & Medicine, 38, 85-92 (2005)   Heme oxygenases (HO-1 and HO-2) catalyze the NADPH-cytochrome P₄₅₀ reductase (CPR)-dependent degradation of heme into iron, carbon monoxide, and biliverdin, which is reduced into bilirubin. Under basal conditions, HO-1 is often undetected and can be induced by numerous stress conditions. Although HO-2 is constitutively expressed, its activity appears to be regulated by post-translational modifications. HO activity has been associated with cellular protection, by which it degrades heme, a prooxidant, into bioactive metabolites. Under given circumstances, overexpression of HO-1 can render cells more sensitive to free radicals. Here, we investigated the properties of human HO isoforms that protect against oxidative stress. Considering that CPR can be a limiting factor for optimal HO activity, we tested stable HO-1 and HO-2 cell lines that derived from the CPR cells. Results indicate that the HO-1 and HO-2 cells are more resistant than controls to hemin and to the organic tert-butyl hydroperoxide, t-BuOOH. However, HO-1 cells are less resistant than HO-2 cells to hydrogen peroxide (H₂O₂). The levels of oxidatively modified proteins of HO-1 and HO-2 cells in response to t-BuOOH toxicity are identical, but the level of oxidatively modified proteins of HO-2 cells is less than that of HO-1 cells in response to H₂O₂ toxicity. Performing subcellular fractionations revealed that HO-2 and CPR are found together in the microsomal fractions, whereas HO-1 is partially present in the microsome and also found in other fractions, such as the cytosol. These same findings were observed in non-transfected primary neurons where HO-1 proteins were chemically induced with 15-deoxy-Δ^12,14-prostaglandin J₂ (15dPGJ₂). The differences in subcellular localization of HO-1 and HO-2 could explain some of the discrepancies in their cellular activity and enzymatic protective mechanisms.  

3.587           Calpain and other cytosolic proteases can contribute to the degradation of retro-translocated prion protein in the cytosol

Wang, X., Wang, F., Sy, M-S- and Ma, J.

1. Biol. Chem., 280(1), 317-325 (2005)

  PrP, a cell surface-localized N-linked glycoprotein, is required for the pathogenesis of prion diseases. Recent studies have revealed that prion protein (PrP) becomes neurotoxic and prone to aggregation when it is in the cytosol, suggesting that cytosolic PrP may play a role in the pathogenesis of prion disease. Retro-translocation of PrP from the endoplasmic reticulum to the cytosol for proteasome degradation offers a natural route for PrP to enter the cytosol, but whether PrP is subject to retrotranslocation is controversial. In this study, we investigated the metabolism of endogenous wild-type PrP in several cell lines and in primary mouse cortical neurons. Our results suggest that a portion of the endogenous wild-type PrP is retro-translocated to the cytosol and degraded by the proteasome. Moreover, we also found that calpain and other cytosolic proteases could degrade PrP in the cytosol when the proteasome activity is compromised. These results provide the foundation for the hypothesis that cytosolic PrP may be involved in the pathogenesis of prion disease.  

3.588           Exogenous antigens are processed through the endoplasmic reticulum-associated degradatin (ERAD) in cross-presentation by dendritic cells

Imai, J., Hasegawa, H., Maruya, M., Koyasu, S. and Yahara, I. Int. Immunol., 17(1), 45-53 (2005)   Antigen cross-presentation is critical in infectious and tumor immunity where cytotoxic T lymphocytes are induced by dendritic cells specifically equipped with cellular machineries to present exogenous antigens with major histocompatibility complex (MHC) class I molecules. To examine molecular mechanisms of antigen cross-presentation, we employed as a model system a murine dendritic cell line DC2.4 capable of presenting soluble antigens such as ovalbumin (OVA) with MHC class I. Here, we demonstrate that exogenously added OVA is accumulated in the endoplasmic reticulum (ER) and late endosomes followed by retrograde transport to the cytoplasm through the Sec61 transporter complexes, and that CHIP functions as an E3 ubiquitin–ligase for OVA degradation by proteasomes. This mechanism is essentially the same as that known as the ER-associated degradation (ERAD) in the quality control of secretary and membrane proteins.  

3.589           Distinct localization of lipid rafts and externalized phosphotidylserine at the surface of apoptotic cells

Ishii, H. et al Biochem. Biophys. Res. Comm., 327, 94-99 (2005)   Externalization of phosphatidylserine (PS) takes place in apoptotic cells as well as in viable cells under certain circumstances. Recent studies showed that externalized PS is localized at the lipid raft in viable activated immune cells. We found that lipid rafts and PS existed in a mutually exclusive manner in apoptotic cells. The number of PS-exposing apoptotic cells decreased when lipid rafts were disrupted. BCθ, which binds selectively to cholesterol in a cholesterol-rich region, did not effectively recognize lipid rafts of apoptotic cells. Lipid rafts rich in GM1 were successfully prepared from apoptotic cells, but the lipid raft protein LAT was not enriched in the preparation. Furthermore, the amount of PS and phosphatidylethanolamine but not of cholesterol in lipid rafts appeared to change after induction of apoptosis. These results suggest that lipid rafts are structurally modified during apoptosis and, despite being localized differently from PS, are involved in the externalization of PS.      

3.590           FAT/CD36-mediated long-chain fatty acid uptake in adipocytes requires plasma membrane rafts

Pohl, J., Ring, A., Korkmaz, U., Ehehalt, R and Stremmel, W. Mol. Biol. Cell, 16(1), 24-31 (2005)   We previously reported that lipid rafts are involved in long-chain fatty acid (LCFA) uptake in 3T3-L1 adipocytes. The present data show that LCFA uptake does not depend on caveolae endocytosis because expression of a dominant negative mutant of dynamin had no effect on uptake of [³H]oleic acid, whereas it effectively prevented endocytosis of cholera toxin. Isolation of detergent-resistant membranes (DRMs) from 3T3-L1 cell homogenates revealed that FAT/CD36 was expressed in both DRMs and detergent-soluble membranes (DSMs), whereas FATP1 and FATP4 were present only in DSMs but not DRMs. Disruption of lipid rafts by cyclodextrin and specific inhibition of FAT/CD36 by sulfo-N-succinimidyl oleate (SSO) significantly decreased uptake of [³H]oleic acid, but simultaneous treatment had no additional or synergistic effects, suggesting that both treatments target the same mechanism. Indeed, subcellular fractionation demonstrated that plasma membrane fatty acid translocase (FAT/CD36) is exclusively located in lipid rafts, whereas intracellular FAT/CD36 cofractionated with DSMs. Binding assays confirmed that [³H]SSO predominantly binds to FAT/CD36 within plasma membrane DRMs. In conclusion, our data strongly suggest that FAT/CD36 mediates raft-dependent LCFA uptake. Plasma membrane lipid rafts might control LCFA uptake by regulating surface availability of FAT/CD36.  

3.591           Ypt3132 GTPases and their novel F-Box effector protein Rcy1 regulate protein recycling

Chen, S.H. et al Mol. Biol. Cell, 16(1), 178-192 (2005)   Ypt/Rab GTPases control various aspects of vesicle formation and targeting via their diverse effectors. We report a new role for these GTPases in protein recycling through a novel effector. The F-box protein Rcy1, which mediates plasma membrane recycling, is identified here as a downstream effector of the Ypt31/32 GTPase pair because it binds active GTP-bound Ypt31/32 and colocalizes with these GTPases on late Golgi and endosomes. Furthermore, Ypt31/32 regulates the polarized localization and half-life of Rcy1. This suggests that Ypt/Rabs can regulate the protein level of their effectors, in addition to the established ways by which they control their effectors. We show that like Rcy1, Ypt31/32 regulate the coupled phosphorylation and recycling of the plasma membrane v-SNARE Snc1. Moreover, Ypt31/32 and Rcy1 regulate the recycling of the furin-homolog Kex2 to the Golgi. Therefore, Ypt31/32 and Rcy1 mediate endosome-to-Golgi transport, because this is the only step shared by Snc1 and Kex2. Finally, we show that Rcy1 physically interacts with Snc1. Based on this result and because F-box proteins serve as adaptors between specific substrates and ubiquitin ligases, we propose that Ypt31/32 GTPases regulate the function of Rcy1 in the phosphorylation and/or ubiquitination of proteins that recycle through the Golgi.  

3.592           Trafficking and localization of platinum complexes in cisplatin-resistant cell lines monitored by fluorescence-labeled platinum

Liang, X-J. et al

1. Cell. Physiol., 202, 635-641 (2005)

  Cisplatin is a chemotherapeutic agent commonly used in the treatment of a wide variety of malignant tumors. Resistance to cisplatin represents a major obstacle to effective cancer therapy because clinically significant levels of resistance quickly emerge after treatment. Based on previous studies indicating abnormal plasma membrane protein trafficking in cisplatin-resistant (CP-r) cells, Fluorescence (Alexa Fluor)-labeled cisplatin was used to determine whether this defect altered the trafficking and localization of cisplatin by comparing drug sensitive KB-3-1 and KB-CP-r cells. Alexa Fluor-cisplatin was readily internalized and localized throughout the KB-3-1 cells, but overall fluorescence decreased in KB-CP-r cells, as detected by flow cytometry (FACS) and confocal microscopy. Only punctate cytoplasmic staining was observed in KB-CP-r cells with less fluorescence observed in the nucleus. Colocalization experiments with a Golgi-selective stain indicate the involvement of Golgi-like vesicles in initial intracellular processing of Alexa Fluor conjugated cisplatin complexes. As detected using an antibody to Alexa Fluor-cisplatin, cisplatin complex-binding proteins (CCBPs) were reduced in membrane fractions of single-step cisplatin-resistant KB-CP.5 cells, and increased in the cytoplasm of KB-CP.5 cells compared to KB-3-1 cells. CCBPs localized to lower density fractions in KB-CP.5 cells than in KB-3-1 cells as determined by iodixanol gradient centrifugation. In summary, inappropriate trafficking of CCBPs might explain resistance to cisplatin in cultured cancer cells, presumably because membrane binding proteins for cisplatin are not properly located on the cell surface in these cells, but are instead trapped in low density vesicles within the cytoplasm.  

3.593           Fragile X protein functions with LgI and the PAR complex in flies and mice

Zarnescu, D.C. et al Developmental Cell, 8, 43-52 (2005)   Fragile X syndrome, the most common form of inherited mental retardation, is caused by loss of function for the Fragile X Mental Retardation 1 gene (FMR1). FMR1 protein (FMRP) has specific mRNA targets and is thought to be involved in their transport to subsynaptic sites as well as translation regulation. We report a saturating genetic screen of the Drosophila autosomal genome to identify functional partners of dFmr1. We recovered 19 mutations in the tumor suppressor lethal (2) giant larvae (dlgl) gene and 90 mutations at other loci. dlgl encodes a cytoskeletal protein involved in cellular polarity and cytoplasmic transport and is regulated by the PAR complex through phosphorylation. We provide direct evidence for a Fmrp/Lgl/mRNA complex, which functions in neural development in flies and is developmentally regulated in mice. Our data suggest that Lgl may regulate Fmrp/mRNA sorting, transport, and anchoring via the PAR complex.  

3.594           Kaposi’s sarcoma-associated herpesvirus modulates microtubele dynamics via RhoA-GTP-diaphanous 2 signaling and utilizes the dynein motors to deliver its DNA to the nucleus

Naranatt, P.P., Krishnan, H.H., Smith, M.S. and Chandran, B.

1. Virol., 79(2), 1191-1206 (2005)

  Human herpesvirus 8 (HHV-8; also called Kaposi's sarcoma-associated herpesvirus), which is implicated in the pathogenesis of Kaposi's sarcoma (KS) and lymphoproliferative disorders, infects a variety of target cells both in vivo and in vitro. HHV-8 binds to several in vitro target cells via cell surface heparan sulfate and utilizes the 3ß1 integrin as one of its entry receptors. Interactions with cell surface molecules induce the activation of host cell signaling cascades and cytoskeletal changes (P. P. Naranatt, S. M. Akula, C. A. Zien, H. H. Krishnan, and B. Chandran, J. Virol. 77:1524-1539, 2003). However, the mechanism by which the HHV-8-induced signaling pathway facilitates the complex events associated with the internalization and nuclear trafficking of internalized viral DNA is as yet undefined. Here we examined the role of HHV-8-induced cytoskeletal dynamics in the infectious process and their interlinkage with signaling pathways. The depolymerization of microtubules did not affect HHV-8 binding and internalization, but it inhibited the nuclear delivery of viral DNA and infection. In contrast, the depolymerization of actin microfilaments did not have any effect on virus binding, entry, nuclear delivery, or infection. Early during infection, HHV-8 induced the acetylation of microtubules and the activation of the RhoA and Rac1 GTPases. The inactivation of Rho GTPases by Clostridium difficile toxin B significantly reduced microtubular acetylation and the delivery of viral DNA to the nucleus. In contrast, the activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor significantly augmented the nuclear delivery of viral DNA. Among the Rho GTPase-induced downstream effector molecules known to stabilize the microtubules, the activation of RhoA-GTP-dependent diaphanous 2 was observed, with no significant activation in the Rac- and Cdc42-dependent PAK1/2 and stathmin molecules. The nuclear delivery of viral DNA increased in cells expressing a constitutively active RhoA mutant and decreased in cells expressing a dominant-negative mutant of RhoA. HHV-8 capsids colocalized with the microtubules, as observed by confocal microscopic examination, and the colocalization was abolished by the destabilization of microtubules with nocodazole and by the phosphatidylinositol 3-kinase inhibitor affecting the Rho GTPases. These results suggest that HHV-8 induces Rho GTPases, and in doing so, modulates microtubules and promotes the trafficking of viral capsids and the establishment of infection. This is the first demonstration of virus-induced host cell signaling pathways in the modulation of microtubule dynamics and in the trafficking of viral DNA to the infected cell nucleus. These results further support our hypothesis that HHV-8 manipulates the host cell signaling pathway to create an appropriate intracellular environment that is conducive to the establishment of a successful infection.  

3.595           Analyses of murine Postsynaptic density-95 identify novel isoforms and potential translational control elements

Bence, M., Arbuckle, M.I., Dickson, K.S. and Grant, S.G.N. Mol. Brain res., 133(1), 143-152 (2005)   Postsynaptic density-95 (PSD-95) is an evolutionarily conserved synaptic adaptor protein that is known to bind many proteins including the NMDA receptor. This observation has implicated it in many NMDA receptor-dependent processes including spatial learning and synaptic plasticity. We have cloned and characterised the murine PSD-95 gene. In addition, we have identified two previously uncharacterised splice variants of the major murine PSD-95 transcript (PSD-95α): PSD-95α-2b results from an extension of exon 2 and PSD-95α-Δ18 from the temporal exclusion of exon 18. The presence of PSD-95α-2b sequences in other PSD-95 family members implicates this peptide stretch as functionally significant. Another potential transcript (PSD-95γ) was also identified based on examination of EST databases. Immunoprecipitation assays demonstrate that proteins corresponding in size to PSD-95α-Δ18 and PSD-95γ interact with the NMDA receptor, suggesting an important biological role for these isoforms. Finally, we have performed bioinformatics analyses of the PSD-95 mRNA untranslated regions, identifying multiple translational control elements that suggest protein production could be regulated post-transcriptionally. The variety of mRNA isoforms and regulatory elements identified provides for a high degree of diversity in the structure and function of PSD-95 proteins.  

3.596           Isoform-specific regulation of adenylyl cylase function by disruption of membrane trafficking

Ding, Q., Gros, R., Chorazyczewski, J., Ferguson, S.S.g. and Feldman, R.D. Mol. Pharmacol., 6782), 564-571 (2005)   Oligomerization plays an important role in endoplasmic reticulumprocessing and membrane insertion (and ultimately in regulationof function) of a number of transmembrane spanning proteins.Furthermore, it is known that adenylyl cyclases (ACs), criticalregulators of cellular functions, associate into higher order(dimeric) forms. However, the importance of these higher orderaggregates in regulating adenylyl cyclase activity or traffickingto the cell membrane is unclear. Therefore, we examined thepotential role of oligomerization in the membrane traffickingof adenylyl cyclase. For this purpose, the ability of full-lengthadenylyl cyclase and various truncation mutants to self-assembleand to be targeted to the cell membrane was assessed. A truncationmutant comprised of the initial six transmembrane spanning domainsand half of the C1 catalytic domain coimmunoprecipitated withfull-length AC VI. Using both biotinylation assays and assessmentof enzyme distribution using sucrose density gradients, we demonstratethat expression of this mutant in human embryonic kidney 293cells impaired the ability of AC VI to traffic to the plasmamembrane. Furthermore, mutant expression resulted in a significantreduction in adenylyl cyclase activity. The decrease in AC VImembrane expression was not caused by alterations in enzymetranscription. The effect of the mutant was specific for theAC V and VI isoforms and expression of the transmembrane M1domain but not the C1a domain was required for the mutant toaffect adenylyl cyclase activity. In aggregate, these data suggestthat alterations in the ability of adenylyl cyclases to formhigher order forms regulate both enzyme trafficking and enzymeactivity.    

3.597           Fractionation of the epithelial apical junctional complex: reassessment of protein distributions in different substructures

Vogelmann, R. And Nelson, W.J. Mol. Biol. Cell, 16, 701-716 (2005)   The epithelial apical junctional complex (AJC) is an important regulator of cell structure and function. The AJC is compartmentalized into substructures comprising the tight and adherens junctions, and other membrane complexes containing the membrane proteins nectin, junctional adhesion molecule, and crumbs. In addition, many peripheral membrane proteins localize to the AJC. Studies of isolated proteins indicate a complex map of potential binding partners in which there is extensive overlap in the interactions between proteins in different AJC substructures. As an alternative to a direct search for specific protein-protein interactions, we sought to separate membrane substructures of the AJC in iodixanol density gradients and define their protein constituents. Results show that the AJC can be fractured into membrane substructures that contain specific membrane and peripheral membrane proteins. The composition of each substructure reveals a more limited overlap in common proteins than predicted from the inventory of potential interactions; some of the overlapping proteins may be involved in stepwise recruitment and assembly of AJC substructures.  

3.598           Roles of the ITAM and PY motifs of Epstein-Barr virus latent membrane protein 2A in the inhibition of epithelial cell differentiation and activation of b-catenin signaling

Morrison, J.A. and Raab-Traub, N.

1. Virol., 79(4), 2375-2382 (2005)

  Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is important for maintenance of latency in infected B lymphocytes. Through its immunoreceptor tyrosine-based activation motif (ITAM) and PY motifs, LMP2A is able to block B-cell receptor (BCR) signaling, bind BCR-associated kinases, and manipulate the turnover of itself and these kinases via a PY-mediated interaction with the Nedd4 family of ubiquitin ligases. In epithelial cells, LMP2A has been shown to activate the phosphatidylinositol 3'-OH kinase/Akt and ß-catenin signaling pathways. In the present study, the biological consequences of LMP2A expression in the normal human foreskin keratinocyte (HFK) cell line were investigated and the importance of the ITAM and PY motifs for LMP2A signaling effects in HFK cells was ascertained. The ITAM was essential for the activation of Akt by LMP2A in HFK cells, while both the ITAM and PY motifs contributed to LMP2A-mediated accumulation and nuclear translocation of the oncoprotein ß-catenin. LMP2A inhibited induction of differentiation in an assay conducted with semisolid methylcellulose medium, and the PY motifs were critical for this inhibition. LMP2A is expressed in the EBV-associated epithelial malignancies nasopharyngeal carcinoma and gastric carcinoma, and these data indicate that LMP2A affects cellular processes that likely contribute to carcinogenesis.  

3.599           Alteration of the extracellular matrix interferes with raft association of neurofascin in oligodendrocytes. Potential significance for multiple sclerosis

Maier, O. Et al Mol. Cell. Neurosci., 28(2), 390-401 (2005)   Remyelination, as potential treatment for demyelinating diseases like multiple sclerosis (MS), requires the formation of new axoglial interactions by differentiating oligodendrocyte progenitor cells. Since the oligodendrocyte-specific isoform of neurofascin, NF155 (neurofascin isoform of 155 kDa), may be important for establishing axoglial interactions, we analyzed whether its expression is changed in chronic relapsing experimental allergic encephalomyelinitis (EAE). Although overall expression of NF155 was not changed, immunoreactivity of NF155 was dramatically increased in EAE lesion sites indicating an enhanced accessibility of NF155 epitopes. As this may be due to infiltrating plasma components, for example, fibronectin, we analyzed whether fibronectin affects the intracellular distribution and membrane association of NF155 in primary oligodendrocytes. In oligodendrocytes cultivated on polylysine, NF155 was recruited to membrane microdomains (rafts) during development and became enriched in secondary and tertiary processes. Fibronectin perturbed localization and raft association of NF155 and inhibited the morphological differentiation of oligodendrocytes. Consistent with the in vitro data, raft association of NF155 was reduced in spinal cord of EAE rats. The results suggest that the association of NF155 to microdomains in the oligodendrocyte membrane is required for its participation in intermolecular interactions, which are important for myelination and/or myelin integrity.  

3.600           Characterizing the sphingolipid signaling pathway that remediates defects associated with loss of the yeast amphiphysin-like orthologs, Rvs161p and Rvs167p

Germann, M., Swain, E., Bergman, L. and Nickels, Jr. J.T.

1. Biol. Chem., 280(6), 4270-4278 (2005)

  Loss of function of either the RVS161 or RVS167 Saccharomyces cerevisiae amphiphysin-like gene confers similar growth phenotypes that can be suppressed by mutations in sphingolipid biosynthesis. We performed a yeast two-hybrid screen using Rvs161p as bait to uncover proteins involved in this sphingolipid-dependent suppressor pathway. In the process, we have demonstrated a direct physical interaction between Rvs167p and the two-hybrid interacting proteins, Acf2p, Gdh3p, and Ybr108wp, while also elucidating the Rvs167p amino acid domains to which these proteins bind. By using subcellular fractionation, we demonstrate that Rvs167p, Ybr108wp, Gdh3p, and Acf2p all localize to Rvs161p-containing lipid rafts, thus placing them within a single compartment that should facilitate their interactions. Moreover, our results suggest that Acf2p and Gdh3p functions are needed for suppressor pathway activity. To determine pathway mechanisms further, we examined the localization of Rvs167p in suppressor mutants. These studies reveal roles for Rvs161p and the very long chain fatty acid elongase, Sur4p, in the localization and/or stability of Rvs167p. Previous yeast studies showed that rvs defects could be suppressed by changes in sphingolipid metabolism brought about by deleting SUR4 (Desfarges, L., Durrens, P., Juguelin, H., Cassagne, C., Bonneu, M., and Aigle, M. (1993) Yeast 9, 267–277). Using rvs167 sur4 and rvs161 sur4 double null cells as models to study suppressor pathway activity, we demonstrate that loss of SUR4 does not remediate the steady-state actin cytoskeletal defects of rvs167 or rvs161 cells. Moreover, suppressor activity does not require the function of the actin-binding protein, Abp1p, or Sla1p, a protein that is thought to regulate assembly of the cortical actin cytoskeleton. Based on our results, we suggest that sphingolipid-dependent suppression of rvs defects may not work entirely through regulating changes in actin organization.  

3.601           Internalization of renal type Iic Na-P_i cotransporter in response to a high-phosphate diet

Segawa, H. et al Am. J. Renal Physiol., 288, F587-F596 (2005)   Dietary phosphate levels regulate the renal brush-border type IIa Na-P_i cotransporter. Another Na-P_i cotransporter, type IIc, colocalizes with type IIa Na-P_i cotransporter in the apical membrane of renal proximal tubular cells. The goal of the present study was to determine whether dietary phosphate levels also rapidly regulate the type IIc Na-P_i cotransporter. Type IIa and type IIc transporter protein levels were increased in rats chronically fed a low-P_i diet compared with those fed a normal-P_i diet. Two hours after beginning a high-P_i diet, type IIa transporter levels were decreased, whereas type IIc protein levels remained unchanged. Western blot analysis of brush-border membrane prepared 4 h after beginning a high-P_i diet showed a significant reduction in type IIc transporter protein levels, and immunohistochemistry showed translocation of the type IIc-immunoreactive signal from the entire brush border to subapical membrane. Membrane fractionation studies revealed a decrease in apical membrane type IIc protein without changes in total cortical type IIc protein, which is compatible with redistribution of type IIc protein from the apical membrane to the dense membrane fraction. The microtubule-disrupting reagent colchicine prevented this reduction in apical type IIc transporter at the apical membrane but had no effect on type IIa transporter levels. These data suggest that the type IIc Na-P_i cotransporter level is rapidly regulated by rapid adaptation to dietary P_i in a microtubule-dependent manner. Furthermore, the mechanisms of the internalization of the type IIc transporter are distinct from those of the type IIa transporter.  

3.602           Phosphorylation-induced autoinhibition regulates the cytoskeletal protein lethal (2) giant larvae

Betschinger, J., Eisenhaber, F. and Knoblich, J.A. Current Biol., 15(3), 276-282 (2005)   During asymmetric cell division, cell fate determinants localize asymmetrically and segregate into one of the two daughter cells. In Drosophila neuroblasts, the asymmetric localization of cell fate determinants to the basal cell cortex requires aPKC. aPKC localizes to the apical cell cortex and phosphorylates the cytoskeletal protein Lethal (2) giant larvae (Lgl). Upon phosphorylation, Lgl dissociates from the cytoskeleton and becomes inactive. Here, we show that phosphorylation regulates Lgl by allowing an autoinhibitory interaction of the N terminus with the C terminus of the protein. We demonstrate that interaction with the cytoskeleton is mediated by a C-terminal domain while the N terminus is not required. Instead, the N terminus can bind to the C terminus and can compete for binding to the cytoskeleton. Interaction between the N- and C-terminal domains requires phosphorylation of Lgl by aPKC. Our results suggest that unphosphorylated, active Lgl exists in an open conformation that interacts with the cytoskeleton while phosphorylation changes the protein to an autoinhibited state.  

3.603           Annexin 2 promotes the formation of lipid microdomains required for calcium-regulated exocytosis of dense-core vesicles

Chasserot-Golaz, S. et al Mol. Biol. Cell, 16, 1108-1119 (2005)   Annexin 2 is a calcium-dependent phospholipid-binding protein that has been implicated in a number of membranerelated events, including regulated exocytosis. In chromaffin cells, we previously reported that catecholamine secretion requires the translocation and formation of the annexin 2 tetramer near the exocytotic sites. Here, to obtain direct evidence for a role of annexin 2 in exocytosis, we modified its expression level in chromaffin cells by using the Semliki Forest virus expression system. Using a real-time assay for individual cells, we found that the reduction of cytosolic annexin 2, and the consequent decrease of annexin 2 tetramer at the cell periphery, strongly inhibited exocytosis, most likely at an early stage before membrane fusion. Secretion also was severely impaired in cells expressing a chimera that sequestered annexin 2 into cytosolic aggregates. Moreover, we demonstrate that secretagogue-evoked stimulation triggers the formation of lipid rafts in the plasma membrane, essential for exocytosis, and which can be attributed to the annexin 2 tetramer. We propose that annexin 2 acts as a calcium-dependent promoter of lipid microdomains required for structural and spatial organization of the exocytotic machinery.  

3.604           The deubiquitinating enzyme Ubp1 affects sorting of the ATP-binding cassette-transporter Ste6 in the endocytic pathway

Schmitz, C., Kinner, A. and Kölling, R. Mol. Biol. Cell, 16, 1319-1329 (2005)   Deubiquitinating enzymes (Dubs) are potential regulators of ubiquitination-dependent processes. Here, we focus on a member of the yeast ubiquitin-specific processing protease (Ubp) family, the Ubp1 protein. We could show that Ubp1 exists in two forms: a longer membrane-anchored form (mUbp1) and a shorter soluble form (sUbp1) that seem to be independently expressed from the same gene. The membrane-associated mUbp1 variant could be localized to the endoplasmic reticulum (ER) membrane by sucrose density gradient centrifugation and by immunofluorescence microscopy. Overexpression of the soluble Ubp1 variant stabilizes the ATP-binding cassette-transporter Ste6, which is transported to the lysosome-like vacuole for degradation, and whose transport is regulated by ubiquitination. Ste6 stabilization was not the result of a general increase in deubiquitination activity, because overexpression of Ubp1 had no effect on the degradation of the ER-associated degradation substrate carboxypeptidase Y^* and most importantly on Ste6 ubiquitination itself. Also, overexpression of another yeast Dub, Ubp3, had no effect on Ste6 turnover. This suggests that the Ubp1 target is a component of the protein transport machinery. On Ubp1 overexpression, Ste6 accumulates at the cell surface, which is consistent with a role of Ubp1 at the internalization step of endocytosis or with enhanced recycling to the cell surface from an internal compartment.  

3.605           A domain at the C-terminus of PS1 is required for presenilinase and g-secretase activities

Brunkan, A.L., Martinez, M., Wang, J., Walker, E.S. and Goate, A.M.

1. Neurochem., 92(5), 1158-1169 (2005)

  The structural requirements for presenilin (PS) to produce active presenilinase and -secretase enzymes are poorly understood. Here we investigate the role the cytoplasmic C-terminal region of PS1 plays in PS1 activity. Deletion or addition of residues at the PS C-terminus has been reported to inhibit presenilinase endoproteolysis of PS and alter -secretase activity. In this study, we use a sensitive assay in PS1/2KO MEFs to define a domain at the extreme C-terminus of PS1 that is essential for both presenilinase and -secretase activities. Progressive deletion of the C-terminus demonstrated that removal of nine residues produces a PS1 molecule (458ST) that lacks both presenilinase processing and -secretase cleavage of Notch and APP substrates. In contrast, removal of four or five residues had no effect (462ST, 463ST), while intermediate truncations partially inhibited PS1 activity. The 458ST mutant was unable to replace endogenous wtPS1 in HEK293 cells. Although 458ST was able to form a -secretase complex, this complex was not matured, illustrated by mutant PS1 instability, lack of endoproteolysis, and little production of mature Nicastrin. These data indicate that the C-terminal end of PS1 is essential for Nicastrin trafficking and modification as well as the replacement of endogenous PS1 by PS1 transgenes.  

3.606           Novel role of ARF6 in vascular endothelial growth factor-induced signaling and angiogenesis

Ikeda, S. et al Cir. Res., 96, 467-475 (2005)   Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that VEGF stimulates a Rac1-dependent NAD(P)H oxidase to produce reactive oxygen species (ROS) that are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. The small GTPase ARF6 is involved in membrane trafficking and cell motility; however, its roles in VEGF signaling and physiological responses in ECs are unknown. In this study, we show that overexpression of dominant-negative ARF6 [ARF6(T27N)] almost completely inhibits VEGF-induced Rac1 activation, ROS production, and VEGFR2 autophosphorylation in ECs. Fractionation of caveolae/lipid raft membranes demonstrates that ARF6, Rac1, and VEGFR2 are localized in caveolin-enriched fractions basally. VEGF stimulation results in the release of VEGFR2 from caveolae/lipid rafts and caveolin-1 without affecting localization of ARF6, Rac1, or caveolin-1 in these fractions. The egress of VEGFR2 from caveolae/lipid rafts is contemporaneous with the tyrosine phosphorylation of caveolin-1 (Tyr14) and VEGFR2 and with their association with each other. ARF6(T27N) significantly inhibits both VEGF-induced responses. Immunofluorescence studies show that activated VEGFR2 and phosphocaveolin colocalize at focal complexes/adhesions after VEGF stimulation. Both overexpression of ARF6(T27N) and mutant caveolin-1(Y14F), which cannot be phosphorylated, block VEGF-stimulated EC migration and proliferation. Moreover, ARF6 expression is markedly upregulated in association with an increase in capillary density in a mouse hindlimb ischemia model of angiogenesis. Thus, ARF6 is involved in the temporal-spatial organization of caveolae/lipid rafts– and ROS-dependent VEGF signaling in ECs as well as in angiogenesis in vivo.  

3.607           Stable plasma membrane levels of hCTR1 mediate cellular copper uptake

Eisses, J.F., Chi, Y. and Kaplan, J.H.

1. Biol. Chem., 280(10), 9635-9639 (2005)

  The human copper transporter 1 (hCtr1), when heterologously overexpressed in insect cells, mediates saturable Cu uptake. In mammalian expression systems, a rapid Cu-dependent internalization of hCtr1 has been reported in cells that overexpress epitope-tagged hCtr1 when exposed to Cu in the external medium. This finding led to the suggestion that such internalization may be a step in the hCtr1 transmembrane Cu transport mechanism. We have demonstrated that preincubation in Cu-containing media of sf9 cells stably expressing hCtr1 has no effect on the initial rate of Cu transport. Furthermore, Western blot analyses of fractionated sf9 cell membranes show no evidence of a regulatory Cu-dependent internalization from the plasma membrane. In similar studies on human embryonic kidney (HEK) 293 cells, we showed that incubation with Cu does not alter the initial rate of Cu uptake mediated by endogenous levels of hCtr1 compared with untreated cells. Confirmation that hCtr1 mediates this transport is provided by specific small interfering RNA-dependent decreases in hCtr1 protein levels and in Cu transport rates. Western blot analysis and confocal microscopy of human embryonic kidney 293 cells showed that the majority of hCtr1 protein is localized at the plasma membrane and no significant internalization is detected upon Cu treatment. We concluded that internalization of hCtr1 is not a required step in the transport pathway; we suggest that oligomeric hCtr1 acts as a conventional transporter providing a permeation pathway for Cu through the membrane and that internalization of endogenous hCtr1 in response to elevated extracellular Cu levels does not play a significant regulatory role in Cu homeostasis.  

3.608           Membrane localization of the U2 domain of protein 4.1B is necessary and sufficient for meningioma growth suppression

Robb, V.A., Gerber, M.A., Hart-Mahon, E.K. and Gutmann, D.H. Oncogene, 24, 1946-1957 (2005)   Meningiomas are common central nervous system tumors; however, the molecular mechanisms underlying their pathogenesis are largely undefined. Previous work has implicated Protein 4.1B as an important tumor suppressor involved in the development of these neoplasms. In this report, we demonstrate that the U2 domain is necessary and sufficient for the ability of Protein 4.1B to function as a meningioma growth suppressor. Using a series of truncation and deletion constructs of DAL-1 (a fragment of Protein 4.1B that retains all the growth suppressive properties), we narrowed the domain required for 4.1B growth suppression to a fragment containing a portion of the FERM domain and the U2 domain using clonogenic assays on meningioma cells. Deletion of the U2 domain in the context of the full-length DAL-1 molecule eliminated growth suppressor function, as measured by thymidine incorporation and caspase-3 activation. Moreover, targeting the U2 domain to the plasma membrane using a membrane localization signal (MLS) reduced cell proliferation, similar to wild-type DAL-1. Collectively, the data suggest that the U2 domain, when properly targeted to the plasma membrane, contains all the residues necessary for mediating Protein 4.1B growth suppression.  

3.609           Nuclear location-dependent role of peripheral benzodiapine receptor (PBR) in hepatic tumoral cell lines proliferation

Corsi, L., Geminiani, E., Avallone, R. and Baraldi, M. Life Sciences, 76(22), 2523-2533 (2005)   PBR is involved in numerous biological functions, including steroid biosynthesis, mitochondrial oxidative phosphorylation and cell proliferation. The presence of PBR at the perinuclear/nuclear subcellular level has been demonstrated in aggressive breast cancer cell lines and human glioma cells where it seems to be involved in cell proliferation. In our study we investigated the presence of perinuclear/nuclear PBR in different hepatic tumor cell lines with regard to binding to [³H] PK 11195 and protein analysis. The results obtained by saturation binding experiments and scatchard analysis of perinuclear/nuclear PBR density in parallel with the results on the growth curves of the cell lines tested, indicate that the perinuclear/nuclear PBR density correlates inversely with cell doubling time. Moreover, the cell line with high perinuclear/nuclear PBR proliferated in response to PBR ligand, whereas that with low perinuclear/nuclear PBR did not. Our results reinforce the idea that the subcellular localisation of PBR defines its function and that this receptor could be a possible target for new strategies against cancer.  

3.610           Two modes of exocytosis at hippocampal synapses revealed by rate of FM1-43 efflux from individual vesicles

Richards, D.A., Bai, J. and Chapman, E.R.

1. Cell Biol., 168(6), 929-939 (2005)

  We have examined the kinetics by which FM1-43 escapes from individual synaptic vesicles during exocytosis at hippocampal boutons. Two populations of exocytic events were observed; small amplitude events that lose dye slowly, which made up more than half of all events, and faster, larger amplitude events with a fluorescence intensity equivalent to single stained synaptic vesicles. These populations of destaining events are distinct in both brightness and kinetics, suggesting that they result from two distinct modes of exocytosis. Small amplitude events show tightly clustered rate constants of dye release, whereas larger events have a more scattered distribution. Kinetic analysis of the association and dissociation of FM1-43 with membranes, in combination with a simple pore permeation model, indicates that the small, slowly destaining events may be mediated by a narrow 1-nm fusion pore.  

3.611           Neutral sphingomyelinase 2 (smpd3) in the control of postnatal growth and development

Stoffel, W., Jenke, B., Blöck, B., Zumbansen, M. And Koebke, J. PNAS, 102(12), 4554-4559 (2005)   Neutral sphingomyelinases sphingomyelin phosphodiesterase (SMPD)2 and -3 hydrolyze sphingomyelin to phosphocholine and ceramide. smpd2 is expressed ubiquitously, and smpd3 is expressed predominantly in neurons of the CNS. Their activation and the functions of the released ceramides have been associated with signaling pathways in cell growth, differentiation, and apoptosis. However, these cellular responses remain poorly understood. Here we describe the generation and characterization of the smpd3^–/– and smpd2^–/–smpd3^–/– double mutant mouse, which proved to be devoid of neutral sphingomyelinase activity. SMPD3 plays a pivotal role in the control of late embryonic and postnatal development: the smpd3-null mouse develops a novel form of dwarfism and delayed puberty as part of a hypothalamus-induced combined pituitary hormone deficiency. Our studies suggest that SMPD3 is segregated into detergent-resistant subdomains of Golgi membranes of hypothalamic neurosecretory neurons, where its transient activation modifies the lipid bilayer, an essential step in the Golgi secretory pathway. The smpd3^–/– mouse might mimic a form of human combined pituitary hormone deficiency.  

3.612           Caveolae targeting and regulation of large conductance Ca²⁺ -activated K⁺ channels in vascular endothelial cells

Wang, X-L. et al

1. Biol. Chem., 280(12), 11656-11664 (2005)

  The vascular endothelium is richly endowed with caveolae, which are specialized membrane microdomains that facilitate the integration of specific cellular signal transduction processes. We found that the large conductance Ca²⁺-activated K⁺ (BK) channels are associated with caveolin-1 in bovine aortic endothelial cells (BAECs). OptiPrep gradient cell fractionation demonstrated that BK channels were concentrated in the caveolae-rich fraction in BAECs. Immunofluorescence imaging showed co-localization of caveolin-1 and BK channels in the BAEC membrane. Immunoprecipitation and glutathione S-transferase pull-down assay results indicated that caveolin-1 and BK channels are physically associated. However, whole cell patch clamp recordings could not detect BK (iberiotoxin-sensitive) currents in cultured BAECs under baseline conditions, even though the presence of BK mRNA and protein expression was confirmed by reverse transcription-PCR and Western blots. Cholesterol depletion redistributed the BK channels to non-caveolar fractions of BAECs, resulting in BK channel activation (7.3 ± 1.6 pA/picofarad (pF), n = 5). BK currents were also activated by isoproterenol (ISO, 1 µM, 6.9 ± 2.4 pA/pF, n = 6). Inclusion of a caveolin-1 scaffolding domain peptide (10 µM) in the pipette solution completely abrogated the effects of ISO on BK channel activation, whereas inclusion of the scrambled control peptide (10 µM) did not inhibit the ISO effects. We have also found that caveolin-1 knockdown by small interference RNA activated BK currents (5.3 ± 1.4 pA/pF, n = 6). We conclude that: 1) BK channels are targeted to caveolae microdomains in vascular endothelial cells; 2) caveolin-1 interacts with BK channels and exerts a negative regulatory effect on channel functions; and 3) BK channels are inactive under control conditions but can be activated by cholesterol depletion, knockdown of caveolin-1 expression, or ISO stimulation. These novel findings may have important implications for the role of BK channels in the regulation of endothelial function.  

3.613           Aph-1 contributes to the stabilization and trafficking of the g-secretase complex through mechanisms involving intermolecular and intramolecular interactions

Niimura, M. Et al

1. Biol. Chem., 280(13), 12967-12975 (2005)

  g-Secretase cleaves type I transmembrane proteins, including -amyloid precursor protein and Notch, and requires the formation of a protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2 for its activity. Aph-1 is implicated in the stabilization of this complex, although its precise mechanistic role remains unknown. Substitution of the first glycine within the transmembrane GXXXG motif of Aph-1 causes a loss-of-function phenotype in Caenorhabditis elegans. Here, using an untranslated region-targeted RNA interference/rescue strategy in Drosophila Schneider 2 cells, we show that Aph-1 contributes to the assembly of the -secretase complex by multiple mechanisms involving intermolecular and intramolecular interactions depending on or independent of the conserved glycines. Aph-1 binds to nicastrin forming an early subcomplex independent of the conserved glycines within the endoplasmic reticulum. Certain mutations in the conserved GXXXG motif affect the interaction of the Aph-1·nicastrin subcomplex with presenilin that mediates trafficking of the presenilin·Aph-1·nicastrin tripartite complex to the Golgi. The same mutations decrease the stability of Aph-1 polypeptides themselves, possibly by affecting intramolecular associations through the transmembrane domains. Our data suggest that the proper assembly of the Aph-1·nicastrin subcomplex with presenilin is the prerequisite for the trafficking as well as the enzymatic activity of the -secretase complex and that Aph-1 functions as a stabilizing scaffold in the assembly of this complex.  

3.614           Molecular mechanisms of cholesterol absorption and transport in the intestine

Hui, D.Y. and Howles, P.N. Seminars in Cell & Developmental Biol., 16(2), 183-192 (2005)   Many enzymes and transport proteins participate in cholesterol absorption. This review summarizes recent results on several proteins that are important for each step of the cholesterol absorption pathway, including the important roles of: (i) pancreatic triglyceride lipase (PTL), carboxyl ester lipase (CEL), and ileal bile acid transporter in determining the rate of cholesterol absorption; (ii) ATP binding cassette (ABC) transporters and the Niemann-Pick C-1 like-1 (NPC1L1) protein as intestinal membrane gatekeepers for cholesterol efflux and influx; and (iii) intracellular membrane vesicles and transport proteins in lipid trafficking through intracellular compartments prior to lipoprotein assembly and secretion to plasma circulation.  

3.615           Adeno-associated virus-mediated gene transfer of the heart/muscle adenine nucleotide translocator (ANT) in mouse

Flierl, A., Chen, Y., Coskun, P.E., Samulski, R.J. and Wallace, D.C. Gen. Ther., 12, 570-578 (2005)   Mitochondrial myopathy, associated with muscle weakness and progressive external ophthalmoplegia, is caused by mutations in mitochondria oxidative phosphorylation genes including the heart-muscle isoform of the mitochondrial adenine nucleotide translocator (ANT1). To develop therapies for mitochondrial disease, we have prepared a recombinant adeno-associated viral vector (rAAV) carrying the mouse Ant1 cDNA. This vector has been used to transduce muscle cells and muscle from Ant1 mutant mice, which manifest mitochondrial myopathy. AAV-ANT1 transduction resulted in long-term, stable expression of the Ant1 transgene in muscle precursor cells as well as differentiated muscle fibers. The transgene ANT1 protein was targeted to the mitochondrion, was inserted into the mitochondrial inner membrane, formed a functional ADP/ATP carrier, increased the mitochondrial export of ATP and reversed the histopathological changes associated with the mitochondrial myopathy. Thus, AAV transduction has the potential of providing symptomatic relief for the ophthalmoplegia and ptosis resulting from paralysis of the extraocular eye muscles cause by mutations in the Ant1 gene.  

3.616           Nulear localization anti-DNA antibodies enter cells via caveoli and modulate expression of caveolin and p53

Yanase, K. and Madaio, M.P.

1. Autoimmun., 24, 145-151 (2005)

  After administration to normal mice, a subset of monoclonal (m) anti-DNA antibodies (Ab) derived from MRL-lpr/lpr mice was identified that enter cells, in vivo. In the kidneys, this was associated with glomerular hypercellularity and proteinuria. In cultured cells, the same mAb bound to myosin 1 on the cell surface, prior to internalization, nuclear localization and inhibition of apoptosis. The present study focuses on the mechanisms underlying the observed functional effects. Subcellular localization studies revealed that following internalization, a prototypic, nuclear localizing, m antibody (Ab; termed H7) co-localized with myosin 1, shortly after internalization, within caveolae, near the cell membrane. Cell fractionation studies confirmed the presence of both H7 and myosin within the caveolar fraction. Since variations in caveolin protein expression have been associated with apoptotic events in cancer cells, through p53 dependent and independent pathways, modulation of caveolin by intracellular H7 was evaluated. Cellular entry of the anti-DNA Ab resulted in an increase in caveolin protein expression. Furthermore, after exposure of cells to dexamethasone to induce apoptosis, the usual increase in p53 was inhibited in the presence of intracellular H7. Taken together, the results suggest that upregulation of caveolin and inhibition of p53 induction are involved in H7-induced, inhibition of apoptosis. Furthermore, they suggest that this inhibition contributes to the glomerular hypercellularity observed in normal mice with intranuclear H7. The results also raise the possibility that inhibition of apoptotic pathways during inflammation or/and autoimmunity could influence subsequent disease events. The novel mechanism of cellular perturbation is indirect and dependent on apoptotic stimuli, and it may account for the presence of intranuclear antibodies in inflammatory and normal tissues of individuals with lupus.  

3.617           A simplified method for the preparation of detergent-free lipid rafts

Macdonald, J.L. and Pike, L.J.

1. Lipid. Res., 46, 1061-1067 (2005)

  Lipid rafts are small plasma membrane domains that contain highlevels of cholesterol and sphingolipids. Traditional methodsfor the biochemical isolation of lipid rafts involve the extractionof cells with nonionic detergents followed by the separationof a low-density, detergent-resistant membrane fraction on densitygradients. Because of concerns regarding the possible introductionof artifacts through the use of detergents, it is importantto develop procedures for the isolation of lipid rafts thatdo not involve detergent extraction. We report here a simplified method for the purification of detergent-freelipid rafts that requires only one short density gradient centrifugation,but yields a membrane fraction that is highly enriched in cholesteroland protein markers of lipid rafts, with no contamination fromnonraft plasma membrane or intracellular membranes.  

3.618           Studies on rabbit natural and recombinant tissue factors: intracellular retention and regulation of surface expression in cultured cells

Fortin, J-P., Rivard, G.E., Adam, A. and Marceau, F. Am. J. Physiol., 288, H2192-H2202 (2005)   Tissue factor (TF) is the most important trigger of blood coagulation in vascular pathology. Rabbit TF, with or without ( C) its COOH-terminal intracellular tail, has been conjugated to green fluorescent protein (GFP) to study subcellular localization and other functions of TF. TF-GFP and TF C-GFP are associated with Na₂CO₃-resistant buoyant fractions in HEK-293 cells (lipid rafts); there is no morphological difference in the surface distribution of these or other GFP-labeled membrane proteins present in or excluded from rafts (confocal microscopy, HEK-293 cells). Endogenous TF expressed by rabbit aortic smooth muscle cells (SMCs) is also raft associated. Membranes from HEK-293 cells expressing recombinant TF-GFP or wild-type TF were equipotent to clot human plasma; however, TF C-GFP was 20-fold more active (per membrane weight). Immunoblot confirmed that the deletion mutant is more abundantly expressed, and confocal microscopy showed that it has preferential membrane localization, whereas TF-GFP is mainly intracellular (nuclear lining and multiple granules). With a similar half-life (<4 h), the two constructions differ by their intracellular retention, lower for TF C-GFP. In serum-starved SMCs, the expression of endogenous TF was upregulated by interleukin-1 and/or FBS treatment (immunoblot, immunofluorescence, clotting assay). However, TF secretion or surface expression was not regulated by stimuli of physiological intensity (such as stimulation of the coexpressed kinin B₁ receptors), although a calcium ionophore was highly active in this respect. TF is a raft-associated molecule whose surface expression (secretion) is apparently retarded or impaired by structural determinant(s) located in its COOH-terminal tail.  

3.619           Proteomic profiling of hepatic endoplasmic reticulum-associated proteins in an animal model of insulin resistance and metabolic dyslipidemia

Morand, J-P. F., Macri, J. and Adeli, K.

1. Biol. Chem., 280(18), 17626-17633 (2005)

  Hepatic insulin resistance and lipoprotein overproduction are common features of the metabolic syndrome and insulin-resistant states. A fructose-fed, insulin-resistant hamster model was recently developed to investigate mechanisms linking the development of hepatic insulin resistance and overproduction of atherogenic lipoproteins. Here we report a systematic analysis of protein expression profiles in the endoplasmic reticulum (ER) fractions isolated from livers of fructose-fed hamsters with the intention of identifying new candidate proteins involved in hepatic complications of insulin resistance and lipoprotein dysregulation. We have profiled hepatic ER-associated proteins from chow-fed (control) and fructose-fed (insulin-resistant) hamsters using two-dimensional gel electrophoresis and mass spectrometry. A total of 26 large scale two-dimensional gels of hepatic ER were used to identify 34 differentially expressed hepatic ER protein spots observed to be at least 2-fold differentially expressed with fructose feeding and the onset of insulin resistance. Differentially expressed proteins were identified by matrix-assisted laser desorption ionization-quadrupole time of flight (MALDI-Q-TOF), MALDI-TOF-postsource decay, and database mining using ProteinProspector MS-fit and MS-tag or the PROWL ProFound search engine using a focused rodent or mammalian search. Hepatic ER proteins ER60, ERp46, ERp29, glutamate dehydrogenase, and TAP1 were shown to be more than 2-fold down-regulated, whereas -glucosidase, P-glycoprotein, fibrinogen, protein disulfide isomerase, GRP94, and apolipoprotein E were all found to be up-regulated in the hepatic ER of the fructose-fed hamster. Seven isoforms of ER60 in the hepatic ER were all shown to be down-regulated at least 2-fold in hepatocytes from fructosefed/insulin-resistant hamsters. Implications of the differential expression of positively identified protein factors in the development of hepatic insulin resistance and lipoprotein abnormalities are discussed.  

3.620           The low density lipoprotein receptor-related protein (LRP) is a novel b-secretase (BACE1) substrate

Von Armin, C.A.F. et al

1. Biol. Chem., 280(18), 17777-17785 (2005)

  BACE is a transmembrane protease with -secretase activity that cleaves the amyloid precursor protein (APP). After BACE cleavage, APP becomes a substrate for -secretase, leading to release of amyloid- peptide (A ), which accumulates in senile plaques in Alzheimer disease. APP and BACE are co-internalized from the cell surface to early endosomes. APP is also known to interact at the cell surface and be internalized by the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic and signaling receptor. Using a new fluorescence resonance energy transfer (FRET)-based assay of protein proximity, fluorescence lifetime imaging (FLIM), and co-immunoprecipitation we demonstrate that the light chain of LRP interacts with BACE on the cell surface in association with lipid rafts. Surprisingly, the BACE-LRP interaction leads to an increase in LRP C-terminal fragment, release of secreted LRP in the media and subsequent release of the LRP intracellular domain from the membrane. Taken together, these data suggest that there is a close interaction between BACE and LRP on the cell surface, and that LRP is a novel BACE substrate.  

3.621           Disturbance of sphingolipid biosynthesis abrogates the signaling of Mss4, phosphatidylinositol-4-phosphate 5-kinase, in yeast

Kobayashi, T., Takematsu, H., Yamaji, T., Hiramoto, S. and Kozutsumi, Y.

1. Biol. Chem., 289(18), 18087-18094 (2005)

  The functional relationships between phosphoinositides and sphingolipids have not been well characterized to date. ISP-1/myriocin is a potent inhibitor of sphingolipid biosynthesis and induces severe growth defects in eukaryotic cells because of the sphingolipid deprivation. We characterized a novel multicopy suppressor gene of ISP-1-mediated cell death in yeast, MSS4. MSS4 encodes a phosphatidylinositol-4-phosphate 5-kinase that synthesizes phosphatidylinositol (4,5)-bisphosphate (PI4,5P₂). We demonstrate here that ISP-1 treatment of yeast causes defects both in the activity and subcellular localization of Mss4. The effect of the Mss4 defect on the downstream signaling was examined, because interaction between the Mss4 product, PI4,5P₂, and the pleckstrin-homology domain of Rom2 mediates recruitment of Rom2 to the membrane, which is the crucial step for subsequent Rho1/2 activation. Indeed, failure of Rom2 recruitment was observed in ISP-1-treated cells as well as in csg2-deleted cells, which have reduced mannosylated inositolphosphorylceramide. These data suggested that proper sphingolipids are required for the signaling pathway involving Mss4.  

3.622           Actin depolymerization transduces the strength of B-cell receptor stimulation

Hao, S. and August, A. Mol. Biol. Cell, 16, 2275-2294 (2005)   Polymerization of the actin cytoskeleton has been found to be essential for B-cell activation. We show here, however, that stimulation of BCR induces a rapid global actin depolymerization in a BCR signal strength-dependent manner, followed by polarized actin repolymerization. Depolymerization of actin enhances and blocking actin depolymerization inhibits BCR signaling, leading to altered BCR and lipid raft clustering, ERK activation, and transcription factor activation. Furthermore actin depolymerization by itself induces altered lipid raft clustering and ERK activation, suggesting that F-actin may play a role in separating lipid rafts and in setting the threshold for cellular activation.  

3.623           Myosin-1a is critical for normal brush border structure and composition

Tyska, M. Et al Mol. Biol. Cell, 16, 2443-2457 (2005)   To develop our understanding of myosin-1a function in vivo, we have created a mouse line null for the myosin-1a gene. Myosin-1a knockout mice demonstrate no overt phenotypes at the whole animal level but exhibit significant perturbations and signs of stress at the cellular level. Among these are defects in microvillar membrane morphology, distinct changes in brush-border organization, loss of numerous cytoskeletal and membrane components from the brush border, and redistribution of intermediate filament proteins into the brush border. We also observed significant ectopic recruitment of another short-tailed class I motor, myosin-1c, into the brush border of knockout enterocytes. This latter finding, a clear demonstration of functional redundancy among vertebrate myosins-I, may account for the lack of a whole animal phenotype. Nevertheless, these results indicate that myosin-1a is a critical multifunctional component of the enterocyte, required for maintaining the normal composition and highly ordered structure of the brush border.  

3.624           Cell cycle-regulated microtubeli-independent organelle division in Cyanidioschyzon merolae

Hishida, K., Yagisawa, F., Kuroiwa, H., Nagata, T. and Kuroiwa, T. Mol. Biol. Cell, 16, 2493-2502 (2005)   Mitochondrial and chloroplast division controls the number and morphology of organelles, but how cells regulate organelle division remains to be clarified. Here, we show that each step of mitochondrial and chloroplast division is closely associated with the cell cycle in Cyanidioschyzon merolae. Electron microscopy revealed direct associations between the spindle pole bodies and mitochondria, suggesting that mitochondrial distribution is physically coupled with mitosis. Interconnected organelles were fractionated under microtubule-stabilizing condition. Immunoblotting analysis revealed that the protein levels required for organelle division increased before microtubule changes upon cell division, indicating that regulation of protein expression for organelle division is distinct from that of cytokinesis. At the mitochondrial division site, dynamin stuck to one of the divided mitochondria and was spatially associated with the tip of a microtubule stretching from the other one. Inhibition of microtubule organization, proteasome activity or DNA synthesis, respectively, induced arrested cells with divided but shrunk mitochondria, with divided and segregated mitochondria, or with incomplete mitochondrial division restrained at the final severance, and repetitive chloroplast division. The results indicated that mitochondrial morphology and segregation but not division depend on microtubules and implied that the division processes of the two organelles are regulated at distinct checkpoints.  

3.625           Presenilin function and g-secretase activity

Brunkan, A.L. and Goate, A.M.

1. Neurochem., 93, 769-792 (2005)

  Alzheimer's disease (AD) is the most common form of dementia and is characterized pathologically by the accumulation of -amyloid (A ) plaques and neurofibrillary tangles in the brain. Genetic studies of AD first highlighted the importance of the presenilins (PS). Subsequent functional studies have demonstrated that PS form the catalytic subunit of the -secretase complex that produces the A peptide, confirming the central role of PS in AD biology. Here, we review the studies that have characterized PS function in the -secretase complex in Caenorhabditis elegans, mice and in in vitro cell culture systems, including studies of PS structure, PS interactions with substrates and other -secretase complex members, and the evidence supporting the hypothesis that PS are aspartyl proteases that are active in intramembranous proteolysis. A thorough knowledge of the mechanism of PS cleavage in the context of the -secretase complex will further our understanding of the molecular mechanisms that cause AD, and may allow the development of therapeutics that can alter A production and modify the risk for AD.  

3.626           Amphotropic murine leukaemia virus envelope protein is associated with cholesterol-rich microdomains

Beer, C., Pedersen, L. and Wirth, M. Virology J., 2(36), 1-9 (2005)   Background Cholesterol-rich microdomains like lipid rafts were recently identified as regions within the plasma membrane, which play an important role in the assembly and budding of different viruses, e.g., measles virus and human immunodeficiency virus. For these viruses association of newly synthesized viral proteins with lipid rafts has been shown. Results Here we provide evidence for the association of the envelope protein (Env) of the 4070A isolate of amphotropic murine leukaemia virus (A-MLV) with lipid rafts. Using density gradient centrifugation and immunocytochemical analyses, we show that Env co-localizes with cholesterol, ganglioside GM1 and caveolin-1 in these specific regions of the plasma membrane. Conclusions These results show that a large amount of A-MLV Env is associated with lipid rafts and suggest that cholesterol-rich microdomains are used as portals for the exit of A-MLV.  

3.627           Role of photoreceptor-specific retinal dehydrogenase in the retinoid cycle in vivo

Maeda, A. et al

1. Biol. Chem., 280(19), 18822-18832 (2005)

  The retinoid cycle is a recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. Photoreceptor-specific retinol dehydrogenase (prRDH) catalyzes reduction of all-trans-retinal to all-trans-retinol and is thought to be a key enzyme in the retinoid cycle. We disrupted mouse prRDH (human gene symbol RDH8) gene expression by targeted recombination and generated a homozygous prRDH knock-out (prRDH–/–) mouse. Histological analysis and electron microscopy of retinas from 6- to 8-week-old prRDH–/– mice revealed no structural differences of the photoreceptors or inner retina. For brief light exposure, absence of prRDH did not affect the rate of 11-cis-retinal regeneration or the decay of Meta II, the activated form of rhodopsin. Absence of prRDH, however, caused significant accumulation of all-trans-retinal following exposure to bright lights and delayed recovery of rod function as measured by electroretinograms and single cell recordings. Retention of all-trans-retinal resulted in slight overproduction of A2E, a condensation product of all-trans-retinal and phosphatidylethanolamine. We conclude that prRDH is an enzyme that catalyzes reduction of all-trans-retinal in the rod outer segment, most noticeably at higher light intensities and prolonged illumination, but is not an essential enzyme of the retinoid cycle.  

3.628           Acidocacisomes – conserved from bacteria to man

Docampo, R., de Souza, W., Miranda, K., Rohloff, P. and Moreno, S.N.J. Nature Rev. Microbiol., 3, 251-261 (2005)   Recent work has shown that acidocalcisomes, which are electron-dense acidic organelles rich in calcium and polyphosphate, are the only organelles that have been conserved during evolution from prokaryotes to eukaryotes. Acidocalcisomes were first described in trypanosomatids and have been characterized in most detail in these species. Acidocalcisomes have been linked with several functions, including storage of cations and phosphorus, polyphosphate metabolism, calcium homeostasis, maintenance of intracellular pH homeostasis and osmoregulation. Here, we review acidocalcisome ultrastructure, composition and function in different trypanosomatids and other organisms.  

3.629           C18:3-GM1a induces apoptosis in Neuro2 cells: enzymatic remodeling of fatty acyl chains of glycosphingolipids

Nakagawa, T. Et al

1. Lipi. Res., 46, 1103-1112 (2005)

  GM1a [Galß1-3GalNAcß1-4(NeuAc 2-3)Galß1-4Glcß1-1Cer] is known to support and protect neuronal functions. However, we report that -linolenic acid-containing GM1a (C18:3-GM1a), which was prepared using the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase, induced apoptosis in neuronalcells. Intranucleosomal DNA fragmentation, chromatin condensation,and caspase activation, all typical features of apoptosis, wereobserved when mouse neuroblastoma Neuro2a cells were culturedwith C18:3-GM1a but not GM1a containing stearic acid (C18:0)or oleic acid (C18:1). The phenotype of Neuro2a cells inducedby C18:3-GM1a was similar to that evoked by lyso-GM1a. However,lyso-GM1a caused a complete disruption of lipid microdomainsof Neuro2a cells and hemolysis of sheep erythrocytes, whereasC18:3-GM1a did neither. C18:3-GM1a, but not lyso-GM1a, was foundto be abundant in lipid microdomains after the removal of looselybound GM1a by BSA. The activation of stress-activated proteinkinase/c-Jun N-terminal kinase in Neuro2a cells was observedwith lyso-GM1a but not C18:3-GM1a. These results indicate that the mechanism of apoptosis inducedby C18:3-GM1a is distinct from that caused by lyso-GM1a. Thisstudy also clearly shows that fatty acid composition of gangliosidessignificantly affected their pharmacological activities whenadded to the cell cultures and suggests why naturally occurringgangliosides do not possess polyunsaturated fatty acids as amajor constituent.  

3.630           Truncated prion protein and doppel are myelinotoxic in the absence of oligodendrocytic PrP^C

Radovanovic, I. Et al

1. Neurosci., 25(19), 4879-4888 (2005)

  The cellular prion protein PrP^C confers susceptibility to transmissible spongiform encephalopathies, yet its normal function is unknown. Although PrP^C-deficient mice develop and live normally, expression of amino proximally truncated PrP^C ( PrP) or of its structural homolog Doppel (Dpl) causes cerebellar degeneration that is prevented by coexpression of full-length PrP^C. We now report that mice expressing PrP or Dpl suffer from widespread leukoencephalopathy. Oligodendrocyte-specific expression of full-length PrP^C under control of the myelin basic protein (MBP) promoter repressed leukoencephalopathy and vastly extended survival but did not prevent cerebellar granule cell (CGC) degeneration. Conversely, neuron-specific PrP^C expression under control of the neuron-specific enolase (NSE) promoter antagonized CGC degeneration but not leukoencephalopathy. PrP^C was found in purified myelin and in cultured oligodendrocytes of both wild-type and MBP-PrP transgenic mice but not in NSE-PrP mice. These results identify white-matter damage as an extraneuronal PrP-associated pathology and suggest a previously unrecognized role of PrP^C in myelin maintenance.  

3.631           Differential effect of phosphatidylethanolamine depletion on raft proteins. Further evidence for diversity of rafts in Saccharomyces cerevisiae

Opekarova, M., Malinska, K., Novakova, L. and Tanner, W. Biochim. Biophys. Acta, 1711(1), 87-95 (2005)   A considerable amount of evidence supports the idea that lipid rafts are involved in many cellular processes, including protein sorting and trafficking. We show that, in this process, also a non-raft lipid, phosphatidylethanolamine (PE), has an indispensable function. The depletion of this phospholipid results in an accumulation of a typical raft-resident, the arginine transporter Can1p, in the membranes of Golgi, while the trafficking of another plasma membrane transporter, Pma1p, is interrupted at the level of the ER. Both these transporters associate with a Triton (TX-100) resistant membrane fraction before their intracellular transport is arrested in the respective organelles. The Can1p undelivered to the plasma membrane is fully active when reconstituted to a PE-containing vesicle system in vitro. We further demonstrate that, in addition to the TX-100 resistance at 4 °C, Can1p and Pma1pa exhibit different accessibility to nonyl glucoside (NG), which points to distinct intimate lipid surroundings of these two proteins. Also, at 20 °C, these two proteins are extracted by TX-100 differentially. The features above suggest that Pma1p and Can1p are associated with different compartments. This is independently supported by the observations made by confocal microscopy. In addition we show that PE is involved in the stability of Can1p–raft association.  

3.632           Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells

Beronja, S. et al

1. Cell Biol., 169(4), 635-646 (2005)

  Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs.  

3.633           Myosin Va transports dense core secretory vesicles in pancreatic MIN6 b-cells

Varadi, A., Tsuboi, T. And Rutter, G.A. Mol. Biol. Cell, 16, 2670-2680 (2005)   The role of unconventional myosins in neuroendocrine cells is not fully understood, with involvement suggested in the movement of both secretory vesicles and mitochondria. Here, we demonstrate colocalization of myosin Va (MyoVa) with insulin in pancreatic -cells and show that MyoVa copurifies with insulin in density gradients and with the vesicle marker phogrin-enhanced green fluorescent protein upon fluorescence-activated sorting of vesicles. By contrast, MyoVa immunoreactivity was poorly colocalized with mitochondrial or other markers. Demonstrating an important role for MyoVa in the recruitment of secretory vesicles to the cell surface, a reduction of MyoVa protein levels achieved by RNA interference caused a significant decrease in glucose- or depolarization-stimulated insulin secretion. Similarly, expression of the dominant-negative–acting globular tail domain of MyoVa decreased by 50% the number of vesicles docked at the plasma membrane and by 87% the number of depolarization-stimulated exocytotic events detected by total internal reflection fluorescence microscopy. We conclude that MyoVa-driven movements of vesicles along the cortical actin network are essential for the terminal stages of regulated exocytosis in -cells.  

3.634           Acute and chronic changes in cholesterol modulate Na-P_i cotransport activity in OK cells

Breusegem, S.Y. et al Am. J. Physiol., 289, FF154-F165 (2005)   We previously showed an inverse correlation between membrane cholesterol content and Na-P_i cotransport activity during the aging process and adaptation to alterations in dietary P_i in the rat (Levi M, Jameson DM, and van der Meer BW. Am J Physiol Renal Fluid Electrolyte Physiol 256: F85–F94, 1989). The purpose of the present study was to determine whether alterations in cholesterol content per se modulate Na-P_i cotransport activity and apical membrane Na-P_i protein expression in opossum kidney (OK) cells. Acute cholesterol depletion achieved with -methyl cyclodextrin ( -MCD) resulted in a significant increase in Na-P_i cotransport activity accompanied by a moderate increase in apical membrane Na-P_i protein abundance and no alteration of total cellular Na-P_i protein abundance. Conversely, acute cholesterol enrichment achieved with -MCD/cholesterol resulted in a significant decrease in Na-P_i cotransport activity with a moderate decrease in apical membrane Na-Pi protein abundance and no change of the total cellular Na-P_i protein abundance. In contrast, chronic cholesterol depletion, achieved by growing cells in lipoprotein-deficient serum (LPDS), resulted in parallel and significant increases in Na-P_i cotransport activity and apical membrane and total cellular Na-P_i protein abundance. Cholesterol depletion also resulted in a significant increase in membrane lipid fluidity and alterations in lipid microdomains as determined by laurdan fluorescence spectroscopy and imaging. Chronic cholesterol enrichment, achieved by growing cells in LPDS followed by loading with low-density lipoprotein, resulted in parallel and significant decreases in Na-P_i cotransport activity and apical membrane and total cellular Na-P_i protein abundance. Our results indicate that in OK cells acute and chronic alterations in cholesterol content per se modulate Na-P_i cotransport activity by diverse mechanisms that also include significant interactions of Na-P_i protein with lipid microdomains.  

3.635           Water and solute permeability of rat lung caveolae: high permeabilities explained by acyl chain unsaturation

Hill, W.G., Almasri, E., Ruiz, W.G., Apodaca, G. and Zeidel, M.L. Am. J. Physiol., 289, C33-C41 (2005)   Caveolae are invaginated membrane structures with high levels of cholesterol, sphingomyelin, and caveolin protein that are predicted to exist as liquid-ordered domains with low water permeability. We isolated a caveolae-enriched membrane fraction without detergents from rat lung and characterized its permeability properties to nonelectrolytes and protons. Membrane permeability to water was 2.85 ± 0.41 x 10^–3 cm/s, a value 5–10 times higher than expected based on comparisons with other cholesterol and sphingolipid-enriched membranes. Permeabilities to urea, ammonia, and protons were measured and found to be moderately high for urea and ammonia at 8.85 ± 2.40 x 10^–7and 6.84 ± 1.03 x 10^–2 respectively and high for protons at 8.84 ± 3.06 x 10^–2 cm/s. To examine whether caveolin or other integral membrane proteins were responsible for high permeabilities, liposomes designed to mimic the lipids of the inner and outer leaflets of the caveolar membrane were made. Osmotic water permeability to both liposome compositions were determined and a combined inner/outer leaflet water permeability was calculated and found to be close to that of native caveolae at 1.58 ± 1.1 x 10^–3 cm/s. In caveolae, activation energy for water flux was high (19.4 kcal/mol) and water permeability was not inhibited by HgCl₂; however, aquaporin 1 was detectable by immunoblotting. Immunostaining of rat lung with AQP1 and caveolin antisera revealed very low levels of colocalization. We conclude that aquaporin water channels do not contribute significantly to the observed water flux and that caveolae have relatively high water and solute permeabilities due to the high degree of unsaturation in their fatty acyl chains.  

3.636           Use of phospho-specific antibodies to determine the phosphorylation of endogenous Na⁺/H⁺ exchanger NHE3 at PKA consensus sites

Kocinsky, H.S. et al Am. J. Physiol., 289, F249-F258 (2005)   Transfection studies using mutant constructs have implicated one or both protein kinase A (PKA) consensus phosphorylation sites [serines 552 and 605 in rat Na⁺/H⁺ exchanger type 3 (NHE3)] as critical for mediating inhibition of NHE3 in response to several stimuli including dopamine. However, whether one or both of these sites is actually phosphorylated in endogenous NHE3 in proximal tubule cells is unknown. The purpose of this study was to generate phosphospecific antibodies so that the state of phosphorylation of these serine residues in endogenous NHE3 could be assessed in vitro and in vivo. To this end, polyclonal and monoclonal phosphospecific peptide antibodies were generated against each PKA consensus site. Phosphospecificity was established by ELISA and Western blot assays. We then used these antibodies in vitro to evaluate the effect of dopamine on phosphorylation of the corresponding PKA sites (serines 560 and 613) in NHE3 endogenously expressed in opossum kidney cells. Baseline phosphorylation of both sites was detected that was significantly increased by dopamine. Next, we determined the baseline phosphorylation state of each serine in rat kidney NHE3 in vivo. We found that serine 552 of NHE3 is phosphorylated to a much greater extent than serine 605 at baseline in vivo. Moreover, we detected a distinct subcellular localization for NHE3 phosphorylated at serine 552 compared with total NHE3. Specifically, NHE3 phosphorylated at serine 552 localized to the coated pit region of the brush-border membrane, where NHE3 is inactive, while total NHE3 was found throughout the brush-border membrane. These findings strongly suggest that phosphorylation of NHE3 plays a role in its subcellular trafficking in vivo. In conclusion, we successfully generated phosphospecific antibodies that should be useful to assess the phosphorylation of endogenous NHE3 at its two PKA consensus sites under a variety of physiological conditions in vitro and in vivo.  

3.637           Interaction of presenilins with FKBP38 promotes apoptosis by reducing mitochondrial Bcl-2

Wang, H-Q. et al Hum. Mol. Genet., 14(13), 1889-1902 (2005)   Presenilins 1 and 2 (PS1/2), causative molecules for familial Alzheimer's disease (FAD), are multipass transmembrane proteins localized predominantly in the endoplasmic reticulum (ER) and Golgi apparatus. Heteromeric protein complexes containing PS1/2 are thought to participate in several functions, including intramembrane proteolysis mediated by their -secretase activities. Previous studies have shown that PS1/2 are also involved in the regulation of apoptotic cell death, although the underlying mechanism remains unknown. Here, we demonstrate that FKBP38, an immunophilin family member residing in the mitochondrial membrane, is an authentic PS1/2-interacting protein. PS1/2 and FKBP38 form macromolecular complexes together with anti-apoptotic Bcl-2. PS1/2 promote the degradation of FKBP38 and Bcl-2 and sequester these proteins in the ER/Golgi compartments, thereby inhibiting FKBP38-mediated mitochondrial targeting of Bcl-2 via a -secretase-independent mechanism. Thus, PS1/2 increase the susceptibility to apoptosis by antagonizing the anti-apoptotic function of FKBP38. In contrast, C-terminal fragments of caspase-processed PS1/2 redistribute Bcl-2 to the mitochondria by abrogating the activity of full-length PS1/2, resulting in a dominant-negative anti-apoptotic effect. In cultured cells and mutant PS1-knockin mice brains, FAD-linked PS1/2 mutants enhance the pro-apoptotic activity by causing a more efficient reduction in mitochondrial Bcl-2 than wild-type PS1/2. These results suggest a novel molecular mechanism for the regulation of mitochondria-mediated apoptosis by competition between PS1/2 and FKBP38 for subcellular targeting of Bcl-2. Excessive pro-apoptotic activity of PS1/2 may play a role in the pathogenesis of FAD.  

3.638           Analysis of proteome bound to D-loop region of mitochondrial DNA by DNA-linked affinity chromatography and reverse-phase liquid chromatography/tandem mass spectrometry

Choi, Y-S., Ryu, B-K., Min, H-K., Lee, S-W. and Pak, Y.K. Ann. N.Y. Acad. Sci., 1042, 88-100 (2005)   Mitochondrial dysfunction has been suggested as a causal factor for insulin resistance and diabetes. Previously we have shown a decrease of mitochondrial DNA (mtDNA) content in tissues of diabetic patients. The mitochondrial proteins, which regulate the mitochondrial biogensis, including transcription and replication of mtDNA, are encoded by nuclear DNA. Despite the potential function of the proteins bound to the D-loop region of mtDNA in regulating mtDNA transcription/replication, only a few proteins are known to bind the D-loop region of mtDNA. The functional association of these known proteins with insulin resistance is weak. In this study, we applied proteomic analysis to identify a group of proteins (proteome) that physically bind to D-loop DNA of mtDNA. We amplified D-loop DNA (1.1 kb) by PCR and conjugated the PCR fragments to CNBr-activated sepharose. Mitochondria fractions were isolated by both differential centrifugation and Optiprep-gradient ultracentrifugation. The D-loop DNA binding proteome fractions were enriched via this affinity chromatography and analyzed by SDS-PAGE. The proteins on the gel were transferred onto PVDF membrane and the peptide sequences of each band were subsequently analyzed by capillary reverse-phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS). We identified many D-loop DNA binding proteins, including mitochondrial transcription factor A (mtTFA, Tfam) and mitochondrial single-stranded DNA binding protein (mtSSBP) which were known to bind to mtDNA. We also report the possibility of novel D-loop binding proteins such as histone family proteins and high-mobility group proteins.  

3.639           Intravesicular localization and exocytosis of a-synuclein and its aggregates

Lee, H.J., Patel, S. and Lee, S-J.

1. Neurosci., 25(25), 6016-6024 (2005)

  a-Synuclein ( -syn), particularly in its aggregated forms, is implicated in the pathogenesis of Parkinson's disease and other related neurological disorders. However, the normal biology of -syn and how it relates to the aggregation of the protein are not clearly understood. Because of the lack of the signal sequence and its predominant localization in the cytosol, -syn is generally considered exclusively an intracellular protein. Contrary to this assumption, here, we show that a small percentage of newly synthesized -syn is rapidly secreted from cells via unconventional, endoplasmic reticulum/Golgi-independent exocytosis. Consistent with this finding, we also demonstrate that a portion of cellular -syn is present in the lumen of vesicles. Importantly, the intravesicular -syn is more prone to aggregation than the cytosolic protein, and aggregated forms of -syn are also secreted from cells. Furthermore, secretion of both monomeric and aggregated -syn is elevated in response to proteasomal and mitochondrial dysfunction, cellular defects that are associated with Parkinson's pathogenesis. Thus, intravesicular localization and secretion are part of normal life cycle of -syn and might also contribute to pathological function of this protein.  

3.640           Analysis of human immunodeficiency virus type 1 Gag ubiquitination

Gottwein, E. And Kräusslich, H-G.

1. Virol., 79(14), 9134-9144 (2005)

  Ubiquitin is important for the release of human immunodeficiency virus type 1 (HIV-1) and several other retroviruses, but the functional significance of Gag ubiquitination is unknown. To address this problem, we decided to analyze Gag ubiquitination in detail. A low percentage of the HIV-1 p6 protein has previously been shown to be ubiquitinated, and published mutagenesis data suggested that Gag ubiquitination is largely lost upon mutation of the two lysine residues in p6. In this study, we show that Gag proteins lacking the p6 domain or the two lysine residues within p6 are ubiquitinated at levels comparable to those of the wild-type Gag protein. We detected monoubiquitinated forms of the matrix (MA), capsid (CA), and nucleocapsid (NC) proteins in mature virus preparations. Protease digestion of Gag polyproteins extracted from immature virions indicated that ubiquitinated MA, CA, and possibly NC are as abundant as ubiquitinated p6. The HIV-1 late-domain motifs PTAP and LRSLF were not required for Gag ubiquitination, and mutation of the PTAP motif even resulted in an increase in the amount of Gag-Ub conjugates detected. Finally, at steady state, ubiquitinated Gag proteins were not enriched in either membrane-associated or virus-derived Gag fractions. In summary, these results indicate that HIV-1 Gag can be monoubiquitinated in all domains and that ubiquitination of lysine residues outside p6 may thus contribute to viral release and/or infectivity.  

3.641           Hyperhomocysteinemia, a cardiac metabolic disease role of nitric oxide and the p22^phox subunit of NADPH oxidase

Becker, J.S. et al Circulation, 111, 2112-2118 (2005)   Background— Hyperhomocysteinemia (HHcy) is a reliable indicator of cardiovascular disease, in part because of the production of superoxide and scavenging of nitric oxide (NO). The present study assessed the impact of HHcy on the NO-dependent control of cardiac O₂ consumption and examined enzymatic sourcesof superoxide. Methods and Results— Rats and mice were fed methionine in drinking water for 5 to 9 weeks to increase plasma homocysteine, a process that did not cause significant changes in hemodynamic function. The ability of the NO agonists bradykinin and carbachol to reduce myocardial O₂ consumption in vitro was impaired by 40% in methionine-fed rats, and this impairment was proportional to their individual plasma homocysteine concentration. However, responses were restored in the presence of ascorbic acid, tempol, and apocynin, which inhibits NADPH oxidase assembly. Western blots showed no difference in Cu/Zn or Mn superoxide dismutase, endothelial NO synthase, or inducible NO synthase protein, but HHcy caused a 100% increase in the p22^phox subunit of NADPH oxidase. Western blots with plasma membrane–enriched fractions of cell lysate detected elevated levels of p22^phox, p67^phox, and rac-1, which indicates increased oxidase assembly. Finally, mice lacking a functional gp91^phox subunit of NADPH oxidase demonstrated normal NO-dependent regulation of myocardial O₂consumption after methionine feeding. Conclusions— In HHcy, superoxide produced by NADPH oxidasereduces the ability of NO to regulate mitochondrial functionin the myocardium. The severity of this effect is proportionalto the increase in homocysteine.  

3.642           Ceramide 1-phosphate, a mediator of phagocytosis

Hinkovska-Galcheva, V. Et al

1. Biol. Chem., 280(28), 26612-26621 (2005)

  The agonist-stimulated metabolism of membrane lipids produces potent second messengers that regulate phagocytosis. We studied whether human ceramide kinase (hCERK) activity and ceramide 1-phosphate formation could lead to enhanced phagocytosis through a mechanism involving modulation of the membrane-structural order parameter. hCERK was stably transfected into COS-1 cells that were stably transfected with the Fc RIIA receptor. hCERK-transfected cells displayed a significant increase in phagocytic index in association with increased ceramide kinase activation and translocation to lipid rafts after activation with opsonized erythrocytes. When challenged with opsonized erythrocytes, hCERK-transfected cells increased phagocytosis by 1.5-fold compared with vector control and simultaneously increased ceramide 1-phosphate levels 2-fold compared with vector and unstimulated control cells. Control and hCERK-transfected cells were subjected to cellular fractionation. Utilizing an antibody against hCERK, we observed that CERK translocates during activation from the cytosol to a lipid raft fraction. The plasma membrane-structural order parameter of the transfectants was measured by labeling cells with Laurdan. Cells transfected with hCERK showed a higher liquid crystalline order than control cells with stimulation, conditions that are favorable for the promotion of membrane fusion at the sites of phagocytosis. The change in the structural order parameter of the lipid rafts probably contributes to phagocytosis by promoting phagosome formation.  

3.643           Murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release

Choi, K.S., Aizaki, H. and Lai, M.M.

1. Virol., 79(15), 9862-9871 (2005)

  Thorp and Gallagher first reported that depletion of cholesterol inhibited virus entry and cell-cell fusion of mouse hepatitis virus (MHV), suggesting the importance of lipid rafts in MHV replication (E. B. Thorp and T. M. Gallagher, J. Virol. 78:2682-2692, 2004). However, the MHV receptor is not present in lipid rafts, and anchoring of the MHV receptor to lipid rafts did not enhance MHV infection; thus, the mechanism of lipid rafts involvement is not clear. In this study, we defined the mechanism and extent of lipid raft involvement in MHV replication. We showed that cholesterol depletion by methyl ß-cyclodextrin or filipin did not affect virus binding but reduced virus entry. Furthermore, MHV spike protein bound to nonraftraft membrane at 4°C but shifted to lipid rafts at 37°C, indicating a redistribution of membrane following virus binding. Thus, the lipid raft involvement in MHV entry occurs at a step following virus binding. We also found that the viral spike protein in the plasma membrane of the infected cells was associated with lipid rafts, whereas that in the Golgi membrane, where MHV matures, was not. Moreover, the buoyant density of the virion was not changed when MHV was produced from the cholesterol-depleted cells, suggesting that MHV does not incorporate lipid rafts into the virion. These results indicate that MHV release does not involve lipid rafts. However, MHV spike protein has an inherent ability to associate with lipid rafts. Correspondingly, cell-cell fusion induced by MHV was retarded by cholesterol depletion, consistent with the association of the spike protein with lipid rafts in the plasma membrane. These findings suggest that MHV entry requires specific interactions between the spike protein and lipid rafts, probably during the virus internalization step.  

3.644           Epidermal growth factor receptors are localized to lipid rafts that contain a balance of inner and outer leaflet lipids

Pike, L.J., Han, X. and Gross, R.W.

1. Biol. Chem., 280(29), 26796-26804

  The epidermal growth factor (EGF) receptor partitions into lipid rafts made using a detergent-free method, but is extracted from low density fractions by Triton X-100. By screening several detergents, we identified Brij 98 as a detergent in which the EGF receptor is retained in detergent-resistant membrane fractions. To identify the difference in lipid composition between those rafts that harbored the EGF receptor (detergent-free and Brij 98-resistant) and those that did not (Triton X-100-resistant), we used multidimensional electrospray ionization mass spectrometry to perform a lipidomics study on these three raft preparations. Although all three raft preparations were similarly enriched in cholesterol, the EGF receptor-containing rafts contained more ethanolamine glycerophospholipids and less sphingomyelin than did the non-EGF receptor-containing Triton X-100 rafts. As a result, the detergent-free and Brij 98-resistant rafts exhibited a balance of inner and outer leaflet lipids, whereas the Triton X-100 rafts contained a preponderance of outer leaflet lipids. Furthermore, in all raft preparations, the outer leaflet phospholipid species were significantly different from those in the bulk membrane, whereas the inner leaflet lipids were quite similar to those found in the bulk membrane. These findings indicate that the EGF receptor is retained only in rafts that exhibit a lipid distribution compatible with a bilayer structure and that the selection of phospholipids for inclusion into rafts occurs mainly on the outer leaflet lipids.  

3.645           Light-dependent redistribution of arrestin in vertebrate rods is an energy-independent process governed by protein-protein interactions

Nair, K.S. et al Neuron, 46, 555-567 (2005)   In rod photoreceptors, arrestin localizes to the outer segment (OS) in the light and to the inner segment (IS) in the dark. Here, we demonstrate that redistribution of arrestin between these compartments can proceed in ATP-depleted photoreceptors. Translocation of transducin from the IS to the OS also does not require energy, but depletion of ATP or GTP inhibits its reverse movement. A sustained presence of activated rhodopsin is required for sequestering arrestin in the OS, and the rate of arrestin relocalization to the OS is determined by the amount and the phosphorylation status of photolyzed rhodopsin. Interaction of arrestin with microtubules is increased in the dark. Mutations that enhance arrestin-microtubule binding attenuate arrestin translocation to the OS. These results indicate that the distribution of arrestin in rods is controlled by its dynamic interactions with rhodopsin in the OS and microtubules in the IS and that its movement occurs by simple diffusion.  

3.646           Role of fission yeast myosin I in organization of sterol-rich membrane domains

Takeda, T. and Chang, F. Current Biol., 15, 1331-1336 (2005)   Specialized membrane domains containing lipid rafts are thought to be important for membrane processes such as signaling and trafficking [1 and 2]. An unconventional type I myosin has been shown to reside in lipid rafts and function to target a disaccharidase to rafts in brush borders of intestinal mammalian cells [3]. In the fission yeast Schizosaccharomyces pombe, distinct sterol-rich membrane domains are formed at the cell division site and sites of polarized cell growth at cell tips [4]. Here, we show that the sole S. pombe myosin I, myo1p, is required for proper organization of these membrane domains. myo1 mutants lacking the TH1 domain exhibit a uniform distribution of sterol-rich membranes all over the plasma membrane throughout the cell cycle. These effects are independent of endocytosis because myo1 mutants exhibit no endocytic defects. Conversely, overexpression of myo1p induces ectopic sterol-rich membrane domains. Myo1p localizes to nonmotile foci that cluster in sterol-rich plasma membrane domains and fractionates with detergent-resistant membranes. Because the myo1p TH1 domain may bind directly to acidic phospholipids, these findings suggest a model for how type I myosin contributes to the organization of specialized membrane domains.  

3.647           Relationship between Alzheimer’s disease clinical stage and Gq/11 in subcellular fractions of frontal cortex

Kelly, J.F. et al

1. Neural Transm., 112, 1049-1056 (2005)

  Alzheimer’s disease (AD) is associated with impaired coupling of cell surface muscarinic cholinergic receptors to G proteins of the Gq/11 class in brain. This alteration may contribute to progression of cognitive impairment during the course of the disease. We hypothesized that increasing severity of cognitive impairment would be related to decreased levels of Gq/11 detected in key subcellular fractions made from postmortem brain tissue. In this study, we used Western blotting to determine the quantity of Gq/11α in P2, synaptic plasma membrane, cytoplasm, microsomal membrane, and lipid raft fractions prepared from superior frontal cortex gray matter of 25 patients with clinical AD confirmed by post-mortem examination. Multiple linear regression analysis that adjusted for age, sex, and education showed a linear relationship between frontal cortex synaptic plasma membrane Gq/11α levels and severity of cognitive impairment determined by Mini Mental State score measured proximate to death.  

3.648           Two domains within the first putative transmembrane domain of presenilin 1 differentially influence presenilinase and g-secretase activity

Brunkan, A.L. et al

1. Neurochem., 94, 1315-1328 (2005)

  Presenilins (PS) are thought to contain the active site for presenilinase endoproteolysis of PS and -secretase cleavage of substrates. The structural requirements for PS incorporation into the -secretase enzyme complex, complex stability and maturation, and appropriate presenilinase and -secretase activity are poorly understood. We used rescue assays to identify sequences in transmembrane domain one (TM1) of PS1 required to support presenilinase and -secretase activities. Swap mutations identified an N-terminal TM1 domain that is important for -secretase activity only and a C-terminal TM1 domain that is essential for both presenilinase and -secretase activities. Exchange of residues 95 98 of PS1 (sw95 98) completely abolishes both activities while the familial Alzheimer's disease mutation V96F significantly inhibits both activities. Reversion of residue 96 back to valine in the sw95 98 mutant rescues PS function, identifying V96 as the critical residue in this region. The TM1 mutants do not bind to an aspartyl protease transition state analog -secretase inhibitor, indicating a conformational change induced by the mutations that abrogates catalytic activity. TM1 mutant PS1 molecules retain the ability to interact with -secretase substrates and -secretase complex members, although Nicastrin stability is decreased by the presence of these mutants. -Secretase complexes that contain V96F mutant PS1 molecules display a partial loss of function for -secretase that alters the ratio of amyloid- peptide species produced, leading to the amyloid- peptide aggregation that causes familial Alzheimer's disease.  

3.649           Effects of a mosquitocidal toxin on a mammalian epithelial cell line expressing its target receptor

Pauchet, Y. Et al Cell. Microbiol., 7(9), 1335-1344 (2005)   The spread of diseases transmitted by Anopheles and Culex mosquitoes, such as malaria and West Nile fever, is a growing concern for human health. Bacillus sphaericus binary toxin (Bin) is one of the few available bioinsecticides able to control populations of these mosquitoes efficiently. We previously showed that Bin binds to Cpm1, an -glucosidase located on the apical side of Culex larval midgut epithelium. We analysed the effects of Bin by expressing a construct encoding Cpm1 in the mammalian epithelial MDCK cell line. Cpm1 is targeted to the apical side of polarized MDCK, where it is anchored by glycosylphosphatidylinositol (GPI) and displays -glucosidase activity. Bin bound to transfected cells and induced a non-specific current presumably related to the opening of pores. The formation of these pores may be related to the location of the toxin/receptor complex in lipid raft microdomains. Finally, Bin promoted the time-dependent appearance of intracytoplasmic vacuoles but did not drive cell lysis. Thus, the dual functionality (enzyme/toxin receptor) of Cpm1 is fully conserved in MDCK cells and Cpm1 is an essential target protein for Bin cytotoxicity in Culex mosquitoes.  

3.650           The golgin lava lamp mediates dynein-based Golgi movements during Drosophila cellularization

Papoulas, O., Hays, T.S. and Sisson, J.C. Nature Cell Biol., 7(8), 612-618 (2005)   Drosophila melanogaster cellularization is a dramatic form of cytokinesis in which a membrane furrow simultaneously encapsulates thousands of cortical nuclei of the syncytial embryo to generate a polarized cell layer. Formation of this cleavage furrow depends on Golgi-based secretion and microtubules^{1, 2, 3}. During cellularization, specific Golgi move along microtubules, first to sites of furrow formation and later to accumulate within the apical cytoplasm of the newly forming cells³. Here we show that Golgi movements and furrow formation depend on cytoplasmic dynein. Furthermore, we demonstrate that Lava lamp (Lva), a golgin protein that is required for cellularization, specifically associates with dynein, dynactin, cytoplasmic linker protein-190 (CLIP-190) and Golgi spectrin, and is required for the dynein-dependent targeting of the secretory machinery. The Lva domains that bind these microtubule-dependent motility factors inhibit Golgi movement and cellularization in a live embryo injection assay. Our results provide new evidence that golgins promote dynein-based motility of Golgi membranes.  

3.651           Functional role of the AAA peroxins in dislocation of the cycling PTS1 receptor back to the cytosol

Platta, H.W., Grunau, S., Rosenkrantz, K., Girzalsky, W. and Erdmann, R. Nature Cell Biol., 7(8), 817-822 (2005)   Peroxisomal import receptors bind their cargo proteins in the cytosol and target them to docking and translocation machinery at the peroxisomal membrane (reviewed in ref. 1). The receptors release the cargo proteins into the peroxisomal lumen and, according to the model of cycling receptors, they are supposed to shuttle back to the cytosol. This shuttling of the receptors has been assigned to peroxins including the AAA peroxins Pex1p and Pex6p, as well as the ubiquitin-conjugating enzyme Pex4p (reviewed in ref. 2). One possible target for Pex4p is the PTS1 receptor Pex5p, which has recently been shown to be ubiquitinated^{3, 4, 5}. Pex1p and Pex6p are both cytosolic and membrane-associated AAA ATPases of the peroxisomal protein import machinery, the exact function of which is still unknown. Here we demonstrate that the AAA peroxins mediate the ATP-dependent dislocation of the peroxisomal targeting signal-1 (PTS1) receptor from the peroxisomal membrane to the cytosol.

3.652           Exogenous nitric oxide reduces glucose transporters translocation and lactate production in ischemic myocardium in vivo

Lei, B. Et al PNAS, 102, 6966-6971 (2005)   Nitric oxide (NO) inhibits myocardial glucose transport and metabolism, although the underlying mechanism(s) and functional consequences of this effect are not clearly understood. We tested the hypothesis that NO inhibits the activation of AMP-activated protein kinase (AMPK) and translocation of cardiac glucose transporters (GLUTs; GLUT-4) and reduces lactate production. Ischemia was induced in open-chest dogs by a 66% flow reduction in the left anterior descending coronary artery (LAD). During ischemia, dogs were untreated (control) or treated by direct LAD infusion of (i) nitroglycerin (NTG) (0.5 µg·kg^–1·min^–1); (ii) 8-Br-cGMP (50 µg·kg^–1·min^–1); or (iii) NO synthase inhibitor L-nitro-argininemethylester (40 µg·kg^–1·min^–1; n = 9 per group). Cardiac substrate oxidation was measured with isotopic tracers. There were no differences in myocardial blood flow or oxygen delivery among groups; however, at 45 min of ischemia, the activation of AMPK was significantly less in NTG (77 ± 12% vs. nonischemic myocardium) and 8-Br-cGMP (104 ± 13%), compared with control (167 ± 17%). Similarly, GLUT-4 translocation was significantly reduced in NTG (74 ± 7%) and 8-Br-cGMP (120 ± 11%), compared with control (165 ± 17%). Glucose uptake and lactate output were 30% and 60% lower in NTG compared with control. Inhibition of NO synthesis stimulated glucose oxidation (67% increase compared with control) but did not affect AMPK phosphorylation, GLUT-4 translocation and glucose uptake. Contractile function in the ischemic region was significantly improved by NTG and L-nitro-argininemethylester. In conclusion, in ischemic myocardium an NO donor inhibits glucose uptake and lactate production via a reduction in AMPK stimulation of GLUT-4 translocation, revealing a mechanism of metabolic modulation and myocardial protection activated by NO donors.  

3.653           Role of membrane microdomains in PTH-mediated down-regulation of NaPi-Iia in opossum kidney cells

Nashiki, K. Et al Kidney Int., 68, 1137-1147 (2005)   Background. Parathyroid hormone (PTH) rapidly down-regulates type IIa sodium-dependent phosphate transporter (NaPi-IIa) via an endocytic pathway. Since the relationship between PTH signaling and NaPi-IIa endocytosis has not been explored, we investigated the role of membrane microdomains in this process. Methods. We examined the submembrane localization of NaPi-IIa in opossum kidney (OK-N2) cells that stably expressed human NaPi-IIa, and searched for a PTH-induced specific phosphorylating substrate on their membrane microdomains by immunoblotting with specific antibody against phospho substrates of protein kinases. Results. We found that NaPi-IIa was primarily localized in low-density membrane (LDM) domains of the plasma membrane; PTH reduced the levels of immunoreactive NaPi-IIa in these domains. Furthermore, PTH activated both protein kinase A (PKA) and protein kinase C (PKCa) and increased the phosphorylation of 250 kD and 80 kD substrates; this latter substrate was identified as ezrin, which a member of the ezrin-radixin-moesin (ERM) protein family. In response to PTH, ezrin was phosphorylated by both PKA and PKC. Dominant negative ezrin blocked the reduction in NaPi-IIa expression in the LDM domains that was induced by PTH. Conclusion. These data suggest that NaPi-IIa and PTH-induced phosphorylated proteins that include ezrin are compartmentalized in LDM microdomains. This compartmentalization may play an important role in the down-regulation of NaPi-IIa via endocytosis.  

3.654           B-cell receptor translocation to lipid rafts and associated signaling differ between prognostically important subgroups of chronic lymphocytic leukemia

Alsup, D.J. et al Cancer Res., 65(16), 7328-7337 (2005)   Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease in which interaction of the malignant cells with antigen is thought to play a key role. Individual CLL-cell clones markedly differ in their ability to respond to B-cell receptor ligation, but the mechanism underlying the frequent hyporesponsiveness is incompletely understood. Our aim was to further clarify the extent and cause of the B-cell receptor signaling abnormality in CLL and to assign pathophysiologic relevance to the presence or absence of B-cell receptor responsiveness. We show that extracellular signal-regulated kinase-2 phosphorylation, intracellular Ca²⁺ increases, CD79a phosphorylation, and translocation of the B-cell receptor to lipid rafts in response to ligation with anti–immunoglobulin M (as a surrogate for antigen) are features of CLL cells with relatively unmutated VH genes (<5% deviation from germ line) and a poor prognosis. B-cell receptor stimulation in these cases also promoted cell survival. In clones with mutated VH genes (>5% deviation from germ line), surface immunoglobulin M ligation failed to induce receptor translocation to rafts or to prolong cell survival. This failure of receptor translocation observed in mutated CLL cells was associated with the constitutive exclusion of the B-cell receptor from rafts by a mechanism involving src-dependent interactions between the B-cell receptor and the actin cytoskeleton. We conclude that exposure to antigen promotes the survival of unmutated CLL clones, contributing to the poor prognosis of this group. In contrast, hyporesponsive mutated CLL clones may have developed into a stage where continuous exposure to antigen results in relative tolerance to antigenic stimulation mediated by the exclusion of the B-cell receptor from lipid rafts.  

3.655           Overexpression of OSBP-related protein 2 (ORP2) induces changes in cellular cholesterol metabolism and enhances endocytosis

Hynynen, R. Et al Biochem. J., 273-283 (2005)   ORP2 [OSBP (oxysterol-binding protein)-related protein 2] belongs to the 12-member mammalian ORP gene/protein family. We characterize in the present study the effects of inducible ORP2 overexpression on cellular cholesterol metabolism in HeLa cells and compare the results with those obtained for CHO cells (Chinese-hamster ovary cells) that express ORP2 constitutively. In both cell systems, the prominent phenotype is enhancement of [¹⁴C]cholesterol efflux to all extracellular acceptors, which results in a reduction of cellular free cholesterol. No change was observed in the plasma membrane cholesterol content or distribution between raft and non-raft domains upon ORP2 expression. However, elevated HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase activity and LDL (low-density lipoprotein) receptor expression, as well as enhanced transport of newly synthesized cholesterol to a cyclodextrin-accessible pool, suggest that the ORP2 expression stimulates transport of cholesterol out of the endoplasmic reticulum. In contrast with ORP2/CHO cells, the inducible ORP2/HeLa cells do not show down-regulation of cholesterol esterification, suggesting that this effect represents an adaptive response to long-term cholesterol depletion in the CHO cell model. Finally, we provide evidence that ORP2 binds PtdIns(3,4,5)P₃ and enhances endocytosis, phenomena that are probably interconnected. Our results suggest a function of ORP2 in both cholesterol trafficking and control of endocytic membrane transport.  

3.656           Phagocytic signaling molecules in lipid rafts of COS-1 cells transfected with FcgRIIA

Mansfield, P.J., Hinkovska-Galcheva, V., Borofsky, M.S., Shayman, J.A. and Boxer, L.A. Biochem. Biophys. Res. Comm., 331, 132-138 (2005)   COS-1 cells bearing FcγRIIA were used as a model to demonstrate co-localization of several enzymes previously shown to regulate neutrophil phagocytosis. In COS-1 cells, phospholipase D (PLD) in the membrane fraction was activated during phagocytosis. PLD was found almost exclusively in lipid rafts, along with RhoA and ARF1. Protein kinase C-δ (PKCδ) and Raf-1 translocated to lipid rafts. In neutrophils, ceramide levels increase during phagocytosis, indicating that FcγRIIA engagement initiates ceramide generation. Applying this model, we transfected COS-1 cells with FcγRIIA that had been mutated in the ITAM region, rendering them unable to ingest particles. When the mutant receptors were engaged, ceramide was generated and MAPK was activated normally, thus these processes did not require actual ingestion of particles. These results indicate that signaling proteins for phagocytosis are either constitutively present in, or are recruited to, lipid rafts where they are readily available to activate one another.  

3.657           Caveolin-1 is essential for activation of Rac1 and NAD(P)H oxidase after angiotensin II type 1 receptor stimulation in vascular smooth muscle cells

Zuo, L. et al Arterioscler. Thromb. Vasc. Biol., 25, 1824-1830 (2005)   Objective— Angiotensin II (Ang II) is a potent mediator of vascular hypertrophy in vascular smooth muscle cells (VSMCs). These effects are mediated through the Ang II type 1 receptor (AT₁R) and require its trafficking through caveolin-1 (Cav1)–enriched lipid rafts and reactive oxygen species (ROS) derived from Rac1-dependent NAD(P)H oxidase. The specific role(s) of Cav1 in AT₁R signalingis incompletely understood. Methods and Results— Knockdown of Cav1 protein by small interfering RNA (siRNA) inhibits Ang II–stimulated Rac1 activation and membrane translocation, H₂O₂ production, ROS-dependent epidermal growth factor receptor (EGF-R) transactivation, and subsequent phosphorylation of Akt without affecting ROS-independent extracellular signal-regulated kinase 1/2 phosphorylation. Ang II stimulates tyrosine phosphorylation of Sos-1, a Rac–guanine nucleotide exchange factor, which is inhibited by Cav1 siRNA, demonstrating involvement of Cav1 in Rac1 activation. Detergent-free fractionation showed that EGF-Rs are found basally in Cav1-enriched lipid raft membranes and associate with Cav1. Ang II stimulates AT₁R movement into these microdomains contemporaneously with the egress of EGF-R. Both aspects of this bidirectional receptor trafficking are inhibited by Cav1 siRNA. Moreover, Cav1 siRNA inhibits Ang II–induced vascular hypertrophy. Conclusions— Cav1 plays an essential role in AT₁R targetinginto Cav1-enriched lipid rafts and Rac1 activation, which arerequired for proper organization of ROS-dependent Ang II signalinglinked to VSMC hypertrophy. Angiotensin II (Ang II)–induced vascular hypertrophy is dependent on caveolae/lipid rafts and reactive oxygen species (ROS) derived from NAD(P)H oxidase. Using caveolin-1 siRNA, we demonstrate that caveolin-1 plays an essential role in AT₁receptor targeting into caveolae/lipid rafts and Rac1 activation,which are required for ROS-dependent, growth-related Ang IIsignaling.  

3.658           Maturation of Borma virus disease virus glycoprotein

Richt, J.A. and Garten, W. FEBS Lett., 579, 4751-4756 (2005)   The maturation of Borna disease virus (BDV) glycoprotein GP was studied in regard to intracellular compartmentalization, compartmentalization signal-domains, proteolytic processing, and packaging into virus particles. Our data show that BDV-GP is (i) predominantly located in the endoplasmic reticulum (ER), (ii) partially exists in the ER already as cleaved subunits GP-N and GP-C, (iii) is directed to the ER/cis-Golgi region by its transmembrane and/or cytoplasmic domains in CD8-BDV-GP hybrid constructs and (iv) is incorporated in the virus particles as authentic BDV glycoprotein exclusively in the cleaved form decorated with N-glycans of the complex type. Downregulation of BDV-glycoproteins on the cell surface, their limited proteolytic processing, and protection of antigenic epitopes on the viral glycoproteins by host-identical N-glycans are different strategies for persistent virus infections.  

3.659           PAK5 kinase is an inhibitor of MARK/Par-1, which leads to stable microtubules and dynamic actin

Matenia, D. Et al Mol. Biol. Cell, 16, 4410-4422 (2005)   MARK/Par-1 is a kinase involved in development of embryonic polarity. In neurons, MARK phosphorylates tau protein and causes its detachment from microtubules, the tracks of axonal transport. Because the target sites of MARK on tau occur at an early stage of Alzheimer neurodegeneration, we searched for interaction partners of MARK. Here we report that MARK2 is negatively regulated by PAK5, a neuronal member of the p21-activated kinase family. PAK5 suppresses the activity of MARK2 toward its target, tau protein. The inhibition requires the binding between the PAK5 and MARK2 catalytic domains, but does not require phosphorylation. In transfected Chinese hamster ovary (CHO) cells both kinases show a vesicular distribution with partial colocalization on endosomes containing AP-1/2. Although MARK2 transfected alone destabilizes microtubules and stabilizes actin stress fibers, PAK5 keeps microtubules stable through the down-regulation of MARK2 but destabilizes the F-actin network so that stress fibers and focal adhesions disappear and cells develop filopodia. The results point to an inverse relationship between actin- and microtubule-related signaling by the PAK5 and MARK2 pathways that affect both cytoskeletal networks.  

3.660           The novel fission yeast protein Pal1p interacts with Hip1-related Sla2p/End4p and is involved in cellular morphogenesis

Ge, W., Chew, T.G., Wachtler, V., Naqvi, S.N. and Balasubramanian, M.K. Mol. Biol. Cell, 16, 4124-4138 (2005)   The establishment and maintenance of characteristic cellular morphologies is a fundamental property of all cells. Here we describe Schizosaccharomyces pombe Pal1p, a protein important for maintenance of cylindrical cellular morphology. Pal1p is a novel membrane-associated protein that localizes to the growing tips of interphase cells and to the division site in cells undergoing cytokinesis in an F-actin- and microtubule-independent manner. Cells deleted for pal1 display morphological defects, characterized by the occurrence of spherical and pear-shaped cells with an abnormal cell wall. Pal1p physically interacts and displays overlapping localization with the Huntingtin-interacting-protein (Hip1)-related protein Sla2p/End4p, which is also required for establishment of cylindrical cellular morphology. Sla2p is important for efficient localization of Pal1p to the sites of polarized growth and appears to function upstream of Pal1p. Interestingly, spherical pal1 mutants polarize to establish a pearlike morphology before mitosis in a manner dependent on the kelch-repeat protein Tea1p and the cell cycle inhibitory kinase Wee1p. Thus, overlapping mechanisms involving Pal1p, Tea1p, and Sla2p contribute to the establishment of cylindrical cellular morphology, which is important for proper spatial regulation of cytokinesis.  

3.661           Membrane rafts segregate pro- from anti-apoptotic insulin-like growth factor-I receptor signaling in colon carcinoma cells stimulated by members of the tumor necrosis factor superfamily

Remacle-Bonnet, M. et al Am. J. Pathol., 167(3), 761-773 (2005)   In the tumor microenvironment, autocrine/paracrine loops of insulin-like growth factors (IGFs) contribute to cancer cell survival. However, we report here that IGF-I can send contradictory signals that interfere with cell death induced by different ligands of the tumor necrosis factor (TNF) superfamily. IGF-I protected human colon carcinoma cells from TNF- -induced apoptosis, but it enhanced the apoptotic response to anti-Fas antibody and TNF-related apoptosis inducing ligand stimulation. This proapoptotic effect of IGF-I, observed in several but not all tested colon cancer cell lines, was mediated via the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway. Furthermore, IGF-I receptors (IGF-IR) were located in and out of membrane lipid rafts and were tyrosine autophosphorylated in response to IGF-I. However, disruption of rafts by acute cholesterol depletion shifted IGF-IR to non-raft domains, abolished the IGF-I-mediated proapoptotic effect, and inhibited the IGF-I-dependent IRS-1 and Akt recruitment into and phosphorylation/activation within lipid rafts. Replenishing cell membranes with cholesterol reversed these effects. Activation of extracellular-regulated kinase-1/2 and p38 mitogen-activated protein kinase, which convey the IGF-I anti-apoptotic effect, occurred independently of lipid rafts. Thus, we propose that segregation of IGF-IR in and out of lipid rafts may dynamically regulate the pro- and anti-apoptotic effects of IGF-I on apoptosis induced by TNF superfamily members.  

3.662           The actin cytoskeleton differentially regulates platelet a-granule and dense-granule secretion

Flaumenahft, R. Et al Blood, 105(10), 3879-3887 (2005)   Stimulation of platelets with strong agonists results in centralization of cytoplasmic organelles and secretion of granules. These observations have led to the supposition that cytoskeletal contraction facilitates granule release by promoting the interaction of granules with one another and with membranes of the open canalicular system. Yet, the influence of the actin cytoskeleton in controlling the membrane fusion events that mediate granule secretion remains largely unknown. To evaluate the role of the actin cytoskeleton in platelet granule secretion, we have assessed the effects of latrunculin A and cytochalasin E on granule secretion. Exposure of platelets to low concentrations of these reagents resulted in acceleration and augmentation of agonist-induced -granule secretion with comparatively modest effects on dense granule secretion. In contrast, exposure of platelets to high concentrations of latrunculin A inhibited agonist-induced -granule secretion but stimulated dense granule secretion. Incubation of permeabilized platelets with low concentrations of latrunculin A primed platelets for Ca²⁺- or guanosine triphosphate (GTP)- -S-induced -granule secretion. Latrunculin A-dependent -granule secretion was inhibited by antibodies directed at vesicle-associated membrane protein (VAMP), demonstrating that latrunculin A supports soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent membrane fusion. These results indicate that the actin cytoskeleton interferes with platelet exocytosis and differentially regulates -granule and dense granule secretion.  

3.663           Plant and animal homeodomains use convergent mechanisms for intercellular transfer

Tasetto, M., Maizel, A., Osorio, J. and Joliot. A. EMBO Reports, 6(9), 885-890 (2005)   Homeoproteins are defined by the structure of their DNA-binding domain, the homeodomain. Intercellular transfer of homeoprotein was observed ex vivo between animal cells and in vivo in higher plants. In the latter case, transfer is through intercytoplasmic channels that connect plant cells, but these do not exist in animals. Here, we show that the homeodomain of KNOTTED1, a maize homeoprotein, is transferred between animal cells and that a mutation in the homeodomain blocking the intercellular transfer of KNOTTED1 in plants also inhibits the transfer of the KNOTTED1 homeodomain in animal cells. This mutation decreases nuclear addressing, and its effect on nuclear import and intercellular transfer is reverted by the addition of an ectopic nuclear localization signal. We propose that, despite evolutionary distance and the differences in multicellular organization, similar mechanisms are at work for intercellular transfer of homeoprotein in plants and animals. Furthermore, our results suggest that, at least in animals, homeodomain secretion requires passage through the nucleus.    

3.664           Purification of neuronal inclusions of patients with Huntington’s disease reveals a broad range of N-terminal fragments of expanded huntingtin and insoluble polymers

Hoffner, G., Island, M-L. and Djian, P.

1. Neurochem., 95, 125-136 (2005)

  Huntington's disease resulting from huntingtin containing an expanded polyglutamine is associated with aggregates largely confined to neuronal inclusions, and with neuronal death. Inclusions are thought to originate from discrete N-terminal fragments of expanded huntingtin produced by specific endopeptidases. We have now purified the neuronal inclusions of Huntington's disease brain. When incubated in concentrated formic acid, purified inclusions release a polymer, an oligomer and a broad range of N-terminal fragments of expanded huntingtin. The fragments and the polymeric forms are linked to each other by non-covalent bonds as they are both released by formic acid, whereas the polymeric forms themselves are presumably stabilized by covalent bonds, as they are resistant to formic acid. We also demonstrate the presence in affected areas of the brain but not in unaffected areas of a broad range of soluble N-terminal fragments of expanded huntingtin not yet associated with the inclusions and which are likely to be the precursors of the inclusions. Fragmentation of expanded huntingtin in Huntington's disease must result from the operation of multiple proteolytic activities with little specificity and not from that of a specific endopeptidase; subsequent aggregation of the fragments by covalent and non-covalent bonds leads to the formation of the inclusions.  

3.665           Neuronal sorting protein-related receptor sorLA/LR11 regulates processing of the amyloid precursor protein

Andersen, O.M. et al PNAS, 102(38), 13461-13466 (2005)   sorLA (sorting protein-related receptor) is a type-1 membrane protein of unknown function that is expressed in neurons. Its homology to sorting receptors that shuttle between the plasma membrane, endosomes, and the Golgi suggests a related function in neuronal trafficking processes. Because expression of sorLA is reduced in the brain of patients with Alzheimer's disease (AD), we tested involvement of this receptor in intracellular transport and processing of the amyloid precursor protein (APP) to the amyloid -peptide (A ), the principal component of senile plaques. We demonstrate that sorLA interacts with APP in vitro and in living cells and that both proteins colocalize in endosomal and Golgi compartments. Overexpression of sorLA in neurons causes redistribution of APP to the Golgi and decreased processing to A , whereas ablation of sorLA expression in knockout mice results in increased levels of A in the brain similar to the situation in AD patients. Thus, sorLA acts as a sorting receptor that protects APP from processing into A and thereby reduces the burden of amyloidogenic peptide formation. Consequently, reduced receptor expression in the human brain may increase A production and plaque formation and promote spontaneous AD.  

3.666           Subcellular localization of iron regulatory proteins to Golgi and ER membranes

Patton, S.M., Pinero, D.J., Surguladze, N., Beard, J. and Connor, J.R.

1. Cell Sci., 118, 4365-4373 (2005)

  Interaction between iron regulatory proteins and iron responsive elements on certain mRNAs is at the core of regulation of intracellular iron homeostasis. Previous results suggested that in cultured cells iron regulatory proteins (IRPs) exist in cytosolic and microsomal subcellular locations and that this distribution is affected by cellular iron status. In this study, we tested the hypothesis that the membrane-associated fractions of iron regulatory proteins are specifically in the endoplasmic reticulum and Golgi membranes. Confocal microscopy revealed that IRP1 could be co-localized to the endoplasmic reticulum and the Golgi apparatus. To examine the intracellular distribution of IRPs biochemically, we used rats fed normal or iron-deficient diets. As expected, the IRPs were found predominantly in the cytosolic fraction. However, subfractionation of crude microsomal preparations revealed IRP1 in the Golgi apparatus. In animals fed an iron-deficient diet, IRP1 was found in the Golgi apparatus and the endoplasmic reticulum. To identify the mechanisms and factors involved in the localization of iron regulatory proteins in the cytosol and membrane fractions, cells were treated with a phorbol ester, a protein kinase C inhibitor (chelerythrine), hydrogen peroxide, interleukin-1ß, and 1,2-bis-(o-aminophenoxy)-ethane-N,N,-N'N'-tetraacetic acid tetraacetoxy-methyl ester. The results indicate that iron-regulatory-protein-binding activity in the membrane fraction can be altered by cell stress or iron status and that phosphorylation plays a role in the translocation. As a result of this study we propose a novel model for intracellular distribution of IRPs and identify differences between the two iron regulatory proteins.  

3.667           LRP1B functions as a receptor for pseudomonas exotoxin

Pastrana, D.V., Hanson, A.J., Knisely, J., Bu, G. and FitzGerald, D.J. Biochim. Biophys. Acta, 1741, 234-239 (2005)   Pseudomonas aeruginosa is an opportunistic pathogen that produces several virulence factors, among them Pseudomonas Exotoxin A (PE). Previously, low-density lipoprotein receptor-related protein 1 (LRP1) was shown to be the primary receptor for PE. In this report, we show that a close family member, LRP1B, can also function as a receptor.  

3.668           Helicobacter pylori VacA cytotoxin: a probe for a clathrin-independent and Cdc42-dependent pinocytic pathway routed to late endosomes

Gauthier, N.C. et al Mol. Biol. Cell, 16, 4852-4866 (2005)   The vacuolating cytotoxin VacA is a major virulence factor of Helicobacter pylori, a bacterium responsible for gastroduodenal ulcers and cancer. VacA associates with lipid rafts, is endocytosed, and reaches the late endocytic compartment where it induces vacuolation. We have investigated the endocytic and intracellular trafficking pathways used by VacA, in HeLa and gastric AGS cells. We report here that VacA was first bound to plasma-membrane domains localized above F-actin structures that were controlled by the Rac1 GTPase. VacA was subsequently pinocytosed by a clathrin-independent mechanism into cell peripheral early endocytic compartments lacking caveolin 1, the Rab5 effector early endosomes antigen-1 (EEA1) and transferrin. These compartments took up fluid-phase (as evidenced by the accumulation of fluorescent dextran) and glycosylphosphatidylinositol-anchored proteins (GPI-APs). VacA pinocytosis was controlled by Cdc42 and did not require cellular tyrosine kinases, dynamin 2, ADP-ribosylating factor 6, or RhoA GTPase activities. VacA was subsequently routed to EEA1-sorting endosomes and then sorted to late endosomes. During all these different endocytic steps, VacA was continuously associated with detergent resistant membrane domains. From these results we propose that VacA might be a valuable probe to study raft-associated molecules, pinocytosed by a clathrin-independent mechanism, and routed to the degradative compartment.  

3.669           Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by SIP₁ receptor, PI3 kinase, Tiam1/Rac1, and a-actinin

Singleton, P.A., Dudek, S.M., Chiang, E.T. and Garcia, J.G.N. FASEB J., 19, 1646-1656 (2005)   Endothelial cell (EC) barrier dysfunction results in increased vascular permeability observed in inflammation, tumor angiogenesis, and atherosclerosis. The platelet-derived phospholipid sphingosine-1-phosphate (S1P) decreases EC permeability in vitro and in vivo and thus has obvious therapeutic potential. We examined S1P-mediated human pulmonary artery EC signaling and barrier regulation in caveolin-enriched microdomains (CEM). Immunoblotting from S1P-treated EC revealed S1P-mediated rapid recruitment (1 µM, 5 min) to CEMs of the S1P receptors S1P₁ and S1P₃, p110 PI3 kinase and ß catalytic subunits, the Rac1 GEF, Tiam1, and -actinin isoforms 1 and 4. Immunoprecipitated p110 PI3 kinase catalytic subunits from S1P-treated EC exhibited PIP₃ production in CEMs. Immunoprecipitation of S1P receptors from CEM fractions revealed complexes containing Tiam1 and S1P₁. PI3 kinase inhibition (LY294002) attenuated S1P-induced Tiam1 association with S1P₁, Tiam1/Rac1 activation, -actinin-1/4 recruitment, and EC barrier enhancement. Silencing of either S1P₁ or Tiam1 expression resulted in the loss of S1P-mediated Rac1 activation and -actinin-1/4 recruitment to CEM. Finally, silencing S1P₁, Tiam1, or both -actinin isoforms 1/4 inhibits S1P-induced cortical F-actin rearrangement and S1P-mediated barrier enhancement. Taken together, these results suggest that S1P-induced recruitment of S1P₁ to CEM fractions promotes PI3 kinase-mediated Tiam1/Rac1 activation required for -actinin-1/4-regulated cortical actin rearrangement and EC barrier enhancement.—Singleton, P. A., Dudek, S. M., Chiang, E. T., Garcia, J. G. N. Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P₁ receptor, PI3 kinase, Tiam1/Rac1 and -actinin.      

3.670           Centriolin anchoring of exocyst and SNARE complexes at the midbody is required for secretory-vesicle-mediated abscission

Gromley, A. et al Cell, 123, 75-87 (2005)   The terminal step in cytokinesis, called abscission, requires resolution of the membrane connection between two prospective daughter cells. Our previous studies demonstrated that the coiled-coil protein centriolin localized to the midbody during cytokinesis and was required for abscission. Here we show that centriolin interacts with proteins of vesicle-targeting exocyst complexes and vesicle-fusion SNARE complexes. These complexes require centriolin for localization to a unique midbody-ring structure, and disruption of either complex inhibits abscission. Exocyst disruption induces accumulation of v-SNARE-containing vesicles at the midbody ring. In control cells, these v-SNARE vesicles colocalize with a GFP-tagged secreted polypeptide. The vesicles move to the midbody ring asymmetrically from one prospective daughter cell; the GFP signal is rapidly lost, suggesting membrane fusion; and subsequently the cell cleaves at the site of vesicle delivery/fusion. We propose that centriolin anchors protein complexes required for vesicle targeting and fusion and integrates membrane-vesicle fusion with abscission.  

3.671           Identification of AQP5 in lipid rafts and its translocation to apical membranes by activation of M₃ mAChRs in interlobular ducts of rat parotid gland

Ishikawa, Y. Et al Am. J. Physiol Cell Physiol., 289, C1303-C1311 (2005)   Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. M₃ muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and M₃ mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular Ca²⁺ signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands.  

3.672           Loss- and gain-of-function analysis of the lipid raft proteins reggie/flotillin in Drosphilia: they are posttranslationally regulated, and misexpression interferes with wing and eye development

Hoehne, M., de Couet, H.G., Stuermer, C.A.O. and Fischbach, K-F. Mol. Cell. Neurosci., 30, 326-338 (2005)   Reggie/Flotillin proteins are upregulated after optic nerve dissection and evolutionary highly conserved components of lipid rafts. Whereas many biochemical and cell culture studies suggest an involvement in the assembly of multiprotein complexes at cell contact sites, not much is known about their biological in vivo functions. We therefore set out to study the expression pattern and the effects of loss- and gain-of-function in the Drosophila melanogaster model system. We found that in flies these proteins are mainly expressed in axons at the root of fiber tracts, in places where strong fasciculation is required, e.g. at the neck of the peduncle of the mushroom bodies and in the optic chiasms. Despite their evolutionary conservation which implies fundamental and important functions, a P-element-induced null mutant (KG00210) of reggie1/flotillin2 (reggie1/flo2) in D. melanogaster shows no apparent phenotypic defects. This was even more surprising as we show that in this reggie1/flo2 null mutant the paralogous Reggie2/Flo1 protein is unstable and degraded, while the transcript is still present. The requirement of Reggie1/Flo2 for Reggie2/Flo1 stabilization is confirmed by misexpression experiments. Reggie2/Flo1 can only be misexpressed when Reggie1/Flo2 is provided as well. Conversely, Reggie1/Flo2 immunoreactivity can be detected, when its transgene is misexpressed alone. Using appropriate Gal4 driver lines, misexpression of Reggie1/Flo2 alone or together with Reggie2/Flo1 in the eye imaginal disc results in a specific and severe mislocalization of cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) (while DE-Cadherin is unaffected) and in differentiation defects pointing to impaired signaling. In the wing imaginal disc, global overexpression of Reggie/Flotillin proteins leads to a significant extension of the Wingless signal and severely disrupts normal wing development. Our data support the notion that Reggie/Flotillin proteins are implicated in signaling processes at cellular contact sites.  

3.673           Comparative genomic analysis reveals a novel mitochondrial isoform of human rTS protein and unusual phylogenetic distribution of the rTS gene

Liang, P., Nair, J.R., Song, L., McGuire, J.J. and Dolnick, B.J. BMC Genomics, 6, 125 (2005)   Background The rTS gene (ENOSF1), first identified in Homo sapiens as a gene complementary to the thymidylate synthase (TYMS) mRNA, is known to encode two protein isoforms, rTSα and rTSβ. The rTSβ isoform appears to be an enzyme responsible for the synthesis of signaling molecules involved in the down-regulation of thymidylate synthase, but the exact cellular functions of rTS genes are largely unknown. Results Through comparative genomic sequence analysis, we predicted the existence of a novel protein isoform, rTS, which has a 27 residue longer N-terminus by virtue of utilizing an alternative start codon located upstream of the start codon in rTSβ. We observed that a similar extended N-terminus could be predicted in all rTS genes for which genomic sequences are available and the extended regions are conserved from bacteria to human. Therefore, we reasoned that the protein with the extended N-terminus might represent an ancestral form of the rTS protein. Sequence analysis strongly predicts a mitochondrial signal sequence in the extended N-terminal of human rTSγ, which is absent in rTSβ. We confirmed the existence of rTS in human mitochondria experimentally by demonstrating the presence of both rTSγ and rTSβ proteins in mitochondria isolated by subcellular fractionation. In addition, our comprehensive analysis of rTS orthologous sequences reveals an unusual phylogenetic distribution of this gene, which suggests the occurrence of one or more horizontal gene transfer events. Conclusion The presence of two rTS isoforms in mitochondria suggests that the rTS signaling pathway may be active within mitochondria. Our report also presents an example of identifying novel protein isoforms and for improving gene annotation through comparative genomic analysis.  

3.674           Brome mosaic virus 1a nucleoside triphosphatase/helicase domain plays crucial roles in recruiting RNA replication templates

Wang, X. et al

1. Virol., 79(21), 13747-13758 (2005)

  Positive-strand RNA virus RNA replication is invariably membrane associated and frequently involves viral proteins with nucleoside triphosphatase (NTPase)/helicase motifs or activities. Brome mosaic virus (BMV) encodes two RNA replication factors: 1a has a C-terminal NTPase/helicase-like domain, and 2a^pol has a central polymerase domain. 1a accumulates on endoplasmic reticulum membranes, recruits 2a^pol, and induces 50- to 70-nm membrane invaginations (spherules) serving as RNA replication compartments. 1a also recruits BMV replication templates such as genomic RNA3. In the absence of 2a^pol, 1a dramatically stabilizes RNA3 by transferring RNA3 to a membrane-associated, nuclease-resistant state that appears to correspond to the interior of the 1a-induced spherules. Prior results show that the 1a NTPase/helicase-like domain contributes to RNA recruitment. Here, we tested mutations in the conserved helicase motifs of 1a to further define the roles of this domain in RNA template recruitment. All 1a helicase mutations tested showed normal 1a accumulation, localization to perinuclear endoplasmic reticulum membranes, and recruitment of 2a^pol. Most 1a helicase mutants also supported normal spherule formation. Nevertheless, these mutations severely inhibited RNA replication and 1a-induced stabilization of RNA3 in vivo. For such 1a mutants, the membrane-associated RNA3 pool was both reduced and highly susceptible to added nuclease. Thus, 1a recruitment of viral RNA templates to a membrane-associated, nuclease-resistant state requires additional functions beyond forming spherules and recruiting RNA to membranes, and these functions depend on the 1a helicase motifs. The possibility that, similar to some double-stranded RNA viruses, the 1a NTPase/helicase-like domain may be involved in importing viral RNAs into a preformed replication compartment is discussed.  

3.675           Nef is physically recruited into the immunological synapse and potentiates T cell activation early after TCR engagemant

Fenard, D. et al

1. Immunol., 175, 6050-6057 (2005)

  The HIV-1 protein Nef enhances viral pathogenicity and accelerates disease progression in vivo. Nef potentiates T cell activation by an unknown mechanism, probably by optimizing the intracellular environment for HIV replication. Using a new T cell reporter system, we have found that Nef more than doubles the number of cells expressing the transcription factors NF- B and NFAT after TCR stimulation. This Nef-induced priming of TCR signaling pathways occurred independently of calcium signaling and involved a very proximal step before protein kinase C activation. Engagement of the TCR by MHC-bound Ag triggers the formation of the immunological synapse by recruiting detergent-resistant membrane microdomains, termed lipid rafts. Approximately 5–10% of the total cellular pool of Nef is localized within lipid rafts. Using confocal and real-time microscopy, we found that Nef in lipid rafts was recruited into the immunological synapse within minutes after Ab engagement of the TCR/CD3 and CD28 receptors. This recruitment was dependent on the N-terminal domain of Nef encompassing its myristoylation. Nef did not increase the number of cell surface lipid rafts or immunological synapses. Recently, studies have shown a specific interaction of Nef with an active subpopulation of p21-activated kinase-2 found only in the lipid rafts. Thus, the corecruitment of Nef and key cellular partners (e.g., activated p21-activated kinase-2) into the immunological synapse may underlie the increased frequency of cells expressing transcriptionally active forms of NF- B and NFAT and the resultant changes in T cell activation.  

3.676           g-Secretase is a functional component of phagosomes

Jutras, I. et al

1. Biol. Chem., 280(43), 36310-36317 (2005)

  g-Secretase is a high molecular mass protein complex that catalyzes the intramembrane cleavage of its protein substrates. Two proteins involved in phagocytosis, CD44 and the low density lipoprotein receptor-related protein, are -secretase substrates, suggesting that this complex might regulate some aspects of phagocytosis. Our results indicate that the four components of -secretase, viz. presenilin, nicastrin, APH-1, and PEN-2, are present and enriched on phagosome membranes from both murine macrophages and Drosophila S2 phagocytes. The -secretase components form high molecular mass complexes in lipid microdomains of the phagosome membrane with the topology expected for the functional enzyme. In contrast to the majority of the phagosome proteins studied so far, which appear to associate transiently with this organelle, -secretase resides on newly formed phagosomes and remains associated throughout their maturation into phagolysosomes. Finally, our results indicate that interferon- stimulates -secretase-dependent cleavages on phagosomes and that -secretase activity may be involved in the phagocytic response of macrophages to inflammatory cytokines.  

3.677           The blood-brain barrier transmigrating single domain antibody: mechanism of transport and antigenic epitopes in human brain endothelial cells

Abulrop, A., Sprong, H., Van Bergen en Henegouwen P. and Stanimirovic, D.

1. Neurochem., 95, 1201-1214 (2005)

  Antibodies against receptors that undergo transcytosis across the blood brain barrier (BBB) have been used as vectors to target drugs or therapeutic peptides into the brain. We have recently discovered a novel single domain antibody, FC5, which transmigrates across human cerebral endothelial cells in vitro and the BBB in vivo. The purpose of this study was to characterize mechanisms of FC5 endocytosis and transcytosis across the BBB and its putative receptor on human brain endothelial cells. The transport of FC5 across human brain endothelial cells was polarized, charge independent and temperature dependent, suggesting a receptor-mediated process. FC5 taken up by human brain endothelial cells co-localized with clathrin but not with caveolin-1 by immunochemistry and was detected in clathrin-enriched subcellular fractions by western blot. The transendothelial migration of FC5 was reduced by inhibitors of clathrin-mediated endocytosis, K⁺ depletion and chlorpromazine, but was insensitive to caveolae inhibitors, filipin, nystatin or methyl- -cyclodextrin. Following internalization, FC5 was targeted to early endosomes, bypassed late endosomes/lysosomes and remained intact after transcytosis. The transcytosis process was inhibited by agents that affect actin cytoskeleton or intracellular signaling through PI3-kinase. Pretreatment of human brain endothelial cells with wheatgerm agglutinin, sialic acid, (2,3)-neuraminidase or Maackia amurensis agglutinin that recognizes (2,3)-, but not with Sambucus nigra agglutinin that recognizes (2,6) sialylgalactosyl residues, significantly reduced FC5 transcytosis. FC5 failed to recognize brain endothelial cells-derived lipids, suggesting that it binds luminal (2,3)-sialoglycoprotein receptor which triggers clathrin-mediated endocytosis. This putative receptor may be a new target for developing brain-targeting drug delivery vectors.  

3.678           Lipids as modulators of proteolytic activity of BACE. Involvement of cholesterol, glycosphingolipids, and anionic phospholipids in vitro

Kalvodova, L. et al

1. Biol. Chem., 280(44), 36815-36823 (2005)

  The -secretase, BACE, is a membrane spanning aspartic protease, which cleaves the amyloid precursor protein (APP) in the first step of proteolytic processing leading to the formation of the neurotoxic -amyloid peptide (A ). Previous results have suggested that the regulation of -secretase and BACE access to APP is lipid dependent, and involves lipid rafts. Using the baculovirus expression system, we have expressed recombinant human full-length BACE in insect cells and purified milligram amounts to homogeneity. We have studied partitioning of fluorophor-conjugated BACE between the liquid ordered and disordered phases in giant (10–150 µm) unilamellar vesicles, and found 20% to associate with the raft-like, liquid-ordered phase; the fraction associated with liquid-ordered phase increased upon cross-linking of raft lipids. To examine involvement of individual lipid species in modulating BACE activity, we have reconstituted the purified BACE in large ( 100 nm) unilamellar vesicles, and determined its specific activity in vesicles of various lipid compositions. We have identified 3 groups of lipids that stimulate proteolytic activity of BACE: 1) neutral glycosphingolipids (cerebrosides), 2) anionic glycerophospholipids, and 3) sterols (cholesterol).  

3.679           Cholesterol-dependent syntaxin-4 and SNAP-23 clustering regulates caveolar fusion with the endothelial plasma

Predescu, S.A., Predescu,, D.N., Shimizu, K., Klein, I.K. and Malik, A.B.

1. Biol. Chem., 280(44), 37130-37138 (2005)

  We determined the organization of target (t) SNARE proteins on the basolateral endothelial plasma membrane (PM) and their role in the mechanism of caveolar fusion. Studies were performed in a cell-free system involving endothelial PM sheets and isolated biotin-labeled caveolae. We monitored the fusion of caveolae with the PM by the detection of biotin-streptavidin complexes using correlative high resolution fluorescence microscopy and gold labeling electron microscopy on ultrathin sections of PM sheets. Imaging of PM sheets demonstrated and biochemical findings showed that the t-SNARE proteins present in endothelial cells (SNAP-23 and syntaxin-4) formed cholesterol-dependent clusters in discrete areas of the PM. Upon fusion of caveolae with the target PM, 50% of the caveolae co-localized with the t-SNARE clusters, indicating that these caveolae were at the peak of the fusion reaction. Fluorescent streptavidin staining of PM sheets correlated with the ultrastructure in the same area. These findings demonstrate that t-SNARE clusters in the endothelial target PM serve as the fusion sites for caveolae during exocytosis.  

3.680           Association of human immunodeficiency virus type 1 Gag with membrane does not require highly basic sequences in the nucleocapsid: use of a novel Gag multimerization assay

Ono, A., Waheed, A.A., Joshi, A.a and Freed, E.O.

1. Virol., 79(22), 14131-14140 (2005)

  Human immunodeficiency virus type 1 (HIV-1) particle production, a process driven by the Gag polyprotein precursor, occurs on the plasma membrane in most cell types. The plasma membrane contains cholesterol-enriched microdomains termed lipid rafts, which can be isolated as detergent-resistant membrane (DRM). Previously, we and others demonstrated that HIV-1 Gag is associated with DRM and that disruption of Gag-raft interactions impairs HIV-1 particle production. However, the determinants of Gag-raft association remain undefined. In this study, we developed a novel epitope-based Gag multimerization assay to examine whether Gag assembly is essential for its association with lipid rafts. We observed that membrane-associated, full-length Gag is poorly detected by immunoprecipitation relative to non-membrane-bound Gag. This poor detection is due to assembly-driven masking of Gag epitopes, as denaturation greatly improves immunoprecipitation. Gag mutants lacking the Gag-Gag interaction domain located in the N terminus of the nucleocapsid (NC) were efficiently immunoprecipitated without denaturation, indicating that the epitope masking is caused by higher-order Gag multimerization. We used this assay to examine the relationship between Gag assembly and Gag binding to total cellular membrane and DRM. Importantly, a multimerization-defective NC mutant displayed wild-type levels of membrane binding and DRM association, indicating that NC-mediated Gag multimerization is dispensable for association of Gag with membrane or DRM. We also demonstrate that different properties of sucrose and iodixanol membrane flotation gradients may explain some discrepancies regarding Gag-raft interactions. This report offers new insights into the association of HIV-1 Gag with membrane and with lipid rafts.  

3.681           A role for Fis1 in both mitochondrial and peroxisomal fission in mammalian cells

Koch, A., Yoon, Y., Bonekamp, N.A., McNiven, M.A. and Schrader, M. Mol. Biol. Cell, 16, 5077-5086 (2005)   The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.  

3.682           GLUT8 contains a [DE]XXXL[LI] sorting motif and localizes to a late endosomal/lysosomal compartment

Augustin, R., Riley, J. and Moley, K.H. Traffic, 6, 1196-1212 (2005)   Glucose transporter 8 (GLUT8) contains a cytoplasmic N-terminal dileucine motif and localizes to a thus far unidentified intracellular compartment. Translocation of GLUT8 to the plasma membrane (PM) was found in insulin-treated mouse blastocysts. Using overexpression of GLUT8 in adipocytes and neuronal cells however, insulin treatment or depolarization of the cells did not lead to GLUT8 PM translocation in other studies. In addition, other experiments showing dynamin-dependent endocytosis of GLUT8 suggested that GLUT8 recycles between an endosomal compartment and the PM. To reveal the functional/physiological role of GLUT8, we studied its subcellular localization in 3T3L1, HEK293 and CHO cells. We show that GLUT8 does not co-localize with GLUT4 and does not redistribute to the PM after treatment with insulin, ionophores or okadaic acid in these cell lines. Once endocytosed, GLUT8 does not recycle to the PM. GLUT8 localizes to late endosomes and lysosomes. An interspecies GLUT8 - sequence alignment revealed the presence of a highly conserved late endosomal/lysosomal-targeting motif ([DE]XXXL[LI]). Changing the glutamate to arginine as found in GLUT4 (RRXXXLL) alters GLUT8 endocytosis and retains the transporter at the PM. Furthermore, sorting GLUT8 to late endosomes/lysosomes does not require prior presence of GLUT8 at the PM followed by its endocytosis. In summary, GLUT8 does not reside in a recycling vesicle pool and is distinct from GLUT4. From our data, we postulate a role for GLUT8 in transport of hexoses across intracellular membranes, for example in specific compartments of GLUT8 expression such as the acrosome of mature spermatozoa or secretory granules in neurons. Furthermore, a role for GLUT8 in hexose transport across the lysosomal membrane, a transport mechanism that has long been suggested but unexplained, is discussed.  

3.683           The VirE1VirE2 complex of Agrobacterium tumefaciens interacts with single-stranded DNA and forms channels

Duckely, M. et al Mol. Microbiol., 58(4), 1130-1142 (2005)   The VirE2 protein is crucial for the transfer of single-stranded DNA (ssDNA) from Agrobacterium tumefaciens to the nucleus of the plant host cell because of its ssDNA binding activity, assistance in nuclear import and putative ssDNA channel activity. The native form of VirE2 in Agrobacterium's cytoplasm is in complex with its specific chaperone, VirE1. Here, we describe the ability of the VirE1VirE2 complex to both bind ssDNA and form channels. The affinity of the VirE1VirE2 complex for ssDNA is slightly reduced compared with VirE2, but the kinetics of binding to ssDNA are unaffected by the presence of VirE1. Upon binding of VirE1VirE2 to ssDNA, similar helical structures to those reported for the VirE2 ssDNA complex were observed by electron microscopy. The VirE1VirE2 complex can release VirE1 once the VirE2 ssDNA complexes assembled. VirE2 exhibits a low affinity for small unilamellar vesicles composed of bacterial lipids and a high affinity for lipid vesicles containing sterols and sphingolipids, typical components of animal and plant membranes. In contrast, the VirE1VirE2 complex associated similarly with all kind of lipids. Finally, black lipid membrane experiments revealed the ability of the VirE1VirE2 complex to form channels. However, the majority of the channels displayed a conductance that was a third of the conductance of VirE2 channels. Our results demonstrate that the binding of VirE1 to VirE2 does not inhibit VirE2 functions and that the effector chaperone complex is multifunctional.  

3.684           Biochemical characterization of intracellular membranes bearing Trk neurotrophin receptors

Yano, H. and Chao, M.V. Neurochem. Res., 30(6/7), 767-777 (2005)   Neurotrophin receptor trafficking plays an important role in directing cellular communication in developing as well as mature neurons. However, little is known about the requirements for intracellular localization of the neurotrophin receptors in neurons. To isolate the subcellular membrane compartments containing the Trk neurotrophin receptor, we performed biochemical subcellular fractionation experiments using primary cortical neurons and rat PC12 pheochromocytoma cells. By differential centrifugation and density gradient centrifugation, we have isolated Trk-bearing compartments, suggesting distinct membranous localization of Trk receptors. A number of Trk-interacting proteins, such as GIPC and dynein light chain Tctex-1 were found in these fractions. Additionally, membranes enriched in phosphorylated activated forms of Trk receptors were found upon ligand treatment in primary neurons and PC12 cells. Interestingly, density gradient centrifugation experiments showed that Trk receptors from PC12 cells are present in heavy membrane fractions, while Trk from primary neurons are fractionated in lighter membrane fractions. These results suggest that the intracellular membrane localization of Trk can differ according to cell type. Taken together, these biochemical approaches allowed separation of distinct Trk-bearing membrane pools, which may be involved in different functions of neurotrophin receptor signaling and trafficking.  

3.685           Cytotoxicity of an anti-cancer lysophospholipid through selective modification of lipid raft composition

Zaremberg, V., Gajate, C., Cacharro, L.M., Mollinedo, F. and McMaster, C.R.

1. Biol. Chem., 280(45), 38047-38058 (2005)

  Edelfosine is a prototypical member of the alkylphosphocholine class of antitumor drugs. Saccharomyces cerevisiae was used to screen for genes that modulate edelfosine cytotoxicity and identified sterol and sphingolipid pathways as relevant regulators. Edelfosine addition to yeast resulted in the selective partitioning of the essential plasma membrane protein Pma1p out of lipid rafts. Microscopic analysis revealed that Pma1p moved from the plasma membrane to intracellular punctate regions and finally localized to the vacuole. Consistent with altered sterol and sphingolipid synthesis resulting in increased edelfosine sensitivity, mislocalization of Pma1p was preceded by the movement of sterols out of the plasma membrane. Cells with enfeebled endocytosis and vacuolar protease activities prevented edelfosine-mediated (i) mobilization of sterols, (ii) loss of Pma1p from lipid rafts, and (iii) cell death. The activities of proteins and signaling processes are meaningfully altered by changes in lipid raft biophysical properties. This study points to a novel mode of action for an anti-cancer drug through modification of plasma membrane lipid composition resulting in the displacement of an essential protein from lipid rafts.    

3.686           Thermally induced changes in lipid composition of raft and non-raft regions of hepatocyte plasma membranes of rainbow trout

Zehmer, J.K. and Hazel, J.R.

1. Exp. Biol., 208, 4283-4290 (2005)

  In poikilotherms, increases in plasma membrane (PM) cholesterol and an increase in the degree of lipid acyl chain saturation commonly accompany an increase in growth temperature. This has typically been interpreted in terms of membrane fluidity/order homeostasis, but these changes would also be expected to stabilize the structure of PM rafts against thermal perturbation. Rafts are microdomains that organize the molecules of many signaling cascades and are formed as a result of interactions between lipids with saturated acyl chains and cholesterol. No study to date has examined the thermally induced compositional changes of raft and non-raft regions of the PM separately. In this study we have measured the phospholipid class composition and fatty acid composition of raft-enriched (raft) and raft-depleted PM (RDPM) of hepatocytes from trout Oncorhynchus mykiss acclimated to 5°C and 20°C. In the raft, warm acclimation was associated with a reduction in the proportion of phosphatidylcholine from 56% to 30% while phosphatidylserine and phosphatidylinositol each increased from 8% to approximately 20% of the total phospholipid. Additionally, there were significantly fewer unsaturated fatty acids in the raft lipids from warm-acclimated (61%) than from the cold-acclimated trout (68%). In contrast, there were no significant changes in phospholipid class or acyl chain unsaturation in the RDPM. These data suggest that changes in raft lipid composition, rather than the PM as a whole, are particularly important during thermal acclimation.  

3.687           A primate virus generates transformed human cells by fusion

Duelli, D.M., Hearn, S., Myers, M.P. and Lazebnik, Y.

1. Cell Biol., 171(3), 493-503 (2005)

  Amodel that explains both the origin and sporadic nature of cancer argues that cancer cells are a chance result of events that cause genomic and epigenetic variability. The prevailing view is that these events are mutations that affect chromosome segregation or stability. However, genomic and epigenetic variability is also triggered by cell fusion, which is often caused by viruses. Yet, cells fused by viruses are considered harmless because they die. We provide evidence that a primate virus uses both viral and exosomal proteins involved in cell fusion to produce transformed proliferating human cells. Although normal cells indeed fail to proliferate after fusion, expression of an oncogene or a mutated tumor suppressor p53 in just one of the fusion partners is sufficient to produce heterogeneous progeny. We also show that this virus can produce viable oncogenically transformed cells by fusing cells that are otherwise destined to die. Therefore, we argue that viruses can contribute to carcinogenesis by fusing cells.  

3.688           Plasma membrane and lysosomal localization of CB1 cannabinoid receptor are dependent on lipid rafts and regulated by anandamide in human breast cancer cells

Sarnataro, D. et al FEBS Lett., 579, 6343-6349 (2005)   In this report we show, by confocal analysis of indirect immunofluorescence, that the type-1 cannabinoid receptor (CB1R), which belongs to the family of G-protein-coupled receptors, is expressed on the plasma membrane in human breast cancer MDA-MB-231 cells. However, a substantial proportion of the receptor is present in lysosomes. We found that CB1R is associated with cholesterol- and sphyngolipid-enriched membrane domains (rafts). Cholesterol depletion by methyl-β-cyclodextrin (MCD) treatment strongly reduces the flotation of the protein on the raft-fractions (DRM) of sucrose density gradients suggesting that CB1 raft-association is cholesterol dependent. Interestingly binding of the agonist, anandamide (AEA) also impairs DRM-association of the receptor suggesting that the membrane distribution of the receptor is dependent on rafts and is possibly regulated by the agonist binding. Indeed MCD completely blocked the clustering of CB1R at the plasma membrane. On the contrary the lysosomal localization of CB1R was impaired by this treatment only after AEA binding.  

3.689           Effect of cholesterol depletion on mitogenesis and survival: the role of caveolar and noncaveolar domains in insulin-like growth factor-mediated cellular function

Matthews, L.C., Taggart, M.J. and Westwood, M. Endocrinol., 146(12), 5463-5473 (2005)   The type 1 IGF receptor (IGF-IR) is thought to localize to a subset of lipid rafts, known as caveolae, but the impact on IGF signaling remains controversial. We investigated this potential regulatory mechanism by assessing IGF function in caveolae-positive (3T3L1 and NWTb3) and -negative (HepG2) cells. Coimmunoprecipitation studies demonstrated that IGF-IR and insulin receptor substrate 1 associated with caveolin, a caveolar marker, in 3T3L1 and NWTb3 cells. Subcellular fractionation showed that methyl-cyclodextrin, which disrupts lipid rafts by sequestration of cholesterol, disrupted the colocalization of caveolin and the IGF-IR at the plasma membrane. Methyl-cyclodextrin did not alter IGF-I-induced 3T3L1 or NWTb3 proliferation but significantly impaired the ability of IGF-I to protect these cells from apoptosis. Immunoblotting revealed that methyl-cyclodextrin had no effect on IGF-I-induced activation of the IGF-IR or insulin receptor substrate 1 but increased and decreased the phosphorylation of MAPK and protein kinase B, respectively. In caveolae-negative HepG2 cells, the effect of methyl-cyclodextrin on IGF signaling and cellular function was similar to that observed in caveolae-positive 3T3L1 and NWTb3 cells. Furthermore, transfecting caveolin into HepG2 cells to give morphologically identifiable caveolae made no difference to IGF action, despite a demonstrable interaction between caveolin and the IGF-IR. This suggests that although IGF-IR localizes to caveolin-rich subcellular fractions and coimmunoprecipitates with caveolin, caveolae may not be obligatory for IGF signaling.  

3.690           The ubiquitously expressed Csk adaptor protein Cbp is dispensable for embryogenesis and T-cell development and function

Dobenecker, M-W., Schmedt, C., Okada, M. And Tarakhovsky, A. Mol. Cell. Biol., 25(23), 10533-10542 (2005)   Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of "lipid rafts" is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.  

3.691           Different effects of intracochlear sensory and neuronal injury stimulation on expression of synaptic N-methyl-D-aspartate receptors in the auditory cortex of rats in vivo

Wang, Z., Ruan, Q. and Wang, D. Acta Oto-Laryngologica, 125, 1145-1151 (2005)   Conclusions. The expression of synaptic N-methyl-D-aspartate (NMDA) receptors in the auditory cortex is dynamic and is bidirectionally regulated by auditory activity. Furthermore, the time course of changes in the level of NR2A protein differs after sensory and neuronal injury stimulation, which modulate different changes in synaptic plasticity. Objective. To examine the effects of different types of auditory activity on the expression of synaptic NMDA receptors (NMDARs) in the auditory cortex of rats. Material and methods. We prepared synaptosomes from the auditory cortices of postnatal Day 28 ototoxic-deafened Sprague–Dawley rats and postnatal Day 28 Sprague–Dawley rats subjected to noise trauma that were given various treatments and compared them to the synaptosomes of 1–6-week-old normal Sprague–Dawley rats. The expression of different NMDAR subunits in the synaptosomes was investigated by means of Western blotting. Results. Changes in NR1 and NR2B proteins were not significant during different types of auditory activity. The level of NR2A protein increased remarkably during postnatal development and as a result of electrical intracochlear stimulation, auditory deprivation and noise trauma. Seventy-two h after a 2-h period of sensory electrical intracochlear stimulation, the expression of NR2A protein returned to the level caused by auditory deprivation. Seventy-two h after a 3-h period of noise trauma, elevation of the level of NR2A protein was unchanged. We also confirmed that elevation of the level of synaptic NR2A protein was sensitive to protein synthesis inhibitor and NMDAR antagonist. However, transcription inhibitor had no effect on NR2A protein expression.    

3.692           Defective insulin receptor activation and altered lipid rafts in Niemann-Pick type C diseases hepatocytes

Vainio, S. et al Biochem. J., 391, 465-472 (2005)   Niemann–Pick type C (NPC) disease is a neuro-visceral cholesterol storage disorder caused by mutations in the NPC-1 or NPC-2 gene. In the present paper, we studied IR (insulin receptor) activation and the plasma-membrane lipid assembly in primary hepatocytes from control and NPC1–/– mice. We have previously reported that, in hepatocytes, IR activation is dependent on cholesterol–sphingolipid rafts [Vainio, Heino, Mansson, Fredman, Kuismanen, Vaarala and Ikonen (2002) EMBO Rep. 3, 95–100]. We found that, in NPC hepatocytes, IR levels were up-regulated and the receptor activation was compromised. Defective IR activation was reproduced in isolated NPC plasma-membrane preparations, which displayed an increased cholesterol content and saturation of major phospholipids. The NPC plasma membranes were less fluid than control membranes as indicated by increased DPH (1,6-diphenyl-1,3,5-hexatriene) fluorescence anisotropy values. Both in NPC hepatocytes and plasma-membrane fractions, the association of IR with low-density DRMs (detergent-resistant membranes) was increased. Moreover, the detergent resistance of both cholesterol and phosphatidylcholine were increased in NPC membranes. Finally, cholesterol removal inhibited IR activation in control membranes but restored IR activation in NPC membranes. Taken together, the results reveal a lipid imbalance in the NPC hepatocyte, which increases lipid ordering in the plasma membrane, alters the properties of lipid rafts and interferes with the function of a raft-associated plasma-membrane receptor. Such a mechanism may participate in the pathogenesis of NPC disease and contribute to insulin resistance in other disorders of lipid metabolism.  

3.693           Malonyl-CoA decarboxylase is present in the cytosolic, mitochondrial and peroxisomal compartments of rat hepatocytes

Joly, E. et al FEBS Lett., 579(29), 6581-6586 (2005)   A role for cytosolic malonyl-CoA decarboxylase (MCD) as a regulator of fatty acid oxidation has been postulated. However, there is no direct evidence that MCD is present in the cytosol. To address this issue, we performed cell fractionation and electron microscopic colloidal gold studies of rat liver to determine the location and activity of MCD. By both methods, substantial amounts of MCD protein and activity were found in the cytosol, mitochondria and peroxisomes, the latter with the highest specific activity. MCD species with different electrophoretic mobility were observed in the three fractions. The data demonstrate that active MCD is present in the cytosol, mitochondria and peroxisomes of rat liver, consistent with the view that MCD participates in the regulation of cytosolic malonyl-CoA levels and of hepatic fatty acid oxidation.  

3.694           Defects in structural integrity of ergosterol and the Cdc50p-Drs2p putative phospholipid translocase cause accumulation of endocytic membranes, onto which actin parches are assembled in yeast

Kishimoto, T., Yamamoto, T. and Tanaka, K. Mol. Biol. Cell, 16, 5592-5609 (2005)   Specific changes in membrane lipid composition are implicated in actin cytoskeletal organization, vesicle formation, and control of cell polarity. Cdc50p, a membrane protein in the endosomal/trans-Golgi network compartments, is a noncatalytic subunit of Drs2p, which is implicated in translocation of phospholipids across lipid bilayers. We found that the cdc50 mutation is synthetically lethal with mutations affecting the late steps of ergosterol synthesis (erg2 to erg6). Defects in cell polarity and actin organization were observed in the cdc50 erg3 mutant. In particular,actin patches, which are normally found at cortical sites, wereassembled intracellularly along with their assembly factors,including Las17p, Abp1p, and Sla2p. The exocytic SNARE Snc1p,which is recycled by an endocytic route, was also intracellularlyaccumulated, and inhibition of endocytic internalization suppressedthe cytoplasmic accumulation of both Las17p and Snc1p. Simultaneousloss of both phospholipid asymmetry and sterol structural integritycould lead to accumulation of endocytic intermediates capableof initiating assembly of actin patches in the cytoplasm.        

3.695           A genome-wide visual screen reveals a role for sphingolipids and ergosterol in cell surface delivery in yeast

Proszynski, T.J. et al PNAS, 102(50), 17981-17986 (2005)   Recently synthesized proteins are sorted at the trans-Golginetwork into specialized routes for exocytosis. Surprisinglylittle is known about the underlying molecular machinery. Here,we present a visual screen to search for proteins involved incargo sorting and vesicle formation. We expressed a GFP-taggedplasma membrane protein in the yeast deletion library and identifiedmutants with altered marker localization. This screen revealeda requirement of several enzymes regulating the synthesis ofsphingolipids and ergosterol in the correct and efficient deliveryof the marker protein to the cell surface. Additionally, weidentified mutants regulating the actin cytoskeleton (Rvs161pand Vrp1p), known membrane traffic regulators (Kes1p and Chs5p),and several unknown genes. This visual screening method cannow be used for different cargo proteins to search in a genome-widefashion for machinery involved in post-Golgi sorting.  

3.696           Expression and activity of NOX5 in the circulating malignant B cells of hairy cell leukemia

Kamiguti, A.S. et al

1. Immunol., 175, 8424-8430 (2005)

  Hairy cells (HCs) are mature malignant B cells that contain a number of constitutively active signaling molecules including GTP-bound Rac1, protein kinase C, and Src family kinases. Because Rac1 is a component of the reactive oxidant species (ROS)-generating NADPH oxidase system, we investigated the role of this GTPase in ROS production in HCs. In this study, we show that ROS production in HCs involves a flavin-containing oxidase dependent on Ca²⁺, but not on GTP-Rac1 or protein kinase C. This suggests the involvement of the nonphagocytic NADPH oxidase NOX5, an enzyme found in lymphoid tissues, but not in circulating lymphocytes. By using RT-PCR and Southern and Western blotting and by measuring superoxide anion production in membrane fractions in the absence of cytosolic components, we demonstrate for the first time that HCs (but not circulating normal B cells or some other lymphoid cell types) express NOX5. We also demonstrate that inhibition of NADPH oxidase in HCs results in a selective increase in the activity of Src homology region 2 domain-containing phosphatase 1 (SHP-1). Furthermore, SHP-1 in HCs coimmunoprecipitates with tyrosine phosphorylated CD22 and localizes in the same cellular compartment as NOX5. This allows the inactivation of SHP-1 by NOX5-generated ROS and contributes to the maintenance of the constitutive activation of HCs.  

3.697           Solute traffic across mammalian peroxisomal membrane – single-channel conductance monitoring reveals pore-forming activities in peroxisomes

Antonenkov, V.D., Rokka, A., Sormunen, R.T., Benz, R. And Hiltunen, J.K. Cell. Mol. Life Sci., 62, 2886-2895 (2005)   Mouse liver peroxisomes were isolated by centrifugation in a self-generated Percoll gradient followed by an Optiprep density gradient centrifugation. Peroxisomes contributed 90–96% of the total protein content in the fraction, as confirmed by marker enzyme assays, protein pattern in SDS-PAGE, immunoblotting, and electron microscopy. Solubilized peroxisomal membrane proteins were reconstituted into a planar lipid bilayer. A single-channel conductance monitoring of the reconstituted lipid bilayer revealed the presence of two pore-forming components with a conductance in 1 M KCl of 1.3 nS and 2.5 nS. Control experiments with fractions enriched in mitochondria, lysosomes, and fragments of endoplasmic reticulum showed that the peroxisomal channel-forming activities were not due to admixture of isolated peroxisomes with other cellular organelles. The peroxisomal channels were well preserved in membrane preparations but became unstable after solubilization from the membranes by detergent.  

3.698           The compartmentalization of prolactin signaling in the mouse mammary gland

Bolander Jr., F.F. Mol. Cell. Endocrinol., 245(1-2), 105-110 (2005)   In mammary epithelial cells, prolactin (PRL) activates at least two signaling pathways: Jak/Stat and nitric oxide (NO). The former induces differentiation as measured by α-lactalbumin accumulation, while experiments with sodium nitroprusside (SNP) show that NO inhibits differentiation. In order to resolve this apparent contradiction, the kinetics, inducibility, and cellular localization of NO production and sensitivity in mammary cells were examined. First, mammary cells remained responsive to PRL throughout the incubation with respect to NO production. Second, although desensitization occurred with continuous PRL exposure, recovery began as quickly as 30 min after PRL withdrawal. Since PRL is secreted in pulses in vivo, complete desensitization was not a likely explanation for the cells’ escape from NO inhibition. Finally, the cellular site of transduction was examined using the caveolar disrupting agent, methyl-β-cyclodextrin (MBCD). MBCD inhibited the accumulation of PRL-induced NO but not α-lactalbumin. This finding was confirmed by membrane fractionation studies where the PRL-induced NO production occurred primarily in caveolae and PRL-stimulated tyrosine phosphorylation of Stat5, which transcribes the α-lactalbumin gene, occurred predominantly in noncaveolar membranes. Finally, endogenous elevations of NO by arginine did not inhibit differentiation. As such, the inhibition seen with SNP appeared to be an artifact of the ubiquitous generation of NO from SNP. Physiologically, PRL induces NO only in caveolae and this restricted distribution does not interfere with differentiation.  

3.699           Oestrogen-mediated tyrosine phosphorylation of caveolin-1 and its effect on the oestrogen receptor localization: an in vivo study

Kiss, A.L. et al Mol. Cell. Endocrinol., 245(1-2), 128-137 (2005)   Recently, it has been shown that 17β estradiol (E2) induces a rapid and transient activation of the Src ERK phosphorylation cascade: a clear indication that the α oestrogen receptor (ERα) is able to associate with the plasma membrane. Increasing evidence suggests that caveolae, which are caveolin-1 containing, highly hydrophobic membrane domains, play an important role in E2 induced signal transduction. Caveolae can accumulate signalling molecules preferentially; thus, they may have a regulatory role in signalling processes. Results from previous experiments have shown that E2 treatment decreased the number of surface connected caveolae significantly in uterine smooth muscle cells and also downregulated the expression of caveolin-1. In addition to providing further evidence that ERα interacts with caveolin/caveolae in uterine smooth muscle cells, this study also shows that the interaction between caveolin-1 and ERα is actually facilitated by E2. One of the signal transduction components found to accumulate in caveolae is Src kinase in an amount that increases simultaneously with increases in the amount of ERα. Upon E2 treatment, Src kinase is tyrosine phosphorylated, which, in turn, stimulates Src kinase to phosphorylate caveolin-1. Phosphorylation of caveolin-1 can drive caveolae to pinch off from the plasma membrane, thereby decreasing the amount of plasma membrane-associated caveolin-1. This loss of caveolin/caveolae activates the signal cascade that triggers cell proliferation.  

3.700           Sorghum Genome Sequencing by Methylation Filtration

Bedell, J.A. et al PloS Biol., 3(1), 103-115 (2005)   Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF) technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis.  

3.701           T cell receptor-induced lipid raft recruitment of the IκB kinase complex is necessary and sufficient for NF-κB activation occurring in the cytosol

Sebald, A., Mattioli, I. and Schmitz, M.L. Eur. J. Immunol., 35(1), 318-325 (2005)   TCR-induced NF-κB activation is necessary for the innate immune response and involves induced lipid raft recruitment of the IκB kinase (IKK) complex. In this study, we systematically investigated lipid raft recruitment of members of the NF-κB activation pathway in human T cells. All upstream components leading to IKK activation were found constitutively or inducibly in lipid rafts, while the NF-κB/IκB complex and phosphorylated forms of IKK /β, IκB and p65 are exclusively found in the cytosolic fraction. Disruption of raft organization precluded NF-κB activation induced by T cell costimulation, but IL-1-triggered NF-κB activation remained unaffected. Targeting of the IKK complex to lipid rafts caused constitutive IKK activation and NF-κB DNA binding, which was further triggered upon T cell costimulation. Various experimental approaches revealed that costimulation-induced IKK /β activation loop phosphorylation is independent from IKKβ-mediated transautophosphorylation, but rather involves phosphorylation by the IKK-interacting protein NIK and its upstream activator COT.  

3.702           Leishmania donovani lipophosphoglycan disrupts phagosome microdomains in J774 macrophages

Dermine. J-F., Goyette, G., Houde, M., Turco, S.J. and Desjardins, M. Cell. Microbiol., 7(9), 1263-1270 (2005)   Clearance of pathogens by phagocytosis and their killing in phagolysosomes is a key aspect of our innate ability to fight infectious agents. Leishmania parasites have evolved ways to survive and replicate in macrophages by inhibiting phagosome maturation and avoiding the harsh environment of phagolysosomes. We describe here that during this process Leishmania donovani uses a novel strategy involving its surface lipophosphoglycan (LPG), a virulence factor impeding many host functions, to prevent the formation or disrupt lipid microdomains on the phagosome membrane. LPG acts locally on the membrane and requires its repetitive carbohydrate moieties to alter the organization of microdomains. Targeting and disruption of functional foci, where proteins involved in key aspects of phagolysosome biogenesis assemble, is likely to confer a survival advantage to the parasite.  

3.703           Intragranular Vesiculotubular Compartments are Involved in Piecemeal Degranulation by Activated Human Eosinophils

Melo, R.C.N., Perez, S.A.C., Spencer, L.A., Dvorak, A.M. and Weller, P.F. Traffic, 6(10), 866-879 (2005)   Eosinophils, leukocytes involved in allergic, inflammatory and immunoregulatory responses, have a distinct capacity to rapidly secrete preformed granule-stored proteins through piecemeal degranulation (PMD), a secretion process based on vesicular transport of proteins from within granules for extracellular release. Eosinophil-specific granules contain cytokines and cationic proteins, such as major basic protein (MBP). We evaluated structural mechanisms responsible for mobilizing proteins from within eosinophil granules. Human eosinophils stimulated for 30–60 min with eotaxin, regulated on activation, normal, T-cell expressed and secreted (RANTES) or platelet activating factor exhibited ultrastructural features of PMD (e.g. losses of granule contents) and extensive vesiculotubular networks within emptying granules. Brefeldin A inhibited granule emptying and collapsed intragranular vesiculotubular networks. By immunonanogold ultrastructural labelings, CD63, a tetraspanin membrane protein, was localized within granules and on vesicles outside of granules, and mobilization of MBP into vesicles within and extending from granules was demonstrated. Electron tomography with three dimension reconstructions revealed granule internal membranes to constitute an elaborate tubular network able to sequester and relocate granule products upon stimulation. We provide new insights into PMD and identify eosinophil specific granules as organelles whose internal tubulovesicular networks are important for the capacity of eosinophils to secrete, by vesicular transport, their content of preformed and granule-stored cytokines and cationic proteins.    

3.704           Peroxisomal proteomics, a new tool for risk assessment of peroxisome proliferating pollutants in the marine environment

Mi, J., Orbea, A., Syme, N., Ahmed, M., Cajaraville, M.P. and Cristobal, S. Proteomics, 5(15), 3954-2965 (2005)   In an attempt to improve the detection of peroxisome proliferation as a biomarker in environmental pollution assessment, we have applied a novel approach based on peroxisomal proteomics. Peroxisomal proteins from digestive glands of mussels Mytilus galloprovincialis were analyzed using 2-DE and MS. We have generated a reference 2-DE map from samples obtained in a well-studied reference area and compared this with peroxisomal proteomes from other sequenced genomes. In addition, by comparing 2-DE maps from control samples with samples obtained in a polluted area, we have characterized the peroxisome proliferation expression pattern associated with exposure to a polluted environment. Over 100 spots were reproducibly resolved per 2-DE map; 55 differentially expressed spots were quantitatively detected and analyzed, and 14 of these showed an increase in protein expression of more than fourfold. Epoxide hydrolase, peroxisomal antioxidant enzyme, and sarcosine oxidase (SOX) have been identified by ESI MS/MS, and acyl-CoA oxidase, multifunctional protein, and Cu,Zn-superoxide dismutase were immunolocalized by Western blotting. Our results indicate that a peroxisomal protein pattern associated to marine pollutant exposure can be generated, and this approach may have a greater potential as biomarker than traditional, single-protein markers.  

3.705           Altered regulation of EGF receptor signaling following a partial hepatectomy

Skarpen, E., Oksvold, M.P., Grøsvik, H., Widnes, C. and Huitfeldt, H.S.

1. Cell. Physiol., 202(3), 707-716 (2005)

  We have studied epidermal growth factor receptor (EGFR) phosphorylation and localization in the pre-replicative phase of liver regeneration induced by a 70% partial hepatectomy (PH), and how a PH affects EGFR activation and trafficking. When Western blotting was performed on livers after PH with antibodies raised against activated forms of EGFR autophosphorylation sites, no marked increase in EGFR tyrosine phosphorylation was observed. However, events associated with attenuation of EGFR signals were observed. Two hours after PH, we found increased EGFR ubiquitination and internalization, followed by receptor downregulation. Furthermore, EGFR phosphorylation following an injection of EGF was reduced after PH. This reduction correlated with an increased activation of PKC and a distinct augmentation in the phosphorylation of the PKC-regulated T654-site of EGFR. When primary cultured hepatocytes were treated with tetradecanoylphorbol acetate (TPA) to induce T654-phosphorylation of EGFR, we found colocalization of a fraction of EGFR with EEA1, downregulation of EGF-mediated EGFR autophosphorylation, altered ligand-induced intracellular sorting of EGFR, and increased mitogenic signaling through the EGFR-Ras-Raf-ERK pathway. Further, we found that both TPA and a PH enhanced EGF-induced proliferation of hepatocytes. In conclusion, our results suggest that hepatocyte priming involves modulation of EGFR that enhances its ability to mediate growth factor responses without an increase in its receptor tyrosine kinase-activity. This may be a pre-replicative competence event that increases growth factor effects during G1 progression.  

3.706           Characterization of myosin-II binding to Golgi stacks in vitro

Fath, K.R. Cell Motil. Cytoskeleton, 60(4), 222-235 (2005)   In addition to important roles near the actin-rich cell cortex, ample evidence indicates that multiple myosins are also involved in membrane movements in the endomembrane system. Nonmuscle myosin-II has been shown to have roles in anterograde and retrograde trafficking at the Golgi. Myosin-II is present on Golgi stacks isolated from intestinal epithelial cells and has been localized to the Golgi in several polarized and unpolarized cell lines. An understanding of roles of myosin-II in Golgi physiology will be facilitated by understanding the molecular arrangement of myosin-II at the Golgi. Salt-washing removes endogenous myosin-II from isolated Golgi and purified brush border myosin-II can bind in vitro. Brush border myosin-II binds to a tightly bound Golgi peripheral membrane protein with a K_1/2 of 75 nM and binding is saturated at 0.7 pmol myosin/μg Golgi. Binding studies using papain cleavage fragments of brush border myosin-II show that the 120-kDa rod domain, but not the head domain, of myosin heavy chain can bind directly to Golgi stacks. The 120-kDa domain does not bind to Golgi membranes when phosphorylated in vitro with casein kinase-II. These results suggest that phosphorylation in the rod domain may regulate the binding and/or release of myosin-II from the Golgi. These data support a model in which myosin-II is tethered to the Golgi membrane by its tail and actin filaments by its head. Thus, translocation along actin filaments may extend Golgi membrane tubules and/or vesicles away from the Golgi complex.  

3.707           The unconventional myosin-VIIa associates with lysosomes

Soni, L.E., Warren, C.M., Bucci, C., Orten, D.J. and Hasson, T. Cell Motil. Cytoskeleton, 62(1), 13-26 (2005)   Mutations in the myosin-VIIa (MYO7a) gene cause human Usher disease, characterized by hearing impairment and progressive retinal degeneration. In the retina, myosin-VIIa is highly expressed in the retinal pigment epithelium, where it plays a role in the positioning of melanosomes and other digestion organelles. Using a human cultured retinal pigmented epithelia cell line, ARPE-19, as a model system, we have found that a population of myosin-VIIa is associated with cathepsin D- and Rab7-positive lysosomes. Association of myosin-VIIa with lysosomes was Rab7 independent, as dominant negative and dominant active versions of Rab7 did not disrupt myosin-VIIa recruitment to lysosomes. Association of myosin-VIIa with lysosomes was also independent of the actin and microtubule cytoskeleton. Myosin-VIIa copurified with lysosomes on density gradients, and fractionation and extraction experiments suggested that it was tightly associated with the lysosome surface. These studies suggest that myosin-VIIa is a lysosome motor.    

3.708           Release of the type I secreted a-haemolysin via outer membrane vesicles from Escherichia coli

Balsalobre, C. et al Mol. Microbiol., 59(1), 99-112 (2006)   The α-haemolysin is an important virulence factor commonly expressed by extraintestinal pathogenic Escherichia coli. The secretion of the α-haemolysin is mediated by the type I secretion system and the toxin reaches the extracellular space without the formation of periplasmic intermediates presumably in a soluble form. Surprisingly, we found that a fraction of this type I secreted protein is located within outer membrane vesicles (OMVs) that are released by the bacteria. The α-haemolysin appeared very tightly associated with the OMVs as judged by dissociation assays and proteinase susceptibility tests. The α-haemolysin in OMVs was cytotoxically active and caused lysis of red blood cells. The OMVs containing the α-haemolysin were distinct from the OMVs not containing α-haemolysin, showing a lower density. Furthermore, they differed in protein composition and one component of the type I secretion system, the TolC protein, was found in the lower density vesicles. Studies of natural isolates of E. coli demonstrated that the localization of α-haemolysin in OMVs is a common feature among haemolytic strains. We propose an alternative pathway for the transport of the type I secreted α-haemolysin from the bacteria to the host cells during bacterial infections.  

3.709           Nox2 and Rac1 regulate H₂O₂-dependent recruitment of TRAFg to endosomal interleukin-1 receptor complexes

Li, Q. et al Mol. Cell. Biol., 26(1), 140-154 (2006)   Reactive oxygen species (ROS) generated by NADPH oxidases (Nox) have been implicated in the regulation of signal transduction. However, the cellular mechanisms that link Nox activation with plasma membrane receptor signaling remain poorly defined. We have found that Nox2-derived ROS influence the formation of an active interleukin-1 (IL-1) receptor complex in the endosomal compartment by directing the H₂O₂-dependent binding of TRAF6 to the IL-1R1/MyD88 complex. Clearance of both superoxide and H₂O₂ from within the endosomal compartment significantly abrogated IL-1ß-dependent IKK and NF- B activation. MyD88-dependent endocytosis of IL-1R1 following IL-1ß binding was required for the redox-dependent formation of an active endosomal receptor complex competent for IKK and NF- B activation. Small interfering RNAs to either MyD88 or Rac1 inhibited IL-1ß induction of endosomal superoxide and NF- B activation. However, MyD88 and Rac1 appear to be recruited independently to IL-1R1 following ligand stimulation. In this context, MyD88 binding was required for inducing endocytosis of IL-1R1 following ligand binding, while Rac1 facilitated the recruitment of Nox2 into the endosomal compartment and subsequent redox-dependent recruitment of TRAF6 to the MyD88/IL-1R1 complex. The identification of Nox-active endosomes helps explain how subcellular compartmentalization of redox signals can be used to direct receptor activation from the plasma membrane.  

3.710           Lipid raft clustering and redox signaling platform formation in coronary arterial endothelial cells

Zhung, A.Y., Yi, F., Zhang, G., Gulbins, E. and Li, P-L. Hypertension, 47, 74-80 (2006)   Recent studies have indicated that lipid rafts (LRs) in the cell membrane are clustered in response to different stimuli to form signaling platforms for transmembrane transduction. It remains unknown whether this LR clustering participates in redox signaling in endothelial cells. The present study tested a hypothesis that clustering of LRs on the membrane of coronary endothelial cells produces aggregation and activation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, thereby forming a redox signaling platform. By confocal microscopic analysis of agonist-stimulated rafts patch formation, we found that several death receptor ligands or apoptotic factors, including tumor necrosis factor [alpha], Fas ligand, or endostatin, stimulated the clustering and trafficking of individual LRs on the plasma membrane of coronary endothelial cells. Interestingly, double labeling of a membrane-bound NADPH oxidase subunit, gp91phox, and LRs showed that gp91phox colocalized within the LR patches when endothelial cells were stimulated by Fas ligand. In isolated LR fractions from Fas-stimulated endothelial cells, gp91phox, p47phox (a crucial cytosolic regulatory subunit of NADPH oxidase), and Rac GTPase were markedly increased and blocked by nystatin, a compound that disrupts LRs. These clustered LRs contained high NADPH oxidase activity, which increased in response to Fas stimulation. Functionally, Fas ligand-induced inhibition of endothelium-dependent vasorelaxation was reduced if LRs were disrupted or NADPH oxidase was inhibited. These results suggest that LR clustering occurs in coronary endothelial cells. The formation of redox signaling platforms on the cell membrane mediates transmembrane signaling of death receptors, resulting in endothelial dysfunction.  

3.711           Significance of sterol structural specificity

Vainio, S. et al

1. Biol. Chem., 281(1), 348-355 (2006)

  Desmosterol is an immediate precursor of cholesterol in the Bloch pathway of sterol synthesis and an abundant membrane lipid in specific cell types. The significance of the difference between the two sterols, an additional double bond at position C24 in the tail of desmosterol, is not known. Here, we provide evidence that the biophysical and functional characteristics of the two sterols differ and that this is because the double bond at C24 significantly weakens the sterol ordering potential. In model membranes, desmosterol was significantly weaker than cholesterol in promoting the formation or stability of ordered domains, and in mammalian cell membranes, desmosterol associated less avidly than cholesterol with detergent-resistant membranes. Atomic scale molecular dynamics simulations showed that the double bond gives rise to additional stress in the tail, creating a rigid structure between C24 and C27 and favoring tilting of desmosterol distinct from cholesterol. Functional effects of desmosterol in cell membranes were assessed upon acutely exchanging 70% of cholesterol to desmosterol. This led to impaired raft-dependent signaling via the insulin receptor, whereas non-raft-dependent protein secretion was not affected. We suggest that the choice of cholesterol synthesis route may provide a physiological mechanism to modulate raft-dependent functions in cells.  

3.712           Detection of a raft-located estrogen receptor-like protein distinct from ERa

Heberden, C. et al Int. J. Biochem. Cell Biol., 38, 376-391 (2006)   17β-Estradiol (17β-E2) elicits at the cell membrane rapid actions that remain insensitive to the inhibitory effect of ICI 182,780, a pure estrogen antagonist, and therefore cannot be attributed to the classic nuclear receptors. We addressed the question of the identity of the protein involved in these rapid actions. We first examined the responses of several cell lines for intracellular calcium mobilization, an effect not inhibited by ICI 182,780, tamoxifen and raloxifen. We then demonstrated the presence of binding sites in the membranes, by incubating them with antibodies directed against different domains of ERα, and by flow cytometry analysis. The membrane proteins were eluted by affinity chromatography using E2 conjugated to bovine serum albumin as a ligand. Western blots of the elution fractions using an antibody directed against the ligand binding site of ERα showed the existence of a protein of 50 kDa. The protein was concentrated in the lipid rafts, together with another heavier form of 66 kDa. The 50 kDa protein was immunoprecipitable, and co-immunoprecipitation experiments showed that it was associated with the Gβ_1–4 protein, but not with caveolin-1. The protein was expressed in ERα-null cells, like HO-23 and Cos-7 cells. Therefore, in the lipid rafts, there exists a protein, similar to, but molecularly distinct from ERα..  

3.713           Identification of a switch in neurotrophin signaling by selective tyrosine phosphorylation

Arevalo, J., Pereira, D.B., Yano, H., Teng, K.K. and Chao, M.V.

1. Biol. Chem., 281(2), 1001-1007 (2006)

  Neurotrophins, such as nerve growth factor and brain-derived neurotrophic factor, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed by autophosphorylation and recruitment of intracellular signaling molecules. The intracellular pathways used by neurotrophins share many common protein substrates that are used by other receptor tyrosine kinases (RTK), such as Shc, Grb2, FRS2, and phospholipase C- . Here we describe a novel RTK mechanism that involves a 220-kilodalton membrane tetraspanning protein, ARMS/Kidins220, which is rapidly tyrosine phosphorylated in primary neurons after neurotrophin treatment. ARMS/Kidins220 undergoes multiple tyrosine phosphorylation events and also serine phosphorylation by protein kinase D. We have identified a single tyrosine (Tyr¹⁰⁹⁶) phosphorylation event in ARMS/Kidins220 that plays a critical role in neurotrophin signaling. A reassembled complex of ARMS/Kidins220 and CrkL, an upstream component of the C3G-Rap1-MAP kinase cascade, is SH3-dependent. However, Tyr¹⁰⁹⁶ phosphorylation enables ARMS/Kidins220 to recruit CrkL through its SH2 domain, thereby freeing the CrkL SH3 domain to engage C3G for MAP kinase activation in a neurotrophin dependent manner. Accordingly, mutation of Tyr¹⁰⁹⁶ abolished CrkL interaction and sustained MAPK kinase activity, a response that is not normally observed in other RTKs. Therefore, Trk receptor signaling involves an inducible switch mechanism through an unconventional substrate that distinguishes neurotrophin action from other growth factor receptors.  

3.714           From assembly to virus particle budding: pertince of the detergent resisitant membranes

Gosselin-Grenet, A-S., Mottet-Osman, G. and Roux, L. Virology, 344(2), 296-303 (2006)   Detergent resistant membranes (DRMs) are the site of assembly for a variety of viruses. Here, we make use of Sendai virus mutant proteins that are not packaged into virus particles to determine the involvement of this assembly for the virus particle production. We found that, in the context of an infection, (1) all the Sendai virus proteins associated in part with DRMs, (2) mutant HN and M proteins not packaged into virus particles were similarly part of this association, (3) after M protein suppression resulting in a significant reduction of virus production, the floatation profile of the other viral proteins was not altered and finally (4) cellular cholesterol depletion did not decrease the virus particle production, although it somehow reduced their virus infectivity. These results led us to conclude that the assembly complex found in DRM fractions does not constitute a direct precursor of virus particle budding.  

3.715           Roles of ZO-1, accludin, and actin in axidant-induced barrier disruption

Musch, M.W., Walsh-Reitz, M.W. and Chang, E.B. Am. J. Physiol., 290, G222-G231 (2006)   Oxidants such as monochloramine (NH₂Cl) decrease epithelial barrier function by disrupting perijunctional actin and possibly affecting the distribution of tight junctional proteins. These effects can, in theory, disturb cell polarization and affect critical membrane proteins by compromising molecular fence function of the tight junctions. To examine these possibilities, we investigated the actions of NH₂Cl on the distribution, function, and integrity of barrier-associated membrane, cytoskeletal, and adaptor proteins in human colonic Caco-2 epithelial monolayers. NH₂Cl causes a time-dependent decrease in both detergent-insoluble and -soluble zonula occludens (ZO)-1 abundance, more rapidly in the former. Decreases in occludin levels in the detergent-insoluble fraction were observed soon after the fall of ZO-1 levels. The actin depolymerizer cytochalasin D resulted in a decreased transepithelial resistance (TER) more quickly than NH₂Cl but caused a more modest and slower reduction in ZO-1 levels and in occludin redistribution. No changes in the cellular distribution of claudin-1, claudin-5, or ZO-2 were observed after NH₂Cl. However, in subsequent studies, the immunofluorescent cellular staining pattern of all these proteins was altered by NH₂Cl. The actin-stabilizing agent phalloidin did not prevent NH₂Cl-induced decreases in TER or increases of apical to basolateral flux of the paracellular permeability marker mannitol. However, it partially blocked changes in ZO-1 and occludin distribution. Tight junctional fence function was also compromised by NH₂Cl, observed as a redistribution of the -subunit of basolateral Na⁺-K⁺-ATPase to the apical membrane, an effect not found with the apical membrane protein Na⁺/H⁺ exchanger isoform 3. In conclusion, oxidants not only disrupt perijunctional actin but also cause redistribution of tight junctional proteins, resulting in compromised intestinal epithelial barrier and fence function. These effects are likely to contribute to the development of malabsorption and dysfunction associated with mucosal inflammation of the digestive tract.  

3.716           Focal adhesion kinase is critical for entry of Kaposi’s sarcoma-associated herpesvirus into target cells

Krisham, H.H., Sharma-Walia, N., Streblow, D.N., Naranatt, P.P. and Chandran, B.

1. Virol., 80(3), 1167-1180 (2006)

  Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) interacts with cell surface 3ß1 integrin early during in vitro infection of human endothelial cells and fibroblasts and activates the focal adhesion kinase (FAK) that is immediately downstream in the outside-in signaling pathway by integrins, leading to the activation of several downstream signaling molecules. In this study, using real-time DNA and reverse transcription-PCR assays to measure total internalized viral DNA, viral DNA associated with infected nuclei, and viral gene expression, we examined the stage of infection at which FAK plays the most significant role. Early during KSHV infection, FAK was phosphorylated in FAK-positive Du17 mouse embryonic fibroblasts. The absence of FAK in Du3 (FAK^–/–) cells resulted in about 70% reduction in the internalization of viral DNA, suggesting that FAK plays a role in KSHV entry. Expression of FAK in Du3 (FAK^–/–) cells via an adenovirus vector augmented the internalization of viral DNA. Expression of the FAK dominant-negative mutant FAK-related nonkinase (FRNK) in Du17 cells significantly reduced the entry of virus. Virus entry in Du3 cells, albeit in reduced quantity, delivery of viral DNA to the infected cell nuclei, and expression of KSHV genes suggested that in the absence of FAK, another molecule(s) may be partially compensating for FAK function. Infection of Du3 cells induced the phosphorylation of the FAK-related proline-rich tyrosine kinase (Pyk2) molecule, which has been shown to complement some of the functions of FAK. Expression of an autophosphorylation site mutant of Pyk2 in which Y402 is mutated to F (F402 Pyk2) reduced viral entry in Du3 cells, suggesting that Pyk2 facilitates viral entry moderately in the absence of FAK. These results suggest a critical role for KSHV infection-induced FAK in the internalization of viral DNA into target cells.  

3.717           Quality control of a mutant plasma membrane ATPase: ubiquitylation prevents cell-surface stability

Liu, Y. and Chang, A.

1. Cell Sci., 119, 360-369 (2006)

  The plasma membrane ATPase, Pma1, has remarkable longevity at the cell surface. In contrast to the wild-type protein, the temperature-sensitive mutant Pma1-10 is misfolded and undergoes rapid removal from the cell surface for vacuolar degradation. At the restrictive temperature, Pma1-10 becomes ubiquitylated before or upon arrival at the plasma membrane. Internalization from the plasma membrane and vacuolar degradation of Pma1-10 is dependent on the ubiquitin-interacting motif (UIM) of the epsin Ent1, suggesting recognition of ubiquitylated substrate by the endocytic machinery. Surprisingly, ubiquitylation of Pma1-10 is reversed when its internalization is blocked in an end3 mutant. Under these conditions, Pma1-10 acquires association with detergent-insoluble, glycolipid-enriched complexes (DIGs) which has been suggested to promote stability of wild-type Pma1. Ubiquitylation does not cause DIG exclusion because a Pma1-Ub fusion protein is not significantly excluded from DIGs. We suggest that ubiquitylation of Pma1-10 represents a component of a quality control mechanism that targets the misfolded protein for removal from the plasma membrane. Rapid internalization of Pma1-10 caused by its ubiquitylation may preempt establishment of stabilizing interactions.  

3.718           Protein kinase Ce interacts with cytochrome c oxidase subunit IV and enhances cytochrome c oxidase activity in neonatal cardiac myocyte preconditioning

Ogbi, M. and Johnson, J.A. Biochem. J., 393(1), 191-199 (2006)   We have previously identified a phorbol ester-induced PKCe (protein kinase Ce) interaction with the (~18 kDa) COIV [CO (cytochrome c oxidase) subunit IV] in NCMs (neonatal cardiac myocytes). Since PKCe has been implicated as a key mediator of cardiac PC (preconditioning), we examined whether hypoxic PC could induce PKCe–COIV interactions. Similar to our recent study with phorbol esters [Ogbi, Chew, Pohl, Stuchlik, Ogbi and Johnson (2004) Biochem. J. 382, 923–932], we observed a time-dependent increase in the in vitro phosphorylation of an approx. 18 kDa protein in particulate cell fractions isolated from NCMs subjected to 1–60 min of hypoxia. Introduction of a PKCe-selective translocation inhibitor into cells attenuated this in vitro phosphorylation. Furthermore, when mitochondria isolated from NCMs exposed to 30 min of hypoxia were subjected to immunoprecipitation analyses using PKCe-selective antisera, we observed an 11.1-fold increase in PKCe–COIV co-precipitation. In addition, we observed up to 4-fold increases in CO activity after brief NCM hypoxia exposures that were also attenuated by introducing a PKCe-selective translocation inhibitor into the cells. Finally, in Western-blot analyses, we observed a >2-fold PC-induced protection of COIV levels after 9 h index hypoxia. Our studies suggest that a PKCe–COIV interaction and an enhancement of CO activity occur in NCM hypoxic PC. We therefore propose novel mechanisms of PKCe-mediated PC involving enhanced energetics, decreased mitochondrial reactive oxygen species production and the preservation of COIV levels.  

3.719           CD16b associates with high-density, detergent-resistant membranes in human neutrophils

Fernandes, M.J.G. et al Biochem. J., 393(2), 351-359 (2006)   CD16b is unique in that it is the only Fc receptor linked to the plasma membrane by a GPI (glycosylphosphatidylinositol) anchor. GPI-anchored proteins often preferentially localize to DRMs (detergent-resistant membranes) that are rich in sphingolipids and cholesterol and play an important role in signal transduction. Even though the responses to CD16b engagement have been intensively investigated, the importance of DRM integrity for CD16b signalling has not been characterized in human neutrophils. We provide direct evidence that CD16b constitutively partitions with both low- and high-density DRMs. Moreover, upon CD16b engagement, a significant increase in the amount of the receptor is observed in high-density DRMs. Similarly to CD16b, CD11b also resides in low- and high-density DRMs. In contrast with CD16b, the partitioning of CD11b in DRMs does not change in response to CD16b engagement. We also provide evidence for the implication of Syk in CD16b signalling and its partitioning to DRMs in resting and activated PMNs (polymorphonuclear neutrophils). Additionally, DRM-disrupting agents, such as nystatin and methyl-b-cyclodextrin, alter cellular responses to CD16b receptor ligation. Notably, a significant increase in the mobilization of intracellular Ca²⁺ and in tyrosine phosphorylation of intracellular substrates after CD16b engagement is observed. Altogether, the results of this study provide evidence that high-density DRMs play a role in CD16b signalling in human neutrophils.  

3.720           Receptor palmitoylation and ubiquitination regulate anthrax toxin endocytosis

Abrami, L., Leppla, S.H., Gisou van der Gaat, F.

1. Cell Biol., 171(2), 309-320 (2006)

  The anthrax toxin is composed of three independent polypeptidechains. Successful intoxication only occurs when heptamerizationof the receptor-binding polypeptide, the protective antigen(PA), allows binding of the two enzymatic subunits before endocytosis.We show that this tailored behavior is caused by two counteractingposttranslational modifications in the cytoplasmic tail of PAreceptors. The receptor is palmitoylated, and this unexpectedlyprevents its association with lipid rafts and, thus, its prematureubiquitination. This second modification, which is mediatedby the E3 ubiquitin ligase Cbl, only occurs in rafts and isrequired for rapid endocytosis of the receptor. As a consequence,cells expressing palmitoylation-defective mutant receptors areless sensitive to anthrax toxin because of a lower number ofsurface receptors as well as premature internalization of PAwithout a requirement for heptamerization.  

3.721           Deactivation of phosporylated and nonphosphorylated rhodopsin by arrestin splice variants

Burns, M.E. et al

1. Neurosci., 26(3), 1036-1044 (2006)

  Arrestins constitute a family of small cytoplasmic proteins that mediate deactivation of G-protein-coupled receptors (GPCRs) and are known to be essential for cascade inactivation and receptor desensitization. Alternative splicing produces an array of arrestin gene products that have widely different specificities for their cognate receptors in vitro, but the differential functions of these splice variants in vivo are essentially unknown. Bovine rod photoreceptors express two splice variants of visual arrestin (p44 and p48) that display different affinities for the GPCR rhodopsin. To determine the functions of these splice variants in intact cells, we expressed a transgene encoding either a truncated form of murine arrestin (mArr^1–369, or m44) or the long (p48) isoform in mouse rods lacking endogenous arrestin (Arr–/–). Morphological analysis showed that expression of either variant attenuated the light-induced degeneration that is thought to result from excessive cascade activity in Arr–/–rods. Suction electrode recordings from individual rods indicated that the expression of either m44 or p48 splice variants could restore normal kinetics to Arr–/– dim flash responses, indicating that both isoforms can bind to and quench phosphorylated rhodopsin rapidly. To our surprise, only the full-length variant was able to alter the kinetics of responses in rods lacking both arrestin and rhodopsin kinase, indicating that p48 can also quench the activity of nonphosphorylated rhodopsin.  

3.722           LMP1 signaling and activation of NF-kB LMP1 transgenic mice

Thornburg, N.J. et al Oncogene, 25, 288-297 (2006)   Transgenic mice expressing Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) under the control of an immunoglobulin heavy-chain promoter and enhancer develop lymphoma at a threefold higher incidence than LMP1-negative mice. In vitro, LMP1 activates numerous signaling pathways including p38, c-Jun N terminal kinase (JNK), phosphatidylinositol 3 kinase (PI3K)/Akt, and NF- B through interactions with tumor necrosis receptor-associated factors (TRAFs). These pathways are frequently activated in EBV-associated malignancies, although their activation cannot be definitively linked to LMP1 expression in vivo. In this study, interactions between LMP1 and TRAFs and the activation of PI3K/Akt, JNK, p38, and NF- B were examined in LMP1 transgenic mice. LMP1 co-immunoprecipitated with TRAFs 1, 2, and 3. Akt, JNK, and p38 were activated in LMP1-positive and -negative splenocytes as well as LMP1-positive and -negative lymphomas. Multiple forms of NF- B were activated in healthy splenocytes from LMP1 transgenic mice, in contrast to healthy splenocytes from LMP1-negative mice. However, in both LMP1-positive and -negative lymphomas, only the oncogenic NF- B c-Rel, was specifically activated. Similarly to EBV-associated malignancies, p53 protein was detected at high levels in the transgenic lymphomas, although mutations were not detected in the p53 gene. These data indicate that NF- B is activated in LMP1-positive healthy splenocytes; however, NF- B c-Rel is specifically activated in both the transgenic lymphomas and in the rare lymphomas that develop in negative mice. The LMP1-mediated activation of NF- B may contribute to the specific activation of c-Rel and lead to the increased development of lymphoma in the LMP1 transgenic mice.  

3.723           Intracytoplasmic maturation of the human immunodeficiency virus type I reverse transcription complexes determines their capacity to integrate into chromatin

Iodanskiy, S., Berro, R., Altieri, M., Kashanchi, F. And Bukrinsky, M. Retrovirology, 3(4), 1-12 (2006)   Background The early events of the HIV-1 life cycle include entry of the viral core into target cell, assembly of the reverse transcription complex (RTCs) performing reverse transcription, its transformation into integration-competent complexes called pre-integration complexes (PICs), trafficking of complexes into the nucleus, and finally integration of the viral DNA into chromatin. Molecular details and temporal organization of these processes remain among the least investigated and most controversial problems in the biology of HIV. Results To quantitatively evaluate maturation and nuclear translocation of the HIV-1 RTCs, nucleoprotein complexes isolated from the nucleus (nRTC) and cytoplasm (cRTC) of HeLa cells infected with MLV Env-pseudotyped HIV-1 were analyzed by real-time PCR. While most complexes completed reverse transcription in the cytoplasm, some got into the nucleus before completing DNA synthesis. The HIV-specific RNA complexes could get into the nucleus when reverse transcription was blocked by reverse transcriptase inhibitor, although nuclear import of RNA complexes was less efficient than of DNA-containing RTCs. Analysis of the RTC nuclear import in synchronized cells infected in the G2/M phase of the cell cycle showed enrichment in the nuclei of RTCs containing incomplete HIV-1 DNA compared to non-synchronized cells, where RTCs with complete reverse transcripts prevailed. Immunoprecipitation assays identified viral proteins IN, Vpr, MA, and cellular Ini1 and PML associated with both cRTCs and nRTCs, whereas CA was detected only in cRTCs and RT was diminished in nRTCs. Cytoplasmic maturation of the complexes was associated with increased immunoreactivity with anti-Vpr and anti-IN antibodies, and decreased reactivity with antibodies to RT. Both cRTCs and nRTCs carried out endogenous reverse transcription reaction in vitro. In contrast to cRTCs, in vitro completion of reverse transcription in nRTCs did not increase their integration into chromatin. Conclusion These results suggest that RTC maturation occurs predominantly in the cytoplasm. Immature RTCs containing RT and incomplete DNA can translocate into the nucleus during mitosis and complete reverse transcription, but are defective for integration.  

3.724           Glycosylphosphatidylinositol-anchored proteins are required for the transport pg detergent-resistant microdomain-associated membrane proteins Tat2p and Fur4p

Okamoto, M., Yoko-o, T., Umemura, M., Nakayama, K-i. and Jigami, Y.

1. Biol. Chem., 281(7), 4013-4023 (2006)

  In eukaryotic cells many cell surface proteins are attached to the membrane via the glycosylphosphatidylinositol (GPI) moiety. In yeast, GPI also plays important roles in the production of mannoprotein in the cell wall. We previously isolated gwt1 mutants and found that GWT1 is required for inositol acylation in the GPI biosynthetic pathway. In this study we isolated a new gwt1 mutant allele, gwt1-10, that shows not only high temperature sensitivity but also low temperature sensitivity. The gwt1-10 cells show impaired acyltransferase activity and attachment of GPI to proteins even at the permissive temperature. We identified TAT2, which encodes a high affinity tryptophan permease, as a multicopy suppressor of cold sensitivity in gwt1-10 cells. The gwt1-10 cells were also defective in the import of tryptophan, and a lack of tryptophan caused low temperature sensitivity. Microscopic observation revealed that Tat2p is not transported to the plasma membrane but is retained in the endoplasmic reticulum in gwt1-10 cells grown under tryptophan-poor conditions. We found that Tat2p was not associated with detergent-resistant membranes (DRMs), which are required for the recruitment of Tat2p to the plasma membrane. A similar result was obtained for Fur4p, a uracil permease localized in the DRMs of the plasma membrane. These results indicate that GPI-anchored proteins are required for the recruitment of membrane proteins Tat2p and Fur4p to the plasma membrane via DRMs, suggesting that some membrane proteins are redistributed in the cell in response to environmental and nutritional conditions due to an association with DRMs that is dependent on GPI-anchored proteins.  

3.725           Involvement of lipid rafts and caveolae in cardiac ion channel function

Maguy, A., Hebert, T.E. and Nattel, S. Cardiovasc. Res., 69(4), 798-807 (2006)   A variety of lipid microdomains, including caveolae, have been shown to play an important role in both protein targetting and in controlling protein–protein interactions. There is increasing evidence for significant ion channel localization in lipid rafts. Cardiac channel subunits known to localize in lipid rafts include Kv1.4, Kv1.5, Kv2.1, Kv4, Kir2, Kir3, K_ATP, Nav and Cav subunits. This article reviews what is known about the occurrence and functional significance of cardiac ion channel/lipid raft interactions. Much remains to be learned about this area of potentially enormous importance to cardiac function in health and disease.  

3.726           Notch, epidermal growth factor receptor, and b1-integrin pathways are coordinated in neural stem cells

Campos, L., Decker, L., Taylor, V. and Skarnes, W.

1. Biol. Chem., 281(8), 5300-5309 (2006)

  Notch1 and 1-integrins are cell surface receptors involved in the recognition of the niche that surrounds stem cells through cell-cell and cell-extracellular matrix interactions, respectively. Notch1 is also involved in the control of cell fate choices in the developing central nervous system (Lewis, J. (1998) Semin. Cell Dev. Biol. 9, 583-589). Here we report that Notch and 1-integrins are co-expressed and that these proteins cooperate with the epidermal growth factor receptor in neural progenitors. We describe data that suggests that 1-integrins may affect Notch signaling through 1) physical interaction (sequestration) of the Notch intracellular domain fragment by the cytoplasmic tail of the 1-integrin and 2) affecting trafficking of the Notch intracellular domain via caveolin-mediated mechanisms. Our findings suggest that caveolin 1-containing lipid rafts play a role in the coordination and coupling of 1-integrin, Notch1, and tyrosine kinase receptor signaling pathways. We speculate that this will require the presence of the adequate 1-activating extracellular matrix or growth factors in restricted regions of the central nervous system and namely in neurogenic niches.  

3.727           Association of brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2) with recycling endosomes during transferring uptake

Shen, X. et al PNAS, 103(8), 2635-2640 (2006)   ADP-ribosylation factors (ARFs) are critical in vesicular trafficking. Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG)1 and BIG2 activate ARFs by accelerating replacement of bound GDP with GTP. Additional and differing functions of these 200-kDa proteins are now being recognized, as are their independent intracellular movements. Here, we describe the localization in COS7 cells by immunofluorescence microscopy of BIG2, but not BIG1, with structures that have characteristics of recycling endosomes during transferrin (Tfn) uptake and Tfn receptor (TfnR) recycling. Cell content of BIG2 and Rab11, but not TfnR, BIG1, Rab4, or Exo70, was increased after 60 min of Tfn uptake. BIG2, but not BIG1, appeared in density-gradient fractions containing TfnR, Rab11, and Exo70 after 60 min of Tfn uptake. Treatment of cells with BIG2 small interfering RNA (siRNA), but not BIG1 or control siRNAs, decreased BIG2 protein >90% without affecting BIG1, ARF, or actin content, whereas TfnR was significantly increased as was its accumulation in perinuclear recycling endosomes. Tfn release appeared unaffected by BIG1 siRNA but was significantly slowed from cells treated with BIG2 siRNA alone or plus BIG1 siRNA. We suggest that BIG2 has an important role in Tfn uptake and TfnR recycling, perhaps through its demonstrated interaction with Exo70 and the exocyst complex.    

3.728           The HIV lipodome: a raft with an unusual composition

Brügger, B. et al PNAS, 103(8), 2641-2646 (2006)   The lipids of enveloped viruses play critical roles in viralmorphogenesis and infectivity. They are derived from the hostmembranes from which virus budding occurs, but the precise lipidcomposition has not been determined for any virus. Employingmass spectrometry, this study provides a quantitative analysisof the lipid constituents of HIV and a comprehensive comparisonwith its host membranes. Both a substantial enrichment of theunusual sphingolipid dihydrosphingomyelin and a loss of viralinfectivity upon inhibition of sphingolipid biosynthesis inhost cells are reported, establishing a critical role for thislipid class in the HIV replication cycle. Intriguingly, theoverall lipid composition of native HIV membranes resemblesdetergent-resistant membrane microdomains and is strikinglydifferent from that of host cell membranes. With this composition,the HIV lipidome provides strong evidence for the existenceof lipid rafts in living cells.  

3.729           Vesicle-associated membrane protein 7 is expressed in intestinal ER

Siddiqi, S., Mathan, J., Siddiqi, S., Gorelick, F.S. and Mansbach, C.M.

1. Cell Sci., 119, 943-950 (2006)

  Intestinal dietary triacylglycerol absorption is a multi-step process. Triacylglycerol exit from the endoplasmic reticulum (ER) is the rate-limiting step in the progress of the lipid from its apical absorption to its basolateral membrane export. Triacylglycerol is transported from the ER to the cis Golgi in a specialized vesicle, the pre-chylomicron transport vesicle (PCTV). The vesicle-associated membrane protein 7 (VAMP7) was found to be more concentrated on PCTVs compared with ER membranes. VAMP7 has been previously identified associated with post-Golgi sites in eukaryotes. To examine the potential role of VAMP7 in PCTV trafficking, antibodies were generated that identified a 25 kDa band consistent with VAMP7 but did not crossreact with VAMP1,2. VAMP7 was concentrated on intestinal ER by immunofluorescence microscopy. Immunoelectron microscopy showed that the ER proteins Sar1 and rBet1 were present on PCTVs and colocalized with VAMP7. Iodixanol gradient centrifugation showed VAMP7 to be isodense with ER and endosomes. Although VAMP7 localized to intestinal ER, it was not present in the ER of liver and kidney. Anti-VAMP7 antibodies reduced the transfer of triacylglycerol, but not newly synthesized proteins, from the ER to the Golgi by 85%. We conclude that VAMP7 is enriched in intestinal ER and that it plays a functional role in the delivery of triacylglycerol from the ER to the Golgi.  

3.730           Conditioned medium from enterohemorrhagic Escherichia coli-infected T84 cells inhibits signal transducer and activator of transcription 1 activation by gamma interferon

Jandu, N. Et al Infect. Immun., 74(3), 1809-1818 (2006)   Gamma interferon (IFN- ) is a cytokine important to host defense which can signal through signal transducer and activator of transcription 1 (Stat1). Enterohemorrhagic Escherichia coli (EHEC) modulates host cell signal transduction to establish infection, and EHEC serotypes O113:H21 and O157:H7 both inhibit IFN- -induced Stat1 tyrosine phosphorylation in vitro. The aim of this study was to delineate both bacterial and host cell factors involved in the inhibition of Stat1 tyrosine phosphorylation. Human T84 colonic epithelial cells were challenged with direct infection, viable EHEC separated from T84 cells by a filter, sodium orthovanadate, isolated flagellin, bacterial culture supernatants, and conditioned medium treated with proteinase K, trypsin, or heat inactivation. Epithelial cells were then stimulated with IFN- and protein extracts were analyzed by immunoblotting. The data showed that IFN- -inducible Stat1 tyrosine phosphorylation was inhibited when EHEC adhered to T84 cells, but not by bacterial culture supernatants or bacteria separated from the epithelial monolayer. Conditioned medium from T84 cells infected with EHEC O157:H7 suppressed Stat1 activation, and this was not reversed by treatment with proteinases or heat inactivation. Use of pharmacological inhibitors showed that time-dependent bacterial, but not epithelial, protein synthesis was involved. Stat1 inhibition was also independent of bacterial flagellin, host proteasome activity, and protein tyrosine phosphatases. Infection led to altered IFN- receptor domain 1 subcellular distribution and decreased expression in cholesterol-enriched membrane microdomains. Thus, suppression of host cell IFN- signaling by production of a contact-dependent, soluble EHEC factor may represent a novel mechanism for this pathogen to evade the host immune system.    

3.731           The association of Shiga-like toxin with detergent-resistant membranes is modulated by glucosylceramide and is an essential requirement in the endoplasmic reticulum for a cytotoxic effect

Smith, D.C. et al Mol. Biol. Cell, 17, 1375-1387 (2006)   Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the productive intoxication of susceptible mammalian cells by Shiga-like toxin-1 (SLTx). Recently, it has been proposed that the observed association of certain ER-directed toxins and viruses with detergent-resistant membranes (DRM) may provide a general mechanism for their retrograde transport to endoplasmic reticulum (ER). Here, we show that DRM recruitment of SLTx bound to its globotriosylceramide (Gb₃) receptor is mediated by the availability of other glycosphingolipids. Reduction in glucosylceramide (GlcCer) levels led to complete protection against SLTx and a reduced cell surface association of bound toxin with DRM. This reduction still allowed efficient binding and transport of the toxin to the ER. However, toxin sequestration within DRM of the ER was abolished under reduced GlcCer conditions, suggesting that an association of toxin with lipid microdomains or rafts in the ER (where these are defined by detergent insolubility) is essential for a later step leading to or involving retro-translocation of SLTx across the ER membrane. In support of this, we show that a number of ER residents, proteins intimately involved in the process of ER dislocation of misfolded proteins, are present in DRM.  

3.732           Cytokine receptor-mediated trafficking of preformed IL-4 in eosinophils identifies an innate immune mechanism of cytokine secretion

Spencer, L.A. et al PNAS, 103(9), 3333-3338 (2006)   Although leukocytes of the innate immune system, including eosinophils, contain within their granules preformed stores of cytokines available for selective and rapid release, little is known about the mechanisms governing the mobilization and secretion of these cytokines. Here we show that a cytokine receptor, the IL-4 receptor chain, mediates eotaxin-stimulated mobilization of preformed IL-4 from eosinophil granules into secretory vesicles. Eosinophils contain substantial intracellular quantities of several granule- and vesicle-associated cytokine receptors, including IL-4, IL-6, and IL-13 receptors as well as CCR3. Both IL-4 and IL-4 receptor chain colocalized in eosinophil granules; and after eotaxin-stimulation, IL-4 receptor chain, bearing bound IL-4, was mobilized into secretory vesicles. These findings indicate that intracellular cytokine receptors within secretory vesicles transport their cognate cytokines requisite for the secretion of cytokines preformed in innate immune leukocytes.  

3.733           Glial cell line-derived neurotrophic factor-dependent recruitment of ret into lipid rafts enhances signaling by partitioning ret from proteasome-dependent degradation

Pierchala, B.A., Milbrandt, J. and Johnson Jr., E.M.

1. Neurosci., 26(10), 2777-2787 (2006)

  The receptor tyrosine kinase (RTK) Ret is activated by the formation of a complex consisting of ligands such as glial cell line-derived neurotrophic factor (GDNF) and glycerophosphatidylinositol-anchored coreceptors termed GFR s. During activation, Ret translocates into lipid rafts, which is critical for functional responses to GDNF. We found that Ret was rapidly ubiquitinated and degraded in sympathetic neurons when activated with GDNF, but, unlike other RTKs that are trafficked to lysosomes for degradation, Ret was degraded predominantly by the proteasome. After GDNF stimulation, the majority of ubiquitinated Ret was located outside of lipid rafts and Ret was lost predominantly from nonraft membrane domains. Consistent with the predominance of Ret degradation outside of rafts, disruption of lipid rafts in neurons did not alter either the GDNF-dependent ubiquitination or degradation of Ret. GDNF-mediated survival of sympathetic neurons was inhibited by lipid raft depletion, and this inhibitory effect of raft disruption on GDNF-mediated survival was reversed if Ret degradation was blocked via proteasome inhibition. Therefore, lipid rafts sequester Ret away from the degradation machinery located in nonraft membrane domains, such as Cbl family E3 ligases, thereby sustaining Ret signaling.  

3.734           Sorting of Pmel17 to melanosomes through the plasma membrane by AP1 and AP2: evidence for the polarized nature of melanocytes

Valencia, J.C. et al

1. Cell Sci., 119, 1080-1091 (2006)

  Adaptor proteins (AP) play important roles in the sorting of proteins from the trans-Golgi network, but how they function in the sorting of various melanosome-specific proteins such as Pmel17, an essential structural component of melanosomes, in melanocytes is unknown. We characterized the processing and trafficking of Pmel17 via adaptor protein complexes within melanocytic cells. Proteomics analysis detected Pmel17, AP1 and AP2, but not AP3 or AP4 in early melanosomes. Real-time PCR, immunolabeling and tissue in-situ hybridization confirmed the coexpression of AP1 isoforms µ1A and µ1B (expressed only in polarized cells) in melanocytes and keratinocytes, but expression of µ1B is missing in some melanoma cell lines. Transfection with AP1 isoforms (µ1A or µ1B) showed two distinct distribution patterns that involved Pmel17, and only µ1B was able to restore the sorting of Pmel17 to the plasma membrane in cells lacking µ1B expression. Finally, we established that expression of µ1B is regulated physiologically in melanocytes by UV radiation or DKK1. These results show that Pmel17 is sorted to melanosomes by various intracellular routes, directly or indirectly through the plasma membrane, and the presence of basolateral elements in melanocytes suggests their polarized nature.  

3.735           Multifunctional analysis of Chlamydia-specific genes in a yeast expression system

Sisko, J.L., Spaeth, K., Kumar, Y. And Valdivia, R.H. Mol. Microbiol., 60(1), 51-66 (2006)   Our understanding of how obligate intracellular pathogens co-opt eukaryotic cellular functions has been limited by their intractability to genetic manipulation and by the abundance of pathogen-specific genes with no known functional homologues. In this report we describe a gene expression system to characterize proteins of unknown function from the obligate intracellular bacterial pathogen Chlamydia trachomatis. We have devised a homologous recombination-based cloning strategy to construct an ordered array of Saccharomyces cerevisiae strains expressing all Chlamydia-specific genes. These strains were screened to identify chlamydial proteins that impaired various yeast cellular functions or that displayed tropism towards eukaryotic organelles. In addition, to identify bacterial factors that are secreted into the host cell, recombinant chlamydial proteins were screened for reactivity towards antisera raised against vacuolar membranes purified from infected mammalian cells. We report the identification of 34 C. trachomatis proteins that impact yeast cellular functions or are tropic for a range of eukaryotic organelles including mitochondria, nucleus and cytoplasmic lipid droplets, and a new family of Chlamydia-specific proteins that are exported from the parasitopherous vacuole. The versatility of molecular manipulations and protein expression in yeast allows for the rapid construction of comprehensive protein expression arrays to explore the function of pathogen-specific gene products from microorganisms that are difficult to genetically manipulate, grow in culture or too dangerous for routine analysis in the laboratory.  

3.736           Rescue of cell growth by sphingosine with disruption of lipid microdomain formation in Saccharomyces cerevisiae deficient in sphingolipid biosynthesis

Tani, M., Khara, A. and Igarashi, Y. Biochem. J., 394, 237-242 (2006)   In the yeast Saccharomyces cerevisiae, sphingolipids are essential for cell growth. Inactivation of sphingolipid biosynthesis, such as by disrupting the serine palmitoyltransferase gene (LCB2), is lethal, but cells can be rescued by supplying an exogenous LCB (long-chain base) like PHS (phytosphingosine) or DHS (dihydrosphingosine). In the present study, supplying SPH (sphingosine), an unnatural LCB for yeast, similarly rescued the Dlcb2 cells, but only when SPH 1-phosphate production was inhibited by deleting the LCB kinase gene LCB4. Exogenously added SPH was adequately converted into phosphoinositol-containing complex sphingolipids. Interestingly, cells carrying SPH-based sphingolipids exhibited a defect in the association of Pma1p with Triton X-100-insoluble membrane fractions, and displayed sensitivities to both Ca²⁺ and hygromycin B. These results suggest that the SPH-based sphingolipids in these cells have properties that differ from those of the PHS- or DHS-based sphingolipids in regard to lipid microdomain formation, leading to abnormal sensitivities towards certain environmental stresses. The present paper is the first report showing that in sphingolipid-deficient S. cerevisiae, the requirement for LCB can be fulfilled by exogenous SPH, although this supplement results in failure of lipid microdomain formation.

3.737           Localization of a portion of the liver isoform of fatty-acid-binding protein (L-FABP) to peroxisomes

Antonenkov, V.D. et al Biochem. J., 394, 475-484 (2006)   The liver isoform of fatty-acid-binding protein (L-FABP) facilitates the cellular uptake, transport and metabolism of fatty acids and is also involved in the regulation of gene expressions and cell differentiation. Consistent with these functions, L-FABP is predominantly present in the cytoplasm and to a lesser extent in the nucleus; however, a significant portion of this protein has also been detected in fractions containing different organelles. More recent observations, notably on L-FABP-deficient mice, indicated a possible direct involvement of L-FABP in the peroxisomal oxidation of long-chain fatty acids. In order to clarify the links between L-FABP and peroxisomal lipid metabolism, we reinvestigated the subcellular distribution of the protein. Analytical subcellular fractionation by a method preserving the intactness of isolated peroxisomes, two-dimensional gel electrophoresis of peroxisomal matrix proteins combined with MS analysis, and immunoelectron microscopy of liver sections demonstrate the presence of L-FABP in the matrix of peroxisomes as a soluble protein. Peroxisomal L-FABP was highly inducible by clofibrate. The induction of L-FABP was accompanied by a marked increase in the binding capacity of peroxisomal matrix proteins for oleic acid and cis-parinaric acid. The peroxisomal b-oxidation of palmitoyl-CoA and acyl-CoA thioesterase activity were stimulated by L-FABP, indicating that the protein modulates the function of peroxisomal lipid-metabolizing enzymes. The possible role of intraperoxisomal L-FABP in lipid metabolism is discussed.  

3.738           Internalized Pseudomonas exotoxin A can exploit multiple pathways to reach the endoplasmic reticulum

Smith, D.C. et al Traffic, 7(4), 379-393 (2006)   Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the intoxication of mammalian cells by Pseudomonas exotoxin A (PEx). The toxin binds the α2-macroglobulin receptor/low-density lipoprotein receptor-related protein. Here, we show that in HeLa cells, PEx recruits a proportion of this receptor to detergent-resistant microdomains (DRMs). Uptake of receptor-bound PEx involves transport steps both directly from early endosomes to the trans-Golgi network (TGN) independently of Rab9 function and from late endosomes to the TGN in a Rab9-dependent manner. Furthermore, treatments that simultaneously perturb both Arf1-dependent and Rab6-dependent retrograde pathways show that PEx can use multiple routes to reach the ER. The Rab6-dependent route has only been described previously for cargo with lipid-sorting signals. These findings suggest that partial localization of PEx within DRM permits a choice of trafficking routes consistent with a model that DRM-associated toxins reach the ER on a lipid-dependent sorting pathway whilst non-DRM-associated PEx exploits the previously characterized KDEL receptor-mediated uptake pathway. Thus, unexpectedly, an ER-directed toxin with a proteinaceous receptor shows promiscuity in its intracellular trafficking pathways, exploiting routes controlled by both lipid- and protein-sorting signals.  

3.739           Dissecting Rotavirus Particle-Raft Interaction with Small Interfering RNAs: Insights into Rotavirus Transit through the Secretory Pathway

Cuadras, M.A., Bordier, B.B., Zambrano, J.L., Ludert, J.E. and Greenberg, H.B.

1. Virol., 80(8), 3935-3946 (2006)

  Studies of rotavirus morphogenesis, transport, and release have shown that although these viruses are released from the apical surface of polarized intestinal cells before cellular lysis, they do not follow the classic exocytic pathway. Furthermore, increasing evidence suggests that lipid rafts actively participate in the exit of rotavirus from the infected cell. In this study, we silenced the expression of VP4, VP7, and NSP4 by using small interfering RNAs (siRNAs) and evaluated the effect of shutting down the expression of these proteins on rotavirus-raft interactions. Silencing of VP4 and NSP4 reduced the association of rotavirus particles with rafts; in contrast, inhibition of VP7 synthesis slightly affected the migration of virions into rafts. We found that inhibition of rotavirus migration into lipid rafts, by either siRNAs or tunicamycin, also specifically blocked the targeting of VP4 to rafts, suggesting that the association of VP4 with rafts is mostly mediated by the formation of viral particles in the endoplasmic reticulum (ER). We showed that two populations of VP4 exist, one small population that is independently targeted to rafts and a second large pool of VP4 whose association with rafts is mediated by particle formation in the ER. We also present evidence to support the hypothesis that assembly of VP4 into mature virions takes place in the late stages of transit through the ER. Finally, we analyzed the progression of rotavirus proteins in the exocytic pathway and found that VP4 and virion-assembled VP7 colocalized with ERGIC-53, suggesting that rotavirus particles transit through the intermediate compartment between the ER and the Golgi complex.  

3.740           Rab1 Defines a Novel Pathway Connecting the Pre-Golgi Intermediate Compartment with the Cell Periphery

Sannerud, R. et al Mol. Biol. Cell, 17, 1514-1526 (2006)   The function of the pre-Golgi intermediate compartment (IC) and its relationship with the endoplasmic reticulum (ER) and Golgi remain only partially understood. Here, we report striking segregation of IC domains in polarized PC12 cells that develop neurite-like processes. Differentiation involves expansion of the IC and movement of Rab1-containing tubules to the growth cones of the neurites, whereas p58- and COPI-positive IC elements, like rough ER and Golgi, remain in the cell body. Exclusion of Rab1 effectors p115 and GM130 from the neurites further indicated that the centrifugal, Rab1-mediated pathway has functions that are not directly related to ER-to-Golgi trafficking. Disassembly of COPI coats did not affect this pathway but resulted in missorting of p58 to the neurites. Live cell imaging showed that green fluorescent protein (GFP)–Rab1A-containing IC elements move bidirectionally both within the neurites and cell bodies, interconnecting different ER exit sites and the cis-Golgi region. Moreover, in nonpolarized cells GFP-Rab1A-positive tubules moved centrifugally towards the cell cortex. Hydroxymethylglutaryl-CoA reductase, the key enzyme of cholesterol biosynthesis, colocalized with slowly sedimenting, Rab1-enriched membranes when the IC subdomains were separated by velocity sedimentation. These results reveal a novel pathway directly connecting the IC with the cell periphery and suggest that this Rab1-mediated pathway is linked to the dynamics of smooth ER.  

3.741           Lysosomal trafficking and cysteine protease metabolism confer target-specific cytotoxicity by peptide-linked anti-CD30-auristatin conjugates

Kung, M.S. et al

1. Biol. Chem., 281(15), 10540-10547 (2006)

  The chimeric anti-CD30 monoclonal antibody cAC10, linked to the antimitotic agents monomethyl auristatin E (MMAE) or F (MMAF), produces potent and highly CD30-selective anti-tumor activity in vitro and in vivo. These drugs are appended via a valine-citrulline (vc) dipeptide linkage designed for high stability in serum and conditional cleavage and putative release of fully active drugs by lysosomal cathepsins. To characterize the biochemical processes leading to effective drug delivery, we examined the intracellular trafficking, internalization, and metabolism of the parent antibody and two antibody-drug conjugates, cAC10vc-MMAE and cAC10vc-MMAF, following CD30 surface antigen interaction with target cells. Both cAC10 and its conjugates bound to target cells and internalized in a similar manner. Subcellular fractionation and immunofluorescence studies demonstrated that the antibody and antibody-drug conjugates entering target cells migrated to the lysosomes. Trafficking of both species was blocked by inhibitors of clathrin-mediated endocytosis, suggesting that drug conjugation does not alter the fate of antibody-antigen complexes. Incubation of cAC10vc-MMAE or cAC10vc-MMAF with purified cathepsin B or with enriched lysosomal fractions prepared by subcellular fractionation resulted in the release of active, free drug. Cysteine protease inhibitors, but not aspartic or serine protease inhibitors, blocked antibody-drug conjugate metabolism and the ensuing cytotoxicity of target cells and yielded enhanced intracellular levels of the intact conjugates. These findings suggest that in addition to trafficking to the lysosomes, cathepsin B and perhaps other lysosomal cysteine proteases are requisite for drug release and provide a mechanistic basis for developing antibody-drug conjugates cleavable by intracellular proteases for the targeted delivery of anti-cancer therapeutics.  

3.742           Reproducibility of LC-MS-based protein identification

Berg, M., Parbel, A., Pettersen, H., Fenyo, D. and Björkesten, L.

1. Exp. Botany, 57(7), 1509-1514 (2006)

  Traditional analysis of liquid chromatography-mass spectrometry (LC-MS) data, typically performed by reviewing chromatograms and the corresponding mass spectra, is both time-consuming and difficult. Detailed data analysis is therefore often omitted in proteomics applications. When analysing multiple proteomics samples, it is usually only the final list of identified proteins that is reviewed. This may lead to unnecessarily complex or even contradictory results because the content of the list of identified proteins depends heavily on the conditions for triggering the collection of tandem mass spectra. Small changes in the signal intensity of a peptide in different LC-MS experiments can lead to the collection of a tandem mass spectrum in one experiment but not in another. Also, the quality of the tandem mass spectrometry experiments can vary, leading to successful identification in some cases but not in others. Using a novel image analysis approach, it is possible to achieve repeat analysis with a very high reproducibility by matching peptides across different LC-MS experiments using the retention time and parent mass over charge (m/z). It is also easy to confirm the final result visually. This approach has been investigated by using tryptic digests of integral membrane proteins from organelle-enriched fractions from Arabidopsis thaliana and it has been demonstrated that very highly reproducible, consistent, and reliable LC-MS data interpretation can be made.  

3.743           The nuclear microspherule protein 58 is a novel RNA-binding protein that interacts with fragile X mental retardation in polyribosomal mRNPs from neurons

Davidovic, L. et al Hum. Mol. Genet., 15(9), 1525-1538 (2006)   The fragile X syndrome, the leading cause of inherited mental retardation, is due to the inactivation of the fragile mental retardation 1 gene (FMR1) and the subsequent absence of its gene product FMRP. This RNA-binding protein is thought to control mRNA translation and its absence in fragile X cells leads to alteration in protein synthesis. In neurons, FMRP is thought to repress specific mRNAs during their transport as silent ribonucleoparticles (mRNPs) from the cell body to the distant synapses which are the sites of local synthesis of neuro-specific proteins. The mechanism by which FMRP sorts out its different mRNAs targets might be tuned by the intervention of different proteins. Using a yeast two-hybrid system, we identified MicroSpherule Protein 58 (MSP58) as a novel FMRP-cellular partner. In cell cultures, we found that MSP58 is predominantly present in the nucleus where it interacts with the nuclear isoform of FMRP. However, in neurons but not in glial cells, MSP58 is also present in the cytoplasmic compartment, as well as in neurites, where it co-localizes with FMRP. Biochemical evidence is given that MSP58 is associated with polyribosomal poly(A)+ mRNPs. We also show that MSP58, similar to FMRP, is present on polyribosomes prepared from synaptoneurosomes and that it behaves as an RNA-binding protein with a high affinity to the G-quartet structure. We propose that this novel cellular partner for FMRP escorts FMRP-containing mRNP from the nucleus and nucleolus to the somato-dendritic compartment where it might participate in neuronal translation regulation.  

3.744           Overloading of stable and exclusion of unstable human superoxide dismutase-1 variants in mitochondria of murine amyotrophic lateral sclerosis models

Bergemalm, D. et al

1. Neurosci., 26(16), 4147-4154 (2006)

  Mutants of human superoxide dismutase-1 (hSOD1) cause amyotrophic lateral sclerosis (ALS), and mitochondria are thought to be primary targets of the cytotoxic action. The high expression rates of hSOD1s in transgenic ALS models give high levels of the stable mutants G93A and D90A as well as the wild-type human enzyme, significant proportions of which lack Cu and the intrasubunit disulfide bond. The endogenous murine SOD1 (mSOD1) also lacks Cu and is disulfide reduced but is active and oxidized in mice expressing the low-level unstable mutants G85R and G127insTGGG. The possibility that the molecular alterations may cause artificial loading of the stable hSOD1s into mitochondria was explored. Approximately 10% of these hSOD1s were localized to mitochondria, reaching levels 100-fold higher than those of mSOD1 in control mice. There was no difference between brain and spinal cord and between stable mutants and the wild-type hSOD1. mSOD1 was increased fourfold in mitochondria from high-level hSOD1 mice but was normal in those with low levels, suggesting that the Cu deficiency and disulfide reduction cause mitochondrial overloading. The levels of G85R and G127insTGGG mutant hSOD1s in mitochondria were 100- and 1000-fold lower than those of stable mutants. Spinal cords from symptomatic mice contained hSOD1 aggregates covering the entire density gradient, which could contaminate isolated organelle fractions. Thus, high hSOD1 expression rates can cause artificial loading of mitochondria. Unstable low-level hSOD1s are excluded from mitochondria, indicating other primary locations of injury. Such models may be preferable for studies of ALS pathogenesis.  

3.745           Cytoplasmic tails of SialT2 and GalNacT impose their respective proximal and distal Golgi localization

Uliana, A.S., Giraudo, C.G. and Maccioni, H.J.F. Traffic, 7, 604-612 (2006)   Complex glycolipid synthesis is catalyzed by different glycosyltransferases resident of the Golgi complex. Most of them are type II membrane proteins comprising a lumenal, C-terminal domain linked to an N-terminal domain (Ntd) constituted by a short cytoplasmic tail (ct), a transmembrane, and a lumenal stem regions. They concentrate selectively in different sub-Golgi compartments, in an overlapped manner, acting in succession in the addition of sugars to acceptor glycolipids. The Ntds are sufficient to localize glycosyltransferases in the Golgi complex, but it is not clear whether they also confer selective concentration in sub-Golgi compartments. Here, we studied whether the Ntd of SialT2, localized in the proximal Golgi, and the one of GalNAcT, a trans/TGN Golgi-concentrated enzyme, concentrate reporter proteins in the corresponding sub-Golgi compartment. The sub-Golgi concentration of the Ntds fused to spectral variants of the GFP was determined in CHO-K1 cells from their behavior upon addition of brefeldin A. Fluorescence microscopy and subcellular fractionation showed that the SialT2 Ntd concentrates in a proximal sub-Golgi compartment – and that of GalNAcT in TGN elements. Exchanging the transmembrane region and the cts of SialT2 and GalNAcT indicates that information for proximal or distal Golgi concentration is associated with the cts.  

3.746           rAAV2 traffics through both the late and the recycling endosomes in a dose-dependent fashion

Ding, W., Zhang, L.N., Yeaman, C. and Engelhardt, J.F. Mol. Ther., 13(4), 671-682 (2006)   Inefficient trafficking of recombinant adeno-associated virus type-2 (rAAV2) to the nucleus is a major barrier for transduction. Using imaging and subcellular fractionation techniques, we evaluated the extent of rAAV2 movement through the late (Rab7) and recycling (Rab11) endosomes. Following rAAV2 infection of HeLa cells, immunoisolation of HA–Rab7- or HA–Rab11-tagged endosomes and intracellular colocalization of Cy3-labeled rAAV2 with EGFP–Rab7 or EGFP–Rab11 markers demonstrated dose-dependent trafficking of rAAV2 through the recycling and late endosomal compartments. At low multiplicities of infection (m.o.i. 100 genomes/cell), rAAV2 predominantly trafficked to the Rab7 compartment. In contrast, rAAV2 predominantly trafficked to the recycling endosome at 100-fold higher m.o.i. siRNA studies inhibiting either Rab7 or Rab11 demonstrated that reducing Rab11 protein levels more significantly inhibited rAAV2 transduction on a per genome basis compared to inhibition of Rab7. Dose–response curves, comparing the m.o.i. of AV2Luc infection to relative transduction, also supported the hypothesis that viral movement through the Rab11 compartment at high m.o.i. is more competent for transgene expression ( 100-fold) than virus that moves through the Rab7 compartment at low m.o.i. These findings suggest that strategies to shunt viral movement from the late to the recycling endosome may be effective at increasing viral transduction for gene therapy.  

3.747           Mapping the Arabidopsis organelle proteome

Dunkley, T.P.J. et al PNAS, 103(17), 6518-6523 (2006)   A challenging task in the study of the secretory pathway is the identification and localization of new proteins to increase our understanding of the functions of different organelles. Previous proteomic studies of the endomembrane system have been hindered by contaminating proteins, making it impossible to assign proteins to organelles. Here we have used the localization of organelle proteins by the isotope tagging technique in conjunction with isotope tags for relative and absolute quantitation and 2D liquid chromatography for the simultaneous assignment of proteins to multiple subcellular compartments. With this approach, the density gradient distributions of 689 proteins from Arabidopsis thaliana were determined, enabling confident and simultaneouslocalization of 527 proteins to the endoplasmic reticulum, Golgiapparatus, vacuolar membrane, plasma membrane, or mitochondriaand plastids. This parallel analysis of endomembrane componentshas enabled protein steady-state distributions to be determined.Consequently, genuine organelle residents have been distinguishedfrom contaminating proteins and proteins in transit throughthe secretory pathway.  

3.748           Opposite effect of caveolin-1 in the metabolism of high-density and low-density lipoproteins

Truong, T.Q., Aubin, D., Bourgeois, P., Falstrault, L. and Brisette, L. Biochim. Biophys. Acta, 1761, 24-36 (2006)   Receptors of the scavenger class B family were reported to be localized in caveolae, the cell surface microdomains rich in free cholesterol and glycosphyngolipids, which are characterized by the presence of caveolin-1. Parenchymal hepatic and hepatoma HepG2 cells express very low levels of caveolin-1. In the present study, stable transformants of HepG2 cells expressing caveolin-1 were generated to address the effect of caveolin-1 on receptor activity. Compared to normal cells, these cells show higher ¹²⁵I-bovine serum albumin (BSA) uptake and cholesterol efflux, two indicators of functional caveolae. By immunoprecipitation, cell fractionation and confocal analyses, we found that caveolin-1 is well colocalized with the cluster of differentiation-36 (CD36) and the low-density lipoprotein (LDL) receptor (LDLr) but to a lesser extent with the scavenger receptor class B type I (SR-BI) in HepG2 cells expressing caveolin-1. However, caveolin-1 expression favors the dimerization of SR-BI. Two clones of cells expressing caveolin-1 were investigated for their lipoprotein metabolism activity. Compared to normal cells, these cells show a 71–144% increase in ¹²⁵I-LDL degradation. The analysis of the cholesteryl esters (CE)-selective uptake (CE association minus protein association) revealed that the expression of caveolin-1 in HepG2 cells decreases by 59%–73% LDL-CE selective uptake and increases high-density lipoprotein (HDL)-CE selective uptake by 44%–66%. We conclude that the expression of caveolin-1 in HepG2 cells moves the balance of LDL degradation/CE selective uptake towards degradation and favors HDL-CE selective uptake. Thus, in the normal hepatic parenchymal situation where caveolin-1 is poorly expressed, LDL-CE selective uptake is the preferred pathway.  

3.749           Nephrin ectodomain engagement results in Src kinase activation, nephrin phosphorylation, Nck recruitment, and actin polymerization

Verma, R. et al

1. Clin. Invest., 116(5), 1346-1359 (2006)

  A properly established and maintained podocyte intercellular junction, or slit diaphragm, is a necessary component of the selective permeability barrier of the kidney glomerulus. The observation that mutation or deletion of the slit diaphragm transmembrane protein nephrin results in failure of podocyte foot process morphogenesis and concomitant proteinuria first suggested the hypothesis that nephrin serves as a component of a signaling complex that directly integrates podocyte junctional integrity with cytoskeletal dynamics. The observations made herein provide the first direct evidence to our knowledge for a phosphorylation-mediated signaling mechanism by which this integrative function is derived. Our data support the model that during podocyte intercellular junction formation, engagement of the nephrin ectodomain induces transient Fyn catalytic activity that results in nephrin phosphorylation on specific nephrin cytoplasmic domain tyrosine residues. We found that this nephrin phosphorylation event resulted in recruitment of the SH2–SH3 domain–containing adapter protein Nck and assembly of actin filaments in an Nck-dependent fashion. Considered in the context of the role of nephrin family proteins in other organisms and the integral relationship of actin dynamics and junction formation, these observations establish a function for nephrin in regulating actin cytoskeletal dynamics.  

3.750           The Interaction between Cytoplasmic Prion Protein and the Hydrophobic Lipid Core of Membrane Correlates with Neurotoxicity

Wang, X., Wang, F., Arterburn, L., Wollmann, R. and Ma, J.

1. Biol. Chem., 281(19), 13559-13565 (2006)

  Prion protein (PrP), normally a cell surface protein, has been detected in the cytosol of a subset of neurons. The appearance of PrP in the cytosol could result from either retro-translocation of misfolded PrP from the endoplasmic reticulum (ER) or impaired import of PrP into the ER. Transgenic mice expressing cytoplasmic PrP (cyPrP) developed neurodegeneration in cerebellar granular neurons, although no detectable pathology was observed in other brain regions. In order to understand why granular neurons in the cerebellum were most susceptible to cyPrP-induced degeneration, we investigated the subcellular localization of cyPrP. Interestingly, we found that cyPrP is membrane-bound. In transfected cells, it binds to the ER and plasma/endocytic vesicular membranes. In transgenic mice, it is associated with synaptic and microsomal membranes. Furthermore, the cerebellar neurodegeneration in transgenic mice correlates with the interaction between cyPrP and the hydrophobic lipid core of the membrane but not with either the aggregation status or the dosage of cyPrP. These results suggest that lipid membrane perturbation could be a cellular mechanism for cyPrP-induced neurotoxicity and explain the seemingly conflicting results concerning cyPrP.  

3.751           Differential expression of nuclear AT1 receptors and angiotensin II within the kidney of the male congenic mRen2.Lewis rat

Pendergrass, K.D., Averill, D.B., Ferrario, C.M., Diz, D.I. and Chappell, M.C. Am. J. Physiol. Renal Physiol., 290, F1497-F1506 (2006)   We established a new congenic model of hypertension, the mRen(2).Lewis rat and assessed the intracellular expression of angiotensin peptides and receptors in the kidney. The congenic strain was established from the backcross of the (mRen2)27 transgenic rat that expresses the mouse renin 2 gene onto the Lewis strain. The 20-wk-old male congenic rats were markedly hypertensive compared with the Lewis controls (systolic blood pressure: 195 ± 2 vs. 107 ± 2 mmHg, P < 0.01). Although plasma ANG II levels were not different between strains, circulating levels of ANG-(1–7) were 270% higher and ANG I concentrations were 40% lower in the mRen2.Lewis rats. In contrast, both cortical (CORT) and medullary (MED) ANG II concentrations were 60% higher in the mRen2.Lewis rats, whereas tissue ANG I was 66 and 84% lower in CORT and MED. For both strains, MED ANG II, ANG I, and ANG-(1–7) were significantly higher than CORT levels. Intracellular ANG II binding distinguished nuclear (NUC) and plasma membrane (PM) receptor using the ANG II radioligand ¹²⁵I-sarthran. Isolated CORT nuclei exhibited a high density (B_max >200 fmol/mg protein) and affinity for the sarthran ligand (K_D<0.5 nM); the majority of these sites (>95%) were the AT₁ receptor subtype. CORT ANG II receptor B_max and K_D values in nuclei were 75 and 50% lower, respectively, for the mRen2.Lewis vs. the Lewis rats. In the MED, the PM receptor density (Lewis: 50 ± 4 vs. mRen2.Lewis: 21 ± 5 fmol/mg protein) and affinity (Lewis: 0.31 ± 0.1 vs. 0.69 ± 0.1 nM) were lower in the mRen2.Lewis rats. In summary, the hypertensive mRen2.Lewis rats exhibit higher ANG II in both CORT and MED regions of the kidney. Evaluation of intracellular ANG II receptors revealed lower CORT NUC and MED PM AT₁ sites in the mRen2.Lewis. The downregulation of AT₁ sites in the mRen2.Lewis rats may reflect a compensatory response to dampen the elevated levels of intrarenal ANG II.  

3.752           MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion

Spinazzola, A. et al Nature Genetics, 38(5), 570-575 (2006)   The mitochondrial (mt) DNA depletion syndromes (MDDS) are genetic disorders characterized by a severe, tissue-specific decrease of mtDNA copy number, leading to organ failure. There are two main clinical presentations: myopathic (OMIM 609560) and hepatocerebral1 (OMIM 251880). Known mutant genes, including TK2 (ref. 2), SUCLA2 (ref. 3), DGUOK (ref. 4) and POLG 5, 6, account for only a fraction of MDDS cases7. We found a new locus for hepatocerebral MDDS on chromosome 2p21-23 and prioritized the genes on this locus using a new integrative genomics strategy. One of the top-scoring candidates was the human ortholog of the mouse kidney disease gene Mpv17 (ref. 8). We found disease-segregating mutations in three families with hepatocerebral MDDS and demonstrated that, contrary to the alleged peroxisomal localization of the MPV17 gene product9, MPV17 is a mitochondrial inner membrane protein, and its absence or malfunction causes oxidative phosphorylation (OXPHOS) failure and mtDNA depletion, not only in affected individuals but also in Mpv17-/- mice.  

3.753           Gas1 Is Related to the Glial Cell-derived Neurotrophic Factor Family Receptors and Regulates Ret Signaling

Cabrera, J.R. et al

1. Biol. Chem., 281(20), 14330-14339 (2006)

  The growth arrest-specific gene 1 (Gas1) protein has been proposed to function during development as an inhibitor of growth and a mediator of cell death and is also re-expressed in adult neurons during excitotoxic insult. Here we have demonstrated that the Gas1 protein shows high structural similarity to the glial cell-derived neurotrophic factor (GDNF) family receptors , which mediate GDNF responses through the receptor tyrosine kinase Ret. We found that Gas1 binds Ret in a ligand-independent manner and sequesters Ret in lipid rafts. Signaling downstream of Ret is thus modified through a mechanism that involves the adaptor protein Shc as well as ERK, eventually blocking Akt activation. Consequently, when Gas1 is induced, Ret-mediated GDNF-dependent survival effects are compromised. The growth arrest-specific gene 1 (Gas1) protein has been proposed to function during development as an inhibitor of growth and a mediator of cell death and is also re-expressed in adult neurons during excitotoxic insult. Here we have demonstrated that the Gas1 protein shows high structural similarity to the glial cell-derived neurotrophic factor (GDNF) family receptors , which mediate GDNF responses through the receptor tyrosine kinase Ret. We found that Gas1 binds Ret in a ligand-independent manner and sequesters Ret in lipid rafts. Signaling downstream of Ret is thus modified through a mechanism that involves the adaptor protein Shc as well as ERK, eventually blocking Akt activation. Consequently, when Gas1 is induced, Ret-mediated GDNF-dependent survival effects are compromised.  

3.754           Sequence Requirements for Localization of Human Cytomegalovirus Tegument Protein pp28 to the Virus Assembly Compartment and for Assembly of Infectious Virus

Seo, J-Y. and Britt, W.J.

1. Virol., 80(11), 5611-5626 (2006)

  The human cytomegalovirus UL99 open reading frame encodes a 190-amino-acid (aa) tegument protein, pp28, that is myristoylated and phosphorylated. pp28 is essential for assembly of infectious virus, and nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with a UL99 deletion. pp28 is localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in transfected cells, while in infected cells, it is localized together with other virion proteins in a juxtanuclear compartment termed the assembly compartment (AC). We investigated the sequence requirements for pp28 trafficking to the AC and assembly of infectious virus. Our studies indicated that the first 30 to 35 aa were required for localization of pp28 to the ERGIC in transfected cells. Mutant forms of pp28 containing only the first 35 aa localized with other virion structural proteins to cytoplasmic compartments early in infection, but localization to the AC at late times required a minimum of 50 aa. In agreement with previous reports, we demonstrated that the deletion of a cluster of acidic amino acids (aa 44 to 59) prevented wild-type trafficking of pp28 and recovery of infectious virus. A recombinant virus expressing only the first 50 aa was replication competent, and this mutant, pp28, localized to the AC in cells infected with this virus. These findings argued that localization of pp28 to the AC was essential for assembly of infectious virus and raised the possibility that amino acids in the amino terminus of pp28 have additional roles in the envelopment and assembly of the virion other than simply localizing pp28 to the AC.  

3.755           Detergent-free caveolae proteome suggests an interaction with ER and mitochondria

McMahon, K-A. et al Proteomics, 6, 143-152 (2006)   Recent proteomic studies of detergent resistant membrane fractions have begun to characterize the protein composition of caveolae and lipid rafts. The methods used in most of these studies, however, are not able to distinguish between plasma membrane and internal membrane lipid domains. Here we used a non-detergent method for obtaining fractions enriched in caveolae derived from the plasma membrane of multiple cell types. Unexpectedly, the proteins in the caveolae proteome suggest these lipid domains may interact with elements of ER and mitochondria. A comparison of the partial proteome we obtained with other published reports identifies 26 proteins that are candidate marker proteins for identifying caveolae in multiple cell types.  

3.756           Deletion of SERP1/RAMP4, a Component of the Endoplasmic Reticulum (ER) Translocation Sites, Leads to ER Stress

Hori, O. et al Mol. Cell. Biol., 26(11), 4257-4267 (2006)   Stress-associated endoplasmic reticulum (ER) protein 1 (SERP1), also known as ribosome-associated membrane protein 4 (RAMP4), is a Sec61-associated polypeptide that is induced by ER stress. SERP1^–/– mice, made by targeted gene disruption, demonstrated growth retardation, increased mortality, and impaired glucose tolerance. Consistent with high levels of SERP1 expression in pancreas, pancreatic islets from SERP1^–/– mice failed to rapidly synthesize proinsulin in response to a glucose load. In addition, reduced size and enhanced ER stress were observed in the anterior pituitary of SERP1^–/– mice, and growth hormone production was slowed in SERP1^–/– pituitary after insulin stimulation. Experiments using pancreatic microsomes revealed aberrant association of ribosomes and the Sec61 complex and enhanced ER stress in SERP1^–/– pancreas. In basal conditions, the Sec61 complex in SERP1^–/– microsomes was more cofractionated with ribosomes, compared with SERP1^+/+ counterparts, in high-salt conditions. In contrast, after glucose stimulation, the complex showed less cofractionation at an early phase (45 min) but more at a later phase (120 min). Although intracellular insulin/proinsulin levels were not significantly changed in both genotypes, these results suggest that subtle changes in translocation efficiency play an important role in the regulation of ER stress and rapid polypeptide synthesis.  

3.757           DHCR24-Knockout Embryonic Fibroblasts Are Susceptible to Serum Withdrawal-Induced Apoptosis Because of Dysfunction of Caveolae and Insulin-Akt-Bad Signaling

Lu, X. et al Endocrinology, 147(6), 3123-3132 (2006)   The DHCR24 gene encodes an enzyme catalyzing the last step of cholesterol biosynthesis, the conversion of desmosterol to cholesterol. To elucidate the physiological significance of cholesterol biosynthesis in mammalian cells, we investigated proliferation of mouse embryonic fibroblasts (MEFs) prepared from DHCR24^–/– mice. Both DHCR24^–/– and wild-type MEFs proliferated in the presence of serum in culture media. However, the inhibition of external cholesterol supply by serum withdrawal induced apoptosis of DHCR24^–/– MEFs, which was associated with a marked decrease in the intracellular and plasma membrane cholesterol levels, Akt inactivation, and Bad dephosphorylation. Insulin is an antiapoptotic factor capable of stimulating the Akt-Bad cascade, and its receptor (IR) is enriched in caveolae, cholesterol-rich microdomains of plasma membrane. We thus analyzed the association of IR and caveolae in the cholesterol-depleted MEFs. Subcellular fractionation and immunocytochemical analyses revealed that the IR and caveolin-1 contents were markedly reduced in the caveolae fraction of the MEFs, suggesting the disruption of caveolae, and that large amounts of IR were present apart from caveolin-1 on plasma membrane, indicating the uncoupling of IR with caveolae. Consistent with these findings, insulin-dependent phosphorylations of insulin receptor substrate-1, Akt, and Bad were impaired in the cholesterol-depleted MEFs. However, this impairment was partial because treatment of the MEFs with insulin restored Akt activation and prevented apoptosis. Cholesterol supply also prevented apoptosis. These results demonstrate that the cellular cholesterol biosynthesis is critical for the activation and maintenance of the Akt-Bad cell survival cascade in response to growth factors such as insulin.    

3.758           Membrane vesicles shed by Legionella pneumophila inhibit fusion of phagosomes with lysosomes

Fernandez-Moreira, E., Helbig, J.H. and Swanson, M.S. Infect. Immun.,74(6),  3285-3295 (2006)   When cultured in broth to the transmissive phase, Legionella pneumophila infects macrophages by inhibiting phagosome maturation, whereas replicative-phase cells are transported to the lysosomes. Here we report that the ability of L. pneumophila to inhibit phagosome-lysosome fusion correlated with developmentally regulated modifications of the pathogen's surface, as judged by its lipopolysaccharide profile and by its binding to a sialic acid-specific lectin and to the hydrocarbon hexadecane. Likewise, the composition of membrane vesicles shed by L. pneumophila was developmentally regulated, based on binding to the lectin and to the lipopolysaccharide-specific monoclonal antibody 3/1. Membrane vesicles were sufficient to inhibit phagosome-lysosome fusion by a mechanism independent of type IV secretion, since only 25% of beads suspended with or coated by vesicles from transmissive phase wild type or dotA secretion mutants colocalized with lysosomal probes, whereas 75% of beads were lysosomal when untreated or presented with vesicles from the L. pneumophila letA regulatory mutant or E. coli. As observed previously for L. pneumophila infection of mouse macrophages, vesicles inhibited phagosome-lysosome fusion only temporarily; by 10 h after treatment with vesicles, macrophages delivered 72% of ingested beads to lysosomes. Accordingly, in the context of the epidemiology of the pneumonia Legionnaires' disease and virulence mechanisms of Leishmania and Mycobacteria, we discuss a model here in which L. pneumophila developmentally regulates its surface composition and releases vesicles into phagosomes that inhibit their fusion with lysosomes.  

3.759           Mutational analysis of the Lem3p-Dnf1p putative phospholipid-translocating P-type ATPase reveals novel regulatory roles for Lem3p and a carboxyl-terminal region of Dnf1p independent of the phospholipid-translocating activity of Dnf1p in yeast

Noji, T. et al Biochem. Biophys. Res. Comm., 344, 323-331 (2006)   Lem3p-Dnf1p is a putative aminophospholipid translocase (APLT) complex that is localized to the plasma membrane; Lem3p is required for Dnf1p localization to the plasma membrane. We have identified lem3 mutations, which did not affect formation or localization of the Lem3p-Dnf1p complex, but caused a synthetic growth defect with the null mutation of CDC50, a structurally and functionally redundant homologue of LEM3. Interestingly, these lem3 mutants exhibited nearly normal levels of NBD-labeled phospholipid internalization across the plasma membrane, suggesting that Lem3p may have other functions in addition to regulation of the putative APLT activity of Dnf1p at the plasma membrane. Similarly, deletion of the COOH-terminal cytoplasmic region of Dnf1p affected neither the localization nor the APLT activity of Dnf1p at the plasma membrane, but caused a growth defect in the cdc50Δ background. Our results suggest that the Lem3p-Dnf1p complex may play a role distinct from its plasma membrane APLT activity when it substitutes for the Cdc50p-Drs2p complex, its redundant partner in the endosomal/trans-Golgi network compartments.  

3.760           The rab exchange factor Sec2p reversibly associates with the exocyst

Medkova, M., France, Y.E., Coleman, J. and Novick, P. Mol. Biol. Cell, 17(6), 2757-2769 (2006)   Activation of the rab GTPase, Sec4p, by its exchange factor, Sec2p, is needed for polarized transport of secretory vesicles to exocytic sites and for exocytosis. A small region in the C-terminal half of Sec2p regulates its localization. Loss of this region results in temperature-sensitive growth and the depolarized accumulation of secretory vesicles. Here, we show that Sec2p associates with the exocyst, an octameric effector of Sec4p involved in tethering secretory vesicles to the plasma membrane. Specifically, the exocyst subunit Sec15p directly interacts with Sec2p. This interaction normally occurs on secretory vesicles and serves to couple nucleotide exchange on Sec4p to the recruitment of the Sec4p effector. The mislocalization of Sec2p mutants correlates with dramatically enhanced binding to the exocyst complex. We propose that Sec2p is normally released from the exocyst after vesicle tethering so that it can recycle onto a new round of vesicles. The mislocalization of Sec2p mutants results from a failure to be released from Sec15p, blocking this recycling pathway.  

3.761           Caveolin-1 is required for fatty acid translocase (FAT/CD36) localization and function at the plasma membrane of mouse embryonic fibroblasts

Ring, A., Le Lay, S., pohl, J., Verkade, P. and Stremmel, W. Biochim. Biophys. Acta, 1761, 416-423 (2006)   Several lines of evidence suggest that lipid rafts are involved in cellular fatty acid uptake and influence fatty acid translocase (FAT/CD36) function. However, it remains unknown whether caveolae, a specialized raft type, are required for this mechanism. Here, we show that wild-type (WT) mouse embryonic fibroblasts (MEFs) and caveolin-1 knockout (KO) MEFs, which are devoid of caveolae, have comparable overall expression of FAT/CD36 protein but altered subcellular FAT/CD36 localization and function. In WT MEFs, FAT/CD36 was isolated with both lipid raft enriched detergent-resistant membranes (DRMs) and detergent-soluble membranes (DSMs), whereas in cav-1 KO cells it was exclusively associated with DSMs. Subcellular fractionation demonstrated that FAT/CD36 in WT MEFs was localized intracellularly and at the plasma membrane level while in cav-1 KO MEFs it was absent from the plasma membrane. This mistargeting of FAT/CD36 in cav-1 KO cells resulted in reduced fatty acid uptake compared to WT controls. Adenoviral expression of caveolin-1 in KO MEFs induced caveolae formation, redirection of FAT/CD36 to the plasma membrane and rescue of fatty acid uptake. In conclusion, our data provide evidence that caveolin-1 is necessary to target FAT/CD36 to the plasma membrane. Caveolin-1 may influence fatty acid uptake by regulating surface availability of FAT/CD36.  

3.762           Phosphorylation of phototransduction GAP RGS9-1 depends on the a isoform of protein kinase C

Wang, Q., Hu, G., Leitges, M. and Wensel, T.G. Invest. Ophthalmol. Vis. Sci., 47, E-abstract 822 (2006)   Purpose:To investigate the regulation of the photoreceptor–specific GTPase Accelerating Protein (GAP) RGS9–1 by PKC . Normal light response kinetics require both RGS9–1 and its membrane anchor R9AP, whose interactions have been previously shown to be modified by a light–inhibited phosphorylation on Ser⁴⁷⁵ of RGS9–1 catalyzed by one or more isozymes of proteinkinase C (PKC). Methods:PKC –/– and wild–type mice were compared. The presence of PKC in mouse rod outer segments was examined by western blot of purified rod outer segments. The localization of PKC to rod outer segments was confirmed by analysis of fractions collected from two successive sucrose gradients and iso–osmotic OptiPrep gradient purification. The amount of PKC in purified intact rod outer segments was determined by quantitative western blot of rod outer segments and retina lysates. In vitro phosphorylation of RGS9–1 in rod outer segments by endogenous PKC was detected by kinase assays. In vivo light–regulated phosphorylation of RGS9–1 at Ser⁴⁷⁵ was assessed by immunoprecipitation of RGS9–1 from mice retinas and followed by western blot using Ser⁴⁷⁵–phosphate–specific antibody. Results:PKC immuno–reactivity is present in wild–type mouse rod outer segments but missing in those of PKC –/– mice. It consistently co–purifies with rod outer segments. Approximately 20% of the total retinal PKC pool is in mouse rod outer segments. PKC knockouts do not exhibit the light–sensitive phosphorylation of RGS9–1 on PKC target site Ser⁴⁷⁵ that is observed in wild–type mice and show slower RGS9–1 Ser⁴⁷⁵ phosphorylation in vitro. Conclusion:PKC immuno–reactivity in photoreceptor cells is genuine and not due to cross–activity and contamination. PKC is essential for light–sensitive RGS9–1 Ser⁴⁷⁵ phosphorylation. These results indicate that rod outer segments contain modest levels of PKC , and this enzyme is responsible for regulation of the R9AP–RGS9–1 membrane complex by Ser⁴⁷⁵ phosphorylation, thus likely regulating the rate–limitingreaction in vision.  

3.763           Regulation of RPE phagocytosis by integrin receptor-tetraspanin surface membrane domains

Finnemann, S.C. and Chang, Y. Invest. Ophthalmol. Vis. Sci., 47, e-abstract 5879 (2006)   Purpose: Retinal pigment epithelial (RPE) cells depend on vß5 integrin receptors and their downstream signaling pathways to maximize their phagocytic activity following circadian photoreceptor outer segment (POS) shedding in the retina. We hypothesize that the activity of vß5 integrin itself may be regulated in the RPE to synchronize phagocytosis. Integrin receptors may associate with proteins of the tetraspanin family in specialized membrane microdomains to control integrin signaling. Here, we test whether tetraspanins functionally interact with vß5 integrin in RPE cells. Methods: RT–PCRs and immunoblotting established tetraspanin expression in RPE cells in vitro and in vivo. We used confocal microscopy following live cell labeling with receptor antibodies and choleratoxin B to co–localize vß5 integrin and tetraspanins to plasma membrane sub–domains at the apical surface of the RPE. We used optiprep gradient fractionation of RPE cell lysates and co–immunoprecipitation assays to further test subcellular co–fractionation of tetraspanins and vß5 integrin. We used isolated POS fragments in quantitative uptake assays to determine effects of surface cholesterol depletion, tetraspanin antibody blocking and tetraspanin overexpression on the phagocytic function of RPE cells in culture. Results: We found that tetraspanins CD9, CD63 and CD81 localize to the apical, phagocytic surface of RPE cells in culture. CD81 partially co–localized with vß5 integrin. Furthermore, we identified CD81 in a complex with vß5 integrin in RPE cells in culture and in intact retina. CD81 co–fractionated with vß5 in low–density membrane domains of RPE cell lysates. Cholesterol depletion, which altered CD81 and vß5 surface distribution, as well as specific inhibition of CD81 reduced POS phagocytosis by human and rat RPE cell cultures. In contrast, CD81 overexpression increased POS phagocytosis. Conclusions: Our results demonstrate that the apical plasma membrane tetraspanin CD81 regulates the phagocytic activity of the RPE. Functional interaction with tetraspanins including CD81 and microdomain recruitment may contribute to the temporal control of vß5 integrin receptor signaling in the RPE that is necessary for rhythmic phagocytosis of shed POS. Ongoing experiments study CD81 and integrin vß5 complex formation and distribution during active RPE phagocytosis in vitro and in vivo.  

3.764           Lipid raft-based membrane compartmentation of a plant transport protein expressed in Saccharomyces cerevisiae

Grossmann, G., Opekarova, M., Novakova, L., Stolz, J. and Tanner, W. Eukaryot. Cell, 5(6), 945-953 (2006)   The hexose-proton symporter HUP1 shows a spotty distribution in the plasma membrane of the green alga Chlorella kessleri. Chlorella cannot be transformed so far. To study the membrane localization of the HUP1 protein in detail, the symporter was fused to green fluorescent protein (GFP) and heterologously expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In these organisms, the HUP1 protein has previously been shown to be fully active. The GFP fusion protein was exclusively targeted to the plasma membranes of both types of fungal cells. In S. cerevisiae, it was distributed nonhomogenously and concentrated in spots resembling the patchy appearance observed previously for endogenous H⁺ symporters. It is documented that the Chlorella protein colocalizes with yeast proteins that are concentrated in 300-nm raft-based membrane compartments. On the other hand, it is completely excluded from the raft compartment housing the yeast H⁺/ATPase. As judged by their solubilities in Triton X-100, the HUP1 protein extracted from Chlorella and the GFP fusion protein extracted from S. cerevisiae are detergent-resistant raft proteins. S. cerevisiae mutants lacking the typical raft lipids ergosterol and sphingolipids showed a homogenous distribution of HUP1-GFP within the plasma membrane. In an ergosterol synthesis (erg6) mutant, the rate of glucose uptake was reduced to less than one-third that of corresponding wild-type cells. In S. pombe, the sterol-rich plasma membrane domains can be stained in vivo with filipin. Chlorella HUP1-GFP accumulated exactly in these domains. Altogether, it is demonstrated here that a plant membrane protein has the property of being concentrated in specific raft-based membrane compartments and that the information for its raft association is retained between even distantly related organisms.  

3.765           Assembly of infectious HIV-1 in human epithelial and T-lymphoblastic cell lines

Grigorov, B., Arcanger, F., Roingeard, P., Darlix, J-L. and Muriaux, D.

1. Mol. Biol., 359, 848-862 (2006)

  The canonical view of the ultimate steps of HIV-1 replication is that virus assembly and budding are taking place at the plasma membrane of infected cells. Surprisingly, recent studies revealed that these steps also occur on endosomal membranes in the interior of infected cells, such as macrophages. This prompted us to revisit the site of HIV-1 assembly in human epithelial-like cells and in infected human T-lymphoblastic cells. To address this question, we investigated the intracellular location of the major viral structural components of HIV-1, namely Gag, Env and the genomic RNA. Using a sub-cellular fractionation method, as well as immuno-confocal and electron microscopy, we show that Gag, the Env glycoproteins and the genomic RNA accumulate in late endosomes that contain infectious HIV-1 particles. In epithelial-like 293T cells, HIV-1 assembles and buds both at the plasma membrane and in endosomes, while in chronically infected human T lymphocytes, viral assembly mostly occurs within the cell where large amounts of infectious virions accumulate in endosomal compartments. In addition, HIV-1 release could be enhanced by ionomycin, a drug stimulating calcium-dependent exocytosis. These results favour the view that newly made Gag molecules associate with the genomic RNA in the cytosol, then viral core complexes can be targeted to late endosomes together with Env, where infectious HIV-1 are made and subsequently released by exocytosis.  

3.766           Thyroid hormone receptor isoforms localize to cardiac mitochondrial matrix with potential for binding to receptor elements on mtDNA

Morrish, F. et al Mitochondrion, 6(3), 143-148 (2006)   Thyroid hormone (T₃) rapidly promotes both nuclear and mitochondrial DNA transcription in cardiomyocytes, suggesting that T3 directly binds and activates mitochondrial genes. We showed for the first time mitochondrial localization for multiple TRα isoforms in heart, including truncated versions. Additionally, we demonstrated novel mitochondrial localization for versions of TRα₂, the dominant negative isoform lacking a functional ligand-binding domain. We also confirmed by electromobility shift assays, that TRα₂ in mitochondrial extracts binds to thyroid receptor response elements present in the 12S rRNA (DRO) and D-loop region (DR2) of mitochondrial DNA. Thus, TRα isoforms may directly regulate T₃ responses at mtDNA in the heart.  

3.767           Intracellular ATP-sensitive K⁺ channels in mouse pancreata beta cells: against a role in organelle cation homeostasis

Varadi, A. et al Diabetologia, 49, 1567-1577 (2006)   Aims/hypothesis  ATP-sensitive K⁺ (K_ATP) channels located on the beta cell plasma membrane play a critical role in regulating insulin secretion and are targets for the sulfonylurea class of antihyperglycaemic drugs. Recent reports suggest that these channels may also reside on insulin-containing dense-core vesicles and mitochondria. The aim of this study was to explore these possibilities and to test the hypothesis that vesicle-resident channels play a role in the control of organellar Ca²⁺ concentration or pH. Methods  To quantify the subcellular distribution of the pore-forming subunit Kir6.2 and the sulfonylurea binding subunit SUR1 in isolated mouse islets and clonal pancreatic MIN6 beta cells, we used four complementary techniques: immunoelectron microscopy, density gradient fractionation, vesicle immunopurification and fluorescence-activated vesicle isolation. Intravesicular and mitochondrial concentrations of free Ca²⁺ were measured in intact or digitonin-permeabilised MIN6 cells using recombinant, targeted aequorins, and intravesicular pH was measured with the recombinant fluorescent probe pHluorin. Results  SUR1 and Kir6.2 immunoreactivity were concentrated on dense-core vesicles and on vesicles plus the endoplasmic reticulum/Golgi network, respectively, in both islets and MIN6 cells. Reactivity to neither subunit was detected on mitochondria. Glibenclamide, tolbutamide and diazoxide all failed to affect Ca²⁺ uptake into mitochondria, and K_ATP channel regulators had no significant effect on intravesicular free Ca²⁺ concentrations or vesicular pH. Conclusions/Interpretation  A significant proportion of Kir6.2 and SUR1 subunits reside on insulin-secretory vesicles and the distal secretory pathway in mouse beta cells but do not influence intravesicular ion homeostasis. We propose that dense-core vesicles may serve instead as sorting stations for the delivery of channels to the plasma membrane.  

3.768           Cholesterol depletion facilitates ubiquitylation of NPC1 and its association with SKD1/Vps4

Ohsaki, Y. et al

1. Cell Sci., 119, 2643-2653 (2006)

  Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2. NPC1 is a polytopic glycoprotein that contains a sterol-sensing domain, whereas NPC2 is a soluble protein that contains an MD-2-like lipid-recognition domain. In the current study, we addressed the hypothesis that ubiquitylation of NPC1 might be regulated by cholesterol. We found that depletion of cellular cholesterol facilitated ubiquitylation of NPC1 expressed in COS cells. A loss-of-function mutant, NPC1(P691S), which contains an amino acid substitution in the sterol-sensing domain, failed to respond to cholesterol depletion. Another mutant, NPC1( LLNF), which lacks the endosomal-targeting motif, also failed to respond. SKD1(E235Q), a dominant-negative mutant of SKD1/Vps4 that inhibits disassembly of the endosomal sorting complex required for transport (ESCRT), caused an accumulation of ubiquitylated NPC1. SKD1(E235Q) associated with NPC1 on the endosomal membrane, whereas wild-type SKD1 associated with NPC1 only when cells were depleted of cholesterol. Similarly, in control human skin fibroblasts, cholesterol depletion facilitated ubiquitylation of endogenous NPC1. In patient cells that lack NPC2 function, NPC1 was ubiquitylated regardless of cellular cholesterol levels, suggesting that NPC2 is required to prevent NPC1 ubiquitylation under cholesterol-rich conditions. These results suggest that ubiquitylation of NPC1 and its association with the ESCRT complex are controlled by endosomal cholesterol levels utilizing a mechanism that involves NPC2.  

3.769           A rat model of human FENIB (familial encephalopathy with neuroserpin inclusion bodies)

Takano, K. et al Biochim. Biophys. Res. Comm, 346(3), 1040-1047 (2006)   FENIB (familial encephalopathy with neuroserpin inclusion bodies) is caused by intracellular accumulation/polymerization of mutant neuroserpins in the endoplasmic reticulum (ER). Transgenic rats overexpressing megsin (Tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic–acid Schiff (PAS)-positive inclusions distributed throughout deeper layers of cerebral cortex, CA1 of the hippocampus, and substantia nigra. Hippocampal extracts from Tg meg rats showed increased expression of ER stress proteins, and activation of caspases-12 and -3, associated with decreased neuronal density. Enhanced ER stress was also observed in dopaminergic neurons in the substantia nigra, in parallel with decreased neuronal viability and motor coordination. In each case, PAS-positive inclusions were also positive for megsin. These data suggest that overexpression of megsin results in ER stress, eventuating in the formation of PAS-positive inclusions. Tg meg rats provide a novel model of FENIB, where accumulation of serpins in the ER induces selective dysfunction/loss of specific neuronal populations.  

3.770           Retrovirus infection strongly enhances scrapie infectivity release in cell culture

Leblanc, P. et al EMBO J., 25, 2674-2685 (2006)   Prion diseases are neurodegenerative disorders associated in most cases with the accumulation in the central nervous system of PrP^Sc (conformationally altered isoform of cellular prion protein (PrP^C); Sc for scrapie), a partially protease-resistant isoform of the PrP^C. PrP^Sc is thought to be the causative agent of transmissible spongiform encephalopathies. The mechanisms involved in the intercellular transfer of PrP^Sc are still enigmatic. Recently, small cellular vesicles of endosomal origin called exosomes have been proposed to contribute to the spread of prions in cell culture models. Retroviruses such as murine leukemia virus (MuLV) or human immunodeficiency virus type 1 (HIV-1) have been shown to assemble and bud into detergent-resistant microdomains and into intracellular compartments such as late endosomes/multivesicular bodies. Here we report that moloney murine leukemia virus (MoMuLV) infection strongly enhances the release of scrapie infectivity in the supernatant of coinfected cells. Under these conditions, we found that PrP^C, PrP^Sc and scrapie infectivity are recruited by both MuLV virions and exosomes. We propose that retroviruses can be important cofactors involved in the spread of the pathological prion agent.    

3.771           Impairment of mitochondrial anti-oxidant defence in SOD1-related motor neuron injury and amelioration by ebselen

Wood-Allum, C.A. et al Brain, 129, 1693-1709 (2006)   There is now compelling evidence of mitochondrial dysfunction in motor neuron disease (MND), but the molecular basis of these abnormalities is unknown. It is also unclear whether the observed mitochondrial dysfunction plays a central role in disease pathogenesis, and if so, whether its amelioration might present therapeutic opportunities. We adopted a candidate generation approach using proteomics to screen for changes in mitochondrial protein expression in a well-validated cell-culture model of superoxide dismutase 1 (SOD1) related familial MND (fMND). Changed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectroscopy. Protein candidates included apoptotic regulators, anti-oxidants and components of the electron transport chain. Confirmatory Western blotting was performed, and validated protein expression changes were further investigated. Peroxiredoxin 3 (Prx3), a mitochondrial thioredoxin-dependent hydroperoxidase, is downregulated in the presence of mutant SOD1 in both our cell-culture model and in the spinal cord mitochondria of mutant SOD1 transgenic mice. We confirm the expression of Prx3 within the mitochondria of spinal motor neurons in mouse and humans by immunohistochemistry. Using quantitative real-time PCR (Q-PCR), we show that Prx3 is also downregulated in spinal motor neurons from patients with both sporadic (sMND) and SOD1-related fMND. In a disease characterized by oxidative stress, this represents a potentially important deficit in mitochondrial anti-oxidant defence. Recent evidence suggests that oxidative stress from aberrant copper chemistry may not play a major part in the pathogenesis of SOD1-related fMND. From the results of this study we propose disruption of mitochondrial anti-oxidant defence as an alternative mechanism whereby mutant SOD1 may generate oxidative stress within motor neurons. We further demonstrate that ebselen, an anti-oxidant drug already safely used in human studies and that acts as a Prx mimic, is able to ameliorate the toxicity of mutant SOD1 in our cell-culture model. We conclude by showing that ebselen is capable of inducing transcription of the anti-oxidant response element (ARE) and postulate that ebselen may act both by the transcriptional upregulation of anti-oxidant proteins, and directly as an anti-oxidant in its own right.  

3.772           Melanosomal sequestration of cytotoxic drugs contributes to the intractability of malignant melanomas

Chen, K.G. et al PNAS, 103(26), 9903-9907 (2006)   Multidrug resistance mechanisms underlying the intractability of malignant melanomas remain largely unknown. In this study, we demonstrate that the development of multidrug resistance in melanomas involves subcellular sequestration of intracellular cytotoxic drugs such as cis-diaminedichloroplatinum II (cisplatin; CDDP). CDDP is initially sequestered in subcellular organelles such as melanosomes, which significantly reduces its nuclear localization when compared with nonmelanoma/KB-3-1 epidermoid carcinoma cells. The melanosomal accumulation of CDDP remarkably modulates melanogenesis through a pronounced increase in tyrosinase activity. The altered melanogenesis manifested an 8-fold increase in both intracellular pigmentation and extracellular transport of melanosomes containing CDDP. Thus, our experiments provide evidence that melanosomes contribute to the refractory properties of melanoma cells by sequestering cytotoxic drugs and increasing melanosome-mediated drug export. Preventing melanosomal sequestration of cytotoxic drugs by inhibiting the functions of melanosomes may have great potential as an approach to improving the chemosensitivity of melanoma cells.  

3.773           A Novel Mechanism of Interaction between α-Synuclein and Biological Membranes

Kim, Y.S., Laurine, E., Woods, W. and Lee, S-J.

1. Mol. Biol., 360(2), 386-397 (2006)

  Conformational abnormalities and aggregation of α-synuclein (α-syn) have been linked to the pathogenesis of Parkinson’s (PD) and related diseases. It has been shown that α-syn can stably bind artificial phospholipid vesicles through α-helix formation in its N-terminal repeat region. However, little is known about the membrane interaction in cells. In the current study, we determined the membrane-binding properties of α-syn to biological membranes by using bi-functional chemical crosslinkers, which allow the detection of transient, but specific, interactions. By utilizing various point mutations and deletions within α-syn, we demonstrated that the membrane interaction of α-syn in cells is also mediated by α-helix formation in the N-terminal repeat region. Moreover, the PD-linked A30P mutation causes reduced membrane binding, which is concordant with the artificial membrane studies. However, contrary to the interaction with artificial membranes, the interaction with biological membranes is rapidly reversible and is not driven by electrostatic attraction. Furthermore, the interaction of α-syn with cellular membranes occurs only in the presence of non-protein and non-lipid cytosolic components, which distinguishes it from the spontaneity of the interaction with artificial membranes. More interestingly, addition of the cytosolic preparation to artificial membranes resulted in the transient, charge-independent binding of α-syn similar to the interaction with biological membranes. These results suggest that in cells, α-syn is engaged in a fundamentally different mode of membrane interaction than the charge-dependent artificial membrane binding, and the mode of interaction is determined by the intrinsic properties of α-syn itself and by the cytoplasmic context.  

3.774           Identification of Proteomic Signatures of Exposure to Marine Pollutants in Mussels (Mytilus edulis)

Apraiz, I., Mi, J. and Cristobal, S. Mol. Cell. Proteomics, 5(7), 1274-1285 (2006)   Bivalves and especially mussels are very good indicators of marine and estuarine pollution, and so they have been widely used in biomonitoring programs all around the world. However, traditional single parameter biomarkers face the problem of high sensitivity to biotic and abiotic factors. In our study, digestive gland peroxisome-enriched fractions of Mytilus edulis (L., 1758) were analyzed by DIGE and MS. We identified several proteomic signatures associated with the exposure to several marine pollutants (diallyl phthalate, PBDE-47, and bisphenol-A). Animals collected from North Atlantic Sea were exposed to the contaminants independently under controlled laboratory conditions. One hundred and eleven spots showed a significant increase or decrease in protein abundance in the two-dimensional electrophoresis maps from the groups exposed to pollutants. We obtained a unique protein expression signature of exposure to each of those chemical compounds. Moreover a set of proteins composed a proteomic signature in common to the three independent exposures. It is remarkable that the principal component analysis of these spots showed a discernible separation between groups, and so did the hierarchical clustering into four classes. The 14 proteins identified by MS participate in - and ß-oxidation pathways, xenobiotic and amino acid metabolism, cell signaling, oxyradical metabolism, peroxisomal assembly, respiration, and the cytoskeleton. Our results suggest that proteomic signatures could become a valuable tool to monitor the presence of pollutants in field experiments where a mixture of pollutants is often present. Further studies on the identified proteins could provide crucial information to understand possible mechanisms of toxicity of single xenobiotics or mixtures of them in marine ecosystems.  

3.775           Outer Membrane Vesicle Production by Escherichia coli Is Independent of Membrane Instability

McBroom, A.J., Johnson, A.P., Vemulapalli, S. and Kuehn, M.J.

1. Bacteriol., 188(15), 5385-5392 (2006)

  It has been long noted that gram-negative bacteria produce outer membrane vesicles, and recent data demonstrate that vesicles released by pathogenic strains can transmit virulence factors to host cells. However, the mechanism of vesicle release has remained undetermined. This genetic study addresses whether these structures are merely a result of membrane instability or are formed by a more directed process. To elucidate the regulatory mechanisms and physiological basis of vesiculation, we conducted a screen in Escherichia coli to identify gene disruptions that caused vesicle over- or underproduction. Only a few low-vesiculation mutants and no null mutants were recovered, suggesting that vesiculation may be a fundamental characteristic of gram-negative bacterial growth. Gene disruptions were identified that caused differences in vesicle production ranging from a 5-fold decrease to a 200-fold increase relative to wild-type levels. These disruptions included loci governing outer membrane components and peptidoglycan synthesis as well as the ^E cell envelope stress response. Mutations causing vesicle overproduction did not result in upregulation of the ompC gene encoding a major outer membrane protein. Detergent sensitivity, leakiness, and growth characteristics of the novel vesiculation mutant strains did not correlate with vesiculation levels, demonstrating that vesicle production is not predictive of envelope instability.  

3.776           Ribosomal Protein S6 Associates with Alphavirus Nonstructural Protein 2 and Mediates Expression from Alphavirus Messages

Montgomery, S.A., Berglund, P., Beard, C.W. and Johnston, R.E.

1. Virol., 80(15), 7729-7739 (2006)

  Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.  

3.777           Cytotoxic Necrotizing Factor Type 1 Delivered by Outer Membrane Vesicles of Uropathogenic Escherichia coli Attenuates Polymorphonuclear Leukocyte Antimicrobial Activity and Chemotaxis

Davis, J.M., Carvalho, H.M., Rasmussen, S.B. and O’Brien, A.D. Infect. Immun., 74(8), 4401-4408 (2006)   Cytotoxic necrotizing factor type 1 (CNF1), a toxin produced by many strains of uropathogenic Escherichia coli (UPEC), constitutively activates small GTPases of the Rho family by deamidating a single amino acid within these target proteins. Such activated GTPases not only stimulate actin polymerization within affected cells but also, as we previously reported, decrease membrane fluidity on mouse polymorphonuclear leukocytes (PMNs). In that same investigation we found that this diminished membrane movement impedes the clustering of the complement receptor CD11b/CD18 on PMNs and, in turn, decreases PMN phagocytic capacity and microbicidal activity on PMNs in direct contact with CNF1-expressing UPEC as well as on those in proximity to wild-type UPEC. The latter observation suggested to us that CNF1 is released from neighboring bacteria, although at the time of initiation of the study described here, no specific mechanism for export of CNF1 from UPEC had been described. Here we present evidence that CNF1 is released from the CNF1-expressing UPEC strain CP9 (serotype O4/H5/K54) in a complex with outer membrane vesicles (OMVs) and that these CNF1-bearing vesicles transfer biologically active CNF1 to PMNs and attenuate phagocyte function. Furthermore, we show that CNF1-bearing vesicles act in a dose-dependent fashion on PMNs to inhibit their chemotactic response to formyl-Met-Leu-Phe, while purified CNF1 does not. We conclude that OMVs provide a means for delivery of CNF1 from a UPEC strain to PMNs and thus negatively affect the efficacy of the acute inflammatory response to these organisms.  

3.778           The Secretory Granule Protein Syncollin Localizes to HL-60 Cells and Neutrophils

Bach, J-P. et al

1. Histochem. Cytochem., 54(8), 877-888 (2006)

  The secretory granule protein syncollin was first identified in the exocrine pancreas where a population of the protein is associated with the luminal surface of the zymogen granule membrane. In this study we provide first morphological and biochemical evidence that, in addition to its pancreatic localization, syncollin is also present in neutrophilic granulocytes of rat and human origin. By immunohistological studies, syncollin was detected in neutrophilic granulocytes of the spleen. Furthermore, syncollin is expressed by the promyelocytic HL-60 cells, where it is stored in azurophilic granules and in a vesicular compartment. These findings were confirmed by fractionation experiments and immunoelectron microscopy. Treatment with a phorbol ester triggered the release of syncollin indicating that in HL-60 cells it is a secretory protein that can be mobilized upon stimulation. A putative role for syncollin in host defense is discussed.  

3.779           Cardiac aquaporin expression in humans, rats, and mice

Butler, T.L. et al Am. J. Physiol. Heart Circ. Physiol., 291, H705-H713 (2006)   Water accumulation in the heart is important in ischemia-reperfusion injury and operations performed by using cardiopulmonary bypass, with cardiac dysfunction associated with myocardial edema being the principal determinant of clinical outcome. As an initial step in determining the role of aquaporin (AQP) water channels in myocardial edema, we have assessed the myocardial expression of AQPs in humans, rats, and mice. RT-PCR revealed expression of AQP-1, -4, -6, -7, -8, and -11 transcripts in the mouse heart. AQP-1, -6, -7, and -11 mRNAs were found in the rat heart as well as low levels of AQP-4 and -9. Human hearts contained AQP-1, -3, -4, -5, -7, -9, -10, and -11 mRNAs. AQP-1 protein expression was confirmed by Western blot analysis in all three species. AQP-4 protein was detected in the mouse heart but not in the rat or human heart. To determine the potential functional consequences of myocardial AQP expression, water permeability was measured in plasma membrane vesicles from myocardial cells of wild-type versus various AQP knockout mice. Water permeability was reduced by AQP-1 knockout but not by AQP-4 or AQP-8 knockout. With the use of a model of isolated rat heart perfusion, it was found that osmotic and ischemic stresses are not associated with changes in AQP-1 or AQP-4 expression. These studies support a possible functional role of AQP-1 in myocardium but indicate that early adaptations to osmotic and ischemic stress do not involve transcriptional or posttranslational AQP-1 regulation.  

3.780           More than colocalizing with polycystin-1, polycystin-L is in the centrosome

Bui-Xuan, E-F. et al Am. J. Physiol. Renal Physiol., 291, F395-F406 (2006)   Polycystin-1 and polycystin-2 are involved in autosomal dominant polycystic kidney disease by unknown mechanisms. These two proteins are located in primary cilia where they mediate mechanosensation, suggesting a link between cilia function and renal disease. In this study, we sought to characterize the subcellular localization of polycystin-L, a closely related member of polycystin-2, in epithelial renal cell lines. We have shown that endogenous polycystin-L subcellular distribution is different in proliferative and nonproliferative cultures. Polycystin-L is found mostly in the endoplasmic reticulum in subconfluent cell cultures, while in confluent cells it is redistributed to sites of cell-cell contact and to the primary cilium as is polycystin-1. Subcellular fractionation confirmed a common distribution of polycystin-L and polycystin-1 in the fractions corresponding to those containing the plasma membrane of postconfluent cells. Reciprocal coimmunoprecipitation experiments showed that polycystin-L was associated with polycystin-1 in a common complex in both subconfluent and confluent cell cultures. Interestingly, we also identified a novel site for a polycystin member (polycystin-L) in unciliated cells, the centrosome, which allowed us to reveal an involvement of polycystin-L in cell proliferation.  

3.781           Association of Yeast Transporters with Detergent-Resistant Membranes Correlates with Their Cell-Surface Location

Lauwers, E. and Andre, B. Traffic, 7(8), 1045-1059 (2006)   Detergent-resistant membrane (DRM) fractions enriched in ergosterol and sphingolipids can be isolated from yeast cells and have been proposed to represent the biochemical equivalents of lipid rafts. Most yeast plasma membrane proteins studied for their detergent solubility have been found in DRMs, except for the Hxt1 and Gap1 permeases. We here compared Gap1 detergent solubility in wild-type and various mutant cells under conditions promoting cell surface accumulation or ubiquitin-dependent down-regulation of the permease. We show that Gap1 present at the plasma membrane is associated with DRMs. This association occurs at the Golgi level. In the absence of sphingolipid neosynthesis, Gap1 fails to accumulate at the plasma membrane and is missorted to the vacuolar lumen. Furthermore, the presence of Gap1 at the plasma membrane correlates perfectly with its association with DRMs, whatever the activity or ubiquitination state of the permease and regardless of whether it has reached the cell surface via normal secretion, after recycling, or upon missorting to the vacuole before rerouting to the plasma membrane. Finally, we show that Hxt1 present at the cell surface is also associated with DRMs. We discuss a model where yeast plasma membrane proteins are systematically associated with sphingolipid/ergosterol-enriched microdomains when located at the cell surface.    

3.782           Dynamic Sequestration of the Recycling Compartment by Classical Protein Kinase C

Idkowiak-Baldys, J., Becker, K.P., Kitatani, K. And Hannum, Y.A.

1. Biol. Chem., 281(31), 22321-22331 (2006)

  It has been previously shown that upon sustained stimulation (30-60 min) with phorbol esters, protein kinase C (PKC) and II become sequestered in a juxtanuclear region, the pericentrion. The activation of PKC also results in sequestration of transferrin, suggesting a role for PKC in regulating endocytosis and sequestration of recycling components. In this work we characterize the pericentrion as a PKC-dependent subset of the recycling compartment. We demonstrate that upon sustained stimulation of PKC, both protein (CD59, caveolin) and possibly also lipid (Bodipy-GM1) cargo become sequestered in a PKC-dependent manner. This sequestration displayed a strict temperature requirement and was inhibited below 32 °C. Treatment of cells with phorbol myristate acetate for 60 min led to the formation of a distinct membrane structure. PKC sequestration and pericentrion formation were blocked by hypertonic sucrose as well as by potassium depletion (inhibitors of clathrin-dependent endocytosis) but not by nystatin or filipin, which inhibit clathrin-independent pathways. Interestingly, it was also observed that some molecules that internalize through clathrin-independent pathways (CD59, Bodipy-GM1, caveolin) also sequestered to the pericentrion upon sustained PKC activation, suggesting that PKC acted distal to the site of internalization of endocytic cargo. Together these results suggest that PKC regulates sequestration of recycling molecules into this compartment, the pericentrion.  

3.783           Pathogenic mutations of presenilins enhance pro-apoptotic activity by reducing mitochondrial Bcl-2

Wang, H-Q. et al Alzheimer's and Dementia, 2(3), Supplement 1,  Page S494 (2006)   Background: The mechanism of neuronal loss in Alzheimer’s disease (AD) remains unknown, although circumstantial evidence of apoptosis in postmortem brain tissues has supported the hypothesis that an apoptotic mechanism plays a role in the neurodegeneration in AD. Mutations in presenilins 1 and 2 (PS1/2) are responsible for the majority of familial AD. These proteins are localized predominantly in the endoplasmic reticulum (ER) and Golgi apparatus, and form heteromeric protein complexes to participate in several functions. Previous studies have shown that PS1/2 are involved in the regulation of apoptotic cell death, although the molecular basis is still unresolved. Objective(s): The aim of this study is to clarify the molecular basis by which PS1/2 regulate apoptosis. We also address a question how presenilin mutations modify the regulatory activity. Methods: We performed yeast 2-hybrid screen, co-immunoprecipitation assay, glycerol velocity gradient centrifugation and subcellular fractionation with iodixanol gradient, using HEK293 cells, PS1/2-knockout mouse fibroblasts and I213T-PS1-knockin mouse brains. Results: PS1/2 interacted with FKBP38 that is an immunophilin family member residing in the mitochondrial membrane and inhibits apoptosis by targeting and anchoring antiapoptotic Bcl-2 to the mitochondria. PS1/2 and FKBP38 formed macromolecular complexes together with Bcl-2. This complex formation promoted the degradation of FKBP38 and Bcl-2, and sequestered these proteins in the ER/Golgi compartments, thereby inhibiting FKBP38-mediated mitochondrial targeting of Bcl-2. Thus, full-length PS1/2 increased the susceptibility to apoptosis by antagonizing the anti-apoptotic function of FKBP38. In contrast, C-terminal fragments of caspase-processed PS1/2 redistributed Bcl-2 to the mitochondria by abrogating the activity of full-length PS1/2, resulting in a dominant-negative anti-apoptotic effect. In cultured cells and mutant PS1-knockin mouse brains, familial AD-linked PS1/2 mutants enhanced the pro-apoptotic activity by causing a more efficient reduction in mitochondrial Bcl-2 than wild-type PS1/2. Conclusions: We demonstrated that PS1/2 regulate the susceptibility to apoptosis by interacting with FKBP38. These results suggest a novel molecular mechanism for the regulation of mitochondria-mediated apoptosis by competition between PS1/2 and FKBP38 for subcellular targeting of Bcl-2. Furthermore, familial AD-linked mutations in PS1/2 could lower the threshold for activation of mitochondria-mediated apoptosis pathways inneurons of individuals with familial AD.  

3.784           Accumulation of sphingolipids increases secretion of the amyloid β-peptide by stabilization of the β-amyloid precursor protein

Hampel, H., Sandhoff, K. and Walter, J. Alzheimer's and Dementia, 2(3), Supplement 1, Pages S528-S529 (2006)   Alzheimer’s disease is associated with extracellular deposits in the brain of amyloid-_-peptides (A_) that are generated by proteolytic processing of the _-amyloid precursor protein (APP). It has been shown that membrane lipids, including cholesterol and sphingolipids affect the subcellular transport of APP in the secretory pathway and its proteolytic processing. Previously, we also showed that the inhibition of glycosphingolipid (GSL) biosynthesis reduces the secretion of APP and A_. Sphingolipids are highly enriched in the plasma membrane of cells. From here they are transported to late endosomal/lysosomal compartments where they are degraded. Inherited defects in degradation of these lipids cause sphingolipid storage disorders (SLSDs) marked by accumulation of GSLs in neurons associated with severe neurodegeneration. There is also a common defect in lipid transport along the endocytic pathway in SLSDs. Since processing of APP by secretases also occurs predominantly in post- Golgi secretory and endocytic compartments, we investigated the effect of accumulation of sphingolipids on transport and processing of APP. We used cultured cells and experimentally increased GSL levels by incubation with bovine brain gangliosides. The proteolytic processing of APP and its derivatives was studied by the detection of full length and soluble APP, APP C-terminal fragments (CTFs) as well as A_ and by pulse-chase experiments. Distribution of APP and its processing products was analyzed by iodixanol density gradient. The accumulation of GSLs markedly increased the secretion of endogenous APP and A_. A strong increase in total APP-CTF levels upon addition of GSLs was also observed. Addition of sphingomyelin showed similar effects. However _-secretase activity was not affected by GSLs in in vitro assays. By biochemical and cell biological experiments, we demonstrate that the increased levels of cellular GSLs altered the distribution and stability of APP-CTFs. Similar results were also obtained in independent genetic cellular models of GSL storage. On the other hand, GSL deficient cells showed decreased levels of APP-CTFs. Together, these data demonstrate that GSLs enhance the secretion of A_ likely by stabilizing APP-CTFs, thereby providing more substrate for _-secretases. Our studies suggest a novel role of GSLs in regulation of APP trafficking and A_ generation along the endocytic pathway.  

3.785           YKE4 (YIL023C) Encodes a Bidirectional Zinc Transporter in the Endoplasmic Reticulum of Saccharomyces cerevisiae

Kumanovics, A., Poruk, K.E., Osborn, K.A., Ward, D.M. and Kaplan, J.

1. Biol. Chem., 281(32), 22566-22574 (2006)

  YIL023C encodes a member of the SLC39A, or ZIP, family, which we refer to as yeast KE4 (YKE4) after its mouse ortholog. Yke4p was localized to the endoplasmic reticulum (ER) membrane using Yke4p-specific antiserum. YKE4 is not an essential gene; however, deletion of YKE4 resulted in a sensitivity to calcofluor white and poor growth at 36 °C on respiratory substrates containing high zinc. Overexpression of transition metal transporters Zrc1p and Cot1p or the mouse orthologue mKe4 in yke4 suppressed the poor growth at 36 °C on respiratory substrates. We found that the role of Yke4p depends on the zinc status of the cells. In a zinc-adequate environment, Yke4p transports zinc into the secretory pathway, and the deletion of YKE4 leads to a zinc-suppressible cell wall defect. In high zinc medium, transport of zinc into the secretory pathway through Yke4p is a way to eliminate zinc from the cytosol, and deletion of YKE4 leads to toxic zinc accumulation in the cytosol. Under low cytosolic zinc conditions, however, Yke4p removes zinc from the secretory pathway, and deletion of YKE4 partially compensates for the loss of Msc2p, an ER zinc importer, and therefore helps to alleviate ER stress. In our model, Yke4p balances zinc levels between the cytosol and the secretory pathway, whereas the previously described Msc2p-Zrg17p ER zinc importer complex functions mainly in zinc-depleted conditions to ensure a ready supply of zinc essential for ER functions, such as phospholipid biosynthesis and unfolded protein response.  

3.786           Release of iron from ferritin requires lysosomal activity

Kidane, T.Z., Sauble, E. and Linder, M.C. Am. J. Physiol. Cell Physiol., 291, C445-C455 (2006)   How ferritin-Fe becomes available for cell functions is unknown. Our previous studies with rat hepatoma cells indicated ferritin had to be degraded to release its Fe. In these studies, we investigated whether this occurs in other cell types and whether lysosomes are required. Release of ferritin-Fe was induced with desferoxamine (DFO) in ⁵⁹Fe-preloaded hepatoma, Caco2, and erythroid K562 cells and measured by rocket immunoelectrophoresis and autoradiography. The half-lives for ferritin-⁵⁹Fe and protein were parallel (23, 16, and 11 h for the hepatic, Caco2, and K562 cells, respectively). Co-treatment with 180 µM Fe, leupeptin, chymostatin, or chloroquine markedly decreased rates of ferritin-Fe release and ferritin degradation. Lactacystin had no effect except for a small one in erythroid cells. Fractionation of hepatoma cell lysates on iodixanol gradients showed rapid depletion of cytosolic ferritin by DFO treatment but no accumulation in lysosomes. We conclude that regardless of cell type, release of Fe from ferritin occurs mainly through lysosomal proteolysis.  

3.787           Lipid rafts mediate ultraviolet light-induced Fas aggregation in M624 melanoma cells

Elyassaki, W. and Wu, S. Photochem. Photobiol., 82, 787-792 (2006)   Ultraviolet light (UV) induces aggregation of Fas-receptor through a Fas-ligand-independent pathway. However, the mechanism of ultraviolet light-induced Fas-receptor aggregation is not known. In this report, we show that lipid rafts mediate ultraviolet light-induced aggregation of Fas. Our data show that UV induces a redistribution of Fas-receptor in a 25-5% Optiprep continuous gradient. The amount of Fas-receptorS is significantly increased in a gradient fraction that contain lipid rafts and is associated with an increase of FADD and caspase-8. Our data also show that the active dimeric form of caspase-8 (p44/p41) is increased in the lipid raft fraction. In addition, our data show that cholesterol, a major component of lipid rafts, is significantly reduced in only the lipid raft fractions after UV-irradiation. However, ceramide, another major lipid raft component, is increased evenly in all gradient fractions after UV-irradiation. These results suggest that UV alters the composition of major lipid raft components, which leads to the recruitment of Fas-receptor and FADD, with subsequent activation of caspase-8. Based on our results, we propose a novel mechanism by which UV induces apoptosis through a membrane lipid raft-mediated signaling pathway.  

3.788           Mitchell Medical Lecture: OH radical formation from the lysosomal electron carriers

Yoshida, M. et al Biochim. Biophys. Acta, 1757 (5-6), Suppl. 1, 217 (2006)   No abstract available  

3.789           Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments

Lampugnani, M.G., Orsenigo, F., Gagliani, M.C., Tacchetti, C. and Dejana, E.

1. Cell Biol., 174(4), 593-604 (2006)

  Receptor endocytosis is a fundamental step in controlling the magnitude, duration, and nature of cell signaling events. Confluent endothelial cells are contact inhibited in their growth and respond poorly to the proliferative signals of vascular endothelial growth factor (VEGF). In a previous study, we found that the association of vascular endothelial cadherin (VEC) with VEGF receptor (VEGFR) type 2 contributes to density-dependent growth inhibition (Lampugnani, G.M., A. Zanetti, M. Corada, T. Takahashi, G. Balconi, F. Breviario, F. Orsenigo, A. Cattelino, R. Kemler, T.O. Daniel, and E. Dejana. 2003. J. Cell Biol. 161:793–804). In the present study, we describe the mechanism through which VEC reduces VEGFR-2 signaling. We found that VEGF induces the clathrin-dependent internalization of VEGFR-2. When VEC is absent or not engaged at junctions, VEGFR-2 is internalized more rapidly and remains in endosomal compartments for a longer time. Internalization does not terminate its signaling; instead, the internalized receptor is phosphorylated, codistributes with active phospholipase C– , and activates p44/42 mitogen-activated protein kinase phosphorylation and cell proliferation. Inhibition of VEGFR-2 internalization reestablishes the contact inhibition of cell growth, whereas silencing the junction-associated density-enhanced phosphatase-1/CD148 phosphatase restores VEGFR-2 internalization and signaling. Thus, VEC limits cell proliferation by retaining VEGFR-2 at the membrane and preventing its internalization into signaling compartments.  

3.790           The membrane proximal disulfides of the EGF receptor extracellular domain are required for high affinity binding and signal transduction but do not play a role in the localization of the receptor to lipid rafts

Macdonald, J., Li, Z., Su, W. and Pike, L.J. Biochim. Biophys. Acta, 1763(8), 870-878 (2006)   The EGF receptor is a transmembrane receptor tyrosine kinase that is enriched in lipid rafts. Subdomains I, II and III of the extracellular domain of the EGF receptor participate in ligand binding and dimer formation. However, the function of the cysteine-rich subdomain IV has not been elucidated. In this study, we analyzed the role of the membrane-proximal portion of subdomain IV in EGF binding and signal transduction. A double Cys → Ala mutation that breaks the most membrane-proximal disulfide bond (Cys600 to Cys612), ablated high affinity ligand binding and substantially reduced signal transduction. A similar mutation that breaks the overlapping Cys596 to Cys604 disulfide had little effect on receptor function. Mutation of residues within the Cys600 to Cys612 disulfide loop did not alter the ligand binding or signal transducing activities of the receptor. Despite the fact that the C600,612A EGF receptor was significantly impaired functionally, this receptor as well as all of the other receptors with mutations in the region of residues 596 to 612 localized normally to lipid rafts. These data suggest that the disulfide-bonded structure of the membrane-proximal portion of the EGF receptor, rather than its primary sequence, is important for EGF binding and signaling but is not involved in localizing the receptor to lipid rafts.  

3.791           RGS Expression Rate-Limits Recovery of Rod Photoresponses

Krispel, C.M. et al Neurons, 51(4), 409-416 (2006)   Signaling through G protein-coupled receptors (GPCRs) underlies many cellular processes, yet it is not known which molecules determine the duration of signaling in intact cells. Two candidates are G protein-coupled receptor kinases (GRKs) and Regulators of G protein signaling (RGSs), deactivation enzymes for GPCRs and G proteins, respectively. Here we investigate whether GRK or RGS governs the overall rate of recovery of the light response in mammalian rod photoreceptors, a model system for studying GPCR signaling. We show that overexpression of rhodopsin kinase (GRK1) increases phosphorylation of the GPCR rhodopsin but has no effect on photoresponse recovery. In contrast, overexpression of the photoreceptor RGS complex (RGS9-1·Gβ5L·R9AP) dramatically accelerates response recovery. Our results show that G protein deactivation is normally at least 2.5 times slower than rhodopsin deactivation, resolving a long-standing controversy concerning the mechanism underlying the recovery of rod visual transduction.  

3.792           The Use of GFP to Localize Rho GTPases in Living Cells

Michaelson, D. and Philips, M. Methods in Enzymol., 406, 296-315 (2006)   The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has revolutionized the study of protein localization and dynamics. GFP fusions permit analysis of proteins in living cells and offer distinct advantages over conventional immunofluorescence. Among these are lower background, higher resolution, robust dual color colocalization, and avoidance of fixation artifacts. In the case of Ras and Rho family proteins, GFP fusions have allowed breakthroughs in the understanding of how CAAX proteins are targeted to specific cell membranes and how signaling at different membranes can result in different cellular responses. GFP-tagged Rho proteins have also been informative in analyzing the interactions with the cytosolic chaperone, RhoGDI. The major disadvantages of studying GFP fusion proteins is that they are generally overexpressed relative to endogenous proteins, and the GFP tag can, in principle, affect protein function. Fortunately, in the case of Ras and Rho family proteins, a GFP tag at the N terminus seems to have little effect on protein targeting and function. Nevertheless, it is prudent to confirm GFP fusion protein data with the study of the endogenous protein. This chapter describes the tagging of Rho proteins with GFP and the analysis of GFP–Rho protein localization by epifluorescence and confocal microscopy. It further describes methods of analyzing endogenous Rho proteins as confirmation of data acquired using GFP–Rho fusion proteins. These techniques will be useful for anyone studying Rho protein function and are widely applicable to many cell types and signal transduction systems.    

3.793           Activation process of the mosquitocidal δ-endotoxin Cry39A produced by Bacillus thuringiensis subsp. aizawai BUN1-14 and binding property to Anopheles stephensi BBMV

Ito, T., Bando, H. and Asano, S-i.

1. Invertebrate Pathol., 93(1), 29-35 (2006)

  Most δ-endotoxins produced by Bacillus thuringiensis require proteolytic processing in order to become active. The in vitro and in vivo activation processes of Cry39A, a δ-endotoxin that is highly toxic to Anopheles stephensi, were investigated. Cry39A with a molecular mass of 72 kDa was processed in vitro into a 60 kDa fragment by trypsin and gut extract from A. stephensi larvae. N-terminal amino acid sequencing of the 60 kDa fragment revealed that trypsin and the protease(s) in the gut extract cleaved Cry39A between Arg⁶¹ and Gly⁶². In contrast, 40 and 25 kDa polypeptides were generated in vivo by intramolecular cleavage of the 60 kDa fragment in A. stephensi larvae. Further, a co-precipitation assay was used to investigate the binding property of the activated Cry39A to A. stephensi BBMV. Cry39A bound to A. stephensi BBMV specifically and did not compete with the Cry4Aa toxin. This indicated that the binding molecule(s) for Cry39A might differ from those for Cry4A. In addition, Cry39A preferentially bound to the Triton X-100-insoluble membrane fraction.  

3.794           Endogenous spartin, mutated in hereditary spastic paraplegia, has a complex subcellular localization suggesting diverse roles in neurons

Robay, D., Patel, H., Simpson, M.A., Brown, N.A. and Crosby, A.H. Exp. Cell Res., 312(15), 2764-2777 (2006)   Mutation of spartin (SPG20) underlies a complicated form of hereditary spastic paraplegia, a disorder principally defined by the degeneration of upper motor neurons. Using a polyclonal antibody against spartin to gain insight into the function of the endogenous molecule, we show that the endogenous molecule is present in two main isoforms of 85 kDa and 100 kDa, and 75 kDa and 85 kDa in human and murine, respectively, with restricted subcellular localization. Immunohistochemical studies on human and mouse embryo sections and in vitro cell studies indicate that spartin is likely to possess both nuclear and cytoplasmic functions. The nuclear expression of spartin closely mirrors that of the snRNP (small nuclear ribonucleoprotein) marker α-Sm, a component of the spliceosome. Spartin is also enriched at the centrosome within mitotic structures. Notably we show that spartin protein undergoes dynamic positional changes in differentiating human SH-SY5Y cells. In undifferentiated non-neuronal cells, spartin displays a nuclear and diffuse cytosolic profile, whereas spartin transiently accumulates in the trans-Golgi network and subsequently decorates discrete puncta along neurites in terminally differentiated neuroblastic cells. Investigation of these spartin-positive vesicles reveals that a large proportion colocalizes with the synaptic vesicle marker synaptotagmin. Spartin is also enriched in synaptic-like structures and in synaptic vesicle-enriched fraction.  

3.795           Cholesterol depletion reduces aggregation of amyloid-beta peptide in hippocampal neurons

Scheider, A., Schulz-Schaeffer, W., Hartmann, T., Schulz, J.B. and Simons, M. Neurobiology of Disease, 23, 573-577 (2006)   A key event in the pathogenesis of Alzheimer's disease is the conversion of soluble amyloid Aβ-peptide into toxic aggregates. Here, we studied the effect of cholesterol depletion on the formation of insoluble Aβ. We found that reduction of neuronal cholesterol by 25% reduced the neuronal formation of insoluble Aβ without affecting the secretion of soluble Aβ. Moreover, we demonstrate that Aβ-oligomers from Alzheimer's disease brains associate with a detergent-resistant membrane fraction in a cholesterol-dependent manner. These results suggest a key role for cholesterol in aggregation of Aβ.    

3.796           Cotranslocational Degradation Protects the Stressed Endoplasmic Reticulum from Protein Overload

Oyadomari, S. et al Cell, 126(4), 727-739 (2006)   The ER's capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58^IPK/DNAJC3, a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58^IPK recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58^IPK−/− mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58^IPK is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells.  

3.797           Translocation of Endothelial Nitric-Oxide Synthase Involves a Ternary Complex with Caveolin-1 and NOSTRIN

Schilling, K. et al Mol. Biol. Cell, 17, 3870-3880 (2006)   Recently, we characterized a novel endothelial nitric-oxide synthase (eNOS)-interacting protein, NOSTRIN (for eNOS-trafficking inducer), which decreases eNOS activity upon overexpression and induces translocation of eNOS away from the plasma membrane. Here, we show that NOSTRIN directly binds to caveolin-1, a well-established inhibitor of eNOS. Because this interaction occurs between the N terminus of caveolin (positions 1–61) and the central domain of NOSTRIN (positions 323–434), it allows for independent binding of each of the two proteins to eNOS. Consistently, we were able to demonstrate the existence of a ternary complex of NOSTRIN, eNOS, and caveolin-1 in Chinese hamster ovary (CHO)-eNOS cells. In human umbilical vein endothelial cells (HUVECs), the ternary complex assembles at the plasma membrane upon confluence or thrombin stimulation. In CHO-eNOS cells, NOSTRIN-mediated translocation of eNOS involves caveolin in a process most likely representing caveolar trafficking. Accordingly, trafficking of NOSTRIN/eNOS/caveolin is affected by altering the state of actin filaments or cholesterol levels in the plasma membrane. During caveolar trafficking, NOSTRIN functions as an adaptor to recruit mediators such as dynamin-2 essential for membrane fission. We propose that a ternary complex between NOSTRIN, caveolin-1, and eNOS mediates translocation of eNOS, with important implications for the activity and availability of eNOS in the cell.  

3.798           An Internal EELD Domain Facilitates Mitochondrial Targeting of Mcl-1 via a Tom70-dependent Pathway

Chou, C-H., Lee, R-S., Yang-Yen, H-F. Mol. Biol. Cell, 17, 3952-3963 (2006)   Mcl-1 functions at an apical step in many regulatory programs that control cell death. Although the mitochondrion is one major subcellular organelle where Mcl-1 functions, the molecular mechanism by which Mcl-1 is targeted to mitochondria remains unclear. Here, we demonstrate that Mcl-1 is loosely associated with the outer membrane of mitochondria. Furthermore, we demonstrate that Mcl-1 interacts with the mitochondrial import receptor Tom70, and such interaction requires an internal domain of Mcl-1 that contains an EELD motif. A Tom70 antibody that blocks Mcl-1–Tom70 interaction blocks mitochondrial import of Mcl-1 in vitro. Furthermore, Mcl-1 is significantly less targeted to mitochondria in Tom70 knockdown than in the control cells. Similar targeting preference is also observed for the DM mutant of Mcl-1 whose mutation at the EELD motif markedly attenuates its Tom70 binding activity. Together, our results indicate that the internal EELD domain facilitates mitochondrial targeting of Mcl-1 via a Tom70-dependent pathway.  

3.799           Cumulative Mutations Affecting Sterol Biosynthesis in the Yeast Saccharomyces cerevisiae Result in Synthetic Lethality That Is Suppressed by Alterations in Sphingolipid Profiles

Valachovic, M. et al Genetics, 173, 1893-1908 (2006)   UPC2 and ECM22 belong to a Zn(2)–Cys(6) family of fungal transcription factors and have been implicated in the regulation of sterol synthesis in Saccharomyces cerevisiae and Candida albicans. Previous reports suggest that double deletion of these genes in S. cerevisiae is lethal depending on the genetic background of the strain. In this investigation we demonstrate that lethality of upc2 ecm22 in the S288c genetic background is attributable to a mutation in the HAP1 transcription factor. In addition we demonstrate that strains containing upc2 ecm22 are also inviable when carrying deletions of ERG6 and ERG28 but not when carrying deletions of ERG3, ERG4, or ERG5. It has previously been demonstrated that UPC2 and ECM22 regulate S. cerevisiae ERG2 and ERG3 and that the erg2 upc2 ecm22 triple mutant is also synthetically lethal. We used transposon mutagenesis to isolate viable suppressors of hap1 , erg2 , erg6 , and erg28 in the upc2 ecm22 genetic background. Mutations in two genes (YND1 and GDA1) encoding apyrases were found to suppress the synthetic lethality of three of these triple mutants but not erg2 upc2 ecm22 . We show that deletion of YND1, like deletion of GDA1, alters the sphingolipid profiles, suggesting that changes in sphingolipids compensate for lethality produced by changes in sterol composition and abundance.  

3.800           Membrane Association of the Cycling Peroxisome Import Receptor Pex5p

Kerssen, D. et al

1. Biol. Chem., 281(37), 27003-27015 (2006)

  Peroxisomal proteins carrying a peroxisome targeting signal type 1 (PTS1) are recognized in the cytosol by the cycling import receptor Pex5p. The receptor-cargo complex docks at the peroxisomal membrane where it associates with multimeric protein complexes, referred to as the docking and RING finger complexes. Here we have identified regions within the Saccharomyces cerevisiae Pex5p sequence that interconnect the receptor-cargo complex with the docking complex. Site-directed mutagenesis of the conserved tryptophan residue within a reverse WXXXF motif abolished two-hybrid binding with the N-terminal half of Pex14p. In combination with an additional mutation introduced into the Pex13p-binding site, we generated a Pex5p mutant defective in a stable association not only with the docking complex but also with the RING finger peroxins at the membrane. Surprisingly, PTS1 proteins are still imported into peroxisomes in these mutant cells. Because these mutations had no significant effect on the membrane binding properties of Pex5p, we examined yeast and human Pex5p for intrinsic lipid binding activity. In vitro analyses demonstrated that both proteins have the potential to insert spontaneously into phospholipid membranes. Altogether, these data strongly suggest that a translocation-competent state of the PTS1 receptor enters the membrane via protein-lipid interactions before it tightly associates with other peroxins.  

3.801           Palmitoylation Plays a Role in Targeting Vac8p to Specific Membrane Subdomains

Peng, Y., Tang, F. and Weisman, L.S. Traffic, 7, 1378-1387 (2006)   Vac8p is a multifunctional yeast protein involved in several distinct vacuolar events including vacuole inheritance, vacuole homotypic fusion, nucleus–vacuole junction formation and the cytoplasm to vacuole protein targeting pathway. Vac8p associates with the vacuole membrane via myristoylation and palmitoylation. Vac8p has three putative palmitoylation sites, at Cys 4, 5 and 7. Here, we show that each of these cysteines may serve as a palmitoylation site. Palmitoylation at Cys 7 alone provides partial function of Vac8p, whereas palmitoylation at either Cys 4 or Cys 5 alone is sufficient for Vac8p function. In the former mutant, there is a severe defect in the localization of Vac8p to the vacuole membrane, while in the latter mutants, there is a partial defect in the localization of Vac8p. In addition, our studies provide evidence that palmitoylation targets Vac8p to specific membrane subdomains.  

3.802           Invasion of Host Cells by JC Virus Identifies a Novel Role for Caveolae in Endosomal Sorting of Noncaveolar Ligands

Querbes, W., O’Hara, B.A., Williams, G. and Arwood, W.J.

1. Virol., 80(19), 9402-9413 (2006)

  Invasion of glial cells by the human polyomavirus, JC virus (JCV), leads to a rapidly progressing and uniformly fatal demyelinating disease known as progressive multifocal leukoencephalopathy. The endocytic trafficking steps used by JCV to invade cells and initiate infection are not known. We demonstrated that JCV infection was inhibited by dominant defective and constitutively active Rab5-GTPase mutants that acted at distinct steps in endosomal sorting. We also found that labeled JCV colocalized with labeled cholera toxin B and with caveolin-1 (cav-1) on early endosomes following internalization by clathrin-dependent endocytosis. JCV entry and infection were both inhibited by dominant defective mutants of eps15 and Rab5-GTPase. Expression of a dominant-negative scaffolding mutant of cav-1 did not inhibit entry or infection by JCV. A single-cell knockdown experiment using cav-1 shRNA did not inhibit JCV entry but interfered with a downstream trafficking event important for infection. These data show that JCV enters cells by clathrin-dependent endocytosis, is transported immediately to early endosomes, and is then sorted to a caveolin-1-positive endosomal compartment. This latter step is dependent on Rab5-GTPase, cholesterol, caveolin-1, and pH. This is the first example of a ligand that enters cells by clathrin-dependent endocytosis and is then sorted from early endosomes to caveosomes, indicating that caveolae-derived vesicles play a more important role than previously realized in sorting cargo from early endosomes.  

3.803           Reconstitution of Herpes Simplex Virus Type 1 Nuclear Capsid Egress In Vitro

Remillard-Labrosse, G., Guay, G. and Lippe, R.

1. Virol., 80(19), 9741-9753 (2006)

  Newly assembled herpesvirus capsids travel from the nucleus to the plasma membrane by a mechanism that is poorly understood. Furthermore, the contribution of cellular proteins to this egress has yet to be clarified. To address these issues, an in vitro nuclear egress assay that reproduces the exit of herpes simplex virus type 1 (HSV-1) capsids from nuclei isolated from infected cells was established. As expected, the assay has all the hallmarks of intracellular transport assays, namely, a dependence on time, energy, and temperature. Surprisingly, it is also dependent on cytosol and was slightly enhanced by infected cytosol, suggesting an implication of both host and viral proteins in the process. The capsids escaped these nuclei by budding through the inner nuclear membrane, accumulated as enveloped capsids between the two nuclear membranes, and were released in cytosol exclusively as naked capsids, exactly as in intact cells. This is most consistent with the view that the virus escapes by crossing the two nuclear membranes rather than through nuclear pores. Unexpectedly, nuclei isolated at the nonpermissive temperature from cells infected with a U_L26 thermosensitive protease mutant (V701) supported capsid egress. Although electron microscopy, biochemical, and PCR analyses hinted at a likely reconstitution of capsid maturation, DNA encapsidation could not be confirmed by a traditional SQ test. This assay should prove very useful for identification of the molecular players involved in HSV-1 nuclear egress.  

3.804           Characterization of erasin (UBXD2): a new ER protein that promotes ER-associated protein degradation

Liang, J. et al

1. Cell Sci., 119, 4011-4024 (2006)

  Ubiquitin regulator-X (UBX) is a discrete protein domain that binds p97/valosin-containing protein (VCP), a molecular chaperone involved in diverse cell processes, including endoplasmic-reticulum-associated protein degradation (ERAD). Here we characterize a human UBX-containing protein, UBXD2, that is highly conserved in mammals, which we have renamed erasin. Biochemical fractionation, immunofluorescence and electron microscopy, and protease protection experiments suggest that erasin is an integral membrane protein of the endoplasmic reticulum and nuclear envelope with both its N- and C-termini facing the cytoplasm or nucleoplasm. Localization of GFP-tagged deletion derivatives of erasin in HeLa cells revealed that a single 21-amino-acid sequence located near the C-terminus is necessary and sufficient for localization of erasin to the endoplasmic reticulum. Immunoprecipitation and GST-pulldown experiments confirmed that erasin binds p97/VCP via its UBX domain. Additional immunoprecipitation assays indicated that erasin exists in a complex with other p97/VCP-associated factors involved in ERAD. Overexpression of erasin enhanced the degradation of the ERAD substrate CD3 , whereas siRNA-mediated reduction of erasin expression almost completely blocked ERAD. Erasin protein levels were increased by endoplasmic reticulum stress. Immunohistochemical staining of brain tissue from patients with Alzheimer's disease and control subjects revealed that erasin accumulates preferentially in neurons undergoing neurofibrillary degeneration in Alzheimer's disease. These results suggest that erasin may be involved in ERAD and in Alzheimer's disease.  

3.805           Characterization of Proline-Serine-Rich Carboxyl Terminus in Human Sulfotransferase 2B1b: Immunogenicity, Subcellular Localization, Kinetic Properties, and Phosphorylation

He, D. and Falany, C.N. Drug. Metab. Dispos., 34(10), 1749-1755 (2006)   The human sulfotransferase (SULT) 2B1 gene is a member of the SULT2 gene family and encodes two isoforms, SULT2B1a and SULT2B1b. Although messages for both SULT2B1a and SULT2B1b are detectable in human tissues, only SULT2B1b has been identified immunologically. Compared with other human SULTs, SULT2B1b has an extension at the proline- and serine-rich carboxyl (PSC) end of about 53 amino acids. The structure and function of this unique PSC extension were investigated. Constructs of full-length SULT2B1b as well as truncated SULT2B1b without the PSC extension were expressed in Escherichia coli. Removal of the PSC extension significantlydecreased the thermostability of the expressed enzyme as wellas decreasing the rate of dehydroepiandrosterone sulfation.Rabbit polyclonal antibodies were raised against both the full-lengthand truncated SULT2B1b proteins. Immunoblot analysis showedthat antibodies raised to full-length SULT2B1b immunoreact onlywith full-length SULT2B1b, whereas antibodies raised to truncatedSULT2B1b react with both full-length and truncated SULT2B1b.Unlike full-length SULT2B1b, truncated SULT2B1b was incapableof translocation to nuclei in transfected human BeWo choriocarcinomacells. Phosphorylated serines were detected in the PSC extensionof full-length SULT2B1b expressed in BeWo cells but not in truncatedSULT2B1b. At least one phosphorylated serine was detected inexpressed SULT2B1b via two-dimensional gel electrophoresis,immunoblot analysis, and mass spectroscopic analysis. Bacteriallyexpressed full-length SULT2B1b but not truncated SULT2B1b wasphosphorylated by casein kinase or Cdc2 protein kinase in vitro.This study suggests that the PSC extension of SULT2B1b is animportant site in the immunogenicity, nuclear translocation,kinetic activity, and thermostability of this SULT isoform.  

3.806           The Cannabinoid CB1 Receptor Antagonist Rimonabant (SR141716) Inhibits Human Breast Cancer Cell Proliferation through a Lipid Raft-Mediated Mechanism

Sarnataro, D. Et al Mol. Pharmacol., 70(4), 1298-1306 (2006)   The endocannabinoid system has been shown to modulate key cell-signaling pathways involved in cancer cell growth. In this study, we show that cannabinoid receptor type 1 (CB1) antagonist Rimonabant (SR141716) inhibited human breast cancer cell proliferation, being more effective in highly invasive metastatic MDA-MB-231 cells than in less-invasive T47D and MCF-7 cells. The SR141716 antiproliferative effect was not accompanied by apoptosis or necrosis and was characterized by a G₁/S-phase cell cycle arrest, decreased expression of cyclin D and E, and increased levels of cyclin-dependent kinase inhibitor p27^KIP1. We have also shownthat SR141716 exerted a significant antiproliferative action,in vivo, by reducing the volume of xenograft tumors inducedby MDA-MB-231 injection in mice. On the other hand, at the concentrationrange in which we observed the antiproliferative effect in tumorcells, we did not observe evidence of any genotoxic effect onnormal cells. Our data also indicate that the SR141716 antiproliferativeeffect requires lipid raft/caveolae integrity to occur. Indeed,we found that CB1 receptor (CB1R) is completely displaced fromlipid rafts in SR141716-treated MDA-MB-231 cells, and cholesteroldepletion by methyl--cyclodextrin strongly prevented SR141716-mediatedantiproliferative effect. Taken together, our results suggestthat SR141716 inhibits human breast cancer cell growth via aCB1R lipid raft/caveolae-mediated mechanism.  

3.807           Rab14 is part of the early endosomal clathrin-coated TGN microdomain

Proikas-Cezanne, T., Gaugel, A., Frickey, T. and Nordheim, A. FEBS Lett., 580, 5241-5246 (2006)   Rab14 localizes to the Golgi/TGN and to early endosomes, but its biological function remains unclear. By structural modeling, we identified Rab14-specific residues and established a close relationship between the Rab2/Rab4/Rab14, Rab11/25 and Rab39 sub-groups within the Rab protein family. By quantitative confocal microscopy and by density centrifugation we show that Rab14 is part of the early endosomal AP-1 microdomain. Overexpression of a dominant-negative Rab14 GTP-binding mutant that solely localizes to the Golgi donor compartment accelerated EGF degradation. We suggest that the AP-1 microdomain represents the interconnecting compartment in which Rab14 vesicles cycle between early endosomes and the Golgi cisternae.  

3.808           Use of analogs and inhibitors to study the functional significance of protein palmitoylation

Resh, M.D. Methods, 40, 191-197 (2006)   Covalent attachment of palmitate to proteins is a post-translational modification that exerts diverse effects on protein localization and function. The three key technical approaches required for an investigator to determine the role of palmitoylation of your favorite palmitoylated protein (YFPP) are methods to: (1) detect YFPP palmitoylation; (2) alter or inhibit palmitoylation of YFPP; (3) determine the functional significance of altered YFPP palmitoylation. Here, I describe experimental methods to address these three issues. Both radioactive (radiolabeling with [³H]palmitate or ¹²⁵I-IC16 palmitate) and non-radioactive (chemical labeling and mass spectrometry) methods to detect palmitoylated proteins are presented. Next, techniques to inhibit protein palmitoylation are described. These include site specific mutagenesis, and treatment of cells with inhibitors of protein palmitoylation, including 2-bromopalmitate, cerulenin, and tunicamycin. Alternative methods to replace palmitate with other fatty acids are also presented. Finally, general approaches to determining the effect of altered palmitoylation status on YFPP association with membranes and lipid rafts, as well as signal transduction, are described.  

3.809           Different Routes of Bone Morphogenic Protein (BMP) Receptor Endocytosis Influence BMP Signaling

Hartung, A. et al Mol. Cell. Biol., 26(20), 7791-7805 (2006)   Endocytosis is important for a variety of functions in eukaryotic cells, including the regulation of signaling cascades via transmembrane receptors. The internalization of bone morphogenetic protein (BMP) receptor type I (BRI) and type II (BRII) and its relation to signaling were largely unexplored. Here, we demonstrate that both receptor types undergo constitutive endocytosis via clathrin-coated pits (CCPs) but that only BRII undergoes also caveola-like internalization. Using several complementary approaches, we could show that (i) BMP-2-mediated Smad1/5 phosphorylation occurs at the plasma membrane in nonraft regions, (ii) continuation of Smad signaling resulting in a transcriptional response requires endocytosis via the clathrin-mediated route, and (iii) BMP signaling leading to alkaline phosphatase induction initiates from receptors that fractionate into cholesterol-enriched, detergent-resistant membranes. Furthermore, we show that BRII interacts with Eps15R, a constitutive component of CCPs, and with caveolin-1, the marker protein of caveolae. Taken together, the localization of BMP receptors in distinct membrane domains is prerequisite to their taking different endocytosis routes with specific impacts on Smad-dependent and Smad-independent signaling cascades.  

3.810           Ultrastructural Analysis of ESCRT Proteins Suggests a Role for Endosome-Associated Tubular–Vesicular Membranes in ESCRT Function

Welsch, S. et al Traffic, 7, 1551-1566 (2006)   The endosomal sorting complex required for transport (ESCRT) is thought to support the formation of intralumenal vesicles of multivesicular bodies (MVBs). The ESCRT is also required for the budding of HIV and has been proposed to be recruited to the HIV-budding site, the plasma membrane of T cells and MVBs in macrophages. Despite increasing data on the function of ESCRT, the ultrastructural localization of its components has not been determined. We therefore localized four proteins of the ESCRT machinery in human T cells and macrophages by quantitative electron microscopy. All the proteins were found throughout the endocytic pathway, including the plasma membrane, with only around 10 and 3% of the total labeling in the cytoplasm and on the MVBs, respectively. The majority of the labeling (45%) was unexpectedly found on tubular–vesicular endosomal membranes rather than on endosomes themselves. The ESCRT labeling was twice as concentrated on early and late endosomes/lysosomes in macrophages compared with that in T cells, where it was twice more abundant at the plasma membrane. The ESCRT proteins were not redistributed on HIV infection, suggesting that the amount of ESCRT proteins located at the budding site suffices for HIV release. These results represent the first systematic ultrastructural localization of ESCRT and provide insights into its role in uninfected and HIV-infected cells.  

3.811           Molecular probes for sensing the cholesterol composition of subcellular organelle membranes

Wang, R. et al Biochim. Biophys. Acta, 1761, 1169-1181 82006)   Neuroendocrine cells contain two types of secretagogue-regulated acidic compartments: secretory granules (SGs) and synaptic-like microvesicles (SLMVs), which can be identified by acidotropic probes such as acridine orange (AO) and DAMP. We investigated the accumulation of these probes in SGs and SLMVs as a function of glucose levels in the culture media using a pancreatic β-cell line MIN6. AO was accumulated in the low-glucose condition, but not in the high-glucose condition. The AO accumulation correlated well with the SLMV dynamics by glucose and DAMP was localized in the SGs. Because SG membranes are reportedly high in cholesterol, we prepared liposomes with increasing cholesterol levels. AO is well incorporated into liposomes having a 20 to 40 mol% cholesterol composition, whereas DAMP was so in those having over 40 mol% cholesterol levels. Indeed, when cholesterol was depleted from MIN6 SG membranes, DAMP incorporation decreased, instead AO was incorporated. In PC12 cells, AO incorporation into SGs was significant but DAMP incorporation was limited. Consistently, the cholesterol composition was found 37 to 39 mol% in the SG membrane of PC12 cells. We suggest that cholesterol-sensing probes, AO and DAMP, are useful tools for investigating cholesterol compositions in acidic organelle membranes.  

3.812           Evidence against Calcium as a Mediator of Mitochondrial Dysfunction during Apoptosis Induced by Arachidonic Acid and Other Free Fatty Acids

Maia, R.C., Culver, C.A. and Laster, S.M.

1. Immunol., 177, 6398-6404 (2006)

  Apoptosis is often accompanied by activation of phospholipase A₂, causing release of free fatty acids (FFAs), which in turn are thought to contribute to the loss of mitochondrial transmembrane potential ( _m). In these experiments, we asked whether calcium plays a role as an intermediate in this process. A total of 14 FFAs were compared for their ability to cause loss of _m and for their ability to affect levels of intracellular calcium. Among the FFAs, unsaturated FFAs tended to induce apoptosis while saturated FFAs did not. Arachidonic acid (AA) was most damaging, causing loss of _m and cell death in 8–10 h while linoleic acid, -linolenic acid, and docosapentaenoic also strongly induced apoptosis. Effects of the FFAs on levels of intracellular calcium were very different. Many caused strong calcium responses; however, the ability to induce a strong calcium response was not predictive of ability to induce apoptosis, and overall, we did not find a correlation between apoptosis and calcium induction. Also, verapamil and TMB-8 were able to block the calcium response, but these inhibitors did not prevent loss of _m, indicating that the calcium response is not necessary for FFA-induced loss of _m. In contrast, we found that cyclosporine A could inhibit the AA-induced loss of _m with both whole cells and isolated mitochondria, confirming that the antimitochondrial effects of FFA can stem from direct effects on the mitochondrial permeability transition pore. Finally, we show that the strong apoptosis-inducing activity of AA may stem from its ability to selectively induce its own release.  

3.813           ErbB-4 and TNF-α converting enzyme localization to membrane microd

Thiel, K.W. and Carpenter, G. Biochem. Biophys. Res. Comm., 350(3), 629-633 (2006)   Sequential proteolytic processing of ErbB-4 occurs in response to ligand addition. Here, we assess the localization of cleavable and non-cleavable ErbB-4 isoforms to membrane microdomains using three methodologies: (1) Triton X-100-insolubility, (2) Brij98-insolubility, and (3) detergent-free density gradient centrifugation. Whereas ErbB-4 translocated to a Triton X-100-insoluble fraction upon treatment of T47D cells with heregulin, it constitutively associated with a Brij98-insoluble fraction and a lipid raft fraction isolated using detergent-free methodology. Comparison of cleavable and non-cleavable isoforms of ErbB-4 revealed that both ErbB-4 isoforms are constitutively localized to either a Triton X-100-soluble or Brij98-insoluble fraction. In contrast, addition of heregulin resulted in translocation of the cleavable isoform to a detergent-free lipid raft. Tumor necrosis factor-α converting enzyme (TACE), the ectodomain secretase for ErbB-4, was present predominantly in its mature active form in most microdomains analyzed. These data suggest the assembly of ErbB-4 ectodomain cleavage apparatus in a membrane microdomain.  

3.814           Targeting β2-microglobulin for induction of tumor apoptosis in human hematological malignancies

Yang, J. et al Cancer Cell, 10, 295-307 (2006)   We discovered that monoclonal antibodies (mAbs) specific to human β₂-microglobulin (β₂M) induce apoptosis in vitro and were therapeutic in mouse models of myeloma and other hematological tumor cells. Cell death occurred rapidly, without the need for exogenous immunological effector mechanisms. The mAbs induced cell death via recruiting MHC class I molecules to lipid rafts and activating Lyn and PLCγ2, leading to activated JNK and inhibited PI3K/Akt and ERK, compromised mitochondrial integrity, and caspase-9-dependent cascade activation. Although the expression of β₂M on normal hematopoietic cells is a potential safety concern, the mAbs were selective to tumor-transformed cells and did not induce apoptosis of normal cells. Therefore, such mAbs offer the potential for a therapeutic approach to hematological malignancies.  

3.815           Protein Misfolding Cyclic Amplification for Diagnosis and Prion Propagation Studies

Castilla, J. et al Methods in Enzymol., 412, 3-21 (2006)   Diverse human disorders are thought to arise from the misfolding and aggregation of an underlying protein. Among them, prion diseases are some of the most intriguing disorders that can be transmitted by an unprecedented infectious agent, termed prion, composed mainly (if not exclusively) of the misfolded prion protein. The hallmark event in the disease is the conversion of the native prion protein into the disease-associated misfolded protein. We have recently described a novel technology to mimic the prion conversion process in vitro. This procedure, named protein misfolding cyclic amplification (PMCA), conceptually analogous to DNA amplification by polymerase chain reaction (PCR), has important applications for research and diagnosis. In this chapter we describe the rational behind PMCA and some of the many potential applications of this novel technology. We also describe in detail the technical and methodological aspects of PMCA, as well as its application in automatic and serial modes that have been developed with a view to improving disease diagnosis.  

3.816           Modulation of GalT1 and SialT1 Sub-Golgi Localization by SialT2 Expression Reveals an Organellar Level of Glycolipid Synthesis Control

Uliana, A.S., Crespo, P.M., Martina, J.A., Daniotti, J.L. and Maccioni, H.J.F.

1. Biol. Chem., 281(43), 32852-32860 (2006)

  Ganglioside glycosyltransferases organize as multienzyme complexes that localize in different sub-Golgi compartments. Here we studied whether in CHO-K1 cells lacking CMP-NeuAc: GM3 sialyltransferase (SialT2), the sub-Golgi localization of UDP-Gal:glucosylceramide beta-1,4-galactosyltransferase (GalT1) and CMP-NeuAc:lactosylceramide sialyltransferase (SialT1) complex is affected when SialT2, another member of this complex, is coexpressed. GalT1 and SialT1 sub-Golgi localization was determined by studying the effect of brefeldin A (BFA) and monensin on the synthesis of glycolipids and on the sub-Golgi localization of GalT1(1-52)-CFP (cyan fluorescent protein) and SialT1(1-54)-YFP (yellow fluorescent protein) chimeras by single cell fluorescence microscopy and by isopycnic subfractionation. We found that BFA, and also monensin, impair the synthesis of glycolipids beyond GM3 ganglioside in wild type (WT) cells but beyond GlcCer in SialT2(+) cells. Although BFA redistributed GalT1-CFP and SialT1-YFP to the endoplasmic reticulum in WT cells, a fraction of these chimeras remained associated with a distal Golgi compartment, enriched in trans Golgi network, and recycling endosome markers in SialT2(+) cells. In BFA-treated cells, the percentage of GalT1-CFP and SialT1-YFP associated with Golgi-like membrane fractions separated by isopycnic subfractionation was higher in SialT2(+) cells than in WT cells. These effects were reverted by knocking down the expression of SialT2 with specific siRNA. Results indicate that sub-Golgi localization of glycosyltransferase complexes may change according to the relative levels of the expression of participating enzymes and reveal a capacity of the organelle to adapt the topology of the glycolipid synthesis machinery to functional states of the cell.  

3.817           Kinesin-2 mediates physical and functional interactions between polycystin-2 and fibrocystin

Wu, Y. et al Hum. Mol. Genet., 15(22), 3280-3292 (2006)   Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1, encoding polycystin-1 (PC1), or PKD2 (polycystin-2, PC2). Autosomal recessive PKD (ARPKD) is caused by mutations in PKHD1, encoding fibrocystin/polyductin (FPC). No molecular link between ADPKD and ARPKD has been determined. Here, we demonstrated, by yeast two-hybrid and biochemical assays, that KIF3B, a motor subunit of kinesin-2, associates with PC2 and FPC. Co-immunoprecipitation experiments using Madin-Darby canine kidney (MDCK) and inner medullary collecting duct (IMCD) cells and human kidney revealed that PC2 and KIF3B, FPC and KIF3B and, furthermore, PC2 and FPC are endogenously in the same complex(es), though no direct association between the PC2 and FPC intracellular termini was detected. In vitro binding and Far Western blot experiments demonstrated that PC2 and FPC are in the same complex only if KIF3B is present, presumably by forming a PC2–KIF3B–FPC complex. This was supported by our observation that altering KIF3B level in IMCD cells by over-expression or siRNA significantly affected complexing between PC2 and FPC. Immunofluorescence experiments showed that PC2, FPC and KIF3B partially co-localized in primary cilia of over-confluent and perinuclear regions of sub-confluent cells. Furthermore, KIF3B mediated functional modulation of purified PC2 channels by FPC in a planer lipid bilayer electrophysiology system. The FPC C-terminus substantially stimulated PC2 channel activity in the presence of KIF3B, whereas FPC or KIF3B alone had no effect. Taken together, we discovered that kinesin-2 is a linker between PC2 and FPC and mediates the regulation of PC2 channel function by FPC. Our study may be important for elucidating common molecular pathways for PKD of different genotypes.  

3.818           Lipid Phosphate Phosphatases 1 and 3 Are Localized in Distinct Lipid Rafts

Kai, M. et al

1. Biochem., 140, 677-686 (2006)

  Lipid phosphate phosphatases (LPPs), integral membrane proteins with six transmembrane domains, dephosphorylate a variety of extracellular lipid phosphates. Although LPP3 is already known to bind to Triton X-100–insoluble rafts, we here report that LPP1 is also associated with lipid rafts distinct from those harboring LPP3. We found that LPP1 was Triton X-100–soluble, but CHAPS-insoluble in LNCaP cells endogenously expressing LPP1 and several LPP1 cDNA–transfected cells including NIH3T3 fibroblasts. In addition to the non–ionic detergent insolubility, LPP1 further possessed several properties formulated for raft-localizing proteins as follows: first, the CHAPS-insolubility was resistant to the actin-disrupting drug cytochalasin D; second, the CHAPS-insoluble LPP1 floated in an Optiprep density gradient; third, the CHAPS insolubility of LPP1 was lost by cholesterol depletion; and finally, the subcellular distribution pattern of LPP1 exclusively overlapped with that of a raft marker, cholera toxin B subunit. Interestingly, confocal microscopic analysis showed that LPP1 was distributed to membrane compartments distinct from those of LPP3. Analysis using various LPP1/LPP3 chimeras revealed that their first extracellular regions determine the different Triton X-100 solubilities. These results indicate that LPP1 and LPP3 are distributed in distinct lipid rafts that may provide unique microenvironments defining their non-redundant physiological functions.  

3.819           Aquaporin-5 water channel in lipid rafts of rat parotid glands

Ishikawa, Y., Cho, G., Yuan, Z., Inoue, N. and Nakae, Y. Biochim. Biophys. Acta, 1758(8), 1053-1060 (2006)   Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. The activation of M3 muscarinic acetylcholine receptors (mAChRs) or α1-adrenoceptors on the salivary glands induces salivary fluid secretion. AQP5 localizes in lipid rafts and activation of the M3 mAChRs or α1-adrenoceptors induced its translocation together with the lipid rafts to the APM in the interlobular ducts of rat parotid glands. This review focuses on the mechanisms of AQP5 translocation together with lipid rafts to the APM in the interlobular duct cells of parotid glands of normal rats and the impairment of AQP5 translocation in diabetes and senescence.  

3.820           Myelin basic protein-dependent plasma membrane reorganization in the formation of myelin

Fitzner, D. et al EMBO J., 25, 5037-5048 (2006)   During vertebrate development, oligodendrocytes wrap their plasma membrane around axons to produce myelin, a specialized membrane highly enriched in galactosylceramide (GalC) and cholesterol. Here, we studied the formation of myelin membrane sheets in a neuron–glia co-culture system. We applied different microscopy techniques to visualize lipid packing and dynamics in the oligodendroglial plasma membrane. We used the fluorescent dye Laurdan to examine the lipid order with two-photon microscopy and observed that neurons induce a dramatic lipid condensation of the oligodendroglial membrane. On a nanoscale resolution, using stimulated emission depletion and fluorescence resonance energy transfer microscopy, we demonstrated a neuronal-dependent clustering of GalC in oligodendrocytes. Most importantly these changes in lipid organization of the oligodendroglial plasma membrane were not observed in shiverer mice that do not express the myelin basic protein. Our data demonstrate that neurons induce the condensation of the myelin-forming bilayer in oligodendrocytes and that MBP is involved in this process of plasma membrane rearrangement. We propose that this mechanism is essential for myelin to perform its insulating function during nerve conduction.  

3.821           Very Long-chain Fatty Acid-containing Lipids rather than Sphingolipids per se Are Required for Raft Association and Stable Surface Transport of Newly Synthesized Plasma Membrane ATPase in Yeast

Gaigg, B., Toulmay, A. and Scheiter, R.

1. Biol. Chem., 281(45), 34135-34145 (2006)

  The proton-pumping H⁺-ATPase, Pma1p, is an abundant and very long lived polytopic protein of the yeast plasma membrane. Pma1p constitutes a major cargo of the secretory pathway and thus serves as a model to study plasma membrane biogenesis. Pma1p associates with detergent-resistant membrane domains (lipid "rafts") already in the ER, and a lack of raft association correlates with mistargeting of the protein to the vacuole, where it is degraded. We are analyzing the role of specific lipids in membrane domain formation and have previously shown that surface transport of Pma1p is independent of newly synthesized sterols but that sphingolipids with C26 very long chain fatty acid are crucial for raft association and surface transport of Pma1p (Gaigg, B., Timischl, B., Corbino, L., and Schneiter, R. (2005) J. Biol. Chem. 280, 22515-22522). We now describe a more detailed analysis of the function that sphingolipids play in this process. Using a yeast strain in which the essential function of sphingolipids is substituted by glycerophospholipids containing C26 very long chain fatty acids, we find that sphingolipids per se are dispensable for raft association and surface delivery of Pma1p but that the C26 fatty acid is crucial. We thus conclude that the essential function of sphingolipids for membrane domain formation and stable surface delivery of Pma1p is provided by the C26 fatty acid that forms part of the yeast ceramide.  

3.822           Transactivation of Sphingosine 1-Phosphate Receptors Is Essential for Vascular Barrier Regulation: NOVEL ROLE FOR HYALURONAN AND CD44 RECEPTOR FAMILY

Singleton, P.A., Dudek, S.M., Ma, S-F. and Garcia, J.G.N.

1. Biol. Chem., 281(45), 34381-34393 (2006)

  The role for hyaluronan (HA) and CD44 in vascular barrier regulation is unknown. We examined high and low molecular weight HA (HMW-HA, 1,000 kDa; LMW-HA, 2.5 kDa) effects on human transendothelial monolayer electrical resistance (TER). HMW-HA increased TER, whereas LMW-HA induced biphasic TER changes ultimately resulting in EC barrier disruption. HMW-HA induced the association of the CD44s isoform with, and AKT-mediated phosphorylation of, the barrier-promoting sphingosine 1-phosphate receptor (S1P₁) within caveolin-enriched lipid raft microdomains, whereas LMW-HA induced brief CD44s association with S1P₁ followed by sustained association of the CD44v10 isoform with, and Src and ROCK 1/2-mediated phosphorylation of, the barrier-disrupting S1P₃ receptor. HA-induced EC cytoskeletal reorganization and TER alterations were abolished by either disruption of lipid raft formation, CD44 blocking antibody or siRNA-mediated reductions in expression of CD44 isoforms. Silencing S1P₁, AKT1, or Rac1 blocked the barrier enhancing effects of HA whereas silencing S1P₃, Src, ROCK1/2, or RhoA blocked the barrier disruption induced by LMW-HA. In summary, HA regulates EC barrier function through novel differential CD44 isoform interaction with S1P receptors, S1P receptor transactivation, and RhoA/Rac1 signaling to the EC cytoskeleton.  

3.823           Acylation of CD44 and Its Association with Lipid Rafts Are Required for Receptor and Hyaluronan Endocytosis

Thankamony, S.P. and Knudson, W.

1. Biol. Chem., 281(45), 34601-34609 (2006)

  CD44 is a cell surface receptor for the extracellular matrix macromolecule hyaluronan. In addition, CD44 mediates the endocytosis of hyaluronan leading to its subsequent degradation within lysosomes. Using model systems of COS-7 and Flp-293 cells, we demonstrate that the association of CD44 with lipid rafts is essential for the endocytosis of hyaluronan but not the extracellular binding. Further, we demonstrate that palmitoylation of CD44 on two highly conserved cysteine residues is essential for the association with lipid rafts as determined by density gradient ultracentrifugation. Mutations of either cysteine residues or pretreatment of cells with the palmitic acid analog 2-bromopalmitate, reduced the [³H]palmitic acid incorporation into CD44 and prevented CD44-lipid rafts association. Preventing CD44 palmitoylation had no effect on the binding of hyaluronan but inhibited hyaluronan internalization. The turnover of the CD44 receptor itself was also affected by blocking its association with lipid rafts. Using cycloheximide to prevent de novo protein synthesis, palmitoylation-deficient cysteine mutants underwent slower turnover from cell surface compared with the palmitoylation-intact wild type, as determined by immunofluorescence and Western blotting. These results indicate that palmitoylation of CD44 is a critical driving determinant to CD44 association with lipid rafts and, concomitantly, the rates of hyaluronan endocytosis and CD44 turnover from cell surface.  

3.824           Perturbed Interactions of Mutant Proteolipid Protein/DM20 with Cholesterol and Lipid Rafts in Oligodendroglia: Implications for Dysmyelination in Spastic Paraplegia

Krämer-Alber, E-M., Gehrig-Burger, K., Thiele, C., Trotter, J. and Nave, K-A.

1. Neuroscience, 26(45), 11743-11752 (2006)

  Missense mutations in the human PLP1 gene lead to dysmyelinating diseases with a broad range of clinical severity, ranging from severe Pelizaeus–Merzbacher disease (PMD) to milder spastic paraplegia type 2 (SPG-2). The molecular pathology has been generally attributed to endoplasmic reticulum (ER) retention of misfolded proteolipid protein (PLP) (and its splice isoform DM20) and induction of the unfolded protein response. As opposed to previous studies of heterologous expression systems, we have analyzed PLP/DM20 trafficking in oligodendroglial cells, thereby revealing differences between PMD and SPG-2-associated PLP/DM20 isoforms. PLP^A242V and DM20^A242V (jimpy-msd in mice), associated with severe PMD-like phenotype in vivo, were not only retained in the ER but also interfered with oligodendroglial process formation. In contrast, glial cells expressing SPG-2-associated PLP^I186T or DM20^I186T (rumpshaker in mice) developed processes, and mutant PLP/DM20 reached a late endosomal/lysosomal compartment. Unexpectedly, PLP/DM20 with either substitution exhibited impaired cholesterol binding, and the association with lipid raft microdomains was strongly reduced. Turnover analysis demonstrated that mutant PLP was rapidly degraded in oligodendroglial cells, with half-lives for PLP > PLP^I186T > PLP^A242V. Protein degradation was specifically sensitive to proteasome inhibition, although PLP/DM20^I186T degradation was also affected by inhibition of lysosomal enzymes. We conclude that, in addition to ER retention and unfolded protein response (UPR) induction, impaired cholesterol binding and lipid raft association are characteristic cellular defects of PLP1-missense mutations. Mutant protein is rapidly cleared and does not accumulate in oligodendroglial cells. Whereas UPR-induced cell death governs the PMD phenotype of the msd mutation, we propose that impaired cholesterol and lipid raft interaction of the rsh protein may contribute to the dysmyelination observed in SPG-2.  

3.825           Quantitative Proteomics Analysis of Detergent-resistant Membranes from Chemical Synapses: Evidence for Cholesterol as Spatial Organizer of Synaptic Vesicle Cycling

Jia, J-y. et al Mol. Cell. Proteomics, 5, 2060-2071 (2006)   Synaptic vesicles (SVs) in the central nervous system upon stimulation undergo rapid calcium-triggered exoendocytic cycling within the nerve terminal that at least in part depends on components of the clathrin- and dynamin-dependent endocytosis machinery. How exocytic SV fusion and endocytic retrieval are temporally and spatially coordinated is still an open question. One possibility is that specialized membrane microdomains characterized by their high content in membrane cholesterol may assist in the spatial coordination of synaptic membrane protein recycling. Quantitative proteomics analysis of detergent-resistant membranes (DRMs) isolated from rat brain synapses or cholesterol-depleted control samples by liquid chromatography-tandem mass spectrometry identified a total of 159 proteins. Among these 122 proteins were classified as cholesterol-dependent DRM or DRM-associated proteins, many of which with proven or hypothesized functions in exoendocytic vesicle cycling including clathrin, the clathrin adaptor complex AP-2, and a variety of SV proteins. In agreement with this, SV membrane and endocytic proteins displayed a partial resistance to extraction with cold Triton X-100 in cultured rat hippocampal neurons where they co-localized with labeled cholera toxin B, a marker for cholesterol-enriched DRMs. Moreover SV proteins formed cholesterol-dependent complexes in CHAPS-extracted synaptic membrane lysates. Our combined data suggest that lipid microdomains may act as spatial coordinators for exoendocytic vesicle cycling at synapses.  

3.826           Cellular uptake of fatty acids driven by the ER-localized acyl-CoA synthetase FATP4

Milger, K. et al

1. Cell Sci., 119, 4678-4688 (2006)

  Long-chain fatty acids are important metabolites for the generation of energy and the biosynthesis of lipids. The molecular mechanism of their cellular uptake has remained controversial. The fatty acid transport protein (FATP) family has been named according to its proposed function in mediating this process at the plasma membrane. Here, we show that FATP4 is in fact localized to the endoplasmic reticulum and not the plasma membrane as reported previously. Quantitative analysis confirms the positive correlation between expression of FATP4 and uptake of fatty acids. However, this is dependent on the enzymatic activity of FATP4, catalyzing the esterification of fatty acids with CoA. Monitoring fatty acid uptake at the single-cell level demonstrates that the ER localization of FATP4 is sufficient to drive transport of fatty acids. Expression of a mitochondrial acyl-CoA synthetase also enhances fatty acid uptake, suggesting a general relevance for this mechanism. Our results imply that cellular uptake of fatty acids can be regulated by intracellular acyl-CoA synthetases. We propose that the enzyme FATP4 drives fatty acid uptake indirectly by esterification. It is not a transporter protein involved in fatty acid translocation at the plasma membrane.  

3.827           Identification of a Novel Apical Sorting Motif and Mechanism of Targeting of the M2 Muscarinic Acetylcholine Receptor

Chmelar, R.S. and Nathanson, N.M.

1. Biol. Chem., 281(46), 35381-35396 (2006)

  Previous studies have shown that the M₂ receptor is localized at steady state to the apical domain in Madin-Darby canine kidney (MDCK) epithelial cells. In this study, we identify the molecular determinants governing the localization and the route of apical delivery of the M₂ receptor. First, by confocal analysis of a transiently transfected glycosylation mutant in which the three putative glycosylation sites were mutated, we determined that N-glycans are not necessary for the apical targeting of the M₂ receptor. Next, using a chimeric receptor strategy, we found that two independent sequences within the M₂ third intracellular loop can confer apical targeting to the basolaterally targeted M₄ receptor, Val²⁷⁰-Lys²⁸⁰ and Lys²⁸⁰-Ser³⁵⁰. Experiments using Triton X-100 extraction followed by OptiPrep^TM density gradient centrifugation and cholera toxin -subunit-induced patching demonstrate that apical targeting is not because of association with lipid rafts. ³⁵S-Metabolic labeling experiments with domain-specific surface biotinylation as well as immunocytochemical analysis of the time course of surface appearance of newly transfected confluent MDCK cells expressing FLAG-M₂-GFP demonstrate that the M₂ receptor achieves its apical localization after first appearing on the basolateral domain. Domain-specific application of tannic acid of newly transfected cells indicates that initial basolateral plasma membrane expression is required for subsequent apical localization. This is the first demonstration that a G-protein-coupled receptor achieves its apical localization in MDCK cells via transcytosis.  

3.828           The cell biology of HIV-1 and other retroviruses

Freed, E.O. and Mouland, A.J. Retrovirology, 3(77), 1-10 (2006)   In recognition of the growing influence of cell biology in retrovirus research, we recently organized a Summer conference sponsored by the American Society for Cell Biology (ASCB) on the Cell Biology of HIV-1 and other Retroviruses (July 20–23, 2006, Emory University, Atlanta, Georgia). The meeting brought together a number of leading investigators interested in the interplay between cell biology and retrovirology with an emphasis on presentation of new and unpublished data. The conference was arranged from early to late events in the virus replication cycle, with sessions on viral fusion, entry, and transmission; post-entry restrictions to retroviral infection; nuclear import and integration; gene expression/regulation of retroviral Gag and genomic RNA; and assembly/release. In this review, we will attempt to touch briefly on some of the highlights of the conference, and will emphasize themes and trends that emerged at the meeting.  

3.829           Study of proteins associated with the Eimeria tenella refractile body by a proteomic approach

De Venevelles, P. et al Int. J. Parasitol., 36(13), 1399-1407 (2006)   Refractile bodies (RB), whose function is still unknown, are specific structures of Eimeriidae parasites. In order to study their proteome, RB were purified from Eimeria tenella sporozoites by a new procedure using a reversible fixation followed by centrifugation. RB proteins were resolved by two-dimensional electrophoresis. Around 76 and 89 spots were detected on RB two-dimensional gels using gradients in the 3–10 and 4–7 range, respectively. RB proteins were located mainly between pH 5 and 7. RB gels were then compared with previously established maps of the entire sporozoite proteome. Proteins appearing in new spots were identified by mass spectrometry. Thirty protein isoforms were located in RB. Added to the already known RB proteins such as Eimepsin and SO7′, the new RB proteins were defined as haloacid dehalogenase, hydrolase, subtilase, lactacte dehydrogenase or ubiquitin family proteins. The RB proteome analysis confirmed the hypothesis that this structure is a reservoir for proteins necessary to invasion but also suggests that RB have energetic and metabolic functions.  

3.830           Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response

Baumann, S.J. and Kuehn, M.J. Microbes and Infection, 8(9-10), 2400-2408 (2006)   Considerable lung injury results from the inflammatory response to Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF). The P. aeruginosa laboratory strain PAO1, an environmental isolate, and isolates from CF patients were cultured in vitro and outer membrane vesicles from those cultures were quantitated, purified, and characterized. Vesicles were produced throughout the growth phases of the culture and vesicle yield was strain-independent. Strain-dependent differences in the protein composition of vesicles were quantitated and identified. The aminopeptidase PaAP (PA2939) was highly enriched in vesicles from CF isolates. Vesicles from all strains elicited IL-8 secretion by lung epithelial cells. These results suggest that P. aeruginosa colonizing the CF lung may produce vesicles with a particular composition and that the vesicles could contribute to inflammation.  

3.831           Specific and distinct determinants mediate membrane binding and lipid raft incorporation of HIV-1SF2 Nef

Giese, S.I. et al Virology, 355(2), 175-191 (2006)   Membrane association is believed to be a prerequisite for the biological activity of the HIV-1 pathogenicity factor Nef. Attachment to cellular membranes as well as incorporation into detergent-insoluble microdomains (lipid rafts) require the N-terminal myristoylation of Nef. However, this modification is not sufficient for sustained membrane association and a specific raft-targeting signal for Nef has not yet been identified. Using live cell confocal microscopy and membrane fractionation analyses, we found that the N-terminal anchor domain (aa 1–61) is necessary and sufficient for efficient membrane binding of Nef from HIV-1_SF2. Within this domain, highly conserved lysine and arginine residues significantly contributed to Nef's membrane association and localization. Plasma membrane localization of Nef was also governed by an additional membrane-targeting motif between residues 40 and 61. Importantly, two lysines at positions 4 and 7 were not essential for the overall membrane association but critically contributed to Nef's incorporation into lipid raft domains. Cell surface receptor downmodulation was largely unaffected by mutations of all N-terminal basic residues, while the association of Nef with Pak2 kinase activity and its ability to augment virion infectivity correlated with its lysine-mediated raft incorporation. In contrast, all basic residues were required for efficient HIV-1 replication in primary human T lymphocytes but did not contribute to the incorporation of Nef into HIV-1 virions. Together, these results unravel that Nef's membrane association is governed by a complex pattern of signature motifs that differentially contribute to individual Nef activities. The identification of a critical raft targeting determinant and the functional characterization of a membrane-bound, non-raft-associated Nef variant indicate raft incorporation as a regulatory mechanism that determines the biological activity of distinct subpopulations of Nef in HIV-infected cells.  

3.832           Dual regulation of translation initiation and peptide chain elongation during BDNF-induced LTP in vivo: evidence for compartment-specific translation control

Kanhema, T. et al

1. Neurochem., 99, 1328-1337 (2006)

  Protein synthesis underlying activity-dependent synaptic plasticity is controlled at the level of mRNA translation. We examined the dynamics and spatial regulation of two key translation factors, eukaryotic initiation factor 4E (eIF4E) and elongation factor-2 (eEF2), during long-term potentiation (LTP) induced by local infusion of brain-derived neurotrophic factor (BDNF) into the dentate gyrus of anesthetized rats. BDNF-induced LTP led to rapid, transient phosphorylation of eIF4E and eEF2, and enhanced expression of eIF4E protein in dentate gyrus homogenates. Infusion of the extracellular signal-regulated kinase (ERK) inhibitor U0126 blocked BDNF-LTP and modulation of the translation factor activity and expression. Quantitative immunohistochemical analysis revealed enhanced staining of phospho-eIF4E and total eIF4E in dentate granule cells. The in vitro synaptodendrosome preparation was used to isolate the synaptic effects of BDNF in the dentate gyrus. BDNF treatment of synaptodendrosomes elicited rapid, transient phosphorylation of eIF4E paralleled by enhanced expression of α-calcium/calmodulin-dependent protein kinase II. In contrast, BDNF had no effect on eEF2 phosphorylation state in synaptodendrosomes. The results demonstrate rapid ERK-dependent regulation of the initiation and elongation steps of protein synthesis during BDNF-LTP in vivo. Furthermore, the results suggest a compartment-specific regulation in which initiation is selectively enhanced by BDNF at synapses, while both initiation and elongation are modulated at non-synaptic sites.  

3.833           The heterogeneity of NaPi protein dynamics and NaPi cotransport activity in renal brush border membranes

Bliane, J.T. et al FASEB J., 20, A59 (2006)   Alterations in renal proximal tubule brush border membrane (BBM) cholesterol, sphingomyelin, and glycosphingolipid content play an important role in regulating the activity of the sodium-phosphate cotransporter (NaPi). The molecular mechanisms of how alterations in lipid composition may modulate NaPi activity are not known. We have fractionated BBM prepared from rat kidney using detergent-free density gradient ultracentrifugation (OptiPrep). We have found that the NaPi protein preferentially partitions into lipid rafts. To determine the potential consequences of the partitioning of the NaPi transporter in these lipid domains we have i) measured NaPi transport activity and ii) used fluctuation correlation spectroscopy (FCS) methods to determine NaPi diffusion. Partitioning of NaPi protein into lipid rafts results in i) decreased NaPi cotransport activity and ii) decreased diffusion of NaPi protein. Similar results were obtained in BBM isolated from the superficial cortex (SC) versus the juxtamedullary cortex (JMC). The three-fold decrease in NaPi cotransport activity in JMC-BBM is associated with a 3-fold decrease in NaPi protein diffusion. Our results therefore indicate that partitioning of NaPi protein into lipid rafts results in impairment of its activity and diffusion.  

3.834           APPL1 Associates with TrkA and GIPC1 and Is Required for Nerve Growth Factor-Mediated Signal Transduction

Lin, D.C. et al Mol. Cell. Biol., 26(23), 8928-8941 (2006)   The neurotrophin receptor TrkA plays critical roles in the nervous system by recruiting signaling molecules that activate pathways required for the growth and survival of neurons. Here, we report APPL1 as a TrkA-associated protein. APPL1 and TrkA coimmunoprecipitated in sympathetic neurons. We have identified two routes through which this association can occur. APPL1 was isolated as a binding partner for the TrkA-interacting protein GIPC1 from rat brain lysate by mass spectrometry. The PDZ domain of GIPC1 directly engaged the C-terminal sequence of APPL1. This interaction provides a means through which APPL1 may be recruited to TrkA. In addition, the APPL1 PTB domain bound to TrkA, indicating that APPL1 may associate with TrkA independently of GIPC1. Isolation of endosomal fractions by high-resolution centrifugation determined that APPL1, GIPC1, and phosphorylated TrkA are enriched in the same fractions. Reduction of APPL1 or GIPC1 protein levels suppressed nerve growth factor (NGF)-dependent MEK, extracellular signal-regulated kinase, and Akt activation and neurite outgrowth in PC12 cells. Together, these results indicate that GIPC1 and APPL1 play a role in TrkA function and suggest that a population of endosomes bearing a complex of APPL1, GIPC1, and activated TrkA may transmit NGF signals.  

3.835           Molecular Anatomy of a Trafficking Organelle

Takamori, S. et al Cell, 127, 831-846 (2006)   Membrane traffic in eukaryotic cells involves transport of vesicles that bud from a donor compartment and fuse with an acceptor compartment. Common principles of budding and fusion have emerged, and many of the proteins involved in these events are now known. However, a detailed picture of an entire trafficking organelle is not yet available. Using synaptic vesicles as a model, we have now determined the protein and lipid composition; measured vesicle size, density, and mass; calculated the average protein and lipid mass per vesicle; and determined the copy number of more than a dozen major constituents. A model has been constructed that integrates all quantitative data and includes structural models of abundant proteins. Synaptic vesicles are dominated by proteins, possess a surprising diversity of trafficking proteins, and, with the exception of the V-ATPase that is present in only one to two copies, contain numerous copies of proteins essential for membrane traffic and neurotransmitter uptake.  

3.836           ATP-binding Cassette Transporter A1 Expression Disrupts Raft Membrane Microdomains through Its ATPase-related Functions

Landry, Y.D. et al

1. Biol. Chem., 281(47), 36091-36101 (2006)

  ATP-binding cassette transporter A1 (ABCA1) is known to mediate cholesterol efflux to lipid-poor apolipoprotein A-I. In addition, ABCA1 has been shown to influence functions of the plasma membrane, such as endocytosis and phagocytosis. Here, we report that ABCA1 expression results in a significant redistribution of cholesterol and sphingomyelin from rafts to non-rafts. Caveolin, a raft/caveolae marker also redistributes from punctate caveolae-like structures to the general area of the plasma membrane upon ABCA1 expression. Furthermore, we observed significant reduction of Akt activation in ABCA1-expressing cells, consistent with raft disruption. Cholesterol content in the plasma membrane is, however, not altered. Moreover, we provide evidence that a non-functional ABCA1 with mutation in an ATP-binding domain, A937V, fails to redistribute cholesterol, sphingomyelin, or caveolin. A937V also fails to influence Akt activation. Finally, we show that apolipoprotein A-I preferentially associates with non-raft membranes in ABCA1-expressing cells. Our results thus demonstrate that ABCA1 causes a change in overall lipid packing of the plasma membrane, likely through its ATPase-related functions. Such reorganization by ABCA1 effectively expands the non-raft membrane fractions and, consequentially, pre-conditions cells for cholesterol efflux.  

3.837           Localization and in vitro binding studies suggest that the cytoplasmic/nuclear tobacco lectin can interact in situ with high-mannose and complex N-glyc

Lannoo, N. et al FEBS Lett., 580(27), 6329-6337 (2006)   The possible in vivo interaction of the Nicotiana tabacum agglutinin (Nictaba) with endogenous glycoproteins was corroborated using a combination of confocal/electron microscopy of an EGFP-Nictaba fusion protein expressed in tobacco Bright Yellow-2 (BY-2) cells and biochemical analyses. In vitro binding studies demonstrated that the expressed EGFP-Nictaba possesses carbohydrate-binding activity. Microscopic analyses confirmed the previously reported cytoplasmic/nuclear location of Nictaba in jasmonate-treated tobacco leaves and provided evidence for the involvement of a nuclear localization signal-dependent transport mechanism. In addition, it became evident that the lectin is not uniformly distributed over the nucleus and the cytoplasm of BY-2 cells. Far Western blot analysis of extracts from whole BY-2 cells and purified nuclei revealed that Nictaba interacts in a glycan inhibitable way with numerous proteins including many nuclear proteins. Enzymatic deglycosylation with PNGase F indicated that the observed interaction depends on the presence of N-glycans. Glycan array screening, which showed that Nictaba exhibits a strong affinity for high-mannose and complex N-glycans, provided a reasonable explanation for this observation. The cytoplasmic/nuclear localization of a plant lectin that has a high affinity for high-mannose and complex N-glycans and specifically interacts with conspecific glycoproteins suggests that N-glycosylated proteins might be more important in the cytoplasm and nucleus than is currently believed.  

3.838           The role of myristoylation in the membrane association of the Lassa virus matrix protein Z

Strecker, T. et al Virology, J., 3, 93 (2006) The Z protein is the matrix protein of arenaviruses and has been identified as the main driving force for budding. Both LCMV and Lassa virus Z proteins bud from cells in the absence of other viral proteins as enveloped virus-like particles. Z accumulates near the inner surface of the plasma membrane where budding takes place. Furthermore, biochemical data have shown that Z is strongly membrane associated. The primary sequence of Z lacks a typical transmembrane domain and until now it is not understood by which mechanism Z is able to interact with cellular membranes. In this report, we analyzed the role of N-terminal myristoylation for the membrane binding of Lassa virus Z. We show that disruption of the N-terminal myristoylation signal by substituting the N-terminal glycine with alanine (Z-G2A mutant) resulted in a significant reduction of Z protein association with cellular membranes. Furthermore, removal of the myristoylation site resulted in a relocalization of Z from a punctuate distribution to a more diffuse cellular distribution pattern. Finally, treatment of Lassa virus-infected cells with various myristoylation inhibitors drastically reduced efficient Lassa virus replication. Our data indicate that myristoylation of Z is critical for its binding ability to lipid membranes and thus, for effective virus budding.  

3.839           A Membrane-proximal Tetracysteine Motif Contributes to Assembly of CD3 and CD3 Dimers with the T Cell Receptor

Xu, C., Call, M.E. and Wucherpfennig, K.W.

1. Biol. Chem., 281(48), 36977-36984 (2006)

  Assembly of the T cell receptor (TCR) with its dimeric signaling modules, CD3 , CD3 , and , is organized by transmembrane (TM) interactions. Each of the three assembly steps requires formation of a three-helix interface involving one particular basic TCR TM residue and two acidic TM residues of the respective signaling dimer. The extracellular domains of CD3 and CD3 contribute to assembly, but TCR interaction sites on CD3 dimers have not been defined. The structures of the extracellular domains of CD3 and CD3 demonstrated parallel -strands ending at the first cysteine in the CXXCXEXXX motif present in the stalk segment of each CD3 chain. Mutation of the membrane-proximal cysteines impaired assembly of either CD3 dimer with TCR, and little complex was isolated when all four membrane-proximal cysteines were mutated to alanine. These mutations had, however, no discernable effect on CD3 or CD3 dimerization. CD3 assembled with a TCR mutant that lacked both immunoglobulin domains, but shortening of the TCR connecting peptide reduced assembly, consistent with membrane-proximal TCR -CD3 interactions. Chelation of divalent cations did not affect assembly, indicating that coordination of a cation by the tetracysteine motif was not required. The membrane-proximal cysteines were within close proximity but only formed covalent CD3 dimers when one cysteine was mutated. The four cysteines may thus form two intrachain disulfide bonds integral to the secondary structure of CD3 stalk regions. The three-chain interaction theme first established for the TM domains thus extends into the membrane-proximal domains of TCR -CD3 and TCR -CD3 .  

3.840           Human cytosolic sulfotransferase 2B1: Isoform expression, tissue specificity and subcellular localization

Falany, C.N., He, D., Dumas, N., Frost, A.R. and Falany, J.L.

1. Steroid Biochem. Mol. Biol., 102(1-5), 214-221 (2006)

  Sulfation is an important Phase II conjugation reaction involved in the synthesis and metabolism of steroids in humans. Two different isoforms (2B1a and 2B1b) are encoded by the sulfotransferase (SULT) 2B1 gene utilizing different start sites of transcription resulting in the incorporation of different first exons. SULT2B1a and SULT2B1b are 350 and 365 amino acids in length, respectively, and the last 342 aa are identical. Message for both SULT2B1 isoforms is present in human tissues although SULT2B1b message is generally more abundant. However, to date only SULT2B1b protein has been detected in human tissues or cell lines. SULT2B1b is localized in the cytosol and/or nuclei of human cells. A unique 3′-extension of SULT2B1b is required for nuclear localization in human BeWo placental choriocarcinoma cells. Nuclear localization is stimulated by forskolin treatment in BeWo cells and serine phosphorylation has been identified in the 3′-extension. SULT2B1b is selective for the sulfation of 3β-hydroxysteroids such as dehydroepiandrosterone and pregnenolone, and may also have a role in cholesterol sulfation in human skin. The substrate specificity, nuclear localization, and tissue localization of SULT2B1b suggest a role in regulating the responsiveness of cells to adrenal androgens via their direct inactivation or by preventing their conversion to more potent androgens and estrogens.  

3.841           Respiratory Syncytial Virus F Envelope Protein Associates with Lipid Rafts without a Requirement for Other Virus Proteins

Fleming, E.H., Kolokoltsov, A.A., Davey, R.A., Nichols, J.E. and Roberts Jr., N.J.

1. Virol., 80(24), 12160-12170 (2006)

  Like many enveloped viruses, human respiratory syncytial virus (RSV) assembles at and buds from lipid rafts. Translocation of the envelope proteins to these membrane subdomains is essential for production of infectious virus, but the targeting mechanism is poorly understood and it is not known if other virus proteins are required. Here we demonstrate that F protein of RSV intrinsically targets to lipid rafts without a requirement for any other virus protein, including the SH and G envelope proteins. Recombinant virus deficient in SH and G but retaining F protein expression was used to demonstrate that F protein still localized in rafts in both A549 and HEp-2 cells. Expression of a recombinant F gene by use of plasmid vectors demonstrated that F contains its own targeting domain and localized to rafts in the absence of other virus proteins. The domain responsible for translocation was then mapped. Unlike most other virus envelope proteins, F is unusual since the target signal is not contained within the cytoplasmic domain nor did it involve fatty acid modified residues. Furthermore, exchange of the transmembrane domain with that of the vesicular stomatitis virus G protein, a nonraft protein, did not alter F protein raft localization. Taken together, these data suggest that domains present in the extracellular portion of the protein are responsible for lipid raft targeting of the RSV F protein.  

3.842           Transition of Galactosyltransferase 1 from Trans-Golgi Cisterna to the Trans-Golgi Network Is Signal Mediated

Schaub, B.E., Berger, B., Berger, E.G. and Rohrer, J. Mol. Biol. Cell, 17, 5153-5162 (2006)   The Golgi apparatus (GA) is the organelle where complex glycan formation takes place. In addition, it is a major sorting site for proteins destined for various subcellular compartments or for secretion. Here we investigate 1,4-galactosyltransferase 1 (galT) and 2,6-sialyltransferase 1 (siaT), two trans-Golgi glycosyltransferases, with respect to their different pathways in monensin-treated cells. Upon addition of monensin galT dissociates from siaT and the GA and accumulates in swollen vesicles derived from the trans-Golgi network (TGN), as shown by colocalization with TGN46, a specific TGN marker. We analyzed various chimeric constructs of galT and siaT by confocal fluorescence microscopy and time-lapse videomicroscopy as well as Optiprep density gradient fractionation. We show that the first 13 amino acids of the cytoplasmic tail of galT are necessary for its localization to swollen vesicles induced by monensin. We also show that the monensin sensitivity resulting from the cytoplasmic tail can be conferred to siaT, which leads to the rapid accumulation of the galT–siaT chimera in swollen vesicles upon monensin treatment. On the basis of these data, we suggest that cycling between the trans-Golgi cisterna and the trans-Golgi network of galT is signal mediated.  

3.843           PER1 Is Required for GPI-Phospholipase A2 Activity and Involved in Lipid Remodeling of GPI-anchored Proteins

Fujita, M., Umemura, M., Yoko-o, T. and Jigami, Y. Mol. Biol. Cell, 17, 5253-5264 (2006)   Glycosylphoshatidylinositol (GPI) anchors are remodeled during their transport to the cell surface. Newly synthesized proteins are transferred to a GPI anchor, consisting of diacylglycerol with conventional C16 and C18 fatty acids, whereas the lipid moiety in mature GPI-anchored proteins is exchanged to either diacylglycerol containing a C26:0 fatty acid in the sn-2 position or ceramide in Saccharomyces cerevisiae. Here, we report on PER1, a gene encoding a protein that is required for the GPI remodeling pathway. We found that GPI-anchored proteins could not associate with the detergent-resistant membranes in per1 cells. In addition, the mutant cells had a defect in the lipid remodeling from normal phosphatidylinositol (PI) to a C26 fatty acid–containing PI in the GPI anchor. In vitro analysis showed that PER1 is required for the production of lyso-GPI, suggesting that Per1p possesses or regulates the GPI-phospholipase A₂ activity. We also found that human PERLD1 is a functional homologue of PER1. Our results demonstrate for the first time that PER1 encodes an evolutionary conserved component of the GPI anchor remodeling pathway, highlighting the close connection between the lipid remodeling of GPI and raft association of GPI-anchored proteins..  

3.844           Enhanced Amyloidogenic Metabolism of the Amyloid -Protein Precursor in the X11L-deficient Mouse Brain

Sano, Y. et al

1. Biol. Chem., 281(49), 37853-37860 (2006)

  X11L, a neuronal adaptor protein, associates with the cytoplasmic domain of APP and suppresses APP cellular metabolism. APP is the precursor of A , whose metabolism is strongly implicated in Alzheimer disease pathogenesis. To examine the roles of X11L function in APP metabolism, including the generation of A in the brain, we produced X11L-deficient mutant mice on the C57BL/6 background. The mutant mice did not exhibit histopathological alterations or compensatory changes in the expression of other X11 family proteins, X11 and X11L2. The expression level and distribution of APP in the brain of mutant mice were also identical to those in wild-type mice. However, in the hippocampus, where substantial levels of X11L and APP are expressed, the mutant mice exhibited a significant increase in the level of the C-terminal fragments of APP produced by cleavage with -secretase but not -secretase. The levels of A were increased in the hippocampus of aged mutant mice as compared with age-matched controls. These observations clearly indicate that X11L suppresses the amyloidogenic but not amyloidolytic processing of APP in regions of the brain such as the hippocampus, which express significant levels of X11L.  

3.845           Studies of Optineurin, a Glaucoma Gene: Golgi Fragmentation and Cell Death from Overexpression of Wild-Type and Mutant Optineurin in Two Ocular Cell Types

Park, B-C., Shen, X., Samaraweera, M. and Yue, B.Y.J.T. Am. J: Pathol., 169(6), 1976-1989 (2006)   Optineurin (OPTN) has recently been linked to glaucoma, a major cause of blindness worldwide. Mutations in OPTN such as Glu⁵⁰ Lys (E50K) have been reported in patients, particularly those with normal pressure glaucoma. Here, we show that the endogenous OPTN was not secreted in two ocular cell types, human trabecular meshwork and retinal pigment epithelial cells. It localized instead in the cytoplasm in a diffuse pattern without a distinct association with the Golgi apparatus. When overexpressed, however, wild-type OPTN-green fluorescent protein (GFP) formed foci especially around the Golgi, colocalizing partially with the common endocytic pathway marker transferrin receptor in both cell types. Fragmentation of the Golgi was also observed. On nocodazole treatment, the OPTN foci were dispersed into the cytoplasm. Overexpression of mutant OPTN_E50K-GFP resulted in a greater number (P < 0.0055) and size of the foci, compared with the wild type, and the Golgi alteration was potentiated. Cell loss observed in OPTN-expressing cultures was also more pronounced in OPTN_E50K-GFP compared with that of wild-type OPTN-GFP counterparts (P < 0.01). This study highlights a possible role of OPTN in vesicle trafficking and Golgi integrity. It also provides in-sights into the possible mechanisms why E50K would exhibit a propensity toward the development of glaucoma.  

3.846           RalA-exocyst-dependent Recycling Endosome Trafficking Is Required for the Completion of Cytokinesis

Chen, X-W., Inoue, M., Hsu, S. and Saltiel, A.R.

1. Biol. Chem., 281(50), 38609-38616 (2006)

  In eukaryotic cells, recycling endosome-mediated trafficking contributes to the completion of cytokinesis, in a manner under the control of the centrosome. We report that the exocyst complex and its interacting GTPase RalA play a critical role in this polarized trafficking process. RalA resides in the recycling endosome and relocates from the pericentrosomal region to key cytokinetic structures including the cleavage furrow, and later, the abscission site. This event is coupled to the dynamic redistribution of the exocyst proteins. These associate with the centrosome in interphase and concentrate on the central spindle/midbody during cytokinesis. Disruption of RalA-exocyst function leads to cytokinesis failure in late stages, particularly abscission, resembling the cytokinesis defects induced by loss of centrosome function. These data suggest that RalA and the exocyst may regulate vesicle delivery to the centrosome-related abscission site during the terminal stage of cytokinesis, implicating RalA as a critical regulator of cell cycle progression.  

3.847           Peroxisomal membrane permeability and solute transfer

Antonenkov, V.D. anfd Hiltunen, J.K. Biochim. Biophys. Acta, Mol. Cell Res., 1763 (2006)   The review is dedicated to recent progress in the study of peroxisomal membrane permeability to solutes which has been a matter of debate for more than 40 years. Apparently, the mammalian peroxisomal membrane is freely permeable to small solute molecules owing to the presence of pore-forming channels. However, the membrane forms a permeability barrier for ‘bulky’ solutes including cofactors (NAD/H, NADP/H, CoA, and acetyl/acyl-CoA esters) and ATP. Therefore, peroxisomes need specific protein transporters to transfer these compounds across the membrane. Recent electrophysiological studies have revealed channel-forming activities in the mammalian peroxisomal membrane. The possible involvement of the channels in the transfer of small metabolites and in the formation of peroxisomal shuttle systems is described.  

3.848           Transition of Galactosyltransferase 1 from Trans-Golgi Cisterna to the Trans Golgi Network (and Back) is Signal Mediated

Schaub, B., Berger, B., Berger, E. and Rohrer, J. Mol. Biol. Cell, 17 (Suppl), abstract 1814 (2006)   The Golgi apparatus (GA) is the organelle where complex glycan formation takes place. In addition, it is a major sorting site for proteins destined for various sub-cellular compartments or for secretion. Here we investigate β1,4-galactosyltransferase 1 (galT) and α2,6-sialyltransferase 1 (siaT), two trans-Golgi glycosyltransferases, with respect to their different pathways in monensin treated cells. Upon addition of monensin galT dissociates from siaT and the GA and accumulates in swollen vesicles derived from the trans-Golgi network (TGN), as shown by co-localization with TGN46, a specific TGN marker. We analyzed various chimeric constructs of galT and siaT by confocal fluorescence microscopy and time lapse video-microscopy as well as Optiprep density gradient fractionation. We show that the first 13 amino acids of the cytoplasmic tail of galT are necessary and sufficient for its localization to swollen vesicles induced by monensin. We also show that the monensin sensitivity resulting from these 13 amino acids can be conferred to siaT, which leads to the rapid accumulation of the galT-siaT chimera in swollen vesicles upon monensin treatment. Based on these data we suggest that cycling between the trans-Golgi cisterna and the trans-Golgi network of galT is signal mediated.  

3.849           Characterization of Membrane-associated APC Pools in Motile versus Non-Motile Epithelial Cells

Siemers, K.A., Caro-Gonzales, H.Y., Nelson, W.J. and Barth, A.I.M. Mol. Biol. Cell, 17 (Suppl.), abstract 299 (2006)   The adenomatous polyposis coli (APC) protein has an important role in directed cell migration and localizes to the tip of extending membranes in response to growth factor or integrin-mediated signals. Here we used a biochemical approach to analyze the association of APC and its binding partners with membranes in motile and non-motile epithelial cells. In non-polarized motile Madin Darby Canine kidney (MDCK) epithelial cells, homogenized under conditions that disrupt the microtubule cytoskeleton, the majority of APC and its binding partners Asef and β-catenin were recovered in the membrane fraction whereas tubulin was cytosolic. To further analyze the different cytosolic and membrane-associated APC pool we used a self-forming iodixanol density gradient. APC was enriched in two membrane fractions: a lower density membrane fraction that co-distributes with E-cadherin and β-catenin and a higher density membrane fraction that co-distributes with Asef, IQGAP and Par-3. In polarized non-motile MDCK epithelial cells the majority of APC was enriched in the lower density membrane fraction with E-cadherin. Disruption of cell-cell adhesion causes a shift of APC from this lower density “E-cadherin”-membrane fraction into the higher density “IQGAP/Asef”-membrane fraction. These results indicate that APC complexes that distribute in these higher density fractions may have a role in cell motility.  

3.850           Comprehensive Protein Analysis of Naked2-associated Exocytic Vesicles by LC/MS-MS

Cao, Z. et al Mol. Biol. Cell, 17, (Suppl.), abstract 2621 (2006)   Polarized epithelial cells have developed specialized mechanisms for targeting transGolgi network (TGN)-derived vesicles to the apical or basolateral membrane. We have reported that Naked2, but not Naked1, interacts with Golgi-processed form of TGFα and escorts TGFα-containing exocytic vesicles from the TGN to the basolateral surface of polarized MDCK cells, where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. In myristoylation-deficient (G2A) Naked2-expressing MDCK cells, Naked2-associated TGFα-containing vesicles are trapped in the cytoplasm and TGFα is unable to reach the plasma membrane. To determine the protein compositions of these Naked2-associated vesicles, we performed a biochemical enrichment followed by flow cytometric purification. Using a 10-40% iodixanol gradient centrifugation, we first isolated G2A Naked2-EGFP-containing vesicle fractions de-enriched in plasma membrane markers as measured by western blotting for Naked2 and markers of subcellular compartments. This pool of vesicles was then subjected to dual flow sorting using GFP and DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate) lipid incorporation to achieve 99.3% purity. Proteomic analysis of this flow-sorted population of vesicles by LC/MS-MS has identified 296 proteins using a criterion of 3 or more peptides per protein present in order to be included in the list. This list includes known trafficking proteins as well as new and previously unrecognized potential trafficking proteins. To date, we have validated colocalization of Annexin I and II in both wild-type and G2A Naked2-associated vesicles. To our knowledge, this represents the first example of a comprehensive protein characterization of a subset of exocytic vesicles.  

3.851           Trafficking of Shiga toxin/Shiga-like toxin-1 in human glomerular microvascular endothelial cells and human mesangial cells

Warnier, M. et al Kidney Int., 70(12), 2085-2091 (2006)   This study has determined the intracellular transport route of Shiga-like toxin (Stx) and the highly related Shiga toxin in human glomerular microvascular endothelial cells (GMVECs) and mesangial cells. In addition, the effect of tumor necrosis factor- (TNF- ), which contributes to the pathogenesis of hemolytic-uremic syndrome, was evaluated more profound. Establishing the transport route will provide better understanding of the cytotoxic effect of Stx on renal cells. For our studies, we used receptor-binding B-subunit (StxB), which is identical between Shiga toxin and Stx-1. The transport route of StxB was studied by immunofluorescence microscopy and biochemical assays that allow quantitative analysis of retrograde transport from plasma membrane to Golgi apparatus and endoplasmic reticulum (ER). In both cell types, StxB was detergent-resistant membrane associated and followed the retrograde route. TNF- upregulated Gb3 expression in mesangial cells and GMVECs, without affecting the efficiency of StxB transport to the ER. In conclusion, our study shows that in human GMVECs and mesangial cells, StxB follows the retrograde route to the Golgi apparatus and the ER. TNF- treatment increases the amount of cell-associated StxB, but not retrograde transport as such, making it likely that the strong TNF- -induced sensitization of mesangial cells and GMVECs for the toxic action of Stx is not due to a direct effect on the intracellular trafficking of the toxin.  

3.852           Tankyrase recruitment to the lateral membrane in polarized epithelial cells: regulation by cell–cell contact and protein poly(ADP-ribosyl)ation

Yeh, T-Y. et al Biochem. J., 399(3), 415-425 (2006)   PARsylation [poly(ADP-ribosyl)ation] of proteins is implicated in the regulation of diverse physiological processes. Tankyrase is a molecular scaffold with this catalytic activity and has been proposed as a regulator of vesicular trafficking on the basis, in part, of its Golgi localization in non-polarized cells. Little is known about tankyrase localization in polarized epithelial cells. Using MDCK (Madin–Darby canine kidney) cells as a model, we found that E-cadherin-mediated intercellular adhesion recruits tankyrase from the cytoplasm to the lateral membrane (including the tight junction), where it stably associates with detergent-insoluble structures. This recruitment is mostly completed within 8 h of calcium-induced formation of cell–cell contact. Conversely, when intercellular adhesion is disrupted by calcium deprivation, tankyrase returns from the lateral membrane to the cytoplasm and becomes more soluble in detergents. The PARsylating activity of tankyrase promotes its dissociation from the lateral membrane as well as its ubiquitination and proteasome-mediated degradation, resulting in an apparent protein half-life of ~2 h. Inhibition of tankyrase autoPARsylation using H₂O₂-induced NAD⁺ depletion or PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide hydrochloride] treatment results in tankyrase stabilization and accumulation at the lateral membrane. By contrast, stabilization through proteasome inhibition results in tankyrase accumulation in the cytoplasm. These data suggest that cell–cell contact promotes tankyrase association with the lateral membrane, whereas PARsylating activity promotes translocation to the cytosol, which is followed by ubiquitination and proteasome-mediated degradation. Since the lateral membrane is a sorting station that ensures domain-specific delivery of basolateral membrane proteins, the regulated tankyrase recruitment to this site is consistent with a role in polarized protein targeting in epithelial cells.  

3.853           Quantitative proteomic approach to study subcellular localization of membrane proteins

Sadowski, P.G. et al Nature Protocols, 1(4), 1778-1789 (2006)   As proteins within cells are spatially organized according to their role, knowledge about protein localization gives insight into protein function. Here, we describe the LOPIT technique (localization of organelle proteins by isotope tagging) developed for the simultaneous and confident determination of the steady-state distribution of hundreds of integral membrane proteins within organelles. The technique uses a partial membrane fractionation strategy in conjunction with quantitative proteomics. Localization of proteins is achieved by measuring their distribution pattern across the density gradient using amine-reactive isotope tagging and comparing these patterns with those of known organelle residents. LOPIT relies on the assumption that proteins belonging to the same organelle will co-fractionate. Multivariate statistical tools are then used to group proteins according to the similarities in their distributions, and hence localization without complete centrifugal separation is achieved. The protocol requires approximately 3 weeks to complete and can be applied in a high-throughput manner to material from many varied sources.  

3.854           Physiological Mouse Brain Aβ Levels Are Not Related to the Phosphorylation State of Threonine-668 of Alzheimer's APP

Sano, Y. et al PloS, 1, e51 (2006)   Background Amyloid-β peptide species ending at positions 40 and 42 (Aβ40, Αβ42) are generated by the proteolytic processing of the Alzheimer's amyloid precursor protein (APP). Aβ peptides accumulate in the brain early in the course of Alzheimer's disease (AD), especially Aβ42. The cytoplasmic domain of APP regulates intracellular trafficking and metabolism of APP and its carboxyl-terminal fragments (CTFα, CTFβ). The role of protein phosphorylation in general, and that of the phosphorylation state of APP at threonine-668 (Thr⁶⁶⁸) in particular, has been investigated in detail by several laboratories (including our own). Some investigators have recently proposed that the phosphorylation state of Thr⁶⁶⁸ plays a pivotal role in governing brain Aβ levels, prompting the current study. Methodology In order to evaluate whether the phosphorylation state of Thr⁶⁶⁸ controlled brain Aβ levels, we studied the levels and subcellular distributions of holoAPP, sAPPα, sAPPβ, CTFα, CTFβ, Aβ40 and Aβ42 in brains from “knock-in” mice in which a non-phosphorylatable alanyl residue had been substituted at position 668, replacing the threonyl residue present in the wild-type protein. Conclusions The levels and subcellular distributions of holoAPP, sAPPα, sAPPβ, CTFα, CTFβ, Aβ40 and Aβ42 in the brains of Thr⁶⁶⁸Ala mutant mice were identical to those observed in wild-type mice. These results indicate that, despite speculation to the contrary, the phosphorylation state of APP at Thr⁶⁶⁸ does not play an obvious role in governing the physiological levels of brain Aβ40 or Αβ42 in vivo.  

3.855           Cellular Prion Protein and Caveolin-1 Interaction in a Neuronal Cell Line Precedes Fyn/Erk 1/2 Signal Transduction

Toni, M. et al

1. Biomed. Biotechnol., 206, 1-13 (2006)

  It has been reported that cellular prion protein (PrPc) is enriched in caveolae or caveolae-like domains with caveolin-1 (Cav-1) participating to signal transduction events by Fyn kinase recruitment. By using the Glutathione-S-transferase (GST)-fusion proteins assay, we observed that PrPc strongly interacts in vitro with Cav-1. Thus, we ascertained the PrPc caveolar localization in a hypothalamic neuronal cell line (GN11), by confocal microscopy analysis, flotation on density gradient, and coimmunoprecipitation experiments. Following the anti-PrPc antibody-mediated stimulation of live GN11 cells, we observed that PrPc clustered on plasma membrane domains rich in Cav-1 in which Fyn kinase converged to be activated. After these events, a signaling cascade through p42/44 MAP kinase (Erk 1/2) was triggered, suggesting that following translocations from rafts to caveolae or caveolae-like domains PrPc could interact with Cav-1 and induce signal transduction events.  

3.856           Presenilin-Dependent -Secretase on Plasma Membrane and Endosomes Is Functionally Distinct

Fukumori, A. et al Biochemistry, 45, 4907-4914 (2006)   The presenilin (PS)/ -secretase complex, which contains not only PS but also Aph-1, PEN-2, and nicastrin, mediates proteolysis of the transmembrane domain of -amyloid protein precursor ( APP). Intramembrane proteolysis occurs at the interface between the membrane and cytosol ( -site) and near the middle of the transmembrane domain ( -site), generating the APP intracellular domain (AICD) and Alzheimer disease-associated A , respectively. Both cleavage sites exhibit some diversity. Changes in the precision of -cleavage, which potentially results in secretion of pathogenic A 42, have been intensively studied, while those of -cleavage have not. Although a number of PS-associated factors have been identified, it is unclear whether any of them physiologically regulate the precision of cleavage by PS/ -secretase. Moreover, there is currently no clear evidence of whether PS/ -secretase function differs according to the subcellular site. Here, we show that endocytosis affects the precision of PS-dependent -cleavage in cell culture. Relative production of longer AICD 49 increases on the plasma membrane, whereas that of shorter AICD 51 increases on endosomes; however, this occurs without a concomitant major change in the precision of cleavage at -sites. Moreover, very similar changes in the precision of -cleavage are induced by alteration of the pH. Our findings demonstrate that the precision of -cleavage by PS/ -secretase changes depending upon the conditions and the subcellular location. These results suggest that the precision of cleavage by the PS/ -secretase complex may be physiologically regulated by the subcellular location and conditions.  

3.857           Structure and Cholesterol Dynamics of Caveolae/Raft and Nonraft Plasma Membrane Domains

Gallegos, A.M., Storey, S.M., Kier, A.B., Schroeder, F. and Ball, J.M. Biochemistry, 45, 12100-12116 (2006)   Despite recognition that the plasma membrane (PM) is comprised of lipid raft domains that are key organizing sites of multiple signaling pathways and other cell functions, limited information is available regarding the structure and function in sterol dynamics of these microdomains. To begin to resolve these issues, MDCK membranes were subfractionated by three different techniques to produce (i) detergent-resistant membranes (DRM) and detergent-soluble membranes (DSM), (ii) nondetergent caveolae/rafts (NDCR), and (iii) nondetergent, affinity-purified caveolae/rafts (ACR) and noncaveolae/nonrafts (NR). ACR exhibited the least cross contamination with other PM domains or intracellular membranes, in marked contrast to DRM that contained the highest level of cross contaminants. Spectral properties of dehydroergosterol (DHE), a naturally occurring fluorescent sterol, showed that ACR, NDCR, and NR did not contain crystalline sterol, consistent with the lack of crystalline sterol in PM of intact cells. In contrast, DRM contained significant levels of crystalline sterol. Fluorescence polarization of membrane probes showed that ACR were the least fluid and had the highest transbilayer fluidity gradient, the most liquid ordered phase, and the sterol dynamics most responsive to sterol carrier protein-2 (SCP-2). In contrast, DRM had structural properties similar to those of NR, anomalous (very fast) spontaneous sterol dynamics, and sterol dynamics that were unresponsive to SCP-2. Differences between the structural and functional properties of DRM and those of the nondetergent preparations (ACR and NDCR) were not due to the presence of detergent. A nondetergent, affinity-purified (ACR) lipid domain fraction isolated from MDCK cells for the first time revealed unique structural (noncrystalline sterol, liquid-ordered, high transbilayer fluidity gradient) and functional (cholesterol dynamics) properties of lipid rafts as compared to nonrafts (NR). In summary, this study showed membrane microdomains (rafts/caveolae) isolated by three different methodologies have unique structural, functional, and organizational characteristics.  

3.858           A role for ion channels in glioma cell invasion

McFerrin, M.B. and Sontheimer, H. Neuron Glia Biol., 2(1), 39-49 (2006)   Many cells, including neuronal and glial progenitor cells, stem cells and microglial cells, have the capacity to move through the extracellular spaces of the developing and mature brain. This is particularly pronounced in astrocyte-derived tumors, gliomas, which diffusely infiltrate the normal brain. Although a significant body of literature exists regarding signals that are involved in the guidance of cells and their processes, little attention has been paid to cell-shape and cell-volume changes of migratory cells. However, extracellular spaces in the brain are very narrow and represent a major obstacle that requires cells to dynamically regulate their volume. Recent studies in glioma cells show that this involves the secretion of Cl⁻ and K⁺ with water. Pharmacological inhibition of Cl⁻ channels impairs their ability to migrate and limits tumor progression in experimental tumor models. One Cl⁻-channel inhibitor, chlorotoxin, is currently in Phase II clinical trials to treat malignant glioma. This article reviews our current knowledge of cell-volume changes and the role of ion channels during the migration of glioma cells. It also discusses evidence that supports the importance of channel-mediated cell-volume changes in the migration of immature neurons and progenitor cells during development. New unpublished data is presented, which demonstrates that Cl⁻ and K⁺ channels involved in cell shrinkage localize to lipid-raft domains on the invadipodia of glioma cells and that their presence might be regulated by trafficking of these proteins in and out of lipid rafts.  

3.859           Comparative proteomics of glycosomes from bloodstream form and procyclic culture form Trypanosoma brucei brucei 

Colasante, C., Ellis, M., Ruppert, T. And Voncken, F. Proteomics, 6(11), 3275-3293 (2006)   Peroxisomes are present in nearly every eukaryotic cell and compartmentalize a wide range of important metabolic processes. Glycosomes of Kinetoplastid parasites are peroxisome-like organelles, characterized by the presence of the glycolytic pathway. The two replicating stages of Trypanosoma brucei brucei, the mammalian bloodstream form (BSF) and the insect (procyclic) form (PCF), undergo considerable adaptations in metabolism when switching between the two different hosts. These adaptations involve also substantial changes in the proteome of the glycosome. Comparative (non-quantitative) analysis of BSF and PCF glycosomes by nano LC-ESI-Q-TOF-MS resulted in the validation of known functional aspects of glycosomes and the identification of novel glycosomal constituents.  

3.860           Proteomic Analysis of Mature Melanosomes from the Retinal Pigmented Epithelium

Azarian, S.M., McLeod, I., Lillo, C., Gibbs, D., Yates, J.R. and Williams, D.S.

1. Proteome Res., 5(3), 521-529 (2006)

  The protein content of melanosomes in the retinal pigment epithelium (RPE) was analyzed by mass spectrometry. More than 100 proteins were found to be common to two out of three variations of sample preparation. Some proteins normally associated with other organelles were detected. Several lysosomal enzymes were detected, with the presence of cathepsin D confirmed by immunoelectron microscopy, thus supporting the previously suggested notion that melanosomes may contribute to the degradation of ingested photoreceptor outer segment disks.  

3.861           Insights into the membrane proteome of rat liver peroxisomes: Microsomal glutathione-S-transferase is shared by both subcellular compartments

Islinger, M., Lüers, G.H., Zischka, H., Ueffing, M. and Völkl, A. Proteomics, 6(3), 804-816 (2006)   Peroxisomes are ubiquitous “multipurpose” organelles of eukaryotic cells. Their matrix enzymes catalyze mainly catabolic and anabolic reactions of lipid metabolism, thus contributing to the regulation of lipid homeostasis. Since most metabolites must be actively transported across the peroxisomal membrane and since individual proteins and protein complexes play functional roles in such transport processes, we analyzed the peroxisomal membrane proteome. Benzyldimethyl-n-hexadecylammoniumchloride (16-BAC)/SDS-2-D-PAGE and mass spectrometry were used to characterize the proteomes of highly purified “light” and “heavy” peroxisomes of rat liver obtained by density gradient centrifugation. In both populations, the major integral membrane proteins could be detected in high concentrations, verifying 16-BAC/SDS-2-D-PAGE as a suitable tool for the preparation of membrane proteomes destined for mass spectrometric analysis. Both reliable and reproducible detection of a distinct set of microsomal (ER) membrane proteins, including microsomal glutathione-S-transferase (mGST), in light and heavy peroxisomal fractions was also possible. Compared with the abundance of most microsomal membrane proteins, we found mGST to be specifically enriched in peroxisomal membrane fractions. Furthermore, C terminus epitope-tagged mGST versions were localized at least in part to peroxisomes in different mammalian cell lines. Taken together, these data suggest that the peroxisomal GST is not a mere ER-contaminant, but a bona fide protein comprising the membrane proteome of both intracellular compartments. In addition, we could detect several mitochondrial proteins in light peroxisome fractions. This finding may likely indicate a physical association of light peroxisomes with mitochondria, since the organelles could be partly separated by mechanical stress. Whether this association is of functional importance awaits further investigation.  

3.862           Mitochondrial protein patterns correlating with impaired insulin secretion from INS-1E cells exposed to elevated glucose concentrations

Nyblom, H.K., Thorn, K., Ahmed, M. and Bergsten, P. Proteomics, 6(19), 5193-5198 (2006)   Extended hyperglycaemia leads to impaired glucose-stimulated insulin secretion (GSIS) and eventually β-cell apoptosis in individuals with type 2 diabetes mellitus. In an attempt to dissect mechanisms behind the detrimental effects of glucose, we focused on measuring changes in expression patterns of mitochondrial proteins. Impaired GSIS was observed from INS-1E cells cultured for 5 days at 20 or 27 mM glucose compared to cells cultured at 5.5 or 11 mM glucose. After culture, mitochondria were isolated from the INS-1E cells by differential centrifugation. Proteins of the mitochondrial fraction were bound to a strong anionic surface (SAX2) protein array and mass spectra generated by SELDI-TOF-MS. Analysis of the spectra revealed proteins with expression levels that correlated with the glucose concentration of the culture medium. Indeed, such differentially expressed proteins created patterns of protein changes, which correlated with impairment of GSIS. In conclusion, the study reveals the first glucose-induced differentially expressed patterns of β-cell mitochondrial proteins obtained by SELDI-TOF-MS.  

3.863           Proteomic analysis of detergent-resistant membranes from Candida albicans

Insenser, M., Nombela, C., Molero, G. and Gil, C. Proteomics, 6, Suppl. 1., S74-S81 (2006)   Lipid rafts are membrane microdomains with a higher amount of saturated fatty acids and sterols than the rest of the membrane. They are more resistant to the action of non-anionic detergents, and are called, for this reason, detergent-resistant membranes (DRMs). Lipid rafts are involved in many cellular processes, like signaling, cytokinesis, response to environment, etc., and therefore must contain important proteins. We have obtained a fraction enriched in proteins from Candida albicans DRMs. The sample has been analyzed by SDS-PAGE and 29 proteins have been identified including markers for lipid rafts in Saccharomyces cerevisiae, like Pma1p and a glycosylphosphatidylinositol (GPI)-anchored protein belonging to the Phr family. Ecm33p, a GPI-anchored protein involved in cell wall biogenesis, has been found for the first time in lipid rafts. We have also identified proteins implicated in protein glycosylation, like the mannosyltransferases Mnn7p, Pmt2p and Mnt1p; proteins involved in lipid metabolism, like Erg11p and Scs7p; and heat shock proteins, like Ssa1p and Hsp90p. Most of the proteins identified are located in plasma, mitochondrial, Golgi or ER membranes, supporting the postulated existence of lipid-raft domains in all the membranes.  

3.864           Functionally different pools of Shiga toxin receptor, globotriaosyl ceramide, in HeLa cells

Falguierres, T., Römer, W., Amessou, M., Afonso, C., Wolf, C., Tabet, J-C., Lamaze, C. and Johannes, L. FEBS J., 273(22), 5205-5218 (2006)   Many studies have investigated the intracellular trafficking of Shiga toxin, but very little is known about the underlying dynamics of its cellular receptor, the glycosphingolipid globotriaosyl ceramide. In this study, we show that globotriaosyl ceramide is required not only for Shiga toxin binding to cells, but also for its intracellular trafficking. Shiga toxin induces globotriaosyl ceramide recruitment to detergent-resistant membranes, and subsequent internalization of the lipid. The globotriaosyl ceramide pool at the plasma membrane is then replenished from internal stores. Whereas endocytosis is not affected in the recovery condition, retrograde transport of Shiga toxin to the Golgi apparatus and the endoplasmic reticulum is strongly inhibited. This effect is specific, as cholera toxin trafficking on GM₁ and protein biosynthesis are not impaired. The differential behavior of both toxins is also paralleled by the selective loss of Shiga toxin association with detergent-resistant membranes in the recovery condition, and comparison of the molecular species composition of plasma membrane globotriaosyl ceramide indicates subtle changes in favor of unsaturated fatty acids. In conclusion, this study demonstrates the dynamic behavior of globotriaosyl ceramide at the plasma membrane and suggests that globotriaosyl ceramide-specific determinants, possibly its molecular species composition, are selectively required for efficient retrograde sorting on endosomes, but not for endocytosis.  

3.865           Isolation and analysis of lipid rafts in cell-cell interactions

Landry, A. and Xavier, r. Methods Mol. Biol., 341, 251-282 (2006)   Lipid rafts are dynamic structures made up of proteins and lipids that float freely within the liquid-disordered bilayer of cellular membranes and have the ability to cluster to form larger, more-ordered platforms. These clustered structures have been identified in all cell types and have been shown to play critical roles in signal transduction, cellular transport, and cell-cell communication. Lipid rafts also have been implicated in facilitating bacterial/viral entry into host cells and in human disease, highlighting the significance of understanding the role lipid rafts play in physiological and pathological signaling outcomes. In this chapter, we provide protocols to isolate lipid rafts from polarized and nonpolarized cells and outline novel technologies to analyze signal transduction cascades in vivo.  

3.866           Cardiac mitochondrial connexin 43 regulates apoptosis

Goubaeva, F. et al Biochem. Biophys. Res. Comm., 352(1), 97-103 (2007)   Connexin 43 (Cx43) is thought to be present largely in the plasma membrane and its function solely to provide low resistance electrical connection between myocytes. A recent report suggested the presence of Cx43 in the mitochondria as well. We confirmed the presence of Cx43 in the mitochondria isolated from adult rat ventricles with the Cx43 immunoreactivity fractionating to the outer mitochondrial membrane. Mitochondrial Cx43 is mostly phosphorylated only detected by a phospho-specific antibody. Using a Ca²⁺-sensitive electrode and Western blot, we showed that the gap junction inhibitors 18-β-glycyrrhetinic acid (β-GA), oleamide, and heptanol all induced concomitant release of Ca²⁺ and cytochrome C in isolated mitochondria whereas the inactive analog 18-β-glycyrrhizic acid failed to do so. In low density neonatal myocyte culture with no appreciable cell–cell contacts, β-GA induced apoptosis as assessed by TUNEL staining. Our results suggest a novel role of Cx43 as a regulator of mitochondrial physiology and myocyte apoptosis.  

3.867           Signaling proteins in raft-like microdomains are essential for Ca2+ wave propagation in glial cells

Weerth, S.H., Holtzclaw, L.A. and Russell, J.T. Cell Calsium, 41(1), 155-167 (2007)   The hypothesis that calcium signaling proteins segregate into lipid raft-like microdomains was tested in isolated membranes of rat oligodendrocyte progenitor (OP) cells and astrocytes using Triton X-100 solubilization and density gradient centrifugation. Western blot analysis of gradient fractions showed co-localization of caveolin-1 with proteins involved in the Ca²⁺ signaling cascade. These included agonist receptors, P2Y₁, and M₁, TRPC1, IP₃R2, ryanodine receptor, as well as the G protein Gα_q and Homer. Membranes isolated from agonist-stimulated astrocytes showed an enhanced recruitment of phospholipase C (PLCβ1), IP₃R2 and protein kinase C (PKC-α) into lipid raft fractions. IP₃R2, TRPC1 and Homer co-immunoprecipitated, suggesting protein–protein interactions. Disruption of rafts by cholesterol depletion using methyl-β-cyclodextrin (β-MCD) altered the distribution of caveolin-1 and GM1 to non-raft fractions with higher densities. β-MCD-induced disruption of rafts inhibited agonist-evoked Ca²⁺ wave propagation in astrocytes and attenuated wave speeds. These results indicate that in glial cells, Ca²⁺ signaling proteins might exist in organized membrane microdomains, and these complexes may include proteins from different cellular membrane systems. Such an organization is essential for Ca²⁺ wave propagation.    

3.868           Characterisation of lipofuscin-like lysosomal inclusion bodies from human placenta

Schröder, B., Elsässer, H-P-. Schmidt, B. and Hasilik, A. FEBS Lett., 581(1), 102-108 (2007)   A structural hallmark of lysosomes is heterogeneity of their contents. We describe a method for isolation of particulate materials from human placental lysosomes. After a methionine methyl ester-induced disruption of lysosomes and two density gradient centrifugations we obtained a homogeneous membrane fraction and another one enriched in particulate inclusions. The latter exhibited a yellow-brown coloration and contained bodies lacking a delimiting membrane, which were characterised by a granular pattern and high electron density. The lipofuscin-like inclusion materials were rich in tripeptidyl peptidase I, β-glucuronidase, acid ceramidase and apolipoprotein D and contained proteins originating from diverse subcellular localisations. Here we show that human term placenta contains lipofuscin-like lysosomal inclusions, a phenomenon usually associated with senescence in postmitotic cells. These findings imply that a simple pelleting of a lysosomal lysate is not appropriate for the isolation of lysosomal membranes, as the inclusions tend to be sedimented with the membranes.  

3.869           Activation-induced endocytosis of the raft-associated transmembrane adaptor protein LAB/NTAL in B lymphocytes: evidence for a role in internalization of the B cell receptor

Mutch, C.M. et al Int. Immunol., 19(1), 19-30 (2007)   Linker for activation of B cell (LAB)/non-T cell activation linker (NTAL) and phosphoprotein associated with glycophospholipid-enriched membrane microdomain (PAG)/Csk-binding protein (Cbp) are raft-associated transmembrane adaptor proteins with distinct functions in immediate/early phases of receptor signaling pathways. Heterogeneous rafts are thought to compartmentalize membrane-associated signaling events. In order to investigate the subcellular localization of LAB/NTAL and PAG/Cbp, they were expressed as fluorescent chimeric fusion proteins in a human B cell line and their distribution was examined, along with the corresponding endogenous proteins, before and after B cell receptor (BCR) stimulation. Both adaptors were distributed predominantly at the plasma membrane in resting cells and co-clustered with other raft-associated proteins; however, they distributed differently in buoyant membranes isolated by either detergent resistance or non-detergent methods, indicating that they might localize to distinct rafts. After activation, LAB/NTAL was internalized and co-localized with the BCR while PAG/Cbp remained on the cell surface. BCR internalization was reduced in LAB/NTAL-deficient murine B cells, suggesting a regulatory role for LAB/NTAL in activation-induced internalization of the BCR. The cytoplasmic domain of LAB/NTAL, and not the transmembrane/juxtamembrane region, was found to be essential for its internalization.  

3.870           Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response

McBroom, A.J. and Kuehn, M.J. Mol. Microbiol., 63(2), 545-558 (2007)   Conditions that impair protein folding in the Gram-negative bacterial envelope cause stress. The destabilizing effects of stress in this compartment are recognized and countered by a number of signal transduction mechanisms. Data presented here reveal another facet of the complex bacterial stress response, release of outer membrane vesicles. Native vesicles are composed of outer membrane and periplasmic material, and they are released from the bacterial surface without loss of membrane integrity. Here we demonstrate that the quantity of vesicle release correlates directly with the level of protein accumulation in the cell envelope. Accumulation of material occurs under stress, and is exacerbated upon impairment of the normal housekeeping and stress-responsive mechanisms of the cell. Mutations that cause increased vesiculation enhance bacterial survival upon challenge with stressing agents or accumulation of toxic misfolded proteins. Preferential packaging of a misfolded protein mimic into vesicles for removal indicates that the vesiculation process can act to selectively eliminate unwanted material. Our results demonstrate that production of bacterial outer membrane vesicles is a fully independent, general envelope stress response. In addition to identifying a novel mechanism for alleviating stress, this work provides physiological relevance for vesicle production as a protective mechanism.    

3.871           Down-regulation of dopamine transporter by iron chelation in vitro is mediated by altered trafficking, not synthesis

Wiesinger, J.A.

1. Neurochem., 100, 167-179 (2007)

  Neurological development and functioning of dopamine (DA) neurotransmission is adversely affected by iron deficiency in early life. Iron-deficient rats demonstrate significant elevations in extracellular DA and a reduction in dopamine transporter (DAT) densities in the caudate putamen and nucleus accumbens. To explore possible mechanisms by which cellular iron concentrations control DAT functioning, endogenous DAT-expressing PC12 cells were used to determine the effect of iron chelation on DAT protein and mRNA expression patterns. In addition, we used human DAT (hDAT)-transfected Neuro2a (N2A) cells to examine DAT degradation and trafficking patterns. A 50 µm treatment for 24 h with the iron chelator, desferrioxamine (DFO), significantly decreased dopamine uptake in a dose-dependent manner, with no apparent change in K_m, in both PC12 and N2A cells. Reduced DA uptake was accompanied by concentration- and time-dependent reductions in total DAT protein levels in both cell lines. Exposure to increasing concentrations of DFO did not significantly alter DAT mRNA in either PC12 or N2A cells. However, DAT degradation rates increased three–fivefold in both cell types exposed to 50 µm DFO for 24 h. Biotinylation studies in N2A cells indicate a more dramatic loss of DAT in the membrane fraction, while OptiPrep fractionation experiments revealed an increase in lysosomal DAT with iron chelation. Inhibition of protein kinase C activation with staurosporin prevented the effect of iron chelation on DAT function, suggesting that in vitro iron chelation affects DAT primarily through the effects on trafficking rather than on synthesis.  

3.872           Inhibition of the Secretory Pathway by Foot-and-Mouth Disease Virus 2BC Protein Is Reproduced by Coexpression of 2B with 2C, and the Site of Inhibition Is Determined by the Subcellular Location of 2C

Moffat, K. et al

1. Virol., 81(3), 1129-1139 (2007)

  Infection of cells with picornaviruses can lead to a block in protein secretion. For poliovirus this is achieved by the 3A protein, and the consequent reduction in secretion of proinflammatory cytokines and surface expression of major histocompatibility complex class I proteins may inhibit host immune responses in vivo. Foot-and-mouth disease virus (FMDV), another picornavirus, can cause persistent infection of ruminants, suggesting it too may inhibit immune responses. Endoplasmic reticulum (ER)-to-Golgi apparatus transport of proteins is blocked by the FMDV 2BC protein. The observation that 2BC is processed to 2B and 2C during infection and that individual 2B and 2C proteins are unable to block secretion stimulated us to study the effects of 2BC processing on the secretory pathway. Even though 2BC was processed rapidly to 2B and 2C, protein transport to the plasma membrane was still blocked in FMDV-infected cells. The block could be reconstituted by coexpression of 2B and 2C, showing that processing of 2BC did not compromise the ability of FMDV to slow secretion. Under these conditions, 2C was located to the Golgi apparatus, and the block in transport also occurred in the Golgi apparatus. Interestingly, the block in transport could be redirected to the ER when 2B was coexpressed with a 2C protein fused to an ER retention element. Thus, for FMDV a block in secretion is dependent on both 2B and 2C, with the latter determining the site of the block.  

3.873           Membrane Cholesterol Content Modulates ClC-2 Gating and Sensitivity to Oxidative Stress

Hinzpeter, A. et al

1. Biol. Chem., 282(4), 2423-2432 (2007)

  ClC-2 is a broadly expressed member of the voltage-gated ClC chloride channel family. In this study, we aimed to evaluate the role of the membrane lipid environment in ClC-2 function, and in particular the effect of cholesterol and ClC-2 distribution in membrane microdomains. Detergent-resistant and detergent-soluble microdomains (DSM) were isolated from stably transfected HEK293 cells by a discontinuous OptiPrep gradient. ClC-2 was found concentrated in detergent-insoluble membranes in basal conditions and relocalized to DSM upon cholesterol depletion by methyl- -cyclodextrin. As assessed by patch clamp recordings, relocalization was accompanied by acceleration of the activation kinetics of the channel. A similar distribution and activation pattern were obtained when cells were treated with the oxidant tert-butyl hydroperoxide and after ATP depletion. In both cases activation was prevented by cholesterol enrichment of cells. We conclude that the cholesterol environment regulates ClC-2 activity, and we provide evidence that the increase in ClC-2 activity in response to acute oxidative or metabolic stress involves relocalization of this channel to DSM.  

3.874           Filamin links cell shape and cytoskeletal structure to Rho regulation by controlling accumulation of p190RhoGAP in lipid rafts

Mammoto, A., Huang, S. and Ingber, D.E.

1. Cell Sci., 120, 456-467 (2007)

  Cytoskeleton-dependent changes in the activity of the small GTPase Rho mediate the effects of cell shape on cell function; however, little is known about how cell spreading and related distortion of the cytoskeleton regulate Rho activity. Here we show that rearrangements of the actin cytoskeleton associated with early phases of cell spreading in human microvascular endothelial (HMVE) cells suppress Rho activity by promoting accumulation of p190RhoGAP in lipid rafts where it exerts its Rho inhibitory activity. p190RhoGAP is excluded from lipid rafts and Rho activity increases when cell rounding is induced or the actin cytoskeleton is disrupted, and p190RhoGAP knockdown using siRNA prevents Rho inactivation by cell spreading. Importantly, cell rounding fails to prevent accumulation of p190RhoGAP in lipid rafts and to increase Rho activity in cells that lack the cytoskeletal protein filamin. Moreover, filamin is degraded in spread cells and cells that express a calpain-resistant form of filamin exhibit high Rho activity even when spread. Filamin may therefore represent the missing link that connects cytoskeleton-dependent changes of cell shape to Rho inactivation during the earliest phases of cell spreading by virtue of its ability to promote accumulation of p190RhoGAP in lipid rafts.  

3.875           Lethal recessive myelin toxicity of prion protein lacking its central domain

Baumann, F. et al EMBO J., 26, 538-547 (2007)   PrP^C-deficient mice expressing prion protein variants with large amino-proximal deletions (termed PrP _F) suffer from neurodegeneration, which is rescued by full-length PrP^C. We now report that expression of PrP _CD, a PrP variant lacking 40 central residues (94–134), induces a rapidly progressive, lethal phenotype with extensive central and peripheral myelin degeneration. This phenotype was rescued dose-dependently by coexpression of full-length PrP^C or PrP^C lacking all octarepeats. Expression of a PrP^C variant lacking eight residues (114–121) was innocuous in the presence or absence of full-length PrP^C, yet enhanced the toxicity of PrP _CD and diminished that of PrP _F. Therefore, deletion of the entire central domain generates a strong recessive-negative mutant of PrP^C, whereas removal of residues 114–121 creates a partial agonist with context-dependent action. These findings suggest that myelin integrity is maintained by a constitutively active neurotrophic protein complex involving PrP^C, whose effector domain encompasses residues 94–134.  

3.876           c-Jun Downregulation by HDAC3-Dependent Transcriptional Repression Promotes Osmotic Stress-Induced Cell Apoptosis

Xia, Y. et al Mol. Cell, 25, 219-232 (2007)   c-Jun, a major transcription factor in the activating protein 1 (AP-1) family of regulatory proteins, is activated by many physiologic and pathologic stimuli. However, whether c-jun is regulated by epigenetic modification of chromatin structure is not clear. We showed here that c-jun was transcriptionally repressed in response to osmotic stress via a truncated HDAC3 generated by caspase-7-dependent cleavage at aspartic acid 391. The activation of caspase-7, which is independent of cytochrome c release and activation of caspase-9 and caspase-12, depends on activation of caspase-8, which in turn requires MEK2 activity and secretion of FAS ligand. The cell apoptosis induced by the truncated HDAC3 or enhanced by c-Jun deficiency during osmotic stress was suppressed by exogenous expression of c-Jun, indicating that the downregulation of c-Jun by HDAC3-dependent transcriptional repression plays a role in regulating cell survival and apoptosis.  

3.877           Heterogeneity of Raft-Type Membrane Microdomains Associated with VP4, the Rotavirus Spike Protein, in Caco-2 and MA 104 Cells

Delmas, O. Et al

1. Virol., 81(4), 1610-1618 (2007)

  Previous studies have shown that rotavirus virions, a major cause of infantile diarrhea, assemble within small intestinal enterocytes and are released at the apical pole without significant cell lysis. In contrast, for the poorly differentiated kidney epithelial MA 104 cells, which have been used extensively to study rotavirus assembly, it has been shown that rotavirus is released by cell lysis. The subsequent discovery that rotavirus particles associate with raft-type membrane microdomains (RTM) in Caco-2 cells provided a simple explanation for rotavirus polarized targeting. However, the results presented here, together with those recently published by another group, demonstrate that rotavirus also associates with RTM in MA 104 cells, thus indicating that a simple interaction of rotavirus with rafts is not sufficient to explain its apical targeting in intestinal cells. In the present study, we explore the possibility that RTM may have distinct physicochemical properties that may account for the differences observed in the rotavirus cell cycle between MA 104 and Caco-2 cells. We show here that VP4 association with rafts is sensitive to cholesterol extraction by methyl-ß-cyclodextrin treatment in MA 104 cells and insensitive in Caco-2 cells. Using the VP4 spike protein as bait, VP4-enriched raft subsets were immunopurified. They contained 10 to 15% of the lipids present in total raft membranes. We found that the nature and proportion of phospholipids and glycosphingolipids were different between the two cell lines. We propose that this raft heterogeneity may support the cell type dependency of virus assembly and release.  

3.878           Identification of Differentially Activated Cell-Signaling Networks Associated with Pichinde Virus Pathogenesis by Using Systems Kinomics

Bowick, G.C. et al

1. Virol., 81(4), 1923-1933 (2007)

  Phosphorylation plays a key role in regulating many signaling pathways. Although studies investigating the phosphorylated forms of signaling pathways are now commonplace, global analysis of protein phosphorylation and kinase activity has lagged behind genomics and proteomics. We have used a kinomics approach to study the effect of virus infection on host cell signaling in infected guinea pigs. Delineating the host responses which lead to clearance of a pathogen requires the use of a matched, comparative model system. We have used two passage variants of the arenavirus Pichinde, used as a biosafety level 2 model of Lassa fever virus as it produces similar pathologies in guinea pigs and humans, to compare the host cell responses between infections which lead to either a mild, self-limiting infection or lethal disease. Using this model, we can begin to understand the differences in signaling events which give rise to these markedly different outcomes. By contextualizing these data using pathway analysis, we have identified key differences in cellular signaling matrices. By comparing these differentially involved networks, we have identified a number of key signaling "nodes" which show differential phosphorylations between mild and lethal infections. We believe that these nodes provide potential targets for the development of antiviral therapies by acting at the level of the host response rather than by directly targeting viral proteins.  

3.879           The reduced GM-CSF priming of ROS production in granulocytes from patients with myelodysplasia is associated with an impaired lipid raft formation

Fuhler, G.M., Blom, N.R., Coffer, P.J., Drayer, A.L. and Vellenga, E.

1. Leukoc. Biol., 81, 449-457 (2007)

  Patients with myelodysplasia (MDS) show an impaired reactive oxygen species (ROS) production in response to fMLP stimulation of GM-CSF-primed neutrophils. In this study, we investigated the involvement of lipid rafts in this process and showed that treatment of neutrophils with the lipid raft-disrupting agent methyl-ß-cyclodextrin abrogates fMLP-induced ROS production and activation of ERK1/2 and protein kinase B/Akt, two signal transduction pathways involved in ROS production in unprimed and GM-CSF-primed neutrophils. We subsequently showed that there was a decreased presence of Lyn, gp91^phox, and p22^phox in lipid raft fractions from neutrophils of MDS. Furthermore, the plasma membrane expression of the lipid raft marker GM1, which increases upon stimulation of GM-CSF-primed cells with fMLP, was reduced significantly in MDS patients. By electron microscopy, we showed that the fMLP-induced increase in GM1 expression in GM-CSF-primed cells was a result of de novo synthesis, which was less efficient in MDS neutrophils. Taken together, these data indicate an involvement of lipid rafts in activation of signal transduction pathways leading to ROS production and show that in MDS neutrophils, an impaired lipid raft formation in GM-CSF-primed cells results in an impaired ROS production.    

3.880           Novel peroxisomal protease Tysnd1 processes PTS1- and PTS2-containing enzymes involved in β-oxidation of fatty acids

Kurochkin, I.V. et al EMBO J., 26, 835-845 (2007)   Peroxisomes play an important role in -oxidation of fatty acids. All peroxisomal matrix proteins are synthesized in the cytosol and post-translationally sorted to the organelle. Two distinct peroxisomal signal targeting sequences (PTSs), the C-terminal PTS1 and the N-terminal PTS2, have been defined. Import of precursor PTS2 proteins into the peroxisomes is accompanied by a proteolytic removal of the N-terminal targeting sequence. Although the PTS1 signal is preserved upon translocation, many PTS1 proteins undergo a highly selective and limited cleavage. Here, we demonstrate that Tysnd1, a previously uncharacterized protein, is responsible both for the removal of the leader peptide from PTS2 proteins and for the specific processing of PTS1 proteins. All of the identified Tysnd1 substrates catalyze peroxisomal -oxidation. Tysnd1 itself undergoes processing through the removal of the presumably inhibitory N-terminal fragment. Tysnd1 expression is induced by the proliferator-activated receptor agonist bezafibrate, along with the increase in its substrates. A model is proposed where the Tysnd1-mediated processing of the peroxisomal enzymes promotes their assembly into a supramolecular complex to enhance the rate of -oxidation.  

3.881           Relationships between the Sequence of α-Synuclein and its Membrane Affinity, Fibrillization Propensity, and Yeast Toxicity

Volles, M.J. and Lansbury, P.T.

1. Mol. Biol., 366(5), 1510-1522 (2007)

  To investigate the α-synuclein protein and its role in Parkinson's disease, we screened a library of random point mutants both in vitro and in yeast to find variants in an unbiased way that could help us understand the sequence–phenotype relationship. We developed a rapid purification method that allowed us to screen 59 synuclein mutants in vitro and discovered two double-point mutants that fibrillized slowly relative to wild-type, A30P, and A53T α-synucleins. The yeast toxicity of all of these proteins was measured, and we found no correlation with fibrillization rate, suggesting that fibrillization is not necessary for synuclein-induced yeast toxicity. We found that β-synuclein was of intermediate toxicity to yeast, and γ-synuclein was non-toxic. Co-expression of Parkinson's disease-related genes DJ-1, parkin, Pink1, UCH-L1, or synphilin, with synuclein, did not affect synuclein toxicity. A second screen, of several thousand library clones in yeast, identified 25 non-toxic α-synuclein sequence variants. Most of these contained a mutation to either proline or glutamic acid that caused a defect in membrane binding. We hypothesize that yeast toxicity is caused by synuclein binding directly to membranes at levels sufficient to non-specifically disrupt homeostasis.  

3.882           Asymmetric Localization of Calpain 2 during Neutrophil Chemotaxis

Nuzzi, P.A., Senetar, M.A., Huttenlocher, A. Mol. Biol. Cell, 18, 795-805 (2007)   Chemoattractants induce neutrophil polarization through localized polymerization of F-actin at the leading edge. The suppression of rear and lateral protrusions is required for efficient chemotaxis and involves the temporal and spatial segregation of signaling molecules. We have previously shown that the intracellular calcium-dependent protease calpain is required for cell migration and is involved in regulating neutrophil chemotaxis. Here, we show that primary neutrophils and neutrophil-like HL-60 cells express both calpain 1 and calpain 2 and that chemoattractants induce the asymmetric recruitment of calpain 2, but not calpain 1, to the leading edge of polarized neutrophils and differentiated HL-60 cells. Using time-lapse microscopy, we show that enrichment of calpain 2 at the leading edge occurs during early pseudopod formation and that its localization is sensitive to changes in the chemotactic gradient. We demonstrate that calpain 2 is recruited to lipid rafts and that cholesterol depletion perturbs calpain 2 localization, suggesting that its enrichment at the front requires proper membrane organization. Finally, we show that catalytic activity of calpain is required to limit pseudopod formation in the direction of chemoattractant and for efficient chemotaxis. Together, our findings identify calpain 2 as a novel component of the frontness signal that promotes polarization during chemotaxis.      

3.883           PALS1 Regulates E-Cadherin Trafficking in Mammalian Epithelial Cells

Wang, Q., Chen, X-W. and Margolis, B. Mol. Biol. Cell, 18, 874-885 (2007)   Protein Associated with Lin Seven 1 (PALS1) is an evolutionarily conserved scaffold protein that targets to the tight junction in mammalian epithelia. Prior work in our laboratory demonstrated that the knockdown of PALS1 in Madin Darby canine kidney cells leads to tight junction and polarity defects. We have created new PALS1 stable knockdown cell lines with more profound reduction of PALS1 expression, and a more severe defect in tight junction formation was observed. Unexpectedly, we also observed a severe adherens junction defect, and both defects were corrected when PALS1 wild type and certain PALS1 mutants were expressed in the knockdown cells. We found that the adherens junction structural component E-cadherin was not effectively delivered to the cell surface in the PALS1 knockdown cells, and E-cadherin puncta accumulated in the cell periphery. The exocyst complex was also found to be mislocalized in PALS1 knockdown cells, potentially explaining why E-cadherin trafficking is disrupted. Our results suggest a broad and evolutionarily conserved role for the tight junction protein PALS1 in the biogenesis of adherens junction.  

3.884           Role of the Sec61 Translocon in EGF Receptor Trafficking to the Nucleus and Gene Expression

Liao, H-J. and Carpenter, G. Mol. Biol. Cell, 18, 1064-1072 (2007)   The epidermal growth factor (EGF)-dependent trafficking of the intact EGF receptor to the nucleus and its requirement for growth factor induction of cyclin D and other genes has been reported. Unresolved is the mechanism by which this or other transmembrane proteins are excised from a lipid bilayer before nuclear translocalization. We report that, after the addition of EGF, the cell surface EGF receptor is trafficked to the endoplasmic reticulum (ER) where it associates with Sec61 , a component of the Sec61 translocon, and is retrotranslocated from the ER to the cytoplasm. Abrogation of Sec61 expression prevents EGF-dependent localization of EGF receptors to the nucleus and expression of cyclin D. This indicates that EGF receptors are trafficked from the ER to the nucleus by a novel pathway that involves the Sec61 translocon.  

3.885           Conservation of the TRAPPII-specific subunits of a Ypt/Rab exchanger complex

Cox, R., Chen, S.H., Yoo, E. and Segev, N. BMC Evolutionary Biol., 7(12), 1-15 (2007)   Background Ypt/Rab GTPases and their GEF activators regulate intra-cellular trafficking in all eukaryotic cells. In S. cerivisiae, the modular TRAPP complex acts as a GEF for the Golgi gatekeepers: Ypt1 and the functional pair Ypt31/32. While TRAPPI, which acts in early Golgi, is conserved from fungi to animals, not much is known about TRAPPII, which acts in late Golgi and consists of TRAPPI plus three additional subunits. Results Here, we show a phylogenetic analysis of the three TRAPPII-specific subunits. One copy of each of the two essential subunits, Trs120 and Trs130, is present in almost every fully sequenced eukaryotic genome. Moreover, the primary, as well as the predicted secondary, structure of the Trs120- and Trs130-related sequences are conserved from fungi to animals. The mammalian orthologs of Trs120 and Trs130, NIBP and TMEM1, respectively, are candidates for human disorders. Currently, NIBP is implicated in signaling, and TMEM1 is suggested to have trans-membrane domains (TMDs) and to function as a membrane channel. However, we show here that the yeast Trs130 does not function as a trans-membrane protein, and the human TMEM1 does not contain putative TMDs. The non-essential subunit, Trs65, is conserved only among many fungi and some unicellular eukaryotes. Multiple alignment analysis of each TRAPPII-specific subunit revealed conserved domains that include highly conserved amino acids. Conclusion We suggest that the function of both NIBP and TMEM1 in the regulation of intra-cellular trafficking is conserved from yeast to man. The conserved domains and amino acids discovered here can be used for functional analysis that should help to resolve the differences in the assigned functions of these proteins in fungi and animals.  

3.886           Transcriptome analysis reveals the population of dendritic RNAs and their redistribution by neural activity

Matsumoto, M., Setou, M. and Inokuchi, K. Neurosci. Res., 57, 411-423 (2007)   Subcellular localization of RNA is an efficient way to localize proteins to a specific region of a cell. The dendritic localization of RNAs underlies the establishment and maintenance of the synaptic functions of neuronal cells. A requirement for dendritic RNA localization and subsequent local translation has been demonstrated in several forms of experience-dependent synaptic plasticity. In spite of several attempts to identify these RNAs, the population of RNA species present in dendrites as a whole has not been well described. Here we show the results of microarray analyses with RNAs isolated from heavy portion of polysome (HP) fraction where RNA granules are enriched in and synaptosome fraction, prepared from the rat brain. These analyses revealed the complex nature of the dendritic RNA population, which included RNAs that were not expected to be in the dendrites. Neural activity caused by an electroconvulsive shock triggered a redistribution of the population of dendritic transcriptome towards the area of overlap between the HP and the synaptosome, which is assumed to be neck of spine. This redistribution may accompany some changes in the translatability of those transcriptome, which suggests complex mechanisms of local translation in response to synaptic inputs.  

3.887           Retinoschisin Is a Peripheral Membrane Protein with Affinity for Anionic Phospholipids and Affected by Divalent Cations

Vijayasarathy, C., Takada, Y., Zeng, Y., Bush, R.A. and Sieving, P.A. Invest. Ophtalmol. Vis. Sci., 48, 991-1000 (2007)   PURPOSE. Retinoschisin (RS) is a retina-specific, secreted proteinimplicated in X-linked juvenile retinoschisis and essentialfor the structural and functional integrity of the retina. Thisbiochemical characterization and ultrastructural localizationof RS in intact murine retina was performed to further understandingof the molecular basis of its function. METHODS. Subcellular fractions and fractions enriched in photoreceptorinner and outer segments were prepared from mouse retina bydifferential or density gradient ultracentrifugation. Immunoblotanalysis was used to assess the expression of RS in varioussubcellular compartments and its fractionation into solublephase on treatment of retinal cell membranes with several solubilizingreagents. RS–lipid interactions were evaluated by a protein–lipidoverlay assay that used wild-type and mutant forms of RS discoidindomain glutathione S-transferase (GST) fusion proteins. Thesubcellular localization of RS in mouse retina was visualizedby pre-embedding immunogold electron microscopy. Ultrastructurewas evaluated by transmission electron microscopy. RESULTS. RS was intimately associated with cell membranes of the retina. It was found to cluster on the outer leaflet of the plasma membrane of the photoreceptor inner segments, which synthesize and secrete it. It was released from the membrane at high pH, which is characteristic of a peripheral membrane protein. It was extracted from the membrane by the nonionic detergent NP-40, together with glycerophospholipids. Protein–lipid overlay assays indicated a preferential interaction between RS and anioic phospholipids. Extraction of RS from the membrane was inhibited by divalent cations. Photoreceptor inner segment morphology was markedly affected in RS^–/y mice, whichfailed to express RS protein. CONCLUSIONS. RS in intact retina is a peripheral membrane protein. Although distributed over the two membrane faces, RS is associated primarily with the outer leaflet of the inner segment plasma membrane through anionic phospholipids and divalent cations. RS’s localization in photoreceptors and its biochemical properties suggest a functional role locally, at the site of secretion and membrane adhesion, in maintaining the photoreceptor inner segment stability and architecture.  

3.888           Insulin-stimulated exocytosis of GLUT4 is enhanced by IRAP and its partner tankyrase

Yeh, T-Y.J., Sbodio, J.I., Tsun, Z-Y., Luo, B. and Chi, N-W. Biochem. J., 402, 279-290 (2007)   The glucose transporter GLUT4 and the aminopeptidase IRAP (insulin-responsive aminopeptidase) are the major cargo proteins of GSVs (GLUT4 storage vesicles) in adipocytes and myocytes. In the basal state, most GSVs are sequestered in perinuclear and other cytosolic compartments. Following insulin stimulation, GSVs undergo exocytic translocation to insert GLUT4 and IRAP into the plasma membrane. The mechanisms regulating GSV trafficking are not fully defined. In the present study, using 3T3-L1 adipocytes transfected with siRNAs (small interfering RNAs), we show that insulin-stimulated IRAP translocation remained intact despite substantial GLUT4 knockdown. By contrast, insulin-stimulated GLUT4 translocation was impaired upon IRAP knockdown, indicating that IRAP plays a role in GSV trafficking. We also show that knockdown of tankyrase, a Golgi-associated IRAP-binding protein that co-localizes with perinuclear GSVs, attenuated insulin-stimulated GSV translocation and glucose uptake without disrupting insulin-induced phosphorylation cascades. Moreover, iodixanol density gradient analyses revealed that tankyrase knockdown altered the basal-state partitioning of GLUT4 and IRAP within endosomal compartments, apparently by shifting both proteins toward less buoyant compartments. Importantly, the afore-mentioned effects of tankyrase knockdown were reproduced by treating adipocytes with PJ34, a general PARP (poly-ADP-ribose polymerase) inhibitor that abrogated tankyrase-mediated protein modification known as poly-ADP-ribosylation. Collectively, these findings suggest that physiological GSV trafficking depends in part on the presence of IRAP in these vesicles, and that this process is regulated by tankyrase and probably its PARP activity.  

3.889           p56lck, LFA-1 and PI3K but not SHP-2 interact with GM1- or GM3-enriched microdomains in a CD4–p56lck association-dependent manner

Barbat, C. et al Biochem. J., 402(3), 471-481 (2007)   We previously showed that the association of CD4 and G_M3 ganglioside induced by CD4 ligand binding was required for the down-regulation of adhesion and that aggregation of ganglioside-enriched domains was accompanied by transient co-localization of LFA-1 (lymphocyte function-associated antigen-1), PI3K (phosphoinositide 3-kinase) and CD4. We also showed that these proteins co-localized with the G_M1 ganglioside that partially co-localized with G_M3 in these domains. In the present study, we show that CD4–p56^lck association in CD4 signalling is required for the redistribution of p56^lck, PI3K and LFA-1 in ganglioside-enriched domains, since ganglioside aggregation and recruitment of these proteins were not observed in a T-cell line (A201) expressing the mutant form of CD4 that does not bind p56^lck. In addition, we show that although these proteins associated in different ways with G_M1 and G_M3, all of the associations were dependent on CD4–p56^lck association. Gangliosides could associate with these proteins that differ in affinity binding and could be modified following CD4 signalling. Our results suggest that through these associations, gangliosides transiently sequestrate these proteins and consequently inhibit LFA-1-dependent adhesion. Furthermore, while structural diversity of gangliosides may allow association with distinct proteins, we show that the tyrosine phosphatase SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), also required for the down-regulation of LFA-1-dependent adhesion, transiently and partially co-localized with PI3K and p56^lck in detergent-insoluble membranes without association with G_M1 or G_M3. We propose that CD4 ligation and binding with p56^lck and their interaction with G_M3 and/or G_M1 gangliosides induce recruitment of distinct proteins important for CD4 signalling to form a multimolecular signalling complex.  

3.890           EDEM1 reveals a quality control vesicular transport pathway out of the endoplasmic reticulum not involving the COPII exit sites

Zuber, C. et al PNAS, 104(11), 4407-4412 (2007)   Immature and nonnative proteins are retained in the endoplasmic reticulum (ER) by the quality control machinery. Folding-incompetent glycoproteins are eventually targeted for ER-associated protein degradation (ERAD). EDEM1 (ER degradation-enhancing -mannosidase-like protein 1), a putative mannose-binding protein, targets misfolded glycoproteins for ERAD. We report that endogenous EDEM1 exists mainly as a soluble glycoprotein. By high-resolution immunolabeling and serial section analysis, we find that endogenous EDEM1 is sequestered in buds that form along cisternae of the rough ER at regions outside of the transitional ER. They give rise to 150-nm vesicles scattered throughout the cytoplasm that are lacking a recognizable COPII coat. About 87% of the immunogold labeling was over the vesicles and 11% over the ER lumen. Some of the EDEM1 vesicles also contain Derlin-2 and the misfolded Hong Kong variant of -1-antitrypsin, a substrate for EDEM1 and ERAD. Our results demonstrate the existence of a vesicle budding transport pathway out of the rough ER that does not involve the canonical transitional ER exit sites and therefore represents a previously unrecognized passageway to remove potentially harmful misfolded luminal glycoproteins from the ER.  

3.891           WD40 protein Mda1 is purified with Dnm1 and forms a dividing ring for mitochondria before Dnm1 in Cyanidioschyzon merolae

Nishida, K., Yagisawa, F., Kuroiwa, H., Yoshida, Y. and Kuroiwa, T. PNAS, 104(11), 4736-4741 (2007)   Mitochondria are not produced de novo but are maintained by division. Mitochondrial division is a coordinated process of positioning and constriction of the division site and fission of double membranes, in which dynamin-related protein is believed to mediate outer membrane fission. Part of the mitochondrial division machinery was purified from M phase-arrested Cyanidioschyzon merolae cells through biochemical fractionation. The dynamin-related protein Dnm1 was one of the two major proteins in the purified fraction and was accompanied by a newly identified protein CMR185C, named Mda1. Mda1 contained a predictable coiled-coil region and WD40 repeats, similarly to Mdv1 and Caf4 in yeasts. Immunofluorescence and immunoelectron microscopy showed that Mda1 localizes as a medial belt or ring on the mitochondrial outer surface throughout the division. The ring formation of Mda1 followed the plane of the ring of FtsZ, a protein that resides in the matrix. Dnm1 consistently colocalized with Mda1 only in the late stages of division. Mda1 protein was expressed through S to M phases and was phosphorylated specifically in M phase when Mda1 transformed from belt into foci and became colocalizing with Dnm1. Dephosphorylation of Mda1 in vitro increased its sedimentation coefficient, suggesting conformational changes of the macromolecule. Disassembly of the purified mitochondrial division machinery was performed by adding GTP to independently release Dnm1, suggesting that Mda1 forms a stable homo-oligomer by itself as a core structure of the mitochondrial division machinery.  

3.892           The novel cargo Alcadein induces vesicle association of kinesin-1 motor components and activates axonal transport

Araki, Y. et al EMBO J., 26, 1475-1486 (2007)   Alcadein (Alc ) is an evolutionarily conserved type I membrane protein expressed in neurons. We show here that Alc strongly associates with kinesin light chain (K_D 4–8 10^-9 M) through a novel tryptophan- and aspartic acid-containing sequence. Alc can induce kinesin-1 association with vesicles and functions as a novel cargo in axonal anterograde transport. JNK-interacting protein 1 (JIP1), an adaptor protein for kinesin-1, perturbs the transport of Alc , and the kinesin-1 motor complex dissociates from Alc -containing vesicles in a JIP1 concentration-dependent manner. Alc -containing vesicles were transported with a velocity different from that of amyloid -protein precursor (APP)-containing vesicles, which are transported by the same kinesin-1 motor. Alc - and APP-containing vesicles comprised mostly separate populations in axons in vivo. Interactions of Alc with kinesin-1 blocked transport of APP-containing vesicles and increased -amyloid generation. Inappropriate interactions of Alc- and APP-containing vesicles with kinesin-1 may promote aberrant APP metabolism in Alzheimer's disease.  

3.893           Accumulation of Mutant Neuroserpin Precedes Development of Clinical Symptoms in Familial Encephalopathy with Neuroserpin Inclusion Bodies

Galliciotti, G. et al Am. J. Pathol., 170(4), 1305-1313 (2007)   Intracellular protein deposition due to aggregation caused by conformational alteration is the hallmark of a number of neurodegenerative disorders, including Parkinson’s disease, tauopathies, Huntington’s disease, and familial encephalopathy with neuroserpin inclusion bodies. The latter is an autosomal dominant disorder caused by point mutations in neuroserpin resulting in its destabilization. Mutant neuroserpin polymerizes and forms intracellular aggregates that eventually lead to neurodegeneration. We generated genetically modified mice expressing the late-onset S49P-Syracuse or the early-onset S52R-Portland mutation of neuroserpin in central nervous system neurons. Mice exhibited morphological, biochemical, and clinical features resembling those found in the human disease. Analysis of brains revealed large intraneuronal inclusions composed exclusively of mutant neuroserpin, accumulating long before the development of clinical symptoms in a time-dependent manner. Clinical symptoms and amount of neuroserpin inclusions correlated with the predicted instability of the protein. The presence of inclusion bodies in subclinical mice indicates that in humans the prevalence of the disease could be higher than anticipated. In addition to shedding light on the pathophysiology of the human disorder, these mice provide an excellent model to study mechanisms of neurodegeneration or establish novel therapies for familial encephalopathy with neuroserpin inclusion bodies and other neurodegenerative diseases with intracellular protein deposition.  

3.894           Cytosolic Activation of Cathepsins Mediates Parvovirus H-1-Induced Killing of Cisplatin and TRAIL-Resistant Glioma Cells

Di Piazza, M. et al

1. Virol., 81(8), 4186-4198 (2007)

  Gliomas are often resistant to the induction of apoptotic cell death as a result of the development of survival mechanisms during astrocyte malignant transformation. In particular, the overexpression of Bcl-2-family members interferes with apoptosis initiation by DNA-damaging agents (e.g., cisplatin) or soluble death ligands (e.g., TRAIL). Using low-passage-number cultures of glioma cells, we have shown that parvovirus H-1 is able to induce death in cells resistant to TRAIL, cisplatin, or both, even when Bcl-2 is overexpressed. Parvovirus H-1 triggers cell death through both the accumulation of lysosomal cathepsins B and L in the cytosol of infected cells and the reduction of the levels of cystatin B and C, two cathepsin inhibitors. The impairment of either of these effects protects glioma cells from the viral lytic effect. In normal human astrocytes, parvovirus H-1 fails to induce a killing mechanism. In vivo, parvovirus H-1 infection of rat glioma cells intracranially implanted into recipient animals triggers cathepsin B activation as well. This report identifies for the first time cellular effectors of the killing activity of parvovirus H-1 against malignant brain cells and opens up a therapeutic approach which circumvents their frequent resistance to other death inducers.  

3.895           Knockdown of ACAT-1 reduces amyloidogenic processing of APP

Huttunen, H.J., Greco, C. and Kovacs, D.M. FEBS Lett., 581, 1688-1692 (2007)   Previous studies have shown that acyl-coenzyme A:cholesterol acyl transferase (ACAT), an enzyme that controls cellular equilibrium between free cholesterol and cholesteryl esters, modulates proteolytic processing of APP in cell-based and animal models of Alzheimer’s disease. Here we report that ACAT-1 RNAi reduced cellular ACAT-1 protein by 50% and cholesteryl ester levels by 22% while causing a slight increase in the free cholesterol content of ER membranes. This correlated with reduced proteolytic processing of APP and 40% decrease in Aβ secretion. These data show that even a modest decrease in ACAT activity can have robust suppressive effects on Aβ generation.  

3.896           The Nicastrin-like Protein Nicalin Regulates Assembly and Stability of the Nicalin-Nodal Modulator (NOMO) Membrane Protein Complex

Haffner, C., Dettmer, U., Weiler, T. And Haass, C.

1. Biol. Chem., 282(14), 10632-10638 (2007)

  The assembly of the -secretase complex, an Alzheimer disease-related protease required for -amyloid generation, is tightly regulated and predominantly limited by the stoichiometrical availability of its components. We have identified a novel endoplasmic reticulum-located protein complex that is regulated in a similar fashion. It contains the recently identified Nodal signaling antagonists Nicalin (a distant homolog of the -secretase component Nicastrin) and NOMO (Nodal modulator). Using an RNA interference approach, we found that Nicalin and NOMO became unstable in the absence of the respective binding partner, suggesting that complex formation has a stabilizing effect. Overexpression of Nicalin resulted in an increase in NOMO, whereas endogenous Nicalin was reduced below the detection limit. Both effects were shown to occur at a post-transcriptional level. Thus, NOMO is most likely produced in excess amounts and either stabilized by Nicalin or rapidly degraded. In contrast, Nicalin levels are limited independently of NOMO. We, therefore, propose that Nicalin controls the assembly and stability of the Nicalin-NOMO complex.  

3.897           Free Cholesterol Alters Lipid Raft Structure and Function Regulating Neutrophil Ca2+ Entry and Respiratory Burst: Correlations with Calcium Channel Raft Trafficking

Kannan, K.B., Barlos, D. and Hauser, C.J.

1. Immunol., 178, 5253-5261 (2007)

  Recent studies associate cholesterol excess and atherosclerosis with inflammation. The link between these processes is not understood, but cholesterol is an important component of lipid rafts. Rafts are thought to concentrate membrane signaling molecules and thus regulate cell signaling through G protein-coupled pathways. We used methyl -cyclodextrin to deplete cholesterol from polymorphonuclear neutrophil (PMN) rafts and thus study the effects of raft disruption on G protein-coupled Ca²⁺ mobilization. Methyl -cyclodextrin had no effect on Ca²⁺ store depletion by the G protein-coupled agonists platelet-activating factor or fMLP, but abolished agonist-stimulated Ca²⁺ entry. Free cholesterol at very low concentrations regulated Ca²⁺ entry into PMN via nonspecific Ca²⁺ channels in a biphasic fashion. The specificity of cholesterol regulation for Ca²⁺ entry was confirmed using thapsigargin studies. Responses to cholesterol appear physiologic because they regulate respiratory burst in a proportional biphasic fashion. Investigating further, we found that free cholesterol accumulated in PMN lipid raft fractions, promoting formation and polarization of membrane rafts. Finally, the transient receptor potential calcium channel protein TRPC1 redistributed to raft fractions in response to cholesterol. The uniformly biphasic relationships between cholesterol availability, Ca²⁺ signaling and respiratory burst suggest that Ca²⁺ influx and PMN activation are regulated by the quantitative relationships between cholesterol and other environmental lipid raft components. The association between symptomatic cholesterol excess and inflammation may therefore in part reflect free cholesterol- dependent changes in lipid raft structure that regulate immune cell Ca²⁺ entry. Ca²⁺ entry-dependent responses in other cell types may also reflect cholesterol bioavailability and lipid incorporation into rafts.  

3.898           Association of putative ammonium exporters Ato with detergent-resistant compartments of plasma membrane during yeast colony development: pH affects Ato1p localisation in patches

Ricicova, M., Kucerova, H., Vachova, L. and Palkova, Z. Biochim. Biophys. Acta, 1768, 1170-1178 (2007)   It was proposed that Ato1p, Ato2p and Ato3p have a role in ammonia production by Saccharomyces cerevisiae colonies (Palkova et al., Mol Biol Cell 13: 3901–3914, 2002). In this study, we show that all three Ato proteins localise to the plasma membrane and their appearance correlates with the beginning of ammonia release. The expression of ATO genes is controlled by ammonia. All three Ato–GFP proteins associate with detergent-resistant membranes; two of them, Ato1p–GFP and Ato3p–GFP, localise to patches visible under the fluorescence microscope. In contrast with Ato3p–GFP which forms stable patches, the formation of those of Ato1p–GFP is pH dependent. Ato1p–GFP patches form at pH above 6 and they disappear at pH 5 or lower. Both changes, Ato1p–GFP clustering and patches spreading are reversible. The Ato1p–GFP spreading at low pH is independent on endocytosis. These data suggest that besides the ammonia induction of Ato protein synthesis, pH may rapidly regulate Ato1p function.  

3.899           Polybasic KKR Motif in the Cytoplasmic Tail of Nipah Virus Fusion Protein Modulates Membrane Fusion by Inside-Out Signaling

Aguilar, H et al

1. Virol., 81(9), 4520-4532 (2007)

  The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membrane-adjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3- to 5-fold. These results were corroborated in a reverse-pseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper- or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where 20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P = 0.007, r² = 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms.  

3.900           Participation of Rab5, an Early Endosome Protein, in Hepatitis C Virus RNA Replication Machinery

Stone, M., Jia, S., Do Heo, W., Meyer, T. and Konan, K.V.

1. Virol., 81(9), 4551-4563 (2007)

  Like most positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its genome on the surface of rearranged membranes. We have shown previously that HCV NS4AB, but not the product NS4B, inhibits endoplasmic reticulum (ER)-to-Golgi protein traffic (K. V. Konan, T. H. Giddings, Jr., M. Ikeda, K. Li, S. M. Lemon, and K. Kirkegaard, J. Virol. 77:7843-7855). However, both NS4AB and NS4B can induce "membranous web" formation, first reported by Egger et al. (D. B Egger, R. Gosert, L. Bianchi, H. E. Blum, D. Moradpour, and K. Bienz, J. Virol. 76:5974-5984), which is also observed in HCV-infected cells (Y. Rouille, F. Helle, D. Delgrange, P. Roingeard, C. Voisset, E. Blanchard, S. Belouzard, J. McKeating, A. H. Patel, G. Maertens, T. Wakita, C. Wychowski, and J. Dubuisson, J. Virol. 80:2832-2841) and cells that bear a subgenomic NS5A-green fluorescent protein (GFP) replicon (D. Moradpour, M. J. Evans, R. Gosert, Z. Yuan, H. E. Blum, S. P. Goff, B. D. Lindenbach, and C. M. Rice, J. Virol. 78:7400-7409). To determine the intracellular origin of the web, we examined NS4B colocalization with endogenous cellular markers in the context of the full-length or subgenomic replicon. We found that, in addition to ER markers, early endosome (EE) proteins, including Rab5, were associated with web-inducing protein NS4B. Furthermore, an immunoisolated fraction containing NS4B was found to contain both ER and EE proteins. Using fluorescence microscopy, we showed that wild-type and constitutively active Rab5 proteins were associated with NS4B. Interestingly, expression of dominant-negative Rab5 resulted in significant loss of GFP fluorescence in NS5A-GFP replicon cells. We also found that a small reduction in Rab5 protein expression decreased HCV RNA synthesis significantly. Furthermore, transfection of labeled Rab5 small interfering RNAs into NS5A-GFP replicon cells resulted in a significant decrease in GFP fluorescence. Finally, Rab5 protein was found to coimmunoprecipitate with HCV NS4B. These studies suggest that EE proteins, including Rab5, may play a role in HCV genome replication or web formation.  

3.901           Measles virus nucleocapsid transport to the plasma membrane requires stable expression and surface accumulation of the viral matrix protein

Runkler, N., Pohl, C., Schneider-Schaulies, S., Klenk, H-D. and Maisner, A. Cell. Microbiol., 9(5), 1203-1214 (2007)   In measles virus (MV)-infected cells the matrix (M) protein plays a key role in virus assembly and budding processes at the plasma membrane because it mediates the contact between the viral surface glycoproteins and the nucleocapsids. By exchanging valine 101, a highly conserved residue among all paramyxoviral M proteins, we generated a recombinant MV (rMV) from cloned cDNA encoding for a M protein with an increased intracellular turnover. The mutant rMV was barely released from the infected cells. This assembly defect was not due to a defective M binding to other matrix- or nucleoproteins, but could rather be assigned to a reduced ability to associate with cellular membranes, and more importantly, to a defective accumulation at the plasma membrane which was accompanied by the deficient transport of nucleocapsids to the cell surface. Thus, we show for the first time that M stability and accumulation at intracellular membranes is a prerequisite for M and nucleocapsid co-transport to the plasma membrane and for subsequent virus assembly and budding processes.  

3.902           Establishment of subcellular fractionation techniques to monitor the intracellular fate of polymer therapeutics II. Identification of endosomal and lysosomal compartments in HepG2 cells combining single-step subcellular fractionation with fluorescent imaging

Manunta, M., Izzo, L., Duncan, R. and Jones, A.T.

1. Drug. Target., 15(1), 37-50 (2007)

  As they are often designed for lysosomotropic, endosomotropic and/or transcellular delivery, an understanding of intracellular trafficking pathways is essential to enable optimised design of novel polymer therapeutics. Here, we describe a single-step density gradient subcellular fractionation method combined with fluorescent detection analysis that provides a new tool for characterisation of endocytic traffic of polymer therapeutics. Hepatoma (HepG2) cells were used as a model and cell breakage was optimised using a cell cracker to ensure assay of the whole cell population. After removal of unbroken cells and nuclei, the cell lysate as a post-nuclear supernatant (PNS) was layered onto an iodixanol (OptiPrep) density gradient optimised to 5-20%. Early endosomes, late endosomes and lysosomes were identified from gradient fractions by immunoblotting for marker proteins early endosome antigen 1 (EEA 1) and lysosomal associated membrane protein 1 (LAMP 1) using horseradish peroxidase or fluorescently-labelled secondary antibodies. Lysosomes were also detected using N-acetyl-beta-glucosamindase (Hex A) activity. In addition, cells were incubated with Texas-red labelled transferrin (TxR-Tf) for 5 min to specifically label early endosomes and this was directly detected from SDS-PAGE gels. Internalised macromolecules and colloidal particles can potentially alter vesicle buoyant density. To see if typical macromolecules of interest would alter vesicle density or perturb vesicle traffic, HepG2 cells were incubated with dextran or a polyethyleneglycol (PEG)-polyester dendron G4 (1 mg/ml for 24 h). The PEG-polyester dendron G4 caused a slight redistribution of endocytic structures to lower density fractions but immunofluorescence microscopy showed no obvious dendron effects. In conclusion, the combined subcellular fractionation with fluorescent imaging approach described here can be used as a tool for both fundamental cell biology research and/or the quantitative localisation of polymer therapeutics in the endocytic pathway.  

3.903           Differential stimulation-induced receptor localization in lipid rafts for interleukin-6 family cytokines signaling through the gp130/leukemia inhibitory factor receptor complex

Port, M.D., Gibson, R.-M. and Nathanson, N.M.

1. Neurochem., 101, 782-793 (2007)

  Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are cytokines which signal through receptor complexes that include the receptor subunits glycoprotein 130 (gp130) and the LIF receptor (LIFR), but CNTF also requires the non-signal transducing CNTF receptor (CNTFR) for binding. We show here that in IMR-32 neuronal cells endogenously expressing the receptor subunits for LIF and CNTF, CNTFR, but not gp130 or LIFR, is found in detergent-resistant lipid rafts. In addition, stimulation of these cells with CNTF resulted in a rapid translocation of a portion of gp130 and LIFR into detergent-resistant lipid rafts while an equivalent stimulation with LIF did not. Disruption of lipid rafts by cholesterol depletion of cell membranes blocked the CNTF-induced translocation of LIFR and gp130. Interestingly, while cholesterol-depletion did not inhibit signal transducer and activator of transcription 3 phosphorylation by either CNTF or LIF stimulation, it strongly inhibited both CNTF- and LIF-mediated phosphorylation of extracellular signal-regulated kinases 1 and 2 and Akt. LIF and CNTF generally appear to have redundant effects in cells responsive to both cytokines. Intriguingly, the data presented here suggest a possible mechanism whereby CNTF or other cytokines that signal through CNTFR could generate signals distinct from those elicited by cytokines such as LIF which utilize a LIFR/gp130 heterodimer, via association with or exclusion from lipid rafts.  

3.904           Molecular characterization of GDD1/TMEM16E, the gene product responsible for autosomal dominant gnathodiaphyseal dysplasia

Mizuta, K. et al Biochem. Biophys. Acta, 357, 126-132 (2007)   The human GDD1/TMEM16E gene has been found to be mutated in gnathodiaphyseal dysplasia, an unusual skeletal syndrome with autosomal dominant inheritance. The molecular and biochemical function(s) of GDD1 protein has not yet been elucidated. In this study, we examined the murine GDD1 gene expression pattern during embryonic development, and characterized the cellular and tissue localizations of its gene product using a GDD1-specific antibody. In the developing embryos, GDD1 mRNA expression was principally associated with differentiating and developing somites, with a highly complex spatiotemporal pattern that involved the myotomal and sclerotomal lineages of somites. Biochemical studies indicated that GDD1 protein is an integral membrane glycoprotein that resides predominantly in intracellular vesicles. Immunohistochemical analysis showed a high level of murine GDD1 protein expression in cardiac and skeletal muscle tissues, and in growth-plate chondrocytes and osteoblasts in bone. These observations suggest diverse cellular role(s) of GDD1 in the development of musculoskeletal system.  

3.905           UK114, a YjgF/Yer057p/UK114 family protein highly conserved from bacteria to mammals, is localized in rat liver peroxisomes

Antonenkov, V., Ohlmeier, S., Sormunen, R.T. and Hiltunen, J.K. Biochem. Biophys. Acta, 357, 252-257 (2007)   Mammalian UK114 belongs to a highly conserved family of proteins with unknown functions. Although it is believed that UK114 is a cytosolic or mitochondrial protein there is no detailed study of its intracellular localization. Using analytical subcellular fractionation, electron microscopic colloidal gold technique, and two-dimensional gel electrophoresis of peroxisomal matrix proteins combined with mass spectrometric analysis we show here that a large portion of UK114 is present in rat liver peroxisomes. The peroxisomal UK114 is a soluble matrix protein and it is not inducible by the peroxisomal proliferator clofibrate. The data predict involvement of UK114 in peroxisomal metabolism.  

3.906           Fis1, DLP1, and Pex11p coordinately regulate peroxisome morphogenesis

Kobayashi, S., Tanaka, A. and Fujika, Y. Exp. Cell Res., 313, 1675-1686 (2007)   Dynamin-like protein 1 (DLP1) and Pex11pβ function in morphogenesis of peroxisomes. In the present work, we investigated whether Fis1 is involved in fission of peroxisomes. Endogenous Fis1 was morphologically detected in peroxisomes as well as mitochondria in wild-type CHO-K1 and DLP1-defective ZP121 cells. Subcellular fractionation studies also revealed the presence of Fis1 in peroxisomes. Peroxisomal Fis1 showed the same topology, i.e., C-tail anchored membrane protein, as the mitochondrial one. Furthermore, ectopic expression of FIS1 induced peroxisome proliferation in CHO-K1 cells, while the interference of FIS1 RNA resulted in tubulation of peroxisomes, hence reducing the number of peroxisomes. Fis1 interacted with Pex11pβ, by direct binding apparently involving the C-terminal region of Pex11pβ in the interaction. Pex11pβ also interacted with each other, whereas the binding of Pex11pβ to DLP1 was not detectable. Moreover, ternary complexes comprising Fis1, Pex11pβ, and DLP1 were detected by chemical cross-linking. We also showed that the highly conserved N-terminal domain of Pex11pβ was required for the homo-oligomerization of Pex11pβ and indispensable for the peroxisome-proliferating activity. Taken together, these findings indicate that Fis1 plays important roles in peroxisome division and maintenance of peroxisome morphology in mammalian cells, possibly in a concerted manner with Pex11pβ and DLP1.  

3.907           Opposing Actions of Endocannabinoids on Cholangiocarcinoma Growth: RECRUITMENT OF Fas AND Fas LIGAND TO LIPID RAFTS

DeMorrow, S. et al

1. Biol. Chem., 282(17), 13098-13113 (2007)

  Cholangiocarcinomas are devastating cancers of biliary origin with limited treatment options. Modulation of the endocannabinoid system is being targeted to develop possible therapeutic strategies for a number of cancers; therefore, we evaluated the effects of the two major endocannabinoids, anandamide and 2-arachidonylglycerol, on numerous cholangiocarcinoma cell lines. Although anandamide was antiproliferative and proapoptotic, 2-arachidonylglycerol stimulated cholangiocarcinoma cell growth. Specific inhibitors for each of the cannabinoid receptors did not prevent either of these effects nor did pretreatment with pertussis toxin, a G_i/o protein inhibitor, suggesting that anandamide and 2-arachidonylglycerol did not exert their diametric effects through any known cannabinoid receptor or through any other G_i/o protein-coupled receptor.Using the lipid raft disruptors methyl--cyclodextrin and filipin,we demonstrated that anandamide, but not 2-arachidonylglycerol,requires lipid raft-mediated events to inhibit cellular proliferation.Closer inspection of the lipid raft structures within the cellmembrane revealed that although anandamide treatment had noobservable effect 2-arachidonylglycerol treatment effectivelydissipated the lipid raft structures and caused the lipid raft-associatedproteins lyn and flotillin-1 to disperse into the surroundingmembrane. In addition, anandamide, but not 2-arachidonylglycerol,induced an accumulation of ceramide, which was required foranandamide-induced suppression of cell growth. Finally we demonstratedthat anandamide and ceramide treatment of cholangiocarcinomacells recruited Fas and Fas ligand into the lipid rafts, subsequentlyactivating death receptor pathways. These findings suggest thatmodulation of the endocannabinoid system may be a target forthe development of possible therapeutic strategies for the treatmentof this devastating cancer.  

3.908           RhoB plays an essential role in CXCR2 sorting decisions

Neel, N.F., Lapierre, L.A., Goldenring, J.R. and Richmond, A.

1. Cell Sci., 120, 1559-1571 (2007)

  The CXCR2 chemokine receptor is a G-protein-coupled receptor that undergoes clathrin-mediated endocytosis upon ligand binding. The trafficking of CXCR2 is crucial for cells to maintain a proper chemotactic response. The mechanisms that regulate the recycling/degradation sorting decision are unknown. In this study, we used dominant-negative (T19N) and GTPase-deficient activated (Q63L) RhoB mutants, as well as RhoB small interfering RNA (siRNA) to investigate the role of RhoB in CXCR2 trafficking. Expression of either of the RhoB mutants or transfection of RhoB siRNA impaired CXCR2-mediated chemotaxis. Expression of RhoB T19N and transfection of RhoB siRNA impaired sorting of CXCR2 to the lysosome after 3 hours of CXCL8 stimulation and impaired CXCL8-induced CXCR2 degradation. In cells expressing the RhoB Q63L mutant, CXCR2 recycling through the Rab11a recycling compartment was impaired after 30 minutes of CXCL8 stimulation as was CXCL8-induced CXCR2 degradation. For cells expressing activated RhoB, CXCR2 colocalized with Rab4, a marker for the rapid recycling pathway, and with the mannose-6-phosphate receptor, which traffics between the trans-Golgi network and endosomes. These data suggest that CXCR2 recycles through alternative pathways. We conclude that oscillation of RhoB GTPase activity is essential for appropriate sorting decisions, and for directing CXCR2 degradation and recycling – events that are required for optimal chemotaxis.  

3.909           Tyrosine phosphorylation and lipid raft association of pseudorabies virus glycoprotein E during antibody-mediated capping

Desplanques, A.S., Nauwynck, H.J., Tilleman, K., Deforce, D. and Favoreel, H.W. Virology, 362(1), 60-66 (2007)   In specific cell types infected with the alphaherpesviruses herpes simplex virus and pseudorabies virus (PRV), addition of virus-specific antibodies results in redistribution of cell-surface-anchored viral proteins. This redistribution is triggered by the viral protein gE and consists of the directional movement of the antibody–antigen complexes to one pole of the cell. This viral capping process has been associated with increased antibody-resistant virus spread and strongly resembles immunoreceptor capping, a process that is crucial in activation of different immune cells (e.g. capping of Fcγ-receptors, B and T cell receptors). Here, we report that the PRV gE-mediated viral capping process results in increased Src kinase-mediated tyrosine phosphorylation of the cytoplasmic domain of gE and that a fraction of gE associates with lipid rafts, all very reminiscent of immunoreceptor capping. These results provide evidence that gE-mediated capping is a viral mimicry of immunoreceptor capping.  

3.910           APOBEC3G Multimers Are Recruited to the Plasma Membrane for Packaging into Human Immunodeficiency Virus Type 1 Virus-Like Particles in an RNA-Dependent Process Requiring the NC Basic Linker

Burnett, A. and Spearman, P.

1. Virol., 81(10), 5000-5013 (2007)

  APOBEC3G is an endogenous host restriction factor that inhibits human immunodeficiency virus (HIV) replication. The antiviral activity of APOBEC3G is dependent upon its incorporation into the virus particle. The mechanisms governing incorporation of APOBEC3G into virus particles are not completely understood. In particular, some investigators have reported that APOBEC3G interacts directly with the nucleocapsid (NC) subunit of Gag, while others have found that an RNA intermediate is required for Gag-APOBEC3G interactions. In this study, we confirmed the RNA dependence of APOBEC3G packaging and performed detailed mapping of the determinants within NC that are required for virion incorporation. Surprisingly, APOBEC3G packaging did not correlate well with the presence of the N-terminal "I," or interaction, domain within NC. Specifically, Gag constructs containing only the N-terminal region of NC packaged minimal amounts of APOBEC3G, while significant levels of APOBEC3G packaging were achieved with Gag constructs containing the basic linker region of NC. Furthermore, membrane-binding experiments revealed that the basic linker region was essential for the membrane association of APOBEC3G in a Gag-APOBEC3G complex. Fluorescence resonance energy transfer was detected between labeled APOBEC3G in cells and in particles, indicating that APOBEC3G is packaged as a multimer that is bound to packaged RNA. Regions of APOBEC3G-Gag colocalization at the plasma membrane were detected that were distinct from the punctate cytoplasmic bodies where APOBEC3G accumulates within the cell. Together, our results indicate that APOBEC3G multimerizes in an RNA-dependent fashion and that RNA-APOBEC3G multimers are recruited to the plasma membrane and subsequently into virion particles by Gag.  

3.911           The Tyrosine Kinase Fyn Determines the Localization of TrkB Receptors in Lipid Rafts

Pereira, D.R. and Chao, M.V.

1. Neurosci., 27(18), 4859-4869 (2007)

  Localization of Trk neurotrophin receptors is an important factor in directing cellular communication in developing and mature neurons. One potential site of action is in lipid raft membrane microdomains. Although Trk receptors have been localized to lipid rafts, little is known about how these neurotrophin receptors are directed there or how localization to these membrane microdomains regulates Trk signaling. Here, we report that the TrkB brain-derived neurotrophic factor (BDNF) receptor specifically localized to intracellular lipid rafts in cortical and hippocampal membranes in response to BDNF and that this process was critically dependent on the tyrosine kinase Fyn. BDNF-induced TrkB accumulation at lipid rafts was prevented by blocking the internalization of TrkB. BDNF stimulation also resulted in the association between endogenous TrkB and Fyn. Moreover, in neurons derived from Fyn knock-out mice, the translocation of TrkB to lipid rafts in response to BDNF was compromised, whereas the corticohippocampal region of Fyn mutants displayed lower amounts of TrkB in lipid rafts in vivo. In support of a role for lipid rafts in neurotrophin signaling, inhibiting TrkB translocation to lipid rafts, either by using Fyn knock-out neurons or lipid raft-disturbing agents, prevented the full activation of TrkB and of downstream phospholipase C- . These results indicate that the lipid raft localization of TrkB receptors is regulated by Fyn and represents an important factor in determining the outcome of BDNF signaling in neurons.  

3.912           Coupling of the de Novo Fatty Acid Biosynthesis and Lipoylation Pathways in Mammalian Mitochondria

Witkowski, A., Joshi, A. and Smith, S.

1. Biol. Chem., 282(19), 14178-14185 (2007)

  The objective of this study was to identify the products and possible role of a putative pathway for de novo fatty acid synthesis in mammalian mitochondria. Bovine heart mitochondrial matrix preparations were prepared free from contamination by proteins from other subcellular components and, using a combination of radioisotopic labeling and mass spectrometry, were shown to contain all of the enzymes required for the extension of a 2-carbon precursor by malonyl moieties to saturated acyl-ACP thioesters containing up to 14 carbon atoms. A major product was octanoyl-ACP and, in the presence of the apo-H-protein of the glycine cleavage complex, the newly synthesized octanoyl moieties were translocated to the lipoylation site on the acceptor protein. These studies demonstrate that one of the functions of the de novo fatty acid biosynthetic pathway in mammalian mitochondria is to provide the octanoyl precursor required for the essential protein lipoylation pathway.  

3.913           Separation of cell-cell adhesion complexes by differential centrifugation

Vogelmann, R. and Nelson, W.J. Methods in Mol. Biol., 370, 11-22 (2007)   The number of proteins found associated with cell-cell adhesion substructures is growing rapidly. Based on potential protein-protein interactions, complex protein networks at cell-cell contacts can be modeled. Traditional studies to examine protein-protein interactions include co-immunoprecipitation or pull-down experiments of tagged proteins. These studies provide valuable information that proteins can associate directly or indirectly through other proteins in a complex. However, they do not clarify if a given protein is part of other protein complexes or inform about the specificity of those interactions in the context of adhesion substructures. Thus, it is not clear if models compiled from these types of studies reflect the combination of protein interactions in the adhesion complex in vivo for a specific cell type. Therefore, we present here a method to separate cell-cell contact membrane substructures with their associated protein complexes based on their buoyant behavior in iodixanol density gradients. Analysis of 16 proteins of the apical junctional complex (AJC) in epithelial Madin-Darby canine kidney cells revealed a more simple organization of the AJC adhesion complex than that predicted from the combination of all possible protein-protein interactions defined from co-immunoprecipitation and pull-down experiments.  

3.914           HSV Viral Envelope Proteins Partition With Lipid Rafts in Infected Retinal Ganglion Cell Axons

Cortez, D.A., Sucher, A. and LaVail, J.H. Invest. Ophthalmol. Vis. Sci., 48, E-abstract 3166 (2007)   Purpose:Herpes simplex virus type 1 (HSV) is responsible for recurrentscarring of the corneal epithelium in ocular herpetic keratitis,a common cause of blindness. HSV envelope glycoproteins areessential for cell-cell spread of infection from trigeminalaxons to corneal cells. What has been lacking is details ofhow newly made envelope proteins are transported within axons.By analogy to the transport of synaptic membrane precursors,lipid raft membranes may also support the transport of viralglycoproteins. We have tested the hypothesis that HSV glycoproteinsare transported in association with lipid raft membranes ininfected retinal axons in vivo. Methods:Murine retinal ganglion cells were infected with a wild-type (wt) virus, and after 24 hrs the mice were given Valacyclovir to pulse infect the neurons. The optic pathways were dissected 5 days postinfection, and the tissues were processed for Western blotting using antibodies to gB, gC and gD. Additional animals were infected and treated as above, but the optic pathways were dissected and prepared in an Optiprep flotation assay to separatedetergent-resistant membranes (DRM) and detergent-soluble membranes(DSM). We used GM1 and caveolin as control proteins for DRMand transferrin receptor as a control for DSM. Results:By 5 days postinfection all three glycoproteins were transportedto the OT. However, the transport of gD appeared to be moreefficient than that of gC or gB. In preliminary studies allof the tested glycoproteins, (gB, gC, and gD) as well as GM1and caveolin were present in the DRM fractions. We also foundgB, gC and gD in the DSM fraction. Transferrin receptor wasfound principally in the DSM fraction. Conclusions:By five days after infection the three glycoproteins had been transported to the most distal portion of the retinal axons. The three envelope glycoproteins associated with the lipid raft membrane fraction. Further experiments to define host membrane components that associate with transported viral glycoproteins will be essential to understanding the intracellular localization and mechanisms of transport.  

3.915           Myelin protein zero/P0 phosphorylation and function require an adaptor protein linking it to RACK1 and PKC

Gaboreanu, A-M. et al

1. Cell Biol., 177(4), 707-716 (2007)

  Point mutations in the cytoplasmic domain of myelin protein zero (P0; the major myelin protein in the peripheral nervous system) that alter a protein kinase C (PKC ) substrate motif (198HRSTK201) or alter serines 199 and/or 204 eliminate P0-mediated adhesion. Mutation in the PKC substrate motif (R198S) also causes a form of inherited peripheral neuropathy (Charcot Marie Tooth disease [CMT] 1B), indicating that PKC -mediated phosphorylation of P0 is important for myelination. We have now identified a 65-kD adaptor protein that links P0 with the receptor for activated C kinase 1 (RACK1). The interaction of p65 with P0 maps to residues 179–197 within the cytoplasmic tail of P0. Mutations or deletions that abolish p65 binding reduce P0 phosphorylation and adhesion, which can be rescued by the substitution of serines 199 and 204 with glutamic acid. A mutation in the p65-binding sequence G184R occurs in two families with CMT, and mutation of this residue results in the loss of both p65 binding and adhesion function.  

3.916           Characterization of Mammalian Par 6 as a Dual-Location Protein

Cline, E.G. and Nelson, W.J. Mol. Cell. Biol., 27(12), 4431-4443 (2007)   Par 6 acts as a scaffold protein to facilitate atypical protein kinase C-mediated phosphorylation of cytoplasmic protein complexes, leading to epithelial and neuronal cell polarization. In addition to its location in the cytoplasm, Par 6 is localized to the nucleus. However, its organization and potential functions in the nucleus have not been examined. Using an affinity-purified Par 6 antibody and a chimera of Par 6 and green fluorescent protein, we show that Par 6 localizes precisely to nuclear speckles, but not to other nuclear structures, and displays characteristics of speckle proteins. We show that Par 6 colocalizes in the nucleus with Tax, a transcriptional activator of the human T-cell leukemia virus type 1 long terminal repeat, but multiple lines of evidence show that Par 6 is not directly involved in known functions of speckle proteins, including general transcription, splicing, or mRNA transport. Significantly, however, the structure of nuclear speckles is lost when Par 6 levels are reduced by Par 6-specific small interfering RNA. Therefore, we hypothesize that Par 6 in the nucleus acts as a scaffolding protein in nuclear speckle complexes, similar to its role in the cytoplasm.  

3.917           The Host Protein Staufen1 Participates in Human Immunodeficiency Virus Type 1 Assembly in Live Cells by Influencing pr55Gag Multimerization

Chatel-Chaix, L., Abrahamyan, L., Frechima, C., Mouland, A.J. and DesGroseillers, L.

1. Virol., 81(12), 6216-6230 (2007)

  Human immunodeficiency virus type 1 (HIV-1) requires the sequential activities of virus-encoded proteins during replication. The activities of several host cell proteins and machineries are also critical to the completion of virus assembly and the release of infectious virus particles from cells. One of these proteins, the double-stranded RNA-binding protein Staufen1 (Stau1), selectively associates with the HIV-1 genomic RNA and the viral precursor Gag protein, pr55^Gag. In this report, we tested whether Stau1 modulates pr55^Gag assembly using a new and specific pr55^Gag oligomerization assay based on bioluminescence resonance energy transfer (BRET) in both live cells and extracts after cell fractionation. Our results show that both the overexpression and knockdown of Stau1 increase the pr55^Gag-pr55^Gag BRET levels, suggesting a role for Stau1 in regulating pr55^Gag oligomerization during assembly. This effect of Stau1 on pr55^Gag oligomerization was observed only in membranes, a cellular compartment in which pr55^Gag assembly primarily occurs. Consistently, expression of Stau1 harboring a vSrc myristylation signal led to a 6.5-fold enrichment of Stau1 in membranes and a corresponding enhancement in the Stau1-mediated effect on pr55^Gag-pr55^Gag BRET, demonstrating that Stau1 acts on assembly when targeted to membranes. A role for Stau1 in the formation of particles is further supported by the detection of membrane-associated detergent-resistant pr55^Gag complexes and the increase of virus-like particle release when Stau1 expression levels are modulated. Our results indicate that Stau1 influences HIV-1 assembly by modulating pr55^Gag-pr55^Gag interactions, as shown in a live cell interaction assay. This likely occurs when Stau1 interacts with membrane-associated assembly intermediates.  

3.918           Binding Dynamics of Hepatitis C Virus' NS5A Amphipathic Peptide to Cell and Model Membranes

Cho, N-J., Cheong, K.H., Lee, CH., Frank, C.F. and Glenn, J.S.

1. Virol., 81(12), 6682-6689 (2007)

  Membrane association of the hepatitis C virus NS5A protein is required for viral replication. This association is dependent on an N-terminal amphipathic helix (AH) within NS5A and is restricted to a subset of host cell intracellular membranes. The mechanism underlying this specificity is not known, but it may suggest a novel strategy for developing specific antiviral therapy. Here we have probed the mechanistic details of NS5A AH-mediated binding to both cell-derived and model membranes by use of biochemical membrane flotation and quartz crystal microbalance (QCM) with dissipation. With both assays, we observed AH-mediated binding to model lipid bilayers. When cell-derived membranes were coated on the quartz nanosensor, however, significantly more binding was detected, and the QCM-derived kinetic measurements suggested the existence of an interacting receptor in the target membranes. Biochemical flotation assays performed with trypsin-treated cell-derived membranes exhibited reduced AH-mediated membrane binding, while membrane binding of control cytochrome b5 remained unaffected. Similarly, trypsin treatment of the nanosensor coated with cellular membranes abolished AH peptide binding to the cellular membranes but did not affect the binding of a control lipid-binding peptide. These results therefore suggest that a protein plays a critical role in mediating and stabilizing the binding of NS5A's AH to its target membrane. These results also demonstrate the successful development of a new nanosensor technology ideal both for studying the interaction between a protein and its target membrane and for developing inhibitors of that interaction.  

3.919           Membrane microdomain formation is crucial in epiboly during gastrulation of medaka

Adachi, T., Sato, C. and Kitajima, K. Biochem. Biophys. Res. Comm., 358(3), 848-853 (2007)   Membrane microdomain (microdomain) was isolated from early gastrula embryos. The isolated microdomain was characterized by enrichment of cholesterol and sphingomyelin, and by the presence of huge glycoproteins containing Lewis X structure. Importance of the microdomain in the progress of epiboly was assessed using methyl β-cyclodextrin (MBCD) and C2-ceramide that disrupt microdomains through different mechanisms. Both reagents efficiently disrupted the microdomain structure and concomitantly impaired epiboly. Interestingly, when embryos pretreated with MBCD, a cholesterol-binding molecule, were exogenously supplemented with cholesterol, the embryos underwent not only reconstitution of the microdomain, but also complete restoration to the normal epiboly. Thus, normal or impaired development is reversibly controlled by the cholesterol-dependent formation or disruption of microdomains. The most typical phenotype of the microdomain-disrupted embryos is detachment of cells from the blastoderm, suggesting that a major contribution of microdomains to epiboly is cell adhesion of blastodermal cells.  

3.920           Quantification and regulation of the subcellular distribution of bile acid coenzyme A:amino acid N-acyltransferase activity in rat liver

Styles, N.A., Falany, J.L., Barnes, S. and Falany, C.N.

1. Lipid Res., 48, 1305-1315 (2007)

  Bile acid coenzyme A:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids glycine and taurine. To quantify total BAT activity in liver subcellular organelles, livers from young adult male and female Sprague-Dawley rats were fractionated into multiple subcellular compartments. In male and female rats, 65–75% of total liver BAT activity was found in the cytosol, 15–17% was found in the peroxisomes, and 5–10% was found in the heavy mitochondrial fraction. After clofibrate treatment, male rats displayed an increase in peroxisomal BAT specific activity and a decrease in cytosolic BAT specific activity, whereas females showed an opposite response. However, there was no overall change in BAT specific activity in whole liver homogenate. Treatment with rosiglitazone or cholestyramine had no effect on BAT activity in any subcellular compartment. These experiments indicate that the majority of BAT activity in the rat liver resides in the cytosol. Approximately 15% of BAT activity is present in the peroxisomal matrix. These data support the novel finding that clofibrate treatment does not directly regulate BAT activity but does alter the subcellular localization of BAT.  

3.921           Contact-dependent inhibition of EGFR signaling by Nf2/Merlin

Curto, M., Cole, B.K., Lallemand, D., Liu, C-H. and McClatchey, A.I.

1. Cell Biol., 177(5), 893-903 (2007)

  The neurofibromatosis type 2 (NF2) tumor suppressor, Merlin, is a membrane/cytoskeleton-associated protein that mediates contact-dependent inhibition of proliferation. Here we show that upon cell–cell contact Merlin coordinates the processes of adherens junction stabilization and negative regulation of epidermal growth factor receptor (EGFR) signaling by restraining the EGFR into a membrane compartment from which it can neither signal nor be internalized. In confluent Nf2^–/– cells, EGFR activation persists, driving continued proliferation that is halted by specific EGFR inhibitors. These studies define a new mechanism of tumor suppression, provide mechanistic insight into the poorly understood phenomenon of contact-dependent inhibition of proliferation, and suggest a therapeutic strategy for NF2-mutant tumors.  

3.922           Three-dimensional architecture of murine rod outer segments determined by cryoelectron tomography

Nickel, S., Park, P.S-H., Baumeister, W. and Polczewski, K.

1. Cell Biol., 177(5), 917-925 (2007)

  The rod outer segment (ROS) of photoreceptor cells houses allcomponents necessary for phototransduction, a set of biochemicalreactions that amplify and propagate a light signal. Theoreticalapproaches to quantify this process require precise informationabout the physical boundaries of the ROS. Dimensions of internalstructures within the ROS of mammalian species have yet to bedetermined with the precision required for quantitative considerations.Cryoelectron tomography was utilized to obtain reliable three-dimensionalmorphological information about this important structure frommurine retina. Vitrification of samples permitted imaging ofthe ROS in a minimally perturbed manner and the preservationof substructures. Tomograms revealed the characteristic highlyorganized arrangement of disc membranes stacked on top of oneanother with a surrounding plasma membrane. Distances amongthe various membrane components of the ROS were measured todefine the space available for phototransduction to occur. Reconstructionof segments of the ROS from single-axis tilt series images provideda glimpse into the three-dimensional architecture of this highlydifferentiated neuron. The reconstructions revealed spacersthat likely maintain the proper distance between adjacent discsand between discs and the plasma membrane. Spacers were founddistributed throughout the discs, including regions that aredistant from the rim region of discs.  

3.923           Role of caveolae in the pathogenesis of cholesterol-induced gallbladder muscle hypomotility

Xiao, Z., Schmitz, F., Pricolo, V.E., Biancani, P. and Behar, J. Am. J. Physiol. Gastrointest. Liver Physiol., 292, G1641-G1649 (2007)   Muscle cells from human gallbladders (GB) with cholesterol stones (ChS) exhibit a defective contraction, excess cholesterol (Ch) in the plasma membrane, and lower binding of CCK-1 receptors. These abnormalities improved after muscle cells were incubated with Ch-free liposomes that remove the excess Ch from the plasma membrane. The present studies were designed to investigate the role of caveolin-3 proteins (Cav-3) in the pathogenesis of these abnormalities. Muscle cells from GB with ChS exhibit higher Ch levels in the plasma membrane that were mostly localized in caveolae and associated with parallel increases in the expression of Cav-3 in the caveolae compared with that in GB with pigment stones (PS). The overall number of CCK-1 receptors in the plasma membrane was not different between muscle cells from GB with ChS and PS, but they were increased in the caveolae in muscle cells from GB with ChS. Treatment of muscle cells from GB with ChS with a G _i3 protein fragment increased the total binding of CCK-1 receptors (from 8.3 to 11.2%) and muscle contraction induced by CCK-8 (from 11.2 to 17.3% shortening). However, G _q/11 protein fragment had no such effect. Moreover, neither fragment had any effect on muscle cells from GB with PS. We conclude that the defective contraction of muscle cells with excessive Ch levels in the plasma membrane is due to an increased expression of Cav-3 that results in the sequestration of CCK-1 receptors in the caveolae, probably by inhibiting the functions of G _i3 proteins.  

3.924           Subcellular Localization and Physiological Significance of Intracellular Mannan-binding Protein

Nonaka, M. et al

1. Biol. Chem., 282(24), 17908-17920 (2007)

  Mannan-binding protein (MBP) is a C-type mammalian lectin specific for mannose and N-acetylglucosamine. MBP is mainly synthesized in the liver and occurs naturally in two forms, serum MBP (S-MBP) and intracellular MBP (I-MBP). S-MBP activates complement in association with MBP-associated serine proteases via the lectin pathway. Despite our previous study (Mori, K., Kawasaki, T., and Yamashina, I. (1984) Arch. Biochem. Biophys. 232, 223-233), the subcellular localization of I-MBP and its functional implication have not been clarified yet. Here, as an extension of our previous studies, we have demonstrated that the expression of human MBP cDNA reproduces native MBP differentiation of S-MBP and I-MBP in human hepatoma cells. I-MBP shows distinct accumulation in cytoplasmic granules, and is predominantly localized in the endoplasmic reticulum (ER) and involved in COPII vesicle-mediated ER-to-Golgi transport. However, the subcellular localization of either a mutant (C236S/C244S) I-MBP, which lacks carbohydrate-binding activity, or the wild-type I-MBP in tunicamycin-treated cells shows an equally diffuse cytoplasmic distribution, suggesting that the unique accumulation of I-MBP in the ER and COPII vesicles is mediated by an N-glycan-lectin interaction. Furthermore, the binding of I-MBP with glycoprotein intermediates occurs in the ER, which is carbohydrate- and pH-dependent, and is affected by glucose-trimmed high-mannose-type oligosaccharides. These results strongly indicate that I-MBP may function as a cargo transport lectin facilitating ER-to-Golgi traffic in glycoprotein quality control.  

3.925           Stepwise proteolysis liberates tau fragments that nucleate the Alzheimer-like aggregation of full-length tau in a neuronal cell model

Wang, Y.P., Biernat, J., Pickjardt, M., Mandelkow, E. Amd Mandelkow, E-M. PNAS, 104(24), 10252-10257 (2007)   Tau is a highly soluble protein, yet it aggregates abnormally in Alzheimer's disease. Here, we address the question of proteolytic processing of tau and the nucleation of aggregates by tau fragments. We show in neuronal cell models that fragments of the repeat domain of tau containing mutations of FTDP17 (frontotemporal dementia with parkinsonism linked to chromosome 17), produced by endogenous proteases, can induce the aggregation of full-length tau. Fragments are generated by successive cleavages, first N-terminally between K257 and S258, then C-terminally around residues 353–364; conversely, when the N-terminal cleavage is inhibited, no fragmentation and aggregation takes place. The C-terminal truncation and the coaggregation of fragments with full-length tau depends on the propensity for -structure. The aggregation is modulated by phosphorylation but does not depend on it. Aggregation but not fragmentation as such is toxic to cells; conversely, toxicity can be prevented by inhibiting either aggregation or proteolysis. The results reveal a novel pathway of abnormal tau aggregation in neuronal cells.  

3.926           Lipid Rafts Are Triage Centers for Multimeric and Monomeric Thyrotropin Receptor Regulation

Latif, R., Ando, T. and Davies, T.F. Endocrinology, 148(7), 3164-3175 (2007)   The TSH receptor (TSHR), a heptahelical G protein-coupled receptor on the surface of thyrocytes, is a major autoantigen and physiological regulator of the thyroid gland. Unlike other G protein-coupled receptors, the TSHR undergoes posttranslational cleavage of its ectodomain, leading to the existence of several forms of the receptor on the plasma membrane. We previously hypothesized that to achieve high fidelity and specificity of TSH ligand or TSHR autoantibody signaling, the TSHR may compartmentalize into microdomains within the plasma membrane. In support of this hypothesis we have shown previously that TSHRs reside in GM₁ ganglioside-enriched lipid rafts in the plasma membrane of TSHR-expressing cells. In this study, we further explored the different forms of TSHRs that reside in lipid rafts. We studied both TSHR-transfected cells and rat thyrocytes, using both nondetergent biochemical analyses and receptor-lipid raft colocalization. Using the biochemical approach, we observed that monomeric receptors existed in both raft and nonraft fractions of the cell surface in the steady state. We also demonstrated that the multimeric forms of the receptor were preferentially partitioned into the lipid microdomains. Different TSHR forms, including multimers, were dynamically regulated both by receptor-specific and postreceptor-specific modulators. TSH ligand and TSHR antibody of the stimulating variety induced a decrease of multimeric forms in the raft fractions. In addition, multimeric and monomeric forms of the receptor were both associated with Gs within and without the rafts. Although failure to achieve total lipid raft disruption prevented a conclusion regarding the relative power of TSHR signaling within and without the raft domains, these data showed clearly that not only were a significant proportion of TSHRs residing within lipid microdomains but that constitutive multimerization of TSHRs was actually regulated within the lipid rafts.  

3.927           Correlation of Golgi localization of ZW10 and centrosomal accumulation of dynactin

Arasaki, K., Uemura, T., Tani, K. and Tagaya, M. Biochem. Biophys. Res. Comm., 359(3), 811-816 (2007)   ZW10 participates in the termination of the spindle checkpoint during mitosis by interacting with dynamitin, a subunit of the dynein accessory complex dynactin. We previously showed that ZW10 is attached to the endoplasmic reticulum through RINT-1 in interphase HeLa cells and involved in membrane transport between the endoplasmic reticulum and Golgi. Although a recent study demonstrated that ZW10 is localized in the Golgi in COS7 cells, the mechanism that regulates ZW10 localization remains unknown. In this study we showed a correlation between the Golgi localization of ZW10 and the centrosomal accumulation of dynactin. The amounts of ZW10 associated with dynactin were larger in cells where ZW10 was present in the Golgi than those where ZW10 was not in the Golgi. The targeting of ZW10 to the perinuclear Golgi region was found to depend on the perinuclear accumulation of dynactin, suggesting that dynactin regulates ZW10 localization.  

3.928           Siva is an apoptosis-selective p53 target gene important for neuronal cell death

Jacobs, S.B.R., Basak, S., Murray, J., Pathak, N. and Attardi, L.D. Cell Death and Differentiation, 14, 1374-1385 (2007)   p53 plays a central role in neuronal cell death resulting from acute injury or disease. To define the pathway by which p53 triggers apoptosis, we used microarray analysis to identify p53 target genes specifically upregulated during apoptosis but not cell cycle arrest. This analysis identified a small subset of targets highly selective for the p53 apoptotic response, including Siva, a proapoptotic protein whose function is not well understood. Siva's expression pattern suggests that it plays an instructive role in apoptosis, and accordingly, we demonstrate that Siva is essential for p53-dependent apoptosis in cerebellar granule neurons. In addition, we determine that endogenous Siva is associated with the plasma membrane and that Caspase-8 and Bid are important for neuronal apoptosis. Our studies highlight the participation of membrane signaling events in p53's apoptotic program in primary neurons and have significant implications for understanding the mechanisms underlying pathogenesis after neuronal injury and in neurodegenerative diseases.  

3.929           Identification of an ADAM2-ADAM3 Complex on the Surface of Mouse Testicular Germ Cells and Cauda Epididymal Sperm

Nishimura, H., Myles, D.G. and Primakoff, P.

1. Biol. Chem., 282(24), 17900-17907 (2007)

  Male mice lacking ADAM2 (fertilin ) or ADAM3 (cyritestin) are infertile; cauda epididymal sperm (mature sperm) from these mutant mice cannot bind to the egg zona pellucida. ADAM3 is barely present in Adam2-null sperm, despite normal levels of this protein in Adam2-null testicular germ cells (TGCs; sperm precursor cells). Here, we have explored the molecular basis for the loss of ADAM3 in Adam2-null TGCs to clarify the biosynthetic and functional linkage of ADAM2 and ADAM3. A small portion of total ADAM3 was found present on the surface of wild-type and Adam2^-/- TGCs at similar levels. In the Adam2-null TGCs, however, surface-localized ADAM3 exhibited an increased amount of an endoglycosidase H-resistant form that may be related to instability of ADAM3. Moreover, we found a complex between ADAM2 and ADAM3 on the surface of TGCs and sperm. The intracellular chaperone calnexin was a component of the testicular ADAM2-ADAM3 complex. Our findings suggest that the association with ADAM2 is a key element for stability of ADAM3 in epididymal sperm. The presence of the ADAM2-ADAM3 complex in sperm also suggests a potential role of ADAM2 with ADAM3 in sperm binding to the egg zona pellucida.  

3.930           Productive Human Immunodeficiency Virus Type 1 Assembly Takes Place at the Plasma Membrane

Finzi, A., Orthwein, A., Mercier, J. and Cohen, E.A.

1. Virol., 81(14), 7476-7490 (2007)

  Gag proteins are necessary and sufficient to direct human immunodeficiency virus type 1 (HIV-1) particle assembly and budding. Recent evidence suggests that Gag targeting to late endosomal/multivesicular body (LE/MVB) compartments occurs prior to viral particle budding at the plasma membrane (PM). However, the route that Gag follows before reaching its steady-state destinations still remains a subject of debate. Using a subcellular fractionation method that separates PM from LE/MVB combined with pulse-chase labeling, we analyzed Gag trafficking in HIV-1-producing HEK 293T cells. Our results reveal that the majority of newly synthesized Gag is primarily targeted to the PM. While PM-targeted Gag was efficiently released, a significant fraction of the remaining cell surface-associated Gag was found to be subsequently internalized to LE/MVB, where it accumulated, thus accounting for the majority of LE/MVB-associated Gag. Importantly, this accumulation of Gag in LE/MVB was found to be cholesterol dependent since it was sensitive to the sterol-binding drugs filipin and methyl-ß-cyclodextrin. These results point towards the PM as being the primary site of productive HIV-1 assembly in cells that also support Gag accumulation in intracellular compartments.  

3.931           Pitfalls in isolating lipid rafts

Nothdurfter, C., Rammes, G., Rein, T. and Rupprecht, R. Nature review Neurosci., 8 567 (2007)   The recent Review by Allen et al. on lipid raft microdomains and neurotransmitter receptor signalling¹ provides an excellent overview of important structural and functional aspects of these specific membrane microdomains, with a particular focus on their role in the nervous system. Nevertheless, we would like to emphasize two important aspects. As outlined in the Review, the valid and reproducible isolation of lipid rafts is not trivial, but mandatory to draw correct conclusions. Inconsistencies in raft isolation procedures, for example the type, amount and duration of detergent use, make results difficult to compare and may contribute to some controversies in the field. A particularly important issue is the presentation of both a positive and a negative control. Mostly, caveolin 1 or flotillin 1 are used as raft marker proteins^{2, 3, 4}, whereas the transferrin receptor or other proteins such as the Na⁺/K⁺-ATPase are reported as non-raft proteins^{5, 6, 7}. Only a few studies show convincing data of a clear separation of raft from non-raft proteins^{5, 6, 8}. This separation relies highly on the preparation procedure, in particular the use of detergent, for which the type of detergent, the concentration and the duration of incubation are the main determinants. Furthermore, the preparation procedure should be chosen according to the type of tissue under investigation. An example from our laboratory, in which we isolated caveolin 1 and flotillin 1 as raft-associated proteins and the transferrin receptor as a non-raft protein, illustrates this need to vary conditions for different tissue types (Fig. 1). When investigating the concentration of detergent and incubation time needed to separate raft from non-raft proteins within the range reported in the literature^{5, 6}, we also observed a dose–response relationship⁸. Future studies should therefore clearly demonstrate that the method selected is appropriate for the tissue type under investigation to separate raft from non-raft proteins when claiming raft association of particular proteins.  

3.932           Caspase-8 and c-FLIPL Associate in Lipid Rafts with NF- B Adaptors during T Cell Activation

Misra, R.S. et al

1. Biol. Chem., 282(27), 19365-19374 (2007)

  Humans and mice lacking functional caspase-8 in T cells manifest a profound immunodeficiency syndrome due to defective T cell antigen receptor (TCR)-induced NF- B signaling and proliferation. It is unknown how caspase-8 is activated following T cell stimulation, and what is the caspase-8 substrate(s) that is necessary to initiate T cell cycling. We observe that following TCR ligation, a small portion of total cellular caspase-8 and c-FLIP_L rapidly migrate to lipid rafts where they associate in an active caspase complex. Activation of caspase-8 in lipid rafts is followed by rapid cleavage of c-FLIP_L at a known caspase-8 cleavage site. The active caspase·c-FLIP complex forms in the absence of Fas (CD95/APO1) and associates with the NF- B signaling molecules RIP1, TRAF2, and TRAF6, as well as upstream NF- B regulators PKC , CARMA1, Bcl-10, and MALT1, which connect to the TCR. The lack of caspase-8 results in the absence of MALT1 and Bcl-10 in the active caspase complex. Consistent with this observation, inhibition of caspase activity attenuates NF- B activation. The current findings define a link among TCR, caspases, and the NF- B pathway that occurs in a sequestered lipid raft environment in T cells.  

3.933           Lack of a role of membrane-protein interactions in flow-dependent activation of EnaC

Carattino, M.D., Lin, W., Hill, W.G., Satlin, L.M. and Kleyman, T.R. Am. J. Physiol. Renal Physiol., 293, F316-F324 (2007)   Rates of Na⁺ absorption in the distal nephron increase proportionally with the rates of tubular flow. We tested the hypothesis that the deformation or tension generated in the plasma membrane in response to flow activates the epithelial sodium channel (ENaC). We modified the physical properties of the membrane by changing the temperature and the content of cholesterol. Rates of net Na⁺ absorption measured in cortical collecting ducts (CCDs) perfused at room temperature at slow ( 1) and fast ( 5 nl·min^–1·mm^–1) flow rates were less than those measured at 37°C at the same flow rates, although increases in tubular fluid flow rates led to comparable relative increases in net Na⁺ absorption at both temperatures. Xenopus laevis oocytes expressing ENaC responded to an increase in shear stress at 22–25°C with a discrete delay followed by a monoexponential increase in whole-cell Na⁺ currents. We observed that temperature affected 1) basal currents, 2) delay times, 3) kinetics of activation, and 4) fold-increase in macroscopic currents in response to flow. The magnitude of the response to flow displayed biphasic behavior as a function of temperature, with a minimal value at 25°C. Steady-state fluorescence anisotropic measurements of purified plasma membranes did not show any obvious phase transition behavior over a temperature range from 8.3°C to 36.5°C. Modification of the content of membrane cholesterol did not affect the response to flow. Our results suggest that the flow-dependent activation of ENaC is not influenced by modifications in the intrinsic properties of the plasma membrane.  

3.934           Ceramide transfer protein function is essential for normal oxidative stress response and lifespan

Rao, R.P. et al PNAS, 104(27), 11364-11369 (2007)   Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum to the Golgi complex, a process critical in synthesis and maintenance of normal levels of sphingolipids in mammalian cells. However, how its function is integrated into development and physiology of the animal is less clear. Here, we report the in vivo consequences of loss of functional CERT protein. We generated Drosophila melanogaster mutant flies lacking a functional CERT (Dcert) protein using chemical mutagenesis and a Western blot-based genetic screen. The mutant flies die early between days 10 and 30, whereas controls lived between 75 and 90 days. They display >70% decrease in ceramide phosphoethanolamine (the sphingomyelin analog in Drosophila) and ceramide. These changes resulted in increased plasma membrane fluidity that renders them susceptible to reactive oxygen species and results in enhanced oxidative damage to cellular proteins. Consequently, the flies showed reduced thermal tolerance that was exacerbated with aging and metabolic compromise such as decreasing ATP and increasing glucose levels, reminiscent of premature aging. Our studies demonstrate that maintenance of physiological levels of ceramide phosphoethanolamine by CERT in vivo is required to prevent oxidative damages to cellular components that are critical for viability and normal lifespan of the animal.  

3.935           Regulatory Binding Partners and Complexes of NHE3

Donowitz, M. and Li, X. Physiol. Rev., 87, 825-972 (2007)   NHE3 is the brush-border (BB) Na⁺/H⁺ exchanger of small intestine, colon, and renal proximal tubule which is involved in large amounts of neutral Na⁺ absorption. NHE3 is a highly regulated transporter, being both stimulated and inhibited by signaling that mimics the postprandial state. It also undergoes downregulation in diarrheal diseases as well as changes in renal disorders. For this regulation, NHE3 exists in large, multiprotein complexes in which it associates with at least nine other proteins. This review deals with short-term regulation of NHE3 and the identity and function of its recognized interacting partners and the multiprotein complexes in which NHE3 functions.  

3.936           Filamin-A regulates actin-dependent clustering of HIV receptors

Jimenez-Baranda, S. et al Nature Cell Biol., 9(7), 838-846 (2007)   Human immunodeficiency virus (HIV)-1 infection requires envelope (Env) glycoprotein gp120-induced clustering of CD4 and coreceptors (CCR5 or CXCR4) on the cell surface; this enables Env gp41 activation and formation of a complex that mediates fusion between Env-containing and target-cell membranes1. Kinetic studies show that viral receptors are actively transported to the Env-receptor interface in a process that depends on plasma membrane composition and the actin cytoskeleton2, 3, 4, 5, 6, 7. The mechanisms by which HIV-1 induces F-actin rearrangement in the target cell remain largely unknown. Here, we show that CD4 and the coreceptors interact with the actin-binding protein filamin-A, whose binding to HIV-1 receptors regulates their clustering on the cell surface. We found that gp120 binding to cell receptors induces transient cofilin-phosphorylation inactivation through a RhoA–ROCK-dependent mechanism. Blockade of filamin-A interaction with CD4 and/or coreceptors inhibits gp120-induced RhoA activation and cofilin inactivation. Our results thus identify filamin-A as an adaptor protein that links HIV-1 receptors to the actin cytoskeleton remodelling machinery, which may facilitate virus infection.    

3.937           Cholesterol depletion alters detergent-specific solubility profiles of selected tight junction proteins and the phosphorylation of occluding

Lynch, R.D. et al Exp. Cell Res., 313(2), 2597-2610 (2007)   Differential centrifugation of Triton X-100 or CHAPS lysates from control and cholesterol (CH)-depleted MDCK II cells, segregated integral tight junction (TJ) proteins associated with detergent-resistant membranes (DRMs) into two groups. Group A proteins (occludin, claudin-2 and -3) were detected in large, intermediate and small aggregates in both detergents, whereas group B proteins (claudin-1, -4 and -7) were observed in small aggregates in TX-100 and in intermediate and small aggregates in CHAPS. Depletion of CH altered the distribution of group A and B proteins among the three size categories in a detergent-specific manner. In lysates produced with octyl glucoside, a detergent that selectively extracts proteins from DRMs, group A proteins were undetectable in large aggregates and CH depletion did not alter the distribution of either group A or B proteins in intermediate or small aggregates. Neither occludin (group A) nor claudin-1 (group B) was in intimate enough contact with CH to be cross-linked to [³H]-photo-cholesterol. However, antibodies to either TJ protein co-immunoprecipitated caveolin-1, a CH-binding protein. Unlike claudins, occludin's presence in TJs and DRMs did not require palmitoylation. Equilibrium density centrifugation on discontinuous OptiPrep gradients revealed detergent-related differences in the densities of TJ-bearing DRMs. There was little or no change in those densities after CH depletion. Removing CH from the plasma membrane increased tyrosine and threonine phosphorylation of occludin, and transepithelial electrical resistance (TER) within 30 min. After 2 h of CH efflux, phospho-occludin levels and TER fell below control values. We conclude that the association of integral TJ proteins with DRMS, pelleted at low speeds, is partially CH-dependent. However, the buoyant density of TJ-associated DRMs is a function of the detergent used and is insensitive to decreases in CH.  

3.938

     

3.939           The Pseudomonas aeruginosa Secreted Protein PA2934 Decreases Apical Membrane Expression of the Cystic Fibrosis Transmembrane Conductance Regulator

MacEachran, D.P. et al Infect. Immun., 75(8), 3902-3912 (2007)   We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif, for CFTR inhibitory factor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl^– ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in /ß hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe.  

3.940           Caveolin-associated Accumulation of Globotriaosylceramide in the Vascular Endothelium of -Galactosidase A Null Mice

Shu, L. and Shayman, J.A.

1. Biol. Chem., 282(29), 20960-20967 (2007)

  Cardiovascular complications, including stroke and myocardial infarction, result in premature mortality in patients with Fabry disease, an X-linked deficiency of -galactosidase A ( -Gal A). The enzymatic defect results in the deposition of globotriaosylceramide (Gb3) in the vascular endothelium. To better understand the underlying pathogenesis of Fabry disease, the caveolar lipid content of primary cultured mouse aortic endothelial cells isolated from -Gal A null mice was measured. Lipid mass analysis revealed that the excessive Gb3 in cultured -Gal A-deficient mouse aortic endothelial cells accumulated in endothelial plasma membrane caveolar fractions. The levels of glucosylceramide and lactosylceramide increased in parallel with Gb3 levels in an age-dependent manner, whereas globotetraosylceramide (Gb4) levels reached maximal levels by 6 months of age and then rapidly decreased at older ages. The levels of cholesterol enriched in caveolar membranes declined in parallel with the progressive deposition of Gb3. Depleting Gb3 with recombinant human -Gal A protein or D-threo-ethylenedioxyphenyl-P4, an inhibitor of glucosylceramide synthase, restored cholesterol in cultured -Gal A-deficient mouse aortic endothelial cell caveolae. By contrast, recombinant human -Gal A was less effective in normalizing the cholesterol content. These results demonstrate the caveolar accumulation of glycosphingolipids in an in vitro model of a lysosomal storage disease and raise the possibility that dynamic changes in the composition of plasma membrane lipid microdomains may mediate the endothelial dysfunction seen in Fabry disease.  

3.941           Lipid Rafts Establish Calcium Waves in Hepatocytes

Nagata, J. et al Gastroenterology, 133(1), 256-267 (2007)   Background & Aims: Polarity is critical for hepatocyte function. Ca²⁺ waves are polarized in hepatocytes because the inositol 1,4,5-trisphosphate receptor (InsP3R) is concentrated in the pericanalicular region, but the basis for this localization is unknown. We examined whether pericanalicular localization of the InsP3R and its action to trigger Ca²⁺ waves depends on lipid rafts. Methods: Experiments were performed using isolated rat hepatocyte couplets and pancreatic acini, plus SkHep1 cells as nonpolarized controls. The cholesterol depleting agent methyl-beta-cyclodextrin (mβCD) was used to disrupt lipid rafts. InsP3R isoforms were examined by immunoblot and immunofluorescence. Ca²⁺ waves were examined by confocal microscopy. Results: Type II InsP3Rs initially were localized to only some endoplasmic reticulum fractions in hepatocytes, but redistributed into all fractions in mβCD-treated cells. This InsP3R isoform was concentrated in the pericanalicular region, but redistributed throughout the cell after mβCD treatment. Vasopressin-induced Ca²⁺ signals began as apical-to-basal Ca²⁺ waves, and mβCD slowed the wave speed and prolonged the rise time. MβCD had a similar effect on Ca²⁺ waves in acinar cells but did not affect Ca²⁺ signals in SkHep1 cells, suggesting that cholesterol depletion has similar effects among polarized epithelia, but this is not a nonspecific effect of mβCD. Conclusions: Lipid rafts are responsible for the pericanalicular accumulation of InsP3R in hepatocytes, and for the polarized Ca²⁺ waves that result. Signaling microdomains exist not only in the plasma membrane, but also in the nearby endoplasmic reticulum, which in turn, helps establish and maintain structural and functional polarity.  

3.942           Alzheimer’s presenilin 1 modulates sorting of APP and its carboxyl-terminal fragments in cerebral neurons in vivo

Gandy, S. et al

1. Neurochem., 102, 619-626 (2007)

  Studies in continuously cultured cells have established that familial Alzheimer’s disease (FAD) mutant presenilin 1 (PS1) delays exit of the amyloid precursor protein (APP) from the trans-Golgi network (TGN). Here we report the first description of PS1-regulated APP trafficking in cerebral neurons in culture and in vivo. Using neurons from transgenic mice or a cell-free APP transport vesicle biogenesis system derived from the TGN of those neurons, we demonstrated that knocking-in an FAD-associated mutant PS1 transgene was associated with delayed kinetics of APP arrival at the cell surface. Apparently, this delay was at least partially attributable to impaired exit of APP from the TGN, which was documented in the cell-free APP transport vesicle biogenesis assay. To extend the study to APP and carboxyl terminal fragment (CTF) trafficking to cerebral neurons in vivo, we performed subcellular fractionation of brains from APP transgenic mice, some of which carried a second transgene encoding an FAD-associated mutant form of PS1. The presence of the FAD mutant PS1 was associated with a slight shift in the subcellular localization of both holoAPP and APP CTFs toward iodixanol density gradient fractions that were enriched in a marker for the TGN. In a parallel set of experiments, we used an APP : furin chimeric protein strategy to test the effect of artificially forcing TGN concentration of an APP : furin chimera that could be a substrate for β- and γ-cleavage. This chimeric substrate generated excess Aβ42 when compared with wildtype APP. These data indicate that the presence of an FAD-associated mutant human PS1 transgene is associated with redistribution of the APP and APP CTFs in brain neurons toward TGN-enriched fractions. The chimera experiment suggests that TGN-enrichment of a β-/γ-secretase substrate may play an integral role in the action of mutant PS1 to elevate brain levels of Aβ42.  

3.943           Probing the Membrane Environment of the TOR Kinases Reveals Functional Interactions between TORC1, Actin, and Membrane Trafficking in Saccharomyces cerevisiae

Aronova, S., wedaman, K., Anderson, S., Yates III, J. and Powers, T. Mol. Biol. Cell, 18, 2779-2794 (2007)   The TOR kinases are regulators of growth in eukaryotic cells that assemble into two distinct protein complexes, TORC1 and TORC2, where TORC1 is inhibited by the antibiotic rapamycin. Present models favor a view wherein TORC1 regulates cell mass accumulation, and TORC2 regulates spatial aspects of growth, including organization of the actin cytoskeleton. Here, we demonstrate that in yeast both TORC1 and TORC2 fractionate with a novel form of detergent-resistant membranes that are distinct from detergent-resistant plasma membrane "rafts." Proteomic analysis of these TOR-associated membranes revealed the presence of regulators of endocytosis and the actin cytoskeleton. Genetic analyses revealed a significant number of interactions between these components and TORC1, demonstrating a functional link between TORC1 and actin/endocytosis-related genes. Moreover, we found that inhibition of TORC1 by rapamycin 1) disrupted actin polarization, 2) delayed actin repolarization after glucose starvation, and 3) delayed accumulation of lucifer yellow within the vacuole. By combining our genetic results with database mining, we constructed a map of interactions that led to the identification of additional genetic interactions between TORC1 and components involved in membrane trafficking. Together, these results reveal the broad scope of cellular processes influenced by TORC1, and they underscore the functional overlap between TORC1 and TORC2.  

3.944           Evidence for Coupled Biogenesis of Yeast Gap1 Permease and Sphingolipids: Essential Role in Transport Activity and Normal Control by Ubiquitination

Lauwers, E., Grossmann, G. and Andre, B. Mol. Biol. Cell, 18, 3068-3080 (2007)   Current models for plasma membrane organization integrate the emerging concepts that membrane proteins tightly associate with surrounding lipids and that biogenesis of surface proteins and lipids may be coupled. We show here that the yeast general amino acid permease Gap1 synthesized in the absence of sphingolipid (SL) biosynthesis is delivered to the cell surface but undergoes rapid and unregulated down-regulation. Furthermore, the permease produced under these conditions but blocked at the cell surface is inactive, soluble in detergent, and more sensitive to proteases. We also show that SL biogenesis is crucial during Gap1 production and secretion but that it is dispensable once Gap1 has reached the plasma membrane. Moreover, the defects displayed by cell surface Gap1 neosynthesized in the absence of SL biosynthesis are not compensated by subsequent restoration of SL production. Finally, we show that down-regulation of Gap1 caused by lack of SL biogenesis involves the ubiquitination of the protein on lysines normally not accessible to ubiquitination and close to the membrane. We propose that coupled biogenesis of Gap1 and SLs would create an SL microenvironment essential to the normal conformation, function, and control of ubiquitination of the permease.  

3.945           Naked2 Acts as a Cargo Recognition and Targeting Protein to Ensure Proper Delivery and Fusion of TGF- –containing Exocytic Vesicles at the Lower Lateral Membrane of Polarized MDCK Cells

Li, C. et al Mol. Biol. Cell, 18, 3081-3093 (2007)   Transforming growth factor- (TGF- ) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF- is synthesized and then delivered to the basolateral cell surface. We previously reported that Naked2 interacts with basolateral sorting determinants in the cytoplasmic tail of a Golgi-processed form of TGF- and that TGF- is not detected at the basolateral surface of Madin-Darby canine kidney (MDCK) cells expressing myristoylation-deficient (G2A) Naked2. By high-resolution microscopy, we now show that wild-type, but not G2A, Naked2-associated vesicles fuse at the plasma membrane. We further demonstrate that Naked2-associated vesicles are delivered to the lower lateral membrane of polarized MDCK cells independent of µ1B adaptin. We identify a basolateral targeting segment within Naked2; residues 1-173 redirect NHERF-1 from the apical cytoplasm to the basolateral membrane, and internal deletion of residues 37-104 results in apical mislocalization of Naked2 and TGF- . Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF- and increased cytosolic TGF- immunoreactivity. We propose that Naked2 acts as a cargo recognition and targeting (CaRT) protein to ensure proper delivery, tethering, and fusion of TGF- –containing vesicles to a distinct region at the basolateral surface of polarized epithelial cells.  

3.946           Characterization of the Properties and Trafficking of an Anchorless Form of the Prion Protein

Campana, V. et al

1. Biol. Chem., 282(31), 22747-22756 (2007)

  Conversion of PrP^C into PrP^Sc is the central event in the pathogenesis of transmissible prion diseases. Although the molecular basis of this event and the intracellular compartment where it occurs are not yet understood, the association of PrP with cellular membranes and in particular its presence in detergent-resistant microdomains appears to be of critical importance. In addition it appears that scrapie conversion requires membrane-bound glycosylphosphatidylinositol (GPI)-linked PrP. The GPI anchor may affect either the conformation, the intracellular localization, or the association of the prion protein with specific membrane domains. However, how this occurs is not known. To understand the relevance of the GPI anchor for the cellular behavior of PrP, we have studied the biosynthesis and localization of a PrP version which lacks the GPI anchor attachment signal (PrPΔGPI). We found that PrPΔGPI is tethered to cell membranes and associates to membrane detergent-resistant microdomains but does not assume a transmembrane topology. Differently to PrP^C, this protein does not localize at the cell surface but is mainly released in the culture media in a fully glycosylated soluble form. The cellular behavior of anchorless PrP explains why PrPΔGPI Tg mice can be infected but do not show the classical signs of the disorder, thus indicating that the plasma membrane localization of PrP^C and/or of the converted scrapie form might be necessary for the development of a symptomatic disease.  

3.947           The SUMO Conjugating Enzyme Ubc9 is a Regulator of GLUT4 Turnover and Targeting to the Insulin-Responsive Storage Compartment in 3T3-L1 Adipocytes

Liu, L-B., Omatra, W., Kojima, I. and Shibata, H. Diabetes, 56, 1977-1985 (2007)   The small ubiquitin-related modifier (SUMO) conjugating enzyme Ubc9 has been shown to upregulate GLUT4 in L6 myoblast cells, although the mechanism of action has remained undefined. Here we investigated the physiological significance of Ubc9 in GLUT4 turnover and subcellular targeting by adenovirus vector–mediated overexpression and by small interfering RNA (siRNA)-mediated gene silencing of Ubc9 in 3T3-L1 adipocytes. Overexpression of Ubc9 resulted in an inhibition of GLUT4 degradation and promoted its targeting to the unique insulin-responsive GLUT4 storage compartment (GSC), leading to an increase in GLUT4 amount and insulin-responsive glucose transport in 3T3-L1 adipocytes. Overexpression of Ubc9 also antagonized GLUT4 downregulation and its selective loss in GSC induced by long-term insulin stimulation. By contrast, siRNA-mediated depletion of Ubc9 accelerated GLUT4 degradation and decreased the amount of the transporter, concurrent with its selective loss in GSC, which resulted in attenuated insulin-responsive glucose transport. Intriguingly, overexpression of the catalytically inactive mutant Ubc9-C93A produced effects indistinguishable from those with wild-type Ubc9, suggesting that Ubc9 regulates GLUT4 turnover and targeting to GSC by a mechanism independent of its catalytic activity. Thus, Ubc9 is a pivotal regulator of the insulin sensitivity of glucose transport in adipocytes.  

3.948           Core Protein Machinery for Mammalian Phosphatidylinositol 3,5-Bisphosphate Synthesis and Turnover That Regulates the Progression of Endosomal Transport: NOVEL SAC PHOSPHATASE JOINS THE ArPIKfyve-PIKfyve COMPLEX

Sbrissa, D. et al

1. Biol. Chem., 282(33), 23878-23891 (2007)

  Perturbations in phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂)-synthesizing enzymes result in enlarged endocytic organelles from yeast to humans, indicating evolutionarily conserved function of PtdIns(3,5)P₂ in endosome-related events. This is reinforced by the structural and functional homology of yeast Vac14 and human Vac14 (ArPIKfyve), which activate yeast and mammalian PtdIns(3,5)P₂-producing enzymes, Fab1 and PIKfyve, respectively. In yeast, PtdIns(3,5)P₂-specific phosphatase, Fig4, in association with Vac14, turns over PtdIns(3,5)P₂, but whether such a mechanism operates in mammalian cells and what the identity of mammalian Fig4 may be are unknown. Here we have identified and characterized Sac3, a Sac domain phosphatase, as the Fig4 mammalian counterpart. Endogenous Sac3, a widespread 97-kDa protein, formed a stable ternary complex with ArPIKfyve and PIKfyve. Concordantly, Sac3 cofractionated and colocalized with ArPIKfyve and PIKfyve. The intrinsic Sac3^WT phosphatase activity preferably hydrolyzed PtdIns(3,5)P₂ in vitro, although the other D5-phosphorylated polyphosphoinositides were also substrates. Ablation of endogenous Sac3 by short interfering RNAs elevated PtdIns(3,5)P₂ in ³²P-labeled HEK293 cells. Ectopically expressed Sac3^WT in COS cells colocalized with and dilated EEA1-positive endosomes, consistent with the PtdIns(3,5)P₂ requirement in early endosome dynamics. In vitro reconstitution of carrier vesicle formation from donor early endosomes revealed a gain of function upon Sac3 loss, whereas PIKfyve or ArPIKfyve protein depletion produced a loss of function. These data demonstrate a coupling between the machinery for PtdIns(3,5)P₂ synthesis and turnover achieved through a physical assembly of PIKfyve, ArPIKfyve, and Sac3. We suggest that the tight regulation in PtdIns(3,5)P₂ homeostasis is mechanistically linked to early endosome dynamics in the course of cargo transport.  

3.949           Catalase Takes Part in Rat Liver Mitochondria Oxidative Stress Defense

Salvi, M. et al

1. Biol. Chem., 282(33), 24407-24451 (2007)

  Highly purified rat liver mitochondria (RLM) when exposed to tert-butylhydroperoxide undergo matrix swelling, membrane potential collapse, and oxidation of glutathione and pyridine nucleotides, all events attributable to the induction of mitochondrial permeability transition. Instead, RLM, if treated with the same or higher amounts of H₂O₂ or tyramine, are insensitive or only partially sensitive, respectively, to mitochondrial permeability transition. In addition, the block of respiration by antimycin A added to RLM respiring in state 4 conditions, or the addition of H₂O₂, results in O₂ generation, which is blocked by the catalase inhibitors aminotriazole or KCN. In this regard, H₂O₂ decomposition yields molecular oxygen in a 2:1 stoichiometry, consistent with a catalatic mechanism with a rate constant of 0.0346 s^-1. The rate of H₂O₂ consumption is not influenced by respiratory substrates, succinate or glutamate-malate, nor by N-ethylmaleimide, suggesting that cytochrome c oxidase and the glutathione-glutathione peroxidase system are not significantly involved in this process. Instead, H₂O₂ consumption is considerably inhibited by KCN or aminotriazole, indicating activity by a hemoprotein. All these observations are compatible with the presence of endogenous heme-containing catalase with an activity of 825 ± 15 units, which contributes to mitochondrial protection against endogenous or exogenous H₂O₂. Mitochondrial catalase in liver most probably represents regulatory control of bioenergetic metabolism, but it may also be proposed for new therapeutic strategies against liver diseases. The constitutive presence of catalase inside mitochondria is demonstrated by several methodological approaches as follows: biochemical fractionating, proteinase K sensitivity, and immunogold electron microscopy on isolated RLM and whole rat liver tissue.  

3.950           Cell-Surface Thioredoxin-1: Possible Involvement in Thiol-Mediated Leukocyte-Endothelial Cell Interaction Through Lipid Rafts

Hara, T. et al Antioxidants & Redox Signaling, 9(9), 1427-1437 (2007)   Human thioredoxin-1 (hTrx) exhibits a disulfide reducing activity and was originally identified as a soluble cytokine-like factor secreted from cells of a human T-cell leukemia virus type I (HTLV-I)-transformed cell line. Recent studies have revealed that endogenous Trx plays an important role in cytoprotection against various oxidative stress–associated disorders. However, the function of exogenous Trx is still not fully understood. We report here that a cysteine-modified mutant of recombinant human Trx (rhTrx-C35S) binds to human umbilical vein endothelial cells (HUVECs) as well as stimulated T cells and rapidly enters these cells via lipid rafts. In addition, we found that endogenous Trx is expressed on the surface of HUVECs, including lipid rafts. These events suggest cell-surface Trx as a possible target of rhTrx-C35S. Furthermore, we found that anti-human Trx mouse monoclonal antibody inhibits adherence of LPS-stimulated human peripheral blood polymorphonuclear cells (PMNs) to HUVECs. This adherence was also suppressed by a recombinant human Trx (rhTrx), but not by a mutant rhTrx (rhTrx-C32S/C35S) with no reducing activity. Cell-surface Trx may be involved in the process of interaction between PMNs and HUVECs and a possible target of cysteine-modified exogenous Trx as well as wild-type exogenous Trx through redox regulation.  

3.951           Lipid Raft–Mediated Uptake of Cysteine-Modified Thioredoxin-1: Apoptosis Enhancement by Inhibiting the Endogenous Thioredoxin-1

Kondo, N. et al Antioxidants & Redox Signaling, 9(9), 1439-1448 (2007)   Thioredoxin-1 (TRX) plays important roles in cellular signaling by controlling the redox state of cysteine residues in target proteins. TRX is released in response to oxidative stress and shows various biologic functions from the extracellular environment. However, the mechanism by which extracellular TRX transduces the signal into the cells remains unclear. Here we report that the cysteine modification at the active site of TRX promotes the internalization of TRX into the cells. TRX-C35S, in which the cysteine at residue 35 of the active site was replaced with serine, was internalized more effectively than wild-type TRX in human T-cell leukemia virus–transformed T cells. TRX-C35S bound rapidly to the cell surface and was internalized into the cells dependent on lipid rafts in the plasma membrane. This process was inhibited by wild-type TRX, reducing reagents such as dithiothreitol, and methyl-β-cyclodextrin, which disrupts lipid rafts. Moreover, the internalized TRX-C35S binds to endogenous TRX, resulting in the generation of intracellular reactive oxygen species (ROS) and enhanced cis-diamine-dichloroplatinum (II) (CDDP)-induced apoptosis via a ROS-mediated pathway involving apoptosis signal-regulating kinase-1 (ASK-1) activation. These findings suggest that the cysteine at the active site of TRX plays a key role in the internalization and signal transduction of extracellular TRX into the cells.  

3.952           P-Glycoprotein is not present in mitochondrial membranes

Paterson, J.K. and Gottesman, M.M. Exp. Cell Res., 313(14), 3100-3103 (2007)   Recent reports have indicated the presence of P-glycoprotein in crude mitochondrial membrane fractions, leading to the assumption that P-glycoprotein is present in mitochondrial membranes, and may be involved in transport across these membranes. To determine the validity of this claim, two cell lines overexpressing endogenous P-glycoprotein were investigated. Using various centrifugation steps, mitochondria were purified from these cells and analyzed by Western blot reaction with the anti-P-glycoprotein antibody C219 and organelle-specific antibodies. While P-glycoprotein is present in crude mitochondrial fractions, these fractions are contaminated with plasma membranes. Further purification of the mitochondria to remove plasma membranes revealed that P-glycoprotein is not expressed in mitochondria of the KB-V1 (vinblastine-resistant KB-3-1 cells) or MCF-7^ADR (adriamycin-resistant MCF-7 cells) cell lines. To further substantiate these findings, we used confocal microscopy and the anti-P-glycoprotein antibody 17F9. This demonstrated that in intact cells, P-glycoprotein is not present in mitochondria and is primarily localized to the plasma membrane. These findings are consistent with the role of P-glycoprotein in conferring multidrug resistance by decreasing cellular drug accumulation. Therefore, contrary to previous speculation, P-glycoprotein does not confer cellular protection by residing in mitochondrial membranes.  

3.953           Ubiquitination of Human Immunodeficiency Virus Type 1 Gag Is Highly Dependent on Gag Membrane Association

Jäger, S., Gottwein, E. and  Kräusslich, H-G.

1. Virol., 81(17), 9193-9201 (2007)

  Ubiquitin is important for the release of human immunodeficiency virus 1 (HIV-1) and several other retroviruses. All major domains of the HIV-1 Gag protein are monoubiquitinated, but the modifying machinery and the function of HIV-1 Gag ubiquitination remain unclear. Here, we show that the induction of a late budding arrest by mutation of the HIV-1 PTAP motif or by specific inhibition of selected ESCRT components leads to an increase of Gag-ubiquitin conjugates in cells, which coincides with an accumulation of detergent-insoluble, multimerized Gag at the plasma membrane. Membrane flotation experiments revealed that ubiquitinated Gag is highly enriched in membrane-bound fractions. Based on these findings, we propose that a blocking of virus release results in increased Gag ubiquitination as a consequence of its prolonged membrane association. Consistent with this, ubiquitination of a membrane-binding-defective (G2A)Gag mutant was dramatically reduced and the ubiquitination levels of truncated Gag proteins correlated with their abilities to bind to membranes. We therefore propose that membrane association and multimerization of HIV-1 Gag proteins, rather than a specific motif within Gag, trigger recognition by the cellular ubiquitination machinery.  

3.954           Organellar proteomics to create the cell map

Au, C.E. et al Current Opinion in Cell Biology, 19, 376-385 (2007)   The elucidation of a complete, accurate, and permanent representation of the proteome of the mammalian cell may be achievable piecemeal by an organellar based approach. The small volume of organelles assures high protein concentrations. Providing isolated organelles are homogenous, this assures reliable protein characterization within the sensitivity and dynamic range limits of current mass spec based analysis. The stochastic aspect of peptide selection by tandem mass spectrometry for sequence determination by fragmentation is dealt with by multiple biological replicates as well as by prior protein separation on 1-D gels. Applications of this methodology to isolated synaptic vesicles, clathrin coated vesicles, endosomes, phagosomes, endoplasmic reticulum, and Golgi apparatus, as well as Golgi-derived COPI vesicles, have led to mechanistic insight into the identity and function of these organelles.  

3.955           Cholesterol-Dependent and -Independent CD40 Internalization and Signaling Activation in Cardiovascular Endothelial Cells

Chen, J., Chen, L., Wang, G. and Tang, H. Artioscler. Thtomb. Vasc. Biol., 27, 2005-2013 (2007)   Objective— It remains elusive how CD40 endocytosis orclustering on the cell surface is induced by different formsof CD40 agonist. This study aims to investigate whether lipidrafts differentially regulate CD40 traffic and signaling inproinflammatory activation of cardiovascular endothelial cells(ECs). Methods and Results— Using fluorescent microscopy andflow cytometry, we demonstrated that soluble CD40L and agonisticantibody G28.5 induced CD40 internalization via clathrin-independentpathway. Furthermore, depletion of cholesterol by methyl-ß-cyclodextrin(MCD) or siRNA knockdown of caveolin-1 efficiently blocked CD40internalization, suggesting that caveolae-rafts pathway regulatesCD40 internalization. In contrast, a membrane-bound CD40L mimic(megamer) triggered aggregation of CD40 rafts outside of theconventional cholera toxin B subunit-positive lipid rafts resistantto cholesterol depletion. Finally, both G28.5 and megamer inducedCD40 translocation to Brij58-insoluble, low buoyant densityrafts, a movement insensitive to cholesterol depletion. However,MCD effectively inhibited G28.5 but not megamer-induced CD40activation, and such inhibition could be alleviated by cholesterolreconstitution, suggesting that 2 different raft structuresof CD40 induced by G28.5 or megamer possess differential sensitivityto cellular cholesterol levels in downstream signaling. Conclusions— Depending on different forms of agonist,CD40 uses either a cholesterol-dependent or -independent modefor trafficking and signaling in ECs. Although activated CD40 can translocate to lipid rafts despitecholesterol depletion, different forms of CD40L, either solubleor membrane-bound, required distinct membrane constituents andmicrodomains for CD40 internalization and signaling activation.In particular, stimulation with G28.5, but not membrane-boundCD40L, required cellular cholesterol for CD40 internalizationand downstream signaling.  

3.956           Localization of the Mouse 5-Hydroxytryptamine1A Receptor in Lipid Microdomains Depends on Its Palmitoylation and Is Involved in Receptor-Mediated Signaling

Renner, U. et al Mol. Pharmacol., 72(3), 502-513 (2007)   In the present study, we have used wild-type and palmitoylation-deficient mouse 5-hydroxytryptamine_1A receptor (5-HT1A) receptors fused to the yellow fluorescent protein- and the cyan fluorescent protein (CFP)-tagged _i3 subunit of heterotrimeric G-protein to study spatiotemporal distribution of the 5-HT1A-mediated signaling in living cells. We also addressed the question on the molecular mechanisms by which receptor palmitoylation may regulate communication between receptors and G_i-proteins. Our data demonstrate that activation of the 5-HT1A receptor caused a partial release of G _i protein into the cytoplasm and that this translocation is accompanied by a significant increase of the intracellular Ca²⁺ concentration. In contrast, acylation-deficient 5-HT1A mutants failed to reproduce both G _i3-CFP relocation and changes in [Ca²⁺]_i upon agonist stimulation. By using gradient centrifugation and copatching assays, we also demonstrate that a significant fraction of the 5-HT1A receptor resides in membrane rafts, whereas the yield of the palmitoylation-deficient receptor in these membrane microdomains is reduced considerably. Our results suggest that receptor palmitoylation serves as a targeting signal responsible for the retention of the 5-HT1A receptor in membrane rafts. More importantly, the raft localization of the 5-HT1A receptor seems to be involved in receptor-mediated signaling.  

3.957           Activated Ezrin Promotes Cell Migration through Recruitment of the GEF Dbl to Lipid Rafts and Preferential Downstream Activation of Cdc42

Prag, S. et al Mol. Biol. Cell, 18, 2935-2948 (2007)   Establishment of polarized cell morphology is a critical factor for migration and requires precise spatial and temporal activation of the Rho GTPases. Here, we describe a novel role of the actin-binding ezrin/radixin/moesin (ERM)-protein ezrin to be involved in recruiting Cdc42, but not Rac1, to lipid raft microdomains, as well as the subsequent activation of this Rho GTPase and the downstream effector p21-activated kinase (PAK)1, as shown by fluorescence lifetime imaging microscopy. The establishment of a leading plasma membrane and the polarized morphology necessary for random migration are also dependent on ERM function and Cdc42 in motile breast carcinoma cells. Mechanistically, we show that the recruitment of the ERM-interacting Rho/Cdc42-specific guanine nucleotide exchange factor Dbl to the plasma membrane and to lipid raft microdomains requires the phosphorylated, active conformer of ezrin, which serves to tether the plasma membrane or its subdomains to the cytoskeleton. Together these data suggest a mechanism whereby precise spatial guanine nucleotide exchange of Cdc42 by Dbl is dependent on functional ERM proteins and is important for directional cell migration.  

3.958           Human Papillomavirus Type 31 Uses a Caveolin 1- and Dynamin 2-Mediated Entry Pathway for Infection of Human Keratinocytes

Smith, J.L., Campos, S.K. and Ozbun, M.A.

1. Virol., 81(18), 9922-9931 (2007)

  Papillomaviruses are species-specific and epitheliotropic DNA viruses that cause tumors in their natural hosts. Certain infections with genital human papillomavirus (HPV) types are causally related to cervical cancer development. Most papillomaviruses are thought to infect cells via a clathrin-dependent pathway, yet no studies have determined the entry route in permissive host epithelial cells. Employing fluorescently labeled and native virions, we tested the effects of dominant-negative and biochemical inhibitors of cellular endocytosis pathways. Infections of human keratinocytes, a natural host cell type for HPVs, were assessed visually and by infectious entry assays. We found that HPV type 31 (HPV31) entry and initiation of early infection events require both caveolin 1 and dynamin 2 and occur independently of clathrin-mediated endocytosis. Treatment with chlorpromazine and filipin had opposing effects on HPV31 and HPV16 infection. HPV31 entry was remarkably slow, with a half-time of 14 h, whereas the entry half-time of HPV16 was 4 h. Consistent with a caveola-mediated entry pathway for HPV31, the virions associated with detergent-resistant lipid rafts. During a 16-h microscopic tracking of HPV31 and HPV16 virions, no colocalization of the two viral types was observed. These data suggest that HPV31 and HPV16 virions use distinct routes for host epithelial cell entry.  

3.959           Detergent resistance as a tool in membrane research

Lingwood, D. and Simons, K. Nature Protocols, 2(9), 2159-2165 (2007)   The biological membrane is a complicated matrix wherein different lipid environments are thought to exist. The more ordered or raft environment has been perceived biochemically accessible via its relative resistance to detergent. This paper outlines the protocols developed in our laboratory for the analysis of such detergent-resistant membranes (DRMs). We stress the fact that DRMs are artifactual in nature and should not be equivocated to lipid rafts, their usefulness being limited to assigning raft-association potential most convincingly when changes in DRM composition are induced by biochemically/physiologically relevant events. These protocols are completed in 1–2 d.  

3.960           Light-induced recruitment of INAD-signaling complexes to detergent-resistant lipid rafts in Drosophila photoreceptors

Sanxaridis, P.D. et al Mol. Cell. Neurosci., 36(1), 36-46 (2007)   Here, we reveal a novel feature of the dynamic organization of signaling components in Drosophila photoreceptors. We show that the multi-PDZ protein INAD and its target proteins undergo light-induced recruitment to detergent-resistant membrane (DRM) rafts. Reduction of ergosterol, considered to be a key component of lipid rafts in Drosophila, resulted in a loss of INAD-signaling complexes associated with DRM fractions. Genetic analysis demonstrated that translocation of INAD-signaling complexes to DRM rafts requires activation of the entire phototransduction cascade, while constitutive activation of the light-activated channels resulted in recruitment of complexes to DRM rafts in the dark. Mutations affecting INAD and TRP showed that PDZ4 and PDZ5 domains of INAD, as well as the INAD–TRP interaction, are required for translocation of components to DRM rafts. Finally, selective recruitment of phosphorylated, and therefore activatable, eye-PKC to DRM rafts suggests that DRM domains are likely to function in signaling, rather than trafficking.  

3.961           Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells

Yamamoto, K. et al Am. J. Physiol. Heart Circ. Physiol.,293, H1646-H1653 (2007)   Endothelial cells (ECs) release ATP in response to shear stress, a mechanical force generated by blood flow, and the ATP released modulates EC functions through activation of purinoceptors. The molecular mechanism of the shear stress-induced ATP release, however, has not been fully elucidated. In this study, we have demonstrated that cell surface ATP synthase is involved in shear stress-induced ATP release. Immunofluorescence staining of human pulmonary arterial ECs (HPAECs) showed that cell surface ATP synthase is distributed in lipid rafts and co-localized with caveolin-1, a marker protein of caveolae. Immunoprecipitation indicated that cell surface ATP synthase and caveolin-1 are physically associated. Measurement of the extracellular metabolism of [³H]ADP confirmed that cell surface ATP synthase is activein ATP generation. When exposed to shear stress, HPAECs releasedATP in a dose-dependent manner, and the ATP release was markedlysuppressed by the membrane-impermeable ATP synthase inhibitorsangiostatin and piceatannol and by an anti-ATP synthase antibody.Depletion of plasma membrane cholesterol with methyl--cyclodextrin(MCD) disrupted lipid rafts and abolished co-localization ofATP synthase with caveolin-1, which resulted in a marked reductionin shear stress-induced ATP release. Pretreatment of the cellswith cholesterol prevented these effects of MCD. Downregulationof caveolin-1 expression by transfection of caveolin-1 siRNAalso markedly suppressed ATP-releasing responses to shear stress.Neither MCD, MCD plus cholesterol, nor caveolin-1 siRNA hadany effect on the amount of cell surface ATP synthase. Theseresults suggest that the localization and targeting of ATP synthaseto caveolae/lipid rafts is critical for shear stress-inducedATP release by HPAECs.  

3.962           Lipid Raft–Specific Knockdown of Src Family Kinase Activity Inhibits Cell Adhesion and Cell Cycle Progression of Breast Cancer Cells

Hitosugi, T., Sato, M., Sasaki, K and Umezawa, Y. Cancer Res., (67/17), 8139-8148 (2007)   Src family kinase (SFK) is known to control various cell functions, but the significance of the location of its activation was largely unknown. We herein revealed that SFK activation occurs in lipid rafts. Based on this finding, we have developed a lipid raft–targeted SFK inhibitory fusion protein (LRT-SIFP) that inhibits the SFK activity in lipid rafts. LRT-SIFP has a peptide inhibitor of SFK and a lipid raft–targeting sequence in which two cysteine residues are palmitoylated for clustering in lipid rafts. LRT-SIFP was found to inhibit cell adhesion and cell cycle progression of human breast cancer cell lines MCF-7 and MDA-MB231. On the other hand, the cell functions of MCF-7 cells were found to be not affected with a previously developed peptide inhibitor of SFK that lacks the lipid raft–targeting sequence. In addition, when we replaced the targeting sequence of LRT-SIFP with the consensus sequence for geranylgeranylation to make LRT-SIFP unable to cluster in lipid rafts, this mutated LRT-SIFP did not show any effect on the above cell functions of MCF-7 cells. Furthermore, in contrast to the breast cancer cell lines, LRT-SIFP did not show any inhibitory effect on cell adhesion and cell cycle progression of human normal cell line HEK293. The present lipid raft–specific knockdown of SFK activity would potentially be useful for selective cancer therapy to prevent tumorigenesis and metastasis of breast cancer cells.  

3.963           Intracellular Trafficking of Pseudomonas ExoS, a Type III Cytotoxin

Deng, Q., Zhang, Y. and Barbier, J.T. Traffic, 8, 1331-1345 (2007)   Pseudomonas aeruginosa ExoS is a bifunctional type III cytotoxin that disrupts Ras- and Rho-signaling pathways in mammalian cells. A hydrophobic region (residues 51–77, termed the membrane localization domain) targets ExoS to the plasma membrane (PM) and late endosomes of host cells. In the current study, metabolic inhibitors and dominant-negative proteins that disrupt known vesicle-trafficking pathways were used to define the intracellular trafficking of ExoS. Release of ExoS from PM was independent of dynamin and ADP ribosylation factor 6 but inhibited by methyl-β-cyclodextrin, a cholesterol-depleting reagent, and perinuclear localization of ExoS was disrupted by nocodazole. p50 dynamitin, a dynein inhibitor partially disrupted perinuclear localization of ExoS. Methyl-β-cyclodextrin and nocodazole inhibited the ability of type-III-delivered ExoS to ADP-ribosylated Golgi/endoplasmic reticulum-resident Ras. Methyl-β-cyclodextrin also relocated ExoS from the perinuclear region to the PM, indicating that ExoS can cycle through anterograde as well as through retrograde trafficking pathways. These findings show that ExoS endocytosis is cholesterol dependent, and it utilizes host microtubules, for intracellular trafficking. Understanding how type III cytotoxins enter and traffic within mammalian cells may identify new targets for therapeutic intervention of gram-negative bacterial pathogens.  

3.964           Biochemical consequences of the NOS3 Glu298Asp variation in human endothelium: altered caveolar localization and impaired response to shear

Joshi, M.S., Mineo, C., Shaul, P.W. and Bauer, J.A. FASEB J., 21, 2655-2663 (2007)   Human endothelial nitric oxide synthase (NOS3) gene polymorphism at Exon 7 (Glu298Asp) has been linked to vascular endothelial dysfunction, but the mechanisms are not defined. Shear is a key modulator of NOS3 function in vivo and association with caveolae is important for the control of NOS3 protein activity. Here we tested the hypothesis that altered enrichment of NOS3 in the caveolar membrane defines Glu298Asp genotype-specific responses and NOS3 activity. Basal caveolar membrane enrichment was carried out to quantitate the NOS3 enrichment in caveolae. Cells were subjected to shear and NOS3 protein levels, phosphorylation, enzyme function were investigated. Variant genotypes had lower NOx production pre- and post-shear, but no genotype-dependent alterations in pNOS3 were observed. Asp variants had significantly lower NOS3 enrichment in the caveolar membrane fraction. Further, immunoprecipitation studies demonstrated that Asp variants had substantially less NOS3/Cav-1 association ( 40%) during static conditions. Furthermore, acute shear causes impaired NOS3/Cav-1 dissociation in Asp variants. The results from immunoprecipitation studies were in complete agreement with caveolar membrane preparation findings. Collectively, these data demonstrate functional consequences of the Glu298Asp NOS3 variation and further define disruption of NOS3 caveolar localization and shear-induced mobilization as the primary mechanism responsible for these differences.—Joshi, M. S., Mineo, C., Shaul, P. W., Bauer, J. A. Biochemical consequences of the NOS3 Glu298Asp variation in human endothelium: altered caveolar localization and impaired response to shear.  

3.965           Peroxisomes Contain a Specific Phytanoyl-CoA/Pristanoyl-CoA Thioesterase Acting as a Novel Auxiliary Enzyme in - and -Oxidation of Methyl-branched Fatty Acids in Mouse

Westin, M.A., Hunt, M.C. and Elexson, S.E.H.

1. Biol. Chem., 282(37), 26707-26716 (2007)

  Phytanic acid and pristanic acid are derived from phytol, which enter the body via the diet. Phytanic acid contains a methyl group in position three and, therefore, cannot undergo -oxidation directly but instead must first undergo -oxidation to pristanic acid, which then enters -oxidation. Both these pathways occur in peroxisomes, and in this study we have identified a novel peroxisomal acyl-CoA thioesterase named ACOT6, which we show is specifically involved in phytanic acid and pristanic acid metabolism. Sequence analysis of ACOT6 revealed a putative peroxisomal targeting signal at the C-terminal end, and cellular localization experiments verified it as a peroxisomal enzyme. Subcellular fractionation experiments showed that peroxisomes contain by far the highest phytanoyl-CoA/pristanoyl-CoA thioesterase activity in the cell, which could be almost completely immunoprecipitated using an ACOT6 antibody. Acot6 mRNA was mainly expressed in white adipose tissue and was co-expressed in tissues with Acox3 (the pristanoyl-CoA oxidase). Furthermore, Acot6 was identified as a target gene of the peroxisome proliferator-activated receptor (PPAR ) and is up-regulated in mouse liver in a PPAR -dependent manner.  

3.966           Nuclear Import and Export of Venezuelan Equine Encephalitis Virus Nonstructural Protein 2

Montgomery, S.A. and Johnston, R.E.

1. Virol., 81(19), 10268-10279 (2007)

  Many RNA viruses, which replicate predominantly in the cytoplasm, have nuclear components that contribute to their life cycle or pathogenesis. We investigated the intracellular localization of the multifunctional nonstructural protein 2 (nsP2) in mammalian cells infected with Venezuelan equine encephalitis virus (VEE), an important, naturally emerging zoonotic alphavirus. VEE nsP2 localizes to both the cytoplasm and the nucleus of mammalian cells in the context of infection and also when expressed alone. Through the analysis of a series of enhanced green fluorescent protein fusions, a segment of nsP2 that completely localizes to the nucleus of mammalian cells was identified. Within this region, mutation of the putative nuclear localization signal (NLS) PGKMV diminished, but did not obliterate, the ability of the protein to localize to the nucleus, suggesting that this sequence contributes to the nuclear localization of VEE nsP2. Furthermore, VEE nsP2 specifically interacted with the nuclear import protein karyopherin- 1 but not with karyopherin- 2, -3, or -4, suggesting that karyopherin- 1 transports nsP2 to the nucleus during infection. Additionally, a novel nuclear export signal (NES) was identified, which included residues L526 and L528 of VEE nsP2. Leptomycin B treatment resulted in nuclear accumulation of nsP2, demonstrating that nuclear export of nsP2 is mediated via the CRM1 nuclear export pathway. Disruption of either the NLS or the NES in nsP2 compromised essential viral functions. Taken together, these results establish the bidirectional transport of nsP2 across the nuclear membrane, suggesting that a critical function of nsP2 during infection involves its shuttling between the cytoplasm and the nucleus.  

3.967           Association of the Astrovirus Structural Protein VP90 with Membranes Plays a Role in Virus Morphogenesis

Mendez, E., Aguirre-Crespo, G., Zavala, G. and Arias, C.F.

1. Virol., 81(19), 10649-10658 (2007)

  VP90, the capsid polyprotein precursor of human astrovirus Yuc8, is assembled into viral particles, and its processing at the carboxy terminus by cellular caspases, to yield VP70, has been correlated with the cell release of the virus. Here, we characterized the effect of the VP90-VP70 processing on the properties of these proteins, as well as on their intracellular distribution. VP90 was found in membrane-enriched fractions (^mVP90), as well as in fractions enriched in cytosolic proteins (^cVP90), while VP70 was found exclusively in the latter fractions. Upon trypsin activation, infectivity was detected in all VP90-containing fractions, confirming that both ^mVP90 and ^cVP90 are able to assemble into particles; however, the two forms of VP90 showed differential sensitivities to trypsin, especially at their carboxy termini, which in the case of ^mVP90 was shown to remain membrane associated after protease digestion. Structural protein oligomers were detected in purified VP70-containing viruses, as well as in membrane-enriched fractions, but they were less evident in cytosolic fractions. Ultrastructural studies of infected cells revealed different types of viral particles, some of which appeared to be associated with membranes. By immunoelectron microscopy, structural proteins were shown to form virus particles in clusters and to associate with the edges of vesicles induced during infection, which also appear to contain subviral particles inside. Nonstructural proteins and viral RNA colocalized with ^mVP90, but not with ^cVP90, suggesting that ^mVP90 might represent the form of the protein that is initially assembled into particles, at the sites where the virus genome is being replicated.  

3.968           Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers

Grigorov, B. et al Retrovirology, 4(54), 1-12 (2007)   Background The HIV-1 nucleocapsid protein (NC) is formed of two CCHC zinc fingers flanked by highly basic regions. HIV-1 NC plays key roles in virus structure and replication via its nucleic acid binding and chaperoning properties. In fact, NC controls proviral DNA synthesis by reverse transcriptase (RT), gRNA dimerization and packaging, and virion assembly. Results We previously reported a role for the first NC zinc finger in virion structure and replication [1]. To investigate the role of both NC zinc fingers in intracellular Gag trafficking, and in virion assembly, we generated series of NC zinc fingers mutations. Results show that all Zinc finger mutations have a negative impact on virion biogenesis and maturation and rendered defective the mutant viruses. The NC zinc finger mutations caused an intracellular accumulation of Gag, which was found either diffuse in the cytoplasm or at the plasma membrane but not associated with endosomal membranes as for wild type Gag. Evidences are also provided showing that the intracellular interactions between NC-mutated Gag and the gRNA were impaired. Conclusion These results show that Gag oligomerization mediated by gRNA-NC interactions is required for correct Gag trafficking, and assembly in HIV-1 producing cells and the release of infectious viruses.  

3.969           Effects of a New Bioactive Lipid-Based Drug Carrier on Cultured Hepatic Stellate Cells and Liver Fibrosis in Bile Duct-Ligated Rats

Adrian, J.E. et al

1. Pharmacol. Exp. Ther., 321(2), 536-543 (2007)

  In the fibrotic liver, hepatic stellate cells (HSC) produce large amounts of collagen and secrete variety of mediators that promote development of fibrosis in this organ. Therefore, these cells are considered an attractive target for antifibrotic therapies. We incorporated the bioactive lipid dilinoleoylphosphatidylcholine (DLPC) into the membrane of liposomes, and then we evaluated its effect on hepatic stellate cell activation and liver fibrosis. To target DLPC-liposomes to HSC, human serum albumin modified with mannose 6-phosphate (M6P-HSA) was coupled to the surface of these liposomes. In vitro, the effects of the carrier were determined in primary cultures of HSC, Kupffer cells, and liver endothelial cells using real-time reverse transcription-polymerase chain reaction. In vivo DLPC-liposomes were tested in bile duct-ligated rats. Targeted M6P-HSA-DLPC-liposomes and DLPC-liposomes significantly reduced gene expression levels for collagen 1 1, -smooth muscle actin ( -SMA), and transforming growth factor- (TGF- ) in cultured HSC. In fibrotic livers, DLPC-liposomes decreased gene expression for TGF- and collagen 1 1 as well as -SMA and collagen protein expression. In contrast, M6P-HSA-DLPC-liposomes enhanced expression of profibrotic and proinflammatory genes in vivo. In cultured Kupffer and endothelial cells M6P-HSA liposomes influenced the expression of proinflammatory genes. Both types of liposomes increased hepatocyte glycogen content in fibrotic livers, indicating improved functionality of the hepatocytes. We conclude that DLPC-containing liposomes attenuate activation of cultured HSC. In fibrotic livers, M6P-HSA-mediated activation of Kupffer and endothelial cells probably counteracts this beneficial effect of DLPC-liposomes. Therefore, these bioactive drug carriers modulate the activity of all liver cells during liver fibrosis.  

3.970           Characterization of the Intracellular Proteolytic Cleavage of Myocilin and Identification of Calpain II as a Myocilin-processing Protease

Sanchez-Sanchez, F., Martinez-Redondo, F., Aroca-Aguilar, J.D., Coca-Prados, M. and Escrobano, J.

1. Biol. Chem., 282(38), 27810-27824 (2007)

  MYOC, a gene involved in different types of glaucoma, encodes myocilin, a secreted glycoprotein of unknown function, consisting of an N-terminal leucine-zipper-like domain, a central linker region, and a C-terminal olfactomedin-like domain. Recently, we have shown that myocilin undergoes an intracellular endoproteolytic processing. We show herein that the proteolytic cleavage in the linker region splits the two terminal domains. The C-terminal domain is secreted to the culture medium, whereas the N-terminal domain mainly remains intracellularly retained. In transiently transfected 293T cells, the cleavage was prevented by calpain inhibitors, such as calpeptin, calpain inhibitor IV, and calpastatin. Since calpains are calcium-activated proteases, we analyzed how changes in either intra- or extracellular calcium affected the cleavage of myocilin. Intracellular ionomycin-induced calcium uptake enhanced myocilin cleavage, whereas chelation of extracellular calcium by EGTA inhibited the proteolytic processing. Calpains I and II cleaved myocilin in vitro. However, in cells in culture, only RNA interference knockdown of calpain II reduced myocilin processing. Subcellular fractionation and digestion of the obtained fractions with proteinase K showed that full-length myocilin resides in the lumen of the endoplasmic reticulum together with a subpopulation of calpain II. These data revealed that calpain II is responsible for the intracellular processing of myocilin in the lumen of the endoplasmic reticulum. We propose that this cleavage might regulate extracellular interactions of myocilin, contributing to the control of intraocular pressure.  

3.971           HtrA2 Regulates -Amyloid Precursor Protein (APP) Metabolism through Endoplasmic Reticulum-associated Degradation

Huttunen,   H.J. et al

1. Biol. Chem., 282(38), 28285-28295 (2007)

  Alzheimer disease-associated -amyloid peptide is generated from its precursor protein APP. By using the yeast two-hybrid assay, here we identified HtrA2/Omi, a stress-responsive chaperone-protease as a protein binding to the N-terminal cysteinerich region of APP. HtrA2 coimmunoprecipitates exclusively with immature APP from cell lysates as well as mouse brain extracts and degrades APP in vitro. A subpopulation of HtrA2 localizes to the cytosolic side of the endoplasmic reticulum (ER) membrane where it contributes to ER-associated degradation of APP together with the proteasome. Inhibition of the proteasome results in accumulation of retrotranslocated forms of APP and increased association of APP with HtrA2 and Derlin-1 in microsomal membranes. In cells lacking HtrA2, APP holoprotein is stabilized and accumulates in the early secretory pathway correlating with elevated levels of APP C-terminal fragments and increased A secretion. Inhibition of ER-associated degradation (either HtrA2 or proteasome) promotes binding of APP to the COPII protein Sec23 suggesting enhanced trafficking of APP out of the ER. Based on these results we suggest a novel function for HtrA2 as a regulator of APP metabolism through ER-associated degradation.  

3.972           Anionic Lipid Interaction Induces Prion Protein Conformational Change

Wang, F., Wang, X. and Ma, J. FASEB J., 21, 781.3 (2007)   The conversion of the prion protein (PrP) to the pathogenic PrP^Scconformation plays a central role in prion disease. However, the precise mechanism underlying this process remains unclear. Here, we report the conformational conversion of PrP upon interaction with anionic lipids. After the discontinuous iodixanol density gradient centrifugation, we found strong binding between PrP and negatively charged phospholipids, involving both electrostatic and hydrophobic interactions. Under physiologically relevant conditions, interactions with lipid were sufficient to convert full-length, helices rich recombinant mouse PrP to a conformation similar to PrP^Sc, with increased ß sheet content and a PrP^Sc-like proteinase K (PK)-resistant pattern. Conversionis greatly influenced by lipid headgroup structures and lipidvesicle compositions. When lipid vesicles are disrupted by detergent,aggregation is necessary to maintain the PK resistant conformation.Our results imply that the strong lipid-PrP interaction is sufficientto overcome the energy barrier between the two conformationalstates and support the notion that lipid membrane may play arole in PrP conformational change.

3.973           SVIP interacts with Derlin1 and regulates ER-associated degradation

Ballar, P. et al FASEB J., 21, 808.3 (2007)   Production of misfolded proteins in the endoplasmic reticulum (ER) underlies pathogenesis of many diseases. Cells utilize a process, called ER-associated degradation (ERAD) to eliminate misfolded ER proteins, thereby protecting against the toxicity of the defective proteins. However, the molecular mechanisms underlying the process of ERAD remain to be fully understood. Here, we report that the SVIP is specifically and highly expressed in mouse brain as revealed by mouse multi-tissue blotting. By Opti-Prep density gradient fractionation, we demonstrated that SVIP is co-fractionated with ERAD machineries, including Derlin1, Hrd1, and gp78. Co-immunoprecipitation shows that SVIP strongly interacts with Derlin1 and p97/VCP, but weakly with Hrd1 and gp78. SVIP is anchored to the ER membrane probably via myristoylation, since mutation of the putative myristoylation site impaired its ER localization. Further, we found that SVIP is upregulated by tunicamycin-induced ER stress both at mRNA and protein levels. Functionally, SVIP enhances the loading of polyubiquitinated proteins to p97/VCP, suggesting that SVIP may play a role in coupling ubiquitination with retrotranslocation of misfolded proteins during ERAD. Consistently, silencing of SVIP expression by RNA interference stabilizes ERAD substrate tyrosinase(C89R). These results suggest that SVIP may facilitate the coupling of ubiquitination with retrotranslocation during ERAD and might play a protective role in brain.  

3.974           CFTR Inhibitory Factor (CIF) reduces the plasma membrane expression of CFTR by altering intracellular trafficking of CFTR to the lysosomal pathway

Bomberger, J.M. et al FASEB J., 21, 944.4 (2007)   The F508-CFTR mutation, the most common gene mutation in cystic fibrosis (CF), results in diminished plasma membrane expression of CFTR, leading to loss of functional CFTR and altered mucociliary clearance. This impairment promotes chronic infection of CF patients by P. aeruginosa. Previously we reported that a secreted factor from P. aeruginosa (CIF) reduces CFTR-mediated chloride secretion and the plasma membrane expression of CFTR by decreasing endocytic recycling. The aim of the current study was to investigate the mechanism by which CIF reduces the endocytic recycling of CFTR. CIF applied to the apical side of polarized human airway epithelial cells reduced CFTR in the plasma membrane, followed by a subsequent increase in CFTR labeling in the endosomes, then lysosomes, as determined by Optiprep subcellular compartment fractionation experiments. Co-immunoprecipitation studies revealed that CIF decreased the association of CFTR with Rab11a, concurrent with an increase in association of CFTR with Rab4a and Rab7. Lysosomal inhibitors blocked the degradation of CFTR by CIF, whereas proteosomal inhibitors had no effect. These studies demonstrate that CIF induces a redistribution of CFTR trafficking from the endocytic recycling pathway to the degradative pathway. In addition, this data suggests that chronic infection of P. aeruginosa in the CF lung may impact the efficacy of therapeutics developed to increase plasma membrane expression of the F508-CFTR.  

3.975           Cellular spelunking: exploring adipocyte caveolae

Pilch, P.F. et al

1. Lipid Res., 48, 2103-2111 (2007)

  It has been known for decades that the adipocyte cell surface is particularly rich in small invaginations we now know to be caveolae. These structures are common to many cell types but are not ubiquitous. They have generated considerable curiosity, as manifested by the numerous publications on the topic that describe various, sometimes contradictory, caveolae functions. Here, we review the field from an "adipocentric" point of view and suggest that caveolae may have a function of particular use for the fat cell, namely the modulation of fatty acid flux across the plasma membrane. Other functions for adipocyte caveolae that have been postulated include participation in signal transduction and membrane trafficking pathways, and it will require further experimental scrutiny to resolve controversies surrounding these possible activities.  

3.976           Lifeguard/neuronal membrane protein 35 regulates Fas ligand-mediated apoptosis in neurons via microdomain recruitment

Fernandez, M. et al

1. Neurochem., 103, 190-203 (2007)

  Fas ligand (FasL)-receptor system plays an essential role in regulating cell death in the developing nervous system, and it has been implicated in neurodegenerative and inflammatory responses in the CNS. Lifeguard (LFG) is a protein highly expressed in the hippocampus and the cerebellum, and it shows a particularly interesting regulation by being up-regulated during postnatal development and in the adult. We show that over-expression of LFG protected cortical neurons from FasL-induced apoptosis and decreased caspase-activation. Reduction of endogenous LFG expression by small interfering RNA sensitized cerebellar granular neurons to FasL-induced cell death and caspase-8 activation, and also increased sensitivity of cortical neurons. In differentiated cerebellar granular neurons, protection from FasL-induced cell death could be attributed exclusively to LFG and appears to be independent of FLICE inhibitor protein. Thus, LFG is an endogenous inhibitor of FasL-mediated neuronal death and it mediates the FasL resistance of CNS differentiated neurons. Finally, we also demonstrate that LFG is detected in lipid rafts microdomains, where it may interact with Fas receptor and regulate FasL-activated signaling pathways.  

3.977           Mannheimia haemolytica Leukotoxin Binds to Lipid Rafts in Bovine Lymphoblastoid Cells and Is Internalized in a Dynamin-2- and Clathrin-Dependent Manner

Atapatta, D.N. and Czuprynski, C.J. Infect. Immun., 75(10), 4719-4727 (2007)   Mannheimia haemolytica is the principal bacterial pathogen of the bovine respiratory disease complex. Its most important virulence factor is a leukotoxin (LKT), which is a member of the RTX family of exotoxins produced by many gram-negative bacteria. Previous studies demonstrated that LKT binds to the ß₂-integrin LFA-1 (CD11a/CD18) on bovine leukocytes, resulting in cell death. In this study, we demonstrated that depletion of lipid rafts significantly decreases LKT-induced bovine lymphoblastoid cell (BL-3) death. After binding to BL-3 cells, some of the LKT relocated to lipid rafts in an LFA-1-independent manner. We hypothesized that after binding to LFA-1 on BL-3 cells, LKT moves to lipid rafts and clathrin-coated pits via a dynamic process that results in LKT internalization and cytotoxicity. Knocking down dynamin-2 by small interfering RNA reduced both LKT internalization and cytotoxicity. Similarly, expression of dominant negative Eps15 protein expression, which is required for clathrin coat formation, reduced LKT internalization and LKT-mediated cytotoxicity to BL-3 cells. Finally, we demonstrated that inhibiting actin polymerization reduced both LKT internalization and LKT-mediated cytotoxicity. These results suggest that both lipid rafts and clathrin-mediated mechanisms are important for LKT internalization and cytotoxicity in BL-3 cells and illustrate the complex nature of LKT internalization by the cytoskeletal network.  

3.978           Phosphoinositide 3-Kinase-independent Non-genomic Signals Transit from the Androgen Receptor to Akt1 in Membrane Raft Microdomains

Cinar, B., Mukhopadhyay, N.K., Meng, G. and Freeman, M.R.

1. Biol. Chem., 282(40), 29584-29593 (2007)

  The serine-threonine kinase, Akt1/protein kinase B is an important mediator of growth, survival, and metabolic signaling. Recent studies have implicated cholesterol-rich, lipid raft microdomains in survival signals mediated by Akt1. Here we address the role of lipid raft membranes as a potential site of intersection of androgenic and Akt1 signaling. A subpopulation of androgen receptor (AR) was found to localize to a lipid raft subcellular compartment in LNCaP prostate cancer cells. Endogenous AR interacted with endogenous Akt1 preferentially in lipid raft fractions and androgen substantially enhanced the interaction between the two proteins. The association of AR with Akt1 was inhibited by the anti-androgen, bicalutamide, but was not affected by inhibition of phosphoinositide 3-kinase (PI3K). Androgen promoted endogenous Akt1 activity in lipid raft fractions, in a PI3K-independent manner, within 10 min of treatment. Fusion of a lipid raft targeting sequence to AR enhanced localization of the receptor to rafts, and stimulated Akt1 activity in response to androgen, while reducing the cells' dependence on constitutive signaling through PI3K for cell survival. These findings suggest that signals channeled through AR and Akt1 intersect by a mechanism involving formation within lipid raft membranes of an androgen-responsive, extranuclear AR/Akt1 complex. Our results indicate that cholesterol-rich membrane microdomains play a role in transmitting non-genomic signals involving androgen and the Akt pathway in prostate cancer cells.  

3.979           Ablation of a small transmembrane protein of Trypanosoma brucei (TbVTC1) involved in the synthesis of polyphosphate alters acidocalcisome biogenesis and function, and leads to a cytokinesis defect

Fang, J., Rohloff, P., Miranda, K. And Docampo, R. Biochem. J., 407, 161-170 (2007)   Inorganic poly P (polyphosphate) is an abundant component of acidocalcisomes of Trypanosoma brucei. In the present study we report the presence of a protein homologous with the yeast Vtc1p (vacuolar transporter chaperone 1) in T. brucei that is essential for poly P synthesis, acidocalcisome biogenesis and cytokinesis. Localization studies in a cell line expressing a TbVTC1 fused to GFP (green fluorescent protein) revealed its co-localization with the V-H⁺-PPase (vacuolar H⁺-pyrophosphatase), a marker for acidocalcisomes. Western blot analysis of acidocalcisome fractions and immunogold electron microscopy using polyclonal antibodies against a fragment of TbVTC1 confirmed the acidocalcisome localization. Ablation of TbVTC1 expression by RNA interference caused an abnormal morphology of acidocalcisomes, indicating that their biogenesis was disturbed, with a decreased pyrophosphate-driven H⁺ uptake and Ca²⁺ content, a significant decrease in the amount of poly P and a deficient response to hyposmotic stress. Ablation of TbVTC1 expression for longer periods produced marked gross morphological alterations compatible with a defect in cytokinesis, followed by cell death. Overexpression of the TbVTC1 gene caused mild alterations in growth rate, but had no perceptible effect on acidocalcisome morphology. We propose that the PP_i-driven H⁺ pumping deficiency induced by ablation of TbVTC1 leads to alterations in the protonmotive force of acidocalcisomes, which results in deficient fusion or budding of the organelles, decreased H⁺ and Ca²⁺ content, and decreased synthesis of poly P. A decrease in the poly P content would lead to osmotic sensitivity and defects in cytokinesis.  

3.980           Peroxisomal-mitochondrial oxidation in a rodent model of obesity-associated insulin resistance

Noland, R.C. et al Am. J. Physiol. Endocrinol. Metab., 293, E986-E1001 (2007)   Peroxisomal oxidation yields metabolites that are more efficiently utilized by mitochondria. This is of potential clinical importance because reduced fatty acid oxidation is suspected to promote excess lipid accumulation in obesity-associated insulin resistance. Our purpose was to assess peroxisomal contributions to mitochondrial oxidation in mixed gastrocnemius (MG), liver, and left ventricle (LV) homogenates from lean and fatty (fa/fa) Zucker rats. Results indicate that complete mitochondrial oxidation (CO₂ production) using various lipid substrates was increased approximately twofold in MG, unaltered in LV, and diminished 50% in liver of fa/fa rats. In isolated mitochondria, malonyl-CoA inhibited CO₂ production from palmitate 78%, whereas adding isolated peroxisomes reduced inhibition to 21%. These data demonstrate that peroxisomal products may enter mitochondria independently of CPT I, thus providing a route to maintain lipid disposal under conditions where malonyl-CoA levels are elevated, such as in insulin-resistant tissues. Peroxisomal metabolism of lignoceric acid in fa/fa rats was elevated in both liver and MG (LV unaltered), but peroxisomal product distribution varied. A threefold elevation in incomplete oxidation was solely responsible for increased hepatic peroxisomal oxidation (CO₂ unaltered). Alternatively, only CO₂ was detected in MG, indicating that peroxisomal products were exclusively partitioned to mitochondria for complete lipid disposal. These data suggest tissue-specific destinations for peroxisome-derived products and emphasize a potential role for peroxisomes in skeletal muscle lipid metabolism in the obese, insulin-resistant state.  

3.981           Anti–ß2-microglobulin monoclonal antibodies induce apoptosis in myeloma cells by recruiting MHC class I to and excluding growth and survival cytokine receptors from lipid rafts

Yang, J. et al Blood, 110(8), 3028-3035 (2007)   We recently showed that monoclonal antibodies (mAbs) against ß₂-microglobulin (ß₂M) have a remarkably strong apoptotic effect on myeloma cells. The mAbs induced apoptosis by recruiting major histocompatibility complex (MHC) class I to lipid rafts, activated c-Jun N-terminal kinase (JNK), and inhibited phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal–regulated kinase (ERK) pathways. Growth and survival cytokines such as interleukin-6 (IL-6) and insulin-like growth factor-I (IGF-I), which could protect myeloma cells from dexamethasone-induced apoptosis, did not affect mAb-mediated cell death. This study was undertaken to elucidate the mechanisms underlying anti-ß₂M mAb–induced PI3K/Akt and ERK inhibition and the inability of IL-6 and IGF-I to protect myeloma cells from mAb-induced apoptosis. We focused on lipid rafts and confirmed that these membrane microdomains are required for IL-6 and IGF-I signaling. By recruiting MHC class I into lipid rafts, anti-ß₂M mAbs excluded IL-6 and IGF-I receptors and their substrates from the rafts. The mAbs not only redistributed the receptors in cell membrane, but also abrogated IL-6– or IGF-I–mediated Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3), PI3K/Akt, and Ras/Raf/ERK pathway signaling, which are otherwise constitutively activated in myeloma cells. Thus, this study further defines the tumoricidal mechanism of the mAbs and provides strong evidence to support the potential of these mAbs as therapeutic agents for myeloma.  

3.982           Protein kinase C- coimmunoprecipitates with cytochrome oxidase subunit IV and is associated with improved cytochrome-c oxidase activity and cardioprotection

Guo, D. et al Am. J. Physiol. Heart Circ. Physiol., 293, H2219-H2230 (2007)   We have utilized an in situ rat coronary ligation model to establish a PKC- cytochrome oxidase subunit IV (COIV) coimmunoprecipitation in myocardium exposed to ischemic preconditioning (PC). Ischemia-reperfusion (I/R) damage and PC protection were confirmed using tetrazolium-based staining methods and serum levels of cardiac troponin I. Homogenates prepared from the regions at risk (RAR) and not at risk (RNAR) for I/R injury were fractionated into cell-soluble (S), 600 g low-speed centrifugation (L), Percoll/Optiprep density gradient-purified mitochondrial (M), and 100,000 g particulate (P) fractions. COIV immunoreactivity and cytochrome-c oxidase activity measurements estimated the percentages of cellular mitochondria in S, L, M, and P fractions to be 0, 55, 29, and 16%, respectively. We observed 18, 3, and 3% of PKC- , - , and - isozymes in the M fraction under basal conditions. Following PC, we observed a 61% increase in PKC- levels in the RAR M fraction compared with the RNAR M fraction. In RAR mitochondria, we also observed a 2.8-fold increase in PKC- serine 729 phosphoimmunoreactivity (autophosphorylation), indicating the presence of activated PKC- in mitochondria following PC. PC administered before prolonged I/R induced a 1.9-fold increase in the coimmunoprecipitation of COIV, with anti-PKC- antisera and a twofold enhancement of cytochrome-c oxidase activity. Our results suggest that PKC- may interact with COIV as a component of the cardioprotection in PC. Induction of this interaction may provide a novel therapeutic target for protecting the heart from I/R damage.  

3.983           HSP60 in heart failure: abnormal distribution and role in cardiac myocyte apoptosis

Lin, L et al Am. J. Physiol. Heart Circ. Physiol., 293, H2238-H2247 (2007)   Heat shock protein (HSP) 60 is a mitochondrial and cytosolic protein. Previously, we reported that HSP60 doubled in end-stage heart failure, even though levels of the protective HSP72 were unchanged. Furthermore, we observed that acute injury in adult cardiac myocytes resulted in movement of HSP60 to the plasma membrane. We hypothesized that the inflammatory state of heart failure would cause translocation of HSP60 to the plasma membrane and that this would provide a pathway for cardiac injury. Two models were used to test this hypothesis: 1) a rat model of heart failure and 2) human explanted failing hearts. We found that HSP60 localized to the plasma membrane and was also found in the plasma early in heart failure. Plasma membrane HSP60 localized to lipid rafts and was detectable on the cell surface with the use of both flow cytometry and confocal microscopy. Localization of HSP60 to the cell surface correlated with increased apoptosis. In heart failure, HSP60 is in the plasma membrane fraction, on the cell surface, and in the plasma. Membrane HSP60 correlated with increased apoptosis. Release of HSP60 may activate the innate immune system, promoting a proinflammatory state, including an increase in TNF- . Thus abnormal trafficking of HSP60 to the cell surface may be an early trigger for myocyte loss and the progression of heart failure.  

3.984           Metabolism and short-term metabolic effects of conjugated linoleic acids in rat hepatocytes

Priore, P., Giudetti, A.M., Natali, F., Gnoni, G.V. and Geelen, M.J.H. Biochim. Biophys. Acta, 1771(10), 1299-1307 (2007)   Metabolic fate and short-term effects of a 1:1 mixture of cis-9,trans-11 and trans-10,cis-12-conjugated linoleic acids (CLA), compared to linoleic acid (LA), on lipid metabolism was investigated in rat liver. In isolated mitochondria CLA-CoA were poorer substrates than LA-CoA for carnitine palmitoyltransferase-I (CPT-I) activity. However, in digitonin-permeabilized hepatocytes, where interactions among different metabolic pathways can be simultaneously investigated, CLA induced a remarkable stimulatory effect on CPT-I activity. This stimulation can be ascribed to a reduced malonyl-CoA level in turn due to inhibition of acetyl-CoA carboxylase (ACC) activity. The ACC/malonyl-CoA/CPT-I system can therefore represent a coordinate control by which CLA may exert effects on the partitioning of fatty acids between esterification and oxidation. Moreover, the rate of oxidation to CO₂ and ketone bodies was significantly higher from CLA; peroxisomes rather than mitochondria were responsible for this difference. Interestingly, peroxisomal acyl-CoA oxidase (AOX) activity strongly increased by CLA-CoA compared to LA-CoA. CLA, metabolized by hepatocytes at a higher rate than LA, were poorer substrates for cellular and VLDL-triacylglycerol (TAG) synthesis. Overall, our results suggest that increased fatty acid oxidation with consequent decreased fatty acid availability for TAG synthesis is a potential mechanism by which CLA reduce TAG level in rat liver.            

3.985           Effect of hypoxia on the binding and subcellular distribution of iron regulatory proteins

Christova, T. and Templeton, D.M. Mol. Cell. Biochem., 301, 21-32 (2007)   Iron regulatory proteins 1 and 2 (IRP1, IRP2) are key determinants of uptake and storage of iron by the liver, and are responsive to oxidative stress and hypoxia potentially at the level of both protein concentration and mRNA-binding activity. We examined the effect of hypoxia (1% O₂) on IRP1 and IRP2 levels (Western blots) and mRNA-binding activity (gel shift assays) in human hepatoma HepG2 cells, and compared them with HEK 293 cells, a renal cell line known to respond to hypoxia. Total IRP binding to an iron responsive element (IRE) mRNA probe was increased several fold by hypoxia in HEK 293 cells, maximally at 4–8 h. An earlier and more modest increase (1.5- to 2-fold, peaking at 2 h and then declining) was seen in HepG2 cells. In both cell lines, IRP1 made a greater contribution to IRE-binding activity than IRP2. IRP1 protein levels were increased slightly by hypoxia in HEK 293 but not in HepG2 cells. IRP1 was distributed between cytosolic and membrane-bound fractions, and in both cells hypoxia increased both the amount and IRE-binding activity of the membrane-associated IRP1 fraction. Further density gradient fractionation of HepG2 membranes revealed that hypoxia caused an increase in total membrane IRP1, with a shift in the membrane-bound fraction from Golgi to an endoplasmic reticulum (ER)-enriched fraction. Translocation of IRP to the ER has previously been shown to stabilize transferrin receptor mRNA, thus increasing iron availability to the cell. Iron depletion with deferoxamine also caused an increase in ER-associated IRP1. Phorbol ester caused serine phosphorylation of IRP1 and increased its association with the ER. The calcium ionophore ionomycin likewise increased ER-associated IRP1, without affecting total IRE-binding activity. We conclude that IRP1 is translocated to the ER by multiple signals in HepG2 cells, including hypoxia, thereby facilitating its role in regulation of hepatic gene expression.  

3.986           Release of thioredoxin from Saccharomyces cerevisiae with environmental stimuli: solubilization of thioredoxin with ethanol

Takeuchi, Y. et al Appl. Microbiol. Biotechnol., 75, 1393-1399 (2007)   Thioredoxin is crucial for the maintenance of the redox status of cells of all types. Mammalian thioredoxin is secreted from various types of cells, although the mechanism underlying has not yet been clarified. Previously, we demonstrated that thioredoxin was released from Saccharomyces cerevisiae after treatment with ethanol. In this paper, we show that as well as ethanol, low-pH shock and hypoosmotic shock release thioredoxin. Low-molecular-weight proteins in yeast cells were preferentially released by treatment with ethanol and low-pH shock. A cell wall integrity pathway seems partially involved in the hypoosmotic shock-induced release of thioredoxin. Considerable amounts of thioredoxin were present in the insoluble fractions of the cells, a portion of which was associated with lipid microdomains that are resistant to nonionic detergent at 4°C. The intracellular localization of thioredoxin may influence the efficiency of its release from yeast cells with ethanol.  

3.987           Flagellar membrane trafficking in kinetoplastids

Fridberg, A., Buchanan, K.T. and Engman, D.M. Parasitol. Res., 100, 205-212 (2007)   This review was presented at the International Symposium on Vesicle Trafficking in Parasitic Protozoa at Caxambu, Brasil, in November, 2005.  

3.988           Influence of lipid rafts on CD1d presentation by dendritic cells

Peng, W. et al Mol. Membrane Biol., 24(5-6), 475-484 (2007)   Our main objective was to analyze the role of lipid rafts in the activation of V -14^- and V -14⁺ T hybridomas by dendritic cells. We showed that activation of V -14⁺ hybridomas by dendritic cells or other CD1d-expressing cells was altered by disruption of lipid rafts with the cholesterol chelator M CD. However, CD1d presentation to autoreactive V -14^- anti-CD1d hybridomas which do not require the endocytic pathway was not altered. Using partitioning of membrane fractions with Brij98 at 37°C, we confirmed that CD1d was enriched in subcellular fractions corresponding to lipid rafts and we describe that -GalCer enhanced CD1d amount in the low density detergent insoluble fraction. We conclude that the membrane environment of CD1d can influence antigen presentation mainly when the endocytic pathway is required. Flow cytometry analysis can provide additional information on lipid rafts in plasma membranes and allows a dynamics follow-up of lipid rafts partitioning. Using this method, we showed that CD1d plasma membrane expression was sensitive to low concentrations of detergent. This may suggest either that CD1d is associated with lipid rafts mainly in intracellular membranes or that its association with the lipid rafts in the plasma membrane is weak.  

3.989           Lipid Raft Proteomics: More than Just Detergent-Resistant Membranes

Foster, L.J. and Chan, Q.W.T. Subcellular Proteomics, 43, 35-47 (2007)   No abstract available  

3.990           Plasma Membrane Proteomics

Alexandersson, E., Gustavsson, N., Bernfur, K., Kjellbom, P. and Larsson, C. Plant Proteomics, Springer Berlin/Heidelberg, 189-206 (2007)   Proteins residing in the plasma membrane have key functions in transport, signal transduction, vesicle trafficking and many other important processes. To better understand these processes it is necessary to reveal the identity of plasma membrane proteins and to monitor modifications and regulation of their expression. This chapter is an overview of the methods used in plant plasma membrane proteomic studies and the results obtained so far. It focuses on studies using mass spectrometry for identification and includes aspects of plasma membrane fractionation, extraction and washing treatments, assessment of purity, separation methods for plasma membrane proteins and choice of techniques for protein cleavage. Finally, the results of plasma membrane proteomic studies are compared and problems with contaminating proteins are discussed.  

3.991           CD44 Regulates Hepatocyte Growth Factor-mediated Vascular Integrity: ROLE OF c-Met, Tiam1/Rac1, DYNAMIN 2, AND CORTACTIN

Singleton, P.A. et al

1. Biol. Chem., 282(42), 30643-30657 (2007)

  The preservation of vascular endothelial cell (EC) barrier integrity is critical to normal vessel homeostasis, with barrier dysfunction being a feature of inflammation, tumor angiogenesis, atherosclerosis, and acute lung injury. Therefore, agents that preserve or restore vascular integrity have important therapeutic implications. In this study, we explored the regulation of hepatocyte growth factor (HGF)-mediated enhancement of EC barrier function via CD44 isoforms. We observed that HGF promoted c-Met association with CD44v10 and recruitment of c-Met into caveolin-enriched microdomains (CEM) containing CD44s (standard form). Treatment of EC with CD44v10-blocking antibodies inhibited HGF-mediated c-Met phosphorylation and c-Met recruitment to CEM. Silencing CD44 expression (small interfering RNA) attenuated HGF-induced recruitment of c-Met, Tiam1 (a Rac1 exchange factor), cortactin (an actin cytoskeletal regulator), and dynamin 2 (a vesicular regulator) to CEM as well as HGF-induced trans-EC electrical resistance. In addition, silencing Tiam1 or dynamin 2 reduced HGF-induced Rac1 activation, cortactin recruitment to CEM, and EC barrier regulation. We observed that both HGF- and high molecular weight hyaluronan (CD44 ligand)-mediated protection from lipopolysaccharide-induced pulmonary vascular hyperpermeability was significantly reduced in CD44 knock-out mice, thus validating these in vitro findings in an in vivo murine model of inflammatory lung injury. Taken together, these results suggest that CD44 is an important regulator of HGF/c-Met-mediated in vitro and in vivo barrier enhancement, a process with essential involvement of Tiam1, Rac1, dynamin 2, and cortactin.  

3.992           A reversible form of lysine acetylation in the ER and Golgi lumen controls the molecular stabilization of BACE1

Costantini, C., Ko, M.H., Cabell, M., Jonas, M.C. and Puglielli, L. Biochem. J., 407, 383-395 (2007)   The lipid second messenger ceramide regulates the rate of b cleavage of the Alzheimer's disease APP (amyloid precursor protein) by affecting the molecular stability of the b secretase BACE1 (b-site APP cleaving enzyme 1). Such an event is stimulated in the brain by the normal process of aging, and is under the control of the general aging programme mediated by the insulin-like growth factor 1 receptor. In the present study we report that BACE1 is acetylated on seven lysine residues of the N-terminal portion of the nascent protein. This process involves lysine acetylation in the lumen of the ER (endoplasmic reticulum) and is followed by deacetylation in the lumen of the Golgi apparatus, once the protein is fully mature. We also show that specific enzymatic activities acetylate (in the ER) and deacetylate (in the Golgi apparatus) the lysine residues. This process requires carrier-mediated translocation of acetyl-CoA into the ER lumen and is stimulated by ceramide. Site-directed mutagenesis indicates that lysine acetylation is necessary for nascent BACE1 to leave the ER and move ahead in the secretory pathway, and for the molecular stabilization of the protein.  

3.993           A Functional Dynein–Microtubule Network Is Required for NGF Signaling Through the Rap1/MAPK Pathway

Wu, C. et al Traffic, 8, 1503-1520 (2007)   Rap1 transduces nerve growth factor (NGF)/tyrosine receptor kinase A (TrkA) signaling in early endosomes, leading to sustained activation of the p44/p42 mitogen-activated protein kinases (MAPK1/2). However, the mechanisms by which NGF, TrkA and Rap1 are trafficked to early endosomes are poorly defined. We investigated trafficking and signaling of NGF, TrkA and Rap1 in PC12 cells and in cultured rat dorsal root ganglion (DRG) neurons. Herein, we show a role for both microtubule- and dynein-based transport in NGF signaling through MAPK1/2. NGF treatment resulted in trafficking of NGF, TrkA and Rap1 to early endosomes in the perinuclear region of PC12 cells where sustained activation of MAPK1/2 was observed. Disruption of microtubules with nocodazole in PC12 cells had no effect on the activation of TrkA and Ras. However, it disrupted intracellular trafficking of TrkA and Rap1. Moreover, NGF-induced activation of Rap1 and sustained activation of MAPK1/2 were markedly suppressed. Inhibition of dynein activity through overexpression of dynamitin (p50) blocked trafficking of Rap1 and the sustained phase of MAPK1/2 activation in PC12 cells. Remarkably, even in the continued presence of NGF, mature DRG neurons that overexpressed p50 became atrophic and most (>80%) developing DRG neurons died. Dynein- and microtubule-based transport is thus necessary for TrkA signaling to Rap1 and MAPK1/2.  

3.994           Angiotensin II Decreases Glucose Uptake by Downregulation of GLUT1 in the Cell Membrane of the Vascular Smooth Muscle Cell Line A10

Masori, M. et al

1. Cardiovasc. Pharmacol., 50(3), 267-273 (2007)

  Recent evidence suggests a crosstalk between angiotensin II (Ang II) and insulin. However, whether this crosstalk affects glucose uptake, particularly in terms of actin filament involvement, has not yet been studied in vascular smooth muscle cells. Pretreatment of cells with either Ang II or cytochalasin D disarranged actin filaments in a time-dependent manner and inhibited glucose uptake. However, insulin increased actin reorganization and glucose uptake. Membrane fractionation studies showed that Ang II decreased GLUT-1 at the cell membrane, whereas it increased GLUT-1 in the cytoplasm, indicating that Ang II may cause internalization of GLUT-1 via actin disorganization, consequently decreasing glucose uptake. The effects of Ang II on glucose uptake and actin reorganization were blocked by AT1 receptor antagonist, but not by AT2 antagonist. Either P38 or ERK1/2 inhibitors partially reversed the Ang II-inhibited actin reorganization and glucose uptake, suggesting that MAPK signaling pathways could be involved as downstream events in Ang II signaling, and this signaling may interfere with insulin-induced actin reorganization and glucose uptake. These data imply that Ang II induces insulin resistance by decreasing glucose uptake via disarrangement of actin filaments, which provides a novel insight into understanding of insulin resistance by Ang II at the molecular level.  

3.995           BK Channels Are Linked to Inositol 1,4,5-Triphosphate Receptors via Lipid Rafts: A NOVEL MECHANISM FOR COUPLING [Ca2+]i TO ION CHANNEL ACTIVATION

Weaver, A.K., Olsen, M.L., McFerrin, M.B. and Southeimer, H.

1. Biol. Chem., 282(43), 31558-31568 (2007)

  Glioma cells prominently express a unique splice variant of a large conductance, calcium-activated potassium channel (BK channel). These channels transduce changes in intracellular calcium to changes of K⁺ conductance in the cells and have been implicated in growth control of normal and malignant cells. The Ca²⁺ increase that facilitates channel activation is thought to occur via activation of intracellular calcium release pathways or influx of calcium through Ca²⁺-permeable ion channels. We show here that BK channel activation involves the activation of inositol 1,4,5-triphosphate receptors (IP₃R), which localize near BK channels in specialized membrane domains called lipid rafts. Disruption of lipid rafts with methyl- -cyclodextrin disrupts the functional association of BK channel and calcium source resulting in a >50% reduction in K⁺ conductance mediated by BK channels. The reduction of BK current by lipid raft disruption was overcome by the global elevation of intracellular calcium through inclusion of 750 nM Ca²⁺ in the pipette solution, indicating that neither the calcium sensitivity of the channel nor their overall number was altered. Additionally, pretreatment of glioma cells with 2-aminoethoxydiphenyl borate to inhibit IP₃Rs negated the effect of methyl- -cyclodextrin, providing further support that IP₃Rs are the calcium source for BK channels. Taken together, these data suggest a privileged association of BK channels in lipid raft domains and provide evidence for a novel coupling of these Ca²⁺-sensitive channels to their second messenger source.  

3.996           Escherichia coli Signal Recognition Particle Receptor FtsY Contains an Essential and Autonomous Membrane-binding Amphipathic Helix

Parlitz, R. et al

1. Biol. Chem., 282(44), 32176-32184 (2007)

  Escherichia coli membrane protein biogenesis is mediated by a signal recognition particle and its membrane-associated receptor (FtsY). Although crucial for its function, it is still not clear how FtsY interacts with the membrane. Analysis of the structure/function differences between severely truncated active (NG+1) and inactive (NG) mutants of FtsY enabled us to identify an essential membrane-interacting determinant. Comparison of the three-dimensional structures of the mutants, combined with site-directed mutagenesis, modeling, and liposome-binding assays, revealed that FtsY contains a conserved autonomous lipid-binding amphipathic -helix at the N-terminal end of the N domain. Deletion experiments showed that this helix is essential for FtsY function in vivo, thus offering, for the first time, clear evidence for the functionally important, physiologically relevant interaction of FtsY with lipids.  

3.997           Saccharomyces cerevisiae CWH43 Is Involved in the Remodeling of the Lipid Moiety of GPI Anchors to Ceramides

Umemura, M., Fujita, M., Yoko-o., T., Fukamizu, A. and Jigami, Y. Mol. Biol. Cell, 18, 4304-4316 (2007)   The glycosylphosphatidylinositol (GPI)-anchored proteins are subjected to lipid remodeling during their biosynthesis. In the yeast Saccharomyces cerevisiae, the mature GPI-anchored proteins contain mainly ceramide or diacylglycerol with a saturated long-fatty acid, whereas conventional phosphatidylinositol (PI) used for GPI biosynthesis contains an unsaturated fatty acid. Here, we report that S. cerevisiae Cwh43p, whose N-terminal region contains a sequence homologous to mammalian PGAP2, is involved in the remodeling of the lipid moiety of GPI anchors to ceramides. In cwh43 disruptant cells, the PI moiety of the GPI-anchored protein contains a saturated long fatty acid and lyso-PI but not inositolphosphorylceramides, which are the main lipid moieties of GPI-anchored proteins from wild-type cells. Moreover, the C-terminal region of Cwh43p (Cwh43-C), which is not present in PGAP2, is essential for the ability to remodel GPI lipids to ceramides. The N-terminal region of Cwh43p (Cwh43-N) is associated with Cwh43-C, and it enhanced the lipid remodeling to ceramides by Cwh43-C. Our results also indicate that mouse FRAG1 and C130090K23, which are homologous to Cwh43-N and -C, respectively, share these activities.  

3.998           A novel beta-site amyloid precursor protein cleaving enzyme (BACE) isoform regulated by nonsense-mediated mRNA decay and proteasome-dependent degradation

Tanahashi, H. and  Tabira, T. Neurosci. Lett., 428, 103-108 (2007)   Proteolytic cleavage of amyloid beta-peptide (Aβ) from amyloid precursor protein (APP) is a key event in the pathogenesis of Alzheimer's disease. Beta-site amyloid precursor protein cleaving enzyme (BACE) cleaves the APP at the N-terminus of Aβ. We investigated whether particular stress conditions modify the expression and activity of BACE, and found that treatment of human neuroblastoma cells with protein synthesis inhibitors induced expression of a novel splice variant of BACE. This unusual transcript, I-127, is produced by usage of an internal splicing donor site in exon 3. The splicing event leads to a premature termination codon, as well as elimination of one of two conserved aspartic protease active sites, a transmembrane domain, and a C-terminal cytoplasmic tail from BACE. Low levels of this mRNA were found in the human brain. When expressed in cells, I-127 had no effect on Aβ secretion and was retained in the endoplasmic reticulum without propeptide removal. It was also unstable with a turnover t_1/2 of 2 h; normal BACE had a turnover t_1/2 of 8 h. Finally, I-127 was degraded in a proteasome-dependent manner. Thus, I-127 is regulated by both nonsense-mediated mRNA decay (NMD) and proteasome-dependent degradation.  

3.999           Plant organelle proteomics

Lilley, K.S. and Dupree, P. Current Opinion in Plant Biol., 10, 594-599 (2007)   It is important for cell biologists to know the subcellular localization of proteins to understand fully the functions of organelles and the compartmentation of plant metabolism. The accurate description of an organelle proteome requires the ability to identify genuine protein residents. Such accurate assignment is difficult in situations where a pure homogeneous preparation of the organelle cannot be achieved. Practical limitations in both organelle isolation and also analysis of low abundance proteins have resulted in limited datasets from high throughput proteomics approaches. Here, we discuss some examples of quantitative proteomic methods and their use to study plant organelle proteomes, with particular reference to methods designed to give unequivocal assignments to organelles.  

3.1000           Comparison of the contributions of the nuclear and cytoplasmic compartments to global gene expression in human cells

Barthelson, R.A., Lambert, G.M., Vanier, C., Lynch, R.M. and Galbraith, D.W. BMC Genomics, 8(340), 1-15 (2007)   Background In the most general sense, studies involving global analysis of gene expression aim to provide a comprehensive catalog of the components involved in the production of recognizable cellular phenotypes. These studies are often limited by the available technologies. One technology, based on microarrays, categorizes gene expression in terms of the abundance of RNA transcripts, and typically employs RNA prepared from whole cells, where cytoplasmic RNA predominates. Results Using microarrays comprising oligonucleotide probes that represent either protein-coding transcripts or microRNAs (miRNA), we have studied global transcript accumulation patterns for the HepG2 (human hepatoma) cell line. Through subdividing the total pool of RNA transcripts into samples from nuclei, the cytoplasm, and whole cells, we determined the degree of correlation of these patterns across these different subcellular locations. The transcript and miRNA abundance patterns for the three RNA fractions were largely similar, but with some exceptions: nuclear RNA samples were enriched with respect to the cytoplasm in transcripts encoding proteins associated with specific nuclear functions, such as the cell cycle, mitosis, and transcription. The cytoplasmic RNA fraction also was enriched, when compared to the nucleus, in transcripts for proteins related to specific nuclear functions, including the cell cycle, DNA replication, and DNA repair. Some transcripts related to the ubiquitin cycle, and transcripts for various membrane proteins were sorted into either the nuclear or cytoplasmic fractions. Conclusion Enrichment or compartmentalization of cell cycle and ubiquitin cycle transcripts within the nucleus may be related to the regulation of their expression, by preventing their translation to proteins. In this way, these cellular functions may be tightly controlled by regulating the release of mRNA from the nucleus and thereby the expression of key rate limiting steps in these pathways. Many miRNA precursors were also enriched in the nuclear samples, with significantly fewer being enriched in the cytoplasm. Studies of mRNA localization will help to clarify the roles RNA processing and transport play in the regulation of cellular function.  

3.1001           Integral and Associated Lysosomal Membrane Proteins

Schröder, B. et al Traffic, 8(12), 1676-1686 (2007)   We searched for novel proteins in lysosomal membranes, tentatively participating in molecular transport across the membrane and/or in interactions with other compartments. In membranes purified from placental lysosomes, we identified 58 proteins, known to reside at least partially in the lysosomal membrane. These included 17 polypeptides comprising or associated with the vacuolar adenosine triphosphatase. We report on additional 86 proteins that were significantly enriched in the lysosomal membrane fraction. Among these, 12 novel proteins of unknown functions were found. Three were orthologues of rat proteins that have been identified in tritosomes by Bagshaw RD et al. (A proteomic analysis of lysosomal integral membrane proteins reveals the diverse composition of the organelle. Mol Cell Proteomics 2005;4:133–143). Here, the proteins encoded by LOC201931 (FLJ38482) and LOC51622 (C7orf28A) were expressed with an appended fluorescent tag in HeLa cells and found to be present in lysosomal organelles. Among the lysosomally enriched proteins, also 16 enzymes and transporters were detected that had not been assigned to lysosomal membranes previously. Finally, our results identified a particular set of proteins with known functions in signaling and targeting to be at least partially associated with lysosomes.  

3.1002           Function and dynamics of PKD2 in Chlamydomonas reinhardtii flagella

Huang, K. et al

1. Cell Biol., 179(3), 501-514 (2007)

  To analyze the function of ciliary polycystic kidney disease 2 (PKD2) and its relationship to intraflagellar transport (IFT), we cloned the gene encoding Chlamydomonas reinhardtii PKD2 (CrPKD2), a protein with the characteristics of PKD2 family members. Three forms of this protein (210, 120, and 90 kD) were detected in whole cells; the two smaller forms are cleavage products of the 210-kD protein and were the predominant forms in flagella. In cells expressing CrPKD2–GFP, about 10% of flagellar CrPKD2–GFP was observed moving in the flagellar membrane. When IFT was blocked, fluorescence recovery after photobleaching of flagellar CrPKD2–GFP was attenuated and CrPKD2 accumulated in the flagella. Flagellar CrPKD2 increased fourfold during gametogenesis, and several CrPKD2 RNA interference strains showed defects in flagella-dependent mating. These results suggest that the CrPKD2 cation channel is involved in coupling flagellar adhesion at the beginning of mating to the increase in flagellar calcium required for subsequent steps in mating.  

3.1003           SorLA/LR11 Regulates Processing of Amyloid Precursor Protein via Interaction with Adaptors GGA and PACS-1

Schmidt, V. et al

1. Biol. Chem., 282(45), 32956-32964 (2007)

  SorLA has been recognized as a novel sorting receptor that regulates trafficking and processing of the amyloid precursor protein (APP) and that represents a significant risk factor for sporadic Alzheimer disease. Here, we investigated the cellular mechanisms that control intracellular trafficking of sorLA and their relevance for APP processing. We demonstrate that sorLA acts as a retention factor for APP in trans-Golgi compartments/trans-Golgi network, preventing release of the precursor into regular processing pathways. Proper localization and activity of sorLA are dependent on functional interaction with GGA and PACS-1, adaptor proteins involved in protein transport to and from the trans-Golgi network. Aberrant targeting of sorLA to the recycling compartment or the plasma membrane causes faulty APP trafficking and imbalance in non-amyloidogenic and amyloidogenic processing fates. Thus, our findings identified altered routing of sorLA as a major cellular mechanism contributing to abnormal APP processing and enhanced amyloid -peptide formation.  

3.1004           Myristoylation Is Required for Human Immunodeficiency Virus Type 1 Gag-Gag Multimerization in Mammalian Cells

Li, H., Dou, J., Ding, L. and Spearman, P.

1. Virol., 81(23), 12899-12910 (2007)

  The Gag protein of human immunodeficiency virus type 1 directs the virion assembly process. Gag proteins must extensively multimerize during the formation of the spherical immature virion shell. In vitro, virus-like particles can be generated from Gag proteins that lack the N-terminal myristic acid modification or the nucleocapsid (NC) protein. The precise requirements for Gag-Gag multimerization under conditions present in mammalian cells, however, have not been fully elucidated. In this study, a Gag-Gag multimerization assay measuring fluorescence resonance energy transfer was employed to define the Gag domains that are essential for homomultimerization. Three essential components were identified: protein-protein interactions contributed by residues within both the N- and C-terminal domains of capsid (CA), basic residues in NC, and the presence of myristic acid. The requirement of myristic acid for multimerization was reproduced using the heterologous myristoylation sequence from v-src. Only when a leucine zipper dimerization motif was placed in the position of NC was a nonmyristoylated Gag protein able to multimerize. These results support a three-component model for Gag-Gag multimerization that includes membrane interactions mediated by the myristoylated N terminus of Gag, protein-protein interactions between CA domains, and NC-RNA interactions.  

3.1005           Induction of Epidermal Growth Factor Receptor Expression by Epstein-Barr Virus Latent Membrane Protein 1 C-Terminal-Activating Region 1 Is Mediated by NF- B p50 Homodimer/Bcl-3 Complexes

Thornburg, N.J. and Raab-Traub, N.

1. Virol., 81(23), 12954-12961 (2007)

  The Epstein-Barr virus (EBV) is associated with the development of numerous malignancies, including the epithelial malignancy nasopharyngeal carcinoma (NPC). The viral oncoprotein latent membrane protein 1 (LMP1) is expressed in almost all EBV-associated malignancies and has profound effects on gene expression. LMP1 acts as a constitutively active tumor necrosis factor receptor and activates multiple forms of the NF- B family of transcription factors. LMP1 has two domains that both activate NF- B. In epithelial cells, LMP1 C-terminal activating region 1 (CTAR1) uniquely activates p50/p50-, p50/p52-, and p65-containing complexes while CTAR2 activates canonical p50/p65 complexes. CTAR1 also uniquely upregulates the epidermal growth factor receptor (EGFR). In NPC, NF- B p50/p50 homodimers and the transactivator Bcl-3 were detected on the EGFR promoter. In this study, the role of NF- B p50 and Bcl-3 in LMP1-mediated upregulation of EGFR was analyzed. In LMP1-CTAR1-expressing cells, chromatin immunoprecipitation detected p50 and Bcl-3 on the NF- B consensus sites within the egfr promoter. Transient overexpression of p50 and Bcl-3 increased EGFR expression, confirming the regulation of EGFR by these factors. Treatment with p105/p50 siRNA effectively reduced p105/p50 levels but unexpectedly increased Bcl-3 expression and levels of p50/Bcl-3 complexes, resulting in increased EGFR expression. These data suggest that induction of p50/p50/Bcl-3 complexes by LMP1 CTAR1 mediates LMP1-induced EGFR upregulation and that formation of the p50/p50/Bcl-3 complex is negatively regulated by the p105 precursor. The distinct forms of NF- B that are induced by LMP1 CTAR1 likely activate distinct cellular genes.  

3.1006           Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

Brügger, B. et al Retrovirology, 4(70), 1-12   Background The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. Results Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. Conclusion Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo.  

3.1007           Caveolins and intracellular calcium regulation in human airway smooth muscle

Prakash, Y.S. et al Am. J. Physiol. Lung Cell Mol. Physiol., 293, L1118-L1126 (2007)   Regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) is a key factor in airway smooth muscle (ASM) tone. In vascular smooth muscle, specialized membrane microdomains (caveolae) expressing the scaffolding protein caveolin-1 are thought to facilitate cellular signal transduction. In human ASM cells, we tested the hypothesis that caveolae mediate Ca²⁺ responses to agonist stimulation. Fluorescence immunocytochemistry with confocal microscopy, as well as Western blot analysis, was used to determine that agonist receptors (M₃ muscarinic, bradykinin, and histamine) and store-operated Ca²⁺ entry (SOCE)-regulatory mechanisms colocalize with caveolin-1. Although caveolin-2 coexpressed with caveolin-1, caveolin-3 was absent. In fura 2-loaded ASM cells, [Ca²⁺]_i responses to 1 µM ACh, 10 µM histamine, and 10 nM bradykinin, as well as SOCE, were attenuated (each to a different extent) after disruption of caveolae by the cholesterol-chelating drug methyl- -cyclodextrin. Transfection of ASM cells with 50 nM caveolin-1 small interfering RNA significantly weakened caveolin-1 expression and blunted [Ca²⁺]_i responses to bradykinin and histamine, as well as SOCE, but the response to ACh was less intense. These results indicate that caveolae are present in ASM and that caveolin-1 contributes to regulation of [Ca²⁺]_i responses to agonist.  

3.1008           Mitochondrial P450-dependent arachidonic acid metabolism by TCDD-induced hepatic CYP1A5; conversion of EETs to DHETs by mitochondrial soluble epoxide hydrolase

Labitzke, E.M., Diani-Moore, S. and Rifkind, A.B. Arch. Biochem. Biophys., 468, 70-81 (2007)   Several P450 enzymes localized in the endoplasmic reticulum and thought to be involved primarily in xenobiotic metabolism, including mouse and rat CYP1A1 and mouse CYP1A2, have also been found to translocate to mitochondria. We report here that the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces enzymatically active CYP1A4/1A5, the avian orthologs of mammalian CYP1A1/1A2, in chick embryo liver mitochondria as well as in microsomes. P450 proteins and activity levels (CYP1A4-dependent 7-ethoxyresorufin-O-deethylase and CYP1A5-dependent arachidonic acid epoxygenation) in mitochondria were 23–40% of those in microsomes. DHET formation by mitochondria was twice that of microsomes and was attributable to a mitochondrial soluble epoxide hydrolase as confirmed by Western blotting with antiEPHX2, conversion by mitochondria of pure 11,12 and 14,15-EET to the corresponding DHETs and inhibition of DHET formation by the soluble epoxide hydrolase inhibitor, 12(-3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA). TCDD also suppressed formation of mitochondrial and microsomal 20-HETE. The findings newly identify mitochondria as a site of P450-dependent arachidonic acid metabolism and as a potential target for TCDD effects. They also demonstrate that mitochondria contain soluble epoxide hydrolase and underscore a role for CYP1A in endobiotic metabolism.    

3.1009           Improved subcellular fractionation of the heavy mitochondria pellet using Free Flow Electrophoresis system

Aboldzade-Bavil, A. et al Mitochondrion, 7, 419 (2007)   The preparative dissection of cells into their substructure reduces sample complexity and facilitates functional analysis of proteins in a physiological context. According to the high morphological variability of many subcellular compartments, however, cells inhabit different organellar subpopulations supposed to be linked to specialized biological functions or developmental stages. A prerequisite for a fundamental characterization of these organellar subpopulations on the proteome level are highly pure fractions, which could not be obtained by classical separation technologies. We developed a workflow for the subcellular fractionation of the heavy mitochondria pellet using the BD Free Flow Electrophoresis system (FFE). The FFE methodology relies on the net charge of the organelles to be separated, which is caused by protein domains extending from the membrane surface. According to charge and size, particles are deflected differently in an electric field and are separated through a buffer flow perpendicular to the electric field. With this technique a second dimension separation step, relying on physical parameters not utilized in centrifugation techniques, was added to the workflow. Successful isolations of various cellular structures have been obtained by the use of this workflow. For this reason we invented FFE as an additional purification step to purify a 2.860 g fraction of rat liver tissue which mainly contained heavy mitochondria but also peroxisomes and lysosomes, so called ‘‘high density peroxisomes and lysosomes’’. The resulting pellet was loaded on an Optiprep gradient. Thereafter, each density gradient fraction was subjected to FFE. Using this workflow we were able to purify various subcellular fractions. To validate the purity and integrity of sub-fractions we performed enzyme assays for organellar markers and immunoblots with specific antibodies as well as electron microscopy. The subcellular fractionation using the FFE-system indicated that the purification was selective on the organelle level documenting the suitability of this separation technique in proteomic research.  

3.1010           Identification of SVIP as an Endogenous Inhibitor of Endoplasmic Reticulum-associated Degradation

Ballar, P. et al

1. Biol. Chem., 282(47), 33908-33914 (2007)

  Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a process known as ER-associated degradation (ERAD), which starts with misfolded protein recognition, followed by ubiquitination, retrotranslocation to the cytosol, deglycosylation, and targeting to the proteasome for degradation. Actions of multisubunit protein machineries in the ER membrane integrate these steps. We hypothesized that regulation of the multisubunit machinery assembly is a mechanism by which ERAD activity is regulated. To test this hypothesis, we investigated the potential regulatory role of the small p97/VCP-interacting protein (SVIP) on the formation of the ERAD machinery that includes ubiquitin ligase gp78, AAA ATPase p97/VCP, and the putative channel Derlin1. We found that SVIP is anchored to microsomal membrane via myristoylation and co-fractionated with gp78, Derlin1, p97/VCP, and calnexin to the ER. Like gp78, SVIP also physically interacts with p97/VCP and Derlin1. Overexpression of SVIP blocks unassembled CD3 from association with gp78 and p97/VCP, which is accompanied by decreases in CD3 ubiquitination and degradation. Silencing SVIP expression markedly enhances the formation of gp78-p97/VCP-Derlin1 complex, which correlates with increased degradation of CD3 and misfolded Z variant of -1-antitrypsin, established substrates of gp78. These results suggest that SVIP is an endogenous inhibitor of ERAD that acts through regulating the assembly of the gp78-p97/VCP-Derlin1 complex.  

3.1011           The Confluence-dependent Interaction of Cytosolic Phospholipase A2- with Annexin A1 Regulates Endothelial Cell Prostaglandin E2 Generation

Herbert, S.P., Odell, A.F., Ponnambalam, S. and walker, J.H.

1. Biol. Chem., 282(47), 3468-3478 (2007)

  The regulated generation of prostaglandins from endothelial cells is critical to vascular function. Here we identify a novel mechanism for the regulation of endothelial cell prostaglandin generation. Cytosolic phospholipase A₂- (cPLA₂ ) cleaves phospholipids in a Ca²⁺-dependent manner to yield free arachidonic acid and lysophospholipid. Arachidonic acid is then converted into prostaglandins by the action of cyclooxygenase enzymes and downstream synthases. By previously undefined mechanisms, nonconfluent endothelial cells generate greater levels of prostaglandins than confluent cells. Here we demonstrate that Ca²⁺-independent association of cPLA₂ with the Golgi apparatus of confluent endothelial cells correlates with decreased prostaglandin synthesis. Golgi association blocks arachidonic acid release and prevents functional coupling between cPLA₂ and COX-mediated prostaglandin synthesis. When inactivated at the Golgi apparatus of confluent endothelial cells, cPLA₂ is associated with the phospholipid-binding protein annexin A1. Furthermore, the siRNA-mediated knockdown of endogenous annexin A1 significantly reverses the inhibitory effect of confluence on endothelial cell prostaglandin generation. Thus the confluence-dependent interaction of cPLA₂ and annexin A1 at the Golgi acts as a novel molecular switch controlling cPLA₂ activity and endothelial cell prostaglandin generation.  

3.1012           The Nuclear RhoA Exchange Factor Net1 Interacts with Proteins of the Dlg Family, Affects Their Localization, and Influences Their Tumor Suppressor Activity

Garcia-Mata, R. et al Mol. Cell. Biol., 27(24), 8683-8697 (2007)   Net1 is a RhoA-specific guanine nucleotide exchange factor which localizes to the nucleus at steady state. A deletion in its N terminus redistributes the protein to the cytosol, where it activates RhoA and can promote transformation. Net1 contains a PDZ-binding motif at the C terminus which is essential for its transformation properties. Here, we found that Net1 interacts through its PDZ-binding motif with tumor suppressor proteins of the Dlg family, including Dlg1/SAP97, SAP102, and PSD95. The interaction between Net1 and its PDZ partners promotes the translocation of the PDZ proteins to nuclear subdomains associated with PML bodies. Interestingly, the oncogenic mutant of Net1 is unable to shuttle the PDZ proteins to the nucleus, although these proteins still associate as clusters in the cytosol. Our results suggest that the ability of oncogenic Net1 to transform cells may be in part related to its ability to sequester tumor suppressor proteins like Dlg1 in the cytosol, thereby interfering with their normal cellular function. In agreement with this, the transformation potential of oncogenic Net1 is reduced when it is coexpressed with Dlg1 or SAP102. Together, our results suggest that the interaction between Net1 and Dlg1 may contribute to the mechanism of Net1-mediated transformation.  

3.1013           Endocytic Trafficking of Sphingomyelin Depends on Its Acyl Chain Length

Koivusalo, M., Jansen, M., Somerharju, P and Ikonen, E. Mol. Biol. Cell, 18, 5113-5123 (2007)   To study the principles of endocytic lipid trafficking, we introduced pyrene sphingomyelins (PyrSMs) with varying acyl chain lengths and domain partitioning properties into human fibroblasts or HeLa cells. We found that a long-chain, ordered-domain preferring PyrSM was targeted Hrs and Tsg101 dependently to late endosomal compartments and recycled to the plasma membrane in an NPC1- and cholesterol-dependent manner. A short-chain, disordered domain preferring PyrSM recycled more effectively, by using Hrs-, Tsg101- and NPC1-independent routing that was insensitive to cholesterol loading. Similar chain length-dependent recycling was observed for unlabeled sphingomyelins (SMs). The findings 1) establish acyl chain length as an important determinant in the endocytic trafficking of SMs, 2) implicate ESCRT complex proteins and NPC1 in the endocytic recycling of ordered domain lipids to the plasma membrane, and 3) introduce long-chain PyrSM as the first fluorescent lipid tracing this pathway.  

3.1014           Tight junctions contain oligomeric protein assembly critical for maintaining blood–brain barrier integrity in vivo

McCaffrey, G. et al

1. Neurochem., 103, 2540-2555 (2007)

  Tight junctions (TJs) are major components of the blood–brain barrier (BBB) that physically obstruct the interendothelial space and restrict paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS. TJs are dynamic structures whose intricate arrangement of oligomeric transmembrane and accessory proteins rapidly alters in response to external stressors to produce changes in BBB permeability. In this study, we investigate the constitutive trafficking of the TJ transmembrane proteins occludin and claudin-5 that are essential for forming the TJ seal between microvascular endothelial cells that inhibits paracellular diffusion. Using a novel, detergent-free OptiPrep density-gradient method to fractionate rat cerebral microvessels, we identify a plasma membrane lipid raft domain that contains oligomeric occludin and claudin-5. Our data suggest that oligomerization of occludin involves disulfide bond formation within transmembrane regions, and that assembly of the TJ oligomeric protein complex is facilitated by an oligomeric caveolin scaffold. This is the first time that distribution of oligomeric TJ transmembrane proteins within plasma membrane lipid rafts at the BBB has been examined in vivo. The findings reported in this study are critical to understand the mechanism of assembly of the TJ multiprotein complex that is essential for maintaining BBB integrity.  

3.1015           Adaptor Protein LAPF Recruits Phosphorylated p53 to Lysosomes and Triggers Lysosomal Destabilization in Apoptosis

Li, N. et al CancerRes., 67(23), 11176-11185 (2007)   Evidence suggests a functional association between the tumor suppressor p53 and apoptosis-involved organelle lysosome; however, the detailed mechanisms remain poorly understood. We recently reported that a lysosome-targeting protein, LAPF (lysosome-associated and apoptosis-inducing protein containing PH and FYVE domains), could initiate apoptosis of L929 cells through a lysosomal-mitochondrial pathway. In this study, we show that LAPF specifically interacted with phosphorylated p53 (Ser^15/18) both in vitro and in vivo, which could be enhanced by apoptotic stimuli, such as tumor necrosis factor (TNF- ) and ionizing irradiation. The PH domain of LAPF and the transactivation domain of p53 mediated the interaction between both molecules. Phosphorylated p53 (Ser^15/18) could translocate to lysosomes before lysosomal membrane permeabilization (LMP) in LAPF-initiated and TNF-induced apoptosis. Silencing of LAPF expression abrogated lysosomal translocation of phosphorylated p53 (Ser^15/18), whereas silencing of p53 expression had no effect on lysosomal translocation of LAPF. Similar to that of LAPF silencing, silencing of endogenous p53 expression in L929 cells could significantly impair TNF- –induced LMP and apoptosis. However, reexpression of wild-type p53, p53S15D (substitution of Ser¹⁵ to Asp that mimics a phosphorylated state), and p53R175H (a transcription-deficient mutant) in p53-knockdown L929 cells could rescue the decrease in TNF-induced apoptosis. The data suggest that phosphorylated p53 (Ser^15/18) might translocate to lysosome via forming complexes with adaptor protein LAPF and subsequently result in LMP and apoptosis, which might be in a transcription-independent manner.  

3.1016           EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells

Van Rheenen, J. et al

1. Cell Biol., 179(6), 1247-1259 (2007)

  Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP₂; however, this is mainly based on in vitro–binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP₂ in living cells. We show that EGF induces a rapid loss of PIP₂ through PLC activity, resulting in a releaseand activation of a membrane-bound pool of cofilin. Upon release,we find that cofilin binds to and severs F-actin, which is coincidentwith actin polymerization and lamellipod formation. Moreover,our data provide evidence for how PLC is involved in the formationof protrusions in breast carcinoma cells during chemotaxis andmetastasis towards EGF.  

3.1017           The Epithelial Sodium Channel (ENaC) Traffics to Apical Membrane in Lipid Rafts in Mouse Cortical Collecting Duct Cells

Hill, W.G. et al

1. Biol. Chem., 282(52), 37402-37411 (2007)

  We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct (_MPKCCD₁₄) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl-β-cyclodextrin added apically to _MPKCCD₁₄ cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 ± 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of _MPKCCD₁₄ cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.  

3.1018           Age-related subproteomic analysis of mouse liver and kidney peroxisomes

Mi, J., Garcia-Arcos, I., Alvarez, R. and Cristobal, S. Proteome Science, 5, 19- (2007)   Background Despite major recent advances in the understanding of peroxisomal functions and how peroxisomes arise, only scant information is available regarding this organelle in cellular aging. The aim of this study was to characterize the changes in the protein expression profile of aged versus young liver and kidney peroxisome-enriched fractions from mouse and to suggest possible mechanisms underlying peroxisomal aging. Peroxisome-enriched fractions from 10 weeks, 18 months and 24 months C57bl/6J mice were analyzed by quantitative proteomics. Results Peroxisomal proteins were enriched by differential and density gradient centrifugation and proteins were separated by two-dimensional electrophoresis (2-DE), quantified and identified by mass spectrometry (MS). In total, sixty-five proteins were identified in both tissues. Among them, 14 proteins were differentially expressed in liver and 21 proteins in kidney. The eight proteins differentially expressed in both tissues were involved in b-oxidation, a-oxidation, isoprenoid biosynthesis, amino acid metabolism, and stress response. Quantitative proteomics, clustering methods, and prediction of transcription factors, all indicated that there is a decline in protein expression at 18 months and a recovery at 24 months. Conclusions These results indicate that some peroxisomal proteins show a tissue-specific functional response to aging. This response is probably dependent on their differential regeneration capacity. The differentially expressed proteins could lead several cellular effects: such as alteration of fatty acid metabolism that could alert membrane protein functions, increase of the oxidative stress and contribute to decline in bile salt synthesis. The ability to detect age-related variations in the peroxisomal proteome can help in the search for reliable and valid aging biomarkers.  

3.1019           Knockdown of NHERF1 Enhances Degradation of Temperature Rescued F508 CFTR from the Cell Surface of Human Airway Cells

Kwon, S-H., Pollard, H. and Giggino, W.B. Cell. Physiol. Biochem., 20, 763-772 (2007)   DF508 CFTR can be functionally restored in the plasma membrane by exposure of the cell to lower temperature. However, restored F508 CFTR has a much shorter half-life than normal. We studied whether NHERF1, which binds to the PDZ motif of CFTR, might be a critical mediator in the turnover of F508 CFTR from the cell surface. We used RNAi to reduce the expression of NHERF1 in human airway epithelial cells. Knockdown of NHERF1 reversibly reduces surface expression of WT-CFTR without altering its total expression. As expected, temperature correction increased mature C band F508 CFTR (r F508) but unexpectedly allowed immature B band of r F508 to traffic to the cell surface. Both surface and total expression of r F508 in NHERF1 knockdown cells were reduced and degradation of surface localized r F508 was even faster in NHERF1 knockdown cells. Proteasomal and lysosomal inhibitor treatments led to a significant decrease in the accelerated degradation of surface r F508 in NHERF1 knockdown cells. These results indicate that NHERF1 plays a role in the turnover of CFTR at the cell surface, and that r F508 CFTR at the cell surface remains highly susceptible to degradation.      

3.1020           Diversity of Raft-Like Domains in Late Endosomes

Sobo, K., Chevallier, J., Parton, R.G., Gruenberg, J. and Giscou van der Goot, F. PloS One, 4, e391 (2007)   Late endosomes, the last sorting station in the endocytic pathway before lysosomes, are pleiomorphic organelles composed of tubular elements as well as vesicular regions with a characteristic multivesicular appearance, which play a crucial role in intracellular trafficking. Here, we have investigated whether, in addition to these morphologically distinguishable regions, late endosomal membranes are additionally sub-compartmentalized into membrane microdomains. Using sub-organellar fractionation techniques, both with and without detergents, combined with electron microscopy, we found that both the limiting membrane of the organel and the intraluminal vesicles contain raft-type membrane domains. Interestingly, these differentially localized domains vary in protein composition and physico-chemical properties. In addition to the multivesicular organization, we find that late endosomes contain cholesterol rich microdomains both on their limiting membrane and their intraluminal vesicles that differ in composition and properties. Implications of these findings for late endosomal functions are discussed.  

3.1021           Late Endosomal Cholesterol Accumulation Leads to Impaired Intra-Endosomal Trafficking

Sobo, K. et al PloS One, 9, e851 (2007)   Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics. Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2–3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus. These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation.  

3.1022           Hsp90 Inhibition Decreases Mitochondrial Protein Turnover

Margineantu, D.H., Emerson, C.B., Diaz, D. and Hockenbery, D.M. PloS One, 10, e1066 (2007)   Background Cells treated with hsp90 inhibitors exhibit pleiotropic changes, including an expansion of the mitochondrial compartment, accompanied by mitochondrial fragmentation and condensed mitochondrial morphology, with ultimate compromise of mitochondrial integrity and apoptosis. Findings We identified several mitochondrial oxidative phosphorylation complex subunits, including several encoded by mtDNA, that are upregulated by hsp90 inhibitors, without corresponding changes in mRNA abundance. Post-transcriptional accumulation of mitochondrial proteins observed with hsp90 inhibitors is also seen in cells treated with proteasome inhibitors. Detailed studies of the OSCP subunit of mitochondrial F1F0-ATPase revealed the presence of mono- and polyubiquitinated OSCP in mitochondrial fractions. We demonstrate that processed OSCP undergoes retrotranslocation to a trypsin-sensitive form associated with the outer mitochondrial membrane. Inhibition of proteasome or hsp90 function results in accumulation of both correctly targeted and retrotranslocated mitochondrial OSCP. Conclusions Cytosolic turnover of mitochondrial proteins demonstrates a novel connection between mitochondrial and cytosolic compartments through the ubiquitin-proteasome system. Analogous to defective protein folding in the endoplasmic reticulum, a mitochondrial unfolded protein response may play a role in the apoptotic effects of hsp90 and proteasome inhibitors.  

3.1023           Kv11.1 (ERG1) K⁺ Channels Localize in Cholesterol and Sphingolipid Enriched Membranes and Are Modulated by Membrane Cholesterol

Balijepalli, R.C. et al Channels, 14, 263-272 (2007)   The localization of ion channels to specific membrane microdomains can impact the functional properties of channels and their role in cellular physiology. We determined the membrane localization of human Kv11.1 (hERG1) α -subunit protein, which underlies the rapidly activating, delayed rectifier K+ current (IKr) in the heart. Immunocytochemistry and membrane fractionation using discontinuous sucrose density gradients of adult canine ventricular tissue showed that Kv11.1 channel protein localized to both the cell surface and T-tubular sarcolemma. Furthermore, density gradient membrane fractionation using detergent (Triton X-100) and non-detergent (OptiPrep) methods from canine ventricular myocytes or HEK293 cells demonstrated that Kv11.1 protein, along with MiRP1 and Kv7.1 (KCNQ1) proteins, localize in cholesterol and sphingolipid enriched membrane fractions. In HEK293 cells, Kv11.1 channels, but not long QT-associated mutant G601S-Kv11.1 channels, also localized to cholesterol and sphingolipid enriched membrane fractions. Depletion of membrane cholesterol from HEK293 cells expressing Kv11.1 channels using methyl-Mβ -cyclodextrin (Mβ CD) caused a positive shift of the voltage dependence of activation and an acceleration of deactivation kinetics of Kv11.1 current (IKv11.1). Cholesterol loading of HEK293 cells reduced the steep voltage dependence of IKv11.1 activation and accelerated the inactivation kinetics of IKv11.1. Incubation of neonatal mouse myocytes in Mβ CD also accelerated the deactivation kinetics of IKr. We conclude that Kv11.1 protein localizes in cholesterol and sphingolipid enriched membranes and that membrane cholesterol can modulate IKv11.1 and IKr.  

3.1024           Association of Botulinum Neurotoxin Serotypes A and B with Synaptic Vesicle Protein Complexes

Baldwin, M.R. and Barbieri, J.T. Biochemistry, 46(11), 3200-3210 (2007)   Botulinum neurotoxins (BoNTs) elicit flaccid paralysis through cleavage of SNARE proteins within peripheral neurons. There are seven serotypes of the BoNTs, termed A-G, which differ in the SNARE protein and/or site that is cleaved. BoNTs are single-chain toxins that comprise an N-terminal zinc metalloprotease domain that is disulfide linked to the C-terminal translocation/receptor binding domain. SV2 and synaptotagmin have been identified as receptors for BoNT serotypes A and B, respectively. Using affinity chromatography, BoNTs A and B were observed to bind synaptic vesicle protein complexes in synaptosome lysates. Tandem LC-MS/MS identified SV2, synaptotagmin I, synaptophysin, vesicle-associated membrane protein 2 (VAMP2), and the vacuolar proton pump as components of the BoNT-receptor complex. Density gradient analysis showed that BoNT serotypes A and B exhibited unique interactions with the synaptic vesicle protein complexes. The association of BoNT serotypes A and B with synaptic vesicle protein complexes implicates a physiological role for protein complexes in synaptic vesicle biology and provides insight into the interactions of BoNT and neuronal receptors.  

3.1025           Vascular Endothelial Growth Factor Receptor-3 Activity Is Modulated by Its Association with Caveolin-1 on Endothelial Membrane

Galvagni, F. et al Biochemistry, 46(13), 3998-4005 (2007)   Vascular endothelial growth factor receptor-3 (VEGFR-3) is constitutively expressed in lymphatic vessels and transiently in endothelial cells of blood vessels during angiogenesis. Here we report that VEGFR-3 localizes in the caveolae membrane of endothelial cells and co-immunoprecipitates with caveolin-1. Caveolin-1 silencing or its depletion from the cell membrane by cholesterol increases VEGFR-3 autophosphorylation, suggesting that caveolin acts as a negative regulator of VEGFR-3 activity. Receptor activation induces caveolin-1 phosphorylation on tyrosine residues including tyrosine 14. Cell treatment with Src or Abl inhibitors PP2 or STI571, prior to receptor stimulation, affects caveolin-1 phosphorylation without affecting receptor autophosphorylation, suggesting that both Src and Abl are involved in VEGFR-3-dependent caveolin-1 phosphorylation. Caveolin-1 phosphorylation in Src/Fyn/Yes knockout cells demonstrated that Abl phosphorylates caveolin-1 independently from Src family members. These results suggest a functional interaction between VEGFR-3 and caveolin-1 to modulate endothelial cell activation during angiogenesis.  

3.1026           Role of the Hydrophobic Segment of Diacylglycerol Kinase

Dicu, A.O., Topham, M.K., Ottaay, L. and Epand, R.M. Biochemistry, 46(20), 6109-6117 (2007)   Diacylglycerol kinase (DGK ) is unique among mammalian DGK isoforms in having a segment of hydrophobic amino acids. We have evaluated the contributions of this segment to the membrane interactions and functions of this protein. To test the role of the hydrophobic segment, we have compared the properties of DGK with those of a truncated form of the protein (DGK ) lacking the 40 N-terminal amino acids, which includes the hydrophobic segment. The proteins were expressed in COS-7 cells from a gene for human DGK or from a gene for a truncated form (DGK ), both of which had a FLAG tag at the amino terminus. Full-length FLAG-DGK and truncated FLAG-DGK were both more specific for 1-stearoyl-2-arachidonoyl-sn-glycerol than for 1,2-dioleoyl-sn-glycerol. 1-Stearoyl-2-linoleoyl-sn-glycerol exhibited intermediate specificity for both forms of the enzyme. The results show that the truncated form of the enzyme maintains substrate specificity for lipids with an arachidonoyl moiety present at the sn-2 position. The truncation increases the catalytic rate constant for all three substrates and may suggest a role in the negative regulation of this enzyme. A full-length DGK with a C-terminal His tag exhibited substrate specificity similar to that of the other two forms of the enzyme, indicating that the nature and position of the epitope tag did not strongly affect this property. Using an ultracentrifugation floatation assay, we showed that at neutral pH DGK is extracted with 1.5 M KCl while DGK remains essentially fully membrane bound. The full-length protein had a weak tendency to oligomerize in the presence of weak detergents. DGK was monomeric on SDS-PAGE but exhibited partial dimerization with low concentrations of perfluorooctanoic acid. The major conclusions of this work are that the hydrophobic domain of DGK does not contribute to substrate specificity but plays a role in permanently sequestering the enzyme to a membrane.  

3.1027           Lipid Interaction Converts Prion Protein to a PrP^Sc-like Proteinase K-Resistant Conformation under Physiological Conditions

Wang, F. et al Biochemistry, 46(23), 7045-7053 (2007)   The conversion of prion protein (PrP) to the pathogenic PrP^Sc conformation is central to prion disease. Previous studies revealed that PrP interacts with lipids and the interaction induces PrP conformational changes, yet it remains unclear whether in the absence of any denaturing treatment, PrP-lipid interaction is sufficient to convert PrP to the classic proteinase K-resistant conformation. Using recombinant mouse PrP, we analyzed PrP-lipid interaction under physiological conditions and followed lipid-induced PrP conformational change with proteinase K (PK) digestion. We found that the PrP-lipid interaction was initiated by electrostatic contact and followed by hydrophobic interaction. The PrP-lipid interaction converted full-length -helix-rich recombinant PrP to different forms. A significant portion of PrP gained a conformation reminiscent of PrP^Sc, with a PrP^Sc-like PK-resistant core and increased -sheet content. The efficiency for lipid-induced PrP conversion depended on lipid headgroup structure and/or the arrangement of lipids on the surface of vesicles. When lipid vesicles were disrupted by Triton X-100, PrP aggregation was necessary to maintain the lipid-induced PrP^Sc-like conformation. However, the PK resistance of lipid-induced PrP^Sc-like conformation does not depend on amyloid fiber formation. Our results clearly revealed that the lipid interaction can overcome the energy barrier and convert full-length -helix-rich PrP to a PrP^Sc-like conformation under physiological conditions, supporting the relevance of lipid-induced PrP conformational change to in vivo PrP conversion.  

3.1028           Human ABCB6 Localizes to Both the Outer Mitochondrial Membrane and the Plasma Membrane

Paterson, J.K. et al Biochemistry, 46(33), 9443-9452 (2007)   Expression of the ATP-binding cassette transporter ABCB6 has been associated with multiple cellular functions, including resistance to several cytotoxic agents, iron homeostasis, and porphyrin transport. To further elucidate its physiological function and/or role in drug resistance, we determined the subcellular location of ABCB6. Using three novel ABCB6-specific antibodies, Western blot analysis of cells expressing cDNA-derived or endogenous ABCB6 revealed two distinct molecular weight forms. Confocal microscopy indicates that the protein localizes to both mitochondria and the plasma membrane. Differential centrifugation revealed that the lower molecular weight form predominantly resides in the mitochondria, while the larger protein form is more abundant in the plasma membrane. Preliminary studies indicate that ABCB6 is functionally relevant in the plasma membrane, where its expression prevents the accumulation of specific porphyrins in the cell. Digitonin solubilization of mitochondria demonstrated that ABCB6 is present in the outer mitochondrial membrane, while back-titration assays with the ABCB6-specific antibodies reveal that the nucleotide binding domain of ABCB6 is cytoplasmic. These studies are the first to demonstrate that ABCB6 exists in two molecular weight forms, is localized to both the outer mitochondrial membrane and the plasma membrane, and plays a functional role in the plasma membrane.  

3.1029           Subcellular Proteome Analysis of Camptothecin Analogue NSC606985-Treated Acute Myeloid Leukemic Cells

Yu, Y. et al

1. Proteome Res., 6(9), 3808-3818 (2007)

  We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase C . Here, we analyzed protein expression profiles of fractionated nuclei, mitochondria, raw endoplasmic reticula, and cytosols of NSC606985-induced apoptotic AML cell line NB4 cells by two-dimensional electrophoresis combined with MALDI-TOF/TOF tandem mass spectrometry. In total, 90 unique deregulated proteins, including 16 compartment-compartment translocated ones, were identified. They contributed to multiple functional activities such as DNA damage repairing, chromosome assembly, mRNA processing, biosynthesis, modification, and degradation of proteins. More interestingly, several increased oxidative stress-related proteins mainly presented in mitochondria, while upregulated glycolysis proteins mainly occurred in the nuclei. With their functional analyses, the possible roles of these deregulated proteins in NSC606985-induced apoptosis were discussed. Collectively, these discoveries would shed new insights for systematically understanding the mechanisms of the camptothecin-induced apoptosis.  

3.1030           Proteomic Complex Detection Using Sedimentation

Hartman, N.T., Sicilia, F., Lilley, K.S. and Dupree, P. Anal. Chem., 79(5), 2078-2083 (2007)   Protein-protein interactions are important in many cellular processes, but there are still relatively few methods to screen for novel protein complexes. Here we present a quantitative proteomics technique called ProCoDeS (Proteomic Complex Detection using Sedimentation) for profiling the sedimentation of a large number of proteins through a rate zonal centrifugation gradient. Proteins in a putative complex can be identified since they sediment faster than predicted from their monomer molecular weight. Using solubilized mitochondrial membrane proteins from Arabidopsis thaliana, the relative protein abundance in fractions of a rate zonal gradient was measured with the isotopic labeling reagent ICAT and electrospray mass spectrometry. Subunits of the same protein complex had very similar gradient distribution profiles, demonstrating the reproducibility of the quantitation method. The approximate size of the unknown complex can be inferred from its sedimentation rate relative to known protein complexes. ProCoDeS will be of use in screening extracts of tissues, cells, or organelle fractions to identify specific proteins in stable complexes that can be characterized by subsequent targeted techniques such as affinity tagging.  

3.1031           PINK1 Protects against Oxidative Stress by Phosphorylating Mitochondrial Chaperone TRAP1

Pridgeon, J.W., Olzmann, J.A., Chin, L-S. and Li, L. PloS Biology, 5(7), 1494-1503 (2007)   Mutations in the PTEN induced putative kinase 1 (PINK1) gene cause an autosomal recessive form of Parkinson disease (PD). So far, no substrates of PINK1 have been reported, and the mechanism by which PINK1 mutations lead to neurodegeneration is unknown. Here we report the identification of TNF receptor-associated protein 1 (TRAP1), a mitochondrial molecular chaperone also known as heat shock protein 75 (Hsp75), as a cellular substrate for PINK1 kinase. PINK1 binds and colocalizes with TRAP1 in the mitochondria and phosphorylates TRAP1 both in vitro and in vivo. We show that PINK1 protects against oxidative-stress-induced cell death by suppressing cytochrome c release from mitochondria, and this protective action of PINK1 depends on its kinase activity to phosphorylate TRAP1. Moreover, we find that the ability of PINK1 to promote TRAP1 phosphorylation and cell survival is impaired by PD-linked PINK1 G309D, L347P, and W437X mutations. Our findings suggest a novel pathway by which PINK1 phosphorylates downstream effector TRAP1 to prevent oxidative-stress-induced apoptosis and implicate the dysregulation of this mitochondrial pathway in PD pathogenesis.  

3.1032           Distinct role of clathrin-mediated endocytosis in the functional uptake of cholera toxin

Vanden Broeck, D., Lagrou, A.R. and De Wolf, M.J.S. Acta Biochimica Polonica, 54(4), 757-767 (2007)   The involvement of the clathrin-mediated endocytic internalization route in the uptake of cholera toxin (CT) was investigated using different cell lines, including the human intestinal Caco-2 and T84 cell lines, green monkey Vero cells, SH-SY5Y neuroblastoma cells and Madin-Darby canine kidney cells. Suppression of the clathrin-mediated endocytic pathway by classical biochemical procedures, like intracellular acidification and potassium depletion, inhibited cholera toxin uptake by up to about 50% as well as its ability to raise intracellular levels of cAMP. Also prior exposure of these cell types to the cationic amphiphilic drug chlorpromazine reduced the functional uptake of cholera toxin, even to a greater extent. These effects were dose- and cell type-dependent, suggesting an involvement of clathrin-mediated endocytosis in the functional uptake of cholera toxin. For a more straightforward approach to study the role of the clathrin-mediated uptake in the internalization of cholera toxin, a Caco-2^eps- cell line was exploited. These Caco-2^eps- cells constitutively suppress the expression of epsin, an essential accessory protein of clathrin-mediated endocytosis, thereby selectively blocking this internalization route. CT uptake was found to be reduced by over 60% in Caco-2^eps- paralleled by a diminished ability of CT to raise the level of cAMP. The data presented suggest that the clathrin-mediated uptake route fulfils an important role in the functional internalization of cholera toxin in several cell types.  

3.1033           Quantitative proteomic comparison of mouse peroxisomes from liver and kidney

Mi, J., Kirchner, E. and Cristobal, S. Proteomics, 7(11), 1916-1928 (2007)   The peroxisome plays a central role in the catabolic and anabolic pathways that contribute to the lipid homeostasis. Besides this main function, this organelle has gained functional diversity. Although several approaches have been used for peroxisomal proteome analysis, a quantitative protein expression analysis of peroxisomes from different tissues has not been elucidated yet. Here, we applied a 2-DE-based method on mouse liver and kidney peroxisomal enriched fractions to study the tissue-dependent protein expression. Ninety-one spots were identified from the 2-DE maps from pH 3.0–10.0 and 51 spots from the basic range corresponding to 31 peroxisomal proteins, 10 putative peroxisomal, 6 cytosolic, 17 mitochondrial and 1 protein from endoplasmic reticulum. Based on the identification and on the equivalent quality of both tissue preparations, the differences emerging from the comparison could be quantified. In liver, proteins involved in pathways such as - and β-oxidation, isoprenoid biosynthesis, amino acid metabolism and purine and pirimidine metabolism were more abundant whereas in kidney, proteins from the straight-chain fatty acid β-oxidation were highly expressed. These results indicate that tissue-specific functional classes of peroxisomal proteins could be relevant to study peroxisomal cellular responses or pathologies. Finally, a web-based peroxisomal proteomic database was built.  

3.1034           Analysis of the linkage of MYRIP and MYO7A to melanosomes by RAB27A in retinal pigment epithelial cells

Klomp, A.E., teofilo, K., Legacki, E. and Williams, D.S. Cell Motil. Cytoskeleton, 64(6), 474-487 (2007)   The apical region of the retinal pigment epithelium (RPE) typically contains melanosomes. Their apical distribution is dependent on RAB27A and the unconventional myosin, MYO7A. Evidence from studies using in vitro binding assays, melanocyte transfection, and immunolocalization have indicated that the exophilin, MYRIP, links RAB27A on melanosomes to MYO7A, analogous to the manner that melanophilin links RAB27A on melanocyte melanosomes to MYO5A. To test the functionality of this hypothesis in RPE cells, we have examined the relationship among MYRIP, RAB27A and MYO7A with studies of RPE cells in primary culture (including live-cell imaging), analyses of mutant mouse retinas, and RPE cell fractionation experiments. Our results indicate that the retinal distribution of MYRIP is limited to the RPE, mainly the apical region. In RPE cells, RAB27A, MYRIP, and MYO7A were all associated with melanosomes, undergoing both slow and rapid movements. Analyses of mutant mice provide genetic evidence that MYRIP is linked to melanosomes via RAB27A, but show that recruitment of MYRIP to apical RPE is independent of melanosomes and RAB27A. RAB27A and MYRIP also associated with motile small vesicles of unknown origin. The present results provide evidence from live RPE cells that the RAB27A-MYRIP-MYO7A complex functions in melanosome motility. They also demonstrate that RAB27A provides an essential link to the melanosome.  

3.1035           Reduced raft-association of NF155 in active MS-lesions is accompanied by the disruption of the paranodal junction

Maier, O., Baron, W. and Hoekstra, D. GLIA, 55(8), 885-895 (2007)   Neurofascin155 (NF155) is required for the establishment of the paranodal axo-glial junction, the predominant interaction site between myelin and axon. It has been shown that the distribution of NF155 is altered in demyelinating diseases such as multiple sclerosis (MS). However, little is known about the biochemical mechanisms underlying these changes. We therefore compared NF155 in postmortem tissue of active and chronic inactive MS lesions with white matter from healthy controls. Although NF155 showed a very similar expression in all control white matter samples, a strong individual variation was observed in MS-lesions with NF155-levels reduced in most samples. At the same time an NF155-fragment was increased in MS-lesions, suggesting that NF155 is subject to protein degradation in lesion sites. Interestingly, the association of NF155 to membrane microdomains (rafts) was reduced in all lesions, irrespective of the amount of NF155, indicating that membrane association of NF155 was generally affected. Therefore, myelin fractionation experiments were performed to analyze the fate of paranodal proteins during demyelination. Although NF155 was enriched in heavy myelin from both control white matter and active MS-lesions, association of Caspr1/paranodin with heavy myelin was abolished in MS-lesions, demonstrating that paranodal junctions are disrupted. In conclusion, the data support the hypothesis that efficient raft-association of NF155 is essential for the assembly of the paranodal junction and demonstrate that reduced association of NF155 to lipid rafts is accompanied by the disassembly of the paranodal junction and thus contributes to the demyelination process in MS.  

3.1036           Mitochondrial association of myocilin, product of a glaucoma gene, in human trabecular meshwork cells

Sakai, H., Shen, X., Koga, T., Park, B-C., Noskina, Y., Tibudan, M. and Yue, B.Y.J.T.

1. Cell. Physiol., 213(3), 775-784 (2007)

  The trabecular meshwork (TM), an ocular tissue next to the cornea, is a major site for regulation of the aqueous humor outflow. Malfunctioning of this tissue is believed to be responsible for development of glaucoma, a major blinding disease. Myocilin is a gene directly linked to the most common form of glaucoma. Its protein product has been localized to both intra- and extra-cellular sites in TM cells. This study was to investigate the association of myocilin with mitochondria in TM cells. In vitro mitochondrial import assays showed that myocilin was imported to the TM mitochondria, targeting to mitochondrial membranes and/or the intermembrane space. The targeting was mediated mostly via the amino-terminal region of myocilin. When myocilin expression was induced either by treatment with dexamethasone or transfection with a myocilin construct, the mitochondrial membrane potential in TM cells, as assessed by JC-1 staining, was lowered. Subcellular fractionation and Western blot analyses confirmed that a portion of myocilin sedimented with the mitochondrial fractions. Upon anti-Fas treatment to provoke apoptosis, an increase of myocilin distribution in cytosolic fraction was observed, suggesting that myocilin was partially released from mitochondrial compartments. These results confirmed the association of myocilin with TM cell mitochondria and indicated that myocilin may have a proapoptotic role in TM cells.  

3.1037           Assembly and budding of a hepatitis B virus is mediated by a novel type of intracellular vesicles

Mhamdi, M., Funk, A., Hohenberg, H., Will, H. and Sirma, H. Hepatology, 46(1), 95-106 (2007)   Formation of enveloped viruses involves assembly and budding at cellular membranes. In this study, we elucidated the morphogenesis of hepadnaviruses on the ultrastructural and biochemical level using duck hepatitis B virus (DHBV) as a model system. Formation of virus progeny initiates at the endoplasmic reticulum (ER) and is conserved both in vitro and in vivo. The morphogenesis proceeds via membrane-surrounded vesicles containing both virions and subviral particles, indicating a common morphogenetic pathway. The virus particle–containing vesicles (VCVs) are generated and maintained by reorganization of endomembranes accompanied by a striking disorganization of the rough ER (rER). VCVs are novel organelles with unique identity and properties of ER, intermediate compartment, endosomes, and multivesicular bodies. VCVs are dynamic structures whose size and shape are regulated by both membrane fusion and fission. Conclusion: Our data indicate a strong reorganization of endomembranes during DHBV infection, resulting in the biogenesis of novel organelles serving as multifunctional platforms for assembly and budding of virus progeny.  

3.1038           Nondetergent Isolation of Rafts

Shah, M.B. and Sehgal, P.B. Methods Mol. Biol., 398, 21-28 (2007)   Raft and caveolar microdomains have been proposed to participate in numerous cellular functions including signal transduction, cholesterol trafficking, and vesicular sorting. Traditional methods of isolation of rafts from cultured cells and tissue samples have exploited the biochemical properties of these microdomains, i.e., their relative resistance to solubilization by nonionic detergents (at 4°C) and their light buoyant density attributable to their high content of cholesterol and sphingolipids. Thus, a common way to isolate raft microdomains has been their separation on a density gradient in the presence of 0.5–1% Triton X-100 (Bochringer Mannheim Roche Applied Sciences Indianapolis, IN or Sigma-Aldrich, St. Louis, MO). This and other detergent-based methods have been discussed. However, the use of detergents may not be favorable because of artifacts that may arise with their use. (The possibility of rafts solely as detergent-induced artifacts appears to have been diffused by a number of biochemical and biophysical studies that strongly demonstrate the presence of a liquid-ordered phase within biological membranes.) In this chapter, three methods are reviewed to isolate rafts from cultured cells without the use of detergents. Two of these, the sodium carbonate and OptiPrep™ (Sigma-Aldrich St. Louis, MO) methods, are based on gradient separation and can be used to isolate rafts in general, whereas the third is a magnetic-bead immunoisolation approach and might be used to isolate subpopulations of rafts enriched for different markers such as caveolin-1, flotillin (reggie proteins), or other suitable markers. Together these methods allow for a detergent-free isolation of rafts for biochemical, proteomic, and microscopic studies.  

3.1039           Proteomics-Based Method for Risk Assessment of Peroxisome Proliferating Pollutants in the Marine Environment

Cristobal, S. Methods Mol. Biol., 410, 123-135 (2007)   Pollution in aquatic environment is of increasing concern for its impact on both human and natural populations. Applying proteomics to monitor marine pollution is a new approach to evaluate the effects of environmental pollutants on the biota. Aquatic organisms living in coastal and estuarine areas are particularly prone to exposures to a variety of pollutants, some of which can act as peroxisome proliferators. However, peroxisomal responses in particular and biomarker responses in general can be influenced by several biotic and abiotic factors. Utilizing proteomics-based techniques that permit the evaluation of hundreds to thousands of proteins in a single experiment can circumvent those drawbacks. Applying this method, the peroxisomal proteome from digestive glands of mussels Mytilus sp. can be analyzed by two-dimensional electrophoresis (2-DE) and the 2-DE maps from control samples and samples obtained in a polluted area can be compared. The up- and down-regulated proteins compose the protein expression signature (PES) associated with exposure to peroxisome proliferating pollutants. This method generates highly reproducible patterns that can be applied to laboratory or field experiments.  

3.1040           Regulation of Notch Signaling by Dynamic Changes in the Precision of S3 Cleavage of Notch-1

Tagami, S. et al Mol. Cell. Biol., 28(1), 165-176 (2008)   Intramembrane proteolysis by presenilin-dependent -secretase produces the Notch intracellular cytoplasmic domain (NCID) and Alzheimer disease-associated amyloid-β. Here, we show that upon Notch signaling the intracellular domain of Notch-1 is cleaved into two distinct types of NICD species due to diversity in the site of S3 cleavage. Consistent with the N-end rule, the S3-V cleavage produces stable NICD with Val at the N terminus, whereas the S3-S/S3-L cleavage generates unstable NICD with Ser/Leu at the N terminus. Moreover, intracellular Notch signal transmission with unstable NICDs is much weaker than that with stable NICD. Importantly, the extent of endocytosis in target cells affects the relative production ratio of the two types of NICD, which changes in parallel with Notch signaling. Surprisingly, substantial amounts of unstable NICD species are generated from the Val Gly and the Lys Arg mutants, which have been reported to decrease S3 cleavage efficiency in cultured cells. Thus, we suggest that the existence of two distinct types of NICD points to a novel aspect of the intracellular signaling and that changes in the precision of S3 cleavage play an important role in the process of conversion from extracellular to intracellular Notch signaling.

3.1041           Protein-sphingolipid interactions within cellular membranes

Haberkant, P. et al

1. Lipid Res., 49, 251-262 (2008)

  Each intracellular organelle critically depends on maintainingits specific lipid composition that in turn contributes to thebiophysical properties of the membrane. With our knowledge increasingabout the organization of membranes with defined microdomainsof different lipid compositions, questions arise regarding themolecular mechanisms that underlie the targeting to/segregationfrom microdomains of a given protein. In addition to specificlipid-transmembrane segment interactions as a basis for partitioning,the presence in a given microdomain may alter the conformationof proteins and, thus, the activity and availability for regulatorymodifications. However, for most proteins, the specific lipidenvironment of transmembrane segments as well as its relevanceto protein function and overall membrane organization are largelyunknown. To help fill this gap, we have synthesized a novelphotoactive sphingolipid precursor that, together with a precursorfor phosphoglycerolipids and with photo-cholesterol, was investigatedin vivo with regard to specific protein transmembrane span-lipidinteractions. As a proof of principle, we show specific labelingof the ceramide transporter with the sphingolipid probe anddescribe specific in vivo interactions of lipids with caveolin-1,phosphatidylinositol transfer protein β, and the matureform of nicastrin. This novel photolabile sphingolipid probeallows the detection of protein-sphingolipid interactions withinthe membrane bilayer of living cells.  

3.1042           Cytoplasmic Domain of Influenza B Virus BM2 Protein Plays Critical Roles in Production of Infectious Virus

Imai, M., Kawasaki, K. and Odagiri, T.

1. Virol., 82(2), 728-739 (2008)

  Influenza B virus BM2 is a type III integral membrane protein that displays H⁺ ion channel activity. Analysis of BM2 knockout mutants has suggested that this protein is a necessary component for the capture of M1-viral ribonucleoprotein (vRNP) complex at the plasma membrane and for incorporation of vRNP complex into the virion during the assembly process. BM2 comprises 109 amino acid residues and possesses a longer cytoplasmic domain than the other 3 integral membrane proteins (hemagglutinin, neuraminidase, and NB). To explore whether the cytoplasmic domain of BM2 is important for infectious virus production, a series of BM2 deletion mutants lacking three to nine amino acid residues at the carboxyl terminus, BM2 107-109, BM2 104-109, and BM2 101-109, was generated by reverse genetics. Intracellular transport and incorporation into virions were indistinguishable between truncated BM2 proteins and wild-type BM2. The BM2 107-109 mutant produced levels of infectious virus similar to those of wild-type virus and displayed a spherical shape. However, the BM2 104-109 and BM2 101-109 mutants produced viruses containing dramatically reduced vRNP complex, as with BM2 knockout mutants, and formed enlarged, irregularly shaped virions. Moreover, gradient separation of membranes indicated that membrane association of M1 from mutants was greatly affected by carboxyl-terminal truncations of BM2. Studies of alanine substitution mutants further suggested that amino acid sequences in the 98-109 region are variable while those in the 86-97 region are a prerequisite for innate BM2 function. These results indicate that the cytoplasmic domain of the BM2 protein is required for firm association of the M1 protein with lipid membranes, vRNP complex incorporation into virions, and virion morphology.  

3.1043           Mint3/X11 Is an ADP-Ribosylation Factor-dependent Adaptor that Regulates the Traffic of the Alzheimer's Precursor Protein from the Trans-Golgi Network

Shrivastava-Ranjan, P. et al Mol. Biol. Cell, 19, 51-64 (2008)   β-Amyloid peptides (Aβ) are the major component of plaques in brains of Alzheimer's patients, and are they derived from the proteolytic processing of the β-amyloid precursor protein (APP). The movement of APP between organelles is highly regulated, and it is tightly connected to its processing by secretases. We proposed previously that transport of APP within the cell is mediated in part through its sorting into Mint/X11-containing carriers. To test our hypothesis, we purified APP-containing vesicles from human neuroblastoma SH-SY5Y cells, and we showed that Mint2/3 are specifically enriched and that Mint3 and APP are present in the same vesicles. Increasing cellular APP levels increased the amounts of both APP and Mint3 in purified vesicles. Additional evidence supporting an obligate role for Mint3 in traffic of APP from the trans-Golgi network to the plasma membrane include the observations that depletion of Mint3 by small interference RNA (siRNA) or mutation of the Mint binding domain of APP changes the export route of APP from the basolateral to the endosomal/lysosomal sorting route. Finally, we show that increased expression of Mint3 decreased and siRNA-mediated knockdowns increased the secretion of the neurotoxic β-amyloid peptide, Aβ_1-40. Together, our data implicate Mint3 activity as a critical determinant of post-Golgi APP traffic.  

3.1044           Localization of phosphorylated B-crystallin to heart mitochondria during ischemia-reperfusion

Jin, J-K. et al

Am. J. Physiol. Heart Circ. Physiol., 294, H337-H344 (2008)   The cytosolic small heat shock protein B-crystallin ( BC) is a molecular chaperone expressed in large quantities in the heart, where it protects from stresses such as ischemia-reperfusion (I/R). Upon I/R, p38 MAP kinase activation leads to phosphorylation of BC on Ser⁵⁹ (P- BC-S59), which increases its protective ability. BC confers protection, in part, by interacting with and affecting the functions of key components in stressed cells. We investigated the hypothesis that protection from I/R damage in the heart by P- BC-S59 can be mediated by localization to mitochondria. We found that P- BC-S59 localized to mitochondria isolated from untreated mouse hearts and that this localization increased more than threefold when the hearts were subjected to ex vivo I/R. Mitochondrial P- BC-S59 decreased when hearts were treated with the p38 inhibitor SB-202190. Moreover, SB-202190-treated hearts exhibited more tissue damage and less functional recovery upon reperfusion than controls. I/R activates mitochondrial permeability transition (MPT) pore opening, which increases cell damage. We found that mitochondria incubated with a recombinant mutant form of BC that mimics P- BC-S59 exhibited decreased calcium-induced MPT pore opening. These results indicate that mitochondria may be among the key components in stressed cells with which P- BC-S59 interacts and that this localization may protect the myocardium, in part, by modulating MPT pore opening and, thus, reducing I/R injury.  

3.1045           Lpx1p is a peroxisomal lipase required for normal peroxisome morphology

Thoms, S., Debelyy, M.O., Nau, K., Meyer, H.E. and Erdmann, R. FEBS J., 275, 504-514 (2008)   Lpx1p (systematic name: Yor084wp) is a peroxisomal protein from Saccharomyces cerevisiae with a peroxisomal targeting signal type 1 (PTS1) and a lipase motif. Using mass spectrometry, we have identified Lpx1p as present in peroxisomes, and show that Lpx1p import is dependent on the PTS1 receptor Pex5p. We provide evidence that Lpx1p is piggyback-transported into peroxisomes. We have expressed the Lpx1p protein in Escherichia coli, and show that the enzyme exerts acyl hydrolase and phospholipase A activity in vitro. However, the protein is not required for wild-type-like steady-state function of peroxisomes, which might be indicative of a metabolic rather than a biogenetic role. Interestingly, peroxisomes in deletion mutants of LPX1 have an aberrant morphology characterized by intraperoxisomal vesicles or invaginations.    

3.1046           Use of polarized PC12 cells to monitor protein localization in the early biosynthetic pathway

Sannerud, R., Michaël, M., Berger hansen, B. And Saraste, J. Methods in Mol. Biol., 457, 253-265 (2008)   A prerequisite for understanding the cellular functions of an unknown protein is the establishment of its subcellular localization. As increasing numbers of novel proteins of the biosynthetic pathway are currently being identified, accessible new methods are required to facilitate their localization. Differentiating rat pheochromocytoma (PC12) cells reorganize their biosynthetic membrane compartments as they develop neurite-like processes. The authors recently showed that polarization of these cells involves the expansion of the intermediate compartment (IC) between the rough endoplasmic reticulum (RER) and the Golgi apparatus. Tubules emerging from the vacuolar parts of the IC move to the developing neurites accumulating in their growth cones, whereas the vacuoles, like RER and Golgi, remain in the cell body. Thus, polarized PC12 cells enhance the resolution for immunofluorescence microscopic mapping of protein localization in the early biosynthetic pathway. The authors also describe here a rapid cell fractionation protocol employing velocity sedimentation in iodixanol gradients that allows one-step separation of the pre-Golgi vacuoles, tubules, and RER.  

3.1047           Multidrug Transporters CaCdr1p and CaMdr1p of Candida albicans Display Different Lipid Specificities: both Ergosterol and Sphingolipids Are Essential for Targeting of CaCdr1p to Membrane Rafts

Pasrija, R., Panwar, S.L. and Prasad R. Antimicrob.Agents Chemother., 52(2), 694-704 (2008)   In this study, we compared the effects of altered membrane lipid composition on the localization of two membrane drug transporters from different superfamilies of the pathogenic yeast Candida albicans. We demonstrated that in comparison to the major facilitator superfamily multidrug transporter CaMdr1p, ATP-binding cassette transporter CaCdr1p of C. albicans is preferentially localized within detergent-resistant membrane (DRM) microdomains called ‘rafts.’ Both CaCdr1p and CaMdr1p were overexpressed as green fluorescent protein (GFP)-tagged proteins in a heterologous host Saccharomyces cerevisiae, wherein either sphingolipid ( sur4 or fen1 or ipt1) or ergosterol ( erg24 or erg6 or erg4) biosynthesis was compromised. CaCdr1p-GFP, when expressed in the above mutant backgrounds, was not correctly targeted to plasma membranes (PM), which also resulted in severely impaired drug resistance. In contrast, CaMdr1p-GFP displayed no sorting defect in the mutant background and remained properly surface localized and displayed no change in drug resistance. Our data clearly show that CaCdr1p is selectively recruited, over CaMdr1p, to the DRM microdomains of the yeast PM and that any imbalance in the raft lipid constituents results in missorting of CaCdr1p.  

3.1048           Compartmentalization of endocannabinoids into lipid rafts in a dorsal root ganglion cell line

Rimmermann, N. et al Br. J. Pharmacol., 153, 380-389 (2008)   Background and purpose: N-arachidonoyl ethanolamine (AEA) and 2-arachidonoyl glycerol (2-AG) are endogenous cannabinoids binding to the cannabinoid receptors CB₁ and CB₂ to modulate neuronal excitability and synaptic transmission in primary afferent neurons. To investigate the compartmentalization of the machinery for AEA and 2-AG signalling, we studied their partitioning into lipid raft fractions isolated from a dorsal root ganglion X neuroblastoma cell line (F-11). Experimental approach: F-11 cells were homogenized and fractionated using a detergent-free OptiPrep density gradient. All lipids were partially purified from methanolic extracts of the fractions on solid phase cartridges and quantified using liquid chromatography tandem mass spectrometry (LC/MS/MS). Protein distribution was determined by Western blotting. Key results: Under basal conditions, the endogenous cannabinoid AEA was present in both lipid raft and specific non-lipid raft fractions as was one of its biosynthetic enzymes, NAPE-PLD. The 2-AG precursor 1-stearoyl-2-arachidonoyl-sn-glycerol (DAG), diacylglycerol lipase (DAGL ), which cleaves DAG to form 2-AG, and 2-AG were all co-localized with lipid raft markers. CB₁ receptors, previously reported to partition into lipid raft fractions, were not detected in F-11 membranes, but CB₂ receptors were detected at high levels and partitioned into non-lipid raft fractions. Conclusions and implications: The biochemical machinery for the production of 2-AG via the putative diacylglycerol pathway is localized within lipid rafts, suggesting that 2-AG synthesis via DAG occurs within these microdomains. The observed co-localization of AEA, 2-AG, and their synthetic enzymes with the reported localization of CB₁ raises the possibility of intrinsic-autocrine signalling within lipid raft domains and/or retrograde-paracrine signalling.  

3.1049           The 1a-Adrenergic Receptor Occupies Membrane Rafts with Its G Protein Effectors but Internalizes via Clathrin-coated Pits

Morris, D.P., Lei, B., Wu, Y-X., Michelotti, G.-A. and Schwinn, D.A.

1. Biol. Chem., 283(5), 2973-2985 (2008)

  The _1a-adrenergic receptor ( _1aAR) occupies intracellular and plasma membranes in both native and heterologous expression systems. Based on multiple independent lines of evidence, we demonstrate the _1aAR at the cell surface occupies membrane rafts but exits from rafts following stimulation. In non-detergent raft preparations, basal _1aAR is present in low density membrane rafts and colocalizes with its G protein effectors on density gradients. Raft disruption by cholesterol depletion with methyl-β-cyclodextrin eliminates these light rafts. To confirm the presence of the _1aAR in plasma membrane rafts, fluorescence resonance energy transfer measurements were used to demonstrate colocalization of surface receptor and the raft marker, cholera toxin B. This colocalization was largely lost following _1aAR stimulation with phenylephrine. Similarly, receptor stimulation causes exit of the _1aAR from light rafts within 3-10 min in contrast to the G proteins, which largely remain in light rafts. Importantly, this delayed exit of the _1aAR suggests acute receptor signaling and desensitization occur entirely within rafts. Interestingly, both confocal analysis and measurement of surface _1aAR levels indicate modest receptor internalization during the 10 min following stimulation, suggesting most of the receptor has entered non-raft plasma membrane. Nevertheless, activation does increase the rate of receptor internalization as does disruption of rafts with methyl-β-cyclodextrin, suggesting raft exit enables internalization. Confocal analysis of surface-labeled hemagglutinin- _1aAR reveals that basal and stimulated receptor occupies clathrin pits in fixed cells consistent with previous indirect evidence. The evidence presented here strongly suggests the _1aAR is a lipid raft protein under basal conditions and implies agonist-mediated signaling occurs from rafts.  

3.1050           Epithelial polarity requires septin coupling of vesicle transport to polyglutamylated microtubules

Spiliotis, E.T., Hunt, S.J., Hu, Q., Kinoshita, M. And Nelson, W.J.

1. Cell Biol., 180(2), 295-303 (2008)

  In epithelial cells, polarized growth and maintenance of apicaland basolateral plasma membrane domains depend on protein sortingfrom the trans-Golgi network (TGN) and vesicle delivery to theplasma membrane. Septins are filamentous GTPases required forpolarized membrane growth in budding yeast, but whether theyfunction in epithelial polarity is unknown. Here, we show thatin epithelial cells septin 2 (SEPT2) fibers colocalize witha subset of microtubule tracks composed of polyglutamylated(polyGlu) tubulin, and that vesicles containing apical or basolateralproteins exit the TGN along these SEPT2/polyGlu microtubuletracks. Tubulin-associated SEPT2 facilitates vesicle transportby maintaining polyGlu microtubule tracks and impeding tubulinbinding of microtubule-associated protein 4 (MAP4). Significantly,this regulatory step is required for polarized, columnar-shapedepithelia biogenesis; upon SEPT2 depletion, cells become shortand fibroblast-shaped due to intracellular accumulation of apicaland basolateral membrane proteins, and loss of vertically orientedpolyGlu microtubules. We suggest that septin coupling of themicrotubule cytoskeleton to post-Golgi vesicle transport isrequired for the morphogenesis of polarized epithelia.  

3.1051           Dipeptidyl peptidase IV inhibition downregulates Na+-H+ exchanger NHE3 in rat renal proximal tubule

Castello, A. et al Am. J. Physiol. Renal Physiol., 294, F414-F422 (2008)   In the microvillar microdomain of the kidney brush border, sodium hydrogen exchanger type 3 (NHE3) exists in physical complexes with the serine protease dipeptidyl peptidase IV (DPPIV). The purpose of this study was to explore the functional relationship between NHE3 and DPPIV in the intact proximal tubule in vivo. To this end, male Wistar rats were treated with an injection of the reversible DPPIV inhibitor Lys [Z(NO₂)]-pyrrolidide (I40; 60 mg·kg^–1·day^–1 ip) for 7 days. Rats injected with equal amounts of the noninhibitory compound Lys[Z(NO₂)]-OH served as controls. Na⁺-H⁺ exchange activity in isolated microvillar membrane vesicles was 45 ± 5% decreased in rats treated with I40. Membrane fractionation studies using isopycnic centrifugation revealed that I40 provoked redistribution of NHE3 along with a small fraction of DPPIV from the apical enriched microvillar membranes to the intermicrovillar microdomain of the brush border. I40 significantly increased urine output (67 ± 9%; P < 0.01), fractional sodium excretion (63 ± 7%; P < 0.01), as well as lithium clearance (81 ± 9%; P < 0.01), an index of end-proximal tubule delivery. Although not significant, a tendency toward decreased blood pressure and plasma pH/HCO₃^– was noted in I40-treated rats. These findings indicate that inhibition of DPPIV catalytic activity is associated with inhibition of NHE3-mediated NaHCO₃ reabsorption in rat renal proximal tubule. Inhibition of apical Na⁺-H⁺ exchange is due to reduced abundance of NHE3 protein in the microvillar microdomain of the kidney brush border. Moreover, this study demonstrates a physiologically significant interaction between NHE3 and DPPIV in the intact proximal tubule in vivo.    

3.1052           Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

Fridberg, A. et al

1. Cell Sci., 121, 522-535 (2008)

  Sphingolipids and their metabolites have been thought crucial for cell growth and cell cycle progression, membrane and protein trafficking, signal transduction, and formation of lipid rafts; however, recent studies in trypanosomes point to the dispensability of sphingolipids in some of these processes. In this study, we explore the requirements for de novo sphingolipid biosynthesis in the insect life cycle stage of the African trypanosome Trypanosoma brucei by inhibiting the enzyme serine palmitoyltransferase (SPT2) by using RNA interference or treatment with a potent SPT2 inhibitor myriocin. Mass spectrometry revealed that upon SPT2 inhibition, the parasites contained substantially reduced levels of inositolphosphorylceramide. Although phosphatidylcholine and cholesterol levels were increased to compensate for this loss, the cells were ultimately not viable. The most striking result of sphingolipid reduction in procyclic T. brucei was aberrant cytokinesis, characterized by incomplete cleavage-furrow formation, delayed kinetoplast segregation and emergence of cells with abnormal DNA content. Organelle replication continued despite sphingolipid depletion, indicating that sphingolipids act as second messengers regulating cellular proliferation and completion of cytokinesis. Distention of the mitochondrial membrane, formation of multilamellar structures within the mitochondrion and near the nucleus, accumulation of lipid bodies and, less commonly, disruption of the Golgi complex were observed after prolonged sphingolipid depletion. These findings suggest that some aspects of vesicular trafficking may be compromised. However, flagellar membrane targeting and the association of the flagellar membrane protein calflagin with detergent-resistant membranes were not affected, indicating that the vesicular trafficking defects were mild. Our studies indicate that sphingolipid biosynthesis is vital for cell cycle progression and cell survival, but not essential for the normal trafficking of flagellar membrane-associated proteins or lipid raft formation in procyclic T. brucei.  

3.1053           Clustering endothelial E-selectin in clathrin-coated pits and lipid rafts enhances leukocyte adhesion under flow

Setiadi, H. and Mcever, R.P. Blood, 111(4), 1989-1998 (2008)   During inflammation, E-selectin expressed on cytokine-activated endothelial cells mediates leukocyte rolling under flow. E-selectin undergoes endocytosis and may associate with lipid rafts. We asked whether distribution of E-selectin in membrane domains affects its functions. E-selectin was internalized in transfected CHO cells or cytokine-activated human umbilical vein endothelial cells (HUVECs). Confocal microscopy demonstrated colocalization of E-selectin with -adaptin, a clathrin-associated protein. Deleting the cytoplasmic domain of E-selectin or disrupting clathrin-coated pits with hypertonic medium blocked internalization of E-selectin, reduced colocalization of E-selectin with -adaptin, and inhibited E-selectin-mediated neutrophil rolling under flow. Unlike CHO cells, HUVECs expressed a small percentage of E-selectin in lipid rafts. Even fewer neutrophils rolled on E-selectin in HUVECs treated with hypertonic medium and with methyl-β-cyclodextrin, which disrupts lipid rafts. These data demonstrate that E-selectin clusters in both clathrin-coated pits and lipid rafts of endothelial cells but is internalized in clathrin-coated pits. Distribution in both domains markedly enhances E-selectin's ability to mediate leukocyte rolling under flow.  

3.1054           The Vaccinia Virus B5 Protein Requires A34 for Efficient Intracellular Trafficking from the Endoplasmic Reticulum to the Site of Wrapping and Incorporation into Progeny Virions

Earley, A.K., Chan, W.M. and Ward, B.M.

1. Virol., 82(5), 2161-2169 (2008)

  The glycoproteins encoded by the vaccinia virus A34R and B5R genes are involved in intracellular envelope virus formation and are highly conserved among orthopoxviruses. A recombinant virus that has the A34R gene deleted and the B5R gene replaced with a B5R gene fused to the enhanced green fluorescent protein (B5R-GFP) gene was created (vB5R-GFP/ A34R) to investigate the role of A34 during virion morphogenesis. Cells infected with vB5R-GFP/ A34R displayed GFP fluorescence throughout the cytoplasm, which differed markedly from that seen in cells infected with a normal B5R-GFP-expressing virus (vB5R-GFP). Immunofluorescence and subcellular fractionation demonstrated that B5-GFP localizes with the endoplasmic reticulum in the absence of A34. Expression of either full-length A34 or a construct consisting of the lumenal and transmembrane domains restored normal trafficking of B5-GFP to the site of wrapping in the juxtanuclear region. Coimmunoprecipitation studies confirmed that B5 and A34 interact through their luminal domains, and further analysis revealed that in the absence of A34, B5 is not efficiently incorporated into virions released from the cell.  

3.1055           Modulation of ileal bile acid transporter (ASBT) activity by depletion of plasma membrane cholesterol: association with lipid rafts

Annaba, F. et al Am. J. Physiol. Gastrointest. Liver Physiol., 294, G489-G497 (2008)   Apical sodium-dependent bile acid transporter (ASBT) represents a highly efficient conservation mechanism of bile acids via mediation of their active transport across the luminal membrane of terminal ileum. To gain insight into the cellular regulation of ASBT, we investigated the association of ASBT with cholesterol and sphingolipid-enriched specialized plasma membrane microdomains known as lipid rafts and examined the role of membrane cholesterol in maintaining ASBT function. Human embryonic kidney (HEK)-293 cells stably transfected with human ASBT, human ileal brush-border membrane vesicles, and human intestinal epithelial Caco-2 cells were utilized for these studies. Floatation experiments on Optiprep density gradients demonstrated the association of ASBT protein with lipid rafts. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD) significantly reduced the association of ASBT with lipid rafts, which was paralleled by a decrease in ASBT activity in Caco-2 and HEK-293 cells treated with MβCD. The inhibition in ASBT activity by MβCD was blocked in the cells treated with MβCD-cholesterol complexes. Kinetic analysis revealed that MβCD treatment decreased the V_max of the transporter, which was not associated with alteration in the plasma membrane expression of ASBT. Our study illustrates that cholesterol content of lipid rafts is essential for the optimal activity of ASBT and support the association of ASBT with lipid rafts. These findings suggest a novel mechanism by which ASBT activity may be rapidly modulated by alterations in cholesterol content of plasma membrane and thus have important implications in processes related to maintenance of bile acid and cholesterol homeostasis.  

3.1056           Ceramide kinase promotes Ca2+ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells

Hinkovska-Galcheva, V. et al

1. Lipid Res., 49, 531-542 (2008)

  Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). In this study, we evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Stable transfectants of COS-1 cells expressing Fc RIIA or both Fc RIIA/hCERK expression vectors were created. Cell fractionation studies demonstrated that hCERK and the transient receptor potential channel (TRPC-1) were enriched in caveolae fractions. Our data establish that both CERK and TRPC-1 localize to the caveolar microdomains during phagocytosis and that CERK also colocalizes with EIgG in Fc RIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, Fc RIIA/hCERK transfected cells displayed Ca²⁺ sparks around the phagosome. In contrast, cells expressing Fc RIIA under identical conditions displayed little periphagosomal Ca²⁺ signaling. The enhanced Ca²⁺ signals were accompanied by enhanced phagolysosome formation. However, the addition of pharmacological reagents that inhibit store-operated channels (SOCs) reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. The higher Ca²⁺ signal observed in hCERK transfected cells as well as the fact that CERK colocalized with EIgG during phagocytosis support our hypothesis that Ca²⁺ signaling is an important factor for increasing phagocytosis and is regulated by CERK in a manner that involves SOCs/TRPCs.  

3.1057           Cross-talk between PDGF and S1P signalling elucidates the inhibitory effect and potential antifibrotic action of the immunomodulator FTY720 in activated HSC-cultures

Brunati, A.M. et al Biochim. Biophys. Acta, 1783, 347-359 (2008)   Platelet-derived growth factor (PDGF) has been shown to be essential in the activation of hepatic stellate cells (HSCs), contributing to the onset and development of hepatic fibrosis. Recently, sphingosine-1-phosphate (S1P) has been shown to be a mitogen and stimulator of chemotaxis also for HSCs. Since it has been demonstrated in several cell types that cross-talk between PDGF and S1P signalling pathways occurs, our aim was to investigate the potential antifibrotic effect of FTY720, whose phosphorylated form acts as a potent S1P receptor (S1PR) modulator, on HSCs. FTY720 inhibits cell proliferation and migration after PDGF stimulation on HSCs in a concentration range between 0.1 and 1 μM. By using compounds that block S1P signalling (PTX and VPC23019), we assessed that FTY720 also acts in an S1P receptor-independent way by decreasing the level of tyrosine phosphorylation of PDGF receptor, with subsequent inhibition of the PDGF signalling pathway. In addition, inhibition of sphingosine kinase2 (SphK2), which is responsible for FTY720 phosphorylation, by DMS/siRNA unveils a mechanism of action irrespective of its phosphorylation, in particular decreasing the level of S1P₁ on the plasma membrane. These findings led us to hypothesize a potential use of FTY720 as a potential antifibrotic drug for further clinical application.  

3.1058           Lipid rafts regulate ethanol-induced activation of TLR4 signaling in murine macrophages

Fernandez-Lizarbe, S., Pascual, M., Soledad Gascon, M., Blanco, A. and Guerri, C. Mol. Immunol., 45, 2007-2016 (2008)   Toll-like receptors (TLRs) response is critical in innate resistance to infection. Alcohol consumption has been shown to suppress the inflammatory response mediated through TLR4, down regulating the production of inflammatory cytokines. We recently reported that low concentrations of ethanol activate TLR4 signaling in astrocytes and triggers neuroinflammation. Because macrophages are important cells in innate immunity, we investigate whether low concentrations of ethanol could stimulate the TLR4 signaling response in murine RAW 264.7 macrophages, and the mechanism involved in the ethanol-induced TLR4 activation. Our results show that while ethanol, at high concentrations (100 mM) or in the presence of the LPS, suppresses the TLR4 response, low to moderate levels (10–50 mM) activate the TLR4 response and triggers the stimulation of the mitogen-activated protein kinases (MAPKs) and the transcription factor NF-κB pathways, leading to the production of nitric oxide (NO) and inflammatory cytokines. Pre-treatment with anti-TLR4 Abs abolishes the effects of ethanol on the production of cytokines. We also present evidence that stimulation with either ethanol or LPS induces translocation and clustering of TLR4 and signaling molecules (IRAK and MAPKs) into lipid rafts. Treatment with either streptolysin-O or saponin, lipid rafts disrupting agents, abolishes the ethanol-induced activation of the TLR4/IL-1RI signaling pathway. In summary, the present results demonstrate that low to moderate concentrations of ethanol are capable of stimulating TLR4/IL-1RI response, and provide evidence of a novel mechanism by which ethanol, through its interaction with membrane rafts, can promote TLR4/IL-1RI recruitment and signaling.  

3.1059           Human Immunodeficiency Virus Type 1 Nef Recruits the Guanine Exchange Factor Vav1 via an Unexpected Interface into Plasma Membrane Microdomains for Association with p21-Activated Kinase 2 Activity

Rauch, S., Pulkkinen, K., Saksela, K. And Fackler, O.T.

1. Virol., 82(6), 2918-2929 (2008)

  Alterations of T-cell receptor signaling by human immunodeficiency virus type 1 (HIV-1) Nef involve its association with a highly active subpopulation of p21-activated kinase 2 (PAK2) within a dynamic signalosome assembled in detergent-insoluble membrane microdomains. Nef-PAK2 complexes contain the GTPases Rac and Cdc42 as well as a factor providing guanine nucleotide exchange factor (GEF) activity for Rac/Cdc42. However, the identity of this GEF has remained controversial. Previous studies suggested the association of Nef with at least three independent GEFs, Vav, DOCK2/ELMO1, and βPix. Here we used a broad panel of approaches to address which of these GEFs is involved in the functional interaction of Nef with PAK2 activity. Biochemical fractionation and confocal microscopy revealed that Nef recruits Vav1, but not DOCK2/ELMO1 or βPix, to membrane microdomains. Transient RNAi knockdown, analysis of cell lines defective for expression of Vav1 or DOCK2 as well as use of a βPix binding-deficient PAK2 variant confirmed a role for Vav1 but not DOCK2 or βPix in Nef's association with PAK2 activity. Nef-mediated microdomain recruitment of Vav1 occurred independently of the Src homology 3 domain binding PxxP motif, which is known to connect Nef to many cellular signaling processes. Instead, a recently described protein interaction surface surrounding Nef residue F195 was identified as critical for Nef-mediated raft recruitment of Vav1. These results identify Vav1 as a relevant component of the Nef-PAK2 signalosome and provide a molecular basis for the role of F195 in formation of a catalytically active Nef-PAK2 complex.  

3.1060           Signal sequence- and translation-independent mRNA localization to the endoplasmic reticulum

Pyhtila, B. et al RNA, 14, 445-453 (2008)   The process of mRNA localization typically utilizes cis-targeting elements and trans-recognition factors to direct the compartmental organization of translationally suppressed mRNAs. mRNA localization to the endoplasmic reticulum (ER), in contrast, occurs via a co-translational, signal sequence/signal recognition particle (SRP)-dependent mechanism. We have utilized cell fractionation/cDNA microarray analysis, shRNA-mediated suppression of SRP expression, and mRNA reporter construct studies to define the role of the SRP pathway in ER-directed mRNA localization. Cell fractionation studies of mRNA partitioning between the cytosol and ER demonstrated the expected enrichment of cytosolic/nucleoplasmic protein-encoding mRNAs and secretory/integral membrane protein-encoding mRNAs in the cytosol and ER fractions, respectively, and identified a subpopulation of cytosolic/nucleoplasmic protein-encoding mRNAs in the membrane-bound mRNA pool. The latter finding suggests a signal sequence-independent pathway of ER-directed mRNA localization. Extending from these findings, mRNA partitioning was examined in stable SRP54 shRNA knockdown HeLa cell lines. shRNA-directed reductions in SRP did not globally alter mRNA partitioning patterns, although defects in membrane protein processing were observed, further suggesting the existence of multiple pathways for mRNA localization to the ER. ER localization of GRP94-encoding mRNA was observed when translation was disabled by mutation of the start codon/insertion of a 5'UTR stem–loop structure or upon deletion of the encoded signal sequence. Combined, these data indicate that the mRNA localization to the ER can be conferred independent of the signal sequence/SRP pathway and suggest that mRNA localization to the ER may utilize cis-encoded targeting information.  

3.1061           Myo2p, a class V myosin in budding yeast, associates with a large ribonucleic acid–protein complex that contains mRNAs and subunits of the RNA-processing body

Chang, W. et al RNA, 14, 491-502 (2008)   Myo2p is an essential class V myosin in budding yeast with several identified functions in organelle trafficking and spindle orientation. The present study demonstrates that Myo2p is a component of a large RNA-containing complex (Myo2p–RNP) that is distinct from polysomes based on sedimentation analysis and lack of ribosomal subunits in the Myo2p–RNP. Microarray analysis of RNAs that coimmunoprecipitate with Myo2p revealed the presence of a large number of mRNAs in this complex. The Myo2p–RNA complex is in part composed of the RNA processing body (P-body) based on coprecipitation with P-body protein subunits and partial colocalization of Myo2p with P-bodies. P-body disassembly is delayed in the motor mutant, myo2-66, indicating that Myo2p may facilitate the release of mRNAs from the P-body.  

3.1062           Protein quality control: the who’s who, the where’s and therapeutic escapes

Roth, J. et al Histochem. Cell Biol., 129, 163-177 (2008)   In cells the quality of newly synthesized proteins is monitored in regard to proper folding and correct assembly in the early secretory pathway, the cytosol and the nucleoplasm. Proteins recognized as non-native in the ER will be removed and degraded by a process termed ERAD. ERAD of aberrant proteins is accompanied by various changes of cellular organelles and results in protein folding diseases. This review focuses on how the immunocytochemical labeling and electron microscopic analyses have helped to disclose the in situ subcellular distribution pattern of some of the key machinery proteins of the cellular protein quality control, the organelle changes due to the presence of misfolded proteins, and the efficiency of synthetic chaperones to rescue disease-causing trafficking defects of aberrant proteins.  

3.1063           Insulin Internalizes GLUT2 in the Enterocytes of Healthy but Not Insulin-Resistant Mice

Tobin, V. et al Diabetes, 57, 555-562 (2008)   OBJECTIVES—A physiological adaptation to a sugar-richmeal is achieved by increased sugar uptake to match dietaryload, resulting from a rapid transient translocation of thefructose/glucose GLUT2 transporter to the brush border membrane(BBM) of enterocytes. The aim of this study was to define thecontributors and physiological mechanisms controlling intestinalsugar absorption, focusing on the action of insulin and thecontribution of GLUT2-mediated transport. RESEARCH DESIGN AND METHODS—The studies were performedin the human enterocytic colon carcinoma TC7 subclone (Caco-2/TC7)cells and in vivo during hyperinsulinemic-euglycemic clamp experimentsin conscious mice. Chronic high-fructose or high-fat diets wereused to induce glucose intolerance and insulin resistance inmice. RESULTS AND CONCLUSIONS—In Caco-2/TC7 cells, insulin action diminished the transepithelial transfer of sugar and reduced BBM and basolateral membrane (BLM) GLUT2 levels, demonstrating that insulin can target sugar absorption by controlling the membrane localization of GLUT2 in enterocytes. Similarly, in hyperinsulinemic-euglycemic clamp experiments in sensitive mice, insulin abolished GLUT2 (i.e., the cytochalasin B-sensitive component of fructose absorption), decreased BBM GLUT2, and concomitantly increased intracellular GLUT2. Acute insulin treatment before sugar intake prevented the insertion of GLUT2 into the BBM. Insulin resistance in mice provoked a loss of GLUT2 trafficking, and GLUT2 levels remained permanently high in the BBM and low in the BLM. We propose that, in addition to its peripheral effects, insulin inhibits intestinal sugar absorption to prevent excessive blood glucose excursion after a sugar meal. This protective mechanism is lost in the insulin-resistant state induced by high-fat or high-fructose feeding.  

3.1064           Phospholipid actions on PGHS-1 and -2 cyclooxygenase kinetics

Doyen, J.R., Yucer, N., Lichtenberger, L.M. and Kulmacz, R.J. Prostaglandins & Other Lipid Mediators, 85(3-4), 134-143 (2008)   Cyclooxygenase (COX) catalysis by prostaglandin H synthase (PGHS) is a key control step for regulation of prostanoid biosynthesis. Both PGHS isoforms are integral membrane proteins and their substrate fatty acids readily partition into membranes, but the impact of phospholipids and lipid membranes on COX catalysis and the actions of COX inhibitors are not well understood. We have characterized the COX kinetics and ibuprofen inhibition of the purified PGHS isoforms in the presence of phosphatidylcholine (PC) with varying acyl chain structure and physical state. PC was found to directly inhibit COX activity, with non-competitive inhibition by PC monomers binding away from the COX active site and competitive inhibition by micellar/bilayer forms of PC due to sequestration of the arachidonate substrate. Competitive inhibition by native membranes was observed in a comparison of COX kinetics in sheep seminal vesicle microsomes before and after solubilization of PGHS-1. PC liposomes significantly increase the inhibitory potency of ibuprofen against both PGHS isoforms without changing the reversible character of ibuprofen action or requiring binding of PGHS to the liposomes. These results suggest a useful conceptual framework for analyzing the complex interactions among the PGHS proteins, substrates, inhibitors and phospholipid.  

3.1065           Multiple functions of ergosterol in the fission yeast Schizosaccharomyces pombe

Iwaki, T. et al Microbiology, 154, 830-841 (2008)   Sterols are a major class of membrane lipids in eukaryotes. In Schizosaccharomyces pombe, sterol 24-C-methyltransferase (Erg6p), C-8 sterol isomerase (Erg2p), C-5 sterol desaturase (Erg31p, Erg32p), C-22 sterol desaturase (Erg5p) and C-24 (28) sterol reductase (Sts1p/Erg4p) have been predicted, but not yet determined, to catalyse a sequence of reactions from zymosterol to ergosterol. Disruption mutants of these genes were unable to synthesize ergosterol, and most were tolerant to the polyene drugs amphotericin B and nystatin. Disruption of erg31⁺ or erg32⁺ did not cause ergosterol deficiency or tolerance to polyene drugs, indicating that the two C-5 sterol desaturases have overlapping functions. GFP-tagged DRM (detergent-resistant membrane)-associated protein Pma1p localized to the plasma membrane in erg mutants. DRM fractionation revealed that the association between Pma1-GFP and DRM was weakened in erg6 but not in other erg mutants. Several GFP-tagged plasma membrane proteins were tested, and an amino acid permease homologue, SPBC359.03c, was found to mislocalize to intracellular punctate structures in the erg mutants. These results indicate that these proteins are responsible for ergosterol biosynthesis in fission yeast, similar to the situation in Saccharomyces cerevisiae. Furthermore, in fission yeast, ergosterol is important for plasma membrane structure and function and for localization of plasma membrane proteins.  

3.1066           CFTR in a lipid raft-TNFR1 complex modulates gap junctional intercellular communication and IL-8 secretion

Dudez, T. et al Eur. J. Lab. Invest., 38(Suppl.1-2), 20, abstract 54   Background: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause a chronic inflammatory response in the lung of patients with Cystic Fibrosis (CF). Defect in TNF-a signaling through the Src family tyrosine kinases (SFKs) has been reported in CF cells, and shown to unable the regulation of gap junctional communication (GJIC). Here, we sought to elucidate the mechanisms linking TNF-a signaling to the functions of CFTR at the molecular level. Materials and methods: Detergent-resistant membran microdomains (DRMs), or lipid rafts, were isolated using the Optiprep method from MDCKI cells expressing wild-type (WtCFTR) or mutant CFTR lacking its PDZ-interacting motif (CFTR-aTRL), and from the human glandular Calu-3 cell line endogenously expressing CFTR. GJIC was evaluated by dye coupling and IL-8 secretion by dot blots. Results: TNF-a increased the amount of Wt-CFTR but not CFTR-aTRL in DRMs. This recruitment was modulated by SFK activity and associated with DRM localization of TNFR1 and c-Src. Activation of TNFR1 signaling also decreased GJIC and markedly stimulated IL-8 production in WtCFTR cells. The absence of CFTR in DRMs was associated with abnormal TNFR1 signaling as revealed by no recruitment of TNFR1 and c-Src to lipid rafts in CFTR-aTRL cells and loss of regulation of GJIC and IL-8 secretion. We further show in Calu-3 cells that endogen CFTR is present at the cell surface in DRMs, which amount is enhanced by TNF-a and contributes to IL-8 secretion. Conclusions: Localization of CFTR to lipid rafts in association with c-Src and TNFR1 provides a responsive signaling complex to regulate GJIC and cytokine signaling. Disruption of CFTR interactions with component of this complex may lead to abnormalities relevant for the CF pathogenesis.  

3.1067           A Lipid-mediated Quality Control Process in the Golgi Apparatus in Yeast

Pineau, L. et al Mol. Biol. Cell, 9, 807-821 (2008)   When heme biosynthesis is disrupted, the yeast Saccharomyces cerevisiae becomes unable to synthesize its major sterol, ergosterol, and desaturate fatty acids. We took advantage of this physiological peculiarity to evaluate the consequences of ergosterol and/or unsaturated fatty acid (UFA) depletions on the biogenesis of a model polytopic plasma membrane protein, the uracil permease Fur4p. We show that under UFA shortage, which results in low amounts of diunsaturated phospholipid species, and under ergosterol depletion, Fur4p is prematurely routed from the Golgi apparatus to the vacuolar lumen in a process that requires the ubiquitin ligase Rsp5p. Interestingly, this diversion is not correlated to Fur4p exclusion from detergent-resistant membranes. In an independent set of experiments, we show that Fur4p targeting to the plasma membrane depends on phosphatidylethanolamine amounts and more specifically on the propensity of this phospholipid to form a hexagonal phase. In light of recent literature, we propose a model in which ergosterol and diunsaturated phospholipid species maintain optimal membrane curvature for Fur4p to evade the Golgi quality control process and to be properly delivered to its normal destination.  

3.1068           Phospholipid and glycolipid composition of acidocalcisomes of Trypanosoma cruzi

Salto, M.L., Kuhlenschmidt, T., Kuhlenschmidt, M., de Lederkremer, R.M. and Docampo, R. Mol. Biochem. Parasitol., 158, 120-130 (2008)   Highly purified acidocalcisomes from Trypanosoma cruzi epimastigotes were obtained by differential centrifugation and iodixanol gradient ultracentrifugation. Lipid analysis of acidocalcisomes revealed the presence of low amounts of 3β-hydroxysterols and predominance of phospholipids. Alkylacyl phosphatidylinositol (16:0/18:2), diacyl phosphatidylinositol (18:0/18:2), diacyl phosphatidylcholine (16:0/18:2; 16:1/18:2; 16:2/18:2; 18:1/18:2 and 18:2/18:2), and diacyl phosphatidylethanolamine (16:0/18:2 and 16:1/18:2) were the only phospholipids characterized by electrospray ionization-mass spectrometry (ESI-MS). Incubation of epimastigotes with [³H]-mannose and isolation of acidocalcisomes allowed the detection of a glycoinositolphospholipid (GIPL) in these organelles. The sugar content of the acidocalcisomal GIPL was similar to that of the GIPL present in a microsomal fraction but the amount of galactofuranose and inositol with respect to the other monosaccharides was lower, suggesting a different chemical structure. Taken together, these results indicate that acidocalcisomes of T. cruzi have a distinct lipid and carbohydrate composition.  

3.1069           FcγRI (CD64) resides constitutively in lipid rafts

Beekman, J.M., van der Linden, J.A., van de Winkel, J.G.J. and Leusen, J.H.W. Immunol. Lett., 116, 149-155 (2008)   Cellular membranes contain microdomains known as ‘lipid rafts’ or detergent-insoluble microdomains (DRM), enriched in cholesterol and sphingolipids. DRM can play an important role in many cellular processes, including signal transduction, cytoskeletal organization, and pathogen entry. Many receptors like T cell receptors, B cell receptors and IgE receptors have been shown to reside in DRM. The majority of these receptors depend on multivalent ligand interaction to associate with these microdomains. We, here, study association between the high affinity IgG receptor, FcγRI (CD64), and membrane microdomains. FcγRI is a 72 kDa type I glycoprotein that can mediate phagocytosis of opsonized pathogens, but can also effectively capture small immune complexes, and facilitates antigen presentation. We found FcγRI to predominantly reside within detergent-insoluble buoyant membranes, together with FcRγ-chain, but independent of cross-linking ligand. With the use of confocal imaging, FcγRI was found to co-patch with GM1, a microdomain-enriched glycolipid. Depletion of cellular cholesterol, furthermore, modulated FcγRI–ligand interactions. These data indicated FcγRI to reside within lipid rafts without prior triggering of the receptor.  

3.1070           Selective association of misfolded ALS-linked mutant SOD1 with the cytoplasmic face of mitochondria

Vande Velde, C., Miller, T.M., Cashman, N.R. and Cleveland, D.W. PNAS, 105(10), 4022-4027 (2008)   Mutations in copper/zinc superoxide dismutase (SOD1) are causative for dominantly inherited amyotrophic lateral sclerosis (ALS). Despite high variability in biochemical properties among the disease-causing mutants, a proportion of both dismutase-active and -inactive mutants are stably bound to spinal cord mitochondria. This mitochondrial proportion floats with mitochondria rather than sedimenting to the much higher density of protein, thus eliminating coincidental cosedimentation of protein aggregates with mitochondria. Half of dismutase-active and 90% of dismutase-inactive mutant SOD1 is bound to mitochondrial membranes in an alkali- and salt-resistant manner. Sensitivity to proteolysis and immunoprecipitation with an antibody specific for misfolded SOD1 demonstrate that in all mutant SOD1 models, misfolded SOD1 is deposited onto the cytoplasmic face of the outer mitochondrial membrane, increasing antigenic accessibility of the normally structured electrostatic loop. Misfolded mutant SOD1 binding is both restricted to spinal cord and selective for mitochondrial membranes, implicating exposure to mitochondria of a misfolded mutant SOD1 conformer mediated by a unique, tissue-selective composition of cytoplasmic chaperones, components unique to the cytoplasmic face of spinal mitochondria to which misfolded SOD1 binds, or misfolded SOD1 conformers unique to spinal cord that have a selective affinity for mitochondrial membranes.  

3.1071           Carboxypeptidase M and Kinin B1 Receptors Interact to Facilitate Efficient B1 Signaling from B2 Agonists

Zhang, X., Tan, F., Zhang, Y. And Skidgel, R.

1. Biol. Chem., 283(12), 7994-8004 (2008)

  Kinin B1 receptor (B1R) expression is induced by injury or inflammatory mediators, and its signaling produces both beneficial and deleterious effects. Kinins cleaved from kininogen are agonists of the B2R and must be processed by a carboxypeptidase to generate B1R agonists des-Arg⁹-bradykinin or des-Arg¹⁰-kallidin. Carboxypeptidase M (CPM) is a membrane protein potentially well suited for this function. Here we show that CPM expression is required to generate a B1R-dependent increase in [Ca²⁺]_i in cells stimulated with B2R agonists kallidin or bradykinin. CPM and the B1R interact on the cell membrane, as shown by co-immunoprecipitation, cross-linking, and fluorescence resonance energy transfer analysis. CPM and B1R are also co-localized in lipid raft/caveolin-enriched membrane fractions, as determined by gradient centrifugation. Treatment of cells co-expressing CPM and B1R with methyl-β-cyclodextrin to disrupt lipid rafts reduced the B1R-dependent increase in [Ca²⁺]_i in response to B2R agonists, whereas cholesterol treatment enhanced the response. A monoclonal antibody to the C-terminal β-sheet domain of CPM reduced the B1R response to B2R agonists without inhibiting CPM. Cells expressing a novel fusion protein containing CPM at the N terminus of the B1R also increased [Ca²⁺]_i when stimulated with B2R agonists, but the response was not reduced by methyl-β-cyclodextrin or CPM antibody. A B1R- and CPM-dependent calcium signal in response to B2R agonist bradykinin was also found in endothelial cells that express both proteins. Thus, a close relationship of B1Rs and CPM on the membrane is required for efficiently generating B1R signals, which play important roles in inflammation.  

3.1072           Over-expression of mammalian sialidase NEU3 reduces Newcastle disease virus entry and propagation in COS7 cells

Anastasia, L. et al Biochim. Biophys. Acta, 1780(3), 504-512 (2008)   The paramyxovirus Newcastle Disease Virus (NDV) binds to sialic acid-containing glycoconjugates, sialoglycoproteins and sialoglycolipids (gangliosides) of host cell plasma membrane through its hemagglutinin-neuraminidase (sialidase) HN glycoprotein. We hypothesized that the modifications of the cell surface ganglioside pattern determined by over-expression of the mammalian plasma-membrane associated, ganglioside specific, sialidase NEU3 would affect the virus-host cell interactions. Using COS7 cells as a model system, we observed that over-expression of the murine MmNEU3 did not affect NDV binding but caused a marked reduction in NDV infection and virus propagation through cell–cell fusion. Moreover, since GD1a was greatly reduced in COS7 cells following NEU3-over-expression, we added [³H]-labelled GD1a to COS7 cells under conditions that block intralysosomal metabolic processing, and we observed a marked increase of GD1a cleavage to GM1 during NDV infection, indicating a direct involvement of the virus sialidase and host cell GD1a in NDV infectivity. Therefore, the decrease of GD1a in COS7 cell membrane upon MmNEU3 over-expression is likely to be instrumental to NDV reduced infection. Evidence was also provided for the preferential association of NDV-HN at 4 °C to detergent resistant microdomains (DRMs) of COS7 cells plasma membranes.  

3.1073           Fractionation of Subcellular Membrane Vesicles of Epithelial and Nonepithelial Cells by OptiPrep™ Density Gradient Ultracentrifugation

Li, X. and Donowitz, M. Methods Mol. Biol., 440, 97-110 (2008)   Density gradient ultracentrifugation (DGUC) is widely used for physical isolation (enrichment rather than purification) of subcellular membrane vesicles. It has been a valuable tool to study specific subcellular localization and dynamic trafficking of proteins. While sucrose has been the main component of density gradients, a few years ago synthetic OptiPrep™ (iodixanol) began being used for separation of organelles because of its iso-osmotic property. Here, we describe a detailed protocol for density gradient fractionation of various mammalian subcellular vesicles, including endoplasmic reticulum (ER), Golgi apparatus, endosomes, and lipid rafts, as well as apical and basolateral membranes of polarized epithelial cells.  

3.1074           Determination of Genuine Residents of Plant Endomembrane Organelles using Isotope Tagging and Multivariate Statistics

Lilley, K.S. and Dunkley, T.P.J. Methods Mol. Biol., 432, 373-387 (2008)   The knowledge of the localization of proteins to a particular subcellular structure or organelle is an important step towards assigning function to proteins predicted by genome-sequencing projects that have yet to be characterized. Moreover, the localization of novel proteins to organelles also enhances our understanding of the functions of organelles. Many organelles cannot be purified. In several cases where the degree of contamination by organelles with similar physical parameters to the organelle being studied has gone unchecked, this has lead to the mis-localization of proteins. Recently, several techniques have emerged, which depend on characterization of the distribution pattern of organelles partially separated using density centrifugation by quantitative proteomics approaches. Here, we discuss one of these approaches, the localization of organelle proteins by isotope tagging (LOPIT) where the distribution patterns of organelles are assessed by measuring the relative abundance of proteins between fractions along the length of density gradients using stable isotope-coded tags. The subcellular localizations of proteins can be determined by comparing their distributions to those of previously localized proteins by assuming that proteins that belong to the same organelle will cofractionate in density gradients. Analysis of distribution patterns can be achieved by employing multivariate statistical methods such as principal component analysis and partial least squares discriminate analysis. In this chapter, we focus on the use of the LOPIT technique in the assignment of membrane proteins to the plant Golgi apparatus and endoplasmic reticulum.  

3.1075           Quantitative Proteomic Analysis to Profile Dynamic Changes in the Spatial Distribution of Cellular Proteins

Yan, W., Hwang, D. and Aebersold, R. Methods Mol. Biol., 432, 389-401 (2008)   Organelle protein profiles have traditionally been analyzed by subcellular fractionation of a specific organelle followed by the identification of the protein components of specific fractions containing the target organelle(s) using mass spectrometry (MS). However, because of limited resolution of the available fractionation methods, it is often difficult to isolate and thus profile pure organelles. Furthermore, many proteins (e.g., secretory proteins) are often observed to dynamically shuttle between organelles. Therefore, the determination of their true cellular localization requires the concurrent analysis of multiple organelles from the same cell lysate. Here, we report an integrated experimental approach that simultaneously profiles multiple organelles. It is based on the subcellular fractionation of cell lysates by density gradient centrifugation, iTRAQ labeling, and MS analysis of the proteins in selected fractions and principal component analysis (PCA) of the resulting quantitative proteomic data. Quantitative signature patterns of several organelles, including the ribosome, mitochondria, proteasome, lysosome, endoplasmic reticulum (ER), and Golgi apparatus, have been acquired from a single multiplexed proteomic assay using iTRAQ reagents. Through comparison PCA, we compare organelle profiles from cells under different physiological conditions to investigate changes in organelle profiles between control and perturbed samples. Such quantitative proteomics-based subcellular profiling methods thus provide useful tools to dissect the organization of cellular proteins into functional units and to detect dynamic changes in their protein composition.  

3.1076           Free Flow Isoelectric Focusing

A Method for the Separation of Both Hydrophilic and Hydrophobic Proteins of Rat Liver Peroxisomes

Islinger, M. and Weber, G. Methods Mol. Biol., 432, 199-215 (2008)   Peroxisomes take part in various metabolic pathways related to the regulation of lipid homeostasis. Although detailed information on the enzymes involved in the peroxisomal lipid metabolism was acquired in the past, the mechanisms of metabolic exchange between peroxisomes and the cytosol or other organelles still remain an enigma. Therefore, a detailed analysis of the peroxisomal membrane proteome could help identify potential metabolite transporters. However, because of their highly hydrophobic character, membrane proteins tend to precipitate in aqueous media, making their fractionation still a challenging task. To overcome these obstacles, we have elaborated a protocol for the separation of both hydrophilic as well as hydrophobic proteins using free flow isoelectric focusing (FF-IEF). Similar to traditional gel-based isoelectric focusing, a denaturing electrophoresis buffer containing a mixture of urea, thiourea and detergents is applied to keep highly hydrophobic proteins in solution. Electrophoresis is conducted on a BD Free Flow Electrophoresis System with a linear pH gradient from 3 to 10 and sampled into 96 fractions. As a second dimension, sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) is used to further separate and visualize the protein pattern of the peroxisomal subfractions of matrix, peripheral and integral membrane proteins. The identification of the known peroxisomal membrane proteins PMP22, PMP70 as well as mGST in the subsequent matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis of the 100 most prominent protein bands has documented the suitability of this new technique for the analysis of hydrophobic proteins.  

3.1077           Sequencing and analysis of the gene-rich space of cowpea

Timko, M.P. et al BMC Genomics, 9, 103-122 (2008)   Background Cowpea, Vigna unguiculata (L.) Walp., is one of the most important food and forage legumes in the semi-arid tropics because of its drought tolerance and ability to grow on poor quality soils. Approximately 80% of cowpea production takes place in the dry savannahs of tropical West and Central Africa, mostly by poor subsistence farmers. Despite its economic and social importance in the developing world, cowpea remains to a large extent an underexploited crop. Among the major goals of cowpea breeding and improvement programs is the stacking of desirable agronomic traits, such as disease and pest resistance and response to abiotic stresses. Implementation of marker-assisted selection and breeding programs is severely limited by a paucity of trait-linked markers and a general lack of information on gene structure and organization. With a nuclear genome size estimated at ~620 Mb, the cowpea genome is an ideal target for reduced representation sequencing. Results We report here the sequencing and analysis of the gene-rich, hypomethylated portion of the cowpea genome selectively cloned by methylation filtration (MF) technology. Over 250,000 gene-space sequence reads (GSRs) with an average length of 610 bp were generated, yielding ~160 Mb of sequence information. The GSRs were assembled, annotated by BLAST homology searches of four public protein annotation databases and four plant proteomes (A. thaliana, M. truncatula, O. sativa, and P. trichocarpa), and analyzed using various domain and gene modeling tools. A total of 41,260 GSR assemblies and singletons were annotated, of which 19,786 have unique GenBank accession numbers. Within the GSR dataset, 29% of the sequences were annotated using the Arabidopsis Gene Ontology (GO) with the largest categories of assigned function being catalytic activity and metabolic processes, groups that include the majority of cellular enzymes and components of amino acid, carbohydrate and lipid metabolism. A total of 5,888 GSRs had homology to genes encoding transcription factors (TFs) and transcription associated factors (TAFs) representing about 5% of the total annotated sequences in the dataset. Sixty-two (62) of the 64 well-characterized plant transcription factor (TF) gene families are represented in the cowpea GSRs, and these families are of similar size and phylogenetic organization to those characterized in other plants. The cowpea GSRs also provides a rich source of genes involved in photoperiodic control, symbiosis, and defense-related responses. Comparisons to available databases revealed that about 74% of cowpea ESTs and 70% of all legume ESTs were represented in the GSR dataset. As approximately 12% of all GSRs contain an identifiable simple-sequence repeat, the dataset is a powerful resource for the design of microsatellite markers. Conclusion The availability of extensive publicly available genomic data for cowpea, a non-model legume with significant importance in the developing world, represents a significant step forward in legume research. Not only does the gene space sequence enable the detailed analysis of gene structure, gene family organization and phylogenetic relationships within cowpea, but it also facilitates the characterization of syntenic relationships with other cultivated and model legumes, and will contribute to determining patterns of chromosomal evolution in the Leguminosae. The micro and macrosyntenic relationships detected between cowpea and other cultivated and model legumes should simplify the identification of informative markers for marker-assisted trait selection and map-based gene isolation necessary for cowpea improvement.  

3.1078           Endosomal NADPH oxidase regulates c-Src activation following hypoxia/reoxygenation injury

Li, Q., Zhang, Y., Marden, J.J., Banf, B. And Engelhardt, J.F. Biochem. J., 411, 531-541 (2008)   c-Src has been shown to activate NF-κB (nuclear factor κB) following H/R (hypoxia/reoxygenation) by acting as a redox-dependent IκBα (inhibitory κB) tyrosine kinase. In the present study, we have investigated the redox-dependent mechanism of c-Src activation following H/R injury and found that ROS (reactive oxygen species) generated by endosomal Noxs (NADPH oxidases) are critical for this process. Endocytosis following H/R was required for the activation of endosomal Noxs, c-Src activation, and the ability of c-Src to tyrosine-phosphorylate IκBα. Quenching intra-endosomal ROS during reoxygenation inhibited c-Src activation without affecting c-Src recruitment from the plasma membrane to endosomes. However, siRNA (small interfering RNA)-mediated knockdown of Rac1 prevented c-Src recruitment into the endosomal compartment following H/R. Given that Rac1 is a known activator of Nox1 and Nox2, we investigated whether these two proteins were required for c-Src activation in Nox-deficient primary fibroblasts. Findings from these studies suggest that both Nox1 and Nox2 participate in the initial redox activation of c-Src following H/R. In summary, our results suggest that Rac1-dependent Noxs play a critical role in activating c-Src following H/R injury. This signalling pathway may be a useful therapeutic target for ischaemia/reperfusion-related diseases.  

3.1079           Proteomic Analysis of Highly Purified Peroxisomes from Etiolated Soybean Cotyledons

Arai, Y., Hayashi, M. and Nishimura, M. Plant Cell. Physiol., 49(4), 526-539 (2008)   To identify previously unknown peroxisomal proteins, we established an optimized method for isolating highly purified peroxisomes from etiolated soybean cotyledons using Percoll density gradient centrifugation followed by iodixanol density gradient centrifugation. Proteins in highly purified peroxisomes were separated by two-dimensional PAGE. We performed peptide mass fingerprinting of proteins separated in the gel with matrix-assisted laser desorption ionization time-of-flight mass spectrometry and used the peptide mass fingerprints to search a non-redundant soybean expressed sequence tag database. We succeeded in assigning 92 proteins to 70 sequences in the database. Among them, proteins encoded by 30 sequences were judged to be located in peroxisomes. These included enzymes for fatty acid β-oxidation, the glyoxylate cycle, photorespiratoryglycolate metabolism, stress response and metabolite transport.We also show experimental evidence that plant peroxisomes containa short-chain dehydrogenase/reductase family protein, enoyl-CoAhydratase/isomerase family protein, 3-hydroxyacyl-CoA dehydrogenase-likeprotein and a voltage-dependent anion-selective channel protein.  

3.1080           A fluorescent sphingolipid binding domain peptide probe interacts with sphingolipids and cholesterol-dependent raft domains

Hebbar, S. et al

1. Lipid Res., 49, 1077-1089 (2008)

  We have designed a tagged probe [sphingolipid binding domain (SBD)] to facilitate the tracking of intracellular movements of sphingolipids in living neuronal cells. SBD is a small peptide consisting of the SBD of the amyloid precursor protein. It can be conjugated to a fluorophore of choice and exogenously applied to cells, thus allowing for in vivo imaging. Here, we present evidence to describe the characteristics of the SBD association with the plasma membrane. Our experiments demonstrate that SBD binds to isolated raft fractions from human neuroblastomas and insect neuronal cells. In protein-lipid overlay experiments, SBD interacts with a subset of glycosphingolipids and sphingomyelin, consistent with its raft association in neurons. We also provide evidence that SBD is taken up by neuronal cells in a cholesterol- and sphingolipid-dependent manner via detergent-resistant microdomains. Furthermore, using fluorescence correlation spectroscopy to assay the mobility of SBD in live cells, we show that SBD's behavior at the plasma membrane is similar to that of the previously described raft marker cholera toxin B, displaying both a fast and a slow component. Our data suggest that fluorescently tagged SBD can be used to investigate the dynamic nature of glycosphingolipid-rich detergent-resistant microdomains that are cholesterol-dependent.      

3.1081           CFTR in a lipid raft-TNFR1 complex modulates gap junctional intercellular communication and IL-8 secretion

Dudez, T. et al Biochim. Biophys. Acta, 1783, 779-788 (2008)   Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause a chronic inflammatory response in the lung of patients with Cystic Fibrosis (CF). We have showed that TNF- signaling through the Src family tyrosine kinases (SFKs) was defective as determined by an inability of TNF- to regulate gap junctional communication (GJIC) in CF cells. Here, we sought to elucidate the mechanisms linking TNF- signaling to the functions of CFTR at the molecular level. In a MDCKI epithelial cell model expressing wild-type (WtCFTR) or mutant CFTR lacking its PDZ-interacting motif (CFTR-ΔTRL), TNF- increased the amount of WtCFTR but not CFTR-ΔTRL in detergent-resistant membrane microdomains (DRMs). This recruitment was modulated by SFK activity and associated with DRM localization of TNFR1 and c-Src. Activation of TNFR1 signaling also decreased GJIC and markedly stimulated IL-8 production in WtCFTR cells. In contrast, the absence of CFTR in DRMs was associated with abnormal TNFR1 signaling as revealed by no recruitment of TNFR1 and c-Src to lipid rafts in CFTR-ΔTRL cells and loss of regulation of GJIC and IL-8 secretion. These results suggest that localization of CFTR in lipid rafts in association with c-Src and TNFR1 provides a responsive signaling complex to regulate GJIC and cytokine signaling.  

3.1082           Differential membrane compartmentalization of Ret by PTB-adaptor engagement

Lundgren, T.K., Luebke, M., Stenqvist, A. and Ernfors, P. FEBS J., 275, 2055-2066 (2008)   Glial cell line-derived neurotrophic factor family ligands act through the receptor tyrosine kinase Ret, which plays important roles during embryonic development for cell differentiation, survival, and migration. Ret signaling is markedly affected by compartmentalization of receptor complexes into membrane subdomains. Ret can propagate biochemical signaling from within concentrates in cholesterol-rich membrane microdomains or lipid rafts, or outside such regions, but the mechanisms for, and consequences of, Ret translocation between these membrane compartments remain largely unclear. Here we investigate the interaction of Shc and Frs2 phosphotyrosine-binding domain-containing adaptor molecules with Ret and their function in redistributing Ret to specialized membrane compartments. We found that engagement of Ret with the Frs2 adaptor results in an enrichment of Ret in lipid rafts and that signal transduction pathways and chemotaxis responses depend on the integrity of such rafts. The competing Shc adaptor did not promote Ret translocation to equivalent domains, and Shc-mediated effects were less affected by disruption of lipid rafts. However, by expressing a chimeric Shc protein that localizes to lipid rafts, we showed that biochemical signaling downstream of Ret resembled that of Ret signaling via Frs2. We have identified a previously unknown mechanism in which phosphotyrosine-binding domain-containing adaptors, by means of relocating Ret receptor complexes to lipid rafts, segregate diverse signaling and cellular functions mediated by Ret. These results reveal the existence of a novel mechanism that could, by subcellular relocation of Ret, work to amplify ligand gradients during chemotaxis.  

3.1083           Sequestration of NF- B Signaling Complexes in Lipid Rafts Contributes to Repression of NF- B in T Lymphocytes under Hyperthermia Stress

Yan, G., Huang, J., Ruth Jarbadan, N., Jiang, Y. And Cheng, H.

1. Biol. Chem., 283(18), 12489-12500 (2008)

  Sepsis causes extensive apoptosis of lymphocytes, a pathological condition that is frequently associated with hyperthermia. Heat stress has been implicated to repress the activation of an inflammatory mediator, nuclear factor of B (NF- B), which sensitizes cells to apoptosis mediated by inflammatory cytokine, tumor necrosis factor . However, the molecular mechanism of hyperthermia-associated loss of T cells remains unclear. We show that hyperthermia causes rapid translocation of I B kinase (IKK) and NF- B complexes into the plasma membrane-associated lipid rafts in T cells. Heat stress induces aggregation of Carma1 in lipid rafts, which in turn recruits protein kinase C (PKC ) and Bcl10 to the microdomains, causing subsequent membrane translocation of the IKK and NF- B signalosomes. Depletion of Carma1 and inhibition of PKC impair accumulation of NF- B complexes in lipid rafts. Heat stress prohibits I B kinase activity by sequestrating the IKK and NF- B complexes in lipid rafts and by segregating the chaperone protein Hsp90, an essential cofactor for IKK, from the IKK complex. This process ultimately results in functional deficiency of NF- B and renders T cells resistant to tumor necrosis factor -induced activation of IKK, thereby contributing to the apoptotic loss of T lymphocytes in sepsis-associated hyperthermia.

3.1084           MALS-3 regulates polarity and early neurogenesis in the developing cerebral cortex

Srinivasan, K. et al Development, 135, 1781-1790 (2008)   Apicobasal polarity plays an important role in regulating asymmetric cell divisions by neural progenitor cells (NPCs) in invertebrates, but the role of polarity in mammalian NPCs is poorly understood. Here, we characterize the function of the PDZ domain protein MALS-3 in the developing cerebral cortex. We find that MALS-3 is localized to the apical domain of NPCs. Mice lacking all three MALS genes fail to localize the polarity proteins PATJ and PALS1 apically in NPCs, whereas the formation and maintenance of adherens junctions appears normal. In the absence of MALS proteins, early NPCs progressed more slowly through the cell cycle, and their daughter cells were more likely to exit the cell cycle and differentiate into neurons. Interestingly, these effects were transient; NPCs recovered normal cell cycle properties during late neurogenesis. Experiments in which MALS-3 was targeted to the entire membrane resulted in a breakdown of apicobasal polarity, loss of adherens junctions, and a slowing of the cell cycle. Our results suggest that MALS-3 plays a role in maintaining apicobasal polarity and is required for normal neurogenesis in the developing cortex.  

3.1085           Down-regulation of insulin-degrading enzyme by presenilin 1 V97L mutant potentially underlies increased levels of amyloid beta 42

Qin, W. and Jia, J. Eur. J. Neurosci., 27, 2425-2432 (2008)   Amyloid beta (Aβ)42 plays a pivotal role in Alzheimer's disease. We previously reported a novel presenilin (PS)1 mutant (V97L) that was expressed in related patients with early onset Alzheimer's disease. We found that patients with the V97L mutation had increased levels of extracellular and intracellular Aβ42. Here we found that the increased extracellular level of Aβ42 was always accompanied by a reduction of insulin-degrading enzyme (IDE) activity on the plasma membranes. However, increase of intracellular Aβ42 was associated with decreased expression and activity of IDE in the cytosol and endoplasmic reticulum in the PS1 V97L mutant-transfected human SH-SY5Y cell line. These studies indicate that pathological levels of Aβ42 may be caused by the negative effects of PS1 (V97L) on IDE expression and activity. Our findings provide evidence for the molecular basis of familial Alzheimer's disease pathogenesis.  

3.1086           Aberrant Folding of Pathogenic Parkin Mutants: AGGREGATION VERSUS DEGRADATION

Schlehe, J.S. et al

1. Biol. Chem., 283(20), 13771-13779 (2008)

  Loss-of-function mutations in the Parkin gene (PARK2) are responsible for the majority of autosomal recessive Parkinson disease. A growing body of evidence indicates that misfolding and aggregation of Parkin is a major mechanism of Parkin inactivation, accounting for the loss-of-function phenotype of various pathogenic Parkin mutants. Remarkably, wild-type Parkin is also prone to misfolding under certain cellular conditions, suggesting a more general role of Parkin in the pathogenesis of Parkinson disease. We now show that misfolding of Parkin can lead to two phenotypes: the formation of detergent-insoluble, aggregated Parkin, or destabilization of J. Neurochem., 105Parkin resulting in an accelerated proteasomal degradation. By combining two pathogenic Parkin mutations, we could demonstrate that destabilization of Parkin is dominant over the formation of detergent-insoluble Parkin aggregates. Furthermore, a comparative analysis with HHARI, an E3 ubiquitin ligase with an RBR domain highly homologous to that of Parkin, revealed that folding of Parkin is specifically dependent on the integrity of the C-terminal domain, but not on the presence of a putative PDZ-binding motif at the extreme C terminus.  

3.1087           Activated Nuclear Metabotropic Glutamate Receptor mGlu5 Couples to Nuclear Gq/11 Proteins to Generate Inositol 1,4,5-Trisphosphate-mediated Nuclear Ca2+ Release

Kumar, V., Jong, Y-J.I. and O’Malley, K.L.

1. Biol. Chem., 283(20), 14072-14083 (2008)

  Recently we have shown that the metabotropic glutamate 5 (mGlu5) receptor can be expressed on nuclear membranes of heterologous cells or endogenously on striatal neurons where it can mediate nuclear Ca²⁺ changes. Here, pharmacological, optical, and genetic techniques were used to show that upon activation, nuclear mGlu5 receptors generate nuclear inositol 1,4,5-trisphosphate (IP₃) in situ. Specifically, expression of an mGlu5 F767S mutant in HEK293 cells that blocks G_q/11 coupling or introduction of a dominant negative G _q construct in striatal neurons prevented nuclear Ca²⁺ changes following receptor activation. These data indicate that nuclear mGlu5 receptors couple to G_q/11 to mobilize nuclear Ca²⁺. Nuclear mGlu5-mediated Ca²⁺ responses could also be blocked by the phospholipase C (PLC) inhibitor, U73122 [GenBank] , the phosphatidylinositol (PI) PLC inhibitor 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH₃), or by using small interfering RNA targeted against PLCβ1 demonstrating that PI-PLC is involved. Direct assessment of inositol phosphate production using a PIP2/IP₃ "biosensor" revealed for the first time that IP₃ can be generated in the nucleus following activation of nuclear mGlu5 receptors. Finally, both IP₃ and ryanodine receptor blockers prevented nuclear mGlu5-mediated increases in intranuclear Ca²⁺. Collectively, this study shows that like plasma membrane receptors, activated nuclear mGlu5 receptors couple to G_q/11 and PLC to generate IP₃-mediated release of Ca²⁺ from Ca²⁺-release channels in the nucleus. Thus the nucleus can function as an autonomous organelle independent of signals originating in the cytoplasm, and nuclear mGlu5 receptors play a dynamic role in mobilizing Ca²⁺ in a specific, localized fashion.  

3.1088           Phorbol ester induced trafficking-independent regulation and enhanced phosphorylation of the dopamine transporter associated with membrane rafts and cholesterol

Foster, J.D., Adkins, S.D., Lever, J.R. and Vaughan, R.A.

1. Neurochem., 105, 1683-1699 (2008)

  We examined the mechanisms involved in protein kinase C (PKC)-dependent down-regulation of dopamine transporter (DAT) activity and cell surface expression by treating heterologously expressing cells with the clathrin-mediated endocytosis inhibitor concanavalin A (Con A) or the cholesterol depleter/membrane raft disrupter methyl-β-cyclodextrin (MβC) prior to treatment with the PKC activator phorbol 12-myristate, 13-acetate (PMA). Con A blocked PMA-induced surface reductions of DAT but only partially inhibited down-regulation, while MβC partially blocked down-regulation but did not inhibit loss of cell surface DAT, demonstrating that PKC-induced DAT down-regulation occurs by a combination of trafficking and non-trafficking processes. Using density-gradient centrifugation, we found that DATs are distributed approximately equally between Triton-insoluble, cholesterol-rich membrane rafts and Triton-soluble non-raft membranes. DATs in both populations are present at the cell surface and are active for dopamine and cocaine binding. PMA-induced loss of cell surface DAT occurred only from non-raft populations, demonstrating that non-raft DATs are regulated by trafficking events and indicating the likelihood that the cholesterol-dependent non-trafficking regulatory mechanism occurs in rafts. PMA did not affect the DAT raft-non-raft distribution but stimulated the phosphorylation of DAT to a substantially greater level in rafts than non-rafts. These findings reveal a previously unknown role for cholesterol in DAT function and demonstrate the presence of distinct subcellular DAT populations that possess multiple regulatory differences that may impact dopaminergic neurotransmission.  

3.1089           Epstein-Barr Virus Latent Membrane Protein 1 Induces Expression of the Epidermal Growth Factor Receptor through Effects on Bcl-3 and STAT3

Kung,, C-P. and Raab-Traub, N.

1. Virol., 82(11), 5486-5493 (2008)

  Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) activates multiple signaling pathways. Two regions, C-terminal-activating region 1 (CTAR1) and CTAR2, have been identified within the cytoplasmic carboxy terminal domain that activates NF- B. CTAR2 activates the canonical NF- B pathway, which includes p50/p65 complexes. CTAR1 can activate both the canonical and noncanonical pathways to produce multiple distinct NF- B dimers, including p52/p50, p52/p65, and p50/p50. CTAR1 also uniquely upregulates the epidermal growth factor receptor (EGFR) in epithelial cells. Increased p50-Bcl-3 complexes have been detected by chromatin precipitation on the NF- B consensus motifs within the egfr promoter in CTAR1-expressing epithelial cells and nasopharyngeal carcinoma cells. In this study, the mechanism responsible for the increase in Bcl-3 has been further investigated. The data indicate that LMP1-CTAR1 induces Bcl-3 mRNA and increases the nuclear translocation of both Bcl-3 and p50. LMP1-CTAR1 constitutively activates STAT3, and this activation was not due to the induction of interleukin 6 (IL-6). In LMP1-CTAR1-expressing cells, increased levels of activated STAT3 were detected by chromatin immunoprecipitation on STAT-binding sites located within both the promoter and the second intron of Bcl-3. A STAT3 inhibitor significantly reduced the activation of STAT3, as well as the CTAR1-mediated upregulation of Bcl-3 and EGFR. These data suggest that LMP1 activates distinct forms of NF- B through multiple pathways. In addition to activating the canonical and noncanonical pathways, LMP1-CTAR1 constitutively activates STAT3 and increases Bcl-3. The increased nuclear Bcl-3 and p50 homodimer complexes positively regulate EGFR expression. These results indicate that LMP1 likely regulates distinct cellular genes by activating specific NF- B pathways.

3.1090           Involvement of human CD44 during Cryptococcus neoformans infection of brain microvascular endothelial cells

Jong, A. et al Cellular. Microbiol., 10(6), 1313-1326 (2008)   Pathogenic yeast Cryptococcus neoformans causes devastating cryptococcal meningoencephalitis. Our previous studies demonstrated that C. neoformans hyaluronic acid was required for invasion into human brain microvascular endothelial cells (HBMEC), which constitute the blood–brain barrier. In this report, we demonstrate that C. neoformans hyaluronic acid interacts with CD44 on HBMEC. Our results suggest that HBMEC CD44 is a primary receptor during C. neoformans infection, based on the following observations. First, anti-CD44 neutralizing antibody treatment was able to significantly reduce C. neoformans association with HBMEC. Second, C. neoformans association was considerably impaired using either CD44-knock-down HBMEC or C. neoformans hyaluronic acid-deficient strains. Third, overexpression of CD44 in HBMEC increased their association activity towards C. neoformans. Fourth, confocal microscopic images showed that CD44 was enriched at and around the C. neoformans association sites. Fifth, upon C. neoformans and HBMEC engagement, a subpopulation of CD44 and actin translocated to the host membrane rafts. Our results highlight the interactions between C. neoformans hyaluronic acid and host CD44 and the dynamic results of these interactions, which may represent events during the adhesion and entry of C. neoformans at HBMEC membrane rafts.  

3.1091           Evidence for a Superoxide Permeability Pathway in Endosomal Membranes

Mumbengegwi, D.R., Li, Q., Li, C., Bear, C.E. and Engelhardt, J.F. Mol. Cell. Biol., 28(11), 3700-3712 (2008)   The compartmentalized production of superoxide (·O₂^–) by endosomal NADPH oxidase is important in the redox-dependent activation of NF- B following interleukin 1β (IL-1β) stimulation. It remains unclear how ·O₂^– produced within endosomes facilitates redox-dependent signaling events in the cytoplasm. We evaluated ·O₂^– movement out of IL-1β-stimulated endosomes and whether SOD1 at the endosomal surface mediates redox-signaling events required for NF- B activation. The relative outward permeability of NADPH-dependent ·O₂^– from fractionated endosomes was assessed using membrane-permeable (luminol and lucigenin) and -impermeable (isoluminol) luminescent probes for ·O₂^–. In these studies, 60% of ·O₂^– efflux out of endosomes was inhibited by treatment with either of two anion channel blockers, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) or niflumic acid (NFA). Furthermore, radioisotopic electrodiffusion flux assays on endomembrane proteoliposomes suggested that ·O₂^– and Cl^– are transported through the same DIDS-sensitive channel(s). Rab5-based immunoaffinity isolation of IL-1β-stimulated early endosomes demonstrated SOD1 recruitment to endosomes harboring the IL-1 receptor. Finally, SOD1-deficient cells were found to be defective in their ability to activate NF- B following IL-1β stimulation. Together, these results suggest that ·O₂^– exits endosomes through a DIDS-sensitive chloride channel(s) and that SOD1-mediated dismutation of ·O₂^– at the endosomal surface may produce the localized H₂O₂ required for redox-activation of NF- B.  

3.1092           Targeting of Pseudorabies Virus Structural Proteins to Axons Requires Association of the Viral Us9 Protein with Lipid Rafts

Lyman, M.G., Curanovic, D. And Enquist, L.W. PlosPathogens, 4(5), e1000065   The pseudorabies virus (PRV) Us9 protein plays a central role in targeting viral capsids and glycoproteins to axons of dissociated sympathetic neurons. As a result, Us9 null mutants are defective in anterograde transmission of infection in vivo. However, it is unclear how Us9 promotes axonal sorting of so many viral proteins. It is known that the glycoproteins gB, gC, gD and gE are associated with lipid raft microdomains on the surface of infected swine kidney cells and monocytes, and are directed into the axon in a Us9-dependent manner. In this report, we determined that Us9 is associated with lipid rafts, and that this association is critical to Us9-mediated sorting of viral structural proteins. We used infected non-polarized and polarized PC12 cells, a rat pheochromocytoma cell line that acquires many of the characteristics of sympathetic neurons in the presence of nerve growth factor (NGF). In these cells, Us9 is highly enriched in detergent-resistant membranes (DRMs). Moreover, reducing the affinity of Us9 for lipid rafts inhibited anterograde transmission of infection from sympathetic neurons to epithelial cells in vitro. We conclude that association of Us9 with lipid rafts is key for efficient targeting of structural proteins to axons and, as a consequence, for directional spread of PRV from pre-synaptic to post-synaptic neurons and cells of the mammalian nervous system.  

3.1093           Cholesterol Substitution Increases the Structural Heterogeneity of Caveolae

Jansen, M. et al

1. Biol. Chem., 283(21), 14610-14618 (2008)

  Caveolin-1 binds cholesterol and caveola formation involves caveolin-1 oligomerization and cholesterol association. The role of cholesterol in caveolae has so far been addressed by methods that compromise membrane integrity and abolish caveolar invaginations. To study the importance of sterol specificity for the structure and function of caveolae, we replaced cholesterol in mammalian cells with its immediate precursor desmosterol by inhibiting 24-dehydrocholesterol reductase. Desmosterol could substitute for cholesterol in maintaining cell growth, membrane integrity, and preserving caveolar invaginations. However, in desmosterol cells the affinity of caveolin-1 for sterol and the stability of caveolin oligomers were decreased. Moreover, caveolar invaginations became more heterogeneous in dimensions and in the number of caveolin-1 molecules per caveola. Despite the altered caveolar structure, caveolar ligand uptake was only moderately inhibited. We found that in desmosterol cells, Src kinase phosphorylated Cav1 at Tyr¹⁴ more avidly than in cholesterol cells. Taken the role of Cav1 Tyr¹⁴ phosphorylation in caveolarendocytosis, this may help to preserve caveolar uptake in desmosterolcells. We conclude that a sterol C24 double bond interfereswith caveolin-sterol interaction and perturbs caveolar morphologybut facilitates Cav1 Src phosphorylation and allows caveolarendocytosis. More generally, substitution of cholesterol bya structurally closely related sterol provides a method to selectivelymodify membrane protein-sterol affinity, structure and functionof cholesterol-dependent domains without compromising membraneintegrity.  

3.1094           Synaptic Vesicles Are Constitutively Active Fusion Machines that Function Independently of Ca²⁺

Holt, M., Riedel, D., Stein, A., Schuette, C. and Jahn, R. Current Biology, 18, 715-722 (2008)   Background In neurons, release of neurotransmitter occurs through the fusion of synaptic vesicles with the plasma membrane. Many proteins required for this process have been identified, with the SNAREs syntaxin 1, SNAP-25, and synaptobrevin thought to constitute the core fusion machinery. However, there is still a large gap between our understanding of individual protein-protein interactions and the functions of these proteins revealed by perturbations in intact synaptic preparations. To bridge this gap, we have used purified synaptic vesicles, together with artificial membranes containing coreconstituted SNAREs as reaction partners, in fusion assays. Results By using complementary experimental approaches, we show that synaptic vesicles fuse constitutively, and with high efficiency, with proteoliposomes containing the plasma membrane proteins syntaxin 1 and SNAP-25. Fusion is inhibited by clostridial neurotoxins and involves the formation of SNARE complexes. Despite the presence of endogenous synaptotagmin, Ca²⁺ does not enhance fusion, even if phosphatidylinositol 4,5-bisphosphate is present in the liposome membrane. Rather, fusion kinetics are dominated by the availability of free syntaxin 1/SNAP-25 acceptor sites for synaptobrevin. Conclusions Synaptic vesicles are constitutively active fusion machines, needing only synaptobrevin for activity. Apparently, the final step in fusion does not involve the regulatory activities of other vesicle constituents, although these may be involved in regulating earlier processes. This is particularly relevant for the calcium-dependent regulation of exocytosis, which, in addition to synaptotagmin, requires other factors not present in the vesicle membrane. The in vitro system described here provides an ideal starting point for unraveling of the molecular details of such regulatory events.  

3.1095           Caveolae structure and function

Thomas, C.M. and Smart, E.J.

1. Cell. Mol. Med., 12(3), 796-809 (2008)

  Studies on the structure and function of caveolae have revealed how this versatile subcellular organelle can influence numerous signalling pathways. This brief review will discuss a few of the key features of caveolae as it relates to signalling and disease processes.      

3.1096           Segregation and rapid turnover of EDEM1 by an autophagy-like mechanism modulates standard ERAD and folding activities

Cali, T., Galli, C., Olivari, S. And Molinari, M. Biochem. Biophys. Res. Comm., 371, 405-410 (2008)   EDEM1 is a crucial regulator of endoplasmic reticulum (ER)-associated degradation (ERAD) that extracts non-native glycopolypeptides from the calnexin chaperone system. Under normal growth conditions, the intralumenal level of EDEM1 must be low to prevent premature interruption of ongoing folding programs. We report that in unstressed cells, EDEM1 is segregated from the bulk ER into LC3-I-coated vesicles and is rapidly degraded. The rapid turnover of EDEM1 is regulated by a novel mechanism that shows similarities but is clearly distinct from macroautophagy. Cells with defective EDEM1 turnover contain unphysiologically high levels of EDEM1, show enhanced ERAD activity and are characterized by impaired capacity to efficiently complete maturation of model glycopolypeptides. We define as ERAD tuning the mechanisms operating in the mammalian ER at steady state to offer kinetic advantage to folding over disposal of unstructured nascent chains by selective and rapid degradation of ERAD regulators.  

3.1097           Caveolin-1 Expression in Trabecular Meshwork Cells

Nolan, M.J. et al Invest. Ophthalmol. Vis. Sci., 49, E-abstract 1627 (2008)   Purpose:Caveolin-1, a 22-kDa, intergral membrane protein forming oligomersin cells, is a principal component of caveolae in lipid raftsand is also important in regulating vesicle trafficking throughcells. Caveolin-1 is involved, consequently, in signal transduction,tight junction formation, endocytosis, transcytosis, as wellserving as a mechanosensor in endothelial cells. Lipid-modifiedproteins such as endothelial nitric oxide synthase and the Srcfamily of kinases can target to caveolae and interact with caveolin-1to regulate signal transduction. The purpose of this study wasto determine whether trabecular meshwork (TM) cells expresscaveolin-1 and if caveolin-1 expression is influenced by dexamethasone(Dex) treatment. Methods:Human TM cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum (FCS) until confluent, washed twice with PBS, and incubated in DMEM containing 0.1% FCS and 100 µM Dex for 1, 24 and 96 hours. The media was aspirated; the cells were washed with cold PBS, subjected to lysis buffer (Sigma CS0750) containing 1% Triton X-100, and separated by Optiprep density gradient (Sigma D1556). The preparationwas centrifuged at 200,000 x g for 18 hrs; nine 0.5 ml fractionswere pipetted from the top (lightest) to bottom (heaviest).Each fraction was analyzed for protein content, resolved bySDS polyarylamide electrophoresis, and immunoblotted with mousemonoclonal anti-caveolin-1 antibody (Sigma). Results:The distribution of 22-kDa caveolin-1 in TM cells was in fractions4 through 7 and the strongest intensity was in fraction 5; ahigh molecular weight (>300-kDa) was observed in fractions5 through 7. The distribution of 22-kDa caveolin-1 in Dex-treatedTM cells was in fractions 5 through 7 and the strongest intensitywas in fractions 5 and 6, with minor amounts in fraction 8 and9; a high molecular weight (>300-kDa) was also observed infractions 5 through 7. The amount of caveolin-1 in Dex-treatedTM cells was considerably greater, approximately two-fold. Similarly,the amount of caveolin-1 in the media was also increased inthe Dex-treated TM cells. Conclusions:This is the first demonstration of caveolin-1 in TM cells. Dextreatment increased the cell caveolin-1 content and changedcaveolin-1 density gradient distribution as well as increasingthe secretion of media caveolin-1. These results indicate thatcaveolin-1 may play an important role in TM barrier function,cell signaling, and endocytosis.  

3.1098           Overexpression of a host factor TOM1 inhibits tomato mosaic virus propagation and suppression of RNA silencing

Hagiwara-Komoda, Y. et al Virology, 376, 132-139 (2008)   A plant integral membrane protein TOM1 is involved in the multiplication of Tomato mosaic virus (ToMV). TOM1 interacts with ToMV replication proteins and has been suggested to tether the replication proteins to the membranes where the viral RNA synthesis takes place. We have previously demonstrated that inactivation of TOM1 results in reduced ToMV multiplication. In the present study, we show that overexpression of TOM1 in tobacco also inhibits ToMV propagation. TOM1 overexpression led to a decreased accumulation of the soluble form of the replication proteins and interfered with the ability of the replication protein to suppress RNA silencing. The reduced accumulation of the soluble replication proteins was also observed in a silencing suppressor-defective ToMV mutant. Based on these results, we propose that RNA silencing suppression is executed by the soluble form of the replication proteins and that efficient ToMV multiplication requires balanced accumulation of the soluble and membrane-bound replication proteins.  

3.1099           The human SIRT3 protein deacetylase is exclusively mitochondrial

Cooper, H.M: and Spelbrink, J.N. Biochem. J., 411, 279-285 (2008)   It has recently been suggested that perhaps as many as 20% of all mitochondrial proteins are regulated through lysine acetylation while SIRT3 has been implicated as an important mitochondrial protein deacetylase. It is therefore of crucial importance that the mitochondrial localization of potential protein deacetylases is unambiguously established. Although mouse SIRT3 was recently shown to be mitochondrial, HsSIRT3 (human SIRT3) was reported to be both nuclear and mitochondrial and to relocate from the nucleus to the mitochondrion upon cellular stress. In the present study we show, using various HsSIRT3 expression constructs and a combination of immunofluorescence and careful subcellular fractionation, that in contrast with earlier reports HsSIRT3 is exclusively mitochondrial. We discuss possible experimental explanations for these discrepancies. In addition we suggest, on the basis of the analysis of public genome databases, that the full-length mouse SIRT3 protein is a 37 kDa mitochondrial precursor protein contrary to the previously suggested 29 kDa protein.  

3.1100           CLN3p Impacts Galactosylceramide Transport, Raft Morphology, and Lipid Content

Rusyn, E., Mousallem, T., Persaud-Sawin, D-A., Miller, S. and Boustany, R-M.N. Pediatr. Res., 63(6), 625-631 (2008)   Juvenile neuronal ceroid lipofuscinosis (JNCL) belongs to the neuronal ceroid lipofuscinoses characterized by blindness/seizures/motor/cognitive decline and early death. JNCL is caused by CLN3 gene mutations that negatively modulate cell growth/apoptosis. CLN3 protein (CLN3p) localizes to Golgi/Rab4-/Rab11-positive endosomes and lipid rafts, and harbors a galactosylceramide (GalCer) lipid raft-binding domain. Goals are proving CLN3p participates in GalCer transport from Golgi to rafts, and GalCer deficits negatively affect cell growth/apoptosis. GalCer/mutant CLN3p are retained in Golgi, with CLN3p rescuing GalCer deficits in rafts. Diminishing GalCer in normal cells by GalCer synthase siRNA negatively affects cell growth/apoptosis. GalCer restores JNCL cell growth. WT CLN3p binds GalCer, but not mutant CLN3p. Sphingolipid content of rafts/Golgi is perturbed with diminished GalCer in rafts and accumulation in Golgi. CLN3-deficient raft vesicular structures are small by transmission electron microscopy, reflecting altered sphingolipid composition of rafts. CLN1/CLN2/CLN6 proteins bind to lysophosphatidic acid/sulfatide, CLN6/CLN8 proteins to GalCer, and CLN8 protein to ceramide. Sphingolipid composition/morphology of CLN1-/CLN2-/CLN6-/CLN8- and CLN9-deficient rafts are altered suggesting changes in raft structure/lipid stoichiometry could be common themes underlying these diseases.  

3.1101           Lovastatin inhibits amyloid precursor protein (APP) [beta]-cleavage through reduction of APP distribution in Lubrol WX extractable low density lipid rafts

Won, J-S. et al

1. Neurochem., 105, 1536-1549 (2008)

  Previous studies have described that statins (inhibitors of cholesterol and isoprenoid biosynthesis) inhibit the output of amyloid-[beta] (A[beta]) in the animal model and thus decrease risk of Alzheimer's disease. However, their action mechanism(s) in A[beta] precursor protein (APP) processing and A[beta] generation is not fully understood. In this study, we report that lovastatin treatment reduced A[beta] output in cultured hippocampal neurons as a result of reduced APP levels and [beta]-secretase activities in low density Lubrol WX (non-ionic detergent) extractable lipid rafts (LDLR). Rather than altering cholesterol levels in lipid raft fractions and thus disrupting lipid raft structure, lovastatin decreased A[beta] generation through down-regulating geranylgeranyl-pyrophosphate dependent endocytosis pathway. The inhibition of APP endocytosis by treatment with lovastatin and reduction of APP levels in LDLR fractions by treatment with phenylarsine oxide (a general endocytosis inhibitor) support the involvement of APP endocytosis in APP distribution in LDLR fractions and subsequent APP [beta]-cleavage. Moreover, lovastatin-mediated down-regulation of endocytosis regulators, such as early endosomal antigen 1, dynamin-1, and phosphatidylinositol 3-kinase activity, indicates that lovastatin modulates APP endocytosis possibly through its pleiotropic effects on endocytic regulators. Collectively, these data report that lovastatin mediates inhibition of LDLR distribution and [beta]-cleavage of APP in a geranylgeranyl-pyrophosphate and endocytosis-dependent manner.  

3.1102           Synaptotagmin VII Regulates Bone Remodeling by Modulating Osteoclast and Osteoblast Secretion

Zhao, H. et al Developmental Cell, 14, 914-925 (2008)   Maintenance of bone mass and integrity requires a tight balance between resorption by osteoclasts and formation by osteoblasts. Exocytosis of functional proteins is a prerequisite for the activity of both cells. In the present study, we show that synaptotagmin VII, a calcium sensor protein that regulates exocytosis, is associated with lysosomes in osteoclasts and bone matrix protein-containing vesicles in osteoblasts. Absence of synaptotagmin VII inhibits cathepsin K secretion and formation of the ruffled border in osteoclasts and bone matrix protein deposition in osteoblasts, without affecting the differentiation of either cell. Reflecting these in vitro findings, synaptotagmin VII-deficient mice are osteopenic due to impaired bone resorption and formation. Therefore, synaptotagmin VII plays an important role in bone remodeling and homeostasis by modulating secretory pathways functionally important in osteoclasts and osteoblasts.  

3.1103           Multimerization of Tegument Protein pp28 within the Assembly Compartment Is Required for Cytoplasmic Envelopment of Human Cytomegalovirus

Seo, J-Y. and Britt, W.J.

1. Virol., 82(13), 6272-7287 (2008)

  Human cytomegalovirus (HCMV) UL99-encoded pp28 is an essential tegument protein required for envelopment and production of infectious virus. Nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with the UL99 gene deleted. Previous results have suggested that a key function of pp28 in the envelopment of infectious HCMV is expressed after the protein localizes in the assembly compartment (AC). In this study, we investigated the potential role of pp28 multimerization in the envelopment of the infectious virion. Our results indicated that pp28 multimerized during viral infection and that interacting domains responsible for self-interaction were localized in the amino terminus of the protein (amino acids [aa] 1 to 43). The results from transient-expression and/or infection assays indicated that the self-interaction took place in the AC. A mutant pp28 molecule containing only the first 35 aa failed to accumulate in the AC, did not interact with pp28 in the AC, and could not support virus replication. In contrast, the first 50 aa of pp28 was sufficient for the self-interaction within the AC and the assembly of infectious virus. Recombinant viruses encoding an in-frame deletion of aa 26 to 33 of pp28 were replication competent, whereas infectious virus was not recovered from HCMV BACs lacking aa 26 to 43. These findings suggested that the accumulation of pp28 was a prerequisite for multimerization of pp28 within the AC and that pp28 multimerization in the AC represented an essential step in the envelopment and production of infectious virions.  

3.1104           Functional stabilization of Kv1.5 protein by Hsp70 in mammalian cell lines

Hirota, Y. et al Biochem. Biophys. Acta., 372, 469-474 (2008)   The aim of this study was to elucidate the mechanisms for regulations of cardiac Kv1.5 channel expression. We particularly focused on the role of heat shock proteins (Hsps). We tested the effects of Hsps on the stability of Kv1.5 channels using biochemical and electrophysiological techniques: co-expression of Kv1.5 and Hsp family proteins in mammalian cell lines, followed by Western blotting, immunoprecipitation, pulse-chase analysis, immunofluorescence and whole-cell patch clamp. Hsp70 and heat shock factor 1 increased the expression of Kv1.5 protein in HeLa and COS7 cells, whereas either Hsp40, 27 or 90 did not. Hsp70 prolonged the half-life of Kv1.5 protein. Hsp70 was co-immunoprecipitated and co-localized with Kv1.5-FLAG. Hsp70 significantly increased the immunoreactivity of Kv1.5 in the endoplasmic reticulum, Golgi apparatus and on the cell membrane. Hsp70 enhanced Kv1.5 current of transfected cells, which was abolished by pretreatment with brefeldin A or colchicine. Thus, Hsp70, but not other Hsps, stabilizes functional Kv1.5 protein.  

3.1105           Sphingomyelin Functions as a Novel Receptor for Helicobacter pylori VacA

Gupta, V.R. et al PLOSpathogens, 4(5), e1000073 (2008)   The vacuolating cytotoxin (VacA) of the gastric pathogen Helicobacter pylori binds and enters epithelial cells, ultimately resulting in cellular vacuolation. Several host factors have been reported to be important for VacA function, but none of these have been demonstrated to be essential for toxin binding to the plasma membrane. Thus, the identity of cell surface receptors critical for both toxin binding and function has remained elusive. Here, we identify VacA as the first bacterial virulence factor that exploits the important plasma membrane sphingolipid, sphingomyelin (SM), as a cellular receptor. Depletion of plasma membrane SM with sphingomyelinase inhibited VacA-mediated vacuolation and significantly reduced the sensitivity of HeLa cells, as well as several other cell lines, to VacA. Further analysis revealed that SM is critical for VacA interactions with the plasma membrane. Restoring plasma membrane SM in cells previously depleted of SM was sufficient to rescue both toxin vacuolation activity and plasma membrane binding. VacA association with detergent-resistant membranes was inhibited in cells pretreated with SMase C, indicating the importance of SM for VacA association with lipid raft microdomains. Finally, VacA bound to SM in an in vitro ELISA assay in a manner competitively inhibited by lysenin, a known SM-binding protein. Our results suggest a model where VacA may exploit the capacity of SM to preferentially partition into lipid rafts in order to access the raft-associated cellular machinery previously shown to be required for toxin entry into host cells.  

3.1106           Mammalian cell expression of an active site mutant of Pseudomonas exotoxin disrupts LRP1 maturation

Pastrana, D.V., Yun, C.H., McKee, M.L. and FitzGerald, D.J.

1. Biomed. Sci., 15, 427-439 (2008)

  Low density lipoprotein receptor-related protein 1, (LRP1) is a large multifunctional receptor that binds more than 25 physiologic ligands. In addition, it functions as the surface receptor for several Rhinoviruses, HIV-tat and Pseudomonas exotoxin (PE). We report that the expression of PE within mammalian cells can serve as a probe of LRP1 maturation and functionality. To avoid cell killing, an enzymatically inactive form of the toxin (PEΔ553) was expressed. A permanent cell line (termed CY301) was established whereby PEΔ553 was expressed continually into the ER of CHO cells. CY301 cells were 100-fold resistant to exogenously added active PE but exhibited no cross-resistance to other toxins. Our studies indicate that PEΔ553 bound to immature LRP1 in the ER, prevented its maturation to the cell surface and thereby produced a toxin resistant phenotype. By confocal microscopy, cell-associated PEΔ553 was localized to the ER and co-localized with LRP1. Further characterization of CY301 cells indicated that RAP, the chaperone that aids in LRP1 folding, was released to the growth media. Thus the intracellular expression of PEΔ553 appears to be a valuable probe of LRP1 maturation and trafficking.  

3.1107           Engineered Bacterial Outer Membrane Vesicles with Enhanced Functionality

Kim, J-Y. et al

1. Mol. Biol., 380, 51-66 (2008)

  We have engineered bacterial outer membrane vesicles (OMVs) with dramatically enhanced functionality by fusing several heterologous proteins to the vesicle-associated toxin ClyA of Escherichia coli. Similar to native unfused ClyA, chimeric ClyA fusion proteins were found localized in bacterial OMVs and retained activity of the fusion partners, demonstrating for the first time that ClyA can be used to co-localize fully functional heterologous proteins directly in bacterial OMVs. For instance, fusions of ClyA to the enzymes β-lactamase and organophosphorus hydrolase resulted in synthetic OMVs that were capable of hydrolyzing β-lactam antibiotics and paraoxon, respectively. Similarly, expression of an anti-digoxin single-chain Fv antibody fragment fused to the C terminus of ClyA resulted in designer “immuno-MVs” that could bind tightly and specifically to the antibody's cognate antigen. Finally, OMVs displaying green fluorescent protein fused to the C terminus of ClyA were highly fluorescent and, as a result of this new functionality, could be easily tracked during vesicle interaction with human epithelial cells. We expect that the relative plasticity exhibited by ClyA as a fusion partner should prove useful for: (i) further mechanistic studies to identify the vesiculation machinery that regulates OMV secretion and to map the intracellular routing of ClyA-containing OMVs during invasion of host cells; and (ii) biotechnology applications such as surface display of proteins and delivery of biologics.  

3.1108           Cholesterol Depletion Reduces Helicobacter pylori CagA Translocation and CagA-Induced Responses in AGS Cells

Lai, C-H. et al Infect. Immun., 76(7), 3293-3303 (2008)   Infection with Helicobacter pylori cagA-positive strains is associated with gastritis, ulcerations, and gastric cancer. CagA is translocated into infected epithelial cells by a type IV secretion system and can be tyrosine phosphorylated, inducing signal transduction and motogenic responses in epithelial cells. Cellular cholesterol, a vital component of the membrane, contributes to membrane dynamics and functions and is important in VacA intoxication and phagocyte evasion during H. pylori infection. In this investigation, we showed that cholesterol extraction by methyl-β-cyclodextrin reduced the level of CagA translocation and phosphorylation. Confocal microscope visualization revealed that a significant portion of translocated CagA was colocalized with the raft marker GM1 and c-Src during infection. Moreover, GM1 was rapidly recruited into sites of bacterial attachment by live-cell imaging analysis. CagA and VacA were cofractionated with detergent-resistant membranes (DRMs), suggesting that the distribution of CagA and VacA is associated with rafts in infected cells. Upon cholesterol depletion, the distribution shifted to non-DRMs. Accordingly, the CagA-induced hummingbird phenotype and interleukin-8 induction were blocked by cholesterol depletion. Raft-disrupting agents did not influence bacterial adherence but did significantly reduce internalization activity in AGS cells. Together, these results suggest that delivery of CagA into epithelial cells by the bacterial type IV secretion system is mediated in a cholesterol-dependent manner.  

3.1109           Rhesus lymphocryptovirus latent membrane protein 2A activates β-catenin signaling and inhibits differentiation in epithelial cells

Siler, C.A. and Raab-Traub, N. Virology, 377, 273-279 (2008)   Rhesus lymphocryptovirus (LCV) is a γ-herpesvirus closely related to Epstein–Barr virus (EBV). The rhesus latent membrane protein 2A (LMP2A) is highly homologous to EBV LMP2A. EBV LMP2A activates the phosphatidylinositol 3-kinase (PI3K) and β-catenin signaling pathways in epithelial cells and affects differentiation. In the present study, the biochemical and biological properties of rhesus LMP2A in epithelial cells were investigated. The expression of rhesus LMP2A in epithelial cells induced Akt activation, GSK3β inactivation and accumulation of β-catenin in the cytoplasm and nucleus. The nuclear translocation, but not accumulation of β-catenin was dependent on Akt activation. Rhesus LMP2A also impaired epithelial cell differentiation; however, this process was not dependent upon Akt activation. A mutant rhesus LMP2A lacking six transmembrane domains functioned similarly to wild-type rhesus LMP2A indicating that the full number of transmembrane domains is not required for effects on β-catenin or cell differentiation. These results underscore the similarity of LCV to EBV and the suitability of the macaque as an animal model for studying EBV pathogenesis.  

3.1110           Canine MDCK cell lines are refractory to infection with human and mouse prions

Polymenidou, M. et al Vaccine, 26, 2601-2614 (2008)   Influenza vaccine production in embryonated eggs is associated with many disadvantages, and production in cell culture systems is a viable alternative. Madin Darby canine kidney (MDCK) cells are permissive for a variety of orthomyxoviruses and have proven particularly suitable for vaccine mass production. However, mammalian cells harboring the Prnp gene can theoretically acquire prion infections. Here, we have attempted to infect MDCK cells and substrains thereof with prions. We found that MDCK cells did not produce any protease-resistant PrP^Sc upon exposure to brain homogenates derived from humans suffering from Creutzfeldt–Jakob disease (CJD) or from mice infected with Rocky Mountain Laboratory (RML) scrapie prions. Further, transmission of MDCK lysates to N2aPK1 cells did not induce formation of PrP^Sc in the latter. PrP^C biogenesis and processing in MDCK cells were similar to those of prion-sensitive N2aPK1 cells. However, steady-state levels of PrP^C were very low, and PrP^C did not partition with detergent-resistant membranes upon density gradient analysis. These factors may account for their resistance to infection. Alternatively, prion resistance may be related to the specific sequence of canine Prnp, as suggested by the lack of documented prion diseases in dogs.  

3.1111           Differential loss of cytochrome-c oxidase subunits in ischemia-reperfusion injury: exacerbation of COI subunit loss by PKC- inhibition

Yu, Q., Nguyen, T., Ogbi, M., Caldqwell, R.W. and Johnson, J.A. Am. J. Physiol. Heart Circ. Physiol., 294, H2637-H2645 (2008)   We have previously described a PKC- interaction with cytochrome oxidase subunit IV (COIV) that correlates with enhanced CO activity and cardiac ischemic preconditioning (PC). We therefore investigated the effects of PC and ischemia-reperfusion (I/R) injury on CO subunit levels in an anesthetized rat coronary ligation model. Homogenates prepared from the left ventricular regions at risk (RAR) and not at risk (RNAR) for I/R injury were fractionated into cell-soluble (S), 600 g low-speed centrifugation (L), gradient-purified mitochondrial (M), and 100,000 g particulate (P) fractions. In RAR tissue, PC (2 cycles of 5-min ischemia and 5-min reperfusion) decreased the COI in the P fraction ( 29% of total cellular COI), suggesting changes in interfibrillar mitochondria. After 30 min of ischemia and 120 min of reperfusion, total COI levels decreased in the RAR by 72%. Subunit Va was also downregulated by 42% following prolonged I/R in the RAR. PC administered before I/R reduced the loss of COI in the M and P fractions 30% and prevented COVa losses completely. We observed no losses in subunits Vb and VIIa following I/R alone; however, significant losses occurred when PC was administered before prolonged I/R. Delivery of a cell-permeable PKC- translocation inhibitor ( V1-2) to isolated rat hearts before prolonged I/R dramatically increased COI loss, suggesting that PKC- protects COI levels. We propose that additional measures to protect CO subunits when coadministered with PC may improve its cardioprotection against I/R injury.  

3.1112           CD20 Homo-oligomers Physically Associate with the B Cell Antigen Receptor: DISSOCIATION UPON RECEPTOR ENGAGEMENT AND RECRUITMENT OF PHOSPHOPROTEINS AND CALMODULIN-BINDING PROTEINS

Polyak, M.J., Li, H., Shariat, N. And Deans, J.P.

1. Biol. Chem., 283(27), 18545-18552 (2008)

  B cell antigen receptor (BCR) signaling initiates sustained cellular calcium influx necessary for the development, differentiation, and activation of B lymphocytes. CD20 is a B cell-restricted tetraspanning protein organized in the plasma membrane as multimeric molecular complexes involved in BCR-activated calcium entry. Using coprecipitation of native CD20 with tagged or truncated forms of the molecule, we provide here direct evidence of CD20 homo-oligomerization into tetramers. Additionally, the function of CD20 was explored by examining its association with surface-labeled and intracellular proteins before and after BCR signaling. Two major surface-labeled proteins that coprecipitated with CD20 were identified as the heavy and light chains of cell surface IgM, the antigen-binding components of the BCR. After activation, BCR-CD20 complexes dissociated, and phosphoproteins and calmodulin-binding proteins were transiently recruited to CD20. These data provide new evidence of the involvement of CD20 in signaling downstream of the BCR and, together with the previously described involvement of CD20 in calcium influx, the first evidence of physical coupling of the BCR to a calcium entry pathway.  

3.1113           The Subcellular Distribution of Calnexin Is Mediated by PACS-2

Myhill, N. et aql Mol. Cell Biol., 19, 2777-2788 (2008)   Calnexin is an endoplasmic reticulum (ER) lectin that mediates protein folding on the rough ER. Calnexin also interacts with ER calcium pumps that localize to the mitochondria-associated membrane (MAM). Depending on ER homeostasis, varying amounts of calnexin target to the plasma membrane. However, no regulated sorting mechanism is so far known for calnexin. Our results now describe how the interaction of calnexin with the cytosolic sorting protein PACS-2 distributes calnexin between the rough ER, the MAM, and the plasma membrane. Under control conditions, more than 80% of calnexin localizes to the ER, with the majority on the MAM. PACS-2 knockdown disrupts the calnexin distribution within the ER and increases its levels on the cell surface. Phosphorylation by protein kinase CK2 of two calnexin cytosolic serines (Ser554/564) reduces calnexin binding to PACS-2. Consistent with this, a Ser554/564 Asp phosphomimic mutation partially reproduces PACS-2 knockdown by increasing the calnexin signal on the cell surface and reducing it on the MAM. PACS-2 knockdown does not reduce retention of other ER markers. Therefore, our results suggest that the phosphorylation state of the calnexin cytosolic domain and its interaction with PACS-2 sort this chaperone between domains of the ER and the plasma membrane.  

3.1114           Triglyceride-rich lipoprotein lipolysis increases aggregation of endothelial cell membrane microdomains and produces reactive oxygen species

Wang, L., Sapuri-Butti, A.R., Aung, H.H., Parikh, A.N. and Rutlegde, J.C. Am. J. Physiol. Heart Circ. Physiol., 295, H237-H244 (2008)   Triglyceride-rich lipoprotein (TGRL) lipolysis may provide a proinflammatory stimulus to endothelium. Detergent-resistant plasma membrane microdomains (lipid rafts) have a number of functions in endothelial cell inflammation. The mechanisms of TGRL lipolysis-induced endothelial cell injury were investigated by examining endothelial cell lipid rafts and production of reactive oxygen species (ROS). Lipid raft microdomains in human aortic endothelial cells were visualized by confocal microscopy with fluorescein isothiocyanate-labeled cholera toxin B as a lipid raft marker. Incubation of Atto565-labeled TGRL with lipid raft-labeled endothelial cells showed that TGRL colocalized with the lipid rafts, TGRL lipolysis caused clustering and aggregation of lipid rafts, and colocalization of TGRL remnant particles on the endothelial cells aggregated lipid rafts. Furthermore, TGRL lipolysis caused translocation of low-density lipoprotein receptor-related protein, endothelial nitric oxide synthase, and caveolin-1 from raft regions to nonraft regions of the membrane 3 h after treatment with TGRL lipolysis. TGRL lipolysis significantly increased the production of ROS in endothelial cells, and both NADPH oxidase and cytochrome P-450 inhibitors reduced production of ROS. Our studies suggest that alteration of lipid raft morphology and composition and ROS production could contribute to TGRL lipolysis-mediated endothelial cell injury.  

3.1115           Characterization of seipin/BSCL2, a protein associated with spastic paraplegia 17

Ito, D., Fujisawa, T., Iida, H and Suzuki, N. Neurobiol. Disease, 31(2), 266-277 (2008)   Seipin, which is encoded by the BSCL2 gene, is a glycoprotein of unknown biochemical function that is associated with dominant hereditary motor neuron diseases. Mutations in the N-glycosylation site of seipin are associated with the disease states and result in accumulation of unfolded protein in the endoplasmic reticulum (ER), leading to the unfolded protein response (UPR) and cell death, suggesting that these diseases are tightly associated with ER stress. Here, we determined the subcellular localization, functional domains, and distribution of seipin in tissues. Our studies show that the transmembrane domains in seipin are critical for ER retention, ubiquitination, formation of inclusions, and activation of UPR. Using immunohistochemistry, seipin expression is detected in neurons in the spinal cord and in the frontal lobe cortex of the brain. The present study provides new insights into the biology of seipin protein that should help our understanding of the pathogenesis of seipin-related diseases.  

3.1116           EGF induces coalescence of different lipid rafts

Hofman, E.G. et al

1. Cell Sci., 121, 2519-2528 (2008)

  The suggestion that microdomains may function as signaling platforms arose from the presence of growth factor receptors, such as the EGFR, in biochemically isolated lipid raft fractions. To investigate the role of EGFR activation in the organization of lipid rafts we have performed FLIM analyses using putative lipid raft markers such as ganglioside GM1 and glycosylphosphatidylinositol (GPI)-anchored GFP (GPI-GFP). The EGFR was labeled using single domain antibodies from Llama glama that specifically bind the EGFR without stimulating its kinase activity. Our FLIM analyses demonstrate a cholesterol-independent colocalization of GM1 with EGFR, which was not observed for the transferrin receptor. By contrast, a cholesterol-dependent colocalization was observed for GM1 with GPI-GFP. In the resting state no colocalization was observed between EGFR and GPI-GFP, but stimulation of the cell with EGF resulted in the colocalization at the nanoscale level of EGFR and GPI-GFP. Moreover, EGF induced the enrichment of GPI-GFP in a detergent-free lipid raft fraction. Our results suggest that EGF induces the coalescence of the two types of GM1-containing microdomains that might lead to the formation of signaling platforms.        

3.1117           Regulation of the V-ATPase along the Endocytic Pathway Occurs through Reversible Subunit Association and Membrane Localization

Lafourcade, C., Sobo, K., Kieffer-Jaquinod, S., Garin, J. and van der Goot, F.G. PloS One, 3(7), e2758 (2008)   The lumen of endosomal organelles becomes increasingly acidic when going from the cell surface to lysosomes. Luminal pH thereby regulates important processes such as the release of internalized ligands from their receptor or the activation of lysosomal enzymes. The main player in endosomal acidification is the vacuolar ATPase (V-ATPase), a multi-subunit transmembrane complex that pumps protons from the cytoplasm to the lumen of organelles, or to the outside of the cell. The active V-ATPase is composed of two multi-subunit domains, the transmembrane V₀ and the cytoplasmic V₁. Here we found that the ratio of membrane associated V₁/Vo varies along the endocytic pathway, the relative abundance of V₁ being higher on late endosomes than on early endosomes, providing an explanation for the higher acidity of late endosomes. We also found that all membrane-bound V-ATPase subunits were associated with detergent resistant membranes (DRM) isolated from late endosomes, raising the possibility that association with lipid-raft like domains also plays a role in regulating the activity of the proton pump. In support of this, we found that treatment of cells with U18666A, a drug that leads to the accumulation of cholesterol in late endosomes, affected acidification of late endosome. Altogether our findings indicate that the activity of the vATPase in the endocytic pathway is regulated both by reversible association/dissociation and the interaction with specific lipid environments.  

3.1118           Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death

Apostolou, A., Shen, Y., Liang, Y., Luo, J. and Fang, S. Exp. Cell Res., 314, 2454-2467 (2008)   The accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress that initiates the unfolded protein response (UPR). UPR activates both adaptive and apoptotic pathways, which contribute differently to disease pathogenesis. To further understand the functional mechanisms of UPR, we identified 12 commonly UPR-upregulated genes by expression microarray analysis. Here, we describe characterization of Armet/MANF, one of the 12 genes whose function was not clear. We demonstrated that the Armet/MANF protein was upregulated by various forms of ER stress in several cell lines as well as by cerebral ischemia of rat. Armet/MANF was localized in the ER and Golgi and was also a secreted protein. Silencing Armet/MANF by siRNA oligos in HeLa cells rendered cells more susceptible to ER stress-induced death, but surprisingly increased cell proliferation and reduced cell size. Overexpression of Armet/MANF inhibited cell proliferation and improved cell viability under glucose-free conditions and tunicamycin treatment. Based on its inhibitory properties for both proliferation and cell death we have demonstrated, Armet is, thus, a novel secreted mediator of the adaptive pathway of UPR.  

3.1119           Lipoprotein binding preference of CD36 is altered by filipin treatment

Zhang, J., Chu, W. and Crandall, I. Lipids in Health and Disease, 7, 23-31 (2008)   The class B scavenger receptor CD36 binds multiple ligands, including oxidized and native lipoprotein species. CD36 and the related receptor SR-B1 have been localized to caveolae, domains that participate in cell signaling, transcytosis, and regulation of cellular cholesterol homeostasis. Previous work has indicated that the ligand preference of CD36 may depend on the cell type in which it is expressed. To determine if the presence or absence of caveolae is the determining factor for lipoprotein preference, we treated CHO-CD36 and C32 cells with filipin. Filipin treatment rapidly increased the binding capacity of CD36 for the native lipoproteins HDL and LDL, but did not affect the binding capacity of CD36 for oxidized LDL. Filipin treatment affected the distribution of caveolin and CD36 suggesting that the presence caveolae may modulate the ligand preference of CD36. However, its molecular mechanism how CD36 and caveolin interaction in regulating lipoprotein transport remains to be further studied.  

3.1120           Interaction of Hepatitis C Virus Nonstructural Protein 5A with Core Protein Is Critical for the Production of Infectious Virus Particles

Masaki, T. et al

1. Virol., 82(16), 7964-7976 (2008)

  Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) possesses multiple and diverse functions in RNA replication, interferon resistance, and viral pathogenesis. Recent studies suggest that NS5A is involved in the assembly and maturation of infectious viral particles; however, precisely how NS5A participates in virus production has not been fully elucidated. In the present study, we demonstrate that NS5A is a prerequisite for HCV particle production as a result of its interaction with the viral capsid protein (core protein). The efficiency of virus production correlated well with the levels of interaction between NS5A and the core protein. Alanine substitutions for the C-terminal serine cluster in domain III of NS5A (amino acids 2428, 2430, and 2433) impaired NS5A basal phosphorylation, leading to a marked decrease in NS5A-core interaction, disturbance of the subcellular localization of NS5A, and disruption of virion production. Replacing the same serine cluster with glutamic acid, which mimics the presence of phosphoserines, partially preserved the NS5A-core interaction and virion production, suggesting that phosphorylation of these serine residues is important for virion production. In addition, we found that the alanine substitutions in the serine cluster suppressed the association of the core protein with viral genome RNA, possibly resulting in the inhibition of nucleocapsid assembly. These results suggest that NS5A plays a key role in regulating the early phase of HCV particle formation by interacting with core protein and that its C-terminal serine cluster is a determinant of the NS5A-core interaction.  

3.1121           Novel Role of Presenilins in Maturation and Transport of Integrin β1

Zou, K. et al Biochemistry, 47, 3370-3378 (2008)   Presenilins (PSs) play important roles in modulating the trafficking and maturation of several membrane proteins. However, the target membrane proteins whose trafficking and maturation are regulated by PS are largely unknown. By characterizing PS-deficient fibroblasts, we found that integrin β1 maturation is promoted markedly in PS1 and PS2 double-deficient fibroblasts and moderately in PS1- or PS2-deficient fibroblasts; in contrast, nicastrin maturation is completely inhibited in PS1 and PS2 double-deficient fibroblasts. Subcellular fractionation analysis demonstrated that integrin β1 maturation is promoted in the Golgi apparatus. The mature integrin β1 with an increased expression level was delivered to the cell surface, which resulted in an increased cell surface expression level of mature integrin β1 in PS1 and PS2 double-deficient fibroblasts. PS1 and PS2 double-deficient fibroblasts exhibited an enhanced ability to adhere to culture dishes coated with integrin β1 ligands, namely, fibronectin and laminin. The inhibition of γ-secretase activity enhances neither integrin β1 maturation nor the adhesion of wild-type cells. Moreover, PS deficiency also promoted the maturation of integrins α3 and α5 and the cell surface expression of integrin α3. Integrins α3 and α5 were coimmunoprecipitated with integrin β1, suggesting the formation of the functional heterodimers integrins α3β1 and α5β1. Note that integrin β1 exhibited features opposite those of nicastrin in terms of maturation and trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus in PS1 and PS2 double-deficient fibroblasts. Our results therefore suggest that PS regulates the maturation of membrane proteins in opposite directions and cell adhesion by modulating integrin maturation.  

3.1122           Conventional Kinesin Holoenzymes Are Composed of Heavy and Light Chain Homodimers

DeBoer, S.R. et al Biochemistry, 47, 4535-4543 (2008)   Conventional kinesin is a major microtubule-based motor protein responsible for anterograde transport of various membrane-bounded organelles (MBO) along axons. Structurally, this molecular motor protein is a tetrameric complex composed of two heavy (kinesin-1) chains and two light chain (KLC) subunits. The products of three kinesin-1 (kinesin-1A, -1B, and -1C, formerly KIF5A, -B, and -C) and two KLC (KLC1, KLC2) genes are expressed in mammalian nervous tissue, but the functional significance of this subunit heterogeneity remains unknown. In this work, we examine all possible combinations among conventional kinesin subunits in brain tissue. In sharp contrast with previous reports, immunoprecipitation experiments here demonstrate that conventional kinesin holoenzymes are formed of kinesin-1 homodimers. Similar experiments confirmed previous findings of KLC homodimerization. Additionally, no specificity was found in the interaction between kinesin-1s and KLCs, suggesting the existence of six variant forms of conventional kinesin, as defined by their gene product composition. Subcellular fractionation studies indicate that such variants associate with biochemically different MBOs and further suggest a role of kinesin-1s in the targeting of conventional kinesin holoenzymes to specific MBO cargoes. Taken together, our data address the combination of subunits that characterize endogenous conventional kinesin. Findings on the composition and subunit organization of conventional kinesin as described here provide a molecular basis for the regulation of axonal transport and delivery of selected MBOs to discrete subcellular locations.        

3.1123           A Proteomics Approach to Membrane Trafficking

Groen, A.J., de Vries, S.C. and Lilley, K.S. Plant Physiol., 147, 1584-1589 (2008)   Membrane trafficking, including that of integral membrane proteinsas well as peripherally associated proteins, appears to be avital process common to all eukaryotes. An important elementof membrane trafficking is to determine the protein compositionof the various endomembrane compartments. A major issue withsuch a compositional analysis is the difficulty of having todistinguish between resident components involved in specifictasks and the proteins that are in transit through the endomembranesystem. Examples of resident proteins include components ofthe SNARE complex used to target membrane vesicles to differentlocations in the cell. In the case of functionally importantresidents, one would expect such proteins to have a fairly precisesubcellular localization. In the case of proteins "passing through"an endosomal compartment en route to a final destination, onewould expect to find the proteins colocalizing with many membranecompartments. As is evident from several Update articles in this issue, ambiguityexists when employing cytological techniques to identify specificendomembrane compartments, while markers identified based onhomology may behave differently in plant cells. Therefore, aproteomics approach based on proteins that would traffic throughvarious parts of the endomembrane system, such as plasma membrane(PM) receptors, would be a welcome addition to membrane-traffickingstudies. PM receptors are highly dependent on correct traffickingfor their eventual localization, their biological function,and finally their degradation, while recent evidence suggeststhat endocytosis of PM receptors is an integral part of theirbiological function. In this review, first, a short update on endocytosis and endosomal trafficking in Arabidopsis (Arabidopsis thaliana) is provided.In this section, we emphasize trafficking of PM receptors asa proteomics tool by looking at how the PM receptors trafficin a time-dependent fashion in order to determine the relationshipbetween different endosomal compartments. Second, we describethe recent progress in advanced proteomics techniques such aslocalization of organelle proteins by isotope tagging (LOPIT),by which proteins are assigned to different endosomal compartments.  

3.1124           Molecular Identification of a SNAP-25-Like SNARE Protein in Paramecium

Schilde, C., Lutter, K., Kissmehl, R. And Plattner, H. Eukaryot. Cell, 7(8), 1387-1402 (2008)   Using database searches of the completed Paramecium tetraurelia macronuclear genome with the metazoan SNAP-25 homologues, we identified a single 21-kDa Qb/c-SNARE in this ciliated protozoan, named P. tetraurelia SNAP (PtSNAP), containing the characteristic dual heptad repeat SNARE motifs of SNAP-25. The presence of only a single Qb/c class SNARE in P. tetraurelia is surprising in view of the multiple genome duplications and the high number of SNAREs found in other classes of this organism. As inferred from the subcellular localization of a green fluorescent protein (GFP) fusion construct, the protein is localized on a variety of intracellular membranes, and there is a large soluble pool of PtSNAP. Similarly, the PtSNAP that is detected with a specific antibody in fixed cells is associated with a number of intracellular membrane structures, including food vacuoles, the contractile vacuole system, and the sites of constitutive endo- and exocytosis. Surprisingly, using gene silencing, we could not assign a role to PtSNAP in the stimulated exocytosis of dense core vesicles (trichocysts), but we found an increased number of food vacuoles in PtSNAP-silenced cells. In conclusion, we identify PtSNAP as a Paramecium homologue of metazoan SNAP-25 that shows several divergent features, like resistance to cleavage by botulinum neurotoxins.  

3.1125           Reciprocal interaction with G-actin and tropomyosin is essential for aquaporin-2 trafficking

Noda, Y. et al

1. Cell Biol., 182(3), 587-601 (2008)

  Trafficking of water channel aquaporin-2 (AQP2) to the apical membrane and its vasopressin and protein kinase A (PKA)–dependent regulation in renal collecting ducts is critical for body water homeostasis. We previously identified an AQP2 binding protein complex including actin and tropomyosin-5b (TM5b). We show that dynamic interactions between AQP2 and the actin cytoskeleton are critical for initiating AQP2 apical targeting. Specific binding of AQP2 to G-actin in reconstituted liposomes is negatively regulated by PKA phosphorylation. Dual color fluorescence cross-correlation spectroscopy reveals local AQP2 interaction with G-actin in live epithelial cells at single-molecule resolution. Cyclic adenosine monophosphate signaling and AQP2 phosphorylation release AQP2 from G-actin. In turn, AQP2 phosphorylation increases its affinity to TM5b, resulting in reduction of TM5b bound to F-actin, subsequently inducing F-actin destabilization. RNA interference–mediated knockdown and overexpression of TM5b confirm its inhibitory role in apical trafficking of AQP2. These findings indicate a novel mechanism of channel protein trafficking, in which the channel protein itself critically regulates local actin reorganization to initiate its movement.  

3.1126           Sensitization to the Lysosomal Cell Death Pathway by Oncogene-Induced Down-regulation of Lysosome-Associated Membrane Proteins 1 and 2

Fehrenbacher, N. et al Cancer Res., 68(16), 6623-6633 (2008)   Expression and activity of lysosomal cysteine cathepsins correlate with the metastatic capacity and aggressiveness of tumors. Here, we show that transformation of murine embryonic fibroblasts with v-H-ras or c-src^Y527F changes the distribution, density, and ultrastructure of the lysosomes, decreases the levels of lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in an extracellular signal-regulated kinase (ERK)- and cathepsin-dependent manner, and sensitizes the cells to lysosomal cell death pathways induced by various anticancer drugs (i.e., cisplatin, etoposide, doxorubicin, and siramesine). Importantly, K-ras and erbb2 elicit a similar ERK-mediated activation of cysteine cathepsins, cathepsin-dependent down-regulation of LAMPs, and increased drug sensitivity in human colon and breast carcinoma cells, respectively. Notably, reconstitution of LAMP levels by ectopic expression or by cathepsin inhibitors protects transformed cells against the lysosomal cell death pathway. Furthermore, knockdown of either lamp1 or lamp2 is sufficient to sensitize the cells to siramesine-induced cell death and photo-oxidation–induced lysosomal destabilization. Thus, the transformation-associated ERK-mediated up-regulation of cysteine cathepsin expression and activity leads to a decrease in the levels of LAMPs, which in turn contributes to the enhanced sensitivity of transformed cells to drugs that trigger lysosomal membrane permeabilization. These data indicate that aggressive cancers with high cysteine cathepsin levels are especially sensitive to lysosomal cell death pathways and encourage the further development of lysosome-targeting compounds for cancer therapy.  

3.1127           Intravesicular Calcium Release Mediates the Motion and Exocytosis of Secretory Organelles: A STUDY WITH ADRENAL CHROMAFFIN CELLS

Camacho, M., Machado, J.D., Alvarez, J. and Borges, R.

1. Biol. Chem., 283(33), 22383-22389 (2008)

  Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) possesses multiple and diverse functions in RNA replication, interferon resistance, and viral pathogenesis. Recent studies suggest that NS5A is involved in the assembly and maturation of infectious viral particles; however, precisely how NS5A participates in virus production has not been fully elucidated. In the present study, we demonstrate that NS5A is a prerequisite for HCV particle production as a result of its interaction with the viral capsid protein (core protein). The efficiency of virus production correlated well with the levels of interaction between NS5A and the core protein. Alanine substitutions for the C-terminal serine cluster in domain III of NS5A (amino acids 2428, 2430, and 2433) impaired NS5A basal phosphorylation, leading to a marked decrease in NS5A-core interaction, disturbance of the subcellular localization of NS5A, and disruption of virion production. Replacing the same serine cluster with glutamic acid, which mimics the presence of phosphoserines, partially preserved the NS5A-core interaction and virion production, suggesting that phosphorylation of these serine residues is important for virion production. In addition, we found that the alanine substitutions in the serine cluster suppressed the association of the core protein with viral genome RNA, possibly resulting in the inhibition of nucleocapsid assembly. These results suggest that NS5A plays a key role in regulating the early phase of HCV particle formation by interacting with core protein and that its C-terminal serine cluster is a determinant of the NS5A-core interaction.  

3.1128           Lysyl Oxidase Oxidizes Cell Membrane Proteins and Enhances the Chemotactic Response of Vascular Smooth Muscle Cells

Lucero, H.A. et al

1. Biol. Chem., 283(35), 24103-24117 (2008)

  Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by β-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-β (PDGFR-β), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-β. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-β was diminished by 2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-β-dependent signal transduction pathway, including PDGFR-β, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-β.  

3.1129           Intramembrane Processing by Signal Peptide Peptidase Regulates the Membrane Localization of Hepatitis C Virus Core Protein and Viral Propagation

Okamoto, K. et al

1. Virol., 82(17), 8349-8361 (2008)

  Hepatitis C virus (HCV) core protein has shown to be localized in the detergent-resistant membrane (DRM), which is distinct from the classical raft fraction including caveolin, although the biological significance of the DRM localization of the core protein has not been determined. The HCV core protein is cleaved off from a precursor polyprotein at the lumen side of Ala¹⁹¹ by signal peptidase and is then further processed by signal peptide peptidase (SPP) within the transmembrane region. In this study, we examined the role of SPP in the localization of the HCV core protein in the DRM and in viral propagation. The C terminus of the HCV core protein cleaved by SPP in 293T cells was identified as Phe¹⁷⁷ by mass spectrometry. Mutations introduced into two residues (Ile¹⁷⁶ and Phe¹⁷⁷) upstream of the cleavage site of the core protein abrogated processing by SPP and localization in the DRM fraction. Expression of a dominant-negative SPP or treatment with an SPP inhibitor, L685,458, resulted in reductions in the levels of processed core protein localized in the DRM fraction. The production of HCV RNA in cells persistently infected with strain JFH-1 was impaired by treatment with the SPP inhibitor. Furthermore, mutant JFH-1 viruses bearing SPP-resistant mutations in the core protein failed to propagate in a permissive cell line. These results suggest that intramembrane processing of HCV core protein by SPP is required for the localization of the HCV core protein in the DRM and for viral propagation.  

3.1130           Ral-regulated interaction between Sec5 and paxillin targets Exocyst to focal complexes during cell migration

Spiczka, K.S. and Yeaman, C.

1. Cell Sci., 121, 2880-2891 (2008)

  Changes in cellular behavior that cause epithelial cells to lose adhesiveness, acquire a motile invasive phenotype and metastasize to secondary sites are complex and poorly understood. Molecules that normally function to integrate adhesive spatial information with cytoskeleton dynamics and membrane trafficking probably serve important functions in cellular transformation. One such complex is the Exocyst, which is essential for targeted delivery of membrane and secretory proteins to specific plasma membrane sites to maintain epithelial cell polarity. Upon loss of cadherin-mediated adhesion in Dunning R3327-5'A prostate tumor cells, Exocyst localization shifts from lateral membranes to tips of protrusive membrane extensions. Here, it colocalizes and co-purifies with focal complex proteins that regulate membrane trafficking and cytoskeleton dynamics. These sites are the preferred destination of post-Golgi transport vesicles ferrying biosynthetic cargo, such as ₅-integrin, which mediates adhesion of cells to the substratum, a process essential to cell motility. Interference with Exocyst activity impairs integrin delivery to plasma membrane and inhibits tumor cell motility and matrix invasiveness. Localization of Exocyst and, by extension, targeting of Exocyst-dependent cargo, is dependent on Ral GTPases, which control association between Sec5 and paxillin. Overexpression of Ral-uncoupled Sec5 mutants inhibited Exocyst interaction with paxillin in 5'A cells, as did RNAi-mediated reduction of either RalA or RalB. Reduction of neither GTPase significantly altered steady-state levels of assembled Exocyst in these cells, but did change the observed localization of Exocyst proteins.  

3.1131           Multistep, sequential control of the trafficking and function of the multiple sulfatase deficiency gene product, SUMF1 by PDI, ERGIC-53 and ERp44

Fraldi, A. et al Human Mol. Genet., 17(17), 2610-2621 (2008)   Sulfatase modifying factor 1 (SUMF1) encodes for the formylglicinegenerating enzyme, which activates sulfatases by modifying akey cysteine residue within their catalytic domains. SUMF1 ismutated in patients affected by multiple sulfatase deficiency,a rare recessive disorder in which all sulfatase activitiesare impaired. Despite the absence of canonical retention/retrievalsignals, SUMF1 is largely retained in the endoplasmic reticulum(ER), where it exerts its enzymatic activity on nascent sulfatases.Part of SUMF1 is secreted and paracrinally taken up by distantcells. Here we show that SUMF1 interacts with protein disulfideisomerase (PDI) and ERp44, two thioredoxin family members residingin the early secretory pathway, and with ERGIC-53, a lectinthat shuttles between the ER and the Golgi. Functional assaysreveal that these interactions are crucial for controlling SUMF1traffic and function. PDI couples SUMF1 retention and activationin the ER. ERGIC-53 and ERp44 act downstream, favoring SUMF1export from and retrieval to the ER, respectively. SilencingERGIC-53 causes proteasomal degradation of SUMF1, while down-regulatingERp44 promotes its secretion. When over-expressed, each of threeinteractors favors intracellular accumulation. Our results reveala multistep control of SUMF1 trafficking, with sequential interactionsdynamically determining ER localization, activity and secretion.  

3.1132           Pmp-Like Proteins Pls1 and Pls2 Are Secreted into the Lumen of the Chlamydia trachomatis Inclusion

Jorgensen, I. and Valdivia, R.H. Infect. Immun., 76(9), 3940-3950 (2008)   The obligate intracellular pathogen Chlamydia trachomatis secretes effector proteins across the membrane of the pathogen-containing vacuole (inclusion) to modulate host cellular functions. In an immunological screen for secreted chlamydial proteins, we identified CT049 and CT050 as potential inclusion membrane-associated proteins. These acidic, nonglobular proteins are paralogously related to the passenger domain of the polymorphic membrane protein PmpC and, like other Pmp proteins, are highly polymorphic among C. trachomatis ocular and urogenital strains. We generated antibodies to these Pmp-like secreted (Pls) proteins and determined by immunofluorescence microscopy that Pls1 (CT049) and Pls2 (CT050) localized to globular structures within the inclusion lumen and at the inclusion membrane. Fractionation of membranes and cytoplasmic components from infected cells by differential and density gradient centrifugation further indicated that Pls1 and Pls2 associated with membranes distinct from the bulk of bacterial and inclusion membranes. The accumulation of Pls1 and, to a lesser extent, Pls2 in the inclusion lumen was insensitive to the type III secretion inhibitor C1, suggesting that this translocation system is not essential for Pls protein secretion. In contrast, Pls secretion and stability were sensitive to low levels of β-lactam antibiotics, suggesting that a functional cell wall is required for Pls secretion from the bacterial cell. Finally, we tested the requirement for these proteins in Chlamydia infection by microinjecting anti-Pls1 and anti-Pls2 antibodies into infected cells. Coinjection of anti-Pls1 and -Pls2 antibodies partially inhibited expansion of the inclusion. Because Pls proteins lack classical sec-dependent secretion signals, we propose that Pls proteins are secreted into the inclusion lumen by a novel mechanism to regulate events important for chlamydial replication and inclusion expansion.  

3.1133           Subcellular distribution of APP/C99 and amyloid-beta peptide is altered in CHO NPC1-null cells compared to CHOwt

Posavec, M., Boskovic, D., Goate, A., Hecimovic, S. and Boskovic, R. Alzheimer’s and Dementia, 4(4), Suppl. 1, T353 (2008)   Background: A sphingolipid storage disease (SLSD) Niemann Pick type C (NPC) is caused by dysfunction of NPC1 protein which leads to accumulation of free cholesterol and glycosphingolipids in endosomal/lysosomal compartments. It has been recently shown that this defect leads to increased formation of amyloid-beta (Abeta) peptide, and is accompanied by altered localization of presenilin 1 to early/late endosomes. We hypothesized that cholesterol accumulation upon NPC1 loss of function leads to increased APP/C99 localization in endosomes and increased formation of Abeta in these compartments. To test this we monitored subcellular localization of APP/C99 and Abeta between CHO NPC1-null (M12) and CHOwt cells. Methods: The cells were stably transfected with APPsw-6myc construct. Subcellular distribution of APP processing products and organelle markers was analyzed by subcellular fractionation in an Iodixanol gradient. After centrifugation (20h and 100,000xg), fractions were collected from the top and protein (DC Protein Assay, BioRad) and cholesterol concentrations (AmplexRed cholesterol assay, Molecular Probes) were determined in each fraction. After acetone precipitation, APP processing products and organelle markers were detected by western blotting using specific antibodies, while Abeta levels were determined by ELISA assay (BioSource International, Inc.). Results: We observed that the majority of subcellular markers tested (BIP/GRP78, EEA1 and Rab7) showed an altered subcellular distribution in M12 cells compared to CHOwt, indicating that loss of NPC1 leads to altered membrane/protein trafficking. In addition, markedly increased levels of BIP/GRP78 were detected in M12 vs. CHOwt cells, which has been previously reported in another SLSD - GM1 gangliosidosis. Although APP was found in similar fractions of M12 and CHOwt cells, the proportion of APP in early/late endosomes was higher in M12 cells. In these cells we also observed increased levels of Abeta40 in endosomal fractions. Conclusions: Our results suggest that increased formation of Abeta in CHO NPC1-null cells is due to redistribution of APP/C99 to early/late endosomes. We propose that NPC1 dysfunction leads to increased coupling of presenilin 1 and its substrate C99 in endosome compartments generating increased Abeta.  

3.1134           Trafficking of chlamydial antigens to the endoplasmic reticulum of infected epithelial cells

Giles, D.K. and Wyrick, P.B. Microbes and Infection, 10, 1494-1503 (2008)   Confinement of the obligate intracellular bacterium Chlamydia trachomatis to a membrane-bound vacuole, termed an inclusion, within infected epithelial cells neither prevents secretion of chlamydial antigens into the host cytosol nor protects chlamydiae from innate immune detection. However, the details leading to chlamydial antigen presentation are not clear. By immunoelectron microscopy of infected endometrial epithelial cells and in isolated cell secretory compartments, chlamydial major outer membrane protein (MOMP), lipopolysaccharide (LPS) and the inclusion membrane protein A (IncA) were localized to the endoplasmic reticulum (ER) and co-localized with multiple ER markers, but not with markers of the endosomes, lysosomes, Golgi nor mitochondria. Chlamydial LPS was also co-localized with CD1d in the ER. Since the chlamydial antigens, contained in everted inclusion membrane vesicles, were found within the host cell ER, these data raise additional implications for antigen processing by infected uterine epithelial cells for classical and non-classical T cell antigen presentation.  

3.1135           The GET Complex Mediates Insertion of Tail-Anchored Proteins into the ER Membrane

Schuldiner, M. et al Cell, 134, 634-645 (2008)   Tail-anchored (TA) proteins, defined by the presence of a single C-terminal transmembrane domain (TMD), play critical roles throughout the secretory pathway and in mitochondria, yet the machinery responsible for their proper membrane insertion remains poorly characterized. Here we show that Get3, the yeast homolog of the TA-interacting factor Asna1/Trc40, specifically recognizes TMDs of TA proteins destined for the secretory pathway. Get3 recognition represents a key decision step, whose loss can lead to misinsertion of TA proteins into mitochondria. Get3-TA protein complexes are recruited for endoplasmic reticulum (ER) membrane insertion by the Get1/Get2 receptor. In vivo, the absence of Get1/Get2 leads to cytosolic aggregation of Get3-TA complexes and broad defects in TA protein biogenesis. In vitro reconstitution demonstrates that the Get proteins directly mediate insertion of newly synthesized TA proteins into ER membranes. Thus, the GET complex represents a critical mechanism for ensuring efficient and accurate targeting of TA proteins.  

3.1136           Sulfotransferase 2B1b in human breast: Differences in subcellular localization in African American and Caucasian women

Dumas, N.A., He, D., Frost, A.R. and Falany, C.N.

1. Steroid Biochem Mol. Biol., 111, 171-177 (2008)

  Breast cancer (BC) is the most commonly diagnosed cancer among American women; however, the development of post-menopausal BC is significantly lower in African Americans as compared to Caucasians. Hormonal stimulation is important in BC development and differences in the conversion of dehydroepiandrosterone (DHEA) into estrogens may be involved in the lower incidence of post-menopausal BC in African American women. DHEA sulfation by sulfotransferase 2B1b (SULT2B1b) is important in regulating the conversion of DHEA into estrogens in tissues. SULT2B1b is localized in both cytosol and nuclei of some tissues including cancerous and associated-normal breast tissue. Immunohistochemical staining was used to evaluate the total expression and subcellular localization of SULT2B1b in African American and Caucasian breast tissues. Cell fractionation, immunoblot analysis and sulfation assays were used to characterize the subcellular expression and activity of SULT2B1b in BC tissues and T-47D breast adenocarcinoma cells. Immunohistochemical analysis of SULT2B1b showed that African Americans had a significantly greater amount of SULT2B1b in epithelial cells of associated-normal breast tissue as compared to Caucasians. Also, more SULT2B1b in African American associated-normal breast epithelial cells was localized in the nuclei than in Caucasians. Equivalent levels of SULT2B1b were detected in breast adenocarcinoma tissues from both African American and Caucasian women. Nuclei isolation and immunoblot analysis of both BC tissue and human T-47D breast adenocarcinoma cells demonstrated that SULT2B1b is present in nuclei and cytoplasm.  

3.1137           Distinct binding sites for the ATPase and substrate-binding domain of human Hsp70 on the cell surface of antigen presenting cells

Zitzler, S., Hellwig, A., Hartl, F-U., Wieland, F. And Diestelkötter-Bachert, P. Mol. Immunol., 45, 3974-3983 (2008)   Hsp70 has high potential as an immune-adjuvant molecule: it mediates cytokine expression and maturation of antigen presenting cells (APCs) and also elicits a cytotoxic T-lymphocyte (CTL) response to antigenic peptides. How Hsp70 interacts with APCs is only poorly understood. Various surface proteins have been implicated in binding Hsp70 but their role in antigen presentation has remained controversial. The specific aim of this work was to determine the binding and uptake of human full-length Hsp70 as well as its separate ATPase (N70) and substrate-binding domains (C70) by APCs. Using laser scanning microscopy and FACS analysis, we established the existence of at least two distinct receptors for Hsp70, which are localized to distinct microdomains of the APC membrane. These receptors interact with the N70 and C70 domains of Hsp70, respectively. This observation was supported by the finding of a substantial portion of Hsp70 and C70, but not N70, in a detergent resistant membrane fraction. Accordingly, C70 and N70 did not compete with each other for binding. The bound proteins were rapidly internalized, with N70 and C70 localizing to separate endosomal compartments. Similarly, internalized free and peptide-loaded Hsp70 segregated rapidly within the cell. Efficient cross presentation of antigenic peptide bound to Hsp70 or C70 was demonstrated with the B3Z read out system. Consequently, the interaction of C70 with its putative receptor seems to be responsible for Hsp70-mediated cross presentation. Future studies should make use of C70 in identifying the uptake receptor of Hsp70–peptide complexes. In addition we could observe a stimulation of uptake of free peptide by preincubation with Hsp70 and N70, but not C70, whereas an Hsp-dependent cytokine secretion could not be detected. Consequently, by employing the individual domains it may be possible to distinguish between the different outcomes of Hsp70 treatment, like immune stimulation, DC maturation and antigen-specific responses.  

3.1138           The insulin-regulated aminopeptidase IRAP is colocalised with GLUT4 in the mouse hippocampus - potential role in modulation of glucose uptake in neurones?

Fernando, R.N., Albiston, A.L. and Chai, S.Y. Eur. J. Neurosci., 28, 588-598 (2008)   It is proposed that insulin-regulated aminopeptidase (IRAP) is the site of action of two peptides, angiotensin IV and LVV-hemorphin 7, which have facilitatory effects on learning and memory. In fat and muscles, IRAP codistributes with the insulin-responsive glucose transporter GLUT4 in specialised vesicles, where it plays a role in the tethering and/or trafficking of these vesicles. This study investigated whether an analogous system exists in two functionally distinct regions of the brain, the hippocampus and the cerebellum. In the hippocampus, IRAP was found in the pyramidal neurones where it exhibited a high degree of colocalisation with GLUT4. Consistent with the role of GLUT4 in insulin-responsive tissues, the glucose transporter was thought to be responsible for facilitating glucose uptake into these pyramidal neurones in response to potassium-induced depolarisation or cAMP activation as the glucose influx was sensitive to indinavir treatment. Angiotensin IV and LVV-hemorphin 7 enhanced this activity-dependent glucose uptake in hippocampal slices. In contrast, in the cerebellum, where the distribution of IRAP was dissociated from GLUT4, the effect of the peptides on glucose uptake was absent. We propose that the modulation of glucose uptake by angiotensin IV and LVV-hemorphin 7 is region-specific and is critically dependent on a high degree of colocalisation between IRAP and GLUT4. These findings also confirm a role for IRAP and GLUT4 in activity-dependent glucose uptake in hippocampal neurones.  

3.1139           Separable requirements for cytoplasmic domain of PSGL-1 in leukocyte rolling and signaling under flow

Miner, J.J. et al Blood, 112(5), 2035-2045 (2008)   In inflamed venules, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to roll on P-selectin and E-selectin and to activate integrin Lβ2 (lymphocyte function-associated antigen-1, LFA-1) to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Studies in cell lines have suggested that PSGL-1 requires its cytoplasmic domain to localize in membrane domains, to support rolling on P-selectin, and to signal through spleen tyrosine kinase (Syk). We generated " CD" mice that express PSGL-1 without the cytoplasmic domain. Unexpectedly, neutrophils from these mice localized PSGL-1 normally in microvilli, uropods, and lipid rafts. CD neutrophils expressed less PSGL-1 on their surfaces because of inefficient export from the endoplasmic reticulum. Limited digestion of wild-type neutrophils with O-sialoglycoprotein endopeptidase was used to reduce the PSGL-1 density to that on CD neutrophils. At matched PSGL-1 densities, both CD and wild-type neutrophils rolled similarly on P-selectin. However, CD neutrophils rolling on P-selectin did not trigger Syk-dependent activation of LFA-1 to slow rolling on ICAM-1. These data demonstrate that the PSGL-1 cytoplasmic domain is dispensable for leukocyte rolling on P-selectin but is essential to activate β2 integrins to slow rolling on ICAM-1.  

3.1140           The presence of an ER exit signal determines the protein sorting upon ER exit in yeast

Watanabe, R., Castillon, G.A., Meury, A. and Riezman, H. Biochem. J., 414, 237-245 (2008)   In yeast, there are at least two vesicle populations upon ER (endoplasmic reticulum) exit, one containing Gap1p (general aminoacid permease) and a glycosylated α-factor, gpαF (glycosylated proα-factor), and the other containing GPI (glycosylphosphatidylinositol)-anchored proteins, Gas1p (glycophospholipid-anchored surface protein) and Yps1p. We attempted to identify sorting determinants for this protein sorting event in the ER. We found that mutant Gas1 proteins that lack a GPI anchor and/or S/T region (serine- and threonine-rich region), two common characteristic features conserved among yeast GPI-anchored proteins, were still sorted away from Gap1p-containing vesicles. Furthermore, a mutant glycosylated α-factor, gpαGPI, which contains both the GPI anchor and S/T region from Gas1p, still entered Gap1p-containing vesicles, demonstrating that these conserved characteristics do not prevent proteins from entering Gap1p-containing vesicles. gpαF showed severely reduced budding efficiency in the absence of its ER exit receptor Erv29p, and this residual budding product no longer entered Gap1p-containing vesicles. These results suggest that the interaction of gpαF with Erv29p is essential for sorting into Gap1p-containing vesicles. We compared the detergent solubility of Gas1p and the gpαGPI in the ER with that in ER-derived vesicles. Both GPI-anchored proteins similarly partitioned into the DRM (detergent-resistant membrane) in the ER. Based on the fact that they entered different ER-derived vesicles, we conclude that DRM partitioning of GPI-anchored proteins is not the dominant determinant of protein sorting upon ER exit. Interestingly, upon incorporation into the ER-derived vesicles, gpαGPI was no longer detergent-insoluble, in contrast with the persistent detergent insolubility of Gas1p in the ER-derived vesicles. We present different explanations for the different behaviours of GPI-anchored proteins in distinct ER-derived vesicle populations.  

3.1141           Increased basolateral sorting of carcinoembryonic antigen in a polarized colon carcinoma cell line after cholesterol depletion-Implications for treatment of inflammatory bowel disease

Ehehalt, R. et al World J. Gastroenterol., 14(10), 1528-1533 (2008)   AIM: To investigate a possible increase of basolateral expression of carcinoembryonic antigen (CEA) by interfering with the apical transport machinery, we studied the effect of cholesterol depletion on CEA sorting and secretion. METHODS: Cholesterol depletion was performed in polarized Caco-2 cells using lovastatin and methyl-beta-cyclodextrin. RESULTS: We show that CEA is predominantly expressed and secreted at the apical surface. Reduction of the cholesterol level of the cell by 40%-50% with lovastatin and methyl-beta-cyclodextrin led to a significant change of the apical-to-basolateral transport ratio towards the basolateral membrane. CONCLUSION: As basolateral expression of CEA has been suggested to have anti-inflamatory properties, Cholesterol depletion of enterocytes might be a potential approach to influence the course of inflammatory bowel disease.  

3.1142           The Viral Oncoprotein LMP1 Exploits TRADD for Signaling by Masking Its Apoptotic Activity

Schneider, F. et al PloS Biology, 6(1), 86-98 (2008)   The tumor necrosis factor (TNF)-receptor 1–associated death domain protein (TRADD) mediates induction of apoptosis as well as activation of NF-κB by cellular TNF-receptor 1 (TNFR1). TRADD is also recruited by the latent membrane protein 1 (LMP1) oncoprotein of Epstein-Barr virus, but its role in LMP1 signaling has remained enigmatic. In human B lymphocytes, we have generated, to our knowledge, the first genetic knockout of TRADD to investigate TRADD's role in LMP1 signal transduction. Our data from TRADD-deficient cells demonstrate that TRADD is a critical signaling mediator of LMP1 that is required for LMP1 to recruit and activate I-κB kinase β (IKKβ). However, in contrast to TNFR1, LMP1-induced TRADD signaling does not induce apoptosis. Searching for the molecular basis for this observation, we characterized the 16 C-terminal amino acids of LMP1 as an autonomous and unique virus-derived TRADD-binding domain. Replacing the death domain of TNFR1 by LMP1′s TRADD-binding domain converts TNFR1 into a nonapoptotic receptor that activates NF-κB through a TRAF6-dependent pathway, like LMP1 but unlike wild-type TNFR1. Thus, the unique interaction of LMP1 with TRADD encodes the transforming phenotype of viral TRADD signaling and masks TRADD's pro-apoptotic function.  

3.1143           Vesicular Egress of Non-Enveloped Lytic Parvoviruses Depends on Gelsolin Functioning

Bär, S., Daeffler, L., Rommelaere, J. And Nüesch, J.P.F. PloS Pathogens, 4(8), 1-11 (2008)   The autonomous parvovirus Minute Virus of Mice (MVM) induces specific changes in the cytoskeleton filaments of infected permissive cells, causing in particular the degradation of actin fibers and the generation of “actin patches.” This is attributed to a virus-induced imbalance between the polymerization factor N-WASP (Wiscott-Aldrich syndrome protein) and gelsolin, a multifunctional protein cleaving actin filaments. Here, the focus is on the involvement of gelsolin in parvovirus propagation and virus-induced actin processing. Gelsolin activity was knocked-down, and consequences thereof were determined for virus replication and egress and for actin network integrity. Though not required for virus replication or progeny particle assembly, gelsolin was found to control MVM (and related H1-PV) transport from the nucleus to the cell periphery and release into the culture medium. Gelsolin-dependent actin degradation and progeny virus release were both controlled by (NS1)/CKIIα, a recently identified complex between a cellular protein kinase and a MVM non-structural protein. Furthermore, the export of newly synthesized virions through the cytoplasm appeared to be mediated by (virus-modified) lysomal/late endosomal vesicles. By showing that MVM release, like entry, is guided by the cytoskeleton and mediated by vesicles, these results challenge the current view that egress of non-enveloped lytic viruses is a passive process.  

3.1144           Endophilin B1 as a Novel Regulator of Nerve Growth Factor/ TrkA Trafficking and Neurite Outgrowth

Wan, J. et al

1. Neurosci., 28(36), 9002-9012 (2008)

  Neurotrophins and their cognate receptors Trks are important regulators of neuronal survival and differentiation. Recent studies reveal that internalization and trafficking of Trks play a critical role in neurotrophin-mediated signaling. At present, little is known of the molecular events that mediate this process. In the current study, we show that endophilin B1 is a novel regulator of nerve growth factor (NGF) trafficking. We found that endophilin B1 interacts with both TrkA and early endosome marker EEA1. Interestingly, knockdown of endophilin B1 results in enlarged EEA1-positive vesicles in NGF-treated PC12 cells. This is accompanied by increased lysosomal targeting of NGF/TrkA and TrkA degradation, and reduced total TrkA levels. In addition, knockdown of endophilin B1 attenuates Erk1/2 activation in the endosomal fraction after NGF treatment. This is accompanied by a marked inhibition of NGF-induced gene transcription and neurite outgrowth in endophilin B1-knocked down cells. Our observations implicate endophilin B1 as a novel regulator of NGF trafficking, thereby affecting TrkA levels and downstream signaling on endosomes to mediate biological functions of NGF.  

3.1145           Conditioning the heart induces formation of signalosomes that interact with mitochondria to open mitoKATP channels

Quinlan, C.L. et al Am. J. Physiol. Heart Circ. Physiol., 295, H953-H961 (2008)   Perfusion of the heart with bradykinin triggers cellular signaling events that ultimately cause opening of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels, increased H₂O₂ production, inhibition of the mitochondrial permeability transition (MPT), and cardioprotection. We hypothesized that the interaction of bradykinin with its receptor induces the assembly of a caveolar signaling platform (signalosome) that contains the enzymes of the signaling pathway and that migrates to mitochondria to induce mitoK_ATP channel opening. We developed a novel method for isolating and purifying signalosomes from Langendorff-perfused rat hearts treated with bradykinin. Fractions containing the signalosomes were found to open mitoK_ATP channels in mitochondria isolated from untreated hearts via the activation of mitochondrial PKC- . mitoK_ATP channel opening required signalosome-dependent phosphorylation of an outer membrane protein. Immunodetection analysis revealed the presence of the bradykinin B₂ receptor only in the fraction isolated from bradykinin-treated hearts. Immunodetection and immunogold labeling of caveolin-3, as well as sensitivity to cholesterol depletion and resistance to Triton X-100, attested to the caveolar nature of the signalosomes. Ischemic preconditioning, ischemic postconditioning, and perfusion with ouabain also led to active signalosome fractions that opened mitoK_ATP channels in mitochondria from untreated hearts. These results provide initial support for a novel mechanism for signal transmission from a plasma membrane receptor to mitoK_ATP channels.  

3.1146           Use of Fluorescence-activated Vesicle Sorting for Isolation of Naked2-associated, Basolaterally Targeted Exocytic Vesicles for Proteomics Analysis

Cao, Z. et al Mol. Cell. Proteomics, 7(9), 1651-1667 (2008)   By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor- (TGF ), Naked2 coats TGF -containing exocytic vesicles and directs them to the basolateral corner of polarized epithelial cells where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. These TGF -containing Naked2-associated vesicles are not directed to the subapical Sec6/8 exocyst complex as has been reported for other basolateral cargo, and thus they appear to represent a distinct set of basolaterally targeted vesicles. To identify constituents of these vesicles, we exploited our finding that myristoylation-deficient Naked2 G2A vesicles are unable to fuse at the plasma membrane. Isolation of a population of myristoylation-deficient, green fluorescent protein-tagged G2A Naked2-associated vesicles was achieved by biochemical enrichment followed by flow cytometric fluorescence-activated vesicle sorting. The protein content of these plasma membrane de-enriched, flow-sorted fluorescent G2A Naked2 vesicles was determined by LC/LC-MS/MS analysis. Three independent isolations were performed, and 389 proteins were found in all three sets of G2A Naked2 vesicles. Rab10 and myosin IIA were identified as core machinery, and Na⁺/K⁺-ATPase 1 was identified as anadditional cargo within these vesicles. As an initial validationstep, we confirmed their presence and that of three additionalproteins tested (annexin A1, annexin A2, and IQGAP1) in wild-typeNaked2 vesicles. To our knowledge, this is the first large scaleprotein characterization of a population of basolaterally targetedexocytic vesicles and supports the use of fluorescence-activatedvesicle sorting as a useful tool for isolation of cellular organellesfor comprehensive proteomics analysis.  

3.1147           Phosphorylation, lipid raft interaction and traffic of α-synuclein in a yeast model for Parkinson

Zabrocki, P. et al Biochim. Biophys. Acta., 1783(10), 1767-1780 (2008)   Parkinson's disease is a neurodegenerative disorder characterized by the formation of Lewy bodies containing aggregated α-synuclein. We used a yeast model to screen for deletion mutants with mislocalization and enhanced inclusion formation of α-synuclein. Many of the mutants were affected in functions related to vesicular traffic but especially mutants in endocytosis and vacuolar degradation combined inclusion formation with enhanced α-synuclein-mediated toxicity. The screening also allowed for identification of casein kinases responsible for α-synuclein phosphorylation at the plasma membrane as well as transacetylases that modulate the α-synuclein membrane interaction. In addition, α-synuclein was found to associate with lipid rafts, a phenomenon dependent on the ergosterol content. Together, our data suggest that toxicity of α-synuclein in yeast is at least in part associated with endocytosis of the protein, vesicular recycling back to the plasma membrane and vacuolar fusion defects, each contributing to the obstruction of different vesicular trafficking routes.  

3.1148           Isolation and characterization of mutant animal cell line defective in alkyl-dihydroxyacetonephosphate synthase: Localization and transport of plasmalogens to post-Golgi compartments

Honsho, M., Yagita, Y., Kinoshita, N. and Fujuki, Y. Biochim. Biophys. Acta, 1783(10), 1857-1865 (2008)   We herein isolated plasmalogen-deficient Chinese hamster ovary (CHO) mutant, ZPEG251, with a phenotype of normal import of peroxisomal matrix and membrane proteins. In ZPEG251, plasmenylethanolamine (PlsEtn) was severely reduced. Complementation analysis by expression of genes responsible for the plasmalogen biogenesis suggested that alkyl-dihydroxyacetonephosphate synthase (ADAPS), catalyzing the second step of plasmalogen biogenesis, was deficient in ZPEG251. ADAPS mRNA was barely detectable as verified by Northern blot and reverse transcription-PCR analyses. Defect of ADAPS expression was also assessed by immunoblot. As a step toward delineating functional roles of PlsEtn, we investigated its subcellular localization. PlsEtn was localized to post-Golgi compartments and enriched in detergent-resistant membranes. Transport of PlsEtn to post-Golgi compartments was apparently affected by lowering cellular ATP, but not by inhibitors of microtubule assembly and vesicular transport. Partitioning of cholesterol and sphingomyelin, a typical feature of lipid rafts, was not impaired in plasmalogen-deficient cells, including peroxisome assembly-defective mutants, hence suggesting that PlsEtn was not essential for lipid-raft architecture in CHO cells.  

3.1149           Occludin oligomeric assembly at tight junctions of the blood-brain barrier is disrupted by peripheral inflammatory hyperalgesia

McCaffrey, G. et al

1. Neurochem., 106, 2395-2409 (2008)

  Tight junctions (TJs) at the blood-brain barrier (BBB) dynamically alter paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS in response to external stressors, such as pain, inflammation, and hypoxia. In this study, we investigated the effect of λ-carrageenan-induced peripheral inflammatory pain (i.e., hyperalgesia) on the oligomeric assembly of the key TJ transmembrane protein, occludin. Oligomerization of integral membrane proteins is a critical step in TJ complex assembly that enables the generation of tightly packed, large multiprotein complexes capable of physically obliterating the interendothelial space to inhibit paracellular diffusion. Intact microvessels isolated from rat brains were fractionated by detergent-free density gradient centrifugation, and gradient fractions were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis/ Western blot. Injection of λ-carrageenan into the rat hind paw produced after 3 h a marked change in the relative amounts of oligomeric, dimeric, and monomeric occludin isoforms associated with different plasma membrane lipid raft domains and intracellular compartments in endothelial cells at the BBB. Our findings suggest that increased BBB permeability (i.e., leak) associated with λ-carrageenan-induced peripheral inflammatory pain is promoted by the disruption of disulfide-bonded occludin oligomeric assemblies, which renders them incapable of forming an impermeant physical barrier to paracellular transport.  

3.1150           Cathepsin D Is the Main Lysosomal Enzyme Involved in the Degradation of α-Synuclein and Generation of Its Carboxy-Terminally Truncated Species

Sevlever, D., Jiang, P. and Yen, S-H.C. Biochemistry, 47, 9678-9687 (2008)   α-Synuclein is likely to play a key role in the development of Parkinson’s disease as well as other synucleinopathies. In animal models, overexpression of full-length or carboxy-terminally truncated α-synuclein has been shown to produce pathology. Although the proteosome and lysosome have been proposed to play a role in the degradation of α-synuclein, the enzyme(s) involved in α-synuclein clearance and generation of its carboxy-terminally truncated species have not been identified. In this study, the role of cathepsin D and calpain I in these processes was analyzed. In vitro experiments, using either recombinant or endogenous α-synuclein as substrates and purified cathepsin D or lysosomes, demonstrated that cathepsin D degraded α-synuclein very efficiently, and that limited proteolysis resulted in the generation of carboxy-terminally truncated species. Purified calpain I also cleaved α-synuclein, but carboxy-terminally truncated species were not the main cleavage products, and calpain I activity present in cellular lysates was not able to degrade the protein. Knockdown of cathepsin D in cells overexpressing wild-type α-synuclein increased total α-synuclein levels by 28% and lysosomal α-synuclein by 2-fold. In in vitro experiments, pepstatin A completely blocked the degradation of α-synuclein in purified lysosomes. Furthermore, lysosomes isolated from cathepsin D knockdown cells showed a marked reduction in α-synuclein degrading activity, indicating that cathepsin D is the main lysosomal enzyme involved in α-synuclein degradation. Our findings suggest that upregulation of cathepsin D could be an additional therapeutic strategy to lessen α-synuclein burden in synucleinopathies.  

3.1151           Discrimination between exosomes and HIV-1: Purification of both vesicles from cell-free supernatants

Cantin, R., Diou, J., Belanger, D., Tremblay, A.M. and Gilbert, C.

1. Immunol. Methods, 338, 21-30 (2008)

  Although enveloped retroviruses bud from the cell surface of T lymphocytes, they use the endocytic pathway and the internal membrane of multivesicular bodies for their assembly and release from macrophages and dendritic cells (DCs). Exosomes, physiological nanoparticles produced by hematopoietic cells, egress from this same pathway and are similar to retroviruses in terms of size, density, the molecules they incorporate and their ability to activate immune cells. Retroviruses are therefore likely to contaminate in vitro preparations of exosomes and vice versa and sucrose gradients are inefficient at separating them. However, we have found that their sedimentation velocities in an iodixanol (Optiprep™) velocity gradient are sufficiently different to allow separation and purification of both vesicles. Using acetylcholinesterase as an exosome marker, we demonstrate that Optiprep™ velocity gradients are very efficient in separating exosomes from HIV-1 particles produced on 293T cells, primary CD4⁺ T cells, macrophages or DCs, with exosomes collecting at 8.4–12% iodixanol and HIV-1 at 15.6%. We also show that immunodepletion with an anti-acetylcholinesterase antibody rapidly produces highly purified preparations of HIV-1 or exosomes. These findings have applications in fundamental research on exosomes and/or AIDS, as well as in clinical applications where exosomes are involved, more specifically in tumour therapy or in gene therapy using exosomes generated from DCs genetically modified by transfection with virus.  

3.1152           Uptake of long chain fatty acids is regulated by dynamic interaction of FAT/CD36 with cholesterol/sphingolipid enriched microdomains (lipid rafts)

Ehehalt, R. et al BMC Cell Biol., 9, 45-56 (2008)   Background Mechanisms of long chain fatty acid uptake across the plasma membrane are important targets in treatment of many human diseases like obesity or hepatic steatosis. Long chain fatty acid translocation is achieved by a concert of co-existing mechanisms. These lipids can passively diffuse, but certain membrane proteins can also accelerate the transport. However, we now can provide further evidence that not only proteins but also lipid microdomains play an important part in the regulation of the facilitated uptake process. Methods Dynamic association of FAT/CD36 a candidate fatty acid transporter with lipid rafts was analysed by isolation of detergent resistant membranes (DRMs) and by clustering of lipid rafts with antibodies on living cells. Lipid raft integrity was modulated by cholesterol depletion using methyl-β-cyclodextrin and sphingolipid depletion using myriocin and sphingomyelinase. Functional analyses were performed using an [3H]-oleate uptake assay. Results Overexpression of FAT/CD36 and FATP4 increased long chain fatty acid uptake. The uptake of long chain fatty acids was cholesterol and sphingolipid dependent. Floating experiments showed that there are two pools of FAT/CD36, one found in DRMs and another outside of these domains. FAT/CD36 co-localized with the lipid raft marker PLAP in antibody-clustered domains at the plasma membrane and segregated away from the non-raft marker GFP-TMD. Antibody cross-linking increased DRM association of FAT/CD36 and accelerated the overall fatty acid uptake in a cholesterol dependent manner. Another candidate transporter, FATP4, was neither present in DRMs nor co-localized with FAT/CD36 at the plasma membrane. Conclusion Our observations suggest the existence of two pools of FAT/CD36 within cellular membranes. As increased raft association of FAT/CD36 leads to an increased fatty acid uptake, dynamic association of FAT/CD36 with lipid rafts might regulate the process. There is no direct interaction of FATP4 with lipid rafts or raft associated FAT/CD36. Thus, lipid rafts have to be considered as targets for the treatment of lipid disorders.  

3.1153           The unique architecture of Bunyamwera virus factories around the Golgi complex

Fontana, J., Lopez-Montero, N., Elliott, R.M., Fernandez, J.J. and Risco, C. Cell. Microbiol., 10(10), 2112-2028 (2008)   Viral factories are novel structures built by viruses in infected cells. During their construction organelles are recruited and build a large scaffold for viral replication and morphogenesis. We have studied how a bunyavirus uses the Golgi to build the factory. With the help of confocal and 3D ultrastructural imaging together with molecular mapping in situ and in vitro we have characterized a tubular structure that harbours the viral replication complexes in a globular domain. Numerous ribonucleoproteins were released from purified tubes disrupted in vitro. Actin and myosin I were identified by peptide mass fingerprinting in isolated tubes while actin and the viral NSm non-structural protein were detected in the tubes' internal proteinaceous scaffold by immunogold labelling. Studies with NSm deletion mutants and drugs affecting actin showed that both NSm and actin are key factors for tube and virus assembly in Golgi. Three-dimensional reconstructions based on oriented serial sections of infected cells showed that tubes anchor cell organelles to Golgi stacks and make contacts with intracellular viruses. We propose that this new structure, unique among enveloped viruses, assembles in association with the most stable component of Golgi stacks, the actin-containing matrix scaffold, connecting viral replication and morphogenesis inside viral factories.    

3.1154           Absence of 2-Hydroxylated Sphingolipids Is Compatible with Normal Neural Development But Causes Late-Onset Axon and Myelin Sheath Degeneration

Zöller, I. et al

1. Neurosci., 28(39), 9741-9754 (2008)

  Sphingolipids containing 2-hydroxylated fatty acids are among the most abundant lipid components of the myelin sheath and therefore are thought to play an important role in formation and function of myelin. To prove this hypothesis, we generated mice lacking a functional fatty acid 2-hydroxylase (FA2H) gene. FA2H-deficient (FA2H^–/–) mice lacked 2-hydroxylated sphingolipids in the brain and in peripheral nerves. In contrast, nonhydroxylated galactosylceramide was increased in FA2H^–/– mice. However, oligodendrocyte differentiation examined by in situ hybridization with cRNA probes for proteolipid protein and PDGF receptor and the time course of myelin formation were not altered in FA2H^–/– mice compared with wild-type littermates. Nerve conduction velocity measurements of sciatic nerves revealed no significant differences between FA2H^–/– and wild-type mice. Moreover, myelin of FA2H^–/– mice up to 5 months of age appeared normal at the ultrastructural level, in the CNS and peripheral nervous system. Myelin thickness and g-ratios were normal in FA2H^–/– mice. Aged (18-month-old) FA2H^–/– mice, however, exhibited scattered axonal and myelin sheath degeneration in the spinal cord and an even more pronounced loss of stainability of myelin sheaths in sciatic nerves. These results show that structurally and functionally normal myelin can be formed in the absence of 2-hydroxylated sphingolipids but that its long-term maintenance is strikingly impaired. Because axon degeneration appear to start rather early with respect to myelin degenerations, these lipids might be required for glial support of axon function.  

3.1155           Kinesin KIF4 Regulates Intracellular Trafficking and Stability of the Human Immunodeficiency Virus Type 1 Gag Polyprotein

Martinez, N., Xue, X., Berro, R.G., Kreitzer, G. and Resh, M.D.

1. Virol., 82(20), 9937-9950 (2008)

  Retroviral Gag proteins are synthesized as soluble, myristoylated precursors that traffic to the plasma membrane and promote viral particle production. The intracellular transport of human immunodeficiency virus type 1 (HIV-1) Gag to the plasma membrane remains poorly understood, and cellular motor proteins responsible for Gag movement are not known. Here we show that disrupting the function of KIF4, a kinesin family member, slowed temporal progression of Gag through its trafficking intermediates and inhibited virus-like particle production. Knockdown of KIF4 also led to increased Gag degradation, resulting in reduced intracellular Gag protein levels; this phenotype was rescued by reintroduction of KIF4. When KIF4 function was blocked, Gag transiently accumulated in discrete, perinuclear, nonendocytic clusters that colocalized with endogenous KIF4, with Ubc9, an E2 SUMO-1 conjugating enzyme, and with SUMO. These studies identify a novel transit station through which Gag traffics en route to particle assembly and highlight the importance of KIF4 in regulating HIV-1 Gag trafficking and stability.  

3.1156           Fusion of Enhanced Green Fluorescent Protein to the Pseudorabies Virus Axonal Sorting Protein Us9 Blocks Anterograde Spread of Infection in Mammalian Neurons

Lyman, M.G:, Curanovic, D., Brideau, A.D. and Enquist, L.W.

1. Virol., 82(20), 10308-10311 (2008)

  Pseudorabies virus encodes a membrane protein (Us9) that is essential for the axonal sorting of virus particles within neurons and anterograde spread in the mammalian nervous system. Enhanced green fluorescent protein (GFP)-tagged Us9 mimicked the trafficking properties of the wild-type protein in nonneuronal cells. We constructed a pseudorabies virus strain that expressed Us9-GFP and tested its spread capabilities in the rat visual system and in primary neuronal cultures. We report that Us9-EGFP does not promote anterograde spread of infection and may disrupt packing of viral membrane proteins in lipid rafts, an essential step for Us9-mediated axonal sorting.  

3.1157           An epidermal growth factor (EGF) -dependent interaction between GIT1 and sorting nexin 6 promotes degradation of the EGF receptor

Cavet, M.E., Pang, J., Yin, G. And Berk, B.C. FASEB J., 22(10), 3607-3616 (2008)   G-protein coupled receptor (GPCR) kinase-2 interacting protein 1 (GIT1) is a multifunctional scaffolding protein that regulates epidermal growth factor receptor (EGFR) signaling pathways. We demonstrate that GIT1 interacts with sorting nexin 6 (SNX6), a member of the SNX family that increases EGFR trafficking between endosomes and lysosomes, thereby enhancing EGFR degradation. The GIT1-SNX6 interaction is increased 3-fold after treatment with EGF for 60 min. The second coiled-coil domain (CC2; aa 424–474) of GIT1 mediates binding to SNX6. Subcellular fractionation and confocal microscopy data indicate that GIT1 and SNX6 interact in endosomes. Knockdown of GIT1 expression by small interfering RNA decreased the rate of EGF-induced EGFR degradation. Expression of exogenous GIT1 or SNX6 alone did not alter EGFR degradation; however, coexpression of GIT1 and SNX6 decreased EGFR levels both basally and in response to EGF. In contrast, expression of GIT1(CC2 deleted) and SNX6 did not reduce EGFR levels, demonstrating that the interaction between GIT1 and SNX6 was required to regulate EGFR trafficking. Phosphorylation of the EGFR substrate phospholipase C- was decreased by coexpression of GIT1 and SNX6. These data demonstrate an endosomal, EGF-regulated interaction between SNX6 and GIT1 that enhances degradation of the EGFR, and thereby alters EGFR signaling. Our findings suggest a new role for GIT1 in tyrosine kinase receptor trafficking.—Cavet, M. E., Pang, J., Yin, G., Berk, B. C. An epidermal growth factor (EGF)-dependent interaction between GIT1 and sorting nexin 6 promotes degradation of the EGF receptor.  

3.1158           Formation and function of ceramide-enriched membrane platforms with CD38 during M1-receptor stimulation in bovine coronary arterial myocytes

Jia, S-J. et al Am. J. Physiol. Heart Circ. Physiol., 295, H1743-H1752 (2008)   CD38 contains an ADP ribosylcyclase domain that mediates intracellular Ca²⁺ signaling by the production of cyclic ADP-ribose (cADPR), but the mechanisms by which the agonists activate this enzyme remain unclear. The present study tested a hypothesis that a special lipid-raft (LR) form, ceramide-enriched lipid platform, contributes to CD38 activation to produce cADPR in response to muscarinic type 1 (M₁) receptor stimulation in bovine coronary arterial myocytes (CAMs). By confocal microscopic analysis, oxotremorine (Oxo), an M₁ receptor agonist, was found to increase LR clustering on the membrane with the formation of a complex of CD38 and LR components such as GM₁, acid sphingomyelinase (ASMase), and ceramide, a typical ceramide-enriched macrodomain. At 80 µM, Oxo increased LR clustering by 78.8%, which was abolished by LR disruptors, methyl-β-cyclodextrin (MCD), or filipin. With the use of a fluorescence resonance energy transfer (FRET) technique, 15.5 ± 1.9% energy transfer rate (vs. 5.3 ± 0.9% of control) between CD38 and LR component, ganglioside M₁ was detected, further confirming the proximity of both molecules. In the presence of MCD or filipin, there were no FRET signals detected. In floated detergent-resistant membrane fractions, CD38 significantly increased in LR fractions of CAMs treated by Oxo. Moreover, MCD or filipin attenuated Oxo-induced production of cADPR via CD38. Functionally, Oxo-induced intracellular Ca²⁺ release and coronary artery constriction via cADPR were also blocked by LR disruption or ASMase inhibition. These results provide the first evidence that the formation of ceramide-enriched lipid macrodomains is crucial for Oxo-induced activation of CD38 to produce cADPR in CAMs, and these lipid macrodomains mediate transmembrane signaling of M₁ receptor activation to produce second messenger cADPR.  

3.1159           Determination of the Topology of the Hydrophobic Segment of Mammalian Diacylglycerol Kinase Epsilon in a Cell Membrane and Its Relationship to Predictions from Modeling

Decaffmeyer, M. et al

1. Mol. Biol., 383, 797-809 (2008)

    The epsilon isoform of diacylglycerol kinase (DGK ) is unique among mammalian DGKs in having a segment of hydrophobic amino acids comprising approximately residues 20 to 41. Several algorithms predict this segment to be a transmembrane (TM) helix. Using PepLook, we have performed an in silico analysis of the conformational preference of the segment in a hydrophobic environment comprising residues 18 to 42 of DGK . We find that there are two distinct groups of stable conformations, one corresponding to a straight helix that would traverse the membrane and the second corresponding to a bent helix that would enter and leave the same side of the membrane. Furthermore, the calculations predict that substituting the Pro32 residue in the hydrophobic segment with an Ala will cause the hydrophobic segment to favor a TM orientation. We have expressed the P32A mutant of DGK , with a FLAG tag (an N-terminal 3×FLAG epitope tag) at the amino terminus, in COS-7 cells. We find that this mutation causes a large reduction in both k_cat and K_m while maintaining k_cat/K_m constant. Specificity of the P32A mutant for substrates with polyunsaturated acyl chains is retained. The P32A mutant also has higher affinity for membranes since it is more difficult to extract from the membrane with high salt concentration or high pH compared with the wild-type DGK . We also evaluated the topology of the proteins with confocal immunofluorescence microscopy using NIH 3T3 cells. We find that the FLAG tag at the amino terminus of the wild-type enzyme is not reactive with antibodies unless the cell membrane is permeabilized with detergent. We also demonstrate that at least a fraction of the wild-type DGK is present in the plasma membrane and that comparable amounts of the wild-type and P32A mutant proteins are in the plasma membrane fraction. This indicates that in these cells the hydrophobic segment of the wild-type DGK is not TM but takes up a bent conformation. In contrast, the FLAG tag at the amino terminus of the P32A mutant is exposed to antibody both before and after membrane permeabilization. This modeling approach thus provides an explanation, not provided by simple predictive algorithms, for the observed topology of this protein in cell membranes. The work also demonstrates that the wild-type DGK is a monotopic protein.  

3.1160           Cripto recruits Furin and PACE4 and controls Nodal trafficking during proteolytic maturation

Blancet, M-H. et al EMBO J., 27, 2580-2591 (2008)   The glycosylphosphatidylinositol (GPI)-anchored proteoglycan Cripto binds Nodal and its type I receptor Alk4 to activate Smad2,3 transcription factors, but a role during Nodal precursor processing has not been described. We show that Cripto also binds the proprotein convertases Furin and PACE4 and localizes Nodal processing at the cell surface. When coexpressed as in early embryonic cells, Cripto and uncleaved Nodal already associated during secretion, and a Cripto-interacting region in the Nodal propeptide potentiated the effect of proteolytic maturation on Nodal signalling. Disruption of the trans-Golgi network (TGN) by brefeldin A blocked secretion, but export of Cripto and Nodal to the cell surface was not inhibited, indicating that Nodal is exposed to extracellular convertases before entering the TGN/endosomal system. Density fractionation and antibody uptake experiments showed that Cripto guides the Nodal precursor in detergent-resistant membranes to endocytic microdomains marked by GFP–Flotillin. We conclude that Nodal processing and endocytosis are coupled in signal-receiving cells.  

3.1161           Effects of Monoglycerides on P-Glycoprotein: Modulation of the Activity and Expression in Caco-2 Cell Monolayers

Barta, C.A., Sachs-Barrable, K., Feng, F. and Wasan, K.M. Mol. Pharmaceut., 5(5), 863-875 (2008)   The purpose of this study was to analyze the effects of two common monoglyceride components of lipid excipients, 1-monoolein and 1-monostearin, on the activity and expression of P-glycoprotein (P-gp) in Caco-2 cells. Non-cytotoxic concentrations of 1-monoolein and 1-monostearin were determined by assessing membrane permeability and mitochondrial activity in Caco-2 cells, a human colon adenocarcinoma cell line. Concentrations of 500 and 100 μM were used to evaluate P-gp activity through Rh123 accumulation and bifunctional transport studies. The P-gp protein expression levels were quantified through the use of immunoblots. The changes in cell membrane fluidity and nuclear membrane integrity upon the addition of monoglycerides were analyzed by fluorescence anisotropy using DPH and TMA-DPH as the fluorescent labels and by using increasing salt concentrations to release the nuclear contents, respectively. The absorptive flux (apical to basolateral) in the bifunctional transport studies was not found to be statistically significant for the non-cytotoxic concentrations of 1-monoolein and 1-monostearin. However, treatments of 500 and 100 μM of 1-monoolein or 1-monostearin displayed statistically lowered efflux (basolaterial to apical, P < 0.05) compared to the controls (7.9 0.8, 12.9 2.6 × 10⁶ cm/s for 1-monoolein or 11.1 2.0, 11.4 2.3 × 10⁶ cm/s for 1-monostearin, respectively, compared to the untreated control, 21.1 2.9 × 10⁶ cm/s, n = 5). Rh123 accumulation was also found to be enhanced upon 24 h incubation with both concentrations of the monoglycerides; however, only concentrations of 500 μM of the monoglycerides were shown to significantly reduce the P-gp protein expression. The results from this study suggest that these two monoglycerides, common components in various lipid excipients, are inhibitors of P-gp.  

3.1162           CDK5-dependent Phosphorylation of the Rho Family GTPase TC10 Regulates Insulin-stimulated GLUT4 Translocation

Okada, S. et al

1. Biol. Chem., 283(51), 35455-35463 (2008)

  Insulin stimulation results in the activation of cyclin-dependent kinase-5 (CDK5) in lipid raft domains via a Fyn-dependent phosphorylation on tyrosine residue 15. In turn, activated CDK5 phosphorylates the Rho family GTP-binding protein TC10 on threonine 197 that is sensitive to the CDK5 inhibitor olomoucine and blocked by small interfering RNA-mediated knockdown of CDK5. The phosphorylation deficient mutant T197A-TC10 was not phosphorylated and excluded from the lipid raft domain, whereas the phosphorylation mimetic mutant (T197D-TC10 ) was lipid raft localized. Insulin resulted in the GTP loading of T197D-TC10 but not T197A-TC10 and in parallel, T197D-TC10 but not T197A-TC10 depolymerized cortical actin and inhibited insulin-stimulated GLUT4 translocation. These data demonstrate that CDK5-dependent phosphorylation maintains TC10 in lipid raft compartments thereby disrupting cortical actin, whereas subsequent dephosphorylation of TC10 through inactivation of CDK5 allows for the re-assembly of F-actin. Because cortical actin reorganization is required for insulin-stimulated GLUT4 translocation, these data are consistent with a CDK5-dependent TC10 cycling between lipid raft and non-lipid raft compartments.

 

3.1163           Bluetongue Virus Outer Capsid Protein VP5 Interacts with Membrane Lipid Rafts via a SNARE Domain

Bhattacharya, B. and Roy, P.

1. Virol., 82(21), 10600-10612 (2008)

  Bluetongue virus (BTV) is a nonenveloped double-stranded RNA virus belonging to the family Reoviridae. The two outer capsid proteins, VP2 and VP5, are responsible for virus entry. However, little is known about the roles of these two proteins, particularly VP5, in virus trafficking and assembly. In this study, we used density gradient fractionation and methyl beta cyclodextrin, a cholesterol-sequestering drug, to demonstrate not only that VP5 copurifies with lipid raft domains in both transfected and infected cells, but also that raft domain integrity is required for BTV assembly. Previously, we showed that BTV nonstructural protein 3 (NS3) interacts with VP2 and also with cellular exocytosis and ESCRT pathway proteins, indicating its involvement in virus egress (A. R. Beaton, J. Rodriguez, Y. K. Reddy, and P. Roy, Proc. Natl. Acad. Sci. USA 99:13154-13159, 2002; C. Wirblich, B. Bhattacharya, and P. Roy J. Virol. 80:460-473, 2006). Here, we show by pull-down and confocal analysis that NS3 also interacts with VP5. Further, a conserved membrane-docking domain similar to the motif in synaptotagmin, a protein belonging to the SNARE (soluble N-ethylmaleimide-sensitive fusion attachment protein receptor) family was identified in the VP5 sequence. By site-directed mutagenesis, followed by flotation and confocal analyses, we demonstrated that raft association of VP5 depends on this domain. Together, these results indicate that VP5 possesses an autonomous signal for its membrane targeting and that the interaction of VP5 with membrane-associated NS3 might play an important role in virus assembly.  

3.1164           The yeast O-acyltransferase Gup1p interferes in lipid metabolism with direct consequences on the sphingolipid-sterol-ordered domains integrity/assembly

Ferreira, C. and Lucas, C. Biochim. Biophys. Acta, 1778, 2648-2653 (2008)   Saccharomyces cerevisiae Gup1p is a membrane-bound O-acyltransferase. Previous works involved GUP1 in a wide range of crucial processes for cell preservation and functioning. These include cytoskeleton polarization and secretory/endocytic pathway, GPI-anchor remodelling, wall composition and integrity, and membrane lipids, with a reduction in phospholipids and an increase in acylglycerols. DRM fractions were found in considerably lower amounts in gup1Δ than in wt strain. Additionally, the proteins presumably associated with lipid micro domains, Gas1p and Pma1p, were present in much smaller amounts in the mutant DRMs. Pma1p is also found in minor quantities in the whole cells extracts of the gup1Δ mutant. Accordingly, H⁺-ATPase activity was reduced in about 40%. Deletion of GUP1 resulted in higher sensibility to specific sphingolipid biosynthesis inhibitors and a notorious resistance to ergosterol biosynthesis inhibitors. Furthermore, the majority of mutant cells displayed an even (less punctuated) sterol distribution. The present work presents improvements to DRMs extraction methodology and filipin-sterol staining, provides evidence supporting that Gup1p is involved in lipid metabolism and shows the direct consequences of its absence on the plasma membrane sphingolipid-sterol-ordered domains integrity/assembly.  

3.1165           Autocrine activity of tumor-derived membrane vesicles

Hong, B.S., Choi, E.Y., Yoon, Y.J. and Gho, Y.S. Eur. J. Cancer Suppl., 6(12), 96 (2008)   Background: Actively growing tumor cells release membrane vesicles into extracellular milieu, and the rate of shedding increases in malignant tumors. Tumor-derived membrane vesicles (TMVs), enriched in most surface antigens and proteases derived from their originating cells, are nowadays gaining attention as important mediators of cell-to-cell communication facilitating processes such as angiogenesis and immune modulation with paracrine functions. Since TMVs have the potential to affect to tumor cells themselves, the role of TMVs in autocrine signals to tumor cells has been investigated. Material and Methods: We isolated TMVs from SW480, human colorectal adenocarcinoma cells by ultracentrifugation onto sucrose cushion and iodixanol gradients. We characterized TMVs with Western blotting using  membrane vesicles markers, density in an iodixanol gradient, and transmission electron microscopy. Cell proliferation was determined by [3H]- thymidine incorporation and cell migration assay was performed in a 48-well microchemotaxis chamber. Intracellular Ca2+ mobilization was determined with the fluorescent Ca2+ indicator fluo-3/AM, and AlexaFluor 488- conjugated phalloidine was used for imaging actin stress fiber formation. Results: The purified TMVs settled at a density of ~1.110 g/mL, presenting membrane vesicles markers (CD63 and CD81), and almost all TMVs were spherical and bi-layered vesicles ranging from 40 to 150 nm in size. TMVs stimulated proliferation of tumor cells in a dose-dependent manner, with a maximum effect of 2.4±0.2-fold increases upon exposure to 5 mg/ml of TMVs. TMVs increased migration of tumor cells in a dose-dependent manner, with a maximum effect of 4.1±0.9-fold increases upon exposure to 1 mg/ml of TMVs. The treatment of 1 mg/ml of TMVs to fluo-3/AM-loaded tumor cells caused increased intracellular free Ca2+ release into the cytosol. Moreover, formation of actin stress fibers was enhanced by the treatment of 1 mg/ml of TMVs for 30 min. Theses results suggest that TMVs may deliver autocrine signals to target tumor cells. Conclusion: We suggest that TMVs may be involved in promoting autocrine signaling to tumor themselves, neighboring or distant tumor cells for the rapid induction of proliferation, migration, as well as survival. Therefore, modulating biogenesis and functions of TMVs may be potential novel therapeutic strategies for treating pathological states including malignant tumors.  

3.1166           Ceramide and raft signaling are linked with each other in UVA radiation-induced gene expression

Grether-Beck, S. et al Oncogene, 27, 4768-4778 (2008)   Solar ultraviolet A (UVA) (320–400 nm) radiation-induced gene expression in keratinocytes is initiated at the level of the cell membrane via generation of singlet oxyge and subsequent formation of ceramide from sphingomyelin. We now report that the UVA response also involves raft signaling and that ceramide and raft signaling are linked with each other. Upon UVA irradiation, the lipid composition of rafts decreased 40% in sphingomyelinand 60% in cholesterol (Chol). Also, decrease of Chol increased the susceptibility towards UVA-induced gene expression, whereas increase of Chol completely abolished their capacity to generate signaling ceramides and to mount the subsequent UVA response. This inhibition was not associated with UVA-induced Chol oxidation and was also seen after treatment of cells with plant sterols. The UVA responsiveness depended on the ratio of Chol versus ceramide in rafts. A ratio smaller than 1 permitted initiation and transduction of the signaling response, whereas a ratio greater than 1, for example, upon sterol pretreatment, abolished this response, indicating that UVA radiation-induced ceramide signaling is controlled by the lipid composition of rafts.    

3.1167           Involvement of miltefosine-mediated ERK activation in glioma cell apoptosis through Fas regulation

Tewari, R., Sharma, V., Koul, N. and Sen, E.

1. Neurochem., 107(3), 616-627 (2008)

  The anti-neoplastic property of alkyl phospholipids has been tested for the treatment of several malignancies. In this study, we evaluated the efficacy of miltefosine (Hexadecylphosphocholine – an alkyl phospholipids analogue) on glioblastoma multiforme. In this study, we demonstrate that miltefosine-induced apoptosis is accompanied by elevated Fas, Fas-associated death domain (FADD) expression, caspase-8 activity and the increased distribution of Fas and FADD towards lipid raft microdomain to form death inducing signaling complex. Treatment with miltefosine resulted in increase in Ras, extracellular signal-regulated kinase (ERK) and p38MAPK activity. Expression of dominant-negative Ras (Ras N17) attenuated miltefosine-mediated apoptosis. Although inhibition of both ERK and p38MAPK decreased the pro-apoptotic effects of miltefosine, it was the inhibition of ERK and not p38MAPK activation that decreased Fas and FADD expression. An ERK-dependent increase in the expression of γH2AX-involved in response to DNA double-stranded breaks was also observed. Taken together, our findings suggest the involvement of ERK activation in miltefosine-induced glioma cell apoptosis.  

3.1168           Delta Protein Kinase C Interacts with the d Subunit of the F1F0 ATPase in Neonatal Cardiac Myocytes Exposed to Hypoxia or Phorbol Ester: IMPLICATIONS FOR F1F0 ATPase REGULATION

Nguyen, T., Ogbi, M. and Johnson, J.A.

1. Biol. Chem., 283(44), 29831-29840 (2008)

  Mitochondrial protein kinase C isozymes have been reported to mediate both cardiac ischemic preconditioning and ischemia/reperfusion injury. In addition, cardiac preconditioning improves the recovery of ATP levels after ischemia/reperfusion injury. We have, therefore, evaluated protein kinase C modulation of the F₁F₀ ATPase in neonatal cardiac myocytes. Exposure of cells to 3 or 100 nM 4β-phorbol 12-myristate-13-acetate induced co-immunoprecipitation of protein kinase C (but not , , or protein kinase C) with the d subunit of the F₁F₀ ATPase. This co-immunoprecipitation correlated with 40 ± 3% and 72 ± 9% inhibitions of oligomycin-sensitive F₁F₀ ATPase activity, respectively. We observed prominent expression of protein kinase C in cardiac myocyte mitochondria, which was enhanced following a 4-h hypoxia exposure. In contrast, hypoxia decreased mitochondrial PKC levels by 85 ± 1%. Following 4 h of hypoxia, F₁F₀ ATPase activity was inhibited by 75 ± 9% and protein kinase C co-immunoprecipitated with the d subunit of F₁F₀ ATPase. In vitro incubation of protein kinase C with F₁F₀ ATPase enhanced F₁F₀ activity in the absence of protein kinase C activators and inhibited it in the presence of activators. Recombinant protein kinase C also inhibited F₁F₀ ATPase activity. Protein kinase C overlay assays revealed protein kinase C binding to the d subunit of F₁F₀ ATPase, which was modulated by diacylglycerol, phosphatidylserine, and cardiolipin. Our results suggest a novel regulation of the F₁F₀ ATPase by the protein kinase C isozyme.  

3.1169           Transport of LDL-derived cholesterol from the NPC1 compartment to the ER involves the trans-Golgi network and the SNARE protein complex

Urano, Y. et al PNAS, 105(43), 16513-16518 (2008)   Mammalian cells acquire cholesterol mainly from LDL. LDL enter the endosomes, allowing cholesteryl esters to be hydrolyzed by acid lipase. The hydrolyzed cholesterol (LDL-CHOL) enters the Niemann–Pick type C1 (NPC1)-containing endosomal compartment en route to various destinations. Whether the Golgi is involved in LDL-CHOL transport downstream of the NPC1 compartment has not been demonstrated. Using subcellular fractionation and immunoadsorption to enrich for specific membrane fractions, here we show that, when parental Chinese hamster ovary (CHO) cells are briefly exposed to ³H-cholesteryl linoleate (CL) labeled-LDL, newly liberated ³H-LDL-CHOL appears in membranes rich in trans-Golgi network (TGN) long before it becomes available for re-esterification at the endoplasmic reticulum (ER) or for efflux at the plasma membrane. In mutant cells lacking NPC1, the appearance of newly liberated ³H-LDL-CHOL in the TGN-rich fractions is much reduced. We next report a reconstituted transport system that recapitulates the transport of LDL-CHOL to the TGN and to the ER. The transport system requires ATP and cytosolic factors and depends on functionality of NPC1. We demonstrate that knockdown by RNAi of 3 TGN-specific SNAREs (VAMP4, syntaxin 6, and syntaxin 16) reduces ≥50% of the LDL-CHOL transport in intact cells and in vitro. These results show that vesicular trafficking is involved in transporting a significant portion of LDL-CHOL from the NPC1-containing endosomal compartment to the TGN before its arrival at the ER.  

3.1170           Importance of cholesterol-rich membrane microdomains in the interaction of the S protein of SARS-coronavirus with the cellular receptor angiotensin-converting enzyme 2

Glende, J. et al Virology, 381, 215-221 (2008)   Cholesterol present in the plasma membrane of target cells has been shown to be important for the infection by SARS-CoV. We show that cholesterol depletion by treatment with methyl-β-cyclodextrin (mβCD) affects infection by SARS-CoV to the same extent as infection by vesicular stomatitis virus-based pseudotypes containing the surface glycoprotein S of SARS-CoV (VSV-ΔG-S). Therefore, the role of cholesterol for SARS-CoV infection can be assigned to the S protein and is unaffected by other coronavirus proteins. There have been contradictory reports whether or not angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV, is present in detergent-resistant membrane domains. We found that ACE2 of both Vero E6 and Caco-2 cells co-purifies with marker proteins of detergent-resistant membranes supporting the notion that cholesterol-rich microdomains provide a platform facilitating the efficient interaction of the S protein with the cellular receptor ACE2. To understand the involvement of cholesterol in the initial steps of the viral life cycle, we applied a cell-based binding assay with cells expressing the S protein and cells containing angiotensin-converting enzyme 2 (ACE2). Alternatively, we used a soluble S protein as interaction partner. Depletion of cholesterol from the ACE2-expressing cells reduced the binding of S-expressing cells by 50% whereas the binding of soluble S protein was not affected. This result suggests that optimal infection requires a multivalent interaction between viral attachment protein and cellular receptors.  

3.1171           Membrane progestin receptors α and γ in renal epithelium

Lemale, J. et al Biochim. Biophys. Acta, 1783, 2234-2240 (2008)   Sex hormones have broader effects than regulating reproductive functions. Recent identification of membrane progestin receptors expressed in kidney prompted us to investigate their putative involvement in the renal effects of this hormone. We first focused our investigations on mPRα and γ by analyzing three parameters 1/ their distribution along the mouse nephron and their subcellular location in native kidney, 2/ the ability of progesterone to stimulate ERK pathway and/or Ca²⁺ release from internal stores in native kidney structures and 3/ the cellular localization of mPRα and its molecular determinants in heterologous expression system. We observed that 1/ mPRα expression is restricted to proximal tubules of both male and female mice whereas mPRγ exhibits a much broader expression all along the nephron except the glomerulus, 2/ mPRα and γ are not localized at the plasma membrane in native kidney, 3/ this expression does not permit either progesterone-induced ERK phosphorylation or Ca²⁺ release and 4/ in HEK transfected cells, mPRα localizes in the endoplasmic reticulum (ER) due to a C-terminal ER retention motif (− KXX). Therefore, we have characterized mPRs in kidney but their role in renal physiology remains to be elucidated.  

3.1172           Early adhesion induces interaction of FAK and Fyn in lipid domains and activates raft-dependent Akt signaling in SW480 colon cancer cells

Baillat, G., Siret, C., Delamarre, E. and Luis, J. Biochim. Biophys. Acta, 1783, 2323-2331 (2008)   Integrin-dependent interaction of epithelial tumor cells with extracellular matrix (ECM) is critical for their migration, but also for hematogenous dissemination. Elevated expression and activity of Src family kinases (SFKs) in colon cancer cells is often required in the disease progression. In this work, we highlighted how focal adhesion kinase (FAK) and SFKs interacted and we analyzed how PI3K/Akt and MAPK/Erk1/2 signaling pathways were activated in early stages of colon cancer cell adhesion. During the first hour, integrin engagement triggered FAK-Y397 phosphorylation and a fraction of FAK was located in lipid rafts/caveolae domains where it interacted with Fyn. The FAK-Y861 and/or -Y925 phosphorylations led to a subsequently FAK translocation out of lipid domains. In parallel, a PI3K/Akt pathway dependent of lipid microdomain integrity was activated. In contrast, the MAPK/Erk1/2 signaling triggered by adhesion increased during at least 4 h and was independent of cholesterol disturbing. Thus, FAK/Fyn interaction in lipid microdomains and a Akt-1 activation occurred at the same time during early contact with ECM suggesting a specific signaling dependent of lipid rafts/caveolae domains.    

3.1173           Night Blindness and the Mechanism of Constitutive Signaling of Mutant G90D Rhodopsin

Dizhoor, A.M. et al

1. Neurosci., 28(45), 11662-11672 (2008)

  The G90D rhodopsin mutation is known to produce congenital night blindness in humans. This mutation produces a similar condition in mice, because rods of animals heterozygous (D+) or homozygous (D+/+) for this mutation have decreased dark current and sensitivity, reduced Ca²⁺, and accelerated values of _REC and _D, similar to light-adapted wild-type (WT) rods. Our experiments indicate that G90D pigment activates the cascade, producing an equivalent background light of 130 Rh* rod^–1 for D+ and 890 Rh* rod^–1 for D+/+. The active species of the G90D pigment could be unregenerated G90D opsin or G90D rhodopsin, either spontaneously activated (as Rh*) or in some other form. Addition of 11-cis-retinal in lipid vesicles, which produces regeneration of both WT and G90D opsin in intact rods and ROS membranes, had no effect on the waveform or sensitivity of dark-adapted G90D responses, indicating that the active species is not G90D opsin. The noise spectra of dark-adapted G90D and WT rods are similar, and the G90D noise variance is much less than of a WT rod exposed to background light of about the same intensity as the G90D equivalent light, indicating that Rh* is not the active species. We hypothesize that G90D rhodopsin undergoes spontaneous changes in molecular conformation which activate the transduction cascade with low gain. Our experiments provide the first indication that a mutant form of the rhodopsin molecule bound to its 11-cis-chromophore can stimulate the visual cascade spontaneously at a rate large enough to produce visual dysfunction.  

3.1174           Lamellar Bodies of Human Epidermis: Proteomics Characterization by High Throughput Mass Spectrometry and Possible Involvement of CLIP-170 in their Trafficking/Secretion

Raymonds, A-A-. et al Mol. Cell. Proteomics, 7(11), 2151-2175 (2008)   Lamellar bodies (LBs) are tubulovesicular secretory organelles of epithelial cells related to lysosomes. In the epidermis, they play a crucial role in permeability barrier homeostasis, secreting their contents, lipids, a variety of hydrolases, protease inhibitors, and antimicrobial peptides, in the upper keratinocyte layers. The identification of proteins transported in epidermal LBs is still far from complete, and the way their secretion is controlled unknown. In this study, we describe the first proteomics characterization by nano-LC-MS/MS of a fraction enriched in epidermal LBs. We identified 984 proteins, including proteins known or thought to be secreted by LBs. Moreover 31 proteins corresponded to lysosomal components further suggesting that LBs are a new class of secretory lysosomes. Many of the newly found proteins could play a role in the epidermal barrier and desquamation (one acid ceramidase-like protein, apolipoproteins, glycosidases, protease inhibitors, and peptidases) and in LB trafficking (e.g. Rab, Arf, and motor complex proteins). We focus here on CLIP-170/restin, a protein that mediates interactions between organelles and microtubules. Western blotting confirmed the presence of CLIP-170 and its known effectors IQGAP1 and Cdc42 in the LB-enriched fraction. We showed, by confocal microscopy analysis of skin cryosections, that CLIP-170 was expressed in differentiated keratinocytes, first at the periphery of the nucleus then with a granular cytoplasmic labeling evocative of LBs. It was preferentially co-localized with Cdc42 and with the known LB protein cathepsin D. CLIP-170 was also largely co-localized with Rab7. This study strongly suggests a new function for CLIP-170, its involvement together with Cdc42 and/or Rab7 in the intracellular trafficking of LBs, and provides evidence that nano-LC-MS/MS combined with monodimensional electrophoresis separation constitutes a powerful method for identifying proteins in a complex mixture such as subcellular structures.  

3.1175           P2Y1 Receptor Activation Elicits Its Partition out of Membrane Rafts and Its Rapid Internalization from Human Blood Vessels: Implications for Receptor Signaling

Norambuena, A. et al Mol. Pharmacol., 74(6), 1666-1677 (2008)   The nucleotide P2Y₁ receptor (P2Y₁R) is expressed in both the endothelial and vascular smooth muscle cells; however, its plasma membrane microregionalization and internalization in human tissues remain unknown. We report on the role of membrane rafts in P2Y₁R signaling by using sodium carbonate or OptiPrep sucrose density gradients, Western blot analysis, reduction of tissue cholesterol content, and vasomotor assays of endothelium-denuded human chorionic arteries. In tissue extracts prepared either in sodium carbonate or OptiPrep, approximately 20 to 30% of the total P2Y₁R mass consistently partitioned into raft fractions and correlated with vasomotor activity. Vessel treatment with methyl β-cyclodextrin reduced the raft partitioning of the P2Y₁R and obliterated the P2Y₁R-mediated contractions but not the vasomotor responses elicited by either serotonin or KCl. Perfusion of chorionic artery segments with 100 nM 2-methylthio ADP or 10 nM [[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl] 2,3dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS 2365), a selective P2Y₁R agonist, not only displaced within 4 min the P2Y₁R localization out of membrane rafts but also induced its subsequent internalization. 2'-Deoxy-N⁶-methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS 2179), a specific P2Y₁R antagonist, did not cause a similar displacement but blocked the agonist-induced exit from rafts. Neither adenosine nor uridine triphosphate displaced the P2Y₁R from the membrane raft, further evidencing the pharmacodynamics of the receptor-ligand interaction. Vascular reactivity assays showed fading of the ligand-induced vasoconstrictions, a finding that correlated with the P2Y₁R exit from raft domains and internalization. These results demonstrate in intact human vascular smooth muscle the association of the P2Y₁R to membrane rafts, highlighting the role of this microdomain in P2Y₁R signaling.  

3.1176           Hereditary Spastic Paraplegia-Associated Mutations in the NIPA1 Gene and Its Caenorhabditis elegans Homolog Trigger Neural Degeneration In Vitro and In Vivo through a Gain-of-Function Mechanism

Zhao, J., Matthies, D.S., Botzolakis, E.J., Macdonald, R.L., Blakely, R.D. and Hedera, P.

1. Neurosci., 28(51), 13938-13951 (2008)

  We studied the consequences of expression of wild-type (WT) human NIPA1 and two mutant forms of NIPA1 with known HSP-associated mutations (T45R and G106R) on cultured rat cortical neurons and using equivalent substitutions in the Caenorhabditis elegans NIPA1 homolog CeNIPA. WT NIPA1 localized in transfected neuronal and non-neuronal cells to the Golgi complex, a subset of synaptic vesicles, to a subset of early endosomes, and plasma cell membrane. Mutant NIPA1 accumulated in the endoplasmic reticulum (ER) triggering ER stress and features of apoptotic cell death. Flow cytometric analysis of NIPA1 surface expression demonstrated relatively intact trafficking of mutant forms and only the T45R mutant exhibited modestly reduced patterns of surface expression without evidence for a dominant-negative effect. In vivo pan-neuronal expression of the WT C. elegans NIPA1 homolog (CeNIPA) was well tolerated, with no obvious impact on neuronal morphology or behavior. In striking contrast, expression of CeNIPA bearing HSP-associated mutations caused a progressive neural degeneration and a clear motor phenotype. Neuronal loss in these animals began at day 7 and by day 9 animals were completely paralyzed. These effects appeared to arise from activation of the apoptotic program triggered by unfolded protein response (UPR), as we observed marked modifications of motor and cellular phenotype when mutant NIPA1 was expressed in caspase (ced-3)- and UPR (xbp-1)-deficient backgrounds. We propose that HSP-associated mutations in NIPA1 lead to cellular and functional deficits through a gain-of-function mechanism supporting the ER accumulation of toxic NIPA1 proteins.

3.1177           Eosinophil granules function extracellularly as receptor-mediated secretory organelles

Neves, J.S. et al PNAS, 105(47), 18478-18483 (2008)   Intracellular granules in several types of leukocytes contain preformed proteins whose secretions contribute to immune and inflammatory functions of leukocytes, including eosinophils, cells notably associated with asthma, allergic inflammation, and helminthic infections. Cytokines and chemokines typically elicit extracellular secretion of granule proteins by engaging receptors expressed externally on the plasma membranes of cells, including eosinophils. Eosinophil granules, in addition to being intracellular organelles, are found as intact membrane-bound structures extracellularly in tissue sites of eosinophil-associated diseases. Neither the secretory capacities of cell-free eosinophil granules nor the presence of functional cytokine and chemokine receptors on membranes of leukocyte granules have been recognized. Here, we show that granules of human eosinophils express membrane receptors for a cytokine, IFN-γ, and G protein–coupled membrane receptors for a chemokine, eotaxin, and that these receptors function by activating signal-transducing pathways within granules to elicit secretion from within granules. Capacities of intracellular granule organelles to function autonomously outside of eosinophils as independent, ligand-responsive, secretion-competent structures constitute a novel postcytolytic mechanism for regulated secretion of eosinophil granule proteins that may contribute to eosinophil-mediated inflammation and immunomodulation.  

3.1178           Induction of HIV Transcription by Nef Involves Lck Activation and Protein Kinase C Raft Recruitment Leading to Activation of ERK1/2 but Not NF B

Witte, V. et al

1. Immunol., 181, 8425-8432 (2008)

  The Nef protein of HIV-1 is a key promoter of disease progression, owing to its dramatic yet ill-defined impact on viral replication. Previously, we have shown that Nef enhances embryonic ectodermal development Tat-mediated transcription in a manner depending on Lck and the cytoplasmic sequestration of the transcriptional repressor embryonic ectodermal development. In this study, we report that Lck is activated by Nef and targets protein kinase C downstream, leading to the translocation of the kinase into membrane microdomains. Although microdomain-localized protein kinase C is thought to induce the transcription factor NF B, we unexpectedly failed to correlate Nef-induced signaling events with enhanced NF B activity. Instead, we observed an increase in ERK MAPK activity. We conclude that Nef-mediated signaling cooperates with Nef-induced derepression and supports HIV transcription through an ERK MAPK-dependent, but NF B-independent, pathway.    

3.1179           Switch-like Control of SREBP-2 Transport Triggered by Small Changes in ER Cholesterol: A Delicate Balance

Radhakrishnan, A., Goldstein, J.L., McDonald, J.G. and Brown, M.S. Cell Metabolism, 8, 512-521 (2008)   Animal cells control their membrane lipid composition within narrow limits, but the sensing mechanisms underlying this control are largely unknown. Recent studies disclosed a protein network that controls the level of one lipid—cholesterol. This network resides in the endoplasmic reticulum (ER). A key component is Scap, a tetrameric ER membrane protein that binds cholesterol. Cholesterol binding prevents Scap from transporting SREBPs to the Golgi for activation. Using a new method to purify ER membranes from cultured cells, we show that Scap responds cooperatively to ER cholesterol levels. When ER cholesterol exceeds 5% of total ER lipids (molar basis), SREBP-2 transport is abruptly blocked. Transport resumes when ER cholesterol falls below the 5% threshold. The 5% threshold is lowered to 3% when cells overexpress Insig-1, a Scap-binding protein. Cooperative interactions between cholesterol, Scap, and Insig create a sensitive switch that controls the cholesterol composition of cell membranes with remarkable precision.  

3.1180           Mechanosensing machinery for cells under low substratum rigidity

Wei, W-C., Lin, H-H., Shen, M-R. and Tang, M-J. Am. J. Physiol.Cell Physiol., 295, 1579-1589 (2008)   Mechanical stimuli are essential during development and tumorigenesis. However, how cells sense their physical environment under low rigidity is still unknown. Here we show that low rigidity of collagen gel downregulates β₁-integrin activation, clustering, and focal adhesion kinase (FAK) Y397 phosphorylation, which is mediated by delayed raft formation. Moreover, overexpression of autoclustered β₁-integrin (V737N), but not constitutively active β₁-integrin (G429N), rescues FAKY397 phosphorylation level suppressed by low substratum rigidity. Using fluorescence resonance energy transfer to assess β₁-integrin clustering, we have found that substratum rigidity between 58 and 386 Pa triggers β₁-integrin clustering in a dose-dependent manner, which is highly dependent on actin filaments but not microtubules. Furthermore, augmentation of β₁-integrin clustering enhances the interaction between β₁-integrin, FAK, and talin. Our results indicate that contact with collagen fibrils is not sufficient for integrin activation. However, substratum rigidity is required for integrin clustering and activation. Together, our findings provide new insight into the mechanosensing machinery and the mode of action for epithelial cells in response to their physical environment under low rigidity.  

3.1181           Trafficking of chlamydial antigens to the endoplasmic reticulum of infected epithelial cells

Giles, D.K. and Wyrick, P.B. Microbes and Infection, 10, 1494-1503 (2008)   Confinement of the obligate intracellular bacterium Chlamydia trachomatis to a membrane-bound vacuole, termed an inclusion, within infected epithelial cells neither prevents secretion of chlamydial antigens into the host cytosol nor protects chlamydiae from innate immune detection. However, the details leading to chlamydial antigen presentation are not clear. By immunoelectron microscopy of infected endometrial epithelial cells and in isolated cell secretory compartments, chlamydial major outer membrane protein (MOMP), lipopolysaccharide (LPS) and the inclusion membrane protein A (IncA) were localized to the endoplasmic reticulum (ER) and co-localized with multiple ER markers, but not with markers of the endosomes, lysosomes, Golgi nor mitochondria. Chlamydial LPS was also co-localized with CD1d in the ER. Since the chlamydial antigens, contained in everted inclusion membrane vesicles, were found within the host cell ER, these data raise additional implications for antigen processing by infected uterine epithelial cells for classical and non-classical T cell antigen presentation.  

3.1182           The peroxisomal membrane protein import receptor Pex3p is directly transported to peroxisomes by a novel Pex19p- and Pex16p-dependent pathway

Matsuzaki, T. and Fujiki, Y.

1. Cell Biol., 183(7), 1275-1286 (2008)

  Two distinct pathways have recently been proposed for the import of peroxisomal membrane proteins (PMPs): a Pex19p- and Pex3p-dependent class I pathway and a Pex19p- and Pex3p-independent class II pathway. We show here that Pex19p plays an essential role as the chaperone for full-length Pex3p in the cytosol. Pex19p forms a soluble complex with newly synthesized Pex3p in the cytosol and directly translocates it to peroxisomes. Knockdown of Pex19p inhibits peroxisomal targeting of newly synthesized full-length Pex3p and results in failure of the peroxisomal localization of Pex3p. Moreover, we demonstrate that Pex16p functions as the Pex3p-docking site and serves as the peroxisomal membrane receptor that is specific to the Pex3p–Pex19p complexes. Based on these novel findings, we suggest a model for the import of PMPs that provides new insights into the molecular mechanisms underlying the biogenesis of peroxisomes and its regulation involving Pex3p, Pex19p, and Pex16p.  

3.1183           Dendrites of Mammalian Neurons Contain Specialized P-Body-Like Structures That Respond to Neuronal Activation

Cougot, N., Bhattacharrya, S.N., Tapia-Arancibia, L., Bordonne, R., Filipowicz, W., Bertrand, E. and Rage, F.

1. Neurosci., 28(51), 13793-13804 (2008)

  Intracellular mRNA transport and local translation play a key role in neuronal physiology. Translationally repressed mRNAs are transported as a part of ribonucleoprotein (RNP) particles to distant dendritic sites, but the properties of different RNP particles and mechanisms of their repression and transport remain largely unknown. Here, we describe a new class of RNP-particles, the dendritic P-body-like structures (dlPbodies), which are present in the soma and dendrites of mammalian neurons and have both similarities and differences to P-bodies of non-neuronal cells. These structures stain positively for a number of P-body and microRNP components, a microRNA-repressed mRNA and some translational repressors. They appear more heterogeneous than P-bodies of HeLa cells, and they rarely contain the exonuclease Xrn1 but are positive for rRNA. These particles show motorized movements along dendrites and relocalize to distant sites in response to synaptic activation. Furthermore, Dcp1a is stably associated with dlP-bodies in unstimulated cells, but exchanges rapidly on neuronal activation, concomitantly with the loss of Ago2 from dlP-bodies. Thus, dlP-bodies may regulate local translation by storing repressed mRNPs in unstimulated cells, and releasing them on synaptic activation.  

3.1184           Proteomics in Trypanosoma cruzi – localization of novel proteins to various organelles

Ferella, M., Nilsson, D., darban, H., Rodrigues, C., Bontempi, E.J., Docampo, R. and Andersson, B. Proteomics, 8(13), 2735-2749 (2008)   The completion of the genome sequence of Trypanosoma cruzi has been followed by several studies of   protein expression, with the long-term aim to obtain a complete picture of the parasite proteome. We report a proteomic analysis of an organellar cell fraction from T. cruzi CL Brener epimastigotes. A total of 396 proteins were identified by LC-MS/MS. Of these, 138 were annotated as hypothetical in the genome databases and the rest could be assigned to several metabolic and biosynthetic pathways, transport, and structural functions. Comparative analysis with a whole cell proteome study resulted in the validation of the expression of 173 additional proteins. Of these, 38 proteins previously reported in other stages were not found in the only large-scale study of the total epimastigote stage proteome. A selected set of identified proteins was analyzed further to investigate gene copy number, sequence variation, transmembrane domains, and targeting signals. The genes were cloned and the proteins expressed with a c-myc epitope tag in T. cruzi epimastigotes. Immunofluorescence microscopy revealed the localization of these proteins in different cellular compartments such as ER, acidocalcisome, mitochondrion, and putative cytoplasmic transport or delivery vesicles. The results demonstrate that the use of enriched subcellular fractions allows the detection of T. cruzi proteins that are undetected by whole cell proteomic methods.  

3.1185           2-D DIGE analyses of enriched secretory lysosomes reveal heterogeneous profiles of functionally relevant proteins in leukemic and activated human NK cells

Schmidt, H., Gelhaus, C., Nebendahl, M., Lettau, M., Wartzl, C., Kabelitz, D., Leippe, M. and Janssen, O. Proteomics, 8(14), 2911-2925 (2008)   As part of the innate immune system, natural killer (NK) cells detect and lyse tumor and virus-infected cells without prior antigen-dependent recognition and expansion. To this end, they utilize dual-function organelles that combine properties of conventional lysosomes and exocytotic vesicles. Upon stimulation, these secretory lysosomes (SLs) release their cytotoxic molecules into the immunological synapse. In addition, several molecules associated with secretory vesicles become exposed on the plasma membrane. Recent studies often took advantage of the few established NK cell lines, for instance to analyze the exocytotic machinery associated with NK cell vesicles. NK cell lines and primary NK cells differ, however, substantially in the expression of “typical” surface receptors and their requirements to induce target cell lysis. Here, we directly compared the lysosomal compartments of different NK cell populations. We enriched SLs of two leukemic cell lines (YTS and NKL) and IL-2-expanded NK cells by subcellular fractionation and characterized their proteome by 2-D difference gel electrophoresis and MS. Although the overall protein composition of the lysosomal preparations was very similar and more than 90% of the proteins were present at comparable levels, we define a cell line-specific setup of functionally relevant proteins involved in antigen presentation and cytotoxic effector function.  

3.1186           Sub-cellular localization of membrane proteins

Sadowski, P.G., Groen, A.J., Dupree, P. and Lilley, K.S. Proteomics, 8(19), 3991-4011 (2008)   In eukaryotes, numerous complex sub-cellular structures exist. The majority of these are delineated by membranes. Many proteins are trafficked to these in order to be able to carry out their correct physiological function. Assigning the sub-cellular location of a protein is of paramount importance to biologists in the elucidation of its role and in the refinement of knowledge of cellular processes by tracing certain activities to specific organelles. Membrane proteins are a key set of proteins as these form part of the boundary of the organelles and represent many important functions such as transporters, receptors, and trafficking. They are, however, some of the most challenging proteins to work with due to poor solubility, a wide concentration range within the cell and inaccessibility to many of the tools employed in proteomics studies. This review focuses on membrane proteins with particular emphasis on sub-cellular localization in terms of methodologies that can be used to determine the accurate location of membrane proteins to organelles. We also discuss what is known about the membrane protein cohorts of major organelles.  

3.1187           Src-Tyrosine kinases are major agents in mitochondrial tyrosine phosphorylation

Tibaldi, E., Brunati, A.M., Massimino, M.L., Stringaro, A., Colone, M., Agostinelli, e., Arancia, G. and Toninello, A.

1. Cell. Biochem., 104(3), 840-849 (2008)

  Mitochondrial tyrosine phosphorylation is emerging as an important mechanism in regulating mitochondrial function. This article, aimed at identifying which kinases are the major agents in mitochondrial tyrosine phosphorylation, shows that this role should be attributed to Src family members. Indeed, various members of this family, for example, Fgr, Fyn, Lyn, c-Src, are constitutively present in the internal structure of mitochondria as well as Csk, a key enzyme in the regulation of the activity of this family. By means of different approaches, biochemical fractioning, Western blotting and immunogold analysis “in situ” of phosphotyrosine signaling, evidence is reported on the existence of a signal transduction pathway from plasma membrane to mitochondria, resulting in increasing Src-dependent mitochondrial tyrosine phosphorylation. The activation of Src kinases at mitochondrial level is associated with the proliferative status where several mitochondrial proteins are specifically tyrosine-phosphorylated.  

3.1188           Low substratum rigidity of collagen gel promotes ERK phosphorylation via lipid raft to augment cell migration

Wei, W-C., Hsu, Y-C., Chiu, W-T., Wang, C-Z., Wu, C-M., Wang, Y-K., Shen, M-R. and Tang, M-J.

1. Cell. Biochem., 103(4), 1111-1124 (2008)

  Previous study demonstrated that low substratum rigidity down-regulates focal adhesion proteins. In this study we found that cells cultured on collagen gel exhibited higher migration capacity than those cultured on collagen gel-coated dishes. Low rigidity of collagen gel induced delayed but persistent phosphorylation of ERK1/2. Inhibition of collagen gel-induced ERK1/2 phosphorylation by MEK inhibitors and ERK2 kinase mutant induced a rounding up of the cells and prevented collagen gel-induced cell migration. Interestingly, phosphorylated ERK1/2 induced by low rigidity was present in focal adhesion sites and the lipid raft. MβCD (Methyl-β-cyclodextrin), a lipid raft inhibitor, inhibited collagen gel-induced ERK1/2 phosphorylation, and cell migration. Overexpression of FAK C-terminal fragment (FRNK) in MDCK cells triggered ERK phosphorylation. Meanwhile, low substratum rigidity induced degradation of FAK into a 35 kDa C-terminal fragment. A calpain inhibitor that partially rescued FAK degradation also prevented low rigidity-induced ERK phosphorylation. However, MβCD did not prevent low rigidity-induced FAK degradation. Taken together, we demonstrate that the degradation product of FAK induced by collagen gel triggers activation of ERK1/2, which in turn facilitates cell spreading and migration through the lipid raft.  

3.1189           Escherichia coli interaction with human brain microvascular endothelial cells induces signal transducer and activator of transcription 3 association with the C-terminal domain of Ec-gp96, the outer membrane protein A receptor for invasion

Maruvada, R., Argon, Y. and Prasadaroa, N. Cell. Microbiol., 10(11), 2326-2338 (2008)   Our inability to develop new therapeutic strategies to prevent meningitis due to Escherichia coli K1 is attributed to our incomplete understanding of the pathophysiology of the disease. Previously, we demonstrated that outer membrane protein A of E. coli interacts with a gp96 homologue, Ec-gp96, on human brain microvascular endothelial cells (HBMEC) for invasion. However, signalling events mediated by Ec-gp96 that allow internalization of E. coli are incompletely understood. Here, we demonstrate that signal transducer and activator of transcription 3 (Stat3) activation and its interaction with Ec-gp96 were critical for E. coli invasion. The activated Stat3 was colocalized with Ec-gp96 at the actin condensation sites, and overexpressing a dominant negative (DN) form of Stat3 in HBMEC significantly abrogated the invasion. Furthermore, overexpression of Ec-gp96Δ200, the C-terminal 214-amino-acid truncated Ec-gp96, prevented the invasion of E. coli in HBMEC. In contrast, lack of ATP binding by gp96 did not affect the invasion. Overexpression of DN forms of either phosphatidyl inositol-3 kinase (PI3-kinase) subunit p85 or protein kinase C- (PKC- ) had no effect on the activation of Stat3 and its association with Ec-gp96, whereas overexpression of DN-Stat3 abolished the activation of both PI3-kinase and PKC- . Together, our findings identified a novel interaction of Stat3 with Ec-gp96, upstream of PI3-kinase and PKC- activation that is required for the invasion of E. coli into HBMEC.  

3.1190           Functional interactions between anthrax toxin receptors and the WNT signalling protein LRP6

Abrami, L., Kunz, B., Deuquet, J., Bafico, A., Davidson, G and Giscoe van der Goot, F. Cell. Microbiol., 10(12), 2509-2519 (2008)   To exert its activity, anthrax toxin must be endocytosed and its enzymatic toxic subunits delivered to the cytoplasm. It has been proposed that, in addition to the anthrax toxin receptors (ATRs), lipoprotein-receptor-related protein 6 (LRP6), known for its role in Wnt signalling, is also required for toxin endocytosis. These findings have however been challenged. We show that LRP6 can indeed form a complex with ATRs, and that this interaction plays a role both in Wnt signalling and in anthrax toxin endocytosis. We found that ATRs control the levels of LRP6 in cells, and thus the Wnt signalling capacity. RNAi against ATRs indeed led to a drastic decrease in LRP6 levels and a subsequent drop in Wnt signalling. Conversely, LRP6 plays a role in anthrax toxin endocytosis, but is not essential. We indeed found that toxin binding triggered tyrosine phosphorylation of LRP6, induced its redistribution into detergent-resistant domains, and its subsequent endocytosis. RNAis against LRP6 strongly delayed toxin endocytosis. As the physiological role of ATRs is probably to interact with the extracellular matrix, our findings raise the interesting possibility that, through the ATR–LRP6 interaction, adhesion to the extracellular matrix could locally control Wnt signalling.  

3.1191           Caveolin-1 secreting LNCaP cells induce tumor growth of caveolin-1 negative LNCaP cells in vivo

Bartz, R., Zhou, J., Hsieh, J-T., Ying, Y., Li, W. and Liu, P. Int. J. Cancer, 122(3), 520-525 (2008)   Caveolin-1 (Cav-1) was originally identified as a structural protein of caveolae, which is a plasma membrane domain that regulates a variety of signaling pathways involved in cell growth and migration. Here, we show that expression of Cav-1 in the Cav-1-deficient human prostate cancer cell line LNCaP both stimulates cell proliferation and promotes tumor growth in nude mice. Unexpectedly, Cav-1 expressing LNCaP (LNCaP^Cav-1) cells injected into one side of a nude mouse promoted tumor growth of Cav-1 negative LNCaP cells injected on the contralateral side of the same animal. The LNCaP tumors were positive for Cav-1, however, this signal was not caused by migrated LNCaP^Cav-1 cells, but we show that this Cav-1 was secreted by the LNCaP^Cav-1 tumors. We demonstrate that conditioned media from LNCaP^Cav-1 cells contained Cav-1 that was associated with a lipoprotein particle ranging in size from 15 to 30 nm and a density similar to high density lipoprotein particle. These results suggest that LNCaP^Cav-1 cells secreting Cav-1 particle produce an endocrine factor that stimulates tumor growth.  

3.1192           A flow-cytometry method for analyzing the composition of membrane rafts

Morales-Garzia, M.G., Fournie, J-J., Morena-Altamirano, M.M.B., Rodriguez-Luna, G., Mondragon-Flores, R. and Sanchez-Garzia, F.J. Cytometry Part A, 73A(10), 918-925 (2008)   Membrane rafts are involved in a broad variety of biological processes. Their protein composition under growth factor stimulation, anti-inflammatory or proinflammatory microenvironments, or in the course of pathogenic infections still remains to be determined. However, current techniques aimed at the identification of particular proteins on membrane rafts are not devoid of pitfalls. Membrane rafts were obtained by detergent-free based differential centrifugation from Jurkat T cells and J774 macrophages. Membrane rafts were labeled with fluorochrome-labeled antibodies directed against different cell membrane molecules, and with fluorochrome-labeled cholera toxin B that targets GM1 and analyzed by flow cytometry. CD3, CD11a, and GM1 were shown to be differentially expressed on Jurkat T cell-derived membrane rafts, indicating heterogeneity in membrane rafts composition. On the other hand, it was shown in J774 cell-derived membrane rafts that most but not all CD14 is present in the GM1-containing membrane fragments, thus confirming the heterogeneity of membrane rafts composition in other cell lines. The method described here allows the fluorometric assessment of the relative expression of more than one membrane raft component at a time, and at a single vesicle level in a fast and sensitive manner. This method seems to be a suitable approach to evaluate the molecular heterogeneity in membrane rafts composition.  

3.1193           Proteomic and immunologic analyses of brain tumor exosomes

Graner, M.W., Alzate, O., Dechkovskaia, A.M., Keene, J.D., sampson, J.H., Mitchell, D.A. and Bigner, D.D. FASEB J., 23(5), 1541-1557 (2009)   Brain tumors are horrific diseases with almost universally fatal outcomes; new therapeutics are desperately needed and will come from improved understandings of glioma biology. Exosomes are endosomally derived 30–100 nm membranous vesicles released from many cell types into the extracellular milieu; surprisingly, exosomes are virtually unstudied in neuro-oncology. These microvesicles were used as vaccines in other tumor settings, but their immunological significance is unevaluated in brain tumors. Our purpose here is to report the initial biochemical, proteomic, and immunological studies on murine brain tumor exosomes, following known procedures to isolate exosomes. Our findings show that these vesicles have biophysical characteristics and proteomic profiles similar to exosomes from other cell types but that brain tumor exosomes have unique features (e.g., very basic isoelectric points, expressing the mutated tumor antigen EGFRvIII and the putatively immunosuppressive cytokine TGF-β). Administration of such exosomes into syngeneic animals produced both humoral and cellular immune responses in immunized hosts capable of rejecting subsequent tumor challenges but failed to prolong survival in established orthotopic models. Control animals received saline or cell lysate vaccines and showed no antitumor responses. Exosomes and microvesicles isolated from sera of patients with brain tumors also possess EGFR, EGFRvIII, and TGF-β. We conclude that exosomes released from brain tumor cells are biochemically/biophysically like other exosomes and have immune-modulating properties. They can escape the blood-brain barrier, with potential systemic and distal signaling and immune consequences  

3.1194           Use of Bodipy-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells

Marks, D.L., Bittman, r. and pagano, R.E. Histochem. Cell. Biol., 130(5), 819-832 (2008)   Much evidence has accumulated to show that cellular membranes such as the plasma membrane, contain multiple “microdomains” of differing lipid and protein composition and function. These domains are sometimes enriched in cholesterol and sphingolipids and are believed to be important structures for the regulation of many biological and pathological processes. This review focuses on the use of fluorescent (Bodipy) labeled analogs of sphingolipids and cholesterol to study such domains. We discuss the similarities between the behavior of Bodipy-cholesterol and natural cholesterol in artificial bilayers and in cultured cells, and the use of Bodipy-sphingolipid analogs to visualize membrane domains in living cells based on the concentration-dependent monomer-excimer fluorescence properties of the Bodipy-fluorophore. The use of Bodipy-d-erythro-lactosylceramide is highlighted for detection of domains on the plasma membrane and endosome membranes, and the importance of the sphingolipid stereochemistry in modulating domain formation is discussed. Finally, we suggest that Bodipy-sphingolipids may be useful in future studies to examine the relationship between membrane domains at the cell surface and domains enriched in other lipids and proteins on the inner leaflet of the plasma membrane.  

3.1195           A deleted prion protein that is neurotoxic in vivo is localized normally in cultured cells

Christensen, H.M. and Harris, D.A.

1. Neurochem., 108, 44-56 (2009)

  The prion protein (PrP) possesses sequence-specific domains that endow the molecule with neuroprotective and neurotoxic activities, and that may contribute to the pathogenesis of prion diseases. To further define critical neurotoxic determinants within PrP, we previously generated Tg([DELTA]CR) mice that express a form of PrP harboring a deletion of 21 amino acids within the central domain of the protein [ Li et al., EMBO J. 26 (2007), 548]. These animals exhibit a neonatal lethal phenotype that is dose-dependently rescued by co-expression of wild-type PrP. In this study, we examined the localization and cell biological properties of the PrP([DELTA]CR) protein in cultured cells to further understand the mechanism of PrP([DELTA]CR) neurotoxicity. We found that the distribution of PrP([DELTA]CR) was identical to that of wild-type PrP in multiple cell lines of both neuronal and non-neuronal origin, and that co-expression of the two proteins did not alter the localization of either one. Both proteins were found in lipid rafts, and both were localized to the apical surface in polarized epithelial cells. Taken together, our results suggest that PrP([DELTA]CR) toxicity is not a result of mislocalization or aggregation of the protein, and more likely stems from altered binding interactions leading to the activation of deleterious signaling pathways.

3.1196           De novo generation of a transmissible spongiform encephalopathy by mouse transgenesis

Sigurdson, C.J. et al PNAS, 106(1), 304-309 (2009)   Most transmissible spongiform encephalopathies arise either spontaneously or by infection. Mutations of PRNP, which encodes the prion protein, PrP, segregate with phenotypically similar diseases. Here we report that moderate overexpression in transgenic mice of mPrP(170N,174T), a mouse PrP with two point mutations that subtly affect the structure of its globular domain, causes a fully penetrant lethal spongiform encephalopathy with cerebral PrP plaques. This genetic disease was reproduced with 100% attack rate by intracerebral inoculation of brain homogenate to tga20 mice overexpressing WT PrP, and from the latter to WT mice, but not to PrP-deficient mice. Upon successive transmissions, the incubation periods decreased and PrP became more protease-resistant, indicating the presence of a strain barrier that was gradually overcome by repeated passaging. This shows that expression of a subtly altered prion protein, with known 3D structure, efficiently generates a prion disease.  

3.1197           Human eosinophils constitutively express multiple Th1, Th2, and immunoregulatory cytokines that are secreted rapidly and differentially

Spencer, L.A., Szelaq, C.T., Perez, S.A.C., Kirschhofer, C.L., Neves, J.S., Radke, A.L and Weller, P.F.

1. Leukoc. Biol., 85, 117-123 (2009)

  Eosinophils are innate immune leukocytes implicated in the initiation and maintenance of type 2 immune responses, including asthma and allergy. The ability to store and rapidly secrete preformed cytokines distinguishes eosinophils from most lymphocytes, which must synthesize cytokine proteins prior to secretion and may be a factor in the apparent Th2 bias of eosinophils. Multiple studies confirm that human eosinophils from atopic or hypereosinophilic donors can secrete over 30 cytokines with a varying and often opposing immune-polarizing potential. However, it remains unclear whether all of these cytokines are constitutively preformed and available for rapid secretion from eosinophils in the circulation of healthy individuals or are restricted to eosinophils from atopic donors. Likewise, the relative concentrations of cytokines stored within eosinophils have not been studied. Here, we demonstrate that human blood eosinophils are not singularly outfitted with Th2-associated cytokines but rather, constitutively store a cache of cytokines with nominal Th1, Th2, and regulatory capacities, including IL-4, IL-13, IL-6, IL-10, IL-12, IFN- , and TNF- . We demonstrate further rapid and differential release of each cytokine in response to specific stimuli. As agonists, strong Th1 and inflammatory cytokines elicited release of Th2-promoting IL-4 but not Th1-inducing IL-12. Moreover, a large quantity of IFN- was secreted in response to Th1, Th2, and inflammatory stimuli. Delineations of the multifarious nature of preformed eosinophil cytokines and the varied stimulus-dependent profiles of rapid cytokine secretion provide insights into the functions of human eosinophils in mediating inflammation and initiation of specific immunity.  

3.1198           An Amphipathic α-Helix Controls Multiple Roles of Brome Mosaic Virus Protein 1a in RNA Replication Complex Assembly and Function

Liu, L., Westler, W.M., den Boon, J.A., Wang, X., Diaz, A., Steinberg, H.A. and Ahlquist, P. PloSPathogens, 5(3), e1000351 (2009)   Brome mosaic virus (BMV) protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER) membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2a^Pol) and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic α-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a–ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a–membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2a^Pol accumulation. The second class of helix A mutations not only maintains efficient 1a–membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2a^Pol accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.    

3.1199           Analysis of the Differential Host Cell Nuclear Proteome Induced by Attenuated and Virulent Hemorrhagic Arenavirus Infection

Bowick, G.C., Spratt, H.M., Hogg, A.E., Endsley, J.J., Wiktorowicz, J.E., Kurosky, A., Luxon, B.A., Gorenstein, D.G. and Herzog, N.K.

1. Virol., 83(2), 687-700 (2009)

  Arenaviruses are important emerging pathogens and include a number of hemorrhagic fever viruses classified as NIAID category A priority pathogens and CDC potential biothreat agents. Infection of guinea pigs with the New World arenavirus Pichindé virus (PICV) has been used as a biosafety level 2 model for the Lassa virus. Despite continuing research, little is known about the molecular basis of pathogenesis, and this has hindered the design of novel antiviral therapeutics. Modulation of the host response is a potential strategy for the treatment of infectious diseases. We have previously investigated the global host response to attenuated and lethal arenavirus infections by using high-throughput immunoblotting and kinomics approaches. In this report, we describe the differential nuclear proteomes of a murine cell line induced by mock infection and infection with attenuated and lethal variants of PICV, investigated by using two-dimensional gel electrophoresis. Spot identification using tandem mass spectrometry revealed the involvement of a number of proteins that regulate inflammation via potential modulation of NF- B activity and of several heterogeneous nuclear ribonuclear proteins. Pathway analysis revealed a potential role for transcription factor XBP-1, a transcription factor involved in major histocompatibility complex II (MHC-II) expression; differential DNA-binding activity was revealed by electrophoretic mobility shift assay, and differences in surface MHC-II expression were seen following PICV infection. These data are consistent with the results of several previous studies and highlight potential differences between transcriptional and translational regulation. This study provides a number of differentially expressed targets for further research and suggests that key events in pathogenesis may be established early in infection.  

3.1200           Intracellular Signaling Mechanisms and Activities of Human Herpesvirus 8 Interleukin-6

Chen, D., Sandford, G. and Nicholas, J.

1. Virol., 83(2), 722-733 (2009)

  Human herpesvirus 8 (HHV-8)-encoded viral interleukin-6 (vIL-6) has been implicated as a key factor in virus-associated neoplasia because of its proproliferative and survival effects and also in view of its angiogenic properties. A major difference between vIL-6 and human IL-6 (hIL-6) is that vIL-6, uniquely, is largely retained and can signal intracellularly. While vIL-6 is generally considered to be a lytic gene, several reports have noted its low-level expression in latently infected primary effusion lymphoma (PEL) cultures, in the absence of other lytic gene expression. Thus, intracellular autocrine signal transduction by the viral cytokine may be of particular relevance to the growth and survival of latently infected cells and to pathogenesis. Here we report that most intracellular vIL-6 is located in the endoplasmic reticulum (ER), signals via the gp130 signal transducer in this compartment, and does so independently of the gp80 -subunit of the IL-6 receptor, required for hIL-6 signal transduction. Signaling and biological assays incorporating ER-retained vIL-6 and hIL-6 confirmed vIL-6 activity, specifically, in this compartment. Knockdown of vIL-6 expression in PEL cells led to markedly reduced cell growth in normal culture, independently of extracellular cytokines. This could be reversed by reintroduction via virus vector of exclusively ER-retained vIL-6. These data indicate that in virus biology vIL-6 may act to support the growth and survival of cells latently infected with HHV-8 in an autocrine manner via intracrine signaling and that these activities may contribute to the maintenance of latently infected cells and to virus-induced neoplasia.  

3.1201           Novel N-terminal Cleavage of APP Precludes Aβ Generation in ACAT-Defective AC29 Cells

Huttunen, H.J., Puglielli, L., Ellis, B.C., MacKenzie Ingano, L.A. and Kovacs, D.M.

1. Mol. Neurosci., 37(6), 6-15 (2009)

  A common pathogenic event that occurs in all forms of Alzheimer’s disease is the progressive accumulation of amyloid β-peptide (Aβ) in brain regions responsible for higher cognitive functions. Inhibition of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which generates intracellular cholesteryl esters from free cholesterol and fatty acids, reduces the biogenesis of the Aβ from the amyloid precursor protein (APP). Here we have used AC29 cells, defective in ACAT activity, to show that ACAT activity steers APP either toward or away from a novel proteolytic pathway that replaces both α and the amyloidogenic β cleavages of APP. This alternative pathway involves a novel cleavage of APP holoprotein at Glu281, which correlates with reduced ACAT activity and Aβ generation in AC29 cells. This sterol-dependent cleavage of APP occurs in the endosomal compartment after internalization of cell surface APP. The resulting novel C-terminal fragment APP-C470 is destined to proteasomal degradation limiting the availability of APP for the Aβ generating system. The proportion of APP molecules that are directed to the novel cleavage pathway is regulated by the ratio of free cholesterol and cholesteryl esters in cells. These results suggest that subcellular cholesterol distribution may be an important regulator of the cellular fate of APP holoprotein and that there may exist several competing proteolytic systems responsible for APP processing within the endosomal compartment.  

3.1202           Analysis of Viral and Cellular Proteins in HIV-1 Reverse Transcription Complexes by Co-immunoprecipitation

Iordanskiy, S.N. and Bukrinsky, M.I. Methods in Mol. Biol., 485, 121-134 (2009)   Molecular details and temporal organization of the early (preintegration) phase of HIV life cycle remain among the least investigated and most controversial problems in the biology of HIV. To accomplish reverse transcription and intracellular transport of the viral genetic material, HIV forms multi-molecular complexes termed reverse transcription complexes (RTCs). Analysis of the kinetics of reverse transcription and nuclear import of RTCs, as well as assessment of the changes in their protein content in the course of reverse transcription and nuclear translocation is a necessary step in understanding the mechanisms of cytoplasmic maturation and nuclear import of HIV-1 RTCs. Here, we review methods that allow quantitative assessment of the dynamics of the maturation of HIV-1 RTCs and transformations of RTC protein composition associated with nuclear import of the complexes.  

3.1203           Proteomics Study of the Hepatitis C Virus Replication Complex

Chang, K., Wang, T. and Luo, G. Methods in Mol. Biol., 510, 185-193 (2009)   RNA replication of HCV occurs in the multiprotein complexes associated with the endoplasmic reticular (ER) membranes. The HCV NS3 to NS5B proteins are necessary and sufficient for HCV RNA replication in the cell, but cellular proteins in the HCV replication complex (RC) have not been determined. Several methods have been used to isolate the HCV RC, including crude cell extract preparation, subcellular fractionation, and affinity purification. The components of the HCV RC can be separated by two-dimensional electrophoresis and then determined by proteolytical digestion and mass spectrometry analysis in conjunction with peptide/protein database search and immunobiochemistry and functional genomic studies.  

3.1204           Proteins Associated with Immunopurified Granules from a Model Pancreatic Islet β-Cell System: Proteomic Snapshot of an Endocrine Secretory Granule

Hickey, A.J.R., Bradley, J.W.I., Skea, G.L., Middleditch, M.J., Buchanan, C.M., Phillips, A.R.J. and Cooper, G.J.S.

1. Proteome Res., 8(1), 178-186 (2009)

  β-Cell granules contain proteins involved in fuel regulation, which when altered, contribute to metabolic disorders including diabetes mellitus. We analyzed proteins present in purified granules from the INS-1E β-cell model. Fifty-one component proteins were identified by LC-MS/MS including hormones, granins, protein processing components, cellular trafficking components, enzymes implicated in cellular metabolism and chaperone proteins. These findings may increase understanding of granule secretion and the processes leading to protein aggregation and β-cell death in type-2 diabetes.  

3.1205           Ca²⁺ influx mechanisms in caveolae vesicles of pulmonary smooth muscle plasma membrane under inhibition of α₂β₁ isozyme of Na⁺/K⁺-ATPase by ouabain

Ghosh, B., Kar, P., Mandal, A., Dey, K., Chakraborti, T. And Chakraborti, S. Life Sciences, 84, 139-148 (2009)   Aims We sought to determine the mechanisms of an increase in Ca²⁺ level in caveolae vesicles in pulmonary smooth muscle plasma membrane during Na⁺/K⁺-ATPase inhibition by ouabain. Main methods The caveolae vesicles isolated by density gradient centrifugation were characterized by electron microscopic and immunologic studies and determined ouabain induced increase in Na⁺ and Ca²⁺ levels in the vesicles with fluorescent probes, SBFI-AM and Fura2-AM, respectively. Key findings We identified the α₂β₁ and α₁β₁ isozymes of Na⁺/K⁺-ATPase in caveolae vesicles, and only the α₁β₁ isozyme in noncaveolae fraction of the plasma membrane. The α₂-isoform contributes solely to the enzyme inhibition in the caveolae vesicles at 40 nM ouabain. Methylisobutylamiloride (Na⁺/H⁺-exchange inhibitor) and tetrodotoxin (voltage-gated Na⁺-channel inhibitor) pretreatment prevented ouabain induced increase in Na⁺ and Ca²⁺ levels. Ouabain induced increase in Ca²⁺ level was markedly, but not completely, inhibited by KB-R7943 (reverse-mode Na⁺/Ca²⁺-exchange inhibitor) and verapamil (L-type Ca²⁺-channel inhibitor). However, pretreatment with tetrodotoxin in conjunction with KB-R7943 and verapamil blunted ouabain induced increase in Ca²⁺ level in the caveolae vesicles, indicating that apart from Na⁺/Ca⁺-exchanger and L-type Ca²⁺-channels, “slip-mode conductance” of Na⁺ channels could also be involved in this scenario. Significance Inhibition of α₂ isoform of Na⁺/K⁺-ATPase by ouabain plays a crucial role in modulating the Ca²⁺ influx regulatory components in the caveolae microdomain for marked increase in (Ca²⁺)_i in the smooth muscle, which could be important for the manifestation of pulmonary hypertension.  

3.1206           Moesin Regulates the Trafficking of Nascent Clathrin-coated Vesicles

Barroso-Gonzales, J., Machado, J-D., Garcia-Exposito, L. and Valenzuela-Fernandez, A.

1. Biol. Chem., 284(4), 2419-2434 (2009)

  Clathrin-coated vesicles are responsible for the trafficking of several internalized biological cargos. We have observed that the endogenous F-actin-linker moesin co-distributes with constitutive components of clathrin-coated structures. Total internal reflection fluorescence microscopy studies have shown that short interference RNA of moesin enhances the lateral movement of clathrin-coated structures and provokes their abnormal clustering. The aggregation of clathrin-coated structures has also been observed in cells overexpressing N-moesin, a dominant-negative construct unable to bind to F-actin. Only overexpressed moesin constructs with an intact phosphatidylinositol 4,5-bisphosphate-binding domain co-distribute with clathrin-coated structures. Hence, this N-terminal domain is mostly responsible for moesin/clathrin-coated structure association. Biochemical endosome fractioning together with total internal reflection fluorescence microscopy comparative studies, between intact cells and plasma-membrane sheets, indicate that moesin knockdown provokes the accumulation of endocytic rab5-clathrin-coated vesicles carrying the transferrin receptor. The altered trafficking of these endocytic rab5-clathrin-coated vesicles accounts for a transferrin receptor recycling defect that reduces cell-surface expression of the transferrin receptor and increases the amount of sequestered transferrin ligand. Therefore, we propose that moesin is a clathrin-coated vesicle linker that drives cargo trafficking and acts on nascent rab5-clathrin-coated vesicles by simultaneously binding to clathrin-coated vesicle-associated phosphatidylinositol 4,5-bisphosphate and actin cytoskeleton. Hence, functional alterations of moesin may be involved in pathological disorders associated with clathrin-mediated internalization or receptor recycling.  

3.1207           Kinesin Adapter JLP Links PIKfyve to Microtubule-based Endosome-to-Trans-Golgi Network Traffic of Furin

Ikonomov, O.C., Fligger, J., Sbrissa, D., Dondapati, R., Mlak, K., Deebn, R. And Shisheva, A.

1. Biol. Chem., 284(6), 3750-3761 (2009)

  JIPs (c-Jun N-terminal kinase interacting proteins), which scaffold JNK/p38 MAP kinase signaling modules, also bind conventional kinesins and are implicated in microtubule-based membrane trafficking in neuronal cells. Here we have identified a novel splice variant of the Jip4 gene product JLP_L (JNK-interacting leucine zipper protein) in yeast-two hybrid screens with the phosphoinositide kinase PIKfyve. The interaction was confirmed by pulldown and coimmunoprecipitation assays in native cells. It engages the PIKfyve cpn60_TCP1 consensus sequence and the last 75 residues of the JLP C terminus. Subpopulations of both proteins cofractionated and populated similar structures at the cell perinuclear region. Because PIKfyve is essential in endosome-to-trans-Golgi network (TGN) cargo transport, we tested whether JLP is a PIKfyve functional partner in this trafficking pathway. Short interfering RNA (siRNA)-mediated depletion of endogenous JLP or PIKfyve profoundly delayed the microtubule-based transport of chimeric furin (Tac-furin) from endosomes to the TGN in a CHO cell line, which was rescued upon ectopic expression of siRNA-resistant JLP or PIKfyve constructs. Peptides from the contact sites in PIKfyve and JLP, or a dominant-negative PIKfyve mutant introduced into cells by ectopic expression or microinjection, induced a similar defect. Because Tac-TGN38 delivery from endosomes to the TGN, unlike that of Tac-furin, does not require intact microtubules, we monitored the effect of JLP and PIKfyve depletion or the interacting peptides administration on Tac-TGN38 trafficking. Remarkably, neither maneuver altered the Tac-TGN38 delivery to the TGN. Our data indicate that JLP interacts with PIKfyve and that both proteins and their association are required in microtubule-based, but not in microtubule-independent, endosome-to-TGN cargo transport.  

3.1208           Stabilizing effects of eicosapentaenoic acid on Kv1.5 channel protein expressed in mammalian cells

Koshida, S. et al Eur. J. Pharmacol., 604, 93-102 (2009)   We investigated the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the stability of Kv1.5 channel protein. The expression and function of Kv1.5 (Kv1.5-FLAG) in transfected African green monkey kidney fibroblast cells as well as rat atrium were estimated by immunoblotting, immunoprecipitation, immunofluorescence and patch-clamp techniques. Both EPA and DHA immediately blocked Kv1.5 channel current in a dose-dependent manner, accompanied by reduction of their phosphorylation. Chronic treatment (for 12 h) with EPA at lower concentrations (0.3–10 μM) increased the level of Kv1.5-FLAG protein as well as Kv1.5 channel current without changes in its gating kinetics, prolonging its half-life; in contrast, both EPA and DHA at higher concentrations (30–100 μM) decreased the expression of Kv1.5-FLAG. EPA at the higher concentrations also decreased mRNA of Kv1.5 and synapse-associated protein 97 expression. EPA at the lower concentrations increased Kv1.5 expression in the endoplasmic reticulum, Golgi apparatus and cell membrane. EPA-induced increase of Kv1.5 channel expression and current was abolished by pretreatment with the protein transport inhibitor brefeldin A or colchicines, and by the Kv1.5 channel blocker 4-aminopyridine. Oral administration of EPA (30 mg/kg) increased the level of endogenous Kv1.5 in rat atria. These results indicate that chronic treatment with EPA at lower concentrations stabilizes Kv1.5 channel protein in the endoplasmic reticulum and Golgi apparatus thereby enhancing the Kv1.5 channel current on the cell membrane.  

3.1209           The N-terminal half of the receptor domain of botulinum neurotoxin A binds to microdomains of the plasma membrane

Muraro, L., Tosatto, S., Motterlini, L., Rosetto, O. and Montecucco, C. Biochem. Biophys. Res. Comm., 380, 76-80 (2009)   Botulinum neurotoxin type A (BoNT/A) is largely employed in human therapy because of its specific inhibition of peripheral cholinergic nerve terminals. BoNT/A binds to them rapidly and with high specificity via its receptor binding domain termed HC. Recent evidence indicate that BoNT/A interacts specifically with polysialogangliosides and with a luminal loop of the synaptic vesicle protein SV2 via the C-terminal half of HC. Here we show that the N-terminal half of HC binds to sphingomyelin-enriched membrane microdomains and that it has a defined interaction with phosphatidylinositol phosphates (PIP). We have identified a PIP binding site in this half of HC and we show how this interaction could predispose BoNT/A for membrane insertion, which is the step subsequent to binding, in the four-steps route leading BoNT/A inside nerve terminals.  

3.1210           Biochemical and proteomic approaches for the study of membrane microdomains

Zheng, Y.Z. and Foster, L.J.

1. Proteomics, 72, 12-22 (2009)

  Many cellular signaling and communication events take place at the plasma membrane and thus the characterization of the plasma membrane proteome has been a hot research area in the hopes of learning more about these processes. Membrane microdomains are large protein and lipid complexes found on the cell surface membrane, able to concentrate or recruit signaling molecules or factors. The first step of any organelle proteomics study is to get a pure and enriched protein sample yet this has always been problematic in membrane proteomics as it is virtually impossible to purify a specific membrane type to homogeneity. In this review, we summarize the biochemical and proteomic approaches that have been used recently in the isolation and identification of several membrane microdomains and non-typical membrane proteins.  

3.1211           Recruitment of protein phosphatase 2A to dorsal ruffles by platelet-derived growth factor in smooth muscle cells: Dephosphorylation of Hsp27

Berrou, E and Brykaert, M. Exp. Cell Res., 315, 836-848 (2009)   In this study, we investigated the mechanism underlying Hsp27 dephosphorylation in smooth muscle cells. We found that protein phosphatase 2A (PP2A) dephosphorylates Hsp27. In addition, Hsp27 dephosphorylation was regulated by membrane cholesterol content. We showed that PDGF induced a three-fold increase in the proportion of PP2A activity regulated by cholesterol in the Triton-insoluble fraction of cell lysates. Moreover, cholesterol depletion decreased the amount of PP2A recovered in Triton-insoluble fraction. Thus, PDGF might regulate a small pool of PP2A associated with lipid rafts. Isolation of detergent-resistant membrane fragments by Optiprep -gradient density indicated that this pool of PP2A was not associated with caveolae, but was recovered in a higher density fraction (DRM-H) with ganglioside GM1, α-actinin, Hsp27 and p34, a component of Arp2/3 complex. These proteins were also present in dorsal ruffles containing GM1 but not caveolin-1. Phosphorylated Hsp27 levels detected in dorsal ruffles were variable. Cholesterol depletion, which inhibits dorsal ruffle formation, decreased PP2A levels and increased the Hsp27-P to Hsp27 ratio in DRM-H. These findings suggest that Hsp27 is dephosphorylated by PP2A in dorsal ruffles, in non-caveolar lipid raft microdomains. However, similarly to p34, non-phosphorylated Hsp27 is associated to non-raft membrane domains at the leading edge of lamellipodia.        

3.1212           HTLV-1 Tax Is a Critical Lipid Raft Modulator That Hijacks I B Kinases to the Microdomains for Persistent Activation of NF- B

Huang, J., Ren, T., Guan, H., Jiang, Y. And Cheng, H. J.Biol. Chem., 284(10), 6208-6217 (2009)   Upon T cell activation, I B kinases (IKKs) are transiently recruited to the plasma membrane-associated lipid raft microdomains for activation of NF- B in promoting T cell proliferation. Retroviral Tax proteins from human T cell leukemia virus type 1 and type 2 (HTLV-1 and -2) are capable of activating IKK, yet only HTLV-1 infection causes T cell leukemia, which correlates with persistent activation of NF- B induced by Tax1. Here, we show that the Tax proteins exhibit differential modes of IKK activation. The subunits of IKK are constitutively present in lipid rafts in activated forms in HTLV-1-infected T cells that express Tax. Disruption of lipid rafts impairs I B kinase activation by Tax1. We also show that the cytoplasmic Tax1 protein persistently resides in the Golgi-associated lipid raft microdomains. Tax1 directs lipid raft translocation of IKK through selective interaction with IKK and accordingly, depletion of IKK impairs Tax1-directed lipid raft recruitment of IKK and IKKβ. In contrast, Tax2 activates NF- B in a manner independent of lipid raft recruitment of IKK. These findings indicate that Tax1 actively recruits IKK to the lipid raft microdomains for persistent activation of NF- B, thereby contributing to HTLV-1 oncogenesis.  

3.1213           Proteomic profiling of secretory granules of different T cell subpopulations

Schmidt, H., Gelhaus, C., Nebendahl, M., Leippe, M. and Janssen, O. Cell Communication and Signaling, 7 (Suppl 1), A85 (2009)   In cytotoxic T lymphocytes and Natural Killer (NK) cells, effector molecules including granzymes, perforin, granulysin and FasL are stored in specialized granules termed secretory lysosomes (SL). These vesicles represent dual-functional organelles that obviously combine degradative and exocytotic properties [1]. We previously established an enrichment protocol to define the proteome of SL from NK cells. We found that the protein content of SL very much depends on the function of a given cell type or clone, best reflected by crucial differences in functionally relevant proteins in transformed NK cell lines [2]. In order to compare the lysosomal content of different T cell subpopulations, we enriched SL from alpha/beta (CD4 or CD8) and gamma/delta (Vdelta 1 or Vdelta2) T cell lines and clones. To this end, the T cell lysates were separated by density gradient centrifugation on Iodixanol gradients. As described before, for the differential proteome analysis we focused on the fraction that contained most FasL, Lamp1 and Lamp3 (as specific SL or general lysosomal markers) and compared the isolated lysosomal fractions by 2D-DIGE. We found that the protein content of SL of in vitro expanded CD4 and CD8 cells as well as Vdelta1 and Vdelta 2 cells is more similar than for example gamma/delta cells compared to alpha/beta cells. A detailed MALDI-based profile of individual SL proteomes based on more than 1000 picked spots from several DIGE experiments will be presented with a focus on the functionally relevant proteins mentioned above. The observed differences might reveal new aspects of population-specific dynamics of activation/maturation and effector function in the T cell compartment.  

3.1214           The Comprehensive Native Interactome of a Fully Functional Tagged Prion Protein

Rutishauer, D., Mertz, K.D., Moos, R., Brunner, E., Rülicke, T., Calella, A.M. and Aguzzi, A. PloSOne, 4(2), e446 (2009)   The enumeration of the interaction partners of the cellular prion protein, PrP^C, may help clarifying its elusive molecular function. Here we added a carboxy proximal myc epitope tag to PrP^C. When expressed in transgenic mice, PrP_myc carried a GPI anchor, was targeted to lipid rafts, and was glycosylated similarly to PrP^C. PrP_myc antagonized the toxicity of truncated PrP, restored prion infectibility of PrP^C-deficient mice, and was physically incorporated into PrP^Sc aggregates, indicating that it possessed all functional characteristics of genuine PrP^C. We then immunopurified myc epitope-containing protein complexes from PrP_myc transgenic mouse brains. Gentle differential elution with epitope-mimetic decapeptides, or a scrambled version thereof, yielded 96 specifically released proteins. Quantitative mass spectrometry with isotope-coded tags identified seven proteins which co-eluted equimolarly with PrP^C and may represent component of a multiprotein complex. Selected PrP^C interactors were validated using independent methods. Several of these proteins appear to exert functions in axomyelinic maintenance.        

3.1215           Depletion of Kinesin 5B Affects Lysosomal Distribution and Stability and Induces Peri-Nuclear Accumulation of Autophagosomes in Cancer Cells

Cardoso, C.M.P., Groth-Pedersen, L., Høyer-Hansen, M., Kirkegaard, T., Corcelle, E., Andersen, J.S.-, Jäättelä, M. and Nylandsted, J. PloSOne, 4(2), e4424 (2009)   Background Enhanced lysosomal trafficking is associated with metastatic cancer. In an attempt to discover cancer relevant lysosomal motor proteins, we compared the lysosomal proteomes from parental MCF-7 breast cancer cells with those from highly invasive MCF-7 cells that express an active form of the ErbB2 (ΔN-ErbB2). Methodology/Principal Findings Mass spectrometry analysis identified kinesin heavy chain protein KIF5B as the only microtubule motor associated with the lysosomes in MCF-7 cells, and ectopic ΔN-ErbB2 enhanced its lysosomal association. KIF5B associated with lysosomes also in HeLa cervix carcinoma cells as analyzed by subcellular fractionation. The depletion of KIF5B triggered peripheral aggregations of lysosomes followed by lysosomal destabilization, and cell death in HeLa cells. Lysosomal exocytosis in response to plasma membrane damage as well as fluid phase endocytosis functioned, however, normally in these cells. Both HeLa and MCF-7 cells appeared to express similar levels of the KIF5B isoform but the death phenotype was weaker in KIF5B-depleted MCF-7 cells. Surprisingly, KIF5B depletion inhibited the rapamycin-induced accumulation of autophagosomes in MCF-7 cells. In KIF5B-depleted cells the autophagosomes formed and accumulated in the close proximity to the Golgi apparatus, whereas in the control cells they appeared uniformly distributed in the cytoplasm. Conclusions/Significance Our data identify KIF5B as a cancer relevant lysosomal motor protein with additional functions in autophagosome formation.  

3.1216           Passage through the Golgi is necessary for Shiga toxin B subunit to reach the endoplasmic reticulum 

McKinzie, J., Johannes, L., Taguchi, T. and Sheff, D. FEBS J., 276, 1581-1595 (2009)   Both Shiga holotoxin and the isolated B subunit, navigate a retrograde pathway from the plasma membrane to the endoplasmic reticulum (ER) of mammalian cells to deliver catalytic A subunits into the cytosol. This route passes through early/recycling endosomes and then through the Golgi. Although passage through the endosomes takes only 30 min, passage through the Golgi is much slower, taking hours. This suggests that Golgi passage is a key step in retrograde traffic. However, there is no empirical data demonstrating that Golgi passage is required for the toxins to enter the ER. In fact, an alternate pathway bypassing the Golgi is utilized by SV40 virus. Here we find that blocking Shiga toxin B access to the entire Golgi with AlF₄⁻ treatment, temperature block or subcellular surgery prevented Shiga toxin B from reaching the ER. This suggests that there is no direct endosome to ER route available for retrograde traffic. Curiously, when Shiga toxin B was trapped in endosomes, it entered the cytosol directly from the endosomal compartment. Our results suggest that trafficking through the Golgi apparatus is required for Shiga toxin B to reach the ER and that diversion into the Golgi may prevent toxin escape from endosomes into the cytosol.  

3.1217           Channel-forming activities of peroxisomal membrane proteins from the yeast Saccharomyces cerevisiae 

Grunau, S., Mindthoff, S., Rottensteiner, H., Sormuen, R.T., Hitunen, J.K., Erdmann, R. And Antonenkov, V.D. FEBS J., 276, 1698-1708 (2009)   Highly-purified peroxisomes from the yeast Saccharomyces cerevisiae grown on oleic acid were investigated for the presence of channel (pore)-forming proteins in the membrane of these organelles. Solubilized membrane proteins were reconstituted in planar lipid bilayers and their pore-forming activity was studied by means of multiple-channel monitoring or single-channel analysis. Two abundant pore-forming activities were detected with an average conductance of 0.2 and 0.6 nS in 1.0 m KCl, respectively. The high-conductance pore (0.6 nS in 1.0 m KCl) is slightly selective to cations (P_K+/P_Cl− ∼ 1.3) and showed an unusual flickering at elevated (> ±40 mV) holding potentials directed upward relative to the open state of the channel. The data obtained for the properties of the low-conductance pore (0.2 nS in 1.0 m KCl) support the notion that the high-conductance channel represents a cluster of two low-conductance pores. The results lead to conclusion that the yeast peroxisomes contain membrane pore-forming proteins that may aid the transfer of small solutes between the peroxisomal lumen and cytoplasm.  

3.1218           Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells

Bauman, S.J. and Kuehn, M.J. BMC Microbiol., 9, 26-37 (2009)   Background Pseudomonas aeruginosa is the major pathogen associated with chronic and ultimately fatal lung infections in patients with cystic fibrosis (CF). To investigate how P. aeruginosa-derived vesicles may contribute to lung disease, we explored their ability to associate with human lung cells. Results Purified vesicles associated with lung cells and were internalized in a time- and dose-dependent manner. Vesicles from a CF isolate exhibited a 3- to 4-fold greater association with lung cells than vesicles from the lab strain PAO1. Vesicle internalization was temperature-dependent and was inhibited by hypertonic sucrose and cyclodextrins. Surface-bound vesicles rarely colocalized with clathrin. Internalized vesicles colocalized with the endoplasmic reticulum (ER) marker, TRAPα, as well as with ER-localized pools of cholera toxin and transferrin. CF isolates of P. aeruginosa abundantly secrete PaAP (PA2939), an aminopeptidase that associates with the surface of vesicles. Vesicles from a PaAP knockout strain exhibited a 40% decrease in cell association. Likewise, vesicles from PAO1 overexpressing PaAP displayed a significant increase in cell association. Conclusion These data reveal that PaAP promotes the association of vesicles with lung cells. Taken together, these results suggest that P. aeruginosa vesicles can interact with and be internalized by lung epithelial cells and contribute to the inflammatory response during infection.  

3.1219           HIV entry in macrophages is dependent on intact lipid rafts

Carter, G.C., Bernstone, L., Sangani, D., Bee, W.J., Harder, T. and James, W. Virology, 386, 192-202 (2009)   Macrophages are an important natural target cell for HIV-1, but previous studies of virus entry into these cells are limited, and the involvement of membrane cholesterol and lipid rafts is unknown. Cholesterol disruption of macrophage membranes using four pharmacological agents acting by different mechanisms: methyl-β cyclodextrin, nystatin, filipin complex and Lovastatin, all significantly inhibited productive HIV entry and reverse transcription. The inhibitory effects of these drugs resulted in decreased virus release from infected cells, and could be substantially reversed by the addition of water-soluble cholesterol. The virus bound equally to cholesterol-disrupted cells even though HIV receptor expression levels were significantly reduced. Macrophage CD4 and CCR5 were found to partition with the detergent-resistant membranes with a typical raft-associating protein flotillin-1. HIV particles were observed co-localising with a marker of lipid rafts (CTB-FITC) early post infection. These data suggest that macrophage membrane cholesterol is essential for HIV entry, and implicate lipid raft involvement.  

3.1220           Biochemical and functional characterization of the Ror2/BRIb receptor complex

Sammar, M., Sieber, C. and Knaus, P. Biochem. Biophys. Res. Comm., 381, 1-6 (2009)   Ror2 belongs to the Ror family of receptor tyrosine kinases. Two distinct human disorders result from mutations in Ror2 suggesting a role in cartilage formation, chondrocyte differentiation, and joint formation. We have previously demonstrated functional and physical association of Ror2 with the BMP receptor type Ib (BRIb). The interaction site was mapped to the extracellular CRD domain of Ror2. Here we show specific association with and transphosphorylation by BRIb, but not BMP receptors Ia or II. This association is independent of N-glycosylation, excluding the possibility that the interaction is mediated by carbohydrate moieties present in the CRD region of Ror2. The Ror2/BRIb complex proved very stable under high ionic and reducing conditions, yet it appeared sensitive to SDS-treatment. Besides we provide evidence that the Ror2/BRIb complex forms in distinct microdomains at the plasma membrane (DRMs), indicating that Ror2 may interfere with BMP signaling complexes within these membrane domains.        

3.1221           Transmembrane Form Agrin-induced Process Formation Requires Lipid Rafts and the Activation of Fyn and MAPK

Ramseger, R., White, R. and Kröger, S.

1. Biol. Chem., 284(12, 7697-7705 (2009)

  Overexpression or clustering of the transmembrane form of the extracellular matrix heparan sulfate proteoglycan agrin (TM-agrin) induces the formation of highly dynamic filopodia-like processes on axons and dendrites from central and peripheral nervous system-derived neurons. Here we show that the formation of these processes is paralleled by a partitioning of TM-agrin into lipid rafts, that lipid rafts and transmembrane-agrin colocalize on the processes, that extraction of lipid rafts with methyl-β-cyclodextrin leads to a dose-dependent reduction of process formation, that inhibition of lipid raft synthesis prevents process formation, and that the continuous presence of lipid rafts is required for the maintenance of the processes. Association of TM-agrin with lipid rafts results in the phosphorylation and activation of the Src family kinase Fyn and subsequently in the phosphorylation and activation of MAPK. Inhibition of Fyn or MAPK activation inhibits process formation. These results demonstrate that the formation of filopodia-like processes by TM-agrin is the result of the activation of a complex intracellular signaling cascade, supporting the hypothesis that TM-agrin is a receptor or coreceptor on neurons.  

3.1222           Contribution of PIP-5 kinase Iα to raft-based FcγRIIA signaling

Symanska, E., Korzeniowski, M., Raynal, P., Sobota, A. and Kwiatkowska, K. Exp. Cell Res., 315, 981-995 (2009)   Receptor FcγIIA (FcγRIIA) associates with plasma membrane rafts upon activation to trigger signaling cascades leading to actin polymerization. We examined whether compartmentalization of PI(4,5)P₂ and PI(4,5)P₂-synthesizing PIP5-kinase Iα to rafts contributes to FcγRIIA signaling. A fraction of PIP5-kinase Iα was detected in raft-originating detergent-resistant membranes (DRM) isolated from U937 monocytes and other cells. The DRM of U937 monocytes contained also a major fraction of PI(4,5)P₂. PIP5-kinase Iα bound PI(4,5)P₂, and depletion of the lipid displaced PIP5-kinase Iα from the DRM. Activation of FcγRIIA in BHK transfectants led to recruitment of the kinase to the plasma membrane and enrichment of DRM in PI(4,5)P₂. Immunofluorescence studies revealed that in resting cells the kinase was associated with the plasma membrane, cytoplasmic vesicles and the nucleus. After FcγRIIA activation, PIP5-kinase Iα and PI(4,5)P₂ co-localized transiently with the activated receptor at distinct cellular locations. Immunoelectron microscopy studies revealed that PIP5-kinase Iα and PI(4,5)P₂ were present at the edges of electron-dense assemblies containing activated FcγRIIA in their core. The data suggest that activation of FcγRIIA leads to membrane rafts coalescing into signaling platforms containing PIP5-kinase Iα and PI(4,5)P₂.  

3.1223           The tyrosine phosphatase SHP-2 controls urokinase-dependent signaling and functions in human vascular smooth muscle cells

Kiyan, J., Haller, H. and Dumler, I. Exp. Cell Res., 315, 1029-1039 (2009)   The urokinase (uPA)/urokinase receptor (uPAR) multifunctional system is an important mediator of functional behaviour of human vascular smooth muscle cells (VSMC). uPAR associates with platelet-derived growth factor receptor β (PDGFR-β), which serves as a transmembrane adaptor for uPAR in VSMC, to transduce intracellular signaling and initiate functional changes. The precise and rapid propagation of these signaling cascades demands both strict and flexible regulatory mechanisms that remain unexplored. We provide evidence that the tyrosine phosphatase SHP-2 mediates these processes. uPA regulated SHP-2 phosphorylation, catalytic activity, and its co-localization and association with the PDGFR-β. Active PDGFR-β was required for the uPA-induced SHP-2 phosphorylation. uPAR-directed STAT1 pathway was disturbed in cells expressing SHP-2 inactive mutant. Both, cell proliferation and migration were impaired in VSMC with downregulated SHP-2. Elucidating the underlying mechanisms, we found that uPA induced SHP-2 recruitment to lipid rafts. Disruption of rafts abolished uPA-related control of SHP-2 phosphorylation, its association with PDGFR-β and finally the VSMC functional responses. Our results demonstrate that SHP-2 plays an important role in uPA-directed signaling and functional control of human VSMC and suggest that this phosphatase might contribute to the pathogenesis of the uPA-related vascular remodeling.        

3.1224           Flagellar membrane localization via association with lipid rafts

Tyler, K.M., Fridberg, A., Toriello, K.M., Olson, C.L., Cieslak, J.A., Hazlett, T.L. and Engman, D.M.

1. Cell. Sci., 122, 859-866 (2009)

  The eukaryotic flagellar membrane has a distinct composition from other domains of the plasmalemma. Our work shows that the specialized composition of the trypanosome flagellar membrane reflects increased concentrations of sterols and saturated fatty acids, correlating with direct observation of high liquid order by laurdan fluorescence microscopy. These findings indicate that the trypanosome flagellar membrane possesses high concentrations of lipid rafts: discrete regions of lateral heterogeneity in plasma membranes that serve to sequester and organize specialized protein complexes. Consistent with this, a dually acylated Ca²⁺ sensor that is concentrated in the flagellum is found in detergent-resistant membranes and mislocalizes if the lipid rafts are disrupted. Detergent-extracted cells have discrete membrane patches localized on the surface of the flagellar axoneme, suggestive of intraflagellar transport particles. Together, these results provide biophysical and biochemical evidence to indicate that lipid rafts are enriched in the trypanosome flagellar membrane, providing a unique mechanism for flagellar protein localization and illustrating a novel means by which specialized cellular functions may be partitioned to discrete membrane domains.  

3.1225           Identification of a palmitoyl acyltransferase required for protein sorting to the flagellar membrane

Emmer, B.T., Souther, C., Tooriello, K.M., Olson, C.L., Epting, C.L. and Engman, D.M.

1. Cell Sci., 122, 867-874 (2009)

  Protein palmitoylation has diverse effects in regulating protein membrane affinity, localization, binding partner interactions, turnover and function. Here, we show that palmitoylation also contributes to the sorting of proteins to the eukaryotic flagellum. African trypanosomes are protozoan pathogens that express a family of unique Ca²⁺-binding proteins, the calflagins, whichundergo N-terminal myristoylation and palmitoylation. The localizationof calflagins depends on their acylation status. Myristoylationalone is sufficient for membrane association, but, in the absenceof palmitoylation, the calflagins localize to the pellicular(cell body) membrane. Palmitoylation, which is mediated by aspecific palmitoyl acyltransferase, is then required for subsequenttrafficking of calflagin to the flagellar membrane. Coincidentwith the redistribution of calflagin from the pellicular tothe flagellar membrane is their association with lipid rafts,which are highly enriched in the flagellar membrane. Screeningof candidate palmitoyl acyltranferases identified a single enzyme,TbPAT7, that is necessary for calflagin palmitoylation and flagellarmembrane targeting. Our results implicate protein palmitoylationin flagellar trafficking, and demonstrate the conservation andspecificity of palmitoyl acyltransferase activity by DHHC-CRDproteins across kingdoms.  

3.1226           Can we generate new hypotheses about Dent's disease from gene analysis of a mouse model?

Guggino, S.E. Exp. Physiol., 94(2), 191-196 (2009)   In humans, Dent's disease, an X-linked renal tubular disorder, is characterized by low molecular weight proteinuria, aminoaciduria, glycosuria, hyperphosphaturia, hypercalciuria, nephrolithiasis, progressive renal failure and sometimes rickets or osteomalacia. The aetiology of X-linked Dent's disease is established to be caused by mutations of the CLCN5 gene. The protein product of this gene is the voltage-gated chloride–proton exchanger CLC-5. Previous studies by the Johns Hopkins group (Guggino) and the Hamburg group (Jentsch) have established that the Clcn5 knockout mouse recapitulates the renal attributes of Dent's disease. In order to understand the changes in kidney function that accompany the knockout of the Clcn5 gene, we examined gene expression profiles from dissected proximal segment 1 (S1) and segment 2 (S2) tubules of mouse kidneys. Overall, 725 genes are expressed differentially in the proximal tubules of the Dent Clcn5 knockout mouse model compared with those of control wild-type mice. A major finding is the change in the cholesterol synthesis pathway. Some interesting changes also occur in genes encoding transport proteins. One of these transport proteins, the sodium bile cotransporter gene, Slc10a2, has transcripts increased by 17-fold in the Clcn5 knockout mouse. The Clc-3 protein encoded by Clcn3, a chloride–proton exchanger related to Clc-5, has a 1.9-fold increase in transcripts. The Npt2c protein, a proximal tubule sodium phosphate cotransporter encoded by Slc34a3, has a 0.6-fold decrease in the number of transcripts. The sodium–proton exchanger-like protein, Nhe10/sperm, encoded by Slc9a10, has a 0.5-fold decrease intranscript number. These genes are discussed with regard tothe possible physiological outcomes of their transcript or proteinchanges.      

3.1227           The Ubiquitin Ligase c-Cbl Down-Regulates Fc RIIa Activation in Human Neutrophils

Marois, L., Vaillancourt, M., Marois, S., Proulx, S., Pare, G., Rollet-Labelle, E. and Naccache, P.H.

1. Immunol., 182, 2374-2384 (2009)

  Little is known about the mechanisms that arrest Fc RIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. In our previous studies, we observed a loss of immunoreactivity of Abs directed against Fc RIIa following its cross-linking. In this study, we report on the mechanisms involved in this event. A stimulated internalization of Fc RIIa leading to the down-regulation of its surface expression was observed by flow cytometry and confocal microscopy. Immunoprecipitation of the receptor showed that Fc RIIa is ubiquitinated after stimulation. MG132 and clasto-lactacystin β-lactone inhibited the loss of immunoreactivity of Fc RIIa, suggesting that this receptor was down-regulated via the proteasomal pathway. The E3 ubiquitin ligase c-Cbl was found to translocate from the cytosol to the plasma membrane following receptor cross-linking. Furthermore, c-Cbl was recruited to the same subset of high-density, detergent-resistant membrane fractions as stimulated Fc RIIa itself. Silencing the expression of c-Cbl by small interfering RNA decreased Fc RIIa ubiquitination and prevented its degradation without affecting the internalisation process. It also prolonged the stimulation of the tyrosine phosphorylation response to the cross-linking of the receptor. We conclude that c-Cbl mediates the ubiquitination of stimulated Fc RIIa and thereby contributes to the termination of Fc RIIa signaling via its proteasomal degradation, thus leading to the down-regulation of neutrophil signalisation and function (phagocytosis) through this receptor.  

3.1228           Activation of a nuclear-localized SIPK in tobacco cells challenged by cryptogein, an elicitor of plant defence reactions

Dahan, J., Pichereaux, C., Rossignol, M., Blanc, S., Wendenhenne, D., Pugin, A. and Bourque, S. Biochem. J., 418, 191-200 (2009)   When a plant cell is challenged by a well-defined stimulus, complex signal transduction pathways are activated to promote the modulation of specific sets of genes and eventually to develop adaptive responses. In this context, protein phosphorylation plays a fundamental role through the activation of multiple protein kinase families. Although the involvement of protein kinases at the plasma membrane and cytosolic levels are now well-documented, their nuclear counterparts are still poorly investigated. In the field of plant defence reactions, no known study has yet reported the activation of a nuclear protein kinase and/or its nuclear activity in plant cells, although some protein kinases, e.g. MAPK (mitogen-activated protein kinase), are known to be translocated into the nucleus. In the present study, we investigated the ability of cryptogein, a proteinaceous elicitor of tobacco defence reactions, to induce different nuclear protein kinase activities. We found that at least four nuclear protein kinases are activated in response to cryptogein treatment in a time-dependent manner, some of them exhibiting Ca²⁺-dependent activity. The present study focused on one 47 kDa protein kinase with a Ca²⁺-independent activity, closely related to the MAPK family. After purification and microsequencing, this protein kinase was formally identified as SIPK (salicyclic acid-induced protein kinase), a biotic and abiotic stress-activated MAPK of tobacco. We also showed that cytosolic activation of SIPK is not sufficient to promote a nuclear SIPK activity, the latter being correlated with cell death. In that way, the present study provides evidence of a functional nuclear MAPK activity involved in response to an elicitor treatment.  

3.1229           Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection

Cohen, R.N., van der Aa, M.A.E.M., Macaraeg, N., Lee, A.P. and Szoka Jr., F.C.

1. Controlled Release, 135, 166-174 (2009)

  Nuclear uptake of plasmid DNA is one of the many cellular barriers that limit the efficiency of non-viral gene delivery systems. We have determined the number of plasmids that reach the nucleus of a transfected cell using an internally standardized quantitative PCR (qPCR) assay. We isolated nuclei using two different protocols: a density gradient technique and a detergent-based method. The density gradient procedure yielded nuclei with substantially less adhering plasmids on the outside of the nuclei. Using the density gradient protocol we determined that cells transfected with Lipofectamine™ lipoplexes or polyethylenimine polyplexes contained between 75 and 50,000 plasmids/nucleus, depending on the applied plasmid dose. Any increase above 3000 plasmids/nucleus resulted in only marginal increases in transgene expression. Furthermore, lipoplex-delivered plasmids were more efficiently expressed, on the basis of protein expression per plasmid number in the nucleus, than polyplex-delivered plasmids. This indicates that polymer may remain bound to some plasmids in the nucleus. Lastly, by sorting transfected cells into high- and low-expressing sub-populations, we observe that a sub-population of cells contain 3× greater plasmids/nucleus but express nearly 100× more transgene than other cells within a single transfection reaction. Taken together these results suggest the importance of considering the processes downstream from nuclear entry for strategies to improve the efficiency of gene transfer reagents.   Corrigendum to “Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection” Cohen, R.N., van der Aa, M.A.E.M., Macaraeg, N., Lee, A.P. and Szoka Jr., F.C.

1. Controlled Release, 135, 166-174 (2009)

  The authors regret that there was an error in one of the sentences in Section 2.3 of the above published article. Page 167 column 2, the last sentence at the end of that column should read as follows: Nuclei were recovered from the 30/35% iodixanol interface by making a hole in the bottom of the ultracentrifuge tube with a 16-gauge needle and collecting fractions into ultracentrifuge tubes. The authors would like to apologise for any inconvenience this may have caused the readers of the journal.  

3.1230           Expression of reticulon 3 in Alzheimer's disease brain

Kume, H., Konishis, Y., Murayama, K.S., kametani, F. and Araki, W. Neuropathol. and Appl. Neurobiol., 35, 178-188 (2009)   Aims:: Reticulon 3 (RTN3), a member of the reticulon family of proteins, interacts with the β-secretase, β-site amyloid precursor protein-cleaving enzyme 1 (BACE1), and inhibits its activity to produce β-amyloid protein. The aim of the present study was to clarify the biological role of RTN3 in the brain and its potential involvement in the neuropathology of Alzheimer's disease (AD). Methods:: We performed immunohistochemical and biochemical analyses using a specific antibody against RTN3 to investigate the expression and subcellular localization of RTN3 in control and AD brain tissue samples. Results:: Western blot analysis revealed no significant differences in the RTN3 levels between control and AD brains. Immunohistochemical staining showed that RTN3 immunoreactivity was predominantly localized in pyramidal neurones of the cerebral cortex. The patterns of RTN3 immunostaining were similar in control and AD cerebral cortices, and senile plaques were generally negative for RTN3. Biochemical subcellular fractionation disclosed that RTN3 colocalized with BACE1 in various fractions, including the endoplasmic reticulum and the Golgi apparatus. Double-immunofluorescence staining additionally indicated that RTN3 was localized in both endoplasmic reticulum and Golgi compartments in neurones. Conclusions:: These results show that RTN3 is primarily expressed in pyramidal neurones of the human cerebral cortex and that no clear difference of RTN3 immunoreactivity is observable between control and AD brains. Our data also suggest that there is considerable colocalization of RTN3 with BACE1 at a subcellular level.  

3.1231           A high-cholesterol diet increases the association between caveolae and insulin receptors in rat liver

Hahn-Obercyger, M., Graeve, L. and Madar, Z.

1. Lipid Res., 50, 98-107 (2009)

  Caveolin-1, a component of caveolae, regulates signaling pathway compartmentalization by interacting with tyrosine (Tyr) kinase receptors and their substrates. Perturbations in caveolae lipid composition have been shown in vitro to displace proteins from lipid microdomains, thereby altering their functionality and subsequent downstream signaling. The role of caveolin-1 in insulin receptor (IR) signaling has been widely investigated in vitro mainly in 3T3-L1 adipocyte cells. However, in vivo experiments investigating this connection in liver tissue have not been carried out. The objective of the present study was to investigate the effects of a high-cholesterol diet on caveolin-1 expression and IR localization and activity in the rat liver. Compared with a standard diet, rats fed with diet rich in cholesterol significantly altered liver caveolae by increasing both caveolin-1 (66%, P < 0.05) and caveolin-2 (55%, P < 0.05) expression while caveolin-1 mRNA levels were reduced. Concomitantly, a 25% increase in localization of the caveolae-resident signaling protein IR was observed. The distribution of caveolar and noncaveolar phosphorylated IR was unaffected but insulin-induced IR activation was significantly enhanced following consumption of the high-cholesterol diet (120%, P < 0.001). However, the downstream molecules IRS-1 and Akt have shown impaired activity in cholesterol-fed rats suggesting insulin resistance condition. Insulin stimulation failed to induce Tyr phosphorylation of caveolin-1 in cholesterol-fed rats. These findings suggest a mechanism by which a high-cholesterol diet altered caveolin-1 expression in vivo accompanied by altered IR localization and activity.  

3.1232           Membrane microdomains from early gastrula embryos of medaka, Oryzias latipes , are a platform of E-cadherin- and carbohydrate-mediated cell–cell interactions during epiboly

Adachi, T., Sato, C., Kishi, Y., Totani, K., Murata, T., Usui, T. and Kitajima, K. Glycoconj. J., 26, 285-299 (2009)   Formation of membrane microdomain is critical for cell migration (epiboly) during gastrulation of medaka fish [Adachi et al. (Biochem. Biophys. Res. Commun. 358:848–853, 2007)]. In this study, we characterized membrane microdomain from gastrula embryos to understand its roles in epiboly. A cell adhesion molecule (E-cadherin), its associated protein (β-catenin), transducer proteins (PLCγ, cSrc), and a cytoskeleton protein (β-actin) were enriched in the membrane microdomain. Le^X-containing glycolipids and glycoproteins (Le^X-gp) were exclusively enriched in the membrane microdomain. Interestingly, the isolated membrane microdomain had the ability to bind to each other in the presence of Ca²⁺. This membrane microdomain binding was achieved through the E-cadherin homophilic and the Le^X-glycan-mediated interactions. E-cadherin and Le^X-gp were co-localized on the same membrane microdomain, suggesting that these two interactions are operative at the same time. Thus, the membrane microdomain functions as a platform of the E-cadherin- and Le^X-glycan-mediated cell adhesion and signal transduction.  

3.1233           Src Family Kinases Accelerate Prolactin Receptor Internalization, Modulating Trafficking and Signaling in Breast Cancer Cells

Piazza, T.M., Lu, J-C., Carver, K.C. and Schuler, L.A. Mol. Endocrinol., 23, 202-212 (2009)   Despite the growing body of evidence supporting prolactin (PRL) actions in human breast cancer, little is known regarding PRL regulation of its own receptor in these cells. Ligand-initiated endocytosis is a key process in the regulation of receptor availability and signaling cascades that may lead to oncogenic actions. Although exposure to exogenous PRL accelerates degradation of the long isoform of the PRL receptor (lPRLR), neither the signals initiated by PRL that lead to lPRLR internalization and subsequent down-regulation, nor the relationship to downstream pathways are understood in breast cancer cells. In this study, we showed that PRL-induced down-regulation of the lPRLR was reduced by inhibition of src family kinases (SFKs), but not Janus kinase 2, in MCF-7 cells. Inhibition of SFKs also resulted in accumulation of a PRL-induced PRLR fragment containing the extracellular domain, which appeared to be generated from newly synthesized PRLR. lPRLR was constitutively associated with SFKs in lipid rafts. PRL-induced SFK activation led to recruitment of the guanosine triphosphatase, dynamin-2, to an internalization complex, resulting in endocytosis. Inhibition of endocytosis by small interfering RNA-mediated knockdown of dynamin-2 blocked PRL-induced down-regulation of lPRLR, confirming that internalization is essential for this process. Endocytosis also was required for optimal phosphorylation of ERK1/2 and Akt, but not for Janus kinase 2 or signal transducer and activator of transcription 5, indicating that internalization selectively modulates signaling cascades. Together, these data indicate that SFKs are key mediators of ligand-initiated lPRLR internalization, down-regulation, and signal transduction in breast cancer cells, and underscore the importance of target cell context in receptor trafficking and signal transduction.  

3.1234           Downregulation of sodium transporters and NHERF proteins in IBD patients and mouse colitis models: Potential contributors to IBD-associated diarrhea

Sullivan, S., Alex, P., Dassopoulos, T., Zachos, N.C., Iacobuzio-Donahue, C., Donowitz, M., Brant, S.R., Cuffari, C., Harris, M.L., Wu Datta, L., Conklin, L., Chen, Y. and Li, X. Inflamm. Bowel. Dis., 15(2), 261-274 (2009)   Background: One of the most common symptoms among patients with inflammatory bowel disease (IBD) is diarrhea, which is thought to be contributed by changes in electrolyte transport associated with intestinal inflammation. This study was designed to test the hypothesis that intestinal Na+-related transporters/channels and their regulatory proteins may be downregulated as a potential contributor to IBD-associated diarrhea. Methods: SDS-PAGE and Western blotting and/or confocal immunomicroscopy were used to examine the expression of Na+/H+-exchangers 1-3 (NHE1-3), epithelial Na+ channel (ENaC), Na+/K+-ATPase, the intracellular Cl- channel 5 (ClC-5), and NHE3 regulatory factors (NHERF1,2) in ileal and colonic pinch biopsies from IBD patients and noninflammatory controls, as well as from colonic mucosa of dextran sodium sulfate (DSS)- and TNBS-induced acute murine IBD models. Results: NHE1,3 (but not NHE2), -ENaC, Na+/K+-ATPase- , ClC-5, and NHERF1 were all downregulated in sigmoid mucosal biopsies from most cases of active UC and/or CD compared to controls. NHE3 was also decreased in ileal mucosal biopsies of active CD, as well as in 50% of sigmoid biopsies from inactive UC or CD. Importantly, similar downregulation of NHE1,3, -ENaC, and NHERF1,2 was also observed in the mouse colon (but not ileum) of DSS- and TNBS-induced colitis. Conclusions: IBD-associated diarrhea may be due to a coordinated downregulation of multiple Na+ transporter and related regulatory proteins, including NHE1,3, Na+/K+-ATPase, and ENaC, as well as NHERF1,2, and ClC-5, all of which are involved directly or indirectly in intestinal Na+ absorption.        

3.1235           Guanosine Triphosphate Cyclohydrolase I Expression and Enzymatic Activity Are Present in Caveolae of Endothelial Cells

Peterson, T.E., d’Uscio, L.V., Cao, S., Wang, X-L. and Katusic, Z.S. Hypertension, 53, 189-195 (2009)   Tetrahydrobiopterin is an essential cofactor required for the synthesis of NO. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme for tetrahydrobiopterin production in endothelial cells, yet little is known about the subcellular localization of this enzyme. In this study, we demonstrated that GTPCH I is localized to caveolar membrane microdomains along with caveolin-1 and endothelial NO synthase. GTPCH I activity was detected in isolated caveolar membranes from cultured endothelial cells. Confocal and electron microscopy analyses confirmed GTPCH I colocalization with caveolin-1. Consistent with in vitro studies, GTPCH I activity was evident in isolated caveolar microdomains from lung homogenates of wild-type mice. Importantly, a 2-fold increase in GTPCH I activity was detected in the aortas of caveolin-1–deficient mice, suggesting that caveolin-1 may be involved in the control of GTPCH I enzymatic activity. Indeed, overexpression of caveolin-1 inhibits GTPCH I activity, and tetrahydrobiopterin biosynthesis is activated by the disruption of caveolae structure. These studies demonstrate that GTPCH I is targeted to caveolae microdomains in vascular endothelial cells, and tetrahydrobiopterin production occurs in close proximity to endothelial NO synthase. In addition, our findings provide new insights into the regulation of GTPCH I activity by the caveolar coat protein, caveolin-1.  

3.1236           Isolation of rafts from mouse brain tissue by a detergent-free method

Persaud-Sawin, D-A., Lightcap, S. and Harry, G.J.

1. Lipid Res., 50, 759-767 (2009)

  Membrane rafts are rich in cholesterol and sphingolipids and have specific proteins associated with them. Due to their small size, their identification and isolation have proved to be problematic. Their insolubility in nonionic detergents, such as Triton-X 100, at 4°C has been the most common means of isolation. However, detergent presence can produce artifacts or interfere with ganglioside distribution. The direction is therefore toward the use of detergent-free protocols. We report an optimized method of raft isolation from lipid-rich brain tissue using a detergent-free method. We compared this to Triton-X 100-based isolation along sucrose or Optiprep^TM gradients using the following endpoints: low protein content, high cholesterol content, presence of Flotillin 1 (Flot1), and absence of transferrin receptor (TfR) proteins. These criteria were met in raft fractions isolated in a detergent-free buffer along a sucrose gradient of 5%/35%/42.5%. The use of optiprep gave less consistent results with respect to protein distribution. We demonstrate that clean raft fractions with minimal myelin contamination can be reproducibly obtained in the top three low-density fractions along a sucrose step gradient.  

3.1237           Lipid Raft Segregation Modulates TRPM8 Channel Activity

Morenilla-Palao, C., Pertusa, M., Meseguer, V., Cabedo, H. and Viana, F.

1. Biol. Chem., 284(14), 9215-9224 (2009)

  Transient receptor potential channels are a family of cation channels involved in diverse cellular functions. Most of these channels are expressed in the nervous system and play a key role in sensory physiology. TRPM8 (transient receptor potential melastatine 8), a member of this family, is activated by cold, cooling substances such menthol and icilin and voltage. Although TRPM8 is a thermosensitive channel highly expressed in cold sensory neurons, the mechanisms underlying its temperature sensitivity are still poorly understood. Here we show that, in sensory neurons, TRPM8 channel is localized in cholesterol-rich specialized membrane domains known as lipid rafts. We also show that, in heterologous expression systems, lipid raft segregation of TRPM8 is favored by glycosylation at the Asn⁹³⁴ residue of the polypeptide. In electrophysiological and imaging experiments, using cold and menthol as agonists, we also demonstrate that lipid raft association modulates TRPM8 channel activity. We found that menthol- and cold-mediated responses of TRPM8 are potentiated when the lipid raft association of the channel is prevented. In addition, lipid raft disruption shifts the threshold for TRPM8 activation to a warmer temperature. In view of these data, we suggest a role for lipid rafts in the activity and temperature sensitivity of TRPM8. We propose a model wherein different lipid membrane environments affect the cold sensing properties of TRPM8, modulating the response of cold thermoreceptors.        

3.1238           The amino-terminal region of Atg3 is essential for association with phosphatidylethanolamine in Atg8 lipidation

Hanada, T., Satomi, Y., Takao, T. and Ohsumi, Y. FEBS Lett., 583, 1078-1083 (2009)   Autophagy is a bulk degradation process conserved among eukaryotes. In macro-autophagy, autophagosomes sequester cytoplasmic components and deliver their contents to lysosomes/vacuoles. Autophagosome formation requires the conjugation of Atg8, a ubiquitin-like protein, to phosphatidylethanolamine (PE). Here we report that the amino (N)-terminal region of Atg3, an E2-like enzyme for Atg8, plays a crucial role in Atg8–PE conjugation. The conjugating activities of Atg3 mutants lacking the 7 N-terminal amino acid residues or containing a Leu-to-Asp mutation at position 6 were severely impaired both in vivo and in vitro. In addition, the amino-terminal region is critical for interaction with the substrate, PE.  

3.1239           Acute hypertension provokes acute trafficking of distal tubule Na-Cl cotransporter (NCC) to subapical cytoplasmic vesicles

Lee, D.H., Riquier, A.D.M., Yang, L.E., Leong, P.K.K., Maunsbach, A.B. and McDonough, A.A. Am. J. Physiol. Renal Physiol., 296, F810-F818 (2009)   When blood pressure (BP) is elevated above baseline, a pressure natriuresis-diuresis response ensues, critical to volume and BP homeostasis. Distal convoluted tubule Na⁺-Cl^– cotransporter (NCC) is regulated by trafficking between the apical plasma membrane (APM) and subapical cytoplasmic vesicles (SCV). We aimed to determine whether NCC trafficking contributes to pressure diuresis by decreasing APM NCC or compensates for increased volume flow to the DCT by increasing APM NCC. BP was raised 50 mmHg (high BP) in rats by arterial constriction for 5 or 20–30 min, provoking a 10-fold diuresis at both times. Kidneys were excised, and NCC subcellular distribution was analyzed by 1) sorbitol density gradient fractionation and immunoblotting and 2) immunoelectron microscopy (immuno-EM). NCC distribution did not change after 5-min high BP. After 20–30 min of high BP, 20% of NCC redistributed from low-density, APM-enriched fractions to higher density, endosome-enriched fractions, and, by quantitative immuno-EM, pool size of APM NCC decreased 14% and SCV pool size increased. Because of the time lag of the response, we tested the hypothesis that internalization of NCC was secondary to the decrease in ANG II that accompanies high BP. Clamping ANG II at a nonpressor level by coinfusion of captopril (12 µg/min) and ANG II (20 ng·kg^–1·min^–1) during 30-min high BP reduced diuresis to eightfold and prevented redistribution of NCC from APM- to SCV-enriched fractions. We conclude that DCT NCC may participate in pressure natriuresis-diuresis by retraction out of apical plasma membranes and that the retraction is, at least in part, driven by the fall in ANG II that accompanies acute hypertension.  

3.1240           Phosphotyrosine-dependent in vitro reconstitution of recombinant LAT-nucleated multiprotein signalling complexes on liposomes

Sangani, D., Venien-Bryan, C. and Harder, T. Mol. Membrane Biol., 26(2), 159-170 (2009)   Numerous cell surface receptors propagate activation signals to the interior of the cell via tyrosine phosphorylation of transmembrane proteins. This leads to the phosphotyrosine (PiY)-mediated recruitment of cytoplasmic signalling protein complexes which catalyze crucial biochemical signalling reactions. Here we describe the first in vitro reconstitution of such PiY-nucleated protein complexes on an artificial lipid membrane. A tyrosine phosphorylated recombinant variant of the transmembrane adaptor protein Linker for Activation of T cells (PiYLAT) was anchored in liposomes. These PiYLAT proteoliposomes specifically recruited cooperative high avidity signalling protein complexes from Jurkat cytosol. Nucleation of signalling protein assemblies readily occurred on PiYLAT liposomes composed of phosphatidylserine, but not on PiYLAT liposomes composed of phosphatidylcholine. Purified recombinant grb2 alone did not stably associate with tyrosine phosphorylated LAT proteoliposomes. However, when grb2 was presented to the PiYLAT proteoliposomes in the context of Jurkat cytosol it was incorporated into multiprotein signalling complexes. Together the data suggest that these reconstituted high-avidity signalling protein complexes represent a cooperative protein network. This novel in vitro approach offers a novel technology permitting biochemical, structural, and pharmacological analyses of plasma membrane receptor signalling complexes.  

3.1241           Renal NHE3 and NaPi2 partition into distinct membrane domains

Riquuier, A.D.M., Lee, D.H., and McDonough, A.A. Am. J. Physiol. Cell Physiol., 296, C900-C910 (2009)   Hypertension provokes differential trafficking of the renal proximal tubule Na⁺/H⁺ exchanger 3 (NHE3) to the base of the apical microvilli and Na⁺-P_i cotransporter 2 (NaPi2) to endosomes. The resultant diuresis and natriuresis are key to blood pressure control. We tested the hypothesis that this differential trafficking of NHE3 vs. NaPi2 was associated with partitioning to distinct membrane domains. In anesthetized rats, arterial pressure was increased (104 ± 2 to 142 ± 4 mmHg, 15 min) by arterial constriction and urine output increased 23-fold. Renal membranes were fractionated by cold 1% Triton X-100 extraction then centrifugation through OptiPrep flotation gradients. In controls, 84 ± 9% of NHE3 localized to flotillin-enriched lipid raft domains and 69 ± 5% of NaPi2 localized to transferrin receptor-enriched nonrafts. MyosinVI and dipeptidyl peptidase IV, associated with NHE3 regulation, coenriched in lipid rafts with NHE3, while NHE regulatory factor-1 coenriched in nonrafts with NaPi2. Partitioning was not altered by hypertension. Detergent insoluble membranes were pelleted after detergent extraction. NHE3 detergent insolubility decreased as it redistributed from body (80 ± 10% detergent insoluble) to base (75 ± 3%) of the apical microvilli, while NaPi2 partitioned into more insoluble domains as it moved from the microvilli (45 ± 7% detergent insoluble) to endosomes (82 ± 1%). In conclusion, NHE3 and NaPi2, while both localized to apical microvilli, are segregated into domains: NHE3 to lipid rafts and NaPi2 to nonrafts. These domain properties may play a role in the distinct trafficking patterns observed during elevated pressures: NHE3 remains in rafts and settles to the base of the microvilli while NaPi2 is freely endocytosed.  

3.1242           Soybean proteomics and its application to functional analysis

Komatsu, S. and Ahsan, N.

1. Proteomics, 72, 325-336 (2009)

  Complete genome sequences, which are available for rice and Arabidopsis, provide insights into many fundamental aspects of plant biology; they do not, however, address some important aspects of legume biology. Legumes are important for maintenance of human health and as crops for sustainable agriculture. Two model species of legume, Lotus japonicus and Medicago truncatula, have been the focus of projects on genome sequencing and functional genomics. A project aimed at sequencing the genome of the agricultural legume soybean recently began, but functional genomics studies of this plant are in their infancy, and therefore proteomics approaches could be a powerful tool for functional analysis. In this review, we discuss the strengths and weaknesses of proteomics technologies in soybean biology and we examine the limitations of current techniques.  

3.1243           Hydroponics on a chip: Analysis of the Fe deficient Arabidopsis thylakoid membrane proteome

Laganowsky, A., Gomez, S.M., Whitelegge, J.P. and Nishio, J.N.

1. Proteomics, 72(3), 397-415 (2009)

  The model plant Arabidopsis thaliana was used to evaluate the thylakoid membrane proteome under Fe-deficient conditions. Plants were cultivated using a novel hydroponic system, called “hydroponics on a chip”, which yields highly reproducible plant tissue samples for physiological analyses, and can be easily used for in vivo stable isotope labeling. The thylakoid membrane proteome, from intact chloroplasts isolated from Fe-sufficient and Fe-deficient plants grown with hydroponics on a chip, was analyzed using liquid chromatography coupled to mass spectrometry. Intact masses of thylakoid membrane proteins were measured, many for the first time, and several proteins were identified with post-translational modifications that were altered by Fe deficiency; for example, the doubly phosphorylated form of the photosystem II oxygen evolving complex, PSBH, increased under Fe-deficiency. Increased levels of photosystem II protein subunit PSBS were detected in the Fe-deficient samples. Antioxidant enzymes, including ascorbate peroxidase and peroxiredoxin Q, were only detected in the Fe-deficient samples. We present the first biochemical evidence that the two major LHC IIb proteins (LHCB1 and LHCB2) may have significantly different functions in the thylakoid membrane. The study illustrates the utility of intact mass proteomics as an indispensable tool for functional genomics. “Hydroponics on a chip” provides the ability to grow A. thaliana under defined conditions that will be useful for systems biology.    

3.1244           Advancements in plant proteomics using quantitative mass spectrometry

Oeljeklaus, S., Meyer, H.E. and warscheid, B.

1. Proteomics, 72(3), 545-554 (2009)

  Due to innovative advancements in quantitative MS technologies, proteomics has evolved from taking mere “snapshots” of distinct proteomes in a defined state to monitoring, for instance, changes in abundance, location and/or posttranslational modification(s) of proteins under various conditions, thereby facilitating the functional characterization of proteins in large scale experiments. In plant biology, MS-based quantitative proteomics strategies utilizing stable isotope labeling or label-free methods for protein quantification have only recently been started to find increasing application to comparative and functional proteomics analyses. This review summarizes latest trends and applications in MS-based quantitative plant proteomics and provides insight into different technologies available. In addition, the studies presented here illustrate the enormous potential of quantitative MS for the analysis of important functional aspects with the emphasis on organellar and phosphoproteomics as well as dynamics and turnover of proteins in plants.  

3.1245           Long-Distance Delivery of Bacterial Virulence Factors by Pseudomonas aeruginosa Outer Membrane Vesicles

Bomberger, J.M., Maceachran, D.P., Coutermarsh, B.A., Ye, S., O’Toole, G.A. and Stanton, B.A. PloSPathogens, 5(4), e1000382 (2009)   Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane–derived vesicles (OMV) secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including β-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP–mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner.  

3.1246           Characterization of a myristoylated, monomeric HIV Gag protein

Dou, J., Wang, J-J., Chen, X., Li, H., Ding, L. and Spearman, P. Virology, 387, 341-352 (2009)   The process of HIV assembly requires extensive homomultimerization of the Gag polyprotein on cellular membranes to generate the nascent particle bud. Here we generated a full-length, monomeric Gag polyprotein bearing mutations that eliminated multimerization in living cells as indicated by fluorescence resonance energy transfer (FRET). Monomeric Gag resembled non-myristoylated Gag in its weak membrane binding characteristics and lack of association with detergent-resistant membranes (DRMs or lipid rafts). Monomeric Gag failed to assemble virus-like particles, but was inefficiently rescued into particles by wildtype Gag through the influence of the matrix domain. The subcellular distribution of monomeric Gag was remarkably different than either non-myristoylated Gag or wildtype Gag. Monomeric Gag was found on intracellular membranes and at the plasma membrane, where it highlighted plasma membrane extensions and ruffles. This study indicates that monomeric Gag can traffic to assembly sites in the cell, where it interacts weakly with membranes.  

3.1247           Subcellular fractionation of human eosinophils: Isolation of functional specific granules on isoosmotic density gradients

Neves, J.S., Perez, S.A.C., Spencer, L.A., Melo, R.C.N. and Weller, P.F.

1. Immunol., Methods, 344, 64-72 (2009)

  Subcellular fractionation has been an important tool in investigating human eosinophil structure and function, including localizing of cytokine/chemokines within granules, investigating granule protein translocation and intracellular transport during eosinophil secretion, and studying secretory mechanisms of granules. The resolution of organelles obtained by subcellular fractionation was improved considerably after the introduction of nonionic iodinated density-gradient metrizamide and Nycodenz media that, unlike sucrose, exhibit relatively low tonicity throughout the gradient. However, the structure and membrane preservation of isolated organelles were still compromised due to the lack of gradient isoosmolarity. This paper describes a detailed protocol of subcellular fractionation of nitrogen cavitated eosinophils on an isoosmotic iodinated density gradient (iodixanol – OptiPrep) and the isolation of well preserved and functional membrane-bound specific granules.  

3.1248           Mitochondrial degeneration and not apoptosis is the primary cause of embryonic lethality in ceramide transfer protein mutant mice

Wang, X., Rao, R.P., Kasakowska-Cholody, T., Massod, M.A., Southon, E., Zhang, H., Berthel, C., Nagashim, K., Veenstra, T.K., Tessarollo, L., Acharya, U. and Acharya, J.K.

1. Cell Biol., 184(1), 143-158 (2009)

  Ceramide transfer protein (CERT) functions in the transfer ofceramide from the endoplasmic reticulum (ER) to the Golgi. Inthis study, we show that CERT is an essential gene for mousedevelopment and embryonic survival and, quite strikingly, iscritical for mitochondrial integrity. CERT mutant embryos accumulateceramide in the ER but also mislocalize ceramide to the mitochondria,compromising their function. Cells in mutant embryos show abnormaldilation of the ER and degenerating mitochondria. These subcellularchanges manifest as heart defects and cause severely compromisedcardiac function and embryonic death around embryonic day 11.5.In spite of ceramide accumulation, CERT mutant mice do not dieas a result of enhanced apoptosis. Instead, cell proliferationis impaired, and expression levels of cell cycle–associatedproteins are altered. Individual cells survive, perhaps becausecell survival mechanisms are activated. Thus, global compromiseof ER and mitochondrial integrity caused by ceramide accumulationin CERT mutant mice primarily affects organogenesis rather thancausing cell death via apoptotic pathways.  

3.1249           Akt-Mediated Transactivation of the S1P1 Receptor in Caveolin-Enriched Microdomains Regulates Endothelial Barrier Enhancement by Oxidized Phospholipids

Singleton, P.A., Chatchavalvanich, C., Fu, P., Xing, J., Birukova, A.A., Fortune, J.A., Klibanov, A.M., garcia, J.G.N. and Birukov, K.G. Circ. Res., 104, 978-986 (2009)   Endothelial cell (EC) barrier dysfunction results in increased vascular permeability, leading to increased mass transport across the vessel wall and leukocyte extravasation, the key mechanisms in pathogenesis of tissue inflammation and edema. We have previously demonstrated that OxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) significantly enhances vascular endothelial barrier properties in vitro and in vivo and attenuates endothelial hyperpermeability induced by inflammatory and edemagenic agents via Rac and Cdc42 GTPase dependent mechanisms. These findings suggested potential important therapeutic value of barrier-protective oxidized phospholipids. In this study, we examined involvement of signaling complexes associated with caveolin-enriched microdomains (CEMs) in barrier-protective responses of human pulmonary ECs to OxPAPC. Immunoblotting from OxPAPC-treated ECs revealed OxPAPC-mediated rapid recruitment (5 minutes) to CEMs of the sphingosine 1-phosphate receptor (S1P₁), the serine/threonine kinase Akt, and the Rac1 guanine nucleotide exchange factor Tiam1 and phosphorylation of caveolin-1, indicative of signaling activation in CEMs. Abolishing CEM formation (methyl-β-cyclodextrin) blocked OxPAPC-mediated Rac1 activation, cytoskeletal reorganization, and EC barrier enhancement. Silencing (small interfering RNA) Akt expression blocked OxPAPC-mediated S1P₁ activation (threonine phosphorylation), whereas silencing S1P₁ receptor expression blocked OxPAPC-mediated Tiam1 recruitment to CEMs, Rac1 activation, and EC barrier enhancement. To confirm our in vitro results in an in vivo murine model of acute lung injury with pulmonary vascular hyperpermeability, we observed that selective lung silencing of caveolin-1 or S1P₁ receptor expression blocked OxPAPC-mediated protection from ventilator-induced lung injury. Taken together, these results suggest Akt-dependent transactivation of S1P₁ within CEMs is important for OxPAPC-mediated cortical actin rearrangement and EC barrier protection.  

3.1250           TIP47, a Lipid Cargo Protein Involved in Macrophage Triglyceride Metabolism

Buers, I., Robenek, H., Lorkowski, S., Nitschke, Y., Severs, N.J. and Hofnagel, O. Arterioscler. Thromb. Vasc. Biol., 29, 767-773 (2009)   Objective— Uptake of lipids by macrophages (M) leads tolipid droplet accumulation and foam cell formation. The PATfamily proteins are implicated in lipid droplet formation, butthe precise function of the 47-kDa tail interacting protein(TIP47), a member of this family, is poorly defined. The presentstudy was performed to determine the function of TIP47 in Mlipid metabolism. Methods and Results— Freeze-fracture cytochemistry demonstratesthat TIP47 is present in the plasma membrane of M and is aggregatedinto clusters when the cells are incubated with oleate. Suppressionof adipophilin levels using siRNA knockdown leads to migrationof TIP47 from a cytoplasmic pool to the lipid droplet. Further,reduction of TIP47 decreases triglyceride levels, whereas raisingTIP47 levels by expression of EGFP-TIP47 shows the oppositeeffect. Conclusion— Our results show that the TIP47 protein levelsdirectly correlate with triglyceride levels. We propose thatTIP47 may act as a carrier protein for free fatty acids andin this way participates in conversion of M into foam cells. The function of TIP47 on M lipid metabolism was investigated.TIP47 protein levels were found to directly correlate with triglyceridelevels. From this and other experimental evidence, we proposethat TIP47 acts as a carrier protein for free fatty acids andthereby participates in conversion of M into foam cells.  

3.1251           Potential abnormalities in iron metabolism in hyperlipidemia patient fibroblasts

Morrison, C., Sauble, E.N., Nguyen, A., La, A., Bach, G. and Linder, M.C. FASEB J., 23, 105.4 (2009)   Mucolipidosis type IV (MLIV) is an autosomal recessive neurodegenerative disorder that results from a mutation inmucolipin 1, a 580 amino acid non-selective cation channel present on lysosomal membranes. This mutation disruptssorting, transport and/or fusion of endosomes and lysosomes, and subjects suffer from iron deficiency anemia. Toestablish whether lysosomal turnover of endogenous ferritin was disrupted, fibroblasts from normal and MLIVsubjects were pretreated with 59Fe-labeled ferric ammonium citrate (180 μM) for 24h to produce ferritin (Ft) and thenwith desferioxamine to induce Ft turnover in lysosomes (Kidane et al, Am J Physiol 291: C445, 2006). No defects in Ftturnover and Fe release were observed, as demonstrated with iodixanol density gradients separating cytoplasmic andlysosomes/endosomal Ft and its iron. However, when exogenous (cationized) horse spleen Ft was administered,turnover of this iron was slowed and more was held in lysosomes and endosomes in the case of cells from diseased subjects. Accumulation of cytoplasmic Ft protein (measured by ELISA) was also reduced. We conclude that a reduced rate of processing of iron entering by endocytosis may slow recycling of red cell iron and contribute to the development an iron deficiency-like anemia.  

3.1252           Nuclear AT2 receptors mediate angiotensin II-dependent generation of nitric oxide

Gwathmey, T.M., Pendergrass, K.D., Pirro, N.T., Shaltout, H.A., Reid, S.D., Rose, J.C. and Chappell, M.C. FASEB J., 23, 606.9 (2009)   We recently reported the expression of receptors for the hormone angiotensin II [Ang II] in isolated nuclei of the sheep kidney. The renal cortex contained predominantly AT2 receptors [~70%], while the renal medulla expressed essentially the AT1 subtype [>90%]. The present study examined their functional roles in cortical nuclei of adult sheep. Nuclei were isolated from kidneys of adult (1.5 yr old) sheep by Optiprep density gradient and loaded with the fluorescence dye difluorofluorescein diacetate (DAF) to assess the production of nitric oxide (NO). Ang II (1 nM) significantly increased DAF fluorescence above control (80 +/- 11%; P<0.001; N=4); DAF stimulation was abolished by either the AT2 antagonist PD123319 (1μM, P>0.05 vs. control) or the NO synthase inhibitor L-NAME (1mM, P>0.05 vs. control). Treatment with AT1 receptor antagonist losartan (1μM) did not alter Ang II-induced NO generation (P>0.05 vs. Ang II). Protein analysis of isolated cortical nuclei revealed a prominent band for both eNOS/NOSIII (135kDa) and the principal NO receptor soluble guanylate cyclase-&#946; (sGC, 70kDa). We demonstrate that the nuclear AT2 receptor subtype is functionally linked to NO generation and confirm the expression of eNOS and sGC in sheep nuclei. These data suggest that the nucleus contains the necessary signaling components for NO generation and provide further support of a functional intracellular RAS within the kidney.  

3.1253           Mechanisms of iron release from lysosomes

Nguyen, A., Zhao, N., Morrison, C., Gonzales, A., Sauble, E., La, A., Linder, M.C., and Knutson, M. FASEB J., 23, 921.11 (2009)   Stored cellular Fe is made available and recycled at least partly through lysosomal degradation of cytoplasmic ferritin (Ft) after autophagy, but how Fe returns to the cytoplasm is unknown. Divalent metal transporter 1 (DMT1) is associated not just with transferrin-related endosomes but also with lysosomes, and the latter is true for Zip 8, which might also be an Fe transporter. To begin the determine whether one or both DMT1 and Zip8 might be involved, we first established that Zip 8 transfection into HEK cells (with low endogenous levels) enhanced Fe uptake, determined with 59Fe-citrate. We then knocked down expression of Zip 8 with siRNA in rat hepatoma cells, and studied the accumulation of 59Fe in lysosomes separated from cytoplasmic Ft on iodixanol gradients (Kidane et al, Am J Physiol 291: C445, 2006). To track and move Ft into lysosomes, we pretreated cells with 59Fe-ferric ammonium citrate for 24h then induced Fe depletion with deferoxamine (as previously). Compared with scrambled siRNA, 80-90% knockdown of Zip 8 mRNA was associated with about 35% greater retention of 59Fe in lysosomes. Using confocal microscopy on HepG2 cells, we also determined that Fe depletion rapidly increased colocalization of DMT1 with the lysosomal marker (LAMP2). We conclude that both of these transporters could be involved in the return of lysosomal Fe to the cytoplasm.marker (LAMP2). We conclude that both of these transporters could be involved in the return of lysosomal Fe to the cytoplasm.  

3.1254           Protective Role of Endogenous Gangliosides for Lysosomal Pathology in a Cellular Model of Synucleinopathies

Wei, J., Fujita, M., Nakai, M., Waragai, M., Sekigawa, A., Sugama, S., Takenouchi, T., Masliah, E. and Hashimoto, M. Am. J. Pathol., 174(5), 1891-1909 (2009)   Gangliosides may be involved in the pathogenesis of Parkinson’s disease and related disorders, although the precise mechanisms governing this involvement remain unknown. In this study, we determined whether changes in endogenous ganglioside levels affect lysosomal pathology in a cellular model of synucleinopathy. For this purpose, dementia with Lewy body-linked P123H β-synuclein (β-syn) neuroblastoma cells transfected with -synuclein were used as a model system because these cells were characterized as having extensive formation of lysosomal inclusions bodies. Treatment of these cells with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glycosyl ceramide synthase, resulted in various features of lysosomal pathology, including compromised lysosomal activity, enhanced lysosomal membrane permeabilization, and increased cytotoxicity. Consistent with these findings, expression levels of lysosomal membrane proteins, ATP13A2 and LAMP-2, were significantly decreased, and electron microscopy demonstrated alterations in the lysosomal membrane structures. Furthermore, the accumulation of both P123H β-syn and -synuclein proteins was significant in PDMP-treated cells because of the suppressive effect of PDMP on the autophagy pathway. Finally, the detrimental effects of PDMP on lysosomal pathology were significantly ameliorated by the addition of gangliosides to the cultured cells. These data suggest that endogenous gangliosides may play protective roles against the lysosomal pathology of synucleinopathies.  

3.1255           Endosomal Nox2 Facilitates Redox-Dependent Induction of NF-κB by TNF-α

Li, Q., Spencer, N.Y., Oakley, F.D., Buettner, G.R. and Engelhardt, J.F. Antioxidants & redox Signaling, 11(6), 1249-1263 (2009)   Growing evidence suggests that NADPH oxidase (Nox)-derived reactive oxygen species (ROS) play important roles in regulating cytokine signaling. We have explored how TNF-α induction of Nox-dependent ROS influences NF-κB activation. Cellular stimulation by TNF-α induced NADPH-dependent superoxide production in the endosomal compartment, and this ROS was required for IKK-mediated activation of NF-κB. Inhibiting endocytosis reduced the ability of TNF-α to induce both NADPH-dependent endosomal superoxide and NF-κB, supporting the notion that redox-dependent signaling of the receptor occurs in the endosome. Molecular analyses demonstrated that endosomal H₂O₂ was critical for the recruitment of TRAF2 to the TNFR1/TRADD complex after endocytosis. Studies using both Nox2 siRNA and Nox2-knockout primary fibroblasts indicated that Nox2 was critical for TNF-α–mediated induction of endosomal superoxide. Redox-active endosomes that form after TNF-α or IL-1β induction recruit several common proteins (Rac1, Nox2, p67^phox, SOD1), while also retaining specificity for ligand-activated receptor effectors. Our studies suggest that TNF-α and IL-1β signaling pathways both can use Nox2 to facilitate redox activation of their respective receptors at the endosomal level by promoting the redox-dependent recruitment of TRAFs. These studies help to explain how cellular compartmentalization of redox signals can be used to direct receptor activation from the plasma membrane.  

3.1256           Signaling Components of Redox Active Endosomes: The Redoxosomes

Oakley, F.D., Abbott, D., Li, Q. and Engelhardt, J.F. Antioxidants & Redox Signaling, 11(6), 1313-1333 (2009)   Subcellular compartmentalization of reactive oxygen species (ROS) plays a critical role in transmitting cell signals in response to environmental stimuli. In this regard, signals at the plasma membrane have been shown to trigger NADPH oxidase-dependent ROS production within the endosomal compartment and this step can be required for redox-dependent signal transduction. Unique features of redox-active signaling endosomes can include NADPH oxidase complex components (Nox1, Noxo1, Noxa1, Nox2, p47phox, p67phox, and/or Rac1), ROS processing enzymes (SOD1 and/or peroxiredoxins), chloride channels capable of mediating superoxide transport and/or membrane gradients required for Nox activity, and novel redox-dependent sensors that control Nox activity. This review will discuss the cytokine and growth factor receptors that likely mediate signaling through redox-active endosomes, and the common mechanisms whereby they act. Additionally, the review will cover ligand-independent environmental injuries, such as hypoxia/reoxygenation injury, that also appear to facilitate cell signaling through NADPH oxidase at the level of the endosome. We suggest that redox-active endosomes encompass a subset of signaling endosomes that we have termed redoxosomes. Redoxosomes are uniquely equipped with redox-processing proteins capable of transmitting ROS signals from the endosome interior to redox-sensitive effectors on the endosomal surface. In this manner, redoxosomes can control redox-dependent effector functions through the spatial and temporal regulation of ROS as second messengers.  

3.1257           Isolation of Saccharomyces Cerevisiae Mitochondria for Mössbauer, Epr, and Electronic Absorption Spectroscopic Analyses

Lindahl, P.A., Morales, J.G., Miao, R. and Holmes-Hampton, G. Methods in Enzymol., 456, 267-285 (2009)   Methods are presented to aid in the study of iron metabolism in isolated mitochondria. The “iron-ome” of mitochondria, including the type and concentration of all Fe-containing species in the organelle, is evaluated by integrating the results of four spectroscopic methods, including Mössbauer spectroscopy, electron paramagnetic resonance, electronic absorption spectroscopy, and inductively coupled plasma mass spectrometry. Although this systems biology approach only allows groups of Fe centers to be assessed, rather than individual species, it affords new and useful information. There are many considerations in executing this approach, and this chapter focuses on the practical methods that we have developed for this purpose. First, large quantities of mitochondria are required, and so published isolation methods must be scaled up. Second, mitochondria are isolated under strict anaerobic conditions to allow control of redox state and to protect O₂-sensitive Fe-containing proteins from degradation. Third, the importance of packing mitochondria for both spectroscopic and analytical characterizations is developed. By measuring the volume of packed samples and the percentage of mitochondria contained within that volume, absolute Fe and protein concentrations within the organelle can be obtained. Packing samples into spectroscopy holders also affords maximal signal intensities, which are critical for these studies. Custom inserts designed for this purpose are described. Also described are the designs of a 25-L glass bioreactor, a mechanical cell homogenizer, a device for inserting short EPR tubes into the standard Oxford Instruments EPR cryostat, and a device for transferring samples from Mössbauer holders to EPR tubes while maintaining samples at liquid N₂ temperatures. A brief summary of what we have learned by use of these methods is included.  

3.1258           Interruption of Growth Hormone Signaling via SHC and ERK in 3T3-F442A Preadipocytes upon Knockdown of Insulin Receptor Substrate-1

Wang, X., Yang, N., Deng, L., Li, X., Jiang, J., Gan, Y. and Frank, S.J. Mol. Endocrinol., 23(4), 486-496 (2009)   Insulin receptor substrate-1 (IRS-1) is a docking protein tyrosine phosphorylated in response to insulin, IGF-1, GH, and other cytokines. IRS-1 has an N-terminal plekstrin homology domain (which facilitates membrane localization), a phosphotyrosine-binding domain [which associates with tyrosine-phosphorylated insulin receptor or IGF-1 receptor (IGF-1R)], and tyrosine residues that, when phosphorylated, bind signaling molecules. The role of IRS-1 in GH signaling is uncertain. We previously reported that IRS-1 and Janus kinase 2 associate independently of tyrosine phosphorylation via IRS-1’s N terminus and that IRS-1 reconstitution greatly enhances GH-induced ERK, but not STAT5, activation. We now use GH-responsive 3T3-F442A preadipocytes to study the influence of IRS-1 on GH action. We stably transfected cells with vector only (Control) or a vector encoding IRS-1 short hairpin RNA [knockdown (KD)] and compared representative clones. Immunoblotting confirmed more than 80% knockdown of IRS-1 in KD cells. GH caused characteristic Janus kinase 2 and STAT5 activation in both Control and KD cells, but ERK activation was dramatically reduced in KD cells in GH time course and dose-response experiments. Notably, GH-induced Src homology collagen (SHC) activation and SHC-Grb2 association in KD cells were also markedly diminished compared with Control cells. Subcellular fractionation revealed that IRS-1 in Control cells was largely cytosolic, but the component isolated with plasma membranes was highly enriched in lipid raft membranes (LR). In KD cells, GH-induced ERK activation in the LR fraction was particularly diminished compared with Control cells. These data suggest that LR-enriched IRS-1 contributes substantially to GH-induced ERK activation in LR in 3T3-F442A fibroblasts. Furthermore, our results are consistent with IRS-1 residing upstream of SHC in the GH-induced ERK-signaling pathway.  

3.1259           Activation-dependent stabilization of the human thromboxane receptor: role of reactive oxygen species

Wilson, S.J., Cavanagh, C.C., Lesher, A.M., Frey, A.J., Russell, S.E. and Smyth, E.M.

1. Lipid Res., 50, 1047-1056 (2009)

  Thromboxane A₂ (TxA₂), the principle product of platelet COX-1-dependent arachidonic acid metabolism, directs multiple pro-atherogenic processes via its receptor, TP. Oxidative challenge offsets TP degradation, a key component in limiting TxA₂'s actions. Following TP activation, we observed cellular reactive oxygen species (ROS) generation coincident with increased TP expression. We examined the link between TP-evoked ROS and TP regulation. TP expression was augmented in TP -transfected cells treated with a TxA₂ analog [1S-1 ,2β(5Z),3 (1E,3R*),4 ]]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-heptenoic acid (IBOP). This was reduced with a cellular antioxidant, N-acetyl cysteine, or two distinct NADPH oxidase inhibitors, diphenyleneiodonium and apocynin. Homologous upregulation of the native TP was also reduced in apocynin-treated aortic smooth muscle cells (ASMCs) and was absent in ASMCs lacking an NADPH oxidase subunit (p47^–/–). TP transcription was not increased in IBOP-treated cells, indicating a posttranscriptional mechanism. IBOP induced translocation of TP to the Golgi and reduced degradation of the immature form of the receptor. These data are consistent with a ROS-dependent mechanism whereby TP activation enhanced TP stability early in posttranscriptional biogenesis. Given the significant role played by TP and ROS in perturbed cardiovascular function, the convergence of TP on ROS-generating pathways for regulation of TxA₂-dependent events may be critical for cardiovascular disease.  

3.1260           NHE3 regulatory complexes

Donowitz, M., Mohan, S., Zhu, C.X., Chen, T-E., Lin, R., Cha, B., Zachos, N.C., Murtazina, R., Sarker, R. and Li, X.

1. Exp. Biol., 212, 1638-1646 (2009)

  The epithelial brush border Na/H exchanger NHE3 is active under basal conditions and functions as part of neutral NaCl absorption in the intestine and renal proximal tubule, where it accounts for the majority of total Na absorbed. NHE3 is highly regulated. Both stimulation and inhibition occur post-prandially. This digestion related regulation of NHE3 is mimicked by multiple extracellular agonists and intracellular second messengers. The regulation of NHE3 depends on its C-terminal cytoplasmic domain, which acts as a scaffold to bind multiple regulatory proteins and links NHE3 to the cytoskeleton. The cytoskeletal association occurs by both direct binding to ezrin and by indirect binding via ezrin binding to the C-terminus of the multi-PDZ domain containing proteins NHERF1 and NHERF2. This is a review of the domain structure of NHE3 and of the scaffolding function and role in the regulation of NHE3 of the NHE3 C-terminal domain.

3.1261           Genetic evidence for the requirement of the endocytic pathway in the uptake of coenzyme Q6 in Saccharomyces cerevisiae

Padilla-Lopez, S., Jimenez-Hildago, M., Martin-Montalvo, A., Clarke, C.F., Navas, P. and Santos-Ocana, C. Biochim. Biophys. Acta, 1788, 1238-1248 (2009)   Coenzyme Q is an isoprenylated benzoquinone lipid that functions in respiratory electron transport and as a lipid antioxidant. Dietary supplementation with Q is increasingly used as a therapeutic for treatment of mitochondrial and neurodegenerative diseases, yet little is known regarding the mechanism of its uptake. As opposed to other yeast backgrounds, EG103 strains are unable to import exogenous Q₆ to the mitochondria. Furthermore, the distribution of exogenous Q₆ among endomembranes suggests an impairment of the membrane traffic at the level of the endocytic pathway. This fact was confirmed after the detection of defects in the incorporation of FM4-64 marker and CPY delivery to the vacuole. A similar effect was demonstrated in double mutant strains in Q₆ synthesis and several steps of endocytic process; those cells are unable to uptake exogenous Q₆ to the mitochondria and restore the growth on non-fermentable carbon sources. Additional data about the positive effect of peptone presence for exogenous Q₆ uptake support the hypothesis that Q₆ is transported to mitochondria through an endocytic-based system.      

3.1262           Determinants of Secretion and Intracellular Localization of Human Herpesvirus 8 Interleukin-6

Chen, D., Choi, Y.B., Sandford, G. and Nicholas, J.

1. Virol., 83(13), 6874-6882 (2009)

  Human herpesvirus 8 (HHV-8) interleukin-6 (vIL-6) is distinct from human and other cellular IL-6 proteins in that it does not require the nonsignaling -receptor subunit for the formation of gp130-based signal transducing complexes and also is largely retained intracellularly rather than being secreted. We and others have reported that vIL-6 is retained and is active in the endoplasmic reticulum (ER) compartment, and data from our laboratory have demonstrated that intracellular vIL-6 is functional in the autocrine promotion of proliferation and survival of HHV-8 latently infected primary effusion lymphoma cells. It has also been reported that vIL-6 secretion in gp130-deficient cells can be enhanced by introduced gp130, thereby implicating the signal transducer in vIL-6 trafficking to the cell surface. We examine here the requirements for intracellular retention and localization of vIL-6. Using vIL-6-hIL-6 chimeric and point-mutated vIL-6 proteins, we identified regions and residues of vIL-6 influencing vIL-6 secretion. However, there was no correlation between vIL-6 secretion and gp130 interaction. We found that vIL-6, but not hIL-6, could associate stably with ER-resident chaperone protein calnexin. Glycosylation-dependent interaction of vIL-6 with calnexin correlated with proper protein folding, but there was no direct relationship between vIL-6-calnexin interaction and intracellular retention. While calnexin depletion had little influence on absolute amounts of secreted vIL-6, it led to markedly reduced levels of intracellular cytokine. This was reversed by gp130 transduction, which had no detectable effect on vIL-6 secretion, but redistributed vIL-6 into ER-distinct locations in calnexin-depleted cells, specifically. Our data reveal that calnexin plays a role in ER localization of vIL-6 and that gp130 promotes ER exit, but not secretion, of the viral cytokine.  

3.1263           Psychosine Accumulates in Membrane Microdomains in the Brain of Krabbe Patients, Disrupting the Raft Architecture

White, A.B., Givogri, M.I:, Lopez-Rosas, A., Cao, H., van Breemen, R., Thinakaran, g. and Bongarzozne, E.R.

1. Neurosci., 29(19), 6068-6077 (2009)

  Lipid rafts (LRs) are membrane realms characterized by high concentrations of cholesterol and sphingolipids. Often, they are portrayed as scaffolds on which many different signaling molecules can assemble their cascades. The idea of rafts as scaffolds is garnering significant attention as the consequences of LR disruption have been shown to be manifest in multiple signaling pathways. In this study, LRs in the brain of the twitcher (TWI) mouse, a bona-fide model for infant variants of human globoid cell leukodystrophy or Krabbe disease, were investigated. This mouse has deficient activity of GALC (β-galactosylceramidase) that leads to a progressive accumulation of some galactosyl-sphingolipids in the brain. We hypothesized that the accumulation of psychosine (galactosyl-sphingosine) in the TWI CNS may result in the disruption of rafts in different cell populations such as neurons and oligodendrocytes, both cellular targets during disease. In this communication, we demonstrate that psychosine specifically accumulates in LRs in the TWI brain and sciatic nerve and in samples from brains of human Krabbe patients. It is also shown that this accumulation is accompanied by an increase in cholesterol in these domains and changes in the distribution of the LR markers flotillin-2 and caveolin-1. Finally, we show evidence that this phenomenon may provide a mechanism by which psychosine can exert its known inhibitory effect on protein kinase C. This study provides a previously undescribed biophysical aspect for the mechanism of pathogenesis in Krabbe disease.  

3.1264           Cholesterol Regulates the Endoplasmic Reticulum Exit of the Major Membrane Protein P0 Required for Peripheral Myelin Compaction

Saher, G., Quintes, S., Möbius, W., Wehr, M.C., Krämer-Albers, E-M., Bråugger, B. and Nave, K-A.

1. Neurosci., 29(19), 6064-6104 (2009)

  Rapid impulse conduction requires electrical insulation of axons by myelin, a cholesterol-rich extension of the glial cell membrane with a characteristic composition of proteins and lipids. Mutations in several myelin protein genes cause endoplasmic reticulum (ER) retention and disease, presumably attributable to failure of misfolded proteins to pass the ER quality control. Because many myelin proteins partition into cholesterol-rich membrane rafts, their interaction with cholesterol could potentially be part of the ER quality control system. Here, we provide in vitro and in vivo evidence that the major peripheral myelin protein P0 requires cholesterol for exiting the ER and reaching the myelin compartment. Cholesterol dependency of P0 trafficking in heterologous cells is mediated by a cholesterol recognition/interaction amino acid consensus (CRAC) motif. Mutant mice lacking cholesterol biosynthesis in Schwann cells suffer from severe hypomyelination with numerous uncompacted myelin stretches. This demonstrates that high-level cholesterol coordinates P0 export with myelin membrane synthesis, which is required for the correct stoichiometry of myelin components and for myelin compaction.  

3.1265           UBXD4, a UBX-Containing Protein, Regulates the Cell Surface Number and Stability of 3-Containing Nicotinic Acetylcholine Receptors

Rezvani, K., teng, Y., Pan, Y., Dani, J.A., Lindstrom, J., Garcia Gras, E.A., McIntosh, J.M. and De Biasi, M.

1. Neurosci., 29(21), 6883-6896 (2009)

  Adaptor proteins are likely to modulate spatially and temporally the trafficking of a number of membrane proteins, including neuronal nicotinic acetylcholine receptors (nAChRs). A yeast two-hybrid screen identified a novel UBX-containing protein, UBXD4, as one of the cytosolic proteins that interact directly with the 3 and 4 nAChR subunits. The function of UBX-containing proteins is largely unknown. Immunoprecipitation and confocal microscopy confirmed the interaction of UBXD4 with 3-containing nAChRs ( 3* nAChRs) expressed in HEK293 cells, PC12 cells, and rat cortical neurons. Overexpression of UBXD4 in differentiated PC12 cells (dPC12) increased nAChR cell surface expression, especially that of the 3β2 subtype. These findings were corroborated by electrophysiology, immunofluorescent staining, and biotinylation of surface receptors. Silencing of UBXD4 led to a significant reduction of 3* nAChRs in rat cortical neurons and dPC12 cells. Biochemical and immunofluorescence studies of endogenous UBXD4 showed that the protein is located in both the ER and cis-Golgi compartments. Our investigations also showed that the 3 subunit is ubiquitinated and that UBXD4 can interfere with its ubiquitination and consequent degradation by the proteasome. Our data suggest that UBXD4 modulates the distribution of 3* nAChRs between specialized intracellular compartments and the plasma membrane. This effect is achieved by controlling the stability of the 3 subunit and, consequently, the number of receptors at the cell surface.  

3.1266           Association of Fc RIIa (CD32a) with Lipid Rafts Regulates Ligand Binding Activity

Bournazos, S., Hart, S.P., Chamberlain, L.H., Glennie, M.J. and Dransfield, I.

1. Immunol., 182, 8026-8036 (2009)

  Binding of Igs to myeloid cells via FcR is a key event in the control of innate and acquired immunity. Fc RIIa (CD32a) is a receptor for multivalent IgG expressed predominantly by myeloid cells, and its association with microdomains rich in cholesterol and sphingolipids, termed as lipid rafts, has been reported to be essential for efficient signaling. However, for many myeloid cell types, ligand binding to CD32a is suppressed by as yet undefined mechanisms. In this study, we have examined the role of CD32a-lipid raft interactions in the regulation of IgG binding to CD32a. Disruption of lipid raft structure following depletion or sequestration of membrane cholesterol greatly inhibited CD32a-mediated IgG binding. Furthermore, specific CD32a mutants, which show reduced association with lipid rafts (A224S and C241A), displayed decreased levels of IgG binding compared with wild-type CD32a. In contrast, constitutively lipid raft-associated CD32a (GPI-anchored CD32a) exhibited increased capacity for IgG binding compared with the full-length transmembrane CD32a. Our findings clearly suggest a major role for lipid rafts in the regulation of IgG binding and, more specifically, that suppression of CD32a-mediated IgG binding in myeloid cells is achieved by receptor exclusion from lipid raft membrane microdomains.  

3.1267           Myosin IIA Associates with NK Cell Lytic Granules to Enable Their Interaction with F-Actin and Function at the Immunological Synapse

Sanborn, K.B., Rak, G.D., maru, S.Y., Demers, K., Difeo, A., Martignetti, J.A., Betts, M.R., Favier, R., Banerjee, P.P. and Orange, J.S.

1. Immunol., 182, 6969-6984 (2009)

  NK cell cytotoxicity requires the formation of an actin-rich immunological synapse (IS) with a target cell and the polarization of perforin-containing lytic granules toward the IS. Following the polarization of lytic granules, they traverse through the actin-rich IS to join the NK cell membrane in order for directed secretion of their contents to occur. We examined the role of myosin IIA as a candidate for facilitating this prefinal step in lytic NK cell IS function. Lytic granules in and derived from a human NK cell line, or ex vivo human NK cells, were constitutively associated with myosin IIA. When isolated using density gradients, myosin IIA-associated NK cell lytic granules directly bound to F-actin and the interaction was sensitive to the presence of ATP under conditions of flow. In NK cells from patients with a truncation mutation in myosin IIA, NK cell cytotoxicity, lytic granule penetration into F-actin at the IS, and interaction of isolated granules with F-actin were all decreased. Similarly, inhibition of myosin function also diminished the penetration of lytic granules into F-actin at the IS, as well as the final approach of lytic granules to and their dynamics at the IS. Thus, NK cell lytic granule-associated myosin IIA enables their interaction with actin and final transit through the actin-rich IS to the synaptic membrane, and can be defective in the context of naturally occurring human myosin IIA mutation.  

3.1268           Nuclear angiotensin II type 2 (AT2) receptors are functionally linked to nitric oxide production

Gwathmey, T.M., Shaltout, H.A., Pendergrass, K.D., Pirro, N.T., Figueroa, J.P., Rose, J.C., Diz, D.I. and Chappell, M.C. Am. J. Physiol. Renal Physiol., 296, F1484-F1493 (2009)   Expression of nuclear angiotensin II type 1 (AT₁) receptors in rat kidney provides further support for the concept of an intracellular renin-angiotensin system. Thus we examined the cellular distribution of renal ANG II receptors in sheep to determine the existence and functional roles of intracellular ANG receptors in higher order species. Receptor binding was performed using the nonselective ANG II antagonist ¹²⁵I-[Sar¹,Thr⁸]-ANG II (¹²⁵I-sarthran) with the AT₁ antagonist losartan (LOS) or the AT₂ antagonist PD123319 (PD) in isolated nuclei (NUC) and plasma membrane (PM) fractions obtained by differential centrifugation or density gradient separation. In both fetal and adult sheep kidney, PD competed for the majority of cortical NUC ( 70%) and PM ( 80%) sites while LOS competition predominated in medullary NUC ( 75%) and PM ( 70%). Immunodetection with an AT₂ antibody revealed a single 42-kDa band in both NUC and PM extracts, suggesting a mature molecular form of the NUC receptor. Autoradiography for receptor subtypes localized AT₂ in the tubulointerstitium, AT₁ in the medulla and vasa recta, and both AT₁ and AT₂ in glomeruli. Loading of NUC with the fluorescent nitric oxide (NO) detector DAF showed increased NO production with ANG II (1 nM), which was abolished by PD and N-nitro-L-arginine methyl ester, but not LOS. Our studies demonstrate ANG II receptor subtypes are differentially expressed in ovine kidney, while nuclear AT₂ receptors are functionally linked to NO production. These findings provide further evidence of a functional intracellular renin-angiotensin system within the kidney, which may represent a therapeutic target for the regulation of blood pressure.  

3.1269           Caveolin-1 directly interacts with UT-A1 urea transporter: the role of caveolae/lipid rafts in UT-A1 regulation at the cell membrane

Feng, X., Huang, H., Yang, Y., Fröhlich, O., Klein, J.D., Sands, J.M. and Chen, G. Am. J. Physiol. Renala Physiol., 296, F1514-F1520 (2009)   The cell plasma membrane contains specialized microdomains called lipid rafts which contain high amounts of sphingolipids and cholesterol. Lipid rafts are involved in a number of membrane protein functions. The urea transporter UT-A1, located in the kidney inner medullary collecting duct (IMCD), is important for urine concentrating ability. In this study, we investigated the possible role of lipid rafts in UT-A1 membrane regulation. Using sucrose gradient cell fractionation, we demonstrated that UT-A1 is concentrated in the caveolae-rich fraction both in stably expressing UT-A1 HEK293 cells and in freshly isolated kidney IMCD suspensions. In these gradients, UT-A1 at the cell plasma membrane is codistributed with caveolin-1, a major component of caveolae. The colocalization of UT-A1 in lipid rafts/caveolae was further confirmed in isolated caveolae from UT-A1-HEK293 cells. The direct association of UT-A1 and caveolin-1 was identified by immunoprecipitation and GST pull-down assay. Examination of internalized UT-A1 in pEGFP-UT-A1 transfected HEK293 cells fluorescent overlap with labeled cholera toxin subunit B, a marker of the caveolae-mediated endocytosis pathway. Disruption of lipid rafts by methyl-β-cyclodextrin or knocking down caveolin-1 by small-interference RNA resulted in UT-A1 cell membrane accumulation. Functionally, overexpression of caveolin-1 in oocytes decreased UT-A1 urea transport activity and UT-A1 cell surface expression. Our results indicate that lipid rafts/caveolae participate in UT-A1 membrane regulation and this effect is mediated via a direct interaction of caveolin-1 with UT-A1.  

3.1270           The Organelle Proteome of the DT40 Lymphocyte Cell Line

Hall, S.L., Hester, S., Griffin, J.L., Lilley, K.S. and jackson, A.P. Mol. Cell. Proteomics, 8, 1295-1305 (2009)   A major challenge in eukaryotic cell biology is to understand the roles of individual proteins and the subcellular compartments in which they reside. Here, we use the localization of organelle proteins by isotope tagging technique to complete the first proteomic analysis of the major organelles of the DT40 lymphocyte cell line. This cell line is emerging as an important research tool because of the ease with which gene knockouts can be generated. We identify 1090 proteins through the analysis of preparations enriched for integral membrane or soluble and peripherally associated proteins and localize 223 proteins to the endoplasmic reticulum, Golgi, lysosome, mitochondrion, or plasma membrane by matching their density gradient distributions to those of known organelle residents. A striking finding is that within the secretory and endocytic pathway a high proportion of proteins are not uniquely localized to a single organelle, emphasizing the dynamic steady-state nature of intracellular compartments in eukaryotic cells.  

3.1271           Saccharomyces cerevisiae Na+/H+ Antiporter Nha1p Associates with Lipid Rafts and Requires Sphingolipid for Stable Localization to the Plasma Membrane

Mitsui, K., Hatakeyama, K., Matsushita, M. and Kanazawa, H.

1. Biochem., 145(6), 709-720 (2009)

  The plasma membrane-type Na⁺/H⁺ antiporter Nha1p from budding yeast plays an important role in intracellular Na⁺ and pH homeostasis by mediating the exchange of Na⁺ for H⁺ across the plasma membrane. However, the mechanism of intracellular targeting of Nha1p to the plasma membrane remains unknown. Here, we found that Nha1p exists predominantly in detergent-resistant membrane fractions (DRMs) following density gradient centrifugation. When ergosterol was extracted from membranes, Nha1p was transferred to a detergent-soluble fraction, suggesting that Nha1p associates with ergosterol-containing DRMs, also known as lipid rafts. Density gradient centrifugation of cell extracts of yeast mutants that were defective in different stages of the secretory pathway revealed that, unlike previously identified raft proteins, the association of Nha1p with DRMs occurs mainly at the plasma membrane. In lcb1-100 cells, which are temperature-sensitive for sphingolipid synthesis, newly synthesized Nha1p failed to localize to the plasma membrane at the non-permissive temperature. Rather, Nha1p was distributed in an intracellular punctate pattern. The addition of phytosphingosine or the inhibition of endocytosis in lcb1-100 cells restored the targeting of Nha1p to the plasma membrane. The results of the current study suggest that sphingolipids are required for the stable localization of Nha1p to the plasma membrane.  

3.1272           A genome-wide screen for genes affecting eisosomes reveals Nce102 function in sphingolipid signaling

Frölich, F., Moreira, K., Aguilar, P.S., Hubner, N.C., Mann, M., Walter, P. and Walther, T.C.

1. Cell Biol., 185(7), 1227-1242 (2009)

  The protein and lipid composition of eukaryotic plasma membranes is highly dynamic and regulated according to need. The sphingolipid-responsive Pkh kinases are candidates for mediating parts of this regulation, as they affect a diverse set of plasma membrane functions, such as cortical actin patch organization, efficient endocytosis, and eisosome assembly. Eisosomes are large protein complexes underlying the plasma membrane and help to sort a group of membrane proteins into distinct domains. In this study, we identify Nce102 in a genome-wide screen for genes involved in eisosome organization and Pkh kinase signaling. Nce102 accumulates in membrane domains at eisosomes where Pkh kinases also localize. The relative abundance of Nce102 in these domains compared with the rest of the plasma membrane is dynamically regulated by sphingolipids. Furthermore, Nce102 inhibits Pkh kinase signaling and is required for plasma membrane organization. Therefore, Nce102 might act as a sensor of sphingolipids that regulates plasma membrane function.

3.1273           Alterations of Mitochondrial Enzymes Contribute to Cardiac Hypertrophy before Hypertension Development in Spontaneously Hypertensive Rats

Meng, C., Jin, X., Xia, L., Shen, S-M., Wang, X-L., Cai, J., Chen, G-Q., Wang, L-S., and Fang, N-Y.

1. Proteome Res., 8, 2463-2475 (2009)

  Mitochondrial dysfunction is recently thought to be tightly associated with the development of cardiac hypertrophy as well as hypertension. However, the detailed molecular events in mitochondria at early stages of hypertrophic pathogenesis are still unclear. Applying two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) combined with MALDI-TOF/TOF tandem mass spectrometry, here we identified the changed mitochondrial proteins of left ventricular mitochondria in prehypertensive/hypertensive stages of cardiac hypertrophy through comparing spontaneously hypertensive rats (SHR) and the age-matched normotensive Wistar Kyoto (WKY) rats. The results revealed that in the hypertrophic left ventricle of SHR as early as 4 weeks old with normal blood pressure, 33 mitochondrial protein spots presented significant alterations, with 17 down-regulated and 16 up-regulated. Such alterations were much greater than those in 20-week-old SHR with elevated blood pressure. Of the total alterations, the expression of two mitochondrial enzymes, trifunctional enzyme alpha subunit (Hadha) and NADH dehydrogenase 1 alpha subcomplex 10 (Ndufa10), were found to have special expression modification patterns in SHR strain. These data would provide new clues to investigate the potential contribution of mitochondrial dysfunction to the development of cardiac hypertrophy.  

3.1274           The enterocyte microvillus is a vesicle-generating organelle

McConnell, R.E., Higginbotham, J.N., Shifrin, D.A., Tabb, D.L., Coffey, R.J. and Tyska, M.J.

1. Cell Biol., 185(7), 1285-1298 (2009)

  For decades, enterocyte brush border microvilli have been viewedas passive cytoskeletal scaffolds that serve to increase apicalmembrane surface area. However, recent studies revealed thatin the in vitro context of isolated brush borders, myosin-1a(myo1a) powers the sliding of microvillar membrane along coreactin bundles. This activity also leads to the shedding of smallvesicles from microvillar tips, suggesting that microvilli mayfunction as vesicle-generating organelles in vivo. In this study,we present data in support of this hypothesis, showing thatenterocyte microvilli release unilamellar vesicles into theintestinal lumen; these vesicles retain the right side out orientationof microvillar membrane, contain catalytically active brushborder enzymes, and are specifically enriched in intestinalalkaline phosphatase. Moreover, myo1a knockout mice demonstratestriking perturbations in vesicle production, clearly implicatingthis motor in the in vivo regulation of this novel activity.In combination, these data show that microvilli function asvesicle-generating organelles, which enable enterocytes to deploycatalytic activities into the intestinal lumen.  

3.1275            High-Resolution Fractionation of Signaling Endosomes Containing Different Receptors

McCaffrey, G., Welker, J., Scott, J., van der Salm, L. and Grimes, M.L. Traffic, 10(7), 938-950 (2009)   Receptor endocytosis is regulated by ligand binding, and receptors may signal after endocytosis in signaling endosomes. We hypothesized that signaling endosomes containing different types of receptors may be distinct from one another and have different physical characteristics. To test this hypothesis, we developed a high-resolution organelle fractionation method based on mass and density, optimized to resolve endosomes from other organelles. Three different types of receptors undergoing ligand-induced endocytosis were localized predominately in endosomes that were resolved from one another using this method. Endosomes containing activated receptor tyrosine kinases (RTKs), TrkA and EGFR, were similar to one another. Endosomes containing p75^NTR (in the tumor necrosis receptor superfamily) and PAC1 (a G-protein-coupled receptor) were distinct from each other and from RTK endosomes. Receptor-specific endosomes may direct the intracellular location and duration of signal transduction pathways to dictate response to signals and determine cell fate.  

3.1276           Applications of proteomics in the study of inflammatory bowel diseases: Current status and future directions with available technologies

Alex, P., Gucek, M. and Li, X. Inflamm. Bowel Dis., 15(4). 616-629 (2009)   Inflammatory bowel diseases (IBD) are chronic, heterogeneous, and multifactorial intestinal inflammatory disorders. Major challenges in IBD research include identification of major pathogenic alterations of genes/proteins as well as effective biomarkers for early diagnosis, prognosis, and prediction of therapeutic response. Since proteins govern cellular structure and biological function, a wide selection of proteomic approaches enables effective characterization of IBD pathogenesis by investigating the dynamic nature of protein expression, cellular and subcellular distribution, posttranslational modifications, and interactions at both the cellular and subcellular levels. The aims of this review are to 1) highlight the current status of proteomic studies of IBD, and 2) introduce the available and emerging proteomic technologies that have potential applications in the study of IBD. These technologies include various mass spectrometry technologies, quantitative proteomics (2D-PAGE, ICAT, SILAC, iTRAQ), protein/antibody arrays, and multi-epitope-ligand cartography. This review also presents information and methodologies, from sample selection and enrichment to protein identification, that are not only essential but also particularly relevant to IBD research. The potential future application of these technologies is expected to have a significant impact on the discovery of novel biomarkers and key pathogenic factors for IBD.    

3.1277           A Single Conserved Leucine Residue on the First Intracellular Loop Regulates ER Export of G Protein-Coupled Receptors

Duvernay, M.T., Dong, C., Zhang, X., Robitaille, M., Hebert, T.E. and Wu, G. Traffic, 10, 552-566 (2009)   The intrinsic structural determinants for export trafficking of G protein-coupled receptors (GPCRs) have been mainly identified in the termini of the receptors. In this report, we determined the role of the first intracellular loop (ICL1) in the transport from the endoplasmic reticulum (ER) to the cell surface of GPCRs. The [alpha]2B-adrenergic receptor (AR) mutant lacking the ICL1 is unable to traffic to the cell surface and to initiate signaling measured as ERK1/2 activation. Mutagenesis studies identify a single Leu48 residue in the ICL1 modulates [alpha]2B-AR export from the ER. The ER export function of the Leu48 residue can be substituted by Phe, but not Ile, Val, Tyr and Trp, and is unlikely involved in correct folding or dimerization of [alpha]2B-AR in the ER. Importantly, the isolated Leu residue is remarkably conserved in the center of the ICL1s among the family A GPCRs and is also required for the export to the cell surface of [beta]2-AR, [alpha]1B-AR and angiotensin II type 1 receptor. These data indicate a crucial role for a single Leu residue within the ICL1 in ER export of GPCRs.  

3.1278           Cytoplasmic prion protein induces forebrain neurotoxicity

Wang, X., Bowers, S.L., Wang, F., Pu, X-a., Nelson, R.J. and Ma, J. Biochim. Biophys. Acta, 1792, 555-563 (82009)   The prion protein (PrP) is essential for the pathogenesis of prion disease. PrP has been detected in the cytosol of neurons and transgenic mice expressing PrP in the cytosol (cyPrP) under a pan-neuronal promoter developed rapid cerebellar granule neuron degeneration. Yet, it remains unclear whether cyPrP is capable to cause toxicity in other neuronal populations. Here, we report that transgenic mice expressing cyPrP in the forebrain neurons developed behavioral abnormalities including clasping and hyperactivity. These mice had reduced thickness in cortex and developed astrogliosis in hippocampal and cortical regions. Moreover, cyPrP in these mice was recognized by the A11 anti-oligomer antibody and was associated with the hydrophobic lipid core of membranes, indicating that cyPrP oligomer caused membrane perturbation contributes to cyPrP neurotoxicity. Together, our results clearly revealed that cyPrP is able to cause toxicity in different neuronal populations, supporting a role of cyPrP in PrP-mediated neurodegenerative disorders.  

3.1279           Interactions of DNA with Biofilm-Derived Membrane Vesicles

Schooling, S.R., Hubley, A. and Beveridge, T.J.

1. Bacteriol., 191(13), 4097-4102 (2009)

  The biofilm matrix contributes to the chemistry, structure,and function of biofilms. Biofilm-derived membrane vesicles(MVs) and DNA, both matrix components, demonstrated concentration-,pH-, and cation-dependent interactions. Furthermore, MV-DNAassociation influenced MV surface properties. This bears consequencesfor the reactivity and availability for interaction of matrixpolymers and other constituents.  

3.1280           Derlin-dependent accumulation of integral membrane proteins at cell surfaces

Schaheen, B., Dang, H. and Fares, H.

1. Cell Sci., 122, 2228-2239 (2009)

  Quality-control mechanisms of protein folding of transmembrane and secreted proteins is mediated by endoplasmic-reticulum-associated degradation (ERAD), which is used to detect and to degrade misfolded proteins in the ER. The ERAD machinery consists of chaperones, transmembrane proteins and ubiquitin-associated enzymes that detect, modify, and retro-translocate the misfolded proteins to the cytoplasm for degradation by the proteasome. In contrast to ERAD, little is known about the fates of integral membrane and secreted proteins that become misfolded at the plasma membrane or in the extracellular space. Derlin proteins are a family of proteins that are conserved in all eukaryotes, where they function in ERAD. Here, we show that loss of Derlin function in Caenorhabditis elegans and in mouse macrophages results in the accumulation of integral membrane proteins at the plasma membrane. Induction of LDL receptor misfolding at the plasma membrane results in a sharp decrease in its half-life, which can be rescued by proteasomal inhibitors or by reduction of Derlin-1 levels. We also show that Derlin proteins localize to endosomes as well as to the ER. Our data are consistent with a model where Derlin proteins function in a spatially segregated quality control pathway that is used for the recognition and degradation of transmembrane proteins that become misfolded at the plasma membrane and/or in endosomes.  

3.1281           Functional implications of the influence of ABCA1 on lipid microenvironment at the plasma membrane: a biophysical study

Zarubica, A., Plazzo, A.P., Stöckl, M., Trombik, T., Hamon, Y., Müller, P., pomorski, T., Herrmann, A. and Chimini, G. FASEB J., 23, 1775-1785 (2009)   The ABCA1 transporter orchestrates cellular lipid homeostasis by promoting the release of cholesterol to plasmatic acceptors. The molecular mechanism is, however, unknown. We report here on the biophysical analysis in living HeLa cells of the ABCA1 lipid microenvironment at the plasma membrane. The modifications of membrane attributes induced by ABCA1 were assessed at both the outer and inner leaflet by monitoring either the lifetime of membrane inserted fluorescent lipid analogues by fluorescence lifetime imaging microscopy (FLIM) or, respectively, the membrane translocation of cationic sensors. Analysis of the partitioning of dedicated probes in plasma membrane blebs vesiculated from these cells allowed visualization of ABCA1 partitioning into the liquid disordered-like phase and corroborated the idea that ABCA1 destabilizes the lipid arrangement at the membrane. Specificity was demonstrated by comparison with cells expressing an inactive transporter. The physiological relevance of these modifications was finally demonstrated by the reduced membrane mobility and function of transferrin receptors under the influence of an active ABCA1. Collectively, these data assess that the control of both transversal and lateral lipid distribution at the membrane is the primary function of ABCA1 and positions the effluxes of cholesterol from cell membranes downstream to the redistribution of the sterol into readily extractable membrane pools.  

3.1282           In vitro import of peroxisome-targeting signal type 2 (PTS2) receptor Pex7p into peroxisomes

Miyata, N., Hosoi, K-i., Mukai, S. and Fujiki, Y. Biochim. Biophys. Acta, 1793, 860-870 (2009)   Pex7p, the peroxisome-targeting signal type 2 (PTS2) receptor, transports PTS2 proteins to peroxisomes from the cytosol. We here established a cell-free Pex7p translocation system. In assays using post-nuclear supernatant fractions each from wild-type CHO-K1 and pex7 ZPG207 cells, ³⁵S-labeled Pex7p was imported into peroxisomes. ³⁵S-Pex7p import was also evident using rat liver peroxisomes. ³⁵S-Pex7p was not imported into peroxisomal remnants from a pex5 ZPG231 defective in PTS2 import and pex2 Z65. When the import of ³⁵S-Pex5pL was inhibited with an excess amount of recombinant Pex5pS, ³⁵S-Pex7p import was concomitantly abrogated, suggesting that Pex5pL was a transporter for Pex7p, unlike a yeast cochaperone, Pex18p. ³⁵S-Pex7p as well as ³⁵S-Pex5p was imported in an ATP-independent manner, whilst the import of PTS1 and PTS2 cargo-proteins was ATP-dependent. Thereby, ATP-independent import of Pex7p implicated that Pex5p export requiring ATP hydrolysis is not a limiting step for its cargo recruitment to peroxisomes. PTS1 protein import was indeed insensitive to N-ethylmaleimide, whereas Pex5p export was N-ethylmaleimide-sensitive. Taken together, the cargo-protein translocation through peroxisomal membrane more likely involves another ATP-requiring step in addition to the Pex5p export. Moreover, upon concurrent import into peroxisomes, ³⁵S-Pex5pL and ³⁵S-Pex7p were detected at mutually distinct ratios in the immunoprecipitates each of the import machinery peroxins including Pex14p, Pex13p, and Pex2p, hence suggesting that Pex7p as well as Pex5p translocated from the initial docking complex to RING complex on peroxisomes.  

3.1283           Sphingosine Facilitates SNARE Complex Assembly and Activates Synaptic Vesicle Exocytosis

Darios, F. et al Neuron, 62, 683-694 (2009)   Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication. The membrane fusion process is mediated by a conserved set of SNARE proteins: vesicular synaptobrevin and plasma membrane syntaxin and SNAP-25. Recent data suggest that the fusion process may be subject to regulation by local lipid metabolism. Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin. We show that sphingosine, a releasable backbone of sphingolipids, activates synaptobrevin in synaptic vesicles to form the SNARE complex implicated in membrane fusion. Consistent with the role of synaptobrevin in vesicle fusion, sphingosine upregulated exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and hippocampal neurons, but not in neurons obtained from synaptobrevin-2 knockout mice. Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator.  

3.1284           Taking the Scenic Route: Biosynthetic Traffic to the Plasma Membrane in Polarized Epithelial Cells 

Fölsch, H., Mattila, P.E. and Weisz, O.A. Traffic, 10(8), 972-981 (2009)   The maintenance of epithelial cell function requires the establishment and continuous renewal of differentiated apical and basolateral plasma membrane domains with distinct lipid and protein compositions. Newly synthesized proteins destined for either surface domain are processed along the biosynthetic pathway and segregated into distinct subsets of transport carriers emanating from the trans-Golgi network. Recent studies have illuminated additional complexities in the subsequent delivery of these proteins to the cell surface. In particular, multiple routes to the apical and basolateral cell surfaces have been uncovered, and many of these involve indirect passage through endocytic compartments. This review summarizes our current understanding of these routes and discusses open issues that remain to be clarified.  

3.1285           SHMT1 and SHMT2 Are Functionally Redundant in Nuclear De novo Thymidylate Biosynthesis

Anderson, D.D. and Stover, P.J. PloSOne, 4(6), e5839 (2009)   The three enzymes that constitute the de novo thymidylate synthesis pathway in mammals, cytoplasmic serine hydroxymethyltransferase (SHMT1), thymidylate synthase (TYMS) and dihydrofolate reductase (DHFR) undergo sumoylation and nuclear import during S-phase. In this study, we demonstrate that purified intact mouse liver nuclei convert dUMP to dTMP in the presence of NADPH and serine. Neither nuclear extracts nor intact nuclei exposed to aminomethylphosphonate, a SHMT inhibitor, exhibit thymidylate synthesis activity. Nuclei isolated from Shmt1^−/− mouse livers retained 25% of thymidylate synthesis activity exhibited by nuclei isolated from wild type mice. This residual activity was due to the presence of a cytoplasmic/nuclear isozyme of SHMT encoded by Shmt2. Shmt2 is shown to encode two transcripts, one which encodes a protein that localizes exclusively to the mitochondria (SHMT2), and a second transcript that lacks exon 1 and encodes a protein that localizes to the cytoplasm and nucleus during S-phase (SHMT2α). The ability of Shmt2 to encode a cytoplasmic isozyme of SHMT may account for the viability of Shmt1^−/− mice and provide redundancy that permitted the expansion of the human SHMT1 L474F polymorphism that impairs SHMT1 sumoylation and nuclear translocation.  

3.1286           Lipid Rafts and Clathrin Cooperate in the Internalization of PrPC in Epithelial FRT Cells

Sernataro, D., Caputo, A., Casanova, P., Puri, C., Paladino, S., Tovodar, S.S., Campana, V., Tacchetti, C. and Zurzolo, C. PloSOne, 4(6), e5829 (2009)   Background The cellular prion protein (PrPC) plays a key role in the pathogenesis of Transmissible Spongiform Encephalopathies in which the protein undergoes post-translational conversion to the infectious form (PrPSc). Although endocytosis appears to be required for this conversion, the mechanism of PrPC internalization is still debated, as caveolae/raft- and clathrin-dependent processes have all been reported to be involved. Methodology/Principal Findings We have investigated the mechanism of PrPC endocytosis in Fischer Rat Thyroid (FRT) cells, which lack caveolin-1 (cav-1) and caveolae, and in FRT/cav-1 cells which form functional caveolae. We show that PrPC internalization requires activated Cdc-42 and is sensitive to cholesterol depletion but not to cav-1 expression suggesting a role for rafts but not for caveolae in PrPC endocytosis. PrPC internalization is also affected by knock down of clathrin and by the expression of dominant negative Eps15 and Dynamin 2 mutants, indicating the involvement of a clathrin-dependent pathway. Notably, PrPC co-immunoprecipitates with clathrin and remains associated with detergent-insoluble microdomains during internalization thus indicating that PrPC can enter the cell via multiple pathways and that rafts and clathrin cooperate in its internalization. Conclusions/Significance These findings are of particular interest if we consider that the internalization route/s undertaken by PrPC can be crucial for the ability of different prion strains to infect and to replicate in different cell lines.  

3.1287           In Vivo Generation of Neurotoxic Prion Protein: Role for Hsp70 in Accumulation of Misfolded Isoforms

Fernandez-Funez, P., Casas-Tinto, S., Zhang, Y., Gomez-Velazquez, M., Morales-Garza, M.A., Cepeda-Nieto, A.C., Castilla, J., Soto, C., Rincon-Limas, D.E. PloSGenetics, 5(6), e1000507 (2009)   Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrP^C) converts into a misfolded isoform (PrP^Sc) with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrP^C; in older flies, PrP misfolds, acquires biochemical and structural properties of PrP^Sc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrP^Sc-specific conformational epitopes. In contrast to PrP^Sc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrP^Sc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrP^Sc-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrP^Sc is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover the potential therapeutic role of Hsp70 in treating these devastating disorders.  

3.1288           A novel sorting strategy of trichosanthin for hijacking human immunodeficiency virus type 1

Zhao, W-L., Zhang, F., Feng, D., Shan, J.W., Chen, S. and Sui, S-F. Biochem. Biophys. Res. Comm.,384, 347-351 (2009)   Trichosanthin (TCS) is a type I ribosome-inactivating protein that plays dual role of plant toxin and anti-viral peptide. The sorting mechanism of such an exogenous protein is in long pursuit. Here, we examined TCS trafficking in cells expressing the HIV-1 scaffold protein Gag, and we found that TCS preferentially targets the Gag budding sites at plasma membrane or late endosomes depending on cell types. Lipid raft membrane but not the Gag protein mediates the association of TCS with viral components. After Gag budding, TCS is then released in association with the virus-like particles to generate TCS-enriched virions. The resulting TCS-enriched HIV-1 exhibits severely impaired infectivity. Overall, the observations indicate the existence of a unique and elaborate sorting strategy for hijacking HIV-1.  

3.1289           Vesicle-mediated secretion of human eosinophil granule-derived major basic protein

Melo, R.C.N., Spencer, L.A., Perez, S.A.C., Neves, J.S., Bafford, S.P., Morgan, E.S., Dvorak, A.M. and Weller, P.F. Lab. Invest., 89, 769-781 (2009)   Major basic protein (MBP), the predominant cationic protein of human eosinophil specific granules, is stored within crystalloid cores of these granules. Secretion of MBP contributes to the immunopathogenesis of varied diseases. Prior electron microscopy (EM) of eosinophils in sites of inflammation noted losses of granule cores in the absence of granule exocytosis and suggested that eosinophil granule proteins might be released through piecemeal degranulation (PMD), a secretory process mediated by transport vesicles. Because release of eosinophil granule-derived MBP through PMD has not been studied, we evaluated secretion of this cationic protein by human eosinophils. Intracellular localizations of MBP were studied within nonstimulated and eotaxin-stimulated human eosinophils by both immunofluorescence and a pre-embedding immunonanogold EM method that enables optimal epitope preservation and antigen access to membrane microdomains. In parallel, quantification of transport vesicles was assessed in eosinophils from a patient with hypereosinophilic syndrome (HES). Our data demonstrate vesicular trafficking of MBP within eotaxin-stimulated eosinophils. Vesicular compartments, previously implicated in transport from granules to the plasma membrane, including large vesiculotubular carriers termed eosinophil sombrero vesicles (EoSVs), were found to contain MBP. These secretory compartments were significantly increased in numbers within HES eosinophils. Moreover, in addition to granule-stored MBP, even unstimulated eosinophils contained appreciable amounts of MBP within secretory vesicles, as evidenced by immunonanogold EM and immunofluorescent colocalizations of MBP and CD63. These data suggest that eosinophil MBP, with its multiple extracellular activities, can be mobilized from granules by PMD into secretory vesicles and both granule- and secretory vesicle-stored pools of MBP are available for agonist-elicited secretion of MBP from human eosinophils. The recognition of PMD as a secretory process to release MBP is important to understand the pathological basis of allergic and other eosinophil-associated inflammatory diseases.  

3.1290           Reversible Phosphorylation as a Molecular Switch to Regulate Plasma Membrane Targeting of Acylated SH4 Domain Proteins

Tournaviti, S., San Pietro, E., Terjung, S., Schafmeier, T., Wegehingel, S., Ritzerfeld, J., Schulz, J., Smith, D.F., Pepperkok, R. and Nickel, W. Traffic, 10(8), 1047-1060 (2009)   Acylated SH4 domains represent N-terminal targeting signals that anchor peripheral membrane proteins such as Src kinases in the inner leaflet of plasma membranes. Here we provide evidence for a novel regulatory mechanism that may control the levels of SH4 proteins being associated with plasma membranes. Using a fusion protein of the SH4 domain of Leishmania HASPB and GFP as a model system, we demonstrate that threonine 6 is a substrate for phosphorylation. Substitution of threonine 6 by glutamate (to mimic a phosphothreonine residue) resulted in a dramatic redistribution from plasma membranes to intracellular sites with a particular accumulation in a perinuclear region. As shown by both pharmacological inhibition and RNAi-mediated down-regulation of the threonine/ serine-specific phosphatases PP1 and PP2A, recycling back to the plasma membrane required dephosphorylation of threonine 6. We provide evidence that a cycle of phosphorylation and dephosphorylation may also be involved in intracellular targeting of other SH4 proteins such as the Src kinase Yes.  

3.1291           Occludin oligomeric assemblies at tight junctions of the blood–brain barrier are altered by hypoxia and reoxygenation stress

McCaffrey, G., Willis, C.L., Staatz, W.D., Nametz, N., Quigley, C.A., Hom, S., Lochhead, J.J. and Davis, T.P.

1. Neurochem., 110(1), 58-71 (2009)

  Hypoxic (low oxygen) and reperfusion (post-hypoxic reoxygenation) phases of stroke promote an increase in microvascular permeability at tight junctions (TJs) of the blood–brain barrier (BBB) that may lead to cerebral edema. To investigate the effect of hypoxia (Hx) and reoxygenation on oligomeric assemblies of the transmembrane TJ protein occludin, rats were subjected to either normoxia (Nx, 21% O₂, 60 min), Hx (6% O₂, 60 min), or hypoxia/reoxygenation (H/R, 6% O₂, 60 min followed by 21% O₂, 10 min). After treatment, cerebral microvessels were isolated, fractionated by detergent-free density gradient centrifugation, and occludin oligomeric assemblies associated with plasma membrane lipid rafts were solubilized by perfluoro-octanoic acid (PFO) exclusively as high molecular weight protein complexes. Analysis by non-reducing and reducing sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis/western blot of PFO-solubilized occludin revealed that occludin oligomeric assemblies co-localizing with 'TJ-associated' raft domains contained a high molecular weight 'structural core' that was resistant to disassembly by either SDS or a hydrophilic reducing agent ex vivo, and by Hx and H/R conditions in vivo. However, exposure of PFO-solubilized occludin oligomeric assemblies to SDS ex vivo revealed the non-covalent association of a significant amount of dimeric and monomeric occludin isoforms to the disulfide-bonded inner core, and dispersal of these non-covalently attached occludin subunits to lipid rafts of higher density in vivo was differentially promoted by Hx and H/R. Our data suggest a model of isoform interaction within occludin oligomeric assemblies at the BBB that enables occludin to simultaneously perform a structural role in inhibiting paracellular diffusion, and a signaling role involving interactions of dimeric and monomeric occludin isoforms with a variety of regulatory molecules within different plasma membrane lipid raft domains.  

3.1292           Molecular and reverse genetic characterization of NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) genes unravels their function in transcription and nucleotide excision repair in Arabidopsis thaliana

Liu, Z., Zhu, Y., Gao, J., Yu, F., Dong, A. and Shen, W-H. Plant J., 59(1), 27-38 ( 2009)   Compared with the well-studied biochemical function of NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) as a histone chaperone in nucleosome assembly/disassembly, the physiological roles of NAP1 remain largely uncharacterized. Here, we define the NAP1 gene family members in Arabidopsis, examine their molecular properties, and use reverse genetics to characterize their biological roles. We show that the four AtNAP1-group proteins can form homodimers and heterodimers, can bind histone H2A, and are localized abundantly in the cytoplasm and weakly in the nucleus at steady state. AtNAP1;4 differs from the others by showing inhibitor-sensitive nucleocytoplasmic shuttling and tissue-specific expression, restricted to root segments and pollen grains. The other three AtNAP1 genes are ubiquitously expressed in plants and the AtNAP1;3 protein is detected as the major isoform in seedlings. We show that disruption of the AtNAP1-group genes does not affect normal plant growth under our laboratory conditions. Interestingly, two allelic triple mutants, Atnap1;1-1 Atnap1;2-1 Atnap1;3-1 and Atnap1;1-1 Atnap1;2-1 Atnap1;3-2, exhibit perturbed genome transcription, and show hypersensitivity to DNA damage caused by UV-C irradiation. We show that AtNAP1;3 binds chromatin, with enrichment at some genes involved in the nucleotide excision repair (NER) pathway, and that the expression of these genes is downregulated in the triple mutants. Taken together, our results highlight conserved and isoform-specific properties of AtNAP1 proteins, and unravel their function in the NER pathway of DNA damage repair.  

3.1293           Repeated Cocaine Administration Decreases 5-HT_2A Receptor-Mediated Serotonergic Enhancement of Synaptic Activity in Rat Medial Prefrontal Cortex

Huang, C-C., Liang, Y-C., Lee, C-C., Wu, M-Y. and Hsu, K-S. Neuropsychopharmacol., 34, 1979-1992 (2009)   Neural adaptations in the medial prefrontal cortex (mPFC) are thought to be crucial in the development and maintenance of addictive behaviors. The mPFC receives a dense serotonergic (5-hydroxytryptamine, 5-HT) innervation from raphe nuclei and 5-HT exerts complex actions on mPFC pyramidal neurons. The present study, using a rat model of behavioral sensitization to cocaine, was designed to determine whether repeated cocaine exposure in vivo is capable of altering 5-HT-induced regulation of glutamatergic transmission in the mPFC. In layer V pyramidal neurons of the mPFC, application of 5-HT, through activation of 5-HT_2A receptors, induced a massive enhancement of spontaneous excitatory postsynaptic currents (sEPSCs). Repeated cocaine administration for 5 days resulted in an attenuation in the ability of 5-HT to enhance sEPSCs. This effect was prevented when cocaine was co-administered with the selective 5-HT_2A receptor antagonist ketanserin and was mimicked by repeated 5-HT_2A receptor agonist (-)4-iodo-2,5-dimethoxyphenylisopropylamine administration. Repeated cocaine administration is not associated with any changes in the levels of 5-HT_2A receptors or regulator of GTP-binding protein signaling 4. These results suggest that cocaine-induced inhibition of 5-HT_2A receptor-mediated enhancement of glutamatergic transmission in the mPFC may be caused, at least in part, by the impairment of coupling of 5-HT_2A receptors with GTP-binding proteins during cocaine withdrawal. These alterations in 5-HT_2A receptor responsiveness in the mPFC may be relevant to the development of behavioral sensitization and withdrawal effects following repeated cocaine administration.  

3.1294           Caveolin-1-dependent and -independent membrane domains

Lay, S.L., Li, Q., Proschogo, N., Rodriguez, M., Gunaratnam, K., Cartland, S., Rentero, C., Jessup, W., Mitchell, T. and Gaus, K.

1. Lipid Res., 50, 1609-1620 (2009)

  Lipid rafts defined as cholesterol- and sphingomyelin-rich domains have been isolated from different cell types that vary greatly in their lipid profiles. Here, we investigated the contribution of the structural protein caveolin-1 (Cav1) to the overall lipid composition and domain abundance in mouse embryonic fibroblasts (MEFs) from wild-type (WT) or Cav1-deficient (Cav1^–/–) animals. Our findings show that Cav1 expression had no effect on free (membrane-associated) cholesterol levels. However, Cav1^–/–-deficient cells did have a higher proportion of sphingomyelin, decreased abundance of unsaturated phospholipids, and a trend toward shorter fatty acid chains in phosphatidylcholine. We isolated detergent-resistant membranes (DRMs), nondetergent raft domains (NDR), and cholesterol oxidase (CO)-sensitive domains and assessed the abundance of ordered domains in intact cells using the fluorescent dye Laurdan. Despite differences in phospholipid composition, we found that cholesterol levels in DRMs, NDR, and CO-sensitive domains were similar in both cell types. The data suggest that Cav1 is not required to target cholesterol to lipid rafts and that CO does not specifically oxidize caveolar cholesterol. In contrast, the abundance of ordered domains in adherent cells is reduced in Cav1^–/– compared with WT MEFs, suggesting that cell architecture is critical in maintaining Cav1-induced lipid rafts.  

3.1295           Human Dna2 Is a Nuclear and Mitochondrial DNA Maintenance Protein

Duxin, J.P., Dao, B., Martinsson, P., Rajala, N., Guittat, L., Campbell, J.L., Spelbrink, J.N. and Stewart, S.A. Mol. Cell. Biol., 29(15), 4274-4282 (2009)   Dna2 is a highly conserved helicase/nuclease that in yeast participates in Okazaki fragment processing, DNA repair, and telomere maintenance. Here, we investigated the biological function of human Dna2 (hDna2). Immunofluorescence and biochemical fractionation studies demonstrated that hDna2 was present in both the nucleus and the mitochondria. Analysis of mitochondrial hDna2 revealed that it colocalized with a subfraction of DNA-containing mitochondrial nucleoids in unperturbed cells. Upon the expression of disease-associated mutant forms of the mitochondrial Twinkle helicase which induce DNA replication pausing/stalling, hDna2 accumulated within nucleoids. RNA interference-mediated depletion of hDna2 led to a modest decrease in mitochondrial DNA replication intermediates and inefficient repair of damaged mitochondrial DNA. Importantly, hDna2 depletion also resulted in the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that nuclear hDna2 plays a role in genomic DNA stability. Together, our data indicate that hDna2 is similar to its yeast counterpart and is a new addition to the growing list of proteins that participate in both nuclear and mitochondrial DNA maintenance.  

3.1296           Endosomal Trafficking of HIV-1 Gag and Genomic RNAs Regulates Viral Egress

Molle, D., Segura-Morales, C., Camus, G., Berlioz-Torrent, C., Kjems, J., Basyuk, E. and Bertrand, E.

1. Biol. Chem., 284(29), 19727-19743 (2009)

  HIV-1 Gag can assemble and generate virions at the plasma membrane,but it is also present in endosomes where its role remains incompletelycharacterized. Here, we show that HIV-1 RNAs and Gag are transportedon endosomal vesicles positive for TiVamp, a v-SNARE involvedin fusion events with the plasma membrane. Inhibition of endosomaltraffic did not prevent viral release. However, inhibiting lysosomaldegradation induced an accumulation of Gag in endosomes andincreased viral production 7-fold, indicating that transportof Gag to lysosomes negatively regulates budding. This alsosuggested that endosomal Gag-RNA complexes could access retrogradepathways to the cell surface and indeed, depleting cells ofTiVamp-reduced viral production. Moreover, inhibition of endosomaltransport prevented the accumulation of Gag at sites of cellularcontact. HIV-1 Gag could thus generate virions using two pathways,either directly from the plasma membrane or through an endosome-dependentroute. Endosomal Gag-RNA complexes may be delivered at specificsites to facilitate cell-to-cell viral transmission.  

3.1297           Heat shock protein 70 is secreted from endothelial cells by a non-classical pathway involving exosomes

Zhan, R., Leng, X., Liu, X., Wang, X., Gong, J., Yan, L., Wang, L., Wang, Y., Wang, X. and Qian, L-J. Biochem. Biophys. Res. Comm., 387, 229-233 (2009)   Emerging evidence suggests that a high level of circulating heat shock protein 70 (HSP70) correlates with a lower risk of vascular disease; however, the biological significance of this inverse relationship has not been explored. Herein, we report that oxidative low density lipoprotein (Ox-LDL) and homocysteine (Hcy) induce HSP70 release from endothelial cells. In rat endothelial cells, Ox-LDL and Hcy induced robust release of HSP70, independent of the classical route of endoplasmic reticulum/Golgi protein trafficking or the formation of lipid rafts. In contrast, Ox-LDL and Hcy significantly enhanced the exosomal secretory rate and increased the HSP70 content of exosomes. Exogenous HSP70 had no impact on LPS-, Ox-LDL- and Hcy-induced activation of endothelial cells, whereas HSP70 did activate monocytes alone, resulting in monocyte adhesion to endothelial cells. These results indicate that exosome-dependent secretion of HSP70 from endothelial cells provides a novel paracrine mechanism to regulate vascular endothelial functional integrity.  

3.1298           In Vitro Intracellular Trafficking of Virulence Antigen during Infection by Yersinia pestis

DiMezzo, T.L., ruthel, G., Brueggemann, E.E., Hines, H.B., Ribot, W.J., Chapman, C.E., Powell, B.S. and Welkos, S.L. PloSOne, 4(7), e628 (2009)   Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops) and a multifunctional virulence antigen (V). V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS)-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s) of V, time-course experiments were performed with macrophages (MΦs) infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MΦs were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs]) in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MΦs were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1) endosomal proteins between 10–45 min of infection, (2) lysosomal protein(s) between 1–2 h of infection, (3) mitochondrial proteins between 2.5–3 h infection, and (4) Golgi protein(s) between 4–6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V.  

3.1299           Correction of the Disease Phenotype of Myocilin-Causing Glaucoma by a Natural Osmolyte

Jia, L-Y., gong, B., Pang, C-P., Huang, Y., Lam, D., S-C., Wang, N. And Yam, H-F. Invest. Ophthalmol. Vis. Sci., 50, 3743-3749 (2009)   PURPOSE. To characterize a novel Asp384Asn (D384N) mutant myocilin (MYOC) that causes juvenile-onset open-angle glaucoma (JOAG) and investigate the correction of mutant phenotype by a natural osmolyte, trimethylamine N-oxide (TMAO). METHODS. A Chinese JOAG family was recruited and genomic DNA was extracted from peripheral blood obtained from 44 family members. Coding regions of the MYOC were sequenced. Two hundred individuals (>60 years old) without ocular hypertension or glaucoma were the control subjects. Full-length human wild-type MYOC cDNA was cloned in p3xFLAG-myc-CMV-25 and missense mutationwas introduced by site-directed mutagenesis. Transfected humantrabecular meshwork cells were treated with small-molecule chemicalchaperones. Secreted MYOC was analyzed by combined immunoprecipitation-Westernblot analysis. Intracellular myocilin was fractionated intoTriton X-100-soluble and insoluble fractions, and analyzed byWestern blot analysis. Intracellular aggregate and apoptosiswere assayed by immunofluorescence. The effect of TMAO on subcellularmyocilin distribution was analyzed by density gradient fractionation,followed by Western blot analysis. RESULTS. A novel c.1150G>A change of MYOC was identified. Screening of optineurin, WDR36, and CYP1B1 showed an absenceof disease-causing polymorphisms. Mutated D384N myocilin hadreduced solubility and was aggregation-prone and nonsecreted.Treatment of transfected cells with TMAO improved the solubilityof the D384N mutant, which was corrected for secretion in adose–response manner. TMAO reduced the distribution ofthe D384N mutant in the endoplasmic reticulum (ER), alleviatedER stress, and rescued cells from apoptosis. CONCLUSIONS. The results indicate that TMAO, with chaperoningactivity, facilitated the folding and secretion of mutant MYOC.This therapeutic approach assisted by a chemical chaperone canbe developed for treating glaucoma.  

3.1300           Harmonin in the Murine Retina and the Retinal Phenotypes of Ush1c-Mutant Mice and Human USH1C

Williams, D.S., Aleman, T.S., Lillo, C., Lopes, V.S., Hughes, L.C., Stone, E.M. and Jacobsen, S.G. Invest. Ophthalmol. Vis. Sci., 50, 3881-3889 (2009)   PURPOSE. To investigate the expression of harmonin in the mouse retina, test for ultrastructural and physiological mutant phenotypes in the retina of an Ush1c mutant mouse, and define in detailthe retinal phenotype in human USH1C. METHODS. Antibodies were generated against harmonin. Harmonin isoform distribution was examined by Western blot analysis and immunocytochemistry. Retinas of deaf circler (dfcr) mice, which possess mutant Ush1c, were analyzed by microscopy and electroretinography (ERG). Two siblings with homozygous 238_239insC (R80fs) USH1Cmutations were studied with ERG, perimetry, and optical coherencetomography (OCT). RESULTS. Harmonin isoforms a and c, but not b are expressedin the retina. Harmonin is concentrated in the photoreceptorsynapse where the majority is postsynaptic. Dfcr mice do notundergo retinal degeneration and have normal synaptic ultrastructureand ERGs. USH1C patients had abnormal rod and cone ERGs. Rod-and cone-mediated sensitivities and retinal laminar architecturewere normal across 50°–60° of visual field. Atransition zone to severely abnormal function and structurewas present at greater eccentricities. CONCLUSIONS. The largest harmonin isoforms are not expressed in the retina. A major retinal concentration of harmonin is in the photoreceptor synapses, both pre- and post-synaptically. The dfcr mouse retina is unaffected by its mutant Ush1c. Patientswith USH1C retained regions of normal central retina surroundedby degeneration. Perhaps the human disease is simply more aggressivethan that in the mouse. Alternatively, the dfcr mouse may bea model for nonsyndromic deafness, due to the nonpathologiceffect of its mutation on the retinal isoforms.  

3.1301           Slit Diaphragms Contain Tight Junction Proteins

Fukasawa, H., Borheimer, S., Kudlicka, K. And Farquhar, M.G.

1. Am. Soc. Nephrol., 20, 1491-1503 (2009)

  Slit diaphragms are essential components of the glomerular filtration apparatus, as changes in these junctions are the hallmark of proteinuric diseases. Slit diaphragms, considered specialized adherens junctions, contain both unique membrane proteins (e.g., nephrin, podocin, and Neph1) and typical adherens junction proteins (e.g., P-cadherin, FAT, and catenins). Whether slit diaphragms also contain tight junction proteins is unknown. Here, immunofluorescence, immunogold labeling, and cell fractionation demonstrated that rat slit diaphragms contain the tight junction proteins JAM-A (junctional adhesion molecule A), occludin, and cingulin. We found these proteins in the same protein complexes as nephrin, podocin, CD2AP, ZO-1, and Neph1 by cosedimentation, coimmunoprecipitation, and pull-down assays. PAN nephrosis increased the protein levels of JAM-A, occludin, cingulin, and ZO-1 several-fold in glomeruli and loosened their attachment to the actin cytoskeleton. These data extend current information about the molecular composition of slit diaphragms by demonstrating the presence of tight junction proteins, although slit diaphragms lack the characteristic morphologic features of tight junctions. The contribution of these proteins to the assembly of slit diaphragms and potential signaling cascades requires further investigation.  

3.1302           TNF-α-induced up-regulation of pro-inflammatory cytokines is reduced by phosphatidylcholine in intestinal epithelial cells

Treede, I., Braun, A., Jeliaskova, P., Giese, T., Füllekrug, J., Griffiths, G., Stremmel, W. and Ehehalt, R. BMC Gastroenterol., 9, 53-63 (2009)   Background Phosphatidylcholine (PC) is a major lipid of the gastrointestinal mucus layer. We recently showed that mucus from patients suffering from ulcerative colitis has low levels of PC. Clinical studies reveal that the therapeutic addition of PC to the colonic mucus using slow release preparations is beneficial. The positive role of PC in this disease is still unclear; however, we have recently shown that PC has an intrinsic anti-inflammatory property. It could be demonstrated that the exogenous application of PC inhibits membrane-dependent actin assembly and TNF-α-induced nuclear NF-κB activation. We investigate here in more detail the hypothesis that the exogenous application of PC has anti-inflammatory properties. Methods PC species with different fatty acid side chains were applied to differentiated and non-differentiated Caco-2 cells treated with TNF-α to induce a pro-inflammatory response. We analysed TNF-α-induced NF-κB-activation via the transient expression of a NF-κB-luciferase reporter system. Pro-inflammatory gene transcription was detected with the help of a quantitative real time (RT)-PCR analysis. We assessed the binding of TNF-α to its receptor by FACS and analysed lipid rafts by isolating detergent resistant membranes (DRMs). Results The exogenous addition of all PC species tested significantly inhibited TNF-α-induced pro-inflammatory signalling. The expression levels of IL-8, ICAM-1, IP-10, MCP-1, TNF-α and MMP-1 were significantly reduced after PC pre-treatment for at least two hours. The effect was comparable to the inhibition of NF-kB by the NF-kB inhibitor SN 50 and was not due to a reduced binding of TNF-α to its receptor or a decreased surface expression of TNF-α receptors. PC was also effective when applied to the apical side of polarised Caco-2 cultures if cells were stimulated from the basolateral side. PC treatment changed the compartmentation of the TNF-α-receptors 1 and 2 to DRMs. Conclusion PC induces a prolonged inhibition of TNF-α-induced pro-inflammatory signalling. This inhibition may be caused by a shift of the TNF-α receptors at the surface to lipid rafts. Our results may offer a potential molecular explanation for the positive role of PC seen in clinical studies for the treatment of ulcerative colitis.  

3.1303           Raptor Binds the SAIN (Shc and IRS-1 NPXY Binding) Domain of Insulin Receptor Substrate-1 (IRS-1) and Regulates the Phosphorylation of IRS-1 at Ser-636/639 by mTOR

Tzatsos, A.

1. Biol. Chem., 284(34), 22525-22534 (2009)

  In normal physiological states mTOR phosphorylates and activates Akt. However, under diabetic-mimicking conditions mTOR inhibits phosphatidylinositol (PI) 3-kinase/Akt signaling by phosphorylating insulin receptor substrate-1 (IRS-1) at Ser-636/639. The molecular basis for the differential effect of mTOR signaling on Akt is poorly understood. Here, it has been shown that knockdown of mTOR, Raptor, and mLST8, but not Rictor and mSin1, suppresses insulin-stimulated phosphorylation of IRS-1 at Ser-636/639 and stabilizes IRS-1 after long term insulin stimulation. This phosphorylation depends on the PI 3-kinase/PDK1 axis but is Akt-independent. At the molecular level, Raptor binds the SAIN (Shc and IRS-1 NPXY binding) domain of IRS-1 and regulates the phosphorylationof IRS-1 at Ser-636/639 by mTOR. IRS-1 lacking the SAIN domaindoes not interact with Raptor, is not phosphorylated at Ser-636/639,and favorably interacts with PI 3-kinase. Overall, these dataprovide new insights in the molecular mechanisms by which mTORC1inhibits PI 3-kinase/Akt signaling at the level of IRS-1 andsuggest that mTOR signaling toward Akt is scaffold-dependent.  

3.1304           Hypoxia-inducible Factor Prolyl-4-hydroxylase PHD2 Protein Abundance Depends on Integral Membrane Anchoring of FKBP38

Barth, S., Edlich, F., Berchner-Pfannschmidt, U., Gneuss, S., Jahreis, G., Hasgall, P.A., Fandrey, J., Wenger, R.H. and Camenisch, G.

1. Biol. Chem., 284(34), 23046-23058 (2009)

  Prolyl-4-hydroxylase domain (PHD) proteins are 2-oxoglutarate and dioxygen-dependent enzymes that mediate the rapid destruction of hypoxia-inducible factor subunits. Whereas PHD1 and PHD3 proteolysis has been shown to be regulated by Siah2 ubiquitin E3 ligase-mediated polyubiquitylation and proteasomal destruction, protein regulation of the main oxygen sensor responsible for hypoxia-inducible factor regulation, PHD2, remained unknown. We recently reported that the FK506-binding protein (FKBP) 38 specifically interacts with PHD2 and determines PHD2 protein stability in a peptidyl-prolyl cis-trans isomerase-independent manner. Using peptide array binding assays, fluorescence spectroscopy, and fluorescence resonance energy transfer analysis, we defined a minimal linear glutamate-rich PHD2 binding domain in the N-terminal part of FKBP38 and showed that this domain forms a high affinity complex with PHD2. Vice versa, PHD2 interacted with a non-linear N-terminal motif containing the MYND (myeloid, Nervy, and DEAF-1)-type Zn²⁺ finger domain with FKBP38. Biochemical fractionation and immunofluorescence analysis demonstrated that PHD2 subcellular localization overlapped with FKBP38 in the endoplasmic reticulum and mitochondria. An additional fraction of PHD2 was found in the cytoplasm. In cellulo PHD2/FKBP38 association, as well asregulation of PHD2 protein abundance by FKBP38, is dependenton membrane- anchored FKBP38 localization mediated by the C-terminaltransmembrane domain. Mechanistically our data indicate thatPHD2 protein stability is regulated by a ubiquitin-independentproteasomal pathway involving FKBP38 as adaptor protein thatmediates proteasomal interaction. We hypothesize that FKBP38-boundPHD2 is constantly degraded whereas cytosolic PHD2 is stableand able to function as an active prolyl-4-hydroxylase.  

3.1305           Enrichment and analysis of secretory lysosomes from lymphocyte populations

Schmidt, H., Gelhaus, C., Lucius, R., Nebendahl, M., Leippe, M. and Janssen, O. BMC Immunol., 10, 41-52 (2009)   Background In specialized cells, such as mast cells, macrophages, T lymphocytes and Natural Killer cells in the immune system and for instance melanocytes in the skin, secretory lysosomes (SL) have evolved as bifunctional organelles that combine degradative and secretory properties. Mutations in lysosomal storage, transport or sorting molecules are associated with severe immunodeficiencies, autoimmunity and (partial) albinism. In order to analyze the function and content of secretory lysosomes in different cell populations, an efficient enrichment of these organelles is mandatory. Results Based on a combination of differential and density gradient centrifugation steps, we provide a protocol to enrich intact SL from expanded hematopoietic cells, here T lymphocytes and Natural Killer cells. Individual fractions were initially characterized by Western blotting using antibodies against an array of marker proteins for intracellular compartments. As indicated by the presence of LAMP-3 (CD63) and FasL (CD178), we obtained a selective enrichment of SL in one of the resulting organelle fractions. The robustness and reproducibility of the applied separation protocol was examined by a high-resolution proteome analysis of individual SL preparations of different donors by 2D difference gel electrophoresis (2D-DIGE). Conclusion The provided protocol is readily applicable to enrich and isolate intact secretory vesicles from individual cell populations. It can be used to compare SL of normal and transformed cell lines or primary cell populations from healthy donors and patients with lysosomal storage or transport diseases, or from corresponding mutant mice. A subsequent proteome analysis allows the characterization of molecules involved in lysosomal maturation and cytotoxic effector function at high-resolution.  

3.1306           Ebolavirus Glycoprotein GP Masks both Its Own Epitopes and the Presence of Cellular Surface Proteins

Reynard, O., Borowiak, M., Volchkova, V.A., Delpeut, S., Mateo, M. and Volchkov, V.E. J.Virol., 83(18), 9596-9601 (2009)   Ebolavirus (EBOV) is the etiological agent of a severe hemorrhagic fever with a high mortality rate. The spike glycoprotein (GP) is believed to be one of the major determinants of virus pathogenicity. In this study, we demonstrated the molecular mechanism responsible for the downregulation of surface markers caused by EBOV GP expression. We showed that expression of mature GP on the plasma membrane results in the masking of cellular surface proteins, including major histocompatibility complex class I. Overexpression of GP also results in the masking of certain antigenic epitopes on GP itself, causing an illusory effect of disappearance from the plasma membrane.  

3.1307           Retinal Pigment Epithelium Defects in Humans and Mice with Mutations in MYO7A: Imaging Melanosome-Specific Autofluorescence

Gibbs, D., Cideciyan, A.V., Jacobson, S.G. and Williams, D.S. Invest. Ophthalmol. Vis. Sci., 50, 4386-4393 (2009)   PURPOSE. Usher syndrome (USH) is a genetically heterogeneous disease with autosomal recessive deafness and blindness. Gene therapy is under development for use in the most common genetic variant of USH1, USH1B, which is caused by mutations in the MYO7A gene. This study was undertaken to identify an imagingmethod for noninvasively monitoring the RPE component of theUSH1B disease. METHODS. NIR-autofluorescence (NIR-AF) was examined in USH1B patients with scanning laser ophthalmoscopy, and retinal thickness with spectral-domain optical coherence tomography. Myo7a-nullmouse retinas and purified RPE melanosomes were analyzed byspectral deconvolution confocal microscopy. RESULTS. In USH1B patients, NIR-AF was normal in regions of retained photoreceptors and abnormal in regions lacking photoreceptors. Subtle changes in NIR-AF were associated with intermediate photoreceptor loss. In ex vivo mouse retinas, the NIR-AF source was traced to the melanosomes in the RPE and choroid. Purified RPE melanosomes emitted the same signal. Fluorophores, excited by long-wavelength light, were evident throughout the apical RPE of WT mouse eyecups. In Myo7a-null eyecups, these fluorophores had a more restricteddistribution. They were absent from the apical processes ofthe RPE, thus correlating with the melanosome localization defectsdescribed previously by conventional microscopy. CONCLUSIONS. The data indicate that melanosomes in the RPE andchoroid are the dominant source of NIR-AF from the posteriorregion of the eye. NIR-AF is a novel tool that provides sensitiveand label-free imaging of the retina and RPE and is currentlythe only melanosome-specific, noninvasive technique for monitoringRPE disease in new therapeutic initiatives for retinal degenerations.  

3.1308           Mapping Organelle Proteins and Protein Complexes in Drosophila melanogaster

Tan, D.J.L., Dvinge, H., Christoforou, A., Bertone, P., Martinez Arias, A. and Lilley, K.S.

1. Proteeome Res., 8, 2667-2678 (2009)

  Many proteins within eukaryotic cells are organized spatially and functionally into membrane bound organelles and complexes. A protein’s location thus provides information about its function. Here, we apply LOPIT, a mass-spectrometry based technique that simultaneously maps proteins to specific subcellular compartments, to Drosophila embryos. We determine the subcellular distribution of hundreds of proteins, and protein complexes. Our results reveal the potential of LOPIT to provide average snapshots of cells.    

3.1309           Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity

Gibbins, D.J., Ciaudo, C., Erhardt, M. and Voinnet, O. Nature Cell Biol., 11(9), 1143-1149 (2009)   In animals, P-bodies or GW-bodies appear to cause the congregation of proteins involved in microRNA (miRNA)-mediated post-transcriptional silencing. The localization of P-bodies does not overlap with that of known organelles and are thus considered independent of lipid bilayers. Nonetheless, an miRNA effector protein, argonaute 2 (AGO2), was initially identified as membrane-associated, and some miRNAs have been found in secreted vesicles (exosomes) that derive from endo-lysosomal compartments called multivesicular bodies (MVBs). Proteins can be sorted in a ubiquitin-dependent manner into MVBs by three heteromeric subcomplexes, collectively termed ESCRT (endosomal sorting complex required for transport), to be further secreted in exosomes and/or degraded by the lysosome. Here we show that GW-bodies containing GW182 and AGO2, two main components of the RNA-induced silencing complex (RISC), are distinct from P-bodies due to their congregation with endosomes and MVBs. Moreover, miRNAs and miRNA-repressible mRNAs are enriched at these cellular membranes, suggesting that endosomes and/or MVBs are sites of miRNA-loaded RISC (miRISC) accumulation and, possibly, action. We further show that purified exosome-like vesicles secreted by MVBs are considerably enriched in GW182, but not P-body components, AGO2 or miRNA-repressible mRNA. Moreover, cells depleted of some ESCRT components show compromised miRNA-mediated gene silencing and over-accumulate GW182, which associates with ubiquitylated proteins. Therefore, GW182, possibly in association with a fraction of miRNA-loaded AGO2, is sorted into MVBs for secretion and/or lysosomal degradation. We propose that this process promotes continuous assembly or disassembly of membrane-associated miRISCs, which is possibly required for miRNA loading or target recognition and subsequent silencing.  

3.1310           Silencing by small RNAs is linked to endosomal trafficking

Lee, Y.S., Pressman, S., Andress, A.P., Kim, K., White, J.L., Cassidy, J.J., Li, X., Lubell, K., Lim, D.H., Cho, I.S., Nakahara, K., Preall, J.B., Bellare, P., Sontheimer, E.J. and Carthew, R.W. Nature Cell Biol., 11(9), 1150-1156 (2009)   Small RNAs direct RNA-induced silencing complexes (RISCs) to regulate stability and translation of mRNAs^{1, 2}. RISCs associated with target mRNAs often accumulate in discrete cytoplasmic foci known as GW-bodies³. However, RISC proteins can associate with membrane compartments such as the Golgi and endoplasmic reticulum⁴. Here, we show that GW-bodies are associated with late endosomes (multivesicular bodies, MVBs). Blocking the maturation of MVBs into lysosomes by loss of the tethering factor HPS4 (ref. 5) enhances short interfering RNA (siRNA)- and micro RNA (miRNA)-mediated silencing in Drosophila melanogaster and humans. It also triggers over-accumulation of GW-bodies. Blocking MVB formation by ESCRT (endosomal sorting complex required for transport)⁶ depletion results in impaired miRNA silencing and loss of GW-bodies. These results indicate that active RISCs are physically and functionally coupled to MVBs. We further show that MVBs promote the competence of RISCs in loading small RNAs. We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs. It may provide a means to enhance the dynamics of RNA silencing in the cytoplasm.  

3.1311           Rab35 Controls Actin Bundling by Recruiting Fascin as an Effector Protein

Zhang, J., Fonovic, M., Suyama, K., Bogyo, M. and Scott, M.P. Science, 325, 1250-1254 (2009)   Actin filaments are key components of the eukaryotic cytoskeleton that provide mechanical structure and generate forces during cell shape changes, growth, and migration. Actin filaments are dynamically assembled into higher-order structures at specified locations to regulate diverse functions. The Rab family of small guanosine triphosphatases is evolutionarily conserved and mediates intracellular vesicle trafficking. We found that Rab35 regulates the assembly of actin filaments during bristle development in Drosophila and filopodia formation in cultured cells. These effects were mediated by the actin-bundling protein fascin, which directly associated with active Rab35. Targeting Rab35 to the outer mitochondrial membrane triggered actin recruitment, demonstrating a role for an intracellular trafficking protein in localized actin assembly.  

3.1312           Reduced Amyloid Deposition in Mice Overexpressing RTN3 Is Adversely Affected by Preformed Dystrophic Neurites

Shi, Q., Prior, M., He, W., tang, X., Hu, X. and Yan, R.

1. Neurosci., 29(29), 9163-9173 (2009)

  Reticulon 3 (RTN3) was initially identified as a negative modulator of BACE1, an enzyme that cleaves amyloid precursor protein (APP) to release β-amyloid peptide. Interestingly, RTN3 can also form aggregates after accumulation, and increased RTN3 aggregation correlates with the formation of RTN3 immunoreactive dystrophic neurites (RIDNs) in brains of Alzheimer's cases. Transgenic mice expressing RTN3 alone develop RIDNs in their hippocampus but not in their cortex. To determine the in vivo effects of RTN3 and preformed RIDNs on amyloid deposition, we crossed bitransgenic mice expressing APP and presenilin 1 (PS1) mutations with mice overexpressing RTN3. We found that amyloid deposition in cortex, the hippocampal CA3 region, and dentate gyrus was significantly reduced in triple transgenic mice compared with bitransgenic controls. However, reduction of amyloid deposition in the hippocampal CA1 region, where RIDNs predominantly formed before amyloid deposition, was less significant. Hence, preformed RTN3 aggregates in RIDNs clearly offset the negative modulation of BACE1 activity by RTN3. Furthermore, our study indicates that the increased expression of RTN3 could result in an alteration of BACE1 intracellular trafficking by retaining more BACE1 in the endoplasmic reticulum compartment where cleavage of APP by BACE1 is less favored. Our results suggest that inhibition of RTN3 aggregation is likely to be beneficial by reducing both amyloid deposition and the formation RIDNs.  

3.1313           Correction of the Disease Phenotype of Myocilin-Causing Glaucoma by a Natural Osmolyte

Jia, L-Y., Gong, B., Pang, C-P., Huang, Y., Lam, D.S-C., Wang, N. and Yam, G.H-F. Invest. Ophthalmol. Vis. Sci., 50(8), 3743-3749 (2009)   PURPOSE. To characterize a novel Asp384Asn (D384N) mutant myocilin (MYOC) that causes juvenile-onset open-angle glaucoma (JOAG) and investigate the correction of mutant phenotype by a natural osmolyte, trimethylamine N-oxide (TMAO). METHODS. A Chinese JOAG family was recruited and genomic DNA was extracted from peripheral blood obtained from 44 family members. Coding regions of the MYOC were sequenced. Two hundred individuals (>60 years old) without ocular hypertension or glaucoma were the control subjects. Full-length human wild-type MYOC cDNA was cloned in p3xFLAG-myc-CMV-25 and missense mutationwas introduced by site-directed mutagenesis. Transfected humantrabecular meshwork cells were treated with small-molecule chemicalchaperones. Secreted MYOC was analyzed by combined immunoprecipitation-Westernblot analysis. Intracellular myocilin was fractionated intoTriton X-100-soluble and insoluble fractions, and analyzed byWestern blot analysis. Intracellular aggregate and apoptosiswere assayed by immunofluorescence. The effect of TMAO on subcellularmyocilin distribution was analyzed by density gradient fractionation,followed by Western blot analysis. RESULTS. A novel c.1150G>A change of MYOC was identified. Screening of optineurin, WDR36, and CYP1B1 showed an absenceof disease-causing polymorphisms. Mutated D384N myocilin hadreduced solubility and was aggregation-prone and nonsecreted.Treatment of transfected cells with TMAO improved the solubilityof the D384N mutant, which was corrected for secretion in adose–response manner. TMAO reduced the distribution ofthe D384N mutant in the endoplasmic reticulum (ER), alleviatedER stress, and rescued cells from apoptosis. CONCLUSIONS. The results indicate that TMAO, with chaperoningactivity, facilitated the folding and secretion of mutant MYOC.This therapeutic approach assisted by a chemical chaperone canbe developed for treating glaucoma.  

3.1314           Harmonin in the Murine Retina and the Retinal Phenotypes of Ush1c-Mutant Mice and Human USH1C

Williams, D.S., Aleman, T.S., Lillo, C., Lopes, V.S., Hughes, L.C., Stone, E.M. and Jacobson, S.G. Invest. Ophthalmol. Vis. Sci., 50(8), 3881-3889 (2009)   PURPOSE. To investigate the expression of harmonin in the mouse retina, test for ultrastructural and physiological mutant phenotypes in the retina of an Ush1c mutant mouse, and define in detailthe retinal phenotype in human USH1C. METHODS. Antibodies were generated against harmonin. Harmonin isoform distribution was examined by Western blot analysis and immunocytochemistry. Retinas of deaf circler (dfcr) mice, which possess mutant Ush1c, were analyzed by microscopy and electroretinography (ERG). Two siblings with homozygous 238_239insC (R80fs) USH1Cmutations were studied with ERG, perimetry, and optical coherencetomography (OCT). RESULTS. Harmonin isoforms a and c, but not b are expressedin the retina. Harmonin is concentrated in the photoreceptorsynapse where the majority is postsynaptic. Dfcr mice do notundergo retinal degeneration and have normal synaptic ultrastructureand ERGs. USH1C patients had abnormal rod and cone ERGs. Rod-and cone-mediated sensitivities and retinal laminar architecturewere normal across 50°–60° of visual field. Atransition zone to severely abnormal function and structurewas present at greater eccentricities. CONCLUSIONS. The largest harmonin isoforms are not expressed in the retina. A major retinal concentration of harmonin is in the photoreceptor synapses, both pre- and post-synaptically. The dfcr mouse retina is unaffected by its mutant Ush1c. Patientswith USH1C retained regions of normal central retina surroundedby degeneration. Perhaps the human disease is simply more aggressivethan that in the mouse. Alternatively, the dfcr mouse may bea model for nonsyndromic deafness, due to the nonpathologiceffect of its mutation on the retinal isoforms.  

3.1315           Nogo-B Receptor Stabilizes Niemann-Pick Type C2 Protein and Regulates Intracellular Cholesterol Trafficking

Harrison, K.D., Miao, R.Q., Fernandez-Hernando, C.F., Suarez, Y., Davalos, A. and Sessa, W.C. Cell Metabolism, 10, 208-218 (2009)   The Nogo-B receptor (NgBR) is a recently identified receptor for the N terminus of reticulon 4B/Nogo-B. Other than its role in binding Nogo-B, little is known about the biology of NgBR. To elucidate a basic cellular role for NgBR, we performed a yeast two-hybrid screen for interacting proteins, using the C-terminal domain as bait, and identified Niemann-Pick type C2 protein (NPC2) as an NgBR-interacting protein. NPC2 protein levels are increased in the presence of NgBR, and NgBR enhances NPC2 protein stability. NgBR localizes primarily to the endoplasmic reticulum (ER) and regulates the stability of nascent NPC2. RNAi-mediated disruption of NgBR or genetic deficiency in NgBR lead to a decrease in NPC2 levels, increased intracellular cholesterol accumulation, and a loss of sterol sensing, all hallmarks of an NPC2 mutation. These data identify NgBR as an NPC2-interacting protein and provide evidence of a role for NgBR in intracellular cholesterol trafficking.      

3.1316           Contributions of quantitative proteomics to understanding membrane microdomains

Zheng, Y. and Foster, L.J.

1. Lipid Res., 50, 1976-1985 (2009)

  Membrane microdomains, e.g., lipid rafts and caveolae, are crucial cell surface organelles responsible for many cellular signaling and communication events, which makes the characterization of their proteomes both interesting and valuable. They are large cellular complexes comprised of specific proteins and lipids, yet they are simple enough in composition to be amenable to modern LC/MS/MS methods for proteomics. However, the proteomic characterization of membrane microdomains by traditional qualitative mass spectrometry is insufficient for distinguishing true components of the microdomains from copurifying contaminants or for evaluating dynamic changes in the proteome compositions. In this review, we discuss the contributions quantitative proteomics has made to our understanding of the biology of membrane microdomains.  

3.1317           Assembly of Arenavirus Envelope Glycoprotein GPC in Detergent-Soluble Membrane Microdomains

Agnihothram, S.S., Dancho, B., Grant, K.W., Grimes, M.L., Lyles, D.S. and Nunberg, J.H.

1. Virol., 83(19), 9890-9900 (2009)

  The family Arenaviridae includes a number of highly pathogenic viruses that are responsible for acute hemorrhagic fevers in humans. Genetic diversity among arenavirus species in their respective rodent hosts supports the continued emergence of new pathogens. In the absence of available vaccines or therapeutic agents, the hemorrhagic fever arenaviruses remain a serious public health and biodefense concern. Arenaviruses are enveloped virions that assemble and bud from the plasma membrane. In this study, we have characterized the microdomain organization of the virus envelope glycoprotein (GPC) on the cell surface by using immunogold electron microscopy. We find that Junín virus (JUNV) GPC clusters into discrete microdomains of 120 to 160 nm in diameter and that this property of GPC is independent of its myristoylation and of coexpression with the virus matrix protein Z. In cells infected with the Candid#1 strain of JUNV, and in purified Candid#1 virions, these GPC microdomains are soluble in cold Triton X-100 detergent and are thus distinct from conventional lipid rafts, which are utilized by numerous other viruses for assembly. Virion morphogenesis ultimately requires colocalization of viral components, yet our dual-label immunogold staining studies failed to reveal a spatial association of Z with GPC microdomains. This observation may reflect either rapid Z-dependent budding of virus-like particles upon coassociation or a requirement for additional viral components in the assembly process. Together, these results provide new insight into the molecular basis for arenavirus morphogenesis.  

3.1318           Characterization of Hepatitis C Virus Core Protein Multimerization and Membrane Envelopment: Revelation of a Cascade of Core-Membrane Interactions

Ai, L-S., Lee, Y-W. and Chen, S.S.-L.

1. Virol., 83(19), 9923-9939 (2009)

  The molecular basis underlying hepatitis C virus (HCV) core protein maturation and morphogenesis remains elusive. We characterized the concerted events associated with core protein multimerization and interaction with membranes. Analyses of core proteins expressed from a subgenomic system showed that the signal sequence located between the core and envelope glycoprotein E1 is critical for core association with endoplasmic reticula (ER)/late endosomes and the core's envelopment by membranes, which was judged by the core's acquisition of resistance to proteinase K digestion. Despite exerting an inhibitory effect on the core's association with membranes, (Z-LL)2-ketone, a specific inhibitor of signal peptide peptidase (SPP), did not affect core multimeric complex formation, suggesting that oligomeric core complex formation proceeds prior to or upon core attachment to membranes. Protease-resistant core complexes that contained both innate and processed proteins were detected in the presence of (Z-LL)2-ketone, implying that core envelopment occurs after intramembrane cleavage. Mutations of the core that prevent signal peptide cleavage or coexpression with an SPP loss-of-function D219A mutant decreased the core's envelopment, demonstrating that SPP-mediated cleavage is required for core envelopment. Analyses of core mutants with a deletion in domain I revealed that this domain contains sequences crucial for core envelopment. The core proteins expressed by infectious JFH1 and Jc1 RNAs in Huh7 cells also assembled into a multimeric complex, associated with ER/late-endosomal membranes, and were enveloped by membranes. Treatment with (Z-LL)2-ketone or coexpression with D219A mutant SPP interfered with both core envelopment and infectious HCV production, indicating a critical role of core envelopment in HCV morphogenesis. The results provide mechanistic insights into the sequential and coordinated processes during the association of the HCV core protein with membranes in the early phase of virus maturation and morphogenesis.  

3.1319           Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Giles, L.M., Li, L. and Chin, L-S.

1. Biol. Chem., 284(32), 21765-21775 (2009)

  Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA E) in the C-terminal region of the AAA⁺ (ATPasesassociated with a variety of cellular activities) protein torsinA.The pathogenic mechanism by which torsinA E mutation leads todystonia remains unknown. Here we report the identificationand characterization of a 628-amino acid novel protein, printor,that interacts with torsinA. Printor co-distributes with torsinAin multiple brain regions and co-localizes with torsinA in theendoplasmic reticulum. Interestingly, printor selectively bindsto the ATP-free form but not to the ATP-bound form of torsinA,supporting a role for printor as a cofactor rather than a substrateof torsinA. The interaction of printor with torsinA is completelyabolished by the dystonia-associated torsinA E mutation. Ourfindings suggest that printor is a new component of the DYT1pathogenic pathway and provide a potential molecular targetfor therapeutic intervention in dystonia.  

3.1320           Functionally Relevant Domains of the Prion Protein Identified In Vivo

Baumann, F., Pahnke, J., Radovanovic, I., Rülicke, T., Bremer, J., Tolnay, M. And Aguzzi, A. PloSOne, 4(9), e607 (2009)   The prion consists essentially of PrP^Sc, a misfolded and aggregated conformer of the cellular protein PrP^C. Whereas PrP^C deficient mice are clinically healthy, expression of PrP^C variants lacking its central domain (PrP_ΔCD), or of the PrP-related protein Dpl, induces lethal neurodegenerative syndromes which are repressed by full-length PrP. Here we tested the structural basis of these syndromes by grafting the amino terminus of PrP^C (residues 1–134), or its central domain (residues 90–134), onto Dpl. Further, we constructed a soluble variant of the neurotoxic PrP_ΔCD mutant that lacks its glycosyl phosphatidyl inositol (GPI) membrane anchor. Each of these modifications abrogated the pathogenicity of Dpl and PrP_ΔCD in transgenic mice. The PrP-Dpl chimeric molecules, but not anchorless PrP_ΔCD, ameliorated the disease of mice expressing truncated PrP variants. We conclude that the amino proximal domain of PrP exerts a neurotrophic effect even when grafted onto a distantly related protein, and that GPI-linked membrane anchoring is necessary for both beneficial and deleterious effects of PrP and its variants.  

3.1321           Lipid rafts serve as signaling platforms for Tie2 receptor tyrosine kinase in vascular endothelial cells

Katoh, S-Y., Kamimoto, T., Yamakawa, D. and Takakura, N. Exp. Cell Res., 315, 2818-2823 (2009)   The Tie2 receptor tyrosine kinase plays a pivotal role in vascular and hematopoietic development. The major intracellular signaling systems activated by Tie2 in response to Angiopoietin-1 (Ang1) include the Akt and Erk1/2 pathways. Here, we investigated the role of cholesterol-rich plasma membrane microdomains (lipid rafts) in Tie2 regulation. Tie2 could not be detected in the lipid raft fraction of human umbilical vein endothelial cells (HUVECs) unless they were first stimulated with Ang1. After stimulation, a minor fraction of Tie2 associated tightly with the lipid rafts. Treatment of HUVECs with the lipid raft disrupting agent methyl-β-cyclodextrin selectively inhibited Ang1-induced Akt phosphorylation, but not Erk1/2 phosphorylation. It has been reported that inhibition of FoxO activity is an important mechanism for Ang1-stimulated Tie2-mediated endothelial function. Consistent with this, we found that phosphorylation of FoxO mediated by Tie2 activation was attenuated by lipid raft disruption. Therefore, we propose that lipid rafts serve as signaling platforms for Tie2 receptor tyrosine kinase in vascular endothelial cells, especially for the Akt pathway.  

3.1322           Lipid dependence of ABC transporter localization and function

Klappe, K., Hummel, I., Hoekstra, D. And Kok, J.W. Chemistry and Physics of Lipids, 161, 57-64 (2009)   Lipid rafts have been implicated in many cellular functions, including protein and lipid transport and signal transduction. ATP-binding cassette (ABC) transporters have also been localized in these membrane domains. In this review the evidence for this specific localization will be evaluated and discussed in terms of relevance to ABC transporter function. We will focus on three ABC transporters of the A, B and C subfamily, respectively. Two of these transporters are relevant to multidrug resistance in tumor cells (Pgp/ABCB1 and MRP1/ABCC1), while the third (ABCA1) is extensively studied in relation to the reverse cholesterol pathway and cellular cholesterol homeostasis. We will attempt to derive a generalized model of lipid rafts to which they associate based on the use of various different lipid raft isolation procedures. In the context of lipid rafts, modulation of ABC transporter localization and function by two relevant lipid classes, i.e. sphingolipids and cholesterol, will be discussed.  

3.1323           Mitochondrial Expression and Functional Activity of Breast Cancer Resistance Protein in Different Multiple Drug-Resistant Cell Lines

Solazzo, M., Fantappie, O., D’Amico, M., Sassoli, C., Tani, A., Cipriani, G., Bogani, C., Formigli, L. and Mazanti, R. Cancer Res., 69(18), 7235-7242 (2009)   The multidrug resistance (MDR) phenotype is characterized by the overexpression of a few transport proteins at the plasma membrane level, one of which is the breast cancer resistance protein (BCRP). These proteins are expressed in excretory organs, in the placenta and blood-brain barrier, and are involved in the transport of drugs and endogenous compounds. Because some of these proteins are expressed in the mitochondria, this study was designed to determine whether BCRP is expressed at a mitochondrial level and to investigate its function in various MDR and parental drug–sensitive cell lines. By using Western blot analysis, immunofluorescence confocal and electron microscopy, flow cytometry analysis, and the BCRP (ABCG-2) small interfering RNA, these experiments showed that BCRP is expressed in the mitochondrial cristae, in which it is functionally active. Mitoxantrone accumulation was significantly reduced in mitochondria and in cells that overexpress BCRP, in comparison to parental drug–sensitive cells. The specific inhibitor of BCRP, fumitremorgin c, increased the accumulation of mitoxantrone significantly in comparison with basal conditions in both whole cells and in mitochondria of BCRP-overexpressing cell lines. In conclusion, this study shows that BCRP is overexpressed and functionally active in the mitochondria of MDR-positive cancer cell lines. However, its presence in the mitochondria of parental drug–sensitive cells suggests that BCRP can be involved in the physiology of cancer cells.  

3.1324           Regulation of Cell Death by Recycling Endosomes and Golgi Membrane Dynamics via a Pathway Involving Src-family kinases, Cdc42 and Rab11a

Landry, M-L., Sicotte, A., Champagne, C. and Lavoie, J.N. Mol. Biol. Cell, 20, 4091-4106 (2009)   Actin dynamics and membrane trafficking influence cell commitment to programmed cell death through largely undefined mechanisms. To investigate how actin and recycling endosome (RE) trafficking can engage death signaling, we studied the death program induced by the adenovirus early region 4 open reading frame 4 (E4orf4) protein as a model. We found that in the early stages of E4orf4 expression, Src-family kinases (SFKs), Cdc42, and actin perturbed the organization of the endocytic recycling compartment and promoted the transport of REs to the Golgi apparatus, while inhibiting recycling of protein cargos to the plasma membrane. The resulting changes in Golgi membrane dynamics that relied on actin-regulated Rab11a membrane trafficking triggered scattering of Golgi membranes and contributed to the progression of cell death. A similar mobilization of RE traffic mediated by SFKs, Cdc42 and Rab11a also contributed to Golgi fragmentation and to cell death progression in response to staurosporine, in a caspase-independent manner. Collectively, these novel findings suggest that diversion of RE trafficking to the Golgi complex through a pathway involving SFKs, Cdc42, and Rab11a plays a general role in death signaling by mediating regulated changes in Golgi dynamics.  

3.1325           The Small Heat Shock Protein B-Crystallin Is Secreted From Retinal Pigment Epithelial Cells ARPE in Culture

Bhat, S.P., Gangalum, R.K. and Jing, Z. Invest. Ophthalmol. Vis. Sci., 50, abstract 4183 (2009)   Purpose: Elevated expression of the small heat shock protein, aB-crystallin (aB) has been associated with cancer, cardiomyopathies and a large number of neurodegenerative disorders including, Alzheimer’s, Parkinson’s, Alexander’s diseases, multiple sclerosis and retinal degenerations. Interestingly, the expression of this protein in retinal pigment epithelium (RPE) and its presence in ‘drusen’; has been implicated in age-related macular degeneration. In this investigation we have explored the status of aB in ARPE cells in culture with a focus on mechanistic understanding of how aB becomes associated with the  ‘drusen’. Methods: The association of aB with Golgi membranes was studied by sucrose density gradient fractionation and confocal microscopy. Protein transport was studied using various inhibitors. Exosomes were isolated from the culture medium by differential centrifugation, characterized by AChE assay and immuno-electron microscopy. Optiprep gradients were used to isolate lipid rafts from ARPE cells and characterized by the presence of established raft markers. Results: Using synchronized cultures we show that aB is a Golgi -associated protein in ARPE. We demonstrate that aB is secreted out of ARPE and that this secretion is not inhibited by classical protein transport inhibitors such as Brefeldin and Monensin, and glycosylation inhibitor, Tunicamycin and microtubule disrupting agent, Nocodazole. Examination of the culture medium reveals that aB is associated with exosomes and multivesicular bodies (MVB). Interestingly the secretion of this protein is inhibited by methyl a-cyclodextrin (MBC) suggesting lipid raft dependent exocytosis of vesicles containing aB. Conclusion: The small heat shock protein aB is secreted from ARPE cells via a non-conventional secretory pathway that involves the lipid rafts, and biogenesis and release of MVBs and exosomes. While these data point to an involvement of B with membrane trafficking, polarization and cellular secretion, they also suggest a possible route to the presence of this protein in the ‘drusen’.  

3.1326           Omega-3 Protects Trabecular Meshwork From Metabolic Stress

Beverley, R.M., Nolan, M.J., McCarty, R.D., Yue, B.Y.J.T., Qi, L. Invest. Ophthalmol. Vis. Sci., 50, abstract 4866 (2009)   Purpose:Supplemental dietary omega-3 fatty acid reduces IOP in ratsand is neuroprotective against oxidative stress in retinal pigmentepithelial cells. The omega-3 fatty acid docosahexaenoic acid(DHA) is an important omega-3 representing a large componentof total acyl chains in nervous tissue and is concentrated inlipid rafts. The purpose of this study was to determine whethertrabecular meshwork (TM) cell survival is influenced by DHAand the effects of DHA on caveolin/lipid rafts. Methods:Primary cultures of human TM cells were pre-treated with 0.67, 2.01 and 6.03 µM DHA for 24 hrs and then challenged with metabolic stress using 1, 10 and 40 mM lactate for 3 hrs. Cell viability was determined. Lipid rafts in cell lysates were separated by an Optiprep density gradient (Sigma D1556). The preparationwas centrifuged at 200,000 x g for 18 hrs; nine 1.0 ml fractionswere pipetted from the top (lightest) to bottom (heaviest).Each fraction was analyzed for protein content, resolved bySDS polyacrylamide electrophoresis, and immunoblotted with anti-caveolin-1,anti-caveolin-2, and LRP-6 antibodies. Results:There was a statistically significant dose dependent increasein cell survival of TM cells subjected to lactate stress followingDHA treatment; 0.67 µM pretreatment was insignificant,2.01 µM pretreatment was significant (P<0.03), and6.03 µM pretreatment was highly significant (P<0.001).The distribution of 22-kDa caveolin-1 in DHA-treated TM cellswas markedly increased in fractions 1, 2, 4, and 5 comparedto controls. The distribution of caveolin-2 in DHA-treated cellswas increased in fractions 1 and 2, presumably forming homo-and heterodimers with caveolin-1. LRP-6 was strongly upregulatedin fractions 3, 4, 5, and 6 compared to controls. Conclusions:DHA treatment increased cell viability and changed caveolin-1, caveolin-2, and LRP-6 density gradient distribution in TM cells subjected to metabolic stress. These results indicate that DHA modulates the distribution and content of caveolin/lipid rafts which may play an important role in TM cell viability and cell signaling.  

3.1327           Gamma Secretase and Trabecular Meshwork Stress

McCarty, R.D., Nolan, M.J., Beverley, R.M., Quinn, K., Yue, B.Y.J.T., Samples, J.R. and Knepper, P.A. Invest. Ophthalmol. Vis. Sci., 50, abstract 4870 (2009)   Purpose:Primary open-angle glaucoma (POAG) and Alzheimer's disease sharemany similarities in pathogenic mechanisms. -secretase is apresenilin dependent enzyme which mediates the proteolytic cleavageof several transmembrane proteins. Examples of -secretase substratesinclude CD44 to form soluble sCD44, a potential biomarker ofPOAG, amyloid precursor to form amyloid β-peptide, a biomarkerof Alzheimer’s disease, LDL receptor-related protein,E-cadherin and Erb-4. The intracellular domains (ICD) of -secretasesubstrates are liberated and function as nuclear signals. Thepurpose of this study was to determine whether metabolic stressalters -secretase, β-secretase, and MT1-MMP in trabecularmeshwork (TM) cells. Methods:Human TM cells were grown in modified Eagle’s medium containing 10% fetal bovine serum (FBS) until confluent, washed twice with PBS, and incubated in MEM containing 0.1% FBS with 1, 10, and 40 mM lactate for 3, 12, and 24 hrs. The media was aspirated; the cells were washed with cold PBS, subjected to lysis buffer (Sigma CS0750) containing 1% Triton X-100, and separated by an Optiprep density gradient (Sigma D1556). The preparationwas centrifuged at 200,000 x g for 18 hrs; nine 1.0 ml fractionswere pipetted from the top (lightest) to bottom (heaviest).Each fraction was analyzed for protein content, resolved bySDS polyacrylamide electrophoresis, and immunoblotted with anti--secretase, β-secretase, MT1-MMP, and caveolin-1. Results:The distribution of -secretase in lactate stressed TM cellswas in fractions 3 through 9 and a decreased intensity was observedin fractions 6 through 9 compared to controls. In contrast,the distribution of β-secretase was observed in fractions5 through 7 with a robust intensity (increased more than three-fold)in fractions 6 and 7 compared to controls. There was no demonstrablechange in the distribution of MT1-MMP in metabolic stressedTM cells. The content of caveolin-1 was markedly decreased infractions 4 and 5 compared to controls. Conclusions:This is a first demonstration of - and β-secretase, and their modulation by metabolic stress, in TM cells. Both - and β-secretase are concentrated in caveolin lipid rafts, and the decrease in -secretase and increase in β-secretase may be important in the TM response to metabolic stress.  

3.1328           Wnt Signaling and Trabecular Meshwork Lipid Rafts

Knepper, P.A., Beverley, R.M., McCarty, R.D., Nolan, M.J., Yue, B.Y.J.T. and Samples, J.R. Invest. Ophthamol. Vis. Sci., 50, abstract 4875 (2009)   Purpose:Wnt proteins interact with Frizzled receptors and members ofthe low-density-lipoprotein receptor (LRP). LRP serves as anendocytosis receptor for -2-macroglobulin, a mediator of retinalganglion cell death in glaucoma and also for amyloid precursorprotein, a mediator of selected cell death in Alzheimer’sdisease. Secreted frizzled-related protein-1 (sFRP-1) is anantagonist of Wnt signaling that causes a decreased outflowfacility in ex vivo perfusion-of cultured human eyes. To explorepossible signaling mechanisms by which sFRP-1 decreases outflowfacility, we challenged trabecular meshwork (TM) cells withexogenous sFRP-1 and evaluated its effects on cell viabilityand its downstream effects on Wnt pathway of LRP-6 (canonical)and CD44 (non-canonical) content in caveolin/lipid rafts. Methods:Human TM cells cultured in the presence 2.5, 5.0 and 10.0 ug of recombinant human sFRP-1 (R & D Systems) were tested for cell survival. Caveolin enriched lipid rafts were isolated using ice cold Triton X-100 and then separated on an OptiPrepdensity gradient (Sigma D1556). The preparation was centrifugedat 200,000 x g for 18 hrs; nine 1.0 ml fractions were pipettedfrom the top (lightest) to bottom (heaviest). Each fractionwas analyzed for protein content, resolved by SDS polyacrylamideelectrophoresis, and immunoblotted with anti-caveolin-1, CD44,and LRP-6 antibodies. Results:There was a statistically significant (P<0.001) dose dependentdecrease of cell viability in TM cells treated with sFRP-1.The distribution of 22-kDa caveolin-1 in sFRP-1 treated TM cellswas in fractions 3 through 7 with the strongest intensity infractions 5 and 6. The amount of caveolin-1 in sFRP-1 treatedTM cells was considerably greater, approximately two-fold. Thedistribution of CD44 in sFRP-1 treated TM cells remained infractions 1 through 9 but with an increased content of 32 kDaCD44 in fraction 5 compared to controls. The distribution ofLRP-6 in sFRP-1 treated TM cells was in fractions 4 through9 and decreased compared to control TM cells. Conclusions:This is the first demonstration of the effects of exogenoussFRP-1 on the downstream signaling of caveolin, CD44 and LRP-6in TM cells. sFRP-1 increased the cell caveolin-1 content, changedcaveolin-1 density gradient distribution, decreased LRP-6 contentand increased sCD44. These results indicate that caveolin-1,CD44 and/or LRP-6 are downstream effectors of sFRP-1.  

3.1329           Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium

Shevchuk, O., Batzillaa, C., Hägele, S., Kusch, H., Engelmann, S., Hecker, M., Haas, A., Heuner, K., Glöckner, G. and Steinert, M. Int. J. Med. Microbiol., 299, 489-508 (2009)   Legionella pneumophila, the agent of Legionnaires’ disease, replicates intracellularly within specialized phagosomes of human macrophages and amoebae. In this study, we have developed a protocol for the isolation of Legionella-containing phagosomes from Dictyostelium discoideum. Cell fractionation, two-dimensional gel electrophoresis and MALDI-TOF MS combined with genomic data identified 157 phagosome host proteins. In addition to proteins with an evident role in phagosome maturation, we identified proteins for which a function remains to be elucidated. Possible interactions of coronin with cytosolic NADPH oxidase components and protein kinase C inhibitors which together may lead to an inhibition of phagosomal superoxide generation are discussed. Comparative proteomics of phagosomes containing highly virulent L. pneumophila Corby versus less virulent L. hackeliae revealed distinctive protein expression patterns, e.g., an abundance of RhoGDI in L. hackeliae degrading phagosomes versus little RhoGDI in L. pneumophila Corby replicative phagosomes. We present a kinetic dissection of phagosome maturation including the complex alterations of the phagosome protein composition. A reference flow chart suggests so far unrecognized consequences of infection for host cell physiology, actin degradation on phagosomes, and a putative cysteine proteinase inhibitor interference with lysosomal enzyme sorting and activation processes.  

3.1330           Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein

Aligo, J., Jia, S., Manna, D. and Konan, K.V. Virology, 393, 68-83 (2009)   During replication, hepatitis C virus (HCV) NS4B protein rearranges intracellular membranes to form foci, or the web, the putative site for HCV replication. To understand the role of the C-terminal domain (CTD) in NS4B function, mutations were introduced into NS4B alone or in the context of HCV polyprotein. First, we show that the CTD is required for NS4B-induced web structure, but it is not sufficient to form the web nor is it required for NS4B membrane association. Interestingly, all the mutations introduced into the CTD impeded HCV genome replication, but only two resulted in a disruption of NS4B foci. Further, we found that NS4B interacts with NS3 and NS5A, and that mutations causing NS4B mislocalization have a similar effect on these proteins. Finally, we show that the redistribution of Rab5 to NS4B foci requires an intact CTD, suggesting that Rab5 facilitates NS4B foci formation through interaction with the CTD.  

3.1331           Caveolae Contribute to the Apoptosis Resistance Induced by the α_1A-Adrenoceptor in Androgen-Independent Prostate Cancer Cells

Katsogiannou, M., El Boustany, C., Gackiere, F., Delcourt, P., Athias, A., Mariot, P., Dewailly, E., Jouuy, N., Lamaze, C., Bidaux, G., Mauroy, B., Prevarskay, N. and Slomianny, C. PloSOne, 4(9), e7068 (2009)   Background During androgen ablation prostate cancer cells' growth and survival become independent of normal regulatory mechanisms. These androgen-independent cells acquire the remarkable ability to adapt to the surrounding microenvironment whose factors, such as neurotransmitters, influence their survival. Although findings are becoming evident about the expression of α_1A-adrenoceptors in prostate cancer epithelial cells, their exact functional role in androgen-independent cells has yet to be established. Previous work has demonstrated that membrane lipid rafts associated with key signalling proteins mediate growth and survival signalling pathways in prostate cancer cells. Methodology/Principal Findings In order to analyze the membrane topology of the α_1A-adrenoceptor we explored its presence by a biochemical approach in purified detergent resistant membrane fractions of the androgen-independent prostate cancer cell line DU145. Electron microscopy observations demonstrated the colocalisation of the α_1A-adrenoceptor with caveolin-1, the major protein component of caveolae. In addition, we showed that agonist stimulation of the α_1A-adrenoceptor induced resistance to thapsigargin-induced apoptosis and that caveolin-1 was necessary for this process. Further, immunohistofluorescence revealed the relation between high levels of α_1A-adrenoceptor and caveolin-1 expression with advanced stage prostate cancer. We also show by immunoblotting that the TG-induced apoptosis resistance described in DU145 cells is mediated by extracellular signal-regulated kinases (ERK). Conclusions/Significance In conclusion, we propose that α_1A-adrenoceptor stimulation in androgen-independent prostate cancer cells via caveolae constitutes one of the mechanisms contributing to their protection from TG-induced apoptosis.  

3.1332           High extracellular glucose inhibits exocytosis through disruption of syntaxin 1A-containing lipid rafts

Somanath, S., Barg, S., Marshall, C., Silwood, C.J. and Turner, M.D. Biochem. Biophys. Res. Comm., 389, 241-246 (2009)   Diabetes is characterized by high blood glucose which eventually impairs the secretion of insulin. Glucose directly affects cholesterol biosynthesis and may in turn affect cellular structures that depend on the sterol, including lipid rafts that help organize the secretory apparatus. Here, we investigated the long-term effects of glucose upon lipid rafts and secretory granule dynamics in pancreatic β-cells. Raft fractions, identified by the presence of GM1 and flotillin, contained characteristically high levels of cholesterol and syntaxin 1A, the t-SNARE which tethers granules to the plasma membrane. Seventy-two hours exposure to 28 mM glucose resulted in 30% reduction in membrane cholesterol, with consequent redistribution of raft markers and syntaxin 1A throughout the plasma membrane. Live cell imaging indicated loss of syntaxin 1A from granule docking sites, and fewer docked granules. In conclusion, glucose-mediated inhibition of cholesterol biosynthesis perturbs lipid raft stability, resulting in a loss of syntaxin 1A from granule docking sites and inhibition of insulin secretion.  

3.1333           Coassembly of Mgm1 isoforms requires cardiolipin and mediates mitochondrial inner membrane fusion

DeVay, R.M., Dominguez-Ramirez, L., Lackner, L.L., Hoppins, S., Stahlberg, H. and Nunnari, J.

1. Cell Biol., 186(6), 793-803 (2009)

  Two dynamin-related protein (DRP) families are essential forfusion of the outer and inner mitochondrial membranes, Fzo1(yeast)/Mfn1/Mfn2 (mammals) and Mgm1 (yeast)/Opa1 (mammals),respectively. Fzo1/Mfns possess two medial transmembrane domains,which place their critical GTPase and coiled-coil domains inthe cytosol. In contrast, Mgm1/Opa1 are present in cells aslong (l) isoforms that are anchored via the N terminus to theinner membrane, and short (s) isoforms were predicted to besoluble in the intermembrane space. We addressed the roles ofMgm1 isoforms and how DRPs function in membrane fusion. Ouranalysis indicates that in the absence of a membrane, l- ands-Mgm1 both exist as inactive GTPase monomers, but that togetherin trans they form a functional dimer in a cardiolipin-dependentmanner that is the building block for higher-order assemblies.  

3.1334           Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells

Mukai, A., Kurisaki, T., Sato, S.B., Kobayashi, T., Kondoh, G. and Hashimoto, N. Exp. Cell Res., 315, 3052-3063 (2009)   Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, β-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.  

3.1335           Phosphotyrosine protein dynamics in cell membrane rafts of sphingosine-1-phosphate-stimulated human endothelium: Role in barrier enhancement

Zhao, J., Singleton, P.A., Brown, M.E., Dudek, S.M. and Garcia, J.G.N. Cellular Signalling, 21, 1945-1960 (2009)   Sphingosine-1-phosphate (S1P), a lipid growth factor, is critical to the maintenance and enhancement of vascular barrier function via processes highly dependent upon cell membrane raft-mediated signaling events. Anti-phosphotyrosine 2 dimensional gel electrophoresis (2-DE) immunoblots confirmed that disruption of membrane raft formation (via methyl-β-cyclodextrin) inhibits S1P-induced protein tyrosine phosphorylation. To explore S1P-induced dynamic changes in membrane rafts, we used 2-D techniques to define proteins within detergent-resistant cell membrane rafts which are differentially expressed in S1P-challenged (1 μM, 5 min) human pulmonary artery endothelial cells (EC), with 57 protein spots exhibiting > 3-fold change. S1P induced the recruitment of over 20 cell membrane raft proteins exhibiting increasing levels of tyrosine phosphorylation including known barrier-regulatory proteins such as focal adhesion kinase (FAK), cortactin, p85α phosphatidylinositol 3-kinase (p85αPI3K), myosin light chain kinase (nmMLCK), filamin A/C, and the non-receptor tyrosine kinase, c-Abl. Reduced expression of either FAK, MLCK, cortactin, filamin A or filamin C by siRNA transfection significantly attenuated S1P-induced EC barrier enhancement. Furthermore, S1P induced cell membrane raft components, p-caveolin-1 and glycosphingolipid (GM1), to the plasma membrane and enhanced co-localization of membrane rafts with p-caveolin-1 and p-nmMLCK. These results suggest that S1P induces both the tyrosine phosphorylation and recruitment of key actin cytoskeletal proteins to membrane rafts, resulting in enhanced human EC barrier function.  

3.1336           A Novel Role for Nutrition in the Alteration of Functional Microdomains on the Cell Surface

Kim, W., Chapkin, R.S., Barhoumi, R. and Ma, D.W.L. Methods in Mol. Biol., 579, 261-270 (2009)   Membrane rafts are ordered microdomains of the plasma membrane consisting of cholesterol, sphingolipids, and saturated fatty acids which appear to regulate many cellular signaling pathways. One such type of membrane raft is caveolae, which are cave-like invaginations of the plasma membrane. Interestingly, changes in the acyl composition of cellular membranes have been shown to alter the specific localization of membrane raft associated proteins and their function. This is noteworthy because modification of membrane acyl composition is readily accomplished through changes in dietary fat composition. Here we describe a common approach used to fractionate cell membranes to obtain an enriched preparation of caveolae and gas chromatographic techniques to determine fatty acyl composition. In addition, methods used to visualize and quantify lipid rafts using a fluorescent probe Laurdan in living cells will also be described.  

3.1337           Lipid Raft-Redox Signaling Platforms in Plasma Membrane

Yi, F., Jin, S. and Li, P-L. Methods in Mol. Biol., 580, 93-107 (2009)   Membrane lipid rafts (LRs) have been demonstrated to be importantly involved in transmembrane signaling in a variety of mammalian cells. Many receptors can be aggregated within the LR clusters to form signaling platforms. Currently, LRs were reported to be clustered to aggregate, recruit, and assemble NADPH oxidase subunits and related proteins in various cells in response to various stimuli, forming redox signaling platforms. These LR signaling platforms may play important roles in the regulation of cellular activity and cell function, and also in the development of cell dysfunction or injury associated with various pathological stimuli. This LRs clustering-mediated mechanism is considered to take a center stage in redox signaling associated with death receptors. In this chapter, some basic methods and procedures for characterization of LR-redox signaling platforms formation and for determination of the function of these signaling platforms are described in detail, which include identification of LR-redox signaling platforms in cell membrane by using fluorescent or confocal microscopy of LR-redox signaling platforms and fluorescent resonance energy transfer analysis, isolation of LR-redox signaling platforms by flotation of detergent-resistant membranes, and function measurement of LR-redox signaling platforms by electron spin resonance spectroscopy. It is expected that information provided here will help readers to design necessary experiments in their studies on LR signaling platforms and redox regulation of cell function.  

3.1338            Hsp90 Co-localizes with Rab-GDI-1 and Regulates Agonist-induced Amylase Release in AR42J Cells

Raffaniello, R., Fedorova, D., Ip, D. and Rafiq, S. Cell. Physiol. Biochem., 24(5-6), 369-378 (2009)   Rab proteins are small GTPases required for vesicle trafficking through the secretory and endocytic pathways. Rab GDP-dissociation inhibitor (rab-GDI) regulates Rab protein function and localization by maintaining Rab proteins in the GDP-bound conformation. Two isoforms of rab-GDI are present in most mammalian cells: GDI-1 and GDI-2. It has recently been demonstrated that a Heat shock protein 90 (Hsp90) chaperone complex regulates the interactions between Rab proteins and Rab-GDI-1. The AR42J cell line is derived from rat pancreatic exocrine tumor cells and develops an acinar-like phenotype when treated with dexamethasone (Dex). The aim of the present study was to examine the expression of rab-GDI isoforms and Hsp90 in AR42J cells in the presence or absence of Dex. Rab-GDI:Hsp90 interactions were also examined. Both rab-GDI isoforms were detected in AR42J cells by immunoblotting. In Dex-treated cells, quantitative immunoblotting revealed that rab-GDI-1 expression increased by 28%, although this change was not statistically significant. Rab-GDI-2 levels were unaltered by Dex treatment. Approximately 21% rab-GDI-1 was membrane associated, whereas rab-GDI-2 was exclusively cytosolic. Dex treatment did not affect the subcellular distribution of rab-GDI isoforms. Hsp90 was present in the cytosolic and membrane fractions of AR42J cells and co-immunoprecipitated with cytosolic rab-GDI-1. Moreover, density gradient centrifugation of AR42J cell membranes revealed that Hsp90 and rab-GDI-1 co-localize on low- and high-density membrane fractions, including amylase-containing secretory granules. The Hsp90 inhibitor, geldanamycin, inhibited CCK-8-induced amylase release from these cells in a dose-dependent manner. Our results indicate that as AR42J cells differentiate into acinar-like cells, rab-GDI isoform expression and localization is not significantly altered. Moreover, our findings suggest that Hsp90 regulates agonist-induced secretion in exocrine cells by interacting with rab-GDI-1.  

3.1339           The cytosolic domain of PEX3, a protein involved in the biogenesis of peroxisomes, binds membrane lipids

Pinto, M.P., Grou, C.P., Fransen, M., Sa-Miranda, C. And Azevedo, J.E. Biochim. Biophys. Acta, 1793, 1669-1675 (2009)   According to current models, most newly synthesized peroxisomal intrinsic membrane proteins are recognized in the cytosol and targeted to the peroxisomal membrane by PEX19. At the organelle membrane the PEX19-cargo protein complex interacts with PEX3, a protein believed to possess only one transmembrane domain and exposing the majority of its polypeptide chain into the cytosol. In agreement with this topological model, a recombinant protein comprising the cytosolic domain of PEX3 can be purified in a soluble and monomeric form in the absence of detergents or other solubilizing agents. Here, we show that this recombinant protein actually precipitates when incubated with mild detergents, suggesting that this domain of PEX3 interacts with amphipathic molecules. Following this observation, we tested this recombinant protein in lipid-binding assays and found that it interacts strongly with liposomes inducing their flocculation or even partial solubilization. The implications of these findings are discussed.  

3.1340           An involvement of yeast peroxisomal channels in transmembrane transfer of glyoxylate cycle intermediates

Antonenkov, V.D., Mindhoff, S., Grunau, S., Erdmann, R. And Hiltunen, J.K. Int. J. Biochem. Cell. Biol., 41(2), 2546-2554 (2009)   The separate localization of glyoxylate cycle enzymes in the peroxisomes and the cytosol of the yeast Saccharomyces cerevisiae indicates that the peroxisomal membrane must permit the flow of metabolites between the two compartments. The transfer of these metabolites may require peroxisomal membrane channel(s). We used an electrophysiological approach (reconstitution assay in lipid bilayers) to assess the ability of peroxisomal membrane channels to conduct different solutes including metabolites of the glyoxylate cycle. At least two distinct channel-forming activities were detected in peroxisomal preparations. One of these activities was highly inducible by dithiothreitol and showed large-amplitude current increments when 1 M KCl was used as a bath solution. Single-channel analysis revealed that the inducible channel is anion-selective (P_Cl^-/P_K⁺=2.6;  P_citrate/P_K⁺=1.6) and displays flickering at holding potentials over ±30 mV directed upward or downward relative to the main open state of the channel. The channel inducible by DTT facilitates the transfer of solutes with a molecular mass up to 400 Da, sufficient to allow the transmembrane trafficking of glyoxylate cycle intermediates between the peroxisomal lumen and the cytoplasm.  

3.1341           Cryptococcus neoformans cryoultramicrotomy and vesicle fractionation reveals an intimate association between membrane lipids and glucuronoxylomannan

Oliveira, D.., Nimrichter, L., Miranda, K., Frases, S., Faull, K.F., Casadevall, A. And Roddrigues, M.L. Fungal Genetics and Biology, 46, 956-963 (2009)   Cryptococcus neoformans is an encapsulated pathogenic fungus. The cryptococcal capsule is composed of polysaccharides and is necessary for virulence. It has been previously reported that glucuronoxylomannan (GXM), the major capsular component, is synthesized in cytoplasmic compartments and transported to the extracellular space in vesicles, but knowledge on the organelles involved in polysaccharide synthesis and traffic is extremely limited. In this paper we report the GXM distribution in C. neoformans cells sectioned by cryoultramicrotomy and visualized by transmission electron microscopy (TEM) and polysaccharide immunogold staining. Cryosections of fungal cells showed high preservation of intracellular organelles and cell wall structure. Incubation of cryosections with an antibody to GXM revealed that cytoplasmic structures associated to vesicular compartments and reticular membranes are in close proximity to the polysaccharide. GXM was generally found in association with the membrane of intracellular compartments and within different layers of the cell wall. Analysis of extracellular fractions from cryptococcal supernatants by transmission electron microscopy in combination with serologic, chromatographic and spectroscopic methods revealed fractions containing GXM and lipids. These results indicate an intimate association of GXM and lipids in both intracellular and extracellular spaces consistent with polysaccharide synthesis and transport in membrane-associated structures.  

3.1342           Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells

Borot, F., Vieu, D-L., Faure, G., Fritsch, J., Colas, J., Moriceau, S., Baudouin-Legros, M., Brouillard, F., Ayala-Sanmartin, J., Touqui, L., Chanson, M., Edelman, A. and Ollero, M. PloSOne, 4(10), e7116 (2009)   The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2α) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2α. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-α. This was concomitant with increased IL-8 synthesis and cPLA2α activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-β-cyclodextrin induced further cPLA2α activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-α-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2α and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-α-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis.  

3.1343           ABC Transporter Pdr10 Regulates the Membrane Microenvironment of Pdr12 in Saccharomyces cerevisiae

Rockwell, N.C., Wolfger, H., Kuchler, K. and Thorner, J.

1. Membrane Biol., 229, 27-52 (2009)

  The eukaryotic plasma membrane exhibits both asymmetric distribution of lipids between the inner and the outer leaflet and lateral segregation of membrane components within the plane of the bilayer. In budding yeast (Saccharomyces cerevisiae), maintenance of leaflet asymmetry requires P-type ATPases, which are proposed to act as inward-directed lipid translocases (Dnf1, Dnf2, and the associated protein Lem3), and ATP-binding cassette (ABC) transporters, which are proposed to act as outward-directed lipid translocases (Pdr5 and Yor1). The S. cerevisiae genome encodes two other Pdr5-related ABC transporters: Pdr10 (67% identity) and Pdr15 (75% identity). We report the first analysis of Pdr10 localization and function. A Pdr10-GFP chimera was located in discrete puncta in the plasma membrane and was found in the detergent-resistant membrane fraction. Compared to control cells, a pdr10∆ mutant was resistant to sorbate but hypersensitive to the chitin-binding agent Calcofluor White. Calcofluor sensitivity was attributable to a partial defect in endocytosis of the chitin synthase Chs3, while sorbate resistance was attributable to accumulation of a higher than normal level of the sorbate exporter Pdr12. Epistasis analysis indicated that Pdr10 function requires Pdr5, Pdr12, Lem3, and mature sphingolipids. Strikingly, Pdr12 was shifted to the detergent-resistant membrane fraction in pdr10∆ cells. Pdr10 therefore acts as a negative regulator for incorporation of Pdr12 into detergent-resistant membranes, a novel role for members of the ABC transporter superfamily.  

3.1344           Solubilization, purification, and reconstitution of α2β1 isozyme of Na+/K+-ATPase from caveolae of pulmonary smooth muscle plasma membrane: comparative studies with DHPC, C12E8, and Triton X-100

Ghosh, B., Chakraborti, T., Kar, P., Dey, K. and Chakraborti, S. Mol. Cell. Biochem., 323, 169-184 (2009)   We identified α2, α1, and β1 isoforms of Na+/K+-ATPase in caveolae vesicles of bovine pulmonary smooth muscle plasma membrane. The biochemical and biophysical characteristics of the α2β1 isozyme of Na+/K+-ATPase from caveolae vesicles were studied during solubilization and purification using the detergents 1,2-heptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C12E8), and Triton X-100, and reconstitution with the phospholipid dioleoyl-phosphatidylcholine (DOPC). DHPC was determined to be superior to C12E8, whereas C12E8 was better than Triton X-100 in the active enzyme yields and specific activity. Fluorescence studies with DHPC-purified α2β1 isozyme of Na+/K+-ATPase elicited higher E1Na−E2 K transition compared with that of the C12E8- and Triton X-100-purified enzyme. The rate of Na+ efflux in DHPC–DOPC-reconstituted isozyme was higher compared to the C12E8–DOPC- and Triton X100–DOPC-reconstituted enzyme. Circular dichroism analysis suggests that the DHPC-purified α2β1 isozyme of Na+/K+-ATPase possessed more organized secondary structure compared to the C12E8- and Triton X-100-purified isozyme.  

3.1345           Localisation of endothelin B receptor variants to plasma membrane microdomains and its effects on downstream signalling

Grossmann, S.., Higashiyama, S., Oksche, A., Schaefer, M. and Tannert, A. Mol. Membrane Biol., 26(5-7), 279-292 (2009)   The endothelin B (ET_B) receptor can undergo a proteolytic cleavage resulting in an unglycosylated N-terminally truncated receptor. We investigated whether ET_B receptor processing affects caveolar localisation and mitogenic signalling. Distinct subcellular localisations of ET_B receptor constructs and epidermal growth factor (EGF) receptor ligands were analysed performing detergent-free caveolae preparations and total internal reflection fluorescence microscopy. ET_B receptor-induced transactivation of the EGF receptor and its downstream signalling was investigated performing shedding assays and ERK1/2 phosphorylation analyses. In COS7 cells, the N-terminally truncated but not the full-length or glycosylation-deficient ET_B receptor localised to caveolae. In caveolae-free HEK293 cells, only ET_B receptor constructs fused to caveolin-2 localised to membrane microdomains. A caveolar accumulation of the ET_B receptor disfavoured EGF receptor ligand shedding. Nonetheless, the activation of ERK1/2 was efficient and long-lasting. In HEK293 cells, the shedding activity was also impaired by N-terminal truncation. The subsequent ERK1/2 phosphorylation was long-lasting only for the full-length ET_B receptor. We conclude that the ET_B receptor localisation might depend on the presence of caveolae within the cell investigated. The data further suggest that caveolar enrichment of ET_B receptors does not facilitate the release of EGF receptor ligands. However, independent of their localisation, ET_B receptors are able to induce an ERK1/2 phosphorylation.  

3.1346           Functions of lipid raft membrane microdomains at the blood–brain barrier

Dodelet-Devillers, A., cayrol, R., van Horssen, J., Haqqani, A.S., de Vries, H.E., Engelhardt, B., Greenwood, J. and Prat, A.

1. Mol. Med., 87, 765-774 (2009)

  The blood–brain barrier (BBB) is a highly specialized structural and functional component of the central nervous system that separates the circulating blood from the brain and spinal cord parenchyma. Brain endothelial cells (BECs) that primarily constitute the BBB are tightly interconnected by multiprotein complexes, the adherens junctions and the tight junctions, thereby creating a highly restrictive cellular barrier. Lipid-enriched membrane microdomain compartmentalization is an inherent property of BECs and allows for the apicobasal polarity of brain endothelium, temporal and spatial coordination of cell signaling events, and actin remodeling. In this manuscript, we review the role of membrane microdomains, in particular lipid rafts, in the BBB under physiological conditions and during leukocyte transmigration/diapedesis. Furthermore, we propose a classification of endothelial membrane microdomains based on their function, or at least on the function ascribed to the molecules included in such heterogeneous rafts: (1) rafts associated with interendothelial junctions and adhesion of BECs to basal lamina (scaffolding rafts); (2) rafts involved in immune cell adhesion and migration across brain endothelium (adhesion rafts); (3) rafts associated with transendothelial transport of nutrients and ions (transporter rafts).  

3.1347           Lipid microdomain polarization is required for NADPH oxidase-dependent ROS signaling in Picea meyeri pollen tube tip growth

Liu, P., Li, R-L., Wang, Q-L., Niehaus, K., Baluska, F., Samaj, J. And Lin, J-X. Plant J., 60(2), 303-313 (2009)   The polarization of sterol-enriched lipid microdomains has been linked to morphogenesis and cell movement in diverse cell types. Recent biochemical evidence has confirmed the presence of lipid microdomains in plant cells; however, direct evidence for a functional link between these microdomains and plant cell growth is still lacking. Here, we reported the involvement of lipid microdomains in NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) signaling in Picea meyeri pollen tube growth. Staining with di-4-ANEPPDHQ or filipin revealed that sterol-enriched microdomains were polarized to the growing tip of the pollen tube. Sterol sequestration with filipin disrupted membrane microdomain polarization, depressed tip-based ROS formation, dissipated tip-focused cytosolic Ca2+ gradient and thereby arrested tip growth. NOX clustered at the growing tip, and corresponded with the ordered membrane domains. Immunoblot analysis and native gel assays demonstrated that NOX was partially associated with detergent-resistant membranes and, furthermore, that NOX in a sterol-dependent fashion depends on membrane microdomains for its enzymatic activity. In addition, in vivo time-lapse imaging revealed the coexistence of a steep tip-high apical ROS gradient and subapical ROS production, highlighting the reported signaling role for ROS in polar cell growth. Our results suggest that the polarization of lipid microdomains to the apical plasma membrane, and the inclusion of NOX into these domains, contribute, at least in part, to the ability to grow in a highly polarized manner to form pollen tubes.  

3.1348           Hitchhiking of Cu/Zn Superoxide Dismutase to Peroxisomes - Evidence for a Natural Piggyback Import Mechanism in Mammals

Islinger, M., Li, K.W., Seitz, J., Völkl, A. And Lüers, G.H. Traffic, 10, 1711-1721 (2009)   Most newly synthesized peroxisomal proteins are imported in a receptor-mediated fashion, depending on the interaction of a peroxisomal targeting signal (PTS) with its cognate targeting receptor Pex5 or Pex7 located in the cytoplasm. Apart from this classic mechanism, heterologous protein complexes that have been proposed more than a decade ago are also to be imported into peroxisomes. However, it remains still unclear if this so-called piggyback import is of physiological relevance in mammals. Here, we show that Cu/Zn superoxide dismutase 1 (SOD1), an enzyme without an endogenous PTS, is targeted to peroxisomes using its physiological interaction partner 'copper chaperone of SOD1' (CCS) as a shuttle. Both proteins have been identified as peroxisomal constituents by 2D-liquid chromatography mass spectrometry of isolated rat liver peroxisomes. Yet, while a major fraction of CCS was imported into peroxisomes in a PTS1-dependent fashion in CHO cells, overexpressed SOD1 remained in the cytoplasm. However, increasing the concentrations of both CCS and SOD1 led to an enrichment of SOD1 in peroxisomes. In contrast, CCS-mediated SOD1 import into peroxisomes was abolished by deletion of the SOD domain of CCS, which is required for heterodimer formation. SOD1/CCS co-import is the first demonstration of a physiologically relevant piggyback import into mammalian peroxisomes.  

3.1349           Urokinase-receptor-mediated phenotypic changes in vascular smooth muscle cells require the involvement of membrane rafts

Kiyan, J., Smith, G., Haller, H. And Dumler, I. Biochem. J., 423, 343-351 (2009)   The cholesterol-enriched membrane microdomains lipid rafts play a key role in cell activation by recruiting and excluding specific signalling components of cell-surface receptors upon receptor engagement. Our previous studies have demonstrated that the GPI (glycosylphosphatidylinositol)-linked uPAR [uPA (urokinase-type plasminogen activator) receptor], which can be found in lipid rafts and in non-raft fractions, can mediate the differentiation of VSMCs (vascular smooth muscle cells) towards a pathophysiological de-differentiated phenotype. However, the mechanism by which uPAR and its ligand uPA regulate VSMC phenotypic changes is not known. In the present study, we provide evidence that the molecular machinery of uPAR-mediated VSMC differentiation employs lipid rafts. We show that the disruption of rafts in VSMCs by membrane cholesterol depletion using MCD (methyl-β-cyclodextrin) or filipin leads to the up-regulation of uPAR and cell de-differentiation. uPAR silencing by means of interfering RNA resulted in an increased expression of contractile proteins. Consequently, disruption of lipid rafts impaired the expression of these proteins and transcriptional activity of related genes. We provide evidence that this effect was mediated by uPAR. Similar effects were observed in VSMCs isolated from Cav1^−/− (caveolin-1-deficient) mice. Despite the level of uPAR being significantly higher after the disruption of the rafts, uPA/uPAR-dependent cell migration was impaired. However, caveolin-1 deficiency impaired only uPAR-dependent cell proliferation, whereas cell migration was strongly up-regulated in these cells. Our results provide evidence that rafts are required in the regulation of uPAR-mediated VSMC phenotypic modulations. These findings suggest further that, in the context of uPA/uPAR-dependent processes, caveolae-associated and non-associated rafts represent different signalling membrane domains.  

3.1350           Analysis of the dual function of the ESCRT-III protein Snf7 in endocytic trafficking and in gene expression

Weiss, P., Huppert, S. and Kölling, R. Biochem. J., 424, 89-97 (2009)   ESCRT (endosomal sorting complex required for transport)-III mediates the budding and scission of intralumenal vesicles into multivesicular endosomes in yeast. For the main ESCRT-III subunit Snf7, an additional role in activation of the transcription factor Rim101 (the ‘Rim pathway’) is now also firmly established. In the present study, we investigate how these two Snf7 functions are related to each other. By generating SNF7 mutations that severely affect endocytic trafficking, but leave the Rim pathway function intact, we show that the two functions of SNF7 can be separated genetically. We analysed in detail how the SNF7 mutations affect the interaction of Snf7 with its various binding partners. Although the interactions with proteins Rim13 and Rim20, necessary for the Rim-pathway-related functions, were not altered by the mutations, there was a strong effect on interactions with components of the ESCRT pathway. The interactions, as measured by co-immunoprecipitation, with the ESCRT-III subunits Vps20 and Vps24 were strongly increased by the mutations, whereas the interactions with proteins Vps4 and Bro1, acting downstream of ESCRT-III, were reduced. As Vps4 is required for disassembly of ESCRT-III these results suggest that ESCRT-III is more stable in our SNF7 mutants. In line with this notion, a higher fraction of mutant Snf7 protein was detected at the membrane. Upon a shift to alkaline pH, a stronger binding signal for virtually all interaction partners, except Vps4, was observed. This indicates that the ESCRT network at the endosomal membrane is more extensive under these conditions.  

3.1351           Missense mutations in the SH3TC2 protein causing Charcot-Marie-Tooth disease type 4C affect its localization in the plasma membrane and endocytic pathway

Lupo, V., Galindo, M.I., Martinez-Rubio, D., Sevilla, T., Vilchez, J.J., Palau, F. and Espinos, C. Hum. Mol. Genet., 18(23), 4603-4614 (2009)   Mutations in SH3TC2 (KIAA1985) cause Charcot-Marie-Tooth disease (CMT) type 4C, a demyelinating inherited neuropathy characterized by early-onset and scoliosis. Here we demonstrate that the SH3TC2 protein is present in several components of the endocytic pathway including early endosomes, late endosomes and clathrin-coated vesicles close to the trans-Golgi network and in the plasma membrane. Myristoylation of SH3TC2 in glycine 2 is necessary but not sufficient for the proper location of the protein in the cell membranes. In addition to myristoylation, correct anchoring also needs the presence of SH3 and TPR domains. Mutations that cause a stop codon and produce premature truncations that remove most of the TPR domains are expressed as the wild-type protein. In contrast, missense mutations in or around the region of the first-TPR domain are absent from early endosomes, reduced in plasma membrane and late endosomes and are variably present in clathrin-coated vesicles. Our findings suggest that the endocytic and membrane trafficking pathway is involved in the pathogenesis of CMT4C disease. We postulate that missense mutations of SH3TC2 could impair communication between the Schwann cell and the axon causing an abnormal myelin formation.  

3.1352           Motor Protein–Dependent Membrane Trafficking of KCl Cotransporter-4 Is Important for Cancer Cell Invasion

Chen, Y-F., Chou, C-Y., Wilkins, R.J., Ellory, J.C., Mount, D.B. and Shen, M-R. Cancer Res., 69(22), 8585-8593 (2009)   The KCl cotransporter (KCC) is a major determinant of osmotic homeostasis and plays an emerging role in tumor biology. This study stresses the important role of KCC4 in tumor malignant behavior. Real-time reverse transcription-PCR on samples collected by laser microdissection and immunofluorescent stainings with different KCC isoform antibodies indicate that KCC4 is abundant in metastatic cervical and ovarian cancer tissues. Insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulate KCC4 recruitment from a presumably inactive cytoplasmic pool of endoplasmic reticulum and Golgi to plasma membrane along actin cytoskeleton that is significantly inhibited by LY294002 and wortmannin. Throughout the trafficking process, KCC4 is incorporated into lipid rafts that function as a platform for the association between KCC4 and myosin Va, an actin-dependent motor protein. KCC4 and ezrin, a membrane cytoskeleton linker, colocalize at lamellipodia of migratory cancer cells. Interference with KCC activity by either an inhibitor or a dominant-negative loss-of-function mutant profoundly suppressed the IGF-I–induced membrane trafficking of KCC4 and the structural interaction between KCC4 and ezrin near the cell surface. Endogenous cancer cell invasiveness was significantly attenuated by small interfering RNA targeting KCC4, and the residual invasiveness was much less sensitive to IGF-I or EGF stimulation. In the metastatic cancer tissues, KCC4 colocalizes with IGF-I or EGF, indicating a likely in vivo stimulation of KCC4 function by growth factors. Thus, blockade of KCC4 trafficking and surface expression may provide a potential target for the prevention of IGF-I– or EGF-dependent cancer spread.  

3.1353           Fc RI ligation leads to a complex with BLT1 in lipid rafts that enhances rat lung macrophage antimicrobial functions

Serezani, C.H., Aronoff, D.M., Sitrin, R.G. and Peters-Golden, M. Blood, 114(15), 3316-3324 (2009)   Leukotriene (LT) B₄ is generated in response to engagement of the Fc receptor (Fc R) and potently contributes to Fc R-mediated antimicrobial functions in pulmonary alveolar macrophages. In this study, we report that the LTB₄ receptor leukotriene B₄ receptor 1 (BLT1) redistributes from nonlipid raft (LR) to LR membrane microdomains upon immunoglobulin G–red blood cell, but not LTB₄, challenge. Cholesterol depletion to disrupt LRs abolished LTB₄-induced enhancement of phagocytosis, microbicidal activity, and signaling. The dependence on LR integrity for BLT1 signaling correlated with formation of a complex consisting of BLT1, its primary coupled G protein G i3, Src kinase, and Fc RI within LRs. This association was dependent on Src-mediated phosphorylation of BLT1. These data identify a novel form of regulation in which engagement of a macrophage immunoreceptor recruits a stimulatory G protein–coupled receptor into a LR microdomain with resultant enhanced antimicrobial signaling.  

3.1354           Ubiquilin and p97/VCP bind erasin, forming a complex involved in ERAD

Lim, P., Danner, R., Liang, J., Doong, H., harman, C., Srinivasan, D., Rothenberg, C., Wang, H., Ye, Y., Fang, S. And Monteiro, M.J.

1. Cell Biol., 187(2), 201-217 (2009)

  Unwanted proteins in the endoplasmic reticulum (ER) are exported into the cytoplasm and degraded by the proteasome through the ER-associated protein degradation pathway (ERAD). Disturbances in ERAD are linked to ER stress, which has been implicated in the pathogenesis of several human diseases. However, the composition and organization of ERAD complexes in human cells is still poorly understood. In this paper, we describe a trimeric complex that we propose functions in ERAD. Knockdown of erasin, a platform for p97/VCP and ubiquilin binding, or knockdown of ubiquilin in human cells slowed degradation of two classical ERAD substrates. In Caenorhabditis elegans, ubiquilin and erasin are ER stress-response genes that are regulated by the ire-1 branch of the unfolded protein response pathway. Loss of ubiquilin or erasin resulted in activation of ER stress, increased accumulation of polyubiquitinated proteins, and shortened lifespan in worms. Our results strongly support a role for this complex in ERAD and in the regulation of ER stress.  

3.1355           Filamin A Regulates Caveolae Internalization and Trafficking in Endothelial Cells

Sverdlov, M., Shinin, V., Place, A.T., Castellon, M. and Minshall, R.D. Mol. Biol. Cell, 20, 4531-4540 (2009)   Transcytosis via caveolae is critical for maintaining vascular homeostasis by regulating the tissue delivery of macromolecules, hormones, and lipids. In the present study, we test the hypothesis that interactions between F-actin cross-linking protein filamin A and caveolin-1 facilitate the internalization and trafficking of caveolae. Small interfering RNA-mediated knockdown of filamin A, but not filamin B, reduced the uptake and transcytosis of albumin by 35 and 60%, respectively, without altering the actin cytoskeletal structure or cell–cell adherens junctions. Mobility of both intracellular caveolin-1–green fluorescent protein (GFP)-labeled vesicles measured by fluorescence recovery after photobleaching and membrane-associated vesicles measured by total internal reflection-fluorescence microscopy was decreased in cells with reduced filamin A expression. In addition, in melanoma cells that lack filamin A (M2 cells), the majority of caveolin-1-GFP was localized on the plasma membrane, whereas in cells in which filamin A expression was reconstituted (A7 cells and M2 cells transfected with filamin A-RFP), caveolin-1-GFP was concentrated in intracellular vesicles. Filamin A association with caveolin-1 in endothelial cells was confirmed by cofractionation of these proteins in density gradients, as well as by coimmunoprecipitation. Moreover, this interaction was enhanced by Src activation, associated with increased caveolin-1 phosphorylation, and blocked by Src inhibition. Taken together, these data suggest that filamin A association with caveolin-1 promotes caveolae-mediated transport by regulating vesicle internalization, clustering, and trafficking.  

3.1356           Characteristics of alpha/beta interferon induction after infection of murine fibroblasts with wild-type and mutant alphaviruses

Burke, C.W., Gardner, C.L., Steffan, J.J., Ryman, K.D. and Klimstra, W.B. Virology, 395, 121-132 (2009)   We examined the characteristics of interferon alpha/beta (IFN-α/β) induction after alphavirus or control Sendai virus (SeV) infection of murine fibroblasts (MEFs). As expected, SeV infection of wild-type (wt) MEFs resulted in strong dimerization of IRF3 and the production of high levels of IFN-α/β. In contrast, infection of MEFs with multiple alphaviruses failed to elicit detectable IFN-α/β. In more detailed studies, Sindbis virus (SINV) infection caused dimerization and nuclear migration of IRF3, but minimal IFN-β promoter activity, although surprisingly, the infected cells were competent for IFN production by other stimuli early after infection. A SINV mutant defective in host macromolecular synthesis shutoff induced IFN-α/β in the MEF cultures dependent upon the activities of the TBK1 IRF3 activating kinase and host pattern recognition receptors (PRRs) PKR and MDA5 but not RIG-I. These results suggest that wild-type alphaviruses antagonize IFN induction after IRF3 activation but also may avoid detection by host PRRs early after infection.  

3.1357           N-Myristoylation targets dihydroceramide Δ4-desaturase 1 to mitochondria: Partial involvement in the apoptotic effect of myristic acid

Beauchamp, E., Tekpli, X., Marteil, g., Lagadic-Gossmann, D., Legrand, P. And Rioux, V. Biochimie, 91, 1411-1419 (2009)   This study was designed to analyze the effect of myristic acid on ceramide synthesis and its related lipoapoptosis pathway. It was previously observed that myristic acid binds dihydroceramide Δ4-desaturase 1 (DES1) through N-myristoylation and activates this enzyme involved in the final de novo ceramide biosynthesis step. In the present study, we show first by immunofluorescence microscopy and subcellular fractionation that DES1 myristoylation targets part of the recombinant protein to the mitochondria in COS-7 cells. In addition, native dihydroceramide Δ4-desaturase activity was found in both the endoplasmic reticulum and mitochondria in rat hepatocytes. Dihydroceramide conversion to ceramide was increased in COS-7 cells expressing DES1 and incubated with myristic acid. The expression of the wild-type myristoylable DES1-Gly alone, but not the expression of the unmyristoylable mutant DES1-Ala, induced apoptosis of COS-7 cells. Finally, myristic acid alone also increased the production of cellular ceramide and had an apoptotic effect. This effect was potentiated on caspase activity when the myristoylable form of DES1 was expressed. Therefore, these results suggest that the myristoylation of DES1 can target the enzyme to the mitochondria leading to an increase in ceramide levels which in turn contributes to partially explain the apoptosis effect of myristic acid in COS-7 cells.  

3.1358           Outer Membrane Machinery and Alginate Synthesis Regulators Control Membrane Vesicle Production in Pseudomonas aeruginosa

Tashiro, Y., Sakai, R., Toyofuku, M., Sawada, I., Nakajima-Kambe, T., Uchiyama, H. and Nomura, N.

1. Bacteriol., 191(24), 7509-7519 (2009)

  The opportunistic human bacterial pathogen Pseudomonas aeruginosa produces membrane vesicles (MVs) in its surrounding environment. Several features of the P. aeruginosa MV production mechanism are still unknown. We previously observed that depletion of Opr86, which has a role in outer membrane protein (OMP) assembly, resulted in hypervesiculation. In this study, we showed that the outer membrane machinery and alginate synthesis regulatory machinery are closely related to MV production in P. aeruginosa. Depletion of Opr86 resulted in increased expression of the periplasmic serine protease MucD, suggesting that the accumulation of misfolded OMPs in the periplasm is related to MV production. Indeed, the mucD mutant showed a mucoid phenotype and the mucD mutation caused increased MV production. Strains with the gene encoding alginate synthetic regulator AlgU, MucA, or MucB deleted also caused altered MV production. Overexpression of either MucD or AlgW serine proteases resulted in decreased MV production, suggesting that proteases localized in the periplasm repress MV production in P. aeruginosa. Deletion of mucD resulted in increased MV proteins, even in strains with mutations in the Pseudomonas quinolone signal (PQS), which serves as a positive regulator of MV production. This study suggests that misfolded OMPs may be important for MV production, in addition to PQS, and that these regulators act in independent pathways.  

3.1359           Bovine viral diarrhea virus NS4B protein is an integral membrane protein associated with Golgi markers and rearranged host membranes

Weiskircher, E., Aligo, J., Ning, G. and Konan, K.V. Virol. J., 6, 185-199 (2009)   Background Very little is known about BVDV NS4B, a protein of approximately 38 kDa. However, a missense mutation in NS4B has been implicated in changing BVDV from a cytopathic to noncytopathic virus, suggesting that NS4B might play a role in BVDV pathogenesis. Though this is one possible function, it is also likely that NS4B plays a role in BVDV genome replication. For example, BVDV NS4B interacts with NS3 and NS5A, implying that NS4B is part of a complex, which contains BVDV replicase proteins. Other possible BVDV NS4B functions can be inferred by analogy to hepatitis C virus (HCV) NS4B protein. For instance, HCV NS4B remodels host membranes to form the so-called membranous web, the site for HCV genome replication. Finally, HCV NS4B is membrane-associated, implying that HCV NS4B may anchor the virus replication complex to the membranous web structure. Unlike its HCV counterpart, we know little about the subcellular distribution of BVDV NS4B protein. Further, it is not clear whether NS4B is localized to host membrane alterations associated with BVDV infection. Results We show first that release of infectious BVDV correlates with the kinetics of BVDV genome replication in infected cells. Secondly, we found that NS4B subcellular distribution changes over the course of BVDV infection. Further, BVDV NS4B is an integral membrane protein, which colocalizes mainly with the Golgi compartment when expressed alone or in the context of BVDV infection. Additionally, BVDV induces host membrane rearrangement and these membranes contain BVDV NS4B protein. Finally, NS4B colocalizes with replicase proteins NS5A and NS5B proteins, raising the possibility that NS4B is a component of the BVDV replication complex. Interestingly, NS4B was found to colocalize with mitochondria suggesting that this organelle might play a role in BVDV genome replication or cytopathogenicity. Conclusion These results show that BVDV NS4B is an integral membrane protein associated with the Golgi apparatus and virus-induced membranes, the putative site for BVDV genome replication. On the basis of NS4B Colocalization with NS5A and NS5B, we conclude that NS4B protein is an integral component of the BVDV replication complex.  

3.1360           EFFECT OF SIMVASTATIN ON GLIOMA CELL PROLIFERATION, MIGRATION, AND APOPTOSIS

Wu, H., Jiang, H., Lu, D., Xiong, Y., Qu, C., Zhou, D., Mahmood, A. and Chopp, M. Neurosurgery, 65(6), 1087-1097 (2009)   OBJECTIVE: In this study, we investigated the effects of simvastatin on proliferation, migration, and apoptosis in human U251 and U87 glioma cells and the underlying molecular mechanism. METHODS: We used colony formation assay to test the cell proliferation, in vitro scratch assay to examine the cell migration, and caspase-3 activity assay, annexin V staining, and cytochrome C release to evaluate the cell apoptosis. Lipid raft fractions were isolated from glioma cells. Total cholesterol content assay was used to test the change of cholesterol level in lipid raft fractions. Immunocytochemistry staining was performed to detect the changes of lipid rafts in cell membranes. Western blotting analysis was performed to examine the signal transduction both in cells and in lipid raft fractions. RESULTS: Simvastatin inhibited proliferation and migration of U251 and U87 cells dose dependently. Simvastatin induced an increase of caspase-3 activity and annexin V staining, and down-regulated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Simvastatin also decreased cholesterol content in lipid raft fractions, suppressed caveolin-1 expression in the lipid rafts, and induced Fas translocation into lipid rafts, suggesting that simvastatin may inhibit the prosurvival PI3K/Akt pathway and trigger caspase-3-dependent apoptotic cell death through the modulation of lipid rafts. CONCLUSION: These results suggest that modulation of lipid rafts, Fas translocation, and PI3K/Akt/caspase-3 pathway are involved in the antitumor effect of simvastatin and may have a potential role in cancer prevention and treatment.  

3.1361           APP Anterograde Transport Requires Rab3A GTPase Activity for Assembly of the Transport Vesicle

Szodorai, A., Kuan, Y-H., Hunzelmann, S., Engel, U., Sakane, A., Sasaki, T., Takai, Y., Kirsch, J., Müller, U., Beyreuther, K., Brady, S., Morfini, G. and Kins, S.

1. Neurosci., 29(46), 14534-14544 (2009)

  The amyloid precursor protein (APP) is anterogradely transported by conventional kinesin in a distinct transport vesicle, but both the biochemical composition of such a vesicle and the specific kinesin-1 motor responsible for transport are poorly defined. APP may be sequentially cleaved by β- and -secretases leading to accumulation of β-amyloid (Aβ) peptides in brains of Alzheimer's disease patients, whereas cleavage of APP by -secretases prevents Aβ generation. Here, we demonstrate by time-lapse analysis and immunoisolations that APP is a cargo of a vesicle containing the kinesin heavy chain isoform kinesin-1C, the small GTPase Rab3A, and a specific subset of presynaptic protein components. Moreover, we report that assembly of kinesin-1C and APP in this vesicle type requires Rab3A GTPase activity. Finally, we show cleavage of APP in transport vesicles by -secretase activity, likely mediated by ADAM10. Together, these data indicate that maturation of APP transport vesicles, including recruitment of conventional kinesin, requires Rab3 GTPase activity.  

3.1362           Cyclodextrin overcomes deficient lysosome-to-endoplasmic reticulum transport of cholesterol in Niemann-Pick type C cells

Abi-Mosleh, L., Infante, R.E., Radhakrishnan, A., Goldstein, J.L. and Brown, M.S. PNAS, 106(46), 19316-19321 (2009)   A handoff model has been proposed to explain the egress from lysosomes of cholesterol derived from receptor-mediated endocytosis of LDL. Cholesterol is first bound by soluble Niemann-Pick C2 (NPC2) protein, which hands off the cholesterol to the N-terminal domain of membrane-bound NPC1. Cells lacking NPC1 or NPC2 accumulate LDL-derived cholesterol in lysosomes and fail to deliver LDL cholesterol to the endoplasmic reticulum (ER) for esterification by acyl-CoA acyltransferase (ACAT) and for inhibition of sterol regulatory element-binding protein cleavage. Here, we support this model by showing that the cholesterol transport defect in NPC1 mutant cells is restricted to lysosomal export. Other cholesterol transport pathways appear normal, including the movement of cholesterol from the plasma membrane to the ER after treatment of cells with 25-hydroxycholesterol or sphingomyelinase. The NPC1 or NPC2 block in cholesterol delivery to the ER can be overcome by 2-hydroxypropyl-β-cyclodextrin, which leads to a marked increase in ACAT-mediated cholesterol esterification. The buildup of cholesteryl esters in the cytosol is expected to be much less toxic than the buildup of free cholesterol in the lysosomes of patients with mutations in NPC1 or NPC2.  

3.1363           Immobilization of the Glycosylphosphatidylinositol-anchored Gas1 Protein into the Chitin Ring and Septum Is Required for Proper Morphogenesis in Yeast

Rolli, E., Ragni, E., calderon, J., Porello, S., Fascio, U. and Popolo, L. Mol. Biol. Cell, 20, 4856-4870 (2009)   Gas1p is a glucan-elongase that plays a crucial role in yeast morphogenesis. It is predominantly anchored to the plasma membrane through a glycosylphosphatidylinositol, but a fraction was also found covalently bound to the cell wall. We have used fusions with the green fluorescent protein or red fluorescent protein (RFP) to determine its localization. Gas1p was present in microdomains of the plasma membrane, at the mother-bud neck and in the bud scars. By exploiting the instability of RFP-Gas1p, we identified mobile and immobile pools of Gas1p. Moreover, in chs3 cells the chitin ring and the cross-linked Gas1p were missing, but this unveiled an additional unexpected localization of Gas1p along the septum line in cells at cytokinesis. Localization of Gas1p was also perturbed in a chs2 mutant where a remedial septum is produced. Phenotypic analysis of cells expressing a fusion of Gas1p to a transmembrane domain unmasked new roles of the cell wall-bound Gas1p in the maintenance of the bud neck size and in cell separation. We present evidence that Crh1p and Crh2p are required for tethering Gas1p to the chitin ring and bud scar. These results reveal a new mechanism of protein immobilization at specific sites of the cell envelope.  

3.1364           Inhibition of acyl-coenzyme A: cholesterol acyl transferase modulates amyloid precursor protein trafficking in the early secretory pathway

Huttunen, H.J., Peach, C., Bhattacharyya, R., Barren, C., Pettingell, W., Hutter-Paier, B., Windisch, M., Berezovska, O. and Kovacs, D.M. FASEB J., 23, 3819-3828 (2009)   Amyloid β-peptide (Aβ) has a central role in the pathogenesis of Alzheimer’s disease (AD). Cellular cholesterol homeostasis regulates endoproteolytic generation of Aβ from the amyloid precursor protein (APP). Previous studies have identified acyl-coenzyme A: cholesterol acyltransferase (ACAT), an enzyme that regulates subcellular cholesterol distribution, as a potential therapeutic target for AD. Inhibition of ACAT activity decreases Aβ generation in cell- and animal-based models of AD through an unknown mechanism. Here we show that ACAT inhibition retains a fraction of APP molecules in the early secretory pathway, limiting the availability of APP for secretase-mediated proteolytic processing. ACAT inhibitors delayed the trafficking of immature APP molecules from the endoplasmic reticulum (ER) as shown by metabolic labeling and live-cell imaging. This resulted in partial ER retention of APP and enhanced ER-associated degradation of APP by the proteasome, without activation of the unfolded protein response pathway. The ratio of mature APP to immature APP was reduced in brains of mice treated with ACAT inhibitors, and strongly correlated with reduced brain APP-C99 and cerebrospinal fluid Aβ levels in individual animals. Our results identify a novel ACAT-dependent mechanism that regulates secretory trafficking of APP, likely contributing to decreased Aβ generation in vivo.—Huttunen, H. J., Peach, C., Bhattacharyya, R., Barren, C., Pettingell, W., Hutter-Paier, B., Windisch, M., Berezovska, O., Kovacs, D. M. Inhibition of acyl-coenzyme A: cholesterol acyl transferase modulates amyloid precursor protein trafficking in the early secretory pathway.  

3.1365           Peroxisomal proteomics: Biomonitoring in mussels after the Prestige’s oil spill

Apraiz, I., Cajaraville, M.P. and Cristobal, S. Marine Pollution Bulletin, 58, 1815-1826 (2009)   Peroxisomal proteomics was applied to assess possible biological effects after the Prestige’s oil spill. Mussels were sampled in July 2004 and 2005 in four stations in the NW (closest to the spill) and NE coasts of the Iberian Peninsula. Principal components analysis (PCA) suggested differences in protein expression among stations and sampling years. Several proteins were putatively identified by mass spectrometry and immunolocalization. PC1 separated the NW stations in 2004 from the rest of the stations and sampling years mainly due to up-regulation of peroxisomal β-oxidation proteins and PMP70. PC3 separated the NE stations, based on up-regulation of the antioxidant enzyme catalase in 2004 compared to 2005. PC4 separated the stations in the NE and the NW. This work shows that environmental proteomics, together with multivariate data analysis, could provide information to interpret the effects of oil spills at cellular level in mussels.  

3.1366           Palmitoylation of Hepatitis C Virus Core Protein Is Important for Virion Production

Majeau, N., Fromentin, R., Savard, C., Duval, M., Tremblay, M.J. and Leclerc, D.

1. Biol. Chem., 284(49), 33915-33925 (2009)

  Hepatitis C virus core protein is the viral nucleocapsid of hepatitis C virus. Interaction of core with cellular membranes like endoplasmic reticulum (ER) and lipid droplets (LD) appears to be involved in viral assembly. However, how these interactions with different cellular membranes are regulated is not well understood. In this study, we investigated how palmitoylation, a post-translational protein modification, can modulate the targeting of core to cellular membranes. We show that core is palmitoylated at cysteine 172, which is adjacent to the transmembrane domain at the C-terminal end of core. Site-specific mutagenesis of residue Cys¹⁷² showed that palmitoylation is not involved in the maturation process carried out by the signal peptide peptidase or in the targeting of core to LD. However, palmitoylation was shown to be important for core association with smooth ER membranes and ER closely surrounding LDs. Finally, we demonstrate that mutation of residue Cys¹⁷² in the J6/JFH1 virus genome clearly impairs virion production.  

3.1367           Colorectal cancer cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells

Hong, B.S., Cho, J-H., Kim, H., Choi, E-J., Rho, S., Kim, J., Kim, J.H., Choi, D-S., Kim, Y-K., Hwang, D. and Gho, Y.S. BMC Genomics, 10, 556-568 (2009)   Background Various cancer cells, including those of colorectal cancer (CRC), release microvesicles (exosomes) into surrounding tissues and peripheral circulation. These microvesicles can mediate communication between cells and affect various tumor-related processes in their target cells. Results We present potential roles of CRC cell-derived microvesicles in tumor progression via a global comparative microvesicular and cellular transcriptomic analysis of human SW480 CRC cells. We first identified 11,327 microvesicular mRNAs involved in tumorigenesis-related processes that reflect the physiology of donor CRC cells. We then found 241 mRNAs enriched in the microvesicles above donor cell levels, of which 27 were involved in cell cycle-related processes. Network analysis revealed that most of the cell cycle-related microvesicle-enriched mRNAs were associated with M-phase activities. The integration of two mRNA datasets showed that these M-phase-related mRNAs were differentially regulated across CRC patients, suggesting their potential roles in tumor progression. Finally, we experimentally verified the network-driven hypothesis by showing a significant increase in proliferation of endothelial cells treated with the microvesicles. Conclusion Our study demonstrates that CRC cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells, suggesting that microvesicles of cancer cells can be involved in tumor growth and metastasis by facilitating angiogenesis-related processes. This information will help elucidate the pathophysiological functions of tumor-derived microvesicles, and aid in the development of cancer diagnostics, including colorectal cancer.  

3.1368           Cystin Localizes to Primary Cilia via Membrane Microdomains and a Targeting Motif

Tao, B., bu, S., Yang, Z., Siroky, B., Kappes, J.C., Kispert, A. and Guay-Woodford, L.M.

1. Am. Soc. Nephrol., 20, 2570-2580 (2009)

  Primary cilia are dynamic, complex structures that contain >500 proteins, including several related to polycystic kidney disease. How these proteins target to cilia and assemble is unknown. We previously identified Cys1 as the gene responsible for disease in Cys1^cpk mice, a mouse model of autosomal recessive polycystic kidney disease; this gene encodes cystin, a 145–amino acid cilium-associated protein. Here, we characterized the localization of cystin in the embryonic kidney and liver, in isolated renal collecting ducts, and in an inner medullary collecting duct mouse cell line. Because endogenous levels of cystin expression are low, we generated inner medullary collecting duct cell lines that stably express enhanced green fluorescence protein–tagged constructs of wild-type cystin or various truncation mutants. We determined that cystin is myristoylated at its G2 residue and that N-myristoylated cystin fractionates with membrane microdomains. Furthermore, the N-myristoylation signal is necessary but not sufficient to target cystin to the primary cilium. Analysis of deletion and chimeric constructs identified an AxEGG motif that is necessary to target and retain cystin in the cilium. Derangement of these localization motifs may lead to cystic kidney disease.  

3.1369           Oral PEG 15–20 protects the intestine against radiation: role of lipid rafts

Valuckaite, V., Zaborina, O., Long, J., Hauer-Jensen, M., Wang, J., Holbrook, C., Zaborin, A., Drabik, K., Katdare, M., Mauceri, H., Weichselbaum, R., Firestone, M.A., Lee, K.Y., Chang, E.B., Matthews, J. and Alverdy, J.C. Am. J. Physiool. Gastrointest. Liver Physiol., 297, G1041-G1052 (2009)   Intestinal injury following abdominal radiation therapy or accidental exposure remains a significant clinical problem that can result in varying degrees of mucosal destruction such as ulceration, vascular sclerosis, intestinal wall fibrosis, loss of barrier function, and even lethal gut-derived sepsis. We determined the ability of a high-molecular-weight polyethylene glycol-based copolymer, PEG 15–20, to protect the intestine against the early and late effects of radiation in mice and rats and to determine its mechanism of action by examining cultured rat intestinal epithelia. Rats were exposed to fractionated radiation in an established model of intestinal injury, whereby an intestinal segment is surgically placed into the scrotum and radiated daily. Radiation injury score was decreased in a dose-dependent manner in rats gavaged with 0.5 or 2.0 g/kg per day of PEG 15–20 (n = 9–13/group, P < 0.005). Complementary studies were performed in a novel mouse model of abdominal radiation followed by intestinal inoculation with Pseudomonas aeruginosa (P. aeruginosa), a common pathogen that causes lethal gut-derived sepsis following radiation. Mice mortality was decreased by 40% in mice drinking 1% PEG 15–20 (n = 10/group, P < 0.001). Parallel studies were performed in cultured rat intestinal epithelial cells treated with PEG 15–20 before radiation. Results demonstrated that PEG 15–20 prevented radiation-induced intestinal injury in rats, prevented apoptosis and lethal sepsis attributable to P. aeruginosa in mice, and protected cultured intestinal epithelial cells from apoptosis and microbial adherence and possible invasion. PEG 15–20 appeared to exert its protective effect via its binding to lipid rafts by preventing their coalescence, a hallmark feature in intestinal epithelial cells exposed to radiation.  

3.1370           Neural Cell Adhesion Molecule Modulates Dopaminergic Signaling and Behavior by Regulating Dopamine D2 Receptor Internalization

Xiao, M-F., Xu, J-C., Tereshchenko, Y., Novak, D., Schachner, M. and Kleene, R.

1. Neurosci., 29(47), 14752-14763 (2009)

  The dopaminergic system plays an important role in the etiology of schizophrenia, and most antipsychotic drugs exert their functions by blocking dopamine D₂ receptors (D₂Rs). Since the signaling strength mediated by D₂Rs is regulated by internalization and degradation processes, it is crucial to identify molecules that modulate D₂R localization at the cell surface. Here, we show that the neural cell adhesion molecule (NCAM) promotes D₂R internalization/desensitization and subsequent degradation via direct interaction with a short peptide in the third intracellular loop of the D₂R. NCAM deficiency in mice leads to increased numbers of D₂Rs at the cell surface and augmented D₂R signaling as a result of impaired D₂R internalization. Furthermore, NCAM-deficient mice show higher sensitivity to the psychostimulant apomorphine and exaggerated activity of dopamine-related locomotor behavior. These results demonstrate that, in addition to its classical function in cell adhesion, NCAM is involved in regulating the trafficking of the neurotransmitter receptor D₂R as well as receptor-mediated signaling and behavior, thus implicating NCAM as modulator of the dopaminergic system and a potential pharmacological target for dopamine-related neurological and psychiatric disorders.  

3.1371           M-Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex

Hase, K., Kimura, S., Takatsu, H., Ohmae, M., Kawano, S., Kitamura, H., Ito, M., Waterai, H., Clayton Hazelett, C., Yeaman, C. and Ohno, H. Nature Cell Biol., 11(12), 1427-1432 (2009)   Cell–cell communication is essential for the development and homeostasis of multicellular organisms. Recently, a new type of cell–cell communication was discovered that is based on the formation of thin membranous nanotubes between remote cells^{1, 2}. These long membrane tethers, termed tunneling nanotubes (TNTs), form an intercellular conduit and have been shown to enable the transport of various cellular components and signals. However, the molecular basis for TNT formation remains to be elucidated. Here we report that a mammalian protein, M-Sec, induces de novo formation of numerous membrane protrusions extending from the plasma membrane, some of which tether onto adjacent cells and subsequently form TNT-like structures. Depletion of M-Sec by RNA interference (RNAi) greatly reduced endogenous TNT formation as well as intercellular propagation of a calcium flux in a macrophage cell line. Furthermore, blockage of the interaction of M-Sec with Ral and the exocyst complex, which serves as a downstream effector of Ral, attenuated the formation of membrane nanotubes. Our results reveal that M-Sec functions as a key regulator of membrane nanotube formation through interaction with the Ral–exocyst pathway.  

3.1372           Identification of novel proteins in isolated polyphosphate vacuoles in the primitive red alga Cyanidioschyzon merolae

Yagisawa, F., Nishida, K., Yoshida, M., Ohnuma, M., Shimada, T., Fujiwara, T., Yoshida, Y., Misumi, O., Kuroiwa, H. and Kuroiwa, T. Plant. J., 60, 882-893 (2009)   Plant vacuoles are organelles bound by a single membrane, and involved in various functions such as intracellular digestion, metabolite storage, and secretion. To understand their evolution and fundamental mechanisms, characterization of vacuoles in primitive plants would be invaluable. Algal cells often contain polyphosphate-rich compartments, which are thought to be the counterparts of seed plant vacuoles. Here, we developed a method for isolating these vacuoles from Cyanidioschyzon merolae, and identified their proteins by MALDI TOF-MS. The vacuoles were of unexpectedly high density, and were highly enriched at the boundary between 62 and 80% w/v iodixanol by density-gradient ultracentrifugation. The vacuole-containing fraction was subjected to SDS-PAGE, and a total of 46 proteins were identified, including six lytic enzymes, 13 transporters, six proteins for membrane fusion or vesicle trafficking, five non-lytic enzymes, 13 proteins of unknown function, and three miscellaneous proteins. Fourteen proteins were homologous to known vacuolar or lysosomal proteins from seed plants, yeasts or mammals, suggesting functional and evolutionary relationships between C. merolae vacuoles and these compartments. The vacuolar localization of four novel proteins, namely CMP249C (metallopeptidase), CMJ260C (prenylated Rab receptor), CMS401C (ABC transporter) and CMT369C (o-methyltransferase), was confirmed by labeling with specific antibodies or transient expression of hemagglutinin-tagged proteins. The results presented here provide insights into the proteome of C. merolae vacuoles and shed light on their functions, as well as indicating new features.  

3.1373           Stomatin-like Protein-1 Interacts with Stomatin and Is Targeted to Late Endosomes

Mairhofer, M., Steiner, M., Salzer, U. and Prohaska, R.

1. Biol. Chem., 284(42), 29218-29229 (2009)

  The human stomatin-like protein-1 (SLP-1) is a membrane protein with a characteristic bipartite structure containing a stomatin domain and a sterol carrier protein-2 (SCP-2) domain. This structure suggests a role for SLP-1 in sterol/lipid transfer and transport. Because SLP-1 has not been investigated, we first studied the molecular and cell biological characteristics of the expressed protein. We show here that SLP-1 localizes to the late endosomal compartment, like stomatin. Unlike stomatin, SLP-1 does not localize to the plasma membrane. Overexpression of SLP-1 leads to the redistribution of stomatin from the plasma membrane to late endosomes suggesting a complex formation between these proteins. We found that the targeting of SLP-1 to late endosomes is caused by a GYXXΦ (Φ being a bulky, hydrophobic amino acid) sorting signal at the N terminus. Mutation of this signal results in plasma membrane localization. SLP-1 and stomatin co-localize in the late endosomal compartment, they co-immunoprecipitate, thus showing a direct interaction, and they associate with detergent-resistant membranes. In accordance with the proposed lipid transfer function, we show that, under conditions of blocked cholesterol efflux from late endosomes, SLP-1 induces the formation of enlarged, cholesterol-filled, weakly LAMP-2-positive, acidic vesicles in the perinuclear region. This massive cholesterol accumulation clearly depends on the SCP-2 domain of SLP-1, suggesting a role for this domain in cholesterol transfer to late endosomes.  

3.1374           Ceramide kinase regulates phospholipase C and phosphatidylinositol 4, 5, bisphosphate in phototransduction

Dasgupta, U., Bamba, T., Chiantia, S., Karim, P., Tayoun, A.N.A., Yonamine, I., Rawat, S.S., Rao, R.P., Nagashima, K., Fukusaki, E., Puri, V., Dolph, P.J., Schwille, P., Acharya, J.K. and Acharya, U. PNAS, 106(47), 20063-20068 (2009) Phosphoinositide-specific phospholipase C (PLC) is a central effector for many biological responses regulated by G-protein–coupled receptors including Drosophila phototransduction where light sensitive channels are activated downstream of NORPA, a PLCβ homolog. Here we show that the sphingolipid biosynthetic enzyme, ceramide kinase, is a novel regulator of PLC signaling and photoreceptor homeostasis. A mutation in ceramide kinase specifically leads to proteolysis of NORPA, consequent loss of PLC activity, and failure in light signal transduction. The mutant photoreceptors also undergo activity-dependent degeneration. Furthermore, we show that a significant increase in ceramide, resulting from lack of ceramide kinase, perturbs the membrane microenvironment of phosphatidylinositol 4, 5, bisphosphate (PIP₂), altering its distribution. Fluorescence image correlation spectroscopic studies on model membranes suggest that an increase in ceramide decreases clustering of PIP₂ and its partitioning into ordered membrane domains. Thus ceramide kinase–mediated maintenance of ceramide level is important for the local regulation of PIP₂ and PLC during phototransduction.  

3.1375           The amino terminus of tau inhibits kinesin-dependent axonal transport: Implications for filament toxicity

LaPointe, N.E., Morfini, G., Pigino, G., Gaisina, I.N., Kozikowski, A.P., Binder, L.I. and Brady, S.T.

1. Neurosic. Res., 87(2), 440-451 (2009)

  The neuropathology of Alzheimer's disease (AD) and other tauopathies is characterized by filamentous deposits of the microtubule-associated protein tau, but the relationship between tau polymerization and neurotoxicity is unknown. Here, we examined effects of filamentous tau on fast axonal transport (FAT) using isolated squid axoplasm. Monomeric and filamentous forms of recombinant human tau were perfused in axoplasm, and their effects on kinesin- and dynein-dependent FAT rates were evaluated by video microscopy. Although perfusion of monomeric tau at physiological concentrations showed no effect, tau filaments at the same concentrations selectively inhibited anterograde (kinesin-dependent) FAT, triggering the release of conventional kinesin from axoplasmic vesicles. Pharmacological experiments indicated that the effect of tau filaments on FAT is mediated by protein phosphatase 1 (PP1) and glycogen synthase kinase-3 (GSK-3) activities. Moreover, deletion analysis suggested that these effects depend on a conserved 18-amino-acid sequence at the amino terminus of tau. Interestingly, monomeric tau isoforms lacking the C-terminal half of the molecule (including the microtubule binding region) recapitulated the effects of full-length filamentous tau. Our results suggest that pathological tau aggregation contributes to neurodegeneration by altering a regulatory pathway for FAT.  

3.1376           Content of endoplasmic reticulum and Golgi complex membranes positively correlates with the proliferative status of brain cells

Silvestre, D.C., Maccioni, H.J.F. and Caputto, B.L.

1. Neurosci. Res., 87(4), 857-865 (2009)

  Although the molecular and cellular basis of particular events that lead to the biogenesis of membranes in eukaryotic cells has been described in detail, understanding of the intrinsic complexity of the pleiotropic response by which a cell adjusts the overall activity of its endomembrane system to accomplish these requirements is limited. Here we carried out an immunocytochemical and biochemical examination of the content and quality of the endoplasmic reticulum (ER) and Golgi apparatus membranes in two in vivo situations characterized by a phase of active cell proliferation followed by a phase of declination in proliferation (rat brain tissue at early and late developmental stages) or by permanent active proliferation (gliomas and their most malignant manifestation, glioblastomas multiforme). It was found that, in highly proliferative phases of brain development (early embryo brain cells), the content of ER and Golgi apparatus membranes, measured as total lipid phosphorous content, is higher than in adult brain cells. In addition, the concentration of protein markers of ER and Golgi is also higher in early embryo brain cells and in human glioblastoma multiforme cells than in adult rat brain or in nonpathological human brain cells. Results suggest that the amount of endomembranes and the concentration of constituent functional proteins diminish as cells decline in their proliferative activity. Although the molecular and cellular basis of particular events that lead to the biogenesis of membranes in eukaryotic cells has been described in detail, understanding of the intrinsic complexity of the pleiotropic response by which a cell adjusts the overall activity of its endomembrane system to accomplish these requirements is limited. Here we carried out an immunocytochemical and biochemical examination of the content and quality of the endoplasmic reticulum (ER) and Golgi apparatus membranes in two in vivo situations characterized by a phase of active cell proliferation followed by a phase of declination in proliferation (rat brain tissue at early and late developmental stages) or by permanent active proliferation (gliomas and their most malignant manifestation, glioblastomas multiforme). It was found that, in highly proliferative phases of brain development (early embryo brain cells), the content of ER and Golgi apparatus membranes, measured as total lipid phosphorous content, is higher than in adult brain cells. In addition, the concentration of protein markers of ER and Golgi is also higher in early embryo brain cells and in human glioblastoma multiforme cells than in adult rat brain or in nonpathological human brain cells. Results suggest that the amount of endomembranes and the concentration of constituent functional proteins diminish as cells decline in their proliferative activity.  

3.1377           The two-hydrophobic domain tertiary structure of reticulon proteins is critical for modulation of β-secretase BACE1

Kume, H., Murayama, K.S. and Araki,, W.

1. Neurosci. Res., 87(13), 2963-2972 (2009)

  β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is a membrane-bound protease that is essential for the production of β-amyloid protein (Aβ). Given the crucial role of Aβ accumulation in Alzheimer's disease (AD), inhibition of BACE1 activity may represent a feasible therapeutic strategy in the treatment of AD. Recently, we and others identified reticulon 3 (RTN3) and reticulon 4-B/C (RTN4-B/C or Nogo-B/C) as membrane proteins that interact with BACE1 and inhibit its ability to produce Aβ. In this study, we employed various mutants of RTN3 and RTN4-C and C. elegans RTN to investigate the molecular mechanisms by which RTNs regulate BACE1. We found that RTN3 mutants lacking the N-terminal or C-terminal or loop domain as well as a RTN4-C mutant lacking the C-terminal domain bound to BACE1 comparably to wild-type RTN3 and RTN4-C. Furthermore, overexpression of wild-type RTN3, RTN4-C, and these RTN mutants similarly reduced Aβ40 and Aβ42 secretion by cells expressing Swedish mutant APP. C. elegans RTN, which has low homology to human RTNs, also interacted with BACE1 and inhibited Aβ secretion. In contrast, two RTN3 mutants containing deletions of the first or second potential transmembrane domains and an RTN3 swap mutant of the second transmembrane domain bound BACE1 but failed to inhibit Aβ secretion. Collectively, these results suggest that the two-transmembrane-domain tertiary structure of RTN proteins is critical for the ability of RTNs to modulate BACE1 activity, whereas N-terminal, C-terminal and loop regions are not essential for this function.  

3.1378           Proteome of plant peroxisomes: new perspectives on the role of these organelles in cell biology

Palma, J.M., Corpas, F.J. and del Rio, L.A. Proteomics, 9(9), 2301-2312 (2009)   Peroxisomes are cell organelles bounded by a single membrane with a basically oxidative metabolism. Peroxisomes house catalase and H₂O₂-producing flavin-oxidases as the main protein constituents. However, since their discovery in early fifties, a number of new enzymes and metabolic pathways have been reported to be also confined to these organelles. Thus, the presence of exo- and endo-peptidases, superoxide dismutases, the enzymes of the plant ascorbate-glutathione cycle plus ascorbate and glutathione, several NADP-dehydrogenases, and also L-arginine-dependent nitric oxide synthase activity has evidenced the relevant role of these organelles in cell physiology. In recent years, the study of new functions of peroxisomes has become a field of intensive research in cell biology, and these organelles have been proposed to be a source of important signal molecules for different transduction pathways. In plants, peroxisomes participate in seed germination, leaf senescence, fruit maturation, response to abiotic and biotic stress, photomorphogenesis, biosynthesis of the plant hormones jasmonic acid and auxin, and in cell signaling by reactive oxygen and nitrogen species (ROS and RNS, respectively). In order to decipher the nature and specific role of the peroxisomal proteins in these processes, several approaches including in vivo and in vitro import assays and generation of mutants have been used. In the last decade, the development of genomics and the report of the first plant genomes provided plant biologists a powerful tool to assign to peroxisomes those proteins which harbored any of the two peroxisomal targeting signals (PTS, either PTS1 or PTS2) described so far. Unfortunately, those molecular approaches could not give any response to those proteins previously localized in plant peroxisomes by classical biochemical and cell biology methods that did not contain any PTS. However, more recently, proteomic studies of highly purified organelles have provided evidence of the presence in peroxisomes of new proteins not previously reported. Thus, the contribution of proteomic approaches to the biology of peroxisomes is essential, not only for elucidation of the mechanisms involved in the import of the PTS1- and PTS2-independent proteins, but also to the understanding of the role of these organelles in the cell physiology of plant growth and development.    

3.1379           Proteomic identification of proteins translocated to membrane microdomains upon treatment of fibroblasts with the glycosphingolipid, C8-β-D-lactosylceramide

Kim, S-y., Wang, T-k., Singh, R.D., Wheatley, C.L., Marks, D.L. and Pagano, R.E. Proteomics, 9(18), 4321-4328 (2009)   Plasma membrane (PM) microdomains, including caveolae and other cholesterol-enriched subcompartments, are involved in the regulation of many cellular processes, including endocytosis, attachment and signaling. We recently reported that brief incubation of human skin fibroblasts with the synthetic glycosphingolipid, D-erythro-octanoyl-lactosylceramide (C8-D-e-LacCer), stimulates endocytosis via caveolae and induces the appearance of micron-size microdomains on the PM. To further understand the effects of C8-D-e-LacCer treatment on PM microdomains, we used a detergent-free method to isolate microdomain-enriched membranes from fibroblasts treated ±C8-D-e-LacCer, and performed 2-DE and mass spectrophotometry to identify proteins that were altered in their distribution in microdomains. Several proteins were identified in the microdomain-enriched fractions, including lipid transfer proteins and proteins related to the functions of small GTPases. One protein, Rho-associated protein kinase 2 (ROCK2), was verified by Western blotting to occur in microdomain fractions and to increase in these fractions after D-e-LacCer treatment. Immunofluorescence revealed that ROCK2 exhibited an increased localization at or near the PM in C8-D-e-LacCer-treated cells. In contrast, ROCK2 distribution in microdomains was decreased by treatment of cells with C8-L-threo-lactosylceramide, a glycosphingolipid with non-natural stereochemistry. This study identifies new microdomain-associated proteins and provides evidence that microdomains play a role in the regulation of the Rho/ROCK signaling pathway.  

3.1380           Gram-positive bacteria produce membrane vesicles: Proteomics-based characterization of Staphylococcus aureus-derived membrane vesicles

Lee, E-Y., Choi, D-Y., Kim, D-K., Kim, J-W., Park, J O., Kim, S., Kim, S-H., Desiderio, D.M., Kim, Y-K., Kim, K-P- and Gho, Y.S. Proteomics, 9(24), 5425-5436 (2009)   Although archaea, Gram-negative bacteria, and mammalian cells constitutively secrete membrane vesicles (MVs) as a mechanism for cell-free intercellular communication, this cellular process has been overlooked in Gram-positive bacteria. Here, we found for the first time that Gram-positive bacteria naturally produce MVs into the extracellular milieu. Further characterizations showed that the density and size of Staphylococcus aureus-derived MVs are both similar to those of Gram-negative bacteria. With a proteomics approach, we identified with high confidence a total of 90 protein components of S. aureus-derived MVs. In the group of identified proteins, the highly enriched extracellular proteins suggested that a specific sorting mechanism for vesicular proteins exists. We also identified proteins that facilitate the transfer of proteins to other bacteria, as well to eliminate competing organisms, antibiotic resistance, pathological functions in systemic infections, and MV biogenesis. Taken together, these observations suggest that the secretion of MVs is an evolutionally conserved, universal process that occurs from simple organisms to complex multicellular organisms. This information will help us not only to elucidate the biogenesis and functions of MVs, but also to develop therapeutic tools for vaccines, diagnosis, and antibiotics effective against pathogenic strains of Gram-positive bacteria.  

3.1381           TRAPPC2L is a Novel, Highly Conserved TRAPP-Interacting Protein

Scrivens, P.J., Shahrzad, N., Moores, A., Morin, A., Brunet, S. and Sacher, M. Traffic, 10(6), 724-736 (2009)   Mutations in the trafficking protein particle complex C2 protein (TRAPPC2), a mammalian ortholog of yeast Trs20p and a component of the trafficking protein particle (TRAPP) vesicle tethering complex, have been linked to the skeletal disorder spondyloepiphyseal dysplasia tarda (SEDT). Intriguingly, the X-linked TRAPPC2 is just one of a complement of Trs20-related genes in humans. Here we characterize TRAPPC2L, a novel, highly conserved TRAPP-interacting protein related to TRAPPC2 and the uncharacterized yeast open reading frame YEL048c. TRAPPC2L and TRAPPC2 genes are found in pairs across species and show broad and overlapping expression, suggesting they are functionally distinct, a notion supported by yeast complementation studies and biochemical characterization. RNA interference-mediated knockdown of either TRAPPC2L or TRAPPC2 in HeLa cells leads to fragmentation of the Golgi, implicating both proteins in Golgi dynamics. Gradient fractionation of cellular membranes indicates that TRAPPC2L is found with a portion of cellular TRAPP on very low-density membranes whereas the remainder of TRAPP, but not TRAPPC2L, is found associated with Golgi markers. YEL048c displays genetic interactions with TRAPP II-encoding genes and the gene product co-fractionates with and interacts with yeast TRAPP II. Taken together these results indicate that TRAPPC2L and its yeast ortholog YEL048c are novel TRAPP-interacting proteins that may modulate the function of the TRAPP II complex.  

3.1382           Proteomic characterization of lipid raft proteins in amyotrophic lateral sclerosis mouse spinal cord

Zhai, J., Ström, A.L., Kilty, R., Venkatakrishnan, P., White, J., Everson, W.V., Smart, E.J. and Zhu, H. FEBS J., 276(12), 3308-3323 (2009)   Familial amyotrophic lateral sclerosis (ALS) has been linked to mutations in the copper/zinc superoxide dismutase (SOD1) gene. The mutant SOD1 protein exhibits a toxic gain-of-function that adversely affects the function of neurons. However, the mechanism by which mutant SOD1 initiates ALS is unclear. Lipid rafts are specialized microdomains of the plasma membrane that act as platforms for the organization and interaction of proteins involved in multiple functions, including vesicular trafficking, neurotransmitter signaling, and cytoskeletal rearrangements. In this article, we report a proteomic analysis using a widely used ALS mouse model to identify differences in spinal cord lipid raft proteomes between mice overexpressing wild-type (WT) and G93A mutant SOD1. In total, 413 and 421 proteins were identified in the lipid rafts isolated from WT and G93A mice, respectively. Further quantitative analysis revealed a consortium of proteins with altered levels between the WT and G93A samples. Functional classification of the 67 altered proteins revealed that the three most affected subsets of proteins were involved in: vesicular transport, and neurotransmitter synthesis and release; cytoskeletal organization and linkage to the plasma membrane; and metabolism. Other protein changes were correlated with alterations in: microglia activation and inflammation; astrocyte and oligodendrocyte function; cell signaling; cellular stress response and apoptosis; and neuronal ion channels and neurotransmitter receptor functions. Changes of selected proteins were independently validated by immunoblotting and immunohistochemistry. The significance of the lipid raft protein changes in motor neuron function and degeneration in ALS is discussed, particularly for proteins involved in vesicular trafficking and neurotransmitter signaling, and the dynamics and regulation of the plasma membrane-anchored cytoskeleton.  

3.1383           Vezatin, an integral membrane protein of adherens junctions, is required for the sound resilience of cochlear hair cells

Bahloul, A., Simmler, M-C., Michel, V., Leibovici, M., Perfeettini, I., Roux, I., Weil, D., Nouaille, S., Zuo, J., Zadro, C., Licastro, D., GAsparini, P., Avan, P., Hardelin, J-P- and petit, C. EMBO Mol. Med., 1(2), 125-138 (2009)   Loud sound exposure is a significant cause of hearing loss worldwide. We asked whether a lack of vezatin, an ubiquitous adherens junction protein, could result in noise-induced hearing loss. Conditional mutant mice bearing non-functional vezatin alleles only in the sensory cells of the inner ear (hair cells) indeed exhibited irreversible hearing loss after only one minute exposure to a 105 dB broadband sound. In addition, mutant mice spontaneously underwent late onset progressive hearing loss and vestibular dysfunction related to substantial hair cell death. We establish that vezatin is an integral membrane protein with two adjacent transmembrane domains, and cytoplasmic N- and C-terminal regions. Late recruitment of vezatin at junctions between MDCKII cells indicates that the protein does not play a role in the formation of junctions, but rather participates in their stability. Moreover, we show that vezatin directly interacts with radixin in its actin-binding conformation. Accordingly, we provide evidence that vezatin associates with actin filaments at cell–cell junctions. Our results emphasize the overlooked role of the junctions between hair cells and their supporting cells in the auditory epithelium resilience to sound trauma.  

3.1384           Calcium homeostasis in plant cell nuclei

Mazars, C., Bourque, S., Mithöfer, A., Pugin, A. and Ranjeva, R. New Phytologist, 181(2), 261-274 (2009)   In plant cells, calcium-based signaling pathways are involved in a large array of biological processes, including cell division, polarity, growth, development and adaptation to changing biotic and abiotic environmental conditions. Free calcium changes are known to proceed in a nonstereotypical manner and produce a specific signature, which mirrors the nature, strength and frequency of a stimulus. The temporal aspects of calcium signatures are well documented, but their vectorial aspects also have a profound influence on biological output. Here, we will focus on the regulation of calcium homeostasis in the nucleus. We will discuss data and present hypotheses suggesting that, while interacting with other organelles, the nucleus has the potential to generate and regulate calcium signals on its own.

3.1385           GABA receptor proteins within lipid rafts in the AY-9944 model of atypical absence seizures

Huo, J., Cortez, M.A. and Snead III, O.C. Epilepsia, 50(4), 776-788 (2009)   Purpose: The inhibition of cholesterol synthesis with AY-9944 (AY) results in chronic recurrent atypical absence seizures in rodents. We hypothesized that cholesterol inhibition during the course of creating the AY model of atypical absence seizures results in an alteration of the entry of -aminobutyric acid (GABA)_A and GABA_B receptors into lipid rafts that contributes to epileptogenesis in this model. Methods: The cholesterol synthesis inhibitor AY (7.5 mg/kg) was administered on postnatal day (P) 2, P8, P14, and P20 in Long-Evans hooded rats. The incorporation of GABA_A and GABA_B receptor proteins into lipid rafts of the brain was then determined. Results: AY produced a shift of both GABA_A and GABA_B receptors in the examined detergent-resistant membranes (DRMs) and the soluble fractions. The percentage of the GABA_A and GABA_B receptors that shifted out of the DRMs varied between 17% and 50%, but the proportion of receptors in DRMs were decreased to levels around that of P5 animals or even lower. The shift observed in the AY-treated versus control animals was statistically significant (p < 0.01) for both GABA_A and GABA_B receptors. Conclusion: Cholesterol synthesis inhibition during rat brain development that is induced by AY leads to chronic atypical absence seizures and is associated with an alteration of GABA_A and GABA_B receptor proteins within lipid rafts. These data suggest a novel avenue of investigation into the epileptogenesis of experimental chronic atypical absence seizures.  

3.1386           Sorting defects of the tryptophan permease Tat2 in an erg2 yeast mutant

Daicho, K., Makino, N., Hiraki, T., Ueno, M., Uritani, M., Abe, F. and Ushimaru, T. FEMS Microbiol. Lett., 298(2), 218-227 (2009)   Cholesterol (ergosterol in yeast) in conjunction with sphingolipids forms tight-packing microdomains, ‘lipid rafts,’ which are thought to be critical for intracellular protein sorting in eukaryotic cells. When the activity of Erg9 involved in the first step of ergosterol biogenesis, but not that of Erg6 involved in a late step, is compromised, vacuolar degradation of the tryptophan permease Tat2 is promoted. It is unknown whether this difference simply reflects the difference between the inhibition of early and late steps. Here, it is shown that the deletion in ERG2, which encodes sterol C8–C7 isomerase (the next enzymatic step after Erg6), promotes the vacuolar degradation of Tat2. It suggests that the accumulation of specific sterol intermediates may alter lipid raft structures, promoting Tat2 degradation. The erg2Δ-mediated Tat2 degradation required Tat2 ubiquitination. Lipid raft association of Tat2 is compromised in erg2Δ cells. The erg2Δ mutation showed a synthetic growth defect with the trp1 mutation, indicating that Tat2 sorting is preferentially compromised in these mutants. Consistent with this notion, the raft-associated protein Pma1 was associated with detergent-resistant membranes and sorted to the plasma membrane. This study suggests the potential for the pharmacological control of cellular nutrient uptake in humans by regulating enzymes involved in cholesterol biogenesis.  

3.1387           MARCKS regulates lamellipodia formation induced by IGF-I via association with PIP2 and β-actin at membrane microdomains

Yamagushi, H., Shiraishi, M., Fukami, K., Tanabe, A., Ikeda-Matsuo, Y., Naito, Y. and Sasaki, Y.

1. Cell. Physiol., 220(3), 748-755 (2009)

  Myristoylated alanine-rich C kinase substrate (MARCKS) is considered to participate in formation of F-actin-based lamellipodia, which represents the first stage of neurite formation. However, the mechanism of how MARCKS is involved in lamellipodia formation is not precisely unknown. Using SH-SY5Y cells, we demonstrated here that MARCKS was translocated from cytosol to detergent-resistant membrane microdomains, known as lipid rafts, within 30 min after insulin-like growth factor-I (IGF-I) stimulation, which was accompanied by MARCKS dephosphorylation, β-actin accumulation in lipid rafts, and lamellipodia formation. The protein kinase C inhibitor, Ro-31-8220, and Rho-kinase inhibitors, HA1077 and Y27632, themselves decreased basal phosphorylation levels of MARCKS and coincidently elicited translocation of MARCKS to lipid rafts. On the other hand, the phosphoinositide 3-kinase inhibitor, LY294002, abolished IGF-I-induced dephosphorylation, translocation of MARCKS to lipid rafts, and lamellipodia formation. Treatment of cells with neomycin, a PIP2-masking reagent, attenuated the translocation of MARCKS to lipid rafts and the lamellipodia formation induced by IGF-I, although dephosphorylation of MARCKS was not affected. Immunocytochemical and immunoprecipitation analysis indicated that IGF-I stimulation induced the translocation of MARCKS to lipid rafts in the edge of lamellipodia and formation of the complex with PIP2. Moreover, we demonstrated that knockdown of endogenous MARCKS resulted in significant attenuation of IGF-I-induced β-actin accumulation in the lipid rafts and lamellipodia formation. These results suggest a novel role for MARCKS in lamellipodia formation induced by IGF-I via the translocation of MARCKS, association with PIP2, and accumulation of β-actin in the membrane microdomains.  

3.1388           Compartmentalization of epidermal growth factor receptor in liver plasma membrane

Wang, Y., Posner, B.I. and Balbis, A.

1. Cell.Biochem., 107(1), 96-103 (2009)

  We have investigated epidermal growth factor (EGF)-induced compartmentalization and activation of the EGF receptor (EGFR) in rat liver plasma membrane (PM) raft subfractions prepared by three different biochemical methods previously developed to characterize the composition of membrane rafts. Only detergent-resistant membranes (DRMs) possessed the basic characteristics attributed to membrane rafts. Following the administration of a low dose of EGF (1 µg/100 g BW) the content of EGFR in PM–DRMs did not change significantly; whereas after a higher dose of EGF (5 µg/100 g BW) we observed a rapid and marked disappearance of EGFR (around 80%) from both PM and DRM fractions. Interestingly, following the administration of either a low or high dose of EGF, the pool of EGFR in the PM–DRM fraction became highly Tyr-phosphorylated. In accordance with the higher level of EGFR Tyr-Phosphorylation, EGF induced an augmented recruitment of Grb2 and Shc proteins to PM–DRMs compared with whole PM. Furthermore neither high nor low doses of EGF affected the caveolin content in DRMs and PM. These observations suggest that EGFR located in DRMs are competent for signaling, and non-caveolae PM rafts are involved in the compartmentalization and internalization of the EGFR.  

3.1389           Clustering transfers the translocated Escherichia coli receptor into lipid rafts to stimulate reversible activation of c-Fyn

Hayward, R.D., Hume, P.J., Humphreys, D., Phillips, N., Smith, K. and Koronakis, V. Cell. Microbiol., 11(3), 433-441 (2009)   Enteropathogenic Escherichia coli (EPEC) mimic a ligand–receptor interaction to induce ‘pedestal-like’ pseudopodia on mammalian cells, providing a tractable system to study tyrosine kinase signalling to the actin cytoskeleton. EPEC delivers its own receptor (Tir), which is engaged by a bacterial surface ligand (intimin). When Tir delivery and activity are uncoupled, intimin-induced Tir clustering stimulates Tir^Y474 phosphorylation by the Src-family kinase (SFK) c-Fyn, triggering actin polymerization and pedestal formation. How c-Fyn specifically targets Tir and is regulated remains unknown. We show that clustering transfers Tir into cholesterol-rich detergent-resistant microdomains (DRMs), a signal prompting transient c-Fyn accumulation at bacterial adhesion sites. Co-clustering of Tir^Y474 and c-Fyn in DRMs rapidly stimulates robust kinase activation both by induced c-Fyn^Y531 dephosphorylation to unlock the inactive state and by reciprocal c-Fyn^Y417 autophosphorylation to promote activity. After signal induction, c-Fyn dissipates and the resting state restored by Csk-dependent phosphorylation of c-Fyn^Y531. These data illustrate a sophisticated mechanism evolved by a pathogen effector to reversibly regulate SFKs, and resolve early interactions at a model receptor initiating tyrosine kinase signalling.  

3.1390           Vps33a Mediates RANKL Storage in Secretory Lysosomes in Osteoblastic Cells

Kariya, Y., Homma, M., Aoki, S., Chiba, A. and Suzuki, H.

1. Bone Minerald Res., 24(10), 1741-1752 (2009)

  Previous studies have indicated that the amount of RANKL expressed on the cell surface of osteoblasts or bone marrow stromal cells (BMSCs) is considered an important factor determining the extent of osteoclast activation. However, subcellular trafficking of RANKL and its regulatory mechanisms in osteoblastic cells is still unclear. In this study, we showed that RANKL is predominantly localized in lysosomal organelles, but little is found on the cell surface of osteoblastic cells. We also showed that RANKL is relocated to the plasma membrane in response to stimulation with RANK‐Fc–coated beads, indicating that the lysosomal organelles where RANKL is localized function as secretory lysosomes. In addition, using a protein pull‐down method, we identified vacuolar protein sorting (Vps)33a as interacting with the cytoplasmic tail of RANKL. Furthermore, knockdown of Vps33a expression reduced the lysosomal storage of RANKL and caused the accumulation of newly synthesized RANKL in the Golgi apparatus, indicating that Vps33a is involved in transporting RANKL from the Golgi apparatus to secretory lysosomes. We also showed that suppression of Vps33a affects the cell surface expression level of RANKL and disrupts the regulated behavior of RANKL. These results suggest that RANKL storage in secretory lysosomes is important to control osteoclast activation and to maintain bone homeostasis.  

3.1391           Overexpression of Sna3 stabilizes tryptophan permease Tat2, potentially competing for the WW domain of Rsp5 ubiquitin ligase with its binding protein Bul1

Hiraki, T. and Ebe, F. FEBS Lett., 584, 55-60 (2010)   Tryptophan permease Tat2 in Saccharomyces cerevisiae undergoes Rsp5-dependent degradation upon exposure to high hydrostatic pressure and it limits the growth of tryptophan auxotrophs. Overexpression of SNA3 encoding an endosomal/vacuolar protein possessing the PPAY motif allowed growth at 25 MPa, which was potentiated by marked stabilization of Tat2. This appeared to depend on the PPAY motif, which interacted with the WW domain of Rsp5. Subcellular localization of Rsp5 was unchanged by overexpression of either SNA3 or SNA3-AAAY. While the loss of Bul1, a binding protein of Rsp5, or the rsp5-ww3 mutation allowed high-pressure growth, overexpression of BUL1 abolished the Sna3-mediated growth at 25 MPa. These results suggest that Sna3 and Bul1 compete for the WW domain of Rsp5 upon Tat2 ubiquitination.  

3.1392           The B[a]P-increased intercellular communication via translocation of connexin-43 into gap junctions reduces apoptosis

Tekpli, X., Rivedal, E., gorria, M., Landevik, N.E., Rissel, M., Dimache-Boitrel, M.T., Baffet, G., Holme, J.A. and Lagadic-Gossmann, D. Toxicol. and Appl. Pharmacol., 242, 231-240 (2010)   Gap junctions are channels in plasma membrane composed of proteins called connexins. These channels are organized in special domains between cells, and provide for direct gap junctional intercellular communication (GJIC), allowing diffusion of signalling molecules < 1 kD. GJIC regulates cell homeostasis and notably the balance between proliferation, cell cycle arrest, cell survival and apoptosis. Here, we have investigated benzo[a]pyrene (B[a]P) effects on GJIC and on the subcellular localization of the major protein of gap junction: connexin-43 (Cx43). Our results showed that B[a]P increased GJIC between mouse hepatoma Hepa1c1c7 cells via translocation of Cx43 from Golgi apparatus and lipid rafts into gap junction plaques. Interestingly, inhibition of GJIC by chlordane or small interference RNA directed against Cx43 enhanced B[a]P-induced apoptosis in Hepa1c1c7 cells. The increased apoptosis caused by inhibition of GJIC appeared to be mediated by ERK/MAPK pathway. It is suggested that B[a]P could induce transfer of cell survival signal or dilute cell death signal via regulation of ERK/MAPK through GJIC.  

3.1393           A di-arginine motif contributes to the ER localization of the type I transmembrane ER oxidoreductase TMX4

Roth, D., Lynes, E., Riemer, J., Hansen, H.G., Althaus, N., Simmen, T. And Ellgaard, L. Biochem. J., 425, 195-205 (2010)   The thiol-disulfide oxidoreductases of the PDI (protein disulfide isomerase) family assist in disulfide-bond formation in the ER (endoplasmic reticulum). In the present study, we have shown that the previously uncharacterized PDI family member TMX4 (thioredoxin-like transmembrane 4) is an N-glycosylated type I membrane protein that localizes to the ER. We also demonstrate that TMX4 contains a single ER-luminal thioredoxin-like domain, which, in contrast with similar domains in other PDIs, is mainly oxidized in living cells. The TMX4 transcript displays a wide tissue distribution, and is strongly expressed in melanoma cells. Unlike many type I membrane proteins, TMX4 lacks a typical C-terminal di-lysine retrieval signal. Instead, the cytoplasmic tail has a conserved di-arginine motif of the RXR type. We show that mutation of the RQR sequence in TMX4 to KQK interferes with ER localization of the protein. Moreover, whereas the cytoplasmic region of TMX4 confers ER localization to a reporter protein, the KQK mutant of the same protein redistributes to the cell surface. Overall, features not commonly found in other PDIs characterize TMX4 and suggest unique functional properties of the protein.  

3.1394           Doppel and PrPC co-immunoprecipitate in detergent-resistant membrane domains of epithelial FRT cells

Caputo, A., Sarnataro, D., Campana, V.., Costanzo, M., Negro, A., Sorgato, M.C. and Zurzolo, C. Biochem. J., 425, 341-351 (2010)   Dpl (doppel) is a paralogue of the PrP^C (cellular prion protein), whose misfolded conformer (the scrapie prion protein, PrP^Sc) is responsible for the onset of TSEs (transmissible spongiform encephalopathies) or prion diseases. It has been shown that the ectopic expression of Dpl in the brains of some lines of PrP-knockout mice provokes cerebellar ataxia, which can be rescued by the reintroduction of the PrP gene, suggesting a functional interaction between the two proteins. It is, however, still unclear where, and under which conditions, this event may occur. In the present study we addressed this issue by analysing the intracellular localization and the interaction between Dpl and PrP^C in FRT (Fischer rat thyroid) cells stably expressing the two proteins separately or together. We show that both proteins localize prevalently on the basolateral surface of FRT cells, in both singly and doubly transfected clones. Interestingly we found that they associate with DRMs (detergent-resistant membranes) or lipid rafts, from where they can be co-immunoprecipitated in a cholesterol-dependent fashion. Although the interaction between Dpl and PrP^C has been suggested before, our results provide the first clear evidence that this interaction occurs in rafts and is dependent on the integrity of these membrane microdomains. Furthermore, both Dpl and PrP^C could be immunoprecipitated with flotillin-2, a raft protein involved in endocytosis and cell signalling events, suggesting that they share the same lipid environment.  

3.1395           Lyn-mediated mitochondrial tyrosine phosphorylation is required to preserve mitochondrial integrity in early liver regeneration

Gringeri, E., Carraro, A., Tibald, E., D’Amico, F., Mancon, M., Toninello, A., Pagano, M.A., Vio, C., Cillo, U. and Brunati, A.M. Biochem. J., 425, 401-412 (2010)   Functional alterations in mitochondria such as overproduction of ROS (reactive oxygen species) and overloading of calcium, with subsequent change in the membrane potential, are traditionally regarded as pro-apoptotic conditions. Although such events occur in the early phases of LR (liver regeneration) after two-thirds PH (partial hepatectomy), hepatocytes do not undergo apoptosis but continue to proliferate until the mass of the liver is restored. The aim of the present study was to establish whether tyrosine phosphorylation, an emerging mechanism of regulation of mitochondrial function, participates in the response to liver injury following PH and is involved in contrasting mitochondrial pro-apoptotic signalling. Mitochondrial tyrosine phosphorylation, negligible in the quiescent liver, was detected in the early phases of LR with a trend similar to the events heralding mitochondrial apoptosis and was attributed to the tyrosine kinase Lyn, a member of the Src family. Lyn was shown to accumulate in an active form in the mitochondrial intermembrane space, where it was found to be associated with a multiprotein complex. Our results highlight a role for tyrosine phosphorylation in accompanying, and ultimately counteracting, mitochondrial events otherwise leading to apoptosis, hence conveying information required to preserve the mitochondrial integrity during LR.  

3.1396           Angiotensin-(1-7)-Angiotensin-Converting Enzyme 2 Attenuates Reactive Oxygen Species Formation to Angiotensin II Within the Cell Nucleus

Gwathmey, T.Y.M., Pendergrass, K.D., Reid, S.D., Rose, J.C., Diz, D.I. and Chappell, M.C. Hypertension, 55, 166-171 (2010)   The angiotensin (Ang) type 1 receptor (AT₁R) is highly expressed on renal nuclei and stimulates reactive oxygen species (ROS). It is not known whether other functional components of the Ang system regulate the nuclear Ang II-AT₁R ROS pathway. Therefore, we examined the expression of Ang receptors in nuclei isolated from the kidneys of young adult (1.5 years) and older adult (3.0 to 5.0 years) sheep. Binding studies in renal nuclei revealed the AT₂R as the predominant receptor subtype ( 80%) in young sheep, with the Ang-(1-7) (AT₇R; Mas protein) and AT₁R antagonists competing for the remaining sites. Conversely, in older sheep, the AT₁R accounted for 85% of nuclear sites, whereas the Ang type 2 receptor and AT₇R subtypes comprise 20% of remaining sites. Ang II increased nuclear ROS to a greater extent in older (97±22%; n=6) versus young animals (7±2%; P=0.01; n=4), and this was abolished by an AT₁R antagonist. The AT₇R antagonist D-Ala⁷-Ang-(1-7) increased ROS formation to Ang II by 2-fold (174±5% versus 97±22%; P<0.05) in older adults. Immunoblots of renal nuclei revealed protein bands for the AT₇R and Ang-converting enzyme 2 (ACE2), which metabolizes Ang II to Ang-(1-7). The ACE2 inhibitor MLN4760 also exacerbated the Ang II-dependent formation of ROS (156±15%) and abolished the generation of Ang-(1-7) from Ang II. We conclude that an ACE2-Ang-(1-7)-AT₇R pathway modulates Ang II-dependent ROS formation within the nucleus, providing a unique protective mechanism against oxidative stress and cell damage.  

3.1397           Cholesterol efflux stimulates metalloproteinase-mediated cleavage of occludin and release of extracellular membrane particles containing its C-terminal fragments

Casas, E., Barron, C., Francis, S.A., McCormack, J.M., McCarthy, K.M., Schneeberger, E.E. and Lynch, R.D. Exp. Cell Res., 316, 353-365 (2010)   That changes in membrane lipid composition alter the barrier function of tight junctions illustrates the importance of the interactions between tetraspan integral tight junction proteins and lipids of the plasma membrane. Application of methyl-β-cyclodextrin to both apical and basolateral surfaces of MDCK cell monolayers for 2 h, results in an 80% decrease in cell cholesterol, a fall in transepithelial electrical resistance, and a 30% reduction in cell content of occludin, with a smaller reduction in levels of claudins-2, -3, and -7. There were negligible changes in levels of actin and the two non-tight junction membrane proteins GP-135 and caveolin-1. While in untreated control cells breakdown of occludin, and probably other tight junction proteins, is mediated by intracellular proteolysis, our current data suggest an alternative pathway whereby in a cholesterol-depleted membrane, levels of tight junction proteins are decreased via direct release into the intercellular space as components of membrane-bound particles. Occludin, along with two of its degradation products and several claudins, increases in the basolateral medium after incubation with methyl-β-cyclodextrin for 30 min. In contrast caveolin-1 is detected only in the apical medium after adding methyl-β-cyclodextrin. Release of occludin and its proteolytic fragments continues even after removal of methyl-β-cyclodextrin. Sedimentation and ultrastructural studies indicate that the extracellular tight junction proteins are associated with the membrane-bound particles that accumulate between adjacent cells. Disruption of the actin filament network by cytochalasin D did not diminish methyl-β-cyclodextrin-induced release of tight junction proteins into the medium, suggesting that the mechanism underlying their formation is not actin-dependent. The 41- and  48-kDa C-terminal occludin fragments formed during cholesterol depletion result from the action of a GM6001-sensitive metalloproteinase(s) at some point in the path leading to release of the membrane particles.  

3.1398           α-tocopherol β-oxidation localized to rat liver mitochondria

Mustacich, D.J., Leonard, S.W., patel, N.K. and traber, M.G. Free Radical Biology & Medicine, 48, 73-81 (2010)   Approximately 40% of Americans take dietary supplements, including vitamin E (α-tocopherol). Unlike other fat-soluble vitamins, α-tocopherol is not accumulated to toxic levels. Rather tissue levels are tightly regulated, in part via increased hepatic metabolism and excretion that could, theoretically, alter metabolism of drugs, environmental toxins, and other nutrients. To date, in vivo subcellular location(s) of α-tocopherol metabolism have not been identified. The proposed pathway of α-tocopherol metabolism proceeds via ω-hydroxylation to 13′-OH-α-tocopherol, followed by successive rounds of β-oxidation to form α-CEHC. To test the hypothesis that α-tocopherol ω-hydroxylation occurs in microsomes while β-oxidation occurs in peroxisomes, rats received daily injections of vehicle, 10 mg α-tocopherol, or 10 mg trolox/100 g body wt for 3 days, and then microsomes, mitochondria, and peroxisomes were isolated from liver homogenates. Homogenate α-tocopherol levels increased 16-fold in α-tocopherol-injected rats, while remaining unchanged in trolox- or vehicle-injected rats. Total α-tocopherol recovered in the three subcellular fractions represented 93 ± 4% of homogenate α-tocopherol levels. In α-tocopherol-injected rats, microsome α-tocopherol levels increased 28-fold, while mitochondria and peroxisome levels increased 8- and 3-fold, respectively, indicating greater partitioning of α-tocopherol to the microsomes with increasing liver α-tocopherol. In α-tocopherol-injected rats, microsome 13′-OH-α-tocopherol levels increased 24-fold compared to controls, and were 7-fold greater than 13′-OH-α-tocopherol levels in peroxisome and mitochondrial fractions of α-tocopherol-injected rats. An unexpected finding was that α-CEHC, the end product of α-tocopherol metabolism, was found almost exclusively in mitochondria. These data are the first to indicate a mitochondrial role in α-tocopherol metabolism.  

3.1399           Retrograde Neurotrophic Signaling Requires a Protein Interacting with Receptor Tyrosine Kinases via C2H2 Zinc Fingers

Fu, X., Zang, K., Zhou, Z., Reichardt, L.F. and Xu, B. Mol. Biol. Cell, 21, 36-49 (2010)   Neurotrophins at axonal terminals signal to cell bodies to regulate neuronal development via signaling endosomes containing activated Trk receptor tyrosine kinases and mitogen-activated protein kinases (MAPKs). Requirements for the formation of signaling endosomes remain, however, poorly characterized. Here we show that a novel Trk-interacting protein, NTRAP (neurotrophic factor receptor–associated protein), plays a crucial role in this signaling process. NTRAP interacts with the Trk intracellular domain through its C₂H₂ zinc fingers in a kinase-dependent manner. It is associated with vesicles, some of which contain markers for signaling endosomes. Inhibition of NTRAP function suppresses neurotrophin-induced neurite outgrowth in PC12 cells by altering TrkA endocytic traffic, inhibiting the formation of endosomes containing persistently active MAPKs. In compartmentalized sensory neuron cultures, down-regulation of NTRAP abolishes the ability of neurotrophins applied to distal axons to activate the transcription factor adenosine 3',5'-monophosphate response element-binding protein (CREB) and to promote neuronal survival. We propose that NTRAP regulates retrograde neurotrophic signaling by controlling the formation of signaling endosomes.  

3.1400           Sec3-containing Exocyst Complex Is Required for Desmosome Assembly in Mammalian Epithelial Cells

Andersen, N.J. and Yeaman, C. Mol. Biol. Cell, 21, 152-164 (2010)   The Exocyst is a conserved multisubunit complex involved in the docking of post-Golgi transport vesicles to sites of membrane remodeling during cellular processes such as polarization, migration, and division. In mammalian epithelial cells, Exocyst complexes are recruited to nascent sites of cell–cell contact in response to E-cadherin–mediated adhesive interactions, and this event is an important early step in the assembly of intercellular junctions. Sec3 has been hypothesized to function as a spatial landmark for the development of polarity in budding yeast, but its role in epithelial cells has not been investigated. Here, we provide evidence in support of a function for a Sec3-containing Exocyst complex in the assembly or maintenance of desmosomes, adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. We show that Sec3 associates with a subset of Exocyst complexes that are enriched at desmosomes. Moreover, we found that membrane recruitment of Sec3 is dependent on cadherin-mediated adhesion but occurs later than that of the known Exocyst components Sec6 and Sec8 that are recruited to adherens junctions. RNA interference-mediated suppression of Sec3 expression led to specific impairment of both the morphology and function of desmosomes, without noticeable effect on adherens junctions. These results suggest that two different exocyst complexes may function in basal–lateral membrane trafficking and will enable us to better understand how exocytosis is spatially organized during development of epithelial plasma membrane domains.  

3.1401           HIV Nef is Secreted in Exosomes and Triggers Apoptosis in Bystander CD4+ T Cells

Lenassi, M., Cagney, G., Liao, M., Vaupotic, T., Bartholomeeusen, K., Cheng, Y., Krogan, N.J., Plemenitas, A. and Peterlin, B.M. Traffic, 11, 110-122 (2010)   The HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed viral proteins. It is also found in the serum of infected individuals (Caby MP, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C. Exosomal-like vesicles are present in human blood plasma. Int Immunol 2005;17:879-887). Extracellular Nef protein has deleterious effects on CD4+ T cells (James CO, Huang MB, Khan M, Garcia-Barrio M, Powell MD, Bond VC. Extracellular Nef protein targets CD4+ T cells for apoptosis by interacting with CXCR4 surface receptors. J Virol 2004;78:3099-3109), the primary targets of HIV, and can suppress immunoglobulin class switching in bystander B cells (Qiao X, He B, Chiu A, Knowles DM, Chadburn A, Cerutti A. Human immunodeficiency virus 1 Nef suppresses CD40-dependent immunoglobulin class switching in bystander B cells. Nat Immunol 2006;7:302-310). Nevertheless, the mode of exit of Nef from infected cells remains a conundrum. We found that Nef stimulates its own export via the release of exosomes from all cells examined. Depending on its intracellular location, these Nef exosomes form at the plasma membrane, late endosomes or both compartments in Jurkat, SupT1 and primary T cells, respectively. Nef release through exosomes is conserved also during HIV-1 infection of peripheral blood lymphocytes (PBLs). Released Nef exosomes cause activation-induced cell death of resting PBLs in vitro. Thus, HIV-infected cells export Nef in bioactive vesicles, which facilitate the depletion of CD4+ T cells that is a hallmark of acquired immunodeficiency syndrome (AIDS).  

3.1402           How Do Bone Cells Secrete Proteins?

Zhao, H., Ito, Y., Chappel, J., Andrews, N., Ross, F.P. and Teitelbaum, S.L. Adv. In Exp. Med. And Biol., 658, 105-109 (2010)   Osteoclasts (OCs), which are the exclusive bone resorbing cells, degrade skeletal matrix by forming an intimate relationship with the bone surface. Thus, when OCs attach to bone, they produce an actin-rich sealing zone representing a gasket-like structure, which isolates the resorptive milieu from the general extracellular space. This “resorptive microenvironment” contains a ruffled border, the unique bone-degrading organelle of the OC, which consists of a complex, villous-like organization of the plasma membrane. This structure appears only in resorbing cells and is the product of signals derived from the bone matrix. These signals polarize as yet undefined acidified vesicles containing the OC vacuolar H+ATPase towards the bone-apposed plasma membrane, into which they insert, thereby increasing its complexity. The ruffled border is thus the most definitive marker of the resorbing osteoclast.  

3.1403           The Mycobacterium bovis Bacille Calmette-Guérin Phagosome Proteome

Lee, B-Y., Jethwaney, D., Schilling, B., Clemens, D.L., Gibson, B.W. and Horwitz, M.A. Mol. Cell. Proteomics, 9(1), 32-53 (2010)   Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin (BCG) alter the maturation of their phagosomes and reside within a compartment that resists acidification and fusion with lysosomes. To define the molecular composition of this compartment, we developed a novel method for obtaining highly purified phagosomes from BCG-infected human macrophages and analyzed the phagosomes by Western immunoblotting and mass spectrometry-based proteomics. Our purification procedure revealed that BCG grown on artificial medium becomes less dense after growth in macrophages. By Western immunoblotting, LAMP-2, Niemann-Pick protein C1, and syntaxin 3 were readily detectable on the BCG phagosome but at levels that were lower than on the latex bead phagosome; flotillin-1 and the vacuolar ATPase were barely detectable on the BCG phagosome but highly enriched on the latex bead phagosome. Immunofluorescence studies confirmed the scarcity of flotillin on BCG phagosomes and demonstrated an inverse correlation between bacterial metabolic activity and flotillin on M. tuberculosis phagosomes. By mass spectrometry, 447 human host proteins were identified on BCG phagosomes, and a partially overlapping set of 289 human proteins on latex bead phagosomes was identified. Interestingly, the majority of the proteins identified consistently on BCG phagosome preparations were also identified on latex bead phagosomes, indicating a high degree of overlap in protein composition of these two compartments. It is likely that many differences in protein composition are quantitative rather than qualitative in nature. Despite the remarkable overlap in protein composition, we consistently identified a number of proteins on the BCG phagosomes that were not identified in any of our latex bead phagosome preparations, including proteins involved in membrane trafficking and signal transduction, such as Ras GTPase-activating-like protein IQGAP1, and proteins of unknown function, such as FAM3C. Our phagosome purification procedure and initial proteomics analyses set the stage for a quantitative comparative analysis of mycobacterial and latex bead phagosome proteomes.  

3.1404           Peroxisomes from the Heavy Mitochondrial Fraction: Isolation by Zonal Free Flow Electrophoresis and Quantitative Mass Spectrometrical Characterization

Islinger, M., Li, K.W., Loos, M., Liebler, S., Angermüller, S., Eckerskorn, C., Weber, G., Abdolzade, A. and Völkl, A.

1. Proteome Res., 9, 113-124 (2010)

  Peroxisomes are a heterogeneous group of organelles fulfilling reactions in a variety of metabolic pathways. To investigate if functionally different subpopulations can be found within a single tissue, peroxisomes from the heavy mitochondrial fraction (HM-Po) of the rat liver were isolated and compared to “classic” peroxisomes from the light mitochondrial fraction (LM-Po) using iTRAQ tandem mass spectrometry. Peroxisomes represent only a minor although significant proportion of the heavy mitochondrial fraction (2700g_max) precluding a straightforward isolation by standard protocols. Thus, a new fractionation scheme suitable for a subsequent mass spectrometrical analysis was developed using a combination of centrifugation techniques and zonal free flow electrophoresis. On the basis of the iTRAQ-measurement, a variation of the peroxisomal protein pattern between both fractions could be determined and further confirmed by immunoblotting and enzyme activity assays for selected proteins: whereas peroxisomes from the light mitochondrial fraction contain high amounts of β-oxidation enzymes, peroxisomes from the heavy mitochondrial fraction were dominated by enzymes fulfilling other functions. Among other findings, HM-Po was characterized by a high abundance of D-amino acid oxidase. This observation can be mirrored at the ultrastructural level, where tissue sections of liver peroxisomes show a heterogeneous staining for the enzymes activity, when visualized by the cerium technique.  

3.1405           Adeno-associated Virus Gene Therapy With Cholesterol 24-Hydroxylase Reduces the Amyloid Pathology Before or After the Onset of Amyloid Plaques in Mouse Models of Alzheimer's Disease

Hudry,, E., Van Dam, D., Kulik, W., De Deyn, P.P., Stety, F.S., Ahouansou, O., Benraiss, A., Delacourte, A., Bougneres, P., Aubourg, P. and Cartier, N. Molecular Therapy, 18(1), 44-53 (2010)   The development of Alzheimer's disease (AD) is closely connected with cholesterol metabolism. Cholesterol increases the production and deposition of amyloid-β (Aβ) peptides that result in the formation of amyloid plaques, a hallmark of the pathology. In the brain, cholesterol is synthesized in situ but cannot be degraded nor cross the blood–brain barrier. The major exportable form of brain cholesterol is 24S-hydroxycholesterol, an oxysterol generated by the neuronal cholesterol 24-hydroxylase encoded by the CYP46A1 gene. We report that the injection of adeno-associated vector (AAV) encoding CYP46A1 in the cortex and hippocampus of APP23 mice before the onset of amyloid deposits markedly reduces Aβ peptides, amyloid deposits and trimeric oligomers at 12 months of age. The Morris water maze (MWM) procedure also demonstrated improvement of spatial memory at 6 months, before the onset of amyloid deposits. AAV5-wtCYP46A1 vector injection in the cortex and hippocampus of amyloid precursor protein/presenilin 1 (APP/PS) mice after the onset of amyloid deposits also reduced markedly the number of amyloid plaques in the hippocampus, and to a less extent in the cortex, 3 months after the injection. Our data demonstrate that neuronal overexpression of CYP46A1 before or after the onset of amyloid plaques significantly reduces Aβ pathology in mouse models of AD.        

3.1406           Cathepsin B-mediated Autophagy Flux Facilitates the Anthrax Toxin Receptor 2-mediated Delivery of Anthrax Lethal Factor into the Cytoplasm

Ha, S-D., Ham, B., Mogridge, J., Saftig, P., Lin, S. and Kim, S.O.

1. Biol. Chem., 285(3), 2120-2129 (2010)

  Anthrax lethal toxin (LeTx) is a virulence factor secreted by Bacillus anthracis and has direct cytotoxic effects on most cells once released into the cytoplasm. The cytoplasmic delivery of the proteolytically active component of LeTx, lethal factor (LF), is carried out by the transporter component, protective antigen, which interacts with either of two known surface receptors known as anthrax toxin receptor (ANTXR) 1 and 2. We found that the cytoplasmic delivery of LF by ANTXR2 was mediated by cathepsin B (CTSB) and required lysosomal fusion with LeTx-containing endosomes. Also, binding of protective antigen to ANXTR1 or -2 triggered autophagy, which facilitated the cytoplasmic delivery of ANTXR2-associated LF. We found that whereas cells treated with the membrane-permeable CTSB inhibitor CA074-Me- or CTSB-deficient cells had no defect in fusion of LC3-containing autophagic vacuoles with lysosomes, autophagic flux was significantly delayed. These results suggested that the ANTXR2-mediated cytoplasmic delivery of LF was enhanced by CTSB-dependent autophagic flux.  

3.1407           CD147, a γ-secretase associated protein is upregulated in Alzheimer's disease brain and its cellular trafficking is affected by presenilin-2

Nahalkova, J., Volk,mann, I., Aoki, M., Winblad, B., Bogdanovic, N., Tjernberg, L.O. and Behbahani, H. Neurochem. Int., 56, 67-76 (2010)   γ-Secretase activity has been extensively investigated due to its role in Alzheimer's disease. Here, we studied the association of CD147, a transmembrane glycoprotein belonging to the immunoglobulin family, with γ-secretase and its expression in Alzheimer's disease and control tissues. Subcellular fractionation of postmitochondrial supernatant from rat brain on step iodixanol gradient in combination with co-immunoprecipitation using an anti-nicastrin antibody showed association of limited amount of CD147 to γ-secretase. By immunoblotting of postnuclear pellets from Alzheimer's disease and control human brain tissues we showed that CD147 with molecular weight 75 kDa is upregulated in frontal cortex and thalamus of the Alzheimer's disease brains. Immunohistochemistry of brain tissues from Alzheimer's disease and control revealed specific upregulation of CD147 in neurons, axons and capillaries of Alzheimer's disease frontal cortex and thalamus. The effect of presenilin-1 and -2, which are the catalytic subunits of γ-secretase, on CD147 expression and subcellular localization was analyzed by confocal microscopy in combination with flow cytometry and showed that PS2 affected the subcellular localization of CD147 in mouse embryonic fibroblast cells. We suggest that a small fraction of CD147 present in the brain is associated with the γ-secretase, and can be involved in mechanisms dysregulated in Alzheimer's disease brain.  

3.1408           The cytoplasmic tail of fibrocystin contains a ciliary targeting sequence

Follit, J.A., Li, L., Vucica, Y and Pazour, G.J:

1. Cell Biol., 188(1), 21-28 (2010)

  Sensory functions of primary cilia rely on ciliary-localizedmembrane proteins, but little is known about how these receptorsare targeted to the cilium. To further our understanding ofthis process, we dissected the ciliary targeting sequence (CTS)of fibrocystin, the human autosomal recessive polycystic kidneydisease gene product. We show that the fibrocystin CTS is an18-residue motif localized in the cytoplasmic tail. This motifis sufficient to target green fluorescent protein (GFP) to ciliaof ciliated cells and targets GFP to lipid rafts if the cellsare not ciliated. Rab8, but not several other Rabs implicatedin ciliary assembly, binds to the CTS in a coimmunoprecipitationassay. Dominant-negative Rab8 interacts more strongly than wild-typeor constitutively active Rab8, and coexpression of this dominant-negativemutant Rab8 blocks trafficking to the cilium. This suggeststhat the CTS functions by binding regulatory proteins like Rab8to control trafficking through the endomembrane system and onto the cilium.  

3.1409           WAVE1 regulates Bcl-2 localization and phosphorylation in leukemia cells

Kang, R., Tang, D., Yu, Y., Wang, Z., Hu, T., Wang, H. and Cao, L. Leukemia, 24, 177-186 (2010)   Bcl-2 proteins are over-expressed in many tumors and are critically important for cell survival. Their anti-apoptotic activities are determined by intracellular localization and post-translational modifications (such as phosphorylation). Here, we showed that WAVE1, a member of the Wiskott–Aldrich syndrome protein family, was over-expressed in blood cancer cell lines, and functioned as a negative regulator of apoptosis. Further enhanced expression of WAVE1 by gene transfection rendered leukemia cells more resistant to anti-cancer drug-induced apoptosis; whereas suppression of WAVE1 expression by RNA interference restored leukemia cells' sensitivity to anti-drug-induced apoptosis. WAVE1 was found to be associated with mitochondrial Bcl-2, and its depletion led to mitochondrial release of Bcl-2, and phosphorylation of ASK1/JNK and Bcl-2. Furthermore, depletion of WAVE1 expression increased anti-cancer drug-induced production of reactive oxygen species in leukemia cells. Taken together, these results suggest WAVE1 as a novel regulator of apoptosis, and potential drug target for therapeutic intervention of leukemia.  

3.1410           UTP Controls Cell Surface Distribution and Vasomotor Activity of the Human P2Y2 Receptor through an Epidermal Growth Factor Receptor-transregulated Mechanism

Norambuena, A., Palma, F., Poblete, M.I., Domoso, M.V., Pardo, E., Gonzalez, A. and Huidobro-Toro, J.P.

1. Biol. Chem., 285(5), 2940-2950 (2010)

  Extracellular nucleotides transmit signals into the cells through the P2 family of cell surface receptors. These receptors are amply expressed in human blood vessels and participate in vascular tone control; however, their signaling mechanisms remain unknown. Here we show that in smooth muscle cells of isolated human chorionic arteries, the activation of the P2Y₂ receptor (P2Y₂R) induces not only its partition into membrane rafts but also its rapid internalization. Cholesterol depletion with methyl-β-cyclodextrin reduced the association of the agonist-activated receptor into membrane rafts but did not affect either the UTP-mediated vasoconstrictions or the vasomotor responses elicited by both serotonin and KCl. Ex vivo perfusion of human chorionic artery segments with 1–10 μm UTP, a selective P2Y₂R agonist, displaced the P2Y₂R localization into membrane rafts within 1 min, a process preceded by the activation of both RhoA and Rac1 GTPases. AG1478, a selective and potent inhibitor of the epidermal growth factor receptor tyrosine kinase activity, not only blocked the UTP-induced vasomotor activity but also abrogated both RhoA and Rac1 activation, the P2Y₂R association with membrane rafts, and its internalization. Altogether, these results show for the first time that the plasma membrane distribution of the P2Y₂R is transregulated by the epidermal growth factor receptor, revealing an unsuspected functional interplay that controls both the membrane distribution and the vasomotor activity of the P2Y₂R in intact human blood vessels.  

3.1411           Differentiation of human adipose-derived stem cells induced by recombinantly expressed fibroblast growth factor 10 in vitro and in vivo

Zhang, X., Wu, M., Zhang, W., Shen, J. and Liu, H. In vitro Cell. Dev. Biol. – Animal, 46, 60-71 (2010)   The adipogenesis effect of fibroblast growth factor 10 (FGF10) has been demonstrated in many studies. The aim of this study is to render a novel method which can continuously induce hypodermal adipose-derived stem cell (ADSC) differentiation and maturation in vivo and in vitro using FGF10. We constructed a recombinant pcDNA3.0-FGF10-MSC which can continuously express FGF10 by transfected FGF10 into a human mesenchymal stem cell (MSC) clone, and we cultured ADSCs from human subcutaneous resected adipose tissue. An in vitro and in vivo co-culture system of pcDNA3.0-FGF10-MSC and ADSCs was then established. We observed the characteristics of ADSCs, monitored the adipogenesis-related transcription factor CAAT/enhancer binding protein-β, peroxisome proliferator-activated receptor-γ, and measured the adipose tissue layer of carrier animals. The results showed that FGF10 secreted from pcDNA3.0-FGF10-MSC could induce ADSC differentiation into mature adipocytes consistently. The study demonstrated that FGF10 can promote the adipogenesis effect in situ, and the autotransplantation of a carrier continuously secreting FGF10 may be utilized for increasing local subcutaneous adipose tissue in cosmetology.  

3.1412           Mitochondrial Localization of Vitamin D Receptor in Human Platelets and Differentiated Megakaryocytes

Silvagno, F., De Vivo, E., Attanasio, A., gallo, V., Mazzucco, G. and Pescamona, G. PloSOne, 5(1), e8670 (2010)   Background Like other steroid hormones, vitamin D elicits both transcriptional events and rapid non genomic effects. Vitamin D receptor (VDR) localization and mechanisms of VDR-triggered non genomic responses are still controversial. Although anticoagulant effects of vitamin D have been reported and VDR signalling has been characterized in monocytes and vascular cells, nothing is known about VDR expression and functions in human platelets, anucleated fragments of megakaryocytes which are known targets of other steroids. Methodology/Principal Findings In this study we characterized the expression and cellular localization of VDR in human platelets and in a megakaryocyte lineage. Human platelets and their TPA-differentiated precursors expressed a classical 50 kDa VDR protein, which increased with megakaryocytes maturation. By biochemical fractionation studies we demonstrated the presence of the receptor in the soluble and mitochondrial compartment of human platelets, and the observation was confirmed by immunoelectron microscopy analysis. Similar localization was found in mature megakaryocytes, where besides its classical nuclear localization the receptor was evident as soluble and mitochondria resident protein. Conclusions The results reported here suggest that megakaryocytopoiesis and platelet activation, which are calcium-dependent events, might be modulated by a mitochondrial non genomic activity of VDR. These data open challenging future studies on VDR physiological role in platelets and more generally in mitochondria.  

3.1413           Synaptic and Endosomal Localization of Active γ-Secretase in Rat Brain

Frykman, S., Hur, J-Y., Frånberg, J., Aoki, M., Winblad, B., Nahalkova, J., Behbahani, H. and Tjernberg, L.O. PloSOne, 5(1), e8948 (2010)   Background A key player in the development of Alzheimer's disease (AD) is the γ-secretase complex consisting of at least four components: presenilin, nicastrin, Aph-1 and Pen-2. γ-Secretase is crucial for the generation of the neurotoxic amyloid β-peptide (Aβ) but also takes part in the processing of many other substrates. In cell lines, active γ-secretase has been found to localize primarily to the Golgi apparatus, endosomes and plasma membranes. However, no thorough studies have been performed to show the subcellular localization of the active γ-secretase in the affected organ of AD, namely the brain. Principal Findings We show by subcellular fractionation of rat brain that high γ-secretase activity, as assessed by production of Aβ40, is present in an endosome- and plasma membrane-enriched fraction of an iodixanol gradient. We also prepared crude synaptic vesicles as well as synaptic membranes and both fractions showed high Aβ40 production and contained high amounts of the γ-secretase components. Further purification of the synaptic vesicles verified the presence of the γ-secretase components in these compartments. The localization of an active γ-secretase in synapses and endosomes was confirmed in rat brain sections and neuronal cultures by using a biotinylated γ-secretase inhibitor together with confocal microscopy. Significance The information about the subcellular localization of γ-secretase in brain is important for the understanding of the molecular mechanisms of AD. Furthermore, the identified fractions can be used as sources for highly active γ-secretase.  

3.1414           Proteomics Analysis of A33 Immunoaffinity-purified Exosomes Released from the Human Colon Tumor Cell Line LIM1215 Reveals a Tissue-specific Protein Signature

Mathivanan, S., Lim, J.W.E., Tauro, B.J., Ji, H., Moritz, R.L. and Simpson, R. Mol. Cell. Protemics, 9(2), 197-208 (2010)   Exosomes are 40–100-nm-diameter nanovesicles of endocytic origin that are released from diverse cell types. To better understand the biological role of exosomes and to avoid confounding data arising from proteinaceous contaminants, it is important to work with highly purified material. Here, we describe an immunoaffinity capture method using the colon epithelial cell-specific A33 antibody to purify colorectal cancer cell (LIM1215)-derived exosomes. LC-MS/MS revealed 394 unique exosomal proteins of which 112 proteins (28%) contained signal peptides and a significant enrichment of proteins containing coiled coil, RAS, and MIRO domains. A comparative protein profiling analysis of LIM1215-, murine mast cell-, and human urine-derived exosomes revealed a subset of proteins common to all exosomes such as endosomal sorting complex required for transport (ESCRT) proteins, tetraspanins, signaling, trafficking, and cytoskeletal proteins. A conspicuous finding of this comparative analysis was the presence of host cell-specific (LIM1215 exosome) proteins such as A33, cadherin-17, carcinoembryonic antigen, epithelial cell surface antigen (EpCAM), proliferating cell nuclear antigen, epidermal growth factor receptor, mucin 13, misshapen-like kinase 1, keratin 18, mitogen-activated protein kinase 4, claudins (1, 3, and 7), centrosomal protein 55 kDa, and ephrin-B1 and -B2. Furthermore, we report the presence of the enzyme phospholipid scramblase implicated in transbilayer lipid distribution membrane remodeling. The LIM1215-specific exosomal proteins identified in this study may provide insights into colon cancer biology and potential diagnostic biomarkers.  

3.1415           Application of Proteomic Marker Ensembles to Subcellular Organelle Identification

Andreyev, A.Y., Shen, Z., Guan, Z., Ryan, A., Fahy, E., Subramaniam, S., Raetz, C.R.H., Briggs, S. and Dennis, E.A. Mol. Cell. Proteomics, 9(2), 388-402 (2010)   Compartmentalization of biological processes and the associated cellular components is crucial for cell function. Typically, the location of a component is revealed through a co-localization and/or co-purification with an organelle marker. Therefore, the identification of reliable markers is critical for a thorough understanding of cellular function and dysfunction. We fractionated macrophage-like RAW264.7 cells, both in the resting and endotoxin-activated states, into six fractions representing the major organelles/compartments: nuclei, mitochondria, cytoplasm, endoplasmic reticulum, and plasma membrane as well as an additional dense microsomal fraction. The identity of the first five of these fractions was confirmed via the distribution of conventional enzymatic markers. Through a quantitative liquid chromatography/mass spectrometry-based proteomics analysis of the fractions, we identified 50-member ensembles of marker proteins ("marker ensembles") specific for each of the corresponding organelles/compartments. Our analysis attributed 206 of the 250 marker proteins ( 82%) to organelles that are consistent with the location annotations in the public domain (obtained using DAVID 2008, EntrezGene, Swiss-Prot, and references therein). Moreover, we were able to correct locations for a subset of the remaining proteins, thus proving the superior power of analysis using multiple organelles as compared with an analysis using one specific organelle. The marker ensembles were used to calculate the organelle composition of the six above mentioned subcellular fractions. Knowledge of the precise composition of these fractions can be used to calculate the levels of metabolites in the pure organelles. As a proof of principle, we applied these calculations to known mitochondria-specific lipids (cardiolipins and ubiquinones) and demonstrated their exclusive mitochondrial location. We speculate that the organelle-specific protein ensembles may be used to systematically redefine originally morphologically defined organelles as biochemical entities.  

3.1416           Chromogranin B Gene Ablation Reduces the Catecholamine Cargo and Decelerates Exocytosis in Chromaffin Secretory Vesicles

Diaz-Vera, J., Morales, Y.G., Hernandez-Fernaud, J.R., Camacho, M., Montesinos, M.S., Calegari, F., Huttner, W.B., Borges, R. and Machado, J.D.

1. Neurosci., 30(3), 950-957 (2010)

  Chromogranins/secretogranins (Cgs) are the major soluble proteins of large dense-core secretory vesicles (LDCVs). We have recently reported that the absence of chromogranin A (CgA) caused important changes in the accumulation and in the exocytosis of catecholamines (CAs) using a CgA-knock-out (CgA-KO) mouse. Here, we have analyzed a CgB-KO mouse strain that can be maintained in homozygosis. These mice have 36% less adrenomedullary epinephrine when compared to Chgb^+/+ [wild type (WT)], whereas the norepinephrine content was similar. The total evoked release of CA was 33% lower than WT mice. This decrease was not due to a lower frequency of exocytotic events but to less secretion per quantum ( 30%) measured by amperometry; amperometric spikes exhibited a slower ascending but a normal decaying phase. Cell incubation with L-DOPA increased the vesicle CA content of WT but not of the CgB-KO cells. Intracellular electrochemistry, using patch amperometry, showed that L-DOPA overload produced a significantly larger increase in cytosolic CAs in cells from the KO animals than chromaffin cells from the WT. These data indicate that the mechanisms for vesicular accumulation of CAs in the CgB-KO cells were saturated, while there was ample capacity for further accumulation in WT cells. Protein analysis of LDCVs showed the overexpression of CgA as well as other proteins apparently unrelated to the secretory process. We conclude that CgB, like CgA, is a highly efficient system directly involved in monoamine accumulation and in the kinetics of exocytosis from LDCVs.  

3.1417           The Ubiquitin–Proteasome System Regulates the Stability of Neuronal Nicotinic Acetylcholine Receptors

Rezvani, K., Teng, Y. and De Biasi, M.

1. Mol. Neurosci., 40, 177-184 (2010)

  Ubiquitination is a key event for protein degradation by the proteasome system, membrane protein internalization, and protein trafficking among cellular compartments. Few data are available on the role of the ubiquitin–proteasome system (UPS) in the trafficking of neuronal nicotinic acetylcholine receptors (nAChRs). Experiments conducted in neuron-like differentiated rat pheochromocytoma cells (PC12 cells) show that the α3, β2, and β4 nAChR subunits are ubiquitinated and that their ubiquitination is necessary for degradation. A 24-h treatment with the proteasome inhibitor PS-341 increased the total levels of α3 and the two β subunits in both whole cell lysates and fractions enriched for the ER/Golgi compartment. nAChR subunit upregulation was also detected in plasma membrane-enriched fractions. Inhibition of the lysosomal degradation machinery by E-64 had a significantly smaller effect on nAChR turnover. The present data, together with previous results showing that the α7 nAChR subunit is a target of the UPS, point to a prominent role of the proteasome in nAChR trafficking.  

3.1418           Lovastatin-induced cholesterol depletion affects both apical sorting and endocytosis of aquaporin-2 in renal cells

Procino, G., Barbieri, C., carmosino, M., Rizzo, F., Valenti, G. and Svelto, M. Am. J. Physiol. Renal Physiol., 298, F266-F278 (2010)   Vasopressin causes the redistribution of the water channel aquaporin-2 (AQP2) from cytoplasmic storage vesicles to the apical plasma membrane of collecting duct principal cells, leading to urine concentration. The molecular mechanisms regulating the selective apical sorting of AQP2 are only partially uncovered. In this work, we investigate whether AQP2 sorting/trafficking is regulated by its association with membrane rafts. In both MCD4 cells and rat kidney, AQP2 preferentially associated with Lubrol WX-insoluble membranes regardless of its presence in the storage compartment or at the apical membrane. Block-and-release experiments indicate that 1) AQP2 associates with detergent-resistant membranes early in the biosynthetic pathway; 2) strong cholesterol depletion delays the exit of AQP2 from the trans-Golgi network. Interestingly, mild cholesterol depletion promoted a dramatic accumulation of AQP2 at the apical plasma membrane in MCD4 cells in the absence of forskolin stimulation. An internalization assay showed that AQP2 endocytosis was clearly reduced under this experimental condition. Taken together, these data suggest that association with membrane rafts may regulate both AQP2 apical sorting and endocytosis.  

3.1419           Endocytic Rab proteins are required for hepatitis C virus replication complex formation

Manna, D., Aligo, J., Xu, C., Park, W.S., Koc, H., Heo, W.D. and Konan, K.V. Virology, 398, 21-37 (2010)   During infection, hepatitis C virus (HCV) NS4B protein remodels host membranes to form HCV replication complexes (RC) which appear as foci under fluorescence microscopy (FM). To understand the role of Rab proteins in forming NS4B foci, cells expressing the HCV replicon were examined biochemically and via FM. First, we show that an isolated NS4B-bound subcellular fraction is competent for HCV RNA synthesis. Further, this fraction is differentially enriched in Rab1, 2, 5, 6 and 7. However, when examined via FM, NS4B foci appear to be selectively associated with Rab5 and Rab7 proteins. Additionally, dominant negative (DN) Rab6 expression impairs Rab5 recruitment into NS4B foci. Further, silencing of Rab5 or Rab7 resulted in a significant decrease in HCV genome replication. Finally, expression of DN Rab5 or Rab7 led to a reticular NS4B subcellular distribution, suggesting that endocytic proteins Rab5 and Rab7, but not Rab11, may facilitate NS4B foci formation.  

3.1420           Cysteinyl leukotrienes acting via granule membrane-expressed receptors elicit secretion from within cell-free human eosinophil granules

Neves, J., Radke, A.L. and Weller, P.F.

1. Allergy Clin. Immunol., 125, 477-482 (2010)

  Background Cysteinyl leukotrienes (cysLTs) are recognized to act via receptors (cysLTRs) expressed on cell surface plasma membranes. Agents that block cysLT₁ receptor (cysLT₁R) are therapeutics for allergic disorders. Eosinophils contain multiple preformed proteins stored within their intracellular granules. Cell-free eosinophil granules are present extracellularly as intact membrane-bound organelles in sites associated with eosinophil infiltration, including asthma, rhinitis, and urticaria, but have unknown functional capabilities. Objective We evaluated the expression of cysLTRs on eosinophil granule membranes and their functional roles in eliciting protein secretion from within eosinophil granules. Methods We studied secretory responses of human eosinophil granules isolated by subcellular fractionation. Granules were stimulated with cysLTs, and eosinophil cationic protein and cytokines were measured in the supernatants. Receptor expression on granule membranes and eosinophils was evaluated by flow cytometry and Western blot. Results We report that receptors for cysLTs, cysLT₁R, cysLT₂ receptor, and the purinergic P2Y12 receptor, are expressed on eosinophil granule membranes. Leukotriene (LT) C₄ and extracellularly generated LTD₄ and LTE₄ stimulated isolated eosinophil granules to secrete eosinophil cationic protein. MRS 2395, a P2Y12 receptor antagonist, inhibited cysLT-induced eosinophil cationic protein release. Montelukast, likely not solely as an inhibitor of cysLT₁R, inhibited eosinophil cationic protein release elicited by LTC₄ and LTD₄ as well as by LTE₄. Conclusion These studies identify previously unrecognized sites of localization, the membranes of intracellular eosinophil granule organelles, and function for cysLT-responsive receptors that mediate cysteinyl leukotriene-stimulated secretion from within eosinophil granules, including those present extracellularly.  

3.1421           Local translation of dendritic RhoA revealed by an improved synaptoneurosome preparation

Troca-Marin, J.A., Alves-Sampio, A., Tejedor, F.J. and Montesinos, M.L. Mol. Cell. Neurosci., 43, 308-314 (2010)   Changes in dendritic spine morphology, a hallmark of synaptic plasticity, involve remodeling of the actin cytoskeleton, a process that is regulated by Rho GTPases. RhoA, a member of this GTPase family, segregates to dendrites in differentiated neurons. Given the emerging role of dendritic mRNA local translation in synaptic plasticity, we have assessed the possible localization and translation of RhoA mRNA at dendrites. At this end, we have developed and describe here in detail an improved method for isolating hippocampal and neocortical mouse synaptoneurosomes. This synaptoneurosomal preparation is much more enriched in synaptic proteins than those obtained in former methods, exhibits bona fide electron microscopy pre- and postsynaptic morphologies, contains abundant dendritic mRNAs, and is competent for activity-regulated protein synthesis. Using this preparation, we have found that RhoA mRNA is dendritically localized and its local translation is enhanced by BDNF stimulation. These findings suggest that some of the known functions of RhoA on spine morphology may be mediated by regulating its local translation.  

3.1422           Cadherins and Pak1 Control Contact Inhibition of Proliferation by Pak1-βPIX-GIT Complex-Dependent Regulation of Cell-Matrix Signaling

Liu, F., Jia, L., Thompson-Baine, A-M. Puglise, J.M. ter Beest, M.B.A. and Zegers, M.M.P. Mol. Cell. Biol., 30(8), 1971-1983 (2010)   It is crucial for organ homeostasis that epithelia have effective mechanisms to restrict motility and cell proliferation in order to maintain tissue architecture. On the other hand, epithelial cells need to rapidly and transiently acquire a more mesenchymal phenotype, with high levels of cell motility and proliferation, in order to repair epithelia upon injury. Cross talk between cell-cell and cell-matrix signaling is crucial for regulating these transitions. The Pak1-βPIX-GIT complex is an effector complex downstream of the small GTPase Rac1. We previously showed that translocation of this complex from cell-matrix to cell-cell adhesion sites was required for the establishment of contact inhibition of proliferation. In this study, we provide evidence that this translocation depends on cadherin function. Cadherins do not recruit the complex by direct interaction. Rather, we found that inhibition of the normal function of cadherin or Pak1 leads to defects in focal adhesion turnover and to increased signaling by phosphatidylinositol 3-kinase. We propose that cadherins are involved in regulation of contact inhibition by controlling the function of the Pak1-βPIX-GIT complex at focal contacts.    

3.1423           Cell Signaling, Internalization, and Nuclear Localization of the Angiotensin Converting Enzyme in Smooth Muscle and Endothelial Cells

Lucero, H.A., Kintsurashvili,, E., Marketou, M.E. and Gavras, H. J: Biol. Chem., 285(8), 5555-5568 (2010)   The angiotensin converting enzyme (ACE) catalyzes the extracellular formation of angiotensin II, and degradation of bradykinin, thus regulating blood pressure and renal handling of electrolytes. We have previously shown that exogenously added ACE elicited transcriptional regulation independent of its enzymatic activity. Because transcriptional regulation generates from protein-DNA interactions within the cell nucleus we have investigated the initial cellular response to exogenous ACE and the putative internalization of the enzyme in smooth muscle cells (SMC) and endothelial cells (EC). The following phenomena were observed when ACE was added to cells in culture: 1) it bound to SMC and EC with high affinity (K_d = 361.5 ± 60.5 pm) and with a low binding occupancy (B_max = 335.0 ± 14.0 molecules/cell); 2) it triggered cellular signaling resulting in late activation of focal adhesion kinase and SHP2; 3) it modulated platelet-derived growth factor receptor-β signaling; 4) it was endocytosed by SMC and EC; and 5) it transited through the early endosome, partially occupied the late endosome and the lysosome, and was localized to the nuclei. The incorporation of ACE or a fragment of it into the nuclei reached saturation at 120 min, and was preceded by a lag time of 40 min. Internalized ACE was partially cleaved into small fragments. These results revealed that extracellular ACE modulated cell signaling properties, and that SMC and EC have a pathway for delivery of extracellular ACE to the nucleus, most likely involving cell surface receptor(s) and requiring transit through late endosome/lysosome compartments.  

3.1424           Green tea catechin EGCG inhibits ileal apical sodium bile acid transporter ASBT

Annaba, F., Kumar, P., Dudeja, A.K., Saksena, S., Gill, R.K. and Alrefai, W.A.

1. J. Gastrointest. Liver Physiol., 298, G467-G473 (2010)

  Green tea catechins exhibit hypocholesterolemic effects probably via their inhibitory effects on intestinal bile acid absorption. Ileal apical sodium-dependent bile acid transporter (ASBT) is responsible for reabsorption of bile acids. The present studies were, therefore, designed to investigate the modulation of ASBT function and membrane expression by green tea catechins in human embryonic kidney HEK-293 cells stably transfected with ASBT-V5 fusion protein and intestinal Caco-2 monolayers. Our data showed that ASBT activity was significantly decreased by (–)-epigallocatechin-3-gallate (EGCG) but not other green tea catechins. Inhibition of PKC, phosphatidylinositol 3-kinase, and MAPK-dependent pathways failed to block the reduction in ASBT activity by EGCG. Kinetics studies showed a significant decrease in the V_max of the transporter, whereas total ASBT content on the plasma membrane was unaltered by EGCG. Concomitant with the decrease in ASBT function, EGCG significantly reduced ASBT pool in the detergent-insoluble fraction, while increasing its presence in the detergent-soluble fraction of plasma membrane. Furthermore, EGCG decreased the association of ASBT with floating lipid raft fractions of cellular membrane on Optiprep density gradient. In conclusion, our data demonstrate a novel role of lipid rafts in the modulation of ASBT function by the dietary component EGCG, which may underlie the hypocholesterolemic effects of green tea.  

3.1425           Rituximab inhibits B-cell receptor signaling

Kheirallah, S., Caron, P., Gross, E., Quillet-Mary, A., Bertrand-Michel, J., Fournie, J-J., Laurent, G. and Bezombes, C. Blood, 115(5), 985-994 (2010)   Rituximab (RTX), a monoclonal antibody directed against the CD20 protein, is a drug commonly used in the treatment of B-cell–derived lymphoid neoplasias and of antibody-mediated autoimmune diseases. In addition to cell- and complement-mediated B-cell depletion, RTX is thought to inhibit B-cell survival and proliferation through negative regulation of canonical signaling pathways involving Akt, ERK, and mammalian target of rapamycin. However, surprisingly, although B-cell receptor (BCR) signaling has been considered critical for normal and more recently, for neoplastic B cells, the hypothesis that RTX could target BCR has never been investigated. Using follicular lymphoma cell lines as models, as well as normal B cells, we show here, for the first time, that pretreatment with RTX results in a time-dependent inhibition of the BCR-signaling cascade involving Lyn, Syk, PLC 2, Akt, and ERK, and calcium mobilization. The inhibitory effect of RTX correlates with decrease of raft-associated cholesterol, complete inhibition of BCR relocalization into lipid raft microdomains, and down-regulation of BCR immunoglobulin expression. Thus, RTX-mediated alteration of BCR expression, dynamics, and signaling might contribute to the immunosuppressive activity of the drug.  

3.1426           Detergent-resistant microdomains mediate activation of host cell signaling in response to attaching–effacing bacteria

Shen-Tu, G., Schauer, D.B., Jones, N.L. and Sherman, P.M. Lab. Invest., 90(2), 266-281 (2010)   Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes outbreaks of bloody diarrhea and the hemolytic–uremic syndrome. EHEC intimately adheres to epithelial cells, effaces microvilli and induces attaching–effacing (AE) lesions. Detergent-resistant microdomains (lipid rafts) serve as membrane platforms for the recruitment of signaling complexes to mediate host responses to infection. The aim of this study was to define the role of lipid rafts in activating signal transduction pathways in response to AE bacterial pathogens. Epithelial cell monolayers were infected with EHEC (MOI 100:1, 3 h, 37°C) and lipid rafts isolated by buoyant density ultracentrifugation. Phosphoinositide 3-kinase (PI3K) localization to lipid rafts was confirmed using PI3K and anti-caveolin-1 antibodies. Mice with cholesterol storage disease Niemann–Pick, type C were used as in vivo models to confirm the role of lipid rafts in mediating signaling response to AE organisms. In contrast to uninfected cells, PI3K was recruited to lipid rafts in response to EHEC infection. Metabolically active bacteria and cells with intact cholesterol-rich microdomains were necessary for the recruitment of second messengers to lipid rafts. Recruitment of PI3K to lipid rafts was independent of the intimin (eaeA) gene, type III secretion system, and production of Shiga-like toxins. Colonization of NPC−/− colonic mucosa by Citrobacter rodentium and AE lesion formation were both delayed, compared with wild-type mice infected with the murine-specific AE bacterial pathogen. C. rodentium-infected NPC−/− mice had reduced colonic epithelial hyperplasia (64±8.251 vs 112±2.958 μm; P<0.05) and decreased secretion of IFN-γ (17.6±17.6 vs 71±26.3 pg/ml, P<0.001). Lipid rafts mediate host cell signal transduction responses to AE bacterial infections both in vitro and in vivo. These findings advance the current understanding of microbial–eukaryotic cell interactions in response to enteric pathogens that hijack signaling responses mediated through lipid rafts.  

3.1427           Molecular Species of Phosphatidylinositol-Cycle Intermediates in the Endoplasmic Reticulum and Plasma Membrane

Shulga, Y.V., Mmyers, D.S., Ivanova, P.T., Milne, S.B., Brown, H.A., Topham, M.K. and Epand, R.M. Biochemistry, 49(2), 312-317 (2010)   Phosphatidylinositol (PI) turnover is a process requiring both the plasma and ER membranes. We have determined the distribution of phosphatidic acid (PA) and PI and their acyl chain compositions in these two subcellular membranes using mass spectrometry. We assessed the role of PI cycling in determining the molecular species and quantity of these lipids by comparing the compositions of the two membranes isolated from embryonic fibroblasts obtained from diacylglycerol kinase ε (DGKε) knockout (KO) and wild-type (WT) mice. In the KO cells, the conversion of arachidonoyl-rich DAG to PA is blocked by the absence of DGKε, resulting in a reduction in the rate of PI cycling. The acyl chain composition is very similar for PI and PA in the endoplasmic reticulum (ER) versus plasma membrane (PM) and for WT versus KO. However, the acyl chain profile for PI is very different from that for PA. This indicates that DGKε is not facilitating the direct transfer of a specific species of PA between the PM and the ER. Approximately 20% of the PA in the ER membrane has one short acyl chain of 14 or fewer carbons. These species of PA are not converted into PI but may play a role in stabilizing regions of high positive curvature in the ER. There are also PI species in both the ER and PM for which there is no detectable PA precursor, indicating that these species of PI are unlikely to arise via the PI cycle. We find that in the PM of KO cells the levels of PI and of PA are decreased 3-fold in comparison with those in either the PM of WT cells or the ER of KO cells. The PI cycle is slowed in the KO cells; hence, the lipid intermediates of the PI cycle can no longer be interconverted and are depleted from the PI cycle by conversion to other species. There is less of an effect of the depletion in the ER where de novo synthesis of PA occurs in comparison with the PM.  

3.1428           Sphingomyelin-rich domains are sites of lysenin oligomerization: Implications for raft studies

Kulma, M., herec, M., Grudzinski, W., Anderluh, G., Gruszecki, W., Kwiarkowska, K. and Sobota, A. Biochim. Biophys. Acta, 1798, 471-481 (2010)   Lysenin is a self-assembling, pore-forming toxin which specifically recognizes sphingomyelin. Mutation of tryptophan 20 abolishes lysenin oligomerization and cytolytic activity. We studied the interaction of lysenin WT and W20A with sphingomyelin in membranes of various lipid compositions which, according to atomic force microscopy studies, generated either homo- or heterogeneous sphingomyelin distribution. Liposomes composed of SM/DOPC, SM/DOPC/cholesterol and SM/DPPC/cholesterol could bind the highest amounts of GST-lysenin WT, as shown by surface plasmon resonance analysis. These lipid compositions enhanced the release of carboxyfluorescein from liposomes induced by lysenin WT, pointing to the importance of heterogeneous sphingomyelin distribution for lysenin WT binding and oligomerization. Lysenin W20A bound more weakly to sphingomyelin-containing liposomes than did lysenin WT. The same amounts of lysenin W20A bound to sphingomyelin mixed with either DOPC or DPPC, indicating that the binding was not affected by sphingomyelin distribution in the membranes. The mutant lysenin had a limited ability to penetrate hydrophobic region of the membrane as indicated by measurements of surface pressure changes. When applied to detect sphingomyelin on the cell surface, lysenin W20A formed large conglomerates on the membrane, different from small and regular clusters of lysenin WT. Only lysenin WT recognized sphingomyelin pool affected by formation of raft-based signaling platforms. During fractionation of Triton X-100 cell lysates, SDS-resistant oligomers of lysenin WT associated with membrane fragments insoluble in Triton X-100 while monomers of lysenin W20A partitioned to Triton X-100-soluble membrane fractions. Altogether, the data suggest that oligomerization of lysenin WT is a prerequisite for its docking in raft-related domains.  

3.1429           PPAR Ligand 15-Deoxy-delta 12,14-Prostaglandin J2 Sensitizes Human Colon Carcinoma Cells to TWEAK-induced Apoptosis

Dionne, S., Levy, E., Levesque, D. and Seidman, E.G. Anticancer Res., 30, 157-166 (2010)   Background: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) has been shown to induce colon cancer cell apoptosis in the presence of interferon-γ. We hypothesized that co-treatment using TWEAK with other pro-apoptosis agents could sensitize death receptor-resistant colon cancer cells. Materials and Methods: The effects of chemopreventive agents and TWEAK on cell death and apoptosis were determined using propidium iodide (PI) exclusion and M30 CytoDEATH. Results: We found that 15d-PGJ₂ sensitizes colon cancer cells to TWEAK-induced apoptosis. Caspase inhibition reduced 15d-PGJ₂-, but not 15d-PGJ₂+TWEAK-induced apoptosis. 15d-PGJ₂ promoted reactive oxygen species (ROS) production and dissipation of mitochondrial potential (ΔΨ_m) that were more marked with combined treatment. ROS, ΔΨ_m and cell death were partially normalized by the antioxidant N-acetylcysteine. TWEAK induced nuclear factor-kappa B activation, which was attenuated by 15d-PGJ₂. 15d-PGJ₂ reduced the expression of the anti-apoptotic proteins BCL-X_L and MCL-1, while increasing BAX and translocation of cytochrome c and apoptosis-inducing factor. Conclusion: 15d-PGJ₂ sensitized cancer cells to TWEAK-induced apoptosis through an ROS-dependent cell death pathway and may have chemotherapeutic utility as an apoptosis-enhancing agent.  

3.1430           The Matrix Peptide Exporter HAF-1 Signals a Mitochondrial UPR by Activating the Transcription Factor ZC376.7 in C. elegans

Haynes, C.M., Yang, Y., Blais, S.P., Neubert, T.A. and Ron, D. Mol. Cell., 37(4), 529-540 (2010)   Genetic analyses previously implicated the matrix-localized protease ClpP in signaling the stress of protein misfolding in the mitochondrial matrix to activate nuclear-encoded mitochondrial chaperone genes in C. elegans (UPR^mt). Here, we report that haf-1, a gene encoding a mitochondria-localized ATP-binding cassette protein, is required for signaling within the UPR^mt and for coping with misfolded protein stress. Peptide efflux from isolated mitochondria was ATP dependent and required HAF-1 and the protease ClpP. Defective UPR^mt signaling in the haf-1-deleted worms was associated with failure of the bZIP protein, ZC376.7, to localize to nuclei in worms with perturbed mitochondrial protein folding, whereas zc376.7(RNAi) strongly inhibited the UPR^mt. These observations suggest a simple model whereby perturbation of the protein-folding environment in the mitochondrial matrix promotes ClpP-mediated generation of peptides whose haf-1-dependent export from the matrix contributes to UPR^mt signaling across the mitochondrial inner membrane.  

3.1431           R-Ras regulates β1-integrin trafficking via effects on membrane ruffling and endocytosis

Conklin, M.C., Ada-Nguema, A., Parsons, M., Riching, K.M. and Keely, P.J. BMC Cell Biology, 11, 14-28 (2010)   Background Integrin-mediated cell adhesion and spreading is dramatically enhanced by activation of the small GTPase, R-Ras. Moreover, R-Ras localizes to the leading edge of migrating cells, and regulates membrane protrusion. The exact mechanisms by which R-Ras regulates integrin function are not fully known. Nor is much known about the spatiotemporal relationship between these two molecules, an understanding of which may provide insight into R-Ras regulation of integrins. Results GFP-R-Ras localized to the plasma membrane, most specifically in membrane ruffles, in Cos-7 cells. GFP-R-Ras was endocytosed from these ruffles, and trafficked via multiple pathways, one of which involved large, acidic vesicles that were positive for Rab11. Cells transfected with a dominant negative form of GFP-R-Ras did not form ruffles, had decreased cell spreading, and contained numerous, non-trafficking small vesicles. Conversely, cells transfected with the constitutively active form of GFP-R-Ras contained a greater number of ruffles and large vesicles compared to wild-type transfected cells. Ruffle formation was inhibited by knock-down of endogenous R-Ras with siRNA, suggesting that activated R-Ras is not just a component of, but also an architect of ruffle formation. Importantly, β₁-integrin co-localized with endogenous R-Ras in ruffles and endocytosed vesicles. Expression of dominant negative R-Ras or knock down of R-Ras by siRNA prevented integrin accumulation into ruffles, impaired endocytosis of β₁-integrin, and decreased β₁-integrin-mediated adhesion. Knock-down of R-Ras also perturbed the dynamics of another membrane-localized protein, GFP-VSVG, suggesting a more global role for R-Ras on membrane dynamics. However, while R-Ras co-internalized with integrins, it did not traffic with VSVG, which instead moved laterally out of ruffles within the plane of the membrane, suggesting multiple levels of regulation of and by R-Ras. Conclusions Our results suggest that integrin function involves integrin trafficking via a cycle of membrane protrusion, ruffling, and endocytosis regulated by R-Ras, providing a novel mechanism by which integrins are linked to R-Ras through control of membrane dynamics.  

3.1432           Acquisition of Complement Resistance through Incorporation of CD55/Decay-Accelerating Factor into Viral Particles Bearing Baculovirus GP64

Kaname, Y., Tani, H., Kataoka, C., Shiokawa, M., Taguwa, S., Abe, T., Moriishi, K., Kinoshita, T. and Matsuura, Y.

1. Virol., 84(7), 3210-3219 (2010)

  A major obstacle to gene transduction by viral vectors is inactivation by human complement in vivo. One way to overcome this is to incorporate complement regulatory proteins, such as CD55/decay accelerating factor (DAF), into viral particles. Lentivirus vectors pseudotyped with the baculovirus envelope protein GP64 have been shown to acquire more potent resistance to serum inactivation and longer transgene expression than those pseudotyped with the vesicular stomatitis virus (VSV) envelope protein G. However, the molecular mechanisms underlying resistance to serum inactivation in pseudotype particles bearing the GP64 have not been precisely elucidated. In this study, we generated pseudotype and recombinant VSVs bearing the GP64. Recombinant VSVs generated in human cell lines exhibited the incorporation of human DAF in viral particles and were resistant to serum inactivation, whereas those generated in insect cells exhibited no incorporation of human DAF and were sensitive to complement inactivation. The GP64 and human DAF were detected on the detergent-resistant membrane and were coprecipitated by immunoprecipitation analysis. A pseudotype VSV bearing GP64 produced in human DAF knockdown cells reduced resistance to serum inactivation. In contrast, recombinant baculoviruses generated in insect cells expressing human DAF or carrying the human DAF gene exhibited resistance to complement inactivation. These results suggest that the incorporation of human DAF into viral particles by interacting with baculovirus GP64 is involved in the acquisition of resistance to serum inactivation.  

3.1433           Endocytosis of the Anthrax Toxin Is Mediated by Clathrin, Actin and Unconventional Adaptors

Abrami, L., Bischofberger, M., Kubnz, B., Groux, R. and van der Goot, F.G. PloSPathogens, 6(3), e1000792 (2010)   The anthrax toxin is a tripartite toxin, where the two enzymatic subunits require the third subunit, the protective antigen (PA), to interact with cells and be escorted to their cytoplasmic targets. PA binds to cells via one of two receptors, TEM8 and CMG2. Interestingly, the toxin times and triggers its own endocytosis, in particular through the heptamerization of PA. Here we show that PA triggers the ubiquitination of its receptors in a β-arrestin-dependent manner and that this step is required for clathrin-mediated endocytosis. In addition, we find that endocytosis is dependent on the heterotetrameric adaptor AP-1 but not the more conventional AP-2. Finally, we show that endocytosis of PA is strongly dependent on actin. Unexpectedly, actin was also found to be essential for efficient heptamerization of PA, but only when bound to one of its 2 receptors, TEM8, due to the active organization of TEM8 into actin-dependent domains. Endocytic pathways are highly modular systems. Here we identify some of the key players that allow efficient heptamerization of PA and subsequent ubiquitin-dependent, clathrin-mediated endocytosis of the anthrax toxin.  

3.1434           Redox signaling via lipid raft clustering in homocysteine-induced injury of podocytes

Zhang, C., Hu, J-J., Xia, M., Boini, K.M., Brimson, C. and Li, P-L. Biochim. Biophys. Acta, 1803, 482-491 (2010)   Our recent studies have indicated that hyperhomocysteinemia (hHcys) may induce podocyte damage, resulting in glomerulosclerosis. However, the molecular mechanisms mediating hHcys-induced podocyte injury are still poorly understood. In the present study, we first demonstrated that an intact NADPH oxidase system is present in podocytes as shown by detection of its membrane subunit (gp91^phox) and cytosolic subunit (p47^phox). Then, confocal microscopy showed that gp91^phox and p47^phox could be aggregated in lipid raft (LR) clusters in podocytes treated with homocysteine (Hcys), which were illustrated by their colocalization with cholera toxin B, a common LR marker. Different mechanistic LR disruptors, either methyl-β-cyclodextrin (MCD) or filipin abolished such Hcys-induced formation of LR-gp91^phox or LR-p47^phox transmembrane signaling complexes. By flotation of detergent-resistant membrane fractions we found that gp91^phox and p47^phox were enriched in LR fractions upon Hcys stimulation, and such enrichment of NADPH oxidase subunits and increase in its enzyme activity were blocked by MCD or filipin. Functionally, disruption of LR clustering significantly attenuated Hcys-induced podocyte injury, as shown by their inhibitory effects on Hcys-decreased expression of slit diaphragm molecules such as nephrin and podocin. Similarly, Hcys-increased expression of desmin was also reduced by disruption of LR clustering. In addition, inhibition of such LR-associated redox signaling prevented cytoskeleton disarrangement and apoptosis induced by Hcys. It is concluded that NADPH oxidase subunits aggregation and consequent activation of this enzyme through LR clustering is an important molecular mechanism triggering oxidative injury of podocytes induced by Hcys.  

3.1435           Low Density Subcellular Fractions Enhance Disease-specific Prion Protein Misfolding

Graham, J.F., Agarwal, S., Kurian, D., Kirby, L., Pinheiro, T.J. and gill, A.C.

1. Biol. Chem., 285(13), 9868-9880 (2010)

  The production of prion particles in vitro by amplification with or without exogenous seed typically results in infectivity titers less than those associated with PrP^Sc isolated ex vivo and highlights the potential role of co-factors that can catalyze disease-specific prion protein misfolding in vivo. We used a cell-free conversion assay previously shown to replicate many aspects of transmissible spongiform encephalopathy disease to investigate the cellular location of disease-specific co-factors using fractions derived from gradient centrifugation of a scrapie-susceptible cell line. Fractions from the low density region of the gradient doubled the efficiency of conversion of recombinant PrP. These fractions contain plasma membrane and cytoplasmic proteins, and conversion enhancement can be achieved using PrP^Sc derived from two different strains of mouse-passaged scrapie as seed. Equivalent fractions from a second scrapie-susceptible cell line also stimulate conversion. We also show that subcellular fractions enhancing disease-specific prion protein conversion prevent in vitro fibrillization of recombinant prion protein, suggesting the existence of separate, competing mechanisms of disease-specific and nonspecific misfolding in vivo.  

3.1436           Cholesterol–Protein Interaction: Methods and Cholesterol Reporter Molecules

Gimpl, G. Subcellular Biochem., 51, 1-45 (2010)   Cholesterol is a major constituent of the plasma membrane in eukaryotic cells. It regulates the physical state of the phospholipid bilayer and is crucially involved in the formation of membrane microdomains. Cholesterol also affects the activity of several membrane proteins, and is the precursor for steroid hormones and bile acids. Here, methods are described that are used to explore the binding and/or interaction of proteins to cholesterol. For this purpose, a variety of cholesterol probes bearing radio-, spin-, photoaffinity- or fluorescent labels are currently available. Examples of proven cholesterol binding molecules are polyene compounds, cholesterol-dependent cytolysins, enzymes accepting cholesterol as substrate, and proteins with cholesterol binding motifs. Main topics of this report are the localization of candidate membrane proteins in cholesterol-rich microdomains, the issue of specificity of cholesterol– protein interactions, and applications of the various cholesterol probes for these studies.  

3.1437           Vimentin-mediated signalling is required for IbeA+ E. coli K1 invasion of human brain microvascular endothelial cells

Chi, F., Jong, T.D., Wang, L., Ouyang, Y., Wu, C., Li, W. and Huang, S-H. Biochem. J., 427, 79-90 (2010)   IbeA in meningitic Escherichia coli K1 strains has been described previously for its role in invasion of BMECs (brain microvascular endothelial cells). Vimentin was identified as an IbeA-binding protein on the surface of HBMECs (human BMECs). In the present study, we demonstrated that vimentin is a primary receptor required for IbeA+ E. coli K1-induced signalling and invasion of HBMECs, on the basis of the following observations. First, E44 (IbeA+ E. coli K1 strain) invasion was blocked by vimentin inhibitors (withaferin A and acrylamide), a recombinant protein containing the vimentin head domain and an antibody against the head domain respectively. Secondly, overexpression of GFP (green fluorescent protein)–vimentin and GFP–VDM (vimentin head domain deletion mutant) significantly increased and decreased bacterial invasion respectively. Thirdly, bacterial invasion was positively correlated with phosphorylation of vimentin at Ser⁸² by CaMKII (Ca²⁺/calmodulin-dependent protein kinase II) and IbeA+ E. coli-induced phosphorylation of ERK (extracellular-signal-regulated kinase). Blockage of CaMKII by KN93 and inhibition of ERK1/2 phosphorylation by PD098059 resulted in reduced IbeA+ E. coli invasion. Fourthly, IbeA+ E. coli and IbeA-coated beads induced the clustering of vimentin that was correlated with increased entry of bacteria and beads. Lastly, IbeA+ E. coli K1 invasion was inhibited by lipid-raft-disrupting agents (filipin and nystatin) and caveolin-1 siRNA (small interfering RNA), suggesting that caveolae/lipid rafts are signalling platforms for inducing IbeA–vimentin-mediated E. coli invasion of HBMECs. Taken together, the present studies suggest that a dynamic and function-related interaction between IbeA and its primary receptor vimentin at HBMEC membrane rafts leads to vimentin phosphorylation and ERK-mediated signalling, which modulate meningitic E. coli K1 invasion.  

3.1438           Mechanisms of proximal tubule sodium transport regulation that link extracellular fluid volume and blood pressure

McDonough, A.A. Am. J. Physiol. Regyl. Integr. Comp. Physiol., 298, R851-R861 (2010)   One-hundred years ago, Starling articulated the interdependence of renal control of circulating blood volume and effective cardiac performance. During the past 25 years, the molecular mechanisms responsible for the interdependence of blood pressure (BP), extracellular fluid volume (ECFV), the renin-angiotensin system (RAS), and sympathetic nervous system (SNS) have begun to be revealed. These variables all converge on regulation of renal proximal tubule (PT) sodium transport. The PT reabsorbs two-thirds of the filtered Na⁺ and volume at baseline. This fraction is decreased when BP or perfusion pressure is increased, during a high-salt diet (elevated ECFV), and during inhibition of the production of ANG II; conversely, this fraction is increased by ANG II, SNS activation, and a low-salt diet. These variables all regulate the distribution of the Na⁺/H⁺ exchanger isoform 3 (NHE3) and the Na⁺-phosphate cotransporter (NaPi2), along the apical microvilli of the PT. Natriuretic stimuli provoke the dynamic redistribution of these transporters along with associated regulators, molecular motors, and cytoskeleton-associated proteins to the base of the microvilli. The lipid raft-associated NHE3 remains at the base, and the nonraft-associated NaPi2 is endocytosed, culminating in decreased Na⁺ transport and increased PT flow rate. Antinatriuretic stimuli return the same transporters and regulators to the body of the microvilli associated with an increase in transport activity and decrease in PT flow rate. In summary, ECFV and BP homeostasis are, at least in part, maintained by continuous and acute redistribution of transporter complexes up and down the PT microvilli, which affect regulation of PT sodium reabsorption in response to fluctuations in ECFV, BP, SNS, and RAS.  

3.1439           Isoflurane via TGF-β1 release increases caveolae formation and organizes sphingosine kinase signaling in renal proximal tubules

Song, J.H., Kim, M., Park, S.W., Chen, S.W.C., Pitson, S.M. and Lee, H.T. Am. J. Physiol. Renal Physiol., 298, F1041-F1050 (2010)   We previously showed that the inhalational anesthetic isoflurane protects against renal proximal tubule necrosis via isoflurane-mediated stimulation and translocation of sphingosine kinase-1 (SK1) with subsequent synthesis of sphingosine-1-phosphate (S1P) in renal proximal tubule cells (Kim M, Kim M, Kim N, D'Agati VD, Emala CW Sr, Lee HT. Am J Physiol Renal Physiol 293: F1827–F1835, 2007). We also demonstrated that the anti-necrotic and anti-inflammatory effect of isoflurane is due in part to phosphatidylserine (PS) externalization and subsequent release of transforming growth factor-β1 (TGF-β1) (Lee HT, Kim M, Kim J, Kim N, Emala CW. Am J Nephrol 27: 416–424, 2007). In this study, we tested the hypothesis that isoflurane, via TGF-β1 release, increases caveolae formation in the buoyant fraction of the cell membrane of human renal proximal tubule (HK-2) cells to organize SK1 and S1P signaling. To detect SK1 protein in the caveolae/caveolin fractions, we overexpressed human SK1 in HK-2 cells (SK1-HK-2). SK1-HK-2 cells exposed to isoflurane increased caveolae/caveolin formation in the buoyant membrane fractions which contained key signaling intermediates involved in isoflurane-mediated renal tubule protection, including S1P, SK1, ERK MAPK, and TGF-β1 receptors. Furthermore, treating SK1-HK-2 cells with recombinant TGF-β1 or PS liposome mixture increased caveolae formation, mimicking the effects of isoflurane. Conversely, TGF-β1-neutralizing antibody blocked the increase in caveolae formation induced by isoflurane in SK1-HK-2 cells. The increase in SK1 activity in the caveolae-enriched fractions from isoflurane-treated nonlentivirus-infected HK-2 cells, while smaller in magnitude, was qualitatively similar to that found in the SK1-HK-2 cell line. Finally, isoflurane also increased caveolae formation in the kidneys of TGF-β1 +/+ mice but not in TGF-β1 +/– mice (mice with reduced levels of TGF-β1). Our study demonstrates that isoflurane organizes several key cytoprotective signaling intermediates including TGF-β1 receptors, SK1 and ERK, within the caveolae fraction of the plasma membrane. Our findings may help to unravel the cellular signaling pathways of volatile anesthetic-mediated renal protection and lead to new therapeutic applications of inhalational anesthetics during the perioperative period.  

3.1440           Control of Rhodopsin's Active Lifetime by Arrestin-1 Expression in Mammalian Rods

Gross, O.P. and Burns, M.E.

1. Neurosci., 30(9), 3450-3457 (2010)

  In rod photoreceptors, deactivation of the light-activated G-protein-coupled receptor rhodopsin (R*) is initiated by phosphorylation and completed through subsequent binding of visual arrestin (Arr1). The in vivo kinetics of these individual interactions have proven difficult to determine with precision since R* lifetime is much shorter than the lifetimes of downstream G-protein and effector molecules. Here, we have used a transgenic mouse line with accelerated downstream deactivation kinetics to reveal the contribution of Arr1 binding to the overall time course of rhodopsin deactivation. Photoresponses revealed that the lifetime of R* is significantly increased in rods that express half of the normal amount of Arr1, in a manner consistent with a twofold decrease in the rate of Arr1 binding across a wide range of flash strengths. A basic model of photoresponse deactivation consistent with established photoreceptor biochemistry shows that R* phosphorylation and Arr1 binding occur with a time constant of 40 ms in wild-type mouse rods, much faster than previous estimates.  

3.1441           Troubleshooting methods for APP processing in vitro

Sastre, M.

1. Pharmacol. Toxicol methods, 61, 86-91 (2010)

  Introduction The amyloid hypothesis states that Aβ is the main trigger for Alzheimer's disease. This report is focused in the study of the processing of the Amyloid Precursor Protein (APP) as a procedure to investigate the molecular mechanisms that may result in changes in the levels of Aβ. Methods Here we analyse different methodologies for Aβ determination, soluble APP, APP-Carboxy terminus fragments (CTFs) and enzymes for synthesis (secretases) and degradation of Aβ. In addition the advantages and disadvantages of different methodologies are discussed. Discussion The potential value of these procedures is described in the context of the function of APP and the different fragments derived from its cleavage.      

3.1442           The Human Polyoma JC Virus Agnoprotein Acts as a Viroporin

Suzuki, T., Orba, Y., Okada, Y., Sunden, Y., Kimura, T., Tanaka, S., Nagashima, K., Hall, W.W., Sawa, H. PloSPathogens, 6(3), e1000801 (2010)   Virus infections can result in a range of cellular injuries and commonly this involves both the plasma and intracellular membranes, resulting in enhanced permeability. Viroporins are a group of proteins that interact with plasma membranes modifying permeability and can promote the release of viral particles. While these proteins are not essential for virus replication, their activity certainly promotes virus growth. Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease resulting from lytic infection of oligodendrocytes by the polyomavirus JC virus (JCV). The genome of JCV encodes six major proteins including a small auxiliary protein known as agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to viral propagation at various stages in the replication cycle, including transcription, translation, processing of late viral proteins, assembly of virions, and viral propagation. Previous studies from our and other laboratories have indicated that JCV agnoprotein plays an important, although as yet incompletely understood role in the propagation of JCV. Here, we demonstrate that agnoprotein possesses properties commonly associated with viroporins. Our findings demonstrate that: (i) A deletion mutant of agnoprotein is defective in virion release and viral propagation; (ii) Agnoprotein localizes to the ER early in infection, but is also found at the plasma membrane late in infection; (iii) Agnoprotein is an integral membrane protein and forms homo-oligomers; (iv) Agnoprotein enhances permeability of cells to the translation inhibitor hygromycin B; (v) Agnoprotein induces the influx of extracellular Ca²⁺; (vi) The basic residues at amino acid positions 8 and 9 of agnoprotein key are determinants of the viroporin activity. The viroporin-like properties of agnoprotein result in increased membrane permeability and alterations in intracellular Ca²⁺ homeostasis leading to membrane dysfunction and enhancement of virus release.  

3.1443           Mapping of Vps21 and HOPS Binding Sites in Vps8 and Effect of Binding Site Mutants on Endocytic Trafficking

Pawelec, A., Arsic, J. and Krölling, R. Eukaryot. Cell, 9(4), 602-610 (2010)   Vps8 is a subunit of the CORVET tethering complex, which is involved in early-to-late endosome fusion. Here, we examine the role of Vps8 in membrane fusion at late endosomes in Saccharomyces cerevisiae. We demonstrate that Vps8 associates with membranes and that this association is independent of the class C/HOPS core complex and, contrary to a previous report, also independent of the Rab GTPase Vps21. Our data indicate that Vps8 makes multiple contacts with membranes. One of these membrane binding regions could be mapped to the N-terminal part of the protein. By two-hybrid analysis, we obtained evidence for a physical interaction between Vps8 and the Rab5 homologue Vps21. In addition, the interaction with the HOPS core complex was confirmed by immunoprecipitation experiments. By deletion analysis, the Vps21 and HOPS binding sites were mapped in Vps8. Deletions that abrogated HOPS core complex binding had a strong effect on the turnover of the endocytic cargo protein Ste6 and on vacuolar sorting of carboxypeptidase Y. In contrast, deletions that abolished Vps21 binding showed only a modest effect. This suggests that the Vps21 interaction is not essential for endosomal trafficking but may be important for some other aspect of Vps8 function.  

3.1444           Amot Recognizes a Juxtanuclear Endocytic Recycling Compartment via a Novel Lipid Binding Domain

Heller, B., Adu-Gyamfi, E., Smith-Kinnaman, W., Babbey, C., Vora, M., Xue, Y., Bittman, R., Stahelin, R.V. and Wells, C.D.

1. Biol. Chem., 285(16), 12308-12320 (2010)

  Polarity proteins promote the asymmetric organization of cells by orienting intracellular sorting mechanisms, such as protein trafficking and cytoskeletal assembly. The localization of individual polarity proteins in turn is often determined by association with factors that mediate contact with other cells or the substratum. This arrangement for the Par and Crb apical polarity complexes at the tight junction is disrupted by the adaptor protein Amot. Amot directly binds the scaffolding proteins Patj and Mupp1 and redistributes them and their binding partners from the plasma membrane to endosomes. However, the mechanism by which Amot is targeted to endosomes is unknown. Here, a novel lipid binding domain within Amot is shown to selectively bind with high affinity to membranes containing monophosphorylated phosphatidylinositols and cholesterol. With similar lipid specificity, Amot inserts into and tubulates membranes in vitro and enlarges perinuclear endosomal compartments in cells. Based on the similar distribution of Amot with cholesterol, Rab11, and Arf6, such membrane interactions are identified at juxtanuclear endocytic recycling compartments. Taken together, these findings indicate that Amot is targeted along with associated apical polarity proteins to the endocytic recycling compartment via this novel membrane binding domain.  

3.1445           A Role for the C Terminus of Mopeia Virus Nucleoprotein in Its Incorporation into Z Protein-Induced Virus-Like Particles

Shtanko, O., Imai, M., Goto, H., Lakashevich, I.S., Neumann, G., Watanabe, T. and Kawaoka, Y.

1. Virol., 84(10), 5415-5422 (2010)

  Arenaviruses are enveloped, negative-strand RNA viruses. For several arenaviruses, virus-like particle (VLP) formation requires the viral matrix Z protein. However, the mechanism by which viral ribonucleoprotein complexes are incorporated into virions is poorly understood. Here, we show that the expression of the Z protein and nucleoprotein (NP) of Mopeia virus, a close relative of the pathogenic Lassa virus, resulted in the highly selective incorporation of the NP protein into Z protein-induced VLPs. Moreover, the Z protein promoted the association of NP with cellular membranes, suggesting that the association of NP, Z, and the cellular membranes may facilitate the efficient incorporation of NP into VLPs. By employing a series of NP deletion constructs and testing their VLP incorporation, we further demonstrated an important role for the C-terminal half of NP in its incorporation into VLPs.  

3.1446           Myristoylated Naked2 Antagonizes Wnt-β-Catenin Activity by Degrading Dishevelled-1 at the Plasma Membrane

Hu, T., Li, C., Cao, Z., Van Raay, T.J., Smith, J.G., Willert, K., Solnica-Krezel, L. and Coffey, R.J.

1. Biol. Chem., 285(18), 13561-13568 (2010)

  In Drosophila, naked cuticle is an inducible antagonist of the Wnt-β-catenin pathway, likely acting at the level of Dishevelled (Dsh/Dvl), an essential component of this pathway. The mechanism by which naked cuticle and its two vertebrate orthologs, Naked1 (NKD1) and Naked2 (NKD2), inhibit Dvl function is unknown. NKD2 is myristoylated, a co-translational modification that leads to its plasma membrane localization. In contrast, myristoylation-deficient G2A NKD2 is cytoplasmic. Herein we show that the ability of Nkd2/NKD2 to antagonize Wnt-β-catenin activity during zebrafish embryonic development and in mammalian HEK293 cells is myristoylation-dependent. NKD2 and Dvl-1 interact and co-localize at the lateral membrane of polarized epithelial cells. In reciprocal overexpression and siRNA knockdown experiments, NKD2 and Dvl-1 destabilize each other via enhanced polyubiquitylation; this effect is also dependent upon Naked2 myristoylation. Cell fractionation and ubiquitylation assays indicate that endogenous NKD2 interacts with a slower migrating, ubiquitylated form of Dvl-1 in plasma membrane fractions. These results provide a mechanism by which NKD2 antagonizes Wnt signaling: myristoylated NKD2 interacts with Dvl-1 at the plasma membrane, and this interaction leads to their mutual ubiquitin-mediated proteasomal degradation.  

3.1447           Acidic calcium stores open for business: expanding the potential for intracellular Ca²⁺ signaling

Patel, S. and Docompo, R. Trends in cell Biology, 20(5), 277-286 (2010)   Changes in cytosolic calcium concentration are crucial for a variety of cellular processes in all cells. It has long been appreciated that calcium is stored and released from intracellular calcium stores such as the endoplasmic reticulum. However, emerging evidence indicates that calcium is also dynamically regulated by a seemingly disparate collection of acidic organelles. In this paper, we review the defining features of these ‘acidic calcium stores’ and highlight recent progress in understanding the mechanisms of uptake and release of calcium from these stores. We also examine the nature of calcium buffering within the stores, and summarize the physiological and pathophysiological significance of these ubiquitous organelles in calcium signaling.  

3.1448           Differential Pattern of Junctional Complex Lipid Raft Proteins in Human Conventional Outflow

Mccarty, R.D., Beverley, R.M., Giovingo, M.C., Nolan, M.J., Yue, B.Y.J.T., Stamer, W.D. and Knepper, P.A. Invest. Ophthalmol. Vis. Sci., 51, E-Abstract 3204 (2010)   Purpose:The trabecular meshwork (TM) is a specialized tissue forminga biologic filter for aqueous humor. The two dominant cell typesin the TM are Schlemm’s canal (SC) endothelial cells onthe inner wall of the collecting channels and TM cells thatcover the TM beams. SC endothelial cells differ morphologicallyand functionally from TM endothelial cells. Exactly how thesecells, their junctional complexes or their extracellular matrices,regulate aqueous outflow barrier resistance is poorly understood.The purpose of this study was to compare junctional complexlipid raft proteins of SC cells to that of TM cells. Methods:Human SC and TM cells were grown to confluency in T-25 flasks. After two weeks at confluence, cells were lysed in ice cold lysis buffer containing 1% Triton X-100. Caveolin enriched lipid rafts were isolated using an OptiPrep density gradient (SigmaD1556). The preparation was centrifuged at 200,000 x g for 18hrs; nine 1.0 ml fractions were pipetted from the top (lightest)to bottom (heaviest). Each fraction was analyzed for proteincontent. Equal amounts of proteins were resolved by SDS polyacrylamidegel electrophoresis and immunoblotted with a panel of junctionalcomplex antibodies. Densitometry was performed. Results:Profile of junctional complexes in enriched lipid rafts. Conclusions:This is the first identification and comparison of the junctionalcomplexes in SC and TM. Lipid raft proteins in SC cells aredistinct from TM cells. SC cells express more annexin 2 thanTM cells. Both have considerable amounts of occludin, zona occludensand other constituents of the lipid rafts. These differentialexpression patterns likely correspond to differences in functionof TM and SC endothelial cells in modulating aqueous outflowresistance.  

3.1449           Differential Targeting of HSV Structural Proteins to Axons Requires Association of Viral Us9 Protein to Lipid Rafts

LaVail, J.H., Huang, G., Cortez, D.A., Sucher, A. and Draper, J.M. Invest. Ophthalmol. Vis. Sci., 51, E-abstract 3812 (2010)   The Herpes simplex virus type 1 (HSV) envelope contains at least10 glycoproteins, several of which are essential for viral spreadleading to recurrent herpetic keratitis or viral encephalitis.How these glycoproteins are targeted within mature axons aftersynthesis is unclear. Purpose:Our goal is to examine the axonal transport of the HSV envelopeglycoproteins and to determine whether targeting of newly synthesizedglycoproteins requires an association with the lipid rafts inmembranes of host neurons. Further, we tested whether the viralprotein, Us9, facilitates axonal transport of viral structuralproteins, as well as that of viral capsids. Methods:Murine retinas were pulse infected with Us9-null mutant or wild type (wt) HSV. After 5 days the optic pathways were dissected and prepared in an Optiprep® flotation assay to separateraft (detergent-resistant, DRM) and non-raft (detergent-soluble,DSM) membranes. The proteins were separated by PAGE and processedfor Western blotting using antibodies to gB, gD, Us9, VP5 andsynaptophysin (Syn). We used antisera to caveolin and transferrinand biotinolylated cholera toxin B subunit to GM1 as controls. Results:By 5 days after infection with wt HSV, all proteins were transported to the optic tract. Two envelope glycoproteins (gB, gD), one capsid protein, VP5, and one membrane protein, Us9, were present in DRM fractions. In addition, gB, gD, Us9 and Syn, were found in DSM fractions. After infection with Us9-null HSV, the concentrationof gB in both fractions was significantly reduced. Conclusions:Lipid rafts play a role in the axonal transport of some, but not all of the HSV glycoproteins in axons of mature animals. Some glycoproteins are targeted independently of others. Specifically, the axonal transport of gB is impaired after Us9-null infection,but the transport of gD (and possibly gE and gC) is not impaired.Significantly, this impairment may account for the fact thatcell-cell spread of new virus depends on gB expression. Thissuggests that axonal transport of HSV requires targeting ofthree subassemblies: membranes associated with gD, membranesassociated with gB and the nucleocapsid.  

3.1450           Soluble CD44 Increases Outflow Resistance in Porcine Organ CulturesSoluble CD44 Increases Outflow Resistance in Porcine Organ Cultures

Giovingo, M.C., McCarty, R.D., Beverley, R.M., Nolan, M.J., Samples, J.R., Yue, B.Y.J.T. and Knepper, P.A. Invest. Ophthalmol. Vis. Sci., 51, E-abstract 5838 (2010)   Purpose:CD44 plays major roles in multiple physiological processes.The soluble CD44 (sCD44) concentration is significantly increasedin the aqueous humor of primary open-angle glaucoma (POAG).Increased sCD44 in mouse eyes causes an increase in intraocularpressure (IOP). The purpose of this study was to determine ifsCD44 changes outflow resistance in porcine anterior segmentorgan culture and profiles of lipid raft proteins of the trabecularmeshwork (TM). Methods:sCD44 was purified from human serum using anion exchange, hyaluronic acid (HA) affinity chromatography and immunoprecipitation. Anterior segments of porcine eyes were placed in organ culture and perfused with Dulbecco’s modified eagle medium (DMEM). Flow rates were measured in eyes treated with sCD44, HA or DMEM. Perturbation of lipid raft containing proteins was assessed by Optiprep densitygradient and Western blot analysis of dissected TM. Results:Flow rates are expressed as percent change (+/-) from the baseline. Rates significantly decreased from baseline in HA and sCD44 infused eyes. Analysis of lipid raft containing proteinsof sCD44 infused porcine microdissected TMs and primary culturesof TM cells revealed a decrease in annexin 2 and an increasein caveolin-1 compared to that of controls. Conclusions:Infusion of sCD44 significantly decreased outflow facility inporcine eyes. Notably, sCD44 caused a decrease in annexin 2and an increase in caveolin-1 which may influence outflow resistance  

3.1451           Regulation of Rho GTPase crosstalk, degradation and activity by RhoGDI1

Boulter, E., Garcia-Mata, R., Guilluy, C., Dubash, A., Rossi, G., Brennwald, P.J. and Burridge, K. Nature Cell Biol., 12(5), 477-483 (2010)   At steady state, most Rho GTPases are bound in the cytosol to Rho guanine nucleotide dissociation inhibitors (RhoGDIs)¹. RhoGDIs have generally been considered to hold Rho proteins passively in an inactive state within the cytoplasm. Here we describe an evolutionarily conserved mechanism by which RhoGDI1 controls the homeostasis of Rho proteins in eukaryotic cells. We found that depletion of RhoGDI1 promotes misfolding and degradation of the cytosolic geranylgeranylated pool of Rho GTPases while activating the remaining membrane-bound fraction. Because RhoGDI1 levels are limiting, and Rho proteins compete for binding to RhoGDI1, overexpression of an exogenous Rho GTPase displaces endogenous Rho proteins bound to RhoGDI1, inducing their degradation and inactivation. These results raise important questions about the conclusions drawn from studies that manipulate Rho protein levels. In many cases the response observed may arise not simply from the overexpression itself but from additional effects on the levels and activity of other Rho GTPases as a result of competition for binding to RhoGDI1; this may require a re-evaluation of previously published studies that rely exclusively on these techniques.    

3.1452           Osteopotentia regulates osteoblast maturation, bone formation, and skeletal integrity in mice

Sohaskey, M.L., Jiang, Y., Zhao, J.J., Mohr, A., Roemer, f. and Harland, R.M.

1. Cell Biol., 189(3), 511-525 (2010)

  During skeletal development and regeneration, bone-forming osteoblasts respond to high metabolic demand by active expansion of their rough endoplasmic reticulum (rER) and increased synthesis of type I collagen, the predominant bone matrix protein. However, the molecular mechanisms that orchestrate this response are not well understood. We show that insertional mutagenesis of the previously uncharacterized osteopotentia (Opt) gene disrupts osteoblast function and causes catastrophic defects in postnatal skeletal development. Opt encodes a widely expressed rER-localized integral membrane protein containing a conserved SUN (Sad1/Unc-84 homology) domain. Mice lacking Opt develop acute onset skeletal defects that include impaired bone formation and spontaneous fractures. These defects result in part from a cell-autonomous failure of osteoblast maturation and a posttranscriptional decline in type I collagen synthesis, which is concordant with minimal rER expansion. By identifying Opt as a crucial regulator of bone formation in the mouse, our results uncover a novel rER-mediated control point in osteoblast function and implicate human Opt as a candidate gene for brittle bone disorders.  

3.1453           Cholesterol loading in macrophages stimulates formation of ER-derived vesicles with elevated ACAT1 activity

Sakashita, N., Chang, C.C.Y., Lei, X., Fujiwara,, Y., Takeya, M.and Chang, T-Y.

1. Lipid Res., 51, 1263-1272 82010)

  ACAT1 is normally a resident enzyme in the endoplasmic reticulum (ER). We previously showed that treating macrophages with denatured LDL causes a large increase in ER-derived, ACAT1-positive vesicles. Here, we isolated ER membranes and ER-derived vesicles to examine their ACAT enzyme activity in vitro. The results showed that when macrophages are grown under normal conditions, ACAT1 is located in high density ER membrane; its enzymatic activity is relatively low. Loading macrophages with cholesterol did not increase the total cellular ACAT1 protein content significantly but caused more ACAT1 to appear in ER-derived vesicles. These vesicles exhibit lower density and are associated with markers of both ER and the trans-Golgi network. When normalized with equal ACAT1 protein mass, the enzymatic activities of ACAT1 in ER-derived vesicles were 3-fold higher than those present in ER membrane. Results using reconstituted ACAT enzyme assay showed that the increase in enzyme activity in ER-derived vesicles is not due to an increase in the cholesterol content associated with these vesicles. Overall, our results show that macrophages cope with cholesterol loading by using a novel mechanism: they produce more ER-derived vesicles with elevated ACAT1 enzyme activity without having to produce more ACAT1 protein.  

3.1454           Biochemical Monitoring of the Early Endocytic Traffic of the Type I Interferon Receptor

Payelle-Brogard, B. and Pellegrini, S.

1. Interferon & Cytokine Res., 30(2), 89-98 (2010)

  The type I interferon (IFN) receptor consists of two transmembrane chains IFNAR1 and IFNAR2, associated with the tyrosine kinases Tyk2 and Jak1, respectively. Binding of IFN to this receptor complex induces activation of Jak/Stat and non-Stat signaling pathways. Ligand binding also drives receptor internalization and sorting toward degradation or recycling. To gain insights into receptor trafficking and its relation to signaling, we performed subcellular organelle fractionation from IFN-stimulated Daudi cells and defined biochemically an early endosomal antigen-1 (EEA1)-positive compartment bearing the activated IFN receptor. Endosomes were thus purified by immunoaffinity isolation on anti-EEA1 antibodies-coated beads. The content of these purified endosomal fractions was analyzed by Western blot and proteomics. Shortly after IFN stimulation, robustly ubiquitinated IFNAR1 and a small amount of IFNAR2 were found in this endosomal compartment, which also contained tyrosine-phosphorylated Tyk2 and Jak1. These data strongly point to the prolonged interaction during traffic of the tyrosine kinases, still in an activated configuration, with the receptors. Among the major constituents of this EEA1-positive compartment, some proteins that have been implicated in IFN signaling were identified. Altogether, these observations suggest that trafficking of the IFN receptor through endosomes may regulate signaling pathways.      

3.1455           Cellular factors implicated in prion replication

Abid, K., Morales, R. and Soto, C. FEBS Lett., 584, 2409-2414 (2010)   Prions are the unconventional infectious agents responsible for prion diseases, which are composed mainly by the misfolded prion protein (PrP^Sc) that replicates by converting the host associated cellular prion protein (PrP^C). Several lines of evidence suggest that other cellular components participate in prion conversion, however, the identity or even the chemical nature of such factors are entirely unknown. In this article we study the conversion factor activity by complementation of a PMCA procedure employing purified PrP^C and PrP^Sc. Our results show that the conversion factor is present in all major organs of diverse mammalian species, and is predominantly located in the lipid raft fraction of the cytoplasmic membrane. On the other hand, it is not present in the lower organisms tested (yeast, bacteria and flies). Surprisingly, treatments that eliminate the major classes of chemical molecules do not affect conversion activity, suggesting that various different compounds may act as conversion factor in vitro. This conclusion is further supported by experiments showing that addition of various classes of molecules have a small, but detectable effect on enhancing prion replication in vitro. More research is needed to elucidate the identity of these factors, their detailed mechanism of action and whether or not they are essential component of the infectious particle.  

3.1456           A role for caveolin-1 in mechanotransduction of fetal type II epithelial cells

Wang, Y., Maciejewski, B.S., Drouillard, D., Santos, M., Hokenson, M.A., Hawwa, R.L., Huang, Z. and Sanchez-Esteban, J. Am. J. Physiol. Lung Cell Mol. Physiol.,298, L775-L783 (2010)   Mechanical forces are critical for fetal lung development. Using surfactant protein C (SP-C) as a marker, we previously showed that stretch-induced fetal type II cell differentiation is mediated via the ERK pathway. Caveolin-1, a major component of the plasma membrane microdomains, is important as a signaling protein in blood vessels exposed to shear stress. Its potential role in mechanotransduction during fetal lung development is unknown. Caveolin-1 is a marker of type I epithelial cell phenotype. In this study, using immunocytochemistry, Western blotting, and immunogold electron microscopy, we first demonstrated the presence of caveolin-1 in embryonic day 19 (E19) rat fetal type II epithelial cells. By detergent-free purification of lipid raft-rich membrane fractions and fluorescence immunocytochemistry, we found that mechanical stretch translocates caveolin-1 from the plasma membrane to the cytoplasm. Disruption of the lipid rafts with cholesterol-chelating agents further increased stretch-induced ERK activation and SP-C gene expression compared with stretch samples without disruptors. Similar results were obtained when caveolin-1 gene was knocked down by small interference RNA. In contrast, adenovirus overexpression of the wild-type caveolin-1 or delivery of caveolin-1 scaffolding domain peptide inside the cells decreased stretch-induced ERK phosphorylation and SP-C mRNA expression. In conclusion, our data suggest that caveolin-1 is present in E19 fetal type II epithelial cells. Caveolin-1 is translocated from the plasma membrane to the cytoplasm by mechanical stretch and functions as an inhibitory protein in stretch-induced type II cell differentiation via the ERK pathway.    

3.1457           Variation of Physiochemical Properties and Cell Association Activity of Membrane Vesicles with Growth Phase in Pseudomonas aeruginosa

Tashiro, Y., Ichikawa, S., Shimizu, M., Toyofuka, M., Takaya, N., Nakajima-Kambe, T., Uchiyama, H. and Nomura, N. Appl. Envr. Microbiol., 76(11), 3732-3739 (2010)   Pseudomonas aeruginosa and other Gram-negative bacteria release membrane vesicles (MVs) from their surfaces, and MVs have an ability to interact with bacterial cells. Although it has been known that many bacteria have mechanisms that control their phenotypes with the transition from exponential phase to stationary phase, changes of properties in released MVs have been poorly understood. Here, we demonstrate that MVs released by P. aeruginosa during the exponential and stationary phases possess different physiochemical properties. MVs purified from the stationary phase had higher buoyant densities than did those purified from the exponential phase. Surface charge, characterized by zeta potential, of MVs tended to be more negative as the growth shifted to the stationary phase, although the charges of PAO1 cells were not altered. Pseudomonas quinolone signal (PQS), one of the regulators related to MV production in P. aeruginosa, was lower in MVs purified from the exponential phase than in those from the stationary phase. MVs from the stationary phase more strongly associated with P. aeruginosa cells than did those from the exponential phase. Our findings suggest that properties of MVs are altered to readily interact with bacterial cells along with the growth transition in P. aeruginosa. T  

3.1458           The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

El-Kadi, A.M., Bros-Facer, V., Deng, W., Philpott, A., Stoddart, E., Banks, G., Jackson, G.S., Fisher, E.M., Duchen, M.R., Greeensmith, L., Moore, A.L. and Hafezparast, M.

1. Biol. Chem., 285(24), 18627-18639 (2010)

  Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.  

3.1459           Modulation of intracellular trafficking by TGFB and rhoa

Ribe, D. and Stenbeck, G. Bone, 47, Suppl. 1, S130-S131 (2010)   Bone diseases such as osteoarthritis and osteoporosis are characterised by defects in transforming growth factor beta (TGFβ) signalling and matrix protein deposition. We are interested in the immediate early effects of TGFβ in matrix secretion. We are studying the trafficking and secretion of the matrix proteins osteonectin (ON) and osteopontin (OP) in osteoblasts using total internal reflection fluorescence microscopy (TIRFM) and membrane fractionation. ROS 17/2.8 cells were transfected with a plasmid for expression of green fluorescent protein (GFP) –tagged ON and examined by TIRFM. Dynamics of ON-containing vesicles were studied by taking TIRFM images of individual cells at 0.3 s intervals for 30 s. Vesicles were identified and their trajectories tracked in ImageJ using the Particle Tracker plugin. Membrane fractions of non-transfected ROS 17/2.8 cells were separated by ultracentrifugation on an Optiprep density gradient and examined by Western blotting. In unstimulated cells, a minority of ON-containing vesicles displayed sustained fast movement along their trajectories (150 nm in 3 s). Treatment of cells with 25 nM TGFβ increased the number of these faster vesicles by 34% within 10 min, an effect that was sustained 20 min after addition of TGFβ. Vesicle motility reverted to the unstimulated state within 60 min of TGFβ treatment. Pre-treatment of cells for 60 min with 2.5 μM of the ROCK inhibitor Y-27632 reduced the mean vesicle speed and displacement by 28% and 43% respectively. Vesicle speed, displacement and the number of fast vesicles did not recover with addition of 25 nM TGFβ to Y-27632 –treated cells. Treatment with 10 μM nocodazole for 20 min completely inhibited vesicle motility, indicating that the microtubule network is essential for fidelity of membrane trafficking. Western blotting of membrane fractions separated on a density gradient showed that blocking the endogenous TGFβ signal with the monoclonal TGFβ antibody mAb240 (1 μg/ml) for 60 min caused a shift in the distribution of OP from a vesicular localisation towards fractions rich in Golgi markers. These studies show that TGFβ has immediate effects on trafficking of the secreted proteins ON and OP in osteoblasts, which are modulated by the Rho signalling cascade. These immediate early events in TGFβ signalling may play a role not only in matrix protein secretion but also in the propagation of further signals that lead to proliferation and aberrant matrix deposition if uncontrolled.      

3.1460           Towards a membrane proteome in Drosophila: a method for the isolation of plasma membrane

Khanna, M.R., Stanley, B.A. and Thomas, G.H. BMC Genomics, 11, 302-317 (2010)   Background The plasma membrane (PM) is a compartment of significant interest because cell surface proteins influence the way in which a cell interacts with its neighbours and its extracellular environment. However, PM is hard to isolate because of its low abundance. Aqueous two-phase affinity purification (2PAP), based on PEG/Dextran two-phase fractionation and lectin affinity for PM-derived microsomes, is an emerging method for the isolation of high purity plasma membranes from several vertebrate sources. In contrast, PM isolation techniques in important invertebrate genetic model systems, such as Drosophila melanogaster, have relied upon enrichment by density gradient centrifugation. To facilitate genetic investigation of activities contributing to the content of the PM sub-proteome, we sought to adapt 2PAP to this invertebrate model to provide a robust PM isolation technique for Drosophila. Results We show that 2PAP alone does not completely remove contaminating endoplasmic reticulum and mitochondrial membrane. However, a novel combination of density gradient centrifugation plus 2PAP results in a robust PM preparation. To demonstrate the utility of this technique we isolated PM from fly heads and successfully identified 432 proteins using MudPIT, of which 37% are integral membrane proteins from all compartments. Of the 432 proteins, 22% have been previously assigned to the PM compartment, and a further 34% are currently unassigned to any compartment and represent candidates for assignment to the PM. The remainder have previous assignments to other compartments. Conclusion A combination of density gradient centrifugation and 2PAP results in a robust, high purity PM preparation from Drosophila, something neither technique can achieve on its own. This novel preparation should lay the groundwork for the proteomic investigation of the PM in different genetic backgrounds in Drosophila. Our results also identify two key steps in this procedure: The optimization of membrane partitioning in the PEG/Dextran mixture, and careful choice of the correct lectin for the affinity purification step in light of variations in bulk membrane lipid composition and glycosylation patterns respectively. This points the way for further adaptations into other systems.  

3.1461           Epstein-Barr Virus Latent Membrane Protein 1 Modulates Distinctive NF- B Pathways through C-Terminus-Activating Region 1 To Regulate Epidermal Growth Factor Receptor Expression

Kung, C-P. and Raab-Traub, N.

1. Virol., 84(13), 6605-6614 (2010)

  Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1) is required for EBV B-lymphocyte transformation, transforms rodent fibroblasts, and can induce lymphoma and epithelial hyperplasia in transgenic mice. Two domains have been identified within the intracellular carboxy terminus that can activate NF- B, C-terminus-activating region 1 (CTAR1) and CTAR2, through interactions with tumor necrosis receptor-associated factors (TRAFs). CTAR1 can activate both the canonical and noncanonical NF- B pathways and has unique effects on cellular gene expression. The epidermal growth factor receptor (EGFR) is highly induced by LMP1-CTAR1 in epithelial cells through activation of a novel NF- B form containing p50 homodimers and Bcl-3. To further understand the regulation of NF- B in CTAR1-induced EGFR expression, we evaluated the ability of CTAR1 to induce EGFR in mouse embryonic fibroblasts (MEFs) defective for different NF- B effectors. CTAR1-mediated EGFR induction required the NF- B-inducing kinase (NIK) but not the I B kinase (IKK) complex components that regulate canonical or noncanonical NF- B pathways. CTAR1-mediated induction of nuclear p50 occurred in IKKβ-, IKK -, and NIK-defective MEFs, indicating that this induction is not dependent on the canonical or noncanonical NF- B pathways. EGFR and nuclear p50 were expressed at high levels in TRAF2–/– fibroblasts and were not induced by CTAR1. In TRAF3–/– MEFs, CTAR1 induced nuclear p50 but did not affect basal levels of STAT3 serine phosphorylation or induce EGFR expression. EGFR was induced by LMP1 in TRAF6–/– MEFs. These findings suggest that this novel NF- B pathway is differentially regulated by TRAF2 and TRAF3, and that distinct interactions of LMP1 and its effectors regulate LMP1-mediated gene expression.  

3.1462           Demonstration of a Direct Interaction between -1 Receptors and Acid-Sensing Ion Channels

Carnally, S.M., Johannessen, M., Henderson, R.M., Jackson, M.B. and Edwardsen, J.M. Biophys. J., 98, 1182-1191 (2010)   The -1 receptor is a widely expressed protein that interacts with a variety of ion channels, including the acid-sensing ion channel (ASIC) 1a. Here we used atomic force microscopy to determine the architecture of the ASIC1a/ -1 receptor complex. When isolated His₈-tagged ASIC1a was imaged in complex with anti-His₆ antibodies, the angle between pairs of bound antibodies was 135 , consistent with the known trimeric structure of the channel. When ASIC1a was coexpressed with FLAG/His₆-tagged -1 receptor, ASIC1a became decorated with small particles, and pairs of these particles bound at an angle of 131 . When these complexes were incubated with anti-FLAG antibodies, pairs of antibodies bound at an angle of 134 , confirming that the small particles were -1 receptors. Of interest, we found that the -1 receptor ligand haloperidol caused an 50% reduction in ASIC1a/ -receptor binding, suggesting a way in which -1 ligands might modulate channel properties. For the first time, to our knowledge, we have resolved the structure of a complex between the -1 receptor and a target ion channel, and demonstrated that the stoichiometry of the interaction is 1 -1 receptor/1 ASIC1a subunit.  

3.1463           Analysis of SM4 sulfatide as a P-selectin ligand using model membranes

Simonis, D., Schlesinger, M.-, Seelandt, C., Borsig, L. and Bendas, G. Biophys. Chem., 150, 98-104 (2010)   Carcinoma tumor cells express highly glycosylated mucins acting as ligands for selectin adhesion receptors and thus facilitating the metastatic process. Recently, a sulfated galactocerebroside SM4 was detected as solely P-selectin ligand on MC-38 colon carcinoma cells. Here we characterize the functionality of SM4 as selectin ligand using model membrane approaches. SM4 was found concentrated in lipid rafts of MC-38 cells indicating a local clustering that may increase the avidity of P-selectin recognition. To confirm this, SM4 was incorporated at various concentrations into POPC model membranes and lateral clustering was analyzed by fluorescence microscopy and found to be comparable to glycolipids carrying the sLe^x epitope. SM4 containing liposomes were used as cell models, binding to immobilized P-selectin. Quartz crystal microbalance data confirmed SM4/P-selectin liposome binding that was inhibited dose-dependently by heparin. Comparable binding characteristics of SM4 and sLe^x liposomes underscore the similarity of these epitopes. Thus, clustering of SM4 on tumor cells is a principle for binding P-selectin.  

3.1464           Live cell visualization of the interactions between HIV-1 Gag and the cellular RNA-binding protein Staufen1

Milev, M.P., Brown, C.M. and Mouland, A.J. Retrovirology, 7, 41-59 (2010)   Background Human immunodeficiency virus type 1 (HIV-1) uses cellular proteins and machinery to ensure transmission to uninfected cells. Although the host proteins involved in the transport of viral components toward the plasma membrane have been investigated, the dynamics of this process remain incompletely described. Previously we showed that the double-stranded (ds)RNA-binding protein, Staufen1 is found in the HIV-1 ribonucleoprotein (RNP) that contains the HIV-1 genomic RNA (vRNA), Gag and other host RNA-binding proteins in HIV-1-producing cells. Staufen1 interacts with the nucleocapsid domain (NC) domain of Gag and regulates Gag multimerization on membranes thereby modulating HIV-1 assembly. The formation of the HIV-1 RNP is dynamic and likely central to the fate of the vRNA during the late phase of the HIV-1 replication cycle. Results Detailed molecular imaging of both the intracellular trafficking of virus components and of virus-host protein complexes is critical to enhance our understanding of factors that contribute to HIV-1 pathogenesis. In this work, we visualized the interactions between Gag and host proteins using bimolecular and trimolecular fluorescence complementation (BiFC and TriFC) analyses. These methods allow for the direct visualization of the localization of protein-protein and protein-protein-RNA interactions in live cells. We identified where the virus-host interactions between Gag and Staufen1 and Gag and IMP1 (also known as VICKZ1, IGF2BP1 and ZBP1) occur in cells. These virus-host interactions were not only detected in the cytoplasm, but were also found at cholesterol-enriched GM1-containing lipid raft plasma membrane domains. Importantly, Gag specifically recruited Staufen1 to the detergent insoluble membranes supporting a key function for this host factor during virus assembly. Notably, the TriFC experiments showed that Gag and Staufen1 actively recruited protein partners when tethered to mRNA. Conclusions The present work characterizes the interaction sites of key components of the HIV-1 RNP (Gag, Staufen1 and IMP1), thereby bringing to light where HIV-1 recruits and co-opts RNA-binding proteins during virus assembly.  

3.1465           Termination of autophagy and reformation of lysosomes regulated by mTOR

Yu, L., McPhee, C.K., Zheng, L., Mardones, G.A., Rong, Y., Peng, J., Mi, N., Zhao, Y., Liu, Z., Wan, F., Hailey, D.W., Oorscot, V., Klumperman, J., Baehrecke, E.H. and Lenardo, M.J. Nature, 465(17), 942-946 (2010)   Autophagy is an evolutionarily conserved process by which cytoplasmic proteins and organelles are catabolized^{1, 2}. During starvation, the protein TOR (target of rapamycin), a nutrient-responsive kinase, is inhibited, and this induces autophagy. In autophagy, double-membrane autophagosomes envelop and sequester intracellular components and then fuse with lysosomes to form autolysosomes, which degrade their contents to regenerate nutrients. Current models of autophagy terminate with the degradation of the autophagosome cargo in autolysosomes^{3, 4, 5}, but the regulation of autophagy in response to nutrients and the subsequent fate of the autolysosome are poorly understood. Here we show that mTOR signalling in rat kidney cells is inhibited during initiation of autophagy, but reactivated by prolonged starvation. Reactivation of mTOR is autophagy-dependent and requires the degradation of autolysosomal products. Increased mTOR activity attenuates autophagy and generates proto-lysosomal tubules and vesicles that extrude from autolysosomes and ultimately mature into functional lysosomes, thereby restoring the full complement of lysosomes in the cell—a process we identify in multiple animal species. Thus, an evolutionarily conserved cycle in autophagy governs nutrient sensing and lysosome homeostasis during starvation.  

3.1466           Coronaviruses Hijack the LC3-I-Positive EDEMosomes, ER-Derived Vesicles Exporting Short-Lived ERAD Regulators, for Replication

Reggiori, F., Monastyrska, I., Verheiji, M.H., Cali, T., Ulasli, M., Bianchi, S., Bernasconi, R., de Haan, C.A.M. and Molinari, M. Cell Host & Microbe, 7, 500-508 (2010)   Coronaviruses (CoV), including SARS and mouse hepatitis virus (MHV), are enveloped RNA viruses that induce formation of double-membrane vesicles (DMVs) and target their replication and transcription complexes (RTCs) on the DMV-limiting membranes. The DMV biogenesis has been connected with the early secretory pathway. CoV-induced DMVs, however, lack conventional endoplasmic reticulum (ER) or Golgi protein markers, leaving their membrane origins in question. We show that MHV co-opts the host cell machinery for COPII-independent vesicular ER export of a short-living regulator of ER-associated degradation (ERAD), EDEM1, to derive cellular membranes for replication. MHV infection causes accumulation of EDEM1 and OS-9, another short-living ER chaperone, in the DMVs. DMVs are coated with the nonlipidated LC3/Atg8 autophagy marker. Downregulation of LC3, but not inactivation of host cell autophagy, protects cells from CoV infection. Our study identifies the host cellular pathway hijacked for supplying CoV replication membranes and describes an autophagy-independent role for nonlipidated LC3-I.  

3.1467           Lysosomal membrane permeabilization and cathepsin release is a Bax/Bak-dependent, amplifying event of apoptosis in fibroblasts and monocytes

Oberle, C., Huai, J., Reinheckel, T., Tacke, M., Rassner, M., Ekert, P.G., Buellesbach, J. and Borner, C. Cell Death and Differentiation, 17, 1167-1178 (2010)   Apoptotic stimuli have been shown to trigger lysosomal membrane permeability (LMP), leading to the release of cathepsins, which activate death signaling pathways in the cytosol. However, it is unknown whether this process is an initiating or amplifying event in apoptosis. In this study, we used fibroblasts and monocytes exposed to etoposide, ultraviolet light, FasL or deprived of interleukin-3 (IL-3) to show that LMP and the cytosolic release of cathepsins B, L and D consistently depends on Bax/Bak and components of the apoptosome. Neither Bax nor Bak resided on the lysosomes, indicating that lysosomes were not directly perforated by Bax/Bak but by effectors downstream of the apoptosome. Detailed kinetic analysis of cells lacking cathepsin B or L or treated with the cysteine protease inhibitor, E64d, revealed a delay in these cells in etoposide- and IL-3 deprivation-induced caspase-3 activation and apoptosis induction but not clonogenic survival, indicating that cathepsins amplify rather than initiate apoptosis.  

3.1468           Non-classical exocytosis of [alpha]-synuclein is sensitive to folding states and promoted under stress conditions

Jang, A., Lee, H-L., Suk, J-E., Jung, J-W., Kim, K-P. and Lee, S-J.

1. Neurochem., 113(5), 1263-1274 (2010)

  Parkinson's disease is characterized by deposition of misfolded/aggregated [alpha]-synuclein proteins in multiple regions of the brain. Neurons can release [alpha]-synuclein; through this release, pathological forms of [alpha]-synuclein are propagated between neurons, and also cause neuroinflammation. In this study, we demonstrate that release of [alpha]-synuclein is consistently increased under various protein misfolding stress conditions in both neuroblastoma and primary neuron models. This release is mediated by a non-classical, endoplasmic reticulum (ER)/Golgi-independent exocytosis, and stress-induced release coincides with increased translocation of [alpha]-synuclein into vesicles. Both vesicle translocation and secretion were blocked by attachment of a highly stable, globular protein to [alpha]-synuclein, whereas forced protein misfolding resulted in an increase in both of these activities. Mass spectrometry analysis showed a higher degree of oxidative modification in secreted [alpha]-synuclein than in the cellular protein. Together, these results suggest that structurally abnormal, damaged [alpha]-synuclein proteins translocate preferentially into vesicles and are released from neuronal cells via exocytosis.  

3.1469           The fibroblast growth factor receptor substrate 3 adapter is a developmentally regulated microtubule-associated protein expressed in migrating and differentiated neurons

Hryciw, T., MacDonald, J.I.S., Phillips, R., Seah, C., Pasternak, S. and Meakin, S.O.

1. Neurochem., 112, 924-939 (2010)

  Fibroblast growth factor (FGF) mediated signaling is essential to many aspects of neural development. Activated FGF receptors signal primarily through the FGF receptor substrate (Frs) adapters, which include Frs2/Frs2[alpha] and Frs3/Frs2[beta]. While some studies suggest that Frs3 can compensate for the loss of Frs2 in transfected cells, the lack of an effective Frs3 specific antibody has prevented efforts to determine the role(s) of the endogenous protein. To this end, we have generated a Frs3 specific antibody and have characterized the pattern of Frs3 expression in the developing nervous system, its subcellular localization as well as its biochemical properties. We demonstrate that Frs3 is expressed at low levels in the ventricular zone of developing cortex, between E12 and E15, and it co-localizes with nestin and acetylated [alpha]-tubulin in radial processes in the ventricular/subventricular zones as well as with [beta]III tubulin in differentiated cortical neurons. Subcellular fractionation studies demonstrate that endogenous Frs3 is both soluble and plasma membrane associated while Frs3 expressed in 293T cells associates exclusively with lipid rafts. Lastly, we demonstrate that neuronal Frs3 binds microtubules comparable to the microtubule-associated protein, MAP2, while Frs2 does not. Collectively, these data suggest that neuronal Frs3 functions as a novel microtubule binding protein and they provide the first biochemical evidence that neuronal Frs3 is functionally distinct from Frs2/Frs2[alpha].  

3.1470           Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole

Miranda, K., Pace, D.A., Cintron, R., Rodrigues, J.C.F., fang, J., Smith, A., Rohloff, P., Coelho, E., de Haas, F., de Souza, W., Coppens, I., Sibley, L.D. and Moreno, S.N.J. Mol. Microbiol., 76(6), 1358-1375 (2010)   Toxoplasma gondii belongs to the phylum Apicomplexa and is an important cause of congenital disease and infection in immunocompromised patients. Like most apicomplexans, T. gondii possesses several plant-like features, such as the chloroplast-like organelle, the apicoplast. We describe and characterize a novel organelle in T. gondii tachyzoites, which is visible by light microscopy and possesses a broad similarity to the plant vacuole. Electron tomography shows the interaction of this vacuole with other organelles. The presence of a plant-like vacuolar proton pyrophosphatase (TgVP1), a vacuolar proton ATPase, a cathepsin L-like protease (TgCPL), an aquaporin (TgAQP1), as well as Ca2+/H+ and Na+/H+ exchange activities, supports similarity to the plant vacuole. Biochemical characterization of TgVP1 in enriched fractions shows a functional similarity to the respective plant enzyme. The organelle is a Ca2+ store and appears to have protective effects against salt stress potentially linked to its sodium transport activity. In intracellular parasites, the organelle fragments, with some markers colocalizing with the late endosomal marker, Rab7, suggesting its involvement with the endocytic pathway. Studies on the characterization of this novel organelle will be relevant to the identification of novel targets for chemotherapy against T. gondii and other apicomplexan parasites as well.  

3.1471           Rab11 Supports Amphetamine-Stimulated Norepinephrine Transporter Trafficking

Matthies, H.J.G., Moore, J.L., Saunders, C., Matthies, D.S., Lapierre, L.A., Goldenring, J.R., Blakely, R.D. and Galli, A.

1. Neurosci., 30(23), 7863-7877 (2010)

  The norepinephrine transporter (NET) is a presynaptic plasma membrane protein that mediates reuptake of synaptically released norepinephrine. NET is also a major target for medications used for the treatment of depression, attention deficit/hyperactivity disorder, narcolepsy, and obesity. NET is regulated by numerous mechanisms, including catalytic activation and membrane trafficking. Amphetamine (AMPH), a psychostimulant and NET substrate, has also been shown to induce NET trafficking. However, neither the molecular basis nor the nature of the relevant membrane compartments of AMPH-modulated NET trafficking has been defined. Indeed, direct visualization of drug-modulated NET trafficking in neurons has yet to be demonstrated. In this study, we used a recently developed NET antibody and the presence of large presynaptic boutons in sympathetic neurons to examine basal and AMPH-modulated NET trafficking. Specifically, we establish a role for Rab11 in AMPH-induced NET trafficking. First, we found that, in cortical slices, AMPH induces a reduction in surface NET. Next, we observed AMPH-induced accumulation and colocalization of NET with Rab11a and Rab4 in presynaptic boutons of cultured neurons. Using tagged proteins, we demonstrated that NET and a truncated Rab11 effector (FIP2 C2) do not redistribute in synchrony, whereas NET and wild-type Rab11a do. Analysis of various Rab11a/b mutants further demonstrates that Rab11 regulates NET trafficking. Expression of the truncated Rab11a effector (FIP2 C2) attenuates endogenous Rab11 function and prevented AMPH-induced NET internalization as does GDP-locked Rab4 S22N. Our data demonstrate that AMPH leads to an increase of NET in endosomes of single boutons and varicosities in a Rab11-dependent manner.  

3.1472           Nox4-Derived H2O2 Mediates Endoplasmic Reticulum Signaling through Local Ras Activation

Wu, R-F., Ma, Z., Liu, Z. and Terada, L.S. Mol. Cell. Biol., 30(14), 3553-3568 (2010)   The unfolded-protein response (UPR) of the endoplasmic reticulum (ER) has been linked to oxidant production, although the molecular details and functional significance of this linkage are poorly understood. Using a ratiometric H₂O₂ sensor targeted to different subcellular compartments, we demonstrate specific production of H₂O₂ by the ER in response to the stressors tunicamycin and HIV-1 Tat, but not to thapsigargin or dithiothreitol. Knockdown of the oxidase Nox4, expressed on ER endomembranes, or expression of ER-targeted catalase blocked ER H₂O₂ production by tunicamycin and Tat and prevented the UPR following exposure to these two agonists, but not to thapsigargin or dithiothreitol. Tat also triggered Nox4-dependent, sustained activation of Ras leading to ERK, but not phosphatidylinositol 3-kinase (PI3K)/mTOR, pathway activation. Cell fractionation studies and green fluorescent protein (GFP) fusions of GTPase effector binding domains confirmed selective activation of endogenous RhoA and Ras on the ER surface, with ER-associated K-Ras acting upstream of the UPR and downstream of Nox4. Notably, the Nox4/Ras/ERK pathway induced autophagy, and suppression of autophagy unmasked cell death and prevented differentiation of endothelial cells in 3-dimensional matrix. We conclude that the ER surface provides a platform to spatially organize agonist-specific Nox4-dependent oxidative signaling events, leading to homeostatic protective mechanisms rather than oxidative stress.  

3.1473           Lysosomal Proteolysis and Autophagy Require Presenilin 1 and Are Disrupted by Alzheimer-Related PS1 Mutations

Lee, J-H., Yu, W.H., Kumar, A., Lee, S., Mohan, P.S., Peterhoff, C.M., Wolfe, D.M., Martinez-Vicente, M., Massey, A.C., Sovak, G., Uchiyama, Y., Westaway, D., Cuervo, A.M. and Nixon, R.A. Cell, 141, 1146-1158 (2010)   Macroautophagy is a lysosomal degradative pathway essential for neuron survival. Here, we show that macroautophagy requires the Alzheimer's disease (AD)-related protein presenilin-1 (PS1). In PS1 null blastocysts, neurons from mice hypomorphic for PS1 or conditionally depleted of PS1, substrate proteolysis and autophagosome clearance during macroautophagy are prevented as a result of a selective impairment of autolysosome acidification and cathepsin activation. These deficits are caused by failed PS1-dependent targeting of the v-ATPase V0a1 subunit to lysosomes. N-glycosylation of the V0a1 subunit, essential for its efficient ER-to-lysosome delivery, requires the selective binding of PS1 holoprotein to the unglycosylated subunit and the Sec61alpha/oligosaccharyltransferase complex. PS1 mutations causing early-onset AD produce a similar lysosomal/autophagy phenotype in fibroblasts from AD patients. PS1 is therefore essential for v-ATPase targeting to lysosomes, lysosome acidification, and proteolysis during autophagy. Defective lysosomal proteolysis represents a basis for pathogenic protein accumulations and neuronal cell death in AD and suggests previously unidentified therapeutic targets.  

3.1474           Activation of Adenosine A2A Receptors Induces TrkB Translocation and Increases BDNF-Mediated Phospho-TrkB Localization in Lipid Rafts: Implications for Neuromodulation

Assaife-Lopes, N., Sousa, V.C., Pereira, D.B., Ribeiro, J.A., Chao, M.V. and Sebastiao, A.M.

1. Neurosci., 30(25), 8468-8480 (2010)

  Brain-derived neurotrophic factor (BDNF) signaling is critical for neuronal development and transmission. Recruitment of TrkB receptors to lipid rafts has been shown to be necessary for the activation of specific signaling pathways and modulation of neurotransmitter release by BDNF. Since TrkB receptors are known to be modulated by adenosine A_2A receptor activation, we hypothesized that activation of A_2A receptors could influence TrkB receptor localization among different membrane microdomains. We found that adenosine A_2A receptor agonists increased the levels of TrkB receptors in the lipid raft fraction of cortical membranes and potentiated BDNF-induced augmentation of phosphorylated TrkB levels in lipid rafts. Blockade of the clathrin-mediated endocytosis with monodansylcadaverine (100 µM) did not modify the effects of the A_2A receptor agonists but significantly impaired BDNF effects on TrkB recruitment to lipid rafts. The effect of A_2A receptor activation in TrkB localization was mimicked by 5 µM forskolin, an adenylyl cyclase activator. Also, it was blocked by the PKA inhibitors Rp-cAMPs and PKI-(14–22), and by the Src-family kinase inhibitor PP2. Moreover, removal of endogenous adenosine or disruption of lipid rafts reduced BDNF stimulatory effects on glutamate release from cortical synaptosomes. Lipid raft integrity was also required for the effects of BDNF on hippocampal long-term potentiation at CA1 synapses. Our data demonstrate, for the first time, a BDNF-independent recruitment of TrkB receptors to lipid rafts induced by activation of adenosine A_2A receptors, with functional consequences for TrkB phosphorylation and BDNF-induced modulation of neurotransmitter release and hippocampal plasticity.  

3.1475           The Platelet Actin Cytoskeleton Associates with SNAREs and Participates in α-Granule Secretion

Woronowicz, K., Dilks, J.R., Rozenvayn, N., Dowal, L., Blair, P.S., Peters, C.G., Woronowicz, L. and Flaumenhaft, R. Biochemistry, 49(21), 4533-4542 (2010)   Following platelet activation, platelets undergo a dramatic shape change mediated by the actin cytoskeleton and accompanied by secretion of granule contents. While the actin cytoskeleton is thought to influence platelet granule secretion, the mechanism for this putative regulation is not known. We found that disruption of the actin cytoskeleton by latrunculin A inhibited α-granule secretion induced by several different platelet agonists without significantly affecting activation-induced platelet aggregation. In a cell-free secretory system, platelet cytosol was required for α-granule secretion. Inhibition of actin polymerization prevented α-granule secretion in this system, and purified platelet actin could substitute for platelet cytosol to support α-granule secretion. To determine whether SNAREs physically associate with the actin cytoskeleton, we isolated the Triton X-100 insoluble actin cytoskeleton from platelets. VAMP-8 and syntaxin-2 associated only with actin cytoskeletons of activated platelets. Syntaxin-4 and SNAP-23 associated with cytoskeletons isolated from either resting or activated platelets. When syntaxin-4 and SNAP-23 were tested for actin binding in a purified protein system, only syntaxin-4 associated directly with polymerized platelet actin. These data show that the platelet cytoskeleton interacts with select SNAREs and that actin polymerization facilitates α-granule release.  

3.1476           Superoxide dismutase-1 and other proteins in inclusions from transgenic amyotrophic lateral sclerosis model mice

Bergemalm, D., Forsberg, K., Srivastava, V., Graffmo, K.S., Andersen, P.M., Brännström, T., Wingsle, G. and Marklund, S.L.

1. Neurochem.,114, 408-418 (2010)

  Mutant superoxide dismutase-1 (SOD1) causes amyotrophic lateral sclerosis (ALS) through a cytotoxic mechanism of unknown nature. A hallmark in ALS patients and transgenic mouse models carrying human SOD1 (hSOD1) mutations are hSOD1-immunoreactive inclusions in spinal cord ventral horns. The hSOD1 inclusions may block essential cellular functions or cause toxicity through sequestering of other proteins. Inclusions from four different transgenic mouse models were examined after density gradient ultracentrifugation. The inclusions are complex structures with heterogeneous densities and are disrupted by detergents. The aggregated hSOD1 was mainly composed of subunits that lacked the native stabilizing intra-subunit disulfide bond. A proportion of subunits formed hSOD1 oligomers or was bound to other proteins through disulfide bonds. Dense inclusions could be isolated and the protein composition was analyzed using proteomic techniques. Mutant hSOD1 accounted for half of the protein. Ten other proteins were identified. Two were cytoplasmic chaperones, four were cytoskeletal proteins, and 4 were proteins that normally reside in the endoplasmic reticulum (ER). The presence of ER proteins in inclusions containing the primarily cytosolic hSOD1 further supports the notion that ER stress is involved in ALS.  

3.1477           Unconventional Secretion of AcbA in Dictyostelium discoideum through a Vesicular Intermediate

Cabral, M., Anjard, C., Malhotra, V., Loomis, W.F. and Kuspa, A. Eukaryot. Cell, 9(7), 1009-1017 (2010)   The acyl coenzyme A (CoA) binding protein AcbA is secreted unconventionally and processed into spore differentiation factor 2 (SDF-2), a peptide that coordinates sporulation in Dictyostelium discoideum. We report that AcbA is localized in vesicles that accumulate in the cortex of prespore cells just prior to sporulation. These vesicles are not observed after cells are stimulated to release AcbA but remain visible after stimulation in cells lacking the Golgi reassembly stacking protein (GRASP). Acyl-CoA binding is required for the inclusion of AcbA in these vesicles, and the secretion of AcbA requires N-ethylmaleimide-sensitive factor (NSF). About 1% of the total cellular AcbA can be purified within membrane-bound vesicles. The yield of vesicles decreases dramatically when purified from wild-type cells that were stimulated to release AcbA, whereas the yield from GRASP mutant cells was only modestly altered by stimulation. We suggest that these AcbA-containing vesicles are secretion intermediates and that GRASP functions at a late step leading to the docking/fusion of these vesicles at the cell surface.  

3.1478           Reduction of Brain β-Amyloid (Aβ) by Fluvastatin, a Hydroxymethylglutaryl-CoA Reductase Inhibitor, through Increase in Degradation of Amyloid Precursor Protein C-terminal Fragments (APP-CTFs) and Aβ Clearance

Shinohaa, M., Sato, N., Kurinami, H., Takeuchi, D., Takeda, S., Shimamura, M., Yamashita, T., Uchiyama, Y., Rakugi, H. and Morishita, R.

1. Biol. Chem., 285(29), 22091-22102 (2010)

  Epidemiological studies suggest that statins (hydroxymethylglutaryl-CoA reductase inhibitors) could reduce the risk of Alzheimer disease. Although one possible explanation is through an effect on β-amyloid (Aβ) metabolism, its effect remains to be elucidated. Here, we explored the molecular mechanisms of how statins influence Aβ metabolism. Fluvastatin at clinical doses significantly reduced Aβ and amyloid precursor protein C-terminal fragment (APP-CTF) levels among APP metabolites in the brain of C57BL/6 mice. Chronic intracerebroventricular infusion of lysosomal inhibitors blocked these effects, indicating that up-regulation of the lysosomal degradation of endogenous APP-CTFs is involved in reduced Aβ production. Biochemical analysis suggested that this was mediated by enhanced trafficking of APP-CTFs from endosomes to lysosomes, associated with marked changes of Rab proteins, which regulate endosomal function. In primary neurons, fluvastatin enhanced the degradation of APP-CTFs through an isoprenoid-dependent mechanism. Because our previous study suggests additive effects of fluvastatin on Aβ metabolism, we examined Aβ clearance rates by using the brain efflux index method and found its increased rates at high Aβ levels from brain. As LRP1 in brain microvessels was increased, up-regulation of LRP1-mediated Aβ clearance at the blood-brain barrier might be involved. In cultured brain microvessel endothelial cells, fluvastatin increased LRP1 and the uptake of Aβ, which was blocked by LRP1 antagonists, through an isoprenoid-dependent mechanism. Overall, the present study demonstrated that fluvastatin reduced Aβ level by an isoprenoid-dependent mechanism. These results have important implications for the development of disease-modifying therapy for Alzheimer disease as well as understanding of Aβ metabolism.  

3.1479           Modulation of the Protein Kinase C Interaction with the "d" Subunit of F1F0-ATP Synthase in Neonatal Cardiac Myocytes: DEVELOPMENT OF CELL-PERMEABLE, MITOCHONDRIALLY TARGETED INHIBITOR AND FACILITATOR PEPTIDES

Nguyen,, T.T., Ogbi, M., Yu, Q., Fishman, J.B., Thomas, W., Harvey, B.J., Fulton, D. and Johnson, J.A.

1. Biol. Chem., 285(29), 22164-22173 (2010)

  The F₁F₀-ATP synthase provides ∼90% of cardiac ATP, yet little is known regarding its regulation under normal or pathological conditions. Previously, we demonstrated that protein kinase Cδ (PKCδ) inhibits F₁F₀ activity via an interaction with the “d” subunit of F₁F₀-ATP synthase (dF₁F₀) in neonatal cardiac myocytes (NCMs) (Nguyen, T., Ogbi, M., and Johnson, J. A. (2008) J. Biol. Chem. 283, 29831–29840). We have now identified a dF₁F₀-derived peptide (NH₂-²AGRKLALKTIDWVSF¹⁶-COOH) that inhibits PKCδ binding to dF₁F₀ in overlay assays. We have also identified a second dF₁F₀-derived peptide (NH₂-¹¹¹RVREYEKQLEKIKNMI¹²⁶-COOH) that facilitates PKCδ binding to dF₁F₀. Incubation of NCMs with versions of these peptides containing HIV-Tat protein transduction and mammalian mitochondrial targeting sequences resulted in their delivery into mitochondria. Preincubation of NCMs, with 10 nm extracellular concentrations of the mitochondrially targeted PKCδ-dF₁F₀ interaction inhibitor, decreased 100 nm 4β-phorbol 12-myristate 13-acetate (4β-PMA)-induced co-immunoprecipitation of PKCδ with dF₁F₀ by 50 ± 15% and abolished the 30 nm 4β-PMA-induced inhibition of F₁F₀-ATPase activity. A scrambled sequence (inactive) peptide, which contained HIV-Tat and mitochondrial targeting sequences, was without effect. In contrast, the cell-permeable, mitochondrially targeted PKCδ-dF₁F₀ facilitator peptide by itself induced the PKCδ-dF₁F₀ co-immunoprecipitation and inhibited F₁F₀-ATPase activity. In in vitro PKC add-back experiments, the PKCδ-F₁F₀ inhibitor blocked PKCδ-mediated inhibition of F₁F₀-ATPase activity, whereas the facilitator induced inhibition. We have developed the first cell-permeable, mitochondrially targeted modulators of the PKCδ-dF₁F₀ interaction in NCMs. These novel peptides will improve our understanding of cardiac F₁F₀ regulation and may have potential as therapeutics to attenuate cardiac injury.

3.1480           A Toxin-based Probe Reveals Cytoplasmic Exposure of Golgi Sphingomyelin

Bakrac, B., Kladnik, A., Macek, P., McHaffie, G., Werner, A., Lakey, J.H. and Anderluh, G.

1. Biol. Chem., 285(29), 22186-22195 (2010)

  Although sphingomyelin is an important cellular lipid, its subcellular distribution is not precisely known. Here we use a sea anemone cytolysin, equinatoxin II (EqtII), which specifically binds sphingomyelin, as a new marker to detect cellular sphingomyelin. A purified fusion protein composed of EqtII and green fluorescent protein (EqtII-GFP) binds to the SM rich apical membrane of Madin-Darby canine kidney (MDCK) II cells when added exogenously, but not to the SM-free basolateral membrane. When expressed intracellularly within MDCK II cells, EqtII-GFP colocalizes with markers for Golgi apparatus and not with those for nucleus, mitochondria, endoplasmic reticulum or plasma membrane. Colocalization with the Golgi apparatus was confirmed by also using NIH 3T3 fibroblasts. Moreover, EqtII-GFP was enriched in cis-Golgi compartments isolated by gradient ultracentrifugation. The data reveal that EqtII-GFP is a sensitive probe for membrane sphingomyelin, which provides new information on cytosolic exposure, essential to understand its diverse physiological roles.  

3.1481           Targeting intracellular Aβ Oligomers through conformational intrabodies

Lecci, A. and Cattaneo, A., Alzheimer’s and Dementia, 6(4), Suppl. 1, S252-S253 (2010)   Background: Increasing evidence supports the role of AβOligomers (AβOs) in the pathogenesis of Alzheimer's Disease (AD). The use of specific conformational intracellular antibodies (intrabodies) allows to selectively target AβOs inside cells at different points for mechanistic as well as for interference purposes. We previously described the selection of conformation-sensitive and sequence-specific single chain Fv antibody fragments (scFvs), targeting AβOs (Meli et al., 2009). Now, we describe the use of these scFvs as intrabodies for selective subcellular targeting and intracellular knock-down/silencing of AβOs, in well established cellular models for AβOs production and secretion. Methods: Sensitive co-immunoprecipitation methods allowed verifying the binding of the recombinant scFvs to the natural AβOs derived from cell culture media or from human Cerospinal Fluid (CSF). The intracellular expression of the anti-AβOs intrabodies was performed in a well established cell model for AβOs production and secretion, referred to as 7PA2 cells (Walsh et al.,2000;2002), generating cells stably transfected with intrabodies in different formats.The in vivo binding and the modulation of AβOs generation were studied by experiments of subcellular fractionation on discontinuous iodixanol gradients and by intrabody-AβOs co-immunoprecipitation approaches. Results: The recombinant anti-AβOs scFvs selectively bind and immunoprecipitate the natural AβOs from 7PA2 conditioned media and from human Cerospinal Fluid (CSF). The effects of the intracellular expression of the anti-AβOs intrabodies on the Aβ/AβOs formation and APP metabolism were studied by subcellular fractionation. Total microsomes fractionated from 7PA2 or 7PA2 expressing intrabodies showed differential distribution of Aβ/AβOs. Moreover, a sensitive intrabody-AβOs co-immunoprecipitation approach allowed demonstrating a selective immunoprecipitation of specific forms of natural SDS-stable AβOs.The expression of an intrabody with an ER-retention signal (KDEL) showed a significant reduction of AβOs secretion and selective modulation of AβOs accumulation in subcellular fractions. Conclusions: The binding ability of the anti-AβOs scFvs to the natural AβOs in vitro as well as in vivo (in 7PA2 living cells), validates their conformation-specificity and -selectivity. Moreover, the anti-AβOs intrabodies interfere with AβOs intracellular formation and secretion. The intrabody approach is therefore a promising way for interfering with the activity of different intracellular Aβ assemblies (in ways that would not be possible by RNA-interference approaches) exploitable for new recombinant therapeutics.  

3.1482           ELGA stimulates gamma-secretase activity to increase Aβ generation

Jung, S. Alzheimer’s and dementia, 6(4), Suppl. 1, S260 (2010)   Background: Amyloid beta is the main pathologic hallmark of Alzheimer's Disease. This Hydrophobic fragment forms amyloid plaque and causes neuronal damage and memory loss. Sequential processing of APP(amyloid precursor protein) by β-/γ-secetase is required for Aβ production and the γ-secretase activity is the rate limiting step of the reaction. In fact pathogenic environment that regulates this enzyme complex is reported in AD patient case study. Thus, identification of genes that regulate its enzymatic activity is important for the treatment of Alzheimer's disease. Methods: We isolated novel ER localized γ-secretase activator (ELGA) by using cell-based functional screening system. Results: Overexpression of ELGA increased Aβ42 production and γ-secretase-mediated cleavage of APP-CTFβ in SH-SY5Y-APPswe cell. To confirm enzymatic activation effect of γ-secretase, we conducted in vitro cleavage assay and ELGA increased generation of APP-CTFγ. Interestingly, ELGA binds with Aph1 and PS1. In iodixanol gradient fractionation, subcellular localization of Aph1 and PS1-NTF seems to shift from ER to Golgi by ELGA. In addition, ELGA seems not to affect notch cleavage. Conclusions: These results suggest thatγELGA may stimulate γ-secretase assembly to increase the generation of Aβ.  

3.1483           Lipid raft-dependent endocytosis: a new route for hepcidin-mediated regulation of ferroportin in macrophages

Auriac, A., Willemetz, A. and Canonne-Hergaux, F. Maemaatologica, 95(8), 1269-1277 (2010)   Background: Expression of the iron exporter ferroportin at the plasma membraneof macrophages is enhanced by iron loading and is decreasedby hepcidin. We previously showed that ferroportin is presentin specific cell surface domains suggestive of lipid rafts.Herein, we have clarified the localization of ferroportin inmacrophage membranes and tested whether raft-mediated endocytosisplays a role in hepcidin activity. Design and Methods: Raft/detergent-resistant membranes from murine bone marrow-derivedmacrophages and J774a1 cells were analyzed by Western blotting.The effect of lipid raft- or clathrin-dependent endocytosisinhibitors was studied on hepcidin activity. For this purpose,after treatment, ferroportin expression was analyzed by fluorescencemicroscopy, Western blotting of total protein extracts or plasmamembrane protein samples, and by quantitative immunofluorescenceassay (In-Cell-Western). Results: Macrophage ferroportin was mostly detected in detergent-resistantmembranes containing raft markers (caveolin 1, flotillin 1).Interestingly, iron overload strongly increased the presenceof ferroportin in the lightest raft fraction. Moreover, lipidraft breakdown by cholesterol sequestration (filipin) or depletion(methyl-beta-cyclodextrin) decreased hepcidin activity on macrophageferroportin. Cell surface biotinylation and immunofluorescencestudies indicated that the process of both hepcidin mediatedendocytosis and degradation of ferroportin were affected. Bycontrast, the inhibition of clathrin dependent endocytosis didnot interfere with hepcidin effect. Conclusions: Macrophage ferroportin is present in lipid rafts which contributeto hepcidin activity. These observations reveal the existenceof a new cellular pathway in hepcidin mediated degradation offerroportin and open a new area of investigation in mammalianiron homeostasis.  

3.1484           Pgrmc1 (Progesterone Receptor Membrane Component 1) Associates with Epidermal Growth Factor Receptor and Regulates Erlotinib Sensitivity

Ahmed, I.S., Rohe, H.J., Twist, K.E. and Craven, R.J.

1. Biol. Chem., 285(32), 24775-24782 (2010)

  Tumorigenesis requires the concerted action of multiple pathways, including pathways that stimulate proliferation and metabolism. Epidermal growth factor receptor (EGFR) is a transmembrane receptor-tyrosine kinase that is associated with cancer progression, and the EGFR inhibitors erlotinib/tarceva and tyrphostin/AG-1478 are potent anti-cancer therapeutics. Pgrmc1 (progesterone receptor membrane component 1) is a cytochrome b₅-related protein that is up-regulated in tumors and promotes cancer growth. Pgrmc1 and its homologues have been implicated in cell signaling, and we show here that Pgrmc1 increases susceptibility to AG-1478 and erlotinib, increases plasma membrane EGFR levels, and co-precipitates with EGFR. Pgrmc1 co-localizes with EGFR in cytoplasmic vesicles and co-fractionates with EGFR in high density microsomes. The findings have therapeutic potential because a Pgrmc1 small molecule ligand, which inhibits growth in a variety of cancer cell types, de-stabilized EGFR in multiple tumor cell lines. EGFR is one of the most potent receptor-tyrosine kinases driving tumorigenesis, and our data support a role for Pgrmc1 in promoting several cancer phenotypes at least in part by binding EGFR and stabilizing plasma membrane pools of the receptor.  

3.1485           The fast-mobility isoform of mouse Mcl-1 is a mitochondrial matrix-localized protein with attenuated anti-apoptotic activity

Huang, C-R. and  Yang-Yen, H-F. FEBS Lett., 584, 3323-3330 (2010)   The full-length pro-survival protein Mcl-1 predominantly resides on the outer membrane of mitochondria. Here, we identified a mitochondrial matrix-localized isoform of Mcl-1 that lacks 33 amino acid residues at the N-terminus which serve both as a mitochondrial targeting and processing signal. Ectopically-expressed Mcl-1 without the N-terminal 33 residues failed to enter the mitochondrial matrix but retained wt-like activities both for interaction with BH3-only proteins and anti-apoptosis. In contrast, the mitochondrial matrix-localized isoform failed to interact with BH3-only proteins and manifested an attenuated anti-apoptotic activity. This study reveals that import of Mcl-1 into the mitochondrial matrix results in the attenuation of Mcl-1’s anti-apoptotic function.  

3.1486           Mitochondrial DNA sequences are present inside nuclear DNA in rat tissues and increase with age

Caro, P., Gomez, J., Arduini, A., Gonzales-Sanchez, M., Gonzales-Garcia, M., Borras, C., Vina, J., Puertas, M.J:, Sastre, J. and Barja, G. Mitochondrion, 10, 479-486 (2010)   Mitochondrial DNA (mtDNA) mutations increase with age. However, the number of cells with predominantly mutated mtDNA is small in old animals. Here a new hypothesis is proposed: mtDNA fragments may insert into nuclear DNA contributing to aging and related diseases by alterations in the nucleus. Real-time PCR quantification shows that sequences of cytochrome oxidase III and 16S rRNA from mtDNA are present in highly purified nuclei from liver and brain in young and old rats. The sequences of these insertions revealed that they contain single nucleotide polymorphisms identical to those present in mtDNA of the same animal. Interestingly, the amount of mitochondrial sequences in nuclear DNA increases with age in both tissues. In situ hybridization of mtDNA to nuclear DNA confirms the presence of mtDNA sequences inside nuclear DNA in rat hepatocytes. Bone marrow metaphase cells from both young and old rats show mtDNA at centromeric regions in 20 out of the 2n = 40 chromosomes. Consequently, mitochondria can be a major trigger of aging but the final target could also be the nucleus.  

3.1487           Modulation of intracellular trafficking by TGFB and rhoa

Ribe, D. and Stenbeck, G. Bone, 47, Suppl. 1, S130-S131 (2010)   Bone diseases such as osteoarthritis and osteoporosis are characterised by defects in transforming growth factor beta (TGFβ) signalling and matrix protein deposition. We are interested in the immediate early effects of TGFβ in matrix secretion. We are studying the trafficking and secretion of the matrix proteins osteonectin (ON) and osteopontin (OP) in osteoblasts using total internal reflection fluorescence microscopy (TIRFM) and membrane fractionation. ROS 17/2.8 cells were transfected with a plasmid for expression of green fluorescent protein (GFP) –tagged ON and examined by TIRFM. Dynamics of ON-containing vesicles were studied by taking TIRFM images of individual cells at 0.3 s intervals for 30 s. Vesicles were identified and their trajectories tracked in ImageJ using the Particle Tracker plugin. Membrane fractions of non-transfected ROS 17/2.8 cells were separated by ultracentrifugation on an Optiprep density gradient and examined by Western blotting. In unstimulated cells, a minority of ON-containing vesicles displayed sustained fast movement along their trajectories (150 nm in 3 s). Treatment of cells with 25 nM TGFβ increased the number of these faster vesicles by 34% within 10 min, an effect that was sustained 20 min after addition of TGFβ. Vesicle motility reverted to the unstimulated state within 60 min of TGFβ treatment. Pre-treatment of cells for 60 min with 2.5 μM of the ROCK inhibitor Y-27632 reduced the mean vesicle speed and displacement by 28% and 43% respectively. Vesicle speed, displacement and the number of fast vesicles did not recover with addition of 25 nM TGFβ to Y-27632 –treated cells. Treatment with 10 μM nocodazole for 20 min completely inhibited vesicle motility, indicating that the microtubule network is essential for fidelity of membrane trafficking. Western blotting of membrane fractions separated on a density gradient showed that blocking the endogenous TGFβ signal with the monoclonal TGFβ antibody mAb240 (1 μg/ml) for 60 min caused a shift in the distribution of OP from a vesicular localisation towards fractions rich in Golgi markers. These studies show that TGFβ has immediate effects on trafficking of the secreted proteins ON and OP in osteoblasts, which are modulated by the Rho signalling cascade. These immediate early events in TGFβ signalling may play a role not only in matrix protein secretion but also in the propagation of further signals that lead to proliferation and aberrant matrix deposition if uncontrolled.  

3.1488           Sortilin Facilitates Signaling of Ciliary Neurotrophic Factor and Related Helical Type 1 Cytokines Targeting the gp130/Leukemia Inhibitory Factor Receptor β Heterodimer

Vejby Larsen, J., Hansen, M., Møller, B., Madsen, P., Scheller, J., Nielsen, M. and Munck Pedersen, C. Mol. Cell. Biol., 30(17), 4175-4187 (2010)   Sortilin is a member of the Vps10p domain family of neuropeptide and neurotrophin binding neuronal receptors. The family members interact with and partly share a variety of ligands and partake in intracellular sorting and protein transport as well as in transmembrane signal transduction. Thus, sortilin mediates the transport of both neurotensin and nerve growth factor and interacts with their respective receptors to facilitate ligand-induced signaling. Here we report that ciliary neurotrophic factor (CNTF), and related ligands targeting the established CNTF receptor , binds to sortilin with high affinity. We find that sortilin may have at least two functions: one is to provide rapid endocytosis and the removal of CNTF, something which is not provided by CNTF receptor , and the other is to facilitate CNTF signaling through the gp130/leukemia inhibitory factor (LIF) receptor β heterodimeric complex. Interestingly, the latter function is independent of both the CNTF receptor and ligand binding to sortilin but appears to implicate a direct interaction with LIF receptor β. Thus, sortilin facilitates the signaling of all helical type 1 cytokines, which engage the gp130/LIF receptor β complex.  

3.1489           Subcellular organelle lipidomics in TLR-4-activated macrophages

Andreyev, A.Y., Fahy, E., Guan, Z., Kelly, S., Li, X., McDonald, J.G., Milne, S., Myers, D., Park, H., Ryan, A., Thompson, B.M., Wang, E., Zhao, Y., Brown, H.A., Merrill, A.H., Raetz, C.R.H., Russell, D.W., Subramaniam, S. and Dennis, E.A.

1. Lipid Res., 51, 2785-2797 (2010)

  Lipids orchestrate biological processes by acting remotely as signaling molecules or locally as membrane components that modulate protein function. Detailed insight into lipid function requires knowledge of the subcellular localization of individual lipids. We report an analysis of the subcellular lipidome of the mammalian macrophage, a cell type that plays key roles in inflammation, immune responses, and phagocytosis. Nuclei, mitochondria, endoplasmic reticulum (ER), plasmalemma, and cytoplasm were isolated from RAW 264.7 macrophages in basal and activated states. Subsequent lipidomic analyses of major membrane lipid categories identified 229 individual/isobaric species, including 163 glycerophospholipids, 48 sphingolipids, 13 sterols, and 5 prenols. Major subcellular compartments exhibited substantially divergent glycerophospholipid profiles. Activation of macrophages by the Toll-like receptor 4-specific lipopolysaccharide Kdo₂-lipid A caused significant remodeling of the subcellular lipidome. Some changes in lipid composition occurred in all compartments (e.g., increases in the levels of ceramides and the cholesterol precursors desmosterol and lanosterol). Other changes were manifest in specific organelles. For example, oxidized sterols increased and unsaturated cardiolipins decreased in mitochondria, whereas unsaturated ether-linked phosphatidylethanolamines decreased in the ER. We speculate that these changes may reflect mitochondrial oxidative stress and the release of arachidonic acid from the ER in response to cell activation.  

3.1490           The ARMS/Kidins220 scaffold protein modulates synaptic transmission

Arevalo, J.C., Wu, S.H., Takahashi, T., Zhang, H., Yu, T., Yano, H., Milner, T.A., Tessorollo, L., Ninan, I., Arancio, O. and Chao, M.V. Mol. Cell. Neurosci., 45, 92-100 (2010)   Activity-dependent changes of synaptic connections are facilitated by a variety of scaffold proteins, including PSD-95, Shank, SAP97 and GRIP, which serve to organize ion channels, receptors and enzymatic activities and to coordinate the actin cytoskeleton. The abundance of these scaffold proteins raises questions about the functional specificity of action of each protein. Here we report that basal synaptic transmission is regulated in an unexpected manner by the ankyrin repeat-rich membrane-spanning (ARMS/Kidins220) scaffold protein. In particular, decreases in the levels of ARMS/Kidins220 in vivo led to an increase in basal synaptic transmission in the hippocampus, without affecting paired pulse facilitation. One explanation to account for the effects of ARMS/Kidins220 is an interaction with the AMPA receptor subunit, GluA1, which could be observed after immunoprecipitation. Importantly, shRNA and cell surface biotinylation experiments indicate that ARMS/Kidins220 levels have an impact on GluA1 phosphorylation and localization. Moreover, ARMS/Kidins220 is a negative regulator of AMPAR function, which was confirmed by inward rectification assays. These results provide evidence that modulation of ARMS/Kidins220 levels can regulate basal synaptic strength in a specific manner in hippocampal neurons.        

3.1491           CHL1 Is a Selective Organizer of the Presynaptic Machinery Chaperoning the SNARE

Andreyeva, A., Leshchyn’ska, I., Knepper, M., Betzel, C., Redecke, L., Sytnyk, V. and Schachner, M. PloSOne, 5(8), e12018 (2010)   Proteins constituting the presynaptic machinery of vesicle release undergo substantial conformational changes during the process of exocytosis. While changes in the conformation make proteins vulnerable to aggregation and degradation, little is known about synaptic chaperones which counteract these processes. We show that the cell adhesion molecule CHL1 directly interacts with and regulates the activity of the synaptic chaperones Hsc70, CSP and αSGT. CHL1, Hsc70, CSP and αSGT form predominantly CHL1/Hsc70/αSGT and CHL1/CSP complexes in synapses. Among the various complexes formed by CHL1, Hsc70, CSP and αSGT, SNAP25 and VAMP2 induce chaperone activity only in CHL1/Hsc70/αSGT and CHL1/CSP complexes, respectively, indicating a remarkable selectivity of a presynaptic chaperone activity for proteins of the exocytotic machinery. In mice with genetic ablation of CHL1, chaperone activity in synapses is reduced and the machinery for synaptic vesicle exocytosis and, in particular, the SNARE complex is unable to sustain prolonged synaptic activity. Thus, we reveal a novel role for a cell adhesion molecule in selective activation of the presynaptic chaperone machinery.  

3.1492           L1 syndrome mutations impair neuronal L1 function at different levels by divergent mechanisms

Schäfer, M.K.E., Nam, Y-C., Moumen, A., Keglowich, L., Bouche, E., Küffner, M., Bock, H.H., Rathjem, F.G., Raoulo, C. and Frotscher, M. Neurobiology of Disease, 40, 222-237 (2010)   Mutations in the human L1CAM gene cause neurodevelopmental disorders collectively referred to as L1 syndrome. Here, we investigated cellular pathomechanisms underlying two L1 syndrome mutations, R184Q and W1036L. We demonstrate that these mutations cause partial endoplasmic reticulum (ER) retention of L1, reduce L1 cell surface expression, but do not induce ER stress in neuronal NSC-34 cells. We provide evidence that surface trafficking of mutated L1 is affected by defective sorting to ER exit sites and attenuated ER export. However, in differentiated neuronal cultures and long-term cultured hippocampal slices, the L1-R184Q protein is restricted to cell bodies, whereas L1-W1036L also aberrantly localizes to dendrites. These trafficking defects preclude axonal targeting of L1, thereby affecting L1-mediated axon growth and arborization. Our results indicate that L1 syndrome mutations impair neuronal L1 function at different levels, firstly by attenuating ER export and secondly by interfering with polarized neuronal trafficking.  

3.1493           Attenuation of the hypoxia-induced protein kinase Cδ interaction with the ‘d’ subunit of F1Fo-ATP synthase in neonatal cardiac myocytes: implications for energy preservation and survival

Nguyen, T.T., Ogbi, M., Yu, Q. and Johnson, J.A. Biochem. J., 429, 335-345 (2010)   The F₁F_o-ATP synthase provides most of the heart's energy, yet events that alter its function during injury are poorly understood. Recently, we described a potent inhibitory effect on F₁F_o-ATP synthase function mediated by the interaction of PKCδ (protein kinase Cδ) with dF₁F_o (‘d’ subunit of the F₁F_o-ATPase/ATP synthase). We have now developed novel peptide modulators which facilitate or inhibit the PKCδ–dF₁F_o interaction. These peptides include HIV-Tat (transactivator of transcription) protein transduction and mammalian mitochondrial-targeting sequences. Pre-incubation of NCMs (neonatal cardiac myocyte) with 10 nM extracellular concentrations of the mitochondrial-targeted PKCδ–dF₁F_o interaction inhibitor decreased Hx (hypoxia)-induced co-IP (co-immunoprecipitation) of PKCδ with dF₁F_o by 40±9%, abolished Hx-induced inhibition of F₁F_o-ATPase activity, attenuated Hx-induced losses in F₁F_o-derived ATP and protected against Hx- and reperfusion-induced cell death. A scrambled-sequence (inactive) peptide, which contained HIV-Tat and mitochondrial-targeting sequences, was without effect. In contrast, the cell-permeant mitochondrial-targeted PKCδ–dF₁F_o facilitator peptide, which we have shown previously to induce the PKCδ–dF₁F_o co-IP, was found to inhibit F₁F_o-ATPase activity to an extent similar to that caused by Hx alone. The PKCδ–dF₁F_o facilitator peptide also decreased ATP levels by 72±18% under hypoxic conditions in the presence of glycolytic inhibition. None of the PKCδ–dF₁F_o modulatory peptides altered the inner mitochondrial membrane potential. Our studies provide the first evidence that disruption of the PKCδ–dF₁F_o interaction using cell-permeant mitochondrial-targeted peptides attenuates cardiac injury resulting from prolonged oxygen deprivation.    

3.1494           Calcium- and polyphosphate-containing acidic granules of sea urchin eggs are similar to acidocalcisomes, but are not the targets for NAADP

Ramos, I.B., Miranda, K., Pace, D.A., Verbist, K.C., Lins, F-Y., Zhang, Y., Oldfield, E., Machado, E.A., De Souza, W. and Docompo, R. Biochem. J., 429, 485-495 (2010)   Acidocalcisomes are acidic calcium-storage compartments described from bacteria to humans and characterized by their high content in poly P (polyphosphate), a linear polymer of many tens to hundreds of P_i residues linked by high-energy phosphoanhydride bonds. In the present paper we report that millimolar levels of short-chain poly P (in terms of P_i residues) and inorganic PP_i are present in sea urchin extracts as detected using ³¹P-NMR, enzymatic determinations and agarose gel electrophoresis. Poly P was localized to granules randomly distributed in the sea urchin eggs, as shown by labelling with the poly-P-binding domain of Escherichia coli exopolyphosphatase. These granules were enriched using iodixanol centrifugation and shown to be acidic and to contain poly P, as determined by Acridine Orange and DAPI (4′,6′-diamidino-2-phenylindole) staining respectively. These granules also contained large amounts of calcium, sodium, magnesium, potassium and zinc, as detected by X-ray microanalysis, and bafilomycin A₁-sensitive ATPase, pyrophosphatase and exopolyphosphatase activities, as well as Ca²⁺/H⁺ and Na⁺/H⁺ exchange activities, being therefore similar to acidocalcisomes described in other organisms. Calcium release from these granules induced by nigericin was associated with poly P hydrolysis. Although NAADP (nicotinic acid–adenine dinucleotide phosphate) released calcium from the granule fraction, this activity was not significantly enriched as compared with the NAADP-stimulated calcium release from homogenates and was not accompanied by poly P hydrolysis. GPN (glycyl-L-phenylalanine-naphthylamide) released calcium when added to sea urchin homogenates, but was unable to release calcium from acidocalcisome-enriched fractions, suggesting that these acidic stores are not the targets for NAADP.  

3.1495           Dephosphorylation of F-BAR Protein Cdc15 Modulates Its Conformation and Stimulates Its Scaffolding Activity at the Cell Division Site

Roberts-Galbraith, R.H., Ohi, M.D., Ballif, B.A., Chen, J-S., McLeod, I., McDonald, W.H., Gygi, S.P., Yates III, J.R. and Gould, K.L. Mol. Cell, 39, 86-99 (2010)   Cytokinesis in Schizosaccharomyces pombe requires the function of Cdc15, the founding member of the pombe cdc15 homology (PCH) family of proteins. As an early, abundant contractile ring component with multiple binding partners, Cdc15 plays a key role in organizing the ring. We demonstrate that Cdc15 phosphorylation at many sites generates a closed conformation, inhibits Cdc15 assembly at the division site in interphase, and precludes interaction of Cdc15 with its binding partners. Cdc15 dephosphorylation induces an open conformation, oligomerization, and scaffolding activity during mitosis. Cdc15 mutants with reduced phosphorylation precociously appear at the division site in filament-like structures and display increased association with protein partners and the membrane. Our results indicate that Cdc15 phosphoregulation impels both assembly and disassembly of the contractile apparatus and suggest a regulatory strategy that PCH family and BAR superfamily members might broadly employ to achieve temporal specificity in their roles as linkers between membrane and cytoskeleton.  

3.1496           Secreted Monocytic miR-150 Enhances Targeted Endothelial Cell Migration

Zhang, Y. et al Mol. Cell, 39, 133-144 (2010)   MicroRNAs (miRNAs) are a class of noncoding RNAs that regulate target gene expression at the posttranscriptional level. Here, we report that secreted miRNAs can serve as signaling molecules mediating intercellular communication. In human blood cells and cultured THP-1 cells, miR-150 was selectively packaged into microvesicles (MVs) and actively secreted. THP-1-derived MVs can enter and deliver miR-150 into human HMEC-1 cells, and elevated exogenous miR-150 effectively reduced c-Myb expression and enhanced cell migration in HMEC-1 cells. In vivo studies confirmed that intravenous injection of THP-1 MVs significantly increased the level of miR-150 in mouse blood vessels. MVs isolated from the plasma of patients with atherosclerosis contained higher levels of miR-150, and they more effectively promoted HMEC-1 cell migration than MVs from healthy donors. These results demonstrate that cells can secrete miRNAs and deliver them into recipient cells where the exogenous miRNAs can regulate target gene expression and recipient cell function.  

3.1497           SH3TC2, a protein mutant in Charcot–Marie–Tooth neuropathy, links peripheral nerve myelination to endosomal recycling

Stendel, C., Roos, A., Kleine, H., Arnoud, E., Özcelik, M., Sidiropoulos, P.N.M., Zenker, J., Schhüpfer, F., Lehmann, U., Sobota, R.M., Litchfield, D.W., Lüscher, B., Chrast, R., Suter, U. and Senderek, J.   Patients with Charcot–Marie–Tooth neuropathy and gene targeting in mice revealed an essential role for the SH3TC2 gene in peripheral nerve myelination. SH3TC2 expression is restricted to Schwann cells in the peripheral nervous system, and the gene product, SH3TC2, localizes to the perinuclear recycling compartment. Here, we show that SH3TC2 interacts with the small guanosine triphosphatase Rab11, which is known to regulate the recycling of internalized membranes and receptors back to the cell surface. Results of protein binding studies and transferrin receptor trafficking are in line with a role of SH3TC2 as a Rab11 effector molecule. Consistent with a function of Rab11 in Schwann cell myelination, SH3TC2 mutations that cause neuropathy disrupt the SH3TC2/Rab11 interaction, and forced expression of dominant negative Rab11 strongly impairs myelin formation in vitro. Our data indicate that the SH3TC2/Rab11 interaction is relevant for peripheral nerve pathophysiology and place endosomal recycling on the list of cellular mechanisms involved in Schwann cell myelination.  

3.1498           PERK (EIF2AK3) Regulates Proinsulin Trafficking and Quality Control in the Secretory Pathway

Gupta, S., McGrath, B. and Cavener, D.R. Diabetes, 59, 1937-1947 (2010)   OBJECTIVE Loss-of-function mutations in Perk (EIF2AK3) result in permanent neonatal diabetes in humans (Wolcott-Rallison Syndrome) and mice. Previously, we found that diabetes associated with Perk deficiency resulted from insufficient proliferation of β-cells and from defects in insulin secretion. A substantial fraction of PERK-deficient β-cells display a highly abnormal cellular phenotype characterized by grossly distended endoplasmic reticulum (ER) and retention of proinsulin. We investigated over synthesis, lack of ER-associated degradation (ERAD), and defects in ER to Golgi trafficking as possible causes. RESEARCH DESIGN AND METHODS ER functions of PERK were investigated in cell culture and mice in which Perk was impaired or gene dosage modulated. The Ins2^+/Akita mutant mice were used as a model system to test the role of PERK in ERAD. RESULTS We report that loss of Perk function does not lead to uncontrolled protein synthesis but impaired ER-to-Golgi anterograde trafficking, retrotranslocation from the ER to the cytoplasm, and proteasomal degradation. PERK was also shown to be required to maintain the integrity of the ER and Golgi and processing of ATF6. Moreover, decreasing Perk dosage surprisingly ameliorates the progression of the Akita mutants toward diabetes. CONCLUSIONS PERK is a positive regulator of ERAD and proteasomal activity. Reducing PERK activity ameliorates the progression of diabetes in the Akita mouse, whereas increasing PERK dosage hastens its progression. We speculate that PERK acts as a metabolic sensor in the insulin-secreting β-cells to modulate the trafficking and quality control of proinsulin in the ER relative to the physiological demands for circulating insulin.  

3.1499           Cholesterol depletion alters amplitude and pharmacology of vascular calcium-activated chloride channels

Sones, W.R., Davis, A.J., Leblanc, N. and Greenwood, A. Cardiovasc. Res., 87, 476-484 (2010)   Aims Calcium-activated chloride channels (CACCs) share common pharmacological properties with Kcnma1-encoded large conductance K⁺ channels (BK_Ca or K_Ca1.1) and it has been suggested that they may co-exist in a macromolecular complex. As K_Ca1.1 channels are known to localize to cholesterol and caveolin-rich lipid rafts (caveolae), the present study investigated whether Ca²⁺-sensitive Cl⁻ currents in vascular myocytes were affected by the cholesterol depleting agent methyl-β-cyclodextrin (M-βCD). Methods and results Calcium-activated chloride and potassium currents were recorded from single murine portal vein myocytes in whole cell voltage clamp. Western blot was undertaken following sucrose gradient ultracentrifugation using protein lysates from whole portal veins. Ca²⁺-activated Cl⁻ currents were augmented by 3 mg mL⁻¹ M-βCD with a rapid time course (t_0.5 = 1.8 min). M-βCD had no effect on the bi-modal response to niflumic acid or anthracene-9-carboxylate but completely removed the inhibitory effects of the K_Ca1.1 blockers, paxilline and tamoxifen, as well as the stimulatory effect of the K_Ca1.1 activator NS1619. Discontinuous sucrose density gradients followed by western blot analysis revealed that the position of lipid raft markers caveolin and flotillin-2 was altered by 15 min application of 3 mg mL⁻¹ M-βCD. The position of K_Ca1.1 and the newly identified candidate for CACCs, TMEM16A, was also affected by M-βCD.  

3.1500           Do Caveolae Have a Role in the Fidelity and Dynamics of Receptor Activation of G-protein-gated Inwardly Rectifying Potassium Channels?

Schwarzer, S., Nobles, M. and Tinker, A.

1. Biol. Chem., 285(36), 27817-27826 (2010)

  In atrial and nodal cardiac myocytes, M2 muscarinic receptors activate inhibitory G-proteins (G_i/o), which in turn stimulate G-protein-gated inwardly rectifying K⁺ channels through direct binding of the Gβγ subunit. Despite also releasing Gβγ, G_s-coupled receptors such as the β-adrenergic receptor are not able to prominently activate this current. An appealing hypothesis would be if components were sequestered in membrane domains such as caveolae/rafts. Using biochemical fractionation followed by Western blotting and/or radioligand binding experiments, we examined the distribution of the components in stable HEK293 and HL-1 cells, which natively express the transduction cascade. The channel, M2 muscarinic, and A1 adenosine receptors were located in noncaveolar/nonraft fractions. G_iα_1/2 was enriched in both caveolar/raft and noncaveolar/nonraft fractions. In contrast, G_sα was only enriched in caveolar/raft fractions. We constructed YFP-tagged caveolin-2 (YFP-Cav2) and chimeras with the M2 (M2-YFP-Cav2) and A1 (A1-YFP-Cav2) receptors. Analysis of gradient fractions showed that these receptor chimeras were now localized to caveolae-enriched fractions. Microscopy showed that M2-YFP and A1-YFP had a diffuse homogenous membrane signal. YFP-Cav2, M2-YFP-Cav2, and A1-YFP-Cav2 revealed a more punctuate pattern. Finally, we looked at the consequences for signaling. Activation via M2-YFP-Cav2 or A1-YFP-Cav2 revealed substantially slower kinetics compared with M2-YFP or A1-YFP and was reversed by the addition of methyl-β-cyclodextrin. Thus the localization of the channel signal transduction cascade in non-cholesterol rich domains substantially enhances the speed of signaling. The presence of G_sα solely in caveolae may account for signaling selectivity between G_i/o and G_s-coupled receptors.  

3.1501           Neogenin Regulation of BMP-Induced Canonical Smad Signaling and Endochondral Bone Formation

Zhou, Z., Xie, J., Lee, D., Liu, Y., Jung, J., Zhou, L., Xiong, S., Mei, L. and Xiong, W-C. Developmental Cell, 19, 90-102 (2010)   Neogenin has been identified as a receptor for the neuronal axon guidance cues netrins and RGMs (repulsive guidance molecules). Here we provide evidence for neogenin in regulating endochondral bone development and BMP (bone morphogenetic protein) signaling. Neogenin-deficient mice were impaired in digit/limb development and endochondral ossification. BMP2 induction of Smad1/5/8 phosphorylation and Runx2 expression, but not noncanonical p38 MAPK activation, was reduced in chondrocytes from neogenin mutant mice. BMP receptor association with membrane microdomains, which is necessary for BMP signaling to Smad, but not p38 MAPK, was diminished in neogenin-deficient chondrocytes. Furthermore, RGMs appear to mediate neogenin interaction with BMP receptors in chondrocytes. Taken together, our results indicate that neogenin promotes chondrogenesis in vitro and in vivo, revealing an unexpected mechanism underlying neogenin regulation of BMP signaling.  

3.1502           Suppression of the novel ER protein Maxer by mutant ataxin-1 in Bergman glia contributes to non-cell-autonomous toxicity

Shiwaku, H., Yoshimura, N., Tamura, T., Sone, M., Ogisgima, S., Watase, K., Tagawe, K. and Okazawa, H. EMBO J., 29, 2446-2460 (2010)   Non-cell-autonomous effect of mutant proteins expressed in glia has been implicated in several neurodegenerative disorders, whereas molecules mediating the toxicity are currently not known. We identified a novel molecule named multiple α-helix protein located at ER (Maxer) downregulated by mutant ataxin-1 (Atx1) in Bergmann glia. Maxer is an endoplasmic reticulum (ER) membrane protein interacting with CDK5RAP3. Maxer anchors CDK5RAP3 to the ER and inhibits its function of Cyclin D1 transcription repression in the nucleus. The loss of Maxer eventually induces cell accumulation at G1 phase. It was also shown that mutant Atx1 represses Maxer and inhibits proliferation of Bergmann glia in vitro. Consistently, Bergmann glia are reduced in the cerebellum of mutant Atx1 knockin mice before onset. Glutamate-aspartate transporter reduction in Bergmann glia by mutant Atx1 and vulnerability of Purkinje cell to glutamate are both strengthened by Maxer knockdown in Bergmann glia, whereas Maxer overexpression rescues them. Collectively, these results suggest that the reduction of Maxer mediates functional deficiency of Bergmann glia, and might contribute to the non-cell-autonomous pathology of SCA1.  

3.1503           Localization and trafficking of endogenous anterior pharynx-defective 1, a component of Alzheimer's disease related γ-secretase

Sanjo, N., Katayama, T., Hasegawa, H., Jin, H., Duthie, M., Mount, H.T.J., Mizusawa, H., St George-Hyslop, P and Fraser, P.E. Neurosci. Lett., 483, 53-56 (2010)   Anterior pharynx-defective 1 (Aph-1) is a multi-spanning membrane protein and an integral component of the high molecular weight γ-secretase complex that also contains presenilin, nicastrin, and Pen-2. In order to clarify the existence of an endogenous fragment of Aph-1 and dissect the localization and processing of endogenous Aph-1 proteins, we examined cell lines and primary cell cultures with our own carboxyl terminal-specific antibodies for Aph-1aL. Fractionation and immunofluorescence studies indicated that the endogenous full-length Aph-1aL isoform localizes primarily to the endoplasmic reticulum as well as Golgi intermediate compartment, but small amount of it was detected at Golgi apparatus where most of its carboxyl terminal domain fragment existed. In primary neuronal and glial cultures, Aph-1aL was present in the neurites and glial cell processes. Endogenous Aph-1a and its proteolytic fragment have unique properties for cleavage control that may have implications for γ-secretase regulation and intracellular distribution.  

3.1504           α2β1 integrin controls association of Rac with the membrane and triggers quiescence of endothelial cells

Cailleteau, L., Estrach, S., Thyss, R., Boyer, L., Doye, A., Domange, B., Johnsson, N., Rubinstein, E., Boucheix, C., Ebrahimian, T., Silvestre, J-S., Lemichez, E., Meneguzzi, G. and Mettouchi, A.

1. Cell Sci., 123, 2491-2501 (2010)

  Integrin receptors and their extracellular matrix ligands provide cues to cell proliferation, survival, differentiation and migration. Here, we show that 2β1 integrin, when ligated to the basement membrane component laminin-1, triggers a proliferation arrest in primary endothelial cells. Indeed, in the presence of strong growth signals supplied by growth factors and fibronectin, 2β1 engagement alters assembly of mature focal adhesions by 5β1 and leads to impairment of downstream signaling and cell-cycle arrest in the G1 phase. Although the capacity of 5β1 to signal for GTP loading of Rac is preserved, the joint engagement of 2β1 interferes with membrane anchorage of Rac. Adapting the ‘split-ubiquitin’ sensor to screen for membrane-proximal 2 integrin partners, we identified the CD9 tetraspanin and further establish its requirement for destabilization of focal adhesions, control of Rac subcellular localization and growth arrest induced by 2β1 integrin. Altogether, our data establish that 2β1 integrin controls endothelial cell commitment towards quiescence by triggering a CD9-dependent dominant signaling.  

3.1505           NCAM-Induced Neurite Outgrowth Depends on Binding of Calmodulin to NCAM and on Nuclear Import of NCAM and fak Fragments

Kleene, R., Mzoughi, M., Joshi, G., Kalus, I., Bormann, U., Schulze, C., Xiao, M-F., Dityatev, A. and Schachner, M.

1. Neurosci., 30(32), 10784-10798 (2010)

  The neural cell adhesion molecule NCAM plays important functional roles not only during nervous system development, but also in the adult after injury and in synaptic plasticity. Homophilic binding of NCAM triggers intracellular signaling events resulting in cellular responses such as neurite outgrowth that require NCAM palmitoylation-dependent raft localization and activation of the nonreceptor tyrosine kinases fyn and fak. In this study, we show that stimulation of NCAM by a function-triggering NCAM antibody results in proteolytic processing of NCAM and fak. The C-terminal fragment of NCAM, consisting of the intracellular domain, the transmembrane domain, and a stub of the extracellular domain, and the N-terminal fragment of fak are imported into the nucleus. NCAM-stimulated fak activation, generation, and nuclear import of NCAM and fak fragments as well as neurite outgrowth are abolished by mutation of the calmodulin binding motif in the intracellular domain of NCAM that is responsible for the calcium-dependent binding of calmodulin to NCAM. This mutation interferes neither with NCAM cell surface expression, palmitoylation, and raft localization nor with fyn activation. The way by which the transmembrane NCAM fragment reaches the nucleus in a calmodulin- and calcium-dependent manner is by endocytotic transport via the endoplasmic reticulum and the cytoplasm. The generation and nuclear import of NCAM and phosphorylated fak fragments resulting from NCAM stimulation may represent a signal pathway activating cellular responses in parallel or in association with classical kinase- and phosphorylation-dependent signaling cascades.  

3.1506           Docosahexaenoic acid alters epidermal growth factor receptor-related signaling by disrupting its lipid raft association

Rogers, K.R., Kikawa, K.D., Mouradian, M., Hernandez, K., McKinnon, K., Ahwah, S.M. and Pardini, R.S. Carcinogenesis, 31(9), 1523-1530 (2010)   Docosahexaenoic acid (DHA), a 22:6 n-3 polyunsaturated fatty acid, is the longest and most highly unsaturated fatty acid found in most membranes and has been shown to inhibit cancer cell growth in part by modifying cell signaling. In the current study, alterations to epidermal growth factor receptor (EGFR) signaling upon DHA supplementation are examined in A549 lung adenocarcinoma, WiDr colon carcinoma and MDA-MB-231 breast carcinoma cell lines. Interestingly, EGFR phosphorylation, most notably at the tyrosine 1068 residue, is dramatically upregulated, and EGFR association with the Sos1 guanine nucleotide exchange factor is concomitantly increased upon DHA supplementation. However, guanosine triphosphate-bound Ras and phosphorylated extracellular signal-regulated kinase (Erk)1/2 are paradoxically downregulated in the same treatments. Previous reports have noted changes in membrane microdomains upon DHA supplementation, and our findings confirmed that EGFR, but not Ras, is excluded from caveolin-rich lipid raft fractions in DHA-treated cells, resulting in a decreased association of Ras with Sos1 and the subsequent downregulation of Erk signaling. Xenografts of the A549 cell line implanted in athymic mice fed a control high-fat diet or a diet high in DHA confirmed our in vitro data. These results demonstrate for the first time a functional consequence of decreased EGFR protein in lipid raft microdomains as a result of DHA treatment in three different cancer models. In addition, we report the ability of DHA to enhance the efficacy of EGFR inhibitors on anchorage-independent cell growth (soft agar), providing evidence for the potential development of enhanced combination therapies.  

3.1507           Effects of cholesterol on CCK-1 receptors and caveolin-3 proteins recycling in human gallbladder muscle

Cong, P., Pricolo, V., Biancani, P. and Behar, J. Am. J. Physiol. Gastrointest. Liver Physiol., 299, G742-G750 (2010)   The contraction of gallbladders (GBs) with cholesterol stones is impaired due to high cholesterol concentrations in caveolae compared with GBs with pigment stones. The reduced contraction is caused by a lower cholecystokinin (CCK)-8 binding to CCK-1 receptors (CCK-1R) due to caveolar sequestration of receptors. We aimed to examine the mechanism of cholesterol-induced sequestration of receptors. Muscle cells from human and guinea pig GBs were studied. Antibodies were used to examine CCK-1R, antigens of early and recycling endosomes, and total (CAV-3) and phosphorylated caveolar-3 protein (pCAV-3) by Western blots. Contraction was measured in muscle cells transfected with CAV3 mRNA or clathrin heavy-chain small-interfering RNA (siRNA). CCK-1R returned back to the bulk plasma membrane (PM) 30 min after CCK-8 recycled by endosomes, peaking at 5 min in early endosomes and at 20 min in recycling endosomes. Pretreatment with cholesterol-rich liposomes inhibited the transfer of CCK-1R and of CAV-3 in the endosomes by blocking CAV-3 phosphorylation. 4-Amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (inhibitor of tyrosine kinase) reproduced these effects by blocking pCAV-3 formation, increasing CAV-3 and CCK-1R sequestration in the caveolae and impairing CCK-8-induced contraction. CAV-3 siRNA reduced CAV-3 protein expression, decreased CCK-8-induced contraction, and accumulated CCK-1R in the caveolae. Abnormal concentrations of caveolar cholesterol had no effect on met-enkephalin that stimulates a -opioid receptor that internalizes through clathrin. We found that impaired muscle contraction in GBs with cholesterol stones is due to high caveolar levels of cholesterol that inhibits pCAV-3 generation. Caveolar cholesterol increases the caveolar sequestration of CAV-3 and CCK-1R caused by their reduced recycling to the PM.      

3.1508           Hsp12 Is an Intrinsically Unstructured Stress Protein that Folds upon Membrane Association and Modulates Membrane Function

Welker, S., Rudolph, B., Frenzel, E., Hagn, F., Liebisch, G., Schmitz, G., Scheuring, J., Kerth, A., Blume, A., Weinkauf, S., Haslbeck, M., Kessler, H. and Buchner, J. Molecular Cell, 39, 507-520  (2010)   Hsp12 of S. cerevisiae is upregulated several 100-fold in response to stress. Our phenotypic analysis showed that this protein is important for survival of a variety of stress conditions, including high temperature. In the absence of Hsp12, we observed changes in cell morphology under stress conditions. Surprisingly, in the cell, Hsp12 exists both as a soluble cytosolic protein and associated to the plasma membrane. The in vitro analysis revealed that Hsp12, unlike all other Hsps studied so far, is completely unfolded; however, in the presence of certain lipids, it adopts a helical structure. The presence of Hsp12 does not alter the overall lipid composition of the plasma membrane but increases membrane stability.  

3.1509           Misfolded Mutant SOD1 Directly Inhibits VDAC1 Conductance in a Mouse Model of Inherited ALS

Israelson, A., Arbel, N., Da Cruz, S., Lliweva, H., Yamanaka, K., Shoshan-Barmatz, V. and Cleveland, D.W. Cell, 67, 575-587 (2010)   Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by loss of motor neurons. With conformation-specific antibodies, we now demonstrate that misfolded mutant SOD1 binds directly to the voltage-dependent anion channel (VDAC1), an integral membrane protein imbedded in the outer mitochondrial membrane. This interaction is found on isolated spinal cord mitochondria and can be reconstituted with purified components in vitro. ADP passage through the outer membrane is diminished in spinal mitochondria from mutant SOD1-expressing ALS rats. Direct binding of mutant SOD1 to VDAC1 inhibits conductance of individual channels when reconstituted in a lipid bilayer. Reduction of VDAC1 activity with targeted gene disruption is shown to diminish survival by accelerating onset of fatal paralysis in mice expressing the ALS-causing mutation SOD1^G37R. Taken together, our results establish a direct link between misfolded mutant SOD1 and mitochondrial dysfunction in this form of inherited ALS.  

3.1510           Membrane Vesicles: A Common Feature in the Extracellular Matter of Cold-Adapted Antarctic Bacteria

Frias, A., Manresa, A., de Oliveira, E., Lopez-Inglisias, C. and Mercade, E. Microb. Ecol., 59, 476-486 (2010)   Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter, which has potential biotechnology applications. We examined the ultrastructure of extracellular matter from five Antarctic bacteria (Shewanella livingstonensis NF22^T, Shewanella vesiculosa M7^T, Pseudoalteromonas sp. M4.2, Psychrobacter fozii NF23^T, and Marinobacter guineae M3B^T) by transmission electron microscopy after high-pressure freezing and freeze substitution. All analyzed extracellular matter appeared as a netlike mesh composed of a capsular polymer around cells and large numbers of membrane vesicles (MVs), which have not yet been described for members of the genera Psychrobacter and Marinobacter. MVs showed the typical characteristics described for these structures, and seemed to be surrounded by the same capsular polymer as that found around the cells. The analysis of MV proteins from Antarctic strains by SDS-PAGE showed different banding profiles in MVs compared to the outer membrane, suggesting some kind of protein sorting during membrane vesicle formation. For the psychrotolerant bacterium, S. livingstonensis NF22^T, the growth temperature seemed to influence the amount and morphology of MVs. In an initial attempt to elucidate the functions of MVs for this psychrotolerant bacterium, we conducted a proteomic analysis on membrane vesicles from S. livingstonensis NF22^T obtained at 4 and 18°C. At both temperatures, MVs were highly enriched in outer membrane proteins and periplasmic proteins related to nutrient processing and transport in Gram-negative bacteria suggesting that MVs could be related with nutrient sensing and bacterial survival. Differences were observed in the expression of some proteins depending on incubation temperature but further studies will be necessary to define their roles and implications in the survival of bacteria in the extreme Antarctic environment.      

3.1511           Dihydrosphingomyelin Impairs HIV-1 Infection by Rigidifying Liquid-Ordered Membrane Domains

Vieira, C.R. et al Chemistry & Biology, 17, 766-775 (2010)   The lateral organization of lipids in cell membranes is thought to regulate numerous cell processes. Most studies focus on the coexistence of two fluid phases, the liquid crystalline (l_d) and the liquid-ordered (l_o); the putative presence of gel domains (s_o) is not usually taken into account. We show that in phospholipid:sphingolipid:cholesterol mixtures, in which sphingomyelin (SM) promoted fluid l_o domains, dihydrosphingomyelin (DHSM) tended to form rigid domains. Genetic and pharmacological blockade of the dihydroceramide desaturase (Des1), which replaced SM with DHSM in cultured cells, inhibited cell infection by replication-competent and -deficient HIV-1. Increased DHSM levels gave rise to more rigid membranes, resistant to the insertion of the gp41 fusion peptide, thus inhibiting viral-cell membrane fusion. These results clarify the function of dihydrosphingolipids in biological membranes and identify Des1 as a potential target in HIV-1 infection.  

3.1512           COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport

Wang, Y-N., Wang, H., Yamaguchi, H., Lee, H-J., Lee, H-H. and Hung, M-C. Biochem. Biophys. Res. Comm., 399, 498-504 (2010)   Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored. Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH₂-terminus resides within the lumen of Golgi/ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with γ-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.  

3.1513           Exosomes: Extracellular organelles important in intercellular communication

Mathivanan, S., Ji, H. and Simpson, R.J.

1. Proteomics, 73, 1907-1920 (2010)

  In addition to intracellular organelles, eukaryotic cells also contain extracellular organelles that are released, or shed, into the microenvironment. These membranous extracellular organelles include exosomes, shedding microvesicles (SMVs) and apoptotic blebs (ABs), many of which exhibit pleiotropic biological functions. Because extracellular organelle terminology is often confounding, with many preparations reported in the literature being mixtures of extracellular vesicles, there is a growing need to clarify nomenclature and to improve purification strategies in order to discriminate the biochemical and functional activities of these moieties. Exosomes are formed by the inward budding of multivesicular bodies (MVBs) and are released from the cell into the microenvironment following the fusion of MVBs with the plasma membrane (PM). In this review we focus on various strategies for purifying exosomes and discuss their biophysical and biochemical properties. An update on proteomic analysis of exosomes from various cell types and body fluids is provided and host-cell specific proteomic signatures are also discussed. Because the ectodomain of ~ 42% of exosomal integral membrane proteins are also found in the secretome, these vesicles provide a potential source of serum-based membrane protein biomarkers that are reflective of the host cell. ExoCarta, an exosomal protein and RNA database (http://exocarta.ludwig.edu.au), is described.  

3.1514           Palmitoylation of CD36/FAT regulates the rate of its post-transcriptional processing in the endoplasmic reticulum

Thorne, R.F., Ralston, K.J., de Bock, C.E., Nhaidat, N.M., Zhang, X.D., Boyd, A.W. and Burns, G.F. Biochim. Biophys. Acta, 1803, 1298-1307 (2010)   CD36/FAT is a transmembrane glycoprotein that functions in the cellular uptake of long-chain fatty acids and also as a scavenger receptor. As such it plays an important role in lipid homeostasis and, pathophysiologically, in the progression of type 2 diabetes and atherosclerosis. CD36 expression is tightly regulated at the levels of both transcription and translation. Here we show that its expression and location are also regulated post-translationally, by palmitoylation. Although palmitoylation of CD36 was not required for receptor maturation and cell surface expression, inhibition of palmitoylation either pharmacologically with cerulenin or by mutation of the relevant cysteines delayed processing at the ER and trafficking through the secretory pathway. The absence of palmitoylation also reduced the half life of the CD36 protein. Additionally, the CD36 palmitoylation mutant did not incorporate efficiently into lipid rafts, a site known to be required for its function of fatty acid uptake, and this reduced the efficiency of uptake of oxidized low density lipoprotein. These findings provide an added level of sophistication where translocation of CD36 to the plasma membrane may be physiologically regulated by palmitoylation.  

3.1515           Accessibility of Cholesterol in Endoplasmic Reticulum Membranes and Activation of SREBP-2 Switch Abruptly at a Common Cholesterol Threshold

Sokolov, A. and Radhakrishnan, A.

1. Biol. Chem., 285(38), 29480-29490 (2010)

  Recent studies have shown that cooperative interactions in endoplasmic reticulum (ER) membranes between Scap, cholesterol, and Insig result in switch-like control over activation of SREBP-2 transcription factors. This allows cells to rapidly adjust rates of cholesterol synthesis and uptake in response to even slight deviations from physiological set-point levels, thereby ensuring cholesterol homeostasis. In the present study we directly probe for the accessibility of cholesterol in purified ER membranes. Using a soluble cholesterol-binding bacterial toxin, perfringolysin O, we show that cholesterol accessibility increases abruptly at ∼5 mol % ER cholesterol, the same concentration at which SREBP-2 activation is halted. This switch-like change in cholesterol accessibility is observed not only in purified ER membranes but also in liposomes made from ER lipid extracts. The accessibility of cholesterol in membranes is related to its chemical activity. Complex formation between cholesterol and some ER phospholipids can result in sharp changes in cholesterol chemical activity and its accessibility to perfringolysin O or membrane sensors like Scap. The control of the availability of the cholesterol ligand to participate in cooperative Scap/cholesterol/Insig interactions further sharpens the sensitive switch that exerts precise control over cholesterol levels in cell membranes.  

3.1516           Sub-proteome approach to the knowledge of liver

Falcon-Perez, J.M., Lu, S.C. and Mato, J.M. Proteomics Clin. Appl., 4, 407-415 (2010)   In the recent years, global proteomics approaches have been widely used to characterize a number of tissue proteomes including plasma and liver; however, the elevated complexity of these samples in combination with the high abundance of some specific proteins make the study of the lowest abundant proteins difficult. This review is focused on different strategies that have been developed to extend the proteome focused on these two tissues, as, for example, the analysis of sub-cellular proteomes. In this regard, two special kind of extracellular vesicles – exosomes and membrane plasma shedding vesicles – are emerging as excellent biological source both to extend the liver and plasma proteomes and to be applied in the discovery of non-invasive liver-specific disease biomarkers.  

3.1517           Urokinase Plasminogen Activator Receptor and/or Matrix Metalloproteinase-9 Inhibition Induces Apoptosis Signaling through Lipid Rafts in Glioblastoma Xenograft Cells

Chetty, C., Lakka, S.S., Bhoopathi, P., Gondi, C.S., Veeravalli, K.K., Fassett, D., Klopfenstein, J.D., Dinh, D.H., Gujrati, M. and Rao, J.S. Mol. Cancer Ther., 9, 2605-2617 (2010)   Small interfering RNA (siRNA)-mediated transcriptional knockdown of urokinase plasminogen activator receptor (uPAR) and matrix metalloproteinase-9 (MMP-9), alone or in combination, inhibits uPAR and/or MMP-9 expression and induces apoptosis in the human glioblastoma xenograft cell lines 4910 and 5310. siRNA against uPAR (pU-Si), MMP-9 (pM-Si), or both (pUM-Si) induced apoptosis and was associated with the cleavage of caspase-8, caspase-3, and poly(ADP-ribose) polymerase. Furthermore, protein levels of the Fas receptor (APO-1/CD-95) were increased following transcriptional inactivation of uPAR and/or MMP-9. In addition, Fas siRNA against the Fas death receptor blocked apoptosis induced by pU-Si, pM-Si, or pUM-Si, thereby indicating the role for Fas signaling in pU-Si–, pM-Si–, or pUM-Si–mediated apoptotic cell death of human glioma xenograft cells. Thus, transcriptional inactivation of uPAR and/or MMP-9 enhanced localization of Fas death receptor, Fas-associated death domain-containing protein, and procaspase-8 into lipid rafts. Additionally, disruption of lipid rafts with methyl β cyclodextrin prevented Fas clustering and pU-Si–, pM-Si–, or pUM-Si–induced apoptosis, which is indicative of coclustering of Fas death receptor into lipid rafts in the glioblastoma xenograft cell lines 4910 and 5310. These data indicate the crucial role of the clusters of apoptotic signaling molecule-enriched rafts in programmed cell death, acting as concentrators of death receptors and downstream signaling molecules, and as the linchpin from which a potent death signal is launched in uPAR- and/or MMP-9–downregulated cells.  

3.1518           m-Calpain-mediated cleavage of Na⁺/Ca²⁺ exchanger-1 in caveolae vesicles isolated from pulmonary artery smooth muscle

Shaikh, S., Samanta, K., Kar, P., Roy, S., Chakraborti, T. and Chakraborti, S. Mol. Cell. Biochem., 341, 167-180 (2010)   Using m-calpain antibody, we have identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of m-calpain in caveolae vesicles isolated from bovine pulmonary artery smooth muscle plasma membrane. In addition, 78, 35, and 18 kDa immunoreactive bands of m-calpain have also been detected. Casein zymogram studies also revealed the presence of m-calpain in the caveolae vesicles. We have also identified Na⁺/Ca²⁺ exchanger-1 (NCX1) in the caveolae vesicles. Purification and N-terminal sequence analyses of these two proteins confirmed their identities as m-calpain and NCX1, respectively. We further sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of NCX1 in the caveolae vesicles. Treatment of the caveolae vesicles with the calcium ionophore, A23187 (1 μM) in presence of CaCl₂ (1 mM) appears to cleave NCX1 (120 kDa) to an 82 kDa fragment as revealed by immunoblot study using NCX1 monoclonal antibody; while pretreatment with the calpain inhibitors, calpeptin or MDL28170; or the Ca²⁺ chelator, BAPTA-AM did not cause a discernible change in the NCX protein profile. In vitro cleavage of the purified NCX1 by the purified m-calpain supports this finding. The cleavage of NCX1 by m-calpain in the caveolae vesicles may be interpreted as an important mechanism of Ca²⁺ overload, which could arise due to inhibition of Ca²⁺ efflux by the forward-mode NCX and that could lead to sustained Ca²⁺ overload in the smooth muscle leading to pulmonary hypertension.  

3.1519           The Intracellular Localization and Function of the ATP-Sensitive K+ Channel Subunit Kir6.1

Ng, K-E., Schwarzer, S., Duchen, M.R. and Tinker, A.

1. Membrane Biol., 234, 137-147 (2010)

  Our aim was to determine the subcellular localization and functional roles of the K_ATP channel subunit Kir6.1 in intracellular membranes. Specifically, we focused on the potential role of Kir6.1 as a subunit of the mitochondrial ATP-sensitive K⁺ channel. Cell imaging showed that a major proportion of heterologously expressed Kir6.1-GFP and endogenously expressed Kir6.1 was distributed in the endoplasmic reticulum with little in the mitochondria or plasma membrane. We used pharmacological and molecular tools to investigate the functional significance of this distribution. The K_ATP channel opener diazoxide increased reactive oxygen species production, and glibenclamide abolished this effect. However, in cells lacking Kir6.1 or expressing siRNA or dominant negative constructs of Kir6.1, the same effect was seen. Ca²⁺ handling was examined in the muscle cell line C2C12. Transfection of the dominant negative constructs of Kir6.1 significantly reduced the amplitude and rate of rise of [Ca²⁺] _c transients elicited by ATP. This study suggests that Kir6.1 is located in the endoplasmic reticulum and plays a role in modifying Ca²⁺ release from intracellular stores.  

3.1520           Ero1α requires oxidizing and normoxic conditions to localize to the mitochondria-associated membrane (MAM)

Gilady, S., Bui, M., Lynes, E.M., Benson, M.D., Watts, R., Vance, J.E. and Simmen, T. Cell Stress and Chaperones, 15, 619-629 (2010)   Protein secretion from the endoplasmic reticulum (ER) requires the enzymatic activity of chaperones and oxidoreductases that fold polypeptides and form disulfide bonds within newly synthesized proteins. The best-characterized ER redox relay depends on the transfer of oxidizing equivalents from molecular oxygen through ER oxidoreductin 1 (Ero1) and protein disulfide isomerase to nascent polypeptides. The formation of disulfide bonds is, however, not the sole function of ER oxidoreductases, which are also important regulators of ER calcium homeostasis. Given the role of human Ero1α in the regulation of the calcium release by inositol 1,4,5-trisphosphate receptors during the onset of apoptosis, we hypothesized that Ero1α may have a redox-sensitive localization to specific domains of the ER. Our results show that within the ER, Ero1α is almost exclusively found on the mitochondria-associated membrane (MAM). The localization of Ero1α on the MAM is dependent on oxidizing conditions within the ER. Chemical reduction of the ER environment, but not ER stress in general leads to release of Ero1α from the MAM. In addition, the correct localization of Ero1α to the MAM also requires normoxic conditions, but not ongoing oxidative phosphorylation.  

3.1521           Compartmentalization of EGFR in cellular membranes: Role of membrane rafts

Balbis, A. and Posner, B.I.

1. Cell. Biochem., 109, 1103-1108 (2010)

  There is now abundant evidence that the intracellular concentration of the EGFR and many other receptors for peptide hormones and growth factors is important for the temporal and spatial regulation of cell signaling. Spatial control is achieved by the selective compartmentalization of signaling components into endosomes. However further control may be effected by sequestration into sub-domains within a given organelle such as membrane rafts which are dynamic, nano scale structures rich in cholesterol and sphingolipids. Current data suggest the presence of EGFRs in non-caveolae membrane rafts. High doses of EGF seem to promote the sorting of EGFR to late endosomes through a raft/cholesterol dependant mechanism, implicating them in EGFR degradation. However our work and that of others has led us to propose a model in which membrane rafts in late endosomes sequester highly active EGFR leading to the recruitment and activation of MAPK in this compartment.  

3.1522           Long chain-polyunsaturated fatty acids modulate membrane phospholipid composition and protein localization in lipid rafts of neural stem cell cultures

Langelier, B., Linard, A., Bordat, C., Lavialle, M. and Heberden, C.

1. Cell. Biochem., 110, 1356-1364 (2010)

  Rat neural stem cells/neural progenitors (NSC/NP) are generally grown in serum-free medium. In this study, NSC/NP were supplemented with the main long-chain polyunsaturated fatty acids (PUFAs) present in the brain, arachidonic acid (AA), or docosahexaenoic acid (DHA), and were monitored for their growth. Lipid and fatty acid contents of the cells were also determined. Under standard conditions, the cells were characterized by phospholipids displaying a highly saturated profile, and very low levels of PUFAs. When cultured in the presence of PUFAs, the cells easily incorporated them into the phospholipid fraction. We also compared the presence of three membrane proteins in the lipid raft fractions: GFR and connexin 43 contents in the rafts were increased by DHA supplementation, whereas Gβ subunit content was not significantly modified. The restoration of DHA levels in the phospholipids could profoundly affect protein localization and, consequently, their functionalities.  

3.1523           Sphingomyelin is important for the cellular entry and intracellular localization of Helicobacter pylori VacA

Gupta, V.R., Wilson, B.A. and Blanke, S.R. Cellular Microbiol., 12(10), 1517-1533 (2010)   Plasma membrane sphingomyelin (SM) binds the Helicobacter pylori vacuolating toxin (VacA) to the surface of epithelial cells. To evaluate the importance of SM for VacA cellular entry, we characterized toxin uptake and trafficking within cells enriched with synthetic variants of SM, whose intracellular trafficking properties are strictly dependent on the acyl chain lengths of their sphingolipid backbones. While toxin binding to the surface of cells was independent of acyl chain length, cells enriched with 12- or 18-carbon acyl chain variants of SM (e.g. C12-SM or C18-SM) were more sensitive to VacA, as indicated by toxin-induced cellular vacuolation, than those enriched with shorter 2- or 6-carbon variants (e.g. C2-SM or C6-SM). In C18-SM-enriched cells, VacA was taken into cells by a previously described Cdc42-dependent pinocytic mechanism, localized initially to GPI-enriched vesicles, and ultimately trafficked to Rab7/Lamp1 compartments. In contrast, within C2-SM-enriched cells, VacA was taken up at a slower rate by a Cdc42-independent mechanism and trafficked to Rab11 compartments. VacA-associated predominantly with detergent-resistant membranes (DRMs) in cells enriched with C18-SM, but predominantly with non-DRMs in C2-SM-enriched cells. These results suggest that SM is required for targeting VacA to membrane rafts important for subsequent Cdc42-dependent pinocytic cellular entry.  

3.1524           Ttyh1, a Ca²⁺-binding protein localized to the endoplasmic reticulum, is required for early embryonic development

Kumada, T., Yamanaka, Y., Kitano, A., Shibata, M., Awaya, T., Kato, T., Okawa, K., Abe, T., Oshima, N., Nakahata, T. and Heike, T. Developmental Dynamics, 239, 2233-2245 (2010)   Using comprehensive genetic studies on neuronal stem/progenitors cells through genome-wide screening with oligonucleotide arrays, we identified an endoplasmic reticulum (ER) -resident protein, Tweety homologue 1 (ttyh1). Ttyh1 encodes a glycosylated protein composed of five predicted transmembrane segments and a C-terminus that is enriched in negatively charged residues capable of Ca²⁺ binding. Ttyh1-containing membranes changed to segmented tubuloreticular structures during mitosis, suggesting that the ER-containing Ttyh1 could be responsible for Ca²⁺ sequestration and Ca²⁺ concentration regulation during mitosis. Ttyh1 inactivation in mice resulted in early embryonic lethality before organization of the nervous system, revealing that ttyh1 is essential in murine embryonic development. Our findings indicate that Ttyh1 plays an indispensable role during mitosis in early embryogenesis, possibly by maintaining Ca²⁺ homeostasis in the ER.  

3.1525           A proteomic approach towards the identification of the matrix protein content of the two types of microbodies in Neurospora crassa

Managadze, D., Würtz, C., Wiese, S., Meyer, H.E., Niehaus, G., Erdmann, R., Warscheid, B and Rottensteiner, H. Proteomics, 10, 3222-3234 (2010)   Microbodies (peroxisomes) comprise a class of organelles with a similar biogenesis but remarkable biochemical heterogeneity. Here, we purified the two distinct microbody family members of filamentous fungi, glyoxysomes and Woronin bodies, from Neurospora crassa and analyzed their protein content by HPLC/ESI-MS/MS. In the purified Woronin bodies, we unambiguously identified only hexagonal 1 (HEX1), suggesting that the matrix is probably exclusively filled with the HEX1 hexagonal crystal. The proteomic analysis of highly purified glyoxysomes allowed the identification of 191 proteins. Among them were 16 proteins with a peroxisomal targeting signal type 1 (PTS1) and three with a PTS2. The collection also contained the previously described N. crassa glyoxysomal matrix proteins FOX2 and ICL1 that lack a typical PTS. Three PTS1 proteins were identified that likely represent the long sought glyoxysomal acyl-CoA dehydrogenases of filamentous fungi. Two of them were demonstrated by subcellular localization studies to be indeed glyoxysomal. Furthermore, two PTS proteins were identified that are suggested to be involved in the detoxification of nitroalkanes. Since the glyoxysomal localization was experimentally demonstrated for one of these enzymes, a new biochemical reaction is expected to be associated with microbody function.  

3.1526           PLP/DM20 Expression and turnover in a transgenic mouse model of pelizaeus-merzbacher disease

Karim, S.A., Barrie, J., McCulloch, M.C., Montaque, P., Edgar, J.M., Iden, D.L., Anderson, T.J., Nave, K-A., Griffiths, I.R. and McLaughlin, M. Glia, 58, 1727-1738 (2010)   The most common cause of Pelizaeus-Merzbacher (PMD) is due to duplication of the PLP1 gene but it is unclear how increased gene dosage affects PLP turnover and causes dysmyelination. We have studied the dynamics of PLP/DM20 in a transgenic mouse model of PMD with increased gene dosage of the proteolipid protein gene (Plp1). The turnover of PLP/DM20 were investigated using an ex-vivo brain slice system and cultured oligodendrocytes. Homozygous mice have reduced PLP translation, markedly enhanced PLP degradation, and markedly reduced incorporation of PLP into myelin. Proteasome inhibition (MG132) prevented the enhanced degradation. Numerous autophagic vesicles are present in homozygous transgenic mice that may influence protein dynamics. Surprisingly, promoting autophagy with rapamycin decreases the degradation of nascent PLP suggesting autophagic vacuoles serve as a cellular storage compartment. We suggest that there are multiple subcellular fates of PLP/DM20 when overexpressed: the vast majority being degraded by the proteasome, a proportion sequestered into autophagic vacuoles, probably fused with endolysosomes, and only a small proportion entering the myelin sheath, where its association with lipid rafts is perturbed. Transgenic oligodendrocytes have fewer membrane sheets and this phenotype is improved with siRNA-mediated knockdown of PLP expression that promotes the formation of MBP+ myelin-like sheets. This finding suggests that RNAi technology is in principle applicable to improve CNS myelination when compromised by PLP/DM20 overexpression.  

3.1527           The Versatility of Helicobacter pylori CagA Effector Protein Functions: The Master Key Hypothesis

Backert, S., Tegtmeyer, N. and Selbach, M. Helicobacter, 15, 163-176 (2010)   Several bacterial pathogens inject virulence proteins into host target cells that are substrates of eukaryotic tyrosine kinases. One of the key examples is the Helicobacter pylori CagA effector protein which is translocated by a type-IV secretion system. Injected CagA becomes tyrosine-phosphorylated on EPIYA sequence motifs by Src and Abl family kinases. CagA then binds to and activates/inactivates multiple signaling proteins in a phosphorylation-dependent and phosphorylation-independent manner. A recent proteomic screen systematically identified eukaryotic binding partners of the EPIYA phosphorylation sites of CagA and similar sites in other bacterial effectors by high-resolution mass spectrometry. Individual phosphorylation sites recruited a surprisingly high number of interaction partners suggesting that each phosphorylation site can interfere with many downstream pathways. We now count 20 reported cellular binding partners of CagA, which represents the highest quantitiy among all yet known virulence-associated effector proteins in the microbial world. This complexity generates a highly remarkable and puzzling scenario. In addition, the first crystal structure of CagA provided us with new information on the function of this important virulence determinant. Here we review the recent advances in characterizing the multiple binding signaling activities of CagA. Injected CagA can act as a ‘master key’ that evolved the ability to highjack multiple host cell signalling cascades, which include the induction of membrane dynamics, actin-cytoskeletal rearrangements and the disruption of cell-to-cell junctions as well as proliferative, pro-inflammatory and anti-apoptotic nuclear responses. The discovery that different pathogens use this common strategy to subvert host cell functions suggests that more examples will emerge soon.  

3.1528           Disruption of cellular cholesterol transport and homeostasis as a novel mechanism of action of membrane-targeted alkylphospholipid analogues

Carrasco, M:P., Jimenez-Lopez, J.M., Rios-Marco, P., Segovia, J.L. and Marco, C. Br. J. Pharmacol., 160, 355-366 (2010)   Background and purpose: Alkylphospholipid (APL) analogues constitute a new class of synthetic anti-tumour agents that act directly on cell membranes. We have previously demonstrated that hexadecylphosphocholine (HePC) alters intracellular cholesterol traffic and metabolism in HepG2 cells. We now extended our studies to analyse the effects of other clinically relevant APLs, such as edelfosine, erucylphosphocholine and perifosine on intracellular cholesterol homeostasis. Experimental approach: Using radiolabelled substrates we determined the effect of APLs on cholesterol metabolism and cholesterol traffic from the plasma membrane to the endoplasmic reticulum (ER). Protein levels and gene expression of the main proteins involved in cholesterol homeostasis were analysed by Western blot and RT-PCR respectively. Membrane raft and non-raft fractions were isolated from HepG2 cells by a detergent-free method. Key results: All APLs inhibited the transport of cholesterol from the plasma membrane to the ER, which induced a significant cholesterogenic response in HepG2 cells. This response involved an increased gene expression and higher levels of several proteins related to the biosynthesis and the receptor-mediated uptake of cholesterol. Cell exposure to the APL-representative HePC enhanced the content of cholesterol mainly in the membrane raft fractions, compared with the untreated cells. Conclusions and implications: Membrane-targeted APLs exhibited a novel and common mechanism of action, through disruption of cholesterol homeostasis, which in turn affected specific lipid microdomains of cellular membranes.  

3.1529           The Arabidopsis Peroxisomal ABC Transporter, Comatose, Complements the Saccharomyces cerevisiae pxa1 pxa2 Mutant for Metabolism of Long-chain Fatty Acids and Exhibits Fatty Acyl-CoA-stimulated ATPase Activity

Nyathi, Y., De Marcos Lousa, C., van Roermund, C.W., Wanders, R.J.A., Johnson, B., Baldwin, S.A., Theodoulou, F.L. and Baker, A.

1. Biol. Chem., 285(38), 29892-29902 (2010)

  The Arabidopsis ABC transporter Comatose (CTS; AtABCD1) is required for uptake into the peroxisome of a wide range of substrates for β-oxidation, but it is uncertain whether CTS itself is the transporter or if the transported substrates are free acids or CoA esters. To establish a system for its biochemical analysis, CTS was expressed in Saccharomyces cerevisiae. The plant protein was correctly targeted to yeast peroxisomes, was assembled into the membrane with its nucleotide binding domains in the cytosol, and exhibited basal ATPase activity that was sensitive to aluminum fluoride and abrogated by mutation of a conserved Walker A motif lysine residue. The yeast pxa1 pxa2Δ mutant lacks the homologous peroxisomal ABC transporter and is unable to grow on oleic acid. Consistent with its exhibiting a function in yeast akin to that in the plant, CTS rescued the oleate growth phenotype of the pxa1 pxa2Δ mutant, and restored β-oxidation of fatty acids with a range of chain lengths and varying degrees of desaturation. When expressed in yeast peroxisomal membranes, the basal ATPase activity of CTS could be stimulated by fatty acyl-CoAs but not by fatty acids. The implications of these findings for the function and substrate specificity of CTS are discussed.  

3.1530           Agonist-Specific Compartmentation of cGMP Action in Myometrium

Buxton, I.L.O., Milton, D., Barnett, S.D. and Tichenor, S.D.

1. Pharmacol. Exp. Ther., 335(1), 256-263 (2010)

  Nitric oxide relaxes myometrium in a cGMP-independent manner. Although cGMP activates its cognate kinase, this is not required for the inhibitory effect of nitric oxide. Thus, nitric oxide-mediated cGMP elevation does not enjoy the same set of substrates as it does in other smooth muscles. To further understand the regulation of relaxation of uterine muscle by cGMP, we have studied the actions of peptide-mediated cGMP action in guinea pig myometrium. We used both functional and biochemical studies of the action of the particulate guanylyl cyclase activator uroguanylin and its receptor, particulate guanylyl cyclase type C, to address the relationship between cGMP elevation acting in the membrane signaling domain to that of the nonmembrane region of the cell. Uroguanylin relaxed oxytocin-induced contractions in a dose-dependent fashion only in pregnant myometrium. Both relaxation and cGMP accumulation after uroguanylin stimulation were blocked by the putative particulate guanylyl cyclase type C inhibitors 2-chloro-ATP and isatin (1H-indole-2,3-dione), but not by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-A]quinoxalin-1-one (ODQ). Uroguanylin stimulated cGMP accumulation only in the pregnant myometrium. Caveolin-1 expression increased in pregnancy toward term. In the caveolin-1-containing membrane domain, uroguanylin, but not the nitric-oxide donor, led to the elevation of cGMP that was insensitive to ODQ. Particulate guanylyl cyclase C was expressed and prouroguanylin was detected in pregnant myometrium. We conclude that a uroguanylin–particulate cyclase-cGMP relaxation pathway is present and cGMP is compartmented in myometrium. The agonist-mediated selectivity of relaxation to cGMP is of fundamental pharmacological interest in understanding signal transduction in smooth muscle.  

3.1531           Inorganic polyphosphate inhibits an aspartic protease-like activity in the eggs of Rhodnius prolixus (Stahl) and impairs yolk mobilization in vitro

Gomes, F.M., Oliveira, D.M.P., Motta, L.S., Ramos, I.B., Miranda, K.M. and Macchado, E.A.

1. Cell. Physiol., 222, 606-611 (2010)

  Inorganic polyphosphate (poly P) is a polymer of phosphate residues that has been shown to act as modulator of some vertebrate cathepsins. In the egg yolk granules of Rhodnius prolixus, a cathepsin D is the main protease involved in yolk mobilization and is dependent on an activation by acid phosphatases. In this study, we showed a possible role of poly P stored inside yolk granules on the inhibition of cathepsin D and arrest of yolk mobilization during early embryogenesis of these insects. Enzymatic assays detected poly P stores inside the eggs of R. prolixus. We observed that micromolar poly P concentrations inhibited cathepsin D proteolytic activity using both synthetic peptides and homogenates of egg yolk as substrates. Poly P was a substrate for Rhodnius acid phosphatase and also a strong competitive inhibitor of a pNPPase activity. Fusion events have been suggested as important steps towards acid phosphatase transport to yolk granules. We observed that poly P levels in those compartments were reduced after in vitro fusion assays and that the remaining poly P did not have the same cathepsin D inhibition activity after fusion. Our results are consistent with the hypothesis that poly P is a cathepsin D inhibitor and a substrate for acid phosphatase inside yolk granules. It is possible that, once activated, acid phosphatase might degrade poly P, allowing cathepsin D to initiate yolk proteolysis. We, therefore, suggest that degradation of poly P might represent a new step toward yolk mobilization during embryogenesis of R. prolixus.    

3.1532           Acylation-dependent Export of Trypanosoma cruzi Phosphoinositide-specific Phospholipase C to the Outer Surface of Amastigotes

De Paulo Martins, V., Okura, M., Maric, D., Engman, D.M., Vieira, M., Docompo, R. and Moreno, S.N.J.

1. Biol. Chem., 285(40), 30906-30917 (2010)

  Phosphoinositide phospholipase C (PI-PLC) plays an essential role in cell signaling. A unique Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid-modified in its N terminus and localizes to the plasma membrane of amastigotes. Here, we show that TcPI-PLC is located onto the extracellular phase of the plasma membrane of amastigotes and that its N-terminal 20 amino acids are necessary and sufficient to target the fused GFP to the outer surface of the parasite. Mutagenesis of the predicted acylated residues confirmed that myristoylation of a glycine residue in the 2nd position and acyl modification of a cysteine in the 4th but not in the 8th or 15th position of the coding sequence are required for correct plasma membrane localization in T. cruzi epimastigotes or amastigotes. Interestingly, mutagenesis of the cysteine at the 8th position increased its flagellar localization. When expressed as fusion constructs with GFP, the N-terminal 6 and 10 amino acids fused to GFP are predominantly located in the cytosol and concentrated in a compartment that co-localizes with a Golgi complex marker. The N-terminal 20 amino acids of TcPI-PLC associate with lipid rafts when dually acylated. Taken together, these results indicate that N-terminal acyl modifications serve as a molecular addressing system for sending TcPI-PLC to the outer surface of the cell.  

3.1533           Rab32 Modulates Apoptosis Onset and Mitochondria-associated Membrane (MAM) Properties

Bui, M., Gilady, S.Y., Fitzsimmons, R.E.B., Benson, M.D., Lynes, E.M., Gesson, K., Alto, N.M., Strack, S., Scott, J.D. and Simmen, T.

1. Biol. Chem., 285(41), 31590-31602 (2010)

  The mitochondria-associated membrane (MAM) has emerged as an endoplasmic reticulum (ER) signaling hub that accommodates ER chaperones, including the lectin calnexin. At the MAM, these chaperones control ER homeostasis but also play a role in the onset of ER stress-mediated apoptosis, likely through the modulation of ER calcium signaling. These opposing roles of MAM-localized chaperones suggest the existence of mechanisms that regulate the composition and the properties of ER membrane domains. Our results now show that the GTPase Rab32 localizes to the ER and mitochondria, and we identify this protein as a regulator of MAM properties. Consistent with such a role, Rab32 modulates ER calcium handling and disrupts the specific enrichment of calnexin on the MAM, while not affecting the ER distribution of protein-disulfide isomerase and mitofusin-2. Furthermore, Rab32 determines the targeting of PKA to mitochondrial and ER membranes and through its overexpression or inactivation increases the phosphorylation of Bad and of Drp1. Through a combination of its functions as a PKA-anchoring protein and a regulator of MAM properties, the activity and expression level of Rab32 determine the speed of apoptosis onset.  

3.1534           Multivesicular Body Formation Requires OSBP-Related Proteins and Cholesterol

Kobuna, H., Inoue, T., Shibata, M., Gengyo-Ando, K., Yamamoto, A., Mitani, S. and Arai, H. PloS Genetics, 6(8), e1001055 (2010)   In eukaryotes, different subcellular organelles have distinct cholesterol concentrations, which is thought to be critical for biological functions. Oxysterol-binding protein-related proteins (ORPs) have been assumed to mediate nonvesicular cholesterol trafficking in cells; however, their in vivo functions and therefore the biological significance of cholesterol in each organelle are not fully understood. Here, by generating deletion mutants of ORPs in Caenorhabditis elegans, we show that ORPs are required for the formation and function of multivesicular bodies (MVBs). In an RNAi enhancer screen using obr quadruple mutants (obr-1; -2; -3; -4), we found that MVB–related genes show strong genetic interactions with the obr genes. In obr quadruple mutants, late endosomes/lysosomes are enlarged and membrane protein degradation is retarded, although endocytosed soluble proteins are normally delivered to lysosomes and degraded. We also found that the cholesterol content of late endosomes/lysosomes is reduced in the mutants. In wild-type worms, cholesterol restriction induces the formation of enlarged late endosomes/lysosomes, as observed in obr quadruple mutants, and increases embryonic lethality upon knockdown of MVB–related genes. Finally, we show that knockdown of ORP1L, a mammalian ORP family member, induces the formation of enlarged MVBs in HeLa cells. Our in vivo findings suggest that the proper cholesterol level of late endosomes/lysosomes generated by ORPs is required for normal MVB formation and MVB–mediated membrane protein degradation.  

3.1535           A Novel Role for the Centrosomal Protein, Pericentrin, in Regulation of Insulin Secretory Vesicle Docking in Mouse Pancreatic β-cells

Jurczyk, A., Pino, S.C., O’Sullivan-Murphy, B., Addorio, M., Lidstone, E.A., Dilorio, P., Lipson, K.-L., Standley, C., Fogarty, K., Lifshitz, L., Urano, F., Mordes, J.P., Greiner, D.L., Rossini, A.A. and Bortell, R. PloSOne, 5(7), e11812 (2010)   The centrosome is important for microtubule organization and cell cycle progression in animal cells. Recently, mutations in the centrosomal protein, pericentrin, have been linked to human microcephalic osteodysplastic primordial dwarfism (MOPD II), a rare genetic disease characterized by severe growth retardation and early onset of type 2 diabetes among other clinical manifestations. While the link between centrosomal and cell cycle defects may account for growth deficiencies, the mechanism linking pericentrin mutations with dysregulated glucose homeostasis and pre-pubertal onset of diabetes is unknown. In this report we observed abundant expression of pericentrin in quiescent pancreatic β-cells of normal animals which led us to hypothesize that pericentrin may have a critical function in β-cells distinct from its known role in regulating cell cycle progression. In addition to the typical centrosome localization, pericentrin was also enriched with secretory vesicles in the cytoplasm. Pericentrin overexpression in β-cells resulted in aggregation of insulin-containing secretory vesicles with cytoplasmic, but not centrosomal, pericentriolar material and an increase in total levels of intracellular insulin. RNAi- mediated silencing of pericentrin in secretory β-cells caused dysregulated secretory vesicle hypersecretion of insulin into the media. Together, these data suggest that pericentrin may regulate the intracellular distribution and secretion of insulin. Mice transplanted with pericentrin-depleted islets exhibited abnormal fasting hypoglycemia and inability to regulate blood glucose normally during a glucose challenge, which is consistent with our in vitro data. This previously unrecognized function for a centrosomal protein to mediate vesicle docking in secretory endocrine cells emphasizes the adaptability of these scaffolding proteins to regulate diverse cellular processes and identifies a novel target for modulating regulated protein secretion in disorders such as diabetes.  

3.1536           Posttranslational mechanisms associated with reduced NHE3 activity in adult vs. young prehypertensive SHR

Crajoinas, R.O., Lessa, L.M.A., Carraro-Lacroix, L.R., Davel, A.P.C., pachero, B.P.M., Rossoni, L.V., Malnic, G. and Girardi, A.C.C. Am. J. Physiol. Renal Physiol., 299, F872-F881 (2010)   Abnormalities in renal proximal tubular (PT) sodium transport play an important role in the pathophysiology of essential hypertension. The Na⁺/H⁺ exchanger isoform 3 (NHE3) represents the major route for sodium entry across the apical membrane of renal PT cells. We therefore aimed to assess in vivo NHE3 transport activity and to define the molecular mechanisms underlying NHE3 regulation before and after development of hypertension in the spontaneously hypertensive rat (SHR). NHE3 function was measured as the rate of bicarbonate reabsorption by means of in vivo stationary microperfusion in PT from young prehypertensive SHR (Y-SHR; 5-wk-old), adult SHR (A-SHR; 14-wk-old), and age-matched Wistar Kyoto (WKY) rats. We found that NHE3-mediated PT bicarbonate reabsorption was reduced with age in the SHR (1.08 ± 0.10 vs. 0.41 ± 0.04 nmol/cm²xs), while it was increased in the transition from youth to adulthood in the WKY rat (0.59 ± 0.05 vs. 1.26 ± 0.11 nmol/cm²xs). Higher NHE3 activity in the Y-SHR compared with A-SHR was associated with a predominant microvilli confinement and a lower ratio of phosphorylated NHE3 at serine-552 to total NHE3 (P-NHE3/total). After development of hypertension, P-NHE3/total increased and NHE3 was retracted out of the microvillar microdomain along with the regulator dipeptidyl peptidase IV (DPPIV). Collectively, our data suggest that the PT is playing a role in adapting to the hypertension in the SHR. The molecular mechanisms of this adaptation possibly include an increase of P-NHE3/total and a redistribution of the NHE3-DPPIV complex from the body to the base of the PT microvilli, both predicted to decrease sodium reabsorption.  

3.1537           Unexpected Role of the Copper Transporter ATP7A in PDGF-Induced Vascular Smooth Muscle Cell Migration

Ashino, T., Sudhahar, V., Yrao, N., Oshikawa, J., Chen, G-F., Wang, H., Huo, Y., Finney, L., Vogt, S., McKinney, R.D., Maryon, E.B., Kaplan, J.H., Ushio-Fakai, M. and Fukai, T. Circ. Res., 107, 787-799 (2010)   Rationale:Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1 (copper transporter 1) but also by the copper exporter ATP7A (Menkes ATPase), whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN).Platelet-derived growth factor (PDGF) promotes vascular smoothmuscle cell (VSMC) migration, a key component of neointimalformation. Objective:To determine the role of copper transporter ATP7A in PDGF-inducedVSMC migration. Methods and Results:Depletion of ATP7A inhibited VSMC migration in response to PDGFor wound scratch in a CTR1/copper-dependent manner. PDGF stimulationpromoted ATP7A translocation from the TGN to lipid rafts, whichlocalized at the leading edge, where it colocalized with PDGFreceptor and Rac1, in migrating VSMCs. Mechanistically, ATP7Asmall interfering RNA or CTR small interfering RNA preventedPDGF-induced Rac1 translocation to the leading edge, therebyinhibiting lamellipodia formation. In addition, ATP7A depletionprevented a PDGF-induced decrease in copper level and secretorycopper enzyme precursor prolysyl oxidase (Pro-LOX) in lipidraft fraction, as well as PDGF-induced increase in LOX activity.In vivo, ATP7A expression was markedly increased and copperaccumulation was observed by synchrotron-based x-ray fluorescencemicroscopy at neointimal VSMCs in wire injury model. Conclusions:These findings suggest that ATP7A plays an important role incopper-dependent PDGF-stimulated VSMC migration via recruitingRac1 to lipid rafts at the leading edge, as well as regulatingLOX activity. This may contribute to neointimal formation aftervascular injury. Our findings provide insight into ATP7A asa novel therapeutic target for vascular remodeling and atherosclerosis.  

3.1538           Role of EBAG9 protein in coat protein complex I-dependent glycoprotein maturation and secretion processes in tumor cells

Wolf, J., Reimer, T.A., Schuck, S., Rüder, C., Gerlach, K., Müller, E-C., Otto, A., Dörken, B. and Rehm, A. FASEB J., 24, 4000-4019 (2010)   Many proteins mature within the secretory pathway by the acquisition of glycans. Failure to maintain the proper distribution of the glycosylation machinery might lead to disease. High expression levels of the ubiquitous Golgi protein estrogen receptor-binding fragment-associated gene 9 (EBAG9) in human tumors correlate with poor clinical prognosis, and EBAG9 overexpression in epithelial cell lines induces truncated glycans, typical of many carcinomas. Here, we addressed the pathogenetic link between EBAG9 expression and the alteration of the cellular glycome. We applied confocal microscopy, live imaging, pulse-chase labeling in conjunction with immunoprecipitation, and enzymatic activity assays in a variety of EBAG9-overexpressing or depleted epithelial tumor cell lines. EBAG9 shuttles between the ER-Golgi intermediate compartment and the cis-Golgi, and we demonstrate association of EBAG9 with coat protein complex I (COPI)-coated transport vesicles. EBAG9 overexpression imposes delay of endoplasmic reticulum-to-Golgi transport and mislocalizes components of the ER quality control and glycosylation machinery. Conversely, EBAG9 down-regulation accelerates glycoprotein transport through the Golgi and enhances mannosidase activity. Thus, EBAG9 acts as a negative regulator of a COPI-dependent ER-to-Golgi transport pathway in epithelial cells and represents a novel pathogenetic principle in which interference with intracellular membrane trafficking results in the emergence of a tumor-associated glycome.  

3.1539           Pathogenic Lysosomal Depletion in Parkinson's Disease

Dehay, B., Bove, J., Rodriguez-Muela, N., Perier, C., Recases, A., Boya, P. and Vila, M.

1. Neurosci., 30(37), 12535-12544 (2010)

  Mounting evidence suggests a role for autophagy dysregulation in Parkinson's disease (PD). The bulk degradation of cytoplasmic proteins (including -synuclein) and organelles (such as mitochondria) is mediated by macroautophagy, which involves the sequestration of cytosolic components into autophagosomes (AP) and its delivery to lysosomes. Accumulation of AP occurs in postmortem brain samples from PD patients, which has been widely attributed to an induction of autophagy. However, the cause and pathogenic significance of these changes remain unknown. Here we found in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of PD that AP accumulation and dopaminergic cell death are preceded by a marked decrease in the amount of lysosomes within dopaminergic neurons. Lysosomal depletion was secondary to the abnormal permeabilization of lysosomal membranes induced by increased mitochondrial-derived reactive oxygen species. Lysosomal permeabilization resulted in a defective clearance and subsequent accumulation of undegraded AP and contributed directly to neurodegeneration by the ectopic release of lysosomal proteases into the cytosol. Lysosomal breakdown and AP accumulation also occurred in PD brain samples, where Lewy bodies were strongly immunoreactive for AP markers. Induction of lysosomal biogenesis by genetic or pharmacological activation of lysosomal transcription factor EB restored lysosomal levels, increased AP clearance and attenuated 1-methyl-4-phenylpyridinium-induced cell death. Similarly, the autophagy-enhancer compound rapamycin attenuated PD-related dopaminergic neurodegeneration, both in vitro and in vivo, by restoring lysosomal levels. Our results indicate that AP accumulation in PD results from defective lysosomal-mediated AP clearance secondary to lysosomal depletion. Restoration of lysosomal levels and function may thus represent a novel neuroprotective strategy in PD.  

3.1540           NMDA-Mediated Regulation of DSCAM Dendritic Local Translation Is Lost in a Mouse Model of Down's Syndrome

Alves-Sampaio, A., Troca-Marin, J.A. and  Montesinos, M.L.

1. Neurosci., 30(40), 13537-13548 (2010)

  Down's syndrome cell adhesion molecule (DSCAM) belongs to the Down's syndrome critical region of human chromosome 21, and it encodes a cell adhesion molecule involved in dendrite morphology and neuronal wiring. Although the function of DSCAM in the adult brain is unknown, its expression pattern suggests a role in synaptic plasticity. Local mRNA translation is a key process in axonal growth, dendritogenesis, and synaptogenesis during development, and in synaptic plasticity in adulthood. Here, we report the dendritic localization of DSCAM mRNA in the adult mouse hippocampus, where it associates with CPEB1 [cytoplasmic polyadenylation element (CPE) binding protein 1], an important regulator of mRNA transport and local translation. We identified five DSCAM isoforms produced by alternative polyadenylation bearing different combinations of regulatory CPE motifs. Overexpression of DSCAM in hippocampal neurons inhibited dendritic branching. Interestingly, dendritic levels of DSCAM mRNA and protein were increased in hippocampal neurons from Ts1Cje mice, a model of Down's syndrome. Most importantly, DSCAM dendritic translation was rapidly induced by NMDA in wild-type, but not in Ts1Cje neurons. We propose that impairment of the NMDA-mediated regulation of DSCAM translation may contribute to the alterations in dendritic morphology and/or synaptic plasticity in Down's syndrome.  

3.1541           Dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis

Heaton, N.S., Perera, R., Berger, K.L., Khadka, S., LaCount, D.J., Kuhn, R.J. and Randall, G. PNAS, 107(40), 17345-17350 (2010)   Dengue virus (DENV) modifies cellular membranes to establish its sites of replication. Although the 3D architecture of these structures has recently been described, little is known about the cellular pathways required for their formation and expansion. In this report, we examine the host requirements for DENV replication using a focused RNAi analysis combined with validation studies using pharmacological inhibitors. This approach identified three cellular pathways required for DENV replication: autophagy, actin polymerization, and fatty acid biosynthesis. Further characterization of the viral modulation of fatty acid biosynthesis revealed that a key enzyme in this pathway, fatty acid synthase (FASN), is relocalized to sites of DENV replication. DENV nonstructural protein 3 (NS3) is responsible for FASN recruitment, inasmuch as (i) NS3 expressed in the absence of other viral proteins colocalizes with FASN and (ii) NS3 interacts with FASN in a two-hybrid assay. There is an associated increase in the rate of fatty acid biosynthesis in DENV-infected cells, and de novo synthesized lipids preferentially cofractionate with DENV RNA. Finally, purified recombinant NS3 stimulates the activity of FASN in vitro. Taken together, these experiments suggest that DENV co-opts the fatty acid biosynthetic pathway to establish its replication complexes. This study provides mechanistic insight into DENV membrane remodeling and highlights the potential for the development of therapeutics that inhibit DENV replication by targeting the fatty acid biosynthetic pathway.  

3.1542           Organelle Proteomics by Label-Free and SILAC-Based Protein Correlation Profiling

Dengjel, J. Jakobsen, L. and Andersen, J.S. Methods in Mol. Biol., 658, 255-265 (2010)   The ability to purify cell organelles and protein complexes on a large scale, combined with advances in protein identification using mass spectrometry, has provided a wealth of information regarding protein localization and function. A major challenge in these studies has been the ability to identify bona fide organelle components from a background of co-purifying contaminants because none of the available biochemical purification protocols afford pure preparations. Since this situation is unlikely to change alternative strategies have been devised to meet this challenge by making use of the information inherent in the fractionation profile of organelles isolated by density gradient centrifugation. In this chapter we describe strategies based on protein correlation profiling and quantitative mass spectrometry to sort out likely candidates. The organelle inventories defined by these methods are suitable to guide future functional experiments.  

3.1543           Critical role of lipid rafts in virus entry and activation of phosphoinositide 3′ kinase/Akt signaling during early stages of Japanese encephalitis virus infection in neural stem/progenitor cells

Das, S., Chakraborty, S. and Basu, A.

1. Neurochem., 115, 537-549 (2010)

  Japanese encephalitis virus (JEV), the leading cause of acute encephalitis in South-East Asia is a neurotropic virus infecting various CNS cell types. Most Flaviviruses including JEV get internalised into cells by receptor-mediated endocytosis, which involve clathrin and membrane cholesterol. The cholesterol-enriched membrane microdomains referred to as lipid rafts act as portals for virus entry in a number of enveloped viruses, including Flavivirus. However, the precise role played by membrane lipid rafts in JEV internalisation into neural stem cells is still unknown. We have established neural stem/progenitor cells and C17.2 cell line as models of productive JEV infection. Increase in membrane fluidity and clustering of viral envelope proteins in lipid rafts was observed in early time points of infection. Localisation of non-structural proteins to rafts at later infection stages was also observed. Co-localisation of JEV glycoprotein with Cholera toxin B confirmed that JEV internalisation occurs in a lipid-raft dependent manner. Though JEV entry is raft dependent, however, there is requirement of functional clathrin during endocytosis inside the cells. Besides virus entry, the lipid rafts act as signalling platforms for Src tyrosine kinases and result in activation of phosphoinositìde 3′-kinase/Akt signalling during early JEV infection. Disruption of lipid raft formation by cholesterol depletion using Methyl β-cyclodextrin, reduced JEV RNA levels and production of infectious virus particles as well as impaired phosphoinositìde 3′-kinase/Akt signalling during initial infection. Overall, our results implicate the importance of host membrane lipid rafts in JEV entry and life cycle, besides maintaining survival of neural stem/progenitor cells during early infection.  

3.1544           Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol

Zhu, X., Owen, J.S., Wilson, M.D., Li, H., Griffiths, G.L., Thomas, M.J., Hiltbold, E.M., Fessler, M.B. and Parks, J.S.

1. Lipid Res., 51, 3196-3206 (2010)

  We previously showed that macrophages from macrophage-specific ATP-binding cassette transporter A1 (ABCA1) knockout (Abca1^-M/-M) mice had an enhanced proinflammatory response to the Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), compared with wild-type (WT) mice. In the present study, we demonstrate a direct association between free cholesterol (FC), lipid raft content, and hyper-responsiveness of macrophages to LPS in WT mice. Abca1^-M/-M macrophages were also hyper-responsive to specific agonists to TLR2, TLR7, and TLR9, but not TLR3, compared with WT macrophages. We hypothesized that ABCA1 regulates macrophage responsiveness to TLR agonists by modulation of lipid raft cholesterol and TLR mobilization to lipid rafts. We demonstrated that Abca1^-M/-M vs. WT macrophages contained 23% more FC in isolated lipid rafts. Further, mass spectrometric analysis suggested raft phospholipid composition was unchanged. Although cell surface expression of TLR4 was similar between Abca1^-M/-M and WT macrophages, significantly more TLR4 was distributed in membrane lipid rafts in Abca1^-M/-M macrophages. Abca1^-M/-M macrophages also exhibited increased trafficking of the predominantly intracellular TLR9 into lipid rafts in response to TLR9-specific agonist (CpG). Collectively, our data suggest that macrophage ABCA1 dampens inflammation by reducing MyD88-dependent TLRs trafficking to lipid rafts by selective reduction of FC content in lipid rafts.  

3.1545           Biogenesis of Salmonella enterica Serovar Typhimurium Membrane Vesicles Provoked by Induction of PagC

Kitagawa, R., Takaya, A., Ohya, M., Mizunoe, Y., Takade, A., Yoshida, S-i., Isogai, E. and Yamamoto, T. J: Bacteriol., 192(21), 5645-5656 (2010)   Gram-negative bacteria ubiquitously release membrane vesicles (MVs) into the extracellular milieu. Although MVs are the product of growing bacteria, not of cell lysis or death, the regulatory mechanisms underlying MV formation remained unknown. We have found that MV biogenesis is provoked by the induction of PagC, a Salmonella-specific protein whose expression is activated by conditions that mimic acidified macrophage phagosomes. PagC is a major constituent of Salmonella MVs, and increased expression accelerates vesiculation. Expression of PagC is regulated at the posttranscriptional and/or posttranslational level in a sigmaS (RpoS)-dependent manner. Serial quantitative analysis has demonstrated that MV formation can accelerate when the quantity of the MV constituents, OmpX and PagC, rises. Overproduction of PagC dramatically impacts the difference in the relative amount of vesiculation, but the corresponding overproduction of OmpX was less pronounced. Quantitative examination of the ratios of PagC and OmpX in the periplasm, outer membrane, and MVs demonstrates that PagC is preferentially enriched in MVs released from Salmonella cells. This suggests that specific protein sorting mechanisms operate when MVs are formed. The possible role(s) of PagC-MV in host cells is discussed.  

3.1546           Relevant Elements of a Maize -Zein Domain Involved in Protein Body Biogenesis

Llop-Tous, I., Madurga, S., giralt, E., Torrent, M. and Dolors-Ludevid, M.

1. Biol. Chem., 285(46), 35633-35644 (2010)

  The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)₈ of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4–6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys⁷ and Cys⁹) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.  

3.1547           Protein-disulfide Isomerase-associated 3 (Pdia3) Mediates the Membrane Response to 1,25-Dihydroxyvitamin D3 in Osteoblasts

Chen, J., Olivares-Navarrete, R., Wang, Y., Herman, T.R., Boyan, B.D. and Schwartz, Z.

1. Biol. Chem., 285(47), 37041-37050 (2010)

  Protein-disulfide isomerase-associated 3 (Pdia3) is a multifunctional protein hypothesized to be a membrane receptor for 1,25(OH)2D3. In intestinal epithelium and chondrocytes, 1,25(OH)2D3 stimulates rapid membrane responses that are different from genomic effects via the vitamin D receptor (VDR). In this study, we show that 1,25(OH)2D3 stimulates phospholipase A2 (PLA2)-dependent rapid release of prostaglandin E2 (PGE2), activation of protein kinase C (PKC), and regulation of bone-related gene transcription and mineralization in osteoblast-like MC3T3-E1 cells (WT) via a mechanism involving Pdia3. Pdia3 was present in caveolae based on co-localization with lipid rafts and caveolin-1. In Pdia3-silenced (Sh-Pdia3) cells, 1,25(OH)2D3 failed to stimulate PKC and PGE2 responses; in Pdia3-overexpressing cells (Ov-Pdia3), responses to 1,25(OH)2D3 were augmented. Downstream mediators of Pdia3, PLA2-activating protein (PLAA) and arachidonic acid, stimulated similar PKC activation in wild-type, Sh-Pdia3, and Ov-Pdia3 cells supporting the hypothesis that Pdia3 mediates the membrane action of 1,25(OH)2D3. Treatment of MC3T3-E1 cells with 1,25(OH)2D3 for 9 min stimulated rapid phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and increased expression of alkaline phosphatase, MMP-13, and osteopontin but decreased expression of osteocalcin, osteoprotegerin (mRNA and protein), and smad2. These effects were attenuated in Sh-Pdia3 cells. Sh-Pdia3 cells produced higher numbers of von Kossa-positive nodules and alizarin red-positive nodules compared with WT cells with or without 1,25(OH)2D3 treatment whereas Ov-Pdia3 did not show any mineralization. Our data suggest Pdia3 is an important initiator of 1,25(OH)2D3-stimulated membrane signaling pathways, which have both genomic and non genomic effects during osteoblast maturation.  

3.1548           Helicobacter pylori Exploits Cholesterol-Rich Microdomains for Induction of NF- B-Dependent Responses and Peptidoglycan Delivery in Epithelial Cells

Hutton, M.L., Kaparakis-Liaskos, M., Turner, L., cardona, A., Kwok, T. and Ferrero, R.L. Infect. Immun., 78(11)   Infection with Helicobacter pylori cag pathogenicity island (cagPAI)-positive strains is associated with more destructive tissue damage and an increased risk of severe disease. The cagPAI encodes a type IV secretion system (TFSS) that delivers the bacterial effector molecules CagA and peptidoglycan into the host cell cytoplasm, thereby inducing responses in host cells. It was previously shown that interactions between CagL, present on the TFSS pilus, and host 5β1 integrin molecules were critical for CagA translocation and the induction of cytoskeletal rearrangements in epithelial cells. As the 5β1 integrin is found in cholesterol-rich microdomains (known as lipid rafts), we hypothesized that these domains may also be involved in the induction of proinflammatory responses mediated by NOD1 recognition of H. pylori peptidoglycan. Indeed, not only did methyl-β-cyclodextrin depletion of cholesterol from cultured epithelial cells have a significant effect on the levels of NF- B and interleukin-8 (IL-8) responses induced by H. pylori bacteria with an intact TFSS (P < 0.05), but it also interfered with TFSS-mediated peptidoglycan delivery to cells. Both of these effects could be restored by cholesterol replenishment of the cells. Furthermore, we demonstrated for the first time the involvement of 5β1 integrin in the induction of proinflammatory responses by H. pylori. Taking the results together, we propose that 5β1 integrin, which is associated with cholesterol-rich microdomains at the host cell surface, is required for NOD1 recognition of peptidoglycan and subsequent induction of NF- B-dependent responses to H. pylori. These data implicate cholesterol-rich microdomains as a novel platform for TFSS-dependent delivery of bacterial products to cytosolic pathogen recognition molecules.  

3.1549           Dynamin-like protein 1 at the Golgi complex: A novel component of the sorting/targeting machinery en route to the plasma membrane

Bomekamp, N.A., Vormund, K., Jacob, R. and Schrader, M. Exp. Cell Res., 316(20), 3454-3467 (2010)   The final step in the liberation of secretory vesicles from the trans-Golgi network (TGN) involves the mechanical action of the large GTPase dynamin as well as conserved dynamin-independent fission mechanisms, e.g. mediated by Brefeldin A-dependent ADP-ribosylated substrate (BARS). Another member of the dynamin family is the mammalian dynamin-like protein 1 (DLP1/Drp1) that is known to constrict and tubulate membranes, and to divide mitochondria and peroxisomes. Here, we examined a potential role for DLP1 at the Golgi complex. DLP1 localized to the Golgi complex in some but not all cell lines tested, thus explaining controversial reports on its cellular distribution. After silencing of DLP1, an accumulation of the apical reporter protein YFP-GL-GPI, but not the basolateral reporter VSVG-SP-GFP at the Golgi complex was observed. A reduction in the transport of YFP-GL-GPI to the plasma membrane was confirmed by surface immunoprecipitation and TGN-exit assays. In contrast, YFP-GL-GPI trafficking was not disturbed in cells silenced for BARS, which is involved in basolateral sorting and trafficking of VSVG-SP-GFP in COS-7 cells. Our data indicate a new role for DLP1 at the Golgi complex and thus a role for DLP1 as a novel component of the apical sorting machinery at the TGN is discussed.  

3.1550           Visfatin-induced lipid raft redox signaling platforms and dysfunction in glomerular endothelial cells

Boini, K.M., Zhang, C., Xia, M., Han, W-Q., Brimson, C., Poklis, J.L. and Li, P-l. Biochim. Biophys. Acta, 1801, 1294-1304 (2010)   Adipokines have been reported to contribute to glomerular injury during obesity or diabetes mellitus. However, the mechanisms mediating the actions of various adipokines on the kidney remained elusive. The present study was performed to determine whether acid sphingomyelinase (ASM)-ceramide associated lipid raft (LR) clustering is involved in local oxidative stress in glomerular endothelial cells (GECs) induced by adipokines such as visfatin and adiponectin. Using confocal microscopy, visfatin but not adiponectin was found to increase LRs clustering in the membrane of GECs in a dose and time dependent manner. Upon visfatin stimulation ASMase activity was increased, and an aggregation of ASMase product, ceramide and NADPH oxidase subunits, gp91^phox and p47^phox was observed in the LR clusters, forming a LR redox signaling platform. The formation of this signaling platform was blocked by prior treatment with LR disruptor filipin, ASMase inhibitor amitriptyline, ASMase siRNA, gp91^phox siRNA and adiponectin. Corresponding to LR clustering and aggregation of NADPH subunits, superoxide (O₂ ⁻) production was significantly increased (2.7 folds) upon visfatin stimulation, as measured by electron spin resonance (ESR) spectrometry. Functionally, visfatin significantly increased the permeability of GEC layer in culture and disrupted microtubular networks, which were blocked by inhibition of LR redox signaling platform formation. In conclusion, the injurious effect of visfatin, but not adiponectin on the glomerular endothelium is associated with the formation of LR redox signaling platforms via LR clustering, which produces local oxidative stress resulting in the disruption of microtubular networks in GECs and increases the glomerular permeability.    

3.1551           Synphilin-1 Enhances α-Synuclein Aggregation in Yeast and Contributes to Cellular Stress and Cell Death in a Sir2-Dependent Manner

Büttner, S., Delay, C., Franssens, V., Bammens, T., Ruli, D., Zaunschirm, S., Machado de oliveira, R., Outeiro, T.F., Madeo, F., Buee, L., Galas, M-C. and Winderickx, J. PloSONe, 5(10), e13700 (2010)   Background Parkinson's disease is characterized by the presence of cytoplasmic inclusions, known as Lewy bodies, containing both aggregated α-synuclein and its interaction partner, synphilin-1. While synphilin-1 is known to accelerate inclusion formation by α-synuclein in mammalian cells, its effect on cytotoxicity remains elusive. Methodology/Principal Findings We expressed wild-type synphilin-1 or its R621C mutant either alone or in combination with α-synuclein in the yeast Saccharomyces cerevisiae and monitored the intracellular localization and inclusion formation of the proteins as well as the repercussions on growth, oxidative stress and cell death. We found that wild-type and mutant synphilin-1 formed inclusions and accelerated inclusion formation by α-synuclein in yeast cells, the latter being correlated to enhanced phosphorylation of serine-129. Synphilin-1 inclusions co-localized with lipid droplets and endomembranes. Consistently, we found that wild-type and mutant synphilin-1 interacts with detergent-resistant membrane domains, known as lipid rafts. The expression of synphilin-1 did not incite a marked growth defect in exponential cultures, which is likely due to the formation of aggresomes and the retrograde transport of inclusions from the daughter cells back to the mother cells. However, when the cultures approached stationary phase and during subsequent ageing of the yeast cells, both wild-type and mutant synphilin-1 reduced survival and triggered apoptotic and necrotic cell death, albeit to a different extent. Most interestingly, synphilin-1 did not trigger cytotoxicity in ageing cells lacking the sirtuin Sir2. This indicates that the expression of synphilin-1 in wild-type cells causes the deregulation of Sir2-dependent processes, such as the maintenance of the autophagic flux in response to nutrient starvation. Conclusions/Significance Our findings demonstrate that wild-type and mutant synphilin-1 are lipid raft interacting proteins that form inclusions and accelerate inclusion formation of α-synuclein when expressed in yeast. Synphilin-1 thereby induces cytotoxicity, an effect most pronounced for the wild-type protein and mediated via Sir2-dependent processes.  

3.1552           The Translocon Sec61β Localized in the Inner Nuclear Membrane Transports Membrane-embedded EGF Receptor to the Nucleus

Wang, Y-N., Yamaguchi, H., Huo, L., Lee, H-J., Lee, H-H., Wang, H., Hsu, J-M. and Hung, M-C.

1. Biol. Chem., 285(49), 38720-38729 (2010)

  Accumulating evidence indicates that endocytosis plays an essential role in the nuclear transport of the ErbB family members, such as epidermal growth factor receptor (EGFR) and ErbB-2. Nevertheless, how full-length receptors embedded in the endosomal membrane pass through the nuclear pore complexes and function as non-membrane-bound receptors in the nucleus remains unclear. Here we show that upon EGF treatment, the biotinylated cell surface EGFR is trafficked to the inner nuclear membrane (INM) through the nuclear pore complexes, remaining in a membrane-bound environment. We further find that importin β regulates EGFR nuclear transport to the INM in addition to the nucleus/nucleoplasm. Unexpectedly, the well known endoplasmic reticulum associated translocon Sec61β is found to reside in the INM and associate with EGFR. Knocking down Sec61β expression reduces EGFR level in the nucleoplasm portion and accumulates it in the INM portion. Thus, the Sec61β translocon plays an unrecognized role in the release of the membrane-anchored EGFR from the lipid bilayer of the INM to the nucleus. The newly identified Sec61β function provides an alternative pathway for nuclear transport that can be utilized by membrane-embedded proteins such as full-length EGFR.  

3.1553           MAL/VIP17, a New Player in the Regulation of NKCC2 in the Kidney

Carmosino, M., Rizzo, F., Procino, G., Basco, D., Valenti, G., Forbush, B., Schaere-Wiemers, N., Caplan, M.J. and Svelto, M. Mol. Biol. Cell, 21, 3985-3997 (2010)   The renal-specific Na⁺-K⁺-2Cl^– cotransporter (NKCC2) is the major salt transport pathway of the apical membrane of the mammalian thick ascending limb of Henle's loop. Here, we analyze the role of the tetraspan protein myelin and lymphocytes-associated protein (MAL)/VIP17 in the regulation of NKCC2. We demonstrated that 1) NKCC2 and MAL/VIP17 colocalize and coimmunoprecipitate in Lilly Laboratories cell porcine kidney cells (LLC-PK1) as well as in rat kidney medullae, 2) a 150-amino acid stretch of NKCC2 C-terminal tail is involved in the interaction with MAL/VIP17, 3) MAL/VIP17 increases the cell surface retention of NKCC2 by attenuating its internalization, and 4) this coincides with an increase in cotransporter phosphorylation. Interestingly, overexpression of MAL/VIP17 in the kidney of transgenic mice results in cysts formation in distal nephron structures consistent with the hypothesis that MAL/VIP17 plays an important role in apical sorting or in maintaining the stability of the apical membrane. The NKCC2 expressed in these mice was highly glycosylated and phosphorylated, suggesting that MAL/VIP17 also is involved in the stabilization of NKCC2 at the apical membrane in vivo. Thus, the involvement of MAL/VIP17 in the activation and surface expression of NKCC2 could play an important role in the regulated absorption of Na⁺ and Cl^– in the kidney.  

3.1554           Abl Tyrosine Kinase Phosphorylates Nonmuscle Myosin Light Chain Kinase to Regulate Endothelial Barrier Function

Dudek, S.M., Chiang, E.T., Camp, S.M., Guo, Y., Zhao, J., Brown, M.E., Singleton, P.A., Wang, L., Desai, A., Arce, F.T., Lal, R., Van Eyk, J.E., Imam, S.Z. and Garcia, J.G.N. Mol. Biol. Cell, 21, 4042-4056 (2010)   Nonmuscle myosin light chain kinase (nmMLCK), a multi-functional cytoskeletal protein critical to vascular homeostasis, is highly regulated by tyrosine phosphorylation. We identified multiple novel c-Abl–mediated nmMLCK phosphorylation sites by mass spectroscopy analysis (including Y²³¹, Y⁴⁶⁴, Y⁵⁵⁶, Y⁸⁴⁶) and examined their influence on nmMLCK function and human lung endothelial cell (EC) barrier regulation. Tyrosine phosphorylation of nmMLCK increased kinase activity, reversed nmMLCK-mediated inhibition of Arp2/3-mediated actin polymerization, and enhanced binding to the critical actin-binding phosphotyrosine protein, cortactin. EC challenge with sphingosine 1-phosphate (S1P), a potent barrier-enhancing agonist, resulted in c-Abl and phosphorylated nmMLCK recruitment into caveolin-enriched microdomains, rapid increases in Abl kinase activity, and spatial targeting of c-Abl to barrier-promoting cortical actin structures. Conversely, reduced c-Abl expression in EC (siRNA) markedly attenuated S1P-mediated cortical actin formation, reduced the EC modulus of elasticity (assessed by atomic force microscopy), reduced nmMLCK and cortactin tyrosine phosphorylation, and attenuated S1P-mediated barrier enhancement. These studies indicate an essential role for Abl kinase in vascular barrier regulation via posttranslational modification of nmMLCK and strongly support c-Abl-cortactin-nmMLCK interaction as a novel determinant of cortical actin-based cytoskeletal rearrangement critical to S1P-mediated EC barrier enhancement.  

3.1555           High-molecular-weight hyaluronan is a novel inhibitor of pulmonary vascular leakiness

Singleton, P.AQ., Mirzapoiazova, T., Guo, Y., Sammani, S., Mambetsariev, N., Lennon, F.E., Moreno-Vinasco, L. and Garcia, J.G.N. Am. J. Physiol. Lung Cell. Mol. Physiol., 299, L639-L651 (2010)   Endothelial cell (EC) barrier dysfunction results in increased vascular permeability, a perturbation observed in inflammatory states, tumor angiogenesis, atherosclerosis, and both sepsis and acute lung injury. Therefore, agents that enhance EC barrier integrity have important therapeutic implications. We observed that binding of high-molecular-weight hyaluronan (HMW-HA) to its cognate receptor CD44 within caveolin-enriched microdomains (CEM) enhances human pulmonary EC barrier function. Immunocytochemical analysis indicated that HMW-HA promotes redistribution of a significant population of CEM to areas of cell-cell contact. Quantitative proteomic analysis of CEM isolated from human EC demonstrated HMW-HA-mediated recruitment of cytoskeletal regulatory proteins (annexin A2, protein S100-A10, and filamin A/B). Inhibition of CEM formation [caveolin-1 small interfering RNA (siRNA) and cholesterol depletion] or silencing (siRNA) of CD44, annexin A2, protein S100-A10, or filamin A/B expression abolished HMW-HA-induced actin cytoskeletal reorganization and EC barrier enhancement. To confirm our in vitro results in an in vivo model of inflammatory lung injury with vascular hyperpermeability, we observed that the protective effects of HMW-HA on LPS-induced pulmonary vascular leakiness were blocked in caveolin-1 knockout mice. Furthermore, targeted inhibition of CD44 expression in the mouse pulmonary vasculature significantly reduced HMW-HA-mediated protection from LPS-induced hyperpermeability. These data suggest that HMW-HA, via CD44-mediated CEM signaling events, represents a potentially useful therapeutic agent for syndromes of increased vascular permeability.      

3.1556           Shank2 redistributes with NaPilla during regulated endocytosis

Dobrinskikh, E., Giral, H., Caldas, Y.A., Levi, M and Doctor, R.B. Am. J. Physiol. Cell Physiol., 299, C1324-C1334 (2010)   Serum phosphate levels are acutely impacted by the abundance of sodium-phosphate cotransporter IIa (NaPiIIa) in the apical membrane of renal proximal tubule cells. PSD-95/Disks Large/Zonula Occludens (PDZ) domain-containing proteins bind NaPiIIa and likely contribute to the delivery, retention, recovery, and trafficking of NaPiIIa. Shank2 is a distinctive PDZ domain protein that binds NaPiIIa. Its role in regulating NaPiIIa activity, distribution, and abundance is unknown. In the present in vivo study, rats were maintained on a low-phosphate diet, and then plasma phosphate levels were acutely elevated by high-phosphate feeding to induce the recovery, endocytosis, and degradation of NaPiIIa. Western blot analysis of renal cortical tissue from rats given high-phosphate feed showed NaPiIIa and Shank2 underwent degradation. Quantitative immunofluorescence analyses, including microvillar versus intracellular intensity ratios and intensity correlation quotients, showed that Shank2 redistributed with NaPiIIa during the time course of NaPiIIa endocytosis. Furthermore, NaPiIIa and Shank2 trafficked through distinct endosomal compartments (clathrin, early endosomes, lysosomes) with the same temporal pattern. These in vivo findings indicate that Shank2 is positioned to coordinate the regulated endocytic retrieval and downregulation of NaPiIIa in rat renal proximal tubule cells.  

3.1557           Nuclear angiotensin-(1–7) receptor is functionally coupled to the formation of nitric oxide

Gwatmey, T.M., Westwood, B.M., Pirro, N.T., Tang, L., Rose, J.C., Diz, D.I. and Chappell, M.C. Am. J. Physiol. Renal Physiol., 299, F983-F990 (2010)   The kidney is an important target for the actions of the renin-angiotensin system (RAS) and this tissue contains a complete local RAS that expresses the bioactive peptides angiotensin II (ANG II) and Ang-(1–7). We find both angiotensin type 1 (AT₁R) and type 2 (AT₂R) receptors expressed on renal nuclei that stimulate reactive oxygen species and nitric oxide (NO), respectively. Since Ang-(1–7) also exhibits actions within the kidney and the Ang-(1–7)/Mas receptor protein contains a nuclear localization sequence, we determined the expression of Ang-(1–7) receptors in nuclei isolated from the kidneys of young adult sheep. Binding studies with ¹²⁵I-[Sar¹Thr⁸]-ANG II revealed sites sensitive to the Ang-(1–7) antagonist [D-Ala⁷]-Ang-(1–7) (DALA, A779), as well as to AT₂ and AT₁ antagonists. Incubation of Ang-(1–7) [10^–15 to 10^–9 M] with isolated cortical nuclei elicited a dose-dependent increase in the fluorescence of the NO indicator [4-amino-5-methylamino-2',7']-difluorofluorescein diacetate. The NO response to Ang-(1–7) was abolished by the NO inhibitor N-nitro-L-arginine methyl ester and DALA, but not the AT₁ antagonist losartan or the AT₂ blocker PD123319. Immunofluorescent studies utilizing the Ang-(1–7)/Mas receptor antibody revealed immunolabeling of the proximal tubules but not staining within the glomerulus in cortical sections of the sheep kidney. In the nuclear fraction of isolated proximal tubules, immunoblots revealed the precursor angiotensinogen and renin, as well as functional activity for ACE, ACE2, and neprilysin. We conclude that renal nuclei express Ang-(1–7)/Mas receptors that are functionally linked to NO formation. The marked sensitivity of the intracellular NO response to Ang-(1–7) implicates a functional role of the Ang-(1–7) axis within the nucleus. Moreover, evidence for the precursor and enzymatic components of the RAS within the nuclear compartment of the proximal tubules provides a potential pathway for the intracellular generation of Ang-(1–7).  

3.1558           The Huntington's disease mutation impairs Huntingtin's role in the transport of NF- B from the synapse to the nucleus

Marcora, E. and Kennedy, M.B. Human Mol. Genet., 19(22), 4373-4384 (2010)   Expansion of a polyglutamine (polyQ) tract in the Huntingtin (Htt) protein causes Huntington's disease (HD), a fatal inherited neurodegenerative disorder. Loss of the normal function of Htt is thought to be an important pathogenetic component of HD. However, the function of wild-type Htt is not well defined. Htt is thought to be a multifunctional protein that plays distinct roles in several biological processes, including synaptic transmission, intracellular transport and neuronal transcription. Here, we show with biochemical and live cell imaging studies that wild-type Htt stimulates the transport of nuclear factor κ light-chain-enhancer of activated B cells (NF-κB) out of dendritic spines (where NF-κB is activated by excitatory synaptic input) and supports a high level of active NF-κB in neuronal nuclei (where NF-κB stimulates the transcription of target genes). We show that this novel function of Htt is impaired by the polyQ expansion and thus may contribute to the etiology of HD.  

3.1559           Epithelial septate junction assembly relies on melanotransferrin iron binding and endocytosis in Drosophila

Tiklova, K., Senti, K-A., Wang, S., Gräslund, A. and Samakovlis, C. Nature Cell. Biol., 12(11), 1071-1078 (2010)   Iron is an essential element in many biological processes. In vertebrates, serum transferrin is the major supplier of iron to tissues, but the function of additional transferrin-like proteins remains poorly understood. Melanotransferrin (MTf) is a phylogenetically conserved, iron-binding epithelial protein. Elevated MTf levels have been implicated in melanoma pathogenesis. Here, we present a functional analysis of MTf in Drosophila melanogaster. Similarly to its human homologue, Drosophila MTf is a lipid-modified, iron-binding protein attached to epithelial cell membranes, and is a component of the septate junctions that form the paracellular permeability barrier in epithelial tissues. We demonstrate that septate junction assembly during epithelial maturation relies on endocytosis and apicolateral recycling of iron-bound MTf. Mouse MTf complements the defects of Drosophila MTf mutants. Drosophila provides the first genetic model for the functional dissection of MTf in epithelial junction assembly and morphogenesis.  

3.1560           Oxidative stress increases blood–brain barrier permeability and induces alterations in occludin during hypoxia–reoxygenation

Lochhead, J.J., McCaffrey, G., Quigley, C.E., Finch, J., DeMarco, K.M., Nametz, N. and Davis, T.P.

1. Cerebral Blood Flow & Metab., 30, 1625-1636 (2010)

  The blood–brain barrier (BBB) has a critical role in central nervous system homeostasis. Intercellular tight junction (TJ) protein complexes of the brain microvasculature limit paracellular diffusion of substances from the blood into the brain. Hypoxia and reoxygenation (HR) is a central component to numerous disease states and pathologic conditions. We have previously shown that HR can influence the permeability of the BBB as well as the critical TJ protein occludin. During HR, free radicals are produced, which may lead to oxidative stress. Using the free radical scavenger tempol (200 mg/kg, intraperitoneal), we show that oxidative stress produced during HR (6% O₂ for 1 h, followed by room air for 20 min) mediates an increase in BBB permeability in vivo using in situ brain perfusion. We also show that these changes are associated with alterations in the structure and localization of occludin. Our data indicate that oxidative stress is associated with movement of occludin away from the TJ. Furthermore, subcellular fractionation of cerebral microvessels reveals alterations in occludin oligomeric assemblies in TJ associated with plasma membrane lipid rafts. Our data suggest that pharmacological inhibition of disease states with an HR component may help preserve BBB functional integrity.  

3.1561           Cholesterol Lipids of Borrelia burgdorferi Form Lipid Rafts and Are Required for the Bactericidal Activity of a Complement-Independent Antibody

LaRocca, T.J., Crowley, J.T., Cusack, B.J., Pathak, P., Benach, J., London, E., Garcia-Monco, J.C. and Benach, J.L. Cell Host & Microbe, 8(4), 331-342 (2010)   Borrelia burgdorferi, the agent of Lyme disease, is unusual as it contains free cholesterol and cholesterol glycolipids. It is also susceptible to complement-independent bactericidal antibodies, such as CB2, a monoclonal IgG1 against outer surface protein B (OspB). We find that the bactericidal action of CB2 requires the presence of cholesterol glycolipids and cholesterol. Ultrastructural, biochemical, and biophysical analysis revealed that the bacterial cholesterol glycolipids exist as lipid raft-like microdomains in the outer membrane of cultured and mouse-derived B. burgdorferi and in model membranes from B. burgdorferi lipids. The order and size of the microdomains are temperature sensitive and correlate with the bactericidal activity of CB2. This study demonstrates the existence of cholesterol-containing lipid raft-like microdomains in a prokaryote, and we suggest that the temperature dependence of B. burgdorferi lipid raft organization may have significant implications in the transmission cycle of the spirochetes which are exposed to a range of temperatures.          

3.1562           Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1

Klappe, K., Dijkhuis, A-J., Hummel, I., van Dam, A., Ivanova, P.T., Milne, S.B., Myers, D.S., Brown, H.A., Permentier, H. and Kok, J.W. Biochem. J., 430, 519-529 (2010)   We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the glycerophospholipids shows several differences compared with control, most prominently an increase in highly saturated C₁₆ species. The glycerophospholipid headgroup composition is unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts. Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid raft integrity in two cell lines and does not affect the function of the lipid-raft-associated protein MRP1.  

3.1563           Dilysine retrieval signal-containing p24 proteins collaborate in inhibiting -cleavage of amyloid precursor protein

Hasegawa, H., Liu, L. and Nishimura, M.

1. Neurochem., 115(3), 771-781 (2010)

  γ-Secretase mediates intramembranous -cleavage and ε-cleavage of β-amyloid precursor protein (APP) to liberate β-amyloid peptide (Aβ) and APP intracellular domain respectively from the membrane. Although the regulatory mechanism of -secretase cleavage remains unresolved, a member of the p24 cargo protein family, named p24δ₁ or TMP21, has been identified as an activity-modulating component. The p24 family proteins are divided into four subfamilies (p24 , β, δ and ). In contrast to p24δ₁, p24β₁ has reportedly no effect on -cleavage. In this study, we determined whether p24 ₂, p24 ₃ or p24 ₄ modulates APP processing. Knockdown of cellular p24 ₂ induced a significant increase in Aβ generation but not in APP intracellular domain production in cell-based and cell-free assays, whereas p24 ₂ over-expression suppressed Aβ secretion. By contrast, Aβ secretion was not altered by p24 ₃ or p24 ₄ knockdown. Endogenous p24 ₂ co-immunoprecipitated with core components of the -secretase complex, and the anti-p24 ₂ immunoprecipitate exhibited -secretase activity. Mutational disruption of the conserved dilysine ER-retrieval motifs of p24 ₂ and p24δ₁ perturbed inhibition of -cleavage. Simultaneous knockdown, or co-over-expression, of these proteins had no additive or synergistic effect on Aβ generation. Our findings suggest that dilysine ER-retrieval signal-containing p24 proteins, p24 ₂ and p24δ₁, bind with -secretase complexes and collaborate in attenuating -cleavage of APP.  

3.1564           Subcellular fractionation methods and strategies for proteomics

Lee, Y.H., Tan, H.T. and Chung, M.C.M. Proteomics, 10(22), 3935-3956 (2010)   Developments in subcellular fractionation strategies have provided the means to profile and analyze the protein composition of organelles and cellular structures by proteomics. Here, we review the application of classical (e.g. density gradient centrifugation) and emerging sophisticated techniques (fluorescent-assisted organelle sorting) in the fractionation, and statistical/bioinformatics tools for the prediction of protein localization in subcellular proteomics. We also review the validation methods currently used (such as microscopy, RNA interference and multiple reaction monitoring) and discuss the importance of verification of the results obtained in subcellular proteomics. Finally, the numerous challenges facing subcellular proteomics including the dynamics of organelles are being examined. However, complementary approaches such as modern statistics, bioinformatics and large-scale integrative analysis are beginning to emerge as powerful tools to proteomics for analyzing subcellular organelles and structures.    

3.1565           Organelle proteomics experimental designs and analysis

Gatto, L., Vizcaino, J.A., Hermjacob, H., Huber, W. and Lilley, K.S. Proteomics, 10(22), 3957-3969 (2010)   In biology, localisation is function: knowledge of the localisation of proteins is of paramount importance to assess and study their function. This supports the need for reliable protein sub-cellular localisation assignment. Concomitant with recent technological advances in organelle proteomics, there is a requirement for more rigorous experimental and analysis design planning and description. In this review, we present an overview of current experimental designs in qualitative and quantitative organelle proteomics as well as associated data analysis. We also consider the major benefits associated with careful description and dissemination of the experiment and analysis designs, namely (i) comparison and optimisation of experimental designs and analysis pipelines, (ii) data validation, (iii) reproducible research, (iv) efficient repository submission and retrieval and (v) meta analysis. Formalisation of experimental design and analysis work flows is of direct benefit for the organelle proteomics researchers and will result in providing organelle localisation data of highest quality for the wider research community.  

3.1566           Molecular characterization of the endoplasmic reticulum: Insights from proteomic studies

Chen, X., Karnovsky, A., Sans, M.D., Andrews, P.C. and Williams, J.A. Proteomics, 10(22), 4040-4052 (2010)   The endoplasmic reticulum (ER) is a multifunctional intracellular organelle responsible for the synthesis, processing and trafficking of a wide variety of proteins essential for cell growth and survival. Therefore, comprehensive characterization of the ER proteome is of great importance to the understanding of its functions and has been actively pursued in the past decade by scientists in the proteomics field. This review summarizes major proteomic studies published in the past decade that focused on the ER proteome. We evaluate the data sets obtained from two different organs, liver and pancreas each of which contains a primary cell type (hepatocyte and acinar cell) with specialized functions. We also discuss how the nature of the proteins uncovered is related to the methods of organelle purification, organelle purity and the techniques used for protein separation prior to MS. In addition, this review also puts emphasis on the biological insights gained from these studies regarding the molecular functions of the ER including protein synthesis and translocation, protein folding and quality control, ER-associated degradation and ER stress, ER export and membrane trafficking, calcium homeostasis and detoxification and drug metabolism.  

3.1567           The proteome of lysosomes

Schröder, B.A., Wrocklage, C., Hasilik, A. and Saftig, P. Proteomics, 10(22), 4053-4076 (2010)   Lysosomes are organelles of eukaryotic cells that are critically involved in the degradation of macromolecules mainly delivered by endocytosis and autophagocytosis. Degradation is achieved by more than 60 hydrolases sequestered by a single phospholipid bilayer. The lysosomal membrane facilitates interaction and fusion with other compartments and harbours transport proteins catalysing the export of catabolites, thereby allowing their recycling. Lysosomal proteins have been addressed in various proteomic studies that are compared in this review regarding the source of material, the organelle/protein purification scheme, the proteomic methodology applied and the proteins identified. Distinguishing true constituents of an organelle from copurifying contaminants is a central issue in subcellular proteomics, with additional implications for lysosomes as being the site of degradation of many cellular and extracellular proteins. Although many of the lysosomal hydrolases were identified by classical biochemical approaches, the knowledge about the protein composition of the lysosomal membrane has remained fragmentary for a long time. Using proteomics many novel lysosomal candidate proteins have been discovered and it can be expected that their functional characterisation will help to understand functions of lysosomes at a molecular level that have been characterised only phenomenologically so far and to generally deepen our understanding of this indispensable organelle.  

3.1568           Analysis of phagosomal proteomes: From latex-bead to bacterial phagosomes

Li, Q., Jagannath, C., Rao, P.K., Singh, C.R. and Lostumba, G. Proteomics, 10(22), 4098-4116 (2010)   Phagosomal proteome characterization has contributed significantly to the understanding of host–pathogen interaction and the mechanism of infectious diseases caused by intracellular bacteria. The latex bead-containing phagosome has been widely used as a model system to study phagosomal proteomes at a global level. In contrast, the study of bacteria-containing phagosomes at a similar level has just begun. A number of intracellular microbial species are studied for their proteomes during the invasion of a host, providing insight into their metabolic adaptation in host cells and interaction with host-cell antimicrobial environments. In this review, we attempt to summarize the most recent advancements in the proteomic study of microbial phagosomes, especially those originating from mouse or human cells. We also briefly describe the proteomics of latex bead-containing phagosomes because they are often used as model phagosomes for study. We provide descriptions on major biological and technological components in phagosomal proteome studies. We also discuss the role of phagosomal proteome study in the broader horizon of systems biology and the technological challenges in phagosomal proteome characterization.  

3.1569           Localization and Activation of the Drosophila Protease Easter Require the ER-Resident Saposin-like Protein Seele

Stein, D., Charatsi, I., Cho, Y.S., Zhang, Z., Nguyen, J., DeLotto, R., Luschnig, S. and Moussian, B. Current Biol., 20(21), 1953-1958 (2010)   Drosophila embryonic dorsal-ventral polarity is generated by a series of serine protease processing events in the egg perivitelline space. Gastrulation Defective processes Snake, which then cleaves Easter, which then processes Spätzle into the activating ligand for the Toll receptor [1,2,3]. seele was identified in a screen for mutations that, when homozygous in ovarian germline clones, lead to the formation of progeny embryos with altered embryonic patterning; maternal loss of seele function leads to the production of moderately dorsalized embryos [4]. By combining constitutively active versions of Gastrulation Defective, Snake, Easter, and Spätzle with loss-of-function alleles of seele, we find that Seele activity is dispensable for Spätzle-mediated activation of Toll but is required for Easter, Snake, and Gastrulation Defective to exert their effects on dorsal-ventral patterning. Moreover, Seele function is required specifically for secretion of Easter from the developing embryo into the perivitelline space and for Easter processing. Seele protein resides in the endoplasmic reticulum of blastoderm embryos, suggesting a role in the trafficking of Easter to the perivitelline space, prerequisite to its processing and function. Easter transport to the perivitelline space represents a previously unappreciated control point in the signal transduction pathway that controls Drosophila embryonic dorsal-ventral polarity.  

3.1570           Stimulation of apical Cl⁻/HCO₃⁻(OH⁻) exchanger, SLC26A3 by neuropeptide Y is lipid raft dependent

Saksena, S., Tyagi, S., Goyal, S., Gill, R.K., Alrefai, W.A., Ramaswamy, A.K. and Dudeja, P.K. Am. J. Physiol. Gastrintest. Liver Physiol., 299, G1334-G1343 (2010)   Neuropeptide Y (NPY), an important proabsorptive hormone of the gastrointestinal tract has been shown to inhibit chloride secretion and stimulate NaCl absorption. However, mechanisms underlying the proabsorptive effects of NPY are not fully understood. The present studies were designed to examine the direct effects of NPY on apical Cl⁻/HCO₃⁻(OH⁻) exchange activity and the underlying mechanisms involved utilizing Caco2 cells. Our results showed that NPY (100 nM, 30 min) significantly increased Cl⁻/HCO₃⁻(OH⁻) exchange activity (∼2-fold). Selective NPY/Y1 or Y2 receptor agonists mimicked the effects of NPY. NPY-mediated stimulation of Cl⁻/HCO₃⁻(OH⁻) exchange activity involved the ERK1/2 MAP kinase-dependent pathway. Cell surface biotinylation studies showed that NPY does not alter DRA (apical Cl⁻/HCO₃⁻(OH⁻) exchanger) surface expression, ruling out the involvement of membrane trafficking events. Interestingly, DRA was found to be predominantly expressed in the detergent-insoluble (DI) and low-density fractions (LDF) of human colonic apical membrane vesicles (AMVs) representing lipid rafts. Depletion of membrane cholesterol by methyl-β-cyclodextrin (MβCD, 10 mM, 1 h) remarkably decreased DRA expression in the DI fractions. Similar results were obtained in Triton-X 100-treated Caco2 plasma membranes. DRA association with lipid rafts in the DI and LDF fractions of Caco2 cells was significantly enhanced (∼45%) by NPY compared with control. MβCD significantly decreased Cl⁻/HCO₃⁻(OH⁻) exchange activity in Caco2 cells as measured by DIDS- or niflumic acid-sensitive ³⁶Cl⁻ uptake (∼50%). Our results demonstrate that NPY modulates Cl⁻/HCO₃⁻(OH⁻) exchange activity by enhancing the association of DRA with lipid rafts, thereby resulting in an increase in Cl⁻/HCO₃⁻(OH⁻) exchange activity. Our findings suggest that the alteration in the association of DRA with lipid rafts may contribute to the proabsorptive effects of NPY in the human intestine.    

3.1571           Immunospecific Responses to Bacterial Elongation Factor Tu during Burkholderia Infection and Immunization

Nieves, W., Heang, J., Asakrah, S., Höner zu Bentrup, K., Roy, C.J. and Morici, L.A. PloSOne, 5(12), e14361 (2010)   Burkholderia pseudomallei is the etiological agent of melioidosis, a disease endemic in parts of Southeast Asia and Northern Australia. Currently there is no licensed vaccine against infection with this biological threat agent. In this study, we employed an immunoproteomic approach and identified bacterial Elongation factor-Tu (EF-Tu) as a potential vaccine antigen. EF-Tu is membrane-associated, secreted in outer membrane vesicles (OMVs), and immunogenic during Burkholderia infection in the murine model of melioidosis. Active immunization with EF-Tu induced antigen-specific antibody and cell-mediated immune responses in mice. Mucosal immunization with EF-Tu also reduced lung bacterial loads in mice challenged with aerosolized B. thailandensis. Our data support the utility of EF-Tu as a novel vaccine immunogen against bacterial infection.  

3.1572           The Role of Glutamate Release on Voltage-Dependent Anion Channels (VDAC)-Mediated Apoptosis in an Eleven Vessel Occlusion Model in Rats

Park, E., Lee, G-J., Choi, S.., Choi, S-K., Chae, S-J., Kang, S-W., Pak, Y.K. and Park, H-K. PloSOne, 5(12), e15192 (2010)   Voltage-dependent anion channel (VDAC) is the main protein in mitochondria-mediated apoptosis, and the modulation of VDAC may be induced by the excessive release of extracellular glutamate. This study examined the role of glutamate release on VDAC-mediated apoptosis in an eleven vessel occlusion model in rats. Male Sprague-Dawley rats (250–350 g) were used for the 11 vessel occlusion ischemic model, which were induced for a 10-min transient occlusion. During the ischemic and initial reperfusion episode, the real-time monitoring of the extracellular glutamate concentration was measured using an amperometric microdialysis biosensor and the cerebral blood flow (CBF) was monitored by laser-Doppler flowmetry. To confirm neuronal apoptosis, the brains were removed 72 h after ischemia to detect the neuron-specific nuclear protein and pro-apoptotic proteins (cleaved caspase-3, VDAC, p53 and BAX). The changes in the mitochondrial morphology were measured by atomic force microscopy. A decrease in the % of CBF was observed, and an increase in glutamate release was detected after the onset of ischemia, which continued to increase during the ischemic period. A significantly higher level of glutamate release was observed in the ischemia group. The increased glutamate levels in the ischemia group resulted in the activation of VDAC and pro-apoptotic proteins in the hippocampus with morphological alterations to the mitochondria. This study suggests that an increase in glutamate release promotes VDAC-mediated apoptosis in an 11 vessel occlusion ischemic model.  

3.1573           Calcium Ionophore-Induced Tissue Factor (TF) Decryption Induces TF Immobilization Into Lipid Rafts and Negative Regulation of TF Procoagulant Activity

Popescu, N.I., Lupu, C. and Lupu, F. Blood, 116, Abstract 1131 (2010)   Cell exposed tissue factor (TF), the physiologic initiator of blood coagulation, is normally expressed in a low procoagulant, or cryptic conformation, and requires activation, or decryption, to fully exhibit its procoagulant potential. TF decryption is not fully understood and multiple decrypting mechanisms have been proposed including phosphatidylserine (PS) exposure, TF monomerization, association with lipid rafts and redox modulation of TF. Calcium ionophores have been extensively used as TF decrypting agents, and both PS-dependent and independent mechanisms have been associated with ionophore-induced TF decryption. In the present study we analyzed the changes that occur in the lateral mobility of cell exposed TF during calcium ionophore-induced decryption, using a TF chimera with monomeric yellow fluorescent protein (YFP-TF). The YFP-TF expressed by endothelial cells (EC) retains TF procoagulant activity, is mainly exposed on the cell surface and can be decrypted similarly with endogenous TF by the calcium ionophore ionomycin. We analyzed the changes in TF membrane mobility during decryption using live cell imaging of YFP-TF expressed in EC. Fluorescence recovery after photobleaching (FRAP) analysis revealed a decreased mobility of TF in EC treated with the decrypting agent ionomycin. The YFP-TF fluorescence in the region of interest was more easily bleached in ionomycin–treated cells as compared with controls. The observed maximum recovery (R_max) of YFP-TF fluorescence in the bleached region of interest was significantly higher in control cells (80.84% recovery) as compared with ionomycin treated EC (39.29% recovery). These correlated with a decrease in YFP-TF mobile fraction from 50% for the control cells to 18% for the ionomycin treated EC. The lateral diffusion of the YFP-TF mobile fraction was similar between the two conditions, with halftime of fluorescence recovery of 7.69 sec in ionophore-treated cells and 10.69 sec in controls. These results suggest an immobilization of YFP-TF during decryption, which can be achieved by either lipid raft translocation or cytoskeleton floating. Similar to previous observations where TF cytoplasmic domain did not influence TF decryption, deletion of the TF cytoplasmic domain did not affect the lateral mobility of YFP-TF in FRAP analysis. To analyze decryption-induced changes in TF association with lipid domains, membrane fractions were isolated on a discontinuous Opti-Prep density gradient. Ionomycintreatment induced YFP-TF translocation from higher density,non-raft membrane fractions toward higher-buoyancy, raft fractions.Furthermore, the observed TF translocation into lipid raftsoccurs without the formation of the quaternary complex withcoagulation factors FVIIa, FXa and tissue factor pathway inhibitor(TFPI), as previously described. To address the functional modulationof TF procoagulant potential in response to lipid raft translocation,cell membrane cholesterol was either depleted with methyl-β-cyclodextrine(MβCD) or supplemented from an aqueous mixture of cholesterol-MβCD.Membrane cholesterol depletion decrypted TF in EC, likely throughPS exposure, while also enhancing the procoagulant potentialof ionomycin-decrypted TF. In contrast, cholesterol supplementationdecreases the procoagulant potential of ionomycin-decryptedTF. Taken together, these observations support the model oftonic inhibition of TF procoagulant activity by the lipid raftenvironment. In conclusion, by live cell imaging we show thatTF membrane mobility changes during calcium-ionophore induceddecryption resulting in an immobilization of TF in lipid rafts.The immobilization is not influenced by the cytoplasmic domainof TF and does not require the formation of the TF-FVIIa-FXa-TFPIquaternary complex. Translocation into lipid rafts providestonic inhibition of TF procoagulant potential and, as a consequence,we show for the first time that decrypting agents can also initiatenegative regulation of TF procoagulant function. This negativefeedback loop may help convert the decrypted TF back to itscryptic, low coagulant form.  

3.1574           Naturally Produced Outer Membrane Vesicles from Pseudomonas aeruginosa Elicit a Potent Innate Immune Response via Combined Sensing of Both Lipopolysaccharide and Protein Components

Ellis, T.N., Leiman, S.A. and Kuehn, M.J. Infect. Immun., 78(9), 3822-3831 (2010)   Pseudomonas aeruginosa is a prevalent opportunistic human pathogen that, like other Gram-negative pathogens, secretes outer membrane vesicles. Vesicles are complex entities composed of a subset of envelope lipid and protein components that have been observed to interact with and be internalized by host cells. This study characterized the inflammatory responses to naturally produced P. aeruginosa vesicles and determined the contribution of vesicle Toll-like receptor (TLR) ligands and vesicle proteins to that response. Analysis of macrophage responses to purified vesicles by real-time PCR and enzyme-linked immunosorbent assay identified proinflammatory cytokines upregulated by vesicles. Intact vesicles were shown to elicit a profoundly greater inflammatory response than the response to purified lipopolysaccharide (LPS). Both TLR ligands LPS and flagellin contributed to specific vesicle cytokine responses, whereas the CpG DNA content of vesicles did not. Neutralization of LPS sensing demonstrated that macrophage responses to the protein composition of vesicles required the adjuvantlike activity of LPS to elicit strain specific responses. Protease treatment to remove proteins from the vesicle surface resulted in decreased interleukin-6 and tumor necrosis factor alpha production, indicating that the production of these specific cytokines may be linked to macrophage recognition of vesicle proteins. Confocal microscopy of vesicle uptake by macrophages revealed that vesicle LPS allows for binding to macrophage surfaces, whereas vesicle protein content is required for internalization. These data demonstrate that macrophage sensing of both LPS and protein components of outer membrane vesicles combine to produce a bacterial strain-specific response that is distinct from those triggered by individual, purified vesicle components.  

3.1575           Role of Lysyl Oxidase Propeptide in Secretion and Enzyme Activity

Grimsby, J.L., Lucero, H.A., Trackman, P.C., Ravid, K. and Kagan, H.M.

1. Cell. Biochem., 111, 1231-1243 (2010)

  Lysyl oxidase (LOX) is secreted as a proenzyme (proLOX) that is proteolytically processed in the extracellular milieu to release the propeptide and mature, active LOX. LOX oxidizes lysyl residues of a number of protein substrates in the extracellular matrix and on the cell surface, which impacts several physiological and disease states. Although the LOX propeptide (LOX-PP) is glycosylated, little is known about the role of this modification in LOX secretion and activity. To gain insight into this issue, cells were transfected with native, full-length LOX cDNA (pre-pro-LOX), the N-glycosylation null pre-[N/Q]pro-LOX cDNA and the deletion mutant pre-LOX cDNA, referred to as secretory LOX, in which mature LOX is targeted to the secretory pathway without its N-terminal propeptide sequence. The results show that glycosylation of the LOX-PP is not required for secretion and extracellular processing of pro-LOX but it is required for optimal enzyme activity of the resulting mature LOX. Complete deletion of the propeptide sequence prevents mature LOX from exiting the endoplasmic reticulum (ER). Taken together, our study points out the requirement of the LOX-PP for pro-LOX exit from the ER and is the first to highlight the influence of LOX-PP glycosylation on LOX enzyme activity.  

3.1576           Improved sub-cellular resolution via simultaneous analysis of organelle proteomics data across varied experimental conditions

Trotter, M.W.B., Sadowski, P.G., Dunkley, T.P.J., Groen, A.J. and Lilley, K.S. Proteomics, 10(23), 4213-4219 (2010)   Spatial organisation of proteins according to their function plays an important role in the specificity of their molecular interactions. Emerging proteomics methods seek to assign proteins to sub-cellular locations by partial separation of organelles and computational analysis of protein abundance distributions among partially separated fractions. Such methods permit simultaneous analysis of unpurified organelles and promise proteome-wide localisation in scenarios wherein perturbation may prompt dynamic re-distribution. Resolving organelles that display similar behavior during a protocol designed to provide partial enrichment represents a possible shortcoming. We employ the Localisation of Organelle Proteins by Isotope Tagging (LOPIT) organelle proteomics platform to demonstrate that combining information from distinct separations of the same material can improve organelle resolution and assignment of proteins to sub-cellular locations. Two previously published experiments, whose distinct gradients are alone unable to fully resolve six known protein–organelle groupings, are subjected to a rigorous analysis to assess protein–organelle association via a contemporary pattern recognition algorithm. Upon straightforward combination of single-gradient data, we observe significant improvement in protein–organelle association via both a non-linear support vector machine algorithm and partial least-squares discriminant analysis. The outcome yields suggestions for further improvements to present organelle proteomics platforms, and a robust analytical methodology via which to associate proteins with sub-cellular organelles.  

3.1577           Oxysterol Binding Protein-dependent Activation of Sphingomyelin Synthesis in the Golgi Apparatus Requires Phosphatidylinositol 4-Kinase II

Banerji, S., Ngo, M., Lane, C.F., Robinson, C-A., Minogue, S. and Ridgway, N.D. Mol. Biol. Cell, 21, 4141-4150 (2010)   Cholesterol and sphingomyelin (SM) associate in raft domains and are metabolically coregulated. One aspect of coordinate regulation occurs in the Golgi apparatus where oxysterol binding protein (OSBP) mediates sterol-dependent activation of ceramide transport protein (CERT) activity and SM synthesis. Because CERT transfer activity is dependent on its phosphatidylinositol 4 phosphate [PtdIns(4)P]-specific pleckstrin homology domain, we investigated whether OSBP activation of CERT involved a Golgi-associated PtdIns 4-kinase (PI4K). Cell fractionation experiments revealed that Golgi/endosome-enriched membranes from 25-hydroxycholesterol-treated Chinese hamster ovary cells had increased activity of a sterol-sensitive PI4K that was blocked by small interfering RNA silencing of OSBP. Consistent with this sterol-requirement, OSBP silencing also reduced the cholesterol content of endosome/trans-Golgi network (TGN) fractions containing PI4KII . PI4KII , but not PI4KIIIβ, was required for oxysterol-activation of SM synthesis and recruitment of CERT to the Golgi apparatus. However, neither PI4KII nor PI4KIIIβ expression was required for 25-hydroxycholesterol–dependent translocation of OSBP to the Golgi apparatus. The presence of OSBP, CERT, and PI4KII in the TGN of oxysterol-stimulated cells suggests that OSBP couples sterol binding or transfer activity with regulation of PI4KII activity, leading to CERT recruitment to the TGN and increased SM synthesis.  

3.1578           Reduced levels of folate transporters (PCFT and RFC) in membrane lipid rafts result in colonic folate malabsorption in chronic alcoholism

Wani, N.A. and Kaur, J. Cell. Physiol., 226, 579-587 (2011)   We studied the effect of chronic ethanol ingestion on folate transport across the colonic apical membranes (CAM) in rats. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20%) solution orally for 3 months and folate transport was studied in the isolated colon apical membrane vesicles. The folate transport was found to be carrier mediated, saturable, with pH optima at 5.0. Chronic ethanol ingestion reduced the folate transport across the CAM by decreasing the affinity of transporters (high Km) for the substrate and by decreasing the number of transporter molecules (low Vmax) on the colon luminal surface. The decreased transport activity at the CAM was associated with down-regulation of the proton-coupled folate transporter (PCFT) and the reduced folate carrier (RFC) which resulted in decreased PCFT and RFC protein levels in the colon of rats fed alcohol chronically. Moreover, the PCFT and the RFC were found to be distributed in detergent insoluble fraction of the CAM in rats. Floatation experiments on Optiprep density gradients demonstrated the association of the PCFT and the RFC protein with lipid rafts (LR). Chronic alcoholism decreased the PCFT and the RFC protein levels in the CAM LR in accordance with the decreased synthesis. Hence, we propose that downregulation in the expression of the PCFT and the RFC in colon results in reduced levels of these transporters in colon apical membrane LR as a mechanism of folate malabsorption during chronic alcoholism.  

3.1579           The Tethering Arm of the EGF Receptor Is Required for Negative Cooperativity and Signal Transduction

Adak, S., DeAndrade, D. and Pike, L.J.

1. Biol. Chem., 286(2), 1545-1555 (2011)

  The EGF receptor is a classical receptor-tyrosine kinase. In the absence of ligand, the receptor adopts a closed conformation in which the dimerization arm of subdomain II interacts with the tethering arm in subdomain IV. Following the binding of EGF, the receptor opens to form a symmetric, back-to-back dimer. Although it is clear that the dimerization arm of subdomain II is central to the formation of receptor dimers, the role of the tethering arm of subdomain IV (residues 561–585) in this configuration is not known. Here we use ¹²⁵I-EGF binding studies to assess the functional role of the tethering arm in the EGF receptor dimer. Mutation of the three major residues that contribute to tethering (D563A,H566A,K585A-EGF receptor) did not significantly alter either the ligand binding properties or the signaling properties of the EGF receptor. By contrast, breaking the Cys⁵⁵⁸-Cys⁵⁶⁷ disulfide bond through double alanine replacements or deleting the loop entirely led to a decrease in the negative cooperativity in EGF binding and was associated with small changes in downstream signaling. Deletion of the Cys⁵⁷¹-Cys⁵⁹³ disulfide bond abrogated cooperativity, resulting in a high affinity receptor and increased sensitivity of downstream signaling pathways to EGF. Releasing the Cys⁵⁷¹-Cys⁵⁹³ disulfide bond resulted in extreme negative cooperativity, ligand-independent kinase activity, and impaired downstream signaling. These data demonstrate that the tethering arm plays an important role in supporting cooperativity in ligand binding. Because cooperativity implies subunit-subunit interactions, these results also suggest that the tethering arm contributes to intersubunit interactions within the EGF receptor dimer.  

3.1580           Differential roles for SUR subunits in KATP channel membrane targeting and regulation

Hund, T.J. and Mohler, P.J. Am. J. Physiol. Heart Circ. Physiol., 300, H33-H35 (2011)   No abstracts available.  

3.1581           Native IL-32 is released from intestinal epithelial cells via a non-classical secretory pathway as a membrane-associated protein

Hasegawa, H., Thomas, H.J., Schooley, K. and Born, T.L. Cytokine, 53, 74-83 (2011)   Although IL-32 has been shown to be induced under various pathological conditions, a detailed understanding of native IL-32 intracellular distribution and mechanism of release from cells has not been reported. We examined the expression of IL-32 in the intestinal epithelial cell line HT-29 following TNFα and IFNγ co-stimulation. The subcellular localization of induced IL-32 was associated with the membrane of lipid droplet-like structures and vacuolar structures that co-localized with markers of endosomes and lysosomes. Prolonged co-stimulation resulted in cell death and appearance of IL-32 in the culture medium. IL-32 released from co-stimulated HT-29 cells was found in a detergent-sensitive particulate fraction, and in a step density gradient the IL-32 particulate was buoyant, suggesting association with a membrane-bound vesicle. Upon Triton X-114 partitioning, most of the IL-32 partitioned to the detergent phase, suggesting hydrophobic characteristics. When IL-32-containing vesicles were subjected to protease K treatment, a protease resistant 12 kDa fragment was generated from 24 kDa IL-32. We propose that under these conditions, native IL-32 is released via a non-classical secretory route perhaps involving multi-vesicular bodies and exosomes. Demonstration of membrane association for both intracellular and released IL-32 suggests this unique cytokine may have a complex biosynthetic pathway and mechanism of action.  

3.1582           Reconfiguring polylysine architectures for controlling polyplex binding and non-viral transfection

Parelkar, S.S., Chan-Seng, D. and Emrick, T. Biomaterials, 32, 2432-2444 (2011)   Poly(l-lysine) (PLL) is a cationic polyelectrolyte of interest for many applications, including in therapeutic biology for DNA complexation and transfection. Several non-lysine based polycations have been shown to afford more efficient transfection in live cells than has been achieved with PLL. We find that reconfiguring polylysine into short oligolysine grafts, strung from a hydrophobic polymer backbone, gives transfection reagents greatly superior to PLL, despite having the identical cationic functional groups (i.e., exclusively primary amines). Altering the oligolysine graft length modulates DNA-polymer interactions and transfection efficiency, while incorporating the PKKKRKV heptapeptide (the Simian virus SV40 large T-antigen nuclear localization sequence) pendent groups onto the polymer backbone led to even greater transfection efficiency over the oligolysine-grafted structures. Protein expression levels obtained with these novel polymer transfection reagents were higher than, or comparable to, expression seen in the cases of JetPEI™, FuGENE® 6 and Lipofectamine™ 2000, the later being notorious for cytotoxicity that accompanies high transfection efficiency. The relative strength of the polymer-DNA complex is key to the transfection performance, as judged by serum stability and PicoGreen analysis. Moreover, polyplexes formed from our graft copolymer structures exhibit low cytotoxicity, contributing to the therapeutic promise of these novel reagents.  

3.1583           Purification and characterization of HIV–human protein complexes

Jäger, S., Gulbache, N., Cimermancic, P., Kane, J., He, N., Chou, S., D’Orso, I., Fernandes, J., Jang, G., Frankel, A.D., Alber, T. and Zhou, Q. Methods, 53, 13-19 (2011)   To fully understand how pathogens infect their host and hijack key biological processes, systematic mapping of intra-pathogenic and pathogen–host protein–protein interactions (PPIs) is crucial. Due to the relatively small size of viral genomes (usually around 10–100 proteins), generation of comprehensive host–virus PPI maps using different experimental platforms, including affinity tag purification-mass spectrometry (AP-MS) and yeast two-hybrid (Y2H) approaches, can be achieved. Global maps such as these provide unbiased insight into the molecular mechanisms of viral entry, replication and assembly. However, to date, only two-hybrid methodology has been used in a systematic fashion to characterize viral–host protein–protein interactions, although a deluge of data exists in databases that manually curate from the literature individual host–pathogen PPIs. We will summarize this work and also describe an AP-MS platform that can be used to characterize viral-human protein complexes and discuss its application for the HIV genome.  

3.1584           Immunization with Salmonella enterica Serovar Typhimurium-Derived Outer Membrane Vesicles Delivering the Pneumococcal Protein PspA Confers Protection against Challenge with Streptococcus pneumoniae

Muralinath, M., Kuehn, M.J., Roland, K.L. and Curtiss III, R. Infect. Immun., 79(2), 887-894 (2011)   Gram-negative bacteria produce outer membrane vesicles (OMVs) that serve a variety of functions related to survival and pathogenicity. Periplasmic and outer membrane proteins are naturally captured during vesicle formation. This property has been exploited as a method to derive immunogenic vesicle preparations for use as vaccines. In this work, we constructed a Salmonella enterica serovar Typhimurium strain that synthesized a derivative of the pneumococcal protein PspA engineered to be secreted into the periplasmic space. Vesicles isolated from this strain contained PspA in the lumen. Mice intranasally immunized with the vesicle preparation developed serum antibody responses against vesicle components that included PspA and Salmonella-derived lipopolysaccharide and outer membrane proteins, while no detectable responses developed in mice immunized with an equivalent dose of purified PspA. Mucosal IgA responses developed against the Salmonella components, while the response to PspA was less apparent in most mice. Mice immunized with the vesicle preparation were completely protected against a 10x 50% lethal dose (LD50) challenge of Streptococcus pneumoniae and significantly protected against a 200x LD50 challenge, while control mice immunized with purified PspA or empty vesicles were not protected. These results establish that vesicles can be used to mucosally deliver an antigen from a Gram-positive organism and induce a protective immune response.  

3.1585           αB-Crystallin Is Found in Detergent-resistant Membrane Microdomains and Is Secreted via Exosom es from Human Retinal Pigment Epithelial

Gangalum, R., Atanasov, I.C., Zhou,, Z.H. and Bhat, S.P.

1. Biol. Chem., 286(5), 3261-3269 (2011)

  αB-crystallin (αB) is known as an intracellular Golgi membrane-associated small heat shock protein. Elevated levels of this protein have been linked with a myriad of neurodegenerative pathologies including Alzheimer disease, multiple sclerosis, and age-related macular degeneration. The membrane association of αB has been known for more than 3 decades, yet its physiological import has remained unexplained. In this investigation we show that αB is secreted from human adult retinal pigment epithelial cells via microvesicles (exosomes), independent of the endoplasmic reticulum-Golgi protein export pathway. The presence of αB in these lipoprotein structures was confirmed by its susceptibility to digestion by proteinase K only when exosomes were exposed to Triton X-100. Transmission electron microscopy was used to localize αB in immunogold-labeled intact and permeabilized microvesicles. The saucer-shaped exosomes, with a median diameter of 100–200 nm, were characterized by the presence of flotillin-1, α-enolase, and Hsp70, the same proteins that associate with detergent-resistant membrane microdomains (DRMs), which are known to be involved in their biogenesis. Notably, using polarized adult retinal pigment epithelial cells, we show that the secretion of αB is predominantly apical. Using OptiPrep gradients we demonstrate that αB resides in the DRM fraction. The secretion of αB is inhibited by the cholesterol-depleting drug, methyl β-cyclodextrin, suggesting that the physiological function of this protein and the regulation of its export through exosomes may reside in its association with DRMs/lipid rafts.  

3.1586           Fc RIIIb Triggers Raft-dependent Calcium Influx in IgG-mediated Responses in Human Neutrophils

Marois, L., Pare, G., Vaillancourt, M., Rolelt-labelle, E. and Naccache, P.H.

1. Biol. Chem., 286(5), 3509-3519 (2011)

  Human neutrophils constitutively express a unique combination of FcγRs, namely FcγRIIa and FcγRIIIb. Numerous lines of evidence support the concept that these FcγRs generate only partially characterized intracellular signals. However, despite the fact that both receptors are likely to be engaged simultaneously in a physiological setting, no recent publications have investigated the distinct, although partially convergent, results of their joint activation in IgG-dependent responses. To examine the significance of the co-expression of FcγRIIa and FcγRIIIb on human neutrophils, we analyzed the neutrophil responses to stimuli that engage these FcγRs, namely the phagocytosis of human IgG-opsonized zymosan and the responses to heat-aggregated IgGs. Blocking antibodies to either FcγR significantly decreased the phagocytic index and the stimulated production of superoxide anions. Both receptors are required for optimal IgG-dependent responses by human neutrophils. On the other hand, only blocking antibodies to FcγRIIIb, but not to FcγRIIa, inhibited the mobilization of calcium in response to heat-aggregated IgGs. Furthermore, phagocytosis of IgG-opsonized zymosan by human neutrophils required an extracellular influx of calcium that was blocked only by antibodies against FcγRIIIb. We also observed that this calcium influx as well as the IgG-dependent phagocytosis were dependent on the integrity of the plasma membrane detergent-resistant microdomains to which both isoforms were recruited following stimulation by heat-aggregated IgGs. These data clarify the mechanisms that regulate the FcγRs constitutively expressed on human neutrophils, describe a specific contribution of FcγRIIIb at the level of the mobilization of calcium, and provide evidence for a crucial role of detergent-resistant microdomains in this process.  

3.1587           Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1 receptosomes

Edelmann, B., Bertsch, U., Tchikov, V., Winoto-Morbach, S., Perrotta, C., Jakob, M., Adam-Klages, S., Kabelitz, D. and Schütze, S. EMBO J., 30(2), 379-394 (2011)   We previously demonstrated that tumour necrosis factor (TNF)-induced ceramide production by endosomal acid sphingomyelinase (A-SMase) couples to apoptosis signalling via activation of cathepsin D and cleavage of Bid, resulting in caspase-9 and caspase-3 activation. The mechanism of TNF-mediated A-SMase activation within the endolysosomal compartment is poorly defined. Here, we show that TNF-induced A-SMase activation depends on functional caspase-8 and caspase-7 expression. The active forms of all three enzymes, caspase-8, caspase-7 and A-SMase, but not caspase-3, colocalize in internalized TNF receptosomes. While caspase-8 and caspase-3 are unable to induce activation of purified pro-A-SMase, we found that caspase-7 mediates A-SMase activation by direct interaction resulting in proteolytic cleavage of the 72-kDa pro-A-SMase zymogen at the non-canonical cleavage site after aspartate 253, generating an active 57 kDa A-SMase molecule. Caspase-7 down modulation revealed the functional link between caspase-7 and A-SMase, confirming proteolytic cleavage as one further mode of A-SMase activation. Our data suggest a signalling cascade within TNF receptosomes involving sequential activation of caspase-8 and caspase-7 for induction of A-SMase activation by proteolytic cleavage of pro-A-SMase.  

3.1588           The phosphoinositide 3-kinase Vps34p is required for pexophagy in Saccharomyces cerevisiae

Grunau, S., Lay, D., Mindthoff, S., Platta, H.W., Girzalsky, W., Just, W.W. and Erdmann, R. Biochem. J., 434, 161-170 (2011)   PIds (phosphoinositides) are phosphorylated derivatives of the membrane phospholipid PtdIns that have emerged as key regulators of many aspects of cellular physiology. We have discovered a PtdIns3P-synthesizing activity in peroxisomes of Saccharomyces cerevisiae and have demonstrated that the lipid kinase Vps34p is already associated with peroxisomes during biogenesis. However, although Vps34 is required, it is not essential for optimal peroxisome biogenesis. The function of Vps34p-containing complex I as well as a subset of PtdIns3P-binding proteins proved to be mandatory for the regulated degradation of peroxisomes. This demonstrates that PtdIns3P-mediated signalling is required for pexophagy.  

3.1589           An RNA-zipcode-independent mechanism that localizes Dia1 mRNA to the perinuclear ER through interactions between Dia1 nascent peptide and Rho–GTP

Liao, G., ma, X. and Liu, G.

1. Cell Sci., 124, 589-599 (2011)

  Signal-peptide-mediated ER localization of mRNAs encoding for membrane and secreted proteins, and RNA-zipcode-mediated intracellular targeting of mRNAs encoding for cytosolic proteins are two well-known mechanisms for mRNA localization. Here, we report a previously unidentified mechanism by which mRNA encoding for Dia1, a cytosolic protein without the signal peptide, is localized to the perinuclear ER in an RNA-zipcode-independent manner in fibroblasts. Dia1 mRNA localization is also independent of the actin and microtubule cytoskeleton but requires translation and the association of Dia1 nascent peptide with the ribosome–mRNA complex. Sequence mapping suggests that interactions of the GTPase binding domain of Dia1 peptide with active Rho are important for Dia1 mRNA localization. This mechanism can override the β-actin RNA zipcode and redirect β-actin mRNA to the perinuclear region, providing a new way to manipulate intracellular mRNA localization.  

3.1590           Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled -subunits

Valkova, C., Albrizio, M., Röder, I.V., Schwake, M., Betto, R., Rudolf, R. and Kaether, C. PNAS, 108(2), 621-625 (2011)   The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor α-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled α-subunits to the early secretory pathway.        

3.1591           A role for oxysterol-binding protein–related protein 5 in endosomal cholesterol trafficking

Du, X., Kumar, J., Ferguson, C., Schulz, T.A., Ong, Y.S., Hong, W., Prinz, W.A., Parton, R.G., Brown, A.J. and Yang, H.

1. Cell Biol., 192(1), 121-135 (2011)

  Oxysterol-binding protein (OSBP) and its related proteins (ORPs) constitute a large and evolutionarily conserved family of lipid-binding proteins that target organelle membranes to mediate sterol signaling and/or transport. Here we characterize ORP5, a tail-anchored ORP protein that localizes to the endoplasmic reticulum. Knocking down ORP5 causes cholesterol accumulation in late endosomes and lysosomes, which is reminiscent of the cholesterol trafficking defect in Niemann Pick C (NPC) fibroblasts. Cholesterol appears to accumulate in the limiting membranes of endosomal compartments in ORP5-depleted cells, whereas depletion of NPC1 or both ORP5 and NPC1 results in luminal accumulation of cholesterol. Moreover, trans-Golgi resident proteins mislocalize to endosomal compartments upon ORP5 depletion, which depends on a functional NPC1. Our results establish the first link between NPC1 and a cytoplasmic sterol carrier, and suggest that ORP5 may cooperate with NPC1 to mediate the exit of cholesterol from endosomes/lysosomes.  

3.1592           Regulation of vascular endothelial growth factor receptor 2 trafficking and angiogenesis by Golgi localized t-SNARE syntaxin 6

Manickam, V., Tiwari, A., Jung, J-J., Bhattacharya, R., Goel, A., Mukhopadhyay, D. and Choudhury, A. Blood, 117(4), 1425-1435 (2011)   Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in physiologic and pathologic angiogenesis. Plasma membrane (PM) levels of VEGFR2 are regulated by endocytosis and secretory transport through the Golgi apparatus. To date, the mechanism whereby the VEGFR2 traffics through the Golgi apparatus remains incompletely characterized. We show in human endothelial cells that binding of VEGF to the cell surface localized VEGFR2 stimulates exit of intracellular VEGFR2 from the Golgi apparatus. Brefeldin A treatment reduced the level of surface VEGFR2, confirming that VEGFR2 traffics through the Golgi apparatus en route to the PM. Mechanistically, we show that inhibition of syntaxin 6, a Golgi-localized target membrane-soluble N-ethylmaleimide attachment protein receptor (t-SNARE) protein, interferes with VEGFR2 trafficking to the PM and facilitates lysosomal degradation of the VEGFR2. In cell culture, inhibition of syntaxin 6 also reduced VEGF-induced cell proliferation, cell migration, and vascular tube formation. Furthermore, in a mouse ear model of angiogenesis, an inhibitory form of syntaxin 6 reduced VEGF-induced neovascularization and permeability. Our data demonstrate the importance of syntaxin 6 in the maintenance of cellular VEGFR2 levels, and suggest that the inhibitory form of syntaxin 6 has good potential as an antiangiogenic agent.  

3.1593           Effector granules in human T lymphocytes: the luminal proteome of secretory lysosomes from human T cells

Schmidt, H., Gelhaus, C., Nebendahl, M., Lettau, M., Lucius, R., Leippe, M., Kabelitz, D. and Janssen, O. Cell Comm. And Singnalling, 9, 4-18 (2011)   Background Cytotoxic cells of the immune system have evolved a lysosomal compartment to store and mobilize effector molecules. In T lymphocytes and NK cells, the death factor FasL is one of the characteristic marker proteins of these so-called secretory lysosomes, which combine properties of conventional lysosomes and exocytotic vesicles. Although these vesicles are crucial for immune effector function, their protein content in T cells has so far not been investigated in detail. Results In the present study, intact membranous vesicles were enriched from homogenates of polyclonally activated T cells and initially characterized by Western blotting and electron microscopic inspection. The vesicular fraction that contained the marker proteins of secretory lysosomes was subsequently analyzed by 2D electrophoresis and mass spectrometry. The proteome analysis and data evaluation revealed that 70% of the 397 annotated proteins had been associated with different lysosome-related organelles in previous proteome studies. Conclusion We provide the first comprehensive proteome map of T cell-derived secretory lysosomes with only minor contaminations by cytosolic, nuclear or other proteins. This information will be useful to more precisely address the activation-dependent maturation and the specific distribution of effector organelles and proteins in individual T or NK cell populations in future studies.  

3.1594           Reverse Engineering Gene Network Identifies New Dysferlin-interacting Proteins

Cacciottolo, M., Belcastro, v., Laval, S., Bushby, K., di Bernardo, D. and Nigro, v.

1. Biol. Chem., 286(7), 5404-5413 (2011)

  Dysferlin (DYSF) is a type II transmembrane protein implicated in surface membrane repair of muscle. Mutations in dysferlin lead to Limb Girdle Muscular Dystrophy 2B (LGMD2B), Miyoshi Myopathy (MM), and Distal Myopathy with Anterior Tibialis onset (DMAT). The DYSF protein complex is not well understood, and only a few protein-binding partners have been identified thus far. To increase the set of interacting protein partners for DYSF we recovered a list of predicted interacting protein through a systems biology approach. The predictions are part of a “reverse-engineered” genome-wide human gene regulatory network obtained from experimental data by computational analysis. The reverse-engineering algorithm behind the analysis relates genes to each other based on changes in their expression patterns. DYSF and AHNAK were used to query the system and extract lists of potential interacting proteins. Among the 32 predictions the two genes share, we validated the physical interaction between DYSF protein with moesin (MSN) and polymerase I and transcript release factor (PTRF) in mouse heart lysate, thus identifying two novel Dysferlin-interacting proteins. Our strategy could be useful to clarify Dysferlin function in intracellular vesicles and its implication in muscle membrane resealing.  

3.1595           Folding, Quality Control, and Secretion of Pancreatic Ribonuclease in Live Cells

Geiger, R., Gautschi, M., Thor, F., Hayer, A. and Helenius, A.

1. Biol. Chem., 286(7), 5813-5822 (2011)

  Although bovine pancreatic RNase is one of the best characterized proteins in respect to structure and in vitro refolding, little is known about its synthesis and maturation in the endoplasmic reticulum (ER) of live cells. We expressed the RNase in live cells and analyzed its folding, quality control, and secretion using pulse-chase analysis and other cell biological techniques. In contrast to the slow in vitro refolding, the protein folded almost instantly after translation and translocation into the ER lumen (t_½ < 3 min). Despite high stability of the native protein, only about half of the RNase reached a secretion competent, monomeric form and was rapidly transported from the rough ER via the Golgi complex (t_½ = 16 min) to the extracellular space (t_½ = 35 min). The rest remained in the ER mainly in the form of dimers and was slowly degraded. The dimers were most likely formed by C-terminal domain swapping since mutation of Asn¹¹³, a residue that stabilizes such dimers, to Ser increased the efficiency of secretion from 59 to 75%. Consistent with stringent ER quality control in vivo, the secreted RNase in the bovine pancreas was mainly monomeric, whereas the enzyme present in the cells also contained 20% dimers. These results suggest that the efficiency of secretion is not only determined by the stability of the native protein but by multiple factors including the stability of secretion-incompetent side products of folding. The presence of N-glycans had little effect on the folding and secretion process.  

3.1596           Calsenilin is degraded by the ubiquitin–proteasome pathway

Jang, C., Choi, J-K., Kim, E., Park, E-S., Wasco, W., Buxbaum, J.D., Kim, Y-S. and Choi, E-K. Biochem. Biophys. Res. Comm., 405, 180-185 (2011)   Calsenilin, a neuronal calcium binding protein that has been shown to have multiple functions in the cell, interacts with presenilin 1 (PS1) and presenilin 2 (PS2), represses gene transcription and binds to A-type voltage-gated potassium channels. In addition, increased levels of calsenilin are observed in the brains of Alzheimer’s disease and epilepsy patients. The present study was designed to investigate the molecular mechanism of calsenilin degradation pathways in cultured cells. Here, we demonstrate that inhibition of the ubiquitin–proteasomal pathway (UPP) but not lysosomal pathway markedly increased the expression levels of calsenilin. Immunofluorescence analysis revealed that following proteasomal inhibition calsenilin accumulated in the endoplasmic reticulum (ER) and Golgi, while lysosomal inhibition had no effect on calsenilin localization. In addition, we found the change of subcellular localization of PS1 from diffuse pattern to punctuate staining pattern in the ER and perinuclear region in the presence of calsenilin. These findings suggest that calsenilin degradation is primarily mediated by the UPP and that impairment in the UPP may contribute to the involvement of calsenilin in disease-associated neurodegeneration.  

3.1597           Porcine Sialoadhesin (CD169/Siglec-1) Is an Endocytic Receptor that Allows Targeted Delivery of Toxins and Antigens to MacrophagesSialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that sialoadhesin is an endocytic receptor. In this report, we show that porcine sialoadhesin-specific antibodies and F(ab')₂ fragments trigger sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the sialoadhesin endocytosis mechanism, two sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine sialoadhesin-specific immunotoxins efficiently kill sialoadhesin-expressing macrophages. Furthermore, porcine sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand sialoadhesin functionality and show that it can function as an endocytic receptor, a feature that cannot only be misused by sialic acid carrying pathogens, but that may also be used for specific targeting of toxins or antigens to sialoadhesin-expressing macrophages.

Delputte, P.L., Van Gorp, H., Favoreel, H.W., Hoebeke, I., Delrue, I., Dewerchin, H., Verdonck, F., Verhasselt, B., Cox, E. and Nauwynck, H.J. PloSOne, 6(2), e16827 (2011)   Sialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that sialoadhesin is an endocytic receptor. In this report, we show that porcine sialoadhesin-specific antibodies and F(ab')₂ fragments trigger sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the sialoadhesin endocytosis mechanism, two sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine sialoadhesin-specific immunotoxins efficiently kill sialoadhesin-expressing macrophages. Furthermore, porcine sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand sialoadhesin functionality and show that it can function as an endocytic receptor, a feature that cannot only be misused by sialic acid carrying pathogens, but that may also be used for specific targeting of toxins or antigens to sialoadhesin-expressing macrophages.  

3.1598           Glucocorticoid-Induced Fetal Programming Alters the Functional Complement of Angiotensin Receptor Subtypes Within the Kidney

Gwathmey, T.M., Shaltout, H.A., Rose, J.C., Diz, D.I. and Chappell, M.C. Hypertension, 57(2), 620-626 (2011)   We examined the impact of fetal programming on the functional responses of renal angiotensin receptors. Fetal sheep were exposed in utero to betamethasone (BMX; 0.17 mg/kg) or control (CON) at 80 to 81 days gestation with full-term delivery. Renal nuclear and plasma membrane fractions were isolated from sheep age 1.0 to 1.5 years for receptor binding and fluorescence detection of reactive oxygen species (ROS) or nitric oxide (NO). Mean arterial blood pressure and blood pressure variability were significantly higher in the BMX-exposed adult offspring versus CON sheep. The proportion of nuclear AT₁ receptors sensitive to losartan was 2-fold higher (67±6% vs 27±9%; P<0.01) in BMX compared with CON. In contrast, the proportion of AT₂ sites was only one third that of controls (BMX, 25±11% vs CON, 78±4%; P<0.01), with a similar reduction in sites sensitive to the Ang-(1-7) antagonist D-Ala7-Ang-(1-7) with BMX exposure. Functional studies revealed that Ang II stimulated ROS to a greater extent in BMX than in CON sheep (16±3% vs 6±4%; P<0.05); however, NO production to Ang II was attenuated in BMX (26±7% vs 82±14%; P<0.05). BMX exposure was also associated with a reduction in the Ang-(1-7) NO response (75±8% vs 131±26%; P<0.05). Weconclude that altered expression of angiotensin receptor subtypesmay be one mechanism whereby functional changes in NO- and ROS-dependentsignaling pathways may favor the sustained increase in bloodpressure evident in fetal programming.  

3.1599           Multidrug Resistance-Related Protein 1 (MRP1) Function and Localization Depend on Cortical Actin

Hummel, I., Klappe, K., Ercan, C. and Kok, J.W. Mol. Pharmacol., 79(2), 229-240 (2011)   MRP1 (ABCC1) is known to be localized in lipid rafts. Here we show in two different cell lines that localization of Mrp1/MRP1 (Abcc1/ABCC1) in lipid rafts and its function as an efflux pump are dependent on cortical actin. Latrunculin B disrupts both cortical actin and actin stress fibers. This results in partial loss of actin and Mrp1/MRP1 (Abcc1/ABCC1) from detergent-free lipid raft fractions, partial internalization of Mrp1/MRP1 (Abcc1/ABCC1), and reduction of Mrp1/MRP1 (Abcc1/ABCC1)-mediated efflux. Pretreatment with nocodazole prevents latrunculin B-induced loss of cortical actin and all effects of latrunculin B on Mrp1 (Abcc1) localization and activity. However, pretreatment with tyrphostin A23 does not prevent latrunculin B-induced loss of cortical actin, lipid raft association, and efflux activity, but it does prevent latrunculin B-induced internalization of Mrp1 (Abcc1). Cytochalasin D disrupts actin stress fibers but not cortical actin and this inhibitor much less affects Mrp1/MRP1 (Abcc1/ABCC1) localization in lipid rafts, internalization, and efflux activity. In conclusion, cortical actin disruption results in reduced Mrp1/MRP1 (Abcc1/ABCC1) activity concomitant with a partial shift of Mrp1/MRP1 (Abcc1/ABCC1) out of lipid raft fractions and partial internalization of the ABC transporter. The results suggest that reduced Mrp1 (Abcc1) function is correlated to the loss of lipid raft association but not internalization of Mrp1 (Abcc1).  

3.1600           Advances in membranous vesicle and exosome proteomics improving biological understanding and biomarker discovery

Raimondo, F., Morosi, L., Chinello, C., Magni, F. and Pitto, M. Proteomics, 11(4), 709-720 (2011)   Exosomes are membranous vesicles released by cells in extracellular fluids: they have been found and analyzed in blood, urine, amniotic fluid, breast milk, seminal fluid, saliva and malignant effusions, besides conditioned media from different cell lines. Several recent papers show that exosome proteomes of different origin include both a common set of membrane and cytosolic proteins, and specific subsets of proteins, likely correlated to cell-type associated functions. This is particularly interesting in relation to their possible involvement in human diseases. The knowledge of exosome proteomics can help not only in understanding their biological roles but also in supplying new biomarkers to be searched for in patients' fluids. This review offers an overview of technical and analytical issues in exosome proteomics, and it highlights the significance of proteomic studies in terms of biological and clinical usefulness.  

3.1601           Recent advances in the use of Sus scrofa (pig) as a model system for proteomic studies

Verma, N., Rettenmeier, A.W. and Schmitz-Spanke, S. Proteomics, 11(4), 776-793 (2011)   Of the numerous animal models available for proteomic studies only a small number have been successfully used in understanding human biology. To date, rodents have been widely employed in proteomic and genomic studies but often these models do not truly mimic the relevant human conditions. On the other hand, the pig shows similarity in size, shape and physiology to human and has been used as a major mammalian model for many studies concerning xenotransplantation, cardiovascular diseases, blood dynamics, nutrition, general metabolic functions, digestive-related disorders, respiratory diseases, diabetes, kidney and bladder diseases, organ-specific toxicity, dermatology and neurological sequelae. With the substantially improved knowledge of the structure and function of the pig genome in the last two decades it has been found that this animal shares a high sequence and chromosomal structure homology with humans. Nevertheless, in comparison to other available model organisms, very little work has been devoted to pig proteomics until recently. Keeping this in mind, the present review will highlight some of the advantages and disadvantages of pig as a model system for proteomic studies.  

3.1602           Cannabinoid 1 receptor-dependent transactivation of fibroblast growth factor receptor 1 emanates from lipid rafts and amplifies extracellular signal-regulated kinase 1/2 activation in embryonic cortical neurons

Asimaki, O., Leondaritis, G., Lois, G., Sakellaridis, N. and Mangoura, D.

1. Neurochem., 116(5), 866-873 (2011)

  G-protein coupled receptors may mediate their effects on neuronal growth and differentiation through activation of extracellular signal-regulated kinases 1/2 (ERK1/2), often elicited by transactivation of growth factor receptor tyrosine kinases. This elaborate signaling process includes inducible formation and trafficking of multiprotein signaling complexes and is facilitated by pre-ordained membrane microdomains, in particular lipid rafts. In this study, we have uncovered novel signaling interactions of cannabinoid receptors with fibroblast growth factor receptors, which depended on lipid rafts and led to ERK1/2 activation in primary neurons derived from chick embryo telencephalon. More specifically, the cannabinoid 1 receptor (CB1R) agonist methanandamide induced tyrosine phosphorylation and transactivation of fibroblast growth factor receptor (FGFR)1 via Src and Fyn, which drove an amplification wave in ERK1/2 activation. Transactivation of FGFR1 was accompanied by the formation of a protein kinase C ε-dependent multiprotein complex that included CB1R, Fyn, Src, and FGFR1. Recruitment of molecules increased with time of exposure to methanandamide, suggesting that in addition to signaling it also served trafficking of receptors. Upon agonist stimulation we also detected a rapid incorporation of CB1R, as well as activated Src and Fyn, and FGFR1 in lipid rafts. Most importantly, lipid raft integrity was a pre-requisite for CB1R-dependent complex formation. Our data provide evidence that lipid rafts may organize CB1 receptor proximal signaling events, namely activation of Src and Fyn, and transactivation of FGFR1 towards activation of ERK1/2 and induction of neuronal differentiation.  

3.1603           Evidence for functional links between the Rgd1-Rho3 RhoGAP-GTPase module and Tos2, a protein involved in polarized growth in Saccharomyces cerevisiae

Claret, S., Roumanie, O., Prouzet-Mauleon, V., lefebre, F., Thoraval, D., Crouzet, M. and Doignon, F. FEMS Yeast Res., 11(2), 179-191 (2011)   The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Two-hybrid screening identified Tos2p as a candidate Rgd1p-binding protein. Further analyses confirmed that Tos2p binds to the RhoGAP Rgd1p through its C-terminal region. Both Tos2p and Rgd1p are localized to polarized growth sites during the cell cycle and associated with detergent-resistant membranes. We observed that TOS2 overexpression suppressed rgd1Δ sensitivity to a low pH. In the tos2Δ strain, the amount of GTP-bound Rho3p was increased, suggesting an influence of Tos2p on Rgd1p activity in vivo. We also showed a functional interaction between the TOS2 and the RHO3 genes: TOS2 overexpression partially suppressed the growth defect of rho3-V51 cells at a restrictive temperature. We propose that Tos2p, a protein involved in polarized growth and most probably associated with the plasma membrane, modulates the action of Rgd1p and Rho3p in S. cerevisiae.  

3.1604           Interaction of the Tobacco Lectin with Histone Proteins

Schouppe, D., Ghesquiere, B., Menschaert, G., De Vos, W.H., Bourque, S., Trooskens, G., Proost, P., Gevaert, K. and Van Damme, E.J.M. Plant Physiol., 155, 1091-1102 (2011)   The tobacco (Nicotiana tabacum) agglutinin or Nictaba is a member of a novel class of plant lectins residing in the nucleus and the cytoplasm of tobacco cells. Since tobacco lectin expression is only observed after the plant has been subjected to stress situations such as jasmonate treatment or insect attack, Nictaba is believed to act as a signaling protein involved in the stress physiology of the plant. In this paper, a nuclear proteomics approach was followed to identify the binding partners for Nictaba in the nucleus and the cytoplasm of tobacco cv Xanthi cells. Using lectin affinity chromatography and pull-down assays, it was shown that Nictaba interacts primarily with histone proteins. Binding of Nictaba with histone H2B was confirmed in vitro using affinity chromatography of purified calf thymus histone proteins on a Nictaba column. Elution of Nictaba-interacting histone proteins was achieved with 1 M N-acetylglucosamine (GlcNAc). Moreover, mass spectrometry analyses indicated that the Nictaba-interacting histone proteins are modified by O-GlcNAc. Since the lectin-histone interaction was shown to be carbohydrate dependent, it is proposed that Nictaba might fulfill a signaling role in response to stress by interacting with O-GlcNAcylated proteins in the plant cell nucleus.  

3.1605           The Minimal Proteome in the Reduced Mitochondrion of the Parasitic Protist Giardia intestinalis

Jedelsky, P.L., Dolezal, P., Rada, P., Pyrih, J., Smid, O., Hrdy, I., Sedinova, M., Marcincikova, M., Voleman, L., Perry, A.J., Beltran, N.C., Lithgow, TS. And Tachezy, J. PloSOne, 6(2), e17285 (2011)   The mitosomes of Giardia intestinalis are thought to be mitochondria highly-reduced in response to the oxygen-poor niche. We performed a quantitative proteomic assessment of Giardia mitosomes to increase understanding of the function and evolutionary origin of these enigmatic organelles. Mitosome-enriched fractions were obtained from cell homogenate using Optiprep gradient centrifugation. To distinguish mitosomal proteins from contamination, we used a quantitative shot-gun strategy based on isobaric tagging of peptides with iTRAQ and tandem mass spectrometry. Altogether, 638 proteins were identified in mitosome-enriched fractions. Of these, 139 proteins had iTRAQ ratio similar to that of the six known mitosomal markers. Proteins were selected for expression in Giardia to verify their cellular localizations and the mitosomal localization of 20 proteins was confirmed. These proteins include nine components of the FeS cluster assembly machinery, a novel diflavo-protein with NADPH reductase activity, a novel VAMP-associated protein, and a key component of the outer membrane protein translocase. None of the novel mitosomal proteins was predicted by previous genome analyses. The small proteome of the Giardia mitosome reflects the reduction in mitochondrial metabolism, which is limited to the FeS cluster assembly pathway, and a simplicity in the protein import pathway required for organelle biogenesis.  

3.1606           The Small GTPase RhoA Localizes to the Nucleus and Is Activated by Net1 and DNA Damage Signals

Dubash,, A.D., Guilluy, C., Srougi, M.C., Boulter, E., Burridge, K. and Garcia-Mata, R. PloSOne, 6(2), e17380 (2011)   Background Rho GTPases control many cellular processes, including cell survival, gene expression and migration. Rho proteins reside mainly in the cytosol and are targeted to the plasma membrane (PM) upon specific activation by guanine nucleotide exchange factors (GEFs). Accordingly, most GEFs are also cytosolic or associated with the PM. However, Net1, a RhoA-specific GEF predominantly localizes to the cell nucleus at steady-state. Nuclear localization for Net1 has been seen as a mechanism for sequestering the GEF away from RhoA, effectively rendering the protein inactive. However, considering the prominence of nuclear Net1 and the fact that a biological stimulus that promotes Net1 translocation out the nucleus to the cytosol has yet to be discovered, we hypothesized that Net1 might have a previously unidentified function in the nucleus of cells. Principal Findings Using an affinity precipitation method to pulldown the active form of Rho GEFs from different cellular fractions, we show here that nuclear Net1 does in fact exist in an active form, contrary to previous expectations. We further demonstrate that a fraction of RhoA resides in the nucleus, and can also be found in a GTP-bound active form and that Net1 plays a role in the activation of nuclear RhoA. In addition, we show that ionizing radiation (IR) specifically promotes the activation of the nuclear pool of RhoA in a Net1-dependent manner, while the cytoplasmic activity remains unchanged. Surprisingly, irradiating isolated nuclei alone also increases nuclear RhoA activity via Net1, suggesting that all the signals required for IR-induced nuclear RhoA signaling are contained within the nucleus. Conclusions/Significance These results demonstrate the existence of a functional Net1/RhoA signaling pathway within the nucleus of the cell and implicate them in the DNA damage response.  

3.1607           Myelination in the absence of UDP-galactose:ceramide galactosyl-transferase and fatty acid 2 –hydroxylase

Meixner, M., Jungnixkel, J., Grothe, C., Gieselmann, V. and Eckhardt, M. BMC Neuroscience, 12, 22-31 (2011)   Background The sphingolipids galactosylceramide (GalCer) and sulfatide are major myelin components and are thought to play important roles in myelin function. The importance of GalCer and sulfatide has been validated using UDP-galactose:ceramide galactosyltransferase-deficient (Cgt^-/-) mice, which are impaired in myelin maintenance. These mice, however, are still able to form compact myelin. Loss of GalCer and sulfatide in these mice is accompanied by up-regulation of 2-hydroxylated fatty acid containing (HFA)-glucosylceramide in myelin. This was interpreted as a partial compensation of the loss of HFA-GalCer, which may prevent a more severe myelin phenotype. In order to test this hypothesis, we have generated Cgt^-/- mice with an additional deletion of the fatty acid 2-hydroxylase (Fa2h) gene. Results Fa2h^-/-/Cgt^-/- double-deficient mice lack sulfatide, GalCer, and in addition HFA-GlcCer and sphingomyelin. Interestingly, compared to Cgt^-/- mice the amount of GlcCer in CNS myelin was strongly reduced in Fa2h^-/-/Cgt^-/- mice by more than 80%. This was accompanied by a significant increase in sphingomyelin, which was the predominant sphingolipid in Fa2h^-/-/Cgt^-/- mice. Despite these significant changes in myelin sphingolipids, compact myelin was formed in Fa2h^-/-/Cgt^-/- mice, and g-ratios of myelinated axons in the spinal cord of 4-week-old Fa2h^-/-/Cgt^-/- mice did not differ significantly from that of Cgt^-/- mice, and there was no obvious phenotypic difference between Fa2h^-/-/Cgt^-/- and Cgt^-/- mice Conclusions These data show that compact myelin can be formed with non-hydroxylated sphingomyelin as the predominant sphingolipid and suggest that the presence of HFA-GlcCer and HFA-sphingomyelin in Cgt^-/- mice does not functionally compensate the loss of HFA-GalCer.  

3.1608           Norepinephrine Deficiency Is Caused by Combined Abnormal mRNA Processing and Defective Protein Trafficking of Dopamine β-Hydroxylase

Kim, C-H., Leung, A., Huh, Y.H., Yang, E., Kim, D-J., leblanc, P., Ryu, H., Kim, K., Kim, D-W., Garland, E.M., Raj, S.R., Biaggioni, I., Robertson, D. and Kim, K-S.

1. Biol. Chem., 286(11), 9196-9204 (2011)

  Human norepinephrine (NE) deficiency (or dopamine β-hydroxylase (DBH) deficiency) is a rare congenital disorder of primary autonomic failure, in which neurotransmitters NE and epinephrine are undetectable. Although potential pathogenic mutations, such as a common splice donor site mutation (IVS1+2T→C) and various missense mutations, in NE deficiency patients were identified, molecular mechanisms underlying this disease remain unknown. Here, we show that the IVS1+2T→C mutation results in a non-detectable level of DBH protein production and that all three missense mutations tested lead to the DBH protein being trapped in the endoplasmic reticulum (ER). Supporting the view that mutant DBH induces an ER stress response, exogenous expression of mutant DBH dramatically induced expression of BiP, a master ER chaperone. Furthermore, we found that a pharmacological chaperone, glycerol, significantly rescued defective trafficking of mutant DBH proteins. Taken together, we propose that NE deficiency is caused by the combined abnormal processing of DBH mRNA and defective protein trafficking and that this disease could be treated by a pharmacological chaperone(s).  

3.1609           Affinity of PIP-aquaporins to sterol-enriched domains in plasma membrane of the cells of etiolated pea seedlings

Belugin, B.V., Zhestkova, I.M. and Trofimova, M.S. Membrane and Cell Biology, 5(1), 56-63 (2011)   The hypothesis that sterol-enriched domains represent sites of preferred localization of PIP-aquaporins was tested in experiments on plasma membranes isolated from cells of etiolated pea (Pisum sativum L.) seedlings. Plasma membranes were isolated from microsomes by the partition in the aqueous two-phase polymer system and separated into vesicle fractions of different buoyant density by flotation in discontinuous OptiPrep gradient. Two types of plasma membrane preparations were used: one was treated with cold 1% Triton X-100 and the other was not. In untreated preparations, three populations of plasma membrane vesicles were obtained, while in the case of treated preparations, fractions of detergent-resistant membranes (DRM) and solubilized membrane proteins were obtained. In all membrane fractions collected after OptiPrep flotation, the amounts of proteins, sterols, and PIP-aquaporins were determined. The highest sterol content was detected in the membrane fraction with buoyant density 1.098 g/cm³ and in the DRM fraction (1.146 g/cm³). These fractions contained much more PIP-aquaporins than the other ones. Phase state of the lipid bilayer was determined by measuring generalized polarization excitation of fluorescence (GPEX) of laurdan incorporated into the membranes of different fractions. It was revealed that the lipid bilayer of the membranes with density of 1.098 g/cm³ had a higher extent of ordering than that of the fractions with density of ∼1.146 g/cm³. The results indicated that uppermost local concentrations of PIP-aquaporins were associated with tightly packed sterol-enriched domains. Moreover, upon solubilization of plasma membrane with Triton X-100, PIP-aquaporins mainly resided in DRM, thus exhibiting a high affinity to sterols.  

3.1610           Proteomic Analysis of the Pancreatic Islet β-Cell Secretory Granule: Current Understanding and Future Opportunities

Cooper, G.J.S. Systems Biol., 2(3), 327-362 (2011)   The pancreatic islet β-cell granule has been the subject of intense study for decades, in part because it serves as the vehicle for the regulated secretion of insulin and amylin , through which it exerts regulation of metabolism. β-cell granule proteins have been closely linked to disease mechanisms in both major types of diabetes, and recent findings from genome-wide association studies have reinforced the importance of these linkages for understanding disease mechanisms . Granule proteins have also proven to be of major interest in pharmaceutics, since two of them, insulin and amylin , have each served as the basis for the development of anti-diabetic pharmacotherapies . In spite of all the attention this enigmatic granule has received to date, many fundamental questions about its molecular structure and function remain unanswered. In the past few years, high-resolution methodologies have begun to unravel the granule proteome in ever-increasing detail. Emerging data complement the results from the other approaches that have been applied to understand the granule. This chapter will explore the current state of knowledge in the field and the implications of emerging proteomic data for the study of physiological processes and disease mechanisms in diabetes.  

3.1611           Adenosine receptors and membrane microdomains

Laskey, R.D. Biochim. Biophys. Acta, 1808, 1284-1289 (2011)   Adenosine receptors are a member of the large family of seven transmembrane spanning G protein coupled receptors. The four adenosine receptor subtypes—A1, A2a, A2b, A3—exert their effects via the activation of one or more heterotrimeric G proteins resulting in the modulation of intracellular signaling. Numerous studies over the past decade have documented the complexity of G protein coupled receptor signaling at the level of protein–protein interactions as well as through signaling cross talk. With respect to adenosine receptors, the activation of one receptor subtype can have profound direct effects in one cell type but little or no effect in other cells. There is significant evidence that the compartmentation of subcellular signaling plays a physiological role in the fidelity of G protein coupled receptor signaling. This compartmentation is evident at the level of the plasma membrane in the form of membrane microdomains such as caveolae and lipid rafts. This review will summarize and critically assess our current understanding of the role of membrane microdomains in regulating adenosine receptor signaling.    

3.1612           A Point Mutation in the Amino Terminus of TLR7 Abolishes Signaling without Affecting Ligand Binding

Iavarone, C., Ramsauer, K., Kubarenko, A.V., Debasitis, J.C., Leukin, I., Weber, A.N.R., Siggs, O.M., Beutler, B., Zhang, P., Otten, g., D’Oro, U., Valiante, N.M., Mbow, M.L. and Vissintin, A.

1. Immunol., 186, 4213-4222 (2011)

  TLR7 is the mammalian receptor for ssRNA and some nucleotide-like small molecules. We have generated a mouse by N-nitrose-N′-ethyl urea mutagenesis in which threonine 68 of TLR7 was substituted with isoleucine. Cells bearing this mutant TLR7 lost the sensitivity to the small-molecule TLR7 agonist resiquimod, hence the name TLR7^rsq1. In this work, we report the characterization of this mutant protein. Similar to the wild-type counterpart, TLR7^rsq1 localizes to the endoplasmic reticulum and is expressed at normal levels in both primary cells and reconstituted 293T cells. In addition to small-molecule TLR7 agonists, TLR7^rsq1 fails to be activated by ssRNA. Whole-transcriptome analysis demonstrates that TLR7 is the exclusive and indispensable receptor for both classes of ligands, consistent with the fact that both ligands induce highly similar transcriptional signatures in TLR7^wt/wt splenocytes. Thus, TLR7^rsq1 is a bona fide phenocopy of the TLR7 null mouse. Because TLR7^rsq1 binds to ssRNA, our studies imply that the N-terminal portion of TLR7 triggers a yet to be identified event on TLR7. TLR7^rsq1 mice might represent a valuable tool to help elucidate novel aspects of TLR7 biology.  

3.1613           Reciprocal Control of hERG Stability by Hsp70 and Hsc70 With Implication for Restoration of LQT2 Mutant Stability

Li, P.L., Ninomiya, H., Kurata, Y., Kato, M., Miake, J., Yamamoto, Y., Igawa, O., Nakai, A., Higaki, K., Toyoda, F., Wu, J., Horie, M., Matsuura, H., Yoshida, A., Shirayoshi, Y., Hiraoka, M. and Hisatome, I. Circ. Res., 108, 458-468 (2011)   Rationale:The human ether-a-go-go–related gene (hERG) encodes the subunit of the potassium current I_Kr. It is highly expressedin cardiomyocytes and its mutations cause long QT syndrome type2. Heat shock protein (Hsp)70 is known to promote maturationof hERG. Hsp70 and heat shock cognate (Hsc70) 70 has been suggestedto play a similar function. However, Hsc70 has recently beenreported to counteract Hsp70. Objective:We investigated whether Hsc70 counteracts Hsp70 in the controlof wild-type and mutant hERG stability. Methods and Results:Coexpression of Hsp70 with hERG in HEK293 cells suppressed hERG ubiquitination and increased the levels of both immature and mature forms of hERG. Immunocytochemistry revealed increased levels of hERG in the endoplasmic reticulum and on the cell surface. Electrophysiological studies showed increased I_Kr. All these effects of Hsp70 were abolished by Hsc70 coexpression. Heat shock treatment of HL-1 mouse cardiomyocytes induced endogenous Hsp70, switched mouse ERG associated with Hsc70 to Hsp70, increased I_Kr, and shortened action potential duration. Channels withdisease-causing missense mutations in intracellular domainshad a higher binding capacity to Hsc70 than wild-type channelsand channels with mutations in the pore region. Knockdown ofHsc70 by small interfering RNA or heat shock prevented degradationof mutant hERG proteins with mutations in intracellular domains. Conclusions:These results indicate reciprocal control of hERG stabilityby Hsp70 and Hsc70. Hsc70 is a potential target in the treatmentof LQT2 resulting from missense hERG mutations.  

3.1614           Specific elimination of effector memory CD4+ T cells due to enhanced Fas signaling complex formation and association with lipid raft microdomains

Ramaswamy, M., Cruz, A.C., Cleland, S.Y., Deng, M., Price, S., Rao, V.K. and Siegel, R.M. Cell Death and Differentiation, 18, 712-720 (2011)   Elimination of autoreactive CD4⁺ T cells through the death receptor Fas/CD95 is an important mechanism of immunological self-tolerance. Fas deficiency results in systemic autoimmunity, yet does not affect the kinetics of T-cell responses to acute antigen exposure or infection. Here we show that Fas and TCR-induced apoptosis are largely restricted to CD4⁺ T cells with an effector memory phenotype (effector memory T cells (T_EM)), whereas central memory and activated naïve CD4⁺ T cells are relatively resistant to both. Sensitivity of T_EM to Fas-induced apoptosis depends on enrichment of Fas in lipid raft microdomains, and is linked to more efficient formation of the Fas death-inducing signaling complex. These results explain how Fas can cull T cells reactive against self-antigens without affecting acute immune responses. This work also identifies Fas-induced apoptosis as a possible immunotherapeutic strategy to eliminate T_EM linked to the pathogenesis of a number of autoimmune diseases.  

3.1615           Docosahexaenoic Acid Reduces Amyloid β Production via Multiple Pleiotropic Mechanisms

Grimm, M.O., Kuchenbecker, J., Grôsgen, S., Burg, V.K., Hundsdôrfer, B., Rothhaar, T.L., Friess, P., de Wilde, M.C., Broersen, L.M., Penke, B., Peter, M., Vigh, L., Grimm, H.S. and Hartmann, T.

1. Biol. Chem., 286(16), 14028-14039 (2011)

  Alzheimer disease is characterized by accumulation of the β-amyloid peptide (Aβ) generated by β- and γ-secretase processing of the amyloid precursor protein (APP). The intake of the polyunsaturated fatty acid docosahexaenoic acid (DHA) has been associated with decreased amyloid deposition and a reduced risk in Alzheimer disease in several epidemiological trials; however, the exact underlying molecular mechanism remains to be elucidated. Here, we systematically investigate the effect of DHA on amyloidogenic and nonamyloidogenic APP processing and the potential cross-links to cholesterol metabolism in vivo and in vitro. DHA reduces amyloidogenic processing by decreasing β- and γ-secretase activity, whereas the expression and protein levels of BACE1 and presenilin1 remain unchanged. In addition, DHA increases protein stability of α-secretase resulting in increased nonamyloidogenic processing. Besides the known effect of DHA to decrease cholesterol de novo synthesis, we found cholesterol distribution in plasma membrane to be altered. In the presence of DHA, cholesterol shifts from raft to non-raft domains, and this is accompanied by a shift in γ-secretase activity and presenilin1 protein levels. Taken together, DHA directs amyloidogenic processing of APP toward nonamyloidogenic processing, effectively reducing Aβ release. DHA has a typical pleiotropic effect; DHA-mediated Aβ reduction is not the consequence of a single major mechanism but is the result of combined multiple effects.  

3.1616           Disease-associated GPR56 Mutations Cause Bilateral Frontoparietal Polymicrogyria via Multiple Mechanisms

Chiang, N-Y., Hsiao, C-C., Huang, Y-S., Chen, H-Y., Hsieh, I-J., Chang, G-W. and Lin, H-H.

1. Biol. Chem., 286(16), 14215-14225 (2011)

  Loss-of-function mutations in the gene encoding G protein-coupled receptor 56 (GPR56) lead to bilateral frontoparietal polymicrogyria (BFPP), an autosomal recessive disorder affecting brain development. The GPR56 receptor is a member of the adhesion-GPCR family characterized by the chimeric composition of a long ectodomain (ECD), a GPCR proteolysis site (GPS), and a seven-pass transmembrane (7TM) moiety. Interestingly, all identified BFPP-associated missense mutations are located within the extracellular region of GPR56 including the ECD, GPS, and the extracellular loops of 7TM. In the present study, a detailed molecular and functional analysis of the wild-type GPR56 and BFPP-associated point mutants shows that individual GPR56 mutants most likely cause BFPP via different combination of multiple mechanisms. These include reduced surface receptor expression, loss of GPS proteolysis, reduced receptor shedding, inability to interact with a novel protein ligand, and differential distribution of the 7TM moiety in lipid rafts. These results provide novel insights into the cellular functions of GPR56 receptor and reveal molecular mechanisms whereby GPR56 mutations induce BFPP.  

3.1617           CIN85 Modulates the Down-regulation of Fc RIIa Expression and Function by c-Cbl in a PKC-dependent Manner in Human Neutrophils

Marois, L., Vaillancourt, M., Pare, G., Gagne, V., Fernandes, M.J.G., Rollet-Labelle, E. and Naccache, P.H.

1. Biol. Chem., 286(17), 15073-15084 (2011)

  We previously described a non-classical mechanism that arrests FcγRIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. The engagement of FcγRIIa leads to its ubiquitination by the ubiquitin ligase c-Cbl and degradation by the proteasome. Herein, we further examined some of the events regulating this novel pathway. The adaptor protein CIN85 was described in other systems to be involved in the regulation of the c-Cbl-dependent pathway. We found that CIN85 is expressed in human neutrophils and that it translocates like c-Cbl from the cytosol to the plasma membrane following receptor cross-linking. CIN85 was also recruited to the same subset of high density detergent-resistant membrane fractions in which stimulated FcγRIIa partitioned with c-Cbl. The integrity of these microdomains is essential to the FcγRIIa degradation process because the cholesterol-depleting agent methyl-β-cyclodextrin inhibits this event. Silencing the expression of CIN85 by siRNA in dibutyryl cyclic AMP-differentiated PLB 985 cells prevented FcγRIIa degradation and increased IgG-mediated phagocytosis. Confocal microscopy revealed that the presence of CIN85 is essential to the proper sorting of FcγRIIa during endocytosis. We also provide direct evidence that CIN85 is a substrate of serine/threonine kinase PKCs. Classical PKCs positively regulate FcγRIIa ubiquitination and degradation because these events were inhibited by Gö6976, a classical PKC inhibitor. We conclude that the ubiquitination and degradation of stimulated FcγRIIa mediated by c-Cbl are positively regulated by the adaptor protein CIN85 in a PKC-dependent manner and that these events contribute to the termination of FcγRIIa signaling.  

3.1618           Advances in qualitative and quantitative plant membrane proteomics

Kota, U. and Goshe, M.B. Phytochemistry, 72, 1040-1060 (2011)   The membrane proteome consists of integral and membrane-associated proteins that are involved in various physiological and biochemical functions critical for cellular function. It is also dynamic in nature, where many proteins are only expressed during certain developmental stages or in response to environmental stress. These proteins can undergo post-translational modifications in response to these different conditions, allowing them to transiently associate with the membrane or other membrane proteins. Along with their increased size, hydrophobicity, and the additional organelle and cellular features of plant cells relative to mammalian systems, the characterization of the plant membrane proteome presents unique challenges for effective qualitative and quantitative analysis using mass spectrometry (MS) analysis. Here, we present the latest advancements developed for the isolation and fractionation of plant organelles and their membrane components amenable to MS analysis. Separations of membrane proteins from these enriched preparations that have proven effective are discussed for both gel- and liquid chromatography-based MS analysis. In this context, quantitative membrane proteomic analyses using both isotope-coded and label-free approaches are presented and reveal the potential to establish a wider-biological interpretation of the function of plant membrane proteins that will ultimately lead to a more comprehensive understanding of plant physiology and their response mechanisms.      

3.1619           FERM Domain Phosphoinositide Binding Targets Merlin to the Membrane and Is Essential for Its Growth-Suppressive Function

Mani, T., Hennigan, R.F., Foster, L.A., Conrady, D.G., Herr, A.B. and Ip, W. Mol. Cell. Biol., 31(10), 1983-1996 (2011)   The neurofibromatosis type 2 tumor suppressor protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of plasma membrane-actin cytoskeleton linkers. For ezrin, phosphatidylinositol 4,5-bisphosphate (PIP2) binding to the amino-terminal FERM domain is required for its conformational activation, proper subcellular localization, and function, but less is known about the role of phosphoinositide binding for merlin. Current evidence indicates that association with the membrane is important for merlin to function as a growth regulator; however, the mechanisms by which merlin localizes to the membrane are less clear. Here, we report that merlin binds phosphoinositides, including PIP2, via a conserved binding motif in its FERM domain. Abolition of FERM domain-mediated phosphoinositide binding of merlin displaces merlin from the membrane and releases it into the cytosol without altering the folding of merlin. Importantly, a merlin protein whose FERM domain cannot bind phosphoinositide is defective in growth suppression. Retargeting the mutant merlin into the membrane using a dual-acylated amino-terminal decapeptide from Fyn is sufficient to restore the growth-suppressive properties to the mutant merlin. Thus, FERM domain-mediated phosphoinositide binding and membrane association are critical for the growth-regulatory function of merlin.  

3.1620           Motor Protein Myo1c Is a Podocyte Protein That Facilitates the Transport of Slit Diaphragm Protein Neph1 to the Podocyte Membrane

Arif, E., Wagner, M.C., Johnstone, D.B., Wong, H.N., George, W.B., Pruthi, P.A., Lazzara, M.J. and Nihalani, D. Mol. Cell. Biol., 31(10), 2134-2150 (2011)   The podocyte proteins Neph1 and nephrin organize a signaling complex at the podocyte cell membrane that forms the structural framework for a functional glomerular filtration barrier. Mechanisms regulating the movement of these proteins to and from the membrane are currently unknown. This study identifies a novel interaction between Neph1 and the motor protein Myo1c, where Myo1c plays an active role in targeting Neph1 to the podocyte cell membrane. Using in vivo and in vitro experiments, we provide data supporting a direct interaction between Neph1 and Myo1c which is dynamic and actin dependent. Unlike wild-type Myo1c, the membrane localization of Neph1 was significantly reduced in podocytes expressing dominant negative Myo1c. In addition, Neph1 failed to localize at the podocyte cell membrane and cell junctions in Myo1c-depleted podocytes. We further demonstrate that similarly to Neph1, Myo1c also binds nephrin and reduces its localization at the podocyte cell membrane. A functional analysis of Myo1c knockdown cells showed defects in cell migration, as determined by a wound assay. In addition, the ability to form tight junctions was impaired in Myo1c knockdown cells, as determined by transepithelial electric resistance (TER) and bovine serum albumin (BSA) permeability assays. These results identify a novel Myo1c-dependent molecular mechanism that mediates the dynamic organization of Neph1 and nephrin at the slit diaphragm and is critical for podocyte function.  

3.1621           Drosophila Photoreceptor Cells Exploited for the Production of Eukaryotic Membrane Proteins: Receptors, Transporters and Channels

Panneels, V., Kock, I., Krijnse-Locker, J., Rezgaoui, M. and Sinning, I. PloSOne, 6(4), e18478 (2011)   Background Membrane proteins (MPs) play key roles in signal transduction. However, understanding their function at a molecular level is mostly hampered by the lack of protein in suitable amount and quality. Despite impressive developments in the expression of prokaryotic MPs, eukaryotic MP production has lagged behind and there is a need for new expression strategies. In a pilot study, we produced a Drosophila glutamate receptor specifically in the eyes of transgenic flies, exploiting the naturally abundant membrane stacks in the photoreceptor cells (PRCs). Now we address the question whether the PRCs also process different classes of medically relevant target MPs which were so far notoriously difficult to handle with conventional expression strategies. Principal Findings We describe the homologous and heterologous expression of 10 different targets from the three major MP classes - G protein-coupled receptors (GPCRs), transporters and channels in Drosophila eyes. PRCs offered an extraordinary capacity to produce, fold and accommodate massive amounts of MPs. The expression of some MPs reached similar levels as the endogenous rhodopsin, indicating that the PRC membranes were almost unsaturable. Expression of endogenous rhodopsin was not affected by the target MPs and both could coexist in the membrane stacks. Heterologous expression levels reached about 270 to 500 pmol/mg total MP, resulting in 0.2–0.4 mg purified target MP from 1 g of fly heads. The metabotropic glutamate receptor and human serotonin transporter - both involved in synaptic transmission - showed native pharmacological characteristics and could be purified to homogeneity as a prerequisite for further studies. Significance We demonstrate expression in Drosophila PRCs as an efficient and inexpensive tool for the large scale production of functional eukaryotic MPs. The fly eye system offers a number of advantages over conventional expression systems and paves the way for in-depth analyses of eukaryotic MPs that have so far not been accessible to biochemical and biophysical studies.  

3.1622           Calcium Uptake and Proton Transport by Acidocalcisomes of Toxoplasma gondii

Rohloff, P., Miranda, K., Rodrigues, J.F.C., Fang, J., Galizzi, M., Plattner, H., Hentschel, J. and Moreno, S.N.J. PloSOne, 6(4), e18390 (2011)   Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to humans. They possess an acidic matrix that contains several cations bound to phosphates, which are mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca²⁺/H⁺ countertransporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. We report the purification and characterization of an acidocalcisome fraction from T. gondii tachyzoites after subcellular fractionation and further discontinuous iodixanol gradient purification. Proton and calcium transport activities in the fraction were characterized by fluorescence microscopy and spectrophotometric methods using acridine orange and arsenazo III, respectively. This work will facilitate the understanding of the function of acidocalcisomes in Apicomplexan parasites, as we can now isolate highly purified fractions that could be used for proteomic analysis to find proteins that may clarify the biogenesis of these organelles.    

3.1623           The Expression of a Xylanase Targeted to ER-Protein Bodies Provides a Simple Strategy to Produce Active Insoluble Enzyme Polymers in Tobacco Plants

Llop-Tous, I., Ortiz, M., Torrent, M. and Ludevid, M.D. PloSOne, 6(4), e19474 (2011)   Background Xylanases deserve particular attention due to their potential application in the feed, pulp bleaching and paper industries. We have developed here an efficient system for the production of an active xylanase in tobacco plants fused to a proline-rich domain (Zera) of the maize storage protein γ-zein. Zera is a self-assembling domain able to form protein aggregates in vivo packed in newly formed endoplasmic reticulum-derived organelles known as protein bodies (PBs). Methodology/Principal Findings Tobacco leaves were transiently transformed with a binary vector containing the Zera-xylanase coding region, which was optimized for plant expression, under the control of the 35S CaMV promoter. The fusion protein was efficiently expressed and stored in dense PBs, resulting in yields of up to 9% of total protein. Zera-xylanase was post-translationally modified with high-mannose-type glycans. Xylanase fused to Zera was biologically active not only when solubilized from PBs but also in its insoluble form. The resistance of insoluble Zera-xylanase to trypsin digestion demonstrated that the correct folding of xylanase in PBs was not impaired by Zera oligomerization. The activity of insoluble Zera-xylanase was enhanced when substrate accessibility was facilitated by physical treatments such as ultrasound. Moreover, we found that the thermostability of the enzyme was improved when Zera was fused to the C-terminus of xylanase. Conclusion/Significance In the present work we have successfully produced an active insoluble aggregate of xylanase fused to Zera in plants. Zera-xylanase chimeric protein accumulates within ER-derived protein bodies as active aggregates that can easily be recovered by a simple density-based downstream process. The production of insoluble active Zera-xylanase protein in tobacco outlines the potential of Zera as a fusion partner for producing enzymes of biotechnological relevance. Zera-PBs could thus become efficient and low-cost bioreactors for industrial purposes.  

3.1624           Identification of Contractile Vacuole Proteins in Trypanosoma cruzi

Ulrich, P.N., Jimenez, V., Park, M., Martins, V.P., Atwood III, J., Moles, K., Collins, D., Rohloff, P., Tarleton, R., Moreno, S.N.J., Orlando, R. and Docampo, R. PloSOne, 6(3), e18013 (2011)   Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis.  

3.1625           A Pseudomonas aeruginosa Toxin that Hijacks the Host Ubiquitin Proteolytic System

Bomberger, J.M., Ye, S., MacEachran, D.P., Koeppen, K., Barnaby, R.L., O’Toole, G. and Stanton, B.A. PloSPathogens, 7(3), e1001325 (2011)   Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen chronically infecting the lungs of patients with chronic obstructive pulmonary disease (COPD), pneumonia, cystic fibrosis (CF), and bronchiectasis. Cif (PA2934), a bacterial toxin secreted in outer membrane vesicles (OMV) by P. aeruginosa, reduces CFTR-mediated chloride secretion by human airway epithelial cells, a key driving force for mucociliary clearance. The aim of this study was to investigate the mechanism whereby Cif reduces CFTR-mediated chloride secretion. Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB), USP10, thereby reducing USP10-mediated deubiquitination of CFTR and increasing the degradation of CFTR in lysosomes. This is the first example of a bacterial toxin that regulates the activity of a host DUB. These data suggest that the ability of P. aeruginosa to chronically infect the lungs of patients with COPD, pneumonia, CF, and bronchiectasis is due in part to the secretion of OMV containing Cif, which inhibits CFTR-mediated chloride secretion and thereby reduces the mucociliary clearance of pathogens.  

3.1626           Tandem application of cationic colloidal silica and Triton X-114 for plasma membrane protein isolation and purification: Towards developing an MDCK protein database

Mathias, R.A., Chen, Y-S., Goode, R.J.A., Kapp, E.A., Mathivanan, S., Moritz, R.L., Zhu, H-J. and Simpson, R.J. Proteomics, 11, 1238-1253 (2011)   Plasma membrane (PM) proteins are attractive therapeutic targets because of their accessibility to drugs. Although genes encoding PM proteins represent 20–30% of eukaryotic genomes, a detailed characterisation of their encoded proteins is underrepresented, due, to their low copy number and the inherent difficulties in their isolation and purification as a consequence of their high hydrophobicity. We describe here a strategy that combines two orthogonal methods to isolate and purify PM proteins from Madin Darby canine kidney (MDCK) cells. In this two-step method, we first used cationic colloidal silica (CCS) to isolate adherent (Ad) and non-adherent (nAd) PM fractions, and then subjected each fraction to Triton X-114 (TX-114) phase partitioning to further enrich for hydrophobic proteins. While CCS alone identified 255/757 (34%) membrane proteins, CCS/TX-114 in combination yielded 453/745 (61%). Strikingly, of those proteins unique to CCS/TX-114, 277/393 (70%) had membrane annotation. Further characterisation of the CCS/TX-114 data set using Uniprot and transmembrane hidden Markov model revealed that 306/745 (41%) contained one or more transmembrane domains (TMDs), including proteins with 25 and 17 TMDs. Of the remaining proteins in the data set, 69/439 (16%) are known to contain lipid modifications. Of all membrane proteins identified, 93 had PM origin, including proteins that mediate cell adhesion, modulate transmembrane ion transport, and cell–cell communication. These studies reveal that the application of CCS to first isolate Ad and nAd PM fractions, followed by their detergent-phase TX-114 partitioning, to be a powerful method to isolate low-abundance PM proteins, and a useful adjunct for in-depth cell surface proteome analyses.  

3.1627           Toward a definition of the complete proteome of plant peroxisomes: Where experimental proteomics must be complemented by bioinformatics

Reumann, S. Proteomics, 11, 1764-1779 (2011)   In the past few years, proteome analysis of Arabidopsis peroxisomes has been established by the complementary efforts of four research groups and has emerged as the major unbiased approach to identify new peroxisomal proteins on a large scale. Collectively, more than 100 new candidate proteins from plant peroxisomes have been identified, including long-awaited low-abundance proteins. More than 50 proteins have been validated as peroxisome targeted, nearly doubling the number of established plant peroxisomal proteins. Sequence homologies of the new proteins predict unexpected enzyme activities, novel metabolic pathways and unknown non-metabolic peroxisome functions. Despite this remarkable success, proteome analyses of plant peroxisomes remain highly material intensive and require major preparative efforts. Characterization of the membrane proteome or post-translational protein modifications poses major technical challenges. New strategies, including quantitative mass spectrometry methods, need to be applied to allow further identifications of plant peroxisomal proteins, such as of stress-inducible proteins. In the long process of defining the complete proteome of plant peroxisomes, the prediction of peroxisome-targeted proteins from plant genome sequences emerges as an essential complementary approach to identify additional peroxisomal proteins that are, for instance, specific to peroxisome variants from minor tissues and organs or to abiotically stressed model and crop plants.      

3.1628           Transient receptor potential canonical channels are essential for chemotactic migration of human malignant gliomas

Bomben, V.C., Turner, K.L., Barclay, T-T.C. and Sontheimer, H.

1. Cell. Physiol., 226, 1879-1888 (2011)

  The majority of malignant primary brain tumors are gliomas, derived from glial cells. Grade IV gliomas, Glioblastoma multiforme, are extremely invasive and the clinical prognosis for patients is dismal. Gliomas utilize a number of proteins and pathways to infiltrate the brain parenchyma including ion channels and calcium signaling pathways. In this study, we investigated the localization and functional relevance of transient receptor potential canonical (TRPC) channels in glioma migration. We show that gliomas are attracted in a chemotactic manner to epidermal growth factor (EGF). Stimulation with EGF results in TRPC1 channel localization to the leading edge of migrating D54MG glioma cells. Additionally, TRPC1 channels co-localize with the lipid raft proteins, caveolin-1 and β-cholera toxin, and biochemical assays show TRPC1 in the caveolar raft fraction of the membrane. Chemotaxis toward EGF was lost when TRPC channels were pharmacologically inhibited or by shRNA knockdown of TRPC1 channels, yet without affecting unstimulated cell motility. Moreover, lipid raft integrity was required for gliomas chemotaxis. Disruption of lipid rafts not only impaired chemotaxis but also impaired TRPC currents in whole cell recordings and decreased store-operated calcium entry as revealed by ratiomeric calcium imaging. These data indicated that TRPC1 channel association with lipid rafts is essential for glioma chemotaxis in response to stimuli, such as EGF.  

3.1629           Sphingosine 1-Phosphate-Induced Motility and Endocytosis of Dendritic Cells Is Regulated by SWAP-70 through RhoA

Ocana-Morgner, C., Reichardt, P., Chopin, M., Braungart, S., Wahren, C., Gunzer, M. and Jessberger, R.

1. Immunol., 186, 5345-5355 (2011)

 

The phospholipid mediator sphingosine 1-phosphate (S1P) enhances motility and endocytosis of mature dendritic cells (DCs). We show that in vitro migration of Swap-70^−/− bone marrow-derived DCs (BMDCs) in response to S1P and S1P-induced upregulation of endocytosis are significantly reduced. S1P-stimulated movement of Swap-70^−/− BMDCs, specifically retraction of their trailing edge, in a collagen three-dimensional environment is impaired. These in vitro observations correlate with delayed entry into lymphatic vessels and migration to lymph nodes of skin DCs in Swap-70^−/− mice. Expression of S1P receptors (S1P_1–3) by wild-type and Swap-70^−/− BMDCs is similar, but Swap-70^−/− BMDCs fail to activate RhoA and to localize Rac1 and RhoA into areas of actin polymerization after S1P stimulus. The Rho-activating G protein Gα_i interacts with SWAP-70, which also supports the localization of Gα₁₃ to membrane rafts in BMDCs. LPS-matured Swap-70^−/− BMDCs contain significantly more active RhoA than wild-type DCs. Preinhibition of Rho activation restored migration to S1P, S1P-induced upregulation of endocytosis in mature Swap-70^−/− BMDCs, and localization of Gα₁₃ to membrane rafts. These data demonstrate SWAP-70 as a novel regulator of S1P signaling necessary for DC motility and endocytosis.

 

3.1630           Cutting Edge: NKG2D-Dependent Cytotoxicity Is Controlled by Ligand Distribution in the Target Cell Membrane

Martinez, E., Brzostowski, J.A., Long, E.O. and Gross, C.C.

1. Immunol., 186, 5538-5542 (2011)

  Although the importance of membrane microdomains in receptor-mediated activation of lymphocytes has been established, much less is known about the role of receptor ligand distribution on APC and target cells. Detergent-resistant membrane domains, into which GPI-linked proteins partition, are enriched in cholesterol and glycosphingolipids. ULBP1 is a GPI-linked ligand for natural cytotoxicity receptor NKG2D. To investigate how ULBP1 distribution on target cells affects NKG2D-dependent NK cell activation, we fused the extracellular domain of ULBP1 to the transmembrane domain of CD45. Introduction of this transmembrane domain eliminated the association of ULBP1 with the detergent-resistant membrane fraction and caused a significant reduction of cytotoxicity and degranulation by NK cells. Clustering and lateral diffusion of ULBP1 was not affected by changes in the membrane anchor. These results show that the partitioning of receptor ligands in discrete membrane domains of target cells is an important determinant of NK cell activation.    

3.1631           Inhibition of mTOR blocks the anti-inflammatory effects of glucocorticoids in myeloid immune cells

Weichhart, T., Haidinger, M., Katholnig, K., Kopecky, C., poglitsch, M., Lassnig, C., Rosner, M., Zlabinger, G.J., Hengstschlâger, M., Müller, M., Hörl, W.H. and Säemann, M.D. Blood, 117(16), 4273-4283 (2011)   A central role for the mammalian target of rapamycin (mTOR) in innate immunity has been recently defined by its ability to limit proinflammatory mediators. Although glucocorticoids (GCs) exert potent anti-inflammatory effects in innate immune cells, it is currently unknown whether the mTOR pathway interferes with GC signaling. Here we show that inhibition of mTOR with rapamycin or Torin1 prevented the anti-inflammatory potency of GC both in human monocytes and myeloid dendritic cells. GCs could not suppress nuclear factor-κB and JNK activation, the expression of proinflammatory cytokines, and the promotion of Th1 responses when mTOR was inhibited. Interestingly, long-term activation of monocytes with lipopolysaccharide enhanced the expression of TSC2, the principle negative regulator of mTOR, whereas dexamethasone blocked TSC2 expression and reestablished mTOR activation. Renal transplant patients receiving rapamycin but not those receiving calcineurin inhibitors displayed a state of innate immune cell hyper-responsiveness despite the concurrent use of GC. Finally, mTOR inhibition was able to override the healing phenotype of dexamethasone in a murine lipopolysaccharide shock model. Collectively, these data identify a novel link between the glucocorticoid receptor and mTOR in innate immune cells, which is of considerable clinical importance in a variety of disorders, including allogeneic transplantation, autoimmune diseases, and cancer.  

3.1632           Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes

Alvarez-Erviti, L., Seow, Y., Yin, H., Betts, C., Lakhal, S. and Wood, M.J.A. Nature Biotech., 29(4), 341-345 (2011)   To realize the therapeutic potential of RNA drugs, efficient, tissue-specific and nonimmunogenic delivery technologies must be developed. Here we show that exosomes—endogenous nano-vesicles that transport RNAs and proteins^{1, 2}—can deliver short interfering (si)RNA to the brain in mice. To reduce immunogenicity, we used self-derived dendritic cells for exosome production. Targeting was achieved by engineering the dendritic cells to express Lamp2b, an exosomal membrane protein, fused to the neuron-specific RVG peptide³. Purified exosomes were loaded with exogenous siRNA by electroporation. Intravenously injected RVG-targeted exosomes delivered GAPDH siRNA specifically to neurons, microglia, oligodendrocytes in the brain, resulting in a specific gene knockdown. Pre-exposure to RVG exosomes did not attenuate knockdown, and non-specific uptake in other tissues was not observed. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the strong mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease, in wild-type mice.  

3.1633           The transmembrane domain of podoplanin is required for its association with lipid rafts and the induction of epithelial-mesenchymal transition

Fernandez-Munoz, B., Yurrita, M.M., Martin-Villar, E., Carroasco-Ramirez, P., Megias, D., renart, J. and Quintanilla, M. Int. J. Biochem. Cell Biol., 43, 886-896 (2011)   Podoplanin is a transmembrane glycoprotein that is upregulated in cancer and was reported to induce an epithelial-mesenchymal transition (EMT) in MDCK cells. The promotion of EMT was dependent on podoplanin binding to ERM (ezrin, radixin, moesin) proteins through its cytoplasmic (CT) domain, which led to RhoA-associated kinase (ROCK)-dependent ERM phosphorylation. Using detergent-resistant membrane (DRM) assays, as well as transmembrane (TM) interactions and ganglioside GM1 binding, we present evidence supporting the localization of podoplanin in raft platforms important for cell signalling. Podoplanin mutant constructs harbouring a heterologous TM region or lacking the CT tail were unable to associate with DRMs, stimulate ERM phosphorylation and promote EMT or cell migration. Similar effects were observed upon disruption of a GXXXG motif within the TM domain, which is involved in podoplanin self-assembly. In contrast, deletion of the extracellular (EC) domain did not affect podoplanin DRM association. Together, these data suggest that both the CT and TM domains are required for podoplanin localization in raft platforms, and that this association appears to be necessary for podoplanin-mediated EMT and cell migration.      

3.1634           Keratin 23, a novel DPC4/Smad4 target gene which binds 14-3-3ε

Liffers, S-T., Maghnouj, A., Munding, J.B., Jackstadt, R., Herbrand, U., Schulenborg, T., Marcus, K., Klein-Scory, S., Schmiegel, W., Schwarte-Waldhoff, I., Meyer, H.E., Stühler, K. and Hanh, S.A. BMC Cancer, 11:137, (2011)   Background Inactivating mutations of SMAD4 are frequent in metastatic colorectal carcinomas. In previous analyses, we were able to show that restoration of Smad4 expression in Smad4-deficient SW480 human colon carcinoma cells was adequate to suppress tumorigenicity and invasive potential, whereas in vitro cell growth was not affected. Using this cellular model system, we searched for new Smad4 targets comparing nuclear subproteomes derived from Smad4 re-expressing and Smad4 negative SW480 cells. Methods High resolution two-dimensional (2D) gel electrophoresis was applied to identify novel Smad4 targets in the nuclear subproteome of Smad4 re-expressing SW480 cells. The identified candidate protein Keratin 23 was further characterized by tandem affinity purification. Immunoprecipitation, subfractionation and immunolocalization studies in combination with RNAi were used to validate the Keratin 23-14-3-3ε interaction. Results We identified keratins 8 and 18, heat shock proteins 60 and 70, plectin 1, as well as 14-3-3ε and γ as novel proteins present in the KRT23-interacting complex. Co-immunoprecipitation and subfractionation analyses as well as immunolocalization studies in our Smad4-SW480 model cells provided further evidence that KRT23 associates with 14-3-3ε and that Smad4 dependent KRT23 up-regulation induces a shift of the 14-3-3ε protein from a nuclear to a cytoplasmic localization. Conclusion Based on our findings we propose a new regulatory circuitry involving Smad4 dependent up-regulation of KRT23 (directly or indirectly) which in turn modulates the interaction between KRT23 and 14-3-3ε leading to a cytoplasmic sequestration of 14-3-3ε. This cytoplasmic KRT23-14-3-3 interaction may alter the functional status of the well described 14-3-3 scaffold protein, known to regulate key cellular processes, such as signal transduction, cell cycle control, and apoptosis and may thus be a previously unappreciated facet of the Smad4 tumor suppressive circuitry.  

3.1635           BAX supports the mitochondrial network, promoting bioenergetics in nonapoptotic cells

Boohaker, R.J., Zhang, G., Carlson, A.L., Nemec, K.N. and Khaled, A.R.

1. J: Physiol. Cell. Physiol., 300, C1466-C1478 (2011)

  The dual functionality of the tumor suppressor BAX is implied by the nonapoptotic functions of other members of the BCL-2 family. To explore this, mitochondrial metabolism was examined in BAX-deficient HCT-116 cells as well as primary hepatocytes from BAX-deficient mice. Although mitochondrial density and mitochondrial DNA content were the same in BAX-containing and BAX-deficient cells, MitoTracker staining patterns differed, suggesting the existence of BAX-dependent functional differences in mitochondrial physiology. Oxygen consumption and cellular ATP levels were reduced in BAX-deficient cells, while glycolysis was increased. These results suggested that cells lacking BAX have a deficiency in the ability to generate ATP through cellular respiration. This conclusion was supported by detection of reduced citrate synthase activity in BAX-deficient cells. In nonapoptotic cells, a portion of BAX associated with mitochondria and a sequestered, protease-resistant form was detected. Inhibition of BAX with small interfering RNAs reduced intracellular ATP content in BAX-containing cells. Expression of either full-length or COOH-terminal-truncated BAX in BAX-deficient cells rescued ATP synthesis and oxygen consumption and reduced glycolytic activity, suggesting that this metabolic function of BAX was not dependent upon its COOH-terminal helix. Expression of BCL-2 in BAX-containing cells resulted in a subsequent loss of ATP measured, implying that, even under nonapoptotic conditions, an antagonistic interaction exists between the two proteins. These findings infer that a basal amount of BAX is necessary to maintain energy production via aerobic respiration.  

3.1636           Omeprazole Inhibits Proliferation and Modulates Autophagy in Pancreatic Cancer Cells

Udelnow, A., Kreyes, A., Ellinger, S., Landfester, K., Walther, P., Klapperstueck, T., Wohlrab, J., Henne-Bruns, D., Knippschild, U. and Würl, P. PloS One, 6(5), e20143 (2011)   Background Omeprazole has recently been described as a modulator of tumour chemoresistance, although its underlying molecular mechanisms remain controversial. Since pancreatic tumours are highly chemoresistant, a logical step would be to investigate the pharmacodynamic, morphological and biochemical effects of omeprazole on pancreatic cancer cell lines. Methodology/Principal Findings Dose-effect curves of omeprazole, pantoprazole, gemcitabine, 5-fluorouracil and the combinations of omeprazole and 5-fluorouracil or gemcitabine were generated for the pancreatic cancer cell lines MiaPaCa-2, ASPC-1, Colo357, PancTu-1, Panc1 and Panc89. They revealed that omeprazole inhibited proliferation at probably non-toxic concentrations and reversed the hormesis phenomena of 5-fluorouracil. Electron microscopy showed that omeprazole led to accumulation of phagophores and early autophagosomes in ASPC-1 and MiaPaCa-2 cells. Signal changes indicating inhibited proliferation and programmed cell death were found by proton NMR spectroscopy of both cell lines when treated with omeprazole which was identified intracellularly. Omeprazole modulates the lysosomal transport pathway as shown by Western blot analysis of the expression of LAMP-1, Cathepsin-D and β-COP in lysosome- and Golgi complex containing cell fractions. Acridine orange staining revealed that the pump function of the vATPase was not specifically inhibited by omeprazole. Gene expression of the autophagy-related LC3 gene as well as of Bad, Mdr-1, Atg12 and the vATPase was analysed after treatment of cells with 5-fluorouracil and omeprazole and confirmed the above mentioned results. Conclusions We hypothesise that omeprazole interacts with the regulatory functions of the vATPase without inhibiting its pump function. A modulation of the lysosomal transport pathway and autophagy is caused in pancreatic cancer cells leading to programmed cell death. This may circumvent common resistance mechanisms of pancreatic cancer. Since omeprazole use has already been established in clinical practice these results could lead to new clinical applications.  

3.1637           Delayed Phosphorylation of Classical Protein Kinase C (PKC) Substrates Requires PKC Internalization and Formation of the Pericentrion in a Phospholipase D (PLD)-dependent Manner

El-Osta, M.A., Idkowiak-Baldys, J. and Hannun, Y.A.

1. Biol. Chem., 286(22), 19340-19353 (2011)

  It was previously demonstrated that sustained activation (30–60 min) of protein kinase C (PKC) results in translocation of PKC α and βII to the pericentrion, a dynamic subset of the recycling compartment whose formation is dependent on PKC and phospholipase D (PLD). Here we investigated whether the formation of the pericentrion modulates the ability of PKC to phosphorylate substrates, especially if it reduces substrate phosphorylation by sequestering PKC. Surprisingly, using an antibody that detects phosphosubstrates of classical PKCs, the results showed that the majority of PKC phosphosubstrates are phosphorylated with delayed kinetics, correlating with the time frame of PKC translocation to the pericentrion. Substrate phosphorylation was blocked by PLD inhibitors and was not observed in response to activation of a PKC βII mutant (F663D) that is defective in interaction with PLD and in internalization. Phosphorylation was also inhibited by blocking clathrin-dependent endocytosis, demonstrating a requirement for endocytosis for the PKC-dependent major phosphorylation effects. Serotonin receptor activation by serotonin showed a similar response to phorbol 12-myristate 13-acetate, implicating a potential role of delayed kinetics in G protein-coupled receptor signaling. Evaluation of candidate substrates revealed that the phosphorylation of the PKC substrate p70S6K kinase behaved in a similar manner. Gradient-based fractionation revealed that the majority of these PKC substrates reside within the pericentrion-enriched fractions and not in the plasma membrane. Finally, proteomic analysis of the pericentrion-enriched fractions revealed several proteins as known PKC substrates and/or proteins involved in endocytic trafficking. These results reveal an important role for PKC internalization and for the pericentrion as key determinants/amplifiers of PKC action.  

3.1638           Cell death via mitochondrial apoptotic pathway due to activation of Bax by lysosomal photodamage

Liu, L., Zhang, Z. and Xing, D. Free Radical Biology & Medicine, 51, 53-68 (2011)   Lysosomal photosensitizers have been used in photodynamic therapy. The combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. Lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, N-aspartyl chlorin e6 (NPe6), an effective photosensitizer that preferentially accumulates in lysosomes, was used to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells (ASTC-a-1) after NPe6–photodynamic treatment (NPe6–PDT) was studied using real-time single-cell analysis. Our results demonstrated that NPe6–PDT induced rapid generation of reactive oxygen species (ROS). The photodynamically produced ROS caused a rapid destruction of lysosomes, leading to release of cathepsins, and the ROS scavengers vitamin C and NAC prevent the effects. Then the following spatiotemporal sequence of cellular events was observed during cell apoptosis: Bcl-2-associated X protein (Bax) activation, cytochrome c release, and caspase-9/-3 activation. Importantly, the activation of Bax proved to be a crucial event in this apoptotic machinery, because suppressing the endogenous Bax using siRNA could significantly inhibit cytochrome c release and caspase-9/-3 activation and protect the cell from death. In conclusion, this study demonstrates that PDT with lysosomal photosensitizer induces Bax activation and subsequently initiates the mitochondrial apoptotic pathway. Keywords: Lysosomal photodamage; NPe6–PDT; Bax; Apoptosis; Mitochondrial pathway; Free radicals Abbreviations: AIF, apoptosis-inducing factor; Bax, Bcl-2-associated X protein; Bid, BH3-interacting-domain death agonist; CFP, cyan fluorescent protein; ER, endoplasmic reticulum; FACS, fluorescence-activated cell sorter; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; H₂DCFDA, dichlorodihydrofluorescein diacetate; LD₅₀, 50% lethal dose; LD₉₀, 90% lethal dose; NAC, N-acetylcysteine; NPe6, N-aspartyl chlorin e6; OMM, outer mitochondrial membrane; PBS, phosphate-buffered saline; PDT, photodynamic treatment; RFP, red fluorescent protein; RNAi, RNA interference; ROS, reactive oxygen species; ΔΨ_m, mitochondrial membrane potential  

3.1639           Conserved GXXXG- and S/T-Like Motifs in the Transmembrane Domains of NS4B Protein Are Required for Hepatitis C Virus Replication

Han, Q., Aligo, J., Manna, D., Belton, K., Chintapalli, S.V., Hong, Y., Patterson, R.L., van Rossum, D.B. and Konan, K.V.

1. Virol., 85(13), 6464-6479 (2011)

  Hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is an integral membrane protein, which plays an important role in the organization and function of the HCV replication complex (RC). Although much is understood about its amphipathic N-terminal and C-terminal domains, we know very little about the role of the transmembrane domains (TMDs) in NS4B function. We hypothesized that in addition to anchoring NS4B into host membranes, the TMDs are engaged in intra- and intermolecular interactions required for NS4B structure/function. To test this hypothesis, we have engineered a chimeric JFH1 genome containing the Con1 NS4B TMD region. The resulting virus titers were greatly reduced from those of JFH1, and further analysis indicated a defect in genome replication. We have mapped this incompatibility to NS4B TMD1 and TMD2 sequences, and we have defined putative TMD dimerization motifs (GXXXG in TMD2 and TMD3; the S/T cluster in TMD1) as key structural/functional determinants. Mutations in each of the putative motifs led to significant decreases in JFH1 replication. Like most of the NS4B chimeras, mutant proteins had no negative impact on NS4B membrane association. However, some mutations led to disruption of NS4B foci, implying that the TMDs play a role in HCV RC formation. Further examination indicated that the loss of NS4B foci correlates with the destabilization of NS4B protein. Finally, we have identified an adaptive mutation in the NS4B TMD2 sequence that has compensatory effects on JFH1 chimera replication. Taken together, these data underscore the functional importance of NS4B TMDs in the HCV life cycle.  

3.1640           Cholesterol sequestration by nystatin enhances the uptake and activity of endostatin in endothelium via regulating distinct endocytic pathways

Chen, Y., Wang, S., Lu, X., Zhang, H., Fu, Y. and Luo, Y. Blood, 117, 6392-6403 (2011)   Specific internalization of endostatin into endothelial cells has been proved to be important for its biologic functions. However, the mechanism of endostatin internalization still remains elusive. In this study, we report for the first time that both caveolae/lipid rafts and clathrin-coated pits are involved in endostatin internalization. Inhibition of either the caveolae pathway or the clathrin pathway with the use of chemical inhibitors, small interfering RNAs, or dominant-negative mutants alters endostatin internalization in vitro. Intriguingly, cholesterol sequestration by nystatin, a polyene antifungal drug, significantly enhances endostatin uptake by endothelial cells through switching endostatin internalization predominantly to the clathrin-mediated pathway. Nystatin-enhanced internalization of endostatin also increases its inhibitory effects on endothelial cell tube formation and migration. More importantly, combined treatment with nystatin and endostatin selectively enhances endostatin uptake and biodistribution in tumor blood vessels and tumor tissues but not in normal tissues of tumor-bearing mice, ultimately resulting in elevated antiangiogenic and antitumor efficacies of endostatin in vivo. Taken together, our data show a novel mechanism of endostatin internalization and support the potential application of enhancing the uptake and therapeutic efficacy of endostatin via regulating distinct endocytic pathways with cholesterol-sequestering agents.  

3.1641           Caveolin-1 and force regulation in porcine airway smooth muscle

Satish, V., Yang, B., Meuchel, L.W., VanOosten, S.K., Ryu, A.J., Thompson, M.A., Prakash, Y.S. and Pabelick, C. Am. J. Physiol. Lung Cell. Mol. Physiol., 300, L920-L929 (2011)   Caveolae are specialized membrane microdomains expressing the scaffolding protein caveolin-1. We recently demonstrated the presence of caveolae in human airway smooth muscle (ASM) and the contribution of caveolin-1 to intracellular calcium ([Ca²⁺]_i) regulation. In the present study, we tested the hypothesis that caveolin-1 regulates ASM contractility. We examined the role of caveolins in force regulation of porcine ASM under control conditions as well as TNF-α-induced airway inflammation. In porcine ASM strips, exposure to 10 mM methyl-β-cyclodextrin (CD) or 5 μM of the caveolin-1 specific scaffolding domain inhibitor peptide (CSD) resulted in time-dependent decrease in force responses to 1 μM ACh. Overnight exposure to the cytokine TNF-α (50 ng/ml) accelerated and increased caveolin-1 expression and enhanced force responses to ACh. Suppression of caveolin-1 with small interfering RNA mimicked the effects of CD or CSD. Regarding mechanisms by which caveolae contribute to contractile changes, inhibition of MAP kinase with 10 μM PD98059 did not alter control or TNF-α-induced increases in force responses to ACh. However, inhibiting RhoA with 100 μM fasudil or 10 μM Y27632 resulted in significant decreases in force responses, with lesser effects in TNF-α exposed samples. Furthermore, Ca²⁺ sensitivity for force generation was substantially reduced by fasudil or Y27632, an effect even more enhanced in the absence of caveolin-1 signaling. Overall, these results indicate that caveolin-1 is a critical player in enhanced ASM contractility with airway inflammation.  

3.1642           Cholesterol Regulates µ-Opioid Receptor-Induced β-Arrestin 2 Translocation to Membrane Lipid Rafts

Qiu, Y., Wang, Y., Law, P-Y., Chen, H-Z. and Loh, H.H. Mol. Pharmacol., 80(1), 210-218 (2011)   μ-Opioid receptor (OPRM1) is mainly localized in lipid raft microdomains but internalizes through clathrin-dependent pathways. Our previous studies demonstrated that disruption of lipid rafts by cholesterol-depletion reagent blocked the agonist-induced internalization of OPRM1 and G protein-dependent signaling. The present study demonstrated that reduction of cholesterol level decreased and culturing cells in excess cholesterol increased the agonist-induced internalization and desensitization of OPRM1, respectively. Further analyses indicated that modulation of cellular cholesterol level did not affect agonist-induced receptor phosphorylation but did affect membrane translocation of β-arrestins. The translocation of β-arrestins was blocked by cholesterol reduction, and the effect could be reversed by incubating with cholesterol. OptiPrep gradient separation of lipid rafts revealed that excess cholesterol retained more receptors in lipid raft domains and facilitated the recruitment of β-arrestins to these microdomains upon agonist activation. Moreover, excess cholesterol could evoke receptor internalization and protein kinase C-independent extracellular signal-regulated kinases activation upon morphine treatment. Therefore, these results suggest that cholesterol not only can influence OPRM1 localization in lipid rafts but also can effectively enhance the recruitment of β-arrestins and thereby affect the agonist-induced trafficking and agonist-dependent signaling of OPRM1.  

3.1643           ST101 induces a novel 17kDa APP cleavage that precludes Aβ generation in vivo

Green, K.N., Khashwji, K., Estrada, T. and LaFerla, F.M. Ann. Neurol., 69(5), 831-844 (2011)   Objective: Inhibiting Aβ generation is a prime therapeutic goal for preventing or treating Alzheimer disease. Here we sought to identify any disease-modifying properties of an azaindolizinone derivative, spiro[imidazo[1,2-a]pyridine-3,2-idan]-2(3H)-one (ST101 or ZSET1446). Methods: The effects of ST101 were studied in 3xTg-AD mice and young cynomolgus monkeys using a combination of biochemical and histological analyses. Results: Here we describe that ST101 induces cleavage of APP protein at a novel site, generating a 17kDa C-terminal fragment. This 17kDa APP cleavage product does not appear to be a substrate for either α- or β-secretase, and thus bypasses generation of Aβ. ST101 is orally active, efficacious at low doses, improves memory function, and robustly reduces brain Aβ in transgenic mice and nonhuman primates. Interpretation: Using rodent and nonhuman primate models, we show that ST101 represents a novel class of small molecules that reduce central nervous system levels of Aβ by inducing an alternate pathway of APP cleavage.  

3.1644           Heme oxygenase-1 expression protects melanocytes from stress-induced cell death: implications for vitiligo

Elassiuty, Y.E., Klarquist, J., Speiser, J., Yousef, R.M., El Refaee, A.A., Hunter, N.S., Shaker, O.G., Gundeti, M., Nieuweboer-Krobotova, L. and Le Poole, I.C. Exp. Dermatol., 20(6), 496-501 (2011)   To study protection of melanocytes from stress-induced cell death by heme oxygenases during depigmentation and repigmentation in vitiligo, expression of isoforms 1 and 2 was studied in cultured control and patient melanocytes and normal skin explants exposed to UV or bleaching agent 4-TBP. Similarly, expression of heme oxygenases was followed in skin from vitiligo patients before and after PUVA treatment. Single and double immunostainings were used in combination with light and confocal microscopic analysis and Western blotting. Melanocyte expression of heme oxygenase 1 is upregulated, whereas heme oxygenase 2 is reduced in response to UV and 4-TBP. Upregulation of inducible heme oxygenase 1 was also observed in UV-treated explant cultures, in skin of successfully PUVA-treated patients and in melanocytes cultured from vitiligo non-lesional skin. Heme oxygenase encoding genes were subsequently cloned to study consequences of either gene product on cell viability, demonstrating that HO-1 but not HO-2 overexpression offers protection from stress-induced cell death in MTT assays. HO-1 expression by melanocytes may contribute to beneficial effects of UV treatment for vitiligo patients.  

3.1645           Interferon-stimulated gene ISG12b2 is localized to the inner mitochondrial membrane and mediates virus-induced cell death

Lu, M-Y. and Liao, F. Cell Death and Differentiation, 18(6), 925-936 (2011)   Interferons (IFNs) are crucial for host defence against viruses. Many IFN-stimulated genes (ISGs) induced by viral infection exert antiviral effects. Microarray analysis of gene expression induced in liver tissues of mice on dengue virus (DENV) infection has led to identification of the ISG gene ISG12b2. ISG12b2 is also dramatically induced on DENV infection of Hepa 1-6 cells (mouse hepatoma cell line). Here, we performed biochemical and functional analyses of ISG12b2. We demonstrate that ISG12b2 is an inner mitochondrial membrane (IMM) protein containing a cleavable mitochondrial targeting sequence and multiple transmembrane segments. Overexpression of ISG12b2 in Hepa 1-6 induced release of cytochrome c from mitochondria, disruption of the mitochondrial membrane potential, and activation of caspase-9, caspase-3, and caspase-8. Treatment of ISG12b2-overexpressing Hepa 1-6 with inhibitors of pan-caspase, caspase-9, or caspase-3, but not caspase-8, reduced apoptotic cell death, suggesting that ISG12b2 activates the intrinsic apoptotic pathway. Of particular interest, we further demonstrated that ISG12b2 formed oligomers, and that ISG12b2 was able to mediate apoptosis through both Bax/Bak-dependent and Bax/Bak-independent pathways. Our study demonstrates that the ISG12b2 is a novel IMM protein induced by IFNs and regulates mitochondria-mediated apoptosis during viral infection.  

3.1646           Continuous Density Gradients to Study Argonaute and GW182 Complexes Associated with the Endocytic Pathway

Gibbings, D. Methods in Mol. Biol., 725, 63-76 (2011)   Most complexes involved in RNA silencing were thought to be concentrated in cytoplasmic sites called P-bodies in the absence of stress. Accumulating evidence suggests that distinct cellular organelles or sites may be involved in the maturation of RNA-induced silencing complexes (RISC), decapping and deadenylation of miRNA-repressed mRNA, transport of translationally repressed mRNA, and disassembly of RISC complexes. Significant fractions of proteins essential for RNA silencing associate with membranes in general (GW182, AGO, and DICER), or more specifically with endoplasmic reticulum and Golgi (AGO), or endosomes and multivesicular bodies (AGO, GW182). In contrast, mRNA decapping and decay occur mainly in the cytoplasm. Continuous density gradients capable of partitioning these cellular compartments are valuable tools in efforts to decipher the complexes, trafficking and regulation of RISC throughout its biogenesis, action and turnover.  

3.1647           Partial colocalization of retinoschisin (RS1) and the Na+/K+- ATPase subunit in membrane rafts and their role in signaling

Härtinger, T., Walczak, Y., Weber, B.H.F.and  Langmann, T. Medizinische Genetik, 23(1), P-basic-162 (2011)     Purpose Previous studies have shown that Retinoschisin (RS1) binds to the Na+/K+-ATPase within the retina. In other cell types like cardiac myo­cytes, the Na+/K+-ATPase is located in detergent resistant membranes (DRMs), which represent rafts floating on the cell surface. These mem­brane mem­brane domains can serve as platforms for receptor-ligand interactions and intracellular signaling. We hypothesized that RS1 binding to the β2-subunit of the α3β2 Na+/K+-ATPase occurs in retinal membrane rafts and thereby may regulate signaling functions. Methods To isolate DRMs from WERI-RB1 cells and mouse retinas, cells were pelleted and lysed after treatment with 2% Lubrol or Triton-X 100 in TNE buffer, respectively at 4°C. A discontinuous density gradient cen­trifugation was then performed with OptiPrep® solution. After 4 hours of centrifugation at 100.000x g, six fractions were collected from top to bottom. The proteins were precipitated using methanol/chloroform before resuspension in buffer containing 1% SDS. The samples were then analyzed by Western Blot using antibodies against the β2 subunit of the Na+/K+-ATPase, RS1 and flotillin as a DRM-marker. For signal­ing approaches retinal lysates or cryo-sections were stained with anti­bodies against phospho-ERK1/2 and p38 MAPK. Results We could successfully implement a raft isolation procedure from WERI cells and mouse retinas. DRMs were floating up in the low den­sity fractions as demonstrated by strong staining with the marker flo­tillin. Staining for the β2 subunit of the Na+/K+-ATPase also showed high signals in raft fractions from WERI-RB1 cells. In retinal samples, staining for the β2 subunit of the Na+/K+-ATPase was strong in the raft fraction, but was also present in high density fractions. We could further show that RS1 was also partially present in DRMs of WERI cells and wild-type retinas. Our results also suggest that ERK1/2 and p38 MAPK are activated in Retinoschisin-deficient and ATP1b2 knock out mice at postnatal days 14 and 10, respectively. Conclusions Our experiments demonstrate that the α3β2 subunits of Na+/K+- ATPase and RS1 partially colocalize in retinal rafts. This distribution of RS1 implicates that it has a dual function. On the one hand, RS1 may serve as a secreted adhesion molecule and on the other hand it may trigger intracellular signaling by interaction with the Na+/K+-ATPase in rafts as signaling platforms.  

3.1648           Rituximab Targets Podocytes in Recurrent Focal Segmental Glomerulosclerosis

Fornoni, A. et al Science Translational Medicine, 3(85), 85ra46 (2011)   Focal segmental glomerulosclerosis (FSGS) is a glomerular disease characterized by proteinuria, progression to end-stage renal disease, and recurrence of proteinuria after kidney transplantation in about one-third of patients. It has been suggested that rituximab might treat recurrent FSGS through an unknown mechanism. Rituximab not only recognizes CD20 on B lymphocytes, but might also bind sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b) protein and regulate acid sphingomyelinase (ASMase) activity. We hypothesized that rituximab prevents recurrent FSGS and preserves podocyte SMPDL-3b expression. We studied 41 patients at high risk for recurrent FSGS, 27 of whom were treated with rituximab at time of kidney transplant. SMPDL-3b protein, ASMase activity, and cytoskeleton remodeling were studied in cultured normal human podocytes that had been exposed to patient sera with or without rituximab. Rituximab treatment was associated with lower incidence of posttransplant proteinuria and stabilization of glomerular filtration rate. The number of SMPDL-3b⁺ podocytes in postreperfusion biopsies was reduced in patients who developed recurrent FSGS. Rituximab partially prevented SMPDL-3b and ASMase down-regulation that was observed in podocytes treated with the sera of patients with recurrent FSGS. Overexpression of SMPDL-3b or treatment with rituximab was able to prevent disruption of the actin cytoskeleton and podocyte apoptosis induced by patient sera. This effect was diminished in cultured podocytes where SMPDL-3b was silenced. Our study suggests that treatment of high-risk patients with rituximab at time of kidney transplant might prevent recurrent FSGS by modulating podocyte function in an SMPDL-3b–dependent manner.  

3.1649           DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery

Van Gaal, E.V.B., Oosting, R., van Eijk, R., Bakowska, M., Feyen, D., Kok, R.J., Hennink, W.E., Crommelin, D.J.A. and Mastrobattista, E. Pharm. Res., 28(7), 1707-1722 (2011)   Purpose To evaluate if introduction of DNA nuclear Targeting Sequences (DTS; i.e. recognition sequences for endogenous DNA-binding proteins) in plasmid DNA (pDNA) leads to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the pDNA. Methods A set of DTS was identified and cloned into EGFP-reporter plasmids controlled by the CMV-promoter. These pDNA constructs were delivered into A431 and HeLa cells using standard electroporation, pEI-based polyfection or lipofection methods. The amount of pDNA delivered into the nucleus was determined by qPCR; transfection efficiency was determined by flow cytometry. Results Neither of these DTS increased transgene expression. We varied several parameters (mitotic activity, applied dose and delivery strategy), but without effect. Although upregulated transgene expression was observed after stimulation with TNF-α, this effect could be ascribed to non-specific upregulation of transcription rather than enhanced nuclear import. Nuclear copy numbers of plasmids containing or lacking a DTS did not differ significantly after lipofectamine-based transfection in dividing and non-dividing cells. Conclusion No beneficial effects of DTS on gene expression or nuclear uptake were observed in this study.  

3.1650           ER-stress-inducible Herp, facilitates the degradation of immature nicastrin

Marutani, T., Maeda, T., Tanabe, C., Zou, K., Araki, W., Kokame, K., Michikawa, M. and Komano, H.

 Biochim. Biophys. Acta, 1810, 790-798 (2011)           Background Herp is an endoplasmic reticulum (ER)-stress-inducible membrane protein harboring an ubiquitin-like domain (ULD). However, its biological functions are not fully understood. Here, we examined the role of Herp in the degradation of γ-secretase components. Methods Effects of ULD-lacking Herp (ΔUb-Herp) expression on the degradation of γ-secretase components were analyzed. Results The cellular expression of ΔUb-Herp was found to inhibit the degradation of overexpressed immature nicastrin and full-length presenilin. The mechanisms underlying Herp-mediated nicastrin degradation was further analyzed. We found that immature nicastrin accumulates in the ER of ΔUb-Herp overexpressing cells or Herp-deficient cells more than that in the ER of wild-type cells. Further, ΔUb-Herp expression inhibited nicastrin ubiquitination, suggesting that the ULD of Herp is likely involved in nicastrin ubiquitination. Co-immunoprecipitation study showed that Herp as well as ΔUb-Herp potentially interacts with nicastrin, mediating nicastrin interaction with p97, which functions in retranslocation of misfolded proteins from the ER to the cytosol. Conclusions Thus, Herp is likely involved in degradation of immature nicastrin by facilitating p97-dependent nicastrin retranslocation and ubiquitination. General significance: We suggest that Herp could play a role in the elimination of the excess unassembled components of a multimeric complex. Research highlights ► The deletion of the ubiquitin-like domain of Herp impairs the degradation of nicastrin. ► Herp facilitates the ubiquitination of nicastrin. ► Herp plays a role for the elimination of the excess unassembled components of a multimeric complex.  

3.1651           Phr1p, a glycosylphosphatidylinsitol-anchored β(1,3)-glucanosyltransferase critical for hyphal wall formation, localizes to the apical growth sites and septa in Candida albicans

Ragni, E., Calderon, J., Fascio, U., Sipiczki, M., Fonzi, W. and  Popolo, L

Fungal Genetics and Biology, 48, 793-805 (2011)   Cell wall biogenesis is a dynamic process relying on the coordinated activity of several extracellular enzymes. PHR1 is a pH-regulated gene of Candida albicans encoding a glycosylphosphatidylinositol-anchored β(1,3)-glucanosyltransferase of family GH72 which acts as a cell wall remodelling enzyme and is crucial for morphogenesis and virulence. In order to explore the function of Phr1p, we obtained a green fluorescent protein (GFP) fusion to determine its localization. During induction of vegetative growth, Phr1p-GFP was concentrated in the plasma membrane of the growing bud, in the mother-bud neck, and in the septum. Phr1p-GFP was recovered in the detergent-resistant membranes indicating its association with the lipid rafts as the wild type Phr1p. Upon induction of hyphal growth, Phr1p-GFP highly concentrated at the apex of the germ tubes and progressively distributed along the lateral sides of the hyphae. Phr1p-GFP also labelled the hyphal septa, where it colocalized with chitin. Localization to the hyphal septa was perturbed in nocodazole-treated cells, whereas inhibition of actin polymerization hindered the apical localization. Electron Microscopy analysis of the hyphal wall ultrastructure of a PHR1 null mutant showed loss of compactness and irregular organization of the surface layer. These observations indicate that Phr1p plays a crucial role in hyphal wall formation, a highly regulated process on which morphogenesis and virulence rely.  

3.1652           Maturation of BRI2 generates a specific inhibitor that reduces APP processing at the plasma membrane and in endocytic vesicles

Matsuda, S., Matsuda, Y., Snapp, E.L. and D’Adamio, L.

Neurobiology of Aging, 32, 1400-1408 (2011)   Processing of the amyloid-β (Aβ) precursor protein (APP) has been extensively studied since it leads to production of Aβ peptides. Toxic forms of Aβ aggregates are considered the cause of Alzheimer's disease (AD). On the other end, BRI2 is implicated in APP processing and Aβ production. We have investigated the precise mechanism by which BRI2 modulates APP cleavages and have found that BRI2 forms a mature BRI2 polypeptide that is transported to the plasma membrane and endosomes where it interacts with mature APP. Notably, immature forms of APP and BRI2 fail to interact. Mature BRI2 inhibits APP processing by α-, β- and γ-secretases on the plasma membrane and in endocytic compartments. Thus, BRI2 is a specific inhibitor that reduces secretases’ access to APP in the intracellular compartments where APP is normally processed.  

3.1653           Effect of Glycans and the Glycophosphatidylinositol Anchor on Strain Dependent Conformations of Scrapie Prion Protein: Improved Purifications and Infrared Spectra

Baron, G.S., Hughson, A.G., Raymond, G.J., Offerdahl, D.K., Barton, K.A., Raymond, L.D., Dorward, D.W. and Caughey, B. Biochemistry, 50, 4479-4490 (2011)   Mammalian prion diseases involve conversion of normal prion protein, PrP^C, to a pathological aggregated state (PrP^res). The three-dimensional structure of PrP^res is not known, but infrared (IR) spectroscopy has indicated high, strain-dependent β-sheet content. PrP^res molecules usually contain a glycophosphatidylinositol (GPI) anchor and large Asn-linked glycans, which can also vary with strain. Using IR spectroscopy, we tested the conformational effects of these post-translational modifications by comparing wild-type PrP^res with GPI- and glycan-deficient PrP^res produced in GPI-anchorless PrP transgenic mice. These analyses required the development of substantially improved purification protocols. Spectra of both types of PrP^res revealed conformational differences between the 22L, ME7, and Chandler (RML) murine scrapie strains, most notably in bands attributed to β-sheets. These PrP^res spectra were also distinct from those of the hamster 263K scrapie strain. Spectra of wild-type and anchorless 22L PrP^res were nearly indistinguishable. With ME7 PrP^res, modest differences between the wild-type and anchorless spectra were detected, notably an 2 cm^–1 shift in an apparent β-sheet band. Collectively, the data provide evidence that the glycans and anchor do not grossly affect the strain-specific secondary structures of PrP^res, at least relative to the differences observed between strains, but can subtly affect turns and certain β-sheet components. Recently reported H–D exchange analyses of anchorless PrP^res preparations strongly suggested the presence of strain-dependent, solvent-inaccessible β-core structures throughout most of the C-terminal half of PrP^res molecules, with no remaining α-helix. Our IR data provide evidence that similar core structures also comprise wild-type PrP^res.  

3.1654           Enzymatic Properties and Regulation of the Native Isozymes of Retinal Membrane Guanylyl Cyclase (RetGC) from Mouse Photoreceptors

Peshenko, I.V., Olshevskaya, E.V., Savchenko, A.B., Kara, S., Palczewski, K., Baehr, W. and Dizhoor, A.M. Biochemistry, 50, 5590-5600 (2011)   Mouse photoreceptor function and survival critically depend on Ca²⁺-regulated retinal membrane guanylyl cyclase (RetGC), comprised of two isozymes, RetGC1 and RetGC2. We characterized the content, catalytic constants, and regulation of native RetGC1 and RetGC2 isozymes using mice lacking guanylyl cyclase activating proteins GCAP1 and GCAP2 and deficient for either GUCY2F or GUCY2E genes, respectively. We found that the characteristics of both native RetGC isozymes were considerably different from other reported estimates made for mammalian RetGCs: the content of RetGC1 per mouse rod outer segments (ROS) was at least 3-fold lower, the molar ratio (RetGC2:RetGC1) 6-fold higher, and the catalytic constants of both GCAP-activated isozymes between 12- and 19-fold higher than previously measured in bovine ROS. The native RetGC isozymes had different basal activity and were accelerated 5–28-fold at physiological concentrations of GCAPs. RetGC2 alone was capable of contributing as much as 135–165 μM cGMP s^–1 or almost 23–28% to the maximal cGMP synthesis rate in mouse ROS. At the maximal level of activation by GCAP, this isozyme alone could provide a significantly high rate of cGMP synthesis compared to what is expected for normal recovery of a mouse rod, and this can help explain some of the unresolved paradoxes of rod physiology. GCAP-activated native RetGC1 and RetGC2 were less sensitive to inhibition by Ca²⁺ in the presence of GCAP1 (EC_50Ca 132–139 nM) than GCAP2 (EC_50Ca 50–59 nM), thus arguing that Ca²⁺ sensor properties of GCAP in a functional RetGC/GCAP complex are defined not by a particular target isozyme but the intrinsic properties of GCAPs themselves.  

3.1655           KLP6: a newly identified kinesin that regulates the morphology and transport of mitochondria in neuronal cells

Tanaka, K., Sugiura, Y., Ichishita, R., Mihara, K. and Oka, T.

1. Cell Sci., 124(14), 2457-2465 (2011)

  Mitochondria utilize diverse cytoskeleton-based mechanisms to control their functions and morphology. Here, we report a role for kinesin-like protein KLP6, a newly identified member of the kinesin family, in mitochondrial morphology and dynamics. An RNA interference screen using Caenorhabditis elegans led us to identify a C. elegans KLP-6 involved in maintaining mitochondrial morphology. We cloned a cDNA coding for a rat homolog of C. elegans KLP-6, which is an uncharacterized kinesin in vertebrates. A rat KLP6 mutant protein lacking the motor domain induced changes in mitochondrial morphology and significantly decreased mitochondrial motility in HeLa cells, but did not affect the morphology of other organelles. In addition, the KLP6 mutant inhibited transport of mitochondria during anterograde movement in differentiated neuro 2a cells. To date, two kinesins, KIF1Bα and kinesin heavy chain (KHC; also known as KIF5) have been shown to be involved in the distribution of mitochondria in neurons. Expression of the kinesin heavy chain/KIF5 mutant prevented mitochondria from entering into neurites, whereas both the KLP6 and KIF1Bα mutants decreased mitochondrial transport in axonal neurites. Furthermore, both KLP6 and KIF1Bα bind to KBP, a KIF1-binding protein required for axonal outgrowth and mitochondrial distribution. Thus, KLP6 is a newly identified kinesin family member that regulates mitochondrial morphology and transport.  

3.1656           Aldolase directly interacts with ARNO and modulates cell morphology and acidic vesicle distribution

Merkulova, M., Hurtado-Lorenzo, A., Hosokawa, H., Zhuang, Z., Brown, D., Ausiello, D.A. and Marshansky, V. Am. J. Physiol. Cell Physiol., 300, C1442-C1455 (2011)   Previously, we demonstrated that the vacuolar-type H⁺-ATPase (V-ATPase) a2-subunit functions as an endosomal pH sensor that interacts with the ADP-ribosylation factor (Arf) guanine nucleotide exchange factor, ARNO. In the present study, we showed that ARNO directly interacts not only with the a2-subunit but with all a-isoforms (a1–a4) of the V-ATPase, indicating a widespread regulatory interaction between V-ATPase and Arf GTPases. We then extended our search for other ARNO effectors that may modulate V-ATPase-dependent vesicular trafficking events and actin cytoskeleton remodeling. Pull-down experiments using cytosol of mouse proximal tubule cells (MTCs) showed that ARNO interacts with aldolase, but not with other enzymes of the glycolytic pathway. Direct interaction of aldolase with the pleckstrin homology domain of ARNO was revealed by pull-down assays using recombinant proteins, and surface plasmon resonance revealed their high avidity interaction with a dissociation constant: K_D = 2.84 × 10⁻¹⁰ M. MTC cell fractionation revealed that aldolase is also associated with membranes of early endosomes. Functionally, aldolase knockdown in HeLa cells produced striking morphological changes accompanied by long filamentous cell protrusions and acidic vesicle redistribution. However, the 50% knockdown we achieved did not modulate the acidification capacity of endosomal/lysosomal compartments. Finally, a combination of small interfering RNA knockdown and overexpression revealed that the expression of aldolase is inversely correlated with gelsolin levels in HeLa cells. In summary, we have shown that aldolase forms a complex with ARNO/Arf6 and the V-ATPase and that it may contribute to remodeling of the actin cytoskeleton and/or the trafficking and redistribution of V-ATPase-dependent acidic compartments via a combination of protein-protein interaction and gene expression mechanisms.  

3.1657           Subcellular Fractionation of Brain Tissue Using Free-Flow Electrophoresis

Islinger, M., Kirsch, J., Angermüller, S., Rotaru, R., Abdolzade-Bavil, A. and Weber, G. Nueromethods, 57(2), 27-45 (2011)   Accurate annotation of protein identifications in organellar proteomics highly depends on the sample quality with special respect to contaminations from other subcellular compartments. In this respect, Freeflow electrophoresis (FFE) offers a valuable alternative to classical centrifugation techniques, since it relies on quite different physical parameters. During the last years, FFE has been successfully used for the separation of various organelles from different tissues, yet is largely unknown in the field of neurobiology. Here we present two separation schemes for the fractionation of a synaptic preparation from rat brain using different modes of FFE. Isotachophoresis (ITP), a focusing technique separating organelles according to their electrophoretic mobilities, was able to distribute the synaptosome sample into different subfractions: mitochondrial cross contaminations showed the highest electrophoretic mobility and migrated nearest to the anode of the FFE instrument; proximate to these, proteins of the presynaptic compartment accumulated, whereas nearest to the cathode of the instrument postsynaptic marker proteins were predominantly found. As a nonfocusing technique, zonal FFE does not possess a separation capacity comparable to ITP; however, due to a continuous separation mode, it is adapted to process higher sample amounts and can be used for large-scale separations. We applied zonal FFE to the same starting material as in ITP and were able to separate mitochondria from synaptic material of the preparation, thus offering a fast alternative to clean synaptosome preparations from residual mitochondrial contaminations.  

3.1658           Methods to Monitor Cell Surface Expression and Endocytic Trafficking of CFTR in Polarized Epithelial Cells

Bomberger, J.M., Guggino, W.B. and Stanton, B.A. Methods in Mol. Biol., 741(3), 271-283 (2011)   Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride secretion is critical to maintaining airway surface hydration and efficient mucociliary clearance in the upper airways. Mutations in CFTR in cystic fibrosis lead to reduced expression of functional CFTR channels at the apical plasma membrane of the airway epithelium, leading to dehydration of the airway surface liquid and diminished mucociliary clearance. Cell surface CFTR is modulated by changes in CFTR endocytosis and recycling, effectively altering the cell surface abundance of the channel. This chapter examines current methods employed to measure the cell surface expression of CFTR, as well as methods to monitor CFTR movement through the endocytic pathway.  

3.1659           Chemical chaperone therapy: chaperone effect on mutant enzyme and cellular pathophysiology in β-galactosidase deficiency

Higaki, K. et al Hum. Mutat, 32, 843-852 (2011)   β-Galactosidase deficiency is a group of lysosomal lipid storage disorders with an autosomal recessive trait. It causes two clinically different diseases, G_M1-gangliosidosis and Morquio B disease. It is caused by heterogeneous mutations in the GLB1 gene coding for the lysosomal acid β-galactosidase. We have previously reported the chaperone effect of N-octyl-4-epi-β-valienamine (NOEV) on mutant β-galactosidase proteins. In this study, we performed genotype analyses of patients with β-galactosidase deficiency and identified 46 mutation alleles including 9 novel mutations. We then examined the NOEV effect on mutant β-galactosidase proteins by using six strains of patient-derived skin fibroblast. We also performed mutagenesis to identify β-galactosidase mutants that were responsive to NOEV and found that 22 out of 94 mutants were responsive. Computational structural analysis revealed the mode of interaction between human β-galactosidase and NOEV. Moreover, we confirmed that NOEV reduced G_M1 accumulation and ameliorated the impairments of lipid trafficking and protein degradation in β-galactosidase deficient cells. These results provided further evidence to NOEV as a promising chaperone compound for β-galactosidase deficiency  

3.1660           Proteomic analysis of microvesicles derived from human colorectal cancer ascites

Choi, D-S. et al Proteomics, 11(13), 2745-2751 (2011)   The presence of malignant ascites in the peritoneal cavity is a poor prognostic indicator of low survival rate. Various cancer cells, including those of colorectal cancer (CRC), release microvesicles (exosomes) into surrounding tissues and peripheral circulation including malignant ascites. Although recent progress has revealed that microvesicles play multiple roles in tumor progression, the protein composition and the pathological function of malignant ascites-derived microvesicles are still unknown. Here, we report the first global proteomic analyses of highly purified microvesicles derived from human CRC ascites. With 1-D SDS-PAGE and nano-LC-MS/MS analyses, we identified a total of 846 microvesicular proteins from ascites of three CRC patients with high confidence; 384 proteins were identified in at least two patients. We identified proteins that might function in tumor progression via disruption of epithelial polarity, migration, invasion, tumor growth, immune modulation, and angiogenesis. Furthermore, we identified several potential diagnostic markers of CRC including colon-specific surface antigens. Our proteomic analyses will help to elucidate diverse functions of microvesicles in cancer progression and will aid in the development of novel diagnostic tools for CRC.  

3.1661           Dietary Fatty Acid in Rat Intestinal Cytosol is Associated With Caveolae

Siddiqi, S. and Mansbach, C.M. Gastroenterology, 140(5), Suppl. 1, S-452 (2011)   The intestinal absorptive cell has no control over the amount of fatty acid (FA) presented to it. In humans, intestinal luminal FA concentrations after a fatty meal are 28 mM. We show here that rat proximal intestinal sacs incubated with 1 mM oleate have 1.6 μmol FA/ mg cytosolic protein whereas it has been shown that after a fat meal, rats have only 10 and 2 fmol FA/mg cytosolic protein bound to liver (LFABP) and intestinal fatty acid binding proteins (IFABP). This raises the question of another binding mechanism for FA in cytosol to prevent membrane disruption by FA. Caveolin-1 containing lipid rafts, caveolae, present an alternative FA binding mechanism. Caveolae occupy 50% of the microvillus membrane presenting a large potential absorptive area. Methods: Intestinal sacs from rat proximal . intestine were incubated (370C) with TriacinC for 15 min to inhibit triglyceride synthesis, 1 mM 3H-oleate was added for 2 min and the sac placed on ice. Cytosol was isolated from sac enterocytes and chromatographed over a Sephacryl S-100 HR column or placed under an OptiPrep gradient. Results: Only 5% of the 3H-oleate but 60% of the protein was recovered from the Sepharose column with PBS as eluent whereas 95% of the 3H-oleate was recovered with 1% Triton X-100 as eluent. All the dpm were in the early eluting fractions suggesting the 3H-oleate was associated with a detergent resistant membrane (DRM) rather than FABP. On the OptiPrep gradient, 57% of the dpm were in the light fractions confirming a DRM location of oleate. The light fraction contained Caveolin-1,-2,-3, CD36, and intestinal alkaline phosphatase (IAP) but not L or IFABP, rab11, or fatty acid transport protein 4 (FATP4) by immunoblot. Immunodepletion of Caveolin-1 removed 90% of the 3H-oleate from the cytosol whereas CD36 and IAP immunodepletion removed 50%. Caveolin-2,-3, IgG, and clathrin immunodepletion removed only 20%. Immuno-precipitation (IP) of Caveolin-1 co-precipitated CD36 and IAP. IP of CD36 co-precipitated Caveolin-1 and IAP; IP of IAP precipitated CD36 and Caveolin-1. Electron microscopy of cytosol showed multiple 18.9}2.9 nm vesicles that increased to 33.4}1.6 nm (p<0.0001) on exposure of the sacs to 1 mM oleate. Immunogold-EM using anti-Caveolin-1 antibodies showed multiple beads over only vesicles in both the fasted and 1 mM oleate fed cytosol. No beads were seen if IgG was used. Conclusions: Absorbed dietary FA are present in rat intestinal cytosol in Caveolin-1 containing lipid rafts not in FABP as has been proposed previously. The FA are thus sequestered from interacting with enterocyte membranes. We speculate that the FA enter the caveolae at the apical membrane, the caveolae detach from the apical membrane to enter the cytosol and are then targeted to the endoplasmic reticulum (ER) for incorporation into complex lipids.  

3.1662           Effector Granules in Human T Lymphocytes: Proteomic Evidence for Two Distinct Species of Cytotoxic Effector Vesicles

Schmidt, H., Gelhaus, C., Nebendahl, M., Lettau, M., Lucius, R., Leippe, M., Kabelitz, D. and janssen, O.

1. Proteome Res., 10(4), 1603-1620 (2011)

  Cytotoxic T cells mobilize effector proteins from prestored lysosomal compartments. Since different activation signals result in alternative routes of target cell killing, utilizing either FasL or the granzyme B/perforin pathway, the existence of distinct forms of effector granules was recently suggested. Applying a protocol for the separation of intact organelles from activated T lymphoblasts, we noticed that FasL-associated secretory lysosomes (SL) segregate from vesicles containing larger amounts of granzymes and granulysin. We previously analyzed the proteome of secretory lysosomes from NK and T cells and now describe the proteome of granzyme-containing vesicles. Moreover, intact FasL-associated SL and granzyme-containing vesicles were compared by electron microscopy and respective extracts were characterized by Western blotting. With the present report, we provide a comprehensive proteome map of granzyme-containing granules and unequivocally demonstrate that T lymphoblasts contain at least two distinct types of effector vesicles. Moreover, the overall protein content of the two vesicle populations was compared by 2D difference gel electrophoresis. Interestingly, the observed differences in protein distribution were not restricted to effector proteins but also applied to cytoskeleton-associated elements that could argue for a differential transport or initiation of degranulation. To our knowledge, this is the first comprehensive description of distinct effector granules in T cells.  

3.1663           Identification, characterization and regulation studies of the aconitase of Paracoccidioides brasiliensis

De A. Brito, W., Rezende, T.C.V., Parente, A.F., Ricart, C.A.O., de Sousa, M.V., Bao, S.N. and de A. Soares, C.M. Fungal Biol., 115, 697-707 (2011)   A protein species preferentially expressed in yeast cells with a molecular mass of 80 kDa and isoeletric point (pI) of 7.79 was isolated from the proteome of Paracoccidioides brasiliensis and characterized as an aconitase (ACO) (E.C. 4.2.1.3). ACO is an enzyme that catalyzes the isomerization of citrate to isocitrate in both the Krebs cycle and the glyoxylate cycle. We report the cloning and characterization of the cDNA encoding the ACO of P. brasiliensis (PbACO). The cDNA showed a 2361 bp open reading frame (ORF) and encoded a predicted protein with 787 amino acids. Polyclonal antibodies against the purified recombinant PbACO was obtained in order to analyze the subcellular localization of the molecule in P. brasiliensis. The protein is present in the extracellular fluid, cell wall enriched fraction, mitochondria, cytosol and peroxisomes of yeast cells as demonstrated by western blot and immunocytochemistry analysis. The expression analysis of the Pbaco gene was performed by quantitative real-time RT-PCR and results demonstrated an increased expression in yeast cells compared to mycelia. Real-time RT-PCR assays was also used to evaluate the Pbaco expression when the fungus grows on media with acetate and ethanol as sole carbon sources and in different iron levels. The results demonstrated that Pbaco transcript is over expressed in acetate and ethanol as sole carbon sources and in high-iron conditions.  

3.1664           Challenges and solutions for the identification of membrane proteins in non-model plants

Vertommen, A., Panis, B., Swennen, R. and Carpentier, S.C.

1. Proteomics, 74, 1165-1181 (2011)

  The workhorse for proteomics in non-model plants is classical two-dimensional electrophoresis, a combination of iso-electric focusing and SDS-PAGE. However, membrane proteins with multiple membrane spanning domains are hardly detected on classical 2-DE gels because of their low abundance and poor solubility in aqueous media. In the current review, solutions that have been proposed to handle these two problems in non-model plants are discussed. An overview of alternative techniques developed for membrane proteomics is provided together with a comparison of their strong and weak points. Subsequently, strengths and weaknesses of the different techniques and methods to evaluate the identification of membrane proteins are discussed. Finally, an overview of recent plant membrane proteome studies is provided with the used separation technique and the number of identified membrane proteins listed.  

3.1665           Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles

György, B., Szabo, T.G., Pasztoi, M., Pal, Z., Misjak, P., Aradi, B., Laslo, V., Pallinger, E., Pap, E., Kittel, A., Nagy, G., Falus, A. and Buzas, E.I. Celll. Mol. Life Sci., 68(16), 2667-2688 (2011)   Abstract Release of membrane vesicles, a process conserved in both prokaryotes and eukaryotes, represents an evolutionary link, and suggests essential functions of adynamic extracellular vesicular compartment (including exosomes, microparticles or microvesicles and apoptotic bodies). Compelling evidence supports the significance of this compartment in a broad range of physiological and pathological processes. However, classification of membrane vesicles, protocols of their isolation and detection, molecular details of vesicular release, clearance and biological functions are still under intense investigation. Here, we give a comprehensive overview of extracellular vesicles. After discussing the technical pitfalls and potential artifacts of the rapidly emerging field, we compare results from meta-analyses of published proteomic studies on membrane vesicles. We also summarize clinical implications of membrane vesicles. Lessons from this compartment challenge current paradigms concerning the mechanisms of intercellular communication and immune regulation. Furthermore, its clinical implementation may open new perspectives in translational medicine both in diagnostics and therapy.  

3.1666           A Concerted Action of Hepatitis C Virus P7 and Nonstructural Protein 2 Regulates Core Localization at the Endoplasmic Reticulum and Virus Assembly

Boson, B., Granio, O., Bartenschlager, R. and Cosset, F-L. PloS Pathogens, 7(7), e1002144 (2011)   Hepatitis C virus (HCV) assembly remains a poorly understood process. Lipid droplets (LDs) are thought to act as platforms for the assembly of viral components. The JFH1 HCV strain replicates and assembles in association with LD-associated membranes, around which viral core protein is predominantly detected. In contrast, despite its intrinsic capacity to localize to LDs when expressed individually, we found that the core protein of the high-titer Jc1 recombinant virus was hardly detected on LDs of cell culture-grown HCV (HCVcc)-infected cells, but was mainly localized at endoplasmic reticulum (ER) membranes where it colocalized with the HCV envelope glycoproteins. Furthermore, high-titer cell culture-adapted JFH1 virus, obtained after long-term culture in Huh7.5 cells, exhibited an ER-localized core in contrast to non-adapted JFH1 virus, strengthening the hypothesis that ER localization of core is required for efficient HCV assembly. Our results further indicate that p7 and NS2 are HCV strain-specific factors that govern the recruitment of core protein from LDs to ER assembly sites. Indeed, using expression constructs and HCVcc recombinant genomes, we found that p7 is sufficient to induce core localization at the ER, independently of its ion-channel activity. Importantly, the combined expression of JFH1 or Jc1 p7 and NS2 induced the same differential core subcellular localization detected in JFH1- vs. Jc1-infected cells. Finally, results obtained by expressing p7-NS2 chimeras between either virus type indicated that compatibilities between the p7 and the first NS2 trans-membrane domains is required to induce core-ER localization and assembly of extra- and intra-cellular infectious viral particles. In conclusion, we identified p7 and NS2 as key determinants governing the subcellular localization of HCV core to LDs vs. ER and required for initiation of the early steps of virus assembly.  

3.1667           Isolation of detergent-resistant membranes from plant photosynthetic and non-photosynthetic tissues

Carmona-Salazar, L., El Hafidi, M., Enriquez-Arredondo, C., Vazquez-Vazquez, C., Gonzales de la Vera, C. and Gavilanes-Ruiz, M. Anal. Biochem., 417, 220-227 (2011)   Microdomains, or lipid rafts, are transient membrane regions enriched in sphingolipids and sterols that have only recently, but intensively, been studied in plants. In this work, we report a detailed, easy-to-follow, and fast procedure to isolate detergent-resistant membranes (DRMs) from purified plasma membranes (PMs) that was used to obtain DRMs from Phaseolus vulgaris and Nicotiana tabacum leaves and germinating Zea mays embryos. Characterized according to yield, ultrastructure, and sterol composition, these DRM preparations showed similarities to analogous preparations from other eukaryotic cells. Isolation of DRMs from germinating maize embryos reveals the presence of microdomains at very early developmental stages of plants.  

3.1668           An N-terminal Polybasic Domain and Cell Surface Localization Are Required for Mutant Prion Protein Toxicity

Solomon, I.H., Khatri, N., Biasini, E., Massignan, T., Huettner, J.E. and Harris, D.A.

1. Biol. Chem., 286(16), 14724-14736 (2011)

  There is evidence that alterations in the normal physiological activity of PrP^C contribute to prion-induced neurotoxicity. This mechanism has been difficult to investigate, however, because the normal function of PrP^C has remained obscure, and there are no assays available to measure it. We recently reported that cells expressing PrP deleted for residues 105–125 exhibit spontaneous ionic currents and hypersensitivity to certain classes of cationic drugs. Here, we utilize cell culture assays based on these two phenomena to test how changes in PrP sequence and/or cellular localization affect the functional activity of the protein. We report that the toxic activity of Δ105–125 PrP requires localization to the plasma membrane and depends on the presence of a polybasic amino acid segment at the N terminus of PrP. Several different deletions spanning the central region as well as three disease-associated point mutations also confer toxic activity on PrP. The sequence domains identified in our study are also critical for PrP^Sc formation, suggesting that common structural features may govern both the functional activity of PrP^C and its conversion to PrP^Sc.  

3.1669           Ubp15p, a Ubiquitin Hydrolase Associated with the Peroxisomal Export Machinery

Debelyy, M.O., Platta, H.W., Saffian, D., hensel, A., thoms, S., Meyer, H.E., Warschied, B., Gizalsky, W. and Erdmann, R.

1. Biol. Chem., 286(32), 28223-28234 (2011)

  Peroxisomal matrix protein import is facilitated by cycling receptors shuttling between the cytosol and the peroxisomal membrane. One crucial step in this cycle is the ATP-dependent release of the receptors from the peroxisomal membrane. This step is facilitated by the peroxisomal AAA (ATPases associated with various cellular activities) proteins Pex1p and Pex6p with ubiquitination of the receptor being the main signal for its export. Here we report that the AAA complex contains dislocase as well as deubiquitinating activity. Ubp15p, a ubiquitin hydrolase, was identified as a novel constituent of the complex. Ubp15p partially localizes to peroxisomes and is capable of cleaving off ubiquitin moieties from the type I peroxisomal targeting sequence (PTS1) receptor Pex5p. Furthermore, Ubp15p-deficient cells are characterized by a stress-related PTS1 import defect. The results merge into a picture in which removal of ubiquitin from the PTS1 receptor Pex5p is a specific event and might represent a vital step in receptor recycling.  

3.1670           Urban planning of the endoplasmic reticulum (ER): How diverse mechanisms segregate the many functions of the ER

Lynes, E.M. and Simmen, T. Biochim. Biophys. Acta, 1813, 1893-1905 (2011)   The endoplasmic reticulum (ER) is the biggest organelle in most cell types, but its characterization as an organelle with a continuous membrane belies the fact that the ER is actually an assembly of several, distinct membrane domains that execute diverse functions. Almost 20 years ago, an essay by Sitia and Meldolesi first listed what was known at the time about domain formation within the ER. In the time that has passed since, additional ER domains have been discovered and characterized. These include the mitochondria-associated membrane (MAM), the ER quality control compartment (ERQC), where ER-associated degradation (ERAD) occurs, and the plasma membrane-associated membrane (PAM). Insight has been gained into the separation of nuclear envelope proteins from the remainder of the ER. Research has also shown that the biogenesis of peroxisomes and lipid droplets occurs on specialized membranes of the ER. Several studies have shown the existence of specific marker proteins found on all these domains and how they are targeted there. Moreover, a first set of cytosolic ER-associated sorting proteins, including phosphofurin acidic cluster sorting protein 2 (PACS-2) and Rab32 have been identified. Intra-ER targeting mechanisms appear to be superimposed onto ER retention mechanisms and rely on transmembrane and cytosolic sequences. The crucial roles of ER domain formation for cell physiology are highlighted with the specific targeting of the tumor metastasis regulator gp78 to ERAD-mediating membranes or of the promyelocytic leukemia protein to the MAM.  

3.1671           A stable yeast strain efficiently producing cholesterol instead of ergosterol is functional for tryptophan uptake, but not weak organic acid resistance

Souza, C.M., Schwabe, T.M.E., Pichler, H., Ploier, B., Leitner, E., Guan, X.L., Wenk, M.R., Riezman, I. and Riezman, H. Metabolic Engineering, 13, 555-569 (2011)   Sterols are major lipids in eukaryotes and differ in their specific structure between species. Both cholesterol and ergosterol can form liquid ordered domains in artificial membranes. We reasoned that substituting the main sterol ergosterol by cholesterol in yeast should permit domain formation and discriminate between physical and sterol structure-dependent functions. Using a cholesterol-producing yeast strain, we show that solute transporters for tryptophan and arginine are functional, whereas the export of weak organic acids via Pdr12p, a multi-drug resistance family member, is not. The latter reveals a sterol function that is probably dependent upon a precise sterol structure. We present a series of novel yeast strains with different sterol compositions as valuable tools to characterize sterol function and use them to refine the sterol requirements for Pdr12p. These strains will also be improved hosts for heterologous expression of sterol-dependent proteins and safe sources to obtain pure cholesterol and other sterols.  

3.1672           Fatty Acid and Peptide Profiles in Plasma Membrane and Membrane Rafts of PUFA Supplemented RAW264.7 Macrophages

Schumann, J., Leichtle, A., Thiery, J. and Fuhrmann, H. PloS One, 6(8), e24066 (2011)   The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers.  

3.1673           Specific binding of activated Vip3Aa10 to Helicoverpa armigera brush border membrane vesicles results in pore formation

Liu, J-G., Yang, A-Z., Shen, X-H., Hua, B-G. and Shi, G-L.

1. Invertebrate Pathol., 108, 92-97 (2011)

  Helicoverpa armigera is one of the most harmful pests in China. Although it had been successfully controlled by Cry1A toxins, some H. armigera populations are building up resistance to Cry1A toxins in the laboratory. Vip3A, secreted by Bacillus thuringiensis, is another potential toxin against H. armigera. Previous reports showed that activated Vip3A performs its function by inserting into the midgut brush border membrane vesicles (BBMV) of susceptible insects. To further investigate the binding of Vip3A to BBMV of H. armigera, the full-length Vip3Aa10 toxin expressed in Escherichia coli was digested by trypsin or midgut juice extract, respectively. Among the fragments of digested Vip3Aa10, only a 62 kDa fragment (Vip3Aa10-T) exhibited binding to BBMV of H. armigera and has insecticidal activity. Moreover, this interaction was specific and was not affected by the presence of Cry1Ab toxin. Binding of Vip3Aa10-T to BBMV resulted in the formation of an ion channel. Unlike Cry1A toxins, Vip3Aa10-T was just slightly associated with lipid rafts of BBMV. These data suggest that although activated Vip3Aa10 specifically interacts with BBMV of H. armigera and forms an ion channel, the mode of action of it may be different from that of Cry1A toxins.  

3.1674           Biochemical Characterization of APPL Endosomes: The Role of Annexin A2 in APPL Membrane Recruitment

Urbanska, A., Sadowski, L., Kalaidzidis, Y. and Miaczynska, M. Traffic, 12(9), 1227-1241 (2011)   APPL endosomes are a recently identified subpopulation of early endosomes characterized by the presence of two homologous Rab5 effector proteins APPL1 and APPL2. They exhibit only limited colocalization with EEA1, another Rab5 effector and a marker of the canonical early endosomes. Although APPL endosomes appear to play important roles in cargo trafficking and signal transduction, their protein composition and biochemical properties remain largely unknown. Here we employed membrane fractionation methods to characterize APPL endosomes biochemically. We demonstrate that they represent heterogeneous membrane structures which can be discriminated from the canonical EEA1-positive early endosomes by their partly different physical properties and a distinct migration pattern in the continuous density gradients. In search for other potential markers of APPL endosomes we identified Annexin A2 as an interacting partner of both APPL1 and APPL2. Annexin A2 is a Ca²⁺ and phosphatidylinositol 4,5-bisphosphate binding protein, previously implicated in several endocytic steps. We show that Annexin A2 co-fractionates and colocalizes with APPL endosomes. Moreover, silencing of its expression causes solubilization of APPL2 from endosomes. Although Annexin A2 is not an exclusive marker of APPL endosomes, our data suggest that it has an important function in membrane recruitment of APPL proteins, acting in parallel to Rab5.  

3.1675           Curcumin Inhibition of the Functional Interaction between Integrin α6β4 and the Epidermal Growth Factor Receptor

Soung, Y.H: and Chung, J. Mol. Cancer Ther., 10, 883-891 (2011)   The functional interaction between integrin α6β4 and growth factor receptors has been implicated in key signaling pathways important for cancer cell function. However, few attempts have been made to selectively target this interaction for therapeutic intervention. Previous studies showed that curcumin, a yellow pigment isolated from turmeric, inhibits integrin α6β4 signaling important for breast carcinoma cell motility and invasion, but the mechanism is not currently known. To address this issue, we tested the hypothesis that curcumin inhibits the functional interaction between α6β4 and the epidermal growth factor receptor (EGFR). In this study, we found that curcumin disrupts functional and physical interactions between α6β4 and EGFR, and blocks α6β4/EGFR-dependent functions of carcinoma cells expressing the signaling competent form of α6β4. We further showed that curcumin inhibits EGF-dependent mobilization of α6β4 from hemidesmosomes to the leading edges of migrating cells such as lammelipodia and filopodia, and thereby prevents α6β4 distribution to lipid rafts where functional interactions between α6β4 and EGFR occur. These data suggest a novel paradigm in which curcumin inhibits α6β4 signaling and functions by altering intracellular localization of α6β4, thus preventing its association with signaling receptors such as EGFR.  

3.1676           Plant organelle proteomics: Collaborating for optimal cell function

Agrawal, G.K: et al Mass Spectrometry Reviews, 30(5), 772-853 (2011)   Organelle proteomics describes the study of proteins present in organelle at a particular instance during the whole period of their life cycle in a cell. Organelles are specialized membrane bound structures within a cell that function by interacting with cytosolic and luminal soluble proteins making the protein composition of each organelle dynamic. Depending on organism, the total number of organelles within a cell varies, indicating their evolution with respect to protein number and function. For example, one of the striking differences between plant and animal cells is the plastids in plants. Organelles have their own proteins, and few organelles like mitochondria and chloroplast have their own genome to synthesize proteins for specific function and also require nuclear-encoded proteins. Enormous work has been performed on animal organelle proteomics. However, plant organelle proteomics has seen limited work mainly due to: (i) inter-plant and inter-tissue complexity, (ii) difficulties in isolation of subcellular compartments, and (iii) their enrichment and purity. Despite these concerns, the field of organelle proteomics is growing in plants, such as Arabidopsis, rice and maize. The available data are beginning to help better understand organelles and their distinct and/or overlapping functions in different plant tissues, organs or cell types, and more importantly, how protein components of organelles behave during development and with surrounding environments. Studies on organelles have provided a few good reviews, but none of them are comprehensive. Here, we present a comprehensive review on plant organelle proteomics starting from the significance of organelle in cells, to organelle isolation, to protein identification and to biology and beyond. To put together such a systematic, in-depth review and to translate acquired knowledge in a proper and adequate form, we join minds to provide discussion and viewpoints on the collaborative nature of organelles in cell, their proper function and evolution.  

3.1677           Function of MRP1/ABCC1 is not dependent on cholesterol or cholesterol-stabilized lipid rafts

Meszaros, P., Klappe, K., Hummel, I., Hoekstra, D. and Kok, J.W. Biochem. J., 437, 483-491 (2011)   MRP1 (multidrug-resistance-related protein 1)/ABCC1 (ATP-binding cassette transporter C1) has been localized in cholesterol-enriched lipid rafts, which suggests a role for these lipid rafts and/or cholesterol in MRP1 function. In the present study, we have shown for the first time that nearly complete oxidation of free cholesterol in the plasma membrane of BHK-MRP1 (MRP1-expressing baby hamster kidney) cells did not affect MRP1 localization in lipid rafts or its efflux function, using 5-carboxyfluorescein diacetate as a substrate. Inhibition of cholesterol biosynthesis, using lovastatin in combination with RO 48-8071, an inhibitor of oxidosqualene cyclase, resulted in a shift of MRP1 out of lipid raft fractions, but did not affect MRP1-mediated efflux in Neuro-2a (neuroblastoma) cells. Short-term methyl-β-cyclodextrin treatment was equally effective in removing free cholesterol from Neuro-2a and BHK-MRP1 cells, but affected MRP1 function only in the latter. The kinetics of loss of both MRP1 efflux function and lipid raft association during long-term methyl-β-cyclodextrin treatment did not match the kinetics of free cholesterol removal in both cell lines. Moreover, MRP1 activity was measured in vesicles consisting of membranes isolated from BHK-MRP1 cells using the substrate cysteinyl leukotriene C₄ and was not changed when the free cholesterol level of these membranes was either decreased or increased. In conclusion, MRP1 activity is not correlated with the level of free cholesterol or with localization in cholesterol-dependent lipid rafts.  

3.1678           Differential, Type I Interferon-Mediated Autophagic Trafficking of Hepatitis C Virus Proteins in Mouse Liver

Desai, M.M., Gong, B., Chan, T., Davey, R.A., Soong, L., Kolokoltsov, A.A. and Sun, J. Gastroenterology, 141(2), 674-685 (2011)   Background & Aims The hepatitis C virus (HCV) serine protease NS3/4A can cleave mitochondria-associated antiviral signaling protein (MAVS) and block retinoic acid–inducible gene I–mediated interferon (IFN) responses. Although this mechanism is thought to have an important role in HCV-mediated innate immunosuppression, its significance in viral persistence is not clear. Methods We generated transgenic mice that express the HCV NS3/4A proteins specifically in the liver and challenged the animals with a recombinant vesicular stomatitis virus, a synthetic HCV genome, IFN alfa, or IFN beta. We evaluated the effects of HCV serine protease on the innate immune responses and their interactions. Results Expression of HCV NS3/4A resulted in cleavage of intrahepatic MAVS; challenge of transgenic mice with vesicular stomatitis virus or a synthetic HCV genome induced strong, type I IFN-mediated responses that were not significantly lower than those of control mice. Different challenge agents induced production of different ratios of IFN alfa and beta, resulting in different autophagic responses and vesicular trafficking patterns of endoplasmic reticulum- and mitochondria-associated viral proteins. IFN beta promoted degradation of the viral proteins by the autolysosome. Variant isoforms of MAVS were associated with distinct, type I IFN-mediated autophagic responses; these responses have a role in trafficking of viral components to endosomal compartments that contain Toll-like receptor–3. Conclusions IFN beta mediates a distinct autophagic mechanism of antiviral host defense. MAVS has an important role in type I IFN-induced autophagic trafficking of viral proteins.  

3.1679           Positive Feedback Loop Between PI3K-Akt-mTORC1 Signaling and the Lipogenic Pathway Boosts Akt Signaling: Induction of the Lipogenic Pathway by a Melanoma Antigen

Yamauchi, Y., Furukawa, K, Hamamura, K. et al Cancer Res., 71, 4989-4997 (2011)   The lipogenic phenotype is a metabolic hallmark of cancer cells. Sterol regulatory element–binding proteins (SREBP) are key transcriptional factors to regulate biosynthesis of cholesterol and fatty acids. It has been poorly understood how the lipogenic phenotype in cancer cells is regulated and how it augments their malignant properties. Here we describe roles of the melanoma antigen ganglioside GD3 and phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR complex 1 (mTORC1) signaling in the regulation of SREBP activity, cholesterol biosynthesis, and the integrity of lipid rafts in human melanoma cells. GD3 expression induced the activation of both SREBP-1 and SREBP-2. Consequently, HMG-CoA reductase expression and cholesterol biosynthesis increased. The activation of the SREBP pathway was independent of the oncogenic BRAF mutation. On the other hand, it was regulated by PI3K-Akt-mTORC1 signaling in human melanoma cells. Disruption of the signaling pathway resulted in the reduction of cholesterol in lipid rafts. Inhibition of the SREBP pathway attenuated Akt activation in lipid rafts and suppressed the growth of human melanoma cells in vitro and in vivo. These results suggest that PI3K-Akt-mTORC1 signaling is important for the integrity of lipid rafts by regulating SREBP activation and subsequent cholesterogenesis. We thus propose a positive feedback circuit in which PI3K-Akt-mTORC1-SREBP signaling boosts Akt signaling in human melanoma cells expressing GD3.  

3.1680           HIV-1 gp41 ectodomain enhances Cryptococcus neoformans binding to human brain microvascular endothelial cells via gp41 core-induced membrane activities

Huang, S-H., Wu, C-H., Jiang, S., Bahner, I., Lossinsky, A.S. and Jong, A.Y. Biochem. J., 438, 457-466 (2011)   Cryptococcus neoformans causes life-threatening meningoencephalitis, particularly prevalent in AIDS patients. The interrelationship between C. neoformans and HIV-1 is intriguing, as both pathogens elicit severe neuropathological complications. We have previously demonstrated that the HIV-1 gp41 ectodomain fragments gp41-I33 (amino acids 579–611) and gp41-I90 (amino acids 550–639) can enhance C. neoformans binding to HBMECs (human brain microvascular endothelial cells). Both peptides contain the loop region of gp41. In the present study, we used immunofluorescence microscopy and transmission and scanning electron microscopy to explore the underlying mechanisms. Our findings indicated that both C. neoformans and gp41-I90 up-regulated ICAM-1 (intercellular adhesion molecule 1) on the HBMECs and elicited membrane ruffling on the surface of HBMECs. The HIV-1 gp41 ectodomain could also induce CD44 and β-actin redistribution to the membrane lipid rafts, but it could not enhance PKCα (protein kinase Cα) phosphorylation like C. neoformans. Instead, gp41-I90 was able to induce syncytium formation on HBMECs. The results of the present study suggest HIV-1 gp41-enhanced C. neoformans binding to HBMECs via gp41 core domain-induced membrane activities, revealing a potential mechanism of invasion for this pathogenic fungus into the brain tissues of HIV-1-infected patients.  

3.1681           Protein Transfer Into Human Cells by VSV-G-induced Nanovesicles

Mangeot, P-E., Dollet, S., Girard, M., Ciancia, C., Joly, S., Peschanski, M. and Lotteau, V. Molecular Therapy, 19(9), 1656-1666 (2011)   Identification of new techniques to express proteins into mammal cells is of particular interest for both research and medical purposes. The present study describes the use of engineered vesicles to deliver exogenous proteins into human cells. We show that overexpression of the spike glycoprotein of the vesicular stomatitis virus (VSV-G) in human cells induces the release of fusogenic vesicles named gesicles. Biochemical and functional studies revealed that gesicles incorporated proteins from producer cells and could deliver them to recipient cells. This protein-transduction method allows the direct transport of cytoplasmic, nuclear or surface proteins in target cells. This was demonstrated by showing that the TetR transactivator and the receptor for the murine leukemia virus (MLV) envelope [murine cationic amino acid transporter-1 (mCAT-1)] were efficiently delivered by gesicles in various cell types. We further shows that gesicle-mediated transfer of mCAT-1 confers to human fibroblasts a robust permissiveness to ecotropic vectors, allowing the generation of human-induced pluripotent stem cells in level 2 biosafety facilities. This highlights the great potential of mCAT-1 gesicles to increase the safety of experiments using retro/lentivectors. Besides this, gesicles is a versatile tool highly valuable for the nongenetic delivery of functions such as transcription factors or genome engineering agents.  

3.1682           Inhibition of Mucin O-Glycosylation Promotes Endocytosis and Nanoparticle Uptake in Corneal Epithelial Cells

Argeso, P., Guzman-Aranguez, A., Woodward, A. and Pintor, J.J. Invest. Ophthalmol. Vis. Sci., 52, E-abstract 4394 (2011)   Purpose:Recent evidence has shown that O-glycans on cell surface-associatedmucins contribute to maintaining barrier function by interactingwith β-galactoside-binding lectins on the epithelial glycocalyx;however, the mechanisms involved have not been completely characterized.In this work, we have evaluated whether abrogation of O-glycosylationpromotes endocytosis and particle uptake in human corneal epithelial(HCLE) cells. Methods:Downregulation of mucin O-glycosylation in HCLE cells was carried out using a stable tetracycline-inducible RNA interfering system to knockdown c1galt1, a critical galactosyltransferase required for the synthesis of core 1 O-glycans. Subcellular membrane vesicles were fractionated by ultracentrifugation in a 5-20% (w/v) continuous gradient of iodixanol. Fractions were analyzedby western blot using a mouse monoclonal MUC16 antibody. HCLEcells were incubated with 0.1 µm carboxylate-modifiedfluorescent nanospheres, and uptake analyzed by confocal microscopyand fluorometry before and after inhibition of endocytosis.Tight junction integrity was evaluated using transepithelialelectrical resistance and ZO-1 staining. Results:Fractionation of membrane fragments revealed that traffickingof the cell surface mucin MUC16 was altered in cells transfectedwith c1galt1 shRNA. Moreover, in particle internalization studies,c1galt1 shRNA-transfected cells had a 1.63-fold increase innanoparticle uptake as compared to scramble control. Nanoparticleinternalization was dramatically reduced at 4°C, when activetransport processes are blocked, and by sodium azide, a generalinhibitor of endocytic processes. Dynasore and nocodazole significantlyreduced nanoparticle uptake by 37% and 36%, respectively, suggestinga mechanism for coated pit budding and vesicular traffickingin nanoparticle uptake. The involvement of the clathrin-mediatedpathway was supported by a significant decrease in uptake observedwith chlorpromazine (44%) and hypertonic media (53%). Downregulationof c1galt1 expression did not decrease the transepithelial electricalresistance or ZO-1 staining, indicating that increased nanoparticleuptake occurs through the transcellular pathway. Conclusions:These results indicate that mucin O-glycans hinder endocytosisand nanoparticle uptake, and suggest that transient manipulationof the glycocalyx barrier is an alternative approach to deliveringtherapeutic nanoparticles to the cornea.  

3.1683           Cardiac ATP-sensitive K+ channel associates with the glycolytic enzyme complex

Hong, M., Kefaloyianni, E., Bao, L., Malester, B., Delaroche, D., Neubert, T.A. and Coetzee, W.A. FASEB J. 25(7), 2456-2467 (2011)   Being gated by high-energy nucleotides, cardiac ATP-sensitive potassium (K_ATP) channels are exquisitely sensitive to changes in cellular energy metabolism. An emerging view is that proteins associated with the K_ATP channel provide an additional layer of regulation. Using putative sulfonylurea receptor (SUR) coiled-coil domains as baits in a 2-hybrid screen against a rat cardiac cDNA library, we identified glycolytic enzymes (GAPDH and aldolase A) as putative interacting proteins. Interaction between aldolase and SUR was confirmed using GST pulldown assays and coimmunoprecipitation assays. Mass spectrometry of proteins from K_ATP channel immunoprecipitates of rat cardiac membranes identified glycolysis as the most enriched biological process. Coimmunoprecipitation assays confirmed interaction for several glycolytic enzymes throughout the glycolytic pathway. Immunocytochemistry colocalized many of these enzymes with K_ATP channel subunits in rat cardiac myocytes. The catalytic activities of aldolase and pyruvate kinase functionally modulate K_ATP channels in patch-clamp experiments, whereas d-glucose was without effect. Overall, our data demonstrate close physical association and functional interaction of the glycolytic process (particularly the distal ATP-generating steps) with cardiac K_ATP channels.  

3.1684           The Assembly of Proline-rich Membrane Anchor (PRiMA)-linked Acetylcholinesterase Enzyme: GLYCOSYLATION IS REQUIRED FOR ENZYMATIC ACTIVITY BUT NOT FOR OLIGOMERIZATION

Chen, V.P., Choi, R.C.Y., Chan, W.K., Leung, K.W., Guo, A.J.Y., Chan, G.K.L., Luk, W.K.W. and Tsim, K.W.K.

1. Biol. Chem., 286(38), 32948-32961 (2011)

  Acetylcholinesterase (AChE) anchors onto cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric form in vertebrate brain. The assembly of AChE tetramer with PRiMA requires the C-terminal “t-peptide” in AChE catalytic subunit (AChE_T). Although mature AChE is well known N-glycosylated, the role of glycosylation in forming the physiologically active PRiMA-linked AChE tetramer has not been studied. Here, several lines of evidence indicate that the N-linked glycosylation of AChE_T plays a major role for acquisition of AChE full enzymatic activity but does not affect its oligomerization. The expression of the AChE_T mutant, in which all N-glycosylation sites were deleted, together with PRiMA in HEK293T cells produced a glycan-depleted PRiMA-linked AChE tetramer but with a much higher K_m value as compared with the wild type. This glycan-depleted enzyme was assembled in endoplasmic reticulum but was not transported to Golgi apparatus or plasma membrane.  

3.1685           Molecular Determinants of Ciliary Membrane Localization of Trypanosoma cruzi Flagellar Calcium-binding Protein

Maric, D., McGwire, B.S., Buchanan, K.T., Olson, C.L., Emmer, B.T., Epting, C.L. and Engman, D.M.

1. Biol. Chem., 286(38), 33109-33117 (2011)

  The flagellar calcium-binding protein (FCaBP) of Trypanosoma cruzi is localized to the flagellar membrane in all life cycle stages of the parasite. Myristoylation and palmitoylation of the N terminus of FCaBP are necessary for flagellar membrane targeting. Not all dually acylated proteins in T. cruzi are flagellar, however. Other determinants of FCaBP therefore likely contribute to flagellar specificity. We generated T. cruzi transfectants expressing the N-terminal 24 or 12 amino acids of FCaBP fused to GFP. Analysis of these mutants revealed that although amino acids 1–12 are sufficient for dual acylation and membrane binding, amino acids 13–24 are required for flagellar specificity and lipid raft association. Mutagenesis of several conserved lysine residues in the latter peptide demonstrated that these residues are essential for flagellar targeting and lipid raft association. Finally, FCaBP was expressed in the protozoan Leishmania amazonensis, which lacks FCaBP. The flagellar localization and membrane association of FCaBP in L. amazonensis suggest that the mechanisms for flagellar targeting, including a specific palmitoyl acyltransferase, are conserved in this organism.  

3.1686           Triage of oxidation-prone proteins by Sqstm1/p62 within the mitochondria

Lee, M. and Shin, J. Biochem. Biophys. Acta, 413, 122-127 (2011)   As the mitochondrion is vulnerable to oxidative stress, cells have evolved several strategies to maintain mitochondrial integrity, including mitochondrial protein quality control mechanisms and autophagic removal of damaged mitochondria. Involvement of an autophagy adaptor, Sqstm1/p62, in the latter process has been recently described. In the present study, we provide evidence that a portion of p62 directly localizes within the mitochondria and supports stable electron transport by forming heterogeneous protein complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of mitochondrial proteins co-purified with p62 revealed that p62 interacts with several oxidation-prone proteins, including a few components of the electron transport chain complexes, as well as multiple chaperone molecules and redox regulatory enzymes. Accordingly, p62-deficient mitochondria exhibited compromised electron transport, and the compromised function was partially restored by in vitro delivery of p62. These results suggest that p62 plays an additional role in maintaining mitochondrial integrity at the vicinity of target machineries through its function in relation to protein quality control.  

3.1687           Mammalian ACSF3 Protein Is a Malonyl-CoA Synthetase That Supplies the Chain Extender Units for Mitochondrial Fatty Acid Synthesis

Witkowski, A., Thweatt, J. and Smith, S.

1. Biol. Chem., 286(39), 33729-33736 (2011)

  The objective of this study was to identify a source of intramitochondrial malonyl-CoA that could be used for de novo fatty acid synthesis in mammalian mitochondria. Because mammalian mitochondria lack an acetyl-CoA carboxylase capable of generating malonyl-CoA inside mitochondria, the possibility that malonate could act as a precursor was investigated. Although malonyl-CoA synthetases have not been identified previously in animals, interrogation of animal protein sequence databases identified candidates that exhibited sequence similarity to known prokaryotic forms. The human candidate protein ACSF3, which has a predicted N-terminal mitochondrial targeting sequence, was cloned, expressed, and characterized as a 65-kDa acyl-CoA synthetase with extremely high specificity for malonate and methylmalonate. An arginine residue implicated in malonate binding by prokaryotic malonyl-CoA synthetases was found to be positionally conserved in animal ACSF3 enzymes and essential for activity. Subcellular fractionation experiments with HEK293T cells confirmed that human ACSF3 is located exclusively in mitochondria, and RNA interference experiments verified that this enzyme is responsible for most, if not all, of the malonyl-CoA synthetase activity in the mitochondria of these cells. In conclusion, unlike fungi, which have an intramitochondrial acetyl-CoA carboxylase, animals require an alternative source of mitochondrial malonyl-CoA; the mitochondrial ACSF3 enzyme is capable of filling this role by utilizing free malonic acid as substrate.  

3.1688           N-Glycans and Glycosylphosphatidylinositol-Anchor Act on Polarized Sorting of Mouse PrPC in Madin-Darby Canine Kidney Cells

Puig, B., Altmeppen, H.C., Thurm, D., Geissen, M., Conrad, C., Braulke, T. and Glatsel, M. PloS One, 6(9), e24624 (2011)   The cellular prion protein (PrP^C) plays a fundamental role in prion disease. PrP^C is a glycosylphosphatidylinositol (GPI)-anchored protein with two variably occupied N-glycosylation sites. In general, GPI-anchor and N-glycosylation direct proteins to apical membranes in polarized cells whereas the majority of mouse PrP^C is found in basolateral membranes in polarized Madin-Darby canine kidney (MDCK) cells. In this study we have mutated the first, the second, and both N-glycosylation sites of PrP^C and also replaced the GPI-anchor of PrP^C by the Thy-1 GPI-anchor in order to investigate the role of these signals in sorting of PrP^C in MDCK cells. Cell surface biotinylation experiments and confocal microscopy showed that lack of one N-linked oligosaccharide leads to loss of polarized sorting of PrP^C. Exchange of the PrP^C GPI-anchor for the one of Thy-1 redirects PrP^C to the apical membrane. In conclusion, both N-glycosylation and GPI-anchor act on polarized sorting of PrP^C, with the GPI-anchor being dominant over N-glycans.  

3.1689           Restoration of IFNγR Subunit Assembly, IFNγ Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol

Sen, S., Roy, K., Mukherjee, S., Mukhopadhyay, R. and Roym, S. PloS Pathogens, 7(9), e1002229 (2011)   Despite the presence of significant levels of systemic Interferon gamma (IFNγ), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNγ signaling in Leishmania donovani (LD) infected macrophages (LD-MØs); the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFNγ signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNγ receptor (IFNγR) expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-MØs as IL-10 mediated signal transducer and activator of transcription 3 (STAT3) phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-MØs are more fluid than normal MØs due to quenching of membrane cholesterol. The decreased rigidity in LD-MØs was not due to parasite derived lipophosphoglycan (LPG) because purified LPG failed to alter fluidity in normal MØs. IFNγR subunit 1 (IFNγR1) and subunit 2 (IFNγR2) colocalize in raft upon IFNγ stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFNγR1 and IFNγR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET) experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFNγ allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1) in LD-MØs, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V)-X_1–5-Y-X_1–5-(R/K)] in the transmembrane domain of IFNγR1 was also noted. The interaction of peptides representing this motif of IFNγR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of magnitude in respective affinity (K_D: 4.27×10⁻⁹ M versus 2.69×10⁻⁷ M). These observations reinforce the importance of cholesterol in the regulation of function of IFNγR1 proteins. This study clearly demonstrates that during its intracellular life-cycle LD perturbs IFNγR1 and IFNγR2 assembly and subsequent ligand driven signaling by quenching MØ membrane cholesterol.  

3.1690           Network Clustering Revealed the Systemic Alterations of Mitochondrial Protein Expression

Jeon, J., Jeong, J.H., Baek, J-H., Koo, H-J., Park, W-H., Yang, J-S., Yu, M-H., Kim, S. and Pak, Y.K. PloS Computational Biol., 7(6), e1002093 (2011)   The mitochondrial protein repertoire varies depending on the cellular state. Protein component modifications caused by mitochondrial DNA (mtDNA) depletion are related to a wide range of human diseases; however, little is known about how nuclear-encoded mitochondrial proteins (mt proteome) changes under such dysfunctional states. In this study, we investigated the systemic alterations of mtDNA-depleted (ρ⁰) mitochondria by using network analysis of gene expression data. By modularizing the quantified proteomics data into protein functional networks, systemic properties of mitochondrial dysfunction were analyzed. We discovered that up-regulated and down-regulated proteins were organized into two predominant subnetworks that exhibited distinct biological processes. The down-regulated network modules are involved in typical mitochondrial functions, while up-regulated proteins are responsible for mtDNA repair and regulation of mt protein expression and transport. Furthermore, comparisons of proteome and transcriptome data revealed that ρ⁰ cells attempted to compensate for mtDNA depletion by modulating the coordinated expression/transport of mt proteins. Our results demonstrate that mt protein composition changed to remodel the functional organization of mitochondrial protein networks in response to dysfunctional cellular states. Human mt protein functional networks provide a framework for understanding how cells respond to mitochondrial dysfunctions.  

3.1691           Mutations associated with Charcot–Marie–Tooth disease cause SIMPLE protein mislocalization and degradation by the proteasome and aggresome–autophagy pathways

Lee, S.M., Olzmann, J.A., Chin, L-S. and Li, L.

1. Cell Sci., 124(19), 3319-3331 (2011)

  Mutations in SIMPLE cause an autosomal dominant, demyelinating form of peripheral neuropathy termed Charcot–Marie–Tooth disease type 1C (CMT1C), but the pathogenic mechanisms of these mutations remain unknown. Here, we report that SIMPLE is an early endosomal membrane protein that is highly expressed in the peripheral nerves and Schwann cells. Our analysis has identified a transmembrane domain (TMD) embedded within the cysteine-rich (C-rich) region that anchors SIMPLE to the membrane, and suggests that SIMPLE is a post-translationally inserted, C-tail-anchored membrane protein. We found that CMT1C-linked pathogenic mutations are clustered within or around the TMD of SIMPLE and that these mutations cause mislocalization of SIMPLE from the early endosome membrane to the cytosol. The CMT1C-associated SIMPLE mutant proteins are unstable and prone to aggregation, and they are selectively degraded by both the proteasome and aggresome–autophagy pathways. Our findings suggest that SIMPLE mutations cause CMT1C peripheral neuropathy by a combination of loss-of-function and toxic gain-of-function mechanisms, and highlight the importance of both the proteasome and autophagy pathways in the clearance of CMT1C-associated mutant SIMPLE proteins.  

3.1692           Lgl1 Activation of Rab10 Promotes Axonal Membrane Trafficking Underlying Neuronal Polarization

Wang, T., Liu, Y., Xu, X-H., Deng, C-Y., Wu, K-Y., Zhu, J., Fu, X-Q., He, M. and Luo, Z-G. Developmental Cell, 21(3), 431-444 (2011)   Directed membrane trafficking is believed to be crucial for axon development during neuronal morphogenesis. However, the underlying mechanisms are poorly understood. Here, we report a role of Lgl1, the mammalian homolog of Drosophila tumor suppressor Lethal giant larvae, in controlling membrane trafficking underlying axonal growth. We find that Lgl1 is associated with plasmalemmal precursor vesicles and enriched in developing axons. Lgl1 upregulation promoted axonal growth, whereas downregulation attenuated it as well as directional membrane insertion. Interestingly, Lgl1 interacted with and activated Rab10, a small GTPase that mediates membrane protein trafficking, by releasing GDP dissociation inhibitor (GDI) from Rab10. Furthermore, Rab10 lies downstream of Lgl1 in axon development and directional membrane insertion. Finally, both Lgl1 and Rab10 are required for neocortical neuronal polarization in vivo. Thus, the Lgl1 regulation of Rab10 stimulates the trafficking of membrane precursor vesicles, whose fusion with the plasmalemma is crucial for axonal growth.  

3.1693           C2 Domain-Containing Phosphoprotein CDP138 Regulates GLUT4 Insertion into the Plasma Membrane

Xie, X., et al Cell Metabolism, 14(3), 378-389 (2011)   The protein kinase B_β (Akt2) pathway is known to mediate insulin-stimulated glucose transport through increasing glucose transporter GLUT4 translocation from intracellular stores to the plasma membrane (PM). Combining quantitative phosphoproteomics with RNAi-based functional analyses, we show that a previously uncharacterized 138 kDa C2 domain-containing phosphoprotein (CDP138) is a substrate for Akt2, and is required for optimal insulin-stimulated glucose transport, GLUT4 translocation, and fusion of GLUT4 vesicles with the PM in live adipocytes. The purified C2 domain is capable of binding Ca²⁺ and lipid membranes. CDP138 mutants lacking the Ca²⁺-binding sites in the C2 domain or Akt2 phosphorylation site S197 inhibit insulin-stimulated GLUT4 insertion into the PM, a rate-limiting step of GLUT4 translocation. Interestingly, CDP138 is dynamically associated with the PM and GLUT4-containing vesicles in response to insulin stimulation. Together, these results suggest that CDP138 is a key molecule linking the Akt2 pathway to the regulation of GLUT4 vesicle-PM fusion.  

3.1694           The Mechanism of Tail-Anchored Protein Insertion into the ER Membrane

Wang, F., Whynot, A., Tung, M., and Denic, V. Mol. Cell., 43(5), 738-750 (2011)   Tail-anchored (TA) proteins access the secretory pathway via posttranslational insertion of their C-terminal transmembrane domain into the endoplasmic reticulum (ER). Get3 is an ATPase that delivers TA proteins to the ER by interacting with the Get1-Get2 transmembrane complex, but how Get3's nucleotide cycle drives TA protein insertion remains unclear. Here, we establish that nucleotide binding to Get3 promotes Get3-TA protein complex formation by recruiting Get3 to a chaperone that hands over TA proteins to Get3. Biochemical reconstitution and mutagenesis reveal that the Get1-Get2 complex comprises the minimal TA protein insertion machinery with functionally critical cytosolic regions. By engineering a soluble heterodimer of Get1-Get2 cytosolic domains, we uncover the mechanism of TA protein release from Get3: Get2 tethers Get3-TA protein complexes into proximity with the ATPase-dependent, substrate-releasing activity of Get1. Lastly, we show that ATP enhances Get3 dissociation from the membrane, thus freeing Get1-Get2 for new rounds of substrate insertion.  

3.1695           The Chlamydia Protease CPAF Regulates Host and Bacterial Proteins to Maintain Pathogen Vacuole Integrity and Promote Virulence

Jorgensen, I., Bednar, M.M., Amin, V., Davis, B.K., Ting, J.P.Y., McCafferty, D.G. and Valdvia, R.H. Cell Host & Microbe, 10(1), 21-32 (2011)   The obligate intracellular bacterial pathogen Chlamydia trachomatis injects numerous effector proteins into the epithelial cell cytoplasm to manipulate host functions important for bacterial survival. In addition, the bacterium secretes a serine protease, chlamydial protease-like activity factor (CPAF). Although several CPAF targets are reported, the significance of CPAF-mediated proteolysis is unclear due to the lack of specific CPAF inhibitors and the diversity of host targets. We report that CPAF also targets chlamydial effectors secreted early during the establishment of the pathogen-containing vacuole ( inclusion ). We designed a cell-permeable CPAF-specific inhibitory peptide and used it to determine that CPAF prevents superinfection by degrading early Chlamydia effectors translocated during entry into a preinfected cell. Prolonged CPAF inhibition leads to loss of inclusion integrity and caspase-1-dependent death of infected epithelial cells. Thus, CPAF functions in niche protection, inclusion integrity and pathogen survival, making the development of CPAF-specific protease inhibitors an attractive antichlamydial therapeutic strategy.  

3.1696           Exosomes: immune properties and potential clinical implementations

Chaput, N. and Thery, C. Semin. Immunopathol, 33, 419-440 (2011)   To communicate, cells are known to release in their environment proteins which bind to receptors on surrounding cells. But cells also secrete more complex structures, called membrane vesicles, composed of a lipid bilayer with inserted transmembrane proteins, enclosing an internal content of hydrophilic components. Exosomes represent a specific subclass of such secreted membrane vesicles, which, despite having been described more than 20 years ago by two groups studying reticulocyte maturation, have only recently received attention from the scientific community. This renewed interest originated first from the description of exosome secretion by antigen-presenting cells, suggesting a potential role in immune responses, and very recently by the identification of the presence of RNA (both messenger and microRNA) in exosomes, suggesting a potential transfer of genetic information between cells. In this review, we will describe the conclusions of 20 years of studies on the immune properties of exosomes and the most recent advances on their roles and potential uses as markers or as therapeutic tools during pathologies, especially in cancer.  

3.1697           Caveolin-1 in cytokine-induced enhancement of intracellular Ca2+ in human airway smooth muscle

Sathish, V., Abcejo, A.J., VanOosten, S.K., Thompson, M.A., Prakash, Y.S. and pabelick, C.M. Am. J. Physiol. Lung Cell Mol. Physiol., 301(4), L607-L614 (2011)   Diseases such as asthma are characterized by airway hyperresponsiveness. Enhanced airway smooth muscle (ASM) intracellular Ca²⁺ ([Ca²⁺]_i) response to agonist stimulation leading to increased airway constriction has been suggested to contribute to airway hyperresponsiveness. Caveolae are flask-shaped plasma membrane invaginations that express the scaffolding protein caveolin and contain multiple proteins important in [Ca²⁺]_i signaling (e.g., agonist receptors, ion channels). We recently demonstrated that caveolae and caveolin-1 are important in [Ca²⁺]_i regulation in human ASM. Proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-13 modulate [Ca²⁺]_i in ASM. We hypothesized that cytokine upregulation of caveolar signaling in ASM contributes to enhanced agonist-induced [Ca²⁺]_i in inflammation. Enzymatically dissociated human ASM cells were exposed to medium (control), 20 ng/ml TNF-α, or 50 ng/ml IL-13 for 24 h. Caveolae-enriched membrane fractions displayed substantial increase in caveolin-1 and -2 expressions by TNF-α and IL-13. Transfection with caveolin-1-mRed DNA substantially accelerated and increased plasma membrane caveolin-1 expression by TNF-α and to a lesser extent by IL-13. Caveolin-1 enhancement was inhibited by nuclear factor-κB and mitogen-activated protein kinase inhibitors. In fura 2-loaded ASM cells, [Ca²⁺]_i responses to 1 μM ACh, 10 μM histamine, or 10 nM bradykinin were all exaggerated by TNF-α as well as IL-13 exposure. However, disruption of caveolae using caveolin-1 suppression via small-interfering RNA resulted in significant blunting of agonist-induced [Ca²⁺]_i responses of vehicle and TNF-α-exposed cells. These functional data were correlated to the presence of TNFR₁ receptor (but not the IL-4/IL-13 receptor) within caveolae. Overall, these results indicate that caveolin-1 plays an important role in airway inflammation by modulating the effect of specific cytokines on [Ca²⁺]_i.    

3.1698           A Nine Amino Acid Domain Is Essential for Mutant Prion Protein Toxicity

Westergard, L., Turnbaugh, J.A. and Harris, D.A.

1. Neurosci., 31(39), 14005-14017 (2011)

  Transgenic mice expressing prion protein (PrP) molecules with several different internal deletions display spontaneous neurodegenerative phenotypes that can be dose-dependently suppressed by coexpression of wild-type PrP. Each of these deletions, including the largest one (Δ32–134), retains 9 aa immediately following the signal peptide cleavage site (residues 23–31; KKRPKPGGW). These residues have been implicated in several biological functions of PrP, including endocytic trafficking and binding of glycosaminoglycans. We report here on our experiments to test the role of this domain in the toxicity of deleted forms of PrP. We find that transgenic mice expressing Δ23–134 PrP display no clinical symptoms or neuropathology, in contrast to mice expressing Δ32–134 PrP, suggesting that residues 23–31 are essential for the toxic phenotype. Using a newly developed cell culture assay, we narrow the essential region to amino acids 23–26, and we show that mutant PrP toxicity is not related to the role of the N-terminal residues in endocytosis or binding to endogenous glycosaminoglycans. However, we find that mutant PrP toxicity is potently inhibited by application of exogenous glycosaminoglycans, suggesting that the latter molecules block an essential interaction between the N terminus of PrP and a membrane-associated target site. Our results demonstrate that a short segment containing positively charged amino acids at the N terminus of PrP plays an essential role in mediating PrP-related neurotoxicity. This finding identifies a protein domain that may serve as a drug target for amelioration of prion neurotoxicity.  

3.1699           Invasion of Cryptococcus neoformans into Human Brain Microvascular Endothelial Cells Is Mediated through the Lipid Rafts-Endocytic Pathway via the Dual Specificity Tyrosine Phosphorylation-regulated Kinase 3 (DYRK3)

Huang, S-H., Long, M., Wu, C-H., Kwon-Chung, K.J., Chang, Y.C., Chi, F., Lee, S. and Jong, A.

1. Biol. Chem., 286(40),34761-34769 (2011)

  Cryptococcus neoformans is a neurotropic fungal pathogen, which provokes the onset of devastating meningoencephalitis. We used human brain microvascular endothelial cells (HBMEC) as the in vitro model to investigate how C. neoformans traverses across the blood-brain barrier. In this study, we present several lines of evidence indicating that C. neoformans invasion is mediated through the endocytic pathway via lipid rafts. Human CD44 molecules from lipid rafts can directly interact with hyaluronic acid, the C. neoformans ligand. Bikunin, which perturbs CD44 function in the lipid raft, can block C. neoformans adhesion and invasion of HBMEC. The lipid raft marker, ganglioside GM1, co-localizes with CD44 on the plasma membrane, and C. neoformans cells can adhere to the host cell in areas where GM1 is enriched. These findings suggest that C. neoformans entry takes place on the lipid rafts. Upon C. neoformans engagement, GM1 is internalized through vesicular structures to the nuclear membrane. This endocytic redistribution process is abolished by cytochalasin D, nocodazole, or anti-DYRK3 (dual specificity tyrosine-phosphorylation-regulated kinase 3) siRNA. Concomitantly, the knockdown of DYRK3 significantly reduces C. neoformans invasion across the HBMEC monolayer in vitro. Our data demonstrate that the lipid raft-dependent endocytosis process mediates C. neoformans internalization into HBMEC and that the CD44 protein of the hosts, cytoskeleton, and intracellular kinase-DYRK3 are involved in this process.  

3.1700           Elicitation of Epithelial Cell-Derived Immune Effectors by Outer Membrane Vesicles of Nontypeable Haemophilus influenza

Sharpe, S.W., Kuehn, M.J. and Mason, K.M. Infect. Immun., 79(11), 4361-4369 (2011)   Outer membrane vesicles (OMVs) are produced by all Gram-negative microorganisms studied to date. The contributions of OMVs to biological processes are diverse and include mediation of bacterial stress responses, selective packaging and secretion of virulence determinants, modulation of the host immune response, and contributions to biofilm formation and stability. First characterized as transformasomes in Haemophilus, these membranous blebs facilitate transfer of DNA among bacteria. Nontypeable Haemophilus influenzae (NTHI), an opportunistic pathogen of the upper and lower respiratory tracts, produces OMVs in vivo, but there is a paucity of information regarding both the composition and role of OMVs during NTHI colonization and pathogenesis. We demonstrated that purified NTHI vesicles are 20 to 200 nm in diameter and contain DNA, adhesin P5, IgA endopeptidase, serine protease, and heme utilization protein, suggesting a multifaceted role in virulence. NTHI OMVs can bind to human pharyngeal epithelial cells, resulting in a time- and temperature-dependent aggregation on the host cell surface, with subsequent internalization. OMVs colocalize with the endocytosis protein caveolin, indicating that internalization is mediated by caveolae, which are cholesterol-rich lipid raft domains. Upon interaction with epithelial cells, NTHI OMVs stimulate significant release of the immunomodulatory cytokine interleukin-8 (IL-8) as well as the antimicrobial peptide LL-37. Thus, we demonstrated that NTHI OMVs contain virulence-associated proteins that dynamically interact with and invade host epithelial cells. Beyond their ability to mediate DNA transfer in Haemophilus, OMV stimulation of host immunomodulatory cytokine and antimicrobial peptide release supports a dynamic role for vesiculation in NTHI pathogenesis and clinically relevant disease progression.  

3.1701           EBV-gp350 Confers B-Cell Tropism to Tailored Exosomes and Is a Neo-Antigen in Normal and Malignant B Cells—A New Option for the Treatment of B-CLL

Ruiss, R., Jochum, S., Mocikat, R., Hammerschmidt, W. and Zeidler, R. PloS One, 6(10), e25294 (2011)   gp350, the major envelope protein of Epstein-Barr-Virus, confers B-cell tropism to the virus by interacting with the B lineage marker CD21. Here we utilize gp350 to generate tailored exosomes with an identical tropism. These exosomes can be used for the targeted co-transfer of functional proteins to normal and malignant human B cells. We demonstrate here the co-transfer of functional CD154 protein on tailored gp350+ exosomes to malignant B blasts from patients with B chronic lymphocytic leukemia (B-CLL), rendering B blasts immunogenic to tumor-reactive autologous T cells. Intriguingly, engulfment of gp350+ exosomes by B-CLL cells and presentation of gp350-derived peptides also re-stimulated EBV-specific T cells and redirected the strong antiviral cellular immune response in patients to leukemic B cells. In essence, we show that gp350 alone confers B-cell tropism to exosomes and that these exosomes can be further engineered to simultaneously trigger virus- and tumor-specific immune responses. The simultaneous exploitation of gp350 as a tropism molecule for tailored exosomes and as a neo-antigen in malignant B cells provides a novel attractive strategy for immunotherapy of B-CLL and other B-cell malignancies.  

3.1702           The Endosomal Na+/H+ Exchanger Contributes to Multivesicular Body Formation by Regulating the Recruitment of ESCRT-0 Vps27p to the Endosomal Membrane

Mitsui, K., Koshimura, Y., Yoshikawa, Y., matsushita, M. and Kanazawa, H.

1. Biol. Chem., 286(43), 37625-37638 (2011)

  Multivesicular bodies (MVBs) are late endosomal compartments containing luminal vesicles (MVB vesicles) that are formed by inward budding of the endosomal membrane. In budding yeast, MVBs are an important cellular mechanism for the transport of membrane proteins to the vacuolar lumen. This process requires a class E subset of vacuolar protein sorting (VPS) genes. VPS44 (allelic to NHX1) encodes an endosome-localized Na⁺/H⁺ exchanger. The function of the VPS44 exchanger in the context of vacuolar protein transport is largely unknown. Using a cell-free MVB formation assay system, we demonstrated that Nhx1p is required for the efficient formation of MVB vesicles in the late endosome. The recruitment of Vps27p, a class E Vps protein, to the endosomal membrane was dependent on Nhx1p activity and was enhanced by an acidic pH at the endosomal surface. Taken together, we propose that Nhx1p contributes to MVB formation by the recruitment of Vps27p to the endosomal membrane, possibly through Nhx1p antiporter activity.  

3.1703           c-Cbl-Mediated Selective Virus-Receptor Translocations into Lipid Rafts Regulate Productive Kaposi's Sarcoma-Associated Herpesvirus Infection in Endothelial Cells

Chakraborty, S., Veettil, M.V., Sadagopan, S., Paudel, N. and Chandran, B.

1. Virol., 85(23), 12410-12430 (2011)

  During target cell entry and infection, many enveloped and nonenveloped viruses utilize cell surface receptors that translocate into lipid rafts (LRs). However, the mechanism behind this translocation is not known. Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with the human microvascular dermal endothelial (HMVEC-d) cell surface heparan sulfate (HS), integrins α3β1, αVβ3, and αVβ5, and the amino acid transporter x-CT protein and enters via c-Cbl-bleb-mediated macropinocytosis (Veettil et al., J. Virol. 82:12126-12144, 2008; Veettil et al., PLoS Pathog. 6:e1001238, 2010). Here we have demonstrated that very early during infection (1 min postinfection), c-Cbl induced the selective translocation of KSHV into the LR along with the α3β1, αVβ3, and x-CT receptors but not αVβ5. Activated c-Cbl localized with LRs at the junctional base of macropinocytic blebs. LR-translocated α3β1 and αVβ3 were monoubiquitinated, leading to productive macropinocytic entry, whereas non-LR-associated αVβ5 was polyubiquitinated, leading to clathrin entry that was targeted to lysosomes. c-Cbl knockdown blocked the macropinocytosis and receptor translocation and diverted KSHV to a clathrin-lysosomal noninfectious pathway. Similar results were also seen by LR disruption with MβCD. These studies provide the first evidence that c-Cbl regulates selective KSHV-α3β1, -αVβ3, and -x-CT receptor translocations into the LRs and differential ubiquitination of receptors which are critical determinants of the macropinocytic entry route and productive infection of KSHV. Our studies suggest that interventions targeting c-Cbl and LRs are potential avenues to block KSHV infection of endothelial cells.  

3.1704           Endothelial progenitor cell–dependent angiogenesis requires localization of the full-length form of uPAR in caveolae

Margheri, F., Chilla, A., Laurenzana, A., Serrati, S., Mazzanti, B., Saccardi, R., Santosuosso, S., Danza, G., Sturli, N., Rosati, F., Magnelli, L., Papucci, L., Calorini, L., Bianchini, F., Del Rosso, M. and Fibbi, G. Blood, 118(13), 3743-3755 (2011)   Endothelial urokinase-type plasminogen activator receptor (uPAR) is thought to provide a regulatory mechanism in angiogenesis. Here we studied the proangiogenic role of uPAR in endothelial colony-forming cells (ECFCs), a cell population identified in human umbilical blood that embodies all of the properties of an endothelial progenitor cell matched with a high proliferative rate. By using caveolae-disrupting agents and by caveolin-1 silencing, we have shown that the angiogenic properties of ECFCs depend on caveolae integrity and on the presence of full-length uPAR in such specialized membrane invaginations. Inhibition of uPAR expression by antisense oligonucleotides promoted caveolae disruption, suggesting that uPAR is an inducer of caveolae organization. Vascular endothelial growth factor (VEGF) promoted accumulation of uPAR in ECFC caveolae in its undegraded form. We also demonstrated that VEGF-dependent ERK phosphorylation required integrity of caveolae as well as caveolar uPAR expression. VEGF activity depends on inhibition of ECFC MMP12 production, which results in impairment of MMP12-dependent uPAR truncation. Further, MMP12 overexpression in ECFC inhibited vascularization in vitro and in vivo. Our data suggest that intratumor homing of ECFCs suitably engineered to overexpress MMP12 could have the chance to control uPAR-dependent activities required for tumor angiogenesis and malignant cells spreading.  

3.1705           Analysis of Detergent-free Lipid Rafts isolated from a CD4+ T cell line: Interaction with antigen presenting cells promotes coalescing of lipid rafts

Kennedy, C., Nelson, M.D. and Bamezai, A.K. Cell Communication and Signaling, 9, 31-xxx (2011)   Background: Lipid rafts present on the plasma membrane play an important role in spatiotemporal regulation of cell signaling. Physical and chemical characterization of lipid raft size and assessment of their composition before, and after cell stimulation will aid in developing a clear understanding of their regulatory role in cell signaling. We have used visual and biochemical methods and approaches for examining individual and lipid raft sub-populations isolated from a mouse CD4+ T cell line in the absence of detergents. Results: Detergent-free rafts were analyzed before and after their interaction with antigen presenting cells. We provide evidence that the average diameter of lipid rafts isolated from unstimulated T cells, in the absence of detergents, is less than 100 nm. Lipid rafts on CD4+ T cell membranes coalesce to form larger structures, after interacting with antigen presenting cells even in the absence of a foreign antigen. Conclusions: Findings presented here indicate that lipid raft coalescence occurs during cellular interactions prior to sensing a foreign antigen  

3.1706           Lysosomal accumulation of Trk protein in brain of GM1-gangliosidosis mouse and its restoration by chemical chaperone

Takamura, A., Higaki, K., Ninomiya, H., Takai, T., matsuda, J., Iida, M., Ohno, K., Suzuki, Y. and nanba, E.

1. Neurochem., 118(3), 399-406 (2011)

  G_M1-gangliosidosis is a fatal neurodegenerative disorder caused by deficiency of lysosomal acid β-galactosidase (β-gal). Accumulation of its substrate ganglioside G_M1 (G_M1) in lysosomes and other parts of the cell leads to progressive neurodegeneration, but underlying mechanisms remain unclear. Previous studies demonstrated an essential role for interaction of G_M1 with tropomyosin receptor kinase (Trk) receptors in neuronal growth, survival and differentiation. In this study we demonstrate accumulation of G_M1 in the cell-surface rafts and lysosomes of the β-gal knockout (β-gal−/−) mouse brain association with accumulation of Trk receptors and enhancement of its downstream signaling. Immunofluorescence and subcellular fractionation analysis revealed accumulation of Trk receptors in the late endosomes/lysosomes of the β-gal−/− mouse brain and their association with ubiquitin and p62. Administration of a chemical chaperone to β-gal−/− mouse expressing human mutant R201C protein resulted in a marked reduction of intracellular storage of G_M1 and phosphorylated Trk. These findings indicate that G_M1 accumulation in rafts causes activation of Trk signaling, which may participate in the pathogenesis of G_M1-gangliosidosis.  

3.1707           Neuronal glycoprotein M6a induces filopodia formation via association with cholesterol-rich lipid rafts

Scorticati, C., Formoso, K. and Frasch, A.C.

1. Neurochem., 119(3), 521-531 (2011)

  A neuronal integral membrane glycoprotein M6a has been suggested to be involved in a number of biological processes, including neuronal remodeling and differentiation, trafficking of mu-opioid receptors, and Ca²⁺ transportation. Moreover, pathological situations such as chronic stress in animals and depression in humans have been associated with alterations in M6a sequence and expression. The mechanism of action of M6a is essentially unknown. In this work, we analyze the relevance of M6a distribution in plasma membrane, namely its lipid microdomain targeting, for its biological function in filopodia formation. We demonstrate that M6a is localized in membrane microdomains compatible with lipid rafts in cultured rat hippocampal neurons. Removal of cholesterol from neuronal membranes with methyl-β-cyclodextrin decreases M6a-induced filopodia formation, an effect that is reversed by the addition of cholesterol. Inhibition of Src kinases and MAPK prevents filopodia formation in M6a-over-expressing neurons. Src-deficient SYF cells over-expressing M6a fail to promote filopodia formation. Taken together, our findings reveal that the association of M6a with lipid rafts is important for its role in filopodia formation and Src and MAPK kinases participate in M6a signal propagation.  

3.1708           Endocytosis is essential for dynamic translocation of a syntaxin 1 orthologue during fission yeast meiosis

Kashiwazaki, J., Yamasaki, Y., Itadani, A., Teraguchi, E., Maeda, Y., Shimoda, C. and Nakamura, T. Mol. Biol. Cell, 22(19), 3658-3670 (2011)   Syntaxin is a component of the target soluble N-ethylmaleimide–sensitive factor attachment protein receptor complex, which is responsible for fusion of membrane vesicles at the target membrane. The fission yeast syntaxin 1 orthologue Psy1 is essential for both vegetative growth and spore formation. During meiosis, Psy1 disappears from the plasma membrane (PM) and dramatically relocalizes on the nascent forespore membrane, which becomes the PM of the spore. Here we report the molecular details and biological significance of Psy1 relocalization. We find that, immediately after meiosis I, Psy1 is selectively internalized by endocytosis. In addition, a meiosis-specific signal induced by the transcription factor Mei4 seems to trigger this internalization. The internalization of many PM proteins is facilitated coincident with the initiation of meiosis, whereas Pma1, a P-type ATPase, persists on the PM even during the progression of meiosis II. Ergosterol on the PM is also important for the internalization of PM proteins in general during meiosis. We consider that during meiosis in Schizosaccharomyces pombe cells, the characteristics of endocytosis change, thereby facilitating internalization of Psy1 and accomplishing sporulation.  

3.1709           Retrolinkin cooperates with endophilin A1 to mediate BDNF–TrkB early endocytic trafficking and signaling from early endosomes

Fu, X., Yang, Y., Xu, C., Niu, Y., Chen, T., Zhou, Q. and Liu, J-J. Mol. Biol. Cell, 22(19), 3684-3698 (2011)   Brain-derived neurotrophic factor (BDNF) binds to its cell surface receptor TrkB to regulate differentiation, development, synaptic plasticity, and functional maintenance of neuronal cells. Binding of BDNF triggers TrkB dimerization and autophosphorylation, which provides docking sites for adaptor proteins to recruit and activate downstream signaling molecules. The molecular mechanisms underlying BDNF–TrkB endocytic trafficking crucial for spatiotemporal control of signaling pathways remain to be elucidated. Here we show that retrolinkin, a transmembrane protein, interacts with endophilin A1 and mediates BDNF-activated TrkB (pTrk) trafficking and signaling in CNS neurons. We find that activated TrkB colocalizes and interacts with the early endosome marker APPL1. Both retrolinkin and endophilin A1 are required for BDNF-induced dendrite development and acute extracellular signal-regulated kinase activation from early endosomes. Suppression of retrolinkin expression not only blocks BDNF-triggered TrkB internalization, but also prevents recruitment of endophilin A1 to pTrk vesicles trafficking through APPL1-positive endosomes. These findings reveal a novel mechanism for BDNF–TrkB to regulate signaling both in time and space through a specific membrane trafficking pathway.  

3.1710           Distinct Autophagosomal-Lysosomal Fusion Mechanism Revealed by Thapsigargin-Induced Autophagy Arrest

Ganley, I.G., Wong, P-M., Gammoth, N. and Jiang, X. Mol. Cell, 42(6), 731-743 (2011)   Autophagy, a catabolic pathway that delivers cellular components to lysosomes for degradation, can be activated by stressful conditions such as nutrient starvation and endoplasmic reticulum (ER) stress. We report that thapsigargin, an ER stressor widely used to induce autophagy, in fact blocks autophagy. Thapsigargin does not affect autophagosome formation but leads to accumulation of mature autophagosomes by blocking autophagosome fusion with the endocytic system. Strikingly, thapsigargin has no effect on endocytosis-mediated degradation of epidermal growth factor receptor. Molecularly, while both Rab7 and Vps16 are essential regulatory components for endocytic fusion with lysosomes, we found that Rab7 but not Vps16 is required for complete autophagy flux, and that thapsigargin blocks recruitment of Rab7 to autophagosomes. Therefore, autophagosomal-lysosomal fusion must be governed by a distinct molecular mechanism compared to general endocytic fusion.  

3.1711           Conserved Proline-Rich Region of Ebola Virus Matrix Protein VP40 Is Essential for Plasma Membrane Targeting and Virus-Like Particle Release

Reynard, O., Nemirov, K., page, A., mateo, M., Raoul, H., Weissenhorn, W. and Volchkov, V.

1. Infectious Dis., 204, Suppl.3 , S884-S889 (2011)

  The matrix protein VP40 is essential for Ebola virus (EBOV) and Marburg virus assembly and budding at the plasma membrane. In this study we have investigated the effect of single amino acid substitutions in a conserved proline-rich region of the EBOV VP40 located in the carboxy-terminal part of the protein. We demonstrate that substitutions within this region result in an alteration of intracellular VP40 localization and also cause a reduction or a complete block of virus-like particle budding, a benchmark of VP40 function. Furthermore, some mutated VP40s revealed an enhanced binding with cellular Sec24C, a part of the coat protein complex II (COPII) vesicular transport system. Analysis of the 3-dimensional structure of VP40 revealed the spatial proximity of the proline-rich region and an earlier identified site of interaction with Sec24C, thus allowing us to hypothesize that the altered intracellular localization of the VP40 mutants is a consequence of defects in their interaction with COPII-mediated vesicular transport.  

3.1712           A new specialization in astrocytes: Glutamate- and ammonia-induced nuclear size changes

Yang, C.Z., Li, H.L., Zhou, Y., Chai, R.C., Zhao, R., Dong, Y., Xu, Z.Y., Lau, L.T., Yingge, Z., Teng, J., Chen, J and Yu, A.C.H.

1. Neurosci. Res., 89(12), 2041-2051 (2011)

  We observed nuclear swelling in glutamate (Glu)-treated astrocytes that was concomitant with but independent of astrocytic cell swelling. We confirmed Glu-induced nuclear swelling with nuclei isolated from astrocytes. Ammonia is metabolically related to Glu and could induce a nuclear swelling in intact astrocytes but shrinkage in isolated nuclei. Other compounds such as glutamine, aspartate, taurine, glycine, and ATP did not cause any nuclear swelling in isolated nuclei of astrocytes. Surprisingly, Glu and ammonia did not induce nuclear swelling in microglia, C6, HEK 293, or Hep G2 cell lines in cultures and their isolated nuclei. The Glu- and ammonia-induced nuclear size changes appear to be a specific response of astrocytes to these two closely related metabolic compounds  

3.1713           Ndel1, Nudel (  Noodle): Flexible in the cell?

Chansard, M., Hong, J-H., park, Y-U., park, S.K., and  Nguyen, M.D. Cytoskeleton, 68(10), 540-554 (2011)   Nuclear distribution element-like 1 (Ndel1 or Nudel) was firstly described as a regulator of the cytoskeleton in microtubule and intermediate filament dynamics and microtubule-based transport. Emerging evidence indicates that Ndel1 also serves as a docking platform for signaling proteins and modulates enzymatic activities (kinase, ATPase, oligopeptidase, GTPase). Through these structural and signaling functions, Ndel1 plays a role in diverse cellular processes (e.g., mitosis, neurogenesis, neurite outgrowth, and neuronal migration). Furthermore, Ndel1 is linked to the etiology of various mental illnesses and neurodegenerative disorders. In the present review, we summarize the physiological and pathological functions associated with Ndel1. We further advance the concept that Ndel1 interfaces GTPases-mediated processes (endocytosis, vesicles morphogenesis/signaling) and cytoskeletal dynamics to impact cell signaling and behaviors. This putative mechanism may affect cellular functionalities and may contribute to shed light into the causes of devastating human diseases.  

3.1714           Defective photoreceptor phagocytosis in a mouse model of enhanced S-cone syndrome causes progressive retinal degeneration

Mustafi, D., Kevany, B.M., Genoud, C., Okano, K., Cideciyan, A.V., Sumaroka, A., Roman, A.J., Jacobson, S.G., Engel, A., Adams, M.D. and Palczewski, K. FASEB J., 25(9), 3157-3176 (2011)   Enhanced S-cone syndrome (ESCS), featuring an excess number of S cones, manifests as a progressive retinal degeneration that leads to blindness. Here, through optical imaging, we identified an abnormal interface between photoreceptors and the retinal pigment epithelium (RPE) in 9 patients with ESCS. The neural retina leucine zipper transcription factor-knockout (Nrl^−/−) mouse model demonstrates many phenotypic features of human ESCS, including unstable S-cone-positive photoreceptors. Using massively parallel RNA sequencing, we identified 6203 differentially expressed transcripts between wild-type (Wt) and Nrl^−/− mouse retinas, with 6 highly significant differentially expressed genes of the Pax, Notch, and Wnt canonical pathways. Changes were also obvious in expression of 30 genes involved in the visual cycle and 3 key genes in photoreceptor phagocytosis. Novel high-resolution (100 nm) imaging and reconstruction of Nrl^−/− retinas revealed an abnormal packing of photoreceptors that contributed to buildup of photoreceptor deposits. Furthermore, lack of phagosomes in the RPE layer of Nrl^−/− retina revealed impairment in phagocytosis. Cultured RPE cells from Wt and Nrl^−/− mice illustrated that the phagocytotic defect was attributable to the aberrant interface between ESCS photoreceptors and the RPE. Overcoming the retinal phagocytosis defect could arrest the progressive degenerative component of this disease.—Mustafi, D., Kevany, B. M., Genoud, C., Okano, K., Cideciyan, A. V., Sumaroka, A., Roman, A. J., Jacobson, S. G. Engel, A., Adams, M. D., Palczewski, K. Defective photoreceptor phagocytosis in a mouse model of enhanced S-cone syndrome causes progressive retinal degeneration.  

3.1715           FATP4 contributes as an enzyme to the basal and insulin-mediated fatty acid uptake of C2C12 muscle cells

Digel, M., Staffer, S., Ehehalt, F., Stremmel, W., Ehehalt, R. and Füllekrug, J. Am. J. Physiol. Endocrinol. Metab., 301(5), E785-E796 (2011)   The function of membrane proteins in long-chain fatty acid transport is controversial. The acyl-CoA synthetase fatty acid transport protein-4 (FATP4) has been suggested to facilitate fatty acid uptake indirectly by its enzymatic activity, or directly by transport across the plasma membrane. Here, we investigated the function of FATP4 in basal and insulin mediated fatty acid uptake in C₂C₁₂ muscle cells, a model system relevant for fatty acid metabolism. Stable expression of exogenous FATP4 resulted in a twofold higher fatty acyl-CoA synthetase activity, and cellular uptake of oleate was enhanced similarly. Kinetic analysis demonstrated that FATP4 allowed the cells to reach apparent saturation of fatty acid uptake at a twofold higher level compared with control. Short-term treatment with insulin increased fatty acid uptake in line with previous reports. Surprisingly, insulin increased the acyl-CoA synthetase activity of C₂C₁₂ cells within minutes. This effect was sensitive to inhibition of insulin signaling by wortmannin. Affinity purified FATP4 prepared from insulin-treated cells showed an enhanced enzyme activity, suggesting it constitutes a novel target of short-term metabolic regulation by insulin. This offers a new mechanistic explanation for the concomitantly observed enhanced fatty acid uptake. FATP4 was colocalized to the endoplasmic reticulum by double immunofluorescence and subcellular fractionation, clearly distinct from the plasma membrane. Importantly, neither differentiation into myotubes nor insulin treatment changed the localization of FATP4. We conclude that FATP4 functions by its intrinsic enzymatic activity. This is in line with the concept that intracellular metabolism plays a significant role in cellular fatty acid uptake.  

3.1716           Quantitative subproteomic analysis of age-related changes in mouse liver peroxisomes by iTRAQ LC–MS/MS

Amelina, H., Sjödin, M.O.D., Bergquist, J. and Cristobal, S.

1. Chromatography B, 879(10), 3393-3400 (2011)

  Aging is a complex multifactorial phenomenon, which is believed to result from the accumulation of cellular damage to biological macromolecules. Peroxisomes recently emerged as another important source of reactive oxygen species (ROS) production in addition to mitochondria. However, the role of these organelles in the process of aging is still not clear. The aim of this study was to characterize the changes in protein expression profiles of young (10 weeks old) versus old (18 months old) mouse liver peroxisome-enriched fractions. We have applied shotgun proteomic approach based on liquid chromatography and tandem mass spectrometry (LC–MS/MS) combined with iTRAQ (isobaric tags for relative and absolute quantitation) labeling that allows comparative quantitative multiplex analysis. Our analysis led to identification and quantification of 150 proteins, 8 out of which were differentially expressed between two age groups at a statistically significant level (p < 0.05), with folds ranging from 1.2 to 4.1. These proteins involved in peroxisomal β-oxidation, detoxification of xenobiotics and production of ROS. Noteworthy, differences in liver proteome have been observed between as well as within different age groups. In conclusion, our subproteomic quantitative study suggests that mouse liver proteome is sufficiently maintained until certain age.  

3.1717           In vitro nuclear egress of herpes simplex virus type 1 capsids

Remillard-Labrosse, G. and Lippe, R. Methods, 55(2), 153-159 (2011)   During their life cycles, viruses typically undergo many transport events throughout the cell. These events depend on a variety of both viral and host proteins and are often not fully understood. Such studies are often complicated by asynchronous infections and the concurrent presence of various viral intermediates in the cells, making it difficult to molecularly define each step. In the case of the herpes simplex virus type 1, the etiological agent of cold sores and many other illnesses, the viral particles undergo an intricate series of transport steps during its life cycle. Upon entry by fusion with a cellular membrane, they travel to the host cell nucleus where the virus replicates and assembles new viral particles. These particles then travel across the two nuclear envelopes and transit through the trans-Golgi network before finally being transported to and released at the cell surface. Though viral components and some host proteins modulating these numerous transport events have been identified, the details of these processes remain to be elucidated. To specifically address how the virus escapes the nucleus, we set up an in vitro model that reproduces the unconventional route used by herpes simplex type 1 virus to leave nuclei. This has not only allowed us to clarify the route of capsid egress of the virus but is now useful to define it at the molecular level.  

3.1718           GLUT2 Accumulation in Enterocyte Apical and Intracellular Membranes: A Study in Morbidly Obese Human Subjects and ob/ob and High Fat–Fed Mice

Ait-Omar, A. et al Diabetes, 60, 2598-2607 (2011)   OBJECTIVE In healthy rodents, intestinal sugar absorption in response to sugar-rich meals and insulin is regulated by GLUT2 in enterocyte plasma membranes. Loss of insulin action maintains apical GLUT2 location. In human enterocytes, apical GLUT2 location has not been reported but may be revealed under conditions of insulin resistance. RESEARCH DESIGN AND METHODS Subcellular location of GLUT2 in jejunal enterocytes was analyzed by confocal and electron microscopy imaging and Western blot in 62 well-phenotyped morbidly obese subjects and 7 lean human subjects. GLUT2 locations were assayed in ob/ob and ob/+ mice receiving oral metformin or in high-fat low-carbohydrate diet–fed C57Bl/6 mice. Glucose absorption and secretion were respectively estimated by oral glucose tolerance test and secretion of [U-¹⁴C]-3-O-methyl glucose into lumen. RESULTS In human enterocytes, GLUT2 was consistently located in basolateral membranes. Apical GLUT2 location was absent in lean subjects but was observed in 76% of obese subjects and correlated with insulin resistance and glycemia. In addition, intracellular accumulation of GLUT2 with early endosome antigen 1 (EEA1) was associated with reduced MGAT4a activity (glycosylation) in 39% of obese subjects on a low-carbohydrate/high-fat diet. Mice on a low-carbohydrate/high-fat diet for 12 months also exhibited endosomal GLUT2 accumulation and reduced glucose absorption. In ob/ob mice, metformin promoted apical GLUT2 and improved glucose homeostasis. Apical GLUT2 in fasting hyperglycemic ob/ob mice tripled glucose release into intestinal lumen. CONCLUSIONS In morbidly obese insulin-resistant subjects, GLUT2 was accumulated in apical and/or endosomal membranes of enterocytes. Functionally, apical GLUT2 favored and endosomal GLUT2 reduced glucose transepithelial exchanges. Thus, altered GLUT2 locations in enterocytes are a sign of intestinal adaptations to human metabolic pathology.  

3.1719           Motor neuron impairment mediated by a sumoylated fragment of the glial glutamate transporter EAAT2

Foran, E., Bogush, A., Goffredo, M., Roncaglia, P., Gustincich, S., Pasinell, P. and Trotti, D.

Glia, 59(11), 1719-1731 (2011)

Dysregulation of glutamate handling ensuing downregulation of expression and activity levels of the astroglial glutamate transporter EAAT2 is implicated in excitotoxic degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). We previously reported that EAAT2 (a.k.a. GLT-1) is cleaved by caspase-3 at its cytosolic carboxy-terminus domain. This cleavage results in impaired glutamate transport activity and generates a proteolytic fragment (CTE) that we found to be post-translationally conjugated by SUMO1. We show here that this sumoylated CTE fragment accumulates in the nucleus of spinal cord astrocytes of the SOD1-G93A mouse model of ALS at symptomatic stages of disease. Astrocytic expression of CTE, artificially tagged with SUMO1 (CTE-SUMO1) to mimic the native sumoylated fragment, recapitulates the nuclear accumulation pattern of the endogenous EAAT2-derived proteolytic fragment. Moreover, in a co-culture binary system, expression of CTE-SUMO1 in spinal cord astrocytes initiates extrinsic toxicity by inducing caspase-3 activation in motor neuron-derived NSC-34 cells or axonal growth impairment in primary motor neurons. Interestingly, prolonged nuclear accumulation of CTE-SUMO1 is intrinsically toxic to spinal cord astrocytes, although this gliotoxic effect of CTE-SUMO1 occurs later than the indirect, noncell autonomous toxic effect on motor neurons. As more evidence on the implication of SUMO substrates in neurodegenerative diseases emerges, our observations strongly suggest that the nuclear accumulation in spinal cord astrocytes of a sumoylated proteolytic fragment of the astroglial glutamate transporter EAAT2 could participate to the pathogenesis of ALS and suggest a novel, unconventional role for EAAT2 in motor neuron degeneration.

   

3.1720           Characterizing the Escherichia coli O157:H7 Proteome Including Protein Associations with Higher Order Assemblies

Pieper, R., Zhang, Q., Clark, D.J., Hunag, S-T., Suh, M-J., Braisted, J.C., Payne, S.H., Fleischmann, R.D., Peterson, S.N. and Tzipori, S. PloS One, 6(10), e26554 (2011)   Background The recent outbreak of severe infections with Shiga toxin (Stx) producing Escherichia coli (STEC) serotype O104:H4 highlights the need to understand horizontal gene transfer among E. coli strains, identify novel virulence factors and elucidate their pathogenesis. Quantitative shotgun proteomics can contribute to such objectives, allowing insights into the part of the genome translated into proteins and the connectivity of biochemical pathways and higher order assemblies of proteins at the subcellular level. Methodology/Principal Findings We examined protein profiles in cell lysate fractions of STEC strain 86-24 (serotype O157:H7), following growth in cell culture or bacterial isolation from intestines of infected piglets, in the context of functionally and structurally characterized biochemical pathways of E. coli. Protein solubilization in the presence of Triton X-100, EDTA and high salt was followed by size exclusion chromatography into the approximate M_r ranges greater than 280 kDa, 280-80 kDa and 80-10 kDa. Peptide mixtures resulting from these and the insoluble fraction were analyzed by quantitative 2D-LC-nESI-MS/MS. Of the 2521 proteins identified at a 1% false discovery rate, representing 47% of all predicted E. coli O157:H7 gene products, the majority of integral membrane proteins were enriched in the high M_r fraction. Hundreds of proteins were enriched in a M_r range higher than that predicted for a monomer supporting their participation in protein complexes. The insoluble STEC fraction revealed enrichment of aggregation-prone proteins, including many that are part of large structure/function entities such as the ribosome, cytoskeleton and O-antigen biosynthesis cluster. Significance Nearly all E. coli O157:H7 proteins encoded by prophage regions were expressed at low abundance levels or not detected. Comparative quantitative analyses of proteins from distinct cell lysate fractions allowed us to associate uncharacterized proteins with membrane attachment, potential participation in stable protein complexes, and susceptibility to aggregation as part of larger structural assemblies.  

3.1721           Differential Palmit(e)oylation of Wnt1 on C93 and S224 Residues Has Overlapping and Distinct Consequences

Galli, L.M. and Burrus, L.W. PloS One, 6(10), e26636 (2011)   Though the mechanisms by which cytosolic/intracellular proteins are regulated by the post-translational addition of palmitate adducts is well understood, little is known about how this lipid modification affects secreted ligands, such as Wnts. Here we use mutational analysis to show that differential modification of the two known palmit(e)oylated residues of Wnt1, C93 and S224, has both overlapping and distinct consequences. Though the relative roles of each residue are similar with respect to stability and secretion, two distinct biological assays in L cells show that modification of C93 primarily modulates signaling via a ß-catenin independent pathway while S224 is crucial for ß-catenin dependent signaling. In addition, pharmacological inhibition of Porcupine (Porcn), an upstream regulator of Wnt, by IWP1, specifically inhibited ß-catenin dependent signaling. Consistent with these observations, mapping of amino acids in peptide domains containing C93 and S224 demonstrate that acylation of C93 is likely to be Porcn-independent while that of S224 is Porcn-dependent. Cumulatively, our data strongly suggest that C93 and S224 are modified by distinct enzymes and that the differential modification of these sites has the potential to influence Wnt signaling pathway choice.  

3.1722           Rescue of Calcineurin Aα−/− Mice Reveals a Novel Role for the α Isoform in the Salivary Gland

Reddy, R.N., Pena, J.A., Roberts, B.R., Williams, S.R., Price, S.R. and Gooch, J.L. Am. J. Pathol., 178(4), 1605-1613 (2011)   Calcineurin is an important signal transduction mediator in T cells, neurons, the heart, and kidneys. Recent evidence points to unique actions of the two main isoforms of the catalytic subunit. Although the β isoform is required for T-cell development, α is important in the brain and kidney. In addition, mice lacking α but not β suffer from failure to thrive and early mortality. The purpose of this study was to identify the cause of postnatal death of calcineurin α null (CnAα^−/−) mice and to determine the mechanism of α activity that contributes to the phenotype. CnAα^−/− mice and wild-type littermate controls were fed a modified diet and then salivary gland function and histology were examined. In vitro studies were performed to identify the mechanism of α action. Data show that calcineurin is required for normal submandibular gland function and secretion of digestive enzymes. Loss of α does not impair nuclear factor of activated T-cell activity or expression but results in impaired protein trafficking downstream of the inositol trisphosphate receptor. These findings show a novel function of calcineurin in digestion and protein trafficking. Significantly, these data also provide a mechanism to rescue to adulthood a valuable animal model of calcineurin inhibitor-mediated neuronal and renal toxicities.  

3.1723           HAT4, a Golgi Apparatus-Anchored B-Type Histone Acetyltransferase, Acetylates Free Histone H4 and Facilitates Chromatin Assembly

Yang, X., Yu, W., Shi, L., Sun, L., Linag, J., Yi, X., Li, Q., Zhang, Y., Yang, F., Han, X., Zhang, D., Yang, J., Yao, Z. and Shang, Y. Mol. Cell, 44(1), 39-50 (2011)   Histone acetyltransferases (HATs) are an essential regulatory component in chromatin biology. Unlike A-type HATs, which are found in the nucleus and utilize nucleosomal histones as substrates and thus primarily function in transcriptional regulation, B-type HATs have been characterized as cytoplasmic enzymes that catalyze the acetylation of free histones. Here, we report on a member of the GCN5-related N-acetyltransferase superfamily and another B-type HAT, HAT4. Interestingly, HAT4 is localized in the Golgi apparatus and displays a substrate preference for lysine residues of free histone H4, including H4K79 and H4K91, that reside in the globular domain of H4. Significantly, HAT4 depletion impaired nucleosome assembly, inhibited cell proliferation, sensitized cells to DNA damage, and induced cell apoptosis. Our data indicate that HAT4 is an important player in the organization and function of the genome and may contribute to the diversity and complexity of higher eukaryotic organisms.  

3.1724           The Unconventional Role of Acid Sphingomyelinase in Regulation of Retinal Microangiopathy in Diabetic Human and Animal Models

Opreanu, M., Tikhonenko, M., Bozack, S., Lydic, T.A., Reid, G.E., McSorley, K.M., Sochacki, A., Perez, G.I., Esselman, W.J., Kern, T., Kolesnick, R., Grant, M.B. and Busik, J.V. Diabetes, 60, 2370-2378 (2011)   OBJECTIVE Acid sphingomyelinase (ASM) is an important early responder in inflammatory cytokine signaling. The role of ASM in retinal vascular inflammation and vessel loss associated with diabetic retinopathy is not known and represents the goal of this study. RESEARCH DESIGN AND METHODS Protein and gene expression profiles were determined by quantitative RT-PCR and Western blot. ASM activity was determined using Amplex Red sphingomyelinase assay. Caveolar lipid composition was analyzed by nano-electrospray ionization tandem mass spectrometry. Streptozotocin-induced diabetes and retinal ischemia-reperfusion models were used in in vivo studies. RESULTS We identify endothelial caveolae-associated ASM as an essential component in mediating inflammation and vascular pathology in in vivo and in vitro models of diabetic retinopathy. Human retinal endothelial cells (HREC), in contrast with glial and epithelial cells, express the plasma membrane form of ASM that overlaps with caveolin-1. Treatment of HREC with docosahexaenoic acid (DHA) specifically reduces expression of the caveolae-associated ASM, prevents a tumor necrosis factor-α–induced increase in the ceramide-to-sphingomyelin ratio in the caveolae, and inhibits cytokine-induced inflammatory signaling. ASM is expressed in both vascular and neuroretina; however, only vascular ASM is specifically increased in the retinas of animal models at the vasodegenerative phase of diabetic retinopathy. The absence of ASM in ASM^−/− mice or inhibition of ASM activity by DHA prevents acellular capillary formation. CONCLUSIONS This is the first study demonstrating activation of ASM in the retinal vasculature of diabetic retinopathy animal models. Inhibition of ASM could be further explored as a potential therapeutic strategy in treating diabetic retinopathy.  

3.1725           Outer Membrane Vesicles Induce Immune Responses to Virulence Proteins and Protect against Colonization by Enterotoxigenic Escherichia coli

Roy, K., Hamilton, D.J., Munson, G.P. and Fleckenstein, J.M. Clin. Vacc. Immunol., 18(11), 1803-1808 (2011)   Enterotoxigenic Escherichia coli (ETEC) strains are a heterogeneous group of pathogens that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Collectively, these pathogens are responsible for hundreds of thousands of deaths annually in developing countries, particularly in children under the age of 5 years. The heterogeneity of previously investigated molecular targets and the lack of complete sustained protection afforded by antitoxin immunity have impeded progress to date toward a broadly protective vaccine. Many pathogens, including ETEC, have the capacity to form outer membrane vesicles (OMV), which often contain one or more virulence proteins. Prompted by recent studies that identified several immunogenic virulence proteins in outer membrane vesicles of ETEC, we sought to examine the immunogenicity and protective efficacy of these structures in a murine model of infection. Here we demonstrate that immunization with OMV impairs ETEC colonization of the small intestine and stimulates antibodies that recognize the heat-labile toxin and two additional putative virulence proteins, the EtpA adhesin and CexE. Similar to earlier studies with EtpA, vaccination with LT alone also inhibited intestinal colonization. Together, these findings suggest that OMV could be exploited to deliver protective antigens relevant to development of ETEC vaccines.  

3.1726           Screening and Optimization of Ligand Conjugates for Lysosomal Targeting

Meerovich, I., Hoshkaryev, A., Thekkedath, R. and Torchilin, V.P. Bioconjugate Chem., 22(11), 2271-2282 (2011)   The use of lysosome-targeted liposomes may significantly improve the delivery of therapeutic enzymes and chaperones into lysosomes for the treatment of lysosomal storage disorders. The aim of this research was to synthesize new potentially lysosomotropic ligands on a base of Neutral Red and rhodamine B and to study their ability to enhance specific lysosomal delivery of surface-modified liposomes loaded with a model compound, fluorescein isothiocyanate-dextran (FD). The delivery of these liposomes and their content to lysosomes in HeLa cells was investigated by confocal immunofluorescent microscopy, subcellular fractionation, and flow cytometry. Confocal microscopy demonstrated that liposomes modified with derivatives of rhodamine B provide a good rate of colocalization with the specific lysosomal markers. The comparison of fluorescence of FD in lysosomes isolated by subcellular fractionation also showed that the efficiency of lysosomal delivery of the liposomal load by liposomes modified with some of synthesized ligands was significantly higher compared to that with plain liposomes. These results were additionally confirmed by flow cytometry of the intact cells treated with liposomes loaded with 5-dodecanoylaminofluorescein di-β-d-galactopyranoside, a specific substrate for the intralysosomal β-galactosidase, using a number of cell lines, including macrophages with induced phenotype of lysosomal enzyme deficiency; two of the synthesized ligands—rhodamine B DSPE-PEG_2k-amide and 6-(3-(DSPE-PEG_2k)-thioureido) rhodamine B—demonstrated enhanced lysosomal delivery, in some cases, higher than that for commercially available rhodamine B octadecyl ester, with the best results (the enhancement of the lysosomal delivery up to 75% greater in comparison to plain liposomes) shown for the cells with induced lysosomal enzyme deficiency phenotype. Use of liposomes modified with rhodamine B derivatives may be advantageous for the development of drug delivery systems for the treatment of lysosome-associated disorders.  

3.1727           Retinal Degeneration 3 (RD3) Protein Inhibits Catalytic Activity of Retinal Membrane Guanylyl Cyclase (RetGC) and Its Stimulation by Activating Proteins

Peshenko, I.V., Olshevskaya, EW.V., Azadi, S., Molday, L.L., Molday, R.S. and Dizhoor, A.M: Biochemistry, 50(44), 9511-9519 (2011)   Retinal membrane guanylyl cyclase (RetGC) in the outer segments of vertebrate photoreceptors is controlled by guanylyl cyclase activating proteins (GCAPs), responding to light-dependent changes of the intracellular Ca²⁺ concentrations. We present evidence that a different RetGC binding protein, retinal degeneration 3 protein (RD3), is a high-affinity allosteric modulator of the cyclase which inhibits RetGC activity at submicromolar concentrations. It suppresses the basal activity of RetGC in the absence of GCAPs in a noncompetitive manner, and it inhibits the GCAP-stimulated RetGC at low intracellular Ca²⁺ levels. RD3 opposes the allosteric activation of the cyclase by GCAP but does not significantly change Ca²⁺ sensitivity of the GCAP-dependent regulation. We have tested a number of mutations in RD3 implicated in human retinal degenerative disorders and have found that several mutations prevent the stable expression of RD3 in HEK293 cells and decrease the affinity of RD3 for RetGC1. The RD3 mutant lacking the carboxy-terminal half of the protein and associated with Leber congenital amaurosis type 12 (LCA12) is unable to suppress the activity of the RetGC1/GCAP complex. Furthermore, the inhibitory activity of the G57V mutant implicated in cone–rod degeneration is strongly reduced. Our results suggest that inhibition of RetGC by RD3 may be utilized by photoreceptors to block RetGC activity during its maturation and/or incorporation into the photoreceptor outer segment rather than participate in dynamic regulation of the cyclase by Ca²⁺ and GCAPs.  

3.1728           Translation initiation factors and active sites of protein synthesis co-localize at the leading edge of migrating fibroblasts

Willett, M., Brocard, M., Davide, A. and Morley, S.J. Biochem. J., 438, 217-227 (2011)   Cell migration is a highly controlled essential cellular process, often dysregulated in tumour cells, dynamically controlled by the architecture of the cell. Studies involving cellular fractionation and microarray profiling have previously identified functionally distinct mRNA populations specific to cellular organelles and architectural compartments. However, the interaction between the translational machinery itself and cellular structures is relatively unexplored. To help understand the role for the compartmentalization and localized protein synthesis in cell migration, we have used scanning confocal microscopy, immunofluorescence and a novel ribopuromycylation method to visualize translating ribosomes. In the present study we show that eIFs (eukaryotic initiation factors) localize to the leading edge of migrating MRC5 fibroblasts in a process dependent on TGN (trans-Golgi network) to plasma membrane vesicle transport. We show that eIF4E and eIF4GI are associated with the Golgi apparatus and membrane microdomains, and that a proportion of these proteins co-localize to sites of active translation at the leading edge of migrating cells.  

3.1729           Lipid Raft Localization of EGFR Alters the Response of Cancer Cells to the EGFR Tyrosine Kinase

Inhibitor Gefitinib

Irwin, M.E., Mueller, K.L., Bohin, N., Ge, Y. and Boerner, J.L.

1. Cell. Physiol., 226, 2316-2328 (2011)

  Epidermal growth factor receptor (EGFR) is overexpressed in many cancer types including_30% of breast cancers. Several small molecule tyrosine kinase inhibitors (TKIs) targeting EGFR have shown clinical efficacy in lung and colon cancers, but no benefit has been noted in breast cancer. Thirteen EGFR expressing breast cancer cell lines were analyzed for response to EGFR TKIs. Seven were found to be EGFR TKI resistant; while shRNA knockdown of EGFR determined that four of these cell lines retained the requirement of EGFR protein expression for growth. Interestingly, EGFR localized to plasma membrane lipid rafts in all four of these EGFR TKI-resistant cell lines, as determined by biochemical raft isolation and immunofluorescence. When lipid rafts were depleted of cholesterol using lovastatin, all four cell lines were sensitized to EGFR TKIs. In fact, the effects of the cholesterol biosynthesis inhibitor and gefitinib were synergistic. While gefitinib effectively abrogated phosphorylation of Akt- and mitogen-activated protein kinase in an EGFR TKI-sensitive cell line, phosphorylation of Akt persisted in two EGFR TKI-resistant cell lines, however, this phosphorylation was abrogated by lovastatin treatment. Thus, we have shown that lipid raft localization of EGFR correlates with resistance to EGFR TKI-induced growth inhibition and pharmacological depletion of cholesterol from lipid rafts decreases this resistance in breast cancer cell lines. Furthermore, we have presented evidence to suggest that when EGFR localizes to lipid rafts, these rafts provide a platform to facilitate activation of Akt signalling in the absence of EGFR kinase activity.  

3.1730           Activating Transcription Factor 6 Limits Intracellular Accumulation of Mutant α1-Antitrypsin Z and Mitochondrial Damage in Hepatoma Cells

Smith, S.E., Granell, S., Salcedo-Sicilia, L., Baldini, G., Egea, G., Teckman, J.H. and Baldini, G.

1. Biol. Chem., 286(48), 41563-41577 (2011)

  α₁-Antitrypsin is a serine protease inhibitor secreted by hepatocytes. A variant of α₁-antitrypsin with an E342K (Z) mutation (ATZ) has propensity to form polymers, is retained in the endoplasmic reticulum (ER), is degraded by both ER-associated degradation and autophagy, and causes hepatocyte loss. Constant features in hepatocytes of PiZZ individuals and in PiZ transgenic mice expressing ATZ are the formation of membrane-limited globular inclusions containing ATZ and mitochondrial damage. Expression of ATZ in the liver does not induce the unfolded protein response (UPR), a protective mechanism aimed to maintain ER homeostasis in the face of an increased load of proteins. Here we found that in hepatoma cells the ER E3 ligase HRD1 functioned to degrade most of the ATZ before globular inclusions are formed. Activation of the activating transcription factor 6 (ATF6) branch of the UPR by expression of spliced ATF6(1–373) decreased intracellular accumulation of ATZ and the formation of globular inclusions by a pathway that required HRD1 and the proteasome. Expression of ATF6(1–373) in ATZ-expressing hepatoma cells did not induce autophagy and increased the level of the proapoptotic factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) but did not lead to apoptotic DNA fragmentation. Expression of ATF6(1–373) did not cause inhibition of protein synthesis and prevented mitochondrial damage induced by ATZ expression. It was concluded that activation of the ATF6 pathway of the UPR limits ATZ-dependent cell toxicity by selectively promoting ER-associated degradation of ATZ and is thereby a potential target to prevent hepatocyte loss in addition to autophagy-enhancing drugs.  

3.1731           C323 of SR-BI is required for SR-BI-mediated HDL binding and cholesteryl ester uptake

Guo, L., Chen, M., Song, Z., Daugherty, A. and Li, X-A.

1. Lipid Res., 52, 2272-2278 (2011)

  Scavenger receptor BI (SR-BI) is an HDL receptor. It binds HDL and mediates the uptake of cholesteryl ester from HDL. Early studies have pointed out that the extracellular domain of SR-BI is critical for SR-BI-mediated cholesteryl ester uptake. However, the extracellular loop of SR-BI is large: it contains 403 amino acids. The HDL binding site and the modulation of SR-BI-mediated cholesteryl ester uptake remain to be identified. In this study, using C323G mutant SR-BI, we showed that C323G mutant SR-BI lost its HDL binding and cholesteryl ester uptake activity, indicating that the highly conserved C323 is required for SR-BI-mediated HDL binding and cholesteryl ester uptake. Using a blocking antibody against C323 region, we demonstrated that C323 is directly involved in HDL binding and likely an HDL binding site. Using C323G mutant transgenic mouse model, we further demonstrated that C323 of SR-BI is required for regulating plasma cholesterol levels in vivo. Using redox reagents, we showed that physiological relevant levels of H₂O₂ upregulated the SR-BI-mediated cholesteryl ester uptake activity by 65%, whereas GSH or DTT significantly downregulated SR-BI-mediated cholesteryl ester uptake activity by 45%. C323 of SR-BI is critical for SR-BI-mediated HDL binding and cholesteryl ester uptake, and changes in redox status may be a regulatory factor modulating SR-BI-mediated cholesterol transport.  

3.1732           Site-directed mutagenesis of human cytosolic sulfotransferase (SULT) 2B1b to phospho-mimetic Ser348Asp results in an isoform with increased catalytic activity

Salman, E.D., He, D., Runge-Morris, M., Kocarek, T.A. and Falany, C.N.

1. Steroid Biochem. Mol. Biol., 127, 315-323 (2011)

  Human SULT2B1b is distinct from other SULT isoforms due to the presence of unique amino (N)- and carboxy (C)-terminal peptides. Using site-directed mutagenesis, it was determined that phosphorylation of Ser348 was associated with nuclear localization. To investigate the effects of this phosphorylation of Ser348 on activity and cellular localization, an in silico molecular mimic was generated by mutating Ser348 to an Asp. The Asp residue mimics the shape and charge of a phospho-Ser and homology models of SULT2B1b-phospho-S348 and SULT2B1b-S348D suggest a similar significant structural rearrangement in the C-terminal peptide. To evaluate the functional consequences of this post-translational modification and predicted rearrangement, 6His-SULT2B1b-S348D was synthesized, expressed, purified and characterized. The 6His-SULT2B1b-S348D has a specific activity for DHEA sulfation ten-fold higher than recombinant 6His-SULT2B1b (209.6 and 21.8 pmol min−1 mg−1, respectively). Similar to native SULT2B1b, gel filtration chromatography showed SULT2B1b-S348D was enzymatically active as a homodimer. Stability assays comparing SULT2B1b and SUL2B1b-S348 demonstrated that SULT2B1b is 60% less thermostable than SULT2B1b-348D. The increased stability and sulfation activity allowed for better characterization of the sulfation kinetics for putative substrates as well as the determination of dissociation constants that were difficult to obtain with wild-type (WT) 6His-SULT2B1b. The KDs for DHEA and PAPS binding to 6His-SULT2B1b-S348D were 650 ± 7 nM and 265 ± 4 nM, respectively, whereas KDs for binding of substrates to the WT enzyme could not be determined. Characterization of the molecular mimic SULT2B1b-S348D provides a better understanding for the role of the unique structure of SULT2B1b and its effect on sulfation activity, and has allowed for improved kinetic characterization of the SULT2B1b enzyme.  

3.1733           Thyroid-specific knockout of the tumor suppressor mitogen-inducible gene 6 activates epidermal growth factor receptor signaling pathways and suppresses nuclear factor-κB activity

Lin, C-I., Barletta, J.A., Nehs, M.A., Morris, Z.S., Donner, D.B., Whang, E.E., Jeong, J-w., Kimura, S., Moore, F.D. and Ruan, D.T. Surgery, 150(1), 295-302 (2011)   Background Mitogen-inducible gene 6 (Mig-6) is a putative tumor suppressor gene and prognostic biomarker in papillary thyroid cancer. We hypothesized that Mig-6 knockout would activate pro-oncogenic signaling in mouse thyrocytes. Methods We performed a thyroid - specific knockout using the Cre/loxP recombinase system. Results Four knockout and 4 control mouse thyroids were harvested at 2 months of age. Immunoblotting confirmed Mig-6 ablation in knockout mice thyrocytes. Epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK) phosphorylation levels were increased in Mig-6 knockout compared to wild-type mice. Total EGFR levels were similar in knockout and wild-type mice. However, EGFR was absent in the caveolae-containing membrane fraction of knockout mice, indicating that Mig-6 depletion is associated with a change in the membrane distribution of EGFR. Although p65 localized to the nucleus in wild-type mice, it was distributed in both cytoplasm and nucleus in knockouts , suggesting that Mig-6 loss decreases p65 activity. Conclusion Our results confirm the feasibility of targeted, thyroid - specific gene knockout as a strategy for studying the relevance of specific genes in thyroid oncogenesis. We suggest that the loss of Mig-6 alters the membrane distribution of EGFR, which may limit receptor degradation and activate this oncogenic signaling pathway.  

3.1734           Biochemical and Molecular Mechanisms of Folate Transport in Rat Pancreas; Interference with Ethanol Ingestion

Wani, N.A., Nada, R. and Kaur, J. PloS One, 6(12), e28599 (2011)   Folic acid is an essential nutrient that is required for one-carbon biosynthetic processes and for methylation of biomolecules. Deficiency of this micronutrient leads to disturbances in normal physiology of cell. Chronic alcoholism is well known to be associated with folate deficiency which is due, in part to folate malabsorption. The present study deals with the mechanistic insights of reduced folate absorption in pancreas during chronic alcoholism. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20% solution) orally for 3 months and the mechanisms of alcohol associated reduced folate uptake was studied in pancreas. The folate transport system in the pancreatic plasma membrane (PPM) was found to be acidic pH dependent one. The transporters proton coupled folate transporter (PCFT) and reduced folate carrier (RFC) are involved in folate uptake across PPM. The folate transporters were found to be associated with lipid raft microdomain of the PPM. Ethanol ingestion decreased the folate transport by reducing the levels of folate transporter molecules in lipid rafts at the PPM. The decreased transport efficiency of the PPM was reflected as reduced folate levels in pancreas. The chronic ethanol ingestion led to decreased pancreatic folate uptake. The decreased levels of PCFT and RFC expression in rat PPM were due to decreased association of these proteins with lipid rafts (LR) at the PPM.  

3.1735           Nuclear Localization of de Novo Thymidylate Biosynthesis Pathway Is Required to Prevent Uracil Accumulation in DNA

MacFarlane, A.J., Anderson, D.D., Flodby, P., Perry, C.A., Allen, R.H. and Stabler, S.P.

1. Biol. Chem., 286(51), 44015-44022 (2011)

  Uracil accumulates in DNA as a result of impaired folate-dependent de novo thymidylate biosynthesis, a pathway composed of the enzymes serine hydroxymethyltransferase (SHMT), thymidylate synthase (TYMS), and dihydrofolate reductase. In G₁, this pathway is present in the cytoplasm and at S phase undergoes small ubiquitin-like modifier-dependent translocation to the nucleus. It is not known whether this pathway functions in the cytoplasm, nucleus, or both in vivo. SHMT1 generates 5,10-methylenetetrahydrofolate for de novo thymidylate biosynthesis, a limiting step in the pathway, but also tightly binds 5-methyltetrahydrofolate in the cytoplasm, a required cofactor for homocysteine remethylation. Overexpression of SHMT1 in cell cultures inhibits folate-dependent homocysteine remethylation and enhances thymidylate biosynthesis. In this study, the impact of increased Shmt1 expression on folate-mediated one-carbon metabolism was determined in mice that overexpress the Shmt1 cDNA (Shmt1^tg+ mice). Compared with wild type mice, Shmt1^tg+ mice exhibited elevated SHMT1 and TYMS protein levels in tissues and evidence for impaired homocysteine remethylation but surprisingly exhibited depressed levels of nuclear SHMT1 and TYMS, lower rates of nuclear de novo thymidylate biosynthesis, and a nearly 10-fold increase in uracil content in hepatic nuclear DNA when fed a folate- and choline-deficient diet. These results demonstrate that SHMT1 and TYMS localization to the nucleus is essential to prevent uracil accumulation in nuclear DNA and indicate that SHMT1-mediated nuclear de novo thymidylate synthesis is critical for maintaining DNA integrity.  

3.1736           Quantitative proteomics reveals metabolic and pathogenic properties of Chlamydia trachomatis developmental forms

Saka, H.A., Thompson, J.W., Chen, Y-S:, Kumar, Y., Dubois, L.G., Moseley, M.A. and Valdivia, R.H. Mol. Microbiol., 82(5), 1185-1203 (2011)   Chlamydia trachomatis is an obligate intracellular pathogen responsible for ocular and genital infections of significant public health importance. C. trachomatis undergoes a biphasic developmental cycle alternating between two distinct forms: the infectious elementary body (EB), and the replicative but non-infectious reticulate body (RB). The molecular basis for these developmental transitions and the metabolic properties of the EB and RB forms are poorly understood as these bacteria have traditionally been difficult to manipulate through classical genetic approaches. Using two-dimensional liquid chromatography – tandem mass spectrometry (LC/LC-MS/MS) we performed a large-scale, label-free quantitative proteomic analysis of C. trachomatis LGV-L2 EB and RB forms. Additionally, we carried out LC-MS/MS to analyse the membranes of the pathogen-containing vacuole (‘inclusion’). We developed a label-free quantification approaches to measure protein abundance in a mixed-proteome background which we applied for EB and RB quantitative analysis. In this manner, we catalogued the relative distribution of > 54% of the predicted proteins in the C. trachomatis LGV-L2 proteome. Proteins required for central metabolism and glucose catabolism were predominant in the EB, whereas proteins associated with protein synthesis, ATP generation and nutrient transport were more abundant in the RB. These findings suggest that the EB is primed for a burst in metabolic activity upon entry, whereas the RB form is geared towards nutrient utilization, a rapid increase in cellular mass, and securing the resources for an impending transition back to the EB form. The most revealing difference between the two forms was the relative deficiency of cytoplasmic factors required for efficient type III secretion (T3S) in the RB stage at 18 h post infection, suggesting a reduced T3S capacity or a low frequency of active T3S apparatus assembled on a ‘per organism’ basis. Our results show that EB and RB proteomes are streamlined to fulfil their predicted biological functions: maximum infectivity for EBs and replicative capacity for RBs.  

3.1737           Effects of brefeldin A-inhibited guanine nucleotide-exchange (BIG) 1 and KANK1 proteins on cell polarity and directed migration during wound healing

Li, C-C., Kuo,, J-C., Waterman, C.M., Kiyama, R., Moss, J. and Vaughan, M. PNAS, 108(48), 19228-19233 (2011)   Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 activates class I ADP ribosylation factors (ARFs) by accelerating the replacement of bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Among proteins that interact with BIG1, kinesin family member 21A (KIF21A), a plus-end-directed motor protein, moves cargo away from the microtubule-organizing center (MTOC) on microtubules. Because KANK1, a protein containing N-terminal KN, C-terminal ankyrin-repeat, and intervening coiled-coil domains, has multiple actions in cells and also interacts with KIF21A, we explored a possible interaction between it and BIG1. We obtained evidence for a functional and physical association between these proteins, and found that the effects of BIG1 and KANK1 depletion on cell migration in wound-healing assays were remarkably similar. Treatment of cells with BIG1- or KANK1-specific siRNA interfered significantly with directed cell migration and initial orientation of Golgi/MTOC toward the leading edge, which was not mimicked by KIF21A depletion. Although colocalization of overexpressed KANK1 and endogenous BIG1 in HeLa cells was not clear microscopically, their reciprocal immunoprecipitation (IP) is compatible with the presence of small percentages of each protein in the same complexes. Depletion or overexpression of BIG1 protein appeared not to affect KANK1 distribution. Our data identify actions of both BIG1 and KANK1 in regulating cell polarity during directed migration; these actions are consistent with the presence of both BIG1 and KANK1 in dynamic multimolecular complexes that maintain Golgi/MTOC orientation, differ from those that might contain all three proteins (BIG1, KIF21A, and KANK1), and function in directed transport along microtubules.  

3.1738           The Human Immunodeficiency Virus Coat Protein gp120 Promotes Forward Trafficking and Surface Clustering of NMDA Receptors in Membrane Microdomains

Xu, H., Bae, M., Tovar-Romo, L.B., patel, N., Bandaru, V.V.R., Pomerantz, D., Steiner, J.P. and Haughey, N.J.

1. Neurosci., 31(47), 17074-17090 (2011)

  Infection by the human immunodeficiency virus (HIV) can result in debilitating neurological syndromes collectively known as HIV-associated neurocognitive disorders. Although the HIV coat protein gp120 has been identified as a potent neurotoxin that enhances NMDA receptor function, the exact mechanisms for this effect are not known. Here we provide evidence that gp120 activates two separate signaling pathways that converge to enhance NMDA-evoked calcium flux by clustering NMDA receptors in modified membrane microdomains. gp120 enlarged and stabilized the structure of lipid microdomains on dendrites by mechanisms that involved a redox-regulated translocation of a sphingomyelin hydrolase (neutral sphingomyelinase-2) to the plasma membrane. A concurrent pathway was activated that accelerated the forward traffic of NMDA receptors by a PKA-dependent phosphorylation of the NR1 C-terminal serine 897 (masks an ER retention signal), followed by a PKC-dependent phosphorylation of serine 896 (important for surface expression). NMDA receptors were preferentially targeted to synapses and clustered in modified membrane microdomains. In these conditions, NMDA receptors were unable to laterally disperse and did not internalize, even in response to strong agonist induction. Focal NMDA-evoked calcium bursts were enhanced by threefold in these regions. Inhibiting membrane modification or NR1 phosphorylation prevented gp120 from accelerating the surface localization of NMDA receptors. Disrupting the structure of membrane microdomains after gp120 treatments restored the ability of NMDA receptors to disperse and internalize. These findings demonstrate that gp120 contributes to synaptic dysfunction in the setting of HIV infection by interfering with NMDA receptor trafficking.  

3.1739           Analysis of RhoA and Rho GEF activity in whole cells and the cell nucleus

Guilluy, C., Dubash, A. and Garcia-mata, R. Nature Protocols, 6(12), 2050-2060 (2011)   We have recently shown that a fraction of the total cellular pool of the small GTPase RhoA resides in the nucleus, and that the nuclear guanine nucleotide exchange factor (GEF) Net1 has a role in the regulation of its activity. In this protocol, we describe a method to measure both the activities of the nuclear pools of RhoA and Rho GEFs. This process required the development of a nuclear isolation protocol that is both fast and virtually free of cytosolic and membrane contaminants, as well as a redesign of existing RhoA and Rho GEF activity assays so that they work in nuclear samples. This protocol can be also used for other Rho GTPases and Rho GEFs, which have also been found in the nucleus. Completion of the procedure, including nuclear isolation and RhoA or Rho GEF activity assay, takes 1 h 40 min. We also include details of how to perform a basic assay of whole-cell extracts.  

3.1740           A Highly Dynamic ER-Derived Phosphatidylinositol-Synthesizing Organelle Supplies Phosphoinositides to Cellular Membranes

Kim, Y.J., Guzman-Hernandez, M.L. and Balla, T. Developmental Cell, 21, 813-824 (2011)   Polyphosphoinositides are lipid signaling molecules generated from phosphatidylinositol (PtdIns) with critical roles in vesicular trafficking and signaling. It is poorly understood where PtdIns is located within cells and how it moves around between membranes. Here we identify a hitherto-unrecognized highly mobile membrane compartment as the site of PtdIns synthesis and a likely source of PtdIns of all membranes. We show that the PtdIns-synthesizing enzyme PIS associates with a rapidly moving compartment of ER origin that makes ample contacts with other membranes. In contrast, CDP-diacylglycerol synthases that provide PIS with its substrate reside in the tubular ER. Expression of a PtdInsspecific bacterial PLC generates diacylglycerol also in rapidly moving cytoplasmic objects. We propose a model in which PtdIns is synthesized in a highly mobile lipid distribution platform and is delivered to other membranes during multiple contacts by yet-to-be-defined lipid transfer mechanisms.  

3.1741           Saturated Fatty Acids Induce c-Src Clustering within Membrane Subdomains, Leading to JNK Activation

Holzer, R.G., Park, E-J., Li, N., Tran, H., Chen, M., Choi, C., Solinas, G. and Karin, M. Cell, 147(1), 173-184 (2011)   Saturated fatty acids (FA) exert adverse health effects and are more likely to cause insulin resistance and type 2 diabetes than unsaturated FA, some of which exert protective and beneficial effects. Saturated FA, but not unsaturated FA, activate Jun N-terminal kinase (JNK), which has been linked to obesity and insulin resistance in mice and humans. However, it is unknown how saturated and unsaturated FA are discriminated. We now demonstrate that saturated FA activate JNK and inhibit insulin signaling through c-Src activation. FA alter the membrane distribution of c-Src, causing it to partition into intracellular membrane subdomains, where it likely becomes activated. Conversely, unsaturated FA with known beneficial effects on glucose metabolism prevent c-Src membrane partitioning and activation, which are dependent on its myristoylation, and block JNK activation. Consumption of a diabetogenic high-fat diet causes the partitioning and activation of c-Src within detergent insoluble membrane subdomains of murine adipocytes.  

3.1742           Unique Properties of the ATP-Sensitive K+ Channel in the Mouse Ventricular Cardiac Conduction System

Bao, L., Kefaloyianni, E., Lader, J., Hong, M., Morley, G., Fishman, G.I., Sobie, E.A. and Coetzee, W.A. Cirr. Arrhythm. Electrophysiol., 4, 926-935 (2011)   Background—The specialized cardiac conduction system (CCS) expresses a unique complement of ion channels that confer a specific electrophysiological profile. ATP-sensitive potassium (K_ATP) channels in these myocytes have not been systemically investigated. Methods and Results—We recorded K_ATP channels in isolated CCS myocytes using Cntn2-EGFP reporter mice. The CCS K_ATP channels were less sensitive to inhibitory cytosolic ATP compared with ventricular channels and more strongly activated by MgADP. They also had a smaller slope conductance. The 2 types of channels had similar intraburst open and closed times, but the CCS K_ATP channel had a prolonged interburst closed time. CCS K_ATP channels were strongly activated by diazoxide and less by levcromakalim, whereas the ventricular K_ATP channel had a reverse pharmacological profile. CCS myocytes express elevated levels of Kir6.1 but reduced Kir6.2 and SUR2A mRNA compared with ventricular myocytes (SUR1 expression was negligible). SUR2B mRNA expression was higher in CCS myocytes relative to SUR2A. Canine Purkinje fibers expressed higher levels of Kir6.1 and SUR2B protein relative to the ventricle. Numeric simulation predicts a high sensitivity of the Purkinje action potential to changes in ATP:ADP ratio. Cardiac conduction time was prolonged by low-flow ischemia in isolated, perfused mouse hearts, which was prevented by glibenclamide. Conclusions—These data imply a differential electrophysiological response (and possible contribution to arrhythmias) of the ventricular CCS to K_ATP channel opening during periods of ischemia.  

3.1743           Proline-Serine-Threonine Phosphatase-Interacting Protein 2 (PSTPIP2), a Host Membrane-Deforming Protein, Is Critical for Membranous Web Formation in Hepatitis C Virus Replication

Chao, T-C., Su, W-C., Huang, J-Y., Chen, Y-C., Jeng, K-S., Wang, H-D. and Lai, M.M.C.

1. Virol., 86(3), 1739-1749 (2012)

  Hepatitis C virus (HCV) reorganizes intracellular membranes to establish sites of replication. How viral and cellular proteins target, bind, and rearrange specific membranes into the replication factory remains a mystery. We used a lentivirus-based RNA interference (RNAi) screening approach to identify the potential cellular factors that are involved in HCV replication. A protein with membrane-deforming activity, proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2), was identified as a potential factor. Knockdown of PSTPIP2 in HCV subgenomic replicon-harboring and HCV-infected cells was associated with the reduction of HCV protein and RNA expression. PSTPIP2 was localized predominantly in detergent-resistant membranes (DRMs), which contain the RNA replication complex. PSTPIP2 knockdown caused a significant reduction of the formation of HCV- and NS4B-induced membranous webs. A PSTPIP2 mutant defective in inducing membrane curvature failed to support HCV replication, confirming that the membrane-deforming ability of PSTPIP2 is essential for HCV replication. Taking these results together, we suggest that PSTPIP2 facilitates membrane alterations and is a key player in the formation of the membranous web, which is the site of the HCV replication complex.  

3.1744           Cholesterol dependence of Newcastle Disease Virus entry

Martin, J.J., Holguera, J., Sanchez-Felipe, L., Villar, E. and Munoz-Barroso, I. Biochem. Biophys. Acta, 1818, 753-761(2012)   Lipid rafts are membrane microdomains enriched in cholesterol, sphingolipids, and glycolipids that have been implicated in many biological processes. Since cholesterol is known to play a key role in the entry of some other viruses, we investigated the role of cholesterol and lipid rafts in the host cell plasma membrane in Newcastle Disease Virus (NDV) entry. We used methyl-β-cyclodextrin (MβCD) to deplete cellular cholesterol and disrupt lipid rafts. Our results show that the removal of cellular cholesterol partially reduces viral binding, fusion and infectivity. MβCD had no effect on the expression of sialic acid containing molecule expression, the NDV receptors in the target cell. All the above-described effects were reversed by restoring cholesterol levels in the target cell membrane. The HN viral attachment protein partially localized to detergent-resistant membrane microdomains (DRMs) at 4 °C and then shifted to detergent-soluble fractions at 37 °C. These results indicate that cellular cholesterol may be required for optimal cell entry in NDV infection cycle.  

3.1745           S-allyl cysteine in combination with clotrimazole downregulates Fas induced apoptotic events in erythrocytes of mice exposed to lead

Mandal, S., Mukherjee, S., Chowdhury, K.D., Sarkar, A., Basu, K., Paul, S., Karmakar, D., Chatterjee, M., Biswas, T., Sadhukhan, G.C. and Sen, G. Biochim. Biophys. Acta, 1820, 9-23 (2012)   Background Chronic lead (Pb^2 +) exposure leads to the reduced lifespan of erythrocytes. Oxidative stress and K⁺ loss accelerate Fas translocation into lipid raft microdomains inducing Fas mediated death signaling in these erythrocytes. Pathophysiological-based therapeutic strategies to combat against erythrocyte death were evaluated using garlic-derived organosulfur compounds like diallyl disulfide (DADS), S allyl cysteine (SAC) and imidazole based Gardos channel inhibitor clotrimazole (CLT). Methods Morphological alterations in erythrocytes were evaluated using scanning electron microscopy. Events associated with erythrocyte death were evaluated using radio labeled probes, flow cytometry and activity gel assay. Mass spectrometry was used for detection of GSH–4-hydroxy-trans-2-nonenal (HNE) adducts. Fas redistribution into the lipid rafts was studied using immunoblotting technique and confocal microscopy. Results Combination of SAC and CLT was better than DADS and CLT combination and monotherapy with these agents in prolonging the survival of erythrocytes during chronic Pb^2 + exposure. Combination therapy with SAC and CLT prevented redistribution of Fas into the lipid rafts of the plasma membrane and downregulated Fas-dependent death events in erythrocytes of mice exposed to Pb^2 +. Conclusion and general significance Ceramide generation was a critical component of Fas receptor-induced apoptosis, since inhibition of acid sphingomyelinase (aSMase) interfered with Fas-induced apoptosis during Pb^2 + exposure. Combination therapy with SAC and CLT downregulated apoptotic events in erythrocytes by antagonizing oxidative stress and Gardos channel that led to suppression of ceramide-initiated Fas aggregation in lipid rafts. Hence, combination therapy with SAC and CLT may be a potential therapeutic option for enhancing the lifespan of erythrocytes during Pb^2 + toxicity.    

3.1746           Cell surface ceramide controls translocation of transferrin receptor to clathrin-coated pits

Shakor, A.B.A., Atia, M.M., Kwiatkowska, K. and Sobota, A. Cellular Signalling, 24, 677-684 (2012)   Transferrin receptor mediates internalization of transferrin with bound ferric ions through the clathrin-dependent pathway. We found that binding of transferrin to the receptor induced rapid generation of cell surface ceramide which correlated with activation of acid, but not neutral, sphingomyelinase. At the onset of transferrin internalization both ceramide level and acid sphingomyelinase activity returned to their basic levels. Down-regulation of acid sphingomyelinase in cells with imipramine or silencing of the enzyme expression with siRNA stimulated transferrin internalization and inhibited its recycling. In these conditions colocalization of transferrin with clathrin was markedly reduced. Simultaneously, K⁺ depletion of cells which interfered with the assembly of clathrin-coated pits inhibited the uptake of transferrin much less efficiently than it did in control conditions. The down-regulation of acid sphingomyelinase activity led to the translocation of transferrin receptor to the raft fraction of the plasma membrane upon transferrin binding. The data suggest that lack of cell surface ceramide, generated in physiological conditions by acid sphingomyelinase during transferrin binding, enables internalization of transferrin/transferrin receptor complex by clathrin-independent pathway.  

3.1747           Proteostasis of tau. Tau overexpression results in its secretion via membrane vesicles

Simon, D., Garcia-garcia, E., Royo, F., Falcon-Perez, J.M. and Avila, J. FEBS Lett., 586, 47-54 (2012)   Increasing amounts of tau protein were expressed in non-neuronal cells. When intracellular amounts reached a threshold level, tau protein was released to the extracellular culture medium in association with membrane vesicles. Hence, we propose that tau might be secreted through membrane vesicles as a cellular mechanism to eliminate the excess of tau protein, thereby avoiding its toxicity.  

3.1748           Perinuclear Localization of Internalized Outer Membrane Vesicles Carrying Active Cytolethal Distending Toxin from Aggregatibacter actinomycetemcomitans

Rompikuntal, P.K., Thay, B., Khan, M.K., alanko, J., Penttinen, A-M., Asikainen, S.., Wai, S.N. and Oscarsson, J. Infect. Immun., 80(1), 31-42 (2012)   Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similarly to several other Gram-negative species, this organism produces and excretes a cytolethal distending toxin (CDT), a genotoxin associated with cell distention, G₂ cell cycle arrest, and/or apoptosis in many mammalian cell types. In this study, we have identified A. actinomycetemcomitans outer membrane vesicles (OMVs) as a vehicle for simultaneous delivery of multiple proteins, including CDT, into human cells. The OMV proteins were internalized in both HeLa cells and human gingival fibroblasts (HGF) via a mechanism of OMV fusion with lipid rafts in the plasma membrane. The active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. In accordance with a tight association of CdtB with OMVs, vesicles isolated from A. actinomycetemcomitans strain D7SS (serotype a), in contrast to OMVs from a D7SS cdtABC mutant, induced a cytolethal distending effect on HeLa and HGF cells, indicating that OMV-associated CDT was biologically active. Association of CDT with OMVs was also observed in A. actinomycetemcomitans isolates belonging to serotypes b and c, indicating that OMV-mediated release of CDT may be conserved in A. actinomycetemcomitans. Although the role of A. actinomycetemcomitans OMVs in periodontal disease has not yet been elucidated, our present data suggest that OMVs could deliver biologically active CDT and additional virulence factors into susceptible cells of the periodontium.  

3.1749           The Enolase of Borrelia burgdorferi Is a Plasminogen Receptor Released in Outer Membrane Vesicles

Toledo, A., Coleman, J.L., Kuhlow, C.J., Crowley, J.T. and Benach, J.L. Infect Immun., 80(1), 359-368 (2012)   The agent of Lyme disease, Borrelia burgdorferi, has a number of outer membrane proteins that are differentially regulated during its life cycle. In addition to their physiological functions in the organism, these proteins also likely serve different functions in invasiveness and immune evasion. In borreliae, as well as in other bacteria, a number of membrane proteins have been implicated in binding plasminogen. The activation and transformation of plasminogen into its proteolytically active form, plasmin, enhances the ability of the bacteria to disseminate in the host. Outer membrane vesicles of B. burgdorferi contain enolase, a glycolytic-cycle enzyme that catalyzes 2-phosphoglycerate to form phosphoenolpyruvate, which is also a known plasminogen receptor in Gram-positive bacteria. The enolase was cloned, expressed, purified, and used to generate rabbit antienolase serum. The enolase binds plasminogen in a lysine-dependent manner but not through ionic interactions. Although it is present in the outer membrane, microscopy and proteinase K treatment showed that enolase does not appear to be exposed on the surface. However, enolase in the outer membrane vesicles is accessible to proteolytic degradation by proteinase K. Samples from experimentally and tick-infected mice and rabbits as well as from Lyme disease patients exhibit recognition of enolase in serologic assays. Thus, this immunogenic plasminogen receptor released in outer membrane vesicles could be responsible for external proteolysis in the pericellular environment and have roles in nutrition and in enhancing dissemination.    

3.1750           Chromogranins A and B are key proteins in amine accumulation, but the catecholamine secretory pathway is conserved without them

Diaz-Vera, J., Camacho, M., Machado, J.D., Dominguez, N., Montesinos, M.S., Hernandez-Fernaud, J.R., Lujan, R. and Borges, R. FASEB J., 26(1), 430-438 (2012)   Chromogranins are the main soluble proteins in the large dense core secretory vesicles (LDCVs) found in aminergic neurons and chromaffin cells. We recently demonstrated that chromogranins A and B each regulate the concentration of adrenaline in chromaffin granules and its exocytosis. Here we have further studied the role played by these proteins by generating mice lacking both chromogranins. Surprisingly, these animals are both viable and fertile. Although chromogranins are thought to be essential for their biogenesis, LDCVs were evident in these mice. These vesicles do have a somewhat atypical appearance and larger size. Despite their increased size, single-cell amperometry recordings from chromaffin cells showed that the amine content in these vesicles is reduced by half. These data demonstrate that although chromogranins regulate the amine concentration in LDCVs, they are not completely essential, and other proteins unrelated to neurosecretion, such as fibrinogen, might compensate for their loss to ensure that vesicles are generated and the secretory pathway conserved.—Díaz-Vera, J., Camacho, M., Machado, J. D., Domínguez, N., Montesinos, M. S., Hernández-Fernaud, J. R., Luján, R., Borges, R. Chromogranins A and B are key proteins in amine accumulation, but the catecholamine secretory pathway is conserved without them.  

3.1751           Bromovirus RNA Replication Compartment Formation Requires Concerted Action of 1a's Self-Interacting RNA Capping and Helicase Domains

Diaz, A., Gallei, A. and Ahlquist, P.

1. Virol., 86(2), 821-834 (2012)

  All positive-strand RNA viruses replicate their genomes in association with rearranged intracellular membranes such as single- or double-membrane vesicles. Brome mosaic virus (BMV) RNA synthesis occurs in vesicular endoplasmic reticulum (ER) membrane invaginations, each induced by many copies of viral replication protein 1a, which has N-terminal RNA capping and C-terminal helicase domains. Although the capping domain is responsible for 1a membrane association and ER targeting, neither this domain nor the helicase domain was sufficient to induce replication vesicle formation. Moreover, despite their potential for mutual interaction, the capping and helicase domains showed no complementation when coexpressed in trans. Cross-linking showed that the capping and helicase domains each form trimers and larger multimers in vivo, and the capping domain formed extended, stacked, hexagonal lattices in vivo. Furthermore, coexpressing the capping domain blocked the ability of full-length 1a to form replication vesicles and replicate RNA and recruited full-length 1a into mixed hexagonal lattices with the capping domain. Thus, BMV replication vesicle formation and RNA replication depend on the direct linkage and concerted action of 1a's self-interacting capping and helicase domains. In particular, the capping domain's strong dominant-negative effects showed that the ability of full-length 1a to form replication vesicles was highly sensitive to disruption by non-productively titrating lattice-forming self-interactions of the capping domain. These and other findings shed light on the roles and interactions of 1a domains in replication compartment formation and support prior results suggesting that 1a induces replication vesicles by forming a capsid-like interior shell.  

3.1752           Streptococcus suis Capsular Polysaccharide Inhibits Phagocytosis through Destabilization of Lipid Microdomains and Prevents Lactosylceramide-Dependent Recognition

Houde, M., Gottschalk, M., Gagnon, F., Van Calsteren, M-R. and Segura, M. Infect. Immun.,80(2), 506-517 (2012)   Streptococcus suis type 2 is a major swine pathogen and a zoonotic agent, causing meningitis in both swine and humans. S. suis infects the host through the respiratory route, reaches the bloodstream, and persists until breaching into the central nervous system. The capsular polysaccharide (CPS) of S. suis type 2 is considered a key virulence factor of the bacteria. Though CPS allows S. suis to adhere to the membrane of cells of the immune system, it provides protection against phagocytosis. In fact, nonencapsulated mutants are easily internalized and killed by macrophages and dendritic cells. The objective of this work was to study the molecular mechanisms by which the CPS of S. suis prevents phagocytosis. By using latex beads covalently linked with purified CPS, it was shown that CPS itself was sufficient to inhibit entry of both latex beads and bystander fluorescent beads into macrophages. Upon contact with macrophages, encapsulated S. suis was shown to destabilize lipid microdomains at the cell surface, to block nitric oxide (NO) production during infection, and to prevent lactosylceramide accumulation at the phagocytic cup during infection. In contrast, the nonencapsulated mutant was easily internalized via lipid rafts, in a filipin-sensitive manner, leading to lactosylceramide recruitment and strong NO production. This is the first report to identify a role for CPS in lipid microdomain stability and to recognize an interaction between S. suis and lactosylceramide in phagocytes.  

3.1753           HIV-1 Vpu's lipid raft association is dispensable for counteraction of the particle release restriction imposed by CD317/Tetherin

Joëlle, V.F., Tibroni, N., Keppler, O.T. and Fackler, O.T. Virology, 424, 33-44 (2012)   HIV-1 Vpu antagonizes the block to particle release mediated by CD317 (BST-2/HM1.24/Tetherin) via incompletely understood mechanisms. Vpu and CD317 partially reside in cholesterol-rich lipid rafts where HIV-1 budding preferentially occurs. Here we find that lipid raft association of ectopically expressed or endogenous CD317 was unaltered upon co-expression with Vpu or following HIV-1 infection. Similarly, Vpu's lipid raft association remained unchanged upon expression of CD317. We identify amino acids V25 and Y29 of Vpu as crucial for microdomain partitioning and single substitution of these amino acids resulted in Vpu variants with markedly reduced or undetectable lipid raft association. These mutations did not affect Vpu's subcellular distribution and binding capacity to CD317, nor its ability to downmodulate cell surface CD317 and promote HIV-1 release from CD317-positive cells. We conclude that (i) lipid raft incorporation is dispensable for Vpu-mediated CD317 antagonism and (ii) Vpu does not antagonize CD317 by extraction from lipid rafts.  

3.1754           Immune cells and hepatocytes express glycosylphosphatidylinositol-anchored ceruloplasmin at their cell surface

Marques, L., Auriac, A., Willemetz, A., Banha, J., Silva, B., Canonne-Hergaux, F. and Costa, L. Blood Cells, Molecules, and Diseases, 48, 110-120 (2012)   Background Ceruloplasmin is a positive acute-phase protein with both anti- and pro-oxidant activities, thus having still unclear physiological functions in inflammatory processes. Importantly, ceruloplasmin has been implicated in iron metabolism due to its ferroxidase activity, assisting ferroportin on cellular iron efflux. Ceruloplasmin can be expressed as a secreted or as a membrane glycosylphosphatidylinositol-anchored protein (GPI-ceruloplasmin), this latter one being reported as expressed mostly in the brain. Design and methods We studied the expression of both ceruloplasmin isoforms in human peripheral blood lymphocytes, monocytes, mouse macrophages and human hepatocarcinoma cell line HepG2, using immunofluorescence and immunoblotting techniques. Co-localization of ceruloplasmin and ferroportin was also investigated by immunofluorescence in mouse macrophages. Results Ceruloplasmin was detected by immunoblotting and immunofluorescence in membrane and cytosol of all cell types. The cell surface ceruloplasmin was identified as the GPI-isoform and localized in lipid rafts from monocytes, macrophages and HepG2 cells. In macrophages, increased expression levels and co-localization of ferroportin and GPI-ceruloplasmin in cell surface lipid rafts were observed after iron treatment. Such iron upregulation of ceruloplasmin was not observed in HepG2. Conclusions Our results revealed an unexpected ubiquitous expression of the GPI-ceruloplasmin isoform in immune and hepatic cells. Different patterns of regulation of ceruloplasmin in these cells may reflect distinct physiologic functions of this oxidase. In macrophages, GPI-ceruloplasmin and ferroportin likely interact in lipid rafts to export iron from cells. Precise knowledge about ceruloplasmin isoforms expression and function in various cell types will help to clarify the role of ceruloplasmin in many diseases related to iron metabolism, inflammation and oxidative biology.  

3.1755           Baculovirus GP64-Mediated Entry into Mammalian Cells

Kataoka, C., Kaname, Y., Taguwa, S., Abe, T., Fukuhara, T., Tani, H., Moriishi, K and Matsuura, Y.

1. Virol., 86(5), 2610-2620 (2012)

  The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.  

3.1756           Role of protein kinase C in phospholemman mediated regulation of α2β1 isozyme of Na+/K+-ATPase in caveolae of pulmonary artery smooth muscle cells

Dey, K., Roy, S., Ghosh, B. and Chakraborti, S. Biochemie, 94, 991-1000 (2012)   We have recently reported that α₂β₁ and α₁β₁ isozymes of Na⁺/K⁺-ATPase (NKA) are localized in the caveolae whereas only the α₁β₁ isozyme of NKA is localized in the non-caveolae fraction of pulmonary artery smooth muscle cell membrane. It is well known that different isoforms of NKA are regulated differentially by PKA and PKC, but the mechanism is not known in the caveolae of pulmonary artery smooth muscle cells. Herein, we examined whether this regulation occurs through phospholemman (PLM) in the caveolae. Our results suggest that PKC mediated phosphorylation of PLM occurs only when it is associated with the α₂ isoform of NKA, whereas phosphorylation of PLM by PKA occurs when it is associated with the α₁ isoform of NKA. To investigate the mechanism of regulation of α₂ isoform of NKA by PKC-mediated phosphorylation of PLM, we have purified PLM from the caveolae and reconstituted into the liposomes. Our result revealed that (i) in the reconstituted liposomes phosphorylated PLM (PKC mediated) stimulate NKA activity, which appears to be due to an increase in the turnover number of the enzyme; (ii) phosphorylated PLM did not change the affinity of the pump for Na⁺; and (iii) even after phosphorylation by PKC, PLM still remains associated with the α₂ isoform of NKA.  

3.1757           Palmitoylated TMX and calnexin target to the mitochondria-associated membrane

Lynes, E.M., Bui, M., Yap, M.C., Benson, M.D., Schneider, B., Ellgaard, L., Berthiaume, L.C. and Simmen, T. EMBO J., 31(2), 457-470 (2012)   The mitochondria-associated membrane (MAM) is a domain of the endoplasmic reticulum (ER) that mediates the exchange of ions, lipids and metabolites between the ER and mitochondria. ER chaperones and oxidoreductases are critical components of the MAM. However, the localization motifs and mechanisms for most MAM proteins have remained elusive. Using two highly related ER oxidoreductases as a model system, we now show that palmitoylation enriches ER-localized proteins on the MAM. We demonstrate that palmitoylation of cysteine residue(s) adjacent to the membrane-spanning domain promotes MAM enrichment of the transmembrane thioredoxin family protein TMX. In addition to TMX, our results also show that calnexin shuttles between the rough ER and the MAM depending on its palmitoylation status. Mutation of the TMX and calnexin palmitoylation sites and chemical interference with palmitoylation disrupt their MAM enrichment. Since ER-localized heme oxygenase-1, but not cytosolic GRP75 require palmitoylation to reside on the MAM, our findings identify palmitoylation as key for MAM enrichment of ER membrane proteins.  

3.1758           Assurance of mitochondrial integrity and mammalian longevity by the p62–Keap1–Nrf2–Nqo1 cascade

Kwon, J., Han, E., Bui, C-B., Shin, W., Lee, J., Lee, S., Choi, Y-B., Lee, A-H., Lee, K-H., Park, C., Obin, M.S., Park, S.K., Seo, Y.J., Taeg, G., Lee, H-W. and Shin, J. EMBO Reports, 13(2), 150-156 (2012)   Sqstm1/p62 functions in the non-canonical activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, its physiological relevance is not certain. Here, we show that p62 ^−/− mice exhibited an accelerated presentation of ageing phenotypes, and tissues from these mice created a pro-oxidative environment owing to compromised mitochondrial electron transport. Accordingly, mitochondrial function rapidly declined with age in p62 ^−/− mice. In addition, p62 enhanced basal Nrf2 activity, conferring a higher steady-state expression of NAD(P)H dehydrogenase, quinone 1 (Nqo1) to maintain mitochondrial membrane potential and, thereby, restrict excess oxidant generation. Together, the p62–Nrf2–Nqo1 cascade functions to assure mammalian longevity by stabilizing mitochondrial integrity.  

3.1759           A conserved membrane-binding domain targets proteins to organelle contact sites

Toulmay, A. and Printz, W.A.

1. Cell Sci., 125(1), 49-58 (2012)

  Membrane contact sites (MCSs), where the membranes of two organelles are closely apposed, are regions where small molecules such as lipids or calcium are exchanged between organelles. We have identified a conserved membrane-binding domain found exclusively in proteins at MCSs in Saccharomyces cerevisiae. The synaptotagmin-like-mitochondrial-lipid binding protein (SMP) domain is conserved across species. We show that all seven proteins that contain this domain in yeast localize to one of three MCSs. Human proteins with SMP domains also localize to MCSs when expressed in yeast. The SMP domain binds membranes and is necessary for protein targeting to MCSs. Proteins containing this domain could be involved in lipid metabolism. This is the first protein domain found exclusively in proteins at MCSs.  

3.1760           Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane

Gerl, M.J., Sampaio, J.L., Urban, S., Kalvodova, L., Verbavatz, J-M., Binnington, B., Lindemann, D., Lingwood, C.A., Shevchenko, A., Schroder, C. and Simons, K.

1. Cell Biol., 196(2), 213-221 (2012)

  The influenza virus (IFV) acquires its envelope by budding from host cell plasma membranes. Using quantitative shotgun mass spectrometry, we determined the lipidomes of the host Madin–Darby canine kidney cell, its apical membrane, and the IFV budding from it. We found the apical membrane to be enriched in sphingolipids (SPs) and cholesterol, whereas glycerophospholipids were reduced, and storage lipids were depleted compared with the whole-cell membranes. The virus membrane exhibited a further enrichment of SPs and cholesterol compared with the donor membrane at the expense of phosphatidylcholines. Our data are consistent with and extend existing models of membrane raft-based biogenesis of the apical membrane and IFV envelope.  

3.1761           Building Excitable Membranes: Lipid Rafts and Multiple Controls on Trafficking of Electrogenic Molecules

Pristera, A. and Okuse, K. The Neuroscientist, 18(1), 70-81 (2012)   Multiple plasma membrane proteins such as ion transporters and ion channels are involved in electrogenesis by setting resting membrane potentials and triggering/propagating action potentials. Recent findings strongly suggest that some of these membrane proteins are selectively transported into membrane microdomains termed lipid rafts. There appear to be multiple mechanisms for the specific protein translocation to lipid rafts, and many of these proteins exhibit distinct properties when inserted into the raft microdomains. Here the authors review the plasma membrane ion channels specifically localized at membrane lipid rafts in neurons. The mechanisms to selectively translocate these molecules to the lipid rafts and the consequences of the trafficking are also discussed.    

3.1762           Tau deficiency induces parkinsonism with dementia by impairing APP-mediated iron export

Lee, P. et al Nature Med., 18(2), 291-296 (2012)   The microtubule-associated protein tau has risk alleles for both Alzheimer's disease and Parkinson's disease and mutations that cause brain degenerative diseases termed tauopathies^{1, 2, 3, 4}. Aggregated tau forms neurofibrillary tangles in these pathologies^{3, 5}, but little is certain about the function of tau or its mode of involvement in pathogenesis. Neuronal iron accumulation has been observed pathologically in the cortex in Alzheimer's disease^{6, 7}, the substantia nigra (SN) in Parkinson's disease^{8, 9, 10, 11} and various brain regions in the tauopathies^{11, 12}. Here we report that tau-knockout mice develop age-dependent brain atrophy, iron accumulation and SN neuronal loss, with concomitant cognitive deficits and parkinsonism. These changes are prevented by oral treatment with a moderate iron chelator, clioquinol. Amyloid precursor protein (APP) ferroxidase activity couples with surface ferroportin to export iron, but its activity is inhibited in Alzheimer's disease, thereby causing neuronal iron accumulation⁷. In primary neuronal culture, we found loss of tau also causes iron retention, by decreasing surface trafficking of APP. Soluble tau levels fall in affected brain regions in Alzheimer's disease and tauopathies^{13, 14, 15}, and we found a similar decrease of soluble tau in the SN in both Parkinson's disease and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. These data suggest that the loss of soluble tau could contribute to toxic neuronal iron accumulation in Alzheimer's disease, Parkinson's disease and tauopathies, and that it can be rescued pharmacologically.  

3.1763           Uncoupling the roles of synaptotagmin I during endo- and exocytosis of synaptic vesicles

Yao, J., Kwon, S.E., Gaffaney, J.D., Dunning, F.M. and Chapman, E.R. Nature Neurosci., 15(2), 243-250 (2012)   Synaptotagmin I (syt1) is required for normal rates of synaptic vesicle endo- and exocytosis. However, whether the kinetic defects observed during endocytosis in Syt1 knockout neurons are secondary to defective exocytosis or whether syt1 directly regulates the rate of vesicle retrieval remains unknown. To address this question, we sought to dissociate these two activities. We uncoupled the function of syt1 in exo- and endocytosis in mouse neurons either by re-targeting the protein or via mutagenesis of its tandem C2 domains. The effect of these manipulations on exo- and endocytosis were analyzed using electrophysiology, in conjunction with optical imaging of the vesicle cycle. Our results indicate that syt1 is directly involved in endocytosis. Notably, either of the C2 domains of syt1, C2A or C2B, was able to function as a Ca²⁺ sensor for endocytosis. Thus, syt1 functions as a dual Ca²⁺ sensor for both endo- and exocytosis, potentially coupling these two components of the vesicle cycle.  

3.1764           Cullin-3 regulates late endosome maturation

Huotari, J., Meyer-Schaller, N., Hubner, M., Stauffer, S., Katheder, N., Horvath, P., Mancini, r., Heleniius, A. and Peter, M. PNAS, 19(3), 823-828 (2012)   Cullin-3 (Cul3) functions as a scaffolding protein in the Bric-a-brac, Tramtrack, Broad-complex (BTB)–Cul3–Rbx1 ubiquitin E3 ligase complex. Here, we report a previously undescribed role for Cul3 complexes in late endosome (LE) maturation. RNAi-mediated depletion of Cul3 results in a trafficking defect of two cargoes of the endolysosomal pathway, influenza A virus (IAV) and epidermal growth factor receptor (EGFR). IAV is able to reach an acidic endosomal compartment, coinciding with LE/lysosome (LY) markers. However, it remains trapped or the capsid is unable to uncoat after penetration into the cytosol. Similarly, activation and subsequent ubiquitination of EGFR appear normal, whereas downstream EGFR degradation is delayed and its ligand EGF accumulates in LE/LYs. Indeed, Cul3-depleted cells display severe morphological defects in LEs that could account for these trafficking defects; they accumulate acidic LE/LYs, and some cells become highly vacuolated, with enlarged Rab7-positive endosomes. Together, these results suggest a crucial role of Cul3 in regulating late steps in the endolysosomal trafficking pathway.      

3.1765           Tempol modulates changes in xenobiotic permeability and occludin oligomeric assemblies at the blood-brain barrier during inflammatory pain

Lockhead, J.J., McCaffrey, G., Sanchez-Covarrubias, L., Finch, J.D., DeMarco, K.M., Quigley, C.E., Davis, T.P. and Ronaldson, P.T. Am. J. Physiol. Heart Circ. Physiol., 302(3), H582-H593 (2012)   Our laboratory has shown that λ-carrageenan-induced peripheral inflammatory pain (CIP) can alter tight junction (TJ) protein expression and/or assembly leading to changes in blood-brain barrier xenobiotic permeability. However, the role of reactive oxygen species (ROS) and subsequent oxidative stress during CIP is unknown. ROS (i.e., superoxide) are known to cause cellular damage in response to pain/inflammation. Therefore, we examined oxidative stress-associated effects at the blood-brain barrier (BBB) in CIP rats. During CIP, increased staining of nitrosylated proteins was detected in hind paw tissue and enhanced presence of protein adducts containing 3-nitrotyrosine occurred at two molecular weights (i.e., 85 and 44 kDa) in brain microvessels. Tempol, a pharmacological ROS scavenger, attenuated formation of 3-nitrotyrosine-containing proteins in both the hind paw and in brain microvessels when administered 10 min before footpad injection of λ-carrageenan. Similarly, CIP increased 4-hydroxynoneal staining in brain microvessels and this effect was reduced by tempol. Brain permeability to [¹⁴C]sucrose and [³H]codeine was increased, and oligomeric assemblies of occludin, a critical TJ protein, were altered after 3 h CIP. Tempol attenuated both [¹⁴C]sucrose and [³H]codeine brain uptake as well as protected occludin oligomers from disruption in CIP animals, suggesting that ROS production/oxidative stress is involved in modulating BBB functional integrity during pain/inflammation. Interestingly, tempol administration reduced codeine analgesia in CIP animals, indicating that oxidative stress during pain/inflammation may affect opioid delivery to the brain and subsequent efficacy. Taken together, our data show for the first time that ROS pharmacological scavenging is a viable approach for maintaining BBB integrity and controlling central nervous system drug delivery during acute inflammatory pain.  

3.1766           Proteomic Analysis of Microvesicles Derived from Human Mesenchymal Stem Cells

Kim, H-S., Choi, D-Y., Yun, S.J., Choi, S-M., kang, J.W., Jung, J.W., Hwang, D., Kim, K.P. and Kim, D-W.,

1. Proteome Res., 11(2), 839-849 (2012)

  Mesenchymal stem cells (MSCs) have emerged as a promising means for treating degenerative or incurable diseases. Recent studies have shown that microvesicles (MVs) from MSCs (MSC-MVs) contribute to recovery of damaged tissues in animal disease models. Here, we profiled the MSC-MV proteome to investigate their therapeutic effects. LC–MS/MS analysis of MSC-MVs identified 730 MV proteins. The MSC-MV proteome included five positive and two variable known markers of MSCs, but no negative marker, as well as 43 surface receptors and signaling molecules controlling self-renewal and differentiation of MSCs. Functional enrichment analysis showed that cellular processes represented by the MSC-MV proteins include cell proliferation, adhesion, migration, and morphogenesis. Integration of MSC’s self-renewal and differentiation-related genes and the proteome of MSC-conditioned media (MSC-CM) with the MSC-MV proteome revealed potential MV protein candidates that can be associated with the therapeutic effects of MSC-MVs: (1) surface receptors (PDGFRB, EGFR, and PLAUR); (2) signaling molecules (RRAS/NRAS, MAPK1, GNA13/GNG12, CDC42, and VAV2); (3) cell adhesion (FN1, EZR, IQGAP1, CD47, integrins, and LGALS1/LGALS3); and (4) MSC-associated antigens (CD9, CD63, CD81, CD109, CD151, CD248, and CD276). Therefore, the MSC-MV proteome provides a comprehensive basis for understanding the potential of MSC-MVs to affect tissue repair and regeneration.  

3.1767           Angiotensin II Induces Epithelial-to-Mesenchymal Transition in Renal Epithelial Cells through Reactive Oxygen Species/Src/Caveolin-Mediated Activation of an Epidermal Growth Factor Receptor–Extracellular Signal-Regulated Kinase Signaling Pathway

Chen, J., Chen, J-K. and Harris, R.C. Mol. Cell. Biol., 32(5), 981-991 (2012)   Chronic activation of the renin-angiotensin system plays a deleterious role in progressive kidney damage, and the renal proximal tubule is known to play an important role in tubulointerstitial fibrosis; however, the underlying molecular mechanism is unclear. Here we report that in the proximal tubule-like LLCPKcl4 cells expressing angiotensin II (Ang II) type 1 receptor, Ang II induced changes in cell morphology and expression of epithelial-to-mesenchymal transition (EMT) markers, which were inhibited by the miotogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK)-activating kinase (MEK) inhibitor PD98059 or the Src kinase inhibitor PP2. Ang II-stimulated phosphorylation of caveolin-1 (Cav) at Y14 and epidermal growth factor receptor (EGFR) at Y845 and induced association of these phosphoproteins in caveolin-enriched lipid rafts, thereby leading to prolonged EGFR-ERK signaling that was inhibited by Nox4 small interfering RNA (siRNA) and Src siRNA. Two different antioxidants not only inhibited phosphorylation of Src at Y416 but also blocked the EGFR-ERK signaling. Moreover, erlotinib (the EGFR tyrosine kinase inhibitor), EGFR siRNA, and Cav siRNA all inhibited both prolonged EGFR-ERK signaling and phenotypic changes induced by Ang II. Thus, this report provides the first evidence that reactive oxygen species (ROS)/Src-dependent activation of persistent Cav-EGFR-ERK signaling mediates renal tubular cell dedifferentiation and identifies a novel molecular mechanism that may be involved in progressive renal injury caused by chronic exposure to Ang II.  

3.1768           Measuring and evaluating the role of ATP-sensitive K+ channels in cardiac muscle

Kefaloyianni, E., Bao, L., Rindler, M.J., Hong, M., Patel, T., Taskin, E. and Coetzee, W.A.

1. Mol. Cell. Cardiol., 52, 596-607 (2012)

  Since ion channels move electrical charge during their activity, they have traditionally been studied using electrophysiological approaches. This was sometimes combined with mathematical models, for example with the description of the ionic mechanisms underlying the initiation and propagation of action potentials in the squid giant axon by Hodgkin and Huxley. The methods for studying ion channels also have strong roots in protein chemistry (limited proteolysis, the use of antibodies, etc.). The advent of the molecular cloning and the identification of genes coding for specific ion channel subunits in the late 1980s introduced a multitude of new techniques with which to study ion channels and the field has been rapidly expanding ever since (e.g. antibody development against specific peptide sequences, mutagenesis, the use of gene targeting in animal models, determination of their protein structures) and new methods are still in development. This review focuses on techniques commonly employed to examine ion channel function in an electrophysiological laboratory. The focus is on the K_ATP channel, but many of the techniques described are also used to study other ion channels.  

3.1769           Endoplasmic Reticulum Stress Is Important for the Manifestations of α-Synucleinopathy In Vivo

Colla, E., Coune, P., Liu, Y., Pletnikova, O., Troncoso, J.C., Iwatsubo, T., Schneider, B.L. and Lee, M.K.

1. Neurosci., 32(10), 3306-3320 (2012)

  Accumulation of misfolded α-synuclein (αS) is mechanistically linked to neurodegeneration in Parkinson's disease (PD) and other α-synucleinopathies. However, how αS causes neurodegeneration is unresolved. Because cellular accumulation of misfolded proteins can lead to endoplasmic reticulum stress/unfolded protein response (ERS/UPR), chronic ERS could contribute to neurodegeneration in α-synucleinopathy. Using the A53T mutant human αS transgenic (A53TαS Tg) mouse model of α-synucleinopathy, we show that disease onset in the αS Tg model is coincident with induction of ER chaperones in neurons exhibiting αS pathology. However, the neuronal ER chaperone induction was not accompanied by the activation of phospho-eIF2α, indicating that α-synucleinopathy is associated with abnormal UPR that could promote cell death. Induction of ERS/UPR was associated with increased levels of ER/microsomal (ER/M) associated αS monomers and aggregates. Significantly, human PD cases also exhibit higher relative levels of ER/M αS than the control cases. Moreover, αS interacts with ER chaperones and overexpression of αS sensitizes neuronal cells to ERS-induced toxicity, suggesting that αS may have direct impact on ER function. This view is supported by the presence of ERS-activated caspase-12 and the accumulation of ER-associated polyubiquitin. More important, treatment with Salubrinal, an anti-ERS compound, significantly attenuates disease manifestations in both the A53TαS Tg mouse model and the adeno-associated virus-transduced rat model of A53TαS-dependent dopaminergic neurodegeneration. Our data indicate that the accumulation αS within ER leads to chronic ER stress conditions that contribute to neurodegeneration in α-synucleinopathies. Attenuating chronic ERS could be an effective therapy for PD and other α-synucleinopathies.  

3.1770           FBL2 Regulates Amyloid Precursor Protein (APP) Metabolism by Promoting Ubiquitination-Dependent APP Degradation and Inhibition of APP Endocytosis

Watanabe, T., Hikichi, Y., Willuweit, A., Shintani, Y. and Horiguchi, T.

1. Neurosci., 32(10), 3352-3365 (2012)

  The ubiquitin–proteasome pathway is a major protein degradation pathway whose dysfunction is now widely accepted as a cause of neurodegenerative diseases, including Alzheimer's disease. Here we demonstrate that the F-box and leucine rich repeat protein2 (FBL2), a component of the E3 ubiquitin ligase complex, regulates amyloid precursor protein (APP) metabolism through APP ubiquitination. FBL2 overexpression decreased the amount of secreted amyloid β (Aβ) peptides and sAPPβ, whereas FBL2 mRNA knockdown by siRNA increased these levels. FBL2 overexpression also decreased the amount of intracellular Aβ in Neuro2a cells stably expressing APP with Swedish mutation. FBL2 bound with APP specifically at its C-terminal fragment (CTF), which promoted APP/CTF ubiquitination. FBL2 overexpression also accelerated APP proteasome-dependent degradation and decreased APP protein localization in lipid rafts by inhibiting endocytosis. These effects were not observed in an F-box-deleted FBL2 mutant that does not participate in the E3 ubiquitin ligase complex. Furthermore, a reduced insoluble Aβ and Aβ plaque burden was observed in the hippocampus of 7-month-old FBL2 transgenic mice crossed with double-transgenic mice harboring APPswe and PS1_M146V transgenes. These findings indicate that FBL2 is a novel and dual regulator of APP metabolism through FBL2-dependent ubiquitination of APP.  

3.1771           Review: Novel roles of nuclear angiotensin receptors and signaling mechanisms

Gwathmey, T.T.M., Alzayadneh, E.M., Pendergrass, K.D. and Chappell, M.C. Am. J. Physiol. Reegul. Integr. Comp. Physiol., 302, R518-R530 (2012)   The renin-angiotensin system (RAS) constitutes an important hormonal system in the physiological regulation of blood pressure. The dysregulation of the RAS is considered a major influence in the development and progression of cardiovascular disease and other pathologies. Indeed, experimental and clinical evidence indicates that blockade of this system with angiotensin-converting enzyme (ACE) inhibitors or angiotensin type 1 receptor (AT₁R) antagonists is an effective therapy to attenuate hypertension and diabetic renal injury, and to improve heart failure. Originally defined as a circulating system, multiple tissues express a complete RAS, and compelling evidence now favors an intracellular system involved in cell signaling and function. Within the kidney, intracellular expression of the three predominant ANG receptor subtypes is evident in the nuclear compartment. The ANG type 1 receptor (AT₁R) is coupled to the generation of reactive oxygen species (ROS) through the activation of phosphoinositol-3 kinase (PI3K) and PKC. In contrast, both ANG type 2 (AT₂R) and ANG-(1–7) (AT₇R) receptors stimulate nitric oxide (NO) formation, which may involve nuclear endothelial NO synthase (eNOS). Moreover, blockade of either ACE2—the enzyme that converts ANG II to ANG-(1–7)—or the AT₇ receptor exacerbates the ANG II-ROS response on renal nuclei. Finally, in a model of fetal programmed hypertension, the nuclear ROS response to ANG II is enhanced, while both AT₂ and AT₇ stimulation of NO is attenuated, suggesting that an imbalance in the intracellular RAS may contribute to the development of programming events. We conclude that a functional intracellular or nuclear RAS may have important implications in the therapeutic approaches to cardiovascular disease.  

3.1772           Regulation of invadopodia formation and activity by CD147

Grass, G.D., Bratoeva, M. and Toole, B.P.

1. Cell Sci., 125, 777-788 (2012)

  A defining feature of malignant tumor progression is cellular penetration through the basement membrane and interstitial matrices that separate various cellular compartments. Accumulating evidence supports the notion that invasive cells employ specialized structures termed invadopodia to breach these structural barriers. Invadopodia are actin-based, lipid-raft-enriched membrane protrusions containing membrane-type-1 matrix metalloproteinase (MT1-MMP; also known as matrix metalloproteinase 14; MMP14) and several signaling proteins. CD147 (emmprin, basigin), an immunoglobulin superfamily protein that is associated with tumor invasion and metastasis, induces the synthesis of various matrix metalloproteinases in many systems. In this study we show that upregulation of CD147 is sufficient to induce MT1-MMP expression, invasiveness and formation of invadopodia-like structures in non-transformed, non-invasive, breast epithelial cells. We also demonstrate that CD147 and MT1-MMP are in close proximity within these invadopodia-like structures and co-fractionate in membrane compartments with the properties of lipid rafts. Moreover, manipulation of CD147 levels in invasive breast carcinoma cells causes corresponding changes in MT1-MMP expression, invasiveness and invadopodia formation and activity. These findings indicate that CD147 regulates invadopodia formation and activity, probably through assembly of MT1-MMP-containing complexes within lipid-raft domains of the invadopodia.    

3.1773           Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets

Velikkakath, A.K.G., Nishimura, T., Oita, E., Ishihara, N. and Mizushima, N. Mol. Biol. Cell, 2, 896-909 (2012)   Macroautophagy is an intracellular degradation system by which cytoplasmic materials are enclosed by the autophagosome and delivered to the lysosome. Autophagosome formation is considered to take place on the endoplasmic reticulum and involves functions of autophagy-related (Atg) proteins. Here, we report the identification and characterization of mammalian Atg2 homologues Atg2A and Atg2B. Simultaneous silencing of Atg2A and Atg2B causes a block in autophagic flux and accumulation of unclosed autophagic structures containing most Atg proteins. Atg2A localizes on the autophagic membrane, as well as on the surface of lipid droplets. The Atg2A region containing amino acids 1723–1829, which shows relatively high conservation among species, is required for localization to both the autophagic membrane and lipid droplet and is also essential for autophagy. Depletion of both Atg2A and Atg2B causes clustering of enlarged lipid droplets in an autophagy-independent manner. These data suggest that mammalian Atg2 proteins function both in autophagosome formation and regulation of lipid droplet morphology and dispersion.  

3.1774           Impaired CFTR-Dependent Amplification of FSH-Stimulated Estrogen Production in Cystic Fibrosis and PCOS

Chen, H., Guo, J.H., Lu, Y.C., Ding, G.L., Yu, M.K., Tsang, L.L., Fok, K.L., Liu, X.M., Zhang, X.H., Chung, Y.W., Huang, P., Huang, H. and Chan, C.

1. Clin. Endocrinol. Metab., 97(3), 923-932 (2012)

  Context: Estrogens play important roles in a wide range of physiological and pathological processes, and their biosynthesis is profoundly influenced by FSH that regulates the rate-limiting enzyme aromatase-converting estrogens from androgens. Abnormal estrogen levels are often seen in diseases such as ovarian disorders in polycystic ovarian syndrome (PCOS), an endocrine disorder affecting 5–10% of women of reproductive age, and cystic fibrosis (CF), a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). Objectives: We undertook the present study to investigate the mechanism underlying these ovarian disorders, which is not well understood. Results: FSH-stimulated cAMP-responsive element binding protein phosphorylation, aromatase expression, and estradiol production are found to be enhanced by HCO3− and a HCO3− sensor, the soluble adenylyl cyclase, which could be significantly reduced by CFTR inhibition or in ovaries or granulosa cells of cftr knockout/ΔF508 mutant mice. CFTR expression is found positively correlated with aromatase expression in human granulosa cells, supporting its role in regulating estrogen production in humans. Reduced CFTR and aromatase expression is also found in PCOS rodent models and human patients. Conclusions: CFTR regulates ovarian estrogen biosynthesis by amplifying the FSH-stimulated signal via the nuclear soluble adenylyl cyclase. The present findings suggest that defective CFTR-dependent regulation of estrogen production may underlie the ovarian disorders seen in CF and PCOS.  

3.1775           Co-Regulation of Transcellular and Paracellular Leak Across Microvascular Endothelium by Dynamin and Rac

Armstrong, S.M., Khajoee, V., Wang, C., Wang, ST., Tigdi, J., Yin, J., Kuebler, W.M., Gillrie, M., Davis, S.P., Ho, M. and Lee, W.L. Am. J. Pathol., 180(3), 1308-1323 (2012)   Increased permeability of the microvascular endothelium to fluids and proteins is the hallmark of inflammatory conditions such as sepsis. Leakage can occur between (paracellular) or through (transcytosis) endothelial cells, yet little is known about whether these pathways are linked. Understanding the regulation of microvascular permeability is essential for the identification of novel therapies to combat inflammation. We investigated whether transcytosis and paracellular leakage are co-regulated. Using molecular and pharmacologic approaches, we inhibited transcytosis of albumin in primary human microvascular endothelium and measured paracellular permeability. Blockade of transcytosis induced a rapid increase in paracellular leakage that was not explained by decreases in caveolin-1 or increases in activity of nitric oxide synthase. The effect required caveolin-1 but was observed in cells depleted of clathrin, indicating that it was not due to the general inhibition of endocytosis. Inhibiting transcytosis by dynamin blockade increased paracellular leakage concomitantly with the loss of cortical actin from the plasma membrane and the displacement of active Rac from the plasmalemma. Importantly, inhibition of paracellular leakage by sphingosine-1-phosphate, which activates Rac and induces cortical actin, caused a significant increase in transcytosis of albumin in vitro and in an ex vivo whole-lung model. In addition, dominant-negative Rac significantly diminished albumin uptake by endothelia. Our findings indicate that transcytosis and paracellular permeability are co-regulated through a signaling pathway linking dynamin, Rac, and actin.  

3.1776           HIV-1 Nef mobilizes lipid rafts in macrophages through a pathway that competes with ABCA1-dependent cholesterol efflux

Cui, H.L., Grant, A., Mukhamedova, N., Pushkarsky, T., Jenelle, L., Dubrovsky, L., Gaus, K., Fitzgerald, M.L., Sviridov, D. and Bukrinsky, M.   HIV infection, through the actions of viral accessory protein Nef, impairs activity of cholesterol transporter ABCA1, inhibiting cholesterol efflux from macrophages and elevating the risk of atherosclerosis. Nef also induces lipid raft formation. In this study, we demonstrate that these activities are tightly linked and affect macrophage function and HIV replication. Nef stimulated lipid raft formation in macrophage cell line RAW 264.7, and lipid rafts were also mobilized in HIV-1-infected human monocyte-derived macrophages. Nef-mediated transfer of cholesterol to lipid rafts competed with the ABCA1-dependent pathway of cholesterol efflux, and pharmacological inhibition of ABCA1 functionality or suppression of ABCA1 expression by RNAi increased Nef-dependent delivery of cholesterol to lipid rafts. Nef reduced cell-surface accessibility of ABCA1 and induced ABCA1 catabolism via the lysosomal pathway. Despite increasing the abundance of lipid rafts, expression of Nef impaired phagocytic functions of macrophages. The infectivity of the virus produced in natural target cells of HIV-1 negatively correlated with the level of ABCA1. These findings demonstrate that Nef-dependent inhibition of ABCA1 is an essential component of the viral replication strategy and underscore the role of ABCA1 as an innate anti-HIV factor.  

3.1777           Subversion of NPC1 pathway of cholesterol transport by Anaplasma phagocytophilum

Xiong, Q. and Rikihisa, Y. Cell. Microbiol., 14(4), 560-576 (2012)   Intracellular cholesterol amounts, distribution and traffic are tightly regulated to maintain the healthy eukaryotic cell function. However, how intracellular pathogens that require cholesterol, interact with the host cholesterol homeostasis and traffic is not well understood. Anaplasma phagocytophilum is an obligatory intracellular and cholesterol-robbing bacterium, which causes human granulocytic anaplasmosis. Here we found that a subset of cholesterol-binding membrane protein, Niemann-Pick type C1 (NPC1)-bearing vesicles devoid of lysosomal markers were upregulated in HL-60 cells infected with A. phagocytophilum, and trafficked to live bacterial inclusions. The NPC1 localization to A. phagocytophilum inclusions was abolished by low-density lipoprotein (LDL)-derived cholesterol traffic inhibitor U18666A. Studies using NPC1 siRNA and the cell line with cholesterol traffic defect demonstrated that the NPC1 function is required for bacterial cholesterol acquisition and infection. Furthermore, trans-Golgi network-specific soluble N-ethylmaleimide-sensitive factor attachment protein receptors, vesicle-associated membrane protein (VAMP4) and syntaxin 16, which are associated with NPC1 and LDL-derived cholesterol vesicular transport were recruited to A.  phagocytophilum inclusions, and VAMP4 was required for bacteria infection. Taken together, A. phagocytophilum is the first example of a pathogen that subverts the NPC1 pathway of intracellular cholesterol transport and homeostasis for bacterial inclusion membrane biogenesis and cholesterol capture.  

3.1778           Isoform-specific palmitoylation of JNK regulates axonal development

Yang, G., Liu, Y., Yang, K., Liu, R., Zhu, S., Coquinco, A., Wen, W., Kojic, L. and Cynader, M. Cell Death and Differentiation, 19(4), 553-561 (2012)   The c-jun N-terminal kinase (JNK) proteins are encoded by three genes (Jnk1–3), giving rise to 10 isoforms in the mammalian brain. The differential roles of JNK isoforms in neuronal cell death and development have been noticed in several pathological and physiological contexts. However, the mechanisms underlying the regulation of different JNK isoforms to fulfill their specific roles are poorly understood. Here, we report an isoform-specific regulation of JNK3 by palmitoylation, a posttranslational modification, and the involvement of JNK3 palmitoylation in axonal development and morphogenesis. Two cysteine residues at the COOH-terminus of JNK3 are required for dynamic palmitoylation, which regulates JNK3's distribution on the actin cytoskeleton. Expression of palmitoylation-deficient JNK3 increases axonal branching and the motility of axonal filopodia in cultured hippocampal neurons. The Wnt family member Wnt7a, a known modulator of axonal branching and remodelling, regulates the palmitoylation and distribution of JNK3. Palmitoylation-deficient JNK3 mimics the effect of Wnt7a application on axonal branching, whereas constitutively palmitoylated JNK3 results in reduced axonal branches and blocked Wnt7a induction. Our results demonstrate that protein palmitoylation is a novel mechanism for isoform-specific regulation of JNK3 and suggests a potential role of JNK3 palmitoylation in modulating axonal branching.  

3.1779           Novel Role for Non-muscle Myosin Light Chain Kinase (MLCK) in Hyperoxia-induced Recruitment of Cytoskeletal Proteins, NADPH Oxidase Activation, and Reactive Oxygen Species Generation in Lung Endothelium

Usatyuk, P.V., Singleton, P.A., Pendyala, S., Kalari, S.K., He, D., Gorshkova, I.A., Camp, S.M., Moitra, J., Dudek, S.M., Garcia, J.G.N. and Natarajan, V.

1. Biol. Chem., 287(12), 9360-9375 (2012)

  We recently demonstrated that hyperoxia (HO) activates lung endothelial cell NADPH oxidase and generates reactive oxygen species (ROS)/superoxide via Src-dependent tyrosine phosphorylation of p47^phox and cortactin. Here, we demonstrate that the non-muscle ∼214-kDa myosin light chain (MLC) kinase (nmMLCK) modulates the interaction between cortactin and p47^phox that plays a role in the assembly and activation of endothelial NADPH oxidase. Overexpression of FLAG-tagged wild type MLCK in human pulmonary artery endothelial cells enhanced interaction and co-localization between cortactin and p47^phox at the cell periphery and ROS production, whereas abrogation of MLCK using specific siRNA significantly inhibited the above. Furthermore, HO stimulated phosphorylation of MLC and recruitment of phosphorylated and non-phosphorylated cortactin, MLC, Src, and p47^phox to caveolin-enriched microdomains (CEM), whereas silencing nmMLCK with siRNA blocked recruitment of these components to CEM and ROS generation. Exposure of nmMLCK^−/− null mice to HO (72 h) reduced ROS production, lung inflammation, and pulmonary leak compared with control mice. These results suggest a novel role for nmMLCK in hyperoxia-induced recruitment of cytoskeletal proteins and NADPH oxidase components to CEM, ROS production, and lung injury.  

3.1780           Therapeutic Effects of Autologous Tumor-Derived Nanovesicles on Melanoma Growth and Metastasis

Lee, E-Y., Park, K-S., Yoon, Y.J., Lee, J., Moon, H-G., Jang, S.C., Choi, K-H., Kim, Y-K. and Gho, Y.S. PloS One, 7(3), e33330 (2012)   Cancer vaccines with optimal tumor-associated antigens show promise for anti-tumor immunotherapy. Recently, nano-sized vesicles, such as exosomes derived from tumors, were suggested as potential antigen candidates, although the total yield of exosomes is not sufficient for clinical applications. In the present study, we developed a new vaccine strategy based on nano-sized vesicles derived from primary autologous tumors. Through homogenization and sonication of tumor tissues, we achieved high yields of vesicle-bound antigens. These nanovesicles were enriched with antigenic membrane targets but lacked nuclear autoantigens. Furthermore, these nanovesicles together with adjuvant activated dendritic cells in vitro, and induced effective anti-tumor immune responses in both primary and metastatic melanoma mouse models. Therefore, autologous tumor-derived nanovesicles may represent a novel source of antigens with high-level immunogenicity for use in acellular vaccines without compromising safety. Our strategy is cost-effective and can be applied to patient-specific cancer therapeutic vaccination.  

3.1781           NADPH Oxidase-derived Reactive Oxygen Species Increases Expression of Monocyte Chemotactic Factor Genes in Cultured Adipocytes

Han, C.Y., Umemoto, T., Omer, M., Den Hartigh, L.J., Chiba, T., LeBoeuf, R., Buller, C.L., , Sweet, I.R., Pennathur, S., Dale Abel, E. and Chait, A.

1. Biol. Chem., 287(13), 10379-10393 (2012)

  Excess glucose and free fatty acids delivered to adipose tissue causes local inflammation, which contributes to insulin resistance. Glucose and palmitate generate reactive oxygen species (ROS) in adipocytes, leading to monocyte chemotactic factor gene expression. Docosahexaenoate (DHA) has the opposite effect. In this study, we evaluated the potential sources of ROS in the presence of excess nutrients. Differentiated 3T3-L1 adipocytes were exposed to palmitate and DHA (250 μm) in either 5 or 25 mm glucose to evaluate the relative roles of mitochondrial electron transport and NADPH oxidases (NOX) as sources of ROS. Excess glucose and palmitate did not increase mitochondrial oxidative phosphorylation. However, glucose exposure increased glycolysis. Of the NOX family members, only NOX4 was expressed in adipocytes. Moreover, its activity was increased by excess glucose and palmitate and decreased by DHA. Silencing NOX4 inhibited palmitate- and glucose-stimulated ROS generation and monocyte chemotactic factor gene expression. NADPH, a substrate for NOX, and pentose phosphate pathway activity increased with glucose but not palmitate and decreased with DHA exposure. Inhibition of the pentose phosphate pathway by glucose-6-phosphate dehydrogenase inhibitors and siRNA suppressed ROS generation and monocyte chemotactic factor gene expression induced by both glucose and palmitate. Finally, both high glucose and palmitate induced NOX4 translocation into lipid rafts, effects that were blocked by DHA. Excess glucose and palmitate generate ROS via NOX4 rather than by mitochondrial oxidation in cultured adipocytes. NOX4 is regulated by both NADPH generated in the PPP and translocation of NOX4 into lipid rafts, leading to expression of monocyte chemotactic factors.  

3.1782           Rab11-FIP3 is a cell cycle-regulated phosphoprotein

Collins, L.L., Simon, G., Matheson, J., Wu, C., Miller, M.C., Otani, T., Yu, X., Hayashi, S., Prekeris, R. and Gould, G.W. BMC Biology, 13, 4-18 (2012)   Background Rab11 and its effector molecule, Rab11-FIP3 (FIP3), associate with recycling endosomes and traffic into the furrow and midbody of cells during cytokinesis. FIP3 also controls recycling endosome distribution during interphase. Here, we examine whether phosphorylation of FIP3 is involved in these activities. Results We identify four sites of phosphorylation of FIP3 in vivo, S-102, S-280, S-347 and S-450 and identify S-102 as a target for Cdk1-cyclin B in vitro. Of these, we show that S-102 is phosphorylated in metaphase and is dephosphorylated as cells enter telophase. Over-expression of FIP3-S102D increased the frequency of binucleate cells consistent with a role for this phospho-acceptor site in cytokinesis. Mutation of S-280, S-347 or S-450 or other previously identified phospho-acceptor sites (S-488, S-538, S-647 and S-648) was without effect on binucleate cell formation and did not modulate the distribution of FIP3 during the cell cycle. In an attempt to identify a functional role for FIP3 phosphorylation, we report that the change in FIP3 distribution from cytosolic to membrane-associated observed during progression from anaphase to telophase is accompanied by a concomitant dephosphorylation of FIP3. However, the phospho-acceptor sites identified here did not control this change in distribution. Conclusions Our data thus identify FIP3 as a cell cycle regulated phosphoprotein and suggest dephosphorylation of FIP3 accompanies its translocation from the cytosol to membranes during telophase. S102 is dephosphorylated during telophase; mutation of S102 exerts a modest effect on cytokinesis. Finally, we show that de/phosphorylation of the phospho-acceptor sites identified here (S-102, S-280, S-347 and S-450) is not required for the spatial control of recycling endosome distribution or function.  

3.1783           Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling

Steiner, F.A., Talbert, P.B., Kasinathan, S. et al. Genome Res., 22, 766-777 (2012)   An understanding of developmental processes requires knowledge of transcriptional and epigenetic landscapes at the level of tissues and ultimately individual cells. However, obtaining tissue- or cell-type-specific expression and chromatin profiles for animals has been challenging. Here we describe a method for purifying nuclei from specific cell types of animal models that allows simultaneous determination of both expression and chromatin profiles. The method is based on in vivo biotin-labeling of the nuclear envelope and subsequent affinity purification of nuclei. We describe the use of the method to isolate nuclei from muscle of adult Caenorhabditis elegans and from mesoderm of Drosophila melanogaster embryos. As a case study, we determined expression and nucleosome occupancy profiles for affinity-purified nuclei from C. elegans muscle. We identified hundreds of genes that are specifically expressed in muscle tissues and found that these genes are depleted of nucleosomes at promoters and gene bodies in muscle relative to other tissues. This method should be universally applicable to all model systems that allow transgenesis and will make it possible to determine epigenetic and expression profiles of different tissues and cell types.  

3.1784           Annexin A2 Is Involved in the Formation of Hepatitis C Virus Replication Complex on the Lipid Raft

Saxena, V., Lai, C-K., Chao, T-C., Jeng, K-S. and Lai, M.M.C.

1. Virol., 86(8), 4139-4150 (2012)

  The hepatitis C virus (HCV) RNA replicates in hepatic cells by forming a replication complex on the lipid raft (detergent-resistant membrane [DRM]). Replication complex formation requires various viral nonstructural (NS) proteins as well as host cellular proteins. In our previous study (C. K. Lai, K. S. Jeng, K. Machida, and M. M. Lai, J. Virol. 82:8838–8848, 2008), we found that a cellular protein, annexin A2 (Anxa2), interacts with NS3/NS4A. Since NS3/NS4A is a membranous protein and Anxa2 is known as a lipid raft-associated scaffold protein, we postulate that Anxa2 helps in the formation of the HCV replication complex on the lipid raft. Further studies showed that Anxa2 was localized at the HCV-induced membranous web and interacted with NS4B, NS5A, and NS5B and colocalized with them in the perinuclear region. The silencing of Anxa2 decreased the formation of membranous web-like structures and viral RNA replication. Subcellular fractionation and bimolecular fluorescence complementation analysis revealed that Anxa2 was partially associated with HCV at the lipid raft enriched with phosphatidylinositol-4-phosphate (PI4P) and caveolin-2. Further, the overexpression of Anxa2 in HCV-nonsusceptible HEK293 cells caused the enrichment of HCV NS proteins in the DRM fraction and increased the colony-forming ability of the HCV replicon. Since Anxa2 is known to induce the formation of the lipid raft microdomain, we propose that Anxa2 recruits HCV NS proteins and enriches them on the lipid raft to form the HCV replication complex.  

3.1785           Compartmentalization of endocannabinoids into lipid rafts in a microglial cell line devoid of caveolin-1

Rimmermann, N., Bradshaw, H.B., Kozela, E., Levy, R., Juknat, A. and Vogel, Z. Br. J. Pharmacol., 165(8), 2436-2449 (2012)   BACKGROUND AND PURPOSEN-acyl ethanolamines (NAEs) and 2-arachidonoyl glycerol (2-AG) are endogenous cannabinoids and along with related lipids are synthesized on demand from membrane phospholipids. Here, we have studied the compartmentalization of NAEs and 2-AG into lipid raft fractions isolated from the caveolin-1-lacking microglial cell line BV-2, following vehicle or cannabidiol (CBD) treatment. Results were compared with those from the caveolin-1-positive F-11 cell line. EXPERIMENTAL APPROACH BV-2 cells were incubated with CBD or vehicle. Cells were fractionated using a detergent-free continuous OptiPrep density gradient. Lipids in fractions were quantified using HPLC/MS/MS. Proteins were measured using Western blot. KEY RESULTS BV-2 cells were devoid of caveolin-1. Lipid rafts were isolated from BV-2 cells as confirmed by co-localization with flotillin-1 and sphingomyelin. Small amounts of cannabinoid CB₁ receptors were found in lipid raft fractions. After incubation with CBD, levels and distribution in lipid rafts of 2-AG, N-arachidonoyl ethanolamine (AEA), and N-oleoyl ethanolamine (OEA) were not changed. Conversely, the levels of the saturated N-stearoyl ethanolamine (SEA) and N-palmitoyl ethanolamine (PEA) were elevated in lipid raft fractions. In whole cells with growth medium, CBD treatment increased AEA and OEA time-dependently, while levels of 2-AG, PEA and SEA did not change. CONCLUSIONS AND IMPLICATIONS Whereas levels of 2-AG were not affected by CBD treatment, the distribution and levels of NAEs showed significant changes. Among the NAEs, the degree of acyl chain saturation predicted the compartmentalization after CBD treatment suggesting a shift in cell signalling activity.  

3.1786           Apolipoprotein A-I Attenuates Palmitate-Mediated NF-κB Activation by Reducing Toll-Like Receptor-4 Recruitment into Lipid Rafts

Cheng, A.M., handa, P., Tateya, S., Schwartz, J., Tang, C., Mitra, P., Oram, J.F., Chait, A. and Kim, F. PloS One, 7(3), e33917 (2012)   While high-density lipoprotein (HDL) is known to protect against a wide range of inflammatory stimuli, its anti-inflammatory mechanisms are not well understood. Furthermore, HDL's protective effects against saturated dietary fats have not been previously described. In this study, we used endothelial cells to demonstrate that while palmitic acid activates NF-κB signaling, apolipoprotein A–I, (apoA-I), the major protein component of HDL, attenuates palmitate-induced NF-κB activation. Further, vascular NF-κB signaling (IL-6, MCP-1, TNF-α) and macrophage markers (CD68, CD11c) induced by 24 weeks of a diabetogenic diet containing cholesterol (DDC) is reduced in human apoA-I overexpressing transgenic C57BL/6 mice compared to age-matched WT controls. Moreover, WT mice on DDC compared to a chow diet display increased gene expression of lipid raft markers such as Caveolin-1 and Flotillin-1, and inflammatory Toll-like receptors (TLRs) (TLR2, TLR4) in the vasculature. However apoA-I transgenic mice on DDC show markedly reduced expression of these genes. Finally, we show that in endothelial cells TLR4 is recruited into lipid rafts in response to palmitate, and that apoA-I prevents palmitate-induced TLR4 trafficking into lipid rafts, thereby blocking NF-κB activation. Thus, apoA-I overexpression might be a useful therapeutic tool against vascular inflammation.  

3.1787           Erythropoietin Receptor Signaling Is Membrane Raft Dependent

McGraw, K.L., Fuhler, G.M., Johnson, J.O., Clark, J.A., Caceres, G.C., Sokol, L. and List, A.F. PloS One, 7(4), e34477 (2012)   Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3±1.4(SE) vs. 25.6±3.2 aggregates/cell; p≤0.001), accompanied by a >3-fold increase in cluster size (p≤0.001). Raft fraction immunoblotting showed Epo-R translocation to MR after Epo stimulation and was confirmed by fluorescence microscopy in Epo stimulated UT7 cells and primary erythroid bursts. Receptor recruitment into MR was accompanied by incorporation of JAK2, Lyn, and STAT5 and their activated forms. Raft disruption by cholesterol depletion extinguished Epo induced Jak2, STAT5, Akt and MAPK phosphorylation in UT7 cells and erythroid progenitors. Furthermore, inhibition of the Rho GTPases Rac1 or RhoA blocked receptor recruitment into raft fractions, indicating a role for these GTPases in receptor trafficking. These data establish a critical role for MR in recruitment and assembly of Epo-R and signal intermediates into discrete membrane signaling units.  

3.1788           Channel-Forming Activities in the Glycosomal Fraction from the Bloodstream Form of Trypanosoma brucei

Gualdron-Lopez, M., Vapola, M.H., Miinalainen, I:J., Hitunen, J.K., Michels, P.A. and Antenenkov, V.D. PloS One, 7(4), e34530 (2012)   Background Glycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear. Methods/Principal Findings We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (P_K+/P_Cl−~0.31), while the other two types of channels are slightly selective for cations (P_K+/P_Cl− ratios ~1.15 and ~1.27 for the high- and low-conductance channels, respectively). The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel's pore. Conclusions/Significance These results indicate that the membrane of glycosomes apparently contains several types of pore-forming channels connecting the glycosomal lumen and the cytosol.    

3.1789           NGF Causes TrkA to Specifically Attract Microtubules to Lipid Rafts

Pryor, S., McCaffrey, G., Young, L.R. and Grimes, M.L. PloS One, 7(4), e35163 (2012)   Membrane protein sorting is mediated by interactions between proteins and lipids. One mechanism that contributes to sorting involves patches of lipids, termed lipid rafts, which are different from their surroundings in lipid and protein composition. Although the nerve growth factor (NGF) receptors, TrkA and p75^NTR collaborate with each other at the plasma membrane to bind NGF, these two receptors are endocytosed separately and activate different cellular responses. We hypothesized that receptor localization in membrane rafts may play a role in endocytic sorting. TrkA and p75^NTR both reside in detergent-resistant membranes (DRMs), yet they responded differently to a variety of conditions. The ganglioside, GM1, caused increased association of NGF, TrkA, and microtubules with DRMs, but a decrease in p75^NTR. When microtubules were induced to polymerize and attach to DRMs by in vitro reactions, TrkA, but not p75^NTR, was bound to microtubules in DRMs and in a detergent-resistant endosomal fraction. NGF enhanced the interaction between TrkA and microtubules in DRMs, yet tyrosine phosphorylated TrkA was entirely absent in DRMs under conditions where activated TrkA was detected in detergent-sensitive membranes and endosomes. These data indicate that TrkA and p75^NTR partition into membrane rafts by different mechanisms, and that the fraction of TrkA that associates with DRMs is internalized but does not directly form signaling endosomes. Rather, by attracting microtubules to lipid rafts, TrkA may mediate other processes such as axon guidance.  

3.1790           Requirement of translocated lysosomal V1 H+-ATPase for activation of membrane acid sphingomyelinase and raft clustering in coronary endothelial cells

Xu, M., Xia, M., Li, X-X., Han, W-Q., Boini, K.M., Zhang, F., Zhang, Y., Ritter, J.K. and Li, P-L. Mol. Biol. Cell, 23, 1546-1557 (2012)   Acid sphingomyelinase (ASM) mediates the formation of membrane raft (MR) redox signalosomes in a process that depends on a local acid microenvironment in coronary arterial endothelial cells (CAECs). However, it is not known how this local acid microenvironment is formed and maintained. The present study hypothesized that lysosomal V1 H⁺-ATPase provides a hospitable acid microenvironment for activation of ASM when lysosomes traffic and fuse into the cell membrane. Confocal microscopy showed that local pH change significantly affected MRs, with more fluorescent patches under low pH. Correspondingly, the ASM product, ceramide, increased locally in the cell membrane. Electron spin resonance assay showed that local pH increase significantly inhibited NADPH oxidase–mediated production of O₂^−. in CAECs. Direct confocal microscopy demonstrated that Fas ligand resulted in localized areas of decreased pH around CAEC membranes. The inhibitors of both lysosomal fusion and H⁺-ATPase apparently attenuated FasL-caused pH decrease. V1 H⁺-ATPase accumulation and activity on cell membranes were substantially suppressed by the inhibitors of lysosomal fusion or H⁺-ATPase. These results provide the first direct evidence that translocated lysosomal V1 H⁺-ATPase critically contributes to the formation of local acid microenvironment to facilitate activation of ASM and consequent MR aggregation, forming MR redox signalosomes and mediating redox signaling in CAECs.  

3.1791           Host Acyl Coenzyme A Binding Protein Regulates Replication Complex Assembly and Activity of a Positive-Strand RNA Virus

Zhang, J., Diaz, A., Mao, L., Ahlquist, P and Wang, X.

1. Virol., 86(9), 5110-5121 (2012)

  All positive-strand RNA viruses reorganize host intracellular membranes to assemble their replication complexes. Similarly, brome mosaic virus (BMV) induces two alternate forms of membrane-bound RNA replication complexes: vesicular spherules and stacks of appressed double-membrane layers. The mechanisms by which these membrane rearrangements are induced, however, remain unclear. We report here that host ACB1-encoded acyl coenzyme A (acyl-CoA) binding protein (ACBP) is required for the assembly and activity of both BMV RNA replication complexes. ACBP is highly conserved among eukaryotes, specifically binds to long-chain fatty acyl-CoA, and promotes general lipid synthesis. Deleting ACB1 inhibited BMV RNA replication up to 30-fold and resulted in formation of spherules that were ∼50% smaller but ∼4-fold more abundant than those in wild-type (wt) cells, consistent with the idea that BMV 1a invaginates and maintains viral spherules by coating the inner spherule membrane. Furthermore, smaller and more frequent spherules were preferentially formed under conditions that induce layer formation in wt cells. Conversely, cellular karmella structures, which are arrays of endoplasmic reticulum (ER) membranes formed upon overexpression of certain cellular ER membrane proteins, were formed normally, indicating a selective inhibition of 1a-induced membrane rearrangements. Restoring altered lipid composition largely complemented the BMV RNA replication defect, suggesting that ACBP was required for maintaining lipid homeostasis. Smaller and more frequent spherules are also induced by 1a mutants with specific substitutions in a membrane-anchoring amphipathic α-helix, implying that the 1a-lipid interactions play critical roles in viral replication complex assembly.  

3.1792           Lysosome fusion to the cell membrane is mediated by the dysferlin C2A domain in coronary arterial endothelial cells

Han, W-Q., Xia, M., Xu, M., Boini, K.M., Ritter, J.K., Li, N-J. and Li, P-L.

1. Cell Sci., 125, 1225-1234 (2012)

  Dysferlin has recently been reported to participate in cell membrane repair in muscle and other cells through lysosome fusion. Given that lysosome fusion is a crucial mechanism that leads to membrane raft clustering, the present study attempted to determine whether dysferlin is involved in this process and its related signalling, and explores the mechanism underlying dysferlin-mediated lysosome fusion in bovine coronary arterial endothelial cells (CAECs). We found that dysferlin is clustered in membrane raft macrodomains after Fas Ligand (FasL) stimulation as detected by confocal microscopy and membrane fraction flotation. Small-interfering RNA targeted to dysferlin prevented membrane raft clustering. Furthermore, the translocation of acid sphingomyelinase (ASMase) to membrane raft clusters, whereby local ASMase activation and ceramide production – an important step that mediates membrane raft clustering – was attenuated. Functionally, silencing of the dysferlin gene reversed FasL-induced impairment of endothelium-dependent vasodilation in isolated small coronary arteries. By monitoring fluorescence quenching or dequenching, silencing of the dysferlin gene was found to almost completely block lysosome fusion to plasma membrane upon FasL stimulation. Further studies to block C2A binding and silencing of AHNAK (a dysferlin C2A domain binding partner), showed that the dysferlin C2A domain is required for FasL-induced lysosome fusion to the cell membrane, ASMase translocation and membrane raft clustering. We conclude that dysferlin determines lysosome fusion to the plasma membrane through its C2A domain and it is therefore implicated in membrane-raft-mediated signaling and regulation of endothelial function in coronary circulation.  

3.1793           Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes

Tauro, B.J., Greening, D.W., Mathias, R.A., Ji, H., Mathivanan, S., Scott, A.M. and Simpson, R.J. Methods, 56, 293-304 (2012)   Exosomes are 40–100 nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of oncogenic proteins as well as mRNA and miRNA. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. However, all preparations invariably contain varying proportions of other membranous vesicles that co-purify with exosomes such as shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, in this study we performed a comprehensive evaluation of current methods used for exosome isolation including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM coated magnetic beads (IAC-Exos). Notably, all isolations contained 40–100 nm vesicles, and were positive for exosome markers (Alix, TSG101, HSP70) based on electron microscopy and Western blotting. We employed a proteomic approach to profile the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, we found IAC-Exos to be the most effective method to isolate exosomes. For example, Alix, TSG101, CD9 and CD81 were significantly higher (at least 2-fold) in IAC-Exos, compared to UG-Exos and DG-Exos. Application of immunoaffinity capture has enabled the identification of proteins including the ESCRT-III component VPS32C/CHMP4C, and the SNARE synaptobrevin 2 (VAMP2) in exosomes for the first time. Additionally, several cancer-related proteins were identified in IAC-Exos including various ephrins (EFNB1, EFNB2) and Eph receptors (EPHA2–8, EPHB1–4), and components involved in Wnt (CTNNB1, TNIK) and Ras (CRK, GRB2) signalling.      

3.1794           Accumulation of an Antidepressant in Vesiculogenic Membranes of Yeast Cells Triggers Autophagy

Chen, J., Korostyshevsky, D., Lee, S. and Perlstein, E.O. PloS One, 7(4), e34024 (2012)   Many antidepressants are cationic amphipaths, which spontaneously accumulate in natural or reconstituted membranes in the absence of their specific protein targets. However, the clinical relevance of cellular membrane accumulation by antidepressants in the human brain is unknown and hotly debated. Here we take a novel, evolutionarily informed approach to studying the effects of the selective-serotonin reuptake inhibitor sertraline/Zoloft® on cell physiology in the model eukaryote Saccharomyces cerevisiae (budding yeast), which lacks a serotonin transporter entirely. We biochemically and pharmacologically characterized cellular uptake and subcellular distribution of radiolabeled sertraline, and in parallel performed a quantitative ultrastructural analysis of organellar membrane homeostasis in untreated vs. sertraline-treated cells. These experiments have revealed that sertraline enters yeast cells and then reshapes vesiculogenic membranes by a complex process. Internalization of the neutral species proceeds by simple diffusion, is accelerated by proton motive forces generated by the vacuolar H⁺-ATPase, but is counteracted by energy-dependent xenobiotic efflux pumps. At equilibrium, a small fraction (10–15%) of reprotonated sertraline is soluble while the bulk (90–85%) partitions into organellar membranes by adsorption to interfacial anionic sites or by intercalation into the hydrophobic phase of the bilayer. Asymmetric accumulation of sertraline in vesiculogenic membranes leads to local membrane curvature stresses that trigger an adaptive autophagic response. In mutants with altered clathrin function, this adaptive response is associated with increased lipid droplet formation. Our data not only support the notion of a serotonin transporter-independent component of antidepressant function, but also enable a conceptual framework for characterizing the physiological states associated with chronic but not acute antidepressant administration in a model eukaryote.  

3.1795           Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves

Joseph, M., Ludevid, M.D., Torrent, M., Rofidal, V., Tauzin, M., Rossignol, M. and Peltier, J-B. BMC Plant Biol., 12, 36 (2012)   Background The N-terminal proline-rich domain (Zera) of the maize storage protein γ-zein, is able to induce the formation of endoplasmic reticulum (ER)-derived protein bodies (PBs) when fused to proteins of interest. This encapsulation enables a recombinant fused protein to escape from degradation and facilitates its recovery from plant biomass by gradient purification. The aim of the present work was to evaluate if induced PBs encapsulate additional proteins jointly with the recombinant protein. The exhaustive analysis of protein composition of PBs is expected to facilitate a better understanding of PB formation and the optimization of recombinant protein purification approaches from these organelles. Results We analysed the proteome of PBs induced in Nicotiana benthamiana leaves by transient transformation with Zera fused to a fluorescent marker protein (DsRed). Intact PBs with their surrounding ER-membrane were isolated on iodixanol based density gradients and their integrity verified by confocal and electron microscopy. SDS-PAGE analysis of isolated PBs showed that Zera-DsRed accounted for around 85% of PB proteins in term of abundance. Differential extraction of PBs was performed for in-depth analysis of their proteome and structure. Besides Zera-DsRed, 195 additional proteins were identified including a broad range of proteins resident or trafficking through the ER and recruited within the Zera-DsRed polymer. Conclusions This study indicates that Zera-protein fusion is still the major protein component of the new formed organelle in tobacco leaves. The analysis also reveals the presence of an unexpected diversity of proteins in PBs derived from both the insoluble Zera-DsRed polymer formation, including ER-resident and secretory proteins, and a secretory stress response induced most likely by the recombinant protein overloading. Knowledge of PBs protein composition is likely to be useful to optimize downstream purification of recombinant proteins in molecular farming applications.  

3.1796           Targeted Disruption of Core 1 β1,3-galactosyltransferase (C1galt1) Induces Apical Endocytic Trafficking in Human Corneal Keratinocytes

Guzman-Aranguez, A., Woodward, A.M., Pintor, J. and Argüeso, P. PloS One, 7(5), e36628 (2012)   Background Exposed mucosal surfaces limit constitutive endocytosis under physiological conditions to prevent uptake of macromolecules and pathogens and, therefore, cellular damage. It is now accepted that cell surface mucins, a group of high molecular weight glycoproteins on the epithelial glycocalyx, defined by their extensive O-glycosylation, play a major role in maintaining barrier function in these surfaces, but the precise mechanisms are unclear. Methodology/Principal Findings In this work, we utilized a stable tetracycline-inducible RNA interfering system targeting the core 1 ß1,3-galactosyltransferase (C1galt1 or T-synthase), a critical galactosyltransferase required for the synthesis of core 1 O-glycans, to explore the role of mucin-type carbohydrates in apical endocytic trafficking in human corneal keratinocytes. Using cell surface biotinylation and subcellular fractionation, we found increased accumulation of plasma membrane protein in endosomes after C1galt1 depletion. Confocal laser scanning microscopy and fluorometry revealed increased translocation of negatively charged fluorescent nanospheres after C1galt1 knockdown sustained by an active transport process and largely independent of apical intercellular junctions. Internalization of nanospheres could be blocked by dynasore, nocodazole, chlorpromazine, and hyperosmotic sucrose, suggesting a mechanism for clathrin-coated pit budding and vesicular trafficking. This possibility was supported by experiments showing nanosphere colocalization with clathrin heavy chain in the cytoplasm. Conclusions/Significance Together, the data suggest that core 1 O-glycans contribute to maintenance of apical barrier function on exposed mucosal surfaces by preventing clathrin-mediated endocytosis.  

3.1797           Novel Role for Proteinase-activated Receptor 2 (PAR2) in Membrane Trafficking of Proteinase-activated Receptor 4 (PAR4)

Cunningham, M.R., McIntosh, K.A., Pediani, J.D., Robben, J., Cooke, A.E., Nilsson, M., Gould, G.W.., Mundell, S., Milligan, G. and Plevin, R.

1. Biol. Chem., 287(20), 16656-16669 (2012)

  Proteinase-activated receptors 4 (PAR₄) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR₄ remain unknown. Here, we report novel features of the intracellular trafficking of PAR₄ to the plasma membrane. PAR₄ was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR₄ protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R¹⁸³AR → A¹⁸³AA), mutation of which allowed efficient membrane delivery of PAR₄. Interestingly, co-expression with PAR₂ facilitated plasma membrane delivery of PAR₄, an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR₂ and PAR₄. PAR₂ also enhanced glycosylation of PAR₄ and activation of PAR₄ signaling. Our results identify a novel regulatory role for PAR₂ in the anterograde traffic of PAR₄. PAR₂ was shown to both facilitate and abrogate protein interactions with PAR₄, impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR₄ in normal physiology and disease.  

3.1798           Membrane-bound Trafficking Regulates Nuclear Transport of Integral Epidermal Growth Factor Receptor (EGFR) and ErbB-2

Wang, Y-N., Lee, H-H., Lee, H-J., Du, Y., Yamaguchi, H. ansd Hung, M-C.

1. Biol. Chem., 287(20), 16869-16879 (2012)

  Nuclear localization of multiple receptor-tyrosine kinases (RTKs), such as EGF receptor (EGFR), ErbB-2, FGF receptor (FGFR), and many others, has been reported by several groups. We previously showed that cell surface EGFR is trafficked to the nucleus through a retrograde pathway from the Golgi to the endoplasmic reticulum (ER) and that EGFR is then translocated to the inner nuclear membrane (INM) through the INTERNET (integral trafficking from the ER to the nuclear envelope transport) pathway. However, the nuclear trafficking mechanisms of other membrane RTKs, apart from EGFR, remain unclear. The purpose of this study was to compare the nuclear transport of EGFR family proteins with that of FGFR-1. Interestingly, we found that digitonin permeabilization, which selectively releases soluble nuclear transporters from the cytoplasm and has been shown to inhibit nuclear transport of FGFR-1, had no effects on EGFR nuclear transport, raising the possibility that EGFR and FGFR-1 use different pathways to be translocated into the nucleus. Using the subnuclear fractionation assay, we further demonstrated that biotinylated cell surface ErbB-2, but not FGFR-1, is targeted to the INM, associating with Sec61β in the INM, similar to the nuclear trafficking of EGFR. Thus, ErbB-2, but not FGFR-1, shows a similar trafficking pathway to EGFR for translocation to the nucleus, indicating that at least two different pathways of nuclear transport exist for cell surface receptors. This finding provides a new direction for investigating the trafficking mechanisms of various nuclear RTKs.  

3.1799           Raft coalescence and FcγRIIA activation upon sphingomyelin clustering induced by lysenin

Kulma, M., Kwiatkowska, K. and Sobota, A. Cellular Signalling, 24, 1641-1647 (2012)   Activation of immunoreceptor FcγRIIA by cross-linking with antibodies is accompanied by coalescence of sphingolipid/cholesterol-rich membrane rafts leading to the formation of signaling platforms of the receptor. In this report we examined whether clustering of the raft lipid sphingomyelin can reciprocally induce partition of FcγRIIA to rafts. To induce sphingomyelin clustering, cells were exposed to non-lytic concentrations of GST-lysenin which specifically recognizes sphingomyelin. The lysenin/sphingomyelin complexes formed microscale assemblies composed of GST-lysenin oligomers engaging sphingomyelin of rafts. Upon sphingomyelin clustering, non-cross-linked FcγRIIA associated with raft-derived detergent-resistant membrane fractions as revealed by density gradient centrifugation. Pretreatment of cells with GST-lysenin also increased the size of detergent-insoluble molecular complexes of activated FcγRIIA. Sphingomyelin clustering triggered tyrosine phosphorylation of the receptor and its accompanying proteins, Cbl and NTAL, in the absence of receptor ligands and enhanced phosphorylation of these proteins in the ligand presence. These data indicate that clustering of plasma membrane sphingomyelin induces coalescence of rafts and triggers signaling events analogous to those caused by FcγRIIA activation.  

3.1800           Proteomic Analysis Of The Lysosomal Fraction In Trabecular Meshwork Cells Subjected To Chronic Oxidative Stress

Porter, K.M., Jeyabalan, N., Skiba, N.P., Epstein, D.L. and Liton, P.B. Invest. Ophthalmol. Vis. Sci., 53, E-abstract 3248 (2012)   Purpose:Previous work in our laboratory reported impaired lysosomal function in trabecular meshwork (TM) cells subjected to chronic oxidative stress. Here we compare the proteomic composition of isolated lysosomes from TM cells grown under physiological and oxidative stress conditions. Methods:Confluent cultures of porcine TM cells were grown for two weeks under physiological (5% O2) and chronic oxidative stress (40% O2) conditions. The lysosomal fraction was isolated by ultracentrifugation and cell fractioning in OptiPrep gradients. Lysosomal proteins were separated in SDS-PAGE gels, followed by in-gel trypsin digestion. The tryptic-digested peptides were identified by peptide mass finger printing analyses and tandem mass spectrometry. Protein expression levels were quantified by WB analysis using specific antibodies against LAMP1, Rab7, CTSB, CTSD, and CD63. Cathepsin activities were assayed using fluorogenic substrates (z-FR-AMC, z-RR-AMC, z-GPR-AMC, z-VVR-AMC, CTSD/E substrate). Results:Using an acceptance criteria for protein identification of at least two peptides with confidence interval percentage over 95%, we identified a total of 83 proteins. These included lysosomal matrix proteins and lysosomal enzymes, structural glycoproteins (LAMP1, LAMP2, LIMP2, CD63), proteins involved in translocation and lysosomal acidification (vacuolar H+ATPases), and membrane trafficking proteins. The most remarkable difference was the absence of CD63 and lysosomal acid phosphatase peptides in the lysosomal fraction from TM cells grown at 40% O2. Confirming our results obtained using whole lysates, the lysosomal fraction from oxidatively stressed cultures displayed increased LAMP1, CTSB and CTSD protein levels. However, no increased in cathepsin activities were observed. Furthermore, the proteolytic processing of CSTB from single-chain to double-chain was significantly blocked in the stressed cultures. Conclusions:Here we have characterized for the first time the proteome composition of lysosomes from oxidatively stressed TM cells. Our results indicate that chronic exposure to oxidative stress induces changes in the lysosomal proteomic composition and defective lysosomal function. Since the lysosomal system is responsible for the turnover of cellular organelles and degradation of phagocytosed material, diminished lysosomal activity may lead to progressive failure of the cellular TM function with age and contribute to the pathogenesis of primary open angle glaucoma.

3.1801           DHA Increases Outflow Resistance In Porcine Organ Cultures

Giovingo, M., McCarty, R.D., Beverley, R., Nolan, M., Grybauskas, A., Burdi, R.A., Wagner, E. and Knepper, P.A. Invest. Ophthalmol. Vis. Sci., 53, E-abstract 2494 (2012)   Purpose:Caveolae and lipid rafts are highly specialized microdomains of the endothelial plasma membrane which function in signal transduction, endocytosis and transcellular fluid dynamics. Caveolin, a 21 kD protein, is an intergal component in the activity of endothelial cells. Increasing evidence suggests that omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) alter the basic properties of caveolae in endothelia and their function. The purpose of this study was to determine whether DHA changes outflow resistance in porcine anterior segment organ culture and profiles of lipid raft proteins of the trabecular meshwork (TM). Methods:Anterior segments of porcine eyes were placed in organ culture and perfused with Dulbecco's modified eagle medium (DMEM). Flow rates were measured in eyes treated with DHA, cavtratin (a synthetic cell permeable peptide corresponding to amino acids 82 through 101 of caveolin: DGIWKASFTIFTVTKYWFYR which is also known as caveolin scaffolding domain), or DMEM. Perturbation of lipid raft containing proteins was assessed by Optiprep density gradient and western blot analysis of dissected TM. Results:Flow rates are expressed as percentage change (+/-) from the baseline. Rates significantly decreased from baseline in DHA and cavtratin infused eyes.Analysis of lipid raft containing proteins of DHA and primary cultures of TM cells revealed a decrease in caveolin-1 in comparison with controls. Conclusions:Infusion of DHA and cavtratin significantly decreased outflow facility and was time-dependent. DHA alters the lipid raft and caveolae microdomain, thereby influencing outflow facility in organ perfusion culture.  

3.1802           Disruption of the P4-ATPase Aminophospholipid Flippase Atp8a2 Gene Suggests a Role for Phosphatidylserine in Photoreceptor Outer Segments

Coleman, J.A., Djajadi, H.R., Molday, L.L. and Molday, R.S. Invest. Ophthalmol. Vis. Sci., 53, E-abstract 743 (2012)   Purpose:ATP8A2 is a P₄-ATPase which transports phosphatidylserine (PS) from the exocytoplasmic to the cytoplasmic side of photoreceptor outer segment (OS) disc membranes to generate and maintain PS asymmetry. The goal of this study was to determine the importance of ATP8A2 in photoreceptors and other cell types by the targeted disruption of the atp8a2 gene in mice. Methods:Exons 11 - 13 of atp8a2 were replaced using a targeting construct containing a neomycin cassette in hybrid 129 SvEv and C57BL/6 ES cells. Retina membranes were prepared using the Optiprep method. Lipids were separated by thin layer chromatography and quantified by phosphorus assays. Retinoids were analyzed by HPLC. Antibodies specific to OS proteins were used to detect their expression and localization by western blotting and immunofluorescence microscopy. Outer nuclear layer (ONL) thickness was measured by DAPI labeling using the optic nerve as a reference. Ultrathin sections of glutaraldehyde-fixed resin-embedded retina were analyzed by transmission electron microscopy (TEM). Results:Atp8a2 knockout mice are smaller and catatonic compared to wild type littermates. RT-PCR, western blotting, and immunocytochemistry confirmed the absence of ATP8A2 in the retinas of the atp8a2 knockout mice. CDC50A, the β-subunit of ATP8A2, was present at significantly lower levels. Opsin, CNGA1, ABCA4 and other outer segment (OS) proteins were reduced by approximately 50%, but correctly localized to the OS. The OS layer was approximately 50% shorter. OS contained significantly less phosphatidylserine and higher levels of phosphatidylcholine (PC). The number of photoreceptors was reduced by 15% at P23 with no further reduction evident at P50. The ultrastructure of the OS as visualized by EM appeared normal. Spectrophotometry and HPLC measurements revealed that rhodopsin and retinoid levels are 66% lower in the knockout retina. Conclusions:Analysis of atp8a2 knockout mice suggests that PS asymmetry and composition plays a role in photoreceptor OS morphogenesis possibly associated with the trafficking of proteins to the OS. PS asymmetry may also play a crucial role in protein trafficking in other neurons.  

3.1803           Fas Ligand Enhances Malignant Behavior of Tumor Cells through Interaction with Met, Hepatocyte Growth Factor Receptor, in Lipid Rafts

Lin, H-C., Lai, P-Y., Lin, Y-p., Huang, J-H. and Yang, B-C.

1. Biol. Chem., 287(24), 20664-20673 (2012)

  Many late-stage cancer cells express Fas ligand (FasL) and show high malignancy with metastatic potential. We report here a novel signaling mechanism for FasL that hijacks the Met signal pathway to promote tumor metastasis. FasL-expressing human tumor cells express a significant amount of phosphorylated Met. The down-regulation of FasL in these cells led to decreased Met activity and reduced cell motility. Ectopic expression of human FasL in NIH3T3 cells significantly stimulated their migration and invasion. The inhibition of Met and Stat3 activities reverted the FasL-associated phenotype. Notably, FasL variants activated the Met pathway, even though most of their intracellular domain or Fas binding sites were deleted. FasL interacted with Met through the FasL(105–130) extracellular region in lipid rafts, which consequently led to Met activation. Knocking down Met gene expression by RNAi technology reverted the FasL-associated motility to basal levels. Furthermore, treatment with synthetic peptides corresponding to FasL(117–126) significantly reduced the FasL/Met interaction, Met phosphorylation, and cell motility of FasL⁺ transfectants and tumor cells. Finally, the transfectants of truncated FasL showed strong anchorage-independent growth and lung metastasis potential in null mice. Collectively, our results establish the FasL-Met-Stat3 signaling pathway and explains the metastatic phenotype of FasL-expressing tumors.  

3.1804           Lecithin:Cholesterol Acyltransferase Deficiency Protects against Cholesterol-induced Hepatic Endoplasmic Reticulum Stress in Mice

Hager, L., Li, L., Pun, H., Liu, L., Hossain, M.A., Maguire, G.F., Naples, M., Baker, C., Magomedova, L., Tam, J., Adeli, K., Cummins, C.L., Connelly, P.W. and Ng, D.S.

1. Biol. Chem., 287(24), 20775-20768 (2012)

  We recently reported that lecithin:cholesterol acyltransferase (LCAT) knock-out mice, particularly in the LDL receptor knock-out background, are hypersensitive to insulin and resistant to high fat diet-induced insulin resistance (IR) and obesity. We demonstrated that chow-fed Ldlr−/−xLcat+/+ mice have elevated hepatic endoplasmic reticulum (ER) stress, which promotes IR, compared with wild-type controls, and this effect is normalized in Ldlr−/−xLcat−/− mice. In the present study, we tested the hypothesis that hepatic ER cholesterol metabolism differentially regulates ER stress using these models. We observed that the Ldlr−/−xLcat+/+ mice accumulate excess hepatic total and ER cholesterol primarily attributed to increased reuptake of biliary cholesterol as we observed reduced biliary cholesterol in conjunction with decreased hepatic Abcg5/g8 mRNA, increased Npc1l1 mRNA, and decreased Hmgr mRNA and nuclear SREBP2 protein. Intestinal NPC1L1 protein was induced. Expression of these genes was reversed in the Ldlr−/−xLcat−/− mice, accounting for the normalization of total and ER cholesterol and ER stress. Upon feeding a 2% high cholesterol diet (HCD), Ldlr−/−xLcat−/− mice accumulated a similar amount of total hepatic cholesterol compared with the Ldlr−/−xLcat+/+ mice, but the hepatic ER cholesterol levels remained low in conjunction with being protected from HCD-induced ER stress and IR. Hepatic ER stress correlates strongly with hepatic ER free cholesterol but poorly with hepatic tissue free cholesterol. The unexpectedly low ER cholesterol seen in HCD-fed Ldlr−/−xLcat−/− mice was attributable to a coordinated marked up-regulation of ACAT2 and suppressed SREBP2 processing. Thus, factors influencing the accumulation of ER cholesterol may be important for the development of hepatic insulin resistance.    

3.1805           Activation of Myeloid Cell-Specific Adhesion Class G Protein-Coupled Receptor EMR2 via Ligation-Induced Translocation and Interaction of Receptor Subunits in Lipid Raft Microdomains

Huang, Y-S., Chiang, N-Y., Hu, C-H., Hsiao, C-C., Cheng, K-F., Tsai, W-P., Yona, S., Stavey, M., Gordon, S., Chang, XG-W. and Lin, H-H. Mol. Cell. Biol., 32(8), 1408-1420 (2012)   The adhesion class G protein-coupled receptors (adhesion-GPCRs) play important roles in diverse biological processes ranging from immunoregulation to tissue polarity, angiogenesis, and brain development. These receptors are uniquely modified by self-catalytic cleavage at a highly conserved GPCR proteolysis site (GPS) dissecting the receptor into an extracellular subunit (α) and a seven-pass transmembrane subunit (β) with cellular adhesion and signaling functions, respectively. Using the myeloid cell-restricted EMR2 receptor as a paradigm, we exam the mechanistic relevance of the subunit interaction and demonstrate a critical role for GPS autoproteolysis in mediating receptor signaling and cell activation. Interestingly, two distinct receptor complexes are identified as a result of GPS proteolysis: one consisting of a noncovalent α-β heterodimer and the other comprising two completely independent receptor subunits which distribute differentially in membrane raft microdomains. Finally, we show that receptor ligation induces subunit translocation and colocalization within lipid rafts, leading to receptor signaling and inflammatory cytokine production by macrophages. Our present data resolve earlier conflicting results and provide a new mechanism of receptor signaling, as well as providing a paradigm for signal transduction within the adhesion-GPCR family.  

3.1806           Vaccinia Virus A6 Is Essential for Virion Membrane Biogenesis and Localization of Virion Membrane Proteins to Sites of Virion Assembly

Meng, X., Embry, A., Rose, L., Yan, B., Xu, C. and Xiang, Y.

1. Virol., 86(10), 5603-5613 (2012)

  Poxvirus acquires its primary envelope through a process that is distinct from those of other enveloped viruses. The molecular mechanism of this process is poorly understood, but several poxvirus proteins essential for the process have been identified in studies of vaccinia virus (VACV), the prototypical poxvirus. Previously, we identified VACV A6 as an essential factor for virion morphogenesis by studying a temperature-sensitive mutant with a lesion in A6. Here, we further studied A6 by constructing and characterizing an inducible virus (iA6) that could more stringently repress A6 expression. When A6 expression was induced by the inducer isopropyl-β-d-thiogalactoside (IPTG), iA6 replicated normally, and membrane proteins of mature virions (MVs) predominantly localized in viral factories where virions were assembled. However, when A6 expression was repressed, electron microscopy of infected cells showed the accumulation of large viroplasm inclusions containing virion core proteins but no viral membranes. Immunofluorescence and cell fractionation studies showed that the major MV membrane proteins A13, A14, D8, and H3 did not localize to viral factories but instead accumulated in the secretory compartments, including the endoplasmic reticulum. Overall, our results show that A6 is an additional VACV protein that participates in an early step of virion membrane biogenesis. Furthermore, A6 is required for MV membrane protein localization to sites of virion assembly, suggesting that MV membrane proteins or precursors of MV membranes are trafficked to sites of virion assembly through an active, virus-mediated process that requires A6.  

3.1807           Critical Comparison of Multidimensional Separation Methods for Increasing Protein Expression Coverage

Antberg, L., Cifani, P., Sandin, M., Levander, F. and James, P.

1. Proteome Res., 11(5), 2644-2652 (2012)

  We present a comparison of two-dimensional separation methods and how they affect the degree of coverage of protein expression in complex mixtures. We investigated the relative merits of various protein and peptide separations prior to acidic reversed-phase chromatography directly coupled to an ion trap mass spectrometer. The first dimensions investigated were density gradient organelle fractionation of cell extracts, 1D SDS-PAGE protein separation followed by digestion by trypsin or GluC proteases, strong cation exchange chromatography, and off-gel isoelectric focusing of tryptic peptides. The number of fractions from each first dimension and the total data accumulation RP-HPLC–MS/MS time was kept constant and the experiments were run in triplicate. We find that the most critical parameters are the data accumulation time, which defines the level of under-sampling and the avoidance of peptides from high expression level proteins eluting over the entire gradient.  

3.1808           Identification of Core Components and Transient Interactors of the Peroxisomal Importomer by Dual-Track Stable Isotope Labeling with Amino Acids in Cell Culture Analysis

Oeljeklaus, S., Reinartz, B.S., Wolf, J., Wiese, S., Tonillo, J., Podwojski, K., Kuhlmann, K., Stephan, C., Meyer, H.E., Schliebs, W., Brocard, C., Erdmann, R and Warscheid, B.

1. Proteome Res., 11(4), 2567-2580 (2012)

  The importomer complex plays an essential role in the biogenesis of peroxisomes by mediating the translocation of matrix proteins across the organellar membrane. A central part of this highly dynamic import machinery is the docking complex consisting of Pex14p, Pex13p, and Pex17p that is linked to the RING finger complex (Pex2p, Pex10p, Pex12p) via Pex8p. To gain detailed knowledge on the molecular players governing peroxisomal matrix protein import and, thus, the integrity and functionality of peroxisomes, we aimed at a most comprehensive investigation of stable and transient interaction partners of Pex14p, the central component of the importomer. To this end, we performed a thorough quantitative proteomics study based on epitope tagging of Pex14p combined with dual-track stable isotope labeling with amino acids in cell culture-mass spectrometry (SILAC-MS) analysis of affinity-purified Pex14p complexes and statistics. The results led to the establishment of the so far most extensive Pex14p interactome, comprising 9 core and further 12 transient components. We confirmed virtually all known Pex14p interaction partners including the core constituents of the importomer as well as Pex5p, Pex11p, Pex15p, and Dyn2p. More importantly, we identified new transient interaction partners (Pex25p, Hrr25p, Esl2p, prohibitin) that provide a valuable resource for future investigations on the functionality, dynamics, and regulation of the peroxisomal importomer.  

3.1809           Ethanol triggers sphingosine 1-phosphate elevation along with neuroapoptosis in the developing mouse brain

Chakraborty, G., Saito, M., Shah, R., Mao, R-F., Vadasz, C. and Saito, M.

1. Neurochem., 121(5), 806-817 (2012)

  Our previous studies have indicated that de novo ceramide synthesis plays a critical role in ethanol-induced apoptotic neurodegeneration in the 7-day-old mouse brain. In this study, we examined whether the formation of sphingosine 1-phosphate (S1P), a ceramide metabolite, is associated with this apoptotic pathway. Analyses of basal levels of S1P-related compounds indicated that S1P, sphingosine, sphingosine kinase 2, and S1P receptor 1 increased significantly during postnatal brain development. In the 7-day-old mouse brain, sphingosine kinase 2 was localized mainly in neurons. Subcellular fractionation studies of the brain homogenates showed that sphingosine kinase 2 was enriched in the plasma membrane and the synaptic membrane/synaptic vesicle fractions, but not in the nuclear and mitochondrial/lysosomal fractions. Ethanol exposure in 7-day-old mice induced sphingosine kinase 2 activation and increased the brain level of S1P transiently 2–4 h after exposure, followed by caspase 3 activation that peaked around 8 h after exposure. Treatment with dimethylsphingosine, an inhibitor of sphingosine kinases, attenuated the ethanol-induced caspase 3 activation and the subsequent neurodegeneration. These results indicate that ethanol activates sphingosine kinase 2, leading to a transient increase in S1P, which may be involved in neuroapoptotic action of ethanol in the developing brain.  

3.1810           CCL11 elicits secretion of RNases from mouse eosinophils and their cell-free granules

Shamri, R., Melo, R.C.N., Young, K.M., Bivas-Benita, M., Xenakis, J.J., Spencer, L.A. and Weller, P.F. FASEB J., 26(5), 2084-2093 (2012)   Rapid secretion of eosinophil-associated RNases (EARs), such as the human eosinophilic cationic protein (ECP), from intracellular granules is central to the role of eosinophils in allergic diseases and host immunity. Our knowledge regarding allergic inflammation has advanced based on mouse experimental models. However, unlike human eosinophils, capacities of mouse eosinophils to secrete granule proteins have been controversial. To study mechanisms of mouse eosinophil secretion and EAR release, we combined an RNase assay of mouse EARs with ultrastructural studies. In vitro, mouse eosinophils stimulated with the chemokine eotaxin-1 (CCL11) secreted enzymatically active EARs (EC₅₀ 5 nM) by piecemeal degranulation. In vivo, in a mouse model of allergic airway inflammation, increased airway eosinophil infiltration (24-fold) correlated with secretion of active RNases (3-fold). Moreover, we found that eosinophilic inflammation in mice can involve eosinophil cytolysis and release of cell-free granules. Cell-free mouse eosinophil granules expressed functional CCR3 receptors and secreted their granule proteins, including EAR and eosinophil peroxidase in response to CCL11. Collectively, these data demonstrate chemokine-dependent secretion of EARs from both intact mouse eosinophils and their cell-free granules, findings pertinent to understanding the pathogenesis of eosinophil-associated diseases, in which EARs are key factors.  

3.1811           Expression and subcellular distribution of gephyrin in non-neuronal tissues and cells

Nawrotzki, R., Islinger, M., Vogel, I., Völkl, A. and Kirsch, J. Histochem. Cell. Biol., 137(4), 471-482 (2012)   Gephyrin is a scaffolding protein required for the accumulation of inhibitory neurotransmitter receptors at neuronal postsynaptic membranes. In non-neuronal tissues, gephyrin is indispensible for the biosynthesis of molybdenum cofactor, the prosthetic group of oxidoreductases including sulfite oxidase and xanthine oxidase. However, the molecular and cellular basis of gephyrin’s non-neuronal function is poorly understood; in particular, the roles of its splice variants remain enigmatic. Here, we used cDNA screening as well as Northern and immunoblot analyses to show that mammalian liver contains only a limited number of gephyrin splice variants, with the C3-containing variant being the predominant isoform. Using new and established anti-gephyrin antibodies in immunofluorescence and subcellular fractionation studies, we report that gephyrin localizes to the cytoplasm of both tissue hepatocytes and cultured immortalized cells. These findings were corroborated by RNA interference studies in which the cytosolic distribution was found to be abolished. Finally, by blue-native PAGE we show that cytoplasmic gephyrin is part of a ~600 kDa protein complex of yet unknown composition. Our data suggest that the expression pattern of non-neuronal gephyrin is simpler than indicated by previous evidence. In addition, gephyrin’s presence in a cytosolic 600 kDa protein complex suggests that its metabolic and/or other non-neuronal functions are exerted in the cytoplasm and are not confined to a particular subcellular compartment.  

3.1812           Nesca, a novel neuronal adapter protein, links the molecular motor kinesin with the pre-synaptic membrane protein, syntaxin-1, in hippocampal neurons

MacDonald, J.I.S., Dietrich, A., Gamble, S., Hryciw, T., Grant, R.I. and Meakin, S.O.

1. Neurochem., 121(6), 861-880 (2012)

  Vesicular transport in neurons plays a vital role in neuronal function and survival. Nesca is a novel protein that we previously identified and herein describe its pattern of expression, subcellular localization and protein–protein interactions both in vitro and in vivo. Specifically, a large proportion of Nesca is in tight association with both actin and microtubule cytoskeletal proteins. Nesca binds to F-actin, microtubules, βIII and acetylated α-tubulin, but not neurofilaments or the actin-binding protein drebrin, in in vitro-binding assays. Nesca co-immunoprecipitates with kinesin heavy chain (KIF5B) and kinesin light-chain motors as well as with the synaptic membrane precursor protein, syntaxin-1, and is a constituent of the post-synaptic density. Moreover, in vitro-binding assays indicate that Nesca directly binds KIF5B, kinesin light-chain and syntaxin-1. In contrast, Nesca does not co-immunoprecipitate with the kinesin motors KIF1B, KIF3A nor does it bind syntaxin-4 or the synaptosome-associated protein 25 kDa (SNAP-25) in vitro. Nesca expression in neurons is highly punctuate, co-stains with syntaxin-1, and is found in fractions containing markers of early endosomes and Golgi suggesting that it is involved in vesicular transport. Collectively, these data suggest that Nesca functions as an adapter involved in neuronal vesicular transport including vesicles containing soluble N-ethylmaleimide sensitive factor attachment protein receptors that are essential to exocytosis.  

3.1813           Proteasomal degradation of the metabotropic glutamate receptor 1α is mediated by Homer-3 via the proteasomal S8 ATPase

Rezvani, K., Baalman, K., Teng, Y., Mee, M.P., Dawson, S.P., Wang, H., De Biasi, M. and Mayer, R.J.

1. Neurochem., 122(1), 24-37 (2012)

  The metabotropic glutamate receptors (mGluRs) fine-tune the efficacy of synaptic transmission. This unique feature makes mGluRs potential targets for the treatment of various CNS disorders. There is ample evidence to show that the ubiquitin proteasome system mediates changes in synaptic strength leading to multiple forms of synaptic plasticity. The present study describes a novel interaction between post-synaptic adaptors, long Homer-3 proteins, and one of the 26S proteasome regulatory subunits, the S8 ATPase, that influences the degradation of the metabotropic glutamate receptor 1α (mGluR1α). We have shown that the two human long Homer-3 proteins specifically interact with human proteasomal S8 ATPase. We identified that mGluR1α and long Homer-3s immunoprecipitate with the 26S proteasome both in vitro and in vivo. We further found that the mGluR1α receptor can be ubiquitinated and degraded by the 26S proteasome and that Homer-3A facilitates this process. Furthermore, the siRNA mediated silencing of Homer-3 led to increased levels of total and plasma membrane-associated mGluR1α receptors. These results suggest that long Homer-3 proteins control the degradation of mGluR1α receptors by shuttling ubiquitinated mGluR-1α receptors to the 26S proteasome via the S8 ATPase which may modulate synaptic transmission.    

3.1814           Rab3D regulates amylase levels, not agonist-induced amylase release, in AR42J cells

Limi, S., Ojakian, G., and Raffaniello, R. Cell. Mol. Biol. Lett., 17(2), 258-273 (2012)   Rab3D is a low molecular weight GTP-binding protein that associates with secretory granules in exocrine cells. AR42J cells are derived from rat pancreatic exocrine tumor cells and develop an acinar cell-like phenotype when treated with dexamethasone (Dex). In the present study, we examined the role of Rab3D in Dex-treated AR42J cells. Rab3D expression and localization were analyzed by subcellular fractionation and immunoblotting. The role of Rab3D was examined by overexpressing myc-labeled wild-type-Rab3D and a constitutively active form of Rab3D (Rab3D-Q81L) in AR42J cells. We found that Rab3D is predominantly membrane-associated in AR42J cells and co-localizes with zymogen granules (ZG). Following CCK-8-induced exocytosis, amylase-positive ZGs appeared to move towards the periphery of the cell and co-localization between Rab3D and amylase was less complete when compared to basal conditions. Overexpression of WT, but not mutant Rab3D, resulted in an increase in cellular amylase levels. Overexpression of mutant and WT Rab3D did not affect granule morphology, CCK-8-induced secretion, long-term (48 hr) basal amylase release or granule density. We conclude that Rab3D is not involved in agonist-induced exocytosis in AR42J cells. Instead, Rab3D may regulate amylase content in these cells.  

3.1815           Substrate-Immobilized HIV-1 Tat Drives VEGFR2/αvβ3–Integrin Complex Formation and Polarization in Endothelial Cells

Urbinati, C., Ravelli, C., Tanghetti, E., Belleri, M., Giacopuzzi, E., Monti, E., Presta, M. and Rusnati, M. Arterioscler. Thromb. Vasc. Biol., 32, e25-e34 (2012)   Objective—The HIV-1 transactivating factor (Tat) possesses features typical of both cell-adhesive and angiogenic growth factor (AGF) proteins, inducing endothelial cell (EC) adhesion and proangiogenic activation. Tat was exploited to investigate the events triggered by EC adhesion to substrate-bound AGF that lead to proangiogenic activation. Methods and Results—Immobilized Tat induces actin cytoskeleton organization, formation of α_vβ₃ integrin⁺focal adhesion plaques, and recruitment of vascular endothelial growth factor receptor-2 (VEGFR2) in the ventral plasma membrane of adherent ECs. Also, acceptor photobleaching fluorescence resonance energy transfer demonstrated that VEGFR2/α_vβ₃ coupling occurs at the basal aspect of Tat-adherent ECs. Cell membrane fractionation showed that a limited fraction of α_vβ₃ integrin and VEGFR2 does colocalize in lipid rafts at the basal aspect of Tat-adherent ECs. VEGFR2 undergoes phosphorylation and triggers pp60src/ERK_1/2 activation. The use of lipid raft disrupting agents and second messenger inhibitors demonstrated that intact lipid rafts and the VEGFR2/pp60src/ERK_1/2 pathway are both required for cytoskeleton organization and proangiogenic activation of Tat-adherent ECs. Conclusion—Substrate-immobilized Tat causes VEGFR2/α_vβ₃ complex formation and polarization at the basal aspect of adherent ECs, VEGFR2/pp60src/ERK_1/2 phosphorylation, cytoskeleton organization, and proangiogenic activation. These results provide novel insights in the AGF/tyrosine kinase receptor/integrin cross-talk.  

3.1816           Serum albumin disrupts Cryptococcus neoformans and Bacillus anthracis extracellular vesicles

Wolf, J.M., Rivera, J and Casadevall, A. Cell. Microbiol., 14(5), 762-773 (2012)   For both pathogenic fungi and bacteria, extracellular vesicles have been shown to contain many microbial components associated with virulence, suggesting a role in pathogenesis. However, there are many unresolved issues regarding vesicle synthesis and stability, including the fact that vesicular packaging for extracellular factors involved in virulence must also have a mechanism for vesicle unloading. Consequently, we studied the kinetics of vesicle production and stability using [1-¹⁴C] palmitic acid metabolic labelling and dynamic light scattering techniques. Cryptococcus neoformans vesicles were produced throughout all stages of fungal culture growth and they were stable once isolated. Density gradient analysis revealed that only a portion of the vesicle population carried cryptococcal polysaccharide, implying heterogeneity in vesicular cargo. Vesicle incubation with macrophages resulted in rapid vesicle instability, a phenomenon that was ultimately associated with serum albumin. Additionally, albumin, along with mouse serum and murine immunoglobulin destabilized Bacillus anthracis vesicles, but the effect was not observed with ovalbumin or keyhole limpet haemocyanin, demonstrating that this phenomenon is neither host-, microbe- nor protein-specific. Our findings strongly suggest that cryptococcal vesicles are short-lived in vivo and vesicle destabilization is mediated by albumin. The ability of albumin to promote vesicular offload through destabilization indicates a new activity for this abundant serum protein.  

3.1817           Kaposi’s sarcoma-associated herpesvirus interacts with EphrinA2 receptor to amplify signaling essential for productive infection

Chakraborty, S., Valiya, M., Bottero, V. and Chandran, B. PNAS Plus, 109, E1163-E1172 (2012)   Kaposi’s sarcoma-associated herpesvirus (KSHV), etiologically associated with Kaposi’s sarcoma, uses integrins (α3β1, αVβ3, and αVβ5) and associated signaling to enter human dermal microvascular endothelial cells (HMVEC-d), an in vivo target of infection. KSHV infection activated c-Cbl, which induced the selective translocation of KSHV into lipid rafts (LRs) along with the α3β1, αVβ3, and xCT receptors, but not αVβ5. LR-translocated receptors were monoubiquitinated, leading to productive macropinocytic entry, whereas non-LR–associated αVβ5 was polyubiquitinated, leading to clathrin-mediated entry that was targeted to lysosomes. Because the molecule(s) that integrate signal pathways and productive KSHV macropinocytosis were unknown, we immunoprecipitated KSHV-infected LR fractions with anti-α3β1 antibodies and analyzed them by mass spectrometry. The tyrosine kinase EphrinA2 (EphA2), implicated in many cancers, was identified in this analysis. EphA2 was activated by KSHV. EphA2 was also associated with KSHV and integrins (α3β1 and αVβ3) in LRs early during infection. Preincubation of virus with soluble EphA2, knockdown of EphA2 by shRNAs, or pretreatment of cells with anti-EphA2 monoclonal antibodies or tyrosine kinase inhibitor dasatinib significantly reduced KSHV entry and gene expression. EphA2 associates with c-Cbl-myosin IIA and augmented KSHV-induced Src and PI3-K signals in LRs, leading to bleb formation and macropinocytosis of KSHV. EphA2 shRNA ablated macropinocytosis-associated signaling events, virus internalization, and productive nuclear trafficking of KSHV DNA. Taken together, these studies demonstrate that the EphA2 receptor acts as a master assembly regulator of KSHV-induced signal molecules and KSHV entry in endothelial cells and suggest that the EphA2 receptor is an attractive target for controlling KSHV infection.  

3.1818           Generation and Nuclear Translocation of Sumoylated Transmembrane Fragment of Cell Adhesion Molecule L1

Lutz, D., Wolters-Eisfeld, G., Joshi, G., Djogo, N., Jakovcevski, I., Schachner, M. and Kleene, R.

1. Biol. Chem., 287(21), 17161-17175 (2012)

  The functions of the cell adhesion molecule L1 in the developing and adult nervous system are triggered by homophilic and heterophilic interactions that stimulate signal transductions that activate cellular responses. Here, we show that stimulation of signaling by function-triggering L1 antibodies or L1-Fc leads to serine protease-dependent cleavage of full-length L1 at the plasma membrane and generation of a sumoylated transmembrane 70-kDa fragment comprising the intracellular and transmembrane domains and part of the extracellular domain. The 70-kDa transmembrane fragment is transported from the plasma membrane to a late endosomal compartment, released from endosomal membranes into the cytoplasm, and transferred from there into the nucleus by a pathway that depends on importin and chromatin-modifying protein 1. Mutation of the sumoylation site at Lys¹¹⁷² or of the nuclear localization signal at Lys¹¹⁴⁷ abolished L1-stimulated generation or nuclear import of the 70-kDa fragment, respectively. Nuclear import of the 70-kDa fragment may activate cellular responses in parallel or in association with phosphorylation-dependent signaling pathways. Alterations in the levels of the 70-kDa fragment during development and in the adult after spinal cord injury or in a mouse model of Alzheimer disease suggest that this fragment is functionally implicated in development, regeneration, neurodegeneration, tumorigenesis, and possibly synaptic plasticity in the mature nervous system.  

3.1819           Phospholipase A2 activating protein is required for 1α,25-dihydroxyvitamin D3 dependent rapid activation of protein kinase C via Pdia3

Doroudi, M., Schwartz, Z. and Boyan, B.D.

1. Steroid Biochem. Mol. Biol., 132, 48-56 (2012)

  1α,25-Dihydroxyvitamin D₃ (1,25D3) regulates musculoskeletal cells via two different mechanisms: vitamin D receptor (VDR)-dependent gene transcription and rapid membrane-signaling via VDR as well as protein disulfide isomerase, family A, member 3 (Pdia3). In chondrocytes from the costochondral cartilage growth zone (GC), ligand binding to Pdia3 causes a rapid increase in phospholipase A₂ (PLA₂) activity leading to release of arachidonic acid and formation of lysophospholipid (LPL). LPL activates phospholipase C (PLC), and resulting inositol trisphosphate (IP₃) and diacylglycerol contribute to PKCα activation and downstream activation of ERK1/2. PLA₂ activating protein (PLAA) is increased in the growth zone of rat growth plates suggesting that it mediates the 1,25D3-dependent pathway. This study examined the role of PLAA in mediating 1,25D3-dependent PKC activation using GC cells and MC3T3-E1 wild-type and PLAA-silenced osteoblasts as models. PLAA, Pdia3, and caveolin-1 (Cav-1) were detected in plasma membranes and caveolae of GC and MC3T3-E1 cells. Pdia3-immunoprecipitated samples were positive for PLAA only after 1,25D3 treatment. Cav-1 was detected when immunoprecipitated with anti-Pdia3 and anti-PLAA in both vehicle and 1,25D3 treated cells. These observations were confirmed by immunohistochemistry. 1,25D3 failed to activate PLA₂ and PKC or cause PGE₂ release in PLAA-silenced cells. PLAA-antibody successfully blocked the PLAA protein and consequently suppressed PKC activity in GC and MC3T3-E1 cells. Crosslinking studies confirmed the localization of PLAA on the extracellular face on the plasma membrane in untreated MC3T3-E1 cells. Taken together, our results suggest that PLAA is an important mediator of 1α,25(OH)₂D₃ rapid membrane mediated signaling. 1α,25(OH)₂D₃ likely causes conformational changes bringing Pdia3 into proximity with PLAA, and aiding in transducing the signal from caveolae to the plasma membrane.  

3.1820           Polyunsaturated fatty acid supplements modulate mast cell membrane microdomain composition

Basiouni, S., Stöckel, K., Fuhrmann, H. and Schumann, J. Cellular Immunol., 275, 42-46 (2012)   In the present study, the lipid raft composition of a canine mastocytoma cell line (C2) was analyzed. Lipid rafts were well separated from non-raft plasma membranes using a detergent-free isolation technique. To study the influence of n-3 and n-6 polyunsaturated fatty acids (PUFA) on raft fatty acid composition in comparison to non-raft cell membrane, C2 were supplemented with one of the following: α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid or arachidonic acid. Enrichment of the culture medium with a specific PUFA resulted in an increase in the content of this fatty acid both in rafts and non-raft membranes. Contents of cholesterol and protein were found not to be affected by the changes in the fatty acid profiles. In conclusion, our data provide strong evidence that PUFA modulate lipid composition and physiological properties of membrane micro domains of mast cells which in turn may have effects on mast cell function.  

3.1821           Proteomic Analysis of Rta2p-Dependent Raft-Association of Detergent-Resistant Membranes in Candida albicans

Wang, L., Jia, Y., Tang, R-J., Xu, Z., Cao, Y-B., Jia, X-M. and Jiang, Y-Y. Plos One, 7(5), e37768 (2012)   In Candida albicans, lipid rafts (also called detergent-resistant membranes, DRMs) are involved in many cellular processes and contain many important proteins. In our previous study, we demonstrated that Rta2p was required for calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Here, we found that Rta2p was co-localized with raft-constituted ergosterol on the plasma membrane of C. albicans. Furthermore, this membrane expression pattern was totally disturbed by inhibitors of either ergosterol or sphingolipid synthesis. Biochemical fractionation of DRMs together with immunoblot uncovered that Rta2p, along with well-known DRM-associated proteins (Pma1p and Gas1p homologue), was associated with DRMs and their associations were blocked by inhibitors of either ergosterol or sphingolipid synthesis. Finally, we used the proteomic analysis together with immunoblot and identified that Rta2p was required for the association of 10 proteins with DRMs. These 5 proteins (Pma1p, Gas1p homologue, Erg11p, Pmt2p and Ali1p) have been reported to be DRM-associated and also that Erg11p is a well-known target of azoles in C. albicans. In conclusion, our results showed that Rta2p was predominantly localized in lipid rafts and was required for the association of certain membrane proteins with lipid rafts in C. albicans.  

3.1822           Identification of a Wnt-induced protein complex by affinity proteomics using an antibody that recognizes a sub-population of β-catenin

Layton, M.J., Faux, M.C., Church, N:L., Catimel, B., Kershaw, N:J., Kapp, E.A., Nowell, C., Coates, J.L., Burgess, A.W. and Simpson, R.J. Biochim. Biophys. Acta, 1824, 925-937 (2012)   β-catenin is a signaling protein with diverse functions in cell adhesion and Wnt signaling. Although β-catenin has been shown to participate in many protein–protein interactions, it is not clear which combinations of β-catenin-interacting proteins form discrete complexes. We have generated a novel antibody, termed 4B3, which recognizes only a small subset of total cellular β-catenin. Affinity proteomics using 4B3, in combination with subcellular fractionation, has allowed us to define a discrete trimeric complex of β-catenin, α-catenin and the tumor suppressor APC, which forms in the cytoplasm in response to Wnt signaling. Depletion of the limiting component of this complex, APC, implicates the complex in mediating Wnt-induced changes in cell–cell adhesion. APC is also essential for N-terminal phosphorylation of β-catenin within this complex. Each component of β-catenin/APC/α-catenin complex co-exists in other protein complexes, thus use of a selective antibody for affinity proteomics has allowed us to go beyond the generation of a list of potential β-catenin-interacting proteins, and define when and where a specific complex forms.  

3.1823           Impaired neurotransmission in ether lipid-deficient nerve terminals

Brodde, A., Teigler, A., Brugger, B., Lehmann, W.D., Wieland, F., Berger, J. and Just, W.W. Hum. Mol. Genet., 21(12), 2713-2724 (2012)   Isolated defects of ether lipid (EL) biosynthesis in humans cause rhizomelic chondrodysplasia punctata type 2 and type 3, serious peroxisomal disorders. Using a previously described mouse model [Rodemer, C., Thai, T.P., Brugger, B., Kaercher, T., Werner, H., Nave, K.A., Wieland, F., Gorgas, K., and Just, W.W. (2003) Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice. Hum. Mol. Genet., 12, 1881–1895], we investigated the effect of EL deficiency in isolated murine nerve terminals (synaptosomes) on the pre-synaptic release of the neurotransmitters (NTs) glutamate and acetylcholine. Both Ca²⁺-dependent exocytosis and Ca²⁺-independent efflux of the transmitters were affected. EL-deficient synaptosomes respire at a reduced rate and exhibit a lowered adenosin-5′-triphosphate/adenosine diphosphate (ATP/ADP) ratio. Consequently, ATP-driven processes, such as synaptic vesicle cycling and maintenance of Na⁺, K⁺ and Ca²⁺ homeostasis, might be disturbed. Analyzing reactive oxygen species in EL-deficient neural and non-neural tissues revealed that plasmalogens (PLs), the most abundant EL species in mammalian central nervous system, considerably contribute to the generation of the lipid peroxidation product malondialdehyde. Although EL-deficient tissue contains less lipid peroxidation products, fibroblasts lacking ELs are more susceptible to induced oxidative stress. In summary, these results suggest that due to the reduced energy state of EL-deficient tissue, the Ca²⁺-independent efflux of NTs increases while the Ca²⁺-dependent release declines. Furthermore, lack of PLs is mainly compensated for by an increase in the concentration of phosphatidylethanolamine and results in a significantly lowered level of lipid peroxidation products in the brain cortex and cerebellum.  

3.1824           Mutagenesis of the DI/DIII Linker in Dengue Virus Envelope Protein Impairs Viral Particle Assembly

De Wispelaere, M. and Yang, P.L.

1. Virol., 86(13), 7072-7083 (2012)

  The dengue virus (DV) envelope (E) protein is important in mediating viral entry and assembly of progeny virus during cellular infection. Domains I and III (DI and DIII, respectively) of the DV E protein are connected by a highly conserved but poorly ordered region, the DI/DIII linker. Although the flexibility of the DI/DIII linker is thought to be important for accommodating the structural rearrangements undergone by the E protein during viral entry, the function of the linker in the DV infectious cycle is not well understood. In this study, we performed site-directed mutagenesis on conserved residues in the DI/DIII linker of the DV2 E protein and showed that the resulting mutations had little or no effect on the entry process but greatly affected virus assembly. Biochemical fractionation and immunofluorescence microscopy experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the DI/DIII linker mutants express the DV structural proteins at the sites of particle assembly near the ER but fail to form infectious particles. This defect is not due to disruption of E's interaction with prM and pr in immature and mature virions, respectively. Serial passaging of the DV2 mutant E-Y299F led to the identification of a mutation in the membrane-proximal stem region of E that fully compensates for the assembly defect of this DI/DIII linker mutant. Together, our results suggest a critical and previously unidentified role for the E protein DI/DIII linker region during the DV2 assembly process.  

3.1825           Human Herpesvirus 8 Interferon Regulatory Factor-Mediated BH3-Only Protein Inhibition via Bid BH3-B Mimicry

Choi, Y.B., Sandford, G. and Nicholas, J. PloS Pathogens, 8(6), e1002748 (2012)   Viral replication efficiency is in large part governed by the ability of viruses to counteract pro-apoptotic signals induced by infection of host cells. For HHV-8, viral interferon regulatory factor-1 (vIRF-1) contributes to this process in part via inhibitory interactions with BH3-only protein (BOP) Bim, recently identified as an interaction partner of vIRF-1. Here we recognize that the Bim-binding domain (BBD) of vIRF-1 resembles a region (BH3-B) of Bid, another BOP, which interacts intramolecularly with the functional BH3 domain of Bid to inhibit it pro-apoptotic activity. Indeed, vIRF-1 was found to target Bid in addition to Bim and to interact, via its BBD region, with the BH3 domain of each. In functional assays, BBD could substitute for BH3-B in the context of Bid, to suppress Bid-induced apoptosis in a BH3-binding-dependent manner, and vIRF-1 was able to protect transfected cells from apoptosis induced by Bid. While vIRF-1 can mediate nuclear sequestration of Bim, this was not the case for Bid, and inhibition of Bid and Bim by vIRF-1 could occur independently of nuclear localization of the viral protein. Consistent with this finding, direct BBD-dependent inactivation by vIRF-1 of Bid-induced mitochondrial permeabilization was demonstrable in vitro and isolated BBD sequences were also active in this assay. In addition to Bim and Bid BH3 domains, BH3s of BOPs Bik, Bmf, Hrk, and Noxa also were found to bind BBD, while those of both pro- and anti-apoptotic multi-BH domain Bcl-2 proteins were not. Finally, the significance of Bid to virus replication was demonstrated via Bid-depletion in HHV-8 infected cells, which enhanced virus production. Together, our data demonstrate and characterize BH3 targeting and associated inhibition of BOP pro-apoptotic activity by vIRF-1 via Bid BH3-B mimicry, identifying a novel mechanism of viral evasion from host cell defenses.  

3.1826           Regulation in the targeting of TRAIL receptor 1 to cell surface via GODZ for TRAIL sensitivity in tumor cells

Oh, Y., Jeon, Y-J., Hong, G-S., Kim, I., Woo, H-N. and Jung, Y-K. Cell Death and Differentiation, 19(7), 1196-1207 (2012)   Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5), promote the selective clearing of various malignancies by inducing apoptosis, holding the promise as a potent therapeutic agent for anticancer. Though DR4 and DR5 have high sequence similarity, differential regulation of both receptors in human tumor cells remains largely unexplored. Here, we repot that golgi-specific Asp-His-His-Cys (DHHC) zinc finger protein (GODZ) regulates TRAIL/DR4-mediated apoptosis. Using the SOS protein recruitment–yeast two-hybrid screening, we isolated GODZ that interacted with the death domain of DR4. GODZ binds to DR4, but not to DR5, through the DHHC and the C-terminal transmembrane domain. Expression level of GODZ affects apoptosis of tumor cells triggered by TRAIL, but not that induced by TNF-α/cycloheximide (CHX) or DNA-damaging drugs. In parallel, GODZ functions to localize DR4 to the plasma membrane (PM) via DHHC motif. Also, introduction of mutation into the cysteine-rich motif of DR4 results in its mistargeting and attenuates TRAIL- or GODZ-mediated apoptosis. Interestingly, GODZ expression is highly downregulated in Hep-3B tumor cells, which show resistance to TRAIL. However, reconstitution of GODZ expression enhances the targeting of DR4 to cell surface and sensitizes Hep-3B cells to TRAIL. Taken together, these data establish that GODZ is a novel DR4-selective regulator responsible for targeting of DR4 to the PM, and thereby for TRAIL-induced apoptosis.  

3.1827           Loss of P-type ATPase ATP13A2/PARK9 function induces general lysosomal deficiency and leads to Parkinson disease neurodegeneration

Dehay, B., Ramirez, A., Martinez-Vicente, M., Perier, C., Canron, M-H., Doudnikoff, E., Vital, A., Vila, M., Klein, C. and Bezard, E. PNAS, 109(24), 9611-9616 (2011)   Parkinson disease (PD) is a progressive neurodegenerative disorder pathologically characterized by the loss of dopaminergic neurons from the substantia nigra pars compacta and the presence, in affected brain regions, of protein inclusions named Lewy bodies (LBs). The ATP13A2 gene (locus PARK9) encodes the protein ATP13A2, a lysosomal type 5 P-type ATPase that is linked to autosomal recessive familial parkinsonism. The physiological function of ATP13A2, and hence its role in PD, remains to be elucidated. Here, we show that PD-linked mutations in ATP13A2 lead to several lysosomal alterations in ATP13A2 PD patient-derived fibroblasts, including impaired lysosomal acidification, decreased proteolytic processing of lysosomal enzymes, reduced degradation of lysosomal substrates, and diminished lysosomal-mediated clearance of autophagosomes. Similar alterations are observed in stable ATP13A2-knockdown dopaminergic cell lines, which are associated with cell death. Restoration of ATP13A2 levels in ATP13A2-mutant/depleted cells restores lysosomal function and attenuates cell death. Relevant to PD, ATP13A2 levels are decreased in dopaminergic nigral neurons from patients with PD, in which ATP13A2 mostly accumulates within Lewy bodies. Our results unravel an instrumental role of ATP13A2 deficiency on lysosomal function and cell viability and demonstrate the feasibility and therapeutic potential of modulating ATP13A2 levels in the context of PD.  

3.1828           Mo1816 The NHE3 Interacting PDZ Protein NHERF2 is Strongly Lipid Raft-Associated and Determines the Raft Association of the Apical Na+/H+ Exchanger NHE3 in Murine Small Intestinal Brush Border Membrane

Sultan, A., Riederer, B., Xia, W., Lamprecht, G., Lissner, S., Yun, C., de JOnge, H., Gessner, J.E., Donowitz, M. and Seidler, U. Gastroenterology, 142(5), Suppl. 1, S691-S692 (2012)   Abstract not available  

3.1829           The GTPase ARFRP1 controls the lipidation of chylomicrons in the Golgi of the intestinal epithelium

Jaschke, A., Chung, B., Hesse, D., Kluge, R., Zahn, C., Moser, M., Petzke, K-J., Brigelius-Flohe, R., Puchkov, D., Koepsell, H., Heeren, J., Joost, H-G., and Schürmann, A. Hum. Mol. Genet., 21(14), 3128-3142 (2012)   The uptake and processing of dietary lipids by the small intestine is a multistep process that involves several steps including vesicular and protein transport. The GTPase ADP-ribosylation factor-related protein 1 (ARFRP1) controls the ARF-like 1 (ARL1)-mediated Golgi recruitment of GRIP domain proteins which in turn bind several Rab-GTPases. Here, we describe the essential role of ARFRP1 and its interaction with Rab2 in the assembly and lipidation of chylomicrons in the intestinal epithelium. Mice lacking Arfrp1 specifically in the intestine (Arfrp1^vil−/−) exhibit an early post-natal growth retardation with reduced plasma triacylglycerol and free fatty acid concentrations. Arfrp1^vil−/− enterocytes as well as Arfrp1 mRNA depleted Caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triacylglycerol content. In addition, the release of apolipoprotein A-I (ApoA-I) was dramatically decreased, and ApoA-I accumulated in the Arfrp1^vil−/− epithelium, where it predominantly co-localized with Rab2. The release of chylomicrons from Caco-2 was markedly reduced after the suppression of Rab2, ARL1 and Golgin-245. Thus, the GTPase ARFRP1 and its downstream proteins are required for the lipidation of chylo­microns and the assembly of ApoA-I to these particles in the Golgi of intestinal epithelial cells.  

3.1830           The Amyloid Precursor Protein Copper Binding Domain Histidine Residues 149 and 151 Mediate APP Stability and Metabolism

Spoerri, L., Vella, L.J., Pham, C.L.L., Barnham, K.J. and Cappai, R.

1. Biol. Chem., 287(32), 26840-26853 (2012)

  One of the key pathological hallmarks of Alzheimer disease (AD) is the accumulation of the APP-derived amyloid β peptide (Aβ) in the brain. Altered copper homeostasis has also been reported in AD patients and is thought to increase oxidative stress and to contribute to toxic Aβ accumulation and regulate APP metabolism. The potential involvement of the N-terminal APP copper binding domain (CuBD) in these events has not been investigated. Based on the tertiary structure of the APP CuBD, we examined the histidine residues of the copper binding site (His¹⁴⁷, His¹⁴⁹, and His¹⁵¹). We report that histidines 149 and 151 are crucial for CuBD stability and APP metabolism. Co-mutation of the APP CuBD His¹⁴⁹ and His¹⁵¹ to asparagine decreased APP proteolytic processing, impaired APP endoplasmic reticulum-to-Golgi trafficking, and promoted aberrant APP oligomerization in HEK293 cells. Expression of the triple H147N/H149N/H151N-APP mutant led to up-regulation of the unfolded protein response. Using recombinant protein encompassing the APP CuBD, we found that insertion of asparagines at positions 149 and 151 altered the secondary structure of the domain. This study identifies two APP CuBD residues that are crucial for APP metabolism and suggests an additional role of this domain in APP folding and stability besides its previously identified copper binding activity. These findings are of major significance for the design of novel AD therapeutic drugs targeting this APP domain.  

3.1831           Nascent high density lipoproteins formed by ABCA1 resemble lipid rafts and are structurally organized by three apoA-I monomers

Sorci-Thomas, M.G., Owen, J.S., Fulp, B., Bhat, S., Zhu, X., Parks, J.S., Shah, D., Jerome, W.G., Gerelus, M., Zabalawi, M. and Thomas, M.J.

1. Lipid Res., 53, 1890-1909 (2012)

  This report details the lipid composition of nascent HDL (nHDL) particles formed by the action of the ATP binding cassette transporter A1 (ABCA1) on apolipoprotein A-I (apoA-I). nHDL particles of different size (average diameters of ∼12, 10, 7.5, and <6 nm) and composition were purified by size-exclusion chromatography. Electron microscopy suggested that the nHDL were mostly spheroidal. The proportions of the principal nHDL lipids, free cholesterol, glycerophosphocholine, and sphingomyelin were similar to that of lipid rafts, suggesting that the lipid originated from a raft-like region of the cell. Smaller amounts of glucosylceramides, cholesteryl esters, and other glycerophospholipid classes were also present. The largest particles, ∼12 nm and 10 nm diameter, contained ∼43% free cholesterol, 2–3% cholesteryl ester, and three apoA-I molecules. Using chemical cross-linking chemistry combined with mass spectrometry, we found that three molecules of apoA-I in the ∼9–14 nm nHDL adopted a belt-like conformation. The smaller (7.5 nm diameter) spheroidal nHDL particles carried 30% free cholesterol and two molecules of apoA-I in a twisted, antiparallel, double-belt conformation. Overall, these new data offer fresh insights into the biogenesis and structural constraints involved in forming nascent HDL from ABCA1.  

3.1832           The major isoforms of Bim contribute to distinct biological activities that govern the processes of autophagy and apoptosis in interleukin-7 dependent lymphocytes

Ruppert, S.M., Li, W., Zhang, G., Carlson, A.L., Limaye, A., Durum, S.K. and Khaled, A.R. Biochim. Biophys. Acta, 1823, 1877-1893 (2012)   Bim is a BH3-only member of the Bcl-2 family that enables the death of T-cells. Partial rescue of cytokine-deprived T-cells occurs when Bim and the receptor for the T-cell growth factor, interleukin-7, are deleted, implicating Bim as a possible target of interleukin-7-mediated signaling. Alternative splicing yields three major isoforms: BimEL, BimL and BimS. To study the effect of Bim deficiency and define the function of the major isoforms, Bim-containing and Bim-deficient T-cells, dependent on interleukin-7 for growth, were used. Loss of total Bim in interleukin-7-deprived T-cells resulted in delayed apoptosis. However, loss of Bim also impeded the later degradative phase of autophagy. p62, an autophagy-adaptor protein which is normally degraded, accumulated in Bim deficient cells. To explain this, BimL was found to support acidification of lysosomes that later may associate with autophagic vesicles. Key findings showed that inhibition of lysosomal acidification accelerated death upon interleukin-7 withdrawal only in Bim-containing T-cells. intereukin-7 dependent T-cells lacking Bim were less sensitive to inhibition of lysosomal acidification. BimL co-immunoprecipitated with dynein and Lamp1-containing vesicles, indicating BimL could be an adaptor for dynein to facilitate loading of lysosomes. In Bim deficient T-cells, lysosome-tracking probes revealed vesicles of less acidic pH. Over-expression of BimL restored acidic vesicles in Bim deficient T-cells, while other isoforms, BimEL and BimS, promoted intrinsic cell death. These results reveal a novel role for BimL in lysosomal positioning that may be required for the formation of degradative autolysosomes.      

3.1833           Melittin initiates dopamine transporter internalization and recycling in transfected HEK-293 cells

Keith, D.J., Wolfrum, K., Eshleman, A.J. and Janowsky, A. Eur. J. Pharmacol., 690, 13-21 (2012)   The dopamine transporter removes the neurotransmitter from the synapse, regulating dopamine availability. The transporter can be internalized and its function is blocked by cocaine and other ligands. Melittin inhibits dopamine transporter function and causes internalization of the recombinant transporter in stably transfected HEK-293 cells, but the specific pathways for internalization and disposition of the transporter are unknown. Here we report that melittin treatment increased both transporter internalization and colocalization with clathrin, effects that were blocked by pretreatment with cocaine. Density gradient centrifugation revealed that melittin treatment caused the dopamine transporter to associate with a density fraction containing the early endosome marker Rab 5A. Confocal microscopy revealed that melittin treatment also increased transporter colocalization with Rab 5A and decreased colocalization with the late endosome marker Rab 7 and the recycling endosome marker Rab 11. Following 60 min of melittin treatment, the transporter was trafficked back to the membrane. By comparison, phorbol ester treatment increased transporter colocalization with early endosome antigen 1 and Rab 7 in a time-dependent manner. Cocaine treatment alone does not affect transporter trafficking in these cells. Results indicate multiple dopamine transporter internalization and recycling pathways that depend on transporter-ligand interactions and post-translational modifications.  

3.1834           Elucidating the pre- and post-nuclear intracellular processing of 1,4-dihydropyridine based gene delivery carriers

Hyvönen, Z., Hämäläinen, V., Ruponen, M., Lucas, B., Rejman, J., Vercauteren, D., Demeester, J., De Smedt, S. and Braeckmans, K.

1. Controlled Release, 162, 167-175 (2012)

  The low transfection efficacy of non-viral gene delivery systems limits the therapeutic application of these vectors. Besides the inefficient release of the complexes or pDNA from endolysosomes into the cytoplasm or poor nuclear uptake, the nuclear and post-nuclear processing might unfavorably affect the transgene expression. Positively charged amphiphilic 1,4-dihydropyridine (1,4-DHP) derivatives were earlier proposed as a promising tool for the delivery of DNA into target cells in vitro and in vivo. However, the structure/activity relationship of these carriers is poorly understood as yet. In this work we studied the intracellular processing of complexes, composed of three structurally related 1,4-DHP derivatives, in a retinal pigment epithelial (ARPE-19) cell line. The pre- and post-nuclear processing of the complexes was quantified on the nuclear, mRNA and transgene expression level. Here we show that the interaction of 1,4-DHP complexes with the cell membrane temporarily increases the permeability of the ARPE-19 cell membrane for small molecular compounds. However, the main mechanism for internalization of 1,4-DHP complexes is endocytosis. We found that all examined derivatives are able to destabilize endosomal membranes by lipid exchange upon acidification. In addition, the buffering capacity of some of the compounds may contribute to the endosomal escape of the complexes as well through the proton sponge effect. Previously we reported that cellular uptake of 1,4-DHP complexes does not correlate with transgene expression. In this study we surprisingly revealed that there is no correlation between the amount of plasmids taken up by the cell and the amount of plasmids found in the cell nucleus. Furthermore, it was found that a high amount of plasmid in the nucleus does not ensure high mRNA expression, likely due to remaining interactions of the carrier with the plasmids. Neither did the expression of mRNA always result in the production of a functional protein, possibly due to the interaction of free carrier with intracellular components which are involved in the post-translational modification of protein and folding process. Overall, our data suggest that succeeding of both the pre- and the post-nuclear intracellular processes is equally essential for successful transgene expression.  

3.1835           N-linked glycosylation of proline-rich membrane anchor (PRiMA) is not required for assembly and trafficking of globular tetrameric acetylcholinesterase

Chan, W.K.B., Chen, V.P., Luk, W.K.W., Choi, R.C.Y. and Tsim, K.W.K. Neuroscience Lett., 523, 71-75 (2012)   Acetylcholinesterase (AChE) is organized into globular tetramers (G₄) by a structural protein called proline-rich membrane anchor (PRiMA), anchoring it into the cell membrane of neurons in the brain. The assembly of AChE tetramers with PRiMA requires the presence of a C-terminal “t-peptide” in the AChE catalytic subunit (AChE_T). The glycosylation of AChE_T is known to be required for its proper assembly and trafficking; however, the role of PRiMA glycosylation in the oligomer assembly has not been revealed. PRiMA is a glycoprotein containing two putative N-linked glycosylation sites. By using site-directed mutagenesis, the asparagine-43 was identified to be the N-linked glycosylation site of PRiMA. Abolishing glycosylation on mouse PRiMA appeared not to affect its assembly with AChE_T, the enzymatic properties of AChE, and the membrane trafficking of PRiMA-linked AChE tetramers. This result is contrary to the reports that glycosylation is essential for conformation and trafficking of membrane glycoproteins.  

3.1836           Src kinase-mediates androgen receptor-dependent non-genomic activation of signaling cascade leading to endothelial nitric oxide synthase

Yu, J., Akishita, M., Eto, M., Koizumi, H., Hashimoto, R., Ogawa, S., Tanaka, K., Ouchi, Y. and Okabe, T. Biochem. Biophys. Res. Comm., 424, 538-543 (2012)   Our previous study has demonstrated that testosterone rapidly activates endothelial nitric oxide synthase (eNOS), enhancing nitric oxide (NO) release from endothelial cells (ECs) via the phosphatidylinositol 3-kinase/Akt (PI3-kinase/Akt) pathway. The upstream regulators of this pathway are unknown. In this study, we further investigated the non-genomic action of testosterone in human aortic ECs. Acute (30 min) activation of eNOS caused by testosterone was unaffected by pretreatment with a transcriptional inhibitor, actinomycin D. Non-permeable testosterone-BSA rapidly induced Akt and eNOS phosphorylation. In contrast, luciferase reporter assay showed that the transcriptional activity of the androgen-responsive element (ARE) was increased by testosterone, but not by testosterone-BSA at 2 h after stimulation. Immunostaining displayed co-localization of androgen receptor (AR) with caveolin-1. Fractional analysis showed that AR was expressed in caveolae-enriched membrane fractions. Immunoprecipitation assays revealed the association of AR with caveolin-1 and c-Src, suggesting complex formation among them. Testosterone rapidly increased the phosphorylation of c-Src on Tyr416, which was inhibited by an AR antagonist and by siRNA for AR. PP2, a specific-inhibitor of Src kinase, abolished the testosterone-induced phosphorylation of Akt and eNOS. Our data indicate that testosterone induces rapid assembly of a membrane signaling complex among AR, caveolin-1 and c-Src, which then facilitates activation of the c-Src/ PI3-kinase/Akt cascade, resulting in activation of eNOS.  

3.1837           Pseudo half-molecules of the ABC transporter, COMATOSE, bind Pex19 and target to peroxisomes independently but are both required for activity

Nyathi, Y., Zhang, X., Baldwin, J.M., Bernhardt, K., Johnson, B., Baldwin, S.A., Theodoulou, F.L. and Baker, A. FEBS Lett., 586, 2280-2286 (2012)   Peroxisomal ABC transporters of animals and fungi are “half-size” proteins which dimerise to form a functional transporter. However, peroxisomal ABC transporters of land plants are synthesised as a single polypeptide which represents a fused heterodimer. The N- and C-terminal pseudo-halves of COMATOSE (CTS; AtABCD1) were expressed as separate polypeptides which bound Pex19 in vitro and targeted independently to the peroxisome membrane in yeast, where they were stable but not functional. When co-expressed, the pseudo-halves were fully functional as indicated by ATPase activity and rescue of the pxa1pxa2Δ mutant for growth on oleate. The functional significance of heterodimer asymmetry is discussed.  

3.1838           Atg9 vesicles are an important membrane source during early steps of autophagosome formation

Yamamoto, H., Kakuta, S., Watanabe, T.M., Kitamura, A., Sekito, T., Kondo-Kakuta, C., Ichikawa, R., Kinjo, M. and Ohsumi, Y.

1. Cell Biol., 198(2), 219-233 (2012)

  During the process of autophagy, cytoplasmic materials are sequestered by double-membrane structures, the autophagosomes, and then transported to a lytic compartment to be degraded. One of the most fundamental questions about autophagy involves the origin of the autophagosomal membranes. In this study, we focus on the intracellular dynamics of Atg9, a multispanning membrane protein essential for autophagosome formation in yeast. We found that the vast majority of Atg9 existed on cytoplasmic mobile vesicles (designated Atg9 vesicles) that were derived from the Golgi apparatus in a process involving Atg23 and Atg27. We also found that only a few Atg9 vesicles were required for a single round of autophagosome formation. During starvation, several Atg9 vesicles assembled individually into the preautophagosomal structure, and eventually, they are incorporated into the autophagosomal outer membrane. Our findings provide conclusive linkage between the cytoplasmic Atg9 vesicles and autophagosomal membranes and offer new insight into the requirement for Atg9 vesicles at the early step of autophagosome formation.  

3.1839           Sphingolipids Regulate the Yeast High-Osmolarity Glycerol Response Pathway

Tanigawa, M., Kihara, A., Terashima, M., Takahara, T. and Maeda, T. Mol. Cell. Biol., 32(14), 2861-2870 (2012)   The yeast high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase pathway is activated in response to hyperosmotic stress via two independent osmosensing branches, the Sln1 branch and the Sho1 branch. While the mechanism by which the osmosensing machinery activates the downstream MAP kinase cascade has been well studied, the mechanism by which the machinery senses and responds to hyperosmotic stress remains to be clarified. Here we report that inhibition of the de novo sphingolipid synthesis pathway results in activation of the HOG pathway via both branches. Inhibition of ergosterol biosynthesis also induces activation of the HOG pathway. Sphingolipids and sterols are known to be tightly packed together in cell membranes to form partitioned domains called rafts. Raft-enriched detergent-resistant membranes (DRMs) contain both Sln1 and Sho1, and sphingolipid depletion and hyperosmotic stress have similar effects on the osmosensing machinery of the HOG pathway: dissociation of an Sln1-containing protein complex and elevated association of Sho1 with DRMs. These observations reveal the sphingolipid-mediated regulation of the osmosensing machinery of the HOG pathway.  

3.1840           Disruption of Nongenomic Testosterone Signaling in a Model of Spinal and Bulbar Muscular Atrophy

Schindler, M., Fabre, C., de Weille, J., Carreau, S., Mersel, M. and Bakalara, N. Mol. Endocrinol., 26(7), 1102-1116 (2012)   As one of the nine hereditary neurodegenerative polyQ disorders, spinal and bulbar muscular atrophy (SBMA) results from a polyQ tract expansion in androgen receptor (AR). Although protein aggregates are the pathological hallmark of many neurodegenerative diseases, their direct role in the neurodegeneration is more and more questioned. To determine the early molecular mechanisms causing motor neuron degeneration in SBMA, we established an in vitro system based on the tetracycline-inducible expression of normal (AR20Q), the mutated, 51 glutamine-extended (AR51Q), or polyQ-deleted (AR0Q) AR in NSC34, a motor neuron-like cell line lacking endogenous AR. Although no intracellular aggregates were formed, the expression of the AR51Q leads to a loss of function characterized by reduced neurite outgrowth and to a toxic gain of function resulting in decreased cell viability. In this study, we show that both AR20Q and AR51Q are recruited to lipid rafts in response to testosterone stimulation. However, whereas testosterone induces the activation of the c-jun N-terminal kinase/c-jun pathway via membrane-associated AR20Q, it does not so in NSC34 expressing AR51Q. Phosphorylation of c-jun N-terminal kinase plays a crucial role in AR20Q-dependent survival and differentiation of NSC34. Moreover, c-jun protein levels decrease more slowly in AR20Q- than in AR51Q-expressing NSC34 cells. This is due to a rapid and transient inhibition of glycogen synthase kinase 3α occurring in a phosphatidylinositol 3-kinase-independent manner. Our results demonstrate that the deregulation of nongenomic AR signaling may be involved in SBMA establishment, opening new therapeutic perspectives.  

3.1841           The N-Terminal, Polybasic Region of PrPC Dictates the Efficiency of Prion Propagation by Binding to PrPSc

Turnbaugh, J.A., Unterberger, U., Saa, P., Massignan, T., Fluharty, B.R., Bowman, F.P., Miller, M.B., Supattapone, S., Biasini, E. and Harris, D.A.

1. Neurosci., 32(26), 8817-8830 (2012)

  Prion propagation involves a templating reaction in which the infectious form of the prion protein (PrP^Sc) binds to the cellular form (PrP^C), generating additional molecules of PrP^Sc. While several regions of the PrP^C molecule have been suggested to play a role in PrP^Sc formation based on in vitro studies, the contribution of these regions in vivo is unclear. Here, we report that mice expressing PrP deleted for a short, polybasic region at the N terminus (residues 23–31) display a dramatically reduced susceptibility to prion infection and accumulate greatly reduced levels of PrP^Sc. These results, in combination with biochemical data, demonstrate that residues 23–31 represent a critical site on PrP^C that binds to PrP^Sc and is essential for efficient prion propagation. It may be possible to specifically target this region for treatment of prion diseases as well as other neurodegenerative disorders due to β-sheet-rich oligomers that bind to PrP^C.

3.1842           A Cytotoxic Type III Secretion Effector of Vibrio parahaemolyticus Targets Vacuolar H+-ATPase Subunit c and Ruptures Host Cell Lysosomes

Matsuda, S., Okada, N., Kodama, T., Honda, T. and Iida, T. PloS Pathogens, 8(7), e1002803 (2012)   Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1). VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus. However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H⁺-ATPase (V-ATPase), the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepAΔC), which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death.  

3.1843           Alteration of EGFR Spatiotemporal Dynamics Suppresses Signal Transduction

Turk, H.F., Barhoumi, R. and Chapkin, R.S. PloS One, 7(6), e39682 (2012)   The epidermal growth factor receptor (EGFR), which regulates cell growth and survival, is integral to colon tumorigenesis. Lipid rafts play a role in regulating EGFR signaling, and docosahexaenoic acid (DHA) is known to perturb membrane domain organization through changes in lipid rafts. Therefore, we investigated the mechanistic link between EGFR function and DHA. Membrane incorporation of DHA into immortalized colonocytes altered the lateral organization of EGFR. DHA additionally increased EGFR phosphorylation but paradoxically suppressed downstream signaling. Assessment of the EGFR-Ras-ERK1/2 signaling cascade identified Ras GTP binding as the locus of the DHA-induced disruption of signal transduction. DHA also antagonized EGFR signaling capacity by increasing receptor internalization and degradation. DHA suppressed cell proliferation in an EGFR-dependent manner, but cell proliferation could be partially rescued by expression of constitutively active Ras. Feeding chronically-inflamed, carcinogen-injected C57BL/6 mice a fish oil containing diet enriched in DHA recapitulated the effects on the EGFR signaling axis observed in cell culture and additionally suppressed tumor formation. We conclude that DHA-induced alteration in both the lateral and subcellular localization of EGFR culminates in the suppression of EGFR downstream signal transduction, which has implications for the molecular basis of colon cancer prevention by DHA.  

3.1844           A Role for Dendritic Translation of CaMKIIα mRNA in Olfactory Plasticity

Neant-Fery, M., Peres, E., Nashrallah, C., Kessner, M., Gribaudo, S., Greer, C., Didier, A., trembleau, A. and Caille, I. PloS One, 7(6), e40133 (2012)   Local protein synthesis in dendrites contributes to the synaptic modifications underlying learning and memory. The mRNA encoding the α subunit of the calcium/calmodulin dependent Kinase II (CaMKIIα) is dendritically localized and locally translated. A role for CaMKIIα local translation in hippocampus-dependent memory has been demonstrated in mice with disrupted CaMKIIα dendritic translation, through deletion of CaMKIIα 3′UTR. We studied the dendritic localization and local translation of CaMKIIα in the mouse olfactory bulb (OB), the first relay of the olfactory pathway, which exhibits a high level of plasticity in response to olfactory experience. CaMKIIα is expressed by granule cells (GCs) of the OB. Through in situ hybridization and synaptosome preparation, we show that CaMKIIα mRNA is transported in GC dendrites, synaptically localized and might be locally translated at GC synapses. Increases in the synaptic localization of CaMKIIα mRNA and protein in response to brief exposure to new odors demonstrate that they are activity-dependent processes. The activity-induced dendritic transport of CaMKIIα mRNA can be inhibited by an NMDA receptor antagonist and mimicked by an NMDA receptor agonist. Finally, in mice devoid of CaMKIIα 3′UTR, the dendritic localization of CaMKIIα mRNA is disrupted in the OB and olfactory associative learning is severely impaired. Our studies thus reveal a new functional modality for CaMKIIα local translation, as an essential determinant of olfactory plasticity.  

3.1845           The Permeability of Reconstituted Nuclear Pores Provides Direct Evidence for the Selective Phase Model

Hülsmann, B.B., Labokha, A.A. and Görlich, D. Cell, 150(4), 738-751 (2012)   Nuclear pore complexes (NPCs) maintain a permeability barrier between the nucleus and the cytoplasm through FG-repeat-containing nucleoporins (Nups). We previously proposed a selective phase model in which the FG repeats interact with one another to form a sieve-like barrier that can be locally disrupted by the binding of nuclear transport receptors (NTRs), but not by inert macromolecules, allowing selective passage of NTRs and associated cargo. Here, we provide direct evidence for this model in a physiological context. By using NPCs reconstituted from Xenopus laevis egg extracts, we show that Nup98 is essential for maintaining the permeability barrier. Specifically, the multivalent cohesion between FG repeats is required, including cohesive FG repeats close to the anchorage point to the NPC scaffold. Our data exclude alternative models that are based solely on an interaction between the FG repeats and NTRs and indicate that the barrier is formed by a sieve-like FG hydrogel.  

3.1846           Medulloblastoma Exosome Proteomics Yield Functional Roles for Extracellular Vesicles

Epple, L.M., Griffiths, S.G., Dechkovskaia, A.M., Dusto, N.L., White, J., Ouellette, R.J., Anchordoquy, T.J., Bemis, L.T. and Graner, M.W. PloS One, 7(7), e42064 (2012)   Medulloblastomas are the most prevalent malignant pediatric brain tumors. Survival for these patients has remained largely the same for approximately 20 years, and our therapies for these cancers cause significant health, cognitive, behavioral and developmental sequelae for those who survive the tumor and their treatments. We obviously need a better understanding of the biology of these tumors, particularly with regard to their migratory/invasive behaviors, their proliferative propensity, and their abilities to deflect immune responses. Exosomes, virus-sized membrane vesicles released extracellularly from cells after formation in, and transit thru, the endosomal pathway, may play roles in medulloblastoma pathogenesis but are as yet unstudied in this disease. Here we characterized exosomes from a medulloblastoma cell line with biochemical and proteomic analyses, and included characterization of patient serum exosomes. Further scrutiny of the proteomic data suggested functional properties of the exosomes that are relevant to medulloblastoma tumor biology, including their roles as proliferation stimulants, their activities as attractants for tumor cell migration, and their immune modulatory impacts on lymphocytes. Aspects of this held true for exosomes from other medulloblastoma cell lines as well. Additionally, pathway analyses suggested a possible role for the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A); however, inhibition of the protein’s activity actually increased D283MED cell proliferation/clonogenecity, suggesting that HNF4A may act as a tumor suppressor in this cell line. Our work demonstrates that relevant functional properties of exosomes may be derived from appropriate proteomic analyses, which translate into mechanisms of tumor pathophysiology harbored in these extracellular vesicles.  

3.1847           Association between Tetrodotoxin Resistant Channels and Lipid Rafts Regulates Sensory Neuron Excitability

Pristera, A., Baker, M.D. and Okuse, K. PloS One, 7(8), e40079 (2012)   Voltage-gated sodium channels (VGSCs) play a key role in the initiation and propagation of action potentials in neurons. Na_V1.8 is a tetrodotoxin (TTX) resistant VGSC expressed in nociceptors, peripheral small-diameter neurons able to detect noxious stimuli. Na_V1.8 underlies the vast majority of sodium currents during action potentials. Many studies have highlighted a key role for Na_V1.8 in inflammatory and chronic pain models. Lipid rafts are microdomains of the plasma membrane highly enriched in cholesterol and sphingolipids. Lipid rafts tune the spatial and temporal organisation of proteins and lipids on the plasma membrane. They are thought to act as platforms on the membrane where proteins and lipids can be trafficked, compartmentalised and functionally clustered. In the present study we investigated Na_V1.8 sub-cellular localisation and explored the idea that it is associated with lipid rafts in nociceptors. We found that Na_V1.8 is distributed in clusters along the axons of DRG neurons in vitro and ex vivo. We also demonstrated, by biochemical and imaging studies, that Na_V1.8 is associated with lipid rafts along the sciatic nerve ex vivo and in DRG neurons in vitro. Moreover, treatments with methyl-β-cyclodextrin (MβCD) and 7-ketocholesterol (7KC) led to the dissociation between rafts and Na_V1.8. By calcium imaging we demonstrated that the lack of association between rafts and Na_V1.8 correlated with impaired neuronal excitability, highlighted by a reduction in the number of neurons able to conduct mechanically- and chemically-evoked depolarisations. These findings reveal the sub-cellular localisation of Na_V1.8 in nociceptors and highlight the importance of the association between Na_V1.8 and lipid rafts in the control of nociceptor excitability.  

3.1848           Respiratory Syncytial Virus Assembles into Structured Filamentous Virion Particles Independently of Host Cytoskeleton and Related Proteins

Shaikh, F.Y., Utley, T.J., Craven, R.E., Rogers, M.C., Lapierre, L.A., Goldenring, J.R. and Crowe Jr. J.E. PloS One, 7(7), e40826 (2012)   Respiratory syncytial virus (RSV) is a single-stranded RNA virus that assembles into viral filaments at the cell surface. Virus assembly often depends on the ability of a virus to use host proteins to accomplish viral tasks. Since the fusion protein cytoplasmic tail (FCT) is critical for viral filamentous assembly, we hypothesized that host proteins important for viral assembly may be recruited by the FCT. Using a yeast two-hybrid screen, we found that filamin A interacted with FCT, and mammalian cell experiments showed it localized to viral filaments but did not affect viral replication. Furthermore, we found that a number of actin-associated proteins also were excluded from viral filaments. Actin or tubulin cytoskeletal rearrangement was not necessary for F trafficking to the cell surface or for viral assembly into filaments, but was necessary for optimal viral replication and may be important for anchoring viral filaments. These findings suggest that RSV assembly into filaments occurs independently of actin polymerization and that viral proteins are the principal drivers for the mechanical tasks involved with formation of complex, structured RSV filaments at the host cell plasma membrane.  

3.1849           Characterization of the Early Steps of Human Parvovirus B19 Infection

Quattrocchi, S., Ruprecht, N., Bönsch, C., Bieli, S., Zürcher, C., Boller, K., Kempf, C. and Ros, C.

1. Virol., 86(17), 9274-9284 (2012)

  The early steps of human parvovirus B19 (B19V) infection were investigated in UT7/Epo cells. B19V and its receptor globoside (Gb4Cer) associate with lipid rafts, predominantly of the noncaveolar type. Pharmacological disruption of the lipid rafts inhibited infection when the drug was added prior to virus attachment but not after virus uptake. B19V is internalized by clathrin-dependent endocytosis and spreads rapidly throughout the endocytic pathway, reaching the lysosomal compartment within minutes, where a substantial proportion is degraded. B19V did not permeabilize the endocytic vesicles, indicating a mechanism of endosomal escape without apparent membrane damage. Bafilomycin A₁ (BafA1) and NH₄Cl, which raise endosomal pH, blocked the infection by preventing endosomal escape, resulting in a massive accumulation of capsids in the lysosomes. In contrast, in the presence of chloroquine (CQ), the transfer of incoming viruses from late endosomes to lysosomes was prevented; the viral DNA was not degraded; and the infection was boosted. In contrast to the findings for untreated or BafA1-treated cells, the viral DNA was progressively associated with the nucleus in CQ-treated cells, reaching a plateau by 3 h postinternalization, a time coinciding with the initiation of viral transcription. At this time, more than half of the total intracellular viral DNA was associated with the nucleus; however, the capsids remained extranuclear. Our studies provide the first insight into the early steps of B19V infection and reveal mechanisms involved in virus uptake, endocytic trafficking, and nuclear penetration.    

3.1850           ADP ribosylation adapts an ER chaperone response to short-term fluctuations in unfolded protein load

Chambers, J.E., Petrova, K., Tomba, G., Vendruscolo, M. and Ron, D.

1. Cell Biol., 198(3), 371-385 (2012)

  Gene expression programs that regulate the abundance of the chaperone BiP adapt the endoplasmic reticulum (ER) to unfolded protein load. However, such programs are slow compared with physiological fluctuations in secreted protein synthesis. While searching for mechanisms that fill this temporal gap in coping with ER stress, we found elevated levels of adenosine diphosphate (ADP)–ribosylated BiP in the inactive pancreas of fasted mice and a rapid decline in this modification in the active fed state. ADP ribosylation mapped to Arg470 and Arg492 in the substrate-binding domain of hamster BiP. Mutations that mimic the negative charge of ADP-ribose destabilized substrate binding and interfered with interdomain allosteric coupling, marking ADP ribosylation as a rapid posttranslational mechanism for reversible inactivation of BiP. A kinetic model showed that buffering fluctuations in unfolded protein load with a recruitable pool of inactive chaperone is an efficient strategy to minimize both aggregation and costly degradation of unfolded proteins.  

3.1851           Calpains mediate epithelial-cell death during mammary gland involution: mitochondria and lysosomal destabilization

Arnandis, T., Ferrer-Vicens, I., Garcia-Trevijano, E.R., Miralles, V.J., Garcia, C., Torres, L., Vina, J.R. and Zaragoza, R. Cell Death & Differentiation, 19(9), 1536-1548 (2012)   Our aim was to elucidate the physiological role of calpains (CAPN) in mammary gland involution. Both CAPN-1 and -2 were induced after weaning and its activity increased in isolated mitochondria and lysosomes. CAPN activation within the mitochondria could trigger the release of cytochrome c and other pro-apoptotic factors, whereas in lysosomes it might be essential for tissue remodeling by releasing cathepsins into the cytosol. Immunohistochemical analysis localized CAPNs mainly at the luminal side of alveoli. During weaning, CAPNs translocate to the lysosomes processing membrane proteins. To identify these substrates, lysosomal fractions were treated with recombinant CAPN and cleaved products were identified by 2D-DIGE. The subunit b₂ of the v-type H⁺ ATPase is proteolyzed and so is the lysosomal-associated membrane protein 2a (LAMP2a). Both proteins are also cleaved in vivo. Furthermore, LAMP2a cleavage was confirmed in vitro by addition of CAPNs to isolated lysosomes and several CAPN inhibitors prevented it. Finally, in vivo inhibition of CAPN1 in 72-h-weaned mice decreased LAMP2a cleavage. Indeed, calpeptin-treated mice showed a substantial delay in tissue remodeling and involution of the mammary gland. These results suggest that CAPNs are responsible for mitochondrial and lysosomal membrane permeabilization, supporting the idea that lysosomal-mediated cell death is a new hallmark of mammary gland involution.  

3.1852           Caveolae and propofol effects on airway smooth muscle

Grim, K.J., Abcejo, A.J., Barnes, A., Sathish, V., Smelter, D.F., Ford, G.C., Thompson, M.A., prakash, Y.S. and Pabelick, C.M. Br. J. Anaesth., 109(3), 444-453 (2012)   Background The i.v. anaesthetic propofol produces bronchodilatation. Airway relaxation involves reduced intracellular Ca²⁺ ([Ca²⁺]_i) in airway smooth muscle (ASM) and lipid rafts (caveolae), and constitutional caveolin proteins regulate [Ca²⁺]_i. We postulated that propofol-induced bronchodilatation involves caveolar disruption. Methods Caveolar fractions of human ASM cells were tested for propofol content. [Ca²⁺]_i responses of ASM cells loaded with fura-2 were performed in the presence of 10 µM histamine with and without clinically relevant concentrations of propofol (10 and 30 μM and intralipid control). Effects on sarcoplasmic reticulum (SR) Ca²⁺ release were evaluated in zero extracellular Ca²⁺ using the blockers Xestospongin C and ryanodine. Store-operated Ca²⁺ entry (SOCE) after SR depletion was evaluated using established techniques. The role of caveolin-1 in the effect of propofol was tested using small interference RNA (siRNA) suppression. Changes in intracellular signalling cascades relevant to [Ca²⁺]_i and force regulation were also evaluated. Results Propofol was present in ASM caveolar fractions in substantial concentrations. Exposure to 10 or 30 µM propofol form decreased [Ca²⁺]_i peak (but not plateau) responses to histamine by ∼40%, an effect persistent in zero extracellular Ca²⁺. Propofol effects were absent in caveolin-1 siRNA-transfected cells. Inhibition of ryanodine receptors prevented propofol effects on [Ca²⁺]_i, while propofol blunted [Ca²⁺]_i responses to caffeine. Propofol reduced SOCE, an effect also prevented by caveolin-1 siRNA. Propofol effects were associated with decreased caveolin-1 expression and extracellular signal-regulated kinase phosphorylation. Conclusions These novel data suggest a role for caveolae (specifically caveolin-1) in propofol-induced bronchodilatation. Due to its lipid nature, propofol may transiently disrupt caveolar regulation, thus altering ASM [Ca²⁺]_i.  

3.1853           T-Cadherin Is an Auxiliary Negative Regulator of EGFR Pathway Activity in Cutaneous Squamous Cell Carcinoma: Impact on Cell Motility

Kyriakakis, E., Maslova, K., Philippova, M., Pfaff, D., Joshi, M.B., Buechner, S.A., Erne, P. and resink, T.J.

1. Invest. Dermatol., 132(9), 2275-2285 (2012)

  Genetic and epigenetic studies in different cancers, including cutaneous carcinomas, have implicated T-cadherin (T-cad) as a tumor suppressor. Immunohistochemical and in vitro studies have suggested that T-cad loss promotes incipient invasiveness in cutaneous squamous cell carcinoma (SCC). Molecular mechanisms are unknown. This study found that the main consequence of T-cad silencing in SCC is facilitation of ligand-dependent EGFR activation, whereas T-cad overexpression impedes EGFR activation. Gain- and loss-of-function studies in A431 SCC cells demonstrate T-cad-controlled responsiveness to EGF with respect to pharmacological inhibition of EGFR and to diverse signaling and functional events of the EGFR activation cascade (EGFR phosphorylation, internalization, nuclear translocation, cell retraction/de-adhesion, motility, invasion, integrin β1, and Rho small GTPases such as RhoA, Rac1, and Cdc42 activation). Further, T-cad modulates the EGFR pathway activity by influencing membrane compartmentalization of EGFR; T-cad upregulation promotes retention of EGFR in lipid rafts, whereas T-cad silencing releases EGFR from this compartment, rendering EGFR more accessible to ligand stimulation. This study reveals a mechanism for fine-tuning of EGFR activity in SCC, whereby T-cad represents an auxiliary “negative” regulator of the EGFR pathway, which impacts invasion-associated behavioral responses of SCC to EGF. This action of T-cad in SCC may serve as a paradigm explaining other malignancies displaying concomitant T-cad loss and enhanced EGFR activity.  

3.1854           Evidence for a dual function of EphB4 as tumor promoter and suppressor regulated by the absence or presence of the ephrin-B2 ligand

Rutkowski, R., Mertens-Walker, I., Lisle, J.E., herington, A.C. and Stephenson, S-A. Int. J. Cancer, 131(5), E614-E624 (2012)   Overexpression of the receptor tyrosine kinase EphB4 is common in epithelial cancers and linked to tumor progression by promoting angiogenesis, increasing survival and facilitating invasion and migration. However, other studies have reported loss of EphB4 suggesting a tumor suppressor function in some cancers. These opposing roles may be regulated by (i) the presence of the primary ligand ephrin-B2 that regulates pathways involved in tumor suppression or (ii) the absence of ephrin-B2 that allows EphB4 signaling via ligand-independent pathways that contribute to tumor promotion. To explore this theory, EphB4 was overexpressed in the prostate cancer cell line 22Rv1 and the mammary epithelial cell line MCF-10A. Overexpressed EphB4 localized to lipid-rich regions of the plasma membrane and confirmed to be ligand-responsive as demonstrated by increased phosphorylation of ERK1/2 and internalization. EphB4 overexpressing cells demonstrated enhanced anchorage-independent growth, migration and invasion, all characteristics associated with an aggressive phenotype, and therefore supporting the hypothesis that overexpressed EphB4 facilitates tumor promotion. Importantly, these effects were reversed in the presence of ephrin-B2 which led to a reduction in EphB4 protein levels, demonstrating that ligand-dependent signaling is tumor suppressive. Furthermore, extended ligand stimulation caused a significant decrease in proliferation that correlated with a rise in caspase-3/7 and -8 activities. Together, these results demonstrate that overexpression of EphB4 confers a transformed phenotype in the case of MCF-10A cells and an increased metastatic phenotype in the case of 22Rv1 cancer cells and that both phenotypes can be restrained by stimulation with ephrin-B2, in part by reducing EphB4 levels.  

3.1855           Syndecan–syntenin–ALIX regulates the biogenesis of exosomes

Baietti, M.F., Zhang, Z., Mortier, E., Melchior, A., Degeest, G., Geeraerts, A., Ivarsson, Y., Depoortyere, F., Coomans, C., Vermeiren, E., Zimmermann, P. and David, G. Nature Cell Biol., 14(7), 677-685 (2012)   The biogenesis of exosomes, small secreted vesicles involved in signalling processes, remains incompletely understood. Here, we report evidence that the syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin control the formation of exosomes. Syntenin interacts directly with ALIX through LYPX(n)L motifs, similarly to retroviral proteins, and supports the intraluminal budding of endosomal membranes. Syntenin exosomes depend on the availability of heparan sulphate, syndecans, ALIX and ESCRTs, and impact on the trafficking and confinement of FGF signals. This study identifies a key role for syndecan–syntenin–ALIX in membrane transport and signalling processes.  

3.1856           Regulation of Cell Migration by Sphingomyelin Synthases: Sphingomyelin in Lipid Rafts Decreases Responsiveness to Signaling by the CXCL12/CXCR4 Pathway

Asano, S., Kitani, K., Taniguchi, M., Hashimoto, M., Zama, K., Mitsutake, S., Igarashi, Y., Takeya, H., Kigawa, J., Hayashi, A., Umehara, H. and Okazaki, T. Mol. Cell. Biol., 32(16), 3242-3252 (2012)   Sphingomyelin synthase (SMS) catalyzes the formation of sphingomyelin, a major component of the plasma membrane and lipid rafts. To investigate the role of SMS in cell signaling and migration induced by binding of the chemokine CXCL12 to CXCR4, we used mouse embryonic fibroblasts deficient in SMS1 and/or SMS2 and examined the effects of SMS deficiency on cell migration. SMS deficiency promoted cell migration through a CXCL12/CXCR4-dependent signaling pathway involving extracellular signal-regulated kinase (ERK) activation. In addition, SMS1/SMS2 double-knockout cells had heightened sensitivity to CXCL12, which was significantly suppressed upon transfection with the SMS1 or SMS2 gene or when they were treated with exogenous sphingomyelin but not when they were treated with the SMS substrate ceramide. Notably, SMS deficiency facilitated relocalization of CXCR4 to lipid rafts, which form platforms for the regulation and transduction of receptor-mediated signaling. Furthermore, we found that SMS deficiency potentiated CXCR4 dimerization, which is required for signal transduction. This dimerization was significantly repressed by sphingomyelin treatment. Collectively, our data indicate that SMS-derived sphingomyelin lowers responsiveness to CXCL12, thereby reducing migration induced by this chemokine. Our findings provide the first direct evidence for an involvement of SMS-generated sphingomyelin in the regulation of cell migration.  

3.1857           R-SNARE ykt6 resides in membrane-associated protease-resistant protein particles and modulates cell cycle progression when over-expressed

Thayanidhi, N., Liang, Y., Hasegawa, H., Nycz, D.C., oorschot, V., Klumperman, J. and Hay, J.C. Biol. Cell, 104(7), 397-417 (2012)   Background information The arginine-type soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE) ykt6 possesses several atypical properties including selective high expression in neurons, a lipidated C-terminus, localization to punctae that do not correspond with known endomembrane markers, a potent ability to protect the secretory pathway from alpha-synuclein over-expression and specific up-regulation in tumors. We have followed up on several of these features that together suggest nontraditional SNARE structures and functions. Results A significant portion of ykt6 in PC12 cells was found in a protease-resistant state suggestive of a large complex or aggregate. Other endoplasmic reticulum/Golgi SNAREs were not protease resistant, demonstrating that SNARE complexes per se did not cause protease resistance. Mutagenesis indicated that lipidation of the ykt6 C-terminus was also not involved, implicating its longin domain in particle formation. Immunogold electron microscopy revealed ykt6 labeling of ∼100 nm electron densities associated with diverse membranes. Density gradient analysis of the protease-resistant structures confirmed their tight association with membranes. Since excess ykt6 has been correlated with tumorigenesis, we tested whether ykt6 over-expression in normal rat kidney cells that normally express little ykt6 affected the cell cycle. Ykt6 over-expression was found to result in altered cell division cycles as evidenced by significantly smaller cells, a higher mitotic index and increased DNA synthesis. Mutagenesis studies dis-correlated SNARE function with the cell cycle effects; instead, the cell cycle effects correlated better with ykt6 properties related to the longin domain or particle formation. Conclusions The ykt6 particles/aggregates may represent ykt6 engaged in a non-SNARE function(s) or else nonfunctional, stored and/or excess ykt6. Whether the particulate ykt6 structures represent a means of buffering the apparent proliferative activity or are in fact mechanistically related to this activity will be of future interest in neuroscience and cancer biology.  

3.1858           Identification of Autophagosome-associated Proteins and Regulators by Quantitative Proteomic Analysis and Genetic Screens

Dengjel, J., Høyer-hansden, M., Nielsen, M.O., Eisenberg, T., Harder, L.M., Schandorff, S., Farkas, T., Kirkegaard, T., Becker, A.C., Schroeder, S., Vanswelow, K., Lundberg, E., Nielsen, M.M., kristensen, A.R., Akimov, V., Bunkenborg, J., Madeo, F., Jäättelä, M. and Andersen, J.S. Mol. Cell. Proteomics, 11, 1-17 (2012)   Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection.  

3.1859           A Mechanism of Intracellular P2X Receptor Activation

Sivaramakrishnan, V. and Fountain, S.J.

1. Biol. Chem., 287(34), 28315-28326 (2012)

  P2X receptors (P2XRs) are ATP-activated calcium-permeable ligand-gated ion channels traditionally viewed as sensors of extracellular ATP during diverse physiological processes including pain, inflammation, and taste. However, in addition to a cell surface residency P2XRs also populate the membranes of intracellular compartments, including mammalian lysosomes, phagosomes, and the contractile vacuole (CV) of the amoeba Dictyostelium. The function of intracellular P2XRs is unclear and represents a major gap in our understanding of ATP signaling. Here, we exploit the genetic versatility of Dictyostelium to investigate the effects of physiological concentrations of ATP on calcium signaling in isolated CVs. Within the CV, an acidic calcium store, P2XRs are orientated to sense luminal ATP. Application of ATP to isolated vacuoles leads to luminal translocation of ATP and release of calcium. Mechanisms of luminal ATP translocation and ATP-evoked calcium release share common pharmacology, suggesting that they are linked processes. The ability of ATP to mobilize stored calcium is reduced in vacuoles isolated from P2X_AR knock-out amoeba and ablated in cells devoid of P2XRs. Pharmacological inhibition of luminal ATP translocation or depletion of CV calcium attenuates CV function in vivo, manifesting as a loss of regulatory cell volume decrease following osmotic swelling. We propose that intracellular P2XRs regulate vacuole activity by acting as calcium release channels, activated by translocation of ATP into the vacuole lumen.  

3.1860           High insulin levels are required for FAT/CD36 plasma membrane translocation and enhanced fatty acid uptake in obese Zucker rat hepatocytes

Buque, X., Cano, A., Miquilena-Colina, M.E., Garcia-Monzon, C., Ochoa, B. and Aspichueta, P.

Am. J. Physiol. Endocrinol. Metab., 303, E504-E514 (2012)

In myocytes and adipocytes, insulin increases fatty acid translocase (FAT)/CD36 translocation to theplasma membrane (PM), enhancing fatty acid (FA) uptake. Evidence links increased hepatic FAT/CD36 protein amount and gene expression with hyperinsulinemia in animal models and patients with fatty liver, but whether insulin regulates FAT/CD36 expression, amount, distribution, and function in hepatocytes is currently unknown. To investigate this, FAT/CD36 protein content in isolated hepatocytes, subfractions of organelles, and density-gradient isolated membrane subfractions was analyzed in obese and lean Zucker rats by Western blotting in liver sections by immunohistochemistry and in hepatocytes by immunocytochemistry. The uptake of oleate and oleate incorporation into lipids were assessed in hepatocytes at short time points (30–600 s). We found that FAT/CD36 protein amount at the PM was higher in hepatocytes from obese rats than from lean controls. In obese rat hepatocytes, decreased cytoplasmatic content of FAT/CD36 and redistribution from low- to middle- to middle- to high-density subfractions of microsomes were found. Hallmarks of obese Zucker rat hepatocytes were increased amount of FAT/CD36 protein at the PM and enhanced FA uptake and incorporation into triglycerides, which were maintained only when exposed to hyperinsulinemic conditions (80 mU/l). In conclusion, high insulin levels are required for FAT/CD36 translocation to the PM in obese rat hepatocytes to enhance FA uptake and triglyceride synthesis. These results suggest that the hyperinsulinemia found in animal models and patients with insulin resistance and fatty liver might contribute to liver fat accumulation by inducing FAT/CD36 functional presence at the PM of hepatocytes.

 

3.1861           GTP-Binding-Defective ARL4D Alters Mitochondrial Morphology and Membrane Potential

Li, C-C., Wu, T-S., Huang, C-F., Jang, L-T., Liu, Y-T., You, S-T., Liou, G-G. and Lee, F-J.S. PloS One, 7(8), e43552 (2012)   ARL4D, ARL4A, and ARL4C are closely related members of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of GTPases. All three ARL4 proteins contain nuclear localization signals (NLSs) at their C-termini and are primarily found at the plasma membrane, but they are also present in the nucleus and cytoplasm. ARF function and localization depends on their controlled binding and hydrolysis of GTP. Here we show that GTP-binding-defective ARL4D is targeted to the mitochondria, where it affects mitochondrial morphology and function. We found that a portion of endogenous ARL4D and the GTP-binding-defective ARL4D mutant ARL4D(T35N) reside in the mitochondria. The N-terminal myristoylation of ARL4D(T35N) was required for its localization to mitochondria. The localization of ARL4D(T35N) to the mitochondria reduced the mitochondrial membrane potential (ΔΨm) and caused mitochondrial fragmentation. Furthermore, the C-terminal NLS region of ARL4D(T35N) was required for its effect on the mitochondria. This study is the first to demonstrate that the dysfunctional GTP-binding-defective ARL4D is targeted to mitochondria, where it subsequently alters mitochondrial morphology and membrane potential.  

3.1862           N-linked glycosylation of dimeric acetylcholinesterase in erythrocytes is essential for enzyme maturation and membrane targeting

Luk, W.K.W., Chen, V.P., Choi, R.C. and Tsim, K.W.K. FEBS J., 279(12), 3229-3239 (2012)   Acetylcholinesterase (AChE) is well-known for its cholinergic functions in the nervous system; however, this enzyme is also found in other tissues where its function is still not understood. AChE is synthesized through alternative splicing as splicing variants, with isoforms including read-through (AChE_R), tailed (AChE_T) and hydrophobic (AChE_H). In human erythrocytes, AChE_H is a glycophosphatidylinositol-linked dimer on the plasma membrane. Three N-linked glycosylation sites have been identified in the catalytic domain of human AChE. Here, we investigate the roles of glycosylation in assembly and trafficking of human AChE_H. In transfected fibroblasts, expression of AChE_H was able to mimic the function of the dimeric form of AChE on the erythrocyte membrane. A glycan-depleted form was constructed by site-directed mutagenesis. By comparison with the wild-type AChE_H, the mutant had a much lower enzymatic activity and a much higher K_m value. In addition, the mutant was dimerized in the endoplasmic reticulum, but was not trafficked to the Golgi apparatus. The results suggest that the glycosylation may affect AChE_H enzymatic activity and trafficking, but not dimer formation. The present findings indicate the significance of N-glycosylation in controlling the biosynthesis of the AChE_H dimer form.  

3.1863           NEU4L sialidase overexpression promotes β-catenin signaling in neuroblastoma cells, enhancing stem-like malignant cell growth

Tringali, C., Cirillo, F., Lamorte, G., Papini, N., Anastasia, L., Lupo, B., Silvestri, I., Tettaamanti, G. and Venerando, B. Int. J. Cancer, 131(8), 1768-1778 (2012)   Neuroblastoma (NB) is a frequently lethal tumor that occurs in childhood and originates from embryonic neural crest cells. The malignant and aggressive phenotype of NB is strictly related to the deregulation of pivotal pathways governing the proliferation/differentiation status of neural crest precursor cells, such as MYCN, Delta/Notch and Wnt/β-catenin (CTNNB1) signaling. In this article, we demonstrate that sialidase NEU4 long (NEU4L) influences the differentiation/proliferation behavior of NB SK-N-BE cells by determining hyperactivation of the Wnt/β-catenin signaling pathway. NEU4L overexpression in SK-N-BE cells induced significant increases in active, nonphosphorylated β-catenin content, β-catenin/TCF transcriptional activity and β-catenin gene target expression including MYCN, MYC, CCND2 (cyclin D2) and CDC25A. In turn, these molecular features strongly modified the behavior of NEU4L SK-N-BE overexpressing cells, promoting the following: (1) an enhanced proliferation rate, mainly due to a faster transition from G1 to S phase in the cell cycle; (2) a more undifferentiated cell phenotype, which was similar to stem-like NB cells and possibly mediated by an increase of the expression of the pluripotency genes, MYC, NANOG, OCT-4, CD133 and NES (nestin); (3) the failure of NB cell differentiation after serum withdrawal. The molecular link between NEU4L and Wnt/β-catenin signaling appeared to rely most likely on the capability of the enzyme to modify the sialylation level of cell glycoproteins. These findings could provide a new candidate for therapeutic treatment.  

3.1864           The Role of Cholesterol in UV Light B-induced Apoptosis

George, K.S., Elyassaki, W., Wu, Q. and Wu, S. Photchem. Photobiol., 88(5), 1191-1197 (2012)   Modification of major lipid raft components, such as cholesterol and ceramide, plays a role in regulation of programmed cell death under various stimuli. However, the relationship between cholesterol level modification and the activation of apoptotic signaling cascades upon UVB light has not been established. In this report, we demonstrate that upon UVB irradiation cholesterol levels in membrane rafts of skin cells increase, which leads to Fas-receptor (Fas) aggregation in the rafts. Utilizing a continuous velocity floatation technique, we show that Fas accumulated in the lipid rafts of human melanoma M624 cells after UVB irradiation. The subsequent events of death-inducing signaling complex formation were also detected in the lipid raft fractions. Depletion of cholesterol by methyl-β-cyclodextrin reduces Fas aggregation, while overloading increases. Disruption of lipid rafts also prevents Fas death domain-associated protein (Daxx) from dissociating from Fas in the lipid rafts, which is accompanied with a reduced apoptotic, but increased nonapoptotic death of UVB-irradiated human keratinocytes, HaCaT cells. Results indicate that cholesterol located in the plasma membrane of skin cells is required for lipid raft domain formation and activation of UVB-induced apoptosis.  

3.1865           P-glycoprotein trafficking at the blood–brain barrier altered by peripheral inflammatory hyperalgesia

McCaffrey, G., Staatz, W.D., Sanchez-Covarrubias, L., Finch, J., DeMarco, K., Laracuente, M-L., Ronaldson, P.T. and Davis, T.P.

1. Neurochem., 122(5), 962-975 (2012)

  P-glycoprotein (ABCB1/MDR1, EC 3.6.3.44), the major efflux transporter at the blood–brain barrier (BBB), is a formidable obstacle to CNS pharmacotherapy. Understanding the mechanism(s) for increased P-glycoprotein activity at the BBB during peripheral inflammatory pain is critical in the development of novel strategies to overcome the significant decreases in CNS analgesic drug delivery. In this study, we employed the λ-carrageenan pain model (using female Sprague–Dawley rats), combined with confocal microscopy and subcellular fractionation of cerebral microvessels, to determine if increased P-glycoprotein function, following the onset of peripheral inflammatory pain, is associated with a change in P-glycoprotein trafficking which leads to pain-induced effects on analgesic drug delivery. Injection of λ-carrageenan into the rat hind paw induced a localized, inflammatory pain (hyperalgesia) and simultaneously, at the BBB, a rapid change in colocalization of P-glycoprotein with caveolin-1, a key scaffolding/trafficking protein. Subcellular fractionation of isolated cerebral microvessels revealed that the bulk of P-glycoprotein constitutively traffics to membrane domains containing high molecular weight, disulfide-bonded P-glycoprotein-containing structures that cofractionate with membrane domains enriched with monomeric and high molecular weight, disulfide-bonded, caveolin-1-containing structures. Peripheral inflammatory pain promoted a dynamic redistribution between membrane domains of P-glycoprotein and caveolin-1. Disassembly of high molecular weight P-glycoprotein-containing structures within microvascular endothelial luminal membrane domains was accompanied by an increase in ATPase activity, suggesting a potential for functionally active P-glycoprotein. These results are the first observation that peripheral inflammatory pain leads to specific structural changes in P-glycoprotein responsible for controlling analgesic drug delivery to the CNS.  

3.1866           Vaccinia Virus Virion Membrane Biogenesis Protein A11 Associates with Viral Membranes in a Manner That Requires the Expression of Another Membrane Biogenesis Protein, A6

Wu, X., Meng, X., Yan, B., Rose, L., Deng, J. and Xiang, Y.

1. Virol., 86(20), 11276-11286 (2012)

  A group of vaccinia virus (VACV) proteins, including A11, L2, and A6, are required for biogenesis of the primary envelope of VACV, specifically, for the acquisition of viral membrane precursors. However, the interconnection among these proteins is unknown and, with the exception of L2, the connection of these proteins with membranes is also unknown. In this study, prompted by the findings that A6 coprecipitated A11 and that the cellular distribution of A11 was dramatically altered by repression of A6 expression, we studied the localization of A11 in cells by using immunofluorescence and cell fractionation analysis. A11 was found to associate with membranes and colocalize with virion membrane proteins in viral replication factories during normal VACV replication. A11 partitioned almost equally between the detergent and aqueous phases upon Triton X-114 phase separation, demonstrating an intrinsic affinity with lipids. However, in the absence of infection or VACV late protein synthesis, A11 did not associate with cellular membranes. Furthermore, when A6 expression was repressed, A11 did not colocalize with any viral membrane proteins or associate with membranes. In contrast, when virion envelope formation was blocked at a later step by repression of A14 expression or by rifampin treatment, A11 colocalized with virion membrane proteins in the factories. Altogether, our data showed that A11 associates with viral membranes during VACV replication, and this association requires A6 expression. This study provides a physical connection between A11 and viral membranes and suggests that A6 regulates A11 membrane association.  

3.1867           Identification of Transmembrane Protein 134 as a Novel LMP1-Binding Protein by Using Bimolecular Fluorescence Complementation and an Enhanced Retroviral Mutagen

Talaty, P., Emery, A., Holthusen, K. and Everly Jr., D.N.

1. Virol., 86(20), 11345-11355 (2012)

  Latent membrane protein 1 (LMP1) of Epstein-Barr virus induces constitutive signaling in infected cells. LMP1 signaling requires oligomerization of LMP1 via its transmembrane domain, localization to lipid rafts in the membrane, and association of the LMP1 cytoplasmic domain to adaptor proteins, such as the tumor necrosis factor receptor-associated factors (TRAFs). Protein complementation is a novel technique to examine protein-protein interaction through the assembly of functional fluorescent proteins or enzymes from inactive fragments. A previous study in our lab demonstrated the use of bimolecular fluorescence complementation (BiFC) to study the assembly of the LMP1 signaling complexes within the plasma membrane of mammalian cells. In the present study, LMP1 was used as bait in a genome-wide BiFC screen with an enhanced retroviral mutagen to identify new LMP1-binding proteins. Our screen identified a novel LMP1-binding protein, transmembrane protein 134 (Tmem134). Tmem134 is a candidate oncogene that is amplified in breast cancer cell lines. Binding, colocalization, and cofractionation between LMP1 and Tmem134 were confirmed. Finally, Tmem134 affected LMP1-induced NF-κB induction. Together, these data suggest that BiFC is a unique and novel platform to identify proteins recruited to the LMP1-signaling complex.  

3.1868           Loss of Retinoschisin (RS1) Cell Surface Protein in Maturing Mouse Rod Photoreceptors Elevates the Luminance Threshold for Light-Driven Translocation of Transducin But Not Arrestin

Ziccardi, L., Vijayasarathy, C., Bush, R.A. and Sieving, P.A.

1. Neurosci., 32(38), 13010-13021 (2012)

  Loss of retinoschisin (RS1) in Rs1 knock-out (Rs1–KO) retina produces a post-photoreceptor phenotype similar to X-linked retinoschisis in young males. However, Rs1 is expressed strongly in photoreceptors, and Rs1–KO mice have early reduction in the electroretinogram a-wave. We examined light-activated transducin and arrestin translocation in young Rs1–KO mice as a marker for functional abnormalities in maturing rod photoreceptors. We found a progressive reduction in luminance threshold for transducin translocation in wild-type (WT) retinas between postnatal days P18 and P60. At P21, the threshold in Rs1–KO retinas was 10-fold higher than WT, but it decreased to <2.5-fold higher by P60. Light-activated arrestin translocation and re-translocation of transducin in the dark were not affected. Rs1–KO rod outer segment (ROS) length was significantly shorter than WT at P21 but was comparable with WT at P60. These findings suggested a delay in the structural and functional maturation of Rs1–KO ROS. Consistent with this, transcription factors CRX and NRL, which are fundamental to maturation of rod protein expression, were reduced in ROS of Rs1–KO mice at P21 but not at P60. Expression of transducin was 15–30% lower in P21 Rs1–KO ROS and transducin GTPase hydrolysis was nearly twofold faster, reflecting a 1.7- to 2.5-fold increase in RGS9 (regulator of G-protein signaling) level. Transduction protein expression and activity levels were similar to WT at P60. Transducin translocation threshold elevation indicates photoreceptor functional abnormalities in young Rs1–KO mice. Rapid reduction in threshold coupled with age-related changes in transduction protein levels and transcription factor expression are consistent with delayed maturation of Rs1–KO photoreceptors.  

3.1869           Assessing Heterogeneity of Peroxisomes: Isolation of Two Subpopulations from Rat Liver

Islinger, M., Abdolzade-Bavil, A., Liebler, S., Weber, G. and Völkl, A. Methods in Mol. Biol., 909, 83-96 (2012)   Peroxisomes exhibit a heterogeneous morphological appearance in rat liver tissue. In this respect, the isolation and subsequent biochemical characterization of peroxisome species from different subcellular prefractions should help to solve the question of whether peroxisomes indeed diverge into functionally specialized subgroups in one tissue. As a means to address this question, we provide a detailed separation protocol for the isolation of peroxisomes from both the light (LM-Po) and the heavy (HM-Po) mitochondrial prefraction for their subsequent comparative analysis. Both isolation strategies rely on centrifugation in individually adapted Optiprep gradients. In case of the heavy mitochondrial fraction, free flow electrophoresis is appended as an additional separation step to yield peroxisomes of suf ficient purity. In view of their morphology, peroxisomes isolated from both fractions are surrounded by a continuous single membrane and contain a gray-opaque inner matrix. However, beyond this overall similar appearance, HM-Po exhibit a smaller average diameter, fl oat at lower density, and show a more negative average membrane charge when compared to LM-Po.  

3.1870           NADPH oxidase activity in pollen tubes is affected by calcium ions, signaling phospholipids and Rac/Rop GTPases

Potocky, M., Pejhar, P., Gutkowska, M., Jimenez-Quesada, M.J., Potocka, A., de Dios Alche, J., Kost, B. and Zarsky, V.

1. Plant Physiol., 169, 1654-1663 (2012)

  Reactive oxygen species (ROS) generated by NADPH oxidase (NOX) are crucial for tip growth of pollen tubes. However, the regulation of NOX activity in pollen tubes remains unknown. Using purified plasma membrane fractions from tobacco and olive pollen and tobacco BY-2 cells, we demonstrate that pollen NOX is activated by calcium ions and low abundant signaling phospholipids, such as phosphatidic acid and phosphatidylinositol 4,5-bisphosphate in vitro and in vivo. Our data also suggest possible synergism between Ca²⁺ and phospholipid-mediated NOX activation in pollen. Rac/Rop small GTPases are also necessary for normal pollen tube growth and have been proposed to regulate ROS production in root hairs. We show here elevated ROS formation in pollen tubes overexpressing wild-type NtRac5 and constitutively active NtRac5, while overexpression of dominant-negative NtRac5 led to a decrease of ROS in pollen tubes. We also show that PA formed by distinct phospholipases D (PLD) is involved in pathways both upstream and downstream of NOX-mediated ROS generation and identify NtPLDδ as a PLD isoform acting in the ROS response pathway.  

3.1871           Single-Transmembrane Domain IGF-II/M6P Receptor: Potential Interaction with G Protein and Its Association with Cholesterol-Rich Membrane Domains

Amritraj, A., Posse de Chaves, E.I., Hawkes, C., MacDonald, R.G. and Kar, S. Endocrinology, 153(10), 4784-4798 (2012)   The IGF-II/mannose 6-phosphate (M6P) receptor is a single-transmembrane domain glycoprotein that plays an important role in the intracellular trafficking of lysosomal enzymes and endocytosis-mediated degradation of IGF-II. The receptor may also mediate certain biological effects in response to IGF-II binding by interacting with G proteins. However, the nature of the IGF-II/M6P receptor's interaction with the G protein or with G protein-coupled receptor (GPCR) interacting proteins such as β-arrestin remains unclear. Here we report that [¹²⁵I]IGF-II receptor binding in the rat hippocampal formation is sensitive to guanosine-5′-[γ-thio]triphosphate, mastoparan, and Mas-7, which are known to interfere with the coupling of the classical GPCR with G protein. Monovalent and divalent cations also influenced [¹²⁵I]IGF-II receptor binding. The IGF-II/M6P receptor, as observed for several GPCRs, was found to be associated with β-arrestin 2, which exhibits sustained ubiquitination after stimulation with Leu²⁷IGF-II, an IGF-II analog that binds rather selectively to the IGF-II/M6P receptor. Activation of the receptor by Leu²⁷IGF-II induced stimulation of extracellular signal-related kinase 1/2 via a pertussis toxin-dependent pathway. Additionally, we have shown that IGF-II/M6P receptors under normal conditions are associated mostly with detergent-resistant membrane domains, but after stimulation with Leu²⁷IGF-II, are translocated to the detergent-soluble fraction along with a portion of β-arrestin 2. Collectively these results suggest that the IGF-II/M6P receptor may interact either directly or indirectly with G protein as well as β-arrestin 2, and activation of the receptor by an agonist can lead to alteration in its subcellular distribution along with stimulation of an intracellular signaling cascade.  

3.1872           Proteolytic Processing Regulates Toll-like Receptor 3 Stability and Endosomal Localization

Qi, R., Singh, D. and Cheng Kao, C.

1. Biol. Chem., 287(39), 32617-32629 (2012)

  Toll-like receptors (TLRs) 3, 7, and 9 are innate immune receptors that recognize nucleic acids from pathogens in endosomes and initiate signaling transductions that lead to cytokine production. Activation of TLR9 for signaling requires proteolytic processing within the ectodomain by endosome-associated proteases. Whether TLR3 requires similar proteolytic processing to become competent for signaling remains unclear. Herein we report that human TLR3 is proteolytically processed to form two fragments in endosomes. Unc93b1 is required for processing by transporting TLR3 through the Golgi complex and to the endosomes. Proteolytic cleavage requires the eight-amino acid Loop1 within leucine-rich repeat 12 of the TLR3 ectodomain. Proteolytic cleavage is not required for TLR3 signaling in response to poly(I:C), although processing could modulate the degree of response toward viral double-stranded RNAs, especially in mouse cells. Both the full-length and cleaved fragments of TLR3 can bind poly(I:C) and are present in endosomes. However, although the full-length TLR3 has a half-life in HEK293T cells of 3 h, the cleaved fragments have half-lives in excess of 7 h. Inhibition of TLR3 cleavage by either treatment with cathepsin inhibitor or by a mutation in Loop1 decreased the abundance of TLR3 in endosomes targeted for lysosomal degradation.  

3.1873           Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes

Kushnavera, Y., Andreyev, A.Y., Kuwana, T. and Newmeyer, D.D. PloS Biology, 10(9), e1001394 (2012)   Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for “intrinsic” apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the “hit-and-run” hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ~28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event.  

3.1874           An improved procedure for isolation of functional synaptosomes for the transient generation of cybrids from frozen human brain

Castora, F.J. and Trevino, M.B. FASEB J., 26, 586.4 (2012)   Cybrids are cell lines derived from the fusion of mtDNA-depleted cells with cytoplasts or enucleated cells. To date, there are no reports of cybrid fusions from frozen human tissue. In this study, we developed a new method of synaptosome isolation, the "Frozen Brain Synaptosomes" or FBS method. Synaptosomes were isolated from fresh mouse, frozen mouse and frozen human brain by centrifugation in an iodixanol gradient. The effectiveness of our FBS method was compared with current synaptosome isolation procedures that have been utilized to make cybrid cell lines from fresh tissue. The FBS method yielded the most structurally intact synaptosome preparation from frozen tissue as judged by electron microscopy. We assessed cytochrome c oxidase and citrate synthase activity, phospho-synapsin-1 and total ATP levels. The FBS method yielded the greatest enrichment of intact synaptosomes, high protein, mitochondrial enzyme activity and ATP levels, and functional synaptosomes as defined by their indirect response to a phosphatase inhibitor okadaic acid (OKA). We expected that the synaptosomes isolated from frozen postmortem brain would be sufficient in quantity and quality to generate cybrid cells. From ten fusion experiments, we concluded that two putative cybrid cell lines contained the exogenous synaptosomal mitochondria based upon mtDNA sequence analysis. Research support was from the Virginia Center on Aging.  

3.1875           Overexpression of the Coq8 Kinase in Saccharomyces cerevisiae coq Null Mutants Allows for Accumulation of Diagnostic Intermediates of the Coenzyme Q6 Biosynthetic Pathway

Xie, L.X., Ozeir, M., Tang, J.Y., Chen, J.Y., Jaquinod, S-K., Fonteecave, M., Clarke, C.F. ansd Pierrel, F.

1. Biol. Chem., 287(28), 23571-23581 (2012)

  Most of the Coq proteins involved in coenzyme Q (ubiquinone or Q) biosynthesis are interdependent within a multiprotein complex in the yeast Saccharomyces cerevisiae. Lack of only one Coq polypeptide, as in Δcoq strains, results in the degradation of several Coq proteins. Consequently, Δcoq strains accumulate the same early intermediate of the Q₆ biosynthetic pathway; this intermediate is therefore not informative about the deficient biosynthetic step in a particular Δcoq strain. In this work, we report that the overexpression of the protein Coq8 in Δcoq strains restores steady state levels of the unstable Coq proteins. Coq8 has been proposed to be a kinase, and we provide evidence that the kinase activity is essential for the stabilizing effect of Coq8 in the Δcoq strains. This stabilization results in the accumulation of several novel Q₆ biosynthetic intermediates. These Q intermediates identify chemical steps impaired in cells lacking Coq4 and Coq9 polypeptides, for which no function has been established to date. Several of the new intermediates contain a C4-amine and provide information on the deamination reaction that takes place when para-aminobenzoic acid is used as a ring precursor of Q₆. Finally, we used synthetic analogues of 4-hydroxybenzoic acid to bypass deficient biosynthetic steps, and we show here that 2,4-dihydroxybenzoic acid is able to restore Q₆ biosynthesis and respiratory growth in a Δcoq7 strain overexpressing Coq8. The overexpression of Coq8 and the use of 4-hydroxybenzoic acid analogues represent innovative tools to elucidate the Q biosynthetic pathway.  

3.1876           Specific and Nonspecific Regulation of GPCR Function by Cholesterol

Gimpl, G. and Gehrig-Burger, K. Cholesterol Regulation of Ion Channels and Receptors, 205-230 (2012)   Cholesterol regulates the physical state of the phospholipid bilayer and is crucially involved in the formation of membrane microdomains. In view of the abundance of cholesterol in the plasma membrane of eukaryotic cells, any integral membrane protein should always be in close molecular contact with cholesterol. This is particularly true for the heptahelical G-protein-coupled receptors (GPCRs) that form the largest receptor superfamily. Owing to their seven transmembrane helices, large parts of these proteins are embedded in the cholesterol-rich plasma membrane bilayer. Some GPCRs have been shown to be functionally dependent on cholesterol (Table 10.1; Burger et al., 2000; Pucadyil and Chattopadhyay, 2006; Paila and Chattopadhyay, 2010). It is difficult to clarify whether such cholesterol dependence is based on direct interaction with cholesterol or on indirect effects caused by the influence of cholesterol on the biophysical state of the membrane, for example, changes in the membrane fluidity. The following questions (Sections 10.2–10.8) might be addressed in order to prove or to provide evidence whether a candidate GPCR is functionally dependent on cholesterol, and, if so, to what extent this is based on direct cholesterol–receptor interaction.  

3.1877           Rotaviral Enterotoxin Nonstructural Protein 4 Targets Mitochondria for Activation of Apoptosis during Infection

Bhowmick, R., Chandra Halder, U., Chattopadhyay, S., Chanda, S., Nandi, S., Bagchi, P., Nayak, M.K., Chakrabarti, O., Kobayashi, N. and Chawla-Sarkar, M.

1. Biol. Chem., 287(42), 35004-35020 (2012)

  Viruses have evolved to encode multifunctional proteins to control the intricate cellular signaling pathways by using very few viral proteins. Rotavirus is known to express six nonstructural and six structural proteins. Among them, NSP4 is the enterotoxin, known to disrupt cellular Ca²⁺ homeostasis by translocating to endoplasmic reticulum. In this study, we have observed translocation of NSP4 to mitochondria resulting in dissipation of mitochondrial membrane potential during virus infection and NSP4 overexpression. Furthermore, transfection of the N- and C-terminal truncated NSP4 mutants followed by analyzing NSP4 localization by immunofluorescence microscopy identified the 61–83-amino acid region as the shortest mitochondrial targeting signal. NSP4 exerts its proapoptotic effect by interacting with mitochondrial proteins adenine nucleotide translocator and voltage-dependent anion channel, resulting in dissipation of mitochondrial potential, release of cytochrome c from mitochondria, and caspase activation. During early infection, apoptosis activation by NSP4 was inhibited by the activation of cellular survival pathways (PI3K/AKT), because PI3K inhibitor results in early induction of apoptosis. However, in the presence of both PI3K inhibitor and NSP4 siRNA, apoptosis was delayed suggesting that the early apoptotic signal is initiated by NSP4 expression. This proapoptotic function of NSP4 is balanced by another virus-encoded protein, NSP1, which is implicated in PI3K/AKT activation because overexpression of both NSP4 and NSP1 in cells resulted in reduced apoptosis compared with only NSP4-expressing cells. Overall, this study reports on the mechanism by which enterotoxin NSP4 exerts cytotoxicity and the mechanism by which virus counteracts it at the early stage for efficient infection.  

3.1878           Compromised Mitochondrial Fatty Acid Synthesis in Transgenic Mice Results in Defective Protein Lipoylation and Energy Disequilibrium

Smith, S. et al Plos One, 7(10), e47196 (2012)   A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor.  

3.1879           MS-275 sensitizes osteosarcoma cells to Fas ligand-induced cell death by increasing the localization of Fas in membrane lipid rafts

Rao-Lindahl, K., Zhou, Z. and Kleinermann, E.S. Cell Death and Disease, 3, e369 (2012)   Fas expression is inversely correlated with the metastatic potential of osteosarcoma (OS) cells to the lungs. Fas⁺ cells are rapidly eliminated when they enter the lungs via their interaction with constitutive Fas ligand (FasL) on the lung epithelium, whereas Fas⁻ OS cells escape this FasL-induced apoptosis and survive in the lung microenvironment. Upregulation of Fas expression in established OS lung metastases results in tumor regression. Here, we demonstrate that treatment of Fas⁻ OS cells with the histone deacetylase inhibitor MS-275 results in the upregulation of Fas mRNA and sensitizes these cells to FasL-induced apoptosis. However, flow cytometry analysis revealed that Fas cell surface protein expression was not significantly increased. Rather, we observed increased levels of Fas within the membrane lipid rafts, as demonstrated by an increase in Fas expression in detergent-insoluble lipid raft fractions and colocalization with GM1⁺ lipid rafts. We had previously shown that MS-275 treatment inhibited expression of the anti-apoptotic cellular FLICE-inhibitory protein (c-FLIP). Here, we demonstrated that transfection of cells with short hairpin RNA to c-FLIP also resulted in the localization of Fas to lipid rafts. Overall, our studies indicate that MS-275 sensitizes OS cells to FasL by upregulating the expression of Fas in membrane lipid rafts, which correlates with the c-FLIP-dependent distribution of Fas to lipid rafts.  

3.1880           HDL and ApoA-I inhibit antigen presentation-mediated T cell activation by disrupting lipid rafts in antigen presenting cells

Wang, S-h., Yuan, S-g., Peng, D-q. and Zhao, S-p. Atherosclerosis, 225, 105-114 (2012)   Objective Depletion of cholesterol by methyl-β-cyclodextrin (MCD) on peptide-loaded antigen presenting cells (APCs) inhibits antigen presentation and T cell activation. However, whether membrane cholesterol efflux induced by high-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I) also results in inhibition of antigen presentation and T cell activation is still unknown. Methods and results Various types of APCs, including B cells, macrophages and dendritic cells (DCs), were first loaded with antigen, then incubated with HDL and apoA-I to decrease cellular membrane cholesterol content. After being treated with HDL and apoA-I, APCs demonstrated decreased potential to activate T cells, and this decrease correlated with an increase in cholesterol efflux from APCs. Cholesterol repletion reversed the inhibitory effects of HDL and apoA-I, demonstrating that the observed reduction in T cell proliferation is mediated through cholesterol. Furthermore, lipid raft analysis showed that HDL and apoA-I reduced cholesterol and major histocompatibility (MHC) class II protein content in lipid rafts, suggesting that cholesterol efflux from APCs to HDL and apoA-I inhibits antigen presentation and T cell activation by reducing lipid rafts assembly in APCs. Conclusion HDL and apoA-I inhibit the capacity of APCs to stimulate T cell activation, and this inhibition can be attributed to cholesterol efflux and the ensuing disruption of plasma membrane lipid rafts in APCs. Overall, these findings suggest that cholesterol efflux mediated by HDL and apoA-I may serve to link immunity and cardioprotection.  

3.1881           Characterizing Synaptic Vesicle Proteins Using Synaptosomal Fractions and Cultured Hippocampal Neurons

DiGiovanni, J., Sun, T. and Sheng, Z-H. Current Protocols in Neuroscience, Suppl. 59, 2.7.1-2.7.2 (2012)   Cloning and characterization of synaptic vesicle proteins and their binding counterparts on the presynaptic plasma membrane have greatly advanced our understanding of the molecular mechanisms involved in the synaptic vesicle cycle and neurotransmitter release. This unit discusses multidisciplinary approaches to characterize proteins from synaptosome-enriched subcellular fractions and localize them within cultured neurons. The first approach regroups methods used to isolate synaptic vesicles from rat brain synaptosomal preparations, allowing for specific biochemical investigation of synaptic vesicle proteins. The second is a detailed procedure for pre-embedding immunogold staining and electron microscopic observation, which permits the morphological identification of proteins in individual vesicles at intact synapses. Additionally, this chapter proposes methods for light microscopic examination of hippocampal neurons. It includes procedures for embryonic and postnatal hippocampal neuron culture and describes an immunocytochemical staining protocol used to investigate synaptic vesicle protein localization with respect to other proteins or subcellular structures.    

3.1882           Genetic blockage of endocytic pathways reveals differences in the intracellular processing of non-viral gene delivery systems

Ilina, P., Hyvonen, Z., Saura, M., Sandvig, K., Yliperttula, M. and Ruponen, M.

1. Controlled Release, 161, 385-395 (2012)

  Detailed understanding of the uptake mechanisms and intracellular processing of nonviral gene delivery systems will allow design of more effective carriers. This work gets insight into the intracellular kinetics of pDNA delivered by polyethyleneimine (PEI), cationic lipid DOTAP and calcium phosphate (CaP) precipitates. Amount of cell- and nuclear-associated pDNA was quantified by qRT-PCR at multiple time points after transfection. Moreover, the impact of specific endocytic pathways on the cell entry and intracellular kinetics of pDNA was studied by inhibition (blockage) of either clathrin- or dynamin-mediated endocytosis by using both genetically manipulated cell lines and chemical inhibitors of endocytosis. Quantitative analysis of defined kinetic parameters revealed that neither cellular nor nuclear uptake of pDNA correlated with transgene expression, emphasizing the importance of the post-nuclear processes in overall transfection efficacy. Changes in transgene expression observed upon blockage of endocytosis was carrier dependent and correlated relatively well with the changes at the cellular and nuclear uptake levels but not with the amount of cell-associated pDNA. Due to low specificity of chemical inhibitors and activation of alternative endocytosis pathways after genetic blockage of endocytosis neither of these methods is optimal for studying the role of endocytosis. Therefore, one should be careful when interpreting the obtained results from such studies and not to trust the data obtained only from one method.  

3.1883           Identification of Proteins Associated with the Pseudomonas aeruginosa Biofilm Extracellular Matrix

Toyofuku, M., Roschitzki, B., Riedel, K. and Eberl, L.

1. Proteome Res., 11, 4906-4915 (2012)

  Biofilms are surface-associated bacteria that are embedded in a matrix of self-produced polymeric substances (EPSs). The EPS is composed of nucleic acids, polysaccharides, lipids, and proteins. While polysaccharide components have been well studied, the protein content of the matrix is largely unknown. Here we conducted a comprehensive proteomic study to identify proteins associated with the biofilm matrix of Pseudomonas aeruginosa PAO1 (the matrix proteome). This analysis revealed that approximately 30% of the identified matrix proteins were outer membrane proteins, which are also typically found in outer membrane vesicles (OMVs). Electron microscopic inspection confirmed the presence of large amounts of OMVs within the biofilm matrix, supporting previous notions that OMVs are abundant constituents of P. aeruginosa biofilms. Our results demonstrate that while some proteins associated with the P. aeruginosa matrix are derived from secreted proteins and lysed cells, the large majority of the matrix proteins originate from OMVs. Furthermore, we demonstrate that the protein content of planktonic and biofilm OMVs is surprisingly different and may reflect the different physiological states of planktonic and sessile cells.  

3.1884           Pharmacological chaperones for human α-N-acetylgalactosaminidase

Clark, N.E., Metcalf, M.C., Best, D., Fleet, G.W.J. and Garman, S.C. PNAS, 109(43), 17400-17405 (2012)   Schindler/Kanzaki disease is an inherited metabolic disease with no current treatment options. This neurologic disease results from a defect in the lysosomal α-N-acetylgalactosaminidase (α-NAGAL) enzyme. In this report, we show evidence that the iminosugar DGJNAc can inhibit, stabilize, and chaperone human α-NAGAL both in vitro and in vivo. We demonstrate that a related iminosugar DGJ (currently in phase III clinical trials for another metabolic disorder, Fabry disease) can also chaperone human α-NAGAL in Schindler/Kanzaki disease. The 1.4- and 1.5-Å crystal structures of human α-NAGAL complexes reveal the different binding modes of iminosugars compared with glycosides. We show how differences in two functional groups result in >9 kcal/mol of additional binding energy and explain the molecular interactions responsible for the unexpectedly high affinity of the pharmacological chaperones. These results open two avenues for treatment of Schindler/Kanzaki disease and elucidate the atomic basis for pharmacological chaperoning in the entire family of lysosomal storage diseases.  

3.1885           Protein abundance of urea transporters and aquaporin 2 change differently in nephrotic pair-fed vs. non-pair-fed rats

Matar, R.N.M., Malik, B., Wang, X.H., Martin, C.F., Eaton, D.C., Sands, J.M. and Klein, J.D. Am. J. Physiol. Renal Physiol., 302(12), F1545-F1553 (2012)   Salt and water retention is a hallmark of nephrotic syndrome (NS). In this study, we test for changes in the abundance of urea transporters, aquaporin 2 (AQP2), Na-K-2Cl cotransporter 2 (NKCC2), and Na-Cl cotransporter (NCC), in non-pair-fed and pair-fed nephrotic animals. Doxorubicin-injected male Sprague-Dawley rats (n = 10) were followed in metabolism cages. Urinary excretion of protein, sodium, and urea was measured periodically. Kidney inner medulla (IM), outer medulla, and cortex tissue samples were dissected and analyzed for mRNA and protein abundances. At 3 wk, all doxorubicin-treated rats developed features of NS, with a ninefold increase in urine protein excretion (from 144 ± 21 to 1,107 ± 165 mg/day; P < 0.001) and reduced urinary sodium excretion (from 0.17 to 0.12 meq/day; P < 0.001). Urine osmolalities were reduced in the nephrotic animals (1,057 ± 37, treatment vs. 1,754 ± 131, control). Unlike animals fed ad libitum, UT-A1 protein abundance was unchanged in nephrotic pair-fed rats. Glycosylated AQP2 was reduced in the IM base of both nephrotic groups. Abundances of NKCC2 and NCC were consistently reduced (71 ± 7 and 33 ± 13%, respectively) in both nephrotic pair-fed animals and animals fed ad libitum. In pair-fed nephrotic rats, we observed an increase in the cleaved form of membrane-bound γ-epithelial sodium channel (ENaC). However, α- and β-ENaC subunits were unaltered. NKCC2 and AQP2 mRNA levels were similar in treated vs. control rats. We conclude that dietary protein intake affects the response of medullary transport proteins to NS.  

3.1886           Lipid rafts, microdomain heterogeneity and inter-organelle contacts: Impacts on membrane preparation for proteomic studies

Minogue, S. and Waugh, M.G. Biol. Cell, 104(10), 618-627 (2012)   In recent years, there has been considerable interest in mapping the protein content of isolated organelles using mass spectrometry. However, many subcellular compartments are highly dynamic with diverse and intricate architectures that are not always preserved during membrane isolation procedures. Furthermore, lateral heterogeneities in intra-membrane lipid and protein concentrations underlie the formation of membrane microdomains, trafficking vesicles and inter-membrane contacts. These complexities in membrane organisation have important consequences for the design of membrane preparation strategies and test the very concept of organelle purity. We illustrate how some of these biological considerations are relevant to membrane preparation and assess the numerous potential pitfalls in attempting to purify organelles from mammalian cells.  

3.1887           TPC Proteins Are Phosphoinositide- Activated Sodium-Selective Ion Channels in Endosomes and Lysosomes

Wang, X., Zhang, X., Dong, X-p., Samie, M., Li, X., Cheng, X., Goschka, A., Shen, D., Zhou, Y., Harlow, J., Zhu, M.X., Clapham, D.E., Ren, D. and Xu, H. Cell, 151(2), 372-383 (2012)   Mammalian two-pore channel proteins (TPC1, TPC2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double-knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P₂ and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na⁺, not K⁺, as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes and may explain the specificity of PI(3,5)P₂ in regulating the fusogenic potential of intracellular organelles.  

3.1888           Stabilization of Kv1.5 channel protein by bepridil through its action as a chemical chaperone

Suzuki, S., Kurata, Y., Li, P., Notsu, T., Hasegawa, A., Ikeda, N., kato, M., Miake, J., Sakata, S., Shiota, G., Yoshida, A., Ninomiya, H., Higaki, K., Yamamoto, K., Shirayoshi, Y. and Hisatome, I. Eur. J. Pharmacol., 696, 28-34 (2012)   While bepridil has been reported to alter the stability of ion channel proteins, the precise mechanism of action remains unclear. We examined the effect of bepridil on the stability of Kv1.5 channel proteins expressed in COS7 cells. Bepridil at 0.3–30 μM increased the protein level of Kv1.5 channels in a concentration-dependent manner. Chase experiments showed that bepridil delayed the degradation process of Kv1.5 channel proteins in the same manner as a proteasomal inhibitor, MG132, did. Bepridil increased the immunofluorescent signal of Kv1.5 channel proteins in the endoplasmic reticulum (ER) and Golgi apparatus and on the cell surface. The cell fraction experiment also showed bepridil-induced increases in Kv1.5 in the ER, Golgi apparatus, and the cell membrane. Bepridil at a lower concentration of 1 μM had no effect on the proteasome activity in vitro. A blocker of the ultrarapid delayed-rectifier K⁺ channel current, 4-aminopyridine (4AP), abolished bepridil-induced increases in Kv1.5. Kv1.5-medicated membrane currents measured as 4AP-sensitive currents were increased by bepridil. Taken together, we conclude that bepridil stabilizes Kv1.5 proteins at the ER through an action as a chemical chaperone, thereby increasing the density of Kv1.5 channels in the cell membrane.  

3.1889           Review on recent advances in the analysis of isolated organelles

Satori, C.P., Kostal, V. and Arriaga, E.A. Analytica Chimica Acta, 753, 8-18 (2012)   The analysis of isolated organelles is one of the pillars of modern bioanalytical chemistry. This review describes recent developments on the isolation and characterization of isolated organelles both from living organisms and cell cultures. Salient reports on methods to release organelles focused on reproducibility and yield, membrane isolation, and integrated devices for organelle release. New developments on organelle fractionation after their isolation were on the topics of centrifugation, immunocapture, free flow electrophoresis, flow field-flow fractionation, fluorescence activated organelle sorting, laser capture microdissection, and dielectrophoresis. New concepts on characterization of isolated organelles included atomic force microscopy, optical tweezers combined with Raman spectroscopy, organelle sensors, flow cytometry, capillary electrophoresis, and microfluidic devices.  

3.1890           TRAIL-activated EGFR by Cbl-b-regulated EGFR redistribution in lipid rafts antagonises TRAIL-induced apoptosis in gastric cancer cells

Xu, L., Zhang, Y., Liu, J., Qu, J., Hu, X., Zhang, F., Zheng, H., Qu, X. and Liu, Y. Eur. J. Cancer, 48, 3288-3299 (2012)   Most gastric cancer cells are resistant to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Since TRAIL resistance is associated with lipid rafts, in which both death receptors and epidermal growth factor receptors (EGFR) are enriched, our aim is to identify how lipid raft-regulated receptor redistribution influences the sensitivity of TRAIL in gastric cancer cells. In TRAIL-resistant gastric cancer cells, TRAIL did not induce effective death-inducing signalling complex (DISC) formation in lipid rafts, accompanied with EGFR translocation into lipid rafts, and activation of EGFR pathway. Knockdown of casitas B-lineage lymphoma-b (Cbl-b) enhanced TRAIL-induced apoptosis by promoting DISC formation in lipid rafts. However, knockdown of Cbl-b also enhanced EGFR translocation into lipid rafts and EGFR pathway activation induced by TRAIL. Either using inhibitors of EGFR or depletion of EGFR with small interfering RNA (siRNA) prevented EGFR pathway activation, and thus increased TRAIL-induced apoptosis, especially in Cbl-b knockdown clones. Taken together, TRAIL-induced EGFR activation through Cbl-b-regulated EGFR redistribution in lipid rafts antagonised TRAIL-induced apoptosis. The contribution of DISC formation and the inhibition of EGFR signal triggered in lipid rafts are both essential for increasing the sensitivity of gastric cancer cells to TRAIL.  

3.1891           Localisation of a family of complex-forming β-barrels in the T. vaginalis hydrogenosomal membrane

Kay, C., Lawler, K., Self, T.J., Dyall, S.D. and Kerr, I.D. FEBS Lett., 586, 4038-4045 (2012)   Crucial to organellogenesis was the development of membrane translocases responsible for delivering proteins to new cellular compartments. This investigation examines the Trichomonas vaginalis hydrogenosome, a mitochondrially derived organelle. We identify an expanded family of putative β-barrel proteins (THOM A–I) comprising nine related sequences. Sub-cellular localisation by immunofluorescence and biochemical fractionation is consistent with THOMs being localised to the hydrogenosomal membrane. Native gel electrophoresis and chemical cross-linking support the ability of THOM proteins to be components of membrane-bound oligomeric protein complexes, consistent with a role in protein translocation.  

3.1892           Vaspin Is an Adipokine Ameliorating ER Stress in Obesity as a Ligand for Cell-Surface GRP78/MTJ-1 Complex

Nakatsuka, A. et al Diabetes, 61, 2823-2832 (2012)   It is unknown whether adipokines derived from adipose tissues modulate endoplasmic reticulum (ER) stress induced in obesity. Here, we show that visceral adipose tissue–derived serine protease inhibitor (vaspin) binds to cell-surface 78-kDa glucose-regulated protein (GRP78), which is recruited from ER to plasma membrane under ER stress. Vaspin transgenic mice were protected from diet-induced obesity, glucose intolerance, and hepatic steatosis, while vaspin-deficient mice developed glucose intolerance associated with upregulation of ER stress markers. With tandem affinity tag purification using HepG2 cells, we identified GRP78 as an interacting molecule. The complex formation of vaspin, GRP78, and murine tumor cell DnaJ-like protein 1 (MTJ-1) (DnaJ homolog, subfamily C, member 1) on plasma membrane was confirmed by cell-surface labeling with biotin and immunoprecipitation in liver tissues and H-4-II-E-C3 cells. The addition of recombinant human vaspin in the cultured H-4-II-E-C3 cells also increased the phosphorylation of Akt and AMP-activated protein kinase (AMPK) in a dose-dependent manner, and anti-GRP78 antibodies completely abrogated the vaspin-induced upregulation of pAkt and pAMPK. Vaspin is a novel ligand for cell-surface GRP78/MTJ-1 complex, and its subsequent signals exert beneficial effects on ER stress–induced metabolic dysfunctions.  

3.1893           Down-regulation of connexin43 expression reveals the involvement of caveolin-1 containing lipid rafts in human U251 glioblastoma cell invasion

Strale. P-O., Clarhaut, J., Lamiche, C., Cronier, L., Mesnil, M. and Defamie, N. Mol. Carcinogenesis, 51(11), 845-860 (2012)   Glioblastoma cells are characterized by high proliferation and invasive capacities. Tumor development has been associated with a decrease of gap-junctional intercellular communication, but the concrete involvement of gap junction proteins, connexins, remains elusive since they are also suspected to promote cell invasion. In order to better understand how connexins control the glioma cell phenotype, we studied the consequences of inhibiting the intrinsic expression of the major astrocytic connexin, Connexin43, in human U251 glioblastoma cells by the shRNA strategy. The induced down-regulation of Cx43 expression has various effects on the U251 cells such as increased clonogenicity, angiogenesis and decreased adhesion on specific extracellular matrix proteins. We demonstrate that the invasion capacity measured in vitro and ex vivo correlates with Cx43 expression level. For the first time in a cancer cell context, our work demonstrates that Cx43 cofractionates, colocalizes and coimmunoprecipitates with a lipid raft marker, caveolin-1 and that this interaction is inversely correlated to the level of Cx43. This localization of Cx43 in these lipid raft microdomains regulates both homo- and heterocellular gap junctional communications (respectively between U251 cells, or between U251 cells and astrocytes). Moreover, the adhesive and invasive capacities are not dependent, in our model, on Cav-1 expression level. Our results tend to show that heterocellular gap junctional communication between cancer and stroma cells may affect the behavior of the tumor cells. Altogether, our data demonstrate that Cx43 controls the tumor phenotype of glioblastoma U251 cells and in particular, invasion capacity, through its localization in lipid rafts containing Cav-1.  

3.1894           Cryptococcus neoformans-Derived Microvesicles Enhance the Pathogenesis of Fungal Brain Infection

Huang, S-H., Wu, C-H., Chang, Y.C., Kwon-Chung, K.J., Brown, R.J. and Jong, A. PloS One, 7(11), e48570 (2012)   Cryptococcal meningoencephalitis is the most common fungal disease in the central nervous system. The mechanisms by which Cryptococcus neoformans invades the brain are largely unknown. In this study, we found that C. neoformans-derived microvesicles (CnMVs) can enhance the traversal of the blood-brain barrier (BBB) by C. neoformans in vitro. The immunofluorescence imaging demonstrates that CnMVs can fuse with human brain microvascular endothelial cells (HBMECs), the constituents of the BBB. This activity is presumably due to the ability of the CnMVs to activate HBMEC membrane rafts and induce cell fusogenic activity. CnMVs also enhanced C. neoformans infection of the brain, found in both infected brains and cerebrospinal fluid. In infected mouse brains, CnMVs are distributed inside and around C. neoformans-induced cystic lesions. GFAP (glial fibrillary acidic protein)-positive astrocytes were found surrounding the cystic lesions, overlapping with the 14-3-3-GFP (14-3-3-green fluorescence protein fusion) signals. Substantial changes could be observed in areas that have a high density of CnMV staining. This is the first demonstration that C. neoformans-derived microvesicles can facilitate cryptococcal traversal across the BBB and accumulate at lesion sites of C. neoformans-infected brains. Results of this study suggested that CnMVs play an important role in the pathogenesis of cryptococcal meningoencephalitis.  

3.1895           STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma membrane, ER, and ERC

Garbarino, J., Pan, M., Chin, H.F., Lund, F.W., Maxfield, F.R. and Breslow, J.L.

1. Lipid Res., 53, 2716-2725 (2012)

  STARD4, a member of the evolutionarily conserved START gene family, has been implicated in the nonvesicular intracellular transport of cholesterol. However, the direction of transport and the membranes with which this protein interacts are not clear. We present studies of STARD4 function using small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT synthesized cholesteryl esters. Furthermore, D4 KD cells exhibited a reduced rate of sterol transport to the endocytic recycling compartment after cholesterol repletion. Although these cells displayed normal endocytic trafficking in cholesterol-poor and replete conditions, cell surface low density lipoprotein receptor (LDLR) levels were increased and decreased, respectively. We also observed a decrease in NPC1 protein expression, suggesting the induction of compensatory pathways to maintain cholesterol balance. These data indicate a role for STARD4 in nonvesicular transport of cholesterol from the plasma membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well.  

3.1896           Update on Methods and Techniques to Study Endocytosis in Plants

Samajova, O., Takac, T., von Wangenheim, D., Stelzer, E. and Samaj, J. Endocytosis in Plants, 1-36 ( 2012)   The growing interest in the investigation of endocytosis, vesicular transport routes, and corresponding regulatory mechanisms resulted in the exploitation of cell biological, genetic, biochemical, and proteomic approaches. Methods and techniques such as site-directed and T-DNA insertional mutagenesis, RNAi, classical inhibitor treatments, and recombinant GFP technology combined with confocal laser scanning microscopy (CLSM) and electron and immune-electron microscopy were routinely employed for investigation of endocytosis in plant cells. However, new approaches such as high-throughput confocal microscopy screens on mutants and proteomic analyses on isolated vesicular compartments and root cells treated with vesicular trafficking inhibitors (both focused on the identification of new endosomal proteins), together with chemical genomics and advanced microscopy approaches such as Förster resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), light sheet-based fluorescence microscopy, and super-resolution microscopy provided a significant amount of new data and these new methods appear as extremely promising tools in this field.  

3.1897           Sterol C22-Desaturase and Its Biological Roles

Ohta, D. and Mizutani, M. Isoprenoid Synthesis in Plant and M icroorganism, 381-391 (2012)   The C22-unsaturated sterols are primarily found in fungi and plants. The C22-desaturation reaction is catalyzed by independent cytochrome P450 family proteins, CYP61 in fungi, and CYP710 in plants. We describe our extensive characterization studies of plant CYP710 family proteins and discuss possible evolutional relationships of C22-desaturation reactions among eukaryotic organisms. We also discuss possible research directions toward understanding physiological implications of sterols in unidentified brassinosteroid-independent growth/developmental processes.  

3.1898           Proteomic Characterization of Phagosomal Membrane Microdomains During Phagolysosome Biogenesis and Evolution

Goyette, G., Boulais, J., Carruthers, N.J., Landry, C.R., Justras, I., Duclos, S., Dermine, J-F., Michnick, S.W., LaBoissiere, S., Lajoie, G., Barrereiro, L., Thibault, P. and Desjardin, M. Mol. Cell. Proteomics, 11, 1365-1377 (2012)   After their formation at the cell surface, phagosomes become fully functional through a complex maturation process involving sequential interactions with various intracellular organelles. In the last decade, series of data indicated that some of the phagosome functional properties occur in specialized membrane microdomains. The molecules associated with membrane microdomains, as well as the organization of these structures during phagolysosome biogenesis are largely unknown. In this study, we combined proteomics and bioinformatics analyses to characterize the dynamic association of proteins to maturing phagosomes. Our data indicate that groups of proteins shuffle from detergent-soluble to detergent-resistant membrane microdomains during maturation, supporting a model in which the modulation of the phagosome functional properties involves an important reorganization of the phagosome proteome by the coordinated spatial segregation of proteins.  

3.1899           Genetic Depletion of Complement Receptors CD21/35 Prevents Terminal Prion Disease in a Mouse Model of Chronic Wasting Disease

Brady, M., Ferguson, A., Johnson, T., Bender, H., Meyerett-reid, C., Pulford, B., von Teichman, A., Seelig, D., Weis, J.H., Telling, G.C., Aguzzi, A. and Zabel, M.D.

J. Immunol., 189, 4520-4527 (2012)

The complement system has been shown to facilitate peripheral prion pathogenesis. Mice lacking complement receptors CD21/35 partially resist terminal prion disease when infected i.p. with mouse-adapted scrapie prions. Chronic wasting disease (CWD) is an emerging prion disease of captive and free-ranging cervid populations that, similar to scrapie, has been shown to involve the immune system, which probably contributes to their relatively facile horizontal and environmental transmission. In this study, we show that mice overexpressing the cervid prion protein and susceptible to CWD (Tg(cerPrP)5037 mice) but lack CD21/35 expression completely resist clinical CWD upon peripheral infection. CD21/35-deficient Tg5037 mice exhibit greatly impaired splenic prion accumulation and replication throughout disease, similar to CD21/35-deficient murine prion protein mice infected with mouse scrapie. TgA5037;CD21/35−/− mice exhibited little or no neuropathology and deposition of misfolded, protease-resistant prion protein associated with CWD. CD21/35 translocate to lipid rafts and mediates a strong germinal center response to prion infection that we propose provides the optimal environment for prion accumulation and replication. We further propose a potential role for CD21/35 in selecting prion quasi-species present in prion strains that may exhibit differential zoonotic potential compared with the parental strains.

 

3.1900           SMAD versus Non-SMAD Signaling Is Determined by Lateral Mobility of Bone Morphogenetic Protein (BMP) Receptors

Guzman, A., Zelman-Femiak, M., Boergermann, J.H., Paschowsky, S., Kreuzaler, P.A., Fratzl, P., Harms, G.S. and Knaus, P.

1. Biol. Chem., 287(47), 39492-39504 (2012)

  Bone (or body) morphogenetic proteins (BMPs) belong to the TGFβ superfamily and are crucial for embryonic patterning and organogenesis as well as for adult tissue homeostasis and repair. Activation of BMP receptors by their ligands leads to induction of several signaling cascades. Using fluorescence recovery after photobleaching, FRET, and single particle tracking microscopy, we demonstrate that BMP receptor type I and II (BMPRI and BMPRII) have distinct lateral mobility properties within the plasma membrane, which is mandatory for their involvement in different signaling pathways. Before ligand binding, BMPRI and a subpopulation of BMPRII exhibit confined motion, reflecting preassembled heteromeric receptor complexes. A second free diffusing BMPRII population only becomes restricted after ligand addition. This paper visualizes time-resolved BMP receptor complex formation and demonstrates that the lateral mobility of BMPRI has a major impact in stabilizing heteromeric BMPRI-BMPRII receptor complexes to differentially stimulate SMAD versus non-SMAD signaling.  

3.1901           Tollip, an Intracellular Trafficking Protein, Is a Novel Modulator of the Transforming Growth Factor-β Signaling Pathway

Zhu, L., Wang, L., Luo, X., Zhang, Y., Ding, Q., Jiang, X., Wang, X., Pan, Y. and Chen, Y.

1. Biol. Chem., 287(47), 39653-39663 (2012)

  Upon activation, TGF-β type I receptor (TβRI) undergoes active ubiquitination via recruitment of E3 ligases to the receptor complex by Smad7. However, how ubiquitination of TβRI is coupled to intracellular trafficking, and protein degradation remains unclear. We report here that Tollip, an adaptor protein that contains both ubiquitin-associated domains and endosome-targeting domain, plays an important role in modulating trafficking and degradation of TβRI. Tollip was previously demonstrated to possess a functional role in modulating the signaling of interleukin-1 and Toll-like receptors. We identify here that Tollip interacts with Smad7, a major modulatory protein involved in the negative regulation of TGF-β signaling. Overexpression of Tollip antagonizes TGF-β-stimulated transcriptional response, Smad2 phosphorylation, and epithelial-mesenchymal transition. Tollip also interacts with ubiquitinated TβRI, and such interaction requires ubiquitin-associated domains of Tollip. The interaction and intracellular colocalization of Tollip with TβRI is enhanced by Smad7. Overexpression of Tollip accelerates protein degradation of activated TβRI. In addition, Tollip alters subcellular compartmentalization and endosomal trafficking of activated TβRI. Collectively, our studies reveal that Tollip cooperates with Smad7 to modulate intracellular trafficking and degradation of ubiquitinated TβRI, whereby negatively regulates TGF-β signaling pathway.  

3.1902           SIRT5 deacetylates and activates urate oxidase in liver mitochondria of mice

Nakamura, Y., Ogura, M., Ogura, K., Tanaka, D. and Inagaki, N. FEBS Lett., 586, 4076-4081 (2012)   We identified urate oxidase (UOX) as a target of SIRT5 by comparing mitochondrial proteins in livers of SIRT5-overexpressing transgenic (SIRT5 Tg) and wild-type mice by using two-dimensional electrophoresis. Acetylation levels of UOX in liver of SIRT5 Tg mice were approximately half of those in wild-type mice, and UOX activity was significantly increased. Invitro-synthesized UOX protein was acetylated when incubated with mitochondria from wild-type mice liver but the levels were less when incubated with those from SIRT5 Tg mice liver. These results suggest that SIRT5 activates UOX through deacetylation in mouse liver mitochondria.  

3.1903           Potent Inhibition of Late Stages of Hepadnavirus Replication by a Modified Cell Penetrating Peptide

Abdul, F., Ndeboko, B., Buronfosse, T., Zoulim, F., Kann, M., Nielsen, P.E. and Cova, L. PloS One, 7(11), e48721 (2012)   Cationic cell-penetrating peptides (CPPs) and their lipid domain-conjugates (CatLip) are agents for the delivery of (uncharged) biologically active molecules into the cell. Using infection and transfection assays we surprisingly discovered that CatLip peptides were able to inhibit replication of Duck Hepatitis B Virus (DHBV), a reference model for human HBV. Amongst twelve CatLip peptides we identified Deca-(Arg)₈ having a particularly potent antiviral activity, leading to a drastic inhibition of viral particle secretion without detectable toxicity. Inhibition of virion secretion was correlated with a dose-dependent increase in intracellular viral DNA. Deca-(Arg)₈ peptide did neither interfere with DHBV entry, nor with formation of mature nucleocapsids nor with their travelling to the nucleus. Instead, Deca-(Arg)₈ caused envelope protein accumulation in large clusters as revealed by confocal laser scanning microscopy indicating severe structural changes of preS/S. Sucrose gradient analysis of supernatants from Deca-(Arg)₈-treated cells showed unaffected naked viral nucleocapsids release, which was concomitant with a complete arrest of virion and surface protein-containing subviral particle secretion. This is the first report showing that a CPP is able to drastically block hepadnaviral release from infected cells by altering late stages of viral morphogenesis via interference with enveloped particle formation, without affecting naked nucleocapsid egress, thus giving a view inside the mode of inhibition. Deca-(Arg)₈ may be a useful tool for elucidating the hepadnaviral secretory pathway, which is not yet fully understood. Moreover we provide the first evidence that a modified CPP displays a novel antiviral mechanism targeting another step of viral life cycle compared to what has been so far described for other enveloped viruses.  

3.1904           Three-Dimensional Architecture of the Rod Sensory Cilium and Its Disruption in Retinal Neurodegeneration

Gilliam, J.C., Chang, J.T., Sandoval, I.M., Zhang, Y., Li, T., Pittler, S.J., Chiu, W. and Wensel, T.G. Cell, 151(5), 1029-1041 (2012)   Defects in primary cilia lead to devastating disease because of their roles in sensation and developmental signaling but much is unknown about ciliary structure and mechanisms of their formation and maintenance. We used cryo-electron tomography to obtain 3D maps of the connecting cilium and adjacent cellular structures of a modified primary cilium, the rod outer segment, from wild-type and genetically defective mice. The results reveal the molecular architecture of the cilium and provide insights into protein functions. They suggest that the ciliary rootlet is involved in cellular transport and stabilizes the axoneme. A defect in the BBSome membrane coat caused defects in vesicle targeting near the base of the cilium. Loss of the proteins encoded by the Cngb1 gene disrupted links between the disk and plasma membranes. The structures of the outer segment membranes support a model for disk morphogenesis in which basal disks are enveloped by the plasma membrane.  

3.1905           Complete failure of insulin-transmitted signaling, but not obesity-induced insulin resistance, impairs respiratory chain function in muscle

Franko, A., von Kleist-Retzow, J.C., Böse, M., Sanchez-Lasheras, C., Brodesser, S., Krut, O., Kunz, W.S., Wiedermann, D., Hoehn, M., Stöhr, O., Moll, L., Freude, S., Krone, W., Schubert, M. and Wiesner, R.J.

1. Mol. Med., 90, 1145-1160 (2012)

  The role of mitochondrial dysfunction in the development of insulin resistance and type 2 diabetes remains controversial. In order to specifically define the relationship between insulin receptor (InsR) signaling, insulin resistance, hyperglycemia, hyperlipidemia and mitochondrial function, we analyzed mitochondrial performance of insulin-sensitive, slow-oxidative muscle in four different mouse models. In obese but normoglycemic ob/ob mice as well as in obese but diabetic mice under high-fat diet, mitochondrial performance remained unchanged even though intramyocellular diacylglycerols (DAGs), triacylglycerols (TAGs), and ceramides accumulated. In contrast, in muscle-specific InsR knockout (MIRKO) and streptozotocin (STZ)-treated hypoinsulinemic, hyperglycemic mice, levels of mitochondrial respiratory chain complexes and mitochondrial function were markedly reduced. In STZ, but not in MIRKO mice, this was caused by reduced transcription of mitochondrial genes mediated via decreased PGC-1α expression. We conclude that mitochondrial dysfunction is not causally involved in the pathogenesis of obesity-associated insulin resistance under normoglycemic conditions. However, obesity-associated type 2 diabetes and accumulation of DAGs or TAGs is not associated with impaired mitochondrial function. In contrast, chronic hypoinsulinemia and hyperglycemia as seen in STZ-treated mice as well as InsR deficiency in muscle of MIRKO mice lead to mitochondrial dysfunction. We postulate that decreased mitochondrial mass and/or performance in skeletal muscle of non-diabetic, obese or type 2 diabetic, obese patients observed in clinical studies must be explained by genetic predisposition, physical inactivity, or other still unknown factors.    

3.1906           Lysosomal delivery of therapeutic enzymes in cell models of Fabry disease

Marchesan, D., Cox, T.M. and Deegan, P.B.

1. Inherit. Metab. Dis., 35(6), 1107-1117 (2012)

  The success of enzymatic replacement in Gaucher disease has stimulated development of targeted protein replacement for other lysosomal disorders, including Anderson-Fabry disease, which causes fatal cardiac, cerebrovascular and renal injury: deficiency of lysosomal α-Galactosidase A induces accumulation of glycosphingolipids. Endothelial cell storage was the primary endpoint in a clinical trial that led to market authorization. Two α-Galactosidase A preparations are licensed worldwide, but fatal outcomes persist, with storage remaining in many tissues. We compare mechanisms of uptake of α -Galactosidase A into cells relevant to Fabry disease, in order to investigate if the enzyme is targeted to the lysosomes in a mannose-6-phosphate receptor dependent fashion, as generally believed. α -Galactosidase A uptake was examined in fibroblasts, four different endothelial cell models, and hepatic cells in vitro. Uptake of europium-labeled human α -Galactosidase A was measured by time-resolved fluorescence. Ligand-specific uptake was quantified in inhibitor studies. Targeting to the lysosome was determined by precipitation and by confocal microscopy. The quantity and location of cation-independent mannose-6-phosphate receptors in the different cell models were investigated using confocal microscopy. Uptake and delivery of α -Galactosidase A to lysosomes in fibroblasts is mediated by the canonical mannose-6-phosphate receptor pathway, but in endothelial cells in vitro this mechanism does not operate. Moreover, this observation is supported by a striking paucity of expression of cation independent mannose-6-phosphate receptors on the plasma membrane of the four endothelial cell models and by little delivery of enzyme to lysosomes, when compared with fibroblasts. If these observations are confirmed in vivo, alternative mechanisms will be needed to explain the ready clearance of storage from endothelial cells in patients undergoing enzyme replacement therapy.  

3.1907           Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation

Kobuchi, H., Moriya, K., Ogino, T., Fujita, H., Inoue, K., Shuin, T., Yasuda, T., Utsumi, K. and utsumi, T. PloS One, 7(11), e50082 (2012)   Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol.  

3.1908           In-depth analysis of the secretome identifies three major independent secretory pathways in differentiating human myoblasts

Le Bihan, M-C., Bigot, A., Jensen, S.S., Dennis, J.L., Rogowska-Wrzesinska, A., Laine, J., GAche, V., Furling, D., Nørregaard-jensen, O., Voit, T., Mouly, V., Coulton, G.R. and Butler-Browne, G.

1. Proteomics., 77, 344-356 (2012)

  Efficient muscle regeneration requires cross talk between multiple cell types via secreted signaling molecules. However, as yet there has been no comprehensive analysis of this secreted signaling network in order to understand how it regulates myogenesis in humans. Using integrated proteomic and genomic strategies, we show that human muscle cells release not only soluble secreted proteins through conventional secretory mechanisms but also complex protein and nucleic acid cargos via membrane microvesicle shedding. The soluble secretome of muscle cells contains 253 conventionally secreted signaling proteins, including 43 previously implicated in myogenesis, while others are known to modulate various cell types thus implying a much broader role for myoblasts in muscle remodeling. We also isolated and characterized two types of secreted membrane-derived vesicles: nanovesicles harboring typical exosomal features and larger, morphologically distinct, microvesicles. While they share some common features, their distinct protein and RNA cargos suggest independent functions in myogenesis. We further demonstrate that both types of microvesicles can dock and fuse with adjacent muscle cells but also deliver functional protein cargo. Thus, the intercellular signaling networks invoked during muscle differentiation and regeneration may employ conventional soluble signaling molecules acting in concert with muscle derived microvesicles delivering their cargos directly into target cells.  

3.1909           The Ubiquitin Ligase Synoviolin Up-regulates Amyloid β Production by Targeting a Negative Regulator of -Secretase, Rer1, for Degradation

Tanabe, C., Maeda, T., Zou, K., Liu, J., Nakajima, T. and Komano, H.

1. Biol. Chem., 287(53), 44203-44211 (2012)

  Alzheimer's disease is characterized by the deposition of Aβ, which is generated from the amyloid precursor protein through its cleavage by β- and γ-secretases. The γ-secretase complex component nicastrin (NCT) plays significant roles in the assembly and proper trafficking of the γ-secretase complex and in the recognition of amyloid precursor protein. NCT is incorporated into the γ-secretase complex in the endoplasmic reticulum (ER) and glycosylated in the Golgi. In contrast, unassembled NCT is retrieved or retained in the ER by the protein Retention in endoplasmic reticulum 1 (Rer1). We reported previously that synoviolin (Syvn), an E3 ubiquitin ligase, degrades NCT and affects the generation of Aβ. Here, we examined in more detail the effect of Syvn on the generation of Aβ. We found that overexpression of a dominant negative form of Syvn (C307A mutant) and a Syvn-RNAi decreased the generation of Aβ. These results indicate that the ubiquitin ligase activity of Syvn up-regulates the generation of Aβ. We hypothesized, therefore, that Syvn regulates the assembly or localization of the γ-secretase complex by ubiquitinating Rer1, resulting in its subsequent degradation. Our findings that the level of Rer1 was increased in Syvn knockout fibroblasts because of inhibition of its degradation support this hypothesis. Moreover, we found that Rer1 interacts with Syvn in the ER, is ubiquitinated by Syvn, and is then degraded via the proteasome or lysosomal pathways. Finally, we showed that localization of mature NCT to the plasma membrane as well as γ-secretase complex levels are decreased in fibroblasts of Syvn knockout mice. Thus, it is likely that Syvn regulates the assembly of the γ-secretase complex via the degradation of Rer1, which results in the generation of Aβ.  

3.1910           Sphingosine 1-Phosphate (S1P) Carrier-dependent Regulation of Endothelial Barrier: HIGH DENSITY LIPOPROTEIN (HDL)-S1P PROLONGS ENDOTHELIAL BARRIER ENHANCEMENT AS COMPARED WITH ALBUMIN-S1P VIA EFFECTS ON LEVELS, TRAFFICKING, AND SIGNALING OF S1P1

Wilkerson, B.A., Grass, G.D., Wing, S.B., Argraves, W.S. and Argraves, K.M.

1. Biol. Chem., 287(53), 44645-44653 (2012)

  Sphingosine 1-phosphate (S1P) is a blood-borne lysosphingolipid that acts to promote endothelial cell (EC) barrier function. In plasma, S1P is associated with both high density lipoproteins (HDL) and albumin, but it is not known whether the carriers impart different effects on S1P signaling. Here we establish that HDL-S1P sustains EC barrier longer than albumin-S1P. We showed that the sustained barrier effects of HDL-S1P are dependent on signaling by the S1P receptor, S1P1, and involve persistent activation of Akt and endothelial NOS (eNOS), as well as activity of the downstream NO target, soluble guanylate cyclase (sGC). Total S1P1 protein levels were found to be higher in response to HDL-S1P treatment as compared with albumin-S1P, and this effect was not associated with increased S1P1 mRNA or dependent on de novo protein synthesis. Several pieces of evidence indicate that long term EC barrier enhancement activity of HDL-S1P is due to specific effects on S1P1 trafficking. First, the rate of S1P1 degradation, which is proteasome-mediated, was slower in HDL-S1P-treated cells as compared with cells treated with albumin-S1P. Second, the long term barrier-promoting effects of HDL-S1P were abrogated by treatment with the recycling blocker, monensin. Finally, cell surface levels of S1P1 and levels of S1P1 in caveolin-enriched microdomains were higher after treatment with HDL-S1P as compared with albumin-S1P. Together, the findings reveal S1P carrier-specific effects on S1P1 and point to HDL as the physiological mediator of sustained S1P1-PI3K-Akt-eNOS-sGC-dependent EC barrier function.  

3.1911           Vigilin interacts with signal peptide peptidase

Lu, S.H-J., Jeon, A.H.W., Schmitt-Ulms, G., Qamar, S., Dodd, R., McDonald, B., Li, Y., Meadows, W., Cox, K., Bohm, C., Chen, F., fraser, P. and St. George-Hyslop, P. Proteome Science, 10(33) (2012)   Background Signal peptide peptidase (SPP), a member of the presenilin-like intra-membrane cleaving aspartyl protease family, migrates on Blue Native (BN) gels as 100 kDa, 200 kDa and 450 kDa species. SPP has recently been implicated in other non-proteolytic functions such as retro-translocation of MHC Class I molecules and binding of misfolded proteins in the endoplasmic reticulum (ER). These high molecular weight SPP complexes might contain additional proteins that regulate the proteolytic activity of SPP or support its non-catalytic functions. Results In this study, an unbiased iTRAQ-labeling mass spectrometry approach was used to identify SPP-interacting proteins. We found that vigilin, a ubiquitous multi-KH domain containing cytoplasmic protein involved in RNA binding and protein translation control, selectively enriched with SPP. Vigilin interacted with SPP and both proteins co-localized in restricted intracellular domains near the ER, biochemically co-fractionated and were part of the same 450 kDa complex on BN gels. However, vigilin does not alter the protease activity of SPP, suggesting that the SPP-vigilin interaction might be involved in the non-proteolytic functions of SPP. Conclusions We have identified and validated vigilin as a novel interacting partner of SPP that could play an important role in the non-proteolytic functions of SPP. This data adds further weight to the idea that intramembrane-cleaving aspartyl proteases, such as presenilin and SPPs, could have other functions besides the proteolysis of short membrane stubs.  

3.1912           Ligand-independent activation of EphA2 by arachidonic acid induces metastasis-like behaviour in prostate cancer cells

Tawadros, T., Brown, M.D., Hart, C.A. and Clarke, N.W. Br. J. Cancer, 107(10), 1737-1744 (2012)   Background: High intake of omega-6 polyunsaturated fatty acids (PUFA) has been associated with clinical progression in prostate cancer (CaP). This study investigates the signalling mechanism by which the omega-6 PUFA arachidonic acid (AA) induces prostatic cellular migration to bone marrow stroma. Methods: Western blot analysis of the PC-3, PC3-GFP, DU 145 and LNCaP cells or their lipid raft (LR) components post AA stimulation was conducted in association with assays for adhesion and invasion through the bone marrow endothelial monolayers. Results: Arachidonic acid increased transendothelial migration of PC3-GFP cells (adhesion 37%±0.08, P=0.0124; transmigration 270%±0.145, P=0.0008). Akt, Src and focal adhesion kinase (FAK) pathways were induced by AA and integrally involved in transendothelial migration. LR were critical in AA uptake and induced Akt activity. Ephrin receptor A2 (EphA2), localised in LR, is expressed in DU 145 and PC-3 cells. Arachidonic acid induced a rapid increase of EphA2 Akt-dependent/ligand-independent activation, while knockdown of the EphrinA1 ligand decreased AA induced transendothelial migration, with an associated decrease in Src and FAK activity. Arachidonic acid activated Akt in EphA2⁻ LNCaP cells but failed to induce BMEC transendothelial invasion. Conclusion: Arachidonic acid induced stimulation of EphA2 in vitro is associated fundamentally with CaP epithelial migration across the endothelial barrier.  

3.1913           Exosome-mediated delivery of siRNA in vitro and in vivo

El-Andaloussi, S., Lee, Y., Lakhal-Littleton, S., Li, J., Seow, Y., Gardiner, C., Alvarez-Erviti, L., Sargent, I.L. and Wood, M.J.A. Nature Protocols, 7(12), 2112-2126 (2012)   The use of small interfering RNAs (siRNAs) to induce gene silencing has opened a new avenue in drug discovery. However, their therapeutic potential is hampered by inadequate tissue-specific delivery. Exosomes are promising tools for drug delivery across different biological barriers. Here we show how exosomes derived from cultured cells can be harnessed for delivery of siRNA in vitro and in vivo. This protocol first describes the generation of targeted exosomes through transfection of an expression vector, comprising an exosomal protein fused with a peptide ligand. Next, we explain how to purify and characterize exosomes from transfected cell supernatant. Next, we detail crucial steps for loading siRNA into exosomes. Finally, we outline how to use exosomes to efficiently deliver siRNA in vitro and in vivo in mouse brain. Examples of anticipated results in which exosome-mediated siRNA delivery is evaluated by functional assays and imaging are also provided. The entire protocol takes ∼3 weeks.  

3.1914           Down-regulation of alpha-2u globulin in renal mitochondria of STZ-induced diabetic rats observed by a proteomic method

Sun, S-H., Liu, S-Q., Cai, C-P., Cai, R., Chen, L. and Zhang, Q-B. Annales d’Endocrinologie, 73(6), 530-541 (2012)   Aim To identify the changes of mitochondrial protein expression in diabetic renal parenchyma and to characterize their molecular functions and biological processes in diabetes. Methods Mitochondrial proteins extracted from renal parenchyma mitochondria of streptozotocin-induced diabetic rats and normal rats were separated by two-dimensional polyacrylamide gel electrophoresis and identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Results Eleven proteins from 533 visualized protein spots displayed significant different expressions in mitochondria of diabetic kidneys compared with those in normal ones. Among these altered proteins, two proteins with the most obvious changes in protein expression were identified as alpha-2u globulin (mature protein, named A2) and its proteolytically modified form (named A2-fragment) respectively. These proteins were found in mitochondria of male rat renal parenchyma and were proved to be down-regulated in diabetic rats simultaneously. Conclusion Our results suggest that down-regulation of alpha-2u globulin may be associated with an abnormal β-oxidation of long-chain fatty acids during diabetes. The decreased expression of A2-fragment in renal mitochondria of diabetic nephropathy may reduce fatty acid β-oxidation, which leads to a diminished energy supply from mitochondria to kidney tissue and the deposition of a large number of fatty acids in the kidney, ultimately causing and aggravating kidney damage. In conclusion, these findings may be helpful for understanding the molecular mechanism of diabetic nephropathy.  

3.1915           Dual Effects of Statins on Aβ Metabolism: Upregulation of the Degradation of APP-CTF and Aβ Clearance

Sato, N., Shinohara, M., Rakugi, H. and Morishita, R. Neurodegenerative Dis., 10(1-4), 305-308 (2012)   Background/Aims: Retrospective cohort studies have suggested that statin users have a lower prevalence of dementia. On the other hand, a randomized controlled study failed to show beneficial effects on the cognitive decline in Alzheimer’s disease (AD). However, a prospective cohort study demonstrated that users of statins had a lower incidence of AD. One possible interpretation might be that statins could prevent or delay the onset of AD, but not slow cognitive decline once the disease has set in. Given that statins could prevent or delay the onset of AD, what is the responsible mechanism? Methods: We investigated the effect of fluvastatin on Aβ metabolism at a clinically relevant dose in mice. Results: Fluvastatin reduced the brain Aβ level by increased trafficking of the carboxyl terminal fragment of the amyloid precursor protein (APP-CTF), which was mediated by inhibition of protein isoprenylation. Moreover, the statin reduced the brain Aβ level through enhanced Aβ clearance mediated by upregulation of low-density lipoprotein receptor-related protein 1 (LRP-1) expression. The statin increased LRP-1 expression, mediated by inhibition of protein isoprenylation. Conclusion: Statins might prevent the onset of AD through reduced Aβ production by enhancement of APP-CTF degradation and/or upregulation of Aβ clearance. We also showed that promotion of APP-CTF degradation and upregulation of Aβ clearance could be modified by a drug, suggesting possible mechanistic targets for disease-modifying drugs.  

3.1916           Autophagosomes induced by a bacterial Beclin 1 binding protein facilitate obligatory intracellular infection

Niu, H., Xiong, Q., Yamamoto, A., Hayashi-Nishino, M. and Rikihisa, Y. PNAS, 109(51), 20800-20807 (2012)   Autophagy, a cytoplasmic catabolic process, plays a critical role in defense against intracellular infection. In turn, evasion or inhibition of autophagy has emerged as an important virulence factor for intracellular pathogens. However, Anaplasma phagocytophilum, the obligatory intracellular bacterium that causes human granulocytic anaplasmosis, replicates in the membrane-bound compartment resembling early autophagosome. Here, we found that Anaplasma translocated substrate 1 (Ats-1), a type IV secretion effector, binds Beclin 1, a subunit of the class III PI3K and Atg14L, and it nucleates autophagosomes with markers of omegasomes, double FYVE-containing protein 1, Atg14L, and LC3. Ats-1 autophagy induction did not activate the starvation signaling pathway of mammalian target of rapamycin. These autophagy proteins were also localized to the Anaplasma inclusion. Ectopically expressed Ats-1 targeted the Anaplasma inclusions and enhanced infection, whereas host cytoplasmic delivery of anti–Ats-1 or Beclin 1 depletion by siRNA suppressed the infection; beclin 1 heterozygous-deficient mice were resistant to Anaplasma infection. Furthermore, Anaplasma growth arrest by the class III PI3K inhibitor 3-methyladenine was alleviated by essential amino acid supplementation. Thus, Anaplasma actively induces autophagy by secreting Ats-1 that hijacks the Beclin 1-Atg14L autophagy initiation pathway likely to acquire host nutrients for its growth.  

3.1917           Proteome Analysis of Cry4Ba Toxin-interacting Aedes aegypti Lipid Rafts using geLC–MS/MS

Bayyareddy, K., Zhu, X., Orlando, r. and Adang, M.J.

1. Proteome Res., 11(12), 5843-5855 (2012)

  Lipid rafts are microdomains in the plasma membrane of eukaryotic cells. Among their many functions, lipid rafts are involved in cell toxicity caused by pore forming bacterial toxins including Bacillus thuringiensis (Bt) Cry toxins. We isolated lipid rafts from brush border membrane vesicles (BBMV) of Aedes aegypti larvae as a detergent resistant membrane (DRM) fraction on density gradients. Cholesterol, aminopeptidase (APN), alkaline phosphatase (ALP) and the raft marker flotillin were preferentially partitioned into the lipid raft fraction. When mosquitocidal Cry4Ba toxin was preincubated with BBMV, Cry4Ba localized to lipid rafts. A proteomic approach based on one-dimensional gel electrophoresis, in-gel trypsin digestion, followed by liquid chromatography–mass spectrometry (geLC–MS/MS) identified a total of 386 proteins. Of which many are typical lipid raft marker proteins including flotillins and glycosylphosphatidylinositol (GPI)-anchored proteins. Identified raft proteins were annotated in silico for functional and physicochemical characteristics. Parameters such as distribution of isoelectric point, molecular mass, and predicted post-translational modifications relevant to lipid raft proteins (GPI anchorage and myristoylation or palmitoylation) were analyzed for identified proteins in the DRM fraction. From a functional point of view, this study identified proteins implicated in Cry toxin interactions as well as membrane-associated proteins expressed in the mosquito midgut that have potential relevance to mosquito biology and vector management.  

3.1918           MeCP2 Binds to 5hmC Enriched within Active Genes and Accessible Chromatin in the Nervous System

Mellen, M., Ayata, P., Dewell, S., Kriaucionis, S. and Heintz, N. Cell, 151(7), 1417-1430 (2012)   The high level of 5-hydroxymethylcytosine (5hmC) present in neuronal genomes suggests that mechanisms interpreting 5hmC in the CNS may differ from those present in embryonic stem cells. Here, we present quantitative, genome-wide analysis of 5hmC, 5-methylcytosine (5mC), and gene expression in differentiated CNS cell types in vivo. We report that 5hmC is enriched in active genes and that, surprisingly, strong depletion of 5mC is observed over these regions. The contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG-binding protein 2 (MeCP2) as the major 5hmC-binding protein in the brain and demonstrate that MeCP2 binds 5hmC- and 5mC-containing DNA with similar high affinities. The Rett-syndrome-causing mutation R133C preferentially inhibits 5hmC binding. These findings support a model in which 5hmC and MeCP2 constitute a cell-specific epigenetic mechanism for regulation of chromatin structure and gene expression.  

3.1919           Phosphorylation of claudin-2 on serine 208 promotes membrane retention and reduces trafficking to lysosomes

Van Itallie, C.M., Tietgens, A.J., LoGrande, K., Aponte, A., Gucek, M. and Anderson, J.M.

1. Cell Sci., 125(20), 4902-4912 (2012)

  Claudins are critical components of epithelial and endothelial tight junction seals, but their post-transcriptional regulation remains poorly understood. Several studies have implicated phosphorylation in control of claudin localisation and/or function, but these have focused on single sites or pathways with differing results, so that it has been difficult to draw general functional conclusions. In this study, we used mass spectrometry (MS) analysis of purified claudin-2 from MDCK II cells and found that the cytoplasmic tail is multiply phosphorylated on serines, a threonine and tyrosines. Phos-tag SDS PAGE revealed that one site, S208, is heavily constitutively phosphorylated in MDCK II cells and in mouse kidney; this site was targeted for further study. Mutational analysis revealed that the phosphomimetic mutant of claudin-2, S208E, was preferentially localised to the plasma membrane while claudin-2 S208A, which could not be phosphorylated at this site, both immunolocalized and co-fractionated with lysosomal markers. Mutations at sites that were previously reported to interfere with plasma membrane targeting of claudin-2 reduced phosphorylation at S208, suggesting that membrane localisation is required for phosphorylation; however phosphorylation at S208 did not affect binding to ZO-1 or ZO-2 Administration of forskolin or PGE2 resulted in dephosphorylation at S208 and transient small increases in transepithelial electrical resistance (TER). Together these data are consistent with phosphorylation at S208 playing a major role in the retention of claudin-2 at the plasma membrane.  

3.1920           The nuclear translocation of endostatin is mediated by its receptor nucleolin in endothelial cells

Song, N., Ding, Y., Zhuo, W., He, T., Fu, Z., Chen, Y., Song, X., Fu, Y. and Luo, Y. Angiogenesis, 15, 697-711 (2012)   Endostatin, the C-terminal fragment of collagen XVIII, is a potent anti-angiogenic factor that significantly modulates the gene expression pattern in endothelial cells. Upon cell surface binding, endostatin can not only function extracellularly, but also translocate to the nucleus within minutes. However, the mechanism by which this occurs is partially understood. Here we systematically investigated the nuclear translocation mechanism of endostatin. By chemical inhibition and RNA interference, we firstly observed that clathrin-mediated endocytosis, but not caveolae-dependent endocytosis or macropinocytosis, is essential for the nuclear translocation of endostatin. We then indentified that nucleolin and integrin α5β1, two widely accepted endostatin receptors, mediate this clathrin-dependent uptake process, which also involves urokinase plasminogen activator receptor (uPAR). Either mutagenesis study, fluorescence resonance energy transfer assay, or fluorescence cell imaging demonstrates that nucleolin and integrin α5β1 interact with uPAR simultaneously upon endostatin stimulation. Blockade of uPAR decreases not only the interaction between nucleolin and integrin α5β1, but also the uptake process, suggesting that the nucleolin/uPAR/integrin α5β1 complex facilitates the internalization of endostatin. After endocytosis, nucleolin further regulates the nuclear transport of endostatin. RNA interference and mutational analysis revealed that the nuclear translocation of endostatin involves the association of nucleolin with importin α1β1 via the nuclear localization sequence. Taken together, this study reveals the pathway by which endostatin translocates to the nucleus and the importance of nucleolin in this process, providing a new perspective for the functional investigation of the nuclear-translocated endostatin in endothelial cells.  

3.1921           Intracellular Delivery and Trafficking Dynamics of a Lymphoma-Targeting Antibody–Polymer Conjugate

Berguig, G.Y., Convertine, A.J., Shi, J., Palanca-Wessels, M.C., Duvall, C.L., Pun, S.H., Press, O.W. and Stayton, P.S. Mol. Pharmaceutics, 9(12), 3506-3514 (2012)   Ratiometric fluorescence and cellular fractionation studies were employed to characterize the intracellular trafficking dynamics of antibody–poly(propylacrylic acid) (PPAA) conjugates in CD22+ RAMOS-AW cells. The HD39 monoclonal antibody (mAb) directs CD22-dependent, receptor-mediated uptake in human B-cell lymphoma cells, where it is rapidly trafficked to the lysosomal compartment. To characterize the intracellular-release dynamics of the polymer–mAb conjugates, HD39-streptavidin (HD39/SA) was dual-labeled with pH-insensitive Alexa Fluor 488 and pH-sensitive pHrodo fluorophores. The subcellular pH distribution of the HD39/SA–polymer conjugates was quantified as a function of time by live-cell fluorescence microscopy, and the average intracellular pH value experienced by the conjugates was also characterized as a function of time by flow cytometry. PPAA was shown to alter the intracellular trafficking kinetics strongly relative to HD39/SA alone or HD39/SA conjugates with a control polymer, poly(methacryclic acid) (PMAA). Subcellular trafficking studies revealed that after 6 h, only 11% of the HD39/SA–PPAA conjugates had been trafficked to acidic lysosomal compartments with values at or below pH 5.6. In contrast, the average intracellular pH of HD39/SA alone dropped from 6.7 ± 0.2 at 1 h to 5.6 ± 0.5 after 3 h and 4.7 ± 0.6 after 6 h. Conjugation of the control polymer PMAA to HD39/SA showed an average pH drop similar to that of HD39/SA. Subcellular fractionation studies with tritium-labeled HD39/SA demonstrated that after 6 h, 89% of HD39/SA was associated with endosomes (Rab5+) and lysosomes (Lamp2+), while 45% of HD39/SA–PPAA was translocated to the cytosol (lactate dehydrogenase+). These results demonstrate the endosomal-releasing properties of PPAA with antibody–polymer conjugates and detail their intracellular trafficking dynamics and subcellular compartmental distributions over time.  

3.1922           Nitric oxide induces segregation of decay accelerating factor (DAF or CD55) from the membrane lipid-rafts and its internalization in human endometrial cells

Banadakoppa, M., Goluszko, P., Liebenthal, D. and Yallampalli, C. Cell Biol. Int., 36(10), 901-907 (2012)   Recent studies suggest that DAF (decay accelerating factor), a complement regulatory protein, present in lipid rafts, is utilized by Dr fimbriated Escherichia coli for their binding and internalization. Previous studies in our laboratory have shown that NO (nitric oxide) can reduce the invasion of Dr(+) E. coli and the severity of uterine infection in pregnant rats. Also, the expression level of DAF both at the mRNA and protein levels has been shown to be reduced by NO. Therefore NO mediated down-regulation of DAF appears to be an important factor in reducing the susceptibility to E. coli infection. However, it is unclear if NO can actually modulate the membrane association of DAF and therefore initial bacterial binding to cells. We found that NO induces the delocalization of DAF from the G_M1-rich lipid rafts. Using biochemical and cell biological approaches in a uterine epithelial cell model (Ishikawa cells), DAF accumulates in caveolae upon exposure to NO. Interaction of DAF with the caveolar protein, caveolin1, leads to their internalization by endosomes. NO-induced delocalization of DAF from the lipid raft and its accumulation in caveolae are mediated through a cGMP (cyclic guanosine monophosphate) pathway. The acute localized synthesis of NO and its influence on DAF localization may represent an important unrecognized phenomenon of host defence against Dr(+) E. coli bacteria, as well as many disease conditions that involve complement system.  

3.1923           Cis-9,trans-11-conjugated linoleic acid affects lipid raft composition and sensitizes human colorectal adenocarcinoma HT-29 cells to X-radiation

Gradzka, I., Sochanowicz, B., Brzoska, K., Wojciuk, G., Sommer, S., Wojewodzka, M., Gasinska, A., Degen, C., Jahreis, G. and Szumiel, I. Biochim. Biophys. Acta, 1830, 2233-2242 (2013)   Background Investigations concerned the mechanism of HT-29 cells radiosensitization by cis-9,trans-11-conjugated linoleic acid (c9,t11-CLA), a natural component of human diet with proven antitumor activity. Methods The cells were incubated for 24 h with 70 μM c9,t11-CLA and then X-irradiated. The following methods were used: gas chromatography (incorporation of the CLA isomer), flow cytometry (cell cycle), cloning (survival), Western blotting (protein distribution in membrane fractions), and pulse-field gel electrophoresis (rejoining of DNA double-strand breaks). In parallel, DNA-PK activity, γ-H2AX foci numbers and chromatid fragmentation were estimated. Gene expression was analysed by RT-PCR and chromosomal aberrations by the mFISH method. Nuclear accumulation of the EGF receptor (EGFR) was monitored by ELISA. Results and conclusions C9,t11-CLA sensitized HT-29 cells to X-radiation. This effect was not due to changes in cell cycle progression or DNA-repair-related gene expression. Post-irradiation DSB rejoining was delayed, corresponding with the insufficient DNA-PK activation, although chromosomal aberration frequencies did not increase. Distributions of cholesterol and caveolin-1 in cellular membrane fractions changed. The nuclear EGFR translocation, necessary to increase the DNA-PK activity in response to oxidative stress, was blocked. We suppose that c9,t11-CLA modified the membrane structure, thus disturbing the intracellular EGFR transport and the EGFR-dependent pro-survival signalling, both functionally associated with lipid raft properties. General Significance The results point to the importance of the cell membrane interactions with the nucleus after injury inflicted by X -rays. Compounds like c9,t11-CLA, that specifically alter membrane properties, could be used to develop new anticancer strategies.  

3.1924           Cell-surface glycosaminoglycans inhibit intranuclear uptake but promote post-nuclear processes of polyamidoamine dendrimer–pDNA transfection

Ziraksaz, Z., Normani, A., Ruponen, M., Soleimani, M., Tabbakhian, M. and Haririan, I. Eur. J. Pharmaceut. Sci., 48, 55-63 (2013)   Background Interaction of cell-surface glycosaminoglycans (GAGs) with non-viral vectors seems to be an important factor which modifies the intracellular destination of the gene complexes. Intracellular kinetics of polyamidoamine (PAMAM) dendrimer as a non-viral vector in cellular uptake, intranuclear delivery and transgene expression of plasmid DNA with regard to the cell-surface GAGs has not been investigated until now. Methods The physicochemical properties of the PAMAM–pDNA complexes were characterized by photon correlation spectroscopy, atomic force microscopy, zeta measurement and agarose gel electrophoresis. The transfection efficiency and toxicity of the complexes at different nitrogen to phosphate (N:P) ratios were determined using various in vitro cell models such as human embryonic kidney cells, chinese hamster ovary cells and its mutants lacking cell-surface GAGs or heparan sulphate proteoglycans (HSPGs). Cellular uptake, nuclear uptake and transfection efficiency of the complexes were determined using flow cytometry and optimized cell-nuclei isolation with quantitative real-time PCR and luciferase assay. Results Physicochemical studies showed that PAMAM dendrimer binds pDNA efficiently, forms small complexes with high positive zeta potential and transfects cells properly at N:P ratios around 5 and higher. The cytotoxicity could be a problem at N:Ps higher than 10. GAGs elimination caused nearly one order of magnitude higher pDNA nuclear uptake and more than 2.6-fold higher transfection efficiency than CHO parent cells. However, neither AUC of nuclear uptake, nor AUC of transfection affected significantly by only cell-surface HSPGs elimination and interesting data related to the effect of GAGs on intranuclear pDNA using PAMAM as delivery vector have been reported in this study. Conclusion Presented data shows that the rate-limiting step of PAMAM–pDNA complexes transfection is located after delivery to the cell nucleus and GAGs are regarded as an inhibitor of the intranuclear delivery step, while slightly promotes transgene expression.  

3.1925           The function of the ATP-binding cassette (ABC) transporter ABCB1 is not susceptible to actin disruption

Meszaros, P., Hummel, I., Klappe, K., Draghiciu, O., Hoekstra, D. and Kok, J.W. Biochim. Biophys. Acta, 1828, 340-351 (2013)   Previously we have shown that the activity of the multidrug transporter ABCC1 (multidrug resistance protein 1), and its localization in lipid rafts, depends on cortical actin (Hummel I, Klappe K, Ercan C, Kok JW. Mol. Pharm. 2011 79, 229–40). Here we show that the efflux activity of the ATP-binding cassette (ABC) family member ABCB1 (P-glycoprotein), did not depend on actin, neither in ABCB1 over expressing murine National Institutes of Health (NIH) 3T3 MDR1 G185 cells nor in human SK-N-FI cells, which endogenously express ABCB1. Disruption of the actin cytoskeleton, upon treatment of the cells with latrunculin B or cytochalasin D, caused severe changes in cell and membrane morphology, and concomitant changes in the subcellular distribution of ABCB1, as revealed by confocal laser scanning and electron microscopy. Nevertheless, irrespective of actin perturbation, the cell surface pool of ABCB1 remained unaltered. In NIH 3T3 MDR1 G185 cells, ABCB1 is partly localized in detergent-free lipid rafts, which partitioned in two different density gradient regions, both enriched in cholesterol and sphingolipids. Interestingly, disruption of the actin cytoskeleton did not change the density gradient distribution of ABCB1. Our data demonstrate that the functioning of ABCB1 as an efflux pump does not depend on actin, which is due to its distribution in both cell surface-localized non-raft membrane areas and lipid raft domains, which do not depend on actin stabilization.  

3.1926           A silicon cell cycle in a bacterial model of calcium phosphate mineralogenesis

Linton, K.M., Tapping, C.R., Adams, D.G., CarterR, D.H., Shore, R.C. and aaron, J.E: Micron, 44, 419-432 (2013)   The prokaryote Corynebacterium matruchotii produces calcium phosphate (bone salt) and may serve as a convenient model for examining individual factors relevant to vertebrate calcification. A factor of current clinical uncertainty is silicon. To investigate its possible role in biomineralisation advanced optical (digital deconvolution and 3D fluorescent image rendering) and electron microscopy (EDX microanalysis and elemental mapping) were applied to calcifying microbial colonies grown in graded Si concentrations (0–60 mM). Cell viability was confirmed throughout by TO-PRO-3-iodide and SYTO-9 nucleic acid staining. It was observed that calcium accumulated in dense intracellular microspherical objects (types i–iii) as nanoparticles (5 nm, type i), nanospheres (30–50 nm, type ii) and filamentous clusters (0.1–0.5 μm, type iii), with a regular transitory Si content evident. With bacterial colony development (7–28 days) the P content increased from 5 to 60%, while Si was displaced from 60 to 5%, distinguishing the phenomenon from random contamination, and with a significant relationship (p < 0.001) found between calcified object number and Si supplementation (optimum 0.01 mM). The Si-containing, intracellular calcified objects (also positive for Mg and negative with Lysensor blue DND-167 for acidocalcisomes) were extruded naturally in bubble-like chains to complete the cycle by coating the cell surface with discrete mineral particles. These could be harvested by lysis, French press and density fractionation when Si was confirmed in a proportion. It was concluded that the unexplained orthopaedic activity of Si may derive from its special property to facilitate calcium phosphorylation in biological systems, thereby recapitulating an ancient and conserved bacterial cycle of calcification via silicification.  

3.1927           Biogenesis of the Vaccinia Virus Membrane: Genetic and Ultrastructural Analysis of the Contributions of the A14 and A17 Proteins

Unger, B., Mercer, J., Boyle, K.A.a nd Traktman, P.

1. Virol., 87(2), 1083-1097 (2013)

  Vaccinia virus membrane biogenesis requires the A14 and A17 proteins. We show here that both proteins can associate with membranes co- but not posttranslationally, and we perform a structure function analysis of A14 and A17 using inducible recombinants. In the absence of A14, electron-dense virosomes and distinct clusters of small vesicles accumulate; in the absence of A17, small vesicles form a corona around the virosomes. When the proteins are induced at 12 h postinfection (hpi), crescents appear at the periphery of the electron-dense virosomes, with the accumulated vesicles likely contributing to their formation. A variety of mutant alleles of A14 and A17 were tested for their ability to support virion assembly. For A14, biologically important motifs within the N-terminal or central loop region affected crescent maturation and the immature virion (IV)→mature virion (MV) transition. For A17, truncation or mutation of the N terminus of A17 engendered a phenotype consistent with the N terminus of A17 recruiting the D13 scaffold protein to nascent membranes. When N-terminal processing was abrogated, virions attempted to undergo the IV-to-MV transition without removing the D13 scaffold and were therefore noninfectious and structurally aberrant. Finally, we show that A17 is phosphorylated exclusively within the C-terminal tail and that this region is a direct substrate of the viral F10 kinase. In vivo, the biological competency of A17 was reduced by mutations that prevented its serine-threonine phosphorylation and restored by phosphomimetic substitutions. Precleavage of the C terminus or abrogation of its phosphorylation diminished the IV→MV maturation; a block to cleavage spared virion maturation but compromised the yield of infectious virus.  

3.1928           Gangliosides Have a Functional Role during Rotavirus Cell Entry

Martinez, M.A., Lopez, S., Arias, C.F. and Isa, P.

1. Virol., 87(2), 1115-1122 (2013)

  Cell entry of rotaviruses is a complex process, which involves sequential interactions with several cell surface molecules. Among the molecules implicated are gangliosides, glycosphingolipids with one or more sialic acid (SA) residues. The role of gangliosides in rotavirus cell entry was studied by silencing the expression of two key enzymes involved in their biosynthesis—the UDP-glucose:ceramide glucosyltransferase (UGCG), which transfers a glucose molecule to ceramide to produce glucosylceramide GlcCer, and the lactosyl ceramide-α-2,3–sialyl transferase 5 (GM3-s), which adds the first SA to lactoceramide-producing ganglioside GM3. Silencing the expression of both enzymes resulted in decreased ganglioside levels (as judged by GM1a detection). Four rotavirus strains tested (human Wa, simian RRV, porcine TFR-41, and bovine UK) showed a decreased infectivity in cells with impaired ganglioside synthesis; however, their replication after bypassing the entry step was not affected, confirming the importance of gangliosides for cell entry of the viruses. Interestingly, viral binding to the cell surface was not affected in cells with inhibited ganglioside synthesis, but the infectivity of all strains tested was inhibited by preincubation of gangliosides with virus prior to infection. These data suggest that rotaviruses can attach to cell surface in the absence of gangliosides but require them for productive cell entry, confirming their functional role during rotavirus cell entry.  

3.1929           Identification of lysosomal sialidase NEU1 and plasma membrane sialidase NEU3 in human erythrocytes

D’Avila, F., Tringali, C., Papini, N., Anastasia, L., Croci, g., Massaccesi, L., Monti, E., Tettamanti, G. and Venerando, B.

1. Cell. Biochem., 114(1), 204-211 (2013)

  The sialylation level of molecules, sialoglycoproteins and gangliosides, protruding from plasma membranes regulates multiple facets of erythrocyte function, from interaction with endothelium to cell lifespan. Our results demonstrate that: (a) Both sialidases NEU1 and NEU3 are present on erythrocyte plasma membrane; (b) NEU1 is kept on the plasma membrane in absence of the protective protein/cathepsin A (PPCA); (c) NEU1 and NEU3 are retained on the plasma membrane, as peripheral proteins, associated to the external leaflet and released by alkaline treatments; (d) NEU1 and NEU3 are segregated in Triton X-100 detergent-resistant membrane domains (DRMs); (e) NEU3 shows activity also at neutral pH; and (f) NEU1 and NEU3 are progressively lost during erythrocyte life. Interestingly, sialidase activity released from erythrocyte membranes after an alkaline treatment preserves its functionality and recognizes sialoglycoproteins and gangliosides. On the other hand, the weak anchorage of sialidases to the plasma membrane and their loss during erythrocyte life could be a tool to preserve the cellular sialic acid content in order to avoid the early ageing of erythrocyte and processes of cell aggregation in the capillaries.  

3.1930           Sortilin and SorLA Display Distinct Roles in Processing and Trafficking of Amyloid Precursor Protein

Gustavsen, C., Glerup, S., Pallesen, L.T., Olsen, D., Andersen, O.M., Nykjær, A., Madsen, P. and Petersen, C.M.

1. Neurosci., 33(1), 64-71 (2013)

  The development and progression of Alzheimer's disease is linked to excessive production of toxic amyloid-β peptide, initiated by β-secretase cleavage of the amyloid precursor protein (APP). In contrast, soluble APPα (sAPPα) generated by the α-secretase is known to stimulate dendritic branching and enhance synaptic function. Regulation of APP processing, and the shift from neurotrophic to neurotoxic APP metabolism remains poorly understood, but the cellular localization of APP and its interaction with various receptors is considered important. We here identify sortilin as a novel APP interaction partner. Like the related APP receptor SorLA, sortilin is highly expressed in the CNS, but whereas SorLA mainly colocalizes with APP in the soma, sortilin interacts with APP in neurites. The presence of sortilin promotes α-secretase cleavage of APP, unlike SorLA, which inhibits the generation of all soluble products. Also, sortilin and SorLA both bind and mediate internalization of sAPP but to different cellular compartments. The interaction involves the 6A domain of APP, present in both neuronal and non-neuronal APP isoforms. This is important as sAPP receptors described so far only bind the non-neuronal isoforms, leaving SorLA and sortilin as the only receptors for sAPP generated by neurons. Together, our findings establish sortilin, as a novel APP interaction partner that influences both production and cellular uptake of sAPP.  

3.1931           Cyclophilin Inhibitors Block Arterivirus Replication by Interfering with Viral RNA Synthesis

De Wilde, A.H., Li, Y., van der Meer, Y., Vuagniaux, G., Lysek, R., Fang, Y., Snijder, E.J. and van Hemert, M.J.

1. Virol., 87(3), 1454-1464 (2013)

  Virus replication strongly depends on cellular factors, in particular, on host proteins. Here we report that the replication of the arteriviruses equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) is strongly affected by low-micromolar concentrations of cyclosporine A (CsA), an inhibitor of members of the cyclophilin (Cyp) family. In infected cells, the expression of a green fluorescent protein (GFP) reporter gene inserted into the PRRSV genome was inhibited with a half-maximal inhibitory concentration (IC₅₀) of 5.2 μM, whereas the GFP expression of an EAV-GFP reporter virus was inhibited with an IC₅₀ of 0.95 μM. Debio-064, a CsA analog that lacks its undesirable immunosuppressive properties, inhibited EAV replication with an IC₅₀ that was 3-fold lower than that of CsA, whereas PRRSV-GFP replication was inhibited with an IC₅₀ similar to that of CsA. The addition of 4 μM CsA after infection prevented viral RNA and protein synthesis in EAV-infected cells, and CsA treatment resulted in a 2.5- to 4-log-unit reduction of PRRSV or EAV infectious progeny. A complete block of EAV RNA synthesis was also observed in an in vitro assay using isolated viral replication structures. The small interfering RNA-mediated knockdown of Cyp family members revealed that EAV replication strongly depends on the expression of CypA but not CypB. Furthermore, upon fractionation of intracellular membranes in density gradients, CypA was found to cosediment with membranous EAV replication structures, which could be prevented by CsA treatment. This suggests that CypA is an essential component of the viral RNA-synthesizing machinery.  

3.1932           Lipid Exchange between Borrelia burgdorferi and Host Cells

Crowley, J.T., Toledo, A.M., LaRocca, T.J., Coleman, J.L., London, E. and Benah, J.L. PloS One, 9(1), e1003109 (2013)   Borrelia burgdorferi, the agent of Lyme disease, has cholesterol and cholesterol-glycolipids that are essential for bacterial fitness, are antigenic, and could be important in mediating interactions with cells of the eukaryotic host. We show that the spirochetes can acquire cholesterol from plasma membranes of epithelial cells. In addition, through fluorescent and confocal microscopy combined with biochemical approaches, we demonstrated that B. burgdorferi labeled with the fluorescent cholesterol analog BODIPY-cholesterol or ³H-labeled cholesterol transfer both cholesterol and cholesterol-glycolipids to HeLa cells. The transfer occurs through two different mechanisms, by direct contact between the bacteria and eukaryotic cell and/or through release of outer membrane vesicles. Thus, two-way lipid exchange between spirochetes and host cells can occur. This lipid exchange could be an important process that contributes to the pathogenesis of Lyme disease  

3.1933           Modulation of dendritic AMPA receptor mRNA trafficking by RNA splicing and editing

Via, L.L., Bonini, D., Russo, I., Orlandi, C., Barlati, S. and Barbon, A.

Nucleic Acids Research, 41(1), 617-631 (2013)

RNA trafficking to dendrites and local translation are crucial processes for superior neuronal functions. To date, several α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) mRNAs have been detected in dendrites and are subject to local protein synthesis. Here, we report the presence of all AMPAR GluA1-4 mRNAs in hippocampal and cortical rat synaptic spines by synaptoneurosomes analysis. In particular, we showed that dendritic AMPAR mRNAs are present in the Flip versions in the cortex and hippocampus. To further confirm these data, we demonstrate, using in situ hybridization, the dendritic localization of the GluA2 Flip isoform in vitro and in vivo, whereas the Flop variant is restricted mainly to the soma. In addition, we report that dendritic AMPA mRNAs are edited at low levels at their R/G sites; this result was also supported with transfection experiments using chimeric GluA2 DNA vectors, showing that transcripts carrying an unedited nucleotide at the R/G site, in combination with the Flip exon, are more efficiently targeted to dendrites when compared with the edited-Flip versions. Our data show that post-transcriptional regulations such as RNA splicing, editing and trafficking might be mutually coordinated and that the localization of different AMPAR isoforms in dendrites might play a functional role in the regulation of neuronal transmission.

 

3.1934           Membrane localization of Junín virus glycoproteins requires cholesterol and cholesterol rich membranes

Cordo, S.M., Valko, A., Martinez, G.M. and Candurra, N.A. Biochem. Biophys. Res. Comm., 430, 912-917 (2013)   Arenavirus morphogenesis and budding occurs at cellular plasma membrane; however, the nature of membrane assembly sites remains poorly understood. In this study we examined the effect of different cholesterol-lowering agents on Junín virus (JUNV) multiplication. We found that cholesterol cell depletion reduced JUNV glycoproteins (GPs) membrane expression and virus budding. Analysis of membrane protein insolubility in Triton X-100 suggested that JUNV GPs associate with cholesterol enriched membranes. Rafts dissociation conditions as warm detergent extraction and cholesterol removal by methyl-β-cyclodextrin compound showed to impair GPs cholesterol enriched membrane association. Analysis of GPs transfected cells showed similar results suggesting that membrane raft association is independent of other viral proteins.  

3.1935           The Potential Role of HMGB1 Release in Peritoneal Dialysis-Related Peritonitis

Cao, s., Li, S., Li, H., Xiong, L., Zhou, Y., Fan, J., Yu, X. and  Mao, H. Plos One, 8(1), e54647(2013)   High mobility group box 1 (HMGB1), a DNA-binding nuclear protein, has been implicated as an endogenous danger signal in the pathogenesis of infection diseases. However, the potential role and source of HMGB1 in the peritoneal dialysis (PD) effluence of patients with peritonitis are unknown. First, to evaluate HMDB1 levels in peritoneal dialysis effluence (PDE), a total of 61 PD patients were enrolled in this study, including 42 patients with peritonitis and 19 without peritonitis. Demographic characteristics, symptoms, physical examination findings and laboratory parameters were recorded. HMGB1 levels in PDE were determined by Western blot and ELISA. The concentrations of TNF-α and IL-6 in PDE were quantified by ELISA. By animal model, inhibition of HMGB1 with glycyrrhizin was performed to determine the effects of HMGB1 in LPS-induced mice peritonitis. In vitro, a human peritoneal mesothelial cell line (HMrSV5) was stimulated with lipopolysaccharide (LPS), HMGB1 extracellular content in the culture media and intracellular distribution in various cellular fractions were analyzed by Western blot or immunofluorescence. The results showed that the levels of HMGB1 in PDE were higher in patients with peritonitis than those in controls, and gradually declined during the period of effective antibiotic treatments. Furthermore, the levels of HMGB1 in PDE were positively correlated with white blood cells (WBCs) count, TNF-α and IL-6 levels. However, pretreatment with glycyrrhizin attenuated LPS-induced acute peritoneal inflammation and dysfunction in mice. In cultured HMrSV5 cells, LPS actively induced HMGB1 nuclear-cytoplasmic translocation and release in a time and dose-dependent fashion. Moreover, cytosolic HMGB1 was located in lysosomes and secreted via a lysosome-mediated secretory pathway following LPS stimulation. Our study demonstrates that elevated HMGB1 levels in PDE during PD-related peritonitis, at least partially, from peritoneal mesothelial cells, which may be involved in the process of PD-related peritonitis and play a critical role in acute peritoneal dysfunction.  

3.1936           Critical Role of S1PR1 and Integrin β4 in HGF/c-Met-mediated Increases in Vascular Integrity

Ephstein, Y., Singleton, P.A., Chen, W., Wang, L., Salgia, R., Kanteti, P., Dudek, S.M., Garcia, J.G.N. and Jacobson, J.R.

1. Biol. Chem., 288(4), 2191-2200 (2013)

  Vascular endothelial cell (EC) barrier integrity is critical to vessel homeostasis whereas barrier dysfunction is a key feature of inflammatory disorders and tumor angiogenesis. We previously reported that hepatocyte growth factor (HGF)-mediated increases in EC barrier integrity are signaled through a dynamic complex present in lipid rafts involving its receptor, c-Met (1). We extended these observations to confirm that S1PR1 (sphingosine 1-phosphate receptor 1) and integrin β4 (ITGB4) are essential participants in HGF-induced EC barrier enhancement. Immunoprecipitation experiments demonstrated HGF-mediated recruitment of c-Met, ITGB4 and S1PR1 to caveolin-enriched lipid rafts in human lung EC with direct interactions of c-Met with both S1PR1 and ITGB4 accompanied by c-Met-dependent S1PR1 and ITGB4 transactivation. Reduced S1PR1 expression (siRNA) attenuated both ITGB4 and Rac1 activation as well as c-Met/ITGB4 interaction and resulted in decreased transendothelial electrical resistance. Furthermore, reduced ITGB4 expression attenuated HGF-induced c-Met activation, c-Met/S1PR1 interaction, and effected decreases in S1P- and HGF-induced EC barrier enhancement. Finally, the c-Met inhibitor, XL880, suppressed HGF-induced c-Met activation as well as S1PR1 and ITGB4 transactivation. These results support a critical role for S1PR1 and ITGB4 transactivation as rate-limiting events in the transduction of HGF signals via a dynamic c-Met complex resulting in enhanced EC barrier integrity.  

3.1937           Identification of glycosyltransferases involved in cell wall synthesis of wheat endosperm

Suliman, M., Chateigner-Boutin, A.L., Francin-Allami, M., Partier, A., Bouchet, B., Salse, J., Pont, C., Marion, J., Rogniaux, H., Tessier, D., Guillon, F. and Larre, C.

1. Proteomics, 78, 508-521 (2013)

  Plant cell walls are complex structures critical for plant fitness and valuable for human nutrition as dietary fiber and for industrial uses such as biofuel production. The cell wall polysaccharides in wheat endosperm consist of two major polymers, arabinoxylans and beta-glucans, as well as other minor components. Most of these polysaccharides are synthesized in the Golgi apparatus but the mechanisms underlying their synthesis have yet to be fully elucidated and only a few of the enzymes involved have been characterized. To identify actors involved in the wheat endosperm cell wall formation, we used a subcellular fractionation strategy to isolate Golgi-enriched fractions from endosperm harvested during active cell wall deposition. The proteins extracted from these Golgi-enriched fractions were analyzed by LC–MS/MS. We report the identification of 1135 proteins among which 64 glycosyltransferases distributed in 17 families. Their potential function in cell wall synthesis is discussed. In addition, we identified 63 glycosylhydrolases, some of which may be involved in cell wall remodeling. Several glycosyltransferases were validated by showing that when expressed as fusion proteins with a fluorescent reporter, they indeed accumulate in the Golgi apparatus. Our results provide new candidates potentially involved in cell wall biogenesis in wheat endosperm.  

3.1938           Staphylococcus aureus α-Toxin-Dependent Induction of Host Cell Death by Membrane-Derived Vesicles

Thay, B., Wai, S.N. and Oscarsson, J. PloS One, 8(1), e54661 (2013)   Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs), which analogously to outer membrane vesicles (OMVs) of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol-dependent fusion of S. aureus MVs with the plasma membrane represents a route for delivery of a key virulence factor, α-toxin (α-hemolysin; Hla) to human cells. Most S. aureus strains produce this 33-kDa pore-forming protein, which can lyse a wide range of human cells, and induce apoptosis in T-lymphocytes. Our results revealed a tight association of biologically active α-toxin with membrane-derived vesicles isolated from S. aureus strain 8325-4. Concomitantly, α-toxin contributed to HeLa cell cytotoxicity of MVs, and was the main vesicle-associated protein responsible for erythrocyte lysis. In contrast, MVs obtained from an isogenic hla mutant were significantly attenuated with regards to both causing lysis of erythrocytes and death of HeLa cells. This is to our knowledge the first recognition of an S. aureus MV-associated factor contributing to host cell cytotoxicity    

3.1939           Ataxia with Cerebellar Lesions in Mice Expressing Chimeric PrP-Dpl Protein

Lemaire-Vieille, C., Bailly, Y., Erlich, P., Loeuillet, C., Brocard, J., Haeberle, A.M., Bombarde, G., Rak, C., Demais, V., Dumestre-Perard, C., Gagnon, J. and Cesbron, J-Y.

1. Neurosci., 33(4), 1391-1399 (2013)

  Mutations within the central region of prion protein (PrP) have been shown to be associated with severe neurotoxic activity similar to that observed with Dpl, a PrP-like protein. To further investigate this neurotoxic effect, we generated lines of transgenic (Tg) mice expressing three different chimeric PrP-Dpl proteins. Chi1 (amino acids 1–57 of Dpl replaced by amino acids 1–125 of PrP) and Chi2 (amino acids 1–66 of Dpl replaced by amino acids 1–134 of PrP) abrogated the pathogenicity of Dpl indicating that the presence of a N-terminal domain of PrP (23–134) reduced the toxicity of Dpl, as reported. However, when the amino acids 1–24 of Dpl were replaced by amino acids 1–124 of PrP, Chi3 Tg mice, which express the chimeric protein at a very low level, start developing ataxia at the age of 5–7 weeks. This phenotype was not counteracted by a single copy of full-length-PrP^c but rather by its overexpression, indicating the strong toxicity of the chimeric protein Chi3. Chi3 Tg mice exhibit severe cerebellar atrophy with a significant loss of granule cells. We concluded that aa25 to aa57 of Dpl, which are not present in Chi1 and Chi2 constructs, confer toxicity to the protein. We tested this possibility by using the 25–57 Dpl peptide in primary culture of mouse embryo cortical neurons and found a significant neurotoxic effect. This finding identifies a protein domain that plays a role in mediating Dpl-related toxicity.  

3.1940           Acidocalcisomes of Trypanosoma brucei have an inositol 1,4,5-trisphosphate receptor that is required for growth and infectivity

Huang, G., Bartlett, P.J., Thomas, A.P., Moreno, S.N.J. and Docampo, R. PNAS, 110(5), 1887-1892 (2013)   Acidocalcisomes are acidic calcium stores rich in polyphosphate and found in a diverse range of organisms. The mechanism of Ca²⁺ release from these organelles was unknown. Here we present evidence that Trypanosoma brucei acidocalcisomes possess an inositol 1,4,5-trisphosphate receptor (TbIP₃R) for Ca²⁺ release. Localization studies in cell lines expressing TbIP₃R in its endogenous locus fused to an epitope tag revealed its partial colocalization with the vacuolar proton pyrophosphatase, a marker of acidocalcisomes. IP₃ was able to stimulate Ca²⁺ release from a chicken B-lymphocyte cell line in which the genes for all three vertebrate IP₃Rs have been stably ablated (DT40-3KO) and that were stably expressing TbIP₃R, providing evidence of its function. IP₃ was also able to release Ca²⁺ from permeabilized trypanosomes or isolated acidocalcisomes and photolytic release of IP₃ in intact trypanosomes loaded with Fluo-4 elicited a transient Ca²⁺ increase in their cytosol. Ablation of TbIP₃R by RNA interference caused a significant reduction of IP₃-mediated Ca²⁺ release in trypanosomes and resulted in defects in growth in culture and infectivity in mice. Taken together, the data provide evidence of the presence of a functional IP₃R as a Ca²⁺ release channel in acidocalcisomes of trypanosomes and suggest that a Ca²⁺ signaling pathway that involves acidocalcisomes is required for growth and establishment of infection.  

3.1941           Osmotic shock-dependent redistribution of diacylglycerol kinase 1 to non-ionic detergent-resistant membrane via pleckstrin homology and C1 domains

Matsutomo, D., Isozaki, T., Sakai, H. and Sakane, F.

1. Biochem., 153(2), 179-190 (2013)

  Diacylglycerol kinase (DGK) participates in regulating the intracellular concentrations of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKη1 is a type II isozyme that contains a pleckstrin homology (PH) domain and a pair of C1 domains at the N-terminus and separated catalytic domains (catalytic subdomain-a and b). We previously reported that DGKη1 expressed in COS-7 cells is translocated from the cytoplasm to punctate granules that partially include endosomes in response to stress stimuli such as osmotic shock. However, the biochemical properties of the stress-dependent behaviour of DGKη1 remain unknown. Here, we have found that DGKη1 is redistributed from the cytosol to the non-ionic detergent (Nonidet P-40)-resistant membrane (DRM) in response to osmotic shock. Our results strongly suggested that the Nonidet P-40 insolubility of DGKη1 is due to neither cytoskeleton localization nor lipid raft association, implying that DGKη1 is distributed to detergent-resistant membrane microdomains that have a low lipid-to-protein ratio. We revealed, using a series of DGKη1 deletion mutants, that the PH and C1 domains play a pivotal role in osmotic shock-dependent DRM redistribution, whereas catalytic subdomain-a negatively regulates the event.  

3.1942           Pex11α deficiency impairs peroxisome elongation and division and contributes to nonalcoholic fatty liver in mice

Weng, H., Ji, X., Naito, Y., Endo, K., Ma, X., Takahashi, R., Shen, C., Hirokawa, G., Fukushima, Y. and Iwai, N. Am. J. Physiol. Endocrinol. Metab., 304, E187-E196 (2013)   Hepatic triglyceride (TG) accumulation is considered to be a prerequisite for developing nonalcoholic fatty liver (NAFL). Peroxisomes have many important functions in lipid metabolism, including fatty acid β-oxidization. However, the pathogenic link between NAFL and peroxisome biogenesis remains unclear. To examine the molecular and physiological functions of the Pex11α gene, we disrupted this gene in mice. Body weights and hepatic TG concentrations in Pex11α^−/− mice were significantly higher than those in wild-type (WT) mice fed a normal or a high-fat diet. Hepatic TG concentrations in fasted Pex11α^−/− mice were significantly higher than those in fasted WT mice. Plasma TG levels increased at lower rates in Pex11α^−/− mice than in WT mice after treatment with the lipoprotein lipase inhibitor tyloxapol. The number of peroxisomes was lower in the livers of Pex11α^−/− mice than in those of WT mice. Ultrastructural analysis showed that small and regular spherically shaped peroxisomes were more prevalent in Pex11α^−/− mice fed normal chow supplemented without or with fenofibrate. We observed a significantly higher ratio of empty peroxisomes containing only PMP70, a peroxisome membrane protein, but not catalase, a peroxisome matrix protein, in Pex11α^−/− mice. The mRNA expression levels of peroxisomal fatty acid oxidation-related genes (ATP-binding cassette, subfamily D, member 2, and acyl-CoA thioesterase 3) were significantly higher in WT mice than those in Pex11α^−/− mice under fed conditions. Our results demonstrate that Pex11α deficiency impairs peroxisome elongation and abundance and peroxisomal fatty acid oxidation, which contributes to increased lipid accumulation in the liver.  

3.1943           JAK2-V617F-mediated signalling is dependent on lipid rafts and statins inhibit JAK2-V617F-dependent cell growth

Griner, L.N., McGraw, K.L., Johnson, J.O., List, A.F. and Reuther, G.W. Br. J. Hematol., 160(2), 177-187 (2013)   Aberrant JAK2 signalling plays an important role in the aetiology of myeloproliferative neoplasms (MPNs). JAK2 inhibitors, however, do not readily eliminate neoplastic MPN cells and thus do not induce patient remission. Further understanding JAK2 signalling in MPNs may uncover novel avenues for therapeutic intervention. Recent work has suggested a potential role for cellular cholesterol in the activation of JAK2 by the erythropoietin receptor and in the development of an MPN-like disorder in mice. Our study demonstrates for the first time that the MPN-associated JAK2-V617F kinase localizes to lipid rafts and that JAK2-V617F-dependent signalling is inhibited by lipid raft disrupting agents, which target membrane cholesterol, a critical component of rafts. We also show for the first time that statins, 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, widely used to treat hypercholesterolaemia, induce apoptosis and inhibit JAK2-V617F-dependent cell growth. These cells are more sensitive to statin treatment than non-JAK2-V617F-dependent cells. Importantly, statin treatment inhibited erythropoietin-independent erythroid colony formation of primary cells from MPN patients, but had no effect on erythroid colony formation from healthy individuals. Our study is the first to demonstrate that JAK2-V617F signalling is dependent on lipid rafts and that statins may be effective in a potential therapeutic approach for MPNs.  

3.1944           Alterations in ventricular KATP channel properties during aging

Bao, L., Taskin, E., Foster, M., Ray, B., Rosario, R., ananthakrishnan, R., Howlett, S.E., Schmidt, A.M., Ramasamy, R. and Coetzee, W.A. Aging Cell, 12(1), 167-176 (2013)   Coronary heart disease remains the principle cause of mortality in the United States. During aging, the efficiency of the cardiovascular system is decreased and the aged heart is less tolerant to ischemic injury. ATP-sensitive K⁺ (K_ATP) channels protect the myocardium against ischemic damage. We investigated how aging affects cardiac K_ATP channels in the Fischer 344 rat model. Expression of K_ATP channel subunit mRNA and protein levels was unchanged in hearts from 26-month-old vs. 4-month-old rats. Interestingly, the mRNA expression of several other ion channels (> 80) was also largely unchanged, suggesting that posttranscriptional regulatory mechanisms occur during aging. The whole-cell K_ATP channel current density was strongly diminished in ventricular myocytes from aged male rat hearts (also observed in aged C57BL/6 mouse myocytes). Experiments with isolated patches (inside-out configuration) demonstrated that the K_ATP channel unitary conductance was unchanged, but that the inhibitory effect of cytosolic ATP on channel activity was enhanced in the aged heart. The mean patch current was diminished, consistent with the whole-cell data. We incorporated these findings into an empirical model of the K_ATP channel and numerically simulated the effects of decreased cytosolic ATP levels on the human action potential. This analysis predicts lesser activation of K_ATP channels by metabolic impairment in the aged heart and a diminished action potential shortening. This study provides insights into the changes in K_ATP channels during aging and suggests that the protective role of these channels during ischemia is significantly compromised in the aged individual  

3.1945           Role of polymerase η in mitochondrial mutagenesis of Saccharomyces cerevisiae

Chatterje, N., Pabla, R. and Siede, W. Biochem. Biophys. Res. Comm., 431, 270-273 (2013)   DNA polymerase η mostly catalyzes an error-free bypass of the most frequent UV lesions, pyrimidine dimers of the cyclobutane-type. In addition to its nuclear localization, we show here for the first time its mitochondrial localization in budding yeast. In mitochondria, this polymerase improves bypass replication fidelity opposite UV damage as shown in base pair substitution and frameshift assays. For base pair substitutions, polymerase η appears to be related in function and epistatic to DNA polymerase ζ which, however, plays the opposite role in the nucleus.  

3.1946           Mechanistic insights of intestinal absorption and renal conservation of folate in chronic alcoholism

Wani, N.A., Thakur, S., Najar, R.A., Nada, R., Khanduja, K.L. and Kaur, J. Alcohol, 47, 121-130 (2013)   Folate mediated one-carbon metabolism is of fundamental importance for various cellular processes, including DNA synthesis and methylation of biological molecules. Due to the exogenous requirement of folate in mammals, there exists a well developed epithelial folate transport system for regulation of normal folate homeostasis. The intestinal and renal folate uptake is tightly and diversely regulated and disturbances in folate homeostasis like in alcoholism have pathological consequences. The study was sought to delineate the regulatory mechanism of folate uptake in intestine and reabsorption in renal tubular cells that could evaluate insights of malabsorption during alcoholism. The folate transporters PCFT and RFC were found to be associated with lipid rafts of membrane surfaces in intestine and kidney. Importantly, the observed lower intestinal and renal folate uptake was associated with decreased levels of folate transporter viz. PCFT and RFC in lipid rafts of intestinal and renal membrane surfaces. The decreased association of folate transporters in lipid rafts was associated with decreased protein and mRNA levels. In addition, immunohistochemical studies showed that alcoholic conditions deranged that localization of PCFT and RFC. These findings could explain the possible mechanistic insights that may result in folate malabsorption during alcoholism.  

3.1947           Production of Outer Membrane Vesicles and Outer Membrane Tubes by Francisella novicida

McCaig, W.D., Koller, A. and Thanassia, D.G.

1. Bacteriol., 195(6), 1120-1132 (2013)

  Francisella spp. are highly infectious and virulent bacteria that cause the zoonotic disease tularemia. Knowledge is lacking for the virulence factors expressed by Francisella and how these factors are secreted and delivered to host cells. Gram-negative bacteria constitutively release outer membrane vesicles (OMV), which may function in the delivery of virulence factors to host cells. We identified growth conditions under which Francisella novicida produces abundant OMV. Purification of the vesicles revealed the presence of tube-shaped vesicles in addition to typical spherical OMV, and examination of whole bacteria revealed the presence of tubes extending out from the bacterial surface. Recently, both prokaryotic and eukaryotic cells have been shown to produce membrane-enclosed projections, termed nanotubes, which appear to function in cell-cell communication and the exchange of molecules. In contrast to these previously characterized structures, the F. novicida tubes are produced in liquid as well as on solid medium and are derived from the OM rather than the cytoplasmic membrane. The production of the OMV and tubes (OMV/T) by F. novicida was coordinately regulated and responsive to both growth medium and growth phase. Proteomic analysis of purified OMV/T identified known Francisella virulence factors among the constituent proteins, suggesting roles for the vesicles in pathogenesis. In support of this, production of OM tubes by F. novicida was stimulated during infection of macrophages and addition of purified OMV/T to macrophages elicited increased release of proinflammatory cytokines. Finally, vaccination with purified OMV/T protected mice from subsequent challenge with highly lethal doses of F. novicida.  

3.1948           New Type of Outer Membrane Vesicle Produced by the Gram-Negative Bacterium Shewanella vesiculosa M7^T: Implications for DNA Content

Perez-Cruz, C., Carrion, O., Delgado, L., Martinez, G., Lopez-Iglesias, C. and Mercade, e. Appl. Environ. Microbiol., 79(6), 1874-1881 (2013)   Outer membrane vesicles (OMVs) from Gram-negative bacteria are known to be involved in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. An ultrastructural study of the Antarctic psychrotolerant bacterium Shewanella vesiculosa M7^T has revealed that this Gram-negative bacterium naturally releases conventional one-bilayer OMVs through a process in which the outer membrane is exfoliated and only the periplasm is entrapped, together with a more complex type of OMV, previously undescribed, which on formation drag along inner membrane and cytoplasmic content and can therefore also entrap DNA. These vesicles, with a double-bilayer structure and containing electron-dense material, were visualized by transmission electron microscopy (TEM) after high-pressure freezing and freeze-substitution (HPF-FS), and their DNA content was fluorometrically quantified as 1.8 ± 0.24 ng DNA/μg OMV protein. The new double-bilayer OMVs were estimated by cryo-TEM to represent 0.1% of total vesicles. The presence of DNA inside the vesicles was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. In addition, a proteomic study of purified membrane vesicles confirmed the presence of plasma membrane and cytoplasmic proteins in OMVs from this strain. Our data demonstrate the existence of a previously unobserved type of double-bilayer OMV in the Gram-negative bacterium Shewanella vesiculosa M7^T that can incorporate DNA, for which we propose the name outer-inner membrane vesicle (O-IMV).  

3.1949           Caveolin-1 Regulates Endothelial Adhesion of Lung Cancer Cells via Reactive Oxygen Species-Dependen Mechanism

Chanvorachote, P. and Chunhacha, P. PloS One, 8(2), e57466 (2013)   The knowledge regarding the role of caveolin-1 (Cav-1) protein on endothelium adhesion of cancer cells is unclear. The present study revealed that Cav-1 plays a negative regulatory role on cancer-endothelium interaction. Endogenous Cav-1 was shown to down-regulate during cell detachment and the level of such a protein was conversely associated with tumorendothelial adhesion. Furthermore, the ectopic overexpression of Cav-1 attenuated the ability of the cancer cells to adhere to endothelium while shRNA-mediated Cav-1 knock-down exhibited the opposite effect. We found that cell detachment increased cellular hydrogen peroxide and hydroxyl radical generation and such reactive oxygen species (ROS) were responsible for the increasing interaction between cancer cells and endothelial cells through vascular endothelial cell adhesion molecule-1 (VCAM-1). Importantly, Cav-1 was shown to suppress hydrogen peroxide and hydroxyl radical formation by sustaining the level of activated Akt which was critical for the role of Cav-1 in attenuating the cell adhesion. Together, the present study revealed the novel role of Cav-1 and underlying mechanism on tumor adhesion which explain and highlight an important role of Cav-1 on lung cancer cell metastasis.  

3.1950           A Bicyclic 1-Deoxygalactonojirimycin Derivative as a Novel Pharmacological Chaperone for GM₁ Gangliosidosis

Takai, T., Higaki,, K., Aguilar-Moncayo, M., mena-Barragan, T., Hirano, Y., Yura, K., Yu, L., Ninomiiya, H., Garcia-Moreno, M.I., Sakakibara, Y., Ohno, K., Nanba, E., Mellet, C.O., Garcia-Fernandez, J.M. and Suzuki, Y. Molecular Therapy, 21(3), 526-532 (2013)   Lysosomal β-galactosidase (β-Gal) deficiency causes a group of disorders that include neuronopathic GM₁ gangliosidosis and non-neuronopathic Morquio B disease. We have previously proposed the use of small molecule ligands of β-Gal as pharmacological chaperones (PCs) for the treatment of GM₁ gangliosidosis brain pathology. Although it is still under development, PC therapy has yielded promising preclinical results in several lysosomal diseases. In this study, we evaluated the effect of bicyclic 1-deoxygalactonojirimycin (DGJ) derivative of the sp²-iminosugar type, namely 5N,6S-(N′-butyliminomethylidene)-6-thio-1- deoxygalactonojirimycin (6S-NBI-DGJ), as a novel PC for human mutant β-Gal. In vitro, 6S-NBI-DGJ had the ability to inhibit the activity of human β-Gal in a competitive manner and was able to protect this enzyme from heat-induced degradation. Computational analysis supported that the rigid glycone bicyclic core of 6S-NBI-DGJ binds to the active site of the enzyme, with the aglycone N′-butyl substituent, in a precise E-orientation, located at a hydrophobic region nearby. Chaperone potential profiling indicated significant increases of enzyme activity in 24 of 88 β-Gal mutants, including four common mutations. Finally, oral administration of 6S-NBI-DGJ ameliorated the brain pathology of GM₁ gangliosidosis model mice. These results suggest that 6S-NBI-DGJ is a novel PC that may be effective on a broad range of β-Gal mutants.  

3.1951           Sequences within RNA coding for HIV-1 Gag p17 are efficiently targeted to Exosomes

Cabezas, S.C. and Federico, M. Cell. Microbiol., 15(3), 412-429 (2013)   HIV budding requires the interaction with cell factors involved in the biogenesis of exosomes. This implies the possibility that viral products undergo exosome incorporation. While this has been already described for both Gag and Nef HIV-1 proteins, no conclusive results on HIV genome have been produced so far. Here, we report that unspliced, but not single or double spliced, HIV-1 RNA species are incorporated in exosomes. Deletion mutant analysis indicated that the presence of a stretch of sequences within the 5′ end of the Gag p17 open reading frame is sufficient for HIV-1 RNA exosome incorporation. These sequences were found associating with exosomes also out of the HIV-1 context, thus indicating that the diversion towards the vesicular compartment can occur without need of additional HIV-1 sequences. Finally, the incorporation of genomic HIV-1 RNA in exosomes significantly increased when producer cells express HIV-1 defective for viral genome packaging. Manipulating infected cells to favour the selective incorporation in exosomes of genomic HIV-1 RNA might have therapeutic implications.  

3.1952           Visceral Adipose Tissue-derived Serine Proteinase Inhibitor Inhibits Apoptosis of Endothelial Cells as a Ligand for the Cell-Surface GRP78/Voltage-dependent Anion Channel Complex

Nakatsuka, A., Wada, J., Iseda, I., Teshigawara, S., Higashio, K., Murakami, K., Kanzaki, M., Inoue, K., Terami, T., Katayama, A., Hida, K., Eguchi, J., Ogawa, D., Matsuki, Y., Hiramatsu, R., Yagita, H., Kakuta, S., Iwakura, Y. and Makino, H. Circ. Res., 112(5), 771-780 (2013)   Rationale: Visceral adipose tissue-derived serine proteinase inhibitor (vaspin) is an adipokine identified from visceral adipose tissues of genetically obese rats. Objective: The role of vaspin in the diabetic vascular complications remains elusive, and we investigated the effects of vaspin on the vascular function under the diabetic milieu. Methods and Results: Adenovirus carrying the full length of the vaspin gene (Vaspin-Ad) ameliorated intimal proliferation of balloon-injured carotid arteries in diabetic Wistar rats. The expression of Ccl2, Pdgfb, and Pdgfrb genes was significantly reduced by the treatment of Vaspin-Ad. In cuff-injured femoral arteries, the intimal proliferation was ameliorated in vaspin transgenic (Vaspin Tg) mice. The application of recombinant vaspin and Vaspin-Ad promoted the proliferation and inhibited the apoptosis of human aortic endothelial cells. Adenovirus expressing vaspin with calmodulin and streptavidin-binding peptides was applied to human aortic endothelial cells, subjected to tandem tag purification and liquid chromatography-tandem mass spectrometry, and we identified GRP78 (78-kDa glucose-regulated protein) as an interacting molecule. The complex formation of vaspin, GRP78, and voltage-dependent anion channel on the plasma membrane was confirmed by the immunoprecipitation studies using aortas of Vaspin Tg mice. The binding assay using ¹²⁵I-vaspin in human aortic endothelial cells revealed high-affinity binding (dissociation constant = 0.565×10^–9 m) by the treatment of 5 μM thapsigargin, which recruited GRP78 from the endoplasmic reticulum to plasma membrane by inducing endoplasmic reticulum stress. In human aortic endothelial cells, vaspin induced phosphorylation of Akt and inhibited the kringle 5-induced Ca²⁺ influx and subsequent apoptosis. Conclusions: Vaspin is a novel ligand for the cell-surface GRP78/voltage-dependent anion channel complex in endothelial cells and promotes proliferation, inhibits apoptosis, and protects vascular injuries in diabetes mellitus.  

3.1953           Ablation of very long acyl chain sphingolipids causes hepatic insulin resistance in mice due to altered detergent-resistant membranes

Park, J-W., Park, W-J., Kuperman, Y., Boura-Halfon, S., Pewzner-Jung, Y. and Futerman, A.H. Hepatology, 57(2), 525-532 (2013)   Sphingolipids are important structural components of cell membranes and act as critical regulators of cell function by modulating intracellular signaling pathways. Specific sphingolipids, such as ceramide, glucosylceramide, and ganglioside GM3, have been implicated in various aspects of insulin resistance, because they have been shown to modify several steps in the insulin signaling pathway, such as phosphorylation of either protein kinase B (Akt) or of the insulin receptor. We now explore the role of the ceramide acyl chain length in insulin signaling by using a ceramide synthase 2 (CerS2) null mouse, which is unable to synthesize very long acyl chain (C22-C24) ceramides. CerS2 null mice exhibited glucose intolerance despite normal insulin secretion from the pancreas. Both insulin receptor and Akt phosphorylation were abrogated in liver, but not in adipose tissue or in skeletal muscle. The lack of insulin receptor phosphorylation in liver correlated with its inability to translocate into detergent-resistant membranes (DRMs). Moreover, DRMs in CerS2 null mice displayed properties significantly different from those in wild-type mice, suggesting that the altered sphingolipid acyl chain length directly affects insulin receptor translocation and subsequent signaling. Conclusion: We conclude that the sphingolipid acyl chain composition of liver regulates insulin signaling by modifying insulin receptor translocation into membrane microdomains  

3.1954           Sex-specific response of rat costochondral cartilage growth plate chondrocytes to 17β-estradiol involves differential regulation of plasma membrane associated estrogen receptors

Elbaradie, K.B:Y., Wang, Y., Boyan, B.D. and Schwartz, Z. Biochim. Biophys. Acta, 1833, 1165-1172 (2013)   Both male and female rat growth plate chondrocytes express estrogen receptors (ERs); however 17β-estradiol (E₂) induces membrane responses leading to activation of phospholipase A₂ (PLA₂), phospholipase C (PLC), prostaglandin E₂ (PGE₂) production, protein kinase C (PKC), and ultimately mitogen protein kinase (MAPK) only in female cells. This study investigated if these sex-specific responses are due to differences in the actual ERs or in downstream signaling. Western blots and flow cytometry of costochondral cartilage resting zone chondrocytes (RCs) showed 2–3 times more ERα in plasma membranes (PMs) from female cells than male cells. Tunicamycin blocked E₂-dependent ER-translocation to the PM, indicating palmitoylation was required. Co-immunoprecipitation showed E₂ induced complex formation between ER isoforms only in female RCs. To examine if the lack of response in PKC and PGE₂ in males is due to differences in signaling, we examined involvement of ERs and the role of PLC and PLA₂. Selective ERα (propylpyrazole triol, PPT) and ERβ (diarylproprionitrile, DPN) agonists activated PKC in female RCs only. The PLC inhibitor, U73122 blocked E₂'s effect on PKC and the cytosolic PLA₂ inhibitor, AACOCF3 inhibited the effect on PGE₂ in female RCs, confirming involvement of PLC and PLA₂ in the mechanism. The PLC activator, m-3M3FβS activated PKC and PLAA peptide increased PGE₂ levels in male and female RCs, showing that the signaling pathways are present. These data indicate that differences in membrane ER amount, localization, translocation and interaction are responsible for the sexual dimorphic response to E₂.  

3.1955           Protein Kinase Cε Modulates Insulin Receptor Localization and Trafficking in Mouse Embryonic Fibroblasts

Pedersen, D.J., Diakanastasis, B., Stöckli, J. and Schmitz-Peiffer, C. PloS One, 8(3), e58046 (2013)   We have previously shown that deletion of protein kinase C epsilon (PKCε) in mice results in protection against glucose intolerance caused by a high fat diet. This was in part due to reduced insulin uptake by hepatocytes and insulin clearance, which enhanced insulin availability. Here we employed mouse embryonic fibroblasts (MEFs) derived from wildtype (WT) and PKCε-deficient (PKCε^−/−) mice to examine this mechanistically. PKCε^−/− MEFs exhibited reduced insulin uptake which was associated with decreased insulin receptor phosphorylation, while downstream signalling through IRS-1 and Akt was unaffected. Cellular fractionation demonstrated that PKCε deletion changed the localization of the insulin receptor, a greater proportion of which co-fractionated with flotillin-1, a marker of membrane microdomains. Insulin stimulation resulted in redistribution of the receptor in WT cells, while this was markedly reduced in PKCε^−/− cells. These alterations in insulin receptor trafficking were associated with reduced expression of CEACAM1, a receptor substrate previously shown to modulate insulin clearance. Virally-mediated reconstitution of PKCε in MEFs increased CEACAM1 expression and partly restored the sensitivity of the receptor to insulin-stimulated redistribution. These data indicate that PKCε can affect insulin uptake in MEFs through promotion of receptor-mediated endocytosis, and that this may be mediated by regulation of CEACAM1 expression.  

3.1956           A genomic toolkit to investigate kinesin and myosin motor function in cells

Maliga, Z., Junqueira, M., Toyoda, Y., Ettinger, A., Mora-Bermudez, F., Klemm, R.W., Vasilj, A., Guhr, E., Ibarlucea-Benitzer, I., Poser, I., Bonifacio, E., Huttner, W., Shevchenko, A. and Hyman, A.A. Nature Cell Biol., 15(3), 325-334 (2013)   Coordination of multiple kinesin and myosin motors is required for intracellular transport, cell motility and mitosis. However, comprehensive resources that allow systems analysis of the localization and interplay between motors in living cells do not exist. Here, we generated a library of 243 amino- and carboxy-terminally tagged mouse and human bacterial artificial chromosome transgenes to establish 227 stably transfected HeLa cell lines, 15 mouse embryonic stem cell lines and 1 transgenic mouse line. The cells were characterized by expression and localization analyses and further investigated by affinity-purification mass spectrometry, identifying 191 candidate protein–protein interactions. We illustrate the power of this resource in two ways. First, by characterizing a network of interactions that targets CEP170 to centrosomes, and second, by showing that kinesin light-chain heterodimers bind conventional kinesin in cells. Our work provides a set of validated resources and candidate molecular pathways to investigate motor protein function across cell lineages.  

3.1957           Cross-talk between EGFR and T-cadherin: EGFR activation promotes T-cadherin localization to intercellular contacts

Kyriakakis, E., Maslova, K., Frachet, A., Ferri, N., Contini, A., Pfaff, D., Erne, P., Resink, T.J. and Philippova, M. Cellular Signalling, 25, 1044-1053 (2013)   Reciprocal cross-talk between receptor tyrosine kinases (RTKs) and classical cadherins (e.g. EGFR/E-cadherin, VEGFR/VE-cadherin) has gained appreciation as a combinatorial molecular mechanism enabling diversification of the signalling environment and according differential cellular responses. Atypical glycosylphosphatidylinositol (GPI)-anchored T-cadherin (T-cad) was recently demonstrated to function as a negative auxiliary regulator of EGFR pathway activation in A431 squamous cell carcinoma (SCC) cells. Here we investigate the reciprocal impact of EGFR activation on T-cad. In resting A431 T-cad was distributed globally over the cell body. Following EGF stimulation T-cad was redistributed to the sites of cell–cell contact where it colocalized with phosphorylated EGFR^Tyr1068. T-cad redistribution was not affected by endomembrane protein trafficking inhibitor brefeldin A or de novo protein synthesis inhibitor cycloheximide, supporting mobilization of plasma membrane associated T-cad. EGF-induced relocalization of T-cad to cell–cell contacts could be abrogated by specific inhibitors of EGFR tyrosine kinase activity (gefitinib or lapatinib), lipid raft integrity (filipin), actin microfilament polymerization (cytochalasin D or cytochalasin B), p38MAPK (SB203580) or Rac1 (compound4). Erk1/2 inhibitor PD98059 increased phospho-EGFR^tyr1068 levels and not only amplified effects of EGF but also per se promoted some relocalization of T-cad to cell–cell contacts. Rac1 activation by EGF was inhibited by gefitinib, lapatinib or SB203580 but amplified by PD98059. Taken together our data suggest that T-cad translocation to cell–cell contacts is sensitive to the activity status of EGFR, requires lipid raft domain integrity and actin filament polymerization, and crucial intracellular signalling mediators include Rac1 and p38MAPK. The study has revealed a novel aspect of reciprocal cross-talk between EGFR and T-cad.  

3.1958           Proteomics in colorectal cancer translational research: Biomarker discovery for clinical applications

De Wit, M., Fineman, R.J.A., Verheul, H.M.W., Meijer, G.A. and  Jimenez, C.R Clin. Biochem., 46, 466-479 (2013)   Colorectal cancer (CRC) is a major cause of cancer-related death in the western world. Screening to detect the disease in an early stage is the most effective approach to tackle this problem. In addition, better diagnostic tools for assessment of prognosis and prediction of response to drug therapy will allow for personalized therapies and better outcomes. Protein biomarkers that reflect tumor biology have the potential to address a wide range of clinical needs. These include diagnostic (screening) biomarkers for early detection, prognostic biomarkers for estimation of disease outcome, predictive biomarkers for adjuvant treatment stratification, and surveillance biomarkers for disease monitoring and treatment response. An important source for the discovery of potential biomarkers comes from mass spectrometry based proteomics research of the biology of CRC development. Here, we review recent colon cancer proteomics studies directed at identification of biomarker proteins. These include studies that use preclinical models (i.e. cell lines or murine tissues) as well as clinical materials (e.g. tissue and stool samples). We separately highlight some studies that focused on identification of cancer stem cell (CSC) related proteins in tumor spheroids, an in vitro model system for investigating CRC treatment response. Recent proteomics studies have generated many new candidate protein biomarkers. However, the lack of follow-up studies that lead to biomarker verification and/or validation remains a limiting factor in the translation of these candidate biomarkers into clinical applications. This is partly due to technological limitations which are bound to diminish with new technologies, including selected reaction monitoring mass spectrometry (SRM-MS). Antibodies are still required, though, both to perform high-throughput validation as well as to develop cost-effective tests for routine use in a clinical setting.  

3.1959           Role of Us9 Phosphorylation in Axonal Sorting and Anterograde Transport of Pseudorabies Virus

Kratchmarov, R., Taylor, M.P. and Enquist, L.W. PloS One, 8(3), e58776  (2013)   Alphaherpes viruses, such as pseudorabies virus (PRV), undergo anterograde transport in neuronal axons to facilitate anterograde spread within hosts. Axonal sorting and anterograde transport of virions is dependent on the viral membrane protein Us9, which interacts with the host motor protein Kif1A to direct transport. Us9-Kif1A interactions are necessary but not sufficient for these processes, indicating that additional cofactors or post-translational modifications are needed. In this study, we characterized two conserved serine phosphorylation sites (S51 and S53) in the PRV Us9 protein that are necessary for anterograde spread in vivo. We assessed the subcellular localization of phospho-Us9 subspecies during infection of neurons and found that the phospho-form is detectable on the majority, but not all, of axonal vesicles containing Us9 protein. In biochemical assays, phospho-Us9 was enriched in lipid raft membrane microdomains, though Us9 phosphorylation did not require prior lipid raft association. During infections of chambered neuronal cultures, we observed only a modest reduction in anterograde spread capacity for diserine mutant Us9, and no defect for monoserine mutants. Conversely, mutation of the kinase recognition sequence residues adjacent to the phosphorylation sites completely abrogated anterograde spread. In live-cell imaging analyses, anterograde transport of virions was reduced during infection with a recombinant PRV strain expressing GFP-tagged diserine mutant Us9. Phosphorylation was not required for Us9-Kif1A interaction, suggesting that Us9-Kif1A binding is a distinct step from the activation and/or stabilization of the transport complex. Taken together, our findings indicate that, while not essential, Us9 phosphorylation enhances Us9-Kif1A-based transport of virions in axons to modulate the overall efficiency of long-distance anterograde spread of infection.  

3.1960           Methods for the Study of Dopamine Receptors Within Lipid Rafts of Kidney Cells

Yu, P., Villar, V.A. and Jose, P.A. Methods in Mol. Biol., 964, 15-23 (2013)   There is increasing evidence that G protein-coupled receptor (GPCR) signaling is regulated in lipid raft microdomains. GPCRs and GPCR-signaling molecules, including G proteins and protein kinases, have been reported to compartmentalize in these microdomains. Dopamine D₁-like receptors (D₁R and D₅R) belong to a family of GPCRs that are important in the regulation of renal function. These receptors are not only localized and regulated in caveolae that contains caveolin-1 but are also distributed in non-­caveolar lipid rafts which do not contain caveolin-1. This chapter describes detergent- and non-detergent-based methods to obtain lipid raft fractions from renal proximal tubule cells.  

3.1961           Quantitative and Qualitative Preparations of Bacterial Outer Membrane Vesicles

Chutkan, H., MacDonald, I., Manning, A. and Kuehn, M.J. Methods in Mol. Biol., 966, 259-271 (2013)   Gram-negative bacterial outer membrane vesicle production and function have been studied using a variety of quantitative and qualitative methods. These types of analyses can be hampered by the use of impure vesicle preparations. Here we describe a set of techniques that are useful for the quantitative analysis of vesicle production and for preparative yields of highly purified vesicles for studies of vesicle function or composition. Procedures and advice are also included for the purification of vesicles from encapsulated and low-yield strains.    

3.1962           Isolation of Pathogen-Containing Vacuoles

Shechuk, O. and Steinert, M. Methods in Mol. Biol., 983, 419-429 (2013)   Dictyostelium discoideum cells are “professional phagocytes,” as they ingest a large variety of bacteria, yeast, and inert particles. Several bacterial pathogens are able to survive intracellularly within specialized vacuoles of D. discoideum by interfering with host signaling pathways. To better understand the molecular mechanisms underlying these evolutionary conserved processes we have established a method for the isolation of pathogen-containing vacuoles (PCVs). The isolation protocol describes the infection of D. discoideum cells with the intracellular pathogen Legionella pneumophila, loading of the lysosomal compartment with colloidal iron, mechanical lysis of host cells, iodophenylnitrophenyltetrazolium (INT) heavy labeling of mitochondria, removal of nucleic acid by Benzonase treatment, separation of nuclei by low-speed centrifugation, and the magnetic removal of lysosomes. The subcellular fractionation in a discontinuous sucrose density OptiPrep gradient allows the separation of mitochondria and to prepare PCVs with high purity. The proteins isolated from PCVs have been successfully subjected to mass spectrometry and allowed to analyze pathogen-directed maturation processes of vacuoles. The method can also be applied for subsequent protein modification analyses and lipidome comparisons.  

3.1963           Decreased activity of folate transporters in lipid rafts resulted in reduced hepatic folate uptake in chronic alcoholism in rats

Wani, N.A., Nada, R., Khanduja, K.L. and Kaur, J. Genes Nutr., 8, 209-219 (2013)   Folic acid is an essential nutrient that is required for one-carbon biosynthetic processes and for methylation of biomolecules. Deficiency of this micronutrient leads to disturbances in normal physiology of cell. Chronic alcoholism is well known to be associated with folate deficiency, which is due in part to folate malabsorption. The present study deals with the regulatory mechanisms of folate uptake in liver during chronic alcoholism. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20 % solution) orally for 3 months, and the molecular mechanisms of folate uptake were studied in liver. The characterization of the folate transport system in liver basolateral membrane (BLM) suggested it to be a carrier mediated and acidic pH dependent, with the major involvement of proton coupled folate transporter and folate binding protein in the uptake. The folate transporters were found to be associated with lipid raft microdomain of liver BLM. Moreover, ethanol ingestion decreased the folate transport by altering the Vmax of folate transport process and downregulated the expression of folate transporters in lipid rafts. The decreased transporter levels were associated with reduced protein and mRNA levels of these transporters in liver. The deranged folate uptake together with reduced folate transporter levels in lipid rafts resulted in reduced folate levels in liver and thereby to its reduced levels in serum of ethanol-fed rats. The chronic ethanol ingestion led to decreased folate uptake in liver, which was associated with the decreased number of transporter molecules in the lipid rafts that can be ascribed to the reduced synthesis of these transporters.  

3.1964           Facing glycosphingolipid–Shiga toxin interaction: dire straits for endothelial cells of the human vasculature

Bauwens, A., Betz, J., Meisen,I., Kemper, B., Karch, H. and Müthing, J. Cell. Mol. Life Sci., 70, 425-457 (2013)   The two major Shiga toxin (Stx) types, Stx1 and Stx2, produced by enterohemorrhagic Escherichia coli (EHEC) in particular injure renal and cerebral microvascular endothelial cells after transfer from the human intestine into the circulation. Stxs are AB₅ toxins composed of an enzymatically active A subunit and the pentameric B subunit, which preferentially binds to the glycosphingolipid globotriaosylceramide (Gb3Cer/CD77). This review summarizes the current knowledge on Stx-caused cellular injury and the structural diversity of Stx receptors as well as the initial molecular interaction of Stxs with the human endothelium of different vascular beds. The varying lipoforms of Stx receptors and their spatial organization in lipid rafts suggest a central role in different modes of receptor-mediated endocytosis and intracellular destiny of the toxins. The design and development of tailored Stx neutralizers targeting the oligosaccharide–toxin recognition event has become a very real prospect to ameliorate or prevent life-threatening renal and neurological complications.    

3.1965           TLR4–MD-2 complex is negatively regulated by an endogenous ligand, globotetraosylceramide

Kondo, Y. et al PNAS, 110(12), 4714-4719 (2013)   Although endogenous ligands for Toll-like receptor (TLR)4–myeloid differentiation factor 2 (MD2) have not been well-understood, we here report that a globo-series glycosphingolipid, globotetraosylceramide (Gb4), attenuates the toxicity of lipopolysaccharides (LPSs) by binding to TLR4–MD-2. Because α1,4-galactosyltransferase (A4galt)-deficient mice lacking globo-series glycosphingolipids showed higher sensitivity to LPS than wild-type mice, we examined mechanisms by which globo-series glycosphingolipids attenuate LPS toxicity. Cultured endothelial cells lacking A4galt showed higher expression of LPS-inducible genes upon LPS treatment. In turn, introduction of A4galt cDNA resulted in the neo expression of Gb4, leading to the reduced expression of LPS-inducible genes. Exogenous Gb4 induced similar effects. As a mechanism for the suppressive effects of Gb4 on LPS signals, specific binding of Gb4 to the LPS receptor TLR4–MD-2 was demonstrated by coprecipitation of Gb4 with recombinant MD-2 and by native PAGE. A docking model also supported these data. Taken together with colocalization of TLR4–MD-2 with Gb4 in lipid rafts after LPS stimulation, it was suggested that Gb4 competes with LPS for binding to TLR4–MD-2. Finally, administration of Gb4 significantly protected mice from LPS-elicited mortality. These results suggest that Gb4 is an endogenous ligand for TLR4–MD-2 and is capable of attenuating LPS toxicity, indicating the possibility for its therapeutic application in endotoxin shock.  

3.1966           The myosin motor Myo1c is required for VEGFR2 delivery to the cell surface and for angiogenic signaling

Tiwari, A., Jung, J-J., Inamdar, S.M., Nihalani, D. and Choudhury, A. Am. J. Physiol. Heart Circ. Physiol., 304, H687-H696 (2013)   Vascular endothelial growth factor receptor-2 (VEGFR2) is a receptor tyrosine kinase that is expressed in endothelial cells and regulates angiogenic signal transduction under both physiological and pathological conditions. VEGFR2 turnover at the plasma membrane (PM) is regulated by its transport through endocytic and secretory transport pathways. Short-range cargo trafficking along actin filaments is commonly regulated by motor proteins of myosin superfamily. In the current study, performed in primary human endothelial cells, we demonstrate that unconventional myosin 1c (Myo1c; class I family member) regulates the localization of VEGFR2 at the PM. We further demonstrate that the recruitment of VEGFR2 to the PM and its colocalization with Myo1c and caveolin-1 occur in response to VEGF-A (VEGF) stimulation. In addition, VEGF-induced delivery of VEGFR2 to the cell surface requires Myo1c; surface VEGFR2 levels are reduced in the absence of Myo1c and, more importantly, are restored by the overexpression of wild-type but not mutant Myo1c. Subcellular density gradient fractionation revealed that partitioning of VEGFR2 into caveolin-1- and Myo1c-enriched membrane fractions is dependent on VEGF stimulation. Myo1c depletion resulted in increased VEGF-induced VEGFR2 transport to the lysosomes for degradation and was rescued by applying either brefeldin A, which blocks trafficking between the endoplasmic reticulum and the Golgi complex, or dynasore, an inhibitor of dynamin-mediated endocytosis. Myo1c depletion also reduced VEGF-induced VEGFR2 phosphorylation at Y1175 and phosphorylation-dependent activation of ERK1/2 and c-Src kinase, leading to reduced cell proliferation and cell migration. This is the first report demonstrating that Myo1c is an important mediator of VEGF-induced VEGFR2 delivery to the cell surface and plays a role in angiogenic signaling.  

3.1967           Enhancing Mitochondrial Calcium Buffering Capacity Reduces Aggregation of Misfolded SOD1 and Motor Neuron Cell Death without Extending Survival in Mouse Models of Inherited Amyotrophic Lateral Sclerosis

Parone, P., Da Cruz, S., Han, J.S., McAlonis-Downes, M., Vetto, A.P., Lee, S.K., Tseng, E. and Cleveland, D.W.

1. Neurosci., 33(11), 4657-4671 (2013)

  Mitochondria have been proposed as targets for toxicity in amyotrophic lateral sclerosis (ALS), a progressive, fatal adult-onset neurodegenerative disorder characterized by the selective loss of motor neurons. A decrease in the capacity of spinal cord mitochondria to buffer calcium (Ca²⁺) has been observed in mice expressing ALS-linked mutants of SOD1 that develop motor neuron disease with many of the key pathological hallmarks seen in ALS patients. In mice expressing three different ALS-causing SOD1 mutants, we now test the contribution of the loss of mitochondrial Ca²⁺-buffering capacity to disease mechanism(s) by eliminating ubiquitous expression of cyclophilin D, a critical regulator of Ca²⁺-mediated opening of the mitochondrial permeability transition pore that determines mitochondrial Ca²⁺ content. A chronic increase in mitochondrial buffering of Ca²⁺ in the absence of cyclophilin D was maintained throughout disease course and was associated with improved mitochondrial ATP synthesis, reduced mitochondrial swelling, and retention of normal morphology. This was accompanied by an attenuation of glial activation, reduction in levels of misfolded SOD1 aggregates in the spinal cord, and a significant suppression of motor neuron death throughout disease. Despite this, muscle denervation, motor axon degeneration, and disease progression and survival were unaffected, thereby eliminating mutant SOD1-mediated loss of mitochondrial Ca²⁺ buffering capacity, altered mitochondrial morphology, motor neuron death, and misfolded SOD1 aggregates, as primary contributors to disease mechanism for fatal paralysis in these models of familial ALS.  

3.1968           Lipid Peroxidation Product 4-Hydroxy-2-Nonenal Promotes Seeding-Capable Oligomer Formation and Cell-to-Cell Transfer of α-Synuclein

Bae, E-J., Ho, D-H., Park, E., Jung, J.W., Cho, K., Hong, J.H., Lee, H-J., Kim, K.P. and Lee, S-J. Antioxidants & Redox Signaling, 18(7), 770-783 (2013)   Aims: Abnormal accumulation of α-synuclein aggregates is one of the key pathological features of many neurodegenerative movement disorders and dementias. These pathological aggregates propagate into larger brain regions as the disease progresses, with the associated clinical symptoms becoming increasingly severe and complex. However, the factors that induce α-synuclein aggregation and spreading of the aggregates remain elusive. Herein, we have evaluated the effects of the major lipid peroxidation byproduct 4-hydroxy-2-nonenal (HNE) on α-synuclein oligomerization and cell-to-cell transmission of this protein. Results: Incubation with HNE promoted the oligomerization of recombinant human α-synuclein via adduct formation at the lysine and histidine residues. HNE-induced α-synuclein oligomers evidence a little β-sheet structure and are distinct from amyloid fibrils at both conformation and ultrastructure levels. Nevertheless, the HNE-induced oligomers are capable of seeding the amyloidogenesis of monomeric α-synuclein under in vitro conditions. When neuronal cells were treated with HNE, both the translocation of α-synuclein into vesicles and the release of this protein from cells were increased. Neuronal cells can internalize HNE-modified α-synuclein oligomers, and HNE treatment increased the cell-to-cell transfer of α-synuclein proteins. Innovation and Conclusion: These results indicate that HNE induces the oligomerization of α-synuclein through covalent modification and promotes the cell-to-cell transfer of seeding-capable oligomers, thereby contributing to both the initiation and spread of α-synuclein aggregates.  

3.1969           Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast

Cuesta-Marban, A. et al

1. Biol. Chem., 288(12), 8405-8418 (2013)

  The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosine-resistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane.      

3.1970           Alteration of Plasma Membrane Organization by an Anticancer Lysophosphatidylcholine Analogue Induces Intracellular Acidification and Internalization of Plasma Membrane Transporters in Yeast

Czyz, O., Bitew, T., Cuesta-Marban, A., McMaster, C.R., Mollinedo, F. and Zaremberg, V.

1. Biol. Chem., 288(12), 8419-8432 (2013)

  The lysophosphatidylcholine analogue edelfosine is a potent antitumor lipid that targets cellular membranes. The underlying mechanisms leading to cell death remain controversial, although two cellular membranes have emerged as primary targets of edelfosine, the plasma membrane (PM) and the endoplasmic reticulum. In an effort to identify conditions that enhance or prevent the cytotoxic effect of edelfosine, we have conducted genome-wide surveys of edelfosine sensitivity and resistance in Saccharomyces cerevisiae presented in this work and the accompanying paper (Cuesta-Marbán, Á., Botet, J., Czyz, O., Cacharro, L. M., Gajate, C., Hornillos, V., Delgado, J., Zhang, H., Amat-Guerri, F., Acuña, A. U., McMaster, C. R., Revuelta, J. L., Zaremberg, V., and Mollinedo, F. (January 23, 2013) J. Biol. Chem. 288,), respectively. Our results point to maintenance of pH homeostasis as a major player in modulating susceptibility to edelfosine with the PM proton pump Pma1p playing a main role. We demonstrate that edelfosine alters PM organization and induces intracellular acidification. Significantly, we show that edelfosine selectively reduces lateral segregation of PM proteins like Pma1p and nutrient H⁺-symporters inducing their ubiquitination and internalization. The biology associated to the mode of action of edelfosine we have unveiled includes selective modification of lipid raft integrity altering pH homeostasis, which in turn regulates cell growth.  

3.1971           Role of C-terminal Membrane-proximal Basic Residues in Cell Surface Trafficking of HIV Coreceptor GPR15 Protein

Okamoto, Y., Bernstein, J.D. and Shikano, S.

1. Biol. Chem., 288(13), 9189-9199 (2013)

  Cell surface density of G protein-coupled receptors (GPCRs) is controlled by dynamic molecular interactions that often involve recognition of the distinct sequence signals on the cargo receptors. We reported previously that the RXR-type dibasic motif in the distal C-terminal tail of an HIV coreceptor GPR15 negatively regulates the cell surface expression by mediating the coatomer protein I complex-dependent retrograde transport to the endoplasmic reticulum (ER). Here we demonstrate that another pair of basic residues (Arg³¹⁰-Arg³¹¹) in the membrane-proximal region of the C-terminal tail plays a pivotal role in mediating the anterograde trafficking of GPR15. The Ala mutation of the C-terminal membrane-proximal basic residues (MPBRs) (R310/311A) abolished the O-glycosylation and cell surface expression of GPR15. The subcellular fractionation and immunocytochemistry assays indicated that the R310/311A mutant was more localized in the ER but much less in the trans-Golgi when compared with the wild-type GPR15, suggesting the positive role of Arg³¹⁰-Arg³¹¹ in the ER-to-Golgi transport of GPR15. Sequence analysis on human GPCRs showed that the basic residues are frequent in the membrane-proximal region of the C-terminal tail. Similar to GPR15, mutation of the C-terminal MPBRs resulted in a marked reduction of the cell surface expression in multiple different GPCRs. Our results suggest that the C-terminal MPBRs are critically involved in mediating the anterograde trafficking of a broad range of membrane proteins, including GPCRs.  

3.1972           Vps35 loss promotes hyperresorptive osteoclastogenesis and osteoporosis via sustained RANKL signaling

Xia, W-F., Tang, F-L., Xiong, L., Xiong, S., Jung, J-U., Lee, D-H., Li, X-S., Feng, X., Mei, L. and Xiong, W-C.

1. Cell Biol., 200(6), 821-837 (2013)

  Receptor activator of NF-κB (RANK) plays a critical role in osteoclastogenesis, an essential process for the initiation of bone remodeling to maintain healthy bone mass and structure. Although the signaling and function of RANK have been investigated extensively, much less is known about the negative regulatory mechanisms of its signaling. We demonstrate in this paper that RANK trafficking, signaling, and function are regulated by VPS35, a major component of the retromer essential for selective endosome to Golgi retrieval of membrane proteins. VPS35 loss of function altered RANK ligand (RANKL)–induced RANK distribution, enhanced RANKL sensitivity, sustained RANKL signaling, and increased hyperresorptive osteoclast (OC) formation. Hemizygous deletion of the Vps35 gene in mice promoted hyperresorptive osteoclastogenesis, decreased bone formation, and caused a subsequent osteoporotic deficit, including decreased trabecular bone volumes and reduced trabecular thickness and density in long bones. These results indicate that VPS35 critically deregulates RANK signaling, thus restraining increased formation of hyperresorptive OCs and preventing osteoporotic deficits.  

3.1973           Electrophilic nitro-fatty acids inhibit vascular inflammation by disrupting LPS-dependent TLR4 signalling in lipid rafts

Villacorta, L., Chang, L., Salvatore, S.R., Ichikawa, T., Zhang, J., Petrovic-Djergovic, D., Jia, L., Carlsen, H., Schopfer, F.J., Freeman, B.A. and Chen, Y.E. Cardiovasc. Res., 98, 116-124 (2013)   Aims Electrophilic fatty acid nitroalkene derivatives, products of unsaturated fatty acid nitration, exert long-term cardiovascular protection in experimental models of metabolic and cardiovascular diseases. The goal of this study is to examine the effects of nitro-fatty acids in the regulation of upstream signalling events in nuclear factor-κB (NF-κB) activation and determine whether low-dose acute administration of nitro-fatty acids reduces vascular inflammation in vivo. Methods and results Using NF-κB-luciferase transgenic mice, it was determined that pre-emptive treatment with nitro-oleic acid (OA-NO₂), but not oleic acid (OA) inhibits lipopolysaccharide (LPS)-induced NF-κB activation both in vivo and in isolated macrophages. Acute intravenous administration of OA-NO₂ was equally effective to inhibit leukocyte recruitment to the vascular endothelium assessed by intravital microscopy and significantly reduces aortic expression of adhesion molecules. An acute treatment with OA-NO₂ in vivo yielding nanomolar concentrations in plasma, is sufficient to inhibit LPS-induced Toll-like receptor 4 (TLR4)-induced cell surface expression in leukocytes and NF-κB activation. In vitro experiments reveal that OA-NO₂ suppresses LPS-induced TLR4 signalling, inhibitor of κB (IκBα) phosphorylation and ubiquitination, phosphorylation of the IκB kinase (IKK), impairing the recruitment of the TLR4 and TNF receptor associated factor 6 (TRAF6) to the lipid rafts compartments. Conclusion These studies demonstrate that acute administration of nitro-fatty acids is effective to reduce vascular inflammation in vivo. These findings reveal a direct role of nitro-fatty acids in the disruption of the TLR4 signalling complex in lipid rafts, upstream events of the NF-κB pathway, leading to resolution of pro-inflammatory activation of NF-κB in the vasculature.  

3.1974           CG0009, a Novel Glycogen Synthase Kinase 3 Inhibitor, Induces Cell Death through Cyclin D1 Depletion in Breast Cancer Cells

Kim, H.M., Kim, C-S., Lee, J-H., Jang, S.J., Hwang, J.J., Ro, S. and Choi, J. PloS One, 8(4), e60383 (2013)   Glycogen synthase kinase 3α/β (GSK3α/β) is a constitutively active serine/threonine kinase involved in multiple physiological processes, such as protein synthesis, stem cell maintenance and apoptosis, and acts as a key suppressor of the Wnt-β-catenin pathway. In the present study, we examined the therapeutic potential of a novel GSK3 inhibitor, CG0009, in the breast cancer cell lines, BT549, HS578T, MDA-MB-231, NCI/ADR-RES, T47D, MCF7 and MDA-MB-435, from the NCI-60 cancer cell line panel. Assessment of cytotoxicity, apoptosis and changes in estrogen-signaling proteins was performed using cell viability assays, Western blotting and quantitative real-time PCR. CG0009 enhanced the inactivating phosphorylation of GSK3α at Ser21 and GSK3β at Ser9 and simultaneously decreased activating phosphorylation of GSK3β at Tyr216, and induced caspase-dependent apoptosis independently of estrogen receptor α (ERα) expression status, which was not observed with the other GSK3 inhibitors examined, including SB216763, kenpaullone and LiCl. CG0009 treatment (1 µmol/L) completely ablated cyclin D1 expression in a time-dependent manner in all the cell lines examined, except T47D. CG0009 alone significantly activated p53, leading to relocation of p53 and Bax to the mitochondria. GSK3 inhibition by CG0009 led to slight upregulation of the β-catenin target genes, c-Jun and c-Myc, but not cyclin D1, indicating that CG0009-mediated cyclin D1 depletion overwhelms the pro-survival signal of β-catenin, resulting in cell death. Our findings suggest that the novel GSK3 inhibitor, CG0009, inhibits breast cancer cell growth through cyclin D1 depletion and p53 activation, and may thus offer an innovative therapeutic approach for breast cancers resistant to hormone-based therapy.  

3.1975           SH3 interactome conserves general function over specific form

Xin, X. et al Mol. Systems Biol., 9:652 (2013)   Src homology 3 (SH3) domains bind peptides to mediate protein–protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two-hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain-mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form.  

3.1976           Heparanase Regulates Secretion, Composition, and Function of Tumor Cell-derived Exosomes

Thompson, C.A., Purushothaman, A., Ramani, V.C., Vlodavsky, I. and Sanderson, R.D.

1. Biol. Chem., 288(14), 10093-10099 (2013)

  Emerging evidence indicates that exosomes play a key role in tumor-host cross-talk and that exosome secretion, composition, and functional capacity are altered as tumors progress to an aggressive phenotype. However, little is known regarding the mechanisms that regulate these changes. Heparanase is an enzyme whose expression is up-regulated as tumors become more aggressive and is associated with enhanced tumor growth, angiogenesis, and metastasis. We have discovered that in human cancer cells (myeloma, lymphoblastoid, and breast cancer), when expression of heparanase is enhanced or when tumor cells are exposed to exogenous heparanase, exosome secretion is dramatically increased. Heparanase enzyme activity is required for robust enhancement of exosome secretion because enzymatically inactive forms of heparanase, even when present in high amounts, do not dramatically increase exosome secretion. Heparanase also impacts exosome protein cargo as reflected by higher levels of syndecan-1, VEGF, and hepatocyte growth factor in exosomes secreted by heparanase-high expressing cells as compared with heparanase-low expressing cells. In functional assays, exosomes from heparanase-high cells stimulated spreading of tumor cells on fibronectin and invasion of endothelial cells through extracellular matrix better than did exosomes secreted by heparanase-low cells. These studies reveal that heparanase helps drive exosome secretion, alters exosome composition, and facilitates production of exosomes that impact both tumor and host cell behavior, thereby promoting tumor progression.  

3.1977           Analysis of the Early Steps of Herpes Simplex Virus 1 Capsid Tegumentation

Henaff, D., Remillard-Labrosse, G., Loret, S. and Lippe, R.

1. Virol., 87(9), 4895-4906 (2013)

  Herpes simplex virus type 1 particles are multilayered structures with a DNA genome surrounded by a capsid, tegument, and envelope. While the protein content of mature virions is known, the sequence of addition of the tegument and the intracellular compartments where this occurs are intensely debated. To probe this process during the initial stages of egress, we used two approaches: an in vitro nuclear egress assay, which reconstitutes the exit of nuclear capsids to the cytoplasm, and a classical nuclear capsid sedimentation assay. As anticipated, in vitro cytoplasmic capsids did not harbor U_L34, U_L31, or viral glycoproteins but contained U_S3. In agreement with previous findings, both nuclear and in vitro capsids were positive for ICP0 and ICP4. Unexpectedly, nuclear C capsids and cytoplasmic capsids produced in vitro without any cytosolic viral proteins also scored positive for U_L36 and U_L37. Immunoelectron microscopy confirmed that these tegument proteins were closely associated with nuclear capsids. When cytosolic viral proteins were present in the in vitro assay, no additional tegument proteins were detected on the capsids. As previously reported, the tegument was sensitive to high-salt extraction but, surprisingly, was stabilized by exogenous proteins. Finally, some tegument proteins seemed partially lost during egress, while others possibly were added at multiple steps or modified along the way. Overall, an emerging picture hints at the early coating of capsids with up to 5 tegument proteins at the nuclear stage, the shedding of some viral proteins during nuclear egress, and the acquisition of others tegument proteins during reenvelopment.  

3.1978           Bile salt-stimulated phospholipid efflux mediated by ABCB4 localized in nonraft membranes

Morita, S-y., Tsuda, T., Horikami, M., Teraoka, R., Kitagawa, S. and Terada, T.

1. Lipid Res., 54, 1221-1230 (2013)

  ABCB4 is necessary for the secretion of phospholipids from hepatocytes into bile and for the protection of cell membranes against bile salts. Lipid rafts are plasma membrane microdomains containing high contents of cholesterol and sphingolipids, which are separated by Triton X-100 extraction or OptiPrep gradient centrifugation. In this study, we investigated the relationship between the function of ABCB4 and lipid rafts using mouse canalicular membranes and HEK293 cells stably expressing ABCB4. ABCB4 and ABCB1 were mainly distributed in nonraft membranes. The expression of ABCB4, but not ABCB1, led to significant increases in the phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM) contents in nonraft membranes and further enrichment of SM and cholesterol in raft membranes. The ABCB4-mediated efflux of PC, PE, and SM was significantly stimulated by taurocholate, while the efflux of PE and SM was much less than that of PC. This ABCB4-mediated efflux was completely abolished by BODIPY-verapamil, which hardly partitioned into raft membranes. In addition, ABCB1 and ABCB4 mediated the efflux of rhodamine 123 and rhodamine 6G from nonraft membranes, which was not affected by taurocholate. We conclude that ABCB4 located in nonrafts, but not in rafts, is predominantly involved in the efflux of phospholipids and other substrates.  

3.1979           In vitro correction of disorders of lysosomal transport by microvesicles derived from baculovirus-infected Spodoptera cells

Thoene, J., Goss, T., Witcher, M., Mullet, J., N’Kuli, F., Van Der Smissen, P., Courtoy, P and  Hahn, S.H. Mol. Genet. Metabolism, 109, 77-85 (2013)   Infection of Spodoptera frugiperda (Sf9) cells by baculovirus (BV) is well established for transgene expression of soluble proteins, but few correctly folded transmembrane proteins have been so produced. We here report the use of the BV/Sf9 (BVES) method for the expression and transfer, via microvesicles, of the exclusive lysosomal exporters for cystine and sialic acid, human cystinosin and sialin. These proteins and their mRNA are released into the culture medium as very low-density microvesicles (~ 1.05 g/ml), which do not label for lysobisphosphatidic acid. The presence of the human transgene proteins in the vesicles was confirmed by western blotting and confirmed and quantified by mass spectrometry. Addition of vesicles to cultures of human fibroblast lines deficient in either cystinosin or sialin produced a progressive depletion of stored lysosomal cystine or sialic acid, respectively. The depletion effect was slow (T_1/2 ~ 48 h), saturable (down to ~ 40% of initial after 4 days) and stable (> one week). Surprisingly, BV infection of Spodoptera appeared to induce expression and release into microvesicles of the insect orthologue of cystinosin, but not of sialin. We conclude that BVES is an effective method to express and transfer functional transmembrane proteins so as to study their properties in mammalian cells, and has a generic potential for transport protein replacement therapy.  

3.1980           Lipid Droplet-Binding Protein TIP47 Regulates Hepatitis C Virus RNA Replication through Interaction with the Viral NS5A Protein

Vogt, D.A., Camus, G., Herker, E., Webster, B.R., Tsou, C-L., Greene, W.C., Yen, T-S.B. and Ott, M. PloS Pathogens, (9(4), e1003302 (2013)   The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV) infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (membranous web) and assists viral assembly in the close vicinity of lipid droplets (LDs). To identify host proteins that interact with NS5A, we performed a yeast two-hybrid screen with the N-terminus of NS5A (amino acids 1–31), a well-studied α-helical domain important for the membrane tethering of NS5A. Our studies identified the LD-associated host protein, Tail-Interacting Protein 47 (TIP47) as a novel NS5A interaction partner. Coimmunoprecipitation experiments in Huh7 hepatoma cells confirmed the interaction of TIP47 with full-length NS5A. shRNA-mediated knockdown of TIP47 caused a more than 10-fold decrease in the propagation of full-length infectious HCV in Huh7.5 hepatoma cells. A similar reduction was observed when TIP47 was knocked down in cells harboring an autonomously replicating HCV RNA (subgenomic replicon), indicating that TIP47 is required for efficient HCV RNA replication. A single point mutation (W9A) in NS5A that disrupts the interaction with TIP47 but preserves proper subcellular localization severely decreased HCV RNA replication. In biochemical membrane flotation assays, TIP47 cofractionated with HCV NS3, NS5A, NS5B proteins, and viral RNA, and together with nonstructural viral proteins was uniquely distributed to lower-density LD-rich membrane fractions in cells actively replicating HCV RNA. Collectively, our data support a model where TIP47—via its interaction with NS5A—serves as a novel cofactor for HCV infection possibly by integrating LD membranes into the membranous web.  

3.1981           Nuclear Targeting of Human Cytomegalovirus Large Tegument Protein pUL48 Is Essential for Viral Growth

Brock, I., Krüger, M., Mertens, T. and von Einem, J.

1. Virol., 87(10), 6005-6019 (2013)

  We report the identification of a functional nuclear localization signal (NLS) in the human cytomegalovirus (HCMV) large tegument protein pUL48 that is required for nuclear localization in transfected cells and is essential for viral growth. The NLS was mapped to pUL48 amino acid residues 284 to 302. This sequence contains a bipartite NLS comprising two clusters of basic residues (bC1 and bC2) separated by 9 amino acids. Deletion or mutation of bC1 or mutation of bC2 abrogated the nuclear localization of full-length pUL48 in transiently expressing cells, thus strongly implying a bipartite character of the NLS. Nuclear localization could be restored by fusion of a functional NLS together with enhanced green fluorescent protein (EGFP) to the N terminus of these mutants. In HCMV-infected cells, pUL48 was found in both nuclear and cytoplasmic fractions, supporting a function of the NLS during virus infection. NLS mutant viruses, generated by markerless bacterial artificial chromosome mutagenesis, were not viable in cell culture, whereas coexpression of pUL48 complemented growth of these mutants. The fusion of a functional NLS to the N terminus of pUL48 in a nonviable NLS mutant virus partially rescued the growth defect. Furthermore, the replacement of the bipartite pUL48 NLS by the monopartite pUL36 NLS of herpes simplex virus 1 supported viral growth to some extent but still revealed a severe defect in focus formation and release of infectious virus particles. Together, these results show that nuclear targeting of pUL48 is mediated by a bipartite NLS whose function is essential for HCMV growth.  

3.1982           The Receptor Attachment Function of Measles Virus Hemagglutinin Can Be Replaced with an Autonomous Protein That Binds Her2/neu While Maintaining Its Fusion-Helper Function

Rasbach, A., Abel, T., Münch, R.C., Boller, K., Schneider-Schalies, J. and Buchholz, C.J.

1. Virol., 87(11), 6246-6256 (2013)

  Cell entry of enveloped viruses is initiated by attachment to the virus receptor followed by fusion between the virus and host cell membranes. Measles virus (MV) attachment to its receptor is mediated by the hemagglutinin (H), which is thought to produce conformational changes in the membrane fusion protein (F) that trigger insertion of its fusion peptide into the target cell membrane. Here, we uncoupled receptor attachment and the fusion-helper function of H by introducing Y481A, R533A, S548L, and F549S mutations into the viral attachment protein that made it blind to its normal receptors. An artificial receptor attachment protein specific for Her2/neu was incorporated into the membranes of pseudotyped lentivirus particles as a separate transmembrane protein along with the F protein. Surprisingly, these particles entered efficiently into Her2/neu-positive SK-OV-3 as well as CHO-Her2 cells. Cell entry was independent of endocytosis but strictly dependent on the presence of H. H-specific monoclonal antibodies, as well as a mutation in H interfering with H/F cooperation, blocked cell entry. The particles mediated stable and specific transfer of reporter genes into Her2/neu-positive human tumor cells also in vivo, while exhibiting improved infectivity and higher titers than Her2/neu-targeted vectors displaying the targeting domain on H. Extending the current model of MV cell entry, the data suggest that receptor binding of H is not required for its fusion-helper function but that particle-cell contact in general may be sufficient to induce the conformational changes in the H/F complex and activate membrane fusion.  

3.1983           Separation of actin-dependent and actin-independent lipid rafts

Klappe, K., Hummel, I. and Kok, J.W. Anal. Biochem., 438, 133-135 (2013)   Lipid rafts have been isolated on the basis of their resistance to various detergents and more recently by using detergent-free procedures. The actin cytoskeleton is now recognized as a dynamic regulator of lipid raft stability. We carefully analyzed the effects of the cortical actin-disrupting agent latrunculin B on lipid raft markers of both protein and lipid nature and show that two detergent-free membrane subtypes can be isolated and separated from each other on a one-step density gradient combined with pooling of the appropriate gradient fractions. These two subtypes differ in their dependence on the cortical actin cytoskeleton.  

3.1984           Impairment of Mitochondria in Adult Mouse Brain Overexpressing Predominantly Full-Length, N-Terminally Acetylated Human α-Synuclein

Sarafian, T.A., Ryan, C.M., Souda, P., Masliah, E., Kar, U.K., Vinters, H.V.., Mathern, G.W., Faull, K.F., Whitelegge, J.P. and Watson, J.B. PloS One, 8(5), e63557 (2013)   While most forms of Parkinson’s Disease (PD) are sporadic in nature, a small percentage of PD have genetic causes as first described for dominant, single base pair changes as well as duplication and triplication in the α-synuclein gene. The α-synuclein gene encodes a 140 amino acid residue protein that interacts with a variety of organelles including synaptic vesicles, lysosomes, endoplasmic reticulum/Golgi vesicles and, reported more recently, mitochondria. Here we examined the structural and functional interactions of human α-synuclein with brain mitochondria obtained from an early, pre-manifest mouse model for PD over-expressing human α-synuclein (ASOTg). The membrane potential in ASOTg brain mitochondria was decreased relative to wildtype (WT) mitochondria, while reactive oxygen species (ROS) were elevated in ASOTg brain mitochondria. No selective interaction of human α-synuclein with mitochondrial electron transport complexes cI-cV was detected. Monomeric human α-synuclein plus carboxyl terminally truncated forms were the predominant isoforms detected in ASOTg brain mitochondria by 2-dimensional PAGE (Native/SDS) and immunoblotting. Oligomers or fibrils were not detected with amyloid conformational antibodies. Mass spectrometry of human α-synuclein in both ASOTg brain mitochondria and homogenates from surgically resected human cortex demonstrated that the protein was full-length and postranslationally modified by N-terminal acetylation. Overall the study showed that accumulation of full-length, N-terminally acetylated human α-synuclein was sufficient to disrupt brain mitochondrial function in adult mice.  

3.1985           Exosomes for drug delivery — a novel application for the mesenchymal stem cell

Lai, R.C., Yeo, R.W.Y., Tan, K.H. and Lim, S.K. Biotechnolgy Advances, 31, 543-551 (2013)   Exosomes are the most extensively characterized class of secreted membrane vesicles that carry proteins and RNAs for intercellular communication. They are increasingly seen as possible alternatives to liposomes as drug delivery vehicles. Like liposomes, they could deliver their cargo across the plasma membrane and provide a barrier against premature transformation and elimination. In addition, these naturally-occurring secreted membrane vesicles are less toxic and better tolerated in the body as evidenced by their ubiquitous presence in biological fluids, and have an intrinsic homing ability. They are also amenable to in vivo and in vitro loading of therapeutic agents, and membrane modifications to enhance tissue-specific homing. Here we propose human mesenchymal stem cells as the ideal cell source of exosomes for drug delivery. Mesenchymal stem cell transplantation for various disease indications has been extensively tested and shown to be safe in numerous clinical trials. These cells are also prolific producers of immunologically inert exosomes. Immortalization of these cells does not compromise the quantity or quality of exosome production, thus enabling infinite and reproducible exosome production from a single cell clone.    

3.1986           Angiotensin II Impairs Endothelial Nitric-oxide Synthase Bioavailability under Free Cholesterol-enriched Conditions via Intracellular Free Cholesterol-rich Membrane Microdomains

Amiya, E., Watanabe, M., Takeda, N., Saito, T., Shiga, T., Hosoya, Y., Nakao, T., Imai, Y., Manabe, I., Nagai, R., Komuro, I. and Maemura, K.

1. Biol. Chem., 288(20), 14497-14509 (2013)

  Vascular endothelial function is impaired in hypercholesterolemia partly because of injury by modified LDL. In addition to modified LDL, free cholesterol (FC) is thought to play an important role in the development of endothelial dysfunction, although the precise mechanisms remain to be elucidated. The aim of this study was to clarify the mechanisms of endothelial dysfunction induced by an FC-rich environment. Loading cultured human aortic endothelial cells with FC induced the formation of vesicular structures composed of FC-rich membranes. Raft proteins such as phospho-caveolin-1 (Tyr-14) and small GTPase Rac were accumulated toward FC-rich membranes around vesicular structures. In the presence of these vesicles, angiotensin II-induced production of reactive oxygen species (ROS) was considerably enhanced. This ROS shifted endothelial NOS (eNOS) toward vesicle membranes and vesicles with a FC-rich domain trafficked toward perinuclear late endosomes/lysosomes, which resulted in the deterioration of eNOS Ser-1177 phosphorylation and NO production. Angiotensin II-induced ROS decreased the bioavailability of eNOS under the FC-enriched condition.  

3.1987           Estradiol accelerates the effects of fluoxetine on serotonin 1A receptor signaling

Li, Q., Sullivan, N.R., McAllister, C.E., Van de Kar, L.D. and Muma, N.A. Psychoneuroendocrinology, 38, 1145-1157 (2013)   A major problem with current anti-depressant therapy is that it takes on average 6–7 weeks for remission. Since desensitization of serotonin (5-HT)_1A receptor signaling contributes to the anti-depressive response, acceleration of the desensitization may reduce this delay in response to antidepressants. The purpose of the present study was to test the hypothesis that estradiol accelerates fluoxetine-induced desensitization of 5-HT_1A receptor signaling in the paraventricular nucleus of the hypothalamus (PVN) of rats, via alterations in components of the 5-HT_1A receptor signaling pathway. Ovariectomized rats were injected with estradiol and/or fluoxetine, then adrenocorticotropic hormone (ACTH) and oxytocin responses to a 5-HT_1A receptor agonist (+)-8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT) were examined to assess the function of 5-HT_1A receptors in the PVN. Treatment with estradiol for either 2 or 7 days or fluoxetine for 2 days produced at most a partial desensitization of 5-HT_1A receptor signaling, whereas 7 days of fluoxetine produced full desensitization. Combined treatment with estradiol and fluoxetine for 2 days produced nearly a full desensitization, demonstrating an accelerated response compared to either treatment alone. With two days of combined treatments, estradiol prevented the fluoxetine-induced increase in 5-HT_1A receptor protein, which could contribute to the more rapid desensitization. Furthermore, EB treatment for 2 days decreased the abundance of the 35 kD Gαz protein which could contribute to the desensitization response. We found two isoforms of Gαz proteins with molecular mass of 35 and 33 kD, which differentially distributed in the detergent resistant microdomain (DRM) and in Triton X-100 soluble membrane region, respectively. The 35 kD Gαz proteins in the DRM can be sumoylated by SUMO1. Stimulation of 5-HT_1A receptors with 8-OH-DPAT increases the sumoylation of Gαz proteins and reduces the 33 kD Gαz proteins, suggesting that these responses may be related to the desensitization of 5-HT_1A receptors. Treatment with estradiol for 2 days also reduced the levels of the G-protein coupled estrogen receptor GPR30, possibly limiting to the ability of estradiol to produce only a partial desensitization response. These data provide evidence that estradiol may be effective as a short-term adjuvant to SSRIs to accelerate the onset of therapeutic effects.  

3.1988           Rhesus Monkey Rhadinovirus Uses Eph Family Receptors for Entry into B Cells and Endothelial Cells but Not Fibroblasts

Hahn, A.S. and Desrosiers, R.C. PloS One, 9(5), e1003360 (2013)   Cellular Ephrin receptor tyrosine kinases (Ephrin receptors, Ephs) were found to interact efficiently with the gH/gL glycoprotein complex of the rhesus monkey rhadinovirus (RRV). Since EphA2 was recently identified as a receptor for the Kaposi's sarcoma-associated herpesvirus (KSHV) (Hahn et al., Nature Medicine 2012), we analyzed RRV and KSHV in parallel with respect to Eph-binding and Eph-dependent entry. Ten of the 14 Eph proteins, including both A- and B-type, interacted with RRV gH/gL. Two RRV strains with markedly different gH/gL sequences exhibited similar but slightly different binding patterns to Ephs. gH/gL of KSHV displayed high affinity towards EphA2 but substantially weaker binding to only a few other Ephs of the A-type. Productive entry of RRV 26-95 into B cells and into endothelial cells was essentially completely dependent upon Ephs since expression of a GFP reporter cassette from recombinant virus could be blocked to greater than 95% by soluble Eph decoys using these cells. In contrast, entry of RRV into fibroblasts and epithelial cells was independent of Ephs by these same criteria. Even high concentrations and mixtures of soluble Eph decoys were not able to reduce by any appreciable extent the number of fibroblasts and epithelial cells productively entered by RRV. Thus, RRV is similar to its close relative KSHV in the use of Eph family receptors for productive entry into B cells and endothelial cells. However, RRV uses a separate, distinct, Eph-independent pathway for productive entry into fibroblasts and epithelial cells. Whether KSHV also uses an Eph-independent pathway in some circumstances or to some extent remains to be determined.  

3.1989           Autophagic failure promotes the exocytosis and intercellular transfer of α-synuclein

Lee, H-J., Cho, E-D., Lee, K.W., Kim, J-H., Cho, S-G. and Lee, S-J. Exp. Mol. Med., 45, e22  (2013)   The accumulation of abnormal protein aggregates is a major characteristic of many neurodegenerative disorders, including Parkinson’s disease (PD). The intracytoplasmic deposition of α-synuclein aggregates and Lewy bodies, often found in PD and other α-synucleinopathies, is thought to be linked to inefficient cellular clearance mechanisms, such as the proteasome and autophagy/lysosome pathways. The accumulation of α-synuclein aggregates in neuronal cytoplasm causes numerous autonomous changes in neurons. However, it can also affect the neighboring cells through transcellular transmission of the aggregates. Indeed, a progressive spreading of Lewy pathology among brain regions has been hypothesized from autopsy studies. We tested whether inhibition of the autophagy/lysosome pathway in α-synuclein-expressing cells would increase the secretion of α-synuclein, subsequently affecting the α-synuclein deposition in and viability of neighboring cells. Our results demonstrated that autophagic inhibition, via both pharmacological and genetic methods, led to increased exocytosis of α-synuclein. In a mixed culture of α-synuclein-expressing donor cells with recipient cells, autophagic inhibition resulted in elevated transcellular α-synuclein transmission. This increase in protein transmission coincided with elevated apoptotic cell death in the recipient cells. These results suggest that the inefficient clearance of α-synuclein aggregates, which can be caused by reduced autophagic activity, leads to elevated α-synuclein exocytosis, thereby promoting α-synuclein deposition and cell death in neighboring neurons. This finding provides a potential link between autophagic dysfunction and the progressive spread of Lewy pathology.  

3.1990           Lipid Droplets, Perilipins and Cytokeratins – Unravelled Liaisons in Epithelium-Derived Cells

Heid, H., Rickelt, S., Zimbelmann, R., Winter, S., Schumacher, H. and Dörflinger, Y. PloS One, 8(5), e63061 (2013)   Lipid droplets (LDs) are spherical accumulations of apolar lipids and other hydrophobic substances and are generally surrounded by a thin cortical layer of specific amphiphilic proteins (APs). These APs segregate the LDs from the mostly polar components of the cytoplasm. We have studied LDs in epithelium-derived cell cultures and in particular characterized proteins from the perilipin (PLIN) gene family - in mammals consisting of the proteins Perilipin, Adipophilin, TIP47, S3-12 and MLDP/OXPAT (PLIN 1-5). Using a large number of newly generated and highly specific mono- and polyclonal antibodies specific for individual APs, and using improved LD isolation methods, we have enriched and characterized APs in greater detail and purity. The majority of lipid-AP complexes could be obtained in the top layer fractions of density gradient centrifugation separations of cultured cells, but APs could also be detected in other fractions within such separations. The differently sized LD complexes were analyzed using various biochemical methods and mass spectrometry as well as immunofluorescence and electron– in particular immunoelectron-microscopy. Moreover, by immunoprecipitation, protein-protein binding assays and by immunoelectron microscopy we identified a direct linkage between LD-binding proteins and the intermediate-sized filaments (IF) cytokeratins 8 and 18 (also designated as keratins K8 and K18). Specifically, in gradient fractions of higher density supposedly containing small LDs, we received as co-precipitations cytidylyl-, palmitoyl- and cholesterol transferases and other specific enzymes involved in lipid metabolism. So far, common proteomic studies have used LDs from top layer fractions only and did not report on these transferases and other enzymes. In addition to findings of short alternating hydrophobic/hydrophilic segments within the PLIN protein family, we propose and discuss a model for the interaction of LD-coating APs with IF proteins.  

3.1991           The HtrA protease of Borrelia burgdorferi degrades outer membrane protein BmpD and chemotaxis phosphatase CheX

Coleman, J.L., Crowley, J.T., Toledo, A.M. and Benach, J.L. Mol. Microbiol., 88(3), 619-633 (2013)   Borrelia burgdorferi, the spirochaetal agent of Lyme disease, codes for a single HtrA protein, HtrABb (BB0104) that is homologous to DegP of Escherichia coli (41% amino acid identity). HtrABb shows physical and biochemical similarities to DegP in that it has the trimer as its fundamental unit and can degrade casein via its catalytic serine. Recombinant HtrABb exhibits proteolytic activity in vitro, while a mutant (HtrABbS198A) does not. However, HtrABb and DegP have some important differences as well. Native HtrABb occurs in both membrane-bound and soluble forms. Despite its homology to DegP, HtrABb could not complement an E. coli DegP deletion mutant. Late stage Lyme disease patients, as well as infected mice and rabbits developed a robust antibody response to HtrABb, indicating that it is a B-cell antigen. In co-immunoprecipitation studies, a number of potential binding partners for HtrABb were identified, as well as two specific proteolytic substrates, basic membrane protein D (BmpD/BB0385) and chemotaxis signal transduction phosphatase CheX (BB0671). HtrABb may function in regulating outer membrane lipoproteins and in modulating the chemotactic response of B. burgdorferi.      

3.1992           PICK1 and ICA69 Control Insulin Granule Trafficking and Their Deficiencies Lead to Impaired Glucose Tolerance

Cao, M., Mao, Z., Kam, C., Xiao, N., Cao, X., Shen, C., Cheng, K.K.Y., Xu, A., Lee, K-M., Jiang, L: and Xia, J. PloS Biology, 11(4), e1001541 (2013)   Diabetes is a metabolic disorder characterized by hyperglycemia. Insulin, which is secreted by pancreatic beta cells, is recognized as the critical regulator of blood glucose, but the molecular machinery responsible for insulin trafficking remains poorly defined. In particular, the roles of cytosolic factors that govern the formation and maturation of insulin granules are unclear. Here we report that PICK1 and ICA69, two cytosolic lipid-binding proteins, formed heteromeric BAR-domain complexes that associated with insulin granules at different stages of their maturation. PICK1-ICA69 heteromeric complexes associated with immature secretory granules near the trans-Golgi network (TGN). A brief treatment of Brefeldin A, which blocks vesicle budding from the Golgi, increased the amount of PICK1 and ICA69 at TGN. On the other hand, mature secretory granules were associated with PICK1 only, not ICA69. PICK1 deficiency in mice caused the complete loss of ICA69 and led to increased food and water intake but lower body weight. Glucose tolerance tests demonstrated that these mutant mice had high blood glucose, a consequence of insufficient insulin. Importantly, while the total insulin level was reduced in PICK1-deficient beta cells, proinsulin was increased. Lastly, ICA69 knockout mice also displayed similar phenotype as the mice deficient in PICK1. Together, our results indicate that PICK1 and ICA69 are key regulators of the formation and maturation of insulin granules.  

3.1993           Transforming Growth Factor-β1 (TGF-β1)-stimulated Fibroblast to Myofibroblast Differentiation Is Mediated by Hyaluronan (HA)-facilitated Epidermal Growth Factor Receptor (EGFR) and CD44 Co-localization in Lipid Rafts

Midgley, A.C., Rogers, M., Hallett, M.B., Clayton, A., Bowen, T., Phillips, A.O. and Steadman, R.

1. Biol. Chem., 288(21), 14824-14838 (2013)

  Fibroblast to myofibroblast differentiation drives effective wound healing and is largely regulated by the cytokine transforming growth factor-β1 (TGF-β1). Myofibroblasts express α-smooth muscle actin and are present in granulation tissue, where they are responsible for wound contraction. Our previous studies show that fibroblast differentiation in response to TGF-β1 is dependent on and mediated by the linear polysaccharide hyaluronan (HA). Both the HA receptor, CD44, and the epidermal growth factor receptor (EGFR) are involved in this differentiation response. The aim of this study was to understand the mechanisms linking HA-, CD44-, and EGFR-regulated TGF-β1-dependent differentiation. CD44 and EGFR co-localization within membrane-bound lipid rafts was necessary for differentiation, and this triggered downstream mitogen-activated protein kinase (MAPK/ERK) and Ca²⁺/calmodulin kinase II (CaMKII) activation. We also found that ERK phosphorylation was upstream of CaMKII phosphorylation, that ERK activation was necessary for CaMKII signaling, and that both kinases were essential for differentiation. In addition, HA synthase-2 (HAS2) siRNA attenuated both ERK and CaMKII signaling and sequestration of CD44 into lipid rafts, preventing differentiation. In summary, the data suggest that HAS2-dependent production of HA facilitates TGF-β1-dependent fibroblast differentiation through promoting CD44 interaction with EGFR held within membrane-bound lipid rafts. This induces MAPK/ERK, followed by CaMKII activation, leading to differentiation. This pathway is synergistic with the classical TGF-β1-dependent SMAD-signaling pathway and may provide a novel opportunity for intervention in wound healing.  

3.1994           Analysis of subcellular [57Co] cobalamin distribution in SH-SY5Y neurons and brain tissue

Zhao, H., Ruberu, K., Li, H. and garner, B.

1. Neuroscience Methods, 2217, 67-74 (2013)

  Cobalamin (Cbl) utilization as a cofactor for methionine synthase and methylmalonyl-CoA mutase is dependent on the transport of Cbl through lysosomes and its subsequent delivery to the cytosol and mitochondria. We speculated that neuropathological conditions that impair lysosomal function (e.g., age-related lipofuscinosis and specific neurodegenerative diseases) might impair lysosomal Cbl transport. To address this question, an appropriate method to quantify intracellular Cbl transport in neuronal cell types and brain tissue is required. Thus, we developed methods to measure [⁵⁷Co] Cbl levels in lysosomes, mitochondria and cytosol obtained from in vitro and in vivo sources. Human SH-SY5Y neurons or HT1080 fibroblasts were labeled with [⁵⁷Co] Cbl and homogenized using a ball-bearing homogenizer, and the lysates were separated into 10 fractions using ultracentrifugation in an OptiPrep density gradient. Lysosomes were recovered from the top of the gradient (fractions 1–5), which were clearly separated from mitochondria (fractions 7–9) on the basis of the expression of the marker proteins, LAMP2 and VDAC1. The isolated lysosomes were intact based on their colocalization with acid phosphatase activity. The lysosomal and mitochondrial fractions were free of the cytosolic markers beta-actin and methionine synthase. The relative distribution of [⁵⁷Co] Cbl in both neurons and fibroblasts was as follows: 6% in the lysosomes, 14% in the mitochondria and 80% in the cytosol. This technique was also used to fractionate organelles from mouse brain, where marker proteins were detected in the gradient at positions similar to those observed for the cell lines, and the relative distribution of [⁵⁷Co] Cbl was as follows: 12% in the lysosomes, 15% in the mitochondria and 73% in the cytosol. These methods provide a useful tool for the investigation of intracellular Cbl trafficking in a neurobiological setting.  

3.1995           Perilipin-Mediated Lipid Droplet Formation in Adipocytes Promotes Sterol Regulatory Element-Binding Protein-1 Processing and Triacylglyceride Accumulation

Takahashi, Y., Shinoda, A., Furuya, N., Harada, E., Arimura, N., Ichi, I., Fujiwara, Y., Inoue, J. and Sato, R. PloS One, 8(5), e64605 (2013)   Sterol regulatory element-binding protein-1 (SREBP-1) has been thought to be a critical factor that assists adipogenesis. During adipogenesis SREBP-1 stimulates lipogenic gene expression, and peroxisome proliferator-activated receptor γ (PPARγ) enhances perilipin (plin) gene expression, resulting in generating lipid droplets (LDs) to store triacylglycerol (TAG) in adipocytes. Plin coats adipocyte LDs and protects them from lipolysis. Here we show in white adipose tissue (WAT) of plin−/− mice that nuclear active SREBP-1 and its target gene expression, but not nuclear SREBP-2, significantly decreased on attenuated LD formation. When plin−/− mouse embryonic fibroblasts (MEFs) differentiated into adipocytes, attenuated LDs were formed and nuclear SREBP-1 decreased, but enforced plin expression restored them to their original state. Since LDs are largely derived from the endoplasmic reticulum (ER), alterations in the ER cholesterol content were investigated during adipogenesis of 3T3-L1 cells. The ER cholesterol greatly reduced in differentiated adipocytes. The ER cholesterol level in plin−/− WAT was significantly higher than that of wild-type mice, suggesting that increased LD formation caused a change in ER environment along with a decrease in cholesterol. When GFP-SREBP-1 fusion proteins were exogenously expressed in 3T3-L1 cells, a mutant protein lacking the S1P cleavage site was poorly processed during adipogenesis, providing evidence of the increased canonical pathway for SREBP processing in which SREBP-1 is activated by two cleavage enzymes in the Golgi. Therefore, LD biogenesis may create the ER microenvironment favorable for SREBP-1 activation. We describe the novel interplay between LD formation and SREBP-1 activation through a positive feedback loop.  

3.1996           Iron-Induced Changes in the Proteome of Trichomonas vaginalis Hydrogenosomes

Beltran, N.C., Horvathova, L., Jedelsky, P.L., Sedinova, M., Rada, P., Marcincikova, M,., Hrdy, I. and Tachezy, J. PloS One, 8(5), e65148 (2013)   Iron plays a crucial role in metabolism as a key component of catalytic and redox cofactors, such as heme or iron-sulfur clusters in enzymes and electron-transporting or regulatory proteins. Limitation of iron availability by the host is also one of the mechanisms involved in immunity. Pathogens must regulate their protein expression according to the iron concentration in their environment and optimize their metabolic pathways in cases of limitation through the availability of respective cofactors. Trichomonas vaginalis, a sexually transmitted pathogen of humans, requires high iron levels for optimal growth. It is an anaerobe that possesses hydrogenosomes, mitochondrion-related organelles that harbor pathways of energy metabolism and iron-sulfur cluster assembly. We analyzed the proteomes of hydrogenosomes obtained from cells cultivated under iron-rich and iron-deficient conditions employing two-dimensional peptide separation combining IEF and nano-HPLC with quantitative MALDI-MS/MS. We identified 179 proteins, of which 58 were differentially expressed. Iron deficiency led to the upregulation of proteins involved in iron-sulfur cluster assembly and the downregulation of enzymes involved in carbohydrate metabolism. Interestingly, iron affected the expression of only some of multiple protein paralogues, whereas the expression of others was iron independent. This finding indicates a stringent regulation of differentially expressed multiple gene copies in response to changes in the availability of exogenous iron    

3.1997           Apolipoprotein AI and High-Density Lipoprotein Have Anti-Inflammatory Effects on Adipocytes via Cholesterol Transporters: ATP-Binding Cassette A-1, ATP-Binding Cassette G-1, and Scavenger Receptor B-1

Umemoto, T., Han, C.Y., Mitra, P., Averill, M.M., Tang, C., Goodspeed, L., Omer, M., Subramanian, S., Wang, S., Hartigh, L.J.D., Wei, H., Kim, E.J., Kim, J., O’Brien, K.D. and Chait, A. Cir. Res., 112(10), 1345-1354 (2013)   Rationale: Macrophage accumulation in adipose tissue associates with insulin resistance and increased cardiovascular disease risk. We previously have shown that generation of reactive oxygen species and monocyte chemotactic factors after exposure of adipocytes to saturated fatty acids, such as palmitate, occurs via translocation of NADPH oxidase 4 into lipid rafts (LRs). The anti-inflammatory effects of apolipoprotein AI (apoAI) and high-density lipoprotein (HDL) on macrophages and endothelial cells seem to occur via cholesterol depletion of LRs. However, little is known concerning anti-inflammatory effects of HDL and apoAI on adipocytes. Objective: To determine whether apoAI and HDL inhibit inflammation in adipocytes and adipose tissue, and whether this is dependent on LRs. Methods and Results: In 3T3L-1 adipocytes, apoAI, HDL, and methyl-β-cyclodextrin inhibited chemotactic factor expression. ApoAI and HDL also disrupted LRs, reduced plasma membrane cholesterol content, inhibited NADPH oxidase 4 translocation into LRs, and reduced palmitate-induced reactive oxygen species generation and monocyte chemotactic factor expression. Silencing ATP-binding cassette A-1 abrogated the effect of apoAI, but not HDL, whereas silencing ATP-binding cassette G-1 or scavenger receptor B-1 abrogated the effect of HDL but not apoAI. In vivo, apoAI transgenic mice fed a high-fat, high-sucrose, cholesterol-containing diet showed reduced chemotactic factor and proinflammatory cytokine expression and reduced macrophage accumulation in adipose tissue. Conclusions: ApoAI and HDL have anti-inflammatory effects in adipocytes and adipose tissue similar to their effects in other cell types. These effects are consistent with disruption and removal of cholesterol from LRs, which are regulated by cholesterol transporters, such as ATP-binding cassette A-1, ATP-binding cassette G-1, and scavenger receptor B-1.  

3.1998           Tam41 Is a CDP-Diacylglycerol Synthase Required for Cardiolipin Biosynthesis in Mitochondria

Tamura, Y., Harada, Y., Nishikawa, S-i., Yamano, K., Kamiya, M., Shiota, T., Kuroda, T., Kuge, O., Sesaki, H., Imai, K., Tomil, K. and Endo, T. Cell Metabolism, 17(5), 709-718 (2013)   CDP-diacylglycerol (CDP-DAG) is central to the phospholipid biosynthesis pathways in cells. A prevailing view is that only one CDP-DAG synthase named Cds1 is present in both the endoplasmic reticulum (ER) and mitochondrial inner membrane (IM) and mediates generation of CDP-DAG from phosphatidic acid (PA) and CTP. However, we demonstrate here by using yeast Saccharomyces cerevisiae as a model organism that Cds1 resides in the ER but not in mitochondria, and that Tam41, a highly conserved mitochondrial maintenance protein, directly catalyzes the formation of CDP-DAG from PA in the mitochondrial IM. We also find that inositol depletion by overexpressing an arrestin-related protein Art5 partially restores the defects of cell growth and CL synthesis in the absence of Tam41. The present findings unveil the missing step of the cardiolipin synthesis pathway in mitochondria as well as the flexibile regulation of phospholipid biosynthesis to respond to compromised CDP-DAG synthesis in mitochondria.  

3.1999           Bioanalysis of Eukaryotic Organelles

Satori, C.P., Henderson, M.M., Krautkramer, E.A., Kostal, V., Distefano, M.M. and Arriaga, E.A. Chem. Rev., 113(4), 2733-2811 (2013)   No abstract available.  

3.2000           Major histocompatibility complex class-II molecules promote targeting of human immunodeficiency virus type 1 virions in late endosomes by enhancing internalization of nascent particles from the plasma membrane

Finzi, A., Periman, M., Bourgeois-Daigneault, M-C., Thibodeau, J. and Cohen, E.A. Cell. Microbiol., 15(5), 809-822 (2013)   Productive assembly of human immunodeficiency virus type 1 (HIV-1) takes place, primarily, at the plasma membrane. However, depending on the cell types, a significant proportion of nascent virus particles are internalized and routed to late endosomes. We previously reported that expression of human leucocyte antigen (HLA)-DR promoted a redistribution of Gag in late endosomes and an increased detection of mature virions in these compartments in HeLa and human embryonic kidney 293T model cell lines. Although this redistribution of Gag resulted in a marked decrease of HIV-1 release, the underlying mechanism remained undefined. Here, we provide evidence that expression of HLA-DR at the cell surface induces a redistribution of mature Gag products into late endosomes by enhancing nascent HIV-1 particle internalization from the plasma membrane through a process that relies on the presence of intact HLA-DR and β-chain cytosolic tails. These findings raise the possibility that major histocompatibility complex class-II molecules might influence endocytic events at the plasma membrane and as a result promote endocytosis of progeny HIV-1 particles.  

3.2001           Determination and physiological roles of the glycosylphosphatidylinositol lipid remodelling pathway in yeast

Yoko-o, T., Ichikawa, D., Miyagishi, Y., Kato, A., Umemura, M., Takase, K., Ra, M., Ikeda, K., Taguchi, R. and Jigami, Y. Mol. Microbiol., 88(1), 140-155 (2013)   In the yeast Saccharomyces cerevisiae, glycosylphosphatidylinositol (GPI)-anchored proteins play important roles in cell wall biogenesis/assembly and the formation of lipid microdomains. The lipid moieties of mature GPI-anchored proteins in yeast typically contain either ceramide moieties or diacylglycerol. Recent studies have identified that the GPI phospholipase A₂ Per1p and O-acyltransferase Gup1p play essential roles in diacylglycerol-type lipid remodelling of GPI-anchored proteins, while Cwh43p is involved in the remodelling of lipid moieties to ceramide. It has been generally proposed that phosphatidylinositol with diacylglycerol containing a C26 saturated fatty acid, which is generated by the sequential activity of Per1p and Gup1p, is converted to inositolphosphorylceramide by Cwh43p. In this report, we constructed double-mutant strains defective in lipid remodelling and investigated their growth phenotypes and the lipid moieties of GPI-anchored proteins. Based on our analyses of single- and double-mutants of proteins involved in lipid remodelling, we demonstrate that an alternative pathway, in which lyso-phosphatidylinositol generated by Per1p is used as a substrate for Cwh43p, is involved in the remodelling of GPI lipid moieties to ceramide when the normal sequential pathway is inhibited. In addition, mass spectrometric analysis of lipid species of Flag-tagged Gas1p revealed that Gas1p contains ceramide moieties in its GPI anchor.  

3.2002           Motor and sensory neuropathy due to myelin infolding and paranodal damage in a transgenic mouse model of Charcot–Marie–Tooth disease type 1C

Lee, S.M., Sha, D., Mohammed, A.A., Asress, S., Glass, J.D., Chin, L-S. and Li, L. Human Mol. Genet., 22(9), 1755-1770 (2013)   Charcot–Marie–Tooth disease type 1C (CMT1C) is a dominantly inherited motor and sensory neuropathy. Despite human genetic evidence linking missense mutations in SIMPLE to CMT1C, the in vivo role of CMT1C-linked SIMPLE mutations remains undetermined. To investigate the molecular mechanism underlying CMT1C pathogenesis, we generated transgenic mice expressing either wild-type or CMT1C-linked W116G human SIMPLE. Mice expressing mutant, but not wild type, SIMPLE develop a late-onset motor and sensory neuropathy that recapitulates key clinical features of CMT1C disease. SIMPLE mutant mice exhibit motor and sensory behavioral impairments accompanied by decreased motor and sensory nerve conduction velocity and reduced compound muscle action potential amplitude. This neuropathy phenotype is associated with focally infolded myelin loops that protrude into the axons at paranodal regions and near Schmidt–Lanterman incisures of peripheral nerves. We find that myelin infolding is often linked to constricted axons with signs of impaired axonal transport and to paranodal defects and abnormal organization of the node of Ranvier. Our findings support that SIMPLE mutation disrupts myelin homeostasis and causes peripheral neuropathy via a combination of toxic gain-of-function and dominant-negative mechanisms. The results from this study suggest that myelin infolding and paranodal damage may represent pathogenic precursors preceding demyelination and axonal degeneration in CMT1C patients.  

3.2003           Protein Sorting Motifs in the Cytoplasmic Tail of SorCS1 Control Generation of Alzheimer's Amyloid-β Peptide

Lane, R.F., Steele, J.W., Cai, D., Ehrlich, M.E., Attie, A.D. and Gandy, S.

1. Neurosci., 33(16), 7099-7107 (2013)

  Endosomal sorting of the Alzheimer amyloid precursor protein (APP) plays a key role in the biogenesis of the amyloid-β (Aβ) peptide. Genetic lesions underlying Alzheimer's disease (AD) can act by interfering with this physiological process. Specifically, proteins involved in trafficking between endosomal compartments and the trans-Golgi network (TGN) [including the retromer complex (Vps35, Vps26) and its putative receptors (sortilin, SorL1, SorCS1)] have been implicated in the molecular pathology of late-onset AD. Previously, we demonstrated a role for SorCS1 in APP metabolism and Aβ production and, while we implicated a role for the retromer in this regulation, the underlying mechanism remained poorly understood. Here, we provide evidence for a motif within the SorCS1c cytoplasmic tail that, when manipulated, results in perturbed sorting of APP and/or its fragments to endosomal compartments, decreased retrograde TGN trafficking, and increased Aβ production in H4 neuroglioma cells. These perturbations apparently do not involve turnover of the cell surface APP pool, but rather they involve intracellular APP and/or its fragments, downstream of APP endocytosis.  

3.2004           PtdIns(4)P regulates retromer–motor interaction to facilitate dynein–cargo dissociation at the trans-Golgi network

Niu, Y., Zhang, C., Sun, Z., Hong, Z., Li, K., Sun, D.a, yang, Y., Tian, C., Gong, W. and Liu, J-J. Nature Cell Biol., 15(4), 417-429 (2013)   The molecular mechanisms for the retrograde motor dynein–dynactin to unload its cargoes at their final destination remain to be elucidated. In this study, we have investigated the regulatory mechanism underlying release of retromer-associated cargoes at the trans-Golgi network (TGN). We report that phosphotidylinositol-4-phosphate (PtdIns(4)P), a Golgi-enriched phosphoinositide, negatively regulates the protein–protein interaction between the p150^Glued subunit of dynein–dynactin and the retromer component SNX6. We show that PtdIns(4)P specifically facilitates dissociation of retromer-mediated membranous cargoes from the motor at the TGN and uncover an important function for PtdIns(4)P in the spatial control of retrograde vesicular trafficking to the TGN membrane. PtdIns(4)P also regulates SNX4-mediated retrograde vesicular trafficking to the endocytic recycling compartment by modulating its interaction with dynein. These results establish organelle-specific phosphoinositide regulation of motor–cargo interaction as a mechanism for cargo release by molecular motors at target membrane.  

3.2005           Lc3 Over-Expression Improves Survival and Attenuates Lung Injury Through Increasing Autophagosomal Clearance in Septic Mice

Lo, S., Yuan, S-S.F., Hsu, C., Cheng, Y.J., Chang, Y-F., Hsueh, H-W., Lee, P-H., and Hsieh, Y-C. Annals of Surgery, 257(2), 352-363 (2013)   Objective: To clarify the role of autophagy in sepsis-induced lung injury. Background: The role of autophagy as a protective or maladaptive response in lung cells during sepsis has not yet been determined. The lack of specificity of the autophagic process has driven the development of new approaches that assessautophagosomes from formation to fusion with lysosomes. Methods: Sepsis was induced by cecal ligation and puncture (CLP). The autophagic process was manipulated using the pharmacological inhibitors of the autophagy pathway. Green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) transgenic mice were further used to determine the role of autophagy. Results: The formation of autophagosomal protein LC3-II progressively accumulated in the lungs over 24 hours after CLP, with the Lc3 gene expression returning to baseline levels at 24 hours. Autophagosome-lysosome fusion, however, gradually decreased from 8 to 24 hours after CLP, suggesting impaired clearance of autophagosomes rather than upregulation of autophagy in the septic lung. In contrast, transgenic mice overexpressing the Lc3 gene exhibited increased clearance of autophagosomes and improved survival after CLP. This protective effect was also seen in decreased cell death, inflammatory responses, neutrophil accumulation, albumin leakage, and edema formation. However, blockade of autophagosome-lysosome fusion with bafilomycin A1 abolished the protective effects in transgenic mice. This indicates that Lc3 transgene attenuates lung injury/inflammation in sepsis, possibly through increasing the clearance of autophagosomes. Conclusions: Autophagy in the septic lung represents a protective response. However, autophagy, by virtue of excessive autophagosome accumulation, may play a maladaptive role in the late stage of sepsis, leading to acute lung injury.  

3.2006           Negative Regulation of the Novel norpAP24 Suppressor, diehard4, in the Endo-lysosomal Trafficking Underlies Photoreceptor Cell Degeneration

Lee, J., Song, M. and Hong, S. PloS Genetics, 9(6), e1003559 (2013)   Rhodopsin has been used as a prototype system to investigate G protein-coupled receptor (GPCR) internalization and endocytic sorting mechanisms. Failure of rhodopsin recycling upon light activation results in various degenerative retinal diseases. Accumulation of internalized rhodopsin in late endosomes and the impairment of its lysosomal degradation are associated with unregulated cell death that occurs in dystrophies. However, the molecular basis of rhodopsin accumulation remains elusive. We found that the novel norpA^P24 suppressor, diehard4, is responsible for the inability of endo-lysosomal rhodopsin trafficking and retinal degeneration in Drosophila models of retinal dystrophies. We found that diehard4 encodes Osiris 21. Loss of its function suppresses retinal degeneration in norpA^P24, rdgC³⁰⁶, and trp¹, but not in rdgB², suggesting a common cause of photoreceptor death. In addition, the loss of Osiris 21 function shifts the membrane balance between late endosomes and lysosomes as evidenced by smaller late endosomes and the proliferation of lysosomal compartments, thus facilitating the degradation of endocytosed rhodopsin. Our results demonstrate the existence of negative regulation in vesicular traffic between endosomes and lysosomes. We anticipate that the identification of additional components and an in-depth description of this specific molecular machinery will aid in therapeutic interventions of various retinal dystrophies and GPCR-related human diseases.

3.2007           NHERF2 Protein Mobility Rate Is Determined by a Unique C-terminal Domain That Is Also Necessary for Its Regulation of NHE3 Protein in OK Cells

Yang, J., Singh, V., Cha, B., Chen, T-E., Sarker, R., Murtazina, R., Jin, S., Zachos, N.C., Patterson, G.H., Tse, C.M., Kovbasnjuk, O., Li, X. and Donowitz, M.

1. Biol. Chem., 288(23), 16960-16974 (2013)

  Na⁺/H⁺ exchanger regulatory factor (NHERF) proteins are a family of PSD-95/Discs-large/ZO-1 (PDZ)-scaffolding proteins, three of which (NHERFs 1-3) are localized to the brush border in kidney and intestinal epithelial cells. All NHERF proteins are involved in anchoring membrane proteins that contain PDZ recognition motifs to form multiprotein signaling complexes. In contrast to their predicted immobility, NHERF1, NHERF2, and NHERF3 were all shown by fluorescence recovery after photobleaching/confocal microscopy to be surprisingly mobile in the microvilli of the renal proximal tubule OK cell line. Their diffusion coefficients, although different among the three, were all of the same magnitude as that of the transmembrane proteins, suggesting they are all anchored in the microvilli but to different extents. NHERF3 moves faster than NHERF1, and NHERF2 moves the slowest. Several chimeras and mutants of NHERF1 and NHERF2 were made to determine which part of NHERF2 confers the slower mobility rate. Surprisingly, the slower mobility rate of NHERF2 was determined by a unique C-terminal domain, which includes a nonconserved region along with the ezrin, radixin, moesin (ERM) binding domain. Also, this C-terminal domain of NHERF2 determined its greater detergent insolubility and was necessary for the formation of larger multiprotein NHERF2 complexes. In addition, this NHERF2 domain was functionally significant in NHE3 regulation, being necessary for stimulation by lysophosphatidic acid of activity and increased mobility of NHE3, as well as necessary for inhibition of NHE3 activity by calcium ionophore 4-Br-A23187. Thus, multiple functions of NHERF2 require involvement of an additional domain in this protein.  

3.2008           Intestinal caveolin-1 is important for dietary fatty acid absorption

Siddiqi, S., Sheth, A., patel, F., Barnes, M. and Mansbach II, C.M. Biochim. Biophys. Acta, 1831, 1311-1321 (2013)   How dietary fatty acids are absorbed into the enterocyte and transported to the ER is not established. We tested the possibility that caveolin-1 containing lipid rafts and endocytic vesicles were involved. Apical brush border membranes took up 15% of albumin bound ³H-oleate whereas brush border membranes from caveolin-1 KO mice took up only 1%. In brush border membranes, the ³H-oleate was in the detergent resistant fraction of an OptiPrep gradient. On OptiPrep gradients of intestinal cytosol, we also found the ³H-oleate in the detergent resistant fraction, separate from OptiPrep gradients spiked with ³H-oleate or ³H-triacylglycerol. Caveolin-1 immuno-depletion of cytosol removed 91% of absorbed ³H-oleate whereas immuno-depletion using IgG, or anti-caveolin-2 or -3 or anti-clathrin antibodies removed 20%. Electron microscopy showed the presence of caveolin-1 containing vesicles in WT mouse cytosol that were 4 fold increased by feeding intestinal sacs 1 mM oleate. No vesicles were seen in caveolin-1 KO mouse cytosol. Caveolin-1 KO mice gained less weight on a 23% fat diet and had increased fat in their stool compared to WT mice. We conclude that dietary fatty acids are absorbed by caveolae in enterocyte brush border membranes, are endocytosed, and transported in cytosol in caveolin-1 containing endocytic vesicles.  

3.2009           Apical sorting of lysoGPI-anchored proteins occurs independent of association with detergent-resistant membranes but dependent on their N-glycosylation

Castillon, G.A., Michon, L. and Watanabe, R. Mol. Biol. Cell, 24, 2021-2033 (2013)   Most glycosylphosphatidylinositol-anchored proteins (GPI-APs) are located at the apical surface of epithelial cells. The apical delivery of GPI-APs is believed to result from their association with lipid rafts. We find that overexpression of C-terminally tagged PGAP3 caused predominant production of lysoGPI-APs, an intermediate precursor in the GPI lipid remodeling process in Madin–Darby canine kidney cells. In these cells, produced lysoGPI-APs are not incorporated into detergent-resistant membranes (DRMs) but still are delivered apically, suggesting that GPI-AP association with DRMs is not necessary for apical targeting. In contrast, apical transport of both fully remodeled and lyso forms of GPI-APs is dependent on N-glycosylation, confirming a general role of N-glycans in apical protein transport. We also find that depletion of cholesterol causes apical-to-basolateral retargeting not only of fully remodeled GPI-APs, but also of lysoGPI-APs, as well as endogenous soluble and transmembrane proteins that would normally be targeted to the apical membrane. These findings confirm the essential role for cholesterol in the apical protein targeting and further demonstrate that the mechanism of cholesterol-dependent apical sorting is not related to DRM association of GPI-APs.  

3.2010           Stress-Induced Outer Membrane Vesicle Production by Pseudomonas aeruginosa

MacDonald, I.A. and Kuehn, M.J.

1. Bacteriol., 195(13), 2971-2981 (2013)

  As an opportunistic Gram-negative pathogen, Pseudomonas aeruginosa must be able to adapt and survive changes and stressors in its environment during the course of infection. To aid survival in the hostile host environment, P. aeruginosa has evolved defense mechanisms, including the production of an exopolysaccharide capsule and the secretion of a myriad of degradative proteases and lipases. The production of outer membrane-derived vesicles (OMVs) serves as a secretion mechanism for virulence factors as well as a general bacterial response to envelope-acting stressors. This study investigated the effect of sublethal physiological stressors on OMV production by P. aeruginosa and whether the Pseudomonas quinolone signal (PQS) and the MucD periplasmic protease are critical mechanistic factors in this response. Exposure to some environmental stressors was determined to increase the level of OMV production as well as the activity of AlgU, the sigma factor that controls MucD expression. Overexpression of AlgU was shown to be sufficient to induce OMV production; however, stress-induced OMV production was not dependent on activation of AlgU, since stress caused increased vesiculation in strains lacking algU. We further determined that MucD levels were not an indicator of OMV production under acute stress, and PQS was not required for OMV production under stress or unstressed conditions. Finally, an investigation of the response of P. aeruginosa to oxidative stress revealed that peroxide-induced OMV production requires the presence of B-band but not A-band lipopolysaccharide. Together, these results demonstrate that distinct mechanisms exist for stress-induced OMV production in P. aeruginosa.  

3.2011           Lipidome analysis of rotavirus-infected cells confirms the close interaction of lipid droplets with viroplasms

Gaunt, E.R., Zhang, Q., Cheung, W., Wakelam, M.J.O., Lever, A.M. and Desselberger, U.

1. Gen. Virol., 94, 1576-1586 (2013)

  Rotaviruses (RVs) cause acute gastroenteritis in infants and young children, and are globally distributed. Within the infected host cell, RVs establish replication complexes in viroplasms (‘viral factories’) to which lipid droplet organelles are recruited. To further understand this recently discovered phenomenon, the lipidomes of RV-infected and uninfected MA104 cells were investigated. Cell lysates were subjected to equilibrium ultracentrifugation through iodixanol gradients. Fourteen different classes of lipids were differentiated by mass spectrometry. The concentrations of virtually all lipids were elevated in RV-infected cells. Fractions of low density (1.11–1.15 g ml⁻¹), in which peaks of the RV dsRNA genome and lipid droplet- and viroplasm-associated proteins were observed, contained increased amounts of lipids typically found concentrated in the cellular organelle lipid droplets, confirming the close interaction of lipid droplets with viroplasms. A decrease in the ratio of the amounts of surface to internal components of lipid droplets upon RV infection suggested that the lipid droplet–viroplasm complexes became enlarged.  

3.2012           Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain

Uribe, K.B., Etxebarria, A., Martin, C. and Ostolaza, H. PloS One, 8(6), e67648 (2013)   Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT) is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC) domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active “soluble AC”. The calpain-mediated ACT processing allows trafficking of the “soluble AC” domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP “pools”, which would play different roles in the cell pathophysiology.  

3.2013           Proteomic Identification of VEGF-dependent Protein Enrichment to Membrane Caveolar-raft Microdomains in Endothelial Progenitor Cells

Chilla, A., Magherini, F., Margheri, F., Laurenzanat, A., Gamberit, T., Bini, L., Bianchi, L., Danza, G., Mazzanti, B., Serrati, S., Modesti, A., Del Rosso, M. and Fibbi, G. Mol. Cell. Proteomics, 12, 1926-1938 (2013)   Endothelial cell caveolar-rafts are considered functional platforms that recruit several pro-angiogenic molecules to realize an efficient angiogenic program. Here we studied the differential caveolar-raft protein composition of endothelial colony-forming cells following stimulation with VEGF, which localizes in caveolae on interaction with its type-2 receptor. Endothelial colony-forming cells are a cell population identified in human umbilical blood that show all the properties of an endothelial progenitor cell and a high proliferative rate. Two-dimensional gel electrophoresis analysis was coupled with mass spectrometry to identify candidate proteins. The twenty-eight differentially expressed protein spots were grouped according to their function using Gene Ontology classification. In particular, functional categories relative to cell death inhibition and hydrogen peroxide metabolic processes resulted enriched. In these categories, Peroxiredoxin-2 and 6, that control hydrogen peroxide metabolic processes, are the main enriched molecules together with the anti-apoptotic 78 kDa glucose regulated protein. Some of the proteins we identified had never before identified as caveolar-raft components. Other identified proteins include calpain small subunit-1, known to mediates angiogenic response to VEGF, gelsolin, which regulates stress fiber assembly, and annexin A3, an angiogenic mediator that induces VEGF production. We validated the functional activity of the above proteins, showing that the siRNA silencing of these resulted in the inhibition of capillary morphogenesis. Overall, our data show that VEGF stimulation triggers the caveolar-raft recruitment of proteins that warrant a physiological amount of reactive oxygen species to maintain a proper angiogenic function of endothelial colony-forming cells and preserve the integrity of the actin cytoskeleton.  

3.2014           Impaired Very Long-chain Acyl-CoA β-Oxidation in Human X-linked Adrenoleukodystrophy Fibroblasts Is a Direct Consequence of ABCD1 Transporter Dysfunction

Wiesinger, C., Kunze, M., Regelsberger, G., Forss-Petter, S. and Berger, J.

1. Biol. Chem., 288(26), 19269-19279 (2013)

  X-linked adrenoleukodystrophy (X-ALD), an inherited peroxisomal disorder, is caused by mutations in the ABCD1 gene encoding the peroxisomal ATP-binding cassette (ABC) transporter ABCD1 (adrenoleukodystrophy protein, ALDP). Biochemically, X-ALD is characterized by an accumulation of very long-chain fatty acids and partially impaired peroxisomal β-oxidation. In this study, we used primary human fibroblasts from X-ALD and Zellweger syndrome patients to investigate the peroxisomal β-oxidation defect. Our results show that the degradation of C26:0-CoA esters is as severely impaired as degradation of unesterified very long-chain fatty acids in X-ALD and is abolished in Zellweger syndrome. Interestingly, the β-oxidation rates for both C26:0-CoA and C22:0-CoA were similarly affected, although C22:0 does not accumulate in patient fibroblasts. Furthermore, we show that the β-oxidation defect in X-ALD is directly caused by ABCD1 dysfunction as blocking ABCD1 function with a specific antibody reduced β-oxidation to levels observed in X-ALD fibroblasts. By quantification of mRNA and protein levels of the peroxisomal ABC transporters and by blocking with specific antibodies, we found that residual β-oxidation activity toward C26:0-CoA in X-ALD fibroblasts is mediated by ABCD3, although the efficacy of ABCD3 appeared to be much lower than that of ABCD1. Finally, using isolated peroxisomes, we show that β-oxidation of C26:0-CoA is independent of additional CoA but requires a cytosolic factor of >10-kDa molecular mass that is resistant to N-ethylmaleimide and heat inactivation. In conclusion, our findings in human cells suggest that, in contrast to yeast cells, very long-chain acyl-CoA esters are transported into peroxisomes by ABCD1 independently of additional synthetase activity.  

3.2015           Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA

Narayanan, A., Iordanskiy, S., Das, r., Duyne, R., Santos, S., Jaworski, E., Guendel, I., Sampey, G., Dalby, E., Iglesiaas-Ussel, M., Popratiloff, A., Hakami, R., Kehn-Hall, K., Young, M., Subra, C., Gilbert, C., Bailey, C., Romerio, F. and Kashanchi, F.

1. Biol. Chem., 288(27), 20014-20033 (2013)

  Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10⁴–10⁶ copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10³ copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.  

3.2016           The effect of organelle discovery upon sub-cellular protein localisation

Breckels, L.M:, Gatto, L., Christoforou, A., Groen, A.J., Lilley, K.S. and Trotter, M.W.B.

1. Proteomics, 88, 129-140 (2013)

  Prediction of protein sub-cellular localisation by employing quantitative mass spectrometry experiments is an expanding field. Several methods have led to the assignment of proteins to specific subcellular localisations by partial separation of organelles across a fractionation scheme coupled with computational analysis. Methods developed to analyse organelle data have largely employed supervised machine learning algorithms to map unannotated abundance profiles to known protein–organelle associations. Such approaches are likely to make association errors if organelle-related groupings present in experimental output are not included in data used to create a protein–organelle classifier. Currently, there is no automated way to detect organelle-specific clusters within such datasets. In order to address the above issues we adapted a phenotype discovery algorithm, originally created to filter image-based output for RNAi screens, to identify putative subcellular groupings in organelle proteomics experiments. We were able to mine datasets to a deeper level and extract interesting phenotype clusters for more comprehensive evaluation in an unbiased fashion upon application of this approach. Organelle-related protein clusters were identified beyond those sufficiently annotated for use as training data. Furthermore, we propose avenues for the incorporation of observations made into general practice for the classification of protein–organelle membership from quantitative MS experiments. Biological significance Protein sub-cellular localisation plays an important role in molecular interactions, signalling and transport mechanisms. The prediction of protein localisation by quantitative mass-spectrometry (MS) proteomics is a growing field and an important endeavour in improving protein annotation. Several such approaches use gradient-based separation of cellular organelle content to measure relative protein abundance across distinct gradient fractions. The distribution profiles are commonly mapped in silico to known protein–organelle associations via supervised machine learning algorithms, to create classifiers that associate unannotated proteins to specific organelles. These strategies are prone to error, however, if organelle-related groupings present in experimental output are not represented, for example owing to the lack of existing annotation, when creating the protein–organelle mapping. Here, the application of a phenotype discovery approach to LOPIT gradient-based MS data identifies candidate organelle phenotypes for further evaluation in an unbiased fashion. Software implementation and usage guidelines are provided for application to wider protein–organelle association experiments. In the wider context, semi-supervised organelle discovery is discussed as a paradigm with which to generate new protein annotations from MS-based organelle proteomics experiments. This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].  

3.2017           Stx5 is a novel interactor of VLDL-R to affect its intracellular trafficking and processing

Wagner, T., Dieckmann, M., Jaeger, S., Weggen, S. and Pietrzik, C.U. Exp. Cell Res., 319, 1956-1972 (2013)   We identified syntaxin 5 (Stx5), a protein involved in intracellular vesicle trafficking, as a novel interaction partner of the very low density lipoprotein (VLDL)-receptor (VLDL-R), a member of the LDL-receptor family. In addition, we investigated the effect of Stx5 on VLDL-R maturation, trafficking and processing. Here, we demonstrated mutual association of both proteins using several in vitro approaches. Furthermore, we detected a special maturation phenotype of VLDL-R resulting from Stx5 overexpression. We found that Stx5 prevented advanced Golgi-maturation of VLDL-R, but did not cause accumulation of the immature protein in ER, ER to Golgi compartments, or cis-Golgi ribbon, the main expression sites of Stx5. Rather more, abundantly present Stx5 was capable of translocating ER-/N-glycosylated VLDL-R to the plasma membrane, and thus was insensitive to BFA treatment and low temperature. Furthermore, abundant presence of Stx5 significantly interfered with VLDL-R reaching the trans-Golgi network. Based on our findings, we postulate that Stx5 can directly bind to the C-terminal domain of VLDL-R, thereby influencing the receptor′s glycosylation, trafficking and processing characteristics. Resulting from that, we further suggest that Stx5 might play a role in modulating VLDL-R physiology by participating in an abrasively described or completely novel Golgi-bypass pathway.  

3.2018           EphrinA2 Regulates Clathrin Mediated KSHV Endocytosis in Fibroblast Cells by Coordinating Integrin-Associated Signaling and c-Cbl Directed Polyubiquitination

Dutta, D., Chakraborty, S., Bandyopadhyay, C., Veettil, M.V., Ansari, M.A., Singh, V.V. and Chandran, B. PloS One, 9(7), e1003510 (2013)   Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with human dermal endothelial cell surface tyrosine kinase EphrinA2 (EphA2) and integrins (α3β1 and αVβ3) in the lipid raft (LR) region, and EphA2 regulates macropinocytic virus entry by coordinating integrin-c-Cbl associated signaling. In contrast, KSHV enters human foreskin fibroblast (HFF) cells by LR-independent clathrin mediated endocytosis. The present studies conducted to identify the key molecules regulating KSHV entry in HFF cells showed that KSHV induces association with integrins (αVβ5, αVβ3 and α3β1) and EphA2 in non-LR regions early during infection and activates EphA2, which in turn associates with phosphorylated c-Cbl, myosin IIA, FAK, Src, and PI3-K, as well as clathrin and its adaptor AP2 and effector Epsin-15 proteins. EphA2 knockdown significantly reduced these signal inductions, virus internalization and gene expression. c-Cbl knockdown ablated the c-Cbl mediated K63 type polyubiquitination of EphA2 and clathrin association with EphA2 and KSHV. Mutations in EphA2's tyrosine kinase domain (TKD) or sterile alpha motif (SAM) abolished its interaction with c-Cbl. Mutations in tyrosine kinase binding (TKB) or RING finger (RF) domains of c-Cbl resulted in very poor association of c-Cbl with EphA2 and decreased EphA2 polyubiquitination. These studies demonstrated the contributions of these domains in EphA2 and c-Cbl association, EphA2 polyubiquitination and virus-EphA2 internalization. Collectively, these results revealed for the first time that EphA2 influences the tyrosine phosphorylation of clathrin, the role of EphA2 in clathrin mediated endocytosis of a virus, and c-Cbl mediated EphA2 polyubiquitination directing KSHV entry in HFF cells via coordinated signal induction and progression of endocytic events, all of which suggest that targeting EphA2 and c-Cbl could block KSHV entry and infection.  

3.2019           Oncogenic H-Ras Reprograms Madin-Darby Canine Kidney (MDCK) Cell-derived Exosomal Proteins Following Epithelial-Mesenchymal Transition

Tauro, B.J., Mathias, R.A., Greening, D.W., Gopal, S.K., Ji, H., Kapp, E.A., Coleman, B.M., Hill, A.F., Kusebauch, U., Hallows, J.L., Shteynberg, D., Moritz, R.L., Zhu, H-J. and Simpson, R.J. Mol. Cell. Proteomics, 12(8), 2148-2159 (2013)   Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. EMT is associated with increased aggressiveness, invasiveness, and metastatic potential in carcinoma cells. To assess the contribution of extracellular vesicles following EMT, we conducted a proteomic analysis of exosomes released from Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells). Exosomes are 40–100 nm membranous vesicles originating from the inward budding of late endosomes and multivesicular bodies and are released from cells on fusion of multivesicular bodies with the plasma membrane. Exosomes from MDCK cells (MDCK-Exos) and 21D1 cells (21D1-Exos) were purified from cell culture media using density gradient centrifugation (OptiPrep™), and protein content identified by GeLC-MS/MS proteomic profiling. Both MDCK- and 21D1-Exos populations were morphologically similar by cryo-electron microscopy and contained stereotypical exosome marker proteins such as TSG101, Alix, and CD63. In this study we show that the expression levels of typical EMT hallmark proteins seen in whole cells correlate with those observed in MDCK- and 21D1-Exos, i.e. reduction of characteristic inhibitor of angiogenesis, thrombospondin-1, and epithelial markers E-cadherin, and EpCAM, with a concomitant up-regulation of mesenchymal makers such as vimentin. Further, we reveal that 21D1-Exos are enriched with several proteases (e.g. MMP-1, -14, -19, ADAM-10, and ADAMTS1), and integrins (e.g. ITGB1, ITGA3, and ITGA6) that have been recently implicated in regulating the tumor microenvironment to promote metastatic progression. A salient finding of this study was the unique presence of key transcriptional regulators (e.g. the master transcriptional regulator YBX1) and core splicing complex components (e.g. SF3B1, SF3B3, and SFRS1) in mesenchymal 21D1-Exos. Taken together, our findings reveal that exosomes from Ras-transformed MDCK cells are reprogrammed with factors which may be capable of inducing EMT in recipient cells.  

3.2020           Sorting of GLUT4 into its insulin-sensitive store requires the Sec1/Munc18 protein mVps45

Roccisana, J.-, Sadler, J.B.A., Bryant, N.J. and Gould, G.W. Mol. Biol. Cell, 24, 2389-2397 (2013)   Insulin stimulates glucose transport in fat and muscle cells by regulating delivery of the facilitative glucose transporter, glucose transporter isoform 4 (GLUT4), to the plasma membrane. In the absence of insulin, GLUT4 is sequestered away from the general recycling endosomal pathway into specialized vesicles, referred to as GLUT4-storage vesicles. Understanding the sorting of GLUT4 into this store is a major challenge. Here we examine the role of the Sec1/Munc18 protein mVps45 in GLUT4 trafficking. We show that mVps45 is up-regulated upon differentiation of 3T3-L1 fibroblasts into adipocytes and is expressed at stoichiometric levels with its cognate target–soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 16. Depletion of mVps45 in 3T3-L1 adipocytes results in decreased GLUT4 levels and impaired insulin-stimulated glucose transport. Using sub­cellular fractionation and an in vitro assay for GLUT4-storage vesicle formation, we show that mVps45 is required to correctly traffic GLUT4 into this compartment. Collectively our data reveal a crucial role for mVps45 in the delivery of GLUT4 into its specialized, insulin-regulated compartment.  

3.2021           An Inhibitor of the δPKC Interaction with the d Subunit of F1Fo ATP Synthase Reduces Cardiac Troponin I Release from Ischemic Rat Hearts: Utility of a Novel Ammonium Sulfate Precipitation Technique

Ogbi, M., Obi, I. and Johnson, J.A. PloS One, 8(8), e70580 (2013)   We have previously reported protection against hypoxic injury by a cell-permeable, mitochondrially-targeted δPKC-d subunit of F₁Fo ATPase (dF₁Fo) interaction inhibitor [NH₂-YGRKKRRQRRRMLA TRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK​-COOH]in neonatal cardiac myo-cytes. In the present work we demonstrate the partitioning of this peptide to the inner membrane and matrix of mitochondria when it is perfused into isolated rat hearts. We also used ammonium sulfate ((NH₄)₂SO₄) and chloroform/methanol precipitation of heart effluents to demonstrate reduced card-iac troponin I (cTnI) release from ischemic rat hearts perfused with this inhibitor. 50% (NH₄)₂SO₄ saturation of perfusates collected from Langendorff rat heart preparations optimally precipitated cTnI, allowing its detection in Western blots. In hearts receiving 20 min of ischemia followed by 30, or 60 min of reperfusion, the Mean±S.E. (n = 5) percentage of maximal cTnI release was 30±7 and 60±17, respectively, with additional cTnI release occurring after 150 min of reperfusion. Perfusion of hearts with the δPKC-dF₁Fo interaction inhibitor, prior to 20 min of ischemia and 60–150 min of reperfusion, reduced cTnI release by 80%. Additionally, we found that when soybean trypsin inhibitor (SBTI), was added to rat heart effluents, it could also be precipitated using (NH₄)₂SO₄ and detected in western blots. This provided a convenient method for normalizing protein recoveries between groups. Our results support the further development of the δPKC-dF₁Fo inhibitor as a potential therapeutic for combating cardiac ischemic injury. In addition, we have developed an improved method for the detection of cTnI release from perfused rat hearts.  

3.2022           Wip1 suppresses apoptotic cell death through direct dephosphorylation of BAX in response to γ-radiation

Song, J-Y., Ryu, S-H., Cho, Y.M., Kim, Y.S., Lee, B-M., Lee, S-W. and Choi, J. Cell Death and Disease, 4, e744 (2013)   Wild-type p53-induced phosphatase 1 (Wip1) is a p53-inducible serine/threonine phosphatase that switches off DNA damage checkpoint responses by the dephosphorylation of certain proteins (i.e. p38 mitogen-activated protein kinase, p53, checkpoint kinase 1, checkpoint kinase 2, and uracil DNA glycosylase) involved in DNA repair and the cell cycle checkpoint. Emerging data indicate that Wip1 is amplified or overexpressed in various human tumors, and its detection implies a poor prognosis. In this study, we show that Wip1 interacts with and dephosphorylates BAX to suppress BAX-mediated apoptosis in response to γ-irradiation in prostate cancer cells. Radiation-resistant LNCaP cells showed dramatic increases in Wip1 levels and impaired BAX movement to the mitochondria after γ-irradiation, and these effects were reverted by a Wip1 inhibitor. These results show that Wip1 directly interacts with and dephosphorylates BAX. Dephosphorylation occurs at threonines 172, 174 and 186, and BAX proteins with mutations at these sites fail to translocate efficiently to the mitochondria following cellular γ-irradiation. Overexpression of Wip1 and BAX, but not phosphatase-dead Wip1, in BAX-deficient cells strongly reduces apoptosis. Our results suggest that BAX dephosphorylation of Wip1 phosphatase is an important regulator of resistance to anticancer therapy. This study is the first to report the downregulation of BAX activity by a protein phosphatase.  

3.2023           The ER–Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis

Ge, L., Melville, D., Zhang, M. and Schekman, R. eLife, 2, e00947 (2013)   Autophagy is a catabolic process for bulk degradation of cytosolic materials mediated by double-membraned autophagosomes. The membrane determinant to initiate the formation of autophagosomes remains elusive. Here, we establish a cell-free assay based on LC3 lipidation to define the organelle membrane supporting early autophagosome formation. In vitro LC3 lipidation requires energy and is subject to regulation by the pathways modulating autophagy in vivo. We developed a systematic membrane isolation scheme to identify the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) as a primary membrane source both necessary and sufficient to trigger LC3 lipidation in vitro. Functional studies demonstrate that the ERGIC is required for autophagosome biogenesis in vivo. Moreover, we find that the ERGIC acts by recruiting the early autophagosome marker ATG14, a critical step for the generation of preautophagosomal membranes.  

3.2024           Interaction Domains of Sos1/Grb2 Are Finely Tuned for Cooperative Control of Embryonic Stem Cell Fate

Findlay, G.M. et al Cell, 152(5), 1008-1020 (2013)   Metazoan evolution involves increasing protein domain complexity, but how this relates to control of biological decisions remains uncertain. The Ras guanine nucleotide exchange factor (RasGEF) Sos1 and its adaptor Grb2 are multidomain proteins that couple fibroblast growth factor (FGF) signaling to activation of the Ras-Erk pathway during mammalian development and drive embryonic stem cells toward the primitive endoderm (PrE) lineage. We show that the ability of Sos1/Grb2 to appropriately regulate pluripotency and differentiation factors and to initiate PrE development requires collective binding of multiple Sos1/Grb2 domains to their protein and phospholipid ligands. This provides a cooperative system that only allows lineage commitment when all ligand-binding domains are occupied. Furthermore, our results indicate that the interaction domains of Sos1 and Grb2 have evolved so as to bind ligands not with maximal strength but with specificities and affinities that maintain cooperativity. This optimized system ensures that PrE lineage commitment occurs in a timely and selective manner during embryogenesis.  

3.2025           Cell-Cell Communication between Malaria-Infected Red Blood Cells via Exosome-like Vesicles

Regev-Rudzki, N., Wilson, D.W., carvalho, T., Sisquella, X., Coleman, B.M., Rug, M., Bursac, D., Angrisano, F., Gee, M., Hill., A.F. and Baum, J. Cell, 153(5), 1120-1133 (2013)   Cell-cell communication is an important mechanism for information exchange promoting cell survival for the control of features such as population density and differentiation. We determined that Plasmodium falciparum-infected red blood cells directly communicate between parasites within a population using exosome-like vesicles that are capable of delivering genes. Importantly, communication via exosome-like vesicles promotes differentiation to sexual forms at a rate that suggests that signaling is involved. Furthermore, we have identified a P. falciparum protein, PfPTP2, that plays a key role in efficient communication. This study reveals a previously unidentified pathway of P. falciparum biology critical for survival in the host and transmission to mosquitoes. This identifies a pathway for the development of agents to block parasite transmission from the human host to the mosquito.  

3.2026           Large protein complexes retained in the ER are dislocated by non-COPII vesicles and degraded by selective autophagy

Le Fourn, V., Park, S., Jang, I., Gaplovskaa-Kysela, K., Guhl, B., Lee, Y., Cho, J.W:, Zuber, C. and Roth, J. Cell Mol. Life Sci., 70(11), 1985-2002 (2013)   Multisubunit protein complexes are assembled in the endoplasmic reticulum (ER). Existing pools of single subunits and assembly intermediates ensure the efficient and rapid formation of complete complexes. While being kinetically beneficial, surplus components must be eliminated to prevent potentially harmful accumulation in the ER. Surplus single chains are cleared by the ubiquitin–proteasome system. However, the fate of not secreted assembly intermediates of multisubunit proteins remains elusive. Here we show by high-resolution double-label confocal immunofluorescence and immunogold electron microscopy that naturally occurring surplus fibrinogen Aα–γ assembly intermediates in HepG2 cells are dislocated together with EDEM1 from the ER to the cytoplasm in ER-derived vesicles not corresponding to COPII-coated vesicles originating from the transitional ER. This route corresponds to the novel ER exit path we have previously identified for EDEM1 (Zuber et al. Proc Natl Acad Sci USA 104:4407–4412, 2007). In the cytoplasm, detergent-insoluble aggregates of fibrinogen Aα–γ dimers develop that are targeted by the selective autophagy cargo receptors p62/SQSTM1 and NBR1. These aggregates are degraded by selective autophagy as directly demonstrated by high-resolution microscopy as well as biochemical analysis and inhibition of autophagy by siRNA and kinase inhibitors. Our findings demonstrate that different pathways exist in parallel for ER-to-cytoplasm dislocation and subsequent proteolytic degradation of large luminal protein complexes and of surplus luminal single-chain proteins. This implies that ER-associated protein degradation (ERAD) has a broader function in ER proteostasis and is not limited to the elimination of misfolded glycoproteins.  

3.2027           Palmitoylation of Amyloid Precursor Protein Regulates Amyloidogenic Processing in Lipid Rafts

Bhattacharyya, R., Barren, C. and Kovacs, D.M.

1. Neurosci., 33(27), 11169- 11183 (2013)

  Brains of patients affected by Alzheimer's disease (AD) contain large deposits of aggregated amyloid β-protein (Aβ). Only a small fraction of the amyloid precursor protein (APP) gives rise to Aβ. Here, we report that ∼10% of APP undergoes a post-translational lipid modification called palmitoylation. We identified the palmitoylation sites in APP at Cys¹⁸⁶ and Cys¹⁸⁷. Surprisingly, point mutations introduced into these cysteines caused nearly complete ER retention of APP. Thus, either APP palmitoylation or disulfide bridges involving these Cys residues appear to be required for ER exit of APP. In later compartments, palmitoylated APP (palAPP) was specifically enriched in lipid rafts. In vitro BACE1 cleavage assays using cell or mouse brain lipid rafts showed that APP palmitoylation enhanced BACE1-mediated processing of APP. Interestingly, we detected an age-dependent increase in endogenous mouse brain palAPP levels. Overexpression of selected DHHC palmitoyl acyltransferases increased palmitoylation of APP and doubled Aβ production, while two palmitoylation inhibitors reduced palAPP levels and APP processing. We have found previously that acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibition led to impaired APP processing. Here we demonstrate that pharmacological inhibition or genetic inactivation of ACAT decrease lipid raft palAPP levels by up to 76%, likely resulting in impaired APP processing. Together, our results indicate that APP palmitoylation enhances amyloidogenic processing by targeting APP to lipid rafts and enhancing its BACE1-mediated cleavage. Thus, inhibition of palAPP formation by ACAT or specific palmitoylation inhibitors would appear to be a valid strategy for prevention and/or treatment of AD.  

3.2028           Mammalian SOD2 is exclusively located in mitochondria and not present in peroxisomes

Karnati, S., Lüers, G. and Pfreimer, S. Histochem. Cell. Biol., 140(2), 105-117 (2013)   Superoxide dismutases (SODs) are metalloenzymes that belong to the essential antioxidant enzyme systems of virtually all oxygen-respiring organisms. SODs catalyze the dismutation of highly reactive superoxide radicals into hydrogen peroxide and molecular oxygen. For the subcellular localization of the manganese superoxide dismutase (SOD2) in eukaryotic cells, a dual mitochondrial localization and peroxisomal localization were proposed in the literature. However, our own observation from immunofluorescence preparations of human and mouse tissues suggested that SOD2 serves as an excellent marker protein for mitochondria but never co-localized with peroxisomes. To clarify whether our observations were correct, we have carefully reinvestigated the subcellular localization of SOD2 using sensitive double-immunofluorescence methods on frozen and paraffin sections as well as in cell culture preparations. In addition, ultrastructural analyses were performed with post-embedding immunoelectron microscopy on LR White sections as well as labeling of ultrathin cryosections with various immunogold techniques. In all morphological experiments, the SOD2 localization was compared to one of the catalase, a typical marker protein for peroxisomes, solely localized in these organelles. Moreover, biochemical subcellular fractions of mouse liver was used to isolate enriched organelles and highly purified peroxisomal fractions for Western blot analyses of the exact subcellular distributions of SOD2 and catalase. All results with the various methodologies, tissues, and cell types used revealed that catalase and SOD2 were always confined to distinct and separate subcellular compartments. SOD2 was unequivocally in mitochondria, but never present in peroxisomes. Furthermore, our results are supported by accumulating database information on organelle proteomes that also indicate that SOD2 is a pure mitochondrial protein.  

3.2029           Membrane cholesterol modulates the hyaluronan-binding ability of CD44 in T lymphocytes and controls rolling under shear flow

Murai, T., Sato, C., Sato, M., Nishiyama, H., Suga, M., Mio, K. and Kawashima, H.

1. Cell Sci., 126(15), 3284-3294 (2013)

  The adhesion of circulating lymphocytes to the surface of vascular endothelial cells is important for their recruitment from blood to secondary lymphoid organs and to inflammatory sites. CD44 is a key adhesion molecule for this interaction and its ligand-binding ability is tightly regulated. Here we show that the hyaluronan-binding ability of CD44 in T cells is upregulated by the depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD), which disintegrates lipid rafts, i.e. cholesterol- and sphingolipid-enriched membrane microdomains. Increasing concentrations of MβCD led to a dose-dependent decrease in cellular cholesterol content and to upregulation of hyaluronan binding. Additionally, a cholesterol-binding agent filipin also increased hyaluronan binding. Cholesterol depletion caused CD44 to be dispersed from cholesterol-enriched membrane microdomains. Cholesterol depletion also increased the number of cells undergoing rolling adhesion under physiological flow conditions. Our results suggest that the ligand-binding ability of CD44 is governed by its cholesterol-dependent allocation to membrane microdomains at the cell surface. These findings provide novel insight into the regulation of T cell adhesion under blood flow.  

3.2030           Expression profile and mitochondrial colocalization of Tdp1 in peripheral human tissues

Fam, H.K., Chowdhury, M.K., Walton, C., Choi, K., Boerkoel, C.F. and Hendson, G.

1. Mol. Hist., 44(4), 481-494 (2013)

  Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that processes blocked 3′ ends of DNA breaks. Functional loss of Tdp1 causes spinocerebellar ataxia with axonal neuropathy type 1 (SCAN1). Based on the prominent cytoplasmic expression of Tdp1 in the neurons presumably affected in SCAN1, we hypothesized that Tdp1 participates in the repair of mitochondrial DNA. As a step toward testing this hypothesis, we profiled Tdp1 expression in different human tissues by immunohistochemistry and immunofluorescence respectively and determined whether Tdp1 was expressed in the cytoplasm of tissues other than the neurons. We found that Tdp1 was ubiquitously expressed and present in the cytoplasm of many cell types. Within human skeletal muscle and multiple mouse tissues, Tdp1 partially colocalized with the mitochondria. In cultured human dermal fibroblasts, Tdp1 redistributed to the cytoplasm and partially colocalized with mitochondria following oxidative stress. These studies suggest that one role of cytoplasmic Tdp1 is the repair of mitochondrial DNA lesions arising from oxidative stress.  

3.2031           Visual arrestin interaction with clathrin adaptor AP-2 regulates photoreceptor survival in the vertebrate retina

Moaven, H., Koike, Y., Jao, C., Gurevich, V.V., Langen, R. and Chen, J. PNAS, 110(23), 9463-9468 (2013)   Arrestins bind ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) and terminate the activation of G proteins. Additionally, nonvisual arrestin/GPCR complex can initiate G protein-independent intracellular signals through their ability to act as scaffolds that bring other signaling molecules to the internalized GPCR. Like nonvisual arrestins, vertebrate visual arrestin (ARR1) terminates G protein signaling from light-activated, phosphorylated GPCR, rhodopsin. Unlike nonvisual arrestins, its role as a transducer of signaling from internalized rhodopsin has not been reported in the vertebrate retina. Formation of signaling complexes with arrestins often requires recruitment of the endocytic adaptor protein, AP-2. We have previously shown that Lys296 → Glu (K296E), which is a naturally occurring rhodopsin mutation in certain humans diagnosed with autosomal dominant retinitis pigmentosa, causes toxicity through forming a stable complex with ARR1. Here we investigated whether recruitment of AP-2 by the K296E/ARR1 complex plays a role in generating the cell death signal in a transgenic mouse model of retinal degeneration. We measured the binding affinity of ARR1 for AP-2 and found that, although the affinity is much lower than that of the other arrestins, the unusually high concentration of ARR1 in rods would favor this interaction. We further demonstrate that p44, a splice variant of ARR1 that binds light-activated, phosphorylated rhodopsin but lacks the AP-2 binding motif, prevents retinal degeneration and rescues visual function in K296E mice. These results reveal a unique role of ARR1 in a G protein-independent signaling cascade in the vertebrate retina.  

3.2032           Differential effects of cocaine on histone posttranslational modifications in identified populations of striatal neurons

Jordi, E., Heiman, M., Marion-Poll, L., Guermonprez, P., Cheng, S.K., Nairn, A.C., Greengard, P. and Girault, J-A. PNAS, 110(23), 9511-9516 (2013)   Drugs of abuse, such as cocaine, induce changes in gene expression and epigenetic marks including alterations in histone posttranslational modifications in striatal neurons. These changes are thought to participate in physiological memory mechanisms and to be critical for long-term behavioral alterations. However, the striatum is composed of multiple cell types, including two distinct populations of medium-sized spiny neurons, and little is known concerning the cell-type specificity of epigenetic modifications. To address this question we used bacterial artificial chromosome transgenic mice, which express EGFP fused to the N-terminus of the large subunit ribosomal protein L10a driven by the D1 or D2 dopamine receptor (D1R, D2R) promoter, respectively. Fluorescence in nucleoli was used to sort nuclei from D1R- or D2R-expressing neurons and to quantify by flow cytometry the cocaine-induced changes in histone acetylation and methylation specifically in these two types of nuclei. The two populations of medium-sized spiny neurons displayed different patterns of histone modifications 15 min or 24 h after a single injection of cocaine or 24 h after seven daily injections. In particular, acetylation of histone 3 on Lys 14 and of histone 4 on Lys 5 and 12, and methylation of histone 3 on Lys 9 exhibited distinct and persistent changes in the two cell types. Our data provide insights into the differential epigenetic responses to cocaine in D1R- and D2R-positive neurons and their potential regulation, which may participate in the persistent effects of cocaine in these neurons. The method described should have general utility for studying nuclear modifications in different types of neuronal or nonneuronal cell types.

3.2033           Identification and characterization of proteins isolated from microvesicles derived from human lung cancer pleural effusions

Park, J.O., Choi, D-Y., Choi, D-S., Kim, H.J., Kang, J.W., Jung, J.H., Lee, J.H., Kim, J., Freeman, M.R., Lee, K.Y., Gho, Y.S. and Kim, K.P. Proteomics, 13(14), 2125-2134 (2013)   Microvesicles (MVs, also known as exosomes, ectosomes, microparticles) are released by various cancer cells, including lung, colorectal, and prostate carcinoma cells. MVs released from tumor cells and other sources accumulate in the circulation and in pleural effusion. Although recent studies have shown that MVs play multiple roles in tumor progression, the potential pathological roles of MV in pleural effusion, and their protein composition, are still unknown. In this study, we report the first global proteomic analysis of highly purified MVs derived from human nonsmall cell lung cancer (NSCLC) pleural effusion. Using nano-LC–MS/MS following 1D SDS-PAGE separation, we identified a total of 912 MV proteins with high confidence. Three independent experiments on three patients showed that MV proteins from PE were distinct from MV obtained from other malignancies. Bioinformatics analyses of the MS data identified pathologically relevant proteins and potential diagnostic makers for NSCLC, including lung-enriched surface antigens and proteins related to epidermal growth factor receptor signaling. These findings provide new insight into the diverse functions of MVs in cancer progression and will aid in the development of novel diagnostic tools for NSCLC.  

3.2034           The Liver Connexin32 Interactome Is a Novel Plasma Membrane-Mitochondrial Signaling Nexus

Fowler, S.L., Akins, M., Zhou, H., Figeys, D. and Bennett, A.L.

1. Proteome Res., 12(6), 2597-2610 (2013)

  Connexins are the structural subunits of gap junctions and act as protein platforms for signaling complexes. Little is known about tissue-specific connexin signaling nexuses, given significant challenges associated with affinity-purifying endogenous channel complexes to the level required for interaction analyses. Here, we used multiple subcellular fractionation techniques to isolate connexin32-enriched membrane microdomains from murine liver. We show, for the first time, that connexin32 localizes to both the plasma membrane and inner mitochondrial membrane of hepatocytes. Using a combination of immunoprecipitation-high throughput mass spectrometry, reciprocal co-IP, and subcellular fractionation methodologies, we report a novel interactome validated using null mutant controls. Eighteen connexin32 interacting proteins were identified. The majority represent resident mitochondrial proteins, a minority represent plasma membrane, endoplasmic reticulum, or cytoplasmic partners. In particular, connexin32 interacts with connexin26 and the mitochondrial protein, sideroflexin-1, at the plasma membrane. Connexin32 interaction enhances connexin26 stability. Converging bioinformatic, biochemical, and confocal analyses support a role for connexin32 in transiently tethering mitochondria to connexin32-enriched plasma membrane microdomains through interaction with proteins in the outer mitochondrial membrane, including sideroflexin-1. Complex formation increases the pool of sideroflexin-1 that is present at the plasma membrane. Together, these data identify a novel plasma membrane/mitochondrial signaling nexus in the connexin32 interactome.  

3.2035           Proteomic Identification of Unique Photoreceptor Disc Components Reveals the Presence of PRCD, a Protein Linked to Retinal Degeneration

Skiba, N.P:, Spencer, W.J., Salinas, R.Y., Lieu, E.C., Thompson, J.W. and Arshavsky, V.Y.

1. Proteome Res., 12(6), 3010-3018 (2013)

  Visual signal transduction takes place on the surface of flat membrane vesicles called photoreceptor discs, which reside inside the light-sensitive outer segment organelle of vertebrate photoreceptor cells. Although biochemical studies have indicated that discs are built with a handful of highly specialized proteins, proteomic studies have yielded databases consisting of hundreds of entries. We addressed this controversy by employing protein correlation profiling, which allows identification of unique components of organelles that can be fractionated but not purified to absolute homogeneity. We subjected discs to sequential steps of fractionation and identified the relative amounts of proteins in each fraction by label-free quantitative mass spectrometry. This analysis demonstrated that the photoreceptor disc proteome contains only eleven components, which satisfy the hallmark criterion for being unique disc-resident components: the retention of a constant molar ratio among themselves across fractionation steps. Remarkably, one of them is PRCD, a protein whose mutations have been shown to cause blindness, yet cellular localization remained completely unknown. Identification of PRCD as a novel disc-specific protein facilitates understanding its functional role and the pathobiological significance of its mutations. Our study provides a striking example how protein correlation profiling allows a distinction between constitutive components of cellular organelles and their inevitable contaminants.  

3.2036           Investigation of Polyethylenimine/DNA Polyplex Transfection to Cultured Cells Using Radiolabeling and Subcellular Fractionation Methods

Shi, J., Chou, B., Choi, J.L., Ta, A.L. and Pun, S.H. Mol. Pharmaceutics, 10(6), 2145-2156 (2013)   Quantitative analysis of the intracellular trafficking of nonviral vectors provides critical information that can guide the rational design of improved cationic systems for gene delivery. Subcellular fractionation methods, combined with radiolabeling, produce quantitative measurements of the intracellular trafficking of nonviral vectors and the therapeutic payload. In this work, differential and density-gradient centrifugation techniques were used to determine the intracellular distribution of radiolabeled 25 kDa branched polyethylenimine (bPEI)/plasmid DNA complexes (“polyplexes”) in HeLa cells over time. By differential centrifugation, [¹⁴C]bPEI was found mostly in the lighter fractions whereas [³H]DNA was found mostly in the heavier fractions. A majority of the intracellular polymer ( 60%) and DNA ( 90%) were found in the nuclear fraction. Polymer and DNA also differed in their distribution to heavier and denser organelles (lysosomes, mitochondria) in density-gradient centrifugation studies. An unexpected finding from this study was that between 18 and 50% of the DNA applied to the cells became cell-associated (either with the cell membrane and/or internalized), while only 1–6% of the polymer did so, resulting in an effective N/P ratio of less than 1. These results suggest that a significant amount of cationic polymer is dissociated from the DNA cargo early on in the transfection process.  

3.2037           Meigo governs dendrite targeting specificity by modulating Ephrin level and N-glycosylation

Sekine, S.U., haraguchi, S., Chao, K., kato, T., Luo, L., Miura, M. and Chihara, T. Nature Neurosci., 16(6), 683-691 (2013)   Neural circuit assembly requires precise dendrite and axon targeting. We identified an evolutionarily conserved endoplasmic reticulum (ER) protein, Meigo, from a mosaic genetic screen in Drosophila melanogaster. Meigo was cell-autonomously required in olfactory receptor neurons and projection neurons to target their axons and dendrites to the lateral antennal lobe and to refine projection neuron dendrites into individual glomeruli. Loss of Meigo induced an unfolded protein response and reduced the amount of neuronal cell surface proteins, including Ephrin. Ephrin overexpression specifically suppressed the projection neuron dendrite refinement defect present in meigo mutant flies, and ephrin knockdown caused a similar projection neuron dendrite refinement defect. Meigo positively regulated the level of Ephrin N-glycosylation, which was required for its optimal function in vivo. Thus, Meigo, an ER-resident protein, governs neuronal targeting specificity by regulating ER folding capacity and protein N-glycosylation. Furthermore, Ephrin appears to be an important substrate that mediates Meigo's function in refinement of glomerular targeting.  

3.2038           Mechanisms Responsible for the Compositional Heterogeneity of Nascent High Density Lipoprotein

Lund-Katz, S., Lyssenko, N.N., Nickel, M., Nguyen, D., Chetty, P.S., Weibel, G. and Philips, M.C.

1. Biol. Chem., 288(32), 23150-23160 (2013)

  Apolipoprotein (apo) A-I-containing nascent HDL particles produced by the ATP binding cassette transporter A1 have different sizes and compositions. To understand the molecular basis for this heterogeneity, the HDL particles produced by apoA-I-mediated solubilization of phospholipid (PL)/free (unesterified) cholesterol (FC) bilayer membranes in cell and cell-free systems are compared. Incubation of apoA-I with ATP binding cassette transporter A1-expressing baby hamster kidney cells leads to formation of two populations of FC-containing discoidal nascent HDL particles. The larger 11-nm diameter particles are highly FC-enriched (FC/PL = 1.2/1 mol/mol) relative to the smaller 8 nm particles and the cell plasma membrane (FC/PL = 0.4/1). ApoA-I-mediated spontaneous solubilization of either multilamellar or unilamellar vesicles made of a membrane-PL mixture and FC yields discoidal HDL particles with diameters in the range 9–17 nm and, as found with the cell system, the larger particles are relatively enriched in FC despite the fact that all particles are created by solubilization of a common FC/PL membrane domain. The size-dependent distribution of FC among HDL particles is due to varying amounts of PL being sequestered in a boundary layer by interaction with apoA-I at the disc edge. The presence of a relatively large boundary layer in smaller discoidal HDL promotes preferential distribution of phosphatidylserine to such particles. However, phosphatidylcholine and sphingomyelin which are the primary PL constituents of nascent HDL do not exhibit selective incorporation into HDL discs of different sizes. This understanding of the mechanisms responsible for the heterogeneity in lipid composition of nascent HDL particles may provide a basis for selecting subspecies with preferred cardio-protective properties.  

3.2039           The Us2 Gene Product of Herpes Simplex Virus 2 Is a Membrane-Associated Ubiquitin-Interacting Protein

Kang, M-H., Roy, B.B., Finnen, R.L., Le Sage, V., Johnston, S.M., Zhang, H and Banfield, B.W.

1. Virol., 87(17), 9590-9603 (2013)

  The Us2 gene encodes a tegument protein that is conserved in most members of the Alphaherpesvirinae. Previous studies on the pseudorabies virus (PRV) Us2 ortholog indicated that it is prenylated, associates with membranes, and spatially regulates the enzymatic activity of the MAP (mitogen-activated protein) kinase ERK (extracellular signal-related kinase) through direct binding and sequestration of ERK at the cytoplasmic face of the plasma membrane. Here we present an analysis of the herpes simplex virus 2 (HSV-2) Us2 ortholog and demonstrate that, like PRV Us2, HSV-2 Us2 is a virion component and that, unlike PRV Us2, it does not interact with ERK in yeast two-hybrid assays. HSV-2 Us2 lacks prenylation signals and other canonical membrane-targeting motifs yet is tightly associated with detergent-insoluble membranes and localizes predominantly to recycling endosomes. Experiments to identify cellular proteins that facilitate HSV-2 Us2 membrane association were inconclusive; however, these studies led to the identification of HSV-2 Us2 as a ubiquitin-interacting protein, providing new insight into the functions of HSV-2 Us2.  

3.2040           The Cytosolic Adaptor AP-1A Is Essential for the Trafficking and Function of Niemann-Pick Type C Proteins

Poirier, S., Mayer, G., Murphy, S.R., Garver, W.S., chang, T.Y., Schu, P. and Seidah, N.G. Traffic, 14(4), 458-469 (2013)   Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by over-accumulation of low-density lipoprotein-derived cholesterol and glycosphingolipids in late endosomes/lysosomes (LE/L) throughout the body. Human mutations in either NPC1 or NPC2 genes have been directly associated with impaired cholesterol efflux from LE/L. Independent from its role in cholesterol homeostasis and its NPC2 partner, NPC1 was unexpectedly identified as a critical player controlling intracellular entry of filoviruses such as Ebola. In this study, a yeast three-hybrid system revealed that the NPC1 cytoplasmic tail directly interacts with the clathrin adaptor protein AP-1 via its acidic/di-leucine motif. Consequently, a nonfunctional AP-1A cytosolic complex resulted in a typical NPC-like phenotype mainly due to a direct impairment of NPC1 trafficking to LE/L and a partial secretion of NPC2. Furthermore, the mislocalization of NPC1 was not due to cholesterol accumulation in LE/L, as it was not rescued upon treatment with Mβ-cyclodextrin, which almost completely eliminated intracellular free cholesterol. Our cumulative data demonstrate that the cytosolic clathrin adaptor AP-1A is essential for the lysosomal targeting and function of NPC1 and NPC2.  

3.2041           Differential Endosomal Sorting of a Novel P2Y12 Purinoreceptor Mutant

Cunningham, M.R., Nisar, S.P., Cooke, A.E., Emery, E.D. and Mundell, S.J. Traffic, 14(5), 585-598 (2013)   P2Y₁₂ receptor internalization and recycling play an essential role in ADP-induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y₁₂ (P341A) whose P2Y₁₂ receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild-type (WT) P2Y₁₂ recycling and investigated P2Y₁₂-P341A receptor traffic. Treatment with ADP resulted in delayed Rab5-dependent internalization of P341A when compared with WT P2Y₁₂. While WT P2Y₁₂ rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7-positive endosomes with considerable agonist-dependent accumulation in the trans-Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT-P2Y₁₂. Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y₁₂ receptor traffic and explain the compromised receptor function in the platelets of the P2Y₁₂-P341A-expressing patient.  

3.2042           Association of the Vaccinia Virus A11 Protein with the Endoplasmic Reticulum and Crescent Precursors of Immature Virions

Maruri-Avidal, L., Weisberg, A.S. and Moss, B.

1. Virol., 87(18), 10195-10206 (2013)

  The apparent de novo formation of viral membranes within cytoplasmic factories is a mysterious, poorly understood first step in poxvirus morphogenesis. Genetic studies identified several viral proteins essential for membrane formation and the assembly of immature virus particles. Their repression results in abortive replication with the accumulation of dense masses of viroplasm. In the present study, we further characterized one of these proteins, A11, and investigated its association with cellular and viral membranes under normal and abortive replication conditions. We discovered that A11 colocalized in cytoplasmic factories with the endoplasmic reticulum (ER) and L2, another viral protein required for morphogenesis. Confocal microscopy and subcellular fractionation indicated that A11 was not membrane associated in uninfected cells, whereas L2 still colocalized with the ER. Cell-free transcription and translation experiments indicated that both A11 and L2 are tail-anchored proteins that associate posttranslationally with membranes and likely require specific cytoplasmic targeting chaperones. Transmission electron microscopy indicated that A11, like L2, associated with crescent membranes and immature virions during normal infection and with vesicles and tubules near masses of dense viroplasm during abortive infection in the absence of the A17 or A14 protein component of viral membranes. When the synthesis of A11 was repressed, “empty” immature-virion-like structures formed in addition to masses of viroplasm. The immature-virion-like structures were labeled with antibodies to A17 and to the D13 scaffold protein and were closely associated with calnexin-labeled ER. These studies revealed similarities and differences between A11 and L2, both of which may be involved in the recruitment of the ER for virus assembly.  

3.2043           Suppression of Aβ toxicity by puromycin-sensitive aminopeptidase is independent of its proteolytic activity

Kruppa, A.J., Ott, S., Chandraratna, D.S., Irving, J.A., Page, R.M., Speretta, E., Seto, T., Camargo, L:M., Marciniak, S.J., Lomas, D.A. and Crowther, D.C. Biochim. Biophys. Acta, 1832, 2115-2126 (2013)   The accumulation of β-amyloid (Aβ) peptide in the brain is one of the pathological hallmarks of Alzheimer's disease and is thought to be of primary aetiological significance. In an unbiased genetic screen, we identified puromycin-sensitive aminopeptidase (PSA) as a potent suppressor of Aβ toxicity in a Drosophila model system. We established that coexpression of Drosophila PSA (dPSA) in the flies' brains improved their lifespan, protected against locomotor deficits, and reduced brain Aβ levels by clearing the Aβ plaque-like deposits. However, confocal microscopy and subcellular fractionation of amyloid-expressing 7PA2 cells demonstrated that PSA localizes to the cytoplasm. Therefore, PSA and Aβ are unlikely to be in the same cellular compartment; moreover, when we artificially placed them in the same compartment in flies, we could not detect a direct epistatic interaction. The consequent hypothesis that PSA's suppression of Aβ toxicity is indirect was supported by the finding that Aβ is not a proteolytic substrate for PSA in vitro. Furthermore, we showed that the enzymatic activity of PSA is not required for rescuing Aβ toxicity in neuronal SH-SY5Y cells. We investigated whether the stimulation of autophagy by PSA was responsible for these protective effects. However PSA's promotion of autophagosome fusion with lysosomes required proteolytic activity and so its effect on autophagy is not identical to its protection against Aβ toxicity.  

3.2044           Plasma membrane Pdia3 and VDR interact to elicit rapid responses to 1α,25(OH)2D3

Chen, J., Doroudi, M., Cheung, J., Grozier, A.L., Schweartz, Z. and Boyan, B.D. Cellular Signalling, 25, 2362-2373 (2013)   1α,25-Dihydroxyvitamin D3 (1α,25(OH)₂D₃) regulates osteoblasts through genomic and rapid membrane-mediated responses. Here we examined the interaction of protein disulfide isomerase family A, member 3 (Pdia3) and the traditional vitamin D receptor (VDR) in plasma membrane-associated responses to 1α,25(OH)₂D₃. We found that Pdia3 co-localized with VDR and the caveolae scaffolding protein, caveolin-1 on the surface of MC3T3-E1 osteoblasts. Immunoprecipitation showed that both Pdia3 and VDR interacted with caveolin-1. Pdia3 further interacted with phospholipase A2 activating protein (PLAA), whereas VDR interacted with c-Src. 1α,25(OH)₂D₃ changed the interactions and transport of the two receptors and rapidly activated phospholipase A2 (PLA2) and c-Src. Silencing either receptor or caveolin-1 inhibited both PLA2 and c-Src, indicating that the two receptors function interdependently. These two receptor dependent rapid responses to 1α,25(OH)₂D₃ regulated gene expression, proliferation and apoptosis of MC3T3-E1 cells. These data demonstrate the importance of both receptors and caveolin-1 in mediating membrane responses to 1α,25(OH)₂D₃ and subsequently regulating osteoblast biology.  

3.2045           A simple methodology to assess endolysosomal protease activity involved in antigen processing in human primary cells

Vaithikingam, A., Lai, N.Y., Duong, E., Boucau, J., Xu, Y., Shimada, M., Gabdhi, M. and Le Gall, S. BMC Cell  Biol., 14:35 (2013)   Background Endolysosomes play a key role in maintaining the homeostasis of the cell. They are made of a complex set of proteins that degrade lipids, proteins and sugars. Studies involving endolysosome contribution to cellular functions such as MHC class I and II epitope production have used recombinant endolysosomal proteins, knockout mice that lack one of the enzymes or purified organelles from human tissue. Each of these approaches has some caveats in analyzing endolysosomal enzyme functions. Results In this study, we have developed a simple methodology to assess endolysosomal protease activity. By varying the pH in crude lysate from human peripheral blood mononuclear cells (PBMCs), we documented increased endolysosomal cathepsin activity in acidic conditions. Using this new method, we showed that the degradation of HIV peptides in low pH extracts analyzed by mass spectrometry followed similar kinetics and degradation patterns as those performed with purified endolysosomes. Conclusion By using crude lysate in the place of purified organelles this method will be a quick and useful tool to assess endolysosomal protease activities in primary cells of limited availability. This quick method will especially be useful to screen peptide susceptibility to degradation in endolysosomal compartments for antigen processing studies, following which detailed analysis using purified organelles may be used to study specific peptides.  

3.2046           Certain Strongylocentrotus purpuratus sperm mitochondrial proteins co-purify with low density detergent-insoluble membranes and are PKA or PKC-substrates possibly involved in sperm motility regulation

Loza-Huerta, A., Vera-Estrella, R., Darszon, A. and Beltran, C. Biochim. Biophys. Acta, 1830, 5305-5315 (2013)   Background Sea urchin sperm motility is regulated by Speract, a sperm-activating peptide (SAP) secreted from the outer egg coat. Upon binding to its receptor in the sperm flagellum, Speract induces a series of ionic and metabolic changes in Strongylocentrotus purpuratus spermatozoa that regulate their motility. Among these events, protein phosphorylation is one of the most relevant and evidence indicates that some proteins of the Speract signaling cascade localize in low density detergent-insoluble membranes (LD-DIM). Methods LD-DIM-derived proteins from immotile, motile or Speract-stimulated S. purpuratus sperm were resolved in 2-D gels and the PKA and PKC substrates detected with specific antibodies were identified by LC–MS/MS. Results Differential PKA and PKC substrate phosphorylation levels among the LD-DIM isolated from sperm in different motility conditions were found and identified by mass spectrometry as: ATP synthase, creatine kinase, NADH dehydrogenase (ubiquinone) flavoprotein 2, succinyl-CoA ligase and the voltage-dependent anion channel 2 (VDAC2), which are mitochondrial proteins, as well as, the cAMP-dependent protein kinase type II regulatory (PKA RII) subunit, Tubulin β chain and Actin Cy I changed their phosphorylation state. Conclusions Some mitochondrial proteins regulated by PKA or PKC may influence sea urchin sperm motility. General significance The fact that a high percentage (66%) of the PKA or PKC substrates identified in LD-DIM are mitochondrial proteins suggests that the phosphorylation of these proteins modulates sea urchin sperm motility via Speract stimulation by providing sufficient energy to sperm physiology. Those mitochondrial proteins are indeed PKA- or PKC-substrates in the sea urchin spermatozoa.  

3.2047           CD147, CD44, and the Epidermal Growth Factor Receptor (EGFR) Signaling Pathway Cooperate to Regulate Breast Epithelial Cell Invasiveness

Grass, G.D., Tolliver, L.B., Bratoeva, M. and Toole, B.P.

1. Biol. Chem., 288(36), 26089-26104 (2013)

  The immunoglobulin superfamily glycoprotein CD147 (emmprin; basigin) is associated with an invasive phenotype in various types of cancers, including malignant breast cancer. We showed recently that up-regulation of CD147 in non-transformed, non-invasive breast epithelial cells is sufficient to induce an invasive phenotype characterized by membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invadopodia activity (Grass, G. D., Bratoeva, M., and Toole, B. P. (2012) Regulation of invadopodia formation and activity by CD147. J. Cell Sci. 125, 777–788). Here we found that CD147 induces breast epithelial cell invasiveness by promoting epidermal growth factor receptor (EGFR)-Ras-ERK signaling in a manner dependent on hyaluronan-CD44 interaction. Furthermore, CD147 promotes assembly of signaling complexes containing CD147, CD44, and EGFR in lipid raftlike domains. We also found that oncogenic Ras regulates CD147 expression, hyaluronan synthesis, and formation of CD147-CD44-EGFR complexes, thus forming a positive feedback loop that may amplify invasiveness. Last, we showed that malignant breast cancer cells are heterogeneous in their expression of surface-associated CD147 and that high levels of membrane CD147 correlate with cell surface EGFR and CD44 levels, activated EGFR and ERK1, and activated invadopodia. Future studies should evaluate CD147 as a potential therapeutic target and disease stratification marker in breast cancer.  

3.2048           Myosin-1c regulates the dynamic stability of E-cadherin–based cell–cell contacts in polarized Madin–Darby canine kidney cells

Tokuo, H. and Coluccio, L.M. Mol. Biol. Cell, 24, 2820-2832  (2013)   Cooperation between cadherins and the actin cytoskeleton controls the formation and maintenance of cell–cell adhesions in epithelia. We find that the molecular motor protein myosin-1c (Myo1c) regulates the dynamic stability of E-cadherin–based cell–cell contacts. In Myo1c-depleted Madin–Darby canine kidney cells, E-cadherin localization was dis­organized and lateral membranes appeared less vertical with convoluted edges versus control cells. In polarized monolayers, Myo1c-knockdown (KD) cells were more sensitive to reduced calcium concentration. Myo1c separated in the same plasma membrane fractions as E-cadherin, and Myo1c KD caused a significant reduction in the amount of E-cadherin recovered in one peak fraction. Expression of green fluorescent protein (GFP)–Myo1c mutants revealed that the phosphatidylinositol-4,5-bisphosphate–binding site is necessary for its localization to cell–cell adhesions, and fluorescence recovery after photobleaching assays with GFP-Myo1c mutants revealed that motor function was important for Myo1c dynamics at these sites. At 18°C, which inhibits vesicle recycling, Myo1c-KD cells accumulated more E-cadherin–positive vesicles in their cytoplasm, suggesting that Myo1c affects E-cadherin endocytosis. Studies with photoactivatable GFP–E-cadherin showed that Myo1c KD reduced the stability of E-cadherin at cell–cell adhesions. We conclude that Myo1c stabilizes E-cadherin at adherens junctions in polarized epithelial cells and that the motor function and ability of Myo1c to bind membrane are critical.  

3.2049           Regulation of pre-fusion events: recruitment of M-cadherin to microrafts organized at fusion-competent sites of myogenic cells

Mukai, A. and Hashimoto, N. BMC Cell Biol., 14:37 (2013)   Background Previous research indicates that the membrane ruffles and leading edge of lamellipodia of myogenic cells contain presumptive fusion sites. A micrometer-sized lipid raft (microraft) is organized at the presumptive fusion site of mouse myogenic cells in a cell-contact independent way and serves as a platform tethering adhesion proteins that are relevant to cell fusion. However, the mechanisms underlying recruitment of adhesion proteins to lipid rafts and microraft organization remain unknown. Results Here we show that small G-protein Rac1 was required for microraft organization and subsequent cell fusion. However, Rac1 activity was unnecessary for recruitment of M-cadherin to lipid rafts. We found that p120 catenin (p120) binds to M-cadherin exclusively in lipid rafts of differentiating myogenic cells. The Src kinase inhibitor SU6656 prevented p120 binding to M-cadherin and their recruitment to lipid rafts, then suppressed microraft organization, membrane ruffling, and myogenic cell fusion. Suppression of membrane ruffling in SU6656-treated cells was partially restored by pretreatment with the protein tyrosine phosphatase inhibitor vanadate. The present analyses using an antibody to tyrosine phosphorylated p120 suggest that Src family kinases play a role in binding of p120 to M-cadherin and the recruitment of M-cadherin to lipid rafts through phosphorylation of putative substrates other than p120. Conclusions The present study showed that the procedure establishing fusion-competent sites consists of two sequential events: recruitment of adhesion complexes to lipid rafts and organization of microrafts. The recruitment of M-cadherin to lipid rafts depended on interaction with p120 catenin, whereas the organization of microrafts was controlled by a small G protein, Rac1.  

3.2050           Cetuximab enhances TRAIL-induced gastric cancer cell apoptosis by promoting DISC formation in lipid rafts

Xu, L., Hu, X., Qu, X., Hou, K., Zheng, H. and Liu, Y. Biochem. Biophys. Res. Comm., 439, 285-290 (2013)   TRAIL is a member of the tumor necrosis factor family that selectively induces cancer cell apoptosis. However, gastric cancer cells are insensitive to TRAIL. Our and others studies showed that the inhibition of EGFR pathway activation could increase the sensitivity of TRAIL in cancer cells. But the detailed mechanism is not fully understood. In the present study, compared with TRAIL or cetuximab (an anti-EGFR monoclonal antibody) alone, treatment with the TRAIL/cetuximab combination significantly promoted death receptor 4 (DR4) clustering as well as the translocation of both DR4 and Fas-associated death domain-containing protein (FADD) into lipid rafts. This in turn resulted in caspase-8 cleavage and the formation of the death-inducing signaling complex (DISC) in these lipid rafts. Cholesterol-depletion with methyl-β-cyclodextrin partially prevented DR4 clustering and DISC formation, and thus partially reversed apoptosis induced by the TRAIL/cetuximab dual treatment. These results indicate that cetuximab increases TRAIL-induced gastric cancer cell apoptosis at least partially through the promotion of DISC formation in lipid rafts.  

3.2051           Ubiquitination of the glycosomal matrix protein receptor PEX5 in Trypanosoma brucei by PEX4 displays novel features

Gualdron-Lopez, M., Chevalier, N., Van Der Smissen, P., Courtoy, P.J., Rigden, D.J. and Michels, P.A.M. Biochim. Biophys. Acta, 1833, 3076-3092 (2013)   Trypanosomatids contain peroxisome-like organelles called glycosomes. Peroxisomal biogenesis involves a cytosolic receptor, PEX5, which, after its insertion into the organellar membrane, delivers proteins to the matrix. In yeasts and mammalian cells, transient PEX5 monoubiquitination at the membrane serves as the signal for its retrieval from the organelle for re-use. When its recycling is impaired, PEX5 is polyubiquitinated for proteasomal degradation. Stably monoubiquitinated TbPEX5 was detected in cytosolic fractions of Trypanosoma brucei, indicative for its role as physiological intermediate in receptor recycling. This modification's resistance to dithiothreitol suggests ubiquitin conjugation of a lysine residue. T. brucei PEX4, the functional homologue of the ubiquitin-conjugating (UBC) enzyme responsible for PEX5 monoubiquitination in yeast, was identified. It is associated with the cytosolic face of the glycosomal membrane, probably anchored by an identified putative TbPEX22. The involvement of TbPEX4 in TbPEX5 ubiquitination was demonstrated using procyclic ∆PEX4 trypanosomes. Surprisingly, glycosomal matrix protein import was only mildly affected in this mutant. Since other UBC homologues were upregulated, it might be possible that these have partially rescued PEX4's function in PEX5 ubiquitination. In addition, the altered expression of UBCs, notably of candidates involved in cell-cycle control, could be responsible for observed morphological and motility defects of the ∆PEX4 mutant.      

3.2052           Cavin-3 dictates the balance between ERK and Akt signaling

Victor J Hernandez, Jian Weng, Peter Ly, Shanica Pompey, Hongyun Dong, Lopa Mishra, Margaret Schwarz, Richard GW Anderson, and Peter Michaely eLife, 2, e00905 (2013)   Cavin-3 is a tumor suppressor protein of unknown function. Using both in vivo and in vitro approaches, we show that cavin-3 dictates the balance between ERK and Akt signaling. Loss of cavin-3 increases Akt signaling at the expense of ERK, while gain of cavin-3 increases ERK signaling at the expense Akt. Cavin-3 facilitates signal transduction to ERK by anchoring caveolae to the membrane skeleton of the plasma membrane via myosin-1c. Caveolae are lipid raft specializations that contain an ERK activation module and loss of the cavin-3 linkage reduces the abundance of caveolae, thereby separating this ERK activation module from signaling receptors. Loss of cavin-3 promotes Akt signaling through suppression of EGR1 and PTEN. The in vitro consequences of the loss of cavin-3 include induction of Warburg metabolism (aerobic glycolysis), accelerated cell proliferation, and resistance to apoptosis. The in vivo consequences of cavin-3 knockout are increased lactate production and cachexia.  

3.2053           Leucine Carboxyl Methyltransferase 1 (LCMT1)-dependent Methylation Regulates the Association of Protein Phosphatase 2A and Tau Protein with Plasma Membrane Microdomains in Neuroblastoma Cells

Jean-Marie Sontag, Viyada Nunbhakdi-Craig, and Estelle Sontag

1. Biol. Chem., 288(38), 27396-27405 (2013)

  Down-regulation of protein phosphatase 2A (PP2A) methylation occurs in Alzheimer disease (AD). However, the regulation of PP2A methylation remains poorly understood. We have reported that altered leucine carboxyl methyltransferase (LCMT1)-dependent PP2A methylation is associated with down-regulation of PP2A holoenzymes containing the Bα subunit (PP2A/Bα) and subsequent accumulation of phosphorylated Tau in N2a cells, in vivo and in AD. Here, we show that pools of LCMT1, methylated PP2A, and PP2A/Bα are co-enriched in cholesterol-rich plasma membrane microdomains/rafts purified from N2a cells. In contrast, demethylated PP2A is preferentially distributed in non-rafts wherein small amounts of the PP2A methylesterase PME-1 are exclusively present. A methylation-incompetent PP2A mutant is excluded from rafts. Enhanced methylation of PP2A promotes the association of PP2A and Tau with the plasma membrane. Altered PP2A methylation following expression of a catalytically inactive LCMT1 mutant, knockdown of LCMT1, or alterations in one-carbon metabolism all result in a loss of plasma membrane-associated PP2A and Tau in N2a cells. This correlates with accumulation of soluble phosphorylated Tau, a hallmark of AD and other tauopathies. Thus, our findings reveal a distinct compartmentalization of PP2A and PP2A regulatory enzymes in plasma membrane microdomains and identify a novel methylation-dependent mechanism involved in modulating the targeting of PP2A, and its substrate Tau, to the plasma membrane. We propose that alterations in the membrane localization of PP2A and Tau following down-regulation of LCMT1 may lead to PP2A and Tau dysfunction in AD.  

3.2054           Proteomics, transcriptomics and lipidomics of exosomes and ectosomes

Choi, D-S., Kim, D-K., Kim, Y-K. and Gho, Y.S. Proteomics, 13(10-11), 1554-1571 (2013)   Mammalian cells secrete two types of extracellular vesicles either constitutively or in a regulated manner: exosomes (50–100 nm in diameter) released from the intracellular compartment and ectosomes (also called microvesicles, 100–1000 nm in diameter) shed directly from the plasma membrane. Extracellular vesicles are bilayered proteolipids enriched with proteins, mRNAs, microRNAs, and lipids. In recent years, much data have been collected regarding the specific components of extracellular vesicles from various cell types and body fluids using proteomic, transcriptomic, and lipidomic methods. These studies have revealed that extracellular vesicles harbor specific types of proteins, mRNAs, miRNAs, and lipids rather than random cellular components. These results provide valuable information on the molecular mechanisms involved in vesicular cargo-sorting and biogenesis. Furthermore, studies of these complex extracellular organelles have facilitated conceptual advancements in the field of intercellular communication under physiological and pathological conditions as well as for disease-specific biomarker discovery. This review focuses on the proteomic, transcriptomic, and lipidomic profiles of extracellular vesicles, and will briefly summarize recent advances in the biology, function, and diagnostic potential of vesicle-specific components.    

3.2055           Tumor-derived exosomes and microvesicles in head and neck cancer: Implications for tumor biology and biomarker discovery

Principe, S., Hui, A.B-Y., Bruce, J., Sinha, A., Liu, F-F. and Kislinger, T. Proteomics, 13(10-11), 1608-1623 (2013)   Exosomes and microvesicles (MVs) are nanometer-sized, membranous vesicles secreted from many cell types into their surrounding extracellular space and into body fluids. These two classes of extracellular vesicles are regarded as a novel mechanism through which cancer cells, including virally infected cancer cells, regulate their micro-environment via the horizontal transfer of bioactive molecules: proteins, lipids, and nucleic acids (DNA, mRNA, micro-RNAs; oncogenic cargo hence often referred to as oncosomes). In head and neck cancer (HNC), exosomes and MVs have been described in Epstein Barr Virus (EBV)-associated nasopharyngeal cancer (NPC), as well as being positively correlated with oral squamous cell carcinoma (OSCC) progression. It has therefore been suggested that HNC-derived vesicles could represent a useful source for biomarker discovery, enriched in tumor antigens and cargo; hence fundamentally important for cancer progression. This current review offers an overall perspective on the roles of exosomes and MVs in HNC biology, focusing on EBV-associated NPC and OSCC. We also highlight the importance of saliva as a proximal and easily accessible bio-fluid for HNC detection, and propose that salivary vesicles might serve as an alternative model in the discovery of novel HNC biomarkers.  

3.2056           Proteome profiling of exosomes derived from human primary and metastatic colorectal cancer cells reveal differential expression of key metastatic factors and signal transduction components

Ji, H., Greening, D.W., barnes, T.W., Lim, J.W., Tauro, B.J., Rai, A., Xu, R., Adda, C., Mathivanan, S., Zhao, W., Xue, Y., Xu, T., Zhu, H-J. and Simpson, R.J. Proteomics, 13(10-11), 1672-1686 (2013)   Exosomes are small extracellular 40–100 nm diameter membrane vesicles of late endosomal origin that can mediate intercellular transfer of RNAs and proteins to assist premetastatic niche formation. Using primary (SW480) and metastatic (SW620) human isogenic colorectal cancer cell lines we compared exosome protein profiles to yield valuable insights into metastatic factors and signaling molecules fundamental to tumor progression. Exosomes purified using OptiPrep™ density gradient fractionation were 40–100 nm in diameter, were of a buoyant density ∼1.09 g/mL, and displayed stereotypic exosomal markers TSG101, Alix, and CD63. A major finding was the selective enrichment of metastatic factors (MET, S100A8, S100A9, TNC), signal transduction molecules (EFNB2, JAG1, SRC, TNIK), and lipid raft and lipid raft-associated components (CAV1, FLOT1, FLOT2, PROM1) in exosomes derived from metastatic SW620 cells. Additionally, using cryo-electron microscopy, ultrastructural components in exosomes were identified. A key finding of this study was the detection and colocalization of protein complexes EPCAM-CLDN7 and TNIK-RAP2A in colorectal cancer cell exosomes. The selective enrichment of metastatic factors and signaling pathway components in metastatic colon cancer cell-derived exosomes contributes to our understanding of the cross-talk between tumor and stromal cells in the tumor microenvironment.  

3.2057           Interplay between Clathrin and Rab5 Controls the Early Phagocytic Trafficking and Intracellular Survival of Brucella abortus within HeLa cells

Lee, J.J., Kim, D.G., Kim, D.H., Simborio, H.L., Min, W., Lee, H.J., Her, M., Jung, S.C., Watarai, M. and Kim, S.

1. Biol. Chem., 288(39), 28049-28057 (2013)

  Lipid raft-associated clathrin is essential for host-pathogen interactions during infection. Brucella abortus is an intracellular pathogen that circumvents host defenses, but little is known about the precise infection mechanisms that involve interaction with lipid raft-associated mediators. The aim of this study was to elucidate the clathrin-mediated phagocytic mechanisms of B. abortus. The clathrin dependence of B. abortus infection in HeLa cells was investigated using an infection assay and immunofluorescence microscopy. The redistribution of clathrin in the membrane and in phagosomes was investigated using sucrose gradient fractionation of lipid rafts and the isolation of B. abortus-containing vacuoles, respectively. Clathrin and dynamin were concentrated into lipid rafts during B. abortus infection, and the entry and intracellular survival of B. abortus within HeLa cells were abrogated by clathrin inhibition. Clathrin disruption decreased actin polymerization and the colocalization of B. abortus-containing vacuoles with clathrin and Rab5 but not lysosome-associated membrane protein 1 (LAMP-1). Thus, our data demonstrate that clathrin plays a fundamental role in the entry and intracellular survival of B. abortus via interaction with lipid rafts and actin rearrangement. This process facilitates the early intracellular trafficking of B. abortus to safe replicative vacuoles.  

3.2058           Palmitoylation is the switch that assigns calnexin to quality control or ER Ca2+ signaling

Lynes, E.M., Raturi, A., Shenkman, M., Sandoval, C.O., Yap, M.C., Wu, J., Janowicz, A., Myhill, N., Benson, M.D., Campbell, R.E., Berthiaume, L.G., Lederkremer, G.Z. and Simmen, T.

1. Cell Science, 126(17), 3893-3903 (2013)

  The palmitoylation of calnexin serves to enrich calnexin on the mitochondria-associated membrane (MAM). Given a lack of information on the significance of this finding, we have investigated how this endoplasmic reticulum (ER)-internal sorting signal affects the functions of calnexin. Our results demonstrate that palmitoylated calnexin interacts with sarcoendoplasmic reticulum (SR) Ca²⁺ transport ATPase (SERCA) 2b and that this interaction determines ER Ca²⁺ content and the regulation of ER–mitochondria Ca²⁺ crosstalk. In contrast, non-palmitoylated calnexin interacts with the oxidoreductase ERp57 and performs its well-known function in quality control. Interestingly, our results also show that calnexin palmitoylation is an ER-stress-dependent mechanism. Following a short-term ER stress, calnexin quickly becomes less palmitoylated, which shifts its function from the regulation of Ca²⁺ signaling towards chaperoning and quality control of known substrates. These changes also correlate with a preferential distribution of calnexin to the MAM under resting conditions, or the rough ER and ER quality control compartment (ERQC) following ER stress. Our results have therefore identified the switch that assigns calnexin either to Ca²⁺ signaling or to protein chaperoning.  

3.2059           Green tea phenolics inhibit butyrate-induced differentiation of colon cancer cells by interacting with monocarboxylate transporter 1

Sanchez-Tena, S., Vizan, P., Dudeja, P.K., Centelles, J.J. and Casante, M. Biochim. Biophys. Acta, 1832, 2264-2270 (2013)   Diet has a significant impact on colorectal cancer and both dietary fiber and plant-derived compounds have been independently shown to be inversely related to colon cancer risk. Butyrate (NaB), one of the principal products of dietary fiber fermentation, induces differentiation of colon cancer cell lines by inhibiting histone deacetylases (HDACs). On the other hand, (−)-epicatechin (EC) and (−)-epigallocatechin gallate (EGCG), two abundant phenolic compounds of green tea, have been shown to exhibit antitumoral properties. In this study we used colon cancer cell lines to study the cellular and molecular events that take place during co-treatment with NaB, EC and EGCG. We found that (i) polyphenols EC and EGCG fail to induce differentiation of colon adenocarcinoma cell lines; (ii) polyphenols EC and EGCG reduce NaB-induced differentiation; (iii) the effect of the polyphenols is specific for NaB, since differentiation induced by other agents, such as trichostatin A (TSA), was unaltered upon EC and EGCG treatment, and (iv) is independent of the HDAC inhibitory activity of NaB. Also, (v) polyphenols partially reduce cellular NaB; and (vi) on a molecular level, reduction of cellular NaB uptake by polyphenols is achieved by impairing the capacity of NaB to relocalize its own transporter (monocarboxylate transporter 1, MCT1) in the plasma membrane. Our findings suggest that beneficial effects of NaB on colorectal cancer may be reduced by green tea phenolic supplementation. This valuable information should be of assistance in choosing a rational design for more effective diet-driven therapeutic interventions in the prevention or treatment of colorectal cancer.  

3.2060           An abundant LEA protein in the anhydrobiotic midge, PvLEA4, acts as a molecular shield by limiting growth of aggregating protein particles

Hatanaka, R., Hagiwara-Komoda, Y., Furuki, T., Kanamori, Y., Fujitaa, M., Cornette, R., Sakurai, M, Okuda, T. and Kikawada, T. Insect Biochem. Mol. Biol., 43(11), 1055-1067 (2013)   LEA proteins are found in anhydrobiotes and are thought to be associated with the acquisition of desiccation tolerance. The sleeping chironomid Polypedilum vanderplanki, which can survive in an almost completely desiccated state throughout the larval stage, accumulates LEA proteins in response to desiccation and high salinity conditions. However, the biochemical functions of these proteins remain unclear. Here, we report the characterization of a novel chironomid LEA protein, PvLEA4, which is the most highly accumulated LEA protein in desiccated larvae. Cytoplasmic-soluble PvLEA4 showed many typical characteristics of group 3 LEA proteins (G3LEAs), such as desiccation-inducible accumulation, high hydrophilicity, folding into α-helices on drying, and the ability to reduce aggregation of dehydration-sensitive proteins. This last property of LEA proteins has been termed molecular shield function. To further investigate the molecular shield activity of PvLEA4, we introduced two distinct methods, turbidity measurement and dynamic light scattering (DLS). Turbidity measurements demonstrated that both PvLEA4, and BSA as a positive control, reduced aggregation in α-casein subjected to desiccation and rehydration. However, DLS experiments showed that a small amount of BSA relative to α-casein increased aggregate particle size, whereas PvLEA4 decreased particle size in a dose-dependent manner. Trehalose, which is the main heamolymph sugar in most insects but also a protectant as a chemical chaperone in the sleeping chironomid, has less effect on the limitation of aggregate formation. This analysis suggests that molecular shield proteins function by limiting the growth of protein aggregates during drying and that PvLEA4 counteracts protein aggregation in the desiccation-tolerant larvae of the sleeping chironomid.  

3.2061           The Endoplasmic Reticulum Acts as a Platform for Ubiquitylated Components of Nuclear Factor {kappa}B Signaling

Alexia, C., Poales, K., Carvalho, G., Zemirli, N., Dwyer, J., Dubois, S.M., hatchi, E.M., Cordeiro, N., Smith, S.S., Castanier, C., Le Guelte, A., Wan, L., kang, Y., Vazquez, A., Gavard, J., Arnoult, D. and Bidere, N. Science Signaling, 6(291), ra79 (2013)   The innate and adaptive immune responses involve the stimulation of nuclear factor B (NF- B) transcription factors through the Lys⁶³ (K⁶³)–linked ubiquitylation of specific components of NF- B signaling pathways. We found that ubiquitylated components of the NF- B pathway accumulated on the cytosolic leaflet of the endoplasmic reticulum (ER) membrane after the engagement of cell-surface, proinflammatory cytokine receptors or antigen receptors. Through mass spectrometric analysis, we found that the ER-anchored protein metadherin (MTDH) was a partner for these ubiquitylated activators of NF- B and that it directly bound to K⁶³-linked polyubiquitin chains. Knockdown of MTDH inhibited the accumulation of ubiquitylated NF- B signaling components at the ER, reduced the extent of NF- B activation, and decreased the amount of proinflammatory cytokines produced. Our observations highlight an unexpected facet of the ER as a key subcellular gateway for NF- B activation.  

3.2062           Lypd6 Enhances Wnt/β-Catenin Signaling by Promoting Lrp6 Phosphorylation in Raft Plasma Membrane Domains

Özhan, G., Sezgin, E., Wehner, D., Pfister, A.S., Kühl, S.J., Kagermeier-Schenk, B., Kühl, M., Schwille, P. and Weidinger, G. Developmental Cell, 26, 331-345 (2013)   Wnt/β-catenin signaling plays critical roles during embryogenesis, tissue homeostasis, and regeneration. How Wnt-receptor complex activity is regulated is not yet fully understood. Here, we identify the Ly6 family protein LY6/PLAUR domain-containing 6 (Lypd6) as a positive feedback regulator of Wnt/β-catenin signaling. lypd6 enhances Wnt signaling in zebrafish and Xenopus embryos and in mammalian cells, and it is required for wnt8-mediated patterning of the mesoderm and neuroectoderm during zebrafish gastrulation. Lypd6 is GPI anchored to the plasma membrane and physically interacts with the Wnt receptor Frizzled8 and the coreceptor Lrp6. Biophysical and biochemical evidence indicates that Lypd6 preferentially localizes to raft membrane domains, where Lrp6 is phosphorylated upon Wnt stimulation. lypd6 knockdown or mislocalization of the Lypd6 protein to nonraft membrane domains shifts Lrp6 phosphorylation to these domains and inhibits Wnt signaling. Thus, Lypd6 appears to control Lrp6 activation specifically in membrane rafts, which is essential for downstream signaling.  

3.2063           Notch3 is activated by chronic hypoxia and contributes to the progression of human prostate cancer

Danza, G., Di Serio, C., Ambrosio, M.R., Sturli, N., Lonetto, G., Rosati, F., Rocca, B.J., Ventimiglia, G., del Vecchio, M.T., Prudovsky, I., Marchionni, N and Tarantini, F. Int. J. Cancer, 133(11), 2577-2586 (2013)   Prostate cancer (PC) is still the second cause of cancer-related death among men. Although patients with metastatic presentation have an ominous outcome, the vast majority of PCs are diagnosed at an early stage. Nonetheless, even among patients with clinically localized disease the outcome may vary considerably. Other than androgen sensitivity, little is known about which other signaling pathways are deranged in aggressive, localized cancers. The elucidation of such pathways may help to develop innovative therapies aimed at specific molecular targets. We report that in a hormone-sensitive PC cell line, LNCaP, Notch3 was activated by hypoxia and sustained cell proliferation and colony formation in soft agar. Hypoxia also modulated cellular cholesterol content and the number and size of lipid rafts, causing a coalescence of small rafts into bigger clusters; under this experimental condition, Notch3 migrated from the non-raft into the raft compartment where it colocalized with the γ-secretase complex. We also looked at human PC biopsies and found that expression of Notch3 positively correlated with Gleason score and with expression of carbonic anhydrase IX, a marker of hypoxia. In conclusion, hypoxia triggers the activation of Notch3, which, in turn, sustains proliferation of PC cells. Notch3 pathway represents a promising target for adjuvant therapy in patients with PC.  

3.2064           HIV-1 Nef disrupts membrane-microdomain-associated anterograde transport for plasma membrane delivery of selected Src family kinases

Pan, X., Geist, M.M., Rudolph, J.M., Nickel, W. and fackler, O.T. Cell. Microbiol., 15(10), 1605-1621 (2013)   HIV-1 Nef, an essential factor in AIDS pathogenesis, boosts virus replication in vivo. As one of its activities in CD4⁺ T-lymphocytes, Nef potently retargets the Src family kinase (SFK) Lck but not closely related Fyn from the plasma membrane to recycling endosomes and the trans-Golgi network to tailor T-cell activation and optimize virus replication. Investigating the underlying mechanism we find Lck retargeting involves removal of the kinase from membrane microdomains. Moreover, Nef interferes with rapid vesicular transport of Lck to block anterograde transport and plasma membrane delivery of newly synthesized Lck. The sensitivity of Lck to Nef does not depend on functional domains of Lck but requires membrane insertion of the kinase. Surprisingly, the short N-terminal SH4 domain membrane anchor of Lck is necessary and sufficient to confer sensitivity to Nef-mediated anterograde transport block and microdomain extraction. In contrast, the SH4 domain of Fyn is inert to Nef-mediated manipulation. Nef thus interferes with a specialized membrane microdomain-associated pathway for plasma membrane delivery of newly synthesized Lck whose specificity is determined by the affinity of cargo for these sorting platforms. These results provide new insight into the mechanism of Nef action and the pathways used for SFK plasma membrane delivery.  

3.2065           The maize methylome influences mRNA splice sites and reveals widespread paramutation-like switches guided by small RNA

Regulski, M., Lu, Z., Kendall, J. et al Genome Res., 23, 1651-1662 (2013)   The maize genome, with its large complement of transposons and repeats, is a paradigm for the study of epigenetic mechanisms such as paramutation and imprinting. Here, we present the genome-wide map of cytosine methylation for two maize inbred lines, B73 and Mo17. CG (65%) and CHG (50%) methylation (where H = A, C, or T) is highest in transposons, while CHH (5%) methylation is likely guided by 24-nt, but not 21-nt, small interfering RNAs (siRNAs). Correlations with methylation patterns suggest that CG methylation in exons (8%) may deter insertion of Mutator transposon insertion, while CHG methylation at splice acceptor sites may inhibit RNA splicing. Using the methylation map as a guide, we used low-coverage sequencing to show that parental methylation differences are inherited by recombinant inbred lines. However, frequent methylation switches, guided by siRNA, persist for up to eight generations, suggesting that epigenetic inheritance resembling paramutation is much more common than previously supposed. The methylation map will provide an invaluable resource for epigenetic studies in maize.  

3.2066           Use of an anti-apoptotic CHO cell line for transient gene expression

Macaraeg, N.F., Reilly, D.E. and Womg, A.W. Biotechnol. Prog., 29, 1050-1058 (2013)   Transient gene expression in mammalian cells allows for rapid production of recombinant proteins for research and preclinical studies. Here, we describe the development of a polyethylenimine (PEI) transient transfection system using an anti-apoptotic host cell line. The host cell line, referred to as the Double Knockout (DKO), was generated by deleting two pro-apoptotic factors, Bax and Bak, in a CHO-K1 cell line using zinc finger nuclease mediated gene disruption. Optimized DNA and PEI volumes for DKO transfections were 50% and 30% lower than CHO-K1, respectively. During transfection DKO cells produced relatively high levels of lactate, but this was mitigated by a temperature shift to 31°C which further enhanced productivity. DKO cells expressed ∼3- to 4-fold higher antibody titers than CHO-K1 cells. As evidence of their anti-apoptotic properties post-transfection, DKO cells maintained higher viability and had reduced levels of active caspase-3 compared to CHO-K1 cells. Nuclear plasmid DNA copy numbers and message levels were significantly elevated in DKO cells. Although DNA uptake levels, as early as 40 min post-transfection, were higher in DKO cells this was not due to differences in cell surface heparan sulfate (HS) or initial endocytosis mechanism as both cell types utilized caveolae- and clathrin-mediated endocytosis to internalize DNA:PEI complexes. These results suggest that the increased transfection efficiency and titers from DKO cells are attributed to their resistance to transfection-induced apoptosis and not differences in endocytosis mechanism.  

3.2067            LRRK2 secretion in exosomes is regulated by 14-3-3

Fraser, K.B., Moehle, M.S., Daher, J.P.L., Webber, P.J., Williams, J.Y., Stewart, C.A., Yacoubian, T.A., Cowell, r.M., Dokland, T., Ye, t., Chen, D., Siegal, G.P., Galemmo, R.A., Tsika, E., Moore, D.J., Strandaert, D.G., Kojima, K., Mobley, J.A. and West, A.B. Human. Mol. Genet., 22(24), 4988-5000 (2013)   Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset Parkinson's disease (PD). Emerging evidence suggests a role for LRRK2 in the endocytic pathway. Here, we show that LRRK2 is released in extracellular microvesicles (i.e. exosomes) from cells that natively express LRRK2. LRRK2 localizes to collecting duct epithelial cells in the kidney that actively secrete exosomes into urine. Purified urinary exosomes contain LRRK2 protein that is both dimerized and phosphorylated. We provide a quantitative proteomic profile of 1673 proteins in urinary exosomes and find that known LRRK2 interactors including 14-3-3 are some of the most abundant exosome proteins. Disruption of the 14-3-3 LRRK2 interaction with a 14-3-3 inhibitor or through acute LRRK2 kinase inhibition potently blocks LRRK2 release in exosomes, but familial mutations in LRRK2 had no effect on secretion. LRRK2 levels were overall comparable but highly variable in urinary exosomes derived from PD cases and age-matched controls, although very high LRRK2 levels were detected in some PD affected cases. We further characterized LRRK2 exosome release in neurons and macrophages in culture, and found that LRRK2-positive exosomes circulate in cerebral spinal fluid (CSF). Together, these results define a pathway for LRRK2 extracellular release, clarify one function of the LRRK2 14-3-3 interaction and provide a foundation for utilization of LRRK2 as a biomarker in clinical trials.  

3.2068           Vesicular Transport of Progeny Parvovirus Particle through ER and Golgi Regulates Maturation and Cytolysis

Bär, S., Rommelaere, J. and Nüesch, J.P.F PloS Pathogens, 9(9), e1003605 (2013)   Progeny particles of non-enveloped lytic parvoviruses were previously shown to be actively transported to the cell periphery through vesicles in a gelsolin-dependent manner. This process involves rearrangement and destruction of actin filaments, while microtubules become protected throughout the infection. Here the focus is on the intracellular egress pathway, as well as its impact on the properties and release of progeny virions. By colocalization with cellular marker proteins and specific modulation of the pathways through over-expression of variant effector genes transduced by recombinant adeno-associated virus vectors, we show that progeny PV particles become engulfed into COPII-vesicles in the endoplasmic reticulum (ER) and are transported through the Golgi to the plasma membrane. Besides known factors like sar1, sec24, rab1, the ERM family proteins, radixin and moesin play (an) essential role(s) in the formation/loading and targeting of virus-containing COPII-vesicles. These proteins also contribute to the transport through ER and Golgi of the well described analogue of cellular proteins, the secreted Gaussia luciferase in absence of virus infection. It is therefore likely that radixin and moesin also serve for a more general function in cellular exocytosis. Finally, parvovirus egress via ER and Golgi appears to be necessary for virions to gain full infectivity through post-assembly modifications (e.g. phosphorylation). While not being absolutely required for cytolysis and progeny virus release, vesicular transport of parvoviruses through ER and Golgi significantly accelerates these processes pointing to a regulatory role of this transport pathway.  

3.2069           10E,12Z-conjugated linoleic acid impairs adipocyte triglyceride storage by enhancing fatty acid oxidation, lipolysis, and mitochondrial reactive oxygen species

Den Hartigh, L.J., Han, C.Y., Wang, S., Omer, M. and Chait, A.

1. Lipid Res., 54, 2964-2978 (2013)

  Conjugated linoleic acid (CLA) is a naturally occurring dietary trans fatty acid found in food from ruminant sources. One specific CLA isomer, 10E,12Z-CLA, has been associated with health benefits, such as reduced adiposity, while simultaneously promoting deleterious effects, such as systemic inflammation, insulin resistance, and dyslipidemia. The precise mechanisms by which 10E,12Z-CLA exerts these effects remain unknown. Despite potential health consequences, CLA continues to be advertised as a natural weight loss supplement, warranting further studies on its effects on lipid metabolism. We hypothesized that 10E,12Z-CLA impairs lipid storage in adipose tissue by altering the lipid metabolism of white adipocytes. We demonstrate that 10E,12Z-CLA reduced triglyceride storage due to enhanced fatty acid oxidation and lipolysis, coupled with diminished glucose uptake and utilization in cultured adipocytes. This switch to lipid utilization was accompanied by a potent proinflammatory response, including the generation of cytokines, monocyte chemotactic factors, and mitochondrial superoxide. Disrupting fatty acid oxidation restored glucose utilization and attenuated the inflammatory response to 10E,12Z-CLA, suggesting that fatty acid oxidation is critical in promoting this phenotype. With further investigation into the biochemical pathways involved in adipocyte responses to 10E,12Z-CLA, we can discern more information about its safety and efficacy in promoting weight loss.  

3.2070           Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins

Hazenbos, W.L. et al PloS Pathogens, 9(10), e1003653 (2013)   Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.  

3.2071           Combined enrichment of neuromelanin granules and synaptosomes from human substantia nigra pars compacta tissue for proteomic analysis

Plum, A., Helling, S., Theiss, C., Leite, R.E.P., May, C., Jacob-Filho, W., Eisenacher, M., Kuhlmann, K., Meyer, H.E., Riederer, P., Grinberg, L.T., Gerlach, M. and Marcus, K.

1. Proteomics, 94, 202-206 (2013)

  This article gives a detailed description of a protocol using density gradient centrifugation for the enrichment of neuromelanin granules and synaptosomes from low amounts (≥ 0.15 g) of human substantia nigra pars compacta tissue. This has a great advantage compared to already existing methods as it allows for the first time (i) a combined enrichment of neuromelanin granules and synaptosomes and (ii) just minimal amounts of tissue necessary to enable donor specific analysis. Individual specimens were classified as control or diseased according to clinical evaluation and neuropathological examination. For the enrichment of synaptosomes and neuromelanin granules from the same tissue sample density gradient centrifugations using Percoll® and Iodixanol were performed. The purity of resulting fractions was checked by transmission electron microscopy. We were able to establish a reproducible and easy to handle protocol combining two different density gradient centrifugations: using an Iodixanol gradient neuromelanin granules were enriched and in parallel, from the same sample, a fraction of synaptosomes with high purity using a Percoll® gradient was obtained. Our subfractionation strategy will enable a subsequent in depth proteomic characterization of neurodegenerative processes in the substantia nigra pars compacta in patients with Parkinson's disease and dementia with Lewy bodies compared to appropriate controls.  

3.2072           Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles

Kooijimans, S.A.A., Streemersch, S., Braeckmans, K., de Smedt, S.C., Hendrix, A., Wood, M.J.A., Schiffelers, R.M., Raemdonck, K. and Vader, P.

J. Controlled Release, 172, 229-238 (2013)

Extracellular vesicles (EVs) are specialised endogenous carriers of proteins and nucleic acids and are involved in intercellular communication. EVs are therefore proposed as candidate drug delivery systems for the delivery of nucleic acids and other macromolecules. However, the preparation of EV-based drug delivery systems is hampered by the lack of techniques to load the vesicles with nucleic acids. In this work we have now characterised in detail the use of an electroporation method for this purpose. When EVs were electroporated with fluorescently labelled siRNA, siRNA retention was comparable with previously published results (20–25% based on fluorescence spectroscopy and fluorescence fluctuation spectroscopy), and electroporation with unlabelled siRNA resulted in significant siRNA retention in the EV pellet as measured by RT-PCR. Remarkably, when siRNA was electroporated in the absence of EVs, a similar or even greater siRNA retention was measured. Nanoparticle tracking analysis and confocal microscopy showed extensive formation of insoluble siRNA aggregates after electroporation, which could be dramatically reduced by addition of EDTA. Other strategies to reduce aggregate formation, including the use of cuvettes with conductive polymer electrodes and the use of an acidic citrate electroporation buffer, resulted in a more efficient reduction of siRNA precipitation than EDTA. However, under these conditions, siRNA retention was below 0.05% and no significant differences in siRNA retention could be measured between samples electroporated in the presence or absence of EVs. Our results show that electroporation of EVs with siRNA is accompanied by extensive siRNA aggregate formation, which may cause overestimation of the amount of siRNA actually loaded into EVs. Moreover, our data clearly illustrate that electroporation is far less efficient than previously described, and highlight the necessity for alternative methods to prepare siRNA-loaded EVs.

 

3.2073           Protection of a Ceramide Synthase 2 Null Mouse from Drug-induced Liver Injury: ROLE OF GAP JUNCTION DYSFUNCTION AND CONNEXIN 32 MISLOCALIZATION

Park, W-J., Park, J-W., Erez-Roman, R., Kogot-Levin, A., Bame, J.R., Tirosh, B., Saada, A., Merrill Jr., A.H., Pewzner-Jung, Y. and Futerman, H.

1. Biol. Chem., 288(43), 309004-30916 (2013)

  Very long chain (C22-C24) ceramides are synthesized by ceramide synthase 2 (CerS2). A CerS2 null mouse displays hepatopathy because of depletion of C22-C24 ceramides, elevation of C16-ceramide, and/or elevation of sphinganine. Unexpectedly, CerS2 null mice were resistant to acetaminophen-induced hepatotoxicity. Although there were a number of biochemical changes in the liver, such as increased levels of glutathione and multiple drug-resistant protein 4, these effects are unlikely to account for the lack of acetaminophen toxicity. A number of other hepatotoxic agents, such as d-galactosamine, CCl₄, and thioacetamide, were also ineffective in inducing liver damage. All of these drugs and chemicals require connexin (Cx) 32, a key gap junction protein, to induce hepatotoxicity. Cx32 was mislocalized to an intracellular location in hepatocytes from CerS2 null mice, which resulted in accelerated rates of its lysosomal degradation. This mislocalization resulted from the altered membrane properties of the CerS2 null mice, which was exemplified by the disruption of detergent-resistant membranes. The lack of acetaminophen toxicity and Cx32 mislocalization were reversed upon infection with recombinant adeno-associated virus expressing CerS2. We establish that Gap junction function is compromised upon altering the sphingolipid acyl chain length composition, which is of relevance for understanding the regulation of drug-induced liver injury.  

3.2074           Interaction Maps of the Saccharomyces cerevisiae ESCRT-III Protein Snf7

Sciskala, B. and Kölling, R. Eukaryot. Cell, 12(11), 1538-1546 (2013)   The Saccharomyces cerevisiae ESCRT-III protein Snf7 is part of an intricate interaction network at the endosomal membrane. Interaction maps of Snf7 were established by measuring the degree of binding of individual binding partners to putative binding motifs along the Snf7 sequence by glutathione S-transferase (GST) pulldown. For each interaction partner, distinct binding profiles were obtained. The following observations were made. The ESCRT-III subunits Vps20 and Vps24 showed a complementary binding pattern, suggesting a model for the series of events in the ESCRT-III functional cycle. Vps4 bound to individual Snf7 motifs but not to full-length Snf7. This suggests that Vps4 does not bind to the closed conformation of Snf7. We also demonstrate for the first time that the ALIX/Bro1 homologue Rim20 binds to the α6 helix of Snf7. Analysis of a Snf7 α6 deletion mutant showed that the α6 helix is crucial for binding of Bro1 and Rim20 in vivo and is indispensable for the multivesicular body (MVB)-sorting and Rim-signaling functions of Snf7. The Snf7Δα6 protein still appeared to be incorporated into ESCRT-III complexes at the endosomal membrane, but disassembly of the complex seemed to be defective. In summary, our study argues against the view that the ESCRT cycle is governed by single one-to-one interactions between individual components and emphasizes the network character of the ESCRT interactions.  

3.2075           Granzyme B degradation by autophagy decreases tumor cell susceptibility to natural killer-mediated lysis under hypoxia

Baginska, J., Viry, E., Berchem, G., Poli, A., Noman, Z., van Moer, K., Medves, S., Zimmer, J., Oudin, A., Niclou, S.P., Bleackley, R.C., Goping, I.S., Chouaib, S. and Janji, B. PNAS, 110(43), 17450-17455 (2013)   Recent studies demonstrated that autophagy is an important regulator of innate immune response. However, the mechanism by which autophagy regulates natural killer (NK) cell-mediated antitumor immune responses remains elusive. Here, we demonstrate that hypoxia impairs breast cancer cell susceptibility to NK-mediated lysis in vitro via the activation of autophagy. This impairment was not related to a defect in target cell recognition by NK cells but to the degradation of NK-derived granzyme B in autophagosomes of hypoxic cells. Inhibition of autophagy by targeting beclin1 (BECN1) restored granzyme B levels in hypoxic cells in vitro and induced tumor regression in vivo by facilitating NK-mediated tumor cell killing. Together, our data highlight autophagy as a mechanism underlying the resistance of hypoxic tumor cells to NK-mediated lysis. The work presented here provides a cutting-edge advance in our understanding of the mechanism by which hypoxia-induced autophagy impairs NK-mediated lysis in vitro and paves the way for the formulation of more effective NK cell-based antitumor therapies.  

3.2076           Pooled RNAi screen identifies ubiquitin ligase Itch as crucial for influenza A virus release from the endosome during virus entry

Su, W-S., Chen, Y-C., Tseng, C-H., Hsu, P.W-C., Tung, K-F., jeng, K-S. and Lai, M.M.C. PNAS, 110(43), 17516-17521 (2013)   Influenza viruses, like other viruses, rely on host factors to support their life cycle as viral proteins usually “hijack,” or collaborate with, cellular proteins to execute their functions. Identification and understanding of these factors can increase the knowledge of molecular mechanisms manipulated by the viruses and facilitate development of antiviral drugs. To this end, we developed a unique genome-wide pooled shRNA screen to search for cellular factors important for influenza A virus (IAV) replication. We identified an E3 ubiquitin ligase, Itch, as an essential factor for an early step in the viral life cycle. In Itch knockdown cells, the incorporation of viral ribonucleoprotein complex into endosomes was normal, but its subsequent release from endosomes and transport to the nucleus was retarded. In addition, upon virus infection, Itch was phosphorylated and recruited to the endosomes, where virus particles were located. Furthermore, Itch interacted with viral M1 protein and ubiquitinated M1 protein. Collectively, our findings unravel a critical role of Itch in mediating IAV release from the endosome and offer insights into the mechanism for IAV uncoating during virus entry. These findings also highlight the feasibility of pooled RNAi screening for exploring the cellular cofactors of lytic viruses.  

3.2077           A tuberous sclerosis complex signalling node at the peroxisome regulates mTORC1 and autophagy in response to ROS

Zhang, J. et al Nature Cell Biol., 15(10), 1186-1196 (2013)   Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signalling node, TSC1, TSC2 and Rheb, localizes to peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by peroxisomal biogenesis factors 19 and 5 (PEX19 and PEX5), respectively, and peroxisome-localized TSC functioned as a Rheb GTPase-activating protein (GAP) to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, and abrogated peroxisome localization, Rheb GAP activity and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS, and peroxisome-localization-deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for the TSC in responding to ROS at the peroxisome, and identify the peroxisome as a signalling organelle involved in regulation of mTORC1.  

3.2078           Functionally Diverse MicroRNA Effector Complexes Are Regulated by Extracellular Signaling

Wu, P-H., Isaji, M. and carthew, R.W. Molecular Cell, 52(1), 113-123 (2013)   Because microRNAs (miRNAs) influence the expression of many genes in cells, discovering how the miRNA pathway is regulated is an important area of investigation. We found that the Drosophila miRNA-induced silencing complex (miRISC) exists in multiple forms. A constitutive form, called G-miRISC, is comprised of Ago1, miRNA, and GW182. Two distinct miRISC complexes that lack GW182 are regulated by mitogenic signaling. Exposure of cells to serum, lipids, or the tumor promoter PMA suppressed formation of these complexes. P-miRISC is comprised of Ago1, miRNA, and Loqs-PB, and it associates with mRNAs assembled into polysomes. The other regulated Ago1 complex associates with membranous organelles and is likely an intermediate in miRISC recycling. The formation of these complexes is correlated with a 5- to 10-fold stronger repression of target gene expression inside cells. Taken together, these results indicate that mitogenic signaling regulates the miRNA effector machinery to attenuate its repressive activities.  

3.2079           Plant Sterols the Better Cholesterol in Alzheimer's Disease? A Mechanistical Study

Burg, V.K. et al

1. Neurosci., 33(41), 16072-16087 (2013)

  Amyloid-β (Aβ), major constituent of senile plaques in Alzheimer's disease (AD), is generated by proteolytic processing of the amyloid precursor protein (APP) by β- and γ-secretase. Several lipids, especially cholesterol, are associated with AD. Phytosterols are naturally occurring cholesterol plant equivalents, recently been shown to cross the blood–brain-barrier accumulating in brain. Here, we investigated the effect of the most nutritional prevalent phytosterols and cholesterol on APP processing. In general, phytosterols are less amyloidogenic than cholesterol. However, only one phytosterol, stigmasterol, reduced Aβ generation by (1) directly decreasing β-secretase activity, (2) reducing expression of all γ-secretase components, (3) reducing cholesterol and presenilin distribution in lipid rafts implicated in amyloidogenic APP cleavage, and by (4) decreasing BACE1 internalization to endosomal compartments, involved in APP β-secretase cleavage. Mice fed with stigmasterol-enriched diets confirmed protective effects in vivo, suggesting that dietary intake of phytosterol blends mainly containing stigmasterol might be beneficial in preventing AD.  

3.2080           Oncogenic K-ras segregates at spatially distinct plasma membrane signaling platforms according to its phosphorylation status

Barcelo, C., Paco, N., Beckett, A.J., Alvarez-Moya, B., Garrido, E., Gelabert, M., Tebar, F., Jaumot, M., Prior, I. and Agell, N.

1. Cell. Science, 126(20), 4553-4559 (2013)

  Activating mutations in the K-Ras small GTPase are extensively found in human tumors. Although these mutations induce the generation of a constitutively GTP-loaded, active form of K-Ras, phosphorylation at Ser181 within the C-terminal hypervariable region can modulate oncogenic K-Ras function without affecting the in vitro affinity for its effector Raf-1. In striking contrast, K-Ras phosphorylated at Ser181 shows increased interaction in cells with the active form of Raf-1 and with p110α, the catalytic subunit of PI 3-kinase. Because the majority of phosphorylated K-Ras is located at the plasma membrane, different localization within this membrane according to the phosphorylation status was explored. Density-gradient fractionation of the plasma membrane in the absence of detergents showed segregation of K-Ras mutants that carry a phosphomimetic or unphosphorylatable serine residue (S181D or S181A, respectively). Moreover, statistical analysis of immunoelectron microscopy showed that both phosphorylation mutants form distinct nanoclusters that do not overlap. Finally, induction of oncogenic K-Ras phosphorylation – by activation of protein kinase C (PKC) – increased its co-clustering with the phosphomimetic K-Ras mutant, whereas (when PKC is inhibited) non-phosphorylated oncogenic K-Ras clusters with the non-phosphorylatable K-Ras mutant. Most interestingly, PI 3-kinase (p110α) was found in phosphorylated K-Ras nanoclusters but not in non-phosphorylated K-Ras nanoclusters. In conclusion, our data provide – for the first time – evidence that PKC-dependent phosphorylation of oncogenic K-Ras induced its segregation in spatially distinct nanoclusters at the plasma membrane that, in turn, favor activation of Raf-1 and PI 3-kinase.  

3.2081           Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes

Schulze, R.J., Weller, S.G., Schroeder, B., Krueger, E.W., Chi, S., Casey, C.A. and McNiven, M.A.

1. Cell Biol., 203(2), 315-326 (2013)

  Lipid droplets (LDs) are lipid storage organelles that in hepatocytes may be catabolized by autophagy for use as an energy source, but the membrane-trafficking machinery regulating such a process is poorly characterized. We hypothesized that the large GTPase Dynamin 2 (Dyn2), well known for its involvement in membrane deformation and cellular protein trafficking, could orchestrate autophagy-mediated LD breakdown. Accordingly, depletion or pharmacologic inhibition of Dyn2 led to a substantial accumulation of LDs in hepatocytes. Strikingly, the targeted disruption of Dyn2 induced a dramatic four- to fivefold increase in the size of autolysosomes. Chronic or acute Dyn2 inhibition combined with nutrient deprivation stimulated the excessive tubulation of these autolysosomal compartments. Importantly, Dyn2 associated with these tubules along their length, and the tubules vesiculated and fragmented in the presence of functional Dyn2. These findings provide new evidence for the participation of the autolysosome in LD metabolism and demonstrate a novel role for dynamin in the function and maturation of an autophagic compartment.  

3.2082           Polar substitutions in helix 3 of the prion protein produce transmembrane isoforms that disturb vesicle trafficking

Sanchez-garcia, J., Arbelaez, D., Jensen, K., Rincon-Limas, D.E. and Fernandez-Funez, P. Hum. Mol. Genet., 22(21), 4253-4266 (2013)   Prion diseases encompass a diverse group of neurodegenerative conditions characterized by the accumulation of misfolded prion protein (PrP) isoforms. Other conformational variants of PrP have also been proposed to contribute to neurotoxicity in prion diseases, including misfolded intermediates as well as cytosolic and transmembrane isoforms. To better understand PrP neurotoxicity, we analyzed the role of two highly conserved methionines in helix 3 on PrP biogenesis, folding and pathogenesis. Expression of the PrP-M205S and -M205,212S mutants in Drosophila led to hyperglycosylation, intracellular accumulation and widespread conformational changes due to failure of oxidative folding. Surprisingly, PrP-M205S and -M205,212S acquired a transmembrane topology (Ctm) previously linked to mutations in the signal peptide (SP) and the transmembrane domain (TMD). PrP-M205,212S also disrupted the accumulation of key neurodevelopmental proteins in lipid rafts, resulting in shortened axonal projections. These results uncover a new role for the hydrophobic domain in promoting oxidative folding and preventing the formation of neurotoxic Ctm PrP, mechanisms that may be relevant in the pathogenesis of both inherited and sporadic prion diseases.  

3.2083           Wnt5a Directs Polarized Calcium Gradients by Recruiting Cortical Endoplasmic Reticulum to the Cell Trailing Edge

Witze, E.S., Connacher, K.C., Houel, S., Schwartz, M.P., Morphew, M.K., Reid, L., Sacks, D.B., Anseth, K.S. and Ahn, N.G. Developmental Cell, 26(6), 645-657 (2013)   Wnt5a directs the assembly of the Wnt-receptor-actin-myosin-polarity (WRAMP) structure, which integrates cell-adhesion receptors with F-actin and myosin to form a microfilament array associated with multivesicular bodies (MVBs). The WRAMP structure is polarized to the cell posterior, where it directs tail-end membrane retraction, driving forward translocation of the cell body. Here we define constituents of the WRAMP proteome, including regulators of microfilament and microtubule dynamics, protein interactions, and enzymatic activity. IQGAP1, a scaffold for F-actin nucleation and crosslinking, is necessary for WRAMP structure formation, potentially bridging microfilaments and MVBs. Vesicle coat proteins, including coatomer-I subunits, localize to and are required for the WRAMP structure. Electron microscopy and live imaging demonstrate movement of the ER to the WRAMP structure and plasma membrane, followed by elevation of intracellular Ca²⁺. Thus, Wnt5a controls directional movement by recruiting cortical ER to mobilize a rear-directed, localized Ca²⁺ signal, activating actomyosin contraction and adhesion disassembly for membrane retraction.  

3.2084           The biogenesis protein PEX14 is an optimal marker for the identification and localization of peroxisomes in different cell types, tissues, and species in morphological studies

Grant, P., ahlemeyer, B., Karnati, S., Berg, T., Stelzig, I., Nenicu, A., Kuchelmeister, K., Crane, D.I. and Baumgart-Vogt, E. Histochem. Cell. Biol., 140(4), 423-442 (2013)   Catalase and ABCD3 are frequently used as markers for the localization of peroxisomes in morphological experiments. Their abundance, however, is highly dependent on metabolic demands, reducing the validity of analyses of peroxisomal abundance and distribution based solely on these proteins. We therefore attempted to find a protein which can be used as an optimal marker for peroxisomes in a variety of species, tissues, cell types and also experimental designs, independently of peroxisomal metabolism. We found that the biogenesis protein peroxin 14 (PEX14) is present in comparable amounts in the membranes of every peroxisome and is optimally suited for immunoblotting, immunohistochemistry, immunofluorescence, and immunoelectron microscopy. Using antibodies against PEX14, we could visualize peroxisomes with almost undetectable catalase content in various mammalian tissue sections (submandibular and adrenal gland, kidney, testis, ovary, brain, and pancreas from mouse, cat, baboon, and human) and cell cultures (primary cells and cell lines). Peroxisome labeling with catalase often showed a similar tissue distribution to the mitochondrial enzyme mitochondrial superoxide dismutase (both responsible for the degradation of reactive oxygen species), whereas ABCD3 exhibited a distinct labeling only in cells involved in lipid metabolism. We increased the sensitivity of our methods by using QuantumDots™, which have higher emission yields compared to classic fluorochromes and are unsusceptible to photobleaching, thereby allowing more exact quantification without artificial mistakes due to heterogeneity of individual peroxisomes. We conclude that PEX14 is indeed the best marker for labeling of peroxisomes in a variety of tissues and cell types in a consistent fashion for comparative morphometry.  

3.2085           Historical Overview of Autophagy

Dunn Jr., W.A., Schroder, L.A. and Aris, J.P. Current Cancer Res., 8, 1-24 (2013)   This chapter highlights those scientists who founded the field of autophagy (APG) research during its beginnings to those that have made key discoveries to advance the field into the mainstream of science. In the beginning, researchers were interested in lysosome morphology and function and how it related to protein turnover. These early studies were limited to morphological and biochemical methods that were restricted to mammalian cells and organs. APG was thought to be a highly regulated nonselective degradative process that could lead to cell death. When APG was characterized in yeast, a genetic model emerged allowing the identification of APG-related genes. Soon, new protein markers became available to better monitor and characterize APG in yeast, plants, insects, and animals. We now appreciate that APG has a positive role in cellular homeostasis and cell survival by recycling needed nutrients to sustain cellular functions and removing dysfunctional organelles and intracellular pathogens.  

3.2086           Suppression of amyloid-β production by 24S-hydroxycholesterol

Urano, Y., Ochiai, S. and Noguchi, N. FASEB J., 27, 4305-4315 (2013)   Cholesterol can be converted to 24S-hydroxycholesterol (24SOHC) by neuronal cholesterol 24-hydroxylase. In mouse models of Alzheimer's disease (AD), increasing 24SOHC levels reduced AD pathology. However, mechanisms underlying the effects of 24SOHC on amyloid-β (Aβ) production have remained unclear. Here we report that 24SOHC treatment reduces Aβ production and increases endoplasmic reticulum (ER)-resident immature amyloid precursor protein (APP) levels in human neuroblastoma SH-SY5Y cells and CHO cells stably expressing human APP. Treatment with 1–10 μM 24SOHC (equivalent to the concentrations detected in human brain homogenates) diminished Aβ production (IC₅₀=4.6 μM for Aβ₄₀) without affecting secretase activities. To evaluate the intracellular APP transport, we established an in vitro vesicle formation assay. We found that APP budding via COPII vesicles was diminished by 70% in 24SOHC-treated cells. The proteomics and immunoblotting analysis revealed that 24SOHC induced the expression of glucose-regulated protein 78 (GRP78), an ER chaperone, through unfolded protein response pathways, and enhanced the formation of the APP/GRP78 complex. Knockdown of GRP78 diminished the inhibitory effects of 24SOHC on Aβ production. These results suggest that 24SOHC down-regulates APP trafficking via enhancement of the complex formation of APP with up-regulated GRP78 in the ER, resulting in suppression of Aβ production.—Urano, Y., Ochiai, S., Noguchi, N. Suppression of amyloid-β production by 24S-hydroxycholesterovia inhibition of intracellular amyloid precursor protein trafficking.  

3.2087           LMBD1 Protein Serves as a Specific Adaptor for Insulin Receptor Internalization

Tseng, L.T-L., Lin, C-L., Tzen, K-Y., Chang, S.C. and chang, M-F.

1. Biol. Chem., 288(45), 32424-32432 (2013)

  Energy homeostasis is crucial for maintaining normally functioning cells; disturbances in this balance often cause various diseases. The limb region 1 (LMBR1) domain containing 1 gene (lmbrd1) encodes the LMBD1 protein that possesses 9 putative transmembrane domains. LMBD1 has been suggested to be involved in the lysosome in aiding the export of cobalamin. In this study, we determined that LMBD1 plays a regulatory role in the plasma membrane. A micro-positron emission tomography analysis showed that a single-allele knock-out of lmbrd1 increased the ¹⁸F-fluorodeoxyglucose uptake in murine hearts. In addition, the knockdown of lmbrd1 resulted in an up-regulated signaling of the insulin receptor (IR) and its downstream signaling molecule, Akt. Confocal and live total internal reflection fluorescence microscopy showed that LMBD1 co-localized and co-internalized with clathrin and the IR, but not with the transferrin receptor. The results of the mutation analysis and phenotypic rescue experiments indicate that LMBD1 interacts with adaptor protein-2 and is involved in the unique clathrin-mediated endocytosis of the IR. LMBD1 selectively interacts with the IR. The knockdown of lmbrd1 attenuated IR endocytosis, resulting in the perturbation of the IR recycling pathway and consequential enhancement of the IR signaling cascade. In summary, LMBD1 plays an imperative role in mediating and regulating the endocytosis of the IR.  

3.2088           Pseudomonas aeruginosa Outer Membrane Vesicles Modulate Host Immune Responses by Targeting the Toll-Like Receptor 4 Signaling Pathway

Zhao, K., Deng, X., He, C., Yue, B. and Wu, M. Infect. Immun., 83(12), 4509-4518 (2013)   Bacteria can naturally secrete outer membrane vesicles (OMVs) as pathogenic factors, while these vesicles may also serve as immunologic regulators if appropriately prepared. However, it is largely unknown whether Pseudomonas aeruginosa OMVs can activate inflammatory responses and whether immunization with OMVs can provide immune protection against subsequent infection. We purified and identified OMVs, which were then used to infect lung epithelial cells in vitro as well as C57BL/6J mice to investigate the immune response and the underlying signaling pathway. The results showed that OMVs generated from P. aeruginosa wild-type strain PAO1 were more cytotoxic to alveolar epithelial cells than those from quorum-sensing (QS)-deficient strain PAO1-ΔlasR. The levels of Toll-like receptor 4 (TLR4) and proinflammatory cytokines, including interleukin-1β (IL-1β) and IL-6, increased following OMV infection. Compared with lipopolysaccharide (LPS), lysed OMVs in which the membrane structures were broken induced a weak immune response. Furthermore, expression levels of TLR4-mediated responders (i.e., cytokines) were markedly downregulated by the TLR4 inhibitor E5564. Active immunization with OMVs or passive transfer of sera with a high cytokine quantity acquired from OMV-immunized mice could protect healthy mice against subsequent lethal PAO1 challenges (1.5 × 10¹¹ CFU). Collectively, these findings indicate that naturally secreted P. aeruginosa OMVs may trigger significant inflammatory responses via the TLR4 signaling pathway and protect mice against pseudomonal lung infection.  

3.2089           Proteomic techniques for characterisation of mesenchymal stem cell secretome

Skalnikova, H.K. Biochimie, 95, 2196-2211 (2013)   Mesenchymal stem cells (MSCs) are multipotent cells with a substantial potential in human regenerative medicine due to their ability to migrate to sites of injury, capability to suppress immune response and accessibility in large amount from patient's own bone marrow or fat tissue. It has been increasingly observed that the transplanted MSCs did not necessarily engraft and differentiate at the site of injury but might exert their therapeutic effects through secreted trophic signals. The MSCs secrete a variety of autocrine/paracrine factors, called secretome, that support regenerative processes in the damaged tissue, induce angiogenesis, protect cells from apoptotic cell death and modulate immune system. The cell culture medium conditioned by MSCs or osteogenic, chondrogenic as well as adipogenic precursors derived from MSCs has become a subject of intensive proteomic profiling in the search for and identification of released factors and microvesicles that might be applicable in regenerative medicine. Jointly with the methods for MSC isolation, expansion and differentiation, proteomic analysis of MSC secretome was enabled recently mainly due to the extensive development in protein separation techniques, mass spectrometry, immunological methods and bioinformatics. This review describes proteomic techniques currently applied or prospectively applicable in MSC secretomics, with a particular focus on preparation of the secretome sample, protein/peptide separation, mass spectrometry and protein quantification techniques, analysis of posttranslational modifications, immunological techniques, isolation and characterisation of secreted vesicles and exosomes, analysis of cytokine-encoding mRNAs and bioinformatics.  

3.2090           Lipid raft-regulated IGF-1R activation antagonizes TRAIL-induced apoptosis in gastric cancer cells

Xu, L., Qu, X., Hu, X., Zhu, Z., Li, C., Li, E., Ma, Y., Song, N. and Liu, Y. FEBS Lett., 587, 3815-3823 (2013)   Gastric cancer cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the resistance mechanism is not fully understood. In human gastric cancer MGC803 and BGC823 cells, TRAIL induces insulin-like growth factor-1 receptor (IGF-1R) pathway activation. Treatment with IGF-1R inhibitor OSI-906 or small interfering RNAs against IGF-1R, prevents IGF-1R pathway activation and increases TRAIL-induced apoptosis. The TRAIL-induced IGF-1R pathway activation is promoted by IGF-1R translocation into lipid rafts. Moreover, the translocation of IGF-1R into lipid rafts is regulated by Casitas B-lineage lymphoma b (Cbl-b). Taken together, TRAIL-induced IGF-1R activation antagonizes TRAIL-induced apoptosis by Cbl-b-regulated distribution of IGF-1R in lipid rafts.  

3.2091           Ctr2 regulates biogenesis of a cleaved form of mammalian Ctr1 metal transporter lacking the copper- and cisplatin-binding ecto-domain

Ohrvik, H., Nose, Y., Wood, L.K., Kim, B-e., Gleber, S-C., Ralle, M. and Thiele, D.J.

PNAS, 110(46), E4279-E4288 (2013)

Copper is an essential catalytic cofactor for enzymatic activities that drive a range of metabolic biochemistry including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Copper dysregulation is associated with fatal infantile disease, liver, and cardiac dysfunction, neuropathy, and anemia. Here we report that mammals regulate systemic copper acquisition and intracellular mobilization via cleavage of the copper-binding ecto-domain of the copper transporter 1 (Ctr1). Although full-length Ctr1 is critical to drive efficient copper import across the plasma membrane, cleavage of the ecto-domain is required for Ctr1 to mobilize endosomal copper stores. The biogenesis of the truncated form of Ctr1 requires the structurally related, previously enigmatic copper transporter 2 (Ctr2). Ctr2−/− mice are defective in accumulation of truncated Ctr1 and exhibit increased tissue copper levels, and X-ray fluorescence microscopy demonstrates that copper accumulates as intracellular foci. These studies identify a key regulatory mechanism for mammalian copper transport through Ctr2-dependent accumulation of a Ctr1 variant lacking the copper- and cisplatin-binding ecto-domain.

 

3.2092           Adrenergic Regulation of IgE Involves Modulation of CD23 and ADAM10 Expression on Exosomes

Padro, C.J., Shawler, T.M., Gormley, M.G. and Sanders, V.M:

J. Immunol., 191, 5383-5397 (2013)

Soluble CD23 plays a role in the positive regulation of an IgE response. Engagement of the β2 adrenergic receptor (β2AR) on a B cell is known to enhance the level of both soluble CD23 and IgE, although the mechanism by which this occurs is not completely understood. In this study, we report that, in comparison with a CD40 ligand/IL-4–primed murine B cell alone, β2AR engagement on a primed B cell increased gene expression of a disintegrin and metalloproteinase (ADAM)10, which is the primary sheddase of CD23, as well as protein expression of both CD23 and ADAM10, in a protein kinase A– and p38 MAPK–dependent manner, and promoted the localization of these proteins to exosomes as early as 2 d after priming, as determined by both Western blot and flow cytometry and confirmed by electron microscopy. In comparison with isolated exosomes released from primed B cells alone, the transfer of exosomes released from β2AR agonist–exposed primed B cells to cultures of recipient primed B cells resulted in an increase in the level of IgE produced per cell, without affecting the number of cells producing IgE, as determined by ELISPOT. These effects still occurred when a β2AR antagonist was added along with the transfer to block residual agonist, and they failed to occur when exosomes were isolated from β2AR-deficient B cells. These findings suggest that the mechanism responsible for mediating the β2AR-induced increase in IgE involves a shuttling of the β2AR-induced increase in CD23 and ADAM10 proteins to exosomes that subsequently mediate an increase in IgE.

 

3.2093           The Adaptor Protein-1 μ1B Subunit Expands the Repertoire of Basolateral Sorting Signal Recognition in Epithelial Cells

Guo, X., Mattera, R., Ren, X., Chen, Y., Retamal, C., Gonzalez, A. and Bonifacino, J.S.

Developmental Cell, 27(3), 353-366 (2013)

An outstanding question in protein sorting is why polarized epithelial cells express two isoforms of the  1 subunit of the AP-1 clathrin adaptor complex: the ubiquitous  1A and the epithelial-specific  1B. Previous studies led to the notion that  1A and  1B mediate basolateral sorting predominantly from the trans-Golgi network (TGN) and recycling endosomes, respectively. Using improved analytical tools, however, we find that  1A and  1B largely colocalize with each other. They also colocalize to similar extents with TGN and recycling endosome markers, as well as with basolateral cargoes transiting biosynthetic and endocytic-recycling routes. Instead, the two isoforms differ in their signal-recognition specificity. In particular,  1B preferentially binds a subset of signals from cargoes that are sorted basolaterally in a  1B-dependent manner. We conclude that expression of distinct  1 isoforms in epithelial cells expands the repertoire of signals recognized by AP-1 for sorting of a broader range of cargoes to the basolateral surface.

 

3.2094           Syntaxin 16 is a master recruitment factor for cytokinesis

Neto, H., Kaupisch, A., Collins, L.L. and Gould, G.W. Mol. Biol. Cell, 24, 3663-3674 (2013)   Recently it was shown that both recycling endosome and endosomal sorting complex required for transport (ESCRT) components are required for cytokinesis, in which they are believed to act in a sequential manner to bring about secondary ingression and abscission, respectively. However, it is not clear how either of these complexes is targeted to the midbody and whether their delivery is coordinated. The trafficking of membrane vesicles between different intracellular organelles involves the formation of soluble N-ethylmalei­mide–sensitive factor attachment protein receptor (SNARE) complexes. Although membrane traffic is known to play an important role in cytokinesis, the contribution and identity of intracellular SNAREs to cytokinesis remain unclear. Here we demonstrate that syntaxin 16 is a key regulator of cytokinesis, as it is required for recruitment of both recycling endosome–associated Exocyst and ESCRT machinery during late telophase, and therefore that these two distinct facets of cytokinesis are inextricably linked.  

3.2095           Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell–mediated cytotoxicity

Tuli, A., Thiery, J., James, A.M., Michelet, X., Sharma, M., Garg, S., Sanborn, K.B., Orange, J.S., Liberman, J. and Brenner, M.B. Mol. Biol. Cell, 24, 3721-3735 (2013)   Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell–mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity.  

3.2096           Membrane-Anchored Aβ Accelerates Amyloid Formation and Exacerbates Amyloid-Associated Toxicity in Mice

Nagarathinam, A., Höfflinger, P., Bühler, A., Schäfer, C., McCovern, G., Jeffrey, M., Staufenbiel, M., Jucker, M. and Baumann, F.

1. Neurosci., 33(49), 19284-19294 (2013)

  Pathological, genetic, and biochemical hallmarks of Alzheimer's disease (AD) are linked to amyloid-β (Aβ) peptide aggregation. Especially misfolded Aβ₄₂ peptide is sufficient to promote amyloid plaque formation. However, the cellular compartment facilitating the conversion of monomeric Aβ to aggregated toxic Aβ species remains unknown. In vitro models suggest lipid membranes to be the driving force of Aβ conversion. To this end, we generated two novel mouse models, expressing either membrane-anchored or nonanchored versions of the human Aβ₄₂ peptide. Strikingly, membrane-anchored Aβ₄₂ robustly accelerated Aβ deposition and exacerbated amyloid-associated toxicity upon crossing with Aβ precursor protein transgenic mice. These in vivo findings support the hypothesis that Aβ–membrane interactions play a pivotal role in early-onset AD as well as neuronal damage and provide evidence to study Aβ–membrane interactions as therapeutic targets.  

3.2097           Genetic regulation of vesiculogenesis and immunomodulation in Mycobacterium tuberculosis

Rath, P., Huang, C., Wang, T., Wang, T., Li, H., Prados-Rosales, R., Elemento, O., Casadevall, A. and Nathan, C.F. PNAS, 110, E4790-E4797 (2013)   Mycobacterium tuberculosis (Mtb) restrains immune responses well enough to escape eradication but elicits enough immunopathology to ensure its transmission. Here we provide evidence that this host–pathogen relationship is regulated in part by a cytosolic, membrane-associated protein with a unique structural fold, encoded by the Mtb gene rv0431. The protein acts by regulating the quantity of Mtb-derived membrane vesicles bearing Toll-like receptor 2 ligands, including the lipoproteins LpqH and SodC. We propose that rv0431 be named “vesiculogenesis and immune response regulator.”  

3.2098           Impaired endolysosomal function disrupts Notch signalling in optic nerve astrocytes

Valapala, M., Hose, S., Gongora, C., Dong, L., Wawrousek, E.F., Zigler Jr., J.S. and Sinha, D. Nature Communications, 4:1629 (2013)   Astrocytes migrate from the optic nerve into the inner retina, forming a template upon which retinal vessels develop. In the Nuc1 rat, mutation in the gene encoding βA3/A1-crystallin disrupts both Notch signalling in astrocytes and formation of the astrocyte template. Here we show that loss of βA3/A1-crystallin in astrocytes does not impede Notch ligand binding or extracellular cleavages. However, it affects vacuolar-type proton ATPase (V-ATPase) activity, thereby compromising acidification of the endolysosomal compartments, leading to reduced γ-secretase-mediated processing and release of the Notch intracellular domain (NICD). Lysosomal-mediated degradation of Notch is also impaired. These defects decrease the level of NICD in the nucleus, inhibiting the expression of Notch target genes. Overexpression of βA3/A1-crystallin in those same astrocytes restored V-ATPase activity and normal endolysosomal acidification, thereby increasing the levels of γ-secretase to facilitate optimal Notch signalling. We postulate that βA3/A1-crystallin is essential for normal endolysosomal acidification, and thereby, normal activation of Notch signalling in astrocytes.

3.2099           Direct modulation of the outer mitochondrial membrane channel, voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death

Rimmerman, N., Ben-Hail, D., Porat, Z., Juknat, A., Kozela, E., Daniels, M.P., Connelly, P.S., Leishman, E., Bradshaw, H.B., Shoshan-Barmatz, V. and Vogel, Z. Cell Death and Disease, 4, e949 (2013)   Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD.  

3.2100           Interaction of ganglioside GD3 with an EGF receptor sustains the self-renewal ability of mouse neural stem cells in vitro

Wang, J. and Yu, R.K. PNAS, 110(47), 19137-19142 (2013)   Mounting evidence supports the notion that gangliosides serve regulatory roles in neurogenesis; little is known, however, about how these glycosphingolipids function in neural stem cell (NSC) fate determination. We previously demonstrated that ganglioside GD3 is a major species in embryonic mouse brain: more than 80% of the NSCs obtained by the neurosphere method express GD3. To investigate the functional role of GD3 in neurogenesis, we compared the properties of NSCs from GD3-synthase knockout (GD3S-KO) mice with those from their wild-type littermates. NSCs from GD3S-KO mice showed decreased self-renewal ability compared with those from the wild-type animals, and that decreased ability was accompanied by reduced expression of EGF receptor (EGFR) and an increased degradation rate of EGFR and EGF-induced ERK signaling. We also showed that EGFR switched from the low-density lipid raft fractions in wild-type NSCs to the high-density layers in the GD3S-KO NSCs. Immunochemical staining revealed colocalization of EGFR and GD3, and EGFR could be immunoprecipitated from the NSC lysate with an anti-GD3 antibody from the wild-type, but not from the GD3S-KO, mice. Tracking the localization of endocytosed EGFR with endocytosis pathway markers indicated that more EGFR in GD3S-KO NSCs translocated through the endosomal−lysosomal degradative pathway, rather than through the recycling pathway. Those findings support the idea that GD3 interacts with EGFR in the NSCs and that the interaction is responsible for sustaining the expression of EGFR and its downstream signaling to maintain the self-renewal capability of NSCs.  

3.2101           RNA-sequencing from single nuclei

Grindberg, R.V., Yee-Greenbaum, J.L., McConnell, M.J., Novotny, M., O’Shaugnessy, A.L., Lambert, G.M., Arauzo-Bravo, M.J., Lee, J., Fishman, M., Robbins, G.E., Lin, X., Venepally, P., Badger, J.H., Galbraith, D.W., Gage, F.H. and Lasken, R.S. PNAS, 110(49), 19802-19807 (2013)   It has recently been established that synthesis of double-stranded cDNA can be done from a single cell for use in DNA sequencing. Global gene expression can be quantified from the number of reads mapping to each gene, and mutations and mRNA splicing variants determined from the sequence reads. Here we demonstrate that this method of transcriptomic analysis can be done using the extremely low levels of mRNA in a single nucleus, isolated from a mouse neural progenitor cell line and from dissected hippocampal tissue. This method is characterized by excellent coverage and technical reproducibility. On average, more than 16,000 of the 24,057 mouse protein-coding genes were detected from single nuclei, and the amount of gene-expression variation was similar when measured between single nuclei and single cells. Several major advantages of the method exist: first, nuclei, compared with whole cells, have the advantage of being easily isolated from complex tissues and organs, such as those in the CNS. Second, the method can be widely applied to eukaryotic species, including those of different kingdoms. The method also provides insight into regulatory mechanisms specific to the nucleus. Finally, the method enables dissection of regulatory events at the single-cell level; pooling of 10 nuclei or 10 cells obscures some of the variability measured in transcript levels, implying that single nuclei and cells will be extremely useful in revealing the physiological state and interconnectedness of gene regulation in a manner that avoids the masking inherent to conventional transcriptomics using bulk cells or tissues.  

3.2102           Bioinspired Exosome-Mimetic Nanovesicles for Targeted Delivery of Chemotherapeutics to Malignant Tumors

Jang, S.C., Kim, O.Y., Yoon, C.M., Choi, D-S., Roh, T-Y., park, J., Nilsson, J., Lötvall, J., Kim, Y-K. and Gho, Y.S. ACSNano, 7(9), 7698-7710 (2013)   Exosomes, the endogenous nanocarriers that can deliver biological information between cells, were recently introduced as new kind of drug delivery system. However, mammalian cells release relatively low quantities of exosomes, and purification of exosomes is difficult. Here, we developed bioinspired exosome-mimetic nanovesicles that deliver chemotherapeutics to the tumor tissue after systemic administration. The chemotherapeutics-loaded nanovesicles were produced by the breakdown of monocytes or macrophages using a serial extrusion through filters with diminishing pore sizes (10, 5, and 1 μm). These cell-derived nanovesicles have similar characteristics with the exosomes but have 100-fold higher production yield. Furthermore, the nanovesicles have natural targeting ability of cells by maintaining the topology of plasma membrane proteins. In vitro, chemotherapeutic drug-loaded nanovesicles induced TNF-α-stimulated endothelial cell death in a dose-dependent manner. In vivo, experiments in mice showed that the chemotherapeutic drug-loaded nanovesicles traffic to tumor tissue and reduce tumor growth without the adverse effects observed with equipotent free drug. Furthermore, compared with doxorubicin-loaded exosomes, doxorubicin-loaded nanovesicles showed similar in vivo antitumor activity. However, doxorubicin-loaded liposomes that did not carry targeting proteins were inefficient in reducing tumor growth. Importantly, removal of the plasma membrane proteins by trypsinization eliminated the therapeutic effects of the nanovesicles both in vitro and in vivo. Taken together, these studies suggest that the bioengineered nanovesicles can serve as novel exosome-mimetics to effectively deliver chemotherapeutics to treat malignant tumors.  

3.2103           Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma

Kalra, H., Adda, C.G., Liem, M., Ang, C-S., Mechler, A., Simpson, R.J., Hulett, M.D. and Mathivanan, S. Proteomics, 13(22), 3354-3364 (2013)   Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull-down, and OptiPrep^TM density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep^TM density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active.  

3.2104           EGFR inhibitor BIBU induces apoptosis and defective autophagy in glioma cells

Ghildiyal, R., Dixit, D. and Sen, E. Mol. Carcinogenesis, 52(12), 970-982 (2013)   The importance of aberrant EGFR signaling in glioblastoma progression and the promise of EGFR-specific therapies, prompted us to determine the efficacy of novel EGFR inhibitor BIBU-1361 [(3-chloro-4-fluoro-phenyl)-[6-(4-diethylaminomethyl-piperidin-1-yl)-pyrimido [5,4-d]pyrimidin-4-yl]-amine] in affecting glioma survival. BIBU induced apoptosis in a caspase-dependent manner and induced cell cycle arrest in glioma cells. Apoptosis was accompanied by decreased EGFR levels and its increased distribution towards caveolin rich lipid raft microdomains. BIBU inhibited pro-survival pathways Akt/mTOR and gp130/JAK/STAT3; and decreased levels of pro-inflammatory cytokine IL-6. BIBU caused increased LC3-I to LC3-II conversion and triggered the internalization of EGFR within vacuoles along with its increased co-localization with LC3-II. BIBU caused accumulation of p62 and increased levels of cleaved forms of Beclin-1 in all the cell lines tested. Importantly, BIBU failed to initiate execution of autophagy as pharmacological inhibition of autophagy with 3-Methyladenine or Bafilomycin failed to rescue BIBU mediated death. The ability of BIBU to abrogate Akt and STAT3 activation, induce apoptosis and prevent execution of autophagy warrants its investigation as a potent anti-glioma target  

3.2105           Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis

Bielaszewska, M., Rüter, C., Kunsmann, L., Greune, L., Bauwens, A., Zhang, W., Kuczius, T., Kim, K.S., Mellmann, A., Schmidt, M.A.. and Karch, H. PloS Pathogens, 9(12), e1003797 (2013)   Enterohemorrhagic Escherichia coli (EHEC) strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly), a member of the RTX (repeats-in-toxin) family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC) and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to enter host cells in order to target mitochondria.  

3.2106           A Hereditary Spastic Paraplegia Mouse Model Supports a Role of ZFYVE26/SPASTIZIN for the Endolysosomal System

Khundadze, M., Kollmann, K., Koch, N., Biskup, C., Nietzsche, S., Zimmer, G., Hennings, J.C., Huebner, A.K., Symmank, J., Jahic, A., Ilina, E.I., Karle, K., Schöls, L., Kessels, M., Braulke, T., Qualmann, B., Kurth, I., Beetz, C. and Hübner, C.A. PloS Genetics, 9(12), e1003988 (2013)   Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.  

3.2107           Lysosomal NEU1 deficiency affects amyloid precursor protein levels and amyloid-β secretion via deregulated lysosomal exocytosis

Annunziata, I., Patterson, A., Helton, D., Hu, H., Moshiach, S., Gomero, e., Nixon, R. and d’Azzo, A. Nature Communications, 4:2734 (2013)   Alzheimer’s disease (AD) belongs to a category of adult neurodegenerative conditions, which are associated with intracellular and extracellular accumulation of neurotoxic protein aggregates. Understanding how these aggregates are formed, secreted and propagated by neurons has been the subject of intensive research, but so far no preventive or curative therapy for AD is available, and clinical trials have been largely unsuccessful. Here we show that deficiency of the lysosomal sialidase NEU1 leads to the spontaneous occurrence of an AD-like amyloidogenic process in mice. This involves two consecutive events linked to NEU1 loss-of-function—accumulation and amyloidogenic processing of an oversialylated amyloid precursor protein in lysosomes, and extracellular release of Aβ peptides by excessive lysosomal exocytosis. Furthermore, cerebral injection of NEU1 in an established AD mouse model substantially reduces β-amyloid plaques. Our findings identify an additional pathway for the secretion of Aβ and define NEU1 as a potential therapeutic molecule for AD.  

3.2108           sCD44 overexpression increases intraocular pressure and aqueous outflow resistance

Giovingo, M., Nolan, M., McCarty, r., Pang, I-H., Clark, A.F., Beverley, R.M., Schwartz, S., Stamer, W.D., Walker, L., Grybauskas, A., Skuran, K., Kuprys, P.V., Yue, B.Y.J.T. and Knepper, P.A. Mol. Vis., 19, 2151-2164 (2013)   PURPOSE: CD44 plays major roles in multiple physiologic processes. The ectodomain concentration of the CD44 receptor, soluble CD44 (sCD44), is significantly increased in the aqueous humor of primary open-angle glaucoma (POAG). The purpose of this study was to determine if adenoviral constructs of CD44 and isolated 32-kDa sCD44 change intraocular pressure (IOP) in vivo and aqueous outflow resistance in vitro. METHODS: Adenoviral constructs of human standard CD44 (Ad-CD44S), soluble CD44 (Ad-sCD44), and empty viral cDNA were injected into the vitreous of BALB/cJ mice, followed by serial IOP measurements. Overexpression of CD44S and sCD44 was verified in vitro by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Anterior segments of porcine eyes were perfused with the isolated sCD44. sCD44-treated human trabecular meshwork (TM) cells and microdissected porcine TM were examined by confocal microscopy and Optiprep density gradient with western blot analysis to determine changes in lipid raft components. RESULTS: Intravitreous injection of adenoviral constructs with either Ad-CD44S or Ad-sCD44 vectors caused prolonged ocular hypertension in mice. Eight days after vector injection, Ad-CD44S significantly elevated IOP to 28.3±1.2 mmHg (mean±SEM, n=8; p<0.001); Ad-sCD44 increased IOP to 18.5±2.6 mmHg (n=8; p<0.01), whereas the IOP of uninjected eyes was 12.7±0.2 mmHg (n=16). The IOP elevation lasted more than 50 days. Topical administration of a γ-secretase inhibitor normalized Ad-sCD44-induced elevated IOP. sCD44 levels were significantly elevated in the aqueous humor of Ad-CD44S and Ad-sCD44 eyes versus contralateral uninjected eyes (p<0.01). Anterior segment perfusion of isolated 32-kDa sCD44 significantly decreased aqueous outflow rates. Co-administration of isolated sCD44 and CD44 neutralizing antibody or of γ-secretase inhibitor significantly enhanced flow rates. sCD44-treated human TM cells displayed cross-linked actin network formation. Optiprep density gradient and western blot analysis of human TM cells treated with sCD44 showed decreased annexin 2 expression and increased phosphorylated annexin 2 and caveolin 1 expression. CONCLUSIONS: Our data suggest that sCD44 increases outflow resistance in vivo and in vitro. Viral overexpression of both CD44S and sCD44 is sufficient to cause ocular hypertension. Infusion of sCD44 in porcine anterior segment eyes significantly decreased flow rates. Notably, sCD44 enhanced cross-linked actin network formation. The elevated sCD44 levels seen in POAG aqueous humor may play an important causative role in POAG pathogenesis.  

3.2109           Viral Attachment Induces Rapid Recruitment of an Innate Immune Sensor (TRIM5α) to the Plasma Membrane

Ohmine, S., Singh, R.D., Marks, D.L., Meyer, M.A., Pagano, R. and Ukeda, Y.

1. Innate Immun., 5, 414-424 (2013)

  TRIM5α (tripartite motif 5α) acts as a pattern recognition receptor specific for the retrovirus capsid lattice and blocks infection by HIV-1 immediately after entry. However, the precise mechanisms underlying this rapid recognition of viral components remain elusive. Here, we analyzed the influence of viral exposure on TRIM5α. Total internal reflection fluorescence microscopy and lipid flotation assays revealed rapid recruitment of a TRIM5α subpopulation to the plasma membrane (PM) upon exposure to vesicular stomatitis virus-G-pseudotyped HIV-1 viral-like particles (VLPs), but not to envelope (Env)-less HIV-1 VLPs. TRIM5α signals were frequently colocalized with those of HIV-1 capsid at the PM. Exposure to HIV-1 Env-pseudotyped HIV-1 vectors also triggered translocation of endogenous TRIM5α to lipid microdomains within human T cells. Similarly, clustering of lipid microdomains by a glycosphingolipid stereoisomer resulted in rapid TRIM5α recruitment to the PM. Of note, recruitment of endogenous rhesus TRIM5α to the PM prior to HIV-1 infection significantly increased the potency of viral restriction. Our data therefore suggest the importance of TRIM5α recruitment to the PM for TRIM5α-mediated innate immune sensing and restriction of retroviral infection.  

3.2110           The Role of Ect2 Nuclear RhoGEF Activity in Ovarian Cancer Cell Transformation

Huff, L.P., DeCristo, M.J., Trembath, D., Kuan, P.F., Yim, M., Liu, J., Cook, D.R., Miller, C.R., Der, C.J. and Cox, A.D. Genes & Cancer, 4(11-12), 460-475 (2013)   Ect2, a Rho guanine nucleotide exchange factor (RhoGEF), is atypical among RhoGEFs in its predominantly nuclear localization in interphase cells. One current model suggests that Ect2 mislocalization drives cellular transformation by promoting aberrant activation of cytoplasmic Rho family GTPase substrates. However, in ovarian cancers, where Ect2 is both amplified and overexpressed at the mRNA level, we observed that the protein is highly expressed and predominantly nuclear and that nuclear but not cytoplasmic Ect2 increases with advanced disease. Knockdown of Ect2 in ovarian cancer cell lines impaired their anchorage-independent growth without affecting their growth on plastic. Restoration of Ect2 expression rescued the anchorage-independent growth defect, but not if either the DH catalytic domain or the nuclear localization sequences of Ect2 were mutated. These results suggested a novel mechanism whereby Ect2 could drive transformation in ovarian cancer cells by acting as a RhoGEF specifically within the nucleus. Interestingly, Ect2 had an intrinsically distinct GTPase specificity profile in the nucleus versus the cytoplasm. Nuclear Ect2 bound preferentially to Rac1, while cytoplasmic Ect2 bound to RhoA but not Rac. Consistent with nuclear activation of endogenous Rac, Ect2 overexpression was sufficient to recruit Rac effectors to the nucleus, a process that required a functional Ect2 catalytic domain. Furthermore, expression of active nuclearly targeted Rac1 rescued the defect in transformed growth caused by Ect2 knockdown. Our work suggests a novel mechanism of Ect2-driven transformation, identifies subcellular localization as a regulator of GEF specificity, and implicates activation of nuclear Rac1 in cellular transformation.  

3.2111           Thrombomodulin functions as a plasminogen receptor to modulate angiogenesis

Chen, P-K., Chang, B-I., Kuo, C-H., Chen, P-S., Cho, C-F., Chang, C-F., Shi, G-Y. and Wu, H-L. FASEB J., 27(11), 4520-4531 82013)   Urokinase-type plasminogen activator (uPA) activates plasminogen (Plg) through a major pericellular proteolytic system involved in cell migration and angiogenesis; however, the Plg receptor that participates in uPA-mediated Plg activation has not yet been identified. In this study, we demonstrated that thrombomodulin (TM), a type I transmembrane glycoprotein, is a novel Plg receptor that plays a role in pericellular proteolysis and cell migration. Plg activation at the cell surface and the extent of its cell migration- and invasion-promoting effect are cellular TM expression dependent. Direct binding of Plg and the recombinant TM extracellular domain, with a K_D of 0.1−0.3 μM, was determined through surface plasmon resonance analysis. Colocalization of TM, Plg, and the uPA receptor within plasma membrane lipid rafts, at the leading edge of migrating endothelial cells, was demonstrated and was also shown to overlap with areas of major pericellular proteolysis. Moreover, the roles of TM and Plg in neoangiogenesis were demonstrated in vivo through the skin wound-healing model. In conclusion, we propose that TM is a novel Plg receptor that regulates uPA/uPA receptor-mediated Plg activation and pericellular proteolysis within lipid rafts at the leading edge of migrating cells during angiogenesis.

3.2112           Differential Association of the Na+/H+ Exchanger Regulatory Factor (NHERF) Family of Adaptor Proteins with the Raft- and the Non-Raft Brush Border Membrane Fractions of NHE3

Sultan, A., Luo, M., Yu, Q., Riederer, B., Xia, W., Chen, M., Lissner, S., Gessner, J.E., Donowitz, M., Yun, C.C., deJonge, H., Lamprecht, G. and Seidler, U. Cell. Physiol. Biochem., 32(5), 1386-1402 (2013)   Background/Aims: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na⁺/H⁺ exchanger NHE3 is regulated by the Na⁺/H⁺ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Methods: Detergent resistant membranes (“lipid rafts”) were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO₂/HCO₃^- mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results: NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions: The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs.  

3.2113           Autophagy proteins stabilize pathogen-containing phagosomes for prolonged MHC II antigen processing

Romao, S., Gasser, N., Becker, A.C., Guhl, B., Bajagic, M., Vanoaica, D., Ziegler, U., Roesler, J., Dengjel, J., Reichenbach, J. and Münz, C.

J. Cell Biol., 203(5), 757-766 (2013)

Antigen preservation for presentation is a hallmark of potent antigen-presenting cells. In this paper, we report that in human macrophages and dendritic cells, a subset of phagosomes gets coated with Atg8/LC3, a component of the molecular machinery of macroautophagy, and maintains phagocytosed antigens for prolonged presentation on major histocompatibility complex class II molecules. These Atg8/LC3-positive phagosomes are formed around the antigen with TLR2 agonists and require reactive oxygen species production by NOX2 for their generation. A deficiency in the NOX2-dependent formation of these antigen storage phagosomes could contribute to compromise antifungal immune control in chronic granulomatous disease patients

 

3.2114           Interaction of membrane/lipid rafts with the cytoskeleton: Impact on signaling and function ☆: Membrane/lipid rafts, mediators of cytoskeletal arrangement and cell signaling

Head, B.P., Patel, H.H. and Insel, P.A. Biochim. Biophys. Acta, 1838, 532-545 (2014)   The plasma membrane in eukaryotic cells contains microdomains that are enriched in certain glycosphingolipids, gangliosides, and sterols (such as cholesterol) to form membrane/lipid rafts (MLR). These regions exist as caveolae, morphologically observable flask-like invaginations, or as a less easily detectable planar form. MLR are scaffolds for many molecular entities, including signaling receptors and ion channels that communicate extracellular stimuli to the intracellular milieu. Much evidence indicates that this organization and/or the clustering of MLR into more active signaling platforms depends upon interactions with and dynamic rearrangement of the cytoskeleton. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to MLR and help regulate lateral diffusion of membrane proteins and lipids in response to extracellular events (e.g., receptor activation, shear stress, electrical conductance, and nutrient demand). MLR regulate cellular polarity, adherence to the extracellular matrix, signaling events (including ones that affect growth and migration), and are sites of cellular entry of certain pathogens, toxins and nanoparticles. The dynamic interaction between MLR and the underlying cytoskeleton thus regulates many facets of the function of eukaryotic cells and their adaptation to changing environments. Here, we review general features of MLR and caveolae and their role in several aspects of cellular function, including polarity of endothelial and epithelial cells, cell migration, mechanotransduction, lymphocyte activation, neuronal growth and signaling, and a variety of disease settings. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters.  

3.2115           Differentially localized acyl-CoA synthetase 4 isoenzymes mediate the metabolic channeling of fatty acids towards phosphatidylinositol

Küch, E-M., Vellaramkalayil, R., Zhang, I., Lehnen, D., Brügger, B., Stremmel, W., Ehehalt, R., Poppelreuther, M. and Füllekrug, J. Biochem. Biophys. Acta, 1841, 227-239 (2013)   The acyl-CoA synthetase 4 (ACSL4) has been implicated in carcinogenesis and neuronal development. Acyl-CoA synthetases are essential enzymes of lipid metabolism, and ACSL4 is distinguished by its preference for arachidonic acid. Two human ACSL4 isoforms arising from differential splicing were analyzed by ectopic expression in COS cells. We found that the ACSL4_v1 variant localized to the inner side of the plasma membrane including microvilli, and was also present in the cytosol. ACSL4_v2 contains an additional N-terminal hydrophobic region; this isoform was located at the endoplasmic reticulum and on lipid droplets. A third isoform was designed de novo by appending a mitochondrial targeting signal. All three ACSL4 variants showed the same specific enzyme activity. Overexpression of the isoenzymes increased cellular uptake of arachidonate to the same degree, indicating that the metabolic trapping of fatty acids is independent of the subcellular localization. Remarkably, phospholipid metabolism was changed by ACSL4 expression. Labeling with arachidonate showed that the amount of newly synthesized phosphatidylinositol was increased by all three ACSL4 isoenzymes but not by ACSL1. This was dependent on the expression level and the localization of the ACSL4 isoform. We conclude that in our model system exogenous fatty acids are channeled preferentially towards phosphatidylinositol by ACSL4 overexpression. The differential localization of the endogenous isoenzymes may provide compartment specific precursors of this anionic phospholipid important for many signaling processes.  

3.2116           Prolonged Insulin Stimulation Down-regulates GLUT4 through Oxidative Stress-mediated Retromer Inhibition by a Protein Kinase CK2-dependent Mechanism in 3T3-L1 Adipocytes

Ma, J., Nakagawa, Y., Kojima, I. and Shibata, H.

1. Biol. Chem., 289(1), 133-142 (2014)

  Although insulin acutely stimulates glucose uptake by promotion of GLUT4 translocation from intracellular compartments to the plasma membrane in adipocytes and muscles, long term insulin stimulation causes GLUT4 depletion that is particularly prominent in the insulin-responsive GLUT4 storage compartment. This effect is caused mainly by accelerated lysosomal degradation of GLUT4, although the mechanism is not fully defined. Here we show that insulin acutely induced dissociation of retromer components from the low density microsomal membranes of 3T3-L1 adipocytes that was accompanied by disruption of the interaction of Vps35 with sortilin. This insulin effect was dependent on the activity of protein kinase CK2 but not phosphatidylinositol 3-kinase or extracellular signal-regulated kinase 1/2. Knockdown of Vps26 decreased GLUT4 to a level comparable with that with insulin stimulation for 4 h. Vps35 with a mutation in the CK2 phosphorylation motif (Vps35-S7A) was resistant to insulin-induced dissociation from the low density microsomal membrane, and its overexpression attenuated GLUT4 down-regulation with insulin. Furthermore, insulin-generated hydrogen peroxide was an upstream mediator of the insulin action on retromer and GLUT4. These results suggested that insulin-generated oxidative stress switches the GLUT4 sorting direction to lysosomes through inhibition of the retromer function in a CK2-dependent manner.  

3.2117           Pseudomonas aeruginosa Cif Protein Enhances the Ubiquitination and Proteasomal Degradation of the Transporter Associated with Antigen Processing (TAP) and Reduces Major Histocompatibility Complex (MHC) Class I Antigen Presentation

Bomberger, J.M., Ely, K.H., Bangia, N., Ye, S., Green, K.A., Green, W.R., Enelow, R.I. and Stanton, B.A.

1. Biol. Chem., 289(1), 152-162 (2014)

  Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8⁺ T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.  

3.2118           Angiopoietin-2 Secretion by Endothelial Cell Exosomes: REGULATION BY THE PHOSPHATIDYLINOSITOL 3-KINASE (PI3K)/Akt/ENDOTHELIAL NITRIC OXIDE SYNTHASE (eNOS) AND SYNDECAN-4/SYNTENIN PATHWAYS

Ju, R., Zhuang, Z.W., Zhang, J., Lanahan, A.A., Kyriakides, T., Sessa, W.C. and Simons, M.

1. Biol. Chem., 289(1), 510-519 (2014)

  Angiopoietin-2 (Ang2) is an extracellular protein and one of the principal ligands of Tie2 receptor that is involved in the regulation of vascular integrity, quiescence, and inflammation. The mode of secretion of Ang2 has never been established, however. Here, we provide evidence that Ang2 is secreted from endothelial cells via exosomes and that this process is inhibited by the PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling pathway, whereas it is positively regulated by the syndecan-4/syntenin pathway. Vascular defects in Akt1 null mice arise, in part, because of excessive Ang2 secretion and can be rescued by the syndecan-4 knock-out that reduces extracellular Ang2 levels. This novel mechanism connects three critical signaling pathways: angiopoietin/Tie2, PI3K/Akt/eNOS, and syndecan/syntenin, which play important roles in vascular growth and stabilization.  

3.2119           Phospholipase D2 Mediates Survival Signaling through Direct Regulation of Akt in Glioblastoma Cells

Bruntz, R.C., Taylor, H.E., Lindsley, C.W. and Brown, H.A.

1. Biol. Chem., 289(2), 600-616 (2014)

  The lack of innovative drug targets for glioblastoma multiforme (GBM) limits patient survival to approximately 1 year following diagnosis. The pro-survival kinase Akt provides an ideal target for the treatment of GBM as Akt signaling is frequently activated in this cancer type. However, the central role of Akt in physiological processes limits its potential as a therapeutic target. In this report, we show that the lipid-metabolizing enzyme phospholipase D (PLD) is a novel regulator of Akt in GBM. Studies using a combination of small molecule PLD inhibitors and siRNA knockdowns establish phosphatidic acid, the product of the PLD reaction, as an essential component for the membrane recruitment and activation of Akt. Inhibition of PLD enzymatic activity and subsequent Akt activation decreases GBM cell viability by specifically inhibiting autophagic flux. We propose a mechanism whereby phosphorylation of beclin1 by Akt prevents binding of Rubicon (RUN domain cysteine-rich domain containing beclin1-interacting protein), an interaction known to inhibit autophagic flux. These findings provide a novel framework through which Akt inhibition can be achieved without directly targeting the kinase.  

3.2120           Disruption of the Coxsackievirus and Adenovirus Receptor-Homodimeric Interaction Triggers Lipid Microdomain- and Dynamin-dependent Endocytosis and Lysosomal Targeting

Salinas, S., Zussy, C., Loustalot, F., Henaff, D., Menendez, G., Morton, P.E., Parsons, M., Schiavo, G. and Kremer, E.J.

1. Biol. Chem., 289(2), 680-695 (2014)

  The coxsackievirus and adenovirus receptor (CAR) serves as a docking factor for some adenovirus (AdV) types and group B coxsackieviruses. Its role in AdV internalization is unclear as studies suggest that its intracellular domain is dispensable for some AdV infection. We previously showed that in motor neurons, AdV induced CAR internalization and co-transport in axons, suggesting that CAR was linked to endocytic and long-range transport machineries. Here, we characterized the mechanisms of CAR endocytosis in neurons and neuronal cells. We found that CAR internalization was lipid microdomain-, actin-, and dynamin-dependent, and subsequently followed by CAR degradation in lysosomes. Moreover, ligands that disrupted the homodimeric CAR interactions in its D1 domains triggered an internalization cascade involving sequences in its intracellular tail.  

3.2121           Prion Infection Impairs Cholesterol Metabolism in Neuronal Cells

Cui, H., Guo, B., Scicluna, B., Coleman, B.M., Lawson, V.A., Ellett, L., Meikle, P.J., Bukrinsky, M., Mukhamedova, N., Sviridov, D. and Hill, A.F.

1. Biol. Chem., 289(2), 789-802 (2014)

  Conversion of prion protein (PrP^C) into a pathological isoform (PrP^Sc) during prion infection occurs in lipid rafts and is dependent on cholesterol. Here, we show that prion infection increases the abundance of cholesterol transporter, ATP-binding cassette transporter type A1 (ATP-binding cassette transporter type A1), but reduces cholesterol efflux from neuronal cells leading to the accumulation of cellular cholesterol. Increased abundance of ABCA1 in prion disease was confirmed in prion-infected mice. Mechanistically, conversion of PrP^C to the pathological isoform led to PrP^Sc accumulation in rafts, displacement of ABCA1 from rafts and the cell surface, and enhanced internalization of ABCA1. These effects were abolished with reversal of prion infection or by loading cells with cholesterol. Stimulation of ABCA1 expression with liver X receptor agonist or overexpression of heterologous ABCA1 reduced the conversion of prion protein into the pathological form upon infection. These findings demonstrate a reciprocal connection between prion infection and cellular cholesterol metabolism, which plays an important role in the pathogenesis of prion infection in neuronal cells.  

3.2122           Inflammation, caveolae and CD38-mediated calcium regulation in human airway smooth muscle

Sathish, V., Thompson, M.A., Sinha, S., Sieck, G.C., Prakash, Y.S. and Pabelick, C.M: Biochem. Biophys. Acta, 1843, 346-351 (2014)   The pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) increases expression of CD38 (a membrane-associated bifunctional enzyme regulating cyclic ADP ribose), and enhances agonist-induced intracellular Ca^2 + ([Ca^2 +]_i) responses in human airway smooth muscle (ASM). We previously demonstrated that caveolae and their constituent protein caveolin-1 are important for ASM [Ca^2 +]_i regulation, which is further enhanced by TNFα. Whether caveolae and CD38 are functionally linked in mediating TNFα effects is unknown. In this regard, whether the related cavin proteins (cavin-1 and -3) that maintain structure and function of caveolae play a role is also not known. In the present study, we hypothesized that TNFα effects on CD38 expression and function in human ASM involve caveolae. Caveolar fractions from isolated human ASM cells expressed CD38 and its expression was upregulated by exposure to 20 ng/ml TNFα (48 h). ASM cells expressed cavin-1 and cavin-3, which were also upregulated by TNFα. Knockdown of caveolin-1, cavin-1 or cavin-3 (using siRNA) all significantly reduced CD38 expression and ADP-ribosyl cyclase activity in the presence or absence of TNFα. Furthermore, caveolin-1, cavin-1 and cavin-3 siRNAs reduced [Ca^2 +]_i responses to histamine under control conditions, and blunted the enhanced [Ca^2 +]_i responses in TNFα-exposed cells. These data demonstrate that CD38 is expressed within caveolae and its function is linked to the caveolar regulatory proteins caveolin-1, cavin-1 and -3. The link between caveolae and CD38 is further enhanced during airway inflammation demonstrating the important role of caveolae in regulation of [Ca^2 +]_i and contractility in the airway.  

3.2123           Dissecting Functions of the Conserved Oligomeric Golgi Tethering Complex Using a Cell-Free Assay

Cottam, N.P., Wilson, K.M., Ng, B.G., Körner, C., Freeze, H.H. and Ungar, D. Traffic, 15(1), 12-21 (2014)   Vesicle transport sorts proteins between compartments and is thereby responsible for generating the non-uniform protein distribution along the eukaryotic secretory and endocytic pathways. The mechanistic details of specific vesicle targeting are not yet well characterized at the molecular level. We have developed a cell-free assay that reconstitutes vesicle targeting utilizing the recycling of resident enzymes within the Golgi apparatus. The assay has physiological properties, and could be used to show that the two lobes of the conserved oligomeric Golgi tethering complex play antagonistic roles in trans-Golgi vesicle targeting. Moreover, we can show that the assay is sensitive to several different congenital defects that disrupt Golgi function and therefore cause glycosylation disorders. Consequently, this assay will allow mechanistic insight into the targeting step of vesicle transport at the Golgi, and could also be useful for characterizing some novel cases of congenital glycosylation disorders  

3.2124           Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization

Bates, R.C., Fees, C.P., Holland, W.L:, Winger, C.C., Batbayar, K., Ancar, R., Bergren, T., Petkoff, D. and Stich, B.J. Developmental Biol., 386, 165-180 (2014)   We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC-γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca]i). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 min after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca]i and other fertilization events. As compared to 14 other lipids, PA specifically bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca]i, PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca]i release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca]i release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm–egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization.  

3.2125           Comprehensive proteomic profiling of outer membrane vesicles from Campylobacter jejuni

Jang, K-S., Sweredoski, M.J., Graham, R.L.J., Hess, S. and Clemons Jr. W.M.

1. Proteomics, 98, 90-98 (2014)

  Gram-negative bacteria constitutively release outer membrane vesicles (OMVs) during cell growth that play significant roles in bacterial survival, virulence and pathogenesis. In this study, comprehensive proteomic analysis of OMVs from a human gastrointestinal pathogen Campylobacter jejuni NCTC11168 was performed using high-resolution mass spectrometry. The OMVs of C. jejuni NCTC11168 were isolated from culture supernatants then characterized using electron microscopy and dynamic light scattering revealing spherical OMVs of an average diameter of 50 nm. We then identified 134 vesicular proteins using high-resolution LTQ-Orbitrap mass spectrometry. Subsequent functional analysis of the genes revealed the relationships of the vesicular proteins. Furthermore, known N-glycoproteins were identified from the list of the vesicular proteome, implying the potential role of the OMVs as a delivery means for biologically relevant bacterial glycoproteins. These results enabled us to elucidate the overall proteome profile of pathogenic bacterium C. jejuni and to speculate on the function of OMVs in bacterial infections and communication.  

3.2126           CKIP-1 Is an Intrinsic Negative Regulator of T-Cell Activation through an Interaction with CARMA1

Sakamoto, T., Kobayashi, M., Tada, K., Shinohara, M., Io, K., Nagata, K., Iwai, F., Takiuchi, Y., Arai, Y., Yamahita, K., Shindo, K., Kadowaki, N., Koyanagi, Y. and Takaori-Kondo, A. PloS One, 9(1), e85762 (2014)   The transcription factor NF-κB plays a key regulatory role in lymphocyte activation and generation of immune response. Stimulation of T cell receptor (TCR) induces phosphorylation of CARMA1 by PKCθ, resulting in formation of CARMA1-Bcl10-MALT1 (CBM) complex at lipid rafts and subsequently leading to NF-κB activation. While many molecular events leading to NF-κB activation have been reported, it is less understood how this activation is negatively regulated. We performed a cell-based screening for negative regulators of TCR-mediated NF-κB activation, using mutagenesis and complementation cloning strategies. Here we show that casein kinase-2 interacting protein-1 (CKIP-1) suppresses PKCθ-CBM-NF-κB signaling. We found that CKIP-1 interacts with CARMA1 and competes with PKCθ for association. We further confirmed that a PH domain of CKIP-1 is required for association with CARMA1 and its inhibitory effect. CKIP-1 represses NF-κB activity in unstimulated cells, and inhibits NF-κB activation induced by stimulation with PMA or constitutively active PKCθ, but not by stimulation with TNFα. Interestingly, CKIP-1 does not inhibit NF-κB activation induced by CD3/CD28 costimulation, which caused dissociation of CKIP-1 from lipid rafts. These data suggest that CKIP-1 contributes maintenance of a resting state on NF-κB activity or prevents T cells from being activated by inadequate signaling. In conclusion, we demonstrate that CKIP-1 interacts with CARMA1 and has an inhibitory effect on PKCθ-CBM-NF-κB signaling.    

3.2127           Acute Phencyclidine Treatment Induces Extensive and Distinct Protein Phosphorylation in Rat Frontal Cortex

Palmowski, P., Rogowska-Wrzesinska, A., Williamson, J., Beck, H.C., Mikkelsen, J.D., Hansen, H.H. and Jensen, O.N.

1. Proteome Res., 13, 1578-1592 (2014)

  Phencyclidine (PCP), a noncompetitive N-methyl-d-aspartate receptor antagonist, induces psychotomimetic effects in humans and animals. Administration of PCP to rodents is used as a preclinical model for schizophrenia; however, the molecular mechanisms underlying the symptoms remain largely unknown. Acute PCP treatment rapidly induces behavioral and cognitive deficits; therefore, post-translational regulation of protein activity is expected to play a role at early time points. We performed mass-spectrometry-driven quantitative analysis of rat frontal cortex 15, 30, or 240 min after the administration of PCP (10 mg/kg). We identified and quantified 23 548 peptides, including 4749 phosphopeptides, corresponding to 2604 proteins. A total of 352 proteins exhibited altered phosphorylation levels, indicating that protein phosphorylation is involved in the acute response to PCP. Computational assessment of the regulated proteins biological function revealed that PCP perturbs key processes in the frontal cortex including calcium homeostasis, organization of cytoskeleton, endo/exocytosis, and energy metabolism. This study on acute PCP treatment provides the largest proteomics and phosphoproteomics data sets to date of a preclinical model of schizophrenia. Our findings contribute to the understanding of alterations in glutamatergic neurotransmission in schizophrenia and provide a foundation for discovery of novel targets for pharmacological intervention.  

3.2128           A Functional Interplay between the Small GTPase Rab11a and Mitochondria-shaping Proteins Regulates Mitochondrial Positioning and Polarization of the Actin Cytoskeleton Downstream of Src Family Kinases

Landry, M-C., Champagne, C., Boulanger, M-C., Jette, A., Fuchs, M., Dziengelwski, C. and Lavoie, J.N.

1. Biol. Chem., 289(4), 2230-2249 (2014)

  It is believed that mitochondrial dynamics is coordinated with endosomal traffic rates during cytoskeletal remodeling, but the mechanisms involved are largely unknown. The adenovirus early region 4 ORF4 protein (E4orf4) subverts signaling by Src family kinases (SFK) to perturb cellular morphology, membrane traffic, and organellar dynamics and to trigger cell death. Using E4orf4 as a model, we uncovered a functional connection between mitochondria-shaping proteins and the small GTPase Rab11a, a key regulator of polarized transport via recycling endosomes. We found that E4orf4 induced dramatic changes in the morphology of mitochondria along with their mobilization at the vicinity of a polarized actin network typifying E4orf4 action, in a manner controlled by SFK and Rab11a. Mitochondrial remodeling was associated with increased proximity between Rab11a and mitochondrial membranes, changes in fusion-fission dynamics, and mitochondrial relocalization of the fission factor dynamin-related protein 1 (Drp1), which was regulated by the Rab11a effector protein FIP1/RCP. Knockdown of FIP1/RCP or inhibition of Drp1 markedly impaired mitochondrial remodeling and actin assembly, involving Rab11a-mediated mitochondrial dynamics in E4orf4-induced signaling. A similar mobilization of mitochondria near actin-rich structures was mediated by Rab11 and Drp1 in viral Src-transformed cells and contributed to the biogenesis of podosome rosettes. These findings suggest a role for Rab11a in the trafficking of Drp1 to mitochondria upon SFK activation and unravel a novel functional interplay between Rab11a and mitochondria during reshaping of the cell cytoskeleton, which would facilitate mitochondria redistribution near energy-requiring actin-rich structures.  

3.2129           TRPC6 participates in the regulation of cytosolic basal calcium concentration in murine resting platelets

Albarran, L., Berna-Erro, A., Dionisio, N., Redondo, P.C., Lopez, E., Lopez, J.J., Salido, G.M., Sabate, J.M.B. and Rasado, J.A. Biochim. Biophys. Acta, 1843, 789-796 (2014)   Cytosolic-free Ca^2 + plays a crucial role in blood platelet function and is essential for thrombosis and hemostasis. Therefore, cytosolic-free Ca^2 + concentration is tightly regulated in this cell. TRPC6 is expressed in platelets, and an important role for this Ca^2 + channel in Ca^2 + homeostasis has been reported in other cell types. The aim of this work is to study the function of TRPC6 in platelet Ca^2 + homeostasis. The absence of TRPC6 resulted in an 18.73% decreased basal [Ca^2 +]_c in resting platelets as compared to control cells. Further analysis confirmed a similar Ca^2 + accumulation in wild-type and TRPC6-deficient mice; however, passive Ca^2 + leak rates from agonist-sensitive intracellular stores were significantly decreased in TRPC6-deficient platelets. Biotinylation studies indicated the presence of an intracellular TRPC6 population, and subcellular fractionation indicated their presence on endoplasmic reticulum membranes. Moreover, the presence of intracellular calcium release in platelets stimulated with 1-oleoyl-2-acetyl-sn-glycerol further suggested a functional TRPC6 population located on the intracellular membranes surrounding calcium stores. However, coimmunoprecipitation assay confirmed the absence of STIM1–TRPC6 interactions in resting conditions. This findings together with the absence of extracellular Mn^2 + entry in resting wild-type platelets indicate that the plasma membrane TRPC6 fraction does not play a significant role in the maintenance of basal [Ca^2 +]_c in mouse platelets. Our results suggest an active participation of the intracellular TRPC6 fraction as a regulator of basal [Ca^2 +]_c, controlling the passive Ca^2 + leak rate from agonist-sensitive intracellular Ca^2 + stores in resting platelets.  

3.2130           The endoplasmic reticulum–mitochondria connection: One touch, multiple functions

Marchi, S., Patergnani, S. and Pinton, P. Biochim. Biophys. Acta, 1837, 461-469 (2014)   The endoplasmic reticulum (ER) and mitochondria are tubular organelles with a characteristic “network structure” that facilitates the formation of interorganellar connections. The ER and mitochondria join together at multiple contact sites to form specific domains, termed mitochondria-ER associated membranes (MAMs), with distinct biochemical properties and a characteristic set of proteins. The functions of these two organelles are coordinated and executed at the ER–mitochondria interface, which provides a platform for the regulation of different processes. The roles played by the ER–mitochondria interface range from the coordination of calcium transfer to the regulation of mitochondrial fission and inflammasome formation as well as the provision of membranes for autophagy. The novel and unconventional processes that occur at the ER–mitochondria interface demonstrate its multifunctional and intrinsically dynamic nature. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.  

3.2131           CIB1 Synergizes with EphrinA2 to Regulate Kaposi's Sarcoma-Associated Herpesvirus Macropinocytic Entry in Human Microvascular Dermal Endothelial Cells

Bandyopadhyay, C., Valiya-Veettil, M., Dutta, D., Chakraborty, S. and Chandran, B. PloS Pathogens, 10(2), e1003941 (2014)   KSHV envelope glycoproteins interact with cell surface heparan sulfate and integrins, and activate FAK, Src, PI3-K, c-Cbl, and Rho-GTPase signal molecules in human microvascular dermal endothelial (HMVEC-d) cells. c-Cbl mediates the translocation of virus bound α3β1 and αVβ3 integrins into lipid rafts (LRs), where KSHV interacts and activates EphrinA2 (EphA2). EphA2 associates with c-Cbl-myosin IIA and augmented KSHV-induced Src and PI3-K signals in LRs, leading to bleb formation and macropinocytosis of KSHV. To identify the factor(s) coordinating the EphA2-signal complex, the role of CIB1 (calcium and integrin binding protein-1) associated with integrin signaling was analyzed. CIB1 knockdown did not affect KSHV binding to HMVEC-d cells but significantly reduced its entry and gene expression. In contrast, CIB1 overexpression increased KSHV entry in 293 cells. Single virus particle infection and trafficking during HMVEC-d cell entry was examined by utilizing DiI (envelope) and BrdU (viral DNA) labeled virus. CIB1 was associated with KSHV in membrane blebs and in Rab5 positive macropinocytic vesicles. CIB1 knockdown abrogated virus induced blebs, macropinocytosis and virus association with the Rab5 macropinosome. Infection increased the association of CIB1 with LRs, and CIB1 was associated with EphA2 and KSHV entry associated signal molecules such as Src, PI3-K, and c-Cbl. CIB1 knockdown significantly reduced the infection induced EphA2, Src and Erk1/2 activation. Mass spectrometry revealed the simultaneous association of CIB1 and EphA2 with the actin cytoskeleton modulating myosin IIA and alpha-actinin 4 molecules, and CIB1 knockdown reduced EphA2's association with myosin IIA and alpha-actinin 4. Collectively, these studies revealed for the first time that CIB1 plays a role in virus entry and macropinocytosis, and suggested that KSHV utilizes CIB1 as one of the key molecule(s) to coordinate and sustain the EphA2 mediated signaling involved in its entry, and CIB1 is an attractive therapeutic target to block KSHV infection.  

3.2132           A paradigm shift for extracellular vesicles as small RNA carriers: from cellular waste elimination to therapeutic applications

Hagiwara, K., Ochiya, T. and Kosaka, N. Drug Deliv.and Transl. Res., 4:31 (2014)   RNA interference (RNAi) is an important avenue for target-specific gene silencing that is mainly performed by either small interfering RNAs (siRNAs) or microRNAs (miRNAs). This novel method is rapidly becoming a powerful tool for gene therapy. However, the rapid degradation of siRNAs and miRNAs and the limited duration of their action in vivo call for an efficient delivery technology. Recently, increasing attention has been paid to the use of extracellular vesicles (EVs) as delivery systems. The use of EVs as small RNA carriers has multiple advantages over conventional delivery systems. In this review, we summarize recent findings regarding the potential application of EVs as small RNA delivery systems. Moreover, we focus on some of the obstacles to EV-based therapeutics.  

3.2133           Eucommia ulmoides Cortex, Geniposide and Aucubin Regulate Lipotoxicity through the Inhibition of Lysosomal BAX

Lee, G-H., Lee, M-R., Lee, H-Y., Kim, S.H., Kim, H-K., Kim, H-R. and Chae, H-J. PloS One, 9(2), e88017 (2014)   In this study we examined the inhibition of hepatic dyslipidemia by Eucommia ulmoides extract (EUE). Using a screening assay for BAX inhibition we determined that EUE regulates BAX-induced cell death. Among various cell death stimuli tested EUE regulated palmitate-induced cell death, which involves lysosomal BAX translocation. EUE rescued palmitate-induced inhibition of lysosomal V-ATPase, α-galactosidase, α-mannosidase, and acid phosphatase, and this effect was reversed by bafilomycin, a lysosomal V-ATPase inhibitor. The active components of EUE, aucubin and geniposide, showed similar inhibition of palmitate-induced cell death to that of EUE through enhancement of lysosome activity. Consistent with these in vitro findings, EUE inhibited the dyslipidemic condition in a high-fat diet animal model by regulating the lysosomal localization of BAX. This study demonstrates that EUE regulates lipotoxicity through a novel mechanism of enhanced lysosomal activity leading to the regulation of lysosomal BAX activation and cell death. Our findings further indicate that geniposide and aucubin, active components of EUE, may be therapeutic candidates for non-alcoholic fatty liver disease.  

3.2134           Role for Mycobacterium tuberculosis Membrane Vesicles in Iron Acquisition

Prados-Rosales, R., Weinrick, B.C., Pique, D.G., Jacobs Jr, W.R., Casadevall, A. and rodriguez, G.M.

1. Bacteriol., 196(6), 1250-1256 (2014)

  Mycobacterium tuberculosis releases membrane vesicles packed with molecules that can modulate the immune response. Because environmental conditions often influence the production and content of bacterial vesicles, this study examined M. tuberculosis microvesicles released under iron limitation, a common condition faced by pathogens inside the host. The findings indicate that M. tuberculosis increases microvesicle production in response to iron restriction and that these microvesicles contain mycobactin, which can serve as an iron donor and supports replication of iron-starved mycobacteria. Consequently, the results revealed a role of microvesicles in iron acquisition in M. tuberculosis, which can be critical for survival in the host.  

3.2135           On the Formation of Lipid Droplets in Human Adipocytes: The Organization of the Perilipin–Vimentin Cortex

Heid, H., Rickelt, S., Zimbelmann, R., Winter, S., Schumacher, H., Dörflinger, Y., Kuhn, C and Franke, W.W. PloS One, 9(2), e90386 (2014)   We report on the heterogeneity and diversity of lipid droplets (LDs) in early stages of adipogenesis by elucidating the cell and molecular biology of amphiphilic and cytoskeletal proteins regulating and stabilizing the generation of LDs in human adipose cells. A plethora of distinct and differently sized LDs was detected by a brief application of adipocyte differentiation medium and additional short treatment with oleic acid. Using these cells and highly specific antibodies for LD-binding proteins of the perilipin (PLIN) family, we could distinguish between endogenously derived LDs (endogenous LDs) positive for perilipin from exogenously induced LDs (exogenous LDs) positive for adipophilin, TIP47 and S3-12. Having optimized these stimulation conditions, we used early adipogenic differentiation stages to investigate small-sized LDs and concentrated on LD-protein associations with the intermediate-sized filament (IF) vimentin. This IF protein was described earlier to surround lipid globules, showing spherical, cage-like structures. Consequently - by biochemical methods, by immunofluorescence microscopy and by electron- and immunoelectron microscopy - various stages of emerging lipid globules were revealed with perilipin as linking protein between LDs and vimentin. For this LD-PLIN-Vimentin connection, a model is now proposed, suggesting an interaction of proteins via opposed charged amino acid domains respectively. In addition, multiple sheaths of smooth endoplasmic reticulum cisternae surrounding concentrically nascent LDs are shown. Based on our comprehensive localization studies we present and discuss a novel pathway for the LD formation.  

3.2136           Coenzyme Q supplementation or over-expression of the yeast Coq8 putative kinase stabilizes multi-subunit Coq polypeptide complexes in yeast coq null mutants

He, C.H., Xie, L.X., Allan, C.M., Tran, U.C.and Clarke, C.F. Biochim. Biophys. Acta, 1841, 630-644 (2014)   Coenzyme Q biosynthesis in yeast requires a multi-subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over-expression of the putative Coq8 kinase in certain coq null mutants restores steady-state levels of the sensitive Coq polypeptides and promotes the synthesis of late-stage Q-intermediates. Here we show that over-expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria by two-dimensional blue-native/SDS PAGE. The Coq4 polypeptide persists at high molecular mass with over-expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q₆ increased the steady-state levels of Coq4, Coq7, and Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late-stage Q-intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q₆ or over-expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q₆, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q-biosynthetic complex, termed the CoQ-synthome.  

3.2137           Selective Association of Outer Surface Lipoproteins with the Lipid Rafts of Borrelia burgdorferi

Toledo, A., Crowley, J.T., Coleman, J.L. et al mBio, 5(2), e00899 (2014)   Borrelia burgdorferi contains unique cholesterol-glycolipid-rich lipid rafts that are associated with lipoproteins. These complexes suggest the existence of macromolecular structures that have not been reported for prokaryotes. Outer surface lipoproteins OspA, OspB, and OspC were studied for their participation in the formation of lipid rafts. Single-gene deletion mutants with deletions of ∆ospA, ∆ospB, and ∆ospC and a spontaneous gene mutant, strain B313, which does not express OspA and OspB, were used to establish their structural roles in the lipid rafts. All mutant strains used in this study produced detergent-resistant membranes, a common characteristic of lipid rafts, and had similar lipid and protein slot blot profiles. Lipoproteins OspA and OspB but not OspC were shown to be associated with lipid rafts by transmission electron microscopy. When the ability to form lipid rafts in live B. burgdorferi spirochetes was measured by fluorescence resonance energy transfer (FRET), strain B313 showed a statistically significant lower level of segregation into ordered and disordered membrane domains than did the wild-type and the other single-deletion mutants. The transformation of a B313 strain with a shuttle plasmid containing ospA restored the phenotype shared by the wild type and the single-deletion mutants, demonstrating that OspA and OspB have redundant functions. In contrast, a transformed B313 overexpressing OspC neither rescued the FRET nor colocalized with the lipid rafts. Because these lipoproteins are expressed at different stages of the life cycle of B. burgdorferi, their selective association is likely to have an important role in the structure of prokaryotic lipid rafts and in the organism’s adaptation to changing environments.  

3.2138           Proteomic Profiling of Autophagosome Cargo in Saccharomyces cerevisiae

Suzuki, K., Nakamura, S., Morimoto, M., Fujii, K., Noda, N.N., Inagaki, F. and Ohsumi, Y. Plos One, 9(3), e91651 (2014)   Macroautophagy (autophagy) is a bulk protein-degradation system ubiquitously conserved in eukaryotic cells. During autophagy, cytoplasmic components are enclosed in a membrane compartment, called an autophagosome. The autophagosome fuses with the vacuole/lysosome and is degraded together with its cargo. Because autophagy is important for the maintenance of cellular homeostasis by degrading unwanted proteins and organelles, identification of autophagosome cargo proteins (i.e., the targets of autophagy) will aid in understanding the physiological roles of autophagy. In this study, we developed a method for monitoring intact autophagosomes ex vivo by detecting the fluorescence of GFP-fused aminopeptidase I, the best-characterized selective cargo of autophagosomes in Saccharomyces cerevisiae. This method facilitated optimization of a biochemical procedure to fractionate autophagosomes. A combination of LC-MS/MS with subsequent statistical analyses revealed a list of autophagosome cargo proteins; some of these are selectively enclosed in autophagosomes and delivered to the vacuole in an Atg11-independent manner. The methods we describe will be useful for analyzing the mechanisms and physiological significance of Atg11-independent selective autophagy.  

3.2139           GPx8 peroxidase prevents leakage of H2O2 from the endoplasmic reticulum

Ramming, T., Hansen, H.G., Nagata, K., Ellgaard, L. and Appenzeller-Herzog, C. Free Radical Biology and Medicine, 70, 106-116 (2014)   Unbalanced endoplasmic reticulum (ER) homeostasis (ER stress) leads to increased generation of reactive oxygen species (ROS). Disulfide-bond formation in the ER by Ero1 family oxidases produces hydrogen peroxide (H₂O₂) and thereby constitutes one potential source of ER-stress-induced ROS. However, we demonstrate that Ero1α-derived H₂O₂ is rapidly cleared by glutathione peroxidase (GPx) 8. In 293 cells, GPx8 and reduced/activated forms of Ero1α co-reside in the rough ER subdomain. Loss of GPx8 causes ER stress, leakage of Ero1α-derived H₂O₂ to the cytosol, and cell death. In contrast, peroxiredoxin (Prx) IV, another H₂O₂-detoxifying rough ER enzyme, does not protect from Ero1α-mediated toxicity, as is currently proposed. Only when Ero1α-catalyzed H₂O₂ production is artificially maximized can PrxIV participate in its reduction. We conclude that the peroxidase activity of the described Ero1α–GPx8 complex prevents diffusion of Ero1α-derived H₂O₂ within and out of the rough ER. Along with the induction of GPX8 in ER-stressed cells, these findings question a ubiquitous role of Ero1α as a producer of cytoplasmic ROS under ER stress.  

3.2140           Protein Kinase C Mediates Enterohemorrhagic Escherichia coli O157:H7-Induced Attaching and Effacing Lesions

Shen-Tu, G., Kim, H., Liu, M., Johnson-Henry, C. and Sherman, P.M. Infect. Immun., 82(4), 1648-1656 (2014)   Enterohemorrhagic Escherichia coli serotype O157:H7 causes outbreaks of diarrhea, hemorrhagic colitis, and the hemolytic-uremic syndrome. E. coli O157:H7 intimately attaches to epithelial cells, effaces microvilli, and recruits F-actin into pedestals to form attaching and effacing lesions. Lipid rafts serve as signal transduction platforms that mediate microbe-host interactions. The aims of this study were to determine if protein kinase C (PKC) is recruited to lipid rafts in response to E. coli O157:H7 infection and what role it plays in attaching and effacing lesion formation. HEp-2 and intestine 407 tissue culture epithelial cells were challenged with E. coli O157:H7, and cell protein extracts were then separated by buoyant density ultracentrifugation to isolate lipid rafts. Immunoblotting for PKC was performed, and localization in lipid rafts was confirmed with an anti-caveolin-1 antibody. Isoform-specific PKC small interfering RNA (siRNA) was used to determine the role of PKC in E. coli O157:H7-induced attaching and effacing lesions. In contrast to uninfected cells, PKC was recruited to lipid rafts in response to E. coli O157:H7. Metabolically active bacteria and cells with intact lipid rafts were necessary for the recruitment of PKC. PKC recruitment was independent of the intimin gene, type III secretion system, and the production of Shiga toxins. Inhibition studies, using myristoylated PKCζ pseudosubstrate, revealed that atypical PKC isoforms were activated in response to the pathogen. Pretreating cells with isoform-specific PKC siRNA showed that PKCζ plays a role in E. coli O157:H7-induced attaching and effacing lesions. We concluded that lipid rafts mediate atypical PKC signal transduction responses to E. coli O157:H7. These findings contribute further to the understanding of the complex array of microbe-eukaryotic cell interactions that occur in response to infection.  

3.2141           Hijacking of RIG-I Signaling Proteins into Virus-Induced Cytoplasmic Structures Correlates with the Inhibition of Type I Interferon Responses

Santiago, F.W., Covaleda, L.M., Sanchez-Aparicio, M.T., Silvas, J.A., Diaz-Vizarreta, A.C., patel, J.R., Popov, V., Yu, X-j., Garcia-Sastre, A. and Aguilar, P.V.

1. Virol., 88(8), 4572-4585 (2014)

  Recognition of viral pathogens by the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family results in the activation of type I interferon (IFN) responses. To avoid this response, most viruses have evolved strategies that target different essential steps in the activation of host innate immunity. In this study, we report that the nonstructural protein NSs of the newly described severe fever with thrombocytopenia syndrome virus (SFTSV) is a potent inhibitor of IFN responses. The SFTSV NSs protein was found to inhibit the activation of the beta interferon (IFN-β) promoter induced by viral infection and by a RIG-I ligand. Astonishingly, we found that SFTSV NSs interacts with and relocalizes RIG-I, the E3 ubiquitin ligase TRIM25, and TANK-binding kinase 1 (TBK1) into SFTSV NSs-induced cytoplasmic structures. Interestingly, formation of these SFTSV NSs-induced structures occurred in the absence of the Atg7 gene, a gene essential for autophagy. Furthermore, confocal microscopy studies revealed that these SFTSV NSs-induced structures colocalize with Rab5 but not with Golgi apparatus or endoplasmic reticulum markers. Altogether, the data suggest that sequestration of RIG-I signaling molecules into endosome-like structures may be the mechanism used by SFTSV to inhibit IFN responses and point toward a novel mechanism for the suppression of IFN responses.  

3.2142           Improved Functional Expression of Human Cardiac Kv1.5 Channels and Trafficking-Defective Mutants by Low Temperature Treatment

Ding, W-G., Xie, Y., Toyoda, F. and Matsuura, H. Plos One, 9(3), e92923 (2014)   We herein investigated the effect of low temperature exposure on the expression, degradation, localization and activity of human Kv1.5 (hKv1.5). In hKv1.5-expressing CHO cells, the currents were significantly increased when cultured at a reduced temperature (28°C) compared to those observed at 37°C. Western blot analysis indicated that the protein levels (both immature and mature proteins) of hKv1.5 were significantly elevated under the hypothermic condition. Treatment with a proteasome inhibitor, MG132, significantly increased the immature, but not the mature, hKv1.5 protein at 37°C, however, there were no changes in either the immature or mature hKv1.5 proteins at low temperature following MG132 exposure. These observations suggest that the enhancement of the mature hKv1.5 protein at reduced temperature may not result from the inhibition of proteolysis. Moreover, the hKv1.5 fluorescence signal in the cells increased significantly on the cell surface at 28°C versus those cultured at 37°C. Importantly, the low temperature treatment markedly shifted the subcellular distribution of the mature hKv1.5, which showed considerable overlap with the trans-Golgi component. Experiments using tunicamycin, an inhibitor of N-glycosylation, indicated that the N-glycosylation of hKv1.5 is more effective at 28°C than at 37°C. Finally, the hypothermic treatment also rescued the protein expression and currents of trafficking-defective hKv1.5 mutants. These results indicate that low temperature exposure stabilizes the protein in the cellular organelles or on the plasma membrane, and modulates its maturation and trafficking, thus enhancing the currents of hKv1.5 and its trafficking defect mutants.  

3.2143           Herp coordinates compartmentalization and recruitment of HRD1 and misfolded proteins for ERAD

Leitman, J., Shenkman, M., Gofman, Y., Shtern, O. N., Ben-Tal, N., Hendershot, L.M. and lederkremer, G.Z. Mol. Biol. Cell, 25, 1050-1060 (2014)   A functional unfolded protein response (UPR) is essential for endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded secretory proteins, reflecting the fact that some level of UPR activation must exist under normal physiological conditions. A coordinator of the UPR and ERAD processes has long been sought. We previously showed that the PKR-like, ER-localized eukaryotic translation initiation factor 2α kinase branch of the UPR is required for the recruitment of misfolded proteins and the ubiquitin ligase HRD1 to the ER-derived quality control compartment (ERQC), a staging ground for ERAD. Here we show that homocysteine-induced ER protein (Herp), a protein highly upregulated by this UPR branch, is responsible for this compartmentalization. Herp localizes to the ERQC, and our results suggest that it recruits HRD1, which targets to ERAD the substrate presented by the OS-9 lectin at the ERQC. Predicted overall structural similarity of Herp to the ubiquitin-proteasome shuttle hHR23, but including a transmembrane hairpin, suggests that Herp may function as a hub for membrane association of ERAD machinery components, a key organizer of the ERAD complex.  

3.2144           Ubiquitin-like (UBX)-domain-containing protein, UBXN2A, promotes cell death by interfering with the p53-Mortalin interactions in colon cancer cells

Sane, S., Abdullah, A., Boudreau, D.A., Autenried, R.K., Gupta, B.K., Wang, X., Wang, H., Schlenker, E.H., Zhang, D., Telleria, C., Huang, L., Chauhan, S.C. and Rezvani, K. Cell Death and Disease, 5, e1118 (2014)   Mortalin (mot-2) induces inactivation of the tumor suppressor p53’s transcriptional and apoptotic functions by cytoplasmic sequestration of p53 in select cancers. The mot-2-dependent cytoprotective function enables cancer cells to support malignant transformation. Abrogating the p53-mot-2 interaction can control or slow down the growth of cancer cells. In this study, we report the discovery of a ubiquitin-like (UBX)-domain-containing protein, UBXN2A, which binds to mot-2 and consequently inhibits the binding between mot-2 and p53. Genetic analysis showed that UBXN2A binds to mot-2’s substrate binding domain, and it partly overlaps p53’s binding site indicating UBXN2A and p53 likely bind to mot-2 competitively. By binding to mot-2, UBXN2A releases p53 from cytosolic sequestration, rescuing the tumor suppressor functions of p53. Biochemical analysis and functional assays showed that the overexpression of UBXN2A and the functional consequences of unsequestered p53 trigger p53-dependent apoptosis. Cells expressing shRNA against UBXN2A showed the opposite effect of that seen with UBXN2A overexpression. The expression of UBXN2A and its apoptotic effects were not observed in normal colonic epithelial cells and p53−/− colon cancer cells. Finally, significant reduction in tumor volume in a xenograft mouse model in response to UBXN2A expression was verified in vivo. Our results introduce UBXN2A as a home defense response protein, which can reconstitute inactive p53-dependent apoptotic pathways. Inhibition of mot-2-p53 interaction by UBXN2A is an attractive therapeutic strategy in mot-2-elevated tumors.  

3.2145           Mutation of SLC35D3 Causes Metabolic Syndrome by Impairing Dopamine Signaling in Striatal D1 Neurons

Zhang, Z., Hao, C-J., Li, C-G., Zang, D-J., Zhao, J., Li, X-N., Wei, A-H., Wei, Z-B., Yang, L., He, X., Zhen, X-C., Gao, X., Speakman, J.R. and Li, W. PloS Genetics, 10(2), e1004124 (2014)   Obesity is one of the largest health problems facing the world today. Although twin and family studies suggest about two-thirds of obesity is caused by genetic factors, only a small fraction of this variance has been unraveled. There are still large numbers of genes to be identified that cause variations in body fatness and the associated diseases encompassed in the metabolic syndrome (MetS). A locus near a sequence tagged site (STS) marker D6S1009 has been linked to obesity or body mass index (BMI). However, its genetic entity is unknown. D6S1009 is located in the intergenic region between SLC35D3 and NHEG1. Here we report that the ros mutant mice harboring a recessive mutation in the Slc35d3 gene show obesity and MetS and reduced membrane dopamine receptor D1 (D1R) with impaired dopamine signaling in striatal neurons. SLC35D3 is localized to both endoplasmic reticulum (ER) and early endosomes and interacts with D1R. In ros striatal D1 neurons, lack of SLC35D3 causes the accumulation of D1R on the ER to impair its ER exit. The MetS phenotype is reversible by the administration of D1R agonist to the ros mutant. In addition, we identified two mutations in the SLC35D3 gene in patients with MetS, which alter the subcellular localization of SLC35D3. Our results suggest that the SLC35D3 gene, close to the D6S1009 locus, is a candidate gene for MetS, which is involved in metabolic control in the central nervous system by regulating dopamine signaling.  

3.2146           Human cytomegalovirus pUL37x1-induced calcium flux activates PKCα, inducing altered cell shape and accumulation of cytoplasmic vesicles

Sharon-Friling, R. and Shenk, T. PNAS, 111, E1140-E1148 (2014)   The human cytomegalovirus immediate-early protein pUL37x1 induces the release of Ca²⁺ stores from the endoplasmic reticulum into the cytosol. This release causes reorganization of the cellular actin cytoskeleton with concomitant cell rounding. Here we demonstrate that pUL37x1 activates Ca²⁺-dependent protein kinase Cα (PKCα). Both PKCα and Rho-associated protein kinases are required for actin reorganization and cell rounding; however, only PKCα is required for the efficient production of virus progeny, arguing that HCMV depends on the kinase for a second function. PKCα activation is also needed for the production of large (1–5 μm) cytoplasmic vesicles late after infection. The production of these vesicles is blocked by inhibition of fatty acid or phosphatidylinositol-3-phosphate biosynthesis, and the failure to produce vesicles is correlated with substantially reduced production of enveloped virus capsids. These results connect earlier work identifying a requirement for lipid synthesis with specific morphological changes, and support the argument that the PKCα-induced large vesicles are either required for the efficient production of mature virus particles or serve as a marker for the process.    

3.2147           RAB26 coordinates lysosome traffic and mitochondrial localization

Jin, R.U. and Mills, J.C.

1. Cell Sci., 127, 1018-1032 (2014)

  As they mature, professional secretory cells like pancreatic acinar and gastric chief cells induce the transcription factor MIST1 (also known as BHLHA15) to substantially scale up production of large secretory granules in a process that involves expansion of apical cytoplasm and redistribution of lysosomes and mitochondria. How a scaling factor like MIST1 rearranges cellular architecture simply by regulating expression levels of its transcriptional targets is unknown. RAB26 is a MIST1 target whose role in MIST1-mediated secretory cell maturation is also unknown. Here, we confirm that RAB26 expression, unlike most Rabs which are ubiquitously expressed, is tissue specific and largely confined to MIST1-expressing secretory tissues. Surprisingly, functional studies showed that RAB26 predominantly associated with LAMP1/cathepsin D lysosomes and not directly with secretory granules. Moreover, increasing RAB26 expression – by inducing differentiation of zymogen-secreting cells or by direct transfection – caused lysosomes to coalesce in a central, perinuclear region. Lysosome clustering in turn caused redistribution of mitochondria into distinct subcellular neighborhoods. The data elucidate a novel function for RAB26 and suggest a mechanism for how cells could increase transcription of key effectors to reorganize subcellular compartments during differentiation.  

3.2148           Phospholipid flippase ATP8A2 is required for normal visual and auditory function and photoreceptor and spiral ganglion cell survival

Coleman, J.A., Zhu, X., Djajadi, H.R., Molday, L.L., Smith, R.S., Libby, R.T., John, S.W.M. and Molday, R.S.

1. Cell Sci., 127, 1138-1149 (2014)

  ATP8A2 is a P₄-ATPase that is highly expressed in the retina, brain, spinal cord and testes. In the retina, ATP8A2 is localized in photoreceptors where it uses ATP to transport phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the exoplasmic to the cytoplasmic leaflet of membranes. Although mutations in ATP8A2 have been reported to cause mental retardation in humans and degeneration of spinal motor neurons in mice, the role of ATP8A2 in sensory systems has not been investigated. We have analyzed the retina and cochlea of ATP8A2-deficient mice to determine the role of ATP8A2 in visual and auditory systems. ATP8A2-deficient mice have shortened photoreceptor outer segments, a reduction in photoresponses and decreased photoreceptor viability. The ultrastructure and phagocytosis of the photoreceptor outer segment appeared normal, but the PS and PE compositions were altered and the rhodopsin content was decreased. The auditory brainstem response threshold was significantly higher and degeneration of spiral ganglion cells was apparent. Our studies indicate that ATP8A2 plays a crucial role in photoreceptor and spiral ganglion cell function and survival by maintaining phospholipid composition and contributing to vesicle trafficking.  

3.2149           Slit Diaphragm Protein Neph1 and Its Signaling: A NOVEL THERAPEUTIC TARGET FOR PROTECTION OF PODOCYTES AGAINST GLOMERULAR INJURY

Arif, E., Rathore, Y.S., Kumari, b., Ashish, F., Wong, H.N., Holzman, L.B. and Nihalani, D.

1. Biol. Chem., 289(14), 9502-9518 (2014)

  Podocytes are specialized epithelial cells that are critical components of the glomerular filtration barrier, and their dysfunction leads to proteinuria and renal failure. Therefore, preserving podocyte function is therapeutically significant. In this study, we identified Neph1 signaling as a therapeutic target that upon inhibition prevented podocyte damage from a glomerular injury-inducing agent puromycin aminonucleoside (PAN). To specifically inhibit Neph1 signaling, we used a protein transduction approach, where the cytoplasmic domain of Neph1 (Neph1CD) tagged with a protein transduction domain trans-activator of transcription was transduced in cultured podocytes prior to treatment with PAN. The PAN-induced Neph1 phosphorylation was significantly reduced in Neph1CD-transduced cells; in addition, these cells were resistant to PAN-induced cytoskeletal damage. The biochemical analysis using subfractionation studies showed that unlike control cells Neph1 was retained in the lipid raft fractions in the transduced cells following treatment with PAN, indicating that transduction of Neph1CD in podocytes prevented PAN-induced mislocalization of Neph1. In accordance, the immunofluorescence analysis further suggested that Neph1CD-transduced cells had increased ability to retain endogenous Neph1 at the membrane in response to PAN-induced injury. Similar results were obtained when angiotensin was used as an injury-inducing agent. Consistent with these observations, maintaining high levels of Neph1 at the membrane using a podocyte cell line overexpressing chimeric Neph1 increased the ability of podocytes to resist PAN-induced injury and PAN-induced albumin leakage. Using a zebrafish in vivo PAN and adriamycin injury models, we further demonstrated the ability of transduced Neph1CD to preserve glomerular function. Collectively, these results support the conclusion that inhibiting Neph1 signaling is therapeutically significant in preventing podocyte damage from glomerular injury.  

3.2150           Mfge8 promotes obesity by mediating the uptake of dietary fats and serum fatty acids

Khalifeh-Soltani, A., McKleroy,W., Sakuma, S., Cheung, Y.Y., Tharp, K., Qiu, Y., Turner, S.M., Chawla, A., Stahl, A. and Atabai, K. Nature Med., 20(2), 175-183 (2014)   Fatty acids are integral mediators of energy storage, membrane formation and cell signaling. The pathways that orchestrate uptake of fatty acids remain incompletely understood. Expression of the integrin ligand Mfge8 is increased in human obesity and in mice on a high-fat diet, but its role in obesity is unknown. We show here that Mfge8 promotes the absorption of dietary triglycerides and the cellular uptake of fatty acid and that Mfge8-deficient (Mfge8^−/−) mice are protected from diet-induced obesity, steatohepatitis and insulin resistance. Mechanistically, we found that Mfge8 coordinates fatty acid uptake through α_vβ₃ integrin– and α_vβ₅ integrin–dependent phosphorylation of Akt by phosphatidylinositide-3 kinase and mTOR complex 2, leading to translocation of Cd36 and Fatp1 from cytoplasmic vesicles to the cell surface. Collectively, our results imply a role for Mfge8 in regulating the absorption and storage of dietary fats, as well as in the development of obesity and its complications.  

3.2151           Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT)

Amin, N.M., Greco, T.M., Kuchenbrod, L.M., Rigney, M.M., Chung, M-i., Wallingfor, J.B., Cristea, I.M. and Conion, F.L. Development, 141, 962-973 (2014)   The proper dissection of the molecular mechanisms governing the specification and differentiation of specific cell types requires isolation of pure cell populations from heterogeneous tissues and whole organisms. Here, we describe a method for purification of nuclei from defined cell or tissue types in vertebrate embryos using INTACT (isolation of nuclei tagged in specific cell types). This method, previously developed in plants, flies and worms, utilizes in vivo tagging of the nuclear envelope with biotin and the subsequent affinity purification of the labeled nuclei. In this study we successfully purified nuclei of cardiac and skeletal muscle from Xenopus using this strategy. We went on to demonstrate the utility of this approach by coupling the INTACT approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic methodologies to profile proteins expressed in the nuclei of developing hearts. From these studies we have identified the Xenopus orthologs of 12 human proteins encoded by genes, which when mutated in human lead to congenital heart disease. Thus, by combining these technologies we are able to identify tissue-specific proteins that are expressed and required for normal vertebrate organ development.  

3.2152           The FTLD risk factor TMEM106B and MAP6 control dendritic trafficking of lysosomes

Schwenk, B.M., Lang, C.M., Hogl, S., Tahirovic, S., Orozco, D., Rentzsch, K., Lichtenthaler, S.F., Hoogenraad, C.C., capell, A., Haass, C. and edhauer, D. EMBO J., 33(5), 450-467 (2014)   TMEM106B is a major risk factor for frontotemporal lobar degeneration with TDP-43 pathology. TMEM106B localizes to lysosomes, but its function remains unclear. We show that TMEM106B knockdown in primary neurons affects lysosomal trafficking and blunts dendritic arborization. We identify microtubule-associated protein 6 (MAP6) as novel interacting protein for TMEM106B. MAP6 over-expression inhibits dendritic branching similar to TMEM106B knockdown. MAP6 knockdown fully rescues the dendritic phenotype of TMEM106B knockdown, supporting a functional interaction between TMEM106B and MAP6. Live imaging reveals that TMEM106B knockdown and MAP6 overexpression strongly increase retrograde transport of lysosomes in dendrites. Downregulation of MAP6 in TMEM106B knockdown neurons restores the balance of anterograde and retrograde lysosomal transport and thereby prevents loss of dendrites. To strengthen the link, we enhanced anterograde lysosomal transport by expressing dominant-negative Rab7-interacting lysosomal protein (RILP), which also rescues the dendrite loss in TMEM106B knockdown neurons. Thus, TMEM106B/MAP6 interaction is crucial for controlling dendritic trafficking of lysosomes, presumably by acting as a molecular brake for retrograde transport. Lysosomal misrouting may promote neurodegeneration in patients with TMEM106B risk variants.  

3.2153           Agonist-Induced GPCR Shedding from the Ciliary Surface Is Dependent on ESCRT-III and VPS4

Soetedjo, L and Jin, H. Current Biology, 24(5), 509-518 (2014)   Background Membrane trafficking of G protein-coupled receptors (GPCRs) is crucial for temporal and spatial control of cell-surface GPCR signaling. Receptor internalization is a well-documented method cells use for regulating a wide variety of GPCRs following their exposure to agonists. Results We report that, upon agonist stimulation, a GPCR called vasoactive intestinal peptide receptor 2 (VPAC2) is shed, rather than being internalized, in vitro and in vivo, from the membrane of primary cilia—solitary hair-like organelles that project from the cell surface. VPAC2 is released into the extracellular milieu in the form of ciliary ectosomes that are devoid of exosome markers. The agonist-induced VPAC2 shedding is selective, as shown by the fact that other ciliary membrane proteins including two ciliary GPCRs are not shed with VPAC2. VPAC2 ectosome shedding is dependent on several components of endosomal sorting complexes required for transport (ESCRT), including a subset of ESCRT-III, VPS4, and LIP5. Agonist-stimulated VPAC2 is important for ciliary-ectosome generation because it allows VPS4 and LIP5 to transiently accumulate in primary cilia. Shedding of VPAC2 from the ciliary surface results in termination of intracellular VPAC2 signaling. Conclusions Agonist-induced GPCR shedding from the ciliary surface may represent an additional mode of GPCR trafficking and signal regulation.  

3.2154           Preferential secretion of inducible HSP70 by vitiligo melanocytes under stress

Mosenson, J.A., Flood, K., Klarquist, J., Eby, J.M., Koshoffer, A., Boissy, R.E., Overbeck, A., Tung, R.C. and Le poole, L.C. Pigment Cell & Melanoma Res., 27(2), 209-220 (2014)   Inducible HSP70 (HSP70i) chaperones peptides from stressed cells, protecting them from apoptosis. Upon extracellular release, HSP70i serves an adjuvant function, enhancing immune responses to bound peptides. We questioned whether HSP70i differentially protects control and vitiligo melanocytes from stress and subsequent immune responses. We compared expression of HSP70i in skin samples, evaluated the viability of primary vitiligo and control melanocytes exposed to bleaching phenols, and measured secreted HSP70i. We determined whether HSP70i traffics to melanosomes to contact immunogenic proteins by cell fractionation, western blotting, electron microscopy, and confocal microscopy. Viability of vitiligo and control melanocytes was equally affected under stress. However, vitiligo melanocytes secreted increased amounts of HSP70i in response to MBEH, corroborating with aberrant HSP70i expression in patient skin. Intracellular HSP70i colocalized with melanosomes, and more so in response to MBEH in vitiligo melanocytes. Thus, whereas either agent is cytotoxic to melanocytes, MBEH preferentially induces immune responses to melanocytes.  

3.2155           Soybean Proteomics

Hossain, Z. and Komatsu, S. Methods in Mol. Biol., 1072, 315-331 (2014)   Soybean, the world’s most widely grown seed legume, is an important global source of vegetable oil and protein. Though, complete draft genome sequence of soybean is now available, but functional genomics studies remain in their infancy, as this agricultural legume species exhibits genetic constrains like genome duplications and self-incompatibilities. The techniques of proteomics provide much powerful tool for functional analysis of soybean. In the present review, an attempt has been made to summarize all significant contributions in the field of soybean proteomics. Special emphasis is given to subcellular proteomics in response to abiotic stresses for better understanding molecular basis of acquisition of stress tolerance mechanism. Detailed protocols of protein extraction, solubilization, fractionation of subcellular organelle, and proteins identification are explained for soybean proteomics. All this information would not only enrich us in understanding the plants response to environmental stressors but would also enable us to design genetically engineered stress tolerant soybean.    

3.2156           Fluorescence-activated sorting of fixed nuclei: a general method for studying nuclei from specific cell populations that preserves post-translational modifications

Marion-Poll, L., Montalban, E., Munier, A., herve, D. and Giralult, J-A. Eur. J. Neurosci., 39, 1234-1244 (2014)   Long-lasting brain alterations that underlie learning and memory are triggered by synaptic activity. How activity can exert long-lasting effects on neurons is a major question in neuroscience. Signalling pathways from cytoplasm to nucleus and the resulting changes in transcription and epigenetic modifications are particularly relevant in this context. However, a major difficulty in their study comes from the cellular heterogeneity of brain tissue. A promising approach is to directly purify identified nuclei. Using mouse striatum we have developed a rapid and efficient method for isolating cell type-specific nuclei from fixed adult brain (fluorescence-activated sorting of fixed nuclei; FAST-FIN). Animals are quickly perfused with a formaldehyde fixative that stops enzymatic reactions and maintains the tissue in the state it was at the time of death, including nuclear localisation of soluble proteins such as GFP and differences in nuclear size between cell types. Tissue is subsequently dissociated with a Dounce homogeniser and nuclei prepared by centrifugation in an iodixanol density gradient. The purified fixed nuclei can then be immunostained with specific antibodies and analysed or sorted by flow cytometry. Simple criteria allow distinction of neurons and non-neuronal cells. Immunolabelling and transgenic mice that express fluorescent proteins can be used to identify specific cell populations, and the nuclei from these populations can be efficiently isolated, even rare cell types such as parvalbumin-expressing interneurons. FAST-FIN allows the preservation and study of dynamic and labile post-translational protein modifications. It should be applicable to other tissues and species, and allow study of DNA and its modifications.  

3.2157           Membrane vesicles of Clostridium perfringens type A strains induce innate and adaptive immunity

Jiang, Y., Kong, Q., Roland, K.L. and Curtiss III, R. Int. J. Med. Microbiol., 304, 431-443 (2014)   Vesicle shedding from bacteria is a universal process in most Gram-negative bacteria and a few Gram-positive bacteria. In this report, we isolate extracellular membrane vesicles (MVs) from the supernatants of Gram-positive pathogen Clostridium perfringens (C. perfringens). We demonstrated vesicle production in a variety of virulent and nonvirulent type A strains. MVs did not contain alpha-toxin and NetB toxin demonstrated by negative reaction to specific antibody and absence of specific proteins identified by LC–MS/MS. C. perfringens MVs contained DNA components such as 16S ribosomal RNA gene (16S rRNA), alpha-toxin gene (plc) and the perfringolysin O gene (pfoA) demonstrated by PCR. We also identified a total of 431 proteins in vesicles by 1-D gel separation and LC–MS/MS analysis. In vitro studies demonstrated that vesicles could be internalized into murine macrophage RAW264.7 cells without direct cytotoxicity effects, causing release of inflammation cytokines including granulocyte colony stimulating factor (G-CSF), tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1), which could also be detected in mice injected with MVs through intraperitoneal (i.p.) route. Mice immunized with C. perfringens MVs produced high titer IgG, especially IgG1, antibodies against C. perfringens membrane proteins. However, this kind of antibody could not provide protection in mice following challenge, though it could slightly postpone the time of death. Our results indicate that release of MVs from C. perfringens could provide a previously unknown mechanism to induce release of inflammatory cytokines, especially TNF-α, these findings may contribute to a better understanding of the pathogenesis of C. perfringens infection.  

3.2158           Characterization of protective extracellular membrane-derived vesicles produced by Streptococcus pneumonia

Olaya-Abril, A., Prados-Rosales, R., McConnell, M.J., martin-Pena, R., Gonzalez-Reyes, J.A., Jimenez-Munguia, I., Gomez-Gascon, L., Fernandez, J., Luque-garcia, J.L., Garcia-Lidon, C., Estevez, H., pachon, J., Obando, I., Casadevall, A., Profski. L-A. and Rodriguez-Ortega, M.J.

1. Proteomics, 106, 46-60 (2014)

  Extracellular vesicles are produced by many pathogenic microorganisms and have varied functions that include secretion and release of microbial factors, which contribute to virulence. Very little is known about vesicle production by Gram-positive bacteria, as well as their biogenesis and release mechanisms. In this work, we demonstrate the active production of vesicles by Streptococcus pneumoniae from the plasma membrane, rather than being a product from cell lysis. We biochemically characterized them by proteomics and fatty acid analysis, showing that these vesicles and the plasma membrane resemble in essential aspects, but have some differences: vesicles are more enriched in lipoproteins and short-chain fatty acids. We also demonstrate that these vesicles act as carriers of surface proteins and virulence factors. They are also highly immunoreactive against human sera and induce immune responses that protect against infection. Overall, this work provides insights into the biology of this important Gram-positive human pathogen and the role of extracellular vesicles in clinical applications.  

3.2159           Angiogenin interacts with the plasminogen activation system at the cell surface of breast cancer cells to regulate plasmin formation and cell migration

Dutta, S., Bandyopadhyay, C., Bottero, V., Veettil, M.V., Wilson, L., Pins, M.R., Johnson, K.E., Warshall, C. and Chandran, B. Mol. Oncol., 8, 483-507 (2014)   Angiogenin (ANG), a 14-kDa pro-angiogenic secreted protein, has been shown to play a role in cell migration and tumor invasion, which involve proteolytic cleavage of plasminogen to generate plasmin. However, the mechanism by which ANG regulates plasmin formation and cell migration was not known. Our studies here detected elevated levels of secreted and cell surface-bound ANG in highly invasive metastatic breast cancer cells. ANG was also detected at very high levels in the tumor cells in infiltrating ductal carcinomas. By immunofluorescence and immunoprecipitation analysis, ANG was detected at the leading edges of the cell surfaces where it colocalized and interacted with members of the plasminogen activation system (PAS) such as annexin A2 (A2), calpactin (S100-A10) and urokinase plasminogen activator receptor (uPAR). Analysis of lipid raft (LR) and non-lipid raft (NLR) regions of the cell membranes showed the predominance of ANG, A2 and S100-A10 in the LR regions. In contrast, uPAR was detected predominantly in the NLR fractions, suggesting that ANG interacts with uPAR at the junctions of LR and NLR regions. ANG knockdown in T47D and MDA-MB-231 breast cancer cell lines did not affect the cellular expression of A2, S100-A10 and uPAR but decreased cell migration and plasmin formation. Neutralization of ANG with monoclonal antibodies similarly decreased the migration of MDA-MB-231 cells. In the presence of ANG, uPAR was observed to interact with uPA, which is necessary for plasmin formation. Conversely, in the absence of ANG, uPAR did not interact with uPA and FAK and Src kinases were observed to be dephosphorylated. Exogenous addition of recombinant ANG to ANG knocked down MDA-MB-231 cells restored FAK phosphorylation, uPAR interactions with uPA, plasmin formation as well as migration of these cells. Taken together, our results identified a novel role for ANG as a member of the uPAR interactome that facilitates the interaction of uPAR with uPA, leading to plasmin formation and cell migration necessary for tumor invasion and metastasis of breast cancer cells.  

3.2160           Positional Assembly of Enzymes on Bacterial Outer Membrane Vesicles for Cascade Reactions

Park, M., Sun, Q., Liu, F., DeLisa, M.P. and Chen, W. PloS One, 9(5), e97103 (2014)   The systematic organization of enzymes is a key feature for the efficient operation of cascade reactions in nature. Here, we demonstrate a facile method to create nanoscale enzyme cascades by using engineered bacterial outer membrane vesicles (OMVs) that are spheroid nanoparticles (roughly 50 nm in diameter) produced by Gram-negative bacteria during all phases of growth. By taking advantage of the fact that OMVs naturally contain proteins found in the outer cell membrane, we displayed a trivalent protein scaffold containing three divergent cohesin domains for the position-specific presentation of a three-enzyme cascade on OMVs through a truncated ice nucleation protein anchoring motif (INP). The positional assembly of three enzymes for cellulose hydrolysis was demonstrated. The enzyme-decorated OMVs provided synergistic cellulose hydrolysis resulting in 23-fold enhancement in glucose production than free enzymes.  

3.2161           Heterologous Src Homology 4 Domains Support Membrane Anchoring and Biological Activity of HIV-1 Nef

Geist, M.M., Pan, X., Bender, S., Bartenschlager, R., Nickel, W. and Fackler, O.T.

1. Biol. Chem., 289(20), 14030-14044 (2014)

  The HIV-1 pathogenicity factor Nef enhances viral replication by modulation of multiple host cell transport and signaling pathways. Nef associates with membranes via an N-terminal Src homology 4 (SH4) domain, and membrane association is believed to be essential for its biological functions. At which subcellular site(s) Nef exerts its different functions and how kinetics of membrane interactions contribute to its biological activity are unknown. To address how specific characteristics of Nef membrane association affect its biological properties, the SH4 domain of Nef was replaced by heterologous membrane targeting domains. The use of a panel of heterologous SH4 domains resulted in chimeric Nef proteins with distinct steady state subcellular localization, membrane association efficiency, and anterograde transport routes. Irrespective of these modifications, cardinal Nef functions affecting host cell vesicular transport and actin dynamics were fully preserved. In contrast, stable targeting of Nef to the surface of mitochondria, peroxisomes, or the Golgi apparatus, and thus prevention of plasma membrane delivery, caused potent and broad loss of Nef activity. These results support the concept that Nef adopts its active conformation in the membrane-associated state but exclude that membrane-associated Nef simply acts by recruiting soluble factors independently of its local microenvironment. Rather than its steady state subcellular localization or membrane affinity, the ability to undergo dynamic anterograde and internalization cycles appear to determine Nef function. These results reveal that functional membrane interactions of Nef underlie critical spatiotemporal regulation and suggest that delivery to distinct subcellular sites via such transport cycles provides the basis for the multifunctionality of Nef.  

3.2162           Plac8 Links Oncogenic Mutations to Regulation of Autophagy and Is Critical to Pancreatic Cancer Progression

Kinsey, C., Balakrishnan, V., O’Dell, M.R., Huang, J.L., Newman, L., Whitney-Miller, C.L., hezel, A.F. and Land, H. Cell Reports, 7, 1143-1155 (2014)   Mutations in p53 and RAS potently cooperate in oncogenic transformation, and correspondingly, these genetic alterations frequently coexist in pancreatic ductal adenocarcinoma (PDA) and other human cancers. Previously, we identified a set of genes synergistically activated by combined RAS and p53 mutations as frequent downstream mediators of tumorigenesis. Here, we show that the synergistically activated gene Plac8 is critical for pancreatic cancer growth. Silencing of Plac8 in cell lines suppresses tumor formation by blocking autophagy, a process essential for maintaining metabolic homeostasis in PDA, and genetic inactivation in an engineered mouse model inhibits PDA progression. We show that Plac8 is a critical regulator of the autophagic machinery, localizing to the lysosomal compartment and facilitating lysosome-autophagosome fusion. Plac8 thus provides a mechanistic link between primary oncogenic mutations and the induction of autophagy, a central mechanism of metabolic reprogramming, during PDA progression.  

3.2163           Adiponectin is partially associated with exosomes in mouse serum

Phoonsawat, W., Aoki-Yoshida, A., Tsuruta, T. and Sonoyama, K. Biochem. Biophys. Res. Comm., 448, 261-266 (2014)   Exosomes are membrane vesicles 30–120 nm in diameter that are released by many cell types and carry a cargo of proteins, lipids, mRNA, and microRNA. Cultured adipocytes reportedly release exosomes that may play a role in cell-to-cell communication during the development of metabolic diseases. However, the characteristics and function of exosomes released from adipocytes in vivo remain to be elucidated. Clearly, adipocyte-derived exosomes could exist in the circulation and may be associated with adipocyte-specific proteins such as adipocytokines. We isolated exosomes from serum of mice by differential centrifugation and analyzed adiponectin, leptin, and resistin in the exosome fraction. Western blotting detected adiponectin but no leptin and only trace amounts of resistin in the exosome fraction. The adiponectin signal in the exosome fraction was decreased by proteinase K treatment and completely quenched by a combination of proteinase K and Triton X-100. Quantitative ELISA showed that the exosome fraction contains considerable amounts of adiponectin, but not leptin or resistin. The concentration of adiponectin in the serum and the ratio of adiponectin to total protein in the exosome fraction were lower in obese mice than in lean mice. These results suggest that a portion of adiponectin exists as a transmembrane protein in the exosomes in mouse serum. We propose adiponectin as a marker of exosomes released from adipocytes in vivo.  

3.2164           Peroxisomal membrane channel Pxmp2 in the mammary fat pad is essential for stromal lipid homeostasis and for development of mammary gland epithelium in mice

Vapola, M.H., Rokka, A., Sormunen, R.T., Alhonen, L., Schmitz, W., Conzelmann, E., Wärri, A., Grunau, S., Antonenkov, V.D. and Hiltunen, J.K. Developmental Biol., 391, 66-80 (2014)   To understand the functional role of the peroxisomal membrane channel Pxmp2, mice with a targeted disruption of the Pxmp2 gene were generated. These mice were viable, grew and bred normally. However, Pxmp2−/− female mice were unable to nurse their pups. Lactating mammary gland epithelium displayed secretory lipid droplets and milk proteins, but the size of the ductal system was greatly reduced. Examination of mammary gland development revealed that retarded mammary ductal outgrowth was due to reduced proliferation of epithelial cells during puberty. Transplantation experiments established the Pxmp2−/− mammary stroma as a tissue responsible for suppression of epithelial growth. Morphological and biochemical examination confirmed the presence of peroxisomes in the mammary fat pad adipocytes, and functional Pxmp2 was detected in the stroma of wild-type mammary glands. Deletion of Pxmp2 led to an elevation in the expression of peroxisomal proteins in the mammary fat pad but not in liver or kidney of transgenic mice. Lipidomics of Pxmp2−/−mammary fat pad showed a decrease in the content of myristic acid (C₁₄), a principal substrate for protein myristoylation and a potential peroxisomal β-oxidation product. Analysis of complex lipids revealed a reduced concentration of a variety of diacylglycerols and phospholipids containing mostly polyunsaturated fatty acids that may be caused by activation of lipid peroxidation. However, an antioxidant-containing diet did not stimulate mammary epithelial proliferation in Pxmp2−/− mice. The results point to disturbances of lipid metabolism in the mammary fat pad that in turn may result in abnormal epithelial growth. The work reveals impaired mammary gland development as a new category of peroxisomal disorders.  

3.2165           Conformational targeting of intracellular Aβ oligomers demonstrates their pathological oligomerization inside the endoplasmic reticulum

Meli, G., lecci, A., Manca, A., Krako, N., Albertini, V., Benussi, L., Ghidoni, R. and Cattaneo, A. Nature Comm., 5:3867 (2014)   Aβ oligomers (AβOs) are crucially involved in Alzheimer’s Disease (AD). However, the lack of selective approaches for targeting these polymorphic Aβ assemblies represents a major hurdle in understanding their biosynthesis, traffic and actions in living cells. Here, we established a subcellularly localized conformational-selective interference (CSI) approach, based on the expression of a recombinant antibody fragment against AβOs in the endoplasmic reticulum (ER). By CSI, we can control extra- and intracellular pools of AβOs produced in an AD-relevant cell model, without interfering with the maturation and processing of the Aβ precursor protein. The anti-AβOs intrabody selectively intercepts critical AβO conformers in the ER, modulating their assembly and controlling their actions in pathways of cellular homeostasis and synaptic signalling. Our results demonstrate that intracellular Aβ undergoes pathological oligomerization through critical conformations formed inside the ER. This establishes intracellular AβOs as key targets for AD treatment and presents CSI as a potential targeting strategy.  

3.2166           hnRNP L and NF90 Interact with Hepatitis C Virus 5'-Terminal Untranslated RNA and Promote Efficient Replication

Li, Y., Masaki, T., Shimakami, T and Lemon, S.M.

1. Virol., 88(13), 7199-7209 (2014)

  The 5′-terminal sequence of the hepatitis C virus (HCV) positive-strand RNA genome is essential for viral replication. Critical host factors, including a miR-122/Ago2 complex and poly(rC)-binding protein 2 (PCBP2), associate with this RNA segment. We used a biotinylated RNA pulldown approach to isolate host factors binding to the HCV 5′ terminal 47 nucleotides and, in addition to Ago2 and PCBP2, identified several novel proteins, including IGF2BP1, hnRNP L, DHX9, ADAR1, and NF90 (ILF3). PCBP2, IGF2BP1, and hnRNP L bound single-stranded RNA, while DHX9, ADAR1, and NF90 bound a cognate double-stranded RNA bait. PCBP2, IGF2BP1, and hnRNP L binding were blocked by preannealing the single-stranded RNA bait with miR-122, indicating that they bind the RNA in competition with miR-122. However, IGF2BP1 binding was also inhibited by high concentrations of heparin, suggesting that it bound the bait nonspecifically. Among these proteins, small interfering RNA-mediated depletion of hnRNP L and NF90 significantly impaired viral replication and reduced infectious virus yields without substantially affecting HCV internal ribosome entry site-mediated translation. hnRNP L and NF90 were found to associate with HCV RNA in infected cells and to coimmunoprecipitate with NS5A in an RNA-dependent manner. Both also associate with detergent-resistant membranes where viral replication complexes reside. We conclude that hnRNP and NF90 are important host factors for HCV replication, at least in cultured cells, and may be present in the replication complex.  

3.2167           Involvement of Hepatitis C Virus NS5A Hyperphosphorylation Mediated by Casein Kinase I-α in Infectious Virus Production

Masaki, T., Matsunaga, S., Takahashi, H., Nakashima, K., Kimura, Y., Ito, M., Matsuda, M., Murayama, A., Kato, T., Hirano, H., Endo, Y., Lemon, S.M., Wakita, t., Sawasaki, T. and Suzuki, T.

1. Virol.,88(13), 7541-7555 (2014)

  Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) possesses multiple functions in the viral life cycle. NS5A is a phosphoprotein that exists in hyperphosphorylated and basally phosphorylated forms. Although the phosphorylation status of NS5A is considered to have a significant impact on its function, the mechanistic details regulating NS5A phosphorylation, as well as its exact roles in the HCV life cycle, are still poorly understood. In this study, we screened 404 human protein kinases via in vitro binding and phosphorylation assays, followed by RNA interference-mediated gene silencing in an HCV cell culture system. Casein kinase I-α (CKI-α) was identified as an NS5A-associated kinase involved in NS5A hyperphosphorylation and infectious virus production. Subcellular fractionation and immunofluorescence confocal microscopy analyses showed that CKI-α-mediated hyperphosphorylation of NS5A contributes to the recruitment of NS5A to low-density membrane structures around lipid droplets (LDs) and facilitates its interaction with core protein and the viral assembly. Phospho-proteomic analysis of NS5A with or without CKI-α depletion identified peptide fragments that corresponded to the region located within the low-complexity sequence I, which is important for CKI-α-mediated NS5A hyperphosphorylation. This region contains eight serine residues that are highly conserved among HCV isolates, and subsequent mutagenesis analysis demonstrated that serine residues at amino acids 225 and 232 in NS5A (genotype 2a) may be involved in NS5A hyperphosphorylation and hyperphosphorylation-dependent regulation of virion production. These findings provide insight concerning the functional role of NS5A phosphorylation as a regulatory switch that modulates its multiple functions in the HCV life cycle.  

3.2168           The fronds tonoplast quantitative proteomic analysis in arsenic hyperaccumulator Pteris vittata L.

Shen, H., He, Z., Yan, H., Xing, Z., Chen, Y., Xu, W., Xu, W. and Ma, M.

1. Proteomics, 105, 46-57 (2014)

  Pteris vittata, the first known arsenic hyperaccumulating plant, can accumulate very high concentration arsenic in its aboveground tissues, while low in roots. Previous studies have suggested that arsenic vacuole compartmentalization may play an important role in the arsenic-hyperaccumulation in P. vittata, but the mechanism(s) of arsenic transport to vacuole are largely unknown. We obtained tonoplast isolated from fronds of P. vittata sporophyte grown under minus and 1 mM arsenate for 3 weeks by iodixanol step gradient centrifugation method, and then used TMPP protein labeling technology followed by liquid chromatography—a linear ion trap-Orbitrap hybrid mass spectrometer analysis for the quantitative detection of proteins. And we designed and used an “artificial” database for database searching. In total, 56 tonoplast proteins were identified; more than 70% of them were transport proteins. Under arsenate treatment, one TDT transporter protein, a member of the TerC family and a PDR-like protein were upregulated differentially. While V-ATPase subunits c, E, and G, and V-PPase, were downregulated. Additionally, the identified tonoplast proteins in our present study provide an informative basis for arsenic carriers or channels and help to clarify the regulation of tonoplast arsenic transport processes in P. vittata.  

3.2169           Intracellular gene delivery is dependent on the type of non-viral carrier and defined by the cell surface glycosaminoglycans

Normani, A., Hyvönen, Z., Pulkkinen, E., Hiekkala, M. and Ruponen, M.

1. Controlled Release, 187, 59-65 (2014)

  Intracellular limiting steps and molecules involved in internalization and intracellular routing of non-viral gene delivery systems are still poorly understood. In this study, the intracellular kinetics of three different gene delivery systems calcium phosphate precipitates (CaP), polyethyleneimine (PEI) and N-[1-(2,3-dioleyl)propyl]-N,N,N-trimethylammonium chloride (DOTAP)) were quantified at cellular, nuclear, transcriptional and translational levels by using qRT-PCR. Additionally, a role of cell surface glycosaminoglycans (GAGs) was evaluated by performing the aforementioned studies in cells devoid of GAGs (pgsB-618) and cells lacking heparan sulphate (HS). The obtained data showed that the intracellular kinetics was dependent on the type of gene carrier and the weakest intracellular step varied between the carriers; rapid elimination of cell-associated pDNA in CaP, nuclear uptake in DOTAP and transcriptional and translational events in PEI mediated transfections. Overall, neither the amount of cell- nor nuclear associated pDNA correlated with transgene expression but the mRNA expression of the transgene correlated well with the expression at protein level. The nuclear uptake of pDNA in all cases was rapid and efficient thus indicating that the post-nuclear processes including transcription and translation steps have a critical role in defining the efficiency of non-viral gene delivery systems. Our study demonstrated that cell-surface GAGs are not essential for cell surface binding and internalization of gene delivery complexes, but they are able to define the intracellular routing of the complexes by leading them to pathways with high pDNA elimination.  

3.2170           A protocol for the subcellular fractionation of Saccharomyces cerevisiae using nitrogen cavitation and density gradient centrifugation

Wang, Y., Lilley, K.S. and Oliver, S.G. Yeast, 31, 127-135 (2014)   Most protocols for yeast subcellular fractionation involve the use of mechanical shear forces to lyse the spheroplasts produced by the enzymatic digestion of the Saccharomyces cerevisiae cell wall. These mechanical homogenization procedures often involve the manual use of devices such as the Dounce homogenizer, and so are very operator-dependent and, in consequence, lack reproducibility. Here, we report a highly reproducible method of homogenizing yeast cells based on nitrogen cavitation. This has been optimized to allow efficient release of subcellular compartments that show a high degree of integrity. The protocol remains effective and reproducible across a range of sample volumes and buffer environments. The subsequent separation method, which employs both sucrose and iodixanol density gradients, has been developed to resolve the major membrane-bound compartments of S. cerevisiae. We present an integrated protocol that is fast, facile, robust and efficient and that will enable ‘omics’ studies of the subcellular compartments of S. cerevisiae and other yeasts.  

3.2171           HIV-1 protease-induced apoptosis

Rumlova, M., Krizova, I., Keprova, A., Hadravova, R., Dolezal, M., Strohalmova, K., Pichova, I., Hajek, M. and Ruml, T. Retrovirology, 11:37 (2014)   Background Apoptosis is one of the presumptive causes of CD4⁺ T cell depletion during HIV infection and progression to AIDS. However, the precise role of HIV-1 in this process remains unexplained. HIV-1 protease (PR) has been suggested as a possible factor, but a direct link between HIV-1 PR enzymatic activity and apoptosis has not been established. Results Here, we show that expression of active HIV-1 PR induces death in HeLa and HEK-293 cells via the mitochondrial apoptotic pathway. This conclusion is based on in vivo observations of the direct localization of HIV-1 PR in mitochondria, a key player in triggering apoptosis. Moreover, we observed an HIV-1 PR concentration-dependent decrease in mitochondrial membrane potential and the role of HIV-1 PR in activation of caspase 9, PARP cleavage and DNA fragmentation. In addition, in vitro data demonstrated that HIV-1 PR mediates cleavage of mitochondrial proteins Tom22, VDAC and ANT, leading to release of AIF and Hsp60 proteins. By using yeast two-hybrid screening, we also identified a new HIV-1 PR interaction partner, breast carcinoma-associated protein 3 (BCA3). We found that BCA3 accelerates p53 transcriptional activity on the bax promoter, thus elevating the cellular level of pro-apoptotic Bax protein. Conclusion In summary, our results describe the involvement of HIV-1 PR in apoptosis, which is caused either by a direct effect of HIV-1 PR on mitochondrial membrane integrity or by its interaction with cellular protein BCA3.  

3.2172           Potential candidate camelid antibodies for the treatment of protein-misfolding diseases

David, M.A., Jones, D.R. and Tayebi, M.

1. Neuroimmunol., 272, 76-85 (2014)

  Protein-misfolding diseases (PMDs), including Alzheimer's disease would potentially reach epidemic proportion if effective ways to diagnose and treat them were not developed. The quest for effective therapy for PMDs has been ongoing for decades and some of the technologies developed so far show great promise. We report here the development of antibodies by immunization of camelids with prion (PrioV3) and Alzheimer's (PrioAD12, 13 & 120) disease-derived brain material. We show that anti-PrP antibody transmigration across the blood–brain barrier (BBB) was inhibited with phosphatidylinositol-specific phospholipase C (PIPLC). Our camelid anti-prion antibody was also shown to permanently abrogate prion replication in a prion-permissive cell line after crossing the artificial BBB. Furthermore, anti-Aβ/tau antibodies were able to bind their specific immunogens with ELISA and immunohistochemistry. Finally, both PrioV3 and PrioAD12 were shown to co-localize with Lamp-1, a marker of late endosomal/lysosomal compartments. These antibodies could prove to be a valuable tool for the neutralization/clearance of PrP^Sc, Aβ and tau proteins in cellular compartments of affected neurons and could potentially have wider applicability for the treatment of PMDs.  

3.2173           Specific recycling receptors are targeted to the immune synapse by the intraflagellar transport system

Finetti, F., Patrussi, L., Masi, G., Onnis, A., Galgano, D., Lucherini, O.M., Pazour, G. and Baldari, C.T.

1. Cell Sci., 127, 1924-1937 (2014)

  T cell activation requires sustained signaling at the immune synapse, a specialized interface with the antigen-presenting cell (APC) that assembles following T cell antigen receptor (TCR) engagement by major histocompatibility complex (MHC)-bound peptide. Central to sustained signaling is the continuous recruitment of TCRs to the immune synapse. These TCRs are partly mobilized from an endosomal pool by polarized recycling. We have identified IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, as a central regulator of TCR recycling to the immune synapse. Here, we have investigated the interplay of IFT20 with the Rab GTPase network that controls recycling. We found that IFT20 forms a complex with Rab5 and the TCR on early endosomes. IFT20 knockdown (IFT20KD) resulted in a block in the recycling pathway, leading to a build-up of recycling TCRs in Rab5⁺ endosomes. Recycling of the transferrin receptor (TfR), but not of CXCR4, was disrupted by IFT20 deficiency. The IFT components IFT52 and IFT57 were found to act together with IFT20 to regulate TCR and TfR recycling. The results provide novel insights into the mechanisms that control TCR recycling and immune synapse assembly, and underscore the trafficking-related function of the IFT system beyond ciliogenesis.  

3.2174           Parkinson's disease-linked human PARK9/ATP13A2 maintains zinc homeostasis and promotes α-Synuclein externalization via Exosomes

Kong, S.M.Y., Chan, B.K.K., Park, J-S., Hill, K.J., Aitken, J.B., Cottle, L., Farghaian, H., Cole, A.R., Lay, P.A., Sue, C.M. and Cooper, A.A. Hum. Mol. Genet., 23(11), 2816-2833 (2014)   α-Synuclein plays a central causative role in Parkinson's disease (PD). Increased expression of the P-type ATPase ion pump PARK9/ATP13A2 suppresses α-Synuclein toxicity in primary neurons. Our data indicate that ATP13A2 encodes a zinc pump; neurospheres from a compound heterozygous ATP13A2^−/− patient and ATP13A2 knockdown cells are sensitive to zinc, whereas ATP13A2 over-expression in primary neurons confers zinc resistance. Reduced ATP13A2 expression significantly decreased vesicular zinc levels, indicating ATP13A2 facilitates transport of zinc into membrane-bound compartments or vesicles. Endogenous ATP13A2 localized to multi-vesicular bodies (MVBs), a late endosomal compartment located at the convergence point of the endosomal and autophagic pathways. Dysfunction in MVBs can cause a range of detrimental effects including lysosomal dysfunction and impaired delivery of endocytosed proteins/autophagy cargo to the lysosome, both of which have been observed in cells with reduced ATP13A2 function. MVBs also serve as the source of intra-luminal nanovesicles released extracellularly as exosomes that can contain a range of cargoes including α-Synuclein. Elevated ATP13A2 expression reduced intracellular α-Synuclein levels and increased α-Synuclein externalization in exosomes >3-fold whereas ATP13A2 knockdown decreased α-Synuclein externalization. An increased export of exosome-associated α-Synuclein may explain why surviving neurons of the substantia nigra pars compacta in sporadic PD patients were observed to over-express ATP13A2. We propose ATP13A2’s modulation of zinc levels in MVBs can regulate the biogenesis of exosomes capable of containing α-Synuclein. Our data indicate that ATP13A2 is the first PD-associated gene involved in exosome biogenesis and indicates a potential neuroprotective role of exosomes in PD.  

3.2175           Interaction of Integrin β4 With S1P Receptors in S1P- and HGF-Induced Endothelial Barrier Enhancement

Ni, X., Epshtein, Y., Chen, W., Zhou, ts., Xie, L., Garcia, J.G.N. and jacobson, J.R.

1. Cell. Biochem., 115(6), 1187-1195 (2014)

  We previously reported sphingosine 1-phosphate (S1P) and hepatocyte growth factor (HGF) augment endothelial cell (EC) barrier function and attenuate murine acute lung inury (ALI). While the mechanisms underlying these effects are not fully understood, S1P and HGF both transactivate the S1P receptor, S1PR1 and integrin β4 (ITGB4) at membrane caveolin-enriched microdomains (CEMs). In the current study, we investigated the roles of S1PR2 and S1PR3 in S1P/HGF-mediated EC signaling and their associations with ITGB4. Our studies confirmed ITGB4 and S1PR2/3 are recruited to CEMs in human lung EC in response to either S1P (1 µM, 5 min) or HGF (25 ng/ml, 5 min). Co-immunoprecipitation experiments identified an S1P/HGF-mediated interaction of ITGB4 with both S1PR2 and S1PR3. We then employed an in situ proximity ligation assay (PLA) to confirm a direct ITGB4–S1PR3 association induced by S1P/HGF although a direct association was not detectable between S1PR2 and ITGB4. S1PR1 knockdown (siRNA), however, abrogated S1P/HGF-induced ITGB4–S1PR2 associations while there was no effect on ITGB4–S1PR3 associations. Moreover, PLA confirmed a direct association between S1PR1 and S1PR2 induced by S1P and HGF. Finally, silencing of S1PR2 significantly attenuated S1P/HGF-induced EC barrier enhancement as measured by transendothelial resistance while silencing of S1PR3 significantly augmented S1P/HGF-induced barrier enhancement. These results confirm an important role for S1PR2 and S1PR3 in S1P/HGF-mediated EC barrier responses that are associated with their complex formation with ITGB4. Our findings elucidate novel mechanisms of EC barrier regulation that may ultimately lead to new therapeutic targets for disorders characterized by increased vascular permeability including ALI.  

3.2176           Regulation of TrkB receptor translocation to lipid rafts by adenosine A2A receptors and its functional implications for BDNF-induced regulation of synaptic plasticity

Assaife-Lopes, N., Sousa, V.C., pereira, D.B., Ribeiro, J.A. and Sebastiao, A.M. Purinergic Signalling, 10(2), 251-267 (2014)   Brain-derived neurotrophic factor (BDNF) signalling is critical for neuronal development and transmission. Recruitment of TrkB receptors to lipid rafts has been shown to be necessary for the activation of specific signalling pathways and modulation of neurotransmitter release by BDNF. Since TrkB receptors are known to be modulated by adenosine A_2A receptor activation, we hypothesized that activation of A_2A receptors could influence TrkB receptor localization among different membrane microdomains. We found that adenosine A_2A receptor agonists increased the levels of TrkB receptors in the lipid raft fraction of cortical membranes and potentiated BDNF-induced augmentation of phosphorylated TrkB levels in lipid rafts. Blockade of the clathrin-mediated endocytosis with monodansyl cadaverine (100 μM) did not modify the effects of the A_2A receptor agonists, but significantly impaired BDNF effects on TrkB recruitment to lipid rafts. The effect of A_2A receptor activation in TrkB localization was mimicked by 5 μM forskolin, an adenylyl cyclase activator. Also, it was blocked by the PKA inhibitors Rp-cAMPs and PKI-(14-22) and by the Src-family kinase inhibitor PP2. Moreover, removal of endogenous adenosine or disruption of lipid rafts reduced BDNF stimulatory effects on glutamate release from cortical synaptosomes. Lipid raft integrity was also required for the effects of BDNF upon hippocampal long-term potentiation at CA1 synapses. Our data demonstrate, for the first time, a BDNF-independent recruitment of TrkB receptors to lipid rafts, induced by the activation of adenosine A_2A receptors, with functional consequences for TrkB phosphorylation and BDNF-induced modulation of neurotransmitter release and hippocampal plasticity.  

3.2177           Exosomes and their role in CNS viral infections

Sampey, G.C., Meyering, S.S., Zadeh, M.A., Saifuddin, M., Hakami, R. and Kashanchi, F.

1. Neurovirol., 20(3), 199-208 (2014)

  Exosomes are small membrane-bound vesicles that carry biological macromolecules from the site of production to target sites either in the microenvironment or at distant sites away from the origin. Exosomal content of cells varies with the cell type that produces them as well as environmental factors that alter the normal state of the cell such as viral infection. Human DNA and RNA viruses alter the composition of host proteins as well as incorporate their own viral proteins and other cargo into the secreted exosomes. While numerous viruses can infect various cell types of the CNS and elicit damaging neuropathologies, few have been studied for their exosomal composition, content, and function on recipient cells. Therefore, there is a pressing need to understand how DNA and RNA viral infections in CNS control exosomal release. Some of the more recent studies including HIV-1, HTLV-1, and EBV-infected B cells indicate that exosomes from these infections contain viral miRNAs, viral transactivators, and a host of cytokines that can control the course of infection. Finally, because exosomes can serve as vehicles for the cellular delivery of proteins and RNA and given that the blood-brain barrier is a formidable challenge in delivering therapeutics to the brain, exosomes may be able to serve as ideal vehicles to deliver protein or RNA-based therapeutics to the brain.  

3.2178           Sulfatide-mediated control of extracellular matrix-dependent oligodendrocyte maturation

Baron, W., Biljard, M., Nomden, A., de Jonge, J.C., Teunissen, C. and Hoekstra, D. Glia, 62(6), 927-942 (2014)   In the central nervous system, the extracellular matrix (ECM) compound laminin-2, present on developing axons, is essential in regulating oligodendrocyte (OLG) maturation. For example, laminin-2 is involved in mediating interactions between integrins and growth factors, initially localizing in separate membrane microdomains. The galactosphingolipid sulfatide is an important constituent of these microdomains and may serve as a receptor for laminin-2. Here, we investigated whether sulfatide interferes with ECM–integrin interactions and, in this manner, modulates OLG maturation. Our data reveal that disruption of laminin-2–sulfatide interactions impeded OLG differentiation and myelin-like membrane formation. On laminin-2, but not on (re)myelination-inhibiting fibronectin, sulfatide laterally associated with integrin α6 in membrane microdomains. Sulfatide was partly excluded from membrane microdomains on fibronectin, thereby likely precluding laminin-2-mediated myelination. Anti-sulfatide antibodies disrupted integrin α6-PDGFαR interactions on laminin-2 and induced demyelination in myelinated spheroid cultures, but intriguingly stimulated myelin-like membrane formation on fibronectin. Taken together, these findings highlight the importance of laminin–sulfatide interactions in the formation of functional membrane microdomains essential for myelination. Thus, laminin–sulfatide interactions might control the asynchronous localized differentiation of OLGs, thereby allowing myelination to be triggered by axonal demand. Given the accumulation of fibronectin in multiple sclerosis lesions, the findings also provide a molecular rationale for the potential of anti-sulfatide antibodies to trigger quiescent endogenous OLG progenitor cells in axon remyelination.  

3.2179           Mo1751 CGMP-Dependent Kinase 2, Na+/H+ Regulatory Factor 2, and Na+/H+ Exchanger Isoform 3 Dynamically Assemble Within Lipid Rafts in Murine Small Intestinal Brush Border Membrane

Luo, M., Liu, Y., Riederer, B., Patrucco, E., Hofman, F., Donowitz, M., Tian, D., Yun, C., de Jonge, H., Lamprecht, G. and Seidler, U. Gastroenterology, 146(5), Suppl. 1, S651-S652 (2014)   Background: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins, which enables the formation of multiprotein complexes. We previously reported a differential association of the NHERFs to the lipid raft and non-raft fraction of NHE3 in murine intestinal BBM, with NHERF2 being strongly lipid raft associated. Aim: This study was undertaken to explore the association of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII), a key enzyme in hormonal and toxinmediated inhibition of NHE3, within lipid rafts, and to assess the effect of stimulating this signal transduction pathway on the raft assembly of NHE3, NHERF2 and cGKII. Methods: Murine BBM was isolated from wild type, NHE3-deficient, cGMP-kinase II-deficient, and NHERF2-deficient mice before and after intestinal application of a heat-stable Escherichia coli toxin (STA) analogue in vivo. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X solubilised isolated small intestinal BBM. The raft-associated and non-raft proteins were precipitated and studied by Western analysis. Results: NHE3 and NHERF2 were strongly lipid raft associated, and cGMP-dependent kinase II, which together with NHERF2 is essential for guanylin/STa-mediated NHE3 inhibition, was almost exclusively lipid-raft associated. While NHE3 and NHERF2 were always observed in the same lipid raft fraction(s), a part of the cGKII was found in lipid rafts of lower specific weight. The application of an oral STa-analogue to the mice and subsequent small intestinal BBM isolation and lipid raft flotation assay demonstrated a redistribution of cGKII, NHE3 and NHERF2 in a dynamic fashion. Conclusion: The differential association of the NHERFs, as well as kinases, with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is likely one component of the differential and signal-specific NHE3 regulation by the different NHERFs. Many players of the signalling pathway for the guanylin analoges via cGMP-dependent kinase II, leading to NHE3 inhibition, are associated with lipid rafts in the murine small intestine.  

3.2180           Oxysterol-Binding Protein Is a Phosphatidylinositol 4-Kinase Effector Required for HCV Replication Membrane Integrity and Cholesterol Trafficking

Wang, H., Perry, J.W., Lauring, A.S., Neddermann, P., De Francesco, R. and Tai, A.W: Gastroenteerology, 146, 1373-1385 (2014)   Background & Aims Positive-sense RNA viruses remodel intracellular membranes to generate specialized membrane compartments for viral replication. Several RNA viruses, including poliovirus and hepatitis C virus (HCV), require phosphatidylinositol (PI) 4-kinases for their replication. However, it is not known how PI 4-kinases and their product, PI(4)P, facilitate host membrane reorganization and viral replication. In addition, although the HCV replication compartment, known as the membranous web, is believed to be cholesterol enriched, the mechanisms by which this occurs have not been elucidated. We aimed to identify and characterize a PI 4-kinase effector in HCV replication. Methods We used a combination of microscopic and biochemical methods to study HCV replication, web morphology, the distribution of intracellular protein and PI(4)P, along with cholesterol trafficking in HCV-infected cells. PI 4-kinase and oxysterol-binding protein (OSBP) were inhibited using RNA interference or small molecules in cells expressing a full-length genotype 1b replicon or infected with the JFH-1 strain of HCV. Results OSBP was required for HCV replication and membranous web integrity. OSBP was recruited to membranous webs in a PI 4-kinase−dependent manner, and both these factors were found to regulate cholesterol trafficking to the web. We also found OSBP to be required for poliovirus infection but dispensable for dengue virus. Conclusions OSBP is a PI 4-kinase effector in HCV infection, and contributes to the integrity and cholesterol enrichment of the membranous web. OSBP might also be a PI 4-kinase effector in poliovirus infection and could be involved in replication of other viruses that require PI 4-kinases.  

3.2181           Polymer–Peptide Delivery Platforms: Effect of Oligopeptide Orientation on Polymer-Based DNA Delivery

Parelkar, S.S., Letteri, r., Chan-Seng, D., Zolochevska, O., Ellis, J., Figueiredo, M. and Emrick, T. Biomacromolecules, 15(4), 1328-1336 (2014)   The success of nonviral transfection using polymers hinges on efficient nuclear uptake of nucleic acid cargo and overcoming intra- and extracellular barriers. By incorporating PKKKRKV heptapeptide pendent groups as nuclear localization signals (NLS) on a polymer backbone, we demonstrate protein expression levels higher than those obtained from JetPEI and Lipofectamine 2000, the latter being notorious for coupling high transfection efficiency with cytotoxicity. The orientation of the NLS peptide grafts markedly affected transfection performance. Polymers with the sequence attached to the backbone from the valine residue achieved a level of nuclear translocation higher than the levels of those having the NLS groups attached in the opposite orientation. The differences in nuclear localization and DNA complexation strength between the two orientations correlated with a striking difference in protein expression, both in cell culture and in vivo. Polyplexes formed from these comb polymer structures exhibited transfection efficiencies superior to those of Lipofectamine 2000 but with greatly reduced toxicity. Moreover, these novel polymers, when administered by intramuscular ultrasound-mediated delivery, allowed a high level of reporter gene expression in mice, demonstrating their therapeutic promise in vivo.  

3.2182           Deficiency of Sphingosine-1-phosphate Lyase Impairs Lysosomal Metabolism of the Amyloid Precursor Protein

Karaca, I., Tamboli, I.Y., Glebov, K., Richter, J., Fell, L.H., Grimm, M.O., Haupenthal, V.J., hartmann, T., Gräler, M.H., van Echten-Deckert, G. and Walter, J.

1. Biol. Chem., 289(24), 16761-16772 (2014)

  Progressive accumulation of the amyloid β protein in extracellular plaques is a neuropathological hallmark of Alzheimer disease. Amyloid β is generated during sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. In addition to the proteolytic processing by secretases, APP is also metabolized by lysosomal proteases. Here, we show that accumulation of intracellular sphingosine-1-phosphate (S1P) impairs the metabolism of APP. Cells lacking functional S1P-lyase, which degrades intracellular S1P, strongly accumulate full-length APP and its potentially amyloidogenic C-terminal fragments (CTFs) as compared with cells expressing the functional enzyme. By cell biological and biochemical methods, we demonstrate that intracellular inhibition of S1P-lyase impairs the degradation of APP and CTFs in lysosomal compartments and also decreases the activity of γ-secretase. Interestingly, the strong accumulation of APP and CTFs in S1P-lyase-deficient cells was reversed by selective mobilization of Ca²⁺ from the endoplasmic reticulum or lysosomes. Intracellular accumulation of S1P also impairs maturation of cathepsin D and degradation of Lamp-2, indicating a general impairment of lysosomal activity. Together, these data demonstrate that S1P-lyase plays a critical role in the regulation of lysosomal activity and the metabolism of APP.  

3.2183           Quantitation of Physiological and Biochemical Barriers to siRNA Liver Delivery via Lipid Nanoparticle Platform

Xu, Y., Keough, E., Roberts, J., Koeplinger, K., Lyman, M., Fauty, S., Carlini, E., Stern, M., Zhang, R., Yeh, S., Mahan, E., Wang, Y., Slaughter, D., Gindy, M., Raab, C., Thompson, C. and Hochman, J. Mol. Pharmaceutics, 11(5), 1424-1434 (2014)   Effective delivery of small interfering RNA (siRNA) requires efficient cellular uptake and release into cytosol where it forms an active complex with RNAi induced silencing complex (RISC). Despite rapid developments in RNAi therapeutics, improvements in delivery efficiency of siRNA are needed to realize the full potential of this modality in broad therapeutic applications. We evaluated potential physiological and biochemical barrier(s) to the effective liver delivery of siRNA formulated in lipid nanoparticle (LNP) delivery vehicles. The comparative siRNA delivery performance of three LNPs was investigated in rats. They were assembled with either C14- or C18-anchored PEG-lipid(s), cationic lipid(s), and various helper lipid(s) and contained the same siRNA duplex. These LNPs demonstrated differentiated potency with ED₅₀’s ranging from 0.02 to 0.25 mg/kg. The two C14-PEG-LNPs had comparable siRNA exposure in plasma and liver, while the C18-PEG-LNP demonstrated a higher plasma siRNA exposure and a slower but sustained liver uptake. RISC bound siRNA within the liver, a more proximal measure of the pharmacologically active siRNA species, displayed loading kinetics that paralleled the target mRNA knockdown profile, with greater RISC loading associated with more potent LNPs. Liver perfusion and hepatocyte isolation experiments were performed following treatment of rats with LNPs containing VivoTag-fluorescently labeled siRNA. One hour after dosing a majority of the siRNA within the liver was associated with hepatocytes and was internalized (within small subcellular vesicles) with no significant cell surface association, indicating good liver tissue penetration, hepatocellular distribution, and internalization. Comparison of siRNA amounts in hepatocytes and subcellular fractions of the three LNPs suggests that endosomal escape is a significant barrier to siRNA delivery where cationic lipid seems to have a great impact. Quantitation of Ago-2 associated siRNA revealed that after endosomal escape further loss of siRNA occurs prior to RISC loading. This quantitative assessment of LNP-mediated siRNA delivery has highlighted potential barriers with respect to endosomal escape and incomplete RISC loading for delivery optimization efforts.  

3.2184           Nongenomic Thyroid Hormone Signaling Occurs Through a Plasma Membrane–Localized Receptor

Kalyanaraman, H., Schwappacher, r., Joshua, J., Zhuang, S., Scott, B.T., Klos, M., Casteel, D.E., Frangos, J.A., Dillmann, W., Boss, G.R. and Pilz, R.B. Science Signaling, 7(326), ra48 (2014)   Thyroid hormone (TH) is essential for vertebrate development and the homeostasis of most adult tissues, including bone. TH stimulates target gene expression through the nuclear thyroid receptors TRα and TRβ; however, TH also has rapid, transcription-independent (nongenomic) effects. We found a previously uncharacterized plasma membrane–bound receptor that was necessary and sufficient for nongenomic TH signaling in several cell types. We determined that this receptor is generated by translation initiation from an internal methionine of TRα, which produces a transcriptionally incompetent protein that is palmitoylated and associates with caveolin-containing plasma membrane domains. TH signaling through this receptor stimulated a pro-proliferative and pro-survival program by increasing the intracellular concentrations of calcium, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP), which led to the sequential activation of protein kinase G II (PKGII), the tyrosine kinase Src, and extracellular signal–regulated kinase (ERK) and Akt signaling. Hypothyroid mice exhibited a cGMP-deficient state with impaired bone formation and increased apoptosis of osteocytes, which was rescued by a direct stimulator of guanylate cyclase. Our results link nongenomic TH signaling to a previously uncharacterized membrane-bound receptor, and identify NO synthase, guanylate cyclase, and PKGII as TH effectors that activate kinase cascades to regulate cell survival and proliferation.  

3.2185           Sialidase NEU3 Dynamically Associates to Different Membrane Domains Specifically Modifying Their Ganglioside Pattern and Triggering Akt Phosphorylation

Bonardi, D., Papini, N., Pasini, M., Dileo, L., Orizio, F., Monti, E., Caimi, L., Venerando, B. and Bresciani, R. PloS One, 9(6), e99405 (2014)   Lipid rafts are known to regulate several membrane functions such as signaling, trafficking and cellular adhesion. The local enrichment in sphingolipids and cholesterol together with the low protein content allows their separation by density gradient flotation after extraction with non-ionic detergent at low temperature. These structures are also referred to as detergent resistant membranes (DRM). Among sphingolipids, gangliosides play important roles in different biological events, including signal transduction and tumorigenesis. Sialidase NEU3 shows high enzymatic specificity toward gangliosides. Moreover, the enzyme is present both at the cell surface and in endosomal structures and cofractionates with caveolin. Although changes in the expression level of NEU3 have been correlated to different tumors, little is known about the precise distribution of the protein and its ability in modifying the ganglioside composition of DRM and non-DRM, thus regulating intracellular events. By means of inducible expression cell system we found that i) newly synthesized NEU3 is initially associated to non-DRM; ii) at steady state the protein is equally distributed between the two membrane subcompartments, i.e., DRM and non-DRM; iii) NEU3 is degraded via the proteasomal pathway; iv) the enzyme specifically modifies the ganglioside composition of the membrane areas where it resides; and v) NEU3 triggers phosphorylation of Akt, even in absence of exogenously administered EGF. Taken together our data demonstrate that NEU3 regulates the DRM ganglioside content and it can be considered as a modulator of Akt phosphorylation, further supporting the role of this enzyme in cancer and tumorigenesis.  

3.2186           P2X4 Forms Functional ATP-activated Cation Channels on Lysosomal Membranes Regulated by Luminal pH

Huang, P., Zou, Y., Zhong, X.Z., Cao, Q., Zhao, K., Zhu, M.X., Murrell-Lagnado, R and Dong, X-P.

1. Biol. Chem., 289(25), 17658-17667 (2014)

  P2X receptors are commonly known as plasma membrane cation channels involved in a wide variety of cell functions. The properties of these channels have been extensively studied on the plasma membrane. However, studies in amoeba suggest that P2X receptors are also present intracellularly and involved in vesicle fusion with the plasma membrane. Recently, it was shown that in addition to plasma membrane expression, mammalian P2X4 was also localized intracellularly in lysosomes. However, it was not clear whether the lysosomal P2X4 receptors function as channels and how they are activated and regulated. In this paper, we show that both P2X4 and its natural ligand, ATP, are enriched in lysosomes of COS1 and HEK293 cells. By directly recording membrane currents from enlarged lysosomal vacuoles, we demonstrated that lysosomal P2X4 formed channels activated by ATP from the luminal side in a pH-dependent manner. While the acidic pH at the luminal side inhibited P2X4 activity, increasing the luminal pH in the presence of ATP caused P2X4 activation. We further showed that, as for the plasma membrane P2X4, the lysosomal P2X4 was potentiated by ivermectin but insensitive to suramin and PPADS, and it permeated the large cation N-methyl-d-glucamine upon activation. Our data suggest that P2X4 forms functional ATP-activated cation channels on lysosomal membranes regulated by luminal pH. Together with the reported fusion effect of intracellular P2X in lower organisms, we speculate that the lysosome-localized P2X4 may play specific roles in membrane trafficking of acidic organelles in mammalian cells.  

3.2187           GRP78 is a novel receptor initiating a vascular barrier protective response to oxidized phospholipids

Birukova, A.A., Singleton, P.A., Gawlak, G., Tian, X., Mirzapoiazova, T., Mambetsariev, B., Dubrovskyi, O., Oskolkova, O.V., Bochkov, V.N. and Birukov, K.G. Mol. Biol. Cell, 25, 2006-2016 (2014)   Vascular integrity and the maintenance of blood vessel continuity are fundamental features of the circulatory system maintained through endothelial cell–cell junctions. Defects in the endothelial barrier become an initiating factor in several pathologies, including ischemia/reperfusion, tumor angiogenesis, pulmonary edema, sepsis, and acute lung injury. Better understanding of mechanisms stimulating endothelial barrier enhancement may provide novel therapeutic strategies. We previously reported that oxidized phospholipids (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine [OxPAPC]) promote endothelial cell (EC) barrier enhancement both in vitro and in vivo. This study examines the initiating mechanistic events triggered by OxPAPC to increase vascular integrity. Our data demonstrate that OxPAPC directly binds the cell membrane–localized chaperone protein, GRP78, associated with its cofactor, HTJ-1. OxPAPC binding to plasma membrane–localized GRP78 leads to GRP78 trafficking to caveolin-enriched microdomains (CEMs) on the cell surface and consequent activation of sphingosine 1-phosphate receptor 1, Src and Fyn tyrosine kinases, and Rac1 GTPase, processes essential for cytoskeletal reorganization and EC barrier enhancement. Using animal models of acute lung injury with vascular hyperpermeability, we observed that HTJ-1 knockdown blocked OxPAPC protection from interleukin-6 and ventilator-induced lung injury. Our data indicate for the first time an essential role of GRP78 and HTJ-1 in OxPAPC-mediated CEM dynamics and enhancement of vascular integrity.  

3.2188           Rab11 Regulates Trafficking of Trans-sialidase to the Plasma Membrane through the Contractile Vacuole Complex of Trypanosoma cruzi

Niyogi, S., Mucci, J., Campetella, O. and Docompo, R. PloS Pathogens, 10(6), e1004224 (2014)   Trypanosoma cruzi is the etiologic agent of Chagas disease. Although this is not a free-living organism it has conserved a contractile vacuole complex (CVC) to regulate its osmolarity. This obligate intracellular pathogen is, in addition, dependent on surface proteins to invade its hosts. Here we used a combination of genetic and biochemical approaches to delineate the contribution of the CVC to the traffic of glycosylphosphatidylinositol (GPI)-anchored proteins to the plasma membrane of the parasite and promote host invasion. While T. cruzi Rab11 (GFP-TcRab11) localized to the CVC, a dominant negative (DN) mutant tagged with GFP (GFP-TcRab11DN) localized to the cytosol, and epimastigotes expressing this mutant were less responsive to hyposmotic and hyperosmotic stress. Mutant parasites were still able to differentiate into metacyclic forms and infect host cells. GPI-anchored trans-sialidase (TcTS), mucins of the 60–200 KDa family, and trypomastigote small surface antigen (TcTSSA II) co-localized with GFP-TcRab11 to the CVC during transformation of intracellular amastigotes into trypomastigotes. Mucins of the gp35/50 family also co-localized with the CVC during metacyclogenesis. Parasites expressing GFP-TcRab11DN prevented TcTS, but not other membrane proteins, from reaching the plasma membrane, and were less infective as compared to wild type cells. Incubation of these mutants in the presence of exogenous recombinant active, but not inactive, TcTS, and a sialic acid donor, before infecting host cells, partially rescued infectivity of trypomastigotes. Taking together these results reveal roles of TcRab11 in osmoregulation and trafficking of trans-sialidase to the plasma membrane, the role of trans-sialidase in promoting infection, and a novel unconventional mechanism of GPI-anchored protein secretion.  

3.2189           Proteins of the Ciliary Axoneme Are Found on Cytoplasmic Membrane Vesicles during Growth of Cilia

Wood, C.R. and Rosenbaum, J.L. Current Biology, 24, 1114-1120  (2014)   The cilium is a specialized extension of the cell in which many specific proteins are admitted and retained, while many others are excluded or expelled. In order to maintain the organelle, the cell must possess mechanisms for the selective gating of protein entry, as well as for the targeted transport of proteins to the cilium from their sites of synthesis within the cell [1, 2, 3 and 4]. We hypothesized that the cell employs cytoplasmic vesicles as vehicles not only for the transport of proteins destined for the ciliary membrane but also for the transport of axonemal proteins to the cilium by means of peripheral association with vesicles. To test this hypothesis, we employed two different experimental strategies: (1) isolation and biochemical characterization of cytoplasmic vesicles that carry ciliary proteins, and (2) in situ localization of ciliary proteins on cytoplasmic vesicle surfaces using gold labeling and electron microscopy. Our findings indicate that structural proteins destined for the ciliary axoneme are attached to the outer surfaces of cytoplasmic vesicles that carry integral ciliary membrane proteins during the process of ciliary growth.  

3.2190           WLS Retrograde Transport to the Endoplasmic Reticulum during Wnt Secretion

Yu, J., Chia, J., Canning, C.A., Jones, C.M., Bard, F.A. and Virshup, D.M. Developmental Cell, 29, 277-291 (2014)   Wnts are transported to the cell surface by the integral membrane protein WLS (also known as Wntless, Evi, and GPR177). Previous studies of WLS trafficking have emphasized WLS movement from the Golgi to the plasma membrane (PM) and then back to the Golgi via retromer-mediated endocytic recycling. We find that endogenous WLS binds Wnts in the endoplasmic reticulum (ER), cycles to the PM, and then returns to the ER through the Golgi. We identify an ER-targeting sequence at the carboxyl terminus of native WLS that is critical for ER retrograde recycling and contributes to Wnt secretory function. Golgi-to-ER recycling of WLS requires the COPI regulator ARF as well as ERGIC2, an ER-Golgi intermediate compartment protein that is also required for the retrograde trafficking of the KDEL receptor and certain toxins. ERGIC2 is required for efficient Wnt secretion. ER retrieval is an integral part of the WLS transport cycle.  

3.2191           Visualizing active membrane protein complexes by electron cryotomography

Gold, V.A.M., Leva, R., Walter, A., Pfanner, N., van der Laan, M. and Kühlbrandt, W. Nature Communications, 5:4129 (2014)   Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future.  

3.2192           Regulation of Nuclear Translocation of the Myb1 Transcription Factor by TvCyclophilin 1 in the Protozoan Parasite Trichomonas vaginalis

Hsu, H-M., Chu, C-H., Wang, Y-T., Lee, Y., Wei, S-Y., Liu, H-W., Ong, S-J., Chen, C. and Tai, J-H.

1. Biol. Chem., 289(27), 19120-19136 (2014)

  In Trichomonas vaginalis, a Myb1 protein was previously demonstrated to repress transcription of an iron-inducible ap65-1 gene. In this study, a human cyclophilin A homologue, TvCyclophilin 1 (TvCyP1), was identified as a Myb1-binding protein using a bacterial two-hybrid library screening system. The recombinant TvCyP1 exhibited typical peptidyl-prolyl isomerase activity with k_cat/K_m of ∼7.1 μm⁻¹ s⁻¹. In a pulldown assay, the His-tagged Myb1 interacted with a GST-TvCyP1 fusion protein, which had an enzymatic proficiency half that of recombinant TvCyP1. Both the enzymatic proficiency of GST-TvCyP1 and its binding to His-Myb1 were eliminated by mutation of Arg⁶³ in the catalytic motif or inhibited by cyclosporin A. TvCyP1 was primarily localized to the hydrogenosomes by immunofluorescence assay, but it was also co-purified with Myb1 in certain vesicle fractions from differential and gradient centrifugations. Transgenic cells overexpressing HA-TvCyP1 had a higher level of nuclear Myb1 but a much lower level of Myb1 associated with the vesicles than control and those overexpressing HA-TvCyP1(R63A). Myb1 was detected at a much higher level in the HA-TvCyP1 protein complex than in the HA-TvCyP1(R63A) protein complex immunoprecipitated from P15 and P100, but not S100, fractions of postnuclear lysates. A TvCyP1-binding motif, ¹⁰⁵YGPKWNK¹¹¹, was identified in Myb1 in which Gly¹⁰⁶ and Pro¹⁰⁷ were essential for its binding to TvCyP1. Mutation of Gly¹⁰⁶ and Pro¹⁰⁷, respectively, in HA-Myb1 resulted in cytoplasmic retention and elevated nuclear translocation of the overexpressed protein. These results suggest that TvCyP1 may induce the release of Myb1 that is restrained to certain cytoplasmic vesicles prior to its nuclear translocation.  

3.2193           The Lateral Membrane Organization and Dynamics of Myelin Proteins PLP and MBP Are Dictated by Distinct Galactolipids and the Extracellular Matrix

Ozgen, H., Schrimpf, W., Hendrix, J., de Jonge, J.C., Lamb, D.C., Hoekstra, D., Kahya, N. and baron, W. PloS One, 9(7), e101834 (2014)   In the central nervous system, lipid-protein interactions are pivotal for myelin maintenance, as these interactions regulate protein transport to the myelin membrane as well as the molecular organization within the sheath. To improve our understanding of the fundamental properties of myelin, we focused here on the lateral membrane organization and dynamics of peripheral membrane protein 18.5-kDa myelin basic protein (MBP) and transmembrane protein proteolipid protein (PLP) as a function of the typical myelin lipids galactosylceramide (GalC), and sulfatide, and exogenous factors such as the extracellular matrix proteins laminin-2 and fibronectin, employing an oligodendrocyte cell line, selectively expressing the desired galactolipids. The dynamics of MBP were monitored by z-scan point fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), while PLP dynamics in living cells were investigated by circular scanning FCS. The data revealed that on an inert substrate the diffusion rate of 18.5-kDa MBP increased in GalC-expressing cells, while the diffusion coefficient of PLP was decreased in sulfatide-containing cells. Similarly, when cells were grown on myelination-promoting laminin-2, the lateral diffusion coefficient of PLP was decreased in sulfatide-containing cells. In contrast, PLP's diffusion rate increased substantially when these cells were grown on myelination-inhibiting fibronectin. Additional biochemical analyses revealed that the observed differences in lateral diffusion coefficients of both proteins can be explained by differences in their biophysical, i.e., galactolipid environment, specifically with regard to their association with lipid rafts. Given the persistence of pathological fibronectin aggregates in multiple sclerosis lesions, this fundamental insight into the nature and dynamics of lipid-protein interactions will be instrumental in developing myelin regenerative strategies.  

3.2194           Ca2 +–CaM regulating viability of Candida guilliermondii under oxidative stress by acting on detergent resistant membrane proteins

An, B., Chen, Y., Li, B., Qin, G. and Tian, S.

1. Proteomics, 109, 38-49 (2014)

  Reactive oxygen species (ROS) play a vital role in reducing viability of yeast cells. The Ca^2 +–CaM signaling pathways are involved in regulating the intracellular ROS level in yeast cells under stress. Detergent resistant membranes (DRMs), the sterol-rich microdomains, participate in a wide range of cellular processes including growth, trafficking and death in yeast cells. In the present study, we found that Trifluoperazine (TFP), an antagonist of CaM, could increase the viability of Candida guilliermondii cells under H₂O₂ stress. Based on comparative analysis of DRM sub proteomics, a total number of 29 differentially expressed protein spots were identified, among which 8 protein spots belong to the electron transport chain and 7 protein spots belong to transporters. It is suggested that TFP treatment could modulate the intracellular ROS generation in yeast cells. We additionally ascertained that TFP treatment could effectively alleviate the ROS accumulation and protein damage in C. guilliermondii cells under H₂O₂ stress, via investigating the intracellular ROS levels and protein oxidative damage in yeast cells. These findings firstly revealed that the Ca^2 +–CaM signaling pathway is related to the viability of yeast cells under H₂O₂ stress, and provide novel evidences for exploring Ca^2 +–CaM's role in regulating this viability via acting on DRM proteins.  

3.2195           Comparing the different morphotypes of a fish pathogen - implications for key virulence factors in Flavobacterium columnare

Laanto, E., Penttinen, R.K., Bamford, J.K.H. and Sundberg, L-R. BMC Microbiol., 14:170 (2014)   Background Flavobacterium columnare (Bacteroidetes) is the causative agent of columnaris disease in farmed freshwater fish around the world. The bacterium forms three colony morphotypes (Rhizoid, Rough and Soft), but the differences of the morphotypes are poorly known. We studied the virulence of the morphotypes produced by F. columnare strain B067 in rainbow trout (Onconrhynchus mykiss) and used high-resolution scanning electron microscopy to identify the fine structures of the cells grown in liquid and on agar. We also analysed the proteins secreted extracellularly and in membrane vesicles to identify possible virulence factors. Results Only the Rhizoid morphotype was virulent in rainbow trout. Under electron microscopy, the cells of Rhizoid and Soft morphotypes were observed to display an organised structure within the colony, whereas in the Rough type this internal organisation was absent. Planktonic cells of the Rhizoid and Rough morphotypes produced large membrane vesicles that were not seen on the cells of the Soft morphotype. The vesicles were purified and analysed. Two proteins with predicted functions were identified, OmpA and SprF. Furthermore, the Rhizoid morphotype secreted a notable amount of a small, unidentified 13 kDa protein absent in the Rough and Soft morphotypes, indicating an association with bacterial virulence. Conclusions Our results suggest three factors that are associated with the virulence of F. columnare: the coordinated organisation of cells, a secreted protein and outer membrane vesicles. The internal organisation of the cells within a colony may be associated with bacterial gliding motility, which has been suggested to be connected with virulence in F. columnare. The function of the secreted 13 kDa protein by the cells of the virulent morphotype cells remains unknown. The membrane vesicles might be connected with the adhesion of cells to the surfaces and could also carry potential virulence factors. Indeed, OmpA is a virulence factor in several bacterial pathogens, often linked with adhesion and invasion, and SprF is a protein connected with gliding motility and the protein secretion of flavobacteria.  

3.2196           Fluvoxamine alleviates ER stress via induction of Sigma-1 receptor

Omi, T., Tanimukai, H., Kanayama, D., Sakagami, Y., Tagami, S., Okochi, M., Morihara, T., Sato, M., Yanagida, K., Kitasyoji, A., Hara, H., Imaizumi, K., Maurice, T., Chevallier, N., marchal, S., Takeda, M. and Kudo, T. Cell Death and Disease, 5, e1332 (2014)   We recently demonstrated that endoplasmic reticulum (ER) stress induces sigma-1 receptor (Sig-1R) expression through the PERK pathway, which is one of the cell’s responses to ER stress. In addition, it has been demonstrated that induction of Sig-1R can repress cell death signaling. Fluvoxamine (Flv) is a selective serotonin reuptake inhibitor (SSRI) with a high affinity for Sig-1R. In the present study, we show that treatment of neuroblastoma cells with Flv induces Sig-1R expression by increasing ATF4 translation directly, through its own activation, without involvement of the PERK pathway. The Flv-mediated induction of Sig-1R prevents neuronal cell death resulting from ER stress. Moreover, Flv-induced ER stress resistance reduces the infarct area in mice after focal cerebral ischemia. Thus, Flv, which is used frequently in clinical practice, can alleviate ER stress. This suggests that Flv could be a feasible therapy for cerebral diseases caused by ER stress.  

3.2197           The carrying pigeons of the cell: exosomes and their role in infectious diseases caused by human pathogens

Fleming, A., Sampey, G., Chung, M-C., Bailey, C., van Hoek, M.L., Kashanchi, F. and Hakami, R.M. Pathogens and Disease, 71(2), 107-118 (2014)   Exosomes have recently been classified as the newest family members of ‘bioactive vesicles’ that function to promote intercellular communication. Long ignored and thought to be only a mechanism by which cellular waste is removed, exosomes have garnered a huge amount of interest in recent years as their critical functions in maintaining homeostasis through intercellular communication and also in different types of diseases have been demonstrated. Many groundbreaking studies of exosome functions have been performed in the cancer field and the infectious disease areas of study, revealing the importance and also the fascinating complexity of exosomal packaging, targeting, and functions. Selective packaging of exosomes in response to the type of infection, exosomal modulation of the immune response and host signaling pathways, exosomal regulation of pathogen spread, and effects of exosomes on the degree of pathogenesis have all been well documented. In this review, we provide a synthesis of the current understanding of the role of exosomes during infections caused by human pathogens and discuss the implications of these findings for a better understanding of pathogenic mechanisms and future therapeutic and diagnostic applications.  

3.2198           Extracellular vesicles produced by the Gram-positive bacterium Bacillus subtilis are disrupted by the lipopeptide surfactin

Brown, L., Kessler, a., Cabezas-Sanchez, P. Luque-garcia, J.L. and casadevall, A. Mol. Microbiol., 93(1), 183-198 (2014)   Previously, extracellular vesicle production in Gram-positive bacteria was dismissed due to the absence of an outer membrane, where Gram-negative vesicles originate, and the difficulty in envisioning how such a process could occur through the cell wall. However, recent work has shown that Gram-positive bacteria produce extracellular vesicles and that the vesicles are biologically active. In this study, we show that Bacillus subtilis produces extracellular vesicles similar in size and morphology to other bacteria, characterized vesicles using a variety of techniques, provide evidence that these vesicles are actively produced by cells, show differences in vesicle production between strains, and identified a mechanism for such differences based on vesicle disruption. We found that in wild strains of B. subtilis, surfactin disrupted vesicles while in laboratory strains harbouring a mutation in the gene sfp, vesicles accumulated in the culture supernatant. Surfactin not only lysed B. subtilis vesicles, but also vesicles from Bacillus anthracis, indicating a mechanism that crossed species boundaries. To our knowledge, this is the first time a gene and a mechanism has been identified in the active disruption of extracellular vesicles and subsequent release of vesicular cargo in Gram-positive bacteria. We also identify a new mechanism of action for surfactin.  

3.2199           Integrating mitosis, toxicity, and transgene expression in a telecommunications packet-switched network model of lipoplex-mediated gene delivery

Martin, T.M., Wysocki, B.J., Beyersdorf, J.P., Wysocki, T.A. and pannier, A.K. Biotechnol. Bioeng., 111(8), 1659-1671 (2014)   Gene delivery systems transport exogenous genetic information to cells or biological systems with the potential to directly alter endogenous gene expression and behavior with applications in functional genomics, tissue engineering, medical devices, and gene therapy. Nonviral systems offer advantages over viral systems because of their low immunogenicity, inexpensive synthesis, and easy modification but suffer from lower transfection levels. The representation of gene transfer using models offers perspective and interpretation of complex cellular mechanisms, including nonviral gene delivery where exact mechanisms are unknown. Here, we introduce a novel telecommunications model of the nonviral gene delivery process in which the delivery of the gene to a cell is synonymous with delivery of a packet of information to a destination computer within a packet-switched computer network. Such a model uses nodes and layers to simplify the complexity of modeling the transfection process and to overcome several challenges of existing models. These challenges include a limited scope and limited time frame, which often does not incorporate biological effects known to affect transfection. The telecommunication model was constructed in MATLAB to model lipoplex delivery of the gene encoding the green fluorescent protein to HeLa cells. Mitosis and toxicity events were included in the model resulting in simulation outputs of nuclear internalization and transfection efficiency that correlated with experimental data. A priori predictions based on model sensitivity analysis suggest that increasing endosomal escape and decreasing lysosomal degradation, protein degradation, and GFP-induced toxicity can improve transfection efficiency by three-fold. Application of the telecommunications model to nonviral gene delivery offers insight into the development of new gene delivery systems with therapeutically relevant transfection levels  

3.2200           Isolation and characterization of lipid rafts in Emiliania huxleyi: a role for membrane microdomains in host–virus interactions

Rose, S.L., Fulton, J.M., Brown, C.M., natale, f., Van Mooy, B.A.S. and Bidle, K.D. Environmental Microbiol., 16(4), 1150-1166 (2014)   Coccolithoviruses employ a suite of glycosphingolipids (GSLs) to successfully infect the globally important coccolithophore Emiliania huxleyi. Lipid rafts, chemically distinct membrane lipid microdomains that are enriched in GSLs and are involved in sensing extracellular stimuli and activating signalling cascades through protein–protein interactions, likely play a fundamental role in host–virus interactions. Using combined lipidomics, proteomics and bioinformatics, we isolated and characterized the lipid and protein content of lipid rafts from control E. huxleyi cells and those infected with EhV86, the type strain for Coccolithovirus. Lipid raft-enriched fractions were isolated and purified as buoyant, detergent-resistant membranes (DRMs) in OptiPrep density gradients. Transmission electron microscopy of vesicle morphology, polymerase chain reaction amplification of the EhV major capsid protein gene and immunoreactivity to flotillin antisera served as respective physical, molecular and biochemical markers. Subsequent lipid characterization of DRMs via high performance liquid chromatography-triple quadrapole mass spectrometry revealed four distinct GSL classes. Parallel proteomic analysis confirmed flotillin as a major lipid raft protein, along with a variety of proteins affiliated with host defence, programmed cell death and innate immunity pathways. The detection of an EhV86-encoded C-type lectin-containing protein confirmed that infection occurs at the interface between lipid rafts and cellular stress/death pathways via specific GSLs and raft-associated proteins.  

3.2201           Fractionation of Subcellular Membrane Vesicles of Epithelial and Non-epithelial Cells by OptiPrep™ Density Gradient Ultracentrifugation

Li, X. and donowitz, M. Methods in Mol. Biol., 1174, 85-99 (2014)   Density gradient ultracentrifugation (DGUC) is widely used for physical isolation (enrichment rather than purification) of subcellular membrane vesicles. It has been a valuable tool to study specific subcellular localization and dynamic trafficking of proteins. While sucrose has been the main component of density gradients, several years ago, synthetic OptiPrep™ (iodixanol) began being used for separation of organelles due to its iso-osmotic property. Here, we describe a detailed protocol for density gradient fractionation of various mammalian subcellular vesicles, including endoplasmic reticulum (ER), Golgi apparatus, endosomes, and lipid rafts, as well as apical and basolateral membranes of polarized epithelial cells.  

3.2202           A Foundation for Reliable Spatial Proteomics Data Analysis

Gatto, L., Breckels, L.M., Burger, T., Nightingale, D.J.H., Groen, A., Campbell,. C., Nikolovski, N., Mulvey, C.M., Christoforout, A., Ferro, M. and Lilley, K.S. Mol. Cell Proteomics, 13(8), 1937-1952 (2014)   Quantitative mass-spectrometry-based spatial proteomics involves elaborate, expensive, and time-consuming experimental procedures, and considerable effort is invested in the generation of such data. Multiple research groups have described a variety of approaches for establishing high-quality proteome-wide datasets. However, data analysis is as critical as data production for reliable and insightful biological interpretation, and no consistent and robust solutions have been offered to the community so far. Here, we introduce the requirements for rigorous spatial proteomics data analysis, as well as the statistical machine learning methodologies needed to address them, including supervised and semi-supervised machine learning, clustering, and novelty detection. We present freely available software solutions that implement innovative state-of-the-art analysis pipelines and illustrate the use of these tools through several case studies involving multiple organisms, experimental designs, mass spectrometry platforms, and quantitation techniques. We also propose sound analysis strategies for identifying dynamic changes in subcellular localization by comparing and contrasting data describing different biological conditions. We conclude by discussing future needs and developments in spatial proteomics data analysis.  

3.2203

   

3.2204           Signaling components of the 1α,25(OH)2D3-dependent Pdia3 receptor complex are required for Wnt5a calcium-dependent signaling

Doroudi, M., Olivares-Navarrete, R., Hyzy, S., Boyan, B. and Schwartz, Z. Biochim. Biophys. Acta, 1843, 2365-2375 (2014)   Wnt5a and 1α,25(OH)₂D₃ are important regulators of endochondral ossification. In osteoblasts and growth plate chondrocytes, 1α,25(OH)₂D₃ initiates rapid effects via its membrane-associated receptor protein disulfide isomerase A3 (Pdia3) in caveolae, activating phospholipase A₂ (PLA₂)-activating protein (PLAA), calcium/calmodulin-dependent protein kinase II (CaMKII), and PLA₂, resulting in protein kinase C (PKC) activation. Wnt5a initiates its calcium-dependent effects via intracellular calcium release, activating PKC and CaMKII. We investigated the requirement for components of the Pdia3 receptor complex in Wnt5a calcium-dependent signaling. We determined that Wnt5a signals through a CaMKII/PLA₂/PGE₂/PKC cascade. Silencing or blocking Pdia3, PLAA, or vitamin D receptor (VDR), and inhibition of calmodulin (CaM), CaMKII, or PLA₂ inhibited Wnt5a-induced PKC activity. Wnt5a activated PKC in caveolin-1-silenced cells, but methyl-beta-cyclodextrin reduced its stimulatory effect. 1α,25(OH)₂D₃ reduced stimulatory effects of Wnt5a on PKC in a dose-dependent manner. In contrast, Wnt5a had a biphasic effect on 1α,25(OH)₂D₃-stimulated PKC activation; 50 ng/ml Wnt5a caused a 2-fold increase in 1α,25(OH)₂D₃-stimulated PKC but higher Wnt5a concentrations reduced 1α,25(OH)₂D₃-stimulated PKC activation. Western blots showed that Wnt receptors Frizzled2 (FZD2) and Frizzled5 (FZD5), and receptor tyrosine kinase-like orphan receptor 2 (ROR2) were localized to caveolae. Blocking ROR2, but not FZD2 or FZD5, abolished the stimulatory effects of 1α,25(OH)₂D₃ on PKC and CaMKII. 1α,25(OH)₂D₃ membrane receptor complex components (Pdia3, PLAA, caveolin-1, CaM) interacted with Wnt5a receptors/co-receptors (ROR2, FZD2, FZD5) in immunoprecipitation studies, interactions that changed with either 1α,25(OH)₂D₃ or Wnt5a treatment. This study demonstrates that 1α,25(OH)₂D₃ and Wnt5a mediate their effects via similar receptor components and suggests that these pathways may interact.  

3.2205           The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana

Busse-Wicher, M., Gomes, T.C., Tryfona, T., Nikolovski, N., Stott, K., Grantham, N.J., Bolam, D.N., Skaf, M.S. and Dupree, P. Plant J., 79, 492-506 (2014)   The interaction between xylan and cellulose microfibrils is important for secondary cell wall properties in vascular plants; however, the molecular arrangement of xylan in the cell wall and the nature of the molecular bonding between the polysaccharides are unknown. In dicots, the xylan backbone of β-(1,4)-linked xylosyl residues is decorated by occasional glucuronic acid, and approximately one-half of the xylosyl residues are O-acetylated at C-2 or C-3. We recently proposed that the even, periodic spacing of GlcA residues in the major domain of dicot xylan might allow the xylan backbone to fold as a twofold helical screw to facilitate alignment along, and stable interaction with, cellulose fibrils; however, such an interaction might be adversely impacted by random acetylation of the xylan backbone. Here, we investigated the arrangement of acetyl residues in Arabidopsis xylan using mass spectrometry and NMR. Alternate xylosyl residues along the backbone are acetylated. Using molecular dynamics simulation, we found that a twofold helical screw conformation of xylan is stable in interactions with both hydrophilic and hydrophobic cellulose faces. Tight docking of xylan on the hydrophilic faces is feasible only for xylan decorated on alternate residues and folded as a twofold helical screw. The findings suggest an explanation for the importance of acetylation for xylan–cellulose interactions, and also have implications for our understanding of cell wall molecular architecture and properties, and biological degradation by pathogens and fungi. They will also impact strategies to improve lignocellulose processing for biorefining and bioenergy.  

3.2206           Endoplasmic Reticulum-associated Degradation of Niemann-Pick C1: EVIDENCE FOR THE ROLE OF HEAT SHOCK PROTEINS AND IDENTIFICATION OF LYSINE RESIDUES THAT ACCEPT UBIQUITIN

Nakasone, N., Nakamura, Y.S., Higaki, K., Oumi, N., Ohno, K. and Ninomiya, H.

1. Biol. Chem., 289(28), 19714-19725 (2014)

  Most cases with Niemann-Pick disease type C carry mutations in NPC1. Some of the mutations, including the most frequent I1061T, give rise to unstable proteins selected for endoplasmic reticulum-associated degradation. The purpose of the current study was to shed mechanistic insights into the degradation process. A proteasome inhibitor MG132 prolonged the life span of the wild-type NPC1 expressed in COS cells. The expressed protein associated with multiple chaperones including heat shock protein 90 (Hsp90), Hsp70, heat shock cognate protein 70 (Hsc70), and calnexin. Accordingly, expression of an E3 ligase CHIP (carboxyl terminus of Hsp70-interacting protein) enhanced MG132-induced accumulation of ubiquitylated NPC1. Co-expression and RNAi knockdown experiments in HEK cells indicated that Hsp70/Hsp90 stabilized NPC1, whereas Hsc70 destabilized it. In human fibroblasts carrying the I1061T mutation, adenovirus-mediated expression of Hsp70 or treatment with an HSP-inducer geranylgeranylacetone (GGA) increased the level of the mutant protein. In GGA-treated cells, the rescued protein was localized in the late endosome and ameliorated cholesterol accumulation. MALDI-TOF mass spectrometry revealed three lysine residues at amino acids 318, 792, and 1180 as potential ubiquitin-conjugation sites. Substitutions of the three residues with alanine yielded a mutant protein with a steady-state level more than three times higher than that of the wild-type. Introduction of the same substitutions to the I1061T mutant resulted in an increase in its protein level and functional restoration. These findings indicated the role of HSPs in quality control of NPC1 and revealed the role of three lysine residues as ubiquitin-conjugation sites.  

3.2207           SLC17A9 Protein Functions as a Lysosomal ATP Transporter and Regulates Cell Viability

Cao, Q., Zhao, K., Zhong, X.Z., Zou, Y., Yu, H., Huang, P., Xu, T-L. and Dong, X-P.

1. Biol. Chem., 289(33), 23189-23199 (2014)

  Lysosomes contain abundant ATP, which is released through lysosomal exocytosis following exposure to various stimuli. However, the molecular mechanisms underlying lysosomal ATP accumulation remain unknown. The vesicular nucleotide transporter, also known as solute carrier family 17 member 9 (SLC17A9), has been shown to function in ATP transport across secretory vesicles/granules membrane in adrenal chromaffin cells, T cells, and pancreatic cells. Here, using mammalian cell lines, we report that SLC17A9 is highly enriched in lysosomes and functions as an ATP transporter in those organelles. SLC17A9 deficiency reduced lysosome ATP accumulation and compromised lysosome function, resulting in cell death. Our data suggest that SLC17A9 activity mediates lysosomal ATP accumulation and plays an important role in lysosomal physiology and cell viability.  

3.2208           Membrane recruitment of endogenous LRRK2 precedes its potent regulation of autophagy

Schapansky, J., Nardozzi, J.D., Felizia, F. and LaVoie, M.J. Hum. Mol. Genet., 23(16), 4201-4214 (2014)   Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and idiopathic Parkinson's disease. However, the mechanisms for activating its physiological function are not known, hindering identification of the biological role of endogenous LRRK2. The recent discovery that LRRK2 is highly expressed in cells of the innate immune system and genetic association is a risk factor for autoimmune disorders implies an important role for LRRK2 in pathology outside of the central nervous system. Thus, an examination of endogenous LRRK2 in immune cells could provide insight into the protein's function. Here, we establish that stimulation of specific Toll-like receptors results in a complex biochemical activation of endogenous LRRK2, with early phosphorylation of LRRK2 preceding its dimerization and membrane translocation. Membrane-associated LRRK2 co-localized to autophagosome membranes following either TLR4 stimulation or mTOR inhibition with rapamycin. Silencing of endogenous LRRK2 expression resulted in deficits in the induction of autophagy and clearance of a well-described macroautophagy substrate, demonstrating the critical role of endogenous LRRK2 in regulating autophagy. Inhibition of LRRK2 kinase activity also reduced autophagic degradation and suggested the importance of the kinase domain in the regulation of autophagy. Our results demonstrate a well-orchestrated series of biochemical events involved in the activation of LRRK2 important to its physiological function. With similarities observed across multiple cell types and stimuli, these findings are likely relevant in all cell types that natively express endogenous LRRK2, and provide insights into LRRK2 function and its role in human disease.  

3.2209           A systems-level approach to understanding transcriptional regulation by p53 during mammalian hibernation

Pan, P., Treat, M.D. and van Breukelen, F.

1. Exp. Biol., 217, 2489-2498 (2014)

  Presumably to conserve energy, many mammals enter into hibernation during the winter. Homeostatic processes such as transcription and translation are virtually arrested. To further elucidate transcriptional regulation during hibernation, we studied the transcription factor p53. Here, we demonstrate that changes in liver mRNA and protein concentrations of known regulators of p53 are consistent with activation. p53 mRNA and protein concentrations are unrelated. Importantly, p53 protein concentration is increased ~2-fold during the interbout arousal that punctuates bouts of torpor. As a result, both the interbout arousal and the torpid state are characterized by high levels of nuclear-localized p53. Chromatin immunoprecipitation assays indicate that p53 binds DNA during the winter. Furthermore, p53 recruits RNA polymerase II, as indicated by nuclear run-on data. However, and consistent with previous data indicating an arrest of transcriptional elongation during torpor, p53 ‘activity’ does not result in expected changes in target gene transcripts. These data demonstrate the importance of using a systems level-approach in understanding a complex phenotype such as mammalian hibernation. Relying on interpretations of data that are based on steady-state regulation in other systems may be misleading in the context of non-steady-state conditions such as torpor.  

3.2210           Nuclear RhoA signaling regulates MRTF-dependent SMC-specific transcription

Staus, D.P., Weise-Cross, L., magnum, K.D., Medlin, M.D., mangiante, L., Taylor, J.M. and Mack, C.P. Am. J. Physiol. Heart Circ. Physiol., 307, H379-H390 (2014)   We have previously shown that RhoA-mediated actin polymerization stimulates smooth muscle cell (SMC)-specific transcription by regulating the nuclear localization of the myocardin-related transcription factors (MRTFs). On the basis of the recent demonstration that nuclear G-actin regulates MRTF nuclear export and observations from our laboratory and others that the RhoA effector, mDia2, shuttles between the nucleus and cytoplasm, we investigated whether nuclear RhoA signaling plays a role in regulating MRTF activity. We identified sequences that control mDia2 nuclear-cytoplasmic shuttling and used mDia2 variants to demonstrate that the ability of mDia2 to fully stimulate MRTF nuclear accumulation and SMC-specific gene transcription was dependent on its localization to the nucleus. To test whether RhoA signaling promotes nuclear actin polymerization, we established a fluorescence recovery after photobleaching (FRAP)-based assay to measure green fluorescent protein-actin diffusion in the nuclear compartment. Nuclear actin FRAP was delayed in cells expressing nuclear-targeted constitutively active mDia1 and mDia2 variants and in cells treated with the polymerization inducer, jasplakinolide. In contrast, FRAP was enhanced in cells expressing a nuclear-targeted variant of mDia that inhibits both mDia1 and mDia2. Treatment of 10T1/2 cells with sphingosine 1-phosphate induced RhoA activity in the nucleus and forced nuclear localization of RhoA or the Rho-specific guanine nucleotide exchange factor (GEF), leukemia-associated RhoGEF, enhanced the ability of these proteins to stimulate MRTF activity. Taken together, these data support the emerging idea that RhoA-dependent nuclear actin polymerization has important effects on transcription and nuclear structure.  

3.2211           Proteomic Analysis of the EWS-Fli-1 Interactome Reveals the Role of the Lysosome in EWS-Fli-1 Turnover

Elzi, D.J., Song, M., Hakala, K., Weintraub, S.T. and Shiio, Y.

1. Proteome Res., 13(8), 3783-3791 (2014)

  Ewing sarcoma is a cancer of bone and soft tissue in children that is characterized by a chromosomal translocation involving EWS and an Ets family transcription factor, most commonly Fli-1. EWS-Fli-1 fusion accounts for 85% of cases. The growth and survival of Ewing sarcoma cells are critically dependent on EWS-Fli-1. A large body of evidence has established that EWS-Fli-1 functions as a DNA-binding transcription factor that regulates the expression of a number of genes important for cell proliferation and transformation. However, little is known about the biochemical properties of the EWS-Fli-1 protein. We undertook a series of proteomic analyses to dissect the EWS-Fli-1 interactome. Employing a proximity-dependent biotinylation technique, BioID, we identified cation-independent mannose 6-phosphate receptor (CIMPR) as a protein located in the vicinity of EWS-Fli-1 within a cell. CIMPR is a cargo that mediates the delivery of lysosomal hydrolases from the trans-Golgi network to the endosome, which are subsequently transferred to the lysosomes. Further molecular cell biological analyses uncovered a role for lysosomes in the turnover of the EWS-Fli-1 protein. We demonstrate that an mTORC1 active-site inhibitor, torin 1, which stimulates the TFEB-lysosome pathway, can induce the degradation of EWS-Fli-1, suggesting a potential therapeutic approach to target EWS-Fli-1 for degradation.  

3.2212           Extracellular vesicles shed from gefitinib-resistant nonsmall cell lung cancer regulate the tumor microenvironment

Choi, D-Y., You, S., Jung, J.H., Lee, J.C., Rho, J.K., Lee, K.Y., Freeman, M.R., Kim, K.P. and Kim, J. Proteomics, 14(16), 1845-1856 (2014)   Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), including gefitinib, are the first-line treatment of choice for nonsmall cell lung cancer patients who harbor activating EGFR mutations, however, acquired resistance to EGFR-TKIs is inevitable. The main objective of this study was to identify informative protein signatures of extracellular vesicles (EV) derived from gefitinib-resistant nonsmall cell lung cancer cells using proteomics analysis. Nano-LC–MS/MS analysis identified with high confidence (false discovery rate < 0.05, fold change ≥2) 664 EV proteins enriched in PC9R cells, which are resistant to gefitinib due to EGFR T790M mutation. Computational analyses suggested components of several signal transduction mechanisms including the AKT (also PKB, protein kinase B)/mTOR (mechanistic target of rapamycin) pathway are overrepresented in EV from PC9R cells. Treatment of recipient cells with EV harvested from PC9R cells increased phosphorylation of signaling molecules, and enhanced proliferation, invasion, and drug resistance to gefitinib-induced apoptosis. Dose- and time-dependent pharmaceutical inhibition of AKT/mTOR pathway overcame drug resistance of PC9R cells and those of H1975 exhibiting EGFR T790M mutation. Our findings provide new insight into an oncogenic EV protein signature regulating tumor microenvironment, and will aid in the development of novel diagnostic strategies for prediction and assessment of gefitinib resistance.  

3.2213           Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

Daleke-Schermerhorn, M.H: et al Appl. Envir. Microbiol., 80(18), 5854-5865 (2014)   Outer membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of the Mycobacterium tuberculosis antigens ESAT6, Ag85B, and Rv2660c were targeted to the surface of Escherichia coli OMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolR ΔtolA derivative of attenuated Salmonella enterica serovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by the M. tuberculosis antigens and epitopes from Chlamydia trachomatis major outer membrane protein (MOMP). Also, we showed that delivery of Salmonella OMVs displaying Ag85B to antigen-presenting cells in vitro results in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.  

3.2214           Extracellular membrane vesicles secreted by mycoplasma Acholeplasma laidlawii PG8 are enriched in virulence proteins

Chernov, V., Mouzykantov, A.A., baranova, N.B., Medvedeva, E.S., Grygorieva, T.Y., Trushin, M.V., Vishnyakov, I.E., Sabantsev, A.V., Borchsenius, S.N. and Chernova, O.A.

1. Proteomics, 110, 117-128 (2014)

  Mycoplasmas (class Mollicutes), the smallest prokaryotes capable of self-replication, as well as Archaea, Gram-positive and Gram-negative bacteria constitutively produce extracellular vesicles (EVs). However, little is known regarding the content and functions of mycoplasma vesicles. Here, we present for the first time a proteomics-based characterisation of extracellular membrane vesicles from Acholeplasma laidlawii PG8. The ubiquitous mycoplasma is widespread in nature, found in humans, animals and plants, and is the causative agent of phytomycoplasmoses and the predominant contaminant of cell cultures. Taking a proteomics approach using LC–ESI-MS/MS, we identified 97 proteins. Analysis of the identified proteins indicated that A. laidlawii-derived EVs are enriched in virulence proteins that may play critical roles in mycoplasma-induced pathogenesis. Our data will help to elucidate the functions of mycoplasma-derived EVs and to develop effective methods to control infections and contaminations of cell cultures by mycoplasmas. In the present study, we have documented for the first time the proteins in EVs secreted by mycoplasma vesicular proteins identified in this study are likely involved in the adaptation of bacteria to stressors, survival in microbial communities and pathogen–host interactions. These findings suggest that the secretion of EVs is an evolutionally conserved and universal process that occurs in organisms from the simplest wall-less bacteria to complex organisms and indicate the necessity of developing new approaches to control infects.  

3.2215           Outer Membrane Vesicles Mediate Transport of Biologically Active Vibrio cholerae Cytolysin (VCC) from V. cholerae Strains

Elluri, S., Enow, C., Vdovikova, S., Rompikuntal, P.K., Dongre, M., Carlsson, S., Pal, A., Uhlin, B.E. and Wai, S.N. PloS One, 9(9), e106731 (2014)   Background Outer membrane vesicles (OMVs) released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC), is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied. Methodology/Principal Findings OMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor. Conclusion/Significance Biological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and induces toxicity on mammalian cells and furthermore can induce autophagy.  

3.2216           Exosomes from Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Cells License Quiescent CD4+ T Lymphocytes To Replicate HIV-1 through a Nef- and ADAM17-Dependent Mechanism

Arenaccio, C., Chiozzini, C., Columba-Cabezas, S., Manfredi, F., Affabris, E., Baur, A. and Federico, M.

1. Virol., 88(19), 11529-11539 (2014)

  Resting CD4⁺ T lymphocytes resist human immunodeficiency virus (HIV) infection. Here, we provide evidence that exosomes from HIV-1-infected cells render resting human primary CD4⁺ T lymphocytes permissive to HIV-1 replication. These results were obtained with transwell cocultures of HIV-1-infected cells with quiescent CD4⁺ T lymphocytes in the presence of inhibitors of exosome release and were confirmed using exosomes purified from supernatants of HIV-1-infected primary CD4⁺ T lymphocytes. We found that the expression of HIV-1 Nef in exosome-producing cells is both necessary and sufficient for cell activation as well as HIV-1 replication in target CD4⁺ T lymphocytes. We also identified a Nef domain important for the effects we observed, i.e., the ⁶²EEEE⁶⁵ acidic cluster domain. In addition, we observed that ADAM17, i.e., a disintegrin and metalloprotease converting pro-tumor necrosis factor alpha (TNF-α) in its mature form, associates with exosomes from HIV-1-infected cells, and plays a key role in the HIV-1 replication in quiescent CD4⁺ T lymphocytes. Treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in resting CD4⁺ T lymphocytes. TNF-α is the downstream effector of ADAM17 since the treatment of resting lymphocytes with anti-TNF-α antibodies blocked the HIV-1 replication. The data presented here are consistent with a model where Nef induces intercellular communication through exosomes to activate bystander quiescent CD4⁺ T lymphocytes, thus stimulating viral spread.  

3.2217           Diagnostic and Prognostic Potential of Extracellular Vesicles in Peripheral Blood

Revenfeld, A.L.S., Bæk, R., Hjuler Nielsen, M., Stensballe, A., Varming, K. and Jørgensen, M. Clinical Therapeutics, 36(6), 830-846 (2014)   Purpose Extracellular vesicles (EVs) are small, membrane-enclosed entities released from cells in many different biological systems. These vesicles play an important role in cellular communication by virtue of their protein, RNA, and lipid content, which can be transferred among cells. The complement of biomolecules reflects the parent cell, and their characterization may provide information about the presence of an aberrant process. Peripheral blood is a rich source of circulating EVs, which are easily accessible through a blood sample. An analysis of EVs in peripheral blood could provide access to unparalleled amounts of biomarkers of great diagnostic and prognostic value. The objectives of this review are to briefly present the current knowledge about EVs and to introduce a toolbox of selected techniques, which can be used to rapidly characterize clinically relevant properties of EVs from peripheral blood. Methods Several techniques exist to characterize the different features of EVs, including size, enumeration, RNA cargo, and protein phenotype. Each technique has a number of advantages and pitfalls. However, with the techniques presented in this review, a possible platform for EV characterization in a clinical setting is outlined. Findings Although EVs have great diagnostic and prognostic potential, a lack of standardization regarding EV analysis hampers the full use of this potential. Nevertheless, the analysis of EVs in peripheral blood has several advantages compared with traditional analyses of many soluble molecules in blood. Implications Overall, the use of EV analysis as a diagnostic and prognostic tool has prodigious clinical potential.  

3.2218           Production of Outer Membrane Vesicles by the Plague Pathogen Yersinia pestis

Eddy, J.L., Gielda, L.M., Caulfield, A.J., Rangel, S.M. and Lathem, W.W. PloS One, 9(9), e107002 (2014)   Many Gram-negative bacteria produce outer membrane vesicles (OMVs) during cell growth and division, and some bacterial pathogens deliver virulence factors to the host via the release of OMVs during infection. Here we show that Yersinia pestis, the causative agent of the disease plague, produces and releases native OMVs under physiological conditions. These OMVs, approximately 100 nm in diameter, contain multiple virulence-associated outer membrane proteins including the adhesin Ail, the F1 outer fimbrial antigen, and the protease Pla. We found that OMVs released by Y. pestis contain catalytically active Pla that is competent for plasminogen activation and α2-antiplasmin degradation. The abundance of OMV-associated proteins released by Y. pestis is significantly elevated at 37°C compared to 26°C and is increased in response to membrane stress and mutations in RseA, Hfq, and the major Braun lipoprotein (Lpp). In addition, we show that Y. pestis OMVs are able to bind to components of the extracellular matrix such as fibronectin and laminin. These data suggest that Y. pestis may produce OMVs during mammalian infection and we propose that dispersal of Pla via OMV release may influence the outcome of infection through interactions with Pla substrates such as plasminogen and Fas ligand.  

3.2219           Identification and Characterization of Outer Membrane Vesicle-Associated Proteins in Salmonella enterica Serovar Typhimurium

Bai, J., Kim, S.I., Ryu, S. and Yoon, H. Infect. Immun., 82(10), 4001-4010 (2014)   Salmonella enterica serovar Typhimurium is a primary cause of enteric diseases and has acquired a variety of virulence factors during its evolution into a pathogen. Secreted virulence factors interact with commensal flora and host cells and enable Salmonella to survive and thrive in hostile environments. Outer membrane vesicles (OMVs) released from many Gram-negative bacteria function as a mechanism for the secretion of complex mixtures, including virulence factors. We performed a proteomic analysis of OMVs that were isolated under standard laboratory and acidic minimal medium conditions and identified 14 OMV-associated proteins that were observed in the OMV fraction isolated only under the acidic minimal medium conditions, which reproduced the nutrient-deficient intracellular milieu. The inferred roles of these 14 proteins were diverse, including transporter, enzyme, and transcriptional regulator. The absence of these proteins influenced Salmonella survival inside murine macrophages. Eleven of these proteins were predicted to possess secretion signal sequences at their N termini, and three (HupA, GlnH, and PhoN) of the proteins were found to be translocated into the cytoplasm of host cells. The comparative proteomic profiling of OMVs performed in this study revealed different protein compositions in the OMVs isolated under the two different conditions, which indicates that the OMV cargo depends on the growth conditions and provides a deeper insight into how Salmonella utilizes OMVs to adapt to environmental changes.  

3.2220           Retroviral Retention Activates a Syk-Dependent HemITAM in Human Tetherin

Galao, R.P., Pickering, S., Curnock, R. and Neil, S.J.D. Cell Host & Microbe, 16, 291-303 (2014)   Tetherin (BST2/CD317) restricts the release of enveloped viral particles from infected cells. Coupled to this virion retention, hominid tetherins induce proinflammatory gene expression via activating NF-κB. We investigated the events initiating this tetherin-induced signaling and show that physical retention of retroviral particles induces the phosphorylation of conserved tyrosine residues in the cytoplasmic tails of tetherin dimers. This phosphorylation induces the recruitment of spleen tyrosine kinase (Syk), which is required for downstream NF-κB activation, indicating that the tetherin cytoplasmic tail resembles the hemi-immunoreceptor tyrosine-based activation motifs (hemITAMs) found in C-type lectin pattern recognition receptors. Retroviral-induced tetherin signaling is coupled to the cortical actin cytoskeleton via the Rac-GAP-containing protein RICH2 (ARHGAP44), and a naturally occurring tetherin polymorphism with reduced RICH2 binding exhibits decreased phosphorylation and NF-κB activation. Thus, upon virion retention, this linkage to the actin cytoskeleton likely triggers tetherin phosphorylation and subsequent signal transduction to induce an antiviral state.  

3.2221           Epoxide-Mediated Differential Packaging of Cif and Other Virulence Factors into Outer Membrane Vesicles

Ballok, A.E., Filkins, L.M., Bomberger, J.M., Staton, B.A. and O’Toole, G.A.

1. Bacteriol., 196(20), 3633-3642 (2014)

  Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or host-relevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions.  

3.2222           Leishmania donovani activates SREBP2 to modulate macrophage membrane cholesterol and mitochondrial oxidants for establishment of infection

Mukherjee, M., Ball, W.B. and Das, P.K. Int. J. Biochem. Cell Biol., 55, 196-208 (2014)   Establishment of infection by an intracellular pathogen depends on successful internalization with a concomitant neutralization of host defense machinery. Leishmania donovani, an intramacrophage pathogen, targets host SREBP2, a critical transcription factor, to regulate macrophage plasma membrane cholesterol and mitochondrial reactive oxygen species generation, favoring parasite invasion and persistence. Leishmania infection triggered membrane-raft reorientation-dependent Lyn-PI3K/Akt pathway activation which in turn deactivated GSK3β to stabilize nuclear SREBP2. Moreover, cells perceiving less available intracellular cholesterol due to its sequestration at the plasma membrane resulted in the deregulation of the ER-residing SCAP-SREBP2-Insig circuit thereby assisting increased nuclear translocation of SREBP2. Both increased nuclear transport and stabilization of SREBP2 caused HMGCR-catalyzed cholesterol biosynthesis-mediated plasma membrane cholesterol enrichment leading to decreased membrane-fluidity and plausibly assisting delay in phagosomal acidification. Parasite survival ensuing entry was further ensured by SREBP2-dependent trasnscriptional up-regulation of UCP2, which suppressed mitochondrial ROS generation, one of the primary microbicidal molecules in macrophages recognized for its efficacy against Leishmania. Functional knock-down of SREBP2 both in vitro and in vivo was associated with reduction in macrophage plasma membrane cholesterol, increased ROS production and lower parasite survival. To our knowledge, this study, for the first time, reveals that Leishmania exploits macrophage cholesterol-dependent SREBP2 circuit to facilitate its entry and survival within the host.  

3.2223           A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes

Karunanithi, S., iong, T., Uhm, M., Leto, D., Sun, J., Chen, X-W. and Saltiel, A.R. Mol. Biol. Cell, 25, 3059-3069 (2014)   Insulin-stimulated glucose uptake in fat and muscle is mediated by the major facilitative glucose transporter Glut4. Insulin controls the trafficking of Glut4 to the plasma membrane via regulation of a series of small G proteins, including RalA and Rab10. We demonstrate here that Rab10 is a bona fide target of the GTPase-activating protein AS160, which is inhibited after phosphorylation by the protein kinase Akt. Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2. Rab10 and RalA reside in the same pool of Glut4-storage vesicles in untreated cells, and, together with Rlf, they ensure maximal glucose transport. Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10. Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake.  

3.2224           Isolation and Functional Assessment of Eosinophil Crystalloid Granules

Baptista-dos-Reis, R., Muniz, V.S. and Neves, J.S.

Methods in Mol. Biol., 1178, 93-100 (2014)

Cell-free granules, upon extrusion from human eosinophils, remain fully competent to secrete granule-derived proteins in receptor-mediated processes in response to different stimuli. However, in order to avoid the shrinkage and damage of granules, as well as preserve their structure, properties, and functionality, the use of an optimized process of subcellular fractionation using an isoosmotic density gradient is needed. Here, we describe a detailed protocol of subcellular fractionation of nitrogen-cavitated eosinophils on an isoosmotic iodinated density gradient (iodixanol) and the isolation of well-preserved and functional membrane-bound specific granules.

 

3.2225           Role of LARP6 and Nonmuscle Myosin in Partitioning of Collagen mRNAs to the ER Membrane

Wang, H. and Stefanovic, B. PloS One, 9(10), e108870 (2014)   Type I collagen is extracellular matrix protein composed of two α1(I) and one α2(I) polypeptides that fold into triple helix. Collagen polypeptides are translated in coordination to synchronize the rate of triple helix folding to the rate of posttranslational modifications of individual polypeptides. This is especially important in conditions of high collagen production, like fibrosis. It has been assumed that collagen mRNAs are targeted to the membrane of the endoplasmic reticulum (ER) after translation of the signal peptide and by signal peptide recognition particle (SRP). Here we show that collagen mRNAs associate with the ER membrane even when translation is inhibited. Knock down of LARP6, an RNA binding protein which binds 5′ stem-loop of collagen mRNAs, releases a small amount of collagen mRNAs from the membrane. Depolimerization of nonmuscle myosin filaments has a similar, but stronger effect. In the absence of LARP6 or nonmuscle myosin filaments collagen polypeptides become hypermodified, are poorly secreted and accumulate in the cytosol. This indicates lack of coordination of their synthesis and retro-translocation due to hypermodifications and misfolding. Depolimerization of nonmuscle myosin does not alter the secretory pathway through ER and Golgi, suggesting that the role of nonmuscle myosin is primarily to partition collagen mRNAs to the ER membrane. We postulate that collagen mRNAs directly partition to the ER membrane prior to synthesis of the signal peptide and that LARP6 and nonmuscle myosin filaments mediate this process. This allows coordinated initiation of translation on the membrane bound collagen α1(I) and α2(I) mRNAs, a necessary step for proper synthesis of type I collagen.  

3.2226           The Get1/2 transmembrane complex is an endoplasmic-reticulum membrane protein insertase

Wang, F., Chan, C., Weir, N.R. and Denic, V. Nature Letter, 512, 441-444 (2014)   Hundreds of tail-anchored proteins, including soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs) involved in vesicle fusion, are inserted post-translationally into the endoplasmic reticulum membrane by a dedicated protein-targeting pathway^{1, 2, 3, 4}. Before insertion, the carboxy-terminal transmembrane domains of tail-anchored proteins are shielded in the cytosol by the conserved targeting factor Get3 (in yeast; TRC40 in mammals)^{5, 6, 7}. The Get3 endoplasmic-reticulum receptor comprises the cytosolic domains of the Get1/2 (WRB/CAML) transmembrane complex, which interact individually with the targeting factor to drive a conformational change that enables substrate release and, as a consequence, insertion^{8, 9, 10, 11}. Because tail-anchored protein insertion is not associated with significant translocation of hydrophilic protein sequences across the membrane, it remains possible that Get1/2 cytosolic domains are sufficient to place Get3 in proximity with the endoplasmic-reticulum lipid bilayer and permit spontaneous insertion to occur^{12, 13}. Here we use cell reporters and biochemical reconstitution to define mutations in the Get1/2 transmembrane domain that disrupt tail-anchored protein insertion without interfering with Get1/2 cytosolic domain function. These mutations reveal a novel Get1/2 insertase function, in the absence of which substrates stay bound to Get3 despite their proximity to the lipid bilayer; as a consequence, the notion of spontaneous transmembrane domain insertion is a non sequitur. Instead, the Get1/2 transmembrane domain helps to release substrates from Get3 by capturing their transmembrane domains, and these transmembrane interactions define a bona fide pre-integrated intermediate along a facilitated route for tail-anchor entry into the lipid bilayer. Our work sheds light on the fundamental point of convergence between co-translational and post-translational endoplasmic-reticulum membrane protein targeting and insertion: a mechanism for reducing the ability of a targeting factor to shield its substrates enables substrate handover to a transmembrane-domain-docking site embedded in the endoplasmic-reticulum membrane.  

3.2227           Membrane vesicle formation is associated with pyocin production under denitrifying conditions in Pseudomonas aeruginosa PAO1

Toyofuku, M., Zhou, S., Sawada, I., Takaya, N., Uchiyama, H. and Nomura, N. Environmental Microbiol., 16(9), 2927-2938 (2014)   Many Gram-negative bacteria produce membrane vesicles (MVs) that serve as vehicles to mediate intraspecies and interspecies interactions. Despite their ubiquity in Gram-negative bacteria and their biological importance, how MV formation is regulated is poorly understood. Pseudomonas aeruginosa is a ubiquitous bacterium that is one of the most extensively studied model organism in MVs. Recent studies highlight the importance of a quorum-sensing signal, Pseudomonas quinolone signal (PQS), in the formation of MVs; however, PQS synthesis requires oxygen and is not produced under anoxic conditions. This situation leads to the question of MV production under anoxic conditions. Here, we examined whether MVs are produced under denitrifying conditions and what kind of factors are involved in the MV production under such condition. Under denitrifying condition, P. aeruginosa PAO1 produced a considerable amount of MVs. Interestingly, pyocin components were found to be accumulated in the isolated MVs. Pyocin-related protein mutants produced less MVs compared with the wild type. We further indicate that pyocin production is activated by nitric oxide, in which the SOS response is involved. This study presents a regulatory mechanism where pyocin is associated with MV production, and further implies how the environment impacts MV production in P. aeruginosa.  

3.2228           Concentration of Sec12 at ER exit sites via interaction with cTAGE5 is required for collagen export

Saito, K., Yamashiro, N., Tanabe, T., Kontani, K. and katada, T.

1. Cell Biol., 206(6), 751-762 (2014)

  Mechanisms for exporting variably sized cargo from the endoplasmic reticulum (ER) using the same machinery remain poorly understood. COPII-coated vesicles, which transport secretory proteins from the ER to the Golgi apparatus, are typically 60–90 nm in diameter. However, collagen, which forms a trimeric structure that is too large to be accommodated by conventional transport vesicles, is also known to be secreted via a COPII-dependent process. In this paper, we show that Sec12, a guanine-nucleotide exchange factor for Sar1 guanosine triphosphatase, is concentrated at ER exit sites and that this concentration of Sec12 is specifically required for the secretion of collagen VII but not other proteins. Furthermore, Sec12 recruitment to ER exit sites is organized by its direct interaction with cTAGE5, a previously characterized collagen cargo receptor component, which functions together with TANGO1 at ER exit sites. These findings suggest that the export of large cargo requires high levels of guanosine triphosphate–bound Sar1 generated by Sec12 localized at ER exit sites.  

3.2229           The Atypical Cadherin Fat Directly Regulates Mitochondrial Function and Metabolic State

Ssing, A., Tsatskis, Y., Fabian, L., Hester, I., Rosenfeld, R., Serricchio, M., Yau, N., Bietenhader, M., Shanbhag, R., Jurisicova, A., Brill, J.A., McQuibban, G.A. and McNeill, H. Cell, 158(6), 1293-1308 (2014)   Fat (Ft) cadherins are enormous cell adhesion molecules that function at the cell surface to regulate the tumor-suppressive Hippo signaling pathway and planar cell polarity (PCP) tissue organization. Mutations in Ft cadherins are found in a variety of tumors, and it is presumed that this is due to defects in either Hippo signaling or PCP. Here, we show Drosophila Ft functions in mitochondria to directly regulate mitochondrial electron transport chain integrity and promote oxidative phosphorylation. Proteolytic cleavage releases a soluble 68 kDa fragment (Ft^mito) that is imported into mitochondria. Ft^mito binds directly to NADH dehydrogenase ubiquinone flavoprotein 2 (Ndufv2), a core component of complex I, stabilizing the holoenzyme. Loss of Ft leads to loss of complex I activity, increases in reactive oxygen species, and a switch to aerobic glycolysis. Defects in mitochondrial activity in ft mutants are independent of Hippo and PCP signaling and are reminiscent of the Warburg effect.  

3.2230           Clathrin Assembly Protein CALM Plays a Critical Role in KIT Signaling by Regulating Its Cellular Transport from Early to Late Endosomes in Hematopoietic Cells

Rai, S., Tanaka, H., Suzuki, M., Ogoh, H., Taniguchi, Y., Morita, Y., Shimada, T., Tanimura, A., Matsui, K., Yokota, T., Oritani, K., Tanabe, K., Watanabe, T., Kanakura, Y. and Matsumura, I. PloS One, 9(10), e109441 (2014)   CALM is implicated in the formation of clathrin-coated vesicles, which mediate endocytosis and intracellular trafficking of growth factor receptors and nutrients. We previously found that CALM-deficient mice suffer from severe anemia due to the impaired clathrin-mediated endocytosis of transferrin receptor in immature erythroblast. However, CALM has been supposed to regulate the growth and survival of hematopoietic stem/progenitor cells. So, in this study, we focused on the function of CALM in these cells. We here show that the number of Linage⁻Sca-1⁺KIT⁺ (LSK) cells decreased in the fetal liver of CALM^−/− mice. Also, colony forming activity was impaired in CALM^−/− LSK cells. In addition, SCF, FLT3, and TPO-dependent growth was severely impaired in CALM^−/− LSK cells, while they can normally proliferate in response to IL-3 and IL-6. We also examined the intracellular trafficking of KIT using CALM^−/− murine embryonic fibroblasts (MEFs) engineered to express KIT. At first, we confirmed that endocytosis of SCF-bound KIT was not impaired in CALM^−/− MEFs by the internalization assay. However, SCF-induced KIT trafficking from early to late endosome was severely impaired in CALM^−/− MEFs. As a result, although intracellular KIT disappeared 30 min after SCF stimulation in wild-type (WT) MEFs, it was retained in CALM^−/− MEFs. Furthermore, SCF-induced phosphorylation of cytosolic KIT was enhanced and prolonged in CALM^−/− MEFs compared with that in WT MEFs, leading to the excessive activation of Akt. Similar hyperactivation of Akt was observed in CALM^−/− KIT⁺ cells. These results indicate that CALM is essential for the intracellular trafficking of KIT and its normal functions. Also, our data demonstrate that KIT located in the early endosome can activate downstream molecules as a signaling endosome. Because KIT activation is involved in the pathogenesis of some malignancies, the manipulation of CALM function would be an attractive therapeutic strategy.  

3.2231           Modulation of Hepatitis C Virus Genome Replication by Glycosphingolipids and Four-Phosphate Adaptor Protein 2

Khan, I., Katikaneni, D.S., han, Q., Sanchez-Felipe, L., Hanada, K., Ambrose, R.L., macKenzie, J.M. and Konan, K.V.

1. Virol., 88(21), 12276-12295 (2014)

  Hepatitis C virus (HCV) assembles its replication complex on cytosolic membrane vesicles often clustered in a membranous web (MW). During infection, HCV NS5A protein activates PI4KIIIα enzyme, causing massive production and redistribution of phosphatidylinositol 4-phosphate (PI4P) lipid to the replication complex. However, the role of PI4P in the HCV life cycle is not well understood. We postulated that PI4P recruits host effectors to modulate HCV genome replication or virus particle production. To test this hypothesis, we generated cell lines for doxycycline-inducible expression of short hairpin RNAs (shRNAs) targeting the PI4P effector, four-phosphate adaptor protein 2 (FAPP2). FAPP2 depletion attenuated HCV infectivity and impeded HCV RNA synthesis. Indeed, FAPP2 has two functional lipid-binding domains specific for PI4P and glycosphingolipids. While expression of the PI4P-binding mutant protein was expected to inhibit HCV replication, a marked drop in replication efficiency was observed unexpectedly with the glycosphingolipid-binding mutant protein. These data suggest that both domains are crucial for the role of FAPP2 in HCV genome replication. We also found that HCV significantly increases the level of some glycosphingolipids, whereas adding these lipids to FAPP2-depleted cells partially rescued replication, further arguing for the importance of glycosphingolipids in HCV RNA synthesis. Interestingly, FAPP2 is redistributed to the replication complex (RC) characterized by HCV NS5A, NS4B, or double-stranded RNA (dsRNA) foci. Additionally, FAPP2 depletion disrupts the RC and alters the colocalization of HCV replicase proteins. Altogether, our study implies that HCV coopts FAPP2 for virus genome replication via PI4P binding and glycosphingolipid transport to the HCV RC.  

3.2232           Stearoyl Coenzyme A Desaturase 1 Is Associated with Hepatitis C Virus Replication Complex and Regulates Viral Replication

  Nguuyen, L.N., Lim, Y-S., Pham, L.V., Shin, H-Y., Kim, Y-S. and Hwang, S.B.

1. Virol., 88(21), 12311-12325 (2014)

  The hepatitis C virus (HCV) life cycle is tightly regulated by lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have recently screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet formation using cell culture-grown HCV (HCVcc)-infected cells. We selected and characterized the gene encoding stearoyl coenzyme A (CoA) desaturase 1 (SCD1). siRNA-mediated knockdown or pharmacological inhibition of SCD1 abrogated HCV replication in both subgenomic replicon and Jc1-infected cells, while exogenous supplementation of either oleate or palmitoleate, products of SCD1 activity, resurrected HCV replication in SCD1 knockdown cells. SCD1 was coimmunoprecipitated with HCV nonstructural proteins and colocalized with both double-stranded RNA (dsRNA) and HCV nonstructural proteins, indicating that SCD1 is associated with HCV replication complex. Moreover, SCD1 was fractionated and enriched with HCV nonstructural proteins at detergent-resistant membrane. Electron microscopy data showed that SCD1 is required for NS4B-mediated intracellular membrane rearrangement. These data further support the idea that SCD1 is associated with HCV replication complex and that its products may contribute to the proper formation and maintenance of membranous web structures in HCV replication complex. Collectively, these data suggest that manipulation of SCD1 activity may represent a novel host-targeted antiviral strategy for the treatment of HCV infection.  

3.2233           Proteome investigation of an organellar fraction of Toxoplasma gondii: a preliminary study

Ferreira, D.da S., resende, I.T.M. and Lopez, J.A. BMC Proceedings, 8(Suppl 4), P74 (2014)   Toxoplasma gondii is a ubiquitous Apicomplexan parasite responsible for systemic diseases in both humans and animals. Toxoplasmosis is a major public health problem, infecting one-third of the world's human population and leading to abortion in domestic animals [1]. The search for new chemotherapeutic targets is imperative, due to its increasing resistance to the drugs currently available for combating this parasite [2]. Recent high-throughput Proteomic approaches have provided a wealth of protein expression data on Apicomplexan parasites (e.g., T. gondii, Plasmodium falciparum), while a number of smaller-scale studies have examined specific drug-related hypotheses. Proteomic methods can be applied to study sub-cellular localization, cell function, organelle composition, changes in protein expression patterns in response to drug exposure, drug-protein binding, and validation of data from genomic annotation and transcript expression studies [3]. Organellar structures have therefore become potential targets for the parasite life cycle to control the levels of nutrients or salts that surround them [4]. The aim of this study was to perform a proteomic analysis of an organellar fraction of this Apicomplexan protozoan based on the structural and metabolic aspects.  

3.2234           ABCA1, ABCG1, and ABCG4 Are Distributed to Distinct Membrane Meso-Domains and Disturb Detergent-Resistant Domains on the Plasma Membrane

Sano, O., Ito, S., kato, R., Shimizu, Y., Kobayashi, A., Kimura, Y., Kioka, N., hanada, K., Ueda, K. and Matsuo, M. PloS One, 9(10), e109886 (2014)   ATP-binding cassette A1 (ABCA1), ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters.  

3.2235           Endocytosis of Secreted Carboxyl Ester Lipase in a Syndrome of Diabetes and Pancreatic Exocrine Dysfunction

Torsvik, J., Johansson, B.B., Dalva, M., Marie, M., Fjeld, K., Johansson, S., Bjørkøy, G., Saraste, J., Njølstad, P.R. and Molven, A.

1. Biol. Chem., 289, 29097-29111 (2014)

  Maturity-onset diabetes of the young, type 8 (MODY8) is characterized by a syndrome of autosomal dominantly inherited diabetes and exocrine pancreatic dysfunction. It is caused by deletion mutations in the last exon of the carboxyl ester lipase (CEL) gene, resulting in a CEL protein with increased tendency to aggregate. In this study we investigated the intracellular distribution of the wild type (WT) and mutant (MUT) CEL proteins in cellular models. We found that both CEL-WT and CEL-MUT were secreted via the endoplasmic reticulum and Golgi compartments. However, their subcellular distributions differed, as only CEL-MUT was observed as an aggregate at the cell surface and inside large cytoplasmic vacuoles. Many of the vacuoles were identified as components of the endosomal system, and after its secretion, the mutant CEL protein was re-internalized, transported to the lysosomes, and degraded. Internalization of CEL-MUT also led to reduced viability of pancreatic acinar and beta cells. These findings may have implications for the understanding of how the acinar-specific CEL-MUT protein causes both exocrine and endocrine pancreatic disease.  

3.2236           Hallmarks of Hepatitis C Virus in Equine Hepacivirus

Tanaka, T., Kasai, H., Yamashita, A., Okuyama-Dobashi, K., Yasumoto, J., maekawa, S., Enomoto, N., Okamoto, T., Matsuura, Y., Morimatsu, M., Manabe, N., Ochiai, K., Yamashita, K. and Moriishi, K.

1. Virol., 88(22), 13352-13366 (2014)

  Equine hepacivirus (EHcV) has been identified as a closely related homologue of hepatitis C virus (HCV) in the United States, the United Kingdom, and Germany, but not in Asian countries. In this study, we genetically and serologically screened 31 serum samples obtained from Japanese-born domestic horses for EHcV infection and subsequently identified 11 PCR-positive and 7 seropositive serum samples. We determined the full sequence of the EHcV genome, including the 3′ untranslated region (UTR), which had previously not been completely revealed. The polyprotein of a Japanese EHcV strain showed approximately 95% homology to those of the reported strains. HCV-like cis-acting RNA elements, including the stem-loop structures of the 3′ UTR and kissing-loop interaction were deduced from regions around both UTRs of the EHcV genome. A comparison of the EHcV and HCV core proteins revealed that Ile¹⁹⁰ and Phe¹⁹¹ of the EHcV core protein could be important for cleavage of the core protein by signal peptide peptidase (SPP) and were replaced with Ala and Leu, respectively, which inhibited intramembrane cleavage of the EHcV core protein. The loss-of-function mutant of SPP abrogated intramembrane cleavage of the EHcV core protein and bound EHcV core protein, suggesting that the EHcV core protein may be cleaved by SPP to become a mature form. The wild-type EHcV core protein, but not the SPP-resistant mutant, was localized on lipid droplets and partially on the lipid raft-like membrane in a manner similar to that of the HCV core protein. These results suggest that EHcV may conserve the genetic and biological properties of HCV.  

3.2237           Nuclear Enrichment of Folate Cofactors and Methylenetetrahydrofolate Dehydrogenase 1 (MTHFD1) Protect de Novo Thymidylate Biosynthesis during Folate Deficiency

Field, M.S., Kamynina, E., Agunloye, O.C., Liebenthal, R.P., lamarre, S.G., Brosnan, M.E., Brosnan, J.T. and Stover, P.J.

1. Biol. Chem., 289(43), 29642-29650 (2014)

  Folate-mediated one-carbon metabolism is a metabolic network of interconnected pathways that is required for the de novo synthesis of three of the four DNA bases and the remethylation of homocysteine to methionine. Previous studies have indicated that the thymidylate synthesis and homocysteine remethylation pathways compete for a limiting pool of methylenetetrahydrofolate cofactors and that thymidylate biosynthesis is preserved in folate deficiency at the expense of homocysteine remethylation, but the mechanisms are unknown. Recently, it was shown that thymidylate synthesis occurs in the nucleus, whereas homocysteine remethylation occurs in the cytosol. In this study we demonstrate that methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), an enzyme that generates methylenetetrahydrofolate from formate, ATP, and NADPH, functions in the nucleus to support de novo thymidylate biosynthesis. MTHFD1 translocates to the nucleus in S-phase MCF-7 and HeLa cells. During folate deficiency mouse liver MTHFD1 levels are enriched in the nucleus >2-fold at the expense of levels in the cytosol. Furthermore, nuclear folate levels are resistant to folate depletion when total cellular folate levels are reduced by >50% in mouse liver. The enrichment of folate cofactors and MTHFD1 protein in the nucleus during folate deficiency in mouse liver and human cell lines accounts for previous metabolic studies that indicated 5,10-methylenetetrahydrofolate is preferentially directed toward de novo thymidylate biosynthesis at the expense of homocysteine remethylation during folate deficiency.  

3.2238           β-Elemene against human lung cancer via up-regulation of P53 protein expression to promote the release of exosome

Li, J., Yu, J. and wang, Y. Lung Cancer, 86, 144-150 (2014)   Background β-Elemene, a novel antitumor plant drug extracted from the traditional Chinese medicinal herb Zedoary, has been shown to be effective against a wide variety of tumors. Recent studies have indicated that β-elemene can inhibit the growth of lung cancer cells; however, the exact mechanism of β-element's action in lung cancer remains largely unknown. In the present study, the antitumor effect of β-elemene on human lung cancer cells and the mechanism involved has been investigated. Methods The inhibitory effects of β-elemene on cell growth were measured by Trypan Blue exclusion and MTT assay. Flow cytometric analysis was used to detect the cells’ apoptotic rate. The expression of P53 mRNA and protein were measured by RT-PCR and Western blot analysis, respectively. Exosomes were isolated by differential centrifugation steps and analyzed by electron microscopy and western blotting. P53 knockdown cells were established through transfection with P53 siRNA. To investigate the effect of β-elemene on the tumor growth in vivo, a Xenograft nude mouse model was established by injecting the A549 cells into the back of a BABL/c nude mouse. Results β-Elemene markedly inhibited growth and induced apoptosis in lung cancer cells. The levels of the anti-apoptotic genes Bcl-2 and Bcl-xl in A549 cells decreased, while expression of P53 and production of exosomes increased after β-elemene treatment. Further siRNA studies suggested that the effect of β-elemene on A549 cells is dependent on P53 expression. Exosomes derived from A549 cultured with a human lung cancer cell line exhibited decreased tumor cell proliferation. The in vivo study demonstrated that β-elemene inhibited tumor growth, and up-regulated the expression of P53 and the release of exosome. Conclusion Our results demonstrated β-elemene acts on lung cancer cells in a P53 dependent manner and exosomes are involved in the regulation of cell proliferation.    

3.2239           Apolipoprotein J, a glucose-upregulated molecular chaperone, stabilizes core and NS5A to promote infectious hepatitis C virus virion production

Lin, C-C., Tsai, P., Sun, H-Y., Hsu, M-C., Lee, J-C., Wu, I-C., Tsao, C-W., Chang, T-T. and Young, K-C.

1. Hepatol., 61, 984-993 (2014)

  Background & Aims Hepatitis C virus (HCV) infection leads to glucose abnormality. HCV depends on lipid droplets (LDs) and very-low density lipoproteins for assembly/releasing; however, the components and locations for this process remain unidentified. Apolipoprotein J (ApoJ), upregulated by glucose, functions as Golgi chaperone of secreted proteins and resides abundantly in very-low density lipoproteins. This study investigates the interplay between glucose, ApoJ and HCV virion production. Methods The effects of high glucose on ApoJ expression and HCV production were evaluated with cultivated HuH7.5, primary human hepatocytes, and in treatment naive chronic hepatitis C patients. How ApoJ affects HCV lifecycle was assessed using siRNA knockdown strategy in JFH1 infected and subgenomic replicon cells. The interactions and locations of ApoJ with viral and host components were examined by immunoprecipitation, immunofluorescence and subcellular fractionation experiments. Results HCV infection increased ApoJ expression, which in parallel with HCV infectivity was additionally elevated with high glucose treatment. Serum ApoJ correlated positively with fasting blood glucose concentration and HCV-RNA titre in patients. ApoJ silencing reduced intracellular and extracellular HCV infectivity and extracellular HCV-RNA, but accumulated intracellular HCV-RNA in HCV-infected cells. ApoJ interacted with HCV core and NS5A and stabilized the dual protein complex. HCV infection dispersed cytoplasmic ApoJ from the compact zones of the Golgi to encircle LDs, where co-localization of the core, NS5A, HCV-RNA, subcellular markers for LDs, endoplasmic reticulum (ER), Golgi, and membrane contact sites occurred. Conclusions ApoJ facilitates infectious HCV particle production via stabilization of core/NS5A, which might surround LDs at the ER-Golgi membrane contact site.  

3.2240           HTLV-1 Tax Stabilizes MCL-1 via TRAF6-Dependent K63-Linked Polyubiquitination to Promote Cell Survival and Transformation

Choi, Y.B. and Harhaj, E.W. PloS Pathogens, 10(10), e1004458 (2014)   The human T-cell leukemia virus type 1 (HTLV-1) Tax protein hijacks the host ubiquitin machinery to activate IκB kinases (IKKs) and NF-κB and promote cell survival; however, the key ubiquitinated factors downstream of Tax involved in cell transformation are unknown. Using mass spectrometry, we undertook an unbiased proteome-wide quantitative survey of cellular proteins modified by ubiquitin in the presence of Tax or a Tax mutant impaired in IKK activation. Tax induced the ubiquitination of 22 cellular proteins, including the anti-apoptotic BCL-2 family member MCL-1, in an IKK-dependent manner. Tax was found to promote the nondegradative lysine 63 (K63)-linked polyubiquitination of MCL-1 that was dependent on the E3 ubiquitin ligase TRAF6 and the IKK complex. Tax interacted with and activated TRAF6, and triggered its mitochondrial localization, where it conjugated four carboxyl-terminal lysine residues of MCL-1 with K63-linked polyubiquitin chains, which stabilized and protected MCL-1 from genotoxic stress-induced degradation. TRAF6 and MCL-1 played essential roles in the survival of HTLV-1 transformed cells and the immortalization of primary T cells by HTLV-1. Therefore, K63-linked polyubiquitination represents a novel regulatory mechanism controlling MCL-1 stability that has been usurped by a viral oncogene to precipitate cell survival and transformation.  

3.2241           Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance

Nikolovski, N., Shliaha, P.V., Gatto, L., Dupree, P. and Lilley, K.S. Plant Physiol., 166, 1033-1043 (2014)   The proteomic composition of the Arabidopsis (Arabidopsis thaliana) Golgi apparatus is currently reasonably well documented; however, little is known about the relative abundances between different proteins within this compartment. Accurate quantitative information of Golgi resident proteins is of great importance: it facilitates a better understanding of the biochemical processes that take place within this organelle, especially those of different polysaccharide synthesis pathways. Golgi resident proteins are challenging to quantify because the abundance of this organelle is relatively low within the cell. In this study, an organelle fractionation approach targeting the Golgi apparatus was combined with a label-free quantitative mass spectrometry (data-independent acquisition method using ion mobility separation known as LC-IMS-MS^E [or HDMS^E]) to simultaneously localize proteins to the Golgi apparatus and assess their relative quantity. In total, 102 Golgi-localized proteins were quantified. These data show that organelle fractionation in conjunction with label-free quantitative mass spectrometry is a powerful and relatively simple tool to access protein organelle localization and their relative abundances. The findings presented open a unique view on the organization of the plant Golgi apparatus, leading toward unique hypotheses centered on the biochemical processes of this organelle.  

3.2242           Exosome Analysis: A Promising Biomarker System with Special Attention to Saliva

Zheng, X., Chen, F., Zhang, J., Zhang, Q. and Lin, J.

1. Membrane Biol., 247, 1129-1136 (2014)

  Today, exosome-related studies have become a focus in science and technology. Recently, three scientists won the Nobel Prize for determining the mechanisms of exosomal transport, making exosomes a promising biomarker system for disease diagnosis and treatment. This review provides a general introduction of exosomes and explores the recent progress on the function, application, isolation, and identification of exosomes as biomarkers in blood and other body fluids, especially in saliva. Detailed information of exosomal proteins and RNAs is discussed in the paper because of their ability to determine the function of exosomes. Due to their noninvasive assessment for quick and convenient diagnosis of diseases, salivary exosomes may well be promising biomarkers.  

3.2243           Subcellular localization of coagulation factor II receptor-like 1 in neurons governs angiogenesis

Joyal, J-S. et al Nature Med., 20(10), 1165-1173 (2014)   Neurons have an important role in retinal vascular development. Here we show that the G protein–coupled receptor (GPCR) coagulation factor II receptor-like 1 (F2rl1, previously known as Par2) is abundant in retinal ganglion cells and is associated with new blood vessel formation during retinal development and in ischemic retinopathy. After stimulation, F2rl1 in retinal ganglion cells translocates from the plasma membrane to the cell nucleus using a microtubule-dependent shuttle that requires sorting nexin 11 (Snx11). At the nucleus, F2rl1 facilitates recruitment of the transcription factor Sp1 to trigger Vegfa expression and, in turn, neovascularization. In contrast, classical plasma membrane activation of F2rl1 leads to the expression of distinct genes, including Ang1, that are involved in vessel maturation. Mutant versions of F2rl1 that prevent nuclear relocalization but not plasma membrane activation interfere with Vegfa but not Ang1 expression. Complementary angiogenic factors are therefore regulated by the subcellular localization of a receptor (F2rl1) that governs angiogenesis. These findings may have implications for the selectivity of drug actions based on the subcellular distribution of their targets.  

3.2244           KIF13B regulates angiogenesis through Golgi to plasma membrane trafficking of VEGFR2

Yamada, K.H., nakajima, Y., Geyer, M., Wary, K.K., Ushio-Fukai, M., Komarova, Y. and Malik, A.B.

1. Cell Sci., 127(20), 4518-4530 (2014)

  Although the trafficking of newly synthesized VEGFR2 to the plasma membrane is a key determinant of angiogenesis, the molecular mechanisms of Golgi to plasma membrane trafficking are unknown. Here, we have identified a key role of the kinesin family plus-end molecular motor KIF13B in delivering VEGFR2 cargo from the Golgi to the endothelial cell surface. KIF13B is shown to interact directly with VEGFR2 on microtubules. We also observed that overexpression of truncated versions of KIF13B containing the binding domains that interact with VEGFR2 inhibited VEGF-induced capillary tube formation. KIF13B depletion prevented VEGF-mediated endothelial migration, capillary tube formation and neo-vascularization in mice. Impairment in trafficking induced by knockdown of KIF13B shunted VEGFR2 towards the lysosomal degradation pathway. Thus, KIF13B is an essential molecular motor required for the trafficking of VEGFR2 from the Golgi, and its delivery to the endothelial cell surface mediates angiogenesis.  

3.2245           Lipid rafts couple class A scavenger receptors to phospholipase A2 activation during macrophage adhesion

Vadali, S. and Post, S.R.

1. Leukoc. Biol., 96(5), 873-881 (2014)

  SR-A mediated macrophage adhesion to modified ECM proteins in a process that involves physical attachment of SR-A to modified ECM and activation of Lyn-PI3K and PLA₂-12/15-lipoxygenase signaling pathways. Structurally, SR-A-mediated cell adhesion requires a 6-aa membrane-proximal cytoplasmic motif. However, the mechanism that couples SR-A-mediated adhesion to activation of these distinct signaling pathways is not known. For other adhesion receptors, including integrins, localization in cholesterol-rich LRs is an important mechanism for coupling the receptor with the activation of specific signaling pathways. We hypothesized that SR-A-mediated macrophage adhesion might also involve LRs. Our results demonstrate that SR-A is enriched in LRs in HEK cells that heterologously express SR-A and in macrophages that endogenously expressed the receptor. We further show that a truncated SR-A construct (SR-A_Δ1–49), which mediates cell adhesion but not ligand internalization, is also enriched in LRs, suggesting an association between LRs and SR-A-dependent cell adhesion. To examine this association more directly, we used the cholesterol chelator MβCD to deplete cholesterol and disrupt LR function. We found that cholesterol depletion significantly decreased SR-A-mediated macrophage adhesion. We further show that decreased SR-A-dependent macrophage adhesion following cholesterol depletion results from the inhibition of PLA₂ but not PI3K activation. Overall, our results demonstrate an important role for LRs in selectively coupling SR-A with PLA₂ activation during macrophage adhesion.  

3.2246           Plasma exosomal α-synuclein is likely CNS-derived and increased in Parkinson’s disease

Shi, M. et al Acta Neuropathol., 128(5), 639-650 (2014)   Extracellular α-synuclein is important in the pathogenesis of Parkinson’s disease (PD) and also as a potential biomarker when tested in the cerebrospinal fluid (CSF). The performance of blood plasma or serum α-synuclein as a biomarker has been found to be inconsistent and generally ineffective, largely due to the contribution of peripherally derived α-synuclein. In this study, we discovered, via an intracerebroventricular injection of radiolabeled α-synuclein into mouse brain, that CSF α-synuclein was readily transported to blood, with a small portion being contained in exosomes that are relatively specific to the central nervous system (CNS). Consequently, we developed a technique to evaluate the levels of α-synuclein in these exosomes in individual plasma samples. When applied to a large cohort of clinical samples (267 PD, 215 controls), we found that in contrast to CSF α-synuclein concentrations, which are consistently reported to be lower in PD patients compared to controls, the levels of plasma exosomal α-synuclein were substantially higher in PD patients, suggesting an increased efflux of the protein to the peripheral blood of these patients. Furthermore, although no association was observed between plasma exosomal and CSF α-synuclein, a significant correlation between plasma exosomal α-synuclein and disease severity (r = 0.176, p = 0.004) was observed, and the diagnostic sensitivity and specificity achieved by plasma exosomal α-synuclein were comparable to those determined by CSF α-synuclein. Further studies are clearly needed to elucidate the mechanism involved in the transport of CNS α-synuclein to the periphery, which may lead to a more convenient and robust assessment of PD clinically.  

3.2247           CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling

Platt, R. et al Cell, 159(2), 440-455 (2014)   CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras^G12D mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.  

3.2248           p130Cas Scaffolds the Signalosome To Direct Adaptor-Effector Cross Talk during Kaposi's Sarcoma-Associated Herpesvirus Trafficking in Human Microvascular Dermal Endothelial Cells

Bandyopadhyay, C., Veetil, M.V., Dutta, S. and Chandran, B.

1. Virol., 88(23), 13858-13878 (2014)

  Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface receptors, such as heparan sulfate, integrins (α3β1, αVβ3, and αVβ5), and EphrinA2 (EphA2), and activates focal adhesion kinase (FAK), Src, phosphoinositol 3-kinase (PI3-K), c-Cbl, and RhoA GTPase signal molecules early during lipid raft (LR)-dependent productive macropinocytic entry into human dermal microvascular endothelial cells. Our recent studies have identified CIB1 as a signal amplifier facilitating EphA2 phosphorylation and subsequent cytoskeletal cross talk during KSHV macropinocytosis. Although CIB1 lacks an enzymatic activity and traditional adaptor domain or known interacting sequence, it associated with the KSHV entry signal complex and the CIB1-KSHV association was sustained over 30 min postinfection. To identify factors scaffolding the EphA2-CIB1 signal axis, the role of major cellular scaffold protein p130Cas (Crk-associated substrate of Src) was investigated. Inhibitor and small interfering RNA (siRNA) studies demonstrated that KSHV induced p130Cas in an EphA2-, CIB1-, and Src-dependent manner. p130Cas and Crk were associated with KSHV, LRs, EphA2, and CIB1 early during infection. Live-cell microscopy and biochemical studies demonstrated that p130Cas knockdown did not affect KSHV entry but significantly reduced productive nuclear trafficking of viral DNA and routed KSHV to lysosomal degradation. p130Cas aided in scaffolding adaptor Crk to downstream guanine nucleotide exchange factor phospho-C3G possibly to coordinate GTPase signaling during KSHV trafficking. Collectively, these studies demonstrate that p130Cas acts as a bridging molecule between the KSHV-induced entry signal complex and the downstream trafficking signalosome in endothelial cells and suggest that simultaneous targeting of KSHV entry receptors with p130Cas would be an attractive potential avenue for therapeutic intervention in KSHV infection.  

3.2249           Magnetic Nanoparticles to Recover Cellular Organelles and Study the Time Resolved Nanoparticle-Cell Interactome throughout Uptake

Bertoli, F., Davies, G-L., Monopoli, M.P., Moloney, M., Gun’ko, Y.K., Salvati, A. and Dawson, K.A. Small, 10(16), 3307-3315 (2014)   Nanoparticles in contact with cells and living organisms generate quite novel interactions at the interface between the nanoparticle surface and the surrounding biological environment. However, a detailed time resolved molecular level description of the evolving interactions as nanoparticles are internalized and trafficked within the cellular environment is still missing and will certainly be required for the emerging arena of nanoparticle-cell interactions to mature. In this paper promising methodologies to map out the time resolved nanoparticle-cell interactome for nanoparticle uptake are discussed. Thus silica coated magnetite nanoparticles are presented to cells and their magnetic properties used to isolate, in a time resolved manner, the organelles containing the nanoparticles. Characterization of the recovered fractions shows that different cell compartments are isolated at different times, in agreement with imaging results on nanoparticle intracellular location. Subsequently the internalized nanoparticles can be further isolated from the recovered organelles, allowing the study of the most tightly nanoparticle-bound biomolecules, analogous to the ‘hard corona’ that so far has mostly been characterized in extracellular environments. Preliminary data on the recovered nanoparticles suggest that significant portion of the original corona (derived from the serum in which particles are presented to the cells) is preserved as nanoparticles are trafficked through the cells.  

3.2250           The Quantitative Nuclear Matrix Proteome as a Biochemical Snapshot of Nuclear Organization

Engelke, R., Riede, J., Hegermann, J., Wuerch, A., Eimer, S., Dengjel, J. and Mittler, G.

1. Proteome, 13(9), 3940-3956 (2014)

  The nuclear matrix (NM) is an operationally defined structure of the mammalian cell nucleus that resists stringent biochemical extraction procedures applied subsequent to nuclease-mediated chromatin digestion of intact nuclei. This comprises removal of soluble biomolecules and chromatin by means of either detergent (LIS: lithium diiodosalicylate) or high salt (AS: ammonium sulfate, sodium chloride) treatment. So far, progress toward defining bona fide NM proteins has been hindered by the problem of distinguishing them from copurifying abundant contaminants and extraction-method-intrinsic precipitation artifacts. Here, we present a highly improved NM purification strategy, adding a FACS sorting step for efficient isolation of morphologically homogeneous lamin B positive NM specimens. SILAC-based quantitative proteome profiling of LIS-, AS-, or NaCl-extracted matrices versus the nuclear proteome together with rigorous statistical filtering enables the compilation of a high-quality catalogue of NM proteins commonly enriched among the three different extraction methods. We refer to this set of 272 proteins as the NM central proteome. Quantitative NM retention profiles for 2381 proteins highlight elementary features of nuclear organization and correlate well with immunofluorescence staining patterns reported in the Human Protein Atlas, demonstrating that the NM central proteome is significantly enriched in proteins exhibiting a nuclear body as well as nuclear speckle-like morphology.  

3.2251           Hepatic fatty acid uptake is regulated by the sphingolipid acyl chain length

Park, W-J., park, J-W., Merrill Jr., A.H., Storchm J., Pewzner-Jung, Y. and Futerman, A.H. Biochim.Biophys. Acta, 1841, 1754-1766 (2014)   Ceramide synthase 2 (CerS2) null mice cannot synthesize very-long acyl chain (C22–C24) ceramides resulting in significant alterations in the acyl chain composition of sphingolipids. We now demonstrate that hepatic triacylglycerol (TG) levels are reduced in the liver but not in the adipose tissue or skeletal muscle of the CerS2 null mouse, both before and after feeding with a high fat diet (HFD), where no weight gain was observed and large hepatic nodules appeared. Uptake of both BODIPY-palmitate and [³H]-palmitate was also abrogated in the hepatocytes and liver. The role of a number of key proteins involved in fatty acid uptake was examined, including FATP5, CD36/FAT, FABPpm and cytoplasmic FABP1. Levels of FATP5 and FABP1 were decreased in the CerS2 null mouse liver, whereas CD36/FAT levels were significantly elevated and CD36/FAT was also mislocalized upon insulin treatment. Moreover, treatment of hepatocytes with C22–C24-ceramides down-regulated CD36/FAT levels. Infection of CerS2 null mice with recombinant adeno-associated virus (rAAV)-CerS2 restored normal TG levels and corrected the mislocalization of CD36/FAT, but had no effect on the intracellular localization or levels of FATP5 or FABP1. Together, these results demonstrate that hepatic fatty acid uptake via CD36/FAT can be regulated by altering the acyl chain composition of sphingolipids.  

3.2252           Role of the nucleocapsid region in HIV-1 Gag assembly as investigated by quantitative fluorescence-based microscopy

De Rocquigny, H., El Meshri, S.E., Richert, L., Didier, P., Darlix, J-L. and Mely, Y. Virus Res., 193, 78-88 (2014)   The Gag precursor of HIV-1, formed of the four proteic regions matrix (MA), capsid (CA), nucleocapsid (NC) and p6, orchestrates virus morphogenesis. This complex process relies on three major interactions, NC-RNA acting as a scaffold, CA-CA and MA-membrane that targets assembly to the plasma membrane (PM). The characterization of the molecular mechanism of retroviral assembly has extensively benefited from biochemical studies and more recently an important step forward was achieved with the use of fluorescence-based techniques and fluorescently labeled viral proteins. In this review, we summarize the findings obtained with such techniques, notably quantitative-based approaches, which highlight the role of the NC region in Gag assembly.  

3.2253           Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity

Watenabe, S., Hayakawa, ST., Wakasugi, K. and Yamanaka, K. Cell Death and Disease, 5, e1497 (2014)   Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective and progressive loss of motor neurons. Cystatin C (CysC), an endogenous cysteine protease inhibitor, is a major protein component of Bunina bodies observed in the spinal motor neurons of sporadic ALS and is decreased in the cerebrospinal fluid of ALS patients. Despite prominent deposition of CysC in ALS, the roles of CysC in the central nervous system remain unknown. Here, we identified the neuroprotective activity of CysC against ALS-linked mutant Cu/Zn-superoxide dismutase (SOD1)-mediated toxicity. We found that exogenously added CysC protected neuronal cells including primary cultured motor neurons. Moreover, the neuroprotective property of CysC was dependent on the coordinated activation of two distinct pathways: autophagy induction through AMPK-mTOR pathway and inhibition of cathepsin B. Furthermore, exogenously added CysC was transduced into the cells and aggregated in the cytosol under oxidative stress conditions, implying a relationship between the neuroprotective activity of CysC and Bunina body formation. These data suggest CysC is an endogenous neuroprotective agent and targeting CysC in motor neurons may provide a novel therapeutic strategy for ALS.  

3.2254           A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria

Lahiri, S., Chao, J.T., Tavassoli, S., Wong, A.K.O., Choudhary, V., Young, B.P., Loewen, C.J.R. and Prinz, W.A. PloS Biology, 12(10), e1001969 (2014)   Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER) to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC) has decreased phosphatidylserine (PS) transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER–mitochondria tethering complex called ERMES (the ER–mitochondria encounter structure) are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER–mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM) complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.  

3.2255           In Candida albicans hyphae, Sec2p is physically associated with SEC2 mRNA on secretory vesicles

Caballero-Lima, D., Hautbergue, G.M., Wilson, S.A. and Sudbery, P.E. Mol. Microbiol., 94(4), 828-842 (2014)   Candida albicans hyphae grow in a highly polarized fashion from their tips. This polarized growth requires the continuous delivery of secretory vesicles to the tip region. Vesicle delivery depends on Sec2p, the Guanine Exchange Factor (GEF) for the Rab GTPase Sec4p. GTP bound Sec4p is required for the transit of secretory vesicles from the trans-Golgi to sites of polarized growth. We previously showed that phosphorylation of Sec2p at residue S584 was necessary for Sec2p to support hyphal, but not yeast growth. Here we show that on secretory vesicles SEC2 mRNA is physically associated with Sec2p. Moreover, we show that the phosphorylation of S584 allows SEC2 mRNA to dissociate from Sec2p and we speculate that this is necessary for Sec2p function and/or translation. During hyphal extension, the growing tip may be separated from the nucleus by up to 15 μm. Transport of SEC2 mRNA on secretory vesicles to the tip localizes SEC2 translation to tip allowing a sufficient accumulation of this key protein at the site of polarized growth.  

3.2256           290 Exosome analysis in cancer patients: From the preclinical towards the clinical application: Trial design

Mertens, I., Castiglia, M., Carreca, A.P., baggertman, G., Peeters, M., Pauwels, P. and Rolfo, C. Eur. J. Cancer., 50, Suppl. 6, 96 (2014)   Background: Cancer cells produce a heterogeneous mixture of vesicular, organelle-like structures (extracellular vesicles, EVs) into their surroundings including blood and other body fluids. Exosomes are small (40 to 100 nm) membrane derived vesicles that develop from exophytic budding of the cellular membrane after the fusion of multivesicular bodies or mature endosomes with the cellular membrane. It has been shown that tumour cells exposed to hypoxia secrete exosomes with enhanced angiogenic and metastatic potential. Thus exosomes might be involved in tumor progression and they can potentially be used for prognosis and therapy selection, as they contain a variety of molecules such as signal proteins and/or peptides, microRNAs, mRNAs and lipids, which could be potential biomarkers. Materials and Methods: To evaluate the biomarker potential of the exosome derived RNA and protein content, we first optimized an extraction protocol for exosomes in plasma based on the Optiprep density gradient protocol. After purification, the exosomes are characterized and quantified using Western Blot and Nanosight analysis. Later, the RNA and protein fraction of the extracted exosomes from 60 NSCLC, 60 pancreatic cancer and 60 colorectal cancer patients is compared to 60 healthy controls. The proteome content is evaluated using mass spectrometry based quantitative shotgun proteomics. The RNA profiles are generated using next generation sequencing. After profound bioinformatics analysis, the potential of the RNA mand protein profiles will be evaluated for diagnostic, prognostic and therapy purposes. Results: We are able to purify and characterize exosome material from plasma samples derived from patients. The data from nanosight analysis and Western blot indicate that we are able to work with very pure exosome samples for RNA and protein axtraction. Currently, RNA and protein profiles from different cancer types are being compared to healthy controls. Conclusion: The first step in bringing exosome analysis to the clinic is optimized: exosome purification and characterization. In a next step, RNA and protein profiles are being evaluated as potential biomarkers.  

3.2257           RacGTPase-activating protein 1 interacts with hepatitis C virus polymerase NS5B to regulate viral replication

Wu, M-J., Ke, P-Y. and Horng, J-T. Biochem. Biophys. Res. Comm., 454, 19-24 (2014)   Hepatitis C virus (HCV) is a positive-strand RNA virus responsible for chronic liver disease and hepatocellular carcinoma (HCC). RacGTPase-activating protein 1 (RacGAP1) plays an important role during GTP hydrolysis to GDP in Rac1 and CDC42 protein and has been demonstrated to be upregulated in several cancers, including HCC. However, the molecular mechanism leading to the upregulation of RacGAP1 remains poorly understood. Here, we showed that RacGAP1 levels were enhanced in HCV cell-culture-derived (HCVcc) infection. More importantly, we illustrated that RacGAP1 interacts with the viral protein NS5B in mammalian cells. The small interfering RNA (siRNA)-mediated knockdown of RacGAP1 in human hepatoma cell lines inhibited replication of HCV RNA, protein, and production of infectious particles of HCV genotype 2a strain JFH1. Conversely, these were reversed by the expression of a siRNA-resistant RacGAP1 recombinant protein. In addition, viral protein NS5B polymerase activity was significantly reduced by silencing RacGAP1 and, vice versa, was increased by overexpression of RacGAP1 in a cell-based reporter assay. Our results suggest that RacGAP1 plays a crucial role in HCV replication by affecting viral protein NS5B polymerase activity and holds importance for antiviral drug development.  

3.2258           Endonuclease G preferentially cleaves 5-hydroxymethylcytosine-modified DNA creating a substrate for recombination

Robertson, A.B., Robertson, J., Fusser, M. and Klungland, A. Nucleic Acids Res., 42(21), 13280-13293 (2014)   5-hydroxymethylcytosine (5hmC) has been suggested to be involved in various nucleic acid transactions and cellular processes, including transcriptional regulation, demethylation of 5-methylcytosine and stem cell pluripotency. We have identified an activity that preferentially catalyzes the cleavage of double-stranded 5hmC-modified DNA. Using biochemical methods we purified this activity from mouse liver extracts and demonstrate that the enzyme responsible for the cleavage of 5hmC-modified DNA is Endonuclease G (EndoG). We show that recombinant EndoG preferentially recognizes and cleaves a core sequence when one specific cytosine within that core sequence is hydroxymethylated. Additionally, we provide in vivo evidence that EndoG catalyzes the formation of double-stranded DNA breaks and that this cleavage is dependent upon the core sequence, EndoG and 5hmC. Finally, we demonstrate that the 5hmC modification can promote conservative recombination in an EndoG-dependent manner.  

3.2259           Cyclopamine Modulates {gamma}-Secretase-mediated Cleavage of Amyloid Precursor Protein by Altering Its Subcellular Trafficking and Lysosomal Degradation

Vorobyeva, A.G., Lee, r., Miller, S., Longren, C., Sharoni, M., Kandelwal, P.J. and Saunders, A.J.

1. Biol. Chem., 289(48), 33258-33274 (2014)

  Alzheimer disease (AD) is a progressive neurodegenerative disease leading to memory loss. Numerous lines of evidence suggest that amyloid-β (Aβ), a neurotoxic peptide, initiates a cascade that results in synaptic dysfunction, neuronal death, and eventually cognitive deficits. Aβ is generated by the proteolytic processing of the amyloid precursor protein (APP), and alterations to this processing can result in Alzheimer disease. Using in vitro and in vivo models, we identified cyclopamine as a novel regulator of γ-secretase-mediated cleavage of APP. We demonstrate that cyclopamine decreases Aβ generation by altering APP retrograde trafficking. Specifically, cyclopamine treatment reduced APP-C-terminal fragment (CTF) delivery to the trans-Golgi network where γ-secretase cleavage occurs. Instead, cyclopamine redirects APP-CTFs to the lysosome. These data demonstrate that cyclopamine treatment decreases γ-secretase-mediated cleavage of APP. In addition, cyclopamine treatment decreases the rate of APP-CTF degradation. Together, our data demonstrate that cyclopamine alters APP processing and Aβ generation by inducing changes in APP subcellular trafficking and APP-CTF degradation.  

3.2260           BPIFB3 Regulates Autophagy and Coxsackievirus B Replication through a Noncanonical Pathway Independent of the Core Initiation Machinery

Delorme-Axford, E., Morosky, S., Bomberger, J., Stolz, D.B., Jackson, W.T. and Coyne, C.B. mBio, 5(6), e02147 (2014)   Enteroviruses require autophagy to facilitate the formation of autophagosome (AP)-like double-membrane vesicles that provide the scaffolding for RNA replication. Here, we identify bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) as a gene whose silencing greatly enhances coxsackievirus B (CVB) replication and induces dramatic alterations in the morphology of CVB-induced replication organelles. We show that BPIFB3 is associated with the endoplasmic reticulum (ER), and its silencing by RNA interference enhances basal levels of autophagy and promotes increased autophagy during CVB replication. Conversely, overexpression of BPIFB3 inhibits CVB replication, dramatically alters the morphology of LC3B-positive vesicles, and suppresses autophagy in response to rapamaycin. In addition, we found that, whereas silencing of core autophagy components associated with the initiation of APs in control cells suppressed CVB replication, silencing of these same components had no effect on CVB-induced autophagy or viral replication in cells transfected with BPIFB3 small interfering RNA. Based on these results, taken together, this study reports on a previously uncharacterized regulator of enterovirus infection that controls replication through a noncanonical pathway independent from the core autophagy initiation machinery.  

3.2261           Isolation and identification of membrane vesicle-associated proteins in Gram-positive bacteria and mycobacteria

Prados-Rosales, R., Brown, L., Casadevall, A., Montalvo-Qiros, S. and Luque-garcia, J.L. MethodsX, 1, 124-129 (2014)   Many intracellular bacterial pathogens naturally release membrane vesicles (MVs) under a variety of growth environments. For pathogenic bacteria there are strong evidences that released MVs are a delivery mechanism for the release of immunologically active molecules that contribute to virulence. Identification of membrane vesicle-associated proteins that can act as immunological modulators is crucial for opening up new horizons for understanding the pathogenesis of certain bacteria and for developing novel vaccines. In this protocol, we provide all the details for isolating MVs secreted by either mycobacteria or Gram-positive bacteria and for the subsequent identification of the protein content of the MVs by mass spectrometry. The protocol is adapted from Gram-negative bacteria and involves four main steps: (1) isolation of MVs from the culture media; (2) purification of MVs by density gradient ultrucentrifugation; (3) acetone precipitation of the MVs protein content and in-solution trypsin digestion and (4) mass spectrometry analysis of the generated peptides and protein identification. Our modifications are: Growing Mycobacteria in a chemically defined media to reduce the number of unrelated bacterial components in the supernatant. The use of an ultrafiltration system, which allows concentrating larger volumes. In solution digestion of proteins followed by peptides purification by ziptip.  

3.2262           Exosome-associated hepatitis C virus in cell cultures and patient plasma

Liu, Z., Zhang, X., Yu, Q. and He, J.J. Biochem. Biophys. Res. Comm., 455, 218-222 (2014)   Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell–cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV than exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission.  

3.2263           Soluble adenylyl cyclase of sea urchin spermatozoa

Vacquier, V.D., Loza-Huerta, A., Garcia-Rincon, J., Darszon, A. and Beltran, C. Biochim. Biophys. Acta, 1842, 2621-2628 (2014)   Fertilization, a key step in sexual reproduction, requires orchestrated changes in cAMP concentrations. It is notable that spermatozoa (sperm) are among the cell types with extremely high adenylyl cyclase (AC) activity. As production and consumption of this second messenger need to be locally regulated, the discovery of soluble AC (sAC) has broadened our understanding of how such cells deal with these requirements. In addition, because sAC is directly regulated by HCO₃⁻ it is able to translate CO₂/HCO₃⁻/pH changes into cAMP levels. Fundamental sperm functions such as maturation, motility regulation and the acrosome reaction are influenced by cAMP; this is especially true for sperm of the sea urchin (SU), an organism that has been a model in the study of fertilization for more than 130 years. Here we summarize the discovery and properties of SU sperm sAC, and discuss its involvement in sperm physiology. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.  

3.2264           Phosphatidylinositol 3-kinase and COPII generate LC3 lipidation vesicles from the ER-Golgi intermediate compartment

Ge, L., Zhang, M. and Scheckman, R. eLife, 3, e04135 (2014)   Formation of the autophagosome requires significant membrane input from cellular organelles. However, no direct evidence has been developed to link autophagic factors and the mobilization of membranes to generate the phagophore. Previously, we established a cell-free LC3 lipidation reaction to identify the ER-Golgi intermediate compartment (ERGIC) as a membrane source for LC3 lipidation, a key step of autophagosome biogenesis (Ge et al., eLife 2013; 2:e00947). We now report that starvation activation of autophagic phosphotidylinositol-3 kinase (PI3K) induces the generation of small vesicles active in LC3 lipidation. Subcellular fractionation studies identified the ERGIC as the donor membrane in the generation of small lipidation-active vesicles. COPII proteins are recruited to the ERGIC membrane in starved cells, dependent on active PI3K. We conclude that starvation activates the autophagic PI3K, which in turn induces the recruitment of COPII to the ERGIC to bud LC3 lipidation-active vesicles as one potential membrane source of the autophagosome. - See more at: http://elifesciences.org/content/3/e04135.abstract?maxtoshow=&HITS=10&hits=150&RESULTFORMAT=1&andorexacttitle=and&andorexacttitleabs=and&fulltext=Optiprep%20or%20iodixanol&andorexactfulltext=and&searchid=1&FIRSTINDEX=0&sortspec=date&fdate=1/1/2014&tdate=12/31/2014&resourcetype=HWCIT#sthash.8dxaTbFc.dpuf  

3.2265           Lenalidomide Induces Lipid Raft Assembly to Enhance Erythropoietin Receptor Signaling in Myelodysplastic Syndrome Progenitors

McGraw, K.L., Basiiorka, A.A., Johnson, J.O., Clark, J., Caceres, G., Padron, E., Heaton, R., Ozawa, Y., Wei, S., Sokol, L. and List, A.F. PloS One, 9(12), e11429 (2014)   Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS). Despite appropriate cytokine production and cellular receptor display, erythropoietin receptor (EpoR) signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (p = 0.005, raft number; p = 0.023, raft size). Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS.  

3.2266           Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways

Yoon, Y.J., Kim, D-K., Yoon, C.M., Park, J., Kim, Y-K., Roh, T-Y and Gho, Y.S: PloS One, 9(12), e115170 (2014)   Various mammalian cells, including cancer cells, shed extracellular vesicles (EVs), also known as exosomes and microvesicles, into surrounding tissues. These EVs play roles in tumor growth and metastasis by promoting angiogenesis. However, the detailed mechanism of how cancer-derived EVs elicit endothelial cell activation remains unknown. Here, we provide evidence that early growth response-1 (Egr-1) activation in endothelial cells is involved in the angiogenic activity of colorectal cancer cell-derived EVs. Both RNA interference–mediated downregulation of Egr-1 and ERK1/2 or JNK inhibitor significantly blocked EV-mediated Egr-1 activation and endothelial cell migration. Furthermore, lipid raft-mediated endocytosis inhibitor effectively blocked endothelial Egr-1 activation and migration induced by cancer-derived EVs. Our results suggest that Egr-1 activation in endothelial cells may be a key mechanism involved in the angiogenic activity of cancer-derived EVs. These findings will improve our understanding regarding the proangiogenic activities of EVs in diverse pathological conditions including cancer, cardiovascular diseases, and neurodegenerative diseases.  

3.2267           Proteomic Analysis of the Acidocalcisome, an Organelle Conserved from Bacteria to Human Cells

Huang, G., Ulrich, P.N., Storey, M., Johnson, D., Tischer, J., Tovar, J.A., Moreno, S.N.J., Orlando, R. and Docompo, R. Plos Pathogens, 10(12), e1004555 (2014)   Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the acidocalcisome localization of seven new, putative, acidocalcisome proteins (phosphate transporter, vacuolar H⁺-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that acidocalcisomes are especially adapted for the accumulation of polyphosphate.  

3.2268           New insights in the composition of extracellular vesicles from pancreatic cancer cells: implications for biomarkers and functions

Klein-Scory, S., Tehrani, M.M., Eilert-Micus, C., Adamczyk, K.A., Wojtalewics, N., Schnölzer, M., Hahn, S.A., Schmiegel, Wa. And Schwarte-Waldhoff, I. Proteome Science, 12:50 (2014)   Background Pancreatic cancer development is associated with characteristic alterations like desmoplastic reaction and immune escape which are mediated by the cell-cell communication mechanism and by the microenvironment of the cells. The whole of released components are important determinants in these processes. Especially the extracellular vesicles released by pancreatic cancer cells play a role in cell communication and modulate cell growth and immune responses. Results Here, we present the proteomic description of affinity purified extracellular vesicles from pancreatic tumour cells, compared to the secretome, defined as the whole of the proteins released by pancreatic cancer cells. The proteomic data provide comprehensive catalogues of hundreds of proteins, and the comparison reveals a special proteomic composition of pancreatic cancer cell derived extracellular vesicles. The functional analysis of the protein composition displayed that membrane proteins, glycoproteins, small GTP binding proteins and a further, heterogeneous group of proteins are enriched in vesicles, whereas proteins derived from proteasomes and ribosomes, as well as metabolic enzymes, are not components of the vesicles. Furthermore proteins playing a role in carcinogenesis and modulators of the extracellular matrix (ECM) or cell-cell interactions are components of affinity purified extracellular vesicles. Conclusion The data deepen the knowledge of extracellular vesicle composition by hundreds of proteins that have not been previously described as vesicle components released by pancreatic cancer cells. Extracellular vesicles derived from pancreatic cancer cells show common proteins shared with other vesicles as well as cell type specific proteins indicating biomarker candidates and suggesting functional roles in cancer cell stroma interactions.  

3.2269           Stable Cell Surface Expression of GPI-Anchored Proteins, but not Intracellular Transport, Depends on their Fatty Acid Structure

Jaensch, N., Correa, I.R. and Watanabe, R. Traffic, 15(12), 1305-1329 (2014)   Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a class of lipid anchored proteins expressed on the cell surface of eukaryotes. The potential interaction of GPI-APs with ordered lipid domains enriched in cholesterol and sphingolipids has been proposed to function in the intracellular transport of these lipid anchored proteins. Here, we examined the biological importance of two saturated fatty acids present in the phosphatidylinositol moiety of GPI-APs. These fatty acids are introduced by the action of lipid remodeling enzymes and required for the GPI-AP association within ordered lipid domains. We found that the fatty acid remodeling is not required for either efficient Golgi-to-plasma membrane transport or selective endocytosis via GPI-enriched early endosomal compartment (GEEC)/ clathrin-independent carrier (CLIC) pathway, whereas cholesterol depletion significantly affects both pathways independent of their fatty acid structure. Therefore, the mechanism of cholesterol dependence does not appear to be related to the interaction with ordered lipid domains mediated by two saturated fatty acids. Furthermore, cholesterol extraction drastically releases the unremodeled GPI-APs carrying an unsaturated fatty acid from the cell surface, but not remodeled GPI-APs carrying two saturated fatty acids. This underscores the essential role of lipid remodeling to ensure a stable membrane association of GPI-APs particularly under potential membrane lipid perturbation.  

3.2270           Mammalian CORVET Is Required for Fusion and Conversion of Distinct Early Endosome Subpopulations

Perini, E.D., Schaefer, R., Stöter, M., Kalaidzidis, Y. and Zerial, M. Traffic, 15(12), 1366-1389 (2014)   Early endosomes are organized in a network of vesicles shaped by cycles of fusion, fission, and conversion to late endosomes. In yeast, endosome fusion and conversion are regulated, among others, by CORVET, a hexameric protein complex. In the mammalian endocytic system, distinct subpopulations of early endosomes labelled by the Rab5 effectors APPL1 and EEA1 are present. Here, the function of mammalian CORVET with respect to these endosomal subpopulations was investigated. Tgfbrap1 as CORVET-specific subunit and functional ortholog of Vps3p was identified, demonstrating that it is differentially distributed between APPL1 and EEA1 endosomes. Surprisingly, depletion of CORVET-specific subunits caused fragmentation of APPL1-positive endosomes but not EEA1 endosomes in vivo. These and in vitro data suggest that CORVET plays a role in endosome fusion independently of EEA1. Depletion of CORVET subunits caused accumulation of large EEA1 endosomes indicative of another role in the conversion of EEA1 endosomes into late endosomes. In addition, depletion of CORVET-specific subunits caused alterations in transport depending on both the type of cargo and the specific endosomal subpopulation. These results demonstrate that CORVET plays distinct roles at multiple stages in the mammalian endocytic pathway.  

3.2271           Effects of heat shock protein 90 expression on pectoralis major oxidation in broilers exposed to acute heat stress

Hao, Y. and Gu, X.H. Poultry Science, 93(11), 2709-2717 (2014)   This study was conducted to determine the effects of heat shock protein 90 (HSP90) expression on pH, lipid peroxidation, heat shock protein 70 (HSP70), and glucocorticoid receptor (GR) expression of pectoralis major in broilers exposed to acute heat stress. In total, 90 male broilers were randomly allocated to 3 groups: control (CON), heat stress (HS), or geldanamycin treatment (GA). On d 41, the broilers in the GA group were injected intraperitoneally with GA (5 μg/kg of BW), and the broilers in the CON and HS groups were injected intraperitoneally with saline. Twenty-four hours later, the broilers in the CON group were moved to environmental chambers controlled at 22°C for 2 h, and the broilers in the HS and GA groups were moved to environmental chambers controlled at 40°C for 2 h. The pH values of the pectoralis major after 30 min and 24 h of chilling after slaughter of HS and GA broilers were significantly lower (P < 0.01) than those of the CON broilers. Heat stress caused significant increases in sera corticosterone and lactic dehydrogenase, the activity of malondialdehyde and superoxide dismutase, the expression of HSP90 and HSP70, and nuclear expression of GR protein in the pectoralis major (P < 0.05). Heat stress induced a significant decrease in GR protein expression in the cytoplasm and GR mRNA expression. Furthermore, the low expression of HSP90 significantly increased levels of lactic dehydrogenase and malondialdehyde and GR protein expression in the cytoplasm under heat stress (P < 0.01), and significantly decreased nuclear GR protein expression (P < 0.01). Heat shock protein 90 was positively correlated with corticosterone and superoxide dismutase activities (P < 0.01), and HSP90 mRNA was negatively correlated with pH after chilling for 24 h. The results demonstrated that HSP90 plays a pivotal role in protecting cells from oxidation.  

3.2272           The ESCRT machinery regulates the secretion and long-range activity of Hedgehog

Matusek, T., Wendler, F., Poles, S., Pizette, S., D’Angelo, G., Fürthauer, M. and Therond, P.P Nature, 516, 99-103 (2014)   The conserved family of Hedgehog (Hh) proteins acts as short- and long-range secreted morphogens, controlling tissue patterning and differentiation during embryonic development¹. Mature Hh carries hydrophobic palmitic acid and cholesterol modifications essential for its extracellular spreading². Various extracellular transportation mechanisms for Hh have been suggested, but the pathways actually used for Hh secretion and transport in vivo remain unclear. Here we show that Hh secretion in Drosophila wing imaginal discs is dependent on the endosomal sorting complex required for transport (ESCRT)³. In vivo the reduction of ESCRT activity in cells producing Hh leads to a retention of Hh at the external cell surface. Furthermore, we show that ESCRT activity in Hh-producing cells is required for long-range signalling. We also provide evidence that pools of Hh and ESCRT proteins are secreted together into the extracellular space in vivo and can subsequently be detected together at the surface of receiving cells. These findings uncover a new function for ESCRT proteins in controlling morphogen activity and reveal a new mechanism for the transport of secreted Hh across the tissue by extracellular vesicles, which is necessary for long-range target induction.  

3.2273           The emerging role of extracellular vesicles as biomarkers for urogenital cancers

Nawaz, M., Camussi, G., Valadi, H., Nazarenko, I., Ekström, K., Wang, X., Principe, S., Shah, N., Ashraf, N.M., Fatima, F., Neder, L. and Kislinger, T. Nat. Rev. Urol., 11, 688-701 (2014)   The knowledge gained from comprehensive profiling projects that aim to define the complex genomic alterations present within cancers will undoubtedly improve our ability to detect and treat those diseases, but the influence of these resources on our understanding of basic cancer biology is still to be demonstrated. Extracellular vesicles have gained considerable attention in past years, both as mediators of intercellular signalling and as potential sources for the discovery of novel cancer biomarkers. In general, research on extracellular vesicles investigates either the basic mechanism of vesicle formation and cargo incorporation, or the isolation of vesicles from available body fluids for biomarker discovery. A deeper understanding of the cargo molecules present in extracellular vesicles obtained from patients with urogenital cancers, through high-throughput proteomics or genomics approaches, will aid in the identification of novel diagnostic and prognostic biomarkers, and can potentially lead to the discovery of new therapeutic targets.  

3.2274           Effects of sildenafil on nanostructural and nanomechanical changes in mitochondria in an ischaemia-reperfusion rat model

Lee, K.H., Kwon, S.J., Woo, J-S., Lee, G-J., Lee, S-R., jang, H-H., Kim, H.S., Kim, J.W., Park, H.K., Cho, K.S. and Kim, W. Clinical and Experimental Pharmacology and Physiology, 41(10), 763-768 (2014)   Sildenafil exerts cardioprotective effects by activating the opening of mitochondrial ATP-sensitive potassium channels to attenuate ischaemia–reperfusion (IR) injury. In the present study, we used atomic force microscopy (AFM) to investigate changes in mitochondrial morphology and properties to assess sildenafil-mediated cardioprotection in a rat myocardial infarction model. To investigate the cardioprotective effects of sildenafil, we used an in vivo Sprague-Dawley rat model of IR. Rats were randomly divided into three groups: (i) sham-operated rats (control; n = 5); (ii) IR-injured rats treated with vehicle (normal saline; IR; n = 10); and (iii) IR-injured rats treated with 0.75 mg/kg, i.p., sildenafil (IR + Sil; n = 10). Morphological and mechanical changes to mitochondria were analysed by AFM. Infarct areas were significantly reduced in sildenafil-treated rats (7.8 ± 3.9% vs 20.4 ± 7.0% in the sildenafil-treated and untreated IR groups, respectively; relative reduction 62%; P < 0.001). Analysis of mitochondria by AFM showed that IR injury significantly increased the areas of isolated mitochondria compared with control (24 150 ± 18 289 vs 1495 ± 1139 nm², respectively; P < 0.001), indicative of mitochondrial swelling. Pretreatment with sildenafil before IR injury reduced the mitochondrial areas (7428 ± 3682 nm²; P < 0.001; relative reduction 69.2% compared with the IR group) and ameliorated the adhesion force of mitochondrial surfaces. Together, these results suggest that sildenafil has cardioprotective effects against IR injury in a rat model by improving the morphological and mechanical characteristics of mitochondria.  

3.2275           A Golgi-Localized Pool of the Mitotic Checkpoint Component Mad1 Controls Integrin Secretion and Cell Migration

Wan, J., Zhu, F., Zasadil, L.M., Yu, J., Wang, L., Johnson, A., Berthier, E., Beebe, D.J., Audhya, A. and Weaver, B.A. Current Biology, 24(22), 2687-2692 (2014)   Mitotic arrest deficient 1 (Mad1) plays a well-characterized role in the major cell-cycle checkpoint that regulates chromosome segregation during mitosis, the mitotic checkpoint (also known as the spindle assembly checkpoint). During mitosis, Mad1 recruits Mad2 to unattached kinetochores [ 1, 2 ], where Mad2 is converted into an inhibitor of the anaphase-promoting complex/cyclosome bound to its specificity factor, Cdc20 [ 1, 3–6 ]. During interphase, Mad1 remains tightly bound to Mad2 [ 2, 3, 7, 8 ], and both proteins localize to the nucleus and nuclear pores [ 9, 10 ], where they interact with Tpr (translocated promoter region). Recently, it has been shown that interaction with Tpr stabilizes both proteins [ 11 ] and that Mad1 binding to Tpr permits Mad2 to associate with Cdc20 [ 12 ]. However, interphase functions of Mad1 that do not directly affect the mitotic checkpoint have remained largely undefined. Here we identify a previously unrecognized interphase distribution of Mad1 at the Golgi apparatus. Mad1 colocalizes with multiple Golgi markers and cosediments with Golgi membranes. Although Mad1 has previously been thought to constitutively bind Mad2, Golgi-associated Mad1 is Mad2 independent. Depletion of Mad1 impairs secretion of α5 integrin and results in defects in cellular attachment, adhesion, and FAK activation. Additionally, reduction of Mad1 impedes cell motility, while its overexpression accelerates directed cell migration. These results reveal an unexpected role for a mitotic checkpoint protein in secretion, adhesion, and motility. More generally, they demonstrate that, in addition to generating aneuploidy, manipulation of mitotic checkpoint genes can have unexpected interphase effects that influence tumor phenotypes.  

3.2276           Aberrant Glycosylation and Localization of Polycystin-1 Cause Polycystic Kidney in an AQP11 Knockout Model

Inoue, Y., Sohara, E., Kobayashi, K., Chiga, M., Rai, T., Ishibashi, K., Horie, S., Su, X., Zhou, J., Sasaki, S. and Uchida, S.

1. Am. Soc. Nephrol., 25, 2789-2799 (2014)

  We previously reported that disruption of the aquaporin-11 (AQP11) gene in mice resulted in cystogenesis in the kidney. In this study, we aimed to clarify the mechanism of cystogenesis in AQP11(−/−) mice. To enable the analyses of AQP11 at the protein level in vivo, AQP11 BAC transgenic mice (Tg^AQP11) that express 3×HA-tagged AQP11 protein were generated. This AQP11 localized to the endoplasmic reticulum (ER) of proximal tubule cells in Tg^AQP11 mice and rescued renal cystogenesis in AQP11(−/−) mice. Therefore, we hypothesized that the absence of AQP11 in the ER could result in impaired quality control and aberrant trafficking of polycystin-1 (PC-1) and polycystin-2 (PC-2). Compared with kidneys of wild-type mice, AQP11(−/−) kidneys exhibited increased protein expression levels of PC-1 and decreased protein expression levels of PC-2. Moreover, PC-1 isolated from AQP11(−/−) mice displayed an altered electrophoretic mobility caused by impaired N-glycosylation processing, and density gradient centrifugation of kidney homogenate and in vivo protein biotinylation revealed impaired membrane trafficking of PC-1 in these mice. Finally, we showed that the Pkd1(+/−) background increased the severity of cystogenesis in AQP11(−/−) mouse kidneys, indicating that PC-1 is involved in the mechanism of cystogenesis in AQP11(−/−) mice. Additionally, the primary cilia of proximal tubules were elongated in AQP11(−/−) mice. Taken together, these data show that impaired glycosylation processing and aberrant membrane trafficking of PC-1 in AQP11(−/−) mice could be a key mechanism of cystogenesis in AQP11(−/−) mice.  

3.2277           Dual Proteolytic Pathways Govern Glycolysis and Immune Competence

Lu, W. et al Cell, 159, 1578-1590 (2014)   Proteasomes and lysosomes constitute the major cellular systems that catabolize proteins to recycle free amino acids for energy and new protein synthesis. Tripeptidyl peptidase II (TPPII) is a large cytosolic proteolytic complex that functions in tandem with the proteasome-ubiquitin protein degradation pathway. We found that autosomal recessive TPP2 mutations cause recurrent infections, autoimmunity, and neurodevelopmental delay in humans. We show that a major function of TPPII in mammalian cells is to maintain amino acid levels and that TPPII-deficient cells compensate by increasing lysosome number and proteolytic activity. However, the overabundant lysosomes derange cellular metabolism by consuming the key glycolytic enzyme hexokinase-2 through chaperone-mediated autophagy. This reduces glycolysis and impairs the production of effector cytokines, including IFN-γ and IL-1β. Thus, TPPII controls the balance between intracellular amino acid availability, lysosome number, and glycolysis, which is vital for adaptive and innate immunity and neurodevelopmental health.  

3.2278           Sirtuin 4 Is a Lipoamidase Regulating Pyruvate Dehydrogenase Complex Activity

Mathias, R., Greco, T.M., Oberstein, A., Budayeva, H.G., Chakrabarti, R., Rowland, E.A., Kang, Y., Shenk, T. and Cristea, I.M. Cell, 159, 1615-1625 (2014)   Sirtuins (SIRTs) are critical enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, mitochondrial SIRT4 and SIRT5 have little to no deacetylase activity, and a robust catalytic activity for SIRT4 has been elusive. Here, we establish SIRT4 as a cellular lipoamidase that regulates the pyruvate dehydrogenase complex (PDH). Importantly, SIRT4 catalytic efficiency for lipoyl- and biotinyl-lysine modifications is superior to its deacetylation activity. PDH, which converts pyruvate to acetyl-CoA, has been known to be primarily regulated by phosphorylation of its E1 component. We determine that SIRT4 enzymatically hydrolyzes the lipoamide cofactors from the E2 component dihydrolipoyllysine acetyltransferase (DLAT), diminishing PDH activity. We demonstrate SIRT4-mediated regulation of DLAT lipoyl levels and PDH activity in cells and in vivo, in mouse liver. Furthermore, metabolic flux switching via glutamine stimulation induces SIRT4 lipoamidase activity to inhibit PDH, highlighting SIRT4 as a guardian of cellular metabolism.  

3.2279           An AUTS2–Polycomb complex activates gene expression in the CNS

Gao, Z., Lee, P., Stafford, J.M., von Schimmelmann, M., Schaefer, A. and Reinberg, D. Nature, 516, 349-354 (2014)   Naturally occurring variations of Polycomb repressive complex 1 (PRC1) comprise a core assembly of Polycomb group proteins and additional factors that include, surprisingly, autism susceptibility candidate 2 (AUTS2). Although AUTS2 is often disrupted in patients with neuronal disorders, the mechanism underlying the pathogenesis is unclear. We investigated the role of AUTS2 as part of a previously identified PRC1 complex (PRC1–AUTS2), and in the context of neurodevelopment. In contrast to the canonical role of PRC1 in gene repression, PRC1–AUTS2 activates transcription. Biochemical studies demonstrate that the CK2 component of PRC1–AUTS2 neutralizes PRC1 repressive activity, whereas AUTS2-mediated recruitment of P300 leads to gene activation. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) demonstrated that AUTS2 regulates neuronal gene expression through promoter association. Conditional targeting of Auts2 in the mouse central nervous system (CNS) leads to various developmental defects. These findings reveal a natural means of subverting PRC1 activity, linking key epigenetic modulators with neuronal functions and diseases.   Characterization of physical properties of tissue factor-containing microvesicles and a comparison of ultracentrifuge-based recovery procedures Ettelaie, C., Collier, M.E.W., Maraveyas, A. and Ettelaie, R.

1. Extracellular Vesicles, 3:23592 (2014)

  Microvesicles were isolated from the conditioned media of 3 cell lines (MDA-MB-231, AsPC-1 and A375) by ultracentrifugation at a range of relative centrifugal forces, and the tissue factor (TF) protein and activity, microvesicle number, size distribution and relative density compared. Also, by expressing TF-tGFP in cells and isolating the microvesicles, the relative density of TF-containing microvesicles was established. Nanoparticle tracking analysis (NTA) indicated that the larger-diameter microvesicles (>200 nm) were primarily sedimented at 100,000g and possessed TF-dependent thrombin and factor Xa generation potential, while in the absence of factor VII, all microvesicles possessed some thrombin generation capacity. Immuno-precipitation of TF-containing microvesicles followed by NTA also indicated the range of these microvesicles to be 200–400 nm. Analysis of the microvesicles by gradient density centrifugation showed that lower-density (<1.1 g/ml) microvesicles were mainly present in the samples recovered at 100,000g and were associated with TF antigen and activity. Analysis of these fractions by NTA confirmed that these fractions were principally composed of the larger-diameter microvesicles. Similar analysis of microvesicles from healthy or patient plasma supported those obtained from conditioned media indicating that TF activity was mainly associated with lower-density microvesicles. Furthermore, centrifugation of healthy plasma, supplemented with TF-tGFP-containing microvesicles, resulted in 67% retrieval of the fluorescent microvesicles at 100,000g, but only 26% could be recovered at 20,000g. Pre-centrifugation of conditioned media or plasma at 10,000g improved the speed and yield of recovered TF-containing microvesicles by subsequent centrifugation at either 20,000g or 100,000g. In conclusion, TF appears to be associated with low-density (1.03–1.08 g/ml), larger-diameter (200–350 nm) microvesicles.   The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling Van Deun, J., Mestdagh, P., Sormunen, R., Cocquyt, V., Vermaelen, K., Vandesompele, J., Bracke, M., De Wever, O. and Hendrix, A.

1. Extracellular Vesicles, 3:24858 (2014)

  Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers.  

3.2280           Membrane actions of 1α,25(OH)2D3 are mediated by Ca2+/calmodulin-dependent protein kinase II in bone and cartilage cells

Doroudi, M., Plaisance, M.C., Boyan, B.D. and Schwarz, Z.

1. Steroid Biochem. Mol. Biol., 145, 65-74 (2015)

  1α,25(OH)₂D₃ regulates osteoblasts and chondrocytes via its membrane-associated receptor, protein disulfide isomerase A3 (Pdia3) in caveolae. 1α,25(OH)₂D₃ binding to Pdia3 leads to phospholipase-A₂ (PLA₂)-activating protein (PLAA) activation, stimulating cytosolic PLA₂ and resulting in prostaglandin E2 (PGE₂) release and PKCα activation, subsequently stimulating differentiation. However, how PLAA transmits the signal to cPLA₂ is unknown. Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) activation is required for PLA₂ activation in vascular smooth muscle cells, suggesting a similar role in 1α,25(OH)₂D₃-dependent signaling. The aim of the present study is to evaluate the roles of CaM and CaMKII as mediators of 1α,25(OH)₂D₃-stimulated PLAA-dependent activation of cPLA₂ and PKCα, and downstream biological effects. The results indicated that 1α,25(OH)₂D₃ and PLAA-peptide increased CaMKII activity within 9 min. Silencing Cav-1, Pdia3 or Plaa in osteoblasts suppressed this effect. Similarly, antibodies against Plaa or Pdia3 blocked 1α,25(OH)₂D₃-dependent CaMKII. Caveolae disruption abolished activation of CaMKII by 1α,25(OH)₂D₃ or PLAA. CaMKII-specific and CaM-specific inhibitors reduced cPLA₂ and PKC activities, PGE₂ release and osteoblast maturation markers in response to 1α,25(OH)₂D₃. Camk2a-silenced but not Camk2b-silenced osteoblasts showed comparable effects. Immunoprecipitation showed increased interaction of CaM and PLAA in response to 1α,25(OH)₂D₃. The results indicate that membrane actions of 1α,25(OH)₂D₃ via Pdia3 triggered the interaction between PLAA and CaM, leading to dissociation of CaM from caveolae, activation of CaMKII, and downstream PLA₂ activation, and suggest that CaMKII plays a major role in membrane-mediated actions of 1α,25(OH)₂D₃.  

3.2281           Fatty acid profiles from the plasma membrane and detergent resistant membranes of two plant species

Carmona-Salazar, L., El Hafidi, M., Gutierrez-Najera, N., Noyola-martinez, L., Gonzalez-Solis, A. and Gavilanes-Ruiz, M. Phytochemistry, 109, 25-35 (2015)   It is essential to establish the composition of the plant plasma membrane in order to understand its organization and behavior under continually changing environments. Knowledge of the lipid phase, in particular the fatty acid (FA) complex repertoire, is important since FAs determine many of the physical–chemical membrane properties. FAs are constituents of the membrane glycerolipid and sphingolipid backbones and can also be linked to some sterols. In addition, FAs are components of complex lipids that can constitute membrane micro-domains, and the use of detergent-resistant membranes is a common approach to study their composition. The diversity and cellular allocation of the membrane lipids containing FAs are very diverse and the approaches to analyze them provide only general information. In this work, a detailed FA analysis was performed using highly purified plasma membranes from bean leaves and germinating maize embryos and their respective detergent-resistant membrane preparations. The analyses showed the presence of a significant amount of very long chain FAs (containing 28C, 30C and 32C), in both plasma membrane preparations from bean and maize, that have not been previously reported. Herein is demonstrated that a significant enrichment of very long chain saturated FAs and saturated FAs can occur in detergent-resistant membrane preparations, as compared to the plasma membranes from both plant species. Considering that a thorough analysis of FAs is rarely performed in purified plasma membranes and detergent-resistant membranes, this work provides qualitative and quantitative evidence on the contributions of the length and saturation of FAs to the organization of the plant plasma membrane and detergent-resistant membranes.  

3.2282           Methods of isolation and purification of outer membrane vesicles from gram-negative bacteria

Klimentova, J. and Stulik, J. Microbiol. Res., 170, 1-9 (2015)   Abstract Outer membrane vesicles secreted by gram-negative bacteria play an important role in bacterial physiology as well as in virulence and host–pathogen interaction. Isolated vesicles of some bacteria have also been studied for their immunomodulatory potential in the vaccine development. However, the production of vesicles in sufficient amount, purity and reproducibility remains a critical challenge for subsequent analyses in most bacteria. In the present review methods of production, isolation, purification and quantification of outer membrane vesicles are summarized and discussed.  

3.2283           Downregulation of kinin B1 receptor function by B2 receptor heterodimerization and signaling

Zhang, X., Brovkovych, V., Zhang, Y., Tan, Ff. and Skidgel, R.A. Cellular Signalling, 27, 90-103 (2015)   Signaling through the G protein-coupled kinin receptors B1 (kB1R) and B2 (kB2R) plays a critical role in inflammatory responses mediated by activation of the kallikrein–kinin system. The kB2R is constitutively expressed and rapidly desensitized in response to agonist whereas kB1R expression is upregulated by inflammatory stimuli and it is resistant to internalization and desensitization. Here we show that the kB1R heterodimerizes with kB2Rs in co-transfected HEK293 cells and natively expressing endothelial cells, resulting in significant internalization and desensitization of the kB1R response in cells pre-treated with kB2R agonist. However, pre-treatment of cells with kB1R agonist did not affect subsequent kB2R responses. Agonists of other G protein-coupled receptors (thrombin, lysophosphatidic acid) had no effect on a subsequent kB1R response. The loss of kB1R response after pretreatment with kB2R agonist was partially reversed with kB2R mutant Y129S, which blocks kB2R signaling without affecting endocytosis, or T342A, which signals like wild type but is not endocytosed. Co-endocytosis of the kB1R with kB2R was dependent on β-arrestin and clathrin-coated pits but not caveolae. The sorting pathway of kB1R and kB2R after endocytosis differed as recycling of kB1R to the cell surface was much slower than that of kB2R. In cytokine-treated human lung microvascular endothelial cells, pre-treatment with kB2R agonist inhibited kB1R-mediated increase in transendothelial electrical resistance (TER) caused by kB1R stimulation (to generate nitric oxide) and blocked the profound drop in TER caused by kB1R activation in the presence of pyrogallol (a superoxide generator). Thus, kB1R function can be downregulated by kB2R co-endocytosis and signaling, suggesting new approaches to control kB1R signaling in pathological conditions.  

3.2284           Design, synthesis and biological evaluation of novel pyrenyl derivatives as anticancer agents

Bandyopadhyay, D., Sanchez, J.L., Guerrero, A.M., Chang, F-M., Granados, J.C., Short, J.D. and Banik, B.K. Eur. J. Medicinal Chem., 89, 851-862 (2015)   Polycyclic aromatic hydrocarbons are widespread in nature with a toxicity range from non-toxic to extremely toxic. A series of pyrenyl derivatives has been synthesized following a four-step strategy where the pyrene nucleus is attached with a basic heterocyclic moiety through a carbon linker. Virtual screening of the physicochemical properties and druggability has been carried out. The cytotoxicity of the compounds (1–8) have been evaluated in vitro against a small panel of human cancer cell lines which includes two liver cancer (HepG2 and Hepa 1–6), two colon cancer (HT-29 and Caco-2) and one each for cervical (HeLa) and breast (MCF-7) cancer cell lines. The IC₅₀ data indicate that compound 6 and 8 are the most effective cytotoxic agents in the present set of pyrenyl derivatives, suggesting that having a 4-carbon linker is more effective than a 5-carbon linker and the presence of amide carbonyl groups in the linker severely reduces the efficacy of the compound. The compounds showed selectivity toward cancer cells at lower doses (<5 μM) when compared with the normal hepatocytes. The mechanism of action supports the cell death through apoptosis in a caspase-independent manner without cleavage of poly (ADP-ribose) polymerase (PARP), even though the compounds cause plasma membrane morphological changes. The compounds, whether highly cytotoxic or mildly cytotoxic, localize to the membrane of cells. The compounds with either a piperidine ring (6) or an N-methyl piperazine (8) in the side chain were both capable of circumventing the drug resistance in SKOV3-MDR1-M6/6 ovarian cancer cells overexpressing P-glycoprotein. Qualitative structure-activity relationship has also been studied.  

3.2285           Rotenone impairs autophagic flux and lysosomal functions in Parkinson’s disease

Wu, F., Xu, H.D., Guan, J.J., Hou, Y.S., Gu, J.H., Zhen, X.C. and Qin, Z.H. Neuroscience, 284, 900-911 (2015)   Background Rotenone is an environmental neurotoxin that induces accumulation of α-synuclein and degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc), but the molecular mechanisms are not fully understood. We investigated whether rotenone induced impairment of autophagic flux and lysosomal functions. Methods Autophagy flux, accumulation of α-synuclein, lysosomal membrane integrity and neurodegeneration were assessed in the rotenone-treated rat model and PC12 cells, and the effects of the autophagy inducer trehalose on rotenone’s cytotoxicity were also studied. Results Rotenone administration significantly reduced motor activity and caused a loss of tyrosine hydroxylase in SNpc of Lewis rats. The degeneration of nigral dopaminergic neurons was accompanied by the deposition of α-synuclein aggregates, autophagosomes and redistribution of cathepsin D from lysosomes to the cytosol. In cultured PC12 cells, rotenone also induced increases in protein levels of α-synuclein, microtubule-associated protein 1 light chain 3-II, Beclin 1, and p62. Rotenone increased lysosomal membrane permeability as evidenced by leakage of N-acetyl-beta-d-glucosaminidase and cathepsin D, the effects were blocked by reactive oxygen species scavenger tiron. Autophagy inducer trehalose enhanced the nuclear translocation of transcription factor EB, accelerated the clearance of autophagosomes and α-synuclein and attenuated rotenone-induced cell death of PC12 cells. Meanwhile, administration of trehalose to rats in drinking water (2%) decreased rotenone-induced dopaminergic neurons loss in SNpc. Conclusions These studies indicate that the lysosomal dysfunction contributes to rotenone’s neurotoxicity and restoration of lysosomal function could be a new therapeutic strategy for Parkinson’s disease.  

3.2286           The Major Myelin-Resident Protein PLP Is Transported to Myelin Membranes via a Transcytotic Mechanism: Involvement of Sulfatide

Baron, W., Ozgen, H., Klunder, B., de Jonge, J.C., Nomden, A., Plat, A., Teifilieff, E., de Vries, H. and Hoekstra, D. Mol. Cell. Biol., 35(1), 288-302 (2015)   Myelin membranes are sheet-like extensions of oligodendrocytes that can be considered membrane domains distinct from the cell's plasma membrane. Consistent with the polarized nature of oligodendrocytes, we demonstrate that transcytotic transport of the major myelin-resident protein proteolipid protein (PLP) is a key element in the mechanism of myelin assembly. Upon biosynthesis, PLP traffics to myelin membranes via syntaxin 3-mediated docking at the apical-surface-like cell body plasma membrane, which is followed by subsequent internalization and transport to the basolateral-surface-like myelin sheet. Pulse-chase experiments, in conjunction with surface biotinylation and organelle fractionation, reveal that following biosynthesis, PLP is transported to the cell body surface in Triton X-100 (TX-100)-resistant microdomains. At the plasma membrane, PLP transiently resides within these microdomains and its lateral dissipation is followed by segregation into 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS)-resistant domains, internalization, and subsequent transport toward the myelin membrane. Sulfatide triggers PLP's reallocation from TX-100- into CHAPS-resistant membrane domains, while inhibition of sulfatide biosynthesis inhibits transcytotic PLP transport. Taking these findings together, we propose a model in which PLP transport to the myelin membrane proceeds via a transcytotic mechanism mediated by sulfatide and characterized by a conformational alteration and dynamic, i.e., transient, partitioning of PLP into distinct membrane microdomains involved in biosynthetic and transcytotic transport.  

3.2287           Outer-Inner Membrane Vesicles Naturally Secreted by Gram-Negative Pathogenic Bacteria

Pérez-Cruz C, Delgado L, López-Iglesias C, Mercade E PloS One, 10(1), e0116896 (2015)   Outer-inner membrane vesicles (O-IMVs) were recently described as a new type of membrane vesicle secreted by the Antarctic bacterium Shewanella vesiculosa M7T. Their formation is characterized by the protrusion of both outer and plasma membranes, which pulls cytoplasmic components into the vesicles. To demonstrate that this is not a singular phenomenon in a bacterium occurring in an extreme environment, the identification of O-IMVs in pathogenic bacteria was undertaken. With this aim, a structural study by Transmission Electron Microscopy (TEM) and Cryo-transmission electron microscopy (Cryo-TEM) was carried out, confirming that O-IMVs are also secreted by Gram-negative pathogenic bacteria such as Neisseria gonorrhoeae, Pseudomonas aeruginosa PAO1 and Acinetobacter baumannii AB41, in which they represent between 0.23% and 1.2% of total vesicles produced. DNA and ATP, which are components solely found in the cell cytoplasm, were identified within membrane vesicles of these strains. The presence of DNA inside the O-IMVs produced by N. gonorrhoeae was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. A proteomic analysis of N. gonorrhoeae-derived membrane vesicles identified proteins from the cytoplasm and plasma membrane. This confirmation of O-IMV extends the hitherto uniform definition of membrane vesicles in Gram-negative bacteria and explains the presence of components in membrane vesicles such as DNA, cytoplasmic and inner membrane proteins, as well as ATP, detected for the first time. The production of these O-IMVs by pathogenic Gram-negative bacteria opens up new areas of study related to their involvement in lateral gene transfer, the transfer of cytoplasmic proteins, as well as the functionality and role of ATP detected in these new vesicles.  

3.2288           Mammalian ER mannosidase I resides in quality control vesicles, where it encounters its glycoprotein substrates

Benyair, R., Ogen-Shtern, N., Mazkereth, N., Shai, B., Ehrlich, M and Lederkremer, G.Z. Mol. Biol. Cell, 26, 172-184 (2015)   Endoplasmic reticulum α1,2 mannosidase I (ERManI), a central component of ER quality control and ER-associated degradation (ERAD), acts as a timer enzyme, modifying N-linked sugar chains of glycoproteins with time. This process halts glycoprotein folding attempts when necessary and targets terminally misfolded glycoproteins to ERAD. Despite the importance of ERManI in maintenance of glycoprotein quality control, fundamental questions regarding this enzyme remain controversial. One such question is the subcellular localization of ERManI, which has been suggested to localize to the ER membrane, the ER-derived quality control compartment (ERQC), and, surprisingly, recently to the Golgi apparatus. To try to clarify this controversy, we applied a series of approaches that indicate that ERManI is located, at the steady state, in quality control vesicles (QCVs) to which ERAD substrates are transported and in which they interact with the enzyme. Both endogenous and exogenously expressed ERManI migrate at an ER-like density on iodixanol gradients, suggesting that the QCVs are derived from the ER. The QCVs are highly mobile, displaying dynamics that are dependent on microtubules and COP-II but not on COP-I vesicle machinery. Under ER stress conditions, the QCVs converge in a juxtanuclear region, at the ERQC, as previously reported. Our results also suggest that ERManI is turned over by an active autophagic process. Of importance, we found that membrane disturbance, as is common in immunofluorescence methods, leads to an artificial appearance of ERManI in a Golgi pattern.  

3.2289           Characterization of VAMP isoforms in 3T3-L1 adipocytes: implications for GLUT4 trafficking

Sadler, J.B., Bryant, N.J. and Gould, G.W. Mol. Biol. Cell, 26, 530-536 (2015)   The fusion of GLUT4-containing vesicles with the plasma membrane of adipocytes is a key facet of insulin action. This process is mediated by the formation of functional soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes between the plasma membrane t-SNARE complex and the vesicle v-SNARE or VAMP. The t-SNARE complex consists of Syntaxin4 and SNAP23, and whereas many studies identify VAMP2 as the v-SNARE, others suggest that either VAMP3 or VAMP8 may also fulfil this role. Here we characterized the levels of expression, distribution, and association of all the VAMPs expressed in 3T3-L1 adipocytes to provide the first systematic analysis of all members of this protein family for any cell type. Despite our finding that all VAMP isoforms form SDS-resistant SNARE complexes with Syntaxin4/SNAP23 in vitro, a combination of levels of expression (which vary by >30-fold), subcellular distribution, and coimmunoprecipitation analyses lead us to propose that VAMP2 is the major v-SNARE involved in GLUT4 trafficking to the surface of 3T3-L1 adipocytes.  

3.2290           Cell Type-Specific Affinity Purification of Nuclei for Chromatin Profiling in Whole Animals

Steiner, F.A. and Henikoff, S. Methods in Mol. Biol., 1228, 3-14 (2015)   Analyzing cell differentiation during development in a complex organism requires the analysis of expression and chromatin profiles in individual cell types. Our laboratory has developed a simple and generally applicable strategy to purify specific cell types from whole organisms for simultaneous analysis of chromatin and expression. The method, termed INTACT for Isolation of Nuclei TAgged in specific Cell Types, depends on the expression of an affinity-tagged nuclear envelope protein in the cell type of interest. These nuclei can be affinity-purified from the total pool of nuclei and used as a source for RNA and chromatin. The method serves as a simple and scalable alternative to FACS sorting or laser capture microscopy to circumvent the need for expensive equipment and specialized skills. This chapter provides detailed protocols for the cell-type specific purification of nuclei from Caenorhabditis elegans.  

3.2291           Transferrin receptor expression in serum exosomes as a marker of regenerative anaemia in the horse

Rout, E.D., Webb, T.L., Laurence, H.M., Long, L. and Olver, C.S. Equine Veterinary Journal, 47(1), 101-106 (2015)   Reasons for performing study Evaluation of erythrocyte regeneration in horses is challenging, as they do not release reticulocytes into the peripheral blood. This study investigated transferrin receptor 1 (TfR1) expression in exosomes as a noninvasive method of characterising the regenerative response in anaemic horses. Objectives To quantify TfR1 in ultraprecipitate of serum in horses before and after phlebotomy-induced anaemia, and to identify exosomes as the source of TfR1. The hypothesis was that serum exosomal TfR1 expression would increase during a regenerative response. Study design Experimental model of anaemia. Methods Six horses were phlebotomised to achieve a 25% decrease in packed cell volume. Transferrin receptor 1 quantity in exosomes was determined by western blot and relative densitometry before and after phlebotomy. The size and density of the TfR1-associated particles were confirmed by transmission electron microscopy and density gradient centrifugation, respectively. Results Regenerative anaemia was confirmed by decreased packed cell volumes and decreased myeloid:erythroid ratios in the bone marrow. In all 6 horses, TfR1 expression increased between Days 7 and 10. Mean TfR1 levels peaked on Day 10 and at 3-fold higher than levels on Day 0. Appropriately sized particles were evident on transmission electron microscopy and sucrose density gradient fractions expected to contain exosomes also contained TfR1. Conclusions These data indicate that TfR1 expression in serum exosomes may provide a marker for regeneration in anaemic horses.  

3.2292           Co-operation of TLR4 and raft proteins in LPS-induced pro-inflammatory signaling

Plociennikowska, A., hromada-Judycka, A., Borzecka, K. and Kwiatkowska, K. Cell. Mol. Life Sci., 72, 557-581 (2015)   Toll-like receptor 4 (TLR4) is activated by lipopolysaccharide (LPS), a component of Gram-negative bacteria to induce production of pro-inflammatory mediators aiming at eradication of the bacteria. Dysregulation of the host responses to LPS can lead to a systemic inflammatory condition named sepsis. In a typical scenario, activation of TLR4 is preceded by binding of LPS to CD14 protein anchored in cholesterol- and sphingolipid-rich microdomains of the plasma membrane called rafts. CD14 then transfers the LPS to the TLR4/MD-2 complex which dimerizes and triggers MyD88- and TRIF-dependent production of pro-inflammatory cytokines and type I interferons. The TRIF-dependent signaling is linked with endocytosis of the activated TLR4, which is controlled by CD14. In addition to CD14, other raft proteins like Lyn tyrosine kinase of the Src family, acid sphingomyelinase, CD44, Hsp70, and CD36 participate in the TLR4 signaling triggered by LPS and non-microbial endogenous ligands. In this review, we summarize the current state of the knowledge on the involvement of rafts in TLR4 signaling, with an emphasis on how the raft proteins regulate the TLR4 signaling pathways. CD14-bearing rafts, and possibly CD36-rich rafts, are believed to be preferred sites of the assembly of a multimolecular complex which mediates the endocytosis of activated TLR4.  

3.2293           Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing

Lane, R.E., Korbie, D., Anderson, W., Vaidyanathan, R. and Trau, M. Scientific Reports, 5:7639 (2015)   Exosomes are vesicles which have garnered interest due to their diagnostic and therapeutic potential. Isolation of pure yields of exosomes from complex biological fluids whilst preserving their physical characteristics is critical for downstream applications. In this study, we use 100 nm-liposomes from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol as a model system as a model system to assess the effect of exosome isolation protocols on vesicle recovery and size distribution using a single-particle analysis method. We demonstrate that liposome size distribution and ζ-potential are comparable to extracted exosomes, making them an ideal model for comparison studies. Four different purification protocols were evaluated, with liposomes robustly isolated by three of them. Recovered yields varied and liposome size distribution was unaltered during processing, suggesting that these protocols do not induce particle aggregation. This leads us to conclude that the size distribution profile and characteristics of vesicles are stably maintained during processing and purification, suggesting that reports detailing how exosomes derived from tumour cells differ in size to those from normal cells are reporting a real phenomenon. However, we hypothesize that larger particles present in most purified exosome samples represent co-purified contaminating non-exosome debris. These isolation techniques are therefore likely nonspecific and may co-isolate non-exosome material of similar physical properties.  

3.2294           DRAM1 regulates apoptosis through increasing protein levels and lysosomal localization of BAX

Guan, J.J., Zhang, X.D., Sun, W., Qi, L., Wu, J.C. and Qin, Z.H. Cell Death and Disease, 6, e1624 (2015)   DRAM1 (DNA damage-regulated autophagy modulator 1) is a TP53 target gene that modulates autophagy and apoptosis. We previously found that DRAM1 increased autophagy flux by promoting lysosomal acidification and protease activation. However, the molecular mechanisms by which DRAM1 regulates apoptosis are not clearly defined. Here we report a novel pathway by which DRAM1 regulates apoptosis involving BAX and lysosomes. A549 or HeLa cells were treated with the mitochondrial complex II inhibitor, 3-nitropropionic acid (3NP), or an anticancer drug, doxorubicin. Changes in the protein and mRNA levels of BAX and DRAM1 and the role of DRAM1 in BAX induction were determined. The interaction between DRAM1 and BAX and its effect on BAX degradation, BAX lysosomal localization, the release of cathepsin B and cytochrome c by BAX and the role of BAX in 3NP- or doxorubicin-induced cell death were studied. The results showed that BAX, a proapoptotic protein, was induced by DRAM1 in a transcription-independent manner. BAX was degraded by autophagy under basal conditions; however, its degradation was inhibited when DRAM1 expression was induced. There was a protein interaction between DRAM1 and BAX and this interaction prolonged the half-life of BAX. Furthermore, upregulated DRAM1 recruited BAX to lysosomes, leading to the release of lysosomal cathepsin B and cleavage of BID (BH3-interacting domain death agonist). BAX mediated the release of mitochondrial cytochrome c, activation of caspase-3 and cell death partially through the lysosome-cathepsin B-tBid pathway. These results indicate that DRAM1 regulates apoptosis by inhibiting BAX degradation. In addition to mitochondria, lysosomes may also be involved in BAX-initiated apoptosis.  

3.2295           An oncogenic role of ​Agrin in regulating focal adhesion integrity in hepatocellular carcinoma

Chakraborty, S., Lakshmanan, M., Swa, H.L.F., Chen, J., Zhang, X., Ong, Y.S., Loo, L.S., Akincilar, S.C., Gunaratne, J., tergaonkar, V., Hui, K.M. and Hong, W. Nature Communications, 6:6184 (2015)   Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths globally. The identity and role of cell surface molecules driving complex biological events leading to HCC progression are poorly understood, hence representing major lacunae in HCC therapies. Here, combining SILAC quantitative proteomics and biochemical approaches, we uncover a critical oncogenic role of ​Agrin, which is overexpressed and secreted in HCC. ​Agrin enhances cellular proliferation, migration and oncogenic signalling. Mechanistically, ​Agrin’s extracellular matrix sensor activity provides oncogenic cues to regulate Arp2/3-dependent ruffling, invadopodia formation and epithelial–mesenchymal transition through sustained focal adhesion integrity that drives liver tumorigenesis. Furthermore, ​Agrin signalling through ​Lrp4-muscle-specific tyrosine kinase (​MuSK) forms a critical oncogenic axis. Importantly, antibodies targeting ​Agrin reduced oncogenic signalling and tumour growth in vivo. Together, we demonstrate that ​Agrin is frequently upregulated and important for oncogenic property of HCC, and is an attractive target for antibody therapy.  

3.2296           Proteomic Analysis of Isolated Ciliary Transition Zones Reveals the Presence of ESCRT Proteins

Diener, D.R., Lupetti, P. and Rosenbaum, J.L. Current Biology, 25(3), 379-384 (2015)   The transition zone (TZ) is a specialized region of the cilium characterized by Y-shaped connectors between the microtubules of the ciliary axoneme and the ciliary membrane [ 1 ]. Located near the base of the cilium, the TZ is in the prime location to act as a gate for proteins into and out of the ciliary compartment, a role supported by experimental evidence [ 2–6 ]. The importance of the TZ has been underscored by studies showing that mutations affecting proteins located in the TZ result in cilia-related diseases, or ciliopathies, presenting symptoms including renal cysts, retinal degeneration, and situs inversus [ 7–9 ]. Some TZ proteins have been identified and shown to interact with each other through coprecipitation studies in vertebrate cells [ 4, 10, 11 ] and genetics studies in C. elegans [ 3 ]. As a distinct approach to identify TZ proteins, we have taken advantage of the biology of Chlamydomonas to isolate TZs. Proteomic analysis identified 115 proteins, ten of which were known TZ proteins related to ciliopathies, indicating that the preparation was highly enriched for TZs. Interestingly, six proteins of the endosomal sorting complexes required for transport (ESCRT) were also associated with the TZs. Identification of these and other proteins in the TZ will provide new insights into functions of the TZ, as well as candidate ciliopathy genes.

3.2297           A Protocol for Exosome Isolation and Characterization: Evaluation of Ultracentrifugation, Density-Gradient Separation, and Immunoaffinity Capture Methods

Greening, D.W., Xu, R., Ji, H., Tauro, B.J. and Simpson, R.J.

Methods in Mol. Biol., 1295, 179-209 (2015)

Exosomes are 40–150 nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of tumorigenic proteins, mRNA and miRNA. Exosomes are important regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest their importance for diagnostic and therapeutic applications, and as drug delivery vehicles. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. In this chapter, we reveal the protocol and key insights into the isolation, purification and characterization of exosomes, distinct from shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, a comprehensive evaluation of exosome isolation methods including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM-coated magnetic beads (IAC-Exos) were examined. All exosome isolation methodologies contained 40–150 nm vesicles based on electron microscopy, and positive for exosome markers (Alix, TSG101, HSP70) based on immunoblotting. This protocol employed a proteomic profiling approach to characterize the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method in exosome isolation. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, IAC-Exos was shown to be the most effective method to isolate exosomes. However, the use of density-based separation (DG-Exos) provides significant advantages for exosome isolation when the use of immunoaffinity capture is limited (due to antibody availability and suitability of exosome markers).

 

3.2298           UBC9-dependent Association between Calnexin and Protein Tyrosine Phosphatase 1B (PTP1B) at the Endoplasmic Reticulum

Lee, D., Kraus, A., Prins, D., Groenendyk, J., Aubry, I., Liu, W-X., Li, H-D., Julien, O., Touret, N., Sykes, B.D., Tremblay, M.L. and Michalak, M.

1. Biol. Chem., 290(9), 5725-5738 (2015)

  Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein, molecular chaperone, and a component of the translocon. We discovered a novel interaction between the calnexin cytoplasmic domain and UBC9, a SUMOylation E2 ligase, which modified the calnexin cytoplasmic domain by the addition of SUMO. We demonstrated that calnexin interaction with the SUMOylation machinery modulates an interaction with protein tyrosine phosphatase 1B (PTP1B), an ER-associated protein tyrosine phosphatase involved in the negative regulation of insulin and leptin signaling. We showed that calnexin and PTP1B form UBC9-dependent complexes, revealing a previously unrecognized contribution of calnexin to the retention of PTP1B at the ER membrane. This work shows that the SUMOylation machinery links two ER proteins from divergent pathways to potentially affect cellular protein quality control and energy metabolism.  

3.2299           Lipid-induced NOX2 activation inhibits autophagic flux by impairing lysosomal enzyme activity

Jaishy, B., Zhang, Q., Chung, H.S., Riehle, C., Soto, J., Jenkins, S., Abel, P., Cowart, L.A., Van Eyk, J.E. and Abel, E.D.

1. Lipid Res., 56, 546-561 (2015)

  Autophagy is a catabolic process involved in maintaining energy and organelle homeostasis. The relationship between obesity and the regulation of autophagy is cell type specific. Despite adverse consequences of obesity on cardiac structure and function, the contribution of altered cardiac autophagy in response to fatty acid overload is incompletely understood. Here, we report the suppression of autophagosome clearance and the activation of NADPH oxidase (Nox)2 in both high fat-fed murine hearts and palmitate-treated H9C2 cardiomyocytes (CMs). Defective autophagosome clearance is secondary to superoxide-dependent impairment of lysosomal acidification and enzyme activity in palmitate-treated CMs. Inhibition of Nox2 prevented superoxide overproduction, restored lysosome acidification and enzyme activity, and reduced autophagosome accumulation in palmitate-treated CMs. Palmitate-induced Nox2 activation was dependent on the activation of classical protein kinase Cs (PKCs), specifically PKCβII. These findings reveal a novel mechanism linking lipotoxicity with a PKCβ-Nox2-mediated impairment in pH-dependent lysosomal enzyme activity that diminishes autophagic turnover in CMs.  

3.2300           ABCB4 exports phosphatidylcholine in a sphingomyelin-dependent manner

Zhao, Y., Ishigami, M., Nagao, K., hanada, K., Kono, N., Arai, H., matssuo, M., Kioka, N. and Ueda, K.

1. Lipid Res., 56, 644-652 (2015)

  ABCB4, which is specifically expressed on the canalicular membrane of hepatocytes, exports phosphatidylcholine (PC) into bile. Because SM depletion increases cellular PC content and stimulates PC and cholesterol efflux by ABCA1, a key transporter involved in generation of HDL, we predicted that SM depletion also stimulates PC efflux through ABCB4. To test this prediction, we compared the lipid efflux activity of ABCB4 and ABCA1 under SM depletion induced by two different types of inhibitors for SM synthesis, myriocin and (1R,3S)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide, in human embryonic kidney 293 and baby hamster kidney cells. Unexpectedly, SM depletion exerted opposite effects on ABCB4 and ABCA1, suppressing PC efflux through ABCB4 while stimulating efflux through ABCA1. Both ABCB4 and ABCA1 were recovered from Triton-X-100-soluble membranes, but ABCB4 was mainly recovered from CHAPS-insoluble SM-rich membranes, whereas ABCA1 was recovered from CHAPS-soluble membranes. These results suggest that a SM-rich membrane environment is required for ABCB4 to function. ABCB4 must have evolved to exert its maximum activity in the SM-rich membrane environment of the canalicular membrane, where it transports PC as the physiological substrate.  

3.2301           Identification of a Family of Fatty-Acid-Speciated Sonic Hedgehog Proteins, Whose Members Display Differential Biological Properties

Long, J., Tokhunts, R., Ahn, N.G. and Robbins, D.J: Cell Reports, 10, 1280-1287 (2015)   Hedgehog (HH) proteins are proteolytically processed into a biologically active form that is covalently modified by cholesterol and palmitate. However, most studies of HH biogenesis have characterized protein from cells in which HH is overexpressed. We purified Sonic Hedgehog (SHH) from cells expressing physiologically relevant levels and showed that it was more potent than SHH isolated from overexpressing cells. Furthermore, the SHH in our preparations was modified with a diverse spectrum of fatty acids on its amino termini, and this spectrum of fatty acids varied dramatically depending on the growth conditions of the cells. The fatty acid composition of SHH affected its trafficking to lipid rafts as well as its potency. Our results suggest that HH proteins exist as a family of diverse lipid-speciated proteins that might be altered in different physiological and pathological contexts in order to regulate distinct properties of HH proteins.  

3.2302           Subcellular localization and activation of ADAM proteases in the context of FasL shedding in T lymphocytes

Ebsen, H., Lettau, M:, kabelitz, D. and Janssen, O. Mol. Immunol., 65, 416-428 (2015)   The “A Disintegrin And Metalloproteinases” (ADAMs) form a subgroup of the metzincin endopeptidases. Proteolytically active members of this protein family act as sheddases and govern key processes in development and inflammation by regulating cell surface expression and release of cytokines, growth factors, adhesion molecules and their receptors. In T lymphocytes, ADAM10 sheds the death factor Fas Ligand (FasL) and thereby regulates T cell activation, death and effector function. Although FasL shedding by ADAM10 was confirmed in several studies, its regulation is still poorly defined. We recently reported that ADAM10 is highly abundant on T cells whereas its close relative ADAM17 is expressed at low levels and transiently appears at the cell surface upon stimulation. Since FasL is also stored intracellularly and brought to the plasma membrane upon stimulation, we addressed where the death factor gets exposed to ADAM proteases. We report for the first time that both ADAM10 and ADAM17 are associated with FasL-containing secretory lysosomes. Moreover, we demonstrate that TCR/CD3/CD28-stimulation induces a partial positioning of both proteases and FasL to lipid rafts and only the activation-induced raft-positioning results in FasL processing. TCR/CD3/CD28-induced FasL proteolysis is markedly affected by reducing both ADAM10 and ADAM17 protein levels, indicating that in human T cells also ADAM17 is implicated in FasL processing. Since FasL shedding is affected by cholesterol depletion and by inhibition of Src kinases or palmitoylation, we conclude that it requires mobilization and co-positioning of ADAM proteases in lipid raft-like platforms associated with an activation of raft-associated Src-family kinases.  

3.2303           Host ESCRT Proteins Are Required for Bromovirus RNA Replication Compartment Assembly and Function

Diaz, A., Zhang, J., Ollwerther, A., Wang, X. and Ahlquist, P. PloS Pathogens, 11(3), e1004742 (2015)   Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV) RNA replication occurs on perinuclear endoplasmic reticulum (ER) membranes in ~70 nm vesicular invaginations (spherules). BMV RNA replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport) membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA replication and the frequency of viral replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV) spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance.  

3.2304           Polysome arrest restricts miRNA turnover by preventing exosomal export of miRNA in growth-retarded mammalian cells

Ghosh, S., Bose, M., Ray, A. and Bhattacharyya, S.N. Mol. Biol. Cell, 26, 1072-1083 (2015)   MicroRNAs (miRNAs) are tiny posttranscriptional regulators of gene expression in metazoan cells, where activity and abundance of miRNAs are tightly controlled. Regulated turnover of these regulatory RNAs is important to optimize cellular response to external stimuli. We report that the stability of mature miRNAs increases inversely with cell proliferation, and the increased number of microribonucleoproteins (miRNPs) in growth-restricted mammalian cells are in turn associated with polysomes. This heightened association of miRNA with polysomes also elicits reduced degradation of target mRNAs and impaired extracellular export of miRNA via exosomes. Overall polysome sequestration contributes to an increase of cellular miRNA levels but without an increase in miRNA activity. Therefore miRNA activity and turnover can be controlled by subcellular distribution of miRNPs that may get differentially regulated as a function of cell growth in mammalian cells.  

3.2305           The Assembly of GM1 Glycolipid- and Cholesterol-Enriched Raft-Like Membrane Microdomains Is Important for Giardial Encystation

De Chatterjee, A., Mendez, T.L., Roychowdury, S. and Das, S.

Infect. Immun., 83(5), 2030-2042 (2015)

Although encystation (or cyst formation) is an important step of the life cycle of Giardia, the cellular events that trigger encystation are poorly understood. Because membrane microdomains are involved in inducing growth and differentiation in many eukaryotes, we wondered if these raft-like domains are assembled by this parasite and participate in the encystation process. Since the GM1 ganglioside is a major constituent of mammalian lipid rafts (LRs) and known to react with cholera toxin B (CTXB), we used Alexa Fluor-conjugated CTXB and GM1 antibodies to detect giardial LRs. Raft-like structures in trophozoites are located in the plasma membranes and on the periphery of ventral discs. In cysts, however, they are localized in the membranes beneath the cyst wall. Nystatin and filipin III, two cholesterol-binding agents, and oseltamivir (Tamiflu), a viral neuraminidase inhibitor, disassembled the microdomains, as evidenced by reduced staining of trophozoites with CTXB and GM1 antibodies. GM1- and cholesterol-enriched LRs were isolated from Giardia by density gradient centrifugation and found to be sensitive to nystatin and oseltamivir. The involvement of LRs in encystation could be supported by the observation that raft inhibitors interrupted the biogenesis of encystation-specific vesicles and cyst production. Furthermore, culturing of trophozoites in dialyzed medium containing fetal bovine serum (which is low in cholesterol) reduced raft assembly and encystation, which could be rescued by adding cholesterol from the outside. Our results suggest that Giardia is able to form GM1- and cholesterol-enriched lipid rafts and these raft domains are important for encystation.

 

3.2306           Lipidomic and proteomic characterization of platelet extracellular vesicle subfractions from senescent platelets

Pienimaeki-Roemer, A., Kuhlmann, K., Böttcher, A., Konovalova, T., Black, A., Orso, E., Liebish, G., Ahrens, M., Eisenacher, M., Meyer, H.E. and Schmitz, G. Transfusion, 55(3), 507-521 (2015)   Background Platelets (PLTs) in stored PLT concentrates (PLCs) release PLT extracellular vesicles (PL-EVs) induced by senescence and activation, resembling the PLT storage lesion. No comprehensive classification or molecular characterization of senescence-induced PL-EVs exists to understand PL-EV heterogeneity. Study Design and Methods PL-EVs from 5-day-stored PLCs from healthy individuals were isolated and subfractionated by differential centrifugation, filtration, and density gradient ultracentrifugation into five PLT microvesicle (PL-MV) subfractions (Fraction [F]1-F5) and PLT exosomes (PL-EXs). PL-EV size, concentration, and composition were analyzed by nanoparticle tracking analysis, flow cytometry, and lipid and protein mass spectrometry. Protein data were verified by Western blot. Results PL-EVs showed overlapping mean particle sizes of 180 to 260 nm, but differed significantly in composition. Less dense, intermediate, and dense PL-MVs enriched specific lipidomic and proteomic markers related to the plasma membrane, intracellular membranes, PLT granules, mitochondria, and PLT activation. α-Synuclein (81% of total) accumulated in F1 and F2, amyloid-β (Aβ) precursor protein in F3 and F4 (84%), and apolipoprotein (Apo)E (88%) and ApoJ (92%) in F3 to F5. PL-EXs enriched lipid species and proteins, with high abundance of lipid raft, PLT adhesion, and immune response–related markers. Conclusion Differential lipid and protein compositions of PL-EVs suggest their unique cellular origins and functions, partly overlapping with PLT granule secretion. Dense PL-MVs might represent autophagic vesicles released during PLT activation and apoptosis and PL-EXs resemble lipid rafts, with a potential role in PLT aggregation and immunity. Segregation of α-synuclein and Aβ precursor protein, ApoE, and ApoJ into less dense and dense PL-MVs, respectively, show their differential carrier role of neurologic disease–related cargo.  

3.2307           Phospholipids of tumor extracellular vesicles stratify gefitinib-resistant nonsmall cell lung cancer cells from gefitinib-sensitive cells

Jung, J.H., Lee, M.Y., Choi, D-Y., Lee, J.W., You, S., Lee, K.Y., Kim, J. and Kim, K.P. Proteomics, 15(4), 824-835 (2015)   Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) such as gefitinib are one of gold standard treatment options for nonsmall-cell lung cancer (NSCLC) patients, which eventually fail due to the acquired resistance and relapse because of the development of secondary activating mutations such as T790M in EGFR. Predicting chemo-responsiveness of cancer patients provides a major challenge in chemotherapy. The goal of the present study is to determine whether phospholipid signatures of tumor extracellular vesicles (EV) are associated with gefitinib-resistance of NSCLC. A sophisticated MS-based shotgun lipidomic assays were performed for in-depth analysis of the lipidomes of gefitinib-resistant (PC9R) and responsive (PC9) NSCLC cells and their shed EV from these cell lines (PC9EV or PC9REV). Lipid MALDI-MS analysis showed that EV phospholipid composition was significantly distinct in PC9R, compared to PC9 cells. Following statistical analyses has identified 35 (20 positive and 15 negative ion mode) differentially regulated lipids, which are significantly over- or underexpressed in PC9R EV, compared to PC9 EV (p value < 0.01, fold change > 1.5). Our phospholipid signatures suggest that EV associates with drug sensitivity, which is worthy of additional investigation to assess chemoresistance in patients with NSCLC treated with anti-EGFR TKIs.  

3.2308           Evidence for the functioning of a Cl−/H+ antiporter in the membranes isolated from root cells of the halophyte Suaeda altissima and enriched with Golgi membranes

Shuvalov, A.V., Orlava, J.V., Khalilova, L.A., Myasoedov, N.A., Andreev, I.M., Belyaev, D.V. and Balkonin, Y.V. Russian J. Plant Physiol., 61(1), 45-56 (2015)   Cl⁻/H⁺ exchange activity in the membranes isolated from the root cells of the halophyte Suaeda altissima (L.) Pall. was originally revealed and characterized. The membrane vesicles were isolated by centrifugation of microsomes in a continuous iodixanol density gradient. The highest activity of latent inosine phosphatase, a marker of Golgi membranes, was localized in the upper part of the gradient, indicating its enrichment with Golgi membranes. The same part of the gradient was characterized by the highest Cl⁻/H⁺ exchange rate. The Cl⁻/H⁺ exchange activity was detected as electrogenic ΔpCl-dependent H⁺ transport monitored as changes in differential absorbance of a ΔpH-probe acridine orange, or as changes in fluorescence excitation spectrum of a pH-probe pyranine loaded into the vesicles. Generation of transmembrane electric potential (Δψ) during the Cl⁻/H⁺ exchange was assayed as changes in differential absorbance of a Δψ-probe safranin O. Establishing the transmembrane ΔpCl inward vesicles resulted in H⁺ efflux sensitive to DIDS (4,4′-diisothiocyano-2,2′-stylbene-disulfonic acid), an inhibitor of chloride transporters and channels, and generation of Δψ negative inside. To maintain the ΔpCl-dependent H⁺ efflux from the vesicles, either the presence of a penetrating cation tetraphenylphosphonium neutralizing negative charges inside the vesicles or null K⁺ diffusion potential across the membranes was required. The results demonstrate the activity of an electrogenic Cl⁻/H⁺ antiporter in the fraction enriched with Golgi membranes. We hypothesize that the Cl⁻/H⁺ antiporter is involved into the regulation of cytoplasmic Cl⁻ concentrations by vesicular trafficking of Cl⁻ from the cytoplasm to the vacuole by endosomes, derivatives of Golgi membranes.  

3.2309           Detergent-Resistant Membrane Association of NS2 and E2 during Hepatitis C Virus Replication

Shanmugam, S., Saravanabalaji, D. and Yi, M.

1. Virol., 89(8), 4562-4574 (2015)

  Previously, we demonstrated that the efficiency of hepatitis C virus (HCV) E2-p7 processing regulates p7-dependent NS2 localization to putative virus assembly sites near lipid droplets (LD). In this study, we have employed subcellular fractionations and membrane flotation assays to demonstrate that NS2 associates with detergent-resistant membranes (DRM) in a p7-dependent manner. However, p7 likely plays an indirect role in this process, since only the background level of p7 was detectable in the DRM fractions. Our data also suggest that the p7-NS2 precursor is not involved in NS2 recruitment to the DRM, despite its apparent targeting to this location. Deletion of NS2 specifically inhibited E2 localization to the DRM, indicating that NS2 regulates this process. Treatment of cells with methyl-β-cyclodextrin (MβCD) significantly reduced the DRM association of Core, NS2, and E2 and reduced infectious HCV production. Since disruption of the DRM localization of NS2 and E2, either due to p7 and NS2 defects, respectively, or by MβCD treatment, inhibited infectious HCV production, these proteins' associations with the DRM likely play an important role during HCV assembly. Interestingly, we detected the HCV replication-dependent accumulation of ApoE in the DRM fractions. Taking into consideration the facts that ApoE was shown to be a major determinant for infectious HCV particle production at the postenvelopment step and that the HCV Core protein strongly associates with the DRM, recruitment of E2 and ApoE to the DRM may allow the efficient coordination of Core particle envelopment and postenvelopment events at the DRM to generate infectious HCV production.  

3.2310           Human T-Cell Leukemia Virus Type 1 (HTLV-1) Tax Requires CADM1/TSLC1 for Inactivation of the NF-κB Inhibitor A20 and Constitutive NF-κB Signaling

Pujari, R., Hunte, R., Thomas, R., van der Weyden, L., Rauch, D., Ratner, L., Nyborg, J.K., Ramos, J.C., Takai, Y. and Shembade, N. PloS Pathogens, 11(3), e1004721 (2015)   Persistent activation of NF-κB by the Human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, Tax, is vital for the development and pathogenesis of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). K63-linked polyubiquitinated Tax activates the IKK complex in the plasma membrane-associated lipid raft microdomain. Tax also interacts with TAX1BP1 to inactivate the NF-κB negative regulatory ubiquitin-editing A20 enzyme complex. However, the molecular mechanisms of Tax-mediated IKK activation and A20 protein complex inactivation are poorly understood. Here, we demonstrated that membrane associated CADM1 (Cell adhesion molecule1) recruits Ubc13 to Tax, causing K63-linked polyubiquitination of Tax, and IKK complex activation in the membrane lipid raft. The c-terminal cytoplasmic tail containing PDZ binding motif of CADM1 is critical for Tax to maintain persistent NF-κB activation. Finally, Tax failed to inactivate the NF-κB negative regulator ubiquitin-editing enzyme A20 complex, and activate the IKK complex in the lipid raft in absence of CADM1. Our results thus indicate that CADM1 functions as a critical scaffold molecule for Tax and Ubc13 to form a cellular complex with NEMO, TAX1BP1 and NRP, to activate the IKK complex in the plasma membrane-associated lipid rafts, to inactivate NF-κB negative regulators, and maintain persistent NF-κB activation in HTLV-1 infected cells.  

3.2311           Dietary and biliary phosphatidylcholine activates PKC{zeta} in rat intestine

Siddiqi, S. and Mansbach II, C.M.

1. Lipid Res., 56, 859-870 (2015)

  Chylomicron output by the intestine is proportional to intestinal phosphatidylcholine (PC) delivery. Using five different variations of PC delivery to the intestine, we found that lyso-phosphatidylcholine (lyso-PC), the absorbed form of PC, concentrations in the cytosol (0 to 0.45 nM) were proportional to the input rate. The activity of protein kinase C (PKC)ζ, which controls prechylomicron output rate by the endoplasmic reticulum (ER), correlated with the lyso-PC concentration suggesting that it may be a PKCζ activator. Using recombinant PKCζ, the K_m for lyso-PC activation was 1.49 nM and the V_max 1.12 nM, more than the maximal lyso-PC concentration in cytosol, 0.45 nM. Among the phospholipids and their lyso derivatives, lyso-PC was the most potent activator of PKCζ and the only one whose cytosolic concentration suggested that it could be a physiological activator because other phospholipid concentrations were negligible. PKCζ was on the surface of the dietary fatty acid transport vesicle, the caveolin-1-containing endocytic vesicle. Once activated, PKCζ, eluted off the vesicle. A conformational change in PKCζ on activation was suggested by limited proteolysis. We conclude that PKCζ on activation changes its conformation resulting in elution from its vesicle. The downstream effect of dietary PC is to activate PKCζ, resulting in greater chylomicron output by the ER.  

3.2312           Autophagic bulk sequestration of cytosolic cargo is independent of LC3, but requires GABARAPs

Szalai, P., Korseberg Hagen, L., Sætre, F., Luhr, M., Sponheim, M., Øverbye, A., Mills, I.G., Seglen, P.O. and Engedal, N. Exp. Cell Res., 333, 21-38 (2015)   LC3, a mammalian homologue of yeast Atg8, is assumed to play an important part in bulk sequestration and degradation of cytoplasm (macroautophagy), and is widely used as an indicator of this process. To critically examine its role, we followed the autophagic flux of LC3 in rat hepatocytes during conditions of maximal macroautophagic activity (amino acid depletion), combined with analyses of macroautophagic cargo sequestration, measured as transfer of the cytosolic protein lactate dehydrogenase (LDH) to sedimentable organelles. To accurately determine LC3 turnover we developed a quantitative immunoblotting procedure that corrects for differential immunoreactivity of cytosolic and membrane-associated LC3 forms, and we included cycloheximide to block influx of newly synthesized LC3. As expected, LC3 was initially degraded by the autophagic-lysosomal pathway, but, surprisingly, autophagic LC3-flux ceased after ~2 h. In contrast, macroautophagic cargo flux was well maintained, and density gradient analysis showed that sequestered LDH partly accumulated in LC3-free autophagic vacuoles. Hepatocytic macroautophagy could thus proceed independently of LC3. Silencing of either of the two mammalian Atg8 subfamilies in LNCaP prostate cancer cells exposed to macroautophagy-inducing conditions (starvation or the mTOR-inhibitor Torin1) confirmed that macroautophagic sequestration did not require the LC3 subfamily, but, intriguingly, we found the GABARAP subfamily to be essential.  

3.2313           PTEN secretion in exosomes

Putz, U., Mah, S., Goh, C-P., Low, L-H., Howitt, J. and Tan, S-S. Methods, 77-78, 157-163 (2015)   PTEN was discovered as a membrane-associated tumor suppressor protein nearly two decades ago, but the concept that it can be secreted and taken up by recipient cells is revolutionary. Since then, various laboratories have reported that PTEN is indeed secreted and available for uptake by other cells in at least two different guises. First, PTEN may be packaged and exported within extracellular vesicles (EV) called exosomes. Second, PTEN may also be secreted as a naked protein in a longer isoform called PTEN-long. While the conditions favouring the secretion of PTEN-long remain unknown, PTEN secretion in exosomes is enhanced by the Ndfip1/Nedd4 ubiquitination system. In this report, we describe conditions for packaging PTEN in exosomes and their potential use for mediating non cell-autonomous functions in recipient cells. We suggest that this mode of PTEN transfer may potentially provide beneficial PTEN for tumor suppression, however it may also propagate deleterious versions of mutated PTEN causing tumorigenesis.  

3.2314           Scavenger Receptor SREC-I Mediated Entry of TLR4 into Lipid Microdomains and Triggered Inflammatory Cytokine Release in RAW 264.7 Cells upon LPS Activation

Murshid, A., Gong, J., Prince, T., Borges, T.J. and Calderwood, S.K: PloS One, 10(4), e122529 (2015)   Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation. As our previous studies indicated that SREC-I required intact Toll-like receptor 4 (TLR4) expression for its functions in tumor immunity, we examined potential interactions between these two receptors. We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS) in RAW 264.7 and HEK 293 cells overexpressing these two receptors. The receptors then became internalized together in intracellular endosomes. SREC-I promoted TLR4-induced signal transduction through the NF-kB and MAP kinase pathways, leading to enhanced inflammatory cytokine release. Activation of inflammatory signaling through SREC-I/TLR4 complexes appeared to involve recruitment of the receptors into detergent-insoluble, cholesterol-rich lipid microdomains that contained the small GTPase Cdc42 and the non-receptor tyrosine kinase c-src. Under conditions of SREC-I activation by LPS, TLR4 activity required Cdc42 as well as cholesterol and actin polymerization for signaling through NF-kB and MAP kinase pathways in RAW 264.7 cells. SREC-I appeared to respond differently to another ligand, the molecular chaperone Hsp90 that, while triggering SREC-I-TLR4 binding caused only faint activation of the NF-kB pathway. Our experiments therefore indicated that SREC-I could bind LPS and might be involved in innate inflammatory immune responses to extracellular danger signals in RAW 264.7 cells or bone marrow-derived macrophages.  

3.2315           The extracellular RNA complement of Escherichia coli

Ghosal, A., Upadhyaya, B.B., Fritz, J.V., Heintz-Buschart, A., Desai, M.S., Yusuf, D., Huang, D., Baumuratov, A., Wang, K., Galas, D. and Wilmes, P. MicrobiologyOpen, 4(2), 252-266 (2015%   The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular RNA complement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV-free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA-fimL and ves-spy intergenic regions. Our study provides the first detailed characterization of the extracellular RNA complement of the enteric model bacterium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication.  

3.2316           Subcellular localization and activation of ADAM proteases in thecontext of FasL shedding in T lymphocytes

Ebsen, H., Lettau, M., Kabelitz, D. and Janssen, O. Mol. Immunol., 65, 416-428 (2015)   The “A Disintegrin And Metalloproteinases” (ADAMs) form a subgroup of the metzincin endopeptidases. Proteolytically active members of this protein family act as sheddases and govern key processes in development and inflammation by regulating cell surface expression and release of cytokines, growth factors, adhesion molecules and their receptors. In T lymphocytes, ADAM10 sheds the death factor Fas Ligand (FasL) and thereby regulates T cell activation, death and effector function. Although FasL shedding by ADAM10 was confirmed in several studies, its regulation is still poorly defined. We recently reported that ADAM10 is highly abundant on T cells whereas its close relative ADAM17 is expressed at low levels and transiently appears at the cell surface upon stimulation. Since FasL is also stored intracellularly and brought to the plasma membrane upon stimulation, we addressed where the death factor gets exposed to ADAM proteases. We report for the first time that both ADAM10 and ADAM17 are associated with FasL-containing secretory lysosomes. Moreover, we demonstrate that TCR/CD3/CD28-stimulation induces a partial positioning of both proteases and FasL to lipid rafts and only the activation-induced raft-positioning results in FasL processing. TCR/CD3/CD28-induced FasL proteolysis is markedly affected by reducing both ADAM10 and ADAM17 protein levels, indicating that in human T cells also ADAM17 is implicated in FasL processing. Since FasL shedding is affected by cholesterol depletion and by inhibition of Src kinases or palmitoylation, we conclude that it requires mobilization and co-positioning of ADAM proteases in lipid raft-like platforms associated with an activation of raft-associated Src-family kinases.  

3.2317           Cholesterol Transport through Lysosome-Peroxisome Membrane Contacts

Chu, B-B., Liao, Y-C., Qi, W., Xie, C., Du, X., Wang, J., yang, H., Miao, H-H.., Li, B-L. and Song, B-L. Cell, 161, 291-306 (2015)   Cholesterol is dynamically transported among organelles, which is essential for multiple cellular functions. However, the mechanism underlying intracellular cholesterol transport has remained largely unknown. We established an amphotericin B-based assay enabling a genome-wide shRNA screen for delayed LDL-cholesterol transport and identified 341 hits with particular enrichment of peroxisome genes, suggesting a previously unappreciated pathway for cholesterol transport. We show dynamic membrane contacts between peroxisome and lysosome, which are mediated by lysosomal Synaptotagmin VII binding to the lipid PI(4,5)P₂ on peroxisomal membrane. LDL-cholesterol enhances such contacts, and cholesterol is transported from lysosome to peroxisome. Disruption of critical peroxisome genes leads to cholesterol accumulation in lysosome. Together, these findings reveal an unexpected role of peroxisome in intracellular cholesterol transport. We further demonstrate massive cholesterol accumulation in human patient cells and mouse model of peroxisomal disorders, suggesting a contribution of abnormal cholesterol accumulation to these diseases  

3.2318           Isolation of Extracellular Vesicles for Proteomic Profiling

Choi, D-S. and Gho, Y.S:

Methods in Mol. Biol., 1295, 167-177 (2015)

Extracellular vesicles are nano-sized lipid bilayer vesicles released from most cells, including archaea, bacteria, and eukaryotic cells. These membrane vesicles play multiple roles in cell-to-cell communication, including immune modulation, angiogenesis, and transformation of cells by transferring genetic material and functional proteins. They contain specific subsets of proteins, DNA, RNA, and lipids that represent their cellular status. Furthermore, extracellular vesicles are enriched in cell type- or disease-specific vesicular proteins, especially plasma membrane proteins, which have pathophysiological functions; these vesicular proteins are considered novel diagnostic biomarkers as well as therapeutic targets. To profile the proteome, various purification methods of extracellular vesicles have been developed, but density gradient ultracentrifugation is considered the most promising. In this chapter, we describe the isolation of extracellular vesicles derived from SW480 cells and the preparation of tryptic peptides for mass-spectrometry-based proteomic analysis.

   

3.2319           Lateral Gene Transfer and Gene Duplication Played a Key Role in the Evolution of Mastigamoeba balamuthi Hydrogenosomes

Nyvlota, E., Stairs, C.W., Hrdy, I., Ridl, J., Mach, J., Paces, J., Roger, A.J. and tachezy, J. Mol. Biol. Evol., 32(4), 1039-1055 (2015)   Lateral gene transfer (LGT) is an important mechanism of evolution for protists adapting to oxygen-poor environments. Specifically, modifications of energy metabolism in anaerobic forms of mitochondria (e.g., hydrogenosomes) are likely to have been associated with gene transfer from prokaryotes. An interesting question is whether the products of transferred genes were directly targeted into the ancestral organelle or initially operated in the cytosol and subsequently acquired organelle-targeting sequences. Here, we identified key enzymes of hydrogenosomal metabolism in the free-living anaerobic amoebozoan Mastigamoeba balamuthi and analyzed their cellular localizations, enzymatic activities, and evolutionary histories. Additionally, we characterized 1) several canonical mitochondrial components including respiratory complex II and the glycine cleavage system, 2) enzymes associated with anaerobic energy metabolism, including an unusual D-lactate dehydrogenase and acetyl CoA synthase, and 3) a sulfate activation pathway. Intriguingly, components of anaerobic energy metabolism are present in at least two gene copies. For each component, one copy possesses an mitochondrial targeting sequence (MTS), whereas the other lacks an MTS, yielding parallel cytosolic and hydrogenosomal extended glycolysis pathways. Experimentally, we confirmed that the organelle targeting of several proteins is fully dependent on the MTS. Phylogenetic analysis of all extended glycolysis components suggested that these components were acquired by LGT. We propose that the transformation from an ancestral organelle to a hydrogenosome in the M. balamuthi lineage involved the lateral acquisition of genes encoding extended glycolysis enzymes that initially operated in the cytosol and that established a parallel hydrogenosomal pathway after gene duplication and MTS acquisition.  

3.2320           Prometastatic NEDD9 Regulates Individual Cell Migration via Caveolin-1–Dependent Trafficking of Integrins

Kozyulina, P.Y., Loskutov, Y.V., Kozyreva, V.K: et al Mol. Cancer Res., 13(3), 423-438 (2015)   The dissemination of tumor cells relies on efficient cell adhesion and migration, which in turn depends upon endocytic trafficking of integrins. In the current work, it was found that depletion of the prometastatic protein, NEDD9, in breast cancer cells results in a significant decrease in individual cell migration due to impaired trafficking of ligand-bound integrins. NEDD9 deficiency does not affect the expression or internalization of integrins but heightens caveolae-dependent trafficking of ligand-bound integrins to early endosomes. Increase in mobility of ligand-bound integrins is concomitant with an increase in tyrosine phosphorylation of caveolin-1 (CAV1) and volume of CAV1-vesicles. NEDD9 directly binds to CAV1 and colocalizes within CAV1 vesicles. In the absence of NEDD9, the trafficking of ligand-bound integrins from early to late endosomes is impaired, resulting in a significant decrease in degradation of ligand–integrin complexes and an increase in recycling of ligand-bound integrins from early endosomes back to the plasma membrane without ligand disengagement, thus leading to low adhesion and migration. Reexpression of NEDD9 or decrease in the amount of active, tyrosine 14 phosphorylated (Tyr14) CAV1 in NEDD9-depleted cells rescues the integrin trafficking deficiency and restores cellular adhesion and migration capacity. Collectively, these findings indicate that NEDD9 orchestrates trafficking of ligand-bound integrins through the attenuation of CAV1 activity.  

3.2321           Inhibition of CD40-Induced N-Ras Activation Reduces Leishmania major Infection

Chakraborty, S., Srivastava, A., Jha, M.K., Nair, A., Pandey, S.P., Srivastava, N., Kumari, S., Singh, S., Krishnasastry, M.V. and Saha, B.

1. Immunol., 194(8), 3852-3860 (2015)

  Leishmania major is a parasite that resides and replicates in macrophages. We previously showed that the parasite enhanced CD40-induced Raf-MEK-ERK signaling but inhibited PI3K-MKK-p38MAPK signaling to proleishmanial effects. As Raf and PI3K have a Ras-binding domain but exert opposite effects on Leishmania infection, we examined whether Ras isoforms had differential roles in Leishmania infection. We observed that L. major enhanced N-Ras and H-Ras expression but inhibited K-Ras expression in macrophages. L. major infection enhanced N-Ras activity but inhibited H-Ras and K-Ras activity. TLR2 short hairpin RNA or anti-TLR2 or anti-lipophosphoglycan Abs reversed the L. major–altered N-Ras and K-Ras expressions. Pam₃CSK₄, a TLR2 ligand, enhanced N-Ras expression but reduced K-Ras expression, indicating TLR2-regulated Ras expression in L. major infection. Whereas N-Ras silencing reduced L. major infection, K-Ras and H-Ras silencing enhanced the infection both in macrophages in vitro and in C57BL/6 mice. BALB/c-derived macrophages transduced with lentivirally expressed N-Ras short hairpin RNA and pulsed with L. major–expressed MAPK10 enhanced MAPK10-specific Th1-type response. CD40-deficient mice primed with these macrophages had reduced L. major infection, accompanied by higher IFN-γ but less IL-4 production. As N-Ras is activated by Sos, a guanine nucleotide exchange factor, we modeled the N-Ras–Sos interaction and designed two peptides from their interface. Both the cell-permeable peptides reduced L. major infection in BALB/c mice but not in CD40-deficient mice. These data reveal the L. major–enhanced CD40-induced N-Ras activation as a novel immune evasion strategy and the potential for Ras isoform–targeted antileishmanial immunotherapy and immunoprophylaxis.  

3.2322           Impaired maturation of large dense-core vesicles in muted-deficient adrenal chromaffin cells

Hao, Z., Wei, L., Feng, Y., Chen, X., Du, W., Ma, J., Zhou, Z., Chen, L. and Li, W.

1. Cell Sci., 128(7), 1365-1374 (2015)

  The large dense-core vesicle (LDCV), a type of lysosome-related organelle, is involved in the secretion of hormones and neuropeptides in specialized secretory cells. The granin family is a driving force in LDCV biogenesis, but the machinery for granin sorting to this biogenesis pathway is largely unknown. The mu mutant mouse, which carries a spontaneous null mutation on the Muted gene (also known as Bloc1s5), which encodes a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), is a mouse model of Hermansky–Pudlak syndrome. Here, we found that LDCVs were enlarged in mu adrenal chromaffin cells. Chromogranin A (CgA, also known as CHGA) was increased in mu adrenals and muted-knockdown cells. The increased CgA in mu mice was likely due a failure to export this molecule out of immature LDCVs, which impairs LDCV maturation and docking. In mu chromaffin cells, the size of readily releasable pool and the vesicle release frequency were reduced. Our studies suggest that the muted protein is involved in the selective export of CgA during the biogenesis of LDCVs.  

3.2323           Outer membrane vesicles are vehicles for the delivery of Vibrio tasmaniensis virulence factors to oyster immune cells

Vanhove, A.S., Duperthuy, M., Charriere, G.M., Le Roux, F., Goudenege, D., Gourbal, B., Kieffer-Jaquinod, S., Coute, Y., Wai, S.N. and Destoumieux-garcon, D. Environment. Microbiol.,17(4), 1152-1165 (2015)   Vibrio tasmaniensis LGP32, a facultative intracellular pathogen of oyster haemocytes, was shown here to release outer membrane vesicles (OMVs) both in the extracellular milieu and inside haemocytes. Intracellular release of OMVs occurred inside phagosomes of intact haemocytes having phagocytosed few vibrios as well as in damaged haemocytes containing large vacuoles heavily loaded with LGP32. The OMV proteome of LGP32 was shown to be rich in hydrolases (25%) including potential virulence factors such as proteases, lipases, phospholipases, haemolysins and nucleases. One major caseinase/gelatinase named Vsp for vesicular serine protease was found to be specifically secreted through OMVs in which it is enclosed. Vsp was shown to participate in the virulence phenotype of LGP32 in oyster experimental infections. Finally, OMVs were highly protective against antimicrobial peptides, increasing the minimal inhibitory concentration of polymyxin B by 16-fold. Protection was conferred by OMV titration of polymyxin B but did not depend on the activity of Vsp or another OMV-associated protease. Altogether, our results show that OMVs contribute to the pathogenesis of LGP32, being able to deliver virulence factors to host immune cells and conferring protection against antimicrobial peptides.  

3.2324           Inhibition of iron uptake by ferristatin II is exerted through internalization of DMT1 at the plasma membrane

Yanatori, I., Yasui, Y., Noguchi, Y. and Kishi, F. Cell Biol. Int., 39(4), 427-434 (2015)   Ferristatin II, discovered as an iron transport inhibitor, promotes the internalization and degradation of transferrin receptor 1 (TfR1). DMT1, which mediates iron transport across cell membranes, is located at the plasma membrane of enterocytes and imports dietary iron into the cytosol. TfR1 is not directly engaged in the intestinal absorption of free iron, and iron uptake by DMT1 is attenuated by ferristatin II treatment. In this study, we found another function for ferristatin II in iron uptake. Ferristatin II did not cause degradation of DMT1 but did induce DMT1 internalization from the plasma membrane. Dynasore, a small molecule inhibitor of dynamin, did not inhibit this internalization by ferristatin II, which might occur via a clathrin-independent pathway.  

3.2325           The mGluR5 Positive Allosteric Modulator CDPPB Inhibits SO2-Induced Protein Radical Formation and Mitochondrial Dysfunction Through Activation of Akt in Mouse Hippocampal HT22 Cells

Guan, D-F., Ren, P-Y., Hu, W. and Zhang, Y-L. Cell. Mol. Neurobiol., 35, 573-583 (2015)   Sulfur dioxide (SO₂) is a common gas pollutant that is detrimental to many organs. Previous studies have shown that SO₂ exposure is involved in neurotoxicity and increased risk of many brain disorders; however, our understanding of the mechanisms underlying SO₂-induced cytotoxicity on neuronal cells remains elusive. The group I metabotropic glutamate receptor 5 (mGluR5) can modulate addiction, pain, and neuronal cell death. In the present study, we showed that SO₂ derivatives exposure induced protein radical formation, mitochondrial dysfunction, and apoptotic cell death in neuronal HT22 cells. Pretreatment with 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) (CDPPB), a positive allosteric modulator of mGluR5, significantly attenuated SO₂-induced neurotoxicity, which was fully prevented by the mGluR5 antagonist MPEP. CDPPB reduced the protein radical formation and inducible nitric oxide synthase (iNOS)-derived generation of nitric oxide, and inhibited mitochondrial dysfunction in both HT22 cells and isolated mitochondria after SO₂ treatment. Moreover, CDPPB increased the activation of Akt in the presence and absence of SO₂ treatment. Blocking Akt activation using the selective inhibitor LY294002 partially reversed the CDPPB-induced protection against SO₂-induced neurotoxicity. This study provides mechanistic experimental support for oxidative stress and mitochondrial dysfunction after SO₂ exposure in neuronal cells, and also introduces a novel therapeutic approach for SO₂-induced neurotoxicity.  

3.2326           Lipid rafts and raft-mediated supramolecular entities in the regulation of CD95 death receptor apoptotic signaling

Gajate, C. and Mollinedo, F. Apoptosis, 20, 584-606 (2015)   Membrane lipid rafts are highly ordered membrane domains enriched in cholesterol, sphingolipids and gangliosides that have the property to segregate and concentrate proteins. Lipid and protein composition of lipid rafts differs from that of the surrounding membrane, thus providing sorting platforms and hubs for signal transduction molecules, including CD95 death receptor-mediated signaling. CD95 can be recruited to rafts in a reversible way through S-palmitoylation following activation of cells with its physiological cognate ligand as well as with a wide variety of inducers, including several antitumor drugs through ligand-independent intracellular mechanisms. CD95 translocation to rafts can be modulated pharmacologically, thus becoming a target for the treatment of apoptosis-defective diseases, such as cancer. CD95-mediated signaling largely depends on protein–protein interactions, and the recruitment and concentration of CD95 and distinct downstream apoptotic molecules in membrane raft domains, forming raft-based supramolecular entities that act as hubs for apoptotic signaling molecules, favors the generation and amplification of apoptotic signals. Efficient CD95-mediated apoptosis involves CD95 and raft internalization, as well as the involvement of different subcellular organelles. In this review, we briefly summarize and discuss the involvement of lipid rafts in the regulation of CD95-mediated apoptosis that may provide a new avenue for cancer therapy.  

3.2327           Alterations in Cholesterol and Ganglioside GM1 Content of Lipid Rafts in Platelets From Patients With Alzheimer Disease

Liu, L., Zhang, K., Tan, L., Chen, Y-H. and Cao, Y-P. Alzheimer. Dis. Assoc. Disord., 29, 63-69 (2015)   The aim of this study was to investigate the changes in the protein, cholesterol, and ganglioside GM1 content of lipid rafts in platelets from patients with Alzheimer disease (AD), and identify potential blood biomarkers of the disease. A total of 31 Chinese patients with AD and 31 aged-matched control subjects were selected. Lipid rafts were isolated from platelets using Optiprep gradient centrifugation. The protein content of lipid rafts was evaluated using Micro BCA assay, the cholesterol content using molecular probes, ganglioside GM1 content using colorimetry and dot-blotting analysis. The results showed that the cholesterol and ganglioside GM1 content of lipid rafts from platelets was significantly higher in patients with AD than aged-matched control subjects, whereas the protein content of lipid rafts did not show any differences between the 2 groups. These results indicate that the increases in the cholesterol and ganglioside GM1 content of lipid rafts from the platelets of patients with AD might serve as a biochemical adjunct to the clinical diagnosis of AD.  

3.2328           Delivery of liposomal contents to outer membrane vesicles from gram negative bacteria

Ficurilli, M., Liu, C., Riviello, C., Pozo, M.J. and Meers, P.R. Biophys. J., 108(2), Suppl. 1 408a (2015)   Gram negative bacteria produce small ~50-200 nm vesicles from their outer membranes. These outer membrane vesicles (OMV) have been implicated in activities such as transmission of virulence factors, horizontal gene transfer and development of biofilms. In this investigation, we continue our studies on the association and/or fusion of various liposomes with OMV. The delivery of large encapsulated molecules into OMV from L. enzymogenes C3 was investigated using liposomes with lipid compositions previously observed to be apparently fusogenic (Bartos et al., Biophys. J. 104(2) suppl1, 90a). Liposomes (100 nm) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(10-racglycerol) (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in a 1:3 ratio or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were used to encapsulate dextran conjugates of Texas red averaging 40 kDa. They were incubated with Lysobacter OMV (30_C, 1 hr.), then sedimented through 15% iodixanol, and the fluorescence monitored as indicatiive of transfer of liposomal contents to fused products. Both liposomal compositions showed significant evidence of dextran transfer. Because biofilms also contain OMV, the interaction of these liposomes with E. coli (DH10B) biofilms was also investigated via fluorescence microscopy. Significant penetration and binding within the biofilm mass was observed, as well as possible fusion with OMV, and rarely, evidence of transfer of dextran into whole bacterial cells. Fluorescence resonance energy transfer (FRET)-based assays also demonstrated that liposomes as small as 30 nm could rapidly fuse with Lysobacter OMV, suggesting possible delivery to OMV with smaller perturbation and better biofilm penetration.  

3.2329           Three-Dimensional Architecture of Murine Rod Cilium Revealed by Cryo-EM

Wensel, T. and Gilliam, J.C. Methods in Mol. Biol., 1271, 267-292 (2015)   The connecting cilium of the rod photoreceptor is a tubular structure that bridges two adjacent cellular compartments, the inner segment, the major site of biosynthesis and energy metabolism, and the outer segment, a highly specialized ciliary structure responsible for phototransduction. The connecting cilium allows for active processes of protein sorting and transport to occur between them. Mutations affecting the cargo, their transporters, and the structural components of the primary cilium and basal body lead to aberrant trafficking and photoreceptor cell death. Understanding the overall design of the cilium, its architectural organization, and the function of varied protein complexes within the structural hierarchy of the cilium requires techniques for visualizing their native three-dimensional structures at high magnification. Here we describe methods for isolating retinas from mice, purifying fragments of rod cells that include much of the inner segment and the rod photoreceptor cilia, vitrifying the cell fragments, and determining their structures by cryo-electron tomography.  

3.2330           Structure-Based Mutational Analysis of Several Sites in the E Protein: Implications for Understanding the Entry Mechanism of Japanese Encephalitis Virus

Liu, H., Liu, Y., Wang, S., Zhang, Y., Zu, X., Zhou, Z., Zhang, B. and Xiao, G.-

1. Virol., 89(10), 5668-5686 (2015)

  Japanese encephalitis virus (JEV), which causes viral encephalitis in humans, is a serious risk to global public health. The JEV envelope protein mediates the viral entry pathway, including receptor-binding and low-pH-triggered membrane fusion. Utilizing mutagenesis of a JEV infectious cDNA clone, mutations were introduced into the potential receptor-binding motif or into residues critical for membrane fusion in the envelope protein to systematically investigate the JEV entry mechanism. We conducted experiments evaluating infectious particle, recombinant viral particle, and virus-like particle production and found that most mutations impaired virus production. Subcellular fractionation confirmed that five mutations—in I₀, ij, BC, and FG and the R9A substitution—impaired virus assembly, and the assembled virus particles of another five mutations—in kl and the E373A, F407A, L221S, and W217A substitutions—were not released into the secretory pathway. Next, we examined the entry activity of six mutations yielding infectious virus. The results showed N154 and the DE loop are not the only or major receptor-binding motifs for JEV entry into BHK-21 cells; four residues, H144, H319, T410, and Q258, participating in the domain I (DI)-DIII interaction or zippering reaction are important to maintain the efficiency of viral membrane fusion. By continuous passaging of mutants, adaptive mutations from negatively charged amino acids to positively charged or neutral amino acids, such as E138K and D389G, were selected and could restore the viral entry activity.  

3.2331           A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles

Roier, S., Blume, T., Klug, L., Wagner, G.E., Elhenawy, W., Zangger, K., Prassl, R., reidl, J., Daum, G., Feldman, M.F. and Schild, S. Int. J. Med. Microbiol., 305, 298-309 (2015)   Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections.  

3.2332           Exosomes Are Unlikely Involved in Intercellular Nef Transfer

Luo, X., Fan, Y., park, I-W. and He, J.J. PloS One, 10(4), e0124436 (2015)   HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Several recent studies including ours have demonstrated that Nef can be transferred to neighboring cells and alters the function of these cells. However, how the intercellular Nef transfer occurs is in dispute. In the current study, we attempted to address this important issue using several complementary strategies, a panel of exosomal markers, and human CD4+ T lymphocyte cell line Jurkat and a commonly used cell line 293T. First, we showed that Nef was transferred from Nef-expressing or HIV-infected Jurkat to naïve Jurkat and other non-Jurkat cells and that the transfer required the membrane targeting function of Nef and was cell density-dependent. Then, we showed that Nef transfer was cell-cell contact-dependent, as exposure to culture supernatants or exosomes from HIV-infected Jurkat or Nef-expressing Jurkat and 293T led to little Nef detection in the target cells Jurkat. Thirdly, we demonstrated that Nef was only detected to be associated with HIV virions but not with acetylcholinesterase (AChE+) exosomes from HIV-infected Jurkat and not in the exosomes from Nef-expressing Jurkat. In comparison, when it was over-expressed in 293T, Nef was detected in detergent-insoluble AChE+/CD81^low/TSG101^low exosomes, but not in detergent-soluble AChE-/CD81^high/TSG101^high exosomes. Lastly, microscopic imaging showed no significant Nef detection in exosomal vesicle-like structures in and out 293T. Taken together, these results show that exosomes are unlikely involved in intercellular Nef transfer. In addition, this study reveals existence of two types of exosomes: AChE+/CD81^low/TSG101^low exosomes and AChE-/CD81^high/TSG101^high exosomes.  

3.2333           Endomembrane proteomics reveals putative enzymes involved in cell wall metabolism in wheat grain outer layers

Chateigner-Boutin, A-L., Suliman, M., Bouchet, B., Alvarado, C., Lollier, V., Rogniaux, H., Guillon, F. and Larre, C.

1. Exp. Botany, 66(9), 2649-2658 (2015)

  Cereal grain outer layers fulfil essential functions for the developing seed such as supplying energy and providing protection. In the food industry, the grain outer layers called ‘the bran’ is valuable since it is rich in dietary fibre and other beneficial nutriments. The outer layers comprise several tissues with a high content in cell wall material. The cell wall composition of the grain peripheral tissues was investigated with specific probes at a stage of active cell wall synthesis. Considerable wall diversity between cell types was revealed. To identify the cellular machinery involved in cell wall synthesis, a subcellular proteomic approach was used targeting the Golgi apparatus where most cell wall polysaccharides are synthesized. The tissues were dissected into outer pericarp and intermediate layers where 822 and 1304 proteins were identified respectively. Many carbohydrate-active enzymes were revealed: some in the two peripheral grain fractions, others only in one tissue. Several protein families specific to one fraction and with characterized homologs in other species might be related to the specific detection of a polysaccharide in a particular cell layer. This report provides new information on grain cell walls and its biosynthesis in the valuable outer tissues, which are poorly studied so far. A better understanding of the mechanisms controlling cell wall composition could help to improve several quality traits of cereal products (e.g. dietary fibre content, biomass conversion to biofuel).  

3.2334           The pathogenic human Torsin A in Drosophila activates the unfolded protein response and increases susceptibility to oxidative stress

Kim, A-Y., Seo, J-B., Kim, W-t., Choi, H.J., Kim, S-Y., Morrow, G., Tanguya, R.M., Steller, H. and Koh, Y.H. BMC Genomics, 16:338 (2015)   Background Dystonia1 (DYT1) dystonia is caused by a glutamic acid deletion (ΔE) mutation in the gene encoding Torsin A in humans (HTorA). To investigate the unknown molecular and cellular mechanisms underlying DYT1 dystonia, we performed an unbiased proteomic analysis. Results We found that the amount of proteins and transcripts of an Endoplasmic reticulum (ER) resident chaperone Heat shock protein cognate 3 (HSC3) and a mitochondria chaperone Heat Shock Protein 22 (HSP22) were significantly increased in the HTorA^ΔE– expressing brains compared to the normal HTorA (HTorA^WT) expressing brains. The physiological consequences included an increased susceptibility to oxidative and ER stress compared to normal HTorA^WT flies. The alteration of transcripts of Inositol-requiring enzyme-1 (IRE1)-dependent spliced X box binding protein 1(Xbp1), several ER chaperones, a nucleotide exchange factor, Autophagy related protein 8b (ATG8b) and components of the ER associated degradation (ERAD) pathway and increased expression of the Xbp1-enhanced Green Fluorescence Protein (eGFP) in HTorA^ΔE brains strongly indicated the activation of the unfolded protein response (UPR). In addition, perturbed expression of the UPR sensors and inducers in the HTorA^ΔE Drosophila brains resulted in a significantly reduced life span of the flies. Furthermore, the types and quantities of proteins present in the anti-HSC3 positive microsomes in the HTorA^ΔE brains were different from those of the HTorA^WT brains. Conclusion Taken together, these data show that HTorA^ΔE in Drosophila brains may activate the UPR and increase the expression of HSP22 to compensate for the toxic effects caused by HTorA^ΔE in the brains.  

3.2335           A Voltage-Gated Calcium Channel Regulates Lysosomal Fusion with Endosomes and Autophagosomes and Is Required for Neuronal Homeostasis

Tian, X., Gala, U., Zhang, Y., Shang, W., jaiswal, SD.N., di Ronza, A., Jaiswal, M., Yamamoto, S., Sandoval, H., Duraine, L., Sardiello, M., Sillitoe, R.V., Ventatachalam, K., Fan, H., Bellen, H.J: and Tong, C. PloS Biology, 13(3), e1002103 (2015)   Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.  

3.2336           MPP1 as a Factor Regulating Phase Separation in Giant Plasma Membrane-Derived Vesicles

Podkalicka, J., Biernatowska, A., Majkowski, M., Grzybek, M. and Sikorski, A.F. Biophys. J., 108, 2201-2211 (2015)   The existence of membrane-rafts helps to conceptually understand the spatiotemporal organization of membrane-associated events (signaling, fusion, fission, etc.). However, as rafts themselves are nanoscopic, dynamic, and transient assemblies, they cannot be directly observed in a metabolizing cell by traditional microscopy. The observation of phase separation in giant plasma membrane-derived vesicles from live cells is a powerful tool for studying lateral heterogeneity in eukaryotic cell membranes, specifically in the context of membrane rafts. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. It remains unclear, however, if this lipid-driven process is tuneable in any way by interactions with proteins. Here, we demonstrate that MPP1, a member of the MAGUK family, can modulate membrane properties such as the fluidity and phase separation capability of giant plasma membrane-derived vesicles. Our data suggest that physicochemical domain properties of the membrane can be modulated, without major changes in lipid composition, through proteins such as MPP1.  

3.2337           Prostaglandins regulate nuclear localization of Fascin and its function in nucleolar architecture

Groen, C.M., Jayo, A., parsons, M. and Tottle, T.L. Mol. Biol. Cell, 26, 1901-1917 (2015)   Fascin, a highly conserved actin-bundling protein, localizes and functions at new cellular sites in both Drosophila and multiple mammalian cell types. During Drosophila follicle development, in addition to being cytoplasmic, Fascin is in the nuclei of the germline-derived nurse cells during stages 10B–12 (S10B–12) and at the nuclear periphery during stage 13 (S13). This localization is specific to Fascin, as other actin-binding proteins, Villin and Profilin, do not exhibit the same subcellular distribution. In addition, localization of fascin1 to the nucleus and nuclear periphery is observed in multiple mammalian cell types. Thus the regulation and function of Fascin at these new cellular locations is likely to be highly conserved. In Drosophila, loss of prostaglandin signaling causes a global reduction in nuclear Fascin and a failure to relocalize to the nuclear periphery. Alterations in nuclear Fascin levels result in defects in nucleolar morphology in both Drosophila follicles and cultured mammalian cells, suggesting that nuclear Fascin plays an important role in nucleolar architecture. Given the numerous roles of Fascin in development and disease, including cancer, our novel finding that Fascin has functions within the nucleus sheds new light on the potential roles of Fascin in these contexts.  

3.2338           Muscle Releases Alpha-Sarcoglycan Positive Extracellular Vesicles Carrying miRNAs in the Bloodstream

Guescini, M., Canonico, B., Lucertini, F., Maggio, S., Annibalini, G., Barbieri, E., Luchetti, F., Papa, S. and Stocchi, V. PloS One, 10(5), e0125094 (2015)   In the past few years, skeletal muscle has emerged as an important secretory organ producing soluble factors, called myokines, that exert either autocrine, paracrine or endocrine effects. Moreover, recent studies have shown that muscle releases microRNAs into the bloodstream in response to physical exercise. These microRNAs affect target cells, such as hormones and cytokines. The mechanisms underlying microRNA secretion are poorly characterized at present. Here, we investigated whether muscle tissue releases extracellular vesicles (EVs), which carry microRNAs in the bloodstream under physiological conditions such as physical exercise. Using density gradient separation of plasma from sedentary and physically fit young men we found EVs positive for TSG101 and alpha-sarcoglycan (SGCA), and enriched for miR-206. Cytometric analysis showed that the SGCA+ EVs account for 1–5% of the total and that 60–65% of these EVs were also positive for the exosomal marker CD81. Furthermore, the SGCA-immuno captured sub-population of EVs exhibited higher levels of the miR-206/miR16 ratio compared to total plasma EVs. Finally, a significant positive correlation was found between the aerobic fitness and muscle-specific miRNAs and EV miR-133b and -181a-5p were significantly up-regulated after acute exercise. Thus, our study proposes EVs as a novel means of muscle communication potentially involved in muscle remodeling and homeostasis.  

3.2339           Generation of nanovesicles with sliced cellular membrane fragments for exogenous material delivery

Yoon, J., Jo, W., Jeong, D., Kim, J., jeong, H. and Park, J. Biomaterials, 59, 12-20 (2015)   We propose a microfluidic system that generates nanovesicles (NVs) by slicing living cell membrane with microfabricated 500 nm-thick silicon nitride (Si_xN_y) blades. Living cells were sliced by the blades while flowing through microchannels lined with the blades. Plasma membrane fragments sliced from the cells self-assembled into spherical NVs of ∼100–300 nm in diameter. During self-assembly, the plasma membrane fragments enveloped exogenous materials (here, polystyrene latex beads) from the buffer solution. About 30% of beads were encapsulated in NVs, and the generated NVs delivered the encapsulated beads across the plasma membrane of recipient cells, but bare beads could not penetrate the plasma membrane of recipient cells. This result implicates that the NVs generated using the method in this study can encapsulate and deliver exogenous materials to recipient cells, whereas exosomes secreted by cells can deliver only endogenous cellular materials.  

3.2340           Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid

He, C.H., Black, D.S., Nguyen, T.P.T., Wang, C., Srinivasan, C. and  Clarke, C.F. Biochim. Biophys. Acta, 1851, 1227-1239 (2015)   Coq9 is a polypeptide subunit in a mitochondrial multi-subunit complex, termed the CoQ-synthome, required for biosynthesis of coenzyme Q (ubiquinone or Q). Deletion of COQ9 results in dissociation of the CoQ-synthome, but over-expression of Coq8 putative kinase stabilizes the CoQ-synthome in the coq9 null mutant and leads to the accumulation of two nitrogen-containing Q intermediates, imino-demethoxy-Q₆(IDMQ₆) and 3-hexaprenyl-4-aminophenol (4-AP) when para-aminobenzoic acid (pABA) is provided as a ring precursor. To investigate whether Coq9 is responsible for deamination steps in Q biosynthesis, we utilized the yeast coq5-5 point mutant. The yeast coq5-5 point mutant is defective in the C-methyltransferase step of Q biosynthesis but retains normal steady-state levels of the Coq5 polypeptide. Here, we show that when high amounts of ¹³C₆-pABA are provided, the coq5-5 mutant accumulates both¹³C₆-imino-demethyl-demethoxy-Q₆ (¹³C₆-IDDMQ₆) and ¹³C₆-demethyl-demethoxy-Q₆ (¹³C₆-DDMQ₆). Deletion of COQ9 in the yeast coq5-5 mutant along with Coq8 over-expression and ¹³C₆- pABA labeling leads to the absence of ¹³C₆-DDMQ₆, and the nitrogen-containing intermediates ¹³C₆-4-AP and ¹³C₆-IDDMQ₆ persist. We describe a coq9 temperature-sensitive mutant and show that at the non-permissive temperature, steady-state polypeptide levels of Coq9-ts19 increased, while Coq4, Coq5, Coq6, and Coq7 decreased. The coq9-ts19 mutant had decreased Q₆ content and increased levels of nitrogen-containing intermediates. These findings identify Coq9 as a multi-functional protein that is required for the function of Coq6 and Coq7 hydroxylases, for removal of the nitrogen substituent from pABA-derived Q intermediates, and is an essential component of the CoQ synthome.  

3.2341           Characterisation of detergent-insoluble membranes in pollen tubes of Nicotiana tabacum (L.)

Moscatelli, A., Gagliardi, A., Maneta-Peyret, L., Bini, L., Stroppa, N., Onelli, E., Landi, C., Scali, M., Idilli, A.I. and Moreau, P.

Biology Open, 4, 378-399 (2015)

Pollen tubes are the vehicle for sperm cell delivery to the embryo sac during fertilisation of Angiosperms. They provide an intriguing model for unravelling mechanisms of growing to extremes. The asymmetric distribution of lipids and proteins in the pollen tube plasma membrane modulates ion fluxes and actin dynamics and is maintained by a delicate equilibrium between exocytosis and endocytosis. The structural constraints regulating polarised secretion and asymmetric protein distribution on the plasma membrane are mostly unknown. To address this problem, we investigated whether ordered membrane microdomains, namely membrane rafts, might contribute to sperm cell delivery. Detergent insoluble membranes, rich in sterols and sphingolipids, were isolated from tobacco pollen tubes. MALDI TOF/MS analysis revealed that actin, prohibitins and proteins involved in methylation reactions and in phosphoinositide pattern regulation are specifically present in pollen tube detergent insoluble membranes. Tubulins, voltage-dependent anion channels and proteins involved in membrane trafficking and signalling were also present. This paper reports the first evidence of membrane rafts in Angiosperm pollen tubes, opening new perspectives on the coordination of signal transduction, cytoskeleton dynamics and polarised secretion.

 

3.2342           Mitochondria of a human multidrug-resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane

Fantappie, O., Sassoli, C., Tani, A., Nosi, D., Marchatti, S., Formigli, L. and Mazzanti, R.

1. Cell. Mol. Med., 19(6), 1410-1417 (2015)

  Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P-glycoprotein (P-gp), breast cancer resistant protein and multiple resistance protein-1. The MDR phenotype is associated with the constitutive expression of COX-2 and iNOS, whereas celecoxib, a specific inhibitor of COX-2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX-2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1-positive cells, whereas COX-2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P-gp in mitochondria of MDR cancer cells independently from inhibition of COX-2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells.  

3.2343           Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles

Kieselbach, T., Zijnge, V., Granström, E. and Oscarsson, J. PloS One, 10(9), e0138591 (2015)   Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease.  

3.2344           K63 linked ubiquitin chain formation is a signal for HIF1A degradation by Chaperone-Mediated Autophagy

Ferreira, J.V., Soares, A.R., Ramalho, J.S., Pereira, P. and Girao, H. Scientific Reports, 5:10210 (2015)   Chaperone-Mediated Autophagy is a selective form of autophagy. Recently, the degradation of a newly identified CMA substrate, the HIF1A transcription factor, was found to be regulated by the ubiquitin ligase STUB1. In this study we show, for the first time, that K63 ubiquitination is necessary for CMA degradation of HIF1A in vitro and in vivo. Additionally, STUB1 mediates K63 linked ubiquitination of HIF1A. Our findings add a new regulatory step and increase the specificity of the molecular mechanism involved in CMA degradation of HIF1A, expanding the role of ubiquitination to yet another biological process, since the same mechanism might be applicable to other CMA substrates.  

3.2345           Directional cell movement through tissues is controlled by exosome secretion

Sung, B.H., Ketova, T., Hoshino, D., Zijlstra, A. and Weaver, A.M. Nature Communications, 6:7164 (2015)   Directional cell movement through tissues is critical for multiple biological processes and requires maintenance of polarity in the face of complex environmental cues. Here we use intravital imaging to demonstrate that secretion of exosomes from late endosomes is required for directionally persistent and efficient in vivo movement of cancer cells. Inhibiting exosome secretion or biogenesis leads to defective tumour cell migration associated with increased formation of unstable protrusions and excessive directional switching. In vitro rescue experiments with purified exosomes and matrix coating identify adhesion assembly as a critical exosome function that promotes efficient cell motility. Live-cell imaging reveals that exosome secretion directly precedes and promotes adhesion assembly. Fibronectin is found to be a critical motility-promoting cargo whose sorting into exosomes depends on binding to integrins. We propose that autocrine secretion of exosomes powerfully promotes directionally persistent and effective cell motility by reinforcing otherwise transient polarization states and promoting adhesion assembly.    

3.2346           ELMOD2 is anchored to lipid droplets by palmitoylation and regulates adipocyte triglyceride lipase recruitment

Suzuki, M., Murakami, T., Cheng, J., Kano, H., Fukata, M. and Fujimoto, T. Mol. Biol. Cell, 26, 2333-2342 (2015)   Adipocyte triglyceride lipase (ATGL) is the major enzyme involved in the hydrolysis of triglycerides. The Arf1–coat protein complex I (COPI) machinery is known to be engaged in the recruitment of ATGL to lipid droplets (LDs), but the regulatory mechanism has not been clarified. In the present study, we found that ELMOD2, a putative noncanonical Arf–GTPase activating protein (GAP) localizing in LDs, plays an important role in controlling ATGL transport to LDs. We showed that knockdown of ELMOD2 by RNA interference induced an increase in the amount of ATGL existing in LDs and decreased the total cellular triglycerides. These effects of ELMOD2 knockdown were canceled by transfection of small interfering RNA-resistant cDNA of wild-type ELMOD2 but not by that of mutated ELMOD2 lacking the Arf-GAP activity. ELMOD2 was distributed in the endoplasmic reticulum and mitochondria as well as in LDs, but palmitoylation was required only for distribution to LDs. An ELMOD2 mutant deficient in palmitoylation failed to reconstitute the ATGL transport after the ELMOD2 knockdown, indicating that distribution in LDs is indispensable to the functionality of ELMOD2. These results indicate that ELMOD2 regulates ATGL transport and cellular lipid metabolism by modulating the Arf1-COPI activity in LDs.  

3.2347           Association of Cytokines With Exosomes in the Plasma of HIV-1–Seropositive Individuals

Konadu, K.A., Chu, J., Huang, M.B., Amancha, P.K., Armstrong, W., Powell, M.D., Villinger, F. and Bond, V.C. Journal of Infectious Disease, 211(11), 1712-1716 (2015)   Human immunodeficiency virus (HIV)-infected and viremic individuals exhibit elevated levels of plasma cytokines. Here we show that most cytokines are not in free form but appear associated with exosomes that are distinct from virions. Purified exosomes were analyzed to determine the levels of 21 cytokines and chemokines and compared with exosome-depleted plasma. Most cytokines were markedly enriched in exosomes from HIV-positive individuals relative to negative controls and to plasma. Moreover, exposure of naive peripheral blood mononuclear cells to exosomes purified from HIV-positive patients induced CD38 expression on naive and central memory CD4⁺ and CD8⁺ T cells, probably contributing to inflammation and viral propagation via bystander cell activation.  

3.2348           Novel steps in the autophagic-lysosomal pathway

Sætre, F., Korseberg hagen, L., Engedal, N. and Seglen, P.O. FEBS J., 282(11), 2202-2214 (2015)   Autophagy is the process by which portions of cytoplasm are enclosed by membranous organelles, phagophores, which deliver the sequestered cytoplasm to degradative autophagic vacuoles. Genes and proteins involved in phagophore manufacture have been extensively studied, but little is known about how mature phagophores proceed through the subsequent steps of expansion, closure and fusion. Here we have addressed these issues by combining our unique autophagic cargo sequestration assay (using the cytosolic enzyme lactate dehydrogenase as a cargo marker) with quantitative measurements of the lipidation-dependent anchorage and turnover of the phagophore-associated protein LC3. In isolated rat hepatocytes, amino acid starved to induce maximal autophagic activity, the two unrelated reversible autophagy inhibitors 3-methyladenine (3MA) and thapsigargin (TG) both blocked cargo sequestration completely. However, whereas 3MA inhibited LC3 lipidation, TG did not, thus apparently acting at a post-lipidation step to prevent phagophore closure. Intriguingly, the resumption of cargo sequestration seen upon release from a reversible TG block was completely suppressed by 3MA, revealing that 3MA not only inhibits LC3 lipidation but also (like TG) blocks phagophore closure at a post-lipidation step. 3MA did not, however, prevent the resumption of lysosomal LC3 degradation, indicating that phagophores could fuse directly with degradative autophagic vacuoles without carrying cytosolic cargo. This fusion step was clearly blocked by TG. Furthermore, density gradient centrifugation revealed that a fraction of the LC3-marked phagophores retained by TG could be density-shifted by the acidotropic drug propylamine along with the lysosomal marker cathepsin B, suggesting physical association of some phagophores with lysosomes prior to cargo sequestration.    

3.2349           Tu1405 Apolipoprotein-Av Resists Proteolysis in the Intestinal Tract and Is Absorbed Intact, Controlled by Dietary Phosphatidylcholine

Siddiqi, S., Polly, S., Siddiqi, T. and Mansbach, C.M. Gastroenterology, 148(4), S880-S881 (2015)   Purpose of Study: Apolipoprotein-AV (ApoAV) correlates with serum triacylglycerol (TAG) levels and mutations of the apoAV gene cause otherwise unexplained elevated TAG levels in humans. ApoAV is present in enterocytes but is not synthesized by the intestinal cells suggesting that the apoAV is absorbed intact even though it is a 39 kDa protein. Here wetest the hypothesis that apoAV is endocytosed via caveolin-1 containing endocytic vesicles (CEV). We have proposed that CEV are the major mechanism for dietary fatty acid absorption (BBA 183; 1311, 2013). Methods Used: Brush borders (BB) were prepared by the Mg2+ precipitation method from rat intestine. The purity of the preparation was judged by alkaline phosphatase activity which was 17 fold that of the intestinal homogenate. Cytosol was prepared by standard methods. Caveolae were isolated from Triton X-100 treated BB and CEV from cytosol using an OptiPrep gradient. The CEV appeared in the detergent resistant fraction. Immuno-precipitation was used to identify BB and cytosolic proteins associated with apoAV in vivo using Triton X-100 solubilized BB and cytosol proteins. Proteins were identified by immunoblot. Results: In both BB and cytosol, apoAV strongly bound to CD36 and caveolin-1 in the detergent resistant fraction and weakly to clathrin and intestinal alkaline phosphatase (IAP). These results are consistent with the apoAV being on CEV. ApoAV migrated at 39 kDa on SDS-PAGE in BB and cytosol as expected for apoAV. By contrast, pancreatic cholesterol esterase (CEL), which is known to be absorbed by clathrin coated endocytic vesicles, strongly bound to clathrin and IAP and weakly to caveolin-1 and CD36 in the BB and cytosol. The cytosol from caveolin-1 knock out mice did not contain apoAV but did contain CEL. Rat intestinal fluid harvested post corn oil plus albumin gavage contained intact apoAV but greatly degraded albumin. We progressively increased the amount of phosphatidylcholine (PC) delivered to rat intestine using 5 different models. The amount of apoAV in intestinal cytosol was inversely related to the amount of PC delivered. Incubation of apoAV containing CEV with ER showed a 4 fold increase in ER-apoAV suggesting delivery of apoAV from CEV to the ER. Conclusions: We conclude that apoAV, unlike albumin, resists proteolysis in the intestinal tract. ApoAV is bound to caveolin-1 and CD36 on intestinal BB and is endocytosed via CEV. The CEV transport apoAV to the ER. ApoAV's binding to BB is controlled by PC concentrations in the intestinal lumen. CEL binds to clathrin and IAP and is endocytosed via clathrin coated vesicles, separate from the CEV pathway. Cartoon of absorption of apoAV showing normal uptake via CEV, blockage of uptake by lyso-PC, and delivery of apoAV to the ER on CEV.  

3.2350           Exosome release following activation of the dendritic cell immunoreceptor: A potential role in HIV-1 pathogenesis

Mfunyi, C.M., Vaillancourt, M., Vitry, J., Batomene, T-R. N., Posvandzic, A., lambert, A.A. and Gilbert, C. Virology, 484, 103-112 (2015)   Exosomes are extracellular vesicles (EVs) that play a role in intercellular communication. Stimulation of dendritic cells by the HIV-1 virus triggers their release. HIV-1 binds to dendritic cells via dendritic cell immunoreceptor (DCIR). This study shows that inhibiting the binding to DCIR significantly decreases exosome release by HIV-1-pulsed dendritic cells. In addition, exosome release from Raji-CD4 expressing DCIR cells stimulated by anti-DCIR or HIV-1 is decreased when the immunoreceptor tyrosine-based inhibition motif (ITIM) signaling motif of DCIR is mutated. Unlike the EVs released from Raji-CD4-DCIR cells after antibody stimulation, those released from HIV-1-infected cells contain the pro-apoptotic protein DAP-3. Furthermore, EVs from HIV-1 pulsed dendritic cells increase spontaneous apoptosis in uninfected CD4 T lymphocytes while they decrease it in neutrophils. This study describes for the first time that DCIR plays a role in the release of exosomes strengthening the importance of this receptor and EVs/exosomes in HIV-1 pathogenesis.  

3.2351           Vitamin E: Curse or benefit in Alzheimer’s disease? A systematic investigation of the impact of α-, γ- and δ-tocopherol on Aβ generation and degradation in neuroblastoma cells

Grimm, M.O.W., Stahlmann, C.P., Mett, J., Haupenthal, V.J., Zimmer, V.C., Lehmann, J., Hundsdörfer, B., Endres, K., Grimm, H.S. and Hartmann, T.

1. Nutr. Health Aging, 19(6), 646-654 (2015)

  Objectives The E vitamins are a class of lipophilic compounds including tocopherols, which have high antioxidative properties. Because of the elevated lipid peroxidation and increased reactive oxidative species in Alzheimer’s disease (AD) many attempts have been made to slow down the progression of AD by utilizing the antioxidative action of vitamin E. Beside the mixed results of these studies nothing is known about the impact of vitamin E on the mechanisms leading to amyloid-β production and degradation being responsible for the plaque formation, one of the characteristic pathological hallmarks in AD. Here we systematically investigate the influence of different tocopherols on Aβ production and degradation in neuronal cell lines. Measurements Beside amyloid-β level the mechanisms leading to Aβ production and degradation are examined. Results Surprisingly, all tocopherols have shown to increase Aβ level by enhancing the Aβ production and decreasing the Aβ degradation. Aβ production is enhanced by an elevated activity of the involved enzymes, the β- and γ-secretase. These secretases are not directly affected, but tocopherols increase their protein level and expression. We could identify significant differences between the single tocopherols; whereas α-tocopherol had only minor effects on Aβ production, δ-tocopherol showed the highest potency to increase Aβ generation. Beside Aβ production, Aβ clearance was decreased by affecting IDE, one of the major Aβ degrading enzymes. Conclusions Our results suggest that beside the beneficial antioxidative effects of vitamin E, tocopherol has in respect to AD also a potency to increase the amyloid-β level, which differ for the analysed tocopherols. We therefore recommend that further studies are needed to clarify the potential role of these various vitamin E species in respect to AD and to identify the form which comprises an antioxidative property without having an amyloidogenic potential.  

3.2352           Large oncosomes contain distinct protein cargo and represent a separate functional class of tumor-derived extracellular vesicles

Minciacchi, V.R. et al Oncotarget, 6(13), 11327-11341 (2015)   Large oncosomes (LO) are atypically large (1-10µm diameter) cancer-derived extracellular vesicles (EVs), originating from the shedding of membrane blebs and associated with advanced disease. We report that 25% of the proteins, identified by a quantitative proteomics analysis, are differentially represented in large and nano-sized EVs from prostate cancer cells. Proteins enriched in large EVs included enzymes involved in glucose, glutamine and amino acid metabolism, all metabolic processes relevant to cancer. Glutamine metabolism was altered in cancer cells exposed to large EVs, an effect that was not observed upon treatment with exosomes. Large EVs exhibited discrete buoyant densities in iodixanol (OptiPrep^TM) gradients. Fluorescent microscopy of large EVs revealed an appearance consistent with LO morphology, indicating that these structures can be categorized as LO. Among the proteins enriched in LO, cytokeratin 18 (CK18) was one of the most abundant (within the top 5^th percentile) and was used to develop an assay to detect LO in the circulation and tissues of mice and patients with prostate cancer. These observations indicate that LO represent a discrete EV type that may play a distinct role in tumor progression and that may be a source of cancer-specific markers.  

3.2353           Characterization of extracellular vesicles (exosomes) from HIV-1 infected macrophages treated with HIV-1 protease inhibitor, Ritonavir.

Deshmane, S., Sheffield, J., Khalili, K. and Datta, P.

1. Neurovirol., 21, Suppl. 1, Abstract P37, S1-S87 82015)

  Antiretroviral therapy (ART) prevents HIV-associated neurocognitive disorders (HAND). However, milder forms of HAND are still prevalent despite widespread use of ART. Ritonavir-boosted protease inhibitor monotherapy is a maintenance strategy that prevents nucleoside reverse transcriptase inhibitor toxicity and has been effective in maintaining longterm viral suppression in the majority of patients. In this study, we assessed whether extracellular vesicles (EVs) released from HIV-1 infected monocyte/macrophages cultured in the presence of HIV-1 protease inhibitor, Ritonavir, play a role in neurodegeneration. EVs were derived from conditioned media of U937 cells and U1 cells that are latently infected with HIV-1 and cultured in the presence and absence of HIV-1 protease inhibitor, Ritonavir (2.5 and 5 micro molar) by ultracentrifugation and Iodixanol (optiprep) gradient centrifugation. The EVs were characterized for markers of exosomes such as Tsg101 and Alix, and Acetylcholinesterase enzyme (AchE) activity, viral proteins Nef and Gag, and scanning electron microscopy. We have observed significant increase in Nef and Gag proteins in exosomes derived from ritonavir treated cells. Neuronal cultures treated with U1 and U1-ritonavir exosomes were found to be severely compromised in their ability to maintain existing neuronal network as well as their ability to form neurites in a scratch-wound assay. We observed a significant down regulation of cAMP-responseelement- binding protein (CREB) phosphorylation in neurons treated with U1 exosomes and U1 exosomes derived from Ritonavir treated cells in comparison to exosomes derived from U937 cells. Furthermore, we observed significant down regulation of CREB regulated gene expression in these neurons. Collectively, these observations demonstrate that exosomes derived from HIV-1 infected cell and cells treated with Ritonavir can cause neuronal dysfunction and degeneration by targeting CREB signaling pathway. The studies were funded by a pilot grant to PD (CNAC NIMH Grant Number P30MH092177) and ultilized services offered by core facilities of CNAC.  

3.2354           Overexpression of the Insulin-Like Growth Factor II Receptor Increases β-Amyloid Production and Affects Cell Viability

Wang, Y., Buggia-Prevot, V., Zavorka, M.E., Bleackley, R.C., MacDonald, R.G., Thinakaran, G. and kar, S. Mol. Cell. Biol., 35(14), 2368-2384 (2015)   Amyloid β (Aβ) peptides originating from amyloid precursor protein (APP) in the endosomal-lysosomal compartments play a critical role in the development of Alzheimer's disease (AD), the most common type of senile dementia affecting the elderly. Since insulin-like growth factor II (IGF-II) receptors facilitate the delivery of nascent lysosomal enzymes from the trans-Golgi network to endosomes, we evaluated their role in APP metabolism and cell viability using mouse fibroblast MS cells deficient in the murine IGF-II receptor and corresponding MS9II cells overexpressing the human IGF-II receptors. Our results show that IGF-II receptor overexpression increases the protein levels of APP. This is accompanied by an increase of β-site APP-cleaving enzyme 1 levels and an increase of β- and γ-secretase enzyme activities, leading to enhanced Aβ production. At the cellular level, IGF-II receptor overexpression causes localization of APP in perinuclear tubular structures, an increase of lipid raft components, and increased lipid raft partitioning of APP. Finally, MS9II cells are more susceptible to staurosporine-induced cytotoxicity, which can be attenuated by β-secretase inhibitor. Together, these results highlight the potential contribution of IGF-II receptor to AD pathology not only by regulating expression/processing of APP but also by its role in cellular vulnerability.  

3.2355           Ablation of retinal ciliopathy protein RPGR results in altered photoreceptor ciliary composition

Rao, K.N., Li, L., Anand, M. and Khanna, H. Scientific reports, 5:11137 (2015)   Cilia regulate several developmental and homeostatic pathways that are critical to survival. Sensory cilia of photoreceptors regulate phototransduction cascade for visual processing. Mutations in the ciliary protein RPGR (retinitis pigmentosa GTPase regulator) are a prominent cause of severe blindness disorders due to degeneration of mature photoreceptors. However, precise function of RPGR is still unclear. Here we studied the involvement of RPGR in ciliary trafficking by analyzing the composition of photoreceptor sensory cilia (PSC) in Rpgr^ko retina. Using tandem mass spectrometry analysis followed by immunoblotting, we detected few alterations in levels of proteins involved in proteasomal function and vesicular trafficking in Rpgr^ko PSC, prior to onset of degeneration. We also found alterations in the levels of high molecular weight soluble proteins in Rpgr^ko PSC. Our data indicate RPGR regulates entry or retention of soluble proteins in photoreceptor cilia but spares the trafficking of key structural and phototransduction-associated proteins. Given a frequent occurrence of RPGR mutations in severe photoreceptor degeneration due to ciliary disorders, our results provide insights into pathways resulting in altered mature cilia function in ciliopathies.  

3.2356           Mitochondrial m-calpain opens the mitochondrial permeability transition pore in ischemia–reperfusion

Shintani-Ishida, K. and Yoshida, K-i. Int. J. Cardiol., 197, 26-32 (2015)   Background/objectives : Opening of the mitochondrial permeability transition pore (mPTP) is involved in ischemia–reperfusion injury. Isoforms of Ca^2 +-activated cysteine proteases, calpains, are implicated in the development of myocardial infarction in ischemia–reperfusion. Growing evidence has revealed the presence of calpains in the mitochondria. We aimed to characterize mitochondrial calpains in the rat heart and to investigate the roles of calpains in mPTP opening after ischemia–reperfusion. Methods and results : Western blotting analysis showed the expression of μ-calpain, m-calpain and calpain 10 in mitochondria isolated from male Sprague-Dawley rats, but casein zymography detected only m-calpain activity. Subcellular fractionation of mitochondria demonstrated the distribution of m-calpain to the matrix fraction. Addition of > 500 μM of Ca^2 + to isolated mitochondria induced mitochondrial swelling, reflecting mPTP opening, and calpain activation. Ca^2 +-induced mitochondrial swelling was inhibited partially by the calpain inhibitor calpeptin. These results support a partial contribution of calpain in the opening of the mPTP. The addition of Ca^2 + to the mitochondria induced inactivation of complex I of the electron transport chain, and cleavage of the ND6 complex I subunit, which were inhibited by calpeptin. Mitochondria isolated from rat hearts that underwent 30 min of coronary occlusion followed by 30 min of reperfusion showed activation of mitochondrial calpains, ND6 cleavage, complex I inactivation, and mPTP opening, which were inhibited by pretreatment with calpain inhibitor 1. Conclusions : We demonstrated for the first time the presence of mitochondrial matrix m-calpain, and its contribution to complex I inactivation and mPTP opening after postischemic reperfusion in the rat heart.  

3.2357           Cellular Uptake Mechanism of TCTP-PTD in Human Lung Carcinoma Cells

Kim, H.Y., Kim, S., Pyun, H.J., Maeng, J. and Lee, K. Mol. Pharmaceutics, 12(1), 194-203 (2015)   We reported previously that human translationally controlled tumor protein (TCTP) contains, at its NH₂-terminus, a protein transduction domain (PTD), which we called TCTP-PTD, with the amino acid sequence MIIYRDLISH. In this report we describe how TCTP-PTD penetrates A549 human lung cancer cell membranes and promotes protein internalization. Cellular uptake of fluorescent TCTP-PTD and a recombinant fusion protein consisting of TCTP-PTD and GFP (green fluorescent protein) was analyzed by confocal fluorescence microscopy and flow cytometry. Inhibitor assays using several agents that perturb the internalization process revealed that TCTP-PTD transduces the cells partly via lipid-raft/caveola-dependent endocytosis and partly by macropinocytosis in a dynamin/actin/microtubule-dependent pathway. To trace the pathway followed by the penetration of TCTP-PTD, the localization of PTDs was investigated in the lipid-raft, subcellular, and ER fractions. We found that, after entry, TCTP-PTD is localized in the cytoplasm and cytoskeleton, but not in the nucleus, and is transported into endoplasmic reticulum (ER). Expression levels of caveolin-1 in A549 and HeLa cells are different, and these differences appear to contribute to the sensitivity of TCTP-PTD uptake inhibition, against lipid-raft depleter, nystatin. This elucidation of the underlying mechanism of TCTP-PTD translocation may help the design of approaches that employ TCTP-PTD in the cellular delivery of bioactive molecules.  

3.2358           Bacterial Protoplast-Derived Nanovesicles as Vaccine Delivery System against Bacterial Infection

Kim, O.Y., Choi, S.J., jang, S.C., park, K-S., Kim, S.R., Choi, J.P., Lim, J.H., Lee, S-W., park, J., Di Vizio, D., Lötvall, J., Kim, Y-K. and Gho, Y.S. Nano Lett., 15(1), 266-274 (2015)   The notion that widespread infectious diseases could be best managed by developing potent, adjuvant-free vaccines has resulted in the use of various biological immune-stimulating components as new vaccine candidates. Recently, extracellular vesicles, also known as exosomes and microvesicles in mammalian cells and outer membrane vesicles in Gram-negative bacteria, have gained attention for the next generation vaccine. However, the more invasive and effective the vaccine is in delivery, the more risk it holds for severe immune toxicity. Here, in optimizing the current vaccine delivery system, we designed bacterial protoplast-derived nanovesicles (PDNVs), depleted of toxic outer membrane components to generate a universal adjuvant-free vaccine delivery system. These PDNVs exhibited significantly higher productivity and safety than the currently used vaccine delivery vehicles and induced strong antigen-specific humoral and cellular immune responses. Moreover, immunization with PDNVs loaded with bacterial antigens conferred effective protection against bacterial sepsis in mice. These nonliving nanovesicles derived from bacterial protoplast open up a new avenue for the creation of next generation, adjuvant-free, less toxic vaccines to be used to prevent infectious diseases.  

3.2359           Circulating microRNAs: emerging biomarkers for diagnosis and prognosis in patient with gastrointestinal cancers

Lindner, K., haier, J., Wang, Z., Watson, D.I., Hussey, D.J. and Hummel, R. Clinical Science, 128, 1-15 (2015)   To identify novel non-invasive biomarkers for improved detection, risk assessment and prognostic evaluation of cancer, expression profiles of circulating microRNAs are currently under evaluation. Circulating microRNAs are highly promising candidates in this context, as they present some key characteristics for cancer biomarkers: they are tissue-specific with reproducible expression and consistency among individuals from the same species, they are potentially derived directly from the tumour and therefore might correlate with tumour progression and recurrence, and they are bound to proteins or contained in subcellular particles, such as microvesicles or exosomes, making them highly stable and resistant to degradation. The present review highlights the origin of circulating microRNAs, their stability in blood samples, and techniques to isolate exosomal microRNAs, and then addresses the current evidence supporting potential clinical applications of circulating miRNAs for diagnostic and prognostic purposes.  

3.2360           ANKS1B Gene Product AIDA-1 Controls Hippocampal Synaptic Transmission by Regulating GluN2B Subunit Localization

Tindi, J.O., Chavez, A.E., Cvejic, S., Calvo-Ochoa, E., Castillo, P.E. and Jordan, B.A.

1. Neurosci., 35(24), 8986-8996 (2015)

  NMDA receptors (NMDARs) are key mediators of glutamatergic transmission and synaptic plasticity, and their dysregulation has been linked to diverse neuropsychiatric and neurodegenerative disorders. While normal NMDAR function requires regulated expression and trafficking of its different subunits, the molecular mechanisms underlying these processes are not fully understood. Here we report that the amyloid precursor protein intracellular domain associated-1 protein (AIDA-1), which associates with NMDARs and is encoded by ANKS1B, a gene recently linked to schizophrenia, regulates synaptic NMDAR subunit composition. Forebrain-specific AIDA-1 conditional knock-out (cKO) mice exhibit reduced GluN2B-mediated and increased GluN2A-mediated synaptic transmission, and biochemical analyses show AIDA-1 cKO mice have low GluN2B and high GluN2A protein levels at isolated hippocampal synaptic junctions compared with controls. These results are corroborated by immunocytochemical and electrophysiological analyses in primary neuronal cultures following acute lentiviral shRNA-mediated knockdown of AIDA-1. Moreover, hippocampal NMDAR-dependent but not metabotropic glutamate receptor-dependent plasticity is impaired in AIDA-1 cKO mice, further supporting a role for AIDA-1 in synaptic NMDAR function. We also demonstrate that AIDA-1 preferentially associates with GluN2B and with the adaptor protein Ca²⁺/calmodulin-dependent serine protein kinase and kinesin KIF17, which regulate the transport of GluN2B-containing NMDARs from the endoplasmic reticulum (ER) to synapses. Consistent with this function, GluN2B accumulates in ER-enriched fractions in AIDA-1 cKO mice. These findings suggest that AIDA-1 regulates NMDAR subunit composition at synapses by facilitating transport of GluN2B from the ER to synapses, which is critical for NMDAR plasticity. Our work provides an explanation for how AIDA-1 dysfunction might contribute to neuropsychiatric conditions, such as schizophrenia.  

3.2361           Mass-Spectrometry-Based Molecular Characterization of Extracellular Vesicles: Lipidomics and Proteomics

Kreimer, S., Belov, A.M., Ghiran, I., Murthy, S.K., Frank, D.A. and Ivanov, A.R.

1. Proteome Res., 14(6), 2367-2384 (2015)

  This review discusses extracellular vesicles (EVs), which are submicron-scale, anuclear, phospholipid bilayer membrane enclosed vesicles that contain lipids, metabolites, proteins, and RNA (micro and messenger). They are shed from many, if not all, cell types and are present in biological fluids and conditioned cell culture media. The term EV, as coined by the International Society of Extracellular Vesicles (ISEV), encompasses exosomes (30–100 nm in diameter), microparticles (100–1000 nm), apoptotic blebs, and other EV subsets. EVs have been implicated in cell–cell communication, coagulation, inflammation, immune response modulation, and disease progression. Multiple studies report that EV secretion from disease-affected cells contributes to disease progression, e.g., tumor niche formation and cancer metastasis. EVs are attractive sources of biomarkers due to their biological relevance and relatively noninvasive accessibility from a range of physiological fluids. This review is focused on the molecular profiling of the protein and lipid constituents of EVs, with emphasis on mass-spectrometry-based “omic” analytical techniques. The challenges in the purification and molecular characterization of EVs, including contamination of isolates and limitations in sample quantities, are discussed along with possible solutions. Finally, the review discusses the limited but growing investigation of post-translational modifications of EV proteins and potential strategies for future in-depth molecular characterization of EVs.  

3.2362           Exosomes released by keratinocytes modulate melanocyte pigmentation

Lo Cicero, A., Delevoye, C., Gilles-Marsens, F., Loew, D., Dingli, F., Guere, C., Andre, N., Vie, K., van Niel, G. and Raposo, G. Nature Communications, 6:7506 (2015)   Cells secrete extracellular vesicles (EVs), exosomes and microvesicles, which transfer proteins, lipids and RNAs to regulate recipient cell functions. Skin pigmentation relies on a tight dialogue between keratinocytes and melanocytes in the epidermis. Here we report that exosomes secreted by keratinocytes enhance melanin synthesis by increasing both the expression and activity of melanosomal proteins. Furthermore, we show that the function of keratinocyte-derived exosomes is phototype-dependent and is modulated by ultraviolet B. In sum, this study uncovers an important physiological function for exosomes in human pigmentation and opens new avenues in our understanding of how pigmentation is regulated by intercellular communication in both healthy and diseased states.  

3.2363           Extracellular Vesicles: Composition, Biological Relevance, and Methods of Study

Zaborowski, M.P., Balaj, L., Breakfield, X.O. and Lai, C.P. BioScience, 65(8), 783-797 (2015)   The release of extracellular vesicles (EVs), including exosomes and microvesicles, is a phenomenon shared by many cell types as a means of communicating with other cells and also potentially removing cell contents. The cargo of EVs includes the proteins, lipids, nucleic acids, and membrane receptors of the cells from which they originate. EVs released into the extracellular space can enter body fluids and potentially reach distant tissues. Once taken up by neighboring and/or distal cells, EVs can transfer functional cargo that may alter the status of recipient cells, thereby contributing to both physiological and pathological processes. In this article, we will focus on EV composition, mechanisms of uptake, and their biological effects on recipient cells. We will also discuss established and recently developed methods used to study EVs, including isolation, quantification, labeling and imaging protocols, as well as RNA analysis.  

3.2364           A novel chimeric aequorin fused with caveolin-1 reveals a sphingosine kinase 1-regulated Ca2 + microdomain in the caveolar compartment

Pulli, I., Blom, T., Löf, C., mafnusson, M., Rimessi, A., pinton, P. and Törnquist, K. Biochim. Biophys. Acta, 1853, 2173-2182 (2015)   Caveolae are plasma membrane invaginations enriched in sterols and sphingolipids. Sphingosine kinase 1 (SK1) is an oncogenic protein that converts sphingosine to sphingosine 1-phosphate (S1P), which is a messenger molecule involved in calcium signaling. Caveolae contain calcium responsive proteins, but the effects of SK1 or S1P on caveolar calcium signaling have not been investigated. We generated a Caveolin-1–Aequorin fusion protein (Cav1–Aeq) that can be employed for monitoring the local calcium concentration at the caveolae ([Ca²⁺]cav). In HeLa cells, Cav1–Aeq reported different [Ca²⁺] as compared to the plasma membrane [Ca²⁺] in general (reported by SNAP25–Aeq) or as compared to the cytosolic [Ca²⁺] (reported by cyt-Aeq). The Ca²⁺ signals detected by Cav1–Aeq were significantly attenuated when the caveolar structures were disrupted by methyl-β-cyclodextrin, suggesting that the caveolae are specific targets for Ca²⁺ signaling. HeLa cells overexpressing SK1 showed increased [Ca²⁺]cav during histamine-induced Ca²⁺ mobilization in the absence of extracellular Ca²⁺ as well as during receptor-operated Ca²⁺ entry (ROCE). The SK1-induced increase in [Ca²⁺]cav during ROCE was reverted by S1P receptor antagonists. In accordance, pharmacologic inhibition of SK1 reduced the [Ca²⁺]cav during ROCE. S1P treatment stimulated the [Ca²⁺]cav upon ROCE. The Ca²⁺ responses at the plasma membrane in general were not affected by SK1 expression. In summary, our results show that SK1/S1P-signaling regulates Ca²⁺ signals at the caveolae. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.  

3.2365           A method to investigate protein association with intact sealed mycobacterial membrane vesicles

D’Lima, N.G. and Teschke, C.M. Anal. Biochem., 485, 109-111 (2015)   In mycobacteria, probing the association of cytoplasmic proteins with the membrane itself, as well as with integral or peripheral membrane proteins, is limited by the difficulty in extracting intact sealed membrane vesicles due to the complex cell wall structure. Here we tested the association of Mycobacterium tuberculosis SecA1 and SecA2 proteins with intact membrane vesicles by a flotation assay using iodixanol density gradients. These protocols have wide applications for studying the association of other mycobacterial cytoplasmic proteins with the membrane and membrane-associated proteins.  

3.2366           Klebsiella pneumoniae O antigen loss alters the outer membrane protein composition and the selective packaging of proteins into secreted outer membrane vesicles

Cahill, B.K., seeley, K.W., Gutel, D. and Ellis, T.N. Microbiol Res., 180, 1-10 (2015)   Klebsiella pneumoniae is a nosocomial pathogen which naturally secretes lipopolysaccharide (LPS) and cell envelope associated proteins into the environment through the production of outer membrane vesicles (OMVs). The loss of the LPS O antigen has been demonstrated in other bacterial species to significantly alter the composition of OMVs. Therefore, this study aimed to comprehensively analyze the impact of O antigen loss on the sub-proteomes of both the outer membrane and secreted OMVs from K. pneumoniae. As determined by LC–MS/MS, OMVs were highly enriched with outer membrane proteins involved in cell wall, membrane, and envelope biogenesis as compared to the source cellular outer membrane. Deletion of wbbO, the enzyme responsible for O antigen attachment to LPS, decreased but did not eliminate this enrichment effect. Additionally, loss of O antigen resulted in OMVs with increased numbers of proteins involved in post-translational modification, protein turnover, and chaperones as compared to secreted vesicles from the wild type. This alteration of OMV composition may be a compensatory mechanism to deal with envelope stress. This comprehensive analysis confirms the highly distinct protein composition of OMVs as compared to their source membrane, and provides evidence for a selective sorting mechanism that involves LPS polysaccharides. These data support the hypothesis that modifications to LPS alters both the mechanics of protein sorting and the contents of secreted OMVs and significantly impacts the protein composition of the outer membrane.  

3.2367           Calcium release through P2X4 activates calmodulin to promote endolysosomal membrane fusion

Cao, Q., Zhang, X.Z., Zou, Y., Murrell-Lagnado, R., Zhu, M.X. and Dong, X-P.

1. Cell Biol., 209(6), 879-894 (2015)

  Intra-endolysosomal Ca²⁺ release is required for endolysosomal membrane fusion with intracellular organelles. However, the molecular mechanisms for intra-endolysosomal Ca²⁺ release and the downstream Ca²⁺ targets involved in the fusion remain elusive. Previously, we demonstrated that endolysosomal P2X4 forms channels activated by luminal adenosine triphosphate in a pH-dependent manner. In this paper, we show that overexpression of P2X4, as well as increasing endolysosomal P2X4 activity by alkalinization of endolysosome lumen, promoted vacuole enlargement in cells and endolysosome fusion in a cell-free assay. These effects were prevented by inhibiting P2X4, expressing a dominant-negative P2X4 mutant, and disrupting the P2X4 gene. We further show that P2X4 and calmodulin (CaM) form a complex at endolysosomal membrane where P2X4 activation recruits CaM to promote fusion and vacuolation in a Ca²⁺-dependent fashion. Moreover, P2X4 activation-triggered fusion and vacuolation were suppressed by inhibiting CaM. Our data thus suggest a new molecular mechanism for endolysosomal membrane fusion involving P2X4-mediated endolysosomal Ca²⁺ release and subsequent CaM activation.  

3.2368           CIN85 modulates TGFβ signaling by promoting the presentation of TGFβ receptors on the cell surface

Yakymovych, I., Yakymovych, M., Zang, G., Mu, Y., Bergh, A., Landström, M. and Heldin, C-H.

1. Cell Biol., 210(2), 319-332 (2015)

  Members of the transforming growth factor β (TGFβ) family initiate cellular responses by binding to TGFβ receptor type II (TβRII) and type I (TβRI) serine/threonine kinases, whereby Smad2 and Smad3 are phosphorylated and activated, promoting their association with Smad4. We report here that TβRI interacts with the SH3 domains of the adaptor protein CIN85 in response to TGFβ stimulation in a TRAF6-dependent manner. Small interfering RNA–mediated knockdown of CIN85 resulted in accumulation of TβRI in intracellular compartments and diminished TGFβ-stimulated Smad2 phosphorylation. Overexpression of CIN85 instead increased the amount of TβRI at the cell surface. This effect was inhibited by a dominant-negative mutant of Rab11, suggesting that CIN85 promoted recycling of TGFβ receptors. CIN85 enhanced TGFβ-stimulated Smad2 phosphorylation, transcriptional responses, and cell migration. CIN85 expression correlated with the degree of malignancy of prostate cancers. Collectively, our results reveal that CIN85 promotes recycling of TGFβ receptors and thereby positively regulates TGFβ signaling.  

3.2369           Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis

Deruyffelaere, C., Bouchez, I., Morin, H., Guillot, A., Miquel, M., Froissard, M., Chardot, st. and D’Andrea, S. Plant Cell Physiol., 56(7), 1374-1387 (2015)   In oleaginous seeds, lipids—stored in organelles called oil bodies (OBs)—are degraded post-germinatively to provide carbon and energy for seedling growth. To date, little is known about how OB coat proteins, known as oleosins, control OB dynamics during seed germination. Here, we demonstrated that the sequential proteolysis of the five Arabidopsis thaliana oleosins OLE1–OLE5 begins just prior to lipid degradation. Several post-translational modifications (e.g. phosphorylation and ubiquination) of oleosins were concomitant with oleosin degradation. Phosphorylation occurred only on the minor OLE5 and on an 8 kDa proteolytic fragment of OLE2. A combination of immunochemical and proteomic approaches revealed ubiquitination of the four oleosins OLE1–OLE4 at the onset of OB mobilization. Ubiquitination topology was surprisingly complex. OLE1 and OLE2 were modified by three distinct and predominantly exclusive motifs: monoubiquitin, K48-linked diubiquitin (K48Ub₂) and K63-linked diubiquitin. Ubiquitinated oleosins may be channeled towards specific degradation pathways according to ubiquitination type. One of these pathways was identified as the ubiquitin–proteasome pathway. A proteasome inhibitor (MG132) reduced oleosin degradation and induced cytosolic accumulation of K48Ub₂–oleosin aggregates. These results indicate that K48Ub₂-modified oleosins are selectively extracted from OB coat and degraded by the proteasome. Proteasome inhibition also reduced lipid hydrolysis, providing in vivo evidence that oleosin degradation is required for lipid mobilization.  

3.2370           X chromosome-linked intellectual disability protein PQBP1 associates with and regulates the translation of specific mRNAs

Wan, D., Zhang, Z.C., Zhang, X., Li, Q. and Han, J. Hum. Mol. Genet., 24(16), 4599-4614 (2015)   X chromosome-linked intellectual disability is a common developmental disorder, and mutations of the polyglutamine-binding protein 1 (PQBP1) gene have been linked to this disease. In addition to existing in the nucleus as a splicing factor, PQBP1 is also found in cytoplasmic RNA granules, where it associates with RNA-binding proteins. However, the roles of cytoplasmic PQBP1 are largely unknown. Here, we show that the Drosophila homolog of PQBP1 (dPQBP1) is present in the cytoplasm of photoreceptor cells, and its loss results in defective rhabdomere morphogenesis, which is due to impaired Chaoptin translation. We also show that dPQBP1 regulates mRNA translation by interacting with dFMR1, which binds to specific mRNAs and facilitates their assembly into translating ribosomes, a function that is conserved for human PQBP1 and FMRP. Our findings reveal the conserved function of PQBP1 in mRNA translation and provide molecular insights into the pathogenic mechanisms underlying Renpenning syndrome.  

3.2371           Intracellular Catabolism of an Antibody Drug Conjugate with a Noncleavable Linker

Rock, B.M., Tometsko, M.E., patel, S.K., Hamblett, K.J., Fanslow, W. and Rock, D.A. Drug Metab. Dispos., 43, 1341-1344 (2015)   Antibody drug conjugates are emerging as a powerful class of antitumor agents with efficacy across a range of cancers; therefore, understanding the disposition of this class of therapeutic is crucial. Reported here is a method of enriching a specific organelle (lysosome) to understand the catabolism of an anti-CD70 Ab-MCC-DM1, an antibody drug conjugate with a noncleavable linker. With such techniques a higher degree of concentration-activity relationship can be established for in vitro cell lines; this can aid in understanding the resultant catabolite concentrations necessary to exert activity.  

3.2372           Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA

Duchez, A-C. et al PNAS, 112(27), E3654-E3573 (2015)   Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A₂ IIA (sPLA₂-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA₂-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA₂-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA₂-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.  

3.2373           O-glycans direct selectin ligands to lipid rafts on leukocytes

Shao, B., Yago, T., Setiadi, H., Wang, Y., Mehta-D’souza, P., Fu, J., Crocker, P.R., Rodgers, W., Xia, L and McEver, R.P. PNAS, 112(28), 8661-8666 (2015)   Palmitoylated cysteines typically target transmembrane proteins to domains enriched in cholesterol and sphingolipids (lipid rafts). P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are O-glycosylated proteins on leukocytes that associate with lipid rafts. During inflammation, they transduce signals by engaging selectins as leukocytes roll in venules, and they move to the raft-enriched uropods of polarized cells upon chemokine stimulation. It is not known how these glycoproteins associate with lipid rafts or whether this association is required for signaling or for translocation to uropods. Here, we found that loss of core 1-derived O-glycans in murine C1galt1^−/− neutrophils blocked raft targeting of PSGL-1, CD43, and CD44, but not of other glycosylated proteins, as measured by resistance to solubilization in nonionic detergent and by copatching with a raft-resident sphingolipid on intact cells. Neuraminidase removal of sialic acids from wild-type neutrophils also blocked raft targeting. C1galt1^−/− neutrophils or neuraminidase-treated neutrophils failed to activate tyrosine kinases when plated on immobilized anti–PSGL-1 or anti-CD44 F(ab′)₂. Furthermore, C1galt1^−/− neutrophils incubated with anti–PSGL-1 F(ab′)₂ did not generate microparticles. In marked contrast, PSGL-1, CD43, and CD44 moved normally to the uropods of chemokine-stimulated C1galt1^−/− neutrophils. These data define a role for core 1-derived O-glycans and terminal sialic acids in targeting glycoprotein ligands for selectins to lipid rafts of leukocytes. Preassociation of these glycoproteins with rafts is required for signaling but not for movement to uropods.  

3.2374           Cysteine cathepsins are essential in lysosomal degradation of α-synuclein

McGlinchey, R.P. and Lee, J.C. PNAS, 112(30), 9322-9327 (2015)   A cellular feature of Parkinson’s disease is cytosolic accumulation and amyloid formation of α-synuclein (α-syn), implicating a misregulation or impairment of protein degradation pathways involving the proteasome and lysosome. Within lysosomes, cathepsin D (CtsD), an aspartyl protease, is suggested to be the main protease for α-syn clearance; however, the protease alone only generates amyloidogenic C terminal-truncated species (e.g., 1–94, 5–94), implying that other proteases and/or environmental factors are needed to facilitate degradation and to avoid α-syn aggregation in vivo. Using liquid chromatography–mass spectrometry, to our knowledge, we report the first peptide cleavage map of the lysosomal degradation process of α-syn. Studies of purified mouse brain and liver lysosomal extracts and individual human cathepsins demonstrate a direct involvement of cysteine cathepsin B (CtsB) and L (CtsL). Both CtsB and CtsL cleave α-syn within its amyloid region and circumvent fibril formation. For CtsD, only in the presence of anionic phospholipids can this protease cleave throughout the α-syn sequence, suggesting that phospholipids are crucial for its activity. Taken together, an interplay exists between α-syn conformation and cathepsin activity with CtsL as the most efficient under the conditions examined. Notably, we discovered that CtsL efficiently degrades α-syn amyloid fibrils, which by definition are resistant to broad spectrum proteases. This work implicates CtsB and CtsL as essential in α-syn lysosomal degradation, establishing groundwork to explore mechanisms to enhance their cellular activity and levels as a potential strategy for clearance of α-syn.  

3.2375           Calcium-dependent membrane association of a flagellar calcium sensor does not require calcium binding

Maric, D., Olson, C.L., Xu, X., Ames, J.B. and Engman, D.M. Mol. Biochem. Parasitol., 201(1), 72-75 (2015)   Flagellar calcium-binding protein (FCaBP) is a dually acylated Ca²⁺ sensor in the Trypanosoma cruzi flagellar membrane that undergoes a massive conformational change upon Ca²⁺ binding. It is similar to neuronal Ca²⁺ sensors, like recoverin, which regulate their binding partners through a calcium acyl switch mechanism. FCaBP is washed out of permeabilized cells with buffers containing EDTA, indicating Ca²⁺-dependent flagellar membrane association. We hypothesized that, like recoverin, FCaBP projects its acyl groups in the presence of Ca²⁺, permitting flagellar membrane and binding partner association and that it sequesters the acyl groups in low Ca²⁺, disassociating from the membrane and releasing its binding partner to perform a presumed enzymatic function. The X-ray crystal structure of FCaBP suggests that the acyl groups are always exposed, so we set out to test our hypothesis directly. We generated T. cruzi transfectants expressing FCaBP or Ca²⁺-binding mutant FCaBP^E151Q/E188Q and recombinant wildtype and mutant proteins as well. Both FCaBP and FCaBP^E151Q/E188Q were found to associate with lipid rafts, indicating the Ca²⁺-independence of this association. To our initial surprise, FCaBP^E151Q/E188Q, like wildtype FCaBP, exhibited Ca²⁺-dependent flagellar membrane association, even though this protein does not bind Ca²⁺ itself [16]. One possible explanation for this is that FCaBP^E151Q/E188Q, like some other Ca²⁺ sensors, may form dimers and that dimerization of FCaBP^E151Q/E188Q with endogenous wildtype FCaBP might explain its Ca²⁺-dependent localization. Indeed both proteins are able to form dimers in the presence and absence of Ca²⁺. These results suggest that FCaBP possesses two distinct Ca²⁺-dependent interactions—one involving a Ca²⁺-induced change in conformation and another perhaps involving binding partner association.  

3.2376           Virulence from vesicles: Novel mechanisms of host cell injury by Escherichia coli O104:H4 outbreak strain

Kunsmann, L., Rüter, C., Bauwens, A., Greune, L., Glüder, M., Kemper, B., Fruth, A., Wai, S.N., He, X., Lloubes, R., Schmidt, M.A., Dobrindt, U., Mellmann, A., Karch, H.  and Bielaszewska, M. Scientific Reports, 5: 13253 (2015)   The highly virulent Escherichia coli O104:H4 that caused the large 2011 outbreak of diarrhoea and haemolytic uraemic syndrome secretes blended virulence factors of enterohaemorrhagic and enteroaggregative E. coli, but their secretion pathways are unknown. We demonstrate that the outbreak strain releases a cocktail of virulence factors via outer membrane vesicles (OMVs) shed during growth. The OMVs contain Shiga toxin (Stx) 2a, the major virulence factor of the strain, Shigella enterotoxin 1, H4 flagellin, and O104 lipopolysaccharide. The OMVs bind to and are internalised by human intestinal epithelial cells via dynamin-dependent and Stx2a-independent endocytosis, deliver the OMV-associated virulence factors intracellularly and induce caspase-9-mediated apoptosis and interleukin-8 secretion. Stx2a is the key OMV component responsible for the cytotoxicity, whereas flagellin and lipopolysaccharide are the major interleukin-8 inducers. The OMVs represent novel ways for the E. coli O104:H4 outbreak strain to deliver pathogenic cargoes and injure host cells.  

3.2377           Role for Rab10 in Methamphetamine-Induced Behavior

Vanderwerf, S.M., Buck, D.C., Wilmarth, P.A., Sears, L.M., David, L.L., Morton, D.B. and Neve, K.A. PloS One, 10(8), e0136167 (2015)   Lipid rafts are specialized, cholesterol-rich membrane compartments that help to organize transmembrane signaling by restricting or promoting interactions with subsets of the cellular proteome. The hypothesis driving this study was that identifying proteins whose relative abundance in rafts is altered by the abused psychostimulant methamphetamine would contribute to fully describing the pathways involved in acute and chronic effects of the drug. Using a detergent-free method for preparing rafts from rat brain striatal membranes, we identified density gradient fractions enriched in the raft protein flotillin but deficient in calnexin and the transferrin receptor, markers of non-raft membranes. Dopamine D1- and D2-like receptor binding activity was highly enriched in the raft fractions, but pretreating rats with methamphetamine (2 mg/kg) once or repeatedly for 11 days did not alter the distribution of the receptors. LC-MS analysis of the protein composition of raft fractions from rats treated once with methamphetamine or saline identified methamphetamine-induced changes in the relative abundance of 23 raft proteins, including the monomeric GTP-binding protein Rab10, whose abundance in rafts was decreased 2.1-fold by acute methamphetamine treatment. Decreased raft localization was associated with a selective decrease in the abundance of Rab10 in a membrane fraction that includes synaptic vesicles and endosomes. Inhibiting Rab10 activity by pan-neuronal expression of a dominant-negative Rab10 mutant in Drosophila melanogaster decreased methamphetamine-induced activity and mortality and decreased caffeine-stimulated activity but not mortality, whereas inhibiting Rab10 activity selectively in cholinergic neurons had no effect. These results suggest that activation and redistribution of Rab10 is critical for some of the behavioral effects of psychostimulants.  

3.2378           Lysosome-Related Effector Vesicles in T Lymphocytes and NK Cells

Lettau, M., kabelitz, D. and Janssen, O. Scand. J. Immunol., 82(3), 235-243 (2015)   Lysosome-related secretory organelles combine metabolic functions of conventional lysosomes with an inducible secretory potential. Specialized variants of such bi-functional organelles are present in several haematopoietic cell types that store, mobilize and/or secrete effector proteins, for example in mast cells, macrophages or cytotoxic effector cells. In the case of T lymphocytes and NK cells, it was believed that secretory lysosomes serve as a common storage and transport compartment for the most relevant cytotoxic effector proteins including FasL, perforin, granzymes and granulysin. However, recent observations suggest that cytotoxic effector cells might be able to mobilize two distinct lysosomal entities in order to react to differential stimulation with either FasL surface appearance or degranulation-associated release of perforin and granzymes. This assumption is supported by the proteomic characterization of enriched organelles from T and NK cells. FasL-associated light lysosomes biochemically segregate from morphologically distinct heavy lysosomes that preferentially contain granzymes, perforin and mature granulysin. Here, we briefly summarize the current knowledge about cargo proteins that are stored and transported in secretory vesicles and how these vesicles might be generated and mobilized. In addition, we describe common features and major differences of the two distinct effector organelles and discuss how these observations might expand existing models of cytotoxic effector function.  

3.2379           Enhancement of BACE1 Activity by p25/Cdk5-Mediated Phosphorylation in Alzheimer’s Disease

Song, W-J., Son, M-Y., Lee, H-W., Seo, H., Kim, J.H. and Chung, S-H. PloS One, 10(8), e0136950 (2015)   The activity of beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is elevated during aging and in sporadic Alzheimer’s disease (AD), but the underlying mechanisms of this change are not well understood. p25/Cyclin-dependent kinase 5 (Cdk5) has been implicated in the pathogenesis of several neurodegenerative diseases, including AD. Here, we describe a potential mechanism by which BACE activity is increased in AD brains. First, we show that BACE1 is phosphorylated by the p25/Cdk5 complex at Thr252 and that this phosphorylation increases BACE1 activity. Then, we demonstrate that the level of phospho-BACE1 is increased in the brains of AD patients and in mammalian cells and transgenic mice that overexpress p25. Furthermore, the fraction of p25 prepared from iodixanol gradient centrifugation was unexpectedly protected by protease digestion, suggesting that p25/Cdk5-mediated BACE1 phosphorylation may occur in the lumen. These results reveal a link between p25 and BACE1 in AD brains and suggest that upregulated Cdk5 activation by p25 accelerates AD pathogenesis by enhancing BACE1 activity via phosphorylation.  

3.2380           Isolation of Peroxisomes from Yeast

Cramer, J., Effelsberg, D., Girzalsky, W. and Erdmann, R. Cold Spring Harbor Protocols, pdb.top074500 (2015)   Peroxisomes are multifunctional, dynamic organelles present in nearly all eukaryotic cells. Determining their structural and functional characteristics often requires obtaining isolated and purified peroxisomes via subcellular fractionation. Subcellular fractionation techniques are generally based on a three-step procedure: preparation of a cell-free homogenate (postnuclear supernatant), generation of an organellar pellet by differential centrifugation, and density gradient centrifugation. Here we introduce methods for small-scale isolation of peroxisomes from yeast cells using different gradient media as well as large-scale purification using a two-step gradient centrifugation.  

3.2381           Small-Scale Purification of Peroxisomes for Analytical Applications

Cramer, J., Effelsberg, D., Girzalsky, W. and Erdmann, R. Cold Spring Harbor Protocols, pdb.prot083717 (2015) This protocol describes the isolation of peroxisomes from Saccharomyces cerevisiae by density gradient centrifugation using a sucrose, OptiPrep, or OptiPrep/sucrose gradient. Oleic acid–induced cells are first converted to spheroplasts using lyticase for cell wall digestion. Spheroplasts are homogenized, and nuclei and cell debris are removed by low-speed centrifugation to produce a postnuclear supernatant (PNS). Separation of the PNS by density gradient centrifugation is suitable for many analytical applications; however, to increase the yield of peroxisomes, further fractionation of the PNS is possible. Differential centrifugation of the PNS allows removal of the cytosol and other contaminating organelles, resulting in an organellar pellet (OP) enriched in peroxisomes and mitochondria that can be loaded onto the density gradient. Following density gradient centrifugation of the PNS or OP, fractions are collected from the bottom of the centrifuge tube. The distribution of organelles, including peroxisome peak fractions, is characterized by measurement of marker enzyme activity.  

3.2382           Amaranthus leucocarpus lectin recognizes a moesin-like O-glycoprotein and costimulates murine CD3-activated CD4+ T cells

Arenas-Del Angel, M., Legorreta-Herrera, M., Mendoza-Hernandez, G., garfias, Y., Chavez, R., Zenteno, E. and Lascurain, R. Immunity, Inflammation and Disease, 3(3), 182-195 (2015)   The Galβ1,3GalNAcα1,O-Ser/Thr specific lectin from Amaranthus leucocarpus (ALL) binds a ∼70 kDa glycoprotein on murine T cell surface. We show that in the absence of antigen presenting cells, murine CD4⁺ T cells activated by an anti-CD3 antibody plus ALL enhanced cell proliferation similar to those cells activated via CD3/CD28 at 48 h of culture. Moreover, ALL induced the production of IL-4, IL-10, TNF-alpha, and TGF-beta in CD3-activated cells. Proteomic assay using two-dimensional electrophoresis and far-Western blotting, ALL recognized two prominent proteins associated to the lipid raft microdomains in CD3/CD28-activated CD4⁺ T cells. By mass spectrometry, the peptide fragments from ALL-recognized proteins showed sequences with 33% homology to matricin (gi|347839 NCBInr) and 41% identity to an unnamed protein related to moesin (gi|74186081 NCBInr). Confocal microscopy analysis of CD3/CD28-activated CD4⁺ T cells confirmed that staining by ALL colocalized with anti-moesin FERM domain antibody along the plasma membrane and in the intercellular contact sites. Our findings suggest that a moesin-like O-glycoprotein is the ALL-recognized molecule in lipid rats, which induces costimulatory signals on CD4⁺ T cells.  

3.2383           Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts

Paggetti, J., Haderk, F., Seiffert, M., janji, B., Distler, U., Ammerlaan, W., Kim, Y.J., Adam, J., Lichter, P., Solary, E., Berchem, G. and Moussay, E. Blood, 126(9), 1106-1117 (2015)   Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin–positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts.  

3.2384           The prion protein inhibits monocytic cell migration by stimulating β1 integrin adhesion and uropod formation

Richardson, D.D., Tol, S., Valle-Encinas, E., Pleguezuelos, C., Bierings, R., Geerts, D. and Fernandez-Borja, M.

1. Cell Sci., 128, 3018-3029 (2015)

  The broad tissue distribution and evolutionary conservation of the glycosylphosphatidylinositol (GPI)-anchored prion protein (PrP, also known as PRNP) suggests that it plays a role in cellular homeostasis. Given that integrin adhesion determines cell behavior, the proposed role of PrP in cell adhesion might underlie the various in vitro and in vivo effects associated with PrP loss-of-function, including the immune phenotypes described in PrP^−/− mice. Here, we investigated the role of PrP in the adhesion and (transendothelial) migration of human (pro)monocytes. We found that PrP regulates β1-integrin-mediated adhesion of monocytes. Additionally, PrP controls the cell morphology and migratory behavior of monocytes: PrP-silenced cells show deficient uropod formation on immobilized VCAM and display bleb-like protrusions on the endothelium. Our data further show that PrP regulates ligand-induced integrin activation. Finally, we found that PrP controls the activation of several proteins involved in cell adhesion and migration, including RhoA and its effector cofilin, as well as proteins of the ERM family. We propose that PrP modulates β1 integrin adhesion and migration of monocytes through RhoA-induced actin remodeling mediated by cofilin, and through the regulation of ERM-mediated membrane–cytoskeleton linkage.he prion protein inhibits  

3.2385           Microtubule motors transport phagosomes in the RPE, and lack of KLC1 leads to AMD-like pathogenesis

Jiang, M., Easteve-Rudd, J., Lapes, V.S., Diemer, T., Lillo, C., Rump, A. and Williams, D.S.

1. Cell Biol., 210(4), 595-611 (2015)

  The degradation of phagosomes, derived from the ingestion of photoreceptor outer segment (POS) disk membranes, is a major role of the retinal pigment epithelium (RPE). Here, POS phagosomes were observed to associate with myosin-7a, and then kinesin-1, as they moved from the apical region of the RPE. Live-cell imaging showed that the phagosomes moved bidirectionally along microtubules in RPE cells, with kinesin-1 light chain 1 (KLC1) remaining associated in both directions and during pauses. Lack of KLC1 did not inhibit phagosome speed, but run length was decreased, and phagosome localization and degradation were impaired. In old mice, lack of KLC1 resulted in RPE pathogenesis that was strikingly comparable to aspects of age-related macular degeneration (AMD), with an excessive accumulation of RPE and sub-RPE deposits, as well as oxidative and inflammatory stress responses. These results elucidate mechanisms of POS phagosome transport in relation to degradation, and demonstrate that defective microtubule motor transport in the RPE leads to phenotypes associated with AMD.  

3.2386           Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome

Linder, B., Groszhik, A.V., Olarerin-George, A.O., Meydan, C., Mason, C.E. and Jaffrey, S.R. Nature Methods, 12(8), 767-772 (2015)   N⁶-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate. Current mapping approaches localize m6A residues to transcript regions 100–200 nt long but cannot identify precise m6A positions on a transcriptome-wide level. Here we developed m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) and used it to demonstrate that antibodies to m6A can induce specific mutational signatures at m6A residues after ultraviolet light–induced antibody-RNA cross-linking and reverse transcription. We found that these antibodies similarly induced mutational signatures at N⁶,2′-O-dimethyladenosine (m6Am), a modification found at the first nucleotide of certain mRNAs. Using these signatures, we mapped m6A and m6Am at single-nucleotide resolution in human and mouse mRNA and identified small nucleolar RNAs (snoRNAs) as a new class of m6A-containing non-coding RNAs (ncRNAs).  

3.2387           Neurofilament subunits are integral components of synapses and modulate neurotransmission and behavior in vivo

Yuan, A., Sershen, H., Veeranna, Basavarajappa, B.S., Kumar, A., Hashim, A., Berg, M., Lee, J-H., Sato, Y., Rao, M.V., Mohan, P.S., Dyakin, V., Julien, J-P., Lee, VM-Y. and Nixon, R.A. Molecular Psychiatry, 20(8), 986-994 (2015)   Synaptic roles for neurofilament (NF) proteins have rarely been considered. Here, we establish all four NF subunits as integral resident proteins of synapses. Compared with the population in axons, NF subunits isolated from synapses have distinctive stoichiometry and phosphorylation state, and respond differently to perturbations in vivo. Completely eliminating NF proteins from brain by genetically deleting three subunits (α-internexin, NFH and NFL) markedly depresses hippocampal long-term potentiation induction without detectably altering synapse morphology. Deletion of NFM in mice, but not the deletion of any other NF subunit, amplifies dopamine D1-receptor-mediated motor responses to cocaine while redistributing postsynaptic D1-receptors from endosomes to plasma membrane, consistent with a specific modulatory role of NFM in D1-receptor recycling. These results identify a distinct pool of synaptic NF subunits and establish their key role in neurotransmission in vivo, suggesting potential novel influences of NF proteins in psychiatric as well as neurological states.  

3.2388           Identification of P-glycoprotein co-fractionating proteins and specific binding partners in rat brain microvessels

Tome, M.E., Schaefer, C.P., Jacobs, L.M., Zhang, Y., Herndon, J.M., Matty, F.O. and davis, T.P.

1. Neurochem., 134(2), 200-210 (2015)

  Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P-glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood-brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP-containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post-translationally regulated at the BBB. The goal of the current study was to identify proteins that co-localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co-localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co-fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post-translational regulation of PgP activity at the BBB. This model depicts two types of P-glycoprotein (PgP)-containing caveolae. Rat brain microvessels contain two unique pools of PgP that are of different densities. Each pool co-fractionates with three caveolar proteins suggesting there are two populations of caveolae that contain PgP. The two caveolar populations differ in density indicating they have a different structure, lipoprotein content or intracellular location. Our model includes two newly identified PgP-binding partners, Hsc71 and protein disulfide isomerase, in rat brain microvessels.  

3.2389           Glioblastoma-derived extracellular vesicles modify the phenotype of monocytic cells

De Vrij, J. et al Int. J. Cancer, 137, 1630-1642 (2015)   Glioblastoma multiforme (GBM) is the most common primary brain tumor and is without exception lethal. GBMs modify the immune system, which contributes to the aggressive nature of the disease. Particularly, cells of the monocytic lineage, including monocytes, macrophages and microglia, are affected. We investigated the influence of GBM-derived extracellular vesicles (EVs) on the phenotype of monocytic cells. Proteomic profiling showed GBM EVs to be enriched with proteins functioning in extracellular matrix interaction and leukocyte migration. GBM EVs appeared to skew the differentiation of peripheral blood-derived monocytes to alternatively activated/M2-type macrophages. This was observed for EVs from an established cell line, as well as for EVs from primary cultures of GBM stem-like cells (GSCs). Unlike EVs of non-GBM origin, GBM EVs induced modified expression of cell surface proteins, modified cytokine secretion (e.g., an increase in vascular endothelial growth factor and IL-6) and increased phagocytic capacity of the macrophages. Most pronounced effects were observed upon incubation with EVs from mesenchymal GSCs. GSC EVs also affected primary human microglia, resulting in increased expression of Membrane type 1-matrix metalloproteinase, a marker for GBM microglia and functioning as tumor-supportive factor. In conclusion, GBM-derived EVs can modify cells of the monocytic lineage, which acquire characteristics that resemble the tumor-supportive phenotypes observed in patients.  

3.2390           AP-1/σ1B-Dependent SV Protein Recycling Is Regulated in Early Endosomes and Is Coupled to AP-2 Endocytosis

Kratzke, M., Candiello, E., Schmidt, B., Jahn, O. and Schu, P. Mol. Neurobiol., 52, 142-161 (2015)   Adaptor protein (AP)-1/σ1B^−/− mice have reduced synaptic-vesicle (SV) recycling and increased endosomes. Mutant mice have impaired spatial memory, and σ1B-deficient humans have a severe mental retardation. In order to define these σ1B^−/− ‘bulk’ endosomes and to determine their functions in SV recycling, we developed a protocol to separate them from the majority of the neuronal endosomes. The σ1B^−/− ‘bulk’ endosomes proved to be classic early endosomes with an increase in the phospholipid phosphatidylinositol 3-phosphate (PI-3-P), which recruits proteins mediating protein sorting out of early endosomes into different routes. σ1B deficiency induced alterations in the endosomal proteome reveals two major functions: SV protein storage and sorting into endolysosomes. Alternative endosomal recycling pathways are not up-regulated, but certain SV proteins are misrouted. Tetraspanins are enriched in σ1B^−/− synaptosomes, but not in their endosomes or in their clathrin-coated-vesicles (CCVs), indicating AP-1/σ1B-dependent sorting. Synapses contain also more AP-2 CCV, although it is expected that they contain less due to reduced SV recycling. Coat composition of these AP-2 CCVs is altered, and thus, they represent a subpopulation of AP-2 CCVs. Association of calmodulin-dependent protein kinase (CaMK)-IIα, −δ and casein kinase (CK)-IIα with the endosome/SV pool is altered, as well as 14-3-3η, indicating changes in specific signalling pathways regulating synaptic plasticity. The accumulation of early endosomes and endocytotic AP-2 CCV indicates the regulation of SV recycling via early endosomes by the interdependent regulation of AP-2-mediated endocytosis and AP-1/σ1B-mediated SV reformation.  

3.2391           Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

Lee, J-H., McBrayer, M.K., Wolfe, D.K., Haslett, L.J., Kumar, A., Sato, Y., Lie, P.P.Y., Mohan, P., Coffey, E.E., Kompella, U., Mitchell, C.H., Lloyd-Evans, E. and Nixon, R.A. Cell Reports, 12, 1430-1444 (2015)   Presenilin 1 (PS1) deletion or Alzheimer’s disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO) cells induces abnormal Ca²⁺ efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca²⁺. In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca²⁺ homeostasis, but correcting lysosomal Ca²⁺ deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca²⁺ homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.  

3.2392           PAQR3 modulates cholesterol homeostasis by anchoring Scap/SREBP complex to the Golgi apparatus

Xu, D., Wang, Z., Zhang, Y., Jiang, W., Pan, Y., Song, B-L. and Chen, Y. Nature Communications, 6:8100 (2015)   Cholesterol biosynthesis is regulated by transcription factors SREBPs and their escort protein Scap. On sterol depletion, Scap/SREBP complex is transported from endoplasmic reticulum (ER) to the Golgi apparatus where SREBP is activated. Under cholesterol sufficient condition, Insigs act as anchor proteins to retain Scap/SREBP in the ER. However, the anchor protein of Scap/SREBP in the Golgi is unknown. Here we report that a Golgi-localized membrane protein progestin and adipoQ receptors 3 (PAQR3) interacts with Scap and SREBP and tethers them to the Golgi. PAQR3 promotes Scap/SREBP complex formation, potentiates SREBP processing and enhances lipid synthesis. The mutually exclusive interaction between Scap and PAQR3 or Insig-1 is regulated by cholesterol level. PAQR3 knockdown in liver blunts SREBP pathway and decreases hepatic cholesterol content. Disrupting the interaction of PAQR3 with Scap/SREBP by a synthetic peptide inhibits SREBP processing and activation. Thus, PAQR3 regulates cholesterol homeostasis by anchoring Scap/SREBP to the Golgi and disruption of such function reduces cholesterol biosynthesis.  

3.2393           A Breast Cell Atlas: Organelle analysis of the MDA-MB-231 cell line by density-gradient fractionation using isotopic marking and label-free analysis

Sandin, M., Antberg, L., Levander, F. and James, P. EuPA Open Proteomics, 8, 68-77 (2015)   Protein translocation between organelles in the cell is an important process that regulates many cellular functions. However, organelles can rarely be isolated to purity so several methods have been developed to analyse the fractions obtained by density gradient centrifugation. We present an analysis of the distribution of proteins amongst organelles in the human breast cell line, MDA-MB-231 using two approaches: an isotopic labelling and a label-free approach.  

3.2394           Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells

Ishibashi, D., Homma, T., Nakagaki, T., Fuse, t., sano, K., Takatsuki, H., Atarashi, R. and Nishida, N. PloS One, 10(9), e0137958 (2015)   Prion diseases are neurodegenerative disorders caused by the accumulation of abnormal prion protein (PrP^Sc) in the central nervous system. With the aim of elucidating the mechanism underlying the accumulation and degradation of PrP^Sc, we investigated the role of autophagy in its degradation, using cultured cells stably infected with distinct prion strains. The effects of pharmacological compounds that inhibit or stimulate the cellular signal transduction pathways that mediate autophagy during PrP^Sc degradation were evaluated. The accumulation of PrP^Sc in cells persistently infected with the prion strain Fukuoka-1 (FK), derived from a patient with Gerstmann–Sträussler–Scheinker syndrome, was significantly increased in cultures treated with the macroautophagy inhibitor 3-methyladenine (3MA) but substantially reduced in those treated with the macroautophagy inducer rapamycin. The decrease in FK-derived PrP^Sc levels was mediated, at least in part, by the phosphatidylinositol 3-kinase/MEK signalling pathway. By contrast, neither rapamycin nor 3MA had any apparently effect on PrP^Sc from either the 22L or the Chandler strain, indicating that the degradation of PrP^Sc in host cells might be strain-dependent.  

3.2395           An efficient two-step subcellular fractionation method for the enrichment of insulin granules from INS-1 cells

Hen, Y., Xia, Z., Wang, L., Yu, Y., Liu, P., Song, E. and Xu, T. Biophys. Rep., 1(1), 34-40 (2015)   Insulin is one of the key regulators for blood glucose homeostasis. More than 99% of insulin is secreted from the pancreatic β-cells. Within each β-cell, insulin is packaged and processed in insulin secretary granules (ISGs) before its exocytosis. Insulin secretion is a complicated but well-organized dynamic process that includes the budding of immature ISGs (iISGs) from the trans-Golgi network, iISG maturation, and mature ISG (mISG) fusion with plasma membrane. However, the molecular mechanisms involved in this process are largely unknown. It is therefore crucial to separate and enrich iISGs and mISGs before determining their distinct characteristics and protein contents. Here, we developed an efficient two-step subcellular fractionation method for the enrichment of iISGs and mISGs from INS-1 cells: OptiPrep gradient purification followed by Percoll solution purification. We demonstrated that by using this method, iISGs and mISGs can be successfully distinguished and enriched. This method can be easily adapted to investigate SGs in other cells or tissues, thereby providing a useful tool for elucidating the mechanisms of granule maturation and secretion.  

3.2396           Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells

Murillo, A., Vera-Estrella, R., Barkla, B.J., Mendez, E. and Arias, C.F.

1. Virol., 89(20), 10359-10370 (2015)

  Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles.  

3.2397           UBXN2A regulates nicotinic receptor degradation by modulating the E3 ligase activity of CHIP

Teng, Y., Rezvani, K. and De Biasi, M. Biochem. Pharmacol., 97, 518-530 (2015)   Neuronal nicotinic acetylcholine receptors (nAChRs) containing the α3 subunit are known for their prominent role in normal ganglionic transmission while their involvement in the mechanisms underlying nicotine addiction and smoking-related disease has been emerging only in recent years. The amount of information available on the maturation and trafficking of α3-containing nAChRs is limited. We previously showed that UBXN2A is a p97 adaptor protein that facilitates the maturation and trafficking of α3-containing nAChRs. Further investigation of the mechanisms of UBXN2A actions revealed that the protein interacts with CHIP (carboxyl terminus of Hsc70 interacting protein), whose ubiquitin E3 ligase activity regulates the degradation of several disease-related proteins. We show that CHIP displays E3 ligase activity toward the α3 nAChR subunit and contributes to its ubiquitination and subsequent degradation. UBXN2A interferes with CHIP-mediated ubiquitination of α3 and protects the nicotinic receptor subunit from endoplasmic reticulum associated degradation (ERAD). UBXN2A also cross-talks with VCP/p97 and HSC70/HSP70 proteins in a complex where α3 is likely to be targeted by CHIP. Overall,we identify CHIP as an E3 ligase for α3 and UBXN2A as a protein that may efficiently regulate the stability of CHIP’s client substrates.  

3.2398           Tumour-derived exosomes: Tiny envelopes for big stories

Miller, I.V. and Grunewald, T.G.P. Biol. Cell, 107, 287-305 (2015)   The discovery of exosomes, which are small, 30–100 nm sized extracellular vesicles that are released by virtual all cells, has initiated a rapidly expanding and vibrant research field. Current investigations are mainly directed toward the role of exosomes in intercellular communication and their potential value as biomarkers for a broad set of diseases. By horizontal transfer of molecular information such as micro RNAs, messenger RNAs or proteins, as well as by receptor–cell interactions, exosomes are capable to mediate the reprogramming of surrounding cells. Herein, we review how especially cancer cells take advantage of this mechanism to influence their microenvironment in favour of immune escape, therapy resistance, tumour growth and metastasis. Moreover, we provide a comprehensive microarray analysis (n > 1970) to study the expression patterns of genes known to be intimately involved in exosome biogenesis across 26 different cancer entities and a normal tissue atlas. Consistent with the elevated production of exosomes observed in cancer patient plasma, we found a significant overexpression especially of RAB27A, CHMP4C and SYTL4 in the corresponding cancer entities as compared to matched normal tissues. Finally, we discuss the immune-modulatory and anti-tumorigenic functions of exosomes as well as innovative approaches to specifically target the exosomal circuits in experimental cancer therapy.  

3.2399           Ebola Virus Glycoprotein Promotes Enhanced Viral Egress by Preventing Ebola VP40 From Associating With the Host Restriction Factor BST2/Tetherin

Gustin, J.K., Bai, Y., Moses, A.V. and Douglas, J.L. Journal of Infectious Disease, 212, Suppl.2, S181-S190 (2015)   Background. BST2/tetherin is an innate immune molecule with the unique ability to restrict the egress of human immunodeficiency virus (HIV) and other enveloped viruses, including Ebola virus (EBOV). Coincident with this discovery was the finding that the HIV Vpu protein down-regulates BST2 from the cell surface, thereby promoting viral release. Evidence suggests that the EBOV envelope glycoprotein (GP) also counteracts BST2, although the mechanism is unclear. Results. We find that total levels of BST2 remain unchanged in the presence of GP, whereas surface BST2 is significantly reduced. GP is known to sterically mask surface receptors via its mucin domain. Our evaluation of mutant GP molecules indicate that masking of BST2 by GP is probably responsible for the apparent surface BST2 down-regulation; however, this masking does not explain the observed virus-like particle egress enhancement. We discovered that VP40 coimmunoprecipitates and colocalizes with BST2 in the absence but not in the presence of GP. Conclusions. These results suggest that GP may overcome the BST2 restriction by blocking an interaction between VP40 and BST2. Furthermore, we have observed that GP may enhance BST2 incorporation into virus-like particles. Understanding this novel EBOV immune evasion strategy will provide valuable insights into the pathogenicity of this deadly pathogen.  

3.2400           Rapid multiplex analysis of lipid raft components with single-cell resolution

Schatzlmaier, P., Supper, V., Göschl, L., Zwiritz, A., Eckerstorfer, P., Ellmeier, W., Huppa, J.B. and Stockinger, H. Sci. Signal, 8(395), rs11 (2015)   Lipid rafts are dynamic regions of membranes that are involved in cell signaling but are challenging to study because of their small size and dynamic nature. Schatzlmaier et al. found that lipid raft components became associated with nuclei during lysis of cells as the cells passed through a detergent-containing layer in a gradient. In experiments with lymphocytes, the authors demonstrated that this association enabled the quantitative analysis by flow cytometry of the composition of lipid rafts and of the dynamic association of proteins with these membrane microdomains at single-cell resolution.  

3.2401           PLD1 participates in BDNF-induced signalling in cortical neurons

Ammar, M.R., Thahouly, T., Hanauer, A., Stegner, D., Nieswandt, b. and Vitale, N. Scientific Reports, 5:14778 (2015)   The brain-derived neurotrophic factor BDNF plays a critical role in neuronal development and the induction of L-LTP at glutamatergic synapses in several brain regions. However, the cellular and molecular mechanisms underlying these BDNF effects have not been firmly established. Using in vitro cultures of cortical neurons from knockout mice for Pld1 and Rsk2, BDNF was observed to induce a rapid RSK2-dependent activation of PLD and to stimulate BDNF ERK1/2-CREB and mTor-S6K signalling pathways, but these effects were greatly reduced in Pld1−/− neurons. Furthermore, phospho-CREB did not accumulate in the nucleus, whereas overexpression of PLD1 amplified the BDNF-dependent nuclear recruitment of phospho-ERK1/2 and phospho-CREB. This BDNF retrograde signalling was prevented in cells silenced for the scaffolding protein PEA15, a protein which complexes with PLD1, ERK1/2, and RSK2 after BDNF treatment. Finally PLD1, ERK1/2, and RSK2 partially colocalized on endosomal structures, suggesting that these proteins are part of the molecular module responsible for BDNF signalling in cortical neurons.  

3.2402           Dynamic tubulation of mitochondria drives mitochondrial network formation

Wang, C., Du, W., Su, Q., Zhu, M., Feng, P., Li, Y., Zhou, Y., Mi, N., Zhu, Y., Jiang, D., Zhang, S., Zhang, Z., Sun, Y. and Yu, L. Cell Research, 25, 1108-1120 (2015)   Mitochondria form networks. Formation of mitochondrial networks is important for maintaining mitochondrial DNA integrity and interchanging mitochondrial material, whereas disruption of the mitochondrial network affects mitochondrial functions. According to the current view, mitochondrial networks are formed by fusion of individual mitochondria. Here, we report a new mechanism for formation of mitochondrial networks through KIF5B-mediated dynamic tubulation of mitochondria. We found that KIF5B pulls thin, highly dynamic tubules out of mitochondria. Fusion of these dynamic tubules, which is mediated by mitofusins, gives rise to the mitochondrial network. We further demonstrated that dynamic tubulation and fusion is sufficient for mitochondrial network formation, by reconstituting mitochondrial networks in vitro using purified fusion-competent mitochondria, recombinant KIF5B, and polymerized microtubules. Interestingly, KIF5B only controls network formation in the peripheral zone of the cell, indicating that the mitochondrial network is divided into subzones, which may be constructed by different mechanisms. Our data not only uncover an essential mechanism for mitochondrial network formation, but also reveal that different parts of the mitochondrial network are formed by different mechanisms.  

3.2403           Live-Cell Imaging of Phagosome Motility in Primary Mouse RPE Cells

Hazim, R., Jiang, M., Esteve-Rudd, J., Diemer, T., Lopes, V.S. and Williams, D.S. Exp. Med. Biol., 854, 751-755 (2015)   The retinal pigment epithelium (RPE) is a post-mitotic epithelial monolayer situated between the light-sensitive photoreceptors and the choriocapillaris. Given its vital functions for healthy vision, the RPE is a primary target for insults that result in blinding diseases, including age-related macular degeneration (AMD). One such function is the phagocytosis and digestion of shed photoreceptor outer segments. In the present study, we examined the process of trafficking of outer segment disk membranes in live cultures of primary mouse RPE, using high speed spinning disk confocal microscopy. This approach has enabled us to track phagosomes, and determine parameters of their motility, which are important for their efficient degradation.  

3.2404           Apolipoprotein E Regulates Amyloid Formation within Endosomes of Pigment Cells

Van Niel, G., bergam, P., Di Cicco, A., Hurbain, I., Lo Cicero, A., Dingli, F., Palmulli, R., Fort, C., Potier, M.C., Schurgers, L.J., Loew, D., Levy, D. and Raposo, G. Cell reports, 13(1), 43-51 (2015)   Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV) within endosomes and remain associated with ILVs when they are secreted as exosomes. ApoE functions in the ESCRT-independent sorting mechanism of PMEL onto ILVs and regulates the endosomal formation of PMEL amyloid fibrils in vitro and in vivo. This process secures the physiological formation of amyloid fibrils by exploiting ILVs as amyloid nucleating platforms.  

3.2405           Proteomic analysis of extracellular vesicles derived from Mycobacterium tuberculosis

Lee, J., Kim, S-H., Choi, D-S., Lee, J.S., Kim, D-K., Go, G., Park, S-M., Kim, S.H., Shin, J.H., Chang, C.L. and Gho, Y.S. Proteomics, 15(19), 3331-3337 (2015)   The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exosomes, and microvesicles, is an evolutionarily conserved phenomenon from bacteria to eukaryotes. It has been reported that Mycobacterium tuberculosis releases extracellular vesicles harboring immunologically active molecules, and these extracellular vesicles have been suggested to be applicable in vaccine development and biomarker discovery. However, the comprehensive proteomic analysis has not been performed for M. tuberculosis extracellular vesicles. In this study, we identified a total of 287 vesicular proteins by four LC-MS/MS analyses with high confidence. In addition, we identified several vesicular proteins associated with the virulence of M. tuberculosis. This comprehensive proteome profile will help elucidate the pathogenic mechanism of M. tuberculosis. The data have been deposited to the ProteomeXchange with identifier PXD001160 (http://proteomecentral.proteomexchange.org/dataset/PXD001160).  

3.2406           G206D Mutation of Presenilin-1 Reduces Pen2 Interaction, Increases Aβ42/Aβ40 Ratio and Elevates ER Ca2+ Accumulation

Chen, W-T., Hsieh, Y-F., Huang, Y-J., Lin, C-C., Lin, Y-T., Liu, Y-C., Lien, C-C and Cheng, I-H-J. Mol. Neurobiol.,52, 1835-1849 (2015)   Early-onset familial Alzheimer’s disease (AD) is most commonly associated with the mutations in presenilin-1 (PS1). PS1 is the catalytic component of the γ-secretase complex, which cleaves amyloid precursor protein to produce amyloid-β (Aβ), the major cause of AD. Presenilin enhancer 2 (Pen2) is critical for activating γ-secretase and exporting PS1 from endoplasmic reticulum (ER). Among all the familial AD-linked PS1 mutations, mutations at the G206 amino acid are the most adjacent position to the Pen2 binding site. Here, we characterized the effect of a familial AD-linked PS1 G206D mutation on the PS1-Pen2 interaction and the accompanied alteration in γ-secretase-dependent and -independent functions. We found that the G206D mutation reduced PS1-Pen2 interaction, but did not abolish γ-secretase formation and PS1 endoproteolysis. For γ-secretase-dependent function, the G206D mutation increased Aβ₄₂ production but not Notch cleavage. For γ-secretase-independent function, this mutation disrupted the ER calcium homeostasis but not lysosomal calcium homeostasis and autophagosome maturation. Impaired ER calcium homeostasis may due to the reduced mutant PS1 level in the ER. Although this mutation did not alter the cell survival under stress, both increased Aβ₄₂ ratio and disturbed ER calcium regulation could be the mechanisms underlying the pathogenesis of the familial AD-linked PS1 G206D mutation.  

3.2407           Proteomics in neurodegenerative diseases: Methods for obtaining a closer look at the neuronal proteome

Plum, S., Steinbach, S., Abel, L., Marcus, K., helling, S. and may, C. Proteomics Clin. Appl., 9(9-10), 848-871 (2015)   The analysis of brain function in normal aging and neurodegenerative, psychiatric, and neurological diseases has long been a subject of interest and has historically been investigated through descriptive analysis of macroscopic or microscopic observations. It is now possible to characterize brain cells, such as neurons and glial cells, or even their subcellular components, at the molecular level. This ability enables researchers to more closely examine brain cell specific molecular pathways to elucidate distinct brain functions. Furthermore, the analysis of neuronal maintenance and disease-causing effects is a central component of neurological investigations, which include proteomic approaches. Proteomics allows the identification of thousands of proteins through descriptive and comparative analyses and can provide a detailed overview of a distinct cellular state. Such analyses often require the isolation of individual cell types or subcellular components to investigate specific questions. This review provides an overview of the currently applied state-of-the-art prefractionation strategies in this field.  

3.2408           Proteoglycans support proper granule formation in pancreatic acinar cells

Aroso, M., Agricola, B., Hacker, C. and Schrader, M Histochem. Cell Biol., 144, 331-346 (2015)   Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. The molecular mechanisms of their biogenesis and the sorting of zymogens are still incompletely understood. Here, we investigated the role of proteoglycans in granule formation and secretion of zymogens in pancreatic AR42J cells, an acinar model system. Cupromeronic Blue cytochemistry and biochemical studies revealed an association of proteoglycans primarily with the granule membrane. Removal of proteoglycans by carbonate treatment led to a loss of membrane curvature indicating a supportive role in the maintenance of membrane shape and stability. Chemical inhibition of proteoglycan synthesis impaired the formation of normal electron-dense granules in AR42J cells and resulted in the formation of unusually small granule structures. These structures still contained the zymogen carboxypeptidase, a cargo molecule of secretory granules, but migrated to lighter fractions after density gradient centrifugation. Furthermore, the basal secretion of amylase was increased in AR42J cells after inhibitor treatment. In addition, irregular-shaped granules appeared in pancreatic lobules. We conclude that the assembly of a proteoglycan scaffold at the ZG membrane is supporting efficient packaging of zymogens and the proper formation of stimulus-competent storage granules in acinar cells of the pancreas.  

3.2409           mRNA Targeting to Endoplasmic Reticulum Precedes Ago Protein Interaction and MicroRNA (miRNA)-mediated Translation Repression in Mammalian Cells

Barman, B. and Bhattacharyya, S.N.

1. Biol. Chem., 290(41), 24650-24656 (2015)

  MicroRNA (miRNA) binds to the 3′-UTR of its target mRNAs to repress protein synthesis. Extensive research was done to understand the mechanism of miRNA-mediated repression in animal cells. Considering the progress in understanding the mechanism, information about the subcellular sites of miRNA-mediated repression is surprisingly limited. In this study, using an inducible expression system for an miRNA target message, we have delineated how a target mRNA passes through polysome association and Ago2 interaction steps on rough endoplasmic reticulum (ER) before the miRNA-mediated repression sets in. From this study, de novo formed target mRNA localization to the ER-bound polysomes manifested as the earliest event, which is followed by Ago2 micro-ribonucleoprotein binding, and translation repression of target message. Compartmentalization of this process to rough ER membrane ensures enrichment of miRNA-targeted messages and micro-ribonucleoprotein components on ER upon reaching a steady state.  

3.2410           Role of Pex21p for Piggyback Import of Gpd1p and Pnc1p into Peroxisomes of Saccharomyces cerevisiae

Effelsberg, D., Cruz-Zaragoza, L.D., Tonillo, J., Schliebs, W. and Erdmann, R.

1. Biol. Chem., 290(42), 25333-25342 (2015)

  Proteins designated for peroxisomal protein import harbor one of two common peroxisomal targeting signals (PTS). In the yeast Saccharomyces cerevisiae, the oleate-induced PTS2-dependent import of the thiolase Fox3p into peroxisomes is conducted by the soluble import receptor Pex7p in cooperation with the auxiliary Pex18p, one of two supposedly redundant PTS2 co-receptors. Here, we report on a novel function for the co-receptor Pex21p, which cannot be fulfilled by Pex18p. The data establish Pex21p as a general co-receptor in PTS2-dependent protein import, whereas Pex18p is especially important for oleate-induced import of PTS2 proteins. The glycerol-producing PTS2 protein glycerol-3-phosphate dehydrogenase Gpd1p shows a tripartite localization in peroxisomes, in the cytosol, and in the nucleus under osmotic stress conditions. We show the following: (i) Pex21p is required for peroxisomal import of Gpd1p as well as a key enzyme of the NAD⁺ salvage pathway, Pnc1p; (ii) Pnc1p, a nicotinamidase without functional PTS2, is co-imported into peroxisomes by piggyback transport via Gpd1p. Moreover, the specific transport of these two enzymes into peroxisomes suggests a novel regulatory role for peroxisomes under various stress conditions.  

3.2411           Membrane vesicle-mediated release of bacterial RNA

Sjöström, A.E., Sandblad, L., Uhlin, B.E. and Wai, S.N. Scientific Reports, 5:15329 (2015)   Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions.  

3.2412           Noninvasive imaging of radiolabeled exosome-mimetic nanovesicle using 99mTc-HMPAO

Hwang, D.W., Choi, H., jang, S.C., Yoo, M.Y., park, J.Y., Choi, N.E., Oh, H.J., Ha, S., Lee, Y-S., Jeong, J.M., Gho, Y.S. and Lee, D.S. Scientific Reports, 5:15636 (2015)   Exosomes known as nano-sized extracellular vesicles attracted recent interests due to their potential usefulness in drug delivery. Amid remarkable advances in biomedical applications of exosomes, it is crucial to understand in vivo distribution and behavior of exosomes. Here, we developed a simple method for radiolabeling of macrophage-derived exosome-mimetic nanovesicles (ENVs) with 99mTc-HMPAO under physiologic conditions and monitored in vivo distribution of 99mTc-HMPAO-ENVs using SPECT/CT in living mice. ENVs were produced from the mouse RAW264.7 macrophage cell line and labeled with 99mTc-HMPAO for 1 hr incubation, followed by removal of free 99mTc-HMPAO. SPECT/CT images were serially acquired after intravenous injection to BALB/c mouse. When ENVs were labeled with 99mTc-HMPAO, the radiochemical purity of 99mTc-HMPAO-ENVs was higher than 90% and the expression of exosome specific protein (CD63) did not change in 99mTc-HMPAO-ENVs. 99mTc-HMPAO-ENVs showed high serum stability (90%) which was similar to that in phosphate buffered saline until 5 hr. SPECT/CT images of the mice injected with 99mTc-HMPAO-ENVs exhibited higher uptake in liver and no uptake in brain, whereas mice injected with 99mTc-HMPAO showed high brain uptake until 5 hr. Our noninvasive imaging of radiolabeled-ENVs promises better understanding of the in vivo behavior of exosomes for upcoming biomedical application.  

3.2413           Rhodopsin Forms Nanodomains in Rod Outer Segment Disc Membranes of the Cold-Blooded Xenopus laevis

Rakshit, T., Senapati, S., Sinha, S., Whited, A.M. and Park, P.S-H. PloS One, 10(10), e0141114 (2015)   Rhodopsin forms nanoscale domains (i.e., nanodomains) in rod outer segment disc membranes from mammalian species. It is unclear whether rhodopsin arranges in a similar manner in amphibian species, which are often used as a model system to investigate the function of rhodopsin and the structure of photoreceptor cells. Moreover, since samples are routinely prepared at low temperatures, it is unclear whether lipid phase separation effects in the membrane promote the observed nanodomain organization of rhodopsin from mammalian species. Rod outer segment disc membranes prepared from the cold-blooded frog Xenopus laevis were investigated by atomic force microscopy to visualize the organization of rhodopsin in the absence of lipid phase separation effects. Atomic force microscopy revealed that rhodopsin nanodomains form similarly as that observed previously in mammalian membranes. Formation of nanodomains in ROS disc membranes is independent of lipid phase separation and conserved among vertebrates.  

3.2414           Gut microbe-derived extracellular vesicles induce insulin resistance, thereby impairing glucose metabolism in skeletal muscle

Choi, Y., Kwon, Y., Kim, D-K., Jeon, J., Jang, S.C., Wang, T., Ban, M., Kim, M-H., Jeon, S.G., Kim, M-S., Choi, C.S., Jee, Y-K., Gho, Y.S., Ryu, S.H. and Kim, Y-K. Scientific Reports, 5:15878 (2015)   Gut microbes might influence host metabolic homeostasis and contribute to the pathogenesis of type 2 diabetes (T2D), which is characterized by insulin resistance. Bacteria-derived extracellular vesicles (EVs) have been suggested to be important in the pathogenesis of diseases once believed to be non-infectious. Here, we hypothesize that gut microbe-derived EVs are important in the pathogenesis of T2D. In vivo administration of stool EVs from high fat diet (HFD)-fed mice induced insulin resistance and glucose intolerance compared to regular diet (RD)-fed mice. Metagenomic profiling of stool EVs by 16S ribosomal DNA sequencing revealed an increased amount of EVs derived from Pseudomonas panacis (phylum Proteobacteria) in HFD mice compared to RD mice. Interestingly, P. panacis EVs blocked the insulin signaling pathway in both skeletal muscle and adipose tissue. Moreover, isolated P. panacis EVs induced typical diabetic phenotypes, such as glucose intolerance after glucose administration or systemic insulin injection. Thus, gut microbe-derived EVs might be key players in the development of insulin resistance and impairment of glucose metabolism promoted by HFD.  

3.2415           AIF inhibits tumor metastasis by protecting PTEN from oxidation

Shen, S-M., Guo, M., Xiong, Z., Yu, Y., Zhao, X-Y., Zhang, F-F. and Chen, G-Q. EMBO Rep., 16(11), 1563-1580 (2015)   Apoptosis‐inducing factor (AIF) exerts dual roles on cell death and survival, but its substrates as a putative oxidoreductase and roles in tumorigenesis remain elusive. Here, we report that AIF physically interacts with and inhibits the oxidation of phosphatase and tensin homolog on chromosome ten (PTEN), a tumor suppressor susceptible for oxidation‐mediated inactivation. More intriguingly, we also identify PTEN as a mitochondrial protein and the ectopic expression of mitochondrial targeting sequence‐carrying PTEN almost completely inhibits Akt phosphorylation in PTEN‐deficient cells. AIF knockdown causes oxidation‐mediated inactivation of the lipid phosphatase activity of PTEN, with ensuing activation of Akt kinase, phosphorylation of the Akt substrate GSK‐3β, and activation of β‐catenin signaling in cancer cells. Through its effect on β‐catenin signaling, AIF inhibits epithelial–mesenchymal transition (EMT) and metastasis of cancer cells in vitro and in orthotopically implanted xenografts. Accordingly, the expression of AIF is correlated with the survival of human patients with cancers of multiple origins. These results identify PTEN as the substrate of AIF oxidoreductase and reveal a novel function for AIF in controlling tumor metastasis.  

3.2416           Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes

Taylor, D.D. and Shah, S. Methods, 87, 3-10 (2015)   Viable tumor cells actively release vesicles into the peripheral circulation and other biologic fluids, which exhibit proteins and RNAs characteristic of that cell. Our group demonstrated the presence of these extracellular vesicles of tumor origin within the peripheral circulation of cancer patients and proposed their utility for diagnosing the presence of tumors and monitoring their response to therapy in the 1970s. However, it has only been in the past 10 years that these vesicles have garnered interest based on the recognition that they serve as essential vehicles for intercellular communication, are key determinants of the immunosuppressive microenvironment observed in cancer and provide stability to tumor-derived components that can serve as diagnostic biomarkers. To date, the clinical utility of extracellular vesicles has been hampered by issues with nomenclature and methods of isolation. The term “exosomes” was introduced in 1981 to denote any nanometer-sized vesicles released outside the cell and to differentiate them from intracellular vesicles. Based on this original definition, we use “exosomes” as synonymous with “extracellular vesicles.” While our original studies used ultracentrifugation to isolate these vesicles, we immediately became aware of the significant impact of the isolation method on the number, type, content and integrity of the vesicles isolated. In this review, we discuss and compare the most commonly utilized methods for purifying exosomes for post-isolation analyses. The exosomes derived from these approaches have been assessed for quantity and quality of specific RNA populations and specific marker proteins. These results suggest that, while each method purifies exosomal material, there are pros and cons of each and there are critical issues linked with centrifugation-based methods, including co-isolation of non-exosomal materials, damage to the vesicle’s membrane structure and non-standardized parameters leading to qualitative and quantitative variability. The down-stream analyses of these resulting varying exosomes can yield misleading results and conclusions.  

3.2417           Highly-purified exosomes and shed microvesicles isolated from the human colon cancer cell line LIM1863 by sequential centrifugal ultrafiltration are biochemically and functionally distinct

Xu, R., Greening, D.W., Rai, A., Ji, H. and Simpson, R.J. Methods, 87, 11-25 (2015)   Secretion and exchange of extracellular vesicles (EVs) by most cell types is emerging as a fundamental biological process. Although much is known about EVs, there is still a lack of definition as to how many naturally occurring EV subtypes there are and how their properties and functionalities might differ. This vexing issue is critical if EVs are to be fully harnessed for therapeutic applications. To address this question we have developed and describe here a sequential centrifugal ultrafiltration (SCUF) method to examine, in an unbiased manner, what EV subtypes are released in vitro into cell culture medium using the human colon carcinoma cell line LIM1863 as a model system. Using the culture medium from ∼7.2 × 10⁹ LIM1863 cells, SCUF was performed using hydrophilic PVDF membranes with low protein binding properties (Millipore Durapore™ Ultrafree-CL filters with 0.1, 0.22, 0.45 and 0.65 μm pore size). EV particle sizing was measured using both dynamic light scattering and cryo-electron microscopy. Comparative proteome profiling was performed by GeLC–MS/MS and qualitative protein differences between EV subtypes determined by label-free spectral counting. The results showed essentially two EV subtypes; one subtype (fraction Fn1) comprised heterogeneous EVs with particle diameters of 30–1300 nm, the other (fraction Fn5) being homogeneous EVs of 30–100 nm diameter; based on cryo-EM both EV subtypes were round shaped. Western blot analysis showed Fn5 (SCUF-Exos) contained traditional exosome marker proteins (Alix⁺, TSG101⁺, CD81⁺, CD63⁺), while Fn1 (SCUF-sMVs) lacked these protein markers. These findings were consistent with sMVs isolated by differential centrifugation (10,000g, DC-sMVs) and exosomes (100,000g EVs depleted of 10,000g material). The buoyant density of sMVs determined by OptiPrep™ density gradient centrifugation was 1.18–1.19 g/mL and exosomes 1.10–1.11 g/mL. Comparative protein profiling of SCUF-Exos/-sMVs revealed 354 and 606 unambiguous protein identifications, respectively, with 256 proteins in common. A salient finding was the first report of 350 proteins uniquely identified in sMVs may of which have the potential to enable discrimination of this EV subtype from exosomes (notably, members of the septin family, kinesin-like protein (KIF23), exportin-2/chromosome segregation like-1 protein (CSE1L), and Rac GTPase-activating protein 1 (RACGAP1)). We report for the first time that both SCUF-Exos and SCUF-sMVs isolated from LIM1863 colon cancer cells induce invasion of recipient NIH3T3 cells. Interestingly, the SCUF-sMVs promote invasion to a significantly greater extent (3-fold) than SCUF-Exos. This analytical SCUF method for fractionating EVs is potentially scalable using tangential flow filtration, thereby providing a solid foundation for future in-depth functional studies of EV subtypes using diverse cell types and functional assays.  

3.2418           Human Mitochondrial DNA-Protein Complexes Attach to a Cholesterol-Rich Membrane Structure

Gerhold, J.M., Cansiz-Arda, S., Löhmus, M., Engberg, O., Reyes, A., van Rennes, H., Sanz, A., Holt, I.J., Cooper, H.M. and Spelbrink, J.N. Scientific Reports, 5:15292 (2015)   The helicase Twinkle is indispensable for mtDNA replication in nucleoids. Previously, we showed that Twinkle is tightly membrane-associated even in the absence of mtDNA, which suggests that Twinkle is part of a membrane-attached replication platform. Here we show that this platform is a cholesterol-rich membrane structure. We fractionated mitochondrial membrane preparations on flotation gradients and show that membrane-associated nucleoids accumulate at the top of the gradient. This fraction was shown to be highly enriched in cholesterol, a lipid that is otherwise low abundant in mitochondria. In contrast, more common mitochondrial lipids, and abundant inner-membrane associated proteins concentrated in the bottom-half of these gradients. Gene silencing of ATAD3, a protein with proposed functions related to nucleoid and mitochondrial cholesterol homeostasis, modified the distribution of cholesterol and nucleoids in the gradient in an identical fashion. Both cholesterol and ATAD3 were previously shown to be enriched in ER-mitochondrial junctions, and we detect nucleoid components in biochemical isolates of these structures. Our data suggest an uncommon membrane composition that accommodates platforms for replicating mtDNA, and reconcile apparently disparate functions of ATAD3. We suggest that mtDNA replication platforms are organized in connection with ER-mitochondrial junctions, facilitated by a specialized membrane architecture involving mitochondrial cholesterol.  

3.2419           Signal dependent ER export of lemur tyrosine kinase 2

Butler, E.C. and Bradbury, N.A. BMC Cell Biology, 16:26 (2015)   Background The membrane anchored kinase, LMTK2, is a serine/threonine kinase predominantly localized to endosomal compartments. LMTK2 has been shown to be involved in the trafficking of the CFTR ion channel, the androgen receptor, as well as modulating neurodegeneration. As a membrane anchored protein, LMTK2 must be exported from the ER, yet the mechanisms whereby LMTK2 is sequestered within the ER for efficient export are unknown. Methods Sequence analysis of the carboxyl tail of LMTK2 revealed a putative di-acidic ER export motif. Site-directed mutagenesis was utilized to ablate this potential motif. Subcellular fractionation, immunofluorescence microscopy, and transferrin recycling assays were used to determine the consequence of mutating LMTK2’s export motif. Results Mutation of the di-acidic export motif led to ER retention of LMTK2, and an increase in protein half-life and a concomitant loss of LMTK2 from its appropriate terminal destination. Loss of LMTK2 from endosomal compartments by preventing its release from the ER is linked to a reduction in transferrin recycling. Conclusions We have identified a di-acidic ER export motif within the carboxyl tail of the membrane anchored kinase LMTK2. This sequence is used by LMTK2 for its efficient export from the ER.  

3.2420           Phospholipid methylation controls Atg32‐mediated mitophagy and Atg8 recycling

Sakakibara, K. et al EMBO J., 34(21), 2703-2719 (2015)   Degradation of mitochondria via selective autophagy, termed mitophagy, contributes to mitochondrial quality and quantity control whose defects have been implicated in oxidative phosphorylation deficiency, aberrant cell differentiation, and neurodegeneration. How mitophagy is regulated in response to cellular physiology remains obscure. Here, we show that mitophagy in yeast is linked to the phospholipid biosynthesis pathway for conversion of phosphatidylethanolamine to phosphatidylcholine by the two methyltransferases Cho2 and Opi3. Under mitophagy‐inducing conditions, cells lacking Opi3 exhibit retardation of Cho2 repression that causes an anomalous increase in glutathione levels, leading to suppression of Atg32, a mitochondria‐anchored protein essential for mitophagy. In addition, loss of Opi3 results in accumulation of phosphatidylmonomethylethanolamine (PMME) and, surprisingly, generation of Atg8–PMME, a mitophagy‐incompetent lipid conjugate of the autophagy‐related ubiquitin‐like modifier. Amelioration of Atg32 expression and attenuation of Atg8–PMME conjugation markedly rescue mitophagy in opi3‐null cells. We propose that proper regulation of phospholipid methylation is crucial for Atg32‐mediated mitophagy.  

3.2421           The lipid raft proteome of Borrelia burgdorferi

Toledo, A., Perez, A., Coleman, J.L. and benach, J.L. Proteomics, 15(21), 3662-3675 (2015)   Eukaryotic lipid rafts are membrane microdomains that have significant amounts of cholesterol and a selective set of proteins that have been associated with multiple biological functions. The Lyme disease agent, Borrelia burgdorferi, is one of an increasing number of bacterial pathogens that incorporates cholesterol onto its membrane, and form cholesterol glycolipid domains that possess all the hallmarks of eukaryotic lipid rafts. In this study, we isolated lipid rafts from cultured B. burgdorferi as a detergent resistant membrane (DRM) fraction on density gradients, and characterized those molecules that partitioned exclusively or are highly enriched in these domains. Cholesterol glycolipids, the previously known raft-associated lipoproteins OspA and OpsB, and cholera toxin partitioned into the lipid rafts fraction indicating compatibility with components of the DRM. The proteome of lipid rafts was analyzed by a combination of LC-MS/MS or MudPIT. Identified proteins were analyzed in silico for parameters that included localization, isoelectric point, molecular mass and biological function. The proteome provided a consistent pattern of lipoproteins, proteases and their substrates, sensing molecules and prokaryotic homologs of eukaryotic lipid rafts. This study provides the first analysis of a prokaryotic lipid raft and has relevance for the biology of Borrelia, other pathogenic bacteria, as well as for the evolution of these structures. All MS data have been deposited in the ProteomeXchange with identifier PXD002365  

3.2422           Exogenous DNA Loading into Extracellular Vesicles via Electroporation is Size-Dependent and Enables Limited Gene Delivery

Lamichhane, T.N., Raiker, R.S. and Jay, S.M. Mol Pharmaceut., 12(10), 3650-3657 (2015)   Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Specifically, EVs have been identified as natural carriers of nucleic acids, sparking interest in their use for gene therapy and RNA interference applications. So far, small RNAs (siRNA and miRNA) have been successfully loaded into EVs for a variety of delivery applications, but the potential use of EVs for DNA delivery has scarcely been explored. Here, we report that exogenous linear DNA can be associated with EVs via electroporation in quantities sufficient to yield an average of hundreds of DNA molecules per vesicle. We determined that loading efficiency and capacity of DNA in EVs is dependent on DNA size, with linear DNA molecules less than 1000 bp in length being more efficiently associated with EVs compared to larger linear DNAs and plasmid DNAs using this approach. We further showed that EV size is also determinant with regard to DNA loading, as larger microvesicles encapsulated more linear and plasmid DNA than smaller, exosome-like EVs. Additionally, we confirmed the ability of EVs to transfer foreign DNA loaded via electroporation into recipient cells, although functional gene delivery was not observed. These results establish critical parameters that inform the potential use of EVs for gene therapy and, in agreement with other recent results, suggest that substantial barriers must be overcome to establish EVs as broadly applicable DNA delivery vehicles.  

3.2423           Proteome Profiles of Outer Membrane Vesicles and Extracellular Matrix of Pseudomonas aeruginosa Biofilms

Couto, N., Schooling, S.R., Dutcher, J.R. and Barber, J.

1. Proteome Res., 14(10), 4207-4222 (2015)

  In the present work, two different proteomic platforms, gel-based and gel-free, were used to map the matrix and outer membrane vesicle exoproteomes of Pseudomonas aeruginosa PAO1 biofilms. These two proteomic strategies allowed us a confident identification of 207 and 327 proteins from enriched outer membrane vesicles and whole matrix isolated from biofilms. Because of the physicochemical characteristics of these subproteomes, the two strategies showed complementarity, and thus, the most comprehensive analysis of P. aeruginosa exoproteome to date was achieved. Under our conditions, outer membrane vesicles contribute approximately 20% of the whole matrix proteome, demonstrating that membrane vesicles are an important component of the matrix. The proteomic profiles were analyzed in terms of their biological context, namely, a biofilm. Accordingly relevant metabolic processes involved in cellular adaptation to the biofilm lifestyle as well as those related to P. aeruginosa virulence capabilities were a key feature of the analyses. The diversity of the matrix proteome corroborates the idea of high heterogeneity within the biofilm; cells can display different levels of metabolism and can adapt to local microenvironments making this proteomic analysis challenging. In addition to analyzing our own primary data, we extend the analysis to published data by other groups in order to deepen our understanding of the complexity inherent within biofilm populations.  

3.2424           TRPC6 specifically interacts with APP to inhibit its cleavage by γ-secretase and reduce Aβ production

Wang, J., Lu, R., yang, J., Li, H., He, Z., Jing, N., Wang, X. and Wang, Y. Nature Communications, 6:8876 (2015)   Generation of β-amyloid (Aβ) peptide in Alzheimer’s disease involves cleavage of amyloid precursor protein (APP) by γ-secretase, a protease known to cleave several substrates, including Notch. Finding specific modulators for γ-secretase could be a potential avenue to treat the disease. Here, we report that transient receptor potential canonical (TRPC) 6 specifically interacts with APP leading to inhibition of its cleavage by γ-secretase and reduction in Aβ production. TRPC6 interacts with APP (C99), but not with Notch, and prevents C99 interaction with presenilin 1 (PS1). A fusion peptide derived from TRPC6 also reduces Aβ levels without effect on Notch cleavage. Crossing APP/PS1 mice with TRPC6 transgenic mice leads to a marked reduction in both plaque load and Aβ levels, and improvement in structural and behavioural impairment. Thus, TRPC6 specifically modulates γ-secretase cleavage of APP and preventing APP (C99) interaction with PS1 via TRPC6 could be a novel strategy to reduce Aβ formation.  

3.2425           Integrin α5 Suppresses the Phosphorylation of Epidermal Growth Factor Receptor and Its Cellular Signaling of Cell Proliferation via N-Glycosylation

Hang, Q., Isaji, T., Hou, S., Im, S., Fukuda, T. and Gu, J.

1. Biol. Chem., 290(49), 29345-29360 (2015)

  Integrin α5β1-mediated cell adhesion regulates a multitude of cellular responses, including cell proliferation, survival, and cross-talk between different cellular signaling pathways. Integrin α5β1 is known to convey permissive signals enabling anchorage-dependent receptor tyrosine kinase signaling. However, the effects of integrin α5β1 on cell proliferation are controversial, and the molecular mechanisms involved in the regulation between integrin α5β1 and receptor tyrosine kinase remain largely unclear. Here we show that integrin α5 functions as a negative regulator of epidermal growth factor receptor (EGFR) signaling through its N-glycosylation. Expression of WT integrin α5 suppresses the EGFR phosphorylation and internalization upon EGF stimulation. However, expression of the N-glycosylation mutant integrin α5, S3–5, which contains fewer N-glycans, reversed the suppression of the EGFR-mediated signaling and cell proliferation. In a mechanistic manner, WT but not S3–5 integrin α5 forms a complex with EGFR and glycolipids in the low density lipid rafts, and the complex formation is disrupted upon EGF stimulation, suggesting that the N-glycosylation of integrin α5 suppresses the EGFR activation through promotion of the integrin α5-glycolipids-EGFR complex formation. Furthermore, consistent restoration of those N-glycans on the Calf-1,2 domain of integrin α5 reinstated the inhibitory effects as well as the complex formation with EGFR. Taken together, these data are the first to demonstrate that EGFR activation can be regulated by the N-glycosylation of integrin α5, which is a novel molecular paradigm for the cross-talk between integrins and growth factor receptors.  

3.2426           Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization

Kanemoto, S., Kobayashi, Y., yamashita, T., Miyamoto, T., cui, M., asada, R., Cui, X., Hino, K., kaneko, M., Takai, T., matsuhisa, K., tahahashi, N and Imaizumi, K.

1. Cell Sci., 128, 4353-4365 (2015)

  Luman (also known as CREB3) is a type-II transmembrane transcription factor belonging to the OASIS family that localizes to the endoplasmic reticulum (ER) membrane under normal conditions. In response to ER stress, OASIS-family members are subjected to regulated intramembrane proteolysis (RIP), following which the cleaved N-terminal fragments translocate to the nucleus. In this study, we show that treatment of bone marrow macrophages (BMMs) with cytokines – macrophage colony-stimulating factor (M-CSF) and RANKL (also known as TNFSF11) – causes a time-dependent increase in Luman expression, and that Luman undergoes RIP and becomes activated during osteoclast differentiation. Small hairpin (sh)RNA-mediated knockdown of Luman in BMMs prevented the formation of multinucleated osteoclasts, concomitant with the suppression of DC-STAMP, a protein that is essential for cell–cell fusion in osteoclastogenesis. The N-terminus of Luman facilitates promoter activity of DC-STAMP, resulting in upregulation of DC-STAMP expression. Furthermore, Luman interacts with DC-STAMP, and controls its stability and localization. These results suggest that Luman regulates the multinucleation of osteoclasts by promoting cell fusion of mononuclear osteoclasts through DC-STAMP induction and intracellular distribution during osteoclastogenesis.  

3.2427           α/β Hydrolase Domain-containing 6 (ABHD6) Degrades the Late Endosomal/Lysosomal Lipid Bis(monoacylglycero)phosphate

Pribasnig, M.A. et al

1. Biol. Chem., 290(50), 29869-29881 (2015)

  α/β Hydrolase domain-containing 6 (ABHD6) can act as monoacylglycerol hydrolase and is believed to play a role in endocannabinoid signaling as well as in the pathogenesis of obesity and liver steatosis. However, the mechanistic link between gene function and disease is incompletely understood. Here we aimed to further characterize the role of ABHD6 in lipid metabolism. We show that mouse and human ABHD6 degrade bis(monoacylglycero)phosphate (BMP) with high specific activity. BMP, also known as lysobisphosphatidic acid, is enriched in late endosomes/lysosomes, where it plays a key role in the formation of intraluminal vesicles and in lipid sorting. Up to now, little has been known about the catabolism of this lipid. Our data demonstrate that ABHD6 is responsible for ∼90% of the BMP hydrolase activity detected in the liver and that knockdown of ABHD6 increases hepatic BMP levels. Tissue fractionation and live-cell imaging experiments revealed that ABHD6 co-localizes with late endosomes/lysosomes. The enzyme is active at cytosolic pH and lacks acid hydrolase activity, implying that it degrades BMP exported from acidic organelles or de novo-formed BMP. In conclusion, our data suggest that ABHD6 controls BMP catabolism and is therefore part of the late endosomal/lysosomal lipid-sorting machinery.  

3.2428           N-myristoyltransferase 1 interacts with calnexin at the endoplasmic reticulum

Dudek, E., Millott, R., Liu, W-X., Beauchamp, E., Berthiaume, L.G. and Michalak, M. Biochem. Biophys. Res. Comm., 468, 889-893 (2015)   Calnexin is a type 1 integral endoplasmic reticulum (ER) membrane molecular chaperone with a highly conserved C-terminal domain oriented to the cytoplasm. Protein N-myristoylation plays an important role in a wide variety of cellular signal transduction pathways and it is catalyzed by N-myristoyltransferase (NMT), a cytoplasmic and ER associated enzyme. Here using yeast two-hybrid screen, Western blot analysis, immunoprecipitation, immunolocalization and cellular fractionation we discovered that N-myristoyltransferase 1 interacts with calnexin at the ER. These observations point at a previously unrecognized contribution of calnexin to the retention of NMT1 at the ER membrane.  

3.2429           Bacterial membrane vesicles, an overlooked environmental colloid: Biology, environmental perspectives and applications

Toyofuku, M., Tashiro, Y., Hasegawa, Y., Kurosawa, M. and Nomura, N. Advances in Colloid and Interface Science, 226, 65-77 (2015)   Phospholipid vesicles play important roles in biological systems. Bacteria are one of the most abundant organisms on Earth, and bacterial membrane vesicles (MVs) were first observed 50 years ago. Many bacteria release MVs to the environment that mainly consist of the cell membrane and typically range from 20 to 400 nm in size. Bacterial MVs are involved in several biological functions, such as delivery of cargo, virulence and gene transfer. MVs can be isolated from laboratory culture and directly from the environment, indicating their high abundance in and impact on ecosystems. Many colloidal particles in the environment ranging in size from 1 nm to 1 μm have been reported but not characterized at the molecular level, and MVs remain to be explored. Hence, MVs can be considered terra incognita in environmental colloid research. Although MV biogenesis and biological roles are yet to be fully understood, the accumulation of knowledge has opened new avenues for their applications. Via genetic engineering, the MV yield can be greatly increased, and the components of MVs can be tailored. Recent studies have demonstrated that MVs have promising potential for applications such as drug delivery systems and nanobiocatalysts. For instance, MV vaccines have been extensively studied and have already been approved in Europe. Recent MV studies have evoked great interest in the fields of biology and biotechnology, but fundamental questions, such as their transport in the environment or physicochemical features of MVs, remain to be addressed. In this review, we present the current understanding of bacterial MVs and environmental perspectives and further introduce their applications.  

3.2430           Altered machinery of protein synthesis is region- and stage-dependent and is associated with α-synuclein oligomers in Parkinson’s disease

Garcia-Esparcia, P., Hernandez-Ortega, K., Koneti, A., Gil, L., Delgado-Morales, R., Castano, E., Carmona, M. and Ferrer, I. Acta Neuropathologica Comm., 3:76 (2015)   Introduction Parkinson’s disease (PD) is characterized by the accumulation of abnormal α-synuclein in selected regions of the brain following a gradient of severity with disease progression. Whether this is accompanied by globally altered protein synthesis is poorly documented. The present study was carried out in PD stages 1-6 of Braak and middle-aged (MA) individuals without alterations in brain in the substantia nigra, frontal cortex area 8, angular gyrus, precuneus and putamen. Results Reduced mRNA expression of nucleolar proteins nucleolin (NCL), nucleophosmin (NPM1), nucleoplasmin 3 (NPM3) and upstream binding transcription factor (UBF), decreased NPM1 but not NPM3 nucleolar protein immunostaining in remaining neurons; diminished 18S rRNA, 28S rRNA; reduced expression of several mRNAs encoding ribosomal protein (RP) subunits; and altered protein levels of initiation factor eIF3 and elongation factor eEF2 of protein synthesis was found in the substantia nigra in PD along with disease progression. Although many of these changes can be related to neuron loss in the substantia nigra, selective alteration of certain factors indicates variable degree of vulnerability of mRNAs, rRNAs and proteins in degenerating sustantia nigra. NPM1 mRNA and 18S rRNA was increased in the frontal cortex area 8 at stage 5-6; modifications were less marked and region-dependent in the angular gyrus and precuneus. Several RPs were abnormally regulated in the frontal cortex area 8 and precuneus, but only one RP in the angular gyrus, in PD. Altered levels of eIF3 and eIF1, and decrease eEF1A and eEF2 protein levels were observed in the frontal cortex in PD. No modifications were found in the putamen at any time of the study except transient modifications in 28S rRNA and only one RP mRNA at stages 5-6. These observations further indicate marked region-dependent and stage-dependent alterations in the cerebral cortex in PD. Altered solubility and α-synuclein oligomer formation, assessed in total homogenate fractions blotted with anti-α-synuclein oligomer-specific antibody, was demonstrated in the substantia nigra and frontal cortex, but not in the putamen, in PD. Dramatic increase in α-synuclein oligomers was also seen in fluorescent-activated cell sorter (FACS)-isolated nuclei in the frontal cortex in PD. Conclusions Altered machinery of protein synthesis is altered in the substantia nigra and cerebral cortex in PD being the frontal cortex area 8 more affected than the angular gyrus and precuneus; in contrast, pathways of protein synthesis are apparently preserved in the putamen. This is associated with the presence of α-synuclein oligomeric species in total homogenates; substantia nigra and frontal cortex are enriched, albeit with different band patterns, in α-synuclein oligomeric species, whereas α-synuclein oligomers are not detected in the putamen.  

3.2431           Tannerella forsythia Outer Membrane Vesicles Are Enriched with Substrates of the Type IX Secretion System and TonB-Dependent Receptors

Veith, P.D., Chen, Y-Y., Chen, D., O’Brien-Simpson, N.M., Cecil, J., Holden, J.A., Lenzo, J.C. and Reynolds, E.C.

1. Proteome Res., 14(12), 5355-5366 (2015)

  Tannerella forsythia, a Gram-negative oral bacterium closely associated with chronic periodontitis, naturally produces outer membrane vesicles (OMVs). In this study, OMVs were purified by gradient centrifugation, and the proteome was investigated together with cellular fractions using LC–MS/MS analyses of SDS-PAGE fractions, resulting in the identification of 872 proteins including 297 OMV proteins. Comparison of the OMV proteome with the subcellular proteomes led to the localization of 173 proteins to the vesicle membrane and 61 proteins to the vesicle lumen, while 27 substrates of the type IX secretion system were assigned to the vesicle surface. These substrates were generally enriched in OMVs; however, the stoichiometry of the S-layer proteins, TfsA and TfsB, was significantly altered, potentially to accommodate the higher curvature required of the S-layer around OMVs. A vast number of TonB-dependent receptors related to SusC, together with their associated SusD-like lipoproteins, were identified, and these were also relatively enriched in OMVs. In contrast, other lipoproteins were significantly depleted from the OMVs. This study identified the highest number of membrane-associated OMV proteins to date in any bacterium and conclusively demonstrates cargo sorting of particular classes of proteins, which may have significant impact on the virulence of OMVs.  

3.2432           UNC-45A Is a Nonmuscle Myosin IIA Chaperone Required for NK Cell Cytotoxicity via Control of Lytic Granule Secretion

Iizuka, Y., Cichocki, F., Sieben, A., Sforza, F., karim, R., Coughlin, K., Isaksson Vogel, R., Gavioli, R., McCullar, V., Lenvik, T., Lee, M., Miller, J. and Bazzaro, M.

1. Immunol., 195(10), 4760-4770 (2015)

  NK cell’s killing is a tightly regulated process under the control of specific cytoskeletal proteins. This includes Wiskott–Aldrich syndrome protein, Wiskott–Aldrich syndrome protein–interacting protein, cofilin, Munc13-4, and nonmuscle myosin IIA (NMIIA). These proteins play a key role in controlling NK-mediated cytotoxicity either via regulating the attachment of lytic granules to the actin-based cytoskeleton or via promoting the cytoskeletal reorganization that is requisite for lytic granule release. UNC-45A is a highly conserved member of the UNC-45/CRO1/She4p family of proteins that act as chaperones for both conventional and nonconventional myosin. Although we and others have shown that in lower organisms and in mammalian cells NMIIA-associated functions, such as cytokinesis, cell motility, and organelle trafficking, are dependent upon the presence of UNC-45A, its role in NK-mediated functions is largely unknown. In this article, we describe UNC-45A as a key regulator of NK-mediated cell toxicity. Specifically we show that, in human NK cells, UNC-45A localize at the NK cell immunological synapse of activated NK cells and is part of the multiprotein complex formed during NK cell activation. Furthermore, we show that UNC-45A is disposable for NK cell immunological synapse formation and lytic granules reorientation but crucial for lytic granule exocytosis. Lastly, loss of UNC-45A leads to reduced NMIIA binding to actin, suggesting that UNC-45A is a crucial component in regulating human NK cell cytoskeletal dynamics via promoting the formation of actomyosin complexes.  

3.2433           The COPII complex and lysosomal VAMP7 determine intracellular Salmonella localization and growth

Santos, J.C., Duchateau, M., Fredlund, J., Weiner, A., Mallet, A., Schmitt, C., matondo, M., Hourdel, V., Chamot-Rooke, J. and Enninga, J. Cellular Microbiol., 17(12), 1699-1720 (2015)   Salmonella invades epithelial cells and survives within a membrane-bound compartment, the Salmonella-containing vacuole (SCV). We isolated and determined the host protein composition of the SCV at 30 min and 3 h of infection to identify and characterize novel regulators of intracellular bacterial localization and growth. Quantitation of the SCV protein content revealed 392 host proteins specifically enriched at SCVs, out of which 173 associated exclusively with early SCVs, 124 with maturing SCV and 95 proteins during both time-points. Vacuole interactions with endoplasmic reticulum-derived coat protein complex II vesicles modulate early steps of SCV maturation, promoting SCV rupture and bacterial hyper-replication within the host cytosol. On the other hand, SCV interactions with VAMP7-positive lysosome-like vesicles promote Salmonella-induced filament formation and bacterial growth within the late SCV. Our results reveal that the dynamic communication between the SCV and distinct host organelles affects both intracellular Salmonella localization and growth at successive steps of host cell invasion.  

3.2434           The secret life of extracellular vesicles in metal homeostasis and neurodegeneration

Bellingham, S.A., Guo, B. and Hill, A.F. Biol. Cell, 107(11), 389-418 (2015)   Biologically active metals such as copper, zinc and iron are fundamental for sustaining life in different organisms with the regulation of cellular metal homeostasis tightly controlled through proteins that coordinate metal uptake, efflux and detoxification. Many of the proteins involved in either uptake or efflux of metals are localised and function on the plasma membrane, traffic between intracellular compartments depending upon the cellular metal environment and can undergo recycling via the endosomal pathway. The biogenesis of exosomes also occurs within the endosomal system, with several major neurodegenerative disease proteins shown to be released in association with these vesicles, including the amyloid-β (Aβ) peptide in Alzheimer's disease and the infectious prion protein involved in Prion diseases. Aβ peptide and the prion protein also bind biologically active metals and are postulated to play important roles in metal homeostasis. In this review, we will discuss the role of extracellular vesicles in Alzheimer's and Prion diseases and explore their potential contribution to metal homeostasis.  

3.2435           Redefining the Breast Cancer Exosome Proteome by Tandem Mass Tag Quantitative Proteomics and Multivariate Cluster Analysis

Clark, D.J., Fondrie, W.E., Liao, Z., Hanson, P.I., Fulton, A., Mao, L. and Yang, A.J. Anal. Chem., 87(20), 10462-10469 (2015)   Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into the extracellular environment. With evidence that exosomes can be detected in the blood of patients with various malignancies, the development of a platform that uses exosomes as a diagnostic tool has been proposed. However, it has been difficult to truly define the exosome proteome due to the challenge of discerning contaminant proteins that may be identified via mass spectrometry using various exosome enrichment strategies. To better define the exosome proteome in breast cancer, we incorporated a combination of Tandem-Mass-Tag (TMT) quantitative proteomics approach and Support Vector Machine (SVM) cluster analysis of three conditioned media derived fractions corresponding to a 10 000g cellular debris pellet, a 100 000g crude exosome pellet, and an Optiprep enriched exosome pellet. The quantitative analysis identified 2 179 proteins in all three fractions, with known exosomal cargo proteins displaying at least a 2-fold enrichment in the exosome fraction based on the TMT protein ratios. Employing SVM cluster analysis allowed for the classification 251 proteins as “true” exosomal cargo proteins. This study provides a robust and vigorous framework for the future development of using exosomes as a potential multiprotein marker phenotyping tool that could be useful in breast cancer diagnosis and monitoring disease progression.  

3.2436           In Vivo Differentiation of Therapeutic Insulin-Producing Cells from Bone Marrow Cells via Extracellular Vesicle-Mimetic Nanovesicles

Oh, K., Kim, S.R., Kim, D-K., Seo, M.W., Lee, C., Lee, H.M., Oh, J-E., Choi, E.Y., Lee, D-S., Gho, Y.S. and Park, K.S. ACSNano, 9(12), 11718-11727 (2015)   The current diabetes mellitus pandemic constitutes an important global health problem. Reductions in the mass and function of β-cells contribute to most of the pathophysiology underlying diabetes. Thus, physiological control of blood glucose levels can be adequately restored by replacing functioning β-cell mass. Sources of functional islets for transplantation are limited, resulting in great interest in the development of alternate sources, and recent progress regarding cell fate change via utilization of extracellular vesicles, also known as exosomes and microvesicles, is notable. Thus, this study investigated the therapeutic capacity of extracellular vesicle-mimetic nanovesicles (NVs) derived from a murine pancreatic β-cell line. To differentiate insulin-producing cells effectively, a three-dimensional in vivo microenvironment was constructed in which extracellular vesicle-mimetic NVs were applied to subcutaneous Matrigel platforms containing bone marrow (BM) cells in diabetic immunocompromised mice. Long-term control of glucose levels was achieved over 60 days, and differentiation of donor BM cells into insulin-producing cells in the subcutaneous Matrigel platforms, which were composed of islet-like cell clusters with extensive capillary networks, was confirmed along with the expression of key pancreatic β-cell markers. The resectioning of the subcutaneous Matrigel platforms caused a rebound in blood glucose levels and confirmed the source of functioning β-cells. Thus, efficient differentiation of therapeutic insulin-producing cells was attained in vivo through the use of extracellular vesicle-mimetic NVs, which maintained physiological glucose levels.   Optimized exosome isolation protocol for cell culture supernatant and human plasma Lobb, R.J., Becker, M., Wen, S.W., Wong, C.S.F., Wiegmans, A.P., Leimgruber, A. and Möller, A.

1. Extracellular Vesicles, 4:27031 (2015)

  Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research.   iSRAP – a one-touch research tool for rapid profiling of small RNA-seq data Quek, C., Jung, C-h., Bellingham, S.A., Lonie, A. and Hill, A.F.

1. Extracellular Vesicles, 4:29454 (2015)

  Small non-coding RNAs have been significantly recognized as the key modulators in many biological processes, and are emerging as promising biomarkers for several diseases. These RNA species are transcribed in cells and can be packaged in extracellular vesicles, which are small vesicles released from many biotypes, and are involved in intercellular communication. Currently, the advent of next-generation sequencing (NGS) technology for high-throughput profiling has further advanced the biological insights of non-coding RNA on a genome-wide scale and has become the preferred approach for the discovery and quantification of non-coding RNA species. Despite the routine practice of NGS, the processing of large data sets poses difficulty for analysis before conducting downstream experiments. Often, the current analysis tools are designed for specific RNA species, such as microRNA, and are limited in flexibility for modifying parameters for optimization. An analysis tool that allows for maximum control of different software is essential for drawing concrete conclusions for differentially expressed transcripts. Here, we developed a one-touch integrated small RNA analysis pipeline (iSRAP) research tool that is composed of widely used tools for rapid profiling of small RNAs. The performance test of iSRAP using publicly and in-house available data sets shows its ability of comprehensive profiling of small RNAs of various classes, and analysis of differentially expressed small RNAs. iSRAP offers comprehensive analysis of small RNA sequencing data that leverage informed decisions on the downstream analyses of small RNA studies, including extracellular vesicles such as exosomes.  

3.2437           Extracellular vesicles secreted by Schistosoma mansoni contain protein vaccine candidates

Sotillo, J., Pearson, M., Potriquet, J., Becker, L., Pickering, D., Mulvenna, J. and Loukas, A. Int. J. Parasitol., 46, 1-5 (2016)   Herein we show for the first time that Schistosoma mansoni adult worms secrete exosome-like extracellular vesicles ranging from 50 to 130 nm in size. Extracellular vesicles were collected from the excretory/secretory products of cultured adult flukes and purified by Optiprep density gradient, resulting in highly pure extracellular vesicle preparations as confirmed by transmission electron microscopy and Nanosight tracking analysis. Extracellular vesicle proteomic analysis showed numerous known vaccine candidates, potential virulence factors and molecules implicated in feeding. These findings provide new avenues for the exploration of host–schistosome interactions and offer a potential mechanism by which some vaccine antigens exert their protective efficacy.  

3.2438           Effect of exosome isolation methods on physicochemical properties of exosomes and clearance of exosomes from the blood circulation

Yamashita, T., Takahashi, Y., Nishiwaka, M. and Takakura, Y. Eur. J. Pharmaceutics and Biopharmaceutics, 98, 1-8 (2016)   Exosomes, which are expected to be delivery systems for biomolecules such as nucleic acids, are collected by several methods. However, the effect of exosome isolation methods on the characteristics of exosomes as drug carriers, such as recovery efficiency after sterile filtration and pharmacokinetics, has not been investigated despite the importance of these characteristics for the development of exosome-based delivery systems. In the present study, exosomes collected from murine melanoma B16-BL6 cells by several methods were compared with respect to dispersibility, recovery rate after filtering, and clearance from the blood circulation in mice. The exosomes were collected by three ultracentrifugation-based methods: simple ultracentrifugation/pelleting (pelleting method), ultracentrifugation with an iodixanol cushion (cushion method), and ultracentrifugation on an iodixanol density gradient (gradient method). The isolation methods had little effect on the particle number of exosomes. In contrast, transmission electron microscopy observation and size distribution measurement using tunable resistive pulse sensing indicated that the exosomes of the gradient method were more dispersed than the others. The exosomes were labeled with Gaussia luciferase and intravenously injected into mice. Clearance of injected exosomes from the blood circulation did not significantly change with isolation methods. When the exosomes were filtered using a 0.2-μm filter, the recovery rate was 82% for the exosomes of the gradient method, whereas it was less than 50% for the others. These results indicate that the exosome isolation method markedly affects the dispersibility and filtration efficiency of the exosomes.  

3.2439           βA3/A1-crystallin and persistent fetal vasculature (PFV) disease of the eye

Ziegler, Jr., J.S., Valapala, M., Shang, P., Hose, S., Goldberg, M.F. and Sinha, D. Biochim. Biophys. Acta, 1860, 287-298 (2016)   Background Persistent fetal vasculature (PFV) is a human disease in which the fetal vasculature of the eye fails to regress normally. The fetal, or hyaloid, vasculature nourishes the lens and retina during ocular development, subsequently regressing after formation of the retinal vessels. PFV causes serious congenital pathologies and is responsible for as much as 5% of blindness in the United States. Scope of review The causes of PFV are poorly understood, however there are a number of animal models in which aspects of the disease are present. One such model results from mutation or elimination of the gene (Cryba1) encoding βA3/A1-crystallin. In this review we focus on the possible mechanisms whereby loss of functional βA3/A1-crystallin might lead to PFV. Major conclusions Cryba1 is abundantly expressed in the lens, but is also expressed in certain other ocular cells, including astrocytes. In animal models lacking βA3/A1-crystallin, astrocyte numbers are increased and they migrate abnormally from the retina to ensheath the persistent hyaloid artery. Evidence is presented that the absence of functional βA3/A1-crystallin causes failure of the normal acidification of endolysosomal compartments in the astrocytes, leading to impairment of certain critical signaling pathways, including mTOR and Notch/STAT3.  

3.2440           A fluorescent cholesterol analogue for observation of free cholesterol in the plasma membrane of live cells

Ogawa, Y. and Tanaka, M. Anal. Biochem., 492, 49-55 (2016)   Free cholesterol in mammalian cells resides mostly in the plasma membrane, where it plays an important role in cellular homeostasis. We synthesized a new fluorescent cholesterol analogue that retained an intact alkyl chain and the sterane backbone of cholesterol. The hydroxyl group of cholesterol was converted into an amino group that was covalently linked to the fluorophore tetramethylrhodamine to retain the ability to form hydrogen bonds with adjacent molecules. Incubating live MDCK (Madin–Darby canine kidney) cells with our fluorescent cholesterol analogue resulted in the generation of intense signals that were detected by microscopy at the plasma membrane. Incubation with the analogue exerted minimal, if any, influence on cell growth, indicating that it could serve as a useful tool for analyzing free cholesterol at the plasma membrane.  

3.2441           Identification of EDIL3 on extracellular vesicles involved in breast cancer cell invasion

Lee, J-E., Moon, P-G., Cho, Y-E., Kim, Y-B., Kim, I-S., park, H. and Baek, M-C.

1. Proteomics, 131, 17-28 (2016)

  Cancer cell-derived extracellular vesicles have been linked to the pathogenesis of various cancers; however, the role of extracellular vesicles in tumorigenesis remains unclear. To identify extracellular vesicle proteins involved in cancer metastasis, quantitative proteomic analyses were performed on extracellular vesicles derived from two representative breast cancer cell lines: the less invasive MCF-7 and the invasive MDA-MB-231. Proteomic analysis allowed for the identification of 270 proteins in the extracellular vesicles. Here we report a new function of EDIL3 on extracellular vesicles, which are sufficient for enhancement of cell invasion and for acceleration of lung metastasis in vivo. This invasion is most likely mediated via the integrin-FAK signaling cascade in breast cancer cells. However, these effects are suppressed when EDIL3 is inactivated, providing evidence for a critical role of EDIL3 in development of cancer. Consistently, in human patients with metastatic breast cancer, the levels of EDIL3 on circulating extracellular vesicles are significantly elevated. This information is a remarkable breakthrough in understanding of the molecular mechanism underlying metastasis of breast cancer as well as in the research for cancer biomarkers using circulating extracellular vesicles. Furthermore, targeting EDIL3 on extracellular vesicles may lead to a new therapeutic option for treatment of breast cancer.  

3.2442           Bovine milk-derived exosomes for drug delivery

Munagala, R., Aqil, F., Jeybalan, J. and Gupta, R. Cancer Lett., 371, 48-61 (2016)   Exosomes are biological nanovesicles that are involved in cell–cell communication via the functionally-active cargo (such as miRNA, mRNA, DNA and proteins). Because of their nanosize, exosomes are explored as nanodevices for the development of new therapeutic applications. However, bulk, safe and cost-effective production of exosomes is not available. Here, we show that bovine milk can serve as a scalable source of exosomes that can act as a carrier for chemotherapeutic/chemopreventive agents. Drug-loaded exosomes showed significantly higher efficacy compared to free drug in cell culture studies and against lung tumor xenografts in vivo. Moreover, tumor targeting ligands such as folate increased cancer-cell targeting of the exosomes resulting in enhanced tumor reduction. Milk exosomes exhibited cross-species tolerance with no adverse immune and inflammatory response. Thus, we show the versatility of milk exosomes with respect to the cargo it can carry and ability to achieve tumor targetability. This is the first report to identify a biocompatible and cost-effective means of exosomes to enhance oral bioavailability, improve efficacy and safety of drugs.  

3.2443           Peroxisomal Pex11 is a pore-forming protein homologous to TRPM channels

Mindthoff, S., Grunau, S., Steinfort, L.L., Girzalsky, W., Hiltunen, J.K., Erdmann, R. and Antonenkov, V.D. Biochim. Biophys. Acta, 1863, 271-283 (2016)   More than 30 proteins (Pex proteins) are known to participate in the biogenesis of peroxisomes—ubiquitous oxidative organelles involved in lipid and ROS metabolism. The Pex11 family of homologous proteins is responsible for division and proliferation of peroxisomes. We show that yeast Pex11 is a pore-forming protein sharing sequence similarity with TRPM cation-selective channels. The Pex11 channel with a conductance of Λ = 4.1 nS in 1.0 M KCl is moderately cation-selective (P_K⁺/P_Cl⁻ = 1.85) and resistant to voltage-dependent closing. The estimated size of the channel's pore (r ~ 0.6 nm) supports the notion that Pex11 conducts solutes with molecular mass below 300–400 Da. We localized the channel's selectivity determining sequence. Overexpression of Pex11 resulted in acceleration of fatty acids β-oxidation in intact cells but not in the corresponding lysates. The β-oxidation was affected in cells by expression of the Pex11 protein carrying point mutations in the selectivity determining sequence. These data suggest that the Pex11-dependent transmembrane traffic of metabolites may be a rate-limiting step in the β-oxidation of fatty acids. This conclusion was corroborated by analysis of the rate of β-oxidation in yeast strains expressing Pex11 with mutations mimicking constitutively phosphorylated (S165D, S167D) or unphosphorylated (S165A, S167A) protein. The results suggest that phosphorylation of Pex11 is a mechanism that can control the peroxisomal β-oxidation rate. Our results disclose an unexpected function of Pex11 as a non-selective channel responsible for transfer of metabolites across peroxisomal membrane. The data indicate that peroxins may be involved in peroxisomal metabolic processes in addition to their role in peroxisome biogenesis.  

3.2444           ABCA1-dependent sterol release: sterol molecule specificity and potential membrane domain for HDL biogenesis

Yamauchi, Y., Yokoyama, S. and Chang, T-Y.

1. Lipid Res., 57, 77-88 (2016)

  Mammalian cells synthesize various sterol molecules, including the C30 sterol, lanosterol, as cholesterol precursors in the endoplasmic reticulum. The build-up of precursor sterols, including lanosterol, displays cellular toxicity. Precursor sterols are found in plasma HDL. How these structurally different sterols are released from cells is poorly understood. Here, we show that newly synthesized precursor sterols arriving at the plasma membrane (PM) are removed by extracellular apoA-I in a manner dependent on ABCA1, a key macromolecule for HDL biogenesis. Analysis of sterol molecules by GC-MS and tracing the fate of radiolabeled acetate-derived sterols in normal and mutant Niemann-Pick type C cells reveal that ABCA1 prefers newly synthesized sterols, especially lanosterol, as the substrates before they are internalized from the PM. We also show that ABCA1 resides in a cholesterol-rich membrane domain resistant to the mild detergent, Brij 98. Blocking ACAT activity increases the cholesterol contents of this domain. Newly synthesized C29/C30 sterols are transiently enriched within this domain, but rapidly disappear from this domain with a half-life of less than 1 h. Our work shows that substantial amounts of precursor sterols are transported to a certain PM domain and are removed by the ABCA1-dependent pathway.  

3.2445           A draft map of the mouse pluripotent stem cell spatial proteome

Christoforou, A., Mulvey, C.M., Breckels, L.M., Geladaki, A., Hurrell, T., Hayward, P.C., Naake, T., Gatto, L., Viner, R., Martinez Arias, A. and Lilley, K.S. Nature Communications, 7:8992 (2016)   Knowledge of the subcellular distribution of proteins is vital for understanding cellular mechanisms. Capturing the subcellular proteome in a single experiment has proven challenging, with studies focusing on specific compartments or assigning proteins to subcellular niches with low resolution and/or accuracy. Here we introduce hyperLOPIT, a method that couples extensive fractionation, quantitative high-resolution accurate mass spectrometry with multivariate data analysis. We apply hyperLOPIT to a pluripotent stem cell population whose subcellular proteome has not been extensively studied. We provide localization data on over 5,000 proteins with unprecedented spatial resolution to reveal the organization of organelles, sub-organellar compartments, protein complexes, functional networks and steady-state dynamics of proteins and unexpected subcellular locations. The method paves the way for characterizing the impact of post-transcriptional and post-translational modification on protein location and studies involving proteome-level locational changes on cellular perturbation. An interactive open-source resource is presented that enables exploration of these data.  

3.2446           The proprotein convertase PC1/3 regulates TLR9 trafficking and the associated signaling pathways

Duhamel, M., Rodet, F., Murgoci, A.N., Desjardins, r., Gagnon, H., wisztorsski, M., Fournier, I., day, R. and Salzet, M. Scientific Reports, 6:19360 (2016)   Endosomal TLR9 is considered as a potent anti-tumoral therapeutic target. Therefore, it is crucial to decipher the mechanisms controlling its trafficking since it determines TLR9 activation and signalling. At present, the scarcity of molecular information regarding the control of this trafficking and signalling is noticeable. We have recently demonstrated that in macrophages, proprotein convertase 1/3 (PC1/3) is a key regulator of TLR4 Myd88-dependent signalling. In the present study, we established that PC1/3 also regulates the endosomal TLR9. Under CpG-ODN challenge, we found that PC1/3 traffics rapidly to co-localize with TLR9 in CpG-ODN-containing endosomes with acidic pH. In PC1/3 knockdown macrophages, compartmentalization of TLR9 was altered and TLR9 clustered in multivesicular bodies (MVB) as demonstrated by co-localization with Rab7. This demonstrates that PC1/3 controls TLR9 trafficking. This clustering of TLR9 in MVB dampened the anti-inflammatory STAT3 signalling pathway while it promoted the pro-inflammatory NF-kB pathway. As a result, macrophages from PC1/3 KO mice and rat PC1/3-KD NR8383 macrophages secreted more pro-inflammatory cytokines such as TNF-α, IL6, IL1α and CXCL2. This is indicative of a M1 pro-inflammatory phenotype. Therefore, PC1/3 KD macrophages represent a relevant mean for cell therapy as “Trojan” macrophages.  

3.2447           Exosomes Mediate LTB4 Release during Neutrophil Chemotaxis

Majumdar, R., Tameh, A.T. and Parent, C.A. PloS Biology, 14(1), e1002336 (2016)   Leukotriene B₄ (LTB₄) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB₄ and its synthesizing enzymes localize to intracellular multivesicular bodies that, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in a LTB₄ receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB₄ release. Our findings establish that the exosomal pool of LTB₄ acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments.  

3.2448           Modulation of Mitochondrial Antiviral Signaling by Human Herpesvirus 8 Interferon Regulatory Factor 1

Hwang, K.Y. and Choi, Y.B.

1. Virol., 90(1), 506-520 (2016)

  Mitochondrial lipid raft-like microdomains, experimentally also termed mitochondrial detergent-resistant membrane fractions (mDRM), play a role as platforms for recruiting signaling molecules involved in antiviral responses such as apoptosis and innate immunity. Viruses can modulate mitochondrial functions for their own survival and replication. However, viral regulation of the antiviral responses via mDRM remains incompletely understood. Here, we report that human herpesvirus 8 (HHV-8) gene product viral interferon regulatory factor 1 (vIRF-1) is targeted to mDRM during virus replication and negatively regulates the mitochondrial antiviral signaling protein (MAVS)-mediated antiviral responses. The N-terminal region of vIRF-1 interacts directly with membrane lipids, including cardiolipin. In addition, a GxRP motif within the N terminus of vIRF-1, conserved in the mDRM-targeting region of mitochondrial proteins, including PTEN-induced putative kinase 1 (PINK1) and MAVS, was found to be important for vIRF-1 association with mitochondria. Furthermore, MAVS, which has the potential to promote vIRF-1 targeting to mDRM possibly by inducing cardiolipin exposure on the outer membrane of mitochondria, interacts with vIRF-1, which, in turn, inhibits MAVS-mediated antiviral signaling. Consistent with these results, vIRF-1 targeting to mDRM contributes to promotion of HHV-8 productive replication and inhibition of associated apoptosis. Combined, our results suggest novel molecular mechanisms for negative-feedback regulation of MAVS by vIRF-1 during virus replication.  

3.2449           PTP-PEST controls EphA3 activation and ephrin-induced cytoskeletal remodeling

Mansour, M., Nievergali, E., gegenbauer, K., Llerena, C., Atapattu, L., Halle, M., Tremblay, M.L., Janes, P.W. and lackmann, M.

1. Cell Sci., 129, 277-289 (2016)

  Eph receptors and their corresponding membrane-bound ephrin ligands regulate cell positioning and establish tissue patterns during embryonic and oncogenic development. Emerging evidence suggests that assembly of polymeric Eph signalling clusters relies on cytoskeletal reorganisation and underlies regulation by protein tyrosine phosphatases (PTPs). PTP-PEST (also known as PTPN12) is a central regulator of actin cytoskeletal dynamics. Here, we demonstrate that an N-terminal fragment of PTP-PEST, generated through an ephrinA5-triggered and spatially confined cleavage mediated by caspase-3, attenuates EphA3 receptor activation and its internalisation. Isolation of EphA3 receptor signalling clusters within intact plasma membrane fragments obtained by detergent-free cell fractionation reveals that stimulation of cells with ephrin triggers effective recruitment of this catalytically active truncated form of PTP-PEST together with key cytoskeletal and focal adhesion proteins. Importantly, modulation of actin polymerisation using pharmacological and dominant-negative approaches affects EphA3 phosphorylation in a similar manner to overexpression of PTP-PEST. We conclude that PTP-PEST regulates EphA3 activation both by affecting cytoskeletal remodelling and through its direct action as a PTP controlling EphA3 phosphorylation, indicating its multifaceted regulation of Eph signalling.  

3.2450           Lysosomes serve as a platform for hepatitis A virus particle maturation and nonlytic release

Seggewiss, N., Paulmann, D. and Dotzauer, A. Arch. Virol., 161(1), 43-52 (2016)   Early studies on hepatitis A virus (HAV) in cell culture demonstrated the inclusion of several viral particles in an intracellular lipid-bilayer membrane. However, the origin of these virus-associated membranes and the mechanism for the non-lytic release of HAV into bile are still unknown. Analyzing the association of this virus with cell organelles, we found that newly synthesized HAV particles accumulate in lysosomal organelles and that lysosomal enzymes are involved in the maturation cleavage of the virion. Furthermore, by inhibiting the processes of fusion of lysosomes with the plasma membrane, we found that the nonlytic release of HAV from infected cells occurs via lysosome-related organelles.  

3.2451           Borrelia burgdorferi HtrA: evidence for twofold proteolysis of outer membrane protein p66

Coleman, J.L., Toledo, A. and Benach, J.L. Mol. Microbiol., 99(1), 135-150 (2016)   In prokaryotes, members of the High Temperature Requirement A (HtrA) family of serine proteases function in the periplasm to degrade damaged or improperly folded membrane proteins. Borrelia burgdorferi, the agent of Lyme disease, codes for a single HtrA homolog. Two-dimensional electrophoresis analysis of B. burgdorferi B31A3 and a strain that overexpresses HtrA (A3HtrAOE) identified a downregulated protein in A3HtrAOE with a mass, pI and MALDI-TOF spectrum consistent with outer membrane protein p66. P66 and HtrA from cellular lysates partitioned into detergent-resistant membranes, which contain cholesterol-glycolipid-rich membrane regions known as lipid rafts, suggesting that HtrA and p66 may reside together in lipid rafts also. This agrees with previous work from our laboratory, which showed that HtrA and p66 are constituents of B. burgdorferi outer membrane vesicles. HtrA degraded p66 in vitro and A3HtrAOE expressed reduced levels of p66 in vivo. Fluorescence confocal microscopy revealed that HtrA and p66 colocalize in the membrane. The association of HtrA and p66 establishes that they could interact efficiently and their protease/substrate relationship provides functional relevance to this interaction. A3HtrAOE also showed reduced levels of p66 transcript in comparison with wild-type B31A3, indicating that HtrA-mediated regulation of p66 may occur at multiple levels.  

3.2452           Targeting Viral Proteostasis Limits Influenza Virus, HIV, and Dengue Virus Infection

Heaton, N. et al Immunity, 44(1), 46-58 (2016)   Viruses are obligate parasites and thus require the machinery of the host cell to replicate. Inhibition of host factors co-opted during active infection is a strategy hosts use to suppress viral replication and a potential pan-antiviral therapy. To define the cellular proteins and processes required for a virus during infection is thus crucial to understanding the mechanisms of virally induced disease. In this report, we generated fully infectious tagged influenza viruses and used infection-based proteomics to identify pivotal arms of cellular signaling required for influenza virus growth and infectivity. Using mathematical modeling and genetic and pharmacologic approaches, we revealed that modulation of Sec61-mediated cotranslational translocation selectively impaired glycoprotein proteostasis of influenza as well as HIV and dengue viruses and led to inhibition of viral growth and infectivity. Thus, by studying virus-human protein-protein interactions in the context of active replication, we have identified targetable host factors for broad-spectrum antiviral therapies.  

3.2453           Exosomes from HIV-1-infected Cells Stimulate Production of Pro-inflammatory Cytokines through Trans-activating Response (TAR) RNA

Sampey, G.C., Saifuddin, M., Schwab, A., Barclay, r., Punya, S., Chung, M-C., Hakami, R.M., Zadeh, M.A., Lepene, B., Klase, Z.A., El-Hage, N., Young, M., Iordanskiy, S. and kashanchi, F.

1. Biol. Chem., 291(3), 1251-1266 (2016)

  HIV-1 infection results in a chronic illness because long-term highly active antiretroviral therapy can lower viral titers to an undetectable level. However, discontinuation of therapy rapidly increases virus burden. Moreover, patients under highly active antiretroviral therapy frequently develop various metabolic disorders, neurocognitive abnormalities, and cardiovascular diseases. We have previously shown that exosomes containing trans-activating response (TAR) element RNA enhance susceptibility of undifferentiated naive cells to HIV-1 infection. This study indicates that exosomes from HIV-1-infected primary cells are highly abundant with TAR RNA as detected by RT-real time PCR. Interestingly, up to a million copies of TAR RNA/μl were also detected in the serum from HIV-1-infected humanized mice suggesting that TAR RNA may be stable in vivo. Incubation of exosomes from HIV-1-infected cells with primary macrophages resulted in a dramatic increase of proinflammatory cytokines, IL-6 and TNF-β, indicating that exosomes containing TAR RNA could play a direct role in control of cytokine gene expression. The intact TAR molecule was able to bind to PKR and TLR3 effectively, whereas the 5′ and 3′ stems (TAR microRNAs) bound best to TLR7 and -8 and none to PKR. Binding of TAR to PKR did not result in its phosphorylation, and therefore, TAR may be a dominant negative decoy molecule in cells. The TLR binding through either TAR RNA or TAR microRNA potentially can activate the NF-κB pathway and regulate cytokine expression. Collectively, these results imply that exosomes containing TAR RNA could directly affect the proinflammatory cytokine gene expression and may explain a possible mechanism of inflammation observed in HIV-1-infected patients under cART.  

3.2454           Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions

Purushothaman, A., Bandari, S.K., Liu, J., Mobley, J.A., Brown, E.E. and Sanderson, R.D.

1. Biol. Chem., 291(4), 1652-1663 (2016)

  Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.  

3.2455           A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria

Roier, S., Zingl, F.G., Cakar, F., Durakovic, S., Kohl, P., Eichmann, T.O:, Klug, L., Gadermeier, B., Weinzerl, K., Prassl, R., Lass, G., Daum. G., Reidl, J., Feldman, M.F. and Schild, S. Nature Communications, 7:10515 (2016)   Bacterial outer membrane vesicles (OMVs) have important biological roles in pathogenesis and intercellular interactions, but a general mechanism of OMV formation is lacking. Here we show that the VacJ/Yrb ABC (ATP-binding cassette) transport system, a proposed phospholipid transporter, is involved in OMV formation. Deletion or repression of VacJ/Yrb increases OMV production in two distantly related Gram-negative bacteria, Haemophilus influenzae and Vibrio cholerae. Lipidome analyses demonstrate that OMVs from VacJ/Yrb-defective mutants in H. influenzae are enriched in phospholipids and certain fatty acids. Furthermore, we demonstrate that OMV production and regulation of the VacJ/Yrb ABC transport system respond to iron starvation. Our results suggest a new general mechanism of OMV biogenesis based on phospholipid accumulation in the outer leaflet of the outer membrane. This mechanism is highly conserved among Gram-negative bacteria, provides a means for regulation, can account for OMV formation under all growth conditions, and might have important pathophysiological roles in vivo.  

3.2456           H2AX phosphorylation and DNA damage kinase activity are dispensable for herpes simplex virus replication

Botting, C., Lu, X. and Triezenberg, S.J. Virology J., 13:15 (2016)   Background Herpes simplex virus type 1 (HSV-1) can establish both lytic and latent infections in humans. The phosphorylation of histone H2AX, a common marker of DNA damage, during lytic infection by HSV-1 is well established. However, the role(s) of H2AX phosphorylation in lytic infection remain unclear. Methods Following infection of human foreskin fibroblasts by HSV-1 or HSV-2, we assayed the phosphorylation of H2AX in the presence of inhibitors of transcription, translation, or viral DNA replication, or in the presence of inhibitors of ATM and ATR kinases (KU-55933 and VE-821, respectively). We also assayed viral replication in fibroblasts in the presence of the kinase inhibitors or siRNAs specific for ATM and ATR, as well as in cell lines deficient for either ATR or ATM. Results The expression of viral immediate-early and early proteins (including the viral DNA polymerase), but not viral DNA replication or late protein expression, were required for H2AX phosphorylation following HSV-1 infection. Inhibition of ATM kinase activity prevented HSV-stimulated H2AX phosphorylation but had only a minor effect on DNA replication and virus yield in HFF cells. These results differ from previous reports of a dramatic reduction in viral yield following chemical inhibition of ATM in oral keratinocytes or following infection of ATM^−/− cells. Inhibition of the closely related kinase ATR (whether by chemical inhibitor or siRNA disruption) had no effect on H2AX phosphorylation and reduced viral DNA replication only moderately. During infection by HSV-2, H2AX phosphorylation was similarly dispensable but was dependent on both ATM activity and viral DNA replication. Conclusion H2AX phosphorylation represents a cell type-specific and virus type-specific host response to HSV infection with little impact on viral infection.    

3.2457           Iodixanol Gradient Centrifugation to Separate Components of the Low-Density Membrane Fraction from 3T3-L1 Adipocytes

Sadler, J.B.A., lamb, C.A., Gould, G.W. and Bryant, N.J. Cold Spring Harbor Protoc., pdb prot083709 (2016)   We optimized a set of fractionation techniques to facilitate the isolation of subcellular compartments containing insulin-sensitive glucose transporter isoform 4 (GLUT4), which is mobilized from GLUT4 storage vesicles (GSVs) in fat and muscle cells in response to insulin. In the absence of insulin, GLUT4 undergoes a continuous cycle of GSV formation and fusion with other compartments. Full membrane fractionation of 3T3-L1 adipocytes produces a low-density membrane fraction that contains both the constitutive recycling pool (the endosomal recycling compartments) and the insulin-sensitive pool (the GSVs). These two pools can be separated based on density using iodixanol gradient centrifugation, described here.  

3.2458           Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes

Clark, D.J., Fondrie, W.E., Yang, A. and Mao, L.

1. Proteomics, 133, 161-169 (2016)

  Exosomes are 30–100 nm sized membrane vesicles released by cells into the extracellular space that mediate intercellular communication via transfer of proteins and other biological molecules. To better understand the role of these microvesicles in lung carcinogenesis, we employed a Triple SILAC quantitative proteomic strategy to examine the differential protein abundance between exosomes derived from an immortalized normal bronchial epithelial cell line and two non-small cell lung cancer (NSCLC) cell lines harboring distinct activating mutations in the cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) or epidermal growth factor receptor (EGFR). In total, we were able to quantify 721 exosomal proteins derived from the three cell lines. Proteins associated with signal transduction, including EGFR, GRB2 and SRC, were enriched in NSCLC exosomes, and could actively regulate cell proliferation in recipient cells. This study's investigation of the NSCLC exosomal proteome has identified enriched protein cargo that can contribute to lung cancer progression, which may have potential clinical implications in biomarker development for patients with NSCLC.  

3.2459           Exosome-mediated inflammasome signaling after central nervous system injury

De Rivero Vaccari, J.P., Brand III, F., Adamczak, S., Lee, S.W., Perez-Barcena, J., Wang, M.Y., Bullock, M.R., Dalton Dietrich, W. and Keane, R.W.

1. Neurochem., 136(S1), 39-48 82016)

  Neuroinflammation is a response against harmful effects of diverse stimuli and participates in the pathogenesis of brain and spinal cord injury (SCI). The innate immune response plays a role in neuroinflammation following CNS injury via activation of multiprotein complexes termed inflammasomes that regulate the activation of caspase 1 and the processing of the pro-inflammatory cytokines IL-1β and IL-18. We report here that the expression of components of the nucleotide-binding and oligomerization domain (NOD)-like receptor protein-1 (NLRP-1) inflammasome, apoptosis speck-like protein containing a caspase recruitment domain (ASC), and caspase 1 are significantly elevated in spinal cord motor neurons and cortical neurons after CNS trauma. Moreover, NLRP1 inflammasome proteins are present in exosomes derived from CSF of SCI and traumatic brain-injured patients following trauma. To investigate whether exosomes could be used to therapeutically block inflammasome activation in the CNS, exosomes were isolated from embryonic cortical neuronal cultures and loaded with short-interfering RNA (siRNA) against ASC and administered to spinal cord-injured animals. Neuronal-derived exosomes crossed the injured blood–spinal cord barrier, and delivered their cargo in vivo, resulting in knockdown of ASC protein levels by approximately 76% when compared to SCI rats treated with scrambled siRNA. Surprisingly, siRNA silencing of ASC also led to a significant decrease in caspase 1 activation and processing of IL-1β after SCI. These findings indicate that exosome-mediated siRNA delivery may be a strong candidate to block inflammasome activation following CNS injury. We propose the following signaling cascade for inflammasome activation in peripheral tissues after CNS injury: CNS trauma induces inflammasome activation in the nervous system and secretion of exosomes containing inflammasome protein cargo into cerebral spinal fluid. The inflammasome containing exosomes then fuse with target cells to activate the innate immune response in peripheral tissues. We suggest that these findings may be used to develop new therapeutics to treat the devastating inflammation and cell destruction evoked by CNS injuries. IL-1β and IL-18 = pro-inflammatory cytokines.  

3.2460           The influence of a caveolin-1 mutant on the function of P-glycoprotein

Lee, C-Y., Lai, T-Y., Tsai, M-K., Ou-Yang, P., Tsasi, C-Y., Wu, S-W., Hsu, L-C. and Chen, J-S. Scientific Reports, 6:20486 (2016)   The genetic heterogeneity in cancer cells has an increased chance in the acquisition of new mutant such as drug-resistant phenotype in cancer cells. The phenotype of drug resistance in cancer cells could be evaluated by the number or function of drug transporters on cell membranes, which would lead to decreased intracellular anti-cancer drugs concentration. Caveolae are flask-shaped invaginations on cell membrane that function in membrane trafficking, endocytosis, and as a compartment where receptors and signaling proteins are concentrated. Caveolin-1 (CAV1) is the principal structural protein of caveolae and closely correlates with multidrug resistance in cancer cells. In a systematic study of the ubiquitin-modified proteome, lysine 176 of CAV1 was identified as a potential post-translational modification site for ubiquitination. In this article, we identified a mutation at lysine 176 to arginine (K176R) on CAV1 would interfere with the biogenesis of caveolae and broke the interaction of CAV1 with P-glycoprotein. Functional assays further revealed that K176R mutant of CAV1 in cancer cells increased the transport activity of P-glycoprotein and decreased the killing ability of anti-cancer drugs in non-small-cell lung cancer cell lines.  

3.2461           Exosomes as new diagnostic tools in CNS diseases

Kanninen, K.M., Bister, N., Koistinaho, J. and Malm, T. Biochim. Biophys. Acta, 1862, 403-410 (2016)   Exosomes are small extracellular vesicles that modulate important functions in physiology and under pathological conditions of the central nervous system (CNS). Exosomal contents, proteins, lipids and various RNA species, are altered during disease. The fact that exosomes are released into the blood stream from blood cells and endothelial cells responding to CNS diseases as well as from the brain and spinal cord, and that they express markers which allow their tracking to the cell of origin, makes the use of exosomes for diagnostic purposes and biomarker discovery particularly appealing. While the utilization of exosomes for diagnostics in diseases affecting the CNS are still in the early stages of discovery and development, it is expected that through further research and fervent development of protocols relating to isolation and purification the true potential of exosomes derived from the CNS will be harnessed for more effective clinical disease diagnosis. In this review we begin with a short introduction to the origin, composition and function of exosomes in the CNS. Next we discuss the current status of methodologies related to isolation and detection of CNS exosomes. We end with an account of exosomes in diagnostics and biomarker discovery, which focuses on three diseases of the CNS: Alzheimer's disease, multiple sclerosis, and stroke. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.  

3.2462           Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry

Kormelink, T.G., Arkesteijn, G.J.A., nauwelaers, F.A., van den Engh, G., Nolte-‘t Hoen, E.N.M.and Wauben, M.H.M. Cytometry Part A,89A, 135-147 (2016)   Submicron-sized vesicles released by cells are increasingly recognized for their role in intercellular communication and as biomarkers of disease. Methods for high-throughput, multi-parameter analysis of such extracellular vesicles (EVs) are crucial to further investigate their diversity and function. We recently developed a high-resolution flow cytometry-based method (using a modified BD Influx) for quantitative and qualitative analysis of EVs. The fact that the majority of EVs is <200 nm in size requires special attention with relation to specific conditions of the flow cytometer, as well as sample concentration and event rate. In this study, we investigated how (too) high particle concentrations affect high-resolution flow cytometry-based particle quantification and characterization. Increasing concentrations of submicron-sized particles (beads, liposomes, and EVs) were measured to identify coincidence and swarm effects, caused by the concurrent presence of multiple particles in the measuring spot. As a result, we demonstrate that analysis of highly concentrated samples resulted in an underestimation of the number of particles and an interdependent overestimation of light scattering and fluorescence signals. On the basis of this knowledge, and by varying nozzle size and sheath pressure, we developed a strategy for high-resolution flow cytometric sorting of submicron-sized particles. Using the adapted sort settings, subsets of EVs differentially labeled with two fluorescent antibodies could be sorted to high purity. Moreover, sufficient numbers of EVs could be sorted for subsequent analysis by western blotting. In conclusion, swarm effects that occur when measuring high particle concentrations severely hamper EV quantification and characterization. These effects can be easily overlooked without including proper controls (e.g., sample dilution series) or tools (e.g., oscilloscope). Providing that the event rate is well controlled, the sorting strategy we propose here indicates that high-resolution flow cytometric sorting of different EV subsets is feasible.  

3.2463           Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons

Krishnaswami, S.R. et al Nature Protocols, 11(3), 499-524 (2016)   A protocol is described for sequencing the transcriptome of a cell nucleus. Nuclei are isolated from specimens and sorted by FACS, cDNA libraries are constructed and RNA-seq is performed, followed by data analysis. Some steps follow published methods (Smart-seq2 for cDNA synthesis and Nextera XT barcoded library preparation) and are not described in detail here. Previous single-cell approaches for RNA-seq from tissues include cell dissociation using protease treatment at 30 °C, which is known to alter the transcriptome. We isolate nuclei at 4 °C from tissue homogenates, which cause minimal damage. Nuclear transcriptomes can be obtained from postmortem human brain tissue stored at −80 °C, making brain archives accessible for RNA-seq from individual neurons. The method also allows investigation of biological features unique to nuclei, such as enrichment of certain transcripts and precursors of some noncoding RNAs. By following this procedure, it takes about 4 d to construct cDNA libraries that are ready for sequencing.  

3.2464           Myo19 is an outer mitochondrial membrane motor and effector of starvation-induced filopodia

Shneyer, B.I., Usaj, M. and henn, A.

1. Cell Sci., 129, 543-556 (2016)

  Mitochondria respond to environmental cues and stress conditions. Additionally, the disruption of the mitochondrial network dynamics and its distribution is implicated in a variety of neurodegenerative diseases. Here, we reveal a new function for Myo19 in mitochondrial dynamics and localization during the cellular response to glucose starvation. Ectopically expressed Myo19 localized with mitochondria to the tips of starvation-induced filopodia. Corollary to this, RNA interference (RNAi)-mediated knockdown of Myo19 diminished filopodia formation without evident effects on the mitochondrial network. We analyzed the Myo19–mitochondria interaction, and demonstrated that Myo19 is uniquely anchored to the outer mitochondrial membrane (OMM) through a 30–45-residue motif, indicating that Myo19 is a stably attached OMM molecular motor. Our work reveals a new function for Myo19 in mitochondrial positioning under stress.  

3.2465           CD44-mediated monocyte transmigration across Cryptococcus neoformans-infected brain microvascular endothelial cells is enhanced by HIV-1 gp41-I90 ectodomain

He, X., Shi, X., Puthiyakunnon, S., Zhang, L., Zeng, Q., Li, Y., Boddu, S., Qiu, J., Lai, Z., Ma, C., Xie, Y., Long, M., Du, M., Huang, S-H. and Cao, H.

1. Biomed. Sci., 23:28 (2016)

  Background Cryptococcus neoformans (Cn) is an important opportunistic pathogen in the immunocompromised people, including AIDS patients, which leads to fatal cryptococcal meningitis with high mortality rate. Previous researches have shown that HIV-1 gp41-I90 ectodomain can enhance Cn adhesion to and invasion of brain microvascular endothelial cell (BMEC), which constitutes the blood brain barrier (BBB). However, little is known about the role of HIV-1 gp41-I90 in the monocyte transmigration across Cn-infected BBB. In the present study, we provide evidence that HIV-1 gp41-I90 and Cn synergistically enhance monocytes transmigration across the BBB in vitro and in vivo. The underlying mechanisms for this phenomenon require further study. Methods In this study, the enhancing role of HIV-1 gp41-I90 in monocyte transmigration across Cn-infected BBB was demonstrated by performed transmigration assays in vitro and in vivo. Results Our results showed that the transmigration rate of monocytes are positively associated with Cn and/or HIV-1 gp41-I90, the co-exposure (HIV-1 gp41-I90 + Cn) group showed a higher THP-1 transmigration rate (P < 0.01). Using CD44 knock-down HBMEC or CD44 inhibitor Bikunin in the assay, the facilitation of transmigration rates of monocyte enhanced by HIV-1 gp41-I90 was significantly suppressed. Western blotting analysis and biotin/avidin enzyme-linked immunosorbent assays (BA-ELISAs) showed that Cn and HIV-1 gp41-I90 could increase the expression of CD44 and ICAM-1 on the HBMEC. Moreover, Cn and/or HIV-1 gp41-I90 could also induce CD44 redistribution to the membrane lipid rafts. By establishing the mouse cryptococcal meningitis model, we found that HIV-1 gp41-I90 and Cn could synergistically enhance the monocytes transmigration, increase the BBB permeability and injury in vivo. Conclusions Collectively, our findings suggested that HIV-1 gp41-I90 ectodomain can enhance the transmigration of THP-1 through Cn-infected BBB, which may be mediated by CD44. This novel study enlightens the future prospects to elaborate the inflammatory responses induced by HIV-1 gp41-I90 ectodomain and to effectively eliminate the opportunistic infections in AIDS patients.  

3.2466           Characterization and Vaccine Potential of Outer Membrane Vesicles Produced by Haemophilus parasuis

McCaig, W.D., Loving, C.L., Hughes, H.R. and Brockmeier, S.L. PloS One, 11(3), e0149132 (2016)   Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swine and is capable of causing a systemic infection, resulting in high morbidity and mortality. H. parasuis isolates display a wide range of virulence and virulence factors are largely unknown. Commercial bacterins are often used to vaccinate swine against H. parasuis, though strain variability and lack of cross-reactivity can make this an ineffective means of protection. Outer membrane vesicles (OMV) are spherical structures naturally released from the membrane of bacteria and OMV are often enriched in toxins, signaling molecules and other bacterial components. Examination of OMV structures has led to identification of virulence factors in a number of bacteria and they have been successfully used as subunit vaccines. We have isolated OMV from both virulent and avirulent strains of H. parasuis, have examined their protein content and assessed their ability to induce an immune response in the host. Vaccination with purified OMV derived from the virulent H. parasuis Nagasaki strain provided protection against challenge with a lethal dose of the bacteria.  

3.2467           Gestational Diabetes Mellitus Is Associated With Changes in the Concentration and Bioactivity of Placenta-Derived Exosomes in Maternal Circulation Across Gestation

Salomon, C., Scholz-Romero, K., Sarker, S., Sweeney, E., Kobayashi, M., Correa, P., Longo, S., Duncombe, G., Mitchell, M.D., Rice, G.E. and Illanes, S.E. Diabetes, 65, 598-609 (2016)   Although there is significant interest in elucidating the role of placenta-derived exosomes (PdEs) during pregnancy, the exosomal profile in pregnancies complicated by gestational diabetes mellitus (GDM) remains to be established. The aim of this study was to compare the gestational-age profile of PdEs in maternal plasma of GDM with normal pregnancies and to determine the effect of exosomes on cytokine release from human umbilical vein endothelial cells. A prospective cohort of patients was sampled at three time points during pregnancy for each patient (i.e., 11–14, 22–24, and 32–36 weeks' gestation). A retrospective stratified study design was used to quantify exosomes present in maternal plasma of normal (n = 13) and GDM (n = 7) pregnancies. Gestational age and pregnancy status were identified as significant factors contributing to variation in plasma exosome concentration (ANOVA, P < 0.05). Post hoc analyses established that PdE concentration increased during gestation in both normal and GDM pregnancies; however, the increase was significantly greater in GDM (∼2.2-fold, ∼1.5-fold, and ∼1.8-fold greater at each gestational age compared with normal pregnancies). Exosomes isolated from GDM pregnancies significantly increased the release of proinflammatory cytokines from endothelial cells. Although the role of exosomes during GDM remains to be fully elucidated, exosome profiles may be of diagnostic utility for screening asymptomatic populations.  

3.2468           Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes

Kowal, J., Arras, G., Colombo, M., Jouve, M., Morath, J.P., Primdal-Bengtson, B., Dingli, F., Loew, D., Tkach, M. and Thery, C. PNAS, 113, E968-E977 (2016)   Extracellular vesicles (EVs) have become the focus of rising interest because of their numerous functions in physiology and pathology. Cells release heterogeneous vesicles of different sizes and intracellular origins, including small EVs formed inside endosomal compartments (i.e., exosomes) and EVs of various sizes budding from the plasma membrane. Specific markers for the analysis and isolation of different EV populations are missing, imposing important limitations to understanding EV functions. Here, EVs from human dendritic cells were first separated by their sedimentation speed, and then either by their behavior upon upward floatation into iodixanol gradients or by immuno-isolation. Extensive quantitative proteomic analysis allowing comparison of the isolated populations showed that several classically used exosome markers, like major histocompatibility complex, flotillin, and heat-shock 70-kDa proteins, are similarly present in all EVs. We identified proteins specifically enriched in small EVs, and define a set of five protein categories displaying different relative abundance in distinct EV populations. We demonstrate the presence of exosomal and nonexosomal subpopulations within small EVs, and propose their differential separation by immuno-isolation using either CD63, CD81, or CD9. Our work thus provides guidelines to define subtypes of EVs for future functional studies.  

3.2469           Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

Zhang, J., Zhang, Z., Chukkapalli, V., Nchoutmboube, J.A., Li, J., Randall, G., Belov, G.A. and Wang, X. PNAS, 113(8), E1064-E1073 (2016)   All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting the CHO2 gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes.  

3.2470           Shp2 Associates with and Enhances Nephrin Tyrosine Phosphorylation and Is Necessary for Foot Process Spreading in Mouse Models of Podocyte Injury

Verma, R., Venkatareddy, M., Kalinowski, A., Patel, S.R., Salant, D.J. and Garg, P. Mol. Cell. Biol., 36(4), 596-614 (2016)   In most forms of glomerular diseases, loss of size selectivity by the kidney filtration barrier is associated with changes in the morphology of podocytes. The kidney filtration barrier is comprised of the endothelial lining, the glomerular basement membrane, and the podocyte intercellular junction, or slit diaphragm. The cell adhesion proteins nephrin and neph1 localize to the slit diaphragm and transduce signals in a Src family kinase Fyn-mediated tyrosine phosphorylation-dependent manner. Studies in cell culture suggest nephrin phosphorylation-dependent signaling events are primarily involved in regulation of actin dynamics and lamellipodium formation. Nephrin phosphorylation is a proximal event that occurs both during development and following podocyte injury. We hypothesized that abrogation of nephrin phosphorylation following injury would prevent nephrin-dependent actin remodeling and foot process morphological changes. Utilizing a biased screening approach, we found nonreceptor Src homology 2 (sh2) domain-containing phosphatase Shp2 to be associated with phosphorylated nephrin. We observed an increase in nephrin tyrosine phosphorylation in the presence of Shp2 in cell culture studies. In the human glomerulopathies minimal-change nephrosis and membranous nephropathy, there is an increase in Shp2 phosphorylation, a marker of increased Shp2 activity. Mouse podocytes lacking Shp2 do not develop foot process spreading when subjected to podocyte injury in vivo using protamine sulfate or nephrotoxic serum (NTS). In the NTS model, we observed a lack of foot process spreading in mouse podocytes with Shp2 deleted and smaller amounts of proteinuria. Taken together, these results suggest that Shp2-dependent signaling events are necessary for changes in foot process structure and function following injury.      

3.2471           A Role of TMEM16E Carrying a Scrambling Domain in Sperm Motility

Gyobu, S., Miyata, H., Ikawa, M., Yamazaki, D., Takeshima, H., Suzuki, J. and nagata, S. Mol. Cell. Biol., 36(4), 645-659 (2016)   Transmembrane protein 16E (TMEM16E) belongs to the TMEM16 family of proteins that have 10 transmembrane regions and appears to localize intracellularly. Although TMEM16E mutations cause bone fragility and muscular dystrophy in humans, its biochemical function is unknown. In the TMEM16 family, TMEM16A and -16B serve as Ca²⁺-dependent Cl⁻ channels, while TMEM16C, -16D, -16F, -16G, and -16J support Ca²⁺-dependent phospholipid scrambling. Here, we show that TMEM16E carries a segment composed of 35 amino acids homologous to the scrambling domain in TMEM16F. When the corresponding segment of TMEM16A was replaced by this 35-amino-acid segment of TMEM16E, the chimeric molecule localized to the plasma membrane and supported Ca²⁺-dependent scrambling. We next established TMEM16E-deficient mice, which appeared to have normal skeletal muscle. However, fertility was decreased in the males. We found that TMEM16E was expressed in germ cells in early spermatogenesis and thereafter and localized to sperm tail. TMEM16E^−/− sperm showed no apparent defect in morphology, beating, mitochondrial function, capacitation, or binding to zona pellucida. However, they showed reduced motility and inefficient fertilization of cumulus-free but zona-intact eggs in vitro. Our results suggest that TMEM16E may function as a phospholipid scramblase at inner membranes and that its defect affects sperm motility.  

3.2472           Isolation and Analysis of Detergent-Resistant Membrane Fractions

Aureli, M., Grassi, S., Sonnino, S. and Prinetti, A. Methods in Mol. Biol., 1376, 107-131 (2016)   The hypothesis that the Golgi apparatus is capable of sorting proteins and sending them to the plasma membrane through “lipid rafts,” membrane lipid domains highly enriched in glycosphingolipids, sphingomyelin, ceramide, and cholesterol, was formulated by van Meer and Simons in 1988 and came to a turning point when it was suggested that lipid rafts could be isolated thanks to their resistance to solubilization by some detergents, namely Triton X-100. An incredible number of papers have described the composition and properties of detergent-resistant membrane fractions. However, the use of this method has also raised the fiercest criticisms. In this chapter, we would like to discuss the most relevant methodological aspects related to the preparation of detergent-resistant membrane fractions, and to discuss the importance of discriminating between what is present on a cell membrane and what we can prepare from cell membranes in a laboratory tube.  

3.2473           Extensible Multiplex Real-time PCR of MicroRNA Using Microparticles

Jung, S., Kim, J., Lee, D.J., Oh, E.H., Lim, H., Kim, K.P., Choi, N., Kim, T.S. and Kim, S.K. Scientific Reports, 6;22975 (2016)   Multiplex quantitative real-time PCR (qPCR), which measures multiple DNAs in a given sample, has received significant attention as a mean of verifying the rapidly increasing genetic targets of interest in single phenotype. Here we suggest a readily extensible qPCR for the expression analysis of multiple microRNA (miRNA) targets using microparticles of primer-immobilized networks as discrete reactors. Individual particles, 200~500 μm in diameter, are identified by two-dimensional codes engraved into the particles and the non-fluorescent encoding allows high-fidelity acquisition of signal in real-time PCR. During the course of PCR, the amplicons accumulate in the volume of the particles with high reliability and amplification efficiency over 95%. In a quick assay comprising of tens of particles holding different primers, each particle brings the independent real-time amplification curve representing the quantitative information of each target. Limited amount of sample was analyzed simultaneously in single chamber through this highly multiplexed qPCR; 10 kinds of miRNAs from purified extracellular vesicles (EVs).  

3.2474           Up-regulation of Hnf1α gene expression in the liver of rats with experimentally induced chronic renal failure – A possible link between circulating PCSK9 and triacylglycerol concentrations

Sucajtys-Szulc, E., Szolkiewicz, M. and Swierczynski, J. Atherosclerosis, 248, 17-26 (2016)   Background The aim of this study was to verify if an increase in Hnf1α gene expression could be a possible link between circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) and TAGs concentrations in chronic renal failure (CRF). Methods Rats underwent 5/6 nephrectomy or a sham surgery. Liver expressions of Pcsk9, Mttp, ApoB-100, Hnf1α, Hnf4α, lipogenic enzymes and β-actin genes were quantified by qPCR. Liver levels of proteins coding by these genes were analyzed by Western blotting. Serum apoB-100 and PCSK9 concentration were estimated with an immunoassay. Results CRF rats showed an increase in circulating concentrations of TAGs, VLDL, apoB-100 and PCSK9, along with an enhanced liver VLDL-TAG secretion rate and a coordinated liver up-regulation of genes coding: a) lipogenic enzymes; b) Mttp and ApoB-100; c) Pcsk9; d) Hnf1α and Hnf4α. Positive correlations were found between serum creatinine concentrations and: a) the liver levels of HNF1α mRNA (r = 0.79, p < 0.01) and HNF4α (r = 0.76, p < 0.01); b) the liver levels of PCSK9 mRNA (r = 0.88, p < 0.01) and serum PCSK9 concentrations (r = 0.73, p < 0.01); c) the liver levels of apoB-100 mRNA (r = 0.83, p < 0.01) and serum apoB-100 concentrations (r = 0.87, p < 0.01). Clofibrate treatment was shown to concomitantly decrease the liver levels of HNF1α, HNF4α and PCSK9 mRNA, as well as serum PCSK9, TAGs and total cholesterol concentrations in CRF rats. Conclusion The results presented are consistent with a cause-effect relationship between the enhanced liver expression of Hnf1α gene and its target genes the products of which are involved in synthesis, assembly and secretion of VLDL, as well as Pcsk9 gene in CRF rats. This may at least in part explain an association between circulating PCSK9 and TAGs in CRF rats and possibly also in humans with chronic kidney disease (CKD).  

3.2475           Annexin A2 Limits Neutrophil Transendothelial Migration by Organizing the Spatial Distribution of ICAM-1

Heemskerk, N., Asimuddin, A., Oort, c., van Rijssel, J. and van Buul, J.D.

1. Immunol., 196(6), 2767-2778 (2016)

  ICAM-1 is required for firm adhesion of leukocytes to the endothelium. However, how the spatial organization of endothelial ICAM-1 regulates leukocyte adhesion is not well understood. In this study, we identified the calcium-effector protein annexin A2 as a novel binding partner for ICAM-1. ICAM-1 clustering promotes the ICAM-1–annexin A2 interaction and induces translocation of ICAM-1 into caveolin-1–rich membrane domains. Depletion of endothelial annexin A2 using RNA interference enhances ICAM-1 membrane mobility and prevents the translocation of ICAM-1 into caveolin-1–rich membrane domains. Surprisingly, this results in increased neutrophil adhesion and transendothelial migration under flow conditions and reduced crawling time, velocity, and lateral migration distance of neutrophils on the endothelium. In conclusion, our data show that annexin A2 limits neutrophil transendothelial migration by organizing the spatial distribution of ICAM-1.  

3.2476           Disturbance of proteasomal and autophagic protein degradation pathways by amyotrophic lateral sclerosis-linked mutations in ubiquilin 2

Osaka, M., Ito, D. and Suzuki, N. Biochem. Biophys. Res. Comm., 1472, 324-331 (2016)   Ubiquilin (UBQLN), a member of the ubiquitin-like (UBL)-ubiquitin-associated (UBA) family, is a dual regulator of both the proteasomal and autophagic branches of the cellular protein degradation system. Mutations in the UBQLN2 gene encoding ubiquilin 2 cause X-linked amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), and UBQLN2-positive inclusions have been identified in ALS patients with UBQLN2 mutations as well as in cases of both familial and sporadic ALS without UBQLN2 mutations. Compelling evidence links UBQLN2 to disturbance of the protein quality control network in neurons, but the pathomechanisms remain obscure. This study aimed to clarify how ALS-linked mutations in UBQLN2 affect the protein degradation system. Overexpression of a UBQLN2 with ALS-associated mutations resulted in the accumulation of polyubiquitinated proteins in neuronal cells, including the ALS-associated protein TDP-43. This effect was dependent on the UBA domain but not on inclusion formation. Immunocytochemistry and protein fractionation analysis of IVm-UBQLN2 cellular distribution indicated that it sequesters ubiquitinated substrates from both the proteasomal and autophagic branches of the protein degradation system, resulting in accumulation of polyubiquitinated substrates. These findings provide a molecular basis for the development of ALS/FTD-associated proteinopathy and establish novel therapeutic targets for ALS.  

3.2477           Enterovirus 71 induces dsRNA/PKR-dependent cytoplasmic redistribution of GRP78/BiP to promote viral replication

Jheng, J-R., Wang, S-C., Jheng, C-R. and Horng, J-T. Emerging Microbes and Infections, 5, e23 (2016)   GRP78/BiP is an endoplasmic reticulum (ER) chaperone protein with the important function of maintaining ER homeostasis, and the overexpression of GRP78/BiP alleviates ER stress. Our previous studies showed that infection with enterovirus 71 (EV71), a (+)RNA picornavirus, induced GRP78/BiP upregulation; however, ectopic GRP78/BiP overexpression in ER downregulates virus replication and viral particle formation. The fact that a virus infection increases GRP78/BiP expression, which is unfavorable for virus replication, is counterintuitive. In this study, we found that the GRP78/BiP protein level was elevated in the cytoplasm instead of in the ER in EV71-infected cells. Cells transfected with polyinosinic–polycytidylic acid, a synthetic analog of replicative double-stranded RNA (dsRNA), but not with viral proteins, also exhibited upregulation and elevation of GRP78/BiP in the cytosol. Our results further demonstrate that EV71 infections induce the dsRNA/protein kinase R-dependent cytosolic accumulation of GRP78/BiP. The overexpression of a GRP78/BiP mutant lacking a KDEL retention signal failed to inhibit both dithiothreitol-induced eIF2α phosphorylation and viral replication in the context of viral protein synthesis and viral titers. These data revealed that EV71 infection might cause upregulation and aberrant redistribution of GRP78/BiP to the cytosol, thereby facilitating virus replication.  

3.2478           Residual matrix from different separation techniques impacts exosome biological activity

Paolini, L., Zendrini, A., Di Noto, G., Busatto, S., Lottini, E., Radeghieri, A., Dossi, A., Caneschi, A., Ricotta, D. and Bergese, P. Scientific Reports, 6:23550 (2016)   Exosomes are gaining a prominent role in research due to their intriguing biology and several therapeutic opportunities. However, their accurate purification from body fluids and detailed physicochemical characterization remain open issues. We isolated exosomes from serum of patients with Multiple Myeloma by four of the most popular purification methods and assessed the presence of residual contaminants in the preparations through an ad hoc combination of biochemical and biophysical techniques - including Western Blot, colloidal nanoplasmonics, atomic force microscopy (AFM) and scanning helium ion microscopy (HIM). The preparations obtained by iodixanol and sucrose gradients were highly pure. To the contrary, those achieved with limited processing (serial centrifugation or one step precipitation kit) resulted contaminated by a residual matrix, embedding the exosomes. The contaminated preparations showed lower ability to induce NfkB nuclear translocation in endothelial cells with respect to the pure ones, probably because the matrix prevents the interaction and fusion of the exosomes with the cell membrane. These findings suggest that exosome preparation purity must be carefully assessed since it may interfere with exosome biological activity. Contaminants can be reliably probed only by an integrated characterization approach aimed at both the molecular and the colloidal length scales.  

3.2479           SGEF Is Regulated via TWEAK/Fn14/NF-κB Signaling and Promotes Survival by Modulation of the DNA Repair Response to Temozolomide

Ensign, S.P.F., Roos, A., Mathews, I.T., Dhruv, H.D., Tuncali, S., Sarkaria, J.N., Symons, M.H., Loftus, J.C., Berens, M.E. and Tran, N.L.

Mol. Cancer Res., 14(3), 302-312 (2016)

Glioblastoma (GB) is the highest grade and most common form of primary adult brain tumors. Despite surgical removal followed by concomitant radiation and chemotherapy with the alkylating agent temozolomide, GB tumors develop treatment resistance and ultimately recur. Impaired response to treatment occurs rapidly, conferring a median survival of just fifteen months. Thus, it is necessary to identify the genetic and signaling mechanisms that promote tumor resistance to develop targeted therapies to combat this refractory disease. Previous observations indicated that SGEF (ARHGEF26), a RhoG-specific guanine nucleotide exchange factor (GEF), is overexpressed in GB tumors and plays a role in promoting TWEAK-Fn14–mediated glioma invasion. Here, further investigation revealed an important role for SGEF in glioma cell survival. SGEF expression is upregulated by TWEAK-Fn14 signaling via NF-κB activity while shRNA-mediated reduction of SGEF expression sensitizes glioma cells to temozolomide-induced apoptosis and suppresses colony formation following temozolomide treatment. Nuclear SGEF is activated following temozolomide exposure and complexes with the DNA damage repair (DDR) protein BRCA1. Moreover, BRCA1 phosphorylation in response to temozolomide treatment is hindered by SGEF knockdown. The role of SGEF in promoting chemotherapeutic resistance highlights a heretofore unappreciated driver, and suggests its candidacy for development of novel targeted therapeutics for temozolomide-refractory, invasive GB cells.

 

3.2480           Vesicle Size Regulates Nanotube Formation in the Cell

Su, Q.P., Su, W., Ji, Q., Xue, B., Jiang, D., Zhu, Y., Lou, J., Yu, L. and Sun, Y. Scientific Reports, 6:24002 (2016)   Intracellular membrane nanotube formation and its dynamics play important roles for cargo transportation and organelle biogenesis. Regarding the regulation mechanisms, while much attention has been paid on the lipid composition and its associated protein molecules, effects of the vesicle size has not been studied in the cell. Giant unilamellar vesicles (GUVs) are often used for in vitro membrane deformation studies, but they are much larger than most intracellular vesicles and the in vitro studies also lack physiological relevance. Here, we use lysosomes and autolysosomes, whose sizes range between 100 nm and 1 μm, as model systems to study the size effects on nanotube formation both in vivo and in vitro. Single molecule observations indicate that driven by kinesin motors, small vesicles (100–200 nm) are mainly transported along the tracks while a remarkable portion of large vesicles (500–1000 nm) form nanotubes. This size effect is further confirmed by in vitro reconstitution assays on liposomes and purified lysosomes and autolysosomes. We also apply Atomic Force Microscopy (AFM) to measure the initiation force for nanotube formation. These results suggest that the size-dependence may be one of the mechanisms for cells to regulate cellular processes involving membrane-deformation, such as the timing of tubulation-mediated vesicle recycling.  

3.2481           Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens

Cecil, J.D., O’Brien-Simpson, N., Lenzo, J.C., Holden, J.A., Chen, Y-Y., Singleton, W., gause, K.T., Yan, Y., Caruso, F. and Reynolds, E.C. PloS One, 11(4), e0151967 (2016)   Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis.  

3.2482           Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology

Lantos, A.B., Carlevaro, G., Araoz, B., Diaz, P.R., de los Milagros Camara, M., Buscaglia, C.A., Bossi, M., Yu, H., Chen, X., Bertozzi, C.R., Mucci, J. and Campetella, O. PloS Pathogens, 12(4), e1005550 (2016)   Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form.  

3.2483           Extracellular vesicles during Herpes Simplex Virus type 1 infection: an inquire

Kalamvoki, M. and Deschamps, T. Virol. J., 13:63 (2016)   Extracellular vesicles are defined as a heterogeneous group of vesicles that are released by prokaryotic to higher eukaryotic cells and by plant cells in an evolutionary conserved manner. The significance of these vesicles lies in their capacity to transfer selected cargo composed of proteins, lipids and nucleic acids to both recipient and parent cells and to influence various physiological and pathological functions. Microorganisms such as parasites, fungi and protozoa and even single cell organisms such as bacteria generate extracellular vesicles. In addition, several viruses have evolved strategies to hijack the extracellular vesicles for egress or to alter the surrounding environment. The thesis of this article is that: a) during HSV-1 infection vesicles are delivered from infected to uninfected cells that influence the infection; b) the cargo of these vesicles consists of viral and host transcripts (mRNAs, miRNAs and non-coding RNAs) and proteins including innate immune components, such as STING; and c) the viral vesicles carry the tetraspanins CD9, CD63 and CD81, which are considered as markers of exosomes. Therefore, we assume that the STING-carrying vesicles, produced during HSV-1 infection, are reminiscent to exosomes. The presumed functions of the exosomes released from HSV-1 infected cells include priming the recipient cells and accelerating antiviral responses to control the dissemination of the virus. This may be one strategy used by the virus to prevent the elimination by the host and establish persistent infection. In conclusion, the modification of the cargo of exosomes appears to be part of the strategy that HSV-1 has evolved to establish lifelong persistent infections into the human body to ensure successful dissemination between individuals.  

3.2484           Identification of the novel activity-driven interaction between synaptotagmin 1 and presenilin 1 links calcium, synapse, and amyloid beta

Kuzuya, A., Zoltowska, K.M., Post, K.L., Arimon, M., Li, X., Svirsky, S., Maesako, M., Muzikansky, A., Gautam, V., Kovacs, D., Hyman, T. and Berezovska, O. BMC Biology, 14:25 (2016)   Background Synaptic loss strongly correlates with memory deterioration. Local accumulation of amyloid β (Aβ) peptide, and neurotoxic Aβ42 in particular, due to abnormal neuronal activity may underlie synaptic dysfunction, neurodegeneration, and memory impairments. To gain an insight into molecular events underlying neuronal activity-regulated Aβ production at the synapse, we explored functional outcomes of the newly discovered calcium-dependent interaction between Alzheimer’s disease-associated presenilin 1 (PS1)/γ-secretase and synaptic vesicle proteins. Results Mass spectrometry screen of mouse brain lysates identified synaptotagmin 1 (Syt1) as a novel synapse-specific PS1-binding partner that shows Ca²⁺-dependent PS1 binding profiles in vitro and in vivo. We found that Aβ level, and more critically, conformation of the PS1 and the Aβ_42/40 ratio, are affected by Syt1 overexpression or knockdown, indicating that Syt1 and its interaction with PS1 might regulate Aβ production at the synapse. Moreover, β-secretase 1 (BACE1) stability, β- and γ-secretase activity, as well as intracellular compartmentalization of PS1 and BACE1, but not of amyloid precursor protein (APP), nicastrin (Nct), presenilin enhancer 2 (Pen-2), or synaptophysin (Syp) were altered in the absence of Syt1, suggesting a selective effect of Syt1 on PS1 and BACE1 trafficking. Conclusions Our findings identify Syt1 as a novel Ca²⁺-sensitive PS1 modulator that could regulate synaptic Aβ, opening avenues for novel and selective synapse targeting therapeutic strategies.  

3.2485           Sng1 associates with Nce102 to regulate the yeast Pkh-Ypk signalling module in response to sphingolipid status

Garcia-marques, S., Randez-Gil, F., Dupont, S., Garre, E. and Prieto, J.A.  Biochim. Biophys. Acta, 1863, 1319-1333 (2016)   All cells are delimited by biological membranes, which are consequently a primary target of stress-induced damage. Cold alters membrane functionality by decreasing lipids fluidity and the activity of membrane proteins. In Saccharomyces cerevisiae, evidence links sphingolipid homeostasis and membrane phospholipid asymmetry to the activity of the Ypk1/2 proteins, the yeast orthologous of the mammalian SGK1-3 kinases. Their regulation is mediated by different protein kinases, including the PDK1 orthologous Pkh1/2p, and requires the function of protein effectors, among them Nce102p, a component of the sphingolipid sensor machinery. Nevertheless, the mechanisms and the actors involved in Pkh/Ypk regulation remain poorly defined. Here, we demonstrate that Sng1, a transmembrane protein, is an effector of the Pkh/Ypk module and identify the phospholipids asymmetry as key for yeast cold adaptation. Overexpression of SNG1 impairs phospholipid flipping, reduces reactive oxygen species (ROS) and improves, in a Pkh-dependent manner, yeast growth in myriocin-treated cells, suggesting that excess Sng1p stimulates the Pkh/Ypk signalling. Furthermore, we link these effects to the association of Sng1p with Nce102p. Indeed, we found that Sng1p interacts with Nce102p both physically and genetically. Moreover, mutant nce102 ∆ sng1 ∆ cells show features of impaired Pkh/Ypk signalling, including increased ROS accumulation, reduced life span and defects in Pkh/Ypk-controlled regulatory pathways. Finally, myriocin-induced hyperphosphorylation of Ypk1 and Orm2, which controls sphingolipid homeostasis, does not occur in nce102 ∆ sng1 ∆ cells. Hence, both Nce102p and Sng1p participate in a regulatory circuit that controls the activity of the Pkh/Ypk module and their function is required in response to sphingolipid status.  

3.2486           Inflammatory properties of inhibitor of DNA binding 1 secreted by synovial fibroblasts in rheumatoid arthritis

Edhayan, G., Ohara, R.A., Stinson, W.A., Amin, M.A., Isozaki, T., Ha, C.M., Haines III, G.K., Morgan, R., Campbell, P.L., Arbab, A.S., Friday, S.C., Fox, D.A. and Ruth, J.H. Arthritis Res. & Therapy, 18:87 (2016)   Background Inhibitor of DNA binding 1 (Id1) is a nuclear protein containing a basic helix-loop-helix (bHLH) domain that regulates cell growth by selective binding and prevention of gene transcription. Sources of Id1 production in rheumatoid arthritis synovial tissue (RA ST) and its range of functional effects in RA remain to be clarified. Methods We analyzed Id1 produced from synovial fibroblasts and endothelial cells (ECs) with histology and real-time polymerase chain reaction (RT-PCR). Fibroblast supernatants subjected to differential centrifugation to isolate and purify exosomes were measured for Id1 by enzyme-linked immunosorbent assay (ELISA). Western blotting of Id1-stimulated ECs was performed to determine the kinetics of intracellular protein phosphorylation. EC intracellular signaling pathways induced by Id1 were subsequently targeted with silencing RNA (siRNA) for angiogenesis inhibition. Results By PCR and histologic analysis, we found that the primary source of Id1 in STs is from activated fibroblasts that correlate with inflammatory scores in human RA ST and in joints from K/BxN serum-induced mice. Normal (NL) and RA synovial fibroblasts increase Id1 production with stimulation by transforming growth factor beta (TGF-β). Most of the Id1 released by RA synovial fibroblasts is contained within exosomes. Endothelial progenitor cells (EPCs) and human dermal microvascular ECs (HMVECs) activate the Jnk signaling pathway in response to Id1, and Jnk siRNA reverses Id1-induced HMVEC vessel formation in Matrigel plugs in vivo. Conclusions Id1 is a pleotropic molecule affecting angiogenesis, vasculogenesis, and fibrosis. Our data shows that Id1 is not only an important nuclear protein, but also can be released from fibroblasts via exosomes. The ability of extracellular Id1 to activate signaling pathways expands the role of Id1 in the orchestration of tissue inflammation.  

3.2487           Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms

Turnbull, L. et al Nature Communications, 7:11220 (2016)   Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs.  

3.2488           Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection

Sodar, B.W. et al Scientific Reports, 6:24316 (2016)   Circulating extracellular vesicles have emerged as potential new biomarkers in a wide variety of diseases. Despite the increasing interest, their isolation and purification from body fluids remains challenging. Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, we found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. We identified most of these particles as lipoproteins (predominantly low-density lipoprotein, LDL) which mimicked the characteristics of extracellular vesicles and also co-purified with them. Based on biophysical properties of LDL this finding was highly unexpected. Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates. Furthermore, transmission electron microscopy showed an association of LDL with isolated vesicles upon in vitro mixing. This is the first study to show co-purification and in vitro association of LDL with extracellular vesicles and its interference with vesicle analysis. Our data point to the importance of careful study design and data interpretation in studies using blood-derived extracellular vesicles with special focus on potentially co-purified LDL.  

3.2489           Impairment of extramitochondrial oxidative phosphorylation in mouse rod outer segments by blue light irradiation

Calzia, D., Panfoli, I., heinig, N., Schumann, U., Ader, M., Traverso, C.E., Funk, R.H.W. and roehlecke, C. Biochimie, 125, 171-178 (2016)   Exposure to short wavelength light causes increased reactive oxygen intermediates production in the outer retina, particularly in the rod Outer Segments (OS). Consistently, the OS were shown to conduct aerobic ATP production through the ectopic expression of the electron transfer chain complexes I–IV and F₁F_o-ATP synthase. These facts prompted us to verify if the oxidative phosphorylation in the OS is implied in the oxidative damage of the blue-light (BL) treated OS, in an organotypic model of mouse retina. Whole mouse eyeball cultures were treated with short wavelength BL (peak at 405 nm, output power 1 mW/cm²) for 6 h. Immunogold transmission electron microscopy confirmed the expression of Complex I and F₁F_o-ATP synthase in the OS. In situ histochemical assays on unfixed sections showed impairment of respiratory Complexes I and II after BL exposure, both in the OS and IS, utilized as a control. Basal O₂ consumption and ATP synthesis were impaired in the OS purified from blue-light irradiated eyeball cultures. Electron transfer capacity between Complex I and II as well as activity of Complexes I and II was decreased in blue-light irradiated purified OS. The severe malfunctioning of the OS aerobic respiratory capacity after 6 h BL treatment may be the consequence of a self-induced damage. BL exposure would cause an initial over-functioning of both the phototransduction and respiratory chain, with reactive oxygen species production. In a self-renewal vicious cycle, membrane and protein oxidative damage, proton leakage and uncoupling, would impair redox chains, perpetuating the damage and causing hypo-metabolism with eventual apoptosis of the rod. Data may shed new light on the rod-driven retinopathies such as Age Related Macular Degeneration, of which blue-light irradiated retina represents a model.  

3.2490           Identification of CiaR Regulated Genes That Promote Group B Streptococcal Virulence and Interaction with Brain Endothelial Cells

Mu, R., Cutting, A.S., Del Rosario, Y., Villarino, N., Stewart, L., Weston, T.A., Patras, K.A. and Doran, K.S. PloS One, 11(4), e0153891 (2016)   Group B Streptococcus (GBS) is a major causative agent of neonatal meningitis due to its ability to efficiently cross the blood-brain barrier (BBB) and enter the central nervous system (CNS). It has been demonstrated that GBS can invade human brain microvascular endothelial cells (hBMEC), a primary component of the BBB; however, the mechanism of intracellular survival and trafficking is unclear. We previously identified a two component regulatory system, CiaR/H, which promotes GBS intracellular survival in hBMEC. Here we show that a GBS strain deficient in the response regulator, CiaR, localized more frequently with Rab5, Rab7 and LAMP1 positive vesicles. Further, lysosomes isolated from hBMEC contained fewer viable bacteria following initial infection with the ΔciaR mutant compared to the WT strain. To characterize the contribution of CiaR-regulated genes, we constructed isogenic mutant strains lacking the two most down-regulated genes in the CiaR-deficient mutant, SAN_2180 and SAN_0039. These genes contributed to bacterial uptake and intracellular survival. Furthermore, competition experiments in mice showed that WT GBS had a significant survival advantage over the Δ2180 and Δ0039 mutants in the bloodstream and brain.  

3.2491           Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions

Geri, M.J., Bittl, V., Kirchner, S., Sachenheimer, T., Brunner, H.L., Lüchtenborg, C., Özbalci, C., Wiedemann, H., Wegehingel, S., Nickel, w., haberkant, P., Schultz, C., Krüger, M. and Brügger, B. PloS One, 11(4), e0153009 (2016)   Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.  

3.2492           Phosphorylation of Ago2 and Subsequent Inactivation of let-7a RNP-Specific MicroRNAs Control Differentiation of Mammalian Sympathetic Neurons

Patranabis, S. and Bhattacharyya, S.N. Mol. Cell. Biol., 36(8), 1260-1271 (2016)   MicroRNAs (miRNAs) are small regulatory RNAs that regulate gene expression posttranscriptionally by base pairing to the target mRNAs in animal cells. KRas, an oncogene known to be repressed by let-7a miRNAs, is expressed and needed for the differentiation of mammalian sympathetic neurons and PC12 cells. We documented a loss of let-7a activity during this differentiation process without any significant change in the cellular level of let-7a miRNA. However, the level of Ago2, an essential component that is associated with miRNAs to form RNP-specific miRNA (miRNP) complexes, shows an increase with neuronal differentiation. In this study, differentiation-induced phosphorylation and the subsequent loss of miRNA from Ago2 were noted, and these accounted for the loss of miRNA activity in differentiating neurons. Neuronal differentiation induces the phosphorylation of mitogen-activated protein kinase p38 and the downstream kinase mitogen- and stress-activated protein kinase 1 (MSK1). This in turn upregulates the phosphorylation of Ago2 and ensures the dissociation of miRNA from Ago2 in neuronal cells. MSK1-mediated miRNP inactivation is a prerequisite for the differentiation of neuronal cells, where let-7a miRNA gets unloaded from Ago2 to ensure the upregulation of KRas, a target of let-7a. We noted that the inactivation of let-7a is both necessary and sufficient for the differentiation of sympathetic neurons.  

3.2493           P53- and mevalonate pathway–driven malignancies require Arf6 for metastasis and drug resistance

Hashimoto, A., Oikawa, T., Hashimoto, S., Sugino, H., Yoshikawa, A., Otsuka, Y., Handa, H., Onodera, Y., Nam, J-M., Oneyama, C., Okada, M., Fukuda, M. and Sabe, H.

1. Cell Biol., 213(1), 81-95 (2016)

  Drug resistance, metastasis, and a mesenchymal transcriptional program are central features of aggressive breast tumors. The GTPase Arf6, often overexpressed in tumors, is critical to promote epithelial–mesenchymal transition and invasiveness. The metabolic mevalonate pathway (MVP) is associated with tumor invasiveness and known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. We show here that MVP requires the Arf6-dependent mesenchymal program. The MVP enzyme geranylgeranyl transferase II (GGT-II) and its substrate Rab11b are critical for Arf6 trafficking to the plasma membrane, where it is activated by receptor tyrosine kinases. Consistently, mutant p53, which is known to support tumorigenesis via MVP, promotes Arf6 activation via GGT-II and Rab11b. Inhibition of MVP and GGT-II blocked invasion and metastasis and reduced cancer cell resistance against chemotherapy agents, but only in cells overexpressing Arf6 and components of the mesenchymal program. Overexpression of Arf6 and mesenchymal proteins as well as enhanced MVP activity correlated with poor patient survival. These results provide insights into the molecular basis of MVP-driven malignancy.  

3.2494           A branched-chain amino acid metabolite drives vascular fatty acid transport and causes insulin resistance

Jang, C. et al Nature Med., 22(4), 421-426 (2016)   Epidemiological and experimental data implicate branched-chain amino acids (BCAAs) in the development of insulin resistance, but the mechanisms that underlie this link remain unclear^{1, 2, 3}. Insulin resistance in skeletal muscle stems from the excess accumulation of lipid species⁴, a process that requires blood-borne lipids to initially traverse the blood vessel wall. How this trans-endothelial transport occurs and how it is regulated are not well understood. Here we leveraged PPARGC1a (also known as PGC-1α; encoded by Ppargc1a), a transcriptional coactivator that regulates broad programs of fatty acid consumption, to identify 3-hydroxyisobutyrate (3-HIB), a catabolic intermediate of the BCAA valine, as a new paracrine regulator of trans-endothelial fatty acid transport. We found that 3-HIB is secreted from muscle cells, activates endothelial fatty acid transport, stimulates muscle fatty acid uptake in vivo and promotes lipid accumulation in muscle, leading to insulin resistance in mice. Conversely, inhibiting the synthesis of 3-HIB in muscle cells blocks the ability of PGC-1α to promote endothelial fatty acid uptake. 3-HIB levels are elevated in muscle from db/db mice with diabetes and from human subjects with diabetes, as compared to those without diabetes. These data unveil a mechanism in which the metabolite 3-HIB, by regulating the trans-endothelial flux of fatty acids, links the regulation of fatty acid flux to BCAA catabolism, providing a mechanistic explanation for how increased BCAA catabolic flux can cause diabetes.  

3.2495           Ca2+-regulated lysosome fusion mediates angiotensin II-induced lipid raft clustering in mesenteric endothelial cells

Han, W-Q., Chen, W-D., Zhang, K., Liu, J-J., Wu, Y-J. and Gao, P-J. Hypertension Res., 39, 227-236 (2016)   It has been reported that intracellular Ca²⁺ is involved in lysosome fusion and membrane repair in skeletal cells. Given that angiotensin II (Ang II) elicits an increase in intracellular Ca²⁺ and that lysosome fusion is a crucial mediator of lipid raft (LR) clustering, we hypothesized that Ang II induces lysosome fusion and activates LR formation in rat mesenteric endothelial cells (MECs). We found that Ang II acutely increased intracellular Ca²⁺ content, an effect that was inhibited by the extracellular Ca²⁺ chelator ethylene glycol tetraacetic acid (EGTA) and the inositol 1,4,5-trisphosphate (IP3)-induced Ca²⁺ release inhibitor 2-aminoethoxydiphenyl borate (2-APB). Further study showed that EGTA almost completely blocked Ang II-induced lysosome fusion, the translocation of acid sphingomyelinase (ASMase) to LR clusters, ASMase activation and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase activation. In contrast, 2-APB had a slight inhibitory effect. Functionally, both the lysosome inhibitor bafilomycin A1 and the ASMase inhibitor amitriptyline reversed Ang II-induced impairment of vasodilation. We conclude that Ca²⁺-regulated lysosome fusion mediates the Ang II-induced regulation of the LR-redox signaling pathway and mesenteric endothelial dysfunction.  

3.2496           Vaginal epithelial cells regulate membrane adhesiveness to co-ordinate bacterial adhesion

Younes, J.A., Klappe, K., Kok, J.W., Busscher, H.J., Reid, G. and van der Mei, H.C. Cell. Microbiol., 18(4), 605-614 (2016)   Vaginal epithelium is colonized by different bacterial strains and species. The bacterial composition of vaginal biofilms controls the balance between health and disease. Little is known about the relative contribution of the epithelial and bacterial cell surfaces to bacterial adhesion and whether and how adhesion is regulated over cell membrane regions. Here, we show that bacterial adhesion forces with cell membrane regions not located above the nucleus are stronger than with regions above the nucleus both for vaginal pathogens and different commensal and probiotic lactobacillus strains involved in health. Importantly, adhesion force ratios over membrane regions away from and above the nucleus coincided with the ratios between numbers of adhering bacteria over both regions. Bacterial adhesion forces were dramatically decreased by depleting the epithelial cell membrane of cholesterol or sub-membrane cortical actin. Thus, epithelial cells can regulate membrane regions to which bacterial adhesion is discouraged, possibly to protect the nucleus.  

3.2497           Cellular cholesterol accumulation modulates high fat high sucrose (HFHS) diet-induced ER stress and hepatic inflammasome activation in the development of non-alcoholic steatohepatitis

Bashiri, A., Nesan, D., Tavallaee, G., Sue-Chue-Lam, I., Chien, K., maguire, G.F., Naples, M., Zhang, J., Magomedova, L., Adeli, K., Cummins, C.L. and Ng, D.S. Biocheim. Biophys. Acta, 1861, 594-605 (2016)   Non-alcoholic steatohepatitis (NASH), is the form of non-alcoholic fatty liver disease posing risk to progress into serious long term complications. Human and pre-clinical models implicate cellular cholesterol dysregulation playing important role in its development. Mouse model studies suggest synergism between dietary cholesterol and fat in contributing to NASH but the mechanisms remain poorly understood. Our laboratory previously reported the primary importance of hepatic endoplasmic reticulum cholesterol (ER-Chol) in regulating hepatic ER stress by comparing the responses of wild type, Ldlr −/− xLcat +/+ and Ldlr −/− xLcat −/− mice, to a 2% high cholesterol diet (HCD). Here we further investigated the roles of ER-Chol and ER stress in HFHS diet-induced NASH using the same strains. With HFHS diet feeding, both WT and Ldlr −/− xLcat +/+ accumulate ER-Chol in association with ER stress and inflammasome activation but the Ldlr −/− xLcat −/− mice are protected. By contrast, all three strains accumulate cholesterol crystal, in correlation with ER-Chol, albeit less so in Ldlr −/− xLcat −/− mice. By comparison, HCD feeding per se (i) is sufficient to promote steatosis and activate inflammasomes, and (ii) results in dramatic accumulation of cholesterol crystal which is linked to inflammasome activation in Ldlr −/− xLcat −/− mice, independent of ER-Chol. Our data suggest that both dietary fat and cholesterol each independently promote steatosis, cholesterol crystal accumulation and inflammasome activation through distinct but complementary pathways. In vitro studies using palmitate-induced hepatic steatosis in HepG2 cells confirm the key roles by cellular cholesterol in the induction of steatosis and inflammasome activations. These novel findings provide opportunities for exploring a cellular cholesterol-focused strategy for treatment of NASH.  

3.2498           Comparing exosome-like vesicles with liposomes for the functional cellular delivery of small RNAs

Stremersch, S., Vandenbroucke, R.E., Van Wonterghem, E., hendrix, A., De Smedt, S. and Raemdonck, K.

1. Controlled Release, 232, 51-61 (2016)

  Exosome-like vesicles (ELVs) play an important role in intercellular communication by acting as natural carriers for biomolecule transfer between cells. This unique feature rationalizes their exploitation as bio-inspired drug delivery systems. However, the therapeutic application of ELVs is hampered by the lack of efficient and reproducible drug loading methods, in particular for therapeutic macromolecules. To overcome this limitation, we present a generic method to attach siRNA to the surface of isolated ELVs by means of a cholesterol anchor. Despite a feasible uptake in both a dendritic and lung epithelial cell line, B16F10- and JAWSII-derived ELVs were unable to functionally deliver the associated small RNAs, neither exogenous cholesterol-conjugated siRNA nor endogenous miRNA derived from the melanoma producer cell. The latter results were confirmed both for purified ELVs and ELVs delivered via a transwell co-culture set-up. In contrast, simple anionic fusogenic liposomes were able to induce a marked siRNA-mediated gene knockdown under equal experimental conditions, both indicating successful cytosolic delivery of surface-bound cholesterol-conjugated siRNA and further underscoring the incapacity of the here evaluated ELVs to guide cytosolic delivery of small RNAs. In conclusion, we demonstrate that a more in-depth understanding of the biomolecular delivery mechanism and specificity is required before ELVs can be envisioned as a generic siRNA carrier.  

3.2499           Identification of TBK1 and IKKε, the non-canonical IκB kinases, as crucial pro-survival factors in HTLV-1-transformed T lymphocytes

Hang, H., Chen, L., Cai, S-H. and Cheng, H. Leukemia Res., 46, 37-44 (2016)   Persistent activation of NF-κB is a prerequisite for development of adult T cell leukemia-lymphoma (ATL) caused by human T cell leukemia virus type 1 (HTLV-1). HTLV-1 genome encodes a viral transforming protein named Tax, which constitutively activates the canonical IκB kinases (IKK), the central regulator of NF-κB signaling. However, the role of the non-canonical IκB kinases, TBK1 and IKKε, in the pathogenesis of HTLV-1-associated leukemia has not been evaluated. We here show that TBK1/IKKε are crucial pro-survival molecules by maintaining persistent activity of Stat3. Consistent with this finding, silencing Stat3 by the specific shRNA or by the chemical inhibitor ruxolitinib results in drastic impediment of leukemia cell growth. We further find that in HTLV-1-transformed T cells expressing Tax, TBK1 co-localizes with the canonical IκB kinases and Tax in the lipid raft microdomains. The wild type Tax, but not the Tax mutant defective in activating the canonical IKK, promotes the lipid raft translocation of TBK1. This phenomenon correlates with Tax activation of both NF-κB and Stat3. Tax does not interact directly with TBK1/IKKε, and it rather engages a molecular crosstalk between the canonical IKKs and TBK1/IKKε. Our data, therefore, demonstrate a key role of TBK1/IKKε in the survival and proliferation of HTLV-1-transformed T cells and implicate a potential therapy targeting TBK1/IKKε and Stat3 in controlling HTLV-1-mediated oncogenesis.  

3.2500           Localization and signaling of GPCRs in lipid rafts

Villar, Van Anthony M., Cuevas, S., Zheng, X. and Jose, P.A. Methods in Cell Biol., 132, 3-23 (2016)   The understanding of how biological membranes are organized and how they function has evolved. Instead of just serving as a medium in which certain proteins are found, portions of the lipid bilayer have been demonstrated to form specialized platforms that foster the assembly of signaling complexes by providing a microenvironment that is conducive for effective protein–protein interactions. G protein-coupled receptors (GPCRs) and relevant signaling molecules, including the heterotrimeric G proteins, key enzymes such as kinases and phosphatases, trafficking proteins, and secondary messengers, preferentially partition to these highly organized cell membrane microdomains, called lipid rafts. As such, lipid rafts are crucial for the trafficking and signaling of GPCRs. The study of GPCR biology in the context of lipid rafts involves the localization of the GPCR of interest in lipid rafts, at the basal state and upon receptor agonism, and the evaluation of the biological functions of the GPCR in appropriate cell lines. The lack of standardized methodology to study lipid rafts, in general, and of the workings of GPCRs in lipid rafts, in particular, and the inherent drawbacks of current methods have hampered the complete understanding of the underlying molecular mechanisms. Newer methodologies that allow the study of GPCRs in their native form are needed. The use of complementary approaches that produce mutually supportive results appear to be the best way for drawing conclusions with regards to the distribution and activity of GPCRs in lipid rafts.  

3.2501           Cytotoxic and Inflammatory Responses Induced by Outer Membrane Vesicle-Associated Biologically Active Proteases from Vibrio cholera

Mondal, A., Tapader, R., Chatterjee, N.S.C., Ghosh, A., Sinha, R., Koley, H., Saha, D.R., Chakrabarti, M.K., Wai, S.N. and Pal, A. Infect. Immun. 84(5), 1478-1490 (2016)   Proteases in Vibrio cholerae have been shown to play a role in its pathogenesis. V. cholerae secretes Zn-dependent hemagglutinin protease (HAP) and calcium-dependent trypsin-like serine protease (VesC) by using the type II secretion system (TIISS). Our present studies demonstrated that these proteases are also secreted in association with outer membrane vesicles (OMVs) and transported to human intestinal epithelial cells in an active form. OMV-associated HAP induces dose-dependent apoptosis in Int407 cells and an enterotoxic response in the mouse ileal loop (MIL) assay, whereas OMV-associated VesC showed a hemorrhagic fluid response in the MIL assay, necrosis in Int407 cells, and an increased interleukin-8 (IL-8) response in T84 cells, which were significantly reduced in OMVs from VesC mutant strain. Our results also showed that serine protease VesC plays a role in intestinal colonization of V. cholerae strains in adult mice. In conclusion, our study shows that V. cholerae OMVs secrete biologically active proteases which may play a role in cytotoxic and inflammatory responses.  

3.2502           Membrane-association of mRNA decapping factors is independent of stress in budding yeast

Huch, S., Gommlich, J., Muppavarapu, M., Beckham, C. and Nissan, T. Scientific Reports, 6:25477 (2016)   Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation.  

3.2503           Trehalose Alters Subcellular Trafficking and the Metabolism of the Alzheimer-associated Amyloid Precursor Protein

Tien, N.T., karaca, I., Tamboli, I.Y. and Walter, J.

1. Biol Chem., 291(20), 10528-10540 (2016)

  The disaccharide trehalose is commonly considered to stimulate autophagy. Cell treatment with trehalose could decrease cytosolic aggregates of potentially pathogenic proteins, including mutant huntingtin, α-synuclein, and phosphorylated tau that are associated with neurodegenerative diseases. Here, we demonstrate that trehalose also alters the metabolism of the Alzheimer disease-related amyloid precursor protein (APP). Cell treatment with trehalose decreased the degradation of full-length APP and its C-terminal fragments. Trehalose also reduced the secretion of the amyloid-β peptide. Biochemical and cell biological experiments revealed that trehalose alters the subcellular distribution and decreases the degradation of APP C-terminal fragments in endolysosomal compartments. Trehalose also led to strong accumulation of the autophagic marker proteins LC3-II and p62, and decreased the proteolytic activation of the lysosomal hydrolase cathepsin D. The combined data indicate that trehalose decreases the lysosomal metabolism of APP by altering its endocytic vesicular transport.  

3.2504           MAL Is a Regulator of the Recruitment of Myelin Protein PLP to Membrane Microdomains

Bijlard, M., de Jonge, J.C., Klunder, B., Nomden, A., Hoekstra, D. and Baron, W. PloS One, 11(5), e0155317 (2016)   In oligodendrocytes (OLGs), an indirect, transcytotic pathway is mediating transport of de novo synthesized PLP, a major myelin specific protein, from the apical-like plasma membrane to the specialized basolateral-like myelin membrane to prevent its premature compaction. MAL is a well-known regulator of polarized trafficking in epithelial cells, and given its presence in OLGs it was therefore of interest to investigate whether MAL played a similar role in PLP transport in OLGs, taking into account its timely expression in these cells. Our data revealed that premature expression of mCherry-MAL in oligodendrocyte progenitor cells interfered with terminal OLG differentiation, although myelin membrane formation per se was not impaired. In fact, also PLP transport to myelin membranes via the cell body plasma membrane was unaffected. However, the typical shift of PLP from TX-100-insoluble membrane domains to CHAPS-resistant, but TX-100-soluble membrane domains, seen in the absence of MAL expression, is substantially reduced upon expression of the MAL protein. Interestingly, not only in vitro, but also in developing brain a strongly diminished shift from TX-100 resistant to TX-100 soluble domains was observed. Consistently, the MAL-expression mediated annihilation of the typical membrane microdomain shift of PLP is also reflected by a loss of the characteristic surface expression profile of conformation-sensitive anti-PLP antibodies. Hence, these findings suggest that MAL is not involved in vesicular PLP trafficking to either the plasma membrane and/or the myelin membrane as such. Rather, we propose that MAL may regulate PLP’s distribution into distinct membrane microdomains that allow for lateral diffusion of PLP, directly from the plasma membrane to the myelin membrane once the myelin sheath has been assembled.  

3.2505           Blood-Based Biomarkers for Metabolic Syndrome

O’Neill, S., Bphl, M., Gregersen, S., Hermansen, K. and O’Driscoll, L. Trends in Endocrinology & Metabolism, 27(6), 363-374 (2016)   Metabolic syndrome (MetS) is a constellation of factors increasing the risk of type 2 diabetes mellitus (T2DM), cardiovascular disease (CVD), and cancer. MetS diagnosis is cumbersome and the precise diagnosis differs throughout the world. Efforts are underway to find MetS biomarkers that could all be analysed in a single blood sample. Here we review recent advances, including progress on circulating exosomes and microvesicles and their molecular contents, as well as DNA, RNAs, and proteins taken directly from blood samples. While additional research is now warranted to advance upon these findings, there is reason for optimising that such blood-based entities will be beneficial for MetS diagnosis and will help reduce risk of T2DM, CVD, and cancers, contributing both societal and economic benefit.  

3.2506           ABCG1 and ABCG4 Suppress γ-Secretase Activity and Amyloid β Production

Sano, O., Tsujita, M., Shimizu, Y., Kato, R., Kobayashi, A., Kioka, N., Remaley, A.T., Michikawa, M., Ueda, K. and Matsuo, M. PloS One, 11(5), e0155400 (2016)   ATP-binding cassette G1 (ABCG1) and ABCG4, expressed in neurons and glia in the central nervous system, mediate cholesterol efflux to lipid acceptors. The relationship between cholesterol level in the central nervous system and Alzheimer’s disease has been reported. In this study, we examined the effects of ABCG1 and ABCG4 on amyloid precursor protein (APP) processing, the product of which, amyloid β (Aβ), is involved in the pathogenesis of Alzheimer’s disease. Expression of ABCG1 or ABCG4 in human embryonic kidney 293 cells that stably expressed Swedish-type mutant APP increased cellular and cell surface APP levels. Products of cleavage from APP by α-secretase and by β-secretase also increased. The levels of secreted Aβ, however, decreased in the presence of ABCG1 and ABCG4, but not ABCG4-KM, a nonfunctional Walker-A lysine mutant. In contrast, secreted Aβ levels increased in differentiated SH-SY5Y neuron-like cells in which ABCG1 and ABCG4 were suppressed. Furthermore, Aβ42 peptide in the cerebrospinal fluid from Abcg1 null mice significantly increased compared to the wild type mice. To examine the underlying mechanism, we analyzed the activity and distribution of γ-secretase. ABCG1 and ABCG4 suppressed γ-secretase activity and disturbed γ-secretase localization in the raft domains where γ-secretase functions. These results suggest that ABCG1 and ABCG4 alter the distribution of γ-secretase on the plasma membrane, leading to the decreased γ-secretase activity and suppressed Aβ secretion. ABCG1 and ABCG4 may inhibit the development of Alzheimer’s disease and can be targets for the treatment of Alzheimer’s disease.  

3.2507           Do Src Kinase and Caveolin Interact Directly with Na,K-ATPase?

Yosef, E., Katz, A., Peleg, Y., Mehlman, T. and Karlish, J.D:

1. Biol. Chem., 291(22), 11736-11750 (2016)

  Much evidence points to a role of Na,K-ATPase in ouabain-dependent signal transduction. Based on experiments with different cell lines and native tissue membranes, a current hypothesis postulates direct interactions between the Na,K-ATPase and Src kinase (non-receptor tyrosine kinase). Na,K-ATPase is proposed to bind Src kinase and inhibit its activity, whereas ouabain, the specific Na,K-ATPase inhibitor, binds and stabilizes the E2 conformation, thus exposing the Src kinase domain and its active site Tyr-418 for activation. Ouabain-dependent signaling is thought to be mediated within caveolae by a complex consisting of Na,K-ATPase, caveolin, and Src kinase. In the current work, we have looked for direct interactions utilizing purified recombinant Na,K-ATPase (human α1β1FXYD1 or porcine α1D369Nβ1FXYD1) and purified human Src kinase and human caveolin 1 or interactions between these proteins in native membrane vesicles isolated from rabbit kidney. By several independent criteria and techniques, no stable interactions were detected between Na,K-ATPase and purified Src kinase. Na,K-ATPase was found to be a substrate for Src kinase phosphorylation at Tyr-144. Clear evidence for a direct interaction between purified human Na,K-ATPase and human caveolin was obtained, albeit with a low molar stoichiometry (1:15–30 caveolin 1/Na,K-ATPase). In native renal membranes, a specific caveolin 1_4–5 oligomer (95 kDa) was found to be in direct interaction with Na,K-ATPase. We inferred that a small fraction of the renal Na,K-ATPase molecules is in a ∼1:1 complex with a caveolin 1_4–5 oligomer. Thus, overall, whereas a direct caveolin 1/Na,K-ATPase interaction is confirmed, the lack of direct Src kinase/Na,K-ATPase binding requires reassessment of the mechanism of ouabain-dependent signaling.  

3.2508           Preparation of Gap Junctions in Membrane Microdomains for Immunoprecipitation and Mass Spectrometry Interactome Analysis

Fowler, S., Akins, M. and Bennett, S.A.L. Methods in Mol. Biol., 1437, 113-132 (2016)   Protein interaction networks at gap junction plaques are increasingly implicated in a variety of intracellular signaling cascades. Identifying protein interactions of integral membrane proteins is a valuable tool for determining channel function. However, several technical challenges exist. Subcellular fractionation of the bait protein matrix is usually required to identify less abundant proteins in complex homogenates. Sufficient solvation of the lipid environment without perturbation of the protein interactome must also be achieved. The present chapter describes the flotation of light and heavy liver tissue membrane microdomains to facilitate the identification and analysis of endogenous gap junction proteins and includes technical notes for translation to other integral membrane proteins, tissues, or cell culture models. These procedures are valuable tools for the enrichment of gap junction membrane compartments and for the identification of gap junction signaling interactomes.  

3.2509           Lysosomal cholesterol accumulation in macrophages leading to coronary atherosclerosis in CD38−/− mice

Xu, X., Yuan, X., Li, N., Dewey, W.L., Li, P-L. and Zhang, F.

1. Cell. Mol. Med., 20(6), 1001-1013 (2016)

  The disruption in transportation of oxLDL-derived cholesterol and the subsequent lipid accumulation in macrophages are the hallmark events in atherogenesis. Our recent studies demonstrated that lysosomal Ca²⁺ messenger of nicotinic acid adenine dinucleotide phosphate (NAADP), an enzymatic product of CD38 ADP-ribosylcyclase (CD38), promoted lipid endocytic trafficking in human fibroblast cells. The current studies are designed to examine the functional role of CD38/NAADP pathway in the regulation of lysosomal cholesterol efflux in atherosclerosis. Oil red O staining showed that oxLDL concentration-dependently increased lipid buildup in bone marrow-derived macrophages from both wild type and CD38^−/−, but to a significant higher extent with CD38 gene deletion. Bodipy 493/503 fluorescence staining found that the deposited lipid in macrophages was mainly enclosed in lysosomal organelles and largely enhanced with the blockade of CD38/NAADP pathway. Filipin staining and direct measurement of lysosome fraction further revealed that the free cholesterol constituted a major portion of the total cholesterol segregated in lysosomes. Moreover, in situ assay disclosed that both lysosomal lumen acidity and the acid lipase activity were reduced upon cholesterol buildup in lysosomes. In CD38^−/− mice, treatment with Western diet (12 weeks) produced atherosclerotic damage in coronary artery with striking lysosomal cholesterol sequestration in macrophages. These data provide the first experimental evidence that the proper function of CD38/NAADP pathway plays an essential role in promoting free cholesterol efflux from lysosomes and that a defection of this signalling leads to lysosomal cholesterol accumulation in macrophages and results in coronary atherosclerosis in CD38^−/− mice.  

3.2510           Demonstration of an oligosaccharide-diphosphodolichol diphosphatase activity whose subcellular localization is different than those of dolichyl-phosphate-dependent enzymes of the dolichol cycle

Massarweh, A., Bosco, M., iatmanen-harbi, S., tessier, C., Auberger, N., Busca, P., Chantret, I., Gravier-Pelletier, C. and Moore, S.E.H.

1. Lipid Res., 57, 1029-1042 (2016)

  Oligosaccharyl phosphates (OSPs) are hydrolyzed from oligosaccharide-diphosphodolichol (DLO) during protein N-glycosylation by an uncharacterized process. An OSP-generating activity has been reported in vitro, and here we asked if its biochemical characteristics are compatible with a role in endoplasmic reticulum (ER)-situated DLO regulation. We demonstrate a Co²⁺-dependent DLO diphosphatase (DLODP) activity that splits DLO into dolichyl phosphate and OSP. DLODP has a pH optimum of 5.5 and is inhibited by vanadate but not by NaF. Polyprenyl diphosphates inhibit [³H]OSP release from [³H]DLO, the length of their alkyl chains correlating positively with inhibition potency. The diphosphodiester GlcNAc₂-PP-solanesol is hydrolyzed to yield GlcNAc₂-P and inhibits [³H]OSP release from [³H]DLO more effectively than the diphosphomonoester solanesyl diphosphate. During subcellular fractionation of liver homogenates, DLODP codistributes with microsomal markers, and density gradient centrifugation revealed that the distribution of DLODP is closer to that of Golgi apparatus-situated UDP-galactose glycoprotein galactosyltransferase than those of dolichyl-P-dependent glycosyltransferases required for DLO biosynthesis in the ER. Therefore, a DLODP activity showing selectivity toward lipophilic diphosphodiesters such as DLO, and possessing properties distinct from other lipid phosphatases, is identified. Separate subcellular locations for DLODP action and DLO biosynthesis may be required to prevent uncontrolled DLO destruction.  

3.2511           Phosphatidylinositol-3-phosphate is light-regulated and essential for survival in retinal rods

He, F., Agosto, M.A., Anastassov, I.A., Tse, D.Y., Wu, S.M. and Wensel, T.G. Scientific Reports, 6:26978 (2016)   Phosphoinositides play important roles in numerous intracellular membrane pathways. Little is known about the regulation or function of these lipids in rod photoreceptor cells, which have highly active membrane dynamics. Using new assays with femtomole sensitivity, we determined that whereas levels of phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate were below detection limits, phosphatidylinositol-3-phosphate (PI(3)P) levels in rod inner/outer segments increased more than 30-fold after light exposure. This increase was blocked in a rod-specific knockout of the PI-3 kinase Vps34, resulting in failure of endosomal and autophagy-related membranes to fuse with lysosomes, and accumulation of abnormal membrane structures. At early ages, rods displayed normal morphology, rhodopsin trafficking, and light responses, but underwent progressive neurodegeneration with eventual loss of both rods and cones by twelve weeks. The degeneration is considerably faster than in rod knockouts of autophagy genes, indicating defects in endosome recycling or other PI(3)P-dependent membrane trafficking pathways are also essential for rod survival.  

3.2512           Adjuvant-Loaded Subcellular Vesicles Derived From Disrupted Cancer Cells for Cancer Vaccination

Cheung, A.S., Koshy, S.T., Stafford, A.G., Bastings, M.M.C. and Mooney, D.J. Small, 12(17), 2321-2333 (2016)   Targeted subunit vaccines for cancer immunotherapy do not capture tumor antigenic complexity, and approaches employing tumor lysate are often limited by inefficient antigen uptake and presentation, and low immunogenicity. Here, whole cancer cells are processed to generate antigen-rich, membrane-enclosed subcellular particles, termed “reduced cancer cells”, that reflect the diversity and breadth of the parent cancer cell antigen repertoire, and can be loaded with disparate adjuvant payloads. These vesicular particles enhance the uptake of the adjuvant payload, and potentiate the activation of primary dendritic cells in vitro. Similarly, reduced cancer cell-associated antigens are more efficiently presented by primary dendritic cells in vitro than their soluble counterparts or lysate control. In mice, vaccination using adjuvant-loaded reduced cancer cells facilitates the induction of antigen-specific cellular and humoral immune responses. Taken together, these observations demonstrate that adjuvant-loaded reduced cancer cells could be utilized in cancer vaccines as an alternative to lysate.  

3.2513           Enhancement of β-catenin activity by BIG1 plus BIG2 via Arf activation and cAMP signals

Li, C-C., Le, K., kato, J., Moss, J. and Vaughan, M. PNAS, 113(21), 5946-5941 (2016)   Multifunctional β-catenin, with critical roles in both cell–cell adhesion and Wnt-signaling pathways, was among HeLa cell proteins coimmunoprecipitated by antibodies against brefeldin A-inhibited guanine nucleotide-exchange factors 1 and 2 (BIG1 or BIG2) that activate ADP-ribosylation factors (Arfs) by accelerating the replacement of bound GDP with GTP. BIG proteins also contain A-kinase anchoring protein (AKAP) sequences that can act as scaffolds for multimolecular assemblies that facilitate and limit cAMP signaling temporally and spatially. Direct interaction of BIG1 N-terminal sequence with β-catenin was confirmed using yeast two-hybrid assays and in vitro synthesized proteins. Depletion of BIG1 and/or BIG2 or overexpression of guanine nucleotide-exchange factor inactive mutant, but not wild-type, proteins interfered with β-catenin trafficking, leading to accumulation at perinuclear Golgi structures. Both phospholipase D activity and vesicular trafficking were required for effects of BIG1 and BIG2 on β-catenin activation. Levels of PKA-phosphorylated β-catenin S675 and β-catenin association with PKA, BIG1, and BIG2 were also diminished after BIG1/BIG2 depletion. Inferring a requirement for BIG1 and/or BIG2 AKAP sequence in PKA modification of β-catenin and its effect on transcription activation, we confirmed dependence of S675 phosphorylation and transcription coactivator function on BIG2 AKAP-C sequence.  

3.2514           Extracellular vesicles in cardiovascular disease: are they Jedi or Sith?

Osteikoetxea, X., Nemeth, A., Sodar, B.W., Vukman, K.V. and Buzas, E.I.

1. Physiol., 594(11), 2881-2894 (2016)

  In the recent past, extracellular vesicles have become recognized as important players in cell biology and biomedicine. Extracellular vesicles, including exosomes, microvesicles and apoptotic bodies, are phospholipid bilayer-enclosed structures found to be secreted by most if not all cells. Extracellular vesicle secretion represents a universal and highly conserved active cellular function. Importantly, increasing evidence supports that extracellular vesicles may serve as biomarkers and therapeutic targets or tools in human diseases. Cardiovascular disease undoubtedly represents one of the most intensely studied and rapidly growing areas of the extracellular vesicle field. However, in different studies related to cardiovascular disease, extracellular vesicles have been shown to exert diverse and sometimes discordant biological effects. Therefore, it might seem a puzzle whether these vesicles are in fact beneficial or detrimental to cardiovascular health. In this review we provide a general introduction to extracellular vesicles and an overview of their biological roles in cardiovascular diseases. Furthermore, we aim to untangle the various reasons for the observed discrepancy in biological effects of extracellular vesicles in cardiovascular diseases. To this end, we provide several examples that demonstrate that the observed functional diversity is in fact due to inherent differences among various types of extracellular vesicles.  

3.2515           Sequential steps of macroautophagy and chaperone-mediated autophagy are involved in the irreversible process of posterior silk gland histolysis during metamorphosis of Bombyx mori

Shiba, H., yabu, T., Sudayama, M., Mano, N., Arai, N., Nakanishi, T. and Hosono, K.

1. Exp. Biol., 219, 1146-1153 (2016)

  To elucidate the degradation process of the posterior silk gland during metamorphosis of the silkworm Bombyx mori, tissues collected on the 6th day after entering the 5th instar (V6), prior to spinning (PS), during spinning (SP) and after cocoon formation (CO) were used to analyze macroautophagy, chaperone-mediated autophagy (CMA) and the adenosine triphosphate (ATP)-dependent ubiquitin proteasome. Immediately after entering metamorphosis stage PS, the levels of ATP and phosphorylated p70S6 kinase protein decreased spontaneously and continued to decline at SP, followed by a notable restoration at CO. In contrast, phosphorylated AMP-activated protein kinase α (AMPKα) showed increases at SP and CO. Most of the Atg8 protein was converted to form II at all stages. The levels of ubiquitinated proteins were high at SP and CO, and low at PS. The proteasome activity was high at V6 and PS but low at SP and CO. In the isolated lysosome fractions, levels of Hsc70/Hsp70 protein began to increase at PS and continued to rise at SP and CO. The lysosomal cathepsin B/L activity showed a dramatic increase at CO. Our results clearly demonstrate that macroautophagy occurs before entering the metamorphosis stage and strongly suggest that the CMA pathway may play an important role in the histolysis of the posterior silk gland during metamorphosis  

3.2516           Reciprocal regulation of actin cytoskeleton remodelling and cell migration by Ca2+ and Zn2+: role of TRPM2 channels

Li, F., Abuarab, N. and Sivaprasadarao, A.

1. Cell Sci., 129(10), 2016-2029 (2016)

  Cell migration is a fundamental feature of tumour metastasis and angiogenesis. It is regulated by a variety of signalling molecules including H₂O₂ and Ca²⁺. Here, we asked whether the H₂O₂-sensitive transient receptor potential melastatin 2 (TRPM2) Ca²⁺ channel serves as a molecular link between H₂O₂ and Ca²⁺. H₂O₂-mediated activation of TRPM2 channels induced filopodia formation, loss of actin stress fibres and disassembly of focal adhesions, leading to increased migration of HeLa and prostate cancer (PC)-3 cells. Activation of TRPM2 channels, however, caused intracellular release of not only Ca²⁺ but also of Zn²⁺. Intriguingly, elevation of intracellular Zn²⁺ faithfully reproduced all of the effects of H₂O₂, whereas Ca²⁺ showed opposite effects. Interestingly, H₂O₂ caused increased trafficking of Zn²⁺-enriched lysosomes to the leading edge of migrating cells, presumably to impart polarisation of Zn²⁺ location. Thus, our results indicate that a reciprocal interplay between Ca²⁺ and Zn²⁺ regulates actin remodelling and cell migration; they call for a revision of the current notion that implicates an exclusive role for Ca²⁺ in cell migration.  

3.2517           Heparin interacts with the adhesion GPCR GPR56, reduces receptor shedding, and promotes cell adhesion and motility

Chiang, N-Y., Chang, G-W., Huang, Y-S., Peng, Y-M., Hsiao, C-C., Kuo, M-L. and Lin, H-H.

1. Cell Sci., 129(11), 2156-2169 (2016)

  GPR56 is an adhesion-class G-protein-coupled receptor responsible for bilateral frontoparietal polymicrogyria (BFPP), a severe disorder of cortical formation. Additionally, GPR56 is involved in biological processes as diverse as hematopoietic stem cell generation and maintenance, myoblast fusion, muscle hypertrophy, immunoregulation and tumorigenesis. Collagen III and tissue transglutaminase 2 (TG2) have been revealed as the matricellular ligands of GPR56 involved in BFPP and melanoma development, respectively. In this study, we identify heparin as a glycosaminoglycan interacting partner of GPR56. Analyses of truncated and mutant GPR56 proteins reveal two basic-residue-rich clusters, R²⁶GHREDFRFC³⁵ and L¹⁹⁰KHPQKASRRP²⁰⁰, as the major heparin-interacting motifs that overlap partially with the collagen III- and TG2-binding sites. Interestingly, the GPR56–heparin interaction is modulated by collagen III but not TG2, even though both ligands are also heparin-binding proteins. Finally, we show that the interaction with heparin reduces GPR56 receptor shedding, and enhances cell adhesion and motility. These results provide novel insights into the interaction of GPR56 with its multiple endogenous ligands and have functional implications in diseases such as BFPP and cancer  

3.2518           Kinesin 1 Drives Autolysosome Tubulation

Du, W., Su, Q.P., Chen, Y. et al Developmental Cell, 37(4), 326-336 (2016)   Autophagic lysosome reformation (ALR) plays an important role in maintaining lysosome homeostasis. During ALR, lysosomes are reformed by recycling lysosomal components from autolysosomes. The most noticeable step of ALR is autolysosome tubulation, but it is currently unknown how the process is regulated. Here, using an approach combining in vivo studies and in vitro reconstitution, we found that the kinesin motor protein KIF5B is required for autolysosome tubulation and that KIF5B drives autolysosome tubulation by pulling on the autolysosomal membrane. Furthermore, we show that KIF5B directly interacts with PtdIns(4,5)P₂. Kinesin motors are recruited and clustered on autolysosomes via interaction with PtdIns(4,5)P₂ in a clathrin-dependent manner. Finally, we demonstrate that clathrin promotes formation of PtdIns(4,5)P₂-enriched microdomains, which are required for clustering of KIF5B. Our study reveals a mechanism by which autolysosome tubulation was generated.  

3.2519           Plasticity of sarcolemmal KATP channel surface expression: relevance during ischemia and ischemic preconditioning

Yang, H-Q., Foster, M.N., jana, K., Ho, J., Rindler, M.J. and Coetzee, W.A. Am. J. Physiol. Heart Circ. Physiol., 310(11), H1558-H1566 (2016)   Myocardial ischemia remains the primary cause of morbidity and mortality in the United States. Ischemic preconditioning (IPC) is a powerful form of endogenous protection against myocardial infarction. We studied alterations in K_ATP channels surface density as a potential mechanism of the protection of IPC. Using cardiac-specific knockout of Kir6.2 subunits, we demonstrated an essential role for sarcolemmal K_ATP channels in the infarct-limiting effect of IPC in the mouse heart. With biochemical membrane fractionation, we demonstrated that sarcolemmal K_ATP channel subunits are distributed both to the sarcolemma and intracellular endosomal compartments. Global ischemia causes a loss of sarcolemmal K_ATP channel subunit distribution and internalization to endosomal compartments. Ischemia-induced internalization of K_ATP channels was prevented by CaMKII inhibition. K_ATP channel subcellular redistribution was also observed with immunohistochemistry. Ischemic preconditioning before the index ischemia reduced not only the infarct size but also prevented K_ATP channel internalization. Furthermore, not only did adenosine mimic IPC by preventing infarct size, but it also prevented ischemia-induced K_ATP channel internalization via a PKC-mediated pathway. We show that preventing endocytosis with dynasore reduced both K_ATP channel internalization and strongly mitigated infarct development. Our data demonstrate that plasticity of K_ATP channel surface expression must be considered as a potentially important mechanism of the protective effects of IPC and adenosine  

3.2520           Control of chylomicron export from the intestine

Mansbach II, C.M. and Siddiqi, S. Am. J. Gastrointest. Liver Physiol., 310(9), G659-G668 (2016)   The control of chylomicron output by the intestine is a complex process whose outlines have only recently come into focus. In this review we will cover aspects of chylomicron formation and prechylomicron vesicle generation that elucidate potential control points. Substrate (dietary fatty acids and monoacylglycerols) availability is directly related to the output rate of chylomicrons. These substrates must be converted to triacylglycerol before packaging in prechylomicrons by a series of endoplasmic reticulum (ER)-localized acylating enzymes that rapidly convert fatty acids and monoacylglycerols to triacylglycerol. The packaging of the prechylomicron with triacylglycerol is controlled by the microsomal triglyceride transport protein, another potential limiting step. The prechylomicrons, once loaded with triacylglycerol, are ready to be incorporated into the prechylomicron transport vesicle that transports the prechylomicron from the ER to the Golgi. Control of this exit step from the ER, the rate-limiting step in the transcellular movement of the triacylglycerol, is a multistep process involving the activation of PKCζ, the phosphorylation of Sar1b, releasing the liver fatty acid binding protein from a heteroquatromeric complex, which enables it to bind to the ER and organize the prechylomicron transport vesicle budding complex. We propose that control of PKCζ activation is the major physiological regulator of chylomicron output.  

3.2521           GPRC5A suppresses protein synthesis at the endoplasmic reticulum to prevent radiation-induced lung tumorigenesis

Wang, J., Farris, A.B., Xu, K., Wang, P., Zhang, X., duong, D.M., Yi, H., Shu, H-K., Sun, S-Y. and Wang, Y. Nature Communications, 7:11795 (2016)   GPRC5A functions as a lung tumour suppressor to prevent spontaneous and environmentally induced lung carcinogenesis; however, the underlying mechanism remains unclear. Here we reveal that GPRC5A at the endoplasmic reticulum (ER) membrane suppresses synthesis of the secreted or membrane-bound proteins including a number of oncogenes, the most important one being Egfr. The ER-located GPRC5A disturbs the assembly of the eIF4F-mediated translation initiation complex on the mRNA cap through directly binding to the eIF4F complex with its two middle extracellular loops. Particularly, suppression of EGFR by GPRC5A contributes significantly to preventing ionizing radiation (IR)-induced lung tumorigenesis. Thus, GPRC5A deletion enhances IR-promoted EGFR expression through an increased translation rate, thereby significantly increasing lung tumour incidence in Gprc5a^−/− mice. Our findings indicate that under-expressed GPRC5A during lung tumorigenesis enhances any transcriptional stimulation through an active translational status, which can be used to control oncogene expression and potentially the resulting related disease.  

3.2522           Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis

Zhang, Y. et al Nature Communications, 7:11656 (2016)   As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus.  

3.2523           Cross-talk between Dopachrome Tautomerase and Caveolin-1 Is Melanoma Cell Phenotype-specific and Potentially Involved in Tumor Progression

Popa, I.L., Milac, A.L., Sima, L.E., Alexandru, P.R., Pastrami, F., Munteanu, C.V.A. and Negroiu, G.

1. Biol. Cehm., 291(24), 12481-12500 (2016)

  l-Dopachrome tautomerase (l-DCT), also called tyrosinase-related protein-2 (TRP-2), is a melanoma antigen overexpressed in most chemo-/radiotherapeutic stress-resistant tumor clones, and caveolin-1 (CAV1) is a main regulator of numerous signaling processes. A structural and functional relationship between DCT and CAV1 is first presented here in two human amelanotic melanoma cell lines, derived from vertical growth phase (MelJuSo) and metastatic (SKMel28) melanomas. DCT co-localizes at the plasma membrane with CAV1 and Cavin-1, another molecular marker for caveolae in both cell phenotypes. Our novel structural model proposed for the DCT-CAV1 complex, in addition to co-immunoprecipitation and mass spectrometry data, indicates a possible direct interaction between DCT and CAV1. The CAV1 control on DCT gene expression, DCT post-translational processing, and subcellular distribution is cell phenotype-dependent. DCT is a modulator of CAV1 stability and supramolecular assembly in both cell phenotypes. During autocrine stimulation, the expressions of DCT and CAV1 are oppositely regulated; DCT increases while CAV1 decreases. Sub-confluent MelJuSo clones DCT^high/CAV1^low are proliferating and acquire fibroblast-like morphology, forming massive, confluent clusters as demonstrated by immunofluorescent staining and TissueFAXS quantitative image cytometry analysis. CAV1 down-regulation directly contributes to the expansion of MelJuSo DCT^high subtype. CAV1 involved in the perpetuation of cell phenotype-overexpressing anti-stress DCT molecule supports the concept that CAV1 functions as a tumor suppressor in early stages of melanoma. DCT is a regulator of the CAV1-associated structures.  

3.2524           KRAS-MEK Signaling Controls Ago2 Sorting into Exosomes

McKenzie, A.J., Hoshino, D., Hong, N.H., Cha, D.J., Franklin, J.F., Coffey, R.J., Patton, J.G. and Weaver, A.M. Cell Reports, 15, 978-987 (2016)   Secretion of RNAs in extracellular vesicles is a newly recognized form of intercellular communication. A potential regulatory protein for microRNA (miRNA) secretion is the critical RNA-induced silencing complex (RISC) component Argonaute 2 (Ago2). Here, we use isogenic colon cancer cell lines to show that overactivity of KRAS due to mutation inhibits localization of Ago2 to multivesicular endosomes (MVEs) and decreases Ago2 secretion in exosomes. Mechanistically, inhibition of mitogen-activated protein kinase kinases (MEKs) I and II, but not Akt, reverses the effect of the activating KRAS mutation and leads to increased Ago2-MVE association and increased exosomal secretion of Ago2. Analysis of cells expressing mutant Ago2 constructs revealed that phosphorylation of Ago2 on serine 387 prevents Ago2-MVE interactions and reduces Ago2 secretion into exosomes. Furthermore, regulation of Ago2 exosomal sorting controls the levels of three candidate miRNAs in exosomes. These data identify a key regulatory signaling event that controls Ago2 secretion in exosomes.  

3.2525           FLCN Maintains the Leucine Level in Lysosome to Stimulate mTORC1

Wu, X., Zhao, L., Xhen, Z., Ji, X., Qiao, X., Jin, Y. and Liu, W. PloS One, 11(6), e0157100 (2016)   The intracellular amino acid pool within lysosome is a signal that stimulates the nutrient-sensing mTORC1 signalling pathway. The signal transduction cascade has garnered much attention, but little is known about the sequestration of the signalling molecules within the lysosome. Using human HEK293 cells as a model, we found that suppression of the BHD syndrome gene FLCN reduced the leucine level in lysosome, which correlated with decreased mTORC1 activity. Both consequences could be reversed by supplementation with high levels of leucine, but not other tested amino acids. Conversely, overexpressed FLCN could sequester lysosomal leucine and stimulate mTORC1 in an amino acid limitation environment. These results identify a novel function of FLCN: it controls mTORC1 by modulating the leucine signal in lysosome. Furthermore, we provided evidence that FLCN exerted this role by inhibiting the accumulation of the amino acid transporter PAT1 on the lysosome surface, thereby maintaining the signal level within the organelle.  

3.2526           DIETARY FATTY ACID UTILIZES THE CAVEOLIN-1 CONTAINING ENDOCYTIC VESICLES (CEV) AS A VEHICLE FOR TRANSPORT INTO THE INTESTINAL ENDOPLASMIC RETICULUM (ER)

Morris, L., Siddiqi, T., mansbach, C.M.and Siddiqi, S.

1. Investig. Med., 64, abstract 404, 488-726 (2016)

    Purpose of Study How dietary fatty acids (FA) are transported to the ER is not well established. Here we tested the hypothesis that the caveolin-1 containing endocytic vesicle (CEV) that we proposed as the major mechanism for dietary fatty acid absorption (BBA 183; 1311, 2013) also transports fatty acids into the intestinal endoplasmic reticulum (ER). Methods Used Native ER and 3H-cytosol were obtained from the enterocytes of wild type (WT) and caveolin-1 (Cav-1) knockout mice. CEV were isolated from 1% Triton X-100 treated cytosol using an OptiPrep gradient, known to be able to separate detergent resistant membranes (DRM) from detergent soluble membranes (DSM). An in vitro binding assay was performed using these fractions with native ER, and then re-isolated ER was analyzed for lipid and protein content. Summary of Results In WT cytosol, 60% of 3H oleate appeared in the CEV, while 20% was associated with DSM. In Cav-1 KO cytosol, no 3H oleate was found in DRM, whereas 54% of 3H oleate was associated with DSM. When WT ER was incubated with CEV, 68% of the 3H oleate was transported to the ER; greater than the ER incubated with whole cytosol (36%) and DSM (17%). In the Cav-1 KO mice, 21% of 3H oleate was transferred to the ER when incubated with whole cytosol or DSM. Dietary lipid analysis of the re-isolated ER with both genotypes showed that 89% of the FA had been metabolized into triacyl glycerol (TAG) and diacyl glycerol (DAG) by the end of the 15 min incubations. The amount of Caveolin-1 (Cav-1) and (fatty acid translocase) CD36 showed incremental increases in the ER after binding with CEV; while liver fatty acid binding protein (FABP1) was not increased. Conclusions The most dietary oleate is absorbed by associating with caveolae in apical BB. The caveolae are endocytosed and appear in cytosol as CEV (BBA 183; 1311, 2013). We conclude that CEVs deliver the dietary FA to the intestinal ER for esterification to TAG, independentof FABP.  

3.2527           Identification of Sirtuin4 (SIRT4) Protein Interactions: Uncovering Candidate Acyl-Modified Mitochondrial Substrates and Enzymatic Regulators

Mathias, R.A., Greco, T.M. and Cristea, I.M. Methods in Mol. Biol., 1436, 213-239 (2016)   Recent studies have highlighted the three mitochondrial human sirtuins (SIRT3, SIRT4, and SIRT5) as critical regulators of a wide range of cellular metabolic pathways. A key factor to understanding their impact on metabolism has been the discovery that, in addition to their ability to deacetylate substrates, mitochondrial sirtuins can have other prominent enzymatic activities. SIRT4, one of the least characterized mitochondrial sirtuins, was shown to be the first known cellular lipoamidase, removing lipoyl modifications from lysine residues of substrates. Specifically, SIRT4 was found to delipoylate and modulate the activity of the pyruvate dehydrogenase complex (PDH), a protein complex critical for the production of acetyl-CoA. Furthermore, SIRT4 is well known to have ADP-ribosyltransferase activity and to regulate the activity of the glutamate dehydrogenase complex (GDH). Adding to its impressive range of enzymatic activities are its ability to deacetylate malonyl-CoA decarboxylase (MCD) to regulate lipid catabolism, and its newly recognized ability to remove biotinyl groups from substrates that remain to be defined. Given the wide range of enzymatic activities and the still limited knowledge of its substrates, further studies are needed to characterize its protein interactions and its impact on metabolic pathways. Here, we present several proven protocols for identifying SIRT4 protein interaction networks within the mitochondria. Specifically, we describe methods for generating human cell lines expressing SIRT4, purifying mitochondria from crude organelles, and effectively capturing SIRT4 with its interactions and substrates.  

3.2528           Shaping the endoplasmic reticulum in vitro

Ferencz, C-M., Guigas, G., Veres, A., Neumann, B., Stemmann, O. and Weiss, M. Biochim. Biophys. Acta, 1858, 2035-2040 (2016)   Organelles in eukaryotic cells often have complex shapes that deviate significantly from simple spheres. A prime example is the endoplasmic reticulum (ER) that forms an extensive network of membrane tubules in many mammalian cell types and in reconstitution assays in vitro. Despite the successful hunt for molecular determinants of ER shape we are still far from having a comprehensive understanding of ER network morphogenesis. Here, we have studied the hitherto neglected influence of the host substrate when reconstituting ER networks in vitro as compared to ER networks in vivo. In culture cells we observed cytoplasm-spanning ER networks with tubules being connected almost exclusively by three-way junctions and segment lengths being narrowly distributed around a mean length of about 1 μm. In contrast, networks reconstituted from purified ER microsomes on flat glass or gel substrates of varying stiffness showed significantly broader length distributions with an up to fourfold larger mean length. Self-assembly of ER microsomes on small oil droplets, however, yielded networks that resembled more closely the native ER network of mammalian cells. We conclude from these observations that the ER microsomes' inherent self-assembly capacity is sufficient to support network formation with a native geometry if the influence of the host substrate's surface chemistry becomes negligible. We hypothesize that under these conditions the networks' preference for three-way junctions follows from creating ‘starfish-shaped’ vesicles when ER microsomes with a protein-induced spontaneous curvature undergo fusion.    

3.2529           Defined spatiotemporal features of RAS-ERK signals dictate cell fate in MCF-7 mammary epithelial cells

Herrero, A., Casar, B., Colon-Bolea. P., Aguda-Ibanez, L. and Crespo, P. Mol. Biol. Cell, 27, 1958-1968 (2016)   Signals conveyed through the RAS-ERK pathway are essential for the determination of cell fate. It is well established that signal variability is achieved in the different microenvironments in which signals unfold. It is also known that signal duration is critical for decisions concerning cell commitment. However, it is unclear how RAS-ERK signals integrate time and space in order to elicit a given biological response. To investigate this, we used MCF-7 cells, in which EGF-induced transient ERK activation triggers proliferation, whereas sustained ERK activation in response to heregulin leads to adipocytic differentiation. We found that both proliferative and differentiating signals emanate exclusively from plasma membrane–disordered microdomains. Of interest, the EGF signal can be transformed into a differentiating stimulus by HRAS overexpression, which prolongs ERK activation, but only if HRAS localizes at disordered membrane. On the other hand, HRAS signals emanating from the Golgi complex induce apoptosis and can prevent heregulin-induced differentiation. Our results indicate that within the same cellular context, RAS can exert different, even antagonistic, effects, depending on its sublocalization. Thus cell destiny is defined by the ability of a stimulus to activate RAS at the appropriate sublocalization for an adequate period while avoiding switching on opposing RAS signals.  

3.2530           A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles

Koeppenen, K., Hampton, T.H., jarek, M., Scharfe, M., Gerber, S.A., Mielcarz, D.W., Demers, E.G., Dolben, E.L., Hammond, J.H., Hogan, D.A. and Stanton, B.A. PloS Pathogens, 12(6), e1005672 (2016)   Bacterial outer membrane vesicle (OMV)-mediated delivery of proteins to host cells is an important mechanism of host-pathogen communication. Emerging evidence suggests that OMVs contain differentially packaged short RNAs (sRNAs) with the potential to target host mRNA function and/or stability. In this study, we used RNA-Seq to characterize differentially packaged sRNAs in Pseudomonas aeruginosa OMVs, and to show transfer of OMV sRNAs to human airway cells. We selected one sRNA for further study based on its stable secondary structure and predicted mRNA targets. Our candidate sRNA (sRNA52320), a fragment of a P. aeruginosa methionine tRNA, was abundant in OMVs and reduced LPS-induced as well as OMV-induced IL-8 secretion by cultured primary human airway epithelial cells. We also showed that sRNA52320 attenuated OMV-induced KC cytokine secretion and neutrophil infiltration in mouse lung. Collectively, these findings are consistent with the hypothesis that sRNA52320 in OMVs is a novel mechanism of host-pathogen interaction whereby P. aeruginosa reduces the host immune response.  

3.2531           Neuronal expression of ILEI/FAM3C and its reduction in Alzheimer’s disease

Liu, L., Watanabe, N., Akatsu, H. and Nishimura, M. Neuroscience, 330, 236-246 (2016)   Decrease in brain amyloid-β (Aβ) accumulation is a leading strategy for treating Alzheimer’s disease (AD). However, the intrinsic mechanism of the regulation of brain Aβ production is largely unknown. Previously, we reported that ILEI (also referred to as FAM3C) binds to the γ-secretase complex and suppresses Aβ production without inhibiting γ-secretase activity. In this study, we examined ILEI expression in mouse brain using immunohistochemistry and subcellular fractionation. Brain ILEI showed widespread expression in neurons and ependymal cells but not in glial and vascular endothelial cells. Neuronal ILEI resided in perinuclear vesicular structures, which were positive for a marker protein of the trans-Golgi network. Although ILEI immunostaining was negative at synaptic terminals, synaptosome fractionation analysis suggested that ILEI was enriched in presynaptic terminals, particularly in the active zone-docked synaptic vesicles. ILEI expression levels in brain peaked during the postnatal period and declined with age. In comparison with age-matched control brains, the number of ILEI-immunoreactive neurons decreased in AD brains, although the subcellular localization was unaltered. Our results suggest that a decline of ILEI expression may cause accumulation of Aβ in the brain and the eventual development of AD.  

3.2532           Monomerization and ER Relocalization of GRASP Is a Requisite for Unconventional Secretion of CFTR

Kim, J., Noh, S.H., Piao, H., Kim, D.H., Kim, K., Cha, J.S., Chung, Y., Cho, H-S., Kim, J.Y. and Lee, M.G. Traffic, 17(7), 733-753 (2016)   Induction of endoplasmic reticulum (ER)-to-Golgi blockade or ER stress induces Golgi reassembly stacking protein (GRASP)-mediated, Golgi-independent unconventional cell-surface trafficking of the folding-deficient ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR). However, molecular mechanisms underlying this process remain elusive. Here, we show that phosphorylation-dependent dissociation of GRASP homotypic complexes and subsequent relocalization of GRASP to the ER play a critical role in the unconventional secretion of CFTR. Immunolocalization analyses of mammalian cells revealed that the Golgi protein GRASP55 was redistributed to the ER by stimuli that induce unconventional secretion of ΔF508-CFTR, such as induction of ER-to-Golgi blockade by the Arf1 mutant. Notably, the same stimuli also induced phosphorylation of regions near the C-terminus of GRASP55 and dissociation of GRASP homomultimer complexes. Furthermore, phosphorylation-mimicking mutations of GRASP55 induced the monomerization and ER relocalization of GRASP55, and these changes were nullified by phosphorylation-inhibiting mutations. These results provide mechanistic insights into how GRASP accesses the ER-retained ΔF508-CFTR and mediates the ER stress-induced unconventional secretion pathway.  

3.2533           Identification of Individual Exosome-Like Vesicles by Surface Enhanced Raman Spectroscopy

Stremersch, S., Marro, M., Pinchasik, B-El., Baatsen, P., Hendrix, A., De Smedt, S.C., Loza-Alvarez, P., Skirtach, A.G., Raemdonck, K. and Braeckmans, K. Small, 12(24), 3292-3301 (2016)   Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention for the detection of cancer at an early stage. In this study the feasibility of using a surface enhanced Raman spectroscopy (SERS) based method to distinguish between ELVs derived from different cellular origins is evaluated. A gold nanoparticle based shell is deposited on the surface of ELVs derived from cancerous and healthy cells, which enhances the Raman signal while maintaining a colloidal suspension of individual vesicles. This nanocoating allows the recording of SERS spectra from single vesicles. By using partial least squares discriminant analysis on the obtained spectra, vesicles from different origin can be distinguished, even when present in the same mixture. This proof-of-concept study paves the way for noninvasive (cancer) diagnostic tools based on exosomal SERS fingerprinting in combination with multivariate statistical analysis.  

3.2534           Quantification of age-related changes of α-tocopherol in lysosomal membranes in murine tissues and human fibroblasts

König, J., Besoke, F., Stuetz, W., Malaarski, A., Jahreis, G., Grune, T. and Höhn, A. BioFactors, 42(3), 307-315 (2016)   Considering the biological function of α-tocopherol (α-Toc) as a potent protective factor against oxidative stress, this antioxidant is in the focus of aging research. To understand the role of α-Toc during aging we investigated α-Toc concentrations in young and aged primary human fibroblasts after supplementation with RRR-α-Toc. Additionally, α-Toc contents were determined in brain, kidney, and liver tissue of 10 week-, 18 month-, and 24 month-old mice, which were fed a standard diet containing 100 mg/kg dl-α-tocopheryl acetate. α-Toc concentrations in isolated lysosomes and the expression of the α-Toc transport proteins Niemann Pick C1 (NPC1), Niemann Pick C2 (NPC2), and lipoprotein lipase were also analyzed. Obtained data show a significant age-related increase of α-Toc in murine liver, kidney, and brain tissue as well as in human dermal fibroblasts. Also liver and kidney lysosomes are marked by elevated α-Toc contents with aging. NPC1 and NPC2 protein amounts are significantly decreased in adult and aged murine kidney tissue. Also aged human dermal fibroblasts show decreased NPC1 amounts. Supplementation of young and aged fibroblasts led also to decreased NPC1 amounts, suggesting a direct role of this protein in α-Toc distribution. Our results indicate an age-dependent increase of α-Toc in different murine tissues as well as in human fibroblasts. Furthermore saturation and intracellular distribution of α-Toc seem to be strongly dependent on the availability of this vitamin as well as on the presence of the lysosomal protein NPC1.  

3.2535           RNAi delivery by exosome-mimetic nanovesicles – Implications for targeting c-Myc in cancer

Lunavat, T.R., Jang, S.C., Nilsson, L., Park, H.T., Repiska, G., Lässer, C., Nilsson, J.A., Gho, Y.S. and Lötvall, J. Biomaterials, 102, 231-238 (2016)   To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called “Extracellular Vesicles”) have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm. To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called “Extracellular Vesicles”) have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm.  

3.2536           Cathepsin Protease Controls Copper and Cisplatin Accumulation via Cleavage of the Ctr1 Metal-binding Ectodomain

Öhrvik, H., Logeman, B., Turk, B., Reinheckel, T and Thiele, D.J.

1. Biol. Chem., 291(27), 13905-13916 (2016)

  Copper is an essential metal ion for embryonic development, iron acquisition, cardiac function, neuropeptide biogenesis, and other critical physiological processes. Ctr1 is a high affinity Cu⁺ transporter on the plasma membrane and endosomes that exists as a full-length protein and a truncated form of Ctr1 lacking the methionine- and histidine-rich metal-binding ectodomain, and it exhibits reduced Cu⁺ transport activity. Here, we identify the cathepsin L/B endolysosomal proteases functioning in a direct and rate-limiting step in the Ctr1 ectodomain cleavage. Cells and mice lacking cathepsin L accumulate full-length Ctr1 and hyper-accumulate copper. As Ctr1 also transports the chemotherapeutic drug cisplatin via direct binding to the ectodomain, we demonstrate that the combination of cisplatin with a cathepsin L/B inhibitor enhances cisplatin uptake and cell killing. These studies identify a new processing event and the key protease that cleaves the Ctr1 metal-binding ectodomain, which functions to regulate cellular Cu⁺ and cisplatin acquisition.  

3.2537           iTRAQ-based quantitative proteomic analysis reveals the role of the tonoplast in fruit senescence

Liu, R., Wang, Y., Qin, G. and Tian, S.

1. Proteomics, 146, 80-89 (2016)

  The vacuole is by far the largest multifunctional organelle in fruits and plays a functional role in fruit development and fruit quality. Despite its significance, little information exists pertaining to the role of the vacuolar membrane (tonoplast) in the process of fruit senescence. In the present study, an iTRAQ-based quantitative proteomic approach was used to characterize the dynamic alterations in the tonoplast proteome during fruit senescence. Tonoplasts were purified from apple fruit at various stages of senescence using an iodixanol step gradient protocol. A total of 345 tonoplast-related proteins were identified with diverse functions such as transporters and proton pumps, signal transduction, membrane fusion or vesicle trafficking, cellular metabolic process, defense response, protein folding and degradation, and cytoskeleton. Changes in protein abundance during storage were characterized for the identified proteins. A total of 22 proteins displayed differential levels of abundance during storage. The senescence-related tonoplast proteins mostly function in the transportation of metabolites, signal transduction, membrane trafficking, and stress response. RT-qPCR analysis was used to quantify the level of expression of nine genes encoding some of the differentially abundant proteins. The results of this study provide new information regarding the function of the tonoplast during fruit senescence.  

3.2538           Exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and future challenges

Ha, D., Yang, N., Nadithe, V. Acta Pharmaceutica Sinica B, 6(4), 287-296 (2016)   Exosomes are small intracellular membrane-based vesicles with different compositions that are involved in several biological and pathological processes. The exploitation of exosomes as drug delivery vehicles offers important advantages compared to other nanoparticulate drug delivery systems such as liposomes and polymeric nanoparticles; exosomes are non-immunogenic in nature due to similar composition as body׳s own cells. In this article, the origin and structure of exosomes as well as their biological functions are outlined. We will then focus on specific applications of exosomes as drug delivery systems in pharmaceutical drug development. An overview of the advantages and challenges faced when using exosomes as a pharmaceutical drug delivery vehicles will also be discussed.  

3.2539           Membrane Isolation Methods

Stillwell, W. An Introduction to Biological Membranes, 247-271 (2016)   Isolating biological membrane is as much of an art form as it is a science. The procedures are tedious and require patience, precision, and organization. Since every membrane has its own peculiarities, countless procedures have been published. This chapter will consider a few of the problems encountered in isolating membranes and some of the more common techniques that have been employed.  

3.2540           Membrane Reconstitution

Stillwell, W. An Introduction to Biological Membranes, 273-312 (2016)   Membranes are unimaginably complex. They consist of hundreds of different proteins floating in a bewildering and crowded sea of a 1000 or more different lipids and this mixture is in constant flux. A membrane is not homogeneous but exists in fleeting patches that exhibit both lateral and transmembrane asymmetry. Also, countless reactions occur simultaneously in and around the membrane. Can the membrane problem be simplified? In this chapter we will examine how one membrane protein can be isolated away from the others and reconstituted into a model lipid bilayer membrane in order to determine how the protein functions. Finally, we will investigate the isolation and properties of an important membrane domain involved in cell signaling, the lipid raft.  

3.2541           Cytoskeleton and Intracellular Motility

Casem, M.L. Case Studies in Cell Biology, 127-156 (2016)   A cell’s cytoskeleton provides the structural support necessary to maintain cell shape or anchor the cell to the extracellular matrix. At the same time, the cytoskeleton creates a dynamic scaffold for the movement of organelles, chromosomes, and the cell itself. The potential for integration between the different distinct cytoskeletal filament systems is investigated in the case study, “Plakins: Keeping the Cytoskeleton Safe,” (Yang Y, Bauer C, Strasser G, Wollman R, Julien J-P, Fuchs E. Integrators of the cytoskeleton that stabilize microtubules. Cell 1999;98:229–238). The discovery of the microtubule motor protein kinesin (Vale RD, Schnapp BJ, Reese TS, Sheetz MP. Organelle, bead and microtubule translocations promoted by soluble factors from the squid giant axon. Cell 1985;40:559–569) sets the stage for the case study, “The Moving Story of a Microtubule Motor Protein.” The case studies, ”The WASP and the Barbed End” (Co C, Wong DT, Gierke S, Chang V, Taunton J. Mechanism of actin network attachment to moving membranes: barbed end capture by N-WASP WH2 domains. Cell 2007;128: 901–913) and “Cilia Grow Where Vesicles Go” (Wood CR, Rosenbaum JL. Proteins of the ciliary axoneme are found on cytoplasmic membrane vesicles during growth of cilia. Curr Biol 2014; 24: 1114–1120.) investigate different aspects of the dynamic nature of the cytoskeleton.  

3.2542           Role of Lipid Rafts and the Underlying Filamentous-Actin Cytoskeleton in Cannabinoid Receptor 1 Signaling

Mangoura, D., Asimaki, O., Tsirimonaki, E. and Sakellaridis, N. Neuropathology of Drug Addictions and Substance Misuse, (Volume 1), 689-701 (2016)   The cannabinoid 1 receptor, CB1, has evolved as a major regulatory molecule for almost all known aspects of the development and function of the central nervous system (CNS), with biological actions ranging from proper CNS cellularity to complex behaviors such as fear, appetite, and addiction. It is therefore critical to understand the mechanisms and intracellular signal transduction pathways that CB1 utilizes for its acute and long-term actions, in particular activation of the major effector extracellular signal-regulated kinase (ERK) in discrete amplification waves. This highly complex regulation of CB1 signaling to ERK is facilitated by specialized membrane microdomains, the lipid rafts. Integral components of rafts are required for proper CB1 presentation at the plasma membrane as shown by confocal analysis, while the dynamic and hierarchic activation of its major proximal effectors protein kinase Cε, Src, and Fyn requires raft integrity, additionally causing fibroblast growth factor receptor transactivation. Thus, lipid rafts constitute the plasma membrane platform on which CB1 signaling is initiated and organized.  

3.2543           Septin 9 induces lipid droplets growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV

Akil, A. et al Nature Communications, 7:12203 (2016)   The accumulation of lipid droplets (LD) is frequently observed in hepatitis C virus (HCV) infection and represents an important risk factor for the development of liver steatosis and cirrhosis. The mechanisms of LD biogenesis and growth remain open questions. Here, transcriptome analysis reveals a significant upregulation of septin 9 in HCV-induced cirrhosis compared with the normal liver. HCV infection increases septin 9 expression and induces its assembly into filaments. Septin 9 regulates LD growth and perinuclear accumulation in a manner dependent on dynamic microtubules. The effects of septin 9 on LDs are also dependent on binding to PtdIns5P, which, in turn, controls the formation of septin 9 filaments and its interaction with microtubules. This previously undescribed cooperation between PtdIns5P and septin 9 regulates oleate-induced accumulation of LDs. Overall, our data offer a novel route for LD growth through the involvement of a septin 9/PtdIns5P signalling pathway.  

3.2544           AP-1/σ1A and AP-1/σ1B adaptor-proteins differentially regulate neuronal early endosome maturation via the Rab5/Vps34-pathway

Candiello, E., Kratzke, M., Wenzel, d., Cassel, D. and Schu, P. Scientific Reports, 6:29950 (2015)   The σ1 subunit of the AP-1 clathrin-coated-vesicle adaptor-protein complex is expressed as three isoforms. Tissues express σ1A and one of the σ1B and σ1C isoforms. Brain is the tissue with the highest σ1A and σ1B expression. σ1B-deficiency leads to severe mental retardation, accumulation of early endosomes in synapses and fewer synaptic vesicles, whose recycling is slowed down. AP-1/σ1A and AP-1/σ1B regulate maturation of these early endosomes into multivesicular body late endosomes, thereby controlling synaptic vesicle protein transport into a degradative pathway. σ1A binds ArfGAP1, and with higher affinity brain-specific ArfGAP1, which bind Rabex-5. AP-1/σ1A-ArfGAP1-Rabex-5 complex formation leads to more endosomal Rabex-5 and enhanced, Rab5GTP-stimulated Vps34 PI3-kinase activity, which is essential for multivesicular body endosome formation. Formation of AP-1/σ1A-ArfGAP1-Rabex-5 complexes is prevented by σ1B binding of Rabex-5 and the amount of endosomal Rabex-5 is reduced. AP-1 complexes differentially regulate endosome maturation and coordinate protein recycling and degradation, revealing a novel molecular mechanism by which they regulate protein transport besides their established function in clathrin-coated-vesicle formation.  

3.2545           Limited ER quality control for GPI-anchored proteins

Sikorska, N., Lemus, L., Aguilera-Romero, A., Monzano-Lopez, J., Riezman, H., Muniz, M. and Goder, V.

1. Cell Biol., 213(6), 693-704 (2016)

  Endoplasmic reticulum (ER) quality control mechanisms target terminally misfolded proteins for ER-associated degradation (ERAD). Misfolded glycophosphatidylinositol-anchored proteins (GPI-APs) are, however, generally poor ERAD substrates and are targeted mainly to the vacuole/lysosome for degradation, leading to predictions that a GPI anchor sterically obstructs ERAD. Here we analyzed the degradation of the misfolded GPI-AP Gas1* in yeast. We could efficiently route Gas1* to Hrd1-dependent ERAD and provide evidence that it contains a GPI anchor, ruling out that a GPI anchor obstructs ERAD. Instead, we show that the normally decreased susceptibility of Gas1* to ERAD is caused by canonical remodeling of its GPI anchor, which occurs in all GPI-APs and provides a protein-independent ER export signal. Thus, GPI anchor remodeling is independent of protein folding and leads to efficient ER export of even misfolded species. Our data imply that ER quality control is limited for the entire class of GPI-APs, many of them being clinically relevant.  

3.2546           Cortactin promotes exosome secretion by controlling branched actin dynamics

Sinha, S., Hoshino, D., Hong, N.H., Kirkbride, K.C., Grega-larson, E., Seiki, M., Tyska, M.J. and Weaver, A.M.

1. Cell Biol., 214(2), 197-213 (2016)

  Exosomes are extracellular vesicles that influence cellular behavior and enhance cancer aggressiveness by carrying bioactive molecules. The mechanisms that regulate exosome secretion are poorly understood. Here, we show that the actin cytoskeletal regulatory protein cortactin promotes exosome secretion. Knockdown or overexpression of cortactin in cancer cells leads to a respective decrease or increase in exosome secretion, without altering exosome cargo content. Live-cell imaging revealed that cortactin controls both trafficking and plasma membrane docking of multivesicular late endosomes (MVEs). Regulation of exosome secretion by cortactin requires binding to the branched actin nucleating Arp2/3 complex and to actin filaments. Furthermore, cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin MVE docking sites and exosome secretion. Functionally, the addition of purified exosomes to cortactin-knockdown cells rescued defects of those cells in serum-independent growth and invasion. These data suggest a model in which cortactin promotes exosome secretion by stabilizing cortical actin-rich MVE docking sites.  

3.2547           Effect of Rhodopsin Phosphorylation on Dark Adaptation in Mouse Rods

Berry, J., Frederiksen, R., Yao, Y., Nymark, S., Chen, J. and Cornwall, C.

1. Neurosci., 36(26), 6973-6987 (2016)

  Rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that is activated when its 11-cis-retinal moiety is photoisomerized to all-trans retinal. This step initiates a cascade of reactions by which rods signal changes in light intensity. Like other GPCRs, rhodopsin is deactivated through receptor phosphorylation and arrestin binding. Full recovery of receptor sensitivity is then achieved when rhodopsin is regenerated through a series of steps that return the receptor to its ground state. Here, we show that dephosphorylation of the opsin moiety of rhodopsin is an extremely slow but requisite step in the restoration of the visual pigment to its ground state. We make use of a novel observation: isolated mouse retinae kept in standard media for routine physiologic recordings display blunted dephosphorylation of rhodopsin. Isoelectric focusing followed by Western blot analysis of bleached isolated retinae showed little dephosphorylation of rhodopsin for up to 4 h in darkness, even under conditions when rhodopsin was completely regenerated. Microspectrophotometeric determinations of rhodopsin spectra show that regenerated phospho-rhodopsin has the same molecular photosensitivity as unphosphorylated rhodopsin and that flash responses measured by trans-retinal electroretinogram or single-cell suction electrode recording displayed dark-adapted kinetics. Single quantal responses displayed normal dark-adapted kinetics, but rods were only half as sensitive as those containing exclusively unphosphorylated rhodopsin. We propose a model in which light-exposed retinae contain a mixed population of phosphorylated and unphosphorylated rhodopsin. Moreover, complete dark adaptation can only occur when all rhodopsin has been dephosphorylated, a process that requires >3 h in complete darkness.  

3.2548           Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies

Valentine, J.L., Chen, L., Perregaux, E.C. et al Cell Chem. Biol., 23(6), 655-665 (2016)   The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens.  

3.2549           Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies

Chen, L. et al PNAS, 113(26), E3609-E3618 (2016)   The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS–specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.  

3.2550           ATP: The crucial component of secretory vesicles

Estevez-herrera, J., Dominguez, N., pardo, M.R., Gonzalez-Santana, A., Westhead, E.W., Borges, R. and Machado, J.D. PNAS, 113(28), E4098-E4106 (2016)   The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.  

3.2551           Reversible HuR-microRNA binding controls extracellular export of miR-122 and augments stress response

Mukherjee, K., Ghoshai, B., Ghosh, S., Chakrabarty, Y., Shwetha, S., Das, S. and Bhattacharyya, S.N. EMBO Reports, 17(8), 1184-1203 (2016)   microRNAs (miRNAs), the tiny but stable regulatory RNAs in metazoan cells, can undergo selective turnover in presence of specific internal and external cues to control cellular response against the changing environment. We have observed reduction in cellular miR‐122 content, due to their accelerated extracellular export in human hepatic cells starved for small metabolites including amino acids. In this context, a new role of human ELAV protein HuR has been identified. HuR, a negative regulator of miRNA function, accelerates extracellular vesicle (EV)‐mediated export of miRNAs in human cells. In stressed cells, HuR replaces miRNPs from target messages and is both necessary and sufficient for the extracellular export of corresponding miRNAs. HuR could reversibly bind miRNAs to replace them from Ago2 and subsequently itself gets freed from bound miRNAs upon ubiquitination. The ubiquitinated form of HuR is predominantly associated with multivesicular bodies (MVB) where HuR‐unbound miRNAs also reside. These MVB‐associated pool of miRNAs get exported out via EVs thereby delimiting cellular miR‐122 level during starvation. Therefore, by modulating extracellular export of miR‐122, HuR could control stress response in starved human hepatic cells.  

3.2552           Spatial control of lipid droplet proteins by the ERAD ubiquitin ligase Doa10

Ruggiano, A., Mora, G., Buxo, L. and Carvalho, P. EMBO J., 35(15), 1644-1655 (2016)   The endoplasmic reticulum (ER) plays a central role in the biogenesis of most membrane proteins. Among these are proteins localized to the surface of lipid droplets (LDs), fat storage organelles delimited by a phospholipid monolayer. The LD monolayer is often continuous with the membrane of the ER allowing certain membrane proteins to diffuse between the two organelles. In these connected organelles, how some proteins concentrate specifically at the surface of LDs is not known. Here, we show that the ERAD ubiquitin ligase Doa10 controls the levels of some LD proteins. Their degradation is dependent on the localization to the ER and appears independent of the folding state. Moreover, we show that by degrading the ER pool of these LD proteins, ERAD contributes to restrict their localization to LDs. The signals for LD targeting and Doa10‐mediated degradation overlap, indicating that these are competing events. This spatial control of protein localization is a novel function of ERAD that might contribute to generate functional diversity in a continuous membrane system.  

3.2553           Brefeldin A promotes the appearance of oligosaccharyl phosphates derived from Glc3Man9GlcNAc2-PP-dolichol within the endomembrane system of HepG2 cells

Massarweh, A., Bosco, M., Iatmanen-harbi, S., Tessier, C., Amana, L., Busca, P., Chantret, I., Gravier-Pelletier, C. and Moore, S.E.H.

1. Lipid Res., 57, 1477-1491 (2016)

  We reported an oligosaccharide diphosphodolichol (DLO) diphosphatase (DLODP) that generates dolichyl-phosphate and oligosaccharyl phosphates (OSPs) from DLO in vitro. This enzyme could underlie cytoplasmic OSP generation and promote dolichyl-phosphate recycling from truncated endoplasmic reticulum (ER)-generated DLO intermediates. However, during subcellular fractionation, DLODP distribution is closer to that of a Golgi apparatus (GA) marker than those of ER markers. Here, we examined the effect of brefeldin A (BFA), which fuses the GA with the ER on OSP metabolism. In order to increase the steady state level of truncated DLO while allowing formation of mature DLO (Glc₃Man₉GlcNAc₂-PP-dolichol), dolichyl-P-mannose Man₇GlcNAc₂-PP-dolichol mannosyltransferase was partially downregulated in HepG2 cells. We show that BFA provokes GA endomannosidase trimming of Glc₃Man₉GlcNAc₂-PP-dolichol to yield a Man₈GlcNAc₂-PP-dolichol structure that does not give rise to cytoplasmic Man₈GlcNAc₂-P. BFA also strikingly increased OSP derived from mature DLO within the endomembrane system without affecting levels of Man₇GlcNAc₂-PP-dolichol or cytoplasmic Man₇GlcNAc₂-P. The BFA-provoked increase in endomembrane-situated OSP is sensitive to nocodazole, and BFA causes partial redistribution of DLODP activity from GA- to ER-containing regions of density gradients. These findings are consistent with BFA-provoked microtubule-dependent GA-to-ER transport of a previously reported DLODP that acts to generate a novel endomembrane-situated OSP population.  

3.2554           Caspase-cleaved Tau-D421 is colocalized with the immunophilin FKBP52 in the autophagy-endolysosomal system of Alzheimer's disease neurons

Meduri, G., Guillemeau, K., Dounane, O., Sazdovitch, V., Duyckaerts, C., Chambraud, B., Baulieu, E.E. and Giustiniani, J. Neurobiology of Aging, 46, 124-137 (2016)   Pathologic modifications of the Tau protein leading to neurofibrillary tangle (NFT) formation are a common feature of a wide range of neurodegenerative diseases known as tauopathies, which include Alzheimer's disease (AD). We previously showed that the immunophilin FKBP52 physically and functionally interacts with Tau, and we recently reported that FKBP52 levels are abnormally low in AD patients' brains. To decipher the mechanism of FKBP52 decrease in AD brains, we performed multiple labeling immunohistofluorescence and lysosomal purification using postmortem brain samples of healthy controls (n = 8) and AD (n = 20) patients. Confocal analysis revealed that FKBP52 localizes to the endolysosomal system. We also report FKBP52 colocalization with the truncated Tau-D⁴²¹ in the autophagy-endolysosomal system in some AD neurons and that the decrease of FKBP52 correlates with NFT formation. Additional experiments of autophagy inhibition in Tau-inducible SH-SY5Y cells allowed demonstrating FKBP52 release in the extracellular milieu. Our findings point out the possibility that FKBP52 could be abnormally released from NFTs negative neurons in AD brains in correlation with the early pathologic Tau-D⁴²¹ neuronal accumulation.  

3.2555           Translocation of the ABC transporter ABCD4 from the endoplasmic reticulum to lysosomes requires the escort protein LMBD1

Kawaguchi, K., Okamoto, T., Morita, M. and Imanaka, T. Scientific Reports, 6:30183 (2016)   We previously demonstrated that ABCD4 does not localize to peroxisomes but rather, the endoplasmic reticulum (ER), because it lacks the NH2-terminal hydrophilic region required for peroxisomal targeting. It was recently reported that mutations in ABCD4 result in a failure to release vitamin B12 from lysosomes. A similar phenotype is caused by mutations in LMBRD1, which encodes the lysosomal membrane protein LMBD1. These findings suggested to us that ABCD4 translocated from the ER to lysosomes in association with LMBD1. In this report, it is demonstrated that ABCD4 interacts with LMBD1 and then localizes to lysosomes, and this translocation depends on the lysosomal targeting ability of LMBD1. Furthermore, endogenous ABCD4 was localized to both lysosomes and the ER, and its lysosomal localization was disturbed by knockout of LMBRD1. To the best of our knowledge, this is the first report demonstrating that the subcellular localization of the ABC transporter is determined by its association with an adaptor protein.  

3.2556           Acetylation modification regulates GRP78 secretion in colon cancer cells

Li, Z., Zhuang, M., Zhang, L., Zheng, X., Yang, P. and Li, Z. Scientific Reports, 6:30406 (2016)   High glucose-regulated protein 78 (GRP78) expression contributes to the acquisition of a wide range of phenotypic cancer hallmarks, and the pleiotropic oncogenic functions of GRP78 may result from its diverse subcellular distribution. Interestingly, GRP78 has been reported to be secreted from solid tumour cells, participating in cell-cell communication in the tumour microenvironment. However, the mechanism underlying this secretion remains elusive. Here, we report that GRP78 is secreted from colon cancer cells via exosomes. Histone deacetylase (HDAC) inhibitors blocked GRP78 release by inducing its aggregation in the ER. Mechanistically, HDAC inhibitor treatment suppressed HDAC6 activity and led to increased GRP78 acetylation; acetylated GRP78 then bound to VPS34, a class III phosphoinositide-3 kinase, consequently preventing the sorting of GRP78 into multivesicular bodies (MVBs). Of note, we found that mimicking GRP78 acetylation by substituting the lysine at residue 633, one of the deacetylated sites of HDAC6, with a glutamine resulted in decreased GRP78 secretion and impaired tumour cell growth in vitro. Our study thus reveals a hitherto-unknown mechanism of GRP78 secretion and may also provide implications for the therapeutic use of HDAC inhibitors.  

3.2557           Legionella pneumophila Type IV Effectors YlfA and YlfB Are SNARE-Like Proteins that Form Homo- and Heteromeric Complexes and Enhance the Efficiency of Vacuole Remodeling

Campodonico, E.M., Roy, C.R. and Ninio, S. Plos One, 11(7), e0159698 (2016)   Legionella pneumophila is a Gram-negative bacterium that can colonize both freshwater protozoa and human alveolar macrophages, the latter infection resulting in Legionnaires’ disease. The intracellular lifecycle of L. pneumophila requires extensive manipulation of its host cell, which is carried out by effector proteins that are translocated into the host cell through the Dot/Icm type IV secretion system. This study focuses on a pair of highly similar type IV substrates called YlfA/LegC7 and YlfB/LegC2 that were initially identified in a screen for proteins that cause growth inhibition in yeast. Analysis of truncation mutants revealed that the hydrophobic residues in the Ylf amino termini were required for localization of each protein to the membranes of host cells. Central and carboxy terminal coiled coil domains were found to mediate binding of YlfA and YlfB to themselves and to each other. In vivo, a ΔylfA ΔylfB double mutant strain of L. pneumophila was shown to be defective in establishing a vacuole that supports bacterial replication. This phenotype was subsequently correlated with a decrease in the association of endoplasmic reticulum (ER)-derived vesicles with vacuoles containing ΔylfA ΔylfB mutant bacteria. These data suggest that the Ylf proteins are membrane-associated effectors that enhance remodeling of the L. pneumophila -containing vacuole by promoting association and possibly fusion of ER-derived membrane vesicles with the bacterial compartment.  

3.2558           Uropathogenic Escherichia coli Releases Extracellular Vesicles That Are Associated with RNA

Blenkiron, C., Simonov, D., Muthukaruppan, A., Tsai, P., Dauros, P., Green, S., hong, J., Print, C.G., Swift, S. and Phillips, A. PloS One, 11(8), e0160440 (2016)   Background Bacterium-to-host signalling during infection is a complex process involving proteins, lipids and other diffusible signals that manipulate host cell biology for pathogen survival. Bacteria also release membrane vesicles (MV) that can carry a cargo of effector molecules directly into host cells. Supported by recent publications, we hypothesised that these MVs also associate with RNA, which may be directly involved in the modulation of the host response to infection. Methods and Results Using the uropathogenic Escherichia coli (UPEC) strain 536, we have isolated MVs and found they carry a range of RNA species. Density gradient centrifugation further fractionated and characterised the MV preparation and confirmed that the isolated RNA was associated with the highest particle and protein containing fractions. Using a new approach, RNA-sequencing of libraries derived from three different ‘size’ RNA populations (<50nt, 50-200nt and 200nt+) isolated from MVs has enabled us to now report the first example of a complete bacterial MV-RNA profile. These data show that MVs carry rRNA, tRNAs, other small RNAs as well as full-length protein coding mRNAs. Confocal microscopy visualised the delivery of lipid labelled MVs into cultured bladder epithelial cells and showed their RNA cargo labelled with 5-EU (5-ethynyl uridine), was transported into the host cell cytoplasm and nucleus. MV RNA uptake by the cells was confirmed by droplet digital RT-PCR of csrC. It was estimated that 1% of MV RNA cargo is delivered into cultured cells. Conclusions These data add to the growing evidence of pathogenic bacterial MV being associated a wide range of RNAs. It further raises the plausibility for MV-RNA-mediated cross-kingdom communication whereby they influence host cell function during the infection process.  

3.2559           181 VESICLE-ASSOCIATED MEMBRANE PROTEIN 7 IS CRUCIAL FOR LIPID ABSORPTION

Mansbach, C.M., Siddiqi, S. and Mahan, J.

1. Investig. Med., 54, S288 (2016)

  The rate-limiting step in lipid absorption is the exit of triacylglycerols (TAG) from the endoplasmic reticulum (ER) in a specialized transport vesicle, the prechylomicron transport vesicle (PCTV). SNARE proteins direct vesicles to target membranes. Newly synthesized proteins in vesicles constantly move to the cis Golgi. We questioned if intermittent, meal-derived PCTV contained a unique SNARE. Vesicle-associated membrane protein 7 (VAMP7), a SNARE protein previously found only in the post Golgi compartment, was identified by 2D gels in PCTV. We sought its presence in its parent, intestinal ER, and questioned its functionality in lipid transport. Methods We used our novel antirat VAMP7 antibody that identifies a 25 kDa protein on a 2D gel immunoblot (IB) to identify VAMP7. We isolated ER both by sucrose density and iodixanol gradients. Immunohistochemistry (IH) resolved by deconvolution was used to colocalize marker proteins with VAMP7 and immunoelectron microscopy (IEM) was used to colocalize VAMP7 to ER marker proteins on PCTV. Results Our intestinal, liver and kidney ER preparations were not contaminated by endosomes or Golgi as judged by the lack of rab11, syntaxin8, and GOS28 in the area of the gradient occupied by the ER proteins calreticulin and sec22. Intestinal ER but not liver or kidney ER contained VAMP7 by IB. ER-derived PCTV contained VAMP7 and the ER proteins Sar1 and rBet1 by IEM. IH showed VAMP7 to be colocalized with the ER protein PDI. To test the functionality of VAMP7 in TAG transport to the Golgi, we incubated intestinal ER with anti-VAMP7 antibody and observed its effect on delivery of ER-¹⁴ C-TAG and protein to the Golgi. Only 10% of the ¹⁴ C-TAG was transported to the Golgi on anti-VAMP7 antibody treatment as compared to preincubation of the ER with IgG. Newly synthesized ³ H-protein transport was unaffected by anti-VAMP7 antibody as was ER to Golgi TAG transport if the Golgi was preincubated with the antibody. In coimmunoprecipitation studies, VAMP7 was associated with apolipoprotein B48 (apoB48). VAMP7 does not bind to apoB48 in intestinal ER treated with proteinase K, which left a 170 kDa apoB48 fragment. Conclusions We have localized VAMP7 to intestinal ER by IH and IEM and by cell organelle separation techniques and shown that intestine but not liver or kidney ER contains VAMP7. We have also shown that VAMP7 in intestinal ER is functional in the movement of TAG in PCTV from ER to the Golgi. We speculate that VAMP7 binds to a cytosolic exposed portion of apoB48, which enables selection of the prechylomicron for inclusion in PCTV.  

3.2560           Mannosidase IA is in Quality Control Vesicles and Participates in Glycoprotein Targeting to ERAD

Ogen-Shtern, N., Avezov, E., Shenkman, M., Benyair, R. and Lederkremer, G.Z.

1. Mol. Biol., 428, 3194-3205 (2016)

  Endoplasmic reticulum-associated degradation (ERAD) of a misfolded glycoprotein in mammalian cells requires the removal of 3–4 alpha 1,2 linked mannose residues from its N-glycans. The trimming and recognition processes are ascribed to ER Mannosidase I, the ER-degradation enhancing mannosidase-like proteins (EDEMs), and the lectins OS-9 and XTP3-B, all residing in the ER, the ER-derived quality control compartment (ERQC), or quality control vesicles (QCVs). Folded glycoproteins with untrimmed glycans are transported from the ER to the Golgi complex, where they are substrates of other alpha 1,2 mannosidases, IA, IB, and IC. The apparent redundancy of these enzymes has been puzzling for many years. We have now determined that, surprisingly, mannosidase IA is not located in the Golgi but resides in QCVs. We had recently described this type of vesicles, which carry ER α1,2 mannosidase I (ERManI). We show that the overexpression of alpha class I α1,2 mannosidase IA (ManIA) significantly enhances the degradation of ERAD substrates and its knockdown stabilizes it. Our results indicate that ManIA trims mannose residues from Man₉GlcNAc₂ down to Man₅GlcNAc₂, acting in parallel with ERManI and the EDEMs, and targeting misfolded glycoproteins to ERAD.  

3.2561           Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations

Iwai, K., Minamisawa, T., Suga, K., Yajima, Y. and Shiba, K.

1. Extracellular Vesicles, 5:30829 (2016)

  Diagnostic methods that focus on the extracellular vesicles (EVs) present in saliva have been attracting great attention because of their non-invasiveness. EVs contain biomolecules such as proteins, messenger RNA (mRNA) and microRNA (miRNA), which originate from cells that release EVs, making them an ideal source for liquid biopsy. Although there have been many reports on density-based fractionation of EVs from blood and urine, the number of reports on EVs from saliva has been limited, most probably because of the difficulties in separating EVs from viscous saliva using density gradient centrifugation. This article establishes a protocol for the isolation of EVs from human saliva using density gradient centrifugation. The fractionated salivary EVs were characterized by atomic force microscopy, western blot and reverse transcription polymerase chain reaction. The results indicate that salivary EVs have a smaller diameter (47.8±12.3 nm) and higher density (1.11 g/ml) than EVs isolated from conditioned cell media (74.0±23.5 nm and 1.06 g/ml, respectively). Additionally, to improve the throughput of density-based fractionation of EVs, the original protocol was further modified by using a fixed angle rotor instead of a swinging rotor. It was also confirmed that several miRNAs were expressed strongly in the EV-marker-expressing fractions.    

3.2562           Exosomal miRNAs as cancer biomarkers and therapeutic targets

Thind, A. and Wilson, C.

1. Extracellular Vesicles, 5:31292 (2016)

  Intercommunication between cancer cells and with their surrounding and distant environments is key to the survival, progression and metastasis of the tumour. Exosomes play a role in this communication process. MicroRNA (miRNA) expression is frequently dysregulated in tumour cells and can be reflected by distinct exosomal miRNA (ex-miRNA) profiles isolated from the bodily fluids of cancer patients. Here, the potential of ex-miRNA as a cancer biomarker and therapeutic target is critically analysed. Exosomes are a stable source of miRNA in bodily fluids but, despite a number of methods for exosome extraction and miRNA quantification, their suitability for diagnostics in a clinical setting is questionable. Furthermore, exosomally transferred miRNAs can alter the behaviour of recipient tumour and stromal cells to promote oncogenesis, highlighting a role in cell communication in cancer. However, our incomplete understanding of exosome biogenesis and miRNA loading mechanisms means that strategies to target exosomes or their transferred miRNAs are limited and not specific to tumour cells. Therefore, if ex-miRNA is to be employed in novel non-invasive diagnostic approaches and as a therapeutic target in cancer, two further advances are necessary: in methods to isolate and detect ex-miRNA, and a better understanding of their biogenesis and functions in tumour-cell communication.  

3.2563           Nanoparticle analysis sheds budding insights into genetic drivers of extracellular vesicle biogenesis

Hurwitz, S.N., Conlon, M.M., Rider, M.A., Brownstein, N.C. and Meckes Jr., D.G.

1. Extracellular Vesicles, 5:31295 (2016)

  Background: Extracellular vesicles (EVs) are important mediators of cell-to-cell communication in healthy and pathological environments. Because EVs are present in a variety of biological fluids and contain molecular signatures of their cell or tissue of origin, they have great diagnostic and prognostic value. The ability of EVs to deliver biologically active proteins, RNAs and lipids to cells has generated interest in developing novel therapeutics. Despite their potential medical use, many of the mechanisms underlying EV biogenesis and secretion remain unknown. Methods: Here, we characterized vesicle secretion across the NCI-60 panel of human cancer cells by nanoparticle tracking analysis. Using CellMiner, the quantity of EVs secreted by each cell line was compared to reference transcriptomics data to identify gene products associated with vesicle secretion. Results: Gene products positively associated with the quantity of exosomal-sized vesicles included vesicular trafficking classes of proteins with Rab GTPase function and sphingolipid metabolism. Positive correlates of larger microvesicle-sized vesicle secretion included gene products involved in cytoskeletal dynamics and exocytosis, as well as Rab GTPase activation. One of the identified targets, CD63, was further evaluated for its role in vesicle secretion. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 knockout of the CD63 gene in HEK293 cells resulted in a decrease in small vesicle secretion, suggesting the importance of CD63 in exosome biogenesis. Conclusion: These observations reveal new insights into genes involved in exosome and microvesicle formation, and may provide a means to distinguish EV sub-populations. This study offers a foundation for further exploration of targets involved in EV biogenesis and secretion.  

3.2564           Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles

Boere, J., van de Lest, C.H.A., Libregts, S.F.W.M., Arkesteijn, G.J.A., Geerts, W.J.C., Nolte-t’ Hoen, E.N.M., malda, J., van Weeren, P.R. and Wauben, M.H.M.

1. Extracellular Vesicles, 5:31751 (2016)

  Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) – a prominent extracellular matrix component – it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20–200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery.  

3.2565           Regional Regulation of Purkinje Cell Dendritic Spines by Integrins and Eph/Ephrins

Heintz, T.G., Eva, R. And Fawcett, J.W.

PloS One, 11(8), e0158558 (2016)

Climbing fibres and parallel fibres compete for dendritic space on Purkinje cells in the cerebellum. Normally, climbing fibres populate the proximal dendrites, where they suppress the multiple small spines typical of parallel fibres, leading to their replacement by the few large spines that contact climbing fibres. Previous work has shown that ephrins acting via EphA4 are a signal for this change in spine type and density. We have used an in vitro culture model in which to investigate the ephrin effect on Purkinje cell dendritic spines and the role of integrins in these changes. We found that integrins α3, α5 and β4 are present in many of the dendritic spines of cultured Purkinje cells. pFAK, the main downstream signalling molecule from integrins, has a similar distribution, although the intenstity of pFAK staining and the percentage of pFAK+ spines was consistently higher in the proximal dendrites. Activating integrins with Mg2+ led to an increase in the intensity of pFAK staining and an increase in the proportion of pFAK+ spines in both the proximal and distal dendrites, but no change in spine length, density or morphology. Blocking integrin binding with an RGD-containing peptide led to a reduction in spine length, with more stubby spines on both proximal and distal dendrites. Treatment of the cultures with ephrinA3-Fc chimera suppressed dendritic spines specifically on the proximal dendrites and there was also a decrease of pFAK in spines on this domain. This effect was blocked by simultaneous activation of integrins with Mn2+. We conclude that Eph/ephrin signaling regulates proximal dendritic spines in Purkinje cells by inactivating integrin downstream signalling.

 

3.2566           Abstract LB-266: Large oncosomes reprogram prostate fibroblasts toward a pro-angiogenic phenotype

Minciacchi, V., Spinelli, C., Reis-Sobreiro, M., Zandian, M., Adam, R.M., Posadas, E.M., Micheal, F.R., Cocucci, E., Bhowmich, N.and  Di Vizio, D. Cancer Res., 76(14), LB-266 (2016)   Introduction: Cancer cells communicate with different cells in the tumor microenvironment, establishing a supportive stroma that sustains tumor development and facilitates the first steps of metastasis. Extracellular vesicles (EVs) have emerged as key functional mediators of this process. Aim of this study was to determine the mechanism of intercellular communication mediated by the atypically large EVs produced by highly migratory and metastatic tumor cells, referred to as large oncosomes (LO), and prostate fibroblasts (NAF). Methods: Filtration, differential centrifugation followed by iodixanol gradient; flow cytometry and confocal imaging; RNA-seq; kinase assay; TF array; luciferase assay; tube formation; siRNA; RT-qPCR. Results: Active AKT1 is significantly more expressed and functional in LO than in exosomes (Exo). Patients with metastatic disease express abundant active AKT1 in plasma LO. Uptake of LO harboring active AKT1 by NAF results in AKT1 and c-MYC activation. Conditioned media from LO-treated NAF, but not from Exo-treated NAF, promoted endothelial morphogenesis. The Dynamin (DNM) inhibitor Dynasore (Dyn) inhibited LO-uptake, as well as MYC activation and tube formation. Transient silencing of DNM2 significantly reduced LO uptake, suggesting that uptake occurs by phagocytosis. LO treatment increased levels of MYC targets in NAF, suggesting that MYC is involved in LO-induced reprogramming of NAF. Accordingly, MYC expression was higher in activated fibroblasts than NAF, and MYC overexpression in NAF induced hyperplasia in normal prostate epithelium in mice, suggesting MYC activation is an early event in cancer development. Summary/Conclusion: Our results indicate that tumor-derived LO induce a novel, c-MYC mediated, pro-tumorigenic reprogramming of fibroblasts that can be reverted by selectively inhibiting LO uptake.  

3.2567           The potential of endurance exercise-derived exosomes to treat metabolic diseases

Safddar, A., Saleemi, A. and Tarnopolsky, M.A. Nature Endocrinol., 12(9), 504-517 (2016)   Endurance exercise-mediated multisystemic adaptations are known to mitigate metabolism-related disorders such as obesity and type 2 diabetes mellitus (T2DM). However, the underlying molecular mechanisms that promote crosstalk between organs and orchestrate the pro-metabolic effects of endurance exercise remain unclear. Exercise-induced release of peptides and nucleic acids from skeletal muscle and other organs (collectively termed 'exerkines') has been implicated in mediating these systemic adaptations. Given that the extracellular milieu is probably not a hospitable environment for labile exerkines, a lipid vehicle-based mode of delivery has originated over the course of evolution. Two types of extracellular vesicles, exosomes and microvesicles, have been shown to contain proteins and nucleic acids that participate in a variety of physiological and pathological processes. Exosomes, in particular, have been shown to facilitate the exchange of peptides, microRNA, mRNA and mitochondrial DNA between cells and tissues. Intriguingly, circulatory extracellular vesicle content increases in an intensity-dependant manner in response to endurance exercise. We propose that the systemic benefits of exercise are modulated by exosomes and/or microvesicles functioning in an autocrine, paracrine and/or endocrine manner. Furthermore, we posit that native or modified exosomes, and/or microvesicles enriched with exerkines will have therapeutic utility in the treatment of obesity and T2DM.  

3.2568           Activation of lysosomal P2X4 by ATP transported into lysosomes via VNUT/SLC17A9 using V-ATPase generated voltage gradient as the driving force

Zhong, X.Z., Cao, Q., Sun, X. and Dong, X-P.

1. Physiol., 594(15), 4253-4266 (2016)

  The lysosome contains abundant ATP which plays important roles in lysosome functions and in cell signalling. Recently, solute carrier family 17 member 9 (SLC17A9, also known as VNUT for vesicular nucleotide transporter) proteins were suggested to function as a lysosomal ATP transporter responsible for lysosomal ATP accumulation, and P2X4 receptors were suggested to be lysosomal ion channels that are activated by luminal ATP. However, the molecular mechanism of SLC17A9 transporting ATP and the regulatory mechanism of lysosomal P2X4 are largely unknown. In this study, we report that SLC17A9-mediated ATP transport across lysosomal membranes is suppressed by Bafilomycin A1, the V-ATPase inhibitor. By measuring P2X4 activity, which is indicative of ATP transport across lysosomal membranes, we further demonstrated that SLC17A9 mainly uses voltage gradient but not pH gradient as the driving force to transport ATP into lysosomes. This study provides a molecular mechanism for lysosomal ATP transport mediated by SLC17A9. It also suggests a regulatory mechanism of lysosomal P2X4 by SLC17A9.  

3.2569           TMX1 determines cancer cell metabolism as a thiol-based modulator of ER–mitochondria Ca2+ flux

Raturi, A. et al

1. Cell Biol., 214(4), 433-444 (2016)

  The flux of Ca²⁺ from the endoplasmic reticulum (ER) to mitochondria regulates mitochondria metabolism. Within tumor tissue, mitochondria metabolism is frequently repressed, leading to chemotherapy resistance and increased growth of the tumor mass. Therefore, altered ER–mitochondria Ca²⁺ flux could be a cancer hallmark, but only a few regulatory proteins of this mechanism are currently known. One candidate is the redox-sensitive oxidoreductase TMX1 that is enriched on the mitochondria-associated membrane (MAM), the site of ER–mitochondria Ca²⁺ flux. Our findings demonstrate that cancer cells with low TMX1 exhibit increased ER Ca²⁺, accelerated cytosolic Ca²⁺ clearance, and reduced Ca²⁺ transfer to mitochondria. Thus, low levels of TMX1 reduce ER–mitochondria contacts, shift bioenergetics away from mitochondria, and accelerate tumor growth. For its role in intracellular ER–mitochondria Ca²⁺ flux, TMX1 requires its thioredoxin motif and palmitoylation to target to the MAM. As a thiol-based tumor suppressor, TMX1 increases mitochondrial ATP production and apoptosis progression.  

3.2570           Plasma exosome profiles from dairy cows with divergent fertility phenotypes

Mitchell, M.D., Scholz-Romero, K., Ree, S., Peiris, H.N., Koh, Y.Q., Meier, S., Walker, C.G., Burke, C.R., Roche, J.R., Rice, G. and Salomon, C.

1. Dairy Sci., 99(9), 7590-7601 (2016)

  Cell-to-cell communication in physiological and pathological conditions may be influenced by neighboring cells, distant tissues, or local environmental factors. Exosomes are specific subsets of extracellular vesicles that internalize and deliver their content to near and distant sites. Exosomes may play a role in the maternal-embryo crosstalk vital for the recognition and maintenance of a pregnancy; however, their role in dairy cow reproduction has not been established. This study aimed to characterize the exosome profile in the plasma of 2 strains of dairy cow with divergent fertility phenotypes. Plasma was obtained and characterized on the basis of genetic ancestry as fertile (FERT; <23% North American genetics, New Zealand Holstein-Friesian strain, n = 8) or subfertile (SUBFERT; >92% North American genetics, North American Holstein-Friesian strain, n = 8). Exosomes were isolated by differential and buoyant density centrifugation and characterized by size distribution (nanoparticle tracking analysis, NanoSight NS500, NanoSight Ltd., Amesbury, UK), the presence of CD63 (Western blot), and their morphology (electron microscopy). The total number of exosomes was determined by quantifying the immunoreactive CD63 (ExoELISA kit, System Biosciences), and the protein content established by mass spectrometry. Enriched exosome fractions were identified as cup-shape vesicles with diameters around 100 nm and positive for the CD63 marker. The concentration of exosomes was 50% greater in FERT cows. Mass spectrometry identified 104 and 117 proteins in FERT and SUBFERT cows, of which 23 and 36 were unique, respectively. Gene ontology analysis revealed enrichment for proteins involved in immunomodulatory processes and cell-to-cell communication. Although the role of exosomes in dairy cow reproduction remains to be elucidated, their quantification and content in models with divergent fertility phenotypes could provide novel information to support both physiological and genetic approaches to improving dairy cow fertility.  

3.2571           Short communication: Proteins from circulating exosomes represent metabolic state in transition dairy cows

Crookenden, M.A., walker, C.G., peiris, H., Koh, Y., Heiser, A., Loor, J.J., Moyes, K.M., Murray, A., Dukkipati, V.S.R., Kay, K., Meier, S., Roche, J.R. and Mitchell, M.D.

1. Dairy Sci., 99(9), 7661-7668 (2016)

  Biomarkers that identify prepathological disease could enhance preventive management, improve animal health and productivity, and reduce costs. Circulating extracellular vesicles, particularly exosomes, are considered to be long-distance, intercellular communication systems in human medicine. Exosomes provide tissue-specific messages of functional state and can alter the cellular activity of recipient tissues through their protein and microRNA content. We hypothesized that exosomes circulating in the blood of cows during early lactation would contain proteins representative of the metabolic state of important tissues, such as liver, which play integral roles in regulating the physiology of cows postpartum. From a total of 150 cows of known metabolic phenotype, 10 cows were selected with high (n = 5; high risk) and low (n = 5; low risk) concentrations of nonesterified fatty acids, β-hydroxybutyrate, and liver triacylglycerol during wk 1 and 2 after calving. Exosomes were extracted from blood on the day of calving (d 0) and postcalving at wk 1 and wk 4, and their protein composition was determined by mass spectroscopy. Extracellular vesicle protein concentration and the number of exosome vesicles were not affected by risk category; however, the exosome protein cargo differed between the groups, with proteins at each time point identified as being unique to the high- and low-risk groups. The proteins α-2 macroglobulin, fibrinogen, and oncoprotein-induced transcript 3 were unique to the high-risk cows on d 0 and have been associated with metabolic syndrome and liver function in humans. Their presence may indicate a more severe inflammatory state and a greater degree of liver dysfunction in the high-risk cows than in the low-risk cows, consistent with the high-risk cows’ greater plasma β-hydroxybutyrate and liver triacylglycerol concentrations. The commonly shared proteins and those unique to the low-risk category indicate a role for exosomes in immune function. The data provide preliminary evidence of a potential role for exosomes in the immune function in transition dairy cows and exosomal protein cargo as biomarkers of metabolic state.  

3.2572           The coming of age of the mitochondria–ER contact: a matter of thickness

Giacomello, M. and Pellegrini, L. Cell Death and Differentiation, 23(9), 1417-1427 (2016)   The sites of near-contact between the mitochondrion and the endoplasmic reticulum (ER) have earned a lot of attention due to their key role in the maintenance of lipid and calcium (Ca²⁺) homeostasis, in the initiation of autophagy and mitochondrial division, and in sensing metabolic shifts. At these sites, typically called MAMs (mitochondria-associated ER membranes) or MERCs (mitochondria–ER contacts), the organelles juxtapose at a distance that can range from ~10 to ~50 nm. The multifunctional role of this subcellular compartment is puzzling; further, recent studies have shown that mitochondria–ER contacts are highly plastic structures that remodel upon metabolic transitions and that their activity in controlling lipid homeostasis could be involved in Alzheimer’s disease pathogenesis. This review aims at integrating the functions of this subcellular compartment to its most characterizing and unexplored structural parameter, their ‘thickness’: that is, the width of the cleft that separates the cytosolic face of the outer mitochondrial membrane from that of the ER. We describe and discuss the reasons why the thickness of a MERC should be considered a regulated structural parameter of the cell that defines and controls its function. Further, we propose a MERC classification that will help organize the expanding field of MERCs biology and of their role in cell physiology and human disease.  

3.2573           N-Heterocyclic Carbene–Polyethylenimine Platinum Complexes with Potent in Vitro and in Vivo Antitumor Efficacy

Chekkat, N., Dahm, G., Chardon, E., Wantz, M., Sitz, J., Decossas, M., Lambert, O., Frisch, B., Rubbiani, R., Gasser, G., Guichard, G., Fournel, S. and Bellemin-Laponnaz, S. Bioconjugate Chem., 27(8), 1942-1948 (2016)   The current interest for platinum N-heterocyclic carbene complexes in cancer research stems from their impressive toxicity reported against a range of different human cancer cells. To date, the demonstration of their in vivo efficacy relative to that of established platinum-based drugs has not been specifically addressed. Here, we introduce an innovative approach to increase the NHC-Pt complex potency whereby multiple NHC-Pt(II) complexes are coordinated along a polyethylenimine polymer (PEI) chain. We show that such NHC-Pt(II)-PEI conjugates induce human cancer cell death in vitro and in vivo in a xenograft mouse model with no observable side effects in contrast to oxaliplatin. Additional studies indicate nucleus and mitochondria targeting and suggest various mechanisms of action compared to classical platinum-based anticancer drugs.  

3.2574           Lysosomal Acid Lipase Hydrolyzes Retinyl Ester and Affects Retinoid Turnover

Grumet, L. et al

1. Biol. Chem., 291(34), 17977-17987 (2016)

  Lysosomal acid lipase (LAL) is essential for the clearance of endocytosed cholesteryl ester and triglyceride-rich chylomicron remnants. Humans and mice with defective or absent LAL activity accumulate large amounts of cholesteryl esters and triglycerides in multiple tissues. Although chylomicrons also contain retinyl esters (REs), a role of LAL in the clearance of endocytosed REs has not been reported. In this study, we found that murine LAL exhibits RE hydrolase activity. Pharmacological inhibition of LAL in the human hepatocyte cell line HepG2, incubated with chylomicrons, led to increased accumulation of REs in endosomal/lysosomal fractions. Furthermore, pharmacological inhibition or genetic ablation of LAL in murine liver largely reduced in vitro acid RE hydrolase activity. Interestingly, LAL-deficient mice exhibited increased RE content in the duodenum and jejunum but decreased RE content in the liver. Furthermore, LAL-deficient mice challenged with RE gavage exhibited largely reduced post-prandial circulating RE content, indicating that LAL is required for efficient nutritional vitamin A availability. In summary, our results indicate that LAL is the major acid RE hydrolase and required for functional retinoid homeostasis.  

3.2575           Dehydroepiandrosterone-induced changes in mitochondrial proteins contribute to phenotypic alterations in hepatoma cells

Cheng, M-L., Chi, L-M., Wu, P-R. and Ho, H-Y.

Biochem. Pharmacol., 117, 20-34 (2016)

Dehydroepiandrosterone (DHEA)-induced growth arrest of hepatoma cells is associated with metabolic disturbance. Our previous study has suggested that DHEA may cause cellular energy drain. It is possible that mitochondrial dysfunction may be mechanistically implicated in DHEA-induced changes in cellular phenotype. Treatment of SK-Hep-1 cells with DHEA caused significant reduction in proliferation, colony formation, and growth in semi-solid medium. Such changes in cellular phenotype were associated with mitochondrial depolarization, increase in mitochondrial mass, and decrease in respiratory activity. Level of reactive oxygen species (ROS) increased in DHEA-treated cells. To explore the mechanistic aspect of DHEA-induced mitochondrial dysfunction, we employed SILAC approach to study the changes in the mitoproteome of SK-Hep-1 cells after DHEA treatment. Respiratory chain complex proteins such as NDUFB8 and SDHB were differentially expressed. Of mitochondrial proteins with altered expression, FAST kinase domain-containing protein 2 (FASTKD2) showed significantly reduced expression. Exogenous expression of FASTKD2 in SK-Hep-1 cells increased their resistance to growth-inhibitory effect of DHEA, though it alone did not affect cell growth. FASTKD2 expression partially reversed the effect of DHEA on mitochondria, and reduced DHEA-induced ROS generation. Our results suggest that DHEA induces changes in mitochondrial proteins and respiratory activity, and contributes to growth arrest. FASTKD2 may be an important regulator of mitochondrial physiology, and represent a downstream target for DHEA.

3.2576           The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing

Pourcelot, M., Zemirli, N., Da Costa, L.S., Loyant, R., Garcin, D., Vitour, D., Munitic, I., Vazquez, A. and Arnoult, D. BMC Biology, 14:69 (2016)   Background After viral infection and the stimulation of some pattern-recognition receptors, TANK-binding kinase I (TBK1) is activated by K63-linked polyubiquitination followed by trans-autophosphorylation. While the activated TBK1 induces type I interferon production by phosphorylating the transcription factor IRF3, the precise molecular mechanisms underlying TBK1 activation remain unclear. Results We report here the localization of the ubiquitinated and phosphorylated active form of TBK1 to the Golgi apparatus after the stimulation of RIG-I-like receptors (RLRs) or Toll-like receptor-3 (TLR3), due to TBK1 K63-linked ubiquitination on lysine residues 30 and 401. The ubiquitin-binding protein optineurin (OPTN) recruits ubiquitinated TBK1 to the Golgi apparatus, leading to the formation of complexes in which TBK1 is activated by trans-autophosphorylation. Indeed, OPTN deficiency in various cell lines and primary cells impairs TBK1 targeting to the Golgi apparatus and its activation following RLR or TLR3 stimulation. Interestingly, the Bluetongue virus NS3 protein binds OPTN at the Golgi apparatus, neutralizing its activity and thereby decreasing TBK1 activation and downstream signaling. Conclusions Our results highlight an unexpected role of the Golgi apparatus in innate immunity as a key subcellular gateway for TBK1 activation after RNA virus infection.  

3.2577           Characterisation of mouse epididymosomes reveals a complex profile of microRNAs and a potential mechanism for modification of the sperm epigenome

Reilly, J.N., McLaughlin, E.A., Stanger, S.J., Anderson, A.L., Hutcheon, K., Church, K., Mihalas, B.P., Tyagi, S., Holt, J.E., Eamens, A.L. and Nixon, B, Scientific Reports, 6:31794 (2016)   Recent evidence has shown that the sperm epigenome is vulnerable to dynamic modifications arising from a variety of paternal environment exposures and that this legacy can serve as an important determinant of intergenerational inheritance. It has been postulated that such exchange is communicated to maturing spermatozoa via the transfer of small non-protein-coding RNAs (sRNAs) in a mechanism mediated by epididymosomes; small membrane bound vesicles released by the soma of the male reproductive tract (epididymis). Here we confirm that mouse epididymosomes encapsulate an impressive cargo of >350 microRNAs (miRNAs), a developmentally important sRNA class, the majority (~60%) of which are also represented by the miRNA signature of spermatozoa. This includes >50 miRNAs that were found exclusively in epididymal sperm and epididymosomes, but not in the surrounding soma. We also documented substantial changes in the epididymosome miRNA cargo, including significant fold changes in almost half of the miRNAs along the length of the epididymis. Finally, we provide the first direct evidence for the transfer of several prominent miRNA species between mouse epididymosomes and spermatozoa to afford novel insight into a mechanism of intercellular communication by which the sRNA payload of sperm can be selectively modified during their post-testicular maturation.  

3.2578           Isolation of Intact and Functional Melanosomes from the Retinal Pigment Epithelium

Pelkonen, L., Reinsalo, M., Morin-Picardat, E., Kidron, H. and Urtti, A. PloS One, 11(8), e0160352 (2016)   Melanosomes of retinal pigment epithelium (RPE) have many vision supporting functions. Melanosome research would benefit from a method to isolate pure and characterized melanosomes. Sucrose gradient centrifugation is the most commonly used method for isolation of RPE melanosomes, but the isolated products are insufficiently characterized and their quality is unclear. Here we introduce a new gentle method for fractionation of porcine RPE that produces intact functional melanosomes with minimal cross-contamination from other cell organelles. The characterization of isolated organelles was conducted with several methods confirming the purity of the isolated melanosomal fraction (transmission electron microscopy, immunoblotting) and presence of the melanosomal membrane (fluorescence staining of melanosomal membrane, zeta potential measurement). We demonstrate that our isolation method produces RPE melanosomes with the ability to generate free phosphate (Pi) from ATP thereby proving that many membrane proteins remain functional after isolation. The isolated porcine RPE melanosomes represented V-type H+ATPase activity that was demonstrated with bafilomycin A1, a specific V-ATPase inhibitor. We anticipate that the isolation method described here can easily be optimized for the isolation of stage IV melanosomes from other pigmented cell types and tissues.  

3.2579           Tylophorine Analog DCB-3503 Inhibited Cyclin D1 Translation through Allosteric Regulation of Heat Shock Cognate Protein 70

Wang, Y., Lam, W., Chen, S-R., Guan, F-L., Dutchman, G.E., Francis, s., Baker, D.C. and Cheng, Y-C. Scientific Reports, 6:32832 (2016)   Tylophorine analog DCB-3503 is a potential anticancer and immunosuppressive agent that suppresses the translation of cellular regulatory proteins, including cyclin D1, at the elongation step. However, the molecular mechanism underlying this phenomenon remains unknown. This study demonstrates that DCB-3503 preferentially binds to heat shock cognate protein 70 (HSC70), which is a determinant for cyclin D1 translation by binding to the 3′-untranslated region (3′ UTR) of its mRNA. DCB-3503 allosterically regulates the ATPase and chaperone activities of HSC70 by promoting ATP hydrolysis in the presence of specific RNA binding motifs (AUUUA) of cyclin D1 mRNA. The suppression of cyclin D1 translation by DCB-3503 is not solely caused by perturbation of the homeostasis of microRNAs, although the microRNA processing complex is dissociated with DCB-3503 treatment. This study highlights a novel regulatory mechanism of protein translation with AUUUA motifs in the 3′ UTR of mRNA by HSC70, and its activity can be allosterically modulated by DCB-3503. DCB-3503 may be used to treat malignancies, such as hepatocellular carcinoma or breast cancer with elevated expression of cyclin D1.  

3.2580           Restriction of Aerobic Metabolism by Acquired or Innate Arylsulfatase B Deficiency: A New Approach to the Warburg Effect

Bhattacharyya, S., Feferman, L. and Tobocman, J.K. Scientific Reports, 6:32885 (2016)   Aerobic respiration is required for optimal efficiency of metabolism in mammalian cells. Under circumstances when oxygen utilization is impaired, cells survive by anerobic metabolism. The malignant cell has cultivated the use of anerobic metabolism in an aerobic environment, the Warburg effect, but the explanation for this preference is not clear. This paper presents evidence that deficiency of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase), either innate or acquired, helps to explain the Warburg phenomenon. ARSB is the enzyme that removes 4-sulfate groups from the non-reducing end of chondroitin 4-sulfate and dermatan sulfate. Previous reports indicated reduced ARSB activity in malignancy and replication of the effects of hypoxia by decline in ARSB. Hypoxia reduced ARSB activity, since molecular oxygen is needed for post-translational modification of ARSB. In this report, studies were performed in human HepG2 cells and in hepatocytes from ARSB-deficient and normal C57BL/6J control mice. Decline of ARSB, in the presence of oxygen, profoundly reduced the oxygen consumption rate and increased the extracellular acidification rate, indicating preference for aerobic glycolysis. Specific study findings indicate that decline in ARSB activity enhanced aerobic glycolysis and impaired normal redox processes, consistent with a critical role of ARSB and sulfate reduction in mammalian metabolism.  

3.2581           Functional Study and Mapping Sites for Interaction with the Target Enzyme in Retinal Degeneration 3 (RD3) Protein

Peshenko, I.V., Olshevskaya, E.V. and Dizhoor, A.M.

1. Biol. Chem., 291(37), 19713-19723 (2016)

  Retinal degeneration 3 (RD3) protein, essential for normal expression of retinal membrane guanylyl cyclase (RetGC) in photoreceptor cells, blocks RetGC catalytic activity and stimulation by guanylyl cyclase-activating proteins (GCAPs). In a mouse retina, RD3 inhibited both RetGC1 and RetGC2 isozymes. Photoreceptors in the rd3/rd3 mouse retinas lacking functional RD3 degenerated more severely than in the retinas lacking both RetGC isozymes, consistent with a hypothesis that the inhibitory activity of RD3 has a functional role in photoreceptors. To map the potential target-binding site(s) on RD3, short evolutionary conserved regions of its primary structure were scrambled and the mutations were tested for the RD3 ability to inhibit RetGC1 and co-localize with the cyclase in co-transfected cells. Substitutions in 4 out of 22 tested regions, ⁸⁷KIHP⁹⁰, ⁹³CGPAI⁹⁷, ⁹⁹RFRQ¹⁰², and ¹¹⁹RSVL¹²², reduced the RD3 apparent affinity for the cyclase 180–700-fold. Changes of amino acid sequences outside the Lys⁸⁷–Leu¹²² central portion of the molecule either failed to prevent RD3 binding to the cyclase or had a much smaller effect. Mutations in the ⁹³CGPAI⁹⁷ portion of a predicted central α-helix most drastically suppressed the inhibitory activity of RD3 and disrupted RD3 co-localization with RetGC1 in HEK293 cells. Different side chains replacing Cys⁹³ profoundly reduced RD3 affinity for the cyclase, irrespective of their relative helix propensities. We conclude that the main RetGC-binding interface on RD3 required for the negative regulation of the cyclase localizes to the Lys⁸⁷–Leu¹²² region.  

3.2582           Microvesicles from brain-extract—treated mesenchymal stem cells improve neurological functions in a rat model of ischemic stroke

Lee, J.Y., Kim, E., Choi, S-M., Kim, D-W., Kim, K.P., Lee, I. and Kimm, H-S. Scientific Reports, 6:33038 (2016)   Transplantation of mesenchymal stem cells (MSCs) was reported to improve functional outcomes in a rat model of ischemic stroke, and subsequent studies suggest that MSC-derived microvesicles (MVs) can replace the beneficial effects of MSCs. Here, we evaluated three different MSC-derived MVs, including MVs from untreated MSCs (MSC-MVs), MVs from MSCs treated with normal rat brain extract (NBE-MSC-MVs), and MVs from MSCs treated with stroke-injured rat brain extract (SBE-MSC-MVs), and tested their effects on ischemic brain injury induced by permanent middle cerebral artery occlusion (pMCAO) in rats. NBE-MSC-MVs and SBE-MSC-MVs had significantly greater efficacy than MSC-MVs for ameliorating ischemic brain injury with improved functional recovery. We found similar profiles of key signalling proteins in NBE-MSC-MVs and SBE-MSC-MVs, which account for their similar therapeutic efficacies. Immunohistochemical analyses suggest that brain-extract—treated MSC-MVs reduce inflammation, enhance angiogenesis, and increase endogenous neurogenesis in the rat brain. We performed mass spectrometry proteomic analyses and found that the total proteomes of brain-extract—treated MSC-MVs are highly enriched for known vesicular proteins. Notably, MSC-MV proteins upregulated by brain extracts tend to be modular for tissue repair pathways. We suggest that MSC-MV proteins stimulated by the brain microenvironment are paracrine effectors that enhance MSC therapy for stroke injury.  

3.2583           Chromatin remodeling during the in vivo glial differentiation in early Drosophila embryos

Ye, Y., Gu, L., Chen, X., Shi, J., Zhang, X. and Jiang, C. Scientific Reports, 6:33422 (2016)   Chromatin remodeling plays a critical role in gene regulation and impacts many biological processes. However, little is known about the relationship between chromatin remodeling dynamics and in vivo cell lineage commitment. Here, we reveal the patterns of histone modification change and nucleosome positioning dynamics and their epigenetic regulatory roles during the in vivo glial differentiation in early Drosophila embryos. The genome-wide average H3K9ac signals in promoter regions are decreased in the glial cells compared to the neural progenitor cells. However, H3K9ac signals are increased in a group of genes that are up-regulated in glial cells and involved in gliogenesis. There occurs extensive nucleosome remodeling including shift, loss, and gain. Nucleosome depletion regions (NDRs) form in both promoters and enhancers. As a result, the associated genes are up-regulated. Intriguingly, NDRs form in two fashions: nucleosome shift and eviction. Moreover, the mode of NDR formation is independent of the original chromatin state of enhancers in the neural progenitor cells.  

3.2584           Quantitative Lipid Droplet Proteome Analysis Identifies Annexin A3 as a Cofactor for HCV Particle Production

Rösch, K., Kwiatkowski, M., Hofman, S. et al Cell Reports, 16, 3219-3231 (2016)   Lipid droplets are vital to hepatitis C virus (HCV) infection as the putative sites of virion assembly, but morphogenesis and egress of virions remain ill defined. We performed quantitative lipid droplet proteome analysis of HCV-infected cells to identify co-factors of that process. Our results demonstrate that HCV disconnects lipid droplets from their metabolic function. Annexin A3 (ANXA3), a protein enriched in lipid droplet fractions, strongly impacted HCV replication and was characterized further: ANXA3 is recruited to lipid-rich fractions in HCV-infected cells by the viral core and NS5A proteins. ANXA3 knockdown does not affect HCV RNA replication but severely impairs virion production with lower specific infectivity and higher density of secreted virions. ANXA3 is essential for the interaction of viral envelope E2 with apolipoprotein E (ApoE) and for trafficking, but not lipidation, of ApoE in HCV-infected cells. Thus, we identified ANXA3 as a regulator of HCV maturation and egress.  

3.2585           Interaction of the Human Papillomavirus E6 Oncoprotein with Sorting Nexin 27 Modulates Endocytic Cargo Transport Pathways

Ganti, K., Massimi, P., Monzo-merino, J., Tomaic, V., Pim, D., Playford, M.P., Lizano, M., Roberts, S., Kranjec, C., Doorbar, J. and Banks, L. PloS Pathogens, 12(9), e1005854 (2016)   A subset of high-risk Human Papillomaviruses (HPVs) are the causative agents of a large number of human cancers, of which cervical is the most common. Two viral oncoproteins, E6 and E7, contribute directly towards the development and maintenance of malignancy. A characteristic feature of the E6 oncoproteins from cancer-causing HPV types is the presence of a PDZ binding motif (PBM) at its C-terminus, which confers interaction with cellular proteins harbouring PDZ domains. Here we show that this motif allows E6 interaction with Sorting Nexin 27 (SNX27), an essential component of endosomal recycling pathways. This interaction is highly conserved across E6 proteins from multiple high-risk HPV types and is mediated by a classical PBM-PDZ interaction but unlike many E6 targets, SNX27 is not targeted for degradation by E6. Rather, in HPV-18 positive cell lines the association of SNX27 with components of the retromer complex and the endocytic transport machinery is altered in an E6 PBM-dependent manner. Analysis of a SNX27 cargo, the glucose transporter GLUT1, reveals an E6-dependent maintenance of GLUT1 expression and alteration in its association with components of the endocytic transport machinery. Furthermore, knockdown of E6 in HPV-18 positive cervical cancer cells phenocopies the loss of SNX27, both in terms of GLUT1 expression levels and its vesicular localization, with a concomitant marked reduction in glucose uptake, whilst loss of SNX27 results in slower cell proliferation in low nutrient conditions. These results demonstrate that E6 interaction with SNX27 can alter the recycling of cargo molecules, one consequence of which is modulation of nutrient availability in HPV transformed tumour cells.  

3.2586           Immunomodulatory effects of exosomes produced by virus-infected cells

Petrik, J.

Transfusion and Apheresis Science, 55, 84-91 (2016)

Viruses have developed a spectrum of ways to modify cellular pathways to hijack the cell machinery for the synthesis of their nucleic acid and proteins. Similarly, they use intracellular vesicular mechanisms of trafficking for their assembly and eventual release, with a number of viruses acquiring their envelope from internal or plasma cell membranes. There is an increasing number of reports on viral exploitation of cell secretome pathways to avoid recognition and stimulation of the immune response. Extracellular vesicles (EV) containing viral particles have been shown to shield viruses after exiting the host cell, in some cases challenging the boundaries between viral groups traditionally characterised as enveloped and non-enveloped. Apart from viral particles, EV can spread the virus also carrying viral genome and can modify the target cells through their cargo of virus-coded miRNAs and proteins as well as selectively packaged cellular mRNAs, miRNAs, proteins and lipids, differing in composition and quantities from the cell of origin.

 

3.2587           Localization, Shedding, Regulation and Function of Aminopeptidase N/CD13 on Fibroblast like Synoviocytes

Morgan, R.L., Behbahani-Nejad, N., Endres, J., Amin, M.A., Lepore, N.J:, Du, Y., Urqehart, A., Chung, K.C. and Fox, D.A. PloS One, 11(9), e0162008 (2016)   Aminopeptidase N/CD13 is highly expressed by fibroblast like synoviocytes (FLS) and may play a role in rheumatoid arthritis (RA). CD13 was previously detected in human synovial fluid where it was significantly increased in RA compared to osteoarthritis. In this study we found that CD13 in biological fluids (plasma, synovial fluid, FLS culture supernatant) is present as both a soluble molecule and on extracellular vesicles, including exosomes, as assessed by differential ultracentrifugation and density gradient separation. Having determined CD13 could be released as a soluble molecule from FLS, we examined potential mechanisms by which CD13 might be shed from the FLS membrane. The use of protease inhibitors revealed that CD13 is cleaved from the FLS surface by metalloproteinases. siRNA treatment of FLS revealed one of those proteases to be MMP14. We determined that pro-inflammatory cytokines (TNFα, IFNγ, IL-17) upregulated CD13 mRNA in FLS, which may contribute to the increased CD13 in RA synovium and synovial fluid. Inhibition of CD13 function by either inhibitors of enzymatic activity or anti-CD13 antibodies resulted in decreased growth and diminished migration of FLS. This suggests that CD13 may be involved in the pathogenic hyperplasia of RA FLS. This data expands potential roles for CD13 in the pathogenesis of RA.  

3.2588           Vimentin, a Novel NF-κB Regulator, Is Required for Meningitic Escherichia coli K1-Induced Pathogen Invasion and PMN Transmigration across the Blood-Brain Barrier

Huang, S-H., Chi, F., Peng, L., Bo, T., Zhang, B., Liu, L-Q., Wu, X., Mor-Vaknin, N., Markovitz, D.M., Cao, H.and Zhou, Y-H. PloS One, 11(9), e0162641 (2016)   Background NF-κB activation, pathogen invasion, polymorphonuclear leukocytes (PMN) transmigration (PMNT) across the blood-brain barrier (BBB) are the pathogenic triad hallmark features of bacterial meningitis, but the mechanisms underlying these events remain largely unknown. Vimentin, which is a novel NF-κB regulator, is the primary receptor for the major Escherichia coli K1 virulence factor IbeA that contributes to the pathogenesis of neonatal bacterial sepsis and meningitis (NSM). We have previously shown that IbeA-induced NF-κB signaling through its primary receptor vimentin as well as its co-receptor PTB-associated splicing factor (PSF) is required for pathogen penetration and leukocyte transmigration across the BBB. This is the first in vivo study to demonstrate how vimentin and related factors contributed to the pathogenic triad of bacterial meningitis. Methodology/Principal Findings The role of vimentin in IbeA+ E. coli K1-induced NF-κB activation, pathogen invasion, leukocyte transmigration across the BBB has now been demonstrated by using vimentin knockout (KO) mice. In the in vivo studies presented here, IbeA-induced NF-κB activation, E. coli K1 invasion and polymorphonuclear neutrophil (PMN) transmigration across the BBB were significantly reduced in Vim-/- mice. Decreased neuronal injury in the hippocampal dentate gyrus was observed in Vim-/- mice with meningitis. The major inflammatory regulator α7 nAChR and several signaling molecules contributing to NF-κB activation (p65 and p-CamKII) were significantly reduced in the brain tissues of the Vim-/- mice with E. coli meningitis. Furthermore, Vim KO resulted in significant reduction in neuronal injury and in α7 nAChR-mediated calcium signaling. Conclusion/Significance Vimentin, a novel NF-κB regulator, plays a detrimental role in the host defense against meningitic infection by modulating the NF-κB signaling pathway to increase pathogen invasion, PMN recruitment, BBB permeability and neuronal inflammation. Our findings provide the first evidence for Vim-dependent mechanisms underlying the pathogenic triad of bacterial meningitis.  

3.2589           Isolation of human trophoblastic extracellular vesicles and characterization of their cargo and antiviral activity

Ouyang, Y., Bayer, A., Chu, T., Tyurin, V., Kagan, V., Morelli, A.E., Coyne, C.B. and Sadovsky, Y. Placenta, 47, 86-95 (2016)   Introduction Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs. Methods We isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs. Results We found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts. Discussion When compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication.  

3.2590           A novel affinity-based method for the isolation of highly purified extracellular vesicles

Nakai, W., Yoshida, T., Diez, D., Miyatake, Y., Nishibu, T., Imawaka, N., Narusem K., Sadamura, Y. and Hanayama, R. Scientifica Reports, 6:33935 (2016)   Extracellular vesicles (EVs) such as exosomes and microvesicles serve as messengers of intercellular network, allowing exchange of cellular components between cells. EVs carry lipids, proteins, and RNAs derived from their producing cells, and have potential as biomarkers specific to cell types and even cellular states. However, conventional methods (such as ultracentrifugation or polymeric precipitation) for isolating EVs have disadvantages regarding purity and feasibility. Here, we have developed a novel method for EV purification by using Tim4 protein, which specifically binds the phosphatidylserine displayed on the surface of EVs. Because the binding is Ca2+-dependent, intact EVs can be easily released from Tim4 by adding Ca2+ chelators. Tim4 purification, which we have applied to cell conditioned media and biofluids, is capable of yielding EVs of a higher purity than those obtained using conventional methods. The lower contamination found in Tim4-purified EV preparations allows more EV-specific proteins to be detected by mass spectrometry, enabling better characterization and quantification of different EV populations’ proteomes. Tim4 protein can also be used as a powerful tool for quantification of EVs in both ELISA and flow cytometry formats. Thus, the affinity of Tim4 for EVs will find abundant applications in EV studies.  

3.2591           Phosphatidic Acid Produced by RalA-activated PLD2 Stimulates Caveolae-mediated Endocytosis and Trafficking in Endothelial Cells

Jiang, Y., Sverdlov, M.S., Toth, P.T., Huang, L.S., Du, G., Liu, Y., Natarajan, V. and Minshall, R.D:

1. Biol. Chem., 291(39), 20729-20738 (2016)

  Caveolae are the primary route for internalization and transendothelial transport of macromolecules, such as insulin and albumin. Caveolae-mediated endocytosis is activated by Src-dependent caveolin-1 (Cav-1) phosphorylation and subsequent recruitment of dynamin-2 and filamin A (FilA), which facilitate vesicle fission and trafficking, respectively. Here, we tested the role of RalA and phospholipase D (PLD) signaling in the regulation of caveolae-mediated endocytosis and trafficking. The addition of albumin to human lung microvascular endothelial cells induced the activation of RalA within minutes, and siRNA-mediated down-regulation of RalA abolished fluorescent BSA uptake. Co-immunoprecipitation studies revealed that albumin induced the association between RalA, Cav-1, and FilA; however, RalA knockdown with siRNA did not affect FilA recruitment to Cav-1, suggesting that RalA was not required for FilA and Cav-1 complex formation. Rather, RalA probably facilitates caveolae-mediated endocytosis by activating downstream effectors. PLD2 was shown to be activated by RalA, and inhibition of PLD2 abolished Alexa-488-BSA uptake, indicating that phosphatidic acid (PA) generated by PLD2 may facilitate caveolae-mediated endocytosis. Furthermore, using a PA biosensor, GFP-PASS, we observed that BSA induced an increase in PA co-localization with Cav-1-RFP, which could be blocked by a dominant negative PLD2 mutant. Total internal reflection fluorescence microscopy studies of Cav-1-RFP also showed that fusion of caveolae with the basal plasma membrane was dependent on PLD2 activity. Thus, our results suggest that the small GTPase RalA plays an important role in promoting invagination and trafficking of caveolae, not by potentiating the association between Cav-1 and FilA but by stimulating PLD2-mediated generation of phosphatidic acid.  

3.2592           Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation

Wong, M-T. and Chen, S.S.

1. Virol., 90(20), 9075-9095 (2016)

  Hepatitis C virus (HCV) infection reorganizes cellular membranes to create an active viral replication site named the membranous web (MW). The role that human choline kinase-α (hCKα) plays in HCV replication remains elusive. Here, we first showed that hCKα activity, not the CDP-choline pathway, promoted viral RNA replication. Confocal microscopy and subcellular fractionation of HCV-infected cells revealed that a small fraction of hCKα colocalized with the viral replication complex (RC) on the endoplasmic reticulum (ER) and that HCV infection increased hCKα localization to the ER. In the pTM-NS3-NS5B model, NS3-NS5B expression increased the localization of the wild-type, not the inactive D288A mutant, hCKα on the ER, and hCKα activity was required for effective trafficking of hCKα and NS5A to the ER. Coimmunoprecipitation showed that hCKα was recruited onto the viral RC presumably through its binding to NS5A domain 1 (D1). hCKα silencing or treatment with CK37, an hCKα activity inhibitor, abolished HCV-induced MW formation. In addition, hCKα depletion hindered NS5A localization on the ER, interfered with NS5A and NS5B colocalization, and mitigated NS5A-NS5B interactions but had no apparent effect on NS5A-NS4B and NS4B-NS5B interactions. Nevertheless, hCKα activity was not essential for the binding of NS5A to hCKα or NS5B. These findings demonstrate that hCKα forms a complex with NS5A and that hCKα activity enhances the targeting of the complex to the ER, where hCKα protein, not activity, mediates NS5A binding to NS5B, thereby promoting functional membranous viral RC assembly and viral RNA replication.  

3.2593           The impact of various preanalytical treatments on the phenotype of small extracellular vesicles in blood analyzed by protein microarray

Bæk, R., Søndergaard, E.K.L., varming, K. and Jørgensen, M.M.

1. Immunol. Methods, 438, 11-20 (2016)

  The research field of extracellular vesicles (EVs) is increasing immensely and the potential uses of EVs seem endless. They are found in large numbers in various body fluids, and blood samples may well serve as liquid biopsies. However, these small membrane-derived entities of cellular origin are not straightforward to work with in regard to isolation and characterization. A broad range of relevant preanalytical issues was tested, with a focus on the phenotypic impact of smaller EVs. The influences of the i) blood collection tube used, ii) incubation time before the initial centrifugation, iii) transportation/physical stress, iv) storage temperature and time (short term and long term), v) choice of centrifugation protocol, vi) freeze-thaw cycles, and vii) exosome isolation procedure (ExoQuick™) were examined. To identify the impact of the preanalytical treatments, the relative amounts (detected signal intensities of CD9-, CD63- and/or CD81-positive) and phenotypes of small EVs were analyzed using the multiplexed antibody-based microarray technology, termed the EV Array. The analysis encompassed 15 surface- or surface-related markers, including CD9, CD63, CD81, CD142, and Annexin V. This study revealed that samples collected in different blood collection tubes suffered to varying degrees from the preanalytical treatments tested here. There is no unequivocal answer to the questions asked. However, in general, the period of time and prospective transportation before the initial centrifugation, choice of centrifugation protocol, and storage temperature were observed to have major impacts on the samples. On the contrary, long-term storage and freeze-thawing seemed to not have a critical influence. Hence, there are pros and cons of any choice regarding sample collection and preparation and may very well be analysis dependent. However, to compare samples and results, it is important to ensure that all samples are of the same type and have been handled similarly.  

3.2594           Membrane Binding by CHMP7 Coordinates ESCRT-III-Dependent Nuclear Envelope Reformation

Olmos, Y., Perdrix-Rosell, A. and Carlton, J.G. Current Biology, 26, 2635-2641 (2016)   In addition to its role in membrane abscission during cytokinesis, viral budding, endosomal sorting, and plasma membrane repair [1], the endosomal sorting complex required for transport-III (ESCRT-III) machinery has recently been shown to seal holes in the reforming nuclear envelope (NE) during mitotic exit [2 and 3]. ESCRT-III also acts during interphase to repair the NE upon migration-induced rupture [4 and 5], highlighting its key role as an orchestrator of membrane integrity at this organelle. While NE localization of ESCRT-III is dependent upon the ESCRT-III component CHMP7 [3], it is unclear how this complex is able to engage nuclear membranes. Here we show that the N terminus of CHMP7 acts as a novel membrane-binding module. This membrane-binding ability allows CHMP7 to bind to the ER, an organelle continuous with the NE, and it provides a platform to direct NE recruitment of ESCRT-III during mitotic exit. CHMP7’s N terminus comprises tandem Winged-Helix domains [6], and, by using homology modeling and structure-function analysis, we identify point mutations that disrupt membrane binding and prevent both ER localization of CHMP7 and its subsequent enrichment at the reforming NE. These mutations also prevent assembly of downstream ESCRT-III components at the reforming NE and proper establishment of post-mitotic nucleo-cytoplasmic compartmentalization. These data identify a novel membrane-binding activity within an ESCRT-III subunit that is essential for post-mitotic nuclear regeneration.  

3.2595           Analysis of Yeast Extracellular Vesicles

Rodrigues, M.L., Oliveira, D.L., vargas, G., Girard-Dias, W., Franzen, A.J., Frases, S., Miranda, K. and Nimrichter, L. Methods in Mol. Biol., 1459, 175-190 (2016)   Extracellular vesicles (EV) are important carriers of biologically active components in a number of organisms, including fungal cells. Experimental characterization of fungal EVs suggested that these membranous compartments are likely involved in the regulation of several biological events. In fungal pathogens, these events include mechanisms of disease progression and/or control, suggesting potential targets for therapeutic intervention or disease prophylaxis. In this manuscript we describe methods that have been used in the last 10 years for the characterization of EVs produced by yeast forms of several fungal species. Experimental approaches detailed in this chapter include ultracentrifugation methods for EV fractionation, chromatographic approaches for analysis of EV lipids, microscopy techniques for analysis of both intracellular and extracellular vesicular compartments, interaction of EVs with host cells, and physical chemical analysis of EVs by dynamic light scattering.  

3.2596           Functional exosome-mimic for delivery of siRNA to cancer: in vitro and in vivo evaluation

Yang, Z., Xie, J., Zhu, J., kang, C., Chiang, C., Wang, X., Wang, X., Kuang,k T., Chen, F., Chen, Z., Zhang, A., Yu, B., Lee, R.J., Teng, L.and Lee, L.J.

1. Controlled Release, 243, 160-171 (2016)

  Exosomes, the smallest subgroup of extracellular vesicles, have been recognized as extracellular organelles that contain genetic and proteomic information for long distance intercellular communication. Exosome-based drug delivery is currently a subject of intensive research. Here, we report a novel strategy to produce nanoscale exosome-mimics (EMs) in sufficient quantity for gene delivery in cancer both in vitro and in vivo. Size-controllable EMs were generated at a high yield by serial extrusion of non-tumorigenic epithelial MCF-10A cells through filters with different pore sizes. siRNA was then encapsulated into the EMs by electroporation. Biosafety and uptake efficiency of the EMs were evaluated both in vitro and in vivo. The mechanism underlying their cellular endocytosis was also studied.  

3.2597           Outer Membrane Vesicle Production Facilitates LPS Remodeling and Outer Membrane Maintenance in Salmonella during Environmental Transitions

Bonnington, K. and Kuehn, M.J. mBio, 7(5), e01532-16 (2016)   The ability of Gram-negative bacteria to carefully modulate outer membrane (OM) composition is essential to their survival. However, the asymmetric and heterogeneous structure of the Gram-negative OM poses unique challenges to the cell’s successful adaption to rapid environmental transitions. Although mechanisms to recycle and degrade OM phospholipid material exist, there is no known mechanism by which to remove unfavorable lipopolysaccharide (LPS) glycoforms, except slow dilution through cell growth. As all Gram-negative bacteria constitutively shed OM vesicles (OMVs), we propose that cells may utilize OMV formation as a way to selectively remove environmentally disadvantageous LPS species. We examined the native kinetics of OM composition during physiologically relevant environmental changes in Salmonella enterica, a well-characterized model system for activation of PhoP/Q and PmrA/B two-component systems (TCSs). In response to acidic pH, toxic metals, antimicrobial peptides, and lack of divalent cations, these TCSs modify the LPS lipid A and core, lengthen the O antigen, and upregulate specific OM proteins. An environmental change to PhoP/Q- and PmrA/B-activating conditions simultaneously induced the addition of modified species of LPS to the OM, downregulation of previously dominant species of LPS, greater OMV production, and increased OMV diameter. Comparison of the relative abundance of lipid A species present in the OM and the newly budded OMVs following two sets of rapid environmental shifts revealed the retention of lipid A species with modified phosphate moieties in the OM concomitant with the selective loss of palmitoylated species via vesiculation following exposure to moderately acidic environmental conditions.  

3.2598           Generation, Quantification, and Tracing of Metabolically Labeled Fluorescent Exosomes

Coscia, C., Parolini, I., Sanchez, M., Biffoni, M., Boussadia, Z., Zanetti, C., Fiani, M.L. and Sargiacomo, M. Methods in Mol. Biol., 1448, 217-235 (2016)   Over the last 10 years, the constant progression in exosome (Exo)-related studies highlighted the importance of these cell-derived nano-sized vesicles in cell biology and pathophysiology. Functional studies on Exo uptake and intracellular trafficking require accurate quantification to assess sufficient and/or necessary Exo particles quantum able to elicit measurable effects on target cells. We used commercially available BODIPY^® fatty acid analogues to label a primary melanoma cell line (Me501) that highly and spontaneously secrete nanovesicles. Upon addition to cell culture, BODIPY fatty acids are rapidly incorporated into major phospholipid classes ultimately producing fluorescent Exo as direct result of biogenesis. Our metabolic labeling protocol produced bright fluorescent Exo that can be examined and quantified with conventional non-customized flow cytometry (FC) instruments by exploiting their fluorescent emission rather than light-scattering detection. Furthermore, our methodology permits the measurement of single Exo-associated fluorescence transfer to cells making quantitative the correlation between Exo uptake and activation of cellular processes. Thus the protocol presented here appears as an appropriate tool to who wants to investigate mechanisms of Exo functions in that it allows for direct and rapid characterization and quantification of fluorescent Exo number, intensity, size, and eventually evaluation of their kinetic of uptake/secretion in target cells.  

3.2599           Mechanism of Fibronectin Binding to Human Trabecular Meshwork Exosomes and Its Modulation by Dexamethasone

Dismuke, W.M., Klingeborn, M. and Stamer, W.D. PloS One, 11(10), e0165326 (2016)   Exosomes are emerging as important mediators of cell-matrix interactions by means of specific adhesion proteins. Changes in the tissue-specific exosomal protein expression may underlie pathological conditions whereby extracellular matrix turnover and homeostasis is disrupted. Ocular hypertension due to extracellular matrix accumulation in the trabecular meshwork is a hallmark of glucocorticoid-induced glaucoma. In the trabecular meshwork, exosomal fibronectin mediates cell matrix interactions at cellular structures called “invadosomes”. Trabecular meshwork cells use invadosomes to turn over their surrounding matrix and maintain passageways for flow of aqueous humor. In this study, we observed that human trabecular meshwork explants treated with dexamethasone released exosomes with significantly reduced amounts of fibronectin bound per exosome. Further, we found that exosome-fibronectin binding is heparan sulfate-dependent, consistent with our observation that trabecular meshwork exosomes are enriched in the heparin/heparan sulfate binding annexins A2 and A6. In this way, dexamethasone-treated explants released exosomes with a significant reduction in annexin A2 and A6 per exosome. Interestingly, we did not detect exosomal matrix metalloproteinases, but we identified abundant dipeptidyl peptidase 4, a serine protease whose activity was reduced on exosomes isolated from dexamethasone-treated explants. Together, our findings demonstrate mechanistically how corticosteroid-induced alterations in exosomal adhesion cargo and properties can account for the pathological matrix accumulation seen in many glaucoma patients.  

3.2600           Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice

Nagano, M., Ishikawa, T., Fujiwara, M., Fukao, Y., Kawano, Y., Kawai-Yamada, M. and Shimamoto, K. Plant Cell, 28(8), 1966-1983 (2016)   Numerous plant defense-related proteins are thought to congregate in plasma membrane microdomains, which consist mainly of sphingolipids and sterols. However, the extent to which microdomains contribute to defense responses in plants is unclear. To elucidate the relationship between microdomains and innate immunity in rice (Oryza sativa), we established lines in which the levels of sphingolipids containing 2-hydroxy fatty acids were decreased by knocking down two genes encoding fatty acid 2-hydroxylases (FAH1 and FAH2) and demonstrated that microdomains were less abundant in these lines. By testing these lines in a pathogen infection assay, we revealed that microdomains play an important role in the resistance to rice blast fungus infection. To illuminate the mechanism by which microdomains regulate immunity, we evaluated changes in protein composition, revealing that microdomains are required for the dynamics of the Rac/ROP small GTPase Rac1 and respiratory burst oxidase homologs (Rbohs) in response to chitin elicitor. Furthermore, FAHs are essential for the production of reactive oxygen species (ROS) after chitin treatment. Together with the observation that RbohB, a defense-related NADPH oxidase that interacts with Rac1, is localized in microdomains, our data indicate that microdomains are required for chitin-induced immunity through ROS signaling mediated by the Rac1-RbohB pathway.  

3.2601           Hepatitis B virus inhibits insulin receptor signaling and impairs liver regeneration via intracellular retention of the insulin receptor

Barthel, S.R., Medvedev, R., Heinrich, T., Büchner, S.M., Kettern, N. and Hildt, E. Cell. Mol. Life Sci., 73(21), 4121-4140 (2016)   Hepatitis B virus (HBV) causes severe liver disease but the underlying mechanisms are incompletely understood. During chronic HBV infection, the liver is recurrently injured by immune cells in the quest for viral elimination. To compensate tissue injury, liver regeneration represents a vital process which requires proliferative insulin receptor signaling. This study aims to investigate the impact of HBV on liver regeneration and hepatic insulin receptor signaling. After carbon tetrachloride-induced liver injury, liver regeneration is delayed in HBV transgenic mice. These mice show diminished hepatocyte proliferation and increased expression of fibrosis markers. This is in accordance with a reduced activation of the insulin receptor although HBV induces expression of the insulin receptor via activation of NF-E2-related factor 2. This leads to increased intracellular amounts of insulin receptor in HBV expressing hepatocytes. However, intracellular retention of the receptor simultaneously reduces the amount of functional insulin receptors on the cell surface and thereby attenuates insulin binding in vitro and in vivo. Intracellular retention of the insulin receptor is caused by elevated amounts of α-taxilin, a free syntaxin binding protein, in HBV expressing hepatocytes preventing proper targeting of the insulin receptor to the cell surface. Consequently, functional analyses of insulin responsiveness revealed that HBV expressing hepatocytes are less sensitive to insulin stimulation leading to delayed liver regeneration. This study describes a novel pathomechanism that uncouples HBV expressing hepatocytes from proliferative signals and thereby impedes compensatory liver regeneration after liver injury.  

3.2602           Endogenous macrophage migration inhibitory factor reduces the accumulation and toxicity of misfolded SOD1 in a mouse model of ALS

Leyton-Jaimes, M.F., Benaim, C., Abu-hamad, S., Kahn, J., Guetta, A., Bucala, R. and Israelson, A. PNAS, 113(36), 10198-10293 (2016)   Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons in the brain and spinal cord. It has been suggested that the toxicity of mutant SOD1 results from its misfolding and accumulation on the cytoplasmic faces of intracellular organelles, including the mitochondria and endoplasmic reticulum (ER) of ALS-affected tissues. Recently, macrophage migration inhibitory factor (MIF) was shown to directly inhibit the accumulation of misfolded SOD1 and its binding to intracellular membranes, but the role of endogenous MIF in modulating SOD1 misfolding in vivo remains unknown. To elucidate this role, we bred MIF-deficient mice with SOD1^G85R mice, which express a dismutase-inactive mutant of SOD1 and are considered a model of familial ALS. We found that the accumulation of misfolded SOD1, its association with mitochondrial and ER membranes, and the levels of sedimentable insoluble SOD1 aggregates were significantly higher in the spinal cords of SOD1^G85R-MIF^−/− mice than in their SOD1^G85R-MIF^+/+ littermates. Moreover, increasing MIF expression in neuronal cultures inhibited the accumulation of misfolded SOD1 and rescued from mutant SOD1-induced cell death. In contrast, the complete elimination of endogenous MIF accelerated disease onset and late disease progression and shortened the lifespan of the SOD1^G85R mutant mice. These findings indicate that MIF plays a significant role in the folding and misfolding of SOD1 in vivo, and they have implications for the potential therapeutic role of up-regulating MIF within the nervous system to modulate the selective accumulation of misfolded SOD1.  

3.2603           Polycomb repressive complex 2 (PRC2) silences genes responsible for neurodegeneration

Von Schimmelmann, M., Feinberg, P.A., Sullivan, J.M., Ku, S.M., Badimon, A., Duff, M.K., Wang, Z., Lachmann, A., Dewell, S., Ma’ayan, A., Han, M-H., Tarakhovsky, A. and Schaefer, A. Nature Neurosci., 19(10), 1321-1330 (2016)   Normal brain function depends on the interaction between highly specialized neurons that operate within anatomically and functionally distinct brain regions. Neuronal specification is driven by transcriptional programs that are established during early neuronal development and remain in place in the adult brain. The fidelity of neuronal specification depends on the robustness of the transcriptional program that supports the neuron type-specific gene expression patterns. Here we show that polycomb repressive complex 2 (PRC2), which supports neuron specification during differentiation, contributes to the suppression of a transcriptional program that is detrimental to adult neuron function and survival. We show that PRC2 deficiency in striatal neurons leads to the de-repression of selected, predominantly bivalent PRC2 target genes that are dominated by self-regulating transcription factors normally suppressed in these neurons. The transcriptional changes in PRC2-deficient neurons lead to progressive and fatal neurodegeneration in mice. Our results point to a key role of PRC2 in protecting neurons against degeneration.  

3.2604           Protein kinase C controls lysosome biogenesis independently of mTORC1

Li, Y. et al Nature Cell Biol., 18(10), 1065-1077 (2016)   Lysosomes respond to environmental cues by controlling their own biogenesis, but the underlying mechanisms are poorly understood. Here we describe a protein kinase C (PKC)-dependent and mTORC1-independent mechanism for regulating lysosome biogenesis, which provides insights into previously reported effects of PKC on lysosomes. By identifying lysosome-inducing compounds we show that PKC couples activation of the TFEB transcription factor with inactivation of the ZKSCAN3 transcriptional repressor through two parallel signalling cascades. Activated PKC inactivates GSK3β, leading to reduced phosphorylation, nuclear translocation and activation of TFEB, while PKC activates JNK and p38 MAPK, which phosphorylate ZKSCAN3, leading to its inactivation by translocation out of the nucleus. PKC activation may therefore mediate lysosomal adaptation to many extracellular cues. PKC activators facilitate clearance of aggregated proteins and lipid droplets in cell models and ameliorate amyloid β plaque formation in APP/PS1 mouse brains. Thus, PKC activators are viable treatment options for lysosome-related disorders.  

3.2605           The lysosomal membrane protein SCAV-3 maintains lysosome integrity and adult longevity

Li, Y., Chen, B., Zou, W., Wang, X., Wu, Y., Zhao, D., Sun, Y., Liu, Y., Chen, L., Miao, L., Yang, C. and Wang, X.

1. Cell Biol., 215(2), 167-185 (2016)

  Lysosomes degrade macromolecules and recycle metabolites as well as being involved in diverse processes that regulate cellular homeostasis. The lysosome is limited by a single phospholipid bilayer that forms a barrier to separate the potent luminal hydrolases from other cellular constituents, thus protecting the latter from unwanted degradation. The mechanisms that maintain lysosomal membrane integrity remain unknown. Here, we identified SCAV-3, the Caenorhabditis elegans homologue of human LIMP-2, as a key regulator of lysosome integrity, motility, and dynamics. Loss of scav-3 caused rupture of lysosome membranes and significantly shortened lifespan. Both of these phenotypes were suppressed by reinforced expression of LMP-1 or LMP-2, the C. elegans LAMPs, indicating that longevity requires maintenance of lysosome integrity. Remarkably, reduction in insulin/insulin-like growth factor 1 (IGF-1) signaling suppressed lysosomal damage and extended the lifespan in scav-3(lf) animals in a DAF-16–dependent manner. Our data reveal that SCAV-3 is essential for preserving lysosomal membrane stability and that modulation of lysosome integrity by the insulin/IGF-1 signaling pathway affects longevity.  

3.2606           Dual-Wavelength Surface Plasmon Resonance for Determining the Size and Concentration of Sub-Populations of Extracellular Vesicles

Rupert, D., Shelke, G.V., Emilsson, G., Claudio, V., Block, S., Lässer, C., Dahlin, A., Lötvall, J.O., Bally, M., Zhdanov, V.P. and Höök, F. Anal. Chem., 88(20), 9980-9988 (2016)   Accurate concentration determination of subpopulations of extracellular vesicles (EVs), such as exosomes, is of importance both in the context of understanding their fundamental biological role and of potentially using them as disease biomarkers. In principle, this can be achieved by measuring the rate of diffusion-limited mass uptake to a sensor surface modified with a receptor designed to only bind the subpopulation of interest. However, a significant error is introduced if the targeted EV subpopulation has a size, and thus hydrodynamic diffusion coefficient, that differs from the mean size and diffusion coefficient of the whole EV population and/or if the EVs become deformed upon binding to the surface. We here demonstrate a new approach to determine the mean size (or effective film thickness) of bound nanoparticles, in general, and EV subpopulation carrying a marker of interest, in particular. The method is based on operating surface plasmon resonance simultaneously at two wavelengths with different sensing depths and using the ratio of the corresponding responses to extract the particle size on the surface. By estimating in this way the degree of deformation of adsorbed EVs, we markedly improved their bulk concentration determination and showed that EVs carrying the exosomal marker CD63 correspond to not more than around 10% of the EV sample.  

3.2607           Aminochrome induces dopaminergic neuronal dysfunction: a new animal model for Parkinson’s disease

Herrera, A., Munoz, P., paris, I., Diaz-Veliz, G., Mora, S., Inzunza, J., Hultenby, K., Caardenas, C., Jana, F., Raisman-Vozari, R., Gysling, K., Abarca, J., Steinbusch, H.W. and Segura-Aguilar, J. Cell. Mol. Life Sci., 73(18), 3583-3597 (2016)   l-Dopa continues to be the gold drug in Parkinson’s disease (PD) treatment from 1967. The failure to translate successful results from preclinical to clinical studies can be explained by the use of preclinical models which do not reflect what happens in the disease since these induce a rapid and extensive degeneration; for example, MPTP induces a severe Parkinsonism in only 3 days in humans contrasting with the slow degeneration and progression of PD. This study presents a new anatomy and develops preclinical model based on aminochrome which induces a slow and progressive dysfunction of dopaminergic neurons. The unilateral injection of aminochrome into rat striatum resulted in (1) contralateral rotation when the animals are stimulated with apomorphine; (2) absence of significant loss of tyrosine hydroxylase-positive neuronal elements both in substantia nigra and striatum; (3) cell shrinkage; (4) significant reduction of dopamine release; (5) significant increase in GABA release; (6) significant decrease in the number of monoaminergic presynaptic vesicles; (7) significant increase of dopamine concentration inside of monoaminergic vesicles; (8) significant increase of damaged mitochondria; (9) significant decrease of ATP level in the striatum (10) significant decrease in basal and maximal mitochondrial respiration. These results suggest that aminochrome induces dysfunction of dopaminergic neurons where the contralateral behavior can be explained by aminochrome-induced ATP decrease required both for anterograde transport of synaptic vesicles and dopamine release. Aminochrome could be implemented as a new model neurotoxin to study Parkinson’s disease.  

3.2608           Sortilin regulates sorting and secretion of Sonic hedgehog

Campbell, C., Beug, S., Nickerson, P.E.B., peng, J., Mazerolle, C., Bassett, E.A., Ringuette, R., Jama, F.A., Morales, C., Christ, A. and Wallace, A.

1. Cell Sci., 129(20), 3832-3844 (2016)

  Sonic Hedgehog (Shh) is a secreted morphogen that is an essential regulator of patterning and growth. The Shh full-length protein undergoes autocleavage in the endoplasmic reticulum to generate the biologically active N-terminal fragment (ShhN), which is destined for secretion. We identified sortilin (Sort1), a member of the VPS10P-domain receptor family, as a new Shh trafficking receptor. We demonstrate that Sort–Shh interact by performing coimmunoprecipitation and proximity ligation assays in transfected cells and that they colocalize at the Golgi. Sort1 overexpression causes re-distribution of ShhN and, to a lesser extent, of full-length Shh to the Golgi and reduces Shh secretion. We show loss of Sort1 can partially rescue Hedgehog-associated patterning defects in a mouse model that is deficient in Shh processing, and we show that Sort1 levels negatively regulate anterograde Shh transport in axons in vitro and Hedgehog-dependent axon–glial interactions in vivo. Taken together, we conclude that Shh and Sort1 can interact at the level of the Golgi and that Sort1 directs Shh away from the pathways that promote its secretion  

3.2609           A Portrait of the Human Organelle Proteome In Space and Time during Cytomegalovirus Infection

Beltran, P.M., Mathias, R.A. and Cristea, I.M. Cell Systems, 3(4), 361-373 (2016)   The organelles within a eukaryotic host are manipulated by viruses to support successful virus replication and spread of infection, yet the global impact of viral infection on host organelles is poorly understood. Integrating microscopy, subcellular fractionation, mass spectrometry, and functional analyses, we conducted a cell-wide study of organelles in primary fibroblasts throughout the time course of human cytomegalovirus (HCMV) infection. We used label-free and isobaric-labeling proteomics to characterize nearly 4,000 host and 100 viral proteins, then classified their specific subcellular locations over time using machine learning. We observed a global reorganization of proteins across the secretory pathway, plasma membrane, and mitochondria, including reorganization and processing of lysosomal proteins into distinct subpopulations and translocations of individual proteins between organelles at specific time points. We also demonstrate that MYO18A, an unconventional myosin that translocates from the plasma membrane to the viral assembly complex, is necessary for efficient HCMV replication. This study provides a comprehensive resource for understanding host and virus biology during HCMV pathogenesis.  

3.2610           Commercial Dairy Cow Milk microRNAs Resist Digestion under Simulated Gastrointestinal Tract Conditions

Benmoussa, A., Lee, C.H.C., laffont, B., Savard, P., laugier, J., Boilard, E., Gilbert, C., Fliss, I. and provost, P.

1. Nutr., 146, 2206-2215 (2016)

  Background: MicroRNAs are small, gene-regulatory noncoding RNA species present in large amounts in milk, where they seem to be protected against degradative conditions, presumably because of their association with exosomes. Objective: We monitored the relative stability of commercial dairy cow milk microRNAs during digestion and examined their associations with extracellular vesicles (EVs). Methods: We used a computer-controlled, in vitro, gastrointestinal model TNO intestinal model-1 (TIM-1) and analyzed, by quantitative polymerase chain reaction, the concentration of 2 microRNAs within gastrointestinal tract compartments at different points in time. EVs within TIM-1 digested and nondigested samples were studied by immunoblotting, dynamic light scattering, quantitative polymerase chain reaction, and density measurements. Results: A large quantity of dairy milk Bos taurus microRNA-223 (bta-miR-223) and bta-miR-125b (∼10⁹–10¹⁰ copies/300 mL milk) withstood digestion under simulated gastrointestinal tract conditions, with the stomach causing the most important decrease in microRNA amounts. A large quantity of these 2 microRNAs (∼10⁸–10⁹ copies/300 mL milk) was detected in the upper small intestine compartments, which supports their potential bioaccessibility. A protocol optimized for the enrichment of dairy milk exosomes yielded a 100,000 × g pellet fraction that was positive for the exosomal markers tumor susceptibility gene-101 (TSG101), apoptosis-linked gene 2–interacting protein X (ALIX), and heat shock protein 70 (HSP70) and containing bta-miR-223 and bta-miR-125b. This approach, based on successive ultracentrifugation steps, also revealed the existence of ALIX⁻, HSP70^−/low, and TSG101^−/low EVs larger than exosomes and 2–6 times more enriched in bta-miR-223 and bta-miR-125b (P < 0.05). Conclusions: Our findings indicate that commercial dairy cow milk contains numerous microRNAs that can resist digestion and are associated mostly with ALIX⁻, HSP70^−/low, and TSG101^−/low EVs. Our results support the existence of interspecies transfer of microRNAs mediated by milk consumption and challenge our current view of exosomes as the sole carriers of milk-derived microRNAs.  

3.2611           Sphingosine Kinase 1 Cooperates with Autophagy to Maintain Endocytic Membrane Trafficking

Yuong, M.M., Takahashi, Y., Fox, T.E., Yun, J.K., kester, M. and Wang, H-G. Cell Reports, 17, 1532-1545 (2016)   Sphingosine kinase 1 (Sphk1) associates with early endocytic membranes during endocytosis; however, the role of sphingosine or sphingosine-1-phosphate as the critical metabolite in endocytic trafficking has not been established. Here, we demonstrate that the recruitment of Sphk1 to sphingosine-enriched endocytic vesicles and the generation of sphingosine-1-phosphate facilitate membrane trafficking along the endosomal pathway. Exogenous sphingosine and sphingosine-based Sphk1 inhibitors induce the Sphk1-dependent fusion of endosomal membranes to accumulate enlarged late endosomes and amphisomes enriched in sphingolipids. Interestingly, Sphk1 also appears to facilitate endosomal fusion independent of its catalytic activity, given that catalytically inactive Sphk1^G82D is recruited to endocytic membranes by sphingosine or sphingosine-based Sphk1 inhibitor and promotes membrane fusion. Furthermore, we reveal that the clearance of enlarged endosomes is dependent on the activity of ceramide synthase, lysosomal biogenesis, and the restoration of autophagic flux. Collectively, these studies uncover intersecting roles for Sphk1, sphingosine, and autophagic machinery in endocytic membrane trafficking.  

3.2612           Characterization of Heparan Sulfate Proteoglycan-positive Recycling Endosomes Isolated from Glioma Cells

Podyma-Inoue, K.A., Morikawi, T., Rajapakshe, A.A., Terasawa, K. and hara-Yokoyama, M. Cancer Genomics Proteomics, 13, 443-452 (2016)   Background: Heparan sulfate proteoglycans (HSPGs)-dependent endocytic events have been involved in glioma progression. Thus, comprehensive understanding of the intracellular trafficking complexes formed in presence of HSPGs would be important for development of glioma treatments. Materials and Methods: Subcellular fractionation was used to separate vesicles containing HSPGs from the rat C6 glioma cell line. Isolated HSPG-positive vesicles were further characterized with liquid chromatography-mass spectrometry. Results: The HSPG-positive vesicular fractions, distinct from plasma membrane-derived material, were enriched in endocytic marker, Rab11. Proteomic analysis identified more than two hundred proteins to be associated with vesicular membrane, among them, over eighty were related to endosomal uptake, recycling or vesicular transport. Conclusion: Part of HSPGs in glioma cells is internalized through clathrin-dependent endocytosis and undergo recycling. The development of compounds regulating HSPG-mediated trafficking will likely enable design of effective glioma treatment.  

3.2613           FREE

   

3.2614           Trehalose prevents aggregation of exosomes and cryodamage

Bosch, S., de Beaurepaire, L., Allard, M., Mosser, M., Heichette, C., Chretien, D., Jegou, D. and Bach, J-M. Scientific Reports, 6:36263 (2016)   Exosomes are important mediators in intercellular communication. Released by many cell types, they transport proteins, lipids, and nucleic acids to distant recipient cells and contribute to important physiopathological processes. Standard current exosome isolation methods based on differential centrifugation protocols tend to induce aggregation of particles in highly concentrated suspensions and freezing of exosomes can induce damage and inconsistent biological activity. Trehalose is a natural, non-toxic sugar widely used as a protein stabilizer and cryoprotectant by the food and drug industry. Here we report that addition of 25 mM trehalose to pancreatic beta-cell exosome-like vesicle isolation and storage buffer narrows the particle size distribution and increases the number of individual particles per microgram of protein. Repeated freeze-thaw cycles induce an increase in particle concentration and in the width of the size distribution for exosome-like vesicles stored in PBS, but not in PBS 25 mM trehalose. No signs of lysis or incomplete vesicles were observed by cryo-electron tomography in PBS and trehalose samples. In macrophage immune assays, beta-cell extracellular vesicles in trehalose show consistently higher TNF-alpha cytokine secretion stimulation indexes suggesting improved preservation of biological activity. The addition of trehalose might be an attractive means to standardize experiments in the field of exosome research and downstream applications.  

3.2615           Detailed Analysis of Protein Topology of Extracellular Vesicles–Evidence of Unconventional Membrane Protein Orientation

Cvjetkovic, A., Jang, S.C., Konecna, B., Höög, J.L., Sihlbom, C., Lässer, C. and Lötvall, J. Scientific Reports, 6:36338 (2016)   Extracellular vesicles (EVs) are important mediators of intercellular communication that change the recipient cell by shuttling lipids, RNA, or protein cargo between cells. Here, we investigate the topology of the protein cargo found in EVs, as this topology can fundamentally influence the biological effects of EVs. A multiple proteomics approach, combining proteinase treatment and biotin tagging, shows that many proteins of cytosolic origin are localized on the surface of EVs. A detailed analysis of the EV proteome at the peptide level revealed that a number of EV membrane proteins are present in a topologically reversed orientation compared to what is annotated. Two examples of such proteins, SCAMP3 and STX4, were confirmed to have a reversed topology. This reversed typology was determined using flow cytometry and fluorescent microscopy with antibodies directed toward their cytoplasmic epitopes. These results describe a novel workflow to define the EV proteome and the orientation of each protein, including membrane protein topology. These data are fundamentally important to understanding the EV proteome and required to fully explain EV biogenesis as well as biological function in recipient cells.  

3.2616           Characterization of exosomal release in bovine endometrial intercaruncular stromal cells

Koh, Y.Q., Peiris, H.N., Vaswani, K., Reed, S., Rice, G.E., Salomon, C. and Mitchell, M.D: Reprod.Biol. Endocrinol., 14:478 (2016)   Cell-to-cell communication between the blastocyst and endometrium is critical for implantation. In recent years, evidence has emerged from studies in humans and several other animal species that exosomes are secreted from the endometrium and trophoblast cells and may play an important role in cell-to-cell communication maternal-fetal interface during early pregnancy. Exosomes are stable extracellular lipid bilayer vesicles that encapsulate proteins, miRNAs, and mRNAs, with the ability to deliver their cargo to near and distant sites, altering cellular function(s). Furthermore, the exosomal cargo can be altered in response to environmental cues (e.g. hypoxia). The current study aims to develop an in vitro system to evaluate maternal-embryo interactions via exosomes (and exosomal cargo) produced by bovine endometrial stromal cells (ICAR) using hypoxia as a known stimulus associated with the release of exosomes and alterations to biological responses (e.g. cell proliferation). Methods ICAR cells cultured under 8 % O2 or 1 % O2 for 48 h and changes in cell function (i.e. migration, proliferation and apoptosis) were evaluated. Exosome release was determined following the isolation (via differential centrifugation) and characterization of exosomes from ICAR cell-conditioned media. Exosomal proteomic content was evaluated by mass spectrometry. Results Under hypoxic conditions (i.e. 1 % O2), ICAR cell migration and proliferation was decreased (~20 and ~32 %, respectively) and apoptotic protein caspase-3 activation was increased (∼1.6 fold). Hypoxia increased exosome number by ~3.6 fold compared with culture at 8 % O2. Mass spectrometry analysis identified 128 proteins unique to exosomes of ICAR cultured at 1 % O2 compared with only 46 proteins unique to those of ICAR cultured at 8 % O2. Differential production of proteins associated with specific biological processes and molecular functions were identified, most notably ADAM10, pantetheinase and kininogen 2. Conclusions In summary, we have shown that a stimulus such as hypoxia can alter both the cellular function and exosome release of ICAR cells. Alterations to exosome release and exosomal content in response to stimuli may play a crucial role in maternal-fetal crosstalk and could also affect placental development.  

3.2617           Membrane Interactions of the Mason-Pfizer Monkey Virus Matrix Protein and Its Budding Deficient Mutants

Kroupa,, T., langerova, H., Dolezal, M., Prchal, J., Spiwok, V., Hunter, E., Rumlova, M., Hrabal, R. and Ruml, T.

1. Mol. Biol., 428, 4708-4722 (2016)

  Matrix proteins (MAs) play a key role in the transport of retroviral proteins inside infected cells and in the interaction with cellular membranes. In most retroviruses, retroviral MAs are N-terminally myristoylated. This modification serves as a membrane targeting signal and also as an anchor for membrane interaction. The aim of this work was to characterize the interactions anchoring retroviral MA at the plasma membrane of infected cell. To address this issue, we compared the structures and membrane affinity of the Mason-Pfizer monkey virus (M-PMV) wild-type MA with its two budding deficient double mutants, that is, T41I/T78I and Y28F/Y67F. The structures of the mutants were determined using solution NMR spectroscopy, and their interactions with water-soluble phospholipids were studied. Water-soluble phospholipids are widely used models for studying membrane interactions by solution NMR spectroscopy. However, this approach might lead to artificial results due to unnatural hydrophobic interactions. Therefore, we used a new approach based on the measurement of the loss of the ¹H NMR signal intensity of the protein sample induced by the addition of the liposomes containing phospholipids with naturally long fatty acids. HIV-1 MA was used as a positive control because its ability to interact with liposomes has already been described. We found that in contrast to HIV-1, the M-PMV MA interacted with the liposomes differently and much weaker. In our invivo experiments, the M-PMV MA did not co-localize with lipid rafts. Therefore, we concluded that M-PMV might adopt a different membrane binding mechanism than HIV-1.  

3.2618           Outer membrane vesicles containing signalling molecules and active hydrolytic enzymes released by a coral pathogen Vibrio shilonii AK1

Li, J., Azam, F. and Zhang, S. Environmental Microbiol., 18(11), 3850-3866 (2016)   Production and release of outer-membrane vesicles (OMVs) is known in many bacteria including human pathogens. To date, OMV release has not been reported in coral-associated bacteria. We discovered that Vibrio shilonii AK1, a well-studied coral pathogen, produces OMVs in culture. Transmission electron microscopy showed that V. shilonii cultures release two types of vesicles, with a single membrane or two membranes, as well as vesicle chain-like morphotype in purified vesicle fraction. No significant difference was observed in the amount of OMVs produced by cultures grown at 20°C or 30°C. OMV proteomic analysis, never before done in a coral isolate, showed that a large number of low abundance proteins were exclusively detected in OMVs released by 20°C cultures. Further, the OMVs purified from AK1 cultures grown at both 20°C and 30°C carry N-acylhomoserine lactone quorum sensing signals, as well as alkaline phosphatase, lipase and chitinase activities. Our results show that V. shilonii OMVs are conduits of signalling molecules, active enzymes and other proteins to its environment. These findings suggest important ecophysiological roles of OMVs in coral reef environment. We discuss the importance of OMV release for V. shilonii fitness and propose several hypotheses as well as a conceptual model.  

3.2619           N-linked glycosylation at Asn152 on CD147 affects protein folding and stability: promoting tumour metastasis in hepatocellular carcinoma

Li, J-H., Huang, W., Lin, P., Wu, B., Fu, Z-G., Shen, H-M., Jing, L., Liu, Z-Y., Zhou, Y., Meng, Y., Xu, B-Q., Chen, Z-N. and Jiang, J.L. Scientific Reports, 6:35210 (2016)   Cluster of differentiation 147 (CD147), also known as extracellular matrix metalloproteinase inducer, is a transmembrane glycoprotein that mediates oncogenic processes partly through N-glycosylation modifications. N-glycosylation has been demonstrated to be instrumental for the regulation of CD147 function during malignant transformation. However, the role that site-specific glycosylation of CD147 plays in its defective function in hepatocellular carcinomacells needs to be determined. Here, we demonstrate that the modification of N-glycosylation at Asn152 on CD147 strongly promotes hepatocellular carcinoma (HCC) invasion and migration. After the removal of N-glycans at Asn152, CD147 was more susceptible to degradation by ER-localized ubiquitin ligase-mediated endoplasmic reticulum-associated degradation (ERAD). Furthermore, N-linked glycans at Asn152 were required for CD147 to acquire and maintain proper folding in the ER. Moreover, N-linked glycans at Asn152 functioned as a recognition motif that was directly mediated by the CNX quality control system. Two phases in the retention-based ER chaperones system drove ER-localized CD147 trafficking to degradation. Deletion of N-linked glycosylation at Asn152 on CD147 significantly suppressed in situ tumour metastasis. These data could potentially shed light on the molecular regulation of CD147 through glycosylation and provide a valuable means of developing drugs that target N-glycans at Asn152 on CD147.  

3.2620           Cerebral vascular amyloid seeds drive amyloid β-protein fibril assembly with a distinct anti-parallel structure

Xu, F., Fu, Z., Dass, S., Kotarba, A.E., Davis, J., Smith, S.O. and Van Nostrand, W.E. Nature Communications, 7:13527 (2016)   Cerebrovascular accumulation of amyloid β-protein (Aβ), a condition known as cerebral amyloid angiopathy (CAA), is a common pathological feature of patients with Alzheimer’s disease. Familial Aβ mutations, such as Dutch-E22Q and Iowa-D23N, can cause severe cerebrovascular accumulation of amyloid that serves as a potent driver of vascular cognitive impairment and dementia. The distinctive features of vascular amyloid that underlie its unique pathological properties remain unknown. Here, we use transgenic mouse models producing CAA mutants (Tg-SwDI) or overproducing human wild-type Aβ (Tg2576) to demonstrate that CAA-mutant vascular amyloid influences wild-type Aβ deposition in brain. We also show isolated microvascular amyloid seeds from Tg-SwDI mice drive assembly of human wild-type Aβ into distinct anti-parallel β-sheet fibrils. These findings indicate that cerebrovascular amyloid can serve as an effective scaffold to promote rapid assembly and strong deposition of Aβ into a unique structure that likely contributes to its distinctive pathology.  

3.2621           The Warburg Effect Mediator Pyruvate Kinase M2 Expression and Regulation in the Retina

Rajala, R.V.S., Rajala, A., Kooker, C., Wang, Y. and Anderson, R.E. Scientific Reports, 6:37727 (2016)   The tumor form of pyruvate kinase M2 (PKM2) undergoes tyrosine phosphorylation and gives rise to the Warburg effect. The Warburg effect defines a pro-oncogenic metabolism switch such that cancer cells take up more glucose than normal tissue and favor incomplete oxidation of glucose, even in the presence of oxygen. Retinal photoreceptors are highly metabolic and their energy consumption is equivalent to that of a multiplying tumor cell. In the present study, we found that PKM2 is the predominant isoform in both rod- and cone-dominant retina, and that it undergoes a light-dependent tyrosine phosphorylation. We also discovered that PKM2 phosphorylation is signaled through photobleaching of rhodopsin. Our findings suggest that phosphoinositide 3-kinase activation promotes PKM2 phosphorylation. Light and tyrosine phosphorylation appear to regulate PKM2 to provide a metabolic advantage to photoreceptor cells, thereby promoting cell survival.  

3.2622           Outer membrane vesicles derived from Salmonella Typhimurium mutants with truncated LPS induce cross-protective immune responses against infection of Salmonella enterica serovars in the mouse model

Liu, Q., Liu, Q., Yi, J., Liang, K., Liu, T., Roland, K.L., Jiang, Y. and Kong, Q. Int. J. Med. Microbiol., 306, 697-706 (2016)   Salmonella enterica cause diarrheal and systemic diseases and are of considerable concern worldwide. Vaccines that are cross-protective against multiple serovars could provide effective control of Salmonella-mediated diseases. Bacteria-derived outer membrane vesicles (OMVs) are highly immunogenic and are capable of eliciting protective immune responses. Alterations in lipopolysaccharide (LPS) length can result in outer membrane remodeling and composition of outer membrane proteins (OMPs) changing. In this study, we investigated the impact of truncated LPS on both the production and immunogenicity of Salmonella OMVs, including the ability of OMVs to elicit cross-protection against challenge by heterologous Salmonella strains. We found that mutations in waaJ and rfbP enhanced vesiculation, while mutations in waaC, waaF and waaG inhibited this process. Animal experiments indicated that OMVs from waaC, rfaH and rfbP mutants induced stronger serum immune responses compared to OMVs from the parent strain, while all elicited protective responses against the wild-type S. Typhimurium challenge. Furthermore, intranasal or intraperitoneal immunization with OMVs derived from the waaC and rfbP mutants elicited significantly higher cross-reactive IgG responses and provided enhanced cross-protection against S. Choleraesuis and S. Enteritidis challenge than the wild-type OMVs. These results indicate that truncated-LPS OMVs are capable of conferring cross protection against multiple serotypes of Salmonella infection.  

3.2623           Extracellular vesicles for drug delivery

Vader, P., Mol., E.A., Pasterkamp, G. and Schiffelers, R.M. Adv. Drug Delivery Reviews, 106, 148-156 (2016)   Extracellular vesicles (EVs) are cell-derived membrane vesicles, and represent an endogenous mechanism for intercellular communication. Since the discovery that EVs are capable of functionally transferring biological information, the potential use of EVs as drug delivery vehicles has gained considerable scientific interest. EVs may have multiple advantages over currently available drug delivery vehicles, such as their ability to overcome natural barriers, their intrinsic cell targeting properties, and stability in the circulation. However, therapeutic applications of EVs as drug delivery systems have been limited due to a lack of methods for scalable EV isolation and efficient drug loading. Furthermore, in order to achieve targeted drug delivery, their intrinsic cell targeting properties should be tuned through EV engineering. Here, we review and discuss recent progress and remaining challenges in the development of EVs as drug delivery vehicles.  

3.2624           Therapeutic and diagnostic applications of extracellular vesicles

Stremersch, S., De Smedt, S.C. and Raemdonck, K.

1. Controlled Release, 244, 167-183 (2016)

  During the past two decades, extracellular vesicles (EVs) have been identified as important mediators of intercellular communication, enabling the functional transfer of bioactive molecules from one cell to another. Consequently, it is becoming increasingly clear that these vesicles are involved in many (patho)physiological processes, providing opportunities for therapeutic applications. Moreover, it is known that the molecular composition of EVs reflects the physiological status of the producing cell and tissue, rationalizing their exploitation as biomarkers in various diseases. In this review the composition, biogenesis and diversity of EVs is discussed in a therapeutic and diagnostic context. We describe emerging therapeutic applications, including the use of EVs as drug delivery vehicles and as cell-free vaccines, and reflect on future challenges for clinical translation. Finally, we discuss the use of EVs as a biomarker source and highlight recent studies and clinical successes.  

3.2625           A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

Li, Z., Schulze, R.J., Weller, S.G., Krueger, E.W., Schott, M.B., Zhang, X., Casey, C.A., Liu, J., Stöckli, J., James, D.E. and McNiven, M.A. Sci. Adv., 2, e1601470 (2016)   The autophagic digestion of lipid droplets (LDs) through lipophagy is an essential process by which most cells catabolize lipids as an energy source. However, the cellularmachinery used for the envelopment of LDs during autophagy is poorly understood.We report a novel function for a small Rab guanosine triphosphatase (GTPase) in the recruitment of adaptors required for the engulfment of LDs by the growing autophagosome. In hepatocytes stimulated to undergo autophagy, Rab10 activity is amplified significantly, concomitant with its increased recruitment to nascent autophagic membranes at the LD surface. Disruption of Rab10 function by small interfering RNA knockdown or expression of a GTPase-defective variant leads to LD accumulation. Finally, Rab10 activation during autophagy is essential for LC3 recruitment to the autophagosome and stimulates its increased association with the adaptor protein EHBP1 (EH domain binding protein 1) and the membrane-deforming adenosine triphosphatase EHD2 (EH domain containing 2) that, together, are essential in driving the activated “engulfment” of LDs during lipophagy in hepatocytes.  

3.2626           Extracellular Vesicles Move Toward Use in Clinical Laboratories

Strotman, L.N. and Linder, M.W. Clin. Lab. Med., 36(3), 587-602 (2016)   Extracellular vesicles (EVs) are membranous particles found in a variety of biofluids that encapsulate molecular information from the cell, from which they originate.   This rich source of information that is easily obtained can then be mined to find diagnostic biomarkers.   The biological function of EVs are still evolving, but include, intercellular communication, coagulation, inflammation, immune response modulations, waste management and disease progression.   Interest in EVs has expanded but much still needs to be done for EVs to reach their full potential as a source material in laboratory developed tests for personalized medicine, especially in oncology.  

3.2627           Calcium- and Nitric Oxide-Dependent Nuclear Accumulation of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Response to Long Chain Bases in Tobacco BY-2 Cells

Testard, A., Da Silva, D., Ormancey, M., Pichereaux, C., Pouzet, C., Jauneau, A., Grat, S., Robe, E., Briere, C., Cotelle, V., Mazars, C. and Thuleau, P. Plant Cell Physiol., 57(10), 2221-2231 (2016)   Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid long chain bases (LCBs) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in plants. In addition, in tobacco BY-2 cells, it has been shown that DHS triggers a rapid production of H₂O₂ and nitric oxide (NO). Recently, in analogy to what is known in the animal field, plant cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), a ubiquitous enzyme involved in glycolysis, has been suggested to fulfill other functions associated with its oxidative post-translational modifications such as S-nitrosylation on cysteine residues. In particular, in mammals, stress signals inducing NO production promote S-nitrosylation of GAPC and its subsequent translocation into the nucleus where the protein participates in the establishment of apoptosis. In the present study, we investigated the behavior of GAPC in tobacco BY-2 cells treated with DHS. We found that upon DHS treatment, an S-nitrosylated form of GAPC accumulated in the nucleus. This accumulation was dependent on NO production. Two genes encoding GAPCs, namely Nt(BY-2)GAPC1 and Nt(BY-2)GAPC2, were cloned. Transient overexpression of Nt(BY-2)GAPC–green fluorescent protein (GFP) chimeric constructs indicated that both proteins localized in the cytoplasm as well as in the nucleus. Mutating into serine the two cysteine residues thought to be S-nitrosylated in response to DHS did not modify the localization of the proteins, suggesting that S-nitrosylation of GAPCs was probably not necessary for their nuclear relocalization. Interestingly, using Förster resonance energy transfer experiments, we showed that Nt(BY-2)GAPCs interact with nucleic acids in the nucleus. When GAPCs were mutated on their cysteine residues, their interaction with nucleic acids was abolished, suggesting a role for GAPCs in the protection of nucleic acids against oxidative stress.  

3.2628           Extracellular vesicles in diagnosis and therapy of kidney diseases

Zhang, W., Zhou, X., Xhang, H., Yao, Q., Liu, Y. and Dong, Z. Am. J. Physiol. Renal Physiol., 311(5), F844-F851 (2016)   Extracellular vesicles (EV) are endogenously produced, membrane-bound vesicles that contain various molecules. Depending on their size and origins, EVs are classified into apoptotic bodies, microvesicles, and exosomes. A fundamental function of EVs is to mediate intercellular communication. In kidneys, recent research has begun to suggest a role of EVs, especially exosomes, in cell-cell communication by transferring proteins, mRNAs, and microRNAs to recipient cells as nanovectors. EVs may mediate the cross talk between various cell types within kidneys for the maintenance of tissue homeostasis. They may also mediate the cross talk between kidneys and other organs under physiological and pathological conditions. EVs have been implicated in the pathogenesis of both acute kidney injury and chronic kidney diseases, including renal fibrosis, end-stage renal disease, glomerular diseases, and diabetic nephropathy. The release of EVs with specific molecular contents into urine and plasma may be useful biomarkers for kidney disease. In addition, EVs produced by cultured cells may have therapeutic effects for these diseases. However, the role of EVs in kidney diseases is largely unclear, and the mechanism underlying EV production and secretion remains elusive. In this review, we introduce the basics of EVs and then analyze the present information about the involvement, diagnostic value, and therapeutic potential of EVs in major kidney diseases.  

3.2629           Comparative Analysis of Ciliary Membranes and Ectosomes

Long, H., Zhang, F., Xu, N., Liu, G., Diener, D.R., Rosenbaum, J.L. and Huang, K. Current Biology, 26(24), 3327-3335 (2016)   Primary and motile cilia/flagella function as cellular antennae, receiving signals from the environment and subsequently activating signaling pathways that are critical for cellular homeostasis and differentiation [ 1–3 ]. Recent work with the green alga Chlamydomonas and the nematode C. elegans demonstrated that ectosomes can be released from the cilium and can mediate the intercellular communication [ 4–9 ]. To better understand the function of flagellar ectosomes, we have compared their protein composition to that of the flagellar membrane from which they are derived. Ectosomes released from flagella have a unique protein composition, being enriched in a subset of flagellar membrane proteins, proteases, proteins from the endosomal sorting complex required for transport (ESCRT) [ 10–12 ], small GTPases, and ubiquitinated proteins. Live imaging showed that an ESCRT-related protein (PDCD6) was enriched in ectosomes released from flagella during gamete activation. We devised a sensitive and rapid assay to monitor ectosome release using luciferase fused to PDCD6 and a mutated ubiquitin. Ectosome release increased when cells underwent flagellar resorption. Knockdown of two ESCRT-related proteins, PDCD6 and VPS4, attenuated ectosome release during flagellar shortening and shortening was slowed. These data suggest that the ESCRT proteins mediate ectosome release and thereby influence flagellar shortening in Chlamydomonas. In addition, the prevalence of receptors such as agglutinin and ubiquitinated proteins in ciliary ectosomes suggests that they are involved in cell signaling and turnover of ciliary proteins.  

3.2630           PIKfyve inhibition increases exosome release and induces secretory autophagy

Pettersen Hessvik, N., Øverbye, A., Brech, A., Lyngaas Torgersen, M., Seim jacobsen, I., Sandvig, K. And Llorente, A. Cell. Mol. Life Sci., 73(24), 4717-4737 (2016)   Exosomes are vesicles released from cells by fusion of multivesicular bodies (MVBs) with the plasma membrane. This study aimed to investigate whether the phosphoinositide kinase PIKfyve affects this process. Our results show that in PC-3 cells inhibition of PIKfyve by apilimod or depletion by siRNA increased the secretion of the exosomal fraction. Moreover, quantitative electron microscopy analysis showed that cells treated with apilimod contained more MVBs per cell and more intraluminal vesicles per MVB. Interestingly, mass spectrometry analysis revealed a considerable enrichment of autophagy-related proteins (NBR1, p62, LC3, WIPI2) in exosomal fractions released by apilimod-treated cells, a result that was confirmed by immunoblotting. When the exosome preparations were investigated by electron microscopy a small population of p62-labelled electron dense structures was observed together with CD63-containing exosomes. The p62-positive structures were found in less dense fractions than exosomes in density gradients. Inside the cells, p62 and CD63 were found in the same MVB-like organelles. Finally, both the degradation of EGF and long-lived proteins were shown to be reduced by apilimod. In conclusion, inhibition of PIKfyve increases secretion of exosomes and induces secretory autophagy, showing that these pathways are closely linked. We suggest this is due to impaired fusion of lysosomes with both MVBs and autophagosomes, and possibly increased fusion of MVBs with autophagosomes, and that the cells respond by secreting the content of these organelles to maintain cellular homeostasis.  

3.2631           Silver nanoparticle–protein interactions in intact rainbow trout gill cells

Yue, Y., Behra, R., Sigg, L., Suter, M.J-F., Pillai, S. and Schirmer, K. Environ. Sci. Nano, 3, 1174-1185 (2016)   Upon contact with biota, nanoparticles can bind to proteins, which coat the nanoparticles and form a nanoparticle-protein corona. Knowledge of corona proteins is therefore important for a mechanistic understanding of how nanoparticles interact with biomolecules in cells. Here we present the first study to reveal the identity of corona proteins from silver nanoparticle (AgNPs)-exposed living vertebrate cells. The cells are from a rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, representing the interface between the aquatic environment and one of its model species. Subcellular fractionation allowed AgNP-protein corona complexes to be recovered from intact subcellular compartments and proteins lysed from the AgNPs to be detected by mass spectrometry. The identified proteins mark the trail of AgNPs processing in the cells like a forensic fingerprint: the cells take up the AgNPs via endocytic processes and store the particles in endosomal/lysosomal compartments. Moreover, stress response proteins were recovered in the AgNPs protein corona. In this way, we established a list of AgNPs susceptible proteins which can be investigated further in targeted nanoparticle–protein interaction. As a proof of principle, we demonstrate that Na⁺/K⁺-ATPase, identified from the corona and a known key protein in ion regulation in gill cells, is inhibited in its activity by AgNPs, confirming previously published in vivo experiments. The developed methodology is broadly applicable to other nanoparticles and cell types, representing a valuable tool for mechanistic nanoparticle–cell interaction studies, ranging from environmental and human risk assessment to biomedicine. In this way, our research also contributes to safer particle design.  

3.2632           Pex9p is a new yeast peroxisomal import receptor for PTS1-containing proteins

Effelsberg, D., Cruz-Zaragoza, L.D., Schliebs, W. and Erdmann, R.

1. Cell Sci., 129, 4057-4066 (2016)

  Peroxisomal proteins carrying a type 1 peroxisomal targeting signal (PTS1) are recognized by the well-conserved cycling import receptor Pex5p. The yeast YMR018W gene encodes a Pex5p paralog and newly identified peroxin that is involved in peroxisomal import of a subset of matrix proteins. The new peroxin was designated Pex9p, and it interacts with the docking protein Pex14p and a subclass of PTS1-containing peroxisomal matrix enzymes. Unlike Pex5p, Pex9p is not expressed in glucose- or ethanol-grown cells, but it is strongly induced by oleate. Under these conditions, Pex9p acts as a cytosolic and membrane-bound peroxisome import receptor for both malate synthase isoenzymes, Mls1p and Mls2p. The inducible Pex9p-dependent import pathway provides a mechanism for the oleate-inducible peroxisomal targeting of malate synthases. The existence of two distinct PTS1 receptors, in addition to two PTS2-dependent import routes, contributes to the adaptive metabolic capacity of peroxisomes in response to environmental changes and underlines the role of peroxisomes as multi-purpose organelles. The identification of different import routes into peroxisomes contributes to the molecular understanding of how regulated protein targeting can alter the function of organelles according to cellular needs  

3.2633           N-3 vs. n-6 fatty acids differentially influence calcium signalling and adhesion of inflammatory activated monocytes: impact of lipid rafts

Schaefer, M.B. et al Inflamm. Res., 65(11), 881-894 (2016)   Background Anti-inflammatory n-3 fatty acids (FA) like docosahexaenoic acid (DHA) opposed to the pro-inflammatory n-6 FA arachidonic acid (AA) might modulate lipid rafts within the cell membrane by differential incorporation. In inflammation, monocyte adhesion to endothelial cells is a crucial step mediated by intracellular calcium changes. We investigated whether lipid rafts mediate FA-induced modulation of adhesion and intracellular calcium. Methods In isolated human monocytes and monocytic U937 cells we measured adhesion to human umbilical vein endothelial cells (HUVEC) using a parallel flow chamber and a static assay, adhesion molecules by FACScan, and intracellular calcium by fluorescence. Monocyte lipid rafts were isolated by ultracentrifugation and submitted to gas chromatography for FA analysis. Results Pre-incubation with AA or DHA resulted in a predominant incorporation of the respective FA into raft compared to non-raft fraction. DHA as compared to AA significantly reduced monocyte adhesion and calcium release after stimulation with TNF-α while expression of adhesion molecules remained unchanged. Pre-treatment with a calcium chelator abolished the effect of FA on calcium and adhesion. Disruption of lipid rafts prevented FA-induced modulations. Conclusion Incorporation of FA into lipid rafts seem to be crucial for modulation of adhesion under inflammatory conditions.  

3.2634           Mast Cell Degranulation Is Accompanied by the Release of a Selective Subset of Extracellular Vesicles That Contain Mast Cell–Specific Proteases

Groot Kormelink, T., Arkesteijn, G.J.A., van de Lest, C.H.A., Geerts, J.C., Goerdayal, S.S., Altelaar, M.A.F., Redegeld, F.A., Nolte-t’ Hoen, E.N.M. and Wauben, M.H.M.

1. Immunol., 197(8), 3382-3392 (2016)

  Mast cells (MC) are well known for their effector role in allergic disorders; moreover, they are associated with diverse modulatory effects in innate and adaptive immunity. It is largely unclear how MC exert these modulating functions. In this article, we show that IgE-mediated MC degranulation leads to a rapid release of high quantities of extracellular vesicles (EV), comparable to the release of preformed mediators. EV are submicron structures composed of lipid bilayers, proteins, and nucleic acids that are released by cells in a regulated fashion and are involved in intercellular communication. Primary murine mucosal-type MC and connective tissue–type MC released phenotypically different EV populations depending on the stimulus they received. Although unstimulated MC constitutively released CD9⁺ EV, degranulation was accompanied by the release of CD63⁺ EV, which correlated with release of the soluble mediator β-hexosaminidase. This CD63⁺ EV subset was smaller and exhibited a higher buoyant density and distinct phospholipid composition compared with CD9⁺ EV. Marked differences were observed for phosphatidylinositol, phosphatidic acid, and bis(monoacylglycero)phosphate species. Strikingly, proteomic analysis of CD63⁺ EV from connective tissue–type MC unveiled an abundance of MC-specific proteases. With regard to carboxypeptidase A3, it was confirmed that the enzyme was EV associated and biologically active. Our data demonstrate that, depending on their activation status, MC release distinct EV subsets that differ in composition and protease activity and are indicative of differential immunological functions. Concerning the strategic tissue distribution of MC and the presence of degranulated MC in various (allergic) disorders, MC-derived EV should be considered potentially important immune regulators.  

3.2635           Exosome-associated release, uptake, and neurotoxicity of HIV-1 Tat protein

Rahimian, P. and He, J.J.

1. Neurovirol., 22(6), 774-788 (2016)

  HIV-1 Tat is an indispensible transactivator for HIV gene transcription and replication. It has been shown to exit cells as a free protein and enter neighboring cells or interact with surface receptors of neighboring cells to regulate gene expression and cell function. In this study, we report, for the first time, exosome-associated Tat release and uptake. Using a HIV-1 LTR-driven luciferase reporter-based cell assay and Western blotting or in combination with exosome inhibitor, OptiPrep gradient fractionation, and exosome depletion, we demonstrated significant presence of HIV-1 Tat in exosomes derived from Tat-expressing primary astrocytes, Tat-transfected U373.MG and 293T, and HIV-infected MT4. We further showed that exosome-associated Tat from Tat-expressing astrocytes was capable of causing neurite shortening and neuron death, further supporting that this new form of extracellular Tat is biologically active. Lastly, we constructed a Tat mutant deleted of its basic domain and determined the role of the basic domain in Tat trafficking into exosomes. Basic domain-deleted Tat exhibited no apparent effects on Tat trafficking into exosomes, while maintained its dominant-negative function in Tat-mediated LTR transactivation. Taken together, these results show a significant fraction of Tat is secreted and present in the form of exosomes and may contribute to the stability of extracellular Tat and broaden the spectrum of its target cells.    

3.2636           Proteomic analysis of exosomal cargo: the challenge of high purity vesicle isolation

Abramowicz, A., Widlak, P and Pietrowska, M. Molecular Biosystems, 12, 1407-1419 (2016)   The re-discovery of exosomes as intercellular messengers with high potential for diagnostic and therapeutic utility has led to them becoming a popular topic of research in recent years. One of the essential research areas in this field is the characterization of exosomal cargo, which includes numerous non-randomly packed proteins and nucleic acids. Unexpectedly, a very challenging aspect of exploration of extracellular vesicles has turned out to be their effective and selective isolation. The plurality of developed protocols leads to qualitative and quantitative variability in terms of the obtained exosomes, which significantly affects the results of downstream analyses and makes them difficult to compare, reproduce and interpret between research groups. Currently, there is a general consensus among the exosome-oriented community concerning the urgent need for the optimization and standardization of methods employed for the purification of these vesicles. Hence, we review here several strategies for exosome preparation including ultracentrifugation, chemical precipitation, affinity capturing and filtration techniques. The advantages and disadvantages of different approaches are discussed with special emphasis being placed on their adequacy for proteomics applications, which are particularly sensitive to sample quality. We conclude that certain methods, exemplified by ultracentrifugation combined with iodixanol density gradient centrifugation or gel filtration, although labor-intensive, provide superior quality exosome preparations suitable for reliable analysis by mass spectrometry.  

3.2637           Translocon component Sec62 acts in endoplasmic reticulum turnover during stress recovery

Fumagelli, F. et al Nature Cell Biol., 18(11), 1173-1184 (2016)   The endoplasmic reticulum (ER) is a site of protein biogenesis in eukaryotic cells. Perturbing ER homeostasis activates stress programs collectively called the unfolded protein response (UPR). The UPR enhances production of ER-resident chaperones and enzymes to reduce the burden of misfolded proteins. On resolution of ER stress, ill-defined, selective autophagic programs remove excess ER components. Here we identify Sec62, a constituent of the translocon complex regulating protein import in the mammalian ER, as an ER-resident autophagy receptor. Sec62 intervenes during recovery from ER stress to selectively deliver ER components to the autolysosomal system for clearance in a series of events that we name recovER-phagy. Sec62 contains a conserved LC3-interacting region in the C-terminal cytosolic domain that is required for its function in recovER-phagy, but is dispensable for its function in the protein translocation machinery. Our results identify Sec62 as a critical molecular component in maintenance and recovery of ER homeostasis.  

3.2638           L1-associated genomic regions are deleted in somatic cells of the healthy human brain

Erwin, J.A., paquola, A., Singer, T., Gallina, I., Novotny, M., Quayle, C., Bedrosian, T.A., Alves, F.I.A., Butcher, C.R., Herdy, J.R., Sarkar, A., Lasken, R.S., Muotri, A.R. and Gage F.H. Nature Neurosci., 19(12), 1583-1591 (2016)   healthy human brain is a mosaic of varied genomes. Long interspersed element-1 (LINE-1 or L1) retrotransposition is known to create mosaicism by inserting L1 sequences into new locations of somatic cell genomes. Using a machine learning-based, single-cell sequencing approach, we discovered that somatic L1-associated variants (SLAVs) are composed of two classes: L1 retrotransposition insertions and retrotransposition-independent L1-associated variants. We demonstrate that a subset of SLAVs comprises somatic deletions generated by L1 endonuclease cutting activity. Retrotransposition-independent rearrangements in inherited L1s resulted in the deletion of proximal genomic regions. These rearrangements were resolved by microhomology-mediated repair, which suggests that L1-associated genomic regions are hotspots for somatic copy number variants in the brain and therefore a heritable genetic contributor to somatic mosaicism. We demonstrate that SLAVs are present in crucial neural genes, such as DLG2 (also called PSD93), and affect 44–63% of cells of the cells in the healthy brain.  

3.2639           p62- and ubiquitin-dependent stress-induced autophagy of the mammalian 26S proteasome

Cohen-Kaplan, V., Livneh, I., Avni, N., Fabre, B., Ziv, T., Kwon, Y.T. PNAS, 113(47), E7490-E7499 (2016)   The ubiquitin-proteasome system and autophagy are the two main proteolytic systems involved in, among other functions, the maintenance of cell integrity by eliminating misfolded and damaged proteins and organelles. Both systems remove their targets after their conjugation with ubiquitin. An interesting, yet incompletely understood problem relates to the fate of the components of the two systems. Here we provide evidence that amino acid starvation enhances polyubiquitination on specific sites of the proteasome, a modification essential for its targeting to the autophagic machinery. The uptake of the ubiquitinated proteasome is mediated by its interaction with the ubiquitin-associated domain of p62/SQSTM1, a process that also requires interaction with LC3. Importantly, deletion of the PB1 domain of p62, which is important for the targeting of ubiquitinated substrates to the proteasome, has no effect on stress-induced autophagy of this proteolytic machinery, suggesting that the domain of p62 that binds to the proteasome determines the function of p62 in either targeting substrates to the proteasome or targeting the proteasome to autophagy.  

3.2640           Fas/CD95 prevents autoimmunity independently of lipid raft localization and efficient apoptosis induction

Cruz, A.C. et al Nature Communications, 7:13895 (2016)   Mutations affecting the apoptosis-inducing function of the Fas/CD95 TNF-family receptor result in autoimmune and lymphoproliferative disease. However, Fas can also costimulate T-cell activation and promote tumour cell growth and metastasis. Palmitoylation at a membrane proximal cysteine residue enables Fas to localize to lipid raft microdomains and induce apoptosis in cell lines. Here, we show that a palmitoylation-defective Fas C194V mutant is defective in inducing apoptosis in primary mouse T cells, B cells and dendritic cells, while retaining the ability to enhance naive T-cell differentiation. Despite inability to efficiently induce cell death, the Fas C194V receptor prevents the lymphoaccumulation and autoimmunity that develops in Fas-deficient mice. These findings indicate that induction of apoptosis through Fas is dependent on receptor palmitoylation in primary immune cells, and Fas may prevent autoimmunity by mechanisms other than inducing apoptosis.  

3.2641           Glucocorticoid Cell Priming Enhances Transfection Outcomes in Adult Human Mesenchymal Stem Cells

Kelly, A.M., Plautz, S.A., Zempleni, J. and Pannier, A.K. Molecular Therapy, 24(2), 331-341 (2016)   Human mesenchymal stem cells (hMSCs) are one of the most widely researched stem cell types with broad applications from basic research to therapeutics, the majority of which require introduction of exogenous DNA. However, safety and scalability issues hinder viral delivery, while poor efficiency hinders nonviral gene delivery, particularly to hMSCs. Here, we present the use of a pharmacologic agent (glucocorticoid) to overcome barriers to hMSC DNA transfer to enhance transfection using three common nonviral vectors. Glucocorticoid priming significantly enhances transfection in hMSCs, demonstrated by a 3-fold increase in efficiency, 4–15-fold increase in transgene expression, and prolonged transgene expression when compared to transfection without glucocorticoids. These effects are dependent on glucocorticoid receptor binding and caused in part by maintenance of normal metabolic function and increased cellular (5-fold) and nuclear (6–10-fold) DNA uptake over hMSCs transfected without glucocorticoids. Results were consistent across five human donors and in cells up to passage five. Glucocorticoid cell priming is a simple and effective technique to significantly enhance nonviral transfection of hMSCs that should enhance their clinical use, accelerate new research, and decrease reliance on early passage cells.  

3.2642           Genome-wide analysis of mRNAs associated with mouse peroxisomes

Yarmishyn, A.A., Kremenskøy, M., Batagov, A.O., Preuss, A., Wong, J.H. and Kurochkin, I.V. BMC Genomics, 17, Suppl 17:1028 (2016)   Background RNA is often targeted to be localized to the specific subcellular compartments. Specific localization of mRNA is believed to be an important mechanism for targeting their protein products to the locations, where their function is required. Results In this study we performed the genome wide transcriptome analysis of peroxisome preparations from the mouse liver using microarrays. We demonstrate that RNA is absent inside peroxisomes, however it is associated at their exterior via the noncovalent contacts with the membrane proteins. We detect enrichment of specific sets of transcripts in two preparations of peroxisomes, purified with different degrees of stringency. Importantly, among these were mRNAs encoding bona fide peroxisomal proteins, such as peroxins and peroxisomal matrix enzymes involved in beta-oxidation of fatty acids and bile acid biosynthesis. The top-most enriched mRNA, whose association with peroxisomes we confirm microscopically was Hmgcs1, encoding 3-hydroxy-3-methylglutaryl-CoA synthase, a crucial enzyme of cholesterol biosynthesis pathway. We observed significant representation of mRNAs encoding mitochondrial and secreted proteins in the peroxisomal fractions. Conclusions This is a pioneer genome-wide study of localization of mRNAs to peroxisomes that provides foundation for more detailed dissection of mechanisms of RNA targeting to subcellular compartments.  

3.2643           Lipid Raft Integrity Is Required for Survival of Triple Negative Breast Cancer Cells

Badana, A., Chintala, M., Varikuti, G., Pudi, N., Kumari, S., Kappala, V.R. and Malla, R.R.

1. Breast Cancer, 19(4), 372-384 (2016)

  PURPOSE: Lipid rafts are cholesterol enriched microdomains that colocalize signaling pathways involved in cell proliferation, metastasis, and angiogenesis. We examined the effect of methyl-β-cyclodextrin (MβCD)-mediated cholesterol extraction on the proliferation, adhesion, invasion, and angiogenesis of triple negative breast cancer (TNBC) cells. METHODS: We measured cholesterol and estimated cell toxicity. Detergent resistant membrane (DRM) and non-DRM fractions were separated using the OptiPrep gradient method. Cell cycles stages were analyzed by flow cytometry, apoptosis was assessed using the TdT-mediated dUTP nick end-labeling assay, and metastasis was determined using a Matrigel invasion assay. Neo-vessel pattern and levels of angiogenic modulators were determined using an in vitro angiogenesis assay and an angiogenesis array, respectively. RESULTS: The present study found that the cholesterol-depleting agent MβCD, efficiently depleted membrane cholesterol and caused concentration dependent (0.1-0.5 mM) cytotoxicity compared to nystatin and filipin III in TNBC cell lines, MDA-MB 231 and MDA-MB 468. A reduced proportion of caveolin-1 found in DRM fractions indicated a cholesterol extraction-induced disruption of lipid raft integrity. MβCD inhibited 52% of MDA-MB 231 cell adhesion on fibronectin and 56% of MDA-MB 468 cell adhesion on vitronectin, while invasiveness of these cells was decreased by 48% and 52% respectively, following MβCD treatment (48 hours). MβCD also caused cell cycle arrest at the G₂M phase and apoptosis in MDA-MB 231 cells (25% and 58% cells, respectively) and in MDA-MB 468 cells (30% and 38% cells, respectively). We found that MβCD treated cells caused a 52% and 58% depletion of neovessel formation in both MDA-MB 231 and MDA-MB 468 cell lines, respectively. This study also demonstrated that MβCD treatment caused a respective 2.6- and 2.5-fold depletion of tyrosine protein kinase receptor (TEK) receptor tyrosine kinase levels in both TNBC cell lines. CONCLUSION: MβCD-induced cholesterol removal enhances alterations in lipid raft integrity, which reduces TNBC cell survival.  

3.2644           Self-Renewal of Bone Marrow Stem Cells by Nanovesicles Engineered from Embryonic Stem Cells

Jo, W., Jeong, D., Kim, J. and Park, J. Adv. Healthcare Mater., 5(24), 3148-3156 (2016)   Extracellular vesicles can enhance cell proliferation by stimulating signal transduction and delivering genetic materials, and thus may have applications in regenerative medicine and other therapeutic applications. The processes employed to isolate extracellular vesicles, however, are complex and achieve low yield. To overcome these obstacles, a large-scale, micropore device for generating extracellular vesicle-mimetic nanovesicles that have characteristics similar to those of extracellular vesicles is fabricated. The nanovesicles are generated through the self-assembly capability of cell membrane fragments in an aqueous solution. The nanovesicles enhance the proliferation of murine mesenchymal stem cells (MSCs), stimulate the signal pathway related to cell proliferation, and do not influence the characteristics of murine MSCs. Therefore, these nanovesicles could provide stable MSCs for regenerative medicine and other therapeutic applications.    

3.2645           Flow cytometry analysis of DNA ploidy levels and protein profiles distinguish between populations of Lumbriculus (Annelida: Clitellata)

Tweeten, K.A. and Morris, S.J. Invertebrate Biology, 135(4), 385-399 (2016)   Variations in DNA ploidy have been observed in Lumbriculus, a freshwater annelid, as well as in other clitellates. Interpretation and application of experimental results using these animals may be impacted as ploidy levels affect the protein expression, reproductive behavior, and response to stressors. Ploidy is typically determined by chromosome spreads, a time-consuming and inefficient method. We adapted flow cytometry protocols used on vertebrates and plants to determine the ploidy levels in different populations of Lumbriculus, including a laboratory strain (Environmental Protection Agency), a commercial strain (Aquatic Foods), and worms collected from natural habitats. To isolate nuclei, worms were homogenized, filtered to remove cell debris, and centrifuged through Optiprep™ density gradients. Nuclei were recovered, treated with RNAse, and stained with propidium iodide. Flow cytometry of the labeled nuclei showed that Lumbriculus from natural habitats in Minnesota and Iowa were diploid, with an estimated genome size of 2.7 pg. Populations from natural habitats in California and Oregon were highly polyploid, as were the laboratory and commercial strains. Chromosome spreads verified the high ploidy levels indicated by flow cytometry results, but also suggested that flow cytometry may be underestimating the DNA content levels. Staining of nuclei with diamidino-2-phenylindole indicated that this may be due to high levels of heterochromatin in nuclei from polyploid forms of Lumbriculus. To further compare the populations, proteins in worm homogenates were subjected to isoelectrofocusing gel electrophoresis. Distinct protein profiles were seen; one was shared in common by the diploid worms, the other was characteristic of polyploid populations. Diploid worms could also be distinguished from polyploid worms based on differences in hemoglobin linker proteins. The results further support taxonomic classification of the diploid and polyploid forms of Lumbriculus as distinct species.  

3.2646           Nitric Oxide Interacts with Caveolin-1 to Facilitate Autophagy-Lysosome-Mediated Claudin-5 Degradation in Oxygen-Glucose Deprivation-Treated Endothelial Cells

Liu, J., Weaver, J., Jin, X., Zhang, Y., Xu, J., Liu, Ke.J., Li, W. and Liu, W. Mol. Neurobiol., 53(9), 5935-5947 (2016)   Using in vitro oxygen-glucose deprivation (OGD) model, we have previously demonstrated that 2-h OGD induces rapid, caveolin-1-mediated dissociation of claudin-5 from the cellular cytoskeletal framework and quick endothelial barrier disruption. In this study, we further investigated the fate of translocated claudin-5 and the mechanisms by which OGD promotes caveolin-1 translocation. Exposure of bEND3 cells to 4-h OGD, but not 2-h OGD plus 2-h reoxygenation, resulted in claudin-5 degradation. Inhibition of autophagy or the fusion of autophagosome with lysosome, but not proteasome, blocked OGD-induced claudin-5 degradation. Moreover, knockdown of caveolin-1 with siRNA blocked OGD-induced claudin-5 degradation. Western blot analysis showed a transient colocalization of caveolin-1, claudin-5, and LC3B in autolysosome or lipid raft fractions at 2-h OGD. Of note, inhibiting autophagosome and lysosome fusion sustained the colocalization of caveolin-1, claudin-5, and LC3B throughout the 4-h OGD exposure. EPR spin trapping showed increased nitric oxide (NO) generation in 2-h OGD-treated cells, and inhibiting NO with its scavenger C-PTIO or inducible nitric oxide synthase (iNOS) inhibitor 1400W prevented OGD-induced caveolin-1 translocation and claudin-5 degradation. Taken together, our data provide a novel mechanism underlying endothelial barrier disruption under prolonged ischemic conditions, in which NO promotes caveolin-1-mediated delivery of claudin-5 to the autophagosome for autophagy-lysosome-dependent degradation.  

3.2647           Vps35-dependent recycling of Trem2 regulates microglial function

Yin, J., Liu, X., He, Q., Zhou, L., Yan, Z.and Zhao, S. Traffic, 17(12), 1286-1296 (2016)   Triggering receptor expressed on myeloid cells 2 (Trem2), an immune-modulatory receptor, is preferentially expressed in microglia of central nervous system. Trem2 might be involved in the development of Alzheimer's disease (AD) through regulating the inflammatory responses and phagocytosis of microglia. However, the intracellular trafficking of Trem2 remains unclear. In this study, we showed that Trem2 in the plasma membrane underwent endocytosis and recycling. Trem2 is internalized in a clathrin-dependent manner and then recycled back to the plasma membrane through vacuolar protein sorting 35 (Vps35), the key component of cargo recognition core of retromer complex, but not Rab11. When Vps35 is knocked down, Trem2 accumulated in the lysosomes but was not degraded. More importantly, Vps35 deficiency leads to excessive lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression and IL-6 production, which can be abolished by Trem2 overexpression. Furthermore, R47H Trem2, an AD-associated mutant, failed to interact with Vps35 and became unstable compared with wild-type Trem2. Our study suggests that Vps35/retromer is responsible for recycling of Trem2 in the regulation of microglial function such as proinflammatory responses, whereas R47H mutation impairs Trem2 trafficking, which might contribute to AD.  

3.2648           Methods for the physical characterization and quantification of extracellular vesicles in biological samples

Rupert, D., Claudio, V., L¨sser, C. and Bally, M. Biochim. Biophys. Acta, 1861, 3164-3179 (2017)   Background Our body fluids contain a multitude of cell-derived vesicles, secreted by most cell types, commonly referred to as extracellular vesicles. They have attracted considerable attention for their function as intercellular communication vehicles in a broad range of physiological processes and pathological conditions. Extracellular vesicles and especially the smallest type, exosomes, have also generated a lot of excitement in view of their potential as disease biomarkers or as carriers for drug delivery. In this context, state-of-the-art techniques capable of comprehensively characterizing vesicles in biological fluids are urgently needed. Scope of review This review presents the arsenal of techniques available for quantification and characterization of physical properties of extracellular vesicles, summarizes their working principles, discusses their advantages and limitations and further illustrates their implementation in extracellular vesicle research. Major conclusions The small size and physicochemical heterogeneity of extracellular vesicles make their physical characterization and quantification an extremely challenging task. Currently, structure, size, buoyant density, optical properties and zeta potential have most commonly been studied. The concentration of vesicles in suspension can be expressed in terms of biomolecular or particle content depending on the method at hand. In addition, common quantification methods may either provide a direct quantitative measurement of vesicle concentration or solely allow for relative comparison between samples. General significance The combination of complementary methods capable of detecting, characterizing and quantifying extracellular vesicles at a single particle level promises to provide new exciting insights into their modes of action and to reveal the existence of vesicle subpopulations fulfilling key biological tasks.  

3.2649           Mitochondrial peroxiredoxins are essential in regulating the relationship between Drosophila immunity and aging

Odnojoz, O., nakatsuka, K., Klichko, V.I., Nguyen, J., Solis, L.C., Ostling, K., Badinloo, M., Orr, W.C. and Radyuk, S.N. Biochim. Biophys Acta, 1863, 68-80 (2017)   Previously, we have shown that flies under-expressing the two mitochondrial peroxiredoxins (Prxs), dPrx3 and dPrx5, display increases in tissue-specific apoptosis and dramatically shortened life span, associated with a redox crisis, manifested as changes in GSH:GSSG and accumulation of protein mixed disulfides. To identify specific pathways responsible for the observed biological effects, we performed a transcriptome analysis. Functional clustering revealed a prominent group enriched for immunity-related genes, including a considerable number of NF-kB-dependent antimicrobial peptides (AMP) that are up-regulated in the Prx double mutant. Using qRT-PCR analysis we determined that the age-dependent changes in AMP levels in mutant flies were similar to those observed in controls when scaled to percentage of life span. To further clarify the role of Prx-dependent mitochondrial signaling, we expressed different forms of dPrx5, which unlike the uniquely mitochondrial dPrx3 is found in multiple subcellular compartments, including mitochondrion, nucleus and cytosol. Ectopic expression of dPrx5 in mitochondria but not nucleus or cytosol partially extended longevity under normal or oxidative stress conditions while complete restoration of life span occurred when all three forms of dPrx5 were expressed from the wild type dPrx5 transgene. When dPrx5 was expressed in mitochondria or in all three compartments, it substantially delayed the development of hyperactive immunity while expression of cytosolic or nuclear forms had no effect on the immune phenotype. The data suggest a critical role of mitochondria in development of chronic activation of the immune response triggered by impaired redox control.  

3.2650           Bacterial protoplast-derived nanovesicles for tumor targeted delivery of chemotherapeutics

Kim, O.Y., Dinh, N.T.H., Park, H.T., Choi. S.J., Hong, K. and Gho, Y.S. Biomaterials, 113, 68-79 (2017)   Increasing incidents of patients diagnosed with cancer have brought massive improvement in the delivery technologies to help patients receiving chemotherapy. However, tumor specific targeting of the chemotherapeutics still remains as a challenge mainly due to the difficulties in the conjugation and manipulation of bio-specific molecules on the surface. Herein, we genetically engineered bacterial protoplast to develop nanovesicles having no toxic outer membrane components that can specifically target and deliver chemotherapeutics to tumor tissues. The bacterial protoplast nanovesicles expressing tumor-targeting moieties on the surface were prepared by serial extrusions through nano-sized membrane filters. The nano-sized vesicular structure of protoplast nanovesicles offers passive targeting to solid tumor site and expression of tumor-targeting moiety enhance tumor-specific uptake via receptor-mediated targeting. Chemotherapeutics-loaded in the nanovesicles induce dose-dependent cytotoxicity in tumor cells in vitro. Moreover, specific trafficking of drug-loaded nanovesicles to the tumor tissue and efficient prevention of tumor growth in tumor xenografted mice are shown. Importantly, this tumor growth suppression of protoplast nanovesicles has shown to reduce the chemotherapeutics-induced adverse effects after systemic administration to mice. This study offers great potential of protoplast nanovesicles as effective and safe delivery system to optimize and contribute to the development of advanced chemotherapy.  

3.2651           Isolation of detergent-resistant membranes (DRMs) from Escherichia coli

GuzmanFlores, J.E., Alvarez, A.F., Poggio, S., Gavilanes-Ruiz, M. and georgellis, D. Anal. Biochem., 518, 1-8 (2017)   Lipid rafts or membrane microdomains have been proposed to compartmentalize cellular processes by spatially organizing diverse molecules/proteins in eukaryotic cells. Such membrane microdomains were recently reported to also exist in a few bacterial species. In this work, we report the development of a procedure for membrane microdomain isolation from Escherichia coli plasma membranes as well as a method to purify the latter. The method here reported could easily be adapted to other gram-negative bacteria, wherein the isolation of this kind of sub-membrane preparation imposes special difficulties. The analysis of isolated membrane microdomains might provide important information on the nature and function of these bacterial structures and permit their comparison with the ones of eukaryotic cells.  

3.2652           Dataset of the proteome of purified outer membrane vesicles from the human pathogen Aggregatibacter actinomycetemcomintans

Kieselbach, T. and Oscarsson, J. Data in Brief, 10, 426-431 (2017)   The Gram-negative bacterium Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen, which is linked to aggressive forms of periodontitis and can be associated with endocarditis. The outer membrane vesicles (OMVs) of this species contain effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA), which they can deliver into human host cells. The OMVs can also activate innate immunity through NOD1- and NOD2-active pathogen-associated molecular patterns. This dataset provides a proteome of highly purified OMVs from A. actinomycetemcomitans serotype e strain 173. The experimental data do not only include the raw data of the LC-MS/MS analysis of four independent preparations of purified OMVs but also the mass lists of the processed data and the Mascot.dat files from the database searches. In total 501 proteins are identified, of which 151 are detected in at least three of four independent preparations. In addition, this dataset contains the COG definitions and the predicted subcellular locations (PSORTb 3.0) for the entire genome of A. actinomycetemcomitans serotype e strain SC1083, which is used for the evaluation of the LC-MS/MS data. These data are deposited in ProteomeXchange in the public dataset PXD002509. In addition, a scientific interpretation of this dataset by Kieselbach et al. (2015) [2] is available at http://dx.doi.org/10.1371/journal.pone.0138591.  

3.2653           Microfluidic approaches for isolation, detection, and characterization of extracellular vesicles: Current status and future directions

Gholizadeh, S., Draz, M.S., Zarghooni, M., Sanati-Nezhad, A., Ghavami, S., Shafiee, H. and Akbari, M. Biosensors and Bioelectronics, 91, 588-605 (2017)   Extracellular vesicles (EVs) are cell-derived vesicles present in body fluids that play an essential role in various cellular processes, such as intercellular communication, inflammation, cellular homeostasis, survival, transport, and regeneration. Their isolation and analysis from body fluids have a great clinical potential to provide information on a variety of disease states such as cancer, cardiovascular complications and inflammatory disorders. Despite increasing scientific and clinical interest in this field, there are still no standardized procedures available for the purification, detection, and characterization of EVs. Advances in microfluidics allow for chemical sampling with increasingly high spatial resolution and under precise manipulation down to single molecule level. In this review, our objective is to give a brief overview on the working principle and examples of the isolation and detection methods with the potential to be used for extracellular vesicles. This review will also highlight the integrated on-chip systems for isolation and characterization of EVs.  

3.2654           Influence of maternal BMI on the exosomal profile during gestation and their role on maternal systemic inflammation

Elfeky, O., Longo, S., lai, A., Rice, G.E. and Salomom, C. Placenta, 50, 60-69 (2017)   Recent studies report that 35% of women are either overweight or obese at reproductive age. The placenta continuously releases exosomes across gestation and their concentration is higher in pregnancy complications. While there is considerable interest in elucidating the role of exosomes during gestation, important questions remain to be answered: i) Does maternal BMI affect the exosomal profile across gestation? and ii) What is the contribution of placenta-derived exosomes to the total number of exosomes present in maternal plasma across gestation? Plasma samples were classified according to the maternal BMI into three groups (n = 15 per group): Lean, overweight, and obese. Total exosomes and specific placenta-derived exosomes were determined by Nanoparticle Tracking Analysis (NanoSight™) using quantum dots coupled with CD63 or PLAP antibodies. The effect of exosomes on cytokine (IL-6, IL-8, IL-10 and TNF-α) release from endothelial cells was established by cytokine array analysis (Bioplex-200). The total number of exosomes present in maternal circulation was strongly correlated with maternal BMI. Between ∼12% and ∼25% of circulating exosomes in maternal blood are of placental origin during gestation, and the contribution of placental exosomes to the total exosomal population decreases with higher maternal BMI across gestation. Exosomes increase IL-6, IL-8 and TNF-α release from endothelial cells, an effect even higher when exosomes were isolated from obese women compared to lean and overweight. This study established that maternal BMI is a factor that explains a significant component of the variation in the exosomes data. Exosomes may contribute to the maternal systemic inflammation during pregnancy.  

3.2655           Isolation of bacterial compartments to track movement of protein synthesis factors

Zhao, H. and martinis, S.A. Methods, 113, 120-126 (2017)   Aminoacyl-tRNA synthetases (AARSs) comprise an enzyme family that generates and maintains pools of aminoacylated tRNAs, which serve as essential substrates for protein synthesis. Many protein synthesis factors, including tRNA and AARSs also have non-canonical functions. Particularly in mammalian cells, alternate functions of AARSs have been associated with re-distribution in the cell to sites that are removed from translation. Sub-fractionation methods for E. coli were designed and optimized to carefully investigate re-localization of bacterial AARSs and tRNA that might aid in conferring alternate activities. Cell fractionation included isolation of the cytoplasm, periplasm, membrane, outer membrane vesicles, and extracellular media. Specific endogenous proteins and RNAs were probed respectively within each fraction via Western blots using antibodies and by Northern blots with primers to unique regions of the nucleic acid.  

3.2656           The new obesity-associated protein, neuronal growth regulator 1 (NEGR1), is implicated in Niemann-Pick disease Type C (NPC2)-mediated cholesterol trafficking

Kim, H., Chun, Y., Che, L., Kim, J., Lee, S. and Lee, S. Biochem. Biophys. Res. Comm., 482, 1367-1374 (2017)   Neuronal growth regulator 1 (NEGR1) is a newly identified raft-associated protein, which has recently been spotlighted as a new locus related to human obesity. Niemann-Pick disease Type C2 (NPC2) protein functions as a key player in the intracellular cholesterol trafficking, and its defect is linked to a fatal human neurodegenerative disease, NPC. In this study, we identified that NEGR1 interacts with NPC2 and increases its protein stability. Ectopically expressed NEGR1 proteins relieved an abnormal cholesterol accumulation in endosomal compartments. Importantly, NEGR1-defective mouse embryonic fibroblast cells exhibit increased cholesterol levels and triglyceride contents. These findings provide the first insight into the role of NEGR1 in intracellular cholesterol homeostasis, possibly explaining the missing link between NEGR1 with human obesity.  

3.2657           MicroRNAs in extracellular vesicles: potential cancer biomarkers

Kinoshita, T., Yip, K.W., Spence, T. and Liu, F-F.

1. Hum. Genet., 62(1), 67-74 (2017)

  Extracellular vesicles (EV) are small membrane-bound structures that are secreted by various cell types, including tumor cells. Recent studies have shown that EVs are important for cell-to-cell communication, locally and distantly; horizontally transferring DNA, mRNA, microRNA (miRNA), proteins and lipids. In the context of cancer biology, tumor-derived EVs are capable of modifying the microenvironment, promoting tumor progression, immune evasion, angiogenesis and metastasis. miRNAs contained within EVs are functionally associated with cancer progression, metastasis and aggressive tumor phenotypes. These factors, along with their stability in bodily fluids, have led to extensive investigations on the potential role of circulating EV-derived miRNAs as tumor biomarkers. In this review, we summarize the current understanding of circulating EV miRNAs in human cancer, and discuss their clinical utility and challenges in functioning as biomarkers.  

3.2658           Extracellular Vesicles Isolated from the Leaf Apoplast Carry Stress-Response Proteins

Rutter, B.D. and Innes, R.W. Plant Physiol., 173, 728-741 (2017)   Exosomes are extracellular vesicles (EVs) that play a central role in intercellular signaling in mammals by transporting proteins and small RNAs. Plants are also known to produce EVs, particularly in response to pathogen infection. The contents of plant EVs have not been analyzed, however, and their function is unknown. Here, we describe a method for purifying EVs from the apoplastic fluids of Arabidopsis (Arabidopsis thaliana) leaves. Proteomic analyses of these EVs revealed that they are highly enriched in proteins involved in biotic and abiotic stress responses. Consistent with this finding, EV secretion was enhanced in plants infected with Pseudomonas syringae and in response to treatment with salicylic acid. These findings suggest that EVs may represent an important component of plant immune responses.  

3.2659           Bicarbonate-sensing soluble adenylyl cyclase is present in the cell cytoplasm and nucleus of multiple shark tissues

Roa, J.N. and Tresguerres, M. Physiol Rep., 5(2), e13090 (2017)   The enzyme soluble adenylyl cyclase (sAC) is directly stimulated by bicarbonate (HCO₃⁻) to produce the signaling molecule cyclic adenosine monophosphate (cAMP). Because sAC and sAC‐related enzymes are found throughout phyla from cyanobacteria to mammals and they regulate cell physiology in response to internal and external changes in pH, CO₂, and HCO₃⁻, sAC is deemed an evolutionarily conserved acid‐base sensor. Previously, sAC has been reported in dogfish shark and round ray gill cells, where they sense and counteract blood alkalosis by regulating the activity of V‐type H⁺‐ ATPase. Here, we report the presence of sAC protein in gill, rectal gland, cornea, intestine, white muscle, and heart of leopard shark Triakis semifasciata. Co‐expression of sAC with transmembrane adenylyl cyclases supports the presence of cAMP signaling microdomains. Furthermore, immunohistochemistry on tissue sections, and western blots and cAMP‐activity assays on nucleus‐enriched fractions demonstrate the presence of sAC protein in and around nuclei. These results suggest that sAC modulates multiple physiological processes in shark cells, including nuclear functions.  

3.2660           MicroRNA Profiling of Exosomes

Daly, M. and O’Driscoll, L. Methods in Mol. Biol., 1509, 37-46 (2017   Exosomes are nano-sized membrane-bound vesicles released by a range of different cell types. Exosomes have been shown to specifically package certain membrane and cytosolic proteins and nucleic acids. Furthermore, it has been shown that their contents can be transferred to secondary cells, affecting the recipient cells’ cellular processes. Exosomes are present in a multitude of body fluids and so represent a novel source of circulating biomarkers. Here, we describe ultracentrifugation methods suitable for the isolation of exosomes from serum and plasma. We also detail transmission electron microscopy, nanoparticle tracking analysis, and immunoblotting methods suitable for the characterization of exosomes.  

3.2661           The Trypanosoma cruzi Surface, a Nanoscale Patchwork Quilt

Mucci, J., Lantos, A.B., Buscaglia, C.A., Leguizamon, M.S. and Campetella, O. Trends in Parasitol., 33(2), 102-112 (2017)   The Trypanosoma cruzi trypomastigote membrane provides a major protective role against mammalian host-derived defense mechanisms while allowing the parasite to interact with different cell types and trigger pathogenesis. This surface has been historically appreciated as a rather unstructured ‘coat’, mainly consisting of a continuous layer of glycolipids and heavily O-glycosylated mucins, occasionally intercalated with different developmentally regulated molecules displaying adhesive and/or enzymatic properties. Recent findings, however, indicate that the trypomastigote membrane is made up of multiple, densely packed and discrete 10–150 nm lipid-driven domains bearing different protein composition; hence resembling a highly organized ‘patchwork quilt’ design. Here, we discuss different aspects underlying the biogenesis, assembly, and dynamics of this cutting-edge fashion outfit, as well as its functional implications.  

3.2662           Localization of a Trypanosome Peroxin to the Endoplasmic Reticulum

Bauer, S.T., McQueeney, K.E., patel, T and Morris, M.T.

1. Eurkaryotic Microbiol., 64, 97-105 (2017)

  Trypanosoma brucei is the causative agent of diseases that affect 30,000–50,000 people annually. Trypanosoma brucei harbors unique organelles named glycosomes that are essential to parasite survival, which requires growth under fluctuating environmental conditions. The mechanisms that govern the biogenesis of these organelles are poorly understood. Glycosomes are evolutionarily related to peroxisomes, which can proliferate de novo from the endoplasmic reticulum or through the growth and division of existing organelles depending on the organism and environmental conditions. The effect of environment on glycosome biogenesis is unknown. Here, we demonstrate that the glycosome membrane protein, TbPex13.1, is localized to glycosomes when cells are cultured under high glucose conditions and to the endoplasmic reticulum in low glucose conditions. This localization in low glucose was dependent on the presence of a C-terminal tripeptide sequence. Our findings suggest that glycosome biogenesis is influenced by extracellular glucose levels and adds to the growing body of evidence that de novo glycosome biogenesis occurs in trypanosomes. Because the movement of peroxisomal membrane proteins is a hallmark of ER-dependent peroxisome biogenesis, TbPex13.1 may be a useful marker for the study such processes in trypanosomes.  

3.2663           Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy

Kimura, T., Jia, J., Kumar, S., Choi, S.W., Gu, Y., Mudd, M., Dupont, N., Jiang, S., Peters, R., Farzam, F., Jain, A., Lidke, K.A., Adams, C.M., Johansen, T. and Deretic, V. EMBO J., 36, 42-60 (2017)   Autophagy is a process delivering cytoplasmic components to lysosomes for degradation. Autophagy may, however, play a role in unconventional secretion of leaderless cytosolic proteins. How secretory autophagy diverges from degradative autophagy remains unclear. Here we show that in response to lysosomal damage, the prototypical cytosolic secretory autophagy cargo IL-1β is recognized by specialized secretory autophagy cargo receptor TRIM16 and that this receptor interacts with the R-SNARE Sec22b to recruit cargo to the LC3-II⁺ sequestration membranes. Cargo secretion is unaffected by downregulation of syntaxin 17, a SNARE promoting autophagosome–lysosome fusion and cargo degradation. Instead, Sec22b in combination with plasma membrane syntaxin 3 and syntaxin 4 as well as SNAP-23 and SNAP-29 completes cargo secretion. Thus, secretory autophagy utilizes a specialized cytosolic cargo receptor and a dedicated SNARE system. Other unconventionally secreted cargo, such as ferritin, is secreted via the same pathway.  

3.2664           Simvastatin promotes NPC1-mediated free cholesterol efflux from lysosomes through CYP7A1/LXRα signalling pathway in oxLDL-loaded macrophages

Xu, X., Zhang, A., Halquist, M.S., Yuan, X., Henderson, S.C., Dewey, W.L., Li, P-L., Li, N. and Zhang, F.

1. Cell. Mol. Med., 21(2), 364-374 (2017)

  Statins, 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors, are the first-line medications prescribed for the prevention and treatment of coronary artery diseases. The efficacy of statins has been attributed not only to their systemic cholesterol-lowering actions but also to their pleiotropic effects that are unrelated to cholesterol reduction. These pleiotropic effects have been increasingly recognized as essential in statins therapy. This study was designed to investigate the pleiotropic actions of simvastatin, one of the most commonly prescribed statins, on macrophage cholesterol homeostasis with a focus on lysosomal free cholesterol egression. With simultaneous nile red and filipin staining, analysis of confocal/multi-photon imaging demonstrated that simvastatin markedly attenuated unesterified (free) cholesterol buildup in macrophages loaded with oxidized low-density lipoprotein but had little effect in reducing the sizes of cholesteryl ester-containing lipid droplets; the reduction in free cholesterol was mainly attributed to decreases in lysosome-compartmentalized cholesterol. Functionally, the egression of free cholesterol from lysosomes attenuated pro-inflammatory cytokine secretion. It was determined that the reduction of lysosomal free cholesterol buildup by simvastatin was due to the up-regulation of Niemann-Pick C1 (NPC1), a lysosomal residing cholesterol transporter. Moreover, the enhanced enzymatic production of 7-hydroxycholesterol by cytochrome P450 7A1 and the subsequent activation of liver X receptor α underscored the up-regulation of NPC1. These findings reveal a novel pleiotropic effect of simvastatin in affecting lysosomal cholesterol efflux in macrophages and the associated significance in the treatment of atherosclerosis.  

3.2665           Isolation and characterization of exosomes derived from fertile sheep hydatid cysts

Siles-Lucas, M., Sanchez-Ovejero, C., Gonzalez-Sanchez, M., Gonzalez, E., Falcon-Perez, J., Boufana, B., Fratini, F., Casulli, A. and Manzano-Roman, R. Vet. Parasitol., 236, 23-33 (2017)   Cystic echinococcosis (CE) is a chronic and complex zoonotic disease. Information on the mechanisms involved in parasite establishment, growth and persistence remain limited. These may be modulated by a crosstalk between extracellular vesicles (EVs). EVs including exosomes and microvesicles are able to carry developmental signaling proteins which coordinate growth and establishment of several parasites. Here, an exosome enriched EV fraction was isolated from hydatid fluid (HF) of fertile sheep cysts. A proteomic analysis of this fraction identified a number of parasite-derived vesicle-membrane associated proteins as well as cytosolic proteinsAdditionally, the exosomal enriched fraction contained proteins of host origin. Specific proteins −antigen B2 and TSPAN14- in the exosomal fraction were further assayed by immunoblot and transmission electron microscopy. To the best of our knowledge, this is the first report on the presence of parasite exosomes in fertile hydatid cyst fluid. Further characterization of the exosome cargo will allow the discovery of new markers for the detection of CE in humans and animals, and the treatment of CE patients, and provide new insights regarding the role of these EVs in the establishment and persistence of hydatid cysts.  

3.2666           Endosulfine-alpha inhibits membrane-induced α-synuclein aggregation and protects against α-synuclein neurotoxicity

Ysselstein, D., Dehay, B., Costantino, I.M., McCabe, G.P., Frosch, M.P., George, J.M., Bezard, E. and Rochet, J-C. Acta Neuropathologica Comm., 5:3 (2017)   Neuropathological and genetic findings suggest that the presynaptic protein α-synuclein (aSyn) is involved in the pathogenesis of synucleinopathy disorders, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy. Evidence suggests that the self-assembly of aSyn conformers bound to phospholipid membranes in an aggregation-prone state plays a key role in aSyn neurotoxicity. Accordingly, we hypothesized that protein binding partners of lipid-associated aSyn could inhibit the formation of toxic aSyn oligomers at membrane surfaces. To address this hypothesis, we characterized the protein endosulfine-alpha (ENSA), previously shown to interact selectively with membrane-bound aSyn, in terms of its effects on the membrane-induced aggregation and neurotoxicity of two familial aSyn mutants, A30P and G51D. We found that wild-type ENSA, but not the non-aSyn-binding S109E variant, interfered with membrane-induced aSyn self-assembly, aSyn-mediated vesicle disruption and aSyn neurotoxicity. Immunoblotting analyses revealed that ENSA was down-regulated in the brains of synucleinopathy patients versus non-diseased individuals. Collectively, these results suggest that ENSA can alleviate neurotoxic effects of membrane-bound aSyn via an apparent chaperone-like activity at the membrane surface, and a decrease in ENSA expression may contribute to aSyn neuropathology in synucleinopathy disorders. More generally, our findings suggest that promoting interactions between lipid-bound, amyloidogenic proteins and their binding partners is a viable strategy to alleviate cytotoxicity in a range of protein misfolding disorders.  

3.2667           The effects of glomerular and tubular renal progenitors and derived extracellular vesicles on recovery from acute kidney injury

Ranghino, A., Bruno, S., Bussolati, B., Moggio, A., Dimuccio, V., Tapporo, M., Biancone, L., Gontero, P., Frea, B. and Camussi, G. Stem Cell Res. Ther., 8:24 (2017)   Background Mesenchymal stromal cells (MSCs) and renal stem/progenitors improve the recovery of acute kidney injury (AKI) mainly through the release of paracrine mediators including the extracellular vesicles (EVs). Several studies have reported the existence of a resident population of MSCs within the glomeruli (Gl-MSCs). However, their contribution towards kidney repair still remains to be elucidated. The aim of the present study was to evaluate whether Gl-MSCs and Gl-MSC-EVs promote the recovery of AKI induced by ischemia-reperfusion injury (IRI) in SCID mice. Moreover, the effects of Gl-MSCs and Gl-MSC-EVs were compared with those of CD133⁺ progenitor cells isolated from human tubules of the renal cortical tissue (T-CD133⁺ cells) and their EVs (T-CD133⁺-EVs). Methods IRI was performed in mice by clamping the left renal pedicle for 35 minutes together with a right nephrectomy. Immediately after reperfusion, the animals were divided in different groups to be treated with: Gl-MSCs, T-CD133⁺ cells, Gl-MSC-EVs, T-CD133⁺-EVs or vehicle. To assess the role of vesicular RNA, EVs were either isolated by floating to avoid contamination of non-vesicles-associated RNA or treated with a high dose of RNase. Mice were sacrificed 48 hours after surgery. Results Gl-MSCs, and Gl-MSC-EVs both ameliorate kidney function and reduce the ischemic damage post IRI by activating tubular epithelial cell proliferation. Furthermore, T-CD133⁺ cells, but not their EVs, also significantly contributed to the renal recovery after IRI compared to the controls. Floating EVs were effective while RNase-inactivated EVs were ineffective. Analysis of the EV miRnome revealed that Gl-MSC-EVs selectively expressed a group of miRNAs, compared to EVs derived from fibroblasts, which were biologically ineffective in IRI. Conclusions In this study, we demonstrate that Gl-MSCs may contribute in the recovery of mice with AKI induced by IRI primarily through the release of EVs.  

3.2668           TAX1BP1 Restrains Virus-Induced Apoptosis by Facilitating Itch-Mediated Degradation of the Mitochondrial Adaptor MAVS

Choi, Y.B., Shembade, N., parvatiyar, K., Balachandran, S. and Harhaj, E.W. Mol. Cell. Biol., 37(1), e00422-16 (2017)   The host response to RNA virus infection consists of an intrinsic innate immune response and the induction of apoptosis as mechanisms to restrict viral replication. The mitochondrial adaptor molecule MAVS plays critical roles in coordinating both virus-induced type I interferon production and apoptosis; however, the regulation of MAVS-mediated apoptosis is poorly understood. Here, we show that the adaptor protein TAX1BP1 functions as a negative regulator of virus-induced apoptosis. TAX1BP1-deficient cells are highly sensitive to apoptosis in response to infection with the RNA viruses vesicular stomatitis virus and Sendai virus and to transfection with poly(I·C). TAX1BP1 undergoes degradation during RNA virus infection, and loss of TAX1BP1 is associated with apoptotic cell death. TAX1BP1 deficiency augments virus-induced activation of proapoptotic c-Jun N-terminal kinase (JNK) signaling. Virus infection promotes the mitochondrial localization of TAX1BP1 and concomitant interaction with the mitochondrial adaptor MAVS. TAX1BP1 recruits the E3 ligase Itch to MAVS to trigger its ubiquitination and degradation, and loss of TAX1BP1 or Itch results in increased MAVS protein expression. Together, these results indicate that TAX1BP1 functions as an adaptor molecule for Itch to target MAVS during RNA virus infection and thus restrict virus-induced apoptosis.  

3.2669           Spatiotemporal Uncoupling of MicroRNA-Mediated Translational Repression and Target RNA Degradation Controls MicroRNP Recycling in Mammalian Cells

Bose, M., Baarman, B., Goswami, A. and Bhattacharyya, S.N. Mol. Cell. Biol., 37(4), e00464-16 (2017)   MicroRNA (miRNA)-mediated repression controls expression of more than half of protein-coding genes in metazoan animals. Translation repression is associated with target mRNA degradation initiated by decapping and deadenylation of the repressed mRNAs. Earlier evidence suggests the endoplasmic reticulum (ER) as the site where microRNPs (miRNPs) interact with their targets before translation repression sets in, but the subcellular location of subsequent degradation of miRNA-repressed messages is largely unidentified. Here, we explore the subcellular distribution of essential components of degradation machineries of miRNA-targeted mRNAs. We have noted that interaction of target mRNAs with AGO2 protein on the ER precedes the relocalization of repressed messages to multivesicular bodies (MVBs). The repressed messages subsequently get deadenylated, lose their interaction with AGO2, and become decapped. Blocking maturation of endosomes to late endosome and MVBs by targeting the endosomal protein HRS uncouples miRNA-mediated translation repression from target RNA degradation. HRS is also targeted by the intracellular parasite Leishmania donovani, which curtails the HRS level in infected cells to prevent uncoupling of mRNA-AGO2 interaction, preventing degradation of translationally repressed messages, and thus stops recycling of miRNPs preengaged in repression.  

3.2670           CD63 Regulates Epstein-Barr Virus LMP1 Exosomal Packaging, Enhancement of Vesicle Production, and Noncanonical NF-κB Signaling

Hurwitz, S.N., Nkosi, D., Conlon, M.N., York, S.B., Liu, X., Tremblay, D.C. and Meckes Jr., D.G.

1. Virol., 91(5), e02251-16 (2017)

  Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. Recently, LMP1 was shown to be copurified with CD63, a conserved tetraspanin protein enriched in late endosomal and lysosomal compartments. Here, we demonstrate the importance of CD63 presence for exosomal packaging of LMP1. Nanoparticle tracking analysis and gradient purification revealed an increase in extracellular vesicle secretion and exosomal proteins following LMP1 expression. Immunoisolation of CD63-positive exosomes exhibited accumulation of LMP1 in this vesicle population. Functionally, CRISPR/Cas9 knockout of CD63 resulted in a reduction of LMP1-induced particle secretion. Furthermore, LMP1 packaging was severely impaired in CD63 knockout cells, concomitant with a disruption in the perinuclear localization of LMP1. Importantly, LMP1 trafficking to lipid rafts and activation of NF-κB and PI3K/Akt pathways remained intact following CD63 knockout, while mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and noncanonical NF-κB activation were observed to be increased. These results suggest that CD63 is a critical player in LMP1 exosomal trafficking and LMP1-mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling.  

3.2671           Comprehensive proteome profiling of glioblastoma-derived extracellular vesicles identifies markers for more aggressive disease

Mallawaarratchy, D.M., Hallal, S., Russell, B., Ly, L., Wbrahimkhani, S., Wei, H., Christopherson, R.I., Buckland, M.E. and Kaufman, K.L.

1. Neurooncol., 131, 233-244 (2017)

  Extracellular vesicles (EVs) play key roles in glioblastoma (GBM) biology and represent novel sources of biomarkers that are detectable in the peripheral circulation. Despite this notionally non-invasive approach to assess GBM tumours in situ, a comprehensive GBM EV protein signature has not been described. Here, EVs secreted by six GBM cell lines were isolated and analysed by quantitative high-resolution mass spectrometry. Overall, 844 proteins were identified in the GBM EV proteome, of which 145 proteins were common to EVs secreted by all cell lines examined; included in the curated EV compendium (Vesiclepedia_559; http://microvesicles.org). Levels of 14 EV proteins significantly correlated with cell invasion (invadopodia production; r² > 0.5, p < 0.05), including several proteins that interact with molecules responsible for regulating invadopodia formation. Invadopodia, actin-rich membrane protrusions with proteolytic activity, are associated with more aggressive disease and are sites of EV release. Gene levels corresponding to invasion-related EV proteins showed that five genes (annexin A1, actin-related protein 3, integrin-β1, insulin-like growth factor 2 receptor and programmed cell death 6-interacting protein) were significantly higher in GBM tumours compared to normal brain in silico, with common functions relating to actin polymerisation and endosomal sorting. We also show that Cavitron Ultrasonic Surgical Aspirator (CUSA) washings are a novel source of brain tumour-derived EVs, demonstrated by particle tracking analysis, TEM and proteome profiling. Quantitative proteomics corroborated the high levels of proposed invasion-related proteins in EVs enriched from a GBM compared to low-grade astrocytoma tumour. Large-scale clinical follow-up of putative biomarkers, particularly the proposed survival marker annexin A1, is warranted.  

3.2672           High-density lipoprotein and apolipoprotein A-I inhibit palmitate-induced translocation of toll-like receptor 4 into lipid rafts and inflammatory cytokines in 3T3-L1 adipocytes

Yamada, H., Umemoto, T., kawano, M., Kawakami, M., Kakei, M., Momomura, S-i., Ishikawa, S-e and Hara, K. Biochem. Biophys. Res. Comm., 484, 403-408 (2017)   Saturated fatty acids (SFAs) activate toll-like receptor 4 (TLR4) signal transduction in macrophages and are involved in the chronic inflammation accompanying obesity. High-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I) produce anti-inflammatory effects via reverse cholesterol transport. However, the underlying mechanisms by which HDL and apoA-I inhibit inflammatory responses in adipocytes remain to be determined. Here we examined whether palmitate increases the translocation of TLR4 into lipid rafts and whether HDL and apoA-I inhibit inflammation in adipocytes. Palmitate exposure (250 μM, 24 h) increased interleukin-6 and tumor necrosis factor-α gene expressions and translocation of TLR4 into lipid rafts in 3T3-L1 adipocytes. Pretreatment with HDL and apoA-I (50 μg/mL, 6 h) suppressed palmitate-induced inflammatory cytokine expression and TLR4 translocation into lipid rafts. Moreover, HDL and apoA-I inhibited palmitate-induced phosphorylation of nuclear factor-kappa B. HDL showed an anti-inflammatory effect via ATP-binding cassette transporter G1 and scavenger receptor class B, member 1, whereas apoA-I showed an effect via ATP-binding cassette transporter A1. These results demonstrated that HDL and apoA-I reduced palmitate-potentiated TLR4 trafficking into lipid rafts and its related inflammation in adipocytes via these specific transporters.  

3.2673           Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury

Bielaszewska, M. et al PloS Pathogens, 13(2), e1006159 (2017)   Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors, we provide new insights into the pathogenesis of EHEC O157 infections. Our data have implications for considering O157 OMVs as vaccine candidates.  

3.2674           Extracellular Vesicles: Unique Intercellular Delivery Vehicles

Maas, S.L.N., Breakfield, X.O. and Weaver, A.M. Trends in Cell Biol., 27(3), 172-188 (2017)   Extracellular vesicles (EVs) are a heterogeneous collection of membrane-bound carriers with complex cargoes including proteins, lipids, and nucleic acids. While the release of EVs was previously thought to be only a mechanism to discard nonfunctional cellular components, increasing evidence implicates EVs as key players in intercellular and even interorganismal communication. EVs confer stability and can direct their cargoes to specific cell types. EV cargoes also appear to act in a combinatorial manner to communicate directives to other cells. This review focuses on recent findings and knowledge gaps in the area of EV biogenesis, release, and uptake. In addition, we highlight examples whereby EV cargoes control basic cellular functions, including motility and polarization, immune responses, and development, and contribute to diseases such as cancer and neurodegeneration.  

3.2675           Glycan region of GPI anchored-protein is required for cytocidal oligomerization of an anticancer parasporin-2, Cry46Aa1 protein, from Bacillus thuringiensis strain A1547

Abe, Y., Inoue, H., Ashida, H., Maeda, Y., Kinoshita, T. and Kitada, S.

1. Inverterbrate Pathol., 142, 71-81 (2017)

  Parasporin-2 (PS2), alternatively named Cry46Aa1, an anticancer protein derived from Bacillus thuringiensis strain A1547, causes specific cell damage via PS2 oligomerization in the cell membrane. Although PS2 requires glycosylphosphatidylinositol (GPI)-anchored proteins for its cytocidal action, their precise role is unknown. Here, we report that the glycan of GPI induces PS2 oligomerization, which causes cell death. Cytotoxicity, cell-binding and oligomerization of the toxin were not observed in GPI-anchored protein-deficient Chinese hamster ovary cells. Expression and protease-treatment analyses showed that the actions of the toxin were dependent on the glycan core, not the polypeptide moiety, of GPI-anchored proteins. However, surface expression of some GPI-anchored proteins is observed in PS2-insensitive cells. These data suggest that GPI-anchored proteins do not determine the target specificity, but instead function as a kind of coreceptor, in the cytocidal action of PS2.  

3.2676           Inhibition of Transient Receptor Potential Channel Mucolipin-1 (TRPML1) by Lysosomal Adenosine Involved in Severe Combined Immunodeficiency Diseases

Zhong, X.Z., Zou, Y., Sun, X., Dong, G., Cao, Q., Pandey, A., Rainey, J.K., Zhu, X. and Dong, X-P. J.Biol. Chem., 292(8), 3445-3455 (2017)   Impaired adenosine homeostasis has been associated with numerous human diseases. Lysosomes are referred to as the cellular recycling centers that generate adenosine by breaking down nucleic acids or ATP. Recent studies have suggested that lysosomal adenosine overload causes lysosome defects that phenocopy patients with mutations in transient receptor potential channel mucolipin-1 (TRPML1), a lysosomal Ca²⁺ channel, suggesting that lysosomal adenosine overload may impair TRPML1 and then lead to subsequent lysosomal dysfunction. In this study, we demonstrate that lysosomal adenosine is elevated by deleting adenosine deaminase (ADA), an enzyme responsible for adenosine degradation. We also show that lysosomal adenosine accumulation inhibits TRPML1, which is rescued by overexpressing ENT3, the adenosine transporter situated in the lysosome membrane. Moreover, ADA deficiency results in lysosome enlargement, alkalinization, and dysfunction. These are rescued by activating TRPML1. Importantly, ADA-deficient B-lymphocytes are more vulnerable to oxidative stress, and this was rescued by TRPML1 activation. Our data suggest that lysosomal adenosine accumulation impairs lysosome function by inhibiting TRPML1 and subsequently leads to cell death in B-lymphocytes. Activating TRPML1 could be a new therapeutic strategy for those diseases.  

3.2677           Sequences within the C Terminus of the Metabotropic Glutamate Receptor 5 (mGluR5) Are Responsible for Inner Nuclear Membrane Localization

Sergin, I., Jong, Y-J.I., Harmon, S.K., Kumar, V. and O’Malley, K.L.

1. Biol. Chem., 292(9), 3637-3655 (2017)

  Traditionally, G-protein-coupled receptors (GPCR) are thought to be located on the cell surface where they transmit extracellular signals to the cytoplasm. However, recent studies indicate that some GPCRs are also localized to various subcellular compartments such as the nucleus where they appear required for various biological functions. For example, the metabotropic glutamate receptor 5 (mGluR5) is concentrated at the inner nuclear membrane (INM) where it mediates Ca²⁺ changes in the nucleoplasm by coupling with G_q/11. Here, we identified a region within the C-terminal domain (amino acids 852–876) that is necessary and sufficient for INM localization of the receptor. Because these sequences do not correspond to known nuclear localization signal motifs, they represent a new motif for INM trafficking. mGluR5 is also trafficked to the plasma membrane where it undergoes re-cycling/degradation in a separate receptor pool, one that does not interact with the nuclear mGluR5 pool. Finally, our data suggest that once at the INM, mGluR5 is stably retained via interactions with chromatin. Thus, mGluR5 is perfectly positioned to regulate nucleoplasmic Ca²⁺ in situ.  

3.2678           Methods to isolate extracellular vesicles for diagnosis

Kang, H., Kim, J. and Park, J. Micro and Nano Syst. Lett., 5:15 82017)   Extracellular vesicles (EVs) are small membrane-bound bodies that are released into extracellular space by diverse cells, and are found in body fluids like blood, urine and saliva. EVs contain RNA, DNA and proteins, which can be biomarkers for diagnosis. EVs can be obtained by minimally-invasive biopsy, so they are useful in disease diagnosis. High yield and purity contribute to precise diagnosis of disease, but damaged EVs and impurities can cause confu sed results. However, EV isolation methods have different yields and purities. Furthermore, the isolation method that is most suitable to maximize EV recovery efficiency depends on the experimental conditions. This review focuses on merits and demerits of several types of EV isolation methods, and provides examples of how to diagnose disease by exploiting information obtained by analysis of EVs.  

3.2679           Misrouting of v-ATPase subunit V0a1 dysregulates lysosomal acidification in a neurodegenerative lysosomal storage disease model

Bagh, M.B., Peng, S., Chandra, G., Zhang, Z., Singh, S.P., pattabiraman, N., Liu, A. and Mukherjee, A.B. Nature Communications, 8:14612 (2017)   Defective lysosomal acidification contributes to virtually all lysosomal storage disorders (LSDs) and to common neurodegenerative diseases like Alzheimer’s and Parkinson’s. Despite its fundamental importance, the mechanism(s) underlying this defect remains unclear. The v-ATPase, a multisubunit protein complex composed of cytosolic V1-sector and lysosomal membrane-anchored V0-sector, regulates lysosomal acidification. Mutations in the CLN1 gene, encoding PPT1, cause a devastating neurodegenerative LSD, INCL. Here we report that in Cln1−/− mice, which mimic INCL, reduced v-ATPase activity correlates with elevated lysosomal pH. Moreover, v-ATPase subunit a1 of the V0 sector (V0a1) requires palmitoylation for interacting with adaptor protein-2 (AP-2) and AP-3, respectively, for trafficking to the lysosomal membrane. Notably, treatment of Cln1−/− mice with a thioesterase (Ppt1)-mimetic, NtBuHA, ameliorated this defect. Our findings reveal an unanticipated role of Cln1 in regulating lysosomal targeting of V0a1 and suggest that varying factors adversely affecting v-ATPase function dysregulate lysosomal acidification in other LSDs and common neurodegenerative diseases.  

3.2680           Sorting Nexin 9 facilitates podocin endocytosis in the injured podocyte

Sasaki, Y., Hidaka, T., Ueno, T., Akiba-Takagi, M., Trejo, J.A.O., Seki, T., Nagai-Hosoe, Y., Tanaka, E., Horikoshi, S., Tomino, Y., Suzuki, Y. and Asanuma, K. Scientific Reports, 7:43921 (2017)   The irreversibility of glomerulosclerotic changes depends on the degree of podocyte injury. We have previously demonstrated the endocytic translocation of podocin to the subcellular area in severely injured podocytes and found that this process is the primary disease trigger. Here we identified the protein sorting nexin 9 (SNX9) as a novel facilitator of podocin endocytosis in a yeast two-hybrid analysis. SNX9 is involved in clathrin-mediated endocytosis, actin rearrangement and vesicle transport regulation. Our results revealed and confirmed that SNX9 interacts with podocin exclusively through the Bin–Amphiphysin–Rvs (BAR) domain of SNX9. Immunofluorescence staining revealed the expression of SNX9 in response to podocyte adriamycin-induced injury both in vitro and in vivo. Finally, an analysis of human glomerular disease biopsy samples demonstrated strong SNX9 expression and co-localization with podocin in samples representative of severe podocyte injury, such as IgA nephropathy with poor prognosis, membranous nephropathy and focal segmental glomerulosclerosis. In conclusion, we identified SNX9 as a facilitator of podocin endocytosis in severe podocyte injury and demonstrated the expression of SNX9 in the podocytes of both nephropathy model mice and human patients with irreversible glomerular disease.  

3.2681           Defining the purity of exosomes required for diagnostic profiling of small RNA suitable for biomarker discovery

Quek, C., Bellingham, S.A., Jung, C-H., Scicluna, B.J., Shambrook, M.C., Sharples, R.A., Cheng, L. and Hill, A.F. RNA Biol., 14(2), 245-258 (2017)   Small non-coding RNAs (ncRNA), including microRNAs (miRNA), enclosed in exosomes are being utilised for biomarker discovery in disease. Two common exosome isolation methods involve differential ultracentrifugation or differential ultracentrifugation coupled with Optiprep gradient fractionation. Generally, the incorporation of an Optiprep gradient provides better separation and increased purity of exosomes. The question of whether increased purity of exosomes is required for small ncRNA profiling, particularly in diagnostic and biomarker purposes, has not been addressed and highly debated. Utilizing an established neuronal cell system, we used next-generation sequencing to comprehensively profile ncRNA in cells and exosomes isolated by these 2 isolation methods. By comparing ncRNA content in exosomes from these two methods, we found that exosomes from both isolation methods were enriched with miRNAs and contained a diverse range of rRNA, small nuclear RNA, small nucleolar RNA and piwi-interacting RNA as compared with their cellular counterparts. Additionally, tRNA fragments (30-55 nucleotides in length) were identified in exosomes and may act as potential modulators for repressing protein translation. Overall, the outcome of this study confirms that ultracentrifugation-based method as a feasible approach to identify ncRNA biomarkers in exosomes.  

3.2682           Plasmalogen biosynthesis is spatiotemporally regulated by sensing plasmalogens in the inner leaflet of plasma membranes

Honsho, M., Abe, Y. and Fujiki, Y. Scientific Reports, 7:43936 (2017)   Alkenyl ether phospholipids are a major sub-class of ethanolamine- and choline-phospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. Biosynthesis of ethanolamine-containing alkenyl ether phospholipids, plasmalogens, is regulated by modulating the stability of fatty acyl-CoA reductase 1 (Far1) in a manner dependent on the level of cellular plasmalogens. However, precise molecular mechanisms underlying the regulation of plasmalogen synthesis remain poorly understood. Here we show that degradation of Far1 is accelerated by inhibiting dynamin-, Src kinase-, or flotillin-1-mediated endocytosis without increasing the cellular level of plasmalogens. By contrast, Far1 is stabilized by sequestering cholesterol with nystatin. Moreover, abrogation of the asymmetric distribution of plasmalogens in the plasma membrane by reducing the expression of CDC50A encoding a β-subunit of flippase elevates the expression level of Far1 and plasmalogen synthesis without reducing the total cellular level of plasmalogens. Together, these results support a model that plasmalogens localised in the inner leaflet of the plasma membranes are sensed for plasmalogen homeostasis in cells, thereby suggesting that plasmalogen synthesis is spatiotemporally regulated by monitoring cellular level of plasmalogens.  

3.2683           Critical role of quorum sensing-dependent glutamate metabolism in homeostatic osmolality and outer membrane vesiculation in Burkholderia glumae

Kang, Y., Goo, E., Kim, J. and Hwang, I. Scientific Reports, 7:44195 (2017)   Metabolic homeostasis in cooperative bacteria is achieved by modulating primary metabolism in a quorum sensing (QS)-dependent manner. A perturbed metabolism in QS mutants causes physiological stress in the rice bacterial pathogen Burkholderia glumae. Here, we show that increased bacterial osmolality in B. glumae is caused by unusually high cellular concentrations of glutamate and betaine generated by QS deficiencies. QS negatively controls glutamate uptake and the expression of genes involved in the glutamine synthetase and glutamine oxoglutarate aminotransferase cycles. Thus, cellular glutamate levels were significantly higher in the QS mutants than in the wild type, and they caused hyperosmotic cellular conditions. Under the hypotonic conditions of the periplasm in the QS mutants, outer membrane bulging and vesiculation were observed, although these changes were rescued by knocking out the gltI gene, which encodes a glutamate transporter. Outer membrane modifications were not detected in the wild type. These results suggest that QS-dependent glutamate metabolism is critical for homeostatic osmolality. We suggest that outer membrane bulging and vesiculation might be the outcome of a physiological adaptation to relieve hypotonic osmotic stress in QS mutants. Our findings reveal how QS functions to maintain bacterial osmolality in a cooperative population.  

3.2684           Localization of Protein Kinase NDR2 to Peroxisomes and Its Role in Ciliogenesis

Abe, S., Nagai, T., Masukawa, M., Okumoto, K., Homma, Y., Fujiki, Y. and Mizuno, K.

1. Biol. Chem., 292(10), 4089-4098 (2017)

  Nuclear Dbf2-related (NDR) kinases, comprising NDR1 and NDR2, are serine/threonine kinases that play crucial roles in the control of cell proliferation, apoptosis, and morphogenesis. We recently showed that NDR2, but not NDR1, is involved in primary cilium formation; however, the mechanism underlying their functional difference in ciliogenesis is unknown. To address this issue, we examined their subcellular localization. Despite their close sequence similarity, NDR2 exhibited punctate localization in the cytoplasm, whereas NDR1 was diffusely distributed within the cell. Notably, NDR2 puncta mostly co-localized with the peroxisome marker proteins, catalase and CFP-SKL (cyan fluorescent protein carrying the C-terminal typical peroxisome-targeting signal type-1 (PTS1) sequence, Ser-Lys-Leu). NDR2 contains the PTS1-like sequence, Gly-Lys-Leu, at the C-terminal end, whereas the C-terminal end of NDR1 is Ala-Lys. An NDR2 mutant lacking the C-terminal Leu, NDR2(ΔL), exhibited almost diffuse distribution in cells. Additionally, NDR2, but neither NDR1 nor NDR2(ΔL), bound to the PTS1 receptor Pex5p. Together, these findings indicate that NDR2 localizes to the peroxisome by using the C-terminal GKL sequence. Intriguingly, topology analysis of NDR2 suggests that NDR2 is exposed to the cytosolic surface of the peroxisome. The expression of wild-type NDR2, but not NDR2(ΔL), recovered the suppressive effect of NDR2 knockdown on ciliogenesis. Furthermore, knockdown of peroxisome biogenesis factor genes (PEX1 or PEX3) partially suppressed ciliogenesis. These results suggest that the peroxisomal localization of NDR2 is implicated in its function to promote primary cilium formation.  

3.2685           Comparative Characterization of Phosphatidic Acid Sensors and Their Localization during Frustrated Phagocytosis

Kassas, N., Tanguy, E., Thahouly, T., Fouillen, L., Heintz, D., Chasserot-Golaz, S., Bader, M-F., grant, N.J. and Vitale, N.

1. Biol. Chem., 292(10), 4266-4279 (2017)

  Phosphatidic acid (PA) is the simplest phospholipid naturally existing in living organisms, but it constitutes only a minor fraction of total cell lipids. PA has attracted considerable attention because it is a phospholipid precursor, a lipid second messenger, and a modulator of membrane shape, and it has thus been proposed to play key cellular functions. The dynamics of PA in cells and in subcellular compartments, however, remains an open question. The recent generation of fluorescent probes for PA, by fusing GFP to PA-binding domains, has provided direct evidence for PA dynamics in different intracellular compartments. Here, three PA sensors were characterized in vitro, and their preferences for different PA species in particular lipidic environments were compared. In addition, the localization of PA in macrophages during frustrated phagocytosis was examined using these PA sensors and was combined with a lipidomic analysis of PA in intracellular compartments. The results indicate that the PA sensors display some preferences for specific PA species, depending on the lipid environment, and the localization study in macrophages revealed the complexity of intracellular PA dynamics.  

3.2686           Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array

Zetsche, B. et al Nature Biotechnology, 35(1), 31-34 (2017)   Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.  

3.2687           Tyrosine phosphorylation modulates mitochondrial chaperonin Hsp60 and delays rotavirus NSP4-mediated apoptotic signaling in host cells

Chattopadhyay, S., Mukherjee, A., Patra, U., Bhowmick, R., Basak, T., Sengupta, S. and Chawla-Sarkar, M. Cell. Microbiol., 19(3), e12670 (2017)   Phosphoproteomics-based platforms have been widely used to identify post translational dynamics of cellular proteins in response to viral infection. The present study was undertaken to assess differential tyrosine phosphorylation during early hours of rotavirus (RV) SA11 infection. Heat shock proteins (Hsp60) were found to be enriched in the data set of RV-SA11 induced differentially tyrosine-phosphorylated proteins at 2 hr post infection (hpi). Hsp60 was further found to be phosphorylated by an activated form of Src kinase on 227th tyrosine residue, and tyrosine phosphorylation of mitochondrial chaperonin Hsp60 correlated with its proteasomal degradation at 2–2.5hpi. Interestingly, mitochondrial Hsp60 positively influenced translocation of the rotaviral nonstructural protein 4 to mitochondria during RV infections. Phosphorylation and subsequent transient degradation of mitochondrial Hsp60 during early hours of RV-SA11 infection resulted in inhibition of premature import of nonstructural protein 4 into mitochondria, thereby delaying early apoptosis. Overall, the study highlighted one of the many strategies rotavirus undertakes to prevent early apoptosis and subsequent reduced viral progeny yield.  

3.2688           Extracellular vesicles: their role in cancer biology and epithelial-mesenchymal transition

Gopal, S.K., Greening, D.W., Rai, A., Chen, M., Xu, R., Shafiq, A., Mathias, R.A., Zhu, H-J., and Simpson, R.J. Biochemical J., 474(1), 21-45 (2017)   Cell–cell communication is critical across an assortment of physiological and pathological processes. Extracellular vesicles (EVs) represent an integral facet of intercellular communication largely through the transfer of functional cargo such as proteins, messenger RNAs (mRNAs), microRNA (miRNAs), DNAs and lipids. EVs, especially exosomes and shed microvesicles, represent an important delivery medium in the tumour micro-environment through the reciprocal dissemination of signals between cancer and resident stromal cells to facilitate tumorigenesis and metastasis. An important step of the metastatic cascade is the reprogramming of cancer cells from an epithelial to mesenchymal phenotype (epithelial–mesenchymal transition, EMT), which is associated with increased aggressiveness, invasiveness and metastatic potential. There is now increasing evidence demonstrating that EVs released by cells undergoing EMT are reprogrammed (protein and RNA content) during this process. This review summarises current knowledge of EV-mediated functional transfer of proteins and RNA species (mRNA, miRNA, long non-coding RNA) between cells in cancer biology and the EMT process. An in-depth understanding of EVs associated with EMT, with emphasis on molecular composition (proteins and RNA species), will provide fundamental insights into cancer biology.  

3.2689           RasGRF Couples Nox4-Dependent Endoplasmic Reticulum Signaling to Ras

Wu, R.F., Liao, C., Hatoum, H., Fu, G., Ochoa, C.D. and Terada, L.S. Arterioscler. Thromb. Vasc. Biol., 37(1), 98-107 (2017)   Objectives—In response to endoplasmic reticulum (ER) stress, endothelial cells initiate corrective pathways such as the unfolded protein response. Recent studies suggest that reactive oxygen species produced on the ER may participate in homeostatic signaling through Ras in response to ER stress. We sought to identify mechanisms responsible for this focal signaling pathway. Approach and Results—In endothelial cells, we found that ER stress induced by tunicamycin activates the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 focally on the ER surface but not on the plasma membrane. Ras activation is also restricted to the ER, occurs downstream of Nox4, and is required for activation of the unfolded protein response. In contrast, treatment with the growth factor VEGF (vascular endothelial growth factor) results in Ras activation and reactive oxygen species production confined instead to the plasma membrane and not to the ER, demonstrating local coupling of reactive oxygen species and Ras signals. We further identify the calcium-responsive, ER-resident guanyl exchange factors RasGRF1 and RasGRF2 as novel upstream mediators linking Nox4 with Ras activation in response to ER stress. Oxidation of the sarcoendoplasmic reticulum calcium ATPase and increases in cytosolic calcium caused by ER stress are blocked by Nox4 knockdown, and reduction in cytosolic free calcium prevents both Ras activation and the unfolded protein response. Conclusions—ER stress triggers a localized signaling module on the ER surface involving Nox4-dependent calcium mobilization, which directs local Ras activation through ER-associated, calcium-responsive RasGRF.  

3.2690           Chapter Two – Rationally Designed Peptide Probes for Extracellular Vesicles

Tamura, R. and Yin, H. Adv. Clin. Chem., 79, 25-41 (2017)   Extracellular vesicles (EVs) are submicroscopic lipid vesicles secreted from cells and play significant roles in cell-to-cell communication by transporting varieties of cell signaling molecules like proteins, DNA, mRNA, and microRNA. Recent studies showed that EVs are highly correlated with cancer progression and metastasis. However, there are some difficulties in probing each vesicle using popular analytical methods because of their small sizes and heterogeneous origins. These obstacles may be overcome by using a novel approach that senses highly curved membrane and negatively charged membrane lipids. In this chapter, we highlight the basic biological concepts of EVs, isolation, and quantification methods, and recent advent of peptide probes for EVs.  

3.2691           Membrane vesicles in sea water: heterogeneous DNA content and implications for viral abundance estimates

Biller, S.J., McDaniel, L.D., Breitbart, M., Rogers, E., Paul, J.H. and Chrisholm, S.W. ISME J., 11, 394-404 (2017)   Diverse microbes release membrane-bound extracellular vesicles from their outer surfaces into the surrounding environment. Vesicles are found in numerous habitats including the oceans, where they likely have a variety of functional roles in microbial ecosystems. Extracellular vesicles are known to contain a range of biomolecules including DNA, but the frequency with which DNA is packaged in vesicles is unknown. Here, we examine the quantity and distribution of DNA associated with vesicles released from five different bacteria. The average quantity of double-stranded DNA and size distribution of DNA fragments released within vesicles varies among different taxa. Although some vesicles contain sufficient DNA to be visible following staining with the SYBR fluorescent DNA dyes typically used to enumerate viruses, this represents only a small proportion (<0.01–1%) of vesicles. Thus DNA is packaged heterogeneously within vesicle populations, and it appears that vesicles are likely to be a minor component of SYBR-visible particles in natural sea water compared with viruses. Consistent with this hypothesis, chloroform treatment of coastal and offshore seawater samples reveals that vesicles increase epifluorescence-based particle (viral) counts by less than an order of magnitude and their impact is variable in space and time.  

3.2692           Large-scale isolation and cytotoxicity of extracellular vesicles derived from activated human natural killer cells

Jong, A.Y., Wu, C-H., Li, J., Sun, J., Fabbri, M., Wayne, A.S. and Seeger, R.C.

1. Extracellular Vesicles, 6:1, 1294368 (2017)

  Extracellular vesicles (EVs) have been the focus of great interest, as they appear to be involved in numerous important cellular processes. They deliver bioactive macromolecules such as proteins, lipids, and nucleic acids, allowing intercellular communication in multicellular organisms. EVs are secreted by all cell types, including immune cells such as natural killer cells (NK), and they may play important roles in the immune system. Currently, a large-scale procedure to obtain functional NK EVs is lacking, limiting their use clinically. In this report, we present a simple, robust, and cost-effective method to isolate a large quantity of NK EVs. After propagating and activating NK cells ex vivo and then incubating them in exosome-free medium for 48 h, EVs were isolated using a polymer precipitation method. The isolated vesicles contain the tetraspanin CD63, an EV marker, and associated proteins (fibronectin), but are devoid of cytochrome C, a cytoplasmic marker. Nanoparticle tracking analysis showed a size distribution between 100 and 200 nm while transmission electron microscopy imaging displayed vesicles with an oval shape and comparable sizes, fulfilling the definition of EV. Importantly, isolated EV fractions were cytotoxic against cancer cells. Furthermore, our results demonstrate for the first time that isolated activated NK (aNK) cell EVs contain the cytotoxic proteins perforin, granulysin, and granzymes A and B, incorporated from the aNK cells. Activation of caspase -3, -7 and -9 was detected in cancer cells incubated with aNK EVs, and caspase inhibitors blocked aNK EV-induced cytotoxicity, suggesting that aNK EVs activate caspase pathways in target cells. The ability to isolate functional aNK EVs on a large scale may lead to new clinical applications.  

3.2693           Testosterone promotes tube formation of endothelial cells isolated from veins via activation of Smad1 protein

Liu, P., Li, X., Song, F., Li, P., Wei, J., Yan, Q., Xu, X., Yang, J., Li, C. and Fu, X. Mol. Cell. Endocrinol., 446, 21-31 (2017)   Testosterone (T) deficiency is positively correlated with the increased incidence of cardiovascular disease. However, the effects of T on vascular endothelial cells remain obscure. Tube formation capacity is critical for vascular regeneration/repair and Smad1 plays an important role in these events. In this study, we investigated the effects of T on Smad1 activation and tube formation of cultured human umbilical endothelial cells (HUVECs). Our results showed that T rapidly increased endothelial Smad1 phosphorylation. This effect was mimicked by cell-impermeable T-BSA conjugates and was not altered by transcriptional inhibitor actinomycin D or translational inhibitor cycloheximide. T-induced Smad1 phosphorylation was blocked by ERK1/2 and c-Src inhibitors or their specific siRNAs, while it was reinforced by ERK1/2 or c-Src overexpression. Indeed, T rapidly activated ERK1/2 and c-Src signalings and c-Src was confirmed as the upstream of ERK1/2. Moreover, caveolae disruptor methyl-β-cyclodextrin (β-MCD) blocked Smad1 activation induced by T. The association of caveolin-1 with androgen receptor (AR) or c-Src was detected by immunoprecipitation and it was significantly increased by rapid T stimulation. Furthermore, fractional analysis showed that AR and c-Src were expressed in caveolae-enriched membrane fractions. T promoted tube formation of HUVECs, which was blocked by c-Src and ERK1/2 inhibitors or by the knockdown of Smad1. In conclusion, T increased tube formation of endothelial cells isolated from veins by stimulating Smad1 phosphorylation in a nongenomic manner, which was mediated by signals from AR/c-Src located in caveolae to ERK1/2 cascade. These findings may shed new light on the relevance of T to its vascular functions.  

3.2694           LOXL2 drives epithelial-mesenchymal transition via activation of IRE1-XBP1 signalling pathway

Cuevas, E.P., Eraso, P., Mazon, M.J:, Santos, V., Moreno-Bueno, G., Cano, A. and Portillo, F. Scientific Reports, 7:44988 (2017)   Epithelial-to-Mesenchymal Transition (EMT) is a key process contributing to the aggressiveness of cancer cells. EMT is triggered by activation of different transcription factors collectively known as EMT-TFs. Different cellular cues and cell signalling networks activate EMT at transcriptional and posttranscriptional level in different biological and pathological situations. Among them, overexpression of LOXL2 (lysyl oxidase-like 2) induces EMT independent of its catalytic activity. Remarkably, perinuclear/cytoplasmic accumulation of LOXL2 is a poor prognosis marker of squamous cell carcinomas and is associated to basal breast cancer metastasis by mechanisms no yet fully understood. Here, we report that overexpression of LOXL2 promotes its accumulation in the Endoplasmic Reticulum where it interacts with HSPA5 leading to activation of the IRE1-XBP1 signalling pathway of the ER-stress response. LOXL2-dependent IRE1-XBP1 activation induces the expression of several EMT-TFs: SNAI1, SNAI2, ZEB2 and TCF3 that are direct transcriptional targets of XBP1. Remarkably, inhibition of IRE1 blocks LOXL2-dependent upregulation of EMT-TFs thus hindering EMT induction.  

3.2695           623 – Purification of urinary extracellular vesicles for uro-oncological biomarker studies using an iodixanol (Optiprep™) density gradient

Dhondt, B., Vergauwen, G., Van Deun, J., Geeurickx, E., Claeys, T., Poelaert, F., Buelens, s., Hendrix, A., De Wever, O. and Lumen, N. Eur. Urol. Suppl., 16(3), e1078 (2017)   INTRODUCTION & OBJECTIVES: Extracellular vesicles (EVs), are bilayered, nanometer-sized vesicles released by cells into the extracellular space after fusion of multivesicular endosomes with the plasma membrane or by direct budding. They play a role in intercellular communication by transporting proteins and nucleic acids to target cells. Their molecular content is a fingerprint of the releasing cells. In addition, EVs are released into easily accessible body fluids such as urine. EV analyses can therefore be considered as a real-time “liquid biopsy” for patients with cancer. Consequently, urinary EVs (uEVs) might be used to diagnose urological cancers or to monitor progression and therapy response. The isolation of highly purified uEVs is a prerequisite to obtain reliable omics data for biomarker studies. Tamm-Horsfall protein (THP; uromodulin) polymers in urine are co-isolated as a contaminant when the current EV isolation methods are applied, hindering accurate downstream analysis by masking of low abundance proteins or by entrapment of non-EV associated extracellular RNA molecules. For biomarker studies, the MISEV (Minimal Information for Studies on Extracellular Vesicles) criteria encourage the use of density gradients to at least validate the association of the identified molecules with EVs. This study evaluates the ability of an iodixanol (OptiprepTM) density gradient to separate uEVs from THP contamination. MATERIAL & METHODS: Urine samples of prostate cancer patients were collected, pooled and centrifuged for 10 minutes at 1000g before storage at -80°C. Upon use, pooled samples of 50 ml were thawed at room temperature and concentrated using a Centricon® Plus-70 Centrifugal Filter Device with a 10kD MWCO. Using an OptiprepTM density gradient (ODG), uEVs were isolated, applying both a topdown (sedimentation) and a bottom-up (floatation) approach. Yield and size distribution of the isolated particles were assessed by nanoparticle tracking analysis (NTA). The successful isolation of uEVs was evaluated by western blot (WB) analysis, assessing the presence of the EV-associated protein markers ALIX, TSG101 and CD9. THP contamination was assessed by Ponceau S staining and confirmed by WB. RESULTS: In top-down ODG, Ponceau S staining identified THP as a smear over the different ODG fractions. This was verified and supported by WB. Surprisingly, the highest amount of THP was seen in the ODG fractions containing the uEVs. In a bottom-up ODG approach, the abundance of THP was found in the bottom fractions of the gradient. Ponceau S staining did not identify the presence of THP in the EV rich fraction of the bottom-up ODG. Conversely, WB analysis did identify residual presence of THP in the EV rich fraction. In addition, WB demonstrated the presence of EV-associated proteins in the bottom fractions, indicating uEV entrapment by THP complexes. CONCLUSIONS: Bottom-up ODG results in an efficient, but not complete, separation of uEVs from THP contamination. Further research should evaluate if adding additional purification steps results in obtaining highly purified EVs from urine in order to conduct reliable uro-oncological biomarker studies.  

3.2696           Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells – Liquid biopsies for monitoring complications of pregnancy

Truong, G., Guanzon, D., Kinhal, V., Elfeky, O., Lai, A., Longo, S., Nuzhat, Z., Palma, C., Scholz-Romero, K., Menon, R., Mol, B.W., Rice, G.E. and Salomon, C. PloS One, 12(3), e0174514 (2017)   Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT) cells, modifying their bioactivity on endothelial cells (EC). Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot^®) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte^™) was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (<18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-α released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications.  

3.2697           Sorting nexin-4 regulates β-amyloid production by modulating β-site-activating cleavage enzyme-1

Kim, N-Y., Cho, M-H., Won, S-H., Kang, H-J., Yoon, S-Y. and Kim, D-H.

Alzheimer Res. & Therapy, 9:4 (2017)

Background

Amyloid precursor protein (APP) is cleaved by β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) to produce β-amyloid (Aβ), a critical pathogenic peptide in Alzheimer’s disease (AD). Aβ generation can be affected by the intracellular trafficking of APP or its related secretases, which is thus important to understanding its pathological alterations. Although sorting nexin (SNX) family proteins regulate this trafficking, the relevance and role of sorting nexin-4 (SNX4) regarding AD has not been studied yet.

Methods

In this study, human brain tissue and APP/PS1 mouse brain tissue were used to check the disease relevance of SNX4. To investigate the role of SNX4 in AD pathogenesis, several experiments were done, such as coimmunoprecipitation, Western blotting, immunohistochemistry, and gradient fractionation.

Results

We found that SNX4 protein levels changed in the brains of patients with AD and of AD model mice. Overexpression of SNX4 significantly increased the levels of BACE1 and Aβ. Downregulation of SNX4 had the opposite effect. SNX4 interacts with BACE1 and prevents BACE1 trafficking to the lysosomal degradation system, resulting in an increased half-life of BACE1 and increased production of Aβ.

Conclusions

We show that SNX4 regulates BACE1 trafficking. Our findings suggest novel therapeutic implications of modulating SNX4 to regulate BACE1-mediated β-processing of APP and subsequent Aβ generation.

 

3.2698           Mitochondria are devoid of amyloid β-protein (Aβ)-producing secretases: Evidence for unlikely occurrence within mitochondria of Aβ generation from amyloid precursor protein

Mamada, N., Tanokashira, D., Ishii, K., Tamaoka, A. and Araki, W. Biochem. Biophys. Res.Comm., 486, 321-328 (2017)   Mitochondrial dysfunction is implicated in the pathological mechanism of Alzheimer's disease (AD). Amyloid β-protein (Aβ), which plays a central role in AD pathogenesis, is reported to accumulate within mitochondria. However, a question remains as to whether Aβ is generated locally from amyloid precursor protein (APP) within mitochondria. We investigated this issue by analyzing the expression patterns of APP, APP-processing secretases, and APP metabolites in mitochondria separated from human neuroblastoma SH-SY5Y cells and those expressing Swedish mutant APP. APP, BACE1, and PEN-2 protein levels were significantly lower in crude mitochondria than microsome fractions while those of ADAM10 and the other γ-secretase complex components (presenilin 1, nicastrin, and APH-1) were comparable between fractions. The crude mitochondrial fraction containing substantial levels of cathepsin D, a lysosomal marker, was further separated via iodixanol gradient centrifugation to obtain mitochondria- and lysosome-enriched fractions. Mature APP, BACE1, and all γ-secretase complex components (in particular, presenilin 1 and PEN-2) were scarcely present in the mitochondria-enriched fraction, compared to the lysosome-enriched fraction. Moreover, expression of the β-C-terminal fragment (β-CTF) of APP was markedly low in the mitochondria-enriched fraction. Additionally, immunocytochemical analysis showed very little co-localization between presenilin 1 and Tom20, a marker protein of mitochondria. In view of the particularly low expression levels of BACE1, γ-secretase complex proteins, and β-CTF in mitochondria, we propose that it is unlikely that Aβ generation from APP occurs locally within this organelle.  

3.2699           Glycoconjugates from extracellular vesicles: Structures, functions and emerging potential as cancer biomarkers

Costa, J. BBA – Reviews on Cancer, 1868, 157-166 (2017)   Extracellular vesicles (EVs) are released by virtually all cells, carry cellular molecules to the extracellular environment, and may interact with other cells. They are found in body fluids, therefore, constituting useful target sources for the identification of disease biomarkers, for example, in cancer. EVs originate from the plasma membrane or from multivesicular endosomes. They have the same topology as the plasma membrane and are rich in glycoconjugates, displaying specific glycosignatures. Surface glycoconjugates play important roles in EVs biogenesis and in their interaction with other cells. Changes in glycosylation constitute a hallmark of different types of cancer, therefore, the study of glycoconjugates and glycosignatures of EVs appear as promising candidates to identify novel cancer biomarkers and to increase the specificity and sensitivity of the existing clinical biomarkers, many of which are glycosylated.  

3.2700           Assessment of Caveolae/Lipid Rafts in Isolated Cells

Callera, G.E., Bruder-Nascimento, T. and Touyz, R.M. Methods in Mol. Biol., 1527, 251-269 (2017)   This chapter outlines protocols to evaluate protein localization, recruitment or phosphorylation levels in cholesterol/sphingolipids-enriched cell membrane domains and recommends experimental designs with pharmacological tolls to evaluate potential cell functions associated with these domains. We emphasize the need for the combination of several approaches towards understanding the protein components and cellular functions attributed to these distinct microdomains.  

3.2701           A Protocol for Isolation and Proteomic Characterization of Distinct Extracellular Vesicle Subtypes by Sequential Centrifugal Ultrafiltration

Xu, R., Simpson, R.J. and Greening, D.W. Methods in Mol. Biol., 1545, 91-116 (2017)   Scientific and clinical interest in extracellular vesicles (EVs) has increased rapidly as evidence mounts that they may constitute a new signaling paradigm. Recent studies have highlighted EVs carry preassembled complex biological information that elicit pleiotropic responses in target cells. It is well recognized that cells secrete essentially two EV subtypes that can be partially separated by differential centrifugation (DC): the larger size class (referred to as “microvesicles” or “shed microvesicles,” sMVs) is heterogeneous (100–1500 nm), while the smaller size class (referred to as “exosomes”) is relatively homogeneous in size (50–150 nm). A key issue hindering progress in understanding underlying mechanisms of EV subtype biogenesis and cargo selectivity has been the technical challenge of isolating homogeneous EV subpopulations suitable for molecular analysis. In this protocol we reveal a novel method for the isolation, purification, and characterization of distinct EV subtypes: exosomes and sMVs. This method, based on sequential centrifugal ultrafiltration (SCUF), affords unbiased isolation of EVs from conditioned medium from a human colon cancer cell model. For both EV subtypes, this protocol details extensive purification and characterization based on dynamic light scattering, cryoelectron microscopy, quantitation, immunoblotting, and comparative label-free proteome profiling. This analytical SCUF method developed is potentially scalable using tangential flow filtration and provides a solid foundation for future in-depth functional studies of EV subtypes from diverse cell types.  

3.2702           Isolation of Exosomes from HTLV-Infected Cells

Barclay, R.A., Pleet, M.L., Akpamagbo, Y., Noor, K., Mathiesen, A. and Kashanchi, F. Methods in Mol. Biol., 1582, 57-75 (2017)   Exosomes are small vesicles, approximately 30–100 nm in diameter, that transport various cargos, such as proteins and nucleic acids, between cells. It has been previously shown that exosomes can also transport viral proteins, such as the HTLV protein Tax, and viral RNAs, potentially contributing to disease pathogenesis. Therefore, it is important to understand their impact on recipient cells. Here, we describe methods of isolating and purifying exosomes from cell culture or tissue through ultracentrifugation, characterizing exosomes by surface biomarkers, and assays that evaluate the effect of exosomes on cells.  

3.2703           Quantitative Analysis of Exosomal miRNA via qPCR and Digital PCR

Bellingham, S.A., Shambrook, M. and Hill, A.F. Methods in Mol. Biol., 1545, 55-70 (2017)   Extracellular vesicles, such as exosomes and microvesicles, have been shown to contain potential microRNA (miRNA) biomarkers that may be utilized in the diagnosis of various diseases from cancer to neurological disorders. The unique nature of the extracellular vesicle bilayer allows miRNA to be protected from degradation making it an ideal source of material for biomarkers discovery from both fresh and archived samples. Here we describe the quantitative analysis of miRNA isolated from exosomes by quantitative PCR and digital PCR.  

3.2704           Synthesis and characterization of a novel rhodamine labeled cholesterol reporter

Maiwald, A., Bauer, O. and Gimpl, G. Biochim. Biophys. Acta, 1859, 1099-1113 (2017)   We introduce the novel fluorescent cholesterol probe RChol in which a sulforhodamine group is linked to the sixth carbon atom of the steroid backbone of cholesterol. The same position has recently been selected to generate the fluorescent reporter 6-dansyl-cholestanol (DChol) and the photoreactive 6-azi-cholestanol. In comparison with DChol, RChol is brighter, much more photostable, and requires less energy for excitation, i.e. favorable conditions for microscopical imaging. RChol easily incorporates into methyl-β-cyclodextrin forming a water-soluble inclusion complex that acts as an efficient sterol donor for cells and membranes. Like cholesterol, RChol possesses a free 3′OH group, a prerequisite to undergo intracellular esterification. RChol was also able to support the growth of cholesterol auxotrophic cells and can therefore substitute for cholesterol as a major component of the plasma membrane. According to subcellular fractionation, slight amounts of RChol (~ 12%) were determined in low-density Triton-insoluble fractions whereas the majority of RChol was localized in non-rafts fractions. In phase-separated giant unilamellar vesicles, RChol preferentially partitions in liquid-disordered membrane domains. Intracellular RChol was transferred to extracellular sterol acceptors such as high density lipoproteins in a dose-dependent manner. Unlike DChol, RChol was not delivered to the cholesterol storage pathway. Instead, it translocated to endosomes/lysosomes with some transient contacts to peroxisomes. Thus, RChol is considered as a useful probe to study the endosomal/lysosomal pathway of cholesterol.  

3.2705           Achieving the Promise of Therapeutic Extracellular Vesicles: The Devil is in Details of Therapeutic Loading

Sutaria, D.S., Badawi, M., Phelps, M.A. and Schmittgen, T.D. Pharm. Res., 34(5), 1053-1066 (2017)   Extracellular vesicles (EVs) represent a class of cell secreted organelles which naturally contain biomolecular cargo such as miRNA, mRNA and proteins. EVs mediate intercellular communication, enabling the transfer of functional nucleic acids from the cell of origin to the recipient cells. In addition, EVs make an attractive delivery vehicle for therapeutics owing to their increased stability in circulation, biocompatibility, low immunogenicity and toxicity profiles. EVs can also be engineered to display targeting moieties on their surfaces which enables targeting to desired tissues, organs or cells. While much has been learned on the role of EVs as cell communicators, the field of therapeutic EV application is currently under development. Critical to the future success of EV delivery system is the description of methods by which therapeutics can be successfully and efficiently loaded within the EVs. Two methods of loading of EVs with therapeutic cargo exist, endogenous and exogenous loading. We have therefore focused this review on describing the various published approaches for loading EVs with therapeutics.  

3.2706           Exosome and Microvesicle-Enriched Fractions Isolated from Mesenchymal Stem Cells by Gradient Separation Showed Different Molecular Signatures and Functions on Renal Tubular Epithelial Cells

Collino, F., Pomatto, M., Bruno, S., Lindoso, R.S., Tapparo, M., Sicheng, W., Quesenberry, P. and Camussi, G. Stem Cell Rev. and Rep., 13(2), 226-243 (2017)   Several studies have suggested that extracellular vesicles (EVs) released from mesenchymal stem cells (MSCs) may mediate MSC paracrine action on kidney regeneration. This activity has been, at least in part, ascribed to the transfer of proteins/transcription factors and different RNA species. Information on the RNA/protein content of different MSC EV subpopulations and the correlation with their biological activity is currently incomplete. The aim of this study was to evaluate the molecular composition and the functional properties on renal target cells of MSC EV sub-populations separated by gradient floatation. The results demonstrated heterogeneity in quantity and composition of MSC EVs. Two peaks of diameter were observed (90–110 and 170–190 nm). The distribution of exosomal markers and miRNAs evaluated in the twelve gradient fractions showed an enrichment in fractions with a flotation density of 1.08–1.14 g/mL. Based on this observation, we evaluated the biological activity on renal cell proliferation and apoptosis resistance of low (CF1), medium (CF2) and high (CF3) floatation density fractions. EVs derived from all fractions, were internalized by renal cells, CF1 and CF2 but not CF3 fraction stimulated significant cell proliferation. CF2 also inhibited apoptosis on renal tubular cells submitted to ischemia-reperfusion injury. Comparative miRNomic and proteomic profiles reveal a cluster of miRNAs and proteins common to all three fractions and an enrichment of selected molecules related to renal regeneration in CF2 fraction. In conclusion, the CF2 fraction enriched in exosomal markers was the most active on renal tubular cell proliferation and protection from apoptosis.  

3.2707           Defining the dynamin-based ring organizing center on the peroxisome-dividing machinery isolated from Cyanidioschyzon merolae

Imoto, Y., Abe, Y., Okumoto, K., Honsbo, M., Kuroiwa, H., Kuroiwa, T. and Fujiki, Y.

1. Cell Sci., 130(5), 853-867 (2017)

  Organelle division is executed through contraction of a ring-shaped supramolecular dividing machinery. A core component of the machinery is the dynamin-based ring conserved during the division of mitochondrion, plastid and peroxisome. Here, using isolated peroxisome-dividing (POD) machinery from a unicellular red algae, Cyanidioschyzon merolae, we identified a dynamin-based ring organizing center (DOC) that acts as an initiation point for formation of the dynamin-based ring. C. merolae contains a single peroxisome, the division of which can be highly synchronized by light–dark stimulation; thus, intact POD machinery can be isolated in bulk. Dynamin-based ring homeostasis is maintained by the turnover of the GTP-bound form of the dynamin-related protein Dnm1 between the cytosol and division machinery via the DOC. A single DOC is formed on the POD machinery with a diameter of 500–700 nm, and the dynamin-based ring is unidirectionally elongated from the DOC in a manner that is dependent on GTP concentration. During the later step of membrane fission, the second DOC is formed and constructs the double dynamin-based ring to make the machinery thicker. These findings provide new insights to define fundamental mechanisms underlying the dynamin-based membrane fission in eukaryotic cells.  

3.2708           The VDAC2–BAK axis regulates peroxisomal membrane permeability

Hosoi, K-i., Miyata, N., Mukai, S., Furuki, S., Okumoto, K., Cheng, E.H. and Fujiki, Y.

1. Cell Biol., 216(3), 709-721 (2017)

  Peroxisomal biogenesis disorders (PBDs) are fatal genetic diseases consisting of 14 complementation groups (CGs). We previously isolated a peroxisome-deficient Chinese hamster ovary cell mutant, ZP114, which belongs to none of these CGs. Using a functional screening strategy, VDAC2 was identified as rescuing the peroxisomal deficiency of ZP114 where VDAC2 expression was not detected. Interestingly, knockdown of BAK or overexpression of the BAK inhibitors BCL-X_L and MCL-1 restored peroxisomal biogenesis in ZP114 cells. Although VDAC2 is not localized to the peroxisome, loss of VDAC2 shifts the localization of BAK from mitochondria to peroxisomes, resulting in peroxisomal deficiency. Introduction of peroxisome-targeted BAK harboring the Pex26p transmembrane region into wild-type cells resulted in the release of peroxisomal matrix proteins to cytosol. Moreover, overexpression of BAK activators PUMA and BIM permeabilized peroxisomes in a BAK-dependent manner. Collectively, these findings suggest that BAK plays a role in peroxisomal permeability, similar to mitochondrial outer membrane permeabilization.  

3.2709           Chromatin remodeling during in vivo neural stem cells differentiating to neurons in early Drosophila embryos

Ye, Y., Li, M., Gu, L., Chen, X., Shi, J., Zhang, X. and Jiang, C. Cell Death and Differentiation, 24(3), 409-420 (2017)   Neurons are a key component of the nervous system and differentiate from multipotent neural stem cells (NSCs). Chromatin remodeling has a critical role in the differentiation process. However, its in vivo epigenetic regulatory role remains unknown. We show here that nucleosome depletion regions (NDRs) form in both proximal promoters and distal enhancers during NSCs differentiating into neurons in the early Drosophila embryonic development. NDR formation in the regulatory regions involves nucleosome shift and eviction. Nucleosome occupancy in promoter NDRs is inversely proportional to the gene activity. Genes with promoter NDR formation during differentiation are enriched for functions related to neuron development and maturation. Active histone-modification signals (H3K4me3 and H3K9ac) in promoters are gained in neurons in two modes: de novo establishment to high levels or increase from the existing levels in NSCs. The gene sets corresponding to the two modes have different neuron-related functions. Dynamic changes of H3K27ac and H3K9ac signals in enhancers and promoters synergistically repress genes associated with neural stem or progenitor cell-related pluripotency and upregulate genes associated with neuron projection morphogenesis, neuron differentiation, and so on. Our results offer new insights into chromatin remodeling during in vivo neuron development and lay a foundation for its epigenetic regulatory mechanism study of other lineage specification.  

3.2710           Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites

Boson, B., Denolly, S., Turlure, F., Chamot, C., Dreux, M. and Cosset, F-L. Gastroenterol., 152(4), 895-907 (2017)   Background & Aims Daclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs, and inhibition of viral particle assembly. Methods Cell-culture−derived HCV particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity and production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy. Results Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B, and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes. Conclusions In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins because viral particles and replication complex vesicles cannot form or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection.  

3.2711           LC–MS/MS Based Quantitation of ABC and SLC Transporter Proteins in Plasma Membranes of Cultured Primary Human Retinal Pigment Epithelium Cells and Immortalized ARPE19 Cell Line

Mol. Pharmeceut., 14(3), 605-613 (2017)

The retinal pigment epithelium (RPE) forms the outer blood–retinal barrier between neural retina and choroid. The RPE has several important vision supporting functions, such as transport mechanisms that may also modify pharmacokinetics in the posterior eye segment. Expression of plasma membrane transporters in the RPE cells has not been quantitated. The aim of this study was to characterize and compare transporter protein expression in the ARPE19 cell line and hfRPE (human fetal RPE) cells by using quantitative targeted absolute proteomics (QTAP). Among 41 studied transporters, 16 proteins were expressed in hfRPE and 13 in ARPE19 cells. MRP1, MRP5, GLUT1, 4F2hc, TAUT, CAT1, LAT1, and MATE1 proteins were detected in both cell lines within 4-fold differences. MPR7, OAT2 and RFC1 were detected in the hfRPE cells, but their expression levels were below the limit of quantification in ARPE19 cells. PCFT was detected in both studied cell lines, but the expression was over 4-fold higher in hfRPE cells. MCT1, MCT4, MRP4, and Na+/K+ ATPase were upregulated in the ARPE19 cell line showing over 4-fold differences in the quantitative expression values. Expression levels of 25 transporters were below the limit of quantification in both cell models. In conclusion, we present the first systematic and quantitative study on transporter protein expression in the plasma membranes of ARPE19 and hfRPE cells. Overall, transporter expression in the ARPE19 and hfRPE cells correlated well and the absolute expression levels were similar, but not identical. The presented quantitative expression levels could be a useful basis for further studies on drug permeation in the outer blood–retinal barrier.

 

3.2712           Immunoactive Clostridial Membrane Vesicle Production Is Regulated by a Sporulation Factor

Obana, N., Nakao, R., Nagayama, K., nakamura, K., Senpuku, H. and Nomura, N. Infect. Immun., 85(5), e00096-17 (2017)   Recently, many Gram-positive bacteria as well as Gram-negative bacteria have been reported to produce membrane vesicles (MVs), but little is known regarding the regulators involved in MV formation. We found that a Gram-positive anaerobic pathogen, Clostridium perfringens, produces MVs predominantly containing membrane proteins and cell wall components. These MVs stimulated proinflammatory cytokine production in mouse macrophage-like cells. We suggested that MVs induced interleukin-6 production through the Toll-like receptor 2 (TLR2) signaling pathway. Thus, the MV could have a role in the bacterium-host interaction and bacterial infection pathogenesis. Moreover, we found that the sporulation master regulator gene spo0A was required for vesiculogenesis. A conserved, phosphorylated aspartate residue of Spo0A was indispensable for MV production, suggesting that the phosphorylation of Spo0A triggers MV production. Multiple orphan sensor kinases necessary for sporulation were also required to maximize MV production. These findings imply that C. perfringens actively produces immunoactive MVs in response to the environment changing, as recognized by membrane-spanning sensor kinases and by modulating the phosphorylation level of Spo0A.  

3.2713           Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells

Costello, J.L. et al

1. Cell Sci., 130, 1675-1687 (2017)

  Tail-anchored (TA) proteins contain a single transmembrane domain (TMD) at the C-terminus that anchors them to the membranes of organelles where they mediate critical cellular processes. Accordingly, mutations in genes encoding TA proteins have been identified in a number of severe inherited disorders. Despite the importance of correctly targeting a TA protein to its appropriate membrane, the mechanisms and signals involved are not fully understood. In this study, we identify additional peroxisomal TA proteins, discover more proteins that are present on multiple organelles, and reveal that a combination of TMD hydrophobicity and tail charge determines targeting to distinct organelle locations in mammals. Specifically, an increase in tail charge can override a hydrophobic TMD signal and re-direct a protein from the ER to peroxisomes or mitochondria and vice versa. We show that subtle changes in those parameters can shift TA proteins between organelles, explaining why peroxisomes and mitochondria have many of the same TA proteins. This enabled us to associate characteristic physicochemical parameters in TA proteins with particular organelle groups. Using this classification allowed successful prediction of the location of uncharacterized TA proteins for the first time.  

3.2714           Localization of the placental BCRP/ABCG2 transporter to lipid rafts: Role for cholesterol in mediating efflux activity

Szilagyi, J.T., Vetrano, A.M., Laskin, J.D. and Aleksunes, L.M. Placenta, 55, 29-36 (2017)   Introduction The breast cancer resistance protein (BCRP/ABCG2) is an efflux transporter in the placental barrier. By transporting chemicals from the fetal to the maternal circulation, BCRP limits fetal exposure to a range of drugs, toxicants, and endobiotics such as bile acids and hormones. The purpose of the present studies was to 1) determine whether BCRP localizes to highly-ordered, cholesterol-rich lipid raft microdomains in placenta microvillous membranes, and 2) determine the impact of cholesterol on BCRP-mediated placental transport in vitro. Methods BCRP expression was analyzed in lipid rafts isolated from placentas from healthy, term pregnancies and BeWo trophoblasts by density gradient ultracentrifugation. BeWo cells were also tested for their ability to efflux BCRP substrates after treatment with the cholesterol sequestrant methyl-β-cyclodextrin (MβCD, 5 mM, 1 h) or the cholesterol synthesis inhibitor pravastatin (200 μM, 48 h). Results and discussion BCRP was found to co-localize with lipid raft proteins in detergent-resistant, lipid raft-containing fractions from placental microvillous membranes and BeWo cells. Treatment of BeWo cells with MβCD redistributed BCRP protein into higher density non-lipid raft fractions. Repletion of the cells with cholesterol restored BCRP localization to lipid raft-containing fractions. Treatment of BeWo cells with MβCD or pravastatin increased cellular retention of two BCRP substrates, the fluorescent dye Hoechst 33342 and the mycotoxin zearalenone. Repletion with cholesterol restored BCRP transporter activity. Taken together, these data demonstrate that cholesterol may play a critical role in the post-translational regulation of BCRP in placental lipid rafts.  

3.2715           Helicobacter pylori-derived extracellular vesicles increased in the gastric juices of gastric adenocarcinoma patients and induced inflammation mainly via specific targeting of gastric epithelial cells

Choi, H-I., Choi, J-P., Seo, J., Kim, B.J., Rho, M., Han, J.K. and Kim, J.G. Exp. Mol. Med., 49, e330 (2017)   Evidence indicates that Helicobacter pylori is the causative agent of chronic gastritis and perhaps gastric malignancy. Extracellular vesicles (EVs) play an important role in the evolutional process of malignancy due to their genetic material cargo. We aimed to evaluate the clinical significance and biological mechanism of H. pylori EVs on the pathogenesis of gastric malignancy. We performed 16S rDNA-based metagenomic analysis of gastric juices either from endoscopic or surgical patients. From each sample of gastric juices, the bacteria and EVs were isolated. We evaluated the role of H. pylori EVs on the development of gastric inflammation in vitro and in vivo. IVIS spectrum and confocal microscopy were used to examine the distribution of EVs. The metagenomic analyses of the bacteria and EVs showed that Helicobacter and Streptococcus are the two major bacterial genera, and they were significantly increased in abundance in gastric cancer (GC) patients. H. pylori EVs are spherical and contain CagA and VacA. They can induce the production of tumor necrosis factor-α, interleukin (IL)-6 and IL-1β by macrophages, and IL-8 by gastric epithelial cells. Also, EVs induce the expression of interferon gamma, IL-17 and EV-specific immunoglobulin Gs in vivo in mice. EVs were shown to infiltrate and remain in the mouse stomach for an extended time. H. pylori EVs, which are abundant in the gastric juices of GC patients, can induce inflammation and possibly cancer in the stomach, mainly via the production of inflammatory mediators from gastric epithelial cells after selective uptake by the cells.  

3.2716           Visualization of a Mammalian Mitochondrion by Coherent X-ray Diffractive Imaging

Kim, Y., Kim, C., Kwon, O.Y., Nam, D., Kim, S.S., Park, J.H., Kim, S., Gallagher-Jones,. M., Kohmura, Y., Ishikawa, T., Song, C., Tae, G. and Noh, D.Y. Scientific Reports, 7:1850 (2017)   We report a three dimensional (3D) quantitative visualization of a mammalian mitochondrion by coherent x-ray diffractive imaging (CXDI) using synchrotron radiation. The internal structures of a mitochondrion from a mouse embryonic fibroblast cell line (NIH3T3) were visualized by tomographic imaging at approximately 60 nm resolution without the need for sectioning or staining. The overall structure consisted of a high electron density region, composed of the outer and inner membranes and the cristae cluster, which enclosed the lower density mitochondrial matrix. The average mass density of the mitochondrion was about 1.36 g/cm3. Sectioned images of the cristae reveal that they have neither a baffle nor septa shape but were instead irregular. In addition, a high resolution, about 14 nm, 2D projection image was captured of a similar mitochondrion with the aid of strongly scattering Au reference objects. Obtaining 3D images at this improved resolution will allow CXDI to be an effective and nondestructive method for investigating the innate structure of mitochondria and other important life supporting organelles.  

3.2717           SorLA in Interleukin-6 Signaling and Turnover

Larsen, J.V. and Petersen, C.M. Mol. Cell. Biol., 37(11), e00641 (2017)   Interleukin-6 (IL-6) is a multifunctional cytokine with important functions in various physiologic processes. Mice lacking IL-6 exhibit multiple phenotypic abnormalities, such as an inadequate immune and acute-phase response, and elevated levels of circulating IL-6 have been found to accompany several pathological conditions. IL-6 binds the nonsignaling IL-6 receptor (IL-6R), which is expressed as a transmembrane, as well as a secreted circulating protein, before it engages homodimeric gp130 for signaling. Complex formation between IL-6 and the membrane-bound IL-6 receptor gives rise to classic cis signaling, whereas complex formation between IL-6 and the soluble IL-6R results in trans signaling. Here, we report that the endocytic receptor SorLA targets IL-6 and IL-6R. We present evidence that SorLA mediates efficient cellular uptake of both IL-6 and the circulating IL-6R in astrocytes. We further show that SorLA interacts with the membrane-bound IL-6R at the cell surface and thereby downregulates IL-6 cis signaling. Finally, we find that the SorLA ectodomain, released from the cell membrane upon enzymatic cleavage of full-length SorLA, may act as an IL-6 carrier protein that stabilizes IL-6 and its capacity for trans signaling. Interleukin-6 (IL-6) is a multifunctional cytokine with important functions in various physiologic processes. Mice lacking IL-6 exhibit multiple phenotypic abnormalities, such as an inadequate immune and acute-phase response, and elevated levels of circulating IL-6 have been found to accompany several pathological conditions. IL-6 binds the nonsignaling IL-6 receptor (IL-6R), which is expressed as a transmembrane, as well as a secreted circulating protein, before it engages homodimeric gp130 for signaling. Complex formation between IL-6 and the membrane-bound IL-6 receptor gives rise to classic cis signaling, whereas complex formation between IL-6 and the soluble IL-6R results in trans signaling. Here, we report that the endocytic receptor SorLA targets IL-6 and IL-6R. We present evidence that SorLA mediates efficient cellular uptake of both IL-6 and the circulating IL-6R in astrocytes. We further show that SorLA interacts with the membrane-bound IL-6R at the cell surface and thereby downregulates IL-6 cis signaling. Finally, we find that the SorLA ectodomain, released from the cell membrane upon enzymatic cleavage of full-length SorLA, may act as an IL-6 carrier protein that stabilizes IL-6 and its capacity for trans signaling.  

3.2718           Gamma-glutamyltransferase activity in exosomes as a potential marker for prostate cancer

Kawakami, K., Fujita, Y., matsuda, Y., Arai, T., Horie, K., kameyama, K., Kato, T., Masunaga, K., Kasuya, Y., Tanaka, M., Mizutani, K., Deguchi, T. and Ito, M. BMC Cancer, 17:316 (2017)   Background Exosomes or extracellular vesicles have the potential as a diagnostic marker for various diseases including cancer. In order to identify novel exosomal markers for prostate cancer (PC), we performed proteomic analysis of exosomes isolated from PC cell lines and examined the usefulness of the marker in patients. Methods Exosomes isolated by differential centrifugation from the culture medium of androgen-dependent LNCaP prostate cancer cell line and its sublines of partially androgen-independent C4, androgen-independent C4–2 and bone metastatic C4–2B were subjected to iTRAQ-based proteomic analysis. Exosomes were also isolated by immunocapture and separated by size exclusion chromatography and density gradient centrifugation. Protein expression was determined by Western blot analysis. GGT activity was measured using a fluorescent probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG). Immunohistochemical analysis of tissues was performed using anti-GGT1 antibody. Results Among proteins upregulated in C4–2 and C4–2B cells than in LNCaP cells, we focused on gamma-glutamyltransferase 1 (GGT1), a cell-surface enzyme that regulates the catabolism of extracellular glutathione. The levels of both GGT1 large and small subunits were elevated in exosomes isolated from C4–2 and C4–2B cells by differential centrifugation and by immunocapture with anti-CD9 or -prostate-specific membrane antigen (PSMA) antibody. In cell lysates and exosomes, GGT1 expression correlated with GGT activity. Size exclusion chromatography of human serum demonstrated the presence of GGT activity and GGT1 subunits in fractions positive for CD9. Density gradient centrifugation revealed the co-presence of GGT1 subunits with CD9 in exosomes isolated by differential centrifugation from human serum. Since GGT activity correlated with GGT1 expression in serum exosomes isolated by differential centrifugation, we measured serum exosomal GGT activity in patients. Unexpectedly, we found that serum exosomal GGT activity was significantly higher in PC patients than in benign prostatic hyperplasia (BPH) patients. In support of this finding, immunohistochemical analysis showed increased GGT1 expression in PC tissues compared with BPH tissues. Conclusions Our results suggest that serum exosomal GGT activity could be a useful biomarker for PC.  

3.2719           Review: Bio-compartmentalization of microRNAs in exosomes during gestational diabetes mellitus

Iljas, J.D., Guanzon, D., Elfeky, O., Rice, G.E. and Solomon, C. Placenta, 54, 76-82 (2017)   Analysis of the human genome revealed that only 1.2% encoded for proteins, which raised questions regarding the biological significance of the remaining genome. We now know that approximately 80% of the genome serves at least one biochemical function within the cell. A portion of this 80% consists of a family of non-coding regulatory RNAs, one important member being microRNAs (miRNAs). miRNAs can be detected in tissues and biofluids, where miRNAs in the latter can be bound to proteins or encapsulated within lipid vesicles such as exosomes. Gestational diabetes mellitus (GDM) is a complication of pregnancy, which has harmful health impacts on both the fetus as well as the mother. The incidence of GDM worldwide varies, but reached 18% in the HAPO cohort using the new International Association of Diabetes and Pregnancy Study Groups (IADPSG) criteria. Not only has GDM been associated with increased risks of further complications during pregnancy, but also poses long-term risks for both the mother and the baby. Thus, understanding the pathophysiology of GDM is important from a public health perspective. Literature has demonstrated that GDM is associated with elevated levels of circulating exosomes in maternal circulation. However, there is a paucity of data defining the expression, role, and diagnostic utility of miRNAs in GDM. This review briefly summarizes recent advances in the function and quantification of intracellular and extracellular miRNAs in GDM.  

3.2720           Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research

Vergauwen, G., Dhondt, B., Van Deun, J., De Smedt, E., Berx, G., Timmerman, E., Gevaert, K., Miinalainen, I., Cocquyt, V., Braems, G., Ven den Broecke, R., Denys, H., De Wevwer, O. and Hendrix, A. Scientific Reports, 7:2704 (2017)   Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications.  

3.2721           Prions on the run: How extracellular vesicles serve as delivery vehicles for self-templating protein aggregates

Liu, S., Hossinger, A., Göbbels, S. and Vorberg, I.M. Prion, 11, 98-112 (2017)   Extracellular vesicles (EVs) are actively secreted, membrane-bound communication vehicles that exchange biomolecules between cells. EVs also serve as dissemination vehicles for pathogens, including prions, proteinaceous infectious agents that cause transmissible spongiform encephalopathies (TSEs) in mammals. Increasing evidence accumulates that diverse protein aggregates associated with common neurodegenerative diseases are packaged into EVs as well. Vesicle-mediated intercellular transmission of protein aggregates can induce aggregation of homotypic proteins in acceptor cells and might thereby contribute to disease progression. Our knowledge of how protein aggregates are sorted into EVs and how these vesicles adhere to and fuse with target cells is limited. Here we review how TSE prions exploit EVs for intercellular transmission and compare this to the transmission behavior of self-templating cytosolic protein aggregates derived from the yeast prion domain Sup 35 NM. Artificial NM prions are non-toxic to mammalian cell cultures and do not cause loss-of-function phenotypes. Importantly, NM particles are also secreted in association with exosomes that horizontally transmit the prion phenotype to naive bystander cells, a process that can be monitored with high accuracy by automated high throughput confocal microscopy. The high abundance of mammalian proteins with amino acid stretches compositionally similar to yeast prion domains makes the NM cell model an attractive model to study self-templating and dissemination properties of proteins with prion-like domains in the mammalian context.  

3.2722           Isolation of membrane vesicles from prokaryotes: a technical and biological comparison reveals heterogeneity

Singorenko, P.D., Chang, V., Whitcombe, A., Simonov, D., Hong, J., Phillips, A., Swift, S. and Blenkiron, C.

1. Extracellular Vesicles, 6, 1324731 (2017)

  Prokaryotes release membrane vesicles (MVs) with direct roles in disease pathogenesis. MVs are heterogeneous when isolated from bacterial cultures so Density Gradient Centrifugation (DGC) is valuable for separation of MV subgroups from contaminating material. Here we report the technical variability and natural biological heterogeneity seen between DGC preparations of MVs for Mycobacterium smegmatis and Escherichia coli and compare these DGC data with size exclusion chromatography (SEC) columns. Crude preparations of MVs, isolated from cultures by ultrafiltration and ultracentrifugation were separated by DGC with fractions manually collected as guided by visible bands. Yields of protein, RNA and endotoxin, protein banding and particle counts were analysed in these. DGC and SEC methods enabled separation of molecularly distinct MV populations from crude MVs. DGC banding profiles were unique for each of the two species of bacteria tested and further altered by changing culture conditions, for example with iron supplementation. SEC is time efficient, reproducible and cost effective method that may also allow partial LPS removal from Gram-negative bacterial MVs. In summary, both DGC and SEC are suitable for the separation of mixed populations of MVs and we advise trials of both, coupled with complete molecular and single vesicle characterisation prior to downstream experimentation.  

3.2723           High-throughput sequencing of two populations of extracellular vesicles provides an mRNA signature that can be detected in the circulation of breast cancer patients

Conley, A., Minciacchi, V.R., Lee, D.H., Knudsen, B.S., karlan, B.Y., Citrigno, L., Viglietto, G., Tewari, M., Freeman, M.R., Demichelis, F. and Di Vizio, D. RNA Biology, 14(3), 305-316 (2017)   Extracellular vesicles (EVs) contain a wide range of RNA types with a reported prevalence of non-coding RNA. To date a comprehensive characterization of the protein coding transcripts in EVs is still lacking. We performed RNA-Sequencing (RNA-Seq) of 2 EV populations and identified a small fraction of transcripts that were expressed at significantly different levels in large oncosomes and exosomes, suggesting they may mediate specialized functions. However, these 2 EV populations exhibited a common mRNA signature that, in comparison to their donor cells, was significantly enriched in mRNAs encoding E2F transcriptional targets and histone proteins. These mRNAs are primarily expressed in the S-phase of the cell cycle, suggesting that they may be packaged into EVs during S-phase. In silico analysis using subcellular compartment transcriptome data from the ENCODE cell line compendium revealed that EV mRNAs originate from a cytoplasmic RNA pool. The EV signature was independently identified in plasma of patients with breast cancer by RNA-Seq. Furthermore, several transcripts differentially expressed in EVs from patients versus controls mirrored differential expression between normal and breast cancer tissues. Altogether, this largest high-throughput profiling of EV mRNA demonstrates that EVs carry tumor-specific alterations and can be interrogated as a source of cancer-derived cargo.    

3.2724           Unique molecular profile of exosomes derived from primary human proximal tubular epithelial cells under diseased conditions

Wang, X., Wilkinson, R., Kildey, K., Potriquet, J., Mulvenna, J., Lobb, R.J., Möller, A., Cloonan, N., Mukhopadhyay, P., Kassianos, A.J. and Healy, H.

1. Extracellular Vesicles, 6(1), 1314073 (2017)

  Human proximal tubular epithelial cells (PTEC) of the kidney are known to respond to and mediate the disease process in a wide range of kidney diseases, yet their exosomal production and exosome molecular cargo remain a mystery. Here we investigate, for the first time, the production and molecular content of exosomes derived from primary human PTEC cultured under normal and diseased conditions representing a spectrum of in vivo disease severity from early inflammation, experienced in multiple initial kidney disease states, through to hypoxia, frequently seen in late stage chronic kidney disease (CKD) due to fibrosis and vascular compromise. We demonstrate a rapid reproducible methodology for the purification of PTEC-derived exosomes, identify increased numbers of exosomes from disease-state cultures and identify differential expression levels of both known and unique miRNA and protein species from exosomes derived from different disease-culture conditions. The validity of our approach is supported by the identification of miRNA, proteins and pathways with known CKD associations, providing a rationale to further evaluate these novel and known pathways as targets for therapeutic intervention.  

3.2725           Phosphatidylserine synthesis at membrane contact sites promotes its transport out of the ER

Kannan, M., Lahiri, S., Liu, L-K., Choudhary, V. and Prinz, W.A.

1. Lipid Res., 58(3), 553-562 (2017)

  Close contacts between organelles, often called membrane contact sites (MCSs), are regions where lipids are exchanged between organelles. Here, we identify a novel mechanism by which cells promote phospholipid exchange at MCSs. Previous studies have shown that phosphatidylserine (PS) synthase activity is highly enriched in portions of the endoplasmic reticulum (ER) in contact with mitochondria. The objective of this study was to determine whether this enrichment promotes PS transport out of the ER. We found that PS transport to mitochondria was more efficient when PS synthase was fused to a protein in the ER at ER-mitochondria contacts than when it was fused to a protein in all portions of the ER. Inefficient PS transport to mitochondria was corrected by increasing tethering between these organelles. PS transport to endosomes was similarly enhanced by PS production in regions of the ER in contact with endosomes. Together, these findings indicate that PS production at MCSs promotes PS transport out of the ER and suggest that phospholipid production at MCSs may be a general mechanism of channeling lipids to specific cellular compartments.  

3.2726           γ2 and γ1AP-1 complexes: Different essential functions and regulatory mechanisms in clathrin-dependent protein sorting

Zizioli, D., Geumann, C., Kratzke, M., Mishra, R., Borsani, G., Finazzi, D., Candiello, E. and Schu, P. Eur. J. Cell Biol., 96, 356-368 (2017)   γ2 adaptin is homologous to γ1, but is only expressed in vertebrates while γ1 is found in all eukaryotes. We know little about γ2 functions and their relation to γ1. γ1 is an adaptin of the heterotetrameric AP-1 complexes, which sort proteins in and do form clathrin-coated transport vesicles and they also regulate maturation of early endosomes. γ1 knockout mice develop only to blastocysts and thus γ2 does not compensate γ1-deficiency in development. γ2 has not been classified as a clathrin-coated vesicle adaptor protein in proteome analyses and functions for monomeric γ2 in endosomal protein sorting have been proposed, but adaptin interaction studies suggested formation of heterotetrameric AP-1/γ2 complexes. We detected γ2 at the trans-Golgi network, on peripheral vesicles and identified γ2 clathrin-coated vesicles in mice. Ubiquitous σ1A and tissue-specific σ1B adaptins bind γ2 and γ1. σ1B knockout in mice does not effect γ1/σ1A AP-1 levels, but γ2/σ1A AP-1 levels are increased in brain and adipocytes. Also γ2 is essential in development. In zebrafish AP-1/γ2 and AP-1/γ1 fulfill different, essential functions in brain and the vascular system.  

3.2727           The role of extracellular vesicles in malaria biology and pathogenesis

Sampaio, N.G., Cheng, L. and Eriksson, E.M. Malaria J., 16:245 (2017)   In the past decade, research on the functions of extracellular vesicles in malaria has expanded dramatically. Investigations into the various vesicle types, from both host and parasite origin, has revealed important roles for extracellular vesicles in disease pathogenesis and susceptibility, as well as cell–cell communication and immune responses. Here, work relating to extracellular vesicles in malaria is reviewed, and the areas that remain unknown and require further investigations are highlighted.  

3.2728           Exosomes in Cancer Nanomedicine and Immunotherapy: Prospects and Challenges

Syn, N.L., Wang, L., Chow, E.K-H., Lim, C.T. and Goh, B-C, Trends in Biotechnology, 35(7), 665-676 (2017)   Exosomes (versatile, cell-derived nanovesicles naturally endowed with exquisite target-homing specificity and the ability to surmount in vivo biological barriers) hold substantial promise for developing exciting approaches in drug delivery and cancer immunotherapy. Specifically, bioengineered exosomes are being successfully deployed to deliver potent tumoricidal drugs (siRNAs and chemotherapeutic compounds) preferentially to cancer cells, while a new generation of exosome-based therapeutic cancer vaccines has produced enticing results in early-phase clinical trials. Here, we review the state-of-the-art technologies and protocols, and discuss the prospects and challenges for the clinical development of this emerging class of therapeutics.  

3.2729           Measurement of Cholesterol Transfer from Lysosome to Peroxisome Using an In Vitro Reconstitution Assay

Luo, J., Liao, Y-C., Xiao, J. and Song, B-L. Methods in Mol. Biol., 1583, 141-161 (2017)   Low-density lipoproteins (LDLs) are taken up by the cell mainly through receptor-mediated endocytosis. LDL-derived cholesterol leaves lysosome and further transports to downstream organelles for specific cellular needs. We recently report that cholesterol transfers from lysosome to peroxisome through lysosome–peroxisome membrane contact (LPMC). Here, we use iodixanol density gradient centrifugation to isolate lysosomes and peroxisomes separately for the in vitro reconstitution of LPMC. We also apply ³H-cholesterol-labeled lysosomes and peroxisomes in vitro to measure ³H-cholesterol transfer through LPMC.  

3.2730           Isolation of Peroxisomes from Rat Liver and Cultured Hepatoma Cells by Density Gradient Centrifugation

Manner, A. and Islinger, M. Methods in Mol. Biol., 1595, 1-11 (2017)   Subcellular fractionation is still a valuable technique to unravel organelle-specific proteomes, validate the location of uncharacterized proteins, or to functionally analyze import and metabolism in individual subcellular compartments. In this respect, density gradient centrifugation still represents a very classic, indispensable technique to isolate and analyze peroxisomes. Here, we present two independent protocols for the purification of peroxisomes from either liver tissue or the HepG2 hepatoma cell line. While the former permits the isolation of highly pure peroxisomes suitable for, e.g., subcellular proteomics experiments, the latter protocol yields peroxisomal fractions from considerably less purity but allows to easily modify metabolic conditions in the culture medium or to genetically manipulate the peroxisomal compartment. In this respect, both purification methods represent alternative tools to be applied in experiments investigating peroxisome physiology.  

3.2731           Extracellular vesicle mimetics: Novel alternatives to extracellular vesicle-based theranostics, drug delivery, and vaccines

Kim, O.Y., Lee, J. and Gho, Y.S. Seminars in Cell & Development Biol., 67, 74-82 (2017)   Extracellular vesicles are nano-sized spherical bilayered proteolipids encasing various components. Cells of all domains of life actively release these vesicles to the surroundings including various biological fluids. These extracellular vesicles are known to play pivotal roles in numerous pathophysiological functions. Extracellular vesicles have distinct characteristics, like high biocompatibility, safety, and nano-sized diameters that allow efficient drug loading capacity and long blood circulation half-life. These characteristics of extracellular vesicles have engrossed many scientists to harness them as new tools for novel delivery systems. This review will highlight the current state of the arts and problems of such extracellular vesicle-based theranostics, drug delivery and vaccines, and introduce “extracellular vesicle mimetics” as the novel alternative of extracellular vesicles. We hope to provide insights into the potential of extracellular vesicle mimetics as superior substitute to the natural extracellular vesicles that can be applied to theranostics, drug delivery, and vaccines against various diseases.  

3.2732           Selective inhibition of sterolO-acyltransferase 1 isozyme by beauveriolide III in intact cells

Ohshiro, T., Kobayashi, K., Ohba, M., Matsuda, D., Rudel, L.L., Takahashi, T., Doi, T. and Tomoda, H. Scientific Reports, 7:4163 (2017)   Beauveriolide III (BeauIII) inhibited sterol O-acyltransferases 1 and 2 (SOAT1 and SOAT2), which are endoplasmic reticulum (ER) membrane proteins, in an enzyme-based assay, and selectively inhibited SOAT1 in a cell-based assay using SOAT1-/SOAT2-CHO cells. This discrepancy in SOAT inhibition by BeauIII was investigated. In the enzyme-based assay, BeauIII inhibited SOAT1 and SOAT2 to a similar extent using microsomes prepared from cells disrupted under the strongest sonication condition. In semi-intact SOAT1-/SOAT2-CHO cells prepared by a treatment with digitonin (plasma membrane permeabilized), BeauIII selectively inhibited SOAT1 (IC₅₀; 5.0 µM (SOAT1) vs >90 µM (SOAT2)), while in those treated with saponin (plasma membrane and ER membrane permeabilized), BeauIII inhibited SOAT1 (IC₅₀, 1.8 µM) and SOAT2 (5.9 µM). SOAT1-selective inhibition by BeauIII was reproduced in intact ER fractions prepared from SOAT1/SOAT2-CHO cells. A Western blotting analysis revealed that biotin-labeled beauveriolide bound to the SOAT1 protein prepared from SOAT1-CHO cells. We concluded that BeauIII binds to a putative active site responsible for SOAT1 that is located on the cytosolic side of the ER, while BeauIII is not accessible to the corresponding active site for SOAT2 located on the luminal side.  

3.2733           The role of exosomes in cancer metastasis

Steinbichler, T.B., Dudas, J., Riechelmann, H., Skvortsova, I-I. Seminars in Cancer Biol., 44, 170-181 (2017)   Exosomes are small membrane vesicles with a size ranging from 40 to 100 nm. They can serve as functional mediators in cell interaction leading to cancer metastasis. Metastasis is a complex multistep process of cancer cell invasion, survival in blood vessels, attachment to and colonization of the host organ. Exosomes influence every step of this cascade and can be targeted by oncological treatment. This review highlights the role of exosomes in the various steps of the metastatic cascade and how exosome dependent pathways can be targeted as therapeutic approach or used for liquid biopsies.  

3.2734           Exosomes purified from a single cell type have diverse morphology

Zabeo, D., Cvjetkovic, A., Lässer, C., Schorb, M., L¨tvall, J. and Höög, J.L.

1. Extracellular Vesicles, 6:1329476 (2017)

  Extracellular vesicles (EVs) are produced by all known organisms and are important for cell communication and physiology. Great morphological diversity has been described regarding EVs found in body fluids such as blood plasma, breast milk, and ejaculate. However, a detailed morphological analysis has never been performed on exosomes when purified from a single cell type. In this study we analysed and quantified, via multiple electron microscopy techniques, the morphology of exosomes purified from the human mast cell line HMC-1. The results revealed a wide diversity in exosome morphology, suggesting that subpopulations of exosomes with different and specific functions may exist. Our findings imply that a new, more efficient way of defining exosome subpopulations is necessary. A system was proposed where exosomes were classified into nine different categories according to their size and shape. Three additional morphological features were also found in exosomes regardless of their morphological classification. These findings show that exosomes purified from a single cell line are also morphologically diverse, similar to previous observations for EVs in body fluids. This knowledge can help to improve the interpretation of experimental results and widen our general understanding of the biological functions of exosomes.  

3.2735           Synapsins regulate brain-derived neurotrophic factor-mediated synaptic potentiation and axon elongation by acting on membrane rafts

Kao, H-T., Ryoo, K., Lin, A., janoschka, S.R., Augustiine, G.J. and Porton, B. Eur. J. Neurosci., 45(8), 1085-1101 (2017)   In neurons, intracellular membrane rafts are essential for specific actions of brain-derived neurotrophic factor (BDNF), which include the regulation of axon outgrowth, growth cone turning and synaptic transmission. Virtually, all the actions of BDNF are mediated by binding to its receptor, TrkB. The association of TrkB with the tyrosine kinase, Fyn, is critical for its localization to intracellular membrane rafts. Here, we show that synapsins, a family of highly amphipathic neuronal phosphoproteins, regulate membrane raft lipid composition and consequently, the ability of BDNF to regulate axon/neurite development and potentiate synaptic transmission. In the brains of mice lacking all synapsins, the expression of both BDNF and TrkB were increased, suggesting that BDNF/TrkB-mediated signaling is impaired. Consistent with this finding, synapsin-depleted neurons exhibit altered raft lipid composition, deficient targeting of Fyn to rafts, attenuated TrkB activation, and abrogation of BDNF-stimulated axon outgrowth and synaptic potentiation. Conversely, overexpression of synapsins in neuroblastoma cells results in corresponding reciprocal changes in raft lipid composition, increased localization of Fyn to rafts and promotion of BDNF-stimulated neurite formation. In the presence of synapsins, the ratio of cholesterol to estimated total phospholipids converged to 1, suggesting that synapsins act by regulating the ratio of lipids in intracellular membranes, thereby promoting lipid raft formation. These studies reveal a mechanistic link between BDNF and synapsins, impacting early development and synaptic transmission.  

3.2736           Autophagosome formation is initiated at phosphatidylinositol synthase‐enriched ER subdomains

Nishimura, T., Tamura, N., Kono, N., Shimanaka, Y., Arai, H., Yamamoto, H. and Mizushima, N. EMBO J., 36(12), 1719-1735 (2017)   The autophagosome, a double‐membrane structure mediating degradation of cytoplasmic materials by macroautophagy, is formed in close proximity to the endoplasmic reticulum (ER). However, how the ER membrane is involved in autophagy initiation and to which membrane structures the autophagy‐initiation complex is localized have not been fully characterized. Here, we were able to biochemically analyze autophagic intermediate membranes and show that the autophagy‐initiation complex containing ULK and FIP200 first associates with the ER membrane. To further characterize the ER subdomain, we screened phospholipid biosynthetic enzymes and found that the autophagy‐initiation complex localizes to phosphatidylinositol synthase (PIS)‐enriched ER subdomains. Then, the initiation complex translocates to the ATG9A‐positive autophagosome precursors in a PI3P‐dependent manner. Depletion of phosphatidylinositol (PI) by targeting bacterial PI‐specific phospholipase C to the PIS domain impairs recruitment of downstream autophagy factors and autophagosome formation. These findings suggest that the autophagy‐initiation complex, the PIS‐enriched ER subdomain, and ATG9A vesicles together initiate autophagosome formation.  

3.2737           Characterization of extracellular membrane vesicles of an Antarctic bacterium, Shewanella livingstonensis Ac10, and their enhanced production by alteration of phospholipid composition

Yokoyama, F., Kuwamoto, J., Imai, T. and Kurihara, T. Extremophiles, 21(4), 723-731 (2017)   A cold-adapted bacterium, Shewanella livingstonensis Ac10, which produces eicosapentaenoic acid (EPA) as a component of its membrane phospholipids, is useful as a model to study the function of EPA and as a host for heterologous production of thermolabile proteins at low temperatures. In this study, we characterized extracellular membrane vesicles (EMVs) of this bacterium to examine the involvement of EPA in the biogenesis of EMVs and for the future application of EMVs to extracellular protein production. We found that this strain produced EMVs from the cell surface. Cryo-electron microscopic observation showed that the majority of the EMVs had a single-bilayer structure with an average diameter of 110 nm, though EMVs with double-bilayer membranes and other diverse structures were also observed. Quantitative analysis demonstrated that the EMV production was significantly increased (3–5 fold) by the depletion of EPA-containing phospholipids. The lack of EPA also altered the protein composition of EMVs. In particular, incorporation of one of the cold-inducible outer membrane proteins, OmpC176, was significantly increased in EMVs after the depletion of EPA. These results provide a basis for the construction of an EMV-based, low-temperature protein production system and show the involvement of EPA in the regulation of EMV biogenesis.  

3.2738           Macrophage-derived exosomes induce inflammatory factors in endothelial cells under hypertensive conditions

Osada-Oka, M., Shiota, M., Izumi, Y., Nishiyama, M., Tanaka, M., Yamaguchi, T., Sakurai, E., Miura, K. and Iwao, H. Hypertension Res., 40(4), 353-360 (2017)   Hypertension is one of the most important cardiovascular risk factors and results in macrophage infiltration of blood vessels. However, how macrophages coordinate inflammatory responses with endothelial cells (ECs) remains unclear. In this study, we investigated whether exosomes upregulate the expression of inflammatory factors in ECs under hypertensive conditions. Hypertension was induced in rats by continuous infusion of angiotensin II (Ang II). Exosomes were purified from rat serum by density gradient and ultracentrifugation and used to stimulate human coronary artery ECs (HCAECs). Moreover, the interactions between HCAECs and exosomes from human THP-1-derived macrophages were analyzed. Administration of Ang II enhanced the expression of CD68, a macrophage marker, in rat hearts, suggesting enhanced infiltration of macrophages. In addition, the expression of intracellular adhesion molecule-1 (ICAM1) and plasminogen activator inhibitor-1 (PAI-1), a proinflammatory factor, was increased in hypertensive rat hearts compared with control rats. CD68 protein expression and an increase in the expression of some exosome markers were detected in exosomes from hypertensive rat serum. Moreover, the exosomes upregulated the expression levels of ICAM1 and PAI-1 in HCAECs. The level of miR-17, a negative regulator of ICAM1 expression, was markedly decreased in exosomes from hypertensive rat serum compared with exosomes from control rats. Interestingly, Ang II-stimulated THP-1-derived exosomes also enhanced the expression of ICAM1 and PAI-1 and contained reduced levels of miR-17 compared with exosomes from unstimulated cells. These results suggest that inflammation of ECs under hypertensive conditions is caused, at least in part, by macrophage-derived exosomes.  

3.2739           The emerging role of exosome and microvesicle- (EMV-) based cancer therapeutics and immunotherapy

Moore, C., Kosgodage, U., lange, S. and Inal, J.M. Int. J. Cancer, 141(3), 428-436 (2017)   There is an urgent need to develop new combination therapies beyond existing surgery, radio- and chemo-therapy, perhaps initially combining chemotherapy with the targeting specificities of immunotherapy. For this, strategies to limit inflammation and immunosuppression and evasion in the tumour microenvironment are also needed. To devise effective new immunotherapies we must first understand tumour immunology, including the roles of T cells, macrophages, myeloid suppressor cells and of exosomes and microvesicles (EMVs) in promoting angiogenesis, tumour growth, drug resistance and metastasis. One promising cancer immunotherapy discussed uses cationic liposomes carrying tumour RNA (RNA-lipoplexes) to provoke a strong anti-viral-like (cytotoxic CD8⁺) anti-tumour immune response. Mesenchymal stem cell-derived EMVs, with their capacity to migrate towards inflammatory areas including solid tumours, have also been used. As tumour EMVs clearly exacerbate the tumour microenvironment, another therapy option could involve EMV removal. Affinity-based methods to deplete EMVs, including an immunodepletion, antibody-based affinity substrate, are therefore considered. Finally EMV and exosome-mimetic nanovesicles (NVs) delivery of siRNA or chemotherapeutic drugs that target tumours using peptide ligands for cognate receptors on the tumour cells are discussed. We also touch upon the reversal of drug efflux in EMVs from cancer cells which can sensitize cells to chemotherapy. The use of immunotherapy in combination with the advent of EMVs provides potent therapies to various cancers.  

3.2740           Effect of circulating exosomes from transition cows on Madin-Darby bovine kidney cell function

Crookenden, M.A. et al

1. Dairy Sci., 100(7), 5687-5700 (2017)

  The greatest risk of metabolic and infectious disease in dairy cows is during the transition from pregnancy to lactating (i.e., the transition period). The objective of this experiment was to determine the effects of extracellular vesicles (microvesicles involved in cell-to-cell signaling) isolated from transition cows on target cell function. We previously identified differences in the protein profiles of exosomes isolated from cows divergent in metabolic health status. Therefore, we hypothesized that these exosomes would affect target tissues differently. To investigate this, 2 groups of cows (n = 5/group) were selected based on the concentration of β-hydroxybutyrate and fatty acids in plasma and triacylglycerol concentration in liver at wk 1 and 2 postcalving. Cows with high concentrations of β-hydroxybutyrate, fatty acids, and triacylglycerol were considered at increased risk of clinical disease during the transition period (high-risk group; n = 5) and were compared with cows that had low concentrations of the selected health indicators (low-risk group; n = 5). At 2 time points during the transition period (postcalving at wk 1 and 4), blood was sampled and plasma exosomes were isolated from the high-risk and low-risk cows. The exosomes were applied at concentrations of 10 and 1 µg/mL to 5 × 10³ Madin-Darby bovine kidney cells grown to 50% confluence in 96-well plates. Results indicate a numerical increase in cell proliferation when exosomes from high-risk cows were applied compared with those from low-risk cows. Consistent with an effect on cell proliferation, quantitative reverse transcriptase PCR indicated a trend for upregulation of 3 proinflammatory genes (granulocyte colony-stimulating factor, ciliary neurotrophic factor, and CD27 ligand) with the application of high-risk exosomes, which are involved in cellular growth and survival. Proteomic analysis indicated 2 proteins in the low-risk group that were not identified in the high-risk group (endoplasmin and catalase), which may also be indicative of the metabolic state of origin. It is likely that the metabolic state of the transition cow affects cellular function through exosomal messaging; however, more in-depth research into cross-talk between exosomes and target cells is required to determine whether exosomes influence Madin-Darby bovine kidney cells in this manner.  

3.2741           The role of lipid raft translocation of prohibitin in regulation of Akt and Raf-protected apoptosis of HaCaT cells upon ultraviolet B irradiation

Wu, Q. and Wu, S. Mol. Carcinogenesis, 56(7), 1789-1797 (2017)   Prohibitin (PHB) plays a role in regulation of ultraviolet B light (UVB)-induced apoptosis of human keratinocytes, HaCaT cells. The regulatory function of PHB appears to be associated with its lipid raft translocation. However, the detailed mechanism for PHB-mediated apoptosis of these keratinocytes upon UVB irradiation is not clear. In this report, we determined the role of lipid raft trans location of PHB in regulation of UVB-induced apoptosis. Our data show that upon UVB irradiation PHB is translocated from the non-raft membrane to the lipid rafts, which is correlated with a release of both Akt and Raf from membrane. Overexpression of Akt and/or Raf impedes UVB-induced lipid raft translocation of PHB. Immunoprecipitation analysis indicates that UVB alters the interactions among PHB, Akt, and Raf. Reduced expression of PHB leads to a decreased phosphorylation of Akt and ERK, as well as a decreased activity of Akt, and increased apoptosis of the cells upon UVB irradiation. These results suggest that PHB regulates UVB-induced apoptosis of keratinocytes via a mechanism that involves detachment from Akt and Raf on the plasma membrane, and sequential lipid raft translocation.  

3.2742           NCAM-140 Translocation into Lipid Rafts Mediates the Neuroprotective Effects of GDNF

Li, L., Chen, H., Wang, M., Chen, F., Gao, J., Sun, S., Li, Y. and Gao, D. Mol. Neurobiol., 54(4), 2739-2751 (2017)   Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for substantia nigra dopaminergic (DA) neuronal cells. Recent studies have demonstrated that neural cell adhesion molecule functions as a signal transduction receptor for GDNF. The purpose of this study is to reveal whether neural cell adhesion molecule (NCAM) mediates the protective effects of GDNF on DA neuronal cells and further explore the mechanisms involved. We utilized SH-SY5Y cell line to establish a model of 6-hydroxydopamine (6-OHDA)-injured DA neuronal cells. Lentiviral vectors were constructed to knockdown or overexpress NCAM-140, and a density gradient centrifugation method was employed to separate membrane lipid rafts. 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), flow cytometric analysis, and western blotting were used to evaluate the protective effects of GDNF. The results showed that GDNF could protect 6-OHDA-injured SH-SY5Y cells via improving cell viability and decreasing the cell death rate and cleaved caspase-3 expression. NCAM-140 knockdown decreased cell viability and increased the cell death rate and cleaved caspase-3 expression, while its overexpression had the opposite effects. Notably, the amount of NCAM-140 located in lipid rafts increased after GDNF treatment. Pretreatment with 2-bromopalmitate, a specific inhibitor of protein palmitoylation, suppressed NCAM-140 translocation to lipid rafts and reduced the NCAM-mediated protective effects of GDNF on injured DA neuronal cells. Our results suggest that GDNF have the protective effects on injured DA cells by influencing NCAM-140 translocation into lipid rafts.  

3.2743           Using hyperLOPIT to perform high-resolution mapping of the spatial proteome

Mulvey, C.M., Breckels, L.M., Geladaki, A., Britovsek, N.K., Nightingale, D.J.H., Christoforou, A., Elzek, M., Deery, M.J., Gatto, L. and Lilley, K.S. Nature Protocols, 12(6), 1110-1135 (2017)   The organization of eukaryotic cells into distinct subcompartments is vital for all functional processes, and aberrant protein localization is a hallmark of many diseases. Microscopy methods, although powerful, are usually low-throughput and dependent on the availability of fluorescent fusion proteins or highly specific and sensitive antibodies. One method that provides a global picture of the cell is localization of organelle proteins by isotope tagging (LOPIT), which combines biochemical cell fractionation using density gradient ultracentrifugation with multiplexed quantitative proteomics mass spectrometry, allowing simultaneous determination of the steady-state distribution of hundreds of proteins within organelles. Proteins are assigned to organelles based on the similarity of their gradient distribution to those of well-annotated organelle marker proteins. We have substantially re-developed our original LOPIT protocol (published by Nature Protocols in 2006) to enable the subcellular localization of thousands of proteins per experiment (hyperLOPIT), including spatial resolution at the suborganelle and large protein complex level. This Protocol Extension article integrates all elements of the hyperLOPIT pipeline, including an additional enrichment strategy for chromatin, extended multiplexing capacity of isobaric mass tags, state-of-the-art mass spectrometry methods and multivariate machine-learning approaches for analysis of spatial proteomics data. We have also created an open-source infrastructure to support analysis of quantitative mass-spectrometry-based spatial proteomics data (http://bioconductor.org/packages/pRoloc) and an accompanying interactive visualization framework (http://www. bioconductor.org/packages/pRolocGUI). The procedure we outline here is applicable to any cell culture system and requires ~1 week to complete sample preparation steps, ~2 d for mass spectrometry data acquisition and 1–2 d for data analysis and downstream informatics.  

3.2744           Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer

Kamerkar, S., LebLeu, V.S., Sugimoto, H., Yang, S., Ruivo, C.F., Melo, S.A., Lee, J.J. and Kalhuri, R. Nature, 546, 498-503 (2017)   The mutant form of the GTPase KRAS is a key driver of pancreatic cancer but remains a challenging therapeutic target. Exosomes are extracellular vesicles generated by all cells, and are naturally present in the blood. Here we show that enhanced retention of exosomes, compared to liposomes, in the circulation of mice is likely due to CD47-mediated protection of exosomes from phagocytosis by monocytes and macrophages. Exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry short interfering RNA or short hairpin RNA specific to oncogenic Kras^G12D, a common mutation in pancreatic cancer. Compared to liposomes, the engineered exosomes (known as iExosomes) target oncogenic KRAS with an enhanced efficacy that is dependent on CD47, and is facilitated by macropinocytosis. Treatment with iExosomes suppressed cancer in multiple mouse models of pancreatic cancer and significantly increased overall survival. Our results demonstrate an approach for direct and specific targeting of oncogenic KRAS in tumours using iExosomes.  

3.2745           The Glycosyltransferase QUA1 Regulates Chloroplast-Associated Calcium Signaling During Salt and Drought Stress in Arabidopsis

Zheng, Y., Liao, C., Zhao, S., Wang, C. and Guo, Y. Plant Cell Physiol., 58(2), 329-341 (2017)   Cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) elevation induced by various signals is responsible for appropriate downstream responses. Through a genetic screen of Arabidopsis thaliana mutants defective in stress-induced [Ca²⁺]_cyt elevation, the glycosyltransferase QUASIMODO1 (QUA1) was identified as a regulator of [Ca²⁺]_cyt in response to salt stress. Compared with the wild type, the qua1-4 mutant exhibited a dramatically greater increase in [Ca²⁺]_cyt under NaCl treatment. Functional analysis showed that QUA1 is a novel chloroplast protein that regulates cytoplasmic Ca²⁺ signaling. QUA1 was detected in chloroplast thylakoids, and the qua1-4 mutant exhibited irregularly stacked grana. The observed greater increase in [Ca²⁺]_cyt was inhibited upon recovery of chloroplast function in the qua1-4 mutant. Further analysis showed that CAS, a thylakoid-localized calcium sensor, also displayed irregularly stacked grana, and the chloroplasts of the qua1-4 cas-1 double mutant were similar to those of cas-1 plants. In QUA1-overexpressing plants, the protein level of CAS was decreased, and CAS was readily degraded under osmotic stress. When CAS was silenced in the qua1-4 mutant, the large [Ca²⁺]_cyt increase was blocked, and the higher expression of PLC3 and PLC4 was suppressed. Under osmotic stress, the qua1-4 mutant showed an even greater elevation in [Ca²⁺]_cyt and was hypersensitive to drought stress. However, this sensitivity was inhibited when the increase in [Ca²⁺]_cyt was repressed in the qua1-4 mutant. Collectively, our data indicate that QUA1 may function in chloroplast-dependent calcium signaling under salt and drought stresses. Additionally, CAS may function downstream of QUA1 to mediate these processes.  

3.2746           A voltage-dependent K+ channel in the lysosome is required for refilling lysosomal Ca2+ stores

Wang, W., Zhang, X., Gao, Q., Lawas, M., Yu, L., Cheng, X., Gu, M., Sahoo, N., Li, X., Li, P., Ireland, S., Meredith, A. and Xu, H.

1. Cell Biol., 216(6), 1715-1730 (2017)

  The resting membrane potential (Δψ) of the cell is negative on the cytosolic side and determined primarily by the plasma membrane’s selective permeability to K⁺. We show that lysosomal Δψ is set by lysosomal membrane permeabilities to Na⁺ and H⁺, but not K⁺, and is positive on the cytosolic side. An increase in juxta-lysosomal Ca²⁺ rapidly reversed lysosomal Δψ by activating a large voltage-dependent and K⁺-selective conductance (LysoK_VCa). LysoK_VCa is encoded molecularly by SLO1 proteins known for forming plasma membrane BK channels. Opening of single LysoK_VCa channels is sufficient to cause the rapid, striking changes in lysosomal Δψ. Lysosomal Ca²⁺ stores may be refilled from endoplasmic reticulum (ER) Ca²⁺ via ER–lysosome membrane contact sites. We propose that LysoK_VCa serves as the perilysosomal Ca²⁺ effector to prime lysosomes for the refilling process. Consistently, genetic ablation or pharmacological inhibition of LysoK_VCa, or abolition of its Ca²⁺ sensitivity, blocks refilling and maintenance of lysosomal Ca²⁺ stores, resulting in lysosomal cholesterol accumulation and a lysosome storage phenotype.  

3.2747           COPII-coated membranes function as transport carriers of intracellular procollagen I

Gorur, A., Yuan, L., Kenny, S.J., Baba, S., Xu, K. and Schekman, R.

1. Cell Biol., 216(6), 1745-1759 (2017)

  The coat protein complex II (COPII) is essential for the transport of large cargo, such as 300-nm procollagen I (PC1) molecules, from the endoplasmic reticulum (ER) to the Golgi. Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles at ER exit sites to more than 300 nm in diameter and accelerates the secretion of PC1. However, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy, correlated light electron microscopy, and live-cell imaging, we demonstrate the existence of mobile COPII-coated vesicles that completely encapsulate the cargo PC1 and are physically separated from ER. We also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude that large COPII vesicles are bona fide carriers of PC1.  

3.2748           Schistosomal MicroRNAs Isolated From Extracellular Vesicles in Sera of Infected Patients: A New Tool for Diagnosis and Follow-up of Human Schistosomiasis

Meningher, T., Lerman, G., Regev-Rudzki, N., Gold, D., Ben-Dov, I.Z., Sidi, Y., Avni, D. and Schwartz, E.

1. Infect. Dis., 215(3), 378-386 (2017)

  Background. Schistosomiasis traditionally has been diagnosed by detecting eggs in stool or urine. However, the sensitivity of these examinations is limited, especially in travelers with a low worm burden. Serologic tests have a greater sensitivity, but their results remain positive regardless of treatment and thus cannot be used for follow-up of patients. We hypothesized that detection of worm microRNAs (miRNAs) in serum can overcome the drawbacks of the existing diagnostic methods. Methods and Results. Twenty-six returning travelers with schistosomiasis (based on positive results of serologic tests or detection of ova) and 17 healthy controls were included in the study. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) amplification of miRNA extracted directly from 500 µL of serum had limited sensitivity and specificity. However, qRT-PCR analysis of RNA extracted from 200 μL of serum extracellular vesicles detected 4 schistosomal miRNAs; the sensitivity and specificity of the 2 highest expressed miRNAs (bantam and miR-2c-3p) were 86% and 84%, respectively. In 7 patients with posttreatment serum available for analysis, we observed outcomes ranging from a reduction in the schistosomal miRNA level to full recovery from disease. Conclusions. qRT-PCR of pathogen miRNAs isolated from extracellular vesicles in sera from infected individuals may provide a new tool for diagnosing schistosomiasis in patients with a low parasite burden. This assay could also be used for evaluating the outcome of therapy, as well as disease-control programs.  

3.2749           Caveolins and cavins in the trafficking, maturation, and degradation of caveolae: implications for cell physiology

Busija, A.R., Patel, H.H. and Insel, P.A. Am. J. Physiol. Cell Physiol., 312, C459-C477 (2017)   Caveolins (Cavs) are ~20 kDa scaffolding proteins that assemble as oligomeric complexes in lipid raft domains to form caveolae, flask-shaped plasma membrane (PM) invaginations. Caveolae (“little caves”) require lipid-lipid, protein-lipid, and protein-protein interactions that can modulate the localization, conformational stability, ligand affinity, effector specificity, and other functions of proteins that are partners of Cavs. Cavs are assembled into small oligomers in the endoplasmic reticulum (ER), transported to the Golgi for assembly with cholesterol and other oligomers, and then exported to the PM as an intact coat complex. At the PM, cavins, ~50 kDa adapter proteins, oligomerize into an outer coat complex that remodels the membrane into caveolae. The structure of caveolae protects their contents (i.e., lipids and proteins) from degradation. Cellular changes, including signal transduction effects, can destabilize caveolae and produce cavin dissociation, restructuring of Cav oligomers, ubiquitination, internalization, and degradation. In this review, we provide a perspective of the life cycle (biogenesis, degradation), composition, and physiologic roles of Cavs and caveolae and identify unanswered questions regarding the roles of Cavs and cavins in caveolae and in regulating cell physiology.¹  

3.2750           β-Adrenergic induction of lipolysis in hepatocytes is inhibited by ethanol exposure

Schott, M.B., Rasineni, K., Weller, S.W., Schulze, R.J., Sletten, A.C., Casey, C.A. and McNiven, M.A.

1. Biol. Chem., 292(28), 11815-11828 (2017)

  In liver steatosis (i.e. fatty liver), hepatocytes accumulate many large neutral lipid storage organelles known as lipid droplets (LDs). LDs are important in the maintenance of energy homeostasis, but the signaling mechanisms that stimulate LD metabolism in hepatocytes are poorly defined. In adipocytes, catecholamines target the β-adrenergic (β-AR)/cAMP pathway to activate cytosolic lipases and induce their recruitment to the LD surface. Therefore, the goal of this study was to determine whether hepatocytes, like adipocytes, also undergo cAMP-mediated lipolysis in response to β-AR stimulation. Using primary rat hepatocytes and human hepatoma cells, we found that treatment with the β-AR agent isoproterenol caused substantial LD loss via activation of cytosolic lipases adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). β-Adrenergic stimulation rapidly activated PKA, which led to the phosphorylation of ATGL and HSL and their recruitment to the LD surface. To test whether this β-AR-dependent lipolysis pathway was altered in a model of alcoholic fatty liver, primary hepatocytes from rats fed a 6-week EtOH-containing Lieber-DeCarli diet were treated with cAMP agonists. Compared with controls, EtOH-exposed hepatocytes showed a drastic inhibition in β-AR/cAMP-induced LD breakdown and the phosphorylation of PKA substrates, including HSL. This observation was supported in VA-13 cells, an EtOH-metabolizing human hepatoma cell line, which displayed marked defects in both PKA activation and isoproterenol-induced ATGL translocation to the LD periphery. In summary, these findings suggest that β-AR stimulation mobilizes cytosolic lipases for LD breakdown in hepatocytes, and perturbation of this pathway could be a major consequence of chronic EtOH insult leading to fatty liver.  

3.2751           Hepatitis C Virus Lipoviroparticles Assemble in the Endoplasmic Reticulum (ER) and Bud off from the ER to the Golgi Compartment in COPII Vesicles

Syed, G.H., Khan, M., Yang, S. and Siddiqui, A.

1. Virol., 91(15), e00499-17 (2017)

  Hepatitis C virus (HCV) exists as a lipoprotein-virus hybrid lipoviroparticle (LVP). In vitro studies have demonstrated the importance of apolipoproteins in HCV secretion and infectivity, leading to the notion that HCV coopts the secretion of very-low-density lipoprotein (VLDL) for its egress. However, the mechanisms involved in virus particle assembly and egress are still elusive. The biogenesis of VLDL particles occurs in the endoplasmic reticulum (ER), followed by subsequent lipidation in the ER and Golgi compartment. The secretion of mature VLDL particles occurs through the Golgi secretory pathway. HCV virions are believed to latch onto or fuse with the nascent VLDL particle in either the ER or the Golgi compartment, resulting in the generation of LVPs. In our attempt to unravel the collaboration between HCV and VLDL secretion, we studied HCV particles budding from the ER en route to the Golgi compartment in COPII vesicles. Biophysical characterization of COPII vesicles fractionated on an iodixanol gradient revealed that HCV RNA is enriched in the highly buoyant COPII vesicle fractions and cofractionates with apolipoprotein B (ApoB), ApoE, and the HCV core and envelope proteins. Electron microscopy of immunogold-labeled microsections revealed that the HCV envelope and core proteins colocalize with apolipoproteins and HCV RNA in Sec31-coated COPII vesicles. Ultrastructural analysis also revealed the presence of HCV structural proteins, RNA, and apolipoproteins in the Golgi stacks. These findings support the hypothesis that HCV LVPs assemble in the ER and are transported to the Golgi compartment in COPII vesicles to embark on the Golgi secretory route.  

3.2752           Exosomes from metastatic cancer cells transfer amoeboid phenotype to non-metastatic cells and increase endothelial permeability: their emerging role in tumor heterogeneity

Schillaci, O., Fontana, s., Monteleone, F., Taverna, S., Di Bella, M.A., Di Vizio, D. And Alessandro, R. Scientific Reports, 7:4711 (2017)   The goal of this study was to understand if exosomes derived from high-metastatic cells may influence the behavior of less aggressive cancer cells and the properties of the endothelium. We found that metastatic colon cancer cells are able to transfer their amoeboid phenotype to isogenic primary cancer cells through exosomes, and that this morphological transition is associated with the acquisition of a more aggressive behavior. Moreover, exosomes from the metastatic line (SW620Exos) exhibited higher ability to cause endothelial hyperpermeability than exosomes from the non metastatic line (SW480Exos). SWATH-based quantitative proteomic analysis highlighted that SW620Exos are significantly enriched in cytoskeletal-associated proteins including proteins activating the RhoA/ROCK pathway, known to induce amoeboid properties and destabilization of endothelial junctions. In particular, thrombin was identified as a key mediator of the effects induced by SW620Exos in target cells, in which we also found a significant increase of RhoA activity. Overall, our results demonstrate that in a heterogeneous context exosomes released by aggressive sub-clones can contribute to accelerate tumor progression by spreading malignant properties that affect both the tumor cell plasticity and the endothelial cell behavior.  

3.2753           Directional Exosome Proteomes Reflect Polarity-Specific Functions in Retinal Pigmented Epithelium Monolayers

Klingeborn, M., Dismuke, W.M., Skiba, N.P., Kelly, U., Stamer, W.D. and Bowes Rickman, C. Scientific Reports, 7:4901 (2017)   The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its polarity is responsible for directional secretion and uptake of proteins, lipoprotein particles and extracellular vesicles (EVs). Such a secretional division dictates directed interactions between the systemic circulation (basolateral) and the retina (apical). Our goal is to define the polarized proteomes and physical characteristics of EVs released from the RPE. Primary cultures of porcine RPE cells were differentiated into polarized RPE monolayers on permeable supports. EVs were isolated from media bathing either apical or basolateral RPE surfaces, and two subpopulations of small EVs including exosomes, and dense EVs, were purified and processed for proteomic profiling. In parallel, EV size distribution and concentration were determined. Using protein correlation profiling mass spectrometry, a total of 631 proteins were identified in exosome preparations, 299 of which were uniquely released apically, and 94 uniquely released basolaterally. Selected proteins were validated by Western blot. The proteomes of these exosome and dense EVs preparations suggest that epithelial polarity impacts directional release. These data serve as a foundation for comparative studies aimed at elucidating the role of exosomes in the molecular pathophysiology of retinal diseases and help identify potential therapeutic targets and biomarkers.  

3.2754           DGKδ triggers endoplasmic reticulum release of IFT88-containing vesicles destined for the assembly of primary cilia

Ding, J., Shao, L., Yao, Y., Tong, X., Liu, H., Yue, S., Xie, L. and Cheng, S.Y. Scientific Reports, 7:5296 (2017)   The morphogenic factor Sonic hedgehog (Shh) signals through the primary cilium, which relies on intraflagellar transport to maintain its structural integrity and function. However, the process by which protein and lipid cargos are delivered to the primary cilium from their sites of synthesis still remains poorly characterized. Here, we report that diacylglycerol kinase δ (DGKδ), a residential lipid kinase in the endoplasmic reticulum, triggers the release of IFT88-containing vesicles from the ER exit sites (ERES), thereby setting forth their movement to the primary cilium. Encoded by the gene whose mutations originally implicated the primary cilium as the venue of Shh signaling, IFT88 is known to be part of the complex B that drives the anterograde transport within cilia. We show that IFT88 interacts with DGKδ, and is associated with COPII-coated vesicles at the ERES. Using a combination of RNAi silencing and gene knockout strategies, we further show that DGKδ is required for supporting Shh signaling both in vitro and in vivo, demonstrating the physiological significance of this regulation.    

3.2755           Bovine milk-derived exosomes from colostrum are enriched with proteins implicated in immune response and growth

Samuel, M., Chisanga, D., Liern, M., Keerthikumar, S., Anand, S., Ang, C-S., Adda, C.G., Versteegen, E., Jois, M. and Mathivanan, S. Scientific Reports, 7:5933 (2017)   Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. They contain cell-state specific cargos which often reflects the (patho)physiological condition of the cells/organism. Milk contains high amounts of exosomes and it is unclear whether their cargo is altered based on the lactation stage of the organism. Here, we isolated exosomes from bovine milk that were obtained at various stages of lactation and examined the content by quantitative proteomics. Exosomes were isolated by OptiPrep density gradient centrifugation from milk obtained from cow after 24, 48 and 72 h post calving. As control, exosomes were also isolated from cows during mid-lactation period which has been referred to as mature milk (MM). Biochemical and biophysical characterization of exosomes revealed the high abundance of exosomes in colostrum and MM samples. Quantitative proteomics analysis highlighted the change in the proteomic cargo of exosomes based on the lactation state of the cow. Functional enrichment analysis revealed that exosomes from colostrum are significantly enriched with proteins that can potentially regulate the immune response and growth. This study highlights the importance of exosomes in colostrum and hence opens up new avenues to exploit these vesicles in the regulation of the immune response and growth.  

3.2756           Loss of Calreticulin Uncovers a Critical Role for Calcium in Regulating Cellular Lipid Homeostasis

Wang, W-A., Liu, W-X., Durnaoglu, S., Lee, S-K., Lian, J., Lehner, R., Ahnn, J., Agellon, L.B. and Michalak, M. Scientific Reports, 7:5941 (2017)   A direct link between Ca²⁺ and lipid homeostasis has not been definitively demonstrated. In this study, we show that manipulation of ER Ca²⁺ causes the re-distribution of a portion of the intracellular unesterified cholesterol to a pool that is not available to the SCAP-SREBP complex. The SREBP processing pathway in ER Ca²⁺ depleted cells remained fully functional and responsive to changes in cellular cholesterol status but differed unexpectedly in basal activity. These findings establish the role of Ca²⁺ in determining the reference set-point for controlling cellular lipid homeostasis. We propose that ER Ca²⁺ status is an important determinant of the basal sensitivity of the sterol sensing mechanism inherent to the SREBP processing pathway.  

3.2757           Sphingomimetic multiple sclerosis drug FTY720 activates vesicular synaptobrevin and augments neuroendocrine secretion

Darios, F.D., Jorgacevski, J., Flasker, A., Zorec, R., Garcia-Martinez, V., Villanueva, J., Gutierrez, L.M., Leese, C., Bal, M., Nosyreva, E., Kavalali, E.T. and Davletov, B. Scientific Reports, 7:5958 (2017)   Neurotransmission and secretion of hormones involve a sequence of protein/lipid interactions with lipid turnover impacting on vesicle trafficking and ultimately fusion of secretory vesicles with the plasma membrane. We previously demonstrated that sphingosine, a sphingolipid metabolite, promotes formation of the SNARE complex required for membrane fusion and also increases the rate of exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and in hippocampal neurons. Recently a fungi-derived sphingosine homologue, FTY720, has been approved for treatment of multiple sclerosis. In its non-phosphorylated form FTY720 accumulates in the central nervous system, reaching high levels which could affect neuronal function. Considering close structural similarity of sphingosine and FTY720 we investigated whether FTY720 has an effect on regulated exocytosis. Our data demonstrate that FTY720 can activate vesicular synaptobrevin for SNARE complex formation and enhance exocytosis in neuroendocrine cells and neurons.  

3.2758           Primary fibroblasts from CSPα mutation carriers recapitulate hallmarks of the adult onset neuronal ceroid lipofuscinosis

Benitez, B. and Sands, M.S. Scientific Reports, 7:6332 (2017)   Mutations in the co- chaperone protein, CSPα, cause an autosomal dominant, adult-neuronal ceroid lipofuscinosis (AD-ANCL). The current understanding of CSPα function exclusively at the synapse fails to explain the autophagy-lysosome pathway (ALP) dysfunction in cells from AD-ANCL patients. Here, we demonstrate unexpectedly that primary dermal fibroblasts from pre-symptomatic mutation carriers recapitulate in vitro features found in the brains of AD-ANCL patients including auto-fluorescent storage material (AFSM) accumulation, CSPα aggregates, increased levels of lysosomal proteins and lysosome enzyme activities. AFSM accumulation correlates with CSPα aggregation and both are susceptible to pharmacological modulation of ALP function. In addition, we demonstrate that endogenous CSPα is present in the lysosome-enriched fractions and co-localizes with lysosome markers in soma, neurites and synaptic boutons. Overexpression of CSPα wild-type (WT) decreases lysotracker signal, secreted lysosomal enzymes and SNAP23-mediated lysosome exocytosis. CSPα WT, mutant and aggregated CSPα are degraded mainly by the ALP but this disease-causing mutation exhibits a faster rate of degradation. Co-expression of both WT and mutant CSPα cause a block in the fusion of autophagosomes/lysosomes. Our data suggest that aggregation‐dependent perturbation of ALP function is a relevant pathogenic mechanism for AD-ANCL and supports the use of AFSM or CSPα aggregation as biomarkers for drug screening purposes.  

3.2759           Outer membrane vesicle-associated lipase FtlA enhances cellular invasion and virulence in Francisella tularensis LVS

Chen, F., Cui, G., Wang, S., Nair, M.K.M., He, L., Qi, X., Han, X., Zhang, H., Zhang, J-R. and Su, J. Emerging Microbes & Infections, 6, e66 (2017)   Francisella tularensis is a highly infectious intracellular pathogen that infects a wide range of host species and causes fatal pneumonic tularemia in humans. ftlA was identified as a potential virulence determinant of the F. tularensis live vaccine strain (LVS) in our previous transposon screen, but its function remained undefined. Here, we show that an unmarked deletion mutant of ftlA was avirulent in a pneumonia mouse model with a severely impaired capacity to infect host cells. Consistent with its sequence homology with GDSL lipase/esterase family proteins, the FtlA protein displayed lipolytic activity in both E. coli and F. tularensis with a preference for relatively short carbon-chain substrates. FtlA thus represents the first F. tularensis lipase to promote bacterial infection of host cells and in vivo fitness. As a cytoplasmic protein, we found that FtlA was secreted into the extracellular environment as a component of outer membrane vesicles (OMVs). Further confocal microscopy analysis revealed that the FtlA-containing OMVs isolated from F. tularensis LVS attached to the host cell membrane. Finally, the OMV-associated FtlA protein complemented the genetic deficiency of the ΔftlA mutant in terms of host cell infection when OMVs purified from the parent strain were co-incubated with the mutant bacteria. These lines of evidence strongly suggest that the FtlA lipase promotes F. tularensis adhesion and internalization by modifying bacterial and/or host molecule(s) when it is secreted as a component of OMVs.  

3.2760           Extracellular vesicles from human liver stem cells restore argininosuccinate synthase deficiency

Sanchez, M.B.H., Previdi, S., Bruno, S., Fonsato, V., Deregibus, M.C., Kholia, S., petrillo, S., Tolosano, E., Critelli, R., Spada, M., Romagnoli, R., Salizzoni, M., Tetta, C. and Camussi, G. Stem Cell Res. Ther., 8:176 (2017)   Argininosuccinate synthase (ASS)1 is a urea cycle enzyme that catalyzes the conversion of citrulline and aspartate to argininosuccinate. Mutations in the ASS1 gene cause citrullinemia type I, a rare autosomal recessive disorder characterized by neonatal hyperammonemia, elevated citrulline levels, and early neonatal death. Treatment for this disease is currently restricted to liver transplantation; however, due to limited organ availability, substitute therapies are required. Recently, extracellular vesicles (EVs) have been reported to act as intercellular transporters carrying genetic information responsible for cell reprogramming. In previous studies, we isolated a population of stem cell-like cells known as human liver stem cells (HLSCs) from healthy liver tissue. Moreover, EVs derived from HLSCs were reported to exhibit regenerative effects on the liver parenchyma in models of acute liver injury. The aim of this study was to evaluate whether EVs derived from normal HLSCs restored ASS1 enzymatic activity and urea production in hepatocytes differentiated from HLSCs derived from a patient with type I citrullinemia.  

3.2761           Saccharomyces cerevisiae cells lacking Pex3 contain membrane vesicles that harbor a subset of peroxisomal membrane proteins

Wroblewska, J.P., Cruz-Zaragoza, L.D., Yan, W., Schummer, A., Chuartzman, S.G., de Boer, R., Oeljekaus, S., Schuldiner, M., Zalckvar, E., Warscheid, B., Erdmann, R. and van der Klei, I.J. BBA – Mol Cell Res., 1864, 1656-1667 (2017)   Pex3 has been proposed to be important for the exit of peroxisomal membrane proteins (PMPs) from the ER, based on the observation that PMPs accumulate at the ER in Saccharomyces cerevisiae pex3 mutant cells. Using a combination of microscopy and biochemical approaches, we show that a subset of the PMPs, including the receptor docking protein Pex14, localizes to membrane vesicles in S. cerevisiae pex3 cells. These vesicles are morphologically distinct from the ER and do not co-sediment with ER markers in cell fractionation experiments. At the vesicles, Pex14 assembles with other peroxins (Pex13, Pex17, and Pex5) to form a complex with a composition similar to the PTS1 import pore in wild-type cells. Fluorescence microscopy studies revealed that also the PTS2 receptor Pex7, the importomer organizing peroxin Pex8, the ubiquitin conjugating enzyme Pex4 with its recruiting PMP Pex22, as well as Pex15 and Pex25 co-localize with Pex14. Other peroxins (including the RING finger complex and Pex27) did not accumulate at these structures, of which Pex11 localized to mitochondria. In line with these observations, proteomic analysis showed that in addition to the docking proteins and Pex5, also Pex7, Pex4/Pex22 and Pex25 were present in Pex14 complexes isolated from pex3 cells. However, formation of the entire importomer was not observed, most likely because Pex8 and the RING proteins were absent in the Pex14 protein complexes. Our data suggest that peroxisomal membrane vesicles can form in the absence of Pex3 and that several PMPs can insert in these vesicles in a Pex3 independent manner.  

3.2762           Adaptations in rod outer segment disc membranes in response to environmental lighting conditions

Rakshit, T., Senapati, S., Parmar, V.M., Sahu, B., Maeda, A. and Park, P.S-H. BBA – Mol. Cell Res., 1864, 1691-1702 (2017)   The light-sensing rod photoreceptor cell exhibits several adaptations in response to the lighting environment. While adaptations to short-term changes in lighting conditions have been examined in depth, adaptations to long-term changes in lighting conditions are less understood. Atomic force microscopy was used to characterize the structure of rod outer segment disc membranes, the site of photon absorption by the pigment rhodopsin, to better understand how photoreceptor cells respond to long-term lighting changes. Structural properties of the disc membrane changed in response to housing mice in constant dark or light conditions and these adaptive changes required output from the phototransduction cascade initiated by rhodopsin. Among these were changes in the packing density of rhodopsin in the membrane, which was independent of rhodopsin synthesis and specifically affected scotopic visual function as assessed by electroretinography. Studies here support the concept of photostasis, which maintains optimal photoreceptor cell function with implications in retinal degenerations.  

3.2763           Recent advances on extracellular vesicles in therapeutic delivery: Challenges, solutions, and opportunities

Lu, M., Xing, H., Yang, Z., Sun, Y., Yang, T., Zhao, X., Cai, C., Wang, D. and Ding, P. Eur. J. Pharmaceut. Biopharmaceut., 119, 381-395 (2017)   Extracellular vesicles (EVs) are intrinsic mediators of intercellular communication in our body, allowing functional transfer of biomolecules (lipids, proteins, and nucleic acid) between diverse locations. Such an instrumental role evokes a surge of interest within the drug delivery community in tailoring EVs for therapeutic delivery. These vesicles represent a novel generation of drug delivery systems, providing high delivery efficiency, intrinsic targeting properties, and low immunogenicity. In the recent years, considerable research efforts have been directed toward developing safe and efficient EV-based delivery vehicles. Although EVs are shown to harbor great promise in therapeutic delivery, substantial improvements in exploring standardized isolation techniques with high efficiency and robust yield, scalable production, standard procedures for EV storage, efficient loading methods without damaging EV integrity, understanding their in vivo trafficking, and developing novel EV-based nanocarriers are still required before their clinical transformation. In this review, we seek to summarize the recent advance on harnessing EVs for drug delivery with focus on state-of-the-art solutions for overcoming major challenges.  

3.2764           Phytol-induced pathology in 2-hydroxyacyl-CoA lyase (HACL1) deficient mice. Evidence for a second non-HACL1-related lyase

Mezzar, S., De Schryver, E., Asselberghs, S., Meyhi, E., Morvay, P.L., Baes, M. and Van Veldhoven, P.P. BBA – Mol. Cell Biol. Lipids, 1862, 972-990 (2017)   2-Hydroxyacyl-CoA lyase (HACL1) is a key enzyme of the peroxisomal α-oxidation of phytanic acid. To better understand its role in health and disease, a mouse model lacking HACL1 was investigated. Under normal conditions, these mice did not display a particular phenotype. However, upon dietary administration of phytol, phytanic acid accumulated in tissues, mainly in liver and serum of KO mice. As a consequence of phytanic acid (or a metabolite) toxicity, KO mice displayed a significant weight loss, absence of abdominal white adipose tissue, enlarged and mottled liver and reduced hepatic glycogen and triglycerides. In addition, hepatic PPARα was activated. The central nervous system of the phytol-treated mice was apparently not affected. In addition, 2OH-FA did not accumulate in the central nervous system of HACL1 deficient mice, likely due to the presence in the endoplasmic reticulum of an alternate HACL1-unrelated lyase. The latter may serve as a backup system in certain tissues and account for the formation of pristanic acid in the phytol-fed KO mice. As the degradation of pristanic acid is also impaired, both phytanoyl- and pristanoyl-CoA levels are increased in liver, and the ω-oxidized metabolites are excreted in urine. In conclusion, HACL1 deficiency is not associated with a severe phenotype, but in combination with phytanic acid intake, the normal situation in man, it might present with phytanic acid elevation and resemble a Refsum like disorder.  

3.2765           Roles of exosomes in the normal and diseased eye

Klingeborn, M., Dismuke, W.M., Bowes Rickman, C.and Stamer, W.D. Progress in Retinal and Eye Res., 59, 158-177 (2017)   Exosomes are nanometer-sized vesicles that are released by cells in a controlled fashion and mediate a plethora of extra- and intercellular activities. Some key functions of exosomes include cell-cell communication, immune modulation, extracellular matrix turnover, stem cell division/differentiation, neovascularization and cellular waste removal. While much is known about their role in cancer, exosome function in the many specialized tissues of the eye is just beginning to undergo rigorous study. Here we review current knowledge of exosome function in the visual system in the context of larger bodies of data from other fields, in both health and disease. Additionally, we discuss recent advances in the exosome field including use of exosomes as a therapeutic vehicle, exosomes as a source of biomarkers for disease, plus current standards for isolation and validation of exosome populations. Finally, we use this foundational information about exosomes in the eye as a platform to identify areas of opportunity for future research studies.  

3.2766           Extracellular vesicles

Simpson, R.J. Seminars in Cell and Develop. Biol., 67, 1-2 (2017)   Over the past decade extracellular vesicles (EVs) have been the subject of intense interest by investigators from diverse fields of biology. This has been largely due to their role in intercellular communication and the presence of diverse cargo such as proteins (including oncoproteins, tumor suppressors, transcriptional regulators, splicing factors etc), RNAs (microRNA, mRNA, lncRNA etc), DNA, and lipids [1]. It is now recognized that the function of EVs is dependent on the cargo they carry, which upon uptake by recipient cells can induce profound phenotypic changes. This horizontal transfer of bioactive molecules plays a vital role in tumor invasion and metastasis, immune modulation within the tumor microenvironment (TME), inflammation, epithelial-mesenchymal transition, neurobiology, pathogen dissemination – to name a few. It is now widely recognized that there are two major classes of EVs – shed microvesicles (sMVs), that are also referred to as microparticles, and exosomes [2]. sMVs are formed by the outward budding and abscission of the plasma membrane, whereas exosomes originate from preformed multivesicular bodies that traffic to the plasma membrane whereupon fusion with the plasma membrane they release their contents (exosomes) into the extracellular environment [1]. sMVs and exosomes exhibit different protein and RNA profiles and biophysical properties. While sMVs are heterogeneous in size (100 nm to > 1300 nm), exosomes are essentially homogeneous with a mean diameter ∼36 nm [3]; the buoyant densities of sMVs (1.1–1.2 g/mL) and exosomes (1.1 g/mL), assessed by iodixanol density gradient centrifugation, tend to overlap. However, an interesting development in the EV field over the past 5 years has been the recognition that exosomes and sMVs can be further fractionated into discrete sub-populations, based upon cell polarity [4,5], buoyant density [6,7] and mechanism of biogenesis [8], that display different bioactive molecule compositions. At this juncture it has been difficult to discern functional differences between EV subtypes.  

3.2767           Leishmania donovani restricts mitochondrial dynamics to enhance miRNP stability and target RNA repression in host macrophages

Chakrabarty, Y. and Bhattacharyya, S.N. Mol. Biol. Cell, 28, 2091-2105 (2017)   MicroRNAs (miRNAs), the tiny regulatory RNAs, form complexes with Argonaute (Ago) proteins and inhibit gene expression in metazoan cells. While studying parasite-invaded macrophages, we identify a unique mode of gene regulation in which the parasite Leishmania donovani (Ld) causes mitochondrial depolarization, reduces mitochondrial dynamics, and restricts turnover of cellular microRNA ribonucleoprotein (miRNP) complexes in infected host cells. This leads to increased stability of miRNPs along with elevated levels of Ago2-bound cytokine mRNA in Ld-infected macrophages. Thus the increase of miRNP stability in Ld-infected cells curtails production of proinflammatory cytokines, which are otherwise detrimental for survival of the parasite within the infected macrophages. Loss of mitochondrial membrane potential is accompanied by reduced juxtaposition of endoplasmic reticulum (ER) and mitochondria as well as endosomes. This is likely coupled with enhanced sequestration and stabilization of ER- associated miRNPs observed in infected macrophage cells. Mitofusin 2 (Mfn2), a membrane protein implicated in ER–mitochondria tethering, also shows reduced expression in Ld-infected cells. A mitochondrial role in Ld-induced alteration of miRNA activity and stability is further corroborated by impaired compartmentalization and stabilization of miRNP components in Mfn2-depleted mammalian cells.  

3.2768           Drosophila TG-A transglutaminase is secreted via an unconventional Golgi-independent mechanism involving exosomes and two types of fatty acylations

Shibata, T., Hadano, J., Kawasaki, D., Dong, X. and Kawabata, S-i.

1. Biol. Chem., 292(25), 10723-10734 (2017)

  Transglutaminases (TGs) play essential intracellular and extracellular roles by covalently cross-linking many proteins. Drosophila TG is encoded by one gene and has two alternative splicing-derived isoforms, TG-A and TG-B, which contain distinct N-terminal 46- and 38-amino acid sequences, respectively. The TGs identified to date do not have a typical endoplasmic reticulum (ER)-signal peptide, and the molecular mechanisms of their secretion under physiologic conditions are unclear. Immunocytochemistry revealed that TG-A localizes to multivesicular-like structures, whereas TG-B localizes to the cytosol. We also found that TG-A, but not TG-B, was modified concomitantly by N-myristoylation and S-palmitoylation, and N-myristoylation was a pre-requisite for S-palmitoylation. Moreover, TG-A, but not TG-B, was secreted in response to calcium signaling induced by Ca²⁺ ionophores and uracil, a pathogenic bacteria-derived substance. Brefeldin A and monensin, inhibitors of the ER/Golgi-mediated conventional pathway, did not suppress TG-A secretion, whereas inhibition of S-palmitoylation by 2-bromopalmitate blocked TG-A secretion. Ultracentrifugation, electron microscopy analyses, and treatments with inhibitors of multivesicular body formation revealed that TG-A was secreted via exosomes together with co-transfected mammalian CD63, an exosomal marker, and the secreted TG-A was taken up by other cells. The 8-residue N-terminal fragment of TG-A containing the fatty acylation sites was both necessary and sufficient for the exosome-dependent secretion of TG-A. In conclusion, TG-A is secreted through an unconventional ER/Golgi-independent pathway involving two types of fatty acylations and exosomes.  

3.2769           Contribution of neuroblastoma-derived exosomes to the production of pro-tumorigenic signals by bone marrow mesenchymal stromal cells

Nakata, R., Shimada, H., Fernandez, G.E., Fanter, R., Fabbri, M., malvar, J., Zimmermann, P. and DeClerk, Y.A.

1. Extracellular Vesicles, 6:1, 1332941 (2017)

  The bone marrow (BM) niche is a microenvironment promoting survival, dormancy and therapeutic resistance in tumor cells. Central to this function are mesenchymal stromal cells (MSCs). Here, using neuroblastoma (NB) as a model, we demonstrate that NB cells release an extracellular vesicle (EVs) whose protein cargo is enriched in exosomal proteins but lacks cytokines and chemokines. Using three different purification methods, we then demonstrate that NB-derived exosomes were captured by MSCs and induced the production of pro-tumorigenic cytokines and chemokines, including interleukin-6 (IL-6), IL-8/CXCL8, vascular endothelial cell growth factor and monocyte-chemotactic protein-1, with exosomes prepared by size exclusion chromatography having the highest activity. We found no correlation between the IL-6 and IL-8/CXCL8 stimulatory activity of exosomes from eight NB cell lines and their origin, degree of MYCN amplification, drug resistance and disease status. We then demonstrate that the uptake of NB exosomes by MSCs was associated with a rapid increase in ERK1/2 and AKT activation, and that blocking ERK1/2 but not AKT activation inhibited the IL-6 and IL-8/CXCL8 production by MSCs without affecting exosome uptake. Thus, we describe a new mechanism by which NB cells induce in MSCs an inflammatory reaction that contributes to a favorable microenvironment in the BM.  

3.2770           GSK3β and ERK regulate the expression of 78 kDa SG2NA and ectopic modulation of its level affects phases of cell cycle

Pandey, S., Talukdar, I., Jain, B.P. and Goswami, S.K. Scientific Reports, 7, 7555 (2017)   Striatin and SG2NA are essential constituents of the multi-protein STRIPAK assembly harbouring protein phosphatase PP2A and several kinases. SG2NA has several isoforms generated by mRNA splicing and editing. While the expression of striatin is largely restricted to the striatum in brain, that of SG2NAs is ubiquitous. In NIH3T3 cells, only the 78 kDa isoform is expressed. When cells enter into the S phase, the level of SG2NA increases; reaches maximum at the G2/M phase and declines thereafter. Downregulation of SG2NA extends G1 phase and its overexpression extends G2. Ectopic expression of the 35 kDa has no effects on the cell cycle. Relative abundance of phospho-SG2NA is high in the microsome and cytosol and the nucleus but low in the mitochondria. Okadoic acid, an inhibitor of PP2A, increases the level of SG2NA which is further enhanced upon inhibition of proteasomal activity. Phospho-SG2NA is thus more stable than the dephosphorylated form. Inhibition of GSK3β by LiCl reduces its level, but the inhibition of ERK by PD98059 increases it. Thus, ERK decreases the level of phospho-SG2NA by inhibiting GSK3β. In cells depleted from SG2NA by shRNA, the levels of pGSK3β and pERK are reduced, suggesting that these kinases and SG2NA regulate each other’s expression.  

3.2771           Isolation of mouse chromaffin secretory vesicles and their division into 12 fractions

Pardo, M.R., Estevez-Herrera, J., Castaneyra, L., Borges, R. and Machado, J.D. Anal. Biochem., 536, 1-7 (2017)   The study of chromaffin secretory vesicles (SVs) has contributed immensely to our understanding of exocytosis. These organelles, also called chromaffin granules, are a specific type of large dense secretory vesicle found in many endocrine cells and neurons. Traditionally, they have been isolated from bovine adrenal glands due to the large number of SVs that can be obtained from this tissue. However, technical advances now make it possible to obtain very pure preparations of SVs from mice, which is particular interesting for functional studies given the availability of different genetically modified strains of mice. Despite the small size of the mouse adrenal medulla (400–500 μm and less than 2 mg in weight), we have successfully carried out functional studies on SVs isolated from WT and knockout mice. As such, we present here our method to purify crude vesicles and to fractionate mouse chromaffin SVs, along with examples of their functional characterization.  

3.2772           The iron chaperone poly(rC)-binding protein 2 forms a metabolon with the heme oxygenase 1/cytochrome P450 reductase complex for heme catabolism and iron transfer

Yanatori, I., Richaaardson, D.r., Toyokuni, S. and Kishi, F.

1. Biol. Chem., 292(32), 13205-13229 (2017)

  Mammals incorporate a major proportion of absorbed iron as heme, which is catabolized by the heme oxygenase 1 (HO1)–NADPH-cytochrome P450 reductase (CPR) complex into biliverdin, carbon monoxide, and ferrous iron. Moreover, intestinal iron is incorporated as ferrous iron, which is transported via the iron importer, divalent metal transporter 1 (DMT1). Recently, we demonstrated that the iron chaperone poly(rC)-binding protein 2 (PCBP2) can directly receive ferrous iron from DMT1 or transfer iron to the iron exporter, ferroportin 1. To promote intracellular iron flux, an iron chaperone may be essential for receiving iron generated by heme catabolism, but this hypothesis is untested so far. Herein, we demonstrate that HO1 binds to PCBP2, but not to other PCBP family members, namely PCBP1, PCBP3, or PCBP4. Interestingly, HO1 formed a complex with either CPR or PCBP2, and it was demonstrated that PCBP2 competes with CPR for HO1 binding. Using PCBP2-deletion mutants, we demonstrated that the PCBP2 K homology 3 domain is important for the HO1/PCBP2 interaction. In heme-loaded cells, heme prompted HO1–CPR complex formation and decreased the HO1/PCBP2 interaction. Furthermore, in vitro reconstitution experiments with purified recombinant proteins indicated that HO1 could bind to PCBP2 in the presence of heme, whereas loading of PCBP2 with ferrous iron caused PCBP2 to lose its affinity for HO1. These results indicate that ferrous iron released from heme can be bound by PCBP2 and suggest a model for an integrated heme catabolism and iron transport metabolon.  

3.2773           Serum-derived extracellular vesicles (EVs) impact on vascular remodeling and prevent muscle damage in acute hind limb ischemia

Cavallari, C., Ranghino, A., Tapparo, M., Cedrino, M., Figliolini, F., Grange, C., Giannachi, V., Garneri, P., Deregibus, M.C., Collino, F., Rispoli, P., Camussi, G. and Brizzi, M.F. Scientific Reports, 7:8180 (2017)   Serum is an abundant and accessible source of circulating extracellular vesicles (EVs). Serum-EV (sEV) pro-angiogenic capability and mechanisms are herein analyzed using an in vitro assay which predicts sEV angiogenic potential in vivo. Effective sEVs (e-sEVs) also improved vascular remodeling and prevented muscle damage in a mouse model of acute hind limb ischemia. e-sEV angiogenic proteomic and transcriptomic analyses show a positive correlation with matrix-metalloproteinase activation and extracellular matrix organization, cytokine and chemokine signaling pathways, Insulin-like Growth Factor and platelet pathways, and Vascular Endothelial Growth Factor signaling. A discrete gene signature, which highlights differences in e-sEV and ineffective-EV biological activity, was identified using gene ontology (GO) functional analysis. An enrichment of genes associated with the Transforming Growth Factor beta 1 (TGFβ1) signaling cascade is associated with e-sEV administration but not with ineffective-EVs. Chromatin immunoprecipitation analysis on the inhibitor of DNA binding I (ID1) promoter region, and the knock-down of small mother against decapentaplegic (SMAD)1–5 proteins confirmed GO functional analyses. This study demonstrates sEV pro-angiogenic activity, validates a simple, sEV pro-angiogenic assay which predicts their biological activity in vivo, and identifies the TGFβ1 cascade as a relevant mediator. We propose serum as a readily available source of EVs for therapeutic purposes.  

3.2774           Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA

Nemeth, A. et al Scientific Reports, 7:8202 (2017)   Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes.  

3.2775           Generation and intracellular trafficking of a polysialic acid-carrying fragment of the neural cell adhesion molecule NCAM to the cell nucleus

Westphal, N., Loers, G., Lutz, D., Theis, T., Kleene, R. and Schachner, M. Scientific Reports, 7:8622 (2017)   Polysialic acid (PSA) and its major protein carrier, the neural cell adhesion molecule NCAM, play important roles in many nervous system functions during development and in adulthood. Here, we show that a PSA-carrying NCAM fragment is generated at the plasma membrane by matrix metalloproteases and transferred to the cell nucleus via endosomes and the cytoplasm. Generation and nuclear import of this fragment in cultured cerebellar neurons is induced by a function-triggering NCAM antibody and a peptide comprising the effector domain (ED) of myristoylated alanine-rich C kinase substrate (MARCKS) which interacts with PSA within the plane of the plasma membrane. These treatments lead to activation of the fibroblast growth factor (FGF) receptor, phospholipase C (PLC), protein kinase C (PKC) and phosphoinositide-3-kinase (PI3K), and subsequently to phosphorylation of MARCKS. Moreover, the NCAM antibody triggers calmodulin-dependent activation of nitric oxide synthase, nitric oxide (NO) production, NO-dependent S-nitrosylation of matrix metalloprotease 9 (MMP9) as well as activation of matrix metalloprotease 2 (MMP2) and MMP9, whereas the ED peptide activates phospholipase D (PLD) and MMP2, but not MMP9. These results indicate that the nuclear PSA-carrying NCAM fragment is generated by distinct and functionally defined signal transducing mechanisms.  

3.2776           Regulated Polarization of Tumor-Associated Macrophages by miR-145 via Colorectal Cancer–Derived Extracellular Vesicles

Shinohara, H., Kuranaga, Y., Kumazaki, M., Sugito, N., Yoshikawa, Y., Takai, T., Taniguchi, K., Ito, Y. and Akao, Y.

1. Immunol., 199(4), 1505-1515 (2017)

  Macrophages are polarized into functional classically activated and alternatively activated (M2) phenotypes depending on their microenvironment, and these cells play an important role in the immune system. M2-like polarization of tumor-associated macrophages (TAMs) is activated by various secretions from cancer cells; however, the interaction between cancer cells and TAMs is not well understood. Recent studies showed that cancer cell–derived extracellular vesicles (EVs) contribute to tumor development and modulation of the tumor microenvironment. In the current study, we investigated colorectal cancer–derived EVs containing miR-145 with respect to the polarization of TAMs. Colorectal cancer cells positively secreted miR-145 via EVs, which were taken up by macrophage-like cells. Interestingly, colorectal cancer–derived EVs polarized macrophage-like cells into the M2-like phenotype through the downregulation of histone deacetylase 11. An in vivo study showed that EV-treated macrophages caused significant enlargement of the tumor volumes. These findings suggest that colorectal cancer cells use miR-145 within EVs to efficiently modulate M2-like macrophage polarization and tumor progression.  

3.2777           Surface LAMP-2 Is an Endocytic Receptor That Diverts Antigen Internalized by Human Dendritic Cells into Highly Immunogenic Exosomes

Leone, D.A., Peschel, A., Brown, M., Schachner, H., Ball, M.J., Gyuraszova, M., Salzer-Muhar, U.

1. Immunol., 199(2), 531-546 (2017)

  The lysosome-associated membrane protein (LAMP) family includes the dendritic cell endocytic receptors DC-LAMP and CD68, as well as LAMP-1 and LAMP-2. In this study we identify LAMP-1 (CD107a) and LAMP-2 (CD107b) on the surface of human monocyte-derived dendritic cells (MoDC) and show only LAMP-2 is internalized after ligation by specific Abs, including H4B4, and traffics rapidly but transiently to the MHC class II loading compartment, as does Ag conjugated to H4B4. However, pulsing MoDC with conjugates of primary (keyhole limpet hemocyanin; KLH) and recall (Bet v 1) Ags (H4B4*KLH and H4B4*Bet v 1) induced significantly less CD4 cell proliferation than pulsing with native Ag or Ag conjugated to control mAb (ISO*KLH and ISO*Bet v 1). In H4B4*KLH-pulsed MoDC, the duration of KLH residence in MHC class II loading compartments was significantly reduced, as were surface HLA-DR and DR-bound KLH-derived peptides. Paradoxically, MoDC pulsed with H4B4*KLH, but not the other KLH preparations, induced robust proliferation of CD4 cells separated from them by a transwell membrane, indicating factors in the supernatant were responsible. Furthermore, extracellular vesicles from supernatants of H4B4*KLH-pulsed MoDC contained significantly more HLA-DR and KLH than those purified from control MoDC, and KLH was concentrated specifically in exosomes that were a uniquely effective source of Ag in standard T cell proliferation assays. In summary, we identify LAMP-2 as an endocytic receptor on human MoDC that routes cargo into unusual Ag processing pathways, which reduces surface expression of Ag-derived peptides while selectively enriching Ag within immunogenic exosomes. This novel pathway has implications for the initiation of immune responses both locally and at distant sites.  

3.2778           Caspase-8 controls the secretion of inflammatory lysyl-tRNA synthetase in exosomes from cancer cells

Kim, S.B. et al

1. Cell Biol.,216(7), 2201-2216 (2017)

  Aminoacyl-tRNA synthetases (ARSs), enzymes that normally control protein synthesis, can be secreted and have different activities in the extracellular space, but the mechanism of their secretion is not understood. This study describes the secretion route of the ARS lysyl-tRNA synthetase (KRS) and how this process is regulated by caspase activity, which has been implicated in the unconventional secretion of other proteins. We show that KRS is secreted from colorectal carcinoma cells within the lumen of exosomes that can trigger an inflammatory response. Caspase-8 cleaved the N-terminal of KRS, thus exposing a PDZ-binding motif located in the C terminus of KRS. Syntenin bound to the exposed PDZ-binding motif of KRS and facilitated the exosomic secretion of KRS dissociated from the multi-tRNA synthetase complex. KRS-containing exosomes released by cancer cells induced macrophage migration, and their secretion of TNF-α and cleaved KRS made a significant contribution to these activities, which suggests a novel mechanism by which caspase-8 may promote inflammation.  

3.2779           Acinetobacter baumannii transfers the blaNDM-1 gene via outer membrane vesicles

Chatterjee, S., Mondal, A., Mitra, S. and Basu, S.

1. Antimicrob. Chemother., 72(8), 2201-2207 (2017)

  Objectives: To investigate the transmission of the gene encoding New Delhi metallo-β-lactamase-1 (bla_NDM-1) through outer membrane vesicles (OMVs) released from an Acinetobacter baumannii strain (A_115). Methods: Isolation and purification of OMVs by density gradient from a carbapenem-resistant clinical strain of A. baumannii harbouring plasmid-mediated bla_NDM-1 and aac(6′)-Ib-cr genes was performed. DNA was purified from the OMVs and used for PCR and dot-blot analysis. Vesicles treated with DNase I and proteinase K were used to transform A. baumannii ATCC 19606 and Escherichia coli JM109 strains. MIC values for the transformants were determined, followed by PCR and restriction digestion of plasmids. PFGE was done for A_115 and transformants of ATCC 19606 and JM109. Results: The A. baumannii strain (ST 1462) released vesicles (25–100 nm) during in vitro growth at late log phase. PCR and dot-blot analysis confirmed the presence of bla_NDM-1 and aac(6′)-Ib-cr genes in intravesicular DNA. bla_NDM-1 and aac(6′)-Ib-cr genes were transferred to both the A. baumannii ATCC 19606 and E. coli JM109 recipient cells. The transformation frequency of the purified OMVs was in the range of 10⁻⁵–10⁻⁶ and gradually reduced with storage of OMVs. The sizes of the plasmids in the transformants and their restriction digestion patterns were identical to the plasmid in A_115. The transformants showed elevated MIC values of the β-lactam group of antibiotics, which confirmed the presence of a bla_NDM-1-harbouring plasmid. Conclusions: This is the first experimental evidence of intra- and inter-species transfer of a plasmid harbouring a bla_NDM-1 gene in A. baumannii via OMVs with high transformation frequency.  

3.2780           Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments

Sezgin, E., Azbazdar, Y., Ng, X.W., The, C., Simons, K., Weidinger, G., Wohland, T., Eggeling, C. and Ozhan, G. FEBS J., 284(15), 2513-2526 (2017)   While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt–receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/β-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann–Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity.  

3.2781           S100-A9 protein in exosomes from chronic lymphocytic leukemia cells promotes NF-κB activity during disease progression

Prieto, D., Sotelo, N., Seija, N., Sernbo, S., Abreu, C., Duran, R., Gil, M., Sicco, E., irigoin, V., Oliver, C., Landoni, A.I., gabus, R., Dighiero, G. and Opezzo, P. Blood, 130(6), 777-788 (2017)   Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clonal B lymphocytes, resulting from a complex balance between cell proliferation and apoptotic death. Continuous crosstalk between cancer cells and local/distant host environment is required for effective tumor growth. Among the main actors of this dynamic interplay between tumoral cells and their microenvironment are the nano-sized vesicles called exosomes. Emerging evidence indicates that secretion, composition, and functional capacity of exosomes are altered as tumors progress to an aggressive phenotype. In CLL, no data exist exploring the specific changes in the proteomic profile of plasma-derived exosomes from patients during disease evolution. We hereby report for the first time different proteomic profiles of plasma exosomes, both between indolent and progressive CLLs as well as within the individual patients at the onset of disease and during its progression. Next, we focus on the changes of the exosome protein cargoes, which are found exclusively in patients with progressive CLL after disease progression. The alterations in the proteomic cargoes underline different networks specific for leukemia progression related to inflammation, oxidative stress, and NF-κB and phosphatidylinositol 3-kinase/AKT pathway activation. Finally, our results suggest a preponderant role for the protein S100-A9 as an activator of the NFκB pathway during CLL progression and suggest that the leukemic clone can generate an autoactivation loop through S100-A9 expression, NF-κB activation, and exosome secretion. Collectively, our data propose a new pathway for NF-κB activation in CLL and highlight the importance of exosomes as extracellular mediators promoting tumor progression in CLL.  

3.2782           A complex of Neuroplastin and Plasma Membrane Ca2+ ATPase controls T cell activation

Korthals, M. et al Scientific Reports, 7:8358 (2017)   The outcome of T cell activation is determined by mechanisms that balance Ca²⁺ influx and clearance. Here we report that murine CD4 T cells lacking Neuroplastin (Nptn^−/−), an immunoglobulin superfamily protein, display elevated cytosolic Ca²⁺ and impaired post-stimulation Ca²⁺ clearance, along with increased nuclear levels of NFAT transcription factor and enhanced T cell receptor-induced cytokine production. On the molecular level, we identified plasma membrane Ca²⁺ ATPases (PMCAs) as the main interaction partners of Neuroplastin. PMCA levels were reduced by over 70% in Nptn^−/− T cells, suggesting an explanation for altered Ca²⁺ handling. Supporting this, Ca²⁺ extrusion was impaired while Ca²⁺ levels in internal stores were increased. T cells heterozygous for PMCA1 mimicked the phenotype of Nptn^−/− T cells. Consistent with sustained Ca²⁺ levels, differentiation of Nptn^−/− T helper cells was biased towards the Th1 versus Th2 subset. Our study thus establishes Neuroplastin-PMCA modules as important regulators of T cell activation.  

3.2783           Degradation of cofilin is regulated by Cbl, AIP4 and Syk resulting in increased migration of LMP2A positive nasopharyngeal carcinoma cells

Gainullin, M.R., Zhukov, I.Y., Zhou, X., Mo, Y., Astakhova, L., Ernberg, I. and Matskova, L. Scientific Reports, 7:9012 (2017)   Expression of cofilin is directly associated with metastatic activity in many tumors. Here, we studied the role of Latent Membrane Protein 2 A (LMP2A) of Epstein-Barr Virus (EBV) in the accumulation of cofilin observed in nasopharyngeal cancer (NPC) tumor cells. We used LMP2A transformed NPC cell lines to analyze cofilin expression. We used mutation analysis, ectopic expression and down-regulation of Cbl, AIP4 and Syk in these cell lines to determine the effect of the LMP2A viral protein on cofilin degradation and its role in the assembly of a cofilin degrading protein complex. The LMP2A of EBV was found to interfer with cofilin degradation in NPC cells by accelerating the proteasomal degradation of Cbl and Syk. In line with this, we found significantly higher cofilin expression in NPC tumor samples as compared to the surrounding epithelial tissues. Cofilin, as an actin severing protein, influences cellular plasticity, and facilitates cellular movement in response to oncogenic stimuli. Thus, under relaxed cellular control, cofilin facilitates tumor cell movement and dissemination. Interference with its degradation may enhance the metastatic potential of NPC cells.  

3.2784           Sterol targeting drugs reveal life cycle stage-specific differences in trypanosome lipid rafts

Sharma, A.I., Olson, C.L., Marmede, J.I., Gazos-Lopes, F., Epting, C.L., Almeida, I.C. and Engman, D.M. Scientific Reports, 7:9105 (2017)   Cilia play important roles in cell signaling, facilitated by the unique lipid environment of a ciliary membrane containing high concentrations of sterol-rich lipid rafts. The African trypanosome Trypanosoma brucei is a single-celled eukaryote with a single cilium/flagellum. We tested whether flagellar sterol enrichment results from selective flagellar partitioning of specific sterol species or from general enrichment of all sterols. While all sterols are enriched in the flagellum, cholesterol is especially enriched. T. brucei cycles between its mammalian host (bloodstream cell), in which it scavenges cholesterol, and its tsetse fly host (procyclic cell), in which it both scavenges cholesterol and synthesizes ergosterol. We wondered whether the insect and mammalian life cycle stages possess chemically different lipid rafts due to different sterol utilization. Treatment of bloodstream parasites with cholesterol-specific methyl-β-cyclodextrin disrupts both membrane liquid order and localization of a raft-associated ciliary membrane calcium sensor. Treatment with ergosterol-specific amphotericin B does not. The opposite results were observed with ergosterol-rich procyclic cells. Further, these agents have opposite effects on flagellar sterol enrichment and cell metabolism in the two life cycle stages. These findings illuminate differences in the lipid rafts of an organism employing life cycle-specific sterols and have implications for treatment.  

3.2785           Phosphatidylserine externalization, “necroptotic bodies” release, and phagocytosis during necroptosis

Zargarian, S., Shlomovitz, I., Erlich, Z., Hourizadeh, A., Ofir-Birin, Y.O., Croker, B.A., Regev-Rudzki, N., Edry-Botzer, L. and Gerlic, M. PloS Biology, 15(6), e2002711 (2017)   Necroptosis is a regulated, nonapoptotic form of cell death initiated by receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL) proteins. It is considered to be a form of regulated necrosis, and, by lacking the “find me” and “eat me” signals that are a feature of apoptosis, necroptosis is considered to be inflammatory. One such “eat me” signal observed during apoptosis is the exposure of phosphatidylserine (PS) on the outer plasma membrane. Here, we demonstrate that necroptotic cells also expose PS after phosphorylated mixed lineage kinase-like (pMLKL) translocation to the membrane. Necroptotic cells that expose PS release extracellular vesicles containing proteins and pMLKL to their surroundings. Furthermore, inhibition of pMLKL after PS exposure can reverse the process of necroptosis and restore cell viability. Finally, externalization of PS by necroptotic cells drives recognition and phagocytosis, and this may limit the inflammatory response to this nonapoptotic form of cell death. The exposure of PS to the outer membrane and to extracellular vesicles is therefore a feature of necroptotic cell death and may serve to provide an immunologically-silent window by generating specific “find me” and “eat me” signals.  

3.2786           Mitochondrial metabolic regulation by GRP78

Prasad, M., Pawlak, K.J., Burak, W.E., Perry, E.E., Marshall, B., Whittal, R.M. and Bose, H.S. Sci. Adv., 3, e1602038 (2017)   Steroids, essential for mammalian survival, are initiated by cholesterol transport by steroidogenic acute regulatory protein (StAR). Appropriate protein folding is an essential requirement of activity. Endoplasmic reticulum (ER) chaperones assist in folding of cytoplasmic proteins, whereas mitochondrial chaperones fold only mitochondrial proteins. We show that glucose regulatory protein 78 (GRP78), a master ER chaperone, is also present at the mitochondria-associated ER membrane (MAM), where it folds StAR for delivery to the outer mitochondrial membrane. StAR expression and activity are drastically reduced following GRP78 knockdown. StAR folding starts at the MAM region; thus, its cholesterol fostering capacity is regulated by GRP78 long before StAR reaches the mitochondria. In summary, GRP78 is an acute regulator of steroidogenesis at the MAM, regulating the intermediate folding of StAR that is crucial for its activity.  

3.2787           Reconstitution of calcium-mediated exocytosis of dense-core vesicles

Kreutzberger, A.J.B., Kiessling, V., Linag, B., Seelheim, P., Jakhanwal, S., Jahn, R., Castle, J.D. and Tamm, L.K. Sci.Adv., 3, e1603208 (2017)   Regulated exocytosis is a process by which neurotransmitters, hormones, and secretory proteins are released from the cell in response to elevated levels of calcium. In cells, secretory vesicles are targeted to the plasma membrane, where they dock, undergo priming, and then fuse with the plasma membrane in response to calcium. The specific roles of essential proteins and how calcium regulates progression through these sequential steps are currently incompletely resolved. We have used purified neuroendocrine dense-core vesicles and artificial membranes to reconstruct in vitro the serial events that mimic SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor)–dependent membrane docking and fusion during exocytosis. Calcium recruits these vesicles to the target membrane aided by the protein CAPS (calcium-dependent activator protein for secretion), whereas synaptotagmin catalyzes calcium-dependent fusion; both processes are dependent on phosphatidylinositol 4,5-bisphosphate. The soluble proteins Munc18 and complexin-1 are necessary to arrest vesicles in a docked state in the absence of calcium, whereas CAPS and/or Munc13 are involved in priming the system for an efficient fusion reaction.  

3.2788           MYC Mediates Large Oncosome-Induced Fibroblast Reprogramming in Prostate Cancer

Minciacchi, V.R: et al Cancer Res., 77(9), 2306-2317 (2017)   Communication between cancer cells and the tumor microenvironment results in the modulation of complex signaling networks that facilitate tumor progression. Here, we describe a new mechanism of intercellular communication originating from large oncosomes (LO), which are cancer cell–derived, atypically large (1–10 μm) extracellular vesicles (EV). We demonstrate that, in the context of prostate cancer, LO harbor sustained AKT1 kinase activity, nominating them as active signaling platforms. Active AKT1 was detected in circulating EV from the plasma of metastatic prostate cancer patients and was LO specific. LO internalization induced reprogramming of human normal prostate fibroblasts as reflected by high levels of α-SMA, IL6, and MMP9. In turn, LO-reprogrammed normal prostate fibroblasts stimulated endothelial tube formation in vitro and promoted tumor growth in mice. Activation of stromal MYC was critical for this reprogramming and for the sustained cellular responses elicited by LO, both in vitro and in vivo in an AKT1-dependent manner. Inhibition of LO internalization prevented activation of MYC and impaired the tumor-supporting properties of fibroblasts. Overall, our data show that prostate cancer–derived LO powerfully promote establishment of a tumor-supportive environment by inducing a novel reprogramming of the stroma. This mechanism offers potential alternative options for patient treatment.  

3.2789           Sigma1 Targeting to Suppress Aberrant Androgen Receptor Signaling in Prostate Cancer

Thomas, J.D., Longen, C.G., Oyer, H.M., Chen, N., Maher, C.M., Salvino, J.M., Kania, B., Anderson, K.N., Ostrander, W.F., Knudsen, K.E. and Kim, F.J. Cancer Res.,77(9), 2439-2452 (2017)   Suppression of androgen receptor (AR) activity in prostate cancer by androgen depletion or direct AR antagonist treatment, although initially effective, leads to incurable castration-resistant prostate cancer (CRPC) via compensatory mechanisms including resurgence of AR and AR splice variant (ARV) signaling. Emerging evidence suggests that Sigma1 (also known as sigma-1 receptor) is a unique chaperone or scaffolding protein that contributes to cellular protein homeostasis. We reported previously that some Sigma1-selective small molecules can be used to pharmacologically modulate protein homeostasis pathways. We hypothesized that these Sigma1-mediated responses could be exploited to suppress AR protein levels and activity. Here we demonstrate that treatment with a small-molecule Sigma1 inhibitor prevented 5α- dihydrotestosterone-mediated nuclear translocation of AR and induced proteasomal degradation of AR and ARV, suppressing the transcriptional activity and protein levels of both full-length and splice-variant AR. Consistent with these data, RNAi knockdown of Sigma1 resulted in decreased AR levels and transcriptional activity. Furthermore, Sigma1 physically associated with ARV7 and AR^v567es as well as full-length AR. Treatment of mice xenografted with ARV-driven CRPC tumors with a drug-like small-molecule Sigma1 inhibitor significantly inhibited tumor growth associated with elimination of AR and ARV7 in responsive tumors. Together, our data show that Sigma1 modulators can be used to suppress AR/ARV–driven prostate cancer cells via regulation of pharmacologically responsive Sigma1-AR/ARV interactions, both in vitro and in vivo.  

3.2790           Differential Protein Expression Marks the Transition From Infection With Opisthorchis viverrini to Cholangiocarcinoma

Khoontawad, J., Pairojkul, C., Rucksaken, R., Pinlaor, P., WOngkham, C., Yongvanit, P., Pugkhem, A., Jones, A., Plieskatt, J., Potriquet, J., Bethony, J., Pinlaor, S. and Mulvenna, J. Mol. Cell. Proteomics, 16(5), 911-923 (2017)   Parts of Southeast Asia have the highest incidence of intrahepatic cholangiocarcinoma (CCA) in the world because of infection by the liver fluke Opisthorchis viverrini (Ov). Ov-associated CCA is the culmination of chronic Ov-infection, with the persistent production of the growth factors and cytokines associated with persistent inflammation, which can endure for years in Ov-infected individuals prior to transitioning to CCA. Isobaric labeling and tandem mass spectrometry of liver tissue from a hamster model of CCA was used to compare protein expression profiles from inflammed tissue (Ovinfected but not cancerous) versus cancerous tissue (Ov-induced CCA). Immunohistochemistry and immunoblotting were used to verify dysregulated proteins in the animal model and in human tissue. We identified 154 dysregulated proteins that marked the transition from Ov-infection to Ov-induced CCA, i.e. proteins dysregulated during carcinogenesis but not Ov-infection. The verification of dysregulated proteins in resected liver tissue from humans with Ov-associated CCA showed the numerous parallels in protein dysregulation between human and animal models of Ov-induced CCA. To identify potential circulating markers for CCA, dysregulated proteins were compared with proteins isolated from exosomes secreted by a human CCA cell line (KKU055) and 27 proteins were identified as dysregulated in CCA and present in exosomes. These data form the basis of potential diagnostic biomarkers for human Ov-associated CCA. The profile of protein dysregulation observed during chronic Ovinfection and then in Ov-induced CCA provides insight into the etiology of an infection-induced inflammation-related cancer.  

3.2791           Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus

Gracida-Jimenez, V., Mondragon-Gonzalez, R., Velez-Aguilera, G., Vasquez, A., Laredo-Cisneros, M.S., de Dios Gomez-Lopez, J., Vaca, L., Gourlay, S.C., Jacobs, L.A., Winder, S.J. and Cisneros, B. Scientific Reports, 7:9906 (2017)   β-Dystroglycan (β-DG) is a transmembrane protein with critical roles in cell adhesion, cytoskeleton remodeling and nuclear architecture. This functional diversity is attributed to the ability of β-DG to target to, and conform specific protein assemblies at the plasma membrane (PM) and nuclear envelope (NE). Although a classical NLS and importin α/β mediated nuclear import pathway has already been described for β-DG, the intracellular trafficking route by which β-DG reaches the nucleus is unknown. In this study, we demonstrated that β-DG undergoes retrograde intracellular trafficking from the PM to the nucleus via the endosome-ER network. Furthermore, we provided evidence indicating that the translocon complex Sec61 mediates the release of β-DG from the ER membrane, making it accessible for importins and nuclear import. Finally, we show that phosphorylation of β-DG at Tyr890 is a key stimulus for β-DG nuclear translocation. Collectively our data describe the retrograde intracellular trafficking route that β-DG follows from PM to the nucleus. This dual role for a cell adhesion receptor permits the cell to functionally connect the PM with the nucleus and represents to our knowledge the first example of a cell adhesion receptor exhibiting retrograde nuclear trafficking and having dual roles in PM and NE.  

3.2792           New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes

Fernando, M.R., Jiang, C., Krzyanowski, G.D. and Ryan, W.L. PloS One, 12(8), e0183915 (2017)   Cell-free DNA (cfDNA) in blood is used as a source of genetic material for noninvasive prenatal and cancer diagnostic assays in clinical practice. Recently we have started a project for new biomarker discovery with a view to developing new noninvasive diagnostic assays. While reviewing literature, it was found that exosomes may be a rich source of biomarkers, because exosomes play an important role in human health and disease. While characterizing exosomes found in human blood plasma, we observed the presence of cfDNA in plasma exosomes. Plasma was obtained from blood drawn into K₃EDTA tubes. Exosomes were isolated from cell-free plasma using a commercially available kit. Sizing and enumeration of exosomes were done using electron microscopy and NanoSight particle counter. NanoSight and confocal microscopy was used to demonstrate the association between dsDNA and exosomes. DNA extracted from plasma and exosomes was measured by a fluorometric method and a droplet digital PCR (ddPCR) method. Size of extracellular vesicles isolated from plasma was heterogeneous and showed a mean value of 92.6 nm and a mode 39.7 nm. A large proportion of extracellular vesicles isolated from plasma were identified as exosomes using a fluorescence probe specific for exosomes and three protein markers, Hsp70, CD9 and CD63, that are commonly used to identify exosome fraction. Fluorescence dye that stain dsDNA showed the association between exosomes and dsDNA. Plasma cfDNA concentration analysis showed more than 93% of amplifiable cfDNA in plasma is located in plasma exosomes. Storage of a blood sample showed significant increases in exosome count and exosome DNA concentration. This study provide evidence that a large proportion of plasma cfDNA is localized in exosomes. Exosome release from cells is a metabolic energy dependent process, thus suggesting active release of cfDNA from cells as a source of cfDNA in plasma.  

3.2793           Tylophorine Analogs Allosterically Regulates Heat Shock Cognate Protein 70 And Inhibits Hepatitis C Virus Replication

Wang, Y. et al Scientific Reports, 7:10037 (2017)   Tylophorine analogs have been shown to exhibit diverse activities against cancer, inflammation, arthritis, and lupus in vivo. In this study, we demonstrated that two tylophorine analogs, DCB-3503 and rac-cryptopleurine, exhibit potent inhibitory activity against hepatitis C virus (HCV) replication in genotype 1b Con 1 isolate. The inhibition of HCV replication is at least partially mediated through cellular heat shock cognate protein 70 (Hsc70). Hsc70 associates with the HCV replication complex by primarily binding to the poly U/UC motifs in HCV RNA. The interaction of DCB-3503 and rac-cryptopleurine with Hsc70 promotes the ATP hydrolysis activity of Hsc70 in the presence of the 3′ poly U/UC motif of HCV RNA. Regulating the ATPase activity of Hsc70 may be one of the mechanisms by which tylophorine analogs inhibit HCV replication. This study demonstrates the novel anti-HCV activity of tylophorine analogs. Our results also highlight the importance of Hsc70 in HCV replication.  

3.2794           Concise Review: Developing Best-Practice Models for the Therapeutic Use of Extracellular Vesicles

Reiner, A.T. et al Stem Cell Trans. Med., 6, 1730-1739 (2017)   Growing interest in extracellular vesicles (EVs, including exosomes and microvesicles) as therapeutic entities, particularly in stem cell-related approaches, has underlined the need for standardization and coordination of development efforts. Members of the International Society for Extracellular Vesicles and the Society for Clinical Research and Translation of Extracellular Vesicles Singapore convened a Workshop on this topic to discuss the opportunities and challenges associated with development of EV-based therapeutics at the preclinical and clinical levels. This review outlines topic-specific action items that, if addressed, will enhance the development of best-practice models for EV therapies.  

3.2795           The Prohormone VGF Regulates β Cell Function via Insulin Secretory Granule Biogenesis

Stephens, S.B., Edwards, R.J., Sadahiro, M., Lin, W-J., Jiang, C., Salton, S.R. and Newgard, C.B. Cell Reports, 20, 2480-2489 (2017)   The prohormone VGF is expressed in neuroendocrine and endocrine tissues and regulates nutrient and energy status both centrally and peripherally. We and others have shown that VGF-derived peptides have direct action on the islet β cell as secretagogues and cytoprotective agents; however, the endogenous function of VGF in the β cell has not been described. Here, we demonstrate that VGF regulates secretory granule formation. VGF loss-of-function studies in both isolated islets and conditional knockout mice reveal a profound decrease in stimulus-coupled insulin secretion. Moreover, VGF is necessary to facilitate efficient exit of granule cargo from the trans-Golgi network and proinsulin processing. It also functions to replenish insulin granule stores following nutrient stimulation. Our data support a model in which VGF operates at a critical node of granule biogenesis in the islet β cell to coordinate insulin biosynthesis with β cell secretory capacity.  

3.2796           Prophage-triggered membrane vesicle formation through peptidoglycan damage in Bacillus subtilis

Toyofuku, M., Carcamo-Oyarce, G., Yamamoto, T., Eisenstein, F., Hsiao, C-C., Kurosawa, M., Gademann, K., Pilhofer, M., Nomura, N. and Eberl, L. Nature Communications, 8:481 (2017)   Bacteria release membrane vesicles (MVs) that play important roles in various biological processes. However, the mechanisms of MV formation in Gram-positive bacteria are unclear, as these cells possess a single cytoplasmic membrane that is surrounded by a thick cell wall. Here we use live cell imaging and electron cryo-tomography to describe a mechanism for MV formation in Bacillus subtilis. We show that the expression of a prophage-encoded endolysin in a sub-population of cells generates holes in the peptidoglycan cell wall. Through these openings, cytoplasmic membrane material protrudes into the extracellular space and is released as MVs. Due to the loss of membrane integrity, the induced cells eventually die. The vesicle-producing cells induce MV formation in neighboring cells by the enzymatic action of the released endolysin. Our results support the idea that endolysins may be important for MV formation in bacteria, and this mechanism may potentially be useful for the production of MVs for applications in biomedicine and nanotechnology.  

3.2797           Exosomes expressing carbonic anhydrase 9 promote angiogenesis

Horie, K., Kawakami, K., Fujita, Y., Sugaya, M., Kameyama, K., Mizutani, K., Deguchi, T. and Ito, M. Biochem. Biophys. Res. Comm., 492, 356-361 (2017)   Exosomes or microvesicles that are secreted from cells are considered to play important roles in tumor microenvironment. Carbonic anhydrase 9 (CA9), which is induced by hypoxia-inducible factor 1 (HIF1) in response to hypoxia, is overexpressed in many types of cancer including renal cell carcinoma (RCC). We examined the expression level of CA9 in several RCC cell lines and found that the basal level of CA9 was much higher in OSRC-2 cells than in Caki-1, KMRC-1 and 786-O cells. Consistent with the intracellular expression levels, CA9 was abundantly detected in exosomes isolated by ultracentrifugation from OSRC-2 cells. Density gradient centrifugation of OSRC-2 and 786-O exosomes confirmed the co-presence of CA9 with exosomal markers. Upon hypoxia and treatment with CoCl₂, a hypoxia mimic agent, the CA9 level in exosomes was increased for all cell lines. In order to examine the effects of CA9 exosomes on angiogenesis, we generated stably transfected HEK293 cells expressing CA9. Immunocytochemical staining demonstrated the uptake of CA9 exosomes by human umbilical vein endothelial cells (HUVEC). In vitro angiogenesis assays using HUVEC revealed that CA9 exosomes promoted migration and tube formation. Lastly, MMP2 expression was increased by treatment with CA9 exosomes in HUVEC. Taken together, our results suggest the possibility that CA9 exosomes released from hypoxic RCC may enhance angiogenesis in microenvironment, thereby contributing to cancer progression.  

3.2798           Excess Translation of Epigenetic Regulators Contributes to Fragile X Syndrome and Is Alleviated by Brd4 Inhibition

Korb, E., Herre, M., Zucker-Scharff, I., Gresack, J., Allis, C.D. and Darnell, R.B. Cell, 170, 1209-1223 (2017)   Fragile X syndrome (FXS) is a leading genetic cause of intellectual disability and autism. FXS results from the loss of function of fragile X mental retardation protein (FMRP), which represses translation of target transcripts. Most of the well-characterized target transcripts of FMRP are synaptic proteins, yet targeting these proteins has not provided effective treatments. We examined a group of FMRP targets that encode transcriptional regulators, particularly chromatin-associated proteins. Loss of FMRP in mice results in widespread changes in chromatin regulation and aberrant gene expression. To determine if targeting epigenetic factors could reverse phenotypes associated with the disorder, we focused on Brd4, a BET protein and chromatin reader targeted by FMRP. Inhibition of Brd4 function alleviated many of the phenotypes associated with FXS. We conclude that loss of FMRP results in significant epigenetic misregulation and that targeting transcription via epigenetic regulators like Brd4 may provide new treatments for FXS.  

3.2799           PI3K-C2α knockdown decreases autophagy and maturation of endocytic vesicles

Merill, N., Schipper, J.L., Karnes, J.B., Kauffman, A.L., Martin, K.R. and macKeigan, J.P. PloS One, 12(9), e0184909 (2017)   Phosphoinositide 3-kinase (PI3K) family members are involved in diverse cellular fates including cell growth, proliferation, and survival. While many molecular details are known about the Class I and III PI3Ks, less is known about the Class II PI3Ks. To explore the function of all eight PI3K isoforms in autophagy, we knock down each gene individually and measure autophagy. We find a significant decrease in autophagy following siRNA-mediated PIK3C2A (encoding the Class 2 PI3K, PI3K-C2α) knockdown. This defective autophagy is rescued by exogenous PI3K-C2α, but not kinase-dead PI3K-C2α. Using confocal microscopy, we probe for markers of endocytosis and autophagy, revealing that PI3K-C2α colocalizes with markers of endocytosis. Though endocytic uptake is intact, as demonstrated by transferrin labeling, PIK3C2A knockdown results in vesicle accumulation at the recycling endosome. We isolate distinct membrane sources and observe that PI3K-C2α interacts with markers of endocytosis and autophagy, notably ATG9. Knockdown of either PIK3C2A or ATG9A/B, but not PI3KC3, results in an accumulation of transferrin-positive clathrin coated vesicles and RAB11-positive vesicles at the recycling endosome. Taken together, these results support a role for PI3K-C2α in the proper maturation of endosomes, and suggest that PI3K-C2α may be a critical node connecting the endocytic and autophagic pathways.  

3.2800           Bacterial outer membrane vesicles suppress tumor by interferon-γ-mediated antitumor response

Kim, O.Y., Park, H.T., Dinh, N.T.H., Choi, S.J., Lee, J., Kim, J.H., Lee, S-W. and Gho, Y.S. Nature Communications, 8:626 (2017)   Gram-negative bacteria actively secrete outer membrane vesicles, spherical nano-meter-sized proteolipids enriched with outer membrane proteins, to the surroundings. Outer membrane vesicles have gained wide interests as non-living complex vaccines or delivery vehicles. However, no study has used outer membrane vesicles in treating cancer thus far. Here we investigate the potential of bacterial outer membrane vesicles as therapeutic agents to treat cancer via immunotherapy. Our results show remarkable capability of bacterial outer membrane vesicles to effectively induce long-term antitumor immune responses that can fully eradicate established tumors without notable adverse effects. Moreover, systematically administered bacterial outer membrane vesicles specifically target and accumulate in the tumor tissue, and subsequently induce the production of antitumor cytokines CXCL10 and interferon-γ. This antitumor effect is interferon-γ dependent, as interferon-γ-deficient mice could not induce such outer membrane vesicle-mediated immune response. Together, our results herein demonstrate the potential of bacterial outer membrane vesicles as effective immunotherapeutic agent that can treat various cancers without apparent adverse effects.  

3.2801           New views on phototransduction from atomic force microscopy and single molecule force spectroscopy on native rods

Maity, S., Ilieva, N., Laio, A., Torre, V. and Mazzolini, M. Scientific Report, 7:12000 (2017)   By combining atomic force microscopy (AFM) imaging and single-molecule force spectroscopy (SMFS), we analyzed membrane proteins of the rod outer segments (OS). With this combined approach we were able to study the membrane proteins in their natural environment. In the plasma membrane we identified native cyclic nucleotide-gated (CNG) channels which are organized in single file strings. We also identified rhodopsin located both in the discs and in the plasma membrane. SMFS reveals strikingly different mechanical properties of rhodopsin unfolding in the two environments. Molecular dynamic simulations suggest that this difference is likely to be related to the higher hydrophobicity of the plasma membrane, due to the higher cholesterol concentration. This increases rhodopsin mechanical stability lowering the rate of transition towards its active form, hindering, in this manner, phototransduction.  

3.2802           Confounding factors in extracellular vesicle ultrafiltration and protein analysis

Vergauwen, G., Dhondt, B., Van Deun, J., Timmerman, E., gaevert, K., Braems, G., Ven den Broecke, R., Cocquyt, V., Denys, H., De Wever, O. and Hendrix, A.

7. Extracellular Vesicles, 6, Suppl. 1, abstract OT7.02 (2017)

  Introduction: Identification and validation of extracellular vesicle (EV)-associated functions and biomarkers requires robust isolation and characterisation protocols. We assessed the impact of commonly implemented but modified analytical variables on EV analysis. Methods: We compared five different centrifugal filters that are often used to reduce large volume biofluids or concentrate EVs on three sample types: plasma, urine and EV-spiked PBS. Protein and nanoparticle tracking analysis was performed on the concentrate, membrane and flow through to determine EV recovery. Next, we compared three colorimetric and three fluorometric protein assay kits for their efficiency in measuring protein concentration of EV samples. In all protein assay kits the same sample volume of 5 μL EVs (1 × 1010 particles) was used. The presence and influence of OptiprepTM remnants in EV samples was assessed by DC protein assay kit-based interference of OptiprepTM at 750 nm and Q-Exactive protein analysis respectively. Results: Regenerated cellulose with 10k pore size generated highestparticle and protein recovery of EV-spiked PBS. Other centrifugal membranes did not efficiently recover EVs with 80% reduction in particle concentration and protein concentration measurements below detection threshold due to aspecific adherence of EVs to the centrifugal membranes. Similar findings were observed for plasma and urine, however the differences were less pronounced, probably due to abundant proteins masking centrifugal filter membranes. The Qubit® protein assay kit obtained a respectively 1.5-fold and 2-fold higher protein concentration measurement with the least variance as compared to microBCA and Bradford. The OptiprepTM concentration of EV samples obtained by pelleting density fractions was estimated 1.5–2.5%, whereas no OptiprepTM remnants were detected after EV retrieval from density fractions by size-exclusion chromatography. In addition, removal of OptiprepTM remnants from EV samples improved protein identification by 40-fold as measured by number of unique proteins identified. Conclusion: The choice of centrifugal filters and protein assay kits as well as residuals of EV isolation media can confound EV analysis and should be carefully considered when performing omics approaches and functional assays.  

3.2803           Exosomes from bovine milk reduce the tumour burden and attenuates cancer cachexia

Samuel, M., Jois, M. and Mathivanan, S.

9. Extracellular Vesicles, 6, Suppl. 1, abstract OT9.02 (2017)

  Introduction: Milk has long been associated with good health and is one of the most consumed beverages throughout the world. Exosomes are 30–150 nm membranous vesicles of endocytic origin that are released by all cell types and are also detected in bodily fluids including milk. Whether these milk-derived exosomes can serve as cross-species messengers and have a biological effect on host organism has been poorly understood. Here, we examined the stability of bovine milk exosomes in degrading conditions and studied their biodistribution using mouse models and IVIS imaging after oral administration. We also unravel the role of bovine milk derived exosomes in colon cancer progression. Methods: Milk exosomes were isolated using differential centrifugation and OptiPrepTM density gradient centrifugation. They were further characterised and examined for stability under harsh conditions using western blotting and nanoparticle tracking analysis. IVIS imaging system was used to study th biodistribution of these exosomes on oral gavaging. Mice models were used to understand the role of milk exosomes in cancer progression. Result: On examining the stability of bovine milk exosomes in harsh conditions, it was concluded that these exosomes are remarkably stable in both acidic and high temperature conditions while colorectal cancer cell-derived exosomes were not. Next, we studied the biodistribution of bovine milk exosomes which suggested that orally administered milk exosomes can survive the harsh intestinal environment and can be trafficked to various organs. Interestingly, after 24 h, the milk-derived exosomes reached multiple organs including liver and spleen in the mice. To understand the role of milk-exosomes in cancer progression, in vivo mouse models implanted with colorectal cancer were orally administered with milk-derived exosomes. Remarkably, exosomes isolated from both raw and commercial (grocery store) milk significantly reduced the tumour burden. Furthermore, orally administered milk exosomes prolonged the survival of the mice by inhibition of tumourinduced weight loss in cancer cachexia mice models. Summary: Thus this study provides new insights on the significance of milk exosomes in context of mammalian physiology as well as prompt their use as drug delivery vehicles in therapeutic interventions.  

3.2804           Hepatocyte-derived exosome enrichment and cell culture methods optimisation for the identification of novel DILI biomarkers

Thacker, S., Nautiyal, M., Holman, N., Otieno, M., Watkins, P. and Mosedale, M.

6. Extracellular Vesicles, 6, Suppl. 1, abstract PT06.06 (2017)

  Introduction:We have previously demonstrated that hepatotoxicants induce alterations in hepatocyte-derived exosomes (HDE) prior to overt necrosis, supporting a role for HDE in the pathogenesis of drug-induced liver injury (DILI). Because HDE contain liver-specific mRNAs, miRNAs, and proteins, they may have value as sensitive and specific biomarkers of DILI. In order to explore the DILI biomarker potential of HDE, the objectives of this study were to (1) identify the best method for enrichment and (2) optimise cell culture methods to compare the number and content of HDE released from primary human hepatocytes (PHH) in response to DILI compounds. Methods: To evaluate exosome enrichment, vesicles were isolated from the culture medium of HepG2 cells using ultracentrifugation (UC), OptiPrep density gradient ultracentrifugation (ODG), and ExoQuick-TC™ (EQ). To evaluate the effect of a Matrigel® overlay on exosome release, exosomes were enriched from the culture medium of HepaRG cells using UC. Nanoparticle tracking analysis was performed to assess vesicle number and size. Total RNA extracted from vesicles was used to determine the quantity (Quant-iT™ RiboGreen®) and fraction of miRNA that was vesicular vs. AGO2 bound (immunoprecipitation). Total protein was quantified and exosomal protein enrichment was evaluated via Western blotting. Results: EQ resulted in a significantly higher number of exosome-sized particles than UC (p < 0.001) or ODG (p < 0.0001). Particle size and variation using UC and EQ were similar (~100 ± 10 nm), however ODG enriched for particles significantly larger in size (p < 0.05). EQ and UC resulted in comparable levels of vesicular RNA and protein, however UC had significantly more vesicular RNA and CD63 protein when compared to EQ or ODG (p < 0.05). No significant differences in particle number were observed across Matrigel concentrations ranging from 0–0.25 mg/mL. Conclusion: These data suggest that both UC and EQ enrichment result in significantly more HDE than ODG, but UC produces a purer population of HDE. Matrigel overlay does not inhibit the release ofHDE.We conclude that UC-based enrichment provides the optimal combination of HDE quantity and purity and Matrigel overlay can be used in PHH culture for the identification of novel exosome-based biomarkers for DILI.  

3.2805           Proteomic analysis of mouse lung tissue-derived vesicles, a comparison of ultracentrifugation and density flotation isolation

Lässer, C., Suzuki, S., park, K-S., Shelke, G., Hovhannisyan, L., Crescitelli, R. and Lötvall, J.

7. Extracellular Vesicles, 6, Suppli. A, abstract PT07.08 (2017)

  Introduction: Analysis of the proteome of extracellular vesicles (EVs) is of great importance both to identify biomarkers of disease but also to understand cell-to-cell communication in diseased tissue. The aim of this study was to establish an isolation method that isolates lung vesicles of high purity for proteomic analysis. Methods: A mouse model for allergic asthma was used by sensitization and challenge of C57BL/6 mice to ovalbumin (OVA). Animals were sacrificed and lungs were removed and chopped in to smaller pieces that were incubated in medium for 30 minutes at 37°C and 5% CO2. Vesicles were isolated from medium either by a differential ultracentrifugation protocol (UCF) or by an Optiprep density gradient protocol (OD). Isolated vesicles were evaluated by electron microscopy (EM) and the proteome was analysed with mass spectrometry (LC-MS/MS). Results: EM showed that both protocols isolated vesicles that where on average 40–200 nm in size. LC-MS/MS identified 1223 and 1383 proteins in the UCF and OD vesicles, respectively. Out of these, 989 proteins were detected in both samples and 88 of the top 100 exosomal proteins from the database EVpedia was identified here. Using GO Term finder it was shown that the 989 common proteins were most significantly associated with the cellular component, “extracellular exosome”, “focal adhesion” and “membrane”. The 398 uniquely identified proteins in the OD vesicles were associated with “extracellular exosome” and “membrane”, while the 234 uniquely identified proteins in the UCF vesicles were associated with “proteasome complex” and “cytoplasm”. Conclusion: This study shows that EVs can be isolated directly from lung tissue, and these vesicles contain previously identified EV proteins. Both protocols can be used for the isolation of tissue-derived vesicles. However, flotation removes a number of contaminant proteins, including those related to the proteasome and furthermore it enriches for protein associated with membrane.  

3.2806           The impact of oncogenic EGFRvIII on the proteome of extracellular vesicles released from glioblastoma cells

Choi, D-S., Montermini, L. and Rak, J.

7. Extracellular Vesicles, 6, Suppl. 1, abstract PT07.10 (2017)

  Glioblastoma multiforme (GBM) is the most common, highly invasive, and aggressive astrocytic brain tumour associated with poor prognosis. EGFR is amplified in a subset of GBMs and influences the invasion and proliferation of tumour cells. EGFR amplification is also often accompanied by gene rearrangements leading to the expression of constitutively active oncogenic mutant, EGFR variant III (EGFRvIII). In addition to intrinsic transformation of GBM cells themselves, EGFRvIII may also act in a non-cell-autonomous manner by virtue of intercellular trafficking of this receptor between cellular populations as cargo of extracellular vesicles (EVs). Notably, EGFRvIII may also influence EV biogenesis and alters the expression of multiple genes, but links between these events are poorly understood. To better understand how EGFRvIII contributes to tumour aggressiveness mediated by EVs, we investigated the effect of this oncogene on the EV protein composition. Thus, we employed the quantitative proteomics to analyse EVs derived from indolent parental U373 glioma cells and their EGFRvIII-expressing isogenic counterparts (U373vIII). EVs were purified using Optiprep density gradient ultracentrifugation and analysed with an UHPLC-Orbitrap Fusion Tribrid mass spectrometer. Compilation of three experimental replicates revealed remarkable changes in the expression profiles of the EV proteins, as well as changes in the release rate and concentrations of secreted EVs. For example, U373vIII-derived EVs exhibited a distinct profile of integrin expression, including elevated content of integrin α6β4, known to direct EVs to the lung. In contrast, parental U373 derived EVs carried integrin αVβ5, known to direct EVs to the liver. Thus, while GBMs generally do not metastasise to these respective organs their EVs may home to these sites and contribute, in an oncogene-specific manner, to systemic pathologies associated with brain tumours (inflammation, thrombosis). Moreover, U373vIII cells secreted EVs contained high levels of other invasion-promoting proteins including CD44, CD151, BSG. In conclusion, our results suggest that oncogenic EGFRvIII profoundly impacts the proteome of EVs released by GBM cells, and may define their biological activities beyond the content of EGFRvIII oncoprotein itself.  

3.2807           Chloride intracellular channel protein 4 (CLIC4) is a serological cancer biomarker released from tumour epithelial cells via extracellular vesicles

Sanchez, V.C., Craig-Lucas, A., Wei, B-R., Shukla, A., Read, A., Lou, J., Simpson, M., Hunter, K. and Yuspa, S.

1. Extracellular Vesicles, 6, Suppl.1, abstract PF01.12 (2017)

  CLIC4 is a highly conserved metamorphic protein originally described as an ion channel. It translocates to the nucleus serving as an integral component of TGF-β signalling. In multiple cancers, CLIC4 is a tumour suppressor, excluded from the nucleus and lost from the cytoplasm of progressing cancer cells. In contrast, CLIC4 is upregulated in the tumour stroma in response to TGF-β. CLIC4 lacks a secretory sequence, but recent reports indicate that CLIC4 is detected in the circulation of cancer patients serving a possible biomarker and has been detected in extracellular vesicles (EVs). EVs from cell culture supernatants or biological fluids from SKOV3/ SCID xenograft ovarian and 6DT1 orthograft breast cancer models, were isolated by differential centrifugation, following ultracentrifugation and Optiprep density gradients. EV size distribution and concentration were analysed by NTA and TEM. The presence of markers and CLIC4 were analysed by immunoblot. We validated the presence of CLIC4 in EVs released into supernatants from primary normal and multiple ovarian tumour cell lines. Substantial increases in CLIC4 were measured in EVs of tumour cells when compared to normal cells. TGF-β-induced myofibroblasts also increased CLIC4 in both the cells and the EVs they released. Immunostaining analysis of human ovarian cancer tissue arrays show CLIC4 preferentially expressed in tumour stroma of multiple subtypes with the exception of ovarian serous adenocarcinomas, where it is upregulated in both compartments. In vivo, CLIC4 levels increased in EVs released into the peritoneal cavity as tumour burden increased in a heterotopic xenograft ovarian cancer model. Moreover, CLIC4 levels in EVs isolated from plasma increased with tumour burden and lung metastatic load in an orthotopic syngeneic mouse breast cancer model. To dissect the contribution of stromal vs. tumour epithelial  compartments as the source of the EVs, CLIC4 was deleted in breast cancer cell lines by CRISPR/Cas9. CLIC4 in circulating EVs is reduced in CLIC4 KO tumour-bearing mice when compared to WT, indicating that the major contribution of CLIC4 into circulation is from tumour epithelium. CLIC4 levels in EVs from biological fluids may have value as a cancer biomarker, in conjunction with other markers, to detect or analyse tumour progression or recurrence.  

3.2808           Galectin-3 binding protein present at the surface of tumour exosomes contributes to their capture by stromal cells

Nakata, R., Sarte, L., Zimmermann, P. and DeClerck, Y.A.

4. Extracellular Vesicles, 6, Auppl. 1, abstract PH04.13 (2017)

  Introduction: Galectin-3 binding protein (Gal-3BP/LGALS3BP aka: MAC2-binding protein) is a 90 kDa secreted sialoglycoprotein that is commonly present in the cargo of exosomes and is among the 25 common cancer proteins associated with extracellular vesicles (EVs) secretion in all NCI-60 cancer cell lines (1). Here we have examined its presence and function in exosomes from human neuroblastoma cells that we had previously reported to secrete Gal-3BP (2). Methods: The expression of Gal-3BP was examined in exosomes from 10 human NB cell lines by western blot analysis. Exosomes were prepared by differential ultracentrifugation (DUC), Optiprep density gradient centrifugation (ODGC) and size exclusion chromatography (SEC). Gal-3BP localisation in cells and exosomes was performed by confocal microscopy, flow cytometry and electron microscopy. Its role in exosome biogenesis and capture by stromal cells was examined in NB cells in which the LGAL3SBP gene was removed by CRISPR-Cas9 knock out. Results: Gal-3BP was consistently present in all preparations of exosomes obtained from 10 NB cell lines. It was also present in exosomes from the plasma of patients with NB. It was consistently associated with exosome protein markers like CD-63, syntenin and ALIX in exosomes obtained by DUC, ODGC and SEC, in addition to being present in a soluble form in the culture medium of NB cells. However in NB cells Gal-3BP was clearly segregated from CD-63, suggesting its absence in mulitivesicular bodies and an absence of involvement in exosome biogenesis. This was further supported by the demonstration that syntenin knock down in NB cells did not affect the presence of Gal-3BP in exosomes. We then demonstrated by a combination of flow cytometry and enzymatic digestion, that Gal-3BP is present on the surface of exosomes. To better understand its function, LGALS3BP was knocked out in NB cells. Whereas Gal-3BP KO did not affect the production of exosomes in NB cells, it inhibited their capture by stroma cells. Conclusion: Our data bring insight into the function of a protein commonly identified in the cargo of cancer cell exosomes, suggesting an absence of involvement in exosome biogenesis and a role in exosome uptake by stromal cells. References

1. Hurwitz et al., Oncotarget 2016; 7: 86999–87015.
2. Silverman et al., Cancer Res. 2012; 72: 2228–2238.

 

3.2809           Histone flow: from nucleus to extracellular vesicles

Nair, R.R., Mazza, D., Agresti, A. and Bianchi, M.

24. Extracellular Vesicles, 6, Suppl.1, abstract OS24.04 (2017)

  Introduction: Histones play a central role in DNA packaging and epigenetic regulation. Interestingly, histones are also found as soluble molecules in the blood of sepsis patients (1). Until recently researchers viewedhistone content in each and every cell as fixed. Recent reports indicate that histone content decreases in senescent cells. Our group had shown that macrophages treated with LPS decrease their nucleosome content by approximately 20% in 4 h (2). Our aim was to determine the fate of the 20% “missing” histones in macrophages stimulated with LPS. Methods and Results: First, we evaluated whether stimulated macrophages reorganise their chromatin structure, at a global level. Using quantitative super-resolution microscopy (STORM) we observed that after LPS stimulation of macrophages, nucleosomes clutches (2) reduce both their size and density, suggesting that histones are evicted from chromatin. Evicted histones can have two possible fates: they can be degraded or secreted out of the cells. To test for histone degradation we collected the cells together with their medium, but we found no difference before and after stimulation. In contrast, histones amount in the medium increased after stimulation. These data imply that histones are not degraded but secreted. The medium of stimulated macrophages was subjected to ultracentrifugation on an Optiprep density gradient. We found more histones both in extracellular vesicles (EVs) and in the soluble fraction. This result was confirmed using knock-in mice expressing H2B-GFP macrophages which were found to secrete microvesicles containing H2B-GFP. We excluded that EVs originate from membrane blebbing occurring during apoptosis and necrosis, since there is no significant apoptosis or necrosis in LPS-stimulated macrophages. However, we observed a high level of H3K4 trimethylation in the secreted histones, suggesting that they originate from the nucleus. We next investigated the localisation of histones in microvesicles: inside or outside the membrane. Biochemical experiments and STROM images indicate that histones are mostly on the outer surface of the vesicles. Conclusion: Our data show that the nuclear histones can be evicted out of chromatin and be expelled either as soluble protein or microvesicle–associated proteins. References

1. Chen R et al., Cell Death Dis. 2014; 5: e1370.
2. De Toma I et al., J Intern Med. 2014; 276: 454–469.

 

3.2810           Amoeboid cancer cells shed extracellular vesicles enriched with nuclear derived material

Sobreiro, M.R., Chen, J-F., Morley, S., You, S., Steadman, K., Gill, N.K., Chu, G.C-Y., Chung, L.W.K., Tanaka, H., Yang, W., Rowat, A.C., Tseng, H-R., Posadas, E.M., Di Vizio, D. and Freeman, M.R.

1. Extracellular Vesicles, 6, Suppl. 1, abstract OPT01.01 =PT01.04 (2017)

  Introduction: Deformation of the nucleus is required for migrating cells to pass through interstitial tissue spaces. However, it remains unexplored how cells modify nuclear stiffness during metastasis. Cancer cells exhibiting an “amoeboid” phenotype migrate in a manner that resembles neutrophil movement, in which nuclear deformation plays a critical role. Amoeboid tumour cells are characterised by their plasticity, ability to rapidly move through extracellular matrixes and high rates of shedding of extracellular vesicles (EVs). Methods: Mass spectrometry, flow cytometry, differential centrifugation, iodixanol gradient, confocal 3D imaging, time lapse video microscopy, western blot, NanoVelcro Chip. Results: Here we demonstrate that stable disruption of nuclear structure by silencing DIAPH3, emerin, or lamin A/C promotes conversion to the highly metastatic amoeboid phenotype in prostate and breast cancer cells. These amoeboid cells produced vesicles from nuclear blebs, underwent shedding of non-apoptotic EVs containing DNA, and exhibited increased sensitivity to inhibitors of DNA damage repair. Amoeboid features were detected in high grade prostate cancer, and capture of circulating tumour cells in mice and patients with metastatic prostate cancer. Conclusion: These findings suggest the potential of incorporating the use of biomarkers of amoeboid tumour cells into clinical strategies for precision medicine.  

3.2811           Attempts to re-define cellular components specifically incorporated in HIV as compared to sEVs and exosomes secreted by infected cells

Martin-Jaular, L., Liao, Z., Gerber, P.P., Ostrowski, M., Witwer, K. and Thery, C.

18. Extracellular Vesicles, 6, Suppl. 1, abstract OF18.02 (2017)

  Introduction: HIV, exosomes and/or other small extracellular vesicles (sEVs) share biogenesis aspects and physicochemical characteristics, making their separation difficult. Some cellular proteins are described as excluded from virions (e.g. CD45), whereas others are incorporated (e.g. CD63). We re-evaluated these results in light of our recent demonstration that many subtypes of sEVs are co-isolated by a protocol of EV isolation similar to that used for HIV isolation, and of our recently published sets of protein combinations distinguishing exosomal and non-exosomal sEVs (1). Our goal is to obtain HIV-free sEVs to allow assessing their functional properties. Methods: Medium of Jurkat cells infected or not with VSV-G–pseudotyped NL4-3-IRES-EGFP was subjected to differential centrifugation, and velocity top-to-bottom iodixanol gradient was used to separate sEVs from virus in the 100,000g pellet (100 K). Gradient fractions were analysed by WB for the presence of different markers and by AChE assay. Results: Differential centrifugation showed that CD45 is more abundant in large/medium EVs than in sEVs from both uninfected and infected cells. Velocity gradients revealed at least two types of sEVs in the 100 K pellet. Fractions from the top of the tube contained CD9 and some CD45 but little or no CD63 (i.e. non-exosomal sEVs), whereas intermediate fractions contained CD9, CD63, and syntenin-1, hence probably exosomes. Gag and CD63 but little or no CD9, Syntenin-1 and CD45 were detected in bottom fractions of infected cells’ 100 K pellet. Importantly, AChE activity was found in fractions different from those enriched in Gag but also from those enriched for the other sEVs/exosome markers. Conclusions: Despite exclusion from virus containing fractions, neither AChE activity nor CD45 are satisfying markers to distinguish HIV from exosomes. Velocity gradients achieve some separation of sEVs/exosome or virus markers, but overlap of distribution makes it difficult to use them for unbiased proteomic comparisons. Further work will be required to identify, if they exist, sEV and/or exosomal components specifically excluded from HIV virions. Reference

1. Kowal et al., PNAS 2016; 113: E968.

 

3.2812           Characterisation of extracellular vesicles with milk fat globule membrane-like properties that carry most microRNAs in commercial dairy cow milk

Abderrahim, B., Sophia, L., Ting, S.S., Laugier, J., Boilard, E., Caroline, G. and Provost, P.

21. Extracellular Vesicles, 6, Suppl. 1, abstract OS21.02 (2017)

  Introduction: MicroRNAs are short (~22 nucleotides), non-coding RNAs that play an essential role in post-transcriptional gene regulation. Found in several biological fluids, including milk, they are often associated with extracellular vesicles (EVs), like exosomes. In a previous study, we found that commercial dairy cow milk microRNAs resist digestion in vitro. Surprisingly, we observed thatmost of them sediment at low centrifugation speed, thereby challenging their association with exosomes in commercial milk. Methods: We used differential ultracentrifugation and iodixanol density gradient (IDG) to isolate milk EVs, which we analysed for microRNA enrichment by reverse transcription and quantitative polymerase chain reaction (RT-qPCR) and for EV-associated proteins by western blot. We further characterised these EVs by density measurements, fluorescence RNA labelling, mass spectrometry (LC-MS/MS), dynamic light scattering (DLS), flow cytometry, transmission electron microscopy (TEM) and proteinase K assay. Results: We found no correlation between bta-miR-223 and bta-miR-125b and exosome-associated proteins found in low speed ultracentrifugation pellets (i.e. 12,000g and 35,000g), but a positive correlation (p < 0.05) between bta-miR-125b and xanthine dehydrogenase (XDH). Two IDG fractions were highly enriched in double stranded RNAs and microRNAs, contained several exosome-associated proteins and most of the exosomelike EVs found in these gradients. However, proteinase K assay and subsequent LC-MS/MS analysis challenged the exosome nature of these EVs, as all exosome-enriched proteins were digested during the assay and these digested EVs were found to contain milk fat globule membrane (MFGM)-enriched proteins, including immunomodulatory XDH, butyrophilin 1A1 (BTN1A1), mucin (MUC-1) and lactadherin (MFG-E8). Conclusion: Our results suggest the presence of exosome-like EVs with MFGM-like properties in commercial milk and their association with the majority of milk microRNAs. Considering their resistance to proteinase K digestion and bioaccessibility in vitro, these EVs may contribute to interspecies transfer of dietary microRNAs and immune regulation by milk EVs, which require further investigations.  

3.2813           Recipient cell organelle separation for EV uptake studies: Tracking of extracellular vesicles

Shelke, G. and Lötvall, J.

1. Extracellular Vesicles, 6, Suppl. 1, abstract LBP.45 (2017)

  Background: Extracellular vesicles (EVs) such as exosomes and microvesicle are known to delivery cargo like proteins, lipids, RNA, and DNA to the recipient cells. Transfer of EVs to recipient cells to deliver these cargos is essential to induce cellular phenotypic changes. Current methods to localize EVs in recipient cells are restricted to imaging of cells using co-localization of fluorescent probes. We propose a physical method that provides high-resolution separation of organelles that can be associated with EVs recipient cell trafficking. Methods: EVs were isolated from mast cell line (HMC1.2) by differential centrifugation (16,500´g 20 min and 120,000´g 3 hr) followed by flotation on iodixanol gradient (182,300´g for 16 hours; SW40-Ti rotor). EVs were biotinylated by incubating it with EZ-Link Sulfo-NHS-Biotin (Thermo Scientific) and free biotin was removed by dialysis (3.5 kDa filter) as per the manufacturer recommendations. Biotinylated-EVs were later incubated with HEK-293T cells for 60 min, after which cells were lysed (High salt, high pH buffer and sonication) to obtain crude organelles. Crude organelles carrying biotinylated EVs were further separated on iodixanol density gradient with two consecutive ultracentrifugation steps. Various iodixanol fractions were analyzed using immunoblotting for lysosomal (LAMP1) and endosomal protein (EEA1), as well as streptavidin-HRP based detection of EVs-biotin. Results: High resolution separation of endosomal and lysosomal organelles fraction was obtained using this method. We found that biotinylated EV proteins were enriched in the endosomal fraction. A small quantity of biotinylated-EV proteins were also present in lysosomal enriched fraction. Summary/Conclusion: Endosomal and lysosomal localization of EVs can be performed in recipient cell by iodixanol density gradient centrifugation. EVs were primarily enriched in the endosomal compartment, and only traces were detected in the endo-lysosomal compartment at the time point studied.  

3.2814           Isolation of Lipid Raft Proteins from CD133+ Cancer Stem Cells

Gupta, V.K. and Banerjee, S. Methods in Mol. Biol., 1609, 25-31 (2017)   Pancreatic cancer cells expressing the surface markers CD133 have been widely reported as cancer stem cells and mainly responsible for tumor recurrence and chemoresistance in pancreatic cancer. In spite of its role as a stem cell marker in pancreatic cancer, its function remains elusive. CD133 (also known as prominin-1) is a pentaspan glycoprotein predominantly localized in lipid rafts, specialized membrane microdomains enriched in crucial signaling proteins. Coexistence of CD133 with these signaling proteins can modulate various signaling pathways that might be responsible for aggressive phenotype of CD133+ cells. This chapter describes a detailed protocol to isolate lipid rafts from CD133+ tumor initiating cells. Purified lipid rafts can be investigated further for protein or lipid composition by mass spectrometry that can shed some light on functional role of CD133 protein in these cancer stem cells.  

3.2815           Analysis of Ras/ERK Compartmentalization by Subcellular Fractionation

Agudo-Ibanez, L., Crespo, P and Casar, B. Methods in Mol. Biol., 1487, 151-162 (2017)   A vast number of stimuli use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their cognate receptors, in order to regulate multiple cellular functions, including key processes such as proliferation, cell cycle progression, differentiation, and survival. The duration, intensity and specificity of the responses are, in part, controlled by the compartmentalization/subcellular localization of the signaling intermediaries. Ras proteins are found in different plasma membrane microdomains and endomembranes. At these localizations, Ras is subject to site-specific regulatory mechanisms, distinctively engaging effector pathways and switching-on diverse genetic programs to generate a multitude of biological responses. The Ras effector pathway leading to ERKs activation is also subject to space-related regulatory processes. About half of ERK1/2 substrates are found in the nucleus and function mainly as transcription factors. The other half resides in the cytosol and other cellular organelles. Such subcellular distribution enhances the complexity of the Ras/ERK cascade and constitutes an essential mechanism to endow variability to its signals, which enables their participation in the regulation of a broad variety of functions. Thus, analyzing the subcellular compartmentalization of the members of the Ras/ERK cascade constitutes an important factor to be taken into account when studying specific biological responses evoked by Ras/ERK signals. Herein, we describe methods for such purpose.  

3.2816           Extracellular vesicles from mesenchymal stem cells activates VEGF receptors and accelerates recovery of hindlimb ischemiaGangadaran, P., Rajendran, R.L., Lee, H.W., kalimuthu, S., Hong, C.M., Jeong, S.Y., Lee, S-W., Lee, J. and Ahn, B-C.

J. Controlled Release, 264, 112-126 (2017)

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are potential therapies for various diseases, but their angiogenic mechanisms of therapeutic efficacy remain unclear. Here, we describe how MSC-EVs, activates VEGF receptors and downstream angiogenesis pathways. Mouse MSC-EVs were isolated from cell culture medium and characterized using transmission electron microscopy, nanoparticle analysis, and western blotting. In vitro migration, proliferation, and tube formation assays using endothelial cells were used to assess the angiogenic potential of MSC-EVs, and revealed higher levels of cellular migration, proliferation, and tube formation after treatment. qRT-PCR and western blotting (WB) revealed higher protein and mRNA expression of the angiogenic genes VEGFR1 and VEGFR2 in mouse SVEC-4 endothelial cells after MSC-EVs treatment. Additionally, other vital pro-angiogenic pathways (SRC, AKT, and ERK) were activated by in vitro MSC-EV treatment. WB and qRT-PCR revealed enriched presence of VEGF protein and miR-210-3p in MSC-EV. The hindlimb ischemia mouse model was established and MSC-EVs with or without Matrigel (EV-MSC + Gel) were injected into the ischemic area and blood reperfusion was monitored using molecular imaging techniques. The in vivo administration of MSC-EVs increased both blood reperfusion and the formation of new blood vessels in the ischemic limb, with the addition of matrigel enhancing this effect further by releasing EVs slowly. MSC-EVs enhance angiogenesis in ischemic limbs, most likely via the overexpression of VEGFR1 and VEGFR2 in endothelial cells. These findings reveal a novel mechanism of activating receptors by MSC-EVs influence the angiogenesis.

3.2817           Fractionation Techniques to Examine Effector Translocation

Olson, R.M. and Anderson, D.M. Methods in Mol. Biol., 1531, 101-109 (2017)   Many Gram-negative bacterial pathogens use type III secretion systems to export proteins that act directly on the host and aid in the infectious process. Extracellular bacteria primarily rely upon the type III secretion system to insert or inject effector proteins into the cytosol of their host cell in order to perturb intracellular signaling events and aid in pathogenesis. Intracellular bacteria can also depend on the T3SS translocation of effector proteins from vacuolar compartments into the vacuolar membrane or host cell cytosol where they can modulate intracellular trafficking and/or signaling pathways necessary for their growth and survival. Biochemical fractionation of infected cells in vitro enables detection of these events, making it possible to identify relevant protein–protein interactions, characterize phenotypes of mutant strains and understand how these effector proteins impact host cells. In this chapter we provide methods for the analysis of translocated effector proteins using biochemical and mechanical fractionation procedures.  

3.2818           Analysis of Mitochondrial Membrane Protein Complexes by Electron Cryo-tomography

Gold, V.A.M., Brandt, T., Cavellini, L., Cohen, M.M., Leva, R. and van der Laan, M. Methods in Mol. Biol., 1567, 315-336 (2017)   The visualization of membrane protein complexes in their natural membrane environment is a major goal in an emerging area of research termed structural cell biology. Such approaches provide important information on the spatial distribution of protein complexes in their resident cellular membrane systems and on the structural organization of multi-subunit membrane protein assemblies. We have developed a method to specifically label active membrane protein complexes in their native membrane environment with electron-dense nanoparticles coupled to an activating ligand, in order to visualize them by electron cryo-tomography. As an example, we describe here the depiction of preprotein import sites of mitochondria, formed by the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). Active import sites are selectively labeled via a biotinylated, quantum dot-coupled preprotein that is arrested in translocation across the outer and inner mitochondrial membranes. Additionally, a related method is described for direct labeling of mitochondrial outer membrane proteins that does not depend on binding of a ligand.  

3.2819           Analysis of Fatty Acid and Cholesterol Content from Detergent-Resistant and Detergent-Free Membrane Microdomains

McClellan, M.E. and Eliiott, M.H. Methods in Mol. Biol., 1609, 185-194 (2017)   The compartmentalization of cellular membranes into discrete membrane microdomains (known as lipid rafts) challenged the original definition of membranes as containing randomly distributed lipid and protein components. The lipid microdomain hypothesis has generated significant controversy and rigorous inquiry based on the attractive idea that such domains concentrate machinery to mediate cellular events such as signaling and endocytosis. As such, numerous studies have used biochemical, cell biological, and biophysical methodologies to define the composition of such domains in a variety of experimental contexts. In this chapter, we describe methodologies to isolate membranes from cell or tissue sources with biophysical/biochemical properties of membrane microdomains that are amenable to subsequent classical or mass spectrometry-based lipid analytical approaches.  

3.2820           Isolation of Extracellular Vesicles by Ultracentrifugation

Momen-Heravi, F. Methods in Mol. Biol., 1660, 25-32 (2017)   Extracellular vesicles (EVs) represent a group of heterogeneous vesicles that can be obtained from almost all biofluids. EVs, including microvesicles, exosomes, and apoptotic bodies, can deliver bioactive cargos and signaling molecules. Various physiological roles and pathophysiological roles for EVs in diseases such as cancer, infectious diseases, endocrine diseases, and neurodegenerative disorders have been recognized. These observations highlight EVs as potential novel biomarkers and targets for therapeutic intervention. One of the major limitations in the use of EVs for diagnosis and therapeutic purposes is the lack of standardization of isolation techniques. Here, we describe protocols for ultracentrifugation and sucrose gradient isolation methods, which are the current gold standard, and are the most studied methods for EV isolation.  

3.2821           Purification Protocols for Extracellular Vesicles

Lane, R.E., Korbie, D., Trau, M. And Hill, M.M. Methods in Mol. Biol., 1660, 111-130 (2017)   This chapter provides a description of some of the standard methods used for the isolation of extracellular vesicles (EVs) from a variety of biological fluids, including cell culture media, urine, plasma and serum. The methods presented include ultracentrifugation, ultrafiltration, proprietary polymer-based reagents, size exclusion chromatography, density gradient separation, and immunoaffinity capture. Ultracentrifugation methods use high speed centrifugation to pellet vesicles, whilst polymer-based reagents are added to the sample to facilitate vesicle precipitation using lower speeds. Ultrafiltration involves the concentration of vesicles from a large volume of biological fluid using a centrifugal filter unit. Size exclusion chromatography and density gradient separation are both designed to allow the separation of vesicles from other nonvesicular debris. Immunoaffinity capture methods use antibody-coated beads to selectively isolate vesicles displaying a surface marker of interest. Ultimately, the choice of purification method for an individual experiment is influenced by time, cost, and equipment considerations, as well as the sample requirements for any downstream analyses.  

3.2822           Extraction and Analysis of Extracellular Vesicle-Associated miRNAs Following Antibody-Based Extracellular Vesicle Capture from Plasma Samples

Zocco, D. and Zarovni, N. Methods in Mol. Biol., 1660, 269-285 (2017)   Extracellular vesicle (EV)-associated RNAs (EV-RNA) are under intense investigation due to their potential role in health and disease. Several approaches are currently employed to isolate blood-derived EVs for RNA analysis, most of which are either time-consuming and expensive, such as methods based on EVs physical properties (ultracentrifugation and Optiprep density gradient), or also copurify blood contaminants, mostly protein aggregates and immune complexes, (such as chemical precipitation). In addition, there is a lack of standardized protocols for the extraction of EV-RNA and very little consensus on the technological platforms and normalization tools for assessing the expression levels of different RNA species. These methodological issues complicate the comparison between independent data sets, potentially biasing results and conclusions. In this book chapter we propose a protocol that might overcome some of the abovementioned issues through antibody-based isolation of blood-derived EVs followed by extraction and expression analysis of small-RNA species (miRNA) by reverse transcriptase quantitative PCR (RT-qPCR). The advantages of immunoaffinity approaches over other isolation methods are multiple and include: (1) the selective enrichment of specific EV subpopulations with restricted tissue/cell origin, (2) reduction of matrix effects and blood contaminants that may confound miRNA profiling from complex biological fluids and (3) easy coupling to conventional quantitative assays (e.g., RT-qPCR). In conclusion, we describe a protocol for standard enrichment and quantitative analysis of EV-miRNAs from blood and we warrant for technological improvements, such as the use of novel biomaterials, surface chemistries, binding agents and assay/sensor design that may further improve it.  

3.2823           An Adaptable Polyethylene Glycol-Based Workflow for Proteomic Analysis of Extracellar Vesicles

Hurwitz, S.N. and Meckes Jr., D.G. Methods in Mol. Biol., 1660, 303-317 (2017)   Extracellular vesicles (EVs), including exosomes are endocytically derived nanovesicles expelled from cells that contain molecular information in the form of lipids, proteins, and nucleic acids. Transfer of this information to other cells in local or distant microenvironments facilitates cell-to-cell communication. Importantly, diseased cells release exosomes containing specific cargo that may contribute to pathology and can be harnessed for diagnostic or prognostic use. The broad potential medical utility of exosomes has fueled rapidly expanding research on understanding the composition and functions of exosomes in normal and pathological conditions. Here, we provide a complete workflow for purifying exosome-sized vesicles from biological fluids for in-depth proteomic analyses. Moreover, this polyethylene glycol-based method is efficient, highly adaptable, and compatible with a variety of downstream applications.  

3.2824           Isolation of Extracellular Vesicles in Saliva Using Density Gradient Ultracentrifugation

Iwai, K., Yamamoto, S., Yoshida, M. and Shiba, K. Methods in Mol. Biol., 1660, 343-350 (2017)   This chapter describes a method for isolating human salivary extracellular vesicles (EVs) using density gradient ultracentrifugation. Standard protocols established for isolation of EVs from blood or a conditioned medium of cultured cells do not work for whole saliva, due to its viscosity. Therefore, procedures including a pretreatment step and utilizing iodixanol as a gradient material enable EVs to be concentrated to a 1.1 g/ml density. This protocol is compatible with both swing and angle rotors. By employing an angle rotor, which enables high g-force, the centrifugation time was reduced to 4 h from the 17 h required when using a swing rotor.  

3.2825           Microcapillary Chip-Based Extracellular Vesicle Profiling System

Akagi, T. and Ichiki, T. Methods in Mol. Biol., 1660, 209-217 (2017)   A microcapillary chip-based particle electrophoresis system developed for characterizing extracellular vesicles (EVs) is described. So far, it is technologically difficult to analyze or identify a heterogeneous population of particles ranging from several tens to one hundred nanometers, and hence, there is a growing demand for a new analytical method of nanoparticles among researchers working on extracellular vesicles. The analytical platform presented in this chapter allows detection of individual nanoparticles or nanovesicles of less than 50 nm in diameter and enables the characterization of nanoparticles based on multiple indexes such as concentration, diameter, zeta potential, and surface antigenicity. This platform will provide a useful and easy-to-use solution for obtaining both quantitative and qualitative information on EV samples used in research and development of exosome biology and medicine.  

3.2826           Purification of LAT-Containing Membranes from Resting and Activated T Lymphocytes

Hivroz, C., larghi, P., Jouve, M. and Ardouin, L. Methods in Mol. Biol., 1584, 355-368 (2017)   In T lymphocytes, the immune synapse is an active zone of vesicular traffic. Directional transport of vesicular receptors and signaling molecules from or to the immune synapse has been shown to play an important role in T-cell receptor (TCR) signal transduction. However, how vesicular trafficking is regulating the activation of T cells is still a burning question, and the characterization of these intracellular compartments remains the first step to understand this process. We describe herein a protocol, which combines a separation of membranes on flotation gradient with an affinity purification of Strep-tagged fusion transmembrane proteins with Strep-Tactin^® resin, allowing the purification of membranes containing the Strep-tagged molecule of interest. By keeping the membranes intact, this protocol leads to the purification of molecules physically associated with the Strep-tagged protein as well as of molecules present in the same membrane compartment: transmembrane proteins, proteins strongly associated with the membranes, and luminal proteins. The example shown herein is the purification of membrane compartment prepared from T lymphocytes expressing LAT fused to a Strep-tag.  

3.2827           Surface Profiling of Extracellular Vesicles from Plasma or Ascites Fluid Using DotScan Antibody Microarrays

Belov, L., Hallal, S., Matic, K., Zhou, J., Wissmueller, S., Ahmed, N., Tanjil, S., Mulligan, S.P., Best, O.G., Simpson, R.J. and Christopherson, R.I. Methods in Mol. Biol., 1619, 263-301 (2017)   DotScan antibody microarrays were initially developed for the extensive surface profiling of live leukemia and lymphoma cells. DotScan’s diagnostic capability was validated with an extensive clinical trial using mononuclear cells from the blood or bone marrow of leukemia or lymphoma patients. DotScan has also been used for the profiling of surface proteins on peripheral blood mononuclear cells (PBMC) from patients with HIV, liver disease, and stable and progressive B-cell chronic lymphocytic leukemia (CLL). Fluorescence multiplexing allowed the simultaneous profiling of cancer cells and leukocytes from disaggregated colorectal and melanoma tumor biopsies after capture on DotScan. In this chapter, we have used DotScan for the surface profiling of extracellular vesicles (EV) recovered from conditioned growth medium of cancer cell lines and the blood of patients with CLL. The detection of captured EV was performed by enhanced chemiluminescence (ECL) using biotinylated antibodies that recognized antigens expressed on the surface of the EV subset of interest. DotScan was also used to profile EV from the blood of healthy individuals and the ascites fluid of ovarian cancer patients. DotScan binding patterns of EV from human plasma and other body fluids may yield diagnostic or prognostic signatures for monitoring the incidence, treatment, and progression of cancers.  

3.2828           A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress

Kant, S., Standen, C.L., Morel, C., Swat, W., Flavell, R.A. and Davis, R.J: Cell Reports, 20, 2775-2783 (2017)   Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA) activation of a non-receptor tyrosine kinase (SRC)-dependent cJun NH₂-terminal kinase (JNK) signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway.  

3.2829           Identification of syntaxin 4 as an essential factor for the hepatitis C virus life cycle

Ren, H., Elgner, F., Himmelsbach, K., Akhras, S., Jiang, B., Medvedev, R., Ploen, D. and Hildt, E. Eur. J. Cell Biol., 96, 542-552 (2017)   Although there is evidence that multivesicular bodies (MVBs) are involved in the release of hepatitis C virus (HCV), many aspects of HCV release are still not fully understood. The amount of α-taxilin that prevents SNARE (soluble N-ethylmaleimidesensitive factor attachment protein receptor) complex formation by binding to free syntaxin 4 is reduced in HCV-positive cells. Therefore, it was analyzed whether the t-SNARE protein syntaxin 4 which mediates vesicles fusion is involved in the HCV life cycle. HCV-positive cells possess an increased amount of syntaxin 4 protein, although the amount of syntaxin 4-specific transcripts is decreased in HCV-positive Huh7.5 cells and in HCV-infected primary human hepatocytes. In HCV-positive cells a significant longer half-life of syntaxin 4 was found that overcompensates for the decreased expression and leads to the elevated level of syntaxin 4. Overexpression of syntaxin 4 reduces the intracellular amount of infectious viral particles by facilitating viral release, while silencing of syntaxin 4 expression using specific siRNAs inhibits the release of HCV particles and so leads to an increase in the intracellular amount of infectious viral particles. This indicates that HCV uses a SNARE-dependent pathway for viral release. Confocal immunofluorescence microscopy revealed a colocalization of syntaxin 4 with a MVB-specific marker, exosomes and HCV core, which suggests a fraction of syntaxin 4 is associated with exosomes loaded with HCV. Altogether, it is assumed that syntaxin 4 is a novel essential cellular factor for the release of HCV.  

3.2830           Flow Cytometric and Sorting Analyses for Nuclear DNA Content, Nucleotide Sequencing, and Interphase FISH

Kaeser, G. and Chun, J. Neuromethods, 131, 43-55 (2017)   The study of genomic mosaicism among human brain cells is challenging. The human brain contains hundreds of billions of cells that are intricately connected and difficult to separate as intact, single cells. Additional challenges are encountered when interrogating small, seemingly random changes within single-cell genomes. Flow cytometric analysis (FCM), and fluorescence-activated nuclear sorting (FANS), has expanded our assessment capabilities for global and specific genomic and transcriptomic changes in human brain cells. The general approach is being utilized in a variety of downstream applications by many laboratories. Here we provide detailed methods of nuclear DNA content assessment and sorting that reports population averages as well as single-cell nuclear DNA content from cells of the human brain. We highlight protocol modifications that allow the same nuclear preparation to be used for subpopulation-specific FANS (also see chapter “Single-Cell Whole Genome Amplification and Sequencing to Study Neuronal Mosaicism and Diversity”) in downstream analyses such as fluorescent in situ hybridization (FISH) (see chapters “FISH-Based Assays for Detecting Genomic (Chromosomal) Mosaicism in Human Brain Cells,” “FISH Analysis of Aging-Associated Aneuploidy in Neurons and Non-neuronal Brain Cells” and “Using Fluorescence In Situ Hybridization (FISH) Analysis to Measure Chromosome Instability and Mosaic Aneuploidy in Neurodegenerative Diseases”), and single-cell genomic and transcriptomic sequencing (see chapters “Flow Cytometric Quantification, Isolation, and Subsequent Epigenetic Analysis of Tetraploid Neurons,” “Single Cell CNV Detection in Human Neuronal Nuclei,” “Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) for the Analysis of DNA Copy Number Variation,” and “Single-Cell Whole Genome Amplification and Sequencing to Study Neuronal Mosaicism and Diversity”). Other downstream techniques include, but are not limited to, single-cell qPCR (see chapter “Competitive PCR for Copy Number Assessment by Restricting dNTPs”) and estimation of line-1 copy number (see chapters “Analysis of LINE-1 Retrotransposition in Neural Progenitor Cells and Neurons,” “Estimation of LINE-1 Copy Number in the Brain Tissue and Isolated Neuronal Nuclei,” and “Analysis of Somatic LINE-1 Insertions in Neurons”).  

3.2831           Single-Cell CNV Detection in Human Neuronal Nuclei

Wierman, M.B., Burbulis, I.E., Chronister, W.D., Bekiranov, S. and McConnell, M.J. Neuromethods, 131, 109-131 (2017)   Genomic mosaicism is prevalent throughout human somatic tissues and is much more common than previously thought. Here, we describe step-by-step methods to isolate neuronal nuclei from human brain and identify megabase-scale copy number variants (CNVs) in single nuclei. The approach detailed herein includes use of CellRaft technology for single-nucleus isolation, the PicoPLEX approach to whole-genome amplification and library preparation, and a pooled library purification protocol, termed Gel2Gel, which has been developed in our laboratory. These methods are focused toward neuroscience research, but are adaptable to many biomedical fields.  

3.2832           Identification of Low Allele Frequency Mosaic Mutations in Alzheimer Disease

Frigerio, C.S., Fiers, M., Voet, T. and De Strooper, B. Neuromethods, 131, 361-378 (2017)   Germline mutations ofAPP,PSEN1, andPSEN2 genes cause autosomal dominant Alzheimer disease (AD). Somatic variants of the same genes may underlie pathogenesis in sporadic AD, which is the most prevalent form of the disease. Importantly, such somatic variants may be present at very low allelic frequency, confined to the brain, and are thus very difficult or impossible to detect in blood-derived DNA. Ever-refined methodologies to identify mutations present in a fraction of the DNA of the original tissue are rapidly transforming our understanding of DNA mutation and their role in complex pathologies such as tumors. These methods stand poised to test to what extend somatic variants may play a role in AD and other neurodegenerative diseases.  

3.2833           Single-Cell Whole Genome Amplification and Sequencing to Study Neuronal Mosaicism and Diversity

Reed, P.J., Wang, M., Erwin, J.A., Paquola, A.C.M. and Gage, F.H. Neuromethods, 131, 253-268 (2017)   Neuronal mosaicism describes the extent of intercellular genotypic diversity within a single human brain. This somatic variability is driven by numerous mechanisms including errors in DNA replication acquired throughout development and by the activity of endogenous retrotransposons. The study of retrotransposition in neuronal mosaicism may prove crucial to understanding the true complexity of normal and aberrant brain function. Specifically, numerous lines of evidence suggest that retrotransposition specific aspects of neuronal mosaicism may contribute to the unresolved etiology of many neurologic and neuropsychiatric disorders. Here, we describe the SLAV-Seq method, a recent advancement in the field over previous approaches used to study the diversity of LINE-1 based neuronal mosaicism at the single-cell level. We describe in detail, methodology for the isolation of single cells from bulk tissue by FACS, the amplification of single-cell genomic DNA by multiple displacement amplification (MDA), the targeted enrichment of LINE-1 somatic events, and the sequencing of the LINE-1 enriched library. Finally, we discuss methods for the quantification and analysis of the neuronal mosaicism identified by SLAV-Seq and some of the current technical limitations.  

3.2834           Therapeutic Applications of Extracellular Vesicles: Perspectives from Newborn Medicine

Willis, G.R., Kourembanas, S. and Mitsialis, S.A. Methods in Mol. Biol., 1660, 409-432 (2017)   With the advancements in antenatal steroid therapies and surfactant replacement, current clinical practices in neonatal intensive care units allow the survival of infants at very low gestational age. Despite these advances, there continues to be significant morbidity associated with extreme preterm birth that includes both short-term and long-term cardiorespiratory impairment. With no effective single therapy in preventing or treating developmental lung injuries, the need for new tools to treat and reduce risk of complications associated with extreme preterm birth is urgent. Stem cell-based therapies, in particular therapies utilizing mesenchymal stem (stromal) cells (MSCs), have shown promise in a number of animal models of lung pathologies relevant to neonatology. Recent studies in this field have consolidated the concept that the therapeutic mechanism of MSC action is paracrine, and this led to wide acceptance of the concept that the delivery of the MSC secretome rather than live cells may provide an alternative therapeutic approach for many complex diseases. Here, we summarize the significance and application of cell-free based therapies in preclinical models of neonatal lung injury. We emphasize the development of extracellular vesicle (EV)-based therapeutics and focus on the challenges that remain to be addressed before their application to clinical practice.  

3.2835           Designer outer membrane vesicles as immunomodulatory systems – Reprogramming bacteria for vaccine delivery

Gnopo, Y.M.D., Watkins, H.C., Stevenson, T.C., DeLisa, M.P. and Putnam, D. Adv. Drug Delivery Reviews, 114, 132-142 (2017)   Vaccines often require adjuvants to be effective. Traditional adjuvants, like alum, activate the immune response but in an uncontrolled way. Newer adjuvants help to direct the immune response in a more coordinated fashion. Here, we review the opportunity to use the outer membrane vesicles (OMVs) of bacteria as a way to modulate the immune response toward making more effective vaccines. This review outlines the different types of OMVs that have been investigated for vaccine delivery and how they are produced. Because OMVs are derived from bacteria, they have compositions that may not be compatible with parenteral delivery in humans; therefore, we also review the strategies brought to bear to detoxify OMVs while maintaining an adjuvant profile. OMV-based vaccines can be derived from the pathogens themselves, or can be used as surrogate constructs to mimic a pathogen through the heterologous expression of specific antigens in a desired host source strain, and approaches to doing so are reviewed. Additionally, the emerging area of engineered pathogen-specific carbohydrate sequences, or glycosylated OMVs is reviewed and contrasted with protein antigen delivery. Existing OMV-based vaccines as well as their routes of administration round out the text. Overall, this is an exciting time in the OMV field as it matures and leads to more effective and targeted ways to induce desired pathogen-specific immune responses.  

3.2836           Male hormones activate EphA2 to facilitate Kaposi’s sarcoma-associated herpesvirus infection: Implications for gender disparity in Kaposi’s sarcoma

Wang, X., Zou, Z., Deng, Z., Liang, D., Zhou, X., Sun, R. and Lan, K. PloS Pathogens, 13(9), e1006580 (2017)   There is increasing consensus that males are more vulnerable than females to infection by several pathogens. However, the underlying mechanism needs further investigation. Here, it was showed that knockdown of androgen receptor (AR) expression or pre-treatment with 5α-dihydrotestosterone, the AR agonist, led to a considerably dysregulated Kaposi’s sarcoma-associated herpesvirus (KSHV) infection. In endothelial cells, membrane-localized AR promoted the endocytosis and nuclear trafficking of KSHV. The AR interacted with ephrin receptor A2 (EphA2) and increased its phosphorylation at residue Ser897, which was specifically upregulated upon KSHV infection. This phosphorylation resulted from the AR-mediated recruitment of Src, which resulted in the activation of p90 ribosomal S6 kinase 1 (RSK1), which directly phosphorylates EphA2 at Ser897. Finally, the EphA2-mediated entry of KSHV was abolished in a Ser897Asn EphA2 mutant. Taken together, membrane-localized AR was identified as a KSHV entry factor that cooperatively activates Src/RSK1/EphA2 signaling, which subsequently promotes KSHV infection of both endothelial and epithelial cells.  

3.2837           Erdj3 Has an Essential Role for Z Variant Alpha-1-Antitrypsin Degradation

Khodayari, N., marek, G., Lu, Y., Krotova, K., Wang, R.L. and Brantly, M.

1. Cell. Biochem., 118, 3090-3101 (2017)

  Alpha-1-antitrypsin deficiency (AATD) is an inherited disease characterized by emphysema and liver disease. AATD is most often caused by a single amino acid substitution at amino acid 342 in the mature protein, resulting in the Z mutation of the alpha-1-antitrypsin gene (ZAAT). This substitution is associated with misfolding and accumulation of ZAAT in the endoplasmic reticulum (ER) of hepatocytes and monocytes, causing a toxic gain of function. Retained ZAAT is eliminated by ER-associated degradation and autophagy. We hypothesized that alpha-1-antitrypsin (AAT)-interacting proteins play critical roles in quality control of human AAT. Using co-immunoprecipitation, we identified ERdj3, an ER-resident Hsp40 family member, as a part of the AAT trafficking network. Depleting ERdj3 increased the rate of ZAAT degradation in hepatocytes by redirecting ZAAT to the ER calreticulin-EDEM1 pathway, followed by autophagosome formation. In the Huh7.5 cell line, ZAAT ER clearance resulted from enhancing ERdj3-mediated ZAAT degradation by silencing ERdj3 while simultaneously enhancing autophagy. In this context, ERdj3 suppression may eliminate the toxic gain of function associated with polymerization of ZAAT, thus providing a potential new therapeutic approach to the treatment of AATD-related liver disease.  

3.2838           Visualization of cytosolic ribosomes on the surface of mitochondria by electron cryo-tomography

Gold, V.A.M., Chroscicki, P., Bragoszewski, P. and Chacinska, A. EMBO Reports, 18(10), 1786-1800 (2017)   We employed electron cryo-tomography to visualize cytosolic ribosomes on the surface of mitochondria. Translation-arrested ribosomes reveal the clustered organization of the TOM complex, corroborating earlier reports of localized translation. Ribosomes are shown to interact specifically with the TOM complex, and nascent chain binding is crucial for ribosome recruitment and stabilization. Ribosomes are bound to the membrane in discrete clusters, often in the vicinity of the crista junctions. This interaction highlights how protein synthesis may be coupled with transport. Our work provides unique insights into the spatial organization of cytosolic ribosomes on mitochondria.  

3.2839           Fatty acid binding protein (Fabp) 5 interacts with the calnexin cytoplasmic domain at the endoplasmic reticulum

Jung, J., Wang, J., Groenendyk, J., Lee, D., Michalak, m. and Agellon, L.B. Biochem. Biophys. Res. Comm., 493, 202-206 (2017)   Calnexin is a type 1 integral endoplasmic reticulum membrane molecular chaperone with an endoplasmic reticulum luminal chaperone domain and a highly conserved C-terminal domain oriented to the cytoplasm. Fabp5 is a cytoplasmic protein that binds long-chain fatty acids and other lipophilic ligands. Using a yeast two-hybrid screen, immunoprecipitation, microscale thermophoresis analysis and cellular fractionation, we discovered that Fabp5 interacts with the calnexin cytoplasmic C-tail domain at the endoplasmic reticulum. These observations identify Fabp5 as a previously unrecognized calnexin binding partner.  

3.2840           Heterogeneity in non-epitope loop sequence and outer membrane protein complexes alters antibody binding to the major porin protein PorB in serogroup B Neisseria meningitides

Matthias, K.A., Strader, M.B., Nawar, H., Gao, Y.S., Lee, J., Patel, D.S., Im, W. and Bash, M.C. Mol. Microbiol., 105(6), 934-053 (2017)   PorB is a well-characterized outer membrane protein that is common among Neisseria species and is required for survival. A vaccine candidate, PorB induces antibody responses that are directed against six variable surface-exposed loops that differ in sequence depending on serotype. Although Neisseria meningitidis is naturally competent and porB genetic mosaicism provides evidence for strong positive selection, the sequences of PorB serotypes commonly associated with invasive disease are often conserved, calling into question the interaction of specific PorB loop sequences in immune engagement. In this report, we provide evidence that antibody binding to a PorB epitope can be altered by sequence mutations in non-epitope loops. Through the construction of hybrid PorB types and PorB molecular dynamics simulations, we demonstrate that loops both adjacent and non-adjacent to the epitope loop can enhance or diminish antibody binding, a phenotype that correlates with serum bactericidal activity. We further examine the interaction of PorB with outer membrane-associated proteins, including PorA and RmpM. Deletion of these proteins alters the composition of PorB-containing native complexes and reduces antibody binding and serum killing relative to the parental strain, suggesting that both intramolecular and intermolecular PorB interactions contribute to host adaptive immune evasion.  

3.2841           Skewing of the population balance of lymphoid and myeloid cells by secreted and intracellular osteopontin

Kanayama, M., Xu, S., Danzaki, K., Gibson, J.R., Inoue, M., Gregory, S.G. and Shinohara, M.L. Nature Immunol., 18(9), 973-984 (2017)   The balance of myeloid populations and lymphoid populations must be well controlled. Here we found that osteopontin (OPN) skewed this balance during pathogenic conditions such as infection and autoimmunity. Notably, two isoforms of OPN exerted distinct effects in shifting this balance through cell-type-specific regulation of apoptosis. Intracellular OPN (iOPN) diminished the population size of myeloid progenitor cells and myeloid cells, and secreted OPN (sOPN) increase the population size of lymphoid cells. The total effect of OPN on skewing the leukocyte population balance was observed as host sensitivity to early systemic infection with Candida albicans and T cell–mediated colitis. Our study suggests previously unknown detrimental roles for two OPN isoforms in causing the imbalance of leukocyte populations.  

3.2842           TRIM37, a novel E3 ligase for PEX5-mediated peroxisomal matrix protein import

Wang, W., Xia, Z-J., Farre, J-C. and Subramami, S.

1. Cell Biol., 216(9), 2843-2858 (2017)

  Most proteins destined for the peroxisomal matrix depend on the peroxisomal targeting signals (PTSs), which require the PTS receptor PEX5, whose deficiency causes fatal human peroxisomal biogenesis disorders (PBDs). TRIM37 gene mutations cause muscle–liver–brain–eye (mulibrey) nanism. We found that TRIM37 localizes in peroxisomal membranes and ubiquitylates PEX5 at K464 by interacting with its C-terminal 51 amino acids (CT51), which is required for PTS protein import. PEX5 mutations (K464A or ΔCT51), or TRIM37 depletion or mutation, reduce PEX5 abundance by promoting its proteasomal degradation, thereby impairing its functions in cargo binding and PTS protein import in human cells. TRIM37 or PEX5 depletion induces apoptosis and enhances sensitivity to oxidative stress, underscoring the cellular requirement for functional peroxisomes. Therefore, TRIM37-mediated ubiquitylation stabilizes PEX5 and promotes peroxisomal matrix protein import, suggesting that mulibrey nanism is a new PBD.  

3.2843           Placental Exosomes as Early Biomarker of Preeclampsia: Potential Role of Exosomal MicroRNAs Across Gestation

Salomon, C., Guanzon, D., Scholz-Romero, K., Longo, S., Correa, P., Illanes, S.E. and Rice, G.E.

1. Clin. Endocrinol. Metab., 102(9), 3182-3194 (2017)

  Context There is a need to develop strategies for early prediction of patients who will develop preeclampsia (PE) to establish preventive strategies to reduce the prevalence and severity of the disease and their associated complications. Objective The objective of this study was to investigate whether exosomes and their microRNA cargo present in maternal circulation can be used as early biomarker for PE. Design, Setting, Patients, and Interventions A retrospective stratified study design was used to quantify total exosomes and placenta-derived exosomes present in maternal plasma of normal (n = 32 per time point) and PE (n = 15 per time point) pregnancies. Exosomes present in maternal circulation were determined by nanoparticle tracking analysis. An Illumina TruSeq^® Small RNA Library Prep Kit was used to construct a small RNA library from exosomal RNA obtained from plasma samples. Results In presymptomatic women, who subsequently developed PE, the concentration of total exosomes and placenta-derived exosomes in maternal plasma was significantly greater than those observed in controls, throughout pregnancy. The area under the receiver operating characteristic curves for total exosome and placenta-derived exosome concentrations were 0.745 ± 0.094 and 0.829 ± 0.077, respectively. In total, over 300 microRNAs were identified in exosomes across gestation, where hsa-miR-486-1-5p and hsa-miR-486-2-5p were identified as the candidate microRNAs. Conclusions Although the role of exosomes during PE remains to be fully elucidated, we suggest that the concentration and content of exosomes may be of diagnostic utility for women at risk for developing PE.  

3.2844           Initial autophagic protection switches to disruption of autophagic flux by lysosomal instability during cadmium stress accrual in renal NRK-52E cells

Lee, W-K., Probst, S., Santoyo-Sanchez, M.P., Al-Hamdani, W., Diebels, I., von Sivers, J.K., Kerek, E., Prenner, E.J. and Thevenod, F. Arch. Toxicol., 91(10), 3225-3245 (2017)   The renal proximal tubule (PT) is the major target of cadmium (Cd²⁺) toxicity where Cd²⁺ causes stress and apoptosis. Autophagy is induced by cell stress, e.g., endoplasmic reticulum (ER) stress, and may contribute to cell survival or death. The role of autophagy in Cd²⁺-induced nephrotoxicity remains unsettled due to contradictory results and lack of evidence for autophagic machinery damage by Cd²⁺. Cd²⁺-induced autophagy in rat kidney PT cell line NRK-52E and its role in cell death was investigated. Increased LC3-II and decreased p62 as autophagy markers indicate rapid induction of autophagic flux by Cd²⁺ (5–10 µM) after 1 h, accompanied by ER stress (increased p-PERK, p-eIF2α, CHOP). Cd²⁺ exposure exceeding 3 h results in p62/LC3-II accumulation, but diminished effect of lysosomal inhibitors (bafilomycin A1, pepstatin A +E-64d) on p62/LC3-II levels, indicating decreased autophagic flux and cargo degradation. At 24 h exposure, Cd²⁺ (5–25 µM) activates intrinsic apoptotic pathways (Bax/Bcl-2, PARP-1), which is not evident earlier (≤6 h) although cell viability by MTT assay is decreased. Autophagy inducer rapamycin (100 nM) does not overcome autophagy inhibition or Cd²⁺-induced cell viability loss. The autophagosome–lysosome fusion inhibitor liensinine (5 μM) increases CHOP and Bax/Bcl-2-dependent apoptosis by low Cd²⁺ stress, but not by high Cd²⁺. Lysosomal instability by Cd²⁺ (5 μM; 6 h) is indicated by increases in cellular sphingomyelin and membrane fluidity and decreases in cathepsins and LAMP1. The data suggest dual and temporal impact of Cd²⁺ on autophagy: Low Cd²⁺ stress rapidly activates autophagy counteracting damage but Cd²⁺ stress accrual disrupts autophagic flux and lysosomal stability, possibly resulting in lysosomal cell death.  

3.2845           5-hydroxymethylcytosine accumulation in postmitotic neurons results in functional demethylation of expressed genes

Mellen, M., Ayata, P. and Heintz, N. PNAS, 114(37), E7812-E7821 (2017)   5-hydroxymethylcytosine (5hmC) occurs at maximal levels in postmitotic neurons, where its accumulation is cell-specific and correlated with gene expression. Here we demonstrate that the distribution of 5hmC in CG and non-CG dinucleotides is distinct and that it reflects the binding specificity and genome occupancy of methylcytosine binding protein 2 (MeCP2). In expressed gene bodies, accumulation of 5hmCG acts in opposition to 5mCG, resulting in “functional” demethylation and diminished MeCP2 binding, thus facilitating transcription. Non-CG hydroxymethylation occurs predominantly in CA dinucleotides (5hmCA) and it accumulates in regions flanking active enhancers. In these domains, oxidation of 5mCA to 5hmCA does not alter MeCP2 binding or expression of adjacent genes. We conclude that the role of 5-hydroxymethylcytosine in postmitotic neurons is to functionally demethylate expressed gene bodies while retaining the role of MeCP2 in chromatin organization.  

3.2846           TrkB neurotrophic activities are blocked by α-synuclein, triggering dopaminergic cell death in Parkinson’s disease

Kang, S.S., Zhang, Z., Liu, X., Manfredsson, F.P., Benskey, M.J., Cao, Z., Xu, J., Sun, Y.E. and Ye, K. PNAS, 114(40), 10773-10778 (2017)   BDNF/TrkB neurotrophic signaling is essential for dopaminergic neuronal survival, and the activities are reduced in the substantial nigra (SN) of Parkinson’s disease (PD). However, whether α-Syn (alpha-synuclein) aggregation, a hallmark in the remaining SN neurons in PD, accounts for the neurotrophic inhibition remains elusive. Here we show that α-Syn selectively interacts with TrkB receptors and inhibits BDNF/TrkB signaling, leading to dopaminergic neuronal death. α-Syn binds to the kinase domain on TrkB, which is negatively regulated by BDNF or Fyn tyrosine kinase. Interestingly, α-Syn represses TrkB lipid raft distribution, decreases its internalization, and reduces its axonal trafficking. Moreover, α-Syn also reduces TrkB protein levels via up-regulation of TrkB ubiquitination. Remarkably, dopamine’s metabolite 3,4-Dihydroxyphenylacetaldehyde (DOPAL) stimulates the interaction between α-Syn and TrkB. Accordingly, MAO-B inhibitor rasagiline disrupts α-Syn/TrkB complex and rescues TrkB neurotrophic signaling, preventing α-Syn–induced dopaminergic neuronal death and restoring motor functions. Hence, our findings demonstrate a noble pathological role of α-Syn in antagonizing neurotrophic signaling, providing a molecular mechanism that accounts for its neurotoxicity in PD.  

3.2847           Organelle-specific single-molecule imaging of α4β2 nicotinic receptors reveals the effect of nicotine on receptor assembly and cell-surface trafficking

Fox-Loe, A.M., Moonschi, F.H. and Richrads, C.I.

1. Biol. Chem., 292(51), 21159-21169 (2017)

  Nicotinic acetylcholine receptors (nAChRs) assemble in the endoplasmic reticulum (ER) and traffic to the cell surface as pentamers composed of α and β subunits. Many nAChR subtypes can assemble with varying subunit ratios, giving rise to multiple stoichiometries exhibiting different subcellular localization and functional properties. In addition to the endogenous neurotransmitter acetylcholine, nicotine also binds and activates nAChRs and influences their trafficking and expression on the cell surface. Currently, no available technique can specifically elucidate the stoichiometry of nAChRs in the ER versus those in the plasma membrane. Here, we report a method involving single-molecule fluorescence measurements to determine the structural properties of these membrane proteins after isolation in nanoscale vesicles derived from specific organelles. These cell-derived nanovesicles allowed us to separate single membrane receptors while maintaining them in their physiological environment. Sorting the vesicles according to the organelle of origin enabled us to determine localized differences in receptor structural properties, structural influence on transport between organelles, and changes in receptor assembly within intracellular organelles. These organelle-specific nanovesicles revealed that one structural isoform of the α4β2 nAChR was preferentially trafficked to the cell surface. Moreover, nicotine altered nAChR assembly in the ER, resulting in increased production of the receptor isoform that traffics more efficiently to the cell surface. We conclude that the combined effects of the increased assembly of one nAChR stoichiometry and its preferential trafficking likely drive the up-regulation of nAChRs on the cell surface upon nicotine exposure.  

3.2848           Cell-Engineered Nanovesicle as a Surrogate Inducer of Contact-Dependent Stimuli

Kim, J., Han, C., Jo, W., Kang, S., Cho, S., Jeong, D., Gho, Y.S. and Park, J. Adv. Healthcare Mater., 6(17), 1700381 (2017)   Heterotypic interactions between cells are crucial in various biological phenomena. Particularly, stimuli that regulate embryonic stem cell (ESC) fate are often provided from neighboring cells. However, except for feeder cultures, no practical methods are identified that can provide ESCs with contact-dependent cell stimuli. To induce contact-dependent cell stimuli in the absence of living cells, a novel method that utilizes cell-engineered nanovesicles (CNVs) that are made by extruding living cells through microporous membranes is described. Protein compositions of CNVs are similar to their originating cells, as well as freely diffusible and precisely scalable. Treatment of CNVs produced from three different stromal cells successfully induces the same effect as feeder cultures. The results suggest that the effects of CNVs are mainly mediated by membrane-associated components. The use of CNVs might constitute a novel and efficient tool for ESC research.  

3.2849           Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2

Liu, P., Huang, J., Zheng, Q., Xie, l., Lu, X., Jin, J. and Wang, G. Protein & Cell, 8(10), 735-749 (2017)   Mammalian mitochondrial genome encodes a small set of tRNAs, rRNAs, and mRNAs. The RNA synthesis process has been well characterized. How the RNAs are degraded, however, is poorly understood. It was long assumed that the degradation happens in the matrix where transcription and translation machineries reside. Here we show that contrary to the assumption, mammalian mitochondrial RNA degradation occurs in the mitochondrial intermembrane space (IMS) and the IMS-localized RNASET2 is the enzyme that degrades the RNAs. This provides a new paradigm for understanding mitochondrial RNA metabolism and transport.  

3.2850           Modulation of Autophagy by BDNF Underlies Synaptic Plasticity

Nikoletopoulou, V., Sidiropoulou, K., Kallergi, E., Dalezios, Y. and Tavernarakis, N. Cell Metabolism, 26, 230-242 (2017)   Autophagy is crucial for neuronal integrity. Loss of key autophagic components leads to progressive neurodegeneration and structural defects in pre- and postsynaptic morphologies. However, the molecular mechanisms regulating autophagy in the brain remain elusive. Similarly, while it is widely accepted that protein turnover is required for synaptic plasticity, the contribution of autophagy to the degradation of synaptic proteins is unknown. Here, we report that BDNF signaling via the tropomyosin receptor kinase B (TrkB) and the phosphatidylinositol-3′ kinase (PI3K)/Akt pathway suppresses autophagy in vivo. In addition, we demonstrate that suppression of autophagy is required for BDNF-induced synaptic plasticity and for memory enhancement under conditions of nutritional stress. Finally, we identify three key remodelers of postsynaptic densities as cargo of autophagy. Our results establish autophagy as a pivotal component of BDNF signaling, which is essential for BDNF-induced synaptic plasticity. This molecular mechanism underlies behavioral adaptations that increase fitness in times of scarcity.    

3.2851           Expression and Subcellular Localization of the Kaposi's Sarcoma-Associated Herpesvirus K15P Protein during Latency and Lytic Reactivation in Primary Effusion Lymphoma Cells

Smith, C.G., Kharkwal, H. and Wilson, D.W.

1. Virol., 91(21), e01370-17 (2017)

  The K15P membrane protein of Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with multiple cellular signaling pathways and is thought to play key roles in KSHV-associated endothelial cell angiogenesis, regulation of B-cell receptor (BCR) signaling, and the survival, activation, and proliferation of BCR-negative primary effusion lymphoma (PEL) cells. Although full-length K15P is ∼45 kDa, numerous lower-molecular-weight forms of the protein exist as a result of differential splicing and poorly characterized posttranslational processing. K15P has been reported to localize to numerous subcellular organelles in heterologous expression studies, but there are limited data concerning the sorting of K15P in KSHV-infected cells. The relationships between the various molecular weight forms of K15P, their subcellular distribution, and how these may differ in latent and lytic KSHV infections are poorly understood. Here we report that a cDNA encoding a full-length, ∼45-kDa K15P reporter protein is expressed as an ∼23- to 24-kDa species that colocalizes with the trans-Golgi network (TGN) marker TGN46 in KSHV-infected PEL cells. Following lytic reactivation by sodium butyrate, the levels of the ∼23- to 24-kDa protein diminish, and the full-length, ∼45-kDa K15P protein accumulates. This is accompanied by apparent fragmentation of the TGN and redistribution of K15P to a dispersed peripheral location. Similar results were seen when lytic reactivation was stimulated by the KSHV protein replication and transcription activator (RTA) and during spontaneous reactivation. We speculate that expression of different molecular weight forms of K15P in distinct cellular locations reflects the alternative demands placed upon the protein in the latent and lytic phases.  

3.2852           Expression and Characterization of Membrane-Type 4 Matrix Metalloproteinase (MT4-MMP) and its Different Forms in Melanoma

Hieronimus, B., Pfohl, J., Busch, C. and Graeve, L. Cell. Physiol. Biochem., 42, 198-210 (2017)   Background/Aims: Membrane-type matrix metalloproteinases (MT-MMPs) are expressed on the cell surface and hydrolyze extracellular matrix components and signaling molecules by which they influence cancer cell migration and metastasis. Two of the six known MT-MMPs are anchored to the plasma membrane via a GPI anchor, one of which is MT4-MMP. Only little is known about MT4-MMP expression, synthesis, regulation and degradation. Methods: We analyzed several human cancer cell lines as well as tissue homogenates using Western blotting and quantitative PCR for the expression of MT4-MMP. Organelles of SK-Mel-28 cells were separated using continuous Iodixanol gradients. Glycosylation of the SK-Mel-28 protein was studied via glucosidases and site directed mutagenesis of the MT4-MMP cDNA prior to transfection. Results: We found the MT4-MMP highly expressed in human melanoma cell lines as well as skin and melanoma tissue samples. Three forms of MT4-MMP with molecular masses of 45 kDa, 58 kDa and 69 kDa were detected. Further, we demonstrate that the 58 kDa form is the mature protein in the cell membrane, while the 69 kDa form is its precursor found in intracellular compartments. The 69 kDa forms are processed by furin cleavage in the Golgi apparatus. Moreover, we identified Asn³¹⁸ as the single N-glycosylation site of MT4-MMP. Conclusion: We demonstrate the novel expression of MT4-MMP in melanocytic tissues and propose a precursor/product-relationship of the different forms of MT4-MMP in melanoma cells.  

3.2853           Organelle membrane derived patches: reshaping classical methods for new targets

Shapalov, G., Ritaine, A., Bidaux, G., Slomianny, C., Borowiec, A-S., Gordienko, D., Bultynck, G., Skryma, R. and Prevarskaya, N. Scientific Reports, 7:14082 (2017)   Intracellular ion channels are involved in multiple signaling processes, including such crucial ones as regulation of cellular motility and fate. With 95% of the cellular membrane belonging to intracellular organelles, it is hard to overestimate the importance of intracellular ion channels. Multiple studies have been performed on these channels over the years, however, a unified approach allowing not only to characterize their activity but also to study their regulation by partner proteins, analogous to the patch clamp “golden standard”, is lacking. Here, we present a universal approach that combines the extraction of intracellular membrane fractions with the preparation of patchable substrates that allows to characterize these channels in endogenous protein environment and to study their regulation by partner proteins. We validate this method by characterizing activity of multiple intracellular ion channels localized to different organelles and by providing detailed electrophysiological characterization of the regulation of IP₃R activity by endogenous Bcl-2. Thus, after synthesis and reshaping of the well-established approaches, organelle membrane derived patch clamp provides the means to assess ion channels from arbitrary cellular membranes at the single channel level.  

3.2854           Exenatide Prevents Morphological and Structural Changes of Mitochondria Following Ischaemia-Reperfusion Injury

Lee, K.H., Ha, S.J., Woo, J-S., Lee, G-J., Lee, S-R., Kim, J.W., Park, H.K. and Kim, W. Heart, Lung and Circulation, 26, 519-523 (2017)   Background Exenatide exerts cardioprotective effects by attenuating ischaemic reperfusion (IR) injury, possibly through activating the opening of mitochondrial ATP-sensitive potassium channels. We used atomic force microscopy (AFM) to investigate changes in mitochondrial morphology and properties in order to assess exenatide-mediated cardioprotection in IR injury. Methods We used an in vivo Sprague-Dawley rat IR model and ex vivo Langendorff injury model. In the left anterior descending artery (LAD) occlusion model, animals were randomly divided into three groups: sham-operated rats (Sham, n = 5), IR-injured rats treated with placebo (IR, n = 6), and IR-injured treated with exenatide (IR + EXE, n = 6). For the Langendorff model, rats were randomly divided into two groups: IR injury with placebo (IR, n = 4) and IR injury with exenatide (IR+EXE, n = 4). Morphological and mechanical changes of mitochondria were analysed by AFM. Results Exenatide pre-treatment improved cardiac function as evidenced by improvement in echocardiographic results. The ratio of infarct area (IA) to risk area (RA) was significantly reduced in exenatide-treated rats. According to AFM, IR significantly increased the area of isolated mitochondria, indicative of mitochondrial swelling. Treatment with exenatide reduced the mitochondrial area and ameliorated the adhesion force of mitochondrial surfaces. Conclusions Exenatide pre-treatment improves morphological and mechanical characteristics of mitochondria in response to IR injury in a rat model. These alterations in mitochondrial characteristics appear to play a cardioprotective role against IR injury.  

3.2855           Extracellular vesicles as emerging targets in cancer: Recent development from bench to bedside

Wu, K., Xing, F., Wu, S-Y. and Watabe, K. BBA – Review on Cancer, 1868. 538-563 (2017)   Extracellular vesicles (EVs) have emerged as important players of cancer initiation and progression through cell-cell communication. They have been recognized as critical mediators of extracellular communications, which promote transformation, growth invasion, and drug-resistance of cancer cells. Interestingly, the secretion and uptake of EVs are regulated in a more controlled manner than previously anticipated. EVs are classified into three groups, (i) exosomes, (ii) microvesicles (MVs), and (iii) apoptotic bodies (ABs), based on their sizes and origins, and novel technologies to isolate and distinguish these EVs are evolving. The biologically functional molecules harbored in these EVs, including nucleic acids, lipids, and proteins, have been shown to induce key signaling pathways in both tumor and tumor microenvironment (TME) cells for exacerbating tumor development. While tumor cell-derived EVs are capable of reprogramming stromal cells to generate a proper tumor cell niche, stromal-derived EVs profoundly affect the growth, resistance, and stem cell properties of tumor cells. This review summarizes and discusses these reciprocal communications through EVs in different types of cancers. Further understanding of the pathophysiological roles of different EVs in tumor progression is expected to lead to the discovery of novel biomarkers in liquid biopsy and development of tumor specific therapeutics. This review will also discuss the translational aspects of EVs and therapeutic opportunities of utilizing EVs in different cancer types.  

3.2856           Adipose Tissue Macrophage-Derived Exosomal miRNAs Can Modulate In Vivo and In Vitro Insulin Sensitivity

Ying, W., Riopel, M., Bandyopadhyay, G., Dong, Y., Birmingham, A., Seo, J.B., Ofrecio, J.M., Wollam, J., Hernandez-Carretero, A., Fu, W., Li, P. and Olefsky, J.M. Cell, 171(2), 372-384 (2017)   MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis.  

3.2857           Sortilin limits EGFR signaling by promoting its internalization in lung cancer

Al-Akhrass, H., Naves, T., Vincent, F., Magnaudeix, A., Durand, K., Bertin, F., Melloni, B., Jauberteau, M-O. and Lalloue, F. Nature Communications, 8:1182 (2017)   Tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR) transduce information from the microenvironment into the cell and activate homeostatic signaling pathways. Internalization and degradation of EGFR after ligand binding limits the intensity of proliferative signaling, thereby helping to maintain cell integrity. In cancer cells, deregulation of EGFR trafficking has a variety of effects on tumor progression. Here we report that sortilin is a key regulator of EGFR internalization. Loss of sortilin in tumor cells promoted cell proliferation by sustaining EGFR signaling at the cell surface, ultimately accelerating tumor growth. In lung cancer patients, sortilin expression decreased with increased pathologic grade, and expression of sortilin was strongly correlated with survival, especially in patients with high EGFR expression. Sortilin is therefore a regulator of EGFR intracellular trafficking that promotes receptor internalization and limits signaling, which in turn impacts tumor growth.  

3.2858           The lipid raft-dwelling protein US9 can be manipulated to target APP compartmentalization, APP processing, and neurodegenerative disease pathogenesis

Brandimarti, R., Hill, G.S., Geiger, J.D. and Meucci, O. Scientific Reports, 7:15103 (2017)   The trafficking behavior of the lipid raft-dwelling US9 protein from Herpes Simplex Virus strikingly overlaps with that of the amyloid precursor protein (APP). Both US9 and APP processing machinery rely on their ability to shuttle between endosomes and plasma membranes, as well as on their lateral accumulation in lipid rafts. Therefore, repurposing US9 to track/modify these molecular events represents a valid approach to investigate pathological states including Alzheimer’s disease and HIV-associated neurocognitive disorders where APP misprocessing to amyloid beta formation has been observed. Accordingly, we investigated the cellular localization of US9-driven cargo in neurons and created a US9-driven functional assay based on the exogenous enzymatic activity of Tobacco Etch Virus Protease. Our results demonstrate that US9 can direct and control cleavage of recombinant proteins exposed on the luminal leaflet of transport vesicles. Furthermore, we confirmed that US9 is associated with lipid-rafts and can target functional enzymes to membrane microdomains where pathologic APP-processing is thought to occur. Overall, our results suggest strongly that US9 can serve as a molecular driver that targets functional cargos to the APP machinery and can be used as a tool to study the contribution of lipid rafts to neurodegenerative disease conditions where amyloidogenesis has been implicated.  

3.2859           Exosomes in Prion Diseases

Hartmann, A., Altmeppen, H., Krasemann, S. and Glatzel, M. Neuromethods, 129, 197-207 (2017)   Dementias are characterized by generation and tissue deposition of proteins altered in their secondary or tertiary structure. Prion diseases are prominent and well-studied examples of these diseases. Initiation of prion disease is associated to the conversion of the cellular prion protein (PrP^C) to its pathogenic isoform (PrP^Sc). Spread of PrP^Sc throughout the central nervous system leads to disease progression and is achieved by cell-to-cell transfer, axonal or nanotube-mediated transport or exosomes. In this chapter we describe how to isolate, purify, and quality control exosomes, and provide helpful notes for practical guidance and troubleshooting in these techniques.  

3.2860           Technical Aspects for the Evaluation of Exosomes and Their Content

Fontana, S., Giallombardo, M. and Alessandro, R. Liquid Biopsy in Cancer Patients, 61-70 (2017)   Liquid biopsy is a precious source of exosomes, nanometer-sized vesicles (40–100 nm diameter) that play a relevant role in the cell-cell communication, strongly depending on the nature of the transported molecules (proteins, mRNAs, miRNAs, and lipids). Since a significant body of literature has demonstrated that exosomes released by cancer cells carry tumor-specific RNAs and proteins, they are widely considered very attractive targets for diagnostic application. This chapter focuses on the isolation and study of exosomes from liquid biopsies and summarizes the recent exosomal miRNA and protein profiling data supporting the potential role of tumor-derived exosomes as biomarkers and their potential application in clinical settings.  

3.2861           PIP1 aquaporins, sterols, and osmotic water permeability of plasma membranes from etiolated pea seedlings

Belugin, B.V., Zhestkova, I.M., Piotrovskii, M.S., Lapshin, N.K. and Trofimova, M.S. Biochemistry (Moscow), Suppl: Membrane and Cell Biol., 11(2), 168-176 (2017)   Plasma membrane isolated from microsomal membranes of pea seedling root and shoot cells by means of aqueous two-phase polymer system was separated by flotation in discontinuous OptiPrep gradient into “light” (≤1.146 g/cm³) and “heavy” (≥1.146 g/cm³) fractions. Osmotic water permeability of plasma membrane and its two fractions was investigated by inducing transmembrane osmotic gradient on the vesicle membrane and recording the kinetics of vesicle osmotic shrinkage by the stopped-flow method. Rate constants of osmotic shrinkage and coefficients of osmotic water permeability of the membranes were estimated on the basis of the kinetic curve approximation by exponential dependencies and using electron microscope data on vesicles sizes. In plasma membrane and its fractions the content of sterols and PIP1 aquaporins was determined. It was found that in “light” PM fractions from both roots and shoots the content of PIP1 aquaporins and sterols was higher and the osmotic water permeability coefficient was lower than in “heavy” fractions of plasma membrane. The results indicate that plasma membrane of roots and shoots is heterogeneous in osmotic water permeability. This heterogeneity may be related with the presence of microdomains with different content of aquaporins and sterols in the membrane.  

3.2862           Exploiting the Gastric Epithelial Barrier: Helicobacter pylori’s Attack on Tight and Adherens Junctions

Backert, S., Schmidt, T.P., harrer, A. and Wessler, S. Current Topics in Microbiol. Immunol., 400, 195-226 (2017)   Highly organized intercellular tight and adherens junctions are crucial structural components for establishing and maintenance of epithelial barrier functions, which control the microbiota and protect against intruding pathogens in humans. Alterations in these complexes represent key events in the development and progression of multiple infectious diseases as well as various cancers. The gastric pathogen Helicobacter pylori exerts an amazing set of strategies to manipulate these epithelial cell-to-cell junctions, which are implicated in changing cell polarity, migration and invasive growth as well as pro-inflammatory and proliferative responses. This chapter focuses on the H. pylori pathogenicity factors VacA, CagA, HtrA and urease, and how they can induce host cell signaling involved in altering cell-to-cell permeability. We propose a stepwise model for how H. pylori targets components of tight and adherens junctions in order to disrupt the gastric epithelial cell layer, giving fresh insights into the pathogenesis of this important bacterium.  

3.2863           Bacteriophage Transcytosis Provides a Mechanism To Cross Epithelial Cell Layers

Nguyen, S., Baker, K., PAdman, M.S., Patwa, R., Dunstan, R.A., Weston, T.A., Schlosser, K., Bailey, B., Lithgow, T., Lazarou, M., Luzue, A., Rohwer,m F., Blumberg, R.S. and Barr, J.J. mBio, 8(6), e01874-17 (2017)   viruses are among the most numerous biological entities within the human body. These viruses are found within regions of the body that have conventionally been considered sterile, including the blood, lymph, and organs. However, the primary mechanism that bacterial viruses use to bypass epithelial cell layers and access the body remains unknown. Here, we used in vitro studies to demonstrate the rapid and directional transcytosis of diverse bacteriophages across confluent cell layers originating from the gut, lung, liver, kidney, and brain. Bacteriophage transcytosis across cell layers had a significant preferential directionality for apical-to-basolateral transport, with approximately 0.1% of total bacteriophages applied being transcytosed over a 2-h period. Bacteriophages were capable of crossing the epithelial cell layer within 10 min with transport not significantly affected by the presence of bacterial endotoxins. Microscopy and cellular assays revealed that bacteriophages accessed both the vesicular and cytosolic compartments of the eukaryotic cell, with phage transcytosis suggested to traffic through the Golgi apparatus via the endomembrane system. Extrapolating from these results, we estimated that 31 billion bacteriophage particles are transcytosed across the epithelial cell layers of the gut into the average human body each day. The transcytosis of bacteriophages is a natural and ubiquitous process that provides a mechanistic explanation for the occurrence of phages within the body.  

3.2864           Isolation of exosomes from whole blood by integrating acoustics and microfluidics

Wu, M., Ouyang, Y., Wang, Z., Zhang, R., Huang, P-H., Chen, C., Li, H., Li, P., Quinn, D., Dao, M., Suresh, S., Sadovsky, Y. and Huang, T.J. PNAS, 114(40), 10584-10589 (2017)   Exosomes are nanoscale extracellular vesicles that play an important role in many biological processes, including intercellular communications, antigen presentation, and the transport of proteins, RNA, and other molecules. Recently there has been significant interest in exosome-related fundamental research, seeking new exosome-based biomarkers for health monitoring and disease diagnoses. Here, we report a separation method based on acoustofluidics (i.e., the integration of acoustics and microfluidics) to isolate exosomes directly from whole blood in a label-free and contact-free manner. This acoustofluidic platform consists of two modules: a microscale cell-removal module that first removes larger blood components, followed by extracellular vesicle subgroup separation in the exosome-isolation module. In the cell-removal module, we demonstrate the isolation of 110-nm particles from a mixture of micro- and nanosized particles with a yield greater than 99%. In the exosome-isolation module, we isolate exosomes from an extracellular vesicle mixture with a purity of 98.4%. Integrating the two acoustofluidic modules onto a single chip, we isolated exosomes from whole blood with a blood cell removal rate of over 99.999%. With its ability to perform rapid, biocompatible, label-free, contact-free, and continuous-flow exosome isolation, the integrated acoustofluidic device offers a unique approach to investigate the role of exosomes in the onset and progression of human diseases with potential applications in health monitoring, medical diagnosis, targeted drug delivery, and personalized medicine.  

3.2865           A novel intracellular pool of LFA-1 is critical for asymmetric CD8+ T cell activation and differentiation

Capece, T., Walling, B.L., Lim, K., Kim, K-D., bae, S., Chung, H-L., Topham, D.J. and Kim, M.

1. Cell Biol., 216(11), 3817-3829 (2017)

  The integrin lymphocyte function–associated antigen 1 (LFA-1; CD11a/CD18) is a key T cell adhesion receptor that mediates stable interactions with antigen-presenting cell (APC), as well as chemokine-mediated migration. Using our newly generated CD11a-mYFP knock-in mice, we discovered that naive CD8⁺ T cells reserve a significant intracellular pool of LFA-1 in the uropod during migration. Intracellular LFA-1 quickly translocated to the cell surface with antigenic stimulus. Importantly, the redistribution of intracellular LFA-1 at the contact with APC was maintained during cell division and led to an unequal inheritance of LFA-1 in divided T cells. The daughter CD8⁺ T cells with disparate LFA-1 expression showed different patterns of migration on ICAM-1, APC interactions, and tissue retention, as well as altered effector functions. In addition, we identified Rab27 as an important regulator of the intracellular LFA-1 translocation. Collectively, our data demonstrate that an intracellular pool of LFA-1 in naive CD8⁺ T cells plays a key role in T cell activation and differentiation.  

3.2866           Nef is secreted in exosomes from Nef.GFP-expressing and HIV-1-infected human astrocytes

Diminkus, P.P., Ferdin, J., Plemenitas, A., Peterlin, B.M. and Lenassi, M.

1. Neurovirol., 23, 713-724 (2017)

  HIV-1 infection of the central nervous system causes HIV-associated neurocognitive disorders, even in aviremic patients. Although astrocyte malfunction was associated to these disorders, their implication is overshadowed by contributions of microglia and macrophages. Astrocytes are infected with HIV-1 in vivo and express a relevant amount of viral protein Nef. Nef was shown to stimulate its own release in exosomes from diverse cell types, which in turn have damaging effects on neighboring cells. Using immunoblotting and electron microscopy, we showed that human astrocytes expressing Nef.GFP similarly release Nef in exosomes. Importantly, Nef.GFP expression increases the secretion of exosomes from human astrocytes up to 5.5-fold, as determined by total protein content and nanoparticle tracking analysis. Protein analysis of exosomes and viruses separated on iodixanol gradient further showed that native or pseudotyped HIV-1-infected human astrocytes release exosomes, which contain Nef. Our results provide the basis for future studies of the damaging role of Nef-exosomes produced by HIV-infected astrocytes on the central nervous system.  

3.2867           SG2NA is a regulator of endoplasmic reticulum (ER) homeostasis as its depletion leads to ER stress

Jain, B.P., Pandey, S., Saleem, N., tanti, G.T., Mishra, s. and Goswami, S.K: Cell Stress and Chaperones, 22, 853-866 (2017)   SG2NA belongs to a three-member striatin subfamily of WD40 repeat superfamily of proteins. It has multiple protein-protein interaction domains involved in assembling supramolecular signaling complexes. Earlier, we had demonstrated that there are at least five variants of SG2NA generated by alternative splicing, intron retention, and RNA editing. Such versatile and dynamic mode of regulation implicates it in tissue development. In order to shed light on its role in cell physiology, total proteome analysis was performed in NIH3T3 cells depleted of 78 kDa SG2NA, the only isoform expressing therein. A number of ER stress markers were among those modulated after knockdown of SG2NA. In cells treated with the ER stressors thapsigargin and tunicamycin, expression of SG2NA was increased at both mRNA and protein levels. The increased level of SG2NA was primarily in the mitochondria and the microsomes. A mouse injected with thapsigargin also had an increase in SG2NA in the liver but not in the brain. Cell cycle analysis suggested that while loss of SG2NA reduces the level of cyclin D1 and retains a population of cells in the G1 phase, concurrent ER stress facilitates their exit from G1 and traverse through subsequent phases with concomitant cell death. Thus, SG2NA is a component of intrinsic regulatory pathways that maintains ER homeostasis.  

3.2868           Characterization of a novel role for the dynamin mechanoenzymes in the regulation of human sperm acrosomal exocytosis

Zhou, W., Anderson, A.L., Turner, A.P., De lullis, G.N., McCluskey, A., Mclaughlin, E.A. and Nixon, B. Mol. Hum. Reprod., 23(10), 657-673 (2017)   STUDY QUESTION Does dynamin regulate human sperm acrosomal exocytosis? SUMMARY ANSWER Our studies of dynamin localization and function have implicated this family of mechanoenzymes in the regulation of progesterone-induced acrosomal exocytosis in human spermatozoa. WHAT IS KNOWN ALREADY Completion of an acrosome reaction is a prerequisite for successful fertilization in all studied mammalian species. It follows that failure to complete this unique exocytotic event represents a common aetiology in the defective spermatozoa of male infertility patients that have failed IVF in a clinical setting. Recent studies have implicated the dynamin family of mechanoenzymes as important regulators of the acrosome reaction in murine spermatozoa. The biological basis of this activity appears to rest with the ability of dynamin to polymerize around newly formed membrane vesicles and subsequently regulate the rate of fusion pore expansion. To date, however, the dynamin family of GTPases have not been studied in the spermatozoa of non-rodent species. Here, we have sought to examine the presence and functional significance of dynamin in human spermatozoa. STUDY DESIGN, SIZE, DURATION Dynamin expression was characterized in the testis and spermatozoa of several healthy normozoospermic individuals. In addition, we assessed the influence of selective dynamin inhibition on the competence of human spermatozoa to undergo a progesterone-induced acrosome reaction. A minimum of five biological and technical replicates were performed to investigate both inter- and intra-donor variability in dynamin expression and establish statistical significance in terms of the impact of dynamin inhibition. PARTICIPANTS/MATERIALS, SETTING, METHODS The expression and the localization of dynamin in the human testis, epididymis and mature spermatozoa were determined through the application of immunofluorescence, immunoblotting and/or electron microscopy. Human semen samples were fractionated via density gradient centrifugation and the resultant populations of good and poor quality spermatozoa were induced to capacitate and acrosome react in the presence or absence of selective dynamin inhibitors. The acrosome integrity of live spermatozoa was subsequently assessed via the use of fluorescently conjugated Arachis hypogea lectin (PNA). The influence of dynamin phosphorylation and the regulatory kinase(s) responsible for this modification in human spermatozoa were also assessed via the use of in situ proximity ligation assays and pharmacological inhibition. In all experiments, ≥100 spermatozoa were assessed/treatment group and all graphical data are presented as the mean values ± SEM, with statistical significance being determined by ANOVA. MAIN RESULTS AND THE ROLE OF CHANCE Dynamin 1 (DNM1) and DNM2, but not DNM3, were specifically localized to the acrosomal region of the head of human spermatozoa, an ideal position from which to regulate acrosomal exocytosis. In keeping with this notion, pharmacological inhibition of DNM1 and DNM2 was able to significantly suppress the rates of acrosomal exocytosis stimulated by progesterone. Furthermore, our comparison of dynamin expression in good and poor quality spermatozoa recovered from the same ejaculate, revealed a significant reduction in the amount of DNM2 in the latter subpopulation of cells. In contrast, DNM1 was detected at equivalent levels in both subpopulations of spermatozoa. Such findings are of potential significance given that the poor quality spermatozoa proved refractory to the induction of a progesterone stimulated acrosome reaction. In seeking to identify the regulatory influence of progesterone on DNM2 function, we were able to establish that the protein is a substrate for CDK1-dependent phosphorylation. The functional significance of DNM2 phosphorylation was illustrated by the fact that pharmacological inhibition of CDK1 elicited a concomitant suppression of both DNM2-Ser764 phosphorylation and the overall rates of progesterone-induced acrosomal exocytosis.  

3.2869           Albuminuria is not associated with elevated urinary vesicle concentration but can confound nanoparticle tracking analysis

McNicholas, K., Li, J.Y., Michael, M.Z. and Gleadle, J.M: Nephrology, 22(11), 854-863 (2017)   Aim Extracellular vesicles, such as exosomes, are present in urine with reports of roles in intercellular signalling and diagnostic utility. However, the extent to which the concentration and characteristics of urinary vesicles are altered in albuminuric renal disease has not been well characterized. In this study, we examined the number and characteristics of extracellular vesicles in albuminuric urine. Methods Vesicles were isolated from the urine of 32 patients with varying levels of albuminuria using ultracentrifugation and density gradient purification and were examined using nanoparticle tracking analysis, immunoblotting and transmission electron microscopy. The size profile of particles in these urine preparations was compared with albumin-containing solutions. Results Overall, there were no substantial differences in the number, or characteristics, of vesicles released into proteinuric urine. Analysis of albumin-containing solutions showed particles of exosome-like size, suggesting that such particles can mimic exosomes in standard nanoparticle tracking analysis. Albumin and IgG depletion of proteinuric urine resulted in a substantial reduction in the concentration of particles detected by nanoparticle tracking analysis. Conclusion There was no increase in urinary vesicle concentration in patients with albuminuria. Furthermore, these results demonstrate the need for cautious interpretation of nanoparticle tracking analysis of vesicle concentration in biological fluids containing protein and for sophisticated preparative methods in vesicle purification from urine.  

3.2870           Proteomics Insights into Autophagy

Cudjoe, Jr., E.K., Saleh, T., Hawkridge, A.M. and Gewirtz, D.A. Proteomics, 17, 1700022 (2017)   Autophagy, a conserved cellular process by which cells recycle their contents either to maintain basal homeostasis or in response to external stimuli, has for the past two decades become one of the most studied physiological processes in cell biology. The 2016 Nobel Prize in Medicine and Biology awarded to Dr. Ohsumi Yoshinori, one of the first scientists to characterize this cellular mechanism, attests to its importance. The induction and consequent completion of the process of autophagy results in wide ranging changes to the cellular proteome as well as the secretome. MS-based proteomics affords the ability to measure, in an unbiased manner, the ubiquitous changes that occur when autophagy is initiated and progresses in the cell. The continuous improvements and advances in mass spectrometers, especially relating to ionization sources and detectors, coupled with advances in proteomics experimental design, has made it possible to study autophagy, among other process, in great detail. Innovative labeling strategies and protein separation techniques as well as complementary methods including immuno-capture/blotting/staining have been used in proteomics studies to provide more specific protein identification. In this review, we will discuss recent advances in proteomics studies focused on autophagy.  

3.2871           Extracellular vesicles in the tumor microenvironment: Therapeutic resistance, clinical biomarkers, and targeting strategies

Han, L., Xu, J., Xu, Q., Zhang, B., lam, E.W-F. and Sun, Y. Med. Res. Rev., 37(6), 1318-1349 (2017)   Numerous studies have proved that cell-nonautonomous regulation of neoplastic cells is a distinctive and essential characteristic of tumorigenesis. Two way communications between the tumor and the stroma, or within the tumor significantly influence disease progression and modify treatment responses. In the tumor microenvironment (TME), malignant cells utilize paracrine signaling initiated by adjacent stromal cells to acquire resistance against multiple types of anticancer therapies, wherein extracellular vesicles (EVs) substantially promote such events. EVs are nanoscaled particles enclosed by phospholipid bilayers, and can mediate intercellular communications between cancerous cells and the adjacent microenvironment to accelerate pathological proceeding. Here we review the most recent studies of EV biology and focus on key cell lineages of the TME and their EV cargoes that are biologically active and responsible for cancer resistance, including proteins, RNAs, and other potentially essential components. Since EVs are emerging as novel but critical elements in establishing and maintaining hallmarks of human cancer, timely and insightful understanding of their molecular properties and functional mechanisms would pave the road for clinical diagnosis, prognosis, and effective targeting in the global landscape of precision medicine. Further, we address the potential of EVs as promising biomarkers in cancer clinics and summarize the technical improvements in EV preparation, analysis, and imaging. We highlight the practical issues that should be exercised with caution to guide the development of targeting agents and therapeutic methodologies to minimize cancer resistance driven by EVs, thereby allowing to effectively control the early steps of disease exacerbation.  

3.2872           Accumulation of undegraded autophagosomes by expression of dominant-negative STX17 (syntaxin 17) mutants

Uematsu, M., Nishimura, T., Sakamaki, Y., Yamamoto, H. and Mizushima, N. Autophagy, 13(8), 1452-1464 (2017)   Macroautophagy/autophagy, which is one of the main degradation systems in the cell, is mediated by a specialized organelle, the autophagosome. Purification of autophagosomes before fusion with lysosomes is important for both mechanistic and physiological studies of the autophagosome. Here, we report a simple method to accumulate undigested autophagosomes. Overexpression of the autophagosomal Qa-SNARE STX17 (syntaxin 17) lacking the N-terminal domain (NTD) or N-terminally tagged GFP-STX17 causes accumulation of autophagosomes. A HeLa cell line, which expresses GFP-STX17ΔNTD or full-length GFP-STX17 under the control of the tetracycline-responsive promoter, accumulates a large number of undigested autophagosomes devoid of lysosomal markers or early autophagy factors upon treatment with doxycycline. Using this inducible cell line, nascent autophagosomes can be easily purified by OptiPrep density-gradient centrifugation and immunoprecipitation. This novel method should be useful for further characterization of nascent autophagosomes.  

3.2873           Targeting of Cellular Organelles by Fluorescent Plasmid DNA Nanoparticles

Costa, D., Costa, C., Caldeira, M., Cortes, L., Queiroz, J.A. and Cruz, C. Biomacromolecules, 18(9), 2928-2936 (2017)   The development of a suitable delivery system and the targeting of intracellular organelles are both essential for the success of drug and gene therapies. The conception of fluorescent ligands, displaying targeting specificity together with low toxicity, is an emerging and reliable tool to develop innovative delivery systems. Biocompatible BSA or pDNA/ligand nanoparticles were synthesized by a coprecipitation method and were shown to display adequate sizes and morphology for delivery purposes, and positive surface charges. Additionally, these fluorescent vectors can target specific intracellular organelles. In vitro transfection mediated by BSA or pDNA based carriers can result in the accumulation of BSA in the cytosol, lysosomes, and mitochondria or the expression of the plasmid-encoded protein, respectively. Moreover, the therapeutic effect of pDNA/ligand vectors in cancer gene therapy instigates further research aiming clinical translation.  

3.2874           Renal Regenerative Potential of Different Extracellular Vesicle Populations Derived from Bone Marrow Mesenchymal Stromal Cells free access

Bruno, S., Tapparo, M., Collino, F., Chiabotto, G., Deregibus, M.C., Lindoso, R.S., Neri, F., Kholia, S., Giunti, S., Wen, S., Qusenberry, P. and Camussi, G. Tissue Engineering: Part A, 23(21-22), 1262-1273 (2017)   Extracellular vesicles (EVs) derived from human bone marrow mesenchymal stromal cells (MSCs) promote the regeneration of kidneys in different animal models of acute kidney injury (AKI) in a manner comparable with the cells of origin. However, due to the heterogeneity observed in the EVs isolated from MSCs, it is unclear which population is responsible for the proregenerative effects. We therefore evaluated the effect of various EV populations separated by differential ultracentrifugation (10K population enriched with microvesicles and 100K population enriched with exosomes) on AKI recovery. Only the exosomal-enriched population induced an improvement of renal function and morphology comparable with that of the total EV population. Interestingly, the 100K EVs exerted a proproliferative effect on murine tubular epithelial cells, both in vitro and in vivo. Analysis of the molecular content from the different EV populations revealed a distinct profile. The 100K population, for instance, was enriched in specific mRNAs (CCNB1, CDK8, CDC6) reported to influence cell cycle entry and progression; miRNAs involved in regulating proliferative/antiapoptotic pathways and growth factors (hepatocyte growth factor and insulin-like growth factor-1) that could explain the effect of renal tubular cell proliferation. On the other hand, the EV population enriched in microvesicles (10K) was unable to induce renal regeneration and had a molecular profile with lower expression of proproliferative molecules. In conclusion, the different molecular composition of exosome- and microvesicle-enriched populations may explain the regenerative effect of EVs observed in AKI.  

3.2875           Phosphorylation of LAMP2A by p38 MAPK couples ER stress to chaperone-mediated autophagy

Li, W., Zhu, J., Dou, J., She, H., tao, K., Xu, H., yang, Q. and Mao, Z. Nature Communications, 8:1763 (2017)   Endoplasmic reticulum (ER) and lysosomes coordinate a network of key cellular processes including unfolded protein response (UPR) and autophagy in response to stress. How ER stress is signaled to lysosomes remains elusive. Here we find that ER disturbance activates chaperone-mediated autophagy (CMA). ER stressors lead to a PERK-dependent activation and recruitment of MKK4 to lysosomes, activating p38 MAPK at lysosomes. Lysosomal p38 MAPK directly phosphorylates the CMA receptor LAMP2A at T211 and T213, which causes its membrane accumulation and active conformational change, activating CMA. Loss of ER stress-induced CMA activation sensitizes cells to ER stress-induced death. Neurotoxins associated with Parkinson’s disease fully engages ER-p38 MAPK–CMA pathway in the mouse brain and uncoupling it results in a greater loss of SNc dopaminergic neurons. This work identifies the coupling of ER and CMA as a critical regulatory axis fundamental for physiological and pathological stress response.  

3.2876           A subset of extracellular vesicles carries the bulk of microRNAs in commercial dairy cow’s milk

Benmoussa, A., Ly, S., Shan, S.T., Laugier, J., Boilard, E., Gilbert, C. and Provost, P.

1. Extracellular Vesicles, 6, 1401897 (2017)

  MicroRNAs are small gene-regulatory RNAs that are found in various biological fluids, including milk, where they are often contained inside extracellular vesicles (EVs), like exosomes. In a previous study, we reported that commercial dairy cow’s milk microRNAs resisted simulated digestion and were not exclusively associated with canonical exosomes. Here, we report the characterization of a milk EV subset that sediments at lower ultracentrifugation speeds and that contains the bulk of microRNAs. Milk EVs were isolated by differential ultracentrifugation and Iodixanol density gradient (IDG), and analysed for (1) microRNA enrichment by reverse transcription and quantitative polymerase chain reaction (RT-qPCR), and (2) EV-associated proteins by Western blot. Milk EVs were characterized further by dynamic light scattering (DLS), density measurements, fluorescent DiR and RNA labelling, high-sensitivity flow cytometry (HS-FCM), transmission electron microscopy (TEM), proteinase K and RNase A assay, and liquid chromatography tandem-mass spectrometry (LC-MS/MS). We found that the bulk of milk microRNAs (e.g., bta-miR-125b, bta-miR-148a, etc.) sediment at 12,000 g and 35,000 g. Their distribution pattern was different from that of exosome-enriched proteins, but similar to that of several proteins commonly found in milk fat globule membranes (MFGM), including xanthine dehydrogenase (XDH). These low-speed ultracentrifugation pellets contained cytoplasm-enclosing phospholipid bilayered membrane vesicles of a density comprised between 1.11 and 1.14 g/mL in Iodixanol. This milk EV subset of ~100 nm in diameter/~200 nm hydrodynamic size resisted to proteinase K digestion and protected their microRNA content from RNase A digestion. Our results support the existence of a milk EV subset pelleting at low ultracentrifugations speeds, with a protein coating comparable with MFGM, which contains and protects the bulk of milk microRNAs from degradation. This milk EV subset may represent a new EV population of interest, whose content in microRNAs and proteins supports its potential bioactivity.  

3.2877           ORAI channels are critical for receptor-mediated endocytosis of albumin

Zeng, B. et al Nature Communications, 8:1920 (2017)   Impaired albumin reabsorption by proximal tubular epithelial cells (PTECs) has been highlighted in diabetic nephropathy (DN), but little is known about the underlying molecular mechanisms. Here we find that ORAI1-3, are preferentially expressed in PTECs and downregulated in patients with DN. Hyperglycemia or blockade of insulin signaling reduces the expression of ORAI1-3. Inhibition of ORAI channels by BTP2 and diethylstilbestrol or silencing of ORAI expression impairs albumin uptake. Transgenic mice expressing a dominant-negative Orai1 mutant (E108Q) increases albuminuria, and in vivo injection of BTP2 exacerbates albuminuria in streptozotocin-induced and Akita diabetic mice. The albumin endocytosis is Ca2+-dependent and accompanied by ORAI1 internalization. Amnionless (AMN) associates with ORAIs and forms STIM/ORAI/AMN complexes after Ca2+ store depletion. STIM1/ORAI1 colocalizes with clathrin, but not with caveolin, at the apical membrane of PTECs, which determines clathrin-mediated endocytosis. These findings provide insights into the mechanisms of protein reabsorption and potential targets for treating diabetic proteinuria.  

3.2878           Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors

Sisquella, X. et al Nature Communications, 8, 1985 (2017)   STING is an innate immune cytosolic adaptor for DNA sensors that engage malaria parasite (Plasmodium falciparum) or other pathogen DNA. As P. falciparum infects red blood cells and not leukocytes, how parasite DNA reaches such host cytosolic DNA sensors in immune cells is unclear. Here we show that malaria parasites inside red blood cells can engage host cytosolic innate immune cell receptors from a distance by secreting extracellular vesicles (EV) containing parasitic small RNA and genomic DNA. Upon internalization of DNA-harboring EVs by human monocytes, P. falciparum DNA is released within the host cell cytosol, leading to STING-dependent DNA sensing. STING subsequently activates the kinase TBK1, which phosphorylates the transcription factor IRF3, causing IRF3 to translocate to the nucleus and induce STING-dependent gene expression. This DNA-sensing pathway may be an important decoy mechanism to promote P. falciparum virulence and thereby may affect future strategies to treat malaria.  

3.2879           Endocytosis regulates TDP-43 toxicity and turnover

Liu, G., Coyne, A.N., Pei, F., Vaughan, S., Chaung, M., Zarnescu, D.C. and Buchan, J.R. Nature Communications, 8:2092 (2017)   Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease. ALS-affected motor neurons exhibit aberrant localization of a nuclear RNA binding protein, TDP-43, into cytoplasmic aggregates, which contributes to pathology via unclear mechanisms. Here, we demonstrate that TDP-43 turnover and toxicity depend in part upon the endocytosis pathway. TDP-43 inhibits endocytosis, and co-localizes strongly with endocytic proteins, including in ALS patient tissue. Impairing endocytosis increases TDP-43 toxicity, aggregation, and protein levels, whereas enhancing endocytosis reverses these phenotypes. Locomotor dysfunction in a TDP-43 ALS fly model is also exacerbated and suppressed by impairment and enhancement of endocytic function, respectively. Thus, endocytosis dysfunction may be an underlying cause of ALS pathology.  

3.2880           The small vesicular culprits: the investigation of extracellular vesicles as new targets for cancer treatment

Urabe, F., Kosaka, N., Yoshioka, Y., Egawa, S. and Ochiya, T. Clin. Trans. Med., 6:45 (2017)   Extracellular vesicles (EVs) are membranous vesicles released from almost all type of cells including cancer cells. EVs transfer their components, such as microRNAs (miRNAs), messenger RNAs, lipids and proteins, from one cell to another, affecting the target cells. Emerging evidence suggests that reciprocal interactions between cancer cells and the cells in their microenvironment via EVs drive disease progression and therapy resistance. Therefore, understanding the roles of EVs in cancer biology will provide us with new opportunities to treat patients. EVs are also useful for monitoring disease processes. EVs have been found in many kinds of biological fluids such as blood, urine, saliva and semen. Because of their accessibility, EVs offer ease of collection with minimal discomfort to patients and are preferred for serial collection. In addition, they reflect and carry dynamic changes in disease, allowing us to access crucial molecular information about the disease status. Therefore, EVs hold great possibility as clinically useful biomarkers to provide multiple non-invasive snapshots of primary and metastatic tumors. In this review, we summarize current knowledge of miRNAs in EVs in cancer biology and as biomarkers. Furthermore, we discuss the potential of miRNAs in EVs for clinical application.  

3.2881           A method for the isolation and enrichment of purified bovine milk exosomes

Vaswani, K., Koh, Y.Q., Almughlliq, F.B. and Peiris, H.N.’ Reprod. Biol., 17, 341-348 (2017)   Exosomes are nanovesicles that play important roles in intercellular communication as they carry information to target cells. Isolation of high purity exosomes will aid in studying the exosomal cargo and quantity as well as how cell-specific messages are carried. We describe a new method incorporating size exclusion chromatography (SEC) to enrich milk-derived exosomes from extracellular vesicles (EVs). This involved the initial isolation of EVs from bovine milk via milk processing and ultracentrifugation; followed by a new method to enrich exosomes using SEC. This method was compared to buoyant density gradient centrifugation, a widely used method of enrichment. Exosomes were characterised by particle concentration and size (nanoparticle tracking analysis, NTA), morphology (transmission electron microscopy, TEM), presence of exosomal markers (immunoblotting) and protein concentration (bicinchoninic acid assay, BCA). Proteomic profiles of exosomal fractions were analyzed by mass spectrometry using Information Dependant Acquisition. Milk exosomal fractions were shown to contain exosomal markers flotillin-1 (FLOT-1) and tumor susceptibility gene-101 (TSG-101). The new method produced a higher yield of exosomes compared to buoyant density gradient centrifugation. Pooled exosomal fractions exhibited intact morphology by TEM. The use of SEC confirmed the fractionation of exosomes based on size while minimizing the interference with proteins. Tetraspanins CD9 and CD81 were observed via mass spectrometry in exosomal fractions. This new and efficient method confirmed the signatures for exosomes derived from unpasteurized bovine milk. Purification of exosomes is a foundational technique in the study of biomarkers for pathological conditions and effective drug delivery systems.          

3.2882           An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues

Corces, M.R. et al Nature Methods, 14(10), 959-962 (2017)   We present Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling that works across multiple applications with substantial improvement of signal-to-background ratio and information content. The Omni-ATAC protocol generates chromatin accessibility profiles from archival frozen tissue samples and 50-μm sections, revealing the activities of disease-associated DNA elements in distinct human brain structures. The Omni-ATAC protocol enables the interrogation of personal regulomes in tissue context and translational studies.  

3.2883           Communication via extracellular vesicles enhances viral infection of a cosmopolitan alga

Schatz, D., Rosenwasser, S., Malitsky, S.M., Wolf, S.G., Feldmesser, E. and Vardi, A. Nature Microbiol., 2, 1485-1492 (2017)   Communication between microorganisms in the marine environment has immense ecological impact by mediating trophic-level interactions and thus determining community structure1. Extracellular vesicles (EVs) are produced by bacteria2,3, archaea4, protists5 and metazoans, and can mediate pathogenicity6 or act as vectors for intercellular communication. However, little is known about the involvement of EVs in microbial interactions in the marine environment7. Here we investigated the signalling role of EVs produced during interactions between the cosmopolitan alga Emiliania huxleyi and its specific virus (EhV, Phycodnaviridae)8, which leads to the demise of these large-scale oceanic blooms9,10. We found that EVs are highly produced during viral infection or when bystander cells are exposed to infochemicals derived from infected cells. These vesicles have a unique lipid composition that differs from that of viruses and their infected host cells, and their cargo is composed of specific small RNAs that are predicted to target sphingolipid metabolism and cell-cycle pathways. EVs can be internalized by E. huxleyi cells, which consequently leads to a faster viral infection dynamic. EVs can also prolong EhV half-life in the extracellular milieu. We propose that EVs are exploited by viruses to sustain efficient infectivity and propagation across E. huxleyi blooms. As these algal blooms have an immense impact on the cycling of carbon and other nutrients11,12, this mode of cell–cell communication may influence the fate of the blooms and, consequently, the composition and flow of nutrients in marine microbial food webs.  

3.2884           Genetic variation in glia–neuron signalling modulates ageing rate

Yin, J-A., gao, G., Liu, X-J., Hao, Z-Q., Li, K., kang, X-L., Li, H., Shan, Y-H., Hu, W-L., Li, H-P. and Cai, S-Q. Nature, 551, 198-203 (2017)   The rate of behavioural decline in the ageing population is remarkably variable among individuals. Despite the considerable interest in studying natural variation in ageing rate to identify factors that control healthy ageing, no such factor has yet been found. Here we report a genetic basis for variation in ageing rates in Caenorhabditis elegans. We find that C. elegans isolates show diverse lifespan and age-related declines in virility, pharyngeal pumping, and locomotion. DNA polymorphisms in a novel peptide-coding gene, named regulatory-gene-for-behavioural-ageing-1 (rgba-1), and the neuropeptide receptor gene npr-28 influence the rate of age-related decline of worm mating behaviour; these two genes might have been subjected to recent selective sweeps. Glia-derived RGBA-1 activates NPR-28 signalling, which acts in serotonergic and dopaminergic neurons to accelerate behavioural deterioration. This signalling involves the SIR-2.1-dependent activation of the mitochondrial unfolded protein response, a pathway that modulates ageing. Thus, natural variation in neuropeptide-mediated glia–neuron signalling modulates the rate of ageing in C. elegans.  

3.2885           Multiple marker abundance profiling: combining selected reaction monitoring and data-dependent acquisition for rapid estimation of organelle abundance in subcellular samples

Hooper, C.M., Stevens, T.J., Saukkonen, A., Castleden, I.R., Singh, P., Mann, G.W., Fabre, B., Ito, J., Deery, M.J., Lilley, K.S., Petzold, C.J., Millar, A.H., Heazlewood, J.L. and Parsons, H.T. Plant J., 92(6), 1202-1217 (2017)   Measuring changes in protein or organelle abundance in the cell is an essential, but challenging aspect of cell biology. Frequently-used methods for determining organelle abundance typically rely on detection of a very few marker proteins, so are unsatisfactory. In silico estimates of protein abundances from publicly available protein spectra can provide useful standard abundance values but contain only data from tissue proteomes, and are not coupled to organelle localization data. A new protein abundance score, the normalized protein abundance scale (NPAS), expands on the number of scored proteins and the scoring accuracy of lower-abundance proteins in Arabidopsis. NPAS was combined with subcellular protein localization data, facilitating quantitative estimations of organelle abundance during routine experimental procedures. A suite of targeted proteomics markers for subcellular compartment markers was developed, enabling independent verification of in silico estimates for relative organelle abundance. Estimation of relative organelle abundance was found to be reproducible and consistent over a range of tissues and growth conditions. In silico abundance estimations and localization data have been combined into an online tool, multiple marker abundance profiling, available in the SUBA4 toolbox (http://suba.live).  

3.2886           Molecular and cellular reorganization of neural circuits in the human lineage

Sousa, A.M.M. et al Science, 358(6366), 1027-1032 (2017)   To better understand the molecular and cellular differences in brain organization between human and nonhuman primates, we performed transcriptome sequencing of 16 regions of adult human, chimpanzee, and macaque brains. Integration with human single-cell transcriptomic data revealed global, regional, and cell-type–specific species expression differences in genes representing distinct functional categories. We validated and further characterized the human specificity of genes enriched in distinct cell types through histological and functional analyses, including rare subpallial-derived interneurons expressing dopamine biosynthesis genes enriched in the human striatum and absent in the nonhuman African ape neocortex. Our integrated analysis of the generated data revealed diverse molecular and cellular features of the phylogenetic reorganization of the human brain across multiple levels, with relevance for brain function and disease.  

3.2887           Proceedings of the 2017 ISEV symposium on “HIV, NeuroHIV, drug abuse, & EVs”

Hu, G., Yelamanchili, S., Kashanchi, F., haughey, N., Bond, V.C., Witwer, K., pulliam, l. and Buch, S.

1. Neurovirol., 23(6), 935-940 (2017)

  Despite the success of combination antiretroviral therapy (cART), there is increased prevalence of HIV-associated neurocognitive disorders (HAND) in HIV-1-infected individuals on cART, which poses a major health care challenge. Adding further complexity to this long-term antiretroviral use is the comorbidity with drugs of abuse such as morphine, cocaine, and methamphetamine, which can in turn, exacerbate neurologic and cognitive deficits associated with HAND. Furthermore, HIV proteins, such as the transactivator of transcription (Tat) and the envelope protein (gp120), as well as antiretrovirals themselves can also contribute to the progression of neurodegeneration underlying HAND. In the field of NeuroHIV and drug addiction, EVs hold the potential to serve as biomarkers of cognitive dysfunction, targets of therapy, and as vehicles for therapeutic delivery of agents that can ameliorate disease pathogenesis. Based on the success of a previous Satellite Symposium in 2015 at the ISEV meeting in Washington, experts again expanded on their latest research findings in the field, shedding light on the emerging trends in the field of Extracellular Vesicle (EV) biology in NeuroHIV and drug abuse. The satellite symposium sought to align experts in the fields of NeuroHIV and drug abuse to share their latest insights on the role of EVs in regulating neuroinflammation, neurodegeneration, peripheral immune response, and HIV latency in HIV-infected individuals with or without the comorbidity of drug abuse.  

3.2888           Radically truncated MeCP2 rescues Rett syndrome-like neurological defects

Tillotson, R., Selfridge, J., Koerner, M.V., Gadalla, K.K.E., Guy, J., De Soussa, D., Hector, R.D., Cobb, S.R. and Bird, A. Nature, 550, 398-401 (2017)   Heterozygous mutations in the X-linked MECP2 gene cause the neurological disorder Rett syndrome1. The methyl-CpG-binding protein 2 (MeCP2) protein is an epigenetic reader whose binding to chromatin primarily depends on 5-methylcytosine2,3. Functionally, MeCP2 has been implicated in several cellular processes on the basis of its reported interaction with more than 40 binding partners4, including transcriptional co-repressors (for example, the NCoR/SMRT complex5), transcriptional activators6, RNA7, chromatin remodellers8,9, microRNA-processing proteins10 and splicing factors11. Accordingly, MeCP2 has been cast as a multi-functional hub that integrates diverse processes that are essential in mature neurons12. At odds with the concept of broad functionality, missense mutations that cause Rett syndrome are concentrated in two discrete clusters coinciding with interaction sites for partner macromolecules: the methyl-CpG binding domain13 and the NCoR/SMRT interaction domain5. Here we test the hypothesis that the single dominant function of MeCP2 is to physically connect DNA with the NCoR/SMRT complex, by removing almost all amino-acid sequences except the methyl-CpG binding and NCoR/SMRT interaction domains. We find that mice expressing truncated MeCP2 lacking both the N- and C-terminal regions (approximately half of the native protein) are phenotypically near-normal; and those expressing a minimal MeCP2 additionally lacking a central domain survive for over one year with only mild symptoms. This minimal protein is able to prevent or reverse neurological symptoms when introduced into MeCP2-deficient mice by genetic activation or virus-mediated delivery to the brain. Thus, despite evolutionary conservation of the entire MeCP2 protein sequence, the DNA and co-repressor binding domains alone are sufficient to avoid Rett syndrome-like defects and may therefore have therapeutic utility.  

3.2889           Prion protein inhibits fast axonal transport through a mechanism involving casein kinase 2

Zamponi, E.,Buratti, F., Cataldi, G., Caicedo, H-H., Song, Y., Jungbauer, L.M., LaDu, M.J., Bisbal, M., Lorenzo, A., Ma, J., Helguera, P.R., Morfini, G.A., Brady, S.T. and Pigino, G.F. PloS One, 12(12), e0188340 (2017)   Prion diseases include a number of progressive neuropathies involving conformational changes in cellular prion protein (PrP^c) that may be fatal sporadic, familial or infectious. Pathological evidence indicated that neurons affected in prion diseases follow a dying-back pattern of degeneration. However, specific cellular processes affected by PrP^c that explain such a pattern have not yet been identified. Results from cell biological and pharmacological experiments in isolated squid axoplasm and primary cultured neurons reveal inhibition of fast axonal transport (FAT) as a novel toxic effect elicited by PrP^c. Pharmacological, biochemical and cell biological experiments further indicate this toxic effect involves casein kinase 2 (CK2) activation, providing a molecular basis for the toxic effect of PrP^c on FAT. CK2 was found to phosphorylate and inhibit light chain subunits of the major motor protein conventional kinesin. Collectively, these findings suggest CK2 as a novel therapeutic target to prevent the gradual loss of neuronal connectivity that characterizes prion diseases.  

3.2890           Autophagosomal Content Profiling Reveals an LC3C-Dependent Piecemeal Mitophagy Pathway

Le Guerroue, F., Eck, F., Jung, J., Starzetz, T., Mittelbronn, M., Kaulich, M. and Behrends, C. Mol. Cell, 68(4), 786-796 (2017)   Autophagy allows the degradation of cytosolic endogenous and exogenous material in the lysosome. Substrates are engulfed by double-membrane vesicles, coined autophagosomes, which subsequently fuse with lysosomes. Depending on the involvement of specific receptor proteins, autophagy occurs in a selective or nonselective manner. While this process is well understood at the level of bulky cargo such as mitochondria and bacteria, we know very little about individual proteins and protein complexes that are engulfed and degraded by autophagy. In contrast to the critical role of autophagy in balancing proteostasis, our current knowledge of the autophagic degradome is very limited. Here, we combined proximity labeling with quantitative proteomics to systematically map the protein inventory of autophagosomes. Using this strategy, we uncovered a basal, housekeeping mitophagy pathway that involves piecemeal degradation of mitochondrial proteins in a LC3C- and p62-dependent manner and contributes to mitochondrial homeostasis maintenance when cells rely on oxidative phosphorylation.  

3.2891           A Link between Linearmycin Biosynthesis and Extracellular Vesicle Genesis Connects Specialized Metabolism and Bacterial Membrane Physiology

Hoefler, B.C., Stubbendieck, R.M., Josyula, N.K., Moisan, S.M., Schulze, E.M. and Straight, P.D. Cell Chem. Biol., 24(10), 1238-1249 (2017)   Specialized metabolites support bacterial competitive fitness as antibiotics, signals, pigments, and metal scavengers. Little is known about how specialized metabolites are processed and trafficked for their diverse competitive functions. Linearmycins A and B are linear polyketides with antifungal and antibacterial activity but are colony-localized in imaging mass spectrometry of Streptomyces sp. Mg1 (S. sp. Mg1). To decipher a connection between colony localization and antibiotic activity, we identified the linearmycin gene cluster and investigated linearmycin production and distribution by S. sp. Mg1. Our results uncover a large family of variant linearmycins with limited solubility in aqueous solution. We hypothesized that extracellular vesicles may traffic the lipid-like linearmycins. We found that vesicles isolated from culture supernatants contained linearmycins. Surprisingly, abolishing production of linearmycins in S. sp. Mg1 also diminished extracellular vesicle production. Our results reveal integration of linearmycin biosynthesis with production of extracellular vesicles, suggesting a deep connection between specialized metabolism and bacterial membrane physiology.    

3.2892           A Carbon Nanotube Optical Reporter Maps Endolysosomal Lipid Flux

Jena, P.V., Roxbury, D., Galassi, T.V., Akkari, L., Horoszko, C.P., Iaea, D.B., Budhathoki-Uprety, J., Pipalia, N., Haka, A.S., Harvey, J.D., Mittal, J., Maxfield, F.R., Joyce, J.A. and Heller, D.A. ACS Nano, 11, 10689-10703 (2017)   Lipid accumulation within the lumen of endolysosomal vesicles is observed in various pathologies including atherosclerosis, liver disease, neurological disorders, lysosomal storage disorders, and cancer. Current methods cannot measure lipid flux specifically within the lysosomal lumen of live cells. We developed an optical reporter, composed of a photoluminescent carbon nanotube of a single chirality, that responds to lipid accumulation via modulation of the nanotube’s optical band gap. The engineered nanomaterial, composed of short, single-stranded DNA and a single nanotube chirality, localizes exclusively to the lumen of endolysosomal organelles without adversely affecting cell viability or proliferation or organelle morphology, integrity, or function. The emission wavelength of the reporter can be spatially resolved from within the endolysosomal lumen to generate quantitative maps of lipid content in live cells. Endolysosomal lipid accumulation in cell lines, an example of drug-induced phospholipidosis, was observed for multiple drugs in macrophages, and measurements of patient-derived Niemann–Pick type C fibroblasts identified lipid accumulation and phenotypic reversal of this lysosomal storage disease. Single-cell measurements using the reporter discerned subcellular differences in equilibrium lipid content, illuminating significant intracellular heterogeneity among endolysosomal organelles of differentiating bone-marrow-derived monocytes. Single-cell kinetics of lipoprotein-derived cholesterol accumulation within macrophages revealed rates that differed among cells by an order of magnitude. This carbon nanotube optical reporter of endolysosomal lipid content in live cells confers additional capabilities for drug development processes and the investigation of lipid-linked diseases.  

3.2893           Qualitative differences in T‐cell activation by dendritic cell‐derived extracellular vesicle subtypes

Tkach, m., Kowal, j., Zucchetti, A.E., Enserink, l., Jouve, M., Lankar, D., Saitakis, M., Martin-Jaular, l. and Thery, C. EMBO J., 36(20), 3012-3028 (2017)   Exosomes, nano‐sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC‐peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4⁺ T‐cell activation in vitro. When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN‐γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN‐γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles.  

3.2894           Loss of native α-synuclein multimerization by strategically mutating its amphipathic helix causes abnormal vesicle interactions in neuronal cells

Dettmer, U., Ramalingam, N., von Saucken, V.E., Kim, T-E., Newman, A.J., Terry-Kantor, E., Nuber, S., Ericksson, M., Fanning, S., Bartels, T., Lindquist, S., Levy, O.A. and Selkoe, D. Hum. Mol. Genet., 26(18), 3466-3481 (2017)   α-Synuclein (αS) forms round cytoplasmic inclusions in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Evidence suggests a physiological function of αS in vesicle trafficking and release. In contrast to earlier tenets, recent work indicates that αS normally exists in cells in a dynamic equilibrium between monomers and tetramers/multimers. We engineered αS mutants incapable of multimerization, leading to excess monomers at vesicle membranes. By EM, such mutants induced prominent vesicle clustering, leading to round cytoplasmic inclusions. Immunogold labeling revealed abundant αS intimately associated with vesicles of varied size. Fluorescence microscopy with marker proteins showed that the αS-associated vesicles were of diverse endocytic and secretory origin. An αS ‘3K’ mutant (E35K + E46K + E61K) that amplifies the PD/DLB-causing E46K mutation induced αS-rich vesicle clusters resembling the vesicle-rich areas of Lewy bodies, supporting pathogenic relevance. Mechanistically, E46K can increase αS vesicle binding via membrane-induced amphipathic helix formation, and ‘3K’ further enhances this effect. Another engineered αS variant added hydrophobicity to the hydrophobic half of αS helices, thereby stabilizing αS-membrane interactions. Importantly, substituting charged for uncharged residues within the hydrophobic half of the stabilized helix not only reversed the strong membrane interaction of the multimer-abolishing αS variant but also restored multimerization and prevented the aberrant vesicle interactions. Thus, reversible αS amphipathic helix formation and dynamic multimerization regulate a normal function of αS at vesicles, and abrogating multimers has pathogenic consequences.  

3.2895           A plasmid from an Antarctic haloarchaeon uses specialized membrane vesicles to disseminate and infect plasmid-free cells

Erdmann, S., Tschitischko, B., Zhong, L., Raftery, M.-J. and Cavicchioli, R. Nature Microbiol., 2, 1446-1455 (2017)   The major difference between viruses and plasmids is the mechanism of transferring their genomic information between host cells. Here, we describe the archaeal plasmid pR1SE from an Antarctic species of haloarchaea that transfers via a mechanism similar to a virus. pR1SE encodes proteins that are found in regularly shaped membrane vesicles, and the vesicles enclose the plasmid DNA. The released vesicles are capable of infecting a plasmid-free strain, which then gains the ability to produce plasmid-containing vesicles. pR1SE can integrate and replicate as part of the host genome, resolve out with fragments of host DNA incorporated or portions of the plasmid left behind, form vesicles and transfer to new hosts. The pR1SE mechanism of transfer of DNA could represent the predecessor of a strategy used by viruses to pass on their genomic DNA and fulfil roles in gene exchange, supporting a strong evolutionary connection between plasmids and viruses.  

3.2896           Analysis of the small non-protein-coding RNA profile of mouse spermatozoa reveals specific enrichment of piRNAs within mature spermatozoa

Hutcheon, K., McLaughlin, E.A., Stanger, S.J., Bernstein, I.R., Dun, M.D., Eamens, A.L. and Nixon, B. RNA Biology, 14(12), 1776-2790 (2017)   Post-testicular sperm maturation and storage within the epididymis is a key determinant of gamete quality and fertilization competence. Here we demonstrate that mouse spermatozoa possess a complex small non-protein-coding RNA (sRNA) profile, the composition of which is markedly influenced by their epididymal transit. Thus, although microRNAs (miRNAs) are highly represented in the spermatozoa of the proximal epididymis, this sRNA class is largely diminished in mature spermatozoa of the distal epididymis. Coincident with this, a substantial enrichment in Piwi-interacting RNA (piRNA) abundance in cauda spermatozoa was detected. Further, features of cauda piRNAs, including; predominantly 29–31 nts in length; preference for uracil at their 5' terminus; no adenine enrichment at piRNA nt 10, and; predominantly mapping to intergenic regions of the mouse genome, indicate that these piRNAs are generated by the PIWIL1-directed primary piRNA production pathway. Accordingly, PIWIL1 was detected via immunoblotting and mass spectrometry in epididymal spermatozoa. These data provide insight into the complexity and dynamic nature of the sRNA profile of spermatozoa and raise the intriguing prospect that piRNAs are generated in situ in maturing spermatozoa. Such information is of particular interest in view of the potential role for paternal sRNAs in influencing conception, embryo development and intergenerational inheritance.  

3.2897           Exosome secretion promotes chemotaxis of cancer cells

Sung, B.H. and Weaver, A.M. Cell Adhesion and Migration, 11(2), 187-195 (2017)   Migration of cells toward chemical cues, or chemotaxis, is important for many biologic processes such as immune defense, wound healing and cancer metastasis. Although chemotaxis is thought to occur in cancer cells, it is less well characterized than chemotaxis of professional immune cells such as neutrophils. Here, we show that cancer cell chemotaxis relies on secretion of exosome-type extracellular vesicles. Migration of fibrosarcoma cells toward a gradient of exosome-depleted serum was diminished by knockdown of the exosome secretion regulator Rab27a. Rescue experiments in which chemotaxis chambers were coated with purified extracellular vesicles demonstrate that exosomes but not microvesicles affect both speed and directionality of migrating cells. Chamber coating with purified fibronectin and fibronectin-depleted exosomes demonstrates that the exosome cargo fibronectin promotes cell speed but cannot account for the role of exosomes in promoting directionality of fibrosarcoma cell movement during chemotaxis. These experiments indicate that exosomes contain multiple motility-promoting cargoes that contribute to different aspects of cell motility.  

3.2898           The isolation of morphologically intact and biologically active extracellular vesicles from the secretome of cancer-associated adipose tissue

Jeurissen, S., Vergauwen, G., Van Deun, J., Lapeire, L., Depoorter, V, Miinalainen, I et al. Cell Adhesion & Migration, 11(2), 196-204 (2017)   Breast cancer cells closely interact with different cell types of the surrounding adipose tissue to favor invasive growth and metastasis. Extracellular vesicles (EVs) are nanometer-sized vesicles secreted by different cell types that shuttle proteins and nucleic acids to establish cell-cell communication. To study the role of EVs released by cancer-associated adipose tissue in breast cancer progression and metastasis a standardized EV isolation protocol that obtains pure EVs and maintains their functional characteristics is required. We implemented differential ultracentrifugation as a pre-enrichment step followed by OptiPrep density gradient centrifugation (dUC-ODG) to isolate EVs from the conditioned medium of cancer-associated adipose tissue. A combination of immune-electron microscopy, nanoparticle tracking analysis (NTA) and Western blot analysis identified EVs that are enriched in flotillin-1, CD9 and CD63, and sized between 20 and 200 nm with a density of 1.076–1.125 g/ml. The lack of protein aggregates and cell organelle proteins confirmed the purity of the EV preparations. Next, we evaluated whether dUC-ODG isolated EVs are functionally active. ZR75.1 breast cancer cells treated with cancer-associated adipose tissue-secreted EVs from breast cancer patients showed an increased phosphorylation of CREB. MCF-7 breast cancer cells treated with adipose tissue-derived EVs exhibited a stronger propensity to form cellular aggregates. In conclusion, dUC-ODG purifies EVs from conditioned medium of cancer-associated adipose tissue, and these EVs are morphologically intact and biologically active.   3.2898a Decreased Fatty Acid β-Oxidation Is the Main Cause of Fatty Liver Induced by Polyunsaturated Fatty Acid Deficiency in Mice Nakajima, T., Yang, Y., Lu, Y., Kamijo, Y., Yamada, Y., Nakamura, K., Koyama, M., Yamaguchi, S., Sugiyama, E., Tanaka, N. and Aoyama, T. Tohoku J. Exp. Med., 242, 229-239 (2017)   Insufficient intake of polyunsaturated fatty acids (PUFA) causes fatty liver. The mechanism responsible is primarily related to increased lipogenesis and decreased FA degradation based on rodent studies. However, these studies were limited by the fact that the typical PUFA-deficient diets contained insufficient amounts of long-chain FA, the PUFA-containing diets were primarily composed of n-3 PUFA-enriched oil, and the intake of PUFA was excessive compared with the physiological requirement. To address these issues, mice were fed a PUFA-deficient diet containing long-chain FA at a standard fed level and then were orally fed a n-3/n-6-balanced PUFA-containing oil [PUFA (+)] or a PUFA-deficient oil [PUFA (−)] at physiological relevant levels (0.1 mL/mouse/2d). We compared these groups and examined whether fatty liver in PUFA deficiency was attributable to both the effects of increased lipogenesis and decreased FA catabolism. Compared with the PUFA (+) group, the PUFA (−) group showed increases in liver triglyceride and serum FA content. Hepatic gene expression of several mitochondrial β-oxidation enzymes, the serum 3-hydroxybutyrate level, and DNA-binding ability of peroxisome proliferator-activated receptor α (PPARα) were increased in the PUFA (+) group, whereas these adaptive responses were significantly attenuated in the PUFA (−) group. The hepatic expression of typical lipogenesis genes did not differ between the groups. Therefore, fatty liver in PUFA deficiency is attributable to suppression of the FA-degrading system probably from decreased PPARα adaptive responsiveness, and PUFA may be an essential factor for PPARα functioning. This finding is helpful for managing clinical situations having a risk of PUFA deficiency.  

3.2899           Exosomes as secondary inductive signals involved in kidney organogenesis

Krause, M., Rak-Raszewska, A., Naillat, F., Saarela, U., Schmidt, C., Ronkainen, V-P., Bart, G., Ylä-Herttuala, S. and Vainio, S.J.

1. Extracellular Vesicles, 7(1), 1422675 (2018)

  The subfraction of extracellular vesicles, called exosomes, transfers biological molecular information not only between cells but also between tissues and organs as nanolevel signals. Owing to their unique properties such that they contain several RNA species and proteins implicated in kidney development, exosomes are putative candidates to serve as developmental programming units in embryonic induction and tissue interactions. We used the mammalian metanephric kidney and its nephron-forming mesenchyme containing the nephron progenitor/stem cells as a model to investigate if secreted exosomes could serve as a novel type of inductive signal in a process defined as embryonic induction that controls organogenesis. As judged by several characteristic criteria, exosomes were enriched and purified from a cell line derived from embryonic kidney ureteric bud (UB) and from primary embryonic kidney UB cells, respectively. The cargo of the UB-derived exosomes was analysed by qPCR and proteomics. Several miRNA species that play a role in Wnt pathways and enrichment of proteins involved in pathways regulating the organization of the extracellular matrix as well as tissue homeostasis were identified. When labelled with fluorescent dyes, the uptake of the exosomes by metanephric mesenchyme (MM) cells and the transfer of their cargo to the cells can be observed. Closer inspection revealed that besides entering the cytoplasm, the exosomes were competent to also reach the nucleus. Furthermore, fluorescently labelled exosomal RNA enters into the cytoplasm of the MM cells. Exposure of the embryonic kidney-derived exosomes to the whole MM in an ex vivo organ culture setting did not lead to an induction of nephrogenesis but had an impact on the overall organization of the tissue. We conclude that the exosomes provide a novel signalling system with an apparent role in secondary embryonic induction regulating organogenesis.  

3.2900           Characterization of Trichuris muris secreted proteins and extracellular vesicles provides new insights into host–parasite communication

Eichenberger, R.M., Talukder, M.H., Field, M.A., Wangchuk, P., Giacomin, P., Loukas, A. and Sotillo, J.

1. Extracellular Vesicles, 7(1), 1428004 (2017)

  Whipworms are parasitic nematodes that live in the gut of more than 500 million people worldwide. Owing to the difficulty in obtaining parasite material, the mouse whipworm Trichuris muris has been extensively used as a model to study human whipworm infections. These nematodes secrete a multitude of compounds that interact with host tissues where they orchestrate a parasitic existence. Herein we provide the first comprehensive characterization of the excretory/secretory products of T. muris. We identify 148 proteins secreted by T. muris and show for the first time that the mouse whipworm secretes exosome-like extracellular vesicles (EVs) that can interact with host cells. We use an Optiprep® gradient to purify the EVs, highlighting the suitability of this method for purifying EVs secreted by a parasitic nematode. We also characterize the proteomic and genomic content of the EVs, identifying >350 proteins, 56 miRNAs (22 novel) and 475 full-length mRNA transcripts mapping to T. muris gene models. Many of the miRNAs putatively mapped to mouse genes are involved in regulation of inflammation, implying a role in parasite-driven immunomodulation. In addition, for the first time to our knowledge, colonic organoids have been used to demonstrate the internalization of parasite EVs by host cells. Understanding how parasites interact with their host is crucial to develop new control measures. This first characterization of the proteins and EVs secreted by T. muris provides important information on whipworm–host communication and forms the basis for future studies.  

3.2901           Salmonella enterica Serovar Typhimurium Alters the Extracellular Proteome of Macrophages and Leads to the Production of Proinflammatory Exosomes

Hui, W.W., Hercik, K., Belsare, S., Alugubelly, N., Clapp, B., Rinaldi, C. and Edelmann, M.J. Infect. Immun., 86(2), e00386-17 (2018)   Salmonella enterica serovar Typhimurium is a Gram-negative bacterium, which can invade and survive within macrophages. Pathogenic salmonellae induce the secretion of specific cytokines from these phagocytic cells and interfere with the host secretory pathways. In this study, we describe the extracellular proteome of human macrophages infected with S. Typhimurium, followed by analysis of canonical pathways of proteins isolated from the extracellular milieu. We demonstrate that some of the proteins secreted by macrophages upon S. Typhimurium infection are released via exosomes. Moreover, we show that infected macrophages produce CD63⁺ and CD9⁺ subpopulations of exosomes at 2 h postinfection. Exosomes derived from infected macrophages trigger the Toll-like receptor 4-dependent release of tumor necrosis factor alpha (TNF-α) from naive macrophages and dendritic cells, but they also stimulate secretion of such cytokines as RANTES, IL-1ra, MIP-2, CXCL1, MCP-1, sICAM-1, GM-CSF, and G-CSF. Proinflammatory effects of exosomes are partially attributed to lipopolysaccharide, which is encapsulated within exosomes. In summary, we show for the first time that proinflammatory exosomes are formed in the early phase of macrophage infection with S. Typhimurium and that they can be used to transfer cargo to naive cells, thereby leading to their stimulation.  

3.2902           Exosomes in Cancer Liquid Biopsy: A Focus on Breast Cancer

Halvaei, S., Daryani, S., Eslami-S, Z., Samadi, T., Jafarbeik-Iravani, N., Bakhshayesh, T.O., Majidzadeh-A, K. and Esmaeili, R. Molecular Therapy – Nucleic Acids, 10, 131-141 (2018)   The important challenge about cancer is diagnosis in primary stages and proper treatment. Although classical clinico-pathological features of the tumor have major prognostic value, the advances in diagnosis and treatment are indebted to discovery of molecular biomarkers and control of cancer in the pre-invasive state. Moreover, the efficiency of available therapeutic options is highly diminished, and chemotherapy is still the main treatment due to lack of enough specific targets. Accordingly, finding the new noninvasive biomarkers for cancer is still an important clinical challenge that is not achieved yet. There are current technologies to screen, diagnose, prognose, and treat cancer, but the limitations of these implements and procedures are undeniable. Liquid biopsy as a noninvasive method has a promising future in the field of cancer, and exosomes as one of the recent areas have drawn much attention. In this review, the potential capability of exosomes is summarized in cancer with the special focus on breast cancer as the second cause of cancer mortality in women all around the world. It discusses reasons to choose exosomes for liquid biopsy and the studies related to different potential biomarkers found in the exosomes. Moreover, exosome studies on milk as a specific biofluid are also discussed. At last, because choosing the method for exosome studies is very challenging, a summary of different techniques is provided.  

3.2903           Proteolytic maturation of Drosophila Neuroligin 3 by tumor necrosis factor α-converting enzyme in the nervous system

Wu, J., Tao, Y., Tian, Y., Xing, G., Lv, H., Han, J., Lin, C. and Xie, W. BBA – General Subjects, 1862, 440-450 (2018)   Background The functions of autism-associated Neuroligins (Nlgs) are modulated by their post-translational modifications, such as proteolytic cleavage. A previous study has shown that there are different endogenous forms of DNlg3 in Drosophila, indicating it may undergo proteolytic processing. However, the molecular mechanism underlying DNlg3 proteolytic processing is unknown. Here, we report a novel proteolytic mechanism that is essential for DNlg3 maturation and function in the nervous system. Methods Molecular cloning, cell culture, immunohistochemistry, western blotting and genetic studies were employed to map the DNlg3 cleavage region, identify the protease and characterize the cleavage manner. Behavior analysis, immunohistochemistry and genetic manipulations were employed to study the functions of different DNlg3 forms in the nervous system and neuromuscular junction (NMJs). Results Tumor necrosis factor α-converting enzyme (TACE) cleaved DNlg3 exclusively at its extracellular acetylcholinesterase-like domain to generate the N-terminal fragment and the short membrane-anchored fragment (sDNlg3). DNlg3 was constitutively processed in an activity-independent manner. Interestingly, DNlg3 was cleaved intracellularly in the Golgi apparatus before it arrived at the cell surface, a unique cleavage mechanism that is distinct from ‘conventional’ ectodomain shedding of membrane proteins, including rodent Nlg1. Genetic studies showed that sDNlg3 was essential for maintaining proper locomotor activity in Drosophila. Conclusions Our results revealed a unique cleavage mechanism of DNlg3 and a neuron-specific role for DNlg3 maturation which is important in locomotor activity.  

3.2904           Intercellular transfer of pathogenic α-synuclein by extracellular vesicles is induced by the lipid peroxidation product 4-hydroxynonenal

Zhang, S., Eitan, E., Wu, T-Y. and mattson, M.P. Neurobiology of Aging, 61, 52-65 (2018)   Parkinson's disease (PD) is characterized by accumulations of toxic α-synuclein aggregates in vulnerable neuronal populations in the brainstem, midbrain, and cerebral cortex. Recent findings suggest that α-synuclein pathology can be propagated transneuronally, but the underlying molecular mechanisms are unknown. Advances in the genetics of rare early-onset familial PD indicate that increased production and/or reduced autophagic clearance of α-synuclein can cause PD. The cause of the most common late-onset PD is unclear, but may involve metabolic compromise and oxidative stress upstream of α-synuclein accumulation. As evidence, the lipid peroxidation product 4-hydroxynonenal (HNE) is elevated in the brain during normal aging and moreso in brain regions afflicted with α-synuclein pathology. Here, we report that HNE increases aggregation of endogenous α-synuclein in primary neurons and triggers the secretion of extracellular vesicles (EVs) containing cytotoxic oligomeric α-synuclein species. EVs released from HNE-treated neurons are internalized by healthy neurons which as a consequence degenerate. Levels of endogenously generated HNE are elevated in cultured cells overexpressing human α-synuclein, and EVs released from those cells are toxic to neurons. The EV-associated α-synuclein is located both inside the vesicles and on their surface, where it plays a role in EV internalization by neurons. On internalization, EVs harboring pathogenic α-synuclein are transported both anterogradely and retrogradely within axons. Focal injection of EVs containing α-synuclein into the striatum of wild-type mice results in spread of synuclein pathology to anatomically connected brain regions. Our findings suggest a scenario for late-onset PD in which lipid peroxidation promotes intracellular accumulation and then extrusion of EVs containing toxic α-synuclein species; the EVs are then internalized by adjacent neurons, so propagating the neurodegenerative process.  

3.2905           Chromosome 19 microRNAs exert antiviral activity independent from type III interferon signaling

Bayer, A., lennemann, N.J., Ouyang, Y., Sadovsky, E., Sheridan, M.A., Roberts, R.M., Coyne, C.B. and Sadovsky, Y. Placenta, 61, 33-38 (2018)   Introduction Cultured primary human trophoblasts (PHT), derived from term placentas, are relatively resistant to infection by diverse viruses. The resistance can be conferred to non-trophoblastic cells by pre-exposing them to medium that was conditioned by PHT cells. This antiviral effect is mediated, at least in part, by microRNAs (miRNA) expressed from the chromosome 19 microRNA cluster (C19MC). Recently we showed that PHT cells and cells pre-exposed to PHT medium are also resistant to infection by Zika virus (ZIKV), an effect mediated by the constitutive release of the type III interferons (IFN) IFN lambda-1 and IFN lambda-2 in trophoblastic medium. We hypothesized that trophoblastic C19MC miRNA are active against ZIKV, and assessed the interaction of this pathway with IFN lambda-1 - mediated resistance. Methods Term PHT cells were cultured using standard techniques. An osteosarcoma cell line (U2OS) was used as non-trophoblastic cells, which were infected with either ZIKV or vesicular stomatitis virus (VSV). Trophoblastic extracellular vesicles (EVs) were produced by gradient ultracentrifugation. RT-qPCR was used to determine viral infection, cellular or medium miRNA levels and the expression of interferon-stimulated genes. Results We showed that C19MC miRNA attenuate infection of U2OS cells by ZIKV, and that C19MC miRNA or exosomes that contain C19MC miRNA did not influence the type III IFN pathway. Similarly, cell exposure to recombinant IFN lambda-1 had no effect on miRNA expression, and these pathways did not exhibit synergistic interaction. Discussion PHT cells exert antiviral activity by at least two independent mechanisms, mediated by C19MC miRNA and by type III IFNs.  

3.2906           DCF1 subcellular localization and its function in mitochondria

Chen, Y., Feng, R., Luo, G., Guo, J., Wang, Y., Sun, Y., Zheng, L. and Wen, T. Biochemie, 144, 50-55 (2018)   Dendritic cell factor 1 (DCF1) is a transmembrane protein that plays important roles in regulating neural stem cell differentiation and dendritic spine formation. Apart from its cytoplasmic functions, DCF1 plays a role in autophagy during the regulation of amyloid precursor proteins. However, the subcellular localization of DCF1 remains unknown. Therefore, in this study, DCF1 tagged with green fluorescent protein was transiently expression in HelaS3 and HEK293T cells. The results showed that DCF1 was widely expressed in different organelles, including the mitochondria, Golgi apparatus, endoplasmic reticulum, endosomes and lysosomes. An iodixanol step gradient further confirmed that DCF1 is localized to the mitochondria, endosomes, lysosomes, endoplasmic reticulum, and proteasome. Finally, functional analysis of the mitochondria revealed that DCF1 affected the expression and localization of MGST1. This study presents a comprehensive evaluation of the subcellular localization of DCF1, which provides important information on complex functions mediated by DCF1.  

3.2907           Characterization of outer membrane vesicles of Acetobacter pasteurianus NBRC3283

Hashimoto, M., matsumoto, T., Tamura-Nakano, M., Ozono, m., Hashiguchi, S. and Suda, Y.

1. Bioscience and Bioenginering, 125(4), 425-431 (2018)

  Acetobacter pasteurianus is characterized as a fermenting bacterium of kurozu, which is a common traditional Japanese black vinegar. Recently, we separated acid-resistant and low Toll-like receptor 4 (TLR4)-stimulatory lipopolysaccharides (LPS) from A. pasteurianus. We also showed that their lipid A parts possessed a novel sugar backbone that is responsible for the low TLR4-stimulatory and acid–resistant properties of the LPS. Outer membrane vesicles (OMVs) are nano-sized spherical structures secreted from many gram-negative bacteria. OMVs contain LPS and act as immunomodulants such as vaccines. In this study, we investigated OMVs secreted from A. pasteurianus. OMV secretion from A. pasteurianus NBRC 3283 cells was observed after 2 days in culture by transmission electron microscopy imaging. Thus OMVs were separated from the culture supernatants by ultracentrifugation and then purified by OptiPrep density gradient centrifugation. The OMVs contained several proteins including outer membrane proteins, and several sugars as components of LPS. The OMVs weakly stimulated TLR4 in accordance with the activity of A. pasteurianus LPS. Additionally, the TLR2-stimulating activity of the OMVs was significantly potent, indicating the existence of lipoproteins. Furthermore OMV-like spherical particles were observed in kurozu. Some of these particles are probably derived from A. pasteurianus. These data suggest that A. pasteurianus produce OMVs that contain LPS and probably lipoproteins, and can modulate the innate immune system.  

3.2908           Glycolysis promotes caspase-3 activation in lipid rafts in T cells

Secinaro, M.A., Fortner, K.A., Dienz, O., Logan, A., Murphy, M.P., Anathy, V., Boyson, J.E. and Budd, R.C. Cell Death & Disease, 9:62 (2018)   Resting T cells undergo a rapid metabolic shift to glycolysis upon activation in the presence of interleukin (IL)-2, in contrast to oxidative mitochondrial respiration with IL-15. Paralleling these different metabolic states are striking differences in susceptibility to restimulation-induced cell death (RICD); glycolytic effector T cells are highly sensitive to RICD, whereas non-glycolytic T cells are resistant. It is unclear whether the metabolic state of a T cell is linked to its susceptibility to RICD. Our findings reveal that IL-2-driven glycolysis promotes caspase-3 activity and increases sensitivity to RICD. Neither caspase-7, caspase-8, nor caspase-9 activity is affected by these metabolic differences. Inhibition of glycolysis with 2-deoxyglucose reduces caspase-3 activity as well as sensitivity to RICD. By contrast, IL-15-driven oxidative phosphorylation actively inhibits caspase-3 activity through its glutathionylation. We further observe active caspase-3 in the lipid rafts of glycolytic but not non-glycolytic T cells, suggesting a proximity-induced model of self-activation. Finally, we observe that effector T cells during influenza infection manifest higher levels of active caspase-3 than naive T cells. Collectively, our findings demonstrate that glycolysis drives caspase-3 activity and susceptibility to cell death in effector T cells independently of upstream caspases. Linking metabolism, caspase-3 activity, and cell death provides an intrinsic mechanism for T cells to limit the duration of effector function.  

3.2909           Current knowledge on exosome biogenesis and release

Hessvik, N.P. and Llorente, A. Cell. Mol. Life Sci., 75(2), 193-208 (2018)   Exosomes are nanosized membrane vesicles released by fusion of an organelle of the endocytic pathway, the multivesicular body, with the plasma membrane. This process was discovered more than 30 years ago, and during these years, exosomes have gone from being considered as cellular waste disposal to mediate a novel mechanism of cell-to-cell communication. The exponential interest in exosomes experienced during recent years is due to their important roles in health and disease and to their potential clinical application in therapy and diagnosis. However, important aspects of the biology of exosomes remain unknown. To explore the use of exosomes in the clinic, it is essential that the basic molecular mechanisms behind the transport and function of these vesicles are better understood. We have here summarized what is presently known about how exosomes are formed and released by cells. Moreover, other cellular processes related to exosome biogenesis and release, such as autophagy and lysosomal exocytosis are presented. Finally, methodological aspects related to exosome release studies are discussed.  

3.2910           Type A viral hepatitis: A summary and update on the molecular virology, epidemiology, pathogenesis and prevention

Lemon, S.M., Ott, J., Van Damme, P. and Shouval, D.

1. Hepatol., 68(1), 167-184 (2018)

  Although epidemic jaundice was well known to physicians of antiquity, it is only in recent years that medical science has begun to unravel the origins of hepatitis A virus (HAV) and the unique pathobiology underlying acute hepatitis A in humans. Improvements in sanitation and the successful development of highly efficacious vaccines have markedly reduced the worldwide occurence of this enterically-transmitted infection over the past quarter century, yet the virus persists in vulnerable populations and those without HAV immunity and remains a common cause of food-borne disease outbreaks in economically-advantaged societies. Reductions in HAV incidence have led to increases in the median age at which infection occurs, often resulting in more severe disease in affected persons and paradoxical increases in disease burden in some developing nations. Here, we summarize recent advances in the molecular virology and epidemiology of HAV, an atypical member of the Picornaviridae family, survey what is known of the pathogenesis of hepatitis A in humans and the host-pathogen interactions that typify the infection. The article also reviews medical and public health aspects of HAV vaccination and disease prevention.  

3.2911           Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

Zhou, W., Woodson, m., Neupane, B., bai, F., Sherman, M.B., Choi, K.H., Neelakanta, G. and Sultana, H. PloS Pathogens, 14(1), e1006764 (2018)   Molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, we show for the first time that a cell line from medically important arthropods, such as ticks, secretes extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Our study shows that tick-borne Langat virus (LGTV), a model pathogen closely related to tick-borne encephalitis virus (TBEV), profusely uses arthropod exosomes for transmission of viral RNA and proteins to the human- skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed the presence of purified arthropod/neuronal exosomes with the size range of 30 to 200 nm in diameter. Both positive and negative strands of LGTV RNA and viral envelope-protein were detected inside exosomes derived from arthropod, murine and human cells. Detection of Nonstructural 1 (NS1) protein in arthropod and neuronal exosomes further suggested that exosomes contain viral proteins. Viral RNA and proteins in exosomes derived from tick and mammalian cells were secured, highly infectious and replicative in all tested evaluations. Treatment with GW4869, a selective inhibitor that blocks exosome release affected LGTV loads in both arthropod and mammalian cell-derived exosomes. Transwell-migration assays showed that exosomes derived from infected-brain-microvascular endothelial cells (that constitute the blood-brain barrier) facilitated LGTV RNA and protein transmission, crossing of the barriers and infection of neuronal cells. Neuronal infection showed abundant loads of both tick-borne LGTV and mosquito-borne West Nile virus RNA in exosomes. Our data also suggest that exosome-mediated LGTV viral transmission is clathrin-dependent. Collectively, our results suggest that flaviviruses uses arthropod-derived exosomes as a novel means for viral RNA and protein transmission from the vector, and the vertebrate exosomes for dissemination within the host that may subsequently allow neuroinvasion and neuropathogenesis.  

3.2912           Neuronal lysosomal dysfunction releases exosomes harboring APP C-terminal fragments and unique lipid signatures

Miranda, A.M., Lasiecka, Z.M., Xu, Y., Neufeld, J., Shahriar, S., Simoes, S., Chan, R.B., Oliveira, T.G., Small, S.A. and Di Paolo, G. Nature Communications, 9:291 (2018)   Defects in endolysosomal and autophagic functions are increasingly viewed as key pathological features of neurodegenerative disorders. A master regulator of these functions is phosphatidylinositol-3-phosphate (PI3P), a phospholipid synthesized primarily by class III PI 3-kinase Vps34. Here we report that disruption of neuronal Vps34 function in vitro and in vivo impairs autophagy, lysosomal degradation as well as lipid metabolism, causing endolysosomal membrane damage. PI3P deficiency also promotes secretion of unique exosomes enriched for undigested lysosomal substrates, including amyloid precursor protein C-terminal fragments (APP-CTFs), specific sphingolipids, and the phospholipid bis(monoacylglycero)phosphate (BMP), which normally resides in the internal vesicles of endolysosomes. Secretion of these exosomes requires neutral sphingomyelinase 2 and sphingolipid synthesis. Our results reveal a homeostatic response counteracting lysosomal dysfunction via secretion of atypical exosomes eliminating lysosomal waste and define exosomal APP-CTFs and BMP as candidate biomarkers for endolysosomal dysfunction associated with neurodegenerative disorders.  

3.2913           Exosome-Mimetic Nanovesicles from Hepatocytes promote hepatocyte proliferation in vitro and liver regeneration in vivo

Wu, J-Y., Ji, A-L., Wang, Z-x., Qiang, G-H., Qu, Z., Wu, J-H. and Jiang, C-P. Science Reports, 8:2471 (2018)   The liver has great regenerative capacity after functional mass loss caused by injury or disease. Many studies have shown that primary hepatocyte-derived exosomes, which can deliver biological information between cells, promote the regenerative process of the liver. However, the yield of exosomes is very limited. Recent studies have demonstrated that exosome-mimetic nanovesicles (NVs) can be prepared from cells with almost 100 times the production yield compared with exosomes. Thus, this study investigated the therapeutic capacity of exosome-mimetic NVs from primary hepatocytes in liver regeneration. Exosome-mimetic NVs were prepared by serial extrusions of cells through polycarbonate membranes, and the yield of these NVs was more than 100 times that of exosomes. The data indicated that the NVs could promote hepatocyte proliferation and liver regeneration by significantly enhancing the content of sphingosine kinase 2 in recipient cells. To the best of our knowledge, this is the first time that exosome-mimetic NVs from primary hepatocytes have been prepared, and these NVs have components similar to exosomes from primary hepatocytes and, in some respects, biofunctions similar to exosomes. Strategies inspired by this study may lead to substitution of exosomes with exosome-mimetic NVs for biofunctional purposes, including utilization in tissue repair and regeneration.  

3.2914           Acidic organelles mediate TGF-β1-induced cellular fibrosis via (pro)renin receptor and vacuolar ATPase trafficking in human peritoneal mesothelial cells

Oba-yabana, I., Mori, T., Takahashi, C., Hirose, T., Ohsaki, Y., Kinugasa, S., Muroya, Y., Sato, E., Nguyen, G., Piedagnel, R., Ronco, P.M., Totsune, K. and Ito, S. Scientific Reports, 8:2648 (2018)   TGF-β1, which can cause renal tubular injury through a vacuolar-type H⁺-ATPase (V-ATPase)-mediated pathway, is induced by the glucose degradation product methylglyoxal to yield peritoneal injury and fibrosis. The present study investigated the roles of V-ATPase and its accessory protein, the (pro)renin receptor, in peritoneal fibrosis during peritoneal dialysis. Rats daily administered 20 mM methylglyoxal intraperitoneally developed significant peritoneal fibrosis after 7 days with increased expression of TGF-β and V-ATPase, which was reduced by the inhibition of V-ATPase with co-administration of 100 mM bafilomycin A1. The (pro)renin receptor and V-ATPase were expressed in acidic organelles and cell membranes of human peritoneal mesothelial cells. TGF-β1 upregulated the expression of collagens, α-SMA, and EDA-fibronectin, together with ERK1/2 phosphorylation, which was reduced by inhibition of V-ATPase, (pro)renin receptor, or the MAPK pathway. Fibronectin and the soluble (pro)renin receptor were excreted from cells by acidic organelle trafficking in response to TGF-β1; this excretion was also suppressed by inhibition of V-ATPase. Soluble (pro)renin receptor concentrations in effluents of patients undergoing peritoneal dialysis were associated with the dialysate-to-plasma ratio of creatinine. Together, these results demonstrate a novel fibrosis mechanism through the (pro)renin receptor and V-ATPase in the acidic organelles of peritoneal mesothelial cells.  

3.2915           Optical and surface plasmonic approaches to characterize extracellular vesicles. A review

Shpacovitch, V. and Hergenröder, R. Anal. Chim. Acta, 1005, 1-15 (2018)   Extracellular vesicles (EVs) have been recognized as messengers delivering various active molecules between cells. This feature of EVs drew the attention of clinicians as well as researchers from different fields. However, exciting ideas to employ EVs as means of drug delivery or to test them as biomarkers of cellular status require very thoughtful and attentive approaches to the selection of analytical techniques for EV characterization. Optical and surface plasmonic analytical methods offer a researcher an invaluable opportunity to use already sized and/or quantified EVs in further functional cell-based assays and in focused biochemical tests (nucleic acid and protein arrays, etc.). Moreover, a high sensitivity and relative flexibility of surface plasmonic sensors open a possibility to develop instruments performing quantitative, metrical and EV surface/content analysis in a single device. This review aims to consider the applicability of established and modern optical techniques as well as novel surface plasmonic approaches for different aspects of EV analysis.  

3.2916           Tetraspanin CD63 Bridges Autophagic and Endosomal Processes To Regulate Exosomal Secretion and Intracellular Signaling of Epstein-Barr Virus LMP1

Hurwitz, S.N., Cheerathodi, M.R., Nkosi, D., York, S.B. and Meckes Jr., D.G.

1. Virol., 92(5), e1969-17 (2018)

  The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of cross talk between endosomes and autophagosomes and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here, we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether, these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity in both noninfected and virally infected cells.  

3.2917           Legionella effector AnkX interacts with host nuclear protein PLEKHN1

Yu, X., Noll, R.R., Duenas, B.P., Allgood, S.C., barker, K., Caplan, J.L., machner, M.P., LaBaer, J., Qiu, J. and Neunuebel, M.R. BMC Microbiol., 18:5 (2018)   Background The intracellular bacterial pathogen Legionella pneumophila proliferates in human alveolar macrophages, resulting in a severe pneumonia termed Legionnaires’ disease. Throughout the course of infection, L. pneumophila remains enclosed in a specialized membrane compartment that evades fusion with lysosomes. The pathogen delivers over 300 effector proteins into the host cell, altering host pathways in a manner that sets the stage for efficient pathogen replication. The L. pneumophila effector protein AnkX targets host Rab GTPases and functions in preventing fusion of the Legionella-containing vacuole with lysosomes. However, the current understanding of AnkX’s interaction with host proteins and the means through which it exerts its cellular function is limited. Results Here, we investigated the protein interaction network of AnkX by using the nucleic acid programmable protein array (NAPPA), a high-density platform comprising 10,000 unique human ORFs. This approach facilitated the discovery of PLEKHN1 as a novel interaction partner of AnkX. We confirmed this interaction through multiple independent in vitro pull-down, co-immunoprecipitation, and cell-based assays. Structured illumination microscopy revealed that endogenous PLEKHN1 is found in the nucleus and on vesicular compartments, whereas ectopically produced AnkX co-localized with lipid rafts at the plasma membrane. In mammalian cells, HaloTag-AnkX co-localized with endogenous PLEKHN1 on vesicular compartments. A central fragment of AnkX (amino acids 491–809), containing eight ankyrin repeats, extensively co-localized with endogenous PLEKHN1, indicating that this region may harbor a new function. Further, we found that PLEKHN1 associated with multiple proteins involved in the inflammatory response. Conclusions Altogether, our study provides evidence that in addition to Rab GTPases, the L. pneumophila effector AnkX targets nuclear host proteins and suggests that AnkX may have novel functions related to manipulating the inflammatory response.  

3.2918           Exosomal microRNAs (exomiRs): Small molecules with a big role in cancer

Bhome, R., Del Vecchio, F., Lee, G-H., Buillock, M.D., Primrose, J.N., Sayan, A.E. and Mirnezami, A.H. Cancer Lett., 420, 228-235 (2018)   Exosomes are secreted vesicles which can transmit molecular cargo between cells. Exosomal microRNAs (exomiRs) have drawn much attention in recent years because there is increasing evidence to suggest that loading of microRNAs into exosomes is not a random process. Preclinical studies have identified functional roles for exomiRs in influencing many hallmarks of cancer. Mechanisms underpinning their actions, such as exomiR receptors (“miRceptors”), are now becoming apparent. Even more exciting is the fact that exomiRs are highly suitable candidates for use as non-invasive biomarkers in an era of personalized cancer medicine.  

3.2919           Preparation of Highly Enriched ER Membranes Using Free-Flow Electrophoresis

Parsons, H.T. Methods in Mol. Biol., 1691, 103-115 (2018)   Free-flow electrophoresis (FFE) is a technique for separation of proteins, peptides, organelles, and cells. With zone electrophoresis (ZE-FFE), organelles are separated according to surface charge. The ER is the only remaining major cellular compartment in Arabidopsis not to have been isolated using density centrifugation, immune-isolation, or any other method previously applied to purification of plant membranes. By using continuous-flow electrophoresis ER vesicles of similar surface charge, which may have been fragmented during cell lysis, can be focused. A large portion of these vesicles are of sufficiently different surface charge that separation from the majority of Golgi and other contaminants is possible. Here we adapt an earlier ZE-FFE Golgi isolation protocol for the isolation of highly pure ER vesicles and for tracking the migration of peripheral ER vesicles. Isolating ER vesicles of homogenous surface charge allows multi-'omic analyses to be performed on the ER. This facilitates investigations into structure–function relationships within the ER.  

3.2920           Methods to Enrich Exosomes from Conditioned Media and Biological Fluids

Sharma, S., Scholz-Romero, K., Rice, G.E. and Salomon, C. Methods in Mol. Biol., 1710, 103-115 (2018)   Exosomes are nano-vesicles which can transport a range of molecules including but not limited to proteins and miRNA. This ability of exosomes renders them useful in cellular communication often resulting in biological changes. They have several functions in facilitating normal biological processes such as immune responses and an involvement in pregnancy. However, they have also been linked to pathological conditions including cancer and pregnancy complications such as preeclampsia. An understanding for the role of exosomes in preeclampsia is based on the ability to purify and characterize exosomes. There have been several techniques proposed for the enrichment of exosomes such as ultracentrifugation, density gradient separation, and ultrafiltration although there is no widely accepted optimized technique. Here we describe a workflow for isolating exosomes from cell-conditioned media and biological fluids using a combination of centrifugation, buoyant density, and ultrafiltration approaches.  

3.2921           Overview of Protocols for Studying Extracellular RNA and Extracellular Vesicles

Small, J., Alexander, R. and Balaj, L. Methods in Mol. Biol., 1740, 17-21 (2018)   Understanding the role of extracellular RNA (exRNA) has emerged as an exciting avenue for biomarker, therapeutic, as well as basic cell–cell communication applications and discoveries. Multiple protocols, kits, and procedures have been developed in the last decade to allow fractionation as well as isolation of subpopulations of macromolecules of interest found in biofluids. Here, we introduce the protocols decision tree developed by the Extracellular RNA Communication Consortium and available on their website (exRNA portal), and compare all methods currently available to the exRNA field and report pros and cons for each platform.  

3.2922           Extracellular RNA Isolation from Cell Culture Supernatant

Bakr, S., Simonson, B., Danielson, K.M. and Das, S. Methods in Mol. Biol., 1740, 23-34 (2018)   Extracellular RNAs are emerging as novel biomarkers and mediators of intercellular communication. Various methods to isolate RNA from biofluids and cell culture supernatants have been previously used by investigators. Here, we describe several standardized protocols for the isolation of RNAs from cell culture supernatants that utilize commercially available kits and reagents.  

3.2923           Cushioned–Density Gradient Ultracentrifugation (C-DGUC): A Refined and High Performance Method for the Isolation, Characterization, and Use of Exosomes

Li, K., Wong, D.K., Hong, K-Y. and Raffia, R.L. Methods in Mol. Biol., 1740, 69-83 (2018)   Exosomes represent one class of extracellular vesicles that are thought to be shed by all cell types. Although the exact nature of exosome biogenesis and function remains incompletely understood, they are increasingly recognized as a source of intercellular communication in health and disease. Recent observations of RNA exchange via donor cell-derived exosomes that exert genetic regulation in recipient cells have led to a boon into exosome research. The excitement and promise of exosomes as a new therapeutic avenue for human pathologies remain limited by challenges associated with their isolation from culture media and biofluids. The introduction of new methodologies to facilitate the isolation of exosomes has simultaneously raised concerns related to the reproducibility of studies describing exosome effector functions. Even high-speed ultracentrifugation, the first and long considered gold standard approach for exosome isolation has recently been noted to be subject to uncontrolled variables that could impact functional readouts of exosome preparations. This chapter describes principles and methods that attempt to overcome such limitations by first concentrating exosomes in a liquid cushion and subsequently resolving them using density gradient ultracentrifugation. Our approach avoids possible complications associated with direct pelleting onto plastic tubes and allows for further purification of exosomes from dense protein aggregates.  

3.2924           Dimerization leads to changes in APP (amyloid precursor protein) trafficking mediated by LRP1 and SorLA

Eggert, S., Gonzalez, A.C., Thomas, C., Schilling, S., Schwarz, S.M., Tischer, C., Adam, V., Strecker, P., Schmidt, V., Willnow, T.E., Hermey, G., Pietrzik, C.U., Koo, E.H. and Kins, S. Cell Mol. Life Sci., 75, 301.322 (2018)   Proteolytic cleavage of the amyloid precursor protein (APP) by α-, β- and γ-secretases is a determining factor in Alzheimer’s disease (AD). Imbalances in the activity of all three enzymes can result in alterations towards pathogenic Aβ production. Proteolysis of APP is strongly linked to its subcellular localization as the secretases involved are distributed in different cellular compartments. APP has been shown to dimerize in cis-orientation, affecting Aβ production. This might be explained by different substrate properties defined by the APP oligomerization state or alternatively by altered APP monomer/dimer localization. We investigated the latter hypothesis using two different APP dimerization systems in HeLa cells. Dimerization caused a decreased localization of APP to the Golgi and at the plasma membrane, whereas the levels in the ER and in endosomes were increased. Furthermore, we observed via live cell imaging and biochemical analyses that APP dimerization affects its interaction with LRP1 and SorLA, suggesting that APP dimerization modulates its interplay with sorting molecules and in turn its localization and processing. Thus, pharmacological approaches targeting APP oligomerization properties might open novel strategies for treatment of AD.  

3.2925           Lipid composition of membrane microdomains isolated detergent-free from PUFA supplemented RAW264.7 macrophages

Hellwwing, C., Tigistu-Sahle, F., Fuhrmann, H., Käkelä, R. and Schumann, J.

1. Cell Physiol., 233(3), 2602-2612 (2018)

  Profound alterations in the lipid profile of raft and non-raft plasma membrane microdomains were found when RAW264.7 macrophages were supplemented with polyunsaturated fatty acids (PUFAs) in physiologically relevant concentrations. For the first time lipids in the detergent-free isolated membrane domains of phagocytic immune cells were characterized by mass spectrometry. The extent of remodeling of the membrane lipids differed with different n3 and n6 PUFA supplements. The mildest effects were detected for α-linolenic acid (LNA) and linoleic acid (LA), the C18 precursors of the n3 and n6 families, respectively. When the effects of highly unsaturated PUFAs were compared, eicosapentaenoic acid (EPA) caused more extensive restructuring of membrane lipids than docosahexaenoic acid (DHA) or arachidonic acid (AA). The supplements altered the lipid species composition of both the raft and non-raft membrane fractions. The rafts containing elevated proportions of highly unsaturated lipid species may relocate sterically incompatible lipids and proteins originally belonging to this microdomain. Such effect was evident for sphingomyelin, which favored non-rafts instead of rafts after EPA supplementation. The current work suggests that the different functional consequences found previously when supplementing macrophages with either EPA or DHA have their origin in the different effects of these PUFAs on membrane architecture.  

3.2926           TMD1 domain and CRAC motif determine the association and disassociation of MxIRT1 with detergent-resistant membranes

Tan, S., Zhang, P., Xiao, W., Feng, B., Chen, L-Y., Li, S., Li, P., Zhao, W-Z., Qi, X-T. and Yin, L-P. Traffic, 19(2), 122-137 (2018)   Iron is essential for most living organisms. The iron-regulated transporter1 (IRT1) plays a major role in iron uptake in roots, and its trafficking from endoplasmic reticulum (ER) to plasma membrane (PM) is tightly coordinated with changes in iron environment. However, studies on the IRT1 response are limited. Here, we report that Malus xiaojinesis IRT1 (MxIRT1) associates with detergent-resistant membranes (DRMs, a biochemical counterpart of PM microdomains), whereas the PM microdomains are known platforms for signal transduction in the PM. Depending on the shift of MxIRT1 from microdomains to homogeneous regions in PM, MxIRT1-mediated iron absorption is activated by the cholesterol recognition/interaction amino acid consensus (CRAC) motif of MxIRT1. MxIRT1 initially associates with DRMs in ER via its transmembrane domain 1 (TMD1), and thus begins DRMs-dependent intracellular trafficking. Subsequently, MxIRT1 is sequestered in COPII vesicles via the ER export signal sequence in MxIRT1. These studies suggest that iron homeostasis is influenced by the CRAC motif and TMD1 domain due to their determination of MxIRT1-DRMs association.  

3.2927           The hydrophobic region of the Leishmania peroxin 14: requirements for association with a glycosome mimetic membrane

Cyr, N., Smith, T.K., Boisselier, E., Leroux, L-P., Kottarampatel, A.H., Davidsen, A., Salesse, C. and Jardim, A. Biochem. J., 475, 511-529 (2018)   Protein import into the Leishmania glycosome requires docking of the cargo-loaded peroxin 5 (PEX5) receptor to the peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14–membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149–179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5–PTS1 receptor–cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.  

3.2928           The MTM1–UBQLN2–HSP complex mediates degradation of misfolded intermediate filaments in skeletal muscle

Gavrilidis, C., Laredj, L., Solinhac, R., Messaddeq, n., Viaud, J., laporte, J., Sumara, I. and Hnia, K. Nature Cell Biol., 20, 198-210 (2018)   The ubiquitin proteasome system and autophagy are major protein turnover mechanisms in muscle cells, which ensure stemness and muscle fibre maintenance. Muscle cells contain a high proportion of cytoskeletal proteins, which are prone to misfolding and aggregation; pathological processes that are observed in several neuromuscular diseases called proteinopathies. Despite advances in deciphering the mechanisms underlying misfolding and aggregation, little is known about how muscle cells manage cytoskeletal degradation. Here, we describe a process by which muscle cells degrade the misfolded intermediate filament proteins desmin and vimentin by the proteasome. This relies on the MTM1–UBQLN2 complex to recognize and guide these misfolded proteins to the proteasome and occurs prior to aggregate formation. Thus, our data highlight a safeguarding function of the MTM1–UBQLN2 complex that ensures cytoskeletal integrity to avoid proteotoxic aggregate formation.  

3.2929           HIV Activates the Tyrosine Kinase Hck to Secrete ADAM Protease-Containing Extracellular Vesicles

Lee, J-H., Ostalecki, C., Zhao, Z., Kesti, T., Bruns, H., Simon, B., Harrer, T., Saksela, K. and Baur, A.S. EBioMed., 28, 151-161 (2018)   HIV-Nef activates the myeloid cell-typical tyrosine kinase Hck, but its molecular role in the viral life cycle is not entirely understood. We found that HIV plasma extracellular vesicles (HIV pEV) containing/10 proteases and Nef also harbor Hck, and analyzed its role in the context of HIV pEV secretion. Myeloid cells required Hck for the vesicle-associated release of ADAM17. This could be induced by the introduction of Nef and implied that HIV targeted Hck for vesicle-associated ADAM17 secretion from a myeloid compartment. The other contents of HIV-pEV, however, including miRNA and effector protein profiles, as well as the presence of haptoglobin suggested hepatocytes as a possible cellular source. HIV liver tissue analysis supported this assumption, revealing induction of Hck translation, evidence for ADAM protease activation and HIV infection. Our findings suggest that HIV targets Hck to induce pro-inflammatory vesicles release and identifies hepatocytes as a possible host cell compartment.  

3.2930           Exosome and MiRNA in Stroke

Bihl, J., Wang, J., Ma, X., Yang, Y., Zhao, B. and Chen, Y. Cellular and Molecular Approaches to Regeneration and Repair (Springer), 325-361 (2018)   Stroke is one of the leading causes of death and disability worldwide. Various types of stem cells have been applied to treat stroke and have been shown promising potential. The principal mechanism of therapeutic action has been partially ascribed to their strong paracrine capacity. Exosomes are small vesicles released from all kinds of cells and mediate intercellular communication by transferring exosomal protein and microRNA (miRNA) cargoes between cells in the brain. Among these cargoes, miRNAs play a key role in mediating biological function due to their prominent roles in gene regulation. Emerging data suggest that stem cell-released exosomes have advantages over stem cells to treat stroke, because exosomes could cross the blood bran barrier and easily to be modified and handled. Here, we first review the biogenesis, cargoes, and detection of exosomes. Then, we discussed the role of miRNAs in stroke. At last, we highlight the use of stem cell-released exosomes as biomarkers and therapeutic avenues in stroke. Perspectives on the developing role of stem cell-released exosomes mediated transfer of miRNAs as a therapeutic approach will also be discussed.  

3.2931           Mutant p53 cancers reprogram macrophages to tumor supporting macrophages via exosomal miR-1246

Cooks, T., Pateras, J.S., Jenkins, L.M., Patel, K.M., Robles, A.I., Morris, J., Forshew, T., Appella, E., Gorgoulis, V.G. and Harris, C.C. Nature Communications, 9:77 (2018)   TP53 mutants (mutp53) are involved in the pathogenesis of most human cancers. Specific mutp53 proteins gain oncogenic functions (GOFs) distinct from the tumor suppressor activity of the wild-type protein. Tumor-associated macrophages (TAMs), a hallmark of solid tumors, are typically correlated with poor prognosis. Here, we report a non-cell-autonomous mechanism, whereby human mutp53 cancer cells reprogram macrophages to a tumor supportive and anti-inflammatory state. The colon cancer cells harboring GOF mutp53 selectively shed miR-1246-enriched exosomes. Uptake of these exosomes by neighboring macrophages triggers their miR-1246-dependent reprogramming into a cancer-promoting state. Mutp53-reprogammed TAMs favor anti-inflammatory immunosuppression with increased activity of TGF-β. These findings, associated with poor survival in colon cancer patients, strongly support a microenvironmental GOF role for mutp53 in actively engaging the immune system to promote cancer progression and metastasis.  

3.2932           Ursodeoxycholyl lysophosphatidylethanolamide negatively regulates TLRmediated

lipopolysaccharide response in human THP-1-derived macrophages

Horvatova, A., Utaipan, T., Otto, A-C., Zhang, Y., Gan-Schreier, H., Pavek, P., Pathil, A., Stremmel, W. and Chamulitrat, W. Eur. J. Pharmacol., 825, 63-74 (2018)   The bile acid-phospholipid conjugate ursodeoxycholyl oleoyl-lysophophatidylethanolamide (UDCA-18:1LPE) is an anti-inflammatory and anti-fibrotic agent as previously shown in cultured hepatocytes and hepatic stellate cells as well as in in vivo models of liver injury. We hypothesize that UDCA-18:1LPE may directly inhibit the activation of immune cells. We found that UDCA-18:1LPE was capable of inhibiting the migration of phorbol ester-differentiated human THP-1 cells. We examined anti-inflammatory activity of UDCA-18:1LPE during activation of THP1-derived macrophages. Treatment of these macrophages by bacterial lipopolysaccharide (LPS) for 24 h induced the release of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β. This release was markedly inhibited by pretreatment with UDCA-18:1LPE by ~ 65–90%. Derivatives with a different fatty-acid chain in LPE moiety also exhibited anti-inflammatory property. Western blotting and indirect immunofluorescence analyses revealed that UDCA-18:1LPE attenuated the expression of phosphorylated p38, MKK4/MKK7, JNK1/2, and c-Jun as well as nuclear translocation of NF-κB by ~ 22–86%. After LPS stimulation, the Toll-like receptor adaptor proteins, myeloid differentiation factor 88 and TNF receptor associated factor 6, were recruited into lipid rafts and UDCA-18:1LPE inhibited this recruitment by 22% and 58%, respectively. Moreover, LPS treatment caused a decrease of the known cytoprotective lysophosphatidylcholine species containing polyunsaturated fatty acids by 43%, and UDCA-18:1LPE co-treatment reversed this decrease. In conclusion, UDCA-18:1LPE and derivatives inhibited LPS inflammatory response by interfering with Toll-like receptor signaling in lipid rafts leading to an inhibition of MAPK and NF-κB activation. These conjugates may represent a class of lead compounds for development of anti-inflammatory drugs.  

3.2933           Chemotherapy induces secretion of exosomes loaded with heparanase that degrades extracellular matrix and impacts tumor and host cell behavior

Bandari, S.K., Purushothaman, A., Ramani, V.C., Brinkley, G.J., Chandrashekar, D.S., varambally, S., Mobley, J.A., Zhang, Y., Brown, E.E., Vlodavsky, I. and Sanderson, R.D. Matrix Biol., 65, 104-118 (2018)   The heparan sulfate-degrading enzyme heparanase promotes the progression of many cancers by driving tumor cell proliferation, metastasis and angiogenesis. Heparanase accomplishes this via multiple mechanisms including its recently described effect on enhancing biogenesis of tumor exosomes. Because we recently discovered that heparanase expression is upregulated in myeloma cells that survive chemotherapy, we were prompted to investigate the impact of anti-myeloma drugs on exosome biogenesis. When myeloma cells were exposed to the commonly utilized anti-myeloma drugs bortezomib, carfilzomib or melphalan, exosome secretion by the cells was dramatically enhanced. These chemotherapy-induced exosomes (chemoexosomes) have a proteome profile distinct from cells not exposed to drug including a dramatic elevation in the level of heparanase present as exosome cargo. The chemoexosome heparanase was not found inside the chemoexosome, but was present on the exosome surface where it was capable of degrading heparan sulfate embedded within an extracellular matrix. When exposed to myeloma cells, chemoexosomes transferred their heparanase cargo to those cells, enhancing their heparan sulfate degrading activity and leading to activation of ERK signaling and an increase in shedding of the syndecan-1 proteoglycan. Exposure of chemoexosomes to macrophages enhanced their secretion of TNF-α, an important myeloma growth factor. Moreover, chemoexosomes stimulated macrophage migration and this effect was blocked by H1023, a monoclonal antibody that inhibits heparanase enzymatic activity. These data suggest that anti-myeloma therapy ignites a burst of exosomes having a high level of heparanase that remodels extracellular matrix and alters tumor and host cell behaviors that likely contribute to chemoresistance and eventual patient relapse.  

3.2934           Manufacturing Exosomes: A Promising Therapeutic Platform

Colao, I.L., Corteling, R., Bracewell, D. and Wall, I. Trend in Mol. Med., 24(3), 242-256 (2018)   Exosome research has been rejuvenated in recent years, due in part to the evolution in understanding of stem cell mode of action. The paracrine effect of stem cell therapy candidates has been mechanistically linked to inherited, specific functionality in secreted exosome derivatives. Even though exosomes are expected to enter clinical trials imminently, there has been a lack of manufacturing process development work that is needed to generate clinically relevant quantities of exosomes as trials progress towards larger patient numbers. If manufacturing research is not undertaken now, then the advancement of exosomes as a new therapeutic platform will be slowed. Thus, there is an urgent need for technological advancements. Here, we present process options for industrial and academic researchers to consider to translate exosomes into viable therapeutic candidates from a manufacturing perspective.  

3.2935           Proteasome inhibition blocks necroptosis by attenuating death complex aggregation

Ali, M. and Mocarski, E.S. Cell Death & Disease, 9:346 (2018)   Proteasome inhibitors have achieved clinical success because they trigger intrinsic and extrinsic cell death to eliminate susceptible human cancers. The ubiquitin-proteasome protein degradation system regulates signaling pathways by controlling levels of components such as cellular inhibitor of apoptosis (cIAP)1 and cIAP2 in TNF-mediated cell death. Here, we sought to evaluate the contribution of necroptosis to the cell death pattern induced by the specific proteasome inhibitor Carfilzomib (Cf). Proteasome inhibitor-sensitive multiple myeloma cell lines die in response to Cf by apoptosis in combination with serine protease-dependent death, without any contribution of RIPK3-dependent necroptosis. Proteasome inhibition leads to the induction of apoptotic markers such as activated caspase-3 rather than necroptotic markers such as phosphorylated-MLKL in all cell lines tested. In HT-29 cells, Cf attenuates the late RIPK1 interaction with TNFR1 during TNF-induced necroptosis without altering the sensitivity of cIAP antagonists. Cf treatment results in decreased translocation of death signaling components RIPK1, FADD, caspase-8, cFLIP, and RIPK3 to detergent insoluble fractions. Our results show that proteasome inhibition with Cf impairs necroptosis and favors apoptosis even in cells with intact necroptotic machinery. Following the induction of TNFR1-mediated necroptosis, proteasome activity stabilizes effective aggregation and activation of ripoptosome/necrosome complexes.  

3.2936           Extracellular vesicles as a platform for membrane-associated therapeutic protein delivery

Yang, Y., Hong, Y., Cho, E., Kim, G.B. and Kim, I-S.

1. Extracellular Vesicles, 7:1, 1440131 (2018)

  Membrane proteins are of great research interest, particularly because they are rich in targets for therapeutic application. The suitability of various membrane proteins as targets for therapeutic formulations, such as drugs or antibodies, has been studied in preclinical and clinical studies. For therapeutic application, however, a protein must be expressed and purified in as close to its native conformation as possible. This has proven difficult for membrane proteins, as their native conformation requires the association with an appropriate cellular membrane. One solution to this problem is to use extracellular vesicles as a display platform. Exosomes and microvesicles are membranous extracellular vesicles that are released from most cells. Their membranes may provide a favourable microenvironment for membrane proteins to take on their proper conformation, activity, and membrane distribution; moreover, membrane proteins can cluster into microdomains on the surface of extracellular vesicles following their biogenesis. In this review, we survey the state-of-the-art of extracellular vesicle (exosome and small-sized microvesicle)-based therapeutics, evaluate the current biological understanding of these formulations, and forecast the technical advances that will be needed to continue driving the development of membrane protein therapeutics.  

3.2937           The dynamic recruitment of TRBP to neuronal membranes mediates dendritogenesis during development

Antoniou, A., Khudayberdiev, S., Idziak, A., Bicker, S., Jacob, R. and Schratt, G. EMBO Reports, 19, e44853 (2018)   MicroRNAs are important regulators of local protein synthesis during neuronal development. We investigated the dynamic regulation of microRNA production and found that the majority of the microRNA-generating complex, consisting of Dicer, TRBP, and PACT, specifically associates with intracellular membranes in developing neurons. Stimulation with brain-derived neurotrophic factor (BDNF), which promotes dendritogenesis, caused the redistribution of TRBP from the endoplasmic reticulum into the cytoplasm, and its dissociation from Dicer, in a Ca²⁺-dependent manner. As a result, the processing of a subset of neuronal precursor microRNAs, among them the dendritically localized pre-miR16, was impaired. Decreased production of miR-16-5p, which targeted the BDNF mRNA itself, was rescued by expression of a membrane-targeted TRBP. Moreover, miR-16-5p or membrane-targeted TRBP expression blocked BDNF-induced dendritogenesis, demonstrating the importance of neuronal TRBP dynamics for activity-dependent neuronal development. We propose that neurons employ specialized mechanisms to modulate local gene expression in dendrites, via the dynamic regulation of microRNA biogenesis factors at intracellular membranes of the endoplasmic reticulum, which in turn is crucial for neuronal dendrite complexity and therefore neuronal circuit formation and function.  

3.2938           Phagocytosis depends on TRPV2-mediated calcium influx and requires TRPV2 in lipids rafts: alteration in macrophages from patients with cystic fibrosis

Leveque, M., penna, A., Le Trionnaire, S., Belleguic, C., Desrues, B., Brinchault, G., Jouneau, S., Lagadic-Gossmann, D. and Martin-Chouly, C. Scientific Reports, 8:4310 (2018)   Whereas many phagocytosis steps involve ionic fluxes, the underlying ion channels remain poorly defined. As reported in mice, the calcium conducting TRPV2 channel impacts the phagocytic process. Macrophage phagocytosis is critical for defense against pathogens. In cystic fibrosis (CF), macrophages have lost their capacity to act as suppressor cells and thus play a significant role in the initiating stages leading to chronic inflammation/infection. In a previous study, we demonstrated that impaired function of CF macrophages is due to a deficient phagocytosis. The aim of the present study was to investigate TRPV2 role in the phagocytosis capacity of healthy primary human macrophage by studying its activity, its membrane localization and its recruitment in lipid rafts. In primary human macrophages, we showed that P. aeruginosa recruits TRPV2 channels at the cell surface and induced a calcium influx required for bacterial phagocytosis. We presently demonstrate that to be functional and play a role in phagocytosis, TRPV2 might require a preferential localization in lipid rafts. Furthermore, CF macrophage displays a perturbed calcium homeostasis due to a defect in TRPV2. In this context, deregulated TRPV2-signaling in CF macrophages could explain their defective phagocytosis capacity that contribute to the maintenance of chronic infection.  

3.2939           Subpopulations of extracellular vesicles and their therapeutic potential

Lässer, C., Jang, S.C. and Lötvall, J. Mol. Aspects Med.,60, 1-14 (2018)   Extracellular vesicles (EVs), such as exosomes and microvesicles, have over the last 10–15 years been recognized to convey key messages in the molecular communication between cells. Indeed, EVs have the capacity to shuttle proteins, lipids, and nucleotides such as RNA between cells, leading to an array of functional changes in the recipient cells. Importantly, the EV secretome changes significantly in diseased cells and under conditions of cellular stress. More recently, it has become evident that the EV secretome is exceptionally diverse, with many different types of EVs being released by a single cell type, and these EVs can be described in terms of differences in density, molecular cargos, and morphology. This review will discuss the diversity of EVs, will introduce some suggestions for how to categorize them, and will propose how EVs and their subpopulations might be used for very different therapeutic purposes.  

3.2940           Exosome-Mediated Benefits of Cell Therapy in Mouse and Human Models of Duchenne Muscular Dystrophy

Aminzadeh, M.A. et al Stem Cell Reports, 10, 942-955 (2018)   Genetic deficiency of dystrophin leads to disability and premature death in Duchenne muscular dystrophy (DMD), affecting the heart as well as skeletal muscle. Here, we report that clinical-stage cardiac progenitor cells, known as cardiosphere-derived cells (CDCs), improve cardiac and skeletal myopathy in the mdx mouse model of DMD. Injection of CDCs into the hearts of mdx mice augments cardiac function, ambulatory capacity, and survival. Exosomes secreted by human CDCs reproduce the benefits of CDCs in mdx mice and in human induced pluripotent stem cell-derived Duchenne cardiomyocytes. Surprisingly, CDCs and their exosomes also transiently restored partial expression of full-length dystrophin in mdx mice. The findings further motivate the testing of CDCs in Duchenne patients, while identifying exosomes as next-generation therapeutic candidates.  

3.2941           Hepatitis B Virus Subverts the Autophagy Elongation Complex Atg5-12/16L1 and Does Not Require Atg8/LC3 Lipidation for Viral Maturation

Döring, T., Zeyen, l., Bartusch, C. and Prange, R.

1. Virol., 92(7), e01513-17 (2018)

  Previous studies indicated that hepatitis B virus (HBV) stimulates autophagy to favor its production. To understand how HBV co-opts autophagy as a proviral machinery, we studied the roles of key autophagy proteins in HBV-replicating liver cell cultures. RNA interference-mediated silencing of Atg5, Atg12, and Atg16L1, which promote autophagophore expansion and LC3 membrane conjugation, interfered with viral core/nucleocapsid (NC) formation/stability and strongly diminished virus yields. Concomitantly, the core/NC membrane association and their sorting to envelope-positive compartments were perturbed. A close inspection of the HBV/autophagy cross talk revealed that the virus depended on Atg12 covalently conjugated to Atg5. In support of this finding, HBV required the E2-like enzymes Atg10 and Atg3, which catalyze or facilitate Atg5-12 conjugation, respectively. Atg10 and Atg3 knockdowns decreased HBV production, while Atg3 overexpression increased virus yields. Mapping analyses demonstrated that the HBV core protein encountered the Atg5-12/16L1 complex via interaction with the intrinsically disordered region of the Atg12 moiety that is dispensable for autophagy function. The role of Atg12 in HBV replication was confirmed by its incorporation into virions. Although the Atg5-12/16L1 complex and Atg3 are essential for LC3 lipidation and, thus, for autophagosome maturation and closure, HBV propagation did not require LC3. Silencing of LC3B, the most abundant LC3 isoform, did not inhibit but rather augmented virus production. Similar augmenting effects were obtained upon overexpression of a dominant negative mutant of Atg4B that blocked the lipid conjugation of the LC3 isoforms and their GABARAP paralogues. Together, our data indicate that HBV subverts early, nondegradative autophagy components as assembly scaffolds, thereby concurrently avoiding autophagosomal destruction.  

3.2942           Placental exosomes profile in maternal and fetal circulation in intrauterine growth restriction - Liquid biopsies to monitoring fetal growth

Miranda, J., Paules, C., Nair, S., lai, A., Palma, C., Scholz-Romero, K., Rice, G.E., Gratacos, E., Crispi, F. and Salomon, C. Placenta, 64, 34-43 (2018)   Introduction Placenta-derived exosomes may represent an additional pathway by which the placenta communicates with the maternal system to induce maternal vascular adaptations to pregnancy and it may be affected during Fetal growth restriction (FGR). The objective of this study was to quantify the concentration of total and placenta-derived exosomes in maternal and fetal circulation in small fetuses classified as FGR or small for gestational age (SGA). Methods Prospective cohort study in singleton term gestations including 10 normally grown fetuses and 20 small fetuses, sub-classified into SGA and FGR accordingly to birth weight (BW) percentile and fetoplacental Doppler. Exosomes were isolated from maternal and fetal plasma and characterized by morphology, enrichment of exosomal proteins, and size distribution by electron microscopy, western blot, and nanoparticle tracking analysis, respectively. Total and specific placenta-derived exosomes were determined using quantum dots coupled with CD63^+ve and placental-type alkaline phosphatase (PLAP)^+ve antibodies, respectively. Results Maternal concentrations of CD63^+ve and PLAP^+ve exosomes were similar between the groups (all p > 0.05). However, there was a significant positive correlation between the ratio of placental-derived to total exosomes (PLAP^+ve ratio) and BW percentile, [rho = 0.77 (95% CI: 0.57 to 0.89); p = 0.0001]. The contribution of placental exosomes to the total exosome concentration in maternal and fetal circulation showed a significant decrease among cases, with lower PLAP^+ve ratios in FGR compared to controls and SGA cases. Discussion Quantification of placental exosomes in maternal plasma reflects fetal growth and it may be a useful indicator of placental function.  

3.2943           Modulation of key lipid raft constituents in primary rat hepatocytes by fumonisin B1 - Implications for cancer promotion in the liver

Burger, H-M., Abel, S. and Gelderblom, W.C.A. Food Chem. Toxicol., 115, 34-41 (2018)   Fumonisin B₁ (FB₁), a group 2B natural occurring carcinogenic mycotoxin, modulated lipid and fatty acid (FA) constituents of lipid rafts isolated from primary hepatocytes following exposure to a cytotoxic concentration of FB₁ (250 μM). The major effects observed in rafts, included a significant (p < 0.05) increase in raft cholesterol (CHOL) and glycerophospholipid such as phosphatidylethanolamine (PE), whereas sphingomyelin (SM) decreased (p < 0.05). Changes in lipid constituents resulted in the disruption of important membrane fluidity parameters represented as a decreased (p < 0.05) in the phosphatidylcholine (PC)/PE and PC/(PE+SM) ratios and an increase (p < 0.05) in the CHOL/PL (PL=PC+PE) ratio, suggesting the preservation of lipid raft rigidity and integrity. Observed FA changes in the raft PE fraction included a significant (p < 0.05) increase in C18:2ω-6, C20:3ω-6, C20:4ω-6, C22:4ω-6, C22:5ω-3 and C22:6ω-3, with an increase in total ω-6 and ω-3 polyunsaturated fatty acids (PUFAs). Modulation of the FA content in PE, specifically the C20:4ω-6 PC/PE ratio and PUFA levels, together with changes in CHOL and SM are key determinants regulating the integrity and function of lipid rafts. In primary hepatocytes these changes are associated with the inhibition of cell proliferation and induction of apoptosis. A lipogenic mechanism is proposed whereby FB₁ modulates lipid rafts and differentially target cell survival indices of normal and preneoplastic hepatocytes during cancer promotion in the liver.  

3.2944           In vitro gentamicin exposure alters caveolae protein profile in cochlear spiral ligament pericytes

Gheli, E., Grondin, Y., Millet, E., bartos, A., Bortoni, M., dos Santos, C.O.G., Trevino-Villarreal, H.J., Sepulveda, R. and Rogers, R. Proteome Sci., 16:7 (2018)   Background The aminoglycoside antibiotic gentamicin is an ototoxic drug and has been used experimentally to investigate cochlear damage induced by noise. We have investigated the changes in the protein profile associated with caveolae in gentamicin treated and untreated spiral ligament (SL) pericytes, specialized cells in the blood labyrinth barrier of the inner ear microvasculature. Pericytes from various microvascular beds express caveolae, protein and cholesterol rich microdomains, which can undergo endocytosis and transcytosis to transport small molecules in and out the cells. A different protein profile in transport-specialized caveolae may induce pathological changes affecting the integrity of the blood labyrinth barrier and ultimately contributing to hearing loss. Method Caveolae isolation from treated and untreated cells is achieved through ultracentrifugation of the lysates in discontinuous gradients. Mass spectrometry (LC-MS/MS) analysis identifies the proteins in the two groups. Proteins segregating with caveolae isolated from untreated SL pericytes are then compared to caveolae isolated from SL pericytes treated with the gentamicin for 24 h. Data are analyzed using bioinformatic tools. Results The caveolae proteome in gentamicin treated cells shows that 40% of total proteins are uniquely associated with caveolae during the treatment, and 15% of the proteins normally associated with caveolae in untreated cell are suppressed. Bioinformatic analysis of the data shows a decreased expression of proteins involved in genetic information processing, and an increase in proteins involved in metabolism, vesicular transport and signal transduction in gentamicin treated cells. Several Rab GTPases proteins, ubiquitous transporters, uniquely segregate with caveolae and are significantly enriched in gentamicin treated cells. Conclusion We report that gentamicin exposure modifies protein profile of caveolae from SL pericytes. We identified a pool of proteins which are uniquely segregating with caveolae during the treatment, mainly participating in metabolic and biosynthetic pathways, in transport pathways and in genetic information processing. Finally, we show for the first time proteins associated with caveolae SL pericytes linked to nonsyndromic hearing loss.  

3.2945           The mitochondrial outer-membrane location of the EXD2 exonuclease contradicts its direct role in nuclear DNA repair

Hensen, F., Moretton, A., van Esvald, S., Fare, G, and Spelbrink, J.N. Scientific Reports, 8:5368 (2018)   EXD2 is a recently identified exonuclease that has been implicated in nuclear double-strand break repair. Given our long standing interest in mitochondrial DNA maintenance and indications that EXD2 could also be a mitochondrial protein we sought to determine its cellular localization and possible mitochondrial associated functions. Our results show that EXD2 indeed shows mitochondrial localization, but, surprisingly, is found predominantly associated with the mitochondrial outer-membrane. Gradient purified nuclei show only the faintest hint of EXD2 presence while overexpression of the predicted full-length protein shows exclusive mitochondrial localization. Importantly, induction of double-strand DNA breaks via X-irradiation or Zeocin treatment does not support the notion that EXD2 re-locates to the nucleus following double-strand breaks and thus is unlikely to have a direct role in nuclear DNA repair. Knockdown or overexpression of EXD2 affects the cellular distribution of mitochondria. These results suggest that the reported defects in nuclear DNA repair following EXD2 depletion are likely an indirect consequence of altered mitochondrial dynamics and/or function.  

3.2946           “Good things come in small packages”: application of exosome-based therapeutics in neonatal lung injury

Willis, G.R., Mitsialis, S.A. and Kourembanas, S. Pediatric Res., 83(1), 298-307 (2018)   Infants born at very low gestational age contribute disproportionately to neonatal morbidity and mortality. Advancements in antenatal steroid therapies and surfactant replacement have favored the survival of infants with ever-more immature lungs. Despite such advances in medical care, cardiopulmonary and neurological impairment prevail in constituting the major adverse outcomes for neonatal intensive care unit survivors. With no single effective therapy for either the prevention or treatment of such neonatal disorders, the need for new tools to treat and reduce risk of further complications associated with extreme preterm birth is urgent. Mesenchymal stem/stromal cell (MSC)-based approaches have shown promise in numerous experimental models of lung injury relevant to neonatology. Recent studies have highlighted that the therapeutic potential of MSCs is harnessed in their secretome, and that the therapeutic vector therein is represented by the exosomes released by MSCs. In this review, we summarize the development and significance of stem cell-based therapies for neonatal diseases, focusing on preclinical models of neonatal lung injury. We emphasize the development of MSC exosome-based therapeutics and comment on the challenges in bringing these promising interventions to clinic.  

3.2947           Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression

Pi, F., Binzel, D.W., Lee, T.J., Li, Z., Sun, M., Rychahou, P., Li, H., Haque, F., Wang, S., Croce, C.M., Guo, B., Evers, B.M. and Guo, P. Nature Nanotechnol., 13, 82-89 (2018)   Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.  

3.2948           Overcoming Limitations Inherent in Sulfamidase to Improve Mucopolysaccharidosis IIIA Gene Therapy

Chen, Y., Zheng, S., Tecedor, L. and Davidson, B.L. Molecular Therapy, 26(4), 1118-1126 (2018)   Sulfamidase (SGSH) deficiency causes mucopolysaccharidosis type IIIA (MPS IIIA), a lysosomal storage disease (LSD) that affects the CNS. In earlier work in LSD mice and dog models, we exploited the utility of adeno-associated viruses (AAVs) to transduce brain ventricular lining cells (ependyma) for secretion of lysosomal hydrolases into the cerebrospinal fluid (CSF), with subsequent distribution of enzyme throughout the brain resulting in improved cognition and extending lifespan. A critical feature of this approach is efficient secretion of the expressed enzyme from transduced cells, for delivery by CSF to nontransduced cells. Surprisingly, we found that SGSH was poorly secreted from cells, resulting in retention of the expressed product. Using site-directed mutagenesis of native SGSH, we identified an improved secretion variant that also displayed enhanced uptake properties that were mannose-6-phosphate receptor independent. In studies in MPS IIIA-deficient mice, ependymal transduction with AAVs expressing variant SGSH improved spatial learning and reduced memory deficits, substrate accumulation, and astrogliosis. Secondary lysosomal enzyme elevations in the CSF and brain parenchyma were also resolved. In contrast, ependymal transduction with AAVs expressing wild-type SGSH had significantly lower CSF SGSH levels and limited impacts on behavior. These results demonstrate the utility of a previously undescribed SGSH variant for improved MPS IIIA brain gene therapy.  

3.2949           A plant-like mitochondrial carrier family protein facilitates mitochondrial transport of di- and tricarboxylates in Trypanosoma brucei

Colasante, C., Zheng, F., Kemp, C. and Voncken, F. Mol. Biochem. Paarasitol., 221, 36-51 (2018)   The procyclic form of the human parasite Trypanosoma brucei harbors one single, large mitochondrion containing all tricarboxylic acid (TCA) cycle enzymes and respiratory chain complexes present also in higher eukaryotes. Metabolite exchange among subcellular compartments such as the cytoplasm, the mitochondrion, and the peroxisomes is crucial for redox homeostasis and for metabolic pathways whose enzymes are dispersed among different organelles. In higher eukaryotes, mitochondrial carrier family (MCF) proteins transport TCA-cycle intermediates across the inner mitochondrial membrane. Previously, we identified several MCF members that are essential for T. brucei survival. Among these, only one MCF protein, TbMCP12, potentially could transport dicarboxylates and tricarboxylates. Here, we conducted phylogenetic and sequence analyses and functionally characterised TbMCP12 in vivo. Our results suggested that similarly to its homologues in plants, TbMCP12 transports both dicarboxylates and tricarboxylates across the mitochondrial inner membrane. Deleting this carrier in T. brucei was not lethal, while its overexpression was deleterious. Our results suggest that the intracellular abundance of TbMCP12 is an important regulatory element for the NADPH balance and mitochondrial ATP-production.  

3.2950           Purification of Highly Active Alphavirus Replication Complexes Demonstrates Altered Fractionation of Multiple Cellular Membranes

Pietilä, M.K., van Hemert, M.J. and Ahola, T.

1. Virol., 92(8), e01852-17 (2018)

  Positive-strand RNA viruses replicate their genomes in membrane-associated structures; alphaviruses and many other groups induce membrane invaginations called spherules. Here, we established a protocol to purify these membranous replication complexes (RCs) from cells infected with Semliki Forest virus (SFV). We isolated SFV spherules located on the plasma membrane and further purified them using two consecutive density gradients. This revealed that SFV infection strongly modifies cellular membranes. We removed soluble proteins, the Golgi membranes, and most of the mitochondria, but plasma membrane, endoplasmic reticulum (ER), and late endosome markers were retained in the membrane fraction that contained viral RNA synthesizing activity, replicase proteins, and minus- and plus-strand RNA. Electron microscopy revealed that the purified membranes displayed spherule-like structures with a narrow neck. This membrane enrichment was specific to viral replication, as such a distribution of membrane markers was only observed after infection. Besides the plasma membrane, SFV infection remodeled the ER, and the cofractionation of the RC-carrying plasma membrane and ER suggests that SFV recruits ER proteins or membrane to the site of replication. The purified RCs were highly active in synthesizing both genomic and subgenomic RNA. Detergent solubilization destroyed the replication activity, demonstrating that the membrane association of the complex is essential. Most of the newly made RNA was in double-stranded replicative molecules, but the purified complexes also produced single-stranded RNA as well as released newly made RNA. This indicates that the purification established here maintained the functionality of RCs and thus enables further structural and functional studies of active RCs.  

3.2951           Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis

Deo, P., Chow, S.H., hay, I.D., Kleifeld, o., Costin, A., Elglass, K.D., Jiang, J-H., Ramm, G., Gabriel, K., Dougan, G., Lithgow, T., Heinz, E. and Naderer, T. PloS Pathogens, 14(3), e1006945 (2018)   Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity.  

3.2952           Inhibition of acid sphingomyelinase disrupts LYNUS signaling and triggers autophagy

Justice, M.J., Bronova, I., Schweitzer, K.S., Poirier, C., Blum, J.S., Berdyshev, E.V. and Petrache, I.

1. Lipd Res., 59(4), 596-606 (2018)

  Activation of the lysosomal ceramide-producing enzyme, acid sphingomyelinase (ASM), by various stresses is centrally involved in cell death and has been implicated in autophagy. We set out to investigate the role of the baseline ASM activity in maintaining physiological functions of lysosomes, focusing on the lysosomal nutrient-sensing complex (LYNUS), a lysosomal membrane-anchored multiprotein complex that includes mammalian target of rapamycin (mTOR) and transcription factor EB (TFEB). ASM inhibition with imipramine or sphingomyelin phosphodiesterase 1 (SMPD1) siRNA in human lung cells, or by transgenic Smpd1^+/− haploinsufficiency of mouse lungs, markedly reduced mTOR- and P70-S6 kinase (Thr 389)-phosphorylation and modified TFEB in a pattern consistent with its activation. Inhibition of baseline ASM activity significantly increased autophagy with preserved degradative potential. Pulse labeling of sphingolipid metabolites revealed that ASM inhibition markedly decreased sphingosine (Sph) and Sph-1-phosphate (S1P) levels at the level of ceramide hydrolysis. These findings suggest that ASM functions to maintain physiological mTOR signaling and inhibit autophagy and implicate Sph and/or S1P in the control of lysosomal function.  

3.2953           118. Single-Cell Transcriptome of the Depressed and Suicidal Brain

Nagy, C., Maitra, M., Theroux, J-F., Djambazian, H and Turecki, G. Biol. Psychiatry, 83, Abstract 118, S1-S107 (2018)   Background Brain molecular changes are typically measured in tissue homogenates. However, multiple neuronal and glial subtypes with specific gene expression patterns are likely to be distinctly modified in a diseased state. The detection of subtle transcriptomic alterations such as those expected in psychiatric, would benefit greatly from single cell resolution. Methods Using post-mortem brain tissue we conducted single-cell (single-nuclei) transcriptome analysis in 16 individuals who died by suicide during an episode of major depression and 16 matched healthy controls. Bulk nuclei were isolated from BA8/9 using an Optiprep™ gradient and single nuclei were captured using 10x Genomics’ Chromium technology, a droplet based protocol. The tagged libraries were sequenced using Illumina’s HiSeq 4000 platform to approximately 40-50k reads per nucleus. We used a custom Cell Ranger pipeline to produce a gene barcode matrix. Downstream bioinformatics analysis was performed by Seurat. Results Using Seurat to perform unsupervised clustering, we are able to produce well define clusters that can be classified by differential expression of board markers of cell type. Transcriptional profiles were used to further subdivided into clusters for differential analysis between groups. Random forest plots produced differential cluster profiles between groups. Conclusions These data are the stepping stone for identifying cell-specific genes and networks of genes whose expression is dysregulated in depression.  

3.2954           Immunomodulatory Effects of Pneumococcal Extracellular Vesicles on Cellular and Humoral Host Defenses

Codemo, M., Muschiol. S., Iovino, F., Nannapaneni, P., Plant, l., Wai, S.N. and Henriques-Normark, B. mBio, 9(2), e00559-18 (2018)   Gram-positive bacteria, including the major respiratory pathogen Streptococcus pneumoniae, were recently shown to produce extracellular vesicles (EVs) that likely originate from the plasma membrane and are released into the extracellular environment. EVs may function as cargo for many bacterial proteins, however, their involvement in cellular processes and their interactions with the innate immune system are poorly understood. Here, EVs from pneumococci were characterized and their immunomodulatory effects investigated. Pneumococcal EVs were protruding from the bacterial surface and released into the medium as 25 to 250 nm lipid stained vesicles containing a large number of cytosolic, membrane, and surface-associated proteins. The cytosolic pore-forming toxin pneumolysin was significantly enriched in EVs compared to a total bacterial lysate but was not required for EV formation. Pneumococcal EVs were internalized into A549 lung epithelial cells and human monocyte-derived dendritic cells and induced proinflammatory cytokine responses irrespective of pneumolysin content. EVs from encapsulated pneumococci were recognized by serum proteins, resulting in C3b deposition and formation of C5b-9 membrane attack complexes as well as factor H recruitment, depending on the presence of the choline binding protein PspC. Addition of EVs to human serum decreased opsonophagocytic killing of encapsulated pneumococci. Our data suggest that EVs may act in an immunomodulatory manner by allowing delivery of vesicle-associated proteins and other macromolecules into host cells. In addition, EVs expose targets for complement factors in serum, promoting pneumococcal evasion of humoral host defense.  

3.2955           Release of Staphylococcus aureus extracellular vesicles and their application as a vaccine platform

Wang, X., Thompson, C.D., Weidenmaier, C. and Lee, J.C. Nature Communications, 9:1379 (2018)   Secretion of extracellular vesicles (EVs), a process common to eukaryotes, archae, and bacteria, represents a secretory pathway that allows cell-free intercellular communication. Microbial EVs package diverse proteins and influence the host-pathogen interaction, but the mechanisms underlying EV production in Gram-positive bacteria are poorly understood. Here we show that EVs purified from community-associated methicillin-resistant Staphylococcus aureus package cytosolic, surface, and secreted proteins, including cytolysins. Staphylococcal alpha-type phenol-soluble modulins promote EV biogenesis by disrupting the cytoplasmic membrane; whereas, peptidoglycan cross-linking and autolysin activity modulate EV production by altering the permeability of the cell wall. We demonstrate that EVs purified from a S. aureus mutant that is genetically engineered to express detoxified cytolysins are immunogenic in mice, elicit cytolysin-neutralizing antibodies, and protect the animals in a lethal sepsis model. Our study reveals mechanisms underlying S. aureus EV production and highlights the usefulness of EVs as a S. aureus vaccine platform.  

3.2956           New splicing variants of mitochondrial Rho GTPase-1 (Miro1) transport peroxisomes

Okumoto, K., Ono, T., Tayama, R., Shimomura, A., Nagata, A. and Fujiki, Y.

1. Cell. Biol., 217(2), 619-633 (2018)

  Microtubule-dependent long-distance movement of peroxisomes occurs in mammalian cells. However, its molecular mechanisms remain undefined. In this study, we identified three distinct splicing variants of human mitochondrial Rho GTPase-1 (Miro1), each containing amino acid sequence insertions 1 (named Miro1-var2), 2 (Miro1-var3), and both 1 and 2 (Miro1-var4), respectively, at upstream of the transmembrane domain. Miro1-var4 and Miro1-var2 are localized to peroxisomes in a manner dependent on the insertion 1 that is recognized by the cytosolic receptor Pex19p. Exogenous expression of Miro1-var4 induces accumulation of peroxisomes at the cell periphery and augments long-range movement of peroxisomes along microtubules. Depletion of all Miro1 variants by knocking down MIRO1 suppresses the long-distance movement of peroxisomes. Such abrogated movement is restored by reexpression of peroxisomal Miro1 variants. Collectively, our findings identify for the first time peroxisome-localized Miro1 variants as adapter proteins that link peroxisomes to the microtubule-dependent transport complexes including TRAK2 in the intracellular translocation of peroxisomes in mammalian cells.  

3.2957           Unconventional secretion of FABP4 by endosomes and secretory lysosomes

Villeneuve, J., Bassaganyas, L., Lepreux, S., Chiritoiu, M., Costet, p., Ripoche, J., Malhotra, V. and Schekman, R.

1. Cell Biol., 217(2), 649-665 (2018)

  An appreciation of the functional properties of the cytoplasmic fatty acid binding protein 4 (FABP4) has advanced with the recent demonstration that an extracellular form secreted by adipocytes regulates a wide range of physiological functions. Little, however, is known about the mechanisms that mediate the unconventional secretion of FABP4. Here, we demonstrate that FABP4 secretion is mediated by a membrane-bounded compartment, independent of the conventional endoplasmic reticulum–Golgi secretory pathway. We show that FABP4 secretion is also independent of GRASP proteins, autophagy, and multivesicular bodies but involves enclosure within endosomes and secretory lysosomes. We highlight the physiological significance of this pathway with the demonstration that an increase in plasma levels of FABP4 is inhibited by chloroquine treatment of mice. These findings chart the pathway of FABP4 secretion and provide a potential therapeutic means to control metabolic disorders associated with its dysregulated secretion.  

3.2958           Mechanism of Stx17 recruitment to autophagosomes via IRGM and mammalian Atg8 proteins

Kumar, S., Jain, A., Farzam, F., Jia, J., Gu, Y., Choi, S.W., Mudd, M.H., Claude-Taupin, A., Wester, M.J., Lidke, K.A., Rusten, T-E., and Deretic, V.

1. Cell Biol., 217(3), 997-1013 (2018)

  Autophagy is a conserved eukaryotic process with metabolic, immune, and general homeostatic functions in mammalian cells. Mammalian autophagosomes fuse with lysosomes in a SNARE-driven process that includes syntaxin 17 (Stx17). How Stx17 translocates to autophagosomes is unknown. In this study, we show that the mechanism of Stx17 recruitment to autophagosomes in human cells entails the small guanosine triphosphatase IRGM. Stx17 directly interacts with IRGM, and efficient Stx17 recruitment to autophagosomes requires IRGM. Both IRGM and Stx17 directly interact with mammalian Atg8 proteins, thus being guided to autophagosomes. We also show that Stx17 is significant in defense against infectious agents and that Stx17–IRGM interaction is targeted by an HIV virulence factor Nef.  

3.2959           Amino acid deprivation and central carbon metabolism regulate the production of outer membrane vesicles and tubes by Francisella

Sampath, V., McCraig, W.D. and Thanassi, D.G. Mol. Microbiol., 107(4), 523-541 (2018)   Francisella tularensis is a highly virulent Gram‐negative bacterial pathogen that causes the zoonotic disease tularemia. F. novicida, a model tularemia strain, produces spherical outer membrane vesicles (OMV), as well as novel tubular vesicles and extensions of the cell surface. These OMV and tubes (OMV/T) are produced in a regulated manner and contain known virulence factors. Mechanisms by which bacterial vesicles are produced and regulated are not well understood. We performed a genetic screen in F. novicida to decipher the molecular basis for regulated OMV/T formation, and identified both hypo‐ and hyper‐vesiculating mutants. Mutations in fumA and tktA, involved in central carbon metabolism, and in FTN_0908 and FTN_1037, of unknown function, resulted in severe defects in OMV/T production. Cysteine deprivation was identified as the signal that triggers OMV/T formation in F. novicida during growth in rich medium. We also found that fully virulent F. tularensis produces OMV/T in a similarly regulated manner. Further analysis revealed that OMV/T production is responsive to deprivation of essential amino acids in addition to cysteine, and that the hypo‐vesiculating mutants are defective in responding to this signal. Thus, amino acid starvation, such as encountered by Francisella during host cell invasion, regulates the production of membrane‐derived structures.  

3.2960           Preferential capture of EpCAM‐expressing extracellular vesicles on solid surfaces coated with an aptamer‐conjugated zwitterionic polymer

Yoshida, M., Hibino, K., Yamamoto, S., Matsumura, S., Yajima, Y. and Shiba, K. Biotechnol. Bioeng., 115(3), 536-544 (2018)   Extracellular vesicles (EVs) collectively represent small vesicles that are secreted from cells and carry biomolecules (e.g., miRNA, lncRNA, mRNA, proteins, lipids, metabolites, etc.) that originate in those cells. Body fluids, such as blood and saliva, include large numbers of EVs, making them potentially a rich source of diagnostic information. However, these EVs are mixtures of vesicles released from diseased tissues as well as from normal cells. This heterogeneous nature therefore blurs the clinical information obtainable from EV‐based diagnosis. Here, we synthesized an EpCAM‐affinity coating agent, which consists of a peptide aptamer for EpCAM and a zwitterionic MPC polymer, and have shown that this conjugate endowed the surfaces of inorganic materials with the preferential affinity to EpCAM‐expressing EVs. This coating agent, designated as EpiVeta, could be useful as a coating for various diagnostic devices to allow concentration of cancer‐related EVs from heterogeneous EV mixtures.  

3.2961           Localization and functional characterization of the p.Asn965Ser (N965S) ABCA4 variant in mice reveal pathogenic mechanisms underlying Stargardt macular degeneration

Molday, L.L., Wahl, D., Sarunic, M.V. and Molday, R.S. Hum. Mol. Genet., 27(2), 295-306 (2018)   ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) proteins that transports N-retinylidene-phosphatidylethanolamine (N-Ret-PE) across outer segment disc membranes thereby facilitating the removal of potentially toxic retinoid compounds from photoreceptor cells. Mutations in the gene encoding ABCA4 are responsible for Stargardt disease (STGD1), an autosomal recessive retinal degenerative disease that causes severe vision loss. To define the molecular basis for STGD1 associated with the p.Asn965Ser (N965S) mutation in the Walker A motif of nucleotide binding domain 1 (NBD1), we generated a p.Asn965Ser knockin mouse and compared the subcellular localization and molecular properties of the disease variant with wild-type (WT) ABCA4. Here, we show that the p.Asn965Ser ABCA4 variant expresses at half the level of WT ABCA4, partially mislocalizes to the endoplasmic reticulum (ER) of photoreceptors, is devoid of N-Ret-PE activated ATPase activity, and causes an increase in autofluorescence and the bisretinoid A2E associated with lipofuscin deposits in retinal pigment epithelial cells as found in Stargardt patients and Abca4 knockout mice. We also show for the first time that a significant fraction of WT ABCA4 is retained in the inner segment of photoreceptors. On the basis of these studies we conclude that loss in substrate-dependent ATPase activity and protein misfolding are mechanisms underlying STGD1 associated with the p.Asn965Ser mutation in ABCA4. Functional and molecular modeling studies further suggest that similar pathogenic mechanisms are responsible for Tangiers disease associated with the p.Asn935Ser (N935S) mutation in the NBD1 Walker A motif of ABCA1.  

3.2962           Parkinson’s disease-linked DNAJC13 mutation aggravates alpha-synuclein-induced neurotoxicity through perturbation of endosomal trafficking

Yoshida, S., Hasegawa, T., Suzuki, M. et al Hum. Mol. Genet., 27(5), 823-836 (2018)   Mutations in DNAJC13 gene have been linked to familial form of Parkinson’s disease (PD) with Lewy pathology. DNAJC13 is an endosome-related protein and believed to regulate endosomal membrane trafficking. However, the mechanistic link between DNAJC13 mutation and α-synuclein (αSYN) pathology toward neurodegeneration remains poorly understood. In this study, we showed that PD-linked N855S-mutant DNAJC13 caused αSYN accumulation in the endosomal compartment, presumably due to defective cargo trafficking from the early endosome to the late and/or recycling endosome. In vivo experiments using human αSYN transgenic flies showed that mutant DNAJC13 not only increased the amount of insoluble αSYN in fly head but also induced dopaminergic neurodegeneration, rough eye phenotype and age-dependent locomotor impairment. Together, these findings suggest that DNAJC13 mutation perturbs multi-directional endosomal trafficking, resulting in the aberrant endosomal retention of αSYN, which might predispose to the neurodegenerative process that leads to PD.  

3.2963           Photoreceptor-induced RPE phagolysosomal maturation defects in Stargardt-like Maculopathy (STGD3)

Dejos, C., Kuny, S., Han, W.H., Capel, H., Lemieux, H. and Sauve, Y. Scientific Reports, 8:5944 (2018)   For many neurodegenerative disorders, expression of a pathological protein by one cell type impedes function of other cell types, which in turn contributes to the death of the first cell type. In transgenic mice modelling Stargardt-like (STGD3) maculopathy, human mutant ELOVL4 expression by photoreceptors is associated with defects in the underlying retinal pigment epithelium (RPE). To examine how photoreceptors exert cytotoxic effects on RPE cells, transgenic ELOVL4 (TG1–2 line; TG) and wild-type (WT) littermates were studied one month prior (preclinical stage) to onset of photoreceptor loss (two months). TG photoreceptor outer segments presented to human RPE cells are recognized and internalized into phagosomes, but their digestion is delayed. Live RPE cell imaging pinpoints decreased numbers of acidified phagolysomes. In vivo, master regulator of lysosomal genes, transcription factor EB (TFEB), and key lysosomal enzyme Cathepsin D are both unaffected. Oxidative stress, as ruled out with high-resolution respirometry, does not play a role at such an early stage. Upregulation of CRYBA1/A3 and phagocytic cells (microglia/macrophages) interposed between RPE and photoreceptors support adaptive responses to processing delays. Impaired phagolysosomal maturation is observed in RPE of mice expressing human mutant ELOVL4 in their photoreceptors prior to photoreceptor death and associated vision loss.  

3.2964           Calculated cell-specific intracellular hydrogen peroxide concentration: Relevance in cancer cell susceptibility during ascorbate therapy

Erudaitius, D., Mantooth, J., Huang, A., Soliman, J., Doskey, C.M., Buettner, G.R. and Rodgers, V.G.J. Free Radical Bio. Med., 120, 356-367 (2018)   The high extracellular hydrogen peroxide (H₂O₂) concentrations generated during pharmacological ascorbate (P-AscH^-) therapy has been shown to exhibit a high flux into susceptible cancer cells leading to a decrease in clonogenic survival. It is hypothesized that the intracellular H₂O₂ concentration for susceptibility is independent of cell type and that the variation observed in dosing is associated with differences in the cell-specific overall steady-state intracellular H₂O₂ concentration values. The steady-state variation in intracellular H₂O₂ concentration is coupled to a number of cellular specific transport and reaction factors including catalase activity and membrane permeability. Here a lumped-parameter mathematical modeling approach, assuming a catalase-dominant peroxide removal mechanism, is used to calculate intracellular H₂O₂ concentration for several cell lines. Experimental measurements of critical parameters pertaining to the model are obtained. The cell lines investigated are normal pancreatic cells, H6c7, the pancreatic cancer cell line, MIA PaCa-2 and the glioblastoma cell lines, LN-229, T98G, and U-87; all which vary in susceptibility. The intracellular H₂O₂ concentration estimates are correlated with the clonogenic surviving fraction for each cell line, in-vitro. The results showed that, despite the fact that the experimental parameters including catalase concentration and plasma membrane permeability demonstrated significant variability across cell lines, the calculated steady-state intracellular to extracellular H₂O₂ concentration ratio did not vary significantly across cell lines. Thus, the calculated intracellular H₂O₂ concentration is not unique in characterizing susceptibility. These results imply that, although intracellular H₂O₂ concentration plays a key role in cellular susceptibility to P-AscH^- adjuvant therapy, its overall contribution in a unifying mechanism across cell types is complex.  

3.2965           Exosomal proteome analysis of human plasma to monitor sepsis progression

Xu, Y., Ku, X., Wu, C., Cai, C., Tang, J. and Yan, W. Biochem. Biophys. Res. Comm., 499(4), 856-861 (2018)   Exosomes are cell-derived vesicles containing RNA, lipid, and protein, which act in body immune response, intercellular signaling and some other important biological processes. Exosomes have been extensively studied in the past several years on their disease related mechanisms and potential roles to monitor disease progression as biomarkers. Compared with analyzing exosome RNA, comprehensive proteome profiling of exosomes in clinical samples (e.g. blood) are highly demanded but limited mainly due to lack of a reproducible method for efficient exosome extraction. In this study, we evaluated and optimized an exosome preparation approach using one-step ultracentrifugation through an Optiprep™ cushion. Exosomes prepared via this method and analyzed by mass spectrometry using Q-Exactive plus, has led to reproducible identification and quantification of 200 + proteins from human plasma samples of as little as 300 μL. Therefore, such a straightforward exosome extract method has enable us to deeply profile exosome proteomes from human blood at a scale of clinical studies. As a proof of principal, we practiced this approach in analyzing the exosome proteomic profiles of blood samples collected from a sepsis patient during six time points after diagnosis. Among the 238 proteins identified and quantified across the 6 samples, protein SPTLC3 involved in the sphingolipid metabolism, shows a negative correlation (p = 0.02, correlation coefficient = −0.984) with disease progression indicated by body temperature (BD) and C-reactive protein (CRP). Therefore, SPTLC3 could be an interesting target for future study on molecular mechanism of sepsis development, as well as potential classifier to monitor clinical progression of sepsis.  

3.2966           4TM-TRPM8 channels are new gatekeepers of the ER-mitochondria Ca2+ transfer

Bidaux, G., Gordienko, D., Shapovalov, G., Farfariello, V. et al BBA – Mol. Cell Res., 1865, 981-994 (2018)   Calcium (Ca²⁺) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca²⁺ release was associated with the ER Ca²⁺ release channels, inositol 1,4,5‑triphosphate receptor (IP₃R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca²⁺ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8 (TRPM8) channel isoforms as functional ER Ca²⁺ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca²⁺ concentration ([Ca²⁺]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca²⁺ release mechanism distinct from classical Ca²⁺ release channels.  

3.2967           Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation

Carpier, J-M., Zucchetti, A.E., Bataille, l., Dogniaux, S., Shafaq-Zadah, M., Bardin, S., Lucchino, M., Maurin, M., Joannas, L.D., Magalhaes, J.G., Johannes, l., Galli, T., Goud, B. and Hivroz, C.

1. Exp. Med., 215(4), 1245-1265 (2018)

  The adapter molecule linker for activation of T cells (LAT) orchestrates the formation of signalosomes upon T cell receptor (TCR) stimulation. LAT is present in different intracellular pools and is dynamically recruited to the immune synapse upon stimulation. However, the intracellular traffic of LAT and its function in T lymphocyte activation are ill defined. We show herein that LAT, once internalized, transits through the Golgi–trans-Golgi network (TGN), where it is repolarized to the immune synapse. This retrograde transport of LAT depends on the small GTPase Rab6 and the target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) Syntaxin-16, two regulators of the endosome-to-Golgi/TGN retrograde transport. We also show in vitro in Syntaxin-16– or Rab6-silenced human cells and in vivo in CD4⁺ T lymphocytes of the Rab6 knockout mouse that this retrograde traffic controls TCR stimulation. These results establish that the retrograde traffic of LAT from the plasma membrane to the Golgi-TGN controls the polarized delivery of LAT at the immune synapse and T lymphocyte activation.  

3.2968           Endosomal trafficking regulates receptor-mediated transcytosis of antibodies across the blood brain barrier

Haqqani, A., Delaney, C.E., Brunette, E., Baumann, E., Farrington, G.K., Sisk, W., Eldredge, J., Ding, W., Tremblay, T-L. and Stanimirovic, D.B.

1. Cerebral Blood Flow & Metabolism, 38(4), 727-740 (2018)

  Current methods for examining antibody trafficking are either non-quantitative such as immunocytochemistry or require antibody labeling with tracers. We have developed a multiplexed quantitative method for antibody ‘tracking’ in endosomal compartments of brain endothelial cells. Rat brain endothelial cells were co-incubated with blood-brain barrier (BBB)-crossing FC5, monovalent FC5Fc or bivalent FC5Fc fusion antibodies and control antibodies. Endosomes were separated using sucrose-density gradient ultracentrifugation and analyzed using multiplexed mass spectrometry to simultaneously quantify endosomal markers, receptor-mediated transcytosis (RMT) receptors and the co-incubated antibodies in each fraction. The quantitation showed that markers of early endosomes were enriched in high-density fractions (HDF), whereas markers of late endosomes and lysosomes were enriched in low-density fractions (LDF). RMT receptors, including transferrin receptor, showed a profile similar to that of early endosome markers. The in vitro BBB transcytosis rates of antibodies were directly proportional to their partition into early endosome fractions of brain endothelial cells. Addition of the Fc domain resulted in facilitated antibody ‘redistribution’ from LDF into HDF and additionally into multivesicular bodies (MVB). Sorting of various FC5 antibody formats away from late endosomes and lysosomes and into early endosomes and a subset of MVB results in increased antibody transcytosis at the abluminal side of the BBB.  

3.2969           Membrane cholesterol depletion as a trigger of Nav1.9 channel‐mediated inflammatory pain

Amsalem, M., Poilbout, C., Feracci, G., Delmas, p. and Padilla, F. EMBO J., 37, e97349 (2018)   Cholesterol is a major lipid component of the mammalian plasma membrane. While much is known about its metabolism, its transport, and its role in atherosclerotic vascular disease, less is known about its role in neuronal pathophysiology. This study reveals an unexpected function of cholesterol in controlling pain transmission. We show that inflammation lowers cholesterol content in skin tissue and sensory DRG culture. Pharmacological depletion of cellular cholesterol entails sensitization of nociceptive neurons and promotes mechanical and thermal hyperalgesia through the activation of voltage‐gated Nav1.9 channels. Inflammatory mediators enhance the production of reactive oxygen species and induce partitioning of Nav1.9 channels from cholesterol‐rich lipid rafts to cholesterol‐poor non‐raft regions of the membrane. Low‐cholesterol environment enhances voltage‐dependent activation of Nav1.9 channels leading to enhanced neuronal excitability, whereas cholesterol replenishment reversed these effects. Consistently, we show that transcutaneous delivery of cholesterol alleviates hypersensitivity in animal models of acute and chronic inflammatory pain. In conclusion, our data establish that membrane cholesterol is a modulator of pain transmission and shed a new light on the relationship between cholesterol homeostasis, inflammation, and pain.  

3.2970           Extracellular vesicles from early stage Plasmodium falciparum‐infected red blood cells contain PfEMP1 and induce transcriptional changes in human monocytes

Sampaio, N., Emery, S.J., Granham, A.L., Tan, Q.Y., Sisquella, X., Pimentel, M.A., Jex, A.R., Regev-Rudzki, N., Schofield, l. and Eriksson, E.M. Cell. Microbiol., 20, e12822 (2018)   Pathogens can release extracellular vesicles (EVs) for cell–cell communication and host modulation. EVs from Plasmodium falciparum, the deadliest malaria parasite species, can transfer drug resistance genes between parasites. EVs from late‐stage parasite‐infected RBC (iRBC‐EVs) are immunostimulatory and affect endothelial cell permeability, but little is known about EVs from early stage iRBC. We detected the parasite virulence factor PfEMP1, which is responsible for iRBC adherence and a major contributor to disease severity, in EVs, only up to 12‐hr post‐RBC invasion. Furthermore, using PfEMP1 transport knockout parasites, we determined that EVs originated from inside the iRBC rather than the iRBC surface. Proteomic analysis detected 101 parasite and 178 human proteins in iRBC‐EVs. Primary human monocytes stimulated with iRBC‐EVs released low levels of inflammatory cytokines and showed transcriptomic changes. Stimulation with iRBC‐EVs from PfEMP1 knockout parasites induced more gene expression changes and affected pathways involved in defence response, stress response, and response to cytokines, suggesting a novel function of PfEMP1 when present in EVs. We show for the first time the presence of PfEMP1 in early stage P. falciparum iRBC‐EVs and the effects of these EVs on primary human monocytes, uncovering a new mechanism of potential parasite pathogenesis and host interaction.  

3.2971           An Exosome‐Based Vaccine Platform Imparts Cytotoxic T Lymphocyte Immunity Against Viral Antigens

Anticoli, S., Manfredi, f., Chiozzini, C., Arenaccio, C., Olivetta, E., Ferrantelli, F., Capocefalo, A., Falcone, E., Ruggieri, A. and Federico, M. Biotech. J., 13, 1700443 (2018)   Exosomes are 50–150 nm sized nanovesicles released by all eukaryotic cells. The authors very recently described a method to engineer exosomes in vivo with the E7 protein of Human Papilloma Virus (HPV). This technique consists in the intramuscular injection of a DNA vector expressing HPV‐E7 fused at the C‐terminus of an exosome‐anchoring protein, that is, Nef^mut, the authors previously characterized for its high levels of incorporation in exosomes. In this configuration, the ≈11 kDa E7 protein elicited a both strong and effective antigen‐specific cytotoxic T lymphocyte (CTL) immunity. Attempting to establish whether this method could have general applicability, the authors expanded the immunogenicity studies toward an array of viral products of various origin and size including Ebola Virus VP24, VP40 and NP, Influenza Virus NP, Crimean–Congo Hemorrhagic Fever NP, West Nile Virus NS3, and Hepatitis C Virus NS3. All antigens appeared stable upon fusion with Nef^mut, and are uploaded in exosomes at levels comparable to Nef^mut. When injected in mice, DNA vectors expressing the diverse fusion products elicited a well detectable antigen‐specific CD8⁺ T cell response associating with a cytotoxic activity potent enough to kill peptide‐loaded and/or antigen‐expressing syngeneic cells. These data definitely proven both effectiveness and flexibility of this innovative CTL vaccine platform.  

3.2972           Doxycycline-inducible and astrocyte-specific HIV-1 Tat transgenic mice (iTat) as an HIV/neuroAIDS model

Langford, D., oh Kim, B., Zou, W., Fan, Y., Rahimain, P., Liu, Y. and He, J.J.

1. Neurovirol., 24(2), 168-179 (2018)

  HIV-1 Tat is known to be neurotoxic and important for HIV/neuroAIDS pathogenesis. However, the overwhelming majority of the studies involved use of recombinant Tat protein. To understand the contributions of Tat protein to HIV/neuroAIDS and the underlying molecular mechanisms of HIV-1 Tat neurotoxicity in the context of a whole organism and independently of HIV-1 infection, a doxycycline-inducible astrocyte-specific HIV-1 Tat transgenic mouse (iTat) was created. Tat expression in the brains of iTat mice was determined to be in the range of 1–5 ng/ml and led to astrocytosis, loss of neuronal dendrites, and neuroinflammation. iTat mice have allowed us to define the direct effects of Tat on astrocytes and the molecular mechanisms of Tat-induced GFAP expression/astrocytosis, astrocyte-mediated Tat neurotoxicity, Tat-impaired neurogenesis, Tat-induced loss of neuronal integrity, and exosome-associated Tat release and uptake. In this review, we will provide an overview about the creation and characterization of this model and its utilities for our understanding of Tat neurotoxicity and the underlying molecular mechanisms.  

3.2973           Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins

Toledo, A., Huang, Z., Coleman, J.L., London, E. and Benach, J.L. Mol. Microbiol., 108(1), 63-76 (2018)   Lipid rafts are microdomains present in the membrane of eukaryotic organisms and bacterial pathogens. They are characterized by having tightly packed lipids and a subset of specific proteins. Lipid rafts are associated with a variety of important biological processes including signaling and lateral sorting of proteins. To determine whether lipid rafts exist in the inner membrane of Borrelia burgdorferi, we separated the inner and outer membranes and analyzed the lipid constituents present in each membrane fraction. We found that both the inner and outer membranes have cholesterol and cholesterol glycolipids. Fluorescence anisotropy and FRET showed that lipids from both membranes can form rafts but have different abilities to do so. The analysis of the biochemically defined proteome of lipid rafts from the inner membrane revealed a diverse set of proteins, different from those associated with the outer membrane, with functions in protein trafficking, chemotaxis and signaling.  

3.2974           Organellar Omics – A Reviving Strategy to Untangle the Biomolecular Complexity of the Cell

Tharkehwar, A.K., Gevaert, K. and Annaert, W. Proteomics, 18, 1700113 (2018)   A eukaryotic cell encompasses many membrane‐enclosed organelles, each of these holding several types of biomolecules that exhibit tremendous diversity in terms of their localization and expression. Despite the development of increasingly sensitive analytical tools, the enormous biomolecular complexity that exists within a cell cannot yet be fully resolved as low abundant molecules often remain unrecognized. Moreover, a drawback of whole cell analysis is that it does not provide spatial information and therefore it is not capable of assigning distinct biomolecules to specific compartments or analyzing changes in the composition of these compartments. Reduction of the biomolecular complexity of a sample helps to identify low abundant molecules, but such a reductionist approach requires methods that enable proper isolation and purification of individual cellular organelles. Decades of research have led to the development of a plethora of isolation methods for a broad range of subcellular organelles; yet, in particular, intrinsically dynamic compartments belonging to the endocytic machinery, including the plasma membrane, remain difficult to isolate in a sufficiently pure fraction. In this review, we discuss various methods that are commonly used to isolate subcellular organelles from cells and evaluate their advantages and disadvantages.  

3.2975           Endothelial extracellular vesicles modulate the macrophage phenotype: Potential implications in atherosclerosis

He, S., Wu, C., Xiao, J., Li, D., Sun, Z. and Li, M. Scand. J. Immunol., 87(4) e12648 (2018)   Endothelial cells (ECs) and macrophages engage in tight and specific interactions that play critical roles in cardiovascular homeostasis and the pathogenesis of atherosclerosis. Extracellular vesicles (EVs) are circular membrane fragments released from the endosomal compartment as exosomes or shed from the surfaces of the membranes of most cell types. Increasing evidence indicates that EVs play a pivotal role in cell‐to‐cell communication. However, the contribution of EVs, as determine by oxidized low‐density lipoprotein (ox‐LDL)‐exposed and/or Kruppel‐like factor 2 (KLF2)‐transduced ECs in the interaction between vascular ECs and monocytes/macrophages, which is a key event in atherosclerotic plaque development, has remained elusive. This study demonstrates the characteristic impact of EVs from ox‐LDL‐treated and/or KLF2‐transduced ECs on the monocyte/macrophage phenotype in vitro and in vivo.Q‐PCR showed that both the atherosclerosis inducer ox‐LDL and atheroprotective factor KLF2 regulated inflammation‐associated microRNA‐155 (miR‐155) expression in human umbilical vein endothelial cells (HUVECs). Moreover, coculture, immunofluorescence and flow cytometry revealed that miR‐155 was enriched in ox‐LDL‐induced ECs‐EVs and subsequently transferred to human monocytic THP1 cells, in which these vesicles enhance monocyte activation by shifting the monocytes/macrophages balance from anti‐inflammatory M2 macrophages towards proinflammatory M1 macrophages; EVs from KLF2‐expressing ECs suppressed monocyte activation by enhancing immunomodulatory responses and diminishing proinflammatory responses, which indicate the potent anti‐inflammatory activities of these cells. Furthermore, oil red staining showed that atherosclerotic lesions were reduced in mice that received EVs from KLF2‐transduced ECs with decreased proinflammatory M1 macrophages and increased anti‐inflammatory M2 macrophages, and this effect is at least partly due to the decreased expression of inflammation‐associated miR‐155, confirming our in vitro findings. In summary, this study provides novel insights into the pathophysiological effects of altered EV secretion and/or microRNA content and their influence on modulating monocyte activation depending on the environment surrounding EVs‐releasing ECs.  

3.2976           Exosomes as diagnostic biomarkers in cancer

Kim, J-H., Kim, E. and Lee, M.Y. Mol. Cell. Toxicol., 14(2), 113-122 (2018)   Purpose of review Exosomes are extracellular vesicles of 30-150 nm diameter, secreted from nearly all mammalian cells through fusion of multivesicular bodies with the plasma membrane. Owing to the differences in the properties of exosomes and microvesicles released through outward budding of the plasma membrane, exosomes have recently received increasing interest. This review discusses the current status of exosome research for diagnostic biomarkers in cancers. The scope of information that can be acquired from exosomal contents potentially include tumour progression, detection of metastasis, and possible chemotherapeutic resistance, which can facilitate clinical decisions in precision medicine. Recent findings Exosomes protect molecular components including miRNAs and proteins from enzymatic degradation during circulation and serve as stable cargo for them. miRNAs transferred by exosomes have emerged as novel regulators of cellular function in various types of cancers. In addition, exosomes contain numerous plasma membrane and cytosolic proteins and exosome- specific proteins. However, many of the experiments are limited in their methods for the isolation and purification of exosomes. Nevertheless, the physiological and pathological significance of the role of exosomes in miRNA- or protein-based cell-to-cell or tissue-to-tissue communication has been highlighted.  

3.2977           CADM1 is essential for KSHV-encoded vGPCR-and vFLIP-mediated chronic NF-κB activation

Hunte, R., Alonso, P., Thomas, R., Bazile, C.A., Ramos, J.C., van der Weyden, L., Dominguez-Bendala, j., Khan, W.N. and Shembade, N. PloS Pathogens, 14(4), e1006968 (2018)   Approximately 12% of all human cancers worldwide are caused by infections with oncogenic viruses. Kaposi's sarcoma herpesvirus/human herpesvirus 8 (KSHV/HHV8) is one of the oncogenic viruses responsible for human cancers, including Kaposi’s sarcoma (KS), Primary Effusion Lymphoma (PEL), and the lymphoproliferative disorder multicentric Castleman’s disease (MCD). Chronic inflammation mediated by KSHV infection plays a decisive role in the development and survival of these cancers. NF-κB, a family of transcription factors regulating inflammation, cell survival, and proliferation, is persistently activated in KSHV-infected cells. The KSHV latent and lytic expressing oncogenes involved in NF-κB activation are vFLIP/K13 and vGPCR, respectively. However, the mechanisms by which NF-κB is activated by vFLIP and vGPCR are poorly understood. In this study, we have found that a host molecule, Cell Adhesion Molecule 1 (CADM1), is robustly upregulated in KSHV-infected PBMCs and KSHV-associated PEL cells. Further investigation determined that both vFLIP and vGPCR interacted with CADM1. The PDZ binding motif localized at the carboxyl terminus of CADM1 is essential for both vGPCR and vFLIP to maintain chronic NF-κB activation. Membrane lipid raft associated CADM1 interaction with vFLIP is critical for the initiation of IKK kinase complex and NF-κB activation in the PEL cells. In addition, CADM1 played essential roles in the survival of KSHV-associated PEL cells. These data indicate that CADM1 plays key roles in the activation of NF-κB pathways during latent and lytic phases of the KSHV life cycle and the survival of KSHV-infected cells.  

3.2978           Enhanced Production of Exosome-Associated AAV by Overexpression of the Tetraspanin CD9

Schiller, L.T., Lemus-Diaz, N., Ferreira, R.R., Böker, K.O. and Gruber, J.

Molecular Therapy – Methods & Clinical Development, 9, 278-287 (2018)

Research on cell-free vesicles revealed a multitude of characteristics, in particular of microvesicles and exosomes, that range from their potential as biomarkers to a function in horizontal transfer of genetic information from cell to cell and also include supportive functions in viral infection. Exosome-associated adeno-associated viruses (exo-AAVs) are of particular interest for the past couple of years, since they introduced a new source of highly potent recombinant AAVs with improved features, including accelerated transduction rates and more efficient immune escape. However, key factors like the mode of action, efficiency of production or engineering of exo-AAVs remain elusive to a large extent. Here, we used the established system of CD9 over-expression to boost the exosome output of AAV producing HEK-AAV cells. The CD9-powered high-exosome environment was established during exo-AAV1 production, and we could demonstrate that the yield of exo-AAVs dramatically increased when compared to standard exo-AAVs. Furthermore, we report that exo-AAV-CD9GFP was more efficient in transduction of cells in the same titer ranges as standard exo-AAVs. Our results provide a technological approach for the generation of exo-AAVs with superior performance.

 

3.2979           Tumor-originated exosomal lncUEGC1 as a circulating biomarker for early-stage gastric cancer

Lin, L-Y., Yang, L., Zeng, Q., Wang, l., Chen, M-L., Zhao, Z-H., Ye, G-D., Luo, Q-C., Lv, P-Y., Guo, Q-W., Li, B-A., Cai, J-C. and Cai, W-Y. Mol. Cancer, 17:84 (2018)   Conventional tumor markers for non-invasive diagnosis of gastric cancer (GC) exhibit insufficient sensitivity and specificity to facilitate detection of early gastric cancer (EGC). We aimed to identify EGC-specific exosomal lncRNA biomarkers that are highly sensitive and stable for the non-invasive diagnosis of EGC. Hence, in the present study, exosomes from the plasma of five healthy individuals and ten stage I GC patients and from culture media of four human primary stomach epithelial cells and four gastric cancer cells (GCCs) were isolated. Exosomal RNA profiling was performed using RNA sequencing to identify EGC-specific exosomal lncRNAs. A total of 79 and 285 exosomal RNAs were expressed at significantly higher levels in stage I GC patients and GCCs, respectively, than that in normal controls. Through combinational analysis of the RNA sequencing results, we found two EGC-specific exosomal lncRNAs, lncUEGC1 and lncUEGC2, which were further confirmed to be remarkably up-regulated in exosomes derived from EGC patients and GCCs. Furthermore, stability testing demonstrates that almost all the plasma lncUEGC1 was encapsulated within exosomes and thus protected from RNase degradation. The diagnostic accuracy of exosomal lncUEGC1 was evaluated, and lncUEGC1 exhibited AUC values of 0.8760 and 0.8406 in discriminating EGC patients from healthy individuals and those with premalignant chronic atrophic gastritis, respectively, which was higher than the diagnostic accuracy of carcinoembryonic antigen. Consequently, exosomal lncUEGC1 may be promising in the development of highly sensitive, stable, and non-invasive biomarkers for EGC diagnosis.  

3.2980           Urinary extracellular vesicle biomarkers in urological cancers: From discovery towards clinical implementation

Dhondt, B., Van Deun, J., Vermaerke, S., de Marcos, A., Lumen, N., De Wever, O. and Hendrix, A. Int. J. Biochem. Cell Biol., 96, 236-256 (2018)   Urine contains cellular elements, biochemicals, and proteins derived from glomerular filtration of plasma, renal tubule excretion, and urogenital tract secretions that reflect an individual's metabolic and pathophysiologic state. Despite intensive research into the discovery of urinary biomarkers to facilitate early diagnosis, accurate prognosis and prediction of therapy response in urological cancers, none of these markers has reached widespread use. Their implementation into daily clinical practice is hampered by a substantial degree of heterogeneity in performance characteristics and uncertainty about reliability, clinical utility and cost-effectiveness, in addition to several technical limitations. Extracellular vesicles (EV) have raised interest as a potential source of biomarker discovery because of their role in intercellular communication and the resemblance of their molecular content to that of the releasing cells. We review currently used urinary biomarkers in the clinic and attempts that have been made to identify EV-derived biomarkers for urological cancers. In addition, we discuss technical and methodological considerations towards their clinical implementation.  

3.2981           Extracellular vesicles characteristics and emerging roles in atherosclerotic cardiovascular disease

Hafiane, A. and Daskalopoulou, S.S. Metabolism, 85, 213-222 (2018)   The term extracellular vesicles (EVs) describes membrane vesicles released into the extracellular space by most cell types. EVs have been recognized to play an important role in cell-to-cell communication. They are known to contain various bioactive molecules, including proteins, lipids, and nucleic acids. Although the nomenclature of EVs is not entirely standardized, they are considered to include exosomes, microparticles or microvesicles and apoptotic bodies. EVs are believed to play important roles in a wide range of biological processes. Although the pathogenic roles of EVs are largely documented, their protective roles are not as well established. Cardiovascular disease represents one of the most relevant and rapidly growing areas of the EV research. Circulating EVs released from platelets, erythrocytes, leukocytes, and endothelial cells may contain potentially valuable biological information for biomarker development in cardiovascular disease and could serve as a vehicle for therapeutic use. Herein, we provide an overview of the current knowledge in EV in cardiovascular disease, including a discussion on challenges in EV research, EV properties in various cell types, and their importance in atherosclerotic disease.  

3.2982           Myristoylated methionine sulfoxide reductase A is a late endosomal protein

Lim, J.M., Lim, J.C., Kim, G. and Levine, R.L.

1. Biol. Chem., 293(19), 7355-7366 (2018)

  Methionine residues in proteins provide antioxidant defense by reacting with oxidizing species, which oxidize methionine to methionine sulfoxide. Reduction of the sulfoxide back to methionine is catalyzed by methionine sulfoxide reductases, essential for protection against oxidative stress. The nonmyristoylated form of methionine sulfoxide reductase A (MSRA) is present in mitochondria, whereas the myristoylated form has been previously reported to be cytosolic. Despite the importance of MSRA in antioxidant defense, its in vivo binding partners and substrates have not been identified. Starting with a protein array, and followed by immunoprecipitation experiments, colocalization studies, and subcellular fractionation, we identified the late endosomal protein, StAR-related lipid transfer domain–containing 3 (STARD3), as a binding partner of myristoylated MSRA, but not of nonmyristoylated MSRA. STARD3 is known to have both membrane-binding and cytosolic domains that are important in STARD3-mediated transport of cholesterol from the endoplasmic reticulum to the endosome. We found that the STARD3 cytosolic domain localizes MSRA to the late endosome. We propose that the previous conclusion that myristoylated MSRA is strictly a cytosolic protein is artifactual and likely due to vigorous overexpression of MSRA. We conclude that myristoylated MSRA is a late endosomal protein that may play a role in lipid metabolism or may protect endosomal proteins from oxidative damage.    

3.2983           Essential role of ATP6AP2 enrichment in caveolae/lipid raft microdomains for the induction of neuronal differentiation of stem cells

Makdissy, N., Haddad, K., D’arc Albacha, J., Chaker, D., Ismail, B., Azar, A., Oreibi, G., Ayoub, D., Achkar, I., Quilliot, D. and Fajloun, Z. Stem Cell Res. Ther., 9:132 (2018)   Background The subcellular distribution of prorenin receptor and adaptor protein ATP6AP2 may affect neurogenesis. In this study, we hypothesized that ATP6AP2 expression and subcellular relocalization from caveolae/lipid raft microdomains (CLR-Ms) to intracellular sites may correlate with neuronal differentiation (Neu-Dif) of adipose-derived mesenchymal stem cells (ADSCs). Methods Human ADSCs isolated from 24 healthy donors and 24 patients with neurological disorders (ND) were cultured and induced for Neu-Dif. The mechanism of action of ATP6AP2 and the impact of its localization within the plasma membrane (particularly CLR-Ms) and intracellular sites on several pathways (mitogen-activated protein kinase, Wnt(s) signaling and others) and intracellular calcium and exosome release were evaluated. The impact of CLR-Ms on ATP6AP2 or vice versa was determined by pharmacological disruption of CLR-Ms or siATP6AP2 assays. Results In patients with ND, loss of ATP6AP2 from CLR-Ms correlated with an inhibition of Neu-Dif and signaling. However, its relocalization in CLR-Ms was positively correlated to induction of Neu-Dif in healthy subjects. An apparent switch from canonical to noncanonical Wnt signaling as well as from caveolin to flotillin occurs concurrently with the increases of ATP6AP2 expression during neurogenesis. Stimulation by renin activates ERK/JNK/CREB/c-Jun but failed to induce β-catenin. Wnt5a enhanced the renin-induced JNK responsiveness. Gα proteins crosslink ATP6AP2 to caveolin where a switch from Gαi to Gαq is necessary for Neu-Dif. In ATP6AP2-enriched CLR-Ms, the release of exosomes was induced dependently from the intracellular Ca²⁺ and Gαq. Pharmacological disruption of CLR-M formation/stability impairs both ATP6AP2 localization and Neu-Dif in addition to reducing exosome release, indicating an essential role of ATP6AP2 enrichment in CLR-Ms for the induction of Neu-Dif. The mechanism is dependent on CLR-M dynamics, particularly the membrane fluidity. Knockdown of ATP6AP2 inhibited Neu-Dif but increased astrocytic-Dif, depleted ATP6AP2/flotillin/Gαq but accumulated caveolin/Gαi in CLR-Ms, and blocked the activation of JNK/ERK/c-Jun/CREB/exosome release. siATP6AP2 cells treated with sphingomyelinase/methyl-β-cyclodextrin reversed the levels of caveolin/flotillin in CLR-Ms but did not induce Neu-Dif, indicating the crucial relocalization of ATP6AP2 in CLR-Ms for neurogenesis. Treatment of ND-derived cells with nSMase showed reversibility in ATP6AP2 abundance in CLR-Ms and enhanced Neu-Dif. Conclusions This study gives evidence of the determinant role of CLR-M ATP6AP2 localization for neuronal and oligodendrocyte differentiation involving mechanisms of switches from Gαi/caveolin/canonical to Gαq/flotillin/PCP, the ERK/JNK pathway and Ca²⁺-dependent release of exosomes and as a potential target of drug therapy for neurodegenerative disorders.  

3.2984           Antiretroviral Drugs Alter the Content of Extracellular Vesicles from HIV-1-Infected Cells

DeMarino, C., Pleet, M.L., Cowen, M., Barclay, R.A. et al Scientific Reports, 8:7653 (2018)   To date, the most effective treatment of HIV-1 is a combination antiretroviral therapy (cART), which reduces viral replication and reverses pathology. We investigated the effect of cART (RT and protease inhibitors) on the content of extracellular vesicles (EVs) released from HIV-1-infected cells. We have previously shown that EVs contain non-coding HIV-1 RNA, which can elicit responses in recipient cells. In this manuscript, we show that TAR RNA levels demonstrate little change with the addition of cART treatment in cell lines, primary macrophages, and patient biofluids. We determined possible mechanisms involved in the selective packaging of HIV-1 RNA into EVs, specifically an increase in EV-associated hnRNP A2/B1. More recent experiments have shown that several other FDA-approved drugs have the ability to alter the content of exosomes released from HIV-1-infected cells. These findings on cART-altered EV content can also be applied to general viral inhibitors (interferons) which are used to treat other chronic infections. Additionally, we describe unique mechanisms of ESCRT pathway manipulation by antivirals, specifically the targeting of VPS4. Collectively, these data imply that, despite antiretroviral therapy, EVs containing viral products are continually released and may cause neurocognitive and immunological dysfunction.  

3.2985           Tetraspanin blockage reduces exosome‑mediated HIV‑1 entry

Sim, B., Farrow, A.L., Williams, S.D., Bansal, A., Krendelchtchikov., A. and Matthew, Q.L. Arch. Virol., 163, 1683-1689 (2018)   HIV-1 is one of the most studied retroviruses. The role of exosomes in HIV-1 entry and pathogenesis are beginning to be appreciated. Exosomes can incorporate host proteins that are also contained in viruses (e.g., tetraspanins).  

3.2986           Endosomal-Lysosomal Cholesterol Sequestration by U18666A Differentially Regulates Amyloid Precursor Protein (APP) Metabolism in Normal and APP-Overexpressing Cells

Chung, J., Phukari, G., Vergote, D., Mohamed, A., Maulik, M., Stahn, M., Andrew, R.J., Thinakaran, G., de Chaves, E.P. and Kars, S. Mol. Cell. Biol., 38(11), e00529-17 (2018)   Amyloid β (Aβ) peptide, derived from amyloid precursor protein (APP), plays a critical role in the development of Alzheimer's disease. Current evidence indicates that altered levels or subcellular distribution of cholesterol can regulate Aβ production and clearance, but it remains unclear how cholesterol sequestration within the endosomal-lysosomal (EL) system can influence APP metabolism. Thus, we evaluated the effects of U18666A, which triggers cholesterol redistribution within the EL system, on mouse N2a cells expressing different levels of APP in the presence or absence of extracellular cholesterol and lipids provided by fetal bovine serum (FBS). Our results reveal that U18666A and FBS differentially increase the levels of APP and its cleaved products, the α-, β-, and η-C-terminal fragments, in N2a cells expressing normal levels of mouse APP (N2awt), higher levels of human wild-type APP (APPwt), or “Swedish” mutant APP (APPsw). The cellular levels of Aβ_1–40/Aβ_1–42 were markedly increased in U18666A-treated APPwt and APPsw cells. Our studies further demonstrate that APP and its cleaved products are partly accumulated in the lysosomes, possibly due to decreased clearance. Finally, we show that autophagy inhibition plays a role in mediating U18666A effects. Collectively, these results suggest that altered levels and distribution of cholesterol and lipids can differentially regulate APP metabolism depending on the nature of APP expression.  

3.2987           A novel extracellular vesicle-associated endodeoxyribonuclease helps Streptococcus pneumoniae evade neutrophil extracellular traps and is required for full virulence

Jhelum, H., Sori, H. and Sehgal, D. Scientific Reports, 8:7985 (2018)   Streptococcus pneumoniae (pneumococcus) is a major bacterial pathogen that causes pneumonia and septicemia in humans. Pneumococci are cleared from the host primarily by antibody dependent opsonophagocytosis by phagocytes like neutrophils. Neutrophils release neutrophil extracellular traps (NETs) on contacting pneumococci. NETs immobilize pneumococci and restrict its dissemination in the host. One of the strategies utilized by pneumococci to evade the host immune response involves use of DNase(s) to degrade NETs. We screened the secretome of autolysin deficient S. pneumoniae to identify novel DNase(s). Zymogram analysis revealed 3 bands indicative of DNase activity. Mass spectrometric analysis led to the identification of TatD as a potential extracellular DNase. Recombinant TatD showed nucleotide sequence-independent endodeoxyribonuclease activity. TatD was associated with extracellular vesicles. Pneumococcal secretome degraded NETs from human neutrophils. Extracellular vesicle fraction from tatD deficient strain showed little NET degrading activity. Recombinant TatD efficiently degraded NETs. tatD deficient pneumococci showed lower bacterial load in lungs, blood and spleen in a murine sepsis model compared to wildtype strain, and showed less severe lung pathology and compromised virulence. This study provides insights into the role of a novel extracellular DNase in evasion of the innate immune system.  

3.2988           Intrinsic properties and plasma membrane trafficking route of Src family kinase SH4 domains sensitive to retargeting by HIV-1 Nef

Chase, A.J., Wombacher, R. and Fackler, O.T.

1. Biol. Chem., 293(20), 7824-7840 (2018)

  The HIV type 1 pathogenicity factor Nef enhances viral replication by modulating multiple host cell pathways, including tuning the activation state of infected CD4 T lymphocytes to optimize virus spread. For this, Nef inhibits anterograde transport of the Src family kinase (SFK) Lck toward the plasma membrane (PM). This leads to retargeting of the kinase to the trans-Golgi network, whereas the intracellular transport of a related SFK, Fyn, is unaffected by Nef. The 18-amino acid Src homology 4 (SH4) domain membrane anchor of Lck is necessary and sufficient for Nef-mediated retargeting, but other details of this process are not known. The goal of this study was therefore to identify characteristics of SH4 domains responsive to Nef and the transport machinery used. Screening a panel of SFK SH4 domains revealed two groups that were sensitive or insensitive for trans-Golgi network retargeting by Nef as well as the importance of the amino acid at position 8 for determining Nef sensitivity. Anterograde transport of Nef-sensitive domains was characterized by slower delivery to the PM and initial targeting to Golgi membranes, where transport was arrested in the presence of Nef. For Nef-sensitive SH4 domains, ectopic expression of the lipoprotein binding chaperone Unc119a or the GTPase Arl3 or reduction of their endogenous expression phenocopied the effect of Nef. Together, these results suggest that, analogous to K-Ras, Nef-sensitive SH4 domains are transported to the PM by a cycle of solubilization and membrane insertion and that intrinsic properties define SH4 domains as cargo of this Nef-sensitive lipoprotein binding chaperone–GTPase transport cycle.  

3.2989           Extracellular α-synuclein drives sphingosine 1-phosphate receptor subtype 1 out of lipid rafts, leading to impaired inhibitory G-protein signaling

Badawy, S.M.M., Okada, T., Kajimoto, T., Hirase, M., Matovelo, S.A., Makamura, S., Yoshida, D., Ijuin, T. and Nakamura, S-i.

1. Biol. Chem., 293(21), 8208-8216 (2018)

  α-Synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, are thought to be involved in the pathogenesis of Lewy body diseases, such as Parkinson's disease (PD). Although growing evidence suggests that cell-to-cell transmission of α-Syn is associated with the progression of PD and that extracellular α-Syn promotes formation of inclusion bodies, its precise mechanism of action in the extracellular space remains unclear. Here, as indicated by both conventional fractionation techniques and FRET-based protein–protein interaction analysis, we demonstrate that extracellular α-Syn causes expulsion of sphingosine 1-phosphate receptor subtype 1 (S1P₁R) from the lipid raft fractions. S1P₁R regulates vesicular trafficking, and its expulsion involved α-Syn binding to membrane-surface gangliosides. Consequently, the S1P₁R became refractory to S1P stimulation required for activating inhibitory G-protein (G_i) in the plasma membranes. Moreover, the extracellular α-Syn also induced uncoupling of the S1P₁R on internal vesicles, resulting in the reduced amount of CD63 molecule (CD63) in the lumen of multivesicular endosomes, together with a decrease in CD63 in the released exosomes from α-Syn–treated cells. Furthermore, cholesterol-depleting agent–induced S1P₁R expulsion from the rafts also resulted in S1P₁R uncoupling. Taken together, these results suggest that extracellular α-Syn–induced expulsion of S1P₁R from lipid rafts promotes the uncoupling of S1P₁R from G_i, thereby blocking subsequent G_i signals, such as inhibition of cargo sorting into exosomal vesicles in multivesicular endosomes. These findings help shed additional light on PD pathogenesis.  

3.2990           A mechanism for differential sorting of the planar cell polarity proteins Frizzled6 and Vangl2 at the trans-Golgi network

Ma, T., Li, B., Wang, R., Lau, P.K., Huang, Y., Jiang, L. and Schekman, R.

1. Biol. Chem., 293(22), 8410-8427 (2018)

  In planar cell polarity (PCP), the epithelial cells are polarized along the plane of the cell surface perpendicular to the classical apical–basal axis, a process mediated by several conserved signaling receptors. Two PCP-signaling proteins, VANGL planar cell polarity protein 2 (Vangl2) and Frizzled6 (Fzd6), are located asymmetrically on opposite boundaries of the cell. Examining sorting of these two proteins at the trans-Golgi network (TGN), we demonstrated previously that the GTP-binding protein ADP-ribosylation factor–related protein 1 (Arfrp1) and the clathrin-associated adaptor protein complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled6 does not depend on Arfrp1 or AP-1. Here, to further investigate the TGN sorting process in mammalian cells, we reconstituted release of Vangl2 and Frizzled6 from the TGN into vesicles in vitro. Immunoblotting of released vesicles indicated that Vangl2 and Frizzled6 exit the TGN in separate compartments. Knockdown analysis revealed that a clathrin adaptor, epsinR, regulates TGN export of Frizzled6 but not of Vangl2. Protein interaction analysis suggested that epsinR forms a stable complex with clathrin and that this complex interacts with a conserved polybasic motif in the Frizzled6 cytosolic domain to package Frizzled6 into transport vesicles. Moreover, we found that Frizzled6–epsinR binding dissociates epsinR from AP-1, which may separate these two cargo adaptors from each other to perform distinct cargo-sorting functions. Our results suggest that Vangl2 and Frizzled6 are packaged into separate vesicles that are regulated by different clathrin adaptors at the TGN, which may contribute to their asymmetric localizations.  

3.2991           Flotillin proteins recruit sphingosine to membranes and maintain cellular sphingosine-1-phosphate levels

Riento, K., Zhang, Q., Clark, J., Begum, F., Stephens, E., Wakelam, M.J. and Nichols, B. PloS One, 13(5), e0197401 (2018)   Sphingosine-1-phosphate (S1P) is an important lipid signalling molecule. S1P is produced via intracellular phosphorylation of sphingosine (Sph). As a lipid with a single fatty alkyl chain, Sph may diffuse rapidly between cellular membranes and through the aqueous phase. Here, we show that the absence of microdomains generated by multimeric assemblies of flotillin proteins results in reduced S1P levels. Cellular phenotypes of flotillin knockout mice, including changes in histone acetylation and expression of Isg15, are recapitulated when S1P synthesis is perturbed. Flotillins bind to Sph in vitro and increase recruitment of Sph to membranes in cells. Ectopic re-localisation of flotillins within the cell causes concomitant redistribution of Sph. The data suggest that flotillins may directly or indirectly regulate cellular sphingolipid distribution and signalling.  

3.2992           The protein family TcTASV-C is a novel Trypanosoma cruzi virulence factor secreted in extracellular vesicles by trypomastigotes and highly expressed in bloodstream forms

Caeiro, L.D., Alba-Soto, C.D., Rizzi, M., Solana, M.E., Rodriguez, G., CHidichimo, A.M., Rodriguez, M.E., Sanchez, D.O., Levy, G.V. and Tekiel, V. PloS Neglected Tropical Diseases, 12(5), e0006475 (2018)   TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage of Trypanosoma cruzi. We have previously shown that TcTASV-C is located at the parasite surface and secreted to the medium. Here we report that the expression of different TcTASV-C genes occurs simultaneously at the trypomastigote stage and while some secreted and parasite-associated products are found in both fractions, others are different. Secreted TcTASV-C are mainly shedded through trypomastigote extracellular vesicles, of which they are an abundant constituent, despite its scarce expression on culture-derived trypomastigotes. In contrast, TcTASV-C is highly expressed in bloodstream trypomastigotes; its upregulation in bloodstream parasites was observed in different T. cruzi strains and was specific for TcTASV-C, suggesting that some host-molecules trigger TcTASV-C expression. TcTASV-C is also strongly secreted by bloodstream parasites. A DNA prime—protein boost immunization scheme with TcTASV-C was only partially effective to control the infection in mice challenged with a highly virulent T. cruzi strain. Vaccination triggered a strong humoral response that delayed the appearance of bloodstream trypomastigotes at the early phase of the infection. Linear epitopes recognized by vaccinated mice were mapped within the TcTASV-C family motif, suggesting that blockade of secreted TcTASV-C impacts on the settlement of infection. Furthermore, although experimental and naturally T. cruzi-infected hosts did not react with antigens from extracellular vesicles, vaccinated and challenged mice recognized not only TcTASV-C but also other vesicle-antigens. We hypothesize that TcTASV-C is involved in the establishment of the initial T. cruzi infection in the mammalian host. Altogether, these results point towards TcTASV-C as a novel secreted virulence factor of T. cruzi trypomastigotes.  

3.2993           Outer membrane vesicles blebbing contributes to B. vulgatus mpk-mediated immune response silencing

Maerz, J.K., Steimle, A., Lange, A., Bender, A., Fehrenbacher, B. and Frick, J-S. Gut Microbes, 90(1), 1-12 (2018)   The Gram negative intestinal symbiont Bacteroides vulgatus mpk is able to prevent from induction of colonic inflammation in Rag1^−/− mice and promotes immune balance in Il2^−/− mice. These inflammation-silencing effects are associated with B. vulgatus mpk-mediated induction of semi-mature dendritic cells, especially in the colonic lamina propria (cLP). However the beneficial interaction of bacteria with host immune cells is limited due to the existence of a large mucus layer covering the intestinal epithelium. How can intestinal bacteria overcome this physical barrier and contact the host immune system? One mechanism is the production of outer membrane vesicles (OMVs) via ubiquitous blebbing of the outer membrane. These proteoliposomes have the ability to traverse the mucus layer. Hence, OMVs play an important role in immunomodulation and the maintenance of a balanced gut microbiota. Here we demonstrate that the stimulation of bone marrow derived dendritic cells (BMDCs) with isolated OMVs originated from B. vulgatus mpk leads to the induction of a tolerant semi-mature phenotype. Thereby, microbe- associated molecular patterns (MAMPs) delivered by OMVs are crucial for the interaction and the resulting maturation of immune cells. Additional to the binding to host TLR4, a yet unknown ligand to TLR2 is indispensable for the conversion of immature BMDCs into a semi-mature state. Thus, crossing the epithelial mucus layer and directly contact host cells, OMV mediate cross-tolerance via the transport of various Toll-like receptor antigens. These features make OMVs to a key attribute of B. vulgatus mpk for a vigorous acellular prevention and treatment of systemic diseases.  

3.2994           Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next-generation sequencing

Buschmann, D., Kirchner, B., Hermann, S., Märte, M., Wurmser, C., Brandes, F., Kotschote, S., Bonin, M., Steinlein, O.K., Pfaffl, M.W., Schelling, G. and Reithmair, M.

1. Extracellular Vesicles, 7, 1481321 (2018)

  Extracellular vesicles (EVs) are intercellular communicators with key functions in physiological and pathological processes and have recently garnered interest because of their diagnostic and therapeutic potential. The past decade has brought about the development and commercialization of a wide array of methods to isolate EVs from serum. Which subpopulations of EVs are captured strongly depends on the isolation method, which in turn determines how suitable resulting samples are for various downstream applications. To help clinicians and scientists choose the most appropriate approach for their experiments, isolation methods need to be comparatively characterized. Few attempts have been made to comprehensively analyse vesicular microRNAs (miRNAs) in patient biofluids for biomarker studies. To address this discrepancy, we set out to benchmark the performance of several isolation principles for serum EVs in healthy individuals and critically ill patients. Here, we compared five different methods of EV isolation in combination with two RNA extraction methods regarding their suitability for biomarker discovery-focused miRNA sequencing as well as biological characteristics of captured vesicles. Our findings reveal striking method-specific differences in both the properties of isolated vesicles and the ability of associated miRNAs to serve in biomarker research. While isolation by precipitation and membrane affinity was highly suitable for miRNA-based biomarker discovery, methods based on size-exclusion chromatography failed to separate patients from healthy volunteers. Isolated vesicles differed in size, quantity, purity and composition, indicating that each method captured distinctive populations of EVs as well as additional contaminants. Even though the focus of this work was on transcriptomic profiling of EV-miRNAs, our insights also apply to additional areas of research. We provide guidance for navigating the multitude of EV isolation methods available today and help researchers and clinicians make an informed choice about which strategy to use for experiments involving critically ill patients.  

3.2995           Exosome Therapy for Stroke

Chen, J. and Chopp, M. Stroke, 49, 1083-1090 (2018)   Nearly, all cells generate and eject vesicles, and these vesicles constitute major vehicles for intercellular communication. Exosomes, as nanosized vesicles (≈30–100 nm in diameter¹), target cell function by delivering proteins, lipids, and nucleic acids. Exosomes are emerging as a valuable source for disease stage–specific information and as fingerprints of disease progression and as potential biomarkers in different pathophysiological states.^2–5 However, because exosomes provide a major medium of intercellular communication,⁶ they likely also impact the treatment of diseases.^7,8 Recent reports have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles.^6,9 Exosomes harvested from multipotent mesenchymal stromal cells (MSCs) mediate the restorative therapeutic effects of MSCs for stroke.¹⁰ Here, we review the biogenesis of exosomes and their molecular composition and role as messengers of intercellular communication and describe using exosomes for treatment of stroke. We also focus on therapeutic effects and underlying mechanisms of action of exosomes as therapeutic vectors for stroke¹¹ but do not discuss the role of exosomes as disease or injury biomarkers. Capitalizing on the function of exosomes as vehicles for intercellular communication in physiological and pathophysiological conditions such as stroke provides a paradigm shift and enormous potential for safe and effective therapeutic approaches for stroke and for other diseases/injury.  

3.2996           Paracrine mechanisms induced by large oncosomes spontaneously shed by aggressive cells to promote adhesion and invasion of prostate cancer via αv integrin-dependent activation of FAK-AKT

Pathway

Ciardiello, C., Leone, A., Roca, M.S., Moccia, T., Minopoli, M., Vitagliano, C. and Biagio Pucci, S.

1. Extracellular Vesicles, 7, Suppl. 1, abstract OF11-05 (2018)

Background: The identification of new molecular prognostic markers for Prostate Cancer (PCa) is needed to optimize both therapeutic options and follow-up strategies. Thereafter, we characterized a syngeneic model consisting of parental DU145 PCa cells and their derived more aggressive subline DU145R80. A proteomic approach highlighted a small number of differentially expressed proteins between the two cell lines, including key-molecules in cancer progression such as αv-integrin. In the present study, by using the DU145/DUR14580 system, we defined a novel αv-integrin-dependent paracrine effect exerted by large EVs previously described as large oncosomes (LO) to promote tumour cell aggressiveness. Methods: FITC-conjugated cholera toxin B subunit labeled cells highlighted blebbing. Flow cytometry using standard beads identify EVs >1 μM. LO were isolated from cell media by discontinuous 5%–60% OptiPrep™ density gradient ultra-centrifugations. Adherent cells were counted manually. Invasion was evaluated by Boyden chambers. Proteolytic activity was measured by gel zimography. Xenografts of LO-treated/untreated DU145 cells were employed in nude mice. Results: In the present study we found spontaneous blebbing and increased LO shedding from DU145R80 compared to parental DU145 cells. LO from DU145R80 carried increased amount of active metalloproteinase 2 and αv-integrin, compared to LO from DU145. DU145R80-derived LO increased adhesion and invasion in recipient DU145 cells, activating FAK-AKT pathway and increasing proteolytic activity of recipient cells. By blocking αV-integrin on LO surface, using an anti- αv antibody, we reverted the LO-induced effect on adhesion, invasion and MMPs activity in DU145 recipient cells. DU145R80-derived LO promote DU145 tumorogenesis in vivo.  

3.2997           Acetylcholinesterase activity co-isolates minimally with small EVs and does not correlate with particle count

Muth, D.C., Liao, Z., Schøyen, T.H., Seale, T., Martin-Jaular, L., Ostrowski, M., Thery, C. and Witwer, K.

16. Extracellular Veswicles, 7, Suppl. I, abstract OF16.04 (2018)

  Background: Acetylcholinesterase (AChE) activity has been proposed and used as a measure of EV abundance. AChE activity is easily, quickly and cheaply assayed, making it a potentially attractive option for EV quantitation. To evaluate this use of AChE activity, we examined data from different EV isolation methods using multiple cell lines grown in cell culture conditions varying by amounts of serum and serum EVs. Methods: Cell lines were grown in media differing by serum status: EVreplete serum, commercial EV-depleted serum, or serum-free formulations. Cell culture conditioned medium (CCM) was harvested from various leukocyte cell lines, including T-lymphocytic lines H9 and PM1 and the promonocytic line U937. Following a slow spin to remove cells, EVs were isolated from CCM by differential ultracentrifugation (2000, 10,000 and 100,000 ×g) with or without subsequent iodixanol velocity density gradients. Pellets and fractions were assayed for AChE activity by standard colorimetric test; the presence of EV markers (CD63, CD81 and syntenin), and a negative marker (GM130, Golgi) by western blot; and particle count by single particle tracking (ParticleMetrix, NanoSight). Results: AchE activity was highest in replete serum medium. During differential centrifugation, most AChE activity was depleted in the 2000×g and 10,000k ×g steps, with little remaining activity in the 100,000 ×g pellets. When 100,000 ×g pellets were further separated by iodixanol gradient, early AChE activity-enriched fractions overlapped only minimally with tetraspanin-positive EV fractions. AChE activity did notcorrelate significantly (p < 0.05) with measured particle count in any examined condition. Summary/Conclusion: These findings indicate that AChE activity may be mostly associated with debris and/or large particles and is particularly abundant in medium containing undepleted serum. At least for small EVs, high AChE activity may betray contamination, not EV abundance. Additional experiments may be merited to validate these results for primary cells or in biological fluids, but, overall, AChE activity appears to be a poor indicator of EV abundance, echoing a cautionary note sounded in the MISEV2014 guidelines and other publications. Funding: This research was supported in part by the US National Institutes of Health through DA040385 and AG057430 (to KWW).  

3.2998           Identifying novel cellular components specifically incorporated into HIV versus exosomes and other small EVs

Martin-Jaular, L., Liao, Z., Gerber, P., Ostrowski, M., Witwer, K., Borner, G. and Thery, C.

25. Extracellular Vesicles, 7, Suppl. I, abstract OS25.06 (2018)

  Background: HIV buds from infected cells by a mechanism that shares many aspects with the biogenesis of small extracellular vesicles (sEVs). Consequently, sEVs and HIV share many physical and chemical characteristics, which make their separation difficult. For this reason, the function of sEVs during HIV infection remains unclear. Here, we used a novel un-biased approach to identify the cellular components specifically incorporated into either HIV or sEVs Methods: Jurkat cells were infected with VSV-G-pseudotyped NL4-3 virus. EVs were obtained by differential centrifugation of medium conditioned by non-infected and HIV-infected cells. Velocity OptiPrep gradient was used to further separate sEVs from virus. EVs were analysed by Western blotting (WB) for the presence of different markers previously described in sEVs and/or HIV. Fractionation profiling was performed from quantitative proteomic analyses of EVs from Jurkat cells labelled with SILAC amino acids. Results: OptiPrep gradients revealed different types of sEVs in the non-infected and in the HIV-infected cells, with insufficient discrimination achieved by the presence of AChE or CD45, markers that putatively discriminate EVs from HIV. In addition, separation of different particles was not possible due to overlap of markers between fractions. We used a global proteomic approach to identify novel specific markers of the virus or sEV subtypes. Two biological replicates of infected and non-infected samples were analysed. Principal component analysis reveals that the HIV proteins form a cluster very close to several sEV markers. Comparison of fractions from noninfected and HIV-infected cells led us to identify candidate proteins that changed location within the different types of vesicles after ISEV 2018 abstract book 197 infection, either moving towards or away from the HIV cluster. Validation of a short list of candidates wa done by WB after differential centrifugation of conditioned medium. Summary/Conclusion: OptiPrep gradients showed imperfect association of classical protein markers to sEVs or virus. Using a quantitative proteomic approach, we have defined a short list of novel marker candidates that have been validated by WB. Our results will allow obtaining HIV-free sEVs and assessing their function during the course of HIV Infection  

3.2999           DNA outside and inside of EVs and its role in phosphorylation of interferon regulatory factor 3

Lazaro-Ibanez, E., Shelke, G.V., Crescitelli, R., jang, S.C., Garcia, A., Lässer, C. and Lötvall, J.

2. Extracellular Vesicles, 7, Suppl. 1, abstract OT02.02 (2018)

  Background: In contrast to the multiple studies addressing the role or RNAs in extracellular vesicles (EVs), the presence of DNA represents a more unexplored but highly appealing source of information. EV-associated DNA may hold remarkable value in clinical diagnostics as a biomarker, since it can provide information about the characteristics of a disease, but it can also act as an immune sensor activator mediating, e.g. autoimmunity or anti-viral responses. Here, we described that DNA is associated both inside and outside of EVs and that the DNA outside EVs can activate innate immune signalling in recipient cells. Methods: EVs were purified from mast (HMC-1) and erythroleukemic (TF-1) cells by differential centrifugation followed by separation by bottom-loaded iodixanol density gradients. EVs with different density were characterized by western blotting, transmission electron microscopy and particl concentration, followed by total DNA extraction and analysis. The association of the dsDNA inside or outside EVs and its coverage was evaluated by enzymatic DNase treatment followed by whole genome sequencing (WGS) of the DNA inside and outside of EVs. The innate immune activation mediated by EV-DNA in recipient cells was assessed by the phosphorylation of interferon regulatory factor 3 (IRF-3). Results: EV subsets with low and high densities showed differential dsDNA profiles analysed by a bioanalyser. Low-density EVs carried small quantities of dsDNA mainly unprotected from enzymatic degradation. Instead, highdensity EVs contained larger quantities of dsDNA, which was partly protected from enzymatic degradation. WGS results showed that the entire genome was present both in the total DNA and in the DNA protected fromenzymatic degradation. Regardless, from 77% to 97% of the total DNA was removed by DNase treatment, arguing that most of the DNA was present on the outside of the EVs. DNase treatment of the EVs eliminated their ability to induce phosphorylation of IRF-3 in recipient cells. Summary/conclusion: EVs from several cell types carry DNA on their surface, but the inability of DNase to remove all DNA suggests that someof the genetic material is still on the inside of the EVs. EVs with DNA on their surface can induce the phosphorylation of IRF-3, which suggests a role in innate immunity.  

3.3000           Mesenchymal stromal cell extracellular vesicles modulate innate and adaptive immune cells at multi-organ level in a model of bronchopulmonary dysplasia

Reis, M., Willis, G.R., Fernandez-Gonzalez, A., Mansouri, N., Mitsialis, A. and Kourembanas, S.

2. Extracellular Vesicles, 7, Suppl. 1, abstract OT02.04 (2018)

  Background: Bronchopulmonary dysplasia (BPD) is a multifactorial chronic disease that occurs predominantly in preterm infants receiving oxygen therapy and mechanical ventilation, and is characterized by lung growth arrest, diminished alveolar and blood vessel development and impaired pulmonary function. Using a murine model in hyperoxia-induced BPD, we recently showed that a bolus dose of MSC extracellular vesicles (MEx) improved lung architecture and lung function and that this therapeutic effect was associated with modulation of lung macrophage phenotypes. However, BPD is a disease with multi-organ effects. Thus, we extend our studies in this BPD model to investigate the immunomodulatory effects of MEx on the innate and adaptive immune responses at the multiorgan level. Methods: Extracellular vesicles were collected from the conditioned media of human Wharton’s Jelly-MSCs and purified through density flotation in Iodixanol. Newborn mice were exposed to hyperoxia on postnatal day 1 (PN1) (75% O2), treated with MEx on PN4 and returned to room air on PN7. Treated animals and appropriate controls were harvested on PN7 and PN14 for histologic and cytometric assessment of lungs, spleen and thymus. Results: Hyperoxia-exposed mice presented significant lung damage and alveolar simplification as well as medullary involution of the thymus. Injection of MEx into hyperoxic-mice improved lung histology and restored thymic cortico-medullary ratios to levels akin to their normoxic counterparts. At PN7, MEx treatment modulated macrophages into an anti-inflammatory phenotype and mobilized inflammatory LY6ChiCCR2+ monocytes in the lungs and spleens. At PN14, Mex treatment induced a multi-organ reduction of inflammatory monocytes with a shift to a regulatory phenotype. Specifically, MEx altered T-cell subpopulation levels, inducing a reduction in CD8+ lymphocytes and an increase in CD4+ lymphocytes, and promoting the generation of CD4 +CD25hiFoxP3+ regulatory T cells. Summary/conclusion: Using a hyperoxia-induced BPD model, we show that MSC extracellular vesicle treatment results in a profound multiorgan effect on the immune system and promotes a tolerogenic T-cell phenotype that plays a crucial role in the amelioration of BPD-associated pulmonary damage.  

3.3001           A novel strategy to liquid biopsy for early diagnosis of lethal prostate cancer employing palmitoyl-proteomics of extracellular vesicles

Mariscal, J., Zhou, B., De Hoff, P., Pink, D., vagner, T., Zandian, M., Lewis, J.D., Laurent, L.C., Yang, W., Zijlstra, A. and Di Vizio, D.

4. Extracellular Vesicles, 7, Suppl. 1, abstract OT04.02 (2018)

  Background: Efficient risk assessment of prostate cancer patients is crucial for improved management. Tumour extracellular vesicles (EVs) have been shown to be carriers of abundant tumour material which can be used as evidence of disease. Recent discoveries highlight the complexity of the origin and function of EVs. A large subpopulation of EVs, large oncosomes (LO), has been identified so far as the only subtype uniquely secreted by migratory tumour cells, thus making an excellent platform for the discovery of robust biomarkers. In addition, LO cargo shows a significant enrichment in protein susceptible of S-palmitoylation. S-palmitoylation is a post-translational modification involved in vesicle trafficking and protein secretion and whose malfunction has been extensively reported in cancer. Methods: Differential centrifugation, iodixanol gradient, tunable resistive pulse sensing, size exclusion chromatography, electrical sensing zone, micro-flow cytometry, mass spectrometry, palmitoyl-protein  Identification. Results: We refined the methods for the isolation and characterization of EVs to improve specificity and yield. Most of these vesicles have a size of 2–4 μm and are enriched in CK18 and HSPA5 markers in contrast to small EVs (80–120 nm), which are enriched in CD81 and Tsg101 markers. Our preliminary results show a strong association of LO cargo with tumour cell survival mediated by the protein ubiquitination and unfolded protein response pathways. Interestingly, there is a significant enrichment of proteins susceptible of palmitoylation and associated to pro-survival pathways frequently activated in tumour cells. Accordingly, some of these proteins have been previously proposed as biomarkers in a plethora of diseases including cancer but their palmitoylation status have not been considered. Summary/conclusion: Assessment of palmitoylated biomarkers in LO represents a promising strategy in the liquid biopsy of lethal prostate cancer. Funding: National Institutes of Health NIH R01 CA218526  

3.3002           Oral administration of bovine milk-derived extracellular vesicles reduce primary tumour burden but accelerate cancer metastases

Mathivananan, S.

5. Extracellular Vesicles, 7, Suppl. 1, abstract OT05.03 (2018)

  Background: It has been proposed that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs and exert phenotypic changes. However, the concept was challenged by few well-controlled studies emphasizing the instability of nucleic acids which ultimately succumb to the membrane barriers and nucleases of the mammalian gastrointestinal tract. Furthermore, the observations were often criticised as dietresponsive endogenous RNAs or artefacts due to non-adherence of rigorous procedures. Methods: EVs were isolated from raw and commercial milk by ultracentrifugation and OptiPrep density gradient. Quantitative proteomics and RNA-seq of EVs. DIR labelled EVs biodistribution was monitored by IVIS imaging. Quantitative proteomics of mouse liver Tissues. EVs were orally administered to various models including xenograft, cachexic, E-cadherin biosenor and metastatic mice models. Results: Here, we orally administered bovine milk-derived EVs to mice and demonstrated that milk-derived EVs can survive the harsh degrading conditions of the gut and subsequently be detected in multiple organs. Interestingly, oral administration of milk-derived EVs reduced the primary tumour burden in various cancer models and attenuated cancer cachexia. Intriguingly, in spite of the reduction in primary tumour growth, milk-derived exosomes accelerated metastasis in breast and pancreatic cancer mice models. Timing of exosome administration was critical as oral administration after resection of the primary tumour reversed the pro-metastatic effects of milk-derived exosomes in breast cancer.  

3.3003           Exploring the role of extracellular vesicles (EVs) in immune response

Ritz, S., Meira, M., Lagarde, N., Sievers, C., Derfuss, T. And Lindberg, R.

4. Extracellular Vesicles, 7, Suppl. 1, abstract PF04.10 (2018)

  Background: Extracellular vesicles (EVs) play an important role in intercellular communication in physiological (e.g. communication in brain, regulation of immune responses) and in pathological conditions (e.g. cancer, autoimmune diseases). Nearly all cell types, including immune cells, produce exosomes, microparticles and apoptotic bodies, collectively termed EVs. Our goal is to study the functional importance of exosomes in the immunopathogenesis of multiple sclerosis (MS). We specifically aim at characterizing serum-derived exosomes from patients with MS and healthy volunteers (HV) and studying their effects on various immune cells. Methods: Exosomes were isolated from platelet-free serum of HV and MS patients with various disease courses by iodixanol gradient centrifugation (OptiPrep) followed by size-exclusion chromatography (SEC). Nanoparticle Tracking Analysis was used for enumeration and size determination. Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation. Immune cells were separated by MACS technology and stimulated in vitro. Exosomes were added and their interaction with immune cells was determined by ImageStream X. Expression of activation markers was analysed by flow cytometry (Attune NxT). Total RNA was extracted from immune cells, and transcriptional expression was analysed using real-time RT-PCR-based assays. Results: OptiPrep gradient centrifugation, followed by SEC, resulted in a homogenous exosome population. Levels of exosomes in sera from relapsing-remitting (RR) MS patients were significantly higher than in those from HV. Analysis of the interaction between exosomes and immune cells revealed a strong association of exosomes with monocytes, followed by CD4+ T, CD8+ T and B cells. Moreover, application of exosomes impacted on the activation and transcriptional regulation of primary immune cells in vitro. Summary/Conclusion: Increased levels of exosomes in RRMS patients suggest their potential role in the immunopathogenesis of MS. However, further experiments are needed to confirm the functional importance of exosomes in immune regulation of MS. Characterization of exosomes from various disease courses of MS and evaluation of the effects ofcurrent treatments will be performed. Funding: This work was funded by Swiss MS Society, Swiss National Science Foundation.  

3.3004           Efficient isolation of extracellular vesicles from blood plasma based on iodixanol density gradient ultracentrifugation combined with bind-elute chromatography

Brenner, G., Onodi, Z., Nagy, C.T., Kittel, A., Keber, M.M. and Giricz, Z. J.Eextracellular Vesicles, 7, Suppl. 1, abstract PF06.12 (2018)   Background: Blood-derived extracellular vesicles (EVs) are extensively investigated both as biomarkers and therapeutics. However, efficient isolation of EVs from a limited amount of sample is a great challenge. Thus, the aim of this study was to identify a method to isolate the majority of EVs from blood plasma, while eliminating impurities such as lipoprotein particles and soluble proteins. Methods: Rat and human blood samples underwent low-speed centrifugations to remove cells, debris and large particles without prior filtration. Density gradient ultracentrifugation (DGUC) was performed by layering 50%, 30% and 10% iodixanol solutions on top of which sample was loaded and centrifuged at 120,000 ×g for 24 h. Fractions were collected from top to bottom. Fractions with the highest EV content were further purified by ultracentrifugation or size exclusion chromatography. Efficiency and purity were assessed by Western blot. Morphology and size distribution of particles were examined bydynamic ligh scattering (DLS) and electron microscopy (EM). Results: Highest band intensities of EV markers Alix and Tsg101 were detected (60% and 59%, respectively) at a density of 1.13–1.17 g/mL. The presence of EVs was confirmed by EM and DLS, showing particles with a mean diameter of 38 ± 2 nm. By DGUC, 95% of lipoprotein- and 84% of albumin contamination were separated from EV-containing fractions. However, 67% of the total fibrinogen content was present in EV-rich fractions, indicating the need for further purification. After loading 1.3-mL EV-rich fractions of DGUC on HiScreen Capto Core 700 column, the majority of Tsg101 signal was observed in 2 mL eluate in which albumin was not detectable, while the amount of fibrinogen decreased but was not completely removed from EV-rich eluate. Summary/Conclusion: DGUC with iodixanol shows higher efficiency than generally used methods for the isolation of blood-derived exosomes. It separates EVs from the majority of vesicle-like lipoproteins, 152 Friday, 04 May 2018 and reduces the amount of contaminating soluble proteins. Further purification of EV-rich DGUC fractions by chromatography on Capto Core 700 column yields amounts of EVs significantly higher than currently described methods with less contamination by non-EV plasma components. Funding: The project was funded by NKFIH NVKP 16-1-2016-0017. ZG Holds a Bolyai Fellowship from the Hungarian Academy of Sciences.  

3.3005           An optimized workflow for the isolation and purification of extracellular vesicles from small serum volumes

Brennan, K., McGee, M., martin, K.and Richardson, C.

6. Extracellular Vesicles, 7, Suppl. 1, abstract PF06.19 (2018)

  Background: Extracellular vesicles (EVs) are nanometre-scale, membrane- enclosed vesicles that are released from a multitude of cell types and mediate intercellular communication via the transfer of proteins, small RNAs and mRNAs to recipient cells. EVs have gained a lot of interest in the past few years as a source of cancer biomarkers with both diagnostic and prognostic value. A bloodbased cancer screening test is appealing because the specimens can be obtained readily in a non-invasive manner, and poses minimal risk to patients. EVs as a source of blood-based biomarkers present a considerable challenge due to a combination of small sample size, serum viscosity and difficulties in separating EVs from serum proteins and lipoproteins. Methods: In this study, we evaluate particle yield and purity using four isolation methods: differential ultracentrifugation, polymerbased precipitation, size-exclusion chromatography and iodixanol density gradient centrifugation, on their own and in combination, for the isolation of EVs from 100 to 250 μl of human serum. Comprehensive characterization of EV yield and protein content was performed by nanoparticle tracking analysis and Bradford assay following TCA protein precipitation respectively. Furthermore, the relative abundance of EV markers, CD63 and TSG101, and lipoprotein markers, APOB, APOA1 and APOE, was determined by Western blot analysis for each method. Results: Our results demonstrate that polymer-based precipitation recovered the highest number of EVs, while providing the least pure preparations of exosomes. Iodixanol density gradient centrifugation and size-exclusion chromatography provided the best EV/protein ratio by nanoparticle tracking analysis and Bradford assay. Based on Western blotting, we found that the size-exclusion chromatography was superior in isolating EVs devoid of high density lipoprotein. Summary/Conclusion: Our data reveal that a combination of isolation methods is necessary for adequate separation of soluble proteins and lipoproteins from serum EVs.  

3.3006           Proteome analysis of glioma-derived extracellular vesicles isolated from neurosurgical aspirates provide markers for disease stage and progression

Hallal, S., Russell, B., Shivalingam, B., Buckland, M. and Kaufman, K.

5. Extracellular Vesicles, 7, Suppl. 1, abstract PT05.14 (2018)

  Background: Glioblastomas (GBM) are highly lethal brain tumours with limited treatment options available to patients. Non-invasive liquid biopsies that monitor GBM progression are important for developing personalized therapies for GBM. GBM extracellular vesicles (GBM-EVs) play key roles in GBM biology and are detectable in the peripheral circulation. However, profiling GBM-EVs from the blood remains an obstacle as they are a minor subset of the total blood EV population. We investigated whether our previously described in vitro GBM-EV proteome signature could be translated to GBM-EVs isolated from clinical sources that are rich in brain tumour EVs, i.e. Cavitron Ultrasonic Surgical Aspirate (CUSA) fluid. Methods: EVs were harvested from CUSA fluid by ultracentrifugation and enriched on a discontinuous iodixanol/sucrose gradient. Nanoparticle tracking analysis and transmission electron microscopy confirmed the presence of “exosome” sized (~100 nm) and vesicularshaped particles in CUSA fluid, and the proteomes of enriched CUSAEVs from GBM (n = 3) and low-grade astrocytoma (n = 3) were analysed by quantitative label-free LC-MS/MS. SLHD HREC approval was obtained and patients provided informed consent. Results: Multiple proteins were identified in the CUSA-EVs that are associated with glioma biology (EGFR, IDH1, vimentin, CD53). There was a substantial overlap of the CUSA-EV proteins with our in vitro GBM-EV proteomic signature, with GBM CUSA-EVs sharing 76% of GBM signature proteins and low-grade astrocytoma CUSA-EVs sharing 60%. EV proteins previously correlated to GBM cell invasiveness in vitro (ANXA1, IGF2R, ITGB1, PDCD6IP, ACTR3, CALR, IPO5, MVP, PSMD2) were also significantly increased in GBM CUSA-EVs compared to low-grade astrocytomas. Interestingly, significantly higher levels of all molecular chaperone T-Complex Protein 1 Ring Complex (TRiC) subunits, which are associated with multiple oncogenes and play roles in tumour invasion, were identified in GBM CUSA-EVs. ISEV 2018 abstract book 57 Summary/conclusion: CUSA fluid constitutes a novel and rich source of brain tumour EVs, sufficient to elucidate and validate potential prognostic biomarkers. With further study, these targets could offer avenues for tumour staging and monitoring GBM progression via peripheral blood sampling of GBM-EVs. PT05.15  

3.3007           Heat-shock factor 2 associates with cancer-derived extracellular vesicles

Henriksson, E., Luoto, J. and Sistonen, L.

7. Extracellular Vesicles, 7, Suppl. 1, abstract PS07.14 (2018)

  Background: The heat-shock factors (HSF1–4) are transcription factors essential for cellular stress responses and mammalian development. They have also roles in human pathologies, as HSF1 supports malignant growth and HSF2 suppresses cancer invasion. We have previously reported that in the prostate cancer cell line PC3, HSF2 protein levels decline in three-dimensional (3D) organotypic cultures as the organoids become invasive. Silencing of HSF2 accelerates the invasive transformation. The underlying molecular mechanisms are however unclear. We hypothesize that HSF2 is released from cancer cells in extracellular vesicles (EVs) in a cancer-invasion dependent manner. Methods: EVs were isolated from the growth media of PC3 and human U2OS osteosarcoma cells by differential ultracentrifugation or iodixanol density gradient fractionation and analysed by Western blotting. The same cell lines, as well as HSF2-knockout U2OS cells generated using CRISPR-Cas9 technique, were cultivated in Matrigel to produce 3D organotypic cultures. The extracellular proteins were visualized with immunofluorescence microscopy. Results: We found PC3 and U2OS cells to release HSF2 in 2D and 3D cell cultures. The extracellular HSF2 (eHSF2) was present in CD63 and CD81 positive density fractions prepared from 2D culture medium and in 3D cultures the eHSF2 co-localized with CD81 outside the organoids. The characterization of HSF2-carrying EVs is currently being conducted. Summary/Conclusion: Our results show that HSF2 is secreted by cancer cells and the secretion could lea to low intracellular HSF2 levels, enabling an invasive organoid development. This might also apply to cancer patients, as low HSF2 levels in tumours correlate with poor survival.  

3.3008           Human cytomegalovirus-infected cells release extracellular vesicles that carry viral surface proteins

Arakelyan, A., Zicari, Z., Fitzgerald, W., Vanpouille, C., Lebedeva, A., Schmitt, A., Bomsel, M., Britt, W. and Margolia, L.

9. Extracellular Vesicles, 7, Suppl. 1, abstract PF09.10 (2018)

  Background: Extracellular vesicles (EVs) are released by many if not by all cells in the human body. These EVs incorporate, from the cell of origin, various cellular molecules and proteins. If a cell is infected with a virus, EVs can incorporate viral proteins as well. Upon interactions with cells, EVs carrying viral proteins may trigger various physiological responses. Here, we show that EVs carry human cytomegalovirus (HCMV) envelope proteins that are essential for HCMV infectivity. Methods: We isolated EVs from UL32-EGFP-HCMV viral suspension, produced by MRC-5 cells, using an OptiPrep step-gradient. We analysed EVs that were concentrated between 10% and 15% of the OptiPrep gradient for carrying HCMV proteins by staining them with antibodies specific for gB and gH, two viral envelope glycoproteins present on the surface of HCMV. All lipidic particles were labelled with a fluorescent dye (DiI) to distinguish HCMV virions that were GFP-positive/DiIpositive from EVs that were GFP-negative/DiI-positive. Results: Flow analysis demonstrated that EVs constituted 99.7 ± 0.1% (n = 3) of the total events, while 0.3 ± 0.1% (n = 3) were UL32-EGFPHCMV. Next, we analysed DiI-labelled EVs for the presence of HCMV surface proteins by staining with anti-gB AF647 antibodies and with anti-gH PB antibodies or with their isotype controls IgG AF647 and IgG PB. Labelled EVs were analysed with flow cytometer, triggering on DiI fluorescence. On average, 15 ± 3.7% (n = 3) of EVs were positive for gB and 5.3 ± 2.3% (n = 3) were positive for gH HCMV surface proteins and 3.74 ± 1.5% (n = 3) were positive for both gB and gH. Summary/Conclusion: EVs released from HCMV-infected cells carry viral surface proteins. Production by infected cells of EVs carrying various viral proteins is a general phenomenon for various viruses. Understanding of the exact details and molecular mechanisms of this contribution may reveal new therapeutic targets. Funding: The work of AA, SZ, WF, CV, AL and LM was supported by the NICHD/NIH Intramural Program. The work of AL was also supported by the Russian Federation Government grant #14.B25.31.0016 and RFBR grant #16-04-017/16. The work of AS and MB was supported by ANRS (AO2015-2-17046). The work of WB was supported by NIH  

3.3009           A simple method to label vesicles for visualization and in vivo tracking

Salas-Huenuleo, E., Polakovicova, I., varas-Godoy, M., Lobos-Gonzalez, L., Bejarano, J., Corvalan, A.C. and Kogan, M.J.

3. Extracellular Vesicles, 7, PS03.09 (2018)

  Background: Labelling of vesicles for their visualization in vitro or in vivo, involves the use of fluorescent dyes. To obtain labelled vesicles free of unincorporated dye, purification steps are necessary. The standard method is density gradient ultracentrifugation which is not only time consuming, but counts with high sample loss and requires expensive equipment. Here, we established a simple and fast method to acquire labelled vesicles for in vivo tracking and visualization. Methods: Extracellular vesicles (EVs) from cell culture supernatant, synthetic exoliposomes (ELIP) and thermosensitive liposomes (TLIP) were obtained and characterized by nanosight, transmission electron microscopy and zeta potential determinations. Subsequently, the nanostructures were incubated with DiR fluorophore. DiR-labelled vesicles were purified by two different methods, using optiprep density gradient ultracentrifugation or commercial exo-spin columns. The eluates obtained from columns and density gradient fractions were characterized by nanosight, dynamic light scattering, zeta potential, protein content, fluorescence spectroscopy and imaging. Obtained yields of labelled vesicles were compared. Next, purified labelled EVs, ELIP and TLIP were administrated via tail vein injection in mice with an equivalent number of particles and visualized at 48 h using In Vivo imaging system. Organs were extracted, visualized and fluorescence intensity was measured. All animal procedures and care were approved by implicated ethic committees. ISEV 2018 abstract book 217 Results: Using exo-spin column, DiR labelled EVs, ELIP and TLIP were obtained. Profound characterization of every step, column and eluate during the process showed that free DiR was not present in labelled samples. Next, we established that the use of column provides reproducible results with low sample loss. The working time is less than 10 min, significantly less than up to 24 h of the density gradient method. Finally, we used these labelled vesicles to determine and compare their biodistribution in organs of mice. Summary/Conclusion: We compared two methods and established the use of exo-spin column as a tool to obtain labelled vesicles in a reproducible, simple and faster manner, with no need of expensive.  

3.3010           Repeatable, high-purity isolation of urinary extracellular vesicles for uro-oncological biomarker studies

Dhondt, B., Vergauwen, G., Van Deun, J., Geeurickx, E., Tulkens, J., Lippens, L., Miinalainen, I., Rappu, P., Heino, J., Lumen, N., De Wever, O. and Hendrix, A.

4. Extracellular Vesicles, 7, Suppl. 1, abstract PS04.02 (2018)

  Background: Urinary extracellular vesicles (uEV) have raised interest as a potential source of biomarker discovery. Contaminants such as Tamm- Horsfall protein (THP) polymers hinder accurate downstream analysis by masking low abundance proteins or by entrapping non-EV associated extracellular RNA molecules. Methods: Cell-free urine samples from prostate cancer patients were concentrated by ultrafiltration. uEV were isolated using a bottom-up discontinuous Optiprep™ density gradient (ODG) in six technical replicates and characterized by nanoparticle tracking analysis (NTA), transmission electron Microscopy (TEM) and unbiased proteomic analysis (LC–MS/MS). Results: NTA and TEM confirmed the enrichment of 100 nm uEV in density fractions of approximately 1.1 g/ml (EV-rich fractions) and THP contaminants in the high density fractions. Unbiased mass spectrometry-based proteomics identified consistent and biologically relevant EVassociated proteins with high repeatability as analysed by principle component analysis and hierarchical clustering. Volcano plot analysis showed a clear differential protein enrichment between EV rich density fractions and THP high density fractions and gene set enrichment analysis of these proteins demonstrated differential biological functions and cellular origin. Summary/Conclusion: Bottom-up ODG results in an efficient and reproducible separation of uEV from THP, a first step towards biomarker discovery in genitourinary cancer patients. Funding: This study was funded by Kom op tegen Kanker (Stand up toCancer), the Flemisch Cancer Society.  

3.3011           Extracellular vesicles from the parasitic nematode Trichuris muris: new insights into host–parasite communications

Eichenberger, R.M., Talukder, H., Field, M., Wangchuk, P., Giacomin, P.R., Loukas, A., Sotillo, J.S.J.

1. Extracellular Vesicles, 7, Suppl. 1, abstract PT01.09 (2018)

  Background: Trichuris muris is a nematode parasite that lives in the mouse colon and has been widely used to study human whipworm infections, a parasitic disease affecting more than 500 million people worldwide. These nematodes secrete a multitude of compounds that interact with host tissues where they orchestrate a parasitic existence. Until now, there was no evidence that T. muris secreted extracellular vesicles (EVs). Methods: We isolated EVs from the secretory products of T. muris after ultracentrifugation and further purification using Optiprep density gradient. We characterized the proteomic and nucleic acid (miRNA and mRNA) contents of the vesicles and used confocal microscopy to demonstrate the internalisation of parasite EVs by murine colonic organoids. Results: A total of 364 proteins, including tetraspanins and other exosome markers, were identified in T. muris -secreted EVs. In addition, 56 miRNAs and 475 full-length mRNA transcripts mapping to T. muris gene models were also identified. Many of the miRNAs putatively mapped to mouse genes involved in regulation of inflammation, implying a role in parasite-driven immunomodulation. Furthermore, we demonstrated that T. muris EVs can be actively internalized by mouse colonic organoids, suggesting a role in host–parasite communication. Summary/conclusion: Understanding how parasites interact with their hosts is crucial to develop new control measures. This first characterization of the proteins and nucleic acids from the EVs secreted by

1. muris provides important information on whipworm–host communication and forms the basis for future studies.

Funding: This work was supported by a program grant from the National Health and Medical Research Council (NHMRC) [programgrant number 1037304] and a Principal Research fellowship from NHMRC to AL. RME was supported by an Early Postdoc MobilityFellowship (P2ZHP3_161693) from the Swiss National Science Foundation. MHT was supported by an Endeavour Research Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no competing financial interests.  

3.3012           Tumour-derived exosomes contribute to a pro-tumourigenic inflammatory microenvironment in cancer

Sarte, L., Nakata, R., Shimada, H., Fernandez, E. and De Clerk, Y.A.

4. Extracellular Vesicles, 7, Suppl. 1, abstract PT04.05 (2018)

  Background: Inflammation plays an important contributory role in cancer progression through multiple mechanisms. Among those is the ability of tumour cells to induce the expression of pro-tumourigenic cytokines and chemokines by stromal cells in the tumour microenvironment. Here, we have examined the role of tumour-derived exosomes in the induction of inflammation in neuroblastoma (NBL), the second most common solid malignancy in children. Methods: Exosomes from human NBL cells lines were purified by differential ultracentrifugation (DUC), optiprep density gradient centrifugation (ODGC) and size exclusion chromatography (SEC) and characterized by electron microscopy, nanoparticle tracking analysis and western blot analysis (presence of syntenin, ALIX, CD-9, 63 and 81 and absence of GM-130 and calnexin). Exosomes were labelled with green or red lipophilic dyes (PKH67 and PKH 26). Human NBL cells were also engineered to express green fluorescent protein (GFP) labelled exosomes (NBL-X-PACK-GFP). The activity of these tumour-derived exosomes was examined in vitro and their capture in vitro and in vivo Results: NBL-derived exosomes induced the expression of several protumourigenic cytokines such as IL-6, IL-8, MCP-1 and VEGF with exomes purified by SEC having the highest specific activity. The capture of exosomes by stromal cells was affected by Galectin-3-binding protein present at their surface. In vivo, purified NBL-derived exosomes injected intravenously or exosomes produced by NBL-X-Track tumour cells implanted orthotopically in immunodeficient mice were captured by CD-45 and F4/80-positive myeloid cells in the lungs, liver and bone marrow and by few CD-105-positive mesenchymal stromal cells in the bone marrow. Summary/conclusion: Tumour-derived exosomes provide a contactindependent mechanism of communication between tumour cells and stromal cells that contribute to the induction of a pro-inflammatory reaction favourable for cancer progression. Funding: National Cancer Institute/National Institutes of Health USA; St Baldrick’s Foundation.  

3.3013           Chloride intracellular channel protein 4 (CLIC4) is a serological cancer biomarker released from tumour epithelial cells via extracellular vesicles and required for metastasis

Sanchez, V.C., Craig-Licas, A., Wei, B-R., Read, A., Simpson, M., Luo, J., Hunter, K. and Yuspa, S.

5. Extracellular Vesicles, 7, Suppl. 1, abstract PT05.16 (2018)

  Background: CLIC4 is a highly conserved metamorphic protein originally described as an ion channel. It translocates to the nucleus serving as an integral component of TGF-β signalling. In multiple cancers, CLIC4 is a tumour suppressor, excluded from the nucleus and lost from the cytoplasm of progressing cancer cells. In contrast, CLIC4 is upregulated in the tumour stroma acting as a tumour promoter. CLIC4 lacks a secretory sequence, but recent reports indicate that CLIC4 is detected in the circulation of cancer patients serving as possible biomarker and has been detected in extracellular vesicles (EVs). Methods: EVs from cell culture supernatants or biological fluids were isolated by differential centrifugation, following ultracentrifugation and Optiprep density gradients. EV size distribution and concentration were analysed by NTA and TEM. The presence of prototypical markers and CLIC4 was analysed by immunoblot. Results: CLIC4 was present in EVs released from primary normal and multiple ovarian and breast tumour cell lines. Substantial increases in CLIC4 were measured in EVs of tumour cells when compared to normal cells. TGF-β-induced myofibroblasts also increased CLIC4 in both the cells and the EVs they released. In vivo, CLIC4 levels increased in EVs released into the peritoneal cavity as tumour burden increased in a heterotopic xenograft ovarian cancer model. Moreover, CLIC4 levels in EVs isolated from plasma increased with tumour burden and lung metastatic load in orthotopic syngeneic mouse breast cancer models. To dissect the contribution of stromal vs tumour epithelial compartments as the source of the CLIC4-high EVs, CLIC4 was either deleted in tumour cells lines by CRISPR/Cas9 or CLIC4 KO females were implanted CLIC4 WT tumour cells. CLIC4 is reduced in circulating EVs from CLIC4 KO tumour bearing mice when compared to WT and it is present in circulating EVs from CLIC4 KO females bearing WT tumours, indicating that the major contribution of CLIC4 into circulation is from tumour epithelium. Moreover, CLIC4 KO females display no difference in primary tumour size and a significant reduction in both size and number of lung metastases. Summary/conclusion: CLIC4 levels in EVs from biological fluids may have value as a cancer biomarker, in conjunction with other markers, to detect or analyse tumour progression or recurrence.  

3.3014           JIP3 localises to exocytic vesicles and focal adhesions in the growth cones of differentiated PC12 cells

Caswell, P.T. and Dickens, M. Mol. Cell. Biochem., 444, 1-13 (2018)   The JNK-interacting protein 3 (JIP3) is a molecular scaffold, expressed predominantly in neurons, that serves to coordinate the activation of the c-Jun N-terminal kinase (JNK) by binding to JNK and the upstream kinases involved in its activation. The JNK pathway is involved in the regulation of many cellular processes including the control of cell survival, cell death and differentiation. JIP3 also associates with microtubule motor proteins such as kinesin and dynein and is likely an adapter protein involved in the tethering of vesicular cargoes to the motors involved in axonal transport in neurons. We have used immunofluorescence microscopy and biochemical fractionation to investigate the subcellular distribution of JIP3 in relation to JNK and to vesicular and organelle markers in rat pheochromocytoma cells (PC12) differentiating in response to nerve growth factor. In differentiated PC12 cells, JIP3 was seen to accumulate in growth cones at the tips of developing neurites where it co-localised with both JNK and the JNK substrate paxillin. Cellular fractionation of PC12 cells showed that JIP3 was associated with a subpopulation of vesicles in the microsomal fraction, distinct from synaptic vesicles, likely to be an anterograde-directed exocytic vesicle pool. In differentiated PC12 cells, JIP3 did not appear to associate with retrograde endosomal vesicles thought to be involved in signalling axonal injury. Together, these observations indicate that JIP3 may be involved in transporting vesicular cargoes to the growth cones of PC12 cells, possibly targeting JNK to its substrate paxillin, and thus facilitating neurite outgrowth.  

3.3015           Extracellular vesicles as circulating cancer biomarkers: opportunities and challenges

Lane, R.E., Korbie, D., Hill, M.M. and Trau, M. Clin. Transl. Med., 7:14 (2018)   Extracellular vesicles (EVs) are small, lipid-bound particles containing nucleic acid and protein cargo which are excreted from cells under a variety of normal and pathological conditions. EVs have garnered substantial research interest in recent years, due to their potential utility as circulating biomarkers for a variety of diseases, including numerous types of cancer. The following review will discuss the current understanding of the form and function of EVs, their specific role in cancer pathogenesis and their potential for non-invasive disease diagnosis and/or monitoring. This review will also highlight several key issues for this field, including the importance of implementing robust and reproducible sample handling protocols, and the challenge of extracting an EV-specific biomarker signal from a complex biological background.  

3.3016           Control of insulin granule formation and function by the ABC transporters ABCG1 and ABCA1 and by oxysterol binding protein OSBP

Hussain, S.S., Harris, M.T., Kreutzberger, A.J.B., Inouye, C.M., Doyle, C.A., Castle, A.M., Arvan, P. and Castle, J.D. Mol. Cell. Cell, 29(10), 1238-1257 (2018)   In pancreatic β-cells, insulin granule membranes are enriched in cholesterol and are both recycled and newly generated. Cholesterol’s role in supporting granule membrane formation and function is poorly understood. ATP binding cassette transporters ABCG1 and ABCA1 regulate intracellular cholesterol and are important for insulin secretion. RNAi inter­ference–induced depletion in cultured pancreatic β-cells shows that ABCG1 is needed to stabilize newly made insulin granules against lysosomal degradation; ABCA1 is also involved but to a lesser extent. Both transporters are also required for optimum glucose-stimulated insulin secretion, likely via complementary roles. Exogenous cholesterol addition rescues knockdown-induced granule loss (ABCG1) and reduced secretion (both transporters). Another cholesterol transport protein, oxysterol binding protein (OSBP), appears to act proximally as a source of endogenous cholesterol for granule formation. Its knockdown caused similar defective stability of young granules and glucose-stimulated insulin secretion, neither of which were rescued with exogenous cholesterol. Dual knockdowns of OSBP and ABC transporters support their serial function in supplying and concentrating cholesterol for granule formation. OSBP knockdown also decreased proinsulin synthesis consistent with a proximal endoplasmic reticulum defect. Thus, membrane cholesterol distribution contributes to insulin homeostasis at production, packaging, and export levels through the actions of OSBP and ABCs G1 and A1.  

3.3017           Optimized Methods to Explore the Mechanistic and Biomarker Potential of Hepatocyte-Derived Exosomes in Drug-Induced Liver Injury

Thacker, S.E., Nautiyal, M., Otieno, M.A., Watkins, P.B. and Mosedale, M. Toxicol. Sci., 163(1), 92-100 (2018)   Recent evidence supports that alterations in hepatocyte-derived exosomes (HDE) may play a role in the pathogenesis of drug-induced liver injury (DILI). HDE-based biomarkers also hold promise to improve the sensitivity of existing in vitro assays for predicting DILI liability. Primary human hepatocytes (PHH) provide a physiologically relevant in vitro model to explore the mechanistic and biomarker potential of HDE in DILI. However, optimal methods to study exosomes in this culture system have not been defined. Here we use HepG2 and HepaRG cells along with PHH to optimize methods for in vitro HDE research. We compared the quantity and purity of HDE enriched from HepG2 cell culture medium by 3 widely used methods: ultracentrifugation (UC), OptiPrep density gradient ultracentrifugation (ODG), and ExoQuick (EQ)—a commercially available exosome precipitation reagent. Although EQ resulted in the highest number of particles, UC resulted in more exosomes as indicated by the relative abundance of exosomal CD63 to cellular prohibitin-1 as well as the comparative absence of contaminating extravesicular material. To determine culture conditions that best supported exosome release, we also assessed the effect of Matrigel matrix overlay at concentrations ranging from 0 to 0.25 mg/ml in HepaRG cells and compared exosome release from fresh and cryopreserved PHH from same donor. Sandwich culture did not impair exosome release, and freshly prepared PHH yielded a higher number of HDE overall. Taken together, our data support the use of UC-based enrichment from fresh preparations of sandwich-cultured PHH for future studies of HDE in DILI.  

3.3018           Low-input and multiplexed microfluidic assay reveals epigenomic variation across cerebellum and prefrontal cortex

Ma, S., Hsieh, Y-P., Ma, J. and Lu, C. Science Advances, 4(4), eaar8187 (2018)   Extensive effort is under way to survey the epigenomic landscape of primary ex vivo tissues to establish normal reference data and to discern variation associated with disease. The low abundance of some tissue types and the isolation procedure required to generate a homogenous cell population often yield a small quantity of cells for examination. This difficulty is further compounded by the need to profile a myriad of epigenetic marks. Thus, technologies that permit both ultralow input and high throughput are desired. We demonstrate a simple microfluidic technology, SurfaceChIP-seq, for profiling genome-wide histone modifications using as few as 30 to 100 cells per assay and with up to eight assays running in parallel. We applied the technology to profile epigenomes using nuclei isolated from prefrontal cortex and cerebellum of mouse brain. Our cell type–specific data revealed that neuronal and glial fractions exhibited profound epigenomic differences across the two functionally distinct brain regions.  

3.3019           Genome-wide distribution of linker histone H1.0 is independent of MeCP2

Ito-Ishida, A., Yamalanchili, H.K., Shao, Y., Baker, S.A., Heckman, L.D., lavery, L.A., Kim, J-y., Lombardi, L.M., Sun, Y., Liu, Z. and Zoghbi, H.Y. Nature Neurosci., 21, 794-798 (2018)   Previous studies suggested that MeCP2 competes with linker histone H1, but this hypothesis has never been tested in vivo. Here, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) of Flag-tagged-H1.0 in mouse forebrain excitatory neurons. Unexpectedly, Flag-H1.0 and MeCP2 occupied similar genomic regions and the Flag-H1.0 binding was not changed upon MeCP2 depletion. Furthermore, mild overexpression of H1.0 did not alter MeCP2 binding, suggesting that the functional binding of MeCP2 and H1.0 are largely independent.  

3.3020           Small-Molecule Sigma1 Modulator Induces Autophagic Degradation of PD-L1

Maher, C.M., Thomas, J.D., Haas, D.A., Longen, C.G., Oyer, H.M., Tong, J.Y. and Kim, F.J. Mol. Cancer Res., 16(2), 243-255 (2018)   Emerging evidence suggests that Sigma1 (SIGMAR1, also known as sigma-1 receptor) is a unique ligand-regulated integral membrane scaffolding protein that contributes to cellular protein and lipid homeostasis. Previously, we demonstrated that some small-molecule modulators of Sigma1 alter endoplasmic reticulum (ER)–associated protein homeostasis pathways in cancer cells, including the unfolded protein response and autophagy. Programmed death-ligand 1 (PD-L1) is a type I integral membrane glycoprotein that is cotranslationally inserted into the ER and is processed and transported through the secretory pathway. Once at the surface of cancer cells, PD-L1 acts as a T-cell inhibitory checkpoint molecule and suppresses antitumor immunity. Here, we demonstrate that in Sigma1-expressing triple-negative breast and androgen-independent prostate cancer cells, PD-L1 protein levels were suppressed by RNAi knockdown of Sigma1 and by small-molecule inhibition of Sigma1. Sigma1-mediated action was confirmed by pharmacologic competition between Sigma1-selective inhibitor and activator ligands. When administered alone, the Sigma1 inhibitor decreased cell surface PD-L1 expression and suppressed functional interaction of PD-1 and PD-L1 in a coculture of T cells and cancer cells. Conversely, the Sigma1 activator increased PD-L1 cell surface expression, demonstrating the ability to positively and negatively modulate Sigma1 associated PD-L1 processing. We discovered that the Sigma1 inhibitor induced degradation of PD-L1 via autophagy, by a mechanism distinct from bulk macroautophagy or general ER stress–associated autophagy. Finally, the Sigma1 inhibitor suppressed IFNγ-induced PD-L1. Our data demonstrate that small-molecule Sigma1 modulators can be used to regulate PD-L1 in cancer cells and trigger its degradation by selective autophagy. Implications: Sigma1 modulators sequester and eliminate PD-L1 by autophagy, thus preventing functional PD-L1 expression at the cell surface. This posits Sigma1 modulators as novel therapeutic agents in PD-L1/PD-1 blockade strategies that regulate the tumor immune microenvironment.  

3.3021           Proteomic Profiling of Leishmania donovani Promastigote Subcellular Organelles

Jardim, A., Hardie, D.B., Boitz, J. and Borchers, C.H.

1. Proteome Res., 17(3), 1194-12315 (2018)

  To facilitate a greater understanding of the biological processes in the medically important Leishmania donovani parasite, a combination of differential and density-gradient ultracentrifugation techniques were used to achieve a comprehensive subcellular fractionation of the promastigote stage. An in-depth label-free proteomic LC–MS/MS analysis of the density gradients resulted in the identification of ∼50% of the Leishmania proteome (3883 proteins detected), which included ∼645 integral membrane proteins and 1737 uncharacterized proteins. Clustering and subcellular localization of proteins was based on a subset of training Leishmania proteins with known subcellular localizations that had been determined using biochemical, confocal microscopy, or immunoelectron microscopy approaches. This subcellular map will be a valuable resource that will help dissect the cell biology and metabolic processes associated with specific organelles of Leishmania and related kinetoplastids.  

3.3022           Quantitative Proteomics and Cytology of Rice Pollen Sterol-Rich Membrane Domains Reveals Pre-established Cell Polarity Cues in Mature Pollen

Han, B., Yang, N., Pu, H. and Wang, T.

1. Proteome Res., 17(4), 1532-1546 (2018)

  Cell polarity is essential for generating diverse cell functions. The underlying mechanisms of how a cell establishes, maintains, and changes its polarity are poorly understood. Recently, sterol-rich membrane microdomains are found to be associated with these processes. However, both its exact characteristics and importance are still unclear. Here we show microdomains change dynamically in developing and germinating rice pollen with selective enrichment in the aperture and the tip of newly born pollen tubes by use of the sterol-specific probe filipin. Using the sterol extraction sensitivities of microdomain proteins and quantitative proteomics, we identified 237 microdomain-associated proteins from 934 identified pollen detergent resistant membrane proteins. This proteome includes almost all of the known key regulators comprising the polar growth network, and it shows more similarity to front–back polarized HeLa cells than nonpolarized Arabidopsis suspension cells. We immunolocalize flotilin-like protein, a representative of these sterol-dependent proteins and directly visualize microdomains in pollen. These results indicate the presence of microdomains in pollen and pre-established cell polarity around the aperture during pollen maturation. Our findings reveal an atlas of the microdomain-associated proteome in pollen. This work provides useful resources and knowledge needed to further dissect the mechanisms for the establishment and maintenance of cell polarity.  

3.3023           High cholesterol in lipid rafts reduces the sensitivity to EGFR‐TKI therapy in non‐small cell lung cancer

Chen, Q., Pan, Z., Zhao, M., Wang, Q., Qiao, C., Miao, L. and Ding, X.

1. Cell Physiol., 233(9), 6722-6732 (2018)

  Overcoming EGFR‐TKI resistant which has the initial enthusiasm over substantial clinical responses is a formidable challenge on nowadays. In this study, we showed that cholesterol level in lipid rafts in gefitinib resistant non‐small cell lung cancer (NSCLC) cell lines was remarkably higher than gefitinib sensitive cell line, and depletion of cholesterol increased gefitinib sensitivity. Furthermore, cholesterol‐depleted enhanced gefitinib inhibit phosphorylation of EGFR, Akt‐1, MEK1/2, and ERK1/2 and these were reversed in cholesterol add‐back experiments. Gefitinib resistant cell lines showed high affinity of gefitinib and EGFR when cholesterol was depleted. Therefore, targeting cholesterol combined with EGFR‐TKI is potentially a novel therapeutic strategy for gefitinib resistant treatment.  

3.3024           Lymphatic exosomes promote dendritic cell migration along guidance cues

Brown, M., Johnson, L.A., Leone, D.A., Majek, P., Vaahtomeri, K. et al

1. Cell Biol., 217(6), 2205-2221 (2018)

  Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified >1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments.  

3.3025           Localization of palmitoylated and activated G protein α‐subunit in Dictyostelium discoideum

Alamer, S., Kageyama, Y. and Gundersen, R.E.

1. Cell. Biochem., 119(6), 4975-4989 (2018)

  Guanine nucleotide‐binding proteins (G proteins) act as molecular switches to regulate many fundamental cellular processes. The lipid modification, palmitoylation, can be considered as a key factor for proper G protein function and plasma membrane localization. In Dictyostelium discoidum, Gα2 is essential for the chemotactic response to cAMP in their developmental life cycle. However, the regulation of Gα2 with respect to palmitoylation, activation and Gβγ association is less clear. In this study, Gα2 is shown to be palmitoylated on Cys‐4 by [³H]palmitate labeling. Loss of this palmitoylation site results in redistribution of Gα2 within the cell and poor D. discoideum development. Cellular re‐localization is also observed for activated Gα2. In the membrane fraction, Gα2‐wt (YFP) is highly enriched in a low‐density membrane fraction, which is palmitoylation‐dependent. Activated Gα2 monomer and heterotrimer are shifted to two different higher‐density fractions. These results broaden our understanding of how G protein localization and function are regulated inside the cells.  

3.3026           Extracellular Vesicles in Human Reproduction in Health and Disease

Simon, C., Greening, D.W., Bolumar, D., Balaguer, N., Salamonsen, L.A. and Vilella, F. Endocrine Reviews, 39(3), 292-332 (2018)   Extensive evidence suggests that the release of membrane-enclosed compartments, more commonly known as extracellular vesicles (EVs), is a potent newly identified mechanism of cell-to-cell communication both in normal physiology and in pathological conditions. This review presents evidence about the formation and release of different EVs, their definitive markers and cargo content in reproductive physiological processes, and their capacity to convey information between cells through the transfer of functional protein and genetic information to alter phenotype and function of recipient cells associated with reproductive biology. In the male reproductive tract, epididymosomes and prostasomes participate in regulating sperm motility activation, capacitation, and acrosome reaction. In the female reproductive tract, follicular fluid, oviduct/tube, and uterine cavity EVs are considered as vehicles to carry information during oocyte maturation, fertilization, and embryo–maternal crosstalk. EVs via their cargo might be also involved in the triggering, maintenance, and progression of reproductive- and obstetric-related pathologies such as endometriosis, polycystic ovarian syndrome, preeclampsia, gestational diabetes, and erectile dysfunction. In this review, we provide current knowledge on the present and future use of EVs not only as biomarkers, but also as therapeutic targeting agents, mainly as vectors for drug or compound delivery into target cells and tissues.  

3.3027           SIRT5 inhibits peroxisomal ACOX1 to prevent oxidative damage and is downregulated in liver cancer

Chen, X-F., Tian, M-X., Sun, R-Q., Zhang, M. et al EMBO Reports, 19, e5124 (2018)   Peroxisomes account for ~35% of total H₂O₂ generation in mammalian tissues. Peroxisomal ACOX1 (acyl‐CoA oxidase 1) is the first and rate‐limiting enzyme in fatty acid β‐oxidation and a major producer of H₂O₂. ACOX1 dysfunction is linked to peroxisomal disorders and hepatocarcinogenesis. Here, we show that the deacetylase sirtuin 5 (SIRT5) is present in peroxisomes and that ACOX1 is a physiological substrate of SIRT5. Mechanistically, SIRT5‐mediated desuccinylation inhibits ACOX1 activity by suppressing its active dimer formation in both cultured cells and mouse livers. Deletion of SIRT5 increases H₂O₂ production and oxidative DNA damage, which can be alleviated by ACOX1 knockdown. We show that SIRT5 downregulation is associated with increased succinylation and activity of ACOX1 and oxidative DNA damage response in hepatocellular carcinoma (HCC). Our study reveals a novel role of SIRT5 in inhibiting peroxisome‐induced oxidative stress, in liver protection, and in suppressing HCC development.  

3.3028           Survivin Monoclonal Antibodies Detect Survivin Cell Surface Expression and Inhibit Tumor Growth In Vivo

Fenstermaker, R.A., Figel, S.A., Qiu, J., Barone, T.A., Dharma, S.S., Winograd, E.K., Galbo, P.M., Wiltsie, L.M. and Ciesielski, M.J. Clin. Cancer Res., 24(11), 2642-2655 (2018)   Purpose: Survivin is an inhibitor of apoptosis protein (IAP) that is highly expressed in many cancers and represents an attractive molecule for targeted cancer therapy. Although primarily regarded as an intracellular protein with diverse actions, survivin has also been identified in association with circulating tumor exosomes. Experimental Design: We have reported that active, specific vaccination with a long peptide survivin immunogen leads to the development of survivin-specific CD8-mediated tumor cell lysis and prolongation of survival in tumor-bearing mice. In addition to cellular antitumor responses, circulating anti-survivin antibodies are detected in the serum of mice and human glioblastoma patients following vaccination with the survivin immunogen. Results: Here we demonstrate that survivin is present on the outer cell membrane of a wide variety of cancer cell types, including both murine and human glioma cells. In addition, antibodies to survivin that are derived from the immunogen display antitumor activity against murine GL261 gliomas in both flank and intracranial tumor models and against B16 melanoma as well. Conclusions: In addition to immunogen-induced, CD8-mediated tumor cell lysis, antibodies to the survivin immunogen have antitumor activity in vivo. Cell-surface survivin could provide a specific target for antibody-mediated tumor immunotherapeutic approaches.  

3.3029           Rotaviral nonstructural protein 4 triggers dynamin‐related protein 1‐dependent mitochondrial fragmentation during infection

Mukherjee, A., Patra, U., Bhowmick, R. and Chawla-Sarkar, M. Cell. Microbiol., 20(6), e12831 (2018)   Dynamic equilibrium between mitochondrial fission and mitochondrial fusion serves as an important quality control system within cells ensuring cellular vitality and homeostasis. Viruses often target mitochondrial dynamics as a part of their obligatory cellular reprogramming. The present study was undertaken to assess the status and regulation of mitochondrial dynamics during rotavirus infection. Distinct fragmentation of mitochondrial syncytia was observed during late hours of RV (SA11, Wa, A5‐13) infection. RV nonstructural protein 4 (NSP4) was identified as the viral trigger for disrupted mitochondrial morphology. Severance of mitochondrial interconnections was found to be a dynamin‐related protein 1 (Drp1)‐dependent process resulting synergistically from augmented mitochondrial fission and attenuated mitochondrial fusion. Cyclin‐dependent kinase 1 was subsequently identified as the cellular kinase responsible for fission‐active Ser616 phosphorylation of Drp1. In addition to its positive role in mitochondrial fission, Drp1 also resulted in mitochondrial translocation of E3‐ubiquitin ligase Parkin leading to degradation of mitochondrial fusion protein Mitofusin 1. Interestingly, RV‐NSP4 was found to interact with and be involved in recruiting fission‐active pool of Serine 616 phosphoDrp1 (Ser616 pDrp1) to mitochondria independent of accessory adaptors Mitochondrial fission factor and Fission protein 1 (Fis1). Inhibition of either Drp1 or Ser616 pDrp1 resulted in significant decrease in RV‐NSP4‐induced intrinsic apoptotic pathway. Overall, this study underscores an efficient strategy utilised by RV to couple apoptosis to mitochondrial fission facilitating dissemination of viral progeny.  

3.3030           Role of exosomes as a proinflammatory mediator in the development of EBV-associated lymphoma

Higuchi, H., Yamakawa, N., Imadome, K-I., Yahata, T., Kotaki, R. et al Blood, 131(23), 2552-2567 (2018)   Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin’s lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV⁺ B-cell lymphoma.  

3.3031           SNX14 mutations affect endoplasmic reticulum-associated neutral lipid metabolism in autosomal recessive spinocerebellar ataxia 20

Bryant, D., Liu, Y., Datta, S., Hariri, H., Seda, M., Anderson, G., Peskett, E., Demetriou, C., Sousa, S., Jenkins, D., Clayton, P., Bitner-Glindzicz, M., Moore, G.E., Henne, W.M. and Stanier, P. Hum. Mol. Genet., 27(11), 1927-1940 (2018)   Mutations in SNX14 cause the autosomal recessive cerebellar ataxia 20 (SCAR20). Mutations generally result in loss of protein although several coding region deletions have also been reported. Patient-derived fibroblasts show disrupted autophagy, but the precise function of SNX14 is unknown. The yeast homolog, Mdm1, functions in endoplasmic reticulum (ER)-lysosome/vacuole inter-organelle tethering, but functional conservation in mammals is still required. Here, we show that loss of SNX14 alters but does not block autophagic flux. In addition, we find that SNX14 is an ER-associated protein that functions in neutral lipid homeostasis and inter-organelle crosstalk. SNX14 requires its N-terminal transmembrane helices for ER localization, while the Phox homology (PX) domain is dispensable for subcellular localization. Both SNX14-mutant fibroblasts and SNX14^KO HEK293 cells accumulate aberrant cytoplasmic vacuoles, suggesting defects in endolysosomal homeostasis. However, ER-late endosome/lysosome contact sites are maintained in SNX14^KO cells, indicating that it is not a prerequisite for ER-endolysosomal tethering. Further investigation of SNX14^- deficiency indicates general defects in neutral lipid metabolism. SNX14^KO cells display distinct perinuclear accumulation of filipin in LAMP1-positive lysosomal structures indicating cholesterol accumulation. Consistent with this, SNX14^KO cells display a slight but detectable decrease in cholesterol ester levels, which is exacerbated with U18666A. Finally, SNX14 associates with ER-derived lipid droplets (LD) following oleate treatment, indicating a role in ER-LD crosstalk. We therefore identify an important role for SNX14 in neutral lipid homeostasis between the ER, lysosomes and LDs that may provide an early intervention target to alleviate the clinical symptoms of SCAR20.  

3.3032           Differential cell surface recruitment of the superoxide‐producing NADPH oxidases Nox1, Nox2 and Nox5: The role of the small GTPase Sar1

Kiyohara, T., Miyano, K., Kamakura, S., Hayase, J., Chishiki, K., Kohda, A. and Sumimoto, H. Genes Cells, 23, 480-493 (2018)   Transmembrane glycoproteins, synthesized at the endoplasmic reticulum (ER), generally reach the Golgi apparatus in COPII‐coated vesicles en route to the cell surface. Here, we show that the bona fide nonglycoprotein Nox5, a transmembrane superoxide‐producing NADPH oxidase, is transported to the cell surface in a manner resistant to co‐expression of Sar1 (H79G), a GTP‐fixed mutant of the small GTPase Sar1, which blocks COPII vesicle fission from the ER. In contrast, Sar1 (H79G) effectively inhibits ER‐to‐Golgi transport of glycoproteins including the Nox5‐related oxidase Nox2. The trafficking of Nox2, but not that of Nox5, is highly sensitive to over‐expression of syntaxin 5 (Stx5), a t‐SNARE required for COPII ER‐to‐Golgi transport. Thus, Nox2 and Nox5 mainly traffic via the Sar1/Stx5‐dependent and ‐independent pathways, respectively. Both participate in Nox1 trafficking, as Nox1 advances to the cell surface in two differentially N‐glycosylated forms, one complex and one high mannose, in a Sar1/Stx5‐dependent and ‐independent manner, respectively. Nox2 and Nox5 also can use both pathways: a glycosylation‐defective mutant Nox2 is weakly recruited to the plasma membrane in a less Sar1‐dependent manner; N‐glycosylated Nox5 mutants reach the cell surface in part as the complex form Sar1‐dependently, albeit mainly as the high‐mannose form in a Sar1‐independent manner.  

3.3033           New Technologies for Analysis of Extracellular Vesicles

Shao, H., Im, H., Castro, C.M., Breakefield, X., Weissleder, R. and Lee, H. Chem. Rev., 118(4), 1917-1950 (2018)   Extracellular vesicles (EVs) are diverse, nanoscale membrane vesicles actively released by cells. Similar-sized vesicles can be further classified (e.g., exosomes, microvesicles) based on their biogenesis, size, and biophysical properties. Although initially thought to be cellular debris, and thus under-appreciated, EVs are now increasingly recognized as important vehicles of intercellular communication and circulating biomarkers for disease diagnoses and prognosis. Despite their clinical potential, the lack of sensitive preparatory and analytical technologies for EVs poses a barrier to clinical translation. New analytical platforms including molecular ones are thus actively being developed to address these challenges. Recent advances in the field are expected to have far-reaching impact in both basic and translational studies. This article aims to present a comprehensive and critical overview of emerging analytical technologies for EV detection and their clinical applications.  

3.3034           β-Glucocerebrosidase Modulators Promote Dimerization of β-Glucocerebrosidase and Reveal an Allosteric Binding Site

Zheng, J., Chen, L., Skinner, O.S., Ysselstein, D., Remis, J., Lansbury, P., Skerlj, R., Mrosek, M., Heunisch, U., Krapp, S., Charrow, J., Schwake, M., Kelleher, N.L., Silverman, R.B. and Krainc, D.

1. Am. Chem. Soc., 140(18), 5914-5924 (2018)

  β-Glucocerebrosidase (GCase) mutations cause Gaucher’s disease and are a high risk factor in Parkinson’s disease. The implementation of a small molecule modulator is a strategy to restore proper folding and lysosome delivery of degradation-prone mutant GCase. Here, we present a potent quinazoline modulator, JZ-4109, which stabilizes wild-type and N370S mutant GCase and increases GCase abundance in patient-derived fibroblast cells. We then developed a covalent modification strategy using a lysine targeted inactivator (JZ-5029) for in vitro mechanistic studies. By using native top-down mass spectrometry, we located two potentially covalently modified lysines. We obtained the first crystal structure, at 2.2 Å resolution, of a GCase with a noniminosugar modulator covalently bound, and were able to identify the exact lysine residue modified (Lys346) and reveal an allosteric binding site. GCase dimerization was induced by our modulator binding, which was observed by native mass spectrometry, its crystal structure, and size exclusion chromatography with a multiangle light scattering detector. Finally, the dimer form was confirmed by negative staining transmission electron microscopy studies. Our newly discovered allosteric site and observed GCase dimerization provide a new mechanistic insight into GCase and its noniminosugar modulators and facilitate the rational design of novel GCase modulators for Gaucher’s disease and Parkinson’s disease.  

3.3035           Membrane microdomains regulate NLRP10- and NLRP12-dependent signalling in A549 cells challenged with cigarette smoke extract

Singh, D.P., Kaur, G., Bagam, P., Pinkston, R. and Batra, S. Arch. Toxicol., 92(5), 1767-1783 (2018)   Chronic obstructive pulmonary disease (COPD) is predicted to become the third leading cause of death and disability worldwide by 2030; with cigarette smoking (active or passive) being one of the chief cause of its occurrence. Cigarette smoke exposure has been found to result in excessive inflammation and tissue injury, which might lead to COPD, although the exact pathophysiology of the disease remains elusive. While previous studies have demonstrated the role of membrane-bound Toll-like receptors (TLRs) in cigarette smoke (CS)-induced inflammation, scant information is available about the role of cytosolic NOD-like receptors (NLRs) in regulating CS-mediated inflammatory responses. Thus, we investigated the role of NLRP10 and NLRP12 in regulating inflammatory responses in human alveolar type II epithelial cells (A549) and human monocytic cells (THP-1) in response to a challenge with cigarette smoke extract (CSE). We observed CSE-mediated increase in caspase-1 activity; production of IL-1β and IL-18; and expression of NLRP10 and NLRP12 in A549 and THP-1 cells. Interestingly, immunofluorescence imaging results demonstrated an increase in the membrane recruitment of NLRP10 and NLRP12 proteins in CSE-challenged A549 cells. We also observed an increase in the expression of lipid raft proteins (caveolin-1, caveolin-2, and flotillin-1) and an induction of lipid raft assembly following CSE-exposure in A549 cells. Lipid rafts are cholesterol-rich membrane microdomains well known to act as harbours for signalling molecules. Here we demonstrate  the recruitment of NLRP10 and NLRP12 in lipid raft entities as well as the interaction of NLRP12 with the lipid raft protein caveolin-1 in CSE-challenged A549 cells. Furthermore, enrichment of lipid raft entities with poly-unsaturated fatty acids (PUFA) rescued A549 cells from CSE-mediated membrane recruitment of NLRP10 and NLRP12, and also from inflammatory responses and inflammasome activation. Enrichment of membrane microdomains with PUFA was able to reverse filipin (chemical agent used for disrupting lipid rafts)-mediated enhanced inflammation in CSE-challenged A549 cells. Overall, our findings unveil a novel mechanism by identifying an important role of membrane microdomains (lipid rafts) in regulating CSE-induced inflammation and NLRP10/NLRP12-dependent signalling in A549 cells.  

3.3036           Cancer-cell-secreted exosomal miR-105 promotes tumour growth through the MYC-dependent metabolic reprogramming of stromal cells

Yan, W., Wu, X., Zhou, W., Fong, M.Y., Cao, M., Liu, J. et al Nature Cell Biol., 20, 597-609 (2018)   Cancer and other cells residing in the same niche engage various modes of interactions to synchronize and buffer the negative effects of environmental changes. Extracellular microRNAs (miRNAs) have recently been implicated in the intercellular crosstalk. Here we show a mechanistic model involving breast-cancer-secreted, extracellular-vesicle-encapsulated miR-105, which is induced by the oncoprotein MYC in cancer cells and, in turn, activates MYC signalling in cancer-associated fibroblasts (CAFs) to induce a metabolic program. This results in the capacity of CAFs to display different metabolic features in response to changes in the metabolic environment. When nutrients are sufficient, miR-105-reprogrammed CAFs enhance glucose and glutamine metabolism to fuel adjacent cancer cells. When nutrient levels are low and metabolic by-products accumulate, these CAFs detoxify metabolic wastes, including lactic acid and ammonium, by converting them into energy-rich metabolites. Thus, the miR-105-mediated metabolic reprogramming of stromal cells contributes to sustained tumour growth by conditioning the shared metabolic environment.  

3.3037           Polylysine is a Proteostasis Network-Engaging Structural Determinant

Lang, W-H., Calloni, G. and Vabulas, R.M.

1. Proteome Res., 17(5), 1967-1977 (2018)

  C-terminal polylysine (PL) can be synthesized from the polyadenine tail of prematurely cleaved mRNAs or when a read-though of a stop codon happens. Due to the highly positive charge, PL stalls in the electrostatically negative ribosomal exit channel. The stalled polypeptide recruits the Ribosome-associated quality control (RQC) complex which processes and extracts the nascent chain. Dysfunction of the RQC leads to the accumulation of PL-tagged proteins, induction of a stress response, and cellular toxicity. Not much is known about the PL-specific aspect of protein quality control. Using quantitative mass spectrometry, we uncovered the post-ribosomal PL-processing machinery in human cytosol. It encompasses key cytosolic complexes of the proteostasis network, such as chaperonin TCP-1 ring complexes (TRiC) and half-capped 19S-20S proteasomes. Furthermore, we found that the nuclear transport machinery associates with PL, which suggests a novel mechanism by which faulty proteins can be compartmentalized in the cell. The enhanced nuclear import of a PL-tagged polypeptide confirmed this implication, which leads to questions regarding the biological rationale behind it.  

3.3038           Mutant KRAS Exosomes Alter the Metabolic State of Recipient Colonic Epithelial Cells

Zhang, Q., Jeppesen, D.K., Higginbotham, J.N., Beckler, M.D: et al Cell. Mol. Gastroenterol. Hepatol., 5(4), 627-629.e6 (2018)   In colorectal cancer (CRC) cells, mutant Kirsten rat sarcoma (KRAS) cell-autonomously imparts Warburg-like¹ metabolic changes through induction of Glucose transporter 1 (GLUT-1) (SLC2A1).2, 3 We previously reported that mutant KRAS has marked effects on the constituents of CRC exosomes, including proteins and enzymes involved in metabolism and glycolysis.4, 5 The present studies were designed to test whether mutant KRAS exosomes can alter the metabolic state cell-nonautonomously in recipient colonic epithelial cells.  

3.3039           6 – Nucleic Acid Profiling in Tumor Exosomes

Trivedi, M.S. and Abreu, M. Diagnostic and Therapeutic Applications of Exosomes in Cancer, 93-117 (2018)   Exosomes are extracellular membrane vesicles with endocytic origin, and recent studies have demonstrated the spread of oncogenes by exosomes from tumor cells. Hence, the cargo content in the exosomes is of great significance. RNA (coding and noncoding) component of exosomes (evRNA) is of particular diagnostic interest because this provides a message to the neighboring cell, specific for a tumor type. Several methods can characterize such evRNA-based message from tumor cells, and here we discuss a brief overview of such techniques including next-generation sequencing and compare/contrast them for their applications. We discuss some of the caveats and artifacts associated with such techniques including library preparation and present some of ways to overcome such challenges. Lastly, we present some of the potential aspects for the field of evRNA characterization especially at the single EV level. Our main goal is to allow the user for informed selection of a tool for their specific application.  

3.3040           Chapter 11 – Extracellular vesicles as a recipe for design smart drug delivery systems for cancer therapy

Halder, C.V.F., Fonseca, E.M.B., de S. Faria, A.V. and Cierici, S.P. Drug Targeting and Stimuli Sensitive Drug Delivery Systems, 411-455 (2018)   Extracellular vesicles (EVs) are secreted by most cell types and appear ubiquitously in cell culture supernatants and body fluids. EVs are efficient mediators of cell-cell communication, once the key molecules players (e.g., proteins, lipids, RNA, and DNA) are cargoes of EVs and transferred to recipient cells. Based on that, and considering the ability of EVs to cross natural barriers (such as membranes), their inherent cell targeting properties, and stability in the circulation, the utilization of EVs as drug delivery system has gained scientific interest. For instance, EVs have been engineered to incorporate therapeutic miRNAs, siRNAs, or chemotherapeutic agents. Furthermore, examples of protein delivery by exosomes for therapeutic benefit in preclinical models include catalase, prodrug activating enzymes, caspase-1. Despite the potential of EVs as drug delivery, there is still a long way to reach the clinical application. This chapter will focus on the recent progress and challenges in the engineering of exosomes as drug delivery vehicles.  

3.3041           Flaviviruses Exploit the Lipid Droplet Protein AUP1 to Trigger Lipophagy and Drive Virus Production

Zhang, J., Lan, Y., Li, M.Y., Lamers, M.M., Fusade-Boyer, M., Klem, E., Thiele, C., Ashour, J. and Sanyal, S. Cell Host & Microbe, 23(6), 819-831 (2018)   Ubiquitylation is one of the most versatile protein post-translational modifications and is frequently altered during virus infections. Here we employed a functional proteomics screen to identify host proteins that are differentially ubiquitylated upon dengue virus (DENV) infection. Among the several differentially modified proteins identified in infected cells was AUP1, a lipid droplet-localized type-III membrane protein, which exists predominantly in the mono-ubiquitylated form. AUP1 associated with DENV NS4A and relocalized from lipid droplets to autophagosomes upon infection. Virus production was abolished in cells deleted for AUP1 or expressing an AUP1 acyltransferase domain mutant. Ubiquitylation disrupted the AUP1-NS4A interaction, resulting in inhibited acyltransferase activity, defective lipophagy, and attenuated virus production. Our results show that DENV-NS4A exploits the acyltransferase activity of AUP1 to trigger lipophagy, a process regulated by ubiquitylation. This mechanism appears to be a general phenomenon in biogenesis of flaviviruses and underscores the critical role of post-translational modifications in virus infections.  

3.3042           FLIM reveals alternative EV-mediated cellular up-take pathways of paclitaxel

Saari, H., Lisitsyna, E., Rautaniemi, K., Rojalin, T., Niemi, l., Nivaro, o., Laaksonen, T., Yliperttula, M. and Vuorimaa-Laukkanen, E.

1. Controlled release, 284, 133-143 (2018)

  In response to physiological and artificial stimuli, cells generate nano-scale extracellular vesicles (EVs) by encapsulating biomolecules in plasma membrane-derived phospholipid envelopes. These vesicles are released to bodily fluids, hence acting as powerful endogenous mediators in intercellular signaling. EVs provide a compelling alternative for biomarker discovery and targeted drug delivery, but their kinetics and dynamics while interacting with living cells are poorly understood. Here we introduce a novel method, fluorescence lifetime imaging microscopy (FLIM) to investigate these interaction attributes. By FLIM, we show distinct cellular uptake mechanisms of different EV subtypes, exosomes and microvesicles, loaded with anti-cancer agent, paclitaxel. We demonstrate differences in intracellular behavior and drug release profiles of paclitaxel-containing EVs. Exosomes seem to deliver the drug mostly by endocytosis while microvesicles enter the cells by both endocytosis and fusion with cell membrane. This research offers a new real-time method to investigate EV kinetics with living cells, and it is a potential advancement to complement the existing techniques. The findings of this study improve the current knowledge in exploiting EVs as next-generation targeted drug delivery systems.  

3.3043           Cell-type-specific brain methylomes profiled via ultralow-input microfluidics

Ma, S., de la Fuenta Revenga, M., Sun, Z., Sun, C., Murphy, T.W., Xie, H., Gonzalez-Maeso, J. and Lu, C. Nature Biomed. Engineering, 2, 183-194 (2018)   Methylomic analyses typically require substantial amounts of DNA, thus hindering studies involving scarce samples. Here, we show that microfluidic diffusion-based reduced representation bisulfite sequencing (MID-RRBS) permits high-quality methylomic profiling with nanogram-to-single-cell quantities of starting DNA. We used the microfluidic device, which allows for efficient bisulfite conversion with high DNA recovery, to analyse genome-wide DNA methylation in cell nuclei isolated from mouse brains and sorted into NeuN+ (primarily neuronal) and NeuN− (primarily glial) fractions, and to establish cell-type-specific methylomes. Genome-wide methylation and methylation in low-CpG-density promoter regions showed distinct patterns for NeuN+ and NeuN− fractions from the mouse cerebellum. The identification of substantial variations in the methylomic landscapes of the NeuN+ fraction of the frontal cortex of mice chronically treated with an atypical antipsychotic drug suggests that this technology can be broadly used for cell-type-specific drug profiling and for the study of drug–methylome interactions.  

3.3044           Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins

Karimi, N., Cvjetkovic, A., Jang, S.C., Cresitelli, R., Feizi, M.A.H., Nieuwland, R., Lötvall, J. and Lässer, C. Cell. Mol. Life Sci., 75, 2873-2856 (2018)   The isolation of extracellular vesicles (EVs) from blood is of great importance to understand the biological role of circulating EVs and to develop EVs as biomarkers of disease. Due to the concurrent presence of lipoprotein particles, however, blood is one of the most difficult body fluids to isolate EVs from. The aim of this study was to develop a robust method to isolate and characterise EVs from blood with minimal contamination by plasma proteins and lipoprotein particles. Plasma and serum were collected from healthy subjects, and EVs were isolated by size-exclusion chromatography (SEC), with most particles being present in fractions 8–12, while the bulk of the plasma proteins was present in fractions 11–28. Vesicle markers peaked in fractions 7–11; however, the same fractions also contained lipoprotein particles. The purity of EVs was improved by combining a density cushion with SEC to further separate lipoprotein particles from the vesicles, which reduced the contamination of lipoprotein particles by 100-fold. Using this novel isolation procedure, a total of 1187 proteins were identified in plasma EVs by mass spectrometry, of which several proteins are known as EV-associated proteins but have hitherto not been identified in the previous proteomic studies of plasma EVs. This study shows that SEC alone is unable to completely separate plasma EVs from lipoprotein particles. However, combining SEC with a density cushion significantly improved the separation of EVs from lipoproteins and allowed for a detailed analysis of the proteome of plasma EVs, thus making blood a viable source for EV biomarker discovery.  

3.3045           Salivary exosomes as potential biomarkers in cancer

Nair, S., Tang, K.D., Kenny, L. and Punyadeera, C. Oral Oncol., 84, 31-40 (2018)   Over the past decade, there has been emerging research in the field of extracellular vesicles, especially those originating from endosomes, referred to as ‘exosomes. Exosomes are membrane-bound nanovesicles secreted by most cell types upon fusion of multivesicular bodies (MVBs) to the cell plasma membrane. These vesicles are present in almost all body fluids such as blood, urine, saliva, breast milk, cerebrospinal and peritoneal fluids. Exosomes participate in intercellular communication by transferring the biologically active molecules like proteins, nucleic acids, and lipids to neighboring cells. Exosomes are enriched in the tumour microenvironment and growing evidence demonstrates that exosomes mediate cancer progression and metastasis. Given the important biological role played by these nanovesicles in cancer pathogenesis, these can be used as ideal non-invasive biomarkers in detecting and monitoring tumours as well as therapeutic targets. The scope of the current review is to provide an overview of exosomes with a special focus on salivary exosomes as potential biomarkers in head and neck cancers.  

3.3046           Improved molecular karyotyping in glioblastoma

Burbulis, I.E., Wierman, M.B., Wolpert, M., Haakenson, M., Lopes, M-B., Schiff, D., Hicks, j., Loe, j., Ratan, A. and McConnell, M.J. Mutat. Res. Fund. Mol. Mech. Mutagen, 811, 16-26 (2018)   Uneven replication creates artifacts during whole genome amplification (WGA) that confound molecular karyotype assignment in single cells. Here, we present an improved WGA recipe that increased coverage and detection of copy number variants (CNVs) in single cells. We examined serial resections of glioblastoma (GBM) tumor from the same patient and found low-abundance clones containing CNVs in clinically relevant loci that were not observable using bulk DNA sequencing. We discovered extensive genomic variability in this class of tumor and provide a practical approach for investigating somatic mosaicism.  

3.3047           The basic route of the nuclear translocation porcine growth hormone (GH)-growth hormone receptor (GHR) complex (pGH/GHR) in porcine hepatocytes

Hainan, L., Huilin, L., Khan, M., Xin, Z., YuJiang, Y., Hui, Z. and Naiquan, Y. General and Comparative Endocrinol., 266, 101-109 (2018)   Traditional views suggest that growth hormone and the growth hormone receptor (GH/GHR complex) exert their functions only on the plasma membrane. This paradigm, however, has been challenged by recent new findings that the GH/GHR complex could translocate into cell nuclei where they could still exhibit important physiological functions. We also reported the nuclear localization of porcine GH/GHR and their potential functions in porcine hepatocytes. However, the basic path of pGH/GHR’s nuclear translocation remains unclear. Combining previous research results and our current findings, we proposed two basic routes of pGH/GHR’s nuclear transportation as follows: 1) after pGH binding to GHR, pGH/GHR enters into the cytoplasm though clathrin- or caveolin-mediated endocytosis, then the pGH/GHR complex enters into early endosomes (Rab5-positive), and the endosome carries the GH/GHR complex to the endoplasmic reticulum (ER). After endosome docking on the ER, the endosome starts fission, and the pGH/GHR complex enters into the ER lumen. Then the pGH/GHR complex transports into the cytoplasm, possibly by the ERAD pathway. Subsequently, the pGH/GHR complex interacts with IMPα/β, which, in turn, mediates GH/GHR nuclear localization; 2) pGH binds with the GHR on the cell membrane and, subsequently, pGH/GHR internalizes into the cell and enters into the endosome (this endosome may belong to a class of endosomes called envelope-associated endosomes (NAE)). Then, the endosome carries the pGH/GHR to the nuclear membrane. After docking on the nuclear membrane, the pGH/GHR complex fuses with the nuclear membrane and then enters into the cell nucleus.  

3.3048           An optimized method for enrichment of whole brain-derived extracellular vesicles reveals insight into neurodegenerative processes in a mouse model of Alzheimer’s disease

Hurwitz, S.N., Sun, L., Cole, K.Y., Ford III, C.R., Olcese, J.M. and Meckes, Jr., D.G.

1. Neurosci. Methods, 307, 210-220 (2018)

  Background Alzheimer’s disease (AD) is the major cause of dementia that has increased dramatically in prevalence over the past several decades. Yet many questions still surround the etiology of AD. Recently, extracellular vesicles (EVs) that transport protein, lipid, and nucleic acids from cell to cell have been implicated in the clearance and propagation of misfolded proteins. Investigation of EVs in AD progression, and their potential diagnostic utility may contribute to understanding and treating AD. However, the challenges of isolating brain-derived EVs have in part hindered these studies. New method Here, we provide an optimized method for the enrichment of brain-derived EVs by iodixanol floatation density gradient for mass spectrometry analysis. Results We demonstrate the isolation of these vesicles and the enrichment of EV proteins compared to sedimentation gradient isolation of vesicles. Moreover, comparative proteomic analysis of brain-derived EVs from healthy and AD mouse brains revealed differences in vesicular content including proteins involved in aging, immune response, and oxidation-reduction maintenance. These changes provide insight into AD-associated neurodegeneration and potential biomarkers of AD. Comparison with existing methods: Recent techniques have used sedimentation sucrose gradients to isolate EVs from brain tissue. However, here we demonstrate the advantages of floatation iodixanol density gradient isolation of small EVs, and provide evidence of EV enrichment by electron microscopy, immunoblot analysis, and quantitative mass spectrometry. Conclusions Together these findings offer a rigorous technique for enriching whole tissue-derived EVs for downstream analyses, and application of this approach to uncovering molecular changes in AD progression and other neurological conditions.  

3.3049           Maturation of polycistronic mRNAs by the endoribonuclease RNase Y and its associated Y-complex in Bacillus subtilis

DeLoughery, A., Lelanne, J-B., Losick, R. and Li, G-W. PNAS, 115(24), E5585-E5594 (2018)   Endonucleolytic cleavage within polycistronic mRNAs can lead to differential stability, and thus discordant abundance, among cotranscribed genes. RNase Y, the major endonuclease for mRNA decay in Bacillus subtilis, was originally identified for its cleavage activity toward the cggR-gapA operon, an event that differentiates the synthesis of a glycolytic enzyme from its transcriptional regulator. A three-protein Y-complex (YlbF, YmcA, and YaaT) was recently identified as also being required for this cleavage in vivo, raising the possibility that it is an accessory factor acting to regulate RNase Y. However, whether the Y-complex is broadly required for RNase Y activity is unknown. Here, we used end-enrichment RNA sequencing (Rend-seq) to globally identify operon mRNAs that undergo maturation posttranscriptionally by RNase Y and the Y-complex. We found that the Y-complex is required for the majority of RNase Y-mediated mRNA maturation events and also affects riboswitch abundance in B. subtilis. In contrast, noncoding RNA maturation by RNase Y often does not require the Y-complex. Furthermore, deletion of RNase Y has more pleiotropic effects on the transcriptome and cell growth than deletions of the Y-complex. We propose that the Y-complex is a specificity factor for RNase Y, with evidence that its role is conserved in Staphylococcus aureus.  

3.3050           Arthropod EVs mediate dengue virus transmission through interaction with a tetraspanin domain containing glycoprotein Tsp29Fb

Vora, A., Zhou, W., Londono-Renteria, B., Woodson, M., Sherman, M.B., Colpitts, T.M., Neelkanta, G. and Sultana, H. PNAS, 115(28), E6604-E6613 (2018)   Dengue virus (DENV) is a mosquito-borne flavivirus that causes dengue fever in humans, worldwide. Using in vitro cell lines derived from Aedes albopictus and Aedes aegypti, the primary vectors of DENV, we report that DENV2/DENV3-infected cells secrete extracellular vesicles (EVs), including exosomes, containing infectious viral RNA and proteins. A full-length DENV2 genome, detected in arthropod EVs, was infectious to naïve mosquito and mammalian cells, including human-skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed mosquito EVs with a size range from 30 to 250 nm. Treatments with RNase A, Triton X-100, and 4G2 antibody-bead binding assays showed that infectious DENV2-RNA and proteins are contained inside EVs. Viral plaque formation and dilution assays also showed securely contained infectious viral RNA and proteins in EVs are transmitted to human cells. Up-regulated HSP70 upon DENV2 infection showed no role in viral replication and transmission through EVs. In addition, qRT-PCR and immunoblotting results revealed that DENV2 up-regulates expression of a mosquito tetraspanin-domain–containing glycoprotein, designated as Tsp29Fb, in A. aegypti mosquitoes, cells, and EVs. RNAi-mediated silencing and antibody blocking of Tsp29Fb resulted in reduced DENV2 loads in both mosquito cells and EVs. Immunoprecipitation showed Tsp29Fb to directly interact with DENV2 E-protein. Furthermore, treatment with GW4869 (exosome-release inhibitor) affected viral burden, direct interaction of Tsp29Fb with E-protein and EV-mediated transmission of viral RNA and proteins to naïve human cells. In summary, we report a very important finding on EV-mediated transmission of DENV2 from arthropod to mammalian cells through interactions with an arthropod EVs-enriched marker Tsp29Fb.  

3.3051           Determining AMPK Activation via the Lysosomal v-ATPase-Ragulator-AXIN/LKB1 Axis

Zhang, C-S., Li, M., Zong, Y. and Lin, S-C. Methods in Mol. Biol., 1732, 393-411 82018)   Recent studies have revealed how AMPK is activated inside the cell and animal tissues: in response to low glucose, AXIN tethers LKB1, by virtue of their constitutive association, to AMPK located on the surface of late endosome/lysosome. Importantly, the lysosomal v-ATPase (vacuolar ATPase)-Ragulator complex, when primed by glucose starvation or concanamycin A, facilitates AXIN/LKB1 to interact with AMPK. Here, we describe the experimental procedures of the assays for detecting the translocation of AXIN/LKB1 or the assembly of the AXIN-based AMPK-activating complexes on the late endosome/lysosome. The methods in this chapter will be useful for determining whether various metabolic stresses or pharmacological stimuli activate AMPK via the v-ATPase-Ragulator-AXIN/LKB1 axis, which also concomitantly inactivates mTORC1. Detailed protocols for determining the levels of adenylates are also described.  

3.3052           Extraction and Analysis of RNA Isolated from Pure Bacteria-Derived Outer Membrane Vesicles

Habier, J., May, P., Heintz-Buschart, A., Ghosal, A., Wienecke-Baldacchino, A.K., Nolte-‘tHoen, E.N.M., Wilmes, P. and Fritz, J.V. Methods in Mol. Biol., 1737, 213-230 (2018)   Outer membrane vesicles (OMVs) are released by commensal as well as pathogenic Gram-negative bacteria. These vesicles contain numerous bacterial components, such as proteins, peptidoglycans, lipopolysaccharides, DNA, and RNA. To examine if OMV-associated RNA molecules are bacterial degradation products and/or are functionally active, it is necessary to extract RNA from pure OMVs for subsequent analysis. Therefore, we describe here an isolation method of ultrapure OMVs and the subsequent extraction of RNA and basic steps of RNA-Seq analysis. Bacterial culture, extracellular supernatant concentration, OMV purification, and the subsequent RNA extraction out of OMVs are described. Specific pitfalls within the protocol and RNA contamination sources are highlighted.  

3.3053           Experimental Approaches for Defining the Role of the Ca2+-Modulated ROS-GC System in Retinal Rods of Mouse

Makino, C.L., Duda, T., Pertzev, A. and Sharma, R.K. Methods in Mol. Biol., 1753, 129-158 (2018)   Our ability to see is based on the activity of retinal rod and cone photoreceptors. Rods function when there is very little light, while cones operate at higher light levels. Photon absorption by rhodopsin activates a biochemical cascade that converts photic energy into a change in the membrane potential of the cell by decreasing the levels of a second messenger, cGMP, that control the gating of cation channels. But just as important as the activation of the cascade are the shut-off and recovery processes. The timing of shutoff and recovery ultimately affects sensitivity, temporal resolution and even the capacity for counting single photons. An important part of the recovery is restoration of cGMP through the action of rod outer segment membrane guanylate cyclases (ROS-GCs) and guanylate cyclase-activating proteins (GCAPs). In darkness, ROS-GCs catalyze the conversion of GTP to cGMP at a low rate, due to inhibition of cyclase activity by GCAPs. In the light, GCAP enhances ROS-GC activity. Mutations in the ROS-GC system can cause problems in vision, and even result in blindness due to photoreceptor death. The mouse has emerged as a particularly useful subject to study the role of ROS-GC because the technology for the manipulation of their genetics is advanced, making production of mice with targeted mutations much easier. Here we describe some experimental procedures for studying the retinal rods of wild-type and genetically engineered mice: biochemical assays of ROS-GC activity, immunohistochemistry, and single cell recording.  

3.3054           Cell Culture Analysis of the Phagocytosis of Photoreceptor Outer Segments by Primary Mouse RPE Cells

Hazim, R.A. and Williams, D.S. Methods in Mol. Biol., 1753, 63-71 (2018)   The phagocytosis of photoreceptor outer segments (POSs) by the retinal pigment epithelium (RPE) is essential for retinal homeostasis. Defects in this process can be caused by mutations in the photoreceptor cells, the RPE cells, or both cell types. This function can be experimentally investigated by performing an in vitro phagocytosis assay, in which cultured RPE cells are challenged with isolated POSs, and subsequently tested for their ability to degrade the POSs. A significant advantage of this approach is that mutant phenotypes can be attributed either to the photoreceptor or the RPE cells, by experimenting with different permutations of mutant and control photoreceptor and RPE cells. In this chapter, we detail the method for a double-immunofluorescence assay for analysis of the binding, ingestion, and subsequent degradation of isolated mouse POSs by cultured mouse primary RPE cells.  

3.3055           Isolation of Outer Membrane Vesicles Including Their Quantitative and Qualitative Analyses

Kohl, P., Zingl, F.G., Eichmann, T.O. and Schild, S. Methods in Mol. Biol., 1839, 117-134 (2018)   Outer membrane vesicles (OMVs) are naturally secreted from the bacterial cell surface and therefore localized in the cell-free supernatant of bacterial cultures. Here we describe methods for crude and density gradient-purified OMV isolation and protocols for control analyses for protein profiling (SDS-PAGE), detection of indicator proteins (immunoblot analysis), lipid profiling (lipid extraction and LC-MS analysis), vesicle size determination (NanoSight), rough estimation of biomass (TrayCell™), as well as quantifications of defined OMV components, e.g., proteins (Bradford) and LPS (Purpald).  

3.3056           Internalization of NKCC2 is impaired in thick ascending limb of Henle in moesin knockout mice

Kawaguchi, K., Hatano, R., Matsubara, m. and Asano, S. Eur. J. Physiol., 470, 1055-1068 (2018)   Moesin is expressed in several types of cells including epithelial and endothelial cells. Several groups reported that moesin plays important roles in the regulation of the cellular motility, and the process of internalization of membrane proteins. However, the physiological roles of moesin in the kidney still remain unclear. Herein, we examined the physiological function of moesin in the kidney using moesin knockout (Msn ^−/y ) mice. There was no obvious abnormality in the renal morphology of Msn ^−/y mice. However, we found that Msn ^−/y mice exhibited mild hyperchloremia, and reduced glomerular filtration rate compared to wild type (WT) mice. Absolute electrolytes excretions of NaCl in Msn ^−/y mice were not significantly changed compared to WT mice. In the renal medulla, moesin was detected in thick ascending limb of Henle (TALH) as previously reported. To determine the physiological function of moesin in TALH, we examined the expression and subcellular localization of NKCC2 in Msn ^−/y mice. Interestingly, apical surface expression level, but not total expression of NKCC2 was increased in Msn ^−/y mice. Subcellular fractionation of renal medulla lysate and internalization assay using tubular suspension showed that the process of NKCC2 endocytosis is impaired. Since the distribution of NKCC2 in lipid raft fractions was decreased in Msn ^−/y mice, moesin may regulate the NKCC2 distribution to microdomain. These results suggest that moesin regulates the internalization of NKCC2. Furthermore, euhydration by water loading caused hyponatremina in Msn ^−/y mice, suggesting that dysfunction of moesin is associated with the nephrogenic syndrome of inappropriate antidiuresis (NSIAD).  

3.3057           Epigenetic regulation of brain region-specific microglia clearance activity

Ayata, P., Badimon, A., Strasburger, H.J., Duff, M.K. et al Nature Neurosci., 21, 1049-1060 (2018)   The rapid elimination of dying neurons and nonfunctional synapses in the brain is carried out by microglia, the resident myeloid cells of the brain. Here we show that microglia clearance activity in the adult brain is regionally regulated and depends on the rate of neuronal attrition. Cerebellar, but not striatal or cortical, microglia exhibited high levels of basal clearance activity, which correlated with an elevated degree of cerebellar neuronal attrition. Exposing forebrain microglia to apoptotic cells activated gene-expression programs supporting clearance activity. We provide evidence that the polycomb repressive complex 2 (PRC2) epigenetically restricts the expression of genes that support clearance activity in striatal and cortical microglia. Loss of PRC2 leads to aberrant activation of a microglia clearance phenotype, which triggers changes in neuronal morphology and behavior. Our data highlight a key role of epigenetic mechanisms in preventing microglia-induced neuronal alterations that are frequently associated with neurodegenerative and psychiatric diseases.  

3.3058           Neuropilin-1 and platelet-derived growth factor receptors cooperatively regulate intermediate filaments and mesenchymal cell migration during alveolar septation

McGowan, S.E. and McCoy, D.M. Am. J. Physiol. Lung Cell. Mol. Physiol, 315, L102-L115 (2018)   Generation of secondary alveolar septa occurs primarily after birth in humans and is complete in mice postnatally, when mechanical stresses vary as air space pressure oscillates. Alveolar mesenchymal cells deposit elastic fibers, which limit cell strain; although when the elastic fiber network is incomplete, this function is also served by the intracellular cytoskeleton. Intermediate filament proteins support deformation during cell division and migration, which occur during septal elongation. Because platelet-derived growth factor receptor-α (PDGFRα) signaling is essential for alveolar septation, we hypothesized that neuropilin-1 (NRP1) may link PDGFRα to cytoskeletal deformation. During cell migration, NRP1 links receptor tyrosine kinase signaling to cytoskeletal and focal adhesion remodeling. Therefore, we examined the consequences of nrp1 gene deletion in alveolar mesenchymal cells (myofibroblasts and pericytes). NRP1 depletion reduced the proportion of mesenchymal cells that contain nestin and desmin within the subpopulation that lacked PDGFRα but contained PDGFRβ. Desmin was reduced at alveolar entry rings, air spaces were enlarged, and surface area was reduced after NRP1 depletion. PDGFRα and NRP1 colocalized to membrane lipid rafts, which are known to contain Src kinase. NRP1 depletion reduced alveolar mesenchymal cell migration and PDGF-A-mediated activation of Src kinase, which may limit accumulation of desmin at septal tips (alveolar entry rings). Cooperation between NRP1 and PDGF signaling is required for secondary septation, and manipulation of NRP1 could promote alveolar regeneration without producing fibrosis.  

3.3059           Extracellular vesicles and anti-cancer drug resistance

Mc Namee, N. and O’Driscoll, L.

BBA – Reviews on Cancer, 1870, 123-136 (2018)

Extracellular vesicles (EVs) including exosomes, microvesicles, oncosomes, and microparticles have been associated with communicating anti-cancer drug-resistance. The in vitro, pre-clinical in vivo and patients' data linking EVs to drug-resistance (and the specific drugs involved) in breast cancer, prostate cancer, lung cancer, ovarian cancer, haematological malignancies, colorectal cancer, gastric cancer, pancreatic cancer, glioblastoma, neuroblastoma, melanoma, kidney cancer and osteosarcoma. Details of the mechanisms by which the resistance seems to be occurring (e.g. EVs transferring drug-efflux pumps from drug-resistant cancer cells, EVs binding monoclonal antibodies in the peripheral circulation and so reducing their bioavailability, EVs from tumour microenvironment cells, etc.) are outlined, as are efforts to try to block such resistance. Research to date strongly supports EVs as playing a key role in drug-resistance. Further studies including tailored clinical studies are now warranted to determine how best to prevent this occurring, in the interest of patients and also for economic benefit. Furthermore, efforts to exploit safe (non-cancer origin) EVs as anti-cancer drug delivery vehicles that may achieve efficacy with more limited side-effects than free drug, deserve further investigation.

 

3.3060           Exosomal αvβ6 integrin is required for monocyte M2 polarization in prostate cancer

Lu, H., Bowler, N., Harshyne, L.A., Hooper, D.C., Krishn, S.R. et al Matrix Biol., 70, 20-35 (2018)   Therapeutic approaches aimed at curing prostate cancer are only partially successful given the occurrence of highly metastatic resistant phenotypes that frequently develop in response to therapies. Recently, we have described αvβ6, a surface receptor of the integrin family as a novel therapeutic target for prostate cancer; this epithelial-specific molecule is an ideal target since, unlike other integrins, it is found in different types of cancer but not in normal tissues. We describe a novel αvβ6-mediated signaling pathway that has profound effects on the microenvironment. We show that αvβ6 is transferred from cancer cells to monocytes, including β6-null monocytes, by exosomes and that monocytes from prostate cancer patients, but not from healthy volunteers, express αvβ6. Cancer cell exosomes, purified via density gradients, promote M2 polarization, whereas αvβ6 down-regulation in exosomes inhibits M2 polarization in recipient monocytes. Also, as evaluated by our proteomic analysis, αvβ6 down-regulation causes a significant increase in donor cancer cells, and their exosomes, of two molecules that have a tumor suppressive role, STAT1 and MX1/2. Finally, using the Pten^pc−/− prostate cancer mouse model, which carries a prostate epithelial-specific Pten deletion, we demonstrate that αvβ6 inhibition in vivo causes up-regulation of STAT1 in cancer cells. Our results provide evidence of a novel mechanism that regulates M2 polarization and prostate cancer progression through transfer of αvβ6 from cancer cells to monocytes through exosomes.  

3.3061           Nucleic acid loading and fluorescent labeling of isolated extracellular vesicles requires adequate purification

Stremersch, S., Brans, T., Braeckmans, K., De Smedt, S. and Raemdonck, K. Int. J. Pharmaceutics, 548, 783-792 (2018)   Extracellular vesicles (EVs) are nanosized vesicular structures released by cells to communicate with one another. The growing interest in the (patho)physiological function and potential pharmaceutical application of these vesicles is accompanied by a vast number of new research groups entering this research field and a plethora of different protocols to separate EVs from non-vesicular components. This lack of uniformity often generates conflicting or difficult-to-compare results. Here we provide a comparative analysis of different EV isolation strategies, discussing the purity of the final isolate and highlighting the importance of purity on downstream experimental readouts. First, we show that ultracentrifugation (UC) of B16F10 melanoma cell-derived conditioned medium co-purifies proteins or protein complexes with nuclease activity. Such contaminants should be taken into account when aiming to apply EVs as delivery carriers for exogenous nucleic acids. Second, three commonly used purification strategies (i.e. precipitation, UC and density-gradient centrifugation) were evaluated for their ability to remove non-incorporated fluorescent dye (i.e. the lipophilic PKH67 dye), important when probing EV interactions with cells. For both types of impurities, endogenous and exogenous, density gradient purification outperforms the other evaluated methods. Overall, these results demonstrate that the implementation of stringent purification protocols and adequate controls is of pivotal importance to draw reliable conclusions from downstream experiments performed with EV isolates.  

3.3062           α-Tocopherol transfer protein does not regulate the cellular uptake and intracellular distribution of α- and γ-tocopherols and -tocotrienols in cultured liver cells

Irias-Mata, A., Sus, N., Flory, S., Stock, D., Woerner, D., Podszun, M. and Frank, J. Redox Biology, 19, 28-36 (2018)   Liver cells express a cytosolic α-tocopherol transfer protein (αTTP) with high binding affinity for α-tocopherol (αT) and much lower affinities for the non-αT congeners. The role of αTTP in the intracellular distribution of the different vitamin E forms is currently unknown. We therefore investigated the intracellular localization of αT, γ-tocopherol (γT), α-tocotrienol (αT3), and γ-tocotrienol (γT3) in cultured hepatic cells with and without stable expression of αTTP. We first determined cellular uptake of the four congeners and found the methylation of the chromanol ring and saturation of the sidechain to be important factors, with tocotrienols being taken up more efficiently than tocopherols and the γ-congeners more than the α-congeners, irrespective of the expression of αTTP. This, however, could perhaps also be due to an observed higher stability of tocotrienols, compared to tocopherols, in culture media rather than a higher absorption. We then incubated HepG2 cells and αTTP-expressing HepG2 cells with αT, γT, αT3, or γT3, isolated organelle fractions by density gradient centrifugation, and determined the concentrations of the congeners in the subcellular fractions. All four congeners were primarily associated with the lysosomes, endoplasmic reticulum, and plasma membrane, whereas only αT correlated with mitochondria. Neither the chromanol ring methylation or sidechain saturation, nor the expression of αTTP were important factors for the intracellular distribution of vitamin E. In conclusion, αTTP does not appear to regulate the uptake and intracellular localization of different vitamin E congeners in cultured liver cells.  

3.3063           Characterization and applications of extracellular vesicle proteome with post-translational modifications

Zhang, Y., Wu, X. and Tao, W.A. Trends in Anal. Chem., 107, 21-30 (2018)   Extracellular vesicles (EVs) are a diverse population of complex membrane-encapsulated vesicles released by a variety of cell types and exist in most of body fluids. Continuously growing number of reports revealed that EVs participate in multiple biological processes, such as intercellular communication, immune regulation, and dissemination of cancer cells. Accordingly, recent attention has been given to the characterization of extracellular vesicles and their components. This review focuses on state-of-the-art proteomic technologies to analyze proteomes of EVs, especially their post-translational modifications (PTMs). With their strong biological relevance and the relatively noninvasive accessibility from body fluids, the promising potential and early applications of EV proteome and its PTMs as attracting biomarker sources are also evaluated.  

3.3064           Exosomes in cancer: Small transporters with big functions

Li, X., Wang, Y., Wang, Qi., Liu, Y., Bao, W. and Wu, S. Cancer Letters, 435, 55-65 (2018)   Exosomes are nanosized membrane-bound vesicles containing abundant proteins, DNA, mRNA, and non-coding RNAs. Exosomes are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material. Increasing studies have shown that exosomes play an important role in tumour initiation, growth, progression, metastasis, drug resistance and immune escape. In this article, we review recent advances in the biology of exosomes. We elaborate the specific mechanism by which exosomes affect the communication between tumours and the microenvironment. Finally, we report that exosomes may provide promising biomarkers for cancer diagnosis and represent new targets for cancer therapy.  

3.3065           TMEM30A is a candidate interacting partner for the β-carboxyl-terminal fragment of amyloid-β precursor protein in endosomes

Takasugi, N., Araya, R., Kamikubo, Y., Kaneshiro, N., Imaoka, R., Jin, H., Kashiyama, T., Hashimoto, Y., Kurosawa, M., Uehara, T., Nukina, N. and Sakurai, T. PloS One, 13(8), e0200988 (2018)   Although the aggregation of amyloid-β peptide (Aβ) clearly plays a central role in the pathogenesis of Alzheimer’s disease (AD), endosomal traffic dysfunction is considered to precede Aβ aggregation and trigger AD pathogenesis. A body of evidence suggests that the β-carboxyl-terminal fragment (βCTF) of amyloid-β precursor protein (APP), which is the direct precursor of Aβ, accumulates in endosomes and causes vesicular traffic impairment. However, the mechanism underlying this impairment remains unclear. Here we identified TMEM30A as a candidate partner for βCTF. TMEM30A is a subcomponent of lipid flippase that translocates phospholipids from the outer to the inner leaflet of the lipid bilayer. TMEM30A physically interacts with βCTF in endosomes and may impair vesicular traffic, leading to abnormally enlarged endosomes. APP traffic is also concomitantly impaired, resulting in the accumulation of APP-CTFs, including βCTF. In addition, we found that expressed BACE1 accumulated in enlarged endosomes and increased Aβ production. Our data suggested that TMEM30A is involved in βCTF-dependent endosome abnormalities that are related to Aβ overproduction.  

3.3066           Retrograde Transport by Clathrin-Coated Vesicles is Involved in Intracellular Transport of PrPSc in Persistently Prion-Infected Cells

Yamasaki, T., Suzuki, A., Hasebe, R. and Horiuchi, M. Scientific Reports, 8:12241 (2018)   Intracellular dynamics of an abnormal isoform of prion protein (PrPSc) are tightly associated with prion propagation. However, the machineries involved in the intracellular trafficking of PrPSc are not fully understood. Our previous study suggested that PrPSc in persistently prion-infected cells dynamically circulates between endocytic-recycling compartments (ERCs) and peripheral regions of the cells. To investigate these machineries, we focused on retrograde transport from endosomes to the trans-Golgi network, which is one of the pathways involved in recycling of molecules. PrPSc was co-localized with components of clathrin-coated vesicles (CCVs) as well as those of the retromer complex, which are known as machineries for retrograde transport. Fractionation of intracellular compartments by density gradient centrifugation showed the presence of PrPSc and the components of CCVs in the same fractions. Furthermore, PrPSc was detected in CCVs isolated from intracellular compartments of prion-infected cells. Knockdown of clathrin interactor 1, which is one of the clathrin adaptor proteins involved in retrograde transport, did not change the amount of PrPSc, but it altered the distribution of PrPSc from ERCs to peripheral regions, including late endosomes/lysosomes. These data demonstrated that some PrPSc is transported from endosomes to ERCs by CCVs, which might be involved in the recycling of PrPSc.  

3.3067           Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response

Chen, G., Huang, A.C., Zhang, W., Zhang, G., Wu, M., Xu, W. Yang, J. et al Nature, 560, 382-386 (2018)   Tumour cells evade immune surveillance by upregulating the surface expression of programmed death-ligand 1 (PD-L1), which interacts with programmed death-1 (PD-1) receptor on T cells to elicit the immune checkpoint response1,2. Anti-PD-1 antibodies have shown remarkable promise in treating tumours, including metastatic melanoma2,3,4. However, the patient response rate is low4,5. A better understanding of PD-L1-mediated immune evasion is needed to predict patient response and improve treatment efficacy. Here we report that metastatic melanomas release extracellular vesicles, mostly in the form of exosomes, that carry PD-L1 on their surface. Stimulation with interferon-γ (IFN-γ) increases the amount of PD-L1 on these vesicles, which suppresses the function of CD8 T cells and facilitates tumour growth. In patients with metastatic melanoma, the level of circulating exosomal PD-L1 positively correlates with that of IFN-γ, and varies during the course of anti-PD-1 therapy. The magnitudes of the increase in circulating exosomal PD-L1 during early stages of treatment, as an indicator of the adaptive response of the tumour cells to T cell reinvigoration, stratifies clinical responders from non-responders. Our study unveils a mechanism by which tumour cells systemically suppress the immune system, and provides a rationale for the application of exosomal PD-L1 as a predictor for anti-PD-1 therapy.    

3.3068           Production of Extracellular Vesicles Loaded with Therapeutic Cargo

Lamichlane, T.N. and Jay, S.M. Methods in Mol. Biol., 1831, 37-47 (2018)   Extracellular vesicles (EVs) are biological nanoparticles comprising exosomes, microvesicles, and other heterogeneous nanoscopic vesicle populations that are produced by most cell types. In addition to their putative roles as critical mediators of intercellular communication, EVs have begun to be harnessed as drug delivery vehicles, with early evidence indicating they may have significant advantages over synthetic nanoparticle delivery systems for particular applications. Targeted delivery of EV-encapsulated cargo has already been realized and may have broad applicability; however, methods for producing and purifying EVs and loading them with therapeutic molecules have yet to be standardized. In this chapter, we outline steps for EV isolation and characterization and compare current methods for active and passive loading of EVs with payloads of short interfering RNA (siRNA) or small molecules, with the results revealing that active loading via electroporation increases loading efficiency of siRNA but not of Rhodamine B, a model for a small molecule drug, in HEK293T-derived EVs. The methods described here may inform future design of targeted delivery of nucleic acids or small molecules via EVs.  

3.3069           Visual Experience-Dependent Expression of Fn14 Is Required for Retinogeniculate Refinement

Cheadle, L., Tzeng, C.P., Kalish, B.T., Burkley, L.C., Chen, C. and Greenberg, M.E.

Neuron, 99, 525-539 (2018)

Sensory experience influences the establishment of neural connectivity through molecular mechanisms that remain unclear. Here, we employ single-nucleus RNA sequencing to investigate the contribution of sensory-driven gene expression to synaptic refinement in the dorsal lateral geniculate nucleus of the thalamus, a region of the brain that processes visual information. We find that visual experience induces the expression of the cytokine receptor Fn14 in excitatory thalamocortical neurons. By combining electrophysiological and structural techniques, we show that Fn14 is dispensable for early phases of refinement mediated by spontaneous activity but that Fn14 is essential for refinement during a later, experience-dependent period of development. Refinement deficits in mice lacking Fn14 are associated with functionally weaker and structurally smaller retinogeniculate inputs, indicating that Fn14 mediates both functional and anatomical rearrangements in response to sensory experience. These findings identify Fn14 as a molecular link between sensory-driven gene expression and vision-sensitive refinement in the brain.

 

3.3070           Enterococcus faecium produces membrane vesicles containing virulence factors and antimicrobial resistance related proteins

Wagner, T., Joshi, B., Janice, J., Askarian, F., Skalko-Basnet, N., hagestad, O.C., Mekhlif, A., Wai, S.N., Hegstad, K. and Johannessen, M.

1. Proteomics, 187, 28-38 (2018)

  Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resistant to several antimicrobials and has a great ability to acquire new traits. Bacterial membrane vesicles (MVs) are increasingly recognized as a mode of cell-free communication and a way to deliver virulence factors and/or antimicrobial resistance determinants. These features make MVs interesting research targets in research on critical hospital pathogens. This study describes for the first time that E. faecium strains produce MVs. It presents a morphological as well as a proteomic analysis of MVs isolated from four different, clinically relevant E. faecium strains grown under two different conditions and identifies MV-associated proteins in all of them. Interestingly, 11 virulence factors are found among the MV-associated proteins, including biofilm-promoting proteins and extracellular matrix-binding proteins, which may aid in enterococcal colonization. Additionally, 11 antimicrobial resistance-related proteins were MV-associated. Among those, all proteins encoded by the vanA-cluster of a vancomycin resistant strain were found to be MV-associated. This implies that E. faecium MVs may be utilized by the bacterium to release proteins promoting virulence, pathogenicity and antimicrobial resistance.  

3.3071           Comparative lipidomic profiling of the human commensal bacterium Propionibacterium acnes and its extracellular vesicles

Jeon, J., Park, S.C., Her, J., Lee, J.W., Han, J-K., Kim, Y-K., Kim, K.P. and Ban, C. RSC Adv., 8, 15241-15247 (2018)   acnes is a lipophilic commensal bacterium mainly found on the skin and in the gastrointestinal tract. Pathophysiological effects of P. acnes have recently been reported not only in acne progression but in various diseases. As an emerging mode of bacterial communication, extracellular vesicles (EVs) have been demonstrated to conduct critical pathophysiological functions. To provide information on P. acnes lipid composition for the first time, we conducted a comparative lipidomic analysis of P. acnes and P. acnes EVs and identified 214 lipids with high confidence using triplicated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses. P. acnes EVs contained substantially more PCs, DGs, PAs, PEs, LPAs, LPCs, and MGs than P. acnes, and contained fewer PSs, SO1Ps, SA1Ps, LPGs, LPIs, and LPSs. Distinctively, P. acnes EVs possessed a markedly reduced amount of TG. These findings will provide useful clues for understanding the biological and pathophysiological mechanisms of P. acnes and for clinical applications such as vaccine development, diagnostics and therapeutics.  

3.3072           Technical challenges of working with extracellular vesicles

Ramirez, M.I., Arnorim, M.G., Gadelha, C., Milic, I., Welsh, J.A., Freitas, V.M. et al Nanoscale, 10, 881-906 (2018)   Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review.  

3.3073           Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution

Tabara, L-C., Vicente, J-J., Biazik, J., Eskelinen, E-L., Vincent, O. and Escalante, R. Traffic, 19(8), 624-638 (2018)   The multispanning membrane protein vacuole membrane protein 1 (VMP1) marks and regulates endoplasmic reticulum (ER)‐domains associated with diverse ER‐organelle membrane contact sites. A proportion of these domains associate with endosomes during their maturation and remodeling. We found that these VMP1 domains are enriched in choline/ethanolamine phosphotransferase and phosphatidylinositol synthase (PIS1), 2 ER enzymes required for the synthesis of various phospholipids. Interestingly, the lack of VMP1 impairs the formation of PIS1‐enriched ER domains, suggesting a role in the distribution of phosphoinositides. In fact, depletion of VMP1 alters the distribution of PtdIns4P and proteins involved in the trafficking of PtdIns4P. Consistently, in these conditions, defects were observed in endosome trafficking and maturation as well as in Golgi morphology. We propose that VMP1 regulates the formation of ER domains enriched in lipid synthesizing enzymes. These domains might be necessary for efficient distribution of PtdIns4P and perhaps other lipid species. These findings, along with previous reports that involved VMP1 in regulating PtdIns3P during autophagy, expand the role of VMP1 in lipid trafficking and explain the pleiotropic effects observed in VMP1‐deficient mammalian cells and other model systems.  

3.3074           A Fragment of Adhesion Molecule L1 Binds to Nuclear Receptors to Regulate Synaptic Plasticity and Motor Coordination

Kraus, K., Kleene, R., Henis, M., Braren, I., Kataria, H., Sharaf, A., Loers, G., Schachner, M. and Lutz, D. Mol. Neurobiol., 55(9), 7164-7178 (2018)   Proteolytic cleavage of the neuronal isoform of the murine cell adhesion molecule L1, triggered by stimulation of the cognate L1-dependent signaling pathways, results in the generation and nuclear import of an L1 fragment that contains the intracellular domain, the transmembrane domain, and part of the extracellular domain. Here, we show that the LXXLL and FXXLF motifs in the extracellular and transmembrane domain of this L1 fragment mediate the interaction with the nuclear estrogen receptors α (ERα) and β (ERβ), peroxisome proliferator-activated receptor γ (PPARγ), and retinoid X receptor β (RXRβ). Mutations of the LXXLL motif in the transmembrane domain and of the FXXLF motif in the extracellular domain disturb the interaction of the L1 fragment with these nuclear receptors and, when introduced by viral transduction into mouse embryos in utero, result in impaired motor coordination, learning and memory, as well as synaptic connectivity in the cerebellum, in adulthood. These impairments are similar to those observed in the L1-deficient mouse. Our findings suggest that the interplay of nuclear L1 and distinct nuclear receptors is associated with synaptic contact formation and plasticity.  

3.3075           Mobilization of iron from ferritin: new steps and details

La, A., Nguyen, T., Tran, K., Sauble, E., Tu, D., Gonzalez, A., Kidane, T.Z., Soriano, C., Morgan, J., Doan, M., Tran, K., Wang, C-Y., Knutson, M.D. and Linder, M.C. Metallomics, 10, 154-168 (2018)   Much evidence indicates that iron stored in ferritin is mobilized through protein degradation in lysosomes, but concerns about this process have lingered, and the mechanistic details of its aspects are lacking. In the studies presented here, ⁵⁹Fe-labeled ferritin was induced by preloading hepatic (HepG2) cells with radiolabeled Fe. Placing these cells in a medium containing desferrioxamine resulted in the loss of ferritin-⁵⁹Fe, but adding high concentrations of reducing agents or modulating the internal GSH concentration failed to alter the rates of ferritin-⁵⁹Fe release. Confocal microscopy showed that Fe deprivation increased the movement of ferritin into lysosomes and hyperaccumulation was observed when lysosomal proteolysis was inhibited. It also resulted in the rapid movement of DMT1 to lysosomes, which was inhibited by bafilomycin. Ferrihydrite crystals isolated from purified rat liver/spleen ferritin were solubilized at pH 5 and 7 by GSH, ascorbate, citrate and lysosomal fluids obtained from livers and J774a.1 macrophages. The inhibition of DMT1/Nramp2 and siRNA knockdown of Nramp1 each reduced the transfer of ⁵⁹Fe from lysosomes to the cytosol; and hepatocyte-specific knockout of DMT1 in mice prevented the release of Fe from the liver responding to EPO treatment, but did not inhibit lysosomal ferritin degradation. We conclude that ferritin-Fe mobilization does not occur through changes in cellular concentrations of reducing/chelating agents but by the coordinated movement of ferritin and DMT1 to lysosomes, where the ferrihydrite crystals exposed by ferritin degradation dissolve in the lysosomal fluid, and the reduced iron is transported back to the cytosol via DMT1 in hepatocytes, and by both DMT1 and Nramp1 in macrophages, prior to release into the blood or storage in ferritin.  

3.3076           Stimulation of exosome release by extracellular DNA is conserved across multiple cell types

Iliev, D., Strandskog, G., Nepal, A., Aspar, A., Olsen, R., Jørgensen, J., Wolfson, D., Ahluwalia, B.S., Handzhiyski, J. and Mironova, R. FEBS J., 285(16), 3114-3134 (2018)   Exosomes are distinguished from other types of extracellular vesicles by their small and relatively uniform size (30–100 nm) and their composition which reflects their endo‐lysosomal origin. Involvement of these extracellular organelles in intercellular communication and their implication in pathological conditions has fuelled intensive research on mammalian exosomes; however, currently, very little is known about exosomes in lower vertebrates. Here we show that, in primary cultures of head kidney leukocytes from Atlantic salmon (Salmo salar), phosphorothioate CpG oligodeoxynucleotides induce secretion of vesicles with characteristics very similar to these of mammalian exosomes. Further experiments revealed that the oligonucleotide‐induced exosome secretion did not depend on the CpG motifs but it relied on the phosphorothioate modification of the internucleotide linkage. Exosome secretion was also induced by genomic bacterial and eukaryotic DNA in toll‐like receptor 9‐negative piscine and human cell lines demonstrating that this is a phylogenetically conserved phenomenon which does not depend on activation of immune signaling pathways. In addition to exosomes, stimulation with phosphorothioate oligonucleotides and genomic DNA induced secretion of LC3B‐II, an autophagosome marker, which was associated with vesicles of diverse size and morphology, possibly derived from autophagosome‐related intracellular compartments. Overall, this work reveals a previously unrecognized biological activity of phosphorothioate ODNs and genomic DNA – their capacity to induce secretion of exosomes and other types of extracellular vesicles. This finding might help shed light on the side effects of therapeutic phosphorothioate oligodeoxynucleotides and the biological activity of extracellular genomic DNA which is often upregulated in pathological conditions.  

3.3077           Membrane rafts–redox signalling pathway contributes to renal fibrosis via modulation of the renal tubular epithelial–mesenchymal transition

Han, W-Q., Xu, L., Tang, X-F., Chen, W-D., Wu, Y-J. and Gao, P-J.

1. Physiol., 596(16), 3603-3616 (2019)

  The membrane rafts (MRs)–redox pathway is characterized by NADPH oxidase subunit clustering and activation through lysosome fusion, V‐type proton ATPase subunit E2 (encoded by the Atp6v1e2 gene) translocation and sphingomyelin phosphodiesterase 1 (SMPD1, encoded by the SMPD1 gene) activation. In the present study, we hypothesized that the MRs–redox‐derived reactive oxygen species (ROS) are involved in renal inflammation and fibrosis by promoting renal tubular epithelial–mesenchymal transition (EMT). Results show that transforming growth factor‐β1 (TGF‐β1) acutely induced MR formation and ROS production in NRK‐52E cells, a rat renal tubular cell line. In addition, transfection of Atp6v1e2 small hairpin RNAs (shRNA) and SMPD1 shRNA attenuated TGF‐β1‐induced changes in EMT markers, including E‐cadherin, α‐smooth muscle actin (α‐SMA) and fibroblast‐specific protein‐1 (FSP‐1) in NRK‐52E cells. Moreover, Erk1/2 activation may be a downstream regulator of the MRs–redox‐derived ROS, because both shRNAs significantly inhibited TGF‐β1‐induced Erk1/2 phosphorylation. Further in vivo study shows that the renal tubular the MRs–redox signalling pathway was activated in angiotensin II (AngII)‐induced hypertension, as indicated by the increased NADPH oxidase subunit Nox4 fraction in the MR domain, SMPD1 activation and increased ROS content in isolated renal tubular cells. Finally, renal transfection of Atp6v1e2 shRNA and SMPD1 shRNA significantly prevented renal fibrosis and inflammation, as indicated by the decrease of α‐SMA, fibronectin, collagen I, monocyte chemoattractant protein‐1 (MCP‐1), intercellular cell adhesion molecule‐1 (ICAM‐1) and tumour necrosis factor‐α (TNF‐α) in kidneys from AngII‐infused rats. It was concluded that the the MRs–redox signalling pathway is involved in TGF‐β1‐induced renal tubular EMT and renal inflammation/fibrosis in AngII‐induced hypertension.  

3.3078           Human cytomegalovirus-infected cells release extracellular vesicles that carry viral surface proteins

Zicari, S., Arakelyan, A., Palomino, R.A.N., Fitzgerald, W., Vanpoille, C., Lebedeva, A., Scjhmitt, A., Bomsel, M., Britt, W. and Margolis, L. Virology, 524, 97-105 (2018)   Extracellular vesicles (EVs) released by virus-infected cells typically incorporate host and viral components inside the vesicles (cargo molecules). Here, we investigated if human cytomegalovirus (HCMV) proteins are incorporated in EV outer membrane released by HCMV-infected cells. We separated EVs from HCMV using an iodixanol step-gradient and found that the separated vesicles carried EV markers such as the tetraspanin CD63 and Rab27A. Flow analysis of individual EVs demonstrated that on average, 15 ± 3.7% of EVs were positive for gB, 5.3 ± 2.3% were positive for gH and 3.74 ± 1.5% were positive for both gB and gH. In light of previous findings demonstrating HIV envelope proteins in EV membranes, the presence of viral protein at the surface of EVs released by HCMV-infected cells indicated that viral membrane proteins incorporated in EVs released by virus-infected cells may be a general phenomenon.  

3.3079           Rab18 is not necessary for lipid droplet biogenesis or turnover in human mammary carcinoma cells

Jayson, C.K.B., Arit, H., Fischer, A.W., lai, Z.W., Farese Jr., R.V. and Walther, T.C: Mol. Biol. Cell, 29, 2045-2054 (2018)   Rab GTPases recruit peripheral membrane proteins and can define organelle identity. Rab18 localizes to the endoplasmic reticulum (ER) but also to lipid droplets (LDs), where it has been implicated in effector protein recruitment and in defining LD identity. Here, we studied Rab18 localization and function in a human mammary carcinoma cell line. Rab18 localized to the ER and to LD membranes on LD induction, with the latter depending on the Rab18 activation state. In cells lacking Rab18, LDs were modestly reduced in size and numbers, but we found little evidence for Rab18 function in LD formation, LD turnover on cell starvation, or the targeting of several proteins to LDs. We conclude that Rab18 is not a general, necessary component of the protein machinery involved in LD biogenesis or turnover.  

3.3080           Extracellular Vesicles Released by Herpes Simplex Virus 1-Infected Cells Block Virus Replication in Recipient Cells in a STING-Dependent Manner

Deschamps, T. and Kalamvoki, M.

1. Virol., 92(18), e01102-18 (2018)

  Herpes simplex virus 1 (HSV-1)-infected cells release extracellular vesicles (EVs) that deliver to uninfected cells viral factors and host components, such as the stimulator of interferon genes (STING), which activates type I interferon upon foreign DNA sensing. The functions of EVs released by HSV-1-infected cells have remained unknown. Here, we describe a procedure to separate the EVs from HSV-1 virions that is based on an iodixanol/sucrose gradient. STING, along with the EV markers CD63 and CD9, was found in light-density fractions, while HSV components accumulated in heavy-density fractions. HSV-1 infection stimulated the release of EVs from the cells. The EVs derived from infected cells, but not from uninfected cells, activated innate immunity in recipient cells and suppressed viral gene expression and virus replication. Moreover, only the EVs derived from infected cells stimulated the expression of a subset of M1-type markers in recipient macrophages. Conversely, EVs derived from STING-knockdown cells failed to stimulate the expression of these M1-type markers, they activated innate immune responses to a lesser extent in recipient cells, and they did not sustain the inhibition of virus replication. These data suggest that STING from the EV donor cells contributes to the antiviral responses in cells receiving EVs from HSV-1-infected cells. Perturbations in the biogenesis of EVs by silencing CD63 or blocking the activity of the neutral spingomyelinase-2 (nSMase-2) increased the HSV-1 yields. Overall, our data suggest that the EVs released from HSV-1-infected cells negatively impact the infection and could control the dissemination of the virus.  

3.3081           Lipopolysaccharide Upregulates Palmitoylated Enzymes of the Phosphatidylinositol Cycle: An Insight from Proteomic Studies

Sobocinska, J., Roszczenko-Jasinka, P-. Zareba-Kozio M., Hromada-Judycka, A., matveichuk, O.V., Traczyk, G., Lukasiuk, K. and Kwiatkowska, K. Mol. Cell.Proteomics, 17(2), 233-254 (2018)   Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria that induces strong proinflammatory reactions of mammals. These processes are triggered upon sequential binding of LPS to CD14, a GPI-linked plasma membrane raft protein, and to the TLR4/MD2 receptor complex. We have found earlier that upon LPS binding, CD14 triggers generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂], a lipid controlling subsequent proinflammatory cytokine production. Here we show that stimulation of RAW264 macrophage-like cells with LPS induces global changes of the level of fatty-acylated, most likely palmitoylated, proteins. Among the acylated proteins that were up-regulated in those conditions were several enzymes of the phosphatidylinositol cycle. Global profiling of acylated proteins was performed by metabolic labeling of RAW264 cells with 17ODYA, an analogue of palmitic acid functionalized with an alkyne group, followed by detection and enrichment of labeled proteins using biotin-azide/streptavidin and their identification with mass spectrometry. This proteomic approach revealed that 154 fatty-acylated proteins were up-regulated, 186 downregulated, and 306 not affected in cells stimulated with 100 ng/ml LPS for 60 min. The acylated proteins affected by LPS were involved in diverse biological functions, as found by Ingenuity Pathway Analysis. Detailed studies of 17ODYA-labeled and immunoprecipitated proteins revealed that LPS induces S-palmitoylation, hence activation, of type II phosphatidylinositol 4-kinase (PI4KII) β, which phosphorylates phosphatidylinositol to phosphatidylinositol 4-monophosphate, a PI(4,5)P₂ precursor. Silencing of PI4KIIβ and PI4KIIα inhibited LPS-induced expression and production of proinflammatory cytokines, especially in the TRIF-dependent signaling pathway of TLR4. Reciprocally, this LPS-induced signaling pathway was significantly enhanced after overexpression of PI4KIIβ or PI4KIIα; this was dependent on palmitoylation of the kinases. However, the S-palmitoylation of PI4KIIα, hence its activity, was constitutive in RAW264 cells. Taken together the data indicate that LPS triggers S-palmitoylation and activation of PI4KIIβ, which generates PI(4)P involved in signaling pathways controlling production of proinflammatory cytokines  

3.3082           Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17

Matsui, T., Jiang, P., Nakano, S., Sakamaki, Y., Yamamoto, H. and Mizushima, N.

1. Cell Biol., 217(8), 2633-2645 (1018)

  Macroautophagy is an evolutionarily conserved catabolic mechanism that delivers intracellular constituents to lysosomes using autophagosomes. To achieve degradation, lysosomes must fuse with closed autophagosomes. We previously reported that the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein syntaxin (STX) 17 translocates to autophagosomes to mediate fusion with lysosomes. In this study, we report an additional mechanism. We found that autophagosome–lysosome fusion is retained to some extent even in STX17 knockout (KO) HeLa cells. By screening other human SNAREs, we identified YKT6 as a novel autophagosomal SNARE protein. Depletion of YKT6 inhibited autophagosome–lysosome fusion partially in wild-type and completely in STX17 KO cells, suggesting that YKT6 and STX17 are independently required for fusion. YKT6 formed a SNARE complex with SNAP29 and lysosomal STX7, both of which are required for autophagosomal fusion. Recruitment of YKT6 to autophagosomes depends on its N-terminal longin domain but not on the C-terminal palmitoylation and farnesylation that are essential for its Golgi localization. These findings suggest that two independent SNARE complexes mediate autophagosome–lysosome fusion.  

3.3083           Effective Refractive Index and Lipid Content of Extracellular Vesicles Revealed Using Optical Waveguide Scattering and Fluorescence Microscopy

Rupert, D.L.M., Mapar, M., Shelke, G.V., Norling, K., Elmeskog, M., Lötvall, J.O., Block, S., Bally, M., Agnarsson, B. and Höök, F. Langmuir, 34(29), 8522-8531 (2018)   Extracellular vesicles (EVs) are generating a growing interest because of the key roles they play in various biological processes and because of their potential use as biomarkers in clinical diagnostics and as efficient carriers in drug-delivery and gene-therapy applications. Their full exploitation, however, depends critically on the possibility to classify them into different subpopulations, a task that in turn relies on efficient means to identify their unique biomolecular and physical signatures. Because of the large heterogeneity of EV samples, such information remains rather elusive, and there is accordingly a need for new and complementary characterization schemes that can help expand the library of distinct EV features. In this work, we used surface-sensitive waveguide scattering microscopy with single EV resolution to characterize two subsets of similarly sized EVs that were preseparated based on their difference in buoyant density. Unexpectedly, the scattering intensity distribution revealed that the scattering intensity of the high-density (HD) population was on an average a factor of three lower than that of the low-density (LD) population. By further labeling the EV samples with a self-inserting lipid-membrane dye, the scattering and fluorescence intensities from EVs could be simultaneously measured and correlated at the single-particle level. The labeled HD sample exhibited not only lower fluorescence and scattering intensities but also lower effective refractive index (n ≈ 1.35) compared with the LD EVs (n ≈ 1.38), indicating that both the lipid and protein contents were indeed lower in the HD EVs. Although separation in density gradients of similarly sized EVs is usually linked to differences in biomolecular content, we suggest based on these observations that the separation rather reflects the ability of the solute of the gradient to penetrate the lipid membrane enclosing the EVs, that is, the two gradient bands are more likely because of the differences in membrane permeability than to differences in biomolecular content of the EVs.  

3.3084           Quantitative RT-PCR Analysis of Influenza Virus Endocytic Escape

Su, W-C. and Lai, M.M.C. Methods Mol. Biol., 1836, 185-194 (2018)   Although several virus families are internalized into their host cells by direct fusion of the viral envelope with the plasma membrane, most viruses, for example, influenza virus, make use of endocytic pathways for productive entry and infection. After endocytosis, the influenza virus escapes from the endocytic compartment to the cytosol. The distribution of the incoming influenza virus could be traced by detection of the viral RNA in the distinct cellular compartments, including endosome, cytosol, and nucleus. To accomplish this work, we developed a subcellular fractionation method based on density gradient ultracentrifugation and detected the viral RNA using quantitative reverse transcription-polymerase chain reaction analysis. This chapter is devoted to the practical methods and precautions for studying endocytic traffic of virus as well as host cellular factors affecting viral endocytosis.  

3.3085           Therapeutic Efficacy-Potentiated and Diseased Organ-Targeting Nanovesicles Derived from Mesenchymal Stem Cells for Spinal Cord Injury Treatment

Kim, H.Y., Kumar, H., Jo, M-J., Kim, J., Yoon, J-K., Lee, J-R., Kang, M., Choo, Y.W., Song, S.Y., Kwon, S.P., Hyeon, T., Han, I-B. and Kim, B-S. Nano Lett., 18(8), 4965-4975 (2018)   Human mesenchymal stem cell (hMSC)-derived exosomes have been spotlighted as a promising therapeutic agent for cell-free regenerative medicine. However, poor organ-targeting ability and insufficient therapeutic efficacy of systemically injected hMSC-exosomes were identified as critical limitations for their further applications. Therefore, in this study we fabricated iron oxide nanoparticle (IONP)–incorporated exosome-mimetic nanovesicles (NV-IONP) from IONP-treated hMSCs and evaluated their therapeutic efficacy in a clinically relevant model for spinal cord injury. Compared to exosome-mimetic nanovesicles (NV) prepared from untreated hMSCs, NV-IONP not only contained IONPs which act as a magnet-guided navigation tool but also carried greater amounts of therapeutic growth factors that can be delivered to the target cells. The increased amounts of therapeutic growth factors inside NV-IONP were attributed to IONPs that are slowly ionized to iron ions which activate the JNK and c-Jun signaling cascades in hMSCs. In vivo systemic injection of NV-IONP with magnetic guidance significantly increased the amount of NV-IONP accumulating in the injured spinal cord. Accumulated NV-IONP enhanced blood vessel formation, attenuated inflammation and apoptosis in the injured spinal cord, and consequently improved spinal cord function. Taken together, these findings highlight the development of therapeutic efficacy-potentiated extracellular nanovesicles and demonstrate their feasibility for repairing injured spinal cord.  

3.3086           An actin cytoskeletal barrier inhibits lytic granule release from natural killer cells in patients with Chediak-Higashi syndrome

Gil-Krzewska, A., Saeed, M.B., Oszmiana, A., Fischer, E.R., Lagrue, K., Gahl, W.A:, Introne, W.j., Coligan, J.E., Davis, D.M. and Krzewski, K.

1. Allergy Clin. Immunol., 142, 914-927 (2018)

  Background Chediak-Higashi syndrome (CHS) is a rare disorder caused by biallelic mutations in the lysosomal trafficking regulator gene (LYST), resulting in formation of giant lysosomes or lysosome-related organelles in several cell types. The disease is characterized by immunodeficiency and a fatal hemophagocytic lymphohistiocytosis caused by impaired function of cytotoxic lymphocytes, including natural killer (NK) cells. Objective We sought to determine the underlying biochemical cause of the impaired cytotoxicity of NK cells in patients with CHS. Methods We generated a human cell model of CHS using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. We used a combination of classical techniques to evaluate lysosomal function and cell activity in the model system and super-resolution microscopy to visualize F-actin and lytic granules in normal and LYST-deficient NK cells. Results Loss of LYST function in a human NK cell line, NK92mi, resulted in inhibition of NK cell cytotoxicity and reproduced other aspects of the CHS cellular phenotype, including the presence of significantly enlarged lytic granules with defective exocytosis and impaired integrity of endolysosomal compartments. The large granules had an acidic pH and normal activity of lysosomal enzymes and were positive for the proteins essential for lytic granule exocytosis. Visualization of the actin meshwork openings at the immunologic synapse revealed that the cortical actin acts as a barrier for secretion of such large granules at the cell-cell contact site. Decreasing the cortical actin density at the immunologic synapse or decreasing the lytic granule size restored the ability of LYST-deficient NK cells to degranulate and kill target cells. Conclusion The cortical actin and granule size play significant roles in NK cell cytotoxic function. We present evidence that the periodicity of subsynaptic actin is an important factor limiting the release of large lytic granules from NK cells from patients with CHS and could be a novel target for pharmaceutical intervention.  

3.3087           Dysregulated hemolysin liberates bacterial outer membrane vesicles for cytosolic lipopolysaccharide sensing

Chen, S., Yang, d., Wen, Y., Jiang, Z., Zhang, L., Jiang, J., Chen, Y., Hu, T., Wang, Q., Zhang, Y. and Liu, Q. PloS Pathogens, 14(8), e1007240 (2018)   Inflammatory caspase-11/4/5 recognize cytosolic LPS from invading Gram-negative bacteria and induce pyroptosis and cytokine release, forming rapid innate antibacterial defenses. Since extracellular or vacuole-constrained bacteria are thought to rarely access the cytoplasm, how their LPS are exposed to the cytosolic sensors is a critical event for pathogen recognition. Hemolysin is a pore-forming bacterial toxin, which was generally accepted to rupture cell membrane, leading to cell lysis. Whether and how hemolysin participates in non-canonical inflammasome signaling remains undiscovered. Here, we show that hemolysin-overexpressed enterobacteria triggered significantly increased caspase-4 activation in human intestinal epithelial cell lines. Hemolysin promoted LPS cytosolic delivery from extracellular bacteria through dynamin-dependent endocytosis. Further, we revealed that hemolysin was largely associated with bacterial outer membrane vesicles (OMVs) and induced rupture of OMV-containing vacuoles, subsequently increasing LPS exposure to the cytosolic sensor. Accordingly, overexpression of hemolysin promoted caspase-11 dependent IL-18 secretion and gut inflammation in mice, which was associated with restricting bacterial colonization in vivo. Together, our work reveals a concept that hemolysin promotes noncanonical inflammasome activation via liberating OMVs for cytosolic LPS sensing, which offers insights into innate immune surveillance of dysregulated hemolysin via caspase-11/4 in intestinal antibacterial defenses.

3.3088           The Endosomal Protein CEMIP Links WNT Signaling to MEK1–ERK1/2 Activation in Selumetinib-Resistant Intestinal Organoids

Duong, H.Q., Namazanyy, I., Rambow, F., Tang, S.C., Delaunay, S., Tharun, L., Florin, A., Büttner, R., Vandaele, D., Close, P., Marine, J-C., Shostak, K. and Chariot, A. Cancer Res., 78(16), 4533-4548 (2018)   MAPK signaling pathways are constitutively active in colon cancer and also promote acquired resistance to MEK1 inhibition. Here, we demonstrate that BRAF^V600E-mutated colorectal cancers acquire resistance to MEK1 inhibition by inducing expression of the scaffold protein CEMIP through a β-catenin– and FRA-1–dependent pathway. CEMIP was found in endosomes and bound MEK1 to sustain ERK1/2 activation in MEK1 inhibitor–resistant BRAF^V600E-mutated colorectal cancers. The CEMIP-dependent pathway maintained c-Myc protein levels through ERK1/2 and provided metabolic advantage in resistant cells, potentially by sustaining amino acids synthesis. CEMIP silencing circumvented resistance to MEK1 inhibition, partly, through a decrease of both ERK1/2 signaling and c-Myc. Together, our data identify a cross-talk between Wnt and MAPK signaling cascades, which involves CEMIP. Activation of this pathway promotes survival by potentially regulating levels of specific amino acids via a Myc-associated cascade. Targeting this node may provide a promising avenue for treatment of colon cancers that have acquired resistance to targeted therapies.  

3.3089           New Optical Imaging Reporter-labeled Anaplastic Thyroid Cancer-Derived Extracellular Vesicles as a Platform for In Vivo Tumor Targeting in a Mouse Model

Gangadaran, P., Li, X.J., Kalimuthu, S.K., Min, O.J., Hong, C.M., Rajendran, R.L., Lee, H.W., Zhu, L., Baek, S.H., Jeong, S.Y., Lee, S-W., Lee, J. and Ahn, B-C. Scientific Reports, 8:13509 (2018)   Extracellular vesicles (EVs), originating from multivesicular bodies by invagination of the endosomal membrane, are communication channels between distant cells. They are natural carriers of exogeneous cellular materials and have been exploited as drug delivery carriers in various diseases. Here, we found that tumor cell-derived EVs can be used as efficient targets in tumors by monitoring with an optical reporter system. Anaplastic thyroid cancer (CAL62) cell-derived EVs with Renilla luciferase (Rluc) were used to target CAL62 tumors in a mouse model. Optical imaging revealed that cancer cell-derived EVs (EV-CAL62/Rluc) targeted the original tumor (CAL62) in mice within 30 min after systemic injection. Furthermore, fluorescence imaging revealed that EV-CAL62/Rluc were internalized into CAL62 tumors in the mice. Ex vivo Optical imaging further confirmed the in vivo finding. Here, we successfully monitored the tumor targeting ability of tumor cell-derived EVs by optical imaging. Based on these results, tumor cell-derived EVs are highly effective natural carriers for drug delivery for cancer therapies.  

3.3090           Cholesterol impairs autophagy-mediated clearance of amyloid beta while promoting its secretion

Barber-Camps, E., Roca-Agujetas, V., Bartolessis, I., de Dios, C., Fernandez-Checa, J.C., Mari, M., Morales, A., Hartmann, T. and Colell, A. Autophagy, 14(7), 1129-1154 (2018)   Macroautophagy/autophagy failure with the accumulation of autophagosomes is an early neuropathological feature of Alzheimer disease (AD) that directly affects amyloid beta (Aβ) metabolism. Although loss of presenilin 1 function has been reported to impair lysosomal function and prevent autophagy flux, the detailed mechanism leading to autophagy dysfunction in AD remains to be elucidated. The resemblance between pathological hallmarks of AD and Niemann-Pick Type C disease, including endosome-lysosome abnormalities and impaired autophagy, suggests cholesterol accumulation as a common link. Using a mouse model of AD (APP-PSEN1-SREBF2 mice), expressing chimeric mouse-human amyloid precursor protein with the familial Alzheimer Swedish mutation (APP695swe) and mutant presenilin 1 (PSEN1-dE9), together with a dominant-positive, truncated and active form of SREBF2/SREBP2 (sterol regulatory element binding factor 2), we demonstrated that high brain cholesterol enhanced autophagosome formation, but disrupted its fusion with endosomal-lysosomal vesicles. The combination of these alterations resulted in impaired degradation of Aβ and endogenous MAPT (microtubule associated protein tau), and stimulated autophagy-dependent Aβ secretion. Exacerbated Aβ-induced oxidative stress in APP-PSEN1-SREBF2 mice, due to cholesterol-mediated depletion of mitochondrial glutathione/mGSH, is critical for autophagy induction. In agreement, in vivo mitochondrial GSH recovery with GSH ethyl ester, inhibited autophagosome synthesis by preventing the oxidative inhibition of ATG4B deconjugation activity exerted by Aβ. Moreover, cholesterol-enrichment within the endosomes-lysosomes modified the levels and membrane distribution of RAB7A and SNAP receptors (SNAREs), which affected its fusogenic ability. Accordingly, in vivo treatment with 2-hydroxypropyl-β-cyclodextrin completely rescued these alterations, making it a potential therapeutic tool for AD.  

3.3091           Extracellular vesicles from endothelial progenitor cells promote thyroid follicle formation

Degosserie, J., Heymans, C., Spourquet, C., Halbout, M., D’Auria, L., Van Der Smissen, P., Vertommen, D., Courtoy, P.J., Tyteca, D. and Pierreux, C.E.

1. Extracellular Vesicles, 7(1), 1487250 82018)

  Organogenesis is a complex and dynamic process requiring reciprocal communication between different cell types. In the thyroid, thyrocyte progenitors secrete the angiocrine factor, VEGFA, to recruit endothelial cells. In return, endothelial cells promote thyrocyte organisation into spherical follicular structures, which are responsible for thyroid hormone synthesis and storage. Medium conditioned by endothelial progenitor cells (EPCs) can promote follicle formation and lumen expansion (i.e. folliculogenesis) in an ex vivo culture system of thyroid lobes. Here, we postulated that endothelial cells instruct thyrocyte progenitors by producing extracellular vesicles (EVs). We found that medium conditioned by EPCs contain EVs with exosomal characteristics and that these vesicles can be incorporated into thyrocyte progenitors. By mass spectrometry, laminin peptides were abundantly identified in the EV preparations, probably co-sedimenting with EVs. Laminin-α1 silencing in EPC abrogated the folliculogenic effect of EVs. However, density gradient separation of EVs from laminins revealed that both EV-rich and laminin-rich fractions exhibited folliculogenic activity. In conclusion, we suggest that endothelial cells can produce EVs favouring thyrocyte organisation into follicles and lumen expansion, a mechanism promoted by laminin-α1.  

3.3092           Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma

Vagner, T., Spinelli, C., Minciacchi, V.R., Balaj, L., Zandian, M., Conley, A., Zijlstra, A., Freeman, M.R., Demichelis, F., De, S., Posades, E.M., Tanaka, H. and Di Vizio, D.

1. Extracellular Vesicles, 781), 1505403 (2018)

  Cancer-derived extracellular vesicles (EVs) are membrane-enclosed structures of highly variable size. EVs contain a myriad of substances (proteins, lipid, RNA, DNA) that provide a reservoir of circulating molecules, thus offering a good source of biomarkers. We demonstrate here that large EVs (L-EV) (large oncosomes) isolated from prostate cancer (PCa) cells and patient plasma are an EV population that is enriched in chromosomal DNA, including large fragments up to 2 million base pair long. While L-EVs and small EVs (S-EV) (exosomes) isolated from the same cells contained similar amounts of protein, the DNA was more abundant in L-EV, despite S-EVs being more numerous. Consistent with in vitro observations, the abundance of DNA in L-EV obtained from PCa patient plasma was variable but frequently high. Conversely, negligible amounts of DNA were present in the S-EVs from the same patients. Controlled experimental conditions, with spike-ins of L-EVs and S-EVs from cancer cells in human plasma from healthy subjects, showed that circulating DNA is almost exclusively enclosed in L-EVs. Whole genome sequencing revealed that the DNA in L-EVs reflects genetic aberrations of the cell of origin, including copy number variations of genes frequently altered in metastatic PCa (i.e. MYC, AKT1, PTK2, KLF10 and PTEN). These results demonstrate that L-EV-derived DNA reflects the genomic make-up of the tumour of origin. They also support the conclusion that L-EVs are the fraction of plasma EVs with DNA content that should be interrogated for tumour-derived genomic alterations.  

3.3093           Tumor-derived exosomes promote tumor self-seeding in hepatocellular carcinoma by transferring miRNA-25-5p to enhance cell motility

Liu, H., Chen, W., Zhi, X., Chen, E-J., Wei, T., Zhang, J., Shen, J., Hu, L-Q., Zhao, B., Feng, X-H., Bai, X-L. and Liang, T-B. Oncogene, 37, 4964-4978 (2018)   Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The process may breed more aggressive tumor cells, which may contribute to cancer progression. In this study, we observed tumor self-seeding in mouse xenograft models of hepatocellular carcinoma (HCC) for the first time. We confirmed that circulating tumor cell uptake of tumor-derived exosomes, which are increasingly recognized as key instigators of cancer progression by facilitating cell–cell communication, promoted tumor self-seeding by enhancing the invasive and migration capability of recipient HCC cells. Horizontal transfer of exosomal microRNA-25-5p to anoikis-resistant HCC cells significantly enhanced their migratory and invasive abilities, whereas inhibiting microRNA-25-5p alleviated these effects. Our experiments delineate an exosome-based novel pathway employed by functional microRNA from the original tumor cells that can influence the biological fate of circulating tumor cells.  

3.3094           LETM1 couples mitochondrial DNA metabolism and nutrient preference

Durigon, R., Mitchell, A.L., Jones, A.W., Manole, A., Nemmuni, M., Hirst, E.M.A., Houlden, H., Maragni, G., Lattante, S., Doronzio, P.N., Rosa, I.D., Zollino, M., Holt, I.J. and Spinazzola, A. EMBO Mol. Med., 10, e8550 (2018)   The diverse clinical phenotypes of Wolf–Hirschhorn syndrome (WHS) are the result of haploinsufficiency of several genes, one of which, LETM1, encodes a protein of the mitochondrial inner membrane of uncertain function. Here, we show that LETM1 is associated with mitochondrial ribosomes, is required for mitochondrial DNA distribution and expression, and regulates the activity of an ancillary metabolic enzyme, pyruvate dehydrogenase. LETM1 deficiency in WHS alters mitochondrial morphology and DNA organization, as does substituting ketone bodies for glucose in control cells. While this change in nutrient availability leads to the death of fibroblasts with normal amounts of LETM1, WHS‐derived fibroblasts survive on ketone bodies, which can be attributed to their reduced dependence on glucose oxidation. Thus, remodeling of mitochondrial nucleoprotein complexes results from the inability of mitochondria to use specific substrates for energy production and is indicative of mitochondrial dysfunction. However, the dysfunction could be mitigated by a modified diet—for WHS, one high in lipids and low in carbohydrates.  

3.3095           ARL3 subcellular localization and its suspected role in autophagy

Luo, G., Sun, Y., Feng, Y., Zhao, Q. and Wen, T. Biochemie, 154, 187-193 (2018)   ADP-ribosylation factor-like3 (ARL3) is a member of the ADP-ribosylation factor family of GTP-binding proteins that plays important role in regulating Ciliary trafficking. It ubiquitously expressed in normal tissues and tumor cell lines. However, the location and function of ARL3 in organelles are rarely known. In this study, we explored ARL3 subcellular localization in an all-round way in HEK293T, Neuro-2A and U251 cells by density gradient centrifugation and immunofluorescence. The results showed that ARL3 is expressed in most of organelles, and an iodixonal step gradient was further confirmed that ARL3 is mainly localized to the mitochondria, endosomes, lysosomes, and proteasome. By molecular functional analysis, we observed that ARL3 promotes the aggregation of GFP-LC3, up-regulation of LC3-II/LC3-I and down-regulation of SQSMT1/BECN1, and knocking down of ARL3 inbibits autophagy, which suggested that ARL3 is necessary for autophagy. this study presents a comprehensive evaluation of the subcellular localization for ARL3 and provides important on understanding the functions of ARL3.  

3.3096           The Cytotoxic Necrotizing Factor of Yersinia pseudotuberculosis (CNFy) is Carried on Extracellular Membrane Vesicles to Host Cells

Monappa, A.K., Bari, W., Seo, J.K. and Mitchell, R.J. Scientific Reports, 8:14186 (2018)   In this study we show Yersinia pseudotuberculosis secretes membrane vesicles (MVs) that contain different proteins and virulence factors depending on the strain. Although MVs from Y. pseudotuberculosis YPIII and ATCC 29833 had many proteins in common (68.8% of all the proteins identified), those located in the outer membrane fraction differed significantly. For instance, the MVs from Y. pseudotuberculosis YPIII harbored numerous Yersinia outer proteins (Yops) while they were absent in the ATCC 29833 MVs. Another virulence factor found solely in the YPIII MVs was the cytotoxic necrotizing factor (CNFy), a toxin that leads to multinucleation of host cells. The ability of YPIII MVs to transport this toxin and its activity to host cells was verified using HeLa cells, which responded in a dose-dependent manner; nearly 70% of the culture was multinucleated after addition of 5 µg/ml of the purified YPIII MVs. In contrast, less than 10% were multinucleated when the ATCC 29833 MVs were added. Semi-quantification of CNFy within the YPIII MVs found this toxin is present at concentrations of 5 ~ 10 ng per µg of total MV protein, a concentration that accounts for the cellular responses seen.  

3.3097           Enterohemorrhagic Escherichia coli O157 outer membrane vesicles induce interleukin 8 production in human intestinal epithelial cells by signaling via Toll-like receptors TLR4 and TLR5 and activation of the nuclear factor NF-κB

Bielaszewska, M., Marejkova, M., Bauwens, A., Kunsmann-prokscha, L., Mellmann, A. and karch, H. Int. J. Med. Microbiol., 308, 882-889 (2018)   Proinflammatory cytokines play important roles in the pathogenesis of diseases caused by enterohemorrhagic Escherichia coli (EHEC) O157, but the spectrum of bacterial components involved in the proinflammatory responses is not fully understood. Here, we investigated the abilities of outer membrane vesicles (OMVs), nanoparticles released by EHEC O157 during growth, to induce production of proinflammatory cytokines in human intestinal epithelial cells. OMVs from both EHEC O157:H7 and sorbitol-fermenting (SF) EHEC O157:H⁻ induced production of interleukin-8 (IL-8) in Caco-2, HCT-8, and HT-29 intestinal epithelial cell lines. H7 flagellin was the key IL-8-inducing component of EHEC O157:H7 OMVs, whereas cytolethal distending toxin V and O157 lipopolysaccharide (LPS) largely contributed to IL-8 production elicited by flagellin-lacking OMVs from SF EHEC O157:H⁻. The H7 flagellin-mediated signaling via Toll-like receptor (TLR) 5, and O157 LPS-mediated signaling via TLR4/MD-2 complex, which were followed by activation of the nuclear factor NF-κB were major pathways underlying IL-8 production induced by EHEC O157 OMVs. The proinflammatory and immunomodulatory capacities of EHEC O157 OMVs have pathogenetic implications and support the OMVs as suitable vaccine candidates.  

3.3098           Distinct mechanisms enable inward or outward budding from late endosomes/multivesicular bodies

Gireud-Goss, M., Reyes, S., Wilson, M., Farley, M., memarzadeh, K., Srinivasan, S., Sirisaengtaksin, N., Yamashita, S., Tsunoda, S., lang, F.F., Waxham, M.N. and Bean, A.J. Exp. Cell Res., 372(1), 1-15 (2018)   Regulating the residence time of membrane proteins on the cell surface can modify their response to extracellular cues and allow for cellular adaptation in response to changing environmental conditions. The fate of membrane proteins that are internalized from the plasma membrane and arrive at the limiting membrane of the late endosome/multivesicular body (MVB) is dictated by whether they remain on the limiting membrane, bud into internal MVB vesicles, or bud outwardly from the membrane. The molecular details underlying the disposition of membrane proteins that transit this pathway and the mechanisms regulating these trafficking events are unclear. We established a cell-free system that reconstitutes budding of membrane protein cargo into internal MVB vesicles and onto vesicles that bud outwardly from the MVB membrane. Both budding reactions are cytosol-dependent and supported by Saccharomyces cerevisiae (yeast) cytosol. We observed that inward and outward budding from the MVB membrane are mechanistically distinct but may be linked, such that inhibition of inward budding triggers a re-routing of cargo from inward to outward budding vesicles, without affecting the number of vesicles that bud outwardly from MVBs.  

3.3099           Proteomic analyses reveal GNG12 regulates cell growth and casein synthesis by activating the Leu-mediated mTORC1 signaling pathway

Luo, C., Zhao, S., Dai, W., Zheng, N. and Wang, J. BBA – Proteins and Proteomics, 1866(11), 1092-1101 (2018)   In cow mammary epithelial cells (CMECs), cell growth and casein synthesis are regulated by amino acids (AAs), and lysosomes are important organelles in this regulatory process, but the mechanisms remain unclear. Herein, lysosomal membrane proteins (LMPs) in CMECs in the presence (Leu+) and absence (Leu-) of leucine were quantitatively analysed using Sequential Windowed Acquisition of All Theoretical Fragment Ion (SWATH) mass spectrometry. In identified LMPs, Guanine nucleotide-binding protein subunit gamma-12 (GNG12) was a markedly up-regulated protein in Leu+ group. CMECs were treated with Leu+ or Leu−, expression and lysosomal localization of GNG12 were decreased in response to Leu absence. Overexpressing or inhibiting GNG12 demonstrated that cell growth, casein synthesis and activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway were all up-regulated by GNG12. Cell growth, casein synthesis and mTORC1 signaling pathway were decreased in response to Leu absence, but these decreases were partially restored by GNG12 overexpression, and those effects were partially reversed by inhibiting GNG12. Co-immunoprecipitation analysis showed that GNG12 activates the mTORC1 pathway via interaction with Ragulator. Taken together, these results suggest that GNG12 is a positive regulator of the Leu-mediated mTORC1 signaling pathway in CMECs that promotes cell growth and casein synthesis.  

3.3100           StAR-related lipid transfer domain 11 (STARD11)–mediated ceramide transport mediates extracellular vesicle biogenesis

Fukushima, M., Dasgupta, D., Mauer, A.S., Kakazu, E., Nakao, K. and Mali, H.

1. Biol. Chem., 293(39), 15277-15289 (2018)

  Extracellular vesicles are important carriers of cellular materials and have critical roles in cell-to-cell communication in both health and disease. Ceramides are implicated in extracellular vesicle biogenesis, yet the cellular machinery that mediates the formation of ceramide-enriched extracellular vesicles remains unknown. We demonstrate here that the ceramide transport protein StAR-related lipid transfer domain 11 (STARD11) mediates the release of palmitate-stimulated extracellular vesicles having features consistent with exosomes. Using palmitate as a model of lipotoxic diseases and as a substrate for ceramide biosynthesis in human and murine liver cell lines and primary mouse hepatocytes, we found that STARD11-deficient cells release fewer extracellular vesicles. Moreover, STARD11 reciprocally regulated exosome ceramide enrichment and cellular ceramide depletion. We further observed that in STARD11 knockout cells intracellular ceramide accumulates and that this apparent inability to transfer cellular ceramide into extracellular vesicles reduces cellular viability. Using endogenous markers, we uncovered structural and functional colocalization of the endoplasmic reticulum (ER), STARD11, and multivesicular bodies. This colocalization increased following palmitate treatment, suggesting a functional association that may mediate ceramide trafficking from the ER to the multivesicular body. However, the size and number of multivesicular bodies were comparable in WT and STARD11-knockout cells. In conclusion, we propose a model of how STARD11 mediates ceramide trafficking in palmitate-treated cells and stimulates exosome biogenesis.  

3.3101           Arrowtail RNA for Ligand Display on Ginger Exosome-like Nanovesicles to Systemic Deliver siRNA for Cancer Suppression

Li, Z., Wang, H., Yin, H., Bennett, C., Zhang, H-g. and Guo, P. Scientific Reports, 8:14644 (2018) Exosomes have shown increasing potential as delivery vesicles for therapy, but challenges like cost/yield, drug payload, and targeting specificity still exist. Plant derived exosome-like nanoparticles have been reported as a promising substitution and exhibit biocompatibility through oral, intranasal administration; however, systemic delivery of siRNA by exosome-like nanoparticles directly isolated from plants has not been reported. Recently, we reported the control of RNA orientation to decorate human derived exosome with cell targeting ligands for specific delivery of siRNA to tumors. Here, we expand to the application of arrowtail RNA nanoparticles for displaying ligands on ginger derived exosome-like nanovesicles (GDENs) for siRNA delivery and tumor inhibition through IV administration. Cushion ultracentrifugation coupled with equilibrium density gradient ultracentrifugation were used for purifying GDENs that displayed size, density, and morphology similar to human derived exosomes. Folic acid (FA), as a ligand, was displayed on the surface of GDENs for targeted delivery of survivin siRNA to KB cancer models. In vitro gene knockdown efficacy by FA-3WJ/GDENs/siRNA complex was comparable to transfection. We observed inhibition of tumor growth on a xenograft model by intravenous administration, which reveals the potential of GDENs as an economic delivery system for siRNA.  

3.3102           Influenza A Virus Induces Autophagosomal Targeting of Ribosomal Proteins

Becker, A.C., Gannage, m., Giese, S., Hu, Z., Abou-Eid, S., Roubat, C., Paul, P., Bühler, L., Gretzmeier, C., Dumit, V.I., Kaeser-pebernard, S., Schwemmle, M., Münz, C. and Dengjel, J. Mol. Cell. Proteomics, 17(10), 1909-1921 (2018)   Seasonal epidemics of influenza A virus are a major cause of severe illness and are of high socio-economic relevance. For the design of effective antiviral therapies, a detailed knowledge of pathways perturbed by virus infection is critical. We performed comprehensive expression and organellar proteomics experiments to study the cellular consequences of influenza A virus infection using three human epithelial cell lines derived from human lung carcinomas: A549, Calu-1 and NCI-H1299. As a common response, the type I interferon pathway was up-regulated upon infection. Interestingly, influenza A virus infection led to numerous cell line-specific responses affecting both protein abundance as well as subcellular localization. In A549 cells, the vesicular compartment appeared expanded after virus infection. The composition of autophagsomes was altered by targeting of ribosomes, viral mRNA and proteins to these double membrane vesicles. Thus, autophagy may support viral protein translation by promoting the clustering of the respective molecular machinery in autophagosomes in a cell line-dependent manner.  

3.3103           The Impact of Oncogenic EGFRvIII on the Proteome of Extracellular Vesicles Released from Glioblastoma Cells

Choi, D., Montermini, L., Kim, D-K., Meehan, B., Roth, F.P. and Rak, J. Mol. Cell. Proteomics, 17(10), 1948-1964 (2018)   Glioblastoma multiforme (GBM) is a highly aggressive and heterogeneous form of primary brain tumors, driven by a complex repertoire of oncogenic alterations, including the constitutively active epidermal growth factor receptor (EGFRvIII). EGFRvIII impacts both cell-intrinsic and non-cell autonomous aspects of GBM progression, including cell invasion, angiogenesis and modulation of the tumor microenvironment. This is, at least in part, attributable to the release and intercellular trafficking of extracellular vesicles (EVs), heterogeneous membrane structures containing multiple bioactive macromolecules. Here we analyzed the impact of EGFRvIII on the profile of glioma EVs using isogenic tumor cell lines, in which this oncogene exhibits a strong transforming activity. We observed that EGFRvIII expression alters the expression of EV-regulating genes (vesiculome) and EV properties, including their protein composition. Using mass spectrometry, quantitative proteomic analysis and Gene Ontology terms filters, we observed that EVs released by EGFRvIII-transformed cells were enriched for extracellular exosome and focal adhesion related proteins. Among them, we validated the association of pro-invasive proteins (CD44, BSG, CD151) with EVs of EGFRvIII expressing glioma cells, and downregulation of exosomal markers (CD81 and CD82) relative to EVs of EGFRvIII-negative cells. Nano-flow cytometry revealed that the EV output from individual glioma cell lines was highly heterogeneous, such that only a fraction of vesicles contained specific proteins (including EGFRvIII). Notably, cells expressing EGFRvIII released EVs double positive for CD44/BSG, and these proteins also colocalized in cellular filopodia. We also detected the expression of homophilic adhesion molecules and increased homologous EV uptake by EGFRvIII-positive glioma cells. These results suggest that oncogenic EGFRvIII reprograms the proteome and uptake of GBM-related EVs, a notion with considerable implications for their biological activity and properties relevant for the development of EV-based cancer biomarkers.  

3.3104           Vesicular Delivery of the Antifungal Antibiotics of Lysobacter enzymogenes C3

Meers, P.R., Liu, C., Chen, R., Bartos, W., Davis, J., Dziedzic, N., Orciuolo, J., Kutyla, S., Pozo, M.J., Mithrananda, D., Panzera, D. and Wang, S. Appl. Environ. Microbiol., 84(20), e1353-18 (2018)   Lysobacter enzymogenes C3 is a predatory strain of Gram-negative gliding bacteria that produces antifungal antibiotics by the polyketide synthetic pathway. Outer membrane vesicles (OMV) are formed as a stress response and can deliver virulence factors to host cells. The production of OMV by C3 and their role in antifungal activity are reported here. Vesicles in the range of 130 to 150 nm in diameter were discovered in the cell-free supernatants of C3 cultures. These OMV contain molecules characteristic of bacterial outer membranes, such as lipopolysaccharide and phospholipids. In addition, they contain chitinase activity and essentially all of the heat-stable antifungal activity in cell supernatants. We show here that C3 OMV can directly inhibit growth of the yeast Saccharomyces cerevisiae as well as that of the filamentous fungus Fusarium subglutinans. The activity is dependent on physical contact between OMV and the cells. Furthermore, fluorescent lipid labeling of C3 OMV demonstrated transfer of the membrane-associated probe to yeast cells, suggesting the existence of a mechanism of delivery for membrane-associated molecules. Mass spectrometric analysis of C3 OMV extracts indicates the presence of molecules with molecular weights identical to some of the previously identified antifungal products of C3. These data together suggest that OMV act as an important remote mobile component of predation by Lysobacter.  

3.3105           DNA-induced liquid phase condensation of cGAS activates innate immune signaling

Du, M. and Chen, Z.J. Science, 361(6403), 704-709 (2018)   The binding of DNA to cyclic GMP–AMP synthase (cGAS) leads to the production of the secondary messenger cyclic GMP–AMP (cGAMP), which activates innate immune responses. We have shown that DNA binding to cGAS robustly induced the formation of liquidlike droplets in which cGAS was activated. The disordered and positively charged cGAS N terminus enhanced cGAS-DNA phase separation by increasing the valencies of DNA binding. Long DNA was more efficient in promoting cGAS liquid phase separation and cGAS enzyme activity than short DNA. Moreover, free zinc ions enhanced cGAS enzyme activity both in vitro and in cells by promoting cGAS-DNA phase separation. These results demonstrated that the DNA-induced phase transition of cGAS promotes cGAMP production and innate immune signaling.    

3.3106           Extracellular vesicles in cancer — implications for future improvements in cancer care

Xu, R., Rai, A., Chen, M., Suwakulsir, W., Greening, D.W. and Simpson, R.J: Nature Reviews – Clin. Oncol., 15, 617-638 (2018)   The sustained growth, invasion, and metastasis of cancer cells depend upon bidirectional cell–cell communication within complex tissue environments. Such communication predominantly involves the secretion of soluble factors by cancer cells and/or stromal cells within the tumour microenvironment (TME), although these cell types have also been shown to export membrane-encapsulated particles containing regulatory molecules that contribute to cell–cell communication. These particles are known as extracellular vesicles (EVs) and include species of exosomes and shed microvesicles. EVs carry molecules such as oncoproteins and oncopeptides, RNA species (for example, microRNAs, mRNAs, and long non-coding RNAs), lipids, and DNA fragments from donor to recipient cells, initiating profound phenotypic changes in the TME. Emerging evidence suggests that EVs have crucial roles in cancer development, including pre-metastatic niche formation and metastasis. Cancer cells are now recognized to secrete more EVs than their nonmalignant counterparts, and these particles can be isolated from bodily fluids. Thus, EVs have strong potential as blood-based or urine-based biomarkers for the diagnosis, prognostication, and surveillance of cancer. In this Review, we discuss the biophysical properties and physiological functions of EVs, particularly their pro-metastatic effects, and highlight the utility of EVs for the development of cancer diagnostics and therapeutics.  

3.3107           Proteomic analysis of exosomes reveals an association between cell invasiveness and exosomal bioactivity on endothelial and mesenchymal cell migration in vitro

Sharma, S., Alharbi, M., Kobayashi, M., Lai, A., Guanzon, D., Zuniga, F., Ormazabal, V., Palma, C., Scholz-Romwero, K., Rice, G.E., Hooper, J.D. and Salomon, C. Clin. Sci., 132, 2029-2044 (2018)   Ovarian cancer has resulted in over 140 000 deaths reported annually worldwide. This is often attributed to cellular changes in the microenvironment, including increased migration of mesenchymal stem cells (MSCs) and endothelial cells (ECs) to facilitate metastasis. Recently, the ability of exosomes to communicate signals between cells (and promote cancer progression) has been established. In the present study, we explored the effect of exosomes on cells present in the tumour microenvironment. Exosomes were isolated from ovarian cancer cells with different invasive capacity (high = SKOV-3 and low = OVCAR-3) by differential and buoyant density centrifugation and characterised using nanoparticle tracking analysis (NTA), Western blot, and EM. Exosome secretion was positively correlated with invasiveness of releasing cells. Proteomic analyses identified common and unique proteins between exosomes from SKOV-3 and OVCAR-3 with gene ontology analyses revealing that these exosomes are involved in the regulation of cell migration. Since the tumour microenvironment contains multiple cell types, including MSCs and ECs, we examined the effect of these exosomes on MSC and EC migration. Exosomes promoted MSC and EC migration in a time- and concentration-dependent manner. The effect of exosomes isolated from SKOV-3 on cell migration was significantly higher compared with exosomes from OVCAR-3. Thus, we suggest that exosomes from ovarian cancer cells contain a specific set of proteins that are representative of its cell of origin and the invasive capacity.  

3.3108           14-3-3 Proteins Reduce Cell-to-Cell Transfer and Propagation of Pathogenic α-Synuclein

Wang, B., Underwood, R., Kamath, A., Britain, C., McFerrin, M.B., McLean, P.J., Volpicelli-Daley, L.A., Whitaker, R.H., Placzek, W.J., Becker, K., Ma, J. and Yacoubian, T.A.

1. Neurosci., 38(38), 8211-8232 (2018)

  α-Synuclein (αsyn) is the key protein that forms neuronal aggregates in the neurodegenerative disorders Parkinson's disease (PD) and dementia with Lewy bodies. Recent evidence points to the prion-like spread of αsyn from one brain region to another. Propagation of αsyn is likely dependent on release, uptake, and misfolding. Under normal circumstances, this highly expressed brain protein functions normally without promoting pathology, yet the underlying endogenous mechanisms that prevent αsyn spread are not understood. 14-3-3 proteins are highly expressed brain proteins that have chaperone function and regulate protein trafficking. In this study, we investigated the potential role of the 14-3-3 proteins in the regulation of αsyn spread using two models of αsyn spread. In a paracrine αsyn model, 14-3-3θ promoted release of αsyn complexed with 14-3-3θ. Despite higher amounts of released αsyn, extracellular αsyn showed reduced oligomerization and seeding capability, reduced internalization, and reduced toxicity in primary mixed-gender mouse neurons. 14-3-3 inhibition reduced the amount of αsyn released, yet released αsyn was more toxic and demonstrated increased oligomerization, seeding capability, and internalization. In the preformed fibril model, 14-3-3 θ reduced αsyn aggregation and neuronal death, whereas 14-3-3 inhibition enhanced αsyn aggregation and neuronal death in primary mouse neurons. 14-3-3s blocked αsyn spread to distal chamber neurons not exposed directly to fibrils in multichamber, microfluidic devices. These findings point to 14-3-3s as a direct regulator of αsyn propagation, and suggest that dysfunction of 14-3-3 function may promote αsyn pathology in PD and related synucleinopathies.  

3.3109           Widespread expression of perilipin 5 in normal human tissues and in diseases is restricted to distinct lipid droplet subpopulations

Hashani, M., Witzel, H.R., Pawella, L.M., Lehmann-Koch, J., Schumacher, J., Mechtersheimer, G., Schnölzer, M., Schirmacher, P., Roth, W. and Straub, B.K. Cell and Tissue Res., 374, 121-136 (2018)   Diseases associated with the accumulation of lipid droplets are increasing in western countries. Lipid droplet biogenesis, structure and degradation are regulated by proteins of the perilipin family. Perilipin 5 has been shown to regulate basal lipolysis in oxidative tissues. We examine perilipin 5 in normal human tissues and in diseases using protein biochemical and microscopic techniques. Perilipin 5 was constitutively located at small lipid droplets in skeletal myocytes, cardiomyocytes and brown adipocytes. In addition, perilipin 5 was detected in the epithelia of the gastrointestinal and urogenital tract, especially in hepatocytes, the mitochondria-rich parietal cells of the stomach, tubular kidney cells and ductal cells of the salivary gland and pancreas. Granular cytoplasmic expression, without a lipid droplet-bound localization was detected elsewhere. In cardiomyopathies, in skeletal muscle diseases and during hepatocyte steatogenesis, perilipin 5 was upregulated and localized to larger and more numerous lipid droplets. In steatotic human hepatocytes, perilipin 5 was moderately increased and colocalized with perilipins 1 and 2 but not with perilipin 3 at lipid droplets. In liver diseases implicated in alterations of mitochondria, such as mitochondriopathies, alcoholic liver disease, Wilson’s disease and acute liver injury, perilipin 5 was frequently localized to small lipid droplets and less in the cytoplasm. In tumorigenesis, perilipin 5 was especially upregulated in lipo-, leio- and rhabdomyosarcoma and hepatocellular and renal cell carcinoma. In summary, our study provides evidence that perilipin 5 is not restricted to certain cell types but localizes to distinct lipid droplet subpopulations reflecting a possible function in oxidative energy supply in normal tissues and in diseases.  

3.3110           A new ALK isoform transported by extracellular vesicles confers drug resistance to melanoma cells

Cesi, G., Philippidou, D., Kozar, I., Kim, Y.J., Bernardin, F., Van Niel, G., Wenecke-Baldacchino, A., Felten, P., Letellier, E., Dengler, S., Nashan, d., Haan, C. and Kreis, S. Mol. Cancer, 17:145 (2018)   Background Drug resistance remains an unsolved clinical issue in oncology. Despite promising initial responses obtained with BRAF and MEK kinase inhibitors, resistance to treatment develops within months in virtually all melanoma patients. Methods Microarray analyses were performed in BRAF inhibitor-sensitive and resistant cell lines to identify changes in the transcriptome that might play a role in resistance. siRNA approaches and kinase inhibitors were used to assess the involvement of the identified Anaplastic Lymphoma Kinase (ALK) in drug resistance. The capability of extracellular vesicles (EVs) to transfer drug resistant properties was investigated in co-culture assays. Results Here, we report a new mechanism of acquired drug resistance involving the activation of a novel truncated form of ALK. Knock down or inhibition of ALK re-sensitised resistant cells to BRAF inhibition and induced apoptosis. Interestingly, truncated ALK was also secreted into EVs and we show that EVs were the vehicle for transferring drug resistance. Conclusions To our knowledge, this is the first report demonstrating the functional involvement of EVs in melanoma drug resistance by transporting a truncated but functional form of ALK, able to activate the MAPK signalling pathway in target cells. Combined inhibition of ALK and BRAF dramatically reduced tumour growth in vivo. These findings make ALK a promising clinical target in melanoma patients.  

3.3111           Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules

Boussadia, Z., Lamberti, J., Mattel, F., Pizzi, E. et al

1. Exp. Clin. Cancer Res., 37:245 (2018)

  Background Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0–6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions A crucial step of melanoma progression does occur at melanoma intermediate –stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement.  

3.3112           Exosome Research and Co-culture Study

Shimasaki, T., Yamamoto, S. and Arisawa, T. Biol. Pharm. Bull., 41, 1311-1321 (2018)   In biological systems, extracellular vesicles including exosomes have recently been revealed to play a significant role in the communication between various cells, and the number of papers on this subject has dramatically increased. In current conventional exosome studies, the standard research method is to use liquid biopsies to analyze extracts of various disease exosomes. However, exosomes are only one of many key players in natural cellular interactions. Reproducing the phenomena occurring in vivo and investigating the interactions are required in order to examine their role fully. For exosome research, an alternative to the liquid biopsy method for observing natural interactions is the co-culturing technique. It does not require an exosome extraction procedure, and while the technique has been used in many studies thus far, its application to exosome research has been limited. However, the use of co-culturing technologies is necessary to examine the essential interactions of exosomes. An overview of exosome research methodologies and co-culturing systems is thus provided here.  

3.3113           Cortactin and fascin-1 regulate extracellular vesicle release by controlling endosomal trafficking or invadopodia formation and function

Berghein, E., Devriese, D., Van Hoey, E. and Gettemans, J. Scientific Reports, 8:15606 (2018)   Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function.  

3.3114           Chapter 6 - Exosomes: Cellular capsules for drug delivery in Parkinson’s disease

Samal, J., Demir, S. and Pandit, A. Drug Delivery Nanosystems for Biomedical Applications, 91-151 (2018)   Exosomes are nano-sized vesicles secreted by different cell types that play a crucial role in cell-to-cell communication and mediate the transfer of genetic information between different cell types. They are composed of lipids and carry a well-sorted cargo of different types of RNA and proteins, which is reflective of their cellular origin as well as the disease pathophysiology. Their natural ability to deliver these biomolecules across the blood-brain barrier (BBB) makes them an attractive option to be harnessed as drug-delivery vehicles for therapeutic delivery in neurodegenerative disorders. As drug-delivery vehicles, exosomes have been used to deliver small interfering RNAs and proteins to the brain and have shown clear therapeutic effects in animal models of Parkinson’s disease (PD). The presence of biological markers, enhanced stability of cargo, and the amenability of exosomes to surface modifications for targeted delivery of therapeutics to specific cell types makes them promising candidates for the development of novel diagnostic and therapeutic approaches for PD.  

3.3115           A novel in vitro assay reveals SNARE topology and the role of Ykt6 in autophagosome fusion with vacuoles

Gao, J., Reggior, F. and Ungermann, C.

1. Cell Biol., 217(10), 3670-3682 (2018)

  Autophagy is a catabolic pathway that delivers intracellular material to the mammalian lysosomes or the yeast and plant vacuoles. The final step in this process is the fusion of autophagosomes with vacuoles, which requires SNARE proteins, the homotypic vacuole fusion and protein sorting tethering complex, the RAB7-like Ypt7 GTPase, and its guanine nucleotide exchange factor, Mon1-Ccz1. Where these different components are located and function during fusion, however, remains to be fully understood. Here, we present a novel in vitro assay to monitor fusion of intact and functional autophagosomes with vacuoles. This process requires ATP, physiological temperature, and the entire fusion machinery to tether and fuse autophagosomes with vacuoles. Importantly, we uncover Ykt6 as the autophagosomal SNARE. Our assay and findings thus provide the tools to dissect autophagosome completion and fusion in a test tube.  

3.3116           Proteomic Analysis of Exosomes and Its Application in HIV‐1 Infection

Cheruiyot, c., Pataki, Z., Ramratnam, B. and Li, M. Proteomics – Clin. Appl., 12, 1700142 (2018)   Exosomes are 30–100 nm extracellular vesicles secreted from late endosomes by various types of cells. Numerous studies have suggested that exosomes play significant roles in human immunodeficiency virus 1 (HIV‐1) biogenesis. Proteomics coupled with exosome fractionation has been successfully used to identify various exosomal proteins and helped to uncover the interactions between exosomes and HIV‐1. To inform the current progress in the intersection of exosome, proteomics, and HIV‐1, this review is focused on: i) analyzing different exosome isolation, purification methods, and their implications in HIV‐1 studies; ii) evaluating the roles of various proteomic techniques in defining exosomal contents; iii) discussing the research and clinical applications of proteomics and exosome in HIV‐1 biology.  

3.3117           Cancer exosomes induce tumor innervation

Madeo, M., Colbert, P.L., Vermeer, D.W., Lucido, T., Cain, J.T. et al Nature Communications, 9:4284 (2018)   Patients with densely innervated tumors suffer with increased metastasis and decreased survival as compared to those with less innervated tumors. We hypothesize that in some tumors, nerves are acquired by a tumor-induced process, called axonogenesis. Here, we use PC12 cells as an in vitro neuronal model, human tumor samples and murine in vivo models to test this hypothesis. When appropriately stimulated, PC12 cells extend processes, called neurites. We show that patient tumors release vesicles, called exosomes, which induce PC12 neurite outgrowth. Using a cancer mouse model, we show that tumors compromised in exosome release are less innervated than controls. Moreover, in vivo pharmacological blockade of exosome release similarly attenuates tumor innervation. We characterize these nerves as sensory in nature and demonstrate that axonogenesis is potentiated by the exosome-packaged axonal guidance molecule, EphrinB1. These findings indicate that tumor released exosomes induce tumor innervation and exosomes containing EphrinB1 potentiate this activity.  

3.3118           Extracellular Vesicles and the Application of System Biology and Computational Modeling in Cardiac Repair

Garikipati, V.N.S., Shoja-Taheri, F., Davis, M.E. and Koshore, R. Circ. Res., 123, 188-204 (2018)   Recent literature suggests that extracellular vesicles (EVs), secreted from most cells and containing cell-specific cargo of proteins, lipids, and nucleic acids, are major driver of intracellular communication in normal physiology and pathological conditions. The recent evidence on stem/progenitor cell EVs as potential therapeutic modality mimicking their parental cell function is exciting because EVs could possibly be used as a surrogate for the stem cell–based therapy, and this regimen may overcome certain roadblocks identified with the use of stem/progenitor cell themselves. This review provides a comprehensive update on our understanding on the role of EVs in cardiac repair and emphasizes the applications of stem/progenitor cell–derived EVs as therapeutics and discusses the current challenges associated with the EV therapy.  

3.3119           Metastatic breast cancer cells overexpress and secrete miR-218 to regulate type I collagen deposition by osteoblasts

Liu, X., Cao, M., Palomares, M., Wu, X., Li, A., Yan, W., Fong, M.Y., Chan, W-C. and Wang, S.E. Breast Cancer Res., 20:127 (2018)   Background Bone is one of the most frequent metastatic sites of advanced breast cancer. Current therapeutic agents aim to inhibit osteoclast-mediated bone resorption but only have palliative effects. During normal bone remodeling, the balance between bone resorption and osteoblast-mediated bone formation is essential for bone homeostasis. One major function of osteoblast during bone formation is to secrete type I procollagen, which will then be processed before being crosslinked and deposited into the bone matrix. Methods Small RNA sequencing and quantitative real-time PCR were used to detect miRNA levels in patient blood samples and in the cell lysates as well as extracellular vesicles of parental and bone-tropic MDA-MB-231 breast cancer cells. The effects of cancer cell-derived extracellular vesicles isolated by ultracentrifugation and carrying varying levels of miR-218 were examined in osteoblasts by quantitative real-time PCR, Western blot analysis, and P1NP bone formation marker analysis. Cancer cells overexpressing miR-218 were examined by transcriptome profiling through RNA sequencing to identify intrinsic genes and pathways influenced by miR-218. Results We show that circulating miR-218 is associated with breast cancer bone metastasis. Cancer-secreted miR-218 directly downregulates type I collagen in osteoblasts, whereas intracellular miR-218 in breast cancer cells regulates the expression of inhibin β subunits. Increased cancer secretion of inhibin βA results in elevated Timp3 expression in osteoblasts and the subsequent repression of procollagen processing during osteoblast differentiation. Conclusions Here we identify a twofold function of cancer-derived miR-218, whose levels in the blood are associated with breast cancer metastasis to the bone, in the regulation of type I collagen deposition by osteoblasts. The adaptation of the bone niche mediated by miR-218 might further tilt the balance towards osteolysis, thereby facilitating other mechanisms to promote bone metastasis.  

3.3120           Plasma Exosomes Contribute to Microvascular Damage in Diabetic Retinopathy by Activating the Classical Complement Pathway

Huang, C., Fischer, K.P., Hammer, S.S., Navitskaya, S., Blanchard, G.J. and Busik, J.V. Diabetes, 67(8), 1639-1649 (2018)   Diabetic retinopathy (DR) is a microvascular complication of diabetes and is the leading cause of vision loss in working-age adults. Recent studies have implicated the complement system as a player in the development of vascular damage and progression of DR. However, the role and activation of the complement system in DR are not well understood. Exosomes, small vesicles that are secreted into the extracellular environment, have a cargo of complement proteins in plasma, suggesting that they can participate in causing the vascular damage associated with DR. We demonstrate that IgG-laden exosomes in plasma activate the classical complement pathway and that the quantity of these exosomes is increased in diabetes. Moreover, we show that a lack of IgG in exosomes in diabetic mice results in a reduction in retinal vascular damage. The results of this study demonstrate that complement activation by IgG-laden plasma exosomes could contribute to the development of DR.  

3.3121           The Atg2-Atg18 complex tethers pre-autophagosomal membranes to the endoplasmic reticulum for autophagosome formation

Kotani, T., Kirisako, H., Koizumi, M., Ohsumi, Y. and Nakatogawa, H. PNAS, 115(41), 10363-10368 (2018)   The biogenesis of double-membrane vesicles called autophagosomes, which sequester and transport intracellular material for degradation in lysosomes or vacuoles, is a central event in autophagy. This process requires a unique set of factors called autophagy-related (Atg) proteins. The Atg proteins assemble to organize the preautophagosomal structure (PAS), at which a cup-shaped membrane, the isolation membrane (or phagophore), forms and expands to become the autophagosome. The molecular mechanism of autophagosome biogenesis remains poorly understood. Previous studies have shown that Atg2 forms a complex with the phosphatidylinositol 3-phosphate (PI3P)-binding protein Atg18 and localizes to the PAS to initiate autophagosome biogenesis; however, the molecular function of Atg2 remains unknown. In this study, we show that Atg2 has two membrane-binding domains in the N- and C-terminal regions and acts as a membrane tether during autophagosome formation in the budding yeast Saccharomyces cerevisiae. An amphipathic helix in the C-terminal region binds to membranes and facilitates Atg18 binding to PI3P to target the Atg2-Atg18 complex to the PAS. The N-terminal region of Atg2 is also involved in the membrane binding of this protein but is dispensable for the PAS targeting of the Atg2-Atg18 complex. Our data suggest that this region associates with the endoplasmic reticulum (ER) and is responsible for the formation of the isolation membrane at the PAS. Based on these results, we propose that the Atg2-Atg18 complex tethers the PAS to the ER to initiate membrane expansion during autophagosome formation.  

3.3122           A molecular mechanism for calcium-mediated synaptotagmin-triggered exocytosis

Kiessling, V., Kreutzberger, A.J.B., Liang, B., Nyenhuis, S.B., Seelheim, P., Castle, J.D., Cafiso, D.S. and Tamm, L.K. Nature Struct. Mol. Biol., 25, 911-917 (2018)   The regulated exocytotic release of neurotransmitter and hormones is accomplished by a complex protein machinery whose core consists of SNARE proteins and the calcium sensor synaptotagmin-1. We propose a mechanism in which the lipid membrane is intimately involved in coupling calcium sensing to release. We found that fusion of dense core vesicles, derived from rat PC12 cells, was strongly linked to the angle between the cytoplasmic domain of the SNARE complex and the plane of the target membrane. We propose that, as this tilt angle increases, force is exerted on the SNARE transmembrane domains to drive the merger of the two bilayers. The tilt angle markedly increased following calcium-mediated binding of synaptotagmin to membranes, strongly depended on the surface electrostatics of the membrane, and was strictly coupled to the lipid order of the target membrane.  

3.3123           A CRISPR screen identifies IFI6 as an ER-resident interferon effector that blocks flavivirus replication

Richardson, R.B., Ohlson, M.B., Eitson, J.L., Kumar, A., McDougal, M.B., Boys, I.N., Mar, K.B., De La Cruz-Rivera, P.C., Douglas, C., Konopka, G., Xing, C. and Schoggins, J.W. Nature Microbiol., 3, 1214-1223 (2018)   The endoplasmic reticulum (ER) is an architecturally diverse organelle that serves as a membrane source for the replication of multiple viruses. Flaviviruses, including yellow fever virus, West Nile virus, dengue virus and Zika virus, induce unique single-membrane ER invaginations that house the viral replication machinery1. Whether this virus-induced ER remodelling is vulnerable to antiviral pathways is unknown. Here, we show that flavivirus replication at the ER is targeted by the interferon (IFN) response. Through genome-scale CRISPR screening, we uncovered an antiviral mechanism mediated by a functional gene pairing between IFI6 (encoding IFN-α-inducible protein 6), an IFN-stimulated gene cloned over 30 years ago2, and HSPA5, which encodes the ER-resident heat shock protein 70 chaperone BiP. We reveal that IFI6 is an ER-localized integral membrane effector that is stabilized through interactions with BiP. Mechanistically, IFI6 prophylactically protects uninfected cells by preventing the formation of virus-induced ER membrane invaginations. Notably, IFI6 has little effect on other mammalian RNA viruses, including the related Flaviviridae family member hepatitis C virus, which replicates in double-membrane vesicles that protrude outwards from the ER. These findings support a model in which the IFN response is armed with a membrane-targeted effector that discriminately blocks the establishment of virus-specific ER microenvironments that are required for replication.  

3.3124           Polarized Secretion of Extracellular Vesicles by Mammary Epithelia

Chin, A.R., Yan, W., Cao, M., Liu, X. and Wang, S.E.

1. Mammary Gland Biol. Neoplasia, 23, 165-170 (2018)

  Extracellular vesicles (EVs) are secreted by many cell types and are increasingly investigated for their role in human diseases including cancer. Here we focus on the secretion and potential physiological function of non-pathological EVs secreted by polarized normal mammary epithelial cells. Using a transwell system to allow formation of epithelial polarity and EV collection from the apical versus basolateral compartments, we found that impaired secretion of EVs by knockdown of RAB27A or RAB27B suppressed the establishment of mammary epithelial polarity, and that addition of apical but not basolateral EVs suppressed epithelial polarity in a dose-dependent manner. This suggests that apical EV secretion contributes to epithelial polarity, and a possible mechanism is through removal of certain intracellular molecules. In contrast, basolateral but not apical EVs promoted migration of mammary epithelial cells in a motility assay. The protein contents of apical and basolateral EVs from MCF10A and primary human mammary epithelial cells were determined by mass spectrometry proteomic analysis, identifying apical-EV-enriched and basolateral-EV-enriched proteins that may contribute to different physiological functions. Most of these proteins differentially secreted by normal mammary epithelial cells through polarized EV release no longer showed polarized secretion in MCF10A-derived transformed epithelial cells. Our results suggest an essential role of EV secretion in normal mammary epithelial polarization and distinct protein contents and functions in apical versus basolateral EVs secreted by polarized mammary epithelia.    

3.3125           An exploratory study on the effect of daily fruits and vegetable juice on human gut microbiota

Choi, Y.J., Lee, D.H., Kim, H.S. and Kim, Y-K. Food Sci. Biotechnol., 27(5), 1377-1386 82018)   In order to evaluate the effect of daily consumption of fruit and vegetable juice on the human intestinal microbial community, we compared changes in the gut microbiota and extracellular vesicles in human feces and bowel, and skin symptoms at the baseline and 3 weeks post juice consumption of 22 participants. After 3 weeks of juice consumption, a significant increase in the richness of microbiota (α-diversity, P < 0.05) was observed. It was accompanied by an abundance in Faecalibacterium (bacterial: from 1.62 ± 0.80% to 2.14 ± 0.72% and extra vesicle: 2.49 ± 1.49% to 6.06 ± 3.07%; P < 0.05 in all cases). At the end of the study period, there were reductions in body weight regardless of sex (P < 0.05) and improvements of the symptoms including diarrhea, constipation, fatigue, and skin problems. Eating fruits and vegetables could help modulate the profile of the fecal microbiota and alleviate bowel and skin troubles, and fatigue.  

3.3126           Infection-Induced Peroxisome Biogenesis Is a Metabolic Strategy for Herpesvirus Replication

Beltran, P.M.J., Cook, K.C., hashimoto, Y., Galitzine, C., Murray, L.A., Vitek, O. and Cristea, I.M. Cell Host & Microbe, 24, 526-541 (2018)   Viral proteins have evolved to target cellular organelles and usurp their functions for virus replication. Despite the knowledge of these critical functions for several organelles, little is known about peroxisomes during infection. Peroxisomes are primarily metabolic organelles with important functions in lipid metabolism. Here, we discovered that the enveloped viruses human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) induce the biogenesis of and unique morphological changes to peroxisomes to support their replication. Targeted proteomic quantification revealed a global virus-induced upregulation of peroxisomal proteins. Mathematical modeling and microscopy structural analysis show that infection triggers peroxisome growth and fission, leading to increased peroxisome numbers and irregular disc-like structures. HCMV-induced peroxisome biogenesis increased the phospholipid plasmalogen, thereby enhancing virus production. Peroxisome regulation and dependence were not observed for the non-enveloped adenovirus. Our findings uncover a role of peroxisomes in viral pathogenesis, with likely implications for multiple enveloped viruses.  

3.3127           UBXN2A enhances CHIP‐mediated proteasomal degradation of oncoprotein mortalin‐2 in cancer cells

Sane, S., Hafner, A., Srinivasan, R., Masood, D., Slunecka, J.I., Noldner, C.J., Hanson, A.D., Kruisselbrink, T., Wang, X., Wang, Y., Yin, J. and Rezvani, K. Mol. Oncol., 12, 1753-1777 (2018)   Overexpression of oncoproteins is a major cause of treatment failure using current chemotherapeutic drugs. Drug‐induced degradation of oncoproteins is feasible and can improve clinical outcomes in diverse types of cancers. Mortalin‐2 (mot‐2) is a dominant oncoprotein in several tumors, including colorectal cancer (CRC). In addition to inactivating the p53 tumor suppressor protein, mot‐2 enhances tumor cell invasion and migration. Thus, mot‐2 is considered a potential therapeutic target in several cancer types. The current study investigated the biological role of a ubiquitin‐like protein called UBXN2A in the regulation of mot‐2 turnover. An orthogonal ubiquitin transfer technology followed by immunoprecipitation, in vitro ubiquitination, and Magnetic Beads TUBE2 pull‐down experiments revealed that UBXN2A promotes carboxyl terminus of the HSP70‐interacting protein (CHIP)‐dependent ubiquitination of mot‐2. We subsequently showed that UBXN2A increases proteasomal degradation of mot‐2. A subcellular compartmentalization experiment revealed that induced UBXN2A decreases the level of mot‐2 and its chaperone partner, HSP60. Pharmacological upregulation of UBXN2A using a small molecule, veratridine (VTD), decreases the level of mot‐2 in cancer cells. Consistent with the in vitro results, UBXN2A^+/− mice exhibited selective elevation of mot‐2 in colon tissues. An in vitro Anti‐K48 TUBE isolation approach showed that recombinant UBXN2A enhances proteasomal degradation of mot‐2 in mouse colon tissues. Finally, we observed enhanced association of CHIP with the UBXN2A‐mot‐2 complex in tumors in an azoxymethane/dextran sulfate sodium‐induced mouse CRC model. The existence of a multiprotein complex containing UBXN2A, CHIP, and mot‐2 suggests a synergistic tumor suppressor activity of UBXN2A and CHIP in mot‐2‐enriched tumors. This finding validates the UBXN2A‐CHIP axis as a novel and potential therapeutic target in CRC.  

3.3128           Natural T‐cell ligands that are created by genetic variants can be transferred between cells by extracellular vesicles

Kremer, A.N., Zonneveld, M.I., Kremer, A.E., van der Meijden, E.D., Falkenburg, J.H.F., Wauben, M.H.M., Nolte-‘tHoen, E.N.M. and Griffioen, M. Eur. J. Immunol., 48, 1621-1631 (2018)   CD4 T cells play a central role as helper cells in adaptive immunity. Presentation of exogenous antigens in MHC class II by professional antigen‐presenting cells is a crucial step in induction of specific CD4 T cells in adaptive immune responses. For efficient induction of immunity against intracellular threats such as viruses or malignant transformations, antigens from HLA class II‐negative infected or transformed cells need to be transferred to surrounding antigen‐presenting cells to allow efficient priming of naive CD4 T cells. Here we show indirect antigen presentation for a subset of natural HLA class II ligands that are created by genetic variants and demonstrated that (neo)antigens can be transferred between cells by extracellular vesicles. Intercellular transfer by extracellular vesicles was not dependent on the T‐cell epitope, but rather on characteristics of the full‐length protein. This mechanism of (neo)antigen transfer from HLA class II‐negative cells to surrounding antigen‐presenting cells may play a crucial role in induction of anti‐tumor immunity.  

3.3129           Recent Progress in Isolation and Detection of Extracellular Vesicles for Cancer Diagnostics

Wang, W., Luo, J. and Wang, S. Adv. Healthcare Materials, 7:1800484 (2018)   Extracellular vesicles (EVs) are emerging as one of the many new and promising biomarkers for liquid biopsy of cancer due to their loading capability of some specific proteins and nucleic acids that are closely associated with cancer states. As such, the isolation and detection of cancer‐derived EVs offer important information in noninvasive diagnosis of early‐stage cancer and real‐time monitoring of cancer development. In light of the importance of EVs, over the last decade, researchers have made remarkable innovations to advance the development of EV isolation and detection methods by taking advantage of microfluidics, biomolecule probes, nanomaterials, surface plasmon, optics, and so on. This review introduces the basic properties of EVs and common cancer‐derived EV ingredients, and provides a comprehensive overview of EV isolation and detection strategies, with emphasis on liquid biopsies of EVs for cancer diagnostics.  

3.3130           Intracellular sorting and transcytosis of the rat transferrin receptor antibody OX26 across the blood–brain barrier in vitro is dependent on its binding affinity

Haqqani, A.S., Thom, G., Burrell, M., Delaney, C.E., Brunette, E., Baumann, E., Sodja, C., Jezierki, A., Webster, C. and Stanimirovic, D.B.

1. Neurochem., 146, 735-752 (2018)

  The blood–brain barrier (BBB) is a formidable obstacle to the delivery of therapeutics to the brain. Antibodies that bind transferrin receptor (TfR), which is enriched in brain endothelial cells, have been shown to cross the BBB and are being developed as fusion proteins to deliver therapeutic cargos to brain targets. Various antibodies have been developed for this purpose and their in vivo evaluation demonstrated that either low affinity or monovalent receptor binding re‐directs their transcellular trafficking away from lysosomal degradation and toward improved exocytosis on the abluminal side of the BBB. However, these studies have been performed with antibodies that recognize different TfR epitopes and have different binding characteristics, preventing inter‐study comparisons. In this study, the efficiency of transcytosis in vitro and intracellular trafficking in endosomal compartments were evaluated in an in vitro BBB model for affinity variants (K_d from 5 to174 nM) of the rat TfR‐binding antibody, OX26. Distribution in subcellular fractions of the rat brain endothelial cells was determined using both targeted quantitative proteomics‐selected reaction monitoring and fluorescent imaging with markers of early‐ and late endosomes. The OX26 variants with affinities of 76 and 108 nM showed improved trancytosis (P_app values) across the in vitro BBB model compared with a 5 nM OX26. Although ~40% of the 5 nM OX26 and ~35% of TfR co‐localized with late‐endosome/lysosome compartment, 76 and 108 nM affinity variants showed lower amounts in lysosomes and a predominant co‐localization with early endosome markers. The study links bivalent TfR antibody affinity to mechanisms of sorting and trafficking away from late endosomes and lysosomes, resulting in improvement in their transcytosis efficiency.    

3.3131           Sphingolipids regulate neuromuscular synapse structure and function in Drosophila

West, R.J., Briggs, L., Fjelstad, M.P., Ribchester, R.R. and Sweeney, S.T.

1. Comp. Neurol., 526, 1995-2009 (2018)

  Sphingolipids are found in abundance at synapses and have been implicated in regulation of synapse structure, function, and degeneration. Their precise role in these processes, however, remains obscure. Serine Palmitoyl‐transferase (SPT) is the first enzymatic step for synthesis of sphingolipids. Analysis of the Drosophila larval neuromuscular junction (NMJ) revealed mutations in the SPT enzyme subunit, lace/SPTLC2 resulted in deficits in synaptic structure and function. Although NMJ length is normal in lace mutants, the number of boutons per NMJ is reduced to ∼50% of the wild type number. Synaptic boutons in lace mutants are much larger but show little perturbation to the general ultrastructure. Electrophysiological analysis of lace mutant synapses revealed strong synaptic transmission coupled with predominance of depression over facilitation. The structural and functional phenotypes of lace mirrored aspects of Basigin (Bsg), a small Ig‐domain adhesion molecule also known to regulate synaptic structure and function. Mutant combinations of lace and Bsg generated large synaptic boutons, while lace mutants showed abnormal accumulation of Bsg at synapses, suggesting that Bsg requires sphingolipid to regulate structure of the synapse. In support of this, we found Bsg to be enriched in lipid rafts. Our data points to a role for sphingolipids in the regulation and fine‐tuning of synaptic structure and function while sphingolipid regulation of synaptic structure may be mediated via the activity of Bsg.  

3.3132           Exosomes play a role in multiple myeloma bone disease and tumor development by targeting osteoclasts and osteoblasts

Faict, S., Muller, J., De Veirman, K., De Bruyne, E., Maes, K., Vrancken, L., Heusschen, R., De Raeve, H., Schots, R., Vanderkerken, K., Caers, j. and Menu, E. Blood Cancer J., 8:105 (2018)   Progression of multiple myeloma (MM) is largely dependent on the bone marrow (BM) microenvironment wherein communication through different factors including extracellular vesicles takes place. This cross-talk not only leads to drug resistance but also to the development of osteolysis. Targeting vesicle secretion could therefore simultaneously ameliorate drug response and bone disease. In this paper, we examined the effects of MM exosomes on different aspects of osteolysis using the 5TGM1 murine model. We found that 5TGM1 sEVs, or ‘exosomes’, not only enhanced osteoclast activity, they also blocked osteoblast differentiation and functionality in vitro. Mechanistically, we could demonstrate that transfer of DKK-1 led to a reduction in Runx2, Osterix, and Collagen 1A1 in osteoblasts. In vivo, we uncovered that 5TGM1 exosomes could induce osteolysis in a similar pattern as the MM cells themselves. Blocking exosome secretion using the sphingomyelinase inhibitor GW4869 not only increased cortical bone volume, but also it sensitized the myeloma cells to bortezomib, leading to a strong anti-tumor response when GW4869 and bortezomib were combined. Altogether, our results indicate an important role for exosomes in the BM microenvironment and suggest a novel therapeutic target for anti-myeloma therapy.  

3.3133           Neomorphic PDGFRA extracellular domain driver mutations are resistant to PDGFRA targeted therapies

Ip, C.K.M., Ng, P.K.S., Jeong, K.J., Shao, S.H., Ju, Z., Leonard, P.G., Hua, X., Vellano, C., Woessner, R., Sahni, N., Scott, K.L. and Mills, G.B. Nature Communications, 9:4583 (2018)   Activation of platelet-derived growth factor receptor alpha (PDGFRA) by genomic aberrations contributes to tumor progression in several tumor types. In this study, we characterize 16 novel PDGFRA mutations identified from different tumor types and identify three previously uncharacterized activating mutations that promote cell survival and proliferation. PDGFRA Y288C, an extracellular domain mutation, is primarily high mannose glycosylated consistent with trapping in the endoplasmic reticulum (ER). Strikingly, PDGFRA Y288C is constitutively dimerized and phosphorylated in the absence of ligand suggesting that trapping in the ER or aberrant glycosylation is sufficient for receptor activation. Importantly, PDGFRA Y288C induces constitutive phosphorylation of Akt, ERK1/2, and STAT3. PDGFRA Y288C is resistant to PDGFR inhibitors but sensitive to PI3K/mTOR and MEK inhibitors consistent with pathway activation results. Our findings further highlight the importance of characterizing functional consequences of individual mutations for precision medicine.    

3.3134           Emerin Deregulation Links Nuclear Shape Instability to Metastatic Potential

Reis-sobreiro, M., Chen, J-F., Novitskaya, T., You, S., Morley, S. et al Cancer Res., 78(21), 6086-6097 (2018)   Abnormalities in nuclear shape are a well-known feature of cancer, but their contribution to malignant progression remains poorly understood. Here, we show that depletion of the cytoskeletal regulator, Diaphanous-related formin 3 (DIAPH3), or the nuclear membrane–associated proteins, lamin A/C, in prostate and breast cancer cells, induces nuclear shape instability, with a corresponding gain in malignant properties, including secretion of extracellular vesicles that contain genomic material. This transformation is characterized by a reduction and/or mislocalization of the inner nuclear membrane protein, emerin. Consistent with this, depletion of emerin evokes nuclear shape instability and promotes metastasis. By visualizing emerin localization, evidence for nuclear shape instability was observed in cultured tumor cells, in experimental models of prostate cancer, in human prostate cancer tissues, and in circulating tumor cells from patients with metastatic disease. Quantitation of emerin mislocalization discriminated cancer from benign tissue and correlated with disease progression in a prostate cancer cohort. Taken together, these results identify emerin as a mediator of nuclear shape stability in cancer and show that destabilization of emerin can promote metastasis  

3.3135           Secretion and fusion of biogeochemically active archaeal membrane vesicles

Johnson, T.B., Mach, C., Grove, R., Kelly, R., Van Cott, K. and Blum, P. Geobiology, 16, 659-673 (2018)   Microbes belonging to the genus Metallosphaera oxidize sulfidic minerals. These organisms thrive at , 17001lithoautotrophic metabolism, energy availability likely limits their activity raising questions about how they conduct biogeochemical activity. Vesicles are membrane encapsulated structures produced by all biological lineages but using very different mechanisms. Across the Crenarchaeota, it has been proposed that a eukaryotic‐like Endosomal Sorting Complex Required for Transport system promotes formation of these structures but in response to unknown signals and for undefined purposes. To address such questions, Metallosphaera sedula vesicle formation and function were studied under lithoautotrophic conditions. Energy deprivation was evaluated and found to stimulate vesicle synthesis while energy excess repressed vesicle formation. Purified vesicles adhered rapidly to the primary copper ore, chalcopyrite, and formed compact monolayers. These vesicle monolayers catalyzed iron oxidation and solubilization of mineralized copper in a time‐dependent process. As these activities were membrane associated, their potential transfer by vesicle fusion to M. sedula cells was examined. Fluorophore‐loaded vesicles rapidly transferred fluorescence under environmentally relevant conditions. Vesicles from a related archaeal species were also capable of fusion; however, this process was species‐specific as vesicles from different species were incapable of fusion. In addition, vesicles produced by a copper‐resistant M. sedula cell line transferred copper extrusion capacity along with improved viability over mutant M. sedula cells lacking copper transport proteins. Membrane vesicles may therefore play a role in modulating energy‐related traits in geochemical environments by fusion‐mediated protein delivery.  

3.3136           Protein Palmitoylation Plays an Important Role in Trichomonas vaginalis Adherence

Nievas, Y.R., Vashisht, A.A., Corvi, M.M., metz, S., Johnson, P.J., Wohlschlegel, J.A. and de Miguel, N. Mol. Cell. Proteomics, 17(11), 2229-2241 (2018)   The flagellated protozoan parasite Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted infection worldwide. As an obligate extracellular pathogen, adherence to epithelial cells is critical for parasite survival within the human host and a better understanding of this process is a prerequisite for the development of therapies to combat infection. In this sense, recent work has shown S-acylation as a key modification that regulates pathogenesis in different protozoan parasites. However, there are no reports indicating whether this post-translational modification is a mechanism operating in T. vaginalis. In order to study the extent and function of S-acylation in T. vaginalis biology, we undertook a proteomic study to profile the full scope of S-acylated proteins in this parasite and reported the identification of 363 proteins involved in a variety of biological processes such as protein transport, pathogenesis related and signaling, among others. Importantly, treatment of parasites with the palmitoylation inhibitor 2-bromopalmitate causes a significant decrease in parasite: parasite aggregation as well as adherence to host cells suggesting that palmitoylation could be modifying proteins that are key regulators of Trichomonas vaginalis pathogenesis.  

3.3137           β‐elemene increases the sensitivity of gastric cancer cells to TRAIL by promoting the formation of DISC in lipid rafts

Xu, L., Guo, T., Qu, X., Hu, X., Zhang, Y., Che, X. et al Cell Biol. Int., 42(10), 1377-1385 (2018)   β‐Elemene, an anti‐cancer drug extracted from traditional Chinese medicinal herb, showed anti‐tumor effects on gastric cancer cells. Our previous studies reported gastric cancer cells are insensitive to TRAIL. However, whether β‐elemene could enhance anti‐cancer effects of TRAIL on gastric cancer cells is unknown. In our present study, β‐elemene prevented gastric cancer cell viability in dose‐dependent manner, and when combined with TRAIL, obviously inhibited proliferation and promoted apoptosis in gastric cancer cells. Compared to β‐elemene or TRAIL alone, treatment with β‐elemene and TRAIL obviously promoted DR5 clustering as well as translocation of Caspase‐8, DR5 and FADD into lipid rafts. This led to cleavage of Caspase‐8 and the formation of death‐inducing signaling complex (DISC) in lipid rafts. The cholesterol‐sequestering agent nystatin partially reversed DR5 clustering and DISC formation, preventing apoptosis triggered by the combination of β‐elemene and TRAIL. Our results suggest that β‐elemene increases the sensitivity of gastric cancer cells to TRAIL partially by promoting the formation of DISC in lipid rafts.  

3.3138           Mammalian Cell-derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies.

Moonschi, F.H., Fox-Loe, A.M., Fu, X. and Richards, C.I. Bio Protoc., 8(9), e2825 (2018)   Cell-derived vesicles facilitate the isolation of transmembrane proteins in their physiological membrane maintaining their structural and functional integrity. These vesicles can be generated from different cellular organelles producing, housing, or transporting the proteins. Combined with single-molecule imaging, isolated organelle specific vesicles can be employed to study the trafficking and assembly of the embedded proteins. Here we present a method for organelle specific single molecule imaging via isolation of ER and plasma membrane vesicles from HEK293T cells by employing OptiPrep gradients and nitrogen cavitation. The isolation was validated through Western blotting, and the isolated vesicles were used to perform single molecule studies of oligomeric receptor assembly.  

3.3139           Isolation of High-Purity Extracellular Vesicles by the Combination of Iodixanol Density Gradient Ultracentrifugation and Bind-Elute Chromatography From Blood Plasma.

Onodi, Z., Pelyhe, C., Nagy, T., Brenner, G.B., Almasi, L., Kittel, A., Mancek-Keber, M., Ferdinandy, P., Buzas, E.I. and Giricz, Z. Fontiers in Physiol., 9:1479 (2018)   Background: Extracellular vesicles (EVs) (isolated from blood plasma) are currently being extensively researched, both as biomarkers and for their therapeutic possibilities. One challenging aspect to this research is the efficient isolation of high-purity EVs from blood plasma in quantities sufficient for in vivo experiments. In accordance with this challenge, the aim of this study was to develop an isolation method in which to separate the majority of EVs from major impurities such as lipoprotein particles and the abundant plasma proteins albumin and fibrinogen. Methods: Samples of rat blood were centrifuged to remove cells, platelets, large EVs and protein aggregates without prior filtration. Density gradient ultracentrifugation was performed by loading plasma sample onto 50, 30, and 10% iodixanol layers and then centrifuged at 120,000 ×g for 24 h. Ten fractions (F1-10) were collected from top to bottom. Fractions with the highest EV content were further purified by ultracentrifugation, size exclusion, or bind-elute chromatography. Efficiency and purity were assessed by Western blots. Morphology and size distribution of particles were examined by dynamic light scattering and electron microscopy (EM). Results: The highest band intensities of EV markers Alix, Tsg101 and CD81 were detected by Western blot in F6 of small-scale DGUC (61.5 ± 10.4%; 48.1 ± 5.8%; 41.9 ± 3.8%, respectively) at a density of 1.128-1.174 g/mL, where the presence of vesicles with a mean diameter of 38 ± 2 nm was confirmed by EM and DLS. Only 1.4 ± 0.5% of LDL and chylomicron marker, 3.0 ± 1.3% of HDL marker, and 9.9 ± 0.4% of albumin remained in the EV-rich F6. However, 32.8 ± 1.5% of the total fibrinogen beta was found in this fraction. Second-step purification by UC or SEC did not improve EV separation, while after BEC on HiScreen Capto Core 700 albumin and lipoprotein contamination were below detection limit in EV-rich fractions. However, BEC decreased efficiency of EV isolation, and fibrinogen was still present in EV-rich fractions. Conclusion: This is the first demonstration that DGUC is able to markedly reduce the lipoprotein content of EV isolates while it separates EVs with high efficiency. Moreover, isolation of lipoprotein- and albumin-free EVs from blood plasma can be achieved by DGUC followed by BEC, however, on the expense of reduced EV yield.  

3.3140           Staphylococcus aureus Membrane-Derived Vesicles Promote Bacterial Virulence and Confer Protective Immunity in Murine Infection Models

Askarian, F., Lapek Jr, J.D., Dongre, M., Tsai, C-M, T., Kumaraswamy, M., Kousha, A., Valderrama, J.A., Ludviksen, J.A., Cavanagh, J.P., Uchiyama, S., Mollnes, T.E., Gonzalez, D.J., Wai, S.N., Nizet, V. and Johannessen, M. Frontiers in Microbiol., 9:262 (2018)   Staphylococcus aureus produces membrane-derived vesicles (MVs), which share functional properties to outer membrane vesicles. Atomic force microscopy revealed that S. aureus-derived MVs are associated with the bacterial surface or released into the surrounding environment depending on bacterial growth conditions. By using a comparative proteomic approach, a total of 131 and 617 proteins were identified in MVs isolated from S. aureus grown in Luria-Bertani and brain-heart infusion broth, respectively. Purified S. aureus MVs derived from the bacteria grown in either media induced comparable levels of cytotoxicity and neutrophil-activation. Administration of exogenous MVs increased the resistance of S. aureus to killing by whole blood or purified human neutrophils ex vivo and increased S. aureus survival in vivo. Finally, immunization of mice with S. aureus-derived MVs induced production of IgM, total IgG, IgG1, IgG2a, and IgG2b resulting in protection against subcutaneous and systemic S. aureus infection. Collectively, our results suggest S. aureus MVs can influence bacterial–host interactions during systemic infections and provide protective immunity in murine models of infection.  

3.3141           Transmission of Cricket paralysis virus via exosome-like vesicles during infection of Drosophila cells

Kerr, C.H., Dalwadi, U., Scott, N.E., Yip, C.K., Foster, L.J. and Jan, E. Scientific Reports, 8:17353 (2018)   Viruses are classically characterized as being either enveloped or nonenveloped depending on the presence or absence of a lipid bi-layer surrounding their proteinaceous capsid. In recent years, many studies have challenged this view by demonstrating that some nonenveloped viruses (e.g. hepatitis A virus) can acquire an envelope during infection by hijacking host cellular pathways. In this study, we examined the role of exosome-like vesicles (ELVs) during infection of Drosophilia melanogaster S2 cells by Cricket paralysis virus (CrPV). Utilizing quantitative proteomics, we demonstrated that ELVs can be isolated from both mock- and CrPV-infected S2 cells that contain distinct set of proteins compared to the cellular proteome. Moreover, 40 proteins increased in abundance in ELVs derived from CrPV-infected cells compared to mock, suggesting specific factors associate with ELVs during infection. Interestingly, peptides from CrPV capsid proteins (ORF2) and viral RNA were detected in ELVs from infected cells. Finally, ELVs from CrPV-infected cells are infectious suggesting that CrPV may hijack ELVs to acquire an envelope during infection of S2 cells. This study further demonstrates the diverse strategies of nonenveloped viruses from invertebrates to vertebrates to acquire an envelope in order to evade the host response or facilitate transmission.  

3.3142           Presenilin-mediated cleavage of APP regulates synaptotagmin-7 and presynaptic plasticity

Barthet, G., Jorda-Siquier, T., Rumi-Masante, J., Bernadou, F., Müller, U. and Mulle, C. Nature Communications, 9:4780 (2018)   Mutations of the intramembrane protease presenilin (PS) or of its main substrate, the amyloid precursor protein (APP), cause early-onset form of Alzheimer disease. PS and APP interact with proteins of the neurotransmitter release machinery without identified functional consequences. Here we report that genetic deletion of PS markedly decreases the presynaptic levels of the Ca²⁺ sensor synaptotagmin-7 (Syt7) leading to impaired synaptic facilitation and replenishment of synaptic vesicles. The regulation of Syt7 expression by PS occurs post-transcriptionally and depends on γ-secretase proteolytic activity. It requires the substrate APP as revealed by the combined genetic invalidation of APP and PS1, and in particular the APP-Cterminal fragments which interact with Syt7 and accumulate in synaptic terminals under pharmacological or genetic inhibition of γ-secretase. Thus, we uncover a role of PS in presynaptic mechanisms, through APP cleavage and regulation of Syt7, that highlights aberrant synaptic vesicle processing as a possible new pathway in AD.    

3.3143           Proteomic analysis of insulin secretory granules in INS-1 cells by protein correlation profiling

Li, M., Du, W., Zhou, M., Zheng, L., Song, E. and Hou, J. Biophys. Rep., 4(6), 329-338 (2018)   Insulin secretory granules (ISGs), a group of distinguishing organelles in pancreatic β cells, are responsible for the storage and secretion of insulin to maintain blood glucose homeostasis. The molecular mechanisms of ISG biogenesis, maturation, transportation, and exocytosis are still largely unknown because the proteins involved in these distinct steps have not been fully identified. Subcellular fractionation by density gradient centrifugation has been successfully employed to analyze the proteomes of numerous organelles. However, use of this method to elucidate the ISG proteome is limited by co-fractionated contaminants because ISGs are very dynamic and have abundant exchanges or contacts with other organelles, such as the Golgi apparatus, lysosomes, and endosomes. In this study, we developed a new strategy for identifying ISG proteins by protein correlation profiling (PCP)-based proteomics, which included ISG purification by OptiPrep density gradient centrifugation, label-free quantitative proteome, and identification of ISG proteins by correlating fractionation profiles between candidates and known ISG markers. Using this approach, we were able to identify 81 ISG proteins. Among them, TM9SF3, a nine-transmembrane protein, was considered a high confidence ISG candidate protein highlighted in the PCP network. Further biochemical and immunofluorescence assays indicated that TM9SF3 localized in ISGs, suggesting that it is a potential new ISG marker.  

3.3144           The Mechanism behind Bacterial Lipoprotein Release: Phenol-Soluble Modulins Mediate Toll-Like Receptor 2 Activation via Extracellular Vesicle Release from Staphylococcus aureus

Schlatterer, K., Beck, C., Hanzelmann, D., Lebtig, M., Fehrenbacher, B., Schaller, M., Ebner, P., Nega, M., Otto, M., Kretschmer, D. and Peschel, A. mBio, 9(6), e10851-18 (2018)   The innate immune system uses Toll-like receptor (TLR) 2 to detect conserved bacterial lipoproteins of invading pathogens. The lipid anchor attaches lipoproteins to the cytoplasmic membrane and prevents their release from the bacterial cell envelope. How bacteria release lipoproteins and how these molecules reach TLR2 remain unknown. Staphylococcus aureus has been described to liberate membrane vesicles. The composition, mode of release, and relevance for microbe-host interaction of such membrane vesicles have remained ambiguous. We recently reported that S. aureus can release lipoproteins only when surfactant-like small peptides, the phenol-soluble modulins (PSMs), are expressed. Here we demonstrate that PSM peptides promote the release of membrane vesicles from the cytoplasmic membrane of S. aureus via an increase in membrane fluidity, and we provide evidence that the bacterial turgor is the driving force for vesicle budding under hypotonic osmotic conditions. Intriguingly, the majority of lipoproteins are released by S. aureus as components of membrane vesicles, and this process depends on surfactant-like molecules such as PSMs. Vesicle disruption at high detergent concentrations promotes the capacity of lipoproteins to activate TLR2. These results reveal that vesicle release by bacterium-derived surfactants is required for TLR2-mediated inflammation.  

3.3145           Lysosomal Proteases Are a Determinant of Coronavirus Tropism

Zheng, Y., Shang, J., Yang, Y., Liu, C., Wan, Y., Geng, Q., Wang, M., baric, R. and Li, F.

1. Virol., 92(24), e01504-18 (2018)

  Cell entry by coronaviruses involves two principal steps, receptor binding and membrane fusion; the latter requires activation by host proteases, particularly lysosomal proteases. Despite the importance of lysosomal proteases in both coronavirus entry and cell metabolism, the correlation between lysosomal proteases and cell tropism of coronaviruses has not been established. Here, we examined the roles of lysosomal proteases in activating coronavirus surface spike proteins for membrane fusion, using the spike proteins from severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) as the model system. To this end, we controlled the contributions from receptor binding and other host proteases, thereby attributing coronavirus entry solely or mainly to the efficiency of lysosomal proteases in activating coronavirus spike-mediated membrane fusion. Our results showed that lysosomal proteases from bat cells support coronavirus spike-mediated pseudovirus entry and cell-cell fusion more effectively than their counterparts from human cells. Moreover, purified lysosomal extracts from bat cells cleave cell surface-expressed coronavirus spikes more efficiently than their counterparts from human cells. Overall, our study suggests that different lysosomal protease activities from different host species and tissue cells are an important determinant of the species and tissue tropism of coronaviruses.  

3.3146           Extracellular vesicles and their nucleic acids for biomarker discovery

Momen-Heravi, F., Getting, S.J. and Moschos, S.A. Pharmacol. & Therapeutics, 192, 170-187 (2018)   Extracellular vesicles (EVs) are a heterogenous population of vesicles originate from cells. EVs are found in different biofluids and carry different macromolecules, including proteins, lipids, and nucleic acids, providing a snap shot of the parental cells at the time of release. EVs have the ability to transfer molecular cargoes to other cells and can initiate different physiological and pathological processes. Mounting lines of evidence demonstrated that EVs' cargo and machinery is affected in disease states, positioning EVs as potential sources for the discovery of novel biomarkers. In this review, we demonstrate a conceptual overview of the EV field with particular focus on their nucleic acid cargoes. Current knowledge of EV subtypes, nucleic acid cargo and pathophysiological roles are outlined, with emphasis placed on advantages against competing analytes. We review the utility of EVs and their nucleic acid cargoes as biomarkers and critically assess the newly available advances in the field of EV biomarkers and high throughput technologies. Challenges to achieving the diagnostic potential of EVs, including sample handling, EV isolation, methodological considerations, and bioassay reproducibility are discussed. Future implementation of ‘omics-based technologies and integration of systems biology approaches for the development of EV-based biomarkers and personalized medicine are also considered.  

3.3147           Characterization of human mosaic Rett syndrome brain tissue by single-nucleus RNA sequencing

Renthal, W., Boxer, L.D., Hrvatin, S., Li, E., Silberfeld, A., Nagy, M.A., Grifficth, E.C., Vierbuvhen, T. and Greenberg, M.E. Nature Neurosci., 21, 1670-1679 (2018)   In females with X-linked genetic disorders, wild-type and mutant cells coexist within brain tissue because of X-chromosome inactivation, posing challenges for interpreting the effects of X-linked mutant alleles on gene expression. We present a single-nucleus RNA sequencing approach that resolves mosaicism by using single-nucleotide polymorphisms in genes expressed in cis with the X-linked mutation to determine which nuclei express the mutant allele even when the mutant gene is not detected. This approach enables gene expression comparisons between mutant and wild-type cells within the same individual, eliminating variability introduced by comparisons to controls with different genetic backgrounds. We apply this approach to mosaic female mouse models and humans with Rett syndrome, an X-linked neurodevelopmental disorder caused by mutations in the gene encoding the methyl-DNA-binding protein MECP2, and observe that cell-type-specific DNA methylation predicts the degree of gene upregulation in MECP2-mutant neurons. This approach can be broadly applied to study gene expression in mosaic X-linked disorders.  

3.3148           The Toll Pathway in the Central Nervous System of Flies and Mammals

Shmueli, A., Shalit, T., Okun, E. and Shohat-Ophir, G. Neuromol. Med., 20(4), 419-436 (2018)   Toll receptors, first identified to regulate embryogenesis and immune responses in the adult fly and subsequently defined as the principal sensors of infection in mammals, are increasingly appreciated for their impact on the homeostasis of the central as well as the peripheral nervous systems. Whereas in the context of immunity, the fly Toll and the mammalian TLR pathways have been researched in parallel, the expression pattern and functionality have largely been researched disparately. Herein, we provide data on the expression pattern of the Toll homologues, signaling components, and downstream effectors in ten different cell populations of the adult fly central nervous system (CNS). We have compared the expression of the different Toll pathways in the fly to the expression of TLRs in the mouse brain and discussed the implications with respect to commonalities, differences, and future perspectives.  

3.3149           High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis

Ramakrishnan, S., Gohlke, A., Li, F., Coleman, J., Xu, W., Rothman, J.E. and Pincet, F. Langmuir, 34(20), 5849-5859 (2018)   In vivo membrane fusion primarily occurs between highly curved vesicles and planar membranes. A better understanding of fusion entails an accurate in vitro reproduction of the process. To date, supported bilayers have been commonly used to mimic the planar membranes. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that induce membrane fusion usually have limited fluidity when embedded in supported bilayers. This alters the kinetics and prevents correct reconstitution of the overall fusion process. Also, observing content release across the membrane is hindered by the lack of a second aqueous compartment. Recently, a step toward resolving these issues was achieved by using membranes spread on holey substrates. The mobility of proteins was preserved but vesicles were prone to bind to the substrate when reaching the edge of the hole, preventing the observation of many fusion events over the suspended membrane. Building on this recent advance, we designed a method for the formation of pore-spanning lipid bilayers containing t-SNARE proteins on Si/SiO₂ holey chips, allowing the observation of many individual vesicle fusion events by both lipid mixing and content release. With this setup, proteins embedded in the suspended membrane bounced back when they reached the edge of the hole which ensured vesicles did not bind to the substrate. We observed SNARE-dependent membrane fusion with the freestanding bilayer of about 500 vesicles. The time between vesicle docking and fusion is ∼1 s. We also present a new multimodal open-source software, Fusion Analyzer Software, which is required for fast data analysis.  

3.3150           Selective Export into Extracellular Vesicles and Function of tRNA Fragments during T Cell Activation

Chiou, N-T., Kageyama, R. and Ansel, K.M. Cell Reports, 25, 3356-3370 (2018)   The discovery of microRNA (miRNA) sorting into extracellular vesicles (EVs) revealed a novel mode of intercellular communication and uncovered a link between cellular endomembrane compartments and small RNAs in EV-secreting cells. Using a two-step ultracentrifugation procedure to isolate EVs released by T cells, we found that 45% of tRNA fragments (tRFs), but fewer than 1% of miRNAs, were significantly enriched in EVs compared with the corresponding cellular RNA. T cell activation induced the EV-mediated release of a specific set of tRFs derived from the 5′ end and 3′-internal region of tRNAs without variable loops. Inhibition of EV biogenesis pathways specifically led to the accumulation of these activation-induced EV-enriched tRFs within multivesicular bodies (MVBs). Introducing antisense oligonucleotides to inhibit these tRFs enhanced T cell activation. Taken together, these results demonstrate that T cells selectively release tRFs into EVs via MVBs and suggest that this process may remove tRFs that repress immune activation.  

3.3151           Regulation of mitochondrion-associated cytosolic ribosomes by mammalian mitochondrial ribonuclease T2 (RNASET2)

Huang, J., Liu, P. and Wang, G.

1. Biol. Chem., 293(51), 19633-19644 (2018)

  Mitochondrial proteins are encoded in both mitochondrial and nuclear genomes. The expression levels of these two pools of mitochondrial genes are co-regulated and synchronized. Import and assembly of the nucleus-encoded oxidative phosphorylation (OXPHOS) subunits affect protein synthesis in the mitochondrial matrix by engaging the mitochondrial ribosomes. How the ribosomes at the outside of mitochondria are regulated by mitochondria, however, remains mostly unexplored. Here, using an array of biochemical assays and genetic knockdown and overexpression in HEK293 or mouse cells, we show that cytosolic rRNAs that are associated with the mitochondrial outer membrane have very different decay patterns from those of both endoplasmic reticulum–associated and –nonassociated cytosolic rRNAs. Mitochondrial intermembrane space RNase T2 (RNASET2), which has been previously shown to degrade mitochondrial RNAs, is also responsible for selective degradation of the cytosolic rRNAs on the outer membrane. We noted that the degradation activity also has a positive effect on nuclear transcription of rRNAs, suggesting a compensatory feedback mechanism, and affects protein translations in and out of mitochondria. These findings establish a mechanism for the co-regulation of gene expression programs inside and outside of mitochondria in mammalian cells.  

3.3152           The mitochondrial inner membrane protein MPV17 prevents uracil accumulation in mitochondrial DNA

Alonzo, J.R., Venkataraman, C., Field, M.S. and Stover, P.J.

1. Biol. Chem., 293(52), 20285-20294 (2018)

  Mitochondrial proteins are encoded in both mitochondrial and nuclear genomes. The expression levels of these two pools of mitochondrial genes are co-regulated and synchronized. Import and assembly of the nucleus-encoded oxidative phosphorylation (OXPHOS) subunits affect protein synthesis in the mitochondrial matrix by engaging the mitochondrial ribosomes. How the ribosomes at the outside of mitochondria are regulated by mitochondria, however, remains mostly unexplored. Here, using an array of biochemical assays and genetic knockdown and overexpression in HEK293 or mouse cells, we show that cytosolic rRNAs that are associated with the mitochondrial outer membrane have very different decay patterns from those of both endoplasmic reticulum–associated and –nonassociated cytosolic rRNAs. Mitochondrial intermembrane space RNase T2 (RNASET2), which has been previously shown to degrade mitochondrial RNAs, is also responsible for selective degradation of the cytosolic rRNAs on the outer membrane. We noted that the degradation activity also has a positive effect on nuclear transcription of rRNAs, suggesting a compensatory feedback mechanism, and affects protein translations in and out of mitochondria. These findings establish a mechanism for the co-regulation of gene expression programs inside and outside of mitochondria in mammalian cells.  

3.3153           The immunological function of extracellular vesicles in hepatitis B virus-infected hepatocytes

Kakizaki, M., Yamamoto, S., Kurosaki, N., Kagawa, T. and Kotani, A. PloS One, 13(12), e0205886 (2018)   Hepatitis B virus (HBV) generates large amounts of complete and incomplete viral particles. Except for the virion, which acts as infectious particles, the function of those particles remains elusive. Extracellular vesicles (EVs) have been revealed to have biological functions. The EVs which size are less than 100 nm in diameter, were collected from HBV infected-patients. These vesicles contain, complete and incomplete virions, and exosomes, which have been recently shown to be critical as intercellular communicators. Here, the effects of the exosome, the complete, and the incomplete particles on the target cells were investigated. These particles are endocytosed by monocyte/macrophages and function primarily to upregulate PD-L1. The functions and composition of the EVs were affected by nucleotide reverse transcriptase inhibitors (NRTIs), suggesting that the EVs are involved in the pathogenesis of HBV hepatitis and clinical course of those patients treated by NRTIs.  

3.3154           OPEN

 

3.3155           The hnRNP RALY regulates PRMT1 expression and interacts with the ALS-linked protein FUS: implication for reciprocal cellular localization

Gasperini, L., Rossi, A., Cornella. N., Peroni, D., Zuccotti, P., Potrich, V., Quattrone, A. and Macchi, P. Mol. Biol. Cell, 29(26), 3067-3081 (2018)   The RBP associated with lethal yellow mutation (RALY) is a member of the heterogeneous nuclear ribonucleoprotein family whose transcriptome and interactome have been recently characterized. RALY binds poly-U rich elements within several RNAs and regulates the expression as well as the stability of specific transcripts. Here we show that RALY binds PRMT1 mRNA and regulates its expression. PRMT1 catalyzes the arginine methylation of Fused in Sarcoma (FUS), an RNA-binding protein that interacts with RALY. We demonstrate that RALY down-regulation decreases protein arginine N-methyltransferase 1 levels, thus reducing FUS methylation. It is known that mutations in the FUS nuclear localization signal (NLS) retain the protein to the cytosol, promote aggregate formation, and are associated with amyotrophic lateral sclerosis. Confirming that inhibiting FUS methylation increases its nuclear import, we report that RALY knockout enhances FUS NLS mutants’ nuclear translocation, hence decreasing aggregate formation. Furthermore, we characterize the RNA-dependent interaction of RALY with FUS in motor neurons. We show that mutations in FUS NLS as well as in RALY NLS reciprocally alter their localization and interaction with target mRNAs. These data indicate that RALY’s activity is impaired in FUS pathology models, raising the possibility that RALY might modulate disease onset and/or progression.We show that RALY decreases the cytosolic aggregation of FUS mutants that are associated with amyotrophic lateral sclerosis. Mutations in FUS and RALY NLS reciprocally alter their localization and interaction with target mRNAs. RALY’s activity is impaired in FUS pathology models, suggesting that RALY might contribute to the disease progression.  

3.3156           TANGO1 and SEC12 are copackaged with procollagen I to facilitate the generation of large COPII carriers

Yuan, L., Kenny, S.J., Hemmati, j., Xu, K. and Schekman, R. PNAS, 115(52), E12255-E12264 (2018)   Large coat protein complex II (COPII)-coated vesicles serve to convey the large cargo procollagen I (PC1) from the endoplasmic reticulum (ER). The link between large cargo in the lumen of the ER and modulation of the COPII machinery remains unresolved. TANGO1 is required for PC secretion and interacts with PC and COPII on opposite sides of the ER membrane, but evidence suggests that TANGO1 is retained in the ER, and not included in normal size (<100 nm) COPII vesicles. Here we show that TANGO1 is exported out of the ER in large COPII-coated PC1 carriers, and retrieved back to the ER by the retrograde coat, COPI, mediated by the C-terminal RDEL retrieval sequence of HSP47. TANGO1 is known to target the COPII initiation factor SEC12 to ER exit sites through an interacting protein, cTAGE5. SEC12 is important for the growth of COPII vesicles, but it is not sorted into small budded vesicles. We found both cTAGE5 and SEC12 were exported with TANGO1 in large COPII carriers. In contrast to its exclusion from small transport vesicles, SEC12 was particularly enriched around ER membranes and large COPII carriers that contained PC1. We constructed a split GFP system to recapitulate the targeting of SEC12 to PC1 via the luminal domain of TANGO1. The minimal targeting system enriched SEC12 around PC1 and generated large PC1 carriers. We conclude that TANGO1, cTAGE5, and SEC12 are copacked with PC1 into COPII carriers to increase the size of COPII, thus ensuring the capture of large cargo.  

3.3157           Large-scale, cross-flow based isolation of highly pure and endocytosis-competent extracellular vesicles

McNamara, R.P., Caro-Vegas, C.P., Costantini, L.M., Landis, J.T., Griffith, J.D., Damania, B.A. and Dittmer, D.P.

1. Extracellular Vesicles, 7(1), 1541396 (2018)

  Isolation of extracellular vesicles (EVs) from cell culture supernatant or plasma can be accomplished in a variety of ways. Common measures to quantify relative success are: concentration of the EVs, purity from non-EVs associated protein, size homogeneity and functionality of the final product. Here, we present an industrial-scale workflow for isolating highly pure and functional EVs using cross-flow based filtration coupled with high-molecular weight Capto Core size exclusion. Through this combination, EVs loss is kept to a minimum. It outperforms other isolation procedures based on a number of biochemical and biophysical assays. Moreover, EVs isolated through this method can be further concentrated down or directly immunopurified to obtain discreet populations of EVs. From our results, we propose that cross-flow/Capto Core isolation is a robust method of purifying highly concentrated, homogenous, and functionally active EVs from industrial-scale input volumes with few contaminants relative to other methods.  

3.3158           Antidepressants act by inducing autophagy controlled by sphingomyelin–ceramide

Gulbins, A., Schumacher, F., Becker, K.A., Wilker, B., Soddelmann, M., Boldrin, F., Müller, C.P., Edwards, M.J., Goodman, M., Caldwell, C.C., Kleuser, B., Kornhuber, J., Szabo, I. and Gulbins, E Mol. Psychiatry, 23, 2324-2346 (2018)   Major depressive disorder (MDD) is a common and severe disease characterized by mood changes, somatic alterations, and often suicide. MDD is treated with antidepressants, but the molecular mechanism of their action is unknown. We found that widely used antidepressants such as amitriptyline and fluoxetine induce autophagy in hippocampal neurons via the slow accumulation of sphingomyelin in lysosomes and Golgi membranes and of ceramide in the endoplasmic reticulum (ER). ER ceramide stimulates phosphatase 2A and thereby the autophagy proteins Ulk, Beclin, Vps34/Phosphatidylinositol 3-kinase, p62, and Lc3B. Although treatment with amitriptyline or fluoxetine requires at least 12 days to achieve sphingomyelin accumulation and the subsequent biochemical and cellular changes, direct inhibition of sphingomyelin synthases with tricyclodecan-9-yl-xanthogenate (D609) results in rapid (within 3 days) accumulation of ceramide in the ER, activation of autophagy, and reversal of biochemical and behavioral signs of stress-induced MDD. Inhibition of Beclin blocks the antidepressive effects of amitriptyline and D609 and induces cellular and behavioral changes typical of MDD. These findings identify sphingolipid-controlled autophagy as an important target for antidepressive treatment methods and provide a rationale for the development of novel antidepressants that act within a few days.  

3.3159           Abstract 16378: Robust Cardiac Gene Delivery and Evasion of Neutralizing Antibodies by Extracellular Vesicle-Associated AAV Vectors

Adamiak, M., Liang, Y. Mathiyalagan, P. Agarwal, M, Jha, D. Kohlbrenner, E. Chepurko, E Jeong, D. Ceholski, D. Dubois, N. Hajjar, R. and  Sahoo, S.

Circulation, 138, Suppl.1, abstract 16378 (2018)

Introduction: Due to their excellent safety profile in clinical trials, adeno-associated virus (AAV) has become the vector of choice for gene delivery to the myocardium. However, pre-existing immunity to AAV from naturally present neutralizing antibodies (NAbs, present in >60% of the human population) significantly limits the potential candidates for the AAV-based gene therapy. NAbs bind to AAV and block its infection, greatly reducing transduction and clinical efficacy. Thus, development of novel AAV-based vectors that can circumvent the effect of NAbs is essential for clinical administration of AAVs.

Objectives: We investigated the effectiveness of extracellular vesicle (EV)-associated AAV (EV-AAV) in resisting NAbs and enhancing vector transduction in the myocardium.

Methods and Results: We isolated EV-AAV from the conditioned medium of infected 293 cells using a multi-step iodixanol density gradient ultracentrifugation. Electron microscopy, flow cytometry, bioluminescence imaging and echocardiography were used to quantify AAV-mediated gene delivery and transduction efficiency. EV-AAV6-mCherry and EV-AAV9-FLuc were more resistant to antibody-mediated neutralization than conventional AAV and induced significantly higher mCherry or firefly luciferase (FLuc) expression both in vitro and in vivo. To test the therapeutic efficacy, EV-AAV9-SERCA2a or AAV9-SERCA2a were injected intramyocardially in post-MI mice preinjected with human NAbs (IVIg). Remarkably, EV-AAV9-SERCA2a outperformed standard AAV, significantly improving cardiac function both in the presence and absence of NAbs (%EF 57.4 ± 4.7 vs. 29.2± 2.3, respectively; 6 weeks after surgery).

Conclusion: EV-AAV highly outperforms conventional AAV in delivering beneficial genes, such as SERCA2a. The use of naturally enveloped AAV vectors represents a promising approach to evade NAbs, and to facilitate the clinical translation of AAV-based gene therapies to a larger human population.

 

3.3160           Abstracts of Papers Submitted to the 49th Annual Meeting of the American Pancreatic Association, October 31–November 3, 2018, Miami Beach, Florida

 Disordering of Endo-lysosomal System in Pancreatitis

Marenikova, O,.H., Yabukov, I., Gukovsky, I. and Gukovskaya, A.S.

Pancreas, 47(10), abstract, 1370-1436 (2018)   Background: Recent studies in genetic and experimental models indicate a key pathogenic role of impaired autophagy in pancreatitis; however, the underlying mechanisms remain poorly understood. Here we hypothesized that endolysosomal system, which mediates post-Golgi protein trafficking, endocytosis and autophagy, is disordered in acute pancreatitis (AP). Methods: We analyzed the effects of pancreatitis induced in rats by cerulein(CER-AP) or L- arginine (Arg-AP) on the endo-lysosomal system and autophagy by measuring changes in the lysosomal membrane proteins LAMP1 and LAMP2; Rab proteins, key mediators of protein trafficking; and LC3-II, a marker of autophagic vacuoles. Pancreatic levels of these proteins were measured by immunoblot; their cellular localization, by immunofluorescence; and subcellular distribution, by OptiPrep density gradient fractionation. Results: Both CER-AP and Arg-AP caused marked reduction in pancreatic levels of LAMPs and membrane-bound (active) Rabs. In control rats, the density of organelles increased in the order: early endosomes (Rab4, Rab5, Rab11) < lysosomes (LAMP1, LAMP2) < late endosomes (Rab7, Rab9) < zymogen granules (trypsinogen). In pancreatitis the lysosomes became lighter, whereas both early and late endosomes shifted to higher-density fractions, the effect being especially pronounced for early endosomes. The results indicate defective early to late endosomes’ maturation and reduced formation of lysosomes in AP models. Correspondingly, maturation/processing of cathepsins is defective in pancreatitis, as demonstrated by accumulation of the intermediate CatB form (partially processed in endosomes) and a decrease in CatB mature form (fully processed in the lysosome). Endosomal sorting was disordered in ex vivo pancreatitis model, evidenced by transferrin accumulation during its endocytosis in acinar cells. Defective endosomal maturation was associated with impaired autophagy manifested by increased number of autolysosomes. In AP models, trypsin activity co-fractionated with LC3-II and LAMP1, but not with markers of late endosomes. Conclusion: The results implicate defective endosome maturation in disordered autophagy and endocytosis in pancreatitis.  

3.3161           Delivery of an Artificial Transcription Regulator dCas9-VPR by Extracellular Vesicles for Therapeutic Gene Activation

Lainscek, D., Kadunc, L., Keber, M.M., Bratkovic, I.H., Romih, R. and jeerala, R. ACS Synth. Biol., 7, 2715-2725 (2018)   The CRISPR/Cas system has been developed as a potent tool for genome engineering and transcription regulation. However, the efficiency of the delivery of the system into cells, particularly for therapeutic in vivo applications, remains a major bottleneck. Extracellular vesicles (EVs), released by eukaryotic cells, can mediate the transfer of various molecules, including nucleic acids and proteins. We show the packaging and delivery of the CRISPR/Cas system via EVs to the target cells, combining the advantages of both technological platforms. A genome editing with designed extracellular vesicles (GEDEX) system generated by the producer cells can transfer the designed transcriptional regulator dCas9-VPR complexed with appropriate targeting gRNAs enabling activation of gene transcription. We show functional delivery in mammalian cells as well in the animals. The therapeutic efficiency of in vivo delivery of dCas9-VPR/sgRNA GEDEX is demonstrated in a mouse model of liver damage counteracted by upregulation of the endogenous hepatocyte growth factor, demonstrating the potential for therapeutic applications.  

3.3162           M1 Macrophage-Derived Nanovesicles Potentiate the Anticancer Efficacy of Immune Checkpoint Inhibitors

Choo, Y.W., Kang, M., Kim, H.Y., Han, J., Kang, S., Lee, J-R., Jeong, G-J., Kwon, S.P., Song, S.Y., Go, S., Jung, M., Hong, J. and Kim, B-S. ACS Nano, 12, 8977-8993 (2018)   Cancer immunotherapy modulates immune cells to induce antitumor immune responses. Tumors employ immune checkpoints to evade immune cell attacks. Immune checkpoint inhibitors such as anti-PD-L1 antibody (aPD-L1), which is being used clinically for cancer treatments, can block immune checkpoints so that the immune system can attack tumors. However, immune checkpoint inhibitor therapy may be hampered by polarization of macrophages within the tumor microenvironment (TME) into M2 tumor-associated macrophages (TAMs), which suppress antitumor immune responses and promote tumor growth by releasing anti-inflammatory cytokines and angiogenic factors. In this study, we used exosome-mimetic nanovesicles derived from M1 macrophages (M1NVs) to repolarize M2 TAMs to M1 macrophages that release pro-inflammatory cytokines and induce antitumor immune responses and investigated whether the macrophage repolarization can potentiate the anticancer efficacy of aPD-L1. M1NV treatment induced successful polarization of M2 macrophages to M1 macrophages in vitro and in vivo. Intravenous injection of M1NVs into tumor-bearing mice suppressed tumor growth. Importantly, injection of a combination of M1NVs and aPD-L1 further reduced the tumor size, compared to the injection of either M1NVs or aPD-L1 alone. Thus, our study indicates that M1NV injection can repolarize M2 TAMs to M1 macrophages and potentiate antitumor efficacy of the checkpoint inhibitor therapy.  

3.3163           Proteomic Analysis of Lysosomal Membrane Proteins in Bovine Mammary Epithelial Cells Illuminates Potential Novel Lysosome Functions in Lactation

Luo, C., Zhao, S., Dai, W., Zheng, N. and Wang, J.

1. Agric. Food Chem., 66, 13041-13049 (2018)

  Lactation of bovine mammary epithelial cells (BMEC) is a complex biological process that involves in various organelles. Studies have shown that lysosome and lysosomal membrane proteins (LMP) plays an important role in lactation of BMEC. But the LMP of BMEC remains poorly understood. To obtain a global view of the LMP of BMEC and the affect of lysosome on lactation, the LMP of BMEC was identified using sequential windowed acquisition of all theoretical mass spectra (LC-SWATH/MS). 1214 LMP were identified and 559 were reported to be localized on lysosomal membrane for the first time in BMEC. Gene ontology annotation of these identified proteins showed that both previously reported casein synthesis-related LMP, such as LAMTOR1, 2, 3, and rRagC, and newly identified casein and milk fat synthesis-related LMP, such as EIF4E and ACAA1, were found. KEGG pathway analysis of these identified proteins showed that some pathways involved in lactation, such as PI3K-Akt, mTOR, insulin, PPAR, and JAK-STAT pathway, were found. The lysosomal location of five proteins (PRKCA, EIF4E, ACAA1, HRAS, and THBS1) was analyzed by laser confocal microscopy, and all five were associated with the lysosomal membrane. These findings help to elucidate lysosome functions in the regulation of lactation. The results implicate lysosomes as important organelles in regulation of lactation of BMEC that have been previously undervalued.  

3.3164           A pharmaceutical investigation into exosomes

Manandhar, S., Kothandan, V.K., Oh, J., Yoo, S.H., Hwang, J. and Hwang, S.R.

1. Pharm. Investig., 48, 617-626 (2018)

  During the last 5 years, there has been a marked rise in publications regarding exosomes. Exosomes find potential applications as diagnostic biomarkers, therapeutics, drug delivery vehicles, and functional cosmetics and several exosome-based therapies are under clinical trials. Exosomes are nanosized vesicles that contain cell-derived lipid membranes, nucleic acids, and proteins, and are released from the cells of origin, circulate in eukaryotic fluids, and are taken up by recipient cells. The originating cell can be engineered to express specific genes, which enhance immunity, cross-presentation of antigens, or cell targeting by exosomes. Various methods using centrifugation, size-exclusion chromatography, filtration, precipitation, affinity, and microfluidics have been assessed for the isolation and purification of exosomes from biological samples. In addition, exosomes have advantages as drug delivery systems, with biocompatibility, preservation of cargo drugs during circulation in physiological fluids, uptake into the target cells, and drug release after endosomal escape. In this review, we discuss the various aspects of pharmaceutical investigation into exosomes with special focus on engineering, production, characterization, biological activities, applications of exosomes as drug delivery systems, and regulations for evaluation of exosome products.  

3.3165           Molecular mechanisms of biogenesis of apoptotic exosome-like vesicles and their roles as damage-associated molecular patterns

Park, S.J., Kim, J.M., Kim, J., Hur, J., Park, S., Kim, K., Shin, H-J. and Chwae, Y-J. PNAS, 115(50), E1721-E1730 (2018)   Recent research has led to contradictory notions regarding the conventional theory that apoptotic cell death can evoke inflammatory or immunogenic responses orchestrated by released damage-associated patterns (DAMPs). By inducing IL-1β from bone marrow-derived macrophages in an effort to determine the inflammatory mediators released from apoptotic cells, we found that exosomal fractions called “apoptotic exosome-like vesicles” (AEVs) prepared from apoptotic-conditioned medium were the main inflammatory factors. These AEVs showed characteristics of exosomes in their size, density, morphology, and protein expression but had unique marker proteins, sphingosine-1-phosphate receptors 1 and 3 (S1PR1 and 3). Their biogenesis was completely dependent on cellular sphingosine-1-phosphate (S1P)/S1PRs signaling from multiple fine spindles of plasma membrane accompanied by F-actin, S1PR1, S1PR3, and CD63 at the early apoptotic phase and progressing to the maturation of F-actin–guided multivesicular endosomes mediated by G_βγ subunits of S1PRs downstream. S1P-loaded S1PRs on AEVs were critical factors for inducing IL-1β via NF-κB transcriptional factor and p38 MAPK, possibly through the RHOA/NOD2 axis, in differentiating macrophages. The AEVs induced genes of proinflammatory cytokines, chemokines, and mediators in both in vitro and in vivo models. In conclusion, AEVs could be key inflammatory mediators, acting as DAMPs that could explain the pathogeneses of various chronic inflammations, autoimmune diseases, or cancers in the future.  

3.3166           Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids

Negreiros, R.S., Lander, N., Huaang, G., Cordeiro, C.D., Smith, S.A., Morrissey, J.H. an Docompo, R. Mol. Microbiol., 110(6), 973-994 (2018)   polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high‐energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over‐representation of nucleolar and glycosomal proteins. Using the polyphosphate‐binding domain (PPBD) of Escherichia coli exopolyphosphatase (PPX), we localized long‐chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre‐incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration‐dependent and chain length‐dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.  

3.3167           Multiple steps determine CD73 shedding from RPE: lipid raft localization, ARA1 interaction, and MMP-9 up-regulation

Zhang, W., Zhou, S., Liu, G., Kong, F., Chen, S. and Yan, H. Purinergic Signalling, 14, 443-457 (2018)   Physiologically, retinal pigment epithelium (RPE) expresses high levels of CD73 in their membrane, converting AMP to immune suppressive adenosine, mediates an anti-inflammatory effect. However, after being exposed to inflammatory factors, RPE rapidly becomes CD73-negative cells, which render RPE’s immune suppressive function and accelerate local inflammation. Here, we investigated the mechanism leading to the loss of membrane CD73 in RPE. We found the controversy that when membrane CD73 was significantly diminished in inflammatory RPE, Cd73 mRNA levels were not changed at all. It was further verified that, matrix metalloproteinase-9 (MMP-9) mediated the shedding of CD73 from the cell membrane of inflammatory RPE by catalyzing its K547/F548 site. However, MMP-9 could not catalyze uncomplexed CD73, the interaction of CD73 with adenosine receptor A1 subtype (ARA1) is necessary for being catalyzed by MMP-9. After being treated by LPS and TNF-α, the formation of CD73/ARA1 complex in RPE was verified by co-immunoprecipitation and FRET-based assays. It was also revealed that CD73 need to be localized in lipid rafts to be capable of interacting with ARA1, since CD73/ARA1 interaction and CD73 shedding were completely blocked by the addition of lipid raft synthesis inhibitor. As a conclusion, multiple steps are involved in CD73 shedding in RPE, including up-regulation of MMP-9 activity, localization of CD73 in lipid rafts, and the formation of CD73/ARA1 complex. Lipid rafts committed CD73 with high mobility, shuttled CD73 to ARA1 to form a complex, which was capable of being recognized and catalyzed by MMP-9.  

3.3168           Ebola Virus VP40 Modulates Cell Cycle and Biogenesis of Extracellular Vesicles

Pleet, M.L., Erickson, J., Demarino, C., Baarclay,, R.A., Cowen, M., Lepene, B., Liang, J., Kuhn, J.H., Prugar, L., Stonier, S.W., Dye, J.M., Zhou, W., Liotta, L.A., Aman, M.J. and Kashanchi, F.

1. Infect. Dis., 218, Suppl. 5, S365-S387 (2018)

  Background Ebola virus (EBOV) mainly targets myeloid cells; however, extensive death of T cells is often observed in lethal infections. We have previously shown that EBOV VP40 in exosomes causes recipient immune cell death. Methods Using VP40-producing clones, we analyzed donor cell cycle, extracellular vesicle (EV) biogenesis, and recipient immune cell death. Transcription of cyclin D1 and nuclear localization of VP40 were examined via kinase and chromatin immunoprecipitation assays. Extracellular vesicle contents were characterized by mass spectrometry, cytokine array, and western blot. Biosafety level-4 facilities were used for wild-type Ebola virus infection studies. Results VP40 EVs induced apoptosis in recipient T cells and monocytes. VP40 clones were accelerated in growth due to cyclin D1 upregulation, and nuclear VP40 was found bound to the cyclin D1 promoter. Accelerated cell cycling was related to EV biogenesis, resulting in fewer but larger EVs. VP40 EV contents were enriched in ribonucleic acid-binding proteins and cytokines (interleukin-15, transforming growth factor-β1, and interferon-γ). Finally, EBOV-infected cell and animal EVs contained VP40, nucleoprotein, and glycoprotein. Conclusions Nuclear VP40 upregulates cyclin D1 levels, resulting in dysregulated cell cycle and EV biogenesis. Packaging of cytokines and EBOV proteins into EVs from infected cells may be responsible for the decimation of immune cells during EBOV pathogenesis.  

3.3169           Human placental exosomes in gestational diabetes mellitus carry a specific set of miRNAs associated with skeletal muscle insulin sensitivity

Nair, S., Jayabalan, N., Guanzon, D., Palma, C., Scholz-Romero, K., Elfeky, O., Zuniga, F., Ormazabai, V., Diaz, E., Rice, G.E., Duncombe, G., jansson, T., McIntyre, H.D., Lappas, M. and Salomon, C. Clin. Science, 132, 2451-2467 (2018)   There is increasing evidence that miRNAs, which are enriched in nanovesicles called exosomes, are important regulators of gene expression. When compared with normal pregnancies, pregnancies with gestational diabetes mellitus (GDM) are associated with skeletal muscle insulin resistance as well as increased levels of circulating placental exosomes. Here we investigated whether placental exosomes in GDM carry a specific set of miRNAs associated with skeletal muscle insulin sensitivity. Exosomes were isolated from chorionic villous (CV) explants from both women with Normal Glucose Tolerant (NGT) and GDM pregnancies. Using miRNA sequencing, we identified a specific set of miRNAs selectively enriched with exosomes and compared with their cells of origin indicating a specific packaging of miRNAs into exosomes. Gene target and ontology analysis of miRNA differentially expressed in exosomes secreted in GDM compared with NGT are associated with pathways regulating cell migration and carbohydrate metabolism. We determined the expression of a selected set of miRNAs in placenta, plasma, and skeletal muscle biopsies from NGT and GDM. Interestingly, the expression of these miRNAs varied in a consistent pattern in the placenta, in circulating exosomes, and in skeletal muscle in GDM. Placental exosomes from GDM pregnancies decreased insulin-stimulated migration and glucose uptake in primary skeletal muscle cells obtained from patients with normal insulin sensitivity. Interestingly, placental exosomes from NGT increase migration and glucose uptake in response to insulin in skeletal muscle from diabetic subjects. These findings suggest that placental exosomes might have a role in the changes on insulin sensitivity in normal and GDM pregnancies.  

3.3170           In Vitro Dissection of Autophagy

Zhang, M., Liu, D. and Ge, L. Current Protocols in Cell Biology, 77(1), 11-23.1-1123.17 (2018)   Autophagy is an essential cellular process for bulk degradation of cytoplasmic components through the lysosome. Underlying this process is an intricate interaction between protein factors and the cell endomembrane system, leading to a gradual maturation of the autophagic membrane. This structure sequesters a portion of the cytoplasm by the formation of a double‐membrane compartment called the autophagosome. The autophagosome then delivers the cargo to the lysosome to complete degradation. The molecular mechanism accounting for the generation of the autophagic membrane is a longstanding question. Here, a cell‐free approach that has been established to understand the mechanism of early autophagic membrane generation is described. This system has provided insight into the membrane source of the autophagosome, the early protein‐membrane associations, and the membrane remodeling that generates the autophagosomal precursors. The cell‐free assay, in combination with other established approaches (e.g., cell imaging), will facilitate a deeper understanding of the mechanism of autophagy.  

3.3171           Visualizing Autophagic Lysosome Reformation in Cells Using In Vitro Reconstitution Systems

Chen, Y., Su, Q.P., Sun, Y. and Yu, L. Current Protocols in Cell Biology, 78(1), 11.24.1-11.24.24 (2018)   Autophagy is a lysosome‐based degradation pathway. Autophagic lysosome reformation (ALR) is a lysosomal membrane recycling process that marks the terminal step of autophagy. During ALR, LAMP1‐positive tubules, named reformation tubules, are extruded from autolysosomes, and nascent lysosomes are generated from these tubules. By combining proteomic analysis of purified autolysosomes and RNA interference screening of identified candidates, we systematically elucidated the ALR pathway at the molecular level. Based on the key components clathrin, PtdIns(4,5)P₂, and the motor protein KIF5B, among others, we reconstituted this process in vitro. This unit describes a detailed method for visualizing ALR in cells during the autophagy process. This unit also present a protocol for reconstituting the ALR tubular protrusion and elongation process in vitro and three methods for preparing materials for in vitro reconstitution: (1) autolysosome purification from cultured cells, (2) liposome preparation, and (3) KIF5B purification and quality testing.    

3.3172           Seminal exosomes and HIV‐1 transmission

Quattara, L., Anderson, S.M. and Doncol., G.F. Andrologia, 50(11), e13220 (2018)   Exosomes are endosomal‐derived membrane‐confined nanovesicles secreted by many (if not all) cell types and isolated from every human bodily fluid examined up to now including plasma, semen, vaginal secretions and breast milk. Exosomes are thought to represent a new player in cell‐to‐cell communication pathways and immune regulation, and be involved in many physiological and pathological processes. Susceptibility to HIV‐1 infection can be impacted by exosomes, while HIV‐1 pathogenesis can alter exosomal function and composition. Exosomes isolated from semen and vaginal fluid of healthy individuals can inhibit HIV‐1 infection and/or potently block viral transfer in vitro. However, the role of exosomes in HIV‐1 transmission and progression is not fully understood yet and some studies show conflicting results, mainly for exosomes isolated from plasma and breast milk. Determining the composition of exosomes from infected donors and studying their interaction with HIV‐1 in vitro compared to exosomes isolated from uninfected donors will provide insights into the role exosomes play in HIV‐1 transmission during sexual intercourse and breastfeeding.  

3.3173           The Craft of Peroxisome Purification—A Technical Survey Through the Decades

Islinger, M., Manner, A. and Vökl, A. Subcellular Biochemistry, 89, 85-122 (2018)   Purification technologies are one of the working horses in organelle proteomics studies as they guarantee the separation of organelle-specific proteins from the background contamination by other subcellular compartments. The development of methods for the separation of organelles was a major prerequisite for the initial detection and characterization of peroxisome as a discrete entity of the cell. Since then, isolated peroxisomes fractions have been used in numerous studies in order to characterize organelle-specific enzyme functions, to allocate the peroxisome-specific proteome or to unravel the organellar membrane composition. This review will give an overview of the fractionation methods used for the isolation of peroxisomes from animals, plants and fungi. In addition to “classic” centrifugation-based isolation methods, relying on the different densities of individual organelles, the review will also summarize work on alternative technologies like free-flow-electrophoresis or flow field fractionation which are based on distinct physicochemical parameters. A final chapter will further describe how different separation methods and quantitative mass spectrometry have been used in proteomics studies to assign the proteome of PO.  

3.3174           Preparation of Extracellular Vesicles from Mesenchymal Stem Cells

Cruz, F.F., de Castro, L.L. and Rocco, P.R.M. Stem Cell Drugs - A New Generation of Biopharmaceuticals. Stem Cells in Clinical Applications. Springer, Cham, 37-51 (2018)   Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, released from mesenchymal stem cells under different conditions and carry information capable of reestablishing homeostasis. EVs derived from MSCs transfer molecules (such as DNA, proteins/peptides, mRNA, microRNA, and lipids) and/or organelles with reparative and anti-inflammatory properties to recipient cells. EVs present many advantages over MSCs, such as homing ability to target tissue, preventing undesired accumulation in other organs, and absence of any innate toxicity or association with long-term maldifferentiated engrafted cells, tumor generation, or immune rejection after stem cell injection. The paracrine anti-inflammatory effects promoted by MSC-derived EVs have attracted significant interest in the regenerative medicine field, including for potential use in different diseases. This chapter addresses methods used to induce the release of extracellular vesicles from mesenchymal stem cells, the methods of extracting these vesicles, and processing and conditioning techniques used after extraction. We aim to enlighten the reader on how to choose the appropriate protocol and methods to obtain extracellular vesicles, characterize them, and use them in preclinical studies.  

3.3175           The pro-oxidant adaptor p66SHC promotes B cell mitophagy by disrupting mitochondrial integrity and recruiting LC3-II

Onnis, A., Cianfanelli, V., Cassioli, C., Samardzic, D., Pelicci, P.G., Cecconi, F. and Baldari, C.T. Autophagy, 14(12), 2117-2138 (2018)   Macroautophagy/autophagy has emerged as a central process in lymphocyte homeostasis, activation and differentiation. Based on our finding that the p66 isoform of SHC1 (p66SHC) pro-apoptotic ROS-elevating SHC family adaptor inhibits MTOR signaling in these cells, here we investigated the role of p66SHC in B-cell autophagy. We show that p66SHC disrupts mitochondrial function through its CYCS (cytochrome c, somatic) binding domain, thereby impairing ATP production, which results in AMPK activation and enhanced autophagic flux. While p66SHC binding to CYCS is sufficient for triggering apoptosis, p66SHC-mediated autophagy additionally depends on its ability to interact with membrane-associated LC3-II through a specific binding motif within its N terminus. Importantly, p66SHC also has an impact on mitochondria homeostasis by inducing mitochondrial depolarization, protein ubiquitination at the outer mitochondrial membrane, and local recruitment of active AMPK. These events initiate mitophagy, whose full execution relies on the role of p66SHC as an LC3-II receptor which brings phagophore membranes to mitochondria. Importantly, p66SHC also promotes hypoxia-induced mitophagy in B cells. Moreover, p66SHC deficiency enhances B cell differentiation to plasma cells, which is controlled by intracellular ROS levels and the hypoxic germinal center environment. The results identify mitochondrial p66SHC as a novel regulator of autophagy and mitophagy in B cells and implicate p66SHC-mediated coordination of autophagy and apoptosis in B cell survival and differentiation.  

3.3176           Revisiting the role of cholesterol in regulating the pore-formation mechanism of Vibrio cholerae cytolysin, a membrane-damaging β-barrel pore-forming toxin

Kathuria, R., Mondal, A.K., Sharma, R., Bhattacharyya, S. and Chattopadhyay, K. Biochem. J., 475, 3039-3055 (2018)   Vibrio cholerae cytolysin (VCC) is a β-barrel pore-forming toxin with potent membrane-damaging cell-killing activity. Previous studies employing the model membranes of lipid vesicles (liposomes) have shown that pore formation by VCC requires the presence of cholesterol in the liposome membranes. However, the exact role of cholesterol in the mode of action of VCC still remains unclear. Most importantly, implication of cholesterol, if any, in regulating the pore-formation mechanism of VCC in the biomembranes of eukaryotic cells remains unexplored. Here, we show that the presence of cholesterol promotes the interaction of VCC with the membrane lipid bilayer, when non-lipid-dependent interactions are absent. However, in the case of biomembranes of human erythrocytes, where accessory interactions are available, cholesterol appears to play a less critical role in the binding step. Nevertheless, in the absence of an optimal level of membrane cholesterol in the human erythrocytes, membrane-bound fraction of the toxin remains trapped in the form of abortive oligomeric assembly, devoid of functional pore-forming activity. Our study also shows that VCC exhibits a prominent propensity to associate with the cholesterol-rich membrane micro-domains of human erythrocytes. Interestingly, mutation of the cholesterol-binding ability of VCC does not block association with the cholesterol-rich membrane micro-domains on human erythrocytes. Based on these results, we propose that the specific cholesterol-binding ability of VCC does not appear to dictate its association with the cholesterol-rich micro-domains on human erythrocytes. Rather, targeting of VCC toward the membrane micro-domains of human erythrocytes possibly acts to facilitate the cholesterol-dependent pore-formation mechanism of the toxin.  

3.3177           Methods and Technologies for Exosome Isolation and Characterization

Zhang, M., Jin, K., Gao, L., Zhang, Z., Li, F., Zhou, F. and Zhang, L. Small Methods, 2, 1800021 (2018)   Exosomes, a specific subclass of the extracellular vesicles secreted by most cell types, play an important role in cell–cell communication by transporting diverse content including protein, mRNA, miRNA, and DNA. This cargo is closely associated with the pathogenesis of most human malignancies. Therefore, it is becoming an urgent demand to be able to isolate exosomes in a simple, efficient, and economical way for scientific research and clinical diagnosis. Here, several conventional and novel nano‐based techniques of exosome isolation and characterization are summarized, and the advantages and disadvantages among them are compared, with the hope that researchers will be provided with an overview in this field to detect and isolate exosomes in a suitable manner, matching the subsequent experiments.  

3.3178           Acute pain alters P-glycoprotein-containing protein complexes in rat cerebral microvessels: Implications for P-glycoprotein trafficking

Tome, M.E., Jarvis, C.K., Schaefer, C.P., Jacobs, L.M., Herndon, J.M., Hunn, K.C., Arkwright, N.B., Kellohen, K.L., Mierau, P.C. and Davis, T.P:

J. Cerebral Blood Flow & Metabolism, 38(12), 2209-2222 (2018)

P-glycoprotein (PgP) is the major drug efflux pump in human cerebral microvessels. PgP prevents pathogens, toxins and therapeutic drugs from entering the CNS. Understanding the molecular regulation of PgP activity will suggest novel mechanisms to improve CNS drug delivery. Previously, we found that during peripheral inflammatory pain (PIP) (3 h after λ carrageenan injection in the rat paw), PgP traffics to the cortical microvessel endothelial cell plasma membrane concomitant with increased PgP activity. In the current study, we measured the changes in composition of PgP-containing protein complexes after PIP in rat microvessel isolates. We found that a portion of the PgP is contained in a multi-protein complex that also contains the caveolar proteins CAV1, SDPR, PTRF and PRKCDBP. With PIP, total CAV1 bound to PgP was unchanged; however, phosphorylated CAV1 (Y14P-CAV1) in the complex increased. There were few PgP/CAV1 complexes relative to total PgP and CAV1 in the microvessels suggesting CAV1 bound to PgP is unlikely to affect total PgP activity. However, both PgP and CAV1 trafficked away from the nucleus in response to PIP. These data suggest that P-CAV1 bound to PgP potentially regulates PgP trafficking and contributes to the acute PgP activity increase after a PIP stimulus.

3.3179           Long-term 4-AP treatment facilitates functional expression of human Kv1.5 channel

Xie, Y., Ding, W-G., Liu, Y., Yu, M., Sun, X. and Matsuura, H. Eur. J. Pharmacol., 844, 195-203 (2019)   The human Kv1.5 channel (hKv1.5) produces the ultrarapid delayed rectifier potassium current (I_Kur), which is important for determining the repolarization of action potential in the cardiac atrium. However, the expression of I_Kur is reduced in patients with chronic atrial fibrillation. 4-Aminopyridine (4-AP) can specifically suppress I_Kur, suggesting that it modifies hKv1.5 as a chaperone molecule. Herein, the effects of long-term 4-AP treatment on hKv1.5 protein expression and function were investigated in HEK cells. 4-AP treatment (24 h) improved hKv1.5 protein levels, promoted hKv1.5 glycosylation, and facilitated the hKv1.5 current in a time-dependent manner. Long-term 4-AP treatment also markedly enhanced hKv1.5 localization in the cell membrane, endoplasmic reticulum, and Golgi. Importantly, the Ile508 residue located in the hKv1.5 channel pore was found to be important for 4-AP inhibitory activity. These results provide insight into developing hKv1.5 channel blocker that can functionally rescue I_Kur in patients with chronic atrial fibrillation.  

3.3180           Isolation of synaptic vesicles from genetically engineered cultured neurons

McKenzie, C., Spanova, M., Johnson, A., Kainrath, S., Zheden, V., Sitte, H.H. and Janovjak, H.

1. Neurosci. Methods, 312, 114-121 (2019)

  Background Synaptic vesicles (SVs) are an integral part of the neurotransmission machinery, and isolation of SVs from their host neuron is necessary to reveal their most fundamental biochemical and functional properties in in vitro assays. Isolated SVs from neurons that have been genetically engineered, e.g. to introduce genetically encoded indicators, are not readily available but would permit new insights into SV structure and function. Furthermore, it is unclear if cultured neurons can provide sufficient starting material for SV isolation procedures. New method Here, we demonstrate an efficient ex vivo procedure to obtain functional SVs from cultured rat cortical neurons after genetic engineering with a lentivirus. Results We show that ∼10⁸ plated cortical neurons allow isolation of suitable SV amounts for functional analysis and imaging. We found that SVs isolated from cultured neurons have neurotransmitter uptake comparable to that of SVs isolated from intact cortex. Using total internal reflection fluorescence (TIRF) microscopy, we visualized an exogenous SV-targeted marker protein and demonstrated the high efficiency of SV modification. Comparison with existing methods Obtaining SVs from genetically engineered neurons currently generally requires the availability of transgenic animals, which is constrained by technical (e.g. cost and time) and biological (e.g. developmental defects and lethality) limitations. Conclusions These results demonstrate the modification and isolation of functional SVs using cultured neurons and viral transduction. The ability to readily obtain SVs from genetically engineered neurons will permit linking in situ studies to in vitro experiments in a variety of genetic contexts.  

3.3181           Genetic Liver-Specific AMPK Activation Protects against Diet-Induced Obesity and NAFLD

Garcia, D., Hellberg, K., Chaix, A., Dow, L.E., Metallo, C.M. and Shaw, R.J: Cell Reports, 26, 192-208 (2019)   The AMP-activated protein kinase (AMPK) is a highly conserved master regulator of metabolism, whose activation has been proposed to be therapeutically beneficial for the treatment of several metabolic diseases, including nonalcoholic fatty liver disease (NAFLD). NAFLD, characterized by excessive accumulation of hepatic lipids, is the most common chronic liver disease and a major risk factor for development of nonalcoholic steatohepatitis, type 2 diabetes, and other metabolic conditions. To assess the therapeutic potential of AMPK activation, we have generated a genetically engineered mouse model, termed iAMPKCA, where AMPK can be inducibly activated in vivo in mice in a spatially and temporally restricted manner. Using this model, we show that liver-specific AMPK activation reprograms lipid metabolism, reduces liver steatosis, decreases expression of inflammation and fibrosis genes, and leads to significant therapeutic benefits in the context of diet-induced obesity. These findings further support AMPK as a target for the prevention and treatment of NAFLD.  

3.3182           Proteomic Profiling of Mammalian COPII and COPI Vesicles

Adolf, F., Rhiel, M., Hessling, B., Gao, Q., Hellweg, A., Bethune, J. and Wieland, F.T. Cell Reports, 26, 250-265 (2019)   Intracellular transport and homeostasis of the endomembrane system in eukaryotic cells depend on the formation and fusion of vesicular carriers. Coat protein complex (COP) II vesicles export newly synthesized secretory proteins from the endoplasmic reticulum (ER), whereas COPI vesicles facilitate traffic from the Golgi to the ER and intra-Golgi transport. Mammalian cells express various isoforms of COPII and COPI coat proteins. To investigate the roles of coat protein paralogs, we have combined in vitro vesicle reconstitution from semi-intact cells with SILAC-based mass spectrometric analysis. Here, we describe the core proteomes of mammalian COPII and COPI vesicles. Whereas the compositions of COPII vesicles reconstituted with various isoforms of the cargo-binding subunit Sec24 differ depending on the paralog used, all of the isoforms of the COPI coat produce COPI-coated vesicles with strikingly similar protein compositions.  

3.3183           Species-Specific Functional Regions of the Green Alga Gamete Fusion Protein HAP2 Revealed by Structural Studies

Baquero, E., Fedry, J., Legrand, P., Krey, T. and Rey, F.A. Structure, 27, 113-124 (2019)   The cellular fusion protein HAP2, which is structurally homologous to viral class II fusion proteins, drives gamete fusion across several eukaryotic kingdoms. Gamete fusion is a highly controlled process in eukaryotes, and is allowed only between same species gametes. In spite of a conserved architecture, HAP2 displays several species-specific functional regions that were not resolved in the available X-ray structure of the green alga Chlamydomonas reinhardtii HAP2 ectodomain. Here we present an X-ray structure resolving these regions, showing a target membrane interaction surface made by three amphipathic helices in a horseshoe-shaped arrangement. HAP2 from green algae also features additional species-specific motifs inserted in regions that in viral class II proteins are critical for the fusogenic conformational change. Such insertions include a cystine ladder-like module evocative of EGF-like motifs responsible for extracellular protein-protein interactions in animals, and a mucin-like region. These features suggest potential HAP2 interaction sites involved in gamete fusion control.  

3.3184           Cardioprotective effect of 2,3-dehydrosilybin preconditioning in isolated rat heart

Gabrielova, E., Bartosikova, L., Necas, J. and Modriansky, M. Fitoterapia, 132, 12-21 (2019)   2,3-dehydrosilybin (DHS) is a minor component of silymarin, Silybum marianum seed extract, used in some dietary supplements. One of the most promising activities of this compound is its anticancer and cardioprotective activity that results, at least partially, from its cytoprotective, antioxidant, and chemopreventive properties. The present study investigated the cardioprotective effects of DHS in myocardial ischemia and reperfusion injury in rats. Isolated hearts were perfused by the Langendorff technique with low dose DHS (100 nM) prior to 30 min of ischemia induced by coronary artery occlusion. After 60 min of coronary reperfusion infarct size was determined by triphenyltetrazolium staining, while lactatedehydrogenase activity was evaluated in perfusate samples collected at several timepoints during the entire perfusion procedure. Signalosomes were isolated from a heart tissue after reperfusion and involved signalling proteins were detected. DHS reduced the extent of infarction compared with untreated control hearts at low concentration; infarct size as proportion of ischemic risk zone was 7.47 ± 3.1% for DHS versus 75.3 ± 4.8% for ischemia. This protective effect was comparable to infarct limitation induced by ischemic preconditioning (22.3 ± 4.5%). Selective inhibition of Src-family kinases with PP2 (4-Amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine) abrogated the protection afforded by DHS. This study provides experimental evidence that DHS can mediate Src-kinase-dependent cardioprotection against myocardial damage produced by ischemia/reperfusion injury.  

3.3185           Comparison of exosomes purified via ultracentrifugation (UC) and Total Exosome Isolation (TEI) reagent from the serum of Marek’s disease virus (MDV)-vaccinated and tumor-bearing chickens

Neerukonda, S.N., Egan, N.A., patria, J., Assakhi, I., Tavlarides-Hontz, P., Modla, S., Munoz, E.R., Hudson, M.B. and Parcells, M.S:

1. Virol. Methods, 263, 1-9 (2019)

  Extracellular vesicles (EVs) is a collective term used to refer microparticles, exosomes, and apoptotic bodies produced by a variety of cells and released into interstitial spaces and bodily fluids. Serum exosomes can serve as invaluable biomarkers, containing m/miRNAs, lipids, and proteins, indicative of various conditions. There are currently limited studies on the characterization and mutual consensus of biomarker profiles of serum exosomes purified by different methods. Here we compared the advantages and disadvantages of two commonly used serum exosome purification procedures including ultracentrifugation (UC) and Total Exosome Isolation (TEI) reagent, by analyzing exosome size distribution, concentration, morphology and miRNA expression profiles. Serum was obtained from Marek’s disease virus (MDV)-infected chickens that were either vaccinated against Marek’s disease (MD), and thus protected, or unvaccinated and bearing MDV-induced tumors. Nanoparticle tracking analysis (NTA) and Transmission Electron Microscopy (TEM) were performed to evaluate particle size, concentration, and morphological integrity, respectively. Our results indicate that the size distribution of particles purified by either procedure is consistent with that of exosomes (30–150 nm). TEI reagent generated higher yields and co-isolated additional EV populations that are slightly larger (∼180 nm). Based on the miRNA expression profiles from a previous high throughput sequencing experiment of exosome small RNAs, we selected six cellular and four MDV1 miRNAs, to validate their expression in UC- and TEI-purified exosomes. miRNA expression profiles displayed relative correlation between the two procedures, but distinctive differences were observed in abundance with TEI-purified exosomes showing higher miRNA expression consistent with higher yield than those purified by UC. TEI-purified exosomes from vaccinated chickens exhibited greater expression of tumor suppressor miRNA, gga-mir-146b and least expression of oncomiR, gga-mir-21 compared to those obtained from tumor-bearing chickens. We propose that gga-mir-146 and -21 can serve as serum exosome biomarkers for vaccine-induced protection and MD tumors respectively.  

3.3186           Enhancement of antitumor potency of extracellular vesicles derived from natural killer cells by IL-15 priming

Zhu, L., Kalimuthu, S., Oh, J.M., Gangadaan, P., Baek, S.H., Jeong, S.Y., Lee, S-W., Lee, J. and Ahn, B-C. Biomaterials, 190-191, 38-50 (2019)   Purpose Natural killer (NK) cells are the key subset of innate-immunity lymphocytes; they possess antitumor activities and are used for cancer immunotherapy. In a previous study, extracellular vehicles (EVs) from NK-92MI cells were isolated and exploited for their ability to kill human cancer cells in vitro and in vivo (multiple injection methods). Here, the potential of NK-cell–derived EVs (NK-EVs) for immunotherapy was improved by priming with interleukin (IL)-15. Methods NK-EVs were isolated from the culture medium without or with IL-15 (NK-EVs_IL-15) by ultracentrifugation and were purified via density gradient ultracentrifugation. In addition, NK-EVs and NK-EVs_IL-15 were characterized by transmission electron microscopy, nanoparticle-tracking analysis, and western blotting. Flow cytometry, bioluminescence imaging, and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were performed for apoptosis, protein expression, cell proliferation, and cytotoxicity analyses. Furthermore, xenograft tumor–bearing mice were injected with PBS, NK-EVs, or NK-EVs_IL-15 intravenously five times. Tumor growth was monitored using calipers and bioluminescence imaging. Toxicity of the nanoparticles was evaluated by measuring the body weight of the mice. Results NK-EVs_IL-15 showed significantly higher cytolytic activity toward human cancer cell lines (glioblastoma, breast cancer, and thyroid cancer) and simultaneously increased the expression of molecules associated with NK-cell cytotoxicity. When compared with NK-EVs, NK-EVs_IL-15 significantly inhibited the growth of glioblastoma xenograft cells in mice. In addition, both NK-EVs and NK-EVs_IL-15 were not significantly toxic to either normal cells or mice. Conclusion IL-15 may improve the immunotherapeutic effects of NK-EVs, thus improving the applications of NK-EVs in the future.  

3.3187           Exosomes and their implications in central nervous system tumor biology

Mrowczynski, O.D., Zacharia, B.E. and Connor, J.R: Progress in Neurobiol., 172, 71-83 (2019)   Exosomes are 20–100 nm cellular derived vesicles that upon discovery, were thought to be a form of cellular recycling of intracellular contents. More recently, these vesicles are under investigation for their purported significant roles in intercellular communication in both healthy and diseased states. Herein, we focus on the secretion of exosomes associated with glioblastoma, as most exosome studies on brain tumors have been performed in this tumor type. However, we included exosomes secreted from other forms of brain tumors for comparison as available. Exosomes contain intracellular content that can be transferred to other cells in the tumor or to cells of the immune system and endothelial cells. These recipient cells may subsequently take on oncogenic properties, including therapeutic resistance, cancer progression, and angiogenesis. Genetic components (DNA, RNA and miRNA) of the cell of origin may be included in the secreted exosomes. The presence of genetic material in the exosomes could serve as a biomarker for mutations in tumors, potentially leading to novel treatment strategies. In the last decade, exosomes have been identified as having a major impact on multiple aspects of medicine and tumor biology, and appear to be primed for a critical position in cancer diagnosis, prognosis, and treatment.  

3.3188           Distinct shed microvesicle and exosome microRNA signatures reveal diagnostic markers for colorectal cancer

Chen, M., Xu, R., Rai, A., Suwakulsiri, W., Izumikawa, K., Ishikawa, H., Greening, D.W., Takahashi, N. and Simpson, R.J. PloS One, 14(1), e0210003 82019)   Extracellular vesicle (EV) microRNAs are of major interest as potential diagnostic biomarkers in all cancer types. This study aims to identify miRNA profiles of shed microvesicles (sMVs) and exosomes (Exos) secreted from the isogenic colorectal cancer (CRC) cell lines SW480 and SW620 and evaluate their ability to predict CRC. Deep sequencing of miRNAs in parental cell lysates (CLs) and highly-purified sMVs and Exos was performed. We focused on miRNAs enriched in EVs and dysregulated miRNAs in metastatic cells (SW620) relative to primary cancer cells (SW480). We investigated the ability of EV miRNA signatures to predict CRC tumours using 594 tumours (representing different pathological stages) and 11 normal samples obtained from TCGA. In SW480 and SW620 cells we identified 345 miRNAs, of which 61 and 73 were upregulated and downregulated in SW620-CLs compared to SW480-CLs, respectively. Selective distribution of cellular miRNAs into EVs results in distinct miRNA signatures for sMVs and Exos in each cell line. Cross cell line comparisons of EV miRNA profiles reveal a subset of miRNAs critical in CRC progression from primary carcinoma to metastasis. Many miRNAs non-detectable (<5 TPM) in CLs were significantly enriched (>1000 TPM) in secreted EVs. Strikingly, miR-7641 which is non-detectable in SW480-CL but upregulated in SW620-CL is highly enriched in EVs secreted from both cell lines. Pearson correlation analysis demonstrated that EV miRNA profiles can be used to predict CRC tumours with ~96% accuracy. Our findings suggest that EV miRNA profiles from CRC cell lines may allow prediction of CRC tumours, and that miR-7641 may serve as an attractive candidate for the specific, non-invasive diagnosis and prognosis of CRC.  

3.3189           Biogenesis of Extracellular Vesicles during Herpes Simplex Virus 1 Infection: Role of the CD63 Tetraspanin

Dogrammatzis, C., Deschamps, T. and Kalamvoki, M.

1. Virol., 93(2), e01850-18 (2019)

  Herpes simplex virus 1 (HSV-1) infections afflict more than 80% of the population worldwide. The virus primarily infects mucoepithelial cells and establishes latent reservoirs in neurons in sensory ganglia. Frequent reactivation has been linked to severe diseases, especially in immunocompromised individuals. Earlier, we reported that viral and host factors are packaged in extracellular vesicles (EVs) and delivered to uninfected cells, where they activate antiviral responses and restrict virus infection. Here, we interrogated the effect of HSV-1 infection on EV biogenesis. We found that HSV-1 infection causes a decrease in the amount of intracellular CD63 protein with a concomitant increase in extracellular CD63. This observation correlates with our previous finding that infected cells release more CD63-positive EVs than uninfected cells. The stimulation of CD63 exocytosis requires virus replication. CD63 is a member of the tetraspanin family of proteins that traffics between the plasma membrane and endosomal compartments and has a role in sorting cargo into the EVs. Previously, we reported that in cells depleted of CD63, HSV-1 virus yields increased, and here we provide data showing that in cells overexpressing CD63, HSV-1 virus yields decreased. Taken together, our data indicate that CD63 negatively impacts HSV-1 infection and that the CD63-positive EVs could control the dissemination of the virus in the host. Perhaps EV release by HSV-1-infected cells is a mechanism that controls virus dissemination.  

3.3190           Exosomes harbor B cell targets in pancreatic adenocarcinoma and exert decoy function against complement-mediated cytotoxicity

Capello, M., Vykoukal, J.V., katayama, H., bantis, L.E., Wang, H., Kundnani, D.L. et al Nature Communications, 10:254 (2019) Although B cell response is frequently found in cancer, there is little evidence that it alters tumor development or progression. The process through which tumor-associated antigens trigger humoral response is not well delineated. We investigate the repertoire of antigens associated with humoral immune response in pancreatic ductal adenocarcinoma (PDAC) using in-depth proteomic profiling of immunoglobulin-bound proteins from PDAC patient plasmas and identify tumor antigens that induce antibody response together with exosome hallmark proteins. Additional profiling of PDAC cell-derived exosomes reveals significant overlap in their protein content with immunoglobulin-bound proteins in PDAC plasmas, and significant autoantibody reactivity is observed between PDAC cell-derived exosomes and patient plasmas compared to healthy controls. Importantly, PDAC-derived exosomes induce a dose-dependent inhibition of PDAC serum-mediated complement-dependent cytotoxicity towards cancer cells. In summary, we provide evidence that exosomes display a large repertoire of tumor antigens that induce autoantibodies and exert a decoy function against complement-mediated cytotoxicity.  

3.3191           Heroin-based crack induces hyperalgesia through β-arrestin 2 redistribution and phosphorylation of Erk1/2 and JNK in the periaqueductal gray area

Aberoumandi, S.M., Vousooghi, N., Tabrizi, B.A. and Karimi, P. Neurosci. Lett., 698, 133-139 (2019)   Continuous use of crack induces hyperalgesia which is related to drug tolerance. Despite cumulative evidence based on the growth rate of crack abuse, no serious study has been focused on the mechanisms of crack-induced hyperalgesia. This study aimed to elucidate whether extracellular signal-regulated kinases (Erk1/2)/β-arrestin pathways are involved in the crack-induced hyperalgesia. Fifty adult male Wistar rats were randomly divided into five groups: normal saline (NS), crack (0.9 mg/kg/day), heroin (1 mg/kg/day), crack + barbadin (100 μM), and heroin + barbadin groups, which received their intraperitoneal (i.p) treatments for four weeks. The thermal sensitivity was assessed using the hot-plate test. Moreover, phosphorylation of the Erk1/2 and JNK, as well as expression of protein kinase C-alpha (PKC-α), Mu-receptor (MOR), and β-arrestin 2 were determined in the whole lysate and membrane fraction using immunoblotting assay in the periaqueductal gray (PAG) area. The results demonstrated that chronic administration of crack and heroin significantly decreased hind-paw withdrawal latency compared to the NS group. Furthermore, crack as well as heroin administration increased phosphorylated Erk1/2 and JNK in the PAG. In addition, membrane β-arrestin 2 and PKC-α were significantly increased in the crack and heroin-received groups, while membrane MOR expression was decreased in the PAG. Nevertheless, co-administration of barbadin, an inhibitor of β-arrestin, and crack or heroin reversed all these changes. Our findings may partially confirm the role of β-arrestin 2 and PKC rearrangements, Erk1/2 and JNK phosphorylation in crack-induced hyperalgesia and provide potential therapeutic targets to attenuate crack-induced hyperalgesia.  

3.3192           Granulysin species segregate to different lysosome-related effector vesicles (LREV) and get mobilized by either classical or non-classical degranulation

Lettau, M., Dietz, M., Dohmen, K., Leippe, M., Kabelitz, D. and Janssen, O. Mol. Immunol., 107, 44-53 (2019)   Granulysin (GNLY) is a cationic antimicrobial, proinflammatory, and cytotoxic effector protein primarily expressed in human cytotoxic T and NK cells. Its two variants, the 15 kDa precursor and the mature 9 kDa protein processed by proteolysis, act on different microbes or infected and transformed target cells and utilize mechanistically different effector activities. In human peripheral blood lymphocytes of healthy individuals, both forms of GNLY are detected in TCR αβ⁺ (CD4⁺ and CD8⁺) T cells, TCR γδ⁺ T cells, and CD3⁻CD56⁺ NK cells. In general, classical cytotoxic cells (i.e. CD8⁺ TCR αβ⁺ T cells, TCR γδ⁺ T cells, and NK cells) contain effector proteins in higher abundance in more cells of the subset as compared to TCR αβ⁺ CD4⁺ T cells. Imaging flow cytometry analyses demonstrate that the subcellular localization and internal pools of 9 kDa and 15 kDa GNLY are virtually non-overlapping. The 9 kDa form is enriched in dense granules that also contain granzymes (Grz) and carry CD107a, whereas 15 kDa GNLY is associated with CD107a-negative lysosome-related effector vesicles. We further demonstrate that 15 kDa GNLY serves as an additional indicator for non-classical, PKC-dependent degranulation while the liberation of granules containing 9 kDa GNLY requires calcium mobilization. Our studies provide a deeper insight into the subcellular localization and release mechanisms of the individual GNLY species. This information will not only be useful for the interpretation of GNLY-related pathophysiologies, but also for the development of therapeutic interventions employing distinct GNLY effector functions for microbial targeting or immunoregulation.  

3.3193           Herpes simplex virus 1 ICP6 impedes TNF receptor 1–induced necrosome assembly during compartmentalization to detergent-resistant membrane vesicles

Ali, M., Roback, L. and Mocarski, E.S.

1. Biol. Chem., 294(3), 991-1004 (2019)

  Receptor-interacting protein (RIP) kinase 3 (RIPK3)–dependent necroptosis directs inflammation and tissue injury, as well as anti-viral host defense. In human cells, herpes simplex virus 1 (HSV1) UL39-encoded ICP6 blocks RIP homotypic interacting motif (RHIM) signal transduction, preventing this leakage form of cell death and sustaining viral infection. TNF receptor 1 (TNFR1)-induced necroptosis is known to require the formation of a RIPK1–RIPK3–mixed lineage kinase domain–like pseudokinase (MLKL) signaling complex (necrosome) that we find compartmentalizes exclusively to caveolin-1–associated detergent-resistant membrane (DRM) vesicles in HT-29 cells. Translocation proceeds in the presence of RIPK3 kinase inhibitor GSK′840 or MLKL inhibitor necrosulfonomide but requires the kinase activity, as well as RHIM signaling of RIPK1. ICP6 impedes the translocation of RIPK1, RIPK3, and MLKL to caveolin-1–containing DRM vesicles without fully blocking the activation of RIPK3 or phosphorylation of MLKL. Consistent with the important contribution of RIPK1 RHIM-dependent recruitment of RIPK3, overexpression of RHIM-deficient RIPK3 results in phosphorylation of MLKL, but this does not lead to either translocation or necroptosis. Combined, these data reveal a critical role of RHIM signaling in the recruitment of the MLKL-containing necrosome to membrane vesicle–associated sites of aggregation. A similar mechanism is predicted for other RHIM-containing signaling adaptors, Z-nucleic acid–binding protein 1 (ZBP1) (also called DAI and DLM1), and TIR domain–containing adapter–inducing interferon-β (TRIF).  

3.3194           Combining LOPIT with differential ultracentrifugation for high-resolution spatial proteomics

Geladaki, A., Britovsek, N.K., Breckels, L.M., Smith, T.S., Vennard, O.L., Mulvey, C.M., Crook, O.M., Gatto, L. and Lilley, K.S: Nature Communications, 10:331 (2019)   The study of protein localisation has greatly benefited from high-throughput methods utilising cellular fractionation and proteomic profiling. Hyperplexed Localisation of Organelle Proteins by Isotope Tagging (hyperLOPIT) is a well-established method in this area. It achieves high-resolution separation of organelles and subcellular compartments but is relatively time- and resource-intensive. As a simpler alternative, we here develop Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC) and compare this method to the density gradient-based hyperLOPIT approach. We confirm that high-resolution maps can be obtained using differential centrifugation down to the suborganellar and protein complex level. HyperLOPIT and LOPIT-DC yield highly similar results, facilitating the identification of isoform-specific localisations and high-confidence localisation assignment for proteins in suborganellar structures, protein complexes and signalling pathways. By combining both approaches, we present a comprehensive high-resolution dataset of human protein localisations and deliver a flexible set of protocols for subcellular proteomics.  

3.3195           Proteome profiling of secreted and membrane vesicle associated proteins of an invasive and a commensal Staphylococcus haemolyticus isolate

Canavagh, J.P., Askarian, F., Pain, M., Bruun, J-A., Urbanova, I., Wai, S.N., Schmidt, F. and Johannessen, M. Data in Brief, 22, 914-919 (2019)   Bacterial membrane vesicles (MVs) mediate bacterial virulence by enabling secretion and long distance delivery of bacterial effector molecules. Staphylococcus haemolyticus has now been demonstrated to produce membrane vesicles (MVs). The protein content of S. haemolyticus MVs was identified by Mass spectrometry and compared to proteins identified in the total secretome. This information is presented in this data article. Further background and interpretation of the data can be found in the article: Comparative exoproteome profiling of an invasive and a commensal S. haemolyticus isolate (Cavanagh et al., in press). Data are available via Proteome Xchange with identifier PXD010389.  

3.3196           Neurons with Complex Karyotypes Are Rare in Aged Human Neocortex

Chronister, W.D., Burbulis, I.E., Wierman, M.B., Wolpart, M.J., Haakonsen, M.F., Smith, A.C.B., Kleinman, J.E., Hyde, T.M., Weinberger, D.R., Bekiranov, S. and McConnell, M.J. Cell Reports, 26, 825-835 (2019)   A subset of human neocortical neurons harbors complex karyotypes wherein megabase-scale copy-number variants (CNVs) alter allelic diversity. Divergent levels of neurons with complex karyotypes (CNV neurons) are reported in different individuals, yet genome-wide and familial studies implicitly assume a single brain genome when assessing the genetic risk architecture of neurological disease. We assembled a brain CNV atlas using a robust computational approach applied to a new dataset (>800 neurons from 5 neurotypical individuals) and to published data from 10 additional neurotypical individuals. The atlas reveals that the frequency of neocortical neurons with complex karyotypes varies widely among individuals, but this variability is not readily accounted for by tissue quality or CNV detection approach. Rather, the age of the individual is anti-correlated with CNV neuron frequency. Fewer CNV neurons are observed in aged individuals than in young individuals.  

3.3197           High Quality ATAC-Seq Data Recovered from Cryopreserved Breast Cell Lines and Tissue

Fujiwara, S., Baek, S., Varticovski, L., Kim, S. and Hager, G.L. Scientific Reports, 9:516 (2019)   DNA accessibility to transcription regulators varies between cells and modulates gene expression patterns. Several “open” chromatin profiling methods that provide valuable insight into the activity of these regulatory regions have been developed. However, their application to clinical samples has been limited despite the discovery that the Analysis of Transposase-Accessible Chromatin followed by sequencing (ATAC-seq) method can be performed using fewer cells than other techniques. Obtaining fresh rather than stored samples and a lack of adequate optimization and quality controls are major barriers to ATAC’s clinical implementation. Here, we describe an optimized ATAC protocol in which we varied nuclear preparation conditions and transposase concentrations and applied rigorous quality control measures before testing fresh, flash frozen, and cryopreserved breast cells and tissue. We obtained high quality data from small cell number. Furthermore, the genomic distribution of sequencing reads, their enrichment at transcription start sites, and transcription factor footprint analyses were similar between cryopreserved and fresh samples. This updated method is applicable to clinical samples, including cells from fine needle aspiration and tissues obtained via core needle biopsy or surgery. Chromatin accessibility analysis using patient samples will greatly expand the range of translational research and personalized medicine by identification of clinically-relevant epigenetic features.  

3.3198           Anthrax toxin requires ZDHHC5-mediated palmitoylation of its surface-processing host enzymes

Sergeeva, O.A. and van der Goot, F.G. PNAS, 116(4), 1279-1288 (2019)   The protein acyl transferase ZDHHC5 was recently proposed to regulate trafficking in the endocytic pathway. Therefore, we explored the function of this enzyme in controlling the action of bacterial toxins. We found that ZDHHC5 activity is required for two very different toxins: the anthrax lethal toxin and the pore-forming toxin aerolysin. Both of these toxins have precursor forms, the protoxins, which can use the proprotein convertases Furin and PC7 for activation. We show that ZDHHC5 indeed affects the processing of the protoxins to their active forms. We found that Furin and PC7 can both be S-palmitoylated and are substrates of ZDHHC5. The impact of ZDHHC5 on Furin/PC7-mediated anthrax toxin cleavage is dual, having an indirect and a direct component. First, ZDHHC5 affects the homeostasis and trafficking of a subset of cellular proteins, including Furin and PC7, presumably by affecting the endocytic/recycling pathway. Second, while not inhibiting the protease activity per se, ZDHHC5-mediated Furin/PC7 palmitoylation is required for the cleavage of the anthrax toxin. Finally, we show that palmitoylation of Furin and PC7 promotes their association with plasma membrane microdomains. Both the receptor-bound toxin and the convertases are of very low abundance at the cell surface. Their encounter is unlikely on reasonable time scales. This work indicates that palmitoylation drives their encounter in specific domains, allowing processing and thereby intoxication of the cell.  

3.3199           Protocol for eukaryotic plasma membrane isolation using superparamagnetic nanoparticles

Raj, D.B.T.G. and Khan, N.A.

1. Magnetism and Magnetic Materials, 476, 628-631 (2019)

  Superparamagnetic nanoparticles (SPMNP) are used in cell biology for various applications such as subcellular fractionation and for high pure organelle isolation. We simplified previously established methodology with the use of SPMNP for isolation of high pure and high yield plasma membrane from adherent eukaryotic cell line (Thimiri Govinda Raj et al., 2011, 2012, 2016; Tharkeshwar et al., 2014). Here, we show a step by step protocol for SPMNP based plasma membrane fractionation. Our research thus could be used as an alternative method to isolate plasma membrane fraction for cell biology, system biology and immunology research.  

3.3200           Tobramycin reduces key virulence determinants in the proteome of Pseudomonas aeruginosa outer membrane vesicles

Koeppen, K., Barnaby, R., Jackson, A.A., gerber, S.A., Hogan, D.A. and Stanton, B.A. PloS One, 14(1), e0211290 (2019)   Tobramycin is commonly used to treat Pseudomonas aeruginosa lung infections in patients with Cystic Fibrosis (CF). Tobramycin treatment leads to increased lung function and fewer clinical exacerbations in CF patients, and modestly reduces the density of P. aeruginosa in the lungs. P. aeruginosa resides primarily in the mucus overlying lung epithelial cells and secretes outer membrane vesicles (OMVs) that diffuse through the mucus and fuse with airway epithelial cells, thus delivering virulence factors into the cytoplasm that modify the innate immune response. The goal of this study was to test the hypothesis that Tobramycin reduces the abundance of virulence factors in OMVs secreted by P. aeruginosa. Characterization of the proteome of OMVs isolated from control or Tobramycin-exposed P. aeruginosa strain PAO1 revealed that Tobramycin reduced several OMV-associated virulence determinants, including AprA, an alkaline protease that enhances P. aeruginosa survival in the lung, and is predicted to contribute to the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl^- secretion by primary human bronchial epithelial cells. Deletion of the gene encoding AprA reduced the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl^- secretion. Moreover, as predicted by our proteomic analysis, OMVs isolated from Tobramycin treated P. aeruginosa had a diminished inhibitory effect on Phe508del-CFTR Cl^- secretion compared to OMVs isolated from control P. aeruginosa. Taken together, our proteomic analysis of OMVs and biological validation suggest that Tobramycin may improve lung function in CF patients infected with P. aeruginosa by reducing several key virulence factors in OMVs that reduce CFTR Cl^- secretion, which is essential for bacterial clearance from the lungs.  

3.3201           Listeria monocytogenes virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles

Coelho, C., Brown, L., Maryam, M., Vij, R., Smith, D.F.Q., Burnet, M.C.et al

1. Biol. Chem., 294(4), 1202-1217 (2019)

  Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.  

3.3202           A Phytophthora Effector Suppresses Trans-Kingdom RNAi to Promote Disease Susceptibility

Hou, Y., Zhai, Y., Feng, L., Innes, R.W., Zhai, J. and Ma, W. Cell Host & Microbe, 25(1), 153-165 (2019)   RNA silencing (RNAi) has a well-established role in anti-viral immunity in plants. The destructive eukaryotic pathogen Phytophthora encodes suppressors of RNAi (PSRs), which enhance plant susceptibility. However, the role of small RNAs in defense against eukaryotic pathogens is unclear. Here, we show that Phytophthora infection of Arabidopsis leads to increased production of a diverse pool of secondary small interfering RNAs (siRNAs). Instead of regulating endogenous plant genes, these siRNAs are found in extracellular vesicles and likely silence target genes in Phytophthora during natural infection. Introduction of a plant siRNA in Phytophthora leads to developmental deficiency and abolishes virulence, while Arabidopsis mutants defective in secondary siRNA biogenesis are hypersusceptible. Notably, Phytophthora effector PSR2 specifically inhibits secondary siRNA biogenesis in Arabidopsis and promotes infection. These findings uncover the role of siRNAs as antimicrobial agents against eukaryotic pathogens and highlight a defense/counter-defense arms race centered on trans-kingdom gene silencing between hosts and pathogens.  

3.3203           TFEB-driven endocytosis coordinates MTORC1 signaling and autophagy

Nnah, I., Wang, B., Saqcena, C., Weber, G.F., Bonder, E.B., Bagley, D., De Cegli, R., Napolitano, G., Medina, D.L., Ballabio, A. and Dobrowolski, R. Autophagy, 15(1), 153-164 (2019)   The mechanistic target of rapamycin kinase complex 1 (MTORC1) is a central cellular kinase that integrates major signaling pathways, allowing for regulation of anabolic and catabolic processes including macroautophagy/autophagy and lysosomal biogenesis. Essential to these processes is the regulatory activity of TFEB (transcription factor EB). In a regulatory feedback loop modulating transcriptional levels of RRAG/Rag GTPases, TFEB controls MTORC1 tethering to membranes and induction of anabolic processes upon nutrient replenishment. We now show that TFEB promotes expression of endocytic genes and increases rates of cellular endocytosis during homeostatic baseline and starvation conditions. TFEB-mediated endocytosis drives assembly of the MTORC1-containing nutrient sensing complex through the formation of endosomes that carry the associated proteins RRAGD, the amino acid transporter SLC38A9, and activate AKT/protein kinase B (AKT p-T308). TFEB-induced signaling endosomes en route to lysosomes are induced by amino acid starvation and are required to dissociate TSC2, re-tether and activate MTORC1 on endolysosomal membranes. This study characterizes TFEB-mediated endocytosis as a critical process leading to activation of MTORC1 and autophagic function, thus identifying the importance of the dynamic endolysosomal system in cellular clearance.    

3.3204           Isolation and Flow Cytometry Characterization of Extracellular‐Vesicle Subpopulations Derived from Human Mesenchymal Stromal Cells

Gorgun, C., Reverberi, D., Rotta, G., Villa, F., Quarto, . and Tasso, R. Current Protocols in Stem Cell Biology, 48, e76 (2019)   This unit describes protocols for isolating subpopulations of extracellular vesicles (EVs) purified from human adipose tissue–derived mesenchymal stromal cells by density gradient centrifugation and for characterizing them by flow cytometry (FCM). Determining the optimal strategy for isolating EVs is a critical step toward retrieving the maximal amount while ensuring the recovery of different vesicular subtypes. The first protocol details density gradient centrifugation to isolate both exosomes and microvesicles. In the second protocol, characterization of EV subpopulations by FCM is depicted, taking advantage of non‐conventional modalities, in accordance with the latest technical indications. The procedures described here can be easily reproduced and can be employed regardless of the cell type used to obtain EVs.  

3.3205           IgA-enhancing effects of membrane vesicles derived from Lactobacillus sakei subsp. sakei NBRC15893

Yamasaki-Yashiki, S., Miyoshi, Y., Nakauama, T., Kunisawa, J. and Katakura, Y. Bioscience of Microbiota, Food and Health, 38(1), 23-29 (2019)   Immunoglobulin (Ig) A in the mucus of the intestinal tract plays an important role in preventing the invasion of pathogenic microorganisms and regulating the composition of the gut microbiota. Several strains of probiotic lactic acid bacteria (LAB) are known to promote intestinal IgA production. Bacteria are also known to naturally release spherical membrane vesicles (MVs) that are involved in various biological functions such as quorum sensing, pathogenesis, and host immunomodulation. However, the production of MVs by LAB and their effects on host immunity remain poorly understood. In this study, we investigated the MV production by Lactobacillus sakei subsp. sakei NBRC15893 isolated from kimoto, the traditional seed mash used for brewing sake. MVs were separated from the culture broth of L. sakei NBRC15893 through filtration and density gradient ultracentrifugation and were observed by transmission electron microscopy. The MVs showed a spherical morphology, with a diameter of 30–400 nm, and contained proteins and nucleic acids. In addition, both the LAB cells and purified MVs promoted IgA production by murine Peyer’s patch cells. This MV- and cell-induced IgA production was suppressed by neutralization of Toll-like receptor (TLR) 2, which recognizes cell wall components of gram-positive bacteria, using an anti-TLR2 antibody. Collectively, our results indicate that MVs released from L. sakei NBRC15893 enhance IgA production by activating host TLR2 signaling through its cell wall components. Thus, it is important to consider novel interactions between gut microbiota and hosts via MVs, and MVs derived from probiotic bacteria could have promising applications as safe adjuvants.  

3.3206           Proteomic characterization of outer membrane vesicles from gut mucosa-derived fusobacterium nucleatum

Liu, J., Hsieh, C-L., Gelincik, O., Develder, B., Sei, S., Zhang, S., Lipkin, S.M. and Chang, Y-F.

1. Proteomics, 195, 125-137 (2019)

  Fusobacterium nucleatum is a Gram-negative bacterium commonly found in the oral cavity and is often involved in periodontal diseases. Recent studies have shown increased F. nucleatum prevalence in colorectal cancer (CRC) tissues, and causal data has linked this bacterium to CRC tumorigenesis. Immune-based approaches to contain, reduce or eradicate its gut colonization may prevent CRC. Outer membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria, typically contain multiple putative virulence factors and may elicit protective immune responses if used as vaccines. Here, OMVs were isolated from F. nucleatum cultures and purified using gradient centrifugation. Proteins contained within the OMVs were identified by nano LC/MS/MS analysis. Of 98 proteins consistently identified from duplicate analyses, 60 were predicted to localize to the outer membrane or periplasm via signal peptide driven translocation. Of these, six autotransporter proteins, which constitute the majority of protein mass of OMVs, were associated with Type V secretion system. In addition, other putative virulence factor proteins with functional domains, including FadA, MORN2 and YadA-like domain, were identified with multiple exposed epitope sites as determined by in silico analysis. Altogether, the non-replicative OMVs of F. nucleatum contain multiple antigenic virulence factors that may play important roles in the design and development of vaccines against F. nucleatum.  

3.3207           Diurnal Rhythms Spatially and Temporally Organize Autophagy

Ryzhikov, M., Ehlers, A., Steinberg, D., Nakahira, K., Choi, A.M.K. and Haspel, J.A. Cell Reports, 26(7), 1880-1892 (2019)   Circadian rhythms are a hallmark of physiology, but how such daily rhythms organize cellular catabolism is poorly understood. Here, we used proteomics to map daily oscillations in autophagic flux in mouse liver and related these rhythms to proteasome activity. We also explored how systemic inflammation affects the temporal structure of autophagy. Our data identified a globally harmonized rhythm for basal macroautophagy, chaperone-mediated autophagy, and proteasomal activity, which concentrates liver proteolysis during the daytime. Basal autophagy rhythms could be resolved into two antiphase clusters that were distinguished by the subcellular location of targeted proteins. Inflammation induced by lipopolysaccharide reprogrammed autophagic flux away from a temporal pattern that favors cytosolic targets and toward the turnover of mitochondrial targets. Our data detail how daily biological rhythms connect the temporal, spatial, and metabolic aspects of protein catabolism.  

3.3208           Extracellular RNA in renal diseases

Zhou, Y. and Yang, J. ExRNA, 1:5 (2019)   The discovery of extracellular RNA (exRNA) in blood and bodily fluids has expended our knowledge on RNA functions. ExRNAs may be transferred between cells and have functional significance in health and diseases by horizontal gene regulation. Moreover, exRNA may serve as diagnostic biomarkers and therapeutic tools. Thanks to the evolving techniques, exRNAs, particularly those detected from urine have gained great interest in renal diseases. However, most current studies on exRNA in renal diseases were derived from limited numbers of patients or in vitro data. Recent isolation and detection techniques also remained controversial. Nonetheless, the reliable techniques are developing and improving. This review will provide insights into the roles of exRNA in pathogenesis, diagnosis and therapy of renal diseases.  

3.3209           Engineering extracellular vesicles as novel treatment options: exploiting herpesviral immunity in CLL

Gärtner, K., Luckner, m., Wanner, G. and Zeidler, R.

1. Extracellular Vesicles, 8(1), 1573051 (2019)

  Extracellular vesicles (EVs) are important mediators of cell–cell communication. Intriguingly, EVs can be engineered and thus exploited for the targeted transfer of functional proteins of interest. Thus, engineered EVs may constitute attractive tools for the development of novel therapeutic interventions, like cancer immunotherapies, vaccinations or targeted drug delivery. Here, we describe a novel experimental immunotherapeutic approach for the adjuvant treatment of chronic lymphocytic leukaemia (CLL) based on engineered EVs carrying gp350, the major glycoprotein of Epstein–Barr virus (EBV), CD40L, a central immune accessory molecule and pp65, an immunodominant antigen of the human cytomegalovirus (CMV). We show that these engineered EVs specifically interact with malignant B cells from CLL patients and render these cells immunogenic to allogeneic and autologous EBV- and CMV-specific CD4+ and CD8+ T cells. Collectively, co-opting engineered EVs to re-target the strong herpesviral immunity in CLL patients to malignant cells constitutes an attractive strategy for the adjuvant treatment of a still incurable disease.  

3.3210           Slow Release of HIV-1 Protein Nef from Vesicle-like Structures Is Inhibited by Cytosolic Calcium Elevation in Single Human Microglia

Stenovec, M., Lasic, E., Dominkus, P.P., Bobnar, S.T., Zorec, R., Lenassi, M. and Kreft, M. Mol. Neurobiol., 56(1), 102-118 (2019)   Once infected by HIV-1, microglia abundantly produce accessory protein Nef that enhances virus production and infectivity, but little is known about its intracellular compartmentalization, trafficking mode(s), and release from microglia. Here, we transfected immortalized human microglia with a plasmid encoding Nef tagged with green fluorescent protein (Nef.GFP) to biochemically and microscopically identify Nef.GFP-associated cellular compartments and examine their mobility and Nef release from cultured cells. Immunoblotting revealed that Nef.GFP confined to subcellular fractions with a buoyant density similar to organelles positive for lysosomal-associated membrane protein 1 (LAMP1) but structurally segregated from dextran-laden and LysoTracker-laden endo-/lysosomes in live cells. As revealed by confocal microscopy, Nef.GFP-positive vesicle-like structures were smaller than dextran-laden vesicles and displayed slow and non-directional mobility, in contrast to the faster and directional mobility of dextran-laden vesicles. Ionomycin-evoked elevation in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) negligibly affected mobility of Nef.GFP structures but strongly and irrecoverably attenuated mobility of dextran-laden vesicles. A slow time-dependent decrease in the number of Nef.GFP-positive structures was observed in non-stimulated controls (5 ± 1 structures/min), but not in ionomycin-stimulated cells (0 ± 2 structures/min; P < 0.05), indicating that elevated [Ca²⁺]_i inhibits the release of Nef.GFP structures. The latter significantly co-localized with membrane sites immunopositive for the tetraspanins CD9 (36 ± 4%) and CD81 (22 ± 1%). This is the first report to demonstrate that microglial CD9- and CD81-positive plasma membrane-derived compartments are associated with biogenesis and Nef release.  

3.3211           Purification of Leukemia-Derived Exosomes to Study Microenvironment Modulation

Wierz, M., Pierson, S., Gargiulo, E., Guerin, C., Moussay, E. and Paggetti, J. Methods in Mol. Biol., 1884, 231-245 (2019)   Exosomes are membrane-enclosed vesicles released by different cell types into the extracellular space. As mediators of intercellular communication, they are involved in multiple physiological processes, but they are also associated with the pathogenesis of human malignancies including leukemia. Isolation of exosomes enables the characterization of their role in microenvironment modulation as well as their participation in disease pathology. A variety of strategies and techniques exists to purify exosomes from many biological fluids (e.g., blood, urine, and saliva). Here, we describe the efficient production of large quantities of exosomes from leukemic cell lines by using CELLine bioreactors based on two-compartment technology, as well as their isolation and purification by combining differential centrifugation and ultracentrifugation through a density gradient (17% OptiPrep^™ cushion). Thus, exosomes are appropriately prepared for characterization by western blotting to detect exosome markers or imaging flow cytometry (ImageStream), and for downstream analyses such as the internalization in microenvironmental cells by confocal imaging or flow cytometry, methods which are also described in this chapter.  

3.3212           Cyclodextrins reduce the ability of Pseudomonas aeruginosa outer-membrane vesicles to reduce CFTR Cl− secretion

Barnaby, R., Koeppen, K. and Stanton, B.A: Am. J. Physiol. Cell Mol. Physiol., 316(1), L206-L2015 (2019)  

Pseudomonas aeruginosa secretes outer-membrane vesicles (OMVs) that fuse with cholesterol-rich lipid rafts in the apical membrane of airway epithelial cells and decrease wt-CFTR Cl⁻ secretion. Herein, we tested the hypothesis that a reduction of the cholesterol content of CF human airway epithelial cells by cyclodextrins reduces the inhibitory effect of OMVs on VX-809 (lumacaftor)-stimulated Phe508del CFTR Cl⁻ secretion. Primary CF bronchial epithelial cells and CFBE cells were treated with vehicle, hydroxypropyl-β-cyclodextrin (HPβCD), or methyl-β-cyclodextrin (MβCD), and the effects of OMVs secreted by P. aeruginosa on VX-809 stimulated Phe508del CFTR Cl⁻ secretion were measured in Ussing chambers. Neither HPβCD nor MβCD were cytotoxic, and neither altered Phe508del CFTR Cl⁻ secretion. Both cyclodextrins reduced OMV inhibition of VX-809-stimulated Phe508del-CFTR Cl⁻ secretion when added to the apical side of CF monolayers. Both cyclodextrins also reduced the ability of P. aeruginosa to form biofilms and suppressed planktonic growth of P. aeruginosa. Our data suggest that HPβCD, which is in clinical trials for Niemann-Pick Type C disease, and MβCD, which has been approved by the U.S. Food and Drug Administration for use in solubilizing lipophilic drugs, may enhance the clinical efficacy of VX-809 in CF patients when added to the apical side of airway epithelial cells, and reduce planktonic growth and biofilm formation by P. aeruginosa. Both effects would be beneficial to CF patients.

3.3213           Sphingolipid-dependent Dscam sorting regulates axon segregation

Goyal, G., Zheng, J., Adam, E., Steffes, G., jain, M., Klavins, K. and Hummel, T. Nature Communications, 10:813 (2019)   Neurons are highly polarized cells with distinct protein compositions in axonal and dendritic compartments. Cellular mechanisms controlling polarized protein sorting have been described for mature nervous system but little is known about the segregation in newly differentiated neurons. In a forward genetic screen for regulators of Drosophila brain circuit development, we identified mutations in SPT, an evolutionary conserved enzyme in sphingolipid biosynthesis. Here we show that reduced levels of sphingolipids in SPT mutants cause axonal morphology defects similar to loss of cell recognition molecule Dscam. Loss- and gain-of-function studies show that neuronal sphingolipids are critical to prevent aggregation of axonal and dendritic Dscam isoforms, thereby ensuring precise Dscam localization to support axon branch segregation. Furthermore, SPT mutations causing neurodegenerative HSAN-I disorder in humans also result in formation of stable Dscam aggregates and axonal branch phenotypes in Drosophila neurons, indicating a causal link between developmental protein sorting defects and neuronal dysfunction.  

3.3214           Picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential

Van der Grein, S.G., Defourny, K.A.Y., Rabouw, H.H., Galiveti, C.R., Langereis, M.A., Wauben, M.H.M., Arkestejn, G.J.A., van Kuppelveld, F.J.M. and Nolte-t’Hoen, E.N.M. PloS Pathogen,. 15(2), e1007594 (2019)   Several naked virus species, including members of the Picornaviridae family, have recently been described to escape their host cells and spread infection via enclosure in extracellular vesicles (EV). EV are 50–300 nm sized lipid membrane-enclosed particles produced by all cells that are broadly recognized for playing regulatory roles in numerous (patho)physiological processes, including viral infection. Both pro- and antiviral functions have been ascribed to EV released by virus-infected cells. It is currently not known whether this reported functional diversity is a result of the release of multiple virus-containing and non-virus containing EV subpopulations that differ in composition and function. Using encephalomyocarditis virus infection (EMCV, Picornaviridae family), we here provide evidence that EV populations released by infected cells are highly heterogeneous. Virus was contained in two distinct EV populations that differed in physical characteristics, such as sedimentation properties, and in enrichment for proteins indicative of different EV biogenesis pathways, such as the plasma membrane resident proteins Flotillin-1 and CD9, and the autophagy regulatory protein LC3. Additional levels of EV heterogeneity were identified using high-resolution flow cytometric analysis of single EV. Importantly, we demonstrate that EV subsets released during EMCV infection varied largely in potency of transferring virus infection and in their kinetics of release from infected cells. These data support the notion that heterogeneous EV populations released by virus-infected cells can exert diverse functions at distinct time points during infection. Unraveling the compositional, temporal and functional heterogeneity of these EV populations using single EV analysis technologies, as employed in this study, is vital to understanding the role of EV in virus dissemination and antiviral host responses.  

3.3215           Fatty-acid binding protein 5 modulates the SAR1 GTPase cycle and enhances budding of large COPII cargoes

Melville, D., Gorur, A. and Schekman, R. Mol. Biol. Cell, 30(3), 387-399 (2019)   COPII-coated vesicles are the primary mediators of ER-to-Golgi trafficking. Sar1, one of the five core COPII components, is a highly conserved small GTPase, which, upon GTP binding, recruits the other COPII proteins to the ER membrane. It has been hypothesized that the changes in the kinetics of SAR1 GTPase may allow for the secretion of large cargoes. Here we developed a cell-free assay to recapitulate COPII-dependent budding of large lipoprotein cargoes from the ER. We identified fatty-acid binding protein 5 (FABP5) as an enhancer of this budding process. We found that FABP5 promotes the budding of particles ∼150 nm in diameter and modulates the kinetics of the SAR1 GTPase cycle. We further found that FABP5 enhances the trafficking of lipoproteins and of other cargoes, including collagen. These data identify a novel regulator of SAR1 GTPase activity and highlight the importance of this activity for trafficking of large cargoes.  

3.3216           Therapeutic use of mesenchymal stem cell–derived extracellular vesicles in acute lung injury

Lee, J.H., Park, J. and Lee, J-W. Transfusion, 59, 876-883 (2019)   Acute respiratory distress syndrome is a major cause of respiratory failure in critically ill patients. Despite extensive research into its pathophysiology, mortality remains high. No effective pharmacotherapy exists. Based largely on numerous preclinical animal studies, administration of mesenchymal stem or stromal cell (MSC) as a therapeutic for acute lung injury (ALI) holds great promise, and Phase I and II clinical trials are currently under way internationally. However, concern for the use of stem cells, specifically the risk of iatrogenic tumor formation, as well as the prohibitive cost of production, storage, and distribution of cells in bone marrow transplant facilities, may limit access to this lifesaving therapy. Accumulating evidence now suggest that novel stem cell–derived therapies, including MSC‐conditioned medium and extracellular vesicles (EVs) released from MSCs, might constitute compelling alternatives. The current review summarizes the preclinical studies testing MSC EVs as treatment for ALI and other inflammatory lung diseases. While certain logistic obstacles limit the clinical applications of MSC‐conditioned medium such as the volume required for treatment and lack of standardization of what constitutes the components of conditioned medium, the therapeutic application of MSC EVs remains promising, primarily due to ability of EVs to maintain the functional phenotype of the parent cell. However, utilization of MSC EVs will require large‐scale production and standardization concerning identification, characterization, and quantification.  

3.3217           Exosome Release Is Regulated by mTORC1

Zou, W., Lai, M., Zhang, Y., Zheng, L., Xing, Z., Li, T. et al Advanced Sci., 6, 1801313 (2019)   Exosomes are small membrane‐bound vesicles released into extracellular spaces by many types of cells. These nanovesicles carry proteins, mRNA, and miRNA, and are involved in cell waste management and intercellular communication. In the present study, it is shown that exosome release, which leads to net loss of cellular membrane and protein content, is negatively regulated by mechanistic target of rapamycin complex 1 (mTORC1). It is found that in cells and animal models exosome release is inhibited by sustained activation of mTORC1, leading to intracellular accumulation of CD63‐positive exosome precursors. Inhibition of mTORC1 by rapamycin or nutrient and growth factor deprivation stimulates exosome release, which occurs concomitantly with autophagy. The drug‐stimulated release is blocked by siRNA‐mediated downregulation of small GTPase Rab27A. Analysis of the cargo content in exosomes released from rapamycin‐treated cells reveals that inhibition of mTORC1 does not significantly alter its majority protein and miRNA profiles. These observations demonstrate that exosome release, like autophagy, is negatively regulated by mTORC1 in response to changes in nutrient and growth factor conditions.  

3.3218           Tumor‐Derived Extracellular Vesicles as a Novel Source of Protein Biomarkers for Cancer Diagnosis and Monitoring

Ruhen, O. and Meehan, K. Proteomics, 19(1-2), 1800155 (2019)   “Liquid biopsies” have received attention as a complementary tool for traditional tissue biopsies that may enhance the spectrum of analysis for tumor‐derived factors. One such factor gaining prominence in the liquid biopsy field is extracellular vesicles (EVs), membrane‐bound nanovesicles which are secreted by cells into biofluids such as blood, urine, and saliva. EVs are released in both physiological and pathological conditions and can transport a variety of molecules, including proteins, metabolites, RNA, microRNAs, and DNA, to distant sites throughout the body. As such, they are emerging as a promising source of tumor biomarkers for the noninvasive diagnosis, prognosis, and monitoring of cancer patients. In particular, the wealth of tumor‐related information that can be gleaned from the EV proteomic cargo has become apparent through mass spectrometric analysis, which has provided new benchmarks for clinically focused biomarker research. In this review, the current achievements in the use of MS for identifying potential EV‐derived protein biomarkers of cancer are explored, and the techniques and challenges involved in this pursuit are summarized.  

3.3219           Extracellular Vesicles from Neurosurgical Aspirates Identifies Chaperonin Containing TCP1 Subunit 6A as a Potential Glioblastoma Biomarker with Prognostic Significance

Hallal, S., Russell, B.P., Wei, H., Lee, M.Y.T., Toon, C.W., Sy, J., Shivalingam, B., Buckland, M.E. and Kaufman, K.L. Proteomics, 19(1-2), 1800157 (2019)   Glioblastoma, WHO‐grade IV glioma, carries a dismal prognosis owing to its infiltrative growth and limited treatment options. Glioblastoma‐derived extracellular vesicles (EVs; 30–1000 nm membranous particles) influence the microenvironment to mediate tumor aggressiveness and carry oncogenic cargo across the blood–brain barrier into the circulation. As such, EVs are biomarker reservoirs with enormous potential for assessing glioblastoma tumors in situ. Neurosurgical aspirates are rich sources of EVs, isolated directly from glioma microenvironments. EV proteomes enriched from glioblastoma (n = 15) and glioma grade II–III (n = 7) aspirates are compared and 298 differentially‐abundant proteins (p‐value < 0.00496) are identified using quantitative LC–MS/MS. Along with previously reported glioblastoma‐associated biomarkers, levels of all eight subunits of the key molecular chaperone, T‐complex protein 1 Ring complex (TRiC), are higher in glioblastoma‐EVs, including CCT2, CCT3, CCT5, CCT6A, CCT7, and TCP1 (p < 0.00496). Analogous increases in TRiC transcript levels and DNA copy numbers are detected in silico; CCT6A has the greatest induction of expression and amplification in glioblastoma and shows a negative association with survival (p = 0.006). CCT6A is co‐localized with EGFR at 7p11.2, with a strong tendency for co‐amplification (p < 0.001). Immunohistochemistry corroborates the CCT6A proteomics measurements and indicated a potential link between EGFR and CCT6A tissue expression. Putative EV‐biomarkers described here should be further assessed in peripheral blood.  

3.3220           Proteomic Analysis of Extracellular Vesicles for Cancer Diagnostics

Wu, A.Y-T., Ueda, K. and Lai, C.P-K. Proteomics, 19(1-2), 1800162 (2019)   Extracellular vesicles (EVs) including exosomes and microvesicles are lipid bilayer‐encapsulated nanoparticles released by cells, ranging from 40 nm to several microns in diameter. Biological cargoes including proteins, RNAs, and DNAs can be ferried by EVs to neighboring and distant cells via biofluids, serving as a means of cell‐to‐cell communication under normal and pathological conditions, especially cancers. On the other hand, EVs have been investigated as a novel “information capsule” for early disease detection and monitoring via liquid biopsy. This review summarizes current advancements in EV subtype characterization, cancer EV capture, proteomic analysis technologies, as well as possible EV‐based multiomics for cancer diagnostics.  

3.3221           Proteomic Signature of Mesenchymal Stromal Cell‐Derived Small Extracellular Vesicles

Van Balkom, B.W.M., Gremmels, H., Giebel, B. and Lim, S.K. Proteomics, 19(1-2), 18000163 (2019)   Small extracellular vesicles (EVs) are 50–200 nm vesicles secreted by most cells. They are considered as mediators of intercellular communication, and EVs from specific cell types, in particular mesenchymal stem/stromal cells (MSCs), offer powerful therapeutic potential, and can provide a novel therapeutic strategy. They appear promising and safe (as EVs are non‐self‐replicating), and eventually MSC‐derived EVs (MSC‐EVs) may be developed to standardized, off‐the‐shelf allogeneic regenerative and immunomodulatory therapeutics. Promising pre‐clinical data have been achieved using MSCs from different sources as EV‐producing cells. Similarly, a variety EV isolation and characterization methods have been applied. Interestingly, MSC‐EVs obtained from different sources and prepared with different methods show in vitro and in vivo therapeutic effects, indicating that isolated EVs share a common potential. Here, well‐characterized and controlled, publicly available proteome profiles of MSC‐EVs are compared to identify a common MSC‐EV protein signature that might be coupled to the MSC‐EVs’ common therapeutic potential. This protein signature may be helpful in developing MSC‐EV quality control platforms required to confirm the identity and test for the purity of potential therapeutic MSC‐EVs.  

3.3222           Helicobacter pylori Growth Stage Determines the Size, Protein Composition, and Preferential Cargo Packaging of Outer Membrane Vesicles

Zavan, L., Bitto, N.J., Johnston, E.L., Greening, D.W. and Kaparakis-Liaskos, M. Proteomics, 19(1-2), 1800209 (2019)   Gram‐negative bacteria release outer membrane vesicles (OMVs) as part of their normal growth that contain a range of cargo from their parent bacterium, including DNA, RNA, and proteins. The protein content of OMVs is suggested to be similar in composition to various sub‐cellular locations of their parent bacterium. However, very little is known regarding the effect of bacterial growth stage on the size, content, and selective packaging of proteins into OMVs. In this study, the global proteome of Helicobacter pylori and their OMVs throughout bacterial growth are examined to determine if bacterial growth stage affected OMV cargo composition. Analysis of OMVs produced by H. pylori reveals that bacterial growth stage affects the size, composition, and selection of protein cargo into OMVs. Proteomic analysis identifies that the proteome of H. pylori OMVs is vastly different throughout bacterial growth and that OMVs contain a range of proteins compared to their parent bacteria. In addition, bacterial growth stage affects the ability of OMVs to induce the production of IL‐8 by human epithelial cells. Therefore, the findings identify that the size, proteome, and immunogenicity of OMVs produced during various stages of bacterial growth is not comparable. Collectively, these findings highlight the importance of considering the bacterial growth stage from which OMVs are isolated, as this will impact their size, protein composition, and ultimately their biological functions.  

3.3223           Application of Extracellular Vesicles Proteomics to Cardiovascular Disease: Guidelines, Data Analysis, and Future Perspectives

Barrachina, M.N., Calderon-Cruz, B., Fernandez-Rocca, L. and Garcia, A. Proteomics, 19(1-2), 1800247 (2019)   Extracellular vesicles (EVs) are a heterogeneous population of vesicles composed of a lipid bilayer that carry a large repertoire of molecules including proteins, lipids, and nucleic acids. In this review, some guidelines for plasma‐derived EVs isolation, characterization, and proteomic analysis, and the application of the above to cardiovascular disease (CVD) studies are provided. For EVs analysis, blood samples should be collected using a 21‐gauge needle, preferably in citrate tubes, and plasma stored for up to 1 year at −80°, using a single freeze–thaw cycle. For proteomic applications, differential centrifugation (including ultracentrifugation steps) is a good option for EVs isolation. EVs characterization is done by transmission electron microscopy, particle enumeration techniques (nanoparticle‐tracking analysis, dynamic light scattering), and flow cytometry. Regarding the proteomics strategy, a label‐free and gel‐free quantitative method is a good choice due to its accuracy and because it minimizes the amount of sample required for clinical applications. Besides the above, main EVs proteomic findings in cardiovascular‐related diseases are presented and analyzed in this review, paying especial attention to overlapping results between studies. The latter might offer new insights into the clinical relevance and potential of novel EVs biomarkers identified to date in the context of CVD.  

3.3224           PNPLA3 variant M148 causes resistance to starvation‐mediated lipid droplet autophagy in human hepatocytes

Negoita, F., Blomdahl, J., Wasserstrom, S., Winsberg, M.E., Osmark, P., Larsson, S., Stenkula, K.G., Ekstedt, M., Kechaglas, S., Holm, C. and Jones, H.A:

1. Cell. Biochem., 120, 343-356 (2019)

  The mechanism of how patatin‐like phospholipase domain‐containing protein 3 (PNPLA3) variant M148 is associated with increased risk of development of hepatic steatosis is still debated. Here, we propose a novel role of PNPLA3 as a key player during autophagosome formation in the process of lipophagy. A human hepatocyte cell line, HepG2 cells, expressing recombinant I148 or 148M, was used to study lipophagy under energy deprived conditions, and lipid droplet morphology was investigated using florescence microscopy, image analysis and biochemical assays. Autophagic flux was studied using the golden‐standard of LC3‐II turnover in combination with the well characterized GFP‐RFP‐LC3 vector. To discriminate between, perturbed autophagic initiation and lysosome functionality, lysosomes were characterized by Lysotracker staining and LAMP1 protein levels as well as activity and activation of cathepsin B. For validation, human liver biopsies genotyped for I148 and 148M were analyzed for the presence of LC3‐II and PNPLA3 on lipid droplets. We show that the M148‐PNPLA3 variant is associated with lipid droplets that are resistant to starvation‐mediated degradation. M148 expressing hepatocytes reveal decreased autophagic flux and reduced lipophagy. Both I148‐PNPLA3 and M148‐PNPLA3 colocalize and interact with LC3‐II, but the M148‐PNPLA3 variant has lower ability to bind LC3‐II. Together, our data indicate that PNPLA3 might play an essential role in lipophagy in hepatocytes and furthermore that the M148‐PNPLA3 variant appears to display a loss in this activity, leading to decreased lipophagy.  

3.3225           Cell-Free Reconstitution of Autophagic Membrane Formation

Zhang, M. and Ge, L. Methods in Mol. Biol., 1880, 135-148 (2019)   Autophagy is a catabolic pathway for bulk turnover of cytoplasmic components through the lysosome. Completion of autophagy requires a sophisticated membrane remodeling process. The early steps involve autophagic membrane precursor generation from the intracellular membranes. The intricate protein-membrane interactions underlying autophagic membrane precursor generation have been a focus of attention but yet poorly defined. Here, we summarize the procedure of a cell-free system we have established to dissect the molecular mechanism of early autophagic membrane generation.  

3.3226           Nuclear localization and phosphorylation modulate pathological effects of alpha-synuclein

Pinho, R., Paiva, I., Jercic, K.G. et al Hum. Mol. Genet., 28(1), 31-50 (2019)   Alpha-synuclein (aSyn) is a central player in Parkinson’s disease (PD) but the precise molecular mechanisms underlying its pathogenicity remain unclear. It has recently been suggested that nuclear aSyn may modulate gene expression, possibly via interactions with DNA. However, the biological behavior of aSyn in the nucleus and the factors affecting its transcriptional role are not known. Here, we investigated the mechanisms underlying aSyn-mediated transcription deregulation by assessing its effects in the nucleus and the impact of phosphorylation in these dynamics. We found that aSyn induced severe transcriptional deregulation, including the downregulation of important cell cycle-related genes. Importantly, transcriptional deregulation was concomitant with reduced binding of aSyn to DNA. By forcing the nuclear presence of aSyn in the nucleus (aSyn-NLS), we found the accumulation of high molecular weight aSyn species altered gene expression and reduced toxicity when compared with the wild-type or exclusively cytosolic protein. Interestingly, nuclear localization of aSyn, and the effect on gene expression and cytotoxicity, was also modulated by phosphorylation on serine 129. Thus, we hypothesize that the role of aSyn on gene expression and, ultimately, toxicity, may be modulated by the phosphorylation status and nuclear presence of different aSyn species. Our findings shed new light onto the subcellular dynamics of aSyn and unveil an intricate interplay between subcellular location, phosphorylation and toxicity, opening novel avenues for the design of future strategies for therapeutic intervention in PD and other synucleinopathies.  

3.3227           Identification and Regulation of Multimeric Protein Complexes in Autophagy via SILAC-Based Mass Spectrometry Approaches

Kaeser-Pebernard, S., Diedrich, B. and Dengjel, J. Methods in Mol. Biol., 1880, 341-357 (2019)   Mass spectrometry (MS)-based identification and characterization of protein complexes is becoming a prerequisite for in-depth biochemical analyses of intracellular processes. Here, we describe two state-of-the-art MS-based approaches to characterize protein-protein interactions and multi-protein complexes involved in autophagy in mammalian cells. The combination of affinity purification (AP)-MS, which identifies binary protein-protein interactions, with size-exclusion chromatography (SEC)-protein correlation profiling (PCP), which helps monitor protein complex assemblies, is a powerful tool to acquire a full overview of the interlinkage and regulation of novel multi-protein complexes that might play a role in autophagy.  

3.3228           Phosphoinositide Interactions Position cGAS at the Plasma Membrane to Ensure Efficient Distinction between Self- and Viral DNA

Barnett, K.C., Coronas-Serna, J.M., Zhou, W., Ernandes, M.J., Cao, A., Kranzusch, P.J. and Kagan, J.C.

Cell, 176, 1432-1446 (2019)

The presence of DNA in the cytosol of mammalian cells is an unusual event that is often associated with genotoxic stress or viral infection. The enzyme cGAS is a sensor of cytosolic DNA that induces interferon and inflammatory responses that can be protective or pathologic, depending on the context. Along with other cytosolic innate immune receptors, cGAS is thought to diffuse throughout the cytosol in search of its DNA ligand. Herein, we report that cGAS is not a cytosolic protein but rather localizes to the plasma membrane via the actions of an N-terminal phosphoinositide-binding domain. This domain interacts selectively with PI(4,5)P2, and cGAS mutants defective for lipid binding are mislocalized to the cytosolic and nuclear compartments. Mislocalized cGAS induces potent interferon responses to genotoxic stress, but weaker responses to viral infection. These data establish the subcellular positioning of a cytosolic innate immune receptor as a mechanism that governs self-nonself discrimination.

3.3229           Comparative exoproteome profiling of an invasive and a commensal Staphylococcus haemolyticus isolate

Cavanagh, J.P., Pain, M., Askarian, F., Bruun, J-A., Urbarova, I., Wai, S.N., Schmidt, F. and Johannessen, M.

1. Proteomics, 197, 106-114 (2019)

  Staphylococcus haemolyticus is a skin commensal emerging as an opportunistic pathogen. Nosocomial isolates of S. haemolyticus are the most antibiotic resistant members of the coagulase negative staphylococci (CoNS), but information about other S. haemolyticus virulence factors is scarce. Bacterial membrane vesicles (MVs) are one mediator of virulence by enabling secretion and long distance delivery of bacterial effector molecules while protecting the cargo from proteolytic degradation from the environment. We wanted to determine if the MV protein cargo of S. haemolyticus is strain specific and enriched in certain MV associated proteins compared to the totalsecretome. The present study shows that both clinical and commensal S. haemolyticus isolates produce membrane vesicles. The MV cargo of both strains was enriched in proteins involved in adhesion and acquisition of iron. The MV cargo of the clinical strain was further enriched in antimicrobial resistance proteins.  

3.3230           Studying the Fate of Tumor Extracellular Vesicles at High Spatiotemporal Resolution Using the Zebrafish Embryo

Hyenne, V., Ghoroghi, S., Collot, M., Carapito, C., Klymchenko, A.S. and Goetz, J.G. Developmental Cell, 48(4), 554-572 (2019)   Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly to the benefit of tumor progression. Notably, tumor EVs travel in the bloodstream, reach distant organs, and locally modify the microenvironment. However, visualizing these events in vivo still faces major hurdles. Here, we describe an approach for tracking circulating tumor EVs in a living organism: we combine chemical and genetically encoded probes with the zebrafish embryo as an animal model. We provide a first description of tumor EVs’ hemodynamic behavior and document their intravascular arrest. We show that circulating tumor EVs are rapidly taken up by endothelial cells and blood patrolling macrophages and subsequently stored in degradative compartments. Finally, we demonstrate that tumor EVs activate macrophages and promote metastatic outgrowth. Overall, our study proves the usefulness and prospects of zebrafish embryo to track tumor EVs and dissect their role in metastatic niches formation in vivo.  

3.3231           RagA, an mTORC1 activator, interacts with a hedgehog signaling protein, WDR35/IFT121

Sekiguchi, T., Furuno, N., Ishii, T., Hirose, E., Sekiguchi, F., Wang, Y. and Kobayashi, H. Genes to Cells, 24(2), 151-161 (2019)   Small Ras‐like GTPases act as molecular switches for various signal transduction pathways. RagA, RagB/RagC and RagD are small Ras‐like GTPases that play regulatory roles in mTORC1. Lack of proper activation of mTORC1 can lead to diseases, such as cancer and diabetes. In this study, we found an interaction between RagA and WDR35. Mutations of WDR35 may cause genetic diseases including Sensenbrenner syndrome. WDR35 seems to be a hedgehog signaling protein with a possible ciliary function and a possible upstream regulator of RagA. RagB is a homologue of RagA and is also associated with WDR35. WDR35 is present in the endoplasmic reticulum, but usually not in lysosomes, where Rag family proteins act as an mTORC1 switch. Over‐expression of WDR35 results in decreased phosphorylation of ribosome S6 protein in a RagA‐, RagB‐ and RagC‐dependent manner. Thus, WDR35 is associated with RagA, RagB and RagC and might negatively influence mTORC1 activity.  

3.3232           Studying Autophagic Lysosome Reformation in Cells and by an In Vitro Reconstitution System

Chen, Y., Su, Q.P. and Yu, L. Methods in Mol. Biol., 1880, 163-172 (2019)   Autophagic lysosome reformation (ALR) is the terminal step of autophagy. ALR functions to recycle lysosomal membranes and maintain lysosome homeostasis. Maintaining a functional lysosome pool is critical for generating autolysosomes, in which cellular components are degraded and turned over during autophagy. This unit describes methods to visualize ALR in cells. In addition, this unit provides detailed protocols to establish in vitro systems which can be used to reconstitute ALR as well as to reconstitute mitochondrial tubulation/network formation, another process that is driven by motor proteins.  

3.3233           Investigating the Nanodomain Organization of Rhodopsin in Native Membranes by Atomic Force Microscopy

Senapati, S. and Park, P.S-H. Methods in Mol. Biol., 1886, 61-74 (2019)   Membrane proteins play an integral role in cellular communication. They are often organized within the crowded cell membrane into nanoscale domains (i.e., nanodomains), which facilitates their function in cell signaling processes. The visualization of membrane proteins and nanodomains within biological membranes under physiological conditions presents a challenge and is not possible using conventional microscopy methods. Atomic force microscopy (AFM) provides an opportunity to study the organization of membrane proteins within biological membranes with sub-nanometer resolution. An example of a membrane protein organized into nanodomains is rhodopsin. Rhodopsin is expressed in photoreceptor cells of the retina and upon photoactivation initiates a series of biochemical reactions called phototransduction, which represents the first steps of vision. AFM has provided an opportunity to directly visualize the packing of rhodopsin in native retinal membranes and the quantitative analysis of AFM images is beginning to reveal insights about the nanodomain organization of rhodopsin in the membrane. In this report, we outline procedures for imaging rhodopsin nanodomains by AFM and the quantitative analysis of those AFM images.  

3.3234           Exosomes and their importance in metastasis, diagnosis, and therapy of colorectal cancer

Cheshomi, H. and Matin, M.M.

1. Cell. Biochem., 120(2), 2671-2686 (2019)

  Extracellular vesicles are known as actual intermediaries of intercellular communications, such as biological signals and cargo transfer between different cells. A variety of cells release the exosomes as nanovesicular bodies. Exosomes contain different compounds such as several types of nucleic acids and proteins. In this study, we focused on exosomes in colorectal cancer as good tools that can be involved in various cancer‐related processes. Furthermore, we summarize the advantages and disadvantages of exosome extraction methods and review related studies on the role of exosomes in colorectal cancer. Finally, we focus on reports available on relations between mesenchymal stem cell–derived exosomes and colorectal cancer. Several cancer‐related processes such as cancer progression, metastasis, and drug resistance of colorectal cancer are related to the cargoes of exosomes. A variety of molecules, especially proteins, microRNAs, and long noncoding RNAs, play important roles in these processes. The microenvironment features, such as hypoxia, also have very important effects on the properties of the origin cell–derived exosomes. On the other hand, exosomes derived from colorectal cancer cells also interfere with cancer chemoresistance. Furthermore, today it is known that exosomes and their contents can likely be very effective in noninvasive colorectal cancer diagnosis and therapy. Thus, exosomes, and especially their cargoes, play different key roles in various aspects of basic and clinical research related to both progression and therapy of colorectal cancer.  

3.3235           X11 and X11‐like proteins regulate the level of extrasynaptic glutamate receptors

Motodate, R., Saito, H., Sobu, Y., hata, S., Saito, Y., Nakaya, T. and Suzuki, T.

1. Neurochem., 148(4), 480-498 (2019)

  X11/Mint 1 and X11‐like (X11L)/Mint 2 are neuronal adaptor protein to regulate trafficking and/or localization of various membrane proteins. By analyzing the localization of neuronal membrane proteins in X11‐, X11L‐, and X11/X11L doubly deficient mice with membrane fractionation procedures, we found that deficient of X11 and X11L decreased the level of glutamate receptors in non‐PSD fraction. This finding suggests that X11 and X11L regulate the glutamate receptor micro‐localization to the extrasynaptic region. In vitro coimmunoprecipitation studies of NMDA receptors lacking various cytoplasmic regions with X11 and X11L proteins harboring domain deletion suggest that extrasynaptic localization of NMDA receptor may be as a result of the multiple interactions of the receptor subunits with X11 and X11L regulated by protein phosphorylation, while that of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor subunits is not dependent on the binding with X11 and X11L proteins. Because the loss of X11 and X11L tends to impair the exocytosis, but not endocytosis, of glutamate receptors, NMDA receptors are likely to be supplied to the extrasynaptic plasma membrane with a way distinct from the mechanism regulating the localization of NMDA receptors into synaptic membrane region. Reduced localization of NMDA receptor into the extrasynaptic region increased slightly the phosphorylation level of cAMP responsible element binding protein in brain of X11/X11L doubly deficient mice compare to wild‐type mice, suggesting a possible role of X11 and X11L in the regulation of signal transduction pathway through extrasynaptic glutamate receptors.  

3.3236           A toolbox for the immunomagnetic purification of signaling organelles

Fritsch, J., Tchikov, V., Hennig, L., Lucius, R. and Schütze, S. Traffic, 20(3), 246-258 (2019)   Homeostasis and the complex functions of organisms and cells rely on the sophisticated spatial and temporal regulation of signaling in different intra‐ and extracellular compartments and via different mediators. We here present a set of fast and easy to use protocols for the target‐specific immunomagnetic enrichment of receptor containing endosomes (receptosomes), plasma membranes, lysosomes and exosomes. Isolation of subcellular organelles and exosomes is prerequisite for and will advance their detailed subsequent biochemical and functional analysis. Sequential application of the different subprotocols allows isolation of morphological and functional intact organelles from one pool of cells. The enrichment is based on a selective labelling using receptor ligands or antibodies together with superparamagnetic microbeads followed by separation in a patented matrix‐free high‐gradient magnetic purification device. This unique magnetic chamber is based on a focusing system outside of the empty separation column, generating an up to 3 T high‐gradient magnetic field focused at the wall of the column.  

3.3237           Extracellular Vesicles from Wharton's Jelly Mesenchymal Stem Cells Suppress CD4 Expressing T Cells Through Transforming Growth Factor Beta and Adenosine Signaling in a Canine Model

Crain, S.K., Robinson, S.R., Thane, K.E., Davis, A.M., Meola, D.M., Barton, B.A., Yang, V.K. and Hoffman, A.M. Stem Cells and Development, 28(3), 212-226 (2019)   Mesenchymal stem cells (MSCs) are widely investigated as potential therapeutic agents due to their potent immunomodulatory capacity. Although specific mechanisms by which MSC acts on immune cells are emerging, many questions remain, including the potential of extracellular vesicles (EVs) to mediate biological activities. Canine MSCs are of interest for both veterinary and comparative models of disease and have been shown to suppress CD4^pos T cell proliferation. The aim of this study was to determine whether EV isolated from canine Wharton's jelly-derived MSC (WJ-MSC EV) suppresses CD4^pos T cell proliferation using biochemical mechanisms previously ascribed to soluble mediators [transforming growth factor beta (TGF-β) and adenosine]. WJ-MSC EV exhibited mode of 125 nm diameter, low buoyant density (1.1 g/mL), and expression of EV proteins Alix and TSG101. Functionally, EVs inhibited CD4^pos T cell proliferation in a dose-dependent manner, which was absent in EV-depleted samples and EVs from non-MSC fibroblasts. EV suppression of CD4^pos T cell proliferation was inhibited by a TGF-βRI antagonist, neutralizing antibodies to TGF-β, or A2A adenosine receptor blockade. TGF-β was present on EVs as latent complexes most likely tethered to EV membrane by betaglycan. These data demonstrate that canine WJ-MSC EV utilizes TGF-β and adenosine signaling to suppress proliferation of CD4^pos T cell and will enable further investigation into mechanisms of immune cell modulation, as well as refinement of WJ-MSC and their EVs for therapeutic application.    

3.3238           Exosomes mediate Zika virus transmission through SMPD3 neutral Sphingomyelinase in cortical neurons

Zhou, W., Woodson, M., Sherman, M.B., Neelakanta, G. and Sultana, H.- Emerging Microbes & Infection, 8(1), 307-326 (2019)   The harmful effects of ZIKA virus (ZIKV) infection are reflected by severe neurological manifestations such as microcephaly in neonates and other complications associated with Guillain-Barré syndrome in adults. The transmission dynamics of ZIKV in or between neurons, or within the developing brains of the foetuses are not fully understood. Using primary cultures of murine cortical neurons, we show that ZIKV uses exosomes as mediators of viral transmission between neurons. Cryo-electron microscopy showed heterogeneous population of neuronal exosomes with a size range of 30–200 nm. Increased production of exosomes from neuronal cells was noted upon ZIKV infection. Neuronal exosomes contained both ZIKV viral RNA and protein(s) that were highly infectious to naïve cells. RNaseA and neutralizing antibodies treatment studies suggest the presence of viral RNA/proteins inside exosomes. Exosomes derived from time- and dose-dependent incubations showed increasing viral loads suggesting higher packaging and delivery of ZIKV RNA and proteins. Furthermore, we noted that ZIKV induced both activity and gene expression of neutral Sphingomyelinase (nSMase)-2/SMPD3, an important molecule that regulates production and release of exosomes. Silencing of SMPD3 in neurons resulted in reduced viral burden and transmission through exosomes. Treatment with SMPD3 specific inhibitor GW4869, significantly reduced ZIKV loads in both cortical neurons and in exosomes derived from these neuronal cells. Taken together, our results suggest that ZIKV modulates SMPD3 activity in cortical neurons for its infection and transmission through exosomes perhaps leading to severe neuronal death that may result in neurological manifestations such as microcephaly in the developing embryonic brains.  

3.3239           Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine

Matsumura, S., Minamisawa, T., Suga, K., Kishita, H., Akagi, T., Ichiki, T., Ichikawa, Y. and Shiba, K.

1. Extracellular Vesicles, 8(1), 1579541 (2019)

  Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS⁺-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy.    

3.3240           CNS-derived extracellular vesicles from superoxide dismutase 1 (SOD1)G93A ALS mice originate from astrocytes and neurons and carry misfolded SOD1

Silverman, J.M., Christy, D., Shyu, C.C., Moon, K-M., Fernando, S., Gidden, Z., Cowan, C.M., Ban, Y., Stacey, R.G., Grad, L.I., McAlary, L., Mackenzie, I.R., Foster, L.J. and Cashman, N.R.

1. Biol. Chem., 294(10), 3744-3759 (2019)

  Extracellular vesicles (EVs) are secreted by myriad cells in culture and also by unicellular organisms, and their identification in mammalian fluids suggests that EV release also occurs at the organism level. However, although it is clearly important to better understand EVs' roles in organismal biology, EVs in solid tissues have received little attention. Here, we modified a protocol for EV isolation from primary neural cell culture to collect EVs from frozen whole murine and human neural tissues by serial centrifugation and purification on a sucrose gradient. Quantitative proteomics comparing brain-derived EVs from nontransgenic (NTg) and a transgenic amyotrophic lateral sclerosis (ALS) mouse model, superoxide dismutase 1 (SOD1)^G93A, revealed that these EVs contain canonical exosomal markers and are enriched in synaptic and RNA-binding proteins. The compiled brain EV proteome contained numerous proteins implicated in ALS, and EVs from SOD1^G93A mice were significantly depleted in myelin-oligodendrocyte glycoprotein compared with those from NTg animals. We observed that brain- and spinal cord–derived EVs, from NTg and SOD1^G93A mice, are positive for the astrocyte marker GLAST and the synaptic marker SNAP25, whereas CD11b, a microglial marker, was largely absent. EVs from brains and spinal cords of the SOD1^G93A ALS mouse model, as well as from human SOD1 familial ALS patient spinal cord, contained abundant misfolded and nonnative disulfide-cross-linked aggregated SOD1. Our results indicate that CNS-derived EVs from an ALS animal model contain pathogenic disease-causing proteins and suggest that brain astrocytes and neurons, but not microglia, are the main EV source.  

3.3241           1216 - Silibinin induced parallel activation of macroautophagy and inhibition of chaperone-mediated autophagy in bladder cancer

Zeng, J., Hou, T., Chen, Y., He, D. and Li, L. Eur. Urol. Suppl., 18(1), e1630 (2019)   Introduction & Objectives: Macroautophagy and chaperone-mediated autophagy (CMA) represent two best-identified lysosomal degradation processes and often compensate for one another to facilitate cell survival. However, the interplay between macroautophagy and CMA in cancer remains largely unknown. A bulk of evidence from others and us suggests that silibinin, a natural flavonoid from milk thistle, exerts strong pleiotropic anticancer capabilities in various cancers. The aim of this study was to investigate the role of macroautophagy and CMA in silibinin-induced cell death in bladder cancer (BCa). Materials & Methods: Human BCa 5637 and T24 cell lines served as the model system in vitro and in vivo. Cell viability and apoptosis was determined by MTS assay and cell flow cytometry respectively. Lysosomes were isolated from cells by iodixanol-based density gradient centrifugation. The expression of CMA and macroautophagy components were examined by western blot after silibinin treatment. Coimmunoprecipitation was used to determine the interaction between hsc70 and LAMP2A. CMA activity was monitored by in vitro lysosome binding and uptake assay using RNase and GAPDH as substrates. Immunohistochemistry analysis was performed in BCa xenografts after oral silibinin in vivo. Results: We found that CMA receptor LAMP2A was overexpressed and had prognostic value in BCa tissues. Downregulation of hsc70 and LAMP2A on lysosomes and inhibition of hsc70 and LAMP2a interaction were observed after silibinin treatment. Additionally, silibinin treatment decreased the ability of substrates to be taken up by the lysosomes and inhibited lysosomal degradation of substrates, suggesting the suppression of CMA activity. Mechanistically, silibinin decreased LAMP2A stability on lysosome, which resulted in accelerated degradation of LAMP2A on lysosome. On the other hand, exposure to silibinin resulted in activation of AMPK-dependent macroautophagy in BCa. Oral silibinin suppressed the growth of BCa xenografts in vivo, which were accompanied with downregulation of hsc70 and LAMP2A and upregulation of p-AMPK. Interestingly, inhibition of macroautophagy by knocking down Atg5 had no effect on silibinin-induced downregulation of LAMP2A and inhibition of CMA activity. Similarly, augmentation of CMA activity by overexpression of exogenous LAMP2A failed to abolish silibinin-induced activation of macroautophagy, suggesting parallel activation of macroautophagy and inhibition of CMA by silibinin. Conclusions: This study reveals chemically regulation of CMA and macroautophagy as a novel mechanism for silibinin’s anti-proliferative effect, which may have antitumor potential against BCa.  

3.3242           The deubiquitinating enzyme USP25 binds tankyrase and regulates trafficking of the facilitative glucose transporter GLUT4 in adipocytes

Sadler, J.B.A., Lamb, C.A., Welburn, C.R., Adamson, I.S., Kioimourtzoglou, D., Chi, N-W., Gould, G. and Bryant, N.J. Scientific Reports, 9:4710 (2019) Key to whole body glucose homeostasis is the ability of fat and muscle cells to sequester the facilitative glucose transporter GLUT4 in an intracellular compartment from where it can be mobilized in response to insulin. We have previously demonstrated that this process requires ubiquitination of GLUT4 while numerous other studies have identified several molecules that are also required, including the insulin-responsive aminopeptidase IRAP and its binding partner, the scaffolding protein tankyrase. In addition to binding IRAP, Tankyrase has also been shown to bind the deubiquinating enzyme USP25. Here we demonstrate that USP25 and Tankyrase interact, and colocalise with GLUT4 in insulin-sensitive cells. Furthermore depletion of USP25 from adipocytes reduces cellular levels of GLUT4 and concomitantly blunts the ability of insulin to stimulate glucose transport. Collectively, these data support our model that sorting of GLUT4 into its insulin-sensitive store involves a cycle of ubiquitination and subsequent deubiquitination.  

3.3243           Recent advances in extracellular vesicle research for urological cancers: From technology to application

Dong, L., Zieren, R.C., Wang, Y., de Reijke, T.M., Xue, W. and Pienta, K.J. BBA – Reviews on Cancer, 1871, 342-360 (2019)   Urological malignancies, including prostate cancer, bladder cancer and kidney cancer, are major causes of morbidity and mortality worldwide. Because of the high incidence, diversity in biology, and especially direct interaction with urine, urological cancers are an important resource for both scientists and clinicians for novel diagnostic and therapeutic discovery. Extracellular vesicles (EVs) are lipid bilayer encapsulated particles released by cells into the extracellular space. Since EVs work as a safe way to transport important biological information through the whole body, they are now recognized as an important mechanism of cell–cell communication and have opened a new window for us to gain a better understanding of cancer biology, novel diagnostics, and therapeutic options. In recent years, numerous evolutions in EV technologies and novel biological and clinical findings continue to be reported in the research field of urological cancers. This comprehensive review aims to give an update of recent advances in EV technologies and summarize the state-of-the-art knowledge of EVs related to prostate cancer, bladder cancer and kidney cancer, particularly focusing on the potential of EV as biomarkers and their biological roles in promoting cancer and metastasis.  

3.3244           Exosomes in gastric cancer: roles, mechanisms, and applications

Fu, M., Gu, J., Jiang, P., Qian, H., Xu, W. and Zhang, X. Mol. Cancer, 18:41 (2019)   Exosomes are nanosized extracellular vesicles that can be released by almost all types of cells. Initially considered as the garbage bins acting to discard unwanted products of cells, exosomes are now recognized as an important way for cellular communication by transmitting bioactive molecules including proteins, DNA, mRNAs, and non-coding RNAs. The recent studies have shown that exosomes are critically involved in human health and diseases including cancer. Exosomes have been suggested to participate in the promotion of tumorigenesis, tumor growth and metastasis, tumor angiogenesis, tumor immune escape, and tumor therapy resistance. Increasing evidence indicate that exosomes play important roles in gastric cancer development and progression. In this review, we summarized the current understanding of exosomes in gastric cancer with an emphasis on the biological roles of exosomes in gastric cancer and their potential as biomarkers for gastric cancer diagnosis as well as potential targets for gastric cancer therapy.  

3.3245           Pathogenic alpha-synuclein aggregates preferentially bind to mitochondria and affect cellular respiration

Wang, X., Becker, K., Levine, N., Zhang, M., Leberman, A.P., moore, D.J. and Ma, J. Acta Neurophatol. Comm., 7:41 (2019)   Misfolded alpha-synuclein (αSyn) is a major constituent of Lewy bodies and Lewy neurites, which are pathological hallmarks of Parkinson’s disease (PD). The contribution of αSyn to PD is well established, but the detailed mechanism remains obscure. Using a model in which αSyn aggregation in primary neurons was seeded by exogenously added, preformed αSyn amyloid fibrils (PFF), we found that a majority of pathogenic αSyn (indicated by serine 129 phosphorylated αSyn, ps-αSyn) was membrane-bound and associated with mitochondria. In contrast, only a minuscule amount of physiological αSyn was mitochondrial bound. In vitro, αSyn PFF displayed a stronger binding to purified mitochondria than did αSyn monomer, revealing a preferential mitochondria binding by aggregated αSyn. This selective mitochondrial ps-αSyn accumulation was confirmed in other neuronal and animal αSyn aggregation models that do not require exogenously added PFF and, more importantly, in postmortem brain tissues of patients suffering from PD and other neurodegenerative diseases with αSyn aggregation (α-synucleinopathies). We also showed that the mitochondrial ps-αSyn accumulation was accompanied by defects in cellular respiration in primary neurons, suggesting a link to mitochondrial dysfunction. Together, our results show that, contrary to physiological αSyn, pathogenic αSyn aggregates preferentially bind to mitochondria, indicating mitochondrial dysfunction as the common downstream mechanism for α-synucleinopathies. Our findings suggest a plausible model explaining the formation and the peculiar morphology of Lewy body and reveal that disrupting the interaction between ps-αSyn and the mitochondria is a therapeutic target for α-synucleinopathies.  

3.3246           Poloxamer 188 rescues MPTP-induced lysosomal membrane integrity impairment in cellular and mouse models of Parkinson's disease

Dong, H., Qin, Y., Huang, Y., Ji, D. and Wu, F. Neurochem. Int., 126, 178-186 (2019)   Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Rupture of lysosome is a major cellular stress condition leading to cell death in PD. We have previously shown that environmental oxidative toxins could impair autophagic flux and lysosomal functions in PD. Poloxamer 188 (P188) is an amphipathic polymer which has cytoprotective effect in traumatic brain injury and stroke. But whether Dyrk1A could rescue lysosome malfunction-mediated DA neuron death and α-synuclein aggregation in PD is still unknown. In the present study, MPTP mice models and MPP⁺-treated SH-SY5Y cells were used for study, and we found that P188 rescued MPP⁺-induced lysosomal dysfunction and impaired autophagy flux in mild MPP⁺-treated SH-SY5Y cells. P188 administration significantly restored lysosomal membrane integrity and prevented cathepsins leakage from the lysosomes into the cytoplasm, which triggered caspase-dependent apoptotic cell death in sub-acute MPTP mouse model and MPP⁺-treated SH-SY5Y cells. Furthermore, P188 ameliorated α-synuclein accumulation and behavioral impairment in chronic MPTP mouse model with MPTP and probenecid treatment. P188 could alleviate MPTP-induced DA neurons damage by restoring lysosome function.  

3.3247           Species-specific differences in nonlysosomal glucosylceramidase GBA2 function underlie locomotor dysfunction arising from loss-of-function mutations

Woeste, M.A., Stern, S., Raju, D.N., Grahn, E., Dittmann, D., Gutbrod, K. et al

1. Biol. Chem., 294(11), 3853-3871 (2019)

  The nonlysosomal glucosylceramidase β2 (GBA2) catalyzes the hydrolysis of glucosylceramide to glucose and ceramide. Mutations in the human GBA2 gene have been associated with hereditary spastic paraplegia (HSP), autosomal-recessive cerebellar ataxia (ARCA), and the Marinesco-Sjögren–like syndrome. However, the underlying molecular mechanisms are ill-defined. Here, using biochemistry, immunohistochemistry, structural modeling, and mouse genetics, we demonstrate that all but one of the spastic gait locus #46 (SPG46)-connected mutations cause a loss of GBA2 activity. We demonstrate that GBA2 proteins form oligomeric complexes and that protein–protein interactions are perturbed by some of these mutations. To study the pathogenesis of GBA2-related HSP and ARCA in vivo, we investigated GBA2-KO mice as a mammalian model system. However, these mice exhibited a high phenotypic variance and did not fully resemble the human phenotype, suggesting that mouse and human GBA2 differ in function. Whereas some GBA2-KO mice displayed a strong locomotor defect, others displayed only mild alterations of the gait pattern and no signs of cerebellar defects. On a cellular level, inhibition of GBA2 activity in isolated cerebellar neurons dramatically affected F-actin dynamics and reduced neurite outgrowth, which has been associated with the development of neurological disorders. Our results shed light on the molecular mechanism underlying the pathogenesis of GBA2-related HSP and ARCA and reveal species-specific differences in GBA2 function in vivo.  

3.3248           Infection by Anaplasma phagocytophilum Requires Recruitment of Low-Density Lipoprotein Cholesterol by Flotillins

Xiong, Q., Lin, M., Huang, W. and Rikihisa, Y. mBio, 10(2), e02783-18 (2019)   Anaplasma phagocytophilum is an obligatory intracellular bacterium that proliferates in membrane-bound inclusions. A. phagocytophilum is dependent on cholesterol and acquire cholesterol from low-density lipoprotein (LDL) endocytosed by mammalian host cells. The mechanism of cholesterol transport to Anaplasma inclusions, however, is not fully understood. Flotillin-1 (FLOT1) and FLOT2 are cholesterol-associated membrane proteins that form a heterodimer and/or oligomer complex. Here, we found that Anaplasma infection was significantly reduced by small interfering RNA (siRNA) knockdown of FLOT1 or FLOT2. Anaplasma inclusions were encircled with small vesicles containing endogenous FLOT1 or FLOT2 or with ectopically expressed FLOT1-mCherry and FLOT2-green fluorescent protein (FLOT2-GFP). FLOT1- and FLOT2-containing vesicles were enriched with unesterified cholesterol, as indicated by labeling with filipin and aminomethyl coumarin acetic acid-conjugated theonellamide. Localization of FLOT2 to Anaplasma inclusions was dependent on cholesterol, as FLOT2-GFP bearing two mutations in the cholesterol recognition/interaction motif could not target the inclusions. The cholesterol-sequestering agent methyl-β-cyclodextrin abrogated FLOT1 localization to Anaplasma inclusions and cleared infection. FLOT2-GFP also localized to fluorescent 3,3′-dioctadecylindocarbocyanine (DiI)-LDL-containing vesicles, including those surrounding Anaplasma inclusions. FLOT2 siRNA knockdown blocked DiI-LDL trafficking to Anaplasma inclusions and reduced bacteria-associated cholesterol amount, and therefore inhibiting Anaplasma infection. Vesicles containing acid lipase, which hydrolyzes LDL cholesterol esters to free cholesterol, colocalized with FLOT2 and encircled Anaplasma inclusions, while the acid lipase inhibitor orlistat significantly inhibited Anaplasma replication. Together, the data revealed that FLOTs are crucial for Anaplasma replication in host cells, likely by aiding vesicular traffic of LDL-derived free cholesterol to Anaplasma inclusions, and suggest a new way of inhibiting Anaplasma infection.  

3.3249           Highlights of the mini-symposium on extracellular vesicles in inter-organismal communication, held in Munich, Germany, August 2018

Bielska, E., Birch, P.R.J., Buck, A.H., Abreu-Goodger, B.C., Innes, R.W., Jin, H., Pfaffl, M.W., Robatzek, P.S., Regev-Rudzki, N., Tisserant, C., Wang, S. and Weiberg, A.

1. Extracellular Vesicles, 8(1), 1590116 (2019)

  All living organisms secrete molecules for intercellular communication. Recent research has revealed that extracellular vesicles (EVs) play an important role in inter-organismal cell-to-cell communication by transporting diverse messenger molecules, including RNA, DNA, lipids and proteins. These discoveries have raised fundamental questions regarding EV biology. How are EVs biosynthesized and loaded with messenger/cargo molecules? How are EVs secreted into the extracellular matrix? What are the EV uptake mechanisms of recipient cells? As EVs are produced by all kind of organisms, from unicellular bacteria and protists, filamentous fungi and oomycetes, to complex multicellular life forms such as plants and animals, basic research in diverse model systems is urgently needed to shed light on the multifaceted biology of EVs and their role in inter-organismal communications. To help catalyse progress in this emerging field, a mini-symposium was held in Munich, Germany in August 2018. This report highlights recent progress and major questions being pursued across a very diverse group of model systems, all united by the question of how EVs contribute to inter-organismal communication.  

3.3250           Prostate cancer sheds the αvβ3 integrin in vivo through exosomes

Krishn, S.R., Singh, A., Bowler, N., Duffy, A.N., Friedman, A. et al Matrix Biol., 77, 41-57 (2019)   The αvβ3 integrin has been shown to promote aggressive phenotypes in many types of cancers, including prostate cancer. We show that GFP-labeled αvβ3 derived from cancer cells circulates in the blood and is detected in distant lesions in NOD scid gamma (NSG) mice. We, therefore, hypothesized that αvβ3 travels through exosomes and tested its levels in pools of vesicles, which we designate extracellular vesicles highly enriched in exosomes (ExVs), and in exosomes isolated from the plasma of prostate cancer patients. Here, we show that the αvβ3 integrin is found in patient blood exosomes purified by sucrose or iodixanol density gradients. In addition, we provide evidence that the αvβ3 integrin is transferred through ExVs isolated from prostate cancer patient plasma to β3-negative recipient cells. We also demonstrate the intracellular localization of β3-GFP transferred via cancer cell-derived ExVs. We show that the ExVs present in plasma from prostate cancer patients contain higher levels of αvβ3 and CD9 as compared to plasma ExVs from age-matched subjects who are not affected by cancer. Furthermore, using PSMA antibody-bead mediated immunocapture, we show that the αvβ3 integrin is expressed in a subset of exosomes characterized by PSMA, CD9, CD63, and an epithelial-specific marker, Trop-2. Finally, we present evidence that the levels of αvβ3, CD63, and CD9 remain unaltered in ExVs isolated from the blood of prostate cancer patients treated with enzalutamide. Our results suggest that detecting exosomal αvβ3 integrin in prostate cancer patients could be a clinically useful and non-invasive biomarker to follow prostate cancer progression. Moreover, the ability of αvβ3 integrin to be transferred from ExVs to recipient cells provides a strong rationale for further investigating the role of αvβ3 integrin in the pathogenesis of prostate cancer and as a potential therapeutic target.  

3.3251           Autophagy induction via STING trafficking is a primordial function of the cGAS pathway

Giu, X., Yang,k H., Li, T., Tan, X., Shi, P., Li, M., Du, F. and Chen, Z.J. Nature, 567, 262-266 (2019)   Cyclic GMP-AMP (cGAMP) synthase (cGAS) detects infections or tissue damage by binding to microbial or self DNA in the cytoplasm1. Upon binding DNA, cGAS produces cGAMP that binds to and activates the adaptor protein STING, which then activates the kinases IKK and TBK1 to induce interferons and other cytokines2,3,4,5,6. Here we report that STING also activates autophagy through a mechanism that is independent of TBK1 activation and interferon induction. Upon binding cGAMP, STING translocates to the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) and the Golgi in a process that is dependent on the COP-II complex and ARF GTPases. STING-containing ERGIC serves as a membrane source for LC3 lipidation, which is a key step in autophagosome biogenesis. cGAMP induced LC3 lipidation through a pathway that is dependent on WIPI2 and ATG5 but independent of the ULK and VPS34–beclin kinase complexes. Furthermore, we show that cGAMP-induced autophagy is important for the clearance of DNA and viruses in the cytosol. Interestingly, STING from the sea anemone Nematostella vectensis induces autophagy but not interferons in response to stimulation by cGAMP, which suggests that induction of autophagy is a primordial function of the cGAS–STING pathway.  

3.3252           Deubiquitinating enzyme USP30 maintains basal peroxisome abundance by regulating pexophagy

Riccio, V., Demers, N., Hua, R., Vissa, M., Cheng, D.T., Strilchuk, A.W., Wang, Y., McQuibban, G.A. and Kim, P.K.

1. Cell Biol., 218(3), 798-807 (82019)

  The regulation of organelle abundance is critical for cell function and survival; however, the mechanisms responsible are not fully understood. In this study, we characterize a role of the deubiquitinating enzyme USP30 in peroxisome maintenance. Peroxisomes are highly dynamic, changing in abundance in response to metabolic stress. In our recent study identifying the role of USP30 in mitophagy, we observed USP30 to be localized to punctate structures resembling peroxisomes. We report here that USP30, best known as a mitophagy regulator, is also necessary for regulating pexophagy, the selective autophagic degradation of peroxisomes. We find that overexpressing USP30 prevents pexophagy during amino acid starvation, and its depletion results in pexophagy induction under basal conditions. We demonstrate that USP30 prevents pexophagy by counteracting the action of the peroxisomal E3 ubiquitin ligase PEX2. Finally, we show that USP30 can rescue the peroxisome loss observed in some disease-causing peroxisome mutations, pointing to a potential therapeutic target.  

3.3253           Rab11a drives adhesion molecules to the surface of endometrial epithelial cells

Kakar-Bhanot, R., Brahmbhatt, K., Xhauhan, B., katkam, R.R., Bashir, T., Gawde, H., Mayadeo, N., Chaudhari, U.K. and Sachdeva, G. Human Reproduction, 34(3), 519-529 (2019)   STUDY QUESTION Is Rab11a GTPase, a regulator of intracellular trafficking, of significance in endometrial functions? SUMMARY ANSWER Rab11a is an important component of the cascades involved in equipping the endometrial epithelium (EE) with ‘adhesiveness’ and ‘cohesiveness’. WHAT IS KNOWN ALREADY Cell adhesion molecules (CAMs) have been investigated extensively for modulation in their endometrial expression during the peri-implantation phase. However, the mechanisms by which CAMs are transported to the EE surface have not received the same attention. Rab11a facilitates transport of specific proteins to the plasma membrane in endothelial cells, fibroblasts, embryonic ectodermal cells, etc. However, its role in the transport of CAMs in EE remains unexplored. STUDY DESIGN, SIZE, DURATION In-vitro investigations were directed towards deciphering the role of Rab11a in trafficking of CAMs (integrins and E-cadherin) to the cell surface of Ishikawa, an EE cell line. Towards this, Rab11a stable knockdown (Rab-kd) and control clones of Ishikawa were generated. JAr (human trophoblastic cell line) cells were used to form multicellular spheroids. Pre-receptive (n = 6) and receptive (n = 6) phase endometrial tissues from women with proven fertility and receptive phase (n = 6) endometrial tissues from women with unexplained infertility were used. PARTICIPANTS/MATERIALS, SETTING, METHODS Rab-kd and control clones were used for in-vitro assays. Live cells were used for biotinylation, JAr spheroid assays, flow cytometry, trans-epithelial electrical resistance assays and wound-healing assays. Lysosome and Golgi membranes were isolated by ultracentrifugation. Confocal microscopy, immunoblotting, qRT-PCR and immunohistochemistry were employed for assessing the expression of Rab11a, integrins and E-cadherin. MAIN RESULTS AND THE ROLE OF CHANCE shRNA-mediated attenuation of Rab11a expression led to a significant (P < 0.01) decline in the surface localization of αVβ3 integrin. Cell surface protein extracts of Rab-kd clones showed a significant (P < 0.05) reduction in the levels of αV integrin. Further, a significant (P < 0.01) decrease was observed in the percent JAr spheroids attached to Rab-kd clones, compared to control clones. Rab-kd clones also showed a significant (P < 0.001) decline in the total levels of E-cadherin. This was caused neither by reduced transcription nor by increased lysosomal degradation. The role of Rab11a in maintaining the epithelial nature of the cells was evident by a significant increase in the migratory potential, presence of stress-fibres and a decrease in the trans-epithelial resistance in Rab-kd monolayers. Further, the levels of endometrial Rab11a and E-cadherin in the receptive phase were found to be significantly (P < 0.05) lower in women with unexplained infertility compared to that in fertile women. Taken together, these observations hint at a key role of Rab11a in the trafficking of αVβ3 integrin and maintenance of E-cadherin levels at the surface of EE cells.  

3.3254           YAP Partially Reprograms Chromatin Accessibility to Directly Induce Adult Cardiogenesis In Vivo

Monroe, T.O., Hill, M.C., Morikawa, Y., Wehrens, X.H.T., Rodney, G.G. and Martin, J.F. Developmental Cell, 48(6), 765-779 (2019)   Specialized adult somatic cells, such as cardiomyocytes (CMs), are highly differentiated with poor renewal capacity, an integral reason underlying organ failure in disease and aging. Among the least renewable cells in the human body, CMs renew approximately 1% annually. Consistent with poor CM turnover, heart failure is the leading cause of death. Here, we show that an active version of the Hippo pathway effector YAP, termed YAP5SA, partially reprograms adult mouse CMs to a more fetal and proliferative state. One week after induction, 19% of CMs that enter S-phase do so twice, CM number increases by 40%, and YAP5SA lineage CMs couple to pre-existing CMs. Genomic studies showed that YAP5SA increases chromatin accessibility and expression of fetal genes, partially reprogramming long-lived somatic cells in vivo to a primitive, fetal-like, and proliferative state.  

3.3255           ARL13B, a Joubert Syndrome-Associated Protein, Is Critical for Retinogenesis and Elaboration of Mouse Photoreceptor Outer Segments

Dilan, T.I., Moye, A.R., Salido, E.M., Saravanan, T., Kolandaivelu, S., Goldberg, A.F.X. and Ramanurthy, V.

1. Neurosci., 39(8), 1364-1347 (2019)

  Mutations in the Joubert syndrome-associated small GTPase ARL13B are linked to photoreceptor impairment and vision loss. To determine the role of ARL13B in the development, function, and maintenance of ciliated photoreceptors, we generated a pan-retina knock-out (Six3-Cre) and a rod photoreceptor-specific inducible conditional knock-out (Pde6g-Cre^ERT2) of ARL13B using murine models. Embryonic deletion of ARL13B led to defects in retinal development with reduced cell proliferation. In the absence of ARL13B, photoreceptors failed to develop outer segment (OS) membranous discs and axonemes, resulting in loss of function and rapid degeneration. Additionally, the majority of photoreceptor basal bodies did not dock properly at the apical edge of the inner segments. The removal of ARL13B in adult rod photoreceptor cells after maturation of OS resulted in loss of photoresponse and vesiculation in the OS. Before changes in photoresponse, removal of ARL13B led to mislocalization of rhodopsin, prenylated phosphodiesterase-6 (PDE6), and intraflagellar transport protein-88 (IFT88). Our findings show that ARL13B is required at multiple stages of retinogenesis, including early postnatal proliferation of retinal progenitor cells, development of photoreceptor cilia, and morphogenesis of photoreceptor OS discs regardless of sex. Last, our results establish a need for ARL13B in photoreceptor maintenance and protein trafficking.  

3.3256           Oncogenic Regulation of Extracellular Vesicle Proteome and Heterogeneity

Choi, D., Spinelli, C., Montermini, L. and Rak, J. Proteomics, 19, 1800169 (2019)   Mutational and epigenetic driver events profoundly alter intercellular communication pathways in cancer. This effect includes deregulated release, molecular composition, and biological activity of extracellular vesicles (EVs), membranous cellular fragments ranging from a few microns to less than 100 nm in diameter and filled with bioactive molecular cargo (proteins, lipids, and nucleic acids). While EVs are usually classified on the basis of their physical properties and biogenetic mechanisms, recent analyses of their proteome suggest a larger than expected molecular diversity, a notion that is also supported by multicolour nano‐flow cytometry and other emerging technology platforms designed to analyze single EVs. Both protein composition and EV diversity are markedly altered by oncogenic transformation, epithelial to mesenchymal transition, and differentiation of cancer stem cells. Interestingly, only a subset of EVs released from mutant cells may carry oncogenic proteins (e.g., EGFRvIII), hence, these EVs are often referred to as “oncosomes”. Indeed, oncogenic transformation alters the repertoire of EV‐associated proteins, increases the presence of pro‐invasive cargo, and alters the composition of distinct EV populations. Molecular profiling of single EVs may reveal a more intricate effect of transforming events on the architecture of EV populations in cancer and shed new light on their biological role and diagnostic utility.  

3.3257           PAN-INTACT enables direct isolation of lineage-specific nuclei from fibrous tissues

Bhattacharyya, S., Sather, A.A., Bhakta, m., Xing, C. and Munshi, N.V. PloS One, 14(4), e0214677 (2019)   Recent studies have highlighted the extraordinary cell type diversity that exists within mammalian organs, yet the molecular drivers of such heterogeneity remain elusive. To address this issue, much attention has been focused on profiling the transcriptome and epigenome of individual cell types. However, standard cell type isolation methods based on surface or fluorescent markers remain problematic for cells residing within organs with significant connective tissue. Since the nucleus contains both genomic and transcriptomic information, the isolation of nuclei tagged in specific cell types (INTACT) method provides an attractive solution. Although INTACT has been successfully applied to plants, flies, zebrafish, frogs, and mouse brain and adipose tissue, broad use across mammalian organs remains challenging. Here we describe the method, which can be used to isolate cell type specific nuclei from fibrous mouse organs, which are particularly problematic. As a proof-of-concept, we demonstrate successful isolation of cell type-specific nuclei from the mouse heart, which contains substantial connective tissue and harbors multiple cell types, including cardiomyocytes, fibroblasts, endothelial cells, and epicardial cells. Compared to established techniques, PAN-INTACT allows more rapid isolation of cardiac nuclei to facilitate downstream applications. We show cell type-specific isolation of nuclei from the hearts of Nkx2-5^Cre/+; R26^{Sun1-2xsf-GFP-6xmyc/+} mice, which we confirm by expression of lineage markers. Furthermore, we perform Assay for Transposase Accessible Chromatin (ATAC)-Seq to provide high-fidelity chromatin accessibility maps of Nkx2-5⁺ nuclei. To extend the applicability of PAN-INTACT, we also demonstrate successful isolation of Wt1⁺ podocytes from adult kidney. Taken together, our data suggest that PAN-INTACT is broadly applicable for profiling the transcriptional and epigenetic landscape of specific cell types. Thus, we envision that our method can be used to systematically probe mechanistic details of cell type-specific functions within individual organs of intact mice.  

3.3258           Exosome swarms eliminate airway pathogens and provide passive epithelial immunoprotection through nitric oxide

Nocera, A.L., Mueller, S.K., Stephan, J.R., Hing, L., Seifert, P., Han, X., Lin, D.T., Amiji, M:M., Liebermann, T. and Bleier, B.S.

1535. Allergy Clin. Immunol., 143(4), 1525-1535.e1 (2019)

  Background Nasal mucosa–derived exosomes (NMDEs) harbor immunodefensive proteins and are capable of rapid interepithelial protein transfer. Objectives We sought to determine whether mucosal exposure to inhaled pathogens stimulates a defensive swarm of microbiocidal exosomes, which also donate their antimicrobial cargo to adjacent epithelial cells. Methods We performed an institutional review board–approved study of healthy NMDE secretion after Toll-like receptor (TLR) 4 stimulation by LPS (12.5 μg/mL) in the presence of TLR4 inhibitors. Interepithelial transfer of exosomal nitric oxide (NO) synthase and nitric oxide was measured by using ELISAs and NO activity assays. Exosomal antimicrobial assays were performed with Pseudomonas aeruginosa. Proteomic analyses were performed by using SOMAscan. Results In vivo and in vitro LPS exposure induced a 2-fold increase in NMDE secretion along with a 2-fold increase in exosomal inducible nitric oxide synthase expression and function through TLR4 and inhibitor of nuclear factor κB kinase activation. LPS stimulation increased exosomal microbiocidal activity against P aeruginosa by almost 2 orders of magnitude. LPS-stimulated exosomes induced a 4-fold increase in NO production within autologous epithelial cells with protein transfer within 5 minutes of contact. Pathway analysis of the NMDE proteome revealed 44 additional proteins associated with NO signaling and innate immune function. Conclusions We provide direct in vivo evidence for a novel exosome-mediated innate immunosurveillance and defense mechanism of the human upper airway. These findings have implications for lower airway innate immunity, delivery of airway therapeutics, and host microbiome regulation.  

3.3259           Mapping Extracellular RNA Sheds Lights on Distinct Carriers

Lässer, C. Cell, 177(2), 228-230 (2019)   Circulating extracellular RNA can participate in cell-to-cell communication and can be used as a marker of disease. Currently, biological and technical variability prevents the field from reaching its full potential. In this issue of Cell and the April 24 issue of Cell Systems, three studies by the Extracellular RNA Communication Consortium (ERCC) (Murillo et al., 2019, Srinivasan et al., 2019, Rozowsky et al., 2019) address several aspects contributing to this variability and provide knowledge and platforms for empowering future studies.  

3.3260           Reassessment of Exosome Composition

Jeppesen, D.K., Fenix, A.M., Franklin, J.L., Rome, L.H., Burnette, D.T. and Coffey, R.J. Cell, 177(2), 428-445 (2019)   The heterogeneity of small extracellular vesicles and presence of non-vesicular extracellular matter have led to debate about contents and functional properties of exosomes. Here, we employ high-resolution density gradient fractionation and direct immunoaffinity capture to precisely characterize the RNA, DNA, and protein constituents of exosomes and other non-vesicle material. Extracellular RNA, RNA-binding proteins, and other cellular proteins are differentially expressed in exosomes and non-vesicle compartments. Argonaute 1–4, glycolytic enzymes, and cytoskeletal proteins were not detected in exosomes. We identify annexin A1 as a specific marker for microvesicles that are shed directly from the plasma membrane. We further show that small extracellular vesicles are not vehicles of active DNA release. Instead, we propose a new model for active secretion of extracellular DNA through an autophagy- and multivesicular-endosome-dependent but exosome-independent mechanism. This study demonstrates the need for a reassessment of exosome composition and offers a framework for a clearer understanding of extracellular vesicle heterogeneity.  

3.3261           Small RNA Sequencing across Diverse Biofluids Identifies Optimal Methods for exRNA Isolation

Srinivasan, S., Yeri, A., Cheah, P.S., Alexander, R.P., Das, S. and Laurent, L.C: Cell, 177(2), 446-462 (2019)   Poor reproducibility within and across studies arising from lack of knowledge regarding the performance of extracellular RNA (exRNA) isolation methods has hindered progress in the exRNA field. A systematic comparison of 10 exRNA isolation methods across 5 biofluids revealed marked differences in the complexity and reproducibility of the resulting small RNA-seq profiles. The relative efficiency with which each method accessed different exRNA carrier subclasses was determined by estimating the proportions of extracellular vesicle (EV)-, ribonucleoprotein (RNP)-, and high-density lipoprotein (HDL)-specific miRNA signatures in each profile. An interactive web-based application (miRDaR) was developed to help investigators select the optimal exRNA isolation method for their studies. miRDar provides comparative statistics for all expressed miRNAs or a selected subset of miRNAs in the desired biofluid for each exRNA isolation method and returns a ranked list of exRNA isolation methods prioritized by complexity, expression level, and reproducibility. These results will improve reproducibility and stimulate further progress in exRNA biomarker development.  

3.3262           exRNA Atlas Analysis Reveals Distinct Extracellular RNA Cargo Types and Their Carriers Present across Human Biofluids

Murillo, O.D., Thistlewaite, W., Rozowsky, J., Roth, M.E., Gerstein, M.B. and Milosavljevic, A. Cell, 177(2), 463-477 (2019)   To develop a map of cell-cell communication mediated by extracellular RNA (exRNA), the NIH Extracellular RNA Communication Consortium created the exRNA Atlas resource (https://exrna-atlas.org). The Atlas version 4P1 hosts 5,309 exRNA-seq and exRNA qPCR profiles from 19 studies and a suite of analysis and visualization tools. To analyze variation between profiles, we apply computational deconvolution. The analysis leads to a model with six exRNA cargo types (CT1, CT2, CT3A, CT3B, CT3C, CT4), each detectable in multiple biofluids (serum, plasma, CSF, saliva, urine). Five of the cargo types associate with known vesicular and non-vesicular (lipoprotein and ribonucleoprotein) exRNA carriers. To validate utility of this model, we re-analyze an exercise response study by deconvolution to identify physiologically relevant response pathways that were not detected previously. To enable wide application of this model, as part of the exRNA Atlas resource, we provide tools for deconvolution and analysis of user-provided case-control studies.  

3.3263           Vitamin D3 activates the autolysosomal degradation function against Helicobacter pylori through the PDIA3 receptor in gastric epithelial cells

Hu, W., Zhang, L., Li, M.X., Shen, J., Liu, X.D., Xiao, Z.G. et al Autophagy, 15(4), 707-725 (2019)   Helicobacter pylori (H. pylori) is a common human pathogenic bacterium. Once infected, it is difficult for the host to clear this organism using the innate immune system. Increased antibiotic resistance further makes it challenging for effective eradication. However, the mechanisms of immune evasion still remain obscure, and novel strategies should be developed to efficiently eliminate H. pylori infection in stomachs. Here we uncovered desirable anti-H. pylori effect of vitamin D3 both in vitro and in vivo, even against antibiotic-resistant strains. We showed that H. pylori can invade into the gastric epithelium where they became sequestered and survived in autophagosomes with impaired lysosomal acidification. Vitamin D3 treatment caused a restored lysosomal degradation function by activating the PDIA3 receptor, thereby promoting the nuclear translocation of PDIA3-STAT3 protein complex and the subsequent upregulation of MCOLN3 channels, resulting in an enhanced Ca²⁺ release from lysosomes and normalized lysosomal acidification. The recovered lysosomal degradation function drives H. pylori to be eliminated through the autolysosomal pathway. These findings provide a novel pathogenic mechanism on how H. pylori can survive in the gastric epithelium, and a unique pathway for vitamin D3 to reactivate the autolysosomal degradation function, which is critical for the antibacterial action of vitamin D3 both in cells and in animals, and perhaps further in humans.  

3.3264           JC Polyomavirus Uses Extracellular Vesicles To Infect Target Cells

Morris-Love, J., Gee, G.V., O’Hara, B.A., Assetta, B., Atkinson, A.L., Dugan, A.S., haley, S.A. and Atwood, W.J. mBio, 10(2), e00379-19 (2019)   The endemic human JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy in immune-suppressed patients. The mechanisms of virus infection in vivo are not understood because the major target cells for virus in the brain do not express virus receptors and do not bind virus. We found that JCPyV associates with extracellular vesicles (EVs) and can infect target cells independently of virus receptors. Virus particles were found packaged inside extracellular vesicles and attached to the outer side of vesicles. Anti-JCPyV antisera reduced infection by purified virus but had no effect on infection by EV-associated virus. Treatment of cells with the receptor-destroying enzyme neuraminidase inhibited infection with purified virus but did not inhibit infection by EV-associated virus. Mutant pseudoviruses defective in sialic acid receptor binding could not transduce cells as purified pseudovirions but could do so when associated with EVs. This alternative mechanism of infection likely plays a critical role in the dissemination and spread of JCPyV both to and within the central nervous system.  

3.3265           Tumor-derived extracellular vesicles require β1 integrins to promote anchorage-independent growth

DeRita, R.M.-, Sayeed, A., Garcia, V., Rodeck, U., Dicker, A.P. and Linguino, L.R. iScience, 14, 199-209 (2019)   The β1 integrins, known to promote cancer progression, are abundant in extracellular vesicles (EVs). We investigated whether prostate cancer (PrCa) EVs affect anchorage-independent growth and whether β1 integrins are required for this effect. Specifically using a cell-line-based genetic rescue and an in vivo PrCa model, we show that gradient-purified small EVs (sEVs) from either cancer cells or blood from tumor-bearing TRAMP (transgenic adenocarcinoma of the mouse prostate) mice promote anchorage-independent growth of PrCa cells. In contrast, sEVs from cultured PrCa cells harboring a short hairpin RNA to β1, from wild-type mice or from TRAMP mice carrying a β1 conditional ablation in the prostatic epithelium (β1^pc−/−), do not. We find that sEVs, from cancer cells or TRAMP blood, are functional and co-express β1 and sEV markers; in contrast, sEVs from β1^pc−/−/TRAMP or wild-type mice lack β1 and sEV markers. Our results demonstrate that β1 integrins in tumor-cell-derived sEVs are required for stimulation of anchorage-independent growth.  

3.3266           Quantitative Proteomic Analysis of Small and Large Extracellular Vesicles (EVs) Reveals Enrichment of Adhesion Proteins in Small EVs

Jimenez, L., Yu, H., McKenzie, A.J., Franklin, J.L., patton, J.G., Liu, Q. and Weaver, A.M.

1. Proteome Res., 18, 947-959 (2019)

  Extracellular vesicles (EVs) are important mediators of cell–cell communication due to their cargo content of proteins, lipids, and RNAs. We previously reported that small EVs (SEVs) called exosomes promote directed and random cell motility, invasion, and serum-independent growth. In contrast, larger EVs (LEVs) were not active in those assays, but might have unique functional properties. In order to identify protein cargos that may contribute to different functions of SEVs and LEVs, we used isobaric tags for relative and absolute quantitation (iTRAQ)–liquid chromatography (LC) tandem mass spectrometry (MS) on EVs isolated from a colon cancer cell line. Bioinformatics analyses revealed that SEVs are enriched in proteins associated with cell–cell junctions, cell–matrix adhesion, exosome biogenesis machinery, and various signaling pathways. In contrast, LEVs are enriched in proteins associated with ribosome and RNA biogenesis, processing, and metabolism. Western blot analysis of EVs purified from two different cancer cell types confirmed the enrichment of cell–matrix and cell–cell adhesion proteins in SEVs. Consistent with those data, we found that cells exhibit enhanced adhesion to surfaces coated with SEVs compared to an equal protein concentration of LEVs. These data suggest that a major function of SEVs is to promote cellular adhesion.  

3.3267           Localization of Outer Membrane Proteins in Treponema denticola by Quantitative Proteome Analyses of Outer Membrane Vesicles and Cellular Fractions

Veith, P.D., Glew, M.D., Gorasia, D.G., Chen, D., O’Brien-Simpson, N.M. and Reymolds, E.C.

1. Proteome Res., 18, 1567-1581 (2019)

  The identification and localization of outer membrane proteins (Omps) and lipoproteins in pathogenic treponemes such as T. denticola (periodontitis) and T. pallidum (syphilis) has been challenging. In this study, label-free quantitative proteomics using MaxQuant was applied to naturally produced outer membrane vesicles (OMVs) and cellular fractions to identify 1448 T. denticola proteins. Of these, 90 proteins were localized to the outer membrane (OM) comprising 59 lipoproteins, 25 β-barrel proteins, and six other putative OM-associated proteins. Twenty-eight lipoproteins were localized to the inner membrane (IM), and 43 proteins were assigned to the periplasm. The signal cleavage regions of the OM and IM lipoprotein sequences were different and may reveal the signals for their differential localization. Proteins significantly enriched in OMVs included dentilisin, proteins containing leucine-rich repeats, and several lipoproteins containing FGE-sulfatase domains. Blue native PAGE analysis enabled the native size of the dentilisin complex and Msp to be determined and revealed that the abundant β-barrel Omps TDE2508 and TDE1717 formed large complexes. In addition to the large number of integral Omps and potentially surface-located lipoproteins identified in T. denticola, many such proteins were also newly identified in T. pallidum through homology, generating new targets for vaccine development in both species.  

3.3268           Extracellular vesicle-packaged HIF-1α-stabilizing lncRNA from tumour-associated macrophages regulates aerobic glycolysis of breast cancer cells

Chen, F., Chen, J., Yang, L., Liu, J., Zhang, X., Zhang, Y., Tu, Q. et al Nature Cell Biol., 21, 498-510 (2019)   Metabolic reprogramming is a hallmark of cancer. Here, we demonstrate that tumour-associated macrophages (TAMs) enhance the aerobic glycolysis and apoptotic resistance of breast cancer cells via the extracellular vesicle (EV) transmission of a myeloid-specific lncRNA, HIF-1α-stabilizing long noncoding RNA (HISLA). Mechanistically, HISLA blocks the interaction of PHD2 and HIF-1α to inhibit the hydroxylation and degradation of HIF-1α. Reciprocally, lactate released from glycolytic tumour cells upregulates HISLA in macrophages, constituting a feed-forward loop between TAMs and tumour cells. Blocking EV-transmitted HISLA inhibits the glycolysis and chemoresistance of breast cancer in vivo. Clinically, HISLA expression in TAMs is associated with glycolysis, poor chemotherapeutic response and shorter survival of patients with breast cancer. Our study highlights the potential of lncRNAs as signal transducers that are transmitted between immune and tumour cells via EVs to promote cancer aerobic glycolysis.  

3.3269           Vibrio cholerae OmpU Mediates CD36-Dependent Reactive Oxygen Species Generation Triggering an Additional Pathway of MAPK Activation in Macrophages

Prasad, G.V.R.K., Dhar, V. and Mukhopadhaya, A.

1. Immunol., 202, 2431-2450 (2019)

  OmpU, one of the porins of Gram-negative bacteria Vibrio cholerae, induces TLR1/2–MyD88–NF-κB–dependent proinflammatory cytokine production by monocytes and macrophages of human and mouse origin. In this study, we report that in both the cell types, OmpU-induced proinflammatory responses involve activation of MAPKs (p38 and JNK). Interestingly, we observed that in OmpU-treated macrophages, p38 activation is TLR2 dependent, but JNK activation happens through a separate pathway involving reactive oxygen species (ROS) generation by NADPH oxidase complex and mitochondrial ROS. Further, we observed that OmpU-mediated mitochondrial ROS generation probably depends on OmpU translocation to mitochondria and NADPH oxidase–mediated ROS production is due to activation of scavenger receptor CD36. For the first time, to our knowledge, we are reporting that a Gram-negative bacterial protein can activate CD36 as a pattern recognition receptor. Additionally, we found that in OmpU-treated monocytes, both JNK and p38 activation is linked to the TLR2 activation only. Therefore, the ability of macrophages to employ multiple receptors such as TLR2 and CD36 to recognize a single ligand, as in this case OmpU, probably explains the very basic nature of macrophages being more proinflammatory than monocytes.  

3.3270           The potential diagnostic and prognostic role of extracellular vesicles in glioma: current status and future perspectives

Azam, Z., Quillen, V., Wang, G. and To, S-S.T. Acta Oncologica, 58(3), 353-362 (2019)   Lack of appropriate diagnostic/prognostic tools for glioblastoma (GB) is considered one of the major setbacks in the early diagnosis and treatment of this deadly brain tumor. The current gold standard for its diagnosis and staging still relies on invasive biopsy followed by histological examination as well as molecular profiling. Nevertheless, noninvasive approaches are being explored and one example is through the investigation of extracellular vesicles (EVs) in the biofluids of GB patients. EVs are known to carry molecular cargoes such as DNA, mRNA, miRNA, proteins and lipids in almost every type of body fluids. Thus, molecular signature of GB may be present in the EVs derived from these patients. This review focuses on the diagnostic/prognostic potential of EVs in GB, through presenting recent studies on (i) molecular components of EVs, (ii) links between EVs and GB tumor microenvironment, and (iii) clinical potential of EV biomarkers, together with the technical shortcomings researchers need to consider for future studies.  

3.3271           Phosphorylation of Syntaxin 17 by TBK1 Controls Autophagy Initiation

Kumar, S., Gu, Y., Abudu, Y.P., Lidke, K.A., Johansen, T. and Deretic, V. Developmental Cell, 49, 130-144 (2019)   Syntaxin 17 (Stx17) has been implicated in autophagosome-lysosome fusion. Here, we report that Stx17 functions in assembly of protein complexes during autophagy initiation. Stx17 is phosphorylated by TBK1 whereby phospho-Stx17 controls the formation of the ATG13 ⁺FIP200 ⁺ mammalian pre-autophagosomal structure (mPAS) in response to induction of autophagy. TBK1 phosphorylates Stx17 at S202. During autophagy induction, Stx17 ^pS202 transfers from the Golgi, where its steady-state pools localize, to the ATG13 ⁺FIP200 ⁺ mPAS. Stx17 ^pS202 was in complexes with ATG13 and FIP200, whereas its non-phosphorylatable mutant Stx17 ^S202A was not. Stx17 or TBK1 knockouts blocked ATG13 and FIP200 puncta formation. Stx17 or TBK1 knockouts reduced the formation of ATG13 protein complexes with FIP200 and ULK1. Endogenous Stx17 ^pS202 colocalized with LC3B following induction of autophagy. Stx17 knockout diminished LC3 response and reduced sequestration of the prototypical bulk autophagy cargo lactate dehydrogenase. We conclude that Stx17 is a TBK1 substrate and that together they orchestrate assembly of mPAS.  

3.3272           Surface Engineering of Extracellular Vesicles through Chemical and Biological Strategies

Richardson, J.J. and Ejima, H. Chem. Mater., 31(7), 2191-2201 (2019)   Extracellular vesicles, including exosomes, shuttle proteins and genetic information between cells and are recently regarded as a promising class of nanoparticles for biomedical applications. Collecting exosomes from patients and amplifying the signal from the exosome cargo could enable new liquid biopsies owing to the exosome’s strong correlation with disease progression. A promising strategy to develop personalized therapeutic carriers is through surface engineering the exosomes and hijacking their inherent messenger system. However, because of the small size and surface complexity, methods to manipulate exosomes are still in their infancy. The purification, preservation, and engineering of exosomes with high scalability and reproducibility is pivotal for practical applications. By regarding exosomes as a bioderived soft nanoparticle, the amassed knowledge and techniques commonly used in materials chemistry and surface science would contribute to exosome development and exploitation. In this perspective, we first introduce the basics of exosomes (i.e., composition, purification, and analysis), and then we highlight the recent research on surface engineering exosomes from the viewpoint of materials chemistry and discuss possible future directions for the field.  

3.3273           Cushioned-Density Gradient Ultracentrifugation (C-DGUC) improves the isolation efficiency of extracellular vesicles

Duong, P., Chung, A., Bouchareychas, L. and Raffal, R.L. PloS One, 14(4), e0215324 (2019)   Ultracentrifugation (UC) is recognized as a robust approach for the isolation of extracellular vesicles (EVs). However, recent studies have highlighted limitations of UC including low recovery efficiencies and aggregation of EVs that could impact downstream functional analyses. We tested the benefit of using a liquid cushion of iodixanol during UC to address such shortcomings. In this study, we compared the yield and purity of EVs isolated from J774A.1 macrophage conditioned media by conventional UC and cushioned-UC (C-UC). We extended our study to include two other common EV isolation approaches: ultrafiltration (UF) and polyethylene glycol (PEG) sedimentation. After concentrating EVs using these four methods, the concentrates underwent further purification by using OptiPrep density gradient ultracentrifugation (DGUC). Our data show that C-DGUC provides a two-fold improvement in EV recovery over conventional UC-DGUC. We also found that UF-DGUC retained ten-fold more protein while PEG-DGUC achieved similar performance in nanoparticle and protein recovery compared to C-DGUC. Regarding purity as assessed by nanoparticle to protein ratio, our data show that EVs isolated by UC-DGUC achieved the highest purity while C-DGUC and PEG-DGUC led to similarly pure preparations. Collectively, we demonstrate that the use of a high-density iodixanol cushion during the initial concentration step improves the yield of EVs derived from cell culture media compared to conventional UC. This enhanced yield without substantial retention of protein contaminants and without exposure to forces causing aggregation offers new opportunities for the isolation of EVs that can subsequently be used for functional studies.  

3.3274           GLUT1 is associated with sphingolipid-organized, cholesterol-independent domains in L929 mouse fibroblast cells

Rylaarsdam, L.E., Johnecheck, G.N., Looyenga, B.D. and Louters, L.L. Biochemie, 162, 88-96 (2019)   Glucose is a preferred metabolite in most mammalian cells, and proper regulation of uptake is critical for organism homeostasis. The glucose transporter 1 (GLUT1) is responsible for glucose uptake in a wide variety of cells and appears to be regulated in a tissue specific manner. Therefore, a better understanding of GLUT1 regulation within its various cellular environments is essential for developing therapeutic strategies to treat disorders associated with glucose homeostasis. Previous findings suggest that plasma membrane subdomains called lipid rafts may play a role in regulation of GLUT1 uptake activity. While studying this phenomenon in L929 mouse fibroblast cells, we observed that GLUT1 associates with a low density lipid microdomain distinct from traditionally-defined lipid rafts. These structures are not altered by cholesterol removal with methyl-β-cyclodextrin and lack resistance to cold Triton X-100 extraction. Our data indicate that the GLUT1-containing membrane microdomains in L929 cells, as well as GLUT1's basal activity, are instead sphingolipid-dependent, being sensitive to both myriocin and sphingomyelinase treatment. These microdomains appear to be organized primarily by their lipid composition, as disruption of the actin cytoskeleton or microtubules does not alter the association of GLUT1 with them. Furthermore, the association of GLUT1 with these microdomains appears not to require palmitoylation or glycosylation, as pharmacologic inhibition of these processes had no impact on GLUT1 density in membrane fractions. Importantly, we find no evidence that GLUT1 is actively translocated into or out of low density membrane fractions in response to acute activation in L929 cell.  

3.3275           Functional analysis of miR-21-3p, miR-30b-5p and miR-150-5p shuttled by extracellular vesicles from diabetic subjects reveals their association with diabetic retinopathy

Mazzeo, A., Lopatina, T., Gai, C., Trento, M., Porta, M. and Beltramo, E. Exp. Eye Res., 184, 56-63 (2019)   Microvascular dysfunctions due to altered interactions between endothelial cells (ECs) and pericytes are key-events in the pathogenesis of diabetic retinopathy. Extracellular vesicles (EVs) derived from mesenchymal stem cells cultured in diabetic-like conditions enter pericytes, cause their detachment and migration, and stimulate angiogenesis. We recently showed that EVs from diabetic patients with retinopathy have different miRNA profiling patterns from healthy controls, and determine features of retinopathy in in vitro models of retinal microvasculature. In particular, a role for intra-vesicle miR-150-5p, miR-21-3p and miR-30b-5p was hypothesized. In this work, we further characterized EVs from subjects with diabetic retinopathy and investigated miR-150-5p, miR-21-3p and miR-30b-5p functions inside microvascular cells. Human retinal pericytes and ECs were transfected with mimics or inhibitors, as appropriate, of miR-21-3p, miR-30b-5p and miR-150-5p, to evaluate their ability in promoting cell migration and tube formation. mRNA and protein profiling of EVs extracted from diabetic subjects with (DR group) or without retinopathy (noDR group), and healthy controls (CTR group) were also performed. Modulation of miR-150-5p, miR-21-3p and miR-30b-5p inside microvascular cells confirmed their involvement in abnormal angiogenesis. mRNA analysis revealed differing expression of 7 genes involved in angiogenesis, while subsequent protein analysis confirmed increased expression of HIF-1α in DR group. Since all these molecules are involved in the hypoxia-induced retinal damage characteristic of the disease, our data reinforce the hypothesis of a potential use of miR-150-5p, miR-21-3p and miR-30b-5p extracted from circulating EVs as prognostic biomarkers for diabetic retinopathy.  

3.3276           Modulation of calreticulin expression reveals a novel exosome-mediated mechanism of Z variant α1-antitrypsin disposal

Khodayari, N., Ashins, R., Alli, A.A., Tuna, K.M., Holliday, L.S., Krotova, K. and Brantly, M.

1. Biol. Chem., 294(16), 6240-6252 (2019)

  α₁-Antitrypsin deficiency (AATD) is an inherited disease characterized by emphysema and liver disease. AATD is most often caused by a single amino acid substitution at position 342 in the mature protein, resulting in the Z mutation of the AAT gene (ZAAT). This substitution is associated with misfolding and accumulation of ZAAT in the endoplasmic reticulum (ER) of hepatocytes, causing a toxic gain of function. ERdj3 is an ER luminal DnaJ homologue, which, along with calreticulin, directly interacts with misfolded ZAAT. We hypothesize that depletion of each of these chaperones will change the fate of ZAAT polymers. Our study demonstrates that calreticulin modulation reveals a novel ZAAT degradation mechanism mediated by exosomes. Using human PiZZ hepatocytes and K42, a mouse calreticulin-deficient fibroblast cell line, our results show ERdj3 and calreticulin directly interact with ZAAT in PiZZ hepatocytes. Silencing calreticulin induces calcium independent ZAAT–ERdj3 secretion through the exosome pathway. This co-secretion decreases ZAAT aggregates within the ER of hepatocytes. We demonstrate that calreticulin has an inhibitory effect on exosome-mediated ZAAT–ERdj3 secretion. This is a novel ZAAT degradation process that involves a DnaJ homologue chaperone bound to ZAAT. In this context, calreticulin modulation may eliminate the toxic gain of function associated with aggregation of ZAAT in lung and liver, thus providing a potential new therapeutic approach to the treatment of AATD-related liver disease.  

3.3277           PRCD is essential for high-fidelity photoreceptor disc formation

Spencer, W.J., Ding, J-D., Lewis, T.R., Yu, C., Phan, S., Pearring, J.N. et al

PNAS, 116(26), 13087-13096 (2019)

  Progressive rod-cone degeneration (PRCD) is a small protein residing in the light-sensitive disc membranes of the photoreceptor outer segment. Until now, the function of PRCD has remained enigmatic despite multiple demonstrations that its mutations cause blindness in humans and dogs. Here, we generated a PRCD knockout mouse and observed a striking defect in disc morphogenesis, whereby newly forming discs do not properly flatten. This leads to the budding of disc-derived vesicles, specifically at the site of disc morphogenesis, which accumulate in the interphotoreceptor matrix. The defect in nascent disc flattening only minimally alters the photoreceptor outer segment architecture beyond the site of new disc formation and does not affect the abundance of outer segment proteins and the photoreceptor’s ability to generate responses to light. Interestingly, the retinal pigment epithelium, responsible for normal phagocytosis of shed outer segment material, lacks the capacity to clear the disc-derived vesicles. This deficiency is partially compensated by a unique pattern of microglial migration to the site of disc formation where they actively phagocytize vesicles. However, the microglial response is insufficient to prevent vesicular accumulation and photoreceptors of PRCD knockout mice undergo slow, progressive degeneration. Taken together, these data show that the function of PRCD is to keep evaginating membranes of new discs tightly apposed to each other, which is essential for the high fidelity of photoreceptor disc morphogenesis and photoreceptor survival.  

3.3278           Exosomes from N-Myc amplified neuroblastoma cells induce migration and confer chemoresistance to non-N-Myc amplified cells: implications of intra-tumour heterogeneity

Fonseka, P., Liem, M., Ozcitti, C., Adda, C.G., Ang, C-S. and Mathivanan, S.

1. Extracellular Vesicles, 8(1), 1597614 (2019)

  Neuroblastoma accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is a well-established poor prognostic marker for neuroblastoma. Whilst N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressiveness of the disease is poorly understood. Exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. Hence, characterising the exosomal protein components from N-Myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. In this study, a comparative proteomic analysis of exosomes isolated from cells with varying N-Myc amplification status was performed. Label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the neuroblastoma cells. Gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in N-Myc amplified exosomes. Treatment of SH-SY5Y cells with N-Myc amplified SK-N-BE2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. Incubation of exosomes from N-Myc knocked down SK-N-BE2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. These findings suggest that exosomes could play a pivotal role in N-Myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells.  

3.3279           Mechanisms of tethering and cargo transfer during epididymosome-sperm interactions

Zhou, W., Stanger, S.J., Anderson, A.L., Bernstein, I.R., De luliis, G.N., McCluskey, A., Mclaughlin, E.A., Dun, M.D. and Nixon, B. BMC Biology, 17:35 (2019)   Background The mammalian epididymis is responsible for the provision of a highly specialized environment in which spermatozoa acquire functional maturity and are subsequently stored in preparation for ejaculation. Making important contributions to both processes are epididymosomes, small extracellular vesicles released from the epididymal soma via an apocrine secretory pathway. While considerable effort has been focused on defining the cargo transferred between epididymosomes and spermatozoa, comparatively less is known about the mechanistic basis of these interactions. To investigate this phenomenon, we have utilized an in vitro co-culture system to track the transfer of biotinylated protein cargo between mouse epididymosomes and recipient spermatozoa isolated from the caput epididymis; an epididymal segment that is of critical importance for promoting sperm maturation. Results Our data indicate that epididymosome-sperm interactions are initiated via tethering of the epididymosome to receptors restricted to the post-acrosomal domain of the sperm head. Thereafter, epididymosomes mediate the transfer of protein cargo to spermatozoa via a process that is dependent on dynamin, a family of mechanoenzymes that direct intercellular vesicle trafficking. Notably, upon co-culture of sperm with epididymosomes, dynamin 1 undergoes a pronounced relocation between the peri- and post-acrosomal domains of the sperm head. This repositioning of dynamin 1 is potentially mediated via its association with membrane rafts and ideally locates the enzyme to facilitate the uptake of epididymosome-borne proteins. Accordingly, disruption of membrane raft integrity or pharmacological inhibition of dynamin both potently suppress the transfer of biotinylated epididymosome proteins to spermatozoa. Conclusion Together, these data provide new mechanistic insight into epididymosome-sperm interactions with potential implications extending to the manipulation of sperm maturation for the purpose of fertility regulation.  

3.3280           Extracellular Vesicles Released by Glioblastoma Cells Stimulate Normal Astrocytes to Acquire a Tumor-Supportive Phenotype Via p53 and MYC Signaling Pathways

Hallal, S., Mallawaaratchy, D.M., Wei, H., Ebrahimkhani, S., Stringer, B.W., Day, B.W., Boyd, A.W., Guillemin, G.J., Buckland, M.E. and Kaufman, K.L. Mol. Neurobiol., 56, 4566-4581 (2019)   The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES⁺/CD133⁺) and their differentiated (diff) progeny cells (NES⁻/CD133⁻). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFβ1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53β in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression.  

3.3281           Lactate-stimulated ethanol oxidation: Revisiting an old hypothesis

Villalobos-Garcia, D. and Hernandez-Munoz, R. Biochem. Pharmol., 164, 283-288 (2019)   Liver slices from starved rats and incubated without other substrates oxidized ethanol at a rate of 4.1 µmols • h⁻¹ • g⁻¹. Addition of 10 mmols • L⁻¹ lactate increased this rate 2-fold. 4-methylpyrazole (4-MP), an alcohol dehydrogenase (ADH) inhibitor, drastically decreased the rate of ethanol oxidation, but did not inhibit the stimulation due to lactate. In the same context, liver acetaldehyde production, as the main by-product of ethanol oxidation, appeared to be much less inhibited by 4-MP in the presence of lactate. Aminotriazole (a catalase inhibitor), however, completely inhibited the stimulation. Furthermore, 2-hydroxybut-3-ynoate, an alpha-hydroxy acid oxidase inhibitor, completely abolished the stimulated ethanol oxidation promoted by lactate. Moreover, to determine the origin of the H₂O₂ produced, we did liver subcellular fractionation and then analyzed their content in peroxisomes, mitochondria and catalase. We observed that cytoplasm and peroxisomes appears to be the main producers of H₂O₂, and that the acceleration of ethanol oxidation by lactate is completely dependent on catalase. In conclusion, the H₂O₂ necessary to boost the catalase-dependent oxidation of ethanol appears to come from cytoplasm and peroxisomes, and is produced by the enzyme lactate oxidase.  

3.3282           Vibrio cholerae derived outer membrane vesicles modulate the inflammatory response of human intestinal epithelial cells by inducing microRNA-146a

Bitar, A., Aung, K.M., Wai, S.N. and Hammarström, M-L. Scientific Reports, 9:7212 (2019)   The small intestinal epithelium of Vibrio cholerae infected patients expresses the immunomodulatory microRNAs miR-146a and miR-155 at acute stage of disease. V. cholerae release outer membrane vesicles (OMVs) that serve as vehicles for translocation of virulence factors including V. cholerae cytolysin (VCC). The aim was to investigate whether OMVs, with and/or without VCC-cargo could be responsible for induction of microRNAs in intestinal epithelial cells and thereby contribute to immunomodulation. Polarized tight monolayers of T84 cells were challenged with OMVs of wildtype and a VCC deletion mutant of the non-O1/non-O139 (NOVC) V. cholerae strain V:5/04 and with soluble VCC. OMVs, with and without VCC-cargo, caused significantly increased levels of miR-146a. Increase was seen already after 2 hours challenge with OMVs and persisted after 12 hours. Challenge with soluble VCC caused significant increases in interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α), CCL20, IL-1β, and IRAK2 mRNA levels while challenge with OMVs did not cause increases in expression levels of any of these mRNAs. These results suggest that V. cholerae bacteria release OMVs that induce miR-146a in order to pave the way for colonization by reducing the strength of an epithelial innate immune defence reaction and also preventing inflammation in the mucosa that factors like VCC can evoke.  

3.3283           Proteomic characterisation of leech microglia extracellular vesicles (EVs): comparison between differential ultracentrifugation and Optiprep™ density gradient isolation

Arab, T., Raffo-Romero, A., Van Camp, C., Lemaire, Q., Le Marrec-Croq, F., Drago, F., Aboulourd, S., Slomianny, C., Lacoste, A-S., Guigon, I., Touzet, H., Salzet, m., Fournier, I., lefebre, C., Vizioli, J. and Sautiere, P-E.

1. Extracellular Vesicles, 8(1), 1603048 (2019)

  In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.  

3.3284           Mesenchymal stem cell–derived extracellular vesicles: a new impetus of promoting angiogenesis in tissue regeneration

Shi, Y., Shi, H., Nomi, A., Lei-Lei, Z., Zhang, B. and Qian, H. Cytotherapy, 21, 497-508 (2019)   Over the past few decades, extracellular vesicles (EVs) have emerged as crucial mediators of intercellular communication. EVs encapsulate and convey information to surrounding cells or distant cells, where they mediate cellular biological responses. Among their multifaceted roles in the modulation of biological responses, the involvement of EVs in vascular development, growth and maturation has been widely documented and their potential therapeutic application in regenerative medicine or in the treatment of angiogenesis-related diseases is drawing increasing interest. In this review, we have summarized the details about the current knowledge on biogenesis of EVs and conventional isolation methods. Evidence supporting the use of EVs derived from mesenchymal stromal cells (MSCs) to enhance angiogenesis in the development of insufficient angiogenesis, such as chronic wounds, stroke and myocardial infarction, will also be discussed critically. Finally, the main challenges and prerequisites for their therapeutic applications will be evaluated.  

3.3285           Investigation into a novel role for the prolyl isomerase cyclophilin A during Extracellular vesicle signaling in cancer

Wu, Y., Brennan, K. and Mcgea, M.M.

5. Extracellular Vesicles, 8, Suppl. 1, abstract PT05.03 (2019)

  No abstract available  

3.3286           Chloride intracellular channel protein 4 (CLIC4) is a serological cancer biomarker released from tumour epithelial cells via extracellular vesicles and required for metastasis

Sanchez, V.C., Craig-Lucas, A., Lou, J., Hunter, K. and Yusp, S.

1. Extracellulara Vesicles, 8, Suppl. 1, abstract LBF01.06 (2019)

  No abstract available  

3.3287           Distinct extracellular RNA cargo types associate with specific vesicular and non-vesicular RNA carriers across human biofluids

Milosavljevic, A.

19. Extracellular Vesicles, 8, Suppl. 1, OF19.01 (2019)

  No abstract available  

3.3288           Distinct mechanisms of microRNA sorting into cancer cell-derived extracellular vesicle subtypes

Temoche-Diaz, M., Shurleff, m., Nottingham, R., Yaob, J., Lambowitz, A.and Schekman, R.

4. Extracellular Vesicles, 8, Suppl. 1, abstract OT04.06 (2019)

  No abstract available  

3.3289           Shed microvesicles released from human primary and metastatic colorectal cancer cell lines contain key cancer progression proteins and RNA species

Suwalkusiri, W., Rai, A., Xu, R., Chen, M., Greening, D. and Simpson, R.

3. Extracellular Vesicles, 8, Suppl. 1, abstract OWP3.07 (2019)

  No abstract available  

3.3290           Extracellular vesicles provide a capsid-free vector for oncolytic adenoviral DNA delivery

Saari, H., Turunen, T., Turunen, M., Jalasvuoric, M., Butcherd, S., Ylä-Herttuala, S., Viitala, T., Cerullo, V., Siljander, P. and Yliperttula, M.

5. Extracellular Vesicles, 8, Suppl. 1, abstract OT05.01 (2019)

  No abstract available  

3.3291           Protein engineering for loading of Extracellular Vesicles

Osteikoetxe, X., Stein, J., Lazaro-Ibanez, E., O’Driscoll. G., Shatnyeva, O., Davies, R., and Dekker, N.

6. Extracellular Vesicles, 8, Suppl. 1, abstract OT06.06 (2019)

  No abstract available  

3.3292           Analysis of fluorescent labelling efficiency of extracellular vesicles derived from different kingdoms of life with lipid-binding dyes via nano-flow cytometry

Tian, Y., Chen, C., Niu, Q., Zhu, S. and Yan, X.

6. Extracellular Vesicles, 8, Suppl. 1, abstract PF06.06 (2019)

  No abstract available  

3.3293           Quantitative proteomic analysis of trypsin-treated extracellular vesicles to evaluate the real-vesicular proteins

Go, G., Choi, D-S., Kim, D-K., Lee, J. and Ghoa, Y.S.

12. Extracellular Vesicles, 8, Suppl. 1, abstract PF12.03 (2019)

  No abstract available  

3.3294           Exposed aminophospholipids enriched in a subtype of small extracellular vesicles from tumour cell lines

Matsumura, S., Minamisawa, T., Suga, K. and Shiba, K.

21. Extracellular Vesicles, 8, Suppl. 1, abstract OF21.05 (2019)

  No abstract available  

3.3295           Engineering of extracellular vesicles for surface display of targeting ligands

Lazaro-Ibanez, E., Gunnarsson, A., O’Driscoll, G., Shatnyeva, O., Osterkoetxe, X. and Dekker, N.

23. Extracellular Vesicles, 8, Suppl. 1, abstract OS23.03 (2019)

  No abstract available  

3.3296           Virus protein pX facilitates naked particles of hepatitis A virus to acquire an exosome-derived membrane by interacting with ESCRTassociated protein ALIX

Jiang, W., Mab, P., Deng, L. and Long, G.

5. Extracellular Vesicles, 8, Suppl. 1, abstract LB05.03 (2019)

  No abstract available  

3.3297           Modeling tumour: key issues of cell communication by mean of EVs in a three-dimensional environment and the impact on biomarker discovery

Khandury, R., Paniushkina, L., Keller, A., Gajney-Schleicher, T. and Nazarenko, I.

2. Extracellular Vesicles, 8, Suppl. 1, abstract LB02.03 (2019)

  No abstract available  

3.3298           Regulation of Somatostatin Receptor 2 Trafficking by C-Tail Motifs and the Retromer

Olsen, C., Memarzadeh, K., Ulu, A., Carr, H.S., Bean, A.J. and Frost, J.A. Endocrinology, 160(5), 1031-1043 (2019)   The G_i-coupled somatostatin receptor 2 (SST₂) is a G protein–coupled receptor (GPCR) that mediates many of somatostatin’s neuroendocrine actions. Upon stimulation, SST₂ is rapidly internalized and transported to early endosomes before being recycled to the plasma membrane. However, little is known about the intracellular itinerary of SST₂ after it moves to the early endosomal compartment or the cytoplasmic proteins that regulate its trafficking. As postsynaptic density protein/discs large 1/zonula occludens-1 (PDZ) domain interactions often regulate the trafficking and signaling potential of GPCRs, we examined the role of the SST₂ PDZ ligand and additional C-terminal residues in controlling its intracellular trafficking. We determined that SST₂ can recycle to the plasma membrane via multiple pathways, including a LAMP1/Rab7-positive late endosome to the trans-Golgi network (TGN) pathway. Trafficking from the late endosome to the TGN is often regulated by the retromer complex of endosomal coat proteins, and disrupting the retromer components sorting nexins 1/2 inhibits the budding of SST₂ from late endosomes. Moreover, trafficking through the late endosomal/TGN pathway is dependent on an intact PDZ ligand and C-terminal tail, as truncating either the 3 or 10 C-terminal amino acids of SST₂ alters the pathway through which it recycles to the plasma membrane. Moreover, addition of these amino acids to a heterologous receptor is sufficient to redirect it from a degradation pathway to a recycling itinerary. Our results demonstrate that endosomal trafficking of SST₂ is dependent on numerous regulatory mechanisms controlled by its C terminus and the retromer machinery.  

3.3299           PAS domain-containing phosphoglycerate kinase deficiency in Leishmania major results in increased autophagosome formation and cell death

Adhikari, A., Biswas, S., Mukherjee, A., Das, S. and Adak, S.

Biochem. J., 476, 1303-1321 (2019)

Per-Arnt-Sim (PAS) domains are structurally conserved and present in numerous proteins throughout all branches of the phylogenetic tree. Although PAS domain-containing proteins are major players for the adaptation to environmental stimuli in both prokaryotic and eukaryotic organisms, these types of proteins are still uncharacterized in the trypanosomatid parasites, Trypanosome and Leishmania. In addition, PAS-containing phosphoglycerate kinase (PGK) protein is uncharacterized in the literature. Here, we report a PAS domain-containing PGK (LmPAS-PGK) in the unicellular pathogen Leishmania. The modeled structure of N-terminal of this protein exhibits four antiparallel β sheets centrally flanked by α helices, which is similar to the characteristic signature of PAS domain. Activity measurements suggest that acidic pH can directly stimulate PGK activity. Localization studies demonstrate that the protein is highly enriched in the glycosome and its presence can also be seen in the lysosome. Gene knockout, overexpression and complement studies suggest that LmPAS-PGK plays a fundamental role in cell survival through autophagy. Furthermore, the knockout cells display a marked decrease in virulence when host macrophage and BALB/c mice were infected with them. Our work begins to clarify how acidic pH-dependent ATP generation by PGK is likely to function in cellular adaptability of Leishmania.

 

3.3300           Regulation of LC3 lipidation by the autophagy-specific class III phosphatidylinositol-3 kinase complex

Brier, L.W., Ge, L., Stjepaovic, G., Theien, A.M., Hurley, J.H. and Scekman, R. Mol. Biol. Cell, 30(9), 1098-1107 (2019)   Autophagy is a conserved eukaryotic pathway critical for cellular adaptation to changes in nutrition levels and stress. The class III phosphatidylinositol (PI)3-kinase complexes I and II (PI3KC3-C1 and -C2) are essential for autophagosome initiation and maturation, respectively, from highly curved vesicles. We used a cell-free reaction that reproduces a key autophagy initiation step, LC3 lipidation, as a biochemical readout to probe the role of autophagy-related gene (ATG)14, a PI3KC3-C1-specific subunit implicated in targeting the complex to autophagy initiation sites. We reconstituted LC3 lipidation with recombinant PI3KC3-C1, -C2, or various mutant derivatives added to extracts derived from a CRISPR/Cas9-generated ATG14-knockout cell line. Both complexes C1 and C2 require the C-terminal helix of VPS34 for activity on highly curved membranes. However, only complex C1 supports LC3 lipidation through the curvature-targeting amphipathic lipid packing sensor (ALPS) motif of ATG14. Furthermore, the ALPS motif and VPS34 catalytic activity are required for downstream recruitment of WD-repeat domain phosphoinositide-interacting protein (WIPI)2, a protein that binds phosphatidylinositol 3-phosphate and its product phosphatidylinositol 3, 5-bisphosphate, and a WIPI-binding protein, ATG2A, but do not affect membrane association of ATG3 and ATG16L1, enzymes contributing directly to LC3 lipidation. These data reveal the nuanced role of the ATG14 ALPS in membrane curvature sensing, suggesting that the ALPS has additional roles in supporting LC3 lipidation.  

3.3301           V232M substitution restricts a distinct O-glycosylation of PLD3 and its neuroprotective function

Demirev, A.V., Song, H-L., Cho, M-H., Cho, K., Peak, J-J., Yoo, H.J., Kim, D-H. and Yoon, S-Y.

Neurobiology of Disease, 129, 182-194 (2019)

The link between Val232Met variant of phospholipase D3 (PLD3) and late-onset Alzheimer's disease (AD) is still obscure. While it may not affect directly the amyloid precursor protein function, PLD3 could be regulating multiple cellular compartments. Here, we investigated the function of wild-type human PLD3 (PLD3WT) and the Val232Met variant (PLD3VM) in the presence of β-amyloid (Aβ) in a Drosophila melanogaster model of AD. We expressed PLD3WT in CNS of the Aβ-model flies and monitored its effect on the ER stress, cell apoptosis and recovery the Aβ-induced cognitive impairment. The expression reduced ER stress and neuronal apoptosis, which resulted in normalized antioxidative phospholipids levels and brain protection. A specific O-glycosylation at pT271 in PLD3 is essential for its normal trafficking and cellular localization. The V232 M substitution impairs this O-glycosylation, leading to enlarged lysosomes and plausibly aberrant protein recycling. PLD3VM was less neuroprotective, and while, PLD3WT expression enhances the lysosomal functions, V232 M attenuated PLD3's trafficking to the lysosomes. Thus, the V232 M mutation may affect AD pathogenesis. Further understanding of the mechanistic role of PLD3 in AD could lead to developing novel therapeutic agents.

 

3.3302           Extracellular vesicle isolation methods: rising impact of size-exclusion chromatography

Monguio-Tortajada, M., Galvez-Monton, C., Bayes-Genis, A., Roura, S. and Borras, F.E. Cell. Mol. Life Sci., 76(12), 2369-2382 (2019)   Extracellular vesicles (EVs) include a variety of nanosized vesicles released to the extracellular microenvironment by the vast majority of cells transferring bioactive lipids, proteins, mRNA, miRNA or non-coding RNA, as means of intercellular communication. Remarkably, among other fields of research, their use has become promising for immunomodulation, tissue repair and as source for novel disease-specific molecular signatures or biomarkers. However, a major challenge is to define accurate, reliable and easily implemented techniques for EV isolation due to their nanoscale size and high heterogeneity. In this context, differential ultracentrifugation (dUC) has been the most widely used laboratory methodology, but alternative procedures have emerged to allow purer EV preparations with easy implementation. Here, we present and discuss the most used of the different EV isolation methods, focusing on the increasing impact of size exclusion chromatography (SEC) on the resulting EV preparations from in vitro cultured cells-conditioned medium and biological fluids. Comparatively, low protein content and cryo-electron microscopy analysis show that SEC removes most of the overabundant soluble plasma proteins, which are not discarded using dUC or precipitating agents, while being more user friendly and less time-consuming than gradient-based EV isolation. Also, SEC highly maintains the major EVs’ characteristics, including vesicular structure and content, which guarantee forthcoming applications. In sum, together with scaling-up possibilities to increase EV recovery and manufacturing following high-quality standards, SEC could be easily adapted to most laboratories to assist EV-associated biomarker discovery and to deliver innovative cell-free immunomodulatory and pro-regenerative therapies.  

3.3303           Dysregulated haemolysin promotes bacterial outer membrane vesicles‐induced pyroptotic‐like cell death in zebrafish

Wen, Y., Chen, S., Jiang, Z., Wang, Z., Tan, j., Hu, T., Wang, Q., Zhou, X., Zhang, Y., Liu, Q. and Yang, D. Cell. Microbiol., 21(6), e13010 (2019)   Inflammasomes are important innate immune components in mammals. However, the bacterial factors modulating inflammasome activation in fish, and the mechanisms by which they alter fish immune defences, remain to be investigated. In this work, a mutant of the fish pathogen Edwardsiella piscicida (E. piscicida), called 0909I, was shown to overexpress haemolysin, which could induce a robust pyroptotic‐like cell death dependent on caspase‐5‐like activity during infection in fish nonphagocyte cells. E. piscicida haemolysin was found to mainly associate with bacterial outer membrane vesicles (OMVs), which were internalised into the fish cells via a dynamin‐dependent endocytosis and induced pyroptotic‐like cell death. Importantly, bacterial immersion infection of both larvae and adult zebrafish suggested that dysregulated expression of haemolysin alerts the innate immune system and induces intestinal inflammation to restrict bacterial colonisation in vivo. Taken together, these results suggest a critical role of zebrafish innate immunity in monitoring invaded pathogens via detecting the bacterial haemolysin‐associated OMVs and initiating pyroptotic‐like cell death. These new additions to the understanding of haemolysin‐mediated pathogenesis in vivo provide evidence for the existence of noncanonical inflammasome signalling in lower vertebrates.  

3.3304           Glioma exosomes mediate the expansion and function of myeloid‐derived suppressor cells through microRNA‐29a/Hbp1 and microRNA‐92a/Prkar1a pathways

Guo, X., Qiu, W., Wang, J., Liu, Q., Qian, M., Wang, S., Zhang, Z., Gao, X., Chen, Z., Guo, Q., Xu, J., Xue, H. and Li, G. Int. J. Cancer, 144(12), 3111-3126 (2019)   Myeloid‐derived suppressor cells (MDSCs) play a pivotal role in mediating the formation of an immunosuppressive environment and assisting tumors in evading the host immune response. However, the mechanism through which tumors manipulate the differentiation and function of MDSCs remains unclear. Here, we report that hypoxia‐induced glioma cells can stimulate the differentiation of functional MDSCs by transferring exosomal miR‐29a and miR‐92a to MDSCs. Our results showed that glioma‐derived exosomes (GEXs) can enhance the differentiation of functional MDSCs both in vitro and in vivo, and hypoxia‐induced GEXs (H‐GEXs) demonstrated a stronger MDSCs induction ability than did normoxia‐induced GEXs (N‐GEXs). A subsequent miRNA sequencing analysis of N‐GEXs and H‐GEXs revealed that hypoxia‐induced exosomal miR‐29a and miR‐92a expression induced the propagation of MDSCs. miR‐29a and miR‐92a activated the proliferation and function of MDSCs by targeting high‐mobility group box transcription factor 1 (Hbp1) and protein kinase cAMP‐dependent type I regulatory subunit alpha (Prkar1a), respectively. Altogether, the results of our study provide new insights into the role of glioma exosomal miRNAs in mediating the formation of immunosuppressive microenvironments in tumors and elucidate the underlying exosomal miR‐29a/miR‐92a‐based regulatory mechanism responsible for the modulation of functional MDSC induction.  

3.3305           Use of extracellular vesicles from lymphatic drainage as surrogate markers of melanoma progression and BRAFV600E mutation

Garcia-Silva, S., Benito-Martin, A., Sanchez-Redondo, S., Hernandez-Barranco, A. et al

1. Exp. Med., 216(5), 1061-1070 (2019)

  Liquid biopsies from cancer patients have the potential to improve diagnosis and prognosis. The assessment of surrogate markers of tumor progression in circulating extracellular vesicles could be a powerful non-invasive approach in this setting. We have characterized extracellular vesicles purified from the lymphatic drainage also known as exudative seroma (ES) of stage III melanoma patients obtained after lymphadenectomy. Proteomic analysis showed that seroma-derived exosomes are enriched in proteins resembling melanoma progression. In addition, we found that the BRAF^V600E mutation can be detected in ES-derived extracellular vesicles and its detection correlated with patients at risk of relapse  

3.3306           Systematic review of targeted extracellular vesicles for drug delivery

Gudbergsson, J.M., Jønsson, K., Simonsen, J.B. and Johnsen, K.B.

J. Controlled Release, 306, 108-120 (2019)

The idea of using extracellular vesicles (EVs) for targeted drug delivery was first introduced in 2011 and has since then gained increasing attention as promising new candidates in the field. Targeting EVs to areas of disease can be achieved through a complex process of designing and inserting a targeting ligand to the surface of the EVs. Although this can be obtained via chemical conjugation, the most important strategy has been to transfect or modulate the EV-producing cell to endow the EVs with the desired targeting capabilities. However, since EVs are harvested from biological sources, their composition is highly heterogeneous, which makes it difficult to control the purity and quality of the resulting EV-based drug delivery vehicles. In this review, we present a detailed account of EVs in targeted drug delivery based on a systematic literature search. We discuss the potential advantages of EVs compared to synthetic lipid-based nanocarriers, and the methodological and biological limitations associated with their use as targeted drug delivery vehicles.

 

3.3307           Surfaceome of Exosomes Secreted from the Colorectal Cancer Cell Line SW480: Peripheral and Integral Membrane Proteins Analyzed by Proteolysis and TX114

Xu, R., Greening, D.W., Chen, M., Rai, A., Ji, H., Takahashi, N. and Simpson, R.J. Proteomics, 19(8), 1700453 (2019)   Exosomes are important bidirectional cell–cell communicators in normal and pathological physiology. Although exosomal surface membrane proteins (surfaceome) enable target cell recognition and are an attractive source of disease marker, they are poorly understood. Here, a comprehensive surfaceome analysis of exosomes secreted by the colorectal cancer cell line SW480 is described. Sodium carbonate extraction/Triton X‐114 phase separation and mild proteolysis (proteinase K, PK) of intact exosomes is used in combination with label‐free quantitative mass spectrometry to identify 1025 exosomal proteins of which 208 are predicted to be integral membrane proteins (IMPs) according to TOPCONS and GRAVY scores. Interrogation of UniProt database‐annotated proteins reveals 124 predicted peripherally‐associated membrane proteins (PMPs). Surprisingly, 108 RNA‐binding proteins (RBPs)/RNA nucleoproteins (RNPs) are found in the carbonate/Triton X‐114 insoluble fraction. Mild PK treatment of SW480‐GFP labeled exosomes reveal 58 proteolytically cleaved IMPs and 14 exoplasmic PMPs (e.g., CLU/GANAB/LGALS3BP). Interestingly, 18 RBPs/RNPs (e.g., EIF3L/RPL6) appear bound to the outer exosome surface since they are sensitive to PK proteolysis. The finding that outer surface‐localized miRNA Let‐7a‐5p is RNase A–resistant, but degraded by a combination of RNase A/PK treatment suggests exosomal miRNA species also reside on the outer surface of exosomes bound to RBPs/RNPs.  

3.3308           Exosomes Derived from Human Primary and Metastatic Colorectal Cancer Cells Contribute to Functional Heterogeneity of Activated Fibroblasts by Reprogramming Their Proteome

Raj, A., Greening, D.W., Chen, M., Xu, R., Ji, H. and Simpson, R.J. Proteomics, 19(8), 1800148 (2019)   Cancer‐associated fibroblasts (CAFs) are a heterogeneous population of activated fibroblasts that constitute a dominant cellular component of the tumor microenvironment (TME) performing distinct functions. Here, the role of tumor‐derived exosomes (Exos) in activating quiescent fibroblasts into distinct functional subtypes is investigated. Proteomic profiling and functional dissection reveal that early‐ (SW480) and late‐stage (SW620) colorectal cancer (CRC) cell‐derived Exos both activated normal quiescent fibroblasts (α‐SMA⁻, CAV⁺, FAP⁺, VIM⁺) into CAF‐like fibroblasts (α‐SMA⁺, CAV⁻, FAP⁺, VIM⁺). Fibroblasts activated by early‐stage cancer‐exosomes (SW480‐Exos) are highly pro‐proliferative and pro‐angiogenic and display elevated expression of pro‐angiogenic (IL8, RAB10, NDRG1) and pro‐proliferative (SA1008, FFPS) proteins. In contrast, fibroblasts activated by late‐stage cancer‐exosomes (SW620‐Exos) display a striking ability to invade through extracellular matrix through upregulation of pro‐invasive regulators of membrane protrusion (PDLIM1, MYO1B) and matrix‐remodeling proteins (MMP11, EMMPRIN, ADAM10). Conserved features of Exos‐mediated fibroblast activation include enhanced ECM secretion (COL1A1, Tenascin‐C/X), oncogenic transformation, and metabolic reprogramming (downregulation of CAV‐1, upregulation of glycogen metabolism (GAA), amino acid biosynthesis (SHMT2, IDH2) and membrane transporters of glucose (GLUT1), lactate (MCT4), and amino acids (SLC1A5/3A5)). This study highlights the role of primary and metastatic CRC tumor‐derived Exos in generating phenotypically and functionally distinct subsets of CAFs that may facilitate tumor progression.  

3.3309           Proteomic and Post‐Translational Modification Profiling of Exosome‐Mimetic Nanovesicles Compared to Exosomes

Kenari, A.N., Kastaniegaard, K., Greening, D.W., Shambrook, M., Stenballe, A., Cheng, l. and Hill, A.F. Proteomics, 19(8), 1800161 (2019)   Issues associated with upscaling exosome production for therapeutic use may be overcome through utilizing artificial exosomes. Cell‐derived mimetic nanovesicles (M‐NVs) are a potentially promising alternative to exosomes for clinical applicability, demonstrating higher yield without incumbent production and isolation issues. Although several studies have shown that M‐NVs have similar morphology, size and therapeutic potential compared to exosomes, comprehensive characterization and to what extent M‐NVs components mimic exosomes remain elusive. M‐NVs were generated through the extrusion of cells and proteomic profiling demonstrated an enrichment of proteins associated with membrane and cytosolic components. The proteomic data herein reveal a subset of proteins that are highly abundant in M‐NVs in comparison to exosomes. M‐NVs contain proteins that largely represent the parental cell proteome, whereas the profile of exosomal proteins highlight their endosomally derived origin. This advantage of M‐NVs alleviates the necessity of endosomal sorting of endogenous therapeutic proteins or RNA into exosomes. This study also highlights differences in protein post‐translational modifications among M‐NVs, as distinct from exosomes. Overall this study provides key insights into defining the proteome composition of M‐NVs as a distinct from exosomes, and the potential advantage of M‐NVs as an alternative nanocarrier when spontaneous endosomal sorting of therapeutics are limited.  

3.3310           The metabolites NADP+ and NADPH are the targets of the circadian protein Nocturnin (Curled)

Estrella, M.A., Du, J., Chen, L., Rath, S., Prangley, E., Chitrakar, A., Acki, T., Schedl, P., Rabinowitz, J. and Korennykh, A. Nature Communications, 10:2367 (2019)   Nocturnin (NOCT) is a rhythmically expressed protein that regulates metabolism under the control of circadian clock. It has been proposed that NOCT deadenylates and regulates metabolic enzyme mRNAs. However, in contrast to other deadenylases, purified NOCT lacks the deadenylase activity. To identify the substrate of NOCT, we conducted a mass spectrometry screen and report that NOCT specifically and directly converts the dinucleotide NADP⁺ into NAD⁺ and NADPH into NADH. Further, we demonstrate that the Drosophila NOCT ortholog, Curled, has the same enzymatic activity. We obtained the 2.7 Å crystal structure of the human NOCT•NADPH complex, which revealed that NOCT recognizes the chemically unique ribose-phosphate backbone of the metabolite, placing the 2′-terminal phosphate productively for removal. We provide evidence for NOCT targeting to mitochondria and propose that NADP(H) regulation, which takes place at least in part in mitochondria, establishes the molecular link between circadian clock and metabolism.  

3.3311           ARNT2 Tunes Activity-Dependent Gene Expression through NCoR2-Mediated Repression and NPAS4-Mediated Activation

Sharma, N., Pollina, E.A., Nagy, M.A., Hu, L., Lin, C. and Greenberg, M.E: Neuron, 102, 390-406 (2019)   Neuronal activity-dependent transcription is tuned to ensure precise gene induction during periods of heightened synaptic activity, allowing for appropriate responses of activated neurons within neural circuits. The consequences of aberrant induction of activity-dependent genes on neuronal physiology are not yet clear. Here, we demonstrate that, in the absence of synaptic excitation, the basic-helix-loop-helix (bHLH)-PAS family transcription factor ARNT2 recruits the NCoR2 co-repressor complex to suppress neuronal activity-dependent regulatory elements and maintain low basal levels of inducible genes. This restricts inhibition of excitatory neurons, maintaining them in a state that is receptive to future sensory stimuli. By contrast, in response to heightened neuronal activity, ARNT2 recruits the neuronal-specific bHLH-PAS factor NPAS4 to activity-dependent regulatory elements to induce transcription and thereby increase somatic inhibitory input. Thus, the interplay of bHLH-PAS complexes at activity-dependent regulatory elements maintains temporal control of activity-dependent gene expression and scales somatic inhibition with circuit activity.  

3.3312           Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood

Notarangelo, M., Zucal, C., Modelska, A., Pesce, I., Scarduelli, G. et al EBioMed., 43, 114-126 (2019)   Background Extracellular vesicles (EVs) are secreted membranous particles intensively studied for their potential cargo of diagnostic markers. Efficient and cost-effective isolation methods need to be established for the reproducible and high-throughput study of EVs in the clinical practice. Methods We designed the nickel-based isolation (NBI) to rapidly isolate EVs and combined it with newly-designed amplified luminescent proximity homogeneous assay or digital PCR to detect biomarkers of clinical utility. Findings From plasma of 46 healthy donors, we systematically recovered small EV (~250 nm of mean diameter; ~3 × 10¹⁰/ml) and large EV (~560 nm of mean diameter; ~5 × 10⁸/ml) lineages ranging from 50 to 700 nm, which displayed hematopoietic/endothelial cell markers that were also used in spike-in experiments using EVs from tumor cell lines. In retrospective studies, we detected picomolar concentrations of prostate-specific membrane antigen (PSMA) in fractions of EVs isolated from the plasma of prostate cancer patients, discriminating them from control subjects. Directly from oil-encapsulated EVs for digital PCR, we identified somatic BRAF and KRAS mutations circulating in the plasma of metastatic colorectal cancer (CRC) patients, matching 100% of concordance with tissue diagnostics. Importantly, with higher sensitivity and specificity compared with immuno-isolated EVs, we revealed additional somatic alterations in 7% of wild-type CRC cases that were subsequently validated by further inspections in the matched tissue biopsies. Interpretation We propose NBI-combined approaches as simple, fast, and robust strategies to probe the tumor heterogeneity and contribute to the development of EV-based liquid biopsy studies. Fund Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio Trento e Rovereto (CARITRO), and the Italian Ministero Istruzione, Università e Ricerca (Miur).  

3.3313           Different isolation approaches lead to diverse glycosylated extracellular vesicle populations

Freitas, D., Balmana, M., Pocas, J., Campos, D., Osorio, H., Konstantinidi, A., Vakhrushev, S.Y., Magalhäes, A. and Reis, C.A.

1. Extracellular Vesicles, 8, 161131 (2019)

  Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations’ glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrep^TM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates.  

3.3314           In vivo nuclear capture and molecular profiling identifies Gmeb1 as a transcriptional regulator essential for dopamine neuron function

Tuiesta, L.M., Djekidel, M.N., Chen, R., Lu, F., Wang, W., Sabatini, B.L. and Zhang, Y. Nature Communications, 10:2508 (2019)   Midbrain dopamine (mDA) neurons play a central role in reward signaling and are widely implicated in psychiatric and neurodegenerative disorders. To understand how mDA neurons perform these functions, it is important to understand how mDA-specific genes are regulated. However, cellular heterogeneity in the mammalian brain presents a major challenge to obtaining this understanding. To this end, we developed a virus-based approach to label and capture mDA nuclei for transcriptome (RNA-Seq), and low-input chromatin accessibility (liDNase-Seq) profiling, followed by predictive modeling to identify putative transcriptional regulators of mDA neurons. Using this method, we identified Gmeb1, a transcription factor predicted to regulate expression of Th and Dat, genes critical for dopamine synthesis and reuptake, respectively. Gmeb1 knockdown in mDA neurons resulted in downregulation of Th and Dat, as well as in severe motor deficits. This study thus identifies Gmeb1 as a master regulator of mDA gene expression and function, and provides a general method for identifying cell type-specific transcriptional regulators.  

3.3315           Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing—Enabling robust and non-invasive biomarker research

Mussack, V., Wittmann, G. and Pfaffl, M.W. Biomolecular Detection and Quantification, 17, 100089 (2019)   Small extracellular vesicles (EVs) are 50–200 nm sized mediators in intercellular communication that reflect both physiological and pathophysiological changes of their parental cells. Thus, EVs hold great potential for biomarker detection. However, reliable purification methods for the downstream screening of the microRNA (miRNA) cargo carried within urinary EVs by small RNA sequencing have yet to be established. To address this knowledge gap, RNA extracted from human urinary EVs obtained by five different urinary EV purification methods (spin column chromatography, immunoaffinity, membrane affinity, precipitation and ultracentrifugation combined with density gradient) was analyzed by small RNA sequencing. Urinary EVs were further characterized by nanoparticle tracking analysis, Western blot analysis and transmission electron microscopy. Comprehensive EV characterization established significant method-dependent differences in size and concentration as well as variances in protein composition of isolated vesicles. Even though all purification methods captured enough total RNA to allow small RNA sequencing, method-dependent differences were also observed with respect to library sizes, mapping distributions, number of miRNA reads and diversity of transcripts. Whereas EVs obtained by immunoaffinity yielded the purest subset of small EVs, highly comparable with results attained by ultracentrifugation combined with density gradient, precipitation and membrane affinity, sample purification by spin column chromatography indicated a tendency to isolate different subtypes of small EVs, which might also carry a distinct subset of miRNAs. Based on our results, different EV purification methods seem to preferentially isolate different subtypes of EVs with varying efficiencies. As a consequence, sequencing experiments and resulting miRNA profiles were also affected. Hence, the selection of a specific EV isolation method has to satisfy the respective research question and should be well considered. In strict adherence with the MISEV (minimal information for studies of extracellular vesicles) guidelines, the importance of a combined evaluation of biophysical and proteomic EV characteristics alongside transcriptomic results was clearly demonstrated in this present study.  

3.3316           Extracellular Microvesicles as New Industrial Therapeutic Frontiers

Agrahari, V., Agrahari, V., Bumouf, P-A., Chew, C.H. and Bumouf, T. Trend in Biotechnol., 37(7), 707-729 (2019)   Microvesicles (MVs) are subcellular physiological vehicles present in all body fluids that mediate the transfer of intercellular information within biological systems and contribute to healthy conditions. MVs have lipid bilayer membranes decorated with multiple ligands that can interact with receptors on target cells, rendering them as promising candidates for targeted delivery. The biotechnology and cell therapy industries are developing MV-based preparations that use this subcellular therapeutic machinery (in a naïve or modified state) for regenerative medicine, as substitutes for intact cell therapy, and as intelligent targeted drug delivery carriers. However, significant production challenges must be overcome before MVs scale-up development, clinical translation, and routine therapeutic application can be realized. The unique expertise developed in the biotechnology industry should facilitate market access to MV-based therapeutics. In this review, the roles of biotechnology and cell therapy industries to manufacture MVs as inherent therapeutic agents or drug delivery systems are summarized. The manufacturing, development, characterization, and regulatory challenges for successful translation are discussed.  

3.3317           Chapter Fifteen - Monitoring stress-induced autophagic engulfment and degradation of the 26S proteasome in mammalian cells

Cohen-Kaplan, V., Livneh, I., Kwon, Y.T. and Ciechanover, A. Methods in Enzymol., 619, 337-366 (2019)   Almost 70 years after the discovery of the lysosome, and about four decades following the unraveling of ubiquitin as a specific “mark of death,” the field of protein turnover—the numerous processes it regulates, the pathologies resulting from its dysregulation, and the drugs that have been developed to target them—is still growing exponentially. Accordingly, the need for new technologies and methods is ever growing. One interesting question in the field is the mechanism(s) by which the “predators become prey”. We have reported recently that the 26S proteasome, the catalytic arm of the ubiquitin system, is degraded by the autophagy–lysosome machinery, in a process requiring specific ubiquitination of the proteasome, and subsequent recognition by the shuttle protein p62/SQSTM1. Studying the modification(s), recognition sites, engulfment, and breakdown of the 26S proteasome via such “proteaphagy” has required the use of microscopy, subcellular fractionation, ‘classical biochemistry’, and proteomics. In this chapter, we present the essentials of these protocols, with emphasis on the refinements we have introduced in order for them to better suit the particular study of proteaphagy.  

3.3318           Mycobacterium tuberculosis extracellular vesicle-associated lipoprotein LpqH as a potential biomarker to distinguish paratuberculosis infection or vaccination from tuberculosis infection

Palacios, A., Sampedro, L., Sevilla, I.A., Molina, E., Gil, D., Azkargota, M., Elortza, F., Garrido, J.M., Anguita, J. and Prados-Rosales, R. BMC Vet. Res., 15:188 (2019)   Background Both bovine tuberculosis (TB) and paratuberculosis (PTB) are serious and widespread bacterial infections affecting many domestic and wild animal species. However, current vaccines do not confer complete protection and cause interference with other diagnostics tests, including bovine TB. Therefore, the development of “Differentiating Infected from Vaccinated Animals” (DIVA) tests are a pressing need. In this study, we have tested the feasibility of mycobacterial extracellular vesicles (EVs) as potential source of biomarkers to discriminate between Mycobacterium bovis infected, Mycobacterium avium subsp. paratuberculosis (MAP) infected and MAP-vaccinated cows. We have, initially, characterized vesicle production in the two most medically relevant species of mycobacteria for livestock, MAP and M. bovis, for being responsible for tuberculosis (TB) and paratuberculosis (PTB). Results Our results indicate that these two species produce EVs with different kinetics, morphology and size distribution. Analysis of the immunogenicity of both type of EVs showed some cross reactivity with sera from PTB+ and TB+ cows, suggesting a limited diagnostic capacity for both EVs. Conversely, we noticed that Mycobacterium tuberculosis (Mtb) EVs showed some differential reactivity between sera from MAP-vaccinated or PTB+ cows from TB+ ones. Mass spectrometry analysis (MS) identified a 19-kDa EV-associated lipoprotein as the main source of the differential reactivity. Conclusions LpqH could be a good plasma biomarker with capacity to distinguish PTB+ or MAP-vaccinated cows from cows infected with TB.  

3.3319           C-terminal α-synuclein truncations are linked to cysteine cathepsin activity in Parkinson's disease

McGlinchey, R.P., Lacy, S.M., Huffer, K.E., Tayebi, N., Sidransky, E. and Lee, J.C.

1. Biol. Chem., 294(25), 9973-9984 (2019)

  A pathological feature of Parkinson's disease (PD) is Lewy bodies (LBs) composed of α-synuclein (α-syn) amyloid fibrils. α-Syn is a 140 amino acids–long protein, but truncated α-syn is enriched in LBs. The proteolytic processes that generate these truncations are not well-understood. On the basis of our previous work, we propose that these truncations could originate from lysosomal activity attributable to cysteine cathepsins (Cts). Here, using a transgenic SNCA^A53T mouse model, overexpressing the PD-associated α-syn variant A53T, we compared levels of α-syn species in purified brain lysosomes from nonsymptomatic mice with those in age-matched symptomatic mice. In the symptomatic mice, antibody epitope mapping revealed enrichment of C-terminal truncations, resulting from CtsB, CtsL, and asparagine endopeptidase. We did not observe changes in individual cathepsin activities, suggesting that the increased levels of C-terminal α-syn truncations are because of the burden of aggregated α-syn. Using LC-MS and purified α-syn, we identified C-terminal truncations corresponding to amino acids 1–122 and 1–90 from the SNCA^A53T lysosomes. Feeding rat dopaminergic N27 cells with exogenous α-syn fibrils confirmed that these fragments originate from incomplete fibril degradation in lysosomes. We mimicked these events in situ by asparagine endopeptidase degradation of α-syn fibrils. Importantly, the resulting C-terminally truncated fibrils acted as superior seeds in stimulating α-syn aggregation compared with that of the full-length fibrils. These results unequivocally show that C-terminal α-syn truncations in LBs are linked to Cts activities, promote amyloid formation, and contribute to PD pathogenesis.  

3.3320           Ganglioside Synthase Knockout Reduces Prion Disease Incubation Time in Mouse Models

Kobayashi, A., Qi, Z., Shimazaki, T., Munesue, Y., Miyamoto, T., Isoda, n., Sawa, H., Aoshima, K., Kimura, T., Mohri, S., Kitamoto, T., Yamashita, T. and Miyoshi, I. Am. J. Pathol., 189(3), 677-686 (2019)   Localization of the abnormal and normal isoforms of prion proteins to detergent-resistant membrane microdomains, lipid rafts, is important for the conformational conversion. Lipid rafts are enriched in sialic acid–containing glycosphingolipids (namely, gangliosides). Alteration in the ganglioside composition of lipid rafts can affect the localization of lipid raft–associated proteins. To investigate the role of gangliosides in the pathogenesis of prion diseases, we performed intracerebral transmission study of a scrapie prion strain Chandler and a Gerstmann-Sträussler-Scheinker syndrome prion strain Fukuoka-1 using various knockout mouse strains ablated with ganglioside synthase gene (ie, GD2/GM2 synthase, GD3 synthase, or GM3 synthase). After challenge with the Chandler strain, GD2/GM2 synthase knockout mice showed 20% reduction of incubation time, reduced prion protein deposition in the brain with attenuated glial reactions, and reduced localization of prion proteins to lipid rafts. These results raise the possibility that the gangliosides may have an important role in prion disease pathogenesis by affecting the localization of prion proteins to lipid rafts.  

3.3321           Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3

Borghesan, M., Fafian-labora, J., Eleftheriadou, O., Vossenkamper, A., Munoz-Espin, D. and O’Loghlen, A. Cell Reports, 27(13), 3956-3971 (2019)   Senescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence.  

3.3322           Proteomic Profiling of Mouse Epididymosomes Reveals their Contributions to Post-testicular Sperm Maturation

Nixon, B., De Iuliis, G.N., Hart, H.M., Zhou, W., Mathe, A., Bernstein, I.R., Anderson, A.L., Stanger, S.J., Skerrett-Byrne, D.A., Jamaluddin, M.F.B., Almazi, J.G., Bromfield, E.G., Larsen, M.R. and Dun, M.D. Mol. Cell. Proteomics, 18, S91-S108 (2019)   The functional maturation of spermatozoa that is necessary to achieve fertilization occurs as these cells transit through the epididymis, a highly specialized region of the male reproductive tract. A defining feature of this maturation process is that it occurs in the complete absence of nuclear gene transcription or de novo, protein translation in the spermatozoa. Rather, it is driven by sequential interactions between spermatozoa and the complex external milieu in which they are bathed within lumen of the epididymal tubule. A feature of this dynamic microenvironment are epididymosomes, small membrane encapsulated vesicles that are secreted from the epididymal soma. Herein, we report comparative proteomic profiling of epididymosomes isolated from different segments of the mouse epididymis using multiplexed tandem mass tag (TMT) based quantification coupled with high resolution LC-MS/MS. A total of 1640 epididymosome proteins were identified and quantified via this proteomic method. Notably, this analysis revealed pronounced segment-to-segment variation in the encapsulated epididymosome proteome. Thus, 146 proteins were identified as being differentially accumulated between caput and corpus epididymosomes, and a further 344 were differentially accumulated between corpus and cauda epididymosomes (i.e., fold change of ≤ −1.5 or ≥ 1.5; p, < 0.05). Application of gene ontology annotation revealed a substantial portion of the epididymosome proteins mapped to the cellular component of extracellular exosome and to the biological processes of transport, oxidation-reduction, and metabolism. Additional annotation of the subset of epididymosome proteins that have not previously been identified in exosomes revealed enrichment of categories associated with the acquisition of sperm function (e.g., fertilization and binding to the zona pellucida). In tandem with our demonstration that epididymosomes are able to convey protein cargo to the head of maturing spermatozoa, these data emphasize the fundamental importance of epididymosomes as key elements of the epididymal microenvironment responsible for coordinating post-testicular sperm maturation.  

3.3323           Emerging roles of extracellular vesicles in neurodegenerative disorders

You, Y. and Ikezu, T. Neurobiol. of Disease, 130, 104512 (2019)   Extracellular vesicles (EVs) are heterogeneous cell-derived membranous vesicles which carry a large diversity of molecules such as proteins and RNA species. They are now considered to be a general mode of intercellular communication by direct transfer of biomolecules. Emerging evidence demonstrates that EVs are involved in multiple pathological processes of brain diseases including neurodegenerative disorders. In this review, we investigate the current knowledge about EV biology. We also provide an overview of the roles of EVs in related brain diseases, particularly in neurodegenerative disorders. Finally, we discuss their potential applications as novel biomarkers as well as the developments of EV-based therapies.  

3.3324           Hierarchical activation of compartmentalized pools of AMPK depends on severity of nutrient or energy stress

Zong, Y., Zhang, C-S., Li, M., Wang, W., Wang, Z., Hawley, S.A., Ma, T. et al Cell Res., 29, 460-473 (2019)   AMPK, a master regulator of metabolic homeostasis, is activated by both AMP-dependent and AMP-independent mechanisms. The conditions under which these different mechanisms operate, and their biological implications are unclear. Here, we show that, depending on the degree of elevation of cellular AMP, distinct compartmentalized pools of AMPK are activated, phosphorylating different sets of targets. Low glucose activates AMPK exclusively through the AMP-independent, AXIN-based pathway in lysosomes to phosphorylate targets such as ACC1 and SREBP1c, exerting early anti-anabolic and pro-catabolic roles. Moderate increases in AMP expand this to activate cytosolic AMPK also in an AXIN-dependent manner. In contrast, high concentrations of AMP, arising from severe nutrient stress, activate all pools of AMPK independently of AXIN. Surprisingly, mitochondrion-localized AMPK is activated to phosphorylate ACC2 and mitochondrial fission factor (MFF) only during severe nutrient stress. Our findings reveal a spatiotemporal basis for hierarchical activation of different pools of AMPK during differing degrees of stress severity.  

3.3325           Cholesterol Induces Epithelial-to-Mesenchymal Transition of Prostate Cancer Cells by Suppressing Degradation of EGFR through APMAP

Jiang, S., Wang, X., Song, D., Liu, X., Gu, Y. et al

Cancer Res., 79(12), 3063-3075 (2019)

  Cholesterol increases the risk of aggressive prostate cancer and has emerged as a potential therapeutic target for prostate cancer. The functional roles of cholesterol in prostate cancer metastasis are not fully understood. Here, we found that cholesterol induces the epithelial-to-mesenchymal transition (EMT) through extracellular-regulated protein kinases 1/2 pathway activation, which is mediated by EGFR and adipocyte plasma membrane-associated protein (APMAP) accumulation in cholesterol-induced lipid rafts. Mechanistically, APMAP increases the interaction with EGFR substrate 15-related protein (EPS15R) to inhibit the endocytosis of EGFR by cholesterol, thus promoting cholesterol-induced EMT. Both the mRNA and protein levels of APMAP are upregulated in clinical prostate cancer samples. Together, these findings shed light onto an APMAP/EPS15R/EGFR axis that mediates cholesterol-induced EMT of prostate cancer cells.  

3.3326           Single-cell transcriptomic analysis of Alzheimer’s disease

Mathys, H., Velderrain, J.D., Peng, Z., Gao, F., Mohammadi, S., Young, J.Z. et al

Nature, 570, 332-337 (2019)

Alzheimer’s disease is a pervasive neurodegenerative disorder, the molecular complexity of which remains poorly understood. Here, we analysed 80,660 single-nucleus transcriptomes from the prefrontal cortex of 48 individuals with varying degrees of Alzheimer’s disease pathology. Across six major brain cell types, we identified transcriptionally distinct subpopulations, including those associated with pathology and characterized by regulators of myelination, inflammation, and neuron survival. The strongest disease-associated changes appeared early in pathological progression and were highly cell-type specific, whereas genes upregulated at late stages were common across cell types and primarily involved in the global stress response. Notably, we found that female cells were overrepresented in disease-associated subpopulations, and that transcriptional responses were substantially different between sexes in several cell types, including oligodendrocytes. Overall, myelination-related processes were recurrently perturbed in multiple cell types, suggesting that myelination has a key role in Alzheimer’s disease pathophysiology. Our single-cell transcriptomic resource provides a blueprint for interrogating the molecular and cellular basis of Alzheimer’s disease.

 

3.3327           Tethering of vesicles to the Golgi by GMAP210 controls LAT delivery to the immune synapse

Zucchetti, A.E., Bataille, L., Carpier, J-M., Dogniaux, S., Roman-Jouve, M.S., Maurin, M., Stuck, M.W., Rios, R.M., Baldari, C.T., Pazour, G.J. and Hivroz, C. Nature Communications, 102864 (2019)   The T cell immune synapse is a site of intense vesicular trafficking. Here we show that the golgin GMAP210, known to capture vesicles and organize membrane traffic at the Golgi, is involved in the vesicular transport of LAT to the immune synapse. Upon activation, more GMAP210 interact with LAT-containing vesicles and go together with LAT to the immune synapse. Regulating LAT recruitment and LAT-dependent signaling, GMAP210 controls T cell activation. Using a rerouting and capture assay, we show that GMAP210 captures VAMP7-decorated vesicles. Overexpressing different domains of GMAP210, we also show that GMAP210 allows their specific delivery to the immune synapse by tethering LAT-vesicles to the Golgi. Finally, in a model of ectopic expression of LAT in ciliated cells, we show that GMAP210 tethering activity controls the delivery of LAT to the cilium. Hence, our results reveal a function for the golgin GMAP210 conveying specific vesicles to the immune synapse.  

3.3328           Excitotoxic targeting of Kidins220 to the Golgi apparatus precedes calpain cleavage of Rap1-activation complexes

Lopez-Menendez, C., Simon-Garcia, A., Gamir-Morralla, A., Pose-Utrilla, J., Lujan, R., Mochizuki, N., Diaz-Guerra, M. and Iglesias, T. Cell Death & Disease, 10:535 82019)   Excitotoxic neuronal death induced by high concentrations of glutamate is a pathological event common to multiple acute or chronic neurodegenerative diseases. Excitotoxicity is mediated through overactivation of the N-Methyl-D-aspartate type of ionotropic glutamate receptors (NMDARs). Physiological stimulation of NMDARs triggers their endocytosis from the neuronal surface, inducing synaptic activity and survival. However almost nothing is known about the internalization of overactivated NMDARs and their interacting proteins, and how this endocytic process is connected with neuronal death has been poorly explored. Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is a component of NMDAR complexes essential for neuronal viability by the control of ERK activation. Here we have investigated Kidins220 endocytosis induced by NMDAR overstimulation and the participation of this internalization step in the molecular mechanisms of excitotoxicity. We show that excitotoxicity induces Kidins220 and GluN1 traffic to the Golgi apparatus (GA) before Kidins220 is degraded by the protease calpain. We also find that excitotoxicity triggers an early activation of Rap1-GTPase followed by its inactivation. Kidins220 excitotoxic endocytosis and subsequent calpain-mediated downregulation governs this late inactivation of Rap1 that is associated to decreases in ERK activity preceding neuronal death. Furthermore, we identify the molecular mechanisms involved in the excitotoxic shutoff of Kidins220/Rap1/ERK prosurvival cascade that depends on calpain processing of Rap1-activation complexes. Our data fit in a model where Kidins220 targeting to the GA during early excitotoxicity would facilitate Rap1 activation and subsequent stimulation of ERK. At later times, activation of Golgi-associated calpain, would promote the degradation of GA-targeted Kidins220 and two additional components of the specific Rap1 activation complex, PDZ-GEF1, and S-SCAM. In this way, late excitotoxicity would turn off Rap1/ERK cascade and compromise neuronal survival.  

3.3329           Progranulin deficiency leads to reduced glucocerebrosidase activity

Zhou, X., Paushter, D.H., Pagan, M.D., Kim, D., Santos, M.N., Lieberman, R.L., Overkleeft, H.S., Sun, Y., Smolka, M.B. and Hu, F. Plos One, 14(7), e0212382 (2019)   Mutation in the GRN gene, encoding the progranulin (PGRN) protein, shows a dose-dependent disease correlation, wherein haploinsufficiency results in frontotemporal lobar degeneration (FTLD) and complete loss results in neuronal ceroid lipofuscinosis (NCL). Although the exact function of PGRN is unknown, it has been increasingly implicated in lysosomal physiology. Here we report that PGRN interacts with the lysosomal enzyme, glucocerebrosidase (GCase), and is essential for proper GCase activity. GCase activity is significantly reduced in tissue lysates from PGRN-deficient mice. This is further evidence that reduced lysosomal hydrolase activity may be a pathological mechanism in cases of GRN-related FTLD and NCL.  

3.3330           NOX1-Dependent mTORC1 Activation via S100A9 Oxidation in Cancer Stem-like Cells Leads to Colon Cancer Progression

Ohata, H., Shiokawa, D, Obata, Y., Ono, M., nakagama, H. and Okamoto, K. Cell Reports, 28, 1282-1295 (2019)   Cancer stem cells (CSCs) are associated with the refractory nature of cancer, and elucidating the targetable pathways for CSCs is crucial for devising innovative antitumor therapies. We find that the proliferation of CSC-enriched colon spheroids from clinical specimen is dependent on mTORC1 kinase, which is activated by reactive oxygen species (ROS) produced by NOX1, an NADPH oxidase. In the spheroid-derived xenograft tumors, NOX1 is preferentially expressed in LGR5-positive cells. Dependence on NOX1 expression or mTOR kinase activity is corroborated in the xenograft tumors and mouse colon cancer-derived organoids. NOX1 co-localizes with mTORC1 in VPS41-/VPS39-positive lysosomes, where mTORC1 binds to S100A9, a member of S100 calcium binding proteins, in a NOX1-produced ROS-dependent manner. S100A9 is oxidized by NOX1-produced ROS, which facilitates binding to mTORC1 and its activation. We propose that NOX1-dependent mTORC1 activation via S100A9 oxidation in VPS41-/VPS39-positive lysosomes is crucial for colon CSC proliferation and colon cancer progression.  

3.3331           Activation of NIX-mediated mitophagy by an interferon regulatory factor homologue of human herpesvirus

Vo, M.T., Smith, B.J., Nicholas, j. and Choi, Y.B. Nature Communications, 10:3203 (2019)   Viral control of mitochondrial quality and content has emerged as an important mechanism for counteracting the host response to virus infection. Despite the knowledge of this crucial function of some viruses, little is known about how herpesviruses regulate mitochondrial homeostasis during infection. Human herpesvirus 8 (HHV-8) is an oncogenic virus causally related to AIDS-associated malignancies. Here, we show that HHV-8-encoded viral interferon regulatory factor 1 (vIRF-1) promotes mitochondrial clearance by activating mitophagy to support virus replication. Genetic interference with vIRF-1 expression or targeting to the mitochondria inhibits HHV-8 replication-induced mitophagy and leads to an accumulation of mitochondria. Moreover, vIRF-1 binds directly to a mitophagy receptor, NIX, on the mitochondria and activates NIX-mediated mitophagy to promote mitochondrial clearance. Genetic and pharmacological interruption of vIRF-1/NIX-activated mitophagy inhibits HHV-8 productive replication. Our findings uncover an essential role of vIRF-1 in mitophagy activation and promotion of HHV-8 lytic replication via this mechanism.  

3.3332           The generation and use of recombinant extracellular vesicles as biological reference material

Geeurickx, E., Tulkens, J., Dhondt, B., Van Deun, J., Lippens, L. Nature Communications, 10:3288 (2019)   Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications.  

3.3333           IFN-β is a macrophage-derived effector cytokine facilitating the resolution of bacterial inflammation

Satynarayanan, S.K., El Kebir, D., Soboh, S., Butenko, S., Sekheri, M., Saadi, J., Peled, N., Assi, S., Othman, A., Schif-Zuck, S., Feuermann, Y., barkan, D., Sher, n., Filep, J.G. and Ariel, A. Nature Communications, 10:3471 (2019)   The uptake of apoptotic polymorphonuclear cells (PMN) by macrophages is critical for timely resolution of inflammation. High-burden uptake of apoptotic cells is associated with loss of phagocytosis in resolution phase macrophages. Here, using a transcriptomic analysis of macrophage subsets, we show that non-phagocytic resolution phase macrophages express a distinct IFN-β-related gene signature in mice. We also report elevated levels of IFN-β in peritoneal and broncho-alveolar exudates in mice during the resolution of peritonitis and pneumonia, respectively. Elimination of endogenous IFN-β impairs, whereas treatment with exogenous IFN-β enhances, bacterial clearance, PMN apoptosis, efferocytosis and macrophage reprogramming. STAT3 signalling in response to IFN-β promotes apoptosis of human PMNs. Finally, uptake of apoptotic cells promotes loss of phagocytic capacity in macrophages alongside decreased surface expression of efferocytic receptors in vivo. Collectively, these results identify IFN-β produced by resolution phase macrophages as an effector cytokine in resolving bacterial inflammation.  

3.3334           The Edwardsiella piscicida thioredoxin-like protein inhibits ASK1-MAPKs signaling cascades to promote pathogenesis during infection

Yang, D., Liu, X., Xu, W., Gu, Z., yang, C., Zhang, L., Tan, j., Zheng, X., Wang, Z., Quan, S., Zhang, Y. and Liu, Q. PloS Pathogens, 15(7), e1007917 (2019)   It is important that bacterium can coordinately deliver several effectors into host cells to disturb the cellular progress during infection, however, the precise role of effectors in host cell cytosol remains to be resolved. In this study, we identified a new bacterial virulence effector from pathogenic Edwardsiella piscicida, which presents conserved crystal structure to thioredoxin family members and is defined as a thioredoxin-like protein (Trxlp). Unlike the classical bacterial thioredoxins, Trxlp can be translocated into host cells, mimicking endogenous thioredoxin to abrogate ASK1 homophilic interaction and phosphorylation, then suppressing the phosphorylation of downstream Erk1/2- and p38-MAPK signaling cascades. Moreover, Trxlp-mediated inhibition of ASK1-Erk/p38-MAPK axis promotes the pathogenesis of E. piscicida in zebrafish larvae infection model. Taken together, these data provide insights into the mechanism underlying the bacterial thioredoxin as a virulence effector in downmodulating the innate immune responses during E. piscicida infection.  

3.3335           Small Interfering RNA Screening for the Small GTPase Rab Proteins Identifies Rab5B as a Major Regulator of Hepatitis B Virus Production

Inoue, J., Ninomiya, M., Umetsu, T., Makamura, T., Kogure, T., Kakazu, E., Iwata, T. et al

1. Virol., 93(15), e000621-19 (2019)

  Viruses are considered to use vesicular trafficking in infected cells, but the details of assembly/release pathways of hepatitis B virus (HBV) are still unknown. To identify key regulators of HBV production, we performed short interfering RNA (siRNA) screening for Rab proteins, which are considered to act as molecular switches in vesicular trafficking using HepG2.2.15 cells. Among 62 Rab proteins, the suppression of Rab5B most significantly increased HBV DNA in the culture supernatant. Surprisingly, 5 days after the transfection of Rab5B siRNA, HBV DNA in the supernatant was increased more than 30-fold, reflecting the increase of infectious HBV particles. Northern blotting showed that transcription of 2.4/2.1-kb mRNA coding envelope proteins containing large hepatitis B surface protein (LHBs) was increased. Analysis of hepatocyte nuclear factors (HNFs) showed that transcription of HNF4α, which is known to enhance 2.4-kb mRNA transcription, was regulated by Rab5B. Also, it was revealed that LHBs had accumulated in the endoplasmic reticulum (ER) after Rab5B depletion but not in the multivesicular body (MVB), which is thought to be an organelle utilized for HBV envelope formation. Therefore, it was considered that Rab5B is required for the transport of LHBs from the ER to MVB. Immunofluorescent microscopy showed that HBs proteins, including LHBs, colocalized with HBc in the ER of Rab5B-depleted cells, suggesting that HBV envelopment occurs not only in the MVB but also in the ER. In conclusion, Rab5B is a key regulator of HBV production and could be a target of antiviral therapy.  

3.3336           Intercellular Transfer of Chromosomal Antimicrobial Resistance Genes between Acinetobacter baumannii Strains Mediated by Prophages

Wachino, J-i., Jin, W., Kimura, K. and Arakawa, Y. Antimicrop. Agents Chemother., 63(8), e00334-19 (2019)   The spread of antimicrobial resistance genes (ARGs) among Gram-negative pathogens, including Acinetobacter baumannii, is primarily mediated by transferable plasmids; however, ARGs are frequently integrated into its chromosome. How ARG gets horizontally incorporated into the chromosome of A. baumannii, and whether it functions as a cause for further spread of ARG, remains unknown. Here, we demonstrated intercellular prophage-mediated transfer of chromosomal ARGs without direct cell-cell interaction in A. baumannii. We prepared ARG-harboring extracellular DNA (eDNA) components from the culture supernatant of a multidrug-resistant (MDR) A. baumannii NU-60 strain and exposed an antimicrobial-susceptible (AS) A. baumannii ATCC 17978 strain to the eDNA components. The antimicrobial-resistant (AR) A. baumannii ATCC 17978 derivatives appeared to acquire various ARGs, originating from dispersed loci of the MDR A. baumannii chromosome, along with their surrounding regions, by homologous recombination, with the ARGs including armA (aminoglycoside resistance), bla_TEM-1 (β-lactam resistance), tet(B) (tetracycline resistance), and gyrA-81L (nalidixic acid resistance) genes. Notably, the eDNAs conferring antimicrobial resistance were enveloped in specific capsid proteins consisting of phage particles, thereby protecting the eDNAs from detergent and DNase treatments. The phages containing ARGs were likely released into the extracellular space from MDR A. baumannii, thereby transducing ARGs into AS A. baumannii, resulting in the acquisition of AR properties by the recipient. We concluded that the generalized transduction, in which phages were capable of carrying random pieces of A. baumannii genomic DNAs, enabled efficacious intercellular transfer of chromosomal ARGs between A. baumannii strains without direct cell-cell interaction.  

3.3337           The structural unit of melanin in the cell wall of the fungal pathogen Cryptococcus neoformans

Camacho, E., Vij, R., Chrissian, C., Prados-Rosales, R., Gil, D., O’Meally, R., Cordero, R.J.B., Cole, R.N., McCaffery, J.M., Stark, R.E. and Casadevali, A.

1. Biol. Chem., 294(27), 10471-10489 (2019)

  Melanins are synthesized macromolecules that are found in all biological kingdoms. These pigments have a myriad of roles that range from microbial virulence to key components of the innate immune response in invertebrates. Melanins also exhibit unique properties with potential applications in physics and material sciences, ranging from electrical batteries to novel therapeutics. In the fungi, melanins, such as eumelanins, are components of the cell wall that provide protection against biotic and abiotic elements. Elucidation of the smallest fungal cell wall-associated melanin unit that serves as a building block is critical to understand the architecture of these polymers, its interaction with surrounding components, and their functional versatility. In this study, we used isopycnic gradient sedimentation, NMR, EPR, high-resolution microscopy, and proteomics to analyze the melanin in the cell wall of the human pathogenic fungus Cryptococcus neoformans. We observed that melanin is assembled into the cryptococcal cell wall in spherical structures ∼200 nm in diameter, termed melanin granules, which are in turn composed of nanospheres ∼30 nm in diameter, termed fungal melanosomes. We noted that melanin granules are closely associated with proteins that may play critical roles in the fungal melanogenesis and the supramolecular structure of this polymer. Using this structural information, we propose a model for C. neoformans' melanization that is similar to the process used in animal melanization and is consistent with the phylogenetic relatedness of the fungal and animal kingdoms.  

3.3338           Golgi-resident TRIO regulates membrane trafficking during neurite outgrowth

Tao, T., Sun, J., Peng, Y., Li, Y., Wang, P., Chen, X., Zhao, W., Zheng, Y-Y., Wei, L., Wang, W., Zhou, Y., Liu, J., Shi, Y.S. and Zhu, M-S.

1. Biol. Chem., 294(28), 10954-10968 (2019)

  Neurite outgrowth requires coordinated cytoskeletal rearrangements in the growth cone and directional membrane delivery from the neuronal soma. As an essential Rho guanine nucleotide exchange factor (GEF), TRIO is necessary for cytoskeletal dynamics during neurite outgrowth, but its participation in the membrane delivery is unclear. Using co-localization studies, live-cell imaging, and fluorescence recovery after photobleaching analysis, along with neurite outgrowth assay and various biochemical approaches, we here report that in mouse cerebellar granule neurons, TRIO protein pools at the Golgi and regulates membrane trafficking by controlling the directional maintenance of both RAB8 (member RAS oncogene family 8)– and RAB10-positive membrane vesicles. We found that the spectrin repeats in Golgi-resident TRIO confer RAB8 and RAB10 activation by interacting with and activating the RAB GEF RABIN8. Constitutively active RAB8 or RAB10 could partially restore the neurite outgrowth of TRIO-deficient cerebellar granule neurons, suggesting that TRIO-regulated membrane trafficking has an important functional role in neurite outgrowth. Our results also suggest cross-talk between Rho GEF and Rab GEF in controlling both cytoskeletal dynamics and membrane trafficking during neuronal development. They further highlight how protein pools localized to specific organelles regulate crucial cellular activities and functions. In conclusion, our findings indicate that TRIO regulates membrane trafficking during neurite outgrowth in coordination with its GEF-dependent function in controlling cytoskeletal dynamics via Rho GTPases.  

3.3339           Large oncosomes overexpressing integrin alpha-V promote prostate cancer adhesion and invasion via AKT activation

Ciardiello, C., Leone, A., Lanuti, P., Roca, M.S., Minciacchi, V.R. et al

1. Exp. Clin. Cancer Res., 38:317 (2019)

  Background Molecular markers for prostate cancer (PCa) are required to improve the early definition of patient outcomes. Atypically large extracellular vesicles (EVs), referred as “Large Oncosomes” (LO), have been identified in highly migratory and invasive PCa cells. We recently developed and characterized the DU145R80 subline, selected from parental DU145 cells as resistant to inhibitors of mevalonate pathway. DU145R80 showed different proteomic profile compared to parental DU145 cells, along with altered cytoskeleton dynamics and a more aggressive phenotype. Methods Immunofluorescence staining and western blotting were used to identify blebbing and EVs protein cargo. EVs, purified by gradient ultra-centrifugations, were analyzed by tunable resistive pulse sensing and multi-parametric flow cytometry approach coupled with high-resolution imaging technologies. LO functional effects were tested in vitro by adhesion and invasion assays and in vivo xenograft model in nude mice. Xenograft and patient tumor tissues were analyzed by immunohistochemistry. Results We found spontaneous blebbing and increased shedding of LO from DU145R80 compared to DU145 cells. LO from DU145R80, compared to those from DU145, carried increased amounts of key-molecules involved in PCa progression including integrin alpha V (αV-integrin). By incubating DU145 cells with DU145R80-derived LO we demonstrated that αV-integrin on LO surface was functionally involved in the increased adhesion and invasion of recipient cells, via AKT. Indeed either the pre-incubation of LO with an αV-integrin blocking antibody, or a specific AKT inhibition in recipient cells are able to revert the LO-induced functional effects. Moreover, DU145R80-derived LO also increased DU145 tumor engraftment in a mice model. Finally, we identified αV-integrin positive LO-like structures in tumor xenografts as well as in PCa patient tissues. Increased αV-integrin tumor expression correlated with high Gleason score and lymph node status. Conclusions Overall, this study is the first to demonstrate the critical role of αV-integrin positive LO in PCa aggressive features, adding new insights in biological function of these large EVs and suggesting their potential use as PCa prognostic markers.  

3.3340           Mesenchymal stromal cell-derived nanovesicles ameliorate bacterial outer membrane vesicle-induced sepsis via IL-10

Park, K-S., Svennerholm, K., Shelke, G.V., Bandeira, E., Lässer, C., Jang, S.C., Chandode, R., Gribonika, I. and Lötval, J. Stem Cell Res. Ther., 10:231 (2019)   Background Sepsis remains a source of high mortality in hospitalized patients despite proper antibiotic approaches. Encouragingly, mesenchymal stromal cells (MSCs) and their produced extracellular vesicles (EVs) have been shown to elicit anti-inflammatory effects in multiple inflammatory conditions including sepsis. However, EVs are generally released from mammalian cells in relatively low amounts, and high-yield isolation of EVs is still challenging due to a complicated procedure. To get over these limitations, vesicles very similar to EVs can be produced by serial extrusions of cells, after which they are called nanovesicles (NVs). We hypothesized that MSC-derived NVs can attenuate the cytokine storm induced by bacterial outer membrane vesicles (OMVs) in mice, and we aimed to elucidate the mechanism involved. Methods NVs were produced from MSCs by the breakdown of cells through serial extrusions and were subsequently floated in a density gradient. Morphology and the number of NVs were analyzed by transmission electron microscopy and nanoparticle tracking analysis. Mice were intraperitoneally injected with Escherichia coli-derived OMVs to establish sepsis, and then injected with 2 × 10⁹ NVs. Innate inflammation was assessed in peritoneal fluid and blood through investigation of infiltration of cells and cytokine production. The biodistribution of NVs labeled with Cy7 dye was analyzed using near-infrared imaging. Results Electron microscopy showed that NVs have a nanometer-size spherical shape and harbor classical EV marker proteins. In mice, NVs inhibited eye exudates and hypothermia, signs of a systemic cytokine storm, induced by intraperitoneal injection of OMVs. Moreover, NVs significantly suppressed cytokine release into the systemic circulation, as well as neutrophil and monocyte infiltration in the peritoneum. The protective effect of NVs was significantly reduced by prior treatment with anti-interleukin (IL)-10 monoclonal antibody. In biodistribution study, NVs spread to the whole mouse body and localized in the lung, liver, and kidney at 6 h. Conclusions Taken together, these data indicate that MSC-derived NVs have beneficial effects in a mouse model of sepsis by upregulating the IL-10 production, suggesting that artificial NVs may be novel EV-mimetics clinically applicable to septic patients.  

3.3341           Extracellular vesicles in type 2 diabetes mellitus: key roles in pathogenesis, complications, and therapy

Xiao, Y., Zheng, L., Zou, X., Wang, j., Zhong, J. and Zhong, T.

1. Extracellular vesicles, 8(1), 1625677 (2019)

  Type 2 diabetes mellitus (T2DM), a chronic disease, is widely prevalent all over the world. In recent years, the roles of some extracellular vesicles (EVs) in T2DM have attracted much attention. EVs are bilayer membrane vesicles secreted from most cells and can participate in regulating various physiological and pathological processes in vivo by being transported between cells. Recently, it was discovered that some abnormal EVs can contribute to the occurrence of T2DM by inducing insulin resistance and can also participate in the complications of T2DM. In addition, some stem/progenitor cells-derived EVs have a potential application in the therapy of T2DM. This review introduces basic concepts of EVs and summarizes the roles of EVs in the pathogenesis, complications, and therapy of T2DM.  

3.3342           Acetylcholinesterase is not a generic marker of extracellular vesicles

Liao, Z., Jaular, L.M., Soueidi, E., Jouve, M., Muth, D.C., Schøyen, T.H., Seale, T., Haughey, N.J., Ostrowski, M., Thery, C. and Witwer, K.W.

1. Extracellular Vesicles, 8(1), 1628592 (2019)

  Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood.  

3.3343           Analysis of the Escherichia coli extracellular vesicle proteome identifies markers of purity and culture conditions

Hong, J., Dauros-Singorenko, P., Whitcombe, A., Payne, L., Blenkiron, C., Phillips, A. and Swift, S.

1. Extracellular Vesicles, 8(1), 1632099 (2019)

  Bacteria release nano-sized extracellular vesicles (EVs) into the extracellular milieu. Bacterial EVs contain molecular cargo originating from the parent bacterium and have important roles in bacterial survival and pathogenesis. Using 8-plex iTRAQ approaches, we profiled the EV proteome of two Escherichia coli strains, uropathogenic (UPEC) 536 and probiotic Nissle 1917. For these strains, we compared the proteome of crude input EVs prepared by ultracentrifugation alone with EVs purified by either density gradient centrifugation (DGC) or size exclusion chromatography (SEC). We further compared the proteome of EVs from bacterial cultures that were grown in iron-restricted (R) and iron-supplemented (RF) conditions. Overall, outer membrane components were highly enriched, and bacterial inner membrane components were significantly depleted in both UPEC and Nissle EVs, in keeping with an outer membrane origin. In addition, we found enrichment of ribosome-related Gene Ontology terms in UPEC EVs and proteins involved in glycolytic processes and ligase activity in Nissle EVs. We have identified that three proteins (RbsB of UPEC in R; YoeA of UPEC in RF; BamA of Nissle in R) were consistently enriched in the DGC- and SEC-purified EV samples in comparison to their crude input EV, whereas conversely the 60 kDa chaperonin GroEL was enriched in the crude input EVs for both UPEC and Nissle in R condition. Such proteins may have utility as technical markers for assessing the purity of E. coli EV preparations. Several proteins were changed in their abundance depending on the iron availability in the media. Data are available via ProteomeXchange with identifier PXD011345. In summary, we have undertaken a comprehensive characterization of the protein content of E. coli EVs and found evidence of specific EV cargos for physiological activity and conserved protein cargo that may find utility as markers in the future.  

3.3344           Refractive index to evaluate staining specificity of extracellular vesicles by flow cytometry

De Rond, L., Libregts, S.F.W.M., Rikkert, L.G., hau, C.M., van der Pol, E., Nieuwland, R., van Leeuwen, T.G. and Coumans, F.A.W.

1. Extracellular Vesicles, 8(1), 1643671 (2019)

  Extracellular vesicles (EVs) in plasma are commonly identified by staining with antibodies and generic dyes, but the specificity of antibodies and dyes to stain EVs is often unknown. Previously, we showed that platelet-depleted platelet concentrate contains two populations of particles >200 nm, one population with a refractive index (RI) < 1.42 that included the majority of EVs, and a second population with an RI > 1.42, which was thought to include lipoproteins. In this study, we investigated whether EVs can be distinguished from lipoproteins by the RI and whether the RI can be used to determine the specificity of antibodies and generic dyes used to stain plasma EVs. EVs and lipoproteins present in platelet-depleted platelet concentrate were separated by density gradient centrifugation. The density fractions were analyzed by Western blot and transmission electron microscopy, the RI of particles was determined by Flow-SR. The RI was used to evaluate the staining specificity of an antibody against platelet glycoprotein IIIa (CD61) and the commonly used generic dyes calcein AM, calcein violet, di-8-ANEPPS, and lactadherin in plasma. After density gradient centrifugation, EV-enriched fractions (1.12 to 1.07 g/mL) contained the highest concentration of particles with an RI < 1.42, and the lipoprotein-enriched fractions (1.04 to 1.03 g/mL) contained the highest concentration of particles with an RI > 1.42. Application of the RI showed that CD61-APC had the highest staining specificity for EVs, followed by lactadherin and calcein violet. Di-8-ANEPPS stained mainly lipoproteins and calcein AM stained neither lipoproteins nor EVs. Taken together, the RI can be used to distinguish EVs and lipoproteins, and thus allows evaluation of the specificity of antibodies and generic dyes to stain EVs.  

3.3345           Targetome analysis of chaperone-mediated autophagy in cancer cells

Hao, Y., Kacal, M., Ouchida, A.T., Zhang, B., Norberg, E. and Vakifahmetoglu-Norberg, H. Autophagy, 15(9), 1558-1571 (2019)   Chaperone-mediated autophagy (CMA) is a lysosomal degradation pathway of select soluble proteins. Nearly one-third of the soluble proteins are predicted to be recognized by this pathway, yet only a minor fraction of this proteome has been identified as CMA substrates in cancer cells. Here, we undertook a quantitative multiplex mass spectrometry approach to study the proteome of isolated lysosomes in cancer cells during CMA-activated conditions. By integrating bioinformatics analyses, we identified and categorized proteins of multiple cellular pathways that were specifically targeted by CMA. Beyond verifying metabolic pathways, we show that multiple components involved in select biological processes, including cellular translation, was specifically targeted for degradation by CMA. In particular, several proteins of the translation initiation complex were identified as bona fide CMA substrates in multiple cancer cell lines of distinct origin and we show that CMA suppresses cellular translation. We further show that the identified CMA substrates display high expression in multiple primary cancers compared to their normal counterparts. Combined, these findings uncover cellular processes affected by CMA and reveal a new role for CMA in the control of translation in cancer cells.  

3.3346           Extracellular vesicles-based drug delivery system for cancer treatment

Balachandran, B. and Yuana, Y. Cogent Med., 6, 1635806 (2019)   The decrease in cancer mortality indicates an improvement in cancer treatment and management. One strategy that has been a focus in cancer treatment is development of drug delivery systems (DDS). Lipid-based nanoparticles (e.g. liposomes or micelles) has been used in current DDS as vehicles to transport active molecules. Extracellular vesicles (EVs), a new player in DDS, consist of lipid and thus, can be categorized as lipid-based nanoparticles. EVs are derived from cells and harbour various targeting molecules from their origin cells. Therefore, EVs are not foreign to the host immune system and may be more effective and efficient than other synthetic nanoparticles to target solid tumours with a minimum adverse effect, providing an exciting alternative for lipid-based DDS. Active molecules can be loaded into EV endogenously by exposing cells with active molecules to generate EVs carrying these molecules, or exogenous loading using physical or chemical methods. In this review, we summarise the recent developments of EV-based DDS where the choice of donor cells, drug cargo, loading methods, and administration routes are discussed. Further, consideration of the bioavailability and biodistribution of EVs, as well as current challenges concerning the potential biosafety issue and standardized up-scale production of EVs are highlighted.  

3.3347           Latest advances in extracellular vesicles: from bench to bedside

Yamamoto, T., Kosaka, N. and Ochiya, T. Science and Technology of Advanced Materials, 20(1), 746-757 (2019)   Extracellular vesicles (EVs) are small membraned vesicles and approximately 50–150 nm in diameter. Almost all of the type of cells releases the EVs and circulates in the body fluids. EVs contain multiple functional components, such as mRNAs, microRNAs (miRNAs), DNAs, and proteins, which can be transferred to the recipient cells, resulting in phenotypic changes. Recently, EV research has focused on their potential as a drug delivery vehicle and in targeted therapy against specific molecules. Moreover, some surface proteins are specific to particular diseases, and therefore, EVs also have promise as biomarkers. In this concise review, we summarize the latest research focused on EVs, which have the potential to become a promising drug delivery method, biomarker, and new therapeutic target for improving the outcomes of cancer patients.  

3.3348           Data on proteomic profiling of cells and extracellular vesicles of the melittin-resistant Acholeplasma laidlawii strain

Medvedeva, E.S., Mouzykantov, A.A., baranova, N.B., Dramchini, M.A., Chernova, O.A. and Chernov, V.M. Data in brief, 25, 104169 (2019)   Acholeplasma laidlawii (class Mollicutes), a major contaminant of cell cultures, quickly adapts to various classes of antimicrobials, including antimicrobial peptides. The extracellular vesicles of this bacterium can play a significant role in the development of drug-resistance Chernov et al., 2018. We compared the cellular and vesicular proteomes of A. laidlawii strains with differing susceptibility to melittin (an antimicrobial peptide from bee venom), the genomes of which we have previously sequenced. We extracted soluble proteins from cells and extracellular vesicles of the A. laidlawii PG8R_Mel strain showing an increased resistance to melittin, and compared them with the cellular proteome and a previously obtained vesicular proteome of the original (reference) A. laidlawii PG8B strain Chernov et al., 2014. The cellular proteome profile of the A. laidlawii strains differing in susceptibility to melittin was determined by using two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. Here we present the cellular proteins that were differentially expressed. The vesicular proteome profile was determined by using one-dimensional electrophoresis and chromatography-mass spectrometry. A list of the extracellular vesicles proteins of the melittin-resistant A. laidlawii strain is presented here.  

3.3349           Ganglioside GM1 promotes contact inhibition of growth by regulating the localization of epidermal growth factor receptor from glycosphingolipid‐enriched microdomain to caveolae

Zhuop, D. and Guan, F. Cell Prolifiration, 52(4), e12639 (2019)   Objectives Accumulating data show that gangliosides are involved in regulation of cell proliferation. Specific changes in gangliosides expression associated with growth density of cells have been documented in several cell lines. However, the function and the potential mechanism of ganglioside GM1 in contact inhibition of growth are not clear. Materials and Methods EdU incorporation assay and western blot were applied to detect the contact inhibition of growth in human mammary epithelial cells. GM1 manipulation of cell proliferation and epidermal growth factor receptor (EGFR) activation was investigated by immunoprecipitation, OptiPrep density gradient centrifugation and immunofluorescence. The function of GM1 on contact inhibition of growth was further studied by using GM1 stably knockdown and overexpression cells. Results MCF‐10A, MCF‐7 and MDA‐MB‐231 cells showed contact inhibition of growth in high‐density condition. Exogenous addition of GM1 to high‐density cells clearly inhibited cell growth and deactivated EGFR signalling. Compared to normal‐density cells, distribution of EGFR in high‐density cells was decreased in glycosphingolipid‐enriched microdomain (GEM), but more concentrated in caveolae, and incubation with GM1 obviously promoted this translocation. Furthermore, the cell growth and EGFR activation were increased in GM1 stably knockdown cells and decreased in GM1 stably overexpression cells when cultured in high density. Conclusions Our results demonstrated that GM1 suppressed EGFR signalling and promoted contact inhibition of growth by changing the localization of EGFR from GEM to caveolae.  

3.3350           Nudt8 is a novel CoA diphosphohydrolase that resides in the mitochondria

Kerr, E.W., Shumar, S.A. and Leonardi, R. FEBS Lett., 593, 1133-1143 (2019)   CoA regulates energy metabolism and exists in separate pools in the cytosol, peroxisomes, and mitochondria. At the whole tissue level, the concentration of CoA changes with the nutritional state by balancing synthesis and degradation; however, it is currently unclear how individual subcellular CoA pools are regulated. Liver and kidney peroxisomes contain Nudt7 and Nudt19, respectively, enzymes that catalyze CoA degradation. We report that Nudt8 is a novel CoA‐degrading enzyme that resides in the mitochondria. Nudt8 has a distinctive preference for manganese ions and exhibits a broader tissue distribution than Nudt7 and Nudt19. The existence of CoA‐degrading enzymes in both peroxisomes and mitochondria suggests that degradation may be a key regulatory mechanism for modulating the intracellular CoA pools.  

3.3351           Isolation and Characterization of Functionally Active Extracellular Vesicles from Culture Medium Conditioned by Bovine Embryos In Vitro

Pavani, K.C., Hendrix, A., Van Den Broeck, W., Couck, L., Szymanska, K., Lin, X., De Koster, J., Soom, A.V. and Leemans, B. Int. J. Mol. Sci., 20(1), 38 (2019)   Extracellular vesicles (EVs) play a possible role in cell–cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrep^TM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25–230 nm with an average concentration of 236.5 ± 1.27 × 10⁸ particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.Extracellular vesicles (EVs) play a possible role in cell–cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrep^TM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25–230 nm with an average concentration of 236.5 ± 1.27 × 10⁸ particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.Extracellular vesicles (EVs) play a possible role in cell–cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrepTM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25–230 nm with an average concentration of 236.5 ± 1.27 × 108 particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.  

3.3352           Abstract 734: Altered Loading of Protein Cargoes in Tissue-Entrapped Human Vascular and Valvular Extracellular Vesicles

Blaser, M.C., Buffolo, F., Higashi, H., Rogers, M., Lee, L.H., Pham, T. et al Arterioscler. Thromb. Vasc. Biol., 39, Suppl. 1, A734 (2019)   Less than half of all patients develop both vascular and valvular calcification, suggesting differential drivers of disease. Extracellular vesicles (EVs) carry bioactive cargoes that can mediate intercellular signaling and act as early nucleation sites of microcalcification. We studied whether tissue-specific alterations of entrapped EV cargoes exist in cardiovascular disease and establish an innovative method to isolate and characterize EVs from human cardiovascular tissues. EVs were isolated from human carotid endarterectomies (CE) and aortic valves with calcific aortic valve disease (CAVD) by enzymatic digestion, ultracentrifugation, and 15-fraction iodixanol density gradient separation. Global label-free proteomics found an abundance of extracellular matrix (ECM) contaminants in EVs extracted by ultracentrifugation alone. After gradient separation, proteomics revealed that EV markers (e.g. CD9, CD63, CD81, flottilin-1, TSG101) were highly enriched in the four least-dense fractions (F1-F4) of vascular and valvular tissues (n=3 CE and 4 CAVD), while ECM was predominant in all denser fractions (F5-15). Immunogold-transmission electron microscopy confirmed CD63+CD81+ membrane-bound EVs in F1-4, with high levels of globular or fibrous collagen in F5-15. From an additional 4 CE and 4 CAVD donors, F1-4 were isolated and pooled per donor. Mass spectrometry sequenced an EV-specific proteome of 2,041 proteins, of which 372 were differentially enriched between CE and CAVD EVs. 28 KEGG pathways were over-represented in CE EVs, highlighted by ECM/focal adhesion interactions, complement activation, and PI3K-Akt signaling. In contrast, only five were enriched in CAVD EVs, where altered mineral absorption, glycolysis, and Hippo signaling pathways were discovered. Nanoparticle tracking analysis found significant increases in the size of CE vs. CAVD EVs (diameter =230.7±3.3 and 193.5±5.1 nm, respectively; p < 0.001), due perhaps to differences in EV biogenesis or aggregation. While activity of alkaline phosphatase per EV was unchanged, total loading was significantly elevated (>50-fold) in CAVD EVs. The altered EV cargoes described herein identify novel divergent pathogenic pathways for vascular vs. valvular calcification.  

3.3353           The exocyst controls lysosome secretion and antigen extraction at the immune synapse of B cells

Saez, J.J., Diaz, J., Ibanez, J., Bozo, J.P., Reyes, F.C., Alamo, M., Gobert, F-X., Obino, D., Bono, M.R., Lennon-Dumenil, A-M., Yeaman, C. and Yuseff, M-I.

1. Cell Biol., 218(7), 2247-2264 (2019)

  B lymphocytes capture antigens from the surface of presenting cells by forming an immune synapse. Local secretion of lysosomes, which are guided to the synaptic membrane by centrosome repositioning, can facilitate the extraction of immobilized antigens. However, the molecular basis underlying their delivery to precise domains of the plasma membrane remains elusive. Here we show that microtubule stabilization, triggered by engagement of the B cell receptor, acts as a cue to release centrosome-associated Exo70, which is redistributed to the immune synapse. This process is coupled to the recruitment and activation of GEF-H1, which is required for assembly of the exocyst complex, used to promote tethering and fusion of lysosomes at the immune synapse. B cells silenced for GEF-H1 or Exo70 display defective lysosome secretion, which results in impaired antigen extraction and presentation. Thus, centrosome repositioning coupled to changes in microtubule stability orchestrates the spatial-temporal distribution of the exocyst complex to promote polarized lysosome secretion at the immune synapse.    

3.3354           Pharmacological Chaperones for the Treatment of α-Mannosidosis

Riszquez-Cuadro, R., Matsumoto, R., Ortega-Cabellero, F., Nanba, E., Higaki, K., Fernandez, J.M.G. and Mellet, C.O.

1. Med. Chem., 62(12), 5832-5843 (2019)

  α-Mannosidosis (AM) results from deficient lysosomal α-mannosidase (LAMAN) activity and subsequent substrate accumulation in the lysosome, leading to severe pathology. Many of the AM-causative mutations compromise enzyme folding and could be rescued with purpose-designed pharmacological chaperones (PCs). We found that PCs combining a LAMAN glycone-binding motif based on the 5N,6O-oxomethylidenemannojirimycin (OMJ) glycomimetic core and different aglycones, in either mono- or multivalent displays, elicit binding modes involving glycone and nonglycone enzyme regions that reinforce the protein folding and stabilization potential. Multivalent derivatives exhibited potent enzyme inhibition that generally prevailed over the chaperone effect. On the contrary, monovalent OMJ derivatives with LAMAN aglycone binding area-fitting substituents proved effective as activity enhancers for several mutant LAMAN forms in AM patient fibroblasts and/or transfected MAN2B1-KO cells. This translated into a significant improvement in endosomal/lysosomal function, reverting not only the primary LAMAN substrate accumulation but also the additional downstream consequences such as cholesterol accumulation.  

3.3355           Urinary exosome as a potential biomarker for urinary tract infection

Mizutani, K., Kawakami, K., Horie, K., Fujita, Y., Kameyama, K., kato, T., Nakane, K., Tsuchiya, T., Yasuda, M., Masunaga, K., Kasuya, Y., Masuda, Y., Deguchi, T., Koie, T. and Ito, M. Cell. Microbiol., 21, e13020 (2019)   Unlike urinary tract infection (UTI), asymptomatic bacteriuria (ABU) should not be treated, with some exceptions such as pregnant women and patients who will undergo traumatic urologic interventions. However, there has been no clinically available marker for their differential diagnosis. Exosomes or small extracellular vesicles carry proteins contained in cells from which they are derived, thus having the potential as a biomarker of several diseases. On the basis of the hypothesis that the molecular signature of exosomes in urine may differ between UTI and ABU patients, we examined if urinary exosomes could serve as a marker for their differential diagnosis. Exosomes were isolated by ultracentrifugation or affinity‐based method from cell culture medium of monocytic THP‐1 and uroepithelial SV‐HUC‐1 cells and human urine. Protein expression was examined by Western blot analysis, ELISA, and CLEIA. The results showed that the levels of intracellular signalling molecules Akt and ERK and transcription factor NF‐κB increased in exosomes isolated from THP‐1 and SV‐HUC‐1 cells cocultured with Escherichia coli and/or treated with lipopolysaccharide. In urinary exosomes of UTI patients, Akt significantly diminished, and an exosomal marker CD9 showed a trend to decrease after treatment with antimicrobial agents. More importantly, Akt and CD9 levels in urinary exosomes were higher in UTI patients than in ABU patients, which was also observed after correction by urine creatinine. Collectively, these results suggest that Akt and CD9 in urinary exosomes could be useful markers for differential diagnosis of UTI and ABU.  

3.3356           Migrasomes provide regional cues for organ morphogenesis during zebrafish gastrulation

Jiang, D., Jiang, Z., Lu, D., Wang, X., Liang, H., Zhang, J. Meng, Y. et al Nature Cell Biol., 21, 966-077 (2019)   Migrasomes are recently identified vesicular organelles that form on retraction fibres behind migrating cells. Whether migrasomes are present in vivo and, if so, the function of migrasomes in living organisms is unknown. Here, we show that migrasomes are formed during zebrafish gastrulation and signalling molecules, such as chemokines, are enriched in migrasomes. We further demonstrate that Tspan4 and Tspan7 are required for migrasome formation. Organ morphogenesis is impaired in zebrafish MZtspan4a and MZtspan7 mutants. Mechanistically, migrasomes are enriched on a cavity underneath the embryonic shield where they serve as chemoattractants to ensure the correct positioning of dorsal forerunner cells vegetally next to the embryonic shield, thereby affecting organ morphogenesis. Our study shows that migrasomes are signalling organelles that provide specific biochemical information to coordinate organ morphogenesis.  

3.3357           Rab18 Collaborates with Rab7 to Modulate Lysosomal and Autophagy Activities in the Nervous System: an Overlapping Mechanism for Warburg Micro Syndrome and Charcot-Marie-Tooth Neuropathy Type 2B

Nian, F-S., Li, L-L., Cheng, C-Y., Wu, P-C., Lin, Y-T., Tang, C-Y., Ren, B-S., Tai, C-Y., Fann, M-J., Kao, L-S., Hong, C-J. and Tsai, J-W. Mol. Neurobiol., 56(9), 6095-6105 (2019)   Mutations in RAB18, a member of small G protein, cause Warburg micro syndrome (WARBM), whose clinical features include vision impairment, postnatal microcephaly, and lower limb spasticity. Previously, our Rab18^−/− mice exhibited hind limb weakness and spasticity as well as signs of axonal degeneration in the spinal cord and lumbar spinal nerves. However, the cellular and molecular function of RAB18 and its roles in the pathogenesis of WARBM are still not fully understood. Using immunofluorescence staining and expression of Rab18 and organelle markers, we find that Rab18 associates with lysosomes and actively traffics along neurites in cultured neurons. Interestingly, Rab18^−/− neurons exhibit impaired lysosomal transport. Using autophagosome marker LC3-II, we show that Rab18 dysfunction leads to aberrant autophagy activities in neurons. Electron microscopy further reveals accumulation of lipofuscin-like granules in the dorsal root ganglion of Rab18^−/− mice. Surprisingly, Rab18 colocalizes, cofractionates, and coprecipitates with the lysosomal regulator Rab7, mutations of which cause Charcot-Marie-Tooth (CMT) neuropathy type 2B. Moreover, Rab7 is upregulated in Rab18-deficient neurons, suggesting a compensatory effect. Together, our results suggest that the functions of RAB18 and RAB7 in lysosomal and autophagic activities may constitute an overlapping mechanism underlying WARBM and CMT pathogenesis in the nervous system.  

3.3358           Chromogranin B regulates early-stage insulin granule trafficking from the Golgi in pancreatic islet β-cells

Bearrows, S., Bauchle, C.J., Becker, M., Haldemann, J.M., Swaminathan, S. and Stephens, S.B.

1. Cell Sci., 132, jcs231373 (2019)

  Chromogranin B (CgB, also known as CHGB) is abundantly expressed in dense core secretory granules of multiple endocrine tissues and has been suggested to regulate granule biogenesis in some cell types, including the pancreatic islet β-cell, though the mechanisms are poorly understood. Here, we demonstrate a critical role for CgB in regulating secretory granule trafficking in the β-cell. Loss of CgB impairs glucose-stimulated insulin secretion, impedes proinsulin processing to yield increased proinsulin content, and alters the density of insulin-containing granules. Using an in situ fluorescent pulse-chase strategy to track nascent proinsulin, we show that loss of CgB impairs Golgi budding of proinsulin-containing secretory granules, resulting in a substantial delay in trafficking of nascent granules to the plasma membrane with an overall decrease in total plasma membrane-associated granules. These studies demonstrate that CgB is necessary for efficient trafficking of secretory proteins into the budding granule, which impacts the availability of insulin-containing secretory granules for exocytic release     Progressive rod-cone degeneration (PRCD) is a small protein residing in the light-sensitive disc membranes of the photoreceptor outer segment. Until now, the function of PRCD has remained enigmatic despite multiple demonstrations that its mutations cause blindness in humans and dogs. Here, we generated a PRCD knockout mouse and observed a striking defect in disc morphogenesis, whereby newly forming discs do not properly flatten. This leads to the budding of disc-derived vesicles, specifically at the site of disc morphogenesis, which accumulate in the interphotoreceptor matrix. The defect in nascent disc flattening only minimally alters the photoreceptor outer segment architecture beyond the site of new disc formation and does not affect the abundance of outer segment proteins and the photoreceptor’s ability to generate responses to light. Interestingly, the retinal pigment epithelium, responsible for normal phagocytosis of shed outer segment material, lacks the capacity to clear the disc-derived vesicles. This deficiency is partially compensated by a unique pattern of microglial migration to the site of disc formation where they actively phagocytize vesicles. However, the microglial response is insufficient to prevent vesicular accumulation and photoreceptors of PRCD knockout mice undergo slow, progressive degeneration. Taken together, these data show that the function of PRCD is to keep evaginating membranes of new discs tightly apposed to each other, which is essential for the high fidelity of photoreceptor disc morphogenesis and photoreceptor survival.  

3.3359           A single-nuclei RNA sequencing study of Mendelian and sporadic AD in the human brain

Del-Aguila, J.L., Li, Z., Dube, U., Mihindukulasuriya, K.A:, Budde, J.P., Fernandez, M.V., Ibanez, L. et al Alzheimer’s Res. Ther., 11:71 (2019)   Background Alzheimer’s disease (AD) is the most common form of dementia. This neurodegenerative disorder is associated with neuronal death and gliosis heavily impacting the cerebral cortex. AD has a substantial but heterogeneous genetic component, presenting both Mendelian and complex genetic architectures. Using bulk RNA-seq from the parietal lobes and deconvolution methods, we previously reported that brains exhibiting different AD genetic architecture exhibit different cellular proportions. Here, we sought to directly investigate AD brain changes in cell proportion and gene expression using single-cell resolution. Methods We generated unsorted single-nuclei RNA sequencing data from brain tissue. We leveraged the tissue donated from a carrier of a Mendelian genetic mutation, PSEN1 p.A79V, and two family members who suffer from sporadic AD, but do not carry any autosomal mutations. We evaluated alternative alignment approaches to maximize the titer of reads, genes, and cells with high quality. In addition, we employed distinct clustering strategies to determine the best approach to identify cell clusters that reveal neuronal and glial cell types and avoid artifacts such as sample and batch effects. We propose an approach to cluster cells that reduces biases and enable further analyses. Results We identified distinct types of neurons, both excitatory and inhibitory, and glial cells, including astrocytes, oligodendrocytes, and microglia, among others. In particular, we identified a reduced proportion of excitatory neurons in the Mendelian mutation carrier, but a similar distribution of inhibitory neurons. Furthermore, we investigated whether single-nuclei RNA-seq from the human brains recapitulate the expression profile of disease-associated microglia (DAM) discovered in mouse models. We also determined that when analyzing human single-nuclei data, it is critical to control for biases introduced by donor-specific expression profiles. Conclusion We propose a collection of best practices to generate a highly detailed molecular cell atlas of highly informative frozen tissue stored in brain banks. Importantly, we have developed a new web application to make this unique single-nuclei molecular atlas publicly available.  

3.3360           CLIC4 regulates late endosomal trafficking and matrix degradation activity of MMP14 at focal adhesions in RPE cells

Hsu, K-S., Otsu, W., Li, Y., Wang, H-S., Chen, S., Tsang, S.H., Chuang, J-Z. and Sung, C-H. Scientific Reports, 9:12247 (82019)   Dysregulation in the extracellular matrix (ECM) microenvironment surrounding the retinal pigment epithelium (RPE) has been implicated in the etiology of proliferative vitreoretinopathy and age-related macular degeneration. The regulation of ECM remodeling by RPE cells is not well understood. We show that membrane-type matrix metalloproteinase 14 (MMP14) is central to ECM degradation at the focal adhesions in human ARPE19 cells. The matrix degradative activity, but not the assembly, of the focal adhesion is regulated by chloride intracellular channel 4 (CLIC4). CLIC4 is co-localized with MMP14 in the late endosome. CLIC4 regulates the proper sorting of MMP14 into the lumen of the late endosome and its proteolytic activation in lipid rafts. CLIC4 has the newly-identified “late domain” motif that binds to MMP14 and to Tsg101, a component of the endosomal sorting complex required for transport (ESCRT) complex. Unlike the late domain mutant CLIC4, wild-type CLIC4 can rescue the late endosomal sorting defect of MMP14. Finally, CLIC4 knockdown inhibits the apical secretion of MMP2 in polarized human RPE monolayers. These results, taken together, demonstrate that CLIC4 is a novel matrix microenvironment modulator and a novel regulator for late endosomal cargo sorting. Moreover, the late endosomal sorting of MMP14 actively regulates its surface activation in RPE cells.  

3.3361           Toll-Like Receptors 2 and 4 Modulate Pulmonary Inflammation and Host Factors Mediated by Outer Membrane Vesicles Derived from Acinetobacter baumannii

Marion, C.R., Lee, J., Sharma, L., Park, K-S., Lee, C., Liu, W., Liu, P., Feng, J., Gho, Y.S. and Cruz, C.S.D. Infect. Immun., 87(9), e00243-19 (2019)   Pneumonia due to Gram-negative bacteria is associated with high mortality. Acinetobacter baumannii is a Gram-negative bacterium that is associated with hospital-acquired and ventilator-associated pneumonia. Bacteria have been described to release outer membrane vesicles (OMVs) that are capable of mediating systemic inflammation. The mechanism by which A. baumannii OMVs mediate inflammation is not fully defined. We sought to investigate the roles that Toll-like receptors (TLRs) play in A. baumannii OMV-mediated pulmonary inflammation. We isolated OMVs from A. baumannii cultures and intranasally introduced the OMVs into mice. Intranasal introduction of A. baumannii OMVs mediated pulmonary inflammation, which is associated with neutrophil recruitment and weight loss. In addition, A. baumannii OMVs increased the release of several chemokines and cytokines in the mouse lungs. The proinflammatory responses were partially inhibited in TLR2- and TLR4-deficient mice compared to those of wild-type mice. This study highlights the important roles of TLRs in A. baumannii OMV-induced pulmonary inflammation in vivo.  

3.3362           Folate-displaying exosome mediated cytosolic delivery of siRNA avoiding endosome trapping

Zheng, Z., Li, Z., Xu, C., Guo, B. and Guo, P.

1. Controlled Release, 311-312, 43-49 (2019)

  Folate (FA) receptor is a cell surface glycoprotein overexpressed on many cancer cells. It is a high affinity ligand for cancer cell targeting. However, delivery of siRNA directly through folate receptor mediated endocytosis for gene silencing has not, if any, been successful in clinical trial. We have reported the application of RNA nanotechnology to construct FA-displaying exosomes for efficient cell targeting, siRNA delivery and cancer regression (Pi et.al Nature Nanotechnology, 2018:13, 82–89; Li et al., Scientific Report, 2018:8, 14,644). However, the mechanism underlying the efficient therapeutic behavior through folate/exosome complex remains elusive. Here we demonstrate that the efficient cancer suppression with the FA-displaying exosome was due to the receptor-mediated cytosol delivery of the siRNA payload without endosome trapping, as attested by fluorescence colocalization analysis, gene knockdown assay and animal tumor regression. It is expected that the high potency of FA-displaying exosome in cytosolic siRNA delivery will renew the concept and interest in using FA as cancer targeting ligand in human cancer therapy.  

3.3363           In vitro fusion of single synaptic and dense core vesicles reproduces key physiological properties

Kreutzbergeer, A.J.B., Kiessling, V., Stroupe, C., Linag, B., Preobraschenski, J., Ganzella, M., Kreutzberger, M.A.B., nakamoto, R., Jan, R., Castle, J.D. and Tamm, L.K. Nature Communications, 10:3904 (2019)   Regulated exocytosis of synaptic vesicles is substantially faster than of endocrine dense core vesicles despite similar molecular machineries. The reasons for this difference are unknown and could be due to different regulatory proteins, different spatial arrangements, different vesicle sizes, or other factors. To address these questions, we take a reconstitution approach and compare regulated SNARE-mediated fusion of purified synaptic and dense core chromaffin and insulin vesicles using a single vesicle-supported membrane fusion assay. In all cases, Munc18 and complexin are required to restrict fusion in the absence of calcium. Calcium triggers fusion of all docked vesicles. Munc13 (C1C2MUN domain) is required for synaptic and enhanced insulin vesicle fusion, but not for chromaffin vesicles, correlating inversely with the presence of CAPS protein on purified vesicles. Striking disparities in calcium-triggered fusion rates are observed, increasing with curvature with time constants 0.23 s (synaptic vesicles), 3.3 s (chromaffin vesicles), and 9.1 s (insulin vesicles) and correlating with rate differences in cells.  

3.3364           TRPM8-androgen receptor association within lipid rafts promotes prostate cancer cell migration

Grolez, G.P., Gordiendko, D.V., Clarisse, M., Hammadi, M., Desruelles, E., Fromont, G., Prevarskaya, N., Slomianny, C. and Gkika, D. Cell Death & Disease, 10:652 (2019)   In prostate carcinogenesis, androgens are known to control the expression of the transient receptor potential melastatin 8 (TRPM8) protein via activation of androgen receptor (AR). Overexpression and/or activity of TRPM8 channel was shown to suppress prostate cancer (PCa) cell migration. Here we report that at certain concentrations androgens facilitate PCa cell migration. We show that underlying mechanism is inhibition of TRPM8 by activated AR which interacts with the channel within lipid rafts microdomains of the plasma membrane. Thus, our study has identified an additional nongenomic mechanism of the TRPM8 channel regulation by androgens that should be taken into account upon the development of novel therapeutic strategies.  

3.3365           Model System for the Formation of Tick-Borne Encephalitis Virus Replication Compartments without Viral RNA Replication

Yau, W-L., Nguyen-Dinh, V., Larsson, E., Lindqvist, R., Överby. A.K.and Lundmark, R.

1. Virol., 93(18), e00292-19 (2019)

  Flavivirus is a positive-sense, single-stranded RNA viral genus, with members causing severe diseases in humans such as tick-borne encephalitis, yellow fever, and dengue fever. Flaviviruses are known to cause remodeling of intracellular membranes into small cavities, where replication of the viral RNA takes place. Nonstructural (NS) proteins are not part of the virus coat and are thought to participate in the formation of these viral replication compartments (RCs). Here, we used tick-borne encephalitis virus (TBEV) as a model for the flaviviruses and developed a stable human cell line in which the expression of NS proteins can be induced without viral RNA replication. The model system described provides a novel and benign tool for studies of the viral components under controlled expression levels. We show that the expression of six NS proteins is sufficient to induce infection-like dilation of the endoplasmic reticulum (ER) and the formation of RC-like membrane invaginations. The NS proteins form a membrane-associated complex in the ER, and electron tomography reveals that the dilated areas of the ER are closely associated with lipid droplets and mitochondria. We propose that the NS proteins drive the remodeling of ER membranes and that viral RNA, RNA replication, viral polymerase, and TBEV structural proteins are not required.  

3.3366           4-cholesten-3-one decreases breast cancer cell viability and alters membrane raft-localized EGFR expression by reducing lipogenesis and enhancing LXR-dependent cholesterol transporters

Elia, J., Carbonnelle, D., Loge, C., Ory, L., Huvelin, J-M., Tannoury, M., Diab-Assaf, M., Petit, K. and Nazih, H. Lipids in Health and Disease, 18:168 (2019)   Background The alteration of lipid metabolism in cancer cells is recognized as one of the most important metabolic hallmarks of cancer. Membrane rafts defined as plasma membrane microdomains enriched in cholesterol and sphingolipids serve as platforms for signaling regulation in cancer. The main purpose of this study was to evaluate the effect of the cholesterol metabolite, 4-cholesten-3-one, on lipid metabolism and membrane raft integrity in two breast cancer cell lines, MCF-7 and MDA-MB-231. Its ability to reduce cell viability and migration has also been investigated. Methods RT-qPCR was performed to evaluate the expression of enzymes involved in lipogenesis and cholesterol synthesis, and ABCG1 and ABCA1 transporters involved in cholesterol efflux. Its effect on cell viability and migration was studied using the MTT assay, the wound healing assay and the Transwell migration assay, respectively. The effect of 4-cholesten-3-one on membrane rafts integrity was investigated by studying the protein expression of flotillin-2, a membrane raft marker, and raft-enriched EGFR by western blot. Results Interestingly, we found that 4-cholesten-3-one treatment decreased mRNA expression of different enzymes including ACC1, FASN, SCD1 and HMGCR. We further demonstrated that 4-cholesten-3-one increased the expression of ABCG1 and ABCA1. We also found that 4-cholesten-3-one decreased the viability of MCF-7 and MDA-MB-231 cells. This effect was neutralized after treatment with LXR inverse agonist or after LXRβ knockdown by siRNA. As a result, we also demonstrated that 4-cholesten-3-one disrupts membrane rafts and cell migration capacity. Conclusion Our results show that 4-cholesten-3-one exerts promising antitumor activity by altering LXR-dependent lipid metabolism in breast cancer cells without increasing lipogenesis.  

3.3367           Receptor tyrosine kinase inhibitor Sunitinib and integrin antagonist peptide HM-3 show similar lipid raft dependent biphasic regulation of tumor angiogenesis and metastasis

Hu, J., Wang, W., Liu, C., Li, M., Nice, E. and Xu, H.

1. Exp. Clin. Cancer Res., 38:381 (2019)

  Background Anti-angiogenesis remains an attractive strategy for cancer therapy. Some anti-angiogenic reagents have bell-shape dose-response curves with higher than the effective doses yielding lower anti-angiogenic effects. In this study, two different types of anti-angiogenic reagents, a receptor tyrosine kinase inhibitor Sunitinib and an integrin antagonist peptide HM-3, were selected and their effects on tumor angiogenesis and metastasis were compared. The involved molecular mechanisms were investigated. Methods The effect of high dose Sunitinib and HM-3 on tumor angiogenesis and metastasis was investigated with two animal models: metastasis of B16F10 cells in syngeneic mice and metastasis of human MDA-MB-231 cells in nude mice. Furthermore, mechanistic studies were performed with cell migration and invasion assays and with biochemical pull-down assays of intracellular RhoGTPases. Distribution of integrin αvβ3, α5β1, VEGFR2 and the complex of integrin αvβ3 and VEGFR2 inside or outside of lipid rafts was detected with lipid raft isolation and Western-blot analysis. Results Both Sunitinib and HM-3 showed a bell-shape dose-response curve on tumor angiogenesis and metastasis in both animal models. The effects of Sunitinib and HM-3 on endothelial cell and tumor cell proliferation and migration were characterized. Activation of intracellular RhoGTPases and actin stress fiber formation in endothelial and cancer cells following Sunitinib and HM-3 treatment correlated with cell migration analysis. Mechanistic studies confirmed that HM-3 and Sunitinib regulated distribution of integrin αvβ3, α5β1, VEGFR2 and αvβ3-VEGFR2 complexes, both inside and outside of the lipid raft regions to regulate endothelial cell migration and intracellular RhoGTPase activities. Conclusions These data confirmed that a general non-linear dose-effect relationship for these anti-angiogenic drugs exists and their mechanisms are correlative. It also suggests that the effective dose of an anti-angiogenic drug may have to be strictly defined to achieve its optimal clinical effects.  

3.3368           Magnesium transporter 1 (MAGT1) deficiency causes selective defects in N-linked glycosylation and expression of immune-response genes

Matsuda-Lennikov, M., Biancalana, M., Zou, J., Ravell, J.C., Zheng, L., Kanellopoulou, C., Jiang, P., Notarangelo, G., Jing, H., Masutani, E., Oler, A.J., Olano, L.R., Schulz, B.L. and Lenardo, M.J.

1. Biol. Chem., 294(37), 13638-13656 (2019)

  Magnesium transporter 1 (MAGT1) critically mediates magnesium homeostasis in eukaryotes and is highly-conserved across different evolutionary branches. In humans, loss–of–function mutations in the MAGT1 gene cause X-linked magnesium deficiency with Epstein-Barr virus (EBV) infection and neoplasia (XMEN), a disease that has a broad range of clinical and immunological consequences. We have previously shown that EBV susceptibility in XMEN is associated with defective expression of the antiviral natural-killer group 2 member D (NKG2D) protein and abnormal Mg²⁺ transport. New evidence suggests that MAGT1 is the human homolog of the yeast OST3/OST6 proteins that form an integral part of the N-linked glycosylation complex, although the exact contributions of these perturbations in the glycosylation pathway to disease pathogenesis are still unknown. Using MS-based glycoproteomics, along with CRISPR/Cas9-KO cell lines, natural killer cell-killing assays, and RNA-Seq experiments, we now demonstrate that humans lacking functional MAGT1 have a selective deficiency in both immune and nonimmune glycoproteins, and we identified several critical glycosylation defects in important immune-response proteins and in the expression of genes involved in immunity, particularly CD28. We show that MAGT1 function is partly interchangeable with that of the paralog protein tumor-suppressor candidate 3 (TUSC3) but that each protein has a different tissue distribution in humans. We observed that MAGT1-dependent glycosylation is sensitive to Mg²⁺ levels and that reduced Mg²⁺ impairs immune-cell function via the loss of specific glycoproteins. Our findings reveal that defects in protein glycosylation and gene expression underlie immune defects in an inherited disease due to MAGT1 deficiency.    

3.3369           Mitochondrial cysteinyl-tRNA synthetase is expressed via alternative transcriptional initiation regulated by energy metabolism in yeast cells

Nishimura,A., Nasuno, R., Yoshiwaka, Y., Jung, M., Ida, T., Matsunaga, T., Morita, M., Takagi, H., Motohashi, H. and Akaike, T.

1. Biol. Chem., 294(37), 13781-13788 (2019)

  Eukaryotes typically utilize two distinct aminoacyl-tRNA synthetase isoforms, one for cytosolic and one for mitochondrial protein synthesis. However, the genome of budding yeast (Saccharomyces cerevisiae) contains only one cysteinyl-tRNA synthetase gene (YNL247W, also known as CRS1). In this study, we report that CRS1 encodes both cytosolic and mitochondrial isoforms. The 5′ complementary DNA end method and GFP reporter gene analyses indicated that yeast CRS1 expression yields two classes of mRNAs through alternative transcription starts: a long mRNA containing a mitochondrial targeting sequence and a short mRNA lacking this targeting sequence. We found that the mitochondrial Crs1 is the product of translation from the first initiation AUG codon on the long mRNA, whereas the cytosolic Crs1 is produced from the second in-frame AUG codon on the short mRNA. Genetic analysis and a ChIP assay revealed that the transcription factor heme activator protein (Hap) complex, which is involved in mitochondrial biogenesis, determines the transcription start sites of the CRS1 gene. We also noted that Hap complex–dependent initiation is regulated according to the needs of mitochondrial energy production. The results of our study indicate energy-dependent initiation of alternative transcription of CRS1 that results in production of two Crs1 isoforms, a finding that suggests Crs1's potential involvement in mitochondrial energy metabolism in yeast.  

3.3370           Mitochondrial fusion is required for regulation of mitochondrial DNA replication

Ramos, E.S., Motori, E., Brüser, C., Kühl, I., Yeroslaviz, A., Ruzzenenete, B., Kauppila, J.H.K., Buschm, J.D., Hultenby, K., Habermann, B.H., Jacobs, S., Larsson, N-G. and Mourier, A. PloS Genetics, 15(6), e1008085 (2019)   Mitochondrial dynamics is an essential physiological process controlling mitochondrial content mixing and mobility to ensure proper function and localization of mitochondria at intracellular sites of high-energy demand. Intriguingly, for yet unknown reasons, severe impairment of mitochondrial fusion drastically affects mtDNA copy number. To decipher the link between mitochondrial dynamics and mtDNA maintenance, we studied mouse embryonic fibroblasts (MEFs) and mouse cardiomyocytes with disruption of mitochondrial fusion. Super-resolution microscopy revealed that loss of outer mitochondrial membrane (OMM) fusion, but not inner mitochondrial membrane (IMM) fusion, leads to nucleoid clustering. Remarkably, fluorescence in situ hybridization (FISH), bromouridine labeling in MEFs and assessment of mitochondrial transcription in tissue homogenates revealed that abolished OMM fusion does not affect transcription. Furthermore, the profound mtDNA depletion in mouse hearts lacking OMM fusion is not caused by defective integrity or increased mutagenesis of mtDNA, but instead we show that mitochondrial fusion is necessary to maintain the stoichiometry of the protein components of the mtDNA replisome. OMM fusion is necessary for proliferating MEFs to recover from mtDNA depletion and for the marked increase of mtDNA copy number during postnatal heart development. Our findings thus link OMM fusion to replication and distribution of mtDNA.  

3.3371           Mitochondrial protein enriched extracellular vesicles discovered in human melanoma tissues can be detected in patient plasma

Jang, S.C., Crescitelli, R., Cvjetkovic, A., Belgrano, V., Bagge, R.O., Sundfeldt, K., Ochiya, T., Kalluri, R. and Lötvall, J.

1. Extracellular Vesicles, 8(1), 1635420 (2019)

  Extracellular vesicles (EVs), including exosomes and microvesicles, are secreted from all cells, and convey messages between cells in health and disease. However, the diversity of EV subpopulations is only beginning to be explored. Since EVs have been implicated in tumour microenvironmental communication, we started to determine the diversity of EVs specifically in this tissue. To do this, we isolated EVs directly from patient melanoma metastatic tissues. Using EV membrane isolation and mass spectrometry analysis, we discovered enrichment of mitochondrial membrane proteins in the melanoma tissue-derived EVs, compared to non-melanoma-derived EVs. Interestingly, two mitochondrial inner membrane proteins MT-CO2 (encoded by the mitochondrial genome) and COX6c (encoded by the nuclear genome) were highly prevalent in the plasma of melanoma patients, as well as in ovarian and breast cancer patients. Furthermore, this subpopulation of EVs contains active mitochondrial enzymes. In summary, tumour tissues are enriched in EVs with mitochondrial membrane proteins and these mitochondrial membrane proteins can be detected in plasma and are increased in melanoma, ovarian cancer as well as breast cancer.  

3.3372           DNA analysis of low- and high-density fractions defines heterogeneous subpopulations of small extracellular vesicles based on their DNA cargo and topology

Lazaro-Ibanez, E., Lässer, C., Shelke, G.VV., Crescitelli, R., Jang, S.C., Cvjetkovic, A., Garcia-Rodriguez, A. and Lötvall, J.

1. Extracellular Vesicles, 8(1), 1656993 (2019)

  Extracellular vesicles have the capacity to transfer lipids, proteins, and nucleic acids between cells, thereby influencing the recipient cell’s phenotype. While the role of RNAs in EVs has been extensively studied, the function of DNA remains elusive. Here, we distinguished novel heterogeneous subpopulations of small extracellular vesicles (sEVs) based on their DNA content and topology. Low- and high-density sEV subsets from a human mast cell line (HMC-1) and an erythroleukemic cell line (TF-1) were separated using high-resolution iodixanol density gradients to discriminate the nature of the DNA cargo of the sEVs. Paired comparisons of the sEV-associated DNA and RNA molecules showed that RNA was more abundant than DNA and that most of the DNA was present in the high-density fractions, demonstrating that sEV subpopulations have different DNA content. DNA was predominately localised on the outside or surface of sEVs, with only a small portion being protected from enzymatic degradation. Whole-genome sequencing identified DNA fragments spanning all chromosomes and mitochondrial DNA when sEVs were analysed in bulk. Our work contributes to the understanding of how DNA is associated with sEVs and thus provides direction for distinguishing subtypes of EVs based on their DNA cargo and topology.  

3.3373           Answers to naysayers regarding microbial extracellular vesicles

Coelho, C. and Casadevall, A. Biochem. Soc. Trans., 47, 1005-1012 (2019)   It is now over 30 years since the discovery of extracellular vesicles (EVs) in Gram-negative bacteria. However, for cell-walled microbes such as fungi, mycobacteria and Gram-positive bacteria it was thought that EV release would be impossible, since such structures were not believed to cross the thick cell wall. This notion was disproven 10 years ago with the discovery of EVs in fungi, mycobacteria, and gram-positive bacteria. Today, EVs have been described in practically every species tested, ranging from Fungi through Bacteria and Archaea, suggesting that EVs are a feature of every living cell. However, there continues to be skepticism in some quarters regarding EV release and their biological significance. In this review, we list doubts that have been verbalized to us and provide answers to counter them. In our opinion, there is no doubt as to existence and physiological function of EVs and we take this opportunity to highlight the most pressing topics in our understanding of the biological processes underlying these structures.  

3.3374           Hypoxia induces rapid, STAT3 and ROS dependent, mitochondrial translocation of RelA(p65) and IκBα

Ivanova, I.G. and Perkins, N.D. Bioscience Reports, 39, BSR20192101 (2019)   The nuclear factor-κB (NF-κB) family of transcription factors can directly or indirectly regulate many important areas of biology, including immunity, inflammation and cell survival. One intriguing aspect of NF-κB crosstalk with other cell signalling pathways is its regulation of mitochondrial biology, including biogenesis, metabolism and apoptosis. In addition to regulating the expression of mitochondrial genes encoded in the nucleus, NF-κB signalling components are also found within mitochondria themselves and associated with mitochondrial DNA. However, complete biochemical analysis of mitochondrial and sub-mitochondrial localisation of all NF-κB subunits has not been undertaken. Here, we show that only the RelA NF-κB subunit and its inhibitor IκBα reside within mitochondria, whilst p50 is found in the endoplasmic reticulum (ER). Fractionation of mitochondria revealed that only RelA was found in the mitoplast, the location of the mtDNA. We demonstrate that hypoxia leads to a very rapid but transient accumulation of RelA and IκBα in mitochondria. This effect required reactive oxygen species (ROS) but was not dependent on the hypoxia sensing transcription factor subunit HIF1α or intracellular Ca²⁺ release. We also observed rapid mitochondrial localisation of transcription factor STAT3 following hypoxia. Inhibition of STAT3 blocked RelA and IκBα mitochondrial localisation revealing a previously unknown aspect of crosstalk between these key cellular regulators.  

3.3375           Glioblastoma-Associated Microglia Reprogramming Is Mediated by Functional Transfer of Extracellular miR-21

Abels, E.R., Maas, S.L.N., Nieland, L., Krichevsky, A.M., Broekman, M.L.D. and Breakfield, X.O

Cell Reports, 28, 3105-3119 (2019)   Gliomas are primary, diffusely infiltrating brain tumors. Microglia are innate immune cells in the CNS and make up a substantial portion of the tumor mass. Glioma cells shape their microenvironment, communicating with and reprogramming surrounding cells, resulting in enhanced angiogenesis, immune suppression, and remodeling of the extracellular matrix. Glioma cells communicate with microglia, in part by releasing extracellular vesicles (EVs). Mouse glioma cells stably expressing a palmitoylated GFP to label EVs were implanted intracranially into syngeneic miR-21-null mice. Here, we demonstrate functional delivery of miR-21, regulating specific downstream mRNA targets in microglia after uptake of tumor-derived EVs. These findings attest to EV-dependent microRNA delivery as studied in an in vivo-based model and provide insight into the reprograming of microglial cells by tumor cells to create a favorable microenvironment for cancer progression.  

3.3376           Lapatinib-induced annexin A6 upregulation as an adaptive response of triple-negative breast cancer cells to EGFR tyrosine kinase inhibitors

Widatalla, S.E., Korolkova, O.Y., Whalen, D.S., Goodwin, J.S., Williams, K.P., Ochieng, J. and Sakwe, A.M. Carcinogenesis, 40(8), 998-1009 (2019)   The epidermal growth factor receptor (EGFR) is a major oncogene in triple-negative breast cancer (TNBC), but the use of EGFR-targeted tyrosine kinase inhibitors (TKI) and therapeutic monoclonal antibodies is associated with poor response and acquired resistance. Understanding the basis for the acquired resistance to these drugs and identifying biomarkers to monitor the ensuing resistance remain a major challenge. We previously showed that reduced expression of annexin A6 (AnxA6), a calcium-dependent membrane-binding tumor suppressor, not only promoted the internalization and degradation of activated EGFR but also sensitized TNBC cells to EGFR-TKIs. Here, we demonstrate that prolong (>3 days) treatment of AnxA6-low TNBC cells with lapatinib led to AnxA6 upregulation and accumulation of cholesterol in late endosomes. Basal extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation was EGFR independent and significantly higher in lapatinib-resistant MDA-MB-468 (LAP-R) cells. These cells were more sensitive to cholesterol depletion than untreated control cells. Inhibition of lapatinib-induced upregulation of AnxA6 by RNA interference (A6sh) or withdrawal lapatinib from LAP-R cells not only reversed the accumulation of cholesterol in late endosomes but also led to enrichment of plasma membranes with cholesterol, restored EGFR-dependent activation of ERK1/2 and sensitized the cells to lapatinib. These data suggest that lapatinib-induced AnxA6 expression and accumulation of cholesterol in late endosomes constitute an adaptive mechanism for EGFR-expressing TNBC cells to overcome prolong treatment with EGFR-targeted TKIs and can be exploited as an option to inhibit and/or monitor the frequently observed acquired resistance to these drugs.  

3.3377           Multiple BACE1 inhibitors abnormally increase the BACE1 protein level in neurons by prolonging its half-life

Liu, L., Lauro, B.M., Ding, l., Rovere, M., Wolfe, M.S., and Selkoe, D.J. Alzheimer’s & Dementia, 15, 1183-1194 (2019)   Introduction There is keen interest in elucidating the biological mechanisms underlying recent failures of β-site amyloid precursor protein–cleaving enzyme-1 (BACE1) inhibitors in Alzheimer's disease trials. Methods We developed a highly sensitive and specific immunoassay for BACE1 in cell lines and iPSC-derived human neurons to systematically analyze the effects of eight clinically relevant BACE1 inhibitors. Results Seven of 8 inhibitors elevated BACE1 protein levels. Among protease inhibitors tested, the elevation was specific to BACE1 inhibitors. The inhibitors did not increase BACE1 transcription but extended the protein's half-life. BACE1 became elevated at concentrations below the IC₅₀ for amyloid β (Aβ). Discussion Elevation of BACE1 by 7 of 8 BACE1 inhibitors raises new concerns about advancing such β-secretase inhibitors for AD. Chronic elevation could lead to intermittently uninhibited BACE1 when orally dosed inhibitors reach trough levels, abnormally increasing substrate processing. Compounds such as roburic acid that lower Aβ by dissociating β/γ secretase complexes are better candidates because they neither inhibit β- and γ-secretase nor increase BACE1 levels.  

3.3378           Stem cell derived exosomes: microRNA therapy for age-related musculoskeletal disorders

Yao, X., Wei, W., Wang, X., Chenglin, L., Björklund, M. and Ouyang, H. Biomaterials, 224, 119492 (2019)   Age-associated musculoskeletal disorders (MSDs) have been historically overlooked by mainstream biopharmaceutical researchers. However, it has now been recognized that stem and progenitor cells confer innate healing capacity for the musculoskeletal system. Current evidence indicates that exosomes are particularly important in this process as they can mediate sequential and reciprocal interactions between cells to initiate and enhance healing. The present review focuses on stem cells (SCs) derived exosomes as a regenerative therapy for treatment of musculoskeletal disorders. We discuss mechanisms involving exosome-mediated transfer of RNAs and how these have been demonstrated in vitro and in vivo to affect signal transduction pathways in target cells. We envision that standardized protocols for stem cell culture as well as for the isolation and characterization of exosomes enable GMP-compliant large-scale production of SCs-derived exosomes. Hence, potential new treatment for age-related degenerative diseases can be seen in the horizon.  

3.3379           Characterization of artificially re-pigmented ARPE-19 retinal pigment epithelial cell model

Hellinen, L., Hagström, M., Knuutila, H., Ruponen, M., Urtti, A. and Reinisalo, M. Scientific Reports, 9:13761 (2019)   Melanin pigment has a significant role in ocular pharmacokinetics, because many drugs bind at high extent to melanin in the retinal pigment epithelial cells. Most retinal pigment epithelial cell lines lack pigmentation and, therefore, we re-pigmented human ARPE-19 cells to generate a pigmented cell model. Melanosomes from porcine retinal pigment epithelium were isolated and co-incubated with ARPE-19 cells that spontaneously phagocytosed the melanosomes. Internalized melanosomes were functionally integrated to the cellular system as evidenced by correct translocation of cellular Rab27a protein to the melanosomal membranes. The pigmentation was retained during cell cultivation and the level of pigmentation can be controlled by altering the amount of administered melanosomes. We used these cells to study melanosomal uptake of six drugs. The uptake was negligible with low melanin-binders (methotrexate, diclofenac) whereas most of the high melanin-binders (propranolol, chloroquine) were extensively taken up by the melanosomes. This cell line can be used to model pigmentation of the retinal pigment epithelium, while maintaining the beneficial cell line characteristics, such as fast generation of cultures, low cost, long-term maintenance and good reproducibility. The model enables studies at normal and decreased levels of pigmentation to model different retinal conditions.  

3.3380           CAR exosomes derived from effector CAR-T cells have potent antitumour effects and low toxicity

Fu, W., Lei, C., Liu, S., Cui, Y., Wang, C., Qian, K., Li, T., Shen, Y., Fan, X., Lin, F., Ding, M., Pan, M., Ye, X., Yang, Y. and Hu, S. Nature Communications, 10:4355 (2019)   Genetically engineered T cells expressing a chimeric antigen receptor (CAR) are rapidly emerging a promising new treatment for haematological and non-haematological malignancies. CAR-T therapy can induce rapid and durable clinical responses but is associated with unique acute toxicities. Moreover, CAR-T cells are vulnerable to immunosuppressive mechanisms. Here, we report that CAR-T cells release extracellular vesicles, mostly in the form of exosomes that carry CAR on their surface. The CAR-containing exosomes express a high level of cytotoxic molecules and inhibit tumour growth. Compared with CAR-T cells, CAR exosomes do not express Programmed cell Death protein 1 (PD1), and their antitumour effect cannot be weakened by recombinant PD-L1 treatment. In a preclinical in vivo model of cytokine release syndrome, the administration of CAR exosomes is relatively safe compared with CAR-T therapy. This study supports the use of exosomes as biomimetic nanovesicles that may be useful in future therapeutic approaches against tumours.  

3.3381           Endosomal signalling via exosome surface TGFβ-1

Shelke, G.V., Yin, Y., Jang, S.C., Lässer, C., Wennmalm, S., Hoffmann, H.J., Li, L., Gho, Y.S., Nilsson, J.A. and Lötvall, J.

1. Extracellular Vesicles, 8(1), 1650458 (2019)

  Extracellular vesicles such as exosomes convey biological messages between cells, either by surface-to-surface interaction or by shuttling of bioactive molecules to a recipient cell’s cytoplasm. Here we show that exosomes released by mast cells harbour both active and latent transforming growth factor β-1 (TGFβ-1) on their surfaces. The latent form of TGFβ-1 is associated with the exosomes via heparinase-II and pH-sensitive elements. These vesicles traffic to the endocytic compartment of recipient human mesenchymal stem cells (MSCs) within 60 min of exposure. Further, the exosomes-associated TGFβ-1 is retained within the endosomal compartments at the time of signalling, which results in prolonged cellular signalling compared to free-TGFβ-1. These exosomes induce a migratory phenotype in primary MSCs involving SMAD-dependent pathways. Our results show that mast cell-derived exosomes are decorated with latent TGFβ-1 and are retained in recipient MSC endosomes, influencing recipient cell migratory phenotype. We conclude that exosomes can convey signalling within endosomes by delivering bioactive surface ligands to this intracellular compartment.  

3.3382           Targeted Activation of Cystic Fibrosis Transmembrane Conductance Regulator

Villamizar, O., Waters, S.A., Scott, T., Saayman, S., Grepo, N., Urak, R., Davis, A., Jaffe, A. and Morris, K.V. Molecular Therapy, 27(10), 1737-1748 (2019)   Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The majority of CFTR mutations result in impaired chloride channel function as only a fraction of the mutated CFTR reaches the plasma membrane. The development of a therapeutic approach that facilitates increased cell-surface expression of CFTR could prove clinically relevant. Here, we evaluate and contrast two molecular approaches to activate CFTR expression. We find that an RNA-guided nuclease null Cas9 (dCas9) fused with a tripartite activator, VP64-p65-Rta can activate endogenous CFTR in cultured human nasal epithelial cells from CF patients. We also find that targeting BGas, a long non-coding RNA involved in transcriptionally modulating CFTR expression with a gapmer, induced both strong knockdown of BGas and concordant activation of CFTR. Notably, the gapmer can be delivered to target cells when generated as electrostatic particles with recombinant HIV-Tat cell penetrating peptide (CPP), when packaged into exosomes, or when loaded into lipid nanoparticles (LNPs). Treatment of patient-derived human nasal epithelial cells containing F508del with gapmer-CPP, gapmer-exosomes, or LNPs resulted in increased expression and function of CFTR. Collectively, these observations suggest that CRISPR/dCas-VPR (CRISPR) and BGas-gapmer approaches can target and specifically activate CFTR.  

3.3383           Changes in the microsomal proteome of tomato fruit during ripening

Pontiggia, D., Spinelli, F., Fabbri, C., Licursi, V., Negri, R., De Lorenzo, G. and Mattei, B. Scientific Reports, 9:14350 (2019)   The variations in the membrane proteome of tomato fruit pericarp during ripening have been investigated by mass spectrometry-based label-free proteomics. Mature green (MG30) and red ripe (R45) stages were chosen because they are pivotal in the ripening process: MG30 corresponds to the end of cellular expansion, when fruit growth has stopped and fruit starts ripening, whereas R45 corresponds to the mature fruit. Protein patterns were markedly different: among the 1315 proteins identified with at least two unique peptides, 145 significantly varied in abundance in the process of fruit ripening. The subcellular and biochemical fractionation resulted in GO term enrichment for organelle proteins in our dataset, and allowed the detection of low-abundance proteins that were not detected in previous proteomic studies on tomato fruits. Functional annotation showed that the largest proportion of identified proteins were involved in cell wall metabolism, vesicle-mediated transport, hormone biosynthesis, secondary metabolism, lipid metabolism, protein synthesis and degradation, carbohydrate metabolic processes, signalling and response to stress.  

3.3384           Transient Receptor Potential V Channels Are Essential for Glucose Sensing by Aldolase and AMPK

Li, M., Zhang, C-S., Zong, Y., Xie, X-S., Hardie, D.G. and Lin, S.C. Cell Metabolism, 30, 508-524 (2019)   Fructose-1,6-bisphosphate (FBP) aldolase links sensing of declining glucose availability to AMPK activation via the lysosomal pathway. However, how aldolase transmits lack of occupancy by FBP to AMPK activation remains unclear. Here, we show that FBP-unoccupied aldolase interacts with and inhibits endoplasmic reticulum (ER)-localized transient receptor potential channel subfamily V, inhibiting calcium release in low glucose. The decrease of calcium at contact sites between ER and lysosome renders the inhibited TRPV accessible to bind the lysosomal v-ATPase that then recruits AXIN:LKB1 to activate AMPK independently of AMP. Genetic depletion of TRPVs blocks glucose starvation-induced AMPK activation in cells and liver of mice, and in nematodes, indicative of physical requirement of TRPVs. Pharmacological inhibition of TRPVs activates AMPK and elevates NAD⁺ levels in aged muscles, rejuvenating the animals’ running capacity. Our study elucidates that TRPVs relay the FBP-free status of aldolase to the reconfiguration of v-ATPase, leading to AMPK activation in low glucose.  

3.3385           Proteomic analysis of Escherichia coli detergent-resistant membranes (DRM)

Guzman-Flores, J.E., Steinemann-Hernandez, L., Gonzalez de la Vara, L.E., Gavilanes-Ruiz, M., Romeo, T., Alvarez, A.F. and Georgelis, D. PloS One, 14(10), e0223794 (2019)   Membrane microdomains or lipid rafts compartmentalize cellular processes by laterally organizing membrane components. Such sub-membrane structures were mainly described in eukaryotic cells, but, recently, also in bacteria. Here, the protein content of lipid rafts in Escherichia coli was explored by mass spectrometry analyses of Detergent Resistant Membranes (DRM). We report that at least three of the four E. coli flotillin homologous proteins were found to reside in DRM, along with 77 more proteins. Moreover, the proteomic data were validated by subcellular localization, using immunoblot assays and fluorescence microscopy of selected proteins. Our results confirm the existence of lipid raft-like microdomains in the inner membrane of E. coli and represent the first comprehensive profiling of proteins in these bacterial membrane platforms.  

3.3386           Cancer-derived small extracellular vesicles promote angiogenesis by heparin-bound, bevacizumab-insensitive VEGF, independent of vesicle uptake

Ko, S.Y., Lee, W., Kenny, H.A., Dang, L.H., Ellis, L.M., Johasch, E., Lengyel, E. and Naora, H. Communications Biology, 2:386 (2019)   Cancer-derived small extracellular vesicles (sEVs) induce stromal cells to become permissive for tumor growth. However, it is unclear whether this induction solely occurs through transfer of vesicular cargo into recipient cells. Here we show that cancer-derived sEVs can stimulate endothelial cell migration and tube formation independently of uptake. These responses were mediated by the 189 amino acid isoform of vascular endothelial growth factor (VEGF) on the surface of sEVs. Unlike other common VEGF isoforms, VEGF₁₈₉ preferentially localized to sEVs through its high affinity for heparin. Interaction of VEGF₁₈₉ with the surface of sEVs profoundly increased ligand half-life and reduced its recognition by the therapeutic VEGF antibody bevacizumab. sEV-associated VEGF (sEV-VEGF) stimulated tumor xenograft growth but was not neutralized by bevacizumab. Furthermore, high levels of sEV-VEGF were associated with disease progression in bevacizumab-treated cancer patients, raising the possibility that resistance to bevacizumab might stem in part from elevated levels of sEV-VEGF.  

3.3387           PKM2 regulates endothelial cell junction dynamics and angiogenesis via ATP production

Gomez-Escudero, J., Clemente, C., Garcia-Weber, D., Acin-Perez, R., Millan, j., Enriquez, J.A., Bentley, K., Carmeliet, P. and Arroyo, A.G. Scientific Reports, 9:15022 (2019)   Angiogenesis, the formation of new blood vessels from pre-existing ones, occurs in pathophysiological contexts such as wound healing, cancer, and chronic inflammatory disease. During sprouting angiogenesis, endothelial tip and stalk cells coordinately remodel their cell-cell junctions to allow collective migration and extension of the sprout while maintaining barrier integrity. All these processes require energy, and the predominant ATP generation route in endothelial cells is glycolysis. However, it remains unclear how ATP reaches the plasma membrane and intercellular junctions. In this study, we demonstrate that the glycolytic enzyme pyruvate kinase 2 (PKM2) is required for sprouting angiogenesis in vitro and in vivo through the regulation of endothelial cell-junction dynamics and collective migration. We show that PKM2-silencing decreases ATP required for proper VE-cadherin internalization/traffic at endothelial cell-cell junctions. Our study provides fresh insight into the role of ATP subcellular compartmentalization in endothelial cells during angiogenesis. Since manipulation of EC glycolysis constitutes a potential therapeutic intervention route, particularly in tumors and chronic inflammatory disease, these findings may help to refine the targeting of endothelial glycolytic activity in disease.  

3.3388           Muscle-Derived Extracellular Vesicles Influence Motor Neuron Regeneration Accuracy

Madison, R.D. and Robinson, G.A. Neurosci., 419, 46-59 (2019)   Extracellular vesicles are lipid bilayer-enclosed extracellular structures. Although the term extracellular vesicles is quite inclusive, it generally refers to exosomes (< 200 nm), and microvesicles (~ 100–1000 nm). Such vesicles are resistant to degradation and can contain proteins, lipids, and nucleic acids. Although it was previously thought that the primary purpose of such vesicles was to rid cells of unwanted components, it is now becoming increasingly clear that they can function as intercellular messengers, sometimes operating over long distances. As such, there is now intense interest in extracellular vesicles in fields as diverse as immunology, cell biology, cancer, and more recently, neuroscience. The influence that such extracellular vesicles might exert on peripheral nerve regeneration is just beginning to be investigated. In the current studies we show that muscle-derived extracellular vesicles significantly influence the anatomical accuracy of motor neuron regeneration in the rat femoral nerve. These findings suggest a basic cellular mechanism by which target end-organs could guide their own reinnervation following nerve injury.  

3.3389           PKCε-dependent H-Ras activation encompasses the recruitment of the RasGEF SOS1 and of the RasGAP neurofibromin in the lipid rafts of embryonic neurons

Karouzaki, S., Peta, C., Tsirimonaki, T. and Mangoura, D. Neurochem. Int., 131, 104582 (2019)   The spatial organization of plasma membrane proteins is a key factor in the generation of distinct signal outputs, especially for PKC/Ras/ERK signalling. Regulation of activation of the membrane-bound Ras, critical for neuronal differentiation and highly specialized functions, is controlled by exchanges in nucleotides catalyzed by nucleotide exchange factors (GEFs) for GTP loading and Ras activation, and by Ras GTPase Activated Proteins (RasGAPs) that lead to activation of the intrinsic GTPase activity of Ras and thus its inactivation. PKCs are potent Ras activators yet the mechanistic details of these interactions, or the involvement of specific PKC isoforms are now beginning to be addressed. Even less known is the topology where RasGAPs terminate Ras activation. Towards this aim, we isolated lipid rafts from chick embryo neural tissue and primary neuronal cultures when PKCε is the prominent isoform and in combination with in vitro kinase assays, we now show that, in response the PKCε-specific activating peptide ψεRACK, an activated PKCε is recruited to lipid rafts; similar mobility was established when PKCε was physiologically activated with the Cannabinoid receptor 1 (CB1) agonist methanandamide. Activation of H-Ras for both agents was then established for the first time using in vivo RasGAP activity assays, which showed similar temporal profiles of activation and lateral mobility. Moreover, we found that the GEF SOS1, and the major neuronal RasGAP neurofibromin, a specific PKCε substrate, were both transiently significantly enriched in the rafts. Finally, our in silico analysis revealed a highly probable, conserved palmitoylation site adjacent to a CARC motif on neurofibromin, both of which are included only in the RasGAP related domain type I (GRDI) with the known high H-RasGAP activity. Taken together, these results suggest that PKCε activation regulates the spatial plasma membrane enrichments of both SOS1 and neurofibromin, thus controlling the output of activated H-Ras available for downstream signalling in neurons.  

3.3390           Proteomic profiling reveals key cancer progression modulators in shed microvesicles released from isogenic human primary and metastatic colorectal cancer cell lines

Suwakulsiri, W., Rai, A., Xu, R., Chen, M., Greening, D.W. and Simpson, R.J. BBA-Proteins and Proteomics, 1867, 140171 (2019)   Extracellular vesicles comprise two main classes - exosomes and shed microvesicles (sMVs). Whilst much is known about exosome cargo content and functionality, sMVs are poorly understood. Here, we describe the large-scale purification of sMVs released from primary (SW480) and metastatic (SW620) human isogenic colorectal cancer (CRC) cell lines using a combination of differential ultracentrifugation and isopycnic iodixanol density centrifugation. The yield of SW480-sMVs and SW620-sMVs was 0.75 mg and 0.80 mg, respectively. Both SW480-/SW620-sMVs are heterogeneous in size (100–600 nm diameter) and exhibit identical buoyant densities (1.10 g/mL). In contrast to exosomes, sMVs are ALIX⁻, TSG101⁻, CD63⁻ and CD9⁻. Quantitative mass spectrometry identified 1295 and 1300 proteins in SW480-sMVs and SW620-sMVs, respectively. Gene Ontology enrichment analysis identified ‘cell adhesion’ (CDH1, OCLN, CTN families), ‘signalling pathway’ (KRAS, NRAS, MAPK1, MAP2K1), and ‘translation/RNA related’ processes (EIF, RPL, HNRNP families) in both sMV types. Strikingly, SW480- and SW620-sMVs exhibit distinct protein signatures - SW480-sMVs being enriched in ITGA/B, ANXA1, CLDN7, CD44 and EGFR/NOTCH signalling networks, while SW620-sMVs are enriched in PRKCA, MACC1, FGFR4 and MTOR/MARCKS signalling networks. Both SW480- and SW620-sMVs are taken up by NIH3T3 fibroblasts resulting in similar cell invasion capability. This study provides, for the first time, molecular insights into sMVs and CRC biology.  

3.3391           Extracellular vesicles nanoarray technology: Immobilization of individual extracellular vesicles on nanopatterned polyethylene glycol-lipid conjugate brushes

Yokota, S., Kuramochi, H., Okubo, K., Iwaya, A., Tsuchiya, S. and Ichiki, T. PloS One, 14(10), e022409 (2019)   Arraying individual extracellular vesicles (EVs) on a chip is expected one of the promising approaches for investigating their inherent properties. In this study, we immobilized individual EVs on a surface using a nanopatterned tethering chip-based versatile platform. A microfluidic device was used to ensure soft, reproducible exposure of the EVs over the whole chip surface. The device is incorporated with a high-density nanoarray chip patterned with 200-nm diameter nanospots composed of polyethylene glycol (PEG)-lipid conjugate brushes. We present a procedure adopted for fabricating high-density PEG-lipid modified nanospots (200 nmϕ, 5.0 × 10⁵ spots/mm² in 2 × 2 mm² area). This procedure involves nanopatterning using electron beam lithography, followed by multistep selective chemical modification. Aqueous treatment of a silane coupling agent, used as a linker between PEG-lipid molecules and the silicon surface, was the key step that enabled surface modification using a nanopatterned resist film as a mask. The nanoarray chip was removed from the device for subsequent measurements such as atomic force microscopy (AFM). We developed a prototype device and individually immobilized EVs derived from different cell lines (Sk-Br-3 and HEK293) on tethering nanospots. We characterized EV's morphology using AFM and showed the possibility of evaluating the deformability of EVs using the aspect ratio as an indicator.  

3.3392           Advances in exosome isolation methods and their applications in proteomic analysis of biological samples

Hou, R., Li, Y., Sui, Z., Yuan, H., yang, k., Liang, Z., Zhang, l. and Zhang, Y. Anal. Bioanal. Chem., 411, 5351-5361 (2019)   Exosomes are membrane-bound vesicles secreted by cells, and contain various important biological molecules, such as lipids, proteins, messenger RNAs, microRNAs, and noncoding RNAs. Emerging evidence demonstrates that proteomic analysis of exosomes is of great significance in studying metabolic diseases, tumor metastasis, immune regulation, and so forth. However, exosome proteomic analysis has high requirements with regard to the purity of collected exosomes. Here recent advances in the methods for isolating exosomes and their applications in proteomic analysis are summarized.  

3.3393           Nir2 Is an Effector of VAPs Necessary for Efficient Hepatitis C Virus Replication and Phosphatidylinositol 4-Phosphate Enrichment at the Viral Replication Organelle

Wang, H. and Tai, A.W.

1. Virol., 93(22), e00742-19 (2019)

  The endoplasmic reticulum (ER)-resident proteins vesicle-associated membrane protein (VAMP)-associated protein A and B (VAPA and VAPB) have been reported to be necessary for efficient hepatitis C virus (HCV) replication, but the specific mechanisms are not well understood. VAPs are known to recruit lipid transfer proteins to the ER, including oxysterol binding protein (OSBP), which has been previously shown to be necessary for cholesterol delivery to the HCV replication organelle in exchange for phosphatidylinositol 4-phosphate [PI(4)P]. Here, we show that VAPA and VAPB are redundant for HCV infection and that dimerization is not required for their function. In addition, we identify the phosphatidylinositol transfer protein Nir2 as an effector of VAPs to support HCV replication. We propose that Nir2 functions to replenish phosphoinositides at the HCV replication organelle to maintain elevated steady-state levels of PI(4)P, which is removed by OSBP. Thus, Nir2, along with VAPs, OSBP, and the phosphatidylinositol 4-kinase, completes a cycle of phosphoinositide flow between the ER and viral replication organelles to drive ongoing viral replication.  

3.3394           Isolation and characterization of extracellular vesicles from Broncho-alveolar lavage fluid: a review and comparison of different methods

Carnino, J.M., Lee, H. and Jin, Y. Respiratory Res., 20:240 (2019)   Extracellular vesicles (EVs) are cell-derived membranous vesicles secreted by cells into the extracellular space, which play a role in cell to cell communication. EVs are categorized into 3 groups depending on their size, surface marker, and method of release from the host cell. Recently, EVs have become of interest in the study of multiple disease etiologies and are believed to be potential biomarkers for many diseases. Multiple different methods have been developed to isolate EVs from different samples such as cell culture medium, serum, blood, and urine. Once isolated, EVs can be characterized by technology such as nanotracking analysis, dynamic light scattering, and nanoscale flow cytometry. In this review, we summarize the current methods of EV isolation, provide details into the three methods of EV characterization, and provide insight into which isolation approaches are most suitable for EV isolation from bronchoalveolar lavage fluid (BALF).  

3.3395           SF-Assemblin genes in Paramecium: phylogeny and phenotypes of RNAi silencing on the ciliary-striated rootlets and surface organization

Nabi, A., Yano, J., Valentine, M.S., Picariello, T. and Van Houten, J.L. Cilia, 8:2 (2019)   Background Cilia emanate from basal bodies just underneath the cell membrane. Basal bodies must withstand torque from the ciliary beat and be appropriately spaced for cilia to beat in metachronal waves. Basal body rootlets provide stability for motile cilia. Paramecium has three. Our focus is on the largest one, the striated rootlet (SR). Paramecium basal bodies align in straight rows. Previously we found a potential role for the SR in this alignment. Here we present a phylogeny of the Paramecium homologs of the SF-Assemblin gene of the SR of Chlamydomonas, and the organization of these genes. We describe the phenotypes from RNA interference (RNAi) silencing of genes and gene groups. Methods Phenotypes of the RNAi depletions were characterized by immunofluorescence (IF), electron microscopy, and mass spectrometry. Results We found 30 genes for Paramecium SF-Assemblin homologs (SFA) organized into 13 Paralog Groups (further categorized in five Structural Groups). Representatives of Paralog Groups were found in the SRs. Silencing the transcripts of any of the Structural Groups correlates with misaligned rows of basal bodies, SRs, and cortical units. The silencing of Structural Groups was key and gave us the ability to systematically disrupt SR structures and cell surface organization. Conclusions Silencing of SFA genes and Paralog Groups shows no effects on the SR or the cell surface organization. Silencing of the larger Structural Groups has an enormous impact on rows of basal bodies, SRs and cortical units, and SR striations, and length. Misaligned basal bodies have cilia causing the cells to swim in abnormal paths.  

3.3396           Cholesterol transport through the peroxisome-ER membrane contacts tethered by PI(4,5)P2 and extended synaptotagmins

Xiao, J., Luo, J., Hu, A., Xiao, T., Li, M., Kong, Z., Jiang, L., Zhou, Z., Liao, Y., Xie, C., Chu, B., Miao, H., Li, B., Shi, X. and Song, B-L. Science China, 62(9), 1117-1135 (2019)   Most mammalian cells take up cholesterol from low-density lipoproteins (LDLs) via receptor-mediated endocytosis. After reaching lysosomes, LDL-derived cholesterol continues to transport to downstream organelles including the ER for specific structural and functional needs. Peroxisomes are recently found to receive cholesterol from lysosomes through lysosome-peroxisome membrane contacts. However, whether and how cholesterol is conveyed from peroxisomes to the ER remain unknown. Here, by combining high-resolution microscopic analyses and in vitro reconstitution of highly purified organelles or artificial liposomes, we demonstrate that peroxisomes form membrane contacts with the ER through the interaction between peroxisomal PI(4,5)P₂ and ER-resident extended synaptotagmin-1, 2 and 3 (E-Syts). Depletion of peroxisomal PI(4,5)P₂ or E-Syts markedly decreases peroxisome-ER membrane contacts and induces cholesterol accumulation in lysosomes. Furthermore, we show that cholesterol is delivered from ³H-labeled peroxisomes or PI(4,5)P₂-containing liposomes to the ER in vitro, and that the presence of peroxisomes augments cholesterol transfer from lysosomes to the ER. Together, our study reveals a new cholesterol transport pathway along the lysosome-peroxisome-ER membrane contacts in the cell.  

3.3397           Natural and engineered bacterial outer membrane vesicles

Qing, G., Gong, N., Chen, X., Chen, J., Zhang, h., Wang, Y., Wang, R., Zhang, S., Zhang, Z., Zhao, X., Luo, Y. and Liang, X-J. Biophys. Rep., 5(4), 184-198 (2019)   Bacterial outer membrane vesicle (OMV) is a kind of spherical lipid bilayer nanostructure naturally secreted by bacteria, which has diverse functions such as intracellular and extracellular communication, horizontal gene transfer, transfer of contents to host cells, and eliciting an immune response in host cells. In this review, several methods including ultracentrifugation and precipitation for isolating OMVs were summarized. The latest progresses of OMVs in biomedical fields, especially in vaccine development, cancer treatment, infection control, and bioimaging and detection were also summarized in this review. We highlighted the importance of genetic engineering for the safe and effective application and in facilitating the rapid development of OMVs. Finally, we discussed the bottleneck problems about OMVs in preparation and application at present and put forward our own suggestions about them. Some perspectives of OMVs in biomedical field were also provided.  

3.3398           Polyethylene glycol improves current methods for circulating extracellular vesicle-derived DNA isolation

Garcia-Romero, N., Madurga, R., Rackov, G., Palacin-Aliana, i., Nunez-Torres, R., Asensi-Puig, A., Carrion-Navvvvarro, J., Esteban-Rubo, S., Peinado, H., Gonzalez-Neira, A., Gonzalez-Rumayor, V., Belda-Iniesta, C. and Ayuso-Sacido, A.

1. Transl. Med., 17:75 (2019)

  Background Extracellular vesicles (EVs) are small membrane-bound vesicles which play an important role in cell-to-cell communication. Their molecular cargo analysis is presented as a new source for biomarker detection, and it might provide an alternative to traditional solid biopsies. However, the most effective approach for EV isolation is not yet well established. Results Here, we study the efficiency of the most common EV isolation methods-ultracentrifugation, Polyethlyene glycol and two commercial kits, Exoquick^® and PureExo^®. We isolated circulating EVs from the bloodstream of healthy donors, characterized the size and yield of EVs and analyzed their protein profiles and concentration. Moreover, we have used for the first time Digital-PCR to identify and detect specific gDNA sequences, which has several implications for diagnostic and monitoring many types of diseases. Conclusions Our findings present Polyethylene glycol precipitation as the most feasible and less cost-consuming EV isolation technique.  

3.3399           Orally-administered outer-membrane vesicles from Helicobacter pylori reduce H. pylori infection via Th2-biased immune responses in mice

Liu, Q., Li, X., Zhang, Y., Song, Z., Li, R., Ruan, H. and Huang, X. Pathogens and Disease, 77, ftz050 (2019)   As the trend of antibiotic resistance has increased, prevention and treatment of Helicobacter pylori infection have been challenged by the fact that no vaccines preventing H. pylori infection are available. Scientists continue to make sustained efforts to find better vaccine formulations and adjuvants to eradicate this chronic infection. In this study, we systemically analyzed the protein composition and potential vaccine function of outer-membrane vesicles (OMVs) derived from gerbil-adapted H. pylori strain 7.13. In total, we identified 169 proteins in H. pylori OMVs and found that outer-membrane, periplasmic and extracellular proteins (48.9% of the total proteins) were enriched. Furthermore, we evaluated the immune protective response of H. pylori OMVs in a C57BL/6 mouse model, and mice were orally immunized with OMVs or the H. pylori whole cell vaccine (WCV) alone, with or without cholera toxin (CT) as an adjuvant. The data demonstrated that oral immunization with OMVs can elicit a strong humoral and significantly higher mucosal immune response than the group immunized with the WCV plus the CT adjuvant. Moreover, our results also confirmed that OMVs predominantly induced T helper 2 (Th₂)-biased immune responses that can significantly reduce bacterial loads after challenging with the H. pylori Sydney Strain 1 (SS1). In summary, OMVs as new antigen candidates in vaccine design would be of great value in controlling H. pylori infection.  

3.3400           Immediate and deferred epigenomic signatures of in vivo neuronal activation in mouse hippocampus

Fernandez-Albert, J., Lipinski, M., Lopez-Cascales, M.T., Rowley, M.J., Martin-Gonzalez, A.M., del Blanco, B., Corces, V.G. and Barco, A. Nature Neurosci., 22, 1718-17340 (2019)   Activity-driven transcription plays an important role in many brain processes, including those underlying memory and epilepsy. Here we combine genetic tagging of nuclei and ribosomes with RNA sequencing, chromatin immunoprecipitation with sequencing, assay for transposase-accessible chromatin using sequencing and Hi-C to investigate transcriptional and chromatin changes occurring in mouse hippocampal excitatory neurons at different time points after synchronous activation during seizure and sparse activation by novel context exploration. The transcriptional burst is associated with an increase in chromatin accessibility of activity-regulated genes and enhancers, de novo binding of activity-regulated transcription factors, augmented promoter–enhancer interactions and the formation of gene loops that bring together the transcription start site and transcription termination site of induced genes and may sustain the fast reloading of RNA polymerase complexes. Some chromatin occupancy changes and interactions, particularly those driven by AP1, remain long after neuronal activation and could underlie the changes in neuronal responsiveness and circuit connectivity observed in these neuroplasticity paradigms, perhaps thereby contributing to metaplasticity in the adult brain.  

3.3401           Comparative Proteomics Analysis of Four Commonly Used Methods for Identification of Novel Plasma Membrane Proteins

Yoneten, K.K., Kasap, M., Akpinar, G., Kanli, A. and Karaoz, E.

1. Membrane Biol., 252(6), 587-608 (2019)

  Plasma membrane proteins perform a variety of important tasks in the cells. These tasks can be diverse as carrying nutrients across the plasma membrane, receiving chemical signals from outside the cell, translating them into intracellular action, and anchoring the cell in a particular location. When these crucial roles of plasma membrane proteins are considered, the need for their characterization becomes inevitable. Certain characteristics of plasma membrane proteins such as hydrophobicity, low solubility, and low abundance limit their detection by proteomic analyses. Here, we presented a comparative proteomics study in which the most commonly used plasma membrane protein enrichment methods were evaluated. The methods that were utilized include biotinylation, selective CyDye labeling, temperature-dependent phase partition, and density-gradient ultracentrifugation. Western blot analysis was performed to assess the level of plasma membrane protein enrichment using plasma membrane and cytoplasmic protein markers. Quantitative evaluation of the level of enrichment was performed by two-dimensional electrophoresis (2-DE) and benzyldimethyl-n-hexadecylammonium chloride/sodium dodecyl sulfate polyacrylamide gel electrophoresis (16-BAC/SDS-PAGE) from which the protein spots were cut and identified. Results from this study demonstrated that density-gradient ultracentrifugation method was superior when coupled with 16-BAC/SDS-PAGE. This work presents a valuable contribution and provides a future direction to the membrane sub-proteome research by evaluating commonly used methods for plasma membrane protein enrichment.  

3.3402           Emerging role of extracellular vesicles in the regulation of skeletal muscle adaptation

Vechetti, Jr., I J.

1. Appl. Physiol., 127, 645-653 (2019)

  Extracellular vesicles (EVs) were initially characterized as “garbage bags” with the purpose of removing unwanted material from cells. It is now becoming clear that EVs mediate intercellular communication between distant cells through a transfer of genetic material, a process important to the systemic adaptation in physiological and pathological conditions. Although speculative, it has been suggested that the majority of EVs that make it into the bloodstream would be coming from skeletal muscle, since it is one of the largest organs in the human body. Although it is well established that skeletal muscle secretes peptides (currently known as myokines) into the bloodstream, the notion that skeletal muscle releases EVs is in its infancy. Besides intercellular communication and systemic adaptation, EV release could represent the mechanism by which muscle adapts to certain stimuli. This review summarizes the current understanding of EV biology and biogenesis and current isolation methods and briefly discusses the possible role EVs have in regulating skeletal muscle mass.  

3.3403           Lipid droplet size directs lipolysis and lipophagy catabolism in hepatocytes

Schott, M.B., Weller, S.G., Schulze, R.J., Krueger, E.W., Drizyte-Miller, K., Casey, C.A. and McNiven, M.A.

1. Cell Biol., 218(10), 3320-3335 (2019)

  Lipid droplet (LD) catabolism in hepatocytes is mediated by a combination of lipolysis and a selective autophagic mechanism called lipophagy, but the relative contributions of these seemingly distinct pathways remain unclear. We find that inhibition of lipolysis, lipophagy, or both resulted in similar overall LD content but dramatic differences in LD morphology. Inhibition of the lipolysis enzyme adipose triglyceride lipase (ATGL) resulted in large cytoplasmic LDs, whereas lysosomal inhibition caused the accumulation of numerous small LDs within the cytoplasm and degradative acidic vesicles. Combined inhibition of ATGL and LAL resulted in large LDs, suggesting that lipolysis targets these LDs upstream of lipophagy. Consistent with this, ATGL was enriched in larger-sized LDs, whereas lipophagic vesicles were restricted to small LDs as revealed by immunofluorescence, electron microscopy, and Western blot of size-separated LDs. These findings provide new evidence indicating a synergistic relationship whereby lipolysis targets larger-sized LDs to produce both size-reduced and nascently synthesized small LDs that are amenable for lipophagic internalization.  

3.3404           Chondroitin sulfate proteoglycan 4 enhanced melanoma motility and growth requires a cysteine in the core protein transmembrane domain

Yang, J., Price, M.A., Wanshura, L.E.C., He, J., Yi, M., Welch, D.R., Li, G., Conner, S., Sachs, J., Turley, E.A. and McCarthy, J.B. Melanoma Res., 29, 365-375 (2019)   Chondroitin sulfate proteoglycan 4 (CSPG4) is a cell surface proteoglycan that enhances malignant potential in melanoma and several other tumor types. CSPG4 functions as a transmembrane scaffold in melanoma cells to activate oncogenic signaling pathways such as focal adhesion kinase (FAK) and extracellular signal regulated kinases 1,2, that control motility, invasion and anchorage independent growth. Here, we demonstrate that CSPG4 promotes directional motility and anchorage independent growth of melanoma cells by organizing and positioning a signaling complex containing activated FAK to lipid rafts within the plasma membrane of migrating cells. This FAK-containing signal transduction platform, which consists of syntenin-1, active Src and caveolin-1 requires the cytoplasmic domain of CSPG4 for assembly. Enhanced directional motility promoted by this complex also requires a CSPG4 transmembrane cysteine residue C2230. Substituting C2230 with alanine (CSPG4^C2230A) still permits assembly of the signaling complex, however Src remains in an inactive state. CSPG4^C2230A also fails to promote anchorage independent growth and activation of extracellular signal regulated kinases 1,2. Therapies that target the transmembrane domain of CSPG4 could be a novel strategy for limiting progression by disrupting its function as a compartmentalized motogenic and growth-promoting oncogenic signaling node.  

3.3405           Prdx4 limits caspase‐1 activation and restricts inflammasome‐mediated signaling by extracellular vesicles

Lipinski, S., Pfeuffer, S., Arnold, P., Treitz, C., Aden, K., Ebsen, H., Falk-Paulsen, M., Gisch, N., Fazio, A. et al EMBO J., 38, e101266 (2019)   Inflammasomes are cytosolic protein complexes, which orchestrate the maturation of active IL‐1β by proteolytic cleavage via caspase‐1. Although many principles of inflammasome activation have been described, mechanisms that limit inflammasome‐dependent immune responses remain poorly defined. Here, we show that the thiol‐specific peroxidase peroxiredoxin‐4 (Prdx4) directly regulates IL‐1β generation by interfering with caspase‐1 activity. We demonstrate that caspase‐1 and Prdx4 form a redox‐sensitive regulatory complex via caspase‐1 cysteine 397 that leads to caspase‐1 sequestration and inactivation. Mice lacking Prdx4 show an increased susceptibility to LPS‐induced septic shock. This effect was phenocopied in mice carrying a conditional deletion of Prdx4 in the myeloid lineage (Prdx4‐ΔLysMCre). Strikingly, we demonstrate that Prdx4 co‐localizes with inflammasome components in extracellular vesicles (EVs) from inflammasome‐activated macrophages. Purified EVs are able to transmit a robust IL‐1β‐dependent inflammatory response in vitro and also in recipient mice in vivo. Loss of Prdx4 boosts the pro‐inflammatory potential of EVs. These findings identify Prdx4 as a critical regulator of inflammasome activity and provide new insights into remote cell‐to‐cell communication function of inflammasomes via macrophage‐derived EVs.  

3.3406           Tumour exosomal CEMIP protein promotes cancer cell colonization in brain metastasis

Rodrigues, G., Hoshino, A., Lyden, D. et al Nature Cell Biol., 21, 1403-1412 (2019)   The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP⁺ exosomes. Moreover, uptake of CEMIP⁺ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.  

3.3407           Enhanced generation of intraluminal vesicles in neuronal late endosomes in the brain of a Down syndrome mouse model with endosomal dysfunction

D’Acunzo, P., Hargash, T., Pawlik, M., Goulbourne, C.N., Perez-Gonzalez, R. and Levy, E. Develop. Neurobiol., 79(7), 656-663 (2019)   Down syndrome (DS) is a human genetic disease caused by trisomy of chromosome 21 and characterized by early developmental brain abnormalities. Dysfunctional endosomal pathway in neurons is an early event of DS and Alzheimer's disease. Recently, we have demonstrated that exosome secretion is upregulated in human DS postmortem brains, in the brain of the trisomic mouse model Ts[Rb(12.17¹⁶)]2Cje (Ts2) and by DS fibroblasts as compared with disomic controls. High levels of the tetraspanin CD63, a regulator of exosome biogenesis, were observed in DS brains. Partially blocking exosome secretion by DS fibroblasts exacerbated a pre‐existing early endosomal pathology. We thus hypothesized that enhanced CD63 expression induces generation of intraluminal vesicles (ILVs) in late endosomes/multivesicular bodies (MVBs), increasing exosome release as an endogenous mechanism to mitigate endosomal abnormalities in DS. Herein, we show a high‐resolution electron microscopy analysis of MVBs in neurons of the frontal cortex of 12‐month‐old Ts2 mice and littermate diploid controls. Our quantitative analysis revealed that Ts2 MVBs are larger, more abundant, and contain a higher number of ILVs per neuron compared to controls. These findings were further corroborated biochemically by Western blot analysis of purified endosomal fractions showing higher levels of ILVs proteins in the same fractions containing endosomal markers in the brain of Ts2 mice compared to controls. These data suggest that upregulation of ILVs production may be a key homeostatic mechanism to alleviate endosomal dysregulation via the endosomal–exosomal pathway.  

3.3408           Plasma extracellular vesicles as phenotypic biomarkers in prostate cancer patients

Joncas, F-H., Lucien, F., Rouleau, M., Morin, F., Leong, H.S., Pouliot, F., Fradet, Y., Gilbert, C. and Toren, P. The Prostate, 79, 1767-1776 (2019)   Background The development of phenotypic biomarkers to aid the selection of treatment for patients with castrate‐resistant prostate cancer (CRPC) is an important priority. Plasma exosomes have excellent potential as real‐time biomarkers to characterize the tumor because they are easily accessible in the blood and contain DNA, RNA, and protein from the parent cell. This study aims to investigate the characteristics of putative prostate‐specific plasma extracellular vesicle (EV) markers and their relationship with clinical outcomes. Methods and Patients We investigated plasma EVs in a total of 89 patients with prostate cancer (PCa) at different stages of disease progression. EVs were isolated using both precipitation and ultracentrifugation methods; physical characterization was performed using dynamic light scattering, acetylcholinesterase (AChE) activity, and velocity gradients. An immunocapture method was developed for the evaluation of prostate‐specific membrane antigen (PSMA)‐positive exosomes. Exosomal messenger RNA (mRNA) was quantified using droplet digital polymerase chain reaction for the expression of KLK3 and androgen receptor splice variant 7 (AR‐V7) genes, which code prostate‐specific antigen (PSA) and AR‐V7, respectively. Serum sex steroids were measured using liquid chromatography‐tandem mass spectroscopy. Results Isolated exosomes from patients with CRPC had a smaller hydrodynamic size than those isolated from localized patients with PCa, while AChE activity showed no difference. Moreover, no differences were observed after initiation of androgen deprivation therapy in serial patient samples. Velocity gradients identified that PSMA‐positive exosomes occupied a specific fraction of isolated EVs. A total of 35 patients with CRPC had mRNA analyzed from isolated plasma exosomes. Detectable exosomal KLK3 corresponded with higher concomitant serum PSA measurements, as expected (mean, 112.6 vs 26.61 ng/mL; P = .065). Furthermore, detectable levels of AR‐V7 mRNA were associated with a shorter time to progression (median, 16.0 vs 28.0 months; P = .0499). Furthermore, detectable exosomal AR‐V7 was significantly associated with testosterone levels below the lower limit of quantification (<0.1 nM). Conclusions Our results suggest that exosomal AR‐V7 is correlated with lower sex steroid levels in CRPC patients with a poorer prognosis. PSMA immunocapture does not appear sufficient to isolate PCa‐specific exosomes.  

3.3409           Cln3‐mutations underlying juvenile neuronal ceroid lipofuscinosis cause significantly reduced levels of Palmitoyl‐protein thioesterases‐1 (Ppt1)‐protein and Ppt1‐enzyme activity in the lysosome

Appu, A.P., Bagh, M.B., Sadhukhan, T., Mondal, A., Casey, S. and Mukherjee, A.B.

1. Inherit. Metab. Dis., 42(5), 944-954 (2019)

  Mutations in at least 13 different genes (called CLNs) underlie various forms of neuronal ceroid lipofuscinoses (NCLs), a group of the most common neurodegenerative lysosomal storage diseases. While inactivating mutations in the CLN1 gene, encoding palmitoyl‐protein thioesterases‐1 (PPT1), cause infantile NCL (INCL), those in the CLN3 gene, encoding a protein of unknown function, underlie juvenile NCL (JNCL). PPT1 depalmitoylates S‐palmitoylated proteins (constituents of ceroid) required for their degradation by lysosomal hydrolases and PPT1‐deficiency causes lysosomal accumulation of autofluorescent ceroid leading to INCL. Because intracellular accumulation of ceroid is a characteristic of all NCLs, a common pathogenic link for these diseases has been suggested. It has been reported that CLN3‐mutations suppress the exit of cation‐independent mannose 6‐phosphate receptor (CI‐M6PR) from the trans Golgi network (TGN). Because CI‐M6PR transports soluble proteins such as PPT1 from the TGN to the lysosome, we hypothesized that CLN3‐mutations may cause lysosomal PPT1‐insufficiency contributing to JNCL pathogenesis. Here, we report that the lysosomes in Cln3‐mutant mice, which mimic JNCL, and those in cultured cells from JNCL patients, contain significantly reduced levels of Ppt1‐protein and Ppt1‐enzyme activity and progressively accumulate autofluorescent ceroid. Furthermore, in JNCL fibroblasts the V0a1 subunit of v‐ATPase, which regulates lysosomal acidification, is mislocalized to the plasma membrane instead of its normal location on lysosomal membrane. This defect dysregulates lysosomal acidification, as we previously reported in Cln1 ^−/− mice, which mimic INCL. Our findings uncover a previously unrecognized role of CLN3 in lysosomal homeostasis and suggest that CLN3‐mutations causing lysosomal Ppt1‐insuffiiciency may at least in part contribute to JNCL pathogenesis.  

3.3410           Is there a place and role for endocytic TCR signaling?

Saveanu, L., Zucchetti, A.E., Evnouchidou, I., Ardouin, L. and Hivroz, C. Immunological Reviews, 291, 57-74 (2019)   T‐lymphocyte activation relies on the cognate recognition by the TCR of the MHC‐associated peptide ligand (pMHC) presented at the surface of an antigen‐presenting cell (APC). This leads to the dynamic formation of a cognate contact between the T lymphocyte and the APC: the immune synapse (IS). Engagement of the TCR by the pMHC in the synaptic zone induces a cascade of signaling events leading to phosphorylation and dephosphorylation of proteins and lipids, which ultimately shapes the response of T lymphocytes. Although the engagement of the T‐cell receptor (TCR) takes place at the plasma membrane, the TCR/CD3 complexes and the signaling molecules involved in transduction of the TCR signal are also present in intracellular membrane pools. These pools, which are both endocytic and exocytic, have tentatively been characterized by several groups including ours. We will herein summarize what is known on the intracellular pools of TCR signaling components. We will discuss their origin and the mechanisms involved in their mobility at the IS. Finally, we will propose several hypotheses concerning the functional role(s) that these intracellular pools might play in T‐cell activation. We will also discuss the tools that could be used to test these hypotheses.  

3.3411           Pseudomonas aeruginosa Leucine Aminopeptidase Influences Early Biofilm Composition and Structure via Vesicle-Associated Antibiofilm Activity

Esoda, C.N. and Kuehn, M.J. mBio, 10(6), e2548-19 (2019)   Pseudomonas aeruginosa, known as one of the leading causes of disease in cystic fibrosis (CF) patients, secretes a variety of proteases. These enzymes contribute significantly to P. aeruginosa pathogenesis and biofilm formation in the chronic colonization of CF patient lungs, as well as playing a role in infections of the cornea, burn wounds, and chronic wounds. We previously characterized a secreted P. aeruginosa peptidase, PaAP, that is highly expressed in chronic CF isolates. This leucine aminopeptidase is highly expressed during infection and in biofilms, and it associates with bacterial outer membrane vesicles (OMVs), structures known to contribute to virulence mechanisms in a variety of Gram-negative species and one of the major components of the biofilm matrix. We hypothesized that PaAP may play a role in P. aeruginosa biofilm formation. Using a lung epithelial cell/bacterial biofilm coculture model, we show that PaAP deletion in a clinical P. aeruginosa background alters biofilm microcolony composition to increase cellular density, while decreasing matrix polysaccharide content, and that OMVs from PaAP-expressing strains but not PaAP alone or in combination with PaAP deletion strain-derived OMVs could complement this phenotype. We additionally found that OMVs from PaAP-expressing strains could cause protease-mediated biofilm detachment, leading to changes in matrix and colony composition. Finally, we showed that the OMVs could also mediate the detachment of biofilms formed by both nonself P. aeruginosa strains and Klebsiella pneumoniae, another respiratory pathogen. Our findings represent novel roles for OMVs and the aminopeptidase in the modulation of P. aeruginosa biofilm architecture.  

3.3412           Membrane-bound Gaussia luciferase as a tool to track shedding of membrane proteins from the surface of extracellular vesicles

Zaborowaki, M.P., Cheah, P.S., Zhang, X., Bushko, I., Lee, K., Sammarco, A. et al Scientific Reports, 9:17387 (2019)   Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models.  

3.3413           Dynamic constriction and fission of endoplasmic reticulum membranes by reticulon

Espadas, J., Pendin, D., Bocanegra, R., Escalada, A., Misticoni, G. et al Nature Communications, 10:5327 (2019)   The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. Here we find that the ER has an embedded fragmentation mechanism based upon the ability of reticulon to produce fission of elongating network branches. In Drosophila, Rtnl1-facilitated fission is counterbalanced by atlastin-driven fusion, with the prevalence of Rtnl1 leading to ER fragmentation. Ectopic expression of Drosophila reticulon in COS-7 cells reveals individual fission events in dynamic ER tubules. Consistently, in vitro analyses show that reticulon produces velocity-dependent constriction of lipid nanotubes leading to stochastic fission via a hemifission mechanism. Fission occurs at elongation rates and pulling force ranges intrinsic to the ER, thus suggesting a principle whereby the dynamic balance between fusion and fission controlling organelle morphology depends on membrane motility.  

3.3414           Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles

Everaert, C., helsmoortel, H., Decock, A., Hulstraert, E., Van Paemel, R., Verniers, K., Nuytens, J., Anckaert, j., Nijs, N., Tulkens, J., Dhondt, B., Hendrix, A., Mestdagh, P. and Vandesompele, J. Scientific Reports, 9:17574 (2019)   RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer method can be used for unbiased unraveling of the complete transcriptome of a wide range of biofluids and their extracellular vesicles.  

3.3415           Extracellular vesicles and chronic inflammation during HIV infection

Perez, P.S:, Romaniuk, M.A., Duette, G.A., Zhao, Z., Huang, Y., martin-jaular, L., Witwer, K.W., Thery, C. and Ostrowski, M.

1. Extracellular vesicles, 8(1), 1687275 (2019)

  Inflammation is a hallmark of HIV infection. Among the multiple stimuli that can induce inflammation in untreated infection, ongoing viral replication is a primary driver. After initiation of effective combined antiretroviral therapy (cART), HIV replication is drastically reduced or halted. However, even virologically controlled patients may continue to have abnormal levels of inflammation. A number of factors have been proposed to cause inflammation in HIV infection: among others, residual (low-level) HIV replication, production of HIV protein or RNA in the absence of replication, microbial translocation from the gut to the circulation, co-infections, and loss of immunoregulatory responses. Importantly, chronic inflammation in HIV-infected individuals increases the risk for a number of non-infectious co-morbidities, including cancer and cardiovascular disease. Thus, achieving a better understanding of the underlying mechanisms of HIV-associated inflammation in the presence of cART is of utmost importance. Extracellular vesicles have emerged as novel actors in intercellular communication, involved in a myriad of physiological and pathological processes, including inflammation. In this review, we will discuss the role of extracellular vesicles in the pathogenesis of HIV infection, with particular emphasis on their role as inducers of chronic inflammation.  

3.3416           Extracellular vesicles containing oncogenic mutant β-catenin activate Wnt signalling pathway in the recipient cells

Kalra, H., Gangoda, L., Fonseka, P., Chitti, S.V., Liem, M., Keerthikumar, S., Samuel, M., Boukouris, S., Al Saffar, H., Collins, C., Adda, C.G., Ang, C-S. and mathivanan, S.

1. Extracellular Vesicles, 8(1), 1690217 (2019)

  Mutations in β-catenin, especially at the residues critical for its degradation, render it constitutively active. Here, we show that mutant β-catenin can be transported via extracellular vesicles (EVs) and activate Wnt signalling pathway in the recipient cells. An integrative proteogenomic analysis identified the presence of mutated β-catenin in EVs secreted by colorectal cancer (CRC) cells. Follow-up experiments established that EVs released from LIM1215 CRC cells stimulated Wnt signalling pathway in the recipient cells with wild-type β-catenin. SILAC-based quantitative proteomics analysis confirmed the transfer of mutant β-catenin to the nucleus of the recipient cells. In vivo tracking of DiR-labelled EVs in mouse implanted with RKO CRC cells revealed its bio-distribution, confirmed the activation of Wnt signalling pathway in tumour cells and increased the tumour burden. Overall, for the first time, this study reveals that EVs can transfer mutant β-catenin to the recipient cells and promote cancer progression.  

3.3417           Production of outer membrane vesicles from acetic acid bacteria and their properties

Hashimoto, M. Endotoxin and Innate Immunity, 22, 54-57 (2019)   Outer membrane vesicles (OMVs) are mainly composed of lipopolysaccharide (LPS), phospholipids, and outer membrane and periplasmic proteins. Recently OMV vaccines have been developed, because their LPS act as adjuvant. However, attenuation of the toxicity of typical LPS is necessary to reduce adverse effects of OMV vaccine. Previously we found that acetic acid bacteria Acetobacter pasteurianus produces low immunostimulatory LPS. In this study, we separated OMVs from A. pasteurianus and characterized their immunostimulatory effects. A. pasteurianus NBRC 3283 were grown at 27℃ in 804 broth. Vesicle secretion from the cell was observed after 2 days in culture by TEM imaging. The vesicles were separated from culture supernatants after 7 days in culture by ultracentrifugation. We found that the precipitation contains vesicles which can be purified by OptiPrep density gradient centrifugation. Since the vesicles composed of LPS and outer membrane proteins, we concluded they are OMVs and designated as Ap-OMV. We further found that Ap-OMV stimulated TLR2 and weakly TLR4 in TLR expressing cells and TNF-α production in J774A. 1 cells. Furthermore the OMV-like vesicles were also found in Japanese black vinegar, kurozu. These data suggest that A. pasteurianus produce LPS-containing OMVs and can stimulate innate immune system.  

3.3418           Isolation of Redox-Active Endosomes (Redoxosomes) and Assessment of NOX Activity

Shahin, W.S. and Engelhardt, J.F. Methods in Mol. Biol., 1982, 461-472 (2019)   Reactive oxygen species (ROS) convey signals essential for proliferation, maintenance, and senescence of a growing list of cell types. Compartmentalization of these signals is integral to cell viability as well as the signaling pathways ROS direct. Redox-active endosomes (redoxosomes) are formed downstream of several ligand-activated receptors. NADPH oxidase (NOX) is a main component of redoxosomes, which recruits multiple proteins (Rac1, NOX2, p67^phox, SOD1). Isolation of redoxosomes and evaluation of how superoxide (O₂˙⁻) production directs receptor signaling at the level of the endosome have enabled a better understanding of biologic processes controlled by ROS. In this chapter, we will first review the major signaling pathways that utilize redoxosomes and components that control its redox-dependent functions. We will then outline biochemical and biophysical methods for the isolation and characterization of redoxosome properties.  

3.3419           Mapping the Saccharomyces cerevisiae Spatial Proteome with High Resolution Using hyperLOPIT

Nightingale, D.J.H., Oliver, S.G. and Lilley, K.S. Methods in Mol. Biol., 2049, 165-190 (2019)   The subcellular localization of proteins is a posttranslational modification of paramount importance. The ability to study subcellular and organelle proteomes improves our understanding of cellular homeostasis and cellular dynamics. In this chapter, we describe a protocol for the unbiased and high-throughput study of protein subcellular localization in the yeast Saccharomyces cerevisiae: hyperplexed localization of organelle proteins by isotope tagging (hyperLOPIT), which involves biochemical fractionation of Saccharomyces cerevisiae and high resolution mass spectrometry-based protein quantitation using TMT 10-plex isobaric tags. This protocol enables the determination of the subcellular localizations of thousands of proteins in parallel in a single experiment and thereby deep sampling and high-resolution mapping of the spatial proteome.  

3.3420           Stalled developmental programs at the root of pediatric brain tumors

Jessa, S., Blanchet-Cohen, A., Krug, B., Vladoiu, M., Coutelier, M. Faury, D. et al Nature Genetics, 51(12), 1702-1713 (2019)   Childhood brain tumors have suspected prenatal origins. To identify vulnerable developmental states, we generated a single-cell transcriptome atlas of >65,000 cells from embryonal pons and forebrain, two major tumor locations. We derived signatures for 191 distinct cell populations and defined the regional cellular diversity and differentiation dynamics. Projection of bulk tumor transcriptomes onto this dataset shows that WNT medulloblastomas match the rhombic lip-derived mossy fiber neuronal lineage and embryonal tumors with multilayered rosettes fully recapitulate a neuronal lineage, while group 2a/b atypical teratoid/rhabdoid tumors may originate outside the neuroectoderm. Importantly, single-cell tumor profiles reveal highly defined cell hierarchies that mirror transcriptional programs of the corresponding normal lineages. Our findings identify impaired differentiation of specific neural progenitors as a common mechanism underlying these pediatric cancers and provide a rational framework for future modeling and therapeutic interventions.  

3.3421           Control of CD1d-restricted antigen presentation and inflammation by sphingomyelin

Melum, E., Jiang, X., Baker, K.D., Macedo, M.F., Fritsch, J., Dowds, C.M. et al Nature Immunol., 20(12), 1644-1655 (2019)   Invariant natural killer T (iNKT) cells recognize activating self and microbial lipids presented by CD1d. CD1d can also bind non-activating lipids, such as sphingomyelin. We hypothesized that these serve as endogenous regulators and investigated humans and mice deficient in acid sphingomyelinase (ASM), an enzyme that degrades sphingomyelin. We show that ASM absence in mice leads to diminished CD1d-restricted antigen presentation and iNKT cell selection in the thymus, resulting in decreased iNKT cell levels and resistance to iNKT cell-mediated inflammatory conditions. Defective antigen presentation and decreased iNKT cells are also observed in ASM-deficient humans with Niemann–Pick disease, and ASM activity in healthy humans correlates with iNKT cell phenotype. Pharmacological ASM administration facilitates antigen presentation and restores the levels of iNKT cells in ASM-deficient mice. Together, these results demonstrate that control of non-agonistic CD1d-associated lipids is critical for iNKT cell development and function in vivo and represents a tight link between cellular sphingolipid metabolism and immunity.  

3.3422           The Thiol-Rich Interlayer in the Shell/Core Architecture of Mussel Byssal Threads

Valois, E., Hoffman, C., Demartini, D.G. and Waite, J.H. Langmuir, 35(48), 15985-15991 (2019)   The mussel byssus thread is an extremely tough core-shelled fiber that dissipates substantial amounts of energy during tensile loading. The mechanical performance of the shell is critically reliant on 3,4-dihydroxyphenylalanine’s (Dopa) ability to form reversible iron-catecholate complexes at pH 8. However, the formation of these coordinate cross-links is undercut by Dopa’s oxidation to Dopa-quinone, a spontaneous process at seawater conditions. The large mechanical mismatch between the cuticle and the core lends itself to further complications. Despite these challenges, the mussel byssus thread performs its tethering function over long periods of time. Here, we address these two major questions: (1) how does the mussel slow/prevent oxidation in the cuticle, and (2) how is the mechanical mismatch at the core/shell interface mitigated? By combining a number of microscopy and spectroscopy techniques we have discerned a previously undescribed layer. Our results indicate this interlayer is thiol rich and thus will be called the thiol-rich interlayer (TRL). We propose the TRL serves as a long-lasting redox reservoir as well as a mechanical barrier.  

3.3423           Fiber Formation from Liquid Crystalline Collagen Vesicles Isolated from Mussels

Renner-Rao, M., Clark, M. and Harrington, M.J. Langmuir, 35(48), 15992-16001 (2019)   Marine mussels (Mytilus edulis) fabricate byssal threads, high-performance biopolymeric fibers, which exhibit exceptional toughness and self-healing capacity. These properties are associated with collagenous proteins in the fibrous thread core known as preCols that self-organize into a hierarchical semicrystalline structure. Threads assemble individually in a bottom-up process lasting just minutes via secretion of membrane bound vesicles filled with preCols. However, very little is understood about the details and dynamics of this assembly process. Here, we explore the hypothesis that preCols are stored within the vesicles in a liquid crystalline phase, which contributes to fiber assembly by preordering molecules. To achieve this, a protocol was developed for extracting and isolating intact preCol secretory vesicles in high yield and purity. Vesicles were characterized and were manipulated in vitro, clearly indicating the dynamic liquid crystalline nature of the proteins within. Moreover, mechanical shearing of vesicles led to formation of highly birefringent preCol fibers. These findings have relevance for efforts toward sustainable production of advanced polymeric materials, and possibly for engineering biomedical scaffolds based on collagenous proteins.  

3.3424           Improved Label-Free Identification of Individual Exosome-like Vesicles with Au@Ag Nanoparticles as SERS Substrate

Fraire, J.C., Stremersch, S., Bouckaert, D., Monteyne, T., De Beer, T., Wuytens, P., De Rycke, R., Skirtach, A.G., Raemdonck, K., De Smedt, S. and Braeckmans, K. ACS Appl. Mater. Interfaces, 11, 39424-39435 (2019)   Exosome-like vesicles (ELVs) are nanovectors released by cells that are endowed with a variety of molecules, including proteins, nucleic acids, and chemicals that reflect the molecular signature of the producing cell. Given their presence in many biofluids, they form an easily accessible biomarker for early disease detection. Previously we demonstrated the possibility of identifying individual ELVs by analyzing their molecular signatures with surface-enhanced Raman scattering (SERS) after functionalization of ELVs with 4-(dimethylamino)pyridine (DMAP)-stabilized gold nanoparticles (AuNP). Although this strategy was capable of distinguishing ELVs from different cellular origins, the quality of the SERS spectra was suboptimal due to high background coming from the DMAP stabilizing molecules at the AuNP surface. In this study we demonstrate that it is possible to eliminate interfering SERS signals from stabilizing molecules at the AuNP surface by overgrowing in situ the ELV-attached AuNPs with a silver layer so as to form a core–shell nanoparticle (Au@AgNPs) directly at the ELV surface. As such it represents the first known strategy to generate clear SERS spectral fingerprints of delicate biological structures without interference of linker molecules that are needed to ensure colloidal stability of the plasmonic NP and to allow them to associate to the ELV surface. This new strategy using core–shell plasmonic NPs as SERS substrate showed higher near-field enhancements than previous approaches, which resulted in SERS spectra with improved signal-to-noise ratio. This allowed us to discriminate individual vesicles derived from B16F10 melanoma cells and red blood cells (RBC) with an unprecedented sensitivity and specificity >90%. Importantly, thanks to the higher near field enhancement the acquisition time could be reduced by 20-fold in comparison to previously reported strategies, paving the way toward high-throughput label-free single ELV identification.  

3.3425           Common Repository of FBS Proteins (cRFP) To Be Added to a Search Database for Mass Spectrometric Analysis of Cell Secretome

Shun, J., Kwon, Y., Lee, S., Na, S., Hong, E.Y., Jum S., Jung, H-G. eet al

J. Proteome Res., 18, 3800-3806 (2019)

 

We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data search of cell secretomes. cRFP is a small supplementary sequence list of highly abundant fetal bovine serum proteins added to the reference database in use. The aim behind using cRFP is to prevent the contaminant FBS proteins from being misidentified as other proteins in the reference database, just as we would use cRAP (common Repository of Adventitious Proteins) to prevent contaminant proteins present either by accident or through unavoidable contacts from being misidentified as other proteins. We expect it to be widely used in experiments where the proteins are obtained from serum-free media after thorough washing of the cells, or from a complex media such as SILAC, or from extracellular vesicles directly.

 

3.3426           Stiffness and Membrane Anchor Density Modulate DNA-Nanospring-Induced Vesicle Tubulation

Grome, M.W., Zhang, Z. and Lin, C. ACS Appl. Mater. Interfaces, 11, 22987-22992 (2019)   DNA nanotechnology provides an avenue for the construction of rationally designed artificial assemblages with well-defined and tunable architectures. Shaped to mimic natural membrane-deforming proteins and equipped with membrane anchoring molecules, curved DNA nanostructures can reproduce subcellular membrane remodeling events such as vesicle tubulation in vitro. To systematically analyze how structural stiffness and membrane affinity of DNA nanostructures affect the membrane remodeling outcome, here we build DNA-origami curls with varying thickness and amphipathic peptide density, and have them polymerize into nanosprings on the surface of liposomes. We find that modestly reducing rigidity and maximizing the number of membrane anchors not only promote membrane binding and remodeling but also lead to the formation of lipid tubules with better defined diameters, highlighting the ability of programmable DNA-based constructs to controllably deform the membrane.  

3.3427           Investigating the Role of Mitochondria in Type 2 Diabetes – Lessons from Lipidomics and Proteomics Studies of Skeletal Muscle and Liver

Kappler, L., Kollipara, L., Lehmann, R. and Sickmann, A. Adv. in Exp. Med. Biol., 1158, 143-182 (2019)   Mitochondrial dysfunction is discussed as a key player in the pathogenesis of type 2 diabetes mellitus (T2Dm), a highly prevalent disease rapidly developing as one of the greatest global health challenges of this century. Data however about the involvement of mitochondria, central hubs in bioenergetic processes, in the disease development are still controversial. Lipid and protein homeostasis are under intense discussion to be crucial for proper mitochondrial function. Consequently proteomics and lipidomics analyses might help to understand how molecular changes in mitochondria translate to alterations in energy transduction as observed in the healthy and metabolic diseases such as T2Dm and other related disorders. Mitochondrial lipids integrated in a tool covering proteomic and functional analyses were up to now rarely investigated, although mitochondrial lipids might provide a possible lynchpin in the understanding of type 2 diabetes development and thereby prevention. In this chapter state-of-the-art analytical strategies, pre-analytical aspects, potential pitfalls as well as current proteomics and lipidomics-based knowledge about the pathophysiological role of mitochondria in the pathogenesis of type 2 diabetes will be discussed.  

3.3428           Exosomes Released from Rabies Virus-Infected Cells May be Involved in the Infection Process

Wang, J., Wu, F., Liu, C., Dai, W., Teng, Y., Su, W., Kong, W., Gao, F., Cai, l., Hou, A. and Jiang, C. Virologica Sinica, 34, 59-65 (2019)   Exosomes are cell-derived vesicles that are secreted by many eukaryotic cells. It has recently attracted attention as vehicles of intercellular communication. Virus-infected cells release exosomes, which contain viral proteins, RNA, and pathogenic molecules. However, the role of exosomes in virus infection process remains unclear and needs to be further investigated. In this study, we aimed to evaluate the effects of exosomes on rabies virus infection. OptiPrep™ density gradient centrifugation was used to isolate exosomes from rabies virus-infected cell culture supernatants. A rabies virus G protein enzyme-linked immunosorbent assay and acetylcholinesterase activity assays were performed to verify the centrifugation fractions. Exosomes were then characterized using transmission electron microscopy and Western blotting. Our results showed that rabies virus infection increased the release of exosomes. Treatment with GW4869 and si-Rab27a, two exosomal secretion inhibitors, inhibited exosome release. Furthermore, the inhibitors reduced the levels of extracellular and intracellular viral RNA. These data indicated that exosomes may participate in the viral infection process. Moreover, our results establish a basis for future research into the roles of exosomes in rabies virus infection and as potential targets for developing new antiviral strategies.  

3.3429           Truncated BRPF1 Cooperates with Smoothened to Promote Adult Shh Medulloblastoma

Aiello, G., Ballabio, C., Ruggeri, R., Romanel, A., Zippo, A. and Tiberi, L. Cell Reports, 29, 4036-4052 (2019)   The transition of neural progenitors to differentiated postmitotic neurons is mainly considered irreversible in physiological conditions. In the present work, we show that Shh pathway activation through SmoM2 expression promotes postmitotic neurons dedifferentiation, re-entering in the cell cycle and originating medulloblastoma in vivo. Notably, human adult patients present inactivating mutations of the chromatin reader BRPF1 that are associated with SMO mutations and absent in pediatric and adolescent patients. Here, we found that truncated BRPF1 protein, as found in human adult patients, is able to induce medulloblastoma in adult mice upon SmoM2 activation. Indeed, postmitotic neurons re-entered the cell cycle and proliferated as a result of chromatin remodeling of neurons by BRPF1. Our model of brain cancer explains the onset of a subset of human medulloblastoma in adult individuals where granule neuron progenitors are no longer present.  

3.3430           Histone Variant and Cell Context Determine H3K27M Reprogramming of the Enhancer Landscape and Oncogenic State

Nagaraja, S., Queszada, M.A., Gillespie, S.M., Suva, M.L., Nazarian, J. and Monje, M. Molecular Cell, 76, 965-980 (2019)   Development of effective targeted cancer therapies is fundamentally limited by our molecular understanding of disease pathogenesis. Diffuse intrinsic pontine glioma (DIPG) is a fatal malignancy of the childhood pons characterized by a unique substitution to methionine in histone H3 at lysine 27 (H3K27M) that results in globally altered epigenetic marks and oncogenic transcription. Through primary DIPG tumor characterization and isogenic oncohistone expression, we show that the same H3K27M mutation displays distinct modes of oncogenic reprogramming and establishes distinct enhancer architecture depending upon both the variant of histone H3 and the cell context in which the mutation occurs. Compared with non-malignant pediatric pontine tissue, we identify and functionally validate both shared and variant-specific pathophysiology. Altogether, we provide a powerful resource of epigenomic data in 25 primary DIPG samples and 5 rare normal pediatric pontine tissue samples, revealing clinically relevant functional distinctions previously unidentified in DIPG.  

3.3431           Isolation of microglia-derived extracellular vesicles: towards miRNA signatures and neuroprotection

Lemaire, Q., raffo-Romero, A., Arab, T., Van Camp, C., Drago, F., Forte, S., Gimeno, J-P., Begard, S., Colin, M., Vizioli, j., Sautiere, P-E., Salzet, m. and Lefebvre, C.

1. Nanobiotechnol., 17:119 (2019)

  The functional preservation of the central nervous system (CNS) is based on the neuronal plasticity and survival. In this context, the neuroinflammatory state plays a key role and involves the microglial cells, the CNS-resident macrophages. In order to better understand the microglial contribution to the neuroprotection, microglia-derived extracellular vesicles (EVs) were isolated and molecularly characterized to be then studied in neurite outgrowth assays. The EVs, mainly composed of exosomes and microparticles, are an important cell-to-cell communication process as they exhibit different types of mediators (proteins, lipids, nucleic acids) to recipient cells. The medicinal leech CNS was initially used as an interesting model of microglia/neuron crosstalk due to their easy collection for primary cultures. After the microglia-derived EV isolation following successive methods, we developed their large-scale and non-targeted proteomic analysis to (i) detect as many EV protein markers as possible, (ii) better understand the biologically active proteins in EVs and (iii) evaluate the resulting protein signatures in EV-activated neurons. The EV functional properties were also evaluated in neurite outgrowth assays on rat primary neurons and the RNAseq analysis of the microglia-derived EVs was performed to propose the most representative miRNAs in microglia-derived EVs. This strategy allowed validating the EV isolation, identify major biological pathways in EVs and corroborate the regenerative process in EV-activated neurons. In parallel, six different miRNAs were originally identified in microglia-derived EVs including 3 which were only known in plants until now. The analysis of the neuronal proteins under the microglial EV activation suggested possible miRNA-dependent regulation mechanisms. Taken together, this combination of methodologies showed the leech microglial EVs as neuroprotective cargos across species and contributed to propose original EV-associated miRNAs whose functions will have to be evaluated in the EV-dependent dialog between microglia and neurons.  

3.3432           Embryonic stem cell-derived extracellular vesicle-mimetic nanovesicles rescue erectile function by enhancing penile neurovascular regeneration in the streptozotocin-induced diabetic mouse

Kwon, M-H., Song, K-M., Limanjaya, A., Choi, M-J., Ghatak, K., Nguyen, N.M., Ock, J., Yin, G.N., Kang, J-H., Lee, M.R., Gho, Y.S., Ryu, J-K. and Suh, J-K. Scientific Reports, 9:20072 (2019)   Extracellular vesicles (EVs) have attracted particular interest in various fields of biology and medicine. However, one of the major hurdles in the clinical application of EV-based therapy is their low production yield. We recently developed cell-derived EV-mimetic nanovesicles (NVs) by extruding cells serially through filters with diminishing pore sizes (10, 5, and 1 μm). Here, we demonstrate in diabetic mice that embryonic stem cell (ESC)-derived EV-mimetic NVs (ESC-NVs) completely restore erectile function (~96% of control values) through enhanced penile angiogenesis and neural regeneration in vivo, whereas ESC partially restores erectile function (~77% of control values). ESC-NVs promoted tube formation in primary cultured mouse cavernous endothelial cells and pericytes under high-glucose condition in vitro; and accelerated microvascular and neurite sprouting from aortic ring and major pelvic ganglion under high-glucose condition ex vivo, respectively. ESC-NVs enhanced the expression of angiogenic and neurotrophic factors (hepatocyte growth factor, angiopoietin-1, nerve growth factor, and neurotrophin-3), and activated cell survival and proliferative factors (Akt and ERK). Therefore, it will be a better strategy to use ESC-NVs than ESCs in patients with erectile dysfunction refractory to pharmacotherapy, although it remains to be solved for future clinical application of ESC.  

3.3433           Recent advances and challenges in the recovery and purification of cellular exosomes

Ayala-mar, S., Donoso-Quezada, J., Gallo-Villanueva, R.C., Perez-Gonzalez, V.H. and Gonzalez-Valdez, J. Electrophoresis, 40, 3036-3049 (2019)   Exosomes are nanovesicles secreted by most cellular types that carry important biochemical compounds throughout the body with different purposes, playing a preponderant role in cellular communication. Because of their structure, physicochemical properties and stability, recent studies are focusing in their use as nanocarriers for different therapeutic compounds for the treatment of different diseases ranging from cancer to Parkinson's disease. However, current bioseparation protocols and methodologies are selected based on the final exosome application or intended use and present both advantages and disadvantages when compared among them. In this context, this review aims to present the most important technologies available for exosome isolation while discussing their advantages and disadvantages and the possibilities of being combined with other strategies. This is critical since the development of novel exosome‐based therapeutic strategies will be constrained to the effectiveness and yield of the selected downstream purification methodologies for which a thorough understanding of the available technological resources is needed.    

3.3434           Cytosolic aggregates in presence of non‐translocated proteins perturb endoplasmic reticulum structure and dynamics

Mookherjee, D., Majumder, P., Mukherjee, R., Chatterje, D., Kaul, Z., Das, S., Sougrat, R., Chakrabarti, S., Chakrabaru, O. Traffic, 20(12), 943-960 (2019)   Presence of cytosolic protein aggregates and membrane damage are two common attributes of neurodegenerative diseases. These aggregates delay degradation of non‐translocated protein precursors leading to their persistence and accumulation in the cytosol. Here, we find that cells with intracellular protein aggregates (of cytosolic prion protein or huntingtin) destabilize the endoplasmic reticulum (ER) morphology and dynamics when non‐translocated protein load is high. This affects trafficking of proteins out from the ER, relative distribution of the rough and smooth ER and three‐way junctions that are essential for the structural integrity of the membrane network. The changes in ER membranes may be due to high aggregation tendency of the ER structural proteins—reticulons, and altered distribution of those associated with the three‐way ER junctions—Lunapark. Reticulon4 is seen to be enriched in the aggregate fractions in presence of non‐translocated protein precursors. This could be mitigated by improving signal sequence efficiencies of the proteins targeted to the ER. These were observed using PrP variants and the seven‐pass transmembrane protein (CRFR1) with different signal sequences that led to diverse translocation efficiencies. This identifies a previously unappreciated consequence of cytosolic aggregates on non‐translocated precursor proteins—their persistent presence affects ER morphology and dynamics. This may be one of the ways in which cytosolic aggregates can affect endomembranes during neurodegenerative disease.  

3.3435           UNC93B1 recruits syntenin-1 to dampen TLR7 signalling and prevent autoimmunity

Majer, O., Liu, B., Kreuk, S.M., Krogan, N. and Barton, G.M. Nature, 575, 366-370 (2019)   At least two members of the Toll-like receptor (TLR) family, TLR7 and TLR9, can recognize self-RNA and self-DNA, respectively. Despite the structural and functional similarities between these receptors, their contributions to autoimmune diseases such as systemic lupus erythematosus can differ. For example, TLR7 and TLR9 have opposing effects in mouse models of systemic lupus erythematosus—disease is exacerbated in TLR9-deficient mice but attenuated in TLR7-deficient mice1. However, the mechanisms of negative regulation that differentiate between TLR7 and TLR9 are unknown. Here we report a function for the TLR trafficking chaperone UNC93B1 that specifically limits signalling of TLR7, but not TLR9, and prevents TLR7-dependent autoimmunity in mice. Mutations in UNC93B1 that lead to enhanced TLR7 signalling also disrupt binding of UNC93B1 to syntenin-1, which has been implicated in the biogenesis of exosomes2. Both UNC93B1 and TLR7 can be detected in exosomes, suggesting that recruitment of syntenin-1 by UNC93B1 facilitates the sorting of TLR7 into intralumenal vesicles of multivesicular bodies, which terminates signalling. Binding of syntenin-1 requires phosphorylation of UNC93B1 and provides a mechanism for dynamic regulation of TLR7 activation and signalling. Thus, UNC93B1 not only enables the proper trafficking of nucleic acid-sensing TLRs, but also sets the activation threshold of potentially self-reactive TLR7.  

3.3436           Release from UNC93B1 reinforces the compartmentalized activation of select TLRs

Majer, O., Liu, B., Woo, B.J., Kreuk, L.S.M., Van Dis, E. and Barton, G.M. Nature, 575, 371-374 (2019)   Nucleic acid-sensing Toll-like receptors (TLRs) are subject to complex regulation to facilitate the recognition of microbial DNA and RNA while limiting the recognition of an organism’s own nucleic acids1. Failure to properly regulate these TLRs can lead to autoimmune and autoinflammatory diseases2^,3^,4^,5^,6. Intracellular localization of these receptors is thought to be crucial for the discrimination between self and non-self7, but the molecular mechanisms that reinforce compartmentalized activation of intracellular TLRs remain poorly understood. Here we describe a mechanism that prevents the activation of TLR9 from locations other than endosomes. This control is achieved through the regulated release of the receptor from its trafficking chaperone UNC93B1, which occurs only within endosomes and is required for ligand binding and signal transduction. Preventing release of TLR9 from UNC93B1, either by mutations in UNC93B1 that increase affinity for TLR9 or through an artificial tether that impairs release, results in defective signalling. Whereas TLR9 and TLR3 are released from UNC93B1, TLR7 does not dissociate from UNC93B1 in endosomes and is regulated by distinct mechanisms. This work defines a checkpoint that reinforces the compartmentalized activation of TLR9, and provides a mechanism by which activation of individual endosomal TLRs may be distinctly regulated.  

3.3437           Extracellular vesicles in urologic malignancies—Implementations for future cancer care

Wu, Z., Zhang, Z., Xia, W., Cai, J., Li, Y. and Wu, S.

Cell Proliferation, 52, e12659 (2019)

  Extracellular vesicles (Evs), a heterogeneous group of vesicles differing in size and shape, cargo content and function, are membrane‐bound and nano‐sized vesicles that could be released by nearly all variations of cells. Evs have gained considerable attention in the past decades for their functions in modulating intercellular signalling and roles as potential pools for the novel diagnostic and prognostic biomarkers, as well as therapeutic targets in several cancers including urological neoplasms. In general, human and animal cells both can release distinct types of Evs, including exosomes, microvesicles, oncosomes and large oncosomes, and apoptotic bodies, while the content of Evs can be divided into proteins, lipids and nucleic acids. However, the lack of standard methods for isolation and detection platforms rein the widespread usage in clinical applications warranted furthermore investigations in the development of reliable, specific and sensitive isolation techniques. Whether and how the Evs work has become pertinent issues. With the aid of high‐throughput proteomics or genomics methods, a fully understanding of contents contained in Evs from urogenital tumours, beyond all doubt, will improve our ability to identify the complex genomic alterations in the process of cancer and, in turn, contribute to detect potential therapeutic target and then provide personalization strategy for patient.  

3.3438           PLAG enhances macrophage mobility for efferocytosis of apoptotic neutrophils via membrane redistribution of P2Y2

Kim, G.T., Hahn, K.W., Sohn, K-Y., Yoon, S.Y., and Kim, J.W. FEBS J., 286(24), 5016-5029 (2019)   Neutrophil activity, including trapping of damage‐associated molecular patterns by neutrophil extracellular traps (NETs), is an important response to microbial infection. Most activated neutrophils commit to apoptosis and are removed by activated macrophages in the process of efferocytosis. Improper clearance of apoptotic neutrophils often causes an unnecessary and exaggerated immune response and subsequent chronic inflammation. Effective macrophage mobility toward activated neutrophils, which is triggered by binding of ‘find‐me’ signals to receptors such as P2Y2, is a crucial step for the timely clearance of apoptotic neutrophils. In this paper, we investigated the effect of 1‐palmitoyl‐2‐linoleoyl‐3‐acetyl‐rac‐glycerol (PLAG) on efferocytosis and the underlying molecular mechanisms. In a coculture of apoptotic neutrophils with macrophages, PLAG treatment increased levels of efferocytosis of apoptotic neutrophils. PLAG induced faster translocation of P2Y2 from lipid rafts to nonlipid raft plasma membrane domains in macrophages. This repositioning of P2Y2 enables the polarization of the cytoskeleton by association of the receptor with cytoskeletal proteins such as α‐tubulin and actin to improve the mobility of macrophages. The formation of vesicular, chylomicron‐like structures by PLAG was a prerequisite for the induction of this macrophage activity, as none of these effects was seen when the vesicle receptor GPIHBP1 was absent. Taken together, these data showed that PLAG is a powerful immune resolvent that triggers the prompt clearance of apoptotic neutrophils by enhanced efferocytosis activity. PLAG could therefore be an effective lipid‐based efferocytosis enhancer for use as a therapeutic drug to prevent inflammatory disease caused by uncontrolled immune responses.  

3.3439           Insights into the antibiotic resistance dissemination in a wastewater effluent microbiome: bacteria, viruses and vesicles matter

Maestro-Carballa, L., Gomez, M.L., Navarro, A.A., Garcia-Heredia, I., Martinez-Hernandez, F. and Martinez-Garcia, M. Environ. Microbiol., 21(12), 4582-4596 (2019)   Wastewater treatment plants effluents are considered as hotspots for the dispersion of antibiotic resistance genes (ARGs) into natural ecosystems. The bacterial resistome (ARG collection in a metagenome) analyses have provided clues on antibacterial resistance dynamics. However, viruses and vesicles are frequently ignored. Here, we addressed the bacterial, viral and vesicle resistomes from a representative wastewater effluent in natural conditions and amended with polymyxin, which is used as a last resort antibiotic. Metagenomics showed that the natural prokaryotic resistome was vast (9000 ARG hits/Gb metagenome) and diverse, while viral resistome was two orders of magnitude lower (50 ARG hits/Gb metagenome) suggesting that viruses rarely encoded ARGs. After polymyxin amendment, data showed no ARG enrichment – including to polymyxin – in the microbiome. Remarkably, microbiomes responded to polymyxin with a vast release of putative vesicles (threefold increase compared with the control), which might be used as 'traps' to decrease the antibiotic concentration. Intriguingly, although polymyxin resistance genes (PRGs) were rare in the microbiome (0.018% of total ARG found), in the viral and vesicle fractions, PRGs were more abundant (0.5%–0.8% of total ARG found). Our data suggest that vesicles could have a more active role in the context of transmission of antibiotic resistances.  

3.3440           Systemic Exosomal Delivery of shRNA Minicircles Prevents Parkinsonian Pathology

Izco, M., Blesa, J., Schleef, M., Schmeer, M., Porcari, R., Al-Shawi, R. et al Molecular Therapy, 27(12), 2111-2122 (2019)   The development of new therapies to slow down or halt the progression of Parkinson’s disease is a health care priority. A key pathological feature is the presence of alpha-synuclein aggregates, and there is increasing evidence that alpha-synuclein propagation plays a central role in disease progression. Consequently, the downregulation of alpha-synuclein is a potential therapeutic target. As a chronic disease, the ideal treatment will be minimally invasive and effective in the long-term. Knockdown of gene expression has clear potential, and siRNAs specific to alpha-synuclein have been designed; however, the efficacy of siRNA treatment is limited by its short-term efficacy. To combat this, we designed shRNA minicircles (shRNA-MCs), with the potential for prolonged effectiveness, and used RVG-exosomes as the vehicle for specific delivery into the brain. We optimized this system using transgenic mice expressing GFP and demonstrated its ability to downregulate GFP protein expression in the brain for up to 6 weeks. RVG-exosomes were used to deliver anti-alpha-synuclein shRNA-MC therapy to the alpha-synuclein preformed-fibril-induced model of parkinsonism. This therapy decreased alpha-synuclein aggregation, reduced the loss of dopaminergic neurons, and improved the clinical symptoms. Our results confirm the therapeutic potential of shRNA-MCs delivered by RVG-exosomes for long-term treatment of neurodegenerative diseases.  

3.3441           Investigation of somatic CNVs in brains of synucleinopathy cases using targeted SNCA analysis and single cell sequencing

Perez-Rodriguez, D., Kalyva, M., Leja-Salazar, M., Lashley, T., Tarabichi, M. Acta Neuropathol. Commun., 7:219 (2019)   Synucleinopathies are mostly sporadic neurodegenerative disorders of partly unexplained aetiology, and include Parkinson’s disease (PD) and multiple system atrophy (MSA). We have further investigated our recent finding of somatic SNCA (α-synuclein) copy number variants (CNVs, specifically gains) in synucleinopathies, using Fluorescent in-situ Hybridisation for SNCA, and single-cell whole genome sequencing for the first time in a synucleinopathy. In the cingulate cortex, mosaicism levels for SNCA gains were higher in MSA and PD than controls in neurons (> 2% in both diseases), and for MSA also in non-neurons. In MSA substantia nigra (SN), we noted SNCA gains in > 3% of dopaminergic (DA) neurons (identified by neuromelanin) and neuromelanin-negative cells, including olig2-positive oligodendroglia. Cells with CNVs were more likely to have α-synuclein inclusions, in a pattern corresponding to cell categories mostly relevant to the disease: DA neurons in Lewy-body cases, and other cells in the striatonigral degeneration-dominant MSA variant (MSA-SND). Higher mosaicism levels in SN neuromelanin-negative cells may correlate with younger onset in typical MSA-SND, and in cingulate neurons with younger death in PD. Larger sample sizes will, however, be required to confirm these putative findings. We obtained genome-wide somatic CNV profiles from 169 cells from the substantia nigra of two MSA cases, and pons and putamen of one. These showed somatic CNVs in ~ 30% of cells, with clonality and origins in segmental duplications for some. CNVs had distinct profiles based on cell type, with neurons having a mix of gains and losses, and other cells having almost exclusively gains, although control data sets will be required to determine possible disease relevance. We propose that somatic SNCA CNVs may contribute to the aetiology and pathogenesis of synucleinopathies, and that genome-wide somatic CNVs in MSA brain merit further study.  

3.3442           The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis

Bersuker, K., Hendricks, J.M., Li, Z., magtanong, L., Ford, B., Tang, P.H., Roberts, M.A., Tong, B., Maimone, T.J., Zoncu, R., Bassik, M.C., Nomura, D.K., Dixon, S.J. and Olzmann, J.A. Nature, 575, 688-692 (2019)   Ferroptosis is a form of regulated cell death that is caused by the iron-dependent peroxidation of lipids1^,2. The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols3^,4. Ferroptosis has previously been implicated in the cell death that underlies several degenerative conditions2, and induction of ferroptosis by the inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death5. However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines6, which suggests that additional factors govern resistance to ferroptosis. Here, using a synthetic lethal CRISPR–Cas9 screen, we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis-resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q₁₀ (CoQ) (also known as ubiquinone-10), which acts as a lipophilic radical-trapping antioxidant that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumour xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutic agents.  

3.3443           MOWChIP-seq for low-input and multiplexed profiling of genome-wide histone modifications

Zhu, B., Hsieh, Y-P., Murphy, T.W., Zhang, Q., Naler, L.B. and Lu, C. Nature Protocols, 14, 3366-33394 (2019)   Epigenetic mechanisms such as histone modifications play critical roles in adaptive tuning of chromatin structures. Profiling of various histone modifications at the genome scale using tissues from animal and human samples is an important step for functional studies of epigenomes and epigenomics-based precision medicine. Because the profile of a histone mark is highly specific to a cell type, cell isolation from tissues is often necessary to generate a homogeneous cell population, and such operations tend to yield a low number of cells. In addition, high-throughput processing is often desirable because of the multiplexity of histone marks of interest and the large quantity of samples in a hospital setting. In this protocol, we provide detailed instructions for device fabrication, setup, and operation of microfluidic oscillatory washing–based chromatin immunoprecipitation followed by sequencing (MOWChIP-seq) for profiling of histone modifications using as few as 100 cells per assay with a throughput as high as eight assays in one run. MOWChIP-seq operation involves flowing of chromatin fragments through a packed bed of antibody-coated beads, followed by vigorous microfluidic oscillatory washing. Our process is semi-automated to reduce labor and improve reproducibility. Using one eight-unit device, it takes 2 d to produce eight sequencing libraries from chromatin samples. The technology is scalable. We used the protocol to study a number of histone modifications in various types of mouse and human tissues. The protocol can be conducted by a user who is familiar with molecular biology procedures and has basic engineering skills.  

3.3444           Advances in Technologies for Purification and Enrichment of Extracellular Vesicles

Zhang, P., Yeo, J.C. and Lim, C.T. SLAS Technol., 24(5), 477-488 (2019)   Extracellular vesicles (EVs) are lipid bilayer-bound vesicles secreted by cells. Subtypes of EVs such as microvesicles and exosomes are further categorized mainly by their different biogenesis mechanisms. EVs have been revealed to play an important role in disease diagnosis and intercellular communication. Despite the wide interest in EVs, the technologies for the purification and enrichment of EVs are still in their infancy. The isolation of EVs, especially exosomes, is inherently challenging due to their small size and heterogeneity. In this review, we mainly introduce the advances of techniques in isolating microvesicles and exosomes according to their approaches. Also, we discuss the limitations of currently reported technologies in terms of their specificity and efficiency, and provide our thoughts about future developments of EV purification and enrichment technology.  

3.3445           HTLV-1 Extracellular Vesicles Promote Cell-to-Cell Contact

Pinto, D.O., DeMarino, C., Pieet, M.L., Cowen, M., Branscome, H., Al Sharif, S., Jones, j., Dutarte, H., Lepene, B., Liotta, L.A., Mahieux, R. and Kashanchi, F. Frontiers in Microbiol., 10, 2147 (2019)   Human T-cell leukemia virus-1 (HTLV-1) is a neglected and incurable retrovirus estimated to infect 5 to 10 million worldwide. Specific indigenous Australian populations report infection rates of more than 40%, suggesting a potential evolution of the virus with global implications. HTLV-1 causes adult T-cell leukemia/lymphoma (ATLL), and a neurological disease named HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Even though HTLV-1 transmission primarily occurs from cell-to-cell, there is still a gap of knowledge regarding the mechanisms of viral spread and disease progression. We have recently shown that Extracellular Vesicles (EVs) ubiquitously produced by cells may be used by HTLV-1 to transport viral proteins and RNA, and elicit adverse effects on recipient uninfected cells. The viral proteins Tax and HBZ are involved in disease progression and impairment of autophagy in infected cells. Here, we show that activation of HTLV-1 via ionizing radiation (IR) causes a significant increase of intracellular Tax, but not EV-associated Tax. Also, lower density EVs from HTLV-1-infected cells, separated by an Iodixanol density gradient, are positive for gp61+++/Tax+++/HBZ+ proteins (HTLV-1 EVs). We found that HTLV-1 EVs are not infectious when tested in multiple cell lines. However, these EVs promote cell-to-cell contact of uninfected cells, a phenotype which was enhanced with IR, potentially promoting viral spread. We treated humanized NOG mice with HTLV-1 EVs prior to infection and observed an increase in viral RNA synthesis in mice compared to control (EVs from uninfected cells). Proviral DNA levels were also quantified in blood, lung, spleen, liver, and brain post-treatment with HTLV-1 EVs, and we observed a consistent increase in viral DNA levels across all tissues, especially the brain. Finally, we show direct implications of EVs in viral spread and disease progression and suggest a two-step model of infection including the release of EVs from donor cells and recruitment of recipient cells as well as an increase in recipient cell-to-cell contact promoting viral spread.  

3.3446           Neuronal-derived extracellular vesicles are enriched in the brain and serum of HIV-1 transgenic rats

Dagur, R.S., Liao, K., Sil, S., Niu, F., Sun, Z., Lyubchenko, L., Peeples, E.S., Hu, G and Buch, S.

1. Extracellular Vesicles, 9(1), 1703249 (2020)

  Despite the efficacy of combination antiretroviral therapy (ART) in controlling human immunodeficiency virus (HIV-1) replication, cytotoxic viral proteins such as HIV-1 transactivator of transcription (Tat) persist in tissues such as the brain. Although HIV-1 does not infect neuronal cells, it is susceptible to viral Tat protein-mediated toxicity, leading to neuroinflammation that underlies HIV-associated neurocognitive disorders (HAND). Given the role of extracellular vesicles (EVs) in both cellular homoeostasis and under pathological conditions, we sought to investigate the alterations in the quantity of neuronal-derived EVs in the brain – as defined by the presence of cell adhesion molecule L1 (L1CAM) and to evaluate the presence of L1CAM⁺ EVs in the peripheral circulation of HIV-1 transgenic (HIV-1 Tg) rats. The primary goal of this study was to investigate the effect of long-term exposure of HIV-1 viral proteins on the release of neuronal EVs in the brain and their transfer in the systemic compartment. Brain and serum EVs were isolated from both wild type and HIV-1 Tg rats using differential ultracentrifugation with further purification using the Optiprep gradient method. The subpopulation of neuronal EVs was further enriched using immunoprecipitation. The current findings demonstrated increased presence of L1CAM⁺ neuronal-derived EVs both in the brain and serum of HIV-1 Tg rats.  

3.3447           Blood concentrations of small extracellular vesicles are determined by a balance between abundant secretion and rapid clearance

Matsumoto, A., Takahashi, Y., Chang, H-S., Wu, Y-W., Yamamoto, A., Ishihama, Y. and Takakura, Y.

1. Extracellylar Vesicles, 9(1), 1696517 (2020)

  Small extracellular vesicles (sEVs) are important mediators of cell–cell communication with respect to diverse physiological processes. To further understand their physiological roles, understanding blood sEV homoeostasis in a quantitative manner is desired. In this study, we propose novel kinetic approaches to estimate the secretion and clearance of mouse plasma–derived sEVs (MP-sEVs) based on the hypothesis that blood sEV concentrations are determined by a balance between the secretion and clearance of sEVs. Using our specific and sensitive sEV labelling technology, we succeeded in analysing MP-sEV clearance from the blood after intravenous administration into mice. This revealed the rapid disappearance of MP-sEVs with a half-life of approximately 7 min. Moreover, the plasma sEV secretion rate, which is presently impossible to directly evaluate, was calculated as 18 μg/min in mice based on pharmacokinetic (PK) analysis. Next, macrophage-depleted mice were prepared as a model of disrupted sEV homoeostasis with retarded sEV clearance. MP-sEV concentrations were increased in macrophage-depleted mice, which probably reflected a shift in the balance of secretion and clearance. Moreover, the increased MP-sEV concentration in macrophage-depleted mice was successfully simulated using calculated clearance rate constant, secretion rate constant and volume of distribution, suggesting the validity of our PK approaches. These results demonstrate that blood sEV concentration homoeostasis can be explained by the dynamics of rapid secretion/clearance.  

3.3448           Characterization of protein, long noncoding RNA and microRNA signatures in extracellular vesicles derived from resting and degranulated mast cells

Liang, Y., Huang, S., Qiao, L., Peng, X., Li, C., Lin, K., Xie, G., Li, J., Lin, L., Yin, Y., Liao, H.L., Li, Q. and Li, L.

1. Extracellular Vesicles, 9(1), 1697583 (2020)

  Mast cells (MCs) are known to participate in a variety of patho-physiological processes depending largely on the intragranular mediators and the production of cytokines and chemokines during degranulation. Recently, extracellular vesicles (EVs) have been implicated important functions for MCs, but the components of MC-derived EVs have not yet been well-characterized. In this study, we aimed to identify signatures of proteins, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs) in EVs derived from resting (Rest-EV) and degranulated (Sti-EV) MCs by differential ultracentrifugation. Using tandem mass tag (TMT)-based quantitative proteomics technology and RNA sequencing, we identified a total of 1988 proteins, 397 lncRNAs, and 272 miRNAs in Rest-EV and Sti-EV. The proteins include common EVs markers (cytoskeletal proteins), MCs markers (FcεRI and tryptase), and some preformed MCs mediators (lysosomal enzymes) as well. The global expression profiles of lncRNAs and miRNAs identified, for the first time, from Rest-EV and Sti-EV, strongly suggest a potential regulatory function of MC-derived EVs. We have also performed Western blotting and qRT-PCR analysis to further verify some of the proteins, lncRNAs, and miRNAs identified from Rest-EV and Sti-EV. Our findings will help to elucidate the functions of MC-derived EVs, and provide a reference dataset for future translational studies involving MC-derived EVs.  

3.3449           Exosomes cloak the virion to transmit Enterovirus 71 non-lytically

Gu, J.G., Wu, J., Fang, D., Qiu, Y., Zou, X., Jia, X., Yin, Y., Shen, L. and Mao, L.

1. Extracellular Vesicles, 11(1), 32-38 (2020)

  Enterovirus 71 (EV71) is a non-enveloped virus and it can be released from host cells through a traditional cytolytic manner. Now, we showed EV71 could be spread non-lytically between cells during early viral infection. In order to explain this phenomenon, we separated supernatant fluids of rhabdomyosarcoma (RD) cells cultures infected with EV71 by isopycnic gradient centrifugation. Two populations of virus particles were morphology indistinguishable by transmission electron microscope (TEM). It showed that some EV71 particles were wrapped inside extracellular vesicles which were verified to be exosomes by immunoassay and morphologic analysis. In addition, exosomes containing viral RNA were shed in plasma of EV71-infected encephalitis in children. Our findings indicate that the “non-enveloped” EV71 virions could be wrapped within exosomes which promote their spread in the absence of cell lysis.  

3.3450           Heat shock factor 4 regulates lysosome activity by modulating the αB-crystallin-ATP6V1A-mTOR complex in ocular lens

Cui, X., Feng, R., Wang, J., Du, C., Pi, X., Chen, D., Li, J., Li, H., Zhang, J., Zhang, J., Mu, H., Zhang, F., Liu, M. and Hu, Y. BBA-General Subjects, 1864, 129496 (2020)   Background Germline mutations in heat shock factor 4 (HSF4) cause congenital cataracts. Previously, we have shown that HSF4 is involved in regulating lysosomal pH in mouse lens epithelial cell in vitro. However, the underlying mechanism remains unclear. Methods HSF4-deficient mouse lens epithelial cell lines and zebrafish were used in this study. Immunoblotting and quantitative RT-PCR were used for expression analysis. The protein-protein interactions were tested with GST-pull downs. The lysosomes were fractioned by ultracentrifugation. Results HSF4 deficiency or knock down of αB-crystallin elevates lysosomal pH and increases the ubiquitination and degradation of ATP6V1A by the proteasome. αB-crystallin localizes partially in the lysosome and interacts solely with the ATP6V1A protein of the V1 complex of V-ATPase. Furthermore, αB-crystallin can co-precipitate with mTORC1 and ATP6V1A in GST pull down assays. Inhibition of mTORC1 by rapamycin or siRNA can lead to dissociation of αB-crystallin from the ATP6V1A and mTORC1complex, shortening the half-life of ATP6V1A and increasing the lysosomal pH. Mutation of ATP6V1A/S441A (the predicted mTOR phosphorylation site) reduces its association with αB-crystallin. In the zebrafish model, HSF4 deficiency reduces αB-crystallin expression and elevates the lysosomal pH in lens tissues. Conclusion HSF4 regulates lysosomal acidification by controlling the association of αB-crystallin with ATP6V1A and mTOR and regulating ATP6V1A protein stabilization.  

3.3451           Exosomes-mediated synthetic Dicer substrates delivery for intracellular Dicer imaging detection

Dai, W., Su, L., Lu, H., Dong, H. and Zhang, X. Biosensors and Bioelectronics, 151, 111907 (2020)   Ribonuclease Dicer initiates gene-silencing process by cleaving exogenously long RNA duplexes into small interfering RNA (siRNA) or endogenous precursor microRNAs (pre-miRNAs) into mature miRNAs. It holds great promise in cancer diagnosis and therapeutics due to its molecular ruler role. However, the intracellular Dicer detection remains a key challenge and Dicer related gene therapy has never been explored. In this study, we design a fluorescent labeling Dicer substrate and effectively deliver it into cell by exosomes derived from the target parent cells for intracellular Dicer expression level monitor and gene therapy. Using pre-miRNA let-7a as a model, the Dicer substrates with two terminals labeled with fluorescent and quencher group respectively was obtained by T4 RNA mediated ligase reaction from two short RNA sequences. Then, the substrate was packaged into exosomes by electroporation and delivered to target cells for intracellular dicer imaging detection. After packaging substrates into exosomes with little immunogenicity and good innate biocompatibility by electroporation and delivered to target cells, the Dicer mediated substrate cleavage was effectively monitored by the fluorescence recovery, providing a powerful tool for Dicer analysis. Importantly, the cleaved product exhibited significant suppression toward tumor cell growth and regulated cancer cells cycle. This work might open a new avenue for Dicer analysis and Dicer-related clinical application.  

3.3452           Passion fruit-like exosome-PMA/Au-BSA@Ce6 nanovehicles for real-time fluorescence imaging and enhanced targeted photodynamic therapy with deep penetration and superior retention behavior in tumor

Pan, S., Pei, L., Zhang, A., Zhang, Y., Zhang, C., Huang, M., Huang, Z., Liu, B., Wang, L., Ma, L., Zhang, Q. and Cui, D. Biomaterials, 230, 119606 (2020)   Exosomes (Exos) of approximately 30–150 nm in diameters are the promising vehicles for therapeutic drugs. However, several challenges still exist in clinical applications, such as unsatisfied yield of exosomes, complicated labeling procedure and low drug loading efficiency. In this work, the gram-scale amount of high-purity urinary exosomes can be obtained from gastric cancer patients by non-invasive method. Passion fruit-like Exo-PMA/Au-BSA@Ce6 nanovehicles were fabricated by considerable freshly-urinary Exos loaded efficiently with multi-functionalized PMA/Au-BSA@Ce6 nanoparticles via instant electroporation strategy. In this system, prepared Exo-PMA/Au-BSA@Ce6 nanovehicles could be internalized into cancer cells effectively, and could delay the endocytosis of macrophages and prolong blood circulation time owing to its membrane structure and antigens. Under 633 nm laser irradiation and acidic condition, the structures of nanovehicles would be collapsed and tremendous PMA/Au-BSA@Ce6 nanoparticles could be released inside cancer cells, produced considerable singlet oxygen, inhibiting growth of tumor cells. In vivo experiment of MGC-803 tumor-bearing nude mice showed that prepared Exo-PMA/Au-BSA@Ce6 nanovehicles could target tumor cells with deep penetration and superior retention performance in tumors. This work reports a reliable conjugation-free labeling strategy for tracking exosomes harvested from human urine. Moreover, the integration of multifunctional nanoparticles with urinary Exos paves a versatile road for the development of cancer-targeted photodynamic therapy.  

3.3453           White blood cell labeling with Technetium-99m (99mTc) using red blood cell extracellular vesicles-mimetics

Son, S.H., Oh, J.M., Gangadaran, P., Ji, H.D., Lee, H.W., Rajendran, R.L., Baek, S.H., Gopa, A., Kalimuthu, S., Jeong, S.Y., Lee, S-W., Lee, J. and Ahn, B-C. Blood Cells, Molecules and Diseases, 80, 102375 (2020)   Background Extracellular vesicles, have gained increasing attention for their application in drug delivery. Here, we developed a novel method for radiolabeling WBCs with ^99mTc using RBC-derived extracellular vesicles -mimetics (EVMs), and monitored in vivo inflammation tracking of ^99mTc-WBC using gamma camera in acute inflammation mouse model. Methods Engineered EVMs from RBCs were produced by a one-step extrusion method. RBC-EVMs were analyzed by NTA and TEM. Cells were labeled with ^99mTc by using ^99mTc-RBC-EVMs. Inflammation mice model was prepared and confirmed by ¹⁸F-FDG PET/CT. ^99mTc-WBCs were injected in mice, and their biodistribution was analyzed by gamma camera. Finding The radiochemical purity of ^99mTc-RBC-EVMs was 100%. The ^99mTc-labeling did't affect the size and morphology. The ^99mTc in the cytoplasm of RBC-EVMs was successfully confirmed by high angle annular dark field STEM (scanning transmission electron microscope). Cells were successfully labeled with ^99mTc using ^99mTc-RBC-EVMs, and the counts per minute was increased in dose- and time-dependent manners. The ¹⁸F-FDG PET/CT images confirmed establishment of acute inflammation (left mouse foot). ^99mTc-WBCs showed higher uptake in the inflamed foot than non-inflamed foot. Interpretation This novel method for radiolabeling WBCs using RBC-EVMs. ^99mTc labeling may be a feasible method to monitor the in vivo biodistribution of cells.  

3.3454           Traffic-related air pollutants (TRAP-PM) promote neuronal amyloidogenesis through oxidative damage to lipid rafts

Cacciottolo, M., Morgan, T.E., Saffari, A.A., Shirmohammadi, F., Forman, H.J., Sioutas, C. and Finch, C.E. Free Rad. Biol. Med., 147, 242-251 (2020)   Traffic-related air pollution particulate matter (TRAP-PM) is associated with increased risk of Alzheimer Disease (AD). Rodent models respond to nano-sized TRAP-PM (nPM) with increased production of amyloid Aβ peptides, concurrently with oxidative damage. Because pro-Aβ processing of the amyloid precursor protein (APP) occurs on subcellular lipid rafts, we hypothesized that oxidative stress from nPM exposure would alter lipid rafts to favor Aβ production. This hypothesis was tested with J20 mice and N2a cells transgenic for hAPPswe (familial AD). Exposure of J20-APPswe mice to nPM for 150 h caused increased lipid oxidation (4-HNE) and increased the pro-amyloidogenic processing of APP in lipid raft fractions in cerebral cortex; the absence of these changes in cerebellum parallels the AD brain region selectivity for Aβ deposits. In vitro, nPM induced similar oxidative responses in N2a-APPswe cells, with dose-dependent production of NO, oxidative damage (4-HNE, 3NT), and lipid raft alterations of APP with increased Aβ peptides. The antioxidant N-acetyl-cysteine (NAC) attenuated nPM-induced oxidative damage and lipid raft alterations of APP processing. These findings identify neuronal lipid rafts as novel targets of oxidative damage in the pro-amyloidogenic effects of air pollution.  

3.3455           Engineered extracellular vesicles with synthetic lipids via membrane fusion to establish efficient gene delivery

Jhan, Y-Y., Prasca-Chamorro, D., Zuniga, G.P., Moore, D.M., Kumar, S.A., Gaharwar, A.K. and Bishop, C.J. Int. J. Pharmaceut., 573, 118802 (2020)   The low yield of extracellular vesicle (EV) secretion is a major obstacle for mass production and limits their potential for clinical applications as a drug delivery platform. Here, we mass produced engineered extracellular vesicles (eEVs) by fusing the surface composition of EVs with lipid-based materials via a membrane extrusion technique. A library of lipids (DOTAP, POPC, DPPC and POPG) was fused with EVs to form a hybrid-lipid membrane structure. Uniform lamellar vesicles with a controlled size around 100 nm were obtained in this study. Particle number characterization revealed this extrusion method allowed a 6- to 43-fold increase in numbers of vesicles post- isolation. Further, exogenous siRNA was successfully loaded into engineered vesicles with ~ 15% – 20% encapsulation efficiency using electroporation technique. These engineered extracellular vesicles sustained a 14-fold higher cellular uptake to lung cancer cells (A549) and achieved an effective gene silencing effect comparable to commercial Lipofectamine RNAiMax. Our results demonstrate the surface composition and functionality of EVs can be tuned by extrusion with lipids and suggest the engineered vesicles can be a potential substitute as gene delivery carriers while being able to be mass produced to a greater degree with retained targeting capabilities of EVs.  

3.3456           Chapter 2 - Methods for exosome isolation and characterization

Zhou, M., Weber, S.R., Zhao, Y., Chen, H. and Sundstrom, J.M. Exosomes, a Clinical Compendium,  23-28 (2020)   Extracellular vesicles are released from cells under both physiological and pathophysiological conditions. Exosomes are a class of extracellular vesicles that originate from intraluminal membranes, and their diameters vary from 30 to 150 nm. Like hormones and cytokines, exosomes play a critical role in cell-cell communication. Proper methods for exosome isolation and characterization facilitate accurate downstream analyses. This review summarizes both conventional and novel techniques for exosome isolation and characterization as well as their advantages and limitations. Novel methods for fractionating exosome subsets, defining exosomal cargo, tracking exosomes in vivo, and determining whether a protein is associated with the inside or outside of exosomes are also included in this review.  

3.3457           sncRNAs packaged by Helicobacter pylori outer membrane vesicles attenuate IL-8 secretion in human cells

Zhang, H., Zhang, Y., Song, Z., Li, R., Ruan, H. and Liu, Q. Int. J. Med. Microbiol., 310, 151356 (2020)   Bacterial outer membrane vesicles (OMVs) play a vital role in the mechanism of host―pathogen communication, while emerging evidence suggests that OMVs regulate host immune responses through differentially packaged small noncoding RNAs (sncRNAs) to target host mRNA function. Therefore, we identified differentially packaged sncRNAs in Helicobacter pylori OMVs and showed transfer of OMV sncRNAs to human gastric adenocarcinoma cells in this study. Our data revealed that sncRNAs (sR-2509025 and sR-989262) were enriched in OMVs, and reduced lipopolysaccharide or OMV-induced interleukin 8 (IL-8) secretion by cultured AGS cells. Collectively, these findings are consistent with the hypothesis that sncRNAs in H. pylori OMVs play a novel role in the mechanism of host―pathogen interaction, whereby H. pylori evades the host immune response.  

3.3458           How presence of a signal peptide affects human galectins-1 and -4: Clues to explain common absence of a leader sequence among adhesion/growth-regulatory galectins

Kutzner, T.J., Higuero, A.M., Süssmair, M., Kopitz, J., HIngar, M., Diez-Revuelta, N., Caballero, G.G., Kaltner, H., Lindner, I., Abad-Rodriguez, J., Reusch, D. and GAbius, H-J. BBA-General Subjects, 1864, 129449 (2020)   Background Galectins are multifunctional effectors, which all share absence of a signal sequence. It is not clear why galectins belong to the small set of proteins, which avoid the classical export route. Methods Products of recombinant galectin expression in P. pastoris were analyzed by haemagglutination, gel filtration and electrophoresis and lectin blotting as well as mass spectrometry on the level of tryptic peptides and purified glycopeptides(s). Density gradient centrifugation and confocal laser scanning microscopy facilitated localization in transfected human and rat cells, proliferation assays determined activity as growth mediator. Results Directing galectin-1 to the classical secretory pathway in yeast produces N-glycosylated protein that is active. It cofractionates and -localizes with calnexin in human cells, only Gal-4 is secreted. Presence of N-glycan(s) reduces affinity of cell binding and growth regulation by Gal-1. Conclusions Folding and activity of a galectin are maintained in signal-peptide-directed routing, N-glycosylation occurs. This pathway would deplete cytoplasm and nucleus of galectin, presence of N-glycans appears to interfere with lattice formation. General significance Availability of glycosylated galectins facilitates functional assays to contribute to explain why galectins invariably avoid classical routing for export.  

3.3459           Exosomal Th1/Th2 cytokines in preeclampsia and HIV-positive preeclamptic women on highly active anti-retroviral therapy

Pillay, P., Moodley, K., Vatish, M., Moodley, J., Duarte, R. and Mackraj, I. Cytokine, 125, 154795 (2020)   Preeclampsia (PE) is a hypertensive disorder of pregnancy which is a leading cause of maternal and foetal morbidity and mortality. Furthermore, HIV/Highly Active Anti-Retroviral Treatment has been associated with the increased risk of preeclampsia due to maternal immune reconstitution, which complicates the clinical diagnosis of PE in these patients. It is therefore necessary to identify biomarkers involved in the pathology of both disorders with the intent to diagnose. Exosomal cytokines represent ideal biomarkers of PE and inflammatory conditions due to their immunomodulatory role in pregnancy. We therefore quantified exosomal Th1 (IL-2 and TNF-α) and Th2 cytokines (IL-10) in maternal circulation. A significant dysregulation in total exosomes, placental-derived exosomes and exosomal cytokines in PE and HIV-positive PE pregnant woman on Highly Active Antiretroviral Treatment (HAART) was observed (p < 0.01). Additionally, we observed a significant shift towards Th1 immunity in PE which becomes amplified in HIV-positive PE pregnant woman on HAART (p < 0.01). Moreover, we show the potential application of exosomal Tumor necrosis factor alpha (TNF-α) as a biomarker of PE and PE in HIV-positive pregnant women on HAART (CI: 95%, LHR > 10, sensitivity of 100% and specificity of 90%). These findings are in support of exosome release and exosome cytokine encapsulation as a tightly regulated process in favour of maintaining the immune microenvironment, which can orchestrate either normal pregnancy, or the pathogenesis of preeclampsia and preeclampsia in HIV/HAART pregnancies.  

3.3460           Chapter 16 - Potential role of exosomes in reproductive medicine and pregnancy

Nair, S. and Salomom, C. Exsosomes, A Clinical Compendium, 357-381 (2020)   Exosomes are nano-sized (~ 50–150 nm), specialized membrane-bound vesicles, derived from the endocytic compartments of the cell and released into the extracellular space. Recent studies identifies exosomes are important mediators of cell to cell communication by transferring biologically active molecules such as proteins and miRNAs in recipient cells. Exosomes play several roles in regulating the physiological events of pregnancy, essential for successful pregnancy outcomes. The most vital organ in pregnancy, the placenta, extrudes large quantities of extracellular vesicles including exosomes into the maternal circulation. Exosomes derived from placenta are involved in various biological processes including trophoblast invasion, feto-maternal vascular development, maternal immune response and metabolism. Herein, we review the biogenesis, characteristics and biological functions of exosomes in pregnancy, with a special focus on exosomes derived from placenta and highlight their importance in normal and pregnancies with adverse outcomes such as gestational diabetes mellitus, preeclampsia and preterm birth.  

3.3461           Complexity of the microRNA transcriptome of cow milk and milk-derived extracellular vesicles isolated via differential ultracentrifugation

Benmoussa, A., Laugier, J., Beaupariant, C.J., Lambert, M., Droit, A. and Provost, P.

1. Dairy Sci., 103, 16-29 (2020)

  MicroRNAs (miRNAs) are small gene-regulatory noncoding RNA that are highly enriched in cow milk. They are encapsulated in different extracellular vesicle (EV) subsets that protect them from the extracellular milieu and the harsh conditions of the gastrointestinal tract during digestion. Here, we isolated pellets enriched in 4 different EV subsets, via differential ultracentrifugation of commercial cow milk: 12,000 × g (P12K), 35,000 × g (P35K), 70,000 × g (P70K), and 100,000 × g (P100K). Small RNA sequencing (sRNA-Seq) analyses revealed an unprecedented level of diversity in the complete miRNA repertoire and features of unfractionated cow milk and derived EV subsets. Although 5 miRNA sequences represented more than 50% of all miRNAs, milk EV exhibited heterogeneous content of miRNAs and isomeric variants (termed isomiR): P100K EV were enriched in reference miRNA sequences, and P12K and P35K EV in related isomiR. Incubation of milk EV with human cultured HeLa cells led to cellular enrichment in miRNA miR-223, which was concomitant with decreased expression of a reporter gene placed under the control of miR-223, thereby demonstrating the functionality of miR-223. These results suggest that cow milk EV may transfer their miRNAs to human cells and regulate recipient cell gene expression programming in a manner as complex as that of their miRNA transcriptome. The biological activity and relevance of the different milk EV subsets and bioactive mediators, including small noncoding RNA, in health and disease, warrants further investigation.  

3.3462           Analyzing bacterial extracellular vesicles in human body fluids by orthogonal biophysical separation and biochemical characterization

Tulkens, J., De Wever, O. and Hendrix, A. Nature Protocols, 15, 40-67 (2020)   Gram-negative and Gram-positive bacteria release a variety of membrane vesicles through different formation routes. Knowledge of the structure, molecular cargo and function of bacterial extracellular vesicles (BEVs) is primarily obtained from bacteria cultured in laboratory conditions. BEVs in human body fluids have been less thoroughly investigated most probably due to the methodological challenges in separating BEVs from their matrix and host-derived eukaryotic extracellular vesicles (EEVs) such as exosomes and microvesicles. Here, we present a step-by-step procedure to separate and characterize BEVs from human body fluids. BEVs are separated through the orthogonal implementation of ultrafiltration, size-exclusion chromatography (SEC) and density-gradient centrifugation. Size separates BEVs from bacteria, flagella and cell debris in stool; and blood cells, high density lipoproteins (HDLs) and soluble proteins in blood. Density separates BEVs from fibers, protein aggregates and EEVs in stool; and low-density lipoproteins (LDLs), very-low-density lipoproteins (VLDLs), chylomicrons, protein aggregates and EEVs in blood. The procedure is label free, maintains the integrity of BEVs and ensures reproducibility through the use of automated liquid handlers. Post-separation BEVs are characterized using orthogonal biochemical endotoxin and Toll-like receptor-based reporter assays in combination with proteomics, electron microscopy and nanoparticle tracking analysis (NTA) to evaluate BEV quality, abundance, structure and molecular cargo. Separation and characterization of BEVs from body fluids can be done within 72 h, is compatible with EEV analysis and can be readily adopted by researchers experienced in basic molecular biology and extracellular vesicle analysis. We anticipate that this protocol will expand our knowledge on the biological heterogeneity, molecular cargo and function of BEVs in human body fluids and steer the development of laboratory research tools and clinical diagnostic kits.  

3.3463           Hominin-specific regulatory elements selectively emerged in oligodendrocytes and are disrupted in autism patients

Castelijns, B., Baak, M.L., Timpanaro, I.S., Wiggers, C.R.M., Vermunt, M.W., Shang, P., Kondova, I., Geeven, G., Bianchi, V., de Laat, W., Geijsen, N. and Creyghton, M.P. Nature Communications, 11:301 (2020)   Speciation is associated with substantial rewiring of the regulatory circuitry underlying the expression of genes. Determining which changes are relevant and underlie the emergence of the human brain or its unique susceptibility to neural disease has been challenging. Here we annotate changes to gene regulatory elements (GREs) at cell type resolution in the brains of multiple primate species spanning most of primate evolution. We identify a unique set of regulatory elements that emerged in hominins prior to the separation of humans and chimpanzees. We demonstrate that these hominin gains perferentially affect oligodendrocyte function postnatally and are preferentially affected in the brains of autism patients. This preference is also observed for human-specific GREs suggesting this system is under continued selective pressure. Our data provide a roadmap of regulatory rewiring across primate evolution providing insight into the genomic changes that underlie the emergence of the brain and its susceptibility to neural disease.  

3.3464           Mesenchymal stem cell‐derived extracellular vesicles for the treatment of acute respiratory distress syndrome

Abraham, A. and Krasnodembskaya Stem Cells Transl. Med., 9(1), 28-38 (2020)   Acute respiratory distress syndrome (ARDS) is a serious and potentially fatal acute inflammatory lung condition which currently has no specific treatments targeting its pathophysiology. However, mesenchymal stem cells have been shown to have very promising therapeutic potential, and recently, it has been established that their effect is largely due to the transfer of extracellular vesicles (EVs). EVs have been shown to transfer a variety of substances such as mRNA, miRNA, and even organelles such as mitochondria in order to ameliorate ARDS in preclinical models. In addition, the fact that they have been proven to have the same effect as their parent cells combined with their numerous advantages over whole cell administration means that they are a promising candidate for clinical application that merits further research.  

3.3465           Urinary extracellular vesicles: Origin, role as intercellular messengers and biomarkers; efficient sorting and potential treatment options

Svenningsen, p., Sabaratnam, R. and Jensen, B.L. Acta Physiologica, 228(1), e13346 (2020)   Urinary extracellular vesicles (uEVs) are a heterogenous group of vesicles consisting mainly of microvesicles and exosomes that originate predominantly (99.96%) from kidney, the urinary tract epithelium and the male reproductive tract. Secreted EVs contain molecular cargo from parental cells and provide an attractive source for biomarkers, a potential readout of physiological and pathophysiological mechanisms, and events associated with the urinary system. uEVs are readily enriched and isolated from urine samples and we review 6 standard methods that allow for downstream analysis of the uEV cargo. Although the use of uEVs as a surrogate readout for physiological changes in tissue protein levels is widespread, the protein abundance in uEVs is affected significantly by mechanisms that regulate protein sorting and secretion in uEVs. Data suggest that baseline kidney tissue and uEV levels of apical membrane‐associated electrolyte transport proteins are not directly related in human patients. Recent evidence indicates that EVs may contribute to physiological and pathophysiological intercellular signalling and EVs confer protection against renal ischemia‐reperfusion injury. The therapeutic use of EVs as information carriers has mainly been explored in vitro and a major hurdle lies in the translation of the in vitro findings into an in vivo setting. Thus, the EV research field is moving from a technical focus to a more physiological focus, allowing for a deeper understanding of human physiology, development of diagnostic tools and potential treatment strategies for precision medicine.  

3.3466           The significance of exosomes in the development and treatment of hepatocellular carcinoma

Li, X., Li, C., Zhang, L., Wu, M., Cao, K., Jiang, F., Chen, D., Li, N. and Li, W. Molecular Cancer, 9:1 (2020)   Hepatocellular carcinoma (HCC) is the most commonmalignancy. Exsome plays a significant role in the elucidation of signal transduction pathways between hepatoma cells, angiogenesis and early diagnosis of HCC. Exosomes are small vesicular structures that mediate interaction between different types of cells, and contain a variety of components (including DNA, RNA, and proteins). Numerous studies have shown that these substances in exosomes are involved in growth, metastasis and angiogenesis in liver cancer, and then inhibited the growth of liver cancer by blocking the signaling pathway of liver cancer cells. In addition, the exosomal substances could also be used as markers for screening early liver cancer. In this review, we summarized to reveal the significance of exosomes in the occurrence, development, diagnosis and treatment of HCC, which in turn might help us to further elucidate the mechanism of exosomes in HCC, and promote the use of exosomes in the clinical diagnosis and treatment of HCC.  

3.3467           Palmitoylation of Hepatitis C Virus NS2 Regulates Its Subcellular Localization and NS2-NS3 Autocleavage

Wu, M-J., Shanmugam, S., Welsch, C. and Yi, M.

1. Virol., 94(1), e00906-129 (2020)

  Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a multifunctional protein implicated in both HCV RNA replication and virus particle assembly. NS2-encoded cysteine protease is responsible for autoprocessing of NS2-NS3 precursor, an essential step in HCV RNA replication. NS2 also promotes HCV particle assembly by recruiting envelope protein 2 (E2) to the virus assembly sites located at the detergent-resistant membranes (DRM). However, the fundamental mechanism regulating multiple functions of NS2 remains unclear. In this study, we discovered that NS2 is palmitoylated at the position 113 cysteine residue (NS2/C113) when expressed by itself in cells and during infectious-HCV replication. Blocking NS2 palmitoylation by introducing an NS2/C113S mutation reduced NS2-NS3 autoprocessing and impaired HCV RNA replication. Replication of the NS2/C113S mutant was restored by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between NS2 and NS3 to separate the two proteins independently of NS2-mediated autoprocessing. These results suggest that NS2 palmitoylation is critical for HCV RNA replication by promoting NS2-NS3 autoprocessing. The NS2/C113S mutation also impaired infectious-HCV assembly, DRM localization of NS2 and E2, and colocalization of NS2 with Core and endoplasmic reticulum lipid raft-associated protein 2 (Erlin-2). In conclusion, our study revealed that two major functions of NS2 involved in HCV RNA replication and virus assembly, i.e., NS2-NS3 autoprocessing and E2 recruitment to the DRM, are regulated by palmitoylation at NS2/C113. Since S-palmitoylation is reversible, NS2 palmitoylation likely allows NS2 to fine tune both HCV RNA replication and infectious-particle assembly.  

3.3468           Fam20C‐mediated phosphorylation of osteopontin is critical for its secretion but dispensable for its action as a cytokine in the activation of hepatic stellate cells in liver fibrogenesis

Tibaldi, E., Brocca, A., Sticca, A., Gola, E., Pizzi, M., Bordin, L., Pagano, M.A., Mazzorana, m., Dona, G., Violi, P., Marin, O., Romano, A., Angeli, P., Carraro, A. and Brunati, A.M. FASEB J., 34, 1122-1135 (2020)   Osteopontin (OPN) is a phosphoglycoprotein secreted into the extracellular matrix upon liver injury, acting as a cytokine stimulates the deposition of fibrillary collagen in liver fibrogenesis. In livers of mice subjected to bile duct ligation (BDL) and in cultured activated hepatic stellate cells (HSCs), we show that OPN, besides being overexpressed, is substantially phosphorylated by family with sequence similarity 20, member C (Fam20C), formerly known as Golgi casein kinase (G‐CK), which is exclusively resident in the Golgi apparatus. In both experimental models, Fam20C becomes overactive when associated with a 500‐kDa multiprotein complex, as compared with the negligible activity in livers of sham‐operated rats and in quiescent HSCs. Fam20C knockdown not only confirmed the role of Fam20C itself in OPN phosphorylation, but also revealed that phosphorylation was essential for OPN secretion. However, OPN acts as a fibrogenic factor independently of its phosphorylation state, as demonstrated by the increased expression of Collagen‐I by HSCs incubated with either a phosphorylated or nonphosphorylated form of recombinant OPN. Collectively, our results confirm that OPN promotes liver fibrosis and highlight Fam20C as a novel factor driving this process by favoring OPN secretion from HSCs, opening new avenues for deciphering yet unidentified mechanisms underlying liver fibrogenesis.  

3.3469           Hepatitis A virus structural protein pX interacts with ALIX and promotes the secretion of virions and foreign proteins through exosome-like vesicles

Jiang, W., Ma, P., Deng, L., Liu, Z., Wang, X., Liu, X. and Long, G.

1. Extracellular Vesicles, 9(1), 1716513 (2020)

  Hepatitis A virus (HAV), a classic nonenveloped virus, has recently been found to be released mainly in the form of quasi-enveloped HAV (eHAV) by hijacking host endosomal sorting complexes required for transport (ESCRT) complexes. Unlike the nonenveloped virion, eHAV contains the viral protein pX on the surface of the HAV capsid as an extension of VP1. How HAV capsids acquire the host envelope and whether the pX protein is involved in this process were previously unknown. Here, we analyse the role of pX in foreign protein secretion in exosome-like extracellular vesicles (EVs) and the formation of eHAV. Fusion of pX to eGFP guided eGFP into exosome-like EVs through directing eGFP into multivesicular bodies (MVBs), and apoptosis-linked gene 2-interacting protein X (ALIX) release was significantly enhanced. Coimmunoprecipitation (co-IP) demonstrated the interaction between pX and the ALIX V domain. Removal of the C-terminal half of pX abolished eHAV release and reduced the interaction between the HAV virion and ALIX. Finally, the C-terminal half of pX alone was sufficient for loading eGFP into EVs by interacting with ALIX. In conclusion, the C-terminal part of pX is important for eHAV production and may have potential for large protein complex loading into exosome-like EVs for therapeutic purposes.  

3.3470           Techniques Associated with Exosome Isolation for Biomarker Development: Liquid Biopsies for Ovarian Cancer Detection

Sharma, S. and Salomom, C. Methods in Mol. Biol., 2055, 181-199 (2020)   Ovarian cancer is the leading gynecological malignancy worldwide. This is attributed to the fact that the disease is often diagnosed at an advanced stage, where the survival rates drop from approximately 90% (detection at an early stage) to 20%. Furthermore, ovarian cancer is not associated with overt physical symptoms. Thus, there is an urgent need for a highly sensitive and minimally invasive biomarker for the early detection of ovarian cancer. However, this continues to remain an unmet clinical need, as several proposed techniques have shown low sensitivity and specificity, with poor positive and negative predictive values. The quest for an ideal biomarker has bought exosomes to the forefront. Exosomes are small extracellular vesicles of an endocytic origin, which can encapsulate genetic information, in the form of proteins and miRNAs. They are released by multiple cell types and are involved in intercellular communication, through the transfer of their cargo. The process of exosome biogenesis allows for the packaging of molecules from both membranous and cytosolic origins. Therefore, exosomes are representations of the releasing cell, and thus provide an insight into the cellular environment. Furthermore, exosomal encapsulation of molecules such as proteins and miRNAs can prevent degradation, making exosomes an ideal biomarker source. Thus, this chapter provides an overview of ovarian cancer, the potential of exosomes as an early detection biomarker, and the different methods associated with the isolation of different vesicle subpopulations, and exosome enrichment.  

3.3471           Extracellular Vesicles: Recent Developments in Technology and Perspectives for Cancer Liquid Biopsy

Nazarenko, I. Recent Results in Cancer Res., 215, 319-344 (2020)   Extracellular micro- and nanoscale membrane vesicles produced by different cells progressively attract the attention of the scientific community. They function as mediators of intercellular communication and transport genetic material and signaling molecules between the cells. In the context of keeping homeostasis, the extracellular vesicles contribute to the regulation of various systemic and local processes. Vesicles released by the tumor and activated stromal cells exhibit multiple functions including support of tumor growth, preparation of the pre-metastatic niches, and immune suppression. Considerable progress has been made regarding the criteria of classification of the vesicles according to their origin, content, and function: Exosomes, microvesicles, also referred to as microparticles or ectosomes, and large oncosomes were defined as actively released vesicles. Additionally, apoptotic bodies represented by a highly heterogeneous population of particles produced during apoptosis, the programmed cell death, should be considered. Because the majority of isolation techniques do not allow the separation of different types of vesicles, a joined term “extracellular vesicles” (EVs) was recommended by the ISEV community for the definition of vesicles isolated from either the cell culture supernatants or the body fluids. Because EV content reflects the content of the cell of origin, multiple studies on EVs from body fluids in the context of cancer diagnosis, prediction, and prognosis were performed, actively supporting their high potential as a biomarker source. Here, we review the leading achievements in EV analysis from body fluids, defined as EV-based liquid biopsy, and provide an overview of the main EV constituents: EV surface proteins, intravesicular soluble proteins, EV RNA including mRNA and miRNA, and EV DNA as potential biomarkers. Furthermore, we discuss recent developments in technology for quantitative EV analysis in the clinical setting and future perspectives toward miniaturized high-precision liquid biopsy approaches.  

3.3472           MeCP2 Represses Enhancers through Chromosome Topology-Associated DNA Methylation

Clemens, A.W., Wu, D.Y., Moore, J.R., Christian, D.L., Zhao, G. and Gabel, H.W. Molecular Cell, 77, 279-293 (2020)   The genomes of mammalian neurons contain uniquely high levels of non-CG DNA methylation that can be bound by the Rett syndrome protein, MeCP2, to regulate gene expression. How patterns of non-CG methylation are established in neurons and the mechanism by which this methylation works with MeCP2 to control gene expression is unclear. Here, we find that genes repressed by MeCP2 are often located within megabase-scale regions of high non-CG methylation that correspond with topologically associating domains of chromatin folding. MeCP2 represses enhancers found in these domains that are enriched for non-CG and CG methylation, with the strongest repression occurring for enhancers located within MeCP2-repressed genes. These alterations in enhancer activity provide a mechanism for how MeCP2 disruption in disease can lead to widespread changes in gene expression. Hence, we find that DNA topology can shape non-CG DNA methylation across the genome to dictate MeCP2-mediated enhancer regulation in the brain.  

3.3473           Reduction of the therapeutic dose of silencing RNA by packaging it in extracellular vesicles via a pre-microRNA backbone

Reshle, R., Taylor, J.A., Savard, A., Guo, H., Rhym, L.H., Kowalski, P.S., Trung, M.T., Capbell, C., Little, W., Anderson, D.G. and Gibbings, D. Nature Biomedical Engineering, 4, 52-68 (2020)   A small percentage of the short interfering RNA (siRNA) delivered via passive lipid nanoparticles and other delivery vehicles reaches the cytoplasm of cells. The high doses of siRNA and delivery vehicle that are thus required to achieve therapeutic outcomes can lead to toxicity. Here, we show that the integration of siRNA sequences into a Dicer-independent RNA stem–loop based on pre-miR-451 microRNA—which is highly enriched in small extracellular vesicles secreted by many cell types—reduces the expression of the genes targeted by the siRNA in the liver, intestine and kidney glomeruli of mice at siRNA doses that are at least tenfold lower than the siRNA doses typically delivered via lipid nanoparticles. Small extracellular vesicles that efficiently package siRNA can significantly reduce its therapeutic dose.  

3.3474           A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum

Brennan, K., martin, K., Fitzgerald, S.P., O’Sullivan, J., Wu, Y., Blanco, A., Richardson, C. and McGee, M.M.M. Scientific Reports, 10:1039 (20209   Extracellular vesicles (EVs) are nano-sized vesicles containing nucleic acid and protein cargo that are released from a multitude of cell types and have gained significant interest as potential diagnostic biomarkers. Human serum is a rich source of readily accessible EVs; however, the separation of EVs from serum proteins and non-EV lipid particles represents a considerable challenge. In this study, we compared the most commonly used isolation techniques, either alone or in combination, for the isolation of EVs from 200 µl of human serum and their separation from non-EV protein and lipid particles present in serum. The size and yield of particles isolated by each method was determined by nanoparticle tracking analysis, with the variation in particle size distribution being used to determine the relative impact of lipoproteins and protein aggregates on the isolated EV population. Purification of EVs from soluble protein was determined by calculating the ratio of EV particle count to protein concentration. Finally, lipoprotein particles co-isolated with EVs was determined by Western blot analysis of lipoprotein markers APOB and APOE. Overall, this study reveals that the choice of EV isolation procedure significantly impacts EV yield from human serum, together with the presence of lipoprotein and protein contaminants.  

3.3475           Endomembrane Protein Trafficking Regulated by a TvCyP2 Cyclophilin in the Protozoan Parasite, Trichomonas vaginalis

Hsu, H-M., Huang, Y-H., Aryal. S., Liu, H-W-. Chen, C., Chen, S-H., Chu, C-H. and Tai, J-H. Scientific Reports, 10:1275 (2020)   In Trichomonas vaginalis, the TvCyP1-catalyzed conformational switches of two glycinyl-prolyl imide bonds in Myb3 were previously shown to regulate the trafficking of Myb3 from cytoplasmic membrane compartments towards the nucleus. In this study, TvCyP2 was identified as a second cyclophilin that binds to Myb3 at the same dipeptide motifs. The enzymatic proficiency of TvCyP2, but not its binding to Myb3, was aborted by a mutation of Arg⁷⁵ in the catalytic domain. TvCyP2 was localized to the endoplasmic reticulum with a weak signal that extensively extends into the cytoplasm as well as to the plasma membrane according to an immunofluorescence assay. Moreover, TvCyP2 was co-enriched with TvCyP1 and Myb3 in various membrane fractions purified by differential and gradient centrifugation. TvCyP2 was found to proficiently enzymatically regulate the distribution of TvCyP1 and Myb3 among purified membrane fractions, and to localize TvCyP1 in hydrogenosomes and on plasma membranes. Protein complexes immunoprecipitated from lysates of cells overexpressing TvCyP1 and TvCyP2 were found to share some common components, like TvCyP1, TvCyP2, TvBip, Myb3, TvHSP72, and the hydrogenosomal heat shock protein 70 (HSP70). Direct interaction between TvCyP1 and TvCyP2 was confirmed by a GST pull-down assay. Fusion of vesicles with hydrogenosomes was observed by transmission electron microscopy, whereas TvCyP1, TvCyP2, and Myb3 were each detected at the fusion junction by immunoelectron microscopy. These observations suggest that T. vaginalis may have evolved a novel protein trafficking pathway to deliver proteins among the endomembrane compartments, hydrogenosomes and plasma membranes.  

3.3476           PAK Kinases Target Sortilin and Modulate Its Sorting

Pallesen, L.T., Gustafsen, C., Cramer, J.F., Petersen, S.V., Thirup, S.S., Madsen, P. and Petersen, C.M. Mol. Cell. Biol., 40(3), e00411-19 (2020)   The multifunctional type 1 receptor sortilin is involved in endocytosis and intracellular transport of ligands. The short intracellular domain of sortilin binds several cytoplasmic adaptor proteins (e.g., the AP-1 complex and GGA1 to -3), most of which target two well-defined motifs: a C-terminal acidic cluster dileucine motif and a YXXΦ motif in the proximal third of the domain. Both motifs contribute to endocytosis as well as Golgi-endosome trafficking of sortilin. The C-terminal acidic cluster harbors a serine residue, which is subject to phosphorylation by casein kinase. Phosphorylation of this serine residue is known to modulate adaptor binding to sortilin. Here, we show that the cytoplasmic domain of sortilin also engages Rac-p21-activated kinases 1 to 3 (PAK1-3) via a binding segment that includes a tyrosine-based motif, also encompassing a serine residue. We further demonstrate that PAK1-3 specifically phosphorylate this serine residue and that this phosphorylation alters the affinity for AP-1 binding and consequently changes the intracellular localization of sortilin as a result of modulated trafficking. Our findings suggest that trafficking of ligands bound to sortilin is in part regulated by group A PAK kinases, which are downstream effectors of Rho GTPases and are known to affect a variety of processes by remodeling the cytoskeleton and by promoting gene transcription and cell survival.  

3.3477           A Variant of SLC1A5 Is a Mitochondrial Glutamine Transporter for Metabolic Reprogramming in Cancer Cells

Yoo, H.C., Park, S.J., Nam, M., Hwang, G-S., Bang, S. and Han,k J.M. Cell Metabolsim, 31, 267-283 (2020)   Glutamine is an essential nutrient that regulates energy production, redox homeostasis, and signaling in cancer cells. Despite the importance of glutamine in mitochondrial metabolism, the mitochondrial glutamine transporter has long been unknown. Here, we show that the SLC1A5 variant plays a critical role in cancer metabolic reprogramming by transporting glutamine into mitochondria. The SLC1A5 variant has an N-terminal targeting signal for mitochondrial localization. Hypoxia-induced gene expression of the SLC1A5 variant is mediated by HIF-2α. Overexpression of the SLC1A5 variant mediates glutamine-induced ATP production and glutathione synthesis and confers gemcitabine resistance to pancreatic cancer cells. SLC1A5 variant knockdown and overexpression alter cancer cell and tumor growth, supporting an oncogenic role. This work demonstrates that the SLC1A5 variant is a mitochondrial glutamine transporter for cancer metabolic reprogramming.  

3.3478           Proteomic analysis of two populations of Schistosoma mansoni-derived extracellular vesicles: 15k pellet and 120k pellet vesicles

Kifle, D.W:, Peason, M.S:, Becker, L., Pickering, D., Loukas, A. and Sotillo, J. Mol. Biochem. Paeasitol., 236, 111264 (2020)   Helminth parasites secrete extracellular vesicles (EVs) into their environment that have potential roles in host-parasite communication, and thus represent potentially useful targets for novel control strategies. Here, we carried out a comprehensive proteomic analysis of two different populations of EVs – 15k pellet and 120k pellet EVs – from Schistosoma mansoni adult worms. We characterised the proteins present in the membranes of the EVs (including external trypsin-liberated peptides, integral membrane proteins (IMPs) and peripheral membrane proteins (PMPs)), as well as cargo proteins, using LC–MS/MS. A total of 286 and 716 proteins were identified in 15k and 120k pellets, respectively. Some of the most abundant proteins identified from both 15k and 120k pellets include known vaccine candidates such as Sm-TSP-2, saponin B domain-containing proteins, calpain glutathione-S-transferase, Sm29 and cathepsin domain-containing proteins. Other abundant proteins that have not been tested as vaccines include DM9 domain-containing protein, 13 kDa tegumental antigen and histone H4-like protein. Sm23, a member of the tetraspanin family with known vaccine efficacy, was identified in the cargo and IMP compartments of only 15k pellet vesicles. Moreover, a collection of proteins with known or potential relevance in host-parasite communication including proteases, antioxidants and EV biogenesis/trafficking of both vesicle types were identified. Our results provide the first report of a comprehensive compartmental proteomic analysis of adult S. mansoni-derived EVs. Future research should investigate recombinant forms of these proteins as vaccine and serodiagnostic antigens as well as the roles of EV proteins in host-parasite communication.  

3.3479           Exosomes derived from HeLa cells break down vascular integrity by triggering endoplasmic reticulum stress in endothelial cells

Lin, Y., Zhang, C., Xiang, P., Shen, J., Sun, W. and Yu, H.

1. Extracellular Vesicles, 9(1), 1722385 (2020)

  Exosomes play a critical role in intercellular communication since they contain signalling molecules and genetic materials. During tumorigenesis, tumour-derived exosomes have been demonstrated to promote tumour angiogenesis and metastasis. However, how the exosomes facilitate tumour metastasis is not clear. Here we explored the effect of HeLa cell-derived exosomes (Exo^HeLa) on endothelial tight junctions (TJ) and the related mechanisms. After human umbilical vein endothelial cells (HUVEC) were treated with Exo^HeLa, TJ proteins zonula occludens-1 (ZO-1) and Claudin-5 in HUVEC were significantly reduced as compared with that treated with exosomes from human cervical epithelial cells, while mRNA levels of ZO-1 and Claudin-5 remained unchanged. Consequently, permeability of endothelial monolayer was increased after the treatment with Exo^HeLa. Injection of Exo^HeLa into mice also increased vascular permeability and tumour metastasis in vivo. Neither knocking down of Dicer nor use of inhibitors of microRNAs targeting at mRNAs of ZO-1 and Claudin-5 could block the inhibitory effect of Exo^HeLa on ZO-1 and Claudin-5. The expression of genes involved in endoplasmic reticulum (ER) stress was significantly increased in HUVECs after treated with Exo^HeLa. Inhibition of ER stress by knocking down protein kinase RNA-like endoplasmic reticulum kinase prevented the down-regulation of ZO-1 and Claudin-5 by Exo^HeLa. Our study found that HeLa cell-derived exosomes promote metastasis by triggering ER stress in endothelial cells and break down endothelial integrity. Such effect of exosomes is microRNA-independent.  

3.3480           Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation

Crescitelli, R., Lässer, C., jang, S.C., Cvetkovic, A., Malmhäll, C., Karimi, N., Höög, J.L., Johansson, I., Fuchs, J., Thorsell, A., Gho, Y.S., Bagge, R.O. and Lötvall, J.

1. Extracellular Vesicles, 9(1), 1722433 (2020)

  The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lines with little knowledge on how well these represent the characteristics of EVs in vivo. The aim of this study was to establish a method to isolate and categorize subpopulations of EVs isolated directly from tumour tissue. First we established an isolation protocol for subpopulations of EVs from metastatic melanoma tissue, which included enzymatic treatment (collagenase D and DNase). Small and large EVs were isolated with differential ultracentrifugation, and these were further separated into high and low-density (HD and LD) fractions. All EV subpopulations were then analysed in depth using electron microscopy, Bioanalyzer®, nanoparticle tracking analysis, and quantitative mass spectrometry analysis. Subpopulations of EVs with distinct size, morphology, and RNA and protein cargo could be isolated from the metastatic melanoma tissue. LD EVs showed an RNA profile with the presence of 18S and 28S ribosomal subunits. In contrast, HD EVs had RNA profiles with small or no peaks for ribosomal RNA subunits. Quantitative proteomics showed that several proteins such as flotillin-1 were enriched in both large and small LD EVs, while ADAM10 were exclusively enriched in small LD EVs. In contrast, mitofilin was enriched only in the large EVs. We conclude that enzymatic treatments improve EV isolation from dense fibrotic tissue without any apparent effect on molecular or morphological characteristics. By providing a detailed categorization of several subpopulations of EVs isolated directly from tumour tissues, we might better understand the function of EVs in tumour biology and their possible use in biomarker discovery.  

3.3481           Giant Endoplasmic Reticulum vesicles (GERVs), a novel model membrane tool

Grimmer, M. and Bacia, K. Scientific Reports, 10:3100 (2020)   Artificial giant vesicles have proven highly useful as membrane models in a large variety of biophysical and biochemical studies. They feature accessibility for manipulation and detection, but lack the compositional complexity needed to reconstitute complicated cellular processes. For the plasma membrane (PM), this gap was bridged by the establishment of giant PM vesicles (GPMVs). These native membranes have facilitated studies of protein and lipid diffusion, protein interactions, electrophysiology, fluorescence analysis of lateral domain formation and protein and lipid partitioning as well as mechanical membrane properties and remodeling. The endoplasmic reticulum (ER) is key to a plethora of biological processes in any eukaryotic cell. However, its intracellular location and dynamic and intricate tubular morphology makes it experimentally even less accessible than the PM. A model membrane, which will allow the afore-mentioned types of studies on GPMVs to be performed on ER membranes outside the cell, is therefore genuinely needed. Here, we introduce the formation of giant ER vesicles, termed GERVs, as a new tool for biochemistry and biophysics. To obtain GERVs, we have isolated ER membranes from Saccharomyces cerevisiae and fused them by exploiting the atlastin-like fusion protein Sey1p. We demonstrate the production of GERVs and their utility for further studies.  

3.3482           Trypanosoma brucei Pex13.2 Is an Accessory Peroxin That Functions in the Import of Peroxisome Targeting Sequence Type 2 Proteins and Localizes to Subdomains of the Glycosome

Crowe, L.P., Wilkinson, C.L., Nicholson, K.R. and Morris, M.T. mSphere, 5(1), e00744-19 (2020)   Kinetoplastid parasites, including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania, harbor unique organelles known as glycosomes, which are evolutionarily related to peroxisomes. Glycosome/peroxisome biogenesis is mediated by proteins called peroxins that facilitate organelle formation, proliferation, and degradation and import of proteins housed therein. Import of matrix proteins occurs via one of two pathways that are dictated by their peroxisome targeting sequence (PTS). In PTS1 import, a C-terminal tripeptide sequence, most commonly SKL, is recognized by the soluble receptor Pex5. In PTS2 import, a less conserved N-terminal sequence is recognized by Pex7. The soluble receptors deliver their cargo to the import channel consisting minimally of Pex13 and Pex14. While much of the import process is conserved, kinetoplastids are the only organisms to have two Pex13s, Pex13.1 and Pex13.2. It is unclear why trypanosomes require two Pex13s when one is sufficient for most eukaryotes. To interrogate the role of Pex13.2, we have employed biochemical approaches to partially resolve the composition of the Pex13/Pex14 import complexes in T. brucei and characterized glycosome morphology and protein import in Pex13.2-deficient parasites. Here, we show that Pex13.2 is an integral glycosome membrane protein that interacts with Pex13.1 and Pex14. The N terminus of Pex13.2 faces the cytoplasmic side of the membrane, where it can facilitate interactions required for protein import. Two-dimensional gel electrophoresis revealed three glycosome membrane complexes containing combinations of Pex13.1, Pex13.2, and Pex14. The silencing of Pex13.2 resulted in parasites with fewer, larger glycosomes and disrupted glycosome protein import, suggesting the protein is involved in glycosome biogenesis as well as protein import. Furthermore, superresolution microscopy demonstrated that Pex13.2 localizes to discrete foci in the glycosome periphery, indicating that the glycosome periphery is not homogenous.  

3.3483           Deletion of major porins from meningococcal outer membrane vesicle vaccines enhances reactivity against heterologous serogroup B Neisseria meningitidis strains

Matthias, K.A., Reveille, A., Connolly, K.L., Jerse, A.E., Gao, Y.S. and Bash, M.C: Vaccine, 38, 2396-2405 (2020)   Detergent-extracted detoxified outer membrane vesicle (dOMV) vaccines are effective at preventing invasive serogroup B meningococcal (MenB) disease caused by the homologous Neisseria meningitidis strain from which they are produced, but offer limited protection from heterologous strains. Differences in vaccine efficacy are partially due to strain-specific variations in the antigenic sequence types and expression levels of outer membrane proteins (OMPs), including the immunodominant OMP PorA. In this study, dOMV vaccines deficient in major OMPs, including PorA, PorB, and RmpM were isolated and used to immunize rabbits and mice. Serum samples were obtained from each animal and tested for antibody responses against five MenB strains. Immunization with wild type dOMVs elicited antibodies to major antigens including PorA, PorB, RmpM, and lipooligosaccharide (LOS), and demonstrated limited bactericidal activity against heterologous strains. In contrast, OMP-deficient dOMV vaccines elicited broadly cross-reactive bactericidal antibodies, with PorA/PorB-dual deficient dOMVs inducing antibodies exhibiting the greatest cross-reactivity. Enhanced killing of heterologous strains correlated with binding to unique protein bands in immunoblots, suggestive of improved immunogenicity of antigens under-represented in the wild type vaccine.  

3.3484           Single-nucleus RNA-seq identifies Huntington disease astrocyte states

Al-Dalahmah, O., Sosunov, A.A., Shaik, A., Ofori, K., Liu, Y., Vonsattel, J.P., Adorjan, I., Menon, V. and Goldman, J.E. Acta Neurophatol. Comm., 8:19 (2020)   Huntington Disease (HD) is an inherited movement disorder caused by expanded CAG repeats in the Huntingtin gene. We have used single nucleus RNASeq (snRNASeq) to uncover cellular phenotypes that change in the disease, investigating single cell gene expression in cingulate cortex of patients with HD and comparing the gene expression to that of patients with no neurological disease. In this study, we focused on astrocytes, although we found significant gene expression differences in neurons, oligodendrocytes, and microglia as well. In particular, the gene expression profiles of astrocytes in HD showed multiple signatures, varying in phenotype from cells that had markedly upregulated metallothionein and heat shock genes, but had not completely lost the expression of genes associated with normal protoplasmic astrocytes, to astrocytes that had substantially upregulated glial fibrillary acidic protein (GFAP) and had lost expression of many normal protoplasmic astrocyte genes as well as metallothionein genes. When compared to astrocytes in control samples, astrocyte signatures in HD also showed downregulated expression of a number of genes, including several associated with protoplasmic astrocyte function and lipid synthesis. Thus, HD astrocytes appeared in variable transcriptional phenotypes, and could be divided into several different “states”, defined by patterns of gene expression. Ultimately, this study begins to fill the knowledge gap of single cell gene expression in HD and provide a more detailed understanding of the variation in changes in gene expression during astrocyte “reactions” to the disease.  

3.3485           Magnesium protects against sepsis by blocking gasdermin D N-terminal-induced pyroptosis

Wang, D., Zheng, J., Hu, Q., Zhao, C., Shi, P., Chen, Q., Zou, Y., Zou, D., Liu, Q., Pei, J., Wu, X., Gao, X., Ren, J. and Lin, Z. Cell Death & Differentiation, 27, 466-481 (2020)   Hypomagnesemia is a significant risk factor for critically ill patients to develop sepsis, a life-threatening disease with a mortality rate over 25%. Our clinic data analysis showed that hypomagnesemia is associated with a decreased monocyte count in septic patients. At the cellular level, we found that Mg²⁺ inhibits pyroptosis. Specifically, Mg²⁺ limits the oligomerization and membrane localization of gasdermin D N-terminal (GSDMD-NT) upon the activation of either the canonical or noncanonical pyroptotic pathway. Mechanistically, we demonstrated that Ca²⁺ influx is a prerequisite for the function of GSDMD-NT. Mg²⁺ blocks Ca²⁺ influx by inhibiting the ATP-gated Ca²⁺ channel P2X7, thereby impeding the function of GSDMD-NT and inhibiting lipopolysaccharide (LPS)-induced noncanonical pyroptosis. Furthermore, Mg²⁺ administration protects mice from LPS-induced lethal septic shock. Together, our data reveal the underlying mechanism of how Mg²⁺ inhibits pyroptosis and suggest potential clinic applications of magnesium supplementation for sepsis prevention and treatment.  

3.3486           Degranulation of human cytotoxic lymphocytes is a major source of proteolytically active soluble CD26/DPP4

Lettau, M., Dietz, M., Vollmers, S., Armbrust, F., Peters, C., Dang, T.M., Chitadze, G., Kabelitz, D. and Janssen, O. Cell. Mol. Lefe Sci., 77, 751-764 (2020)   Dipeptidyl peptidase 4 (DPP4, CD26) is a serine protease detected on several immune cells and on epithelial cells of various organs. Besides the membrane-bound enzyme, a catalytically active soluble form (sCD26/DPP4) is detected in several body fluids. Both variants cleave off dipeptides from the N-termini of various chemokines, neuropeptides, and hormones. CD26/DPP4 plays a fundamental role in the regulation of blood glucose levels by inactivating insulinotropic incretins and CD26/DPP4 inhibitors are thus routinely used in diabetes mellitus type 2 therapy to improve glucose tolerance. Such inhibitors might also prevent the CD26/DPP4-mediated inactivation of the T-cell chemoattractant CXCL10 released by certain tumors and thus improve anti-tumor immunity and immunotherapy. Despite its implication in the regulation of many (patho-)physiological processes and its consideration as a biomarker and therapeutic target, the cellular source of sCD26/DPP4 remains highly debated and mechanisms of its release are so far unknown. In line with recent reports that activated T lymphocytes could be a major source of sCD26/DPP4, we now demonstrate that CD26/DPP4 is stored in secretory granules of several major human cytotoxic lymphocyte populations and co-localizes with effector proteins such as granzymes, perforin, and granulysin. Upon stimulation, vesicular CD26/DPP4 is rapidly translocated to the cell surface in a Ca²⁺-dependent manner. Importantly, activation-induced degranulation leads to a massive release of proteolytically active sCD26/DPP4. Since activated effector lymphocytes serve as a major source of sCD26/DPP4, these results might explain the observed disease-associated alterations of sCD26/DPP4 serum levels and also indicate a so far unknown role of CD26/DPP4 in lymphocyte-mediated cytotoxicity.  

3.3487           Novel Techniques to Study the Bone-Tumor Microenvironment

Shupp, A.B., Kolb, A.D. and Bussard, K.M. Adv. Exp. Med.Biol., 1225, 1-18 (2020)   Many cancers commonly metastasize to bone. After entering the bone, cancer cells can interact with surrounding stromal cells, which ultimately influences metastasis progression. Extracellular vesicles, direct cell contact and gap junctions, and cytokines are all mechanisms of intercellular communication that have been observed to occur in the bone microenvironment. These methods of cellular crosstalk can occur between cancer cells and a variety of stromal cells, with each interaction having a different impact on cancer progression. Communication between cancer cells and bone-resident cells has previously been implicated in processes such as cancer cell trafficking and arrest in bone, cancer cell dormancy, cancer cell reactivation, and proliferation. In this chapter we review innovative techniques and model systems that can be used to study bidirectional crosstalk between cancer cells and stromal cells in the bone, with an emphasis specifically on bone-metastatic breast cancer. Investigating how metastatic cancer cells interact with, and are influenced by, the bone microenvironment is crucial to better understanding of the progression of bone metastasis.  

3.3488           The intraflagellar transport protein IFT20 controls lysosome biogenesis by regulating the post-Golgi transport of acid hydrolases

Finetti, F., Cassioli, C., Cianfanelli, V., Onnis, A., Paccagnini, E., Kabanova, A. and Baldari, C.T. Cell Death & Differentiation, 27, 310-328 (2020)   The assembly and function of the primary cilium depends on multimolecular intraflagellar transport (IFT) complexes that shuttle their cargo along the axonemal microtubules through their interaction with molecular motors. The IFT system has been moreover recently implicated in a reciprocal interplay between autophagy and ciliogenesis. We have previously reported that IFT20 and other components of the IFT complexes participate in the assembly of the immune synapse in the non-ciliated T cell, suggesting that other cellular processes regulated by the IFT system in ciliated cells, including autophagy, may be shared by cells lacking a cilium. Starting from the observation of a defect in autophagic clearance and an accumulation of lipid droplets in IFT20-deficient T cells, we show that IFT20 is required for lysosome biogenesis and function by controlling the lysosomal targeting of acid hydrolases. This function involves its ability to regulate the retrograde traffic of the cation-independent mannose-6-phosphate receptor (CI-MPR) to the trans-Golgi network, which is achieved by coupling recycling CI-MPRs to the microtubule motor dynein. Consistent with the lysosomal defect, an upregulation of the TFEB-dependent expression of the lysosomal gene network can be observed in IFT20-deficient cells, which is associated with defective tonic T-cell antigen receptor signaling and mTOR activity. We additionally show that the lysosome-related function of IFT20 extends to non-ciliated cells other than T cells, as well as to ciliated cells. Our findings provide the first evidence that a component of the IFT system that controls ciliogenesis is implicated in the biogenesis of lysosomes.  

3.3489           Huntington’s Disease Pathogenesis Is Modified In Vivo by Alfy/Wdfy3 and Selective Macroautophagy

Fox, L.M., Kim, K., Johnson, C.W., Dragich, J.M., Yoo, A.S. and Yamamoto, A. Neuron, 105(5), 813-821 (2020)   Despite being an autosomal dominant disorder caused by a known coding mutation in the gene HTT, Huntington’s disease (HD) patients with similar trinucleotide repeat mutations can have an age of onset that varies by decades. One likely contributing factor is the genetic heterogeneity of patients that might modify their vulnerability to disease. We report that although the heterozygous depletion of the autophagy adaptor protein Alfy/Wdfy3 has no consequence in control mice, it significantly accelerates age of onset and progression of HD pathogenesis. Alfy is required in the adult brain for the autophagy-dependent clearance of proteinaceous deposits, and its depletion in mice and neurons derived from patient fibroblasts accelerates the aberrant accumulation of this pathological hallmark shared across adult-onset neurodegenerative diseases. These findings indicate that selectively compromising the ability to eliminate aggregated proteins is a pathogenic driver, and the selective elimination of aggregates may confer disease resistance.  

3.3490           AMPK, a Regulator of Metabolism and Autophagy, Is Activated by Lysosomal Damage via a Novel Galectin-Directed Ubiquitin Signal Transduction System

Jia, J., Bissa, B., Brecht, L., Hallows, K., Behrends, C. and Deretic, V. Molecular Cell, 77, 951-969 (2020)   AMPK is a central regulator of metabolism and autophagy. Here we show how lysosomal damage activates AMPK. This occurs via a hitherto unrecognized signal transduction system whereby cytoplasmic sentinel lectins detect membrane damage leading to ubiquitination responses. Absence of Galectin 9 (Gal9) or loss of its capacity to recognize lumenal glycans exposed during lysosomal membrane damage abrogate such ubiquitination responses. Proteomic analyses with APEX2-Gal9 have revealed global changes within the Gal9 interactome during lysosomal damage. Gal9 association with lysosomal glycoproteins increases whereas interactions with a newly identified Gal9 partner, deubiquitinase USP9X, diminishes upon lysosomal injury. In response to damage, Gal9 displaces USP9X from complexes with TAK1 and promotes K63 ubiquitination of TAK1 thus activating AMPK on damaged lysosomes. This triggers autophagy and contributes to autophagic control of membrane-damaging microbe Mycobacterium tuberculosis. Thus, galectin and ubiquitin systems converge to activate AMPK and autophagy during endomembrane homeostasis.  

3.3491           Mesenchymal stem cell-derived magnetic extracellular nanovesicles for targeting and treatment of ischemic stroke

Kim, H.Y., Kim, T.J., Kang, L., Kim, Y-J., Kang, M.K., Kim, J., Ryu, J.H., Hyeon, T., Yoon, B-W., Ko, S-B. and Kim, B-S. Biomaterials, 243, 119942 (2020)   Exosomes and extracellular nanovesicles (NV) derived from mesenchymal stem cells (MSC) may be used for the treatment of ischemic stroke owing to their multifaceted therapeutic benefits that include the induction of angiogenesis, anti-apoptosis, and anti-inflammation. However, the most serious drawback of using exosomes and NV for ischemic stroke is the poor targeting on the ischemic lesion of brain after systemic administration, thereby yielding a poor therapeutic outcome. In this study, we show that magnetic NV (MNV) derived from iron oxide nanoparticles (IONP)-harboring MSC can drastically improve the ischemic-lesion targeting and the therapeutic outcome. Because IONP stimulated expressions of therapeutic growth factors in the MSC, MNV contained greater amounts of those therapeutic molecules compared to NV derived from naive MSC. Following the systemic injection of MNV into transient middle-cerebral-artery-occlusion (MCAO)-induced rats, the magnetic navigation increased the MNV localization to the ischemic lesion by 5.1 times. The MNV injection and subsequent magnetic navigation promoted the anti-inflammatory response, angiogenesis, and anti-apoptosis in the ischemic brain lesion, thereby yielding a considerably decreased infarction volume and improved motor function. Overall, the proposed MNV approach may overcome the major drawback of the conventional MSC-exosome therapy or NV therapy for the treatment of ischemic stroke.  

3.3492           Targeting Hsc70-based autophagy to eliminate amyloid β oligomers

Dou, J., Su, P., Xu, C., Wen, Z., Mao, Z. and Li, W. Biochem. Biophys. Res. Comm., 524, 923-928 (2020)   Amyloid β (Aβ) oligomers may be a real culprit in the pathogenesis of Alzheimer’s disease (AD); therefore, the elimination of these toxic oligomers may be of great significance for AD therapy. Autophagy is the catabolic process by which lysosomes degrade cytosolic components, and heat shock cognate 70 kDa protein (Hsc70) binds to proteins with their KFERQ-like motifs [also known as chaperone-mediated autophagy (CMA) motifs] and carries them to lysosomes through CMA or late endosomes through endosomal microautophagy (eMI) for degradation. In this study, our strategy is to make the pathological Aβ become one selective and suitable substrate for CMA and eMI (termed as Hsc70-based autophagy) by tagging its oligomers with multiple CMA motifs. First, we design and synthesize Aβ oligomer binding peptides with three CMA motifs. Second, we determine that the peptide can help Aβ oligomers enter endosomes and lysosomes, which can be further enhanced by ketone. More importantly, we find that the peptide can dramatically reduce Aβ oligomers in induced pluripotent stem cell (iPSC) cortical neurons derived from AD patient fibroblasts and protect primary cultured cortical neurons against the Aβ oligomer-induced neurotoxicity. In conclusion, we demonstrate that the peptide targeting Hsc70-based autophagy can effectively eliminate Aβ oligomers and have superior neuroprotective activity.  

3.3493           Grasp55−/− mice display impaired fat absorption and resistance to high-fat diet-induced obesity

Kim, J., Kim, J., Noh, S.H., Jang, D.G., Park, S-Y., Min, D. Kim, H., Kweon, H-S. eet al Nature Communications, 11:1418 (2020)   The Golgi apparatus plays a central role in the intracellular transport of macromolecules. However, molecular mechanisms of Golgi-mediated lipid transport remain poorly understood. Here, we show that genetic inactivation of the Golgi-resident protein GRASP55 in mice reduces whole-body fat mass via impaired intestinal fat absorption and evokes resistance to high-fat diet induced body weight gain. Mechanistic analyses reveal that GRASP55 participates in the Golgi-mediated lipid droplet (LD) targeting of some LD-associated lipases, such as ATGL and MGL, which is required for sustained lipid supply for chylomicron assembly and secretion. Consequently, GRASP55 deficiency leads to reduced chylomicron secretion and abnormally large LD formation in intestinal epithelial cells upon exogenous lipid challenge. Notably, deletion of dGrasp in Drosophila causes similar defects of lipid accumulation in the midgut. These results highlight the importance of the Golgi complex in cellular lipid regulation, which is evolutionary conserved, and uncover potential therapeutic targets for obesity-associated diseases.  

3.3494           Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine

Dhondt, B., Geeurickx, E., Tulkens, J., Van Deun, J., Vergauwen, G., Lippens, L., Miinalainen, I., Rappu, P., Heino, J., OSt, P., Lumen, N., De Wever, O. and Hendrix, A.

1. Extracellular Vesicles, 9(1), 1736935 (2020)

  Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers.  

3.3495           Fibroblast Growth Factor 2‐Mediated Regulation of Neuronal Exosome Release Depends on VAMP3/Cellubrevin in Hippocampal Neurons

Kumar, R., Tang, Q., Müller, S.A., Gao, P., Mahlstedt, D., Zampagni, S., Tan, Y., Klingl, A., Bötzel, K., Lichtenthaler, S.F., Höglinger, G.U. and Koeglsperger, T. Advanced Science, 7, 1902372 (2020)   Extracellular vesicles (EVs) are endogenous membrane‐derived vesicles that shuttle bioactive molecules between glia and neurons, thereby promoting neuronal survival and plasticity in the central nervous system (CNS) and contributing to neurodegenerative conditions. Although EVs hold great potential as CNS theranostic nanocarriers, the specific molecular factors that regulate neuronal EV uptake and release are currently unknown. A combination of patch‐clamp electrophysiology and pH‐sensitive dye imaging is used to examine stimulus‐evoked EV release in individual neurons in real time. Whereas spontaneous electrical activity and the application of a high‐frequency stimulus induce a slow and prolonged fusion of multivesicular bodies (MVBs) with the plasma membrane (PM) in a subset of cells, the neurotrophic factor basic fibroblast growth factor (bFGF) greatly increases the rate of stimulus‐evoked MVB‐PM fusion events and, consequently, the abundance of EVs in the culture medium. Proteomic analysis of neuronal EVs demonstrates bFGF increases the abundance of the v‐SNARE vesicle‐associated membrane protein 3 (VAMP3, cellubrevin) on EVs. Conversely, knocking‐down VAMP3 in cultured neurons attenuates the effect of bFGF on EV release. The results determine the temporal characteristics of MVB‐PM fusion in hippocampal neurons and reveal a new function for bFGF signaling in controlling neuronal EV release.  

3.3496           Therapeutic potential of extracellular vesicles in preclinical stroke models: a systematic review and meta-analysis

Thomas, J.M., Cunningham, C.J., Lawrence, C.B., Pinteaux, E. and Allan, S.M. BMJ Open Science, 4, e100047 (2020)   Objectives Currently there is a paucity of clinically available regenerative therapies for stroke. Extracellular vesicles (EV) have been investigated for their potential as modulators of regeneration in the poststroke brain. This systematic review and meta-analysis aims to provide a summary of the efficacy of therapeutic EVs in preclinical stroke models, to inform future research in this emerging field. Methods Studies were identified by a comprehensive literature search of two online sources and subsequent screening. Studies using lesion volume or neurological score as outcome measures were included. Standardised mean difference (SMD) and 95% CIs were calculated using a restricted maximum likelihood random effects model. Publication bias was assessed with Egger’s regression and presented as funnel plots with trim and fill analysis. Subgroup analysis was performed to assess the effects of different study variables. Study quality and risk of bias were assessed using the CAMARADES checklist. Results A total of 20 publications were included in the systematic review, of which 19 were assessed in the meta-analysis (43 comparisons). Overall, EV interventions improved lesion volume (SMD: −1.95, 95% CI −2.72 to 1.18) and neurological scores (SMD: −1.26, 95% CI −1.64 to 0.87) compared with control groups. Funnel plots were asymmetrical suggesting publication bias, and trim and fill analysis predicted seven missing studies for lesion volume. Subgroup analysis suggested administration at 0–23 hours after stroke was the most effective timepoint for EV treatment. The median score on the CAMARADES checklist was 7 (IQR: 5–8). Conclusions EVs may offer a promising new avenue for stroke therapies, as EV-based interventions had positive impacts on lesion volume and neurological score in preclinical stroke models.  

3.3497           Structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry

Shang, J., Wan, Y., Liu, C., Yount, B., Gully, K., Yang, Y., Auerbach, A., Peng, G., Baric, R. and Li, F. Plos Pathogens, 16(3), e1008392 (2020)   Coronaviruses recognize a variety of receptors using different domains of their envelope-anchored spike protein. How these diverse receptor recognition patterns affect viral entry is unknown. Mouse hepatitis coronavirus (MHV) is the only known coronavirus that uses the N-terminal domain (NTD) of its spike to recognize a protein receptor, CEACAM1a. Here we determined the cryo-EM structure of MHV spike complexed with mouse CEACAM1a. The trimeric spike contains three receptor-binding S1 heads sitting on top of a trimeric membrane-fusion S2 stalk. Three receptor molecules bind to the sides of the spike trimer, where three NTDs are located. Receptor binding induces structural changes in the spike, weakening the interactions between S1 and S2. Using protease sensitivity and negative-stain EM analyses, we further showed that after protease treatment of the spike, receptor binding facilitated the dissociation of S1 from S2, allowing S2 to transition from pre-fusion to post-fusion conformation. Together these results reveal a new role of receptor binding in MHV entry: in addition to its well-characterized role in viral attachment to host cells, receptor binding also induces the conformational change of the spike and hence the fusion of viral and host membranes. Our study provides new mechanistic insight into coronavirus entry and highlights the diverse entry mechanisms used by different viruses.  

3.3498           Conditional Single Vector CRISPR/SaCas9 Viruses for Efficient Mutagenesis in the Adult Mouse Nervous System

Hunker, A.C., Soden, M.E., Krayushkina, D.,  Heymann, G., Awatramani, R. and Zweifel, L.S. Cell Reports, 30, 4303-4316 (2020)   Mice engineered for conditional, cell type-specific gene inactivation have dominated the field of mouse genetics because of the high efficiency of Cre-loxP-mediated recombination. Recent advances in CRISPR/Cas9 technologies have provided alternatives for rapid gene mutagenesis for loss-of-function (LOF) analysis. Whether these strategies can be streamlined for rapid genetic analysis with the efficiencies comparable with those of conventional genetic approaches has yet to be established. We show that a single adeno-associated viral (AAV) vector containing a recombinase-dependent Staphylococcus aureus Cas9 (SaCas9) and a single guide RNA (sgRNA) are as efficient as conventional conditional gene knockout and can be adapted for use in either Cre- or Flp-driver mouse lines. The efficacy of this approach is demonstrated for the analysis of GABAergic, glutamatergic, and monoaminergic neurotransmission. Using this strategy, we reveal insight into the role of GABAergic regulation of midbrain GABA-producing neurons in psychomotor activation.  

3.3499           TNF receptor–associated factor 6 interacts with ALS-linked misfolded superoxide dismutase 1 and promotes aggregation

Semmler, S., gagne, M., Garg, P., Pickles, S.R., Baudouin, C., Hamon-Keromen, E. et al

1. Biol. Chem., 295(12), 3808-3825 (2020)

  Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the selective loss of motor neurons leading to paralysis. Mutations in the gene encoding superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS, and considerable evidence suggests that these mutations result in an increase in toxicity due to protein misfolding. We previously demonstrated in the SOD1^G93A rat model that misfolded SOD1 exists as distinct conformers and forms deposits on mitochondrial subpopulations. Here, using SOD1^G93A rats and conformation-restricted antibodies specific for misfolded SOD1 (B8H10 and AMF7-63), we identified the interactomes of the mitochondrial pools of misfolded SOD1. This strategy identified binding proteins that uniquely interacted with either AMF7-63 or B8H10-reactive SOD1 conformers as well as a high proportion of interactors common to both conformers. Of this latter set, we identified the E3 ubiquitin ligase TNF receptor–associated factor 6 (TRAF6) as a SOD1 interactor, and we determined that exposure of the SOD1 functional loops facilitates this interaction. Of note, this conformational change was not universally fulfilled by all SOD1 variants and differentiated TRAF6 interacting from TRAF6 noninteracting SOD1 variants. Functionally, TRAF6 stimulated polyubiquitination and aggregation of the interacting SOD1 variants. TRAF6 E3 ubiquitin ligase activity was required for the former but was dispensable for the latter, indicating that TRAF6-mediated polyubiquitination and aggregation of the SOD1 variants are independent events. We propose that the interaction between misfolded SOD1 and TRAF6 may be relevant to the etiology of ALS.  

3.3500           C19orf66 interrupts Zika virus replication by inducing lysosomal degradation of viral NS3

Wu, Y., Yang, X., Yao, Z., Dong, X., Zhang, D., Hu, Y., Zhang, S., Lin, J. et al Plos Neglected Tropical Diseases, 14(3), e0008083 (2020)   The rapidly emerging human health crisis associated with the Zika virus (ZIKV) epidemic and its link to severe complications highlights the growing need to identify the mechanisms by which ZIKV accesses hosts. Interferon response protects host cells against viral infection, while the cellular factors that mediate this defense are the products of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified, only a few have been characterized for their antiviral potential, target specificity and mechanisms of action. In this work, we focused our investigation on the possible antiviral effect of a novel ISG, C19orf66 in response to ZIKV infection and the associated mechanisms. We found that ZIKV infection could induce C19orf66 expression in ZIKV-permissive cells, and such an overexpression of C19orf66 remarkably suppressed ZIKV replication. Conversely, the depletion of C19orf66 led to a significant increase in viral replication. Furthermore, C19orf66 was found to interact and co-localize with ZIKV nonstructural protein 3 (NS3), thus inducing NS3 degradation via a lysosome-dependent pathway. Taken together, this study identified C19orf66 as a novel ISG that exerts antiviral effects against ZIKV by specifically degrading a viral nonstructural protein. These findings uncovered an intriguing mechanism of C19orf66 that targeting NS3 protein of ZIKV, providing clues for understanding the actions of innate immunity, and affording the possible availability of new drug targets that can be used for therapeutic intervention.  

3.3501           The inhibitory effect of ECG and EGCG dimeric procyanidins on colorectal cancer cells growth is associated with their actions at lipid rafts and the inhibition of the epidermal growth factor receptor signaling

Zhu, W., Li, M.C., Wang, F.R., Mackenzie, G.G. and Oteiza, P.L. Biochem. Pharmacol., 175, 113923 (2020)   Colorectal cancer (CRC) is one of the most common cancers worldwide. Epidemiological studies indicate that consumption of fruits and vegetables containing procyanidins is associated with lower CRC risk. This study investigated the capacity of two dimeric procyanidins composed of epicatechin gallate (ECG) or epigallocatechin gallate (EGCG) isolated from persimmons, to inhibit CRC cell growth and promote apoptosis, characterizing the underlying mechanisms. ECG and EGCG dimers reduced the growth of five human CRC cell lines in a concentration (10–60 μM)- and time (24–72 h)-dependent manner, with a 72 h-IC₅₀ value in Caco-2 cells of 10 and 30 μM, respectively. ECG and EGCG dimers inhibited Caco-2 cell proliferation by arresting the cell cycle in G₂/M phase and by inducing apoptosis via the mitochondrial pathway. In addition, ECG and EGCG dimers inhibited cell migration, invasion, and adhesion, decreasing the activity of matrix metalloproteinases (MMP-2/9). Mechanistically, ECG and EGCG dimers inhibited the activation of lipid raft-associated epidermal growth factor (EGF) receptor (EGFR), without affecting its localization at lipid rafts. In particular, ECG and EGCG dimers reduced EGFR phosphorylation at Tyr¹⁰⁶⁸ residue, prevented EGFR dimerization and activation upon stimulation, and induced EGFR internalization both in the absence and presence of EGF. Furthermore, ECG and EGCG dimers increased EGFR phosphorylation at Tyr¹⁰⁴⁵ residue, providing a docking site for ubiquitin ligase c-Cbl and induced EGFR degradation by the proteasome. Downstream of EGFR, ECG and EGCG dimers inhibited the activation of the MEK/ERK1/2 and PI3K/AKT signaling pathways, downregulating proteins involved in the modulation of cell survival. In conclusion, ECG and EGCG dimers reduced CRC cell growth by inhibiting EGFR activation at multiple steps, including the disruption of lipid rafts integrity and promoting EGFR degradation. These results shed light on a potential molecular mechanism on how procyanidins-rich diets may lower CRC risk.  

3.3502           MicroRNA exporter HuR clears the internalized pathogens by promoting pro‐inflammatory response in infected macrophages

Goswami, A., Mukherjee, K., Mazumder, A., Ganguly, S., Mukherjee, I., Chakrabarti, S., Roy, S., Sundar, S., Chattopadhyay, K. and Bhattacharyya, S.N. EMBO Mol. Med., 12, e11011 (2020)   HuR is a miRNA derepressor protein that can act as miRNA sponge for specific miRNAs to negate their action on target mRNAs. Here we have identified how HuR, by inducing extracellular vesicles‐mediated export of miRNAs, ensures robust derepression of miRNA‐repressed cytokines essential for strong pro‐inflammatory response in activated mammalian macrophages. Leishmania donovani, the causative agent of visceral leishmaniasis, on the contrary alters immune response of the host macrophage by a variety of complex mechanisms to promote anti‐inflammatory response essential for the survival of the parasite. We have found that during Leishmania infection, the pathogen targets HuR to promote onset of anti‐inflammatory response in mammalian macrophages. In infected macrophages, Leishmania also upregulate protein phosphatase 2A that acts on Ago2 protein to keep it in dephosphorylated and miRNA‐associated form. This causes robust repression of the miRNA‐targeted pro‐inflammatory cytokines to establish an anti‐inflammatory response in infected macrophages. HuR has an inhibitory effect on protein phosphatase 2A expression, and mathematical modelling of macrophage activation process supports antagonistic miRNA‐modulatory roles of HuR and protein phosphatase 2A which mutually balances immune response in macrophage by targeting miRNA function. Supporting this model, ectopic expression of the protein HuR and simultaneous inhibition of protein phosphatase 2A induce strong pro‐inflammatory response in the host macrophage to prevent the virulent antimonial drug‐sensitive or drug‐resistant form of L. donovani infection. Thus, HuR can act as a balancing factor of immune responses to curtail the macrophage infection process by the protozoan parasite.  

3.3503           Exosome-mimetics as an engineered gene-activated matrix induces in-situ vascularized osteogenesis

Zha, Y., Lin, T., Li, Y., Zhang, X., Wang, Z., Li, Z., Ye, Y., Wang, B., Zhang, S. and Wang, J. Biomaterials, 247, 119985 (2020)   Exosome has been considered as an instructive supplement between complicated cell therapy and single gene/protein drug treatment in the field of regenerative medicine due to its excellent biocompatibility, efficient cellular internalization and large loading capacity. Nevertheless, one major issue that extremely restricts the potential application as gene/drug vehicles is the low yield of nanoscale exosome. Moreover, the intravenous injection of targeted exosomes may cause the obstruction of blood-rich organs. Thus, herein we fabricated a specific exosome-mimetics (EMs) that could come true mass and fast production exhibited the similar size, morphology and membrane protein markers in comparison with conventional exosomes. To bypass the risk of intravenous injection and improve the efficiency of topical therapy, we simultaneously applied the engineered EMs to design a gene-activated matrix (GAM) that could be locally released by encapsulating the plasmid of vascular endothelial growth factor (VEGF) and flexibly binding onto a core-shell nanofiber film. Our findings showed that the well-designed engineered EMs-mediated GAM was able to sustainably deliver VEGF gene and significantly enhance the vascularized osteogenesis in vivo. The current work can not only consolidate the applied foundation of EMs through the breakthrough of high yield, but also provide a local and effective delivery of engineered EMs for the in-situ therapy.  

3.3504           Genome-wide In Vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT Toxicity

Wertz, M.H., Mitchem, M.R., Pinedaa, S.S., hart, T., Doench, J.G. and Heiman, M. Neuron, 106, 76-89 (2020)   Unbiased in vivo genome-wide genetic screening is a powerful approach to elucidate new molecular mechanisms, but such screening has not been possible to perform in the mammalian central nervous system (CNS). Here, we report the results of the first genome-wide genetic screens in the CNS using both short hairpin RNA (shRNA) and CRISPR libraries. Our screens identify many classes of CNS neuronal essential genes and demonstrate that CNS neurons are particularly sensitive not only to perturbations to synaptic processes but also autophagy, proteostasis, mRNA processing, and mitochondrial function. These results reveal a molecular logic for the common implication of these pathways across multiple neurodegenerative diseases. To further identify disease-relevant genetic modifiers, we applied our screening approach to two mouse models of Huntington’s disease (HD). Top mutant huntingtin toxicity modifier genes included several Nme genes and several genes involved in methylation-dependent chromatin silencing and dopamine signaling, results that reveal new HD therapeutic target pathways.  

3.3505           Peroxisome Elevation Induces Stem Cell Differentiation and Intestinal Epithelial Repair

Du, G., Xiong, l., Li, X., Luo, G. and Chen, H. Developmental Cell, 53, 169-184 (2020)   Epithelial-repair-dependent mucosal healing (MH) is associated with a more favorable prognosis for patients with inflammatory bowel disease (IBD). MH is accomplished via repair and regeneration of the intestinal epithelium. However, the mechanism underlying MH is ill defined. We found a striking upregulation of peroxisomes in the injured crypts of IBD patients. By increasing peroxisome levels in Drosophila midguts, we found that peroxisome elevation enhanced RAB7-dependent late endosome maturation, which then promoted stem and/or progenitor-cell differentiation via modulation of Janus Kinase (JAK) and Signal Transducer and Activator of Transcription (STAT)-SOX21A signaling. This in turn enhanced ISC-mediated regeneration. Importantly, RAB7 and SOX21 were upregulated in the crypts of IBD patients. Moreover, administration of drugs that increased peroxisome levels reversed the symptoms of dextran sulfate sodium (DSS)-induced colitis in mice. This study demonstrates a peroxisome-mediated epithelial repair mechanism, which opens a therapeutic avenue for the enhancement of MH in IBD patients.  

3.3506           Deletion of Topoisomerase 1 in excitatory neurons causes genomic instability and early onset neurodegeneration

Fragola, G., Mabb, A.M., Taylor-Blake, B., Niehuas, J.K., Chronister, W.D., Mao, H., Simon, J.M., Yuan, H., Li, Z., McConnell, M.J. and Zylka, M.J. Nature Communications, 11:1962 (2020)   Topoisomerase 1 (TOP1) relieves torsional stress in DNA during transcription and facilitates the expression of long (>100 kb) genes, many of which are important for neuronal functions. To evaluate how loss of Top1 affected neurons in vivo, we conditionally deleted (cKO) Top1 in postmitotic excitatory neurons in the mouse cerebral cortex and hippocampus. Top1 cKO neurons develop properly, but then show biased transcriptional downregulation of long genes, signs of DNA damage, neuroinflammation, increased poly(ADP-ribose) polymerase-1 (PARP1) activity, single-cell somatic mutations, and ultimately degeneration. Supplementation of nicotinamide adenine dinucleotide (NAD⁺) with nicotinamide riboside partially blocked neurodegeneration, and increased the lifespan of Top1 cKO mice by 30%. A reduction of p53 also partially rescued cortical neuron loss. While neurodegeneration was partially rescued, behavioral decline was not prevented. These data indicate that reducing neuronal loss is not sufficient to limit behavioral decline when TOP1 function is disrupted.  

3.3507           Extracellular vesicles spread the RNA interference signal of Tribolium castaneum TcA cells

Mingels, L., Wynant, N., Santos, D., Peeters, p., Gansemans, Y., Billen, J., Van Nieuwerburgh, F. and Broeck, J.V. Insect Biochem. Mol. Biol., 122, 103377 (2020)   The potential utility of RNA interference (RNAi) to control insect pests and viral infections depends largely on the target organism's ability to systemically spread the RNAi response. The efficacy of systemic RNAi varies among insects, though it has been shown to be high in the red flour beetle, Tribolium castaneum. We identified an extracellular RNAi signal that is present in the culture medium of T. castaneum (TcA) cells after treatment with long dsRNA specific for a luciferase reporter gene. Luciferase-specific siRNAs were detected in extracellular vesicles (EVs) that were purified from the culture medium of these dsRNA-treated cells. Furthermore, by measuring the silencing of luciferase expression, we showed that these siRNA-containing EVs can act as an RNAi signal for recipient TcA cells. We have therefore shown that a systemic RNAi response upon dsRNA treatment can be effectively spread through EVs.  

3.3508           Bacterial outer membrane vesicles as a platform for biomedical applications: An update

Li, M., Zhou, H., Yang, C., Wu, Y., Zhou, X., Liu, H. and Wang, Y.

1. Controlled Release, 323, 253-268 (2020)

  Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria both in vitro and in vivo. OMVs are nano-sized spherical vehicles formed by lipid bilayer membranes and contain multiple parent bacteria-derived components. Based on the presence of bacterial antigens, pathogen-associated molecular patterns (PAMPs), adhesins, various proteins and the vesicle structure, OMVs have been developed for biomedical applications as bacterial vaccines, adjuvants, cancer immunotherapy agents, drug delivery vehicles, and anti-bacteria adhesion agents. In this review, we analyze the contributions of the structure and composition of OMVs to their applications, summarize the methods used to isolate and characterize OMVs, and highlight recent progress and future perspectives of OMVs in biomedical applications.  

3.3509           Exploring the potential of engineered exosomes as delivery systems for tumor-suppressor microRNA replacement therapy in ovarian cancer

Kobayashi, M., Sawada, K., Miyamoto, M., Shimizu, A., Yamamoto, M., Kinose, Y., Nakamura, K., Kawamo, M., Kodama, M., Hashimoto, K. and Kimura, T. Biochem. Biophys. Res. Comm., 527, 153-161 (2020)   MicroRNA (miRNA) plays a pivotal role in cancer biology. Therefore, tumor suppressor (TS) miRNAs are an attractive target for cancer therapy. However, clinical trials have failed due to the difficulties in miRNA delivery, warranting the development of a novel drug delivery system (DDS). Exosomes are stable in circulation and selectively picked up by cancer cells, indicating that they can serve as a miRNA carrier. The aim of this study was to explore the possibility of exosomes as a carrier for miRNA replacement therapy for ovarian cancer (OC). First, exosomes were purified from primary-cultured omental fibroblasts of OC patients. miR-199a-3p was selected as a TS miRNA, and the synthesized miR-199a-3p was loaded into exosomes by electroporation. Treatment with miR199a-3p-loaded-exosomes (miR-199a-3p-Exo) drastically increased miR-199a-3p expression level in OC cell lines (CaOV3; 8592-, SKOV3; 67188-, and OVCAR3; 2280-fold). miR-199a-3p-Exo suppressed c-Met expression, a direct target of miR-199a-3p, and thereby inhibited cell proliferation and invasion. In a xenograft study, miR-199a-3p-Exo also drastically inhibited peritoneal dissemination in OC mice model, and diminished c-Met expression, ERK phosphorylation, and MMP2 expression in tumors. These results suggest that miRNA replacement therapy using exosomes shows promise for treatment of OC. Given that omental fibroblasts can be obtained from most OC patients, patient-derived exosomes can be utilized as a DDS for future molecular-targeted therapies.  

3.3510           A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells

Sung, B.H., von Lersner, A., Guerrero, J., Krystofiak, E.S., Inman, D., Pelletier, R., Zijlstra, A., Ponik, S.M. and Weaver, A.M. Nature Communications, 11:2092 (2020)   Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes. Understanding the function of exosomes requires a variety of tools, including live imaging. Our previous live-cell reporter, pHluorin-CD63, allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. However, dim fluorescence and the inability to make stably-expressing cell lines limit its use. We incorporated a stabilizing mutation in the pHluorin moiety, M153R, which now exhibits higher, stable expression in cells and superior monitoring of exosome secretion. Using this improved construct, we visualize secreted exosomes in 3D culture and in vivo and identify a role for exosomes in promoting leader–follower behavior in 2D and 3D migration. Incorporating an additional non-pH-sensitive red fluorescent tag allows visualization of the exosome lifecycle, including multivesicular body (MVB) trafficking, MVB fusion, exosome uptake and endosome acidification. This reporter will be a useful tool for understanding both autocrine and paracrine roles of exosomes.  

3.3511           Unraveling the mechanisms that specify molecules for secretion in extracellular vesicles

Leidal, A.M. and Dednath, J. Methods, 177, 15-26 (2020)   Extracellular vesicles (EVs) are small membrane-bound organelles naturally released from cells and potentially function as vehicles of intercellular communication. Cells release numerous sub-species of EVs, including exosomes and microvesicles, which are formed via distinct cellular pathways and molecular machineries and contain specific proteins, RNAs and lipids. Accumulating evidence indicates that the repertoire of molecules packaged into EVs is shaped by both the physiological state of the cell and the EV biogenesis pathway involved. Although these observations intimate that precisely regulated pathways sort molecules into EVs, the underlying molecular mechanisms that direct molecules for secretion remain poorly defined. Recently, with the advancement of mass spectrometry, next-generation sequencing techniques and molecular biology tools, several mechanisms contributing to EV cargo selection are beginning to be unraveled. This review examines strategies employed to reveal how specific proteins, RNAs and lipids are directed for secretion via EVs.  

3.3512           Methods for loading therapeutics into extracellular vesicles and generating extracellular vesicles mimetic-nanovesicles

Kenari, A.N., Cheng, L. and Hill, A.F. Methods, 177, 103-113 (2020)   Extracellular vesicles (EVs) are membrane bound vesicles released into the extracellular environment by eukaryotic and prokaryotic cells. EVs are enriched in active biomolecules and they can horizontally transfer cargo to recipient cells. In recent years EVs have demonstrated promising clinical applications due to their theragnostic potential. Although EVs have promising therapeutic potential, there are several challenges associated with using EVs before transition from the laboratory to clinical use. Some of these challenges include issues around low yield, isolation and purification methodologies, and efficient engineering (loading) of EVs with therapeutic cargo. Also, to achieve higher therapeutic efficiency, EV architecture and cargo may need to be manipulated prior to clinical application. Some of these issues have been addressed by developing biomimetic EVs. EV mimetic-nanovesicles (M-NVs) are a type of artificial EVs which can be generated from all cell types with comparable characteristics as EVs for an alternative therapeutic modality. In this review, we will discuss current techniques for modifying EVs and methodology used to generate and customize EV mimetic-nanovesicles.  

3.3513           A Translocation Pathway for Vesicle-Mediated Unconventional Protein Secretion

Zhang, M., Liu, L., Lin, X., Lv, X., Zheng, L. and Ge, L. Cell, 181, 637-652 (2020)   Many cytosolic proteins lacking a signal peptide, called leaderless cargoes, are secreted through unconventional secretion. Vesicle trafficking is a major pathway involved. It is unclear how leaderless cargoes enter into the vesicle. Here, we find a translocation pathway regulating vesicle entry and secretion of leaderless cargoes. We identify TMED10 as a protein channel for the vesicle entry and secretion of many leaderless cargoes. The interaction of TMED10 C-terminal region with a motif in the cargo accounts for the selective release of the cargoes. In an in vitro reconstitution assay, TMED10 directly mediates the membrane translocation of leaderless cargoes into the liposome, which is dependent on protein unfolding and enhanced by HSP90s. In the cell, TMED10 localizes on the endoplasmic reticulum (ER)-Golgi intermediate compartment and directs the entry of cargoes into this compartment. Furthermore, cargo induces the formation of TMED10 homo-oligomers which may act as a protein channel for cargo translocation.  

3.3514           Extracellular vesicles from mast cells induce mesenchymal transition in airway epithelial cells

Yin, Y., Shelke, G.V., Lässer, C., Brismar, H. and Lötvall, J. Respiratory Res., 21:101 (2020)   Background In the airways, mast cells are present in close vicinity to epithelial cells, and they can interact with each other via multiple factors, including extracellular vesicles (EVs). Mast cell-derived EVs have a large repertoire of cargos, including proteins and RNA, as well as surface DNA. In this study, we hypothesized that these EVs can induce epithelial to mesenchymal transition (EMT) in airway epithelial cells. Methods In this in-vitro study we systematically determined the effects of mast cell-derived EVs on epithelial A549 cells. We determined the changes that are induced by EVs on A549 cells at both the RNA and protein levels. Moreover, we also analyzed the rapid changes in phosphorylation events in EV-recipient A549 cells using a phosphorylated protein microarray. Some of the phosphorylation-associated events associated with EMT were validated using immunoblotting. Results Morphological and transcript analysis of epithelial A549 cells indicated that an EMT-like phenotype was induced by the EVs. Transcript analysis indicated the upregulation of genes involved in EMT, including TWIST1, MMP9, TGFB1, and BMP-7. This was accompanied by downregulation of proteins such as E-cadherin and upregulation of Slug-Snail and matrix metalloproteinases. Additionally, our phosphorylated-protein microarray analysis revealed proteins associated with the EMT cascade that were upregulated after EV treatment. We also found that transforming growth factor beta-1, a well-known EMT inducer, is associated with EVs and mediates the EMT cascade induced in the A549 cells. Conclusion Mast cell-derived EVs mediate the induction of EMT in epithelial cells, and our evidence suggests that this is triggered through the induction of protein phosphorylation cascades.  

3.3515           Non-pathogenic Escherichia coli acquires virulence by mutating a growth-essential LPS transporter

Kaito, C., yoshikai, H., Wakamatsu, A., Miyashita, A., Matsumoto, Y., Fujiyaki, T., kato, M., Ogura, Y., Hayashi, T., Isogai, T. and Sekimizu, K. PloS Pathogens, 16(4), e1006469 (2020)   The molecular mechanisms that allow pathogenic bacteria to infect animals have been intensively studied. On the other hand, the molecular mechanisms by which bacteria acquire virulence functions are not fully understood. In the present study, we experimentally evaluated the evolution of a non-pathogenic strain of Escherichia coli in a silkworm infection model and obtained pathogenic mutant strains. As one cause of the high virulence properties of E. coli mutants, we identified amino acid substitutions in LptD (G580S) and LptE (T95I) constituting the lipopolysaccharide (LPS) transporter, which translocates LPS from the inner to the outer membrane and is essential for E. coli growth. The growth of the LptD and LptE mutants obtained in this study was indistinguishable from that of the parent strain. The LptD and LptE mutants exhibited increased secretion of outer membrane vesicles containing LPS and resistance against various antibiotics, antimicrobial peptides, and host complement. In vivo cross-linking studies revealed that the conformation of the LptD-LptE complex was altered in the LptD and LptE mutants. Furthermore, several clinical isolates of E. coli carried amino acid substitutions of LptD and LptE that conferred resistance against antimicrobial substances. This study demonstrated an experimental evolution of bacterial virulence properties in an animal infection model and identified functional alterations of the growth-essential LPS transporter that led to high bacterial virulence by conferring resistance against antimicrobial substances. These findings suggest that non-pathogenic bacteria can gain virulence traits by changing the functions of essential genes, and provide new insight to bacterial evolution in a host environment.  

3.3516           Quantitative proteomic analysis of trypsin-treated extracellular vesicles to identify the real-vesicular proteins

Choi, D., Go, GX., Kim, D-K., Lee, J., park, S-M., Di Vizio, D. and Gho, Y.S.

1. Extracellular Vesicles, 9(1), 1757209 (2020)

  Extracellular vesicles (EVs) are nano-sized vesicles surrounded by a lipid bilayer and released into the extracellular milieu by most of cells. Although various EV isolation methods have been established, most of the current methods isolate EVs with contaminated non-vesicular proteins. By applying the label-free quantitative proteomic analyses of human colon cancer cell SW480-derived EVs, we identified trypsin-sensitive and trypsin-resistant vesicular proteins. Further systems biology and protein–protein interaction network analyses based on their cellular localization, we classified the trypsin-sensitive and trypsin-resistant vesicular proteins into two subgroups: 363 candidate real-vesicular proteins and 151 contaminated non-vesicular proteins. Moreover, the protein interaction network analyses showed that candidate real-vesicular proteins are mainly derived from plasma membrane (46.8%), cytosol (36.6%), cytoskeleton (8.0%) and extracellular region (2.5%). On the other hand, most of the contaminated non-vesicular proteins derived from nucleus, Golgi apparatus, endoplasmic reticulum and mitochondria. In addition, ribosomal protein complexes and T-complex proteins were classified as the contaminated non-vesicular proteins. Taken together, our trypsin-digested proteomic approach on EVs is an important advance to identify the real-vesicular proteins that could help to understand EV biogenesis and protein cargo-sorting mechanism during EV release, to identify more reliable EV diagnostic marker proteins, and to decode pathophysiological roles of EVs.  

3.3517           Extracellular vesicles provide a capsid-free vector for oncolytic adenoviral DNA delivery

Saari, H., Turunen, T., Löhmus, A., Turunen, M., Jalasvuori, M., Butcher, S.J., Ylä-Herttuala, S., Viitala, T., Cerullo, V., Siljander, P.R.M. and Yliperttula, M.

1. Extracellular Vesicles, 9(1), 1747206 (2020)

  Extracellular vesicles (EVs) have been showcased as auspicious candidates for delivering therapeutic cargo, including oncolytic viruses for cancer treatment. Delivery of oncolytic viruses in EVs could provide considerable advantages, hiding the viruses from the immune system and providing alternative entry pathways into cancer cells. Here we describe the formation and viral cargo of EVs secreted by cancer cells infected with an oncolytic adenovirus (IEVs, infected cell-derived EVs) as a function of time after infection. IEVs were secreted already before the lytic release of virions and their structure resembled normally secreted EVs, suggesting that they were not just apoptotic fragments of infected cells. IEVs were able to carry the viral genome and induce infection in other cancer cells. As such, the role of EVs in the life cycle of adenoviruses may be an important part of a successful infection and may also be harnessed for cancer- and gene therapy.  

3.3518           GPAT4-Generated Saturated LPAs Induce Lipotoxicity through Inhibition of Autophagy by Abnormal Formation of Omegasomes

Shiozaki, Y., Miyazaki-Anzai, S., Okamura, K., Keenan, A.L., Masuda, M. and Miyazaki, M. iScience, 23, 101105 (2020)   Excessive levels of saturated fatty acids are toxic to vascular smooth muscle cells (VSMCs). We previously reported that mice lacking VSMC-stearoyl-CoA desaturase (SCD), a major enzyme catalyzing the detoxification of saturated fatty acids, develop severe vascular calcification from the massive accumulation of lipid metabolites containing saturated fatty acids. However, the mechanism by which SCD deficiency causes vascular calcification is not completely understood. Here, we demonstrate that saturated fatty acids significantly inhibit autophagic flux in VSMCs, contributing to vascular calcification and apoptosis. Mechanistically, saturated fatty acids are accumulated as saturated lysophosphatidic acids (LPAs) (i.e. 1-stearoyl-LPA) possibly synthesized through the reaction of GPAT4 at the contact site between omegasomes and the MAM. The accumulation of saturated LPAs at the contact site causes abnormal formation of omegasomes, resulting in accumulation of autophagosomal precursor isolation membranes, leading to inhibition of autophagic flux. Thus, saturated LPAs are major metabolites mediating autophagy inhibition and vascular calcification.  

3.3519           Targeting endothelial exosomes for the prevention of cardiovascular disease

Jia, G. and Sowers, J.R. BBA-Mol Basis of Disease, 1866, 165833 (2020)   Exosomes are small lipid bilayer-enclosed 30–140 nm diameter vesicles formed from endosomes. Exosomes are secreted by various cell types including endothelial cells, immune cells and other cardiovascular tissues, and they can be detected in plasma, urine, cerebrospinal fluid, as well as tissues. Exosomes were initially regarded as a disposal mechanism to discard unwanted materials from cells. Recent studies suggest that exosomes play an important role in mediating of intercellular communication through the delivery and transport of cellular components such as nucleic acids, lipids, and proteins and thus regulate cardiovascular disease. Further, the underlying mechanisms by which abnormally released exosomes promote cardiovascular disease are not well understood. This review highlights recent studies involving endothelial exosomes, gives a brief overview of exosome biogenesis and release, isolation and identification of exosomes, and provides a contemporary understanding of the endothelial exosome pathophysiology and potential therapeutic strategies.  

3.3520           Circulating exosomes from patients with peripheral artery disease influence vascular cell migration and contain distinct microRNA cargo

Sorrentino, T., Duong, P., Bouchareychas, L., Chen, M., Chung, A., Schaller, M.S., Oskowitz, A., Raffai, R.L. and Cinte, M.S: JVS: Vascular Sci., 1, 28-41 (2020)   Objective Peripheral artery disease (PAD) is a chronic condition characterized by inflammation. Emerging literature suggests that circulating exosomes and their microRNA (miRNA) contents may influence atherosclerosis and vascular remodeling. We hypothesize that circulating exosomes in patients with PAD directly modulate vascular cell phenotype and contain proinflammatory miRNAs. Methods Exosomes (particle size, 30-150 nm) were isolated from plasma of healthy individuals (n = 6), patients with mild PAD (mPAD; median Rutherford class, 2.5; n = 6), and patients with severe PAD (sPAD; median Rutherford class, 4; n = 5). Exosome identity, size, and concentration were determined by Western blot and nanoparticle tracking analysis. Human vascular smooth muscle cell (VSMC) and endothelial cell (EC) migration was assessed by a standard wound closure assay after exposure to exosome preparations. Monocyte-derived macrophages isolated from healthy volunteers were exposed to exosome preparations, and targeted gene expression was analyzed using quantitative polymerase chain reaction. Exosome miRNA cargos were isolated, and a panel of defined, vascular-active miRNAs was assessed by quantitative polymerase chain reaction. Results There was no difference in overall exosome particle concentration or size between the three groups (one-way analysis of variance [ANOVA], P > .05). Compared with exosomes from healthy individuals, exosomes from mPAD and sPAD patients increased VSMC migration (1.0 ± 0.09-fold vs 1.5 ± 0.09-fold vs 2.0 ± 0.12-fold wound closure; ANOVA, P < .0001) and inhibited EC migration (1.8 ± 0.07-fold vs 1.5 ± 0.04-fold vs 1.3 ± 0.02-fold wound closure; ANOVA, P < .01) in a stepwise fashion. Exosomes also induced changes in monocyte-derived macrophage gene expression that did not appear PAD specific. Hierarchical analysis of exosome miRNA revealed distinct clustering of vascular-active miRNAs between the three groups. Several miRNAs that promote inflammatory pathways in vascular cells were expressed at higher levels in exosomes from sPAD patients. Conclusions Circulating exosomes from individuals with PAD exert in vitro functional effects on VSMCs and ECs that may promote adverse vessel remodeling. Exosomes from healthy individuals, mPAD patients, and sPAD patients contain distinct signatures of immune-regulatory miRNA. Together these data suggest that the proinflammatory cargo of circulating exosomes correlates with atherosclerosis severity in PAD patients and could influence vascular injury and repair.  

3.3521           KAT3-dependent acetylation of cell type-specific genes maintains neuronal identity in the adult mouse brain

Lipnski, M., Munoz-Viana, R., del Blanco, B., Marquez-Galera, A., Medrano-Relinque, J., Carames, J.M., Szczepankiewicz, A.A., Fernandez-Albert, J., Navarron, C.M., Olivares, R., Wilczynski, G.M., Canals, S., Lopez-Atalaya, J.P. and Barco, A. Nature Communications, 11:2588 (2020)   The lysine acetyltransferases type 3 (KAT3) family members CBP and p300 are important transcriptional co-activators, but their specific functions in adult post-mitotic neurons remain unclear. Here, we show that the combined elimination of both proteins in forebrain excitatory neurons of adult mice resulted in a rapidly progressing neurological phenotype associated with severe ataxia, dendritic retraction and reduced electrical activity. At the molecular level, we observed the downregulation of neuronal genes, as well as decreased H3K27 acetylation and pro-neural transcription factor binding at the promoters and enhancers of canonical neuronal genes. The combined deletion of CBP and p300 in hippocampal neurons resulted in the rapid loss of neuronal molecular identity without de- or transdifferentiation. Restoring CBP expression or lysine acetylation rescued neuronal-specific transcription in cultured neurons. Together, these experiments show that KAT3 proteins maintain the excitatory neuron identity through the regulation of histone acetylation at cell type-specific promoter and enhancer regions.  

3.3522           Loss of tumor susceptibility gene 101 (TSG101) perturbs endoplasmic reticulum structure and function

Kaul, Z., Mookherjee, D., Das, S., Chatterjee, D., Chakrabarti, S. and Chakrabarti, O. BBA-Mol. Cell Res., 1867, 118741 (2020)   Tumor susceptibility gene 101 (TSG101), an ESCRT-I protein, is implicated in multiple cellular processes and its functional depletion can lead to blocked lysosomal degradation, cell cycle arrest, demyelination and neurodegeneration. Here, we show that loss of TSG101 results in endoplasmic reticulum (ER) stress and this causes ER membrane remodelling (EMR). This correlates with an expansion of ER, increased vacuolation, altered relative distribution of the rough and smooth ER and disruption of three-way junctions. Blocked lysosomal degradation due to TSG101 depletion leads to ER stress and Ca2+ leakage from ER stores, causing destabilization of actin cytoskeleton. Inhibiting Ca2+ release from the ER by blocking ryanodine receptors (RYRs) with Dantrolene partially rescues the ER stress phenotypes. Hence, in this study we have identified the involvement of TSG101 in modulating ER stress mediated remodelling by engaging the actin cytoskeleton. This is significant because functional depletion of TSG101 effectuates ER-stress, perturbs the structure, mobility and function of the ER, all aspects closely associated with neurodegenerative diseases.  

3.3523           Non-coding RNAs and Exosomes: Their Role in the Pathogenesis of Sepsis

Hashemian, S.M., Pourhanifeh, M.H., Fadaei, S., Velayati, A.A., Mirzaei, H. and Hamblin, M.R. Molecular Therapy-Nucleic Acids, 21, 51-74 (2020)   Sepsis is characterized as an uncontrolled host response to infection, and it represents a serious health challenge, causing excess mortality and morbidity worldwide. The discovery of sepsis-related epigenetic and molecular mechanisms could result in improved diagnostic and therapeutic approaches, leading to a reduced overall risk for affected patients. Accumulating data show that microRNAs, non-coding RNAs, and exosomes could all be considered as novel diagnostic markers for sepsis patients. These biomarkers have been demonstrated to be involved in regulation of sepsis pathophysiology. However, epigenetic modifications have not yet been widely reported in actual clinical settings, and further investigation is required to determine their importance in intensive care patients. Further studies should be carried out to explore tissue-specific or organ-specific epigenetic RNA-based biomarkers and their therapeutic potential in sepsis patients.  

3.3524           Exploiting bacterial outer membrane vesicles as a cross-protective vaccine candidate against avian pathogenic Escherichia coli (APEC)

Hu, R., Li, J., Zhao, Y., Lin, H., Liang, L., Wang, M., Liu, H., Min, Y., Gao, Y. and Yang, M. Microb. Cell Fact., 19:119 (2020)   Background The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its numerous serogroups has promoted the development of biological immunostimulatory materials as new vaccine candidates in poultry farms. Bacterial outer membrane vesicles (OMVs), known as spherical nanovesicles enriched with various immunostimulants, are naturally secreted by Gram-negative bacteria, and have gained much attention for developing effective vaccine candidates. Recent report has demonstrated that OMVs of APEC O78 can induce protective immunity in chickens. Here, a novel multi-serogroup OMVs (MOMVs) vaccine was developed to achieve cross-protection against APEC infection in broiler chickens. Results In this study, OMVs produced by three APEC strains were isolated, purified and prepared into MOMVs by mixing these three OMVs. By using SDS-PAGE and LC–MS/MS, 159 proteins were identified in MOMVs and the subcellular location and biological functions of 20 most abundant proteins were analyzed. The immunogenicity of MOMVs was evaluated, and the results showed that MOMVs could elicit innate immune responses, including internalization by chicken macrophage and production of immunomodulatory cytokines. Vaccination with MOMVs induced specific broad-spectrum antibodies as well as Th1 and Th17 immune responses. The animal experiment has confirmed that immunization with an appropriate dose of MOMVs could not cause any adverse effect and was able to reduce bacteria loads and pro-inflammatory cytokines production, thus providing effective cross-protection against lethal infections induced by multi-serogroup APEC strains in chickens. Further experiments indicated that, although vesicular proteins were able to induce stronger protective efficiency than lipopolysaccharide, both vesicular proteins and lipopolysaccharide are crucial in MOMVs-mediated protection. Conclusions The multi-serogroup nanovesicles produced by APEC strains will open up a new way for the development of next generation vaccines with low toxicity and broad protection in the treatment and control of APEC infection.  

3.3525           Interleukin-6 deficiency exacerbates Huntington’s disease model phenotypes

Wertz, M.H., Pineda, S.S., Lee, H., Kulicke, R., Kellis, M. and Heiman, M. Nolecular Neurodegeneration, 15:29 (2020)   Huntington’s disease (HD) is an incurable neurodegenerative disorder caused by CAG trinucleotide expansions in the huntingtin gene. Markers of both systemic and CNS immune activation and inflammation have been widely noted in HD and mouse models of HD. In particular, elevation of the pro-inflammatory cytokine interleukin-6 (IL-6) is the earliest reported marker of immune activation in HD, and this elevation has been suggested to contribute to HD pathogenesis. To test the hypothesis that IL-6 deficiency would be protective against the effects of mutant huntingtin, we generated R6/2 HD model mice that lacked IL-6. Contrary to our prediction, IL-6 deficiency exacerbated HD-model associated behavioral phenotypes. Single nuclear RNA Sequencing (snRNA-seq) analysis of striatal cell types revealed that IL-6 deficiency led to the dysregulation of various genes associated with synaptic function, as well as the BDNF receptor Ntrk2. These data suggest that IL-6 deficiency exacerbates the effects of mutant huntingtin through dysregulation of genes of known relevance to HD pathobiology in striatal neurons, and further suggest that modulation of IL-6 to a level that promotes proper regulation of genes associated with synaptic function may hold promise as an HD therapeutic target.  

3.3526           The Separation and Characterization of Extracellular Vesicles from Medium Conditioned by Bovine Embryos

Pavani, K.C., Lin, X., Hamacher, J., Van Den Broeck, W., Couck, L., Peelman, L., Hendrix, A. and Van Soom, A. Int. J. Mol. Sci., 21, 2942 (2020)   Extracellular vesicles (EVs) have been identified as one of the communication mechanisms amongst embryos. They are secreted into the embryo culture medium and, as such, represent a source of novel biomarkers for identifying the quality of cells and embryos. However, only small amounts of embryo-conditioned medium are available, which represents a challenge for EV enrichment. Our aim is to assess the suitability of different EV separation methods to retrieve EVs with high specificity and sufficient efficiency. Bovine embryo-conditioned medium was subjected to differential ultracentrifugation (DU), OptiPrep^TM density gradient (ODG) centrifugation, and size exclusion chromatography. Separated EVs were characterized by complementary characterization methods, including Western blot, electron microscopy, and nanoparticle tracking analysis, to assess the efficiency and specificity. OptiPrep^TM density gradient centrifugation outperformed DU and SEC in terms of specificity by substantial removal of contaminating proteins such as ribonucleoprotein complexes (Argonaute-2 (AGO-2)) and lipoproteins (ApoA-I) from bovine embryo-derived EVs (density: 1.02–1.04, 1.20–1.23 g/mL, respectively). In conclusion, ODG centrifugation is the preferred method for identifying EV-enriched components and for improving our understanding of EV function in embryo quality and development.  

3.3527           Profiling Extracellular Long RNA Transcriptome in Human Plasma and Extracellular Vesicles for Biomarker Discovery

Rodosthenous, R.S., Hutchins, E., Reiman, R., Laaurent, L.C., Van Keuren-Jensen, K. and Das, S. iScience, 23, 101182 (2020)   The recent discovery of extracellular RNAs in blood, including RNAs in extracellular vesicles (EVs), combined with low-input RNA-sequencing advances have enabled scientists to investigate their role in human disease. To date, most studies have been focusing on small RNAs, and methodologies to optimize long RNAs measurement are lacking. We used plasma RNA to assess the performance of six long RNA sequencing methods, at two different sites, and we report their differences in reads (%) mapped to the genome/transcriptome, number of genes detected, long RNA transcript diversity, and reproducibility. Using the best performing method, we further compare the profile of long RNAs in the EV- and no-EV-enriched RNA plasma compartments. These results provide insights on the performance and reproducibility of commercially available kits in assessing the landscape of long RNAs in human plasma and different extracellular RNA carriers that may be exploited for biomarker discovery.  

3.3528           Differential adaptations in rod outer segment disc membranes in different models of congenital stationary night blindness

Senapati, S. and Park, P.S-H. BBA-Biomembranes, 1862, 183396 (2020)   Rod photoreceptor cells initiate scotopic vision when the light receptor rhodopsin absorbs a photon of light to initiate phototransduction. These photoreceptor cells are exquisitely sensitive and have adaptive mechanisms in place to maintain optimal function and to overcome dysfunctional states. One adaptation rod photoreceptor cells exhibit is in the packing properties of rhodopsin within the membrane. The mechanism underlying these adaptations is unclear. Mouse models of congenital stationary night blindness with different molecular causes were investigated to determine which signals are important for adaptations in rod photoreceptor cells. Night blindness in these mice is caused by dysfunction in either rod photoreceptor cell signaling or bipolar cell signaling. Changes in the packing of rhodopsin within photoreceptor cell membranes were examined by atomic force microscopy. Mice expressing constitutively active rhodopsin did not exhibit any adaptations, even under constant dark conditions. Mice with disrupted bipolar cell signaling exhibited adaptations, however, they were distinct from those in mice with disrupted phototransduction. These differential adaptations demonstrate that although multiple molecular defects can lead to a similar primary defect causing disease (i.e., night blindness), they can cause different secondary effects (i.e., adaptations). The lighting environment or signaling defects present from birth and during early rearing can condition mice and affect the adaptations occurring in more mature animals. A comparison of effects in wild-type mice, mice with defective phototransduction, and mice with defective bipolar cell signaling, indicated that bipolar cell signaling plays a role in this conditioning but is not required for adaptations in more mature animals.  

3.3529           Extracellular vesicles derived from macrophage promote angiogenesis In vitro and accelerate new vasculature formation In vivo

Gangadaran, P., Rajendran, R.L., Oh, J.M., Hong, C.M., Jeong, S.Y., Lee, S-W., Lee, J. and Ahn, B-C. Exp. Cell Res., 394, 112146 (2020)   Background Ischemia is the partial or complete blockage of blood supply to tissues. Extracellular vesicles (EVs) are emerging as a therapeutic tool for ischemic diseases. Most EV-based ischemia therapies are based on various stem cells. Here, we propose an alternative cell source for the isolation of pro-angiogenic EVs. Methods EVs were isolated from a mouse macrophage cell line (Raw 264.7). The characteristic features of the macrophage-derived EVs (MAC-EVs) were assessed using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting (WB) analysis. WB and qRT-PCR were performed to identify the pro-angiogenic VEGF and Wnt3a proteins and microRNAs (miR-210, miR-126, and miR-130a) in the MAC-EVs. In vitro and in vivo Matrigel plug assays were performed to investigate the capacity of the MAC-EVs for tube (blood vessel-like) formation and new blood vessel formation and assessed by histology. Results The MAC-EVs was positive for ALIX and negative for calnexin, with a round shape and an average size of 189 ± 65.1 nm. WB and qRT-PCR results revealed that VEGF, Wnt3a and miR-130a were more abundant in the MAC-EVs than cells. MAC-EVs treatment resulted in increased endothelial cellular proliferation, migration, and tube formation in vitro. In vivo assay results revealed that MAC-EVs increased the formation of new and larger blood vessels in the Matrigel plug of mice compared to the formation in the control group. Conclusion Our results suggest that MAC-EVs have the potential to induce angiogenesis in vitro and in vivo, could serve as a pro-angiogenic alternative for ischemic diseases.  

3.3530           T2 and T17 cytokines alter the cargo and function of airway epithelium-derived extracellular vesicles

Ax, E., Jevnikar, Z., Cvjetkovic, A., Malmhäll, C., Olsson, H., Råadinger, M. and Lässer, C. Respiratory Res., 21:155 (2020)   Background Asthma is a common and heterogeneous disease that includes subgroups characterized by type 2 (T2) or type 17 (T17) immune responses for which there is a need to identify the underlying mechanisms and biomarkers in order to develop specific therapies. These subgroups can be defined by airway epithelium gene signatures and the airway epithelium has also been implicated to play a significant role in asthma pathology. Extracellular vesicles (EVs) carry functional biomolecules and participate in cell-to-cell communication in both health and disease, properties that are likely to be involved in airway diseases such as asthma. The aim of this study was to identify stimulus-specific proteins and functionality of bronchial epithelium-derived EVs following stimulation with T2 or T17 cytokines. Methods EVs from cytokine-stimulated (T2: IL-4 + IL-13 or T17: IL-17A + TNFα) human bronchial epithelial cells cultured at air-liquid interface (HBEC-ALI) were isolated by density cushion centrifugation and size exclusion chromatography and characterized with Western blotting and electron microscopy. Transcriptomic (cells) and proteomic (EVs) profiling was also performed. Results Our data shows that EVs are secreted and can be isolated from the apical side of HBEC-ALI and that cytokine stimulation increases EV release. Genes upregulated in cells stimulated with T2 or T17 cytokines were increased also on protein level in the EVs. Proteins found in T17-derived EVs were suggested to be involved in pathways related to neutrophil movement which was supported by assessing neutrophil chemotaxis ex vivo. Conclusions Together, the results suggest that epithelial EVs are involved in airway inflammation and that the EV proteome may be used for discovery of disease-specific mechanisms and signatures which may enable a precision medicine approach to the treatment of asthma.  

3.3531           Extracellular vesicle long non-coding RNAs and circular RNAs: Biology, functions and applications in cancer

Li, Z., Zhu, X. and Huang, S. Cancer Lett., 489, 111-120 (2020)   Extracellular vesicles (EVs) have been recognized as vital mediators of intercellular communication that allow horizontal information exchange among tumor cells as well as reciprocal cross-talk between tumor and stromal cells. Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are selectively packaged into EVs then transferred to proximal and distal recipient cells, inducing profound phenotypic changes. Recently, there has been substantial progress in understanding the essential roles of EV-associated lncRNAs and circRNAs in cancer initiation and progression. The distinctive properties of these ncRNAs carried by EVs, including their tissue-specific expression, relative stability, targeted delivery and widespread presence in various biofluids, has also prompted the exploration of their potential in liquid biopsy and therapeutic usage. Herein, we summarized the biology, characteristics and functions of EV-associated lncRNAs and circRNAs, highlighting their vital roles in hallmarks of cancer and their prospective applications in cancer management. The challenges unresolved in this field were additionally pointed out to guide future studies.  

3.3532           Arthropod exosomes as bubbles with message(s) to transmit vector-borne diseases

Sultana, H. and Neelakanta, G. Current Opinion in Insect Sci., 40, 39-47 (2020)   Ticks and mosquitoes are medically important vectors that transmit several pathogens, including arboviruses, to humans. Understanding how these blood-feeding arthropods transmit pathogens to humans requires knowledge on the molecular and cellular interplay at vector-host interface. Recent studies have highlighted the role of tick and mosquito small extracellular vesicles (EVs), including exosomes, facilitating arbovirus transmission within arthropod cells and from arthropod to mammalian cells. In this review, we summarize this emerging line of investigation in understanding the role of tick and mosquito exosomes in vector–pathogen–host tripartite interactions. Understanding the role of arthropod exosomes in pathogen interactions could lead to the discovery of novel therapeutic targets to interfere with the life cycle of several pathogens transmitted by vectors.  

3.3533           Comparison of differential accessibility analysis strategies for ATAC-seq data

Gontarz, P., Fu, S., Xing, X., Liu, S., Miao, B., Bazylianska, V., Sharma, A., Madden, p., Cates, k., Yoo, A., Moszcynska, A., Wang, T. and Zhang, B. Scientific Reports, 10:10150 (2020)   ATAC-seq is widely used to measure chromatin accessibility and identify open chromatin regions (OCRs). OCRs usually indicate active regulatory elements in the genome and are directly associated with the gene regulatory network. The identification of differential accessibility regions (DARs) between different biological conditions is critical in determining the differential activity of regulatory elements. Differential analysis of ATAC-seq shares many similarities with differential expression analysis of RNA-seq data. However, the distribution of ATAC-seq signal intensity is different from that of RNA-seq data, and higher sensitivity is required for DARs identification. Many different tools can be used to perform differential analysis of ATAC-seq data, but a comprehensive comparison and benchmarking of these methods is still lacking. Here, we used simulated datasets to systematically measure the sensitivity and specificity of six different methods. We further discussed the statistical and signal density cut-offs in the differential analysis of ATAC-seq by applying them to real data. Batch effects are very common in high-throughput sequencing experiments. We illustrated that batch-effect correction can dramatically improve sensitivity in the differential analysis of ATAC-seq data. Finally, we developed a user-friendly package, BeCorrect, to perform batch effect correction and visualization of corrected ATAC-seq signals in a genome browser.  

3.3534           Raftlin is recruited by neuropilin-1 to the activated VEGFR2 complex to control proangiogenic signaling

Bayliss, A.L., Sundararaman, A., Granet, C. and Mellor, H. Angiogenesis, 23, 371-383 (2020)   Background VEGFR2 (vascular endothelial growth factor receptor 2) is the major pro-angiogenic receptor in endothelial cells. Compared to other members of the receptor tyrosine kinase family, we know relatively few VEGFR2 signaling partners. Our objective was to use mass spectrometry-based proteomics to identify novel binding partners of activated VEGFR2. Methods We created an endothelial cell line stably expressing GFP-tagged VEGFR2 and isolated activated receptor complexes. Analysis by mass spectrometry identified raftlin as a novel binding partner of VEGFR2. Results We found that raftlin is recruited to the activated VEGFR2 complex via the co-receptor Nrp1 (neuropilin-1). We show that raftlin regulates the surface levels of Nrp1 in endothelial cells, controlling the availability of Nrp1 for VEGFR2 interaction. Raftlin stabilizes active VEGFR2 at the cell surface by inhibiting endocytosis of the activated receptor. Raftlin also promotes recycling of internalized VEGFR2 to the cell surface. Raftlin alters the signaling outcomes of VEGFR2 activation, inhibiting the activation of p38 and FAK (focal adhesion kinases) specifically. Both pathways are linked to cell migration in endothelial cells, and raftlin inhibits endothelial cell migration in response to VEGF. Conclusion Nrp1 is an important co-receptor for VEGFR2; however, its functions are still only partially understood. We show that raftlin works with Nrp1 in endothelial cells to control intracellular trafficking of the activated VEGFR2. This modulates the response to VEGF and controls endothelial cell migration.  

3.3535           Prolonged tau clearance and stress vulnerability rescue by pharmacological activation of autophagy in tauopathy neurons

Silva, M.C., Nandi, G.A., Tentarelli, S., Gurrell, I.K., Jamier, T., Lucente, D., Dickerson, B.C., Brown, D.G., Brandon, N.J. and Haggarty, S.J. Nature Communications, 11:3258 (2020)   Tauopathies are neurodegenerative diseases associated with accumulation of abnormal tau protein in the brain. Patient iPSC-derived neuronal cell models replicate disease-relevant phenotypes ex vivo that can be pharmacologically targeted for drug discovery. Here, we explored autophagy as a mechanism to reduce tau burden in human neurons and, from a small-molecule screen, identify the mTOR inhibitors OSI-027, AZD2014 and AZD8055. These compounds are more potent than rapamycin, and robustly downregulate phosphorylated and insoluble tau, consequently reducing tau-mediated neuronal stress vulnerability. MTORC1 inhibition and autophagy activity are directly linked to tau clearance. Notably, single-dose treatment followed by washout leads to a prolonged reduction of tau levels and toxicity for 12 days, which is mirrored by a sustained effect on mTORC1 inhibition and autophagy. This new insight into the pharmacodynamics of mTOR inhibitors in regulation of neuronal autophagy may contribute to development of therapies for tauopathies.  

3.3536           Small extracellular vesicles modulated by αVβ3 integrin induce neuroendocrine differentiation in recipient cancer cells

Quaglia, F., Krishn, S.R., Daaboul, G.G., Sarker, S., Pippa, R., Domingo-Domenech, J., Kumar, G., Fortina, P., McCue, P., Kelly, W.K., Beltran, H., Liu, Q. and Languino, L.R.

1. Extracellular Vesicles, 9(1), 1761072 (2020)

  The ability of small extracellular vesicles (sEVs) to reprogram cancer cells is well established. However, the specific sEV components able to mediate aberrant effects in cancer cells have not been characterized. Integrins are major players in mediating sEV functions. We have previously reported that the αVβ3 integrin is detected in sEVs of prostate cancer (PrCa) cells and transferred into recipient cells. Here, we investigate whether sEVs from αVβ3-expressing cells affect tumour growth differently than sEVs from control cells that do not express αVβ3. We compared the ability of sEVs to stimulate tumour growth, using sEVs isolated from PrCa C4-2B cells by iodixanol density gradient and characterized with immunoblotting, nanoparticle tracking analysis, immunocapturing and single vesicle analysis. We incubated PrCa cells with sEVs and injected them subcutaneously into nude mice to measure in vivo tumour growth or analysed in vitro their anchorage-independent growth. Our results demonstrate that a single treatment with sEVs shed from C4-2B cells that express αVβ3, but not from control cells, stimulates tumour growth and induces differentiation of PrCa cells towards a neuroendocrine phenotype, as quantified by increased levels of neuroendocrine markers. In conclusion, the expression of αVβ3 integrin generates sEVs capable of reprogramming cells towards an aggressive phenotype.  

3.3537           Variability in the cleavage of exosome-associated transferrin receptor questions the utility of clinically useful soluble transferrin receptor assays for dogs, cats, and horses

Martinez, C.R., Santangelo, K.S. and Olver, C.S. Exp. Hematol., 86, 43-52 (2020)   Whole transferrin receptor (TfR) is present in reticulocyte exosomes. Soluble transferrin receptor (sTfR) is cleaved from whole TfR in human plasma, with the remnant cytoplasmic domain (cTfR) remaining membrane associated. In humans, sTfR is a biomarker that can detect iron deficiency in the presence of inflammatory disease. This condition is still a diagnostic dilemma in veterinary species. We aimed to (1) confirm the presence of exosomes and exosome-associated TfR in the serum of dogs, cats, and horses; and (2) to assess and compare the proportion of cTfR to total (cTfR + whole) in exosomal membranes of healthy and diseased dogs and cats and in healthy horses to indirectly predict their anticipated levels of circulating sTfR. We used discarded serum and whole blood samples from canine and feline patients, separated into healthy and diseased groups based on the health status of each patient, and healthy equine participants from a previous study. Ultracentrifugation, followed in some experiments by OptiPrep discontinuous density gradient fractionation, was used to isolate exosomes. Exosomes and associated TfR were identified using TEM and Western blot for TfR, respectively. Densitometry tracings of Western blots of serum exosomes were used to measure the proportion of cTfR to total TfR. Extracellular vesicles compatible with exosomes were successfully isolated and expressed TfR. The proportion of cTfR in dogs was greater than 50%, indicating that a majority of the whole TfR was cleaved to produce sTfR (and remnant cTfR). There was significant interindividual variation and no significant difference between healthy and diseased animals. The proportion of cTfR in cats was very low at 11%, indicating that very little sTfR was likely produced. There was a small yet significant difference between healthy and diseased cats. Healthy horses do not appear to cleave exosome-associated TfR. Diagnosis of iron deficiency in the presence of inflammatory disease remains a challenge in veterinary medicine. Our results indicate that TfR is poorly or unpredictably cleaved in veterinary species, revealing that there are species differences in exosomal TfR handling. These data suggest that development of an assay for the detection and quantification of sTfR in the species investigated may not be warranted.  

3.3538           Chapter 7 - Extracellular vesicles in the therapy of BPD

Lesage, F. and Thebaud, B. Tantalizing Therapeutics in Bronchopulmonary Dysplasia, 129-148 (2020)   Cell-based therapies are entering the clinical arena to curb complications of extreme preterm birth. While clinical trials are testing the feasibility and safety of first generation mesenchymal stromal cell (MSC) products, next generation cell-free therapies are already on the horizon. Fueled by the realization that MSCs exert their therapeutic benefit by releasing nanosized extracellular vesicles (EVs), a new field of cell-based therapy has blossomed. EVs, contain a rich cargo, making them ideal candidates to treat multifactorial diseases such as bronchopulmonary dysplasia (BPD). EV-based therapies may have significant advantages over the use of whole cells, including product consistency, higher repair potential and manufacturing scalability. As any disruptive technology, numerous iterations and refinements will be required over the coming decade to harness the potential of EVs. This chapter reviews current EV isolation and characterization techniques, summarizes preclinical studies suggesting their lung repair capabilities in experimental models mimicking BPD and highlights future challenges.  

3.3539           Chapter Three - Advances in extracellular vesicles analysis

Vinaiphat, A. and Sze, S.K. Adv. Clin. Chem., 97, 73-116 (2020)   Extracellular vesicles (EVs) play an important role in intercellular communication in normal cellular process and pathological conditions by facilitating the transport of cellular content from one cell to another. EVs as conveyors of various biological molecules with their ability to redirect effects on a target cell physiological function in cell type-specific manner makes EVs an excellent candidate for drug delivery vehicle in disease therapy. Moreover, unique characteristics and contents of EVs which differ depends on cellular origin and physiological state make them a valuable source of diagnostic biomarker. Herein, we review the current progress in extracellular vesicle (EV) analysis, its transition from biomedical research to advancing therapy, and recent pioneered approaches to characterize and quantify EVs' subclasses with an emphasis on the integration of advanced technologies for both qualitative and quantitative analysis of EVs in different clinical tissue/body fluid samples.  

3.3540           The αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis

Keishn, S.R., Salem, I., Quaglia, F., Naranjo, N.M., Agarwal, E., Liu, Q., Sarker, S., Kopenhaver, J., McCue, P.A., Weinreb, P.H., Violette, S.M., Altieri, D.C. and Languino, L.R.

1. Extracellular Vesicles, 9(1), 1763594 (2020)

  Prostate cancer (PrCa) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sEVs). sEVs, as well as large extracellular vesicles (LEVs), isolated via iodixanol density gradients from PrCa cell culture media, express the epithelial-specific αvβ6 integrin, which is known to be induced in cancer. In this study, we show sEV-mediated protein transfer of αvβ6 integrin to microvascular endothelial cells (human microvascular endothelial cells 1 – HMEC1); we demonstrate that de novo αvβ6 integrin expression is not caused by increased mRNA levels. Incubation of HMEC1 with sEVs isolated from PrCa PC3 cells that express the αvβ6 integrin results in a highly significant increase in the number of nodes, junctions and tubules. In contrast, incubation of HMEC1 with sEVs isolated from β6 negative PC3 cells, generated by shRNA against β6, results in a reduction in the number of nodes, junctions and tubules, a decrease in survivin levels and an increase in a negative regulator of angiogenesis, pSTAT1. Furthermore, treatment of HMEC1 with sEVs generated by CRISPR/Cas9-mediated down-regulation of β6, causes up-regulation of pSTAT1. Overall, our findings suggest that αvβ6 integrin in cancer sEVs regulates angiogenesis during PrCa progression.  

3.3541           Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles

Mariscal, J., Vagner, T., Kim, M., Zhou, B., Chin, A., Zandian, M., Freeman, M.R., You, S., Zijlstra, A., Yang, W. and Di Vizio, D.

1. Extracellular Vesicles, 9(1), 1764192 (2020)

  Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, frequently called palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyse the palmitoyl-proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers.  

3.3542           Indoor dust extracellular vesicles promote cancer lung metastasis by inducing tumour necrosis factor-α

Dinh, N.T.H., Lee, J., Lee, L., Kim, S.S., Go, G., Bae, S., Jun, Y.I., Yoon, Y.J., Roh, T-Y. and Gho, Y.S.

1. Extracellular Vesicles, 9(1), 1766821 (2020)

  Indoor pollutants are important problems to public health. Among indoor pollutants, indoor dust contains extracellular vesicles (EVs), which are associated with pulmonary inflammation. However, it has not been reported whether indoor dust EVs affect the cancer lung metastasis. In this study, we isolated indoor dust EVs and investigated their roles in cancer lung metastasis. Upon intranasal administration, indoor dust EVs enhanced mouse melanoma lung metastasis in a dose-dependent manner in mice. Pre-treatment or co-treatment of indoor dust EVs significantly promoted melanoma lung metastasis, whereas post-treatment of the EVs did not. In addition, the lung lysates from indoor dust EV-treated mice significantly increased tumour cell migration in vitro. We observed that tumour necrosis factor-α played important roles in indoor dust EV-mediated promotion of tumour cell migration in vitro and cancer lung metastasis in vivo. Furthermore, Pseudomonas EVs, the main components of indoor dust EVs, and indoor dust EVs showed comparable effects in promoting tumour cell migration in vitro and cancer lung metastasis in vivo. Taken together, our results suggest that indoor dust EVs, at least partly contributed by Pseudomonas EVs, are potential promoting agents of cancer lung metastasis.  

3.3543           A Mouse Model for Infection with Enterovirus A71 in Small Extracellular Vesicles

Gu, J., Wu, J., Cao, Y., Zou, X., Jia, X., Yin, Y., Shen, L., Fang, D. and Mao, L. mSphere, 5(4), e00377-20 (2020)   Enterovirus A71 (EV-A71) is the major pathogen of hand, foot, and mouth disease (HFMD); in some severe cases, it could develop into central nervous system (CNS) disease such as aseptic meningitis, encephalitis, and neurogenic pulmonary edema in children under 5 years. The EV-A71 pathogenesis which is involved with the CNS is unclear due to the lack of a simple and reliable mouse model thus far. Most clinical EV-A71 isolates could not effectively infect the neonatal mouse, which used to be an EV-A71 infection model. The small extracellular vesicles (sEVs) released from clinical EV-A71 isolate-infected cells were infectious in cell lines and could cause a high viral replication in mice. Neonatal ICR mice were injected intraperitoneally with these infectious sEVs and showed more weight loss and higher mortality than those mice injected with the clinical EV-A71 isolate. By using these sEVs, we provided a simple and effective method by which we can generate a stable and valuable animal model for the studies of EV-A71 pathogenesis and therapy.  

3.3544           Schistosomal extracellular vesicle‐enclosed miRNAs modulate host T helper cell differentiation

Meningher, T., Barsheshet, Y., Ofir-Birin, Y., Gold, D., Brant, B., Dekel, E., Sidi, Y., Schwartz, E., Regev-Rudzki, N., Avni, O. and Avni, D. EMBO Reports, 21:e47882 (2020)   During the chronic stage of Schistosoma infection, the female lays fertile eggs, triggering a strong anti‐parasitic type 2 helper T‐cell (Th2) immune response. It is unclear how this Th2 response gradually declines even though the worms live for years and continue to produce eggs. Here, we show that Schistosoma mansoni downregulates Th2 differentiation in an antigen‐presenting cell‐independent manner, by modulating the Th2‐specific transcriptional program. Adult schistosomes secrete miRNA ‐harboring extracellular vesicles that are internalized by Th cells in vitro . Schistosomal miRNA s are found also in T helper cells isolated from Peyer's patches and mesenteric lymph nodes of infected mice. In T helper cells, the schistosomal miR‐10 targets MAP 3K7 and consequently downmodulates NF ‐κB activity, a critical transcription factor for Th2 differentiation and function. Our results explain, at least partially, how schistosomes tune down the Th2 response, and provide further insight into the reciprocal geographic distribution between high prevalence of parasitic infections and immune disorders such as allergy. Furthermore, this worm‐host crosstalk mechanism can be harnessed to develop diagnostic and therapeutic approaches for human schistosomiasis and Th2‐associated diseases.  

3.3545           Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome

Miyamoto, T., Hosoba, K., Itabashi, T., Iwane, A.H., Akutsu, S.N., Ochiai, H., Saito, Y., Yamamoto, T. and Mausuura, S. EMBO J., 39:e103499 (2020)   Primary cilia are antenna‐like organelles on the surface of most mammalian cells that receive sonic hedgehog (Shh) signaling in embryogenesis and carcinogenesis. Cellular cholesterol functions as a direct activator of a seven‐transmembrane oncoprotein called Smoothened (Smo) and thereby induces Smo accumulation on the ciliary membrane where it transduces the Shh signal. However, how cholesterol is supplied to the ciliary membrane remains unclear. Here, we report that peroxisomes are essential for the transport of cholesterol into the ciliary membrane. Zellweger syndrome (ZS ) is a peroxisome‐deficient hereditary disorder with several ciliopathy‐related features and cells from these patients showed a reduced cholesterol level in the ciliary membrane. Reverse genetics approaches revealed that the GTP exchange factor Rabin8, the Rab GTP ase Rab10, and the microtubule minus‐end‐directed kinesin KIFC 3 form a peroxisome‐associated complex to control the movement of peroxisomes along microtubules, enabling communication between peroxisomes and ciliary pocket membranes. Our findings suggest that insufficient ciliary cholesterol levels may underlie ciliopathies.  

3.3546           PagC is involved in salmonella pullorum OMVs production and affects biofilm production

Lu, J., Lianyue, L., Pan, F., Zuo, G., Yu, D., Liu, R., Fan, H. and Ma, Z.

Vet. Microbiol., 247, 108778 (2020)

 

The pagC gene is ubiquitously distributed in Salmonella, but there is limited information regarding its function. Pullorum disease (PD) is a septicemic disease caused by Salmonella Pullorum, which also harbors the pagC gene. In this study, we constructed an S. Pullorum pagC gene deletion strain and its complemented strain. First, we confirmed that the pagC gene does not participate in bacterial growth regulation or environmental pH adaptation. Interestingly, the results of subsequent analyses indicated that the pagC gene defect led to increased bacterial colonization in the intestine (especially in the cecum) and increased biofilm formation, while the number of outer-membrane vesicles (OMVs) in the bacterial culture decreased. Purified OMVs were able to reduce S. Pullorum biofilm formation in vitro. In addition, the results of a mass spectrometry analysis of purified OMVs indicated that some enzymes harbored by OMVs may be involved in biofilm degradation. Based on these results, we conclude that deletion of the pagC gene leads to reduced S. Pullorum OMVs production, which subsequently promotes biofilm stability, increases bacterial colonization in the intestine, and potentially inhibits the switch from sessile to planktonic growth.

 

3.3547           Molecular mechanisms that regulate export of the planar cell-polarity protein Frizzled-6 out of the endoplasmic reticulum

Tang, X., Zhang, l., Ma, T., Wang, M., Li, B., Jiang, L., Yan, Y. and Guo, Y.

1. Biol. Chem., 295(27), 8972-8987 (2020)

  Planar cell polarity (PCP) is a process during which cells are polarized along the plane of the epithelium and is regulated by several transmembrane signaling proteins. After their synthesis, these PCP proteins are delivered along the secretory transport pathway to the plasma membrane, where they perform their physiological functions. However, the molecular mechanisms that regulate PCP protein transport remain largely unclear. Here, we found that the delivery of a PCP protein, Frizzled-6, to the cell surface is regulated by two conserved polybasic motifs: one located in its first intracellular loop and the other in its C-terminal cytosolic domain. We observed that the polybasic motif of Frizzled is also important for its surface localization in the Drosophila wing. Results from a mechanistic analysis indicated that Frizzled-6 packaging into vesicles at the endoplasmic reticulum (ER) is regulated by a direct interaction between the polybasic motif and the Glu-62 and Glu-63 residues on the secretion-associated Ras-related GTPase 1A (SAR1A) subunit of coat protein complex II (COPII). Moreover, we found that newly synthesized Frizzled-6 is associated with another PCP protein, cadherin EGF LAG seven-pass G-type receptor 1 (CELSR1), in the secretory transport pathway, and that this association regulates their surface delivery. Our results reveal insights into the molecular machinery that regulates the ER export of Frizzled-6. They also suggest that the association of CELSR1 with Frizzled-6 is important, enabling efficient Frizzled-6 delivery to the cell surface, providing a quality control mechanism that ensures the appropriate stoichiometry of these two PCP proteins at cell boundaries.  

3.3548           Functionalized exosome harboring bioactive molecules for cancer therapy

Kim, Y.K., Choi, Y., Nam, G-H. and Kim, I-S. Cancer Lett., 489, 155-162 (2020)   Exosomes are nanosized vesicles with a lipid membrane that are secreted by most cells and play a crucial role as intermediates of intercellular communication because they carry bioactive molecules. Exosomes are promising for drug delivery of chemicals, proteins, and nucleic acids owing to their inherent properties such as excellent biocompatibility, high tumor targetability, and prolonged circulation in vivo. In this review, we cover recent approaches and advances made in the field of exosome-mediated delivery of bioactive molecules for cancer therapy and factors that affect the clinical use of exosomes. This review can be used as a guideline for further study in expanding the utility of therapeutic exosomes.  

3.3549           UBR E3 ligases and the PDIA3 protease control degradation of unfolded antibody heavy chain by ERAD

Tang, D., Sandoval, W., Lam, C., Haley, B., Liu, P., Xue, D., Roy, D., Patapoff, T., Louie, S., Snedecor, B. and Misaghi, S.

1. Cell. Biol., 219(7), e201908087 (2020)

  Accumulation of unfolded antibody chains in the ER triggers ER stress that may lead to reduced productivity in therapeutic antibody manufacturing processes. We identified UBR4 and UBR5 as ubiquitin E3 ligases involved in HC ER-associated degradation. Knockdown of UBR4 and UBR5 resulted in intracellular accumulation, enhanced secretion, and reduced ubiquitination of HC. In concert with these E3 ligases, PDIA3 was shown to cleave ubiquitinated HC molecules to accelerate HC dislocation. Interestingly, UBR5, and to a lesser degree UBR4, were down-regulated as cellular demand for antibody expression increased in CHO cells during the production phase, or in plasma B cells. Reducing UBR4/UBR5 expression before the production phase increased antibody productivity in CHO cells, possibly by redirecting antibody molecules from degradation to secretion. Altogether we have characterized a novel proteolysis/proteasome-dependent pathway involved in degradation of unfolded antibody HC. Proteins characterized in this pathway may be novel targets for CHO cell engineering.  

3.3550           Small Extracellular Vesicles Have GST Activity and Ameliorate Senescence-Related Tissue Damage

Fafian-labora, J.A., Rodriguez-Navarro, J.A. and O’Loghlen, A. Cell Metabolism, 32, 71-86 (2020)   Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging.  

3.3551           Optimized method for extraction of exosomes from human primary muscle cells

Gall, L.L., Ouandaogo, Z.G., Anakor, E., Conolly, O., Browne, G.B., Laine, J., Duddu, W. and Duguez, S. Skeletal Muscle, 10:20, (2020)   Skeletal muscle is increasingly considered an endocrine organ secreting myokines and extracellular vesicles (exosomes and microvesicles), which can affect physiological changes with an impact on different pathological conditions, including regenerative processes, aging, and myopathies. Primary human myoblasts are an essential tool to study the muscle vesicle secretome. Since their differentiation in conditioned media does not induce any signs of cell death or cell stress, artefactual effects from those processes are unlikely. However, adult human primary myoblasts senesce in long-term tissue culture, so a major technical challenge is posed by the need to avoid artefactual effects resulting from pre-senescent changes. Since these cells should be studied within a strictly controlled pre-senescent division count (<21 divisions), and yields of myoblasts per muscle biopsy are low, it is difficult or impossible to amplify sufficiently large cell numbers (some 250 × 10⁶ myoblasts) to obtain sufficient conditioned medium for the standard ultracentrifugation approach to exosome isolation. Thus, an optimized strategy to extract and study secretory muscle vesicles is needed. In this study, conditions are optimized for the in vitro cultivation of human myoblasts, and the quality and yield of exosomes extracted using an ultracentrifugation protocol are compared with a modified polymer-based precipitation strategy combined with extra washing steps. Both vesicle extraction methods successfully enriched exosomes, as vesicles were positive for CD63, CD82, CD81, floated at identical density (1.15-1.27 g.ml⁻¹), and exhibited similar size and cup-shape using electron microscopy and NanoSight tracking. However, the modified polymer-based precipitation was a more efficient strategy to extract exosomes, allowing their extraction in sufficient quantities to explore their content or to isolate a specific subpopulation, while requiring >30 times fewer differentiated myoblasts than what is required for the ultracentrifugation method. In addition, exosomes could still be integrated into recipient cells such as human myotubes or iPSC-derived motor neurons. Modified polymer-based precipitation combined with extra washing steps optimizes exosome yield from a lower number of differentiated myoblasts and less conditioned medium, avoiding senescence and allowing the execution of multiple experiments without exhausting the proliferative capacity of the myoblasts.  

3.3552           Rab27a dependent exosome releasing participated in albumin handling as a coordinated approach to lysosome in kidney disease

Feng, Y., Zhong, X., Tang, T-T., Wang, C., Wang, L-T., Li, Z-L., Ni, H-F., Wang, B., Wu, M., Liu, D., Liu, H., Tang, R-N., Liu, B-C. and Lv, L-L. Cell Death & Disease, 11:513 (2020)   Exosomes are increasingly recognized as vehicles of intercellular communication. However, the role of exosome in maintaining cellular homeostasis under stress conditions remained unclear. Here we show that Rab27a expression was upregulated exclusively in tubular epithelial cells (TECs) during proteinuria nephropathy established by adriamycin (ADR) injection and 5/6 nephrectomy as well as in chronic kidney disease patients, leading to the increased secretion of exosomes carrying albumin. The active exosome production promoted tubule injury and inflammation in neighboring and the producing cells. Interferon regulatory factor 1 (IRF-1) was found as the transcription factor contributed to the upregulation of Rab27a. Albumin could be detected in exosome fraction and co-localized with exosome marker CD63 indicating the secretion of albumin into extracellular space by exosomes. Interestingly, inhibition of exosome release accelerated albumin degradation which reversed tubule injury with albumin overload, while lysosome suppression augmented exosome secretion and tubule inflammation. Our findings revealed that IRF-1/Rab27a mediated exosome secretion constituted a coordinated approach to lysosome degradation for albumin handling, which lead to the augment of albumin toxicity as a maladaptive response to maintain cell homeostasis. The findings may suggest a novel therapeutic strategy for proteinuric kidney disease by targeting exosome secretion.  

3.3553           Improved Flow Cytometric Light Scatter Detection of Submicron‐Sized Particles by Reduction of Optical Background Signals

Arkesteijn, G.J.A., Lozano-Andres, E., Libregts, S.F.W.M. and Wauben, M.H.M. Cytometry Part A, 97A, 610-619 (2020)   Flow cytometry allows multiparameter analysis on a single‐cell basis and is currently the method of choice to rapidly assess heterogeneity of cell populations in suspension. With the research field of extracellular vesicles (EV) rapidly expanding, there is an increased demand to address heterogeneity of EV populations in biological samples. Although flow cytometry would be the ideal technique to do so, the available instruments are in general not equipped to optimally detect the dim light scatter signals generated by submicron‐sized particles like EV. Although sideward scatter light and fluorescence are currently used as a threshold signal to identify EV within samples, the forward scatter light (FSC) parameter is often neglected due to the lack of resolution to distinguish EV‐related signals from noise. However, after optimization of FSC detection by adjusting the size of the obscuration bar, we recently showed that certain EV‐subsets could only be identified based on FSC. This observation made us to further study the possibilities to enhance FSC‐detection of submicron‐sized particles. By testing differently sized obscuration bars and differently sized pinholes in the focal plane behind the FSC detection lens, we generated a matrix that allowed us to determine which combination resulted in the lowest optical background in terms of numbers of events regarding FSC detection of submicron‐sized particles. We found that a combination of an 8‐mm obscuration bar and a 200‐μm pinhole reduced optical background in a reproducible manner to such extent that it allowed a robust separation of 100‐nm polystyrene beads from background signals within the FSC channel, and even allowed thresholding on FSC without the interference of massive background signals when both beads and EV were measured. These technical adaptations thus significantly improved FSC detection of submicron‐sized particles and provide an important lead for the further development and design of flow cytometers that aid in detection of submicron‐sized particles.  

3.3554           Mesenchymal stromal cell-derived small extracellular vesicles restore lung architecture and improve exercise capacity in a model of neonatal hyperoxia-induced lung injury

Willis, G.R., Fernandez-Gonzalez, A., Reis, M., Yeung, V., Liu, X., Ericsson, M., Andrews, N.A., Mitsialis, S.A. and Kourembanas, S.

1. Extracellular Vesicles, 9(1), 1790874 (2020)

  Early administration of mesenchymal stromal cell (MSC)-derived small extracellular vesicles (MEx) has shown considerable promise in experimental models of bronchopulmonary dysplasia (BPD). However, the ability of MEx to reverse the long-term pulmonary complications associated with established BPD remains unknown. In this study, MEx were isolated from media conditioned by human Wharton’s Jelly-derived MSC cultures. Newborn mice (FVB strain) were exposed to hyperoxia (HYRX (75% O2)) before returning to room air at postnatal day 14 (PN14). Following prolonged HYRX-exposure, animals received a single MEx dose at PN18 or serial MEx treatments at PN18-39 (“late” intervention). This group was compared to animals that received an early single MEx dose at PN4 (“early” intervention). Animals were harvested at PN28 or 60 for assessment of pulmonary parameters. We found that early and late MEx interventions effectively ameliorated core features of HYRX-induced neonatal lung injury, improving alveolar simplification, pulmonary fibrosis, vascular remodelling and blood vessel loss. Exercise capacity testing and assessment of pulmonary hypertension (PH) showed functional improvements following both early and late MEx interventions. In conclusion, delivery of MEx following prolonged HYRX-exposure improves core features of experimental BPD, restoring lung architecture, decreasing pulmonary fibrosis and vascular muscularization, ameliorating PH and improving exercise capacity. Taken together, delivery of MEx may not only be effective in the immediate neonatal period to prevent the development of BPD but may provide beneficial effects for the management and potentially the reversal of cardiorespiratory complications in infants and children with established BPD.  

3.3555           miRNA- and cytokine-associated extracellular vesicles mediate squamous cell carcinomas

Flemming, J.P., Hill, B.L., Haque, M.W., Raad, J., Bonder, C.S., Harshyne, U., Luginbuhl, A., Wahl III, J.K., Tsai, K.Y., Wermuth, P.J., Overmiller, A.M. and Mahoney, M.G.

1. Extracellular Vesicles, 9(1), 1790159 (2020)

  Exosomes, or small extracellular vesicles (sEVs), serve as intercellular messengers with key roles in normal and pathological processes. Our previous work had demonstrated that Dsg2 expression in squamous cell carcinoma (SCC) cells enhanced both sEV secretion and loading of pro-mitogenic cargo. In this study, using wild-type Dsg2 and a mutant form that is unable to be palmitoylated (Dsg2cacs), we investigated the mechanism by which Dsg2 modulates SCC tumour development and progression through sEVs. We demonstrate that palmitoylation was required for Dsg2 to regulate sub-cellular localisation of lipid raft and endosomal proteins necessary for sEV biogenesis. Pharmacological inhibition of the endosomal pathway abrogated Dsg2-mediated sEV release. In murine xenograft models, Dsg2-expressing cells generated larger xenograft tumours as compared to cells expressing GFP or Dsg2cacs. Co-treatment with sEVs derived from Dsg2-over-expressing cells increased xenograft size. Cytokine profiling revealed, Dsg2 enhanced both soluble and sEV-associated IL-8 and miRNA profiling revealed, Dsg2 down-regulated both cellular and sEV-loaded miR-146a. miR-146a targets IRAK1, a serine-threonine kinase involved in IL-8 signalling. Treatment with a miR-146a inhibitor up-regulated both IRAK1 and IL-8 expression. RNAseq analysis of HNSCC tumours revealed a correlation between Dsg2 and IL-8. Finally, elevated IL-8 plasma levels were detected in a subset of HNSCC patients who did not respond to immune checkpoint therapy, suggesting that these patients may benefit from prior anti-IL-8 treatment. In summary, these results suggest that intercellular communication through cell-cell adhesion, cytokine release and secretion of EVs are coordinated, and critical for tumour growth and development, and may serve as potential prognostic markers to inform treatment options.  

3.3556           Macrophage Exosomes Resolve Atherosclerosis by Regulating Hematopoiesis and Inflammation via MicroRNA Cargo

Bouchaareychas, L., Duong, P., Covarrubias, S., Carpenter, S., Van Keuren-Carpenter, K. and Raffai, R.L. Cell Reports, 32, 1107881 (2020)   Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remains a major therapeutic challenge. Here, we show that exosomes produced by naive bone marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a that are further increased in exosomes produced by BMDM polarized with IL-4 (BMDM-IL-4-exo). These exosomal microRNAs suppress inflammation by targeting NF-κB and TNF-α signaling and foster M2 polarization in recipient macrophages. Repeated infusions of BMDM-IL-4-exo into Apoe^−/− mice fed a Western diet reduce excessive hematopoiesis in the bone marrow and thereby the number of myeloid cells in the circulation and macrophages in aortic root lesions. This also leads to a reduction in necrotic lesion areas that collectively stabilize atheroma. Thus, BMDM-IL-4-exo may represent a useful therapeutic approach for atherosclerosis and other inflammatory disorders by targeting NF-κB and TNF-α via microRNA cargo delivery.  

3.3557           Lactobacillus plantarum-derived extracellular vesicles induce anti-inflammatory M2 macrophage polarization in vitro

Kim, W., Lee, E.J., Bae, I-H., Myoung, K., Kim, S.T., Park, P.J., Lee, K-H., Pham, A.V.Q., Ko, J., Oh, S.H. and Cho, E-G.

1. Extracellular Vesicles, 9(1), 1793514 (2020)

  Probiotics offer various health benefits. Lactobacillus plantarum has been used for decades to enhance human intestinal mucosal immunity and improve skin barrier integrity. Extracellular vesicles (EVs) derived from eukaryotic or prokaryotic cells have been recognized as efficient carriers for delivery of biomolecules to recipient cells, and to efficiently regulate human pathophysiology. However, the mechanism underlying the beneficial effects of probiotic bacteria-derived EVs on human skin is unclear. Herein, we investigated how L. plantarum-derived EVs (LEVs) exert beneficial effects on human skin by examining the effect of LEVs on cutaneous immunity, particularly on macrophage polarization. LEVs promoted differentiation of human monocytic THP1 cells towards an anti-inflammatory M2 phenotype, especially M2b, by inducing biased expression of cell-surface markers and cytokines associated with M2 macrophages. Pre- or post-treatment with LEVs under inflammatory M1 macrophage-favouring conditions, induced by LPS and interferon-γ, inhibited M1-associated surface marker, HLA-DRα expression. Moreover, LEV treatment significantly induced expression of macrophage-characteristic cytokines, IL-1β, GM-CSF and the representative anti-inflammatory cytokine, IL-10, in human skin organ cultures. Hence, LEVs can trigger M2 macrophage polarization in vitro, and induce an anti-inflammatory phenomenon in the human skin, and may be a potent anti-inflammatory strategy to alleviate hyperinflammatory skin conditions.  

3.3558           Alix and Syntenin-1 direct amyloid precursor protein trafficking into extracellular vesicles

Cone, A.S., Hurwitz, S.N., Lee, G.S., Yuan, X., Zhou, Y., Li, Y. and Meckles Jr., D.G. BMC Mol. Cell Biol., 21:58 (2020)   Background Endosomal trafficking and amyloidogenic cleavage of amyloid precursor protein (APP) is believed to play a role in the neurodegeneration observed in Alzheimer’s disease (AD). Recent evidence has suggested that packaging and secretion of APP and its amyloidogenic cleaved products into small extracellular vesicles (EVs) may facilitate uptake of these neurotoxic factors during disease progression. However, the molecular mechanisms underlying trafficking of APP into EVs are poorly understood. Results In this study, the mechanism and impact of APP trafficking into extracellular vesicles (EVs) were assessed by a series of inducible gene knockdowns. We demonstrate that vesicle-associated proteins Alix and Syntenin-1 are essential for proper subcellular localization and efficient EV secretion of APP via an endosomal sorting complexes required for transport (ESCRT)-independent pathway. The neurotoxic C-terminal fragment (CTFβ) of APP is similarly secreted in association with small vesicles. These mechanisms are conserved in terminally differentiated neuron-like cells. Furthermore, knockdown of Alix and Syntenin-1 alters the subcellular localization of APP, sequestering the precursor protein to endoplasmic reticulum and endolysosomal compartments, respectively. Finally, transfer of small EVs containing mutant APP confers an increase in reactive oxygen species production and neurotoxicity to human induced pluripotent stem cell-derived cortical neurons and naïve primary neurons, an effect that is ameliorated by Alix and Syntenin-1 depletion. Conclusions Altogether these findings elucidate a novel mechanism for understanding the intracellular trafficking of APP and CTFβ into secreted extracellular vesicles, and the resultant potential impact on neurotoxicity in the context of Alzheimer’s disease amyloidopathy.  

3.3559           Application of exosomes as liquid biopsy in clinical diagnosis

Zhou, B., Xu, K., Zheng, X., Chen, T., Wang, J., Song, Y., Shao, Y. and Zheng, S. Signal Transduction and targeted Therapy, 5:144 (2020)   Liquid biopsy refers to the sampling and molecular analysis of the biofluids of circulating tumor cells, extracellular vesicles, nucleic acids, and so forth. Exosomes are small extracellular vesicles with sizes between 30–150 nm. They are secreted by multivesicular bodies through exocytosis in live cells and can participate in intercellular communication due to their contents, including nucleic acids, proteins, and lipids. Herein, we investigate publication frequencies on exosomes over the past 10 years, and review recent clinical studies on liquid biopsy of exosomes in the fields of oncology, pregnancy disorders, cardiovascular diseases, and organ transplantation. We also describe the advantages of exosomes as an effective liquid biopsy tool and the progression of exosome extraction methods. Finally, we depict the commercial development of exosome research and discuss the future role of exosomes in liquid biopsy.  

3.3560           Data on proteomic profiling of extracellular vesicles of Acholeplasma laidlawii strains with increased resistance to antibiotics of different classes - ciprofloxacin and tetracycline

Mouzykantov, A., Medvedeva, E., Baranova, N., Chernova, O. and Chernov, V. Data in Brief, 32, 106049 (2020)   To elucidate the regularities of adaptation of the representatives of class Mollicutes to antimicrobials and to identify the promising targets for eradication of mycoplasma infections and contaminations the comparative analysis of the molecular basis of bacterial resistance to antibiotics of different classes is needed. Previously, we presented the data on the whole-genome sequences of Acholeplasma laidlawii strains with different susceptibility to ciprofloxacin (GenBank: LXYB00000000.1), tetracycline (GenBank: NELO00000000.2) and melittin (GenBank: NELN00000000.2) as well as the data on cell and extracellular vesicle proteomes of melittin-resistant A. laidlawii strain [1]. The lists of extracellular vesicle proteins secreted by A. laidlawii strains with the increased resistance to ciprofloxacin (PG8R₁₀) and tetracycline (PG8R_Tet) are presented here. The vesicle proteome profiles were obtained by 1D SDS-PAGE and liquid chromatography-mass spectrometry.  

3.3561           The protein and microRNA cargo of extracellular vesicles from parasitic helminths – current status and research priorities

Sotillo, J., Robinson, M.W., Kimber, M.J., Cucher, M., Ancarola, M.E., Nejsum, P., Marcilla, A., Eichenberger, R.M. and Tritten, L. Int. J. Parasitol., 50, 635-645 (2020)   Helminth parasites have a remarkable ability to persist within their mammalian hosts, which is largely due to their secretion of molecules with immunomodulatory properties. Although the soluble components of helminth secretions have been extensively studied, the discovery that helminths release extracellular vesicles (EVs) has added further complexity to the host-parasite interaction. Whilst several studies have begun to characterise the molecules carried by helminth EVs, work aimed at investigating their biological functions has been hindered by a lack of helminth-specific EV markers. To begin to address this, we summarised helminth EV literature to date. With a focus on the protein and microRNA (miRNA) cargo, we aimed to detect similarities and differences across those major groups of helminths for which data are available; namely nematodes, trematodes and cestodes. Pfam analysis revealed that although there is no universal EV marker for all helminth species, the EF-hand protein family was present in all EV datasets from cestodes and trematodes, and could serve as a platyhelminth EV biomarker. In contrast, M13 metallopeptidases and actin may have potential as markers for nematode EVs. As with proteins, many miRNA families appeared to be species-, stage-, or dataset-specific. Two miRNA families were common to nematode EVs (mir-10 and let-7); the miRNA cargo of EVs secreted by clade I species appeared somewhat different from species from other clades. Five miRNA families (mir-71, mir-10, mir-190, let-7 and mir-2) were shared by all trematode species examined. Our analysis has identified novel markers that may be used in studies aimed at characterising helminth EVs and interrogating their function at the host-parasite interface. In addition, we discuss the heterogeneity of methods used for helminth EV isolation and emphasise the need for a standardised approach in reporting on helminth EV data.  

3.3562           Extracellular vesicles from Heligmosomoides bakeri and Trichuris muris contain distinct microRNA families and small RNAs that could underpin different functions in the host

White, R., Kumar, S., Chow, F.W-N., Robertson, E., Hayes, K.S., Grencis, R.K., Duque-Correa, M.A. and Buck, A.H. Int. J. Parasitol., 50, 719-729 (2020)   Extracellular vesicles (EVs) have emerged as a ubiquitous component of helminth excretory-secretory products that can deliver parasite molecules to host cells to elicit immunomodulatory effects. RNAs are one type of cargo molecule that can underpin EV functions, hence there is extensive interest in characterising the RNAs that are present in EVs from different helminth species. Here we outline methods for identifying all of the small RNAs (sRNA) in helminth EVs and address how different methodologies may influence the sRNAs detected. We show that different EV purification methods introduce relatively little variation in the sRNAs that are detected, and that different RNA library preparation methods yielded larger differences. We compared the EV sRNAs in the gastrointestinal nematode Heligmosomoides bakeri with those in EVs from the distantly related gastrointestinal nematode Trichuris muris, and found that many of the sRNAs in both organisms derive from repetitive elements or intergenic regions. However, only in H. bakeri do these RNAs contain a 5′ triphosphate, and Guanine (G) starting nucleotide, consistent with their biogenesis by RNA-dependent RNA polymerases (RdRPs). Distinct microRNA (miRNA) families are carried in EVs from each parasite, with H. bakeri EVs specific for miR-71, miR-49, miR-63, miR-259 and miR-240 gene families, and T. muris EVs specific for miR-1, miR-1822 and miR-252, and enriched for miR-59, miR-72 and miR-44 families, with the miR-9, miR-10, miR-80 and let-7 families abundant in both. We found a larger proportion of miRNA reads derive from the mouse host in T. muris EVs, compared with H. bakeri EVs. Our report underscores potential biases in the sRNAs sequenced based on library preparation methods, suggests specific nematode lineages have evolved distinct sRNA synthesis/export pathways, and highlights specific differences in EV miRNAs from H. bakeri and T. muris that may underpin functional adaptation to their host niches.  

3.3563           Human ESC-sEVs alleviate age-related bone loss by rejuvenating senescent bone marrow-derived mesenchymal stem cells

Gong, L., Chen, B., Zhang, J., Sun, Y., Yuan, J., Niu, X., Hu, G., Chen, Y., Xie, Z., Deng, Z. Li, Q. and Wang, Y.

1. Extracellular Vesicles, 9(1), 1800971 (2020)

  Tissue-resident stem cell senescence leads to stem cell exhaustion, which is a major cause of physiological and pathological ageing. Stem cell-derived extracellular vesicles (SC-EVs) have been reported in preclinical studies to possess therapeutic potential for diverse diseases. However, whether SC-EVs can rejuvenate senescent tissue stem cells to prevent age-related disorders still remains unknown. Here, we show that chronic application of human embryonic stem cell-derived small extracellular vesicles (hESC-sEVs) rescues the function of senescent bone marrow mesenchymal stem cells (BM-MSCs) and prevents age-related bone loss in ageing mice. Transcriptome analysis revealed that hESC-sEVs treatment upregulated the expression of genes involved in antiaging, stem cell proliferation and osteogenic differentiation in BM-MSCs. Furthermore, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified 4122 proteins encapsulated in hESC-sEVs. Bioinformatics analysis predicted that the protein components in the hESCs-sEVs function in a synergistic way to induce the activation of several canonical signalling pathways, including Wnt, Sirtuin, AMPK, PTEN signalling, which results in the upregulation of antiaging genes in BM-MSCs and then the recovery of senescent BM-MSCs function. Collectively, our findings reveal the effect of hESC-sEVs in reversing BM-MSCs senescence and age-related osteogenic dysfunction, thereby preventing age-related bone loss. Because hESC-sEVs could alleviate senescence of tissue-resident stem cells, they might be promising therapeutic candidates for age-related diseases.  

3.3564           Cancer therapy based on extracellular vesicles as drug delivery vehicles

Calbeza, L., Perazzoli, G., Pena, M., Cepero, A., Luque, C., Melguizo, C. and Prados, J.

1. Controlled Release, 327, 296-315 (2020)

  Extracellular vesicles (EVs) are lipid bilayer vesicles of nanometric size secreted by cells to communicate with other cells, either nearby or remotely. Their physicochemical properties make them a promising nanomedicine for drug transport and release in cancer therapy. In this review, we present the different types and biogenesis of EVs and highlight the importance of adequately selecting the cell of origin in cancer therapy. Furthermore, the main methodologies followed for the isolation of EVs and drug loading, as well as the modification and functionalization of these vesicles to generate EV-based nanocarriers are discussed. Finally, we review some of the main studies using drug-loaded exosomes in tumor therapy both in in vitro and in vivo models (even in resistant tumors). These investigations show promising results, achieving significant improvement in the antitumor effect of drugs in most cases. However, the number of clinical trials and patents based on these nanoformulations is still low, thus further research is still warranted in this area.  

3.3565           The Dichotomous Role of Extracellular Vesicles in the Central Nervous System

Graykowski, D.R., Wang, Y-Z., Upadhyay, A. and Savas, J.N. iScience, 23, 101456 (2020)   Extracellular vesicles (EVs) are important mediators of intercellular communication. Interest in the role of central nervous system (CNS)-derived EVs has been increasing; however, some skepticism of their importance has persisted because many aspects of their biology remain elusive. This ambiguity is largely due to technical barriers that hamper our ability to achieve a comprehensive understanding of their molecular components and mechanisms responsible for their transmission and uptake. However, accumulating evidence supports the notion that EVs play important roles in basic physiological processes within the CNS during neurodevelopment and synaptic plasticity. Interestingly, EVs also act to spread toxic polypeptides in neurodegenerative diseases. Developing a more profound understanding of the role that EVs play in the CNS could lead to the identification of biomarkers and potential vehicles for drug delivery. Here we highlight our current understanding of CNS EVs and summarize our current understanding of their complex role in the CNS.  

3.3566           Inhibition of HDAC increases BDNF expression and promotes neuronal rewiring and functional recovery after brain injury

Sada, N., Fujita, Y., Mizuta, N., Ueno, M., Furukawa, T. and Yamashita, T.

Cell Death and Disease, 11:655 (2020)

Brain injury causes serious motor, sensory, and cognitive disabilities. Accumulating evidence has demonstrated that histone deacetylase (HDAC) inhibitors exert neuroprotective effects against various insults to the central nervous system (CNS). In this study, we investigated the effects of the HDAC inhibition on the expression of brain-derived neurotrophic factor (BDNF) and functional recovery after traumatic brain injury (TBI) in mice. Administration of class I HDAC inhibitor increased the number of synaptic boutons in rewiring corticospinal fibers and improved the recovery of motor functions after TBI. Immunohistochemistry results showed that HDAC2 is mainly expressed in the neurons of the mouse spinal cord under normal conditions. After TBI, HDAC2 expression was increased in the spinal cord after 35 days, whereas BDNF expression was decreased after 42 days. Administration of CI-994 increased BDNF expression after TBI. Knockdown of HDAC2 elevated H4K5ac enrichment at the BDNF promoter, which was decreased following TBI. Together, our findings suggest that HDAC inhibition increases expression of neurotrophic factors, and promote neuronal rewiring and functional recovery following TBI.

 

3.3567           ITGB3-mediated uptake of small extracellular vesicles facilitates intercellular communication in breast cancer cells

Fuentes, P., Sese, M., Guijarro, P.J., Emperador, M., Sanchez-Redondo, S., Peinado, H., Hümmer, S. and Ramon y.Cajal, S. Nature Communications, 11:4261 (2020)   Metastasis, the spread of malignant cells from a primary tumour to distant sites, causes 90% of cancer-related deaths. The integrin ITGB3 has been previously described to play an essential role in breast cancer metastasis, but the precise mechanisms remain undefined. We have now uncovered essential and thus far unknown roles of ITGB3 in vesicle uptake. The functional requirement for ITGB3 derives from its interactions with heparan sulfate proteoglycans (HSPGs) and the process of integrin endocytosis, allowing the capture of extracellular vesicles and their endocytosis-mediated internalization. Key for the function of ITGB3 is the interaction and activation of focal adhesion kinase (FAK), which is required for endocytosis of these vesicles. Thus, ITGB3 has a central role in intracellular communication via extracellular vesicles, proposed to be critical for cancer metastasis.  

3.3568           Dynamic remodelling of the human host cell proteome and phosphoproteome upon enterovirus infection

Giansanti, P., Starting, J.R.P.M., Defourny, K.A.Y., Cesonyte, I., Bottino, A.M.S., Post, H., Viktorova, E.G., Ho, V.Q.T., langereis, M.A., Belov, G.A., Nolte-‘tHoen, E.N.M., Heck, A.J.R. and van Kuppeveld, F.J.M. Nature Communications, 11:4332 (2020)   The group of enteroviruses contains many important pathogens for humans, including poliovirus, coxsackievirus, rhinovirus, as well as newly emerging global health threats such as EV-A71 and EV-D68. Here, we describe an unbiased, system-wide and time-resolved analysis of the proteome and phosphoproteome of human cells infected with coxsackievirus B3. Of the ~3,200 proteins quantified throughout the time course, a large amount (~25%) shows a significant change, with the majority being downregulated. We find ~85% of the detected phosphosites to be significantly regulated, implying that most changes occur at the post-translational level. Kinase-motif analysis reveals temporal activation patterns of certain protein kinases, with several CDKs/MAPKs immediately active upon the infection, and basophilic kinases, ATM, and ATR engaging later. Through bioinformatics analysis and dedicated experiments, we identify mTORC1 signalling as a major regulation network during enterovirus infection. We demonstrate that inhibition of mTORC1 activates TFEB, which increases expression of lysosomal and autophagosomal genes, and that TFEB activation facilitates the release of virions in extracellular vesicles via secretory autophagy. Our study provides a rich framework for a system-level understanding of enterovirus-induced perturbations at the protein and signalling pathway levels, forming a base for the development of pharmacological inhibitors to treat enterovirus infections.  

3.3569           Probiotic Escherichia coli Nissle 1917-derived outer membrane vesicles enhance immunomodulation and antimicrobial activity in RAW264.7 macrophages

Hu, R., Lin, H., Li, J., Zhao, Y., Wang, M., Sun, X., Min, Y., Gao, Y. and Yang, M. BMC Microbiol., 20:268 (2020)   Background Probiotic Escherichia coli Nissle 1917 (EcN) has been widely studied for the treatment of intestinal inflammatory diseases and infectious diarrhea, but the mechanisms by which they communicate with the host are not well-known. Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and deliver microbial molecules to distant target cells in the host, which play a very important role in mediating bacteria-host communication. Here, we aimed to investigate whether EcN-derived OMVs (EcN_OMVs) could mediate immune regulation in macrophages. Results In this study, after the characterization of EcN_OMVs using electron microscopy, nanoparticle tracking and proteomic analyses, we demonstrated by confocal fluorescence microscopy that EcN_OMVs could be internalized by RAW 264.7 macrophages. Stimulation with EcN_OMVs at appropriate concentrations promoted proliferation, immune-related enzymatic activities and phagocytic functions of RAW264.7 cells. Moreover, EcN_OMVs induced more anti-inflammatory responses (IL-10) than pro-inflammatory responses (IL-6 and TNF-α) in vitro, and also modulated the production of Th1-polarizing cytokine (IL-12) and Th2-polarizing cytokine (IL-4). Treatments with EcN_OMVs effectively improved the antibacterial activity of RAW 264.7 macrophages. Conclusions These findings indicated that EcN_OMVs could modulate the functions of the host immune cells, which will enrich the existing body of knowledge of EVs as an important mechanism for the communication of probiotics with their hosts.  

3.3570           The Emerging Role of Exosomes in Diagnosis, Prognosis, and Therapy in Head and Neck Cancer

Hofmann, L., Ludwig, S., Vahl, J.M., Brunner, C., Hoffmann, T.K. and Theodoraki, M-N. Int. J. Mol. Sci., 21, 4072 (2020)   Exosomes, the smallest group of extracellular vesicles, carry proteins, miRNA, mRNA, DNA, and lipids, which they efficiently deliver to recipient cells, generating a communication network. Exosomes strongly contribute to the immune suppressive tumor microenvironment of head and neck squamous cell carcinomas (HNSCC). Isolation of exosomes from HNSCC cell culture or patient’s plasma allows for analyzing their molecular cargo and functional role in immune suppression and tumor progression. Immune affinity-based separation of different exosome subsets, such as tumor-derived or T cell-derived exosomes, from patient’s plasma simultaneously informs about tumor status and immune dysfunction. In this review, we discuss the recent understanding of how exosomes behave in the HNSCC tumor microenvironment and why they are promising liquid biomarkers for diagnosis, prognosis, and therapy in HNSCC.  

3.3571           Targeted Delivery of Mesenchymal Stem Cell-Derived Nanovesicles for Spinal Cord Injury Treatment

Lee, J-R., Kyung, J.W., Kumar, H., Kwon, S.P., Song, S.Y., Han, I-B. and Kim, B-S. Int. J. Mol. Sci., 21, 4185 (2020)   Due to the safety issues and poor engraftment of mesenchymal stem cell (MSC) implantation, MSC-derived exosomes have been spotlighted as an alternative therapy for spinal cord injury (SCI). However, insufficient productivity of exosomes limits their therapeutic potential for clinical application. Moreover, low targeting ability of unmodified exosomes is a critical obstacle for their further applications as a therapeutic agent. In the present study, we fabricated macrophage membrane-fused exosome-mimetic nanovesicles (MF-NVs) from macrophage membrane-fused umbilical cord blood-derived MSCs (MF-MSCs) and confirmed their therapeutic potential in a clinically relevant mouse SCI model (controlled mechanical compression injury model). MF-NVs contained larger quantity of ischemic region-targeting molecules compared to normal MSC-derived nanovesicles (N-NVs). The targeting molecules in MF-NVs, which were derived from macrophage membranes, increased the accumulation of MF-NVs in the injured spinal cord after the in vivo systemic injection. Increased accumulation of MF-NVs attenuated apoptosis and inflammation, prevented axonal loss, enhanced blood vessel formation, decreased fibrosis, and consequently, improved spinal cord function. Synthetically, we developed targeting efficiency-potentiated exosome-mimetic nanovesicles and present their possibility of clinical application for SCI.  

3.3572           The Regulation of Flavivirus Infection by Hijacking Exosome-Mediated Cell–Cell Communication: New Insights on Virus–Host Interactions

Reyes-Ruiz, J.M., Osuna-Ramos, J.F., De Jesus-Gonzalez, L.A., Palacios-Rapalo, S.N., Cordero-Rivera, C.D., Farfan-Morales, C.N., Hurtado-Monzon, A.M., Gallardo-Flores, C.E., Alcaraz-Estrada, S.L., Salas-Benito, J.S. and del Angel, C.N. Viruses, 12, 765 (2020)   The arthropod-borne flaviviruses are important human pathogens, and a deeper understanding of the virus–host cell interaction is required to identify cellular targets that can be used as therapeutic candidates. It is well reported that the flaviviruses hijack several cellular functions, such as exosome-mediated cell communication during infection, which is modulated by the delivery of the exosomal cargo of pro- or antiviral molecules to the receiving host cells. Therefore, to study the role of exosomes during flavivirus infections is essential, not only to understand its relevance in virus–host interaction, but also to identify molecular factors that may contribute to the development of new strategies to block these viral infections. This review explores the implications of exosomes in flavivirus dissemination and transmission from the vector to human host cells, as well as their involvement in the host immune response. The hypothesis about exosomes as a transplacental infection route of ZIKV and the paradox effect or the dual role of exosomes released during flavivirus infection are also discussed here. Although several studies have been performed in order to identify and characterize cellular and viral molecules released in exosomes, it is not clear how all of these components participate in viral pathogenesis. Further studies will determine the balance between protective and harmful exosomes secreted by flavivirus infected cells, the characteristics and components that distinguish them both, and how they could be a factor that determines the infection outcome.  

3.3573           Extracellular Vesicles in Viral Replication and Pathogenesis and Their Potential Role in Therapeutic Intervention

Kumar, A., Kodidela, S., Tadrous, E., Cory, T.J., Walker, C.M., Smith, A.M., Mukherjee, A. and Kumar, S. Viruses, 12, 887 (2020)   Extracellular vesicles (EVs) have shown their potential as a carrier of molecular information, and they have been involved in physiological functions and diseases caused by viral infections. Virus-infected cells secrete various lipid-bound vesicles, including endosome pathway-derived exosomes and microvesicles/microparticles that are released from the plasma membrane. They are released via a direct outward budding and fission of plasma membrane blebs into the extracellular space to either facilitate virus propagation or regulate the immune responses. Moreover, EVs generated by virus-infected cells can incorporate virulence factors including viral protein and viral genetic material, and thus can resemble noninfectious viruses. Interactions of EVs with recipient cells have been shown to activate signaling pathways that may contribute to a sustained cellular response towards viral infections. EVs, by utilizing a complex set of cargos, can play a regulatory role in viral infection, both by facilitating and suppressing the infection. EV-based antiviral and antiretroviral drug delivery approaches provide an opportunity for targeted drug delivery. In this review, we summarize the literature on EVs, their associated involvement in transmission in viral infections, and potential therapeutic implications.  

3.3574           Purification Methods and the Presence of RNA in Virus Particles and Extracellular Vesicles

Zhou, Y., McNamara, R.P. and Dittmer, D.P: Viruses, 12, 917 (2020)   The fields of extracellular vesicles (EV) and virus infections are marred in a debate on whether a particular mRNA or non-coding RNA (i.e., miRNA) is packaged into a virus particle or copurifying EV and similarly, whether a particular mRNA or non-coding RNA is contained in meaningful numbers within an EV. Key in settling this debate, is whether the purification methods are adequate to separate virus particles, EV and contaminant soluble RNA and RNA:protein complexes. Differential centrifugation/ultracentrifugation and precipitating agents like polyethylene glycol are widely utilized for both EV and virus purifications. EV are known to co-sediment with virions and other particulates, such as defective interfering particles and protein aggregates. Here, we discuss how encased RNAs from a heterogeneous mixture of particles can be distinguished by different purification methods. This is particularly important for subsequent interpretation of whether the RNA associated phenotype is contributed solely by virus or EV particles or a mixture of both. We also discuss the discrepancy of miRNA abundance in EV from different input material.  

3.3575           The GABARAP Co-Secretome Identified by APEX2-GABARAP Proximity Labelling of Extracellular Vesicles

Sanwald, J.L., Poschmann, G., Stühler, K., Behrends, C., Hoffmann, S. and Willbold, D. Cell, 9, 1468 (2020)   The autophagy-related ATG8 protein GABARAP has not only been shown to be involved in the cellular self-degradation process called autophagy but also fulfils functions in intracellular trafficking processes such as receptor transport to the plasma membrane. Notably, available mass spectrometry data suggest that GABARAP is also secreted into extracellular vesicles (EVs). Here, we confirm this finding by the immunoblotting of EVs isolated from cell culture supernatants and human blood serum using specific anti-GABARAP antibodies. To investigate the mechanism by which GABARAP is secreted, we applied proximity labelling, a method for studying the direct environment of a protein of interest in a confined cellular compartment. By expressing an engineered peroxidase (APEX2)-tagged variant of GABARAP—which, like endogenous GABARAP, was present in EVs prepared from HEK293 cells—we demonstrate the applicability of APEX2-based proximity labelling to EVs. The biotinylated protein pool which contains the APEX2-GABARAP co-secretome contained not only known GABARAP interaction partners but also proteins that were found in APEX2-GABARAP’s proximity inside of autophagosomes in an independent study. All in all, we not only introduce a versatile tool for co-secretome analysis in general but also uncover the first details about autophagy-based pathways as possible biogenesis mechanisms of GABARAP-containing EVs.  

3.3576           Feasibility of Mechanical Extrusion to Coat Nanoparticles with Extracellular Vesicle Membranes

Van Deun, J., Roux, Q., Deville, S., Van Acker, T., Rappu, P., Miinalainen, I., Heino, J., Vanhaeeecke, F., De Geest, B.G., De Wever, O. and Hendrix, A. Cells, 9, 1797 (2020)   Biomimetic functionalization to confer stealth and targeting properties to nanoparticles is a field of intense study. Extracellular vesicles (EV), sub-micron delivery vehicles for intercellular communication, have unique characteristics for drug delivery. We investigated the top-down functionalization of gold nanoparticles with extracellular vesicle membranes, including both lipids and associated membrane proteins, through mechanical extrusion. EV surface-exposed membrane proteins were confirmed to help avoid unwanted elimination by macrophages, while improving autologous uptake. EV membrane morphology, protein composition and orientation were found to be unaffected by mechanical extrusion. We implemented complementary EV characterization methods, including transmission- and immune-electron microscopy, and nanoparticle tracking analysis, to verify membrane coating, size and zeta potential of the EV membrane-cloaked nanoparticles. While successful EV membrane coating of the gold nanoparticles resulted in lower macrophage uptake, low yield was found to be a significant downside of the extrusion approach. Our data incentivize more research to leverage EV membrane biomimicking as a unique drug delivery approach in the near future.  

3.3577           Proteomic Analysis of Exosomes for Discovery of Protein Biomarkers for Prostate and Bladder Cancer

Wang, Y-T., Shi, T., Srivastava, S., Kegan, J., Liu, T. and Rodland, K.D. Cancers, 12, 2335 (2020)   Extracellular vesicles (EVs) are released by nearly all cell types as part of normal cell physiology, transporting biological cargo, including nucleic acids and proteins, across the cell membrane. In pathological states such as cancer, EV-derived cargo may mirror the altered state of the cell of origin. Exosomes are the smaller, 50–150 nanometer-sized EVs released from fusion of multivesicular endosomes with the plasma membrane. Exosomes play important roles in cell-cell communication and participate in multiple cancer processes, including invasion and metastasis. Therefore, proteomic analysis of exosomes is a promising approach to discover potential cancer biomarkers, even though it is still at an early stage. Herein, we critically review the advances in exosome isolation methods and their compatibility with mass spectrometry (MS)-based proteomic analysis, as well as studies of exosomes in pathogenesis and progression of prostate and bladder cancer, two common urologic cancers whose incidence rates continue to rise annually. As urological tumors, both urine and blood samples are feasible for noninvasive or minimally invasive analysis. A better understanding of the biological cargo and functions of exosomes via high-throughput proteomics will help provide new insights into complex alterations in cancer and provide potential therapeutic targets and personalized treatment for patients.  

3.3578           Development of New Strategies Using Extracellular Vesicles Loaded with Exogenous Nucleic Acid

Orefice, N.S: Pharmaceutics, 12, 705 (2020)   Gene therapy is a therapeutic strategy of delivering foreign genetic material (encoding for an important protein) into a patient’s target cell to replace a defective gene. Nucleic acids are embedded within the adeno-associated virus (AAVs) vectors; however, preexisting immunity to AAVs remains a significant concern that impairs their clinical application. Extracellular vesicles (EVs) hold great potential for therapeutic applications as vectors of nucleic acids due to their endogenous intercellular communication functions through their cargo delivery, including lipids and proteins. So far, small RNAs (siRNA and micro (mi)RNA) have been mainly loaded into EVs to treat several diseases, but the potential use of EVs to load and deliver exogenous plasmid DNA has not been thoroughly described. This review provides a comprehensive overview of the principal methodologies currently employed to load foreign genetic material into EVs, highlighting the need to find the most effective strategies for their successful clinical translation.  

3.3579           Rapid Isolation of Rare Isotype-Switched Hybridoma Variants: Application to the Generation of IgG2a and IgG2b MAb to CD63, a Late Endosome and Exosome Marker

Charrin, S., Palmulli, R., Billard, M., Clay, D., Bouceix, C., Van Niel, G. and Rubenstein, E. Antibodies, 9, 29 (2020)   CD63, a member of the tetraspanin superfamily, is used as a marker of late endosomes and lysosome-related organelles, as well as a marker of exosomes. Here, we selected rare isotype variants of TS63 by sorting hybridoma cells on the basis of their high expression of surface immunoglobulins of the IgG2a and IgG2b subclass. Pure populations of cells secreting IgG2a and IgG2b variants of TS63 (referred to as TS63a and TS63b) were obtained using two rounds of cell sorting and one limited dilution cloning step. We validate that these new TS63 variants are suitable for co-labeling with mAb of the IgG1 subclass directed to other molecules, using anti mouse subclass antibodies, and for the labeling of exosomes through direct binding to protein A-coated gold particles. These mAbs will be useful to study the intracellular localization of various proteins and facilitate electron microscopy analysis of CD63 localization.  

3.3580           Derivation of Cell-Engineered Nanovesicles from Human Induced Pluripotent Stem Cells and Their Protective Effect on the Senescence of Dermal Fibroblasts

Lee, H., Cha, H. and Park, J.H. Int. J. Mol. Sci., 21, 343 (2020)   Stem cells secrete numerous paracrine factors, such as cytokines, growth factors, and extracellular vesicles. As a kind of extracellular vesicle (EV), exosomes produced in the endosomal compartment of eukaryotic cells have recently emerged as a biomedical material for regenerative medicine, because they contain many valuable contents that are derived from the host cells, and can stably deliver those contents to other recipient cells. Although we have previously demonstrated the beneficial effects of human induced potent stem cell-derived exosomes (iPSC-Exo) on the aging of skin fibroblasts, low production yield has remained an obstacle for clinical applications. In this study, we generated cell-engineered nanovesicles (CENVs) by serial extrusion of human iPSCs through membrane filters with diminishing pore sizes, and explored whether the iPSC-CENV ameliorates physiological alterations of human dermal fibroblasts (HDFs) that occur by natural senescence. The iPSC-CENV exhibited similar characteristics to the iPSC-Exo, while the production yield was drastically increased compared to that of iPSC-derived EVs, including exosomes. The proliferation and migration of both young and senescent HDFs were stimulated by the treatment with iPSC-CENVs. In addition, it was revealed that the iPSC-CNEV restored senescence-related alterations of gene expression. Treatment with iPSC-CENVs significantly reduced the activity of senescence-associated-β-galactosidase (SA-β-Gal) in senescent HDFs, as well as suppressing the elevated expression of p53 and p21, key factors involved in cell cycle arrest, apoptosis, and cellular senescence signaling pathways. Taken together, these results suggest that iPSC-CENV could provide an excellent alternative to iPSC-exo, and be exploited as a resource for the treatment of signs of skin aging.  

3.3581           Extracellular Vesicle-Induced Classical Complement Activation Leads to Retinal Endothelial Cell Damage via MAC Deposition

Huang, C., Fisher, K.P., Hammer, S.S. and Busik, J.V. Int. J. Mol. Sci., 21, 1693 (2020)   Several studies have suggested that there is a link between membrane attack complex (MAC) deposition in the retina and the progression of diabetic retinopathy (DR). Our recent investigation demonstrated that circulating IgG-laden extracellular vesicles contribute to an increase in retinal vascular permeability in DR through activation of the complement system. However, the mechanism through which extracellular vesicle-induced complement activation contributes to retinal vascular cytolytic damage in DR is not well understood. In this study, we demonstrate that IgG-laden extracellular vesicles in rat plasma activate the classical complement pathway, and in vitro Streptozotocin (STZ)-induced rat diabetic plasma results in MAC deposition and cytolytic damage in human retinal endothelial cells (HRECs). Moreover, removal of the plasma extracellular vesicles reduced the MAC deposition and abrogated cytolytic damage seen in HRECs. Together, the results of this study demonstrate that complement activation by IgG-laden extracellular vesicles in plasma could lead to MAC deposition and contribute to endothelium damage and progression of DR  

3.3582           Message in a Bottle: Upgrading Cardiac Repair into Rejuvenation

Balbi, C., Costa, A., Barile, l. and Bollini, S. Cells, 9, 724 (2020)   Ischaemic cardiac disease is associated with a loss of cardiomyocytes and an intrinsic lack of myocardial renewal. Recent work has shown that the heart retains limited cardiomyocyte proliferation, which remains inefficient when facing pathological conditions. While broadly active in the neonatal mammalian heart, this mechanism becomes quiescent soon after birth, suggesting loss of regenerative potential with maturation into adulthood. A key question is whether this temporary regenerative window can be enhanced via appropriate stimulation and further extended. Recently the search for novel therapeutic approaches for heart disease has centred on stem cell biology. The “paracrine effect” has been proposed as a promising strategy to boost endogenous reparative and regenerative mechanisms from within the cardiac tissue by exploiting the modulatory potential of soluble stem cell-secreted factors. As such, growing interest has been specifically addressed towards stem/progenitor cell-secreted extracellular vesicles (EVs), which can be easily isolated in vitro from cell-conditioned medium. This review will provide a comprehensive overview of the current paradigm on cardiac repair and regeneration, with a specific focus on the role and mechanism(s) of paracrine action of EVs from cardiac stromal progenitors as compared to exogenous stem cells in order to discuss the optimal choice for future therapy. In addition, the challenges to overcoming translational EV biology from bench to bedside for future cardiac regenerative medicine will be discussed.  

3.3583           Internalization of trophoblastic small extracellular vesicles and detection of their miRNA cargo in P-bodies

Li, H., Pinilla-Macua, P., Ouyang, Y., Sadovsky, E., Kajiwara, K., Sorkin, A. and Sadovsky, Y.

1. Extracellular Vesicles, 9(1), 1812261 (2020)

  Pregnancy is a unique situation, in which placenta-derived small extracellular vesicles (sEVs) may communicate with maternal and foetal tissues. While relevant to homoeostatic and pathological functions, the mechanisms underlying sEV entry and cargo handling in target cells remain largely unknown. Using fluorescently or luminescently labelled sEVs, derived from primary human placental trophoblasts or from a placental cell line, we interrogated the endocytic pathways used by these sEVs to enter relevant target cells, including the neighbouring primary placental fibroblasts and human uterine microvascular endothelial cells. We found that trophoblastic sEVs can enter target cells, where they retain biological activity. Importantly, using a broad series of pharmacological inhibitors and siRNA-dependent silencing approaches, we showed that trophoblastic sEVs enter target cells using macropinocytosis and clathrin-mediated endocytosis pathways, but not caveolin-dependent endocytosis. Tracking their intracellular course, we localized the sEVs to early endosomes, late endosomes, and lysosomes. Finally, we used coimmunoprecipitation to demonstrate the association of the sEV microRNA (miRNA) with the P-body proteins AGO2 and GW182. Together, our data systematically detail endocytic pathways used by placental sEVs to enter relevant fibroblastic and endothelial target cells, and provide support for “endocytic escape” of sEV miRNA to P-bodies, a key site for cytoplasmic RNA regulation.  

3.3584           The cell adhesion molecule L1 interacts with nuclear proteins via its intracellular domain

Minguez, M.G., Wolters-Eisfeld, G., Lutz, D., Buck, F., Schachner, M. and Kleene, R. FASEB J., 34, 9869-9883 (2020)   Proteolytic cleavage of the cell adhesion molecule L1 (L1) in brain tissue and in cultured cerebellar neurons results in the generation and nuclear import of a 30 kDa fragment comprising most of L1's C‐terminal, intracellular domain. In search of molecules that interact with this domain, we performed affinity chromatography with the recombinant intracellular L1 domain and a nuclear extract from mouse brains, and identified potential nuclear L1 binding partners involved in transcriptional regulation, RNA processing and transport, DNA repair, chromatin remodeling, and nucleocytoplasmic transport. By co‐immunoprecipitation and enzyme‐linked immunosorbent assay using recombinant proteins, we verified the direct interaction between L1 and the nuclear binding partners non‐POU domain containing octamer‐binding protein and splicing factor proline/glutamine‐rich. The proximity ligation assay confirmed this close interaction in cultures of cerebellar granule cells. Our findings suggest that L1 fragments regulate multiple nuclear functions in the nervous system. We discuss possible physiological and pathological roles of these interactions in regulation of chromatin structure, gene expression, RNA processing, and DNA repair.  

3.3585           Small but significant: Insights and new perspectives of exosomes in cardiovascular disease

Zhang, J., Cui, X., Guo, J., Cao, C., Zhang, Z., Wangh, B., Zhang, l., Shen, D., Kim, K., Woodffield, T., Tang, J. and Zhang, J.

1. Cell. Mol. Med., 24, 8291-8303 (2020)

  Cardiovascular diseases (CVDs) are a major health problem worldwide, and health professionals are still actively seeking new and effective approaches for CVDs treatment. Presently, extracellular vesicles, particularly exosomes, have gained its popularity for CVDs treatment because of their function as messengers for inter‐ and extra‐cellular communications to promote cellular functions in cardiovascular system. However, as a newly developed field, researchers are still trying to fully understand the role of exosomes, and their mechanism in mediating cardiac repair process. Therefore, a comprehensive review of this topic can be timely and favourable. In this review, we summarized the basic biogenesis and characterization of exosomes and then further extended the focus on the circulating exosomes in cellular communication and stem cell‐derived exosomes in cardiac disease treatment. In addition, we covered interactions between the heart and other organs through exosomes, leading to the diagnostic characteristics of exosomes in CVDs. Future perspectives and limitations of exosomes in CVDs were also discussed with a special focus on exploring the potential delivery routes, targeting the injured tissue and engineering novel exosomes, as well as its potential as one novel target in the metabolism‐related puzzle.  

3.3586           Glutamine deprivation alters the origin and function of cancer cell exosomes

Fan, S-J., Kroeger, B., Marie, P.P., Bridges, E.M., Mason, J.D., McCormick, K., et al EMBO J., 39, e103009 (2020)   Exosomes are secreted extracellular vesicles carrying diverse molecular cargos, which can modulate recipient cell behaviour. They are thought to derive from intraluminal vesicles formed in late endosomal multivesicular bodies (MVBs). An alternate exosome formation mechanism, which is conserved from fly to human, is described here, with exosomes carrying unique cargos, including the GTPase Rab11, generated in Rab11‐positive recycling endosomal MVBs. Release of Rab11‐positive exosomes from cancer cells is increased relative to late endosomal exosomes by reducing growth regulatory Akt/mechanistic Target of Rapamycin Complex 1 (mTORC1) signalling or depleting the key metabolic substrate glutamine, which diverts membrane flux through recycling endosomes. Vesicles produced under these conditions promote tumour cell proliferation and turnover and modulate blood vessel networks in xenograft mouse models in vivo. Their growth‐promoting activity, which is also observed in vitro, is Rab11a‐dependent, involves ERK‐MAPK‐signalling and is inhibited by antibodies against amphiregulin, an EGFR ligand concentrated on these vesicles. Therefore, glutamine depletion or mTORC1 inhibition stimulates release from Rab11a compartments of exosomes with pro‐tumorigenic functions, which we propose promote stress‐induced tumour adaptation.  

3.3587           Characterization and immunostimulatory activity of extracellular vesicles from Filifactor alocis

Kim, H.Y., Lim, Y., An, S-J. and Choi, B-K. Med. Oral Microbiol., 35, 1-9 (2020)   Filifactor alocis, a gram‐positive, obligate anaerobic rod, is an emerging periodontal pathogen that is frequently isolated from patients with periodontitis, peri‐implantitis, and apical periodontitis. Recent studies have shown that extracellular vesicles (EVs) from gram‐negative periodontal pathogens, so‐called outer membrane vesicles (OMVs), harbor various effector molecules responsible for inducing host inflammatory responses. However, there are no reports of EVs from F. alocis. In this study, we purified and characterized the protein profiles of EVs from F. alocis and investigated their immunostimulatory activity on human monocytic THP‐1 and human oral keratinocyte HOK‐16B cell lines. Highly pure EVs were obtained from F. alocis using density gradient ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy showed that F. alocis EVs were between 50 and 270 nm in diameter. Proteome analysis identified 28 proteins, including lipoproteins, autolysins, F. alocis complement inhibitor (FACIN), transporter‐related proteins, metabolism‐related proteins, and ribosomal proteins. Human cytokine array analysis showed that F. alocis EVs remarkably induced the expression of CCL1, CCL2, MIP‐1, CCL5, CXCL1, CXCL10, ICAM‐1, IL‐1β, IL‐1ra, IL‐6, IL‐8, MIF, SerpinE, and TNF‐α in THP‐1 cells and CXCL1, G‐CSF, GM‐CSF, IL‐6, and IL‐8 in HOK‐16B cells. The immunostimulatory activity of F. alocis EVs was similar to that of the whole bacterial cells. Our findings provide new insight into the role of EVs from gram‐positive oral bacteria in periodontal diseases.  

3.3588           Two pools of IRE1α in cardiac and skeletal muscle cells

Wang, Q., Groenendyk, J., Paskevicius, T., Qin, W., Kor, K.C., Liu, Y., Hiess, F., Knollmann, B.C., Chen, S.R.W., Tang, J., Chen, X-Z., Agellon, L.B. and Michalak, M. FASEB J., 33, 8892-8904 (2020)   The endoplasmic reticulum (ER) plays a central role in cellular stress responses via mobilization of ER stress coping responses, such as the unfolded protein response (UPR). The inositol‐requiring 1α (IRE1α) is an ER stress sensor and component of the UPR. Muscle cells also have a well‐developed and highly subspecialized membrane network of smooth ER called the sarcoplasmic reticulum (SR) surrounding myofibrils and specialized for Ca²⁺ storage, release, and uptake to control muscle excitation‐contraction coupling. Here, we describe 2 distinct pools of IRE1α in cardiac and skeletal muscle cells, one localized at the perinuclear ER and the other at the junctional SR. We discovered that, at the junctional SR, calsequestrin binds to the ER luminal domain of IRE1α, inhibiting its dimerization. This novel interaction of IRE1α with calsequestrin, one of the highly abundant Ca²⁺ handling proteins at the junctional SR, provides new insights into the regulation of stress coping responses in muscle cells.  

3.3589           Inhibition of proteasome reveals basal mitochondrial ubiquitination

Sulkshane, P., Duek, I., Ram, J., Thakur, A., Reis, N., Ziv, T. and Glickman, M.H.

1. Proteomics, 229, 103949 (2020)

  Strict quality control for mitochondrial proteins is necessary to ensure cell homeostasis. Two cellular pathways–Ubiquitin Proteasome System (UPS) and autophagy–contribute to mitochondrial homeostasis under stressful conditions. Here, we investigate changes to the mitochondria proteome and to the ubiquitin landscape at mitochondria in response to proteasome inhibition. Treatment of HeLa cells devoid of Parkin, the primary E3 ligase responsible for mitophagy, with proteasome inhibitor MG132 for a few hours caused mitochondrial oxidative stress and fragmentation, reduced energy output, and increased mitochondrial ubiquitination without inducing mitophagy. Overexpression of Parkin did not show any induction of mitophagy in response to MG132 treatment. Analysis of ubiquitin chains on isolated mitochondria revealed predominance of K48, K29 and K63-linked polyubiquitin. Interestingly, of all ubiquitinated mitochondrial proteins detected in response to MG132 treatment, a majority (≥90%) were intramitochondrial irrespective of Parkin expression. However, overall levels of these ubiquitinated mitochondrial proteins did not change significantly upon proteasome inhibition when evaluated by quantitative proteomics (LFQ and SILAC), suggesting that only a small portion are ubiquitinated under basal conditions. Another aspect of proteasome inhibition is significant enrichment of UPS, lysosomal and phagosomal components, and other heat shock proteins associated with isolated mitochondria. Taken together, our study highlights a critical role of UPS for ubiquitinating and removing imported proteins as part of a basal mitochondrial quality control system independent of Parkin.  

3.3590           An Activity-Mediated Transition in Transcription in Early Postnatal Neurons

Stroud, H., Yang, M.G., Tsitohay, Y.N., Hirvatin, S., Ling, E. and Greenberg, M.E. Neuron, 107, 874-890 (2020)   The maturation of the mammalian brain occurs after birth, and this stage of neuronal development is frequently impaired in neurological disorders, such as autism and schizophrenia. However, the mechanisms that regulate postnatal brain maturation are poorly defined. By purifying neuronal subpopulations across brain development in mice, we identify a postnatal switch in the transcriptional regulatory circuits that operates in the maturing mammalian brain. We show that this developmental transition includes the formation of hundreds of cell-type-specific neuronal enhancers that appear to be modulated by neuronal activity. Once selected, these enhancers are active throughout adulthood, suggesting that their formation in early life shapes neuronal identity and regulates mature brain function.  

3.3591           Cell Type-Specific Transcriptomics Reveals that Mutant Huntingtin Leads to Mitochondrial RNA Release and Neuronal Innate Immune Activation

Lee, H., Fenster, R.J., Pineda, S.S., Kellis, M., LaVoie, M:J. and Heiman, M. Neuron, 107, 891-908 (2020)   The mechanisms by which mutant huntingtin (mHTT) leads to neuronal cell death in Huntington’s disease (HD) are not fully understood. To gain new molecular insights, we used single nuclear RNA sequencing (snRNA-seq) and translating ribosome affinity purification (TRAP) to conduct transcriptomic analyses of caudate/putamen (striatal) cell type-specific gene expression changes in human HD and mouse models of HD. In striatal spiny projection neurons, the most vulnerable cell type in HD, we observe a release of mitochondrial RNA (mtRNA) (a potent mitochondrial-derived innate immunogen) and a concomitant upregulation of innate immune signaling in spiny projection neurons. Further, we observe that the released mtRNAs can directly bind to the innate immune sensor protein kinase R (PKR). We highlight the importance of studying cell type-specific gene expression dysregulation in HD pathogenesis and reveal that the activation of innate immune signaling in the most vulnerable HD neurons provides a novel framework to understand the basis of mHTT toxicity and raises new therapeutic opportunities.  

3.3592           Efficient encapsulation of biocompatible nanoparticles in exosomes for cancer theranostics

Tanziela, T., Shaikh, S., Jiang, H., Lu, Z. and Wang, X. Nano Today, 35, 100964 (2020)   Exosomes secrete from various cells and can be separated from all biological fluids, which possess the properties of their origin, and function as cell communication. Moreover, the exosomes have capacity to envelope a widespread range of contents including genetic materials, proteins, drugs and others which make exosomes play a remarkable role in diagnosis and therapy of different diseases including cancers. In this review, we summarized and discussed exosomes’ isolation strategies and the possible combination of some biocompatible nanoparticles with exosomes (Exo-NPs) for biomedical imaging and treatment such as photothermal therapy, magnetic hyperthermia, gene silencing and drug delivery. It is observed that relevant exosomes with characteristics of their origin are promising nanocarriers in the field of oncology to realize cancer theranostics. Attractive features of Exo-NPs such as nano-sized, high penetration, delayed circulation, stability, low immunogenicity, and ability to cross blood brain barriers, distinguished them from other nanocarriers to overcome many hindrances faced in cancer diagnosis and therapy.  

3.3593           Preparation of Multi-omics Grade Extracellular Vesicles by Density-Based Fractionation of Urine

Dhondt, B., Lumenm, N., De Wever, O. and Hendrix, A. Star Protocols, 1, 100073 (2020)   The multidimensional cargo of extracellular vesicles (EV) released in urine is a reflection of the pathophysiological processes occurring within their cells and tissues of origin in the urogenital system. Here, we describe a step-by-step protocol for density-based separation of urinary EV with high specificity and repeatability. The implementation of integrative omics allows the study of the molecular complexity of highly purified urinary EV, supporting the identification of EV-specific functions and biomarkers.  

3.3594           miRNA profile of extracellular vesicles isolated from saliva of Haemaphysalis longicornis tick

Nawaz, M., Malik, M.I., Zhang, H., Gebremedhin, B., Cao, J., Zhou, Y. and Zhou, J. Acta Tropica, 212, 105718 (2020)   Extracellular vesicles (EVs) play a key role in host-parasite interactions. Previous studies have shown that parasites can release microRNA (miRNA) containing EVs, which can transfer their contents to host cells and regulate gene expression in recipient cells. However, a little is known about the secretion of EVs by the ticks. This study was therefore, carried out to examine the saliva of ticks for the presence of miRNA containing EVs. Vesicles were purified from saliva of partially engorged Haemaphysalis longicornis ticks. Transmission electron microscopy (TEM) was carried out to confirm that vesicles within saliva were EVs based on size and morphology. Total RNA was extracted from EVs and was analyzed by deep sequencing to determine miRNA profile. TEM analysis confirmed the presence of extracellular vesicle-like structures within tick saliva. RNA-seq analysis showed that tick-derived EVs contained small non-coding RNA populations including miRNAs. The analysis of tick-derived EVs identified 36 known miRNAs, 34 novel miRNAs and 842 novel Piwi-interacting RNAs (piRNA). The results of this study provide evidence that EVs containing miRNAs can be secreted by the ticks and suggest that vesicles could transfer these miRNAs to modulate host cell functions.  

3.3595           Induction of a proliferative response in the zebrafish retina by injection of extracellular vesicles

Didiano, D., Abner, J.J., Hinger, S.A., Flickinger, Z., Kent, M., Clement, M.A., Balaiya, S., Liu, Q., Dai, X., Levine, E.M. and patton, J.G. Exp. Eye Res., 200, 108254 (2020)   Ongoing research using cell transplantation and viral-mediated gene therapy has been making progress to restore vision by retinal repair, but targeted delivery and complete cellular integration remain challenging. An alternative approach is to induce endogenous Müller glia (MG) to regenerate lost neurons and photoreceptors, as occurs spontaneously in teleost fish and amphibians. Extracellular vesicles (EVs) can transfer protein and RNA cargo between cells serving as a novel means of cell-cell communication. We conducted an in vivo screen in zebrafish to identify sources of EVs that could induce MG to dedifferentiate and generate proliferating progenitor cells after intravitreal injection into otherwise undamaged zebrafish eyes. Small EVs (sEVs) from C6 glioma cells were the most consistent at inducing MG-derived proliferating cells. Ascl1a expression increased after intravitreal injection of C6 sEVs and knockdown of ascl1a inhibited the induction of proliferation. Proteomic and RNAseq analyses of EV cargo content were performed to begin to identify key factors that might target EVs to MG and initiate retina regeneration.  

3.3596           Modern Techniques for the Isolation of Extracellular Vesicles and Viruses

McNamara, R.P. and Dittmer, D.P.

1. Neuroimmun. Pharmacol., 15, 459-472 (2020)

  Extracellular signaling is pivotal to maintain organismal homeostasis. A quickly emerging field of interest within extracellular signaling is the study of extracellular vesicles (EV), which act as messaging vehicles for nucleic acids, proteins, metabolites, lipids, etc. from donor cells to recipient cells. This transfer of biologically active material within a vesicular body is similar to the infection of a cell through a virus particle, which transfers genetic material from one cell to another to preserve an infection state, and viruses are known to modulate EV. Although considerable heterogeneity exists within EV and viruses, this review focuses on those that are small (< 200 nm in diameter) and of relatively low density (< 1.3 g/mL). A multitude of isolation methods for EV and virus particles exist. In this review, we present an update on methods for their isolation, purification, and phenotypic characterization. We hope that the information we provide will be of use to basic science and clinical investigators, as well as biotechnologists in this emerging field.  

3.3597           Rab13 regulates sEV secretion in mutant KRAS colorectal cancer cells

Hinger, S.A., Abner, J.J., Franklin, J.L., Jeppesen, D.K., Coffey, R.J. and Patton, J.G. Scientific Reports, 10:35804 (2020)   Small extracellular vesicles (sEVs), 50–150 nm in diameter, have been proposed to mediate cell–cell communication with important implications in tumor microenvironment interactions, tumor growth, and metastasis. We previously showed that mutant KRAS colorectal cancer (CRC) cells release sEVs containing Rab13 protein and mRNA. Previous work had shown that disruption of intracellular Rab13 trafficking inhibits epithelial cell proliferation and invasiveness. Here, we show that Rab13 additionally regulates the secretion of sEVs corresponding to both traditional exosomes and a novel subset of vesicles containing both β1-integrin and Rab13. We find that exposure of recipient cells to sEVs from KRAS mutant donor cells increases proliferation and tumorigenesis and that knockdown of Rab13 blocks these effects. Thus, Rab13 serves as both a cargo protein and as a regulator of sEV secretion. Our data support a model whereby Rab13 can mediate its effects on cell proliferation and invasiveness via autocrine and paracrine signaling.  

3.3598           Single-Nucleus RNA-Seq Is Not Suitable for Detection of Microglial Activation Genes in Humans

Thrupp, N., Frigerio, C.S., Wolfs, l., Mancuso, R., de Strooper, B. and Fiers, M. Cell Reports, 32, 108189 (2020)   Single-nucleus RNA sequencing (snRNA-seq) is used as an alternative to single-cell RNA-seq, as it allows transcriptomic profiling of frozen tissue. However, it is unclear whether snRNA-seq is able to detect cellular state in human tissue. Indeed, snRNA-seq analyses of human brain samples have failed to detect a consistent microglial activation signature in Alzheimer’s disease. Our comparison of microglia from single cells and single nuclei of four human subjects reveals that, although most genes show similar relative abundances in cells and nuclei, a small population of genes (∼1%) is depleted in nuclei compared to whole cells. This population is enriched for genes previously implicated in microglial activation, including APOE, CST3, SPP1, and CD74, comprising 18% of previously identified microglial-disease-associated genes. Given the low sensitivity of snRNA-seq to detect many activation genes, we conclude that snRNA-seq is not suited for detecting cellular activation in microglia in human disease.  

3.3599           Inhibition of αvβ3 integrin impairs adhesion and uptake of tumor-derived small extracellular vesicles

Altei, W.F., Pachane, B.C., dos Santos, P.K., Ribeiro, L.N.M., Sung, B.H., Weaver, A.M. and Selistre-de-Araujo, H.S. Cell Communications and Signaling, 18:159 (2020)   Background Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells. Methods To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for αvβ3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments. Results We find that SEVs secreted from MDA-MB-231 breast cancer cells carry αvβ3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of αvβ3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content. Conclusion In summary, our findings demonstrate for the first time a key role of αvβ3 integrin in cell-cell communication through SEVs.  

3.3600           Extracellular Vesicles as an Efficient and Versatile System for Drug Delivery

Dang, X.T.T., kavishka, J.M., Zhang, D.X., Prisinu, M. and Le, M.T.N. Cells, 9, 2191 (2020)   Despite the recent advances in drug development, the majority of novel therapeutics have not been successfully translated into clinical applications. One of the major factors hindering their clinical translation is the lack of a safe, non-immunogenic delivery system with high target specificity upon systemic administration. In this respect, extracellular vesicles (EVs), as natural carriers of bioactive cargo, have emerged as a promising solution and can be further modified to improve their therapeutic efficacy. In this review, we provide an overview of the biogenesis pathways, biochemical features, and isolation methods of EVs with an emphasis on their many intrinsic properties that make them desirable as drug carriers. We then describe in detail the current advances in EV therapeutics, focusing on how EVs can be engineered to achieve improved target specificity, better circulation kinetics, and efficient encapsulation of therapeutic payloads. We also identify the challenges and obstacles ahead for clinical translation and provide an outlook on the future perspective of EV-based therapeutics.  

3.3601           Hybrid cellular membrane nanovesicles amplify macrophage immune responses against cancer recurrence and metastasis

Rao, L., Wu, L., Liu, Z., Tian, R., Yu, G., Zhou, Z., Yang, K., Xiong, H-G., Zhang, A. et al Nature Communications, 11, 4909 (2020)   Effectively activating macrophages against cancer is promising but challenging. In particular, cancer cells express CD47, a ‘don’t eat me’ signal that interacts with signal regulatory protein alpha (SIRPα) on macrophages to prevent phagocytosis. Also, cancer cells secrete stimulating factors, which polarize tumor-associated macrophages from an antitumor M1 phenotype to a tumorigenic M2 phenotype. Here, we report that hybrid cell membrane nanovesicles (known as hNVs) displaying SIRPα variants with significantly increased affinity to CD47 and containing M2-to-M1 repolarization signals can disable both mechanisms. The hNVs block CD47-SIRPα signaling axis while promoting M2-to-M1 repolarization within tumor microenvironment, significantly preventing both local recurrence and distant metastasis in malignant melanoma models. Furthermore, by loading a stimulator of interferon genes (STING) agonist, hNVs lead to potent tumor inhibition in a poorly immunogenic triple negative breast cancer model. hNVs are safe, stable, drug loadable, and suitable for genetic editing. These properties, combined with the capabilities inherited from source cells, make hNVs an attractive immunotherapy.  

3.3602           Modification of lipid rafts by extracellular vesicles carrying HIV-1 protein Nef induces redistribution of amyloid precursor protein and Tau, causing neuronal dysfunction

Ditiatkovsky, M., Mukhamedova, N., Dragoljevic, D., Hoang, A., Low, H., Pushkarsky, T., Fu, Y., Carmichael, I., Hill, A.F., Murphy, A.J., Bukrinsky, M. and Sviridov, D.

1. Biol. Chem., 295(38), 13377-13392 (2020)

  HIV-associated neurocognitive disorders (HANDs) are a frequent outcome of HIV infection. Effective treatment of HIV infection has reduced the rate of progression and severity but not the overall prevalence of HANDs, suggesting ongoing pathological process even when viral replication is suppressed. In this study, we investigated how HIV-1 protein Nef secreted in extracellular vesicles (exNef) impairs neuronal functionality. ExNef were rapidly taken up by neural cells in vitro, reducing the abundance of ABC transporter A1 (ABCA1) and thus cholesterol efflux and increasing the abundance and modifying lipid rafts in neuronal plasma membranes. ExNef caused a redistribution of amyloid precursor protein (APP) and Tau to lipid rafts and increased the abundance of these proteins, as well as of Aβ₄₂. ExNef further potentiated phosphorylation of Tau and activation of inflammatory pathways. These changes were accompanied by neuronal functional impairment. Disruption of lipid rafts with cyclodextrin reversed the phenotype. Short-term treatment of C57BL/6 mice with either purified recombinant Nef or exNef similarly resulted in reduced abundance of ABCA1 and elevated abundance of APP in brain tissue. The abundance of ABCA1 in brain tissue of HIV-infected human subjects diagnosed with HAND was lower, and the abundance of lipid rafts was higher compared with HIV-negative individuals. Levels of APP and Tau in brain tissue correlated with the abundance of Nef. Thus, modification of neuronal cholesterol trafficking and of lipid rafts by Nef may contribute to early stages of neurodegeneration and pathogenesis in HAND.  

3.3603           The insufficiency of ATG4A in macroautophagy

Nguyen, N., Olivas, T.J., Mires, A., Jin, J., Yu, S., Luan, L., Nag, S., Kauffman, K.J. and Melia, T.J.

1. Biol. Chem., 295(39), 13584-13600 (2020)

  During autophagy, LC3 and GABARAP proteins become covalently attached to phosphatidylethanolamine on the growing autophagosome. This attachment is also reversible. Deconjugation (or delipidation) involves the proteolytic cleavage of an isopeptide bond between LC3 or GABARAP and the phosphatidylethanolamine headgroup. This cleavage is carried about by the ATG4 family of proteases (ATG4A, B, C, and D). Many studies have established that ATG4B is the most active of these proteases and is sufficient for autophagy progression in simple cells. Here we examined the second most active protease, ATG4A, to map out key regulatory motifs on the protein and to establish its activity in cells. We utilized fully in vitro reconstitution systems in which we controlled the attachment of LC3/GABARAP members and discovered a role for a C-terminal LC3-interacting region on ATG4A in regulating its access to LC3/GABARAP. We then used a gene-edited cell line in which all four ATG4 proteases have been knocked out to establish that ATG4A is insufficient to support autophagy and is unable to support GABARAP proteins removal from the membrane. As a result, GABARAP proteins accumulate on membranes other than mature autophagosomes. These results suggest that to support efficient production and consumption of autophagosomes, additional factors are essential including possibly ATG4B itself or one of its proteolytic products in the LC3 family.  

3.3604           Extracellular vesicles as critical mediators of maternal-fetal communication during pregnancy and their potential role in maternal metabolism

Nair, S. and Salomon, C. Placenta, 8, 60-68 (2020)   Extracellular vesicles (EVs) have been implicated in the pathophysiology of metabolic disorders by transferring biologically active molecules such as miRNAs and proteins to recipient cells, and influencing their metabolic pathways. Pregnancy is one of the greatest metabolic challenges faced by both the mother and the growing fetus, and this is fine-tuned by several factors, including hormones, soluble molecules, and molecules encapsulated in EVs released from the placenta. A wide range of EVs originating from the placenta are present in maternal circulation, and changes in their circulating levels and bioactivity (i.e., capacity to induce changes in the target cells) have been associated with several complications of pregnancies, including gestational diabetes mellitus (GDM), preeclampsia, preterm birth, and fetal growth restriction. Complications of pregnancies are associated with maternal metabolic dysfunction with short- and long-term consequences for both mother and child. However, the potential roles of circulating EVs originating from the placenta and other tissues (e.g. adipose tissue), on changes in maternal metabolism during normal and pregnancy complications have not been fully described. The aim of this brief review, thus, is to discuss the diversity of EVs, and their potential roles in the metabolic alterations during pregnancy, with a special focus on GDM.  

3.3605           Discovery of a novel EGFR ligand DPBA that degrades EGFR and suppresses EGFR-positive NSCLC growth

Yao, N., Wang, C-R., Liu, M-Q., Li, Y-J., Chen, W-M., Li, Z-Q., Qi, Q., Lu, J-J., Fan, C-L., Chen, M-F., Qi, M., Li, X-B., Hong, J., Zhang, D-M. and Ye, W-C. Signal Transduction & Targeted Therapy, 5:214 (2020)   Epidermal growth factor receptor (EGFR) activation plays a pivotal role in EGFR-driven non-small cell lung cancer (NSCLC) and is considered as a key target of molecular targeted therapy. EGFR tyrosine kinase inhibitors (TKIs) have been canonically used in NSCLC treatment. However, prevalent innate and acquired resistances and EGFR kinase-independent pro-survival properties limit the clinical efficacy of EGFR TKIs. Therefore, the discovery of novel EGFR degraders is a promising approach towards improving therapeutic efficacy and overcoming drug resistance. Here, we identified a 23-hydroxybetulinic acid derivative, namely DPBA, as a novel EGFR small-molecule ligand. It exerted potent in vitro and in vivo anticancer activity in both EGFR wild type and mutant NSCLC by degrading EGFR. Mechanistic studies disclosed that DPBA binds to the EGFR extracellular domain at sites differing from those of EGF and EGFR. DPBA did not induce EGFR dimerization, phosphorylation, and ubiquitination, but it significantly promoted EGFR degradation and repressed downstream survival pathways. Further analyses showed that DPBA induced clathrin-independent EGFR endocytosis mediated by flotillin-dependent lipid rafts and unaffected by EGFR TKIs. Activation of the early and late endosome markers rab5 and rab7 but not the recycling endosome marker rab11 was involved in DPBA-induced EGFR lysosomal degradation. The present study offers a new EGFR ligand for EGFR pharmacological degradation and proposes it as a potential treatment for EGFR-positive NSCLC, particularly NSCLC with innate or acquired EGFR TKI resistance. DPBA can also serve as a chemical probe in the studies on EGFR trafficking and degradation.  

3.3606           Transcriptional Reprogramming of Distinct Peripheral Sensory Neuron Subtypes after Axonal Injury

Renthal, W., Tochitsky, I., Yang, l., Kawaguchi, R., Geschwind, D.H. and Woolf, C.J. Neuron, 108, 128-144 (2020)   Primary somatosensory neurons are specialized to transmit specific types of sensory information through differences in cell size, myelination, and the expression of distinct receptors and ion channels, which together define their transcriptional and functional identity. By profiling sensory ganglia at single-cell resolution, we find that all somatosensory neuronal subtypes undergo a similar transcriptional response to peripheral nerve injury that both promotes axonal regeneration and suppresses cell identity. This transcriptional reprogramming, which is not observed in non-neuronal cells, resolves over a similar time course as target reinnervation and is associated with the restoration of original cell identity. Injury-induced transcriptional reprogramming requires ATF3, a transcription factor that is induced rapidly after injury and necessary for axonal regeneration and functional recovery. Our findings suggest that transcription factors induced early after peripheral nerve injury confer the cellular plasticity required for sensory neurons to transform into a regenerative state.  

3.3607           Potential application of mesenchymal stem cells and their exosomes in lung injury: an emerging therapeutic option for COVID-19 patients

Al-Khawaga, S. and Abdelalim, E.M. Stem Cell Research & Therapy, 11:437 (2020)   The COVID-19 pandemic has negatively impacted the global public health and the international economy; therefore, there is an urgent need for an effective therapy to treat COVID-19 patients. Mesenchymal stem cells (MSCs) have been proposed as an emerging therapeutic option for the SARS-CoV-2 infection. Recently, numerous clinical trials have been registered to examine the safety and efficacy of different types of MSCs and their exosomes for treating COVID-19 patients, with less published data on the mechanism of action. Although there is no approved effective therapy for COVID-19 as of yet, MSC therapies showed an improvement in the treatment of some COVID-19 patients. MSC’s therapeutic effect is displayed in their ability to reduce the cytokine storm, enhance alveolar fluid clearance, and promote epithelial and endothelial recovery; however, the safest and most effective route of MSC delivery remains unclear. The use of poorly characterized MSC products remains one of the most significant drawbacks of MSC-based therapy, which could theoretically promote the risk for thromboembolism. Optimizing the clinical-grade production of MSCs and establishing a consensus on registered clinical trials based on cell-product characterization and mode of delivery would aid in laying the foundation for a safe and effective therapy in COVID-19. In this review, we shed light on the mechanistic view of MSC therapeutic role based on preclinical and clinical studies on acute lung injury and ARDS; therefore, offering a unique correlation and applicability in COVID-19 patients. We further highlight the challenges and opportunities in the use of MSC-based therapy.  

3.3608           Lyn kinase regulates egress of flaviviruses in autophagosome-derived organelles

Li, M.Y., Naik, T.S., SIu, L.Y.L., Acuto, O., Spooner, E., Warig, P., Yang, X., Lin, Y., Bruzzone, R., Ashour, J., Evans, M.J. and Sanyal, S. Nature Communications, 11:5189 (2020)   Among the various host cellular processes that are hijacked by flaviviruses, few mechanisms have been described with regard to viral egress. Here we investigate how flaviviruses exploit Src family kinases (SFKs) for exit from infected cells. We identify Lyn as a critical component for secretion of Dengue and Zika infectious particles and their corresponding virus like particles (VLPs). Pharmacological inhibition or genetic depletion of the SFKs, Lyn in particular, block virus secretion. Lyn^−/− cells are impaired in virus release and are rescued when reconstituted with wild-type Lyn, but not a kinase- or palmitoylation-deficient Lyn mutant. We establish that virus particles are secreted in two distinct populations – one as free virions and the other enclosed within membranes. Lyn is critical for the latter, which consists of proteolytically processed, infectious virus progenies within autophagosome-derived vesicles. This process depends on Ulk1, Rab GTPases and SNARE complexes implicated in secretory but not degradative autophagy and occur with significantly faster kinetics than the conventional secretory pathway. Our study reveals a previously undiscovered Lyn-dependent exit route of flaviviruses in LC3+ secretory organelles that enables them to evade circulating antibodies and might affect tissue tropism.  

3.3609           Technologies and Standardization in Research on Extracellular Vesicles

Gandham, S., Su, X., Wood, j., Nocera, A.L., Alli, S.C., Milane, L., Zimmerman, A., Amiji, M. and Ivanov, A.R. Trends in Biotechnology, 38(10), 1066-1098 (2020)   Extracellular vesicles (EVs) are phospholipid bilayer membrane-enclosed structures containing RNAs, proteins, lipids, metabolites, and other molecules, secreted by various cells into physiological fluids. EV-mediated transfer of biomolecules is a critical component of a variety of physiological and pathological processes. Potential applications of EVs in novel diagnostic and therapeutic strategies have brought increasing attention. However, EV research remains highly challenging due to the inherently complex biogenesis of EVs and their vast heterogeneity in size, composition, and origin. There is a need for the establishment of standardized methods that address EV heterogeneity and sources of pre-analytical and analytical variability in EV studies. Here, we review technologies developed for EV isolation and characterization and discuss paths toward standardization in EV research.  

3.3610           Acidocalcisomes and Polyphosphate Granules Are Different Subcellular Structures in Agrobacterium tumefaciens

Frank, C. and Jendrossek, D. Appl. Environ. Microbiol., 86(8), e02759-19 (2020)   Acidocalcisomes are membrane-enclosed, polyphosphate-containing acidic organelles in lower Eukaryota but have also been described for Agrobacterium tumefaciens (M. Seufferheld, M. Vieira, A. Ruiz, C. O. Rodrigues, S. Moreno, and R. Docampo, J Biol Chem 278:29971–29978, 2003, https://doi.org/10.1074/jbc.M304548200). This study aimed at the characterization of polyphosphate-containing acidocalcisomes in this alphaproteobacterium. Unexpectedly, fluorescence microscopic investigation of A. tumefaciens cells using fluorescent dyes and localization of constructed fusions of polyphosphate kinases (PPKs) and of vacuolar H⁺-translocating pyrophosphatase (HppA) with enhanced yellow fluorescent protein (eYFP) suggested that acidocalcisomes and polyphosphate are different subcellular structures. Acidocalcisomes and polyphosphate granules were frequently located close together, near the cell poles. However, they never shared the same position. Mutant strains of A. tumefaciens with deletions of both ppk genes (Δppk1 Δppk2) were unable to form polyphosphate but still showed cell pole-located eYFP-HppA foci and could be stained with MitoTracker. In conclusion, A. tumefaciens forms polyP granules that are free of a surrounding membrane and thus resemble polyP granules of Ralstonia eutropha and other bacteria. The composition, contents, and function of the subcellular structures that are stainable with MitoTracker and harbor eYFP-HppA remain unclear.  

3.3611           Sex Steroids Induce Membrane Stress Responses and Virulence Properties in Pseudomonas aeruginosa

Vidaillac, C., Yong, V.F.L., Aschtgen, M-S., Qu, J., Yang, S., Xu, G., Seng, Z.J., Brown, A.C. et al mBio, 11(5), e01774-20 (2020)   Estrogen, a major female sex steroid hormone, has been shown to promote the selection of mucoid Pseudomonas aeruginosa in the airways of patients with chronic respiratory diseases, including cystic fibrosis. This results in long-term persistence, poorer clinical outcomes, and limited therapeutic options. In this study, we demonstrate that at physiological concentrations, sex steroids, including testosterone and estriol, induce membrane stress responses in P. aeruginosa. This is characterized by increased virulence and consequent inflammation and release of proinflammatory outer membrane vesicles promoting in vivo persistence of the bacteria. The steroid-induced P. aeruginosa response correlates with the molecular polarity of the hormones and membrane fluidic properties of the bacteria. This novel mechanism of interaction between sex steroids and P. aeruginosa explicates the reported increased disease severity observed in females with cystic fibrosis and provides evidence for the therapeutic potential of the modulation of sex steroids to achieve better clinical outcomes in patients with hormone-responsive strains.  

3.3612           Loss of PRCD alters number and packaging density of rhodopsin in rod photoreceptor disc membranes

Schrest, E.R., Murphy, J., Senepati, S., Goldberg, A.F.X., Park, P.S-H. and Kolandaivelu, S. Scientific Reports, 10:17885 (2020)   Progressive rod-cone degeneration (PRCD) is a small protein localized to photoreceptor outer segment (OS) disc membranes. Several mutations in PRCD are linked to retinitis pigmentosa (RP) in canines and humans, and while recent studies have established that PRCD is required for high fidelity disc morphogenesis, its precise role in this process remains a mystery. To better understand the part which PRCD plays in disease progression as well as its contribution to photoreceptor OS disc morphogenesis, we generated a Prcd-KO animal model using CRISPR/Cas9. Loss of PRCD from the retina results in reduced visual function accompanied by slow rod photoreceptor degeneration. We observed a significant decrease in rhodopsin levels in Prcd-KO retina prior to photoreceptor degeneration. Furthermore, ultrastructural analysis demonstrates that rod photoreceptors lacking PRCD display disoriented and dysmorphic OS disc membranes. Strikingly, atomic force microscopy reveals that many disc membranes in Prcd-KO rod photoreceptor neurons are irregular, containing fewer rhodopsin molecules and decreased rhodopsin packing density compared to wild-type discs. This study strongly suggests an important role for PRCD in regulation of rhodopsin incorporation and packaging density into disc membranes, a process which, when dysregulated, likely gives rise to the visual defects observed in patients with PRCD-associated RP.

3.3613           ECM deposition is driven by caveolin-1–dependent regulation of exosomal biogenesis and cargo sorting

Albacete-Albacete, L., Navarro-Lerida, I., Lopez, J.A., Martin-Padura, I., Astudillo, A.M., Ferrerini, A., Van Der-Heyden, M., Balsinde, J., Orend, G., Vazquez, j. and Del Pozo, M.A.

1. Cell Biol., 219(11), e202006178 (2020)

  The composition and physical properties of the extracellular matrix (ECM) critically influence tumor progression, but the molecular mechanisms underlying ECM layering are poorly understood. Tumor–stroma interaction critically depends on cell communication mediated by exosomes, small vesicles generated within multivesicular bodies (MVBs). We show that caveolin-1 (Cav1) centrally regulates exosome biogenesis and exosomal protein cargo sorting through the control of cholesterol content at the endosomal compartment/MVBs. Quantitative proteomics profiling revealed that Cav1 is required for exosomal sorting of ECM protein cargo subsets, including Tenascin-C (TnC), and for fibroblast-derived exosomes to efficiently deposit ECM and promote tumor invasion. Cav1-driven exosomal ECM deposition not only promotes local stromal remodeling but also the generation of distant ECM-enriched stromal niches in vivo. Cav1 acts as a cholesterol rheostat in MVBs, determining sorting of ECM components into specific exosome pools and thus ECM deposition. This supports a model by which Cav1 is a central regulatory hub for tumor–stroma interactions through a novel exosome-dependent ECM deposition mechanism.  

3.3614           Inhibition of class I PI3K enhances chaperone-mediated autophagy

Endicott, S.J., Ziemba, Z.J., Beckmann, L.J., Boynton, D.N. and Miller, R.A.

1. Cell Biol., 219(12), e202001031 (2020)

  Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, where individual peptides, recognized by a consensus motif, are translocated directly across the lysosomal membrane. CMA regulates the abundance of many disease-related proteins, with causative roles in neoplasia, neurodegeneration, hepatosteatosis, and other pathologies relevant to human health and aging. At the lysosomal membrane, CMA is inhibited by Akt-dependent phosphorylation of the CMA regulator GFAP. The INS-PI3K-PDPK1 pathway regulates Akt, but its role in CMA is unclear. Here, we report that inhibition of class I PI3K or PDPK1 activates CMA. In contrast, selective inhibition of class III PI3Ks does not activate CMA. Isolated liver lysosomes from mice treated with either of two orally bioavailable class I PI3K inhibitors, pictilisib or buparlisib, display elevated CMA activity, and decreased phosphorylation of lysosomal GFAP, with no change in macroautophagy. The findings of this study represent an important first step in repurposing class I PI3K inhibitors to modulate CMA in vivo.  

3.3615           Extracellular vesicles engineered with valency-controlled DNA nanostructures deliver CRISPR/Cas9 system for gene therapy

Zhuang, J., Tan, J., Wu, C., Zhang, J., Liu, T., Fan, C., Li, J. and Zhang, Y. Nucleic Acids Res., 48(16), 8870-8882 (2020)   Extracellular vesicles (EVs) hold great promise for transporting CRISPR–Cas9 RNA-guided endonucleases (RNP) throughout the body. However, the cell-selective delivery of EVs is still a challenge. Here, we designed valency-controlled tetrahedral DNA nanostructures (TDNs) conjugated with DNA aptamer, and loaded the valency-controlled TDNs on EV surface via cholesterol anchoring for specific cell targeting. The targeting efficacy of different ratios of aptamer/cholesterol from 1:3 to 3:1 in TDNs on decorating EVs was investigated. TDNs with one aptamer and three cholesterol anchors (TDN1) efficiently facilitated the tumor-specific accumulation of the EVs in cultured HepG2 cells and human primary liver cancer-derived organoids, as well as xenograft tumor models. The intracellular delivery of RNP by TDN1-EVs successfully realized its subsequent genome editing, leading to the downregulation of GFP or WNT10B in specific cells. This system was ultimately applied to reduce the protein expression of WNT10B, which presented remarkable tumor growth inhibition in vitro, ex vivo and in vivo, and could be extended to other therapeutic targets. The present study provides a platform for the directional display of aptamer on surface labeling and the EVs-based Cas9 delivery, which provides a meaningful idea for future cell-selective gene editing.  

3.3616           Fusion-Independent Satellite Cell Communication to Muscle Fibers During Load-Induced Hypertrophy

Murach, K.A:, Vechetti, I.J., Van Pelt, D.W., Crow, S.E., Dungan, C.M., Figueiredo, V.C., Kosmac, K., Fu, X., Richards, C.I., Fry, C.S., McCarthy, J.J. and Peterson, C.A: Function, 1(1), zqaa009 (2020)   The “canonical” function of Pax7+ muscle stem cells (satellite cells) during hypertrophic growth of adult muscle fibers is myonuclear donation via fusion to support increased transcriptional output. In recent years, however, emerging evidence suggests that satellite cells play an important secretory role in promoting load-mediated growth. Utilizing genetically modified mouse models of delayed satellite cell fusion and in vivo extracellular vesicle (EV) tracking, we provide evidence for satellite cell communication to muscle fibers during hypertrophy. Myogenic progenitor cell-EV-mediated communication to myotubes in vitro influences extracellular matrix (ECM)-related gene expression, which is congruent with in vivo overload experiments involving satellite cell depletion, as well as in silico analyses. Satellite cell-derived EVs can transfer a Cre-induced, cytoplasmic-localized fluorescent reporter to muscle cells as well as microRNAs that regulate ECM genes such as matrix metalloproteinase 9 (Mmp9), which may facilitate growth. Delayed satellite cell fusion did not limit long-term load-induced muscle hypertrophy indicating that early fusion-independent communication from satellite cells to muscle fibers is an underappreciated aspect of satellite cell biology. We cannot exclude the possibility that satellite cell-mediated myonuclear accretion is necessary to maintain prolonged growth, specifically in the later phases of adaptation, but these data collectively highlight how EV delivery from satellite cells can directly contribute to mechanical load-induced muscle fiber hypertrophy, independent of cell fusion to the fiber.  

3.3617           Exosomes in Gliomas: Biogenesis, Isolation, and Preliminary Applications in Nanomedicine

Romano, E., Netti, P.A. and Torino, E. Pharmaceuticals, 13, 319 (2020)   Exosomes are phospholipid-based particles endogenously produced by both normal and tumor cells. Initially identified as a pathway for shuttling cellular waste, for a long time they were thought to act as “garbage bags”, and only in the past few years have they emerged as a promising drug delivery system. In this review, we provide an overview of the knowledge about exosome architecture and biogenesis and the recent progress in isolation methods. Furthermore, we describe the mechanisms involved in both extra- and intracellular communication with a focus on glioma brain tumors. Glioma is considered a rare disease and is the most prominent aggressive brain malignancy. How exosomes target glial tumoral cells in vivo remains largely unknown. However, they are able to influence numerous physio-pathological aspects. Here, we discuss the role they play in this heterogeneous and complex microenvironment and their potential applications.  

3.3618           Tiny Actors in the Big Cellular World: Extracellular Vesicles Playing Critical Roles in Cancer

Jurj, A., Pop-Bica, C., Slaby, O., Stefan, C.D., Cho, W.C., Korban, S.S. and Berindan-Neagoe, I. Int. J. Mol. Sci., 27:7688 (2020)   Communications among cells can be achieved either via direct interactions or via secretion of soluble factors. The emergence of extracellular vesicles (EVs) as entities that play key roles in cell-to-cell communication offer opportunities in exploring their features for use in therapeutics; i.e., management and treatment of various pathologies, such as those used for cancer. The potential use of EVs as therapeutic agents is attributed not only for their cell membrane-bound components, but also for their cargos, mostly bioactive molecules, wherein the former regulate interactions with a recipient cell while the latter trigger cellular functions/molecular mechanisms of a recipient cell. In this article, we highlight the involvement of EVs in hallmarks of a cancer cell, particularly focusing on those molecular processes that are influenced by EV cargos. Moreover, we explored the roles of RNA species and proteins carried by EVs in eliciting drug resistance phenotypes. Interestingly, engineered EVs have been investigated and proposed as therapeutic agents in various in vivo and in vitro studies, as well as in several clinical trials.  

3.3619           Differential Protein Expression in Striatal D1- and D2-Dopamine Receptor-Expressing Medium Spiny Neurons

Mmansuri, M.S., Peng, G., Wilson, R.S., Lam, T.T., Zhao, H., Williams, K.R. and Nairn, A.C: Proteomes, 8:27 82020)   Many neurological disorders and diseases including drug addiction are associated with specific neuronal cell types in the brain. The striatum, a region that plays a critically important role in the development of addictive drug-related behavior, provides a good example of the cellular heterogeneity challenges associated with analyses of specific neuronal cell types. Such studies are needed to identify the adaptive changes in neuroproteomic signaling that occur in response to diseases such as addiction. The striatum contains two major cell types, D1 and D2 type dopaminoceptive medium spiny neurons (MSNs), whose cell bodies and processes are intermingled throughout this region. Since little is known about the proteomes of these two neuronal cell populations, we have begun to address this challenge by using fluorescence-activated nuclear sorting (FANS) to isolate nuclei-containing fractions from striatum from D1 and D2 “Translating Ribosome Affinity Purification” (TRAP) mice. This approach enabled us to devise and implement a robust and reproducible workflow for preparing samples from specific MSN cell types for mass spectrometry analyses. These analyses quantified at least 685 proteins in each of four biological replicates of 50 K sorted nuclei from two D1 mice/replicate and from each of four biological replicates of 50 K sorted nuclei from two D2 mice/replicate. Proteome analyses identified 87 proteins that were differentially expressed in D1 versus D2 MSN nuclei and principal component analysis (PCA) of these proteins separated the 8 biological replicates into specific cell types. Central network analysis of the 87 differentially expressed proteins identified Hnrnpd and Hnmpa2b1 in D1 and Cct2 and Cct7 in D2 as potential central interactors. This workflow can now be used to improve our understanding of many neurological diseases including characterizing the short and long-term impact of drugs of abuse on the proteomes of these two dopaminoceptive neuronal populations.  

3.3620           Data on the genome and proteome profiles of ciprofloxacin-resistant Acholeplasma laidlawii strains selected under different conditions in vitro

Mouzykantov, A., Medvedeva, E., baranova, N., Lopuhov, V., Usachev, K., Chernova, O. and Chernov, V. Data in Brief, 33,106412 (2020)   Acholeplasma laidlawii is widespread hypermutable bacteria (class Mollicutes) capable of infecting humans, animals, plants, which is the main contaminant of cell cultures and vaccine preparations. The mechanisms of the development of antimicrobial resistance of this bacterium are associated with the secretion of extracellular vesicles, which can mediate the lateral transfer of antibiotic resistance determinants. We compared the genome profiles of ciprofloxacin-resistant A.laidlawii strains PG8r1 (MIC 10 µg/ml) and PG8r3 (MIC 10 µg/ml) selected under different in vitro conditions - when ciprofloxacin-sensitive (MIC 0.5 µg/ml) A.laidlawii PG8B strain was cultured at increasing concentrations of ciprofloxacin in a broth medium alone, and with vesicles derived from the ciprofloxacin-resistant (MIC 20 µg/ml) A.laidlawii PG8R₁₀c-2 strain, respectively. Genome profiles of PG8c-3 (obtained from a single colony of the strain PG8B) and PG8R₁₀c-2 were analyzed too. Patterns of the quinolone target genes (gyrA, gyrB, parE, parC) containing in extracellular vesicles of PG8c-3, PG8R₁₀c-2, PG8r1 and PG8r3 were determined. Genome sequencing was performed on the NextSeq Illumina platform. Search and annotation of single nucleotide polymorphisms were performed using Samtools and SnpEff, respectively. We also compared cellular proteomes of PG8c-3, PG8r1 and PG8r3. The cellular proteome profiles of the A. laidlawii strains were determined by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. This work presents data on single nucleotide polymorphisms (SNPs) found in the genomes of the ciprofloxacin-resistant strains selected under different in vitro conditions and proteins that were differentially expressed in the cells of ciprofloxacin-resistant strains selected under different conditions in vitro.  

3.3621           Uveal Melanoma-Derived Extracellular Vesicles Display Transforming Potential and Carry Protein Cargo Involved in Metastatic Niche Preparation

Tsering, T., Laskaris, A., Abdouh, M., Bustamante, P., Parent, S., Jin, E., Ferrier, S.T., Arena, G. and Burnier, J.V. Cancers, 12:2923 (2020)   Extracellular vesicles (EVs) carry molecules derived from donor cells and are able to alter the properties of recipient cells. They are important players during the genesis and progression of tumors. Uveal melanoma (UM) is the most common primary intraocular tumor in adults and is associated with a high rate of metastasis, primarily to the liver. However, the mechanisms underlying this process are poorly understood. In the present study, we analyzed the oncogenic potential of UM-derived EVs and their protein signature. We isolated and characterized EVs from five UM cell lines and from normal choroidal melanocytes (NCMs). BRCA1-deficient fibroblasts (Fibro-BKO) were exposed to the EVs and analyzed for their growth in vitro and their reprograming potential in vivo following inoculation into NOD-SCID mice. Mass spectrometry of proteins from UM-EVs and NCM-EVs was performed to determine a protein signature that could elucidate potential key players in UM progression. In-depth analyses showed the presence of exosomal markers, and proteins involved in cell-cell and focal adhesion, endocytosis, and PI3K-Akt signaling pathway. Notably, we observed high expression levels of HSP90, HSP70 and integrin V in UM-EVs. Our data bring new evidence on the involvement of UM-EVs in cancer progression and metastasis.  

3.3622           Exosomal PD-L1 and N-cadherin predict pulmonary metastasis progression for osteosarcoma patients

Wang, J., Zhang, H., Sun, X., Wang, X., Ren, T., Huang, Y., Zhang, R., Zheng, B. and Guo, W.

1. Nanobiotechnol., 18:151 (2020)

  Background Recent studies indicated that exosomal programmed death-ligand 1 (PD-L1) derived from cancers could induce immunosuppression and tumor pathogenesis. However, it is unclear how exosomes influence osteosarcoma (OS) progression and whether PD-L1 also exists in serum exosomes (Sr-exosomes) of patients with osteosarcoma. We examined serum exosomes from 70 OS patients, 9 patients with benign tumors and 22 healthy donors. OS-derived exosomes were functionally evaluated in vivo and in vitro. Results The characteristics of exosomes derived from OS patient serum and OS cell lines were confirmed by several methods. We found OS patients had a higher level of exosomal PD-L1 compared to healthy donors. Meanwhile, OS patients with pulmonary metastasis also showed a relatively higher level of exosomal PD-L1 than patients without metastasis. Next, bioinformatic analysis demonstrated that Sr-exosomes isolated from OS patients may involve in the important process of immune function and cancer pathogenesis for OS patients. Co-expression network centered with PD-L1 among Sr-exosomal differently expressed mRNA demonstrated exosomal N-cadherin had a close relationship with exosomal PD-L1 expression. Then, we confirmed higher level of Sr-exosomal N-cadherin in OS patients with pulmonary metastasis compared to ones without metastasis. Furthermore, we elucidated osteosarcoma-derived exosomes and exosomal-PD-L1 promoted the pulmonary metastasis in metastatic models. ROC (Receiver Operating Characteristic Curve) analysis showed AUC (Area Under Curve) of 0.823 for exosomal PD-L1, 0.806 for exosomal N-cadherin and 0.817 for exosomal N-cadherin/E-cadherin to distinguish OS patients with pulmonary metastasis from ones without metastasis. Conclusions Osteosarcoma stimulates pulmonary metastasis by releasing exosomes, that carry PD-L1 and N-cadherin. Detection of exosomal PD-L1 and N-cadherin from serum of OS patients may predict pulmonary metastasis progression for OS patients.  

3.3623           Single-nucleus transcriptome analysis reveals dysregulation of angiogenic endothelial cells and neuroprotective glia in Alzheimer’s disease

Lau, S-F., Cao, H., Fu, A.K.Y. and Ip, N.Y. PNAS, 117(41), 25800-25809 (2020)   Alzheimer’s disease (AD) is the most common form of dementia but has no effective treatment. A comprehensive investigation of cell type-specific responses and cellular heterogeneity in AD is required to provide precise molecular and cellular targets for therapeutic development. Accordingly, we perform single-nucleus transcriptome analysis of 169,496 nuclei from the prefrontal cortical samples of AD patients and normal control (NC) subjects. Differential analysis shows that the cell type-specific transcriptomic changes in AD are associated with the disruption of biological processes including angiogenesis, immune activation, synaptic signaling, and myelination. Subcluster analysis reveals that compared to NC brains, AD brains contain fewer neuroprotective astrocytes and oligodendrocytes. Importantly, our findings show that a subpopulation of angiogenic endothelial cells is induced in the brain in patients with AD. These angiogenic endothelial cells exhibit increased expression of angiogenic growth factors and their receptors (i.e., EGFL7, FLT1, and VWF) and antigen-presentation machinery (i.e., B2M and HLA-E). This suggests that these endothelial cells contribute to angiogenesis and immune response in AD pathogenesis. Thus, our comprehensive molecular profiling of brain samples from patients with AD reveals previously unknown molecular changes as well as cellular targets that potentially underlie the functional dysregulation of endothelial cells, astrocytes, and oligodendrocytes in AD, providing important insights for therapeutic development.  

3.3624           Staphylococcus aureus secretes immunomodulatory RNA and DNA via membrane vesicles

Rodriguez, B.V. and Kuehn, M.J. Scientific Reports, 10:28293 (2020)   Bacterial-derived RNA and DNA can function as ligands for intracellular receptor activation and induce downstream signaling to modulate the host response to bacterial infection. The mechanisms underlying the secretion of immunomodulatory RNA and DNA by pathogens such as Staphylococcus aureus and their delivery to intracellular host cell receptors are not well understood. Recently, extracellular membrane vesicle (MV) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. S. aureus produce membrane-bound, spherical, nano-sized, MVs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. Here we show that S. aureus secretes RNA and DNA molecules that are mostly protected from degradation by their association with MVs. Importantly, we demonstrate that MVs can be delivered into cultured macrophage cells and subsequently stimulate a potent IFN-β response in recipient cells via activation of endosomal Toll-like receptors. These findings advance our understanding of the mechanisms by which bacterial nucleic acids traffic extracellularly to trigger the modulation of host immune responses.  

3.3625           Cholesterol Efflux-Independent Modification of Lipid Rafts by AIBP (Apolipoprotein A-I Binding Protein)

Low, H., Mukhamedova, N., Capettini, L.S.A., Xia, Y., Carmichael, I., Cody, S.H., Huynh, K., Ditiatkovski, M., Ohkawa, R., Bukrinsky, M., Meikle, P.J., Choi, S-H., Field, S., Miller, U.I. and Sviridov, D. Arterioscler. Thromb. Vasc. Biol., 40, 2346-2359 (2020)   Objective: AIBP (apolipoprotein A-I binding protein) is an effective and selective regulator of lipid rafts modulating many metabolic pathways originating from the rafts, including inflammation. The mechanism of action was suggested to involve stimulation by AIBP of cholesterol efflux, depleting rafts of cholesterol, which is essential for lipid raft integrity. Here we describe a different mechanism contributing to the regulation of lipid rafts by AIBP. Approach and Results: We demonstrate that modulation of rafts by AIBP may not exclusively depend on the rate of cholesterol efflux or presence of the key regulator of the efflux, ABCA1 (ATP-binding cassette transporter A-I). AIBP interacted with phosphatidylinositol 3-phosphate, which was associated with increased abundance and activation of Cdc42 and rearrangement of the actin cytoskeleton. Cytoskeleton rearrangement was accompanied with reduction of the abundance of lipid rafts, without significant changes in the lipid composition of the rafts. The interaction of AIBP with phosphatidylinositol 3-phosphate was blocked by AIBP substrate, NADPH (nicotinamide adenine dinucleotide phosphate), and both NADPH and silencing of Cdc42 interfered with the ability of AIBP to regulate lipid rafts and cholesterol efflux. Conclusions: Our findings indicate that an underlying mechanism of regulation of lipid rafts by AIBP involves PIP-dependent rearrangement of the cytoskeleton.  

3.3626           Comparative Lipidomic Analysis of Extracellular Vesicles Derived from Lactobacillus plantarum APsulloc 331261 Living in Green Tea Leaves Using Liquid Chromatography-Mass Spectrometry

Kim, H., Kim, M., Myoung, K., Kim, W., Ko, J., Kim, K.P. and Cho, E-G. Int. J. Mol. Sci., 21:8076 (2020)   Lactobacillus plantarum is a popular probiotic species due to its safe and beneficial effects on humans; therefore, novel L. plantarum strains have been isolated and identified from various dietary products. Given that bacteria-derived extracellular vesicles (EVs) have been considered as efficient carriers of bioactive materials and shown to evoke cellular responses effectively, L. plantarum-derived EVs are expected to efficiently elicit health benefits. Herein, we identified L. plantarum APsulloc 331261 living in green tea leaves and isolated EVs from the culture medium. We performed quantitative lipidomic analysis of L. plantarum APsulloc 331261 derived EVs (LEVs) using liquid chromatography-mass spectrometry. In comparison to L. plantarum APsulloc 331261, in LEVs, 67 of 320 identified lipid species were significantly increased and 19 species were decreased. In particular, lysophosphatidylserine(18:4) and phosphatidylcholine(32:2) were critically increased, showing over 21-fold enrichment in LEVs. In addition, there was a notable difference between LEVs and the parent cells in the composition of phospholipids. Our results suggest that the lipidomic profile of bacteria-derived EVs is different from that of the parent cells in phospholipid content and composition. Given that lipids are important components of EVs, quantitative and comparative analyses of EV lipids may improve our understanding of vesicle biogenesis and lipid-mediated intercellular communication within or between living organisms.  

3.3627           An optimized method for plasma extracellular vesicles isolation to exclude the copresence of biological drugs and plasma proteins which impairs their biological characterization

Arntz, O.J., Pieters, B.C., van Lent, P.L.E.M., Koenders, M.I., van der Kraan, P.M. and van de Loo, F.A.J. Plos One, 15(7), e0236508 (2020)   Extracellular vesicles (EVs) are cell membrane-derived phospholipid bilayer nanostructures that contain bioactive proteins, enzymes, lipids and polymers of nucleotides. They play a role in intercellular communication and are present in body fluids. EVs can be isolated by methods like ultracentrifugation (UC), polyethylene-glycol-precipitation (PEG) or size exclusion chromatography (SEC). The co-presence of immunoglobulins (Ig) in EV samples isolated from plasma (pEVs) is often reported and this may influence the assessment of the biological function and phenotype of EVs in bio- and immunoassay. Here, we studied the presence of an Ig-based therapeutic (etanercept) in pEV samples isolated from rheumatoid arthritis (RA) patients and improved the isolation method to obtain purer pEVs. From plasma of etanercept (Tumor-necrosis-factor (TNF)-α antibodies)-treated RA patients pEVs were isolated by either UC, PEG or SEC. SEC isolated pEVs showed the highest particle-to-protein ratio. Strong TNF-α inhibition determined in a TNF-α sensitive reporter assay was observed by pEVs isolated by UC and PEG, and to a lesser extent by SEC, suggesting the presence of functional etanercept. SEC isolation of etanercept or labelled immunoglobulin G (IgG) showed co-isolation of these antibodies in the pEV fraction in the presence of plasma or a high protein (albumin) concentration. To minimize the presence of etanercept or immunoglobulins, we extended SEC (eSEC) column length from 56mm to 222mm (total stacking volume unchanged). No effect on the amount of isolated pEVs was observed while protein and IgG content were markedly reduced (90%). Next, from six etanercept- treated RA patients, pEVs were isolated on a eSEC or standard SEC column, in parallel. TNF-α inhibition was again observed in pEVs isolated by conventional SEC but not by eSEC. To confirm the purer pEVs isolated by eSEC the basal IL-8 promoter activation in human monocytes was determined and in 4 out of 5 SEC isolated pEVs activation was observed while eSEC isolated pEVs did not. This study shows that extended SEC columns yielded pEVs without detectable biologicals and with low protein and IgG levels. This isolation method will improve the characterization of pEVs as potential biomarkers and mediators of disease.  

3.3628           Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation

Useckaite, Z., Mukhopadhya, A., Moran, B. and O’Driscoll, L. Scientific Reports, 10:19020 (2020)   MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary.  

3.3629           A Comprehensive Subcellular Atlas of the Toxoplasma Proteome via hyperLOPIT Provides Spatial Context for Protein Functions

Barylyuk, K., Koreny, L., Ke, H., Pain, A., Lilley, K.S. and Waller, R.F. Cell Host & Microbe, 28, 752-766 (2020)   Apicomplexan parasites cause major human disease and food insecurity. They owe their considerable success to highly specialized cell compartments and structures. These adaptations drive their recognition, nondestructive penetration, and elaborate reengineering of the host’s cells to promote their growth, dissemination, and the countering of host defenses. The evolution of unique apicomplexan cellular compartments is concomitant with vast proteomic novelty. Consequently, half of apicomplexan proteins are unique and uncharacterized. Here, we determine the steady-state subcellular location of thousands of proteins simultaneously within the globally prevalent apicomplexan parasite Toxoplasma gondii. This provides unprecedented comprehensive molecular definition of these unicellular eukaryotes and their specialized compartments, and these data reveal the spatial organizations of protein expression and function, adaptation to hosts, and the underlying evolutionary trajectories of these pathogens.  

3.3630           Chromatin Potential Identified by Shared Single-Cell Profiling of RNA and Chromatin

Ma, S., Zhang, B., LaFavre, L.M., Hsu, Y-C., Regev, A. and Buentostro, J.D: Cell, 183, 1103-1116 (2020)   Cell differentiation and function are regulated across multiple layers of gene regulation, including modulation of gene expression by changes in chromatin accessibility. However, differentiation is an asynchronous process precluding a temporal understanding of regulatory events leading to cell fate commitment. Here we developed simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq), a highly scalable approach for measurement of chromatin accessibility and gene expression in the same single cell, applicable to different tissues. Using 34,774 joint profiles from mouse skin, we develop a computational strategy to identify cis-regulatory interactions and define domains of regulatory chromatin (DORCs) that significantly overlap with super-enhancers. During lineage commitment, chromatin accessibility at DORCs precedes gene expression, suggesting that changes in chromatin accessibility may prime cells for lineage commitment. We computationally infer chromatin potential as a quantitative measure of chromatin lineage-priming and use it to predict cell fate outcomes. SHARE-seq is an extensible platform to study regulatory circuitry across diverse cells in tissues.  

3.3631           Mechanosensitivity is an essential component of phototransduction in vertebrate rods

Bocchero, U., Falleroni, F., Motal, S., Li, Y., Cojoc, D., Lamb, T. and Torre, V. PloS Biology, 18(7), e3000750 (2020)   Photoreceptors are specialized cells devoted to the transduction of the incoming visual signals. Rods are able also to shed from their tip old disks and to synthesize at the base of the outer segment (OS) new disks. By combining electrophysiology, optical tweezers (OTs), and biochemistry, we investigate mechanosensitivity in the rods of XenopC19orf66 interus laevis, and we show that 1) mechanosensitive channels (MSCs), transient receptor potential canonical 1 (TRPC1), and Piezo1 are present in rod inner segments (ISs); 2) mechanical stimulation—of the order of 10 pN—applied briefly to either the OS or IS evokes calcium transients; 3) inhibition of MSCs decreases the duration of photoresponses to bright flashes; 4) bright flashes of light induce a rapid shortening of the OS; and 5) the genes encoding the TRPC family have an ancient association with the genes encoding families of protein involved in phototransduction. These results suggest that MSCs play an integral role in rods’ phototransduction.  

3.3632           Tau Pathology Drives Dementia Risk-Associated Gene Networks toward Chronic Inflammatory States and Immunosuppression

Rexach, J., Polioudakis, D., Yin, A., Trojanowski, J.Q., Malhotra, D. and Geschwind, D.H. Cell Reports, 33, 108398 (2020)   To understand how neural-immune-associated genes and pathways contribute to neurodegenerative disease pathophysiology, we performed a systematic functional genomic analysis in purified microglia and bulk tissue from mouse and human AD, FTD, and PSP. We uncover a complex temporal trajectory of microglial-immune pathways involving the type 1 interferon response associated with tau pathology in the early stages, followed by later signatures of partial immune suppression and, subsequently, the type 2 interferon response. We find that genetic risk for dementias shows disease-specific patterns of pathway enrichment. We identify drivers of two gene co-expression modules conserved from mouse to human, representing competing arms of microglial-immune activation (NAct) and suppression (NSupp) in neurodegeneration. We validate our findings by using chemogenetics, experimental perturbation data, and single-cell sequencing in post-mortem brains. Our results refine the understanding of stage- and disease-specific microglial responses, implicate microglial viral defense pathways in dementia pathophysiology, and highlight therapeutic windows.  

3.3633           An OMV-Based Nanovaccine Confers Safety and Protection against Pathogenic Escherichia coli via Both Humoral and Predominantly Th1 Immune Responses in Poultry

Hu, R., Liu, H., Wang, M., Li, J., Lin, H., Liang, M., Gao, Y. and Yang, M. Nanomaterials, 10:2293 (2020)   Avian pathogenic Escherichia coli (APEC) infection in poultry causes enormous economic losses and public health risks. Bacterial outer membrane vesicles (OMVs) and nano-sized proteolipids enriched with various immunogenic molecules have gained extensive interest as novel nanovaccines against bacterial infections. In this study, after the preparation of APEC O2-derived OMVs (APEC_OMVs) using the ultracentrifugation method and characterization of them using electron microscopy and nanoparticle tracking analyses, we examined the safety and vaccination effect of APEC_OMVs in broiler chicks and investigated the underlying immunological mechanism of protection. The results showed that APEC_OMVs had membrane-enclosed structures with an average diameter of 89 nm. Vaccination with 50 μg of APEC_OMVs had no side effects and efficiently protected chicks against homologous infection. APEC_OMVs could be effectively taken up by chicken macrophages and activated innate immune responses in macrophages in vitro. APEC_OMV vaccination significantly improved activities of serum non-specific immune factors, enhanced the specific antibody response and promoted the proliferation of splenic and peripheral blood lymphocytes in response to mitogen. Furthermore, APEC_OMVs also elicited a predominantly IFN-γ-mediated Th1 response in splenic lymphocytes. Our data revealed the involvement of both non-specific immune responses and specific antibody and cytokine responses in the APEC_OMV-mediated protection, providing broader knowledge for the development of multivalent APEC_OMV-based nanovaccine with high safety and efficacy in the future.  

3.3634           The Rab5 activator RME-6 is required for amyloid precursor protein endocytosis depending on the YTSI motif

Eggert, S., Gruebl, T., Rajender, R., Rupp, C., Sander, B., Heesch, A., Zimmermann, M., Hoepfner, S., Zentgraf, H. and Kins, S. Cell. Mol. Life Sci., 77, 5223-5242 (2020)   Endocytosis of the amyloid precursor protein (APP) is critical for generation of β-amyloid, aggregating in Alzheimer's disease. APP endocytosis depending on the intracellular NPTY motif is well investigated, whereas involvement of the YTSI (also termed BaSS) motif remains controversial. Here, we show that APP lacking the YTSI motif (ΔYTSI) displays reduced localization to early endosomes and decreased internalization rates, similar to APP ΔNPTY. Additionally, we show that the YTSI-binding protein, PAT1a interacts with the Rab5 activator RME-6, as shown by several independent assays. Interestingly, knockdown of RME-6 decreased APP endocytosis, whereas overexpression increased the same. Similarly, APP ΔNPTY endocytosis was affected by PAT1a and RME-6 overexpression, whereas APP ΔYTSI internalization remained unchanged. Moreover, we could show that RME-6 mediated increase of APP endocytosis can be diminished upon knocking down PAT1a. Together, our data identify RME-6 as a novel player in APP endocytosis, involving the YTSI-binding protein PAT1a.  

3.3635           Endosomal Dysfunction Induced by Directly Overactivating Rab5 Recapitulates Prodromal and Neurodegenerative Features of Alzheimer’s Disease

Pensalfini, A., Kim, S., Subbanna, S., Smiley, J.F., Basavarajappa, B.S. and Nixon, R.A. Cell Reports, 33, 108420 (2020)   Neuronal endosomal dysfunction, the earliest known pathobiology specific to Alzheimer’s disease (AD), is mediated by the aberrant activation of Rab5 triggered by APP-β secretase cleaved C-terminal fragment (APP-βCTF). To distinguish pathophysiological consequences specific to overactivated Rab5 itself, we activate Rab5 independently from APP-βCTF in the PA-Rab5 mouse model. We report that Rab5 overactivation alone recapitulates diverse prodromal and degenerative features of AD. Modest neuron-specific transgenic Rab5 expression inducing hyperactivation of Rab5 comparable to that in AD brain reproduces AD-related Rab5-endosomal enlargement and mistrafficking, hippocampal synaptic plasticity deficits via accelerated AMPAR endocytosis and dendritic spine loss, and tau hyperphosphorylation via activated glycogen synthase kinase-3β. Importantly, Rab5-mediated endosomal dysfunction induces progressive cholinergic neurodegeneration and impairs hippocampal-dependent memory. Aberrant neuronal Rab5-endosome signaling, therefore, drives a pathogenic cascade distinct from β-amyloid-related neurotoxicity, which includes prodromal and neurodegenerative features of AD, and suggests Rab5 overactivation as a potential therapeutic target.  

3.3636           Growing pains: addressing the pitfalls of plant extracellular vesicle research

Rutter, B.D. and Innes, R.W. New Phytologist, 228, 1505-1510 (2020)   Extracellular vesicles (EVs) are small, membrane‐enclosed compartments that mediate the intercellular transport of proteins and small RNAs. In plants, EVs are thought to play a prominent role in immune responses and are being championed as the long‐sought‐after mechanism for host‐induced gene silencing. However, parallel research on mammalian EVs is raising concerns about potential pitfalls faced by all EV researchers that will need to be addressed in order to convincingly establish that EVs are the primary mediators of small RNA transfer between organisms. Here we discuss these pitfalls in the context of plant EV research, with a focus on experimental approaches required to distinguish bona fide EV cargo from merely co‐purifying contaminants.  

3.3637           Spatial proteomics defines the content of trafficking vesicles captured by golgin tethers

Shin, J.J.H., Crook, O.M., Borgeaud, A.C., Cattin-Ortola, J., Peak-Chew, S.Y., Breckels, L.M., Gillingham, A.K., Chadwick, J., Lilley, K.S. and Munro, S. Nature Communications, 11:5987 (2020)   Intracellular traffic between compartments of the secretory and endocytic pathways is mediated by vesicle-based carriers. The proteomes of carriers destined for many organelles are ill-defined because the vesicular intermediates are transient, low-abundance and difficult to purify. Here, we combine vesicle relocalisation with organelle proteomics and Bayesian analysis to define the content of different endosome-derived vesicles destined for the trans-Golgi network (TGN). The golgin coiled-coil proteins golgin-97 and GCC88, shown previously to capture endosome-derived vesicles at the TGN, were individually relocalised to mitochondria and the content of the subsequently re-routed vesicles was determined by organelle proteomics. Our findings reveal 45 integral and 51 peripheral membrane proteins re-routed by golgin-97, evidence for a distinct class of vesicles shared by golgin-97 and GCC88, and various cargoes specific to individual golgins. These results illustrate a general strategy for analysing intracellular sub-proteomes by combining acute cellular re-wiring with high-resolution spatial proteomics.  

3.3638           Isolation and characterization of exosomes for cancer research

Zhu, L., Sun, H-T., Wang, S., Huang, S-L., Zheng, Y., Wang, C-Q., Hu, B-Y. et al

1. Hematol. Oncol., 13:152 (2020)

  Exosomes are a subset of extracellular vesicles that carry specific combinations of proteins, nucleic acids, metabolites, and lipids. Mounting evidence suggests that exosomes participate in intercellular communication and act as important molecular vehicles in the regulation of numerous physiological and pathological processes, including cancer development. Exosomes are released by various cell types under both normal and pathological conditions, and they can be found in multiple bodily fluids. Moreover, exosomes carrying a wide variety of important macromolecules provide a window into altered cellular or tissue states. Their presence in biological fluids renders them an attractive, minimally invasive approach for liquid biopsies with potential biomarkers for cancer diagnosis, prediction, and surveillance. Due to their biocompatibility and low immunogenicity and cytotoxicity, exosomes have potential clinical applications in the development of innovative therapeutic approaches. Here, we summarize recent advances in various technologies for exosome isolation for cancer research. We outline the functions of exosomes in regulating tumor metastasis, drug resistance, and immune modulation in the context of cancer development. Finally, we discuss prospects and challenges for the clinical development of exosome-based liquid biopsies and therapeutics.  

3.3639           Stimulator of interferon genes (STING) is an essential proviral host factor for human rhinovirus species A and C

McKnight, K.L., Swanson, K.V., Austgen, K., Richards, C., Mitchell, J.K., McGivern, D.R., Fritch, E., Johnson, J., Remlinger, K., Magid-Slav, M., Kapustina, M., You, S. and Lemon, S.M. PNAS, 117(44), 27598-27607 (2020)   Human rhinoviruses (RVs) are positive-strand RNA viruses that cause respiratory tract disease in children and adults. Here we show that the innate immune signaling protein STING is required for efficient replication of members of two distinct RV species, RV-A and RV-C. The host factor activity of STING was identified in a genome-wide RNA interference (RNAi) screen and confirmed in primary human small airway epithelial cells. Replication of RV-A serotypes was strictly dependent on STING, whereas RV-B serotypes were notably less dependent. Subgenomic RV-A and RV-C RNA replicons failed to amplify in the absence of STING, revealing it to be required for a step in RNA replication. STING was expressed on phosphatidylinositol 4-phosphate (PI4P)-enriched membranes and was enriched in RV-A16 compared with RV-B14 replication organelles isolated in isopycnic gradients. The host factor activity of STING was species-specific, as murine STING (mSTING) did not rescue RV-A16 replication in STING-deficient cells. This species specificity mapped primarily to the cytoplasmic, ligand-binding domain of STING. Mouse-adaptive mutations in the RV-A16 2C protein allowed for robust replication in cells expressing mSTING, suggesting a role for 2C in recruiting STING to RV-A replication organelles. Palmitoylation of STING was not required for RV-A16 replication, nor was the C-terminal tail of STING that mediates IRF3 signaling. Despite co-opting STING to promote its replication, interferon signaling in response to STING agonists remained intact in RV-A16 infected cells. These data demonstrate a surprising requirement for a key host mediator of innate immunity to DNA viruses in the life cycle of a small pathogenic RNA virus.  

3.3640           Cerebellar and hepatic alterations in ACBD5-deficient mice are associated with unexpected, distinct alterations in cellular lipid homeostasis

Darwisch, W., von Spangenberg, M., Lehmann, J., Singin, Ö., Deubert, G., Kåuhl, S. et al Communications Biology, 3:713 (2020)   ACBD5 deficiency is a novel peroxisome disorder with a largely uncharacterized pathology. ACBD5 was recently identified in a tethering complex mediating membrane contacts between peroxisomes and the endoplasmic reticulum (ER). An ACBD5-deficient mouse was analyzed to correlate ACBD5 tethering functions with the disease phenotype. ACBD5-deficient mice exhibit elevated very long-chain fatty acid levels and a progressive cerebellar pathology. Liver did not exhibit pathologic changes but increased peroxisome abundance and drastically reduced peroxisome-ER contacts. Lipidomics of liver and cerebellum revealed tissue-specific alterations in distinct lipid classes and subspecies. In line with the neurological pathology, unusual ultra-long chain fatty acids (C > 32) were elevated in phosphocholines from cerebelli but not liver indicating an organ-specific imbalance in fatty acid degradation and elongation pathways. By contrast, ether lipid formation was perturbed in liver towards an accumulation of alkyldiacylglycerols. The alterations in several lipid classes suggest that ACBD5, in addition to its acyl-CoA binding function, might maintain peroxisome-ER contacts in order to contribute to the regulation of anabolic and catabolic cellular lipid pathways.  

3.3641           Paxillin mediates ATP-induced activation of P2X7 receptor and NLRP3 inflammasome

Wang, W., Hu, D., Feng, Y., Wu, C., Song, Y., Liu, W., Li, A. et al BMC Biology, 18:182 (2020)   Background Extracellular adenosine triphosphate (ATP), a key danger-associated molecular pattern (DAMP) molecule, is released to the extracellular medium during inflammation by injured parenchymal cells, dying leukocytes, and activated platelets. ATP directly activates the plasma membrane channel P2X7 receptor (P2X7R), leading to an intracellular influx of K⁺, a key trigger inducing NLRP3 inflammasome activation. However, the mechanism underlying P2X7R-mediated activation of NLRP3 inflammasome is poorly understood, and additional molecular mediators have not been identified. Here, we demonstrate that Paxillin is the molecule connecting the P2X7 receptor and NLRP3 inflammasome through protein interactions. Results We show a distinct mechanism by which Paxillin promotes ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome. Extracellular ATP induces Paxillin phosphorylation and then facilitates Paxillin-NLRP3 interaction. Interestingly, Paxillin enhances NLRP3 deubiquitination and activates NLRP3 inflammasome upon ATP treatment and K⁺ efflux. Moreover, we demonstrated that USP13 is a key enzyme for Paxillin-mediated NLRP3 deubiquitination upon ATP treatment. Notably, extracellular ATP promotes Paxillin and NLRP3 migration from the cytosol to the plasma membrane and facilitates P2X7R-Paxillin interaction and PaxillinNLRP3 association, resulting in the formation of the P2X7R-Paxillin-NLRP3 complex. Functionally, Paxillin is essential for ATP-induced NLRP3 inflammasome activation in mouse BMDMs and BMDCs as well as in human PBMCs and THP-1-differentiated macrophages. Conclusions We have identified paxillin as a mediator of NLRP3 inflammasome activation. Paxillin plays key roles in ATP-induced activation of the P2X7 receptor and NLRP3 inflammasome by facilitating the formation of the P2X7R-Paxillin-NLRP3 complex.  

3.3642           Abiotic stressors impact outer membrane vesicle composition in a beneficial rhizobacterium: Raman spectroscopy characterization

Potter, M., Hanson, C., Anderson, A.J., Vargis, E. and Britt, D.W. Scientific Reports, 10:21289 (2020)   Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have roles in cell-to-cell signaling, biofilm formation, and stress responses. Here, the effects of abiotic stressors on OMV contents and composition from biofilm cells of the plant health-promoting bacterium Pseudomonas chlororaphis O6 (PcO6) are examined. Two stressors relevant to this root-colonizing bacterium were examined: CuO nanoparticles (NPs)-a potential fertilizer and fungicide- and H₂O₂-released from roots during plant stress responses. Atomic force microscopy revealed 40–300 nm diameter OMVs from control and stressed biofilm cells. Raman spectroscopy with linear discriminant analysis (LDA) was used to identify changes in chemical profiles of PcO6 cells and resultant OMVs according to the cellular stressor with 84.7% and 83.3% accuracies, respectively. All OMVs had higher relative concentrations of proteins, lipids, and nucleic acids than PcO6 cells. The nucleic acid concentration in OMVs exhibited a cellular stressor-dependent increase: CuO NP-induced OMVs > H₂O₂-induced OMVs > control OMVs. Biochemical assays confirmed the presence of lipopolysaccharides, nucleic acids, and protein in OMVs; however, these assays did not discriminate OMV composition according to the cellular stressor. These results demonstrate the sensitivity of Raman spectroscopy using LDA to characterize and distinguish cellular stress effects on OMVs composition and contents.  

3.3643           Calcium‐Mediated Liposome Fusion to Engineer Giant Lipid Vesicles with Cytosolic Proteins and Reconstituted Mammalian Proteins

Schmid, Y.R.F., Scheller, L., Buchmann, S. and Dittrich, P.S. Adv. Biosys., 4, 2000153 (2020)   Giant unilamellar lipid vesicles (GUVs) are widely used as model membrane systems and provide an excellent basis to construct artificial cells. To construct more sophisticated artificial cells, proteins—in particular membrane proteins—need to be incorporated in GUVs. However, current methods for protein reconstitution have limited throughput or are not generally applicable for all proteins because they depend on detergent solubilization. This limitation is addressed here by introducing calcium‐mediated membrane fusion to transfer proteins between negatively charged GUVs and cell‐derived plasma membrane vesicles (CDVs), derived from HEK293T cells overexpressing a membrane receptor protein. Fusion conditions are optimized using large unilamellar vesicles and GUVs containing phosphatidylserines and fusogenic lipids. The approach is then applied to induce lipid mixing and subsequent transfer of the overexpressed membrane receptor from CDVs into GUVs. The membrane receptor is detected by immunofluorescence on GUVs that underwent lipid mixing with CDVs. Those GUVs also exhibit esterase activity because cytosolic esterases entrapped in the CDVs are transferred during membrane fusion. Thus, content mixing is demonstrated. Using CDVs circumvents the need to purify or solubilize proteins. Moreover, calcium‐mediated fusion allows transfer of lipids, water‐soluble and membrane bound proteins in one step, resulting in a semi‐synthetic cell.  

3.3644           Human multipotent mesenchymal stromal cells cytokine priming promotes RAB27B-regulated secretion of small extracellular vesicles with immunomodulatory cargo

Cheng, A., Choi, D., Lora, M., Shum-Tim, D., Rak, J. and Colmegna, I. Stem Cell Res. Ther., 11:539 (2020)   Background The paracrine effects of multipotent mesenchymal stromal cells (MSCs) are mediated by their secretome composed by soluble factors (i.e., cytokines, growth factors, hormones) and extracellular vesicles (EVs). EVs promote intercellular communication, and the EV cargoes [e.g., proteins, soluble factors, microRNAs (miRNAs), messenger RNA (mRNA), DNA] reflect the molecular and functional characteristics of their parental cells. MSC-derived EVs (MSC-EVs) are currently evaluated as subcellular therapeutics. A key function of the MSC secretome is its ability to promote immune tolerance (i.e., immunopotency), a property that is enhanced by priming approaches (e.g., cytokines, hypoxia, chemicals) and inversely correlates with the age of the MSC donors. We evaluated mechanisms underlying MSC vesiculation and the effects of inflammation and aging on this process. Methods We evaluated the effects of interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) on human adipose-derived MSC: (a) vesiculation (custom RT² Profiler PCR Array), (b) EV profiles (Nanoparticle Tracking Analysis and Nanoparticle Flow Cytometry), (c) EV cargo (proteomic analysis and Western blot analysis), and (d) immunopotency (standard MSC:CD4 T cell proliferation inhibition assay). We confirmed the role of RAB27B on MSC vesiculation (RAB27B siRNA) and assessed its differential contribution to vesiculation in adult and pediatric MSCs (qPCR). Results Cytokine priming upregulated RAB27B in adipose-derived MSCs increasing their secretion of exosome-like small EVs (sEVs; < 200 nm) containing two key mediators of immunopotency: A20 and TSG-6. These EVs inhibited T cell proliferation in a dose-dependent manner. RAB27B siRNA inhibited MSC vesiculation. Adipose-derived MSCs isolated from pediatric donors exhibited higher RAB27B expression and secreted more sEVs than adult MSCs. Conclusions Cytokine priming is a useful strategy to harvest anti-inflammatory MSC-sEVs for clinical applications. Of relevance, donor age should be considered in the selection of MSC-sEVs for clinical applications.  

3.3645           Function of the SNARE Ykt6 on autophagosomes requires the Dsl1 complex and the Atg1 kinase complex

Gao, J., Kurre, R., Rose, J., Walter, S., Fröhlich, F., Piehler, j., Reggion, F. and Ungermann, C. EMBO Reports, 21:e50733 (2020)   The mechanism and regulation of fusion between autophagosomes and lysosomes/vacuoles are still only partially understood in both yeast and mammals. In yeast, this fusion step requires SNARE proteins, the homotypic vacuole fusion and protein sorting (HOPS) tethering complex, the RAB7 GTPase Ypt7, and its guanine nucleotide exchange factor (GEF) Mon1‐Ccz1. We and others recently identified Ykt6 as the autophagosomal SNARE protein. However, it has not been resolved when and how lipid‐anchored Ykt6 is recruited onto autophagosomes. Here, we show that Ykt6 is recruited at an early stage of the formation of these carriers through a mechanism that depends on endoplasmic reticulum (ER)‐resident Dsl1 complex and COPII‐coated vesicles. Importantly, Ykt6 activity on autophagosomes is regulated by the Atg1 kinase complex, which inhibits Ykt6 through direct phosphorylation. Thus, our findings indicate that the Ykt6 pool on autophagosomal membranes is kept inactive by Atg1 phosphorylation, and once an autophagosome is ready to fuse with vacuole, Ykt6 dephosphorylation allows its engagement in the fusion event.  

3.3646           Daxx maintains endogenous retroviral silencing and restricts cellular plasticity in vivo

Wasylishen, A.R., Sun, C., Moyer, S.M., Qi, Y., Chau, G.P., Aryal, N.K., McAllister, F., Kim, M.P., Barton, M.C., Estrella, J.S., Su, X. and Lozano, G. Sci. Adv., 6:eaba8415 (2020)   Tumor sequencing studies have emphasized the role of epigenetics and altered chromatin homeostasis in cancer. Mutations in DAXX, which encodes a chaperone for the histone 3.3 variant, occur in 25% of pancreatic neuroendocrine tumors (PanNETs). To advance our understanding of physiological functions of Daxx, we developed a conditional Daxx allele in mice. We demonstrate that Daxx loss is well tolerated in the pancreas but creates a permissive transcriptional state that cooperates with environmental stress (inflammation) and other genetic lesions (Men1 loss) to alter gene expression and cell state, impairing pancreas recovery from inflammatory stress in vivo. The transcriptional changes are associated with dysregulation of endogenous retroviral elements (ERVs), and dysregulation of endogenous genes near ERVs is also observed in human PanNETs with DAXX mutations. Our results reveal a physiologic function of DAXX, provide a mechanism associated with impaired tissue regeneration and tumorigenesis, and expand our understanding of ERV regulation in somatic cells.  

3.3647           Nanovesicles derived from iron oxide nanoparticles–incorporated mesenchymal stem cells for cardiac repair

Lee, J-R., Park, B-W., Kim, J., Choo, Y.W., Kim, H.Y., Yoon, J-K. et al Sci. Adv., 6:eaaz0952 (2020)   Because of poor engraftment and safety concerns regarding mesenchymal stem cell (MSC) therapy, MSC-derived exosomes have emerged as an alternative cell-free therapy for myocardial infarction (MI). However, the diffusion of exosomes out of the infarcted heart following injection and the low productivity limit the potential of clinical applications. Here, we developed exosome-mimetic extracellular nanovesicles (NVs) derived from iron oxide nanoparticles (IONPs)–incorporated MSCs (IONP-MSCs). The retention of injected IONP-MSC–derived NVs (IONP-NVs) within the infarcted heart was markedly augmented by magnetic guidance. Furthermore, IONPs significantly increased the levels of therapeutic molecules in IONP-MSCs and IONP-NVs, which can reduce the concern of low exosome productivity. The injection of IONP-NVs into the infarcted heart and magnetic guidance induced an early shift from the inflammation phase to the reparative phase, reduced apoptosis and fibrosis, and enhanced angiogenesis and cardiac function recovery. This approach can enhance the therapeutic potency of an MSC-derived NV therapy.  

3.3648           Paracrine Mechanisms of Mesenchymal Stromal Cells in Angiogenesis

Maacha, S., Sidahmed, H., Jacob, S., Gentilcore, G., Calzone, R., Grivel, J-C. and Cugno, C. Stem Cells Int., 2020:4356359 (2020)   The role of the mesenchymal stromal cell- (MSC-) derived secretome is becoming increasingly intriguing from a clinical perspective due to its ability to stimulate endogenous tissue repair processes as well as its effective regulation of the immune system, mimicking the therapeutic effects produced by the MSCs. The secretome is a composite product secreted by MSC in vitro (in conditioned medium) and in vivo (in the extracellular milieu), consisting of a protein soluble fraction (mostly growth factors and cytokines) and a vesicular component, extracellular vesicles (EVs), which transfer proteins, lipids, and genetic material. MSC-derived secretome differs based on the tissue from which the MSCs are isolated and under specific conditions (e.g., preconditioning or priming) suggesting that clinical applications should be tailored by choosing the tissue of origin and a priming regimen to specifically correct a given pathology. MSC-derived secretome mediates beneficial angiogenic effects in a variety of tissue injury-related diseases. This supports the current effort to develop cell-free therapeutic products that bring both clinical benefits (reduced immunogenicity, persistence in vivo, and no genotoxicity associated with long-term cell cultures) and manufacturing advantages (reduced costs, availability of large quantities of off-the-shelf products, and lower regulatory burden). In the present review, we aim to give a comprehensive picture of the numerous components of the secretome produced by MSCs derived from the most common tissue sources for clinical use (e.g., AT, BM, and CB). We focus on the factors involved in the complex regulation of angiogenic processes.  

3.3649           Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

Tosar, J.P., Segovia, M., Castellano, M., Gambaro, F., Akiyama, Y., Fagundez, P., Olivera, A., Costa, B., Possi, T., Hill, M., Ivanov, P. and Cayota, A. Nucleic Acids Res., 48(22), 12874-12888 (2020)   A major proportion of extracellular RNAs (exRNAs) do not copurify with extracellular vesicles (EVs) and remain in ultracentrifugation supernatants of cell-conditioned medium or mammalian blood serum. However, little is known about exRNAs beyond EVs. We have previously shown that the composition of the nonvesicular exRNA fraction is highly biased toward specific tRNA-derived fragments capable of forming RNase-protecting dimers. To solve the problem of stability in exRNA analysis, we developed a method based on sequencing the size exclusion chromatography (SEC) fractions of nonvesicular extracellular samples treated with RNase inhibitors (RI). This method revealed dramatic compositional changes in exRNA population when enzymatic RNA degradation was inhibited. We demonstrated the presence of ribosomes and full-length tRNAs in cell-conditioned medium of a variety of mammalian cell lines. Their fragmentation generates some small RNAs that are highly resistant to degradation. The extracellular biogenesis of some of the most abundant exRNAs demonstrates that extracellular abundance is not a reliable input to estimate RNA secretion rates. Finally, we showed that chromatographic fractions containing extracellular ribosomes are probably not silent from an immunological perspective and could possibly be decoded as damage-associated molecular patterns.  

3.3650           Extracellular vesicles: new players in regulating vascular barrier function

Chatterjee, V., Yang, X., Ma, M.H., Wu, M.H. and Yuan, S.Y. Am. J. Physiol. Heart Circ. Physiol., 319(6), H1181-H1196 (2020)   Extracellular vesicles (EVs) have attracted rising interests in the cardiovascular field not only because they serve as serological markers for circulatory disorders but also because they participate in important physiological responses to stress and inflammation. In the circulation, these membranous vesicles are mainly derived from blood or vascular cells, and they carry cargos with distinct molecular signatures reflecting the origin and activation state of parent cells that produce them, thus providing a powerful tool for diagnosis and prognosis of pathological conditions. Functionally, circulating EVs mediate tissue-tissue communication by transporting bioactive cargos to local and distant sites, where they directly interact with target cells to alter their function. Recent evidence points to the critical contributions of EVs to the pathogenesis of vascular endothelial barrier dysfunction during inflammatory response to injury or infection. In this review, we provide a brief summary of the current knowledge on EV biology and advanced techniques in EV isolation and characterization. This is followed by a discussion focusing on the role and mechanisms of EVs in regulating blood-endothelium interactions and vascular permeability during inflammation. We conclude with a translational perspective on the diagnostic and therapeutic potential of EVs in vascular injury or infectious diseases, such as COVID-19.  

3.3651           SCYL1 arginine methylation by PRMT1 is essential for neurite outgrowth via Golgi morphogenesis

Amano, G., matsuzaki, S., Mori, Y., Miyoshi, K., Han, S., Shikada, S., Takamura, H., Yoshimura, T. and Katayama, T. Mol. Biol. Cell, 31, 1963-1973 (2020)   Arginine methylation is a common posttranslational modification that modulates protein function. SCY1-like pseudokinase 1 (SCYL1) is crucial for neuronal functions and interacts with γ₂-COP to form coat protein complex I (COPI) vesicles that regulate Golgi morphology. However, the molecular mechanism by which SCYL1 is regulated remains unclear. Here, we report that the γ₂-COP-binding site of SCYL1 is arginine-methylated by protein arginine methyltransferase 1 (PRMT1) and that SCYL1 arginine methylation is important for the interaction of SCYL1 with γ₂-COP. PRMT1 was colocalized with SCYL1 in the Golgi fraction. Inhibition of PRMT1 suppressed axon outgrowth and dendrite complexity via abnormal Golgi morphology. Knockdown of SCYL1 by small interfering RNA (siRNA) inhibited axon outgrowth, and the inhibitory effect was rescued by siRNA-resistant SCYL1, but not SCYL1 mutant, in which the arginine methylation site was replaced. Thus, PRMT1 regulates Golgi morphogenesis via SCYL1 arginine methylation. We propose that SCYL1 arginine methylation by PRMT1 contributes to axon and dendrite morphogenesis in neurons.    

3.3652           PAG1 directs SRC-family kinase intracellular localization to mediate receptor tyrosine kinase-induced differentiation

Foltz, L., Palacios-Moreno, J., Mayfield, M., Kinch, S., Dillon, J., Syrenne, J., Levy, T. and Grimes, M. Mol. Biol. Cell, 31(20), 2269-2282 (2020)   All receptor tyrosine kinases (RTKs) activate similar downstream signaling pathways through a common set of effectors, yet it is not fully understood how different receptors elicit distinct cellular responses to cause cell proliferation, differentiation, or other cell fates. We tested the hypothesis that regulation of SRC family kinase (SFK) signaling by the scaffold protein, PAG1, influences cell fate decisions following RTK activation. We generated a neuroblastoma cell line expressing a PAG1 fragment that lacks the membrane-spanning domain (PAG1^TM-) and localized to the cytoplasm. PAG1^TM- cells exhibited higher amounts of active SFKs and increased growth rate. PAG1^TM- cells were unresponsive to TRKA and RET signaling, two RTKs that induce neuronal differentiation, but retained responses to EGFR and KIT. Under differentiation conditions, PAG1^TM- cells continued to proliferate and did not extend neurites or increase β-III tubulin expression. FYN and LYN were sequestered in multivesicular bodies (MVBs), and dramatically more FYN and LYN were in the lumen of MVBs in PAG1^TM- cells. In particular, activated FYN was sequestered in PAG1^TM- cells, suggesting that disruption of FYN localization led to the observed defects in differentiation. The results demonstrate that PAG1 directs SFK intracellular localization to control activity and to mediate signaling by RTKs that induce neuronal differentiation.  

3.3653           Polarized human cholangiocytes release distinct populations of apical and basolateral small extracellular vesicles

Davies, B.A., Morton, L.O., Jefferson, J.R., Rozeveld, C.N., Doskey, L.C., LaRusso, N.F. and Katzmann, D.J. Mol. Biol. Cell., 31(22), 2463-2474 (2020)   Intercellular communication is critical for organismal homeostasis, and defects can contribute to human disease states. Polarized epithelial cells execute distinct signaling agendas via apical and basolateral surfaces to communicate with different cell types. Small extracellular vesicles (sEVs), including exosomes and small microvesicles, represent an understudied form of intercellular communication in polarized cells. Human cholangiocytes, epithelial cells lining bile ducts, were cultured as polarized epithelia in a Transwell system as a model with which to study polarized sEV communication. Characterization of isolated apically and basolaterally released EVs revealed enrichment in sEVs. However, differences in apical and basolateral sEV composition and numbers were observed. Genetic or pharmacological perturbation of cellular machinery involved in the biogenesis of intralumenal vesicles at endosomes (the source of exosomes) revealed general and domain-specific effects on sEV biogenesis/release. Additionally, analyses of signaling revealed distinct profiles of activation depending on sEV population, target cell, and the function of the endosomal sorting complex required for transport (ESCRT)-associated factor ALG-2–interacting protein X (ALIX) within the donor cells. These results support the conclusion that polarized cholangiocytes release distinct sEV pools to mediate communication via their apical and basolateral domains and suggest that defective ESCRT function may contribute to disease states through altered sEV signaling.  

3.3654           Yeast Rgd3 is a phospho-regulated F-BAR–containing RhoGAP involved in the regulation of Rho3 distribution and cell morphology

Gingras, R.M., Lwin, K.M., Miller, A.M. and Bretscher, A. Mol. Biol. Cell, 31(23), 2570-2582 (2020)   Polarized growth requires the integration of polarity pathways with the delivery of exocytic vesicles for cell expansion and counterbalancing endocytic uptake. In budding yeast, the myosin-V Myo2 is aided by the kinesin-related protein Smy1 in carrying out the essential Sec4-dependent transport of secretory vesicles to sites of polarized growth. Overexpression suppressors of a conditional myo2 smy1 mutant identified a novel F-BAR (Fes/CIP4 homology-Bin-Amphiphysin-Rvs protein)-containing RhoGAP, Rgd3, that has activity primarily on Rho3, but also Cdc42. Internally tagged Rho3 is restricted to the plasma membrane in a gradient corresponding to cell polarity that is altered upon Rgd3 overexpression. Rgd3 itself is localized to dynamic polarized vesicles that, while distinct from constitutive secretory vesicles, are dependent on actin and Myo2 function. In vitro Rgd3 associates with liposomes in a PIP₂-enhanced manner. Further, the Rgd3 C-terminal region contains several phosphorylatable residues within a reported SH3-binding motif. An unphosphorylated mimetic construct is active and highly polarized, while the phospho-mimetic form is not. Rgd3 is capable of activating Myo2, dependent on its phospho state, and Rgd3 overexpression rescues aberrant Rho3 localization and cell morphologies seen at the restrictive temperature in the myo2 smy1 mutant. We propose a model where Rgd3 functions to modulate and maintain Rho3 polarity during growth.  

3.3655           The yeast mitophagy receptor Atg32 is ubiquitinated and degraded by the proteasome

Camougrand, N., Vigie, P., Gonzalez, C., Manon, S. and Bhatia-Kissova, I. PloS One, 15(12), e0241576 (2020)   Mitophagy, the process that degrades mitochondria selectively through autophagy, is involved in the quality control of mitochondria in cells grown under respiratory conditions. In yeast, the presence of the Atg32 protein on the outer mitochondrial membrane allows for the recognition and targeting of superfluous or damaged mitochondria for degradation. Post-translational modifications such as phosphorylation are crucial for the execution of mitophagy. In our study we monitor the stability of Atg32 protein in the yeast S. cerevisiae and show that Atg32 is degraded under normal growth conditions, upon starvation or rapamycin treatment. The Atg32 turnover can be prevented by inhibition of the proteasome activity, suggesting that Atg32 is also ubiquitinated. Mass spectrometry analysis of purified Atg32 protein revealed that at least lysine residue in position 282 is ubiquitinated. Interestingly, the replacement of lysine 282 with alanine impaired Atg32 degradation only partially in the course of cell growth, suggesting that additional lysine residues on Atg32 might also be ubiquitinated. Our results provide the foundation to further elucidate the physiological significance of Atg32 turnover and the interplay between mitophagy and the proteasome.  

3.3656           Epstein-Barr virus LMP1 manipulates the content and functions of extracellular vesicles to enhance metastatic potential of recipient cells

Nkosi, D., Sun, L., Duke, L.C. and Meckes, Jr., D.G. PloS Pathogens, 16(12), e1009023 (2020)   Extracellular vesicles (EV) mediate intercellular communication events and alterations in normal vesicle content contribute to function and disease initiation or progression. The ability to package a variety of cargo and transmit molecular information between cells renders EVs important mediators of cell-to-cell crosstalk. Latent membrane protein 1 (LMP1) is a chief viral oncoprotein expressed in most Epstein-Barr virus (EBV)-associated cancers and is released from cells at high levels in EVs. LMP1 containing EVs have been demonstrated to promote cell growth, migration, differentiation, and regulate immune cell function. Despite these significant changes in recipient cells induced by LMP1 modified EVs, the mechanism how this viral oncogene modulates the recipient cells towards these phenotypes is not well understood. We hypothesize that LMP1 alters EV content and following uptake of the LMP1-modified EVs by the recipient cells results in the activation of cell signaling pathways and increased gene expression which modulates the biological properties of recipient cell towards a new phenotype. Our results show that LMP1 expression alters the EV protein and microRNA content packaged into EVs. The LMP1-modified EVs also enhance recipient cell adhesion, proliferation, migration, invasion concomitant with the activation of ERK, AKT, and NF-κB signaling pathways. The LMP1 containing EVs induced transcriptome reprogramming in the recipient cells by altering gene expression of different targets including cadherins, matrix metalloproteinases 9 (MMP9), MMP2 and integrin-α5 which contribute to extracellular matrix (ECM) remodeling. Altogether, our data demonstrate the mechanism in which LMP1-modified EVs reshape the tumor microenvironment by increasing gene expression of ECM interaction proteins.  

3.3657           A sensitive S-Trap-based approach to the analysis of T cell lipid raft proteome

Chhuon, C., Zhang, S-Y., Jung, V., Lewandowski, D., Lipecka, J., Pawlak, A., Sahali, D., Ollero, M. and Huerrera, I.C:

1. Lipid Res., 61811), 1512-1523 (2020)

  The analysis of T cell lipid raft proteome is challenging due to the highly dynamic nature of rafts and the hydrophobic character of raft-resident proteins. We explored an innovative strategy for bottom-up lipid raftomics based on suspension-trapping (S-Trap) sample preparation. Mouse T cells were prepared from splenocytes by negative immunoselection, and rafts were isolated by a detergent-free method and OptiPrep gradient ultracentrifugation. Microdomains enriched in flotillin-1, LAT, and cholesterol were subjected to proteomic analysis through an optimized protocol based on S-Trap and high pH fractionation, followed by nano-LC-MS/MS. Using this method, we identified 2,680 proteins in the raft-rich fraction and established a database of 894 T cell raft proteins. We then performed a differential analysis on the raft-rich fraction from nonstimulated versus anti-CD3/CD28 T cell receptor (TCR)-stimulated T cells. Our results revealed 42 proteins present in one condition and absent in the other. For the first time, we performed a proteomic analysis on rafts from ex vivo T cells obtained from individual mice, before and after TCR activation. This work demonstrates that the proposed method utilizing an S-Trap-based approach for sample preparation increases the specificity and sensitivity of lipid raftomics.  

3.3658           An implanted device enables in vivo monitoring of extracellular vesicle‐mediated spread of pro‐inflammatory mast cell response in mice

Vukman, K.V., Ferencz, A., Feher, D., Juhos, K., Lörinc, P., Visnovitz, T., Koncz, A., Paloczi, K., Seregelyes, G., Försönits, A., Khamari, D., Galinsoga, A., Drahos, L. and Buzas, E.I.

1. Extracell. Vesicles, 10(1), e12023 (2020)

  Mast cells have been shown to release extracellular vesicles (EVs) in vitro. However, EV‐mediated mast cell communication in vivo remains unexplored. Primary mast cells from GFP‐transgenic and wild type mice, were grown in the presence or absence of lipopolysaccharide (LPS), and the secreted EVs were separated from the conditioned media. Mast cell‐derived EVs were next cultured with LPS‐naïve mast cells, and the induction of TNF‐α expression was monitored. In addition, primary mast cells were seeded in diffusion chambers that were implanted into the peritoneal cavities of mice. Diffusion chambers enabled the release of GFP⁺ mast cell‐derived EVs in vivo into the peritoneal cavity. Peritoneal lavage cells were assessed for the uptake of GFP⁺ EVs and for TNF‐α production. In vitro, LPS‐stimulated mast cell‐derived EVs were efficiently taken up by non‐stimulated mast cells, and induced TNF‐α expression in a TLR4, JNK and P38 MAPK dependent manner. In vivo, using implanted diffusion chambers, we confirmed the release and transmission of mast cell‐derived EVs to other mast cells with subsequent induction of TNF‐α expression. These data show an EV‐mediated spreading of pro‐inflammatory response between mast cells, and provide the first in vivo evidence for the biological role of mast cell‐derived EVs.  

3.3659           Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium

Dong, L., Zieren, R.C., Horie, K., Kim, C-J., Mallick, E., Jing, Y., Feng, M., Kiczler, M.D., Green, J., Amend, S.R., Witwer, K.W., de Reijke, T.M., Cho, Y-K., Pienta, K.J. and Xue, W.

1. Extracell. Vesicles, 10(2), e12044 (2020)

  One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios.  

3.3660           Human Cytomegalovirus Utilizes Extracellular Vesicles To Enhance Virus Spread

Streck, N.T., Zhao, Y., Sundstrom, J.M. and buchovich, N.J.

1. Virol., 94(16), e00609-20 (2020)

  Human cytomegalovirus (HCMV) manipulates cellular processes associated with secretory pathways within an infected cell to facilitate efficient viral replication. However, little is known about how HCMV infection alters the surrounding cellular environment to promote virus spread to uninfected cells. Extracellular vesicles (EVs) are key signaling molecules that are commonly altered in numerous disease states. Previous reports have shown that viruses commonly alter EVs, which can significantly impact infection. This study finds that HCMV modulates EV biogenesis machinery through upregulation of the endosomal sorting complex required for transport (ESCRT) proteins. This regulation appears to increase the activity of EV biogenesis, since HCMV-infected fibroblasts have increased vesicle release and altered vesicle size compared to EVs from uninfected cells. EVs generated through ESCRT-independent pathways are also beneficial to virus spread in fibroblasts, as treatment with the EV inhibitor GW4869 slowed the efficiency of HCMV spread. Importantly, the transfer of EVs purified from HCMV-infected cells enhanced virus spread. This suggests that HCMV modulates the EV pathway to transfer proviral signals to uninfected cells that prime the cellular environment for incoming infection and enhance the efficiency of virus spread.  

3.3661           Multilamellar and Multivesicular Outer Membrane Vesicles Produced by a Buttiauxella agrestis tolB Mutant

Takaki, K., Tahara, Y.O., Nakamichi,  N., Hasefawa, Y., Shintani, M., Ohkuma, M., Miyata, M., Futamata, H. and Tashiro, Y. Appl. Environ. Microbiol., 86(20), e01131-20 (2020)   Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of tolB, which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming Buttiauxella agrestis type strain JCM 1090. The ΔtolB mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the ΔtolB mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of ΔtolB mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the B. agrestis ΔtolB mutant to form unique vesicles.  

3.3662           Hypoxia and extracellular vesicles: A review on methods, vesicular cargo and functions

Bister, N., Pistono, C., Huremagic, B., Jolkkonen, J., Giugno, R. and Malm, T.

1. Extracell. Vesicles, 10(1), e12002 (2020)

  Hypoxia is an essential hallmark of several serious diseases such as cardiovascular and metabolic disorders and cancer. A decline in the tissue oxygen level induces hypoxic responses in cells which strive to adapt to the changed conditions. A failure to adapt to prolonged or severe hypoxia can trigger cell death. While some cell types, such as neurons, are highly vulnerable to hypoxia, cancer cells take advantage of a hypoxic environment to undergo tumour growth, angiogenesis and metastasis. Hypoxia‐induced processes trigger complex intercellular communication and there are now indications that extracellular vesicles (EVs) play a fundamental role in these processes. Recent developments in EV isolation and characterization methodology have increased the awareness of the importance of EV purity in functional and cargo studies. Cell death, a hallmark of severe hypoxia, is a known source of intracellular contaminants in isolated EVs. In this review, methodological aspects of studies investigating hypoxia‐induced EVs are critically evaluated. Key concerns and gaps in the current knowledge are highlighted and future directions for studies are set. To accelerate and advance research, an in‐depth analysis of the functions and cargo of hypoxic EVs, compared to normoxic EVs, is provided with the focus on the altered microRNA contents of the EVs.  

3.3663           Bisecting GlcNAc modification diminishes the pro‐metastatic functions of small extracellular vesicles from breast cancer cells

Tan, Z., Cao, L., Wu, Y., Wang, B., Song, Z., Yang, J., Cheng, L., Yang, X., Zhou, X., Dai, Z., Li, X. and Guan, F.

1. Extracell. Vesicles, 10(1), e12005 (2020)

  Small extracellular vesicles (sEVs) are enriched in glycoconjugates and display specific glycosignatures. Aberrant expression of surface glycoconjugates is closely correlated with cancer progression and metastasis. The essential functions of glycoconjugates in sEVs are poorly understood. In this study, we observed significantly reduced levels of bisecting GlcNAc in breast cancer. Introduction of bisecting GlcNAc into breast cancer cells altered the bisecting GlcNAc status on sEVs, and sEVs with diverse bisecting GlcNAc showed differing functions on recipient cells. Carcinogenesis and metastasis of recipient cells were enhanced by sEVs with low bisecting GlcNAc, and the pro‐metastatic functions of sEVs was diminished by high bisecting GlcNAc modification. We further identified vesicular integrin β1 as a target protein bearing bisecting GlcNAc. Metastasis of recipient cells was strongly suppressed by high bisecting GlcNAc levels on vesicular β1. Our findings demonstrate the important roles of glycoconjugates on sEVs. Modification of sEV glycosylation may contribute to development of novel targets in breast cancer therapy.  

3.3664           Adipose‐derived mesenchymal stem cells reduce autophagy in stroke mice by extracellular vesicle transfer of miR‐25

Kuang, Y., Zheng, X., Zhang, L., Ai, X., Venkataramani, V., Kilic, E., Hermann, D.M., Majid, A., Bähr, M. and Doeppner, T.R.

1. Extracell. Vesicles, 10(1), e12024 (2020)

  Grafted mesenchymal stem cells (MSCs) yield neuroprotection in preclinical stroke models by secreting extracellular vesicles (EVs). The neuroprotective cargo of EVs, however, has not yet been identified. To investigate such cargo and its underlying mechanism, primary neurons were exposed to oxygen‐glucose‐deprivation (OGD) and cocultured with adipose‐derived MSCs (ADMSCs) or ADMSC‐secreted EVs. Under such conditions, both ADMSCs and ADMSC‐secreted EVs significantly reduced neuronal death. Screening for signalling cascades being involved in the interaction between ADMSCs and neurons revealed a decreased autophagic flux as well as a declined p53‐BNIP3 activity in neurons receiving either treatment paradigm. However, the aforementioned effects were reversed when ADMSCs were pretreated with the inhibitor of exosomal secretion GW4869 or when Hrs was knocked down. In light of miR‐25‐3p being the most highly expressed miRNA in ADMSC‐EVs interacting with the p53 pathway, further in vitro work focused on this pathway. Indeed, a miR‐25‐3p oligonucleotide mimic reduced cell death, whereas the anti‐oligonucleotide increased autophagic flux and cell death by modulating p53‐BNIP3 signalling in primary neurons exposed to OGD. Likewise, native ADMSC‐EVs but not EVs obtained from ADMSCs pretreated with the anti‐miR‐25‐3p oligonucleotide (ADMSC‐EVs^{anti‐miR‐25‐3p}) confirmed the aforementioned in vitro observations in C57BL/6 mice exposed to cerebral ischemia. The infarct size was reduced, and neurological recovery was increased in mice treated with native ADMSC‐EVs when compared to ADMSC‐EVs^{anti‐miR‐25‐3p}. ADMSCs induce neuroprotection by improved autophagic flux through secreted EVs containing miR‐25‐3p. Hence, our work uncovers a novel key factor in naturally secreted ADMSC‐EVs for the regulation of autophagy and induction of neuroprotection in a preclinical stroke model.  

3.3665           Emerging Prospects of Exosomes for Cancer Treatment: From Conventional Therapy to Immunotherapy

Nam, G-H., Choi, Y., Kim, G.B., Kim, S., Kim, S.A. and Kim, I-S. Adv. Mater., 32, 2002440 (2020)   Exosomes are a class of extracellular vesicles of around 100 nm in diameter that are secreted by most cells and contain various bioactive molecules reflecting their cellular origin and mediate intercellular communication. Studies of these exosomal features in tumor pathogenesis have led to the development of therapeutic and diagnostic approaches using exosomes for cancer therapy. Exosomes have many advantages for conveying therapeutic agents such as small interfering RNAs, microRNAs, membrane‐associated proteins, and chemotherapeutic compounds; thus, they are considered a prime candidate as a delivery tool for cancer treatment. Since exosomes also provide an optimal microenvironment for the effective function of immunomodulatory factors, exosomes harboring bioactive molecules have been bioengineered as cancer immunotherapies that can effectively activate each stage of the cancer immunity cycle to successfully elicit cancer‐specific immunity. This review discusses the advantages of exosomes for treating cancer and the challenges that must be overcome for their successful clinical development.  

3.3666           In vivo tracking of extracellular vesicles: comparison across radioactive, fluorescence and bioluminescence approaches

Lazaro-Ibaneza, E., Faruqub, F.N., Salehc, A., Silva, A.M., Al-Jamald. K. and Dekker, N.

12. Extracell. Vesicles, 9 (Suppl. 1), abstract OF12.6 (2020)

  Introduction: The development of EVs for therapeutic applications requires an in-depth understanding of their in vivo biodistribution and pharmacokinetic profile. In this study, we have made a comprehensive comparison of nuclear, fluorescent, and bioluminescent imaging technologies to identify the most suitable in vivo EV tracking method. Methods: EVs were purified from Expi293 F cell supernatant by differential centrifugation followed by iodixanol density gradient separation and further characterized following MISEV guidelines. Engineered Expi293 F cells were used to generate EVs carrying mCherry or NanoLuc (Nluc) proteins. The membrane of naïve EV was labelled with Indium111(In111)-DTPA or Xenolight DiR post-EV isolation. CT26 tumour-bearing BALB/c mice were intravenously dosed with 1 × 1011 EVs followed by imaging at 1 h, 4h and 24h using SPEC/CT and IVIS systems. Tissue distribution and blood circulation profile of EVs were analysed from ex vivo samples up to 24h post-injection. Results: Xenolight DiR and (In111)-DTPA were the most suitable EV labels for live whole-body animal imaging, ex vivo organ imaging, and tissue lysate quantification. Nluc was appropriate for ex vivo imaging and tissue lysates quantification, but suboptimal for live imaging with limited sensitivity. mCherry EVs were found not suitable for in vivo tracking studies due to high background signal fluorescence. Ex vivo organ quantification of In111-DTPA and DiR showed that naïve EVs mainly accumulate in liver, followed by spleen, kidneys, and lungs at 24h post-dose, with less than 5% EV exposure to the tumours. Interestingly, NlucEVs accumulated mainly in the lungs, regardless of the small size of the particles injected and the absence of aggregation. Blood circulation profile of In111-DTPA and Nluc EVs showed rapid clearance of vesicles from circulation, with 15% of injected dose detected in blood after 30 min and less than 5% after 16 h. Summary/Conclusion: Radionuclide imaging is an excellent technology to detect EVs in vivo and ex vivo with high resolution and sensitivity but requires advanced infrastructure for radiolabeling. The optical methods have limited tissue penetration and sensitivity but can be improved with the right selection of the dye. These results contribute to the understanding of the biodistribution and pharmacokinetics of EVs and are highly relevant to exploiting their potential for targeted delivery to diseased tissues in vivo.  

3.3667           Extracellular vesicles containing oncogenic mutant β-catenin activate Wnt signalling pathway in the recipient cells

Fonseka, P., Kalrab, H., Gangodab, L. and Mathivananb, S.

20. Extracell. Vesicles, 9 (Suppl. 1), abstract OS20.1 (2020)

  Introduction: Large-scale colorectal cancer (CRC) sequencing studies have shown that 93% of all tumours had at least one mutation in proteins implicated in the Wnt signalling pathway. Mutations in β-catenin have often been associated with the constitutive activation of Wnt signalling pathway and has been established as a major driver of CRC. One of the proposed mechanisms of activating Wnt signalling involves extracellular vesicles (EVs) as cellular couriers to transfer Wnt ligands from one cell to another. However, the association of oncogenic mutant β-catenin with EVs has not been studied. Subpopulations of cancer cells with different mutational loads and behavioural variations lead to intra-tumour heterogeneity Methods: Integrative proteogenomic analysis showed the secretion of mutant β-catenin via EVs. EVs were isolated by ultracentrifugation and OptiPrep density gradient centrifugation. SILAC-based quantitative proteomics analysis, immunofluorescence, biochemical analysis, qPCR and xenograft models were employed to unveiling the role of EVs carrying mutant βcatenin. Results: An integrative proteogenomic analysis identified the presence of mutated β-catenin in EVs secreted by colorectal cancer (CRC) cells. Follow up experiments established that EVs released from LIM1215 CRC cells stimulated Wnt signalling pathway in the recipient cells with wild type β-catenin. SILAC-based quantitative proteomics analysis confirmed the transfer of mutant β-catenin to the nucleus of the recipient (RKO CRC) cells. In vivo tracking of DiR labelled EVs in mouse implanted with RKO CRC cells revealed its bio distribution, confirmed the activation of Wnt signalling pathway in tumour cells and increased the tumour burden. Summary/Conclusion: Overall, for the first time, this study reveals that EVs can transfer mutant β-catenin to the recipient cells and promote cancer progression.  

3.3668           Small extracellular vesicles modulated by the αVβ3 integrin reprogramme recipient cells towards an aggressive neuroendocrine cancer phenotype

Quaglia, F., Krishna, S.R., Daaboulb, G., Sarke, S., McCue, P.A., Kelly, W., Liuc, Q. and Languino, L.R.

21. Extracell. Vesicles, 9 (Suppl. 1), abstract OS21.2 (2020)

  Introduction: The ability of small extracellular vesicles (sEVs) to reprogramme cancer cells is known. Integrins, receptors for extracellular matrix proteins, are major players in mediating sEV functions. Previously, we have reported that the αVβ3 integrin is detected in sEVs of prostate adenocarcinoma (PrCa) cells and transferred into recipient cells in a paracrine fashion; however, its role and expression have never been explored in the most aggressive forms of PrCa, such as neuroendocrine PrCa (NEPrCa). NEPrCa does not express androgen receptor (AR) but does express neuron-specific proteins, such as Aurora kinase A, Synaptophysin and Neuron specific enolase, that activate pro-tumorigenic pathways independently from the AR. Methods: We isolated sEVs from PrCa C4-2B cells using iodixanol density gradients and characterized them by immunoblotting and ExoView. The experiments were performed in vivo by injecting subcutaneously, in nude mice, DU145 cells treated with sEVs expressing or lacking the αVβ3 integrin, and in vitro, by testing anchorage-independent growth of different cell lines treated with the same sEVs. Discarded human tissues from PrCa metastasis were analysed by immunohistochemistry (IHC). Results: We demonstrate that a single treatment of PrCa cells with sEVs significantly stimulates tumour growth and anchorage-independent growth. Moreover, we show that one treatment with sEVs, shed from C4- 2B cells that express αVβ3, but not from the control cells that lack αVβ3, induces differentiation of PrCa cells towards a neuroendocrine phenotype and downregulates AR. Finally, our IHC analysis shows coexpression of αVβ3 integrin and Synaptophysin in NEPrCa metastatic lesions. Summary/Conclusion: In conclusion, our current study shows, for the first time, that αVβ3 integrin expression in donor cells generates sEVs that reprogramme recipient cells towards an aggressive tumour phenotype. Funding: This study was supported by NCI-P01- 140043, R01-224769 to LRL.  

3.3669           Integrated omics reveal conserved and divergent modulation of cardiovascular disease by tissue-entrapped extracellular vesicles

Blasera, M.C., Buffolo, F., Halua, A., Schlotter, F., Higashia, H., Rubinop, L.P., Saddic, L.A., Atkins. S., Rogers, M.A. et al

27. Extracell. Vesicles, 9 (Suppl. 1), abstract OS27.3 (2020)

  Introduction: Fewer than 50% of patients develop both vascular and valvular calcification, implying differential pathogenesis. While circulating extracellular vesicles (EVs) act as biomarkers of cardiovascular diseases, tissue-entrapped EVs are implicated in early mineralization but their contents and function are unstudied. We developed an innovative method to isolate and study EVs from fibrocalcific tissue and investigated entrapped EV cargoes in human cardiovascular diseases. Methods: Human carotid artery endarterectomies and stenotic aortic valves were obtained from 27 donors under IRB-approved informed consent. Tissues underwent enzymatic digestion, ultracentrifugation, and a 15-fraction OptiPrep density gradient. Global proteomics was performed on intact tissue, each OptiPrep fraction, and EV-enriched pooled fractions; the latter also underwent miRNA-seq. Fractionated samples were also studied by CD63 immunogold electron microscopy (TEM) and nanoparticle tracking analysis (NTA). High confidence miR targets were predicted by TargetScan, pathway analyses utilized the BioCarta/KEGG/Reactome databases, and protein-protein interaction networks were built in STRING. Results: Vesicle-associated pathways were increased 5.1x (p < 0.01; 15/30 vesicle-related top GO terms) in proteins common to intact arteries and valves (n = 1,411). Proteomics found 24 EV markers to be highly enriched in the four least-dense OptiPrep fractions of arteries and valves, while extracellular matrix and mitochondria were predominant in denser fractions, as confirmed by TEM/NTA. Proteomics and miRNA-seq of tissue EVs quantified 1,104 proteins and 123 miR cargoes linked to 5,182 target genes. Pathway networks of proteins and miR targets common to artery and valve tissue EVs revealed a shared regulation of Rho GTPase and MAPK intracellular signalling cascades. 179 proteins and 5 miRs were significantly altered between artery and valve EVs (q < 0.05); multi-omics integration found that EVs differentially modulated cellular contraction and p53- mediated transcriptional regulation in vascular and valvular tissue. Summary/Conclusion: Our findings delineate a novel strategy for studying tissue-entrapped EV protein and miR cargoes and identify critical roles that tissue-resident EVs play in mediating cardiovascular disease. Funding: This study was supported by a research grant from Kowa Company (MA) and NIH grants R01 HL136431, R01 HL141917 and R01 HL147095 (EA).  

3.3670           The αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis

Krishna, S.R., Salem, I., Quaglia, F., Naraonja, N.M., Agarwald, E., McCue, P.A., Weinreb, P.H., Violetta, S.M., Altierig, D.C. and Languino, L.R.

2. Extracell. Vesicles, 9 (Suppl.1), abstract OT02.3 (2020)

  Introduction: Prostate cancer (PrCa) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sEVs). sEVs isolated from PrCa cell media, express the epithelial-specific αvβ6 integrin, a surface receptor for fibronectin and vitronectin. The αvβ6 integrin is not detectable in healthy prostate tissues but is highly expressed in PrCa. In this study, we hypothesized that αvβ6 in cancer sEVs plays a crucial role in angiogenesis. Methods: The sEVs isolated from PrCa cell media were characterized by nanoparticle tracking analysis, iodixanol density gradients and expression of sEV markers. The αvβ6-negative endothelial cells (HMEC1) were incubated with αvβ6-positive sEVs from PrCa cells to evaluate the transfer of αvβ6 by immunoblotting (IB) and FACS. The effect of αvβ6-positive sEVs on motility, tube formation and angiogenic signalling were assessed by Boyden chamber, angiogenesis assays and IB in HMEC1. Results: We demonstrate for the first time that the αvβ6 is de novo expressed on endothelial cell surface by sEVmediated protein transfer. PrCa cell-derived αvβ6-positive sEVs, significantly promote the motility and the formation of nodes, junctions and tubules by HMEC1. Mechanistically, we demonstrate that HMEC1 treatment with sEVs from PC3 cells that endogenously express αvβ6, decreases pSTAT1(Y701), a negative regulator of angiogenesis, while upregulating survivin, an inducer of angiogenesis. HMEC1 treatment with sEVs isolated from PC3 cells harbouring CRISPR/Cas9-mediated downregulation of β6, or shRNA-mediated downregulation of β6, results in increased levels of pSTAT1(Y701). This sEV treatment also results in a decrease of survivin in sEVs and HMEC1. Summary/Conclusion: Overall, our findings show that αvβ6 in prostate cancer sEVs regulates a novel proangiogenic signalling pathway. Funding: This study was supported by NCI R01- 224769 (LRL); P01-140043 (LRL and DCA).  

3.3671           Desmoglein 2 enhances squamous cell carcinoma tumour development through extracellular vesicles in an IL-8/miR-146a-dependent mechanism

Flemming, J.P., Haque, M., Hill, B.L., Wahl, J., Tsai, K., Wermuth, P., Overmiller, A. and Mahoney, M.

2. Extracell. Vesicles, 9 (Supp.1), abstract OT02.6 (2020)

  Introduction: The cadherin Dsg2 is a stem cell marker that is upregulated in many different cancers, including SCCs, and its expression correlates with poor prognosis. Dsg2 activates mitogenic signalling and plays a key role in cell proliferation, migration, and survival. We recently showed that Dsg2 enhances EV release, but the mechanism by which these EVs modulate tumorigenesis is not fully understood. Methods: We established SCC cell lines stably expressing wildtype Dsg2 or a palmitoylation deficient mutant, Dsg2cacs. EVs were isolated by sequential ultracentrifugation, iodixanol gradient separation, or qEV Izon column, and analysed by NTA and BCA. Tumour xenografts were established by subcutaneous injection of 106 cells in SCID mice and monitored up to 4 weeks. Cytokine profiling was determined by antibody array. miRNA expression was analysed by RNAseq and confirmed by qPCR. Results: Dsg2 enhanced EV release by 40% and promoted a ~ fivefold increase in tumour size in xenograft models. Tumour growth was increased when control cells were treated with a single 20 µg dose of EVs. Loss of palmitoylation, which altered membrane trafficking of Dsg2, reduced EV release (~55%) as well as tumour development. Plasma EVs from xenograft mice reflected in vitro particle counts from SCC cell lines. A cytokine array analysis was performed revealing that Dsg2-EVs were enriched with pro-inflammatory cytokines including IL-8, a potent chemotactic and angiogenic factor. Most importantly, IL-8 was surface-bound on EVs. Furthermore, RNAseq revealed miR-146a, a negative regulator of IL-8, to be significantly downregulated in response to Dsg2. Treatment with miR-146a mimic or miR-146a inhibitor decreased or increased, IL-8 expression in SCC cells, respectively. Summary/Conclusion: In summary, Dsg2 plays a key role in SCC tumour development by increasing EV biogenesis and downregulating miR-146a, which in turn upregulates IL-8 synthesis and release which can promote invasion, angiogenesis and metastasis. Funding: NIH R01  

3.3672           Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

Tosara, J.P., Segovia, M., Gambaro, F., Akiyama, Y., Fagundez, P., Costa, B., Possic, T., Hill, T., Ivanov, P. and Cayata, A.

9. Extracell. Vesicles, 9 (suppl.1), abstract OT09.1 (2020)

  Introduction: To date, most research involving extracellular RNAs has focused in RNAs encapsulated inside extracellular vesicles (EVs) or in total unfractionated biofluids. It is known that exRNAs also exist outside vesicles or in lipoprotein particles. However, nonvesicular exRNAs remain widely uncharacterized despite being a feasible source of contaminants in EV preparations. Our interest in nonvesicular exRNAs arises from the observation that some small RNAs, such as specific tRNA-derived fragments, have much higher relative representation in this extracellular fraction. At least in part, this enrichment seems to be a consequence of their differential extracellular stability. Methods: To get a representative picture of the whole set of RNAs released to the extracellular nonvesicular space by cultured human cells, we inhibited extracellular degradation by adding recombinant ribonuclease inhibitor to the cell-conditioned media and studied the kinetics of RNA release and degradation. High-resolution iodixanol gradients were used to separate EVs from extracellular RNPs or vesicle-free RNA. The conversion rate between parental ncRNAs and their fragments was studied by high-throughput sequencing and Northern blot. Results: The inhibition of extracellular RNase activity revealed the presence of full-length tRNAs and ribosomes in the extracellular space of a variety of malignant and non-malignant cell lines. Extracellular ribosomes co-isolate with EVs purified by ultracentrifugation or size-exclusion chromatography, but not with EVs purified by density gradients.These ncRNAs are substrates of extracellular RNases, demonstrating an extracellular biogenesis route for the formation of ncRNA-derived fragments, some of which achieve remarkable stability and can be detected in biofluids. We also highlight the immunoregulatory potential of purified RNA-containing extracellular complexes. Summary/Conclusion: In conclusion, ribonuclease inhibition dramatically shapes extracellular RNA profiles and uncovers a population of extracellular ribosomes, tRNAs and other coding and noncoding RNAs which exists outside EVs. Although these RNAs are prone to degradation, some of their fragments can accumulate in cell culture media and in biofluids. This dynamic view of exRNAs impacts our understanding of RNA secretion mechanisms and may offer a window to new molecules with biomarker potential. In contrast, EVs confer an RNase-protected environment and contain more full-length ncRNAs (tRNAs, YRNAs, 7SL RNAs, rRNAs depending on vesicle size) than their fragments. Funding: ANII, Uruguay [FCE_3_2018_1_148745]. CISC-UdelaR, Uruguay [MIA_PAS2019_2_99]. NIH, USA [RO1GM126150 and UG3CA241694, supported by the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director  

3.3673           Role of membrane protein palmitoylation in extracellular vesicle biogenesis in squamous cell carcinoma

Haque, M., Flemming, J.P., Hill, B.L., Wahl, J. and Mahoyney, M.

9. Extracell. Vesicles, 9 (Suppl.1), abstract OT09.3 (2020)

  Introduction: Desmoglein 2 (Dsg2), is a palmitoylated cadherin that is involved in cell-cell adhesion. Interestingly, Dsg2 promotes mitogenic cell signalling and is upregulated in many cancers, including SCC, contributing to poor prognosis and survivability. We recently demonstrated that Dsg2 promotes EV release, but the mechanism by which Dsg2 enhances EV biogenesis and role of palmitoylation is poorly understood. Methods: Pharmacological drug inhibitors 2-bromopalmitate, GW4869, and Bafilomycin A1 were used. Stable SCC cell lines were established by retrovirus infection expressing GFP, wild type Dsg2/GFP, or palmitoylation deficient Dsg2cacs/GFP. EVs were isolated by sequential ultracentrifugation and iodixanol gradient separation and analysed by NTA. Proteins associated with the endocytic pathway were analysed by immunofluorescence and imaged by confocal microscopy or immunoblotting and signals were quantitated using ImageStudio. Results: Here we demonstrate that the effect of Dsg2 on EV release was reduced by the palmitoylation inhibitor 2-bromopalmitate. Furthermore, mutations that prevented palmitoylation (Dsg2cacs) dramatically abrogated EV release by targeting of un-palmitoylated Dsg2 to the lysosomes for degradation. Dsg2 increased expression and subcellular localization of FLOT1, a membrane lipid raft protein critical for membrane invagination. Dsg2 also altered membrane localization of several early (EPS15 and EEA1), but not late (Rab7, Rab11, and HRS), endocytic pathway proteins. Loss of palmitoylation in the Dsg2cacs mutants abrogated these effects. Finally, Dsg2-induced EV release was abrogated by the sphingomyelinase inhibitor GW4869 or augmented by the V-ATPase inhibitor Bafilomycin A1. Summary/Conclusion: The combined results of the drug treatments and functional mutations of Dsg2 suggest that Dsg2 plays a critical role in EV biogenesis by modulating proteins involved in early endosome sorting and is dependent on post-translational palmitoylation. Funding: NIH RO1  

3.3674           Association, structure, and function of fibronectin in extracellular vesicles from hepatocytes

Li, X., Chen, R., Kemper, S.and Brigstock, D.

1. Extracell. Vesicles, 9 (Suppl.1), abstract PF01.05 (2020)

  Introduction: We have shown that extracellular vesicles from normal hepatocytes have anti-fibrogenic activity and that they preferentially bind to hepatic stellate cells (HSCs, the principal fibrosis-causing cell in the liver) and hepatocytes. In this study, our goal was to determine the molecular nature of the EV components involved in cell binding. Fibronectin (FN1) is a key component of extracellular matrix, functioning in processes including cell adhesion, differentiation, and wound healing. Two types of FN1 are present in vertebrates, of which the soluble plasma FN1 is derived principally from hepatocytes, while cell-associated FN1 is produced by numerous cell types. Here we describe a novel function of plasma FN1 in facilitating binding of hepatocyte EVs to target cells. Methods: Differential ultracentrifugation was used to collect EVs released by parental mouse AML12 hepatocytes, FN1 KO AML12 cells in which FN1 was ablated using CRISPR-cas9, primary human or mouse hepatocytes, or human HepG2 cells, or from human or mouse serum. EVs were characterized by nanosight tracking analysis (NTA), Western blot, iodixanol gradient ultracentrifugation, and mass spectrometry. The binding efficiency of PKH26-labelled EVs from parental (EV-Hep) or FN1 KO (EV-HepFN1 KO) AML12 cells was analysed in hepatocytes or HSCs. Swiss Webster mice were injected with CCl4 for five weeks to induce liver fibrosis, with some mice also receiving i. p. administration of EV-Hep or EV-HepFN1 KO over the last two weeks, followed by determination of hepatic fibrogenic genes by qRT-PCR. Results: EV-Hep or EV-HepFN1 KO were 50–200 nm in diameter and positive for common EV markers (CD63, CD9, flotillin-1). Mass spectrometry showed that FN1 was the most abundant protein in EV-Hep and comprised principally the plasma form. The abundant presence of EV FN1 was verified by Western blot and co-immunoprecipitation with anti-CD9 or antiflotillin-1. Western blot showed that FN1 was also abundant in EVs from primary human or mouse hepatocytes, HepG2 cells, and human or mouse serum. FN1 and EV-Hep co-sedimented at a density of ~1.15 g/ml. EV-HepFN1 KO yield and size-range were similar to those of EV-Hep, suggesting that EV biogenesis is FN1-independent. As compared to EV-Hep, the binding of EV-HepFN1 KO to target cells was highly reduced whereas EV binding was independent of FN1 expression by the target cells themselves. Both EVHepFN1 KO and EV-Hep were anti-fibrogenic in vivo but only EV-Hep attenuated collagen 1⍺1 expression in mouse HSCs in vitro. Summary/Conclusion: FN1 is abundantly associated with hepatocyte EVs and facilitates EV binding to target hepatocytes or HSCs. Additional studies are needed to clarify the functional role of FN1 in mediating EV-Hep anti-fibrogenic actions in vitro or in vivo.  

3.3675           Life stage-specific glycosylation of schistosome-derived extracellular vesicles

Kuipers, M.E., Nolte-‘tHoen, E., Nguyena, D.L., Koning, R.I., Smits, H.H.and Hokke, C.H.

3. Extracell. Vesicles, 9 (Suppl.1), abstract PF03.05 (2020)

  Introduction: Glycans play an essential role in pathogen-host interactions. Larvae and adult worms from Schistosoma mansoni release distinct subsets of glycoconjugates as excretory/secretory (ES) products. Extracellular vesicles (EVs) are also among the ES products. We recently found that schistosomuladerived EVs are glycosylated and bind human dendritic cells via C-type lectin receptor (CLR) DC-SIGN, leading to increased IL-10 and IL-12 release. Here we investigated the glycosylation profile of EVs released by S. mansoni adult worms, compared this to schistosomula EVs, and addressed how this may affect parasite-host interactions via CLRs. Methods: EVs from cultured S. mansoni parasites were obtained by ultracentrifugation and purified with iodixanol density gradients. Isolated EVs were analysed by NTA and cryo EM. N-glycan and lipid glycan content was determined by mass spectrometry. Density gradient fractions with EVs were loaded onto SDS-page gels followed by Western Blot (WB) analysis using anti-glycan monoclonal antibodies (mAbs). Results: Cryo EM showed that adult worm EVs lacked the long thin filaments that are characteristic for schistosomula EVs. Additionally, in contrast to schistosomula EVs, glycolipids could not be detected in the adult worm EVs. Mass spectrometry analysis showed that the most abundant N-glycans in the adult worm EVs contained GalNAcβ1–4GlcNAc (LacDiNAc, LDN) motifs, which correspond to previously published overall glycan profiles of this specific life stage. Other differences in EV glycosylation between the two life stages were observed by WB using anti-glycan mAbs: adult worm EVs showed a paucimannosidic glycan motif whereas in the schistosomula EVs Galβ1–4(Fucα1–3) GlcNAc (Lewis X) was detected in line with previous MS analysis. Summary/Conclusion: The adult worm-derived EVs contain life stage-specific N-glycans and show a distinct glycosylation profile compared to the schistosomula EVs. The LDN motifs suggest that adult worm EVs may interact with the macrophage galactose-type lectin (MGL) on host cells and most likely have other immunological consequences than the schistosomula EV-DC-SIGN interaction. Funding: NWO Graduate School Program[022.006.010] (MEK); the European Research Council under the European Union’s Seventh 16 Framework Programme [FP/2007–2013]/ERC Grant Agreement[No. 337581] (EN-‘tH); ZonMW[Vidi 20972] (HHS); Dutch Lung Foundation Consortium[5.1.15.015] (HHS)  

3.3676           Inhibition of extracellular vesicle release in triple negative breast cancer

McNamee, N., Catalano, M., Mukhopadhya, A. and O’Driscoll, L.

8. Extracell. Vesicles, 9 (Suppl.1), abstract PF08.02 (2020)

  Introduction: Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer. Previously we reported that the heterogenous population of EVs released from TNBC cells promotes the growth and aggression of recipient cells. Here we investigated if, by using compounds proposed to inhibit EV release i.e. calpeptin and Y27632 (to block those budding at cell membrane) and GW4869 and manumycin A (to block EVs from MVBs), we could reduce the associated transmission of aggressive phenotype. Methods: EVs were separated from medium conditioned by TNBC cell line Hs578Ts(i)8, using a discontinuous optiprep density gradient, after the cells were treatment for 48 hrs with the compounds listed above. EVs (pooled fractions 3–9 with a density range of 1.03– 1.16 g/mL) were characterised by NTA, BCA, lipid assay, immunoblot, TEM and flow cytometry. To investigate the functional effects of the EVs released, proliferation and migration assays were performed on Hs578T and MDA-MB-468 cells using the EV to cell ratios of 1 × 105 EVs/3x103 cells, 1 × 106 EVs/3x103 cells, 1 × 107 EVs/3x103 cells to evaluate doseresponse. EV-TRACK ID EV190109 (Score of 88%). Results: GW4869 significantly (p = 0.035) decreased EV release from Hs578 Ts(i)8 cells. Manumycin A and a combination of calpeptin and Y27632 (Combo) decreased EV release, but significance was not reached. Conversely, calpeptin and Y27632 actually increased EV release; but not significantly. Of the reduced numbers of EVs released following GW4869 treatment, HLA-DR+ EVs were significantly (p = 0.032) enriched. None of the EVs analysed significantly changed Hs578 T or MDA-MB-468 growth rates. However, EVs from cells treated with calpeptin (p = 0.007), GW4869 (p = 0.025), manumycin A (p = 0.01) and Combo (p = 0.001) caused significant reduction in MDA-MB-468 migration compared to the effects of EVs from untreated cells. Similarly, EV from cells treated with GW4869 (p = 0.011), and Combo (p = 0.018) caused significant reduction in Hs578 T migration. Summary/Conclusion: While GW4869 was the only compound that caused a significant decrease in quantities of EV released, the EVs that continued to be released following treatment with GW4869 or calpeptin and Y27632 significantly reduced migration of both recipient cell lines. Funding: PhD funding: 1252 TCD Scholarship and Carrick Therapeutics Ltd  

3.3677           3D modelling of EV release in progressing prostate cancer

Paniushkina, L., Wolf, M., Vukman, K.V., Bianciardid, L., Zarovni, N., Buzas, E., Dirk, S. and Nazarenko, I.

8. Extracell. Vesicles, 9 (Suppl.1), abstract PF08.05 (2020)

  Introduction: The modelling of cancer progression should be capable to translate acquired knowledge of cell behaviour to the real human body conditions. However, the extracellular vesicles (EVs) isolated from 2D cell models are commonly exploited in research. Taking into account the specificity of the prostate cancer (PC) environment, and a strong need of early diagnosis of castrate-resistance by prostate cancer (CRPC) patients, we suggest in-depth profiling of different EV subtypes isolated from 3D culture as a new tool to model the progressing PC. Methods: Cells from hormone-resistant prostate carcinoma 22-RV1 line were cultured in 2D and 3D conditions, using 3D CoSeedisTM. ACD plasma controlled for haemolysis and remaining platelets was taken from patients with PC and CRPC. The 40 fractions of 4 EV subtypes from cell culture and plasma were obtained by differential centrifugation (DC) followed by iodixanol density gradient purification. Each of the fractions was measured by Nanoparticle Tracking Analysis (NTA), Tunable Resistive Pulse Sensing (TRPS) followed by ELISA. For that, CD63 and CD9 were used as EV markers, ApoB and ApoA1 for lipoprotein contaminants control, and CD3, CD41 and PSMA as tissue-specific biomarkers for determination of fractions containing EVs of different origin. EV-contained fractions were subjected to Next Generation Sequencing (NGS). Results: In 3D conditions, the 22-RV1 cells produce up to 1000-times higher EV number than in 2D. Size and density distribution of EVs derived from 3D cultures but not of 2D resembled plasma EVs. Size distribution and biomarker expression among different EV subtypes allowed distinguishing between PC and CPRCderived samples, indicating a potential to translate these results into clinics for early CPRC detection. Summary/Conclusion: This work demonstrates a new approach to study the secretome of a progressing PC under 3D conditions. The profiles of EV subtypes produced by cancer cells growing in a 3D spatial architecture resemble the profiles of plasma EVs and can serve a useful tool for the establishment of new biomarkers. Funding: European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 722148  

3.3678           Cancer cell-derived EVs containing alphaV beta6 Integrin regulate CD163, IL-6 and IL-10 levels in peripheral blood mononuclear cells

Naranjo, N., Salem, I., Krishn, S.R., Harshyne, L., Hooper, D. and Languino, L.

10. Extracell. Vesicles, 9 (Suppl.1), abstract PF10.02 (2020)

  Introduction: Extracellular vesicles (EVs) mediate communication in the tumour microenvironment and play an important role in cancer progression. Previously, we have shown the enrichment of alphaV beta6 integrin in small extracellular vesicles (sEVs) isolated by differential ultracentrifugation and iodixanol density gradient from PC3 prostate cancer cells. We have also shown in the past that alphaV beta6-positive sEVs induce peripheral blood mononuclear cell (PBMC) polarization by increasing the expression of pro-tumorigenic M2 markers, such as CD163 and CD204. Finally, we have demonstrated that down-regulation of alphaV beta6 integrin up-regulates the STAT1-Interferon Stimulated Genes (ISGs) pathway in cancer cells and in sEVs released by them. Methods: In order to investigate whether prostate cancer cell-derived vesicular STAT1 has a causal effect in PBMC polarization, we down-regulated alphaV beta6 and STAT1 in prostate cancer cells derived sEVs using siRNA as well as CRISPR-Cas9 strategies. The sEVs isolated from these cells were used to analyse M2 polarization by measuring the levels of CD163 in PBMC. Results: The results show that sEVs lacking alphaV beta6 inhibit CD163 levels in PBMC in a STAT1-independent manner. Analysis of cytokines released by PBMC upon incubation with sEVs lacking alphaV beta6, show that PBMC selectively up-regulate the levels of IL-10 and IL-6, which are predominantly anti-tumorigenic cytokines. In contrast, sEVs lacking alphaV beta6 do not upregulate pro-angiogenic cytokines, such as VEGF. Summary/Conclusion: These findings suggest that cancer cell-derived sEVs containing alphaV beta6 integrin promote a pro-tumorigenic PBMC phenotype in the tumour microenvironment by regulating CD163, IL-6 and IL-10 levels. Funding: NIH-R01 CA-224769, P01 CA-140043 (LRL). This project is also funded, in part, under a Commonwealth University Research Enhancement Program grant with the Pennsylvania Department of Health (H.R.) (LRL). Israa Salem, PhD Student, is supported by a fellowship from the Saudi Arabian Ministry of Education  

3.3679           Measuring cholesterol as a high-throughput method for quantifying extracellular vesicles

Sulentic, A., Martin, S., Dibiase, B., Sanchez-Salazar, J., Piard, C., Yee, J.C., Dooley, K. and Finn, J.

4. Extracell. Vesicles, 9 (Suppl.1), abstract PS04.02 (2020)

  Introduction: The extracellular vesicle (EV) field currently lacks a high-throughput method for accurately quantifying EVs in solution. EV quantification has traditionally relied on nanoparticle tracking analysis (NTA), which is time intensive and indiscriminately counts non-EV particles, such as membrane fragments and protein aggregates. We have rigorously assessed two commercially available methods for measuring cholesterol, a major lipid component of the EV lipid bilayer, and evaluated the utility of these assays to quantify EVs in minimally processed samples. Methods: The Amplex® Red Cholesterol Assay and Cedex Bio HT were used to quantify cholesterol in EV samples via enzymatic oxidation, with dynamic ranges of 80–8,000 ng/mL and 4–800 µL/mL, respectively. Samples throughout various stages of purification were analysed, from clarified cell culture medium to highly purified EVs separated on an iodixanol gradient. We evaluated several pre-processing methods, to remove non-EV cholesterol content prior to analysis. Results: The Amplex® and Cedex Bio HT assays were found to perform comparably for quantifying cholesterol in purified EVs (R2 = 0.92). Importantly, cholesterol quantification on purified EV samples, ranging from 1e11 to 4e13 particles/mL, correlated well with NTA measurements (R2 = 0.96). Both 45 µM filtration or an additional 16,000 RCF centrifugation step following clarification removed cholesterol associated with cellular debris or other non-EV sources, allowing for accurate quantification of conditioned medium samples or ultracentrifugation pellets (UCP) instead of needing to rigorously purify samples with an iodixanol density gradient. Summary/Conclusion: Cholesterol quantitation can be used to accurately estimate EV concentration, allowing for rapid characterization of samples from clarified cell culture supernatant to highly purified EVs. This highthroughput analytical capability may enable more comprehensive assessment of methods to boost EV yield through mass screening of cell culture conditions.  

3.3680           A fast and reliable in vitro cell culture system reveals that acidification of bovine milk extracellular vesicles impairs their functionality

Van Herwijnen, M.J.C., Kleinjan, M. and Wauben, M.H.M.

8. Extracell. Vesicles, 9 (Suppl.1), abstract PS08.01 (2020)

  Introduction: The isolation of EVs from milk is technically challenging due to the complexity of milk. Currently used separation procedures allow for the removal of milk fat globules and cells (by low speed centrifugation of fresh milk), removal (by acidification), or disruption (by addition of EDTA) of casein micelles. Using these protocols the integrity, composition and targeting of bovine milk EVs has been evaluated and has led to believe that milk EVs might withstand these conditions. However, the effects on functionality of milk EVs (i.e. immunomodulatory properties) after processing and isolation have not been studied. Therefore, we have set up an in vitro culture system using a human T cell line that allows for the rapid screening of milk EV functionality. Methods: Fresh bovine milk was defatted and cells were removed after 2x3,000 g centrifugation, followed by differential centrifugation at 5,000 g and 10,000 g. This milk was either subjected to acidification with HCL, or EDTA was added, or the milk supernatant remained untouched. Top down Optiprep density gradient separation followed by SEC was used to further purify EVs. These highly purified milk EVs were added to human Jurkat T cells, which were simultaneously stimulated using anti-CD3 and anti-CD28 antibodies. After 24 h T cell activation was measured by IL-2 cytokine production. Results: Precipitation or disruption of casein micelles allowed for the substantial removal of proteins during isolation compared to directly isolated EVs, which aids in the purification of milk EVs. In vitro analysis revealed that in the presence of directly isolated, or EDTA isolated milk EVs, Jurkat cells were suppressed in their activation as measured by IL-2 production. Remarkably, EVs isolated from HCL-acidified milk were impaired in their suppressive capacity to inhibit IL-2 production. Summary/Conclusion: Although casein removal from bovine milk greatly improves purity of isolated milk EVs, the detrimental effects on EV functionality should be considered. Interestingly, EVs exposed to acidic conditions lost their ability to modulate T cell activation, which is in contrast with the general believe that milk EVs could withstand the gastro-intestinal tract. Funding: This work is funded by the European Union’s Horizon 2020 Framework Programme under the grant FETOPEN-801367 evFOUNDRY.  

3.3681           Optimising methods for separation and characterisation of extracellular vesicles from skim milk and infant milk formula

Nukhopadhya, A., Santoro, J., Moran, B., Useckaite, Z. and O’Driscoll, T.

8. Extracell. Vesicles, 9 (Suppl.1), abstract PS08.02 (2020)

  Introduction: Infant milk formula (IMF) is intended to impart nutrition to infants, similar to breast milk. However, although industrial IMF production involves harsh treatment, potential consequences on extracellular vesicles (EV) in IMF are not yet established. This study aimed to optimise methods for separating EVs from IMF and skim milk (SM) and to characterise the EVs in accordance with MISEV2018. Methods: SM and IMF were either not treated (NT) or treated with acetic (AA) or HCl acid (isoelectric precipitation, IP), to remove caseins. Samples were then subjected to differential ultracentrifugation (DUC) or gradient ultracentrifugation using iodixanol solution (GUC). For DUC, 38 mL samples were centrifuged at 12 K g, 35 K g, 75 K g, 100 K g and 200K g sequentially for 75 min each and pellets re-suspended in 1 mL PBS. For bottom-up GUC, increasing iodixanol gradients with 2.3 mL of samples were centrifuged at 186 K g for 18 h. Fractions were then pooled based on densities (1.1–1.2 g/mL). BCA and SDS-PAGE were used to analyse total protein; nanoparticle tracking (NTA) and transmission electron microscopy (TEM) for EV presence; and immunoblotting and imaging flow cytometry (IFCM) to evaluate EV specific markers. (EVTRACK ID: EV190096). Results: Immunoblotting showed absence of Actinin4 from all samples, while CD63 and TSG101 were detected for all samples; apart from IMF_IP. NT_samples were not analysed reliably by NTA and IFCM, due to the high concentration of casein micelles present (~10^14/mL in milk) that otherwise would be co-counted with EVs. As expected, following IP, which most efficiently removed casein micelles, BCA showed that samples had lowest total protein. This was confirmed by SDS-PAGE. Thus, most effects were then focused on the IP casein-depleted samples. IFCM indicated that, post-GUC, SM_IP EVs had significantly (P < 0.05) more CD81-positive particles/mL of milk vs all other GUC and 200 KDUC samples. While there were no significant differences in sizes of EV separated from SM or IMF, directly comparing the IP pre-treated samples, SM had significantly (P < 0.01) higher quantities of EVs when compared to IMF. Additionally, TEM indicated that EVs separated from SM by GUC were intact with limited background debris, whereas those separated from SM by DUC and all IMF EVs were not. Summary/Conclusion: In conclusion, regardless of the method used, IMF has fewer intact EVs compared to SM. Also, to obtain purest SM EVs, IP followed by GUC separation is optimal. Funding: Dept. Agriculture, Food & Marine, Ireland [17/F/234]  

3.3682           A rigorous method for exosome isolation from post-mortem eyes

Klingeborn, M., Hernandez, B., Skiba, N.P., Cady, M.A., Kelly, U., Stamer, W.D. and Bowes, C.

8. Extracell. Vesicles, 9 (Suppl.1), abstract PS08.07 (2020)

  Introduction: In order to determine and validate the tissue-specific content of extracellular vesicles (EVs) in biofluids, robust EV isolation methods from tissues must be developed. However, to date very few rigorous methods to isolate or enrich for intact EVs from tissues have been reported. We present a comprehensive exosome isolation method with a sufficient level of characterization to unequivocally demonstrate true EV identity from ex vivo eyes. Methods: Iodixanol (OptiPrep) buoyant density gradient ultracentrifugation (DGUC), cushioned DGUC (CDGUC), and our newly developed C-DGUC immunocapture (C-DGUC-IP) method were used to compare yield and enrichment of exosomes isolated from porcine eyes between 3 to 5 hours post-mortem. Yield was assessed by Nanoparticle Tracking Analysis (NTA) and immunoblotting for exosomal markers along with total protein quantitation. Enrichment was assessed by comparison of exosomal markers, ocular-specific markers and known contaminant markers, plus in-depth proteomic mass spectrometry analyses. Results: High enrichment of posterior eyecup small EVs (sEV) were achieved by DGUC and C-DGUC, with C-DGUC resulting in an eightfold increase in yield by NTA and two to fivefold increases of exosomal protein markers such as Syntenin-1 and CD81 by immuno-blotting compared to DGUC. Interestingly, in-depth proteomic analyses revealed that a majority of these sEVs with densities of 1.07–1.11 g/ml isolated by DGUC and C-DGUC were likely of endoplasmic reticulum (ER) and Golgi origin, suggesting ER-to-Golgi transport vesicles resulting from post-mortem tissue cell rupture. In order to enrich further for sEVs (including exosomes) we subjected sEVs isolated by C-DGUC to anti-CD81 immunocapture. The resulting sEV proteome was enriched 1.5- to 4-fold for bona fide sEV and exosome markers compared to C-DGUC. Summary/Conclusion: The C-DGUC method provides an enhanced yield and purity of sEVs and exosomes from ex vivo eye tissue. However, to avoid significant contamination with ER and Golgi-derived vesicles from postmortem eyes, a final EV-specific immunocapture step is required to achieve sufficient purity for subsequent analyses. Our highly rigorous method paves the way for identification and validation of ocular-derived exosomes in blood and their potential use as eye disease biomarkers. Funding: This work was supported by NIH grants EY026161 (CBR), EY023287 (WDS), EY022359 (WDS), EY019696 (WDS), a grant from Foundation Fighting Blindness (CBR), a Research to Prevent Blindness (RPB)/International Retinal Research Foundation Catalyst Award (CBR). A Core Grant for Vision Research (P30; EY005722) from NEI (to Duke University) supported mass spectrometric analyses carried out by NPS. In addition, Duke University Department of Ophthalmology is supported by an unrestricted grant to the Duke Eye Centre from RPB.  

3.3683           Exploring Extracellular Vesicles release inhibition in Prostate Cancer

Catalano, M., McNamee, N., Mukhopadhya, A. and O’Driscoll., L.

9. Extracell. Vesicles, 9 (Suppl.1), abstract PS09.08 (2020)

  Introduction: Although pharmacological treatment options are available for prostate cancer, drug resistance can occur leading to limited survival for patients. We previously showed extracellular vesicles (EVs) to be causally involved in transmitting drug resistance. This study aimed to evaluate compounds proposed to reduce/block EV release. Specifically, we selected calpeptin and Y2763 (proposed to inhibit EVs budding from the cell membrane) and manumycin A and GW4869 (proposed to inhibit EVs deriving from MVBs). Associated effects on -and consequences ofEV release were then investigated. Methods: Suitable compounds concentrations that were non-toxic to cells were first selected by performing cytotoxicity assay and flow cytometry (FC). Conditioned medium (CM) was collected from docetaxel-resistant PC3 (PC3 RD) cells after 48 h incubation in dFBS-medium with or without the 4 compounds. EVs were separated from tangential flow filtration concentrated CM using Optiprep density gradient. 1.03–1.16 g/mL fractions were then pooled and washed. EVs were characterised using NTA, immunoblot, TEM and lipid assay and FC. Influences on growth and migration, of EVs continuing to be released (at 1x105EVs/3x103cells, 1x107EVs/3x103cells), were evaluated on recipient DU145 and 22Rv1 cells. EVTRACK ID EV190113, score 88% Results: Calpeptin and Y27632, alone and in combination, did not significantly affect quantities of EVs released. However, GW4869 significantly (p < 0.004) increased quantities of released EVs, of a larger size; very high protein to lipid ratio; and carrying GRP94 compared to control EVs (p < 0.006). This effect was reverted when GW4869 was combined with manumycin A (p < 0.004). Following all compounds treatments, 1x105EVs/ 3x103cells inhibited 22 RV1 proliferation (p < 0.0014), while at 1x107EVs/3x103cells only EVs from manumycin A (p < 0.05) and Y27632 (p < 0.00036) treated cells reduce 22 RV1 proliferation. EVs following GW4869 treatment significantly (p < 0.001) inhibited DU145 migration compared to bulk non-treated control and compared to the effect obtained using the entire pool of EVs (p < 0.001). Summary/Conclusion: While none of the 4 proposed inhibitors significantly reduced EV release, the resulting EVs were less potent in transmitting aggressive behaviour, such as proliferation and migration, to receiving cell lines. Funding: H2020-MSCA-ITN-TRAIN-EV grant [722148]  

3.3684           Subpopulations of tissue-derived extracellular vesicles – methodological evaluation for vesicle size measurement

Crescitelli, R., Lässer, C., Karimic, N., Cvjektkovic, A., Bagge, R.O. and Lötvall, J.

11. Extracell. Vesicles, 9 (Suppl.1), abstract PS11.02 (2020)

  Introduction: Subpopulations of extracellular vesicle (EVs) are commonly classified by their different size, however, the EV size cut off is still under discussion. The aim of the study was to evaluate size range of six EV subpopulations using three methods: electron microscopy (EM), nanoparticle tracking analysis (NTA) and ExoView™. Methods: Methods: EV subpopulations were isolated from melanoma tissues by a centrifugation based protocol recently established in our lab. Large and small EVs (lEVs and sEVs) were isolated with differential ultracentrifugation and these were further separated into low and high-density fractions (LD and HD). Size of EV subpopulations was then evaluated by: EM (N = 8, large EVs and small EVs; N = 2, large and small EVs LD and HD), NTA (N = 7) and ExoView™ (N = 1). Results: Results: Tissue-derived large and small EVs showed difference in size (mean 142 nm vs 75 nm) when examined by EM, whereas NTA and ExoView™ were unable to show a clear difference between the populations (NTA: mean 125.7 nm vs 122 nm, ExoView™: mean 81.7 nm vs 69.2 nm). After iodixanol density gradients, lEVs-LD were significantly larger in size than the sEVs-LD as determined by EM (mean 128.1 nm vs. 67.3 nm) but no difference was observed by NTA and ExoView™ (NTA: mean 134.1 nm vs 124.5 nm, ExoView™: mean 71.1 nm vs 63 nm). HD vesicles from both large and small EVs were the smallest in size when analysed by EM (mean size 41 nm vs 34 nm) but no significant difference was observed by NTA and ExoView™ (NTA: mean 127.8 nm vs 116 nm, ExoView™: mean 98 nm vs 66.4 nm). Summary/Conclusion: Conclusions: Different methods have different limitations: EM preparations can shrink vesicles, NTA can only detected vesicles above 70 nm and ExoView™ only measures vesicles between 50–200 nm. Of the three different methods, EM analysis of single vesicles visualized in a significant number of micrographs was the only one able to distinguish EV subpopulations by size. Funding: Funding: Swedish Research Council (K2014- 85X-22504-01-3), Swedish Heart and Lung Foundation (20120528), Swedish Cancer Foundation (CAN2014/ 844), Knut och Alice Wallenberg Foundation.  

3.3685           Transfection reagent artefact accounts for some reports of extracellular vesicle function

McConnell, R., Youniss, M and Finn, J.

3. Extracell. Vesicles, 9 (Suppl.1), abstract PT03.09 (2020)

  Introduction: Extracellular vesicle (EV) functions are frequently investigated by transiently transfecting cells with plasmid DNA to produce EVs modified with protein(s) or nucleic acid(s) of interest. However, EVs and the DNA-complexes used to transduce cells are physically similar, raising the possibility that they may co-purify during differential ultracentrifugation, the most common EV isolation procedure. Activities attributed to EVs may therefore be due to contaminating DNA -transfection reagent complex. Methods: EV producing cells were transiently transfected with plasmid DNA encoding gene-editing or split enzymes fused to EV-targeting protein sequences. Differential and density gradient ultracentrifugation were used to purify EVs from cell culture supernatant or DNA lipoplexes from cell-free culture media. Protein expression and localization to EVs was confirmed by Western blot. Cell lines stably expressing fluorescent or luminescent reporters were used to assess functional enzyme delivery in recipient reporter cells. Results: Reporter cells treated with ultracentrifuge pellet material (UCP) from media of transiently transfected cells showed robust and reproducible signal, however fractionating the UCP with an iodixanol density gradient revealed that reporter activity was associated with high-density fractions that were depleted in EVs. UCP isolated from identical transfection conditions, but lacking cells (and exosomes), showed identical biological activity levels and distribution in iodixanol gradients, suggesting that the activity was due to contaminating transfection reagent complexes and not EVs. Serial media changes on EV producing cells post-transfection did not significantly reduce UCP activity on reporter cells. Treatment with nucleases did not digest complexed DNA, did not significantly reduce DNA levels in the UCP as measured by qPCR, and did not decrease activity in reporter cells treated with UCP from either transfected cells or no-cell controls. Summary/Conclusion: We find that DNA-transfection reagent complexes are not separated from EVs using differential ultracentrifugation and that common approaches to remove such complexes, including media exchanges and nuclease treatment, are ineffective. Due to the pernicious nature of the DNA-complex in these cellular assays, it is likely that some reports of EV function are likely artefacts produced by contaminating DNA-complexes. We find that density gradient centrifugation can effectively separate EVs and DNA complexes, highlighting the importance of validating elimination of contaminating transfection reagent complexes when using transient transfection to interrogate EV function.  

3.3686           Hypoxia alters the metabolic and miRNA content of breast cancer-derived exosomes

McAndrews, K.M., Cain, M., Kato, N., Melo, S.A., Lovisa, S., Correra de Sampaio, C., Snowden, L., Sugimoto, H., Le Bleu, V. and Kalluric, R.

7. Extracell. Vesicles, 9 (Suppl.1), abstract PT07.07 (2020)

  Introduction: Hypoxia, or low oxygen tension, is a common feature associated with tumour growth and is known to regulate tumour cell function, especially through rewiring of cell metabolism. However, how hypoxia influences tumour cell interactions with surrounding cells is not fully elucidated. We sought to evaluate how hypoxia alters metabolite and metabolism-associated miRNA packaging in exosomes. Methods: Exosomes were isolated from 4T1 breast cancer cells cultured in normoxia (21% O2) and hypoxia (5% O2) via ultracentrifugation, Optiprep gradients, and size exclusion chromatography. Exosomes were further characterized by Nanosight, Qubit protein quantification, and flow cytometry analysis of exosome markers. Metabolite and miRNA profiling was performed on exosomes and exosome-producing cells in normoxia and hypoxia. Results: Secretion of exosomes was increased under hypoxic conditions. Metabolite profiling revealed alterations in metabolites specific to exosomes derived from hypoxic cells. Profiling of exosomal miRNA showed packaging of metabolism-related miRNA into exosomes derived from hypoxic cells. Summary/Conclusion: Hypoxia alters the metabolite and miRNA profiles of cancer cells, with selective packaging of these molecules into exosomes. We identified metabolites and miRNA that are depleted and enriched in exosomes compared to cells. These studies identify hypoxia-associated shifts in exosome cargo, providing insight into exosome cargo packaging with potential implications for understanding how cancer cell-derived exosomes regulate recipient cell function.  

3.3687           Impacts of agricultural dust exposure on human lung-resident mesenchymal stromal/stem cells and their extracellular vesicles

Sveiven, S.N., Adkins, G.B., Zhong, W. and Nordgren, T.M.

9. Extracell. Vesicles, 9 (Suppl.1), abstract PT09.13 (2020)

  Introduction: Agricultural dust is considered a high-risk occupational hazard by the CDC, with impacts reaching throughout the communities surrounding these industries, leading to increased incidence of respiratory illness and disease among individuals within this occupation and these communities. Lung-resident mesenchymal stromal/stem cells (lr-MSC) have an important role in maintaining homoeostasis in the lung, and mediating pro- and anti-inflammatory effects, particularly during exposure to inhaled irritants, like agricultural dust. One way in which these lr-MSC promote lung homoeostasis is through the release of extracellular vesicles (EV), with a variety of cargo that elicit changes among target cells. We hypothesize that exposure to agricultural dust modifies the quantity and cargo of EV released by lr-MSC to promote lung tissue homoeostasis. Methods: Primary human lung-resident mesenchymal stromal cells were exposed to extracts of dusts collected from swine confinement facilities (DE) for 8 or 48hrs and the media from these exposures were collected and enriched for EV by opti-prep density gradient ultracentrifugation. The quantity of these EV were assessed by nanoparticle tracking analysis. Additionally, cytokine and chemokine release by lrMSC were analysed by enzyme-linked immuno assays. Results: As assessed at 48 hr following treatment, DEexposed lr-MSC released pro-inflammatory cytokines, IL-6 and IL-8, with IL-8 release reaching statistical significance at 0.1%, 0.5%, and 1% DE concentrations (p = 0.0367, (p = 0.0367, <0.0001, and <0.0001 respectively) and IL6 trending a similar dose response but only statistically significant at 1% DE (p = <0.0001). DE exposure of lrMSC also induced changes in the lr-MSC-derived EV populations when compared to vehicle control, where lr-MSC released significantly more EV in the 5 and 10% iodixanol fractions (p = <0.0001 and 0.0219, respectively) at 8 hr following DE treatment. Alternatively, there were significantly less EV in the 20 and 40% density fractions in the media of DEexposed lr-MSC versus vehicle control. Summary/Conclusion: Following exposure to agricultural dusts, lr-MSC-derived EV populations more likely consist of exosomes and ectosomes, which play animportant role in promoting lung tissue homoeostasisduring exposure-related pulmonary inflammation.  

3.3688           Beyond stem cells: extracellular vesicles from human induced pluripotent stem cells (hiPSC) and hiPSC-cardiomyocytes as therapeutic approaches for heart failure

Louro, A.F., Almeida, V.A., Gomes-Alves, P.and Serra, M.

11. Extracell. Vesicles, 9 (Suppl.1), abstract PT11.02 (2020)

  Introduction: Heart failure is caused by a variety of underlying diseases, the most common being myocardial infarction. Initially regarded as an alternative to pharmacological approaches, stem cell transplantation has failed to demonstrate clinically meaningful results. Instead, it has become increasingly apparent that the therapeutic effects of transplanted cells are largely mediated by their secretome, while mounting evidence suggests Extracellular Vesicles (EVs) play a major role in cardiac repair. Within this framework, EVs from human induced pluripotent stem cells (hiPSC) and hiPSC-derived cardiomyocytes (hiPSC-CM), hold a tremendous potential to treat cardiovascular disease. We isolated EVs from conditioned culture media at key stages of the hiPSC-CM differentiation and maturation processes, i.e. from hiPSC (hiPSC-EV), cardiac progenitors (CPC-EV), immature (CMi-EV) and mature (CMm-EV) cardiomyocytes, with the aim of studying their potential role as therapeutics, and whether their effectiveness was influenced by the state of their parent cell. Methods: hiPSC were differentiated into cardiomyocytes in a 3D culture approach, using the protocols developed by our group. EV isolation was performed on an iodixanol density gradient, and the EVs were characterized in terms of particle size and particle size distribution, presence of EV-specific markers, and imaging through transmission electron microscopy. Functional studies were performed using human umbilical vein endothelial cells (HUVECs) to evaluate EVuptake, cell migration and angiogenesis. Results: EVs from all hiPSC and cardiac derivatives presented a typical cup-shaped morphology and expressed CD63 and CD81. EV yield varied along differentiation, with a minimum for CPC and a maximum for CMi. PKH26-labelled EVs were uptake by HUVECs, and colocalized with calnexin, a protein from the endoplasmic reticulum. Wound healing assays showed an increased cell migration in HUVECs treated with cardiomyocyte-derived EVs, in comparison with control EVs isolated from foetal bovine serum. Summary/Conclusion: Our findings suggest a different EV secretion profile along CM differentiation and maturation, with preliminary assays showing EV functionality. Ongoing work aims at elucidating the possible differences in function and cargo amongst these types of EVs.  

3.3689           Response to Irradiation of Small Extracellular Vesicles Derived from Prostate Tumors

Garcia, V., Sayeed, A., De Rita, R., Krishn, S.R., McCue, P.A., Dicker, A. and Languino, L.R.

12. Extracell. Vesicles, 9 (Suppl.1), abstract PT12.11 (2020)

  Introduction: Tumor-derived small extracellular vesicles (sEVs) have emerged recently as mediators of tumorigenesis. However, the role of sEVs in response to irradiation, a widely used therapy in prostate cancer, is not fully understood. Methods: Our study involved the TRAMP mouse model of prostate cancer. We used plasma sEVs isolated using differential ultra-centrifugation and further isolated using iodixanol gradient fractionation. We also used Nanoparticle Tracking Analysis (NTA) to analyze sEVs. Mouse pelvises were irradiated using 10 Gy, for 5 consecutive days. Results: We first observed that upon pelvic irradiation of TRAMP mice, the levels of the signaling oncogene c-Src are reduced in plasma-derived sEVs, while the average size of sEVs is increased from 50- 100nms to 70-250nms. Furthermore, we show that the sEVs from irradiated cells lose the ability to stimulate anchorage independent growth and migration of recipient cancer cells. Additionally, sEVs from irradiated mice increase the amount of DNA damage in recipient cancer cells. Summary/Conclusion: Overall, our data show that irradiation of TRAMP mice (and prostate cancer cells) significantly reduces the pro-metastatic and pro-anchorage-independent growth potential of sEVs when tested on human cells. Changes to the composition and behavior of a cancer cell sEV population via radiation therapy offers promise for future therapeutic approaches for prostate cancer. Funding: This study was supported by NIH, P01 CA140043 (to LRL). This project is also funded, in part, under a Commonwealth University Research Enhancement Program grant with the Pennsylvania Department of Health (H.R.); the Department specifically disclaims responsibility for any analyses, interpretations or conclusions.  

3.3690           Bone marrow mesenchymal stem cells-derived exosomes for penetrating and targeted chemotherapy of pancreatic cancer

Zhou, Y., Zhou, W., Chen, X., WWang, Q., Li, C., Chen, Q., Zhang, Y., Lu, Y., Ding, X. and Jiang, C. Acta Pharmaceutica Sinica B, 10(8), 1563-1575 (2020)   Pancreatic ductal adenocarcinoma (PDAC) is one of the most intractable malignancy, with an only 6% 5-year relative survival rate. The dismal therapeutic effect is attributed to the chemotherapy resistance and unique pathophysiology with abundant inflammatory cytokines and abnormal hyperplasia of extracellular matrix (ECM). Based on the theory that bone marrow mesenchymal stem cells (BM-MSCs) can influence the tumorous microenvironment and malignant growth of PDAC, we employed exosomes (Exos) derived from BM-MSCs as PDAC-homing vehicles to surpass the restrictions of pathological ECM and increase the accumulation of therapeutics in tumor site. To overcome chemoresistance of PDAC, paclitaxel (PTX) and gemcitabine monophosphate (GEMP)—an intermediate product of gemcitabine metabolism—were loaded in/on the purified Exos. In this work, the Exo delivery platform showed superiorities in homing and penetrating abilities, which were performed on tumor spheroids and PDAC orthotopic models. Meanwhile, the favorable anti-tumor efficacy in vivo and in vitro, plus relatively mild systemic toxicity, was found. Loading GEMP and PTX, benefitting from the naturally PDAC selectivity, the Exo platform we constructed performs combined functions on excellent penetrating, anti-matrix and overcoming chemoresistance (Scheme 1). Worth expectantly, the Exo platform may provide a prospective approach for targeted therapies of PDAC.  

3.3691           An emerging focus on lipids in extracellular vesicles

Skotland, T., Sagini, K., Sandvig, k. and Llorente, A. Adv. Drug Delivery Reviews, 159, 308-321 (2020)   Extracellular vesicles contain a lipid bilayer membrane that protects the encapsulated material, such as proteins, nucleic acids, lipids and metabolites, from the extracellular environment. These vesicles are released from cells via different mechanisms. During recent years extracellular vesicles have been studied as possible biomarkers for different diseases, as biological nanoparticles for drug delivery, and in basic studies as a tool to understand the structure of biological membranes and the mechanisms involved in vesicular trafficking. Lipids are essential molecular components of extracellular vesicles, but at the moment our knowledge about the lipid composition and the function of lipids in these vesicles is limited. However, the interest of the research community in these molecules is increasing as their role in extracellular vesicles is starting to be acknowledged. In this review, we will present the status of the field and describe what is needed to bring it forward.  

3.3692           2: Extracellular vesicles in regenerative medicine

Romano, M., Zendrini, A., Paoolini, L., Busatto, S., Berardi, A.C., Bergese, P. and Radeghieri, A. Nanomaterials for Theranostics and Tissue Engineering, 29-58 (2020)   Regenerative medicine is a multidisciplinary field aimed at developing methods, molecular agents, and (nano)materials to regrow, repair, or replace damaged, malfunctioning or missing tissues. Current approaches include and combine use of stem cells, tissue engineering based on functional biodegradable scaffolds, and cell-free strategies, with stem cells and their progenitors playing the main role. However, it is now recognized that the therapeutic efficacy of stem cells largely depends on paracrine-secreted soluble factors and extracellular vesicles (EVs). Preclinical and clinical studies indicate that EVs can exert immunomodulatory and regenerative action, thus efficiently recapitulating the therapeutic effects of stem cells. On the other hand, EVs are uncapable of self-replication agents and can be nonimmunogenic, thus offering remarkable advantages and safety over stem cells for therapeutic translation. This chapter, after anintroduction of EV biological and physicochemical properties, will present and discuss advances in EV-based regenerative medicine.  

3.3693           Chapter Five: Imaging intercellular interaction and extracellular vesicle exchange in a co-culture model of chronic lymphocytic leukemia and stromal cells by lattice light-sheet fluorescence microscopy

Elgamal, S., Colombo, F., Cottini, F., Byrd, J.C. and Cocucci, E. Methods in Enzymol., 645, 79-107 (2020)   Recent advances in live cell imaging allow investigating processes that take place over the entire cell volume with unprecedented time and spatial resolution. Here we describe a protocol to study intercellular communication, including extracellular vesicle exchange, between cancer cells and their microenvironment, using lattice light sheet fluorescence microscopy. While the described protocol is intended to study the interactions between chronic lymphocytic leukemia cells and bone marrow stromal cells, many components of it can be applied to study other cancers of hematopoietic or solid tumor origin, as well as to characterize other modalities of intercellular communication.  

3.3694           Adenomyosis-derived extracellular vesicles endow endometrial epithelial cells with an invasive phenotype through epithelial-mesenchymal transition

Chen, D., Qiao, H., Wang, Y., Zhou, L., Yin, N., Fang, L. and Wang, Z. Genes & Diseases, 7, 636-648  (82020)   Extracellular vesicles from highly metastatic tumor cells have been shown to mediate epithelial-mesenchymal transition (EMT)-related events in recipient cells. In endometrial epithelial cells, EMT processes are known to be involved in the development of adenomyosis. We aimed to investigate whether adenomyosis-derived extracellular vesicles (AMEVs) are able to induce an EMT process in endometrial epithelial cells. In this study, AMEVs were isolated from patients with adenomyosis and characterized by transmission electron microscopy, Western blot, and nanoparticle tracking. Primary endometrial epithelial cells (EECs) were derived from normal endometrium tissues from patients with leiomyoma and co-cultured with AMEVs in vitro. AMEV uptake was examined by fluorescence confocal microscopy. The invasion of EECs was confirmed by Transwell assay. Immunohistochemistry, Western blot, and qRT-PCR were performed on EECs to illustrate the expression levels of cytokeratin 19, E-cadherin, vimentin, and zinc finger E-box-binding homeobox 1 (ZEB1). The results indicated that the cellular fluorescence intensity gradually increased after 48 h of co-culture, but decreased after 72 h. After co-culturing with AMEVs for 72 h, EECs expressed significantly lower levels of cytokeratin 19 and E-cadherin, and significantly higher levels of vimentin and ZEB1. Together these results demonstrated that AMEVs induce an EMT process and enhance the invasion of EECs. These changes may contribute to the pathogenesis and progression of adenomyosis.  

3.3695           Partitioning of MLX-Family Transcription Factors to Lipid Droplets Regulates Metabolic Gene Expression

Mejhert, N., Kuruvilla, L., Gabriel, K.R., Danial, N.N., Farese Jr., R.V. and Walther, T.C. Molecular Cell, 77, 1251-1264 (2020)   Lipid droplets (LDs) store lipids for energy and are central to cellular lipid homeostasis. The mechanisms coordinating lipid storage in LDs with cellular metabolism are unclear but relevant to obesity-related diseases. Here we utilized genome-wide screening to identify genes that modulate lipid storage in macrophages, a cell type involved in metabolic diseases. Among ∼550 identified screen hits is MLX, a basic helix-loop-helix leucine-zipper transcription factor that regulates metabolic processes. We show that MLX and glucose-sensing family members MLXIP/MondoA and MLXIPL/ChREBP bind LDs via C-terminal amphipathic helices. When LDs accumulate in cells, these transcription factors bind to LDs, reducing their availability for transcriptional activity and attenuating the response to glucose. Conversely, the absence of LDs results in hyperactivation of MLX target genes. Our findings uncover a paradigm for a lipid storage response in which binding of MLX transcription factors to LD surfaces adjusts the expression of metabolic genes to lipid storage levels.  

3.3696           RhoJ Regulates α5β1 Integrin Trafficking to Control Fibronectin Remodeling during Angiogenesis

Sundararaman, A., Fukushima, Y., Norman, J.C., Uemera, A. and Mellor, H. Current Biology, 30, 2146-2155 (2020)   Rho guanosine triphosphatases (GTPases) are master regulators of cell shape and cell movement [1]. The archetypal family members RhoA, Rac1, and Cdc42 arose early in eukaryotic evolution and coordinate a diverse range of cell morphologies and migrations. Evolution of the vertebrates was paralleled by expansion of this family through gene duplication. Emergence of an adaptive immune system and more complex neural systems presented new roles for Rho GTPases, filled by new family members. Cdc42 underwent gene duplication to produce two related proteins—RhoQ and RhoJ [2]. RhoQ is active in neural dynamics; however, RhoJ is highly expressed in endothelial cells under control of the endothelial-specific promoter ERG [3, 4]. RhoJ is required for angiogenesis [5, 6] and has multiple roles in this process [7, 8]. We recently demonstrated that RhoJ regulates the endosomal trafficking of podocalyxin during angiogenesis to control lumen formation [9]. Here, we use vesicle purification and proteomic analysis to identify the endothelial targets of RhoJ-mediated trafficking. We identify α5β1 integrin as a major RhoJ cargo and show that RhoJ regulates the intracellular trafficking of active α5β1 integrin in endothelial cells to repress fibronectin fibrillogenesis. Accordingly, mice lacking RhoJ show deregulated deposition of fibronectin around vessels during developmental angiogenesis. Intriguingly, we show that RhoJ acts in opposition to Cdc42 in this process through competition for a shared partner, PAK3. These studies identify a critical role for RhoJ in matrix remodeling during blood vessel formation and demonstrate a functional interrelationship between RhoJ and its evolutionary parent.  

3.3697           Extracellular Vesicle-Associated Proteins in Tissue Repair

Roefs, M.T., Sluijter, J.P.G. and Vader, P. Trends in Cell Biol., 30(12), 990-1013 (2020)   The administration of (stem) cell-derived extracellular vesicles (EVs) promotes tissue repair through management of different inflammatory, proliferative and remodeling processes in the body. Despite the widely observed biological and therapeutic roles of EVs in wound healing and tissue repair, knowledge on how EVs activate recipient cells and which EV cargo is responsible for the subsequent functional effects is limited. Recent studies hint toward an important role for proteins as functional EV cargo. Here, we provide an overview of how EV-associated proteins promote tissue repair processes and discuss current challenges in evaluating their contribution to EV function and future directions for translating fundamental insights into clinically relevant EV therapies.  

3.3698           MFSD12 mediates the import of cysteine into melanosomes and lysosomes

Adelmann, C.H., Traunbauer, A.K., Chen, B., Condon, K.J., Chan, S.H., Kunchok, ., Lewis, C.A. and Sabatini, D.M. Nature, 588, 699-704 (2020)   Dozens of genes contribute to the wide variation in human pigmentation. Many of these genes encode proteins that localize to the melanosome—the organelle, related to the lysosome, that synthesizes pigment—but have unclear functions1^,2. Here we describe MelanoIP, a method for rapidly isolating melanosomes and profiling their labile metabolite contents. We use this method to study MFSD12, a transmembrane protein of unknown molecular function that, when suppressed, causes darker pigmentation in mice and humans3^,4. We find that MFSD12 is required to maintain normal levels of cystine—the oxidized dimer of cysteine—in melanosomes, and to produce cysteinyldopas, the precursors of pheomelanin synthesis made in melanosomes via cysteine oxidation5^,6. Tracing and biochemical analyses show that MFSD12 is necessary for the import of cysteine into melanosomes and, in non-pigmented cells, lysosomes. Indeed, loss of MFSD12 reduced the accumulation of cystine in lysosomes of fibroblasts from patients with cystinosis, a lysosomal-storage disease caused by inactivation of the lysosomal cystine exporter cystinosin7^,8^,9. Thus, MFSD12 is an essential component of the cysteine importer for melanosomes and lysosomes.  

3.3699           Coupling of terminal differentiation deficit with neurodegenerative pathology in Vps35-deficient pyramidal neurons

Tang, F-L., Zhao, L., Zhao, Y., Sun, D., Zhu, X-J., Mei, L. and Xiong, W-C. Cell Death & Differentiation, 27, 2099-2116 (2020)   Vps35 (vacuolar protein sorting 35) is a key component of retromer that regulates transmembrane protein trafficking. Dysfunctional Vps35 is a risk factor for neurodegenerative diseases, including Parkinson’s and Alzheimer’s diseases. Vps35 is highly expressed in developing pyramidal neurons, and its physiological role in developing neurons remains to be explored. Here, we provide evidence that Vps35 in embryonic neurons is necessary for axonal and dendritic terminal differentiation. Loss of Vps35 in embryonic neurons results in not only terminal differentiation deficits, but also neurodegenerative pathology, such as cortical brain atrophy and reactive glial responses. The atrophy of neocortex appears to be in association with increases in neuronal death, autophagosome proteins (LC3-II and P62), and neurodegeneration associated proteins (TDP43 and ubiquitin-conjugated proteins). Further studies reveal an increase of retromer cargo protein, sortilin1 (Sort1), in lysosomes of Vps35-KO neurons, and lysosomal dysfunction. Suppression of Sort1 diminishes Vps35-KO-induced dendritic defects. Expression of lysosomal Sort1 recapitulates Vps35-KO-induced phenotypes. Together, these results demonstrate embryonic neuronal Vps35’s function in terminal axonal and dendritic differentiation, reveal an association of terminal differentiation deficit with neurodegenerative pathology, and uncover an important lysosomal contribution to both events.  

3.3700           The gluconeogenic enzyme PCK1 phosphorylates INSIG1/2 for lipogenesis

Xu, D., Wang, Z., Xia, Y., Shao, F., Xia, W., Wei, Y et al Nature, 580, 530-535 (2020)   Cancer cells increase lipogenesis for their proliferation and the activation of sterol regulatory element-binding proteins (SREBPs) has a central role in this process. SREBPs are inhibited by a complex composed of INSIG proteins, SREBP cleavage-activating protein (SCAP) and sterols in the endoplasmic reticulum. Regulation of the interaction between INSIG proteins and SCAP by sterol levels is critical for the dissociation of the SCAP–SREBP complex from the endoplasmic reticulum and the activation of SREBPs1^,2. However, whether this protein interaction is regulated by a mechanism other than the abundance of sterol—and in particular, whether oncogenic signalling has a role—is unclear. Here we show that activated AKT in human hepatocellular carcinoma (HCC) cells phosphorylates cytosolic phosphoenolpyruvate carboxykinase 1 (PCK1), the rate-limiting enzyme in gluconeogenesis, at Ser90. Phosphorylated PCK1 translocates to the endoplasmic reticulum, where it uses GTP as a phosphate donor to phosphorylate INSIG1 at Ser207 and INSIG2 at Ser151. This phosphorylation reduces the binding of sterols to INSIG1 and INSIG2 and disrupts the interaction between INSIG proteins and SCAP, leading to the translocation of the SCAP–SREBP complex to the Golgi apparatus, the activation of SREBP proteins (SREBP1 or SREBP2) and the transcription of downstream lipogenesis-related genes, proliferation of tumour cells, and tumorigenesis in mice. In addition, phosphorylation of PCK1 at Ser90, INSIG1 at Ser207 and INSIG2 at Ser151 is not only positively correlated with the nuclear accumulation of SREBP1 in samples from patients with HCC, but also associated with poor HCC prognosis. Our findings highlight the importance of the protein kinase activity of PCK1 in the activation of SREBPs, lipogenesis and the development of HCC.  

3.3701           Negative feedback control of neuronal activity by microglia

Badimon, A., Strasburger, H.J., Ayata, P., Chen, X., Nair, A. et al Nature, 586, 417-423 (2020)   Microglia, the brain’s resident macrophages, help to regulate brain function by removing dying neurons, pruning non-functional synapses, and producing ligands that support neuronal survival1. Here we show that microglia are also critical modulators of neuronal activity and associated behavioural responses in mice. Microglia respond to neuronal activation by suppressing neuronal activity, and ablation of microglia amplifies and synchronizes the activity of neurons, leading to seizures. Suppression of neuronal activation by microglia occurs in a highly region-specific fashion and depends on the ability of microglia to sense and catabolize extracellular ATP, which is released upon neuronal activation by neurons and astrocytes. ATP triggers the recruitment of microglial protrusions and is converted by the microglial ATP/ADP hydrolysing ectoenzyme CD39 into AMP; AMP is then converted into adenosine by CD73, which is expressed on microglia as well as other brain cells. Microglial sensing of ATP, the ensuing microglia-dependent production of adenosine, and the adenosine-mediated suppression of neuronal responses via the adenosine receptor A₁R are essential for the regulation of neuronal activity and animal behaviour. Our findings suggest that this microglia-driven negative feedback mechanism operates similarly to inhibitory neurons and is essential for protecting the brain from excessive activation in health and disease.  

3.3702           The innate immunity protein IFITM3 modulates γ-secretase in Alzheimer’s disease

Hur, J-Y., Frost, G.R., Wu, X., Crump, C., Pan, S.J. et al Nature, 586, 735-740 (2020)   Innate immunity is associated with Alzheimer’s disease1, but the influence of immune activation on the production of amyloid-β is unknown2^,3. Here we identify interferon-induced transmembrane protein 3 (IFITM3) as a γ-secretase modulatory protein, and establish a mechanism by which inflammation affects the generation of amyloid-β. Inflammatory cytokines induce the expression of IFITM3 in neurons and astrocytes, which binds to γ-secretase and upregulates its activity, thereby increasing the production of amyloid-β. The expression of IFITM3 is increased with ageing and in mouse models that express familial Alzheimer’s disease genes. Furthermore, knockout of IFITM3 reduces γ-secretase activity and the formation of amyloid plaques in a transgenic mouse model (5xFAD) of early amyloid deposition. IFITM3 protein is upregulated in tissue samples from a subset of patients with late-onset Alzheimer’s disease that exhibit higher γ-secretase activity. The amount of IFITM3 in the γ-secretase complex has a strong and positive correlation with γ-secretase activity in samples from patients with late-onset Alzheimer’s disease. These findings reveal a mechanism in which γ-secretase is modulated by neuroinflammation via IFITM3 and the risk of Alzheimer’s disease is thereby increased.  

3.3703           Microglia and macrophages promote corralling, wound compaction and recovery after spinal cord injury via Plexin-B2

Zhou, X., Wahane, S., Fried, M-S., Kluge, M., Friedel, C.C., Avrampou, K., Zachariou, V., Guo, L., Zhang, B., He, X., Friedel, R.H., and Zou, H. Nature Neurosci., 23, 337-350 (2020)   Tissue repair after spinal cord injury requires the mobilization of immune and glial cells to form a protective barrier that seals the wound and facilitates debris clearing, inflammatory containment and matrix compaction. This process involves corralling, wherein phagocytic immune cells become confined to the necrotic core, which is surrounded by an astrocytic border. Here we elucidate a temporally distinct gene signature in injury-activated microglia and macrophages (IAMs) that engages axon guidance pathways. Plexin-B2 is upregulated in IAMs and is required for motor sensory recovery after spinal cord injury. Plexin-B2 deletion in myeloid cells impairs corralling, leading to diffuse tissue damage, inflammatory spillover and hampered axon regeneration. Corralling begins early and requires Plexin-B2 in both microglia and macrophages. Mechanistically, Plexin-B2 promotes microglia motility, steers IAMs away from colliding cells and facilitates matrix compaction. Our data therefore establish Plexin-B2 as an important link that integrates biochemical cues and physical interactions of IAMs with the injury microenvironment during wound healing.  

3.3704           Single-cell epigenomic analyses implicate candidate causal variants at inherited risk loci for Alzheimer’s and Parkinson’s diseases

Corces, M.R., Shcherbina, A., Kundu, S., Gloudemans, M.J., Fresard, L. et al Nature Genet., 52, 1158-1168 (2020)   Genome-wide association studies of neurological diseases have identified thousands of variants associated with disease phenotypes. However, most of these variants do not alter coding sequences, making it difficult to assign their function. Here, we present a multi-omic epigenetic atlas of the adult human brain through profiling of single-cell chromatin accessibility landscapes and three-dimensional chromatin interactions of diverse adult brain regions across a cohort of cognitively healthy individuals. We developed a machine-learning classifier to integrate this multi-omic framework and predict dozens of functional SNPs for Alzheimer’s and Parkinson’s diseases, nominating target genes and cell types for previously orphaned loci from genome-wide association studies. Moreover, we dissected the complex inverted haplotype of the MAPT (encoding tau) Parkinson’s disease risk locus, identifying putative ectopic regulatory interactions in neurons that may mediate this disease association. This work expands understanding of inherited variation and provides a roadmap for the epigenomic dissection of causal regulatory variation in disease.  

3.3705           Exosome mimicry by a HAVCR1–NPC1 pathway of endosomal fusion mediates hepatitis A virus infection

Costafreda, M.I., Abbasi, A., Lu, H. and Kaplan, G. Nature Microbiol., 5, 1096-1106 (2020)   Cell-to-cell communication by exosomes controls normal and pathogenic processes1^,2. Viruses can spread in exosomes and thereby avoid immune recognition3. While biogenesis, binding and uptake of exosomes are well characterized4^,5, delivery of exosome cargo into the cytoplasm is poorly understood3. We report that the phosphatidylserine receptor HAVCR1 (refs. 6^,7) and the cholesterol transporter NPC1 (ref. 8) participate in cargo delivery from exosomes of hepatitis A virus (HAV)-infected cells (exo-HAV) by clathrin-mediated endocytosis. Using CRISPR–Cas9 knockout technology, we show that these two lipid receptors, which interact in the late endosome9, are necessary for the membrane fusion and delivery of RNA from exo-HAV into the cytoplasm. The HAVCR1–NPC1 pathway, which Ebola virus exploits to infect cells9, mediates HAV infection by exo-HAV, which indicates that viral infection via this exosome mimicry mechanism does not require an envelope glycoprotein. The capsid-free viral RNA in the exosome lumen, but not the endosomal uncoating of HAV particles contained in the exosomes, is mainly responsible for exo-HAV infectivity as assessed by methylene blue inactivation of non-encapsidated RNA. In contrast to exo-HAV, infectivity of HAV particles is pH-independent and requires HAVCR1 or another as yet unidentified receptor(s) but not NPC1. Our findings show that envelope-glycoprotein-independent fusion mechanisms are shared by exosomes and viruses, and call for a reassessment of the role of envelope glycoproteins in infection.  

3.3706           Exosomes derived from Vδ2-T cells control Epstein-Barr virus–associated tumors and induce T cell antitumor immunity

Wang, X., Xiang, Z., Liu, Y., Huang, C., Pe,I, Y., Wang, X., Zhi, H. et al Sci. Transl. Med., 12, eaaz3426 (2020)   Treatment of life-threatening Epstein-Barr virus (EBV)–associated tumors remains a great challenge, especially for patients with relapsed or refractory disease. Here, we found that exosomes derived from phosphoantigen-expanded Vδ2-T cells (Vδ2-T-Exos) contained death-inducing ligands (FasL and TRAIL), an activating receptor for natural killer (NK) cells (NKG2D), immunostimulatory ligands (CD80 and CD86), and antigen-presenting molecules (MHC class I and II). Vδ2-T-Exos targeted and efficiently killed EBV-associated tumor cells through FasL and TRAIL pathways and promoted EBV antigen–specific CD4 and CD8 T cell expansion. Administration of Vδ2-T-Exos effectively controlled EBV-associated tumors in Rag2^−/−γc^−/− and humanized mice. Because expanding Vδ2-T cells and preparing autologous Vδ2-T-Exos from cancer patients ex vivo in large scale is challenging, we explored the antitumor activity of allogeneic Vδ2-T-Exos in humanized mouse cancer models. Here, we found that allogeneic Vδ2-T-Exos had more effective antitumor activity than autologous Vδ2-T-Exos in humanized mice; the allogeneic Vδ2-T-Exos increased the infiltration of T cells into tumor tissues and induced more robust CD4 and CD8 T cell–mediated antitumor immunity. Compared with exosomes derived from NK cells (NK-Exos) with direct cytotoxic antitumor activity or dendritic cells (DC-Exos) that induced T cell antitumor responses, Vδ2-T-Exos directly killed tumor cells and induced T cell–mediated antitumor response, thus resulting in more effective control of EBV-associated tumors. This study provided proof of concept for the strategy of using Vδ2-T-Exos, especially allogeneic Vδ2-T-Exos, to treat EBV-associated tumors.  

3.3707           Identification of a degradation signal at the carboxy terminus of SREBP2: A new role for this domain in cholesterol homeostasis

Kober, D.L., Xu, S., Li, S., Bajaj, B., Liang, G., Rosenbaum, D.M and Radhakrishnan, A.

PNAS, 117(45), 28080-28091 (2020)   Lipid homeostasis in animal cells is maintained by sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors whose proteolytic activation requires the cholesterol-sensing membrane protein Scap. In endoplasmic reticulum (ER) membranes, the carboxyl-terminal domain (CTD) of SREBPs binds to the CTD of Scap. When cholesterol levels are low, Scap escorts SREBPs from the ER to the Golgi, where the actions of two proteases release the amino-terminal domains of SREBPs that travel to the nucleus to up-regulate expression of lipogenic genes. The CTD of SREBP remains bound to Scap but must be eliminated so that Scap can be recycled to bind and transport additional SREBPs. Here, we provide insights into how this occurs by performing a detailed molecular dissection of the CTD of SREBP2, one of three SREBP isoforms expressed in mammals. We identify a degradation signal comprised of seven noncontiguous amino acids encoded in exon 19 that mediates SREBP2’s proteasomal degradation in the absence of Scap. When bound to the CTD of Scap, this signal is masked and SREBP2 is stabilized. Binding to Scap requires an arginine residue in exon 18 of SREBP2. After SREBP2 is cleaved in Golgi, its CTD remains bound to Scap and returns to the ER with Scap where it is eliminated by proteasomal degradation. The Scap-binding motif, but not the degradation signal, is conserved in SREBP1. SREBP1’s stability is determined by a degradation signal in a different region of its CTD. These findings highlight a previously unknown role for the CTD of SREBPs in regulating SREBP activity.  

3.3708           Extracellular vesicles as biomarkers and therapeutic targets for cancer

Urabe, F., Kosaka, N., ito, K., Kimura, T., Egawa, S. and Ichiy, T. Am. J. Physiol. Cell Physiol., 318, C29-C39 (2020)   Extracellular vesicles (EVs) are small lipid membrane vesicles that are secreted from almost all kinds of cells into the extracellular space. EVs are widely accepted to be involved in various cellular processes; in particular, EVs derived from cancer cells have been reported to play important roles in modifying the tumor microenvironment and promoting tumor progression. In addition, EVs derived from cancer cells encapsulate various kinds of tumor-specific molecules, such as proteins and RNAs, which contribute to cancer malignancy. Therefore, the unveiling of the precise mechanism of intercellular communication via EVs in cancer patients will provide a novel strategy for cancer treatment. Furthermore, a focus on the contents of EVs could promote the use of EVs in body fluids as clinically useful diagnostic and prognostic biomarkers. In this review, we summarize the current research knowledge on EVs as biomarkers and therapeutic targets and discuss their potential clinical applications.  

3.3709           Circulating extracellular vesicles from patients with valvular heart disease induce neutrophil chemotaxis via FOXO3a and the inhibiting role of dexmedetomidine

Yuan, H-X., Chen, C-Y., Li, Y-Q., Ning, D-S., Li, Y. et al Am. J. Physiol. Endocrinol. Metab., 319, E217-E231 (2020)   We previously demonstrated that circulating extracellular vesicles (EVs) from patients with valvular heart disease (VHD; vEVs) contain inflammatory components and inhibit endothelium-dependent vasodilation. Neutrophil chemotaxis plays a key role in renal dysfunction, and dexmedetomidine (DEX) can reduce renal dysfunction in cardiac surgery. However, the roles of vEVs in neutrophil chemotaxis and effects of DEX on vEVs are unknown. Here, we investigated the impact of vEVs on neutrophil chemotaxis in kidneys and the influence of DEX on vEVs. Circulating EVs were isolated from healthy subjects and patients with VHD. The effects of EVs on chemokine generation, forkhead box protein O3a (FOXO3a) pathway activation and neutrophil chemotaxis on cultured human umbilical vein endothelial cells (HUVECs) and kidneys in mice and the influence of DEX on EVs were detected. vEVs increased FOXO3a expression, decreased phosphorylation of Akt and FOXO3a, promoted FOXO3a nuclear translocation, and activated the FOXO3a signaling pathway in vitro. DEX pretreatment reduced vEV-induced CXCL4 and CCL5 expression and neutrophil chemotaxis in cultured HUVECs via the FOXO3a signaling pathway. vEVs were also found to suppress Akt phosphorylation and activate FOXO3a signaling to increase plasma levels of CXCL4 and CCL5 and neutrophil accumulation in kidney. The overall mechanism was inhibited in vivo with DEX pretreatment. Our data demonstrated that vEVs induced CXCL4-CCL5 to stimulate neutrophil infiltration in kidney, which can be inhibited by DEX via the FOXO3a signaling. Our findings reveal a unique mechanism involving vEVs in inducing neutrophils chemotaxis and may provide a novel basis for using DEX in reducing renal dysfunction in valvular heart surgery.  

3.3710           Phase separation of the Cep63•Cep152 complex underlies the formation of dynamic supramolecular self-assemblies at human centrosomes

Ahn II, J., Park, J-E-., Meng, L., Zhang, L., Kim, T-S., Kruhlak, M.J., Kim, B.Y. and Lee, K.S. Cell Cycle, 19:24, 3437-3457 (2020)   The centrosome is a unique membraneless organelle that plays a pivotal role in the orderly progression of the cell cycle in animal cells. It has been shown that two pericentriolar scaffold proteins, Cep63 and Cep152, generate a heterotetrameric complex to self-assemble into a higher-order cylindrical architecture around a centriole. However, the mechanisms underlying how they reach their threshold concentrations in the vast intracellular space and generate a self-assembled architecture remain mysterious. Here we demonstrate that, like liquid-like assemblies, Cep63 and Cep152 cooperatively generate amorphous aggregates capable of undergoing dynamic turnover and inter-aggregate fusion in vivo and a significant level of internal rearrangemefnt within a condensate in vitro. Consistently, 1,6-hexanediol, a liquid–liquid phase separation disruptor, greatly diminished the ability of endogenous Cep63 and Cep152 to localize to centrosomes. Interestingly, a purified Cep63•Cep152 complex generated either a cylindrical structure or a vesicle-like hollow sphere in a spatially controlled manner. It also formed condensate-like solid spheres in the presence of a macromolecular crowder. At the molecular level, two hydrophobic motifs, one each from Cep63 and Cep152, were required for generating phase-separating condensates and a high molecular–weight assembly. Thus, we propose that the self-assembly of the Cep63•Cep152 complex is triggered by an intrinsic property of the complex undergoing density transition through the hydrophobic-motif-mediated phase separation.  

3.3711           Characterization of GFP-AtPEN1 as a marker protein for extracellular vesicles isolated from Nicotiana benthamiana leaves

Zhang, J., Qiu, Y. and Xu, K. Plant Signaling & Behavior, 15:9, 1791519 (2020)   Plant extracellular vesicles (EVs) are cell-secreted membrane structures enclosing cytosolic components, including pathogenesis-related proteins, tiny RNAs, and microRNAs et al. Their roles are shown to be involved in plant-microbe interactions. Albeit several marker proteins were developed for EVs labeling for Arabidopsis thaliana and other plant species, we lack similar knowledge on EVs isolated from model plant Nicotiana benthamiana, which serves as an excellent host for plant pathogen studies. Here, we transiently expressed two arabidopsis EV markers AtPEN1 and AtTET8 and one ESCRT protein VPS4 in Nicotiana benthamiana leaves and tested for their ability in EV labeling. We found that GFP tagged AtPEN1 expression in Nicotiana benthamiana leaves is more stable than other proteins tested, and GFP-AtPEN1 accumulated in Nicotiana benthamiana EVs. Furthermore, we showed that EVs isolated from Nicotiana benthamiana leaf apoplast have typical EV density and vesicle-like morphology. Our finding demonstrates that GFP-AtPEN1 can be used as an excellent marker protein to label Nicotiana benthamiana EVs.  

3.3712           Improvement, scaling-up, and downstream analysis of exosome production

Jafari, D., Malih, S., Eini, M., jafari, R., Gholipourmalekabadi, M., Sadeghizadeh, M. and Samadikuchsaraei, A. Crit. Rev. Biotechnol., 40:8, 1098-1112 (2020)   Exosomes are the most researched extracellular vesicles. In many biological, physiological, and pathological studies, they have been identified as suitable candidates for treatment and diagnosis of diseases by acting as the carriers of both drugs and genes. Considerable success has been achieved regarding the use of exosomes for tissue regeneration, cancer diagnosis, and targeted drug/gene delivery to specific tissues. While major progress has been made in exosome extraction and purification, extraction of large quantities of exosomes is still a major challenge. This issue limits the scope of both exosome-based research and therapeutic development. In this review, we have aimed to summarize experimental studies focused at increasing the number of exosomes. Biotechnological studies aimed at identifying the pathways of exosome biogenesis to manipulate some genes in order to increase the production of exosomes. Generally, two major strategies are employed to increase the production of exosomes. First, oogenesis pathways are genetically manipulated to overexpress activator genes of exosome biogenesis and downregulate the genes involved in exosome recycling pathways. Second, manipulation of the cell culture medium, treatment with specific drugs, and limiting certain conditions can force the cell to produce more exosomes. In this study, we have reviewed and categorized these strategies. It is hoped that the information presented in this review will provide a better understanding for expanding biotechnological approaches in exosome-based therapeutic development.  

3.3713           Biological Role of Arrestin-1 Oligomerization

Samaranayke, S., Vishnivetskiy, S.A:, Shores, C.R., Thibeault, K.C., Kook, S., Chen, J., Burns, M.E., Gurevich, E.V. and Gurevich, V.V.

1. Neurosci., 40(42), 8055-8069 (2020)

  Members of the arrestin superfamily have great propensity of self-association, but the physiological significance of this phenomenon is unclear. To determine the biological role of visual arrestin-1 oligomerization in rod photoreceptors, we expressed mutant arrestin-1 with severely impaired self-association in mouse rods and analyzed mice of both sexes. We show that the oligomerization-deficient mutant is capable of quenching rhodopsin signaling normally, as judged by electroretinography and single-cell recording. Like wild type, mutant arrestin-1 is largely excluded from the outer segments in the dark, proving that the normal intracellular localization is not due the size exclusion of arrestin-1 oligomers. In contrast to wild type, supraphysiological expression of the mutant causes shortening of the outer segments and photoreceptor death. Thus, oligomerization reduces the cytotoxicity of arrestin-1 monomer, ensuring long-term photoreceptor survival.  

3.3714           MeCP2 Levels Regulate the 3D Structure of Heterochromatic Foci in Mouse Neurons

Ito-Ishida, A., Baker, S.A., Sillitoe, R.V., Sun, Y., Zhou, J., Ono, Y., Iwakiri, J., Yuzaki, M. and Zoghbi, H.Y.

1. Neurosci., 40(45), 8746-8766 (2020)

  Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein critical for normal brain function, and both depletion and overexpression of MeCP2 lead to severe neurodevelopmental disease, Rett syndrome (RTT) and MECP2 multiplication disorder, respectively. However, the molecular mechanism by which abnormal MeCP2 dosage causes neuronal dysfunction remains unclear. As MeCP2 expression is nearly equivalent to that of core histones and because it binds DNA throughout the genome, one possible function of MeCP2 is to regulate the 3D structure of chromatin. Here, to examine whether and how MeCP2 levels impact chromatin structure, we used high-resolution confocal and electron microscopy and examined heterochromatic foci of neurons in mice. Using models of RTT and MECP2 triplication syndrome, we found that the heterochromatin structure was significantly affected by the alteration in MeCP2 levels. Analysis of mice expressing either MeCP2-R270X or MeCP2-G273X, which have nonsense mutations in the upstream and downstream regions of the AT-hook 2 domain, respectively, showed that the magnitude of heterochromatin changes was tightly correlated with the phenotypic severity. Postnatal alteration in MeCP2 levels also induced significant changes in the heterochromatin structure, which underscored importance of correct MeCP2 dosage in mature neurons. Finally, functional analysis of MeCP2-overexpressing mice showed that the behavioral and transcriptomic alterations in these mice correlated significantly with the MeCP2 levels and occurred in parallel with the heterochromatin changes. Taken together, our findings demonstrate the essential role of MeCP2 in regulating the 3D structure of neuronal chromatin, which may serve as a potential mechanism that drives pathogenesis of MeCP2-related disorders.  

3.3715           Intranasal Delivery of lincRNA-Cox2 siRNA Loaded Extracellular Vesicles Decreases Lipopolysaccharide-Induced Microglial Proliferation in Mice

Liao, K., Niu, F., Dagur, R.S., He, M., Tian, C. and Hu, G.

1. Neuroimmun. Pharmacol., 15, 390-399 (2020)

  Long non-coding RNAs (lncRNAs), including long intergenic non-coding RNAs (lincRNAs), play an important regulatory role in controlling various biological processes. Both in vitro and in vivo studies have demonstrated that lincRNA-Cox2 plays a global regulatory role in regulating the expression of immune genes. Extracellular vesicles (EVs) are cell-derived nanosized membrane vesicles that have gained increasing attention in recent years due to their ability to efficiently deliver therapeutics to specific target organs or cell types. In this study, we found that lincRNA-Cox2 controls the expression of a set of cell cycle genes in lipopolysaccharide (LPS)-stimulated microglial cells. Our in vitro study suggested that knocking down lincRNA-Cox2 reversed LPS-induced microglial proliferation. In addition, our in vivo study demonstrated that intranasally delivered lincRNA-Cox2-siRNA loaded EVs could reach the brain resulting in a significant decrease in the expression of lincRNA-Cox2 in the microglia. Importantly, lincRNA-Cox2-siRNA loaded EVs also decreased LPS-induced microglial proliferation in mice. These findings indicate that intranasal delivery of EV-loaded small RNA could be developed as therapeutics for treatment of a multitude of CNS disorders.  

3.3716           Extracellular Vesicles as Drug Delivery Vehicles to the Central Nervous System

Shahjin, F., Chand, S. and Yelamanchili, S.V.

1. Neuroimmun. Pharmacol., 15, 443-458 (2020)

  Effective drug delivery to the CNS to achieve the desired therapeutic response is a significant challenge in the field of drug delivery. In central nervous system (CNS), blood brain barrier (BBB) restricts the desired therapeutic responses due to inefficient targeting, release kinetics, and failure to reach therapeutic concentrations in the brain. Therefore, most potentially beneficial diagnostic and therapeutic agents are not able to reach to the brain upon systemic administration. Despite the existence of many invasive techniques to promote drug deliveries across BBB, novel strategies of drug delivery system which can cross BBB effectively are required, otherwise translation of novel neurotherapeutics from bench to bedside will be difficult to achieve. In this review, we briefly outline the existing and emerging strategies for CNS drug deliveries with a focus on potential and challenges of using extracellular vesicles (EVs) in CNS drug delivery system. EVs are emerging as a promising tool for therapeutic delivery owing to its favorable intrinsic features of biocompatibility, stability, stealth capacity, ability to overcome natural barriers and inherent homing capability. EVs are nanovesicles that allow cell-cell communication. The EVs-cargo reflects the physiological as well as the pathophysiological state of a cell. EVs are shown to play a role in human immunodeficiency virus (HIV) infection and dissemination, which contributes to acquired immune deficiency syndrome (AIDS). In the context of HIV-1 infection, this review also outlines the role of EVs in dissemination, challenges faced in EVs research in HIV-1 co-morbid conditions and potential of nanotechnologies, especially EVs in Neuro-AIDS.  

3.3717           Removal of Trans-2-nonenal Using Hen Egg White Lysosomal-Related Enzymes

Lee, S., Kim, J.H., Nguyen, N-T., Park, R-M., Lee, S-M., Bang, S.H., Jeon, G., Lee, J., Kim, S., Cho, B-K., Kim, Y-H. and Min, J. Mol. Biotechnol., 62, 380-386 (2020)   2-Nonenal is a long-chain aliphatic aldehyde containing nine carbons and an unsaturated bond. 2-Nonenal is the primary cause of odor associated with aging, with an unpleasant greasy and grassy odor. Lysosome, mitochondria, and peroxisome are significant organelles in eukaryotic cells that contain various hydrolases that degrade biomolecules. Proteins in mitochondria and peroxisome also contain aldehyde dehydrogenase. We performed trans-2-nonenal treatment using lysosomal-related enzymes extracted from hen egg white (HEW). As trans-2-nonenal is more structurally stable than cis-2-nonenal, it was selected as the target aldehyde. HEW contains various biologically active proteins and materials such as albumin, ovotransferrin, lysosome, peroxisome, and mitochondria. Here, complementary experiments were conducted to evaluate the role of lysosomal-related enzymes in the treatment of trans-2-nonenal. The activity of lysosomal-related enzymes was confirmed via antimicrobial test against E. coli. HPLC analysis was used to determine the reduction of trans-2-nonenal. The trans-2-nonenal treatment depended on the reaction time and enzyme concentration. Materials considered as an intermediate from trans-2-nonenal treatment were detected by GC/MS spectrometer. Under acidic conditions (pH 6), lysosomal-related enzymes were the most efficient in the treatment of trans-2-nonenal. Furthermore, based on differential pH testing, we found the conditions under which all the 50 ppm trans-2-nonenal was removed. Therefore, our results suggest that the lysosomal-related enzymes reduced trans-2-nonenal, suggesting clinical application as anti-aging deodorants.  

3.3718           Crossing Species Barriers Relies on Structurally Distinct Prion Assemblies and Their Complementation

Igel-Egalon, A., Laferriere, F., Tixador, P., Moudjou, M., Herzog, L., Reine, F., Torres, J.M., Laude, H., Rezaei, H. and Beringue, V. Mol. Neurobiol., 57, 2572-2587 (2020)   Prion replication results from the autocatalytic templated assisted conversion of the host-encoded prion protein PrP^C into misfolded, polydisperse PrP^Sc conformers. Structurally distinct PrP^Sc conformers can give rise to multiple prion strains. Within and between prion strains, the biological activity (replicative efficacy and specific infectivity) of PrP^Sc assemblies is size dependent and thus reflects an intrinsic structural heterogeneity. The contribution of such PrP^Sc heterogeneity across species prion adaptation, which is believed to be based on fit adjustment between PrP^Sc template(s) and host PrP^C, has not been explored. To define the structural-to-fitness PrP^Sc landscape, we measured the relative capacity of size-fractionated PrP^Sc assemblies from different prion strains to cross mounting species barriers in transgenic mice expressing foreign PrP^C. In the absence of a transmission barrier, the relative efficacy of the isolated PrP^Sc assemblies to induce the disease is like the efficacy observed in the homotypic context. However, in the presence of a transmission barrier, size fractionation overtly delays and even abrogates prion pathogenesis in both the brain and spleen tissues, independently of the infectivity load of the isolated assemblies. Altering by serial dilution PrP^Sc assembly content of non-fractionated inocula aberrantly reduces their specific infectivity, solely in the presence of a transmission barrier. This suggests that synergy between structurally distinct PrP^Sc assemblies in the inoculum is requested for crossing the species barrier. Our data support a mechanism whereby overcoming prion species barrier requires complementation between structurally distinct PrP^Sc assemblies. This work provides key insight into the “quasispecies” concept applied to prions, which would not necessarily rely on prion substrains as constituent but on structural PrP^Sc heterogeneity within prion population.  

3.3719           Advances of exosome isolation techniques in lung cancer

Maahgoub, E.O., Razmara, E., Bitaraf, A., Norouzi, F-S., Monzeri, M., Behzadi-Andouhjerdi, R., Falahati, M., Cheng, K., Haik, Y., Hasan, A. and Babashah, S. Mol. Biol. Reports, 47, 7229-7251 (2020)   Lung cancer (LC) is among the leading causes of death all over the world and it is often diagnosed at advanced or metastatic stages. Exosomes, derived from circulating vesicles that are released from the multivesicular body, can be utilized for diagnosis and also the prognosis of LC at early stages. Exosomal proteins, RNAs, and DNAs can help to better discern the prognostic and diagnostic features of LC. To our knowledge, there are various reviews on LC and the contribution of exosomes, but none of them are about the exome techniques and also their efficiency in LC. To fill this gap, in this review, we summarize the recent investigations regarding isolation and also the characterization of exosomes of LC cells. Furthermore, we discuss the noncoding RNAs as biomarkers and their applications in the diagnosis and prognosis of LC. Finally, we compare the efficacy of exosome isolation methods to better fi + 6 + guring out feasible techniques.  

3.3720           Identification of biomarkers associated with extracellular vesicles based on an integrative pan-cancer bioinformatics analysis

Wang, Q. and Yu, C. Med. Oncol., 37:79 (2020)   Extracellular vesicle (EV) has received increasing attention over the last decade. However, biomarkers and mechanisms underlying remain largely limited. Three microarray profiles, GSE78718 (K562 leukemia cell line), GSE45301 (U87-MG glioblastoma cell line), and GSE9589 (SW480 colon cancer cell line), were analyzed for the overlapped differentially expressed genes (DEGs). SurvExpress was used for the prognostic analysis of hub genes signature. Predicted transcription factors networks were built by NetworkAnalysis. Characterization between hub genes and immune cells was analyzed by the tumor immune estimation resources (TIMER) and single-sample gene set enrichment analysis (ssGSEA). The most significantly enriched pathway was lysosome. Hub genes included lysosomal-associated membrane protein 1 (LAMP1), heat shock protein family A (Hsp70) member 5 (HSPA5), lysosomal-associated membrane protein 2 (LAMP2), integrin subunit alpha V (ITGAV), and transmembrane protein 30A (TMEM30A). Significant prognostic values of hub genes signature were identified in glioblastoma (P-value = 0.006), but not colon cancer. In colon cancer, ITGAV displayed remarkably high correlation with tumor immune infiltrating cells. In glioblastoma, the highest correlation was found between HSPA5 and dendritic cell. Moreover, distinct association of immune cells between cell and EV were identified via ssGSEA. This study identified biomarkers in EV with potential immunological insights and clinical values.  

3.3721           Proteomic and metabolic characterization of membrane vesicles derived from Streptococcus mutans at different pH values

Cao, Y., Zhou, Y., Chen, D., Wu, R., Guo, L. and Lin, H. Appl. Microbiol. Biotechnol., 104, 9733-9748 (2020)   Bacterial membrane vesicles (MVs) are used as a tool for intercellular communication and seem essential for bacterial survival. However, few data are available on MVs generated by Streptococcus mutans, which is the main aetiological agent of dental caries. The present study presents an integrated proteomics and metabolomics analysis of MVs isolated from S. mutans at initial pH values of 7.5 and 5.5 and explores their function. The results showed that S. mutans releases more MVs with smaller diameters under acidic conditions than under neutral conditions. Proteomic analysis showed 344 common vesicular proteins, including various virulence factors. The expressions of 140 individual proteins and 37 metabolites were altered as a result of culturing S. mutans at different pH values. Co-analyses of proteomic and metabolomics data indicated that ABC transporters underwent significant changes under acid pressure. We concluded that S. mutans produced MVs at different pH values to carry proteins associated with cariogenesis. Moreover, the alterations of S. mutans MVs under acid pressure were associated with ABC transporters. These results increase our knowledge of S. mutans MVs and imply that S. mutans MVs may play a functional role in carious infection.  

3.3722           Impact of Increased FUT8 Expression on the Extracellular Vesicle Proteome in Prostate Cancer Cells

Clark, D.J., Schnaubelt, M., Hoti, N., Hu, Y., Zhou, Y., Gooya, M and Zhang, H.

1. Proteome Res., 19, 2195-2205 (2020)

  Extracellular vesicles (EVs) are involved in intercellular communication, transporting proteins and nucleic acids to proximal and distal regions. There is evidence of glycosylation influencing protein routing into EVs; however, the impact of aberrant cellular glycotransferase expression on EV protein profiles has yet to be evaluated. In this study, we paired extracellular vesicle characterization and quantitative proteomics to determine the systemic impact of altered α(1,6)fucosyltranferase (FUT8) expression on prostate cancer-derived EVs. Our results showed that increased cellular expression of FUT8 could reduce the number of vesicles secreted by prostate cancer cells as well as increase the abundance of proteins associated with cell motility and prostate cancer metastasis. In addition, overexpression of FUT8 resulted in altered glycans on select EV-derived glycoproteins. This study presents the first evidence of altered cellular glycosylation impacting EV protein profiles and provides further rationale for exploring the functional role of glycosylation in EV biogenesis and biology.  

3.3723           Heterogeneous Subcellular Origin of Exosome-Mimetic Nanovesicles Engineered from Cells

Lee, H., Kang, H., Kang, M., Han, C., Yi, J., Kwon, Y and Park, J. ACS Biomater. Sci., 6, 6063-6068 (2020)   Cell-engineered nanovesicles (CNVs) are considered as an alternative to exosomes, because they can be produced efficiently on a large scale and have been successfully reported in several applied research studies. However, CNVs may originate from various organelles, i.e., some of them may cause adverse effects on recipient cells, and their origin has not yet been identified. In this study, we air-sprayed human embryonic kidney 293 (HEK293) cells into lipid-bilayer CNVs. To identify the subcellular origin of the CNVs, we prepared nine different HEK293 cell lines by transfection with organelle-specific fluorescent protein plasmids that target the plasma membrane, peroxisome, lysosome, early endosome, late endosome, nucleus, mitochondrion, Golgi apparatus, and endoplasmic reticulum. The origin of CNVs were identified by measuring fluorescence expressions for organelle-specific markers using fluorescence nanoparticle tracking analysis (NTA). In the results, we found that CNVs derived from the plasma membrane constituted the largest portion, but CNVs derived from the other organelles comprised a non-negligible portion as well. This information will be useful to guide advanced research on outer membrane vesicles and exosome-mimetic nanovesicles engineered from cells.  

3.3724           Proximity Enzymatic Glyco-Remodeling Enables Direct and Highly Efficient Lipid Raft Imaging on Live Cells

Tao, J., Yu, X., Guo, Y., Wang, G., Ju, H and Dding, L. Anal. Chem., 92, 7232-7239 (2020)   Lipid rafts, highly ordered cell membrane domains mainly composed of cholesterol, sphingolipids, and protein receptors, serve as important functional platforms for regulation of lipid/protein interactions. The major predicament in lipid raft study is the lack of direct and robust visualization tools for in situ tracking raft components. To solve this issue, we herein report a proximity enzymatic glyco-remodeling strategy for direct and highly efficient lipid raft labeling and imaging on live cells. Through cofunctionalization of raft-specific recognition motif and glycan-remodeling enzyme on gold nanoparticles, the fabricated nanoprobe can be specifically guided to the raft domains to perform catalytic remodeling on neighboring glycans. Taking advantage of the abundant glycoconjugates enriched in lipid rafts, this elaborate design achieves the translation of one raft-recognition event to multiple raft-confined labeling operations, thus, significantly increasing the labeling efficiency and imaging sensitivity. The direct covalent labeling also enables in situ and long-term tracking of raft components in live cells. The method possesses broad applicability and potential expansibility, thus, will greatly facilitate the investigations on the complex composition, organization, and dynamics of lipid rafts.  

3.3725           Bacterial Vesicles Mediate Extracellular Electron Transfer

Liu, X., Jing, X., Ye, Y., Ye, J. and Zhou, S. Environ. Technol. Lett., 7, 27-34 (2020)   Many Gram-negative bacteria are known to release outer membrane vesicles (OMVs) into the surrounding environment during normal growth; OMVs perform diverse biological and environmental functions (e.g., virulence factor transport, horizontal gene transfer, quorum signaling, cellular defense, and cell-to-cell communication). However, the production of OMVs has not been reported in Geobacter species, and their role in extracellular electron transfer (EET) is unknown. Here, we demonstrate, for the first time, that Geobacter sulfurreducens releases OMVs containing abundant cytochromes that can promote EET from microbial cells to an anode. OMVs released by Geobacter cells not only promote exoelectrogen EET (1.73-fold higher current density in Shewanella oneidensis MR-1) but also confer electrogenic ability to non-exoelectrogens (G. sulfurreducens mutant strain ΔomcZ and Escherichia coli). These functions are mainly attributed to the abundance of c-type cytochromes bound on or entrapped in OMVs. Our findings suggest that redox-active OMVs can serve as shared mediators facilitating EET in natural ecosystems, representing an ecologically important but overlooked biological electron transfer process.  

3.3726           Cln1-mutations suppress Rab7-RILP interaction and impair autophagy contributing to neuropathology in a mouse model of infantile neuronal ceroid lipofuscinosis

Sarkar, C., Sadhukhan, T., Bagh, M.B., Appu, A.P., Chandra, G., Mondal, A., Saha, A. and Mukherjee, A.B.

1. Inherit. Metab. Dis., 43, 1082-1101 (2020)

  Infantile neuronal ceroid lipofuscinosis (INCL) is a devastating neurodegenerative lysosomal storage disease (LSD) caused by inactivating mutations in the CLN1 gene. CLN1 encodes palmitoyl-protein thioesterase-1 (PPT1), a lysosomal enzyme that catalyzes the deacylation of S-palmitoylated proteins to facilitate their degradation and clearance by lysosomal hydrolases. Despite the discovery more than two decades ago that CLN1 mutations causing PPT1-deficiency underlies INCL, the precise molecular mechanism(s) of pathogenesis has remained elusive. Here, we report that autophagy is dysregulated in Cln1^−/− mice, which mimic INCL and in postmortem brain tissues as well as cultured fibroblasts from INCL patients. Moreover, Rab7, a small GTPase, critical for autophagosome-lysosome fusion, requires S-palmitoylation for trafficking to the late endosomal/lysosomal membrane where it interacts with Rab-interacting lysosomal protein (RILP), essential for autophagosome-lysosome fusion. Notably, PPT1-deficiency in Cln1^−/− mice, dysregulated Rab7-RILP interaction and preventing autophagosome-lysosome fusion, which impaired degradative functions of the autolysosome leading to INCL pathogenesis. Importantly, treatment of Cln1^−/− mice with a brain-penetrant, PPT1-mimetic, small molecule, N-tert (butyl)hydroxylamine (NtBuHA), ameliorated this defect. Our findings reveal a previously unrecognized role of CLN1/PPT1 in autophagy and suggest that small molecules functionally mimicking PPT1 may have therapeutic implications.  

3.3727           Regulation of glucose homeostasis by small extracellular vesicles in normal pregnancy and in gestational diabetes

James-Allen, L.B., Rosario, F.J., Barner, K., Lai, A., Guanzon, D., McIntyre, H.D., Lappas, M., Powell, T.L., Salomon, C. and Jansson, T. Faseb J., 34, 5724-5739 (2020)   The mechanisms underpinning maternal metabolic adaptations to a healthy pregnancy and in gestational diabetes mellitus (GDM) remain poorly understood. We hypothesized that small extracellular vesicles (sEVs) isolated from healthy pregnant women promote islet glucose-stimulated insulin secretion (GSIS) and peripheral insulin resistance in nonpregnant mice and that sEVs from GDM women fail to stimulate insulin secretion and cause exacerbated insulin resistance. Small EVs were isolated from plasma of nonpregnant, healthy pregnant, and GDM women at 24-28 weeks of gestation. We developed a novel approach in nonpregnant mice involving a mini-osmotic pump for continuous 4-day jugular venous infusion of sEVs and determined their effects on glucose tolerance in vivo and islets and skeletal muscle in vitro. Fasting insulin was elevated in mice infused with pregnant sEVs as compared to sEVs from nonpregnant and GDM women. Mice infused with sEVs from GDM women developed glucose intolerance. GSIS was increased in mice infused with healthy pregnancy sEVs compared to mice receiving nonpregnant sEVs. GSIS and muscle basal insulin signaling, and insulin responsiveness were attenuated in mice infused with GDM sEVs. sEVs represent a novel mechanism regulating maternal glucose homeostasis in pregnancy and we speculate that altered sEV content contributes to the development of GDM.  

3.3728           High-Fidelity Determination and Tracing of Small Extracellular Vesicle Cargoes

Chen, X., Jia, M., Liu, L., Qiu, X., Zhang, H., Yu, X., Gu, W., Qing, G., Li, Q. et al Small, 16, 2002800 (2020)   Direct tracing of small extracellular vesicle (sEV) cargoes holds unprecedented importance for elucidating the mechanisms involved in intercellular communication. However, high-fidelity determination of sEVs’ molecular cargoes in situ has yet to be achieved due to the difficulty in transporting molecular probes into intact sEVs. Herein, a fLuorescent Intracellular-Guided Hairpin-Tetrahedron (fLIGHT) nanoprobe is described for direct visualization of sEV microRNAs in situ. Integrating the advantages of nondestructive sEV penetration via DNA origami and single-nucleotide discrimination as well as wash-free fluorescence readout using a hairpin probe, the proposed approach enables high-fidelity fluorescence visualization of sEVs’ microRNA without RNA extraction or leakage, demonstrating the potential of on-site tracing of sEV cargoes. This strategy opens an avenue to establishing universal molecular detection and labeling platforms that can facilitate both sEV-derived fundamental biological studies and molecular diagnostics.  

3.3729           Exosomes and other extracellular vesicles in oral and salivary gland cancers

Zhan, C., yang, X., Yin, X. and Hou, J. Oral Diseases, 26, 865-875 (2020)   Extracellular vesicles (EVs, including exosomes) are a group of heterogeneous nanometer-sized vesicles that are released by all types of cells and serve as functional mediators of cell-to-cell communication. This ability is primarily due to their capacity to package and transport various proteins, lipids, and nucleic acids—namely DNA and messenger RNA (mRNA), but also microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). These contents can influence the function and fate of both recipient and donor cells. More and more studies have shown that EVs are involved in every phase of cancer development, mediating bidirectional cross talk between cancer cells and their tissue microenvironment. More specifically, EVs can promote tumor progression by modifying vesicular contents and establishing a distant premetastatic niche with molecules that favor cancer cell proliferation, migration, invasion, metastasis, angiogenesis, and even drug resistance. Given that the packaging of these molecules is known to be tissue-specific, EVs can not only serve as novel prognostic and diagnostic markers but also be used as potential therapeutic targets and vehicles for drug delivery. The present review discusses the current understanding of the multifaceted roles of EVs in the progression of oral and salivary gland cancers, as well as their potential use in clinical applications.  

3.3730           Synaptotagmin-7 enhances calcium-sensing of chromaffin cell granules and slows discharge of granule cargos

Bendahmane, M., Morales, A., Kreutzberger, A.J., Schenk, N.A., Mohan, R., Bakshi, S., Philippe, J.M., Zhang, S., Kiessling, V., Tamm, L.K., Giovannucci, D.R., Jenkins, P.M. and Anantharam, A.

1. Neurochem., 154, 598-617 (2020)

  Synaptotagmin-7 (Syt-7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin-1 (Syt-1). Despite a broad appreciation for the importance of Syt-7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations—mouse chromaffin cells lacking endogenous Syt-7 (KO cells) and a reconstituted system employing cell-derived granules expressing either Syt-7 or Syt-1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt-7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt-7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt-1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt-7, granules expressing only Syt-7 or Syt-1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt-7 confers substantially greater calcium sensitivity to granule fusion than Syt-1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt-7 plays a central role in regulating secretory output from adrenal chromaffin cells.  

3.3731           Chromobacterium violaceum delivers violacein, a hydrophobic antibiotic, to other microbes in membrane vesicles

Chol, S.Y., Lim, S., Cho, G., Kwon, J., Mun, W., Im, H. and Mitchell, R.J. Environ. Microbiol., 22(2), 705-713 (2020)   This study describes Chromobacterium violaceum's use of extracellular membrane vesicles (MVs) to both solubilize and transport violacein to other microorganisms. Violacein is a hydrophobic bisindole with known antibiotic activities against other microorganisms. Characterization of the MVs found they carried more violacein than protein (1.37 ± 0.19-fold), suggesting they may act as a reservoir for this compound. However, MVs are not produced in response to violacein – a ΔvioA isogenic mutant, which is incapable of making violacein, actually produced significantly more MVs (3.2-fold) than the wild-type strain. Although violacein is insoluble in water (Log P_{octanol:water} = 3.34), 79.5% remained in the aqueous phase when it was present within the C. violaceum MVs, an increase in solubility of 1740-fold. Moreover, tests with a strain of Staphylococcus aureus showed MV-associated violacein is bactericidal, with 3.1 mg/l killing 90% of S. aureus in 6 h. Tests with the ΔvioA MVs found no loss in the S. aureus viability, even when its MVs were added at much higher concentrations, demonstrating violacein is the active component within the wild-type MVs. In conclusion, our study clearly demonstrates C. violaceum produces MVs and uses them as vehicles to solubilize violacein and transport this hydrophobic antibiotic to other microbes.  

3.3732           The Potential of Liquid Biopsy of the Brain Using Blood Extracellular Vesicles: The First Step Toward Effective Neuroprotection Against Neurodegenerative Diseases

Abdel-Haq, H. Mol. Diag. Ther., 24(6), 703-713 (2020)   Early diagnosis and biomarker-based ante-mortem tests are essential in efforts against the development of neurodegenerative diseases and can be considered primary neuroprotective measures. Blood is the ideal biofluid for a routine ante-mortem screening test. However, biomarker discovery in the blood is particularly difficult because of interference from factors both intrinsic and extrinsic to blood with the detection of hallmark neurodegenerative biomarkers, such as the pathological prion protein, amyloid-β, and others. Blood extracellular vesicles (EVs), such as exosomes, are cell-derived vesicles released into the blood from all parts of the body (including the brain and spinal cord). They are an enriched source of neural-derived EVs containing neurodegenerative biomarkers that mirror (in the blood) the condition present in the brain. The feasibility of using, and the reliability of, neural-derived blood EVs (NDBEVs) as a method of diagnosing Alzheimer disease and other neurodegenerative diseases has been assessed in strong proof-of-concept studies. Results from these studies strongly suggest that NDBEVs might represent the right strategy for specific, reliable, and early diagnosis of neurodegenerative diseases. Based on these results, NDBEVs might enable the creation of an ante-mortem blood test (liquid biopsy of the brain) for neurodegenerative diseases. This would enormously accelerate the therapy of neurodegenerative diseases. This review highlights the powerful potential of liquid biopsy of the brain using NDBEVs for early diagnosis and treatment of neurodegenerative diseases, and the challenges and limitations related to the identification of clinically applicable EV (exosomal) biomarkers using blood are discussed.  

3.3733           Matrix-Bound Nanovesicles: The Effects of Isolation Method upon Yield, Purity, and Function

Quijano, L.M., Naranjo, J.D., El-Mossier, S.O., Turner, N.J., Milina, C.P., Bartolacci, J., Zhang, L., White, L., Li, H. and Badylak, S.F. Tissue Engineering: Part C, 26(10), 528-540 (2020)   Identification of matrix-bound nanovesicles (MBV) as ubiquitous components of the extracellular matrix (ECM) raises questions regarding their biologic functions and their potential theranostic application. Unlike liquid-phase extracellular vesicles (e.g., exosomes), MBV are tightly bound to the ECM, which makes their isolation and harvesting more challenging. The indiscriminate use of different methods to harvest MBV can alter or disrupt their structural and/or functional integrity. The objective of the present study was to compare the effect of various MBV harvesting methods upon yield, purity, and biologic activity. Combinations of four methods to solubilize the ECM (collagenase [COL], liberase [LIB], or proteinase K [PK] and nonenzymatic elution with potassium chloride) and four isolation methods (ultracentrifugation, ultrafiltration [UF], density barrier, and size exclusion chromatography [SEC]) were used to isolate MBV from urinary bladder-derived ECM. All combinations of solubilization and isolation methods allowed for the harvesting of MBV, however, distinct differences were noted. The highest yield, purity, cellular uptake, and biologic activity were seen with MBV isolated by a combination of liberase or collagenase followed by SEC. The combination of proteinase K and UF was shown to have detrimental effects on bioactivity. The results show the importance of selecting appropriate MBV harvesting methods for the characterization and evaluation of MBV and for analysis of their potential theranostic application.    

3.3734           Characteristics and Changes of DNA in Extracellular Vesicles

Chang, X., fang, L., Bai, J. and Wang, Z. DNA and Cell Biol., 39(9), 1486-1493 (2020)   Extracellular vesicles (EVs) have been known to carry multiple bioactive molecules, including lipids, mRNA/miRNA, and proteins. However, recent studies show that specific DNAs are also packed into EVs secreted by various cells, which are considered as powerful markers for diagnosis and prognosis of disease. DNAs in EVs are derived from parental cells, representing the mutation and even spanning of the whole genomic DNA of parental cells. Interestingly, increasing numbers of studies have found that the genetic materials in different EVs are not only universal but also random and different, which may be related to the size of EVs. In this review, we discuss the different characteristics of DNAs in EVs and the rules of their variation. We hope our review will trigger the continuing exploration of the origins, characteristics, and variations of DNAs in EVs.  

3.3735           Emerging technologies for profiling extracellular vesicle heterogeneity

Lin, G., Zhu, Y. eet al Lab on a Chip, 24(14), 2423-2437 (2020)   Extracellular vesicles (EVs) are membrane-bound vesicles secreted by most cell types and exist in virtually all bodily fluids. They carry on a wealth of proteomic and genetic information including proteins, lipids, miRNAs, mRNA, non-coding RNA and other molecules from parental cells. Increasing evidence shows that within populations of EVs, their biogenesis, physical characteristics (e.g. size, density, morphology) and cargos (e.g. protein, lipid content, nucleic acids) may vary substantially, which accordingly change their biological properties. To fully exploit the potential of EVs, it requires qualified methods to profile EV heterogeneity. In this review, we survey recent approaches for EV isolation with innovative discoveries in heterogeneity. The main challenges in EV heterogeneity research are identified, and the roles of single cell EV profiling and single EV imaging are highlighted. We further discuss promising opportunities for resolving the underlying complexity of EV heterogeneity.  

3.3736           Diversity of physical properties of bacterial extracellular membrane vesicles revealed through atomic force microscopy phase imaging

Kikuchi, Y., Obana, N., Toyofuku, M., Kodera, N., Soma, T., Ando, T., Fukumori, Y., Nomura, N and Taoka, A. Nanoscale, 12, 7950-7959 (2020)   Bacteria release nanometer-scale extracellular membrane vesicles (MVs) to mediate a variety of biological processes. We analyzed individual MVs under physiological conditions by phase imaging of high-speed atomic force microscopy to assess the physiological heterogeneity of MVs isolated from bacterial cultures. Phase imaging makes it possible to map the physical properties of an individual, fragile MV in an isolated MV population containing a broad variety of vesicle diameters, from 20 to 150 nm. We also developed a method for quantitatively comparing the physical properties of MVs among samples. This allowed for the comparison of the physical properties of MVs isolated from different bacterial species. We compared bacterial MVs isolated from four bacterial species and artificially synthesized liposomes. We demonstrate that each bacterial species generates physically heterogeneous types of MVs, unlike the physical homogeneity displayed by liposomes. These results indicate that the physical heterogeneity of bacterial MVs is mainly caused by compositional differences mediated through biological phenomena and could be unique to each species. We provide a new methodology using phase imaging that would pave the way for single-vesicle analysis of extracellular vesicles of a broad size range.  

3.3737           Exosomes of Malignant Tumors: Prospects of Omiсs Diagnostics

Shushkova, N.A:, Novikova, S.F. and Zgoda, V.G. Biochemistry (Moscow), Supplement Series B Biomedical Chem., 14(2), 105-115 (2020)   Early detection of the disease, prediction of its course, and response to therapy are the main problems in the diagnostics and treatment of malignant tumors. They may be solved by means of identification of biomarkers secreted by tumor cells within extracellular vesicles, particularly, exosomes. The study of exosomal proteins attracts special attention, because exosomal proteins can contain information about tumor identity, and also represent a set of signaling molecules, regulating processes of tumor progression and growth. In addition, analysis of exosomes secreted into the extracellular space corresponds to the promising concept of a liquid biopsy. In this review, we have summarized the current information about molecular studies of exosomes from various types of malignant tumors, including colorectal cancer, lung cancer, ovary, prostate and breast cancers, with special emphasis on omics methods and have considered prospects for their use in diagnostics.  

3.3738           Single-nucleus transcriptomics of the prefrontal cortex in major depressive disorder implicates oligodendrocyte precursor cells and excitatory neurons

Nagy, C., Maitra, M., Tanti, A., Suderman, M., Theroux, J-F., Davoli, M.A., Perlman, K., Yerko, V., Wang, Y.C., Tripathy, S.J., Pavlidis, P., Mechawar, N., Ragoussis, J. and Turecki, G. Nature Neurosci., 23, 771-781 (2020)   Major depressive disorder (MDD) has an enormous impact on global disease burden, affecting millions of people worldwide and ranking as a leading cause of disability for almost three decades. Past molecular studies of MDD employed bulk homogenates of postmortem brain tissue, which obscures gene expression changes within individual cell types. Here we used single-nucleus transcriptomics to examine ~80,000 nuclei from the dorsolateral prefrontal cortex of male individuals with MDD (n = 17) and of healthy controls (n = 17). We identified 26 cellular clusters, and over 60% of these showed differential gene expression between groups. We found that the greatest dysregulation occurred in deep layer excitatory neurons and immature oligodendrocyte precursor cells (OPCs), and these contributed almost half (47%) of all changes in gene expression. These results highlight the importance of dissecting cell-type-specific contributions to the disease and offer opportunities to identify new avenues of research and novel targets for treatment.  

3.3739           Acute and chronic glomerular damage is associated with reduced CD133 expression in urinary extracellular vesicles

Dimuccio, V., Peruzzi, L., Brizzi, M.F., Cocchi, E., Fop, F., Boido, A., Gili, M., Gallo, S., Biancone, L., Camussi, G.a nd Bussolati, B. Am. J. Physiol. Renal Physiol., 318, F486-F495 (2020)   Extracellular vesicles released into urine (uEVs) can represent interesting biomarkers of renal cell damage. CD133, a stem/progenitor cell marker expressed by renal progenitor cells, is highly expressed in uEVs of healthy individuals. In the present study, we evaluated the level of CD133 in the uEVs of patients with acute and chronic glomerular damage by cytofluorimetric analysis. The level of CD133⁺ uEVs was significantly decreased in pediatric patients with acute glomerulonephritis during the acute phase of renal damage, while it was restored after the subsequent recovery. A similar decrease was also observed in patients with chronic glomerulonephritis. Moreover, CD133⁺ uEVs significantly declined in patients with type 2 diabetes, used as validation group, with the lowest levels in patients with albuminuria with diabetic nephropathy. Indeed, receiver-operating characteristic curve analysis indicates the ability of CD133⁺ uEV values to discriminate the health condition from that of glomerular disease. In parallel, a significant decrease of CD133 in renal progenitor cells and in their derived EVs was observed in vitro after cell treatment with a combination of glucose and albumin overload, mimicking the diabetic condition. These data indicate that the level of CD133⁺ uEVs may represent an easily accessible marker of renal normal physiology and could provide information on the “reservoir” of regenerating cells within tubules.  

3.3740           Circulating exosomal microRNAs as emerging non-invasive clinical biomarkers in heart failure: Mega bio-roles of a nano bio-particle

Zhou, R., Wang, l., Zhao, G., Chen, D., Song, X., Momtazi-Borojeni, A.A. and yan, H. IUBMB Life, 72(12), 2546-2562 (2020)   Exosomes are nano-sized extracellular vesicles containing a cell-specific biologically active cargo of proteins and genetic materials. Exosomes are constitutively released from almost all cell-types and affect neighboring or distant cells through a complex intercellular exchange of the genetic information and/or regulation of certain gene expressions that change the function and behavior of recipient cells. Those released into body fluids are the major mediators of intercellular communications. The success of the biological functions of exosomes is highly mediated by the effective transfer of microRNAs (miRs). Exosomes secreted by a damaged or diseased heart can exhibit alterations in the miRs' profile that may reflect the cellular origin and (patho)physiological state, as a “signature” or “fingerprint” of the donor cell. It has been shown that the transportation of cardiac-specific miRs in exosomes can be rapidly detected and measured, holding great potential as biomarkers in heart diseases. Currently, the search for new biomarkers of heart diseases remains a large and increasing enterprise. Notably, circulating exosomal miRs (Exo-miRs) have successfully gained huge interests for their diagnostic and prognostic potentials. The present review highlights circulating Exo-miRs explored for diagnosis/prognosis and outcome prediction in patients with heart failure (HF). To this end, we explain the feasibility of exosomes as clinical biomarkers, discuss the priority of circulating Exo-miRs over non-exosomal ones as a biomarker, and then outline reported circulating Exo-miRs having the biomarker function in HF patients, together with their mechanism of action. In conclusion, circulating Exo-miRs represent emerging diagnostic (Exo-miR-92b-5p, Exo-miR-146a, Exo-miR-181c, and Exo-miR-495) and prognostic (Exo-miR-192, Exo-miR-194, Exo-miR-34a, Exo-miR-425, Exo-miR-744) biomarkers for HF.  

3.3741           Quantitative Proteomic Analysis of Seminal Plasma, Sperm Membrane Proteins, and Seminal Extracellular Vesicles Suggests Vesicular Mechanisms Aid in the Removal and Addition of Proteins to the Ram Sperm Membrane

Leahy, T., Richard, J.P., Pini, T., Gadella, B.M. and de Graaf, S.P. Proteomics, 20(12), 1900289 (2020)   Quantitative proteomic studies are contributing greatly to the understanding of the spermatozoon through the provision of detailed information on the proteins spermatozoa acquire and shed in the acquisition of fertility. Extracellular vesicles (EVs) are thought to aid in the delivery of proteins to spermatozoa in the male reproductive tract. The aim of this study is to isolate, identify and quantify EV proteins isolated from ram seminal plasma. Ram sperm plasma membrane proteins are also isolated using nitrogen cavitation and identified to better understand the interplay of proteins between the sperm membrane and extracellular environment. The categorization of proteins enriched in the EV population according to their function revealed three main groupings: vesicle biogenesis, metabolism, and membrane adhesion and remodeling. The latter group contains many reproduction-specific proteins that show demonstrable links to sperm fertility. Many of these membrane-bound proteins show testicular expression and are shed from the sperm surface during epididymal maturation (e.g., testis expressed 101; TEX101 and lymphocyte Antigen 6 Family Member K; LY6K). Their association with seminal EVs suggests that EVs may not only deliver protein cargo to spermatozoa but also assist in the removal of proteins from the sperm membrane.  

3.3742           Ceramide launches an acute anti-adhesion pro-migration cell signaling program in response to chemotherapy

Canals, D., Salamone, S., Santacreu, B.J., Nemeth, E., Aguilar, D., Hernandez-Corbacho, M.J., Adada, M., Staquicini, D.I., Arap, W., Pasqualini, R., Haley, J., Obeid, L.M. and Hannum, Y.A. FASEB J., 34(6), 7610-7630 (2020)   Chemotherapy has been reported to upregulate sphingomylinases and increase cellular ceramide, often linked to the induction to cell death. In this work, we show that sublethal doses of doxorubicin and vorinostat still increased cellular ceramide, which was located predominantly at the plasma membrane. To interrogate possible functions of this specific pool of ceramide, we used recombinant enzymes to mimic physiological levels of ceramide at the plasma membrane upon chemotherapy treatment. Using mass spectrometry and network analysis, followed by experimental confirmation, the results revealed that this pool of ceramide acutely regulates cell adhesion and cell migration pathways with weak connections to commonly established ceramide functions (eg, cell death). Neutral sphingomyelinase 2 (nSMase2) was identified as responsible for the generation of plasma membrane ceramide upon chemotherapy treatment, and both ceramide at the plasma membrane and nSMase2 were necessary and sufficient to mediate these “side” effects of chemotherapy on cell adhesion and migration. This is the first time a specific pool of ceramide is interrogated for acute signaling functions, and the results define plasma membrane ceramide as an acute signaling effector necessary and sufficient for regulation of cell adhesion and cell migration under chemotherapeutical stress.  

3.3743           A non-canonical rhodopsin-mediated insulin receptor signaling pathway in retinal photoreceptor neurons

Rajala, A. and Rjala, R.V,S, Cell. Biol. Int., 44(4), 1020-1027 (2020)   We previously reported a ligand-independent and rhodopsin-dependent insulin receptor (IR) neuroprotective signaling pathway in both rod and cone photoreceptor cells, which is activated through protein–protein interaction. Our previous studies were performed with either retina or isolated rod or cone outer segment preparations and the expression of IR signaling proteins were examined. The isolation of outer segments with large portions of the attached inner segments is a technical challenge. Optiprep™ density gradient medium has been used to isolate the cells and subcellular organelles, Optiprep™ is a non-ionic iodixanol-based medium with a density of 1.320 g/mL. We employed this method to examine the expression of IR and its signaling proteins, and activation of one of the downstream effectors of the IR in isolated photoreceptor cells. Identification of the signaling complexes will be helpful for therapeutic targeting in disease conditions.  

3.3744           Exosomes Derived from the Human Primary Colorectal Cancer Cell Line SW480 Orchestrate Fibroblast-Led Cancer Invasion

Rai, A., Greening, D.W., Xu, R., Suwakulsiri, W and Simpson, R:J. Proteomics, 20(14), 2000016 (2020)   In localized tumors, basement membrane (BM) prevents invasive outgrowth of tumor cells into surrounding tissues. When carcinomas become invasive, cancer cells either degrade BM or reprogram stromal fibroblasts to breach BM barrier and lead invasion of cancer cells into surrounding tissues in a process called fibroblast-led invasion. However, tumor-derived factors orchestrating fibroblast-led invasion remain poorly understood. Here it is shown that although early-stage primary colorectal adenocarcinoma (SW480) cells are themselves unable to invade Matrigel matrix, they secrete exosomes that reprogram normal fibroblasts to acquire de novo capacity to invade matrix and lead invasion of SW480 cells. Strikingly, cancer cells follow leading fibroblasts as collective epithelial-clusters, thereby circumventing need for epithelial to mesenchymal transition, a key event associated with invasion. Moreover, acquisition of pro-invasive phenotype by fibroblasts treated with SW480-derived exosomes relied on exosome-mediated MAPK pathway activation. Mass spectrometry-based protein profiling reveals that cancer exosomes upregulate fibroblasts proteins implicated in focal adhesion (ITGA2/A6/AV, ITGB1/B4/B5, EGFR, CRK), regulators of actin cytoskeleton (RAC1, ARF1, ARPC3, CYFIP1, NCKAP1, ICAM1, ERM complex), and signalling pathways (MAPK, Rap1, RAC1, Ras) important in pro-invasive remodeling of extracellular matrix. Blocking tumor exosome-mediated signaling to fibroblasts therefore represents an attractive therapeutic strategy in restraining tumors by perturbing stroma-driven invasive outgrowth.  

3.3745           Glial cell line-derived neurotrophic factor alters lipid composition and protein distribution in MPP+-injured differentiated SH-SY5Y cells

Mu, P., Liu, Y., Jiang, S., Gao, J., Sun, S., Li, L. and gao, D.

1. Cell Physiol., 235(12), 9347-9360 (2020)

  Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive loss of dopaminergic neurons in the substantia nigra and striatum. Glial cell line-derived neurotrophic factor (GDNF) can effectively promote the differentiation and survival of many types of neurons, especially dopaminergic neurons, suggesting it could be a treatment for PD. Lipid rafts are highly dynamic cell membrane domains that contain numerous signal protein receptors, providing an important platform for signal transduction. Compelling evidence indicates that alterations in lipid rafts are associated with PD, and some studies have reported that GDNF can regulate the expression of caveolin-1, a lipid raft-marker protein. However, the precise effects of GDNF on lipid rafts remain unknown. We developed a cellular PD model, purified detergent-resistant membranes (membrane rafts), and performed proteomic and lipid metabolomics analyses to examine changes in lipid rafts after GDNF treatment. The results showed considerable protein and lipid alterations in response to GDNF, especially altered levels of dopamine-β-hydroxylase, heat shock 70 kDa protein, neural cell adhesion molecule, cytoskeletal proteins, and long-chain polysaturated/unsaturated fatty acids. These findings reveal a new avenue to explore the relationships between GDNF, lipid rafts, and PD and support the hypothesis that GDNF may be a useful treatment for PD.  

3.3746           Disturbances in PP2A methylation and one-carbon metabolism compromise Fyn distribution, neuritogenesis, and APP regulation

Taleski, G., Schuhmacher, D., Su, H., Sontag, J-M and Sontag, E.

1. Biol. Chem., 296, 100237 (2021)

  The nonreceptor protein tyrosine kinase Fyn and protein Ser/Thr phosphatase 2A (PP2A) are major multifunctional signaling molecules. Deregulation of Fyn and altered PP2A methylation are implicated in cancer and Alzheimer's disease (AD). Here, we tested the hypothesis that the methylation state of PP2A catalytic subunit, which influences PP2A subunit composition and substrate specificity, can affect Fyn regulation and function. Using Neuro-2a (N2a) neuroblastoma cell models, we first show that methylated PP2A holoenzymes containing the Bα subunit coimmunoprecipitate and copurify with Fyn in membrane rafts. PP2A methylation status regulates Fyn distribution and Fyn-dependent neuritogenesis, likely in part by affecting actin dynamics. A methylation-incompetent PP2A mutant fails to interact with Fyn. It perturbs the normal partitioning of Fyn and amyloid precursor protein (APP) in membrane microdomains, which governs Fyn function and APP processing. This correlates with enhanced amyloidogenic cleavage of APP, a hallmark of AD pathogenesis. Conversely, enhanced PP2A methylation promotes the nonamyloidogenic cleavage of APP in a Fyn-dependent manner. Disturbances in one-carbon metabolic pathways that control cellular methylation are associated with AD and cancer. Notably, they induce a parallel loss of membrane-associated methylated PP2A and Fyn enzymes in N2a cells and acute mouse brain slices. One-carbon metabolism also modulates Fyn-dependent process outgrowth in N2a cells. Thus, our findings identify a novel methylation-dependent PP2A/Fyn signaling module. They highlight the underestimated importance of cross talks between essential metabolic pathways and signaling scaffolds that are involved in normal cell homeostasis and currently being targeted for cancer and AD treatment.  

3.3747           Enzymatically active apurinic/apyrimidinic endodeoxyribonuclease 1 is released by mammalian cells through exosomes

Mangiapane, G., Parolini, I., Conte, K., Malfatti, M.C., Corsi, J., Sanchez, M., Pietrantoni, A., D’Agostino, V.D. and Tell, G.

1. Biol. Chem., 296, 100569 (2021)

  The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1), the main AP-endonuclease of the DNA base excision repair pathway, is a key molecule of interest to researchers due to its unsuspected roles in different nonrepair activities, such as: i) adaptive cell response to genotoxic stress, ii) regulation of gene expression, and iii) processing of microRNAs, which make it an excellent drug target for cancer treatment. We and others recently demonstrated that APE1 can be secreted in the extracellular environment and that serum APE1 may represent a novel prognostic biomarker in hepatocellular and non-small-cell lung cancers. However, the mechanism by which APE1 is released extracellularly was not described before. Here, using three different approaches for exosomes isolation: commercial kit, nickel-based isolation, and ultracentrifugation methods and various mammalian cell lines, we elucidated the mechanisms responsible  for APE1 secretion. We demonstrated that APE1 p37 and p33 forms are actively secreted through extracellular vesicles (EVs), including exosomes from different mammalian cell lines. We then observed that APE1 p33 form is generated by proteasomal-mediated degradation and is enzymatically active in EVs. Finally, we revealed that the p33 form of APE1 accumulates in EVs upon genotoxic treatment by cisplatin and doxorubicin, compounds commonly found in chemotherapy pharmacological treatments. Taken together, these findings provide for the first time evidence that a functional Base Excision Repair protein is delivered through exosomes in response to genotoxic stresses, shedding new light into the complex noncanonical biological functions of APE1 and opening new intriguing perspectives on its role in cancer biology.  

3.3748           The aminosterol Claramine inhibits β-secretase 1–mediated insulin receptor cleavage

Babroit, B., Govers, R., Altie, A., Brunel, J.M., Morange, P. and Peiretti, F.

1. Biol. Chem., 297(1), 100818 (2021)

  The cleavage of the insulin receptor by β-secretase 1 (BACE1) in the liver increases during diabetes, which contributes to reduce insulin receptor levels and impair insulin signaling. However, the precise signaling events that lead to this increased cleavage are unclear. We showed that BACE1 cleaves the insulin receptor in the early secretory pathway. Indeed, coimmunoprecipitation experiments reveal the interaction of the proforms of the two proteins. Moreover, fragments of insulin receptor are detected in the early secretory pathway and a mutated form of BACE1 that retains its prodomain cleaves an early secretory pathway-resident form of the insulin receptor. We showed that BACE1 proform levels are regulated by proteasome and/or lysosome-dependent degradation systems whose efficiencies are dependent on the O-GlcNacylation process. Our results showed that enhanced O-GlcNacylation reduces the efficiency of intracellular protein degradation systems, leading to the accumulation of the proform of BACE1 in the early secretory pathway where it cleaves the precursor of the insulin receptor. All these dysregulations are found in the livers of diabetic mice. In addition, we performed a screen of molecules according to their ability to increase levels of the insulin receptor at the surface of BACE1-overexpressing cells. This approach identified the aminosterol Claramine, which accelerated intracellular trafficking of the proform of BACE1 and increased autophagy. Both of these effects likely contribute to the reduced amount of the proform of BACE1 in the early secretory pathway, thereby reducing insulin receptor cleavage. These newly described properties of Claramine are consistent with its insulin sensitizing effect.  

3.3749           CD147 mediates the CD44s-dependent differentiation of myofibroblasts driven by transforming growth factor-β1

Woods, E.L., Grigorieva, I.V., Midgley, A.C., Brown, C.V.M., Lu, Y-a., Phillips, A.O., Bowen, T., Meran, S. and Steadman, R.

1. Biol. Chem., 297(3), 100987 (2021)

  Progressive fibrosis leads to loss of organ function and affects many organs as a result of excessive extracellular matrix production. The ubiquitous matrix polysaccharide hyaluronan (HA) is central to this through association with its primary receptor, CD44, which exists as standard CD44 (CD44s) or multiple splice variants. Mediators such as profibrotic transforming growth factor (TGF)-β1 and proinflammatory interleukin (IL)-1β are widely associated with fibrotic progression. TGF-β1 induces myofibroblast differentiation, while IL-1β induces a proinflammatory fibroblast phenotype that promotes fibroblast binding to monocyte/macrophages. CD44 expression is essential for both responses. Potential CD44 splice variants involved, however, are unidentified. The TGF-β1-activated CD44/epidermal growth factor receptor complex induces differentiation of metastatic cells through interactions with the matrix metalloproteinase inducer, CD147. This study aimed to determine the CD44 variants involved in TGF-β1- and IL-1β-mediated responses and to investigate the potential profibrotic role of CD147. Using immunocytochemistry and quantitative PCR, standard CD44s were shown to be essential for both TGF-β1-induced fibroblast/myofibroblast differentiation and IL-1β-induced monocyte binding. Co-immunoprecipitation identified that CD147 associated with CD44s. Using CD147-siRNA and confocal microscopy, we also determined that incorporation of the myofibroblast marker, αSMA, into F-actin stress fibers was prevented in the absence of CD147 and myofibroblast-dependent collagen gel contraction was inhibited. CD147 did not associate with HA, but removal of HA prevented the association of CD44s with CD147 at points of cell–cell contact. Taken together, our data suggest that CD44s/CD147 colocalization is essential in regulating the mechanical tension required for the αSMA incorporation into F-actin stress fibers that regulates myofibroblast phenotype.  

3.3750           Glyceraldehyde-3-phosphate dehydrogenase present in extracellular vesicles from Leishmania major suppresses host TNF-alpha expression

Das, P., Mukherjee, A. and Adak, S.

1. Biol. Chem., 297(4), 101198 (2021)

  Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills various physiological roles that are unrelated to its glycolytic function. However, to date, the nonglycolytic function of GAPDH in trypanosomal parasites is absent from the literature. Exosomes secreted from Leishmania, like entire parasites, were found to have a significant impact on macrophage cell signaling and function, indicating cross talk with the host immune system. In this study, we demonstrate that the Leishmania GAPDH (LmGAPDH) protein is highly enriched within the extracellular vesicles (EVs) secreted during infection. To understand the function of LmGAPDH in EVs, we generated control, overexpressed, half-knockout (HKO), and complement cell lines. HKO cells displayed lower virulence compared with control cells when macrophages and BALB/c mice were infected with them, implying a crucial role for LmGAPDH in Leishmania infection and disease progression. Furthermore, upon infection of macrophages with HKO mutant Leishmania and its EVs, despite no differences in TNFA mRNA expression, there was a considerable increase in TNF-α protein expression compared with control, overexpressed, and complement parasites as determined by ELISA, RT-PCR, and immunoblot data. In vitro protein translation studies suggest that LmGAPDH-mediated TNF-α suppression occurs in a concentration-dependent manner. Moreover, mRNA binding assays also verified that LmGAPDH binds to the AU-rich 3′-UTR region of TNFA mRNA, limiting its production. Together, these findings confirmed that the LmGAPDH contained in EVs inhibits TNF-α expression in macrophages during infection via posttranscriptional repression.  

3.3751           Rab18 binds PLIN2 and ACSL3 to mediate lipid droplet dynamics

Deng, Y., Zhou, C., Mirza, A.H., Bamigbade, A.T., Zhang, S., Xu, S and Liu, P. BBA-Mol. Cell Biol. Lipids, 1866, 158923 (2021)   Lipid droplet (LD) is a vital organelle governing lipid homeostasis and Rab18 has been linked to lipid metabolism. However, the mechanisms of Rab18-mediated LD dynamics in myoblast cells remain elusive. Here, we report that Rab18 plays an important role in oleic acid (OA)-induced LD accumulation in mouse myoblast C2C12 cells. Rab18 was translocated from the endoplasmic reticulum (ER) to LDs during LD accumulation, which was regulated by perilipin 2 (PLIN2), a major LD protein. LD-associated Rab18 bound with the C terminus of PLIN2 and the LD localization of Rab18 was diminished when PLIN2 was depleted. Moreover, loss of function of Rab18 led to reduced triacylglycerol (TAG) level and fewer but larger LDs. In contrast, overexpression of Rab18 resulted in elevated TAG content and LD number. Furthermore, LD-associated Rab18 interacted with acyl-CoA synthetase long-chain family member 3 (ACSL3), which in turn promoted the LD localization of this protein. These data show that Rab18 interacts with PLIN2 and forms a complex with PLIN2 and ACSL3, which plays a critical role in LD accumulation and dynamics of myoblast cells.  

3.3752           Sex-specific effects of 17β-estradiol and dihydrotestosterone (DHT) on growth plate chondrocytes are dependent on both ERα and ERβ and require palmitoylation to translocate the receptors to the plasma membrane

Cohen, D.J., Elbaradie, K., Boyan, B.D. and Schwartz, Z. BBA-Mol. Cell. Biol. Lipids, 1866, 159028 (2021)   Rat costochondral cartilage growth plate chondrocytes exhibit cell sex-specific responses to 17β-estradiol (E₂), testosterone, and dihydrotestosterone (DHT). Mechanistically, E₂ and DHT stimulate proliferation and extracellular matrix synthesis in chondrocytes from female and male rats, respectively, by signaling through protein kinase C (PKC) and phospholipase C (PLC). Estrogen receptors (ERα; ERβ) and androgen receptors (ARs) are present in both male and female cells, but it is not known whether they interact to elicit sex-specific signaling. We used specific agonists and antagonists of these receptors to examine the relative contributions of ERs and ARs in membrane-mediated E₂ signaling in female chondrocytes and DHT signaling in male chondrocytes. PKC activity in female chondrocytes was stimulated by agonists of ERα and ERβ and required intact caveolae; PKC activity was inhibited by the E₂ enantiomer and by an inhibitor of ERβ. Western blots of cell lysates co-immunoprecipitated for ERα suggested the formation of a complex containing both ERα and ERß with E₂ treatment. DHT and DHT agonists activated PKC in male cells, while AR inhibition blocked the stimulatory effect of DHT on PKC. Inhibition of ERα and ERβ also blocked PKC activation by DHT. Western blots of whole-cell lysates, plasma membranes, and caveolae indicated the translocation of AR to the plasma membrane and specifically to caveolae with DHT treatment. These results suggest that E₂ and DHT promote chondrocyte differentiation via the ability of ARs and ERs to form a complex. The results also indicate that intact caveolae and palmitoylation of the membrane receptor(s) or membrane receptor complex containing ERα and ERβ is required for E₂ and DHT membrane-associated PKC activity in costochondral cartilage cells.  

3.3753           Lipid raft integrity is required for human leukemia Jurkat T-cell migratory activity

Bobkov, D., Yudintceva, N., Lomert, E., Shatrova, A., Kever, L. and Semenova, S. BBA-Mol. Cell. Biol. Lipids, 1866, 158917 (2021)   Lipid rafts are membrane microdomains featuring high cholesterol, sphingolipid, and protein content. These microdomains recruit various receptors, ion channels, and signaling molecules for coordination of various cellular functions, including synaptic transmission, immune response, cytoskeletal organization, adhesion, and migration. Many of these processes also depend on Ca²⁺ intake. We have previously shown in Jurkat cells that activity of transient receptor potential vanilloid, type 6 (TRPV6) calcium channel, and TRPV6-mediated Ca²⁺ influx, depend on lipid raft integrity. In this study, using the transwell cell migration assay and time-lapse video microscopy with Jurkat cells, we found that lipid raft destruction was associated with: inhibited cell adhesion and migration; and decreased mean speed, maximum speed, and trajectory length. Using String Server, we constructed a Protein Interaction Network (PIN). The network indicated that TRPV6 proteins interact with the highest probability (0.9) with Src family kinase members (SFKs) involved in processes related to cell migration. Analysis of detergent-resistant membrane fractions and immunoelectron microscopy data confirmed an association in lipid rafts between TRPV6 and Lck kinase, an SFKs member. Destruction of lipid rafts led to uncoupling of TRPV6 clusters with Lck and their departure from the plasma membrane into the cytosol of the cells. Src family kinases are generally associated with their roles in tumor invasion and progression, epithelial-mesenchymal transitions, angiogenesis, and metastatic development. We suggest that a functional interaction between TRPV6 calcium channels and SFKs members in lipid rafts is one of necessary elements of migration and oncogenic signaling in leukemia cells.  

3.3754           Isolation and characterization of extracellular vesicles produced by cell lines

Wang, F., Cerione, R.A. and Antonyak, M.A. STAR Protocols, 2, 100295 (2021)   Cells produce two broad classes of extracellular vesicles (EVs), exosomes and microvesicles (MVs). Exosomes are 30–150 nm vesicles derived from multivesicular bodies, while MVs are 200–1,000 nm vesicles that pinch off from plasma membranes. Reliable isolation of EVs is crucial to understand their biochemical and functional properties. Here, we describe a protocol to isolate and characterize EVs from conditioned medium from mammalian cell lines. This protocol has been optimized for adherent cells but can also be adapted for suspension cells.  

3.3755           Ultracentrifugal separation, characterization, and functional study of extracellular vesicles derived from serum-free cell culture

Hu, H.T., Nashimura, T. and Suetsugu, S. STAR Protocols, 2, 100625 (2021)   Extracellular vesicles (EVs) play important roles in extracellular trafficking and signaling. Here, we separate EVs by differential centrifugation. EVs separated by this approach are called large EVs (l-EVs) and small EVs (s-EVs), reflecting particle size, which sediment based on different ultracentrifugation forces. The resulting EVs can be quantified and analyzed using nanoparticle tracking analysis, immunoblotting, and functional assays. This protocol was applied to a suspension cell line with high transfection efficiency adapted to a high-density, serum-free culture.  

3.3756           FACS-based isolation of fixed mouse neuronal nuclei for ATAC-seq and Hi-C

Eremenko, E., Golova, A., Stein, D., Einav, M., Khrameeva, M.E.and Toiber, D. STAR Protocols, 2, 100643 (2021)   The organization of chromatin structure plays a crucial role in gene expression, DNA replication, and repair. Chromatin alterations influence gene expression, and modifications could be associated with genomic instability in the cells during aging or diseases. Here, we provide a modified protocol to isolate fixed neuronal nuclei from a single mouse cortex to investigate the spatial organization of chromatin structure on a genome-wide scale by ATAC-seq (the assay for transposase-accessible chromatin with high-throughput sequencing) and chromatin conformation by Hi-C (high-throughput chromosome conformation capture).  

3.3757           Extracellular vesicle separation from milk and infant milk formula using acid precipitation and ultracentrifugation

Mukhopadhya, A., Santoto, J. and O’Driscoll, L. STAR Protocols, 2, 100821 (2021)   Separation of highly enriched extracellular vesicles (EVs) fractions from milk is desirable for quantification, cargo analysis, functional characterization, and investigation as delivery vehicles for nutrients and/or therapeutics. However, a rigorous, reproducible protocol is lacking. This protocol considers a crucial aspect typically overlooked, i.e., that caseins are of similar size to, but more abundant than, EVs in milk. Our protocol combines acid pre-treatment and gradient ultracentrifugation, producing EV-enriched fractions suitable for downstream orthogonal characterization approaches.  

3.3758           Extracellular Vesicle (EV) biohybrid systems for cancer therapy: Recent advances and future perspectives

Ou, Y-H., Liang, J., Czarny, B., Wacker, M.G., Yu, V., Wang, J-W. and Pastorin, G. Seminars in Cancer Biol., 74, 45-61 (2021)   Extracellular vesicles (EVs) are a class of cell-derived lipid-bilayer membrane vesicles secreted by almost all mammalian cells and involved in intercellular communication by shuttling various biological cargoes. Over the last decade, EVs – namely exosomes and microvesicles – have been extensively explored as next-generation nanoscale drug delivery systems (DDSs). This is in large due to their endogenous origin, which enables EVs to circumvent some of the limitations associated with existing cancer therapy approaches (i.e. by preventing recognition by the immune system and improving selectivity towards tumor tissue). However, successful translation of these cell-derived vesicles into clinical applications has been hindered by several factors, among which the loading of exogenous therapeutic molecules still represents a great challenge. In order to address this issue and to further advance these biologically-derived systems as drug carriers, EV-biohybrid nano-DDSs, obtained through the fusion of EVs with conventional synthetic nano-DDSs, have recently been proposed as a valuable alternative as DDSs. Building on the idea of “combining the best of both worlds”, a combination of these two unique entities aims to harness the beneficial properties associated with both EVs and conventional nano-DDSs, while overcoming the flaws of the individual components. These biohybrid systems also provide a unique opportunity for exploitation of new synergisms, often leading to improved therapeutic outcomes, thus paving the way for advancements in cancer therapy. This review aims to describe the recent developments of EV-biohybrid nano-DDSs in cancer therapy, to highlight the most promising results and breakthroughs, as well as to provide a glimpse on the possible intrinsic targeting mechanisms of EVs that can be bequeathed to their hybrid systems. Finally, we also provide some insights in the future perspectives of EV-hybrid DDSs.  

3.3759           Extracellular vesicles in cancer nanomedicine

Tarasov, V.T., Svistunov, A.A., Chubarev, V.N., Dostdar, S.A., Sokolov, A.V., Brzecka, A., Sukocheva, O., Neganova, M.E., Klochkov, S.G., Somasundaram, D.G., Kirkland, C.E. and Aliev, G. Seminars in Cancer Biol., 69, 212-225 (2021)   To date, a lot of nanotechnological optitions are available for targeted drug delivery. Extracellular vesicles (EVs) are membrane structures that cells use for storage, transport, communication, and signaling. Recent research has focused on EVs as natural nanoparticles for drug delivery. This review sheds light on the application of EVs in cancer therapy, such as targeted chemotherapy, gene therapy, and vaccine development. Aspects of biogenesis, isolation, targeting, and loading of EVs are discussed in detail.  

3.3760           Extracellular vesicles, the cornerstone of next-generation cancer diagnosis?

Wang, J., Xiang, X., Ding, l., Wong, L-A., Zeng, Q., Sethi, G., Wang, L., Lee, S.C and Goh, B.C. Seminars in Cancer Biol., 74, 105-120 (2021)   Cancer has risen up to be a major cause of mortality worldwide over the past decades. Despite advancements in cancer screening and diagnostics, a significant number of cancers are still diagnosed at a late stage with poor prognosis. Hence, the discovery of reliable and accurate methods to diagnose cancer early would be of great help in reducing cancer mortality. Extracellular vesicles (EVs) are phospholipid vesicles found in many biofluids and are released by almost all types of cells. In recent years, using EVs as cancer biomarkers has garnered attention as a novel technique of cancer diagnosis. Compared with traditional tissue biopsy, there are many advantages that this novel diagnostic tool presents - it is less invasive, detects early-stage asymptomatic cancers, and allows for monitoring of tumour progression. As such, EV biomarkers have great potential in improving the diagnostic accuracy of cancers and guiding subsequent therapeutic decisions. Efficient isolation and accurate characterization of EVs are essential for reliable outcomes of clinical application. However, these are complicated by the size and biomolecular diversity of EVs. In this review, we present an analysis and evaluation of the current techniques of EV isolation and characterization, as well as discuss the potential EV biomarkers for specific types of cancer. Taken together, EV biomarkers have a lot of potential as a novel method in cancer diagnostics and diagnosis. However, more work is still needed to streamline the purification, characterization and biomarker identification process to ensure optimal outcomes for patients.  

3.3761           Nucleic acid delivery with extracellular vesicles

Schulz-Siegemund, m. and Aigner, A. Adv. Drug Delivery Reviews, 173, 89-111 (2021)   Extracellular vesicles (EVs) are membrane-enclosed particles, heterogeneous in size, shape, contents, biogenesis and structure. They are released by eukaryotic and prokaryotic cells and exert (patho-)physiological roles as mediators for transmitting molecular information from the producer (donor) to a recipient cell. This review focuses on the potential of EVs for delivering nucleic acids, as particularly problematic cargoes with regard to stability/protection and uptake efficacy. It highlights important properties of EVs for nucleic acid delivery and discusses their physiological and pathophysiological roles with regard to various cellular RNA species. It then describes the application of EVs for delivering a broad selection of nucleic acids/oligonucleotides, in particular giving a comprehensive overview of preclinical in vivo studies and the various strategies explored. In this context, different techniques for EV loading are discussed, as well as other important technical aspects related to EV preparation, characterization and in particular, the various approaches of artificial EV modification.  

3.3762           Illuminating RNA trafficking and functional delivery by extracellular vesicles

De Voogt, W.S., Tanenbaum, M.E. and Vader, P. Adv. Drug Delivery Reviews, 174, 250-264 (2021)   RNA-based therapeutics are highly promising for the treatment of numerous diseases, by their ability to tackle the genetic origin in multiple possible ways. RNA molecules are, however, incapable of crossing cell membranes, hence a safe and efficient delivery vehicle is pivotal. Extracellular vesicles (EVs) are endogenously derived nano-sized particles and possess several characteristics which make them excellent candidates as therapeutic RNA delivery agent. This includes the inherent capability to functionally transfer RNAs in a selective manner and an enhanced safety profile compared to synthetic particles. Nonetheless, the fundamental mechanisms underlying this selective inter- and intracellular trafficking and functional transfer of RNAs by EVs are poorly understood. Improving our understanding of these systems is a key element of working towards an EV-based or EV-mimicking system for the functional delivery of therapeutic RNA. In this review, state-of-the-art approaches to detect and visualize RNA in situ and in live cells are discussed, as well as strategies to assess functional RNA transfer, highlighting their potential in studying EV-RNA trafficking mechanisms.  

3.3763           Separation, characterization, and standardization of extracellular vesicles for drug delivery applications

Buschmann, D.,  Mussack, V. and Byrd, J.B.B. Adv. Drug Delivery Reviews, 174, 348-368 (2021)   Extracellular vesicles (EVs) are membranous nanovesicles secreted from living cells, shuttling macromolecules in intercellular communication and potentially possessing intrinsic therapeutic activity. Due to their stability, low immunogenicity, and inherent interaction with recipient cells, EVs also hold great promise as drug delivery vehicles. Indeed, they have been used to deliver nucleic acids, proteins, and small molecules in preclinical investigations. Furthermore, EV-based drugs have entered early clinical trials for cancer or neurodegenerative diseases. Despite their appeal as delivery vectors, however, EV-based drug delivery progress has been hampered by heterogeneity of sample types and methods as well as a persistent lack of standardization, validation, and comprehensive reporting. This review highlights specific requirements for EVs in drug delivery and describes the most pertinent approaches for separation and characterization. Despite residual uncertainties related to pharmacodynamics, pharmacokinetics, and potential off-target effects, clinical-grade, high-potency EV drugs might be achievable through GMP-compliant workflows in a highly standardized environment.  

3.3764           Adherence to minimal experimental requirements for defining extracellular vesicles and their functions

Poupaardin, R., Wolf, M. and Strunk, D. Adv. Drug Delivery Reviews, 176, 113872 (2021)   Rigorous measures are required to cope with the advance of extracellular vesicle (EV) research, from 183 studies published in 2012 to 2,309 studies published in 2020. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines in 2014, updated in 2018, for assuring and improving EV research quality. We performed a systematic review using a text mining approach to assess adherence to MISEV criteria. A keyword search was conducted in 5,093 accessible publications over the period 2012–2020 and analyzed the methodology used for EV isolation and characterization. We found a significant improvement over the years particularly regarding EV characterization where recent papers used a higher number of methods and EV markers to check for quantity and purity. Interestingly, we also found that EV papers using more methods and EV markers were cited more frequently. Papers citing MISEV criteria were more prone to use a higher number of characterization methods. We therefore established a concise checklist summarizing MISEV criteria to support EV researchers towards reaching the highest standards in the field.  

3.3765           Dosing extracellular vesicles

Gupta, D., Zickler, A.M. and El Andaloussi, S. Adv. Drug Delivery Reviews, 178, 113961 (2021)   Extracellular vesicles (EVs) are natural nanoparticles containing biologically active molecules. They are important mediators of intercellular communication and can be exploited therapeutically by various bioengineering approaches. To accurately determine the therapeutic potential of EVs in pre-clinical and clinical settings, dependable dosing strategies are of utmost importance. However, the field suffers from inconsistencies comprising all areas of EV production and characterisation. Therefore, a standardised and well-defined process in EV quantification, key to reliable therapeutic EV dosing, remains to be established. Here, we examined 64 pre-clinical studies for EV-based therapeutics with respect to their applied EV dosing strategies. We identified variations in effective dosing strategies irrespective of the applied EV purification method and cell source. Moreover, we found dose discrepancies depending on the disease model, where EV doses were selected without accounting for published EV pharmacokinetics or biodistribution patterns. We therefore propose to focus on qualitative aspects when dosing EV-based therapeutics, such as the potency of the therapeutic cargo entity. This will ensure batch-to-batch reliability and enhance reproducibility between applications. Furthermore, it will allow for the successful benchmarking of EV-based therapeutics compared to other nanoparticle drug delivery systems, such as viral vector-based or lipid-based nanoparticle approaches.  

3.3766           Pancreatic cancer-targeting exosomes for enhancing immunotherapy and reprogramming tumor microenvironment

Zhou, W., Zhou, Y., Chen, X., Ning, T., Chen, H., Guo, Q., Zhang, Y., Liu, P., Zhang, Y., Li, C., Chu, Y., Sun, T. and Jiang, C. Biomaterials, 268, 120546 (2021)   Immunotherapy has gained increasing focus in treating pancreatic ductal adenocarcinoma (PDAC), since conventional therapies like chemotherapy could not provide satisfactory improvement in overall survival outcome of PDAC patients. However, it is still not the game changing solution due to the unique tumor microenvironment and low cancer immunogenicity of PDAC. Thus, inducing more intratumoral effector immune cells as well as reversing immunosuppression is the core of PDAC treatment. Herein, we demonstrate an exosome-based dual delivery biosystem for enhancing PDAC immunotherapy as well as reversing tumor immunosuppression of M2-like tumor associated macrophages (M2-TAMs) upon disruption of galectin-9/dectin 1 axis. The deliver system is constructed from bone marrow mesenchymal stem cell (BM-MSC) exosomes, electroporation-loaded galectin-9 siRNA, and surficially modified with oxaliplatin (OXA) prodrug as an immunogenic cell death (ICD)-trigger. The use of biomaterials, BM-MSC exosomes, can significantly improve tumor targeting efficacy, thus increasing drug accumulation in the tumor site. The combined therapy (iEXO-OXA) elicits anti-tumor immunity through tumor-suppressive macrophage polarization, cytotoxic T lymphocytes recruitment and Tregs downregulation, and achieves significant therapeutic efficacy in cancer treatment.  

3.3767           Inflammatory tumor microenvironment responsive neutrophil exosomes-based drug delivery system for targeted glioma therapy

Wang, J., Tang, W., Yang, M., Yin, Y., Li, H., Hu, F., Tang, L., Ma, X., Zhang, Y. and Wang, Y. Biomaterials, 273, 120784 (2021)   Clinical treatment of malignant glioma remains a major challenge due to high infiltrative growth and chemotherapeutic resistance of tumors and the presence of the blood brain barrier (BBB). Advanced nanoplatforms that can efficiently cross the BBB and target to brain tumor are urgently needed. Encouraged by the intrinsic inflammatory chemotaxis and excellent BBB-crossing capability of neutrophils, a bioinspired neutrophil-exosomes (NEs-Exos) system for delivering loaded doxorubicin (DOX) drug for glioma treatment is proposed and systematically investigated. In vivo zebrafish and C6-Luc glioma-bearing mice models show that NEs-Exos carrying the drug rapidly penetrate the BBB and migrate into the brain. Additionally, a transwell BBB model and mouse brain inflammatory study show that NEs-Exos can respond chemotactically to inflammatory stimuli and target infiltrating tumor cells in inflamed brain tumors. Moreover, intravenous injection of NEs-Exos/DOX efficiently suppress tumor growth and prolong survival time in a glioma mouse model. On the basis of these results, NEs-Exos are confirmed to have neutrophil-like chemotactic function and BBB penetration. This novel NEs-Exos/DOX delivery platform represents a promising chemotherapeutic approach for clinical treatment of glioma and other solid tumor or brain diseases.  

3.3768           Urinary exosomes-based Engineered Nanovectors for Homologously Targeted Chemo-Chemodynamic Prostate Cancer Therapy via abrogating EGFR/AKT/NF-kB/IkB signaling

Pan, S., Zhang, Y., Huang, M., Deng, Z., Zhang, A., Pei, L., Wang, L., Zhao, W., Ma, L., Zhang, Q. and Cui, D. Biomaterials, 275, 120946 (2021)   Multi-functional nanovectors based on exosomes from cancer cell culture supernatants in vitro has been successfully utilized for tumor-specific targeting and immune escape. However, the labor-intensive purification procedures for rich-dose and high-purity homogeneous exosomes without using targeting ligands are still a challenging task. Herein, we developed a nanovector Exo-PMA/Fe-HSA@DOX through cloaked by urinary exosome membrane as a chemo/chemodynamic theranostic nano-platform for targeted homologous prostate cancer therapy which pertain to the abrogation of Epidermal Growth Factor Receptor (EGFR) and its downstream AKT/NF-kB/IkB signaling instead of ERK signaling cascades. Urinary exosomes-based nanovectors own the same urological cancer cell membrane antigen inclusive of E-cadherin, CD 47 and are free from intracellular substance such as Histone 3 and COX Ⅳ. The targeting properties of the homologous cancer cell are well preserved in Exo-PMA/Fe-HSA@DOX nanovectors in high purity. Meanwhile, the nanovectors based on urinary exosomes from prostate patients deeply penetrated into prostate cancer DU145 3D MCTS, and successfully achieve superior synergistic low-dose chemo/chemodynamic performance in vivo. In addition, the blockage of bypassing EGFR/AKT/NF-kB/IkB signaling pathway is greatly enhanced via elevated intracellular PMA/Fe-HSA@DOX nanoparticles (NPs). It is expected that the rich source and high purity of urinary exosomes offer a reliable solution for mass production of such nanovectors in the future. The targeted homologous cancer therapy based on the urinary exosomes from cancer patients exemplifies a novel targeted anticancer scheme with efficient and facile method.  

3.3769           High-quality milk exosomes as oral drug delivery system

Zhong, J., Xia, B., Shan, S., Zheng, A., Zhang, S., Chen, J. and Liang, X-J. Biomaterials, 277, 121126 (2021)   Many drugs must be administered intravenously instead of oral administration due to their poor oral bioavailability. The cost of repeated infusion treatment for 6 weeks every year is as high as tens of billions of dollars worldwide. Exosomes are nano-sized (30–150 nm) extracellular vesicles secreted by mammalian cells due to environmental stimulation or self-activation. Milk contains abundant exosomes originated from multiple cellular sources. It has been proved that milk exosomes (MEs) could survive with the strongly acidic conditions in the stomach and degradative conditions in the gut. Furthermore, they can cross biological barriers to reach targeted tissues. The ability of MEs to cross the gastrointestinal barrier makes them as a promising drug delivery tool for oral delivery. This review is devoted to the purification of MEs, their biocompatibility and immunogenicity, and prospects for their use as natural drug carriers for oral administration.  

3.3770           Derepression of inflammation-related genes link to microglia activation and neural maturation defect in a mouse model of Kleefstra syndrome

Yamada, A., Hirasawa, T., Nishimura, K., Nikaido, I., Itohara, S. and Shinkai, Y. iScience, 24(7), 102741 (2021)   Haploinsufficiency of EHMT1, which encodes histone H3 lysine 9 (H3K9) methyltransferase G9a-like protein (GLP), causes Kleefstra syndrome (KS), a complex disorder of developmental delay and intellectual disability. Here, we examined whether postnatal supply of GLP can reverse the neurological phenotypes seen in Ehmt1^Δ/+ mice as a KS model. Ubiquitous GLP supply from the juvenile stage ameliorated behavioral abnormalities in Ehmt1^Δ/+ mice. Postnatal neuron-specific GLP supply was not sufficient for the improvement of abnormal behaviors but still reversed the reduction of H3K9me2 and spine number in Ehmt1^Δ/+ mice. Interestingly, some inflammatory genes, including IL-1β (Il1b), were upregulated and activated microglial cells increased in the Ehmt1^Δ/+ brain, and such phenotypes were also reversed by neuron-specific postnatal GLP supply. Il1b inactivation canceled the microglial and spine number phenotypes in the Ehmt1^Δ/+ mice. Thus, H3K9me2 and some neurological phenotypes are reversible, but behavioral abnormalities are more difficult to improve depending on the timing of GLP supply.  

3.3771           High glucose macrophage exosomes enhance atherosclerosis by driving cellular proliferation & hematopoiesis

Bouchereychas, L., Duong, P., Phu, T.A., Gasper, W.J., Van Keuren-Jensen, K. and Raffai, R.L: iScience, 24, 102847 (2021)   We investigated whether extracellular vesicles (EVs) produced under hyperglycemic conditions could communicate signaling to drive atherosclerosis. We did so by treating Apoe^−/− mice with exosomes produced by bone marrow-derived macrophages (BMDM) exposed to high glucose (BMDM–HG-exo) or control. Infusions of BMDM–HG-exo increased hematopoiesis, circulating myeloid cell numbers, and atherosclerotic lesions with an accumulation of macrophage foam and apoptotic cells. Transcriptome-wide analysis of cultured macrophages treated with BMDM–HG-exo or plasma EVs isolated from subjects with type II diabetes revealed a reduced inflammatory state and increased metabolic activity. Furthermore, BMDM–HG-exo induced cell proliferation and reprogrammed energy metabolism by increasing glycolytic activity. Lastly, profiling microRNA in BMDM–HG-exo and plasma EVs from diabetic subjects with advanced atherosclerosis converged on miR-486-5p as commonly enriched and recognized in dysregulated hematopoiesis and Abca1 control. Together, our findings show that EVs serve to communicate detrimental properties of hyperglycemia to accelerate atherosclerosis in diabetes.  

3.3772           Extracellular heat shock proteins and cancer: New perspectives

Albakova, Z., Siam, M.K.S., Sacitharan, P.K., Ziganshin, R.H., Ryazantsev, D.Y. and Sapozhnikov, A.M. Transl. Oncol., 14, 100995 (2021)   Heat shock proteins (HSPs) are a large family of molecular chaperones aberrantly expressed in cancer. The expression of HSPs in tumor cells has been shown to be implicated in the regulation of apoptosis, immune responses, angiogenesis and metastasis. Given that extracellular vesicles (EVs) can serve as potential source for the discovery of clinically useful biomarkers and therapeutic targets, it is of particular interest to study proteomic profiling of HSPs in EVs derived from various biological fluids of cancer patients. Furthermore, a divergent expression of circulating microRNAs (miRNAs) in patient samples has opened new opportunities in exploiting miRNAs as diagnostic tools. Herein, we address the current literature on the expression of extracellular HSPs with particular interest in HSPs in EVs derived from various biological fluids of cancer patients and different types of immune cells as promising targets for identification of clinical biomarkers of cancer. We also discuss the emerging role of miRNAs in HSP regulation for the discovery of blood-based biomarkers of cancer. We outline the importance of understanding relationships between various HSP networks and co-chaperones and propose the model for identification of HSP signatures in cancer. Elucidating the role of HSPs in EVs from the proteomic and miRNAs perspectives may provide new opportunities for the discovery of novel biomarkers of cancer.  

3.3773           Cyclophilin A regulates secretion of tumour-derived extracellular vesicles

Wu, Y., Brennan, K., Fernandez, A.B. and Gee, M.M.Mc. Transl. Oncol., 14, 101112 (2021)   Extracellular Vesicles (EVs) are a heterogenous population of particles that play an important role in cell-cell communication in physiological and pathophysiological situations. In this study we reveal that the peptidyl prolyl isomerase Cyclophilin A (CypA) is enriched in cancer-derived EVs from a range of haematopoietic malignancies. CypA-enriched blood cancer EVs were taken up by normal monocytes independent of EV surface trypsin-sensitive proteins and potently stimulated pro-inflammatory MMP9 and IL-6 secretion. Further characterisation revealed that CypA is intravesicular, however, it is not present in all EVs derived from the haematopoietic cells, instead, it is predominantly located in high density EVs with a range of 1.15–1.18 g/ml. Furthermore, loss of CypA expression in haematological cancer cells attenuates high density EV-induced pro-inflammatory MMP9 and IL-6 secretion from monocytes. Mechanistically, we reveal that homozygous loss or siRNA knockdown of CypA expression significantly reduced the secretion of EVs in the range of 100–200 nm from blood cancer cells under normal and hypoxic conditions. Overall, this work reveals a novel role for CypA in cancer cell EV biogenesis.  

3.3774           Area Postrema Cell Types that Mediate Nausea-Associated Behaviors

Zhang, C., kaye, J.A., Cai, Z., Wang, Y., Prescott, S.L. and Liberles, S.D. Neuron, 109(3), 461-472 (2021)   Nausea, the unpleasant sensation of visceral malaise, remains a mysterious process. The area postrema is implicated in some nausea responses and is anatomically privileged to detect blood-borne signals. To investigate nausea mechanisms, we built an area postrema cell atlas through single-nucleus RNA sequencing, revealing a few neuron types. Using mouse genetic tools for cell-specific manipulation, we discovered excitatory neurons that induce nausea-related behaviors, with one neuron type mediating aversion imposed by multiple poisons. Nausea-associated responses to agonists of identified area postrema receptors were observed and suppressed by targeted cell ablation and/or gene knockout. Anatomical mapping revealed a distributed network of long-range excitatory but not inhibitory projections with subtype-specific patterning. These studies reveal the basic organization of area postrema nausea circuitry and provide a framework toward understanding and therapeutically controlling nausea.  

3.3775           Oligodendrocytes enhance axonal energy metabolism by deacetylation of mitochondrial proteins through transcellular delivery of SIRT2

Chamberlain, K.A., Huang, N., Xie, Y., LiCausi, F., Li, S., Li, Y. and Sheng, Z-H. Neuron, 109(21), 3456-3472 (2021)   Neurons require mechanisms to maintain ATP homeostasis in axons, which are highly vulnerable to bioenergetic failure. Here, we elucidate a transcellular signaling mechanism by which oligodendrocytes support axonal energy metabolism via transcellular delivery of NAD-dependent deacetylase SIRT2. SIRT2 is undetectable in neurons but enriched in oligodendrocytes and released within exosomes. By deleting sirt2, knocking down SIRT2, or blocking exosome release, we demonstrate that transcellular delivery of SIRT2 is critical for axonal energy enhancement. Mass spectrometry and acetylation analyses indicate that neurons treated with oligodendrocyte-conditioned media from WT, but not sirt2-knockout, mice exhibit strong deacetylation of mitochondrial adenine nucleotide translocases 1 and 2 (ANT1/2). In vivo delivery of SIRT2-filled exosomes into myelinated axons rescues mitochondrial integrity in sirt2-knockout mouse spinal cords. Thus, our study reveals an oligodendrocyte-to-axon delivery of SIRT2, which enhances ATP production by deacetylating mitochondrial proteins, providing a target for boosting axonal bioenergetic metabolism in neurological disorders.  

3.3776           Oncogene-regulated release of extracellular vesicles

Killinc, S., Paisner, R., Camarda, R., Perera, R.M., Nomura, D.K. and Gogga, A. Developmental Cell, 56(13), 1989-2006 (2021)   Oncogenes can alter metabolism by changing the balance between anabolic and catabolic processes. However, how oncogenes regulate tumor cell biomass remains poorly understood. Using isogenic MCF10A cells transformed with nine different oncogenes, we show that specific oncogenes reduce the biomass of cancer cells by promoting extracellular vesicle (EV) release. While MYC and AURKB elicited the highest number of EVs, each oncogene selectively altered the protein composition of released EVs. Likewise, oncogenes alter secreted miRNAs. MYC-overexpressing cells require ceramide, whereas AURKB requires ESCRT to release high levels of EVs. We identify an inverse relationship between MYC upregulation and activation of the RAS/MEK/ERK signaling pathway for regulating EV release in some tumor cells. Finally, lysosome genes and activity are downregulated in the context of MYC and AURKB, suggesting that cellular contents, instead of being degraded, were released via EVs. Thus, oncogene-mediated biomass regulation via differential EV release is a new metabolic phenotype.  

3.3777           S-acylation controls SARS-CoV-2 membrane lipid organization and enhances infectivity

Mesquita, F.S., Abrami, L., Sergeeva, L., Trono, D., D’Angelo, G. and van der Goot, F. Developmental Cell, 56(20), 2790-2807 (2021)   SARS-CoV-2 virions are surrounded by a lipid bilayer that contains membrane proteins such as spike, responsible for target-cell binding and virus fusion. We found that during SARS-CoV-2 infection, spike becomes lipid modified, through the sequential action of the S-acyltransferases ZDHHC20 and 9. Particularly striking is the rapid acylation of spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics, and biochemical approaches, we show that this massive lipidation controls spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid-rich lipid nanodomains in the early Golgi, where viral budding occurs. Finally, S-acylation of spike allows the formation of viruses with enhanced fusion capacity. Our study points toward S-acylating enzymes and lipid biosynthesis enzymes as novel therapeutic anti-viral targets.  

3.3778           Protective plant immune responses are elicited by bacterial outer membrane vesicles

McMillan, H.M., Zebell, S.G., Ristaino, J.B., Dong, X. and Kuehn, M.J. Cell Reports, 34, 108645 (2021)   Bacterial outer membrane vesicles (OMVs) perform a variety of functions in bacterial survival and virulence. In mammalian systems, OMVs activate immune responses and are exploited as vaccines. However, little work has focused on the interactions of OMVs with plant hosts. Here, we report that OMVs from Pseudomonas syringae and P. fluorescens activate plant immune responses that protect against bacterial and oomycete pathogens. OMV-mediated immunomodulatory activity from these species displayed different sensitivity to biochemical stressors, reflecting differences in OMV content. Importantly, OMV-mediated plant responses are distinct from those triggered by conserved bacterial epitopes or effector molecules alone. Our study shows that OMV-induced protective immune responses are independent of the T3SS and protein, but that OMV-mediated seedling growth inhibition largely depends on proteinaceous components. OMVs provide a unique opportunity to understand the interplay between virulence and host response strategies and add a new dimension to consider in host-microbe interactions.  

3.3779           Disentangling the complexity of tumor-derived extracellular vesicles

Beltraminelli, T., Perez, C. and De palma, M. Cell Reports, 35, 108960 (2021)   The tumor microenvironment encompasses an intertwined ensemble of both transformed cancer cells and non-transformed host cells, which together establish a signaling network that regulates tumor progression. By conveying both homo- and heterotypic cell-to-cell communication cues, tumor-derived extracellular vesicles (tEVs) modulate several cancer-associated processes, such as immunosuppression, angiogenesis, invasion, and metastasis. Herein we discuss how recent methodological advances in the isolation and characterization of tEVs may help to broaden our understanding of their functions in tumor biology and, potentially, establish their utility as cancer biomarkers.  

3.3780           Inappropriate use of antibiotics exacerbates inflammation through OMV-induced pyroptosis in MDR Klebsiella pneumoniae infection

Ye, C., Li, W., Yang, Y., Qi, J., Huang, W. and Ma, Y. Cell Reports, 36(12), 109750 (2021)   The inappropriate use of antibiotics is a severe public health problem worldwide, contributing to the emergence of multidrug-resistant (MDR) bacteria. To explore the possible impacts of the inappropriate use of antibiotics on the immune system, we use Klebsiella pneumoniae (K. pneumoniae) infection as an example and show that imipenem increases the mortality of mice infected by MDR K. pneumoniae. Further studies demonstrate that imipenem enhances the secretion of outer membrane vesicles (OMVs) with significantly elevated presentation of GroEL, which promotes the phagocytosis of OMVs by macrophages that depends on the interaction between GroEL and its receptor, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). OMVs cause the pyroptosis of macrophages and the release of proinflammatory cytokines, which contribute to exacerbated inflammatory responses. We propose that the inappropriate use of antibiotics in the cases of infection by MDR bacteria such as K. pneumoniae might cause damaging inflammatory responses, which underlines the pernicious effects of inappropriate use of antibiotics.  

3.3781           Acrylamide modulates the mouse epididymal proteome to drive alterations in the sperm small non-coding RNA profile and dysregulate embryo development

Trigg, N.A., Skerett-Byrne, D.A., Xavier, M.J., Roman, S.D., Eamens, A.L. and Nixon, B. Cell Reports, 37(1), 109787 (2021)   Paternal exposure to environmental stressors elicits distinct changes to the sperm sncRNA profile, modifications that have significant post-fertilization consequences. Despite this knowledge, there remains limited mechanistic understanding of how paternal exposures modify the sperm sncRNA landscape. Here, we report the acute sensitivity of the sperm sncRNA profile to the reproductive toxicant acrylamide. Furthermore, we trace the differential accumulation of acrylamide-responsive sncRNAs to coincide with sperm transit of the proximal (caput) segment of the epididymis, wherein acrylamide exposure alters the abundance of several transcription factors implicated in the expression of acrylamide-sensitive sncRNAs. We also identify extracellular vesicles secreted from the caput epithelium in relaying altered sncRNA profiles to maturing spermatozoa and dysregulated gene expression during early embryonic development following fertilization by acrylamide-exposed spermatozoa. These data provide mechanistic links to account for how environmental insults can alter the sperm epigenome and compromise the transcriptomic profile of early embryos.  

3.3782           CRISPR knockdown of Kcnq3 attenuates the M-current and increases excitability of NPY/AgRP neurons to alter energy balance

Stincic, T.L., Bosch, M.A., Hunker, A.C., Juarez, B., Connors, A.M., Zwelfel, L.S., Rønnekleiv, O.K. and Kelly, M.J. Mol. Metabolism, 49, 101218 (2021)   Arcuate nucleus neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons drive ingestive behavior. The M-current, a subthreshold non-inactivating potassium current, plays a critical role in regulating NPY/AgRP neuronal excitability. Fasting decreases while 17β-estradiol increases the M-current by regulating the mRNA expression of Kcnq2, 3, and 5 (Kv7.2, 3, and 5) channel subunits. Incorporating KCNQ3 into heteromeric channels has been considered essential to generate a robust M-current. Therefore, we investigated the behavioral and physiological effects of selective Kcnq3 deletion from NPY/AgRP neurons. Methods We used a single adeno-associated viral vector containing a recombinase-dependent Staphylococcus aureus Cas9 with a single-guide RNA to selectively delete Kcnq3 in NPY/AgRP neurons. Single-cell quantitative measurements of mRNA expression and whole-cell patch clamp experiments were conducted to validate the selective knockdown. Body weight, food intake, and locomotor activity were measured in male mice to assess disruptions in energy balance. Results The virus reduced the expression of Kcnq3 mRNA without affecting Kcnq2 or Kcnq5. The M-current was attenuated, causing NPY/AgRP neurons to be more depolarized, exhibit a higher input resistance, and require less depolarizing current to fire action potentials, indicative of increased excitability. Although the resulting decrease in the M-current did not overtly alter ingestive behavior, it significantly reduced the locomotor activity as measured by open-field testing. Control mice on a high-fat diet exhibited an enhanced M-current and increased Kcnq2 and Kcnq3 expression, but the M-current remained significantly attenuated in KCNQ3 knockdown animals. Conclusions The M-current plays a critical role in modulating the intrinsic excitability of NPY/AgRP neurons that is essential for maintaining energy homeostasis.  

3.3783           A survey of the mouse hindbrain in the fed and fasted states using single-nucleus RNA sequencing

Dowsett, G.K.C., Lam, B.Y.H., Tadross, J.A., Cimino, I., Rimmington, D., Coll, A.P., Polex-Wolf, J., Knudsen, L.B., Pyke, C. and Yoo, G.S.H. Mol. Metabolism, 53, 101240 (2021)   Objective The area postrema (AP) and nucleus tractus solitarius (NTS) located in the hindbrain are key nuclei that sense and integrate peripheral nutritional signals and consequently regulate feeding behaviour. While single-cell transcriptomics have been used in mice to reveal the gene expression profile and heterogeneity of key hypothalamic populations, similar in-depth studies have not yet been performed in the hindbrain. Methods Using single-nucleus RNA sequencing, we provide a detailed survey of 16,034 cells within the AP and NTS of mice in the fed and fasted states. Results Of these, 8,910 were neurons that group into 30 clusters, with 4,289 from mice fed ad libitum and 4,621 from overnight fasted mice. A total of 7,124 nuclei were from non-neuronal cells, including oligodendrocytes, astrocytes, and microglia. Interestingly, we identified that the oligodendrocyte population was particularly transcriptionally sensitive to an overnight fast. The receptors GLP1R, GIPR, GFRAL, and CALCR, which bind GLP1, GIP, GDF15, and amylin, respectively, are all expressed in the hindbrain and are major targets for anti-obesity therapeutics. We characterise the transcriptomes of these four populations and show that their gene expression profiles are not dramatically altered by an overnight fast. Notably, we find that roughly half of cells that express GIPR are oligodendrocytes. Additionally, we profile POMC-expressing neurons within the hindbrain and demonstrate that 84% of POMC neurons express either PCSK1, PSCK2, or both, implying that melanocortin peptides are likely produced by these neurons. Conclusion We provide a detailed single-cell level characterisation of AP and NTS cells expressing receptors for key anti-obesity drugs that are either already approved for human use or in clinical trials. This resource will help delineate the mechanisms underlying the effectiveness of these compounds and also prove useful in the continued search for other novel therapeutic targets.  

3.3784           A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties

Dooley, K., McConnell, R.E., Xu, K., Lewis, N.D., haupt, S., Youniss, M.R., martin, S. et al Molecular Therapy, 29(5), 1729-1743 (2021)   Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two “scaffold” proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.  

3.3785           Rational design for controlled release of Dicer-substrate siRNA harbored in phi29 pRNA-based nanoparticles

Binzel, D.W., Cuo, S., Yin, H., Lee, T.J., Liu, S., Shu, D. and Guo, P. Molecular Therapy-Nucleic Acids, 25, 524-535 (2021)   Small interfering RNA (siRNA) for silencing genes and treating disease has been a dream since ranking as a top Breakthrough of the Year in 2002 by Science. With the recent FDA approval of four siRNA-based drugs, the potential of RNA therapeutics to become the third milestone in pharmaceutical drug development has become a reality. However, the field of RNA interference (RNAi) therapeutics still faces challenges such as specificity in targeting, intracellular processing, and endosome trapping after targeted delivery. Dicer-substrate siRNAs included onto RNA nanoparticles may be able to overcome these challenges. Here, we show that pRNA-based nanoparticles can be designed to efficiently harbor the Dicer-substrate siRNAs in vitro and in vivo to the cytosol of tumor cells and release the siRNA. The structure optimization and chemical modification for controlled release of Dicer-substrate siRNAs in tumor cells were also evaluated through molecular beacon analysis. Studies on the length requirement of the overhanging siRNA revealed that at least 23 nucleotides at the dweller’s arm were needed for dicer processing. The above sequence parameters and structure optimization were confirmed in recent studies demonstrating the release of functional Survivin siRNA from the pRNA-based nanoparticles for cancer inhibition in non-small-cell lung, breast, and prostate cancer animal models.  

3.3786           Neuronal non-CG methylation is an essential target for MeCP2 function

Tillotson, R., Cholewa-Waclaw, J., Chhatbar, K., Lyst, M.J., Kriaucionis, S. and Bird, A. Molecular Cell, 81(6), 1260-1275 (2021)   DNA methylation is implicated in neuronal biology via the protein MeCP2, the mutation of which causes Rett syndrome. MeCP2 recruits the NCOR1/2 co-repressor complexes to methylated cytosine in the CG dinucleotide, but also to sites of non-CG methylation, which are abundant in neurons. To test the biological significance of the dual-binding specificity of MeCP2, we replaced its DNA binding domain with an orthologous domain from MBD2, which can only bind mCG motifs. Knockin mice expressing the domain-swap protein displayed severe Rett-syndrome-like phenotypes, indicating that normal brain function requires the interaction of MeCP2 with sites of non-CG methylation, specifically mCAC. The results support the notion that the delayed onset of Rett syndrome is due to the simultaneous post-natal accumulation of mCAC and its reader MeCP2. Intriguingly, genes dysregulated in both Mecp2 null and domain-swap mice are implicated in other neurological disorders, potentially highlighting targets of relevance to the Rett syndrome phenotype.  

3.3787           Mitocytosis, a migrasome-mediated mitochondrial quality-control process

Jiao, H., Jiang, D., Hu, X., Wei, Y-H., Hu, X. and Yu, L. Cell, 184(11), 2896-2910 (2021)   Damaged mitochondria need to be cleared to maintain the quality of the mitochondrial pool. Here, we report mitocytosis, a migrasome-mediated mitochondrial quality-control process. We found that, upon exposure to mild mitochondrial stresses, damaged mitochondria are transported into migrasomes and subsequently disposed of from migrating cells. Mechanistically, mitocytosis requires positioning of damaged mitochondria at the cell periphery, which occurs because damaged mitochondria avoid binding to inward motor proteins. Functionally, mitocytosis plays an important role in maintaining mitochondrial quality. Enhanced mitocytosis protects cells from mitochondrial stressor-induced loss of mitochondrial membrane potential (MMP) and mitochondrial respiration; conversely, blocking mitocytosis causes loss of MMP and mitochondrial respiration under normal conditions. Physiologically, we demonstrate that mitocytosis is required for maintaining MMP and viability in neutrophils in vivo. We propose that mitocytosis is an important mitochondrial quality-control process in migrating cells, which couples mitochondrial homeostasis with cell migration.  

3.3788           Glioblastoma cell populations with distinct oncogenic programs release podoplanin as procoagulant extracellular vesicles

Tawl, N., Bassawon, R., Meehan, B., Nehme, A., Montermini, L., Gayden, T. De Jay, N. et al Blood Advances, 5(6), 1682-1694 (2021)   Vascular anomalies, including local and peripheral thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of deregulation of the cancer cell genome and epigenome. Although the molecular effectors of these changes are poorly understood, the upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk for venous thromboembolism (VTE) in GBM patients. Therefore, regulation of this platelet-activating protein by transforming events in cancer cells is of considerable interest. We used single-cell and bulk transcriptome data mining, as well as cellular and xenograft models in mice, to analyze the nature of cells expressing PDPN, as well as their impact on the activation of the coagulation system and platelets. We report that PDPN is expressed by distinct (mesenchymal) GBM cell subpopulations and downregulated by oncogenic mutations of EGFR and IDH1 genes, along with changes in chromatin modifications (enhancer of zeste homolog 2) and DNA methylation. Glioma cells exteriorize their PDPN and/or tissue factor (TF) as cargo of exosome-like extracellular vesicles (EVs) shed from cells in vitro and in vivo. Injection of glioma-derived podoplanin carrying extracelluar vesicles (PDPN-EVs) activates platelets, whereas tissue factor carrying extracellular vesicles (TF-EVs) activate the clotting cascade. Similarly, an increase in platelet activation (platelet factor 4) or coagulation (D-dimer) markers occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Coexpression of PDPN and TF by GBM cells cooperatively affects tumor microthrombosis. Thus, in GBM, distinct cellular subsets drive multiple facets of cancer-associated thrombosis and may represent targets for phenotype- and cell type–based diagnosis and antithrombotic intervention.  

3.3789           A novel mechanism of thrombocytopenia by PS exposure through TMEM16F in sphingomyelin synthase 1 deficiency

Fujii, Y., Taniguchi, M., Nagaya, S., Ueda, Y., Hashizume, C., Watanabe, K., Takeya, H., Kosaka, T. and Okazaki, T.

Blood Advances, 5(20), 4265-4277 (2021)   Sphingomyelin synthase 1 (SMS1) contributes to the generation of membrane sphingomyelin (SM) and affects SM-mediated physiological functions. Here, we describe the hematologic phenotypes, such as reduced circulating platelets and dysfunctional hemostasis, in SMS1-deficient (SMS1-KO) mice. SMS1-KO mice display pathologic manifestations related to idiopathic thrombocytopenia (ITP), including relatively high amounts of peripheral blood reticulated platelets, enhanced megakaryopoiesis in the bone marrow and spleen, and splenomegaly. Deficiency of SMS1, but not SMS2, prevented SM production and enhanced phosphatidylserine (PS) externalization on the plasma membranes of platelets and megakaryocytes. Consequently, SMS1-KO platelets were excessively cleared by macrophages in the spleen. Multimer formation in the plasma membrane of TMEM16F, a known calcium (Ca²⁺)-activated nonselective ion channel and Ca²⁺-dependent PS scramblase, was enhanced; the result was PS externalization to outer leaflets through increased Ca²⁺ influx in immortalized mouse embryonic fibroblasts established from SMS1-KO mice (SMS1-KO tMEFs), as seen with SMS1-KO platelets. Thus, SMS1 deficiency changed the TMEM16F distribution on the membrane microdomain, regulating Ca²⁺ influx-dependent PS exposure. SMS1-KO tMEFs in which TMEM16F was knocked out by using the CRISPR/Cas9 system lacked both the Ca²⁺ influx and excess PS exposure seen in SMS1-KO tMEFs. Therefore, SM depletion on platelet membrane microdomains due to SMS1 deficiency enhanced PS externalization via a Ca²⁺ influx through TMEM16F activation, leading to elevated platelet clearance and causing hemostasis dysfunction through thrombocytopenia. Our current findings show that the SM-rich microdomain generated by SMS1 is a potent regulator of thrombocytopenia through TMEM16F, suggesting that its dysfunction may be a novel additional mechanism of ITP.  

3.3790           Exosomes and exosomal RNAs in breast cancer: A status update

Lakshmi, S., Hughes, T.A. and Priya, S. Eur. J. Cancer, 144, 252-268 (2021)   Improved treatment of breast cancer, the world's second most common cancer, requires identification of new sensitive prognostic and diagnostic biomarkers. Exosomes are lipid-bilayer extracellular vesicles of size 30–150 nm, released by all cell types, including breast cancer cells. Cellular communication is the primary function attributed to them. This review discusses the potential utility of exosomes and exosomal RNAs (microRNAs [miRNAs]/long non-coding RNAs [LncRNAs]) in breast cancer biology and treatment. The existing literature shows that exosomes play a significant role in breast tumorigenesis and progression through transfer miRNAs and LncRNAs. These miRNAs and LncRNAs function by post-transcriptionally regulating their target mRNAs, eventually leading to modulation of expression/repression. Over the past two decades, numerous publications point towards diagnostic and therapeutic applications of exosomal miRNAs/LncRNAs. Until now, we do not have clinically approved exosome-based therapeutics. Therefore, it is high time that clinicians and cancer researchers utilise exosome's benefits through randomised clinical trials for better management of breast cancer.  

3.3791           Advances in understanding and in multi-disciplinary methodology used to assess lipid regulation of signalling cascades from the cancer cell plasma membrane

Soteriou, C., Kalli, A.C., Connell, S.D., Tyler, A.I.I. and Thorne, J.L. Progress in Lipid Res., 81, 101080 (2021)   The lipid bilayer is a functional component of cells, forming a stable platform for the initiation of key biological processes, including cell signalling. There are distinct changes in the lipid composition of cell membranes during oncogenic transformation resulting in aberrant activation and inactivation of signalling transduction pathways. Studying the role of the cell membrane in cell signalling is challenging, since techniques are often limited to by timescale, resolution, sensitivity, and averaging. To overcome these limitations, combining ‘computational’, ‘wet-lab’ and ‘semi-dry’ approaches offers the best opportunity to resolving complex biological processes involved in membrane organisation. In this review, we highlight analytical tools that have been applied for the study of cell signalling initiation from the cancer cell membranes through computational microscopy, biological assays, and membrane biophysics. The cancer therapeutic potential of extracellular membrane-modulating agents, such as cholesterol-reducing agents is also discussed, as is the need for future collaborative inter-disciplinary research for studying the role of the cell membrane and its components in cancer therapy.  

3.3792           Hexameric procyanidins inhibit colorectal cancer cell growth through both redox and non-redox regulation of the epidermal growth factor signaling pathway

Daveri, E., Adamo, A.M., Alfine, E., Zhu, W. and Oteiza, P.I. Redox Biology, 38, 101830 (2021)   Dietary proanthocyanidins (PAC) consumption is associated with a decreased risk for colorectal cancer (CRC). Dysregulation of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway is frequent in human cancers, including CRC. We previously showed that hexameric PAC (Hex) exert anti-proliferative and pro-apoptotic actions in human CRC cells. This work investigated if Hex could exert anti-CRC effects through its capacity to regulate the EGFR pathway. In proliferating Caco-2 cells, Hex acted attenuating EGF-induced EGFR dimerization and NADPH oxidase-dependent phosphorylation at Tyr 1068, decreasing EGFR location at lipid rafts, and inhibiting the downstream activation of pro-proliferative and anti-apoptotic pathways, i.e. Raf/MEK/ERK1/2 and PI3K/Akt. Hex also promoted EGFR internalization both in the absence and presence of EGF. While Hex decreased EGFR phosphorylation at Tyr 1068, it increased EGFR Tyr 1045 phosphorylation. The latter provides a docking site for the ubiquitin ligase c-Cbl and promotes EGFR degradation by lysosomes. Importantly, Hex acted synergistically with the EGFR-targeted chemotherapeutic drug Erlotinib, both in their capacity to decrease EGFR phosphorylation and inhibit cell growth. Thus, dietary PAC could exert anti-CRC actions by modulating, through both redox- and non-redox-regulated mechanisms, the EGFR pro-oncogenic signaling pathway. Additionally, Hex could also potentiate the actions of EGFR-targeted drugs.  

3.3793           Bivalent non-typhoidal Salmonella outer membrane vesicles immunized mice sera confer passive protection against gastroenteritis in a suckling mice model

Maiti, S., Howlader, D.R., halder, P., Bhaumik, U., Dutta, M., Dutta, S. and Koley, H. Vaccine, 39, 380-393 (2021)   Invasive non-typhoidal Salmonella (iNTS) serovars, especially Salmonella Typhimurium (ST) and Salmonella Enteritidis (SE), cause gastroenteritis worldwide. Due to the emergence of multi-drug resistance in iNTS, a broad-spectrum vaccine is urgently needed for the prevention of iNTS infection. Currently, there is no effective licensed vaccine against iNTS available in the market. We have formulated an outer membrane vesicles (OMVs) based bivalent immunogen as a vaccine candidate to generate broad-spectrum protective immunity against both recently circulating prevalent ST and SE. We have isolated OMVs from ST and SE and formulated the immunogen by mixing both OMVs (1:1 ratio). Three doses of bivalent immunogen significantly induced humoral immune responses against lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) as well as a cell-mediated immune response in adult mice. We also observed that proteins of OMVs act as an adjuvant for generation of high levels of anti-LPS antibodies through T cell activation. We then characterized the one-day old suckling mice model for both ST and SE mediated gastroenteritis and used the model for a passive protection study. In the passive protection study, we found the passive transfer of bivalent OMVs immunized sera significantly reduced ST and SE mediated colonization and gastroenteritis symptoms in the colon of suckling mice compared to non-immunized sera recipients. The overall study demonstrated that OMVs based bivalent vaccine could generate broad-spectrum immunity against prevalent iNTS mediated gastroenteritis. This study also established the suckling mice model as a suitable animal model for vaccine study against iNTS mediated gastroenteritis.  

3.3794           Evaluation of bovine milk extracellular vesicles for the delivery of locked nucleic acid antisense oligonucleotides

Grossen, P., Portmann, M., Koller, E., Duschmale, M., Minz, T., Sewing, S., Pandya, N.J. et al Eur. J. Pharmaceutics and Bioformaceutics, 158, 198-210 (2021)   The natural capacity of extracellular vesicles (EVs) to transport their payload to recipient cells has raised big interest to repurpose EVs as delivery vehicles for xenobiotics. In the present study, bovine milk-derived EVs (BMEVs) were investigated for their potential to shuttle locked nucleic acid-modified antisense oligonucleotides (LNA ASOs) into the systemic circulation after oral administration. To this end, a broad array of analytical methods including proteomics and lipidomics were used to thoroughly characterize BMEVs. We found that additional purification by density gradients efficiently reduced levels of non-EV associated proteins. The potential of BMEVs to functionally transfer LNA ASOs was tested using advanced in vitro systems (i.e. hPSC-derived neurons and primary human cells). A slight increase in cellular LNA ASO internalization and target gene reduction was observed when LNA ASOs were delivered using BMEVs. When dosed orally in mice, only a small fraction (about 1% of total administered dose) of LNA ASOs was recovered in the peripheral tissues liver and kidney, however, no significant reduction in target gene expression (i.e. functional knockdown) was observed.  

3.3795           Potential of extracellular vesicles in the Parkinson's disease – Pathological mediators and biomarkers

Zhao, Y. and Yang, G. Neurochem. Int., 144, 104974 (2021)   Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by the progressive deterioration of motor function. Histopathologically, it is widely accepted that the progressive death of selected dopaminergic neuronal populations and the accumulation of hallmark Lewy bodies (LBs) composed of α-synuclein (α-syn) might be the two vital pathogenesis. Extracellular vesicles (EVs) are cell-derived membranous vesicles that are liberated from virtually all cell types including neurons, and harbor a variety of proteins, DNA, mRNA, and lipids. The roles of these vesicles include cell-cell signaling, removal of unwanted proteins, and transfer of pathogens (including misfolded proteins) between cells. In PD, EVs not only enhance the spread of α-syn at distant sites and reduce their clearance but also mediate other PD pathogenesis such as the activation of microglia and the dysfunction of autophagy and lysosomal degradation systems. Recently, clinical evidence for the diagnostic performance of EV-associated biomarkers, particularly exosome biomarkers, has merged. In this regard, we reviewed the recent understanding of the biological roles of EVs as important tools for biomarker discovery and pathological regulators of PD, and discuss the main concerns and challenges for the application of EV biomarkers in the clinical setting.  

3.3796           Extracellular vesicles, microRNA and the preimplantation embryo: non-invasive clues of embryo well-being

Hawke, D.C., Watson, A.J. and Betts, D.H. RBMO, 42(1), 39-54 (2021)   Elective single embryo transfer is rapidly becoming the standard of care in assisted reproductive technology for patients under the age of 35 years with a good prognosis. Clinical pregnancy rates have become increasingly dependent on the selection of a single viable embryo for transfer, and diagnostic techniques facilitating this selection continue to develop. Current progress in elucidating the extracellular vesicle and microRNA components of the embryonic secretome is reviewed, and the potential for these findings to improve clinical embryo selection discussed. Key results have shown that extracellular vesicles and microRNAs are rapidly detectable constituents of the embryonic secretome. Evidence suggests that the vesicular population is largely exosomal in nature, secreted at all stages of preimplantation development and capable of traversing the zona pellucida. Both extracellular vesicle and microRNA concentrations within the secretome are elevated for blastocysts with diminished developmental competence, as indicated either by degeneracy or implantation failure, whereas studies have yet to firmly correlate individual microRNA sequences with pregnancy outcome. These emerging correlations support the viability of extracellular vesicles and microRNAs as the basis for a new diagnostic test to supplement or replace morphokinetic assessment.  

3.3797           Tissue-derived extracellular vesicles: Research progress from isolation to application

Qin, B., Hu, X-m., Su, Z-h., Zeng, X-b., Ma, H-y. and Xiong, K. Pathology-Research and Practice, 226, 153604 (2021)   Extracellular vesicles (EVs) are the structures that all cells release into the environment. They are separated by a lipid bilayer and contain the cellular components that release them. To date, most studies have been performed on EVs derived from cell supernatants or different body fluids, while the number of studies on EV isolation directly from tissues is still limited. Studies of EV isolation directly from tissues may provide us with better information. This review summarizes the role of EV in the extracellular matrix, the protocol for isolation of EV in the tissue interstitium, and the application of the protocol in different tissues.  

3.3798           Optimisation and comparison of orthogonal methods for separation and characterisation of extracellular vesicles to investigate how representative infant milk formula is of milk

Mukhopadhya, A., Santoro, J., Moran, B. and Useckaite, Z. Food Chem., 353, 129309 (2021)   Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves harsh treatment. So, we hypothesised that the quantity and/or quality of extracellular vesicles (EVs) in IMF may be reduced. Thus, firstly, we aimed to optimise separation of EVs from IMF and SM and, secondly, we aimed to compare the EV isolates from these two sources. Prior to EV isolation, abundant casein micelles of similar sizes to EVs were removed by treating milk samples with either acetic acid or hydrochloric acid. Samples progressed to differential ultracentrifugation (DUC) or gradient ultracentrifugation (GUC). EV characterisation included BCA, SDS-PAGE, nanoparticle tracking (NTA), electron microscopy (TEM), immunoblotting, and imaging flow cytometry (IFCM). Reduced EV concentrations were found in IMF. SM-derived EVs were intact, while IMF contained disrupted EV-like structures. EV biomarkers were more abundant with isolates from SM, indicating EV proteins in IMF are compromised. Altogether, a suitable method combining acid pre-treatment with GUC for EV separation from milk products was developed. EVs appear to be substantially compromised in IMF compared to SM.  

3.3799           Colchicine reduces extracellular vesicle NLRP3 inflammasome protein levels in chronic coronary disease: A LoDoCo2 biomarker substudy

Silvis, M.J.M., Fiolet, A.T.L., Opstal, T.S.J., Dekker, M., Suquilanda, D., Zivkovic, M., Duyvendak, M., The, S.H.K., Timmers, L., Bax, W.A., Mosterd, A., Cornel, J.H. and de Kleijn, D.P.V. Atherosclerosis, 334, 93-100 (2021)   Background and aims Colchicine reduces the risk of cardiovascular events in patients with coronary disease. Colchicine has broad anti-inflammatory effects and part of the atheroprotective effects have been suggested to be the result of NLRP3 inflammasome inhibition. We studied the effect of colchicine on extracellular vesicle (EV) NLRP3 protein levels and inflammatory markers, high sensitivity-CRP (hs-CRP) and interleukin (IL)-6, in patients with chronic coronary disease. Methods In vitro, the NLRP3 inflammasome was stimulated in PMA-differentiated- and undifferentiated THP-1 cells. In vivo, measurements were performed in serum obtained from 278 participants of the LoDoCo2 trial, one year after randomization to colchicine 0.5 mg once daily or placebo. EVs were isolated using precipitation. NLRP3 protein presence in EVs was confirmed using iodixanol density gradient centrifugation. Levels of NLRP3 protein, hs-CRP and IL-6 were measured using ELISA. Results In vitro, NLRP3 inflammasome stimulation showed an increase of EV NLRP3 protein levels. EV NLRP3 protein levels were lower in patients treated with colchicine (median 1.38 ng/mL), compared to placebo (median 1.58 ng/mL) (p = 0.025). No difference was observed in serum NLRP3 protein levels. Serum hs-CRP levels were lower in patients treated with colchicine (median 0.80 mg/L) compared to placebo (median 1.34 mg/L) (p < 0.005). IL-6 levels were lower in patients treated with colchicine (median 2.07 ng/L) compared to placebo (median 2.59 ng/L), although this was not statistically significant (p = 0.076). Conclusions Colchicine leads to a reduction of EV NLRP3 protein levels. This indicates that inhibitory effects on the NLRP3 inflammasome might contribute to the atheroprotective effects of colchicine in coronary disease.  

3.3800           PROKR1 delivery by cell-derived vesicles restores the myogenic potential of Prokr1-deficient C2C12 myoblasts

Zhang, C., Mok, J., Seong, Y., Lau, H-c., Kim, D., Yoon, J., Oh, S.W., Park, T.S. and Park, J. Nanomed.: Nanotechnol. Biol. Med., 37, 102448 (2021)   Cell-derived vesicles (CDVs) have been investigated as an alternative to exosomes. Here, we generated CDVs from Prokineticin receptor 1 (PROKR1) overexpressing HEK293T cells using micro-extrusion. More than 60 billion PROKR1-enriched CDV (PROKR1^Tg CDVs) particles with canonical exosome properties were recovered from 10⁷ cells. With 25 μg/mL of PROKR1^Tg CDVs, we observed delivery of PROKR1, significant reduction of apoptosis, and myotube formation in C2C12^{Prokr1−/−} myoblasts that have lost their myogenic potential but underwent apoptosis following myogenic commitment. Expression levels of early and late myogenic marker genes and glucose uptake capacity were restored to equivalent levels with wild-type control. Furthermore, PROKR1^Tg CDVs were accumulated in soleus muscle comparable to the liver without significant differences. Therefore, CDVs obtained from genetically engineered cells appear to be an effective method of PROKR1 protein delivery and offer promise as an alternative therapy for muscular dystrophy.  

3.3801           Dextran sulphate inhibits an association of prions with plasma membrane at the early phase of infection

Fuse, T., nakagaki, T., Homma, T., Tange, H., Yamaguchi, N., Atrarashi, R., Ishibashi, D. and Nishida, N. Neurosci. Res., 171, 34-40 (2021)   The defining characteristic of prion diseases is conversion of a cellular prion protein (PrP^C) to an abnormal prion protein (PrP^Sc). The exogenous attachment of PrP^Sc to the surface of a target cell is critical for infection. However, the initial interaction of PrP^Sc with the cell surface is poorly characterized. In the current study, we specifically focused on the association of PrP^Sc with cells during the early phase of infection, using an acute infection model. First, we treated mouse neuroblastoma N2a-58 cells with prion strain 22 L-infected brain homogenates and revealed that PrP^Sc was associated with membrane fractions within three hours, a short exposure time. These results were also observed in PrP^C-deficient hippocampus cell lines. We also demonstrate here that PrP^Sc from 22 L-infected brain homogenates was associated with lipid rafts during the early phase of infection. Furthermore, we revealed that DS500, a glycosaminoglycan mimetic, inhibited both the attachment of PrP^Sc to membrane fractions and subsequent prion transmission, suggesting that the early association of prions with cell surface is important for prion infection.  

3.3802           Purification of membrane vesicles from Gram-positive bacteria using flow cytometry, after iodixanol density-gradient ultracentrifugation

Nasukawa, T., Sugimoto, R., Uchiyama, J., Takemura-Uchiyama, I., Murakami, H., Fukuda, K., Matsuzaki, S. and Sakaguchi, M. Res. Microbiol., 172, 103792 (2021)   Membrane vesicles (MVs) play biologically important roles in Gram-positive bacteria, and purification is essential for their study. Although high-performance flow cytometry has the capability to quantify and isolate specific small particles, it has not been examined for MV isolation. In this study, we used high-performance flow cytometry to analyze MV from Gram-positive bacteria, Staphylococcus aureus and Bacillus subtilis, prepared by iodixanol density-gradient ultracentrifugation. Analysis of the quality of MV samples before and after sorting showed that the flow cytometric sorting provided higher purity and uniformity compared to gradient isolation alone. The MV purification method using flow cytometry should prove useful for applications requiring a very high purity of MV samples such as proteomic, metagenomic or lipidomic studies.  

3.3803           A post-insertion strategy for surface functionalization of bacterial and mammalian cell-derived extracellular vesicles

Jiang, L., Luirink, J., Kooijmans, S.A.A., van Kessel, K.P.M., Jong, W., van Essen, M., Seinen, C.W., de Maat, S., de Jong, O.G., Gitz-Francois, J.F.F., Hennink, W:E., Vader, P. and Schiffelers, R.M. BBA-General Subject, 1865, 129763 (2021)   Extracellular vesicles (EVs) are nanoparticles which are released by cells from all three domains of life: Archaea, Bacteria and Eukarya. They can mediate cell-cell communication by transferring cargoes such as proteins and nucleic acids between cells. EVs receive great interest in both academia and industry as they have the potential to be natural drug carriers or vaccine candidates. However, limitations to their clinical translation exist as efficient isolation, loading, labelling and surface-engineering methods are lacking. In this article, we investigate a ‘post-insertion’ approach, which is commonly used in the functionalization of liposomes in the pharmaceutical field, on two different EV types: mammalian cell-derived EVs and bacteria-derived EVs. We aimed to find an easy and flexible approach to functionalize EVs, thereby improving the labelling, isolation, and surface-engineering.  

3.3804           Hepatic deletion of Mboat7 (LPIAT1) causes activation of SREBP-1c and fatty liver

Xia, M., Chandrasekaran, P., Rong, S., Fu, X. and Mitsche, M.A.

1. Lipid Res., 62, 100031 (2021)

  Genetic variants that increase the risk of fatty liver disease and cirrhosis have recently been identified in the proximity of membrane-bound O-acyltransferase domain-containing 7 (MBOAT7). To elucidate the link between these variants and fatty liver disease, we characterized Mboat7 liver-specific KO mice (Mboat7 LSKO). Chow-fed Mboat7 LSKO mice developed fatty livers and associated liver injury. Lipidomic analysis of liver using MS revealed a pronounced reduction in 20-carbon PUFA content in phosphatidylinositols (PIs) but not in other phospholipids. The change in fatty acid composition of PIs in these mice was associated with a marked increase in de novo lipogenesis because of activation of SREBP-1c, a transcription factor that coordinates the activation of genes encoding enzymes in the fatty acid biosynthesis pathway. Hepatic removal of both SREBP cleavage-activating protein (Scap) and Mboat7 normalized hepatic triglycerides relative to Scap-only hepatic KO, showing that increased SREBP-1c processing is required for Mboat7-induced steatosis. This study reveals a clear relationship between PI fatty acid composition and regulation of hepatic fat synthesis and delineates the mechanism by which mutations in MBOAT7 cause hepatic steatosis.  

3.3805           Chapter One - Exosomes in cancer

Bark, J.M., Kulasinghe, A., Ammenabar, J.M. and Punyadeera, C. Adv. Clin. Chem., 101, 1-40 (2021)   Exosomes are small extracellular vesicles released by cells under physiological and pathological conditions. There is emerging evidence associating exosomes with tumorigenesis. They carry cargo (DNA, RNA, miRNA and protein) pertaining to the cell of origin and play a key role in intercellular communication, influencing several cellular processes. Moreover, exosomes can be shed and found in almost all body fluids, providing a source of biomarkers for tumor diagnosis and prognosis. In addition, the use of exosomes for cancer therapeutics is another research area that is gaining attention. This book chapter aims to explore the role of exosomes in tumor biogenesis, progression and clinical applications, comprehensively compiling the research for three tumor types, namely head and neck cancer, lung cancer and glioblastoma.  

3.3806           Small extracellular vesicles in cancer

Abhange, K., Makler, A., Wen, Y., Ramnauth, N., Mao, W., Asghar, W. and Wan, Y. Bioactive Materials, 6, 3705-3743 (2021)   Extracellular vesicles (EV) are lipid-bilayer enclosed vesicles in submicron size that are released from cells. A variety of molecules, including proteins, DNA fragments, RNAs, lipids, and metabolites can be selectively encapsulated into EVs and delivered to nearby and distant recipient cells. In tumors, through such intercellular communication, EVs can regulate initiation, growth, metastasis and invasion of tumors. Recent studies have found that EVs exhibit specific expression patterns which mimic the parental cell, providing a fingerprint for early cancer diagnosis and prognosis as well as monitoring responses to treatment. Accordingly, various EV isolation and detection technologies have been developed for research and diagnostic purposes. Moreover, natural and engineered EVs have also been used as drug delivery nanocarriers, cancer vaccines, cell surface modulators, therapeutic agents and therapeutic targets. Overall, EVs are under intense investigation as they hold promise for pathophysiological and translational discoveries. This comprehensive review examines the latest EV research trends over the last five years, encompassing their roles in cancer pathophysiology, diagnostics and therapeutics. This review aims to examine the full spectrum of tumor-EV studies and provide a comprehensive foundation to enhance the field. The topics which are discussed and scrutinized in this review encompass isolation techniques and how these issues need to be overcome for EV-based diagnostics, EVs and their roles in cancer biology, biomarkers for diagnosis and monitoring, EVs as vaccines, therapeutic targets, and EVs as drug delivery systems. We will also examine the challenges involved in EV research and promote a framework for catalyzing scientific discovery and innovation for tumor-EV-focused research.  

3.3807           Emerging technologies and commercial products in exosome-based cancer diagnosis and prognosis

Mohammadi, M., Zargartalebi, H., Salahandish, R., Aburashed, R., Yong, K.W. and Sanati-Nexhad, A.S. Biosensors and Bioelectronics, 183, 113176 (2021)   Academic and industrial groups worldwide have reported technological advances in exosome-based cancer diagnosis and prognosis. However, the potential translation of these emerging technologies for research and clinical settings remains unknown. This work overviews the role of exosomes in cancer diagnosis and prognosis, followed by a survey on emerging exosome technologies, particularly microfluidic advances for the isolation and detection of exosomes in cancer research. The advantages and drawbacks of each of the technologies used for the isolation, detection and engineering of exosomes are evaluated to address their clinical challenges for cancer diagnosis and prognosis. Furthermore, commercial platforms for exosomal detection and analysis are introduced, and their performance and impact on cancer diagnosis and prognosis are assessed. Also, the risks associated with the further development of the next generation of exosome devices are discussed. The outcome of this work could facilitate recognizing deliverable Exo-devices and technologies with unprecedented functionality and predictable manufacturability for the next-generation of cancer diagnosis and prognosis.  

3.3808           Chapter One - Urinary extracellular vesicles: single patient analysis for clinical applications

Stanly, C., Fiume, I., Ursic, B., Kralj-Iglic, V., Trepiccione, F., Capasso, G. and Pocsfalvi, G. Adv. Biomembranes and Lipid Self-Assembly, 33, 1-35 (2021)   Urinary extracellular vesicles (uEVs) are a heterogeneous group of membrane-bound vesicles that originate from the urinary system. They are considered to be an informative resource for the non-invasive study of molecular events in health and disease as they carry a variety of molecular constituents from their cells of origin, such as lipids, proteins and RNAs. For early stages of biomarker discovery schemes, untargeted omics-based approaches, which preferably employs uEVs isolated from pooled urine samples, may suit better. However, targeted approaches that preferably uses uEV isolated from individual urine samples are preferred for validation and clinical translation. The volume of urine needed for downstream analytical purposes and the sampling method (i.e. pooled vs. individual samples) are important practical aspects which need to be taken into consideration along with the challenges associated with the different methods of uEVs isolation. Here we provide an overview of the various isolation methods with special focus on the initial sample volumes as well as the sampling method applied. Our original data on the isolation of uEVs by employing salt precipitation and the sucrose/D₂O double cushion ultracentrifugation method is also presented along with the latest trends over the last three years on their clinical applications. In this work, we demonstrate the performance and the reproducibility of the method using small volumes of individual and pooled samples as well as samples of a small group of patients with type 2 diabetes. Our data shows that the isolation method is compatible with both individual and pooled samples-based quantitative proteomics strategies.  

3.3809           Perspectives and challenges in extracellular vesicles untargeted metabolomics analysis

Dudzik, D., Macioszek, S., Struck-Lewicka, W., Kordalewska, M.,Buszewska.Forajta, M., Waszczuk-Jankowska. M. Wawrzyniak, R., Artymowicz, M., Raczak-Gutknecht, J., Siluk, D. and Markuszewski, M.J. Trends in Anal. Chem., 143, 116382 (2021)   The discovery of extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, has opened a new frontier in the study of signal transduction and understanding cell-to-cell communication. EVs play a key role regulating various biological processes such as tissue regeneration, blood coagulation, immunomodulation or carcinogenesis. Therefore, they gain much attention as potentially therapeutic and non-invasive diagnostic targets. In this aspect, a comprehensive characterization of the EVs molecular content and understanding EVs functions are especially relevant. However, routine isolation of a usually small amount of high purity EVs is challenging, and analysis requires highly sensitive advanced analytical solutions. The utility of EVs in clinical settings is still limited due to the lack of standardization in existing protocols. This review focuses on the implication of the state-of-the-art mass-spectrometry based untargeted metabolomics approach for the molecular profiling of EVs, with the emphasis on the analytical methodologies, highlighting potential challenges and limitations.  

3.3810           Delta- and beta- secretases crosstalk amplifies the amyloidogenic pathway in Alzheimer’s disease

Xia, Y., Wang, Z-H., Zhang, Z., Liu, X., Yu, S.P., Wang, J-Z., Wang, X-C. and Ye, K. Progress in Neurobiol., 204, 102113 (2021)   Asparagine endopeptidase (AEP), a newly identified delta-secretase, simultaneously cleaves both APP and Tau, promoting Alzheimer’s disease (AD) pathologies. However, its pathological role in AD remains incompletely understood. Here we show that delta-secretase cleaves BACE1, a rate-limiting protease in amyloid-β (Aβ) generation, escalating its enzymatic activity and enhancing senile plaques deposit in AD. Delta-secretase binds BACE1 and cuts it at N294 residue in an age-dependent manner and elevates its protease activity. The cleaved N-terminal motif is active even under neutral pH and associates with senile plaques in human AD brains. Subcellular fractionation reveals that delta-secretase and BACE1 reside in the endo-lysosomes. Interestingly, truncated BACE1 enzymatic domain (1-294) augments delta-secretase enzymatic activity and accelerates Aβ production, facilitating AD pathologies and cognitive impairments in APP/PS1 AD mouse model. Uncleavable BACE1 (N294A) inhibits delta-secretase activity and Aβ production and decreases AD pathologies in 5XFAD mice, ameliorating cognitive dysfunctions. Hence, delta- and beta- secretases’ crosstalk aggravates each other’s roles in AD pathogenesis.  

3.3811           SCUBE3 loss-of-function causes a recognizable recessive developmental disorder due to defective bone morphogenetic protein signaling

Lin, Y-C., Niceta, M., Muto, V., Vona, B., Pagnamenta, A.T., Maroofian, R., Beetz, C. et al Am. J. Hum. Genet., 108, 115-133 (2021)   Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3^−/− mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.  

3.3812           An ultracentrifugation – hollow-fiber flow field-flow fractionation orthogonal approach for the purification and mapping of extracellular vesicle subtypes

Marassi, V., Maggio, S., battistelli, M., Stocchi, V., Zattoni, A., Reschiglian, P., Guescini, M. and Roda, B.

1. Chromatography A, 1638, 461861 (2021)

  In the course of their life span, cells release a multitude of different vesicles in the extracellular matrix (EVs), constitutively and/or upon stimulation, carrying signals either inside or on their membrane for intercellular communication. As a natural delivery tool, EVs present many desirable advantages, such as biocompatibility and low toxicity. However, due to the complex biogenesis of EVs and their high heterogeneity in size distribution and composition, the characterization and quantification of EVs and their subpopulations still represents an enticing analytical challenge. Centrifugation methods allow to obtain different subpopulations in an easy way from cell culture conditioned medium and biological fluids including plasma, amniotic fluid and urine, but they still present some drawbacks and limitations. An unsatisfactory isolation can limit their downstream analysis and lead to wrong conclusions regarding biological activities. Isolation and characterization of biologically relevant nanoparticles like EVs is crucial to investigate specific molecular and signaling patterns and requires new combined approaches. Our work was focused on HF5 (miniaturized, hollow-fiber flow field-flow fractionation), and its hyphenation to ultracentrifugation techniques, which are the most assessed techniques for vesicle isolation. We exploited model samples obtained from culture medium of murine myoblasts (C2C12), known to release different subsets of membrane-derived vesicles. Large and small EVs (LEVs and SEVs) were isolated by differential ultracentrifugation (UC). Through an HF5 method employing UV, fluorescence and multi-angle laser scattering as detectors, we characterized these subpopulations in terms of size, abundance and DNA/protein content; moreover, we showed that microvesicles tend to hyper-aggregate and partially release nucleic matter. The quali-quantitative information we obtained from the fractographic profiles was improved with respect to Nano Tracking Analysis (NTA) estimation. The SEV population was then further separated using density gradient centrifugation (DGC), and four fractions were submitted again to HF5-multidetection. This technique is based on a fully orthogonal principle, since F4 does not separate by density, and provided uncorrelated information for each of the fractions processed. The “second dimension” achieved with HF5 showed good promise in sorting particles with both different size and content, and allowed to identify the presence of fibrilloid nucleic matter. This analytical bidimensional approach proved to be effective for the characterization of highly complex biological samples such as mixtures of EVs and could provide purified fractions for further biological characterization.  

3.3813           Isolation of extracellular vesicles with combined enrichment methods

Stam, J., bartel, S., Bischoff, R. and WOlters, J.C.

1. Chromatography B, 1169, 122604 (2021)

  Extracellular vesicles (EVs) are currently of tremendous interest in many research disciplines and EVs have potential for development of EV diagnostics or therapeutics. Most well-known single EV isolation methods have their particular advantages and disadvantages in terms of EV purity and EV yield. Combining EV isolation methods provides additional potential to improve the efficacy of both purity and yield. This review assesses the contribution and efficacy of using combined EV isolation methods by performing a two-step systematic literature analysis from all papers applying EV isolation in the year 2019. This resulted in an overview of the various methods being applied for EV isolations. A second database was generated for all studies within the first database that fairly compared multiple EV isolation methods by determining both EV purity and EV yield after isolation. From these databases it is shown that the most used EV isolation methods are not per definition the best methods based on EV purity or EV yield, indicating that more factors play a role in the choice which EV isolation method to choose than only the efficacy of the method. From the included studies it is shown that ~60% of all the included EV isolations were performed with combined EV isolation methods. The majority of EV isolations were performed with differential ultracentrifugation alone or in combination with differential ultrafiltration. When efficacy of EV isolation methods was determined in terms of EV purity and EV yield, combined EV isolation methods clearly outperformed single EV isolation methods, regardless of the type of starting material used. A recommended starting point would be the use of size-exclusion chromatography since this method, especially when combined with low-speed centrifugation, resulted in the highest EV purity, while still providing a reasonable EV yield.  

3.3814           Extracellular Vesicles in Cancer Detection: Hopes and Hypes

Hu, T., Wolfram, J. and Srivastava, S. Trends in Cancer, 7(2), 122-133 (2021)   Early cancer diagnosis is critical for improving patient survival and mortality rates, but most diagnostics on solid tumors rely on imaging tests with limited sensitivity and specificity to identify potential cases, which are then confirmed by tissue biopsies. However, this process is usually not suitable for cancer screening or evaluation of tumor responses to treatment. Liquid biopsies have the potential to bridge this gap, but few such assays have been approved for cancer applications. Extracellular vesicles hold particular promise for liquid biopsy diagnostics but are currently limited by the lack of robust methods for isolation and analysis. New isolation and analysis techniques, however, show promise to improve the clinical utility of extracellular vesicle-based cancer diagnosis.  

3.3815           Extracellular Vesicle-Mediated Bilateral Communication between Glioblastoma and Astrocytes

Nieland, L., Morsett, L.M., Broekman, M.L.D., Breakfield, X.O. and Abels, E.R. Trends in Neurosciences, 44(3), 215-226 (2021)   Glioblastoma the most aggressive form of brain cancer, comprises a complex mixture of tumor cells and nonmalignant stromal cells, including neurons, astrocytes, microglia, infiltrating monocytes/macrophages, lymphocytes, and other cell types. All nonmalignant cells within and surrounding the tumor are affected by the presence of glioblastoma. Astrocytes use multiple modes of communication to interact with neighboring cells. Extracellular vesicle-directed intercellular communication has been found to be an important component of signaling between astrocytes and glioblastoma in tumor progression. In this review, we focus on recent findings on extracellular vesicle-mediated bilateral crosstalk, between glioblastoma cells and astrocytes, highlighting the protumor and antitumor roles of astrocytes in glioblastoma development.  

3.3816           Chapter 15 - Study of microRNAs carried by exosomes

Spada, s. Methods in Cell Biol., 165, 187-197 (2021)   Exosomes are bi-layered vesicles secreted by the cells in physiological and pathological conditions. They are involved in cell-cell communication facilitating the transfer of functional macromolecules, including DNA, RNA, proteins and lipids. In this chapter, we will focus on specific class of RNA, the microRNAs, that are shuttled from the exosome-producing cells to the recipient cells where they affect biological processes. We will describe the recent methodologies developed to detect and isolate exosomal microRNAs providing a suitable workflow that contributes to quickly expand the field of exosomes-derived microRNAs and their potential use as biomarkers.  

3.3817           Single-vesicle imaging and co-localization analysis for tetraspanin profiling of individual extracellular vesicles

Han, C., Kang, H., Yi, J., kang, M., Lee, H., Kwon, Y., Jung, J., Lee, J. and Park, J.

1. Extracell.Vesicles, 10(3), e12047 (2021)

  Extracellular vesicles (EVs) are secreted nano-sized vesicles that contain cellular proteins, lipids, and nucleic acids. Although EVs are expected to be biologically diverse, current analyses cannot adequately characterize this diversity because most are ensemble methods that inevitably average out information from diverse EVs. Here we describe a single vesicle analysis, which directly visualizes marker expressions of individual EVs using a total internal-reflection microscopy and analyzes their co-localization to investigate EV subpopulations. The single-vesicle imaging and co-localization analysis successfully illustrated the diversity of EVs and revealed distinct patterns of tetraspanin expressions. Application of the analysis demonstrated similarities and dissimilarities between the EV fractions that had been acquired from different conventional EV isolation methods. The analysis method developed in this study will provide a new and reliable tool for investigating characteristics of single EVs, and the findings of the analysis might increase understanding of the characteristics of EVs.  

3.3818           Extracellular vesicles from recombinant cell factories improve the activity and efficacy of enzymes defective in lysosomal storage disorders

Seras-Franzoso, J., Diaz-Riascos, Z.V., Corchero, J.L., Gonzalez, P., Garcia-Aranda, N. et al

1. Extracell. Vesicles, 10(5), e12058 (2021)

  In the present study the use of extracellular vesicles (EVs) as vehicles for therapeutic enzymes in lysosomal storage disorders was explored. EVs were isolated from mammalian cells overexpressing alpha-galactosidase A (GLA) or N-sulfoglucosamine sulfohydrolase (SGSH) enzymes, defective in Fabry and Sanfilippo A diseases, respectively. Direct purification of EVs from cell supernatants was found to be a simple and efficient method to obtain highly active GLA and SGSH proteins, even after EV lyophilization. Likewise, EVs carrying GLA (EV-GLA) were rapidly uptaken and reached the lysosomes in cellular models of Fabry disease, restoring lysosomal functionality much more efficiently than the recombinant enzyme in clinical use. In vivo, EVs were well tolerated and distributed among all main organs, including the brain. DiR-labelled EVs were localized in brain parenchyma 1 h after intra-arterial (internal carotid artery) or intravenous (tail vein) administrations. Moreover, a single intravenous administration of EV-GLA was able to reduce globotriaosylceramide (Gb3) substrate levels in clinically relevant tissues, such kidneys and brain. Overall, our results demonstrate that EVs from cells overexpressing lysosomal enzymes act as natural protein delivery systems, improving the activity and the efficacy of the recombinant proteins and facilitating their access to organs neglected by conventional enzyme replacement therapies.  

3.3819           Human milk extracellular vesicles target nodes in interconnected signalling pathways that enhance oral epithelial barrier function and dampen immune responses

Zonneveld, M.I., van Herwijnen, M.J.C., Fernandez-Gutieirrez, M.M., Giovanazzi, A., de Groot, A.M., Kleinjan, M., van Capel, T.M.M., et al

1. Extracell. Vesicles, 10(5), e12071 (2021)

  Maternal milk is nature's first functional food. It plays a crucial role in the development of the infant's gastrointestinal (GI) tract and the immune system. Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer enclosed vesicles released by cells for intercellular communication and are a component of milk. Recently, we discovered that human milk EVs contain a unique proteome compared to other milk components. Here, we show that physiological concentrations of milk EVs support epithelial barrier function by increasing cell migration via the p38 MAPK pathway. Additionally, milk EVs inhibit agonist-induced activation of endosomal Toll like receptors TLR3 and TLR9. Furthermore, milk EVs directly inhibit activation of CD4+ T cells by temporarily suppressing T cell activation without inducing tolerance. We show that milk EV proteins target key hotspots of signalling networks that can modulate cellular processes in various cell types of the GI tract.  

3.3820           Nanoalgosomes: Introducing extracellular vesicles produced by microalgae

Adamo, G., Fierli, D., Romancino, D.P., Piciotto, S., Barone, M.E. et al

1. Extracell. Vesicles, 10(6), e12081 (2021)

  Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production.  

3.3821           Staphylococcus aureus membrane vesicles contain immunostimulatory DNA, RNA and peptidoglycan that activate innate immune receptors and induce autophagy

Bitto, N.J., Cheng, L., Johnston, E.L., Pathirana, R., Phan, T.K., Poon, I.K., O’Brien-Simpson, N.M., Hill, A.F., Stinea, T.P. and Kaparakis-Liaskos, M.

1. Extracell. Vesicles, 10(6), e12080 (2021)

  Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host.  

3.3822           Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer-based precipitation and size exclusion chromatography

Martinez-Greene, J.A., Hernandez-Ortega, K., Quiroz-baez, R., Resendis-Antonio, O., Pichardo-Casas, I., Sinclair, D.A., Budnik, B., Hidalgo-Miranda, A., Uribe-Querol, E., del Pilar Ramos-Godinez, M. and Martinez-martinez, E.

1. Extracell. Vesicles, 10(6), e12087 (2021)

  The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer-based precipitation and size exclusion chromatography (Pre-SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early- and late-eluting EV fractions, we performed a quantitative proteomic analysis of MDA-MB-468-derived EVs. We identified 286 exclusive proteins in early-eluting fractions and 148 proteins with a differential concentration between early- and late-eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies.  

3.3823           Synthetic bacterial vesicles combined with tumour extracellular vesicles as cancer immunotherapy

Park, K-S., Svennerholm, K., Crescitelli, R., Lässer, C., Gribonika, I. and Lötvall, J.

1. Extracell. Vesicles, 10(9), e12120 (2021)

  Bacterial outer membrane vesicles (OMV) have gained attention as a promising new cancer vaccine platform for efficiently provoking immune responses. However, OMV induce severe toxicity by activating the innate immune system. In this study, we applied a simple isolation approach to produce artificial OMV that we have named Synthetic Bacterial Vesicles (SyBV) that do not induce a severe toxic response. We also explored the potential of SyBV as an immunotherapy combined with tumour extracellular vesicles to induce anti-tumour immunity. Bacterial SyBV were produced with high yield by a protocol including lysozyme and high pH treatment, resulting in pure vesicles with very few cytosolic components and no RNA or DNA. These SyBV did not cause systemic pro-inflammatory cytokine responses in mice compared to naturally released OMV. However, SyBV and OMV were similarly effective in activation of mouse bone marrow-derived dendritic cells. Co-immunization with SyBV and melanoma extracellular vesicles elicited tumour regression in melanoma-bearing mice through Th-1 type T cell immunity and balanced antibody production. Also, the immunotherapeutic effect of SyBV was synergistically enhanced by anti-PD-1 inhibitor. Moreover, SyBV displayed significantly greater adjuvant activity than other classical adjuvants. Taken together, these results demonstrate a safe and efficient strategy for eliciting specific anti-tumour responses using immunotherapeutic bacterial SyBV.  

3.3824           Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin

Veerman, R.E., Teewen, L., Czarnewski, P., Akpinar, G.G., Sandberg, A., Cao, X., Pernemalm, M., Orre, L.M., Gabrielsson, S. and Eldh, M.

1. Extracell. Vesicles, 10(9), e12128 (2021)

  Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 μl or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget.

3.3825           Mitochondrial translation deficiency impairs NAD+-mediated lysosomal acidification

Yagi, M., Toshima, T., Amamoto, R., Do, Y., Hirai, H., Setoyama, D., Kang, D.and Uchiumi, T. EMBO J., 40, e105268 (2021)   Mitochondrial translation dysfunction is associated with neurodegenerative and cardiovascular diseases. Cells eliminate defective mitochondria by the lysosomal machinery via autophagy. The relationship between mitochondrial translation and lysosomal function is unknown. In this study, mitochondrial translation-deficient hearts from p32-knockout mice were found to exhibit enlarged lysosomes containing lipofuscin, suggesting impaired lysosome and autolysosome function. These mice also displayed autophagic abnormalities, such as p62 accumulation and LC3 localization around broken mitochondria. The expression of genes encoding for nicotinamide adenine dinucleotide (NAD⁺) biosynthetic enzymes—Nmnat3 and Nampt—and NAD⁺ levels were decreased, suggesting that NAD⁺ is essential for maintaining lysosomal acidification. Conversely, nicotinamide mononucleotide (NMN) administration or Nmnat3 overexpression rescued lysosomal acidification. Nmnat3 gene expression is suppressed by HIF1α, a transcription factor that is stabilized by mitochondrial translation dysfunction, suggesting that HIF1α-Nmnat3-mediated NAD⁺ production is important for lysosomal function. The glycolytic enzymes GAPDH and PGK1 were found associated with lysosomal vesicles, and NAD⁺ was required for ATP production around lysosomal vesicles. Thus, we conclude that NAD⁺ content affected by mitochondrial dysfunction is essential for lysosomal maintenance.  

3.3826           Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV-1 infection

Martin-jaular, L., Nevo, N., Schessner, J.P., Tkach, M., Jouve, M., DIngli, F., Loew, D., Witwer, K.W:, Ostrowski, M., Borner, G.H.H. and Thery, C. EMBO J., 40, e105492 (2021)   Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV-1-infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re-routed to non-viral EVs in a Nef-dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release.  

3.3827           Clinical applications for exosomes: Are we there yet?

Perocheau, D., Touramanidou, L., Gurung, S., Gissen, P. and baruteau, J. Br. J. Pharmacol., 178, 2375-2392 (2021)   Exosomes are a subset of extracellular vesicles essential for cell–cell communication in health and disease with the ability to transport nucleic acids, functional proteins and other metabolites. Their clinical use as diagnostic biomarkers and therapeutic carriers has become a major field of research over recent years, generating rapidly expanding scientific interest and financial investment. Their reduced immunogenicity compared to liposomes or viral vectors and their ability to cross major physiological barriers like the blood–brain barrier make them an appealing and innovative option as biomarkers and therapeutic agents. Here, we review the latest clinical developments of exosome biotechnology for diagnostic and therapeutic purposes, including the most recent COVID-19-related exosome-based clinical trials. We present current exosome engineering strategies for optimal clinical safety and efficacy, and assess the technology developed for good manufacturing practice compliant scaling up and storage approaches along with their limitations in pharmaceutical industry.  

3.3828           Auxilin-like protein MoSwa2 promotes effector secretion and virulence as a clathrin uncoating factor in the rice blast fungus Magnaporthe oryzae

Liu, M., Hu, J., Zhang, A., Dai, Y., Chen, W., He, Y., Zhang, H., Zheng, X. and Zhang, Z. New Phytologist, 230, 720-736 (2021)   Plant pathogens exploit the extracellular matrix (ECM) to inhibit host immunity during their interactions with the host. The formation of ECM involves a series of continuous steps of vesicular transport events. To understand how such vesicle trafficking impacts ECM and virulence in the rice blast fungus Magnaporthe oryzae, we characterised MoSwa2, a previously identified actin-regulating kinase MoArk1 interacting protein, as an orthologue of the auxilin-like clathrin uncoating factor Swa2 of the budding yeast Saccharomyces cerevisiae. We found that MoSwa2 functions as an uncoating factor of the coat protein complex II (COPII) via an interaction with the COPII subunit MoSec24-2. Loss of MoSwa2 led to a deficiency in the secretion of extracellular proteins, resulting in both restricted growth of invasive hyphae and reduced inhibition of host immunity. Additionally, extracellular fluid (ECF) proteome analysis revealed that MoSwa2-regulated extracellular proteins include many redox proteins such as the berberine bridge enzyme-like (BBE-like) protein MoSef1. We further found that MoSef1 functions as an apoplastic virulent factor that inhibits the host immune response. Our studies revealed a novel function of a COPII uncoating factor in vesicular transport that is critical in the suppression of host immunity and pathogenicity of M. oryzae.  

3.3829           Extracellular Vesicles from Child Gut Microbiota Enter into Bone to Preserve Bone Mass and Strength

Liu, J-H., Chen, C-Y., Liu, Z-Z., Luo, Z-W., Rao, S-S. et al Advanced Science, 8, 2004831 (2021)   Recently, the gut microbiota (GM) has been shown to be a regulator of bone homeostasis and the mechanisms by which GM modulates bone mass are still being investigated. Here, it is found that colonization with GM from children (CGM) but not from the elderly (EGM) prevents decreases in bone mass and bone strength in conventionally raised, ovariectomy (OVX)-induced osteoporotic mice. 16S rRNA gene sequencing reveals that CGM reverses the OVX-induced reduction of Akkermansia muciniphila (Akk). Direct replenishment of Akk is sufficient to correct the OVX-induced imbalanced bone metabolism and protect against osteoporosis. Mechanistic studies show that the secretion of extracellular vesicles (EVs) is required for the CGM- and Akk-induced bone protective effects and these nanovesicles can enter and accumulate into bone tissues to attenuate the OVX-induced osteoporotic phenotypes by augmenting osteogenic activity and inhibiting osteoclast formation. The study identifies that gut bacterium Akk mediates the CGM-induced anti-osteoporotic effects and presents a novel mechanism underlying the exchange of signals between GM and host bone.  

3.3830           Conserved arginine residues in synaptotagmin 1 regulate fusion pore expansion through membrane contact

Nyenhuis, S.B., karandikar, N., Kiessling, V., kreutzberger, A.J.B., Thapa, A., Liang, B., Tamm, L.K. and Cafiso, D.S. Nature Comm., 12:761 (2021)   Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca²⁺ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca²⁺ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the fusion probability and kinetics but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.  

3.3831           20S proteasomes secreted by the malaria parasite promote its growth

Dekel, E., Yaffe, D., Rosenhek-Goldian, I., Ben-Nissan, G., Ofir-Birin, Y. et al Nature Comm., 12:1172 (2021)   Mature red blood cells (RBCs) lack internal organelles and canonical defense mechanisms, making them both a fascinating host cell, in general, and an intriguing choice for the deadly malaria parasite Plasmodium falciparum (Pf), in particular. Pf, while growing inside its natural host, the human RBC, secretes multipurpose extracellular vesicles (EVs), yet their influence on this essential host cell remains unknown. Here we demonstrate that Pf parasites, cultured in fresh human donor blood, secrete within such EVs assembled and functional 20S proteasome complexes (EV-20S). The EV-20S proteasomes modulate the mechanical properties of naïve human RBCs by remodeling their cytoskeletal network. Furthermore, we identify four degradation targets of the secreted 20S proteasome, the phosphorylated cytoskeletal proteins β-adducin, ankyrin-1, dematin and Epb4.1. Overall, our findings reveal a previously unknown 20S proteasome secretion mechanism employed by the human malaria parasite, which primes RBCs for parasite invasion by altering membrane stiffness, to facilitate malaria parasite growth.  

3.3832           Cerebellar Kv3.3 potassium channels activate TANK-binding kinase 1 to regulate trafficking of the cell survival protein Hax-1

Zhang, Y., Varela, L., Szigeti-Buck, K., Williams, A., Stoiljkovis, M., Sesta-Pesa, M., Henao-Mejia, J., D’Acunzo, P., Levy, E., Flavell, R.A., Horvath, T.L. and Kaczmarek, L.K. Nature Comm., 12:1731 (2021)   Mutations in KCNC3, which encodes the Kv3.3 potassium channel, cause degeneration of the cerebellum, but exactly how the activity of an ion channel is linked to the survival of cerebellar neurons is not understood. Here, we report that Kv3.3 channels bind and stimulate Tank Binding Kinase 1 (TBK1), an enzyme that controls trafficking of membrane proteins into multivesicular bodies, and that this stimulation is greatly increased by a disease-causing Kv3.3 mutation. TBK1 activity is required for the binding of Kv3.3 to its auxiliary subunit Hax-1, which prevents channel inactivation with depolarization. Hax-1 is also an anti-apoptotic protein required for survival of cerebellar neurons. Overactivation of TBK1 by the mutant channel leads to the loss of Hax-1 by its accumulation in multivesicular bodies and lysosomes, and also stimulates exosome release from neurons. This process is coupled to activation of caspases and increased cell death. Our studies indicate that Kv3.3 channels are directly coupled to TBK1-dependent biochemical pathways that determine the trafficking of cellular constituents and neuronal survival.  

3.3833           Reconstitution of contractile actomyosin rings in vesicles

Litschel, T., Kelley, C.F., Holz, D., Koudehi, M.A., Vogel, S.K., Burbaum, L., Mizuno, N., Vavylonis, D. and Schwille, P. Nature Comm., 12:2254 (2021)   One of the grand challenges of bottom-up synthetic biology is the development of minimal machineries for cell division. The mechanical transformation of large-scale compartments, such as Giant Unilamellar Vesicles (GUVs), requires the geometry-specific coordination of active elements, several orders of magnitude larger than the molecular scale. Of all cytoskeletal structures, large-scale actomyosin rings appear to be the most promising cellular elements to accomplish this task. Here, we have adopted advanced encapsulation methods to study bundled actin filaments in GUVs and compare our results with theoretical modeling. By changing few key parameters, actin polymerization can be differentiated to resemble various types of networks in living cells. Importantly, we find membrane binding to be crucial for the robust condensation into a single actin ring in spherical vesicles, as predicted by theoretical considerations. Upon force generation by ATP-driven myosin motors, these ring-like actin structures contract and locally constrict the vesicle, forming furrow-like deformations. On the other hand, cortex-like actin networks are shown to induce and stabilize deformations from spherical shapes.  

3.3834           Mastocytosis-derived extracellular vesicles deliver miR-23a and miR-30a into pre-osteoblasts and prevent osteoblastogenesis and bone formation

Kim, D-K., Bandara, G., Cho, Y-E., Komarow, H.D., Donahue, D.R., Karim, B., Baek, M-C., Kim, H.M., Metcalfe, D.D. and Olivera, A. Nature Comm, 12:2527 (2021)   Osteoporosis and other manifestations of bone disease are frequent in patients with systemic mastocytosis (SM) in association with the presence of mast cell infiltrates in bone marrow, although the mechanisms behind bone disease remain poorly understood. We find that extracellular vesicles (EVs) released by neoplastic mast cells and present in the serum of patients with SM (SM-EVs) block osteoblast differentiation and mineralization in culture, and when injected into mice diminish the expression of osteoblast markers, and trabecular bone volume and microarchitecture. We demonstrate that miRNA-30a and miRNA-23a, increased in SM-EVs and neoplastic mast cell-derived EVs, attenuate osteoblast maturation by suppressing expression of RUNX2 and SMAD1/5, essential drivers of osteogenesis. Thus, SM-EVs carry and deliver miRNAs that epigenetically interfere with bone formation and can contribute to bone mass reduction in SM. These findings also suggest possibilities for novel approaches to the management of bone disease in mast cell proliferative disorders.  

3.3835           Single nucleus multi-omics regulatory landscape of the murine pituitary

Ruf-Zamojski, F., Zhang, Z., Zamojski, M., Smith, G.R., Mendelev, N. et al Nature Comm., 12:2677 (2021)   To provide a multi-omics resource and investigate transcriptional regulatory mechanisms, we profile the transcriptome, chromatin accessibility, and methylation status of over 70,000 single nuclei (sn) from adult mouse pituitaries. Paired snRNAseq and snATACseq datasets from individual animals highlight a continuum between developmental epigenetically-encoded cell types and transcriptionally-determined transient cell states. Co-accessibility analysis-based identification of a putative Fshb cis-regulatory domain that overlaps the fertility-linked rs11031006 human polymorphism, followed by experimental validation illustrate the use of this resource for hypothesis generation. We also identify transcriptional and chromatin accessibility programs distinguishing each major cell type. Regulons, which are co-regulated gene sets sharing binding sites for a common transcription factor driver, recapitulate cell type clustering. We identify both cell type-specific and sex-specific regulons that are highly correlated with promoter accessibility, but not with methylation state, supporting the centrality of chromatin accessibility in shaping cell-defining transcriptional programs. The sn multi-omics atlas is accessible at snpituitaryatlas.princeton.edu.  

3.3836           NMDAR-dependent long-term depression is associated with increased short term plasticity through autophagy mediated loss of PSD-95

Compans, B., Camus, C., Kallergi, E., Sposini, S., Martineau, M. et al Nature Comm., 12:2849 (2021)   Long-term depression (LTD) of synaptic strength can take multiple forms and contribute to circuit remodeling, memory encoding or erasure. The generic term LTD encompasses various induction pathways, including activation of NMDA, mGlu or P2X receptors. However, the associated specific molecular mechanisms and effects on synaptic physiology are still unclear. We here compare how NMDAR- or P2XR-dependent LTD affect synaptic nanoscale organization and function in rodents. While both LTDs are associated with a loss and reorganization of synaptic AMPARs, only NMDAR-dependent LTD induction triggers a profound reorganization of PSD-95. This modification, which requires the autophagy machinery to remove the T19-phosphorylated form of PSD-95 from synapses, leads to an increase in AMPAR surface mobility. We demonstrate that these post-synaptic changes that occur specifically during NMDAR-dependent LTD result in an increased short-term plasticity improving neuronal responsiveness of depressed synapses. Our results establish that P2XR- and NMDAR-mediated LTD are associated to functionally distinct forms of LTD.  

3.3837           Tissue context determines the penetrance of regulatory DNA variation

Haalow, J.M., Byron, R., Hogan, M.S., Ordonez, R., Groudine, M., Bender, M.A., Stramatoyannopoulos, J.A. and Maurano, M.T. Nature Comm., 12:2850 (2021)   Functional assessment of disease-associated sequence variation at non-coding regulatory elements is complicated by their high degree of context sensitivity to both the local chromatin and nuclear environments. Allelic profiling of DNA accessibility across individuals has shown that only a select minority of sequence variation affects transcription factor (TF) occupancy, yet low sequence diversity in human populations means that no experimental assessment is available for the majority of disease-associated variants. Here we describe high-resolution in vivo maps of allelic DNA accessibility in liver, kidney, lung and B cells from 5 increasingly diverged strains of F1 hybrid mice. The high density of heterozygous sites in these hybrids enables precise quantification of effect size and cell-type specificity for hundreds of thousands of variants throughout the mouse genome. We show that chromatin-altering variants delineate characteristic sensitivity profiles for hundreds of TF motifs. We develop a compendium of TF-specific sensitivity profiles accounting for genomic context effects. Finally, we link maps of allelic accessibility to allelic transcript levels in the same samples. This work provides a foundation for quantitative prediction of cell-type specific effects of non-coding variation on TF activity, which will facilitate both fine-mapping and systems-level analyses of common disease-associated variation in human genomes.  

3.3838           Neuronal genes deregulated in Cornelia de Lange Syndrome respond to removal and re-expression of cohesion

Weiss, F.D., Calderon, L., Wang, Y-F., Georgieva, R., Guo, Y., Cvetesic, N., Kaur, M., Dharmalingam, G., Krantz, I.D., Lenhard, B., Fisher, A.G. and Merkenschlager, M. Nature Comm., 12:2919 (2021)   Cornelia de Lange Syndrome (CdLS) is a human developmental disorder caused by mutations that compromise the function of cohesin, a major regulator of 3D genome organization. Cognitive impairment is a universal and as yet unexplained feature of CdLS. We characterize the transcriptional profile of cortical neurons from CdLS patients and find deregulation of hundreds of genes enriched for neuronal functions related to synaptic transmission, signalling processes, learning and behaviour. Inducible proteolytic cleavage of cohesin disrupts 3D genome organization and transcriptional control in post-mitotic cortical mouse neurons, demonstrating that cohesin is continuously required for neuronal gene expression. The genes affected by acute depletion of cohesin belong to similar gene ontology classes and show significant numerical overlap with genes deregulated in CdLS. Interestingly, reconstitution of cohesin function largely rescues altered gene expression, including the expression of genes deregulated in CdLS.  

3.3839           Analysis of diverse eukaryotes suggests the existence of an ancestral mitochondrial apparatus derived from the bacterial type II secretion system

Horvathova, L., Zarsky, V., Panek, T., Derelle, R., Pyrih, J., Motyckova, A. et al Nature Comm., 12:2947 (2021)   The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.  

3.3840           FtsZ induces membrane deformations via torsional stress upon GTP hydrolysis

Ramirez-Diaz, D.A., Merino-Salomon, A., Meyer, F., Heymann, M., Rivas, g., Bramkamp, M. and Schwille, P. Nature Comm., 12:3310 (2021)   FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far, however, not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we design an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ filaments actively transform these tubes into spring-like structures, where GTPase activity promotes spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, the directional forces that FtsZ-YFP-mts rings exert upon GTP hydrolysis can be estimated to be in the pN range. They are sufficient to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls. We hypothesize that these forces result from torsional stress in a GTPase activity dependent manner.  

3.3841           Vpr counteracts the restriction of LAPTM5 to promote HIV-1 infection in macrophages

Zhao, L., Wang, S., Xu, M., He, Y., Zhang, X., Xiong, Y., Sun, H., Ding, H., Geng, W., Shang, H. and Liang, G. Nature Comm., 12:3691 (2021)   The HIV-1 accessory proteins Vif, Vpu, and Nef can promote infection by overcoming the inhibitory effects of the host cell restriction factors APOBEC3G, Tetherin, and SERINC5, respectively. However, how the HIV-1 accessory protein Vpr enhances infection in macrophages but not in CD4⁺ T cells remains elusive. Here, we report that Vpr counteracts lysosomal-associated transmembrane protein 5 (LAPTM5), a potent inhibitor of HIV-1 particle infectivity, to enhance HIV-1 infection in macrophages. LAPTM5 transports HIV-1 envelope glycoproteins to lysosomes for degradation, thereby inhibiting virion infectivity. Vpr counteracts the restrictive effects of LAPTM5 by triggering its degradation via DCAF1. In the absence of Vpr, the silencing of LAPTM5 precisely phenocopied the effect of Vpr on HIV-1 infection. In contrast, Vpr did not enhance HIV-1 infection in the absence of LAPTM5. Moreover, LAPTM5 was highly expressed in macrophages but not in CD4⁺ T lymphocytes. Re-expressing LAPTM5 reconstituted the Vpr-dependent promotion of HIV-1 infection in primary CD4⁺ T cells, as observed in macrophages. Herein, we demonstrate the molecular mechanism used by Vpr to overcome LAPTM5 restriction in macrophages, providing a potential strategy for anti-HIV/AIDS therapeutics.  

3.3842           Oral administration of bovine milk-derived extracellular vesicles induces senescence in the primary tumor but accelerates cancer metastasis

Samuel, M., Fonseka, P., Sanwlani, R., Gangoda, L., Chee, S.H. et al Nature Comm., 12:3950 (2021)   The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.  

3.3843           Single-nucleus RNA-seq2 reveals functional crosstalk between liver zonation and ploidy

Richter, M.L., Deligiannis, I.K., Yin, K., Danese, A., Lleshi, E., Coupland, P., Vallejos, C.A., Matchett, K:P., Henderson, N.C., Colome-Tatche, M. and Martinez-Jimenez, C.P. Nature Comm., 12:4264 (2021)   Single-cell RNA-seq reveals the role of pathogenic cell populations in development and progression of chronic diseases. In order to expand our knowledge on cellular heterogeneity, we have developed a single-nucleus RNA-seq2 method tailored for the comprehensive analysis of the nuclear transcriptome from frozen tissues, allowing the dissection of all cell types present in the liver, regardless of cell size or cellular fragility. We use this approach to characterize the transcriptional profile of individual hepatocytes with different levels of ploidy, and have discovered that ploidy states are associated with different metabolic potential, and gene expression in tetraploid mononucleated hepatocytes is conditioned by their position within the hepatic lobule. Our work reveals a remarkable crosstalk between gene dosage and spatial distribution of hepatocytes.  

3.3844           Species-specific gamete recognition initiates fusion-driving trimer formation by conserved fusogen HAP2

Zhang, J., Pinello, J.F., Fernandez, I., Baquero, E., Fedry, J., Rey, F.A. and Snell, W:J. Nature Comm., 12:4380 (2021)   Recognition and fusion between gametes during fertilization is an ancient process. Protein HAP2, recognized as the primordial eukaryotic gamete fusogen, is a structural homolog of viral class II fusion proteins. The mechanisms that regulate HAP2 function, and whether virus-fusion-like conformational changes are involved, however, have not been investigated. We report here that fusion between plus and minus gametes of the green alga Chlamydomonas indeed requires an obligate conformational rearrangement of HAP2 on minus gametes from a labile, prefusion form into the stable homotrimers observed in structural studies. Activation of HAP2 to undergo its fusogenic conformational change occurs only upon species-specific adhesion between the two gamete membranes. Following a molecular mechanism akin to fusion of enveloped viruses, the membrane insertion capacity of the fusion loop is required to couple formation of trimers to gamete fusion. Thus, species-specific membrane attachment is the gateway to fusion-driving HAP2 rearrangement into stable trimers.  

3.3845           Stearic acid blunts growth-factor signaling via oleoylation of GNAI proteins

Nuskova, H., Serebryakova, M.V., Ferrer-Caelles, A., Sachsenheimer, T.Luchtenborg, C., Miller, A.K., Brügger, B., Kordyukova, L.V. and Teleman, A.A. Nature Comm., 12:4590 (2021)   Covalent attachment of C16:0 to proteins (palmitoylation) regulates protein function. Proteins are also S-acylated by other fatty acids including C18:0. Whether protein acylation with different fatty acids has different functional outcomes is not well studied. We show here that C18:0 (stearate) and C18:1 (oleate) compete with C16:0 to S-acylate Cys3 of GNAI proteins. C18:0 becomes desaturated so that C18:0 and C18:1 both cause S-oleoylation of GNAI. Exposure of cells to C16:0 or C18:0 shifts GNAI acylation towards palmitoylation or oleoylation, respectively. Oleoylation causes GNAI proteins to shift out of cell membrane detergent-resistant fractions where they potentiate EGFR signaling. Consequently, exposure of cells to C18:0 reduces recruitment of Gab1 to EGFR and reduces AKT activation. This provides a molecular mechanism for the anti-tumor effects of C18:0, uncovers a mechanistic link how metabolites affect cell signaling, and provides evidence that the identity of the fatty acid acylating a protein can have functional consequences.  

3.3846           Interferon lambda 4 impairs hepatitis C viral antigen presentation and attenuates T cell responses

Chen, Q., Coto-Llerena, M., Suslov, A., Teixeira, R.D., Fofana, I., NUciforo, S., Hfomann, M., Thimme, R., Hensel, N., Lohmann, V., Ng, C.K.Y., Rosenberger, G., Wieland, S. and Helm, M.H. Nature Comm., 12:4882 (2021)   Genetic variants of the interferon lambda (IFNL) gene locus are strongly associated with spontaneous and IFN treatment-induced clearance of hepatitis C virus (HCV) infections. Individuals with the ancestral IFNL4-dG allele are not able to clear HCV in the acute phase and have more than a 90% probability to develop chronic hepatitis C (CHC). Paradoxically, the IFNL4-dG allele encodes a fully functional IFNλ4 protein with antiviral activity against HCV. Here we describe an effect of IFNλ4 on HCV antigen presentation. Only minor amounts of IFNλ4 are secreted, because the protein is largely retained in the endoplasmic reticulum (ER) where it induces ER stress. Stressed cells are significantly weaker activators of HCV specific CD8⁺ T cells than unstressed cells. This is not due to reduced MHC I surface presentation or extracellular IFNλ4 effects, since T cell responses are restored by exogenous loading of MHC with HCV antigens. Rather, IFNλ4 induced ER stress impairs HCV antigen processing and/or loading onto the MHC I complex. Our results provide a potential explanation for the IFNλ4–HCV paradox.  

3.3847           Chromatin accessibility associates with protein-RNA correlation in human cancer

Sanghi, A., Gruber, J.I., Metwally, A., Jiang, L., Reynolds, W., Sunwood, J., Orloff, L., Chang, H.Y., Kasowski, M. and Snyder, M.P.

Nature Comm., 12:5732 (2021)

  Although alterations in chromatin structure are known to exist in tumors, how these alterations relate to molecular phenotypes in cancer remains to be demonstrated. Multi-omics profiling of human tumors can provide insight into how alterations in chromatin structure are propagated through the pathway of gene expression to result in malignant protein expression. We applied multi-omics profiling of chromatin accessibility, RNA abundance, and protein abundance to 36 human thyroid cancer primary tumors, metastases, and patient-match normal tissue. Through quantification of chromatin accessibility associated with active transcription units and global protein expression, we identify a local chromatin structure that is highly correlated with coordinated RNA and protein expression. In particular, we identify enhancers located within gene-bodies as predictive of correlated RNA and protein expression, that is independent of overall transcriptional activity. To demonstrate the generalizability of these findings we also identify similar results in an independent cohort of human breast cancers. Taken together, these analyses suggest that local enhancers, rather than distal enhancers, are likely most predictive of cancer gene expression phenotypes. This allows for identification of potential targets for cancer therapeutic approaches and reinforces the utility of multi-omics profiling as a methodology to understand human disease.  

3.3848           Spatiotemporal proteomic profiling of the pro-inflammatory response to lipopolysaccharide in the THP-1 human leukaemia cell line

Mulvey, C.M., Breckels, L.M., Crook, O.M., Sanders, D.J., Ribeiro, A.L.R., Geladaki, A., Christoforou, A., Britovsek, N.K., Hurrell, T., Deery, M.J., Gatto, L., Smith, A.M and Lilley, K.S: Nature Comm., 12:5773 (2021)   Protein localisation and translocation between intracellular compartments underlie almost all physiological processes. The hyperLOPIT proteomics platform combines mass spectrometry with state-of-the-art machine learning to map the subcellular location of thousands of proteins simultaneously. We combine global proteome analysis with hyperLOPIT in a fully Bayesian framework to elucidate spatiotemporal proteomic changes during a lipopolysaccharide (LPS)-induced inflammatory response. We report a highly dynamic proteome in terms of both protein abundance and subcellular localisation, with alterations in the interferon response, endo-lysosomal system, plasma membrane reorganisation and cell migration. Proteins not previously associated with an LPS response were found to relocalise upon stimulation, the functional consequences of which are still unclear. By quantifying proteome-wide uncertainty through Bayesian modelling, a necessary role for protein relocalisation and the importance of taking a holistic overview of the LPS-driven immune response has been revealed. The data are showcased as an interactive application freely available for the scientific community.  

3.3849           A Vaspin–HSPA1L complex protects proximal tubular cells from organelle stress in diabetic kidney disease

Nakatsuka, A., Yamaguchi, S., Eguchi, J., Kakuta, S., Iwakura, Y., Sugiyama, H. and Wada, J. Communications Biol., 4:373 (2021)   Proximal tubular cells (PTCs) are crucial for maintaining renal homeostasis, and tubular injuries contribute to progression of diabetic kidney disease (DKD). However, the roles of visceral adipose tissue-derived serine protease inhibitor (vaspin) in the development of DKD is not known. We found vaspin maintains PTCs through ameliorating ER stress, autophagy impairment, and lysosome dysfunction in DKD. Vaspin−/− obese mice showed enlarged and leaky lysosomes in PTCs associated with increased apoptosis, and these abnormalities were also observed in the patients with DKD. During internalization into PTCs, vaspin formed a complex with heat shock protein family A (Hsp70) member 1 like (HSPA1L) as well as 78 kDa glucose-regulated protein (GRP78). Both vaspin-partners bind to clathrin heavy chain and involve in the endocytosis. Notably, albumin-overload enhanced extracellular release of HSPA1L and overexpression of HSPA1L dissolved organelle stresses, especially autophagy impairment. Thus, vapsin/HSPA1L-mediated pathways play critical roles in maintaining organellar function of PTCs in DKD.  

3.3850           Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles

Rai, A., Greening, D.W., Xu, R., Chen, M., Suwakulsiri, W and Simpson, R.J. Communications Biol., 4:400 (2021)   During the final stages of cell division, newly-formed daughter cells remain connected by a thin intercellular bridge containing the midbody (MB), a microtubule-rich organelle responsible for cytokinetic abscission. Following cell division the MB is asymmetrically inherited by one daughter cell where it persists as a midbody remnant (MB-R). Accumulating evidence shows MB-Rs are secreted (sMB-Rs) into the extracellular medium and engulfed by neighbouring non-sister cells. While much is known about intracellular MB-Rs, sMB-Rs are poorly understood. Here, we report the large-scale purification and biochemical characterisation of sMB-Rs released from colon cancer cells, including profiling of their proteome using mass spectrometry. We show sMB-Rs are an abundant class of membrane-encapsulated extracellular vesicle (200-600 nm) enriched in core cytokinetic proteins and molecularly distinct from exosomes and microparticles. Functional dissection of sMB-Rs demonstrated that they are engulfed by, and accumulate in, quiescent fibroblasts where they promote cellular transformation and an invasive phenotype.  

3.3851           ExoSTING, an extracellular vesicle loaded with STING agonists, promotes tumor immune surveillance

Jang, S.C., Economides, K.D., Moniz, R.J., Sia, C.L., Lewis, N. et al Communications Biol., 4:497 (2021)   Cyclic dinucleotide (CDN) agonists of the STimulator of InterferoN Genes (STING) pathway have shown immune activation and tumor clearance in pre-clinical models. However, CDNs administered intratumorally also promote STING activation leading to direct cytotoxicity of many cell types in the tumor microenvironment (TME), systemic inflammation due to rapid tumor extravasation of the CDN, and immune ablation in the TME. These result in a failure to establish immunological memory. ExoSTING, an engineered extracellular vesicle (EV) exogenously loaded with CDN, enhances the potency of CDN and preferentially activates antigen presenting cells in the TME. Following intratumoral injection, exoSTING was retained within the tumor, enhanced local Th1 responses and recruitment of CD8⁺ T cells, and generated systemic anti-tumor immunity to the tumor. ExoSTING at therapeutically active doses did not induce systemic inflammatory cytokines, resulting in an enhanced therapeutic window. ExoSTING is a novel, differentiated therapeutic candidate that leverages the natural biology of EVs to enhance the activity of CDNs.  

3.3852           Functional differences between TSHR alleles associate with variation in spawning season in Atlantic herring

Chen, J., Bi, H., Petterson, M.E., Sato, D.X., Fuentes-Pardo, A.P., Mo, C., Younis, S., Wallerman, O., Jern, P., Moles, G., Gomez, A., Kleinau, G., Scheerer, P. and Andersson, L.A. Communications Biol., 4:795 (2021)   The underlying molecular mechanisms that determine long day versus short day breeders remain unknown in any organism. Atlantic herring provides a unique opportunity to examine the molecular mechanisms involved in reproduction timing, because both spring and autumn spawners exist within the same species. Although our previous whole genome comparisons revealed a strong association of TSHR alleles with spawning seasons, the functional consequences of these variants remain unknown. Here we examined the functional significance of six candidate TSHR mutations strongly associated with herring reproductive seasonality. We show that the L471M missense mutation in the spring-allele causes enhanced cAMP signaling. The best candidate non-coding mutation is a 5.2 kb retrotransposon insertion upstream of the TSHR transcription start site, near an open chromatin region, which is likely to affect TSHR expression. The insertion occurred prior to the split between Pacific and Atlantic herring and was lost in the autumn-allele. Our study shows that strongly associated coding and non-coding variants at the TSHR locus may both contribute to the regulation of seasonal reproduction in herring.  

3.3853           Actin crosslinker competition and sorting drive emergent GUV size-dependent actin network architecture

Bashirzadeh, Y., Redford, S.A., Lorpaiboon, C., Groaz, A., Moghimianavval, H., Litschel, T., Schwille, P., Hocky, G.M., Dinner, A.R and Liu, A.P. Communications Biol., 4:1136 (2021)   The proteins that make up the actin cytoskeleton can self-assemble into a variety of structures. In vitro experiments and coarse-grained simulations have shown that the actin crosslinking proteins α-actinin and fascin segregate into distinct domains in single actin bundles with a molecular size-dependent competition-based mechanism. Here, by encapsulating actin, α-actinin, and fascin in giant unilamellar vesicles (GUVs), we show that physical confinement can cause these proteins to form much more complex structures, including rings and asters at GUV peripheries and centers; the prevalence of different structures depends on GUV size. Strikingly, we found that α-actinin and fascin self-sort into separate domains in the aster structures with actin bundles whose apparent stiffness depends on the ratio of the relative concentrations of α-actinin and fascin. The observed boundary-imposed effect on protein sorting may be a general mechanism for creating emergent structures in biopolymer networks with multiple crosslinkers.  

3.3854           Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations

Allelein, S., Medina-Perez, P., Lopes, A.L.H., Rau, S., Hause, G., Kölsch, A. and Kuhlmeier, D. Scientific Reports, 11:11585 (2021)   Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples.  

3.3855           Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles

Samoylenko, A., Kögler, M., Zhyvolozhnyi, A., Makieieva, O., Bart, G., Andoh, S.S., Roussey, M., Vainio, S.J. and Hiltunen, J. Scientific Reports, 11:19954 (2021)   Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols.  

3.3856           Single-cell epigenomics reveals mechanisms of human cortical development

Ziffra, R.S., Kim, C.N., Ross, J.M., WIlfert, A., Turner, T.N. et al Nature, 598, 205-213 (2021)   During mammalian development, differences in chromatin state coincide with cellular differentiation and reflect changes in the gene regulatory landscape1. In the developing brain, cell fate specification and topographic identity are important for defining cell identity2 and confer selective vulnerabilities to neurodevelopmental disorders3. Here, to identify cell-type-specific chromatin accessibility patterns in the developing human brain, we used a single-cell assay for transposase accessibility by sequencing (scATAC-seq) in primary tissue samples from the human forebrain. We applied unbiased analyses to identify genomic loci that undergo extensive cell-type- and brain-region-specific changes in accessibility during neurogenesis, and an integrative analysis to predict cell-type-specific candidate regulatory elements. We found that cerebral organoids recapitulate most putative cell-type-specific enhancer accessibility patterns but lack many cell-type-specific open chromatin regions that are found in vivo. Systematic comparison of chromatin accessibility across brain regions revealed unexpected diversity among neural progenitor cells in the cerebral cortex and implicated retinoic acid signalling in the specification of neuronal lineage identity in the prefrontal cortex. Together, our results reveal the important contribution of chromatin state to the emerging patterns of cell type diversity and cell fate specification and provide a blueprint for evaluating the fidelity and robustness of cerebral organoids as a model for cortical development.  

3.3857           TOM20-mediated transfer of Bcl2 from ER to MAM and mitochondria upon induction of apoptosis

Lalier, L., Mignard, V., Joalland, M-P-. Lance, D., Cartron, P-F., Manon, S. and Vallette, F.M. Cell Death & Disease, 12:182 (2021)   In this work, we have explored the subcellular localization of Bcl2, a major antiapoptotic protein. In U251 glioma cells, we found that Bcl2 is localized mainly in the ER and is translocated to MAM and mitochondria upon induction of apoptosis; this mitochondrial transfer was not restricted to the demonstrator cell line, even if cell-specific modulations exist. We found that the Bcl2/mitochondria interaction is controlled by TOM20, a protein that belongs to the protein import machinery of the mitochondrial outer membrane. The expression of a small domain of interaction of TOM20 with Bcl2 potentiates its anti-apoptotic properties, which suggests that the Bcl2–TOM20 interaction is proapoptotic. The role of MAM and TOM20 in Bcl2 apoptotic mitochondrial localization and function has been confirmed in a yeast model in which the ER–mitochondria encounter structure (ERMES) complex (required for MAM stability in yeast) has been disrupted. Bcl2–TOM20 interaction is thus an additional player in the control of apoptosis.  

3.3858           Identification of Neurensin-2 as a novel modulator of emotional behavior

Umschweif, G., Medrihan, L., Guillen-Samander, A., Wang, W., Sagi, Y.and Greengard, P. Mol. Psychiatry, 26, 2872-2885 (2021)   Among the hallmarks of major depressive disorders (MDD) are molecular, functional, and morphological impairments in the hippocampus. Recent studies suggested a key role for hippocampal GABAergic interneurons both in depression and in the response to its treatments. These interneurons highly express the chromatin-remodeler SMARCA3 which mediates the response to chronic antidepressants in an unknown mechanism. Using cell-type-specific molecular and physiological approaches, we report that SMARCA3 mediates the glutamatergic signaling in interneurons by repressing the expression of the neuronal protein, Neurensin-2. This vesicular protein associates with endosomes and postsynaptic proteins and is highly and selectively expressed in subpopulations of GABAergic interneurons. Upregulation of Neurensin-2 in the hippocampus either by stress, viral overexpression, or by SMARCA3 deletion, results in depressive-like behaviors. In contrast, the deletion of Neurensin-2 confers resilience to stress and induces AMPA receptor localization to synapses. This pathway which bidirectionally affects emotional behavior could be involved in neuropsychiatric disorders, and suggests novel therapeutic approaches.  

3.3859           Plasma extracellular vesicle delivery of miR-210-3p by targeting ATG7 to promote sepsis-induced acute lung injury by regulating autophagy and activating inflammation

Li, G., Wang, B., Ding, X., Zhang, X., Tang, J. and Lin, H. Exp. Mol. Med., 53, 1180-1191 (2021)   Extracellular vesicles (EVs) can be used for intercellular communication by facilitating the transfer of miRNAs from one cell to a recipient cell. MicroRNA (miR)-210-3p is released into the blood during sepsis, inducing cytokine production and promoting leukocyte migration. Thus, the current study aimed to elucidate the role of plasma EVs in delivering miR-210-3p in sepsis-induced acute lung injury (ALI). Plasma EVs were isolated from septic patients, after which the expression of various inflammatory factors was measured using enzyme-linked immunosorbent assay. Cell viability and apoptosis were measured via cell counting kit-8 and flow cytometry. Transendothelial resistance and fluorescein isothiocyanate fluorescence were used to measure endothelial cell permeability. Matrigel was used to examine the tubulogenesis of endothelial cells. The targeting relationship between miR-210-3p and ATG7 was assessed by dual-luciferase reporter assays. The expression of ATG7 and autophagy-related genes was determined to examine autophagic activation. A sepsis mouse model was established by cecal ligation and puncture (CLP)-induced surgery. The level of miR-210-3p was highly enriched in septic EVs. MiR-210-3p enhanced THP-1 macrophage inflammation, BEAS-2B cell apoptosis, and HLMVEC permeability while inhibiting angiogenesis and cellular activity. MiR-210-3p overexpression reduced ATG7 and LC3II/LC3I expression and increased P62 expression. Improvements in vascular density and autophagosome formation, increased ATG7 expression, and changes in the ratio of LC3II/LC3I were detected, as well as reduced P62 expression, in adenovirus-anti-miR-210-3p treated mice after CLP injury. Taken together, the key findings of the current study demonstrate that plasma EVs carrying miR-210-3p target ATG7 to regulate autophagy and inflammatory activation in a sepsis-induced ALI model.  

3.3860           Rab22a-NeoF1 fusion protein promotes osteosarcoma lung metastasis through its secretion into exosomes

Zhong, L., Liao, D., Li, J., Liu, W., Wang, J., Zeng, C., Wang, X., Cao, Z., Zhang, R., Li, M., Jiang, K., Zeng, Y-X., Sui, J. and Kang, T. Signal Trandsduction and Targeted Therapy, 6:59 (2021)   It remains unknown for decades how some of the therapeutic fusion proteins positive in a small percentage of cancer cells account for patient outcome. Here, we report that osteosarcoma Rab22a-NeoF1 fusion protein, together with its binding partner PYK2, is sorted into exosomes by HSP90 via its KFERQ-like motif (RVLFLN¹⁴²). The exosomal Rab22a-NeoF1 fusion protein facilitates the pulmonary pre-metastatic niche formation by recruiting bone marrow-derived macrophages. The exosomal PYK2 activates RhoA in its negative recipient osteosarcoma cells and induces signal transducer and activator of transcription 3 activation in its recipient macrophages to increase M2 phenotype. Consequently, lung metastases of its recipient osteosarcoma cells are promoted by this exosomal Rab22a-NeoF1 fusion protein, and this event can be targeted by disrupting its interaction with PYK2 using a designed internalizing RGD peptide.  

3.3861           Specimen-specific drift of densities defines distinct subclasses of extracellular vesicles from human whole saliva

Yamamoto, S., Okamura, K., Fuji, R., kawano, T., Ueda, K., Yajima, Y. and Shiba, K. PloS One, 16(4), e0249526 (2021)   Extracellular vesicles (EVs) in body fluids constitute heterogenous populations, which mirror their diverse parental cells as well as distinct EV-generation pathways. Various methodologies have been proposed to differentiate EVs in order to deepen the current understanding of EV biology. Equilibrium density-gradient centrifugation has often been used to separate EVs based on their buoyant densities; however, the standard conditions used for the method do not necessarily allow all EVs to move to their equilibrium density positions, which complicates the categorization of EVs. Here, by prolonging ultracentrifugation time to 96 h and fractionating EVs both by floating up or spinning down directions, we allowed 111 EV-associated protein markers from the whole saliva of three healthy volunteers to attain equilibrium. Interestingly, the determined buoyant densities of the markers drifted in a specimen-specific manner, and drift patterns differentiated EVs into at least two subclasses. One class carried classical exosomal markers, such as CD63 and CD81, and the other was characterized by the molecules involved in membrane remodeling or vesicle trafficking. Distinct patterns of density drift may represent the differences in generation pathways of EVs.  

3.3862           Comparison of extracellular vesicle isolation and storage methods using high-sensitivity flow cytometry

Deville, S., Berckmans, P., Van Hoof, R., lambrichts, I., Salvati, A. and Nelissen, I. PloS One, 16(2), e0245835 (2021)   Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major limitation for the clinical use of EVs is the lack of standardized methods for the fast and reproducible separation and subsequent detection of EV subpopulations from biofluids, as well as their storage. To advance this application area, fluorescence-based characterization technologies with single-EV resolution, such as high-sensitivity flow cytometry (HS-FCM), are powerful to allow assessment of EV fractionation methods and storage conditions. Furthermore, the use of HS-FCM and fluorescent labeling of EV subsets is expanding due to the potential of high-throughput, multiplex analysis, but requires further method development to enhance the reproducibility of measurements. In this study, we have applied HS-FCM measurements next to standard EV characterization techniques, including nanoparticle tracking analysis, to compare the yield and purity of EV fractions obtained from lipopolysaccharide-stimulated monocytic THP-1 cells by two EV isolation methods, differential centrifugation followed by ultracentrifugation and the exoEasy membrane affinity spin column purification. We observed differences in EV yield and purity. In addition, we have investigated the influence of EV storage at 4°C or -80°C for up to one month on the EV concentration and the stability of EV-associated fluorescent labels. The concentration of the in vitro cell derived EV fractions was shown to remain stable under the tested storage conditions, however, the fluorescence intensity of labeled EV stored at 4°C started to decline within one day.  

3.3863           Human genital antibody-mediated inhibition of Chlamydia trachomatis infection and evidence for ompA genotype-specific neutralization

Ardizzone, C.M., Albritton, H.L., Lillis, R.A., Bagnetto, C.E.L., Shen, L., Cavacini, L.A., Koziowski, P.A and Quayle, A.J. PloS One, 16(10), e0258759 (2021)   The endocervix, the primary site of Chlamydia trachomatis (Ct) infection in women, has a unique repertoire of locally synthesized IgG and secretory IgA (SIgA) with contributions from serum IgG. Here, we assessed the ability of genital and serum-derived IgG and IgA from women with a recent positive Ct test to neutralize Ct elementary bodies (EBs) and inhibit inclusion formation in vitro in human endocervical epithelial cells. We also determined if neutralization was influenced by the major outer membrane protein (MOMP) of the infecting strain, as indicated by ompA gene sequencing and genotyping. At equivalent low concentrations of Ct EB (D/UW-3/Cx + E/UW-5/Cx)-specific antibody, genital-derived IgG and IgA and serum IgA, but not serum IgG, significantly inhibited inclusion formation, with genital IgA being most effective, followed by genital IgG, then serum IgA. The well-characterized Ct genotype D strain, D/UW-3/Cx, was neutralized by serum-derived IgG from patients infected with genotype D strains, genital IgG from patients infected with genotype D or E strains, and by genital IgA from patients infected with genotype D, E, or F strains. Additionally, inhibition of D/UW-3/Cx infection by whole serum, rather than purified immunoglobulin, was associated with levels of serum EB-specific IgG rather than the genotype of infecting strain. In contrast, a Ct genotype Ia clinical isolate, Ia/LSU-56/Cx, was neutralized by whole serum in a genotype and genogroup-specific manner, and inhibition also correlated with EB-specific IgG concentrations in serum. Taken together, these data suggest that (i) genital IgA most effectively inhibits Ct infection in vitro, (ii) human antibody-mediated inhibition of Ct infection is significantly influenced by the ompA genotype of the infecting strain, (iii) the genital antibody repertoire develops or matures differently compared to systemic antibody, and (iv) ompA genotype-specificity of inhibition of infection by whole serum can be overcome by high concentrations of Ct-specific IgG.  

3.3864           Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition

Saffi, G.T., Tang, E., Mamand, S., Inpanathan, S., Fountain, A., Salmena, L. and Botelho, R.J. PloS One, 16(11), e0259313 (2021)   Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and promoted lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H₂O₂ impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H₂O₂ reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, thiol groups, glutathione, or thioredoxin, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS.  

3.3865           Site-specific analysis of N-glycans from different sheep prion strains

Nakic, N., Tran, T.H., Novokmet, M., Andreoletti, O., Lauc, G. and Legname, G. PloS Pathogens, 17(2), e1009232 82021)   Prion diseases are a group of neurodegenerative diseases affecting a wide range of mammalian species, including humans. During the course of the disease, the abnormally folded scrapie prion protein (PrP^Sc) accumulates in the central nervous system where it causes neurodegeneration. In prion disorders, the diverse spectrum of illnesses exists because of the presence of different isoforms of PrP^Sc where they occupy distinct conformational states called strains. Strains are biochemically distinguished by a characteristic three-band immunoblot pattern, defined by differences in the occupancy of two glycosylation sites on the prion protein (PrP). Characterization of the exact N-glycan structures attached on either PrP^C or PrP^Sc is lacking. Here we report the characterization and comparison of N-glycans from two different sheep prion strains. PrP^Sc from both strains was isolated from brain tissue and enzymatically digested with trypsin. By using liquid chromatography coupled to electrospray mass spectrometry, a site-specific analysis was performed. A total of 100 structures were detected on both glycosylation sites. The N-glycan profile was shown to be similar to the one on mouse PrP, however, with additional 40 structures reported. The results presented here show no major differences in glycan composition, suggesting that glycans may not be responsible for the differences in the two analyzed prion strains.  

3.3866           The antiviral sirtuin 3 bridges protein acetylation to mitochondrial integrity and metabolism during human cytomegalovirus infection

Sheng, X. and Cristea, I.M. PloS Pathogens, 17(4), e1009506 (2021)   Regulation of mitochondrial structure and function is a central component of infection with viruses, including human cytomegalovirus (HCMV), as a virus means to modulate cellular metabolism and immune responses. Here, we link the activity of the mitochondrial deacetylase SIRT3 and global mitochondrial acetylation status to host antiviral responses via regulation of both mitochondrial structural integrity and metabolism during HCMV infection. We establish that SIRT3 deacetylase activity is necessary for suppressing virus production, and that SIRT3 maintains mitochondrial pH and membrane potential during infection. By defining the temporal dynamics of SIRT3-substrate interactions during infection, and overlaying acetylome and proteome information, we find altered SIRT3 associations with the mitochondrial fusion factor OPA1 and acetyl-CoA acyltransferase 2 (ACAA2), concomitant with changes in their acetylation levels. Using mutagenesis, microscopy, and virology assays, we determine OPA1 regulates mitochondrial morphology of infected cells and inhibits HCMV production. OPA1 acetylation status modulates these functions, and we establish K834 as a site regulated by SIRT3. Control of SIRT3 protein levels or enzymatic activity is sufficient for regulating mitochondrial filamentous structure. Lastly, we establish a virus restriction function for ACAA2, an enzyme involved in fatty acid beta-oxidation. Altogether, we highlight SIRT3 activity as a regulatory hub for mitochondrial acetylation and morphology during HCMV infection and point to global acetylation as a reflection of mitochondrial health.  

3.3867           Microbiota–host communications: Bacterial extracellular vesicles as a common language

Palomino, R.A.N., Vanpouille, C., Costatini, P.E. and Margolis, L. PloS Pathogens, 17(5), e1009508 (2021)   Both gram-negative and gram-positive bacteria release extracellular vesicles (EVs) that contain components from their mother cells. Bacterial EVs are similar in size to mammalian-derived EVs and are thought to mediate bacteria–host communications by transporting diverse bioactive molecules including proteins, nucleic acids, lipids, and metabolites. Bacterial EVs have been implicated in bacteria–bacteria and bacteria–host interactions, promoting health or causing various pathologies. Although the science of bacterial EVs is less developed than that of eukaryotic EVs, the number of studies on bacterial EVs is continuously increasing. This review highlights the current state of knowledge in the rapidly evolving field of bacterial EV science, focusing on their discovery, isolation, biogenesis, and more specifically on their role in microbiota–host communications. Knowledge of these mechanisms may be translated into new therapeutics and diagnostics based on bacterial EVs.  

3.3868           Nucleotide sugar biosynthesis occurs in the glycosomes of procyclic and bloodstream form Trypanosoma brucei

Guther, M.L.S., Prescott, A.R., Kuettel, S., Tinti, M. and Ferguson, M.A.J. PloS Neglected Tropical Diseases, 15(2), e0009132 (2021)   In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. These biotransformations are catalyzed by thirteen discrete enzymes, five of which possess putative peroxisome targeting sequences. Published experimental analyses using immunofluorescence microscopy and/or digitonin latency and/or subcellular fractionation and/or organelle proteomics have localized eight and six of these enzymes to the glycosomes of bloodstream form and procyclic form T. brucei, respectively. Here we increase these glycosome localizations to eleven in both lifecycle stages while noting that one, phospho-N-acetylglucosamine mutase, also localizes to the cytoplasm. In the course of these studies, the heterogeneity of glycosome contents was also noted. These data suggest that, unlike other eukaryotes, all of nucleotide sugar biosynthesis in T. brucei is compartmentalized to the glycosomes in both lifecycle stages. The implications are discussed.  

3.3869           Exosomes mediate LTB4 release during neutrophil chemotaxis

Majumdar, R., Tameh, A.T., Arya, S.B. and parent, C.A. PloS Biology, 19(7), e3001271 (2021)   Leukotriene B₄ (LTB₄) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB₄ and its synthesizing enzymes localize to intracellular multivesicular bodies, which, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in an LTB₄ receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB₄ release. Our findings establish that the exosomal pool of LTB₄ acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments.  

3.3870           A septin GTPase scaffold of dynein–dynactin motors triggers retrograde lysosome transport

Keisova, I.A., Robinson, B.P. and Spilotis, E.T.

1. Cell Biol., 220(2), e202005219 (2021)

  The metabolic and signaling functions of lysosomes depend on their intracellular positioning and trafficking, but the underlying mechanisms are little understood. Here, we have discovered a novel septin GTPase–based mechanism for retrograde lysosome transport. We found that septin 9 (SEPT9) associates with lysosomes, promoting the perinuclear localization of lysosomes in a Rab7-independent manner. SEPT9 targeting to mitochondria and peroxisomes is sufficient to recruit dynein and cause perinuclear clustering. We show that SEPT9 interacts with both dynein and dynactin through its GTPase domain and N-terminal extension, respectively. Strikingly, SEPT9 associates preferentially with the dynein intermediate chain (DIC) in its GDP-bound state, which favors dimerization and assembly into septin multimers. In response to oxidative cell stress induced by arsenite, SEPT9 localization to lysosomes is enhanced, promoting the perinuclear clustering of lysosomes. We posit that septins function as GDP-activated scaffolds for the cooperative assembly of dynein–dynactin, providing an alternative mechanism of retrograde lysosome transport at steady state and during cellular adaptation to stress.  

3.3871           GSAP regulates lipid homeostasis and mitochondrial function associated with Alzheimer’s disease

Xu, P., Chang, J.C., Zhou, X., Wang, W., Bamkole, M., Wong, E. et al

1. Exp. Med., 218(8), e20202446 (2021)

  Biochemical, pathogenic, and human genetic data confirm that GSAP (γ-secretase activating protein), a selective γ-secretase modulatory protein, plays important roles in Alzheimer’s disease (AD) and Down’s syndrome. However, the molecular mechanism(s) underlying GSAP-dependent pathogenesis remains largely elusive. Here, through unbiased proteomics and single-nuclei RNAseq, we identified that GSAP regulates multiple biological pathways, including protein phosphorylation, trafficking, lipid metabolism, and mitochondrial function. We demonstrated that GSAP physically interacts with the Fe65–APP complex to regulate APP trafficking/partitioning. GSAP is enriched in the mitochondria-associated membrane (MAM) and regulates lipid homeostasis through the amyloidogenic processing of APP. GSAP deletion generates a lipid environment unfavorable for AD pathogenesis, leading to improved mitochondrial function and the rescue of cognitive deficits in an AD mouse model. Finally, we identified a novel GSAP single-nucleotide polymorphism that regulates its brain transcript level and is associated with an increased AD risk. Together, our findings indicate that GSAP impairs mitochondrial function through its MAM localization and that lowering GSAP expression reduces pathological effects associated with AD.  

3.3872           Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

Song, L., Tian, X and Schekman, R.

1. Cell Biol., 220(9), e202101075 (2021)

  Extracellular vesicles (EVs) are thought to mediate the transport of proteins and RNAs involved in intercellular communication. Here, we show dynamic changes in the buoyant density and abundance of EVs that are secreted by PC12 cells stimulated with nerve growth factor (NGF), N2A cells treated with retinoic acid to induce neural differentiation, and mouse embryonic stem cells (mESCs) differentiated into neuronal cells. EVs secreted from in vitro differentiated cells promote neural induction of mESCs. Cyclin D1 enriched within the EVs derived from differentiated neuronal cells contributes to this induction. EVs purified from cells overexpressing cyclin D1 are more potent in neural induction of mESC cells. Depletion of cyclin D1 from the EVs reduced the neural induction effect. Our results suggest that EVs regulate neural development through sorting of cyclin D1.  

3.3873           Acoustofluidic centrifuge for nanoparticle enrichment and separation

Gu, Y., Chen, C., Mao, Z., Bachman, H., Becker, R. et al Science Advances, 7, eabc0467 (2021)   Liquid droplets have been studied for decades and have recently experienced renewed attention as a simplified model for numerous fascinating physical phenomena occurring on size scales from the cell nucleus to stellar black holes. Here, we present an acoustofluidic centrifugation technique that leverages an entanglement of acoustic wave actuation and the spin of a fluidic droplet to enable nanoparticle enrichment and separation. By combining acoustic streaming and droplet spinning, rapid (<1 min) nanoparticle concentration and size-based separation are achieved with a resolution sufficient to identify and isolate exosome subpopulations. The underlying physical mechanisms have been characterized both numerically and experimentally, and the ability to process biological samples (including DNA segments and exosome subpopulations) has been successfully demonstrated. Together, this acoustofluidic centrifuge overcomes existing limitations in the manipulation of nanoscale (<100 nm) bioparticles and can be valuable for various applications in the fields of biology, chemistry, engineering, material science, and medicine.  

3.3874           Mitovesicles are a novel population of extracellular vesicles of mitochondrial origin altered in Down syndrome

D’Acunzo, P., Perez-Gonzalez, R., Kim, Y., Hargash, T., Miller, C. et al Science Advances, 7, eabe5085 (2021)   Mitochondrial dysfunction is an established hallmark of aging and neurodegenerative disorders such as Down syndrome (DS) and Alzheimer’s disease (AD). Using a high-resolution density gradient separation of extracellular vesicles (EVs) isolated from murine and human DS and diploid control brains, we identify and characterize a previously unknown population of double-membraned EVs containing multiple mitochondrial proteins distinct from previously described EV subtypes, including microvesicles and exosomes. We term these newly identified mitochondria-derived EVs “mitovesicles.” We demonstrate that brain-derived mitovesicles contain a specific subset of mitochondrial constituents and that their levels and cargo are altered during pathophysiological processes where mitochondrial dysfunction occurs, including in DS. The development of a method for the selective isolation of mitovesicles paves the way for the characterization in vivo of biological processes connecting EV biology and mitochondria dynamics and for innovative therapeutic and diagnostic strategies.  

3.3875           Mesenchymal growth hormone receptor deficiency leads to failure of alveolar progenitor cell function and severe pulmonary fibrosis

Xie, T., Kulur, V., Liu, N., Deng, N., Wang, Y., Rowan, S.C. et al Science Advances, 7, eabg6005 (2021)   Recent studies have identified impaired type 2 alveolar epithelial cell (ATII) renewal in idiopathic pulmonary fibrosis (IPF) human organoids and severe fibrosis when ATII is defective in mice. ATIIs function as progenitor cells and require supportive signals from the surrounding mesenchymal cells. The mechanisms by which mesenchymal cells promote ATII progenitor functions in lung fibrosis are incompletely understood. We identified growth hormone receptor (GHR) is mainly expressed in mesenchymal cells, and its expression is substantially decreased in IPF lungs. Higher levels of GHR expression correlated with better lung function in patients with IPF. Profibrotic mesenchymal cells retarded ATII growth and were associated with suppressed vesicular GHR expression. Vesicles enriched with Ghr promote ATII proliferation and diminished pulmonary fibrosis in mesenchymal Ghr-deficient mice. Our findings demonstrate a previously unidentified mesenchymal paracrine signaling coordinated by GHR that is capable of supporting ATII progenitor cell renewal and limiting the severity of lung fibrosis.  

3.3876           Nascent fusion pore opening monitored at single-SNAREpin resolution

Heo, P., Coleman, J., Fleury, J-B., Rothman, J.E. and Pincet, F. PNAS, 118(5), e2024922118 (2021)   Vesicle fusion with a target membrane is a key event in cellular trafficking and ensures cargo transport within the cell and between cells. The formation of a protein complex, called SNAREpin, provides the energy necessary for the fusion process. In a three-dimensional microfluidic chip, we monitored the fusion of small vesicles with a suspended asymmetric lipid bilayer. Adding ion channels into the vesicles, our setup allows the observation of a single fusion event by electrophysiology with 10-μs precision. Intriguingly, we identified that small transient fusion pores of discrete sizes reversibly opened with a characteristic lifetime of ∼350 ms. The distribution of their apparent diameters displayed two peaks, at 0.4 ± 0.1 nm and 0.8 ± 0.2 nm. Varying the number of SNAREpins, we demonstrated that the first peak corresponds to fusion pores induced by a single SNAREpin and the second peak is associated with pores involving two SNAREpins acting simultaneously. The pore size fluctuations provide a direct estimate of the energy landscape of the pore. By extrapolation, the energy landscape for three SNAREpins does not exhibit any thermally significant energy barrier, showing that pores larger than 1.5 nm are spontaneously produced by three or more SNAREpins acting simultaneously, and expand indefinitely. Our results quantitatively explain why one SNAREpin is sufficient to open a fusion pore and more than three SNAREpins are required for cargo release. Finally, they also explain why a machinery that synchronizes three SNAREpins, or more, is mandatory to ensure fast neurotransmitter release during synaptic transmission.  

3.3877           GFAP hyperpalmitoylation exacerbates astrogliosis and neurodegenerative pathology in PPT1-deficient mice

Yuan, W., Lu, L., Rao, M., Huang, Y., Liu, C-L.,Liu, S. et al PNAS, 118(13), e2022261118 (2021)   The homeostasis of protein palmitoylation and depalmitoylation is essential for proper physiological functions in various tissues, in particular the central nervous system (CNS). The dysfunction of PPT1 (PPT1-KI, infantile neuronal ceroid lipofuscinosis [INCL] mouse model), which catalyze the depalmitoylation process, results in serious neurodegeneration accompanied by severe astrogliosis in the brain. Endeavoring to determine critical factors that might account for the pathogenesis in CNS by palm-proteomics, glial fibrillary acidic protein (GFAP) was spotted, indicating that GFAP is probably palmitoylated. Questions concerning if GFAP is indeed palmitoylated in vivo and how palmitoylation of GFAP might participate in neural pathology remain unexplored and are waiting to be investigated. Here we show that GFAP is readily palmitoylated in vitro and in vivo; specifically, cysteine-291 is the unique palmitoylated residue in GFAP. Interestingly, it was found that palmitoylated GFAP promotes astrocyte proliferation in vitro. Furthermore, we showed that PPT1 depalmitoylates GFAP, and the level of palmitoylated GFAP is overwhelmingly up-regulated in PPT1-knockin mice, which lead us to speculate that the elevated level of palmitoylated GFAP might accelerate astrocyte proliferation in vivo and ultimately led to astrogliosis in INCL. Indeed, blocking palmitoylation by mutating cysteine-291 into alanine in GFAP attenuate astrogliosis, and remarkably, the concurrent neurodegenerative pathology in PPT1-knockin mice. Together, these findings demonstrate that hyperpalmitoylated GFAP plays critical roles in regulating the pathogenesis of astrogliosis and neurodegeneration in the CNS, and most importantly, pinpointing that cysteine-291 in GFAP might be a valuable pharmaceutical target for treating INCL and other potential neurodegenerative diseases.  

3.3878           Neural Progenitor Cell-Derived Extracellular Vesicles Enhance Blood-Brain Barrier Integrity by NF-κB (Nuclear Factor-κB)-Dependent Regulation of ABCB1 (ATP-Binding Cassette Transporter B1) in Stroke Mice

Zhang, L., Graf, I., Kuang, Y., Zheng, X., Haupt, M., Majid, A., Kilic, E., Hermann, D.M., Psychogios, M-N., Weber, M.S., Ochs, J., Bähr, M. and Doeppner, T.R. Arteroscler. Thromb. Vasc. Biol., 41, 1127-1145 (2021)   Objective: Extracellular vesicles (EVs) derived from neural progenitor cells enhance poststroke neurological recovery, albeit the underlying mechanisms remain elusive. Since previous research described an enhanced poststroke integrity of the blood-brain barrier (BBB) upon systemic transplantation of neural progenitor cells, we examined if neural progenitor cell-derived EVs affect BBB integrity and which cellular mechanisms are involved in the process. Approach and Results: Using in vitro models of primary brain endothelial cell (EC) cultures as well as co-cultures of brain ECs (ECs) and astrocytes exposed to oxygen glucose deprivation, we examined the effects of EVs or vehicle on microvascular integrity. In vitro data were confirmed using a mouse transient middle cerebral artery occlusion model. Cultured ECs displayed increased ABCB1 (ATP-binding cassette transporter B1) levels when exposed to oxygen glucose deprivation, which was reversed by treatment with EVs. The latter was due to an EV-induced inhibition of the NF-κB (nuclear factor-κB) pathway. Using a BBB co-culture model of ECs and astrocytes exposed to oxygen glucose deprivation, EVs stabilized the BBB and ABCB1 levels without affecting the transcellular electrical resistance of ECs. Likewise, EVs yielded reduced Evans blue extravasation, decreased ABCB1 expression as well as an inhibition of the NF-κB pathway, and downstream matrix metalloproteinase 9 (MMP-9) activity in stroke mice. The EV-induced inhibition of the NF-κB pathway resulted in a poststroke modulation of immune responses. Conclusions: Our findings suggest that EVs enhance poststroke BBB integrity via ABCB1 and MMP-9 regulation, attenuating inflammatory cell recruitment by inhibition of the NF-κB pathway.  

3.3879           Therapeutic and Diagnostic Translation of Extracellular Vesicles in Cardiovascular Diseases

Sahoo, S., Adamiak, M., Mathiyalagan, P., Kenneweg, F., Kafert-Kasting, S. and Thum, T. Circulation, 143, 1426-1449 (2021)   Exosomes are small membrane-bound vesicles of endocytic origin that are actively secreted. The potential of exosomes as effective communicators of biological signaling in myocardial function has previously been investigated, and a recent explosion in exosome research not only underscores their significance in cardiac physiology and pathology, but also draws attention to methodological limitations of studying these extracellular vesicles. In this review, we discuss recent advances and challenges in exosome research with an emphasis on scientific innovations in isolation, identification, and characterization methodologies, and we provide a comprehensive summary of web-based resources available in the field. Importantly, we focus on the biology and function of exosomes, highlighting their fundamental role in cardiovascular pathophysiology to further support potential applications of exosomes as biomarkers and therapeutics for cardiovascular diseases.  

3.3880           Exosome Surface Display of IL12 Results in Tumor-Retained Pharmacology with Superior Potency and Limited Systemic Exposure Compared with Recombinant IL12

Lewis, N., Sia, C.L., Kirwin, K., Haupt, S., Mahimkar, G., Zi, T. Xu, T.Z. et al Mol. Cancer Therapeutics, 20(3), 523-534 (2021)   The promise of IL12 as a cancer treatment has yet to be fulfilled with multiple tested approaches being limited by unwanted systemic exposure and unpredictable pharmacology. To address these limitations, we generated exoIL12, a novel, engineered exosome therapeutic that displays functional IL12 on the surface of an exosome. IL12 exosomal surface expression was achieved via fusion to the abundant exosomal surface protein PTGFRN resulting in equivalent potency in vitro to recombinant IL12 (rIL12) as demonstrated by IFNγ production. Following intratumoral injection, exoIL12 exhibited prolonged tumor retention and greater antitumor activity than rIL12. Moreover, exoIL12 was significantly more potent than rIL12 in tumor growth inhibition. In the MC38 model, complete responses were observed in 63% of mice treated with exoIL12; in contrast, rIL12 resulted in 0% complete responses at an equivalent IL12 dose. This correlated with dose-dependent increases in tumor antigen–specific CD8⁺ T cells. Rechallenge studies of exoIL12 complete responder mice showed no tumor regrowth, and depletion of CD8⁺ T cells completely abrogated antitumor activity of exoIL12. Following intratumoral administration, exoIL12 exhibited 10-fold higher intratumoral exposure than rIL12 and prolonged IFNγ production up to 48 hours. Retained local pharmacology of exoIL12 was further confirmed using subcutaneous injections in nonhuman primates. This work demonstrates that tumor-restricted pharmacology of exoIL12 results in superior in vivo efficacy and immune memory without systemic IL12 exposure and related toxicity. ExoIL12 is a novel cancer therapeutic candidate that overcomes key limitations of rIL12 and thereby creates a therapeutic window for this potent cytokine.  

3.3881           COPII mitigates ER stress by promoting formation of ER whorls

Xu, F., Du, W., Zou, Q., Wang, Y., Zhang, X., Xing, X. et al Cell Res., 31, 141-156 (2021)   Cells mitigate ER stress through the unfolded protein response (UPR). Here, we report formation of ER whorls as an effector mechanism of the ER stress response. We found that strong ER stress induces formation of ER whorls, which contain ER-resident proteins such as the Sec61 complex and PKR-like ER kinase (PERK). ER whorl formation is dependent on PERK kinase activity and is mediated by COPII machinery, which facilitates ER membrane budding to form tubular-vesicular ER whorl precursors. ER whorl precursors then go through Sec22b-mediated fusion to form ER whorls. We further show that ER whorls contribute to ER stress-induced translational inhibition by possibly modulating PERK activity and by sequestering translocons in a ribosome-free environment. We propose that formation of ER whorls reflects a new type of ER stress response that controls inhibition of protein translation.  

3.3882           RAB31 marks and controls an ESCRT-independent exosome pathway

Wei, D., Zhan, W., Gao, Y., Huang, L., Gong, R., Wang, W., Zhang, R., Wu, Y., Gao, S. and Kang, T. Cell Res., 31, 157-177 (2021)   Exosomes are generated within the multivesicular endosomes (MVEs) as intraluminal vesicles (ILVs) and secreted during the fusion of MVEs with the cell membrane. The mechanisms of exosome biogenesis remain poorly explored. Here we identify that RAB31 marks and controls an ESCRT-independent exosome pathway. Active RAB31, phosphorylated by epidermal growth factor receptor (EGFR), engages flotillin proteins in lipid raft microdomains to drive EGFR entry into MVEs to form ILVs, which is independent of the ESCRT (endosomal sorting complex required for transport) machinery. Active RAB31 interacts with the SPFH domain and drives ILV formation via the Flotillin domain of flotillin proteins. Meanwhile, RAB31 recruits GTPase-activating protein TBC1D2B to inactivate RAB7, thereby preventing the fusion of MVEs with lysosomes and enabling the secretion of ILVs as exosomes. These findings establish that RAB31 has dual functions in the biogenesis of exosomes: driving ILVs formation and suppressing MVEs degradation, providing an exquisite framework to better understand exosome biogenesis.  

3.3883           Non-Small-Cell Lung Cancer Regression by siRNA Delivered Through Exosomes That Display EGFR RNA Aptamer

Li, Z., Yang, L., Wang, H., Binzel, D.W., Williams, T.M. and Guo, P. Nucleic Acid Therapeutics, 31(5), 364-374 (2021)   Lung cancer is the second most common cancer in both men and women and is the leading cause of cancer death in the United States. The development of drug resistance to commonly used chemotherapeutics in non-small-cell lung cancer (NSCLC) poses significant health risks and there is a dire need to improve patient outcomes. In this study, we report the use of RNA nanotechnology to display ligand on exosome that was loaded with small interfering RNA (siRNA) for NSCLC regression in animal trials. Cholesterol was used to anchor the ligand targeting epidermal growth factor receptor on exosomes that were loaded with siRNA to silence the antiapoptotic factor survivin. The cytosolic delivery of siRNA overcame the problem of endosome trapping, leading to potent gene knockdown, chemotherapy sensitization, and tumor regression, thus achieving a favorable IC₅₀ of 20 nmol/kg siRNA encapsulated by exosome particles in the in vivo gene knockdown assessment.  

3.3884           Chaperone-mediated autophagy controls the turnover of E3 ubiquitin ligase MARCHF5 and regulates mitochondrial dynamics

Nie, T., Tao, K., Zhu, L., Huang, L., Hu, S., Yang, R., Xu, P., Mao, Z. and Yang, Q. Autophagy, 10, 2923-2938 (2021)   As a highly dynamic organelle, mitochondria undergo constant fission and fusion to change their morphology and function, coping with various stress conditions. Loss of the balance between fission and fusion leads to impaired mitochondria function, which plays a critical role in the pathogenesis of Parkinson disease (PD). Yet the mechanisms behind mitochondria dynamics regulation remain to be fully illustrated. Chaperone-mediated autophagy (CMA) is a lysosome-dependent process that selectively degrades proteins to maintain cellular proteostasis. In this study, we demonstrated that MARCHF5, an E3 ubiquitin ligase required for mitochondria fission, is a CMA substrate. MARCHF5 interacted with key CMA regulators and was degraded by lysosomes. Severe oxidative stress compromised CMA activity and stabilized MARCHF5, which facilitated DNM1L translocation and led to excessive fission. Increase of CMA activity promoted MARCHF5 turnover, attenuated DNM1L translocation, and reduced mitochondria fragmentation, which alleviated mitochondrial dysfunction under oxidative stress. Furthermore, we showed that conditional expression of LAMP2A, the key CMA regulator, in dopaminergic (DA) neurons helped maintain mitochondria morphology and protected DA neuronal viability in a rodent PD model. Our work uncovers a critical role of CMA in maintaining proper mitochondria dynamics, and loss of this regulatory control may occur in PD and underlie its pathogenic process.  

3.3885           Extracellular vesicles from methicillin resistant Staphylococcus aureus stimulate proinflammatory cytokine production and trigger IgE-mediated hypersensitivity

Asano, K., Hirose, S., Narita, K., Subsomwong, P., kawai, N., Sukchawalit, R. and Nakane, A. Emerging Microbes &% Infections, 10(1), 1991239 (2021)   Extracellular vesicles (EVs) released from bacteria are enclosed particles carrying biological active molecules. They have been shown to play a role in bacterial communications and delivery of virulence factors to the host cells. Staphylococcus aureus is an opportunistic pathogen causing a variety of infections ranging from impetigo to septicaemia. The EVs released from S. aureus have a high potential to be used for vaccine development against S. aureus infections. However, it is important to clearly understand the impact of SaEVs on the host’s immune response. Our study demonstrated that purified EVs from a clinical isolated methicillin-resistant S. aureus (SaEVs) significantly stimulated proinflammatory cytokine production in mouse immune cells and induced host cell death. An impairment of cytokine production in the Toll-like receptor (TLR)-silenced macrophages suggested that SaEVs stimulate proinflammatory response via TLRs 2, 4 and 9. In mouse infection model, the results demonstrated that SaEV immunization did not provide protective effect. In contrast, all SaEV-immunized mice died within Day 1 after methicillin-resistant S. aureus (MRSA) infection. After MRSA infection for 3 h, the production of IL-6, TNF-α and IL-17 in the spleen of SaEV-immunized mice was significantly higher than that of control mice. On Day 5 after the second immunization, total IgE in the serum was significantly enhanced, and a high titre of Th2-related cytokines was remarkably induced after ex vivo stimulation of the spleen cells with SaEVs. These results suggested that MRSA-derived EVs act as an immunostimulant that induces inflammatory response and IgE-mediated hypersensitivity after MRSA infection.  

3.3886           Mechanism of cargo sorting into small extracellular vesicles

Chen, Y., Zhao, Y., Yin, Y., Jia, X. and Mao, L. Bioengineered, 12(1), 8186-8201 (2021)   Extracellular vesicles (EVs) are special membranous structures released by almost every cell type that carry and protect some biomolecules from being degraded. They transport important signaling molecules involved in cell communication, migration, and numerous physiological processes. EVs can be categorized into two main types according to their size: i) small extracellular vesicles (sEVs), such as exosomes (30–150 nm), released from the fusion of multivesicular bodies (MVBs) with the plasma membrane, and ii) large EVs, such as microvesicles (100–1000 nm). These are no longer considered a waste product of cells, but regulators of intercellular communication, as they can transport specific repertoires of cargos, such as proteins, lipids, and nucleic acids to receptor cells to achieve cell-to-cell communication. This indicates the existence of different mechanisms, which controls the cargos sorting into EVs. This review mainly gives a description about the biological roles of the cargo and the sorting mechanisms of sEVs, especially exosomes.  

3.3887           The composition and function of Enterococcus faecalis membrane vesicles

Afonina, I., Tien, B., Nair, Z., Matysik, A., Lam, L.N., Veleba, M., Jie, A.K.J., Rashid, R., Cazenave-Gassiot, A., Wenk, M., Wai, S.N. and Kline, K.A. Microlife, 2, uqab002 (82021)   Membrane vesicles (MVs) contribute to various biological processes in bacteria, including virulence factor delivery, antimicrobial resistance, host immune evasion and cross-species communication. MVs are frequently released from the surface of both Gram-negative and Gram-positive bacteria during growth. In some Gram-positive bacteria, genes affecting MV biogenesis have been identified, but the mechanism of MV formation is unknown. In Enterococcus faecalis, a causative agent of life-threatening bacteraemia and endocarditis, neither mechanisms of MV formation nor their role in virulence has been examined. Since MVs of many bacterial species are implicated in host–pathogen interactions, biofilm formation, horizontal gene transfer, and virulence factor secretion in other species, we sought to identify, describe and functionally characterize MVs from E. faecalis. Here, we show that E. faecalis releases MVs that possess unique lipid and protein profiles, distinct from the intact cell membrane and are enriched in lipoproteins. MVs of E. faecalis are specifically enriched in unsaturated lipids that might provide membrane flexibility to enable MV formation, providing the first insights into the mechanism of MV formation in this Gram-positive organism.  

3.3888           Reduced Shmt2 Expression Impairs Mitochondrial Folate Accumulation and Respiration, and Leads to Uracil Accumulation in Mouse Mitochondrial DNA

Fiddler, J.L., Xiu, Y.X., Blum, J.E., lamarre, S.G., Phinney, W.N., Stabler, S.P., Brosnan, M.E., Brosnan, J.T., Thalacker-Mercer, A.E. and Field, M.S.

1. Nutr., 151, 2882-2893 (2021)

  Background Adequate cellular thymidylate (dTMP) pools are essential for preservation of nuclear and mitochondrial genome stability. Previous studies have indicated that disruption in nuclear dTMP synthesis leads to increased uracil misincorporation into DNA, affecting genome stability. To date, the effects of impaired mitochondrial dTMP synthesis in nontransformed tissues have been understudied. Objectives This study aimed to determine the effects of decreased serine hydroxymethyltransferase 2 (Shmt2) expression and dietary folate deficiency on mitochondrial DNA (mtDNA) integrity and mitochondrial function in mouse tissues. Methods Liver mtDNA content, and uracil content in liver mtDNA, were measured in Shmt2^+/− and Shmt2^+/+ mice weaned onto either a folate-sufficient control diet (2 mg/kg folic acid; C) or a modified diet lacking folic acid (0 mg/kg folic acid) for 7 wk. Shmt2^+/− and Shmt2^+/+ mouse embryonic fibroblast (MEF) cells were cultured in defined culture medium containing either 0 or 25 nM folate (6S-5-formyl-tetrahydrofolate, folinate) to assess proliferative capacity and mitochondrial function. Chi-square tests, linear mixed models, and 2-factor ANOVA with Tukey post hoc analyses were used to analyze data. Results Shmt2^+/− mice exhibited a 48%–67% reduction in SHMT2 protein concentrations in tissues. Interestingly, Shmt2^+/− mice consuming the folate-sufficient C diet exhibited a 25% reduction in total folate in liver mitochondria. There was also a >20-fold increase in uracil in liver mtDNA in Shmt2^+/− mice consuming the C diet, and dietary folate deficiency also increased uracil content in mouse liver mtDNA from both Shmt2^+/+ and Shmt2^+/− mice. Furthermore, decreased Shmt2 expression in MEF cells reduced cell proliferation, mitochondrial membrane potential, and oxygen consumption rate. Conclusions This study demonstrates that Shmt2 heterozygosity and dietary folate deficiency impair mitochondrial dTMP synthesis in mice, as evidenced by the increased uracil in mtDNA. In addition, Shmt2 heterozygosity impairs mitochondrial function in MEF cells. These findings suggest that elevated uracil in mtDNA may impair mitochondrial function.  

3.3889           Antiandrogens Target TMPRSS2 and Reduce SARS-CoV-2 Virus Entry in Lung Cells

Leach, D.A., Andrea, M., Zwacka, R., Giottis, S., yaates, L., Lloyd, C., Brooke, G.N. and Bevan, C.L.

1. Endocrine. Soc., 5, Issue Suppl. 1, A60 (2021)

  he SARS-CoV-2 coronavirus is the cause of the COVID-19 pandemic. Entry of the virus into host cells, most destructively lung cells, requires two host cell surface proteins, ACE2 and TMPRSS2, downregulation of which is thus a potential therapeutic approach for COVID-19. Both of these cell surface proteins are steroid regulated: TMPRSS2 is a well-characterised androgen-regulated target in prostate cancer. Analysis of sequencing data shows co-expression of the androgen receptor (AR) and TMPRSS2 in key human lung cell types that are targeted by SARS- CoV-2. We show that treatment with antiandrogens such as enzalutamide (a well-tolerated drug widely used in advanced prostate cancer) significantly reduces TMPRSS2 levels in human lung cells and in vivo in mouse lung. We demonstrate that AR binding in the region of the TMPRSS2 gene differs between lung and prostate, identifying distinct regulatory regions. Together, the data and evidence presented supports clinical trials to assess the efficacy of antiandrogens as a treatment option for COVID-19.  

3.3890           Zika Virus Hijacks Extracellular Vesicle Tetraspanin Pathways for Cell-to-Cell Transmission

York, S.B., Sun, L., Cone, A.S., Duke, L.C., Cheerathod, M.R. and Meckes Jr., D.G. mSphere, 6(3), e00192-21 (2021)   Extracellular vesicles (EVs) are membrane-encapsulated structures released by cells which carry signaling factors, proteins, and microRNAs that mediate intercellular communication. Accumulating evidence supports an important role of EVs in the progression of neurological conditions and both the spread and pathogenesis of infectious diseases. It has recently been demonstrated that EVs from hepatitis C virus (HCV)-infected individuals and cells contained replicative-competent viral RNA that was capable of infecting hepatocytes. Being a member of the same viral family, it is likely the Zika virus also hijacks EV pathways to package viral components and secrete vesicles that are infectious and potentially less immunogenic. As EVs have been shown to cross blood-brain and placental barriers, it is possible that Zika virus could usurp normal EV biology to gain access to the brain or developing fetus. Here, we demonstrate that Zika virus-infected cells secrete distinct EV subpopulations with specific viral protein profiles and infectious genomes. Zika virus infection resulted in the enhanced production of EVs with various sizes and densities compared to those released from noninfected cells. We also show that the EV-enriched tetraspanin CD63 regulates the release of EVs and Zika viral genomes and capsids following infection. Overall, these findings provide evidence for an alternative means of Zika virus transmission and demonstrate the role of EV biogenesis and trafficking proteins in the modulation of Zika virus infection and virion morphogenesis.  

3.3891           Dinoroseobacter shibae Outer Membrane Vesicles Are Enriched for the Chromosome Dimer Resolution Site dif

Wang, H., Beier, N., Boedeker, C., Sztajer, H., Henke, P., Neumann-Schaal, M., Mansky, J., Rohde, M., Overmann, J., Petersen, J., Klawonn, F., Kucklick, M., Engelmann, S., Tomasch, J. and Wagner-Döbler, I. mSystems, 6(1), e00693-20 (2021)   Outer membrane vesicles (OMVs) are universally produced by prokaryotes and play important roles in symbiotic and pathogenic interactions. They often contain DNA, but a mechanism for its incorporation is lacking. Here, we show that Dinoroseobacter shibae, a dinoflagellate symbiont, constitutively secretes OMVs containing DNA. Time-lapse microscopy captured instances of multiple OMV production at the septum during cell division. DNA from the vesicle lumen was up to 22-fold enriched for the region around the terminus of replication (ter). The peak of coverage was located at dif, a conserved 28-bp palindromic sequence required for binding of the site-specific tyrosine recombinases XerC/XerD. These enzymes are activated at the last stage of cell division immediately prior to septum formation when they are bound by the divisome protein FtsK. We suggest that overreplicated regions around the terminus have been repaired by the FtsK-dif-XerC/XerD molecular machinery. The vesicle proteome was clearly dominated by outer membrane and periplasmic proteins. Some of the most abundant vesicle membrane proteins were predicted to be required for direct interaction with peptidoglycan during cell division (LysM, Tol-Pal, Spol, lytic murein transglycosylase). OMVs were 15-fold enriched for the saturated fatty acid 16:00. We hypothesize that constitutive OMV secretion in D. shibae is coupled to cell division. The footprint of the FtsK-dif-XerC/XerD molecular machinery suggests a novel potentially highly conserved route for incorporation of DNA into OMVs. Clearing the division site from small DNA fragments might be an important function of vesicles produced during exponential growth under optimal conditions. IMPORTANCE Gram-negative bacteria continually form vesicles from their outer membrane (outer membrane vesicles [OMVs]) during normal growth. OMVs frequently contain DNA, and it is unclear how DNA can be shuffled from the cytoplasm to the OMVs. We studied OMV cargo in Dinoroseobacter shibae, a symbiont of dinoflagellates, using microscopy and a multi-omics approach. We found that vesicles formed during undisturbed exponential growth contain DNA which is enriched for genes around the replication terminus, specifically, the binding site for an enzyme complex that is activated at the last stage of cell division. We suggest that the enriched genes are the result of overreplication which is repaired by their excision and excretion via membrane vesicles to clear the divisome from waste DNA.  

3.3892           Analysis of Cryptococcal Extracellular Vesicles: Experimental Approaches for Studying Their Diversity Among Multiple Isolates, Kinetics of Production, Methods of Separation, and Detection in Cultures of Titan Cells

Reis, F.C.G., Gimenez, B., Josefowicz, L.J., Castelli, R.F., Martins, S., Alves, L.R., de Oliveira, H.C. and Rodrigues, M.L. Microbiol. Spectrum, 9(1), e00125-21 (2021)   Extracellular vesicles (EVs) produced by members of the Cryptococcus genus are associated with fundamental processes of fungal physiology and virulence. However, several questions about the properties of cryptococcal EVs remain unanswered, mostly because of technical limitations. We recently described a fast and efficient protocol of high-yield EV isolation from solid medium. In this study, we aimed at using the solid medium protocol to address some of the open questions about EVs, including the kinetics of EV production, the diversity of EVs produced by multiple isolates under different culture conditions, the separation of vesicles in a density gradient followed by the recovery of functional EVs, the direct detection of EVs in culture supernatants, and the production of vesicles in solid cultures of Titan cells. Our results indicate that the production of EVs is directly impacted by the culture medium and time of growth, resulting in variable detection of EVs per cell and a peak of EV detection at 24 h of growth. Nanoparticle tracking analysis (NTA) of EV samples revealed that multiple isolates produce vesicles with variable properties, including particles of diverging dimensions. EVs were produced in the solid medium in amounts that were separated on a centrifugation density gradient, resulting in the recovery of functional EVs containing the major cryptococcal capsular antigen. We also optimized the solid medium protocol for induction of the formation of Titan cells, and analyzed the production of EVs by NTA and transmission electron microscopy. This analysis confirmed that EVs were isolated from solid cultures of cryptococcal enlarged cells. With these approaches, we expect to implement simple methods that will facilitate the analysis of EVs produced by fungal cells.  

3.3893           GW182 Proteins Restrict Extracellular Vesicle-Mediated Export of MicroRNAs in Mammalian Cancer Cells

Ghosh, S., Mukherjee, K., Chakrabarty, Y., Chatterjee, S., Ghoshal, B. and Bhattacharyya, S.N. Mol. Cell. Biol., 41(5), e00483-20 (2021)   MicroRNAs (miRNAs) are small regulatory RNAs of relatively long half-life in non-proliferative human cells. However, in cancer cells the half-lives of miRNAs are comparatively short. To understand the mechanism of rapid miRNA turnover in cancer cells, we explored the effect of target mRNAs on the abundance of the miRNAs that repress them. We have noted an accelerated extracellular vesicle (EV)-mediated export of miRNAs in presence of their target mRNAs in mammalian cells, and this target-driven miRNA-export process is retarded by Ago2-interacting protein GW182B. The GW182 group of proteins are localized to GW182 bodies or RNA processing bodies in mammalian cells, and GW182B-dependent retardation of miRNA export depends on GW body integrity and is independent of the HuR protein-mediated auxiliary pathway of miRNA export. Our data thus support the existence of a HuR-independent pathway of miRNA export in human cells that can be targeted in MDA-MB-231 cancer cells, to increase the level of cellular let-7a, a known negative regulator of cancer growth.  

3.3894           Dual Pathways of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Trafficking Modulate the Selective Exclusion of Uncleaved Oligomers from Virions

Zhang, S., Nguyen, H.T., Ding, H., Wang, J., Zou, S., Liu, L., Guha, D., Gabuzda, D., Ho, D.D., Kappes, J.C. and Sodroski, J.

1. Virol., 95(3), e01369-20 (2021)

  The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer is transported through the secretory pathway to the infected cell surface and onto virion particles. In the Golgi, the gp160 Env precursor is modified by complex sugars and proteolytically cleaved to produce the mature functional Env trimer, which resists antibody neutralization. We observed mostly uncleaved gp160 and smaller amounts of cleaved gp120 and gp41 Envs on the surface of HIV-1-infected or Env-expressing cells; however, cleaved Envs were relatively enriched in virions and virus-like particles (VLPs). This relative enrichment of cleaved Env in VLPs was observed for wild-type Envs, for Envs lacking the cytoplasmic tail, and for CD4-independent, conformationally flexible Envs. On the cell surface, we identified three distinct populations of Envs: (i) the cleaved Env was transported through the Golgi, was modified by complex glycans, formed trimers that cross-linked efficiently, and was recognized by broadly neutralizing antibodies; (ii) a small fraction of Env modified by complex carbohydrates escaped cleavage in the Golgi; and (iii) the larger population of uncleaved Env lacked complex carbohydrates, cross-linked into diverse oligomeric forms, and was recognized by poorly neutralizing antibodies. This last group of more “open” Env oligomers reached the cell surface in the presence of brefeldin A, apparently bypassing the Golgi apparatus. Relative to Envs transported through the Golgi, these uncleaved Envs were counterselected for virion incorporation. By employing two pathways for Env transport to the surface of infected cells, HIV-1 can misdirect host antibody responses toward conformationally flexible, uncleaved Env without compromising virus infectivity.  

3.3895           Diverse Populations of Extracellular Vesicles with Opposite Functions during Herpes Simplex Virus 1 Infection

Dogrammatzis, C., Saleh, S., Deighan, C. and Kalamvoki, M.

1. Virol., 95(6), e02357-20 (2021)

  Extracellular vesicles (EVs) are released by all types of cells as a means of intercellular communication. Their significance lies in the fact that they can alter recipient cell functions, despite their limited capacity for cargo. We have previously demonstrated that herpes simplex virus 1 (HSV-1) infection influences the cargo and functions of EVs released by infected cells and that these EVs negatively impact a subsequent HSV-1 infection. In the present study, we have implemented cutting-edge technologies to further characterize EVs released during HSV-1 infection. We identified distinct EV populations that were separable through a gradient approach. One population was positive for the tetraspanin CD63 and was distinct from EVs carrying components of the endosomal sorting complexes required for transport (ESCRT). Nanoparticle tracking analysis (NTA) combined with protein analysis indicated that the production of CD63⁺ EVs was selectively induced upon HSV-1 infection. The ExoView platform supported these data and suggested that the amount of CD63 per vesicle is larger upon infection. This platform also identified EV populations positive for other tetraspanins, including CD81 and CD9, whose abundance decreased upon HSV-1 infection. The stimulator of interferon genes (STING) was found in CD63⁺ EVs released during HSV-1 infection, while viral components were found in ESCRT⁺ EVs. Functional characterization of these EVs demonstrated that they have opposite effects on the infection, but the dominant effect was negative. Overall, we have identified the dominant population of EVs, and other EV populations produced during HSV-1 infection, and we have provided information about potential roles.  

3.3896           Sphingolipid-Containing Outer Membrane Vesicles Serve as a Delivery Vehicle To Limit Macrophage Immune Response to Porphyromonas gingivalis

Rocha, F.G., Ottenberg, G.,Eure, Z.G., Davey, M.E. and Gibson III, F.C. Infect. Immun., 89(4), e00614-20 (2021)   Sphingolipids (SLs) are essential structural components of mammalian cell membranes. Our group recently determined that the oral anaerobe Porphyromonas gingivalis delivers its SLs to host cells and that the ability of P. gingivalis to synthesize SLs limits the elicited host inflammatory response during cellular infection. As P. gingivalis robustly produces outer membrane vesicles (OMVs), we hypothesized that OMVs serve as a delivery vehicle for SLs, that the SL status of the OMVs may impact cargo loading to OMVs, and that SL-containing OMVs limit elicited host inflammation similar to that observed by direct bacterial challenge. Transwell cell culture experiments determined that in comparison to the parent strain W83, the SL-null mutant elicited a hyperinflammatory immune response from THP-1 macrophage-like cells with elevated tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and IL-6. Targeted assessment of Toll-like receptors (TLRs) identified elevated expression of TLR2, unchanged TLR4, and elevated expression of the adaptor molecules MyD88 and TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing beta interferon) by SL-null P. gingivalis. No significant differences in gingipain activity were observed in our infection models, and both strains produced OMVs of similar sizes. Using comparative two-dimensional gel electrophoresis, we identified differences in the protein cargo of the OMVs between parent and SL-null strain. Importantly, use of purified OMVs recapitulated the cellular inflammatory response observed in the transwell system with whole bacteria. These findings provide new insights into the role of SLs in P. gingivalis OMV cargo assembly and expand our understanding of SL-OMVs as bacterial structures that modulate the host inflammatory response.  

3.3897           Fusobacterium nucleatum Secretes Outer Membrane Vesicles and Promotes Intestinal Inflammation

Engevik, M.A., Danhof, H.A., Ruan, W., Engevik, A.C., Chang-Graham, A.L. eet al mBio, 12(2), e02706-20 (2021)   Multiple studies have implicated microbes in the development of inflammation, but the mechanisms remain unknown. Bacteria in the genus Fusobacterium have been identified in the intestinal mucosa of patients with digestive diseases; thus, we hypothesized that Fusobacterium nucleatum promotes intestinal inflammation. The addition of >50 kDa F. nucleatum conditioned media, which contain outer membrane vesicles (OMVs), to colonic epithelial cells stimulated secretion of the proinflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor (TNF). In addition, purified F. nucleatum OMVs, but not compounds <50 kDa, stimulated IL-8 and TNF production; which was decreased by pharmacological inhibition of Toll-like receptor 4 (TLR4). These effects were linked to downstream effectors p-ERK, p-CREB, and NF-κB. F. nucleatum >50-kDa compounds also stimulated TNF secretion, p-ERK, p-CREB, and NF-κB activation in human colonoid monolayers. In mice harboring a human microbiota, pretreatment with antibiotics and a single oral gavage of F. nucleatum resulted in inflammation. Compared to mice receiving vehicle control, mice treated with F. nucleatum showed disruption of the colonic architecture, with increased immune cell infiltration and depleted mucus layers. Analysis of mucosal gene expression revealed increased levels of proinflammatory cytokines (KC, TNF, IL-6, IFN-γ, and MCP-1) at day 3 and day 5 in F. nucleatum-treated mice compared to controls. These proinflammatory effects were absent in mice who received F. nucleatum without pretreatment with antibiotics, suggesting that an intact microbiome is protective against F. nucleatum-mediated immune responses. These data provide evidence that F. nucleatum promotes proinflammatory signaling cascades in the context of a depleted intestinal microbiome.  

3.3898           Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering

Thorsen, M.K., Lai, A., Lee, M.W., Hoogerheide, D.P., Wong, G.C. and Freed, J.H. mBio, 12(4), e01548-21 (2021)   During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding.  

3.3899           Bacterial Outer Membrane Vesicles as a Versatile Tool in Vaccine Research and the Fight against Antimicrobial Resistance

Zhu, Z., Antenucci, F., Villumsen, K.R. and Bojesen, A.M. mBio, 12(4), e01707-21 (2021)   Gram-negative bacteria include a number of pathogens that cause disease in humans and animals. Although antibiotics are still effective in treating a considerable range of infections caused by Gram-negative bacteria, the alarming increase of antimicrobial resistance (AMR) induced by excessive use of antibiotics has raised global concerns. Therefore, alternative strategies must be developed to prevent and treat bacterial infections and prevent the advent of a postantibiotic era. Vaccines, one of the greatest achievements in the history of medical science, hold extraordinary potential to prevent bacterial infections and thereby reduce the need for antibiotics. Novel bacterial vaccines are urgently needed, however, and outer membrane vesicles (OMVs), naturally produced by Gram-negative bacteria, represent a promising and versatile tool that can be employed as adjuvants, antigens, and delivery platforms in the development of vaccines against Gram-negative bacteria. Here, we provide an overview of the many roles OMVs can play in vaccine development and the mechanisms behind these applications. Methods to improve OMV yields and a comparison of different strategies for OMV isolation aiming at cost-effective production of OMV-based vaccines are also reviewed.  

3.3900           The versatile role of exosomes in human retroviral infections: from immunopathogenesis to clinical application

Rezaie, J., Aslan, C., Ahmadi, M., Zolbanin, N.M., Kashanchi, F. and Jafari, R. Cell % Bioscience, 11:19 (2021)   Eukaryotic cells produce extracellular vesicles (EVs) mediating intercellular communication. These vesicles encompass many bio-molecules such as proteins, nucleic acids, and lipids that are transported between cells and regulate pathophysiological actions in the recipient cell. Exosomes originate from multivesicular bodies inside cells and microvesicles shed from the plasma membrane and participate in various pathological conditions. Retroviruses such as Human Immunodeficiency Virus -type 1 (HIV-1) and Human T-cell leukemia virus (HTLV)-1 engage exosomes for spreading and infection. Exosomes from virus-infected cells transfer viral components such as miRNAs and proteins that promote infection and inflammation. Additionally, these exosomes deliver virus receptors to target cells that make them susceptible to virus entry. HIV-1 infected cells release exosomes that contribute to the pathogenesis including neurological disorders and malignancy. Exosomes can also potentially carry out as a modern approach for the development of HIV-1 and HTLV-1 vaccines. Furthermore, as exosomes are present in most biological fluids, they hold the supreme capacity for clinical usage in the early diagnosis and prognosis of viral infection and associated diseases. Our current knowledge of exosomes' role from virus-infected cells may provide an avenue for efficient retroviruses associated with disease prevention. However, the exact mechanism involved in retroviruses infection/ inflammation remains elusive and related exosomes research will shed light on the mechanisms of pathogenesis.  

3.3901           Comparative evaluation of methods for isolating small extracellular vesicles derived from pancreatic cancer cells

Wang, J-M., Li, Y-J., Wu, J-Y., Cai, J-X., Wen, J., Xiang, D-X., Hu, X-B. and Li, W-Q. Cell & Bioscience, 11:37 (2021)   Background Small extracellular vesicles (sEVs) are nanosized vesicles involved in cell-to-cell communication. sEVs have been widely studied for clinical applications such as early detection of diseases and as therapeutics. Various methods for sEVs isolation are been using, but different methods may result in different qualities of sEVs and impact downstream analysis and applications. Here, we compared current isolation methods and performed a comparative analysis of sEVs from supernatant of cultured pancreatic cancer cells. Methods Ultracentrifugation, ultrafiltration and co-precipitation as concentration methods were firstly evaluated for yield, size, morphology and protein level of pellets. Then, isolate sEVs obtained by four different purification methods: size exclusion chromatography, density gradient ultracentrifugation, ultracentrifugation, and immunoaffinity capturing, were analysed and compared. Results For the concentration process, ultracentrifugation method obtained high quality and high concentration of pellets. For the purification process, immunoaffinity capturing method obtained the purest sEVs with less contaminants, while density gradient ultracentrifugation-based method obtained sEVs with the smallest size. Proteomic analysis revealed distinct protein contents of purified sEVs from different methods. Conclusions For isolating sEVs derived from supernatant of cultured pancreatic cancer cell line, ultracentrifugation-based method is recommended for concentration of sEVs, density gradient ultracentrifugation-based method may be applied for obtaining purified sEVs with controlled size, immunoaffinity capturing may be suitable for studies requiring sEVs with high purity but may loss subtypes of sEVs without specific protein marker.  

3.3902           Altered oligodendroglia and astroglia in chronic traumatic encephalopathy

Chancellor, K.B., Chancellor, S.E., uke-Cohan, J.E., Huber, B.R., Stein, T.D., Alvarez, V.E., Okaty, B.W., Dymecki, S.M. and McKee, A.C. Acta Neuropathologica, 142, 295-321 (2021)   Chronic traumatic encephalopathy (CTE) is a progressive tauopathy found in contact sport athletes, military veterans, and others exposed to repetitive head impacts. White matter rarefaction and axonal loss have been reported in CTE but have not been characterized on a molecular or cellular level. Here, we present RNA sequencing profiles of cell nuclei from postmortem dorsolateral frontal white matter from eight individuals with neuropathologically confirmed CTE and eight age- and sex-matched controls. Analyzing these profiles using unbiased clustering approaches, we identified eighteen transcriptomically distinct cell groups (clusters), reflecting cell types and/or cell states, of which a subset showed differences between CTE and control tissue. Independent in situ methods applied on tissue sections adjacent to that used in the single-nucleus RNA-seq work yielded similar findings. Oligodendrocytes were found to be most severely affected in the CTE white matter samples; they were diminished in number and altered in relative proportions across subtype clusters. Further, the CTE-enriched oligodendrocyte population showed greater abundance of transcripts relevant to iron metabolism and cellular stress response. CTE tissue also demonstrated excessive iron accumulation histologically. In astrocytes, total cell numbers were indistinguishable between CTE and control samples, but transcripts associated with neuroinflammation were elevated in the CTE astrocyte groups compared to controls. These results demonstrate specific molecular and cellular differences in CTE oligodendrocytes and astrocytes and suggest that white matter alterations are a critical aspect of CTE neurodegeneration.    

3.3903           Cross-platform transcriptional profiling identifies common and distinct molecular pathologies in Lewy body diseases

Feleke, R., Reynolds, R.H., Smith, A.M., Tilley, B., Taliun, S.A.G., Hardy, J., Matthews, P.M., Gentleman, S., Owen, D.R., Johnson, M.R., Srivastava, P.K. and Ryten, M. Acta Neuropathologica, 142, 449-474 (2021)   Parkinson’s disease (PD), Parkinson’s disease with dementia (PDD) and dementia with Lewy bodies (DLB) are three clinically, genetically and neuropathologically overlapping neurodegenerative diseases collectively known as the Lewy body diseases (LBDs). A variety of molecular mechanisms have been implicated in PD pathogenesis, but the mechanisms underlying PDD and DLB remain largely unknown, a knowledge gap that presents an impediment to the discovery of disease-modifying therapies. Transcriptomic profiling can contribute to addressing this gap, but remains limited in the LBDs. Here, we applied paired bulk-tissue and single-nucleus RNA-sequencing to anterior cingulate cortex samples derived from 28 individuals, including healthy controls, PD, PDD and DLB cases (n = 7 per group), to transcriptomically profile the LBDs. Using this approach, we (i) found transcriptional alterations in multiple cell types across the LBDs; (ii) discovered evidence for widespread dysregulation of RNA splicing, particularly in PDD and DLB; (iii) identified potential splicing factors, with links to other dementia-related neurodegenerative diseases, coordinating this dysregulation; and (iv) identified transcriptomic commonalities and distinctions between the LBDs that inform understanding of the relationships between these three clinical disorders. Together, these findings have important implications for the design of RNA-targeted therapies for these diseases and highlight a potential molecular “window” of therapeutic opportunity between the initial onset of PD and subsequent development of Lewy body dementia.  

3.3904           Advances in mesenchymal stem cell exosomes: a review

Taang, Y., Zhou, Y and Li, H-J. Stem Cell Res. Ther., 12:71 (2021)   Stem cells can be used for regenerative medicine and as treatments for disease. The application of tissue engineering-related transplantation, stem cells, and local changes in the microenvironment is expected to solve major medical problems. Currently, most studies focus on tissue repair and regeneration, and mesenchymal stem cells (MSCs) are among the most common research topics. MSCs are applicable as seed cells, and they represent one of the current hot topics in regenerative medicine research. However, due to storage limitations and because cell senescence occurs during in vitro expansion, their clinical application is challenging. Exosomes, which are secreted by MSCs through paracrine signalling, not only have the same effects as MSCs, but they also have the advantages of targeted delivery, low immunogenicity, and high repairability. This article reviews the acquisition methods, characteristics, biological functions, and clinical applications of exosomes.  

3.3905           Comparative proteomic analysis of outer membrane vesicles from Brucella suis, Brucella ovis, Brucella canis and Brucella neotomae

Ruiz-Palma, M.del S., Avila-Calderon, E.D., Aguilera-Arreola, M., Lopez-Merino, A., Ruiz, E.A., Morales-Garcia, M. del R., Lopez-Villegas, E.O., Gomez-Lunar, Z., Arellano-Reynoso, B. and Contreras-Rodriguez, A. Arch. Microbiol., 203, 1611-1626 (2021)   Gram-negative bacteria release nanovesicles, called outer membrane vesicles (OMVs), from their outer membrane. Proteomics has been used to determine their composition. OMVs contain proteins able to elicit an immune response, so they have been proposed as a model to develop acellular vaccines. In this study, OMVs of Brucella suis, B. ovis, B. canis, and B. neotomae were purified and analyzed by SDS-PAGE, transmission electron microscopy and liquid chromatography coupled to mass spectrometry to determine the pan-proteome of these vesicles. In addition, antigenic proteins were detected by western blot with anti-Brucella sera. The in silico analysis of the pan-proteome revealed many homologous proteins, such as Omp16, Omp25, Omp31, SodC, Omp2a, and BhuA. Proteins contained in the vesicles from different Brucella species were detected by anti-Brucella sera. The occurrence of previously described immunogenic proteins derived from OMVs supports the use of these vesicles as candidates to be evaluated as an acellular brucellosis vaccine.    

3.3906           Elucidating Methods for Isolation and Quantification of Exosomes: A Review

Kurian, T.K., Banik, S., Gopal, D., Chakrabarti, S.and Mazumder, N. Mol. Biotechnol., 63, 249-266 (2021)   Exosomes are the smallest extracellular vesicles present in most of the biological fluids. They are found to play an important role in cell signaling, immune response, tumor metastasis, etc. Studies have shown that these vesicles also have diagnostic and therapeutic roles for which their accurate detection and quantification is essential. Due to the complexity in size and structure of exosomes, even the gold standard methods face challenges. This comprehensive review discusses the various standard methods such as ultracentrifugation, ultrafiltration, size-exclusion chromatography, precipitation, immunoaffinity, and microfluidic technologies for the isolation of exosomes. The principle of isolation of each method is described, as well as their specific advantages and disadvantages. Quantification of exosomes by nanoparticle tracking analysis, flow cytometry, tunable resistive pulse sensing, electron microscopy, dynamic light scattering, and microfluidic devices are also described, along with the applications of exosomes in various biomedical domains.  

3.3907           Extracellular vesicles from HTLV-1 infected cells modulate target cells and viral spread

Pinto, D.O., Al Sharif, S., Menssah, G., Cowen, M., Khatkar, P., Erickson, J. et al Retrovirology, 18:6 (2021)   Background The Human T-cell Lymphotropic Virus Type-1 (HTLV-1) is a blood-borne pathogen and etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). HTLV-1 has currently infected up to 10 million globally with highly endemic areas in Japan, Africa, the Caribbean and South America. We have previously shown that Extracellular Vesicles (EVs) enhance HTLV-1 transmission by promoting cell–cell contact. Results Here, we separated EVs into subpopulations using differential ultracentrifugation (DUC) at speeds of 2 k (2000×g), 10 k (10,000×g), and 100 k (100,000×g) from infected cell supernatants. Proteomic analysis revealed that EVs contain the highest viral/host protein abundance in the 2 k subpopulation (2 k > 10 k > 100 k). The 2 k and 10 k populations contained viral proteins (i.e., p19 and Tax), and autophagy proteins (i.e., LC3 and p62) suggesting presence of autophagosomes as well as core histones. Interestingly, the use of 2 k EVs in an angiogenesis assay (mesenchymal stem cells + endothelial cells) caused deterioration of vascular-like-tubules. Cells commonly associated with the neurovascular unit (i.e., astrocytes, neurons, and macrophages) in the blood–brain barrier (BBB) showed that HTLV-1 EVs may induce expression of cytokines involved in migration (i.e., IL-8; 100 k > 2 k > 10 k) from astrocytes and monocyte-derived macrophages (i.e., IL-8; 2 k > 10 k). Finally, we found that EVs were able to promote cell–cell contact and viral transmission in monocytic cell-derived dendritic cell. The EVs from both 2 k and 10 k increased HTLV-1 spread in a humanized mouse model, as evidenced by an increase in proviral DNA and RNA in the Blood, Lymph Node, and Spleen. Conclusions Altogether, these data suggest that various EV subpopulations induce cytokine expression, tissue damage, and viral spread.  

3.3908           Lactobacillus reuteri-derived extracellular vesicles maintain intestinal immune homeostasis against lipopolysaccharide-induced inflammatory responses in broilers

Hu, R., Lin, H., wang, M., Zhao, Y., Liu, H., Min, Y., Yang, X., Gao, Y. and Yang, M.

1. Animal Sci. Biotechnol., 12:25 (2021)

  Background Lactobacillus reuteri strains are widely used as probiotics to prevent and treat inflammatory bowel disease by modulating the host’s immune system. However, the underlying mechanisms by which they communicate with the host have not been clearly understood. Bacterial extracellular vesicles (EVs) have been considered as important mediators of host-pathogen interactions, but their potential role in commensals-host crosstalk has not been widely studied. Here, we investigated the regulatory actions of EVs produced by L. reuteri BBC3, a gut-associated commensal bacterium of Black-Bone chicken, in the development of lipopolysaccharide (LPS)-induced intestinal inflammation in a chicken model using both in vivo and in vitro experiments. Results

1. reuteriBBC3 produced nano-scale membrane vesicles with the size range of 60–250 nm. Biochemical and proteomic analyses showed that L. reuteriBBC3-derived EVs (LrEVs) carried DNA, RNA and several bioactive proteins previously described as mediators of other probiotics’ beneficial effects such as glucosyltransferase, serine protease and elongation factor Tu. In vivo broiler experiments showed that administration of LrEVs exerted similar effects as L. reuteri BBC3 in attenuating LPS-induced inflammation by improving growth performance, reducing mortality and decreasing intestinal injury. LrEVs suppressed the LPS-induced expression of pro-inflammatory genes (TNF-α, IL-1β, IL-6, IL-17 and IL-8), and improved the expression of anti-inflammatory genes (IL-10 and TGF-β) in the jejunum. LrEVs could be internalized by chicken macrophages. In vitro pretreatment with LrEVs reduced the gene expression of TNF-α, IL-1β and IL-6 by suppressing the NF-κB activity, and enhanced the gene expression of IL-10 and TGF-β in LPS-activated chicken macrophages. Additionally, LrEVs could inhibit Th1- and Th17-mediated inflammatory responses and enhance the immunoregulatory cells-mediated immunosuppression in splenic lymphocytes of LPS-challenged chickens through the activation of macrophages. Finally, we revealed that the reduced content of both vesicular proteins and nucleic acids attenuated the suppression of LrEVs on LPS-induced inflammatory responses in ex vivo experiments, suggesting that they are essential for the LrEVs-mediated immunoregulation.

Conclusions We revealed that LrEVs participated in maintaining intestinal immune homeostasis against LPS-induced inflammatory responses in a chicken model. Our findings provide mechanistic insight into how commensal and probiotic Lactobacillus species modulate the host’s immune system in pathogens-induced inflammation.  

3.3909           The iRhom homology domain is indispensable for ADAM17-mediated TNFα and EGF receptor ligand release

Düsterhöft, S., Kahveci-Turkoz, S., Wozniak, J., Seifert, A., Kasparek, P., Ohm, H., Liu, S., Kopkanova, J., Lokau, J., Garbers, C., Preisinger, C., Sediacek, R., Freeman, M. and Ludwig, A. Cell. Mol. Life Sci., 78, 5015-5040 (2021)   Membrane-tethered signalling proteins such as TNFα and many EGF receptor ligands undergo shedding by the metalloproteinase ADAM17 to get released. The pseudoproteases iRhom1 and iRhom2 are important for the transport, maturation and activity of ADAM17. Yet, the structural and functional requirements to promote the transport of the iRhom-ADAM17 complex have not yet been thoroughly investigated. Utilising in silico and in vitro methods, we here map the conserved iRhom homology domain (IRHD) and provide first insights into its structure and function. By focusing on iRhom2, we identified different structural and functional factors within the IRHD. We found that the structural integrity of the IRHD is a key factor for ADAM17 binding. In addition, we identified a highly conserved motif within an unstructured region of the IRHD, that, when mutated, restricts the transport of the iRhom-ADAM17 complex through the secretory pathway in in vitro, ex vivo and in vivo systems and also increases the half-life of iRhom2 and ADAM17. Furthermore, the disruption of this IRHD motif was also reflected by changes in the yet undescribed interaction profile of iRhom2 with proteins involved in intracellular vesicle transport. Overall, we provide the first insights into the forward trafficking of iRhoms which is critical for TNFα and EGF receptor signalling.  

3.3910           Prenatal treatment with rapamycin restores enhanced hippocampal mGluR-LTD and mushroom spine size in a Down’s syndrome mouse model

Urbano-Gamez, J.D., Casanas, J.J., Benito, I and Luz Montesinos, M. Mol. Brain., 14:84 (2021)   Down syndrome (DS) is the most frequent genetic cause of intellectual disability including hippocampal-dependent memory deficits. We have previously reported hippocampal mTOR (mammalian target of rapamycin) hyperactivation, and related plasticity as well as memory deficits in Ts1Cje mice, a DS experimental model. Here we characterize the proteome of hippocampal synaptoneurosomes (SNs) from these mice, and found a predicted alteration of synaptic plasticity pathways, including long term depression (LTD). Accordingly, mGluR-LTD (metabotropic Glutamate Receptor-LTD) is enhanced in the hippocampus of Ts1Cje mice and this is correlated with an increased proportion of a particular category of mushroom spines in hippocampal pyramidal neurons. Remarkably, prenatal treatment of these mice with rapamycin has a positive pharmacological effect on both phenotypes, supporting the therapeutic potential of rapamycin/rapalogs for DS intellectual disability.  

3.3911           Identification and prediction of developmental enhancers in sea urchin embryos

Arenas-Mena, C., Miljovska, S., Rice, E.J., Gurges, J., Shashikant, T., Wang, Z., Ercan, S. and Danko, C.G. BMC Genomics, 22:751 (2021)   Background The transcription of developmental regulatory genes is often controlled by multiple cis-regulatory elements. The identification and functional characterization of distal regulatory elements remains challenging, even in tractable model organisms like sea urchins. Results We evaluate the use of chromatin accessibility, transcription and RNA Polymerase II for their ability to predict enhancer activity of genomic regions in sea urchin embryos. ATAC-seq, PRO-seq, and Pol II ChIP-seq from early and late blastula embryos are manually contrasted with experimental cis-regulatory analyses available in sea urchin embryos, with particular attention to common developmental regulatory elements known to have enhancer and silencer functions differentially deployed among embryonic territories. Using the three functional genomic data types, machine learning models are trained and tested to classify and quantitatively predict the enhancer activity of several hundred genomic regions previously validated with reporter constructs in vivo. Conclusions Overall, chromatin accessibility and transcription have substantial power for predicting enhancer activity. For promoter-overlapping cis-regulatory elements in particular, the distribution of Pol II is the best predictor of enhancer activity in blastula embryos. Furthermore, ATAC- and PRO-seq predictive value is stage dependent for the promoter-overlapping subset. This suggests that the sequence of regulatory mechanisms leading to transcriptional activation have distinct relevance at different levels of the developmental gene regulatory hierarchy deployed during embryogenesis.  

3.3912           KAI1(CD82) is a key molecule to control angiogenesis and switch angiogenic milieu to quiescent state

Lee, J-W., Hur, J., Kwon, Y-W., Chae, C-W., Choi, J-II., Hwang, I. et al

1. Hematol. Oncol., 14:148 (2021)

  Background Little is known about endogenous inhibitors of angiogenic growth factors. In this study, we identified a novel endogenous anti-angiogenic factor expressed in pericytes and clarified its underlying mechanism and clinical significance. Methods Herein, we found Kai1 knockout mice showed significantly enhanced angiogenesis. Then, we investigated the anti-angiogenic roll of Kai1 in vitro and in vivo. Results KAI1 was mainly expressed in pericytes rather than in endothelial cells. It localized at the membrane surface after palmitoylation by zDHHC4 enzyme and induced LIF through the Src/p53 pathway. LIF released from pericytes in turn suppressed angiogenic factors in endothelial cells as well as in pericytes themselves, leading to inhibition of angiogenesis. Interestingly, KAI1 had another mechanism to inhibit angiogenesis: It directly bound to VEGF and PDGF and inhibited activation of their receptors. In the two different in vivo cancer models, KAI1 supplementation significantly inhibited tumor angiogenesis and growth. A peptide derived from the large extracellular loop of KAI1 has been shown to have anti-angiogenic effects to block the progression of breast cancer and retinal neovascularization in vivo. Conclusions KAI1 from PC is a novel molecular regulator that counterbalances the effect of angiogenic factors.  

3.3913           Extracellular vesicle-associated miRNAs are an adaptive response to gestational diabetes mellitus

Nair, S., Guanzon, D., Jayabalan, N., Lai, A., Scholz-Romero, K. et al

1. Transl. Med., 19:360 (2021)

  Background Gestational diabetes mellitus (GDM) is a serious public health issue affecting 9–15% of all pregnancies worldwide. Recently, it has been suggested that extracellular vesicles (EVs) play a role throughout gestation, including mediating a placental response to hyperglycaemia. Here, we investigated the EV-associated miRNA profile across gestation in GDM, assessed their utility in developing accurate, multivariate classification models, and determined the signaling pathways in skeletal muscle proteome associated with the changes in the EV miRNA profile. Methods Discovery: A retrospective, case–control study design was used to identify EV-associated miRNAs that vary across pregnancy and clinical status (i.e. GDM or Normal Glucose Tolerance, NGT). EVs were isolated from maternal plasma obtained at early, mid and late gestation (n = 29) and small RNA sequencing was performed. Validation: A longitudinal study design was used to quantify expression of selected miRNAs. EV miRNAs were quantified by real-time PCR (cases = 8, control = 14, samples at three times during pregnancy) and their individual and combined classification efficiencies were evaluated. Quantitative, data-independent acquisition mass spectrometry was use to establish the protein profile in skeletal muscle biopsies from normal and GDM. Results A total of 2822 miRNAs were analyzed using a small RNA library, and a total of 563 miRNAs that significantly changed (p < 0.05) across gestation and 101 miRNAs were significantly changed between NGT and GDM. Analysis of the miRNA changes in NGT and GDM separately identified a total of 256 (NGT-group), and 302 (GDM-group) miRNAs that change across gestation. A multivariate classification model was developed, based on the quantitative expression of EV-associated miRNAs, and the accuracy to correctly assign samples was > 90%. We identified a set of proteins in skeletal muscle biopsies from women with GDM associated with JAK-STAT signaling which could be targeted by the miRNA-92a-3p within circulating EVs. Interestingly, overexpression of miRNA-92a-3p in primary skeletal muscle cells increase insulin-stimulated glucose uptake. Conclusions During early pregnancy, differently-expressed, EV-associated miRNAs may be of clinical utility in identifying presymptomatic women who will subsequently develop GDM later in gestation. We suggest that miRNA-92a-3p within EVs might be a protected mechanism to increase skeletal muscle insulin sensitivity in GDM.  

3.3914           Artificial exosomes for translational nanomedicine

Li, Y-J., Wu, J-Y., Liu, J., Xu, X., Qiu, X., Huang, S., Hu, X-B., and Xiang, D.X.

1. Nanobiotechnol., 19:242 (2021)

  Exosomes are lipid bilayer membrane vesicles and are emerging as competent nanocarriers for drug delivery. The clinical translation of exosomes faces many challenges such as massive production, standard isolation, drug loading, stability and quality control. In recent years, artificial exosomes are emerging based on nanobiotechnology to overcome the limitations of natural exosomes. Major types of artificial exosomes include ‘nanovesicles (NVs)’, ‘exosome-mimetic (EM)’ and ‘hybrid exosomes (HEs)’, which are obtained by top-down, bottom-up and biohybrid strategies, respectively. Artificial exosomes are powerful alternatives to natural exosomes for drug delivery. Here, we outline recent advances in artificial exosomes through nanobiotechnology and discuss their strengths, limitations and future perspectives. The development of artificial exosomes holds great values for translational nanomedicine.  

3.3915           RNA-sequencing profiling analysis of pericyte-derived extracellular vesicle–mimetic nanovesicles-regulated genes in primary cultured fibroblasts from normal and Peyronie’s disease penile tunica albuginea

Yin, G.N., Piao, S., Liu, Z., Wang, L., Ock, J., Kwon, M-H., Kim, D-k., Gho, Y.S., Suh, J-K. and Ryu, J-K. BMC Urology, 21:103 (2021)   Background Peyronie’s disease (PD) is a severe fibrotic disease of the tunica albuginea that causes penis curvature and leads to penile pain, deformity, and erectile dysfunction. The role of pericytes in the pathogenesis of fibrosis has recently been determined. Extracellular vesicle (EV)–mimetic nanovesicles (NVs) have attracted attention regarding intercellular communication between cells in the field of fibrosis. However, the global gene expression of pericyte-derived EV–mimetic NVs (PC–NVs) in regulating fibrosis remains unknown. Here, we used RNA-sequencing technology to investigate the potential target genes regulated by PC–NVs in primary fibroblasts derived from human PD plaque. Methods Human primary fibroblasts derived from normal and PD patients was cultured and treated with cavernosum pericytes isolated extracellular vesicle (EV)–mimetic nanovesicles (NVs). A global gene expression RNA-sequencing assay was performed on normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC–NVs. Reverse transcription polymerase chain reaction (RT-PCR) was used for sequencing data validation. Results A total of 4135 genes showed significantly differential expression in the normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC–NVs. However, only 91 contra-regulated genes were detected among the three libraries. Furthermore, 20 contra-regulated genes were selected and 11 showed consistent changes in the RNA-sequencing assay, which were validated by RT-PCR. Conclusion The gene expression profiling results suggested that these validated genes may be good targets for understanding potential mechanisms and conducting molecular studies into PD.  

3.3916           Depletion of Mitochondrial Components from Extracellular Vesicles Secreted from Astrocytes in a Mouse Model of Fragile X Syndrome

Ha, B.G., Heo, J-Y., Jang, Y-J., Park, T-S., Choi, J-Y., Jang, W.Y. and Jeong, S-J. Int. J. Mol. Sci., 22:410 (2021)   Mitochondrial dysfunction contributes to neurodegenerative diseases and developmental disorders such as Fragile X syndrome (FXS). The cross-talk between mitochondria and extracellular vesicles (EVs) suggests that EVs may transfer mitochondrial components as intermediators for intracellular communication under physiological and pathological conditions. In the present study, the ability of EVs to transfer mitochondrial components and their role in mitochondrial dysfunction in astrocytes were examined in the brains of Fmr1 knockout (KO) mice, a model of FXS. The amounts of mitochondrial transcription factor NRF-1, ATP synthases ATP5A and ATPB, and the mitochondrial membrane protein VDAC1 in EVs were reduced in cerebral cortex samples and astrocytes from Fmr1 KO mice. These reductions correspond to decreased mitochondrial biogenesis and transcriptional activities in Fmr1 KO brain, along with decreased mitochondrial membrane potential (MMP) with abnormal localization of vimentin intermediate filament (VIF) in Fmr1 KO astrocytes. Our results suggest that mitochondrial dysfunction in astrocytes is associated with the pathogenesis of FXS and can be monitored by depletion of components in EVs. These findings may improve the ability to diagnose developmental diseases associated with mitochondrial dysfunction, such as FXS and autism spectrum disorders (ASD).  

3.3917           Extracellular Vesicles from Follicular and Ampullary Fluid Isolated by Density Gradient Ultracentrifugation Improve Bovine Embryo Development and Quality

Asaadi, A., Dolatabad, N.A., Atashi, H., Raes, A., Van Damme, P., Hoelker, M., Hendrix, A., Pascottini, O.B., Van Soom, A., Kafi, M. and Pavani, K.C. Int. J. Mol. Sci., 22:578 (2021)   Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40–400 nm) and concentrations (2.4 ± 0.2 × 10¹²−1.8 ± 0.2 × 10¹³ particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality.  

3.3918           Human Placenta Exosomes: Biogenesis, Isolation, Composition, and Prospects for Use in Diagnostics

Burkova, E.E., Sedykh, S.E. and Nevinsky, G.A. Int. J. Mol. Sci., 22:2158 (2021)   Exosomes are 40–100 nm nanovesicles participating in intercellular communication and transferring various bioactive proteins, mRNAs, miRNAs, and lipids. During pregnancy, the placenta releases exosomes into the maternal circulation. Placental exosomes are detected in the maternal blood even in the first trimester of pregnancy and their numbers increase significantly by the end of pregnancy. Exosomes are necessary for the normal functioning of the placenta and fetal development. Effects of exosomes on target cells depend not only on their concentration but also on their intrinsic components. The biochemical composition of the placental exosomes may cause various complications of pregnancy. Some studies relate the changes in the composition of nanovesicles to placental dysfunction. Isolation of placental exosomes from the blood of pregnant women and the study of protein, lipid, and nucleic composition can lead to the development of methods for early diagnosis of pregnancy pathologies. This review describes the biogenesis of exosomes, methods of their isolation, analyzes their biochemical composition, and considers the prospects for using exosomes to diagnose pregnancy pathologies.  

3.3919           Immune-Associated Proteins Are Enriched in Lung Tissue-Derived Extracellular Vesicles during Allergen-Induced Eosinophilic Airway Inflammation

Int. J. Mol. Sci., 22:4718 (2021)   Studying the proteomes of tissue-derived extracellular vesicles (EVs) can lead to the identification of biomarkers of disease and can provide a better understanding of cell-to-cell communication in both healthy and diseased tissue. The aim of this study was to apply our previously established tissue-derived EV isolation protocol to mouse lungs in order to determine the changes in the proteomes of lung tissue-derived EVs during allergen-induced eosinophilic airway inflammation. A mouse model for allergic airway inflammation was used by sensitizing the mice intraperitoneal with ovalbumin (OVA), and one week after the final sensitization, the mice were challenged intranasal with OVA or PBS. The animals were sacrificed 24 h after the final challenge, and their lungs were removed and sliced into smaller pieces that were incubated in culture media with DNase I and Collagenase D for 30 min at 37 °C. Vesicles were isolated from the medium by ultracentrifugation and bottom-loaded iodixanol density cushions, and the proteomes were determined using quantitative mass spectrometry. More EVs were present in the lungs of the OVA-challenged mice compared to the PBS-challenged control mice. In total, 4510 proteins were quantified in all samples. Among them, over 1000 proteins were significantly altered (fold change >2), with 614 proteins being increased and 425 proteins being decreased in the EVs from OVA-challenged mice compared to EVs from PBS-challenged animals. The associated cellular components and biological processes were analyzed for the altered EV proteins, and the proteins enriched during allergen-induced airway inflammation were mainly associated with gene ontology (GO) terms related to immune responses. In conclusion, EVs can be isolated from mouse lung tissue, and the EVs’ proteomes undergo changes in response to allergen-induced airway inflammation. This suggests that the composition of lung-derived EVs is altered in diseases associated with inflammation of the lung, which may have implications in type-2 driven eosinophilic asthma pathogenesis.  

3.3920           Exosomal microRNAs as Biomarkers and Therapeutic Targets for Hepatocellular Carcinoma

Sorop, A., Constantinescu, D., Cojocaru, F., Dinischiiotu, A., Cucu, D. and Dima, S.O. Int. J. Mol. Sci., 22:4997 (2021)   Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the second most common cause of cancer-related death globally. This type of liver cancer is frequently detected at a late stage by current biomarkers because of the high clinical and biological heterogeneity of HCC tumours. From a plethora of molecules and cellular compounds, small nanoparticles with an endosomal origin are valuable cancer biomarkers or cargos for novel treatments. Despite their small sizes, in the range of 40–150 nm, these particles are delimited by a lipid bilayer membrane with a specific lipid composition and carry functional information—RNA, proteins, miRNAs, long non-coding RNAs (lncRNAs), or DNA fragments. This review summarizes the role of exosomal microRNA (miRNA) species as biomarkers in HCC therapy. After we briefly introduce the exosome biogenesis and the methods of isolation and characterization, we discuss miRNA’s correlation with the diagnosis and prognosis of HCC, either as single miRNA species, or as specific panels with greater clinical impact. We also review the role of exosomal miRNAs in the tumourigenic process and in the cell communication pathways through the delivery of cargos, including proteins or specific drugs.  

3.3921           INTACT vs. FANS for Cell-Type-Specific Nuclei Sorting: A Comprehensive Qualitative and Quantitative Comparison

Chongtham, M., Butto, T., Mungikar, K., Gerber, S. and Winter, J. Int. J. Mol. Sci., 22:5335 (2021)   Increasing numbers of studies seek to characterize the different cellular sub-populations present in mammalian tissues. The techniques “Isolation of Nuclei Tagged in Specific Cell Types” (INTACT) or “Fluorescence-Activated Nuclei Sorting” (FANS) are frequently used for isolating nuclei of specific cellular subtypes. These nuclei are then used for molecular characterization of the cellular sub-populations. Despite the increasing popularity of both techniques, little is known about their isolation efficiency, advantages, and disadvantages or downstream molecular effects. In our study, we compared the physical and molecular attributes of sfGFP+ nuclei isolated by the two methods—INTACT and FANS—from the neocortices of Arc-CreERT2 × CAG-Sun1/sfGFP animals. We identified differences in efficiency of sfGFP+ nuclei isolation, nuclear size as well as transcriptional (RNA-seq) and chromatin accessibility (ATAC-seq) states. Therefore, our study presents a comprehensive comparison between the two widely used nuclei sorting techniques, identifying the advantages and disadvantages for both INTACT and FANS. Our conclusions are summarized in a table to guide researchers in selecting the most suitable methodology for their individual experimental design.  

3.3922           Urinary Extracellular Vesicles: Uncovering the Basis of the Pathological Processes in Kidney-Related Diseases

Cricri, G., Bellucci, L., Montini, G. and Collino, F. Int. J. Mol. Sci., 22:6507 (2021)   Intercellular communication governs multicellular interactions in complex organisms. A variety of mechanisms exist through which cells can communicate, e.g., cell-cell contact, the release of paracrine/autocrine soluble molecules, or the transfer of extracellular vesicles (EVs). EVs are membrane-surrounded structures released by almost all cell types, acting both nearby and distant from their tissue/organ of origin. In the kidney, EVs are potent intercellular messengers released by all urinary system cells and are involved in cell crosstalk, contributing to physiology and pathogenesis. Moreover, urine is a reservoir of EVs coming from the circulation after crossing the glomerular filtration barrier—or originating in the kidney. Thus, urine represents an alternative source for biomarkers in kidney-related diseases, potentially replacing standard diagnostic techniques, including kidney biopsy. This review will present an overview of EV biogenesis and classification and the leading procedures for isolating EVs from body fluids. Furthermore, their role in intra-nephron communication and their use as a diagnostic tool for precision medicine in kidney-related disorders will be discussed  

3.3923           Extracellular Vesicles in the Fungi Kingdom

Liebana-Jordan, M., Brotons, B., Falcon-Perez, J.M. and Gonzalez, E. Int. J. Mol. Sci., 22:7221 (2021)   Extracellular vesicles (EVs) are membranous, rounded vesicles released by prokaryotic and eukaryotic cells in their normal and pathophysiological states. These vesicles form a network of intercellular communication as they can transfer cell- and function-specific information (lipids, proteins and nucleic acids) to different cells and thus alter their function. Fungi are not an exception; they also release EVs to the extracellular space. The vesicles can also be retained in the periplasm as periplasmic vesicles (PVs) and the cell wall. Such fungal vesicles play various specific roles in the lives of these organisms. They are involved in creating wall architecture and maintaining its integrity, supporting cell isolation and defence against the environment. In the case of pathogenic strains, they might take part in the interactions with the host and affect the infection outcomes. The economic importance of fungi in manufacturing high-quality nutritional and pharmaceutical products and in remediation is considerable. The analysis of fungal EVs opens new horizons for diagnosing fungal infections and developing vaccines against mycoses and novel applications of nanotherapy and sensors in industrial processes.  

3.3924           Stem Cells and Their Derivatives—Implications for Alveolar Bone Regeneration: A Comprehensive Review

Holly, D., Klein, M., Mazreku, M., Zamborsky, R., Polak, S., Danisovic, L. and Czönyeiova, M. Int. J. Mol. Biol., 22:11746 (2021)   Oral and craniofacial bone defects caused by congenital disease or trauma are widespread. In the case of severe alveolar bone defect, autologous bone grafting has been considered a “gold standard”; however, the procedure has several disadvantages, including limited supply, resorption, donor site morbidity, deformity, infection, and bone graft rejection. In the last few decades, bone tissue engineering combined with stem cell-based therapy may represent a possible alternative to current bone augmentation techniques. The number of studies investigating different cell-based bone tissue engineering methods to reconstruct alveolar bone damage is rapidly rising. As an interdisciplinary field, bone tissue engineering combines the use of osteogenic cells (stem cells/progenitor cells), bioactive molecules, and biocompatible scaffolds, whereas stem cells play a pivotal role. Therefore, our work highlights the osteogenic potential of various dental tissue-derived stem cells and induced pluripotent stem cells (iPSCs), the progress in differentiation techniques of iPSCs into osteoprogenitor cells, and the efforts that have been made to fabricate the most suitable and biocompatible scaffold material with osteoinductive properties for successful bone graft generation. Moreover, we discuss the application of stem cell-derived exosomes as a compelling new form of “stem-cell free” therapy.  

3.3925           Identification of Tomato Infecting Viruses That Co-Isolate with Nanovesicles Using a Combined Proteomics and Electron-Microscopic Approach

Mammadova, R., Fiume, I., Bokka, A., Kralj-Iglic, V., Bozic, D., Kisovec, M., Podobnik, M., Zavec, A.B., Hocevar, m., Gellen, G., Schlosser, G. and Pocsfalvi, G. Nanomaterials, 11:1922 (2021)   Plant-derived nanovesicles (NVs) have attracted interest due to their anti-inflammatory, anticancer and antioxidative properties and their efficient uptake by human intestinal epithelial cells. Previously we showed that tomato (Solanum lycopersicum L.) fruit is one of the interesting plant resources from which NVs can be obtained at a high yield. In the course of the isolation of NVs from different batches of tomatoes, using the established differential ultracentrifugation or size-exclusion chromatography methods, we occasionally observed the co-isolation of viral particles. Density gradient ultracentrifugation (gUC), using sucrose or iodixanol gradient materials, turned out to be efficient in the separation of NVs from the viral particles. We applied cryogenic transmission electron microscopy (cryo-TEM), scanning electron microscopy (SEM) for the morphological assessment and LC–MS/MS-based proteomics for the protein identification of the gradient fractions. Cryo-TEM showed that a low-density gUC fraction was enriched in membrane-enclosed NVs, while the high-density fractions were rich in rod-shaped objects. Mass spectrometry–based proteomic analysis identified capsid proteins of tomato brown rugose fruit virus, tomato mosaic virus and tomato mottle mosaic virus. In another batch of tomatoes, we isolated tomato spotted wilt virus, potato virus Y and southern tomato virus in the vesicle sample. Our results show the frequent co-isolation of plant viruses with NVs and the utility of the combination of cryo-TEM, SEM and proteomics in the detection of possible viral contamination.  

3.3926           Exosomes as Naturally Occurring Vehicles for Delivery of Biopharmaceuticals: Insights from Drug Delivery to Clinical Perspectives

Butreddy, A., Kommineni, N. and Dudhipala, N. Nanomaterials, 11:1481 (2021)   Exosomes as nanosized vesicles are emerging as drug delivery systems for therapeutics owing to their natural origin, their ability to mediate intercellular communication, and their potential to encapsulate various biological molecules such as proteins and nucleic acids within the lipid bilayer membrane or in the lumen. Exosomes contain endogenous components (proteins, lipids, RNA) that could be used to deliver cargoes to target cells, offering an opportunity to diagnose and treat various diseases. Owing to their ability to travel safely in extracellular fluid and to transport cargoes to target cells with high efficacy, exosomes offer enhanced delivery of cargoes in vivo. However, several challenges related to the stabilization of the exosomes, the production of sufficient amounts of exosomes with safety and efficacy, the efficient loading of drugs into exosomes, the clearance of exosomes from circulation, and the transition from the bench scale to clinical production may limit their development and clinical use. For the clinical use of exosomes, it is important to understand the molecular mechanisms behind the transport and function of exosome vesicles. This review exploits techniques related to the isolation and characterization of exosomes and their drug delivery potential to enhance the therapeutic outcome and stabilization methods. Further, routes of administration, clinical trials, and regulatory aspects of exosomes will be discussed in this review.  

3.3927           Combining Augmented Radiotherapy and Immunotherapy through a Nano-Gold and Bacterial Outer-Membrane Vesicle Complex for the Treatment of Glioblastoma

Chen, M-H., Liu, T-Y., Chen, Y-C. and Chen, M-H. Nanomaterials, 11:1661 (2021)   Glioblastoma, formerly known as glioblastoma multiforme (GBM), is refractory to existing adjuvant chemotherapy and radiotherapy. We successfully synthesized a complex, Au–OMV, with two specific nanoparticles: gold nanoparticles (AuNPs) and outer-membrane vesicles (OMVs) from E. coli. Au–OMV, when combined with radiotherapy, produced radiosensitizing and immuno-modulatory effects that successfully suppressed tumor growth in both subcutaneous G261 tumor-bearing and in situ (brain) tumor-bearing C57BL/6 mice. Longer survival was also noted with in situ tumor-bearing mice treated with Au–OMV and radiotherapy. The mechanisms for the successful treatment were evaluated. Intracellular reactive oxygen species (ROS) greatly increased in response to Au–OMV in combination with radiotherapy in G261 glioma cells. Furthermore, with a co-culture of G261 glioma cells and RAW 264.7 macrophages, we found that GL261 cell viability was related to chemotaxis of macrophages and TNF-α production.  

3.3928           Periodontal and Dental Pulp Cell-Derived Small Extracellular Vesicles: A Review of the Current Status

Hua, S., Bartold, P.M., Gulati, K., Moran, C.S., Ivanovski, S. and Han, P. Nanomaterials, 11:1858 (2021)   Extracellular vesicles (EVs) are membrane-bound lipid particles that are secreted by all cell types and function as cell-to-cell communicators through their cargos of protein, nucleic acid, lipids, and metabolites, which are derived from their parent cells. There is limited information on the isolation and the emerging therapeutic role of periodontal and dental pulp cell-derived small EVs (sEVs, <200 nm, or exosome). In this review, we discuss the biogenesis of three EV subtypes (sEVs, microvesicles and apoptotic bodies) and the emerging role of sEVs from periodontal ligament (stem) cells, gingival fibroblasts (or gingival mesenchymal stem cells) and dental pulp cells, and their therapeutic potential in vitro and in vivo. A review of the relevant methodology found that precipitation-based kits and ultracentrifugation are the two most common methods to isolate periodontal (dental pulp) cell sEVs. Periodontal (and pulp) cell sEVs range in size, from 40 nm to 2 μm, due to a lack of standardized isolation protocols. Nevertheless, our review found that these EVs possess anti-inflammatory, osteo/odontogenic, angiogenic and immunomodulatory functions in vitro and in vivo, via reported EV cargos of EV–miRNAs, EV–circRNAs, EV–mRNAs and EV–lncRNAs. This review highlights the considerable therapeutic potential of periodontal and dental pulp cell-derived sEVs in various regenerative applications.  

3.3929           Cancer Cells Shuttle Extracellular Vesicles Containing Oncogenic Mutant p53 Proteins to the Tumor Microenvironment

Bhatta, B., Luz, I., Krueger, C., Teo, F.X., lane, D.P. and Sabapathy, K. Cancers, 13:2985 (2021)   Extracellular vesicles (EVs) shed by cancer cells play a major role in mediating the transfer of molecular information by reprogramming the tumor microenvironment (TME). TP53 (encoding the p53 protein) is the most mutated gene across many cancer types. Mutations in TP53 not only result in the loss of its tumor-suppressive properties but also results in the acquisition of novel gain-of-functions (GOF) that promote the growth of cancer cells. Here, we demonstrate that GOF mutant p53 proteins can be transferred via EVs to neighboring cancer cells and to macrophages, thus modulating them to release tumor supportive cytokines. Our data from pancreatic, lung, and colon carcinoma cell lines demonstrate that the mutant p53 protein can be selectively sorted into EVs. More specifically, mutant p53 proteins in EVs can be taken up by neighboring cells and mutant p53 expression is found in non-tumor cells in both human cancers and in non-human tissues in human xenografts. Our findings shed light on the intricate methods in which specific GOF p53 mutants can promote oncogenic mechanisms by reprogramming and then recruiting non-cancerous elements for tumor progression.  

3.3930           Prognostic and Predictive Biomarkers in Non-Small Cell Lung Cancer Patients on Immunotherapy—The Role of Liquid Biopsy in Unraveling the Puzzle

Augustus, E., Zwaenpoel, K., Siozopoulou, V., Raskin, J., Jordaens, S., Baggerman, G., Sorber, l., Roeyen, G., Peeters, m. and Pauwels, P. Cancers, 13:1675 (2021)   In the last decade, immunotherapy has been one of the most important advances in the non-small cell lung cancer (NSCLC) treatment landscape. Nevertheless, only a subset of NSCLC patients benefits from it. Currently, the only Food and Drug Administration (FDA) approved diagnostic test for first-line immunotherapy in metastatic NSCLC patients uses tissue biopsies to determine the programmed death ligand 1 (PD-L1) status. However, obtaining tumor tissue is not always feasible and puts the patient at risk. Liquid biopsy, which refers to the tumor-derived material present in body fluids, offers an alternative approach. This less invasive technique gives real-time information on the tumor characteristics. This review addresses different promising liquid biopsy based biomarkers in NSCLC patients that enable the selection of patients who benefit from immunotherapy and the monitoring of patients during this therapy. The challenges and the opportunities of blood-based biomarkers such as cell-free DNA (cfDNA), circulating tumor cells (CTCs), exosomes, epigenetic signatures, microRNAs (miRNAs) and the T cell repertoire will be addressed. This review also focuses on the less-studied feces-based and breath-based biomarkers.  

3.3931           The C-Circle Biomarker Is Secreted by Alternative-Lengthening-of-Telomeres Positive Cancer Cells inside Exosomes and Provides a Blood-Based Diagnostic for ALT Activity

Chen, Y-Y., Dagg, R., Zhang, Y., Lee, J.H.Y., Lu, R., La Rotta, N.M., Sampl, S., Korkut-Demirbas, M., Holzmann, K., Lau, L.M.S., Reddel, R.R. and Henson, J.D: Cancers, 13:5369 (2021)   C-Circles, self-primed telomeric C-strand templates for rolling circle amplification, are the only known alternative-lengthening-of-telomeres (ALT)-specific molecule. However, little is known about the biology of C-Circles and if they may be clinically useful. Here we show that C-Circles are secreted by ALT+ cancer cells inside exosomes, and that a blood-based C-Circle Assay (CCA) can provide an accurate diagnostic for ALT activity. Extracellular vesicles were isolated by differential centrifugation from the growth media of lung adenocarcinoma, glioblastoma, neuroblastoma, osteosarcoma, and soft tissue sarcoma cell lines, and C-Circles were detected in the exosome fraction from all eleven ALT+ cancer cell lines and not in any extracellular fraction from the eight matching telomerase positive cancer cell lines or the normal fibroblast strain. The existence of C-Circles in ALT+ exosomes was confirmed with exosomes isolated by iodixanol gradient separation and CD81-immunoprecipitation, and C-Circles in the exosomes were protected from nucleases. On average, 0.4% of the total ALT+ intracellular C-Circles were secreted in the exosomes every 24 h. Comparing the serum-based and tumor-based CCAs in 35 high risk neuroblastoma patients divided randomly into ALT+ threshold derivation and validation groups, we found the serum-based CCA to have 100% sensitivity (6/6), 70% specificity (7/10), and 81% concordance (13/16). We conclude that the secretion of C-Circles by ALT+ cancer cells in the exosomes provides a stable blood-based biomarker and a potential clinical diagnostic for ALT activity.  

3.3932           The Role of Copper in the Regulation of Ferroportin Expression in Macrophages

Jonczy, A., Mazgaj, R., Smuda, E., Zelazowska, B., Kopec, Z., Starzynski, R.R. and Lipinski, P. Cells, 10:2259 (2021)   The critical function of ferroportin (Fpn) in maintaining iron homeostasis requires complex and multilevel control of its expression. Besides iron-dependent cellular and systemic control of Fpn expression, other metals also seem to be involved in regulating the Fpn gene. Here, we found that copper loading significantly enhanced Fpn transcription in an Nrf2-dependent manner in primary bone-marrow-derived macrophages (BMDMs). However, prolonged copper loading resulted in decreased Fpn protein abundance. Moreover, CuCl₂ treatment induced Fpn expression in RAW 264.7 macrophages at both the mRNA and protein level. These data suggest that cell-type-specific regulations have an impact on Fpn protein stability after copper loading. Transcriptional suppression of Fpn after lipopolysaccharide (LPS) treatment contributes to increased iron storage inside macrophages and may result in anemia of inflammation. Here, we observed that in both primary BMDMs and RAW 264.7 macrophages, LPS treatment significantly decreased Fpn mRNA levels, but concomitant CuCl₂ stimulation counteracted the transcriptional suppression of Fpn and restored its expression to the control level. Overall, we show that copper loading significantly enhances Fpn transcription in macrophages, while Fpn protein abundance in response to CuCl₂ treatment, depending on macrophage type and factors specific to the macrophage population, can influence Fpn regulation in response to copper loading.  

3.3933           Differential Expression Pattern of Goat Uterine Fluids Extracellular Vesicles miRNAs during Peri-Implantation

Xie, Y., Liu, G., Zang, X., Hu, Q., Zhou, C., Li, Y., Liu, D. and Hong, L. Cells, 10:2308 (2021)   Early pregnancy failure occurs when a mature embryo attaches to an unreceptive endometrium. During the formation of a receptive endometrium, extracellular vesicles (EVs) of the uterine fluids (UFs) deliver regulatory molecules such as small RNAs to mediate intrauterine communication between the embryo and the endometrium. However, profiling of small RNAs in goat UFs’ EVs during pregnancy recognition (day 16) has not been carried out. In this study, EVs were isolated from UFs on day 16 of the estrous cycle or gestation. They were isolated by Optiprep™ Density G radient (ODG) and verified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. Immunostaining demonstrated that CD63 was present both in the endometrial epithelium and glandular epithelium, and stain intensity was greater in the pregnant endometrium compared to the non-pregnant endometrium. Small RNA sequencing revealed that UFs’ EVs contained numerous sRNA families and a total of 106 differentially expressed miRNAs (DEMs). Additionally, 1867 target genes of the DEMs were obtained, and miRNA–mRNA interaction networks were constructed. GO and KEGG analysis showed that miRNAs were significantly associated with the formation of a receptive endometrium and embryo implantation. In addition, the fluorescence in situ hybridization assay (FISH) showed that chi-miR-451-5p was mainly expressed in stromal cells of the endometrium and a higher level was detected in the endometrial luminal epithelium in pregnant states. Moreover, the dual-luciferase reporter assay showed that chi-miR-451-5p directly binds to PSMB8 and may play an important role in the formation of a receptive endometrium and embryo implantation. In conclusion, these results reveal that UFs’ EVs contain various small RNAs that may be vital in the formation of a receptive endometrium and embryo implantation.  

3.3934           Viperin, an IFN-Stimulated Protein, Delays Rotavirus Release by Inhibiting Non-Structural Protein 4 (NSP4)-Induced Intrinsic Apoptosis

Sarkar, R., Nandi, S., Lo, M., Gope, A. and Chawla-Sarkar, M. Viruses, 13:1324 (2021)   Viral infections lead to expeditious activation of the host’s innate immune responses, most importantly the interferon (IFN) response, which manifests a network of interferon-stimulated genes (ISGs) that constrain escalating virus replication by fashioning an ill-disposed environment. Interestingly, most viruses, including rotavirus, have evolved numerous strategies to evade or subvert host immune responses to establish successful infection. Several studies have documented the induction of ISGs during rotavirus infection. In this study, we evaluated the induction and antiviral potential of viperin, an ISG, during rotavirus infection. We observed that rotavirus infection, in a stain independent manner, resulted in progressive upregulation of viperin at increasing time points post-infection. Knockdown of viperin had no significant consequence on the production of total infectious virus particles. Interestingly, substantial escalation in progeny virus release was observed upon viperin knockdown, suggesting the antagonistic role of viperin in rotavirus release. Subsequent studies unveiled that RV-NSP4 triggered relocalization of viperin from the ER, the normal residence of viperin, to mitochondria during infection. Furthermore, mitochondrial translocation of NSP4 was found to be impeded by viperin, leading to abridged cytosolic release of Cyt c and subsequent inhibition of intrinsic apoptosis. Additionally, co-immunoprecipitation studies revealed that viperin associated with NSP4 through regions including both its radical SAM domain and its C-terminal domain. Collectively, the present study demonstrated the role of viperin in restricting rotavirus egress from infected host cells by modulating NSP4 mediated apoptosis, highlighting a novel mechanism behind viperin’s antiviral action in addition to the intricacy of viperin–virus interaction.  

3.3935           Cell-Secreted Vesicles: Novel Opportunities in Cancer Diagnosis, Monitoring and Treatment

Catoni, C., Di Paolo, V., Rossi, E., Quintieri, L. and Zamarchi, R. Diagnostics, 11:1118 (2021)   Extracellular vesicles (EVs) are important mediators of intercellular communication playing a pivotal role in the regulation of physiological and pathological processes, including cancer. In particular, there is significant evidence suggesting that tumor-derived EVs exert an immunosuppressive activity during cancer progression, as well as stimulate tumor cell migration, angiogenesis, invasion and metastasis. The use of EVs as a liquid biopsy is currently a fast-growing area of research in medicine, with the potential to provide a step-change in the diagnosis and treatment of cancer, allowing the prediction of both therapy response and prognosis. EVs could be useful not only as biomarkers but also as drug delivery systems, and may represent a target for anticancer therapy. In this review, we attempted to summarize the current knowledge about the techniques used for the isolation of EVs and their roles in cancer biology, as liquid biopsy biomarkers and as therapeutic tools and targets  

3.3936           Cathepsin B Localizes in the Caveolae and Participates in the Proteolytic Cascade in Trabecular Meshwork Cells. Potential New Drug Target for the Treatment of Glaucoma

Nettesheim, A., Shim, M.S:, Dixon, A., Raychaudhuri, U., Gong, h. and Liton, P.B.

1. Clin. Med., 10:78 (2021)

  Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTB^ko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFβ/Smad signaling in TM cells vitro and in vivo in CTSB^ko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.  

3.3937           Topical Administration of Melatonin-Loaded Extracellular Vesicle-Mimetic Nanovesicles Improves 2,4-Dinitrofluorobenzene-Induced Atopic Dermatitis

Kim, Y.S., Go, G., Yun, C-W., Yea, J-H., Yoon, S., Han, S-Y., Lee, G., Lee, M-Y. and Lee, S.H. Biomolecules, 11:1450 (2021)   Atopic dermatitis (AD) is caused by multiple factors that trigger chronic skin inflammation, including a defective skin barrier, immune cell activation, and microbial exposure. Although melatonin has an excellent biosafety profile and a potential to treat AD, there is limited clinical evidence from controlled trials that support the use of melatonin as an AD treatment. The delivery of melatonin via the transdermal delivery system is also a challenge in designing melatonin-based AD treatments. In this study, we generated melatonin-loaded extracellular vesicle-mimetic nanoparticles (^MelaNVs) to improve the transdermal delivery of melatonin and to evaluate their therapeutic potential in AD. The ^MelaNVs were spherical nanoparticles with an average size of 100 nm, which is the optimal size for the transdermal delivery of drugs. ^MelaNVs showed anti-inflammatory effects by suppressing the release of TNF-α and β-hexosaminidase in LPS-treated RAW264.7 cells and compound 48/80-treated RBL-2H3 cells, respectively. ^MelaNVs showed a superior suppressive effect compared to an equivalent concentration of free melatonin. Treating a 2,4-dinitrofluorobenzene (DNCB)-induced AD-like mouse model with ^MelaNVs improved AD by suppressing local inflammation, mast cell infiltration, and fibrosis. In addition, ^MelaNVs effectively suppressed serum IgE levels and regulated serum IFN-γ and IL-4 levels. Taken together, these results suggest that ^MelaNVs are novel and efficient transdermal delivery systems of melatonin and that ^MelaNVs can be used as a treatment to improve AD.  

3.3938           Bacterial Membrane-Derived Vesicles Attenuate Vancomycin Activity against Methicillin-Resistant Staphylococcus aureus

Kumaraswamy, M., Will, K., Joshi, B., Sakoulas, G., Koushaa, A., Vaaje-Kolstad, G., Johannessen, M., Hegstad, K., Nizet, V. and Askarian, F. Microorganisms, 9:2055 (2021)   Methicillin-resistant Staphylococcus aureus (MRSA) has evolved numerous antimicrobial resistance mechanisms and is identified as a serious public health threat by the World Health Organization and U.S. Centers for Disease Control and Prevention. The glycopeptide vancomycin (VAN) remains a cornerstone of therapy for severe MRSA infections despite increasing reports of therapeutic failure in hospitalized patients with bacteremia or pneumonia. Recently, the role of released bacterial-derived membrane vesicles (MVs) in antibiotic resistance has garnered attention. Here we examined the effect of exogenous MRSA-derived MVs on VAN activity against MRSA in vitro, using minimum inhibitory concentration and checkerboard assays, and ex vivo, incorporating components of host innate immunity such as neutrophils and serum complement present in blood. Additionally, the proteome of MVs from VAN-exposed MRSA was characterized to determine if protein expression was altered. The presence of MVs increased the VAN MIC against MRSA to values where clinical failure is commonly observed. Furthermore, the presence of MVs increased survival of MRSA pre-treated with sub-MIC concentrations of VAN in whole blood and upon exposure to human neutrophils but not human serum. Unbiased proteomic analysis also showed an elevated expression of MV proteins associated with antibiotic resistance (e.g., marR) or proteins that are functionally linked to cell membrane/wall metabolism. Together, our findings indicate MRSA-derived MVs are capable of lowering susceptibility of the pathogen to VAN, whole-blood- and neutrophil-mediated killing, a new pharmacodynamic consideration for a drug increasingly linked to clinical treatment failures    

3.3939           Alcohol-and-HIV-Induced Lysosomal Dysfunction Regulates Extracellular Vesicles Secretion in Vitro and in Liver-Humanized Mice

Dagur, R.S., New-Aaron, M., Ganesan, M., Wang, W., Romanova, S., Kidambi, S., Kharbanda, K.K., Poluektova, L.Y. and Osna, N.A. Biology, 10:29 (2021)   Background: Alcohol abuse is common in people living with HIV-1 and dramatically enhances the severity of HIV-induced liver damage by inducing oxidative stress and lysosomal dysfunction in the liver cells. We hypothesize that the increased release of extracellular vesicles (EVs) in hepatocytes and liver humanized mouse model is linked to lysosome dysfunction. Methods: The study was performed on primary human hepatocytes and human hepatoma RLWXP-GFP (Huh 7.5 cells stably transfected with CYP2E1 and XPack-GFP) cells and validated on ethanol-fed liverhumanized fumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chain knockout (FRG-KO) mice. Cells and mice were infected with HIV-1ADA virus. Results: We observed an increase in the secretion of EVs associated with a decrease in lysosomal activity and expression of lysosomal-associated membrane protein 1. Next-generation RNA sequencing of primary human hepatocytes revealed 63 differentially expressed genes, with 13 downregulated and 50 upregulated genes in the alcohol–HIV-treated group. Upstream regulator analysis of differentially expressed genes through Ingenuity Pathway Analysis identified transcriptional regulators affecting downstream genes associated with increased oxidative stress, lysosomal associated disease, and function and EVs biogenesis. Our in vitro findings were corroborated by in vivo studies on human hepatocytetransplanted humanized mice, indicating that intensive EVs’ generation by human hepatocytes and their secretion to serum was associated with increased oxidative stress and reduction in lysosomal activities triggered by HIV infection and ethanol diet. Conclusion: HIV-and-ethanol-metabolisminduced EVs release is tightly controlled by lysosome status in hepatocytes and participates in the development of double-insult-induced liver injury.  

3.3940           Velocity Gradient Separation Reveals a New Extracellular Vesicle Population Enriched in miR-155 and Mitochondrial DNA

Vaillancourt, M., Hubert, A., Subra, C., Boucher, j., Bazie, W.W. et al Pathogens, 10:526 (2021)   Extracellular vesicles (EVs) and their contents (proteins, lipids, messenger RNA, microRNA, and DNA) are viewed as intercellular signals, cell-transforming agents, and shelters for viruses that allow both diagnostic and therapeutic interventions. EVs circulating in the blood of individuals infected with human immunodeficiency virus (HIV-1) may provide insights into pathogenesis, inflammation, and disease progression. However, distinguishing plasma membrane EVs from exosomes, exomeres, apoptotic bodies, virions, and contaminating proteins remains challenging. We aimed at comparing sucrose and iodixanol density and velocity gradients along with commercial kits as a means of separating EVs from HIV particles and contaminating protein like calprotectin; and thereby evaluating the suitability of current plasma EVs analysis techniques for identifying new biomarkers of HIV-1 immune activation. Multiple analysis have been performed on HIV-1 infected cell lines, plasma from HIV-1 patients, or plasma from HIV-negative individuals spiked with HIV-1. Commercial kits, the differential centrifugation and density or velocity gradients to precipitate and separate HIV, EVs, and proteins such as calprotectin, have been used. EVs, virions, and contaminating proteins were characterized using Western blot, ELISA, RT-PCR, hydrodynamic size measurement, and enzymatic assay. Conversely to iodixanol density or velocity gradient, protein and virions co-sedimented in the same fractions of the sucrose density gradient than AChE-positive EVs. Iodixanol velocity gradient provided the optimal separation of EVs from viruses and free proteins in culture supernatants and plasma samples from a person living with HIV (PLWH) or a control and revealed a new population of large EVs enriched in microRNA miR-155 and mitochondrial DNA. Although EVs and their contents provide helpful information about several key events in HIV-1 pathogenesis, their purification and extensive characterization by velocity gradient must be investigated thoroughly before further use as biomarkers. By revealing a new population of EVs enriched in miR-155 and mitochondrial DNA, this study paves a way to increase our understanding of HIV-1 pathogenesis.    

3.3941           Standardized Methodologies to Utilize Exosome Treatment as Potential Nano Substances in Hearing Loss

Park, D.J.

1. Otorchinolaryngol. Hear Balance Med., 2:6 (2021)

  Recently, studies on the mechanism and clinical application of stem cell-derived exosomes have increased. Although the number of patients with hearing loss is increasing, there is no ideal therapy for the recovery of auditory cells of an independent organ in humans. In this review, we proposed the use of stem cell-derived exosomes for treating hearing loss and summarized the exosome research strategy platform for preclinical studies. It is necessary to select a research direction to assess direct or indirect effects on recipients based on the physiological mechanisms of exosomes that deliver useful molecules (called payloads) to recipient cells or tissues. To apply exosomes in the auditory field, researchers should select a model for assessing the toxicity to the auditory cells and analyzing their mechanisms in the recipient tissue. Such in vitro, ex vivo, and in vivo models have been designed and reported in previous studies. The analytical strategies in various models can evaluate the mechanism of exosomes based on exosome surface markers or the payload, thus helping the researchers in finding evidence regarding the efficacy of exosomes. Here, we propose three strategies for exosome application research in the auditory field.  

3.3942           Diversified transcriptional responses of myeloid and glial cells in spinal cord injury shaped by HDAC3 activity

Wahane, S., Zhou, X., Zhou, X., Friedl, M-S., Kluge, N., Ramakrishnan, A., Shen, L., Friedel, C.C., Zhang, B., Friedel, R.H. and Zou, H. Sci. Adv., 7, eabd8811 (2021)   The innate immune response influences neural repair after spinal cord injury (SCI). Here, we combined myeloid-specific transcriptomics and single-cell RNA sequencing to uncover not only a common core but also temporally distinct gene programs in injury-activated microglia and macrophages (IAM). Intriguingly, we detected a wide range of microglial cell states even in healthy spinal cord. Upon injury, IAM progressively acquired overall reparative, yet diversified transcriptional profiles, each comprising four transcriptional subtypes with specialized tasks. Notably, IAM have both distinct and common gene signatures as compared to neurodegeneration-associated microglia, both engaging phagocytosis, autophagy, and TyroBP pathways. We also identified an immediate response microglia subtype serving as a source population for microglial transformation and a proliferative subtype controlled by the epigenetic regulator histone deacetylase 3 (HDAC3). Together, our data unveil diversification of myeloid and glial subtypes in SCI and an extensive influence of HDAC3, which may be exploited to enhance functional recovery.  

3.3943           Plant-derived exosome-like nanoparticles: A concise review on its extraction methods, content, bioactivities, and potential as functional food ingredient

Suharta, S., barlian, A., Hidajah, A.C., Notobroto, H.B., Ana, I.D., Indariani, S., WUngu, T.D.K. and Wijaya, C.H. Food Science, 86(7), 2838-2850 (2021)   Plant-derived exosome-like nanoparticles (PDENs) are small vesicles released by multivesicular bodies mainly to communicate between cells and regulate immunity against pathogen attack. Current studies have reported that PDENs could modulate gene expression in a cross-kingdom fashion. Therefore, PDENs could be a potential future functional food ingredient as their cross-kingdom communication abilities were reported to exert multiple health benefits. Macrophage and other cells have been reported to absorb PDENs in a manner regulated by the membrane lipid and protein profile and the intactness of the PDENs lipid bilayer. PDENs could be extracted from plant materials by various techniques such as ultracentrifugation, immunoaffinity, size-based isolation, and precipitation, though each method has its pros and cons. PDENs mainly contain lipid, protein, and genetic materials, mainly micro RNAs, which could exert multiple health benefits and functionalities when consumed in sufficient amounts. However, most studies on the health functionalities of PDENs were conducted through in-vitro and in-vivo studies, and its potency to be used as a functional ingredient remains a question as PDENs are sensitive to storage and processing condition and requires costly extraction method. This concise review features various exosome extraction methods, contents of PDENs and their roles, the health functionalities of PDENs, and its potency as a functional food ingredient.  

3.3944           Mammalian hybrid pre-autophagosomal structure HyPAS generates autophagosomes

Kumar, S., Javed, R., Mudd, M., Timmins, G.S., Eskelinen, E-L. and Deretic, V. Cell, 184, 5950-5969 (2021)   The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.  

3.3945           Prolonged epigenomic and synaptic plasticity alterations following single exposure to a psychedelic in mice

De la Fuente Revenga, M., Zhu, B., Guevara, C.A., Huntley, G.W., Lu, C. and Gonzalez-Maeso, J. Cell Reports, 37, 109836 (2021)   Clinical evidence suggests that rapid and sustained antidepressant action can be attained with a single exposure to psychedelics. However, the biological substrates and key mediators of psychedelics’ enduring action remain unknown. Here, we show that a single administration of the psychedelic DOI produces fast-acting effects on frontal cortex dendritic spine structure and acceleration of fear extinction via the 5-HT_2A receptor. Additionally, a single dose of DOI leads to changes in chromatin organization, particularly at enhancer regions of genes involved in synaptic assembly that stretch for days after the psychedelic exposure. These DOI-induced alterations in the neuronal epigenome overlap with genetic loci associated with schizophrenia, depression, and attention deficit hyperactivity disorder. Together, these data support that epigenomic-driven changes in synaptic plasticity sustain psychedelics’ long-lasting antidepressant action but also warn about potential substrate overlap with genetic risks for certain psychiatric conditions.  

3.3946           Engineered extracellular vesicle mimetics from macrophage promotes hair growth in mice and promotes human hair follicle growth

Rajkendran, R.L., Gangadaran, P., Kwack, M.H., Oh, J.M., Hong, C.M., Gopal, A., Sung, Y.K., Lee, J. and Ahn, B-C. Exp. Cell Res., 409, 112887 (2021)   Recent studies clearly show that cell-derived extracellular vesicles (EVs, including exosomes) can promote hair growth. However, large-scale production of EVs remains a big hurdle. Recently, extracellular vesicle mimetics (EMs) engineered by extrusion through various membranes are emerging as a complementary approach for large-scale production. In this study, to investigate their ability to induce hair growth, we generated macrophage-engineered EMs (MAC-EMs) that activated the human dermal papilla (DP) cells in vitro. MAC-EMs intradermally injected into the skin of C57BL/6 mice were retained for up to 72 h. Microscopy imaging revealed that MAC-EMs were predominately internalized into hair follicles. The MAC-EMs treatment induced hair regrowth in mice and hair shaft elongation in a human hair follicle, suggesting the potential of MAC-EMs as an alternative to EVs to overcome clinical limitation.  

3.3947           Wild type, dEX3 and 2B survivin isoforms localize to the tumor cell plasma membrane, are secreted in exosomes, and interact with extracellular tubulin

Figel, S., Birkemeier, M., Dharma, S.S., Barone, T., Steinmetz, E., Ciesielski, M. and Fenstermaker, R. Biochem. Biophys. Reports, 28, 101174 (2021)   The Inhibitor of Apoptosis Protein survivin (svn) is upregulated in nearly all types of cancer and represents a promising therapeutic target. Localization to specific subcellular compartments and interactions with various binding partners allow survivin to play diverse roles in apoptosis resistance and mitosis. Survivin has recently been found in two extracellular compartments: the outer plasma membrane and secreted exosomes. In addition to svn-wt, splice variants svn-dEX3 and svn-2B are also overexpressed in human tumors. Here we show that, similarly to svn-wt, svn-dEX3 and svn-2B can be displayed on the outer plasma membrane, and secreted in exosomes. Additionally, we have identified a novel interaction of all three forms of survivin with secreted tubulin.  

3.3948           The membrane associated accessory protein is an adeno-associated viral egress factor

Elmore, Z.C., havlik, L.P., Oh, D.K., Anderson, L., Daaboul, G., Devlin, G.W., Vincent, H.A: and Asokan, A. Nature Comm., 12:6239 (2021)   Adeno-associated viruses (AAV) rely on helper viruses to transition from latency to lytic infection. Some AAV serotypes are secreted in a pre-lytic manner as free or extracellular vesicle (EV)-associated particles, although mechanisms underlying such are unknown. Here, we discover that the membrane-associated accessory protein (MAAP), expressed from a frameshifted open reading frame in the AAV cap gene, is a novel viral egress factor. MAAP contains a highly conserved, cationic amphipathic domain critical for AAV secretion. Wild type or recombinant AAV with a mutated MAAP start site (MAAPΔ) show markedly attenuated secretion and correspondingly, increased intracellular retention. Trans-complementation with MAAP restored secretion of multiple AAV/MAAPΔ serotypes. Further, multiple processing and analytical methods corroborate that one plausible mechanism by which MAAP promotes viral egress is through AAV/EV association. In addition to characterizing a novel viral egress factor, we highlight a prospective engineering platform to modulate secretion of AAV vectors or other EV-associated cargo.  

3.3949           Single-molecule imaging with cell-derived nanovesicles reveals early binding dynamics at a cyclic nucleotide-gated ion channel

Patel, V.R., Salinas, A.M., Qi, D., Gupta, S., Sidote, D.J. and Goldschen-Ohm, M.P: Nature Comm., 12:6459 (2021)   Ligand binding to membrane proteins is critical for many biological signaling processes. However, individual binding events are rarely directly observed, and their asynchronous dynamics are occluded in ensemble-averaged measures. For membrane proteins, single-molecule approaches that resolve these dynamics are challenged by dysfunction in non-native lipid environments, lack of access to intracellular sites, and costly sample preparation. Here, we introduce an approach combining cell-derived nanovesicles, microfluidics, and single-molecule fluorescence colocalization microscopy to track individual binding events at a cyclic nucleotide-gated TAX-4 ion channel critical for sensory transduction. Our observations reveal dynamics of both nucleotide binding and a subsequent conformational change likely preceding pore opening. Kinetic modeling suggests that binding of the second ligand is either independent of the first ligand or exhibits up to ~10-fold positive binding cooperativity. This approach is broadly applicable to studies of binding dynamics for proteins with extracellular or intracellular domains in native cell membrane.  

3.3950           Epigenetic interaction between UTX and DNMT1 regulates diet-induced myogenic remodeling in brown fat

Li, F.,Jing, J., Movahed, M., Cui, X., Co, Q., Wu, R., Chen, Z., Yu, L., Pan, Y., Shi, H., Shi, H. and Xue, B. Nature Comm., 12:6838 (2021)   Brown adipocytes share the same developmental origin with skeletal muscle. Here we find that a brown adipocyte-to-myocyte remodeling also exists in mature brown adipocytes, and is induced by prolonged high fat diet (HFD) feeding, leading to brown fat dysfunction. This process is regulated by the interaction of epigenetic pathways involving histone and DNA methylation. In mature brown adipocytes, the histone demethylase UTX maintains persistent demethylation of the repressive mark H3K27me3 at Prdm16 promoter, leading to high Prdm16 expression. PRDM16 then recruits DNA methyltransferase DNMT1 to Myod1 promoter, causing Myod1 promoter hypermethylation and suppressing its expression. The interaction between PRDM16 and DNMT1 coordinately serves to maintain brown adipocyte identity while repressing myogenic remodeling in mature brown adipocytes, thus promoting their active brown adipocyte thermogenic function. Suppressing this interaction by HFD feeding induces brown adipocyte-to-myocyte remodeling, which limits brown adipocyte thermogenic capacity and compromises diet-induced thermogenesis, leading to the development of obesity.      

3.3951           Cilia locally synthesize proteins to sustain their ultrastructure and functions

Hao, K., Chen, Y., Yan, X and Zhu, X. Nature Comm., 12:6971 (2021)   Cilia are microtubule-based hair-like organelles propelling locomotion and extracellular liquid flow or sensing environmental stimuli. As cilia are diffusion barrier-gated subcellular compartments, their protein components are thought to come from the cell body through intraflagellar transport or diffusion. Here we show that cilia locally synthesize proteins to maintain their structure and functions. Multicilia of mouse ependymal cells are abundant in ribosomal proteins, translation initiation factors, and RNA, including 18 S rRNA and tubulin mRNA. The cilia actively generate nascent peptides, including those of tubulin. mRNA-binding protein Fmrp localizes in ciliary central lumen and appears to function in mRNA delivery into the cilia. Its depletion by RNAi impairs ciliary local translation and induces multicilia degeneration. Expression of exogenous Fmrp, but not an isoform tethered to mitochondria, rescues the degeneration defects. Therefore, local translation defects in cilia might contribute to the pathology of ciliopathies and other diseases such as Fragile X syndrome.  

3.3952           CircNPHP4 in monocyte-derived small extracellular vesicles controls heterogeneous adhesion in coronary heart atherosclerotic disease

Xiong, F., Mao, R., Zhang, L., Zhao, R., Tan, K., Liu, C., Xu, J., Du, G. and Zhang, T. Cell Death and Disease, 12:948 (2021)   Small extracellular vesicles (sEVs)-derived circular RNAs (circRNAs) could regulate gene expression in recipient cells, and dysregulation of sEVs-derived circRNAs has been implicated in several diseases. However, the expression and function of sEVs-derived circRNAs in coronary heart atherosclerotic disease (CAD) remain unknown. In this study, we investigated global changes in the expression patterns of circRNAs in sEVs from CAD-related monocytes and identified circNPHP4 as a significantly upregulated circRNA. Knockdown of circNPHP4 inhibited heterogeneous adhesion between monocytes and coronary artery endothelial cells and reduced ICAM-1 and VCAM-1 expression. Investigations of the underlying mechanisms revealed that circNPHP4 contains a functional miR-1231-binding site. Mutation of the circNPHP4-binding sites in miR-1231 abolished the interaction, as indicated by a luciferase reporter assay. Furthermore, circNPHP4 affected the expression of miR-1231 and its target gene EGFR. Overexpression of miR-1231 blocked the inhibitory effect of circNPHP4 on heterogeneous adhesion. Moreover, downregulation of miR-1231 restored heterogeneous adhesion upon inhibition by circNPHP4 silencing. Additionally, circNPHP4 overexpression was correlated with aggressive clinicopathological characteristics in CAD patients. A multivariate logistic regression model and bootstrapping validation showed that circNPHP4 overexpression had a good risk prediction capability for CAD. The decision curve analysis revealed that using the CAD nomogram that included circNPHP4 overexpression to predict the risk of CAD was beneficial. Our results suggest that sEVs-derived circNPHP4 can serve as a potential target for CAD treatments or as a potential diagnostic marker for CAD patients.  

3.3953           Extracellular vesicles from hypoxia-preconditioned microglia promote angiogenesis and repress apoptosis in stroke mice via the TGF-β/Smad2/3 pathway

Zhang, L., Wei, W., Ai, X., Kilic, E., Hermann, D.M., Venkataramani, V., Bähr, M. and Doeppner, T.R. Cell Death and Disease, 12:1068 (2021)   Systemic transplantation of oxygen−glucose deprivation (OGD)-preconditioned primary microglia enhances neurological recovery in rodent stroke models, albeit the underlying mechanisms have not been sufficiently addressed. Herein, we analyzed whether or not extracellular vesicles (EVs) derived from such microglia are the biological mediators of these observations and which signaling pathways are involved in the process. Exposing bEnd.3 endothelial cells (ECs) and primary cortical neurons to OGD, the impact of EVs from OGD-preconditioned microglia on angiogenesis and neuronal apoptosis by the tube formation assay and TUNEL staining was assessed. Under these conditions, EV treatment stimulated both angiogenesis and tube formation in ECs and repressed neuronal cell injury. Characterizing microglia EVs by means of Western blot analysis and other techniques revealed these EVs to be rich in TGF-β1. The latter turned out to be a key compound for the therapeutic potential of microglia EVs, affecting the Smad2/3 pathway in both ECs and neurons. EV infusion in stroke mice confirmed the aforementioned in vitro results, demonstrating an activation of the TGF-β/Smad2/3 signaling pathway within the ischemic brain. Furthermore, enriched TGF-β1 in EVs secreted from OGD-preconditioned microglia stimulated M2 polarization of residing microglia within the ischemic cerebral environment, which may contribute to a regulation of an early inflammatory response in postischemic hemispheres. These observations are not only interesting from the mechanistic point of view but have an immediate therapeutic implication as well, since stroke mice treated with such EVs displayed a better functional recovery in the behavioral test analyses. Hence, the present findings suggest a new way of action of EVs derived from OGD-preconditioned microglia by regulating the TGF-β/Smad2/3 pathway in order to promote tissue regeneration and neurological recovery in stroke mice.  

3.3954           Formation of a protein corona on the surface of extracellular vesicles in blood plasma

Toth, E.A., Turiak, L., Visnovitz, T., Cserep, C., Mazlo, A., Sodar, B. et al

1. Extracell. Vesicles, 10, e12140 (2021)

  In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein ‘contamination’ of EV preparations and may add a new perspective to EV research.  

3.3955           Neddylation of Coro1a determines the fate of multivesicular bodies and biogenesis of extracellular vesicles

Fei, X., Li, Z., Yang, D., Kong, X., Lu, X. et al

1. Extracell. Vesicles, 10, e12153 (2021)

  Multivesicular bodies (MVBs) fuse with not only the plasma membranes to release extracellular vesicles (EVs) but also lysosomes for degradation. Rab7 participates in the lysosomal targeting of MVBs. However, the proteins on MVB that directly bind Rab7, causing MVB recruitment of Rab7 remain unidentified. Here, we show that Coro1a undergoes neddylation modification at K233 by TRIM4. Neddylated Coro1a is associated with the MVB membrane and facilitates MVB recruitment and activation of Rab7 by directly binding Rab7. Subsequently, MVBs are targeted to lysosomes for degradation in a Rab7-dependent manner, leading to reduced EV secretion. Furthermore, a decrease in neddylated Coro1a enhances the production of tumour EVs, thereby promoting tumour progression, indicating that neddylated Coro1a is an ideal target for the regulation of EV biogenesis. Altogether, our data identify a novel substrate of neddylation and reveal an unknown mechanism for MVB recruitment of Rab7, thus providing new insight into the regulation of EV biogenesis.  

3.3956           Extracellular vesicles derived from the periodontal pathogen Filifactor alocis induce systemic bone loss through Toll-like receptor 2

Kim, H.Y., Song, M-K., Gho, Y., Kim, H-H. and Choi, B-K.

1. Extracell. Vesicles, 10, e12157 (2021)

  Periodontitis is an inflammatory disease induced by local infection in tooth-supporting tissue. Periodontitis is associated with systemic bone diseases, but little is known about the mechanism of the causal effect of periodontitis on systemic bone resorption. Bacteria-derived extracellular vesicles (EVs) act as natural carriers of virulence factors that are responsible for systemic inflammation. In this study, we investigated the role of EVs derived from Filifactor alocis, a Gram-positive, anaerobic periodontal pathogen, in systemic bone loss and osteoclast differentiation. F. alocis EVs accumulated in the long bones of mice after intraperitoneal administration. These EVs induced proinflammatory cytokines, osteoclastogenesis, and bone resorption via Toll-like receptor 2 (TLR2). The phase separation of F. alocis EVs showed that amphiphilic molecules were responsible for the induced bone resorption and osteoclastogenesis. The osteoclastogenic effects of F. alocis EVs were reduced by lipoprotein lipase. Proteomic analysis of the amphiphilic molecules identified seven lipoproteins. Our results indicate that lipoprotein-like molecules in F. alocis EVs may contribute to systemic bone loss via TLR2.  

3.3957           Visualizing transfer of microbial biomolecules by outer membrane vesicles in microbe-host-communication in vivo

Bittel, M., Reichert, P., Sarfati, H., Dressel, A., Leikam, S. et al

1. Intracell. Vesicles, 10, e12159 (2021)

  The intestinal microbiota influences mammalian host physiology in health and disease locally in the gut but also in organs devoid of direct contact with bacteria such as the liver and brain. Extracellular vesicles (EVs) or outer membrane vesicles (OMVs) released by microbes are increasingly recognized for their potential role as biological shuttle systems for inter-kingdom communication. However, physiologically relevant evidence for the transfer of functional biomolecules from the intestinal microbiota to individual host cells by OMVs in vivo is scarce. By introducing Escherichia coli engineered to express Cre-recombinase (E. coli^Cre) into mice with a Rosa26.tdTomato-reporter background, we leveraged the Cre-LoxP system to report the transfer of bacterial OMVs to recipient cells in vivo. Colonizing the intestine of these mice with E. coli^Cre, resulted in Cre-recombinase induced fluorescent reporter gene-expression in cells along the intestinal epithelium, including intestinal stem cells as well as mucosal immune cells such as macrophages. Furthermore, even far beyond the gut, bacterial-derived Cre induced extended marker gene expression in a wide range of host tissues, including the heart, liver, kidney, spleen, and brain. Together, our findings provide a method and proof of principle that OMVs can serve as a biological shuttle system for the horizontal transfer of functional biomolecules between bacteria and mammalian host cells.  

3.3958           Proteomic dissection of large extracellular vesicle surfaceome unravels interactive surface platform

Rai, A., Fang, H., Claridge, B., Simpson, R. and Greening, D.W:

1. Extracell. Vesicles, 10, e12164 (2021)

  The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra- and extracellular signalling networks, dictates EVs’ capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L-EVs, 100–800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV-subtype that are distinct from small EVs (S-EVs, 30–150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density-gradient purified L-EVs (1.07–1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L-EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell-cell and cell-ECM interactions); and (ii) membrane surface-associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand-receptor analysis of L-EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L-EV proteome revealed enrichment for proteins belonging to COPI/II-coated ER/Golgi-derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L-EVs and S-EVs, our data reveals surfaceome heterogeneity between the two EV-subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L-EVs and molecular leads for future studies seeking to decipher L-EV heterogeneity and function.  

3.3959           Extracellular vesicles released by human retinal pigment epithelium mediate increased polarised secretion of drusen proteins in response to AMD stressors

Flores-Bellver, M., Mighty, J., Aparicio-Domingo, S., Li, K.V., Shi, C. et al

1. Extracell. Vesicles, 10, e12165 (2021)

  Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen-associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen-associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely-tuned mechanism regulating directional apical:basal sorting and secretion of drusen-associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE-derived EVs as a key source of drusen proteins and important contributors to drusen development and growth.  

3.3960           The promise of exosome applications in treating central nervous system diseases

Mattingly, J., Li, Y., Bihl, J. and Wang, J. CNS Neurosci. Ther., 27, 1437-1445 (2021)   Exosomes (EXs), a type of extracellular vesicles, are secreted from virtually all types of cells. EXs serve as cell-to-cell communicators by conveying proteins and nucleic acids with regulatory functions. Increasing evidence shows that EXs are implicated in the pathogenesis of central nervous system (CNS) diseases. Moreover, EXs have recently been highlighted as a new promising therapeutic strategy for in vivo delivery of nucleotides and drugs. Studies have revealed that infusion of EXs elicits beneficial effects on the CNS injury animal models. As compared to cell-based therapy, EXs-based therapy for CNS diseases has unique advantages, opening a new path for neurological medicine. In this review, we summarized the current state of knowledge of EXs, the roles and applications of EXs as a viable pathological biomarker, and EX-based therapy for CNS diseases.  

3.3961           Exogenous loading of miRNAs into small extracellular vesicles

De Abreu, R.C., Ramos, C.V., Becher, C., Lino, M., Jesus, C., da Costa martins, P.A., Martins, P.A.T., Moreno, M.J., Fernandes, H. and Ferreira, L.

1. Extracell. Vesicles, 10, e12111 (2021)

  Small extracellular vesicles (sEVs), through their natural ability to interact with biological membranes and exploit endogenous processing pathways to convey biological information, are quintessential for the delivery of therapeutically relevant compounds, such as microRNAs (miRNAs) and proteins. Here, we used a fluorescently-labelled miRNA to quantify the efficiency of different methods to modulate the cargo of sEVs. Our results showed that, compared with electroporation, heat shock, permeation by a detergent-based compound (saponin) or cholesterol-modification of the miRNA, Exo-Fect was the most efficient method with > 50% transfection efficiency. Furthermore, qRT-PCR data showed that, compared with native sEVs, Exo-Fect modulation led to a > 1000-fold upregulation of the miRNA of interest. Importantly, this upregulation was observed for sEVs isolated from multiple sources. The modulated sEVs were able to delivery miR-155-5p into a reporter cell line, confirming the successful delivery of the miRNA to the target cell and, more importantly, its functionality. Finally, we showed that the membrane of Exo-Fect-loaded sEVs was altered compared with native sEVs and that enhanced the internalization of Exo-Fect-loaded sEVs within the target cells and decreased the interaction of those modulated sEVs with lysosomes.  

3.3962           Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions

Vergauwen, G., Tulkens, J., Pinheiro, C., Cobos, F.A., Dedeyne, S.et al

1. Extracell. Vesicles, 10, e12122 (2021)

  Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size-exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context-dependent and time-dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context-dependent and/or time-dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV.  

3.3963           Extracellular vesicles from in vivo liver tissue accelerate recovery of liver necrosis induced by carbon tetrachloride

Lee, K., Kim, S.R., Lee, C., Jun, Y.I., Bae, S., Yoon, Y.J., Kim, O.Y. and Gho, Y.S.

1. Extracell. Vesicles, 10, e12133 (2021)

  Extracellular vesicles (EVs) are nano-sized vesicles composed of proteolipid bilayers carrying various molecular signatures of the cells. As mediators of intercellular communications, EVs have gained great attention as new therapeutic agents in the field of nanomedicine. Therefore, many studies have explored the roles of cell-derived EVs isolated from cultured hepatocytes or stem cells as inducer of liver proliferation and regeneration under various pathological circumstances. However, study investigating the role of EVs directly isolated from liver tissue has not been performed. Herein, to understand the pathophysiological role and to investigate the therapeutic potential of in vivo liver EVs, we isolated EVs from both normal and carbon tetrachloride (CCl₄)-induced damaged in vivo liver tissues. The in vivo EVs purified from liver tissues display typical features of EVs including spherical morphology, nano-size, and enrichment of tetraspanins. Interestingly, administration of both normal and damaged liver EVs significantly accelerated the recovery of liver tissue from CCl₄-induced hepatic necrosis. This restorative action was through the induction of hepatocyte growth factor at the site of the injury. These results suggest that not only normal liver EVs but also damaged liver EVs play important pathophysiological roles of maintaining homeostasis after tissue damage. Our study, therefore, provides new insight into potentially developing in vivo EV-based therapeutics for preventing and treating liver diseases.  

3.3964           Advanced Nanotechnologies for Extracellular Vesicle-Based Liquid Biopsy

Min, L., Wang, B., Bao, H., Li, X., Zhao, L., Meng, J. and Wang, S. Adv. Sci., 8, 2102789 (2021)   Extracellular vesicles (EVs) are emerging as a new source of biomarkers in liquid biopsy because of their wide presence in most body fluids and their ability to load cargoes from disease-related cells. Owing to the crucial role of EVs in disease diagnosis and treatment, significant efforts have been made to isolate, detect, and analyze EVs with high efficiency. A recent overview of advanced EV detection nanotechnologies is discussed here. First, several key challenges in EV-based liquid biopsies are introduced. Then, the related pivotal advances in nanotechnologies for EV isolation based on physical features, chemical affinity, and the combination of nanostructures and chemical affinity are summarized. Next, a summary of high-sensitivity sensors for EV detection and advanced approaches for single EV detection are provided. Later, EV analysis is introduced in practical clinical scenarios, and the application of machine learning in this field is highlighted. Finally, future opportunities for the development of next-generation nanotechnologies for EV detection are presented.  

3.3965           Plant extracellular vesicles: Trojan horses of cross-kingdom warfare

He, B., Hamby, R. and Jin, H. FASEB BioAdvances, 3, 657-664 (2021)   Plants communicate with their interacting microorganisms through the exchange of functional molecules. This communication is critical for plant immunity, for pathogen virulence, and for establishing and maintaining symbioses. Extracellular vesicles (EVs) are lipid bilayer-enclosed spheres that are released by both the host and the microbe into the extracellular environment. Emerging evidence has shown that EVs play a prominent role in plant–microbe interactions by safely transporting functional molecules, such as proteins and RNAs to interacting organisms. Recent studies revealed that plant EVs deliver fungal gene-targeting small RNAs into fungal pathogens to suppress infection via cross-kingdom RNA interference (RNAi). In this review, we focus on the recent advances in our understanding of plant EVs and their role in plant–microbe interactions.  

3.3966           A Mammalian Target of Rapamycin-Perilipin 3 (mTORC1-Plin3) Pathway is essential to Activate Lipophagy and Protects Against Hepatosteatosis

Garcia-Macia, M., Santos-Ledo, A., Leslie, J., Paish, H.L., Collins, A.L. et al Hapatology, 74(6), 3441-3459 (2021)   Background and Aims NAFLD is the most common hepatic pathology in western countries and no treatment is currently available. NAFLD is characterized by the aberrant hepatocellular accumulation of fatty acids in the form of lipid droplets (LDs). Recently, it was shown that liver LD degradation occurs through a process termed lipophagy, a form of autophagy. However, the molecular mechanisms governing liver lipophagy are elusive. Here, we aimed to ascertain the key molecular players that regulate hepatic lipophagy and their importance in NAFLD. Approach and Results We analyzed the formation and degradation of LD in vitro (fibroblasts and primary mouse hepatocytes), in vivo and ex vivo (mouse and human liver slices) and focused on the role of the autophagy master regulator mammalian target of rapamycin complex (mTORC) 1 and the LD coating protein perilipin (Plin) 3 in these processes. We show that the autophagy machinery is recruited to the LD on hepatic overload of oleic acid in all experimental settings. This led to activation of lipophagy, a process that was abolished by Plin3 knockdown using RNA interference. Furthermore, Plin3 directly interacted with the autophagy proteins focal adhesion interaction protein 200 KDa and autophagy-related 16L, suggesting that Plin3 functions as a docking protein or is involved in autophagosome formation to activate lipophagy. Finally, we show that mTORC1 phosphorylated Plin3 to promote LD degradation. Conclusions These results reveal that mTORC1 regulates liver lipophagy through a mechanism dependent on Plin3 phosphorylation. We propose that stimulating this pathway can enhance lipophagy in hepatocytes to help protect the liver from lipid-mediated toxicity, thus offering a therapeutic strategy in NAFLD.  

3.3967           The multifaceted involvement of exosomes in tumor progression: Induction and inhibition

Gu, W-J., Shen, Y-W., Zhang, L-J., Zhang, H., Nagle, D.G., Luan, X and Liu, S-H. MedComm., 2, 297-314 (2021)   As key performers in intercellular communication, exosomes released by tumor cells play an important role in cancer development, including angiogenesis, cancer-associated fibroblasts activation, epithelial-mesenchymal transformation (EMT), immune escape, and pre-metastatic niche formation. Meanwhile, other cells in tumor microenvironment (TME) can secrete exosomes and facilitate tumor progression. Elucidating mechanisms regarding these processes may offer perspectives for exosome-based antitumor strategies. In this review, we mainly introduce the versatile roles of tumor or stromal cell derived exosomes in cancer development, with a particular focus on the biological capabilities and functionalities of their diverse contents, such as miRNAs, lncRNAs, and circRNAs. The potential clinical application of exosomes as biomarkers in cancer diagnosis and prognosis is also discussed. Finally, the current antitumor strategies based on exosomes in immunotherapy and targeted delivery for chemotherapeutic or biological agents are summarized.  

3.3968           Current Proteomic and Metabolomic Knowledge of Zygotic and Somatic Embryogenesis in Plants

Juarez-Escobar, J., Bojorquez-Velazquez, E., Elizalde-Cobtreras, J.M., Guerrero-Analco, J.A., Loyala-Vargas, V.M., Mata-Rosas, M. and Ruiz-May, E. Int. J. Mol. Sci., 22:11807 (2021)   Embryogenesis is the primary developmental program in plants. The mechanisms that underlie the regulation of embryogenesis are an essential research subject given its potential contribution to mass in vitro propagation of profitable plant species. Somatic embryogenesis (SE) refers to the use of in vitro techniques to mimic the sexual reproduction program known as zygotic embryogenesis (ZE). In this review, we synthesize the current state of research on proteomic and metabolomic studies of SE and ZE in angiosperms (monocots and dicots) and gymnosperms. The most striking finding was the small number of studies addressing ZE. Meanwhile, the research effort focused on SE has been substantial but disjointed. Together, these research gaps may explain why the embryogenic induction stage and the maturation of the somatic embryo continue to be bottlenecks for efficient and large-scale regeneration of plants. Comprehensive and integrative studies of both SE and ZE are needed to provide the molecular foundation of plant embryogenesis, information which is needed to rationally guide experimental strategies to solve SE drawbacks in each species.  

3.3969           Extracellular Vesicles and Their Relationship with the Heart–Kidney Axis, Uremia and Peritoneal Dialysis

Azevedo, C.A.B., da Cunha, R.S., Junho, C.V.C., da Silva, J.V., Moreno-Amaral, A.N., de Morales, T.P., Carneiro-Ramos, M.and Stinghen, A.E.M. Toxins, 13:778 (2021)   Cardiorenal syndrome (CRS) is described as primary dysfunction in the heart culminating in renal injury or vice versa. CRS can be classified into five groups, and uremic toxin (UT) accumulation is observed in all types of CRS. Protein-bound uremic toxin (PBUT) accumulation is responsible for permanent damage to the renal tissue, and mainly occurs in CRS types 3 and 4, thus compromising renal function directly leading to a reduction in the glomerular filtration rate (GFR) and/or subsequent proteinuria. With this decrease in GFR, patients may need renal replacement therapy (RRT), such as peritoneal dialysis (PD). PD is a high-quality and home-based dialysis therapy for patients with end-stage renal disease (ESRD) and is based on the semi-permeable characteristics of the peritoneum. These patients are exposed to factors which may cause several modifications on the peritoneal membrane. The presence of UT may harm the peritoneum membrane, which in turn can lead to the formation of extracellular vesicles (EVs). EVs are released by almost all cell types and contain lipids, nucleic acids, metabolites, membrane proteins, and cytosolic components from their cell origin. Our research group previously demonstrated that the EVs can be related to endothelial dysfunction and are formed when UTs are in contact with the endothelial monolayer. In this scenario, this review explores the mechanisms of EV formation in CRS, uremia, the peritoneum, and as potential biomarkers in peritoneal dialysis.  

3.3970           Elucidating the Role of Extracellular Vesicles in Pancreatic Cancer

Marzan, A.L. and Stewart, S.E: Cancers, 13:5669 (2021)   Pancreatic cancer is one of the deadliest cancers worldwide, with a 5-year survival rate of less than 10%. This dismal survival rate can be attributed to several factors including insufficient diagnostics, rapid metastasis and chemoresistance. To identify new treatment options for improved patient outcomes, it is crucial to investigate the underlying mechanisms that contribute to pancreatic cancer progression. Accumulating evidence suggests that extracellular vesicles, including exosomes and microvesicles, are critical players in pancreatic cancer progression and chemoresistance. In addition, extracellular vesicles also have the potential to serve as promising biomarkers, therapeutic targets and drug delivery tools for the treatment of pancreatic cancer. In this review, we aim to summarise the current knowledge on the role of extracellular vesicles in pancreatic cancer progression, metastasis, immunity, metabolic dysfunction and chemoresistance, and discuss their potential roles as biomarkers for early diagnosis and drug delivery vehicles for treatment of pancreatic cancer.  

3.3971           Special delEVery: Extracellular Vesicles as Promising Delivery Platform to the Brain

Pauwels, M.J., Vandendriessche, C. and Vandenbroucke, R.E. Biomedicines, 9:1734 (2021)   The treatment of central nervous system (CNS) pathologies is severely hampered by the presence of tightly regulated CNS barriers that restrict drug delivery to the brain. An increasing amount of data suggests that extracellular vesicles (EVs), i.e., membrane derived vesicles that inherently protect and transfer biological cargoes between cells, naturally cross the CNS barriers. Moreover, EVs can be engineered with targeting ligands to obtain enriched tissue targeting and delivery capacities. In this review, we provide a detailed overview of the literature describing a natural and engineered CNS targeting and therapeutic efficiency of different cell type derived EVs. Hereby, we specifically focus on peripheral administration routes in a broad range of CNS diseases. Furthermore, we underline the potential of research aimed at elucidating the vesicular transport mechanisms across the different CNS barriers. Finally, we elaborate on the practical considerations towards the application of EVs as a brain drug delivery system.  

3.3972           Extracellular Vesicles from Different Pneumococcal Serotypes Are Internalized by Macrophages and Induce Host Immune Responses

Olaya-Abril, A., Prados-Rosales, R., Gonzalez-Reyes, J.A., Casadevall, A., Pirofski, L-A. and Rodriguez-Ortega, M.J. Pathogens, 10:1530 (2021)   Bacterial extracellular vesicles are membranous ultrastructures released from the cell surface. They play important roles in the interaction between the host and the bacteria. In this work, we show how extracellular vesicles produced by four different serotypes of the important human pathogen, Streptococcus pneumoniae, are internalized by murine J774A.1 macrophages via fusion with the membrane of the host cells. We also evaluated the capacity of pneumococcal extracellular vesicles to elicit an immune response by macrophages. Macrophages treated with the vesicles underwent a serotype-dependent transient loss of viability, which was further reverted. The vesicles induced the production of proinflammatory cytokines, which was higher for serotype 1 and serotype 8-derived vesicles. These results demonstrate the biological activity of extracellular vesicles of clinically important pneumococcal serotypes  

3.3973           Deciphering the Role of Extracellular Vesicles Derived from ZIKV-Infected hcMEC/D3 Cells on the Blood–Brain Barrier System

Fikatas, A., Dehairs, J., Noppen, S., Doijen, J., Vanderhoydone, F., Meyen, E., Swinnen, J.V., Pannecouque, C. and Schols, D. Viruses, 13:2363 (2021)   To date, no vaccines or antivirals are available against Zika virus (ZIKV). In addition, the mechanisms underlying ZIKV-associated pathogenesis of the central nervous system (CNS) are largely unexplored. Getting more insight into the cellular pathways that ZIKV recruits to facilitate infection of susceptible cells will be crucial for establishing an effective treatment strategy. In general, cells secrete a number of vesicles, known as extracellular vesicles (EVs), in response to viral infections. These EVs serve as intercellular communicators. Here, we investigated the role of EVs derived from ZIKV-infected human brain microvascular endothelial cells on the blood–brain barrier (BBB) system. We demonstrated that ZIKV-infected EVs (IEVs) can incorporate viral components, including ZIKV RNA, NS1, and E-protein, and further transfer them to several types of CNS cells. Using label-free impedance-based biosensing, we observed that ZIKV and IEVs can temporally disturb the monolayer integrity of BBB-mimicking cells, possibly by inducing structural rearrangements of the adherent protein VE-cadherin (immunofluorescence staining). Finally, differences in the lipidomic profile between EVs and their parental cells possibly suggest a preferential sorting mechanism of specific lipid species into the vesicles. To conclude, these data suggest that IEVs could be postulated as vehicles (Trojan horse) for ZIKV transmission via the BBB.  

3.3974           The Extracellular Vesicles from the Commensal Staphylococcus Epidermidis ATCC12228 Strain Regulate Skin Inflammation in the Imiquimod-Induced Psoriasis Murine Model

Gomez-Chavez, F., Cedillo-Pelaez, C., Zapi-Colin, L.A., Gutierrez-Gonzalez, G., Martinez-Torres, I. et al Int. J. Mol. Sci., 22:13029 (2021)   Extracellular vesicles (EVs) are evaginations of the cytoplasmic membrane, containing nucleic acids, proteins, lipids, enzymes, and toxins. EVs participate in various bacterial physiological processes. Staphylococcus epidermidis interacts and communicates with the host skin. S. epidermidis’ EVs may have an essential role in this communication mechanism, modulating the immunological environment. This work aimed to evaluate if S. epidermidis’ EVs can modulate cytokine production by keratinocytes in vitro and in vivo using the imiquimod-induced psoriasis murine model. S. epidermidis’ EVs were obtained from a commensal strain (ATC12228EVs) and a clinical isolated strain (983EVs). EVs from both origins induced IL-6 expression in HaCaT keratinocyte cultures; nevertheless, 983EVs promoted a higher expression of the pro-inflammatory cytokines VEGF-A, LL37, IL-8, and IL-17F than ATCC12228EVs. Moreover, in vivo imiquimod-induced psoriatic skin treated with ATCC12228EVs reduced the characteristic psoriatic skin features, such as acanthosis and cellular infiltrate, as well as VEGF-A, IL-6, KC, IL-23, IL-17F, IL-36γ, and IL-36R expression in a more efficient manner than 983EVs; however, in contrast, Foxp3 expression did not significantly change, and IL-36 receptor antagonist (IL-36Ra) was found to be increased. Our findings showed a distinctive immunological profile induction that is dependent on the clinical or commensal EV origin in a mice model of skin-like psoriasis. Characteristically, proteomics analysis showed differences in the EVs protein content, dependent on origin of the isolated EVs. Specifically, in ATCC12228EVs, we found the proteins glutamate dehydrogenase, ornithine carbamoyltransferase, arginine deiminase, carbamate kinase, catalase, superoxide dismutase, phenol-soluble β1/β2 modulin, and polyglycerol phosphate α-glucosyltransferase, which could be involved in the reduction of lesions in the murine imiquimod-induced psoriasis skin. Our results show that the commensal ATCC12228EVs have a greater protective/attenuating effect on the murine imiquimod-induced psoriasis by inducing IL-36Ra expression in comparison with EVs from a clinical isolate of S. epidermidis.  

3.3975           Potential Applications and Functional Roles of Exosomes in Cardiometabolic Disease

Ayala-Mar, S., Rodriguez-Morales, B., Chacon-Ponce, P. and Gonzalez-Valdez, J. Pharmaceutics, 13:2056 (2021)   Despite diagnostic and therapeutic advances, cardiometabolic disease remains the leading cause of death worldwide. Extracellular vesicles (EVs), which include exosomes and microvesicles, have gained particular interest because of their role in metabolic homeostasis and cardiovascular physiology. Indeed, EVs are recognized as critical mediators of intercellular communication in the cardiovascular system. Exosomes are naturally occurring nanocarriers that transfer biological information in the setting of metabolic abnormalities and cardiac dysfunction. The study of these EVs can increase our knowledge on the pathophysiological mechanisms of metabolic disorders and their cardiovascular complications. Because of their inherent properties and composition, exosomes have been proposed as diagnostic and prognostic biomarkers and therapeutics for specific targeting and drug delivery. Emerging fields of study explore the use exosomes as tools for gene therapy and as a cell-free alternative for regenerative medicine. Furthermore, innovative biomaterials can incorporate exosomes to enhance tissue regeneration and engineering. In this work, we summarize the most recent knowledge on the role of exosomes in cardiometabolic pathophysiology while highlighting their potential therapeutic applications.  

3.3976           FACT-seq: profiling histone modifications in formalin-fixed paraffin-embedded samples with low cell numbers

Zhao, L., Xing, P., Polavarapu, V.K., Zhao, M., Valero-Martinez, B., Dang, Y., Maturi, N., Mathot, L., Neves, I., Yildirim, I., Swartling, F.J., Sjöblom, T., Uhrbom, L. and Chen, X. Nucleic Acids Res., 49(21), e125 (2021)   The majority of biopsies in both basic research and translational cancer studies are preserved in the format of archived formalin-fixed paraffin-embedded (FFPE) samples. Profiling histone modifications in archived FFPE tissues is critically important to understand gene regulation in human disease. The required input for current genome-wide histone modification profiling studies from FFPE samples is either 10–20 tissue sections or whole tissue blocks, which prevents better resolved analyses. But it is desirable to consume a minimal amount of FFPE tissue sections in the analysis as clinical tissues of interest are limited. Here, we present FFPE tissue with antibody-guided chromatin tagmentation with sequencing (FACT-seq), the first highly sensitive method to efficiently profile histone modifications in FFPE tissues by combining a novel fusion protein of hyperactive Tn5 transposase and protein A (T7−pA−Tn5) transposition and T7 in vitro transcription. FACT-seq generates high-quality chromatin profiles from different histone modifications with low number of FFPE nuclei. We proved a very small piece of FFPE tissue section containing ∼4000 nuclei is sufficient to decode H3K27ac modifications with FACT-seq. H3K27ac FACT-seq revealed disease-specific super enhancers in the archived FFPE human colorectal and human glioblastoma cancer tissue. In summary, FACT-seq allows decoding the histone modifications in archival FFPE tissues with high sensitivity and help researchers to better understand epigenetic regulation in cancer and human disease.  

3.3977           The BAX-binding protein MOAP1 associates with LC3 and promotes closure of the phagophore

Chang, H-C., Tao, R.N., Tan, C.T., Wu, Y.J., Bay, B.H. and Yu, V.C. Autophagy, 17(11), 3725-3739 (2021)   MOAP1 (modulator of apoptosis 1) is a BAX-binding protein tightly regulated by the ubiquitin-proteasome system. Apoptotic stimuli stabilize MOAP1 protein and facilitate its interaction with BAX to promote apoptosis. Here we show that in contrast to being resistant to apoptotic stimuli, MOAP1-deficient cells are hypersensitive to cell death mediated by starvation rendered by EBSS treatment. MOAP1-deficient cells exhibited impairment in macroautophagy/autophagy signaling induced by EBSS. Mechanistic analysis revealed that MOAP1-deficient cells had no notable defect in the recruitment of the pre-autophagosomal phosphatidylinositol-3-phosphate (PtdIns3P)-binding proteins, ZFYVE1/DFCP1 and WIPI2, nor in the LC3 lipidation mechanism regulated by the ATG12–ATG5-ATG16L1 complex upon EBSS treatment. Interestingly, MOAP1 is required for facilitating efficient closure of phagophore in the EBSS-treated cells. Analysis of LC3-positive membrane structures using Halo-tagged LC3 autophagosome completion assay showed that predominantly unclosed phagophore rather than closed autophagosome was present in the EBSS-treated MOAP1-deficient cells. The autophagy substrate SQSTM1/p62, which is normally contained within the enclosed autophagosome under EBSS condition, was also highly sensitive to degradation by proteinase K in the absence of MOAP1. MOAP1 binds LC3 and the binding is critically dependent on a LC3-interacting region (LIR) motif detected at its N-terminal region. Re-expression of MOAP1, but not its LC3-binding defective mutant, MOAP1-LIR, in the MOAP1-deficient cells, restored EBSS-induced autophagy. Together, these observations suggest that MOAP1 serves a distinct role in facilitating autophagy through interacting with LC3 to promote efficient phagophore closure during starvation.  

3.3978           Loss of Christianson Syndrome Na+/H+ Exchanger 6 (NHE6) Causes Abnormal Endosome Maturation and Trafficking Underlying Lysosome Dysfunction in Neurons

Pescosolido, M.F., Ouyang, Q., Liu, J.S: and Morrow, E.M.

1. Neurosci., 41(44), 9235-9256 (2021)

  Loss-of-function mutations in endosomal Na⁺/H⁺ exchanger 6 (NHE6) cause the X-linked neurologic disorder Christianson syndrome. Patients exhibit symptoms associated with both neurodevelopmental and neurodegenerative abnormalities. While loss of NHE6 has been shown to overacidify the endosome lumen, and is associated with endolysosome neuropathology, NHE6-mediated mechanisms in endosome trafficking and lysosome function have been understudied. Here, we show that NHE6-null mouse neurons demonstrate worsening lysosome function with time in culture, likely as a result of defective endosome trafficking. NHE6-null neurons exhibit overall reduced lysosomal proteolysis despite overacidification of the endosome and lysosome lumen. Akin to Nhx1 mutants in Saccharomyces cerevisiae, we observe decreased endosome-lysosome fusion in NHE6-null neurons. Also, we find premature activation of pH-dependent cathepsin D (CatD) in endosomes. While active CatD is increased in endosomes, CatD activation and CatD protein levels are reduced in the lysosome. Protein levels of another mannose 6-phosphate receptor (M6PR)-dependent enzyme, β-N-acetylglucosaminidase, were also decreased in lysosomes of NHE6-null neurons. M6PRs accumulate in late endosomes, suggesting defective M6PR recycling and retromer function in NHE6-null neurons. Finally, coincident with decreased endosome-lysosome fusion, using total internal reflection fluorescence, we also find a prominent increase in fusion between endosomal multivesicular bodies and the plasma membrane, indicating enhanced exosome secretion from NHE6-null neurons. In summary, in addition to overacidification of endosomes and lysosomes, loss of NHE6 leads to defects in endosome maturation and trafficking, including enhanced exosome release, contributing to lysosome deficiency and potentially leading to neurodegenerative disease.  

3.3979           Schistosoma mansoni Larval Extracellular Vesicle protein 1 (SmLEV1) is an immunogenic antigen found in EVs released from pre-acetabular glands of invading cercariae

Gasan, T.A:, Kuipers, M.E., Roberts, G.H., Padalino, G., Forde-Thomas, J.E:, Wilson, S., Wawrzyniak, J., Tukahebwa, E.M., Hoffmann, K.F: and Chalmers, I.W. PloS Neglected Diseases, 15(11), e0009981 (2021)   Extracellular Vesicles (EVs) are an integral component of cellular/organismal communication and have been found in the excreted/secreted (ES) products of both protozoan and metazoan parasites. Within the blood fluke schistosomes, EVs have been isolated from egg, schistosomula, and adult lifecycle stages. However, the role(s) that EVs have in shaping aspects of parasite biology and/or manipulating host interactions is poorly defined. Herein, we characterise the most abundant EV-enriched protein in Schistosoma mansoni tissue-migrating schistosomula (Schistosoma mansoni Larval Extracellular Vesicle protein 1 (SmLEV1)). Comparative sequence analysis demonstrates that lev1 orthologs are found in all published Schistosoma genomes, yet homologs are not found outside of the Schistosomatidae. Lifecycle expression analyses collectively reveal that smlev1 transcription peaks in cercariae, is male biased in adults, and is processed by alternative splicing in intra-mammalian lifecycle stages. Immunohistochemistry of cercariae using a polyclonal anti-recombinant SmLEV1 antiserum localises this protein to the pre-acetabular gland, with some disperse localisation to the surface of the parasite. S. mansoni—infected Ugandan fishermen exhibit a strong IgG₁ response against SmLEV1 (dropping significantly after praziquantel treatment), with 11% of the cohort exhibiting an IgE response and minimal levels of detectable antigen-specific IgG₄. Furthermore, mice vaccinated with rSmLEV1 show a slightly reduced parasite burden upon challenge infection and significantly reduced granuloma volumes, compared with control animals. Collectively, these results describe SmLEV1 as a Schistosomatidae-specific, EV-enriched immunogen. Further investigations are now necessary to uncover the full extent of SmLEV1’s role in shaping schistosome EV function and definitive host relationships.  

3.3980           Degranulation enhances presynaptic membrane packing, which protects NK cells from perforin-mediated autolysis

Li, Y. and Orange, J.S. PloS Biol., 19(8), e3001328 (2021)   Natural killer (NK) cells kill a target cell by secreting perforin into the lytic immunological synapse, a specialized interface formed between the NK cell and its target. Perforin creates pores in target cell membranes allowing delivery of proapoptotic enzymes. Despite the fact that secreted perforin is in close range to both the NK and target cell membranes, the NK cell typically survives while the target cell does not. How NK cells preferentially avoid death during the secretion of perforin via the degranulation of their perforin-containing organelles (lytic granules) is perplexing. Here, we demonstrate that NK cells are protected from perforin-mediated autolysis by densely packed and highly ordered presynaptic lipid membranes, which increase packing upon synapse formation. When treated with 7-ketocholesterol, lipid packing is reduced in NK cells making them susceptible to perforin-mediated lysis after degranulation. Using high-resolution imaging and lipidomics, we identified lytic granules themselves as having endogenously densely packed lipid membranes. During degranulation, lytic granule–cell membrane fusion thereby further augments presynaptic membrane packing, enhancing membrane protection at the specific sites where NK cells would face maximum concentrations of secreted perforin. Additionally, we found that an aggressive breast cancer cell line is perforin resistant and evades NK cell–mediated killing owing to a densely packed postsynaptic membrane. By disrupting membrane packing, these cells were switched to an NK-susceptible state, which could suggest strategies for improving cytotoxic cell-based cancer therapies. Thus, lipid membranes serve an unexpected role in NK cell functionality protecting them from autolysis, while degranulation allows for the inherent lytic granule membrane properties to create local ordered lipid “shields” against self-destruction.  

3.3981           Integration of feeding behavior by the liver circadian clock reveals network dependency of metabolic rhythms

Greco, C.M., Koronowski, K.B., Smith, J.G., Shi, J., Kunderfranco, P. et al Sci. Adv., 7, eabi7828 (2021)   The mammalian circadian clock, expressed throughout the brain and body, controls daily metabolic homeostasis. Clock function in peripheral tissues is required, but not sufficient, for this task. Because of the lack of specialized animal models, it is unclear how tissue clocks interact with extrinsic signals to drive molecular oscillations. Here, we isolated the interaction between feeding and the liver clock by reconstituting Bmal1 exclusively in hepatocytes (Liver-RE), in otherwise clock-less mice, and controlling timing of food intake. We found that the cooperative action of BMAL1 and the transcription factor CEBPB regulates daily liver metabolic transcriptional programs. Functionally, the liver clock and feeding rhythm are sufficient to drive temporal carbohydrate homeostasis. By contrast, liver rhythms tied to redox and lipid metabolism required communication with the skeletal muscle clock, demonstrating peripheral clock cross-talk. Our results highlight how the inner workings of the clock system rely on communicating signals to maintain daily metabolism.  

3.3982           An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking

Huang, Y., Yin, H., Li, B., Wu, Q., Liu, Q., Poljak, K.Maldutyte, J., Tang, X., Wang, M., Wu, Z., Miller, E.A., Jiang, L., Yao, Z-O. and Guo, Y. PNAS, 118(35), e2101287118 (2021)   The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.  

3.3983           Valosin-Containing Protein/p97 Plays Critical Roles in the Japanese Encephalitis Virus Life Cycle

Sehawat, S., Khasa, R., Deb, A., Prajapat, S.K., Mallick, S., BAsu, A., Surjit, M., Kalia, M. and Vrati, S.

1. Virol., 95(11), e2336-20 (2021)

  Host factors provide critical support for every aspect of the virus life cycle. We recently identified the valosin-containing protein (VCP)/p97, an abundant cellular ATPase with diverse cellular functions, as a host factor important for Japanese encephalitis virus (JEV) replication. In cultured cells, using small interfering RNA (siRNA)-mediated protein depletion and pharmacological inhibitors, we show that VCP is crucial for replication of three flaviviruses, JEV, dengue, and West Nile viruses. An FDA-approved VCP inhibitor, CB-5083, extended survival of mice in the animal model of JEV infection. While VCP depletion did not inhibit JEV attachment on cells, it delayed capsid degradation, potentially through the entrapment of the endocytosed virus in clathrin-coated vesicles (CCVs). Early during infection, VCP-depleted cells showed an increased colocalization of JEV capsid with clathrin and also higher viral RNA levels in purified CCVs. We show that VCP interacts with the JEV nonstructural protein NS5 and is an essential component of the virus replication complex. The depletion of the major VCP cofactor UFD-1 also significantly inhibited JEV replication. Thus, mechanistically, VCP affected two crucial steps of the JEV life cycle—nucleocapsid release and RNA replication. Our study establishes VCP as a common host factor with a broad antiviral potential against flaviviruses.  

3.3984           Exosomes in HIV infection

Hen, J., Li, C., Li, R., Chen, H., Chen, D. and Li, W. Curr.Opin. HIV Aids, 16(5), 262-270 (2021)   Purpose of review  The exosomes play a critical role in HIV infection, which constitute a pathway to release intracellular material and exchange material and information between cells. Exosomes have become a hotspot in the field of AIDS research. This review introduces the formation process of HIV particles and exosomes, and summarizes the role of exosomes in the progression of HIV disease from multiple aspects. Recent findings  Many components of the exosomes involved in HIV transfer and replication affect the occurrence, development, and outcome of AIDS, and are closely related to HIV infection. Exosomes can have a dual impact on HIV infection, and play an important role in activating the latent reservoir of HIV and affecting the chronic inflammation of HIV. The biological information carried by exosomes is also of great significance for the prediction of HIV disease. Summary  The present review summarizes the role of exosomes in HIV disease progression in various aspects in order to further understand the underlying mechanism affecting the infection and providing a new idea for the clinical diagnosis and treatment of AIDS.  

3.3985           Extracellular Vesicles: Novel Opportunities to Understand and Detect Neoplastic Diseases

Bongiovanni, L., Andriessen, A., Wauben, M.H.M., Nolte-’t Hoen, E.N.M. and de Bruin, A. Vet. Pathol., 58(3), 453-471 (2021)   With a size range from 30 to 1000 nm, extracellular vesicles (EVs) are one of the smallest cell components able to transport biologically active molecules. They mediate intercellular communications and play a fundamental role in the maintenance of tissue homeostasis and pathogenesis in several types of diseases. In particular, EVs actively contribute to cancer initiation and progression, and there is emerging understanding of their role in creation of the metastatic niche. This fact underlies the recent exponential growth in EV research, which has improved our understanding of their specific roles in disease and their potential applications in diagnosis and therapy. EVs and their biomolecular cargo reflect the state of the diseased donor cells, and can be detected in body fluids and exploited as biomarkers in cancer and other diseases. Relatively few studies have been published on EVs in the veterinary field. This review provides an overview of the features and biology of EVs as well as recent developments in EV research including techniques for isolation and analysis, and will address the way in which the EVs released by diseased tissues can be studied and exploited in the field of veterinary pathology. Uniquely, this review emphasizes the important contribution that pathologists can make to the field of EV research: pathologists can help EV scientists in studying and confirming the role of EVs and their molecular cargo in diseased tissues and as biomarkers in liquid biopsies.  

3.3986           TRIP12 ubiquitination of glucocerebrosidase contributes to neurodegeneration in Parkinson’s disease

Seo, B.A., Kim, D., Hwang, H., Dawson, V.L., Dawson, T.M. and Ko, H.S. Neuron, 109, 3758-3774 (2021)   Impairment in glucocerebrosidase (GCase) is strongly associated with the development of Parkinson’s disease (PD), yet the regulators responsible for its impairment remain elusive. In this paper, we identify the E3 ligase Thyroid Hormone Receptor Interacting Protein 12 (TRIP12) as a key regulator of GCase. TRIP12 interacts with and ubiquitinates GCase at lysine 293 to control its degradation via ubiquitin proteasomal degradation. Ubiquitinated GCase by TRIP12 leads to its functional impairment through premature degradation and subsequent accumulation of α-synuclein. TRIP12 overexpression causes mitochondrial dysfunction, which is ameliorated by GCase overexpression. Further, conditional TRIP12 knockout in vitro and knockdown in vivo promotes the expression of GCase, which blocks α-synuclein preformed fibrils (α-syn PFFs)-provoked dopaminergic neurodegeneration. Moreover, TRIP12 accumulates in human PD brain and α-synuclein-based mouse models. The identification of TRIP12 as a regulator of GCase provides a new perspective on the molecular mechanisms underlying dysfunctional GCase-driven neurodegeneration in PD.  

3.3987           Engineered exosome-like nanovesicles suppress tumor growth by reprogramming tumor microenvironment and promoting tumor ferroptosis

Hu, S., Ma, J., Su, C., Chen, Y., Shu, Y., Qi, Z., Zhang, B., Shi, G., Zhang, Y., Zhang, Y., Huang, A., Kuang, Y. and Cheng, P. Acta Biomaterialia, 135, 567-581 (2021)   Tumor vaccines that induce effective and sustained antitumor immunity are highly promising for cancer therapy. However, the antitumor potential of these vaccines is weakened due to the immunosuppressive characteristics of the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are the most abundant stromal cells within the TME; they play an important role in tumor growth, metastasis, immunosuppression, and drug resistance. Fibroblast activation protein-α (FAP) is overexpressed in CAFs in more than 90% of human tumor tissues. Further, FAP⁺CAFs are an ideal interstitial target for the immunotherapy of solid tumors. Exosomes derived from tumor cells contain many tumor antigens, which can be used as the basis of tumor vaccines that elicit strong antitumor immunity. Almost all exosome-based cancer vaccines have been designed and developed for tumor parenchymal cells. Moreover, the exosome production is very low and the purification is very difficult, limiting their clinical application as tumor vaccines. In this study, we developed FAP gene–engineered tumor cell–derived exosome-like nanovesicles (eNVs-FAP) as a tumor vaccine that can be prepared easily and in large quantities. The eNVs-FAP vaccine inhibited tumor growth by inducing strong and specific cytotoxic T lymphocyte (CTL) immune responses against tumor cells and FAP⁺CAFs and reprogramming the immunosuppressive TME in the colon, melanoma, lung, and breast cancer models. Moreover, eNVs-FAP vaccine–activated cellular immune responses could promote tumor ferroptosis by releasing interferon-gamma (IFN-γ) from CTLs and depleting FAP⁺CAFs. Thus, eNVs-FAP is a candidate tumor vaccine targeting both the tumor parenchyma and the stroma.  

3.3988           Cytokine Profiling in Low- and High-Density Small Extracellular Vesicles from Epidermoid Carcinoma Cells

Flemming, J.P., Hill, B.L., Anderson-Pullinger, L., harshyne, L.A. and Mahoney, M.G. JID Innovations, 1, 100053 82021)   Exosomes or small extracellular vesicles (sEVs) are membrane-bound nanoparticles that carry various macromolecules and act as autocrine and paracrine signaling messengers. In this study, sEVs from epidermoid carcinoma cells influenced by membrane presentation of the glycoprotein desmoglein 2 and its palmitoylation state were investigated. In this study, sEVs were isolated by sequential ultracentrifugation followed by iodixanol density gradient separation. They were then subjected to multiplex profiling of cytokines associated with the surface of intact sEVs. The results revealed a previously undescribed active sorting of cytokines onto the surface of low-density and high-density sEV subpopulations. Specifically, an altered surface presentation of desmoglein 2 decreased FGF-2 and VEGF in low-density sEVs. In addition, in response to desmoglein 2, IL-8 and RANTES were increased in low-density sEVs but only slightly decreased in high-density sEVs. Finally, IL-6 and G-CSF were increased dramatically in high-density sEVs. This comprehensive analysis of the cytokine production profile by squamous cell carcinoma‒derived sEVs highlights their contribution to immune evasion, pro-oncogenic and proangiogenic activity, and the potential to identify diagnostic disease biomarkers.    

3.3989           Targeted extracellular vesicle delivery systems employing superparamagnetic iron oxide nanoparticles

Zhuo, Z., Wang, J., Luo, Y., Zeng, R., Zhang, C., Zhou, W., Guo, K., Wu, H., Sha, W. and Chen, H. Acta Biomaterialia, 134, 13-31 (2021)   In the past decade, the study of extracellular vesicles (EVs), especially exosomes (50–150 nm) have attracted growing interest in numerous areas of cancer and tissue regeneration due to their unique biological features. A low isolation yield and insufficient targeting abilities limit their therapeutic applicability. Recently, superparamagnetic iron oxide nanoparticles (SPIONs) with magnetic navigation have been exploited to enhance the targeting ability of EVs. To construct targeted EV delivery systems engineered by SPIONs, several groups have pioneered the use of different techniques, such as electroporation, natural incubation, and cell extrusion, to directly internalize SPIONs into EVs. Furthermore, some endogenous ligands, such as transferrins, antibodies, aptamers, and streptavidin, were shown to enable modification of SPIONs, which increases binding with EVs. In this review, we summarized recent advances in targeted EV delivery systems engineered by SPIONs and focused on the key methodological approaches and the current applications of magnetic EVs. This report aims to address the existing challenges and provide comprehensive insights into targeted EV delivery systems.  

3.3990           Extracellular vesicle therapeutics from plasma and adipose tissue

Iannotta, D., Yang, M., Celia, C., Di Marzio, L. and Wolfram, J. Nano Today, 39, 101159 (2021)   Extracellular vesicles (EVs) are cell-released lipid-bilayer nanoparticles that contain biologically active cargo involved in physiological and pathological intercellular communication. In recent years, the therapeutic potential of EVs has been explored in various disease models. In particular, mesenchymal stromal cell-derived EVs have been shown to exert anti-inflammatory, anti-oxidant, anti-apoptotic, and pro-angiogenic properties in cardiovascular, metabolic and orthopedic conditions. However, a major drawback of EV-based therapeutics is scale-up issues due to extensive cell culture requirements and inefficient isolation protocols. An emerging alternative approach to time-consuming and costly cell culture expansion is to obtain therapeutic EVs directly from the body, for example, from plasma and adipose tissue. This review discusses isolation methods and therapeutic applications of plasma and adipose tissue-derived EVs, highlighting advantages and disadvantages compared to cell culture-derived ones.  

3.3991           Chromatin accessibility analysis identifies GSTM1 as a prognostic marker in human glioblastoma patients

Huang, Y-C., Lai, J.C-Y., Peng, P-H., Wei, K-C. and Wu, K-J. Clin. Epigenet., 13:201 (2021)   Background Glioblastoma (GBM) is a malignant human brain tumor that has an extremely poor prognosis. Classic mutations such as IDH (isocitrate dehydrogenase) mutations, EGFR (epidermal growth factor receptor) alternations, and MGMT (O6-methylguanine-methyltransferase) promoter hypermethylation have been used to stratify patients and provide prognostic significance. Epigenetic perturbations have been demonstrated in glioblastoma tumorigenesis. Despite the genetic markers used in the management of glioblastoma patients, new biomarkers that could predict patient survival independent of known biomarkers remain to be identified. Methods ATAC-seq (assay for transposase accessible chromatin followed by sequencing) and RNA-seq have been used to profile chromatin accessible regions using glioblastoma patient samples with short-survival versus long-survival. Cell viability, cell cycle, and Western blot analysis were used to characterize the cellular phenotypes and identify signaling pathways. Results Analysis of chromatin accessibility by ATAC-seq coupled with RNA-seq methods identified the GSTM1 (glutathione S-transferase mu-1) gene, which featured higher chromatin accessibility in GBM tumors with short survival. GSTM1 was confirmed to be a significant prognostic marker to predict survival using a different GBM patient cohort. Knockdown of GSTM1 decreased cell viability, caused cell cycle arrest, and decreased the phosphorylation levels of the NF-kB (nuclear factor kappa B) p65 subunit and STAT3 (signal transducer and activator of transcription 3) (pSer727). Conclusions This report demonstrates the use of ATAC-seq coupled with RNA-seq to identify GSTM1 as a prognostic marker of GBM patient survival. Activation of phosphorylation levels of NF-kB p65 and STAT3 (pSer727) by GSTM1 is shown. Analysis of chromatin accessibility in patient samples could generate an independent biomarker that can be used to predict patient survival.  

3.3992           Plant Sterol-Poor Diet Is Associated with Pro-Inflammatory Lipid Mediators in the Murine Brain

Reinicke, M., Leyh, J., Zimmermann, S., Chey, S., Brkovic, I.B., Wassermann, C., landmann, J., Lütjohann, D., Isermann, B., Bechmann, I and Ceglarek, U. Int. J. Mol. Sci., 22:13207 (2021   Plant sterols (PSs) cannot be synthesized in mammals and are exclusively diet-derived. PSs cross the blood-brain barrier and may have anti-neuroinflammatory effects. Obesity is linked to lower intestinal uptake and blood levels of PSs, but its effects in terms of neuroinflammation—if any—remain unknown. We investigated the effect of high-fat diet-induced obesity on PSs in the brain and the effects of the PSs campesterol and β-sitosterol on in vitro microglia activation. Sterols (cholesterol, precursors, PSs) and polyunsaturated fatty acid-derived lipid mediators were measured in the food, blood, liver and brain of C57BL/6J mice. Under a PSs-poor high-fat diet, PSs levels decreased in the blood, liver and brain (>50%). This effect was reversible after 2 weeks upon changing back to a chow diet. Inflammatory thromboxane B2 and prostaglandin D2 were inversely correlated to campesterol and β-sitosterol levels in all brain regions. PSs content was determined post mortem in human cortex samples as well. In vitro, PSs accumulate in lipid rafts isolated from SIM-A9 microglia cell membranes. In summary, PSs levels in the blood, liver and brain were associated directly with PSs food content and inversely with BMI. PSs dampen pro-inflammatory lipid mediators in the brain. The identification of PSs in the human cortex in comparable concentration ranges implies the relevance of our findings for humans.  

3.3993           Molecular and Functional Characterization of Caveolae in Mixed Cultures of Human NT-2 Neurons and Astrocytes

Sandhu, J.K., Ribecco-Lutkiewicz, M. and Abulrob, A. Neuroglia, 2, 68-88 (2021)   Caveolae are plasma membrane invaginations that are enriched in cholesterol-binding proteins called caveolins. The presence of caveolae and caveolins in mixed cultures of human neurons and glia has not been investigated. Here, we sought to determine the presence of caveolae and caveolins in human NTera-2 (NT2/D1) cells, differentiated with retinoic acid into neuron-like (NT2/N) and astrocyte-like (NT2/A) cells. We found that while caveolin-3 mRNA levels remained relatively constant, caveolin-1 and -2 levels were upregulated in NT2/A and downregulated in NT2/N. No caveolin-1 immunoreactivity was detected in NT2/N. Electron microscopy revealed numerous flask-shaped invaginations (~86–102 nm in diameter) in the plasma membrane of NT2/A and NT2/N cells, while only few were detected in NT2/D1 cells. Immunoelectron microscopy localized caveolin-1 gold particles in the flask-shaped structures on plasmalemma and cytoplasmic vesicles of NT2/A cells. Furthermore, NT2/A endocytosed Alexa 488 conjugated-cholera toxin B subunit (CTX-B) through a caveolae- and clathrin-dependent pathway, whereas NT2/N endocytosed CTX-B through a caveolae-independent pathway. We have established that while NT2/A expressed functional caveolae, the molecular identity of the plasma membrane invaginations in NT2/N is unknown. The expression of caveolin proteins was differentially regulated in these cells. Taken together, our findings support the usefulness of the human NT2 model system to study the role of caveolins in neuron–glia communication, and their involvement in brain health and disease.  

3.3994           Selection of Fluorescent, Bioluminescent, and Radioactive Tracers to Accurately Reflect Extracellular Vesicle Biodistribution in Vivo

Lazaro-Ibanez, E., Faruqu, F.N., Saleh, A.F., Silva, A.M., Wang, J.T-W., Rak, J., Al-Jamal. K.T. and Dekker, N. ACS Nano, 15, 3212-3227 (2021)   The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the in vivo biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with ¹¹¹indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63. To focus specifically on the effect of the tracer, we compared EVs derived from the same cell source and administered systemically by the same route and at equal dose into tumor-bearing BALB/c mice. ¹¹¹Indium and DiR were the most sensitive tracers for in vivo imaging of EVs, providing the most accurate quantification of vesicle biodistribution by ex vivo imaging of organs and analysis of tissue lysates. Specifically, NanoLuc fused to CD63 altered EV distribution, resulting in high accumulation in the lungs, demonstrating that genetic modification of EVs for tracking purposes may compromise their physiological biodistribution. Blood kinetic analysis revealed that EVs are rapidly cleared from the circulation with a half-life below 10 min. Our study demonstrates that radioactivity is the most accurate EV tracking approach for a complete quantitative biodistribution study including pharmacokinetic profiling. In conclusion, we provide a comprehensive comparison of fluorescent, bioluminescent, and radioactivity approaches, including dual labeling of EVs, to enable accurate spatiotemporal resolution of EV trafficking in mice, an essential step in developing EV therapeutics.  

3.3995           Reconstitution of Ultrawide DNA Origami Pores in Liposomes for Transmembrane Transport of Macromolecules

Fragasso, A., De Franceschi, N., Strömmer, P., van der Sluis, E.O., Dietz, H. and Dekker, C. ACS Nano, 15, 12768-12779 (2021)   Molecular traffic across lipid membranes is a vital process in cell biology that involves specialized biological pores with a great variety of pore diameters, from fractions of a nanometer to >30 nm. Creating artificial membrane pores covering similar size and complexity will aid the understanding of transmembrane molecular transport in cells, while artificial pores are also a necessary ingredient for synthetic cells. Here, we report the construction of DNA origami nanopores that have an inner diameter as large as 30 nm. We developed methods to successfully insert these ultrawide pores into the lipid membrane of giant unilamellar vesicles (GUVs) by administering the pores concomitantly with vesicle formation in an inverted-emulsion cDICE technique. The reconstituted pores permit the transmembrane diffusion of large macromolecules, such as folded proteins, which demonstrates the formation of large membrane-spanning open pores. The pores are size selective, as dextran molecules with a diameter up to 28 nm can traverse the pores, whereas larger dextran molecules are blocked. By FRAP measurements and modeling of the GFP influx rate, we find that up to hundreds of pores can be functionally reconstituted into a single GUV. Our technique bears great potential for applications across different fields from biomimetics, to synthetic biology, to drug delivery.  

3.3996           Cell Membrane-Coated Mimics: A Methodological Approach for Fabrication, Characterization for Therapeutic Applications, and Challenges for Clinical Translation

Chugh, V., Krishna, K.V. and Pandit, A. ACS Nano, 15, 17080-18123 (2021)   Cell membrane-coated (CMC) mimics are micro/nanosystems that combine an isolated cell membrane and a template of choice to mimic the functions of a cell. The design exploits its physicochemical and biological properties for therapeutic applications. The mimics demonstrate excellent biological compatibility, enhanced biointerfacing capabilities, physical, chemical, and biological tunability, ability to retain cellular properties, immune escape, prolonged circulation time, and protect the encapsulated drug from degradation and active targeting. These properties and the ease of adapting them for personalized clinical medicine have generated a significant research interest over the past decade. This review presents a detailed overview of the recent advances in the development of cell membrane-coated (CMC) mimics. The primary focus is to collate and discuss components, fabrication methodologies, and the significance of physiochemical and biological characterization techniques for validating a CMC mimic. We present a critical analysis of the two main components of CMC mimics: the template and the cell membrane and mapped their use in therapeutic scenarios. In addition, we have emphasized on the challenges associated with CMC mimics in their clinical translation. Overall, this review is an up to date toolbox that researchers can benefit from while designing and characterizing CMC mimics.  

3.3997           RBD-Modified Bacterial Vesicles Elicited Potential Protective Immunity against SARS-CoV-2

Yang, Z., Hua, L., Yang, M., Liu, S-Q., Shen, J., Li, W. et al Nano Lett., 21, 5920-5930 (2021)   The disease caused by SARS-CoV-2 infection threatens human health. In this study, we used high-pressure homogenization technology not only to efficiently drive the bacterial membrane to produce artificial vesicles but also to force the fusion protein ClyA-receptor binding domain (RBD) to pass through gaps in the bacterial membrane to increase the contact between ClyA-RBD and the membrane. Therefore, the load of ClyA-RBD on the membrane is substantially increased. Using this technology, we constructed a “ring-like” bacterial biomimetic vesicle (BBV) loaded with polymerized RBD (RBD-BBV). RBD-BBVs injected subcutaneously can accumulate in lymph nodes, promote antigen uptake and processing, and elicit SARS-CoV-2-specific humoral and cellular immune responses in mice. In conclusion, we evaluated the potential of this novel bacterial vesicle as a vaccine delivery system and provided a new idea for the development of SARS-CoV-2 vaccines.  

3.3998           Optimized cDICE for Efficient Reconstitution of Biological Systems in Giant Unilamellar Vesicles

Van de Cauter, L.V., Fanalista, F., van Buren, L., De Franceschi, N., Godino, E., Bouw, S., Danelon, C., Dekker, C., Koenderink, G.H. and Ganzinger, K.A. ACS Synth. Biol., 10, 1690-1702 (2021)   Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells, as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of biological systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (protein synthesis using recombinant elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.  

3.3999           Potential Use of Exosomes as Diagnostic Biomarkers and in Targeted Drug Delivery: Progress in Clinical and Preclinical Applications

Huda, M.N., Nafiujjaman, M., Deaguero, I.G., Okonkwo, J., Hill, M.L., Kim, T. and Nurunnabi, M. ACS Biomater. Sci. Eng., 7, 2106-2149 (2021)   Exosomes are cell-derived vesicles containing heterogeneous active biomolecules such as proteins, lipids, mRNAs, receptors, immune regulatory molecules, and nucleic acids. They typically range in size from 30 to 150 nm in diameter. An exosome’s surfaces can be bioengineered with antibodies, fluorescent dye, peptides, and tailored for small molecule and large active biologics. Exosomes have enormous potential as a drug delivery vehicle due to enhanced biocompatibility, excellent payload capability, and reduced immunogenicity compared to alternative polymeric-based carriers. Because of active targeting and specificity, exosomes are capable of delivering their cargo to exosome-recipient cells. Additionally, exosomes can potentially act as early stage disease diagnostic tools as the exosome carries various protein biomarkers associated with a specific disease. In this review, we summarize recent progress on exosome composition, biological characterization, and isolation techniques. Finally, we outline the exosome’s clinical applications and preclinical advancement to provide an outlook on the importance of exosomes for use in targeted drug delivery, biomarker study, and vaccine development.  

3.4000           Surface enhanced Raman scattering of extracellular vesicles for cancer diagnostics despite isolation dependent lipoprotein contamination

Koster, H.J., Rojalin, T., Powell, A., Pham, D., Mizenko, R.R., Birkeland, A.C. and Carney, R.P. Nanoscale, 13, 14760-14776 (2021)   Given the emerging diagnostic utility of extracellular vesicles (EVs), it is important to account for non-EV contaminants. Lipoprotein present in EV-enriched isolates may inflate particle counts and decrease sensitivity to biomarkers of interest, skewing chemical analyses and perpetuating downstream issues in labeling or functional analysis. Using label free surface enhanced Raman scattering (SERS), we confirm that three common EV isolation methods (differential ultracentrifugation, density gradient ultracentrifugation, and size exclusion chromatography) yield variable lipoprotein content. We demonstrate that a dual-isolation method is necessary to isolate EVs from the major classes of lipoprotein. However, combining SERS analysis with machine learning assisted classification, we show that the disease state is the main driver of distinction between EV samples, and largely unaffected by choice of isolation. Ultimately, this study describes a convenient SERS assay to retain accurate diagnostic information from clinical samples by overcoming differences in lipoprotein contamination according to isolation method.  

3.4001           The cell wall polysaccharides of a photosynthetic relative of apicomplexans, Chromera velia

Tortorelli, G., Pettolino, F., Lai, D-H., Tomcala, A., Bacic, A., Obernik, M., Lukes, j. and McFadden, G.I.

1. Phycol., 59(6), 1805-1809 (2021)

  Chromerids are a group of alveolates, found in corals, that show peculiar morphological and genomic features. These organisms are evolutionary placed in-between symbiotic dinoflagellates and parasitic apicomplexans. There are two known species of chromerids: Chromera velia and Vitrella brassicaformis. Here, the biochemical composition of the C. velia cell wall was analyzed. Several polysaccharides adorn this structure, with glucose being the most abundant monosaccharide (approx. 80%) and predominantly 4-linked (approx. 60%), suggesting that the chromerids cell wall is mostly cellulosic. The presence of cellulose was cytochemically confirmed with calcofluor white staining of the algal cell. The remaining wall polysaccharides, assuming structures are similar to those of higher plants, are indicative of a mixture of galactans, xyloglucans, heteroxylans, and heteromannans. The present work provides, for the first time, insights into the outermost layers of the photosynthetic alveolate C. velia.  

3.4002           Biomimetic immunomodulation strategies for effective tissue repair and restoration

Villarreal-Lela, R.A., healey, G.D. and Corradetti, B. Adv. Drug Delivery Reviews, 179, 113913 (2021)   Inflammation plays a central role in wound healing following injury or disease and is mediated by a precise cascade of cellular and molecular events. Unresolved inflammatory processes lead to chronic inflammation and fibrosis, which can result in prolonged wound healing lasting months or years that hampers tissue function. Therapeutic interventions mediated by immunomodulatory drugs, cells, or biomaterials, are therefore most effective during the inflammatory phase of wound healing when a pro-regenerative environment is essential. In this review, we discuss the advantages of exploiting knowledge of the native tissue microenvironment to develop therapeutics capable of modulating the immune response and promoting functional tissue repair. In particular, we provide examples of the most recent biomimetic platforms proposed to accomplish this goal, with an emphasis on those able to induce macrophage polarization towards a pro-regenerative phenotype.  

3.4003           Membrane Domain Localization and Interaction of the Prion-Family Proteins, Prion and Shadoo with Calnexin

Dondapati, D.T., Cingaram, R., Ayaydin, F., Nyeste, A., Kanyo, A., Welker, E. and Fodor, E. Membranes, 11:978 (2021)   The cellular prion protein (PrP^C) is renowned for its infectious conformational isoform PrP^Sc, capable of templating subsequent conversions of healthy PrP^Cs and thus triggering the group of incurable diseases known as transmissible spongiform encephalopathies. Besides this mechanism not being fully uncovered, the protein’s physiological role is also elusive. PrP^C and its newest, less understood paralog Shadoo are glycosylphosphatidylinositol-anchored proteins highly expressed in the central nervous system. While they share some attributes and neuroprotective actions, opposing roles have also been reported for the two; however, the amount of data about their exact functions is lacking. Protein–protein interactions and membrane microdomain localizations are key determinants of protein function. Accurate identification of these functions for a membrane protein, however, can become biased due to interactions occurring during sample processing. To avoid such artifacts, we apply a non-detergent-based membrane-fractionation approach to study the prion protein and Shadoo. We show that the two proteins occupy similarly raft and non-raft membrane fractions when expressed in N2a cells and that both proteins pull down the chaperone calnexin in both rafts and non-rafts. These indicate their possible binding to calnexin in both types of membrane domains, which might be a necessary requisite to aid the inherently unstable native conformation during their lifetime.  

3.4004           Supermeres are functional extracellular nanoparticles replete with disease biomarkers and therapeutic targets

Zhang, Q., Jeppesen, D.K., Higginbotham, J.N., Graves-Deal, R., Trinh, V.Q., Ramirez, M.A. et al Nature Cell Biol., 23, 1240-1254 (2021)   Extracellular vesicles and exomere nanoparticles are under intense investigation as sources of clinically relevant cargo. Here we report the discovery of a distinct extracellular nanoparticle, termed supermere. Supermeres are morphologically distinct from exomeres and display a markedly greater uptake in vivo compared with small extracellular vesicles and exomeres. The protein and RNA composition of supermeres differs from small extracellular vesicles and exomeres. Supermeres are highly enriched with cargo involved in multiple cancers (glycolytic enzymes, TGFBI, miR-1246, MET, GPC1 and AGO2), Alzheimer’s disease (APP) and cardiovascular disease (ACE2, ACE and PCSK9). The majority of extracellular RNA is associated with supermeres rather than small extracellular vesicles and exomeres. Cancer-derived supermeres increase lactate secretion, transfer cetuximab resistance and decrease hepatic lipids and glycogen in vivo. This study identifies a distinct functional nanoparticle replete with potential circulating biomarkers and therapeutic targets for a host of human diseases.  

3.4005           Profiling and promise of supermeres

Clancy, J.W., Boomgarten, A.C. and D’Souza-Schorey, C. Nature Cell Biol., 29, 1216-1223 (2021)   Extracellular vesicles and particles have important roles in physiology and disease. Advances in isolation and characterization technologies have enabled the identification of new particles. Supermeres are the newest addition to the rapidly expanding repertoire of the cell secretome, and provide exciting opportunities for clinical translation.  

3.4006           The Histidine Ammonia Lyase of Trypanosoma cruzi Is Involved in Acidocalcisome Alkalinization and Is Essential for Survival under Starvation Conditions

Mantilla, B.S., Azevedo, C., Denny, P.W., Salardi, A. and Docompo, R. mBio, 12(6), e01981-21 (2021)   Extracellular vesicles (EVs) are released by all types of cells as a means of intercellular communication. Their significance lies in the fact that they can alter recipient cell functions, despite their limited capacity for cargo. We have previously demonstrated that herpes simplex virus 1 (HSV-1) infection influences the cargo and functions of EVs released by infected cells and that these EVs negatively impact a subsequent HSV-1 infection. In the present study, we have implemented cutting-edge technologies to further characterize EVs released during HSV-1 infection. We identified distinct EV populations that were separable through a gradient approach. One population was positive for the tetraspanin CD63 and was distinct from EVs carrying components of the endosomal sorting complexes required for transport (ESCRT). Nanoparticle tracking analysis (NTA) combined with protein analysis indicated that the production of CD63⁺ EVs was selectively induced upon HSV-1 infection. The ExoView platform supported these data and suggested that the amount of CD63 per vesicle is larger upon infection. This platform also identified EV populations positive for other tetraspanins, including CD81 and CD9, whose abundance decreased upon HSV-1 infection. The stimulator of interferon genes (STING) was found in CD63⁺ EVs released during HSV-1 infection, while viral components were found in ESCRT⁺ EVs. Functional characterization of these EVs demonstrated that they have opposite effects on the infection, but the dominant effect was negative. Overall, we have identified the dominant population of EVs, and other EV populations produced during HSV-1 infection, and we have provided information about potential roles.  

3.4007           Chromatin architecture at susceptible gene loci in cerebellar Purkinje cells characterizes DNA damage–induced neurodegeneration

Kwak, Y.D., Shaw, T.I., Downing, S.M., Tewari, A., Aditi, Jin, H., Li, Y., Dumitrache, L.C., Katyal, S., Khodakhah, K., Russell, H.R. and McKinnon, P.J. Sci. Adv., 7, eabg3663 (2021)   The pathogenesis of inherited genome instability neurodegenerative syndromes remains largely unknown. Here, we report new disease-relevant murine models of genome instability–driven neurodegeneration involving disabled ATM and APTX that develop debilitating ataxia. We show that neurodegeneration and ataxia result from transcriptional interference in the cerebellum via aberrant messenger RNA splicing. Unexpectedly, these splicing defects were restricted to only Purkinje cells, disrupting the expression of critical homeostatic regulators including ITPR1, GRID2, and CA8. Abundant genotoxic R loops were also found at these Purkinje cell gene loci, further exacerbating DNA damage and transcriptional disruption. Using ATAC-seq to profile global chromatin accessibility in the cerebellum, we found a notably unique chromatin conformation specifically in Purkinje chromatin at the affected gene loci, thereby promoting susceptibility to DNA damage. These data reveal the pathogenic basis of DNA damage in the nervous system and suggest chromatin conformation as a feature in directing genome instability–associated neuropathology.  

3.4008           Composition, isolation, identification and function of adipose tissue-derived exosomes

Zhao, R., Zhao, T., He, Z., Cai, R. and Pang, W. Adipocyte, 10(1), 587-604 (2021)   Exosomes are nano-sized extracellular vesicles (30–160 nm diameter) with lipid bilayer membrane secrete by various cells that mediate the communication between cells and tissue, which contain a variety of non-coding RNAs, mRNAs, proteins, lipids and other functional substances. Adipose tissue is important energy storage and endocrine organ in the organism. Recent studies have revealed that adipose tissue-derived exosomes (AT-Exosomes) play a critical role in many physiologically and pathologically functions. Physiologically, AT-Exosomes could regulate the metabolic homoeostasis of various organs or cells including liver and skeletal muscle. Pathologically, they could be used in the treatment of disease and or that they may be involved in the progression of the disease. In this review, we describe the basic principles and methods of exosomes isolation and identification, as well as further summary the specific methods. Moreover, we categorize the relevant studies of AT-Exosomes and summarize the different components and biological functions of mammalian exosomes. Most importantly, we elaborate AT-Exosomes crosstalk within adipose tissue and their functions on other tissues or organs from the physiological and pathological perspective. Based on the above analysis, we discuss what remains to be discovered problems in AT-Exosomes studies and prospect their directions needed to be further explored in the future.  

3.4009           Effective methods for isolation and purification of extracellular vesicles from plants

Huang, Y., Wang, S., Cai, Q. and Jin, H. J.Integr. Plant Biol., 63, 2020-2030 (2021)   Plant extracellular vesicles (EVs) play critical roles in the cross-kingdom trafficking of molecules from hosts to interacting microbes, most notably in plant defense responses. However, the isolation of pure, intact EVs from plants remains challenging. A variety of methods have been utilized to isolate plant EVs from apoplastic washing fluid (AWF). Here, we compare published plant EV isolation methods, and provide our recommended method for the isolation and purification of plant EVs. This method includes a detailed protocol for clean AWF collection from Arabidopsis thaliana leaves, followed by EV isolation via differential centrifugation. To further separate and purify specific subclasses of EVs from heterogeneous vesicle populations, density gradient ultracentrifugation and immunoaffinity capture are then utilized. We found that immunoaffinity capture is the most precise method for specific EV subclass isolation when suitable specific EV biomarkers and their corresponding antibodies are available. Overall, this study provides a guide for the selection and optimization of EV isolation methods for desired downstream applications.  

3.4010           CC5.1 Intracellular sorting and EV-mediated release of Y-RNA and Y-RNA binding proteins

Driedonks, T., Ressel, S., Tran, T., Buck, A.H. and Nolte, E.N.M.

5. Extracell. Vesicles, 10, S1, e12083, abstract CC5.1 (2021)

  Introduction: Y-RNA is a non-coding RNA that is abundantly present in EV from various cell types and biofluids such as plasma. EV-associated Y-RNA has been associated with immune-regulatory functions. We previously showed that Toll-like receptor (TLR) activation of primary immune cells alters the abundance of Y-RNA in EV, but not in the cytoplasm, suggesting that Y-RNA shuttling is a regulated process. Y-RNA interacts with various RNA-binding proteins (RBP) in cells. Using subcellular fractionation of THP1 macrophages, we investigated how activation-induced changes in intracellular sorting of Y-RNA and their RBP relate to enhanced release of Y-RNA in EV. Methods: Post-nuclear cytosol of TLR2/1-stimulated and unstimulated THP1 was separated on Optiprep gradients into fractions enriched in ER, Golgi and endo/lysosomes. Y-RNA and RBP in subcellular fractions and EV were analyzed in parallel by Western blot, RT-qPCR and Northern blot. Results: Full-length Y-RNA was mainly detected in subcellular fractions containing endo/lysosomal markers Lamp-1, CD63 and Tsg101. Y-RNA binding proteins Ro60, La, HuR and hnRNP K, but not YBX1, co-localized with Y-RNA in these fractions. This supported the idea that Y-RNA can be sorted into EV at multivesicular endosomes (MVE). Of the tested RBP, only Ro60 was detected in Y-RNA containing EV. While TLR stimulation did not affect cytoplasmic Y-RNA levels, we observed an increase in the local Y-RNA concentration in MVE. This was paralleled with a TLR stimulation-induced increase in Y-RNA and Ro60 incorporation into EV. Summary/Conclusion: Our data exemplify how parallel analysis of subcellular compartments and EV can be used to study cell stimulation-induced changes in the sorting of EV-associated RNAs and their interacting proteins. We demonstrate that full-length Y-RNA and a subset of Y-RNA binding proteins are enriched at EV biogenesis sites. Our results suggest that TLR-activation changes the incorporation of Y-RNA into EV by altering its local concentration at EV-biogenesis sites.  

3.4011           CC6.4 High performance of α-galactosidase A lysosomal enzyme loaded in extracellular vesicles through recombinant protein overexpression

Seras-Franzoso, J., d’Hebron, V., Diaz-Riascos, Z.V., Corchero, J.L., Gonzalez, P. et al

6. Extracell. Vesicles, 10, S1, e12083, abstract CC6.4 (2021)

  Introduction: Fabry disease (FD), is a lysosomal storage disorder (LSD) caused by the mutation of α-galactosidase A (GLA) and enzymatic replacement therapy (ERT) is its preferred therapeutic option. However, ERT requires life-long administration of large amounts of recombinant enzyme and depends highly on mannose-6-phospate (M6P) mediated cell internalization. We propose an alternative system to deliver lysosomal enzymes in EVs simplifying the enzyme downstream processing while protecting the enzyme's activity and improving its bioavailability. Methods: EVs loaded with GLA (EV-GLA) were produced by protein overexpression in CHO cells. EVs were routinely isolated by water exclusion precipitation and further validated by tangential flow filtration and iodixanol gradient ultracentrifugation. EV-GLA activity in vitro was assessed by specific enzymatic activity (EA) assay, GLA substrate (Gb3) accumulation and MP6 competition assays. FD mice models were used to evaluate EV-GLA biodistribution and therapeutic potential. Results: EV-GLA loaded significant amounts of GLA exhibiting 6 times higher EA than free enzyme. In FD in vitro cultures, EV-GLA restored WT levels of Gb3 more efficiently than GLA. In vivo, DiR labeled EV-GLA was detected in liver, lungs and kidneys by ex-vivo imaging but also in difficult-to-target organs such as kidneys and brain. Consequently, single intravenous administration of EV-GLA restored basal Gb3 levels in highly accessible organs like liver or lungs but also reduced Gb3 in kidneys and brain. Cellular uptake revealed that EV internalization was independent on the MP6 route, fact that could partially explain the higher efficacy and bioavailability of EV-GLA compared to the free enzyme. Summary/Conclusion: Our data picture EVs loaded by recombinant lysosomal enzymes as natural drug delivery systems increasing the stability, bioavailability and efficacy of the cargo  

3.4012           CC7.2 Unraveling EV-mediated cardioprotection: EV-dependent and -independent mechanisms?

Roefs, M.T.T., Vader, P. and Sluijter, J.P.G.

7. Extracell. Vesicles, 10, S1, e12083, abstract CC7.2 (2021)

  Introduction: Cardiac progenitor cell (CPC)-derived extracellular vesicles (EVs) have been shown to protect the myocardium against ischemia/reperfusion injury. However, the underlying mechanisms for CPC-EV-mediated cardioprotection remain elusive. By exploring protein-mediated effects of CPC-EVs, we discovered that crude EV preparations activate recipient endothelial cells through EV-dependent and "independent pathways. Methods: CPCs were stimulated with calcium ionophore (ca ion-EVs), previously shown to influence EV release, or vehicle (control-EVs) for 24 hours and crude EVs were isolated using size exclusion chromatography (SEC). EV concentration and size was assessed using NTA and proteomic composition was profiled using mass spectrometry. Following SEC, Optiprep gradient ultracentrifugation was used to separate EVs from free proteins. EV- and protein fractions were functionally characterized based on endothelial cell activation assays. Results: Endothelial cells displayed enhanced phosphorylation of ERK1/2 and AKT and increased wound closure after stimulation with control-EVs, but not with ca ion-EVs. Proteomic analysis identified multiple proteins uniquely expressed or enriched in control-EVs compared with ca ion-EVs. Surprisingly, when investigating the contribution of individual candidate proteins, the extent of endothelial cell activation was found to be influenced by the purity of the EV preparations. EVs isolated using Optiprep gradients lost part of their ability to activate endothelial cells compared to crude EV preparations. In addition, several candidate proteins were found to be present in the free protein fraction instead of the EV fraction. This hints towards a co-stimulatory role of co-isolated proteins in recipient cell activation. Summary/Conclusion: A specific set of EV proteins is identified that may be functionally responsible for the activation of endothelial cells upon exposure to CPC-EVs. It is important to identify if these proteins are EV-associated or represent co-isolated factors that contribute to endothelial cell activation. This may lead to a better mechanistic understanding of CPC-EV-mediated cell activation and translation of EV-mediated therapeutics.

3.4013           OD11.01 Extracellular vesicles are involved in circulation of Hepatitis B Virus RNA in infected cells’ supernatant and patients’ serum

Bousquet, D., Doohyun, F., Adrait, F.A., Coute, F.Y., Bge, I., Martinez, F.M.G. et al

11. Extracell. Vesicles, 10, S1, e12084, abstract OD11.01 (2021)

  Introduction: Despite the availability of effective vaccine, chronic Hepatitis B virus (HBV) infection remains a global health burden. Current antiviral strategies (nucleos(t)ide analogues, NUCs) are unable to eliminate the virus from infected hepatocytes and, thus, achieve a complete cure. Relevant and non-invasive biomarkers are necessary to ameliorate patients’ management and the evaluation of new therapies. In this study, we aim at better characterizing the compartments containing extracellular HBV RNAs, which were recently proposed as a new surrogate marker of intrahepatic viral activity. Methods: Supernatant from HBV-infected HepG2-NTCP cells, treated or not with NUCs, was collected and processed through sucrose/iodixanol gradient separation, to allow physical separation of extracellular vesicles (EVs) and viral particles according to their buoyant density. Viral and EVs-associated proteins were analyzed by Western Blotting and Elisa, while HBV RNAs were detected by specific digital droplet (dd)PCR. NTA and mass spectrometry-based proteomic analyses were used to further characterize the EVs components. Results: Elisa assays for viral surface proteins after gradient separation showed that virions were found in fractions corresponding to a density of 1,21-1,25 g/ml. Western Blotting for CD9 and CD63, markers of exosomes/EVs were detected only in lower density fractions (1,13"1,19 g/m), which were deprived of viral proteins. Interestingly, HBV RNAs were detected not only in virion-like particles but also in lighter gradient fractions, suggesting that EVs could contribute to carry the circulating HBV RNA pool. No significant difference was found in NUC-treated vs untreated samples. To further investigate the nature of EVs detected in light density fractions, NTA analysis was performed, showing that these fractions were indeed containing EVs in size spanning from 30 to 150 nm. Finally, proteomic analyses of the same fractions revealed the presence of specific markers of exosomes (CD63, CD9, TSG101 or HSC70). Summary/Conclusion: Our study will shed light on the molecular biology of serum HBV RNA secretion and will aid the development of serum HBV RNA as a novel biomarker for chronic HBV infection.  

3.4014           PS06.03 Quantitative multi-parameter analysis of individual fecal extracellular vesicle via a laboratory-built nano-flow cytometer

Liu, H., Chen, Y., Qin, Y. and Yan, X.

6. Extracell. Vesicles, 10, S1, e12083, abstract PS06.3 (2021)

  Introduction: Fecal extracellular vesicles (fEVs) has been implicated in physiological processes in various diseases and host immune response. To further explore their prominent biological potential, an in-depth study of fEVs at the single-particle level is important. Employing a laboratory-built nano-flow cytometer (nFCM) that facilitates multiparameter analysis of single EVs as small as 40 nm, here we report quantitative measurement of size distribution, purity, nucleic acids and surface markers of fEVs. Methods: fEVs were isolated from stool samples collected from healthy donors via iodixanol gradient ultracentrifugation. Six fractions of 2 mL each were collected from the top of the tube (F1 " F6). TEM was used to characterize the morphology of fEVs. Monodisperse silica nanoparticles were used as the size reference standards for the size distribution measurement of fEVs via light scattering detection. The purity of fEVs was examined by measuring the particle concentration before and after Triton X-100 treatment. Subpopulation of fEVs expressing specific surface markers, such as CD9, CD63, CD81, CD24, lipopolysaccharide (LPS) and lipoteichoic acid (LTA) were analyzed via immunofluorescent staining. SYTO 16, a cell-permeant stain, was used to stain the DNA of fEVs before and after DNase I treatment. Results: The purity of isolated F1 - F6 fEVs via density gradient UC was ranging from 55.1% - 93.2%. We found that there was almost no expression of CD9, CD63, CD81 and CD24 for fEVs. We also found that ∼20% of fEVs expressing LPS (the marker of EVs from gram-negative bacteria) or LTA (the marker of EVs from gram-positive bacteria). The ratio of fEVs that can be fluorescently stained by SYTO 16 had no obvious change after DNase I treatment, suggesting that all the EV-DNA of fEVs resides in the lumen of EVs. Summary/Conclusion: The laboratory-built nFCM is applicable to the multiparameter biochemical analysis of individual fEV via protein and nucleic acid staining. We expect nFCM will facilitate more in-depth studies of fEVs.  

3.4015           PS10.13 Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin

Veerman, R.E., Czarnewaki, P., Sandberg, A-S., Cao, X., Pernemalm, M., Orre, L., Gabrielsson, S. and Eldh, M.

10. Extracell. Vesicles, 10, S1, e12083, abstract PS10.13 (2021)

  Introduction: Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers for disease, but still EV subtypes are not fully characterised and understood. Moreover, new methods for isolating EVs are emerging leading to discrepancy in isolation methods between published studies and thereby making the comparison of results difficult. Therefore, we conducted a study to compare commercially available methods based on five different principles by isolating EVs from both cell conditioned medium and 250 μl or 3 ml plasma samples. Methods: The used methods included precipitation, membrane affinity, Size-Exclusion Chromatography (SEC), iodixanol gradient and phosphatidylserine affinity. EVs were characterized by electron microscopy, Nanoparticle Tracking Analysis, electrophoresis for RNA quality, flow cytometry and LC-MS/MS. Results: The different methods yield samples of different morphology, particle size, purity, and proteomic profile. For the conditioned medium, SEC with a pore size of 35 isolated most number of EV proteins while membrane affinity isolated the purest samples with enrichment for larger EVs. Also for the plasma samples, membrane affinity isolated the purest samples, while SEC with a pore size of 70 isolated most EV proteins. Furthermore, bioinformatic analyses and comparisons with EV databases and plasma proteins show that the isolation methods reveal different levels of contamination of (lipo-)proteins. Moreover, Gene Set Enrichment Analysis shows that the different isolation methods enrich for distinct subtypes of EVs with signatures derived from diverse cellular compartments. Summary/Conclusion: This study provides information for optimising and designing future EV-studies since our data shows that different methods might be favored depending on interest in EV subtype, sample number and origin, volume, reproducibility, and budget.  

3.4016           PS10.14 Axillary lymphatic exudate from breast cancer patients: evaluation of different methods to isolate extracellular vesicles

Cresitelli, R., Ekström, K., Petursson, I. and Bagge, R.

10. Extracell. Vesicles, 10, S1, e12083, abstract PS10.14 (2021)

  Introduction: Extracellular vesicles (EVs) play a role in cell-cell communication and are also involved in breast cancer development. EVs can be used as biomarkers for early detection, and to monitor disease during and after treatments. A source of EVs can be serous fluid obtained after axillary lymph node dissection (ALND). The aim of this study was to find a suitable method to isolate EVs from lymphatic exudate. Methods: Approximately 50 ml of axillary drain fluid were collected from six breast cancer patients the day after ALND. Three EV isolation methods were tested: ultracentrifugation (UC) (n = 4) to collect large and small EVs, iodixanol cushion (UC-DC) (n = 2) and size exclusion chromatography (SEC) (qEV10/35 nm) (n = 3). EVs were analyzed by TEM and NTA. Seven fractions (F1-7) collected by SEC were further analyzed by western blot. Results: TEM pictures from large and small EVs isolated by UC showed presence of round elements with different size (250 nm vs 100 nm) surrounded by strong background due to contaminants. Similar pictures were observed when the combined large and small EVs were further purified by UC-DC. TEM pictures from single fractions obtained by SEC, showed presence of round elements in the fractions 2, 3 and 4, and an abundance of non-EV elements in fractions 5, 6 and 7. The presence of EVs were confirmed by NTA analysis (UC: large EVs: mean 8.3 × 109 particles/ml, small EVs: mean 3.4 × 1010 particles/ml; UC-DC: mean 4.5 × 109 particles/ml). NTA showed increased number of particles in F2-4 compared to F5-7 (mean 4.5 × 1010 particles/ml vs mean 3.9 × 109 particles/ml). Moreover, western blot analysis indicated that F2-4 were composed by particles positive for EV markers (CD63 and Flotillin-1) compared to F5-7 which mainly contained protein contaminants (Albumin and ApoA1). Summary/Conclusion: All methods were able to isolate EV subpopulations from lymphatic exudate, but SEC was the only one able to separate EVs from protein contaminants.  

3.4017           PS11.02 Characterization of milk Extracellular Vesicles (milk EVs) separated from bovine colostrum, first milk and mature milk over the lactation curve

Santoro, J., Mukhopadhya, A., Oliver, C., Giblin, L., O’Driscoll, L. and Sklodowska, M.

11. Extracell. Vesicles, 10, S1, e12083, abstract PS11.02 (2021)

  Introduction: While cow milk has a specific composition, during the lactation period, this composition differs. Extracellular vesicles (EVs) in milk contribute in regulating the biological processes and cellular communication. Recently, we established an optimal methodology to remove non-EV proteins from complex ‘milk matrix’ by combining isoelectric precipitation followed by gradient ultracentrifugation. In this study, we aim to separate EVs over the lactation curve i.e. from colostrum, first milk and mature milk and characterise them. Methods: Three Holstein-Friesian cows were selected for the study and colostrum (Col) was collected within 24 hours post-calving, first milk (FM) was collected 7-days post calving and mature milk was collected every month up-to month 9 (months 1, 5 and 9 used in this study). All samples were treated with HCl to precipitate casein micelles and protein aggregates were removed by filtration. EVs were separated using Optiprep density gradient using the bottom-up approach. EVs characterisation was performed by BCA to estimate total protein concentration, SDS page to characterise milk proteins; immunoblotting and imaging flow cytometry to investigate EVs specific markers, NTA to evaluate particle size and concentration and TEM to evaluate EVs morphology. Results: No difference in EV size and concentration was observed between Col (1.19E+14 particles/mL) and FM (9.36E+13 particles/mL). Compared to Col and FM, lower concentration of EV particles were observed in mature milk (2.32E+12 particles/mL) (P < 0.01), whereas no difference in size were observed. Particles resembling EV morphology were detected by TEM analysis. Immunoblotting analysis indicated that Actinin4 was absent in all samples, TSG101, CD63 and CD9 were detected in all analysed milk samples. Imaging flow cytometric analysis indicated that there were no differences in samples for HLADR, Col had significantly higher CD9 and CD63 positive particles compared to FM and mature milk (P < 0.05). Summary/Conclusion: Colostrum and first milk samples are enriched in EVs compared to mature milk and colostrum has the highest concentration of EVs which then decrease and is maintained throughout the milking period.  

3.4018           PS11.05 Comparison of different tomato plant explants for the production of extracellular vesicles in suspension culture

Moubarak, M., Kralj-Iglic, V., Bozic, D. and Pocsfalvi, G.

11. Extracell. Vesicles, 10, S1, e12083, abstract PS11.05 (2021)

  Introduction: Comparing to mammalian systems, the biogenesis and biological function of plant extracellular vesicles (EVs) have been less investigated. Only recently has been shown that tomato roots release EVs into the environment. Plant cell suspension culture is grown in a relatively simple mineral containing liquid medium under controlled conditions and thus may be suitable for the production of EVs. Tomato suspension cultures have been used for the advancement of genetic character, studying responses to abiotic stress factors and for biopharming of secondary metabolites, like carotenoids but not for EV production. Here, in the framework of the European greenEV project, we have established tomato (Solanum lycopersicum L) cell suspension cultures to evaluate the feasibility of the production of EVs using different tomato explants. Methods: Seeds of two tomato cv M82 and Microtom were sowed in plant tissue culture basal medium. Leaves, stem and roots explants were collected and cultivated in a medium containing high amount of a synthetic auxin under controlled conditions in dark. Friable callus from each type of explants were inoculated in a liquid medium to establish fine cell suspension cultures. Cell subcultures' supernatants were collected for the evaluation of rate of cells growth. EVs were isolated by gradient ultracentrifugation (gUC). Physical, morphological and molecular characteristics of the different fractions were analyzed by DLS, SEM, BCA assay and SDS-PAGE. Densities of the EV containing fractions were determined by iodixanol gUC. EV yields were calculated based on the protein concentrations and vesicle numbers in each isolates. Results: We have successfully established six tomato suspension cultures. All the suspension cultures studied yielded high amount of vesicles, i.e. from 94 to 233 pg of proteins per cell. Our analysis showed that root suspension cultures from Microtom variant has the highest yields of EVs (233 pg protein/cell) followed by the leaves suspension culture of M82 (146 pg protein/cells). gUC resulted in two visible bands (at low and high densities) between 1.098 and 1.117 g/mL densities. Protein profiles of the root, stem and leaves derived EVs were similar on the SDS-PAGE. Vesicle morphology were visualized by SEM. The two cultivar showed similar SDS-PAGE profiles. Summary/Conclusion: We have successfully set up six batch suspension cultures for two tomato varieties to produce between 30–136 mL culture supernatant and demonstrated that they contain EVs. Our results show that cell suspension culture could be a promising source of EVs generated by plant cells.  

3.4019           PS11.11 Bulk nano-sized vesicles from tomato fruit show discrete populations in gradient ultracentrifugation and dose-dependent pro- and anti-inflammatory effects on THP-1 monocyte cell line

Mammadova, R., Bokka, R., Fiume, I., Kralj-Iglic, V., Bozic, D., Kisovec, M., Podobnik, M. and Pocsfalvi, G.

11. Extracell. Vesicles, 10, S1, e12083, abstract PS11.11 (2021)

  Introduction: Nano-sized membrane bound vesicles can be isolated from a variety of plants, including tomato (S. lycopersicum L). Due to their advantages, such as stability, low toxicity, immunomodulatory, and regenerative properties and the fact that they can be produced from an economically sustainable green resource, they are highly-promising systems in future therapeutic and nutraceutical applications. Methods: Bulk nanovesicles were isolated from tomato fruit by differential ultracentrifugation (dUC). The complex intra and extracellular vesicle populations were separated using discrete sucrose or iodixanol density gradient ultracentrifugation (gUC). Fractions were collected and characterized by their physical and molecular properties, such as density, size-distribution, vesicle and protein concentrations, SDS-PAGE profiles, as well as by scanning electron microscopy (SEM) and cryo-transmission electron microscopy (cryo-TEM). To evaluate the cytotoxicity of the visible fractions, MTT assay using THP-1 monocyte cell line at increasing vesicle concentration (from 5 to 100 ug/mL) for 24h and 48h was performed. Inflammatory effect of the vesicles were tested by RT-qPCR using IL-6 and CxCl10 pro-inflammatory and IL-10 and CD-163 anti-inflammatory mRNA markers. Results: We have isolated tomato fruit-derived vesicles with a high yield using dUC. The bulk vesicle isolates were separated by gUC based on their buoyant density. Two visible bands were obtained at densities 1.08 and 1.13 g/mL. Depending on the fruit quality, vesicle quantity in the two bands was variable and sometimes a third band could also be observed. CyroTEM analysis confirmed the presence of membrane enclosed vesicles with different sizes and forms in both visible bands. SEM and TEM revealed presence of vesicles of heterogenous morphology (50-1000nm) and other amorphous material. We have found that tomato-derived NVs were slightly toxic only at high concentrations. Moreover, both fractions stimulates pro- and anti-inflammatory cytokines in a concentration-dependent manner. Summary/Conclusion: Tomato NVs were isolated with a high yield by dUC and sucrose and iodixanol gUC was applied to separate the EVs into 2–3 visible bands within the EV density range. SEM and cryo-TEM analysis proved the presence of vesicles in the collected fractions. Due to their anti-inflammatory activities, tomato-derived NVs could be explored as potential therapeutic agents or therapeutic vehicles.  

3.4020           PS11.15 Can Extracellular Vesicles release truly be pharmacologically inhibited?

Catalano, M., O’Driscoll, L. and Sklodowska, M.

11. Extracell. Vesicles, 10, S1, e12083, abstract PS11.15 (2021)

  Introduction: We previously reported extracellular vesicles (EVs) to be causally involved in transmitting phenotypic traits of aggressiveness in prostate cancer. We hypothesised that if we could inhibit EV released, we could block many of these serious problems. Thus, this study aimed to evaluate compounds that others have reported to block EV release. Specifically, we selected calpeptin and Y2763 (reported to inhibit EVs budding from cell membrane) and manumycin A and GW4869 (reported to inhibit EVs deriving from MVBs). Methods: To ensure that any effects observed were not simply due to cell death induced by the compounds, suitable concentrations that were non-toxic to cells were first determined by cytotoxicity assay and flow cytometry (FC). Conditioned medium (CM) was then collected from 2 prostate cancer drug-resistant variants (PC3RD, DU145RD) cells after incubation with or without the 4 compounds. Any EVs that continuing to be released following exposure to these compounds, and from controls, were separated from the CM by orthogonal tangential flow filtration and Optiprep density gradient and characterised in line with MISEV2018 guidelines. Results: When working with concentrations of calpeptin, Y2763, manumycin A or GW4869, that did not induce significant cell death, none of the 4 inhibitors (alone or in combination) significantly inhibited EV release from any of the 4 prostate cancer cell line variants, based on nanoparticle tracking analysis, immunoblots for positive and negative EV markers, and transmission electron microscopy analysis. Summary/Conclusion: When used at non-toxic levels, previously reported EV inhibitors proved not to significantly inhibit EV release from a range of prostate cancer cell line variants. This highlights the importance of considering off-target effects in efforts to block EV release.    

3.4021           PS12.06 Effect of radiation on prostate derived, β1 integrin positive small Extracellular Vesicles

Garcia, V., DeRita, R.M., Sayeed, A., Krishn, S., McCue, P., Dicker, A. and Languino, L.R.R.

12. Extracell. Vesicles, 10, S1, e12083, abstract PS12.06 (2021)

  Introduction: EVs are emerging as critical mediators of cell-to-cell communication in response to cancer therapy. Our laboratory has previously reported that prostate irradiation of TRAMP mice significantly blocks tumor growth. Also, our results have demonstrated that β1 integrins in circulating tumor-cell-derived sEVs are required for stimulation of anchorage-independent growth. Methods: Here we characterize the circulating sEVs from TRAMP mice and to add the clinical perspective, we analyzed sEVs from the plasma of PrCa patients. We use plasma sEVs isolated using differential ultra-centrifugation and iodixanol gradient fractionation. We used NTA to analyze sEVs. Mouse pelvises were irradiated using 10 Gy, for 5 consecutive days. Results: Circulating sEVs from the plasma of prostate cancer patients show robust expression of β1 integrins and Src. We also demonstrate a robust expression of β1 integrins and c-Src in sEVs isolated from TRAMP mice which promotes anchorage-independent growth of recipient cells. We observe that upon pelvic irradiation of TRAMP mice, the levels of the β1 integrins and c-Src are reduced in plasma-derived sEVs. In addition, upon irradiation the size of sEVs is increased from 50–100nms to 70–250nms, whereas the concentration of sEVs is not affected. Similarly, upon irradiation, PC3 cell derived sEVs show profound reduction in β1 and c-Src/c-SrcpY416 and in their ability to enhance anchorage-independent growth and cell migration. Summary/Conclusion: Taken together, we have identified a novel effect of irradiation which has profound implications in suppressing sEV-mediated pre-metastatic lesions in cancer patients. These data pave the way to future investigations of circulating EVs from patients who have undergone radiation therapy to evaluate stratification, accuracy of prognosis and efficacy of therapeutic efforts.  

3.4022           PS12.11 In vivo-mimicking 3D cultures secrete distinct extracellular vesicles upon cancer cell invasion

Luoto, J.C., Rato, L.C., Bengs, S., Roininen, J., Eriksson, J., Sistonen, L. and henriksson, E.

12. Extracell. Vesicles, 10, S1, e12083, abstract PS12.11 (2021)

  Introduction: Extracellular vesicles (EVs) are important in intercellular communication and mediate local and long-range signals in cancer metastasis. However, it is currently unknown how the development of the primary tumor and onset of invasion affects the secretion and characteristics of EVs. In this study, we developed an EV production method utilizing in vivo-mimicking extracellular matrix-based 3D cultures, and characterized the EVs over the course of invasive development of tumor organoids. Methods: Human prostate cancer PC3 cells were grown in 3D cultures using ECM-based hydrogel or in standard 2D culture conditions. EVs were isolated with differential centrifugation or high-resolution iodixanol gradient centrifugation. The isolated EVs were characterized with nanoparticle tracking analyses, electron microscopy, immunoblotting and mass spectrometry (MS). Results: Using the ECM-based cell culture method combined with proteomic profiling, we show that PC3 human prostate cancer organoids secrete EVs with previously undefined protein cargo, which substantially differs from EV cargo of 2D cultured cells. Intriguingly, an increase in EV amounts and extensive changes in EV protein composition were detected upon invasive transition of the organoids. Summary/Conclusion: Our results demonstrate that the culture conditions and the developmental status of the organoid cultures have a major impact on EV secretion and cargo loading, highlighting the necessity of in vivo-mimicking conditions for discovery of novel cancer-derived EV components, which can be linked to cancer progression and developed as biomarkers for different stages of cancer.  

3.4023           PS14.06 Proteomic profiling of tissue-derived extracellular vesicles from human pancreatic tumors

Karimi, N., Vilhav, C., Lässer, C. and Lötvall, J.

14. Extracell. Veslicles, 10, S1, e12083, abstract PS14.06 (2021)

  Introduction: Pancreatic cancer is a highly metastatic cancer and one of the major causes of cancer mortality due to its poor prognosis and lack of effective treatments. Profiling of the protein and RNA cargo of EVs has shown promising results in identifying potential diagnostic and prognostic markers and uncovering mechanisms of cancer. In this study, we hypothesized that tissue-derived EVs may identify candidate biomarkers leading to the development of a non-invasive diagnostic tool for the early detection of pancreatic cancer. Methods: Tumor tissue and non-tumor tissue were excised from 3 pancreatic cancer patients. The tissue was sliced into small pieces and incubated with DNase 1 and Collagenase D in cell culture medium for 30 minutes at 37°C (Crescitelli et al JEV 2020). Tissue-derived EVs were then isolated from the media by using ultracentrifugation and an iodixanol density cushion. Isolated EVs were then analysed using quantitative mass spectrometry analysis. Results: In total 6114 proteins were quantified in all samples. Of these proteins, 1010 were differentially expressed between non-tumor tissue-derived EVs and tumor tissue-derived EVs. In total, 837 proteins were significantly upregulated, and 173 proteins were downregulated in tumor tissue-derived EVs compared to non-tumor tissue-derived EVs (p < 0.05; fold change > 1.5). The analysis showed that Common EV markers including CD63, CD81, CD9, and TSG101 were present in both non-tumor tissue-derived EVs and tumor tissue-derived EVs. Gene ontology analysis indicated that “Extracellular Exosome” was the top GO term associated with the proteins identified in both non-tumor tissue-derived EVs and tumor tissue-derived EVs. Moreover, subcellular localizations of quantified proteins showed a major distribution to the membrane which verifies our previous finding (Crescitelli et al JEV 2020).  

3.4024           PS17.05 NGS and Mass Spectrometry Profiles of Different Populations of Cancer-Derived EVs Overlap in Specific Pathways

Vagner, T., Chin, A., Kim, M., Kittel, A., Sagini, K., De Simone, M., Buzas, E., Yang, W., Thery, C., Van Keuren-Jensen, K. and Di Vizio, D.

17. Extracell. Vesicles, 10, S1, e12083, abstract PS17.05 (2021)

  Introduction: Extracellular vesicles (EVs) are important mediators of intercellular communication that can be analyzed via liquid biopsy in patient biological fluids. EVs are typically isolated and analyzed in bulk; however, they are highly heterogeneous in size, cargo, biogenesis, intracellular origin, and function. Specific markers for different EV populations are missing, limiting our understanding of EV functional and molecular diversity. This study stems from the need to identify EV-type-specific as well as general EV markers in 3 EV fractions purified from 3 different cancer cell lines by differential ultracentrifugation followed by discontinuous iodixanol gradient Methods: Differential centrifugation, discontinuous iodixanol density gradient, TRPS, TEM, LC-MS/MS, RNA-Seq Results: We first refined a protocol to separate large oncosomes (EVs >1μm diameter released by highly metastatic cancer cells) from other large EVs of the ectosomal origin. Quantitative proteomic analysis of 3 EV populations (2,8K, 10K, and 100K), obtained from prostate cancer, glioma, and breast cancer cell lines showed that several proteins frequently attributed to exosomes are in fact present in all EVs and can be used as general EV markers. The most distinct protein expression patterns were identified in the fractions containing the largest and the smallest EVs (2,8K and 100K), while the intermediate fraction (10K) represented a transition between the two. Protein and mRNA expression profiles mirrored each other in all EV types, with the 2.8K fraction showing the largest overlap between the corresponding protein and mRNA species. PCA analysis showed well separated clusters containing the EV fractions and their corresponding cells of origin. Additionally, some of the top pathways, such as oxidative phosphorylation and mitochondrial dysfunction, were enriched in the fraction with largest EVs both at the protein and transcript level. We found that ∼80% of the RNA reads were coding RNAs in all EV fractions but the non-coding RNAs seemed to be slightly enriched in 100K. Finally, we identified proteins that might be used as novel markers of EV populations Summary/Conclusion: Comprehensive proteomic and transcriptomic analyses identify specific mRNA and protein profiles in 2,8K and 100K suggesting that cancer pathways are conserved at the protein and RNA level  

3.4025           PS17.15 Antigen-induced eosinophilic respiratory inflammation alters the protein cargo of lung-derived EVs in mice

Lässer, C., Kishino, Y. and Lötvall, J.

17. Extracell. Vesicles, 10, S1, e12083, abstract PS17.15 (2021)

  Introduction: Analysis of the proteome of tissue-derived EVs is of great importance both to identify biomarkers of disease but also to understand cell-to-cell communication in diseased tissue. The aim of this study was to establish an isolation method that isolates lung vesicles of high purity for proteomic analysis and to determine the proteome of lung tissue-derived vesicles during an antigen-induced eosinophilic respiratory inflammation in mice. Methods: A mouse model for allergic asthma was used by sensitization and challenge of BALB/c mice to ovalbumin (OVA). Animals were sacrificed and lungs were removed and chopped in to smaller pieces that were incubated in media with DNase 1 and Collagenase D for 30 minutes at 37°C (Crescitelli et al JEV 2020). Vesicles were isolated from the medium by ultracentrifugation and bottom loaded iodixanol density cushion. Isolated vesicles were evaluated by electron microscopy (EM) and the proteome was analysed with mass spectrometry (LC-MS/MS, N∈-6). Results: Electron microscopy showed that the protocol isolated vesicles that where on average 40–200 nm in size. In total 4510 proteins were quantified in all samples. The identified proteins were analyzed with DAVID to identify enriched cellular components compared to the genome frequency, and the top associated terms were “Extracellular exosome” and “Membrane”. Principle component analysis showed that component 1, representing 40% of the variability, distinguished the OVA-EVs from the PBS-EVs. Over 1000 proteins were significantly altered (fold change >2 and p-value < 0.05), with 614 proteins being up-regulated and 425 proteins being down-regulated in OVA-EVs. The 614 proteins upregulated during allergen-induced inflammation was mainly associated with the GO biological processes terms; “ribosomal units”, “translation”, “mRNA processing”, “immune system processes”, “innate immune response”, “response to virus” and “B cell receptor signaling pathway”. The majority of the top-15 most upregulated proteins were associated the immune related terms. Summary/Conclusion: Extracellular vesicles can be isolated from mouse lung tissue and these vesicles are highly associated with previously identified proteins in extracellular vesicles. In EVs present in OVA/OVA mice immune associated proteins were upregulated reflecting the ongoing antigen-induced eosinophilic respiratory inflammation, suggesting that airway-derived EVs can be altered in diseases with inflammation of the lung, such as asthma.  

3.4026           PS24.06 Empowering the therapeutic potential of clinically expired platelet concentrates: optimization of platelet-derived extracellular vesicles isolation process

Salvador, D., Meliciano, A., Louro, A.F., Mendonca, P., Sousa, A.P. and Serra, M.

24. Extracell. Vesicles, 10, S1, e12083, abstract PS24.06 (2021)

  Introduction: Platelets have become key players in tissue regeneration due to their enriched content in bioactive growth factors and extracellular vesicles (EV), which can be used in several biomedical applications. However, platelet concentrates (PC) derived from whole blood are regularly produced in blood centers with relative short shelf-life for clinical applications. In this work, we used clinically expired PC with no therapeutic value, as a promising source of functional EV with high therapeutic potential. In particular, we evaluated different strategies for EV isolation from PC aiming at improving EV yields and purity. Methods: Three EV isolation strategies were evaluated: i) iodixanol-based gradient ultracentrifugation (DGUC), ii) size exclusion chromatography (SEC), and iii) the combination of these two methodologies (DGUC+SEC). Briefly, PC supernatants were ultracentrifuged and then processed either by DGUC or SEC. DGUC fractions with highest EV contents were further purified by SEC. All EV groups were characterized by nanoparticle tracking analysis, transmission electron microscopy and western blot (WB). Results: Cup-shaped EV with an intact lipid bilayer were obtained in all three strategies. The highest EV yields were attained by DGUC (4.48-1011 particle/mL) when compared to other strategies (9.82-1010 and 1.62-1010 particle/mL for SEC and DGUC+SEC, respectively). WB analysis showed contamination with Apolipoprotein A1 and Argonaute-2 in all EV groups, despite the presence of specific EV markers CD63 and TSG101 which were enriched in the DGUC and SEC samples  

3.4027           PS24.07 Optimized purification of EVs released by blood eating parasites: mini-gradients for EVs from limited sources

Kuipers, M., Hokke, C.H., Smits, H.H.and Nolte-‘tHoen. E.N.

24. Extracell. Vesicles, 10, S1, e12083, abstract PS24.07 (2021)

  Introduction: Isolation of extracellular vesicles (EVs) from blood eating parasites, such as Schistosoma mansoni, is challenging. The complex life cycle and the necessity to use animal hosts for maintaining the parasite limit the available material for EV isolation. Moreover, the biophysical and molecular properties of EVs differ between the parasite's life cycle stages and the immunogenic and toxic byproduct of hemoglobin digestion, hemozoin, contaminates adult worm EV populations enriched by ultracentrifugation. Therefore, we designed optimized protocols for isolation of Schistosoma-derived EVs. Methods: Medium of ex vivo cultured adult worms or in vitro transformed larvae was pre-processed by differential (ultra)centrifugation. We compared SEC and density gradients with sucrose or iodixanol for separation of EVs from non-EV products. Conventional gradients in SW55 (4.5 mL, 16 hours spin) were compared to mini-gradients in TLS55 (1.8 mL, 2 hours spin). Hemozoin was verified visually or by cryo EM. EVs were detected by western blot analysis for Tetraspanin 2. Results: EVs could be separated from non-EV products, including hemozoin, by density gradient centrifugation but not by ultracentrifugation or SEC. Larval EVs that contain filamentous structures on their surface were able to float efficiently into iodixanol but not into sucrose gradients, whereas adult worm EVs floated into both types of gradients. With the mini-gradients, pure EVs could be recovered in substantially reduced volumes, abolishing the need for additional EV concentration steps that decrease EV yield. Summary/Conclusion: Our data indicate that specific parasite EV subsets can display differential migration behavior in density gradients prepared with sucrose or iodixanol. We propose the use of mini-gradients for purifying EVs from limited sources, such as parasites, to increase EV yield while reducing handling time.  

3.4028           Localization of Organelle Proteins by Isotope Tagging: Current status and potential applications in drug discovery research

Elzek, M.A:W., Christopher, J.A., Breckels, L.M. and Lilley, K.S. Drug Discovery Today: Technologies, 39, 57-67 (2021)   Spatial proteomics has provided important insights into the relationship between protein function and subcellular location. Localization of Organelle Proteins by Isotope Tagging (LOPIT) and its variants are proteome-wide techniques, not matched in scale by microscopy-based or proximity tagging-based techniques, allowing holistic mapping of protein subcellular location and re-localization events downstream of cellular perturbations. LOPIT can be a powerful and versatile tool in drug discovery for unlocking important information on disease pathophysiology, drug mechanism of action, and off-target toxicity screenings. Here, we discuss technical concepts of LOPIT with its potential applications in drug discovery and development research.  

3.4029           Polymer-Coated Extracellular Vesicles for Selective Codelivery of Chemotherapeutics and siRNA to Cancer Cells

Jhan, Y-Y., Zuniga, G.P., Singh, K.A., Gaharwar, A.K., Alge, D.L. and Bishop, C.J. ACS Appl. Bio Mater., 4, 1294-1306 (2021)   Combination therapies involving small-interfering RNA (siRNA)-mediated gene silencing and small-molecule drugs are of high interest for cancer treatment. Among the current gene delivery carriers, cell-derived extracellular vesicles (EVs) are particularly promising candidates due to their high biocompatibility, low immunogenicity, in vivo stability, and inherent targeting ability. Here, we developed a multifunctional EV platform capable of selective codelivery of siRNA and doxorubicin (DOX) to cancer cells. siRNA was first loaded into engineered lipid-hybridized EVs (eEVs) to serve as a core. Subsequently, DOX was incorporated into a polyelectrolyte shell surrounding eEVs, which was deposited by layer-by-layer (LbL) assembly. This approach resulted in the production of a stable EV-polymer complex (LbL-eEV) with a diameter of 140.2 ± 9.0 nm and zeta potential of +22.1 ± 0.5 mV. Experiments were performed to assess cellular uptake, cytotoxicity, and gene silencing efficacy in lung adenocarcinoma cells (A549), with noncancerous fibroblast cells (CCL-210) used as a control. The results demonstrated that the LbL-eEV complex can traffic through cells and release siRNA in the cytoplasm, while delivered DOX enters nuclei to induce programmed cell death. Moreover, the inherent selectivity of the particles for cancer cells resulted in effective gene silencing and cancer killing efficiency with reduced cytotoxicity to normal cells. Synchronous delivery of siRNA and DOX was also verified by flow cytometry analysis of single cells. In summary, these data provide a proof of concept for engineering EVs to deliver multiple therapeutics and suggest that LbL-eEVs are a promising drug delivery platform for targeting cancer.  

3.4030           Advances in Analytical Technologies for Extracellular Vesicles

Yan, H., Li, Y., Cheng, S. and Zeng, Y. Anal. Chem., 93, 4739-4774 (2021)   No abstract available  

3.4031           A System-Wide Spatiotemporal Characterization of ErbB Receptor Complexes by Subcellular Fractionation Integrated Quantitative Mass Spectrometry

Wang, S., Zhang, C., Li, M., Zhao, C. and Zheng, Y. Anal. Chem., 93, 7933-7941 (2021)   Precise spatiotemporal regulation of protein complex assembly is essential for cells to achieve a meaningful rely of information flow via intracellular signaling networks in response to extracellular cues, whose disruption would lead to disease. Although various attempts have been made for spatial and/or temporal analysis of protein complexes, it is still a challenge to track cell-wide dynamics of a particular protein complex under physiological conditions. Here we describe a workflow that combines endogenous expression of tagged proteins, organelle marker distribution-directed subcellular fractionation, scaffold protein-mediated receptor complex purification, and targeted proteomics for spatiotemporal quantification of protein complexes in whole cell scale. We applied our method to investigate the assembly kinetics of EGF-dependent ErbB receptor complexes. After fractionation using the density gradient centrifugation and organelle assignment based on organelle markers, endogenous ErbB complex in different subcellular fractionation was efficiently enriched. By using targeted mass spectrometry, ErbB complex components that expressed medium to low level was precisely quantified with in-depth coverage, simultaneously in time and subcellular spaces. Our results revealed a sophisticated scheme of complex behaviors characterized by multiple subcomplexes with distinct molecular composition formed across subcellular fractions enriched with cytosol, plasma membrane, endosome, or mitochondria, implying organelle-specific ErbB functions. Remarkably, our results demonstrated for the first time that activated ErbB receptors might increase their signaling range through promoting a cytosolic, receptor-free subcomplex, consisting of Shc1, Grb2, Arhgef5, Garem1, and Lrrk1. These findings emphasize the potential of our strategy as a powerful tool to study spatiotemporal dynamics of protein complexes.  

3.4032           Self-Assembled Growing DNA Tree Mediated by Exosomes for Amplified Imaging of Messenger RNA in Living Cells

Fang, C., Li, Y., Hu, S., Wang, H., Chen, X. and Zhu, X. Anal. Chem., 93, 8414-8422 (2021)   Sensitive, accurate, and nondestructive probing of endogenous messenger RNA (mRNA) in living cells places extremely high demands on nanocarriers and probes and is still a challenge. In the present study, we describe a target-triggered self-assembled DNA tree for amplified analysis of mRNA in intact living cells. The probes assembled into a DNA tree are transported into cells by exosomes, which is beneficial for reducing cell damage and realizing nondestructive analysis. The probes are l-configured single-stranded DNAs (LDNAs) that can resist the degradation of exonuclease and endonuclease, thus laying the foundation for accurate analysis. Under the induction of the target mRNA, the probes in the cells assemble into a small plantlet and eventually grow into a tree after a few rounds of self-cycling, achieving the exponential amplification of fluorescence signals. Compared with the signal amplification based on one-dimensional DNA trunk self-assembly, the three-dimensional DNA tree shows an excellent sensitivity both ex situ and in situ. In this way, favorable sensitivity, accuracy, and nondestructive analysis are integrated into one system. This DNA tree expands the analysis platform for analyzing more biomarkers on a genetic level in an intracellular, nondestructive, and hypersensitive manner and holds great potential in clinical diagnostic and research applications.  

3.4033           Separation of distinct exosome subpopulations: isolation and characterization approaches and their associated challenges

Singh, K., Nalabotala, R., Koo, K.M., Bose, S., Nayak, R. and Shiddiky, M.J.A. Analyst, 146, 3751-3749 (2021)   Exosomes are nano-sized extracellular vesicles that serve as a communications system between cells and have shown tremendous promise as liquid biopsy biomarkers in diagnostic, prognostic, and even therapeutic use in different human diseases. Due to the natural heterogeneity of exosomes, there is a need to separate exosomes into distinct biophysical and/or biochemical subpopulations to enable full interrogation of exosome biology and function prior to the possibility of clinical translation. Currently, there exists a multitude of different exosome isolation and characterization approaches which can, in limited capacity, separate exosomes based on biophysical and/or biochemical characteristics. While notable reviews in recent years have reviewed these approaches for bulk exosome sorting, we herein present a comprehensive overview of various conventional technologies and modern microfluidic and nanotechnological advancements towards isolation and characterization of exosome subpopulations. The benefits and limitations of these different technologies to improve their use for distinct exosome subpopulations in clinical practices are also discussed. Furthermore, an overview of the most commonly encountered technical and biological challenges for effective separation of exosome subpopulations is presented.    

3.4034           Application of centrifugal microfluidics in immunoassay, biochemical analysis and molecular diagnosis

Shi, Y., Ye, P., Yang, K., Meng, J., Guo, J., Pan, Z., Zhao, W. and Guo, J. Analyst, 146, 5800-5821 (2021)   Rapid diagnosis plays a vital role in daily life and is effective in reducing treatment costs and increasing curability, especially in remote areas with limited availability of resources. Among the various common methods of rapid diagnosis, centrifugal microfluidics has many unique advantages, such as less sample consumption, more precise valve control for sequential loading of samples, and accurately separated module design in a microfluidic network to minimize cross-contamination. Therefore, in recent years, centrifugal microfluidics has been extensively researched, and it has been found to play important roles in biology, chemistry, and medicine. Here, we review the latest developments in centrifugal microfluidic platforms in immunoassays, biochemical analyses, and molecular diagnosis, in recent years. In immunoassays, we focus on the application of enzyme-linked immunosorbent assay (ELISA); in biochemical analysis, we introduce the application of plasma and blood cell separation; and in molecular diagnosis, we highlight the application of nucleic acid amplification tests. Additionally, we discuss the characteristics of the methods under each platform as well as the enhancement of the corresponding performance parameters, such as the limit of detection, separation efficiency, etc. Finally, we discuss the limitations associated with the existing applications and potential breakthroughs that can be achieved in this field in the future.  

3.4035           Long-lived mice with reduced growth hormone signaling have a constitutive upregulation of hepatic chaperone-mediated autophagy

Endicott, S.J., Boynton jr., D.N., Beckmann, L.J. and Miller, R.A. Autophagy, 17(3), 612-625 (2021)   Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis. CMA modulates proteomic organization through selective protein degradation, with targets including metabolic enzymes, cell growth regulators, and neurodegeneration-related proteins. CMA activity is low in ad libitum-fed rodents but is increased by prolonged fasting. AKT negatively regulates CMA at the lysosomal membrane by phosphorylating and inhibiting the CMA regulator GFAP. We have previously reported that long-lived Pou1f1/Pit1 mutant (Snell) mice and ghr (growth hormone receptor) knockout mice (ghr KO) have lower AKT activity when fed compared to littermate controls, suggesting the hypothesis that these mice have increased baseline CMA activity. Here, we report that liver lysosomes from fed Snell dwarf mice and ghr KO mice have decreased GFAP phosphorylation and increased CMA substrate uptake activity. Liver lysosomes isolated from fed Snell dwarf mice and ghr KO mice injected with the protease inhibitor leupeptin had increased accumulation of endogenous CMA substrates, compared to littermate controls, suggesting an increase in CMA in vivo. Mice with liver-specific ablation of GH (growth hormone) signaling did not have increased liver CMA, suggesting that a signaling effect resulting from a loss of growth hormone in another tissue causes enhanced CMA in Snell dwarf and ghr KO mice. Finally, we find Snell dwarf mice have decreased protein levels (in liver and kidney) of CIP2A, a well-characterized CMA target protein, without an associated change in Cip2a mRNA. Collectively, these data suggest that CMA is enhanced downstream of an endocrine change resulting from whole-body ablation of GH signaling.  

3.4036           RETREG1/FAM134B mediated autophagosomal degradation of AMFR/GP78 and OPA1 —a dual organellar turnover mechanism

Mookherjee, D., Das, S., Mukherjee, R., Bera, M., Jana, S.C., Chakrabarti, S. and Chakrabarti, O. Autophagy, 17(7), 1729-1752 (2021)   Turnover of cellular organelles, including endoplasmic reticulum (ER) and mitochondria, is orchestrated by an efficient cellular surveillance system. We have identified a mechanism for dual regulation of ER and mitochondria under stress. It is known that AMFR, an ER E3 ligase and ER-associated degradation (ERAD) regulator, degrades outer mitochondrial membrane (OMM) proteins, MFNs (mitofusins), via the proteasome and triggers mitophagy. We show that destabilized mitochondria are almost devoid of the OMM and generate “mitoplasts”. This brings the inner mitochondrial membrane (IMM) in the proximity of the ER. When AMFR levels are high and the mitochondria are stressed, the reticulophagy regulatory protein RETREG1 participates in the formation of the mitophagophore by interacting with OPA1. Interestingly, OPA1 and other IMM proteins exhibit similar RETREG1-dependent autophagosomal degradation as AMFR, unlike most of the OMM proteins. The “mitoplasts” generated are degraded by reticulo-mito-phagy – simultaneously affecting dual organelle turnover  

3.4037           A tecpr2 knockout mouse exhibits age-dependent neuroaxonal dystrophy associated with autophagosome accumulation

Tamin-Yecheskel, B-C., Fraiberg, M., Kokabi, K., Freud, S., Shatz, O. et al Autophagy, 17(10), 3082-3095 (2021)   Mutations in the coding sequence of human TECPR2 were recently linked to spastic paraplegia type 49 (SPG49), a hereditary neurodegenerative disorder involving intellectual disability, autonomic-sensory neuropathy, chronic respiratory disease and decreased pain sensitivity. Here, we report the generation of a novel CRISPR-Cas9 tecpr2 knockout (tecpr2^−/−) mouse that exhibits behavioral pathologies observed in SPG49 patients. tecpr2^−/− mice develop neurodegenerative patterns in an age-dependent manner, manifested predominantly as neuroaxonal dystrophy in the gracile (GrN) and cuneate nuclei (CuN) of the medulla oblongata in the brainstem and dorsal white matter column of the spinal cord. Age-dependent correlation with accumulation of autophagosomes suggests compromised targeting to lysosome. Taken together, our findings establish the tecpr2 knockout mouse as a potential model for SPG49 and ascribe a new role to TECPR2 in macroautophagy/autophagy-related neurodegenerative disorders.  

3.4038           IFIT3 (interferon induced protein with tetratricopeptide repeats 3) modulates STAT1 expression in small extracellular vesicles

Naranjo, N.M., Salem, I., Harris, M.A. and Linguino, L.R. Biochem. J., 478, 3305-3921 (2021)   We have previously shown that the αvβ6 integrin plays a key role in promoting prostate cancer (PrCa) and it can be transferred to recipient cells via small extracellular vesicles (sEVs). Furthermore, we have reported in a proteomic analysis that αvβ6 integrin down-regulation increases the expression of IFIT3 (interferon induced protein with tetratricopeptide repeats 3) in PrCa cells and their derived sEVs. IFIT3 is a protein well known for being an antiviral effector, but recently its role in cancer has also been elucidated. To study the relationship between IFIT3 and STAT1 (signal transducer and activator of transcription 1), an upstream regulator of IFIT3, in PrCa cells and their released sEVs, we used CRISPR/Cas9 techniques to down-regulate the expression of the β6 integrin subunit, IFIT3 or STAT1. Our results show that IFIT3 and STAT1 are highly expressed in PrCa cells devoid of the β6 integrin subunit. However, IFIT3 but not STAT1, is present in sEVs derived from PrCa cells lacking the β6 integrin subunit. We demonstrate that loss of IFIT3 generates sEVs enriched in STAT1 but reduces the levels of STAT1 in the cells. As expected, IFIT3 is not detectable in STAT1 negative cells or sEVs. We thus propose that the observed STAT1 enrichment in sEVs is a compensatory mechanism for the loss of IFIT3. Overall, these results provide new insights into the intrinsic role of IFIT3 as a regulator of STAT1 expression in sEVs and in intercellular communication in PrCa.  

3.4039           BIG1 mediates sepsis-induced lung injury by modulating lipid raft-dependent macrophage inflammatory responses

Sun, M., Han, X., Zhou, D., Zhong, J., Liu, L., Wang, Y., Ni, J., Shen, X., Liang, C. and Fang, H. Acta Biochim. Biophys. Sin., 53(8), 1088-1097 (2021)   Sepsis is a systemic inflammatory response syndrome with high mortality. It has been reported that brefeldin A-inhibited guanine nucleotide-exchange factor 1 (BIG1) is involved in the pathogenesis of sepsis. However, the mechanism is not fully elucidated. In the present study, we explored the role of BIG1 in mediating lipid raft-dependent macrophage inflammatory response and its impact on lung injury in murine sepsis. In vitro studies revealed that BIG1 deficiency reduces the upregulation and secretion of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-1β and inhibits the activation of the toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88-dependent nuclear factor kappa-B signaling pathway induced by the lipopolysaccharide (LPS) treatment. Further experiments revealed that the inhibitory effects of BIG1 deficiency on LPS-induced inflammation are due to the upregulation of adenosine triphosphate-binding cassette transporter A1. This promotes the free-cholesterol efflux from lipid rafts and results in the reduction of lipid raft TLR4 content. The decrease in TLR4 content in lipid raft thereby inhibits the LPS-induced inflammatory response. Furthermore, using the cecal ligation and puncture-induced polymicrobial sepsis mouse model, we found that conditional knockout (cKO) of the myeloid cell BIG1 significantly reduced the serum concentrations of TNF-α, IL-6, and IL-1β, and downregulated their mRNA expressions in the lungs. Pathological analysis confirmed that the BIG1 cKO alleviated the sepsis-induced lung injury. These results revealed the crucial new role of BIG1 in mediating lipid raft-dependent macrophage inflammatory response. Hence, BIG1 may be a potential promising therapeutic target for the treatment of septic lung injury.  

3.4040           Ginsenoside 20(S)-Rh2 promotes cellular pharmacokinetics and intracellular antibacterial activity of levofloxacin against Staphylococcus aureus through drug efflux inhibition and subcellular stabilization

Chen, X-y., Qian, F., Wang, Y-y., Liu, Y., Sun, Y., Zha, W-b., Hao, K., Zhou, F. Wang, G-j. and Zhang, J-w. Acta Pharmacologica. Sinica., 42, 1930-1941 (2021)   Intracellular Staphylococcus aureus (S. aureus) often causes clinical failure and relapse after antibiotic treatment. We previously found that 20(S)-ginsenoside Rh2 [20(S)-Rh2] enhanced the therapeutic effect of quinolones in a mouse model of peritonitis, which we attributed to the increased concentrations of quinolones within bacteria. In this study, we investigated the enhancing effect of 20(S)-Rh2 on levofloxacin (LVF) from a perspective of intracellular bacteria. In S. aureus 25923-infected mice, coadministration of LVF (1.5 mg/kg, i.v.) and 20(S)-Rh2 (25, 50 mg/kg, i.g.) markedly increased the survival rate, and decreased intracellular bacteria counts accompanied by increased accumulation of LVF in peritoneal macrophages. In addition, 20(S)-Rh2 (1, 5, 10 μM) dose-dependently increased the uptake and accumulation of LVF in peritoneal macrophages from infected mice without drug treatment. In a model of S. aureus 25923-infected THP-1 macrophages, we showed that 20(S)-Rh2 (1, 5, 10 μM) dose-dependently enhanced the intracellular antibacterial activity of LVF. At the cellular level, 20(S)-Rh2 increased the intracellular accumulation of LVF by inhibiting P-gp and BCRP. PK–PD modeling revealed that 20(S)-Rh2 altered the properties of the cell but not LVF. At the subcellular level, 20(S)-Rh2 did not increase the distribution of LVF in lysosomes but exhibited a stronger sensitizing effect in acidic environments. Molecular dynamics (MD) simulations showed that 20(S)-Rh2 improved the stability of the DNA gyrase–LVF complex in lysosome-like acidic conditions. In conclusion, 20(S)-Rh2 promotes the cellular pharmacokinetics and intracellular antibacterial activities of LVF against S. aureus through efflux transporter inhibition and subcellular stabilization, which is beneficial for infection treatment.  

3.4041           Meningeal lymphatics affect microglia responses and anti-Aβ immunotherapy

Da Mesquita, S., Papadopoulos, Z., Dykstra, T., Brase, L., Farias, F.G. et al Nature, 593, 255-260 (2021)   Alzheimer’s disease (AD) is the most prevalent cause of dementia1. Although there is no effective treatment for AD, passive immunotherapy with monoclonal antibodies against amyloid beta (Aβ) is a promising therapeutic strategy2^,3. Meningeal lymphatic drainage has an important role in the accumulation of Aβ in the brain4, but it is not known whether modulation of meningeal lymphatic function can influence the outcome of immunotherapy in AD. Here we show that ablation of meningeal lymphatic vessels in 5xFAD mice (a mouse model of amyloid deposition that expresses five mutations found in familial AD) worsened the outcome of mice treated with anti-Aβ passive immunotherapy by exacerbating the deposition of Aβ, microgliosis, neurovascular dysfunction, and behavioural deficits. By contrast, therapeutic delivery of vascular endothelial growth factor C improved clearance of Aβ by monoclonal antibodies. Notably, there was a substantial overlap between the gene signature of microglia from 5xFAD mice with impaired meningeal lymphatic function and the transcriptional profile of activated microglia from the brains of individuals with AD. Overall, our data demonstrate that impaired meningeal lymphatic drainage exacerbates the microglial inflammatory response in AD and that enhancement of meningeal lymphatic function combined with immunotherapies could lead to better clinical outcomes.  

3.4042           Dysregulation of brain and choroid plexus cell types in severe COVID-19

Yang, A.C., Kern, F., Losada, P.M., Agam, M.R., Maat, C.A., Schmartz, G.P. et al Nature, 595, 565-571 (2021)   Although SARS-CoV-2 primarily targets the respiratory system, patients with and survivors of COVID-19 can suffer neurological symptoms1^,2^,3. However, an unbiased understanding of the cellular and molecular processes that are affected in the brains of patients with COVID-19 is missing. Here we profile 65,309 single-nucleus transcriptomes from 30 frontal cortex and choroid plexus samples across 14 control individuals (including 1 patient with terminal influenza) and 8 patients with COVID-19. Although our systematic analysis yields no molecular traces of SARS-CoV-2 in the brain, we observe broad cellular perturbations indicating that barrier cells of the choroid plexus sense and relay peripheral inflammation into the brain and show that peripheral T cells infiltrate the parenchyma. We discover microglia and astrocyte subpopulations associated with COVID-19 that share features with pathological cell states that have previously been reported in human neurodegenerative disease4^,5^,6. Synaptic signalling of upper-layer excitatory neurons—which are evolutionarily expanded in humans7 and linked to cognitive function8—is preferentially affected in COVID-19. Across cell types, perturbations associated with COVID-19 overlap with those found in chronic brain disorders and reside in genetic variants associated with cognition, schizophrenia and depression. Our findings and public dataset provide a molecular framework to understand current observations of COVID-19-related neurological disease, and any such disease that may emerge at a later date.  

3.4043           Genetic and epigenetic coordination of cortical interneuron development

Allaway,. K.C., Gabitto, M.I., Wapinsky, O., Saldi, G., Wang, C-Y., Bandler, R.C., Wu, S.J., Bonneau, R. and Fishell, G. Nature, 597, 693-697 (2021)   One of the hallmarks of the cerebral cortex is the extreme diversity of interneurons1^,2^,3. The two largest subtypes of cortical interneurons, parvalbumin- and somatostatin-positive cells, are morphologically and functionally distinct in adulthood but arise from common lineages within the medial ganglionic eminence4^,5^,6^,7^,8^,9^,10^,11. This makes them an attractive model for studying the generation of cell diversity. Here we examine how developmental changes in transcription and chromatin structure enable these cells to acquire distinct identities in the mouse cortex. Generic interneuron features are first detected upon cell cycle exit through the opening of chromatin at distal elements. By constructing cell-type-specific gene regulatory networks, we observed that parvalbumin- and somatostatin-positive cells initiate distinct programs upon settling within the cortex. We used these networks to model the differential transcriptional requirement of a shared regulator, Mef2c, and confirmed the accuracy of our predictions through experimental loss-of-function experiments. We therefore reveal how a common molecular program diverges to enable these neuronal subtypes to acquire highly specialized properties by adulthood. Our methods provide a framework for examining the emergence of cellular diversity, as well as for quantifying and predicting the effect of candidate genes on cell-type-specific development.  

3.4044           An endogenous opioid circuit determines state-dependent reward consumption

Castro, D.C., Oswell, C.S., Zhang, E.T., Pedersen, C.E., Piantadosi, S.C., Rossi, M.A., Hunker, A.C., Guglin, A., Moron, J.A., Zweifel, L.S., Stuber, G.D. and Bruchas, M.R. Nature, 598, 646-651 (2021)   µ-Opioid peptide receptor (MOPR) stimulation alters respiration, analgesia and reward behaviour, and can induce substance abuse and overdose1^,2^,3. Despite its evident importance, the endogenous mechanisms for MOPR regulation of consummatory behaviour have remained unknown4. Here we report that endogenous MOPR regulation of reward consumption in mice acts through a specific dorsal raphe to nucleus accumbens projection. MOPR-mediated inhibition of raphe terminals is necessary and sufficient to determine consummatory response, while select enkephalin-containing nucleus accumbens ensembles are engaged prior to reward consumption, suggesting that local enkephalin release is the source of the endogenous MOPR ligand. Selective modulation of nucleus accumbens enkephalin neurons and CRISPR–Cas9-mediated disruption of enkephalin substantiate this finding. These results isolate a fundamental endogenous opioid circuit for state-dependent consumptive behaviour and suggest alternative mechanisms for opiate modulation of reward.  

3.4045           Oestrogen engages brain MC4R signalling to drive physical activity in female mice

Krause, W.C., Rodriguez, R., Gegenhuber, B., Matharu, N., Rodriguez, A.N., Padilla-Roger, A.M., Toma, K., Herber, C.B., Correa, S.M., Duan, X., Ahituv, N. and Tollkuhn, J. Nature, 599, 131-135 (2021)   Oestrogen depletion in rodents and humans leads to inactivity, fat accumulation and diabetes1^,2, underscoring the conserved metabolic benefits of oestrogen that inevitably decrease with age. In rodents, the preovulatory surge in 17β-oestradiol (E2) temporarily increases energy expenditure to coordinate increased physical activity with peak sexual receptivity. Here we report that a subset of oestrogen-sensitive neurons in the ventrolateral ventromedial hypothalamic nucleus (VMHvl)3^,4^,5^,6^,7 projects to arousal centres in the hippocampus and hindbrain, and enables oestrogen to rebalance energy allocation in female mice. Surges in E2 increase melanocortin-4 receptor (MC4R) signalling in these VMHvl neurons by directly recruiting oestrogen receptor-α (ERα) to the Mc4r gene. Sedentary behaviour and obesity in oestrogen-depleted female mice were reversed after chemogenetic stimulation of VMHvl neurons expressing both MC4R and ERα. Similarly, a long-term increase in physical activity is observed after CRISPR-mediated activation of this node. These data extend the effect of MC4R signalling — the most common cause of monogenic human obesity8 — beyond the regulation of food intake and rationalize reported sex differences in melanocortin signalling, including greater disease severity of MC4R insufficiency in women9. This hormone-dependent node illuminates the power of oestrogen during the reproductive cycle in motivating behaviour and maintaining an active lifestyle in women.  

3.4046           Temporal transitions in the post-mitotic nervous system of Caenorhabditis elegans

Sun, H. and Hobert, O. Nature, 600, 93-99 (2021)   In most animals, the majority of the nervous system is generated and assembled into neuronal circuits during embryonic development1. However, during juvenile stages, nervous systems still undergo extensive anatomical and functional changes to eventually form a fully mature nervous system by the adult stage2^,3. The molecular changes in post-mitotic neurons across post-embryonic development and the genetic programs that control these temporal transitions are not well understood4^,5. Here, using the model system Caenorhabditis elegans, we comprehensively characterized the distinct functional states (locomotor behaviour) and the corresponding distinct molecular states (transcriptome) of the post-mitotic nervous system across temporal transitions during post-embryonic development. We observed pervasive, neuron-type-specific changes in gene expression, many of which are controlled by the developmental upregulation of the conserved heterochronic microRNA LIN-4 and the subsequent promotion of a mature neuronal transcriptional program through the repression of its target, the transcription factor lin-14. The functional relevance of these molecular transitions are exemplified by a temporally regulated target gene of the LIN-14 transcription factor, nlp-45, a neuropeptide-encoding gene, which we find is required for several distinct temporal transitions in exploratory activity during post-embryonic development. Our study provides insights into regulatory strategies that control neuron-type-specific gene batteries to modulate distinct behavioural states across temporal, sexual and environmental dimensions of post-embryonic development.  

3.4047           Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through an NGFR-dependent mechanism

Garcia-Silva, S., Benito-Martin, A., Nogues, L., Hernandez-Barranco, A., Mazariegos, M.S. et al Nature Cancer, 2, 1387-1405 (2021)   Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor (NGF) receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR⁺ metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis.  

3.4048           Single-cell analysis reveals transcriptomic remodellings in distinct cell types that contribute to human prostate cancer progression

Chen, S., Zhu, G., Yang, Y., Wang, F., Xiao, Y-T., Zhang, N. et al Nature Cell Biol., 23, 87-98 (2021)   Prostate cancer shows remarkable clinical heterogeneity, which manifests in spatial and clonal genomic diversity. By contrast, the transcriptomic heterogeneity of prostate tumours is poorly understood. Here we have profiled the transcriptomes of 36,424 single cells from 13 prostate tumours and identified the epithelial cells underlying disease aggressiveness. The tumour microenvironment (TME) showed activation of multiple progression-associated transcriptomic programs. Notably, we observed promiscuous KLK3 expression and validated the ability of cancer cells in altering T-cell transcriptomes. Profiling of a primary tumour and two matched lymph nodes provided evidence that KLK3 ectopic expression is associated with micrometastases. Close cell–cell communication exists among cells. We identified an endothelial subset harbouring active communication (activated endothelial cells, aECs) with tumour cells. Together with sequencing of an additional 11 samples, we showed that aECs are enriched in castration-resistant prostate cancer and promote cancer cell invasion. Finally, we created a user-friendly web interface for users to explore the sequenced data.  

3.4049           Quantitative proteomics identifies the core proteome of exosomes with syntenin-1 as the highest abundant protein and a putative universal biomarker

Kugeratski, F.G., Hodge, K., Lilla, S., McAndrews, K.M., Zhou, X., Hwang, R., Zanivan, S. and Kalluri, R. Nature Cell Biol., 23, 631-641 (2021)   Exosomes are extracellular vesicles derived from the endosomal compartment that are potentially involved in intercellular communication. Here, we found that frequently used biomarkers of exosomes are heterogeneous, and do not exhibit universal utility across different cell types. To uncover ubiquitous and abundant proteins, we used an unbiased and quantitative proteomic approach based on super-stable isotope labeling with amino acids in cell culture (super-SILAC), coupled to high-resolution mass spectrometry. In total, 1,212 proteins were quantified in the proteome of exosomes, irrespective of the cellular source or isolation method. A cohort of 22 proteins was universally enriched. Fifteen proteins were consistently depleted in the proteome of exosomes compared to cells. Among the enriched proteins, we identified biogenesis-related proteins, GTPases and membrane proteins, such as CD47 and ITGB1. The cohort of depleted proteins in exosomes was predominantly composed of nuclear proteins. We identified syntenin-1 as a consistently abundant protein in exosomes from different cellular origins. Syntenin-1 is also present in exosomes across different species and biofluids, highlighting its potential use as a putative universal biomarker of exosomes. Our study provides a comprehensive quantitative atlas of core proteins ubiquitous to exosomes that can serve as a resource for the scientific community.  

3.4050           Molecular characterization of selectively vulnerable neurons in Alzheimer’s disease

Leng, K., Li, E., Eser, R., Piergies, A., Sit, R., Tan, M. et al Nature Neurosci., 24, 276-287 (2021)   Alzheimer’s disease (AD) is characterized by the selective vulnerability of specific neuronal populations, the molecular signatures of which are largely unknown. To identify and characterize selectively vulnerable neuronal populations, we used single-nucleus RNA sequencing to profile the caudal entorhinal cortex and the superior frontal gyrus—brain regions where neurofibrillary inclusions and neuronal loss occur early and late in AD, respectively—from postmortem brains spanning the progression of AD-type tau neurofibrillary pathology. We identified RORB as a marker of selectively vulnerable excitatory neurons in the entorhinal cortex and subsequently validated their depletion and selective susceptibility to neurofibrillary inclusions during disease progression using quantitative neuropathological methods. We also discovered an astrocyte subpopulation, likely representing reactive astrocytes, characterized by decreased expression of genes involved in homeostatic functions. Our characterization of selectively vulnerable neurons in AD paves the way for future mechanistic studies of selective vulnerability and potential therapeutic strategies for enhancing neuronal resilience.  

3.4051           A distinct giant coat protein complex II vesicle population in Arabidopsis thaliana

Li, B., Zeng, Y., Cao, W., Zhang, W., Cheng, L., Yin, H., Wu, Q., Wang, X., Huang, Y., Lau, W.C.Y., Yao, Z-P., Guo, Y. and Jiang, L. Nature Plants, 7, 1335-1346 (2021)   Plants live as sessile organisms with large-scale gene duplication events and subsequent paralogue divergence during evolution. Notably, plant paralogues are expressed tissue-specifically and fine-tuned by phytohormones during various developmental processes. The coat protein complex II (COPII) is a highly conserved vesiculation machinery mediating protein transport from the endoplasmic reticulum to the Golgi apparatus in eukaryotes1. Intriguingly, Arabidopsis COPII paralogues greatly outnumber those in yeast and mammals2^,3^,4^,5^,6. However, the functional diversity and underlying mechanism of distinct COPII paralogues in regulating protein endoplasmic reticulum export and coping with various adverse environmental stresses are poorly understood. Here we characterize a novel population of COPII vesicles produced in response to abscisic acid, a key phytohormone regulating abiotic stress responses in plants. These hormone-induced giant COPII vesicles are regulated by an Arabidopsis-specific COPII paralogue and carry stress-related channels/transporters for alleviating stresses. This study thus provides a new mechanism underlying abscisic acid-induced stress responses via the giant COPII vesicles and answers a long-standing question on the evolutionary significance of gene duplications in Arabidopsis.  

3.4052           Recombinant extracellular vesicles as biological reference material for method development, data normalization and assessment of (pre-)analytical variables

Geeurickx, E., Lippens, L., Rappu, P., De Geest, B.G., De Wever, O. and Hendrix, A. Nature Protocols, 16, 603-633 (2021)   The diagnostic and therapeutic use of extracellular vesicles (EV) is under intense investigation and may lead to societal benefits. Reference materials are an invaluable resource for developing, improving and assessing the performance of regulated EV applications and for quantitative and objective data interpretation. We have engineered recombinant EV (rEV) as a biological reference material. rEV have similar biochemical and biophysical characteristics to sample EV and function as an internal quantitative and qualitative control throughout analysis. Spiking rEV in bodily fluids prior to EV analysis maps technical variability of EV applications and promotes intra- and inter-laboratory studies. This protocol, which is an Extension to our previously published protocol (Tulkens et al., 2020), describes the production, separation and quality assurance of rEV, their dilution and addition to bodily fluids, and the detection steps based on complementary fluorescence, nucleic acid and protein measurements. We demonstrate the use of rEV for method development, data normalization and assessment of pre-analytical variables. The protocol can be adopted by researchers with standard laboratory and basic EV separation/characterization experience and requires ~4–5 d.  

3.4053           Isolation and characterization of extracellular vesicle subpopulations from tissues

Crescitelli, R., Lässer, C. and Lötvall, J. Nature Protocols, 16, 1548-1580 (2021)   Extracellular vesicles (EVs) are lipid bilayered membrane structures released by all cells. Most EV studies have been performed by using cell lines or body fluids, but the number of studies on tissue-derived EVs is still limited. Here, we present a protocol to isolate up to six different EV subpopulations directly from tissues. The approach includes enzymatic treatment of dissociated tissues followed by differential ultracentrifugation and density separation. The isolated EV subpopulations are characterized by electron microscopy and RNA profiling. In addition, their protein cargo can be determined with mass spectrometry, western blot and ExoView. Tissue-EV isolation can be performed in 22 h, but a simplified version can be completed in 8 h. Most experiments with the protocol have used human melanoma metastases, but the protocol can be applied to other cancer and non-cancer tissues. The procedure can be adopted by researchers experienced with cell culture and EV isolation.  

3.4054           Extraction of nuclei from archived postmortem tissues for single-nucleus sequencing applications

Maitra, M., Nagy, C., Chawla, A., Wang, Y.C., Nascimento, C., Sudermann, M., Theroux, J-F., Mechawar, N., Ragoussis, J. and Turecki, G. Nature Protocols, 16, 2788-2801 (2021)   Single-cell and single-nucleus sequencing techniques are a burgeoning field with various biological, biomedical and clinical applications. Numerous high- and low-throughput methods have been developed for sequencing the RNA and DNA content of single cells. However, for all these methods, the key requirement is high-quality input of a single-cell or single-nucleus suspension. Preparing such a suspension is the limiting step when working with fragile, archived tissues of variable quality. This hurdle can prevent such tissues from being extensively investigated with single-cell technologies. We describe a protocol for preparing single-nucleus suspensions within the span of a few hours that reliably works for multiple postmortem and archived tissue types using standard laboratory equipment. The stages of the protocol include tissue preparation and dissociation, nuclei extraction, and nuclei concentration assessment and capture. The protocol is comparable to other published protocols but does not require fluorescence-assisted nuclei sorting (FANS) or ultracentrifugation. The protocol can be carried out by a competent graduate student familiar with basic laboratory techniques and equipment. Moreover, these preparations are compatible with single-nucleus (sn)RNA-seq and assay for transposase-accessible chromatin (ATAC)-seq using the 10X Genomics Chromium system. The protocol reliably results in efficient capture of single nuclei for high-quality snRNA-seq libraries.  

3.4055           Ecological significance of extracellular vesicles in modulating host-virus interactions during algal blooms

Schatz, D., Schleyer, G., Saltvedt, M.R., Sandaa, R-A., Feldmesser, e. and Vardi, A. ISME J., 15, 3713-3721 (2021)   Extracellular vesicles are produced by organisms from all kingdoms and serve a myriad of functions, many of which involve cell-cell signaling, especially during stress conditions and host-pathogen interactions. In the marine environment, communication between microorganisms can shape trophic level interactions and population succession, yet we know very little about the involvement of vesicles in these processes. In a previous study, we showed that vesicles produced during viral infection by the ecologically important model alga Emiliania huxleyi, could act as a pro-viral signal, by expediting infection and enhancing the half-life of the virus in the extracellular milieu. Here, we expand our laboratory findings and show the effect of vesicles on natural populations of E. huxleyi in a mesocosm setting. We profile the small-RNA (sRNA) cargo of vesicles that were produced by E. huxleyi during bloom succession, and show that vesicles applied to natural assemblages expedite viral infection and prolong the half-life of this major mortality agent of E. huxleyi. We subsequently reveal that exposure of the natural assemblage to E. huxleyi-derived vesicles modulates not only host-virus dynamics, but also other components of the microbial food webs, thus emphasizing the importance of extracellular vesicles to microbial interactions in the marine environment.  

3.4056           Plasma and vacuolar membrane sphingolipidomes: composition and insights on the role of main molecular species

Carmona-Salazar, L., Cahoon, R.E., Gasca-Pineda, J., Gonzalez-Solis, A., Vera-Estrella, R., Trevino, V., Cahoon, E.B. and Gavilanes-Ruiz, M. Plant Physiology, 186, 624-639 (2021)   Lipid structures affect membrane biophysical properties such as thickness, stability, permeability, curvature, fluidity, asymmetry, and interdigitation, contributing to membrane function. Sphingolipids are abundant in plant endomembranes and plasma membranes (PMs) and comprise four classes: ceramides, hydroxyceramides, glucosylceramides, and glycosylinositolphosphoceramides (GIPCs). They constitute an array of chemical structures whose distribution in plant membranes is unknown. With the aim of describing the hydrophobic portion of sphingolipids, 18 preparations from microsomal (MIC), vacuolar (VM), PM, and detergent-resistant membranes (DRM) were isolated from Arabidopsis (Arabidopsis thaliana) leaves. Sphingolipid species, encompassing pairing of long-chain bases and fatty acids, were identified and quantified in these membranes. Sphingolipid concentrations were compared using univariate and multivariate analysis to assess sphingolipid diversity, abundance, and predominance across membranes. The four sphingolipid classes were present at different levels in each membrane: VM was enriched in glucosylceramides, hydroxyceramides, and GIPCs; PM in GIPCs, in agreement with their key role in signal recognition and sensing; and DRM in GIPCs, as reported by their function in nanodomain formation. While a total of 84 sphingolipid species was identified in MIC, VM, PM, and DRM, only 34 were selectively distributed in the four membrane types. Conversely, every membrane contained a different number of predominant species (11 in VM, 6 in PM, and 17 in DRM). This study reveals that MIC, VM, PM, and DRM contain the same set of sphingolipid species but every membrane source contains its own specific assortment based on the proportion of sphingolipid classes and on the predominance of individual species.  

3.4057           Non-canonical argonaute loading of extracellular vesicle-derived exogenous single-stranded miRNA in recipient cells

Ghoshal, B., Bertrand, E. and Bhattacharyya, S.N.

1. Cell Sci., 134, jcs253914 (2021)

  MicroRNAs (miRNAs), the tiny regulators of gene expression, can be transferred between neighbouring cells via extracellular vesicles (EVs) to control the expression of genes in both donor and recipient cells. How the EV-derived miRNAs are internalized and become functional in target cells is an unresolved question. We have expressed a liver-specific miRNA, miR-122, in non-hepatic cells for packaging in released EVs. With these EVs, we have followed the trafficking of miR-122 to recipient HeLa cells that otherwise do not express this miRNA. We found that EV-associated miR-122 is primarily single-stranded and, to become functional, is loaded onto the recipient cell argonaute proteins without requiring host Dicer1. Following endocytosis, EV-associated miR-122 is loaded onto the host cell argonaute proteins on the endosomal membrane, where the release of internalized miRNAs occurs in a pH-dependent manner, facilitating the formation of the exogenous miRNP pool in the recipient cells. Endosome maturation defects affect EV-mediated entry of exogeneous miRNAs in mammalian cells.  

3.4058           Amyloid-β oligomers block lysosomal targeting of miRNPs to prevent miRNP recycling and target repression in glial cells

De, D. and Bhattacharyya, S.N.

1. Cell Sci., 134, 258360 (2021)

  Upon exposure to amyloid-β oligomers (Aβ_1–42), glial cells start expressing proinflammatory cytokines, despite an increase in levels of repressive microRNAs (miRNAs). Exploring the mechanism of this potential immunity of target cytokine mRNAs against repressive miRNAs in amyloid-β-exposed glial cells, we have identified differential compartmentalization of repressive miRNAs in glial cells that explains this aberrant miRNA function. In Aβ_1–42-treated cells, whereas target mRNAs were found to be associated with polysomes attached to endoplasmic reticulum (ER), the miRNA ribonucleoprotein complexes (miRNPs) were found to be present predominantly with endosomes that failed to recycle to ER-attached polysomes, preventing repression of mRNA targets. Aβ_1–42 oligomers, by masking Rab7a proteins on endosomal surfaces, affected Rab7a interaction with Rab-interacting lysosomal protein (RILP), restricting the lysosomal targeting and recycling of miRNPs. RNA-processing body (P-body) localization of the miRNPs was found to be enhanced in amyloid-β-treated cells as a consequence of enhanced endosomal retention of miRNPs. Interestingly, depletion of P-body components partly rescued the miRNA function in glial cells exposed to amyloid-β and restricted the excess cytokine expression.  

3.4059           Ciliary Ca2+ pumps regulate intraciliary Ca2+ from the action potential and may co-localize with ciliary voltage-gated Ca2+ channels

Yano, J., Wells, R., Lam, Y-W. and Van Houten, J.L.

1. Exp. Biol., 224, jeb232074 (2021)

  Calcium ions (Ca²⁺) entering cilia through the ciliary voltage-gated calcium channels (Ca_V) during the action potential causes reversal of the ciliary power stroke and backward swimming in Paramecium tetraurelia. How calcium is returned to the resting level is not yet clear. Our focus is on calcium pumps as a possible mechanism. There are 23 P. tetraurelia genes for calcium pumps that are members of the family of plasma membrane Ca²⁺ ATPases (PMCAs). They have domains homologous to those found in mammalian PMCAs. Of the 13 pump proteins previously identified in cilia, ptPMCA2a and ptPMCA2b are most abundant in the cilia. We used RNAi to examine which PMCA might be involved in regulating intraciliary Ca²⁺ after the action potential. RNAi for only ptPMCA2a and ptPMCA2b causes cells to significantly prolong their backward swimming, which indicates that Ca²⁺ extrusion in the cilia is impaired when these PMCAs are depleted. We used immunoprecipitations (IP) to find that ptPMCA2a and ptPMCA2b are co-immunoprecipitated with the Ca_V channel α1 subunits that are found only in the cilia. We used iodixanol (OptiPrep) density gradients to show that ptPMCA2a and ptPMCA2b and Ca_V1c are found in the same density fractions. These results suggest that ptPMCA2a and ptPMCA2b are located in the proximity of ciliary Ca_V channels.  

3.4060           Stanniocalcin 1 is a phagocytosis checkpoint driving tumor immune resistance

Lin, H., Kryczek, I., LI, S., Chinnaiyan, A.M., Cieslik, M. and Zou, W. Cancer Cell, 39, 480-493 (2021)   Immunotherapy induces durable clinical responses in a fraction of patients with cancer. However, therapeutic resistance poses a major challenge to current immunotherapies. Here, we identify that expression of tumor stanniocalcin 1 (STC1) correlates with immunotherapy efficacy and is negatively associated with patient survival across diverse cancer types. Gain- and loss-of-function experiments demonstrate that tumor STC1 supports tumor progression and enables tumor resistance to checkpoint blockade in murine tumor models. Mechanistically, tumor STC1 interacts with calreticulin (CRT), an “eat-me” signal, and minimizes CRT membrane exposure, thereby abrogating membrane CRT-directed phagocytosis by antigen-presenting cells (APCs), including macrophages and dendritic cells. Consequently, this impairs APC capacity of antigen presentation and T cell activation. Thus, tumor STC1 inhibits APC phagocytosis and contributes to tumor immune evasion and immunotherapy resistance. We suggest that STC1 is a previously unappreciated phagocytosis checkpoint and targeting STC1 and its interaction with CRT may sensitize to cancer immunotherapy.  

3.4061           Isolation of extracellular vesicles from byproducts of cheesemaking by tangential flow filtration yields heterogeneous fractions of nanoparticles

Sukreet, S., Braga, C.P., An, T.T., Adamec, J., Cui, J., Trible, B. and Zempleni, J.

1. Dairy Sci., 104, 9478-9493 (2021)

  Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (10¹²/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, β-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV.  

3.4062           Receptor-Mediated ER Export of Lipoproteins Controls Lipid Homeostasis in Mice and Humans

Wang, X., Wang, h., Xu, B., Lu, X., Zhu, Y. and Chen, X-W. Cell Metabolism, 33, 350-366 (2021)   Efficient delivery of specific cargos in vivo poses a major challenge to the secretory pathway, which shuttles products encoded by ∼30% of the genome. Newly synthesized protein and lipid cargos embark on the secretory pathway via COPII-coated vesicles, assembled by the GTPase SAR1 on the endoplasmic reticulum (ER), but how lipid-carrying lipoproteins are distinguished from the general protein cargos in the ER and selectively secreted has not been clear. Here, we show that this process is quantitatively governed by the GTPase SAR1B and SURF4, a high-efficiency cargo receptor. While both genes are implicated in lipid regulation in humans, hepatic inactivation of either mouse Sar1b or Surf4 selectively depletes plasma lipids to near-zero and protects the mice from atherosclerosis. These findings show that the pairing between SURF4 and SAR1B synergistically operates a specialized, dosage-sensitive transport program for circulating lipids, while further suggesting a potential translation to treat atherosclerosis and related cardio-metabolic diseases.  

3.4063           Extracellular vesicle-based interorgan transport of mitochondria from energetically stressed adipocytes

Crewe, C., Funcke, J-B., Li, S., Kusminski, C.M., Klein, S. and Scherer, P.E: Cell Metabolism, 33, 1853-1868 (2021)   Adipocytes undergo intense energetic stress in obesity resulting in loss of mitochondrial mass and function. We have found that adipocytes respond to mitochondrial stress by rapidly and robustly releasing small extracellular vesicles (sEVs). These sEVs contain respiration-competent, but oxidatively damaged mitochondrial particles, which enter circulation and are taken up by cardiomyocytes, where they trigger a burst of ROS. The result is compensatory antioxidant signaling in the heart that protects cardiomyocytes from acute oxidative stress, consistent with a preconditioning paradigm. As such, a single injection of sEVs from energetically stressed adipocytes limits cardiac ischemia/reperfusion injury in mice. This study provides the first description of functional mitochondrial transfer between tissues and the first vertebrate example of “inter-organ mitohormesis.” Thus, these seemingly toxic adipocyte sEVs may provide a physiological avenue of potent cardio-protection against the inevitable lipotoxic or ischemic stresses elicited by obesity.  

3.4064           Next-Generation RNA-Sequencing of Serum Small Extracellular Vesicles Discovers Potential Diagnostic Biomarkers for Dementia With Lewy Bodies

Rajkumar, A.P., Psych, M.R.C., Hye, A., Lange, J., Manesch, Y.R., Ballad, C., Fladaby, T. and Aasrsland, D. Am. Geriatr. Psychiatry, 29(6), 573-584 (2021)   Objective There is an urgent clinical need for identifying blood-based diagnostic biomarkers for Dementia with Lewy Bodies (DLB). Transcriptomic studies have reported unique RNA changes in postmortem DLB brains. Small extracellular vesicles (SEV) that transport RNA between brain and peripheral circulation enable identifying molecular changes in living human brain. Hence, we aimed to identify differentially expressed RNA in serum SEVs from people with DLB. Methods We investigated serum SEV total RNA profiles in people with DLB (n = 10) and age and gender matched comparisons (n = 10) using next-generation RNA-sequencing. SEVs were separated by ultracentrifugation with density gradient and were characterized by nanoparticle analysis and western blotting. We verified the differential expression levels of identified differentially expressed genes (DEG) using high-throughput qPCR. Functional implications of identified DEG were evaluated using Ingenuity pathway analyses. Results We identified 846 nominally significant DEG including 30 miRNAs in DLB serum SEVs. We identified significant downregulation of proinflammatory genes, IL1B, CXCL8, and IKBKB. Previously reported postmortem DLB brain DEGs were significantly enriched (χ²=4.99; df=1; p = 0.03) among the identified DEGs, and the differential expression of 40 postmortem DLB brain DEGs could be detected in serum SEVs of people living with DLB. Functional pathway and network analyses highlighted the importance of immunosenescence, ubiquitin proteasome system (UPS) dysfunction, DNA repair, and RNA post-transcriptional modification deficits in DLB pathology. Conclusion Identified DEGs, especially reduced expression levels of inflammation, and UPS-associated RNA, may aid diagnosing DLB, and their biomarker potential warrants further investigation in larger clinical cohorts. Our findings corroborate the absence of chronic neuroinflammation in DLB.  

3.4065           A midbrain dynorphin circuit promotes threat generalization

Fellinger, L., Jo, Y.S., Hunker, A.C., Soden, M.E., Elurn, J., Juarez, B. and Zweifel, L.S. Current Biol., 31, 4388-4396 (2021)   Discrimination between predictive and non-predictive threat stimuli decreases as threat intensity increases. The central mechanisms that mediate the transition from discriminatory to generalized threat responding remain poorly resolved. Here, we identify the stress- and dysphoria-associated kappa opioid receptor (KOR) and its ligand dynorphin (Dyn), acting in the ventral tegmental area (VTA), as a key substrate for regulating threat generalization. We identify several dynorphinergic inputs to the VTA and demonstrate that projections from the bed nucleus of the stria terminalis (BNST) and dorsal raphe nucleus (DRN) both contribute to anxiety-like behavior but differentially affect threat generalization. These data demonstrate that conditioned threat discrimination has an inverted “U” relationship with threat intensity and establish a role for KOR/Dyn signaling in the midbrain for promoting threat generalization.  

3.4066           NPC1-mTORC1 Signaling Couples Cholesterol Sensing to Organelle Homeostasis and Is a Targetable Pathway in Niemann-Pick Type C

Davis, O.B., Shin, H.R., Lim, C-Y., Perera, R.M., Ordonez, M.P. and Zoncu, R. Developmental Cell, 56, 260-276 (2021)   Lysosomes promote cellular homeostasis through macromolecular hydrolysis within their lumen and metabolic signaling by the mTORC1 kinase on their limiting membranes. Both hydrolytic and signaling functions require precise regulation of lysosomal cholesterol content. In Niemann-Pick type C (NPC), loss of the cholesterol exporter, NPC1, causes cholesterol accumulation within lysosomes, leading to mTORC1 hyperactivation, disrupted mitochondrial function, and neurodegeneration. The compositional and functional alterations in NPC lysosomes and nature of aberrant cholesterol-mTORC1 signaling contribution to organelle pathogenesis are not understood. Through proteomic profiling of NPC lysosomes, we find pronounced proteolytic impairment compounded with hydrolase depletion, enhanced membrane damage, and defective mitophagy. Genetic and pharmacologic mTORC1 inhibition restores lysosomal proteolysis without correcting cholesterol storage, implicating aberrant mTORC1 as a pathogenic driver downstream of cholesterol accumulation. Consistently, mTORC1 inhibition ameliorates mitochondrial dysfunction in a neuronal model of NPC. Thus, cholesterol-mTORC1 signaling controls organelle homeostasis and is a targetable pathway in NPC.  

3.4067           Csi-let-7a-5p delivered by extracellular vesicles from a liver fluke activates M1-like macrophages and exacerbates biliary injuries

Yan, C., Zhou, Q-Y., Wu, J., Xu, N., Du, Y., Li, J. et al PNAS, 118(46), e2102206118 (2021)   Chronic infection with liver flukes (such as Clonorchis sinensis) can induce severe biliary injuries, which can cause cholangitis, biliary fibrosis, and even cholangiocarcinoma. The release of extracellular vesicles by C. sinensis (CsEVs) is of importance in the long-distance communication between the hosts and worms. However, the biological effects of EVs from liver fluke on biliary injuries and the underlying molecular mechanisms remain poorly characterized. In the present study, we found that CsEVs induced M1-like activation. In addition, the mice that were administrated with CsEVs showed severe biliary injuries associated with remarkable activation of M1-like macrophages. We further characterized the signatures of miRNAs packaged in CsEVs and identified a miRNA Csi-let-7a-5p, which was highly enriched. Further study showed that Csi-let-7a-5p facilitated the activation of M1-like macrophages by targeting Socs1 and Clec7a; however, CsEVs with silencing Csi-let-7a-5p showed a decrease in proinflammatory responses and biliary injuries, which involved in the Socs1- and Clec7a-regulated NF-κB signaling pathway. Our study demonstrates that Csi-let-7a-5p delivered by CsEVs plays a critical role in the activation of M1-like macrophages and contributes to the biliary injuries by targeting the Socs1- and Clec7a-mediated NF-κB signaling pathway, which indicates a mechanism contributing to biliary injuries caused by fluke infection. However, molecules other than Csi-let-7a-5p from CsEVs that may also promote M1-like polarization and exacerbate biliary injuries are not excluded.  

3.4068           Let-7b-5p in vesicles secreted by human airway cells reduces biofilm formation and increases antibiotic sensitivity of P. aeruginosa

Koeppen, K., NYmon, A., Barnaby, R, Bashor, L., Li, Z., Hampton, T.H., Liefeld, A.E., Kolling, F.W., LaCroix, I.S., Gerber, S.A., Hogan, D.A., Kasetty, S., Nadell, C.D. and Stanton, B.A: PNAS, 118(22), e2105370118 (2021)   Pseudomonas aeruginosa is an opportunistic pathogen that forms antibiotic-resistant biofilms, which facilitate chronic infections in immunocompromised hosts. We have previously shown that P. aeruginosa secretes outer-membrane vesicles that deliver a small RNA to human airway epithelial cells (AECs), in which it suppresses the innate immune response. Here, we demonstrate that interdomain communication through small RNA–containing membrane vesicles is bidirectional and that microRNAs (miRNAs) in extracellular vesicles (EVs) secreted by human AECs regulate protein expression, antibiotic sensitivity, and biofilm formation by P. aeruginosa. Specifically, human EVs deliver miRNA let-7b-5p to P. aeruginosa, which systematically decreases the abundance of proteins essential for biofilm formation, including PpkA and ClpV1-3, and increases the ability of beta-lactam antibiotics to reduce biofilm formation by targeting the beta-lactamase AmpC. Let-7b-5p is bioinformatically predicted to target not only PpkA, ClpV1, and AmpC in P. aeruginosa but also the corresponding orthologs in Burkholderia cenocepacia, another notorious opportunistic lung pathogen, suggesting that the ability of let-7b-5p to reduce biofilm formation and increase beta-lactam sensitivity is not limited to P. aeruginosa. Here, we provide direct evidence for transfer of miRNAs in EVs secreted by eukaryotic cells to a prokaryote, resulting in subsequent phenotypic alterations in the prokaryote as a result of this interdomain communication. Since let-7–family miRNAs are in clinical trials to reduce inflammation and because chronic P. aeruginosa lung infections are associated with a hyperinflammatory state, treatment with let-7b-5p and a beta-lactam antibiotic in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.    

3.4069           Human myeloma cell- and plasma-derived extracellular vesicles contribute to functional regulation of stromal cells

Reale, A., Carmichael, I., Xu, R., Mithraprabhu, S., Khong, T., Chen, M., Fang, H., Savvidou, I., Ramachandran, M., Bingham, N., Simpson, R.J., Greening, D.W. and Spencer, A. Proteomics, 21, 2000119 (2021)   Circulating small extracellular vesicles (sEV) represent promising non-invasive biomarkers that may aid in the diagnosis and risk-stratification of multiple myeloma (MM), an incurable blood cancer. Here, we comprehensively isolated and characterized sEV from human MM cell lines (HMCL) and patient-derived plasma (psEV) by specific EV-marker enrichment and morphology. Importantly, we demonstrate that HMCL-sEV are readily internalised by stromal cells to functionally modulate proliferation. psEV were isolated using various commercial approaches and pre-analytical conditions (collection tube types, storage conditions) assessed for sEV yield and marker enrichment. Functionally, MM-psEV was shown to regulate stromal cell proliferation and migration. In turn, pre-educated stromal cells favour HMCL adhesion. psEV isolated from patients with both pre-malignant plasma cell disorders (monoclonal gammopathy of undetermined significance [MGUS]; smouldering MM [SMM]) and MM have a similar ability to promote cell migration and adhesion, suggesting a role for both malignant and pre-malignant sEV in disease progression. Proteomic profiling of MM-psEV (305 proteins) revealed enrichment of oncogenic factors implicated in cell migration and adhesion, in comparison to non-disease psEV. This study describes a protocol to generate morphologically-intact and biologically functional sEV capable of mediating the regulation of stromal cells, and a model for the characterization of tumour-stromal cross-talk by sEV in MM.  

3.4070           Proteome reprogramming of endometrial epithelial cells by human trophectodermal small extracellular vesicles reveals key insights into embryo implantation

Poh, Q.H., Rai, A., Carmichael, I.I., Salamonsen, L.A. and Greening, D.W. Proteomics, 21, 2000210 (2021)   Embryo implantation into the receptive endometrium is critical in pregnancy establishment, initially requiring reciprocal signalling between outer layer of the blastocyst (trophectoderm cells) and endometrial epithelium; however, factors regulating this crosstalk remain poorly understood. Although endometrial extracellular vesicles (EVs) are known to signal to the embryo during implantation, the role of embryo-derived EVs remains largely unknown. Here, we provide a comprehensive proteomic characterisation of a major class of EVs, termed small EVs (sEVs), released by human trophectoderm cells (Tsc-sEVs) and their capacity to reprogram protein landscape of endometrial epithelium in vitro. Highly purified Tsc-sEVs (30–200 nm, ALIX⁺, TSG101⁺, CD9/63/81⁺) were enriched in known players of implantation (LIFR, ICAM1, TAGLN2, WNT5A, FZD7, ROR2, PRICKLE2), antioxidant activity (SOD1, PRDX1/4/6), tissue integrity (EZR, RAC1, RHOA, TNC), and focal adhesions (FAK, ITGA2/V, ITGB1/3). Functionally, Tsc-sEVs were taken up by endometrial cells, altered transepithelial electrical resistance, and upregulated proteins implicated in embryo attachment (ITGA2/V, ITGB1/3), immune regulation (CD59, CD276, LGALS3), and antioxidant activity (GPX1/3/4, PRDX1/2/4/5/6): processes that are critical for successful implantation. Collectively, we provide critical insights into Tsc-sEV-mediated regulation of endometrial function that contributes to our understanding of the molecular basis of implantation.  

3.4071           Proteomic profiling of human uterine extracellular vesicles reveal dynamic regulation of key players of embryo implantation and fertility during menstrual cycle

Rai, A., Poh, Q.H., Fatmous, M., Fang, H., Gurung, S., Vollenhoven, B., Salamonsen, L.A. and Greening, D.W. Proteomics, 21, 2000211 (2021)   Endometrial extracellular vesicles (EVs) are emerging as important players in reproductive biology. However, how their proteome is regulated throughout the menstrual cycle is not known. Such information can provide novel insights into biological processes critical for embryo development, implantation, and successful pregnancy. Using mass spectrometry-based quantitative proteomics, we show that small EVs (sEVs) isolated from uterine lavage of fertile women (UL-sEV), compared to infertile women, are laden with proteins implicated in antioxidant activity (SOD1, GSTO1, MPO, CAT). Functionally, sEVs derived from endometrial cells enhance antioxidant function in trophectoderm cells. Moreover, there was striking enrichment of invasion-related proteins (LGALS1/3, S100A4/11) in fertile UL-sEVs in the secretory (estrogen plus progesterone-driven, EP) versus proliferative (estrogen-driven, E) phase, with several players downregulated in infertile UL-sEVs. Consistent with this, sEVs from EP- versus E-primed endometrial epithelial cells promote invasion of trophectoderm cells. Interestingly, UL-sEVs from fertile versus infertile women carry known players/predictors of embryo implantation (PRDX2, IDHC), endometrial receptivity (S100A4, FGB, SERPING1, CLU, ANXA2), and implantation success (CAT, YWHAE, PPIA), highlighting their potential to inform regarding endometrial status/pregnancy outcomes. Thus, this study provides novel insights into proteome reprograming of sEVs and soluble secretome in uterine fluid, with potential to enhance embryo implantation and hence fertility.  

3.4072           SEC6 exocyst subunit contributes to multiple steps of growth and development of Physcomitrella (Physcomitrium patens)

Brejskova, L., Hala, M., Rawat, A., Soukupova, H., Cvckova, F., Charlot, F., Nogue, F., Haluska, S. and Zarsky, V. Plant J., 106, 831-843 (2021)   Spatially directed cell division and expansion is important for plant growth and morphogenesis and relies on cooperation between the cytoskeleton and the secretory pathway. The phylogenetically conserved octameric complex exocyst mediates exocytotic vesicle tethering at the plasma membrane. Unlike other exocyst subunits of land plants, the core exocyst subunit SEC6 exists as a single paralog in Physcomitrium patens and Arabidopsis thaliana genomes. Arabidopsis SEC6 (AtSEC6) loss-of-function (LOF) mutation causes male gametophytic lethality. Our attempts to inactivate the P. patens SEC6 gene, PpSEC6, using targeted gene replacement produced two independent partial LOF (‘weak allele’) mutants via perturbation of the PpSEC6 gene locus. These mutants exhibited the same pleiotropic developmental defects: protonema with dominant chloronema stage; diminished caulonemal filament elongation rate; and failure in post-initiation gametophore development. Mutant gametophore buds, mostly initiated from chloronema cells, exhibited disordered cell file organization and cross-wall perforations, resulting in arrested development at the eight- to 10-cell stage. Complementation of both sec6 moss mutant lines by both PpSEC6 and AtSEC6 cDNA rescued gametophore development, including sexual organ differentiation. However, regular sporophyte formation and viable spore production were recovered only by the expression of PpSEC6, whereas the AtSEC6 complementants were only rarely fertile, indicating moss-specific SEC6 functions.  

3.4073           Cocaine Induces Sex-Associated Changes in Lipid Profiles of Brain Extracellular Vesicles

Landfield, Q., Saito, M., Hashim, A., Canals-Baker, S., Sershen, H., Levy, E. and Saito, M. Neurochem. Res., 46, 2909-2922 (2021)   Cocaine is a highly addictive stimulant with diverse effects on physiology. Recent studies indicate the involvement of extracellular vesicles (EVs) secreted by neural cells in the cocaine addiction process. It is hypothesized that cocaine affects secretion levels of EVs and their cargos, resulting in modulation of synaptic transmission and plasticity related to addiction physiology and pathology. Lipids present in EVs are important for EV formation and for intercellular lipid exchange that may trigger physiological and pathological responses, including neuroplasticity, neurotoxicity, and neuroinflammation. Specific lipids are highly enriched in EVs compared to parent cells, and recent studies suggest the involvement of various lipids in drug-induced synaptic plasticity during the development and maintenance of addiction processes. Therefore, we examined interstitial small EVs isolated from the brain of mice treated with either saline or cocaine, focusing on the effects of cocaine on the lipid composition of EVs. We demonstrate that 12 days of noncontingent repeated cocaine (10 mg/kg) injections to mice, which induce locomotor sensitization, cause lipid composition changes in brain EVs of male mice as compared with saline-injected controls. The most prominent change is the elevation of GD1a ganglioside in brain EVs of males. However, cocaine does not affect the EV lipid profiles of the brain in female mice. Understanding the relationship between lipid composition in EVs and vulnerability to cocaine addiction may provide insight into novel targets for therapies for addiction.  

3.4074           Recent Advancement and Technical Challenges in Developing Small Extracellular Vesicles for Cancer Drug Delivery

Geng, T., Pan, P., Leung, E., Chen, Q., Chamley, L. and Wu, Z. Pharm. Res., 38, 179-197 (2021)   Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer membrane-enclosed vesicles and act like ‘messages in a bottle’ in cell-cell communication by transporting their cargoes to recipient cells. Small EVs (sEVs, < 200 nm) are highly researched recently and have been harnessed as novel delivery systems for the treatment of various diseases, including neurodegenerative disorders, cardiovascular diseases, and most importantly cancer primarily because of their non-immunogenicity, tissue penetration and cell-tropism. This review will first provide a comprehensive overview of sEVs regarding the current understanding on their properties, biogenesis, new classification by the ISEV, composition, as well as their roles in cancer development (thereby called “oncosomes”). The primary focus will be given to the current state of sEVs as natural nanocarriers for cancer drug delivery, the technologies and challenges involved in sEV isolation and characterization, therapeutic cargo loading, and surface modification to enhance tumor-targeting. We will also provide examples of sEV products under clinical trials. Furthermore, the current challenges as well as the advance in “sEV mimetics” to address some of the sEVs limitations is briefly discussed. We seek to advance our understanding of sEVs to unlock their full potential as superior drug delivery vehicles in cancer therapy.  

3.4075           Outer membrane vesicles mediated horizontal transfer of an aerobic denitrification gene between Escherichia coli

Qiao, W., Wang, l., Luo, Y. and Miao, J. Biodegradation, 32, 435-448 (2021)   Bacterial genetic material can be horizontally transferred between microorganisms via outer membrane vesicles (OMVs) released by bacteria. Up to now, the application of vesicle-mediated horizontal transfer of “degrading genes” in environmental remediation has not been reported. In this study, the nirS gene from an aerobic denitrification bacterium, Pseudomonas stutzeri, was enclosed in a pET28a plasmid, transformed into Escherichia coli (E. coli) DH5α and expressed in E. coli BL21. The E. coli DH5α released OMVs containing the recombination plasmid pET28a–nirS-EGFP. When compared with the free pET28a–nirS- transformation frequency of 2.76 × 10⁶ CFU/g when the dosage of OMVs was 200 µg under natural conditions, and nirS could express successfully in recipient bacteria. Furthermore, the recipient bacteria that received OMVs containing pET28a–nirS-EGFP could produce 18.16 U/mL activity of nitrite reductase.  

3.4076           Single-molecule manipulation of macromolecules on GUV or SUV membranes using optical tweezers

Wang, Y., Kumar, A., Jin, H. and Zhang, Y. Biophys. J., 120, 5454-5465 (2021)   Despite their wide applications in soluble macromolecules, optical tweezers have rarely been used to characterize the dynamics of membrane proteins, mainly due to the lack of model membranes compatible with optical trapping. Here, we examined optical trapping and mechanical properties of two potential model membranes, giant and small unilamellar vesicles (GUVs and SUVs, respectively) for studies of membrane protein dynamics. We found that optical tweezers can stably trap GUVs containing iodixanol with controlled membrane tension. The trapped GUVs with high membrane tension can serve as a force sensor to accurately detect reversible folding of a DNA hairpin or membrane binding of synaptotagmin-1 C2AB domain attached to the GUV. We also observed that SUVs are rigid enough to resist large pulling forces and are suitable for detecting protein conformational changes induced by force. Our methodologies may facilitate single-molecule manipulation studies of membrane proteins using optical tweezers.  

3.4077           Cancer-Associated Fibroblasts Promote Aggressive Gastric Cancer Phenotypes via Heat Shock Factor 1–Mediated Secretion of Extracellular Vesicles

Grunberg, N., Pevsner-Fischer, M., Goshen-Lago, T., Diment, J., Stein, Y. et al Cancer Res., 81, 1639-1653 (2021)   Gastric cancer is the third most lethal cancer worldwide, and evaluation of the genomic status of gastric cancer cells has not translated into effective prognostic or therapeutic strategies. We therefore hypothesize that outcomes may depend on the tumor microenvironment (TME), in particular, cancer-associated fibroblasts (CAF). However, very little is known about the role of CAFs in gastric cancer. To address this, we mapped the transcriptional landscape of human gastric cancer stroma by microdissection and RNA sequencing of CAFs from patients with gastric cancer. A stromal gene signature was associated with poor disease outcome, and the transcription factor heat shock factor 1 (HSF1) regulated the signature. HSF1 upregulated inhibin subunit beta A and thrombospondin 2, which were secreted in CAF-derived extracellular vesicles to the TME to promote cancer. Together, our work provides the first transcriptional map of human gastric cancer stroma and highlights HSF1 and its transcriptional targets as potential diagnostic and therapeutic targets in the genomically stable tumor microenvironment.  

3.4078           Circulating Small Extracellular Vesicles Activate TYRO3 to Drive Cancer Metastasis and Chemoresistance

Park, M., Kim, J.W., Kim, K.M., Kang, S., Kim, W., Kim, J-K. et al Cancer Res., 81, 3539-3553 (2021)   Extracellular vesicles (EV) in the tumor microenvironment have emerged as crucial mediators that promote proliferation, metastasis, and chemoresistance. However, the role of circulating small EVs (csEV) in cancer progression remains poorly understood. In this study, we report that csEV facilitate cancer progression and determine its molecular mechanism. csEVs strongly promoted the migration of cancer cells via interaction with phosphatidylserine of csEVs. Among the three TAM receptors, TYRO3, AXL, and MerTK, TYRO3 mainly interacted with csEVs. csEV-mediated TYRO3 activation promoted migration and metastasis via the epithelial–mesenchymal transition and stimulation of RhoA in invasive cancer cells. Additionally, csEV–TYRO3 interaction induced YAP activation, which led to increased cell proliferation and chemoresistance. Combination treatment with gefitinib and KRCT-6j, a selective TYRO3 inhibitor, significantly reduced tumor volume in xenografts implanted with gefitinib-resistant non–small cell lung cancer cells. The results of this study show that TYRO3 activation by csEVs facilitates cancer cell migration and chemoresistance by activation of RhoA or YAP, indicating that the csEV/TYRO3 interaction may serve as a potential therapeutic target for aggressive cancers in the clinic.  

3.4079           Exosomes: Isolation, characterization, and biomedical applications

Alzhrani, G.N., Alanazi, S.T., Alsharif, S., Albalawi, A.M., Alsharif, A.A., Abdel-Maksoud, M.-S. and Elsherbiny, N. Cell Biol. Int., 45, 1807-1831 (2021)   Exosomes are nano-sized bioactive vesicles of 30–150 nm in diameter. They are secreted by exocytosis of nearly all type of cells in to the extracellular fluid. Thereby, they can be found in many biological fluids. Exosomes regulate intracellular communication between cells via delivery of their cargo which include lipids, proteins, and nucleic acid. Many desirable features of exosomes made them promising candidates in several therapeutic applications. In this review, we discuss the use of exosomes as diagnostic tools and their possible biomedical applications. Additionally, current techniques used for isolation, purification, and characterization of exosomes from both biological fluids and in vitro cell cultures were discussed.  

3.4080           E-cig vapor condensate alters proteome and lipid profiles of membrane rafts: impact on inflammatory responses in A549 cells

Singh, D.P., Begum, R., Kaur, G., Bagam, P., Kambiranda, D., Singh, R. and Batra, S. Cell. Biol. Toxicol., 37, 773-793 (2021)   Electronic cigarettes (e-cigs) are battery-operated heating devices that aerosolize e-liquid, typically containing nicotine and several other chemicals, which is then inhaled by a user. Over the past decade, e-cigs have gained immense popularity among both smokers and non-smokers. One reason for this is that they are advertised as a safe alternative to conventional cigarettes. However, the recent reports of e-cig use associated lung injury have ignited a considerable debate about the relative harm and benefits of e-cigs. The number of reports about e-cig-induced inflammation and pulmonary health is increasing as researchers seek to better understand the effects of vaping on human health. In line with this, we investigated the molecular events responsible for the e-cig vapor condensate (ECVC)–mediated inflammation in human lung adenocarcinoma type II epithelial cells (A549). In an attempt to limit the variables caused by longer ingredient lists of flavored e-cigs, tobacco-flavored ECVC (TF-ECVC±nicotine) was employed for this study. Interestingly, we observed significant upregulation of cytokines and chemokines (IL-6, IL-8, and MCP-1) in A549 cells following a 48 h TF-ECVC challenge. Furthermore, there was a significant increase in the expression of pattern recognition receptors TLR-4 and NOD-1, lipid raft–associated protein caveolin-1, and transcription factor NF-кB in TF-ECVC with and/or without nicotine-challenged lung epithelial cells. Our results further demonstrate the harboring of TLR-4 and NOD-1 in the caveolae of TF-ECVC-challenged A549 cells. Proteomic and lipidomic analyses of lipid raft fractions from control and challenged cells revealed a distinct protein and lipid profile in TF-ECVC (w/wo nicotine)-exposed A549 cells. Interestingly, the inflammatory effects of TF-ECVC (w/wo nicotine) were inhibited following the caveolin-1 knockdown, thus demonstrating a critical role of caveolae raft–mediated signaling in eliciting inflammatory responses upon TF-ECVC challenge.    

3.4081           Methodologies for Measuring Protein Trafficking across Cellular Membranes

Lyu, Z. and Genereux, J.C: ChemPlusChem, 86, 1397-1415 (2021)   Nearly all proteins are synthesized in the cytosol. The majority of this proteome must be trafficked elsewhere, such as to membranes, to subcellular compartments, or outside of the cell. Proper trafficking of nascent protein is necessary for protein folding, maturation, quality control and cellular and organismal health. To better understand cellular biology, molecular and chemical technologies to properly characterize protein trafficking (and mistrafficking) have been developed and applied. Herein, we take a biochemical perspective to review technologies that enable spatial and temporal measurement of protein distribution, focusing on both the most widely adopted methodologies and exciting emerging approaches.  

3.4082           Role of Extracellular Vesicles in the Diagnosis and Pathogenesis of Barrett’s Esophagus: A Mini-Review

Zhang, Q. and Bansal, A. Digestive Diseases and Sciences, 66, 705-713 (2021)   Esophageal adenocarcinoma (EAC) continues to be a significant public health problem with survival rates that have remained stagnant. Although the population at the highest risk for EAC, i.e., patients with Barrett’s esophagus (BE) has been clearly defined, patients with EAC continue to do poorly due to advanced stage at diagnosis. The field of extracellular vesicles (EV) could have huge application for the management of patients with BE and EAC by allowing timely diagnosis, serial monitoring, and improved understanding of disease biology. EV are actively packaged and actively secreted vesicles and contain microRNAs, proteins, lipids, and DNA. The contents of EV have been shown to provide useful insights into cellular transformation and pro-oncogenic processes. Early work shows promise but suffers from a high degree of technical and biological variation. The current review not only summarizes the current knowledge about EV as diagnostic biomarkers and their role in disease progression of BE and EAC but also provides the reader practical guidance to devise future experiments to perform well-designed studies.  

3.4083           Autolysis-mediated membrane vesicle formation in Bacillus subtilis

Abe, K., Toyofuku, M., Nomura, N. and Obana, N. Environmental Microbiol., 23(5), 2632-2647 (2021)   It is known that Bacillus subtilis releases membrane vesicles (MVs) during the SOS response, which is associated with cell lysis triggered by the PBSX prophage-encoded cell-lytic enzymes XhlAB and XlyA. In this study, we demonstrate that MVs are released under various stress conditions: sucrose fatty acid ester (SFE; surfactant) treatment, cold shock, starvation, and oxygen deficiency. B. subtilis possesses four major host-encoded cell wall-lytic enzymes (autolysins; LytC, LytD, LytE, and LytF). Deletions of the autolysin genes abolished autolysis and the consequent MV production under these stress conditions. In contrast, deletions of xhlAB and xlyA had no effect on autolysis-triggered MV biogenesis, indicating that autolysis is a novel and prophage-independent pathway for MV production in B. subtilis. Moreover, we found that the cell lysis induced by the surfactant treatment was effectively neutralized by the addition of exogenous purified MVs. This result suggests that the MVs can serve as a decoy for the cellular membrane to protect the living cells in the culture from membrane damage by the surfactant. Our results indicate a positive effect of B. subtilis MVs on cell viability and provide new insight into the biological importance of the autolysis phenomenon in B. subtilis.  

3.4084           Proteomic and phospholipidomic characterization of extracellular vesicles inducing tumor microenvironment in Epstein-Barr virus-associated lymphomas

Ito, M., Kudo, K., Higuchi, H., Otsuka, H., Tanaka, M., Fukunishi, N., Araki, T., Takamatsu, M., Ino, Y., Kimura, Y. and Kotani, A. FASEB J., 35, e21505 (2021)   Epstein-Barr virus (EBV) causes malignant carcinomas including B cell lymphomas accompanied by the systemic inflammation. Previously, we observed that phosphatidylserine (PS)-exposing subset of extracellular vesicles (EVs) secreted from an EBV strain Akata-transformed lymphoma (Akata EVs) convert surrounding phagocytes into tumor-associated macrophages (TAMs) via induction of inflammatory response, which is in part mediated by EBV-derived micro RNAs. However, it is still unclear about EV-carried other potential inflammatory factors associated with TAM formation in EBV lymphomas. To this end, we sought to explore proteomic and phospholipidomic profiles of PS-exposing EVs derived from EBV-transformed lymphomas. Mass spectrometric analysis revealed that several immunomodulatory proteins including integrin αLβ2 and fibroblast growth factor 2 (FGF2) were highly expressed in PS-exposing Akata EVs compared with another EBV strain B95-8-transformed lymphoma-derived counterparts which significantly lack TAM-inducing ability. Pharmacological inhibition of either integrin αLβ2 or FGF2 hampered cytokine induction in monocytic cultured cells elicited by PS-exposing Akata EVs, suggesting the involvement of these proteins in EV-mediated TAM induction in EBV lymphomas. In addition, phospholipids containing precursors of immunomodulatory lipid mediators were also enriched in PS-exposing Akata EVs compared with B95-8 counterparts. Phospholipidomic analysis of fractionated Akata EVs by density gradient centrifugation further demonstrated that PS-exposing Akata EVs might be identical to certain Akata EVs in low density fractions containing exosomes. Therefore, we concluded that a variety of immunomodulatory cargo molecules in a certain EV subtype are presumably conducive to the development of EBV lymphomas.  

3.4085           Human fibroblast-derived extracellular vesicles promote hair growth in cultured human hair follicles

Rajendran, R.L., Gangadaran, P., Kwack, M.H., Oh, J.M., Hong, C.M., Sung, Y.K., Lee, J. and Ahn, B-C. FEBS Lett., 595, 942-953 (2021)   Hair loss is a prevalent medical condition affecting both genders. In this study, we investigate the effects of a specific class of extracellular vesicles (EVs), namely human normal fibroblast-derived EVs (hFB-EVs), on human dermal papilla (DP) and outer root sheath (ORS) cells and examine the molecular mechanisms responsible for hair growth in hair follicles (HFs). We find that Wnt3a, which maintains the hair-generating activity of DP cells, is enriched and more strongly associated with hFB-EVs than with fibroblasts. Furthermore, hFB-EV-associated Wnt3a mediated receptor activation in cultured DP cells, leading to an increase in β-catenin in the cytoplasm and its translocation into the nucleus, thereby elevating expression of the target genes Axin2 and Lef1. Additionally, hFB-EVs promoted the migration, proliferation, and differentiation of ORS cells and elongation of the hair shaft in human HFs. These findings revealed a novel mechanism by which hFB-EVs influence hair growth.  

3.4086           Perspectives of bovine and human milk exosomics as health biomarkers for advancing systemic therapeutic potential

Mahala, S., Rai, S., Singh, A., Mehrotra, A., Pandey, H.O. and Kumar, A. Food Biotechnol., 35(4), 273-309 (2021)   The epithelial cells of the mammary gland secrete extracellular nanovesicles known as exosomes that carry and protect microRNAs and other various signaling biomolecules. Milk exosomes are stable during processing and remain protected from digestion in the gastrointestinal tract in order to reach specific target cells including peripheral tissues by crossing biological barriers. Milk exosomal microRNAs have their role as growth-promoting factor, in immunological programming, improving allergy tolerance and epigenetic controller of other mRNAs. However, in contrast, many translational evidence indicated that excessive consumption of bovine milk and continuous exposure of these exosomal microRNAs may be responsible for chronic inflammatory diseases of contemporary societies. Milk exosomes have potential preventive impact on necrotizing enterocolitis, ulcerative colitis, and inflammatory bowel diseases and may be targeted as preventive medicine or in therapeutic diets and also have potential as a nano-vehicle for drug delivery for chemotherapy.  

3.4087           Hybrid Stomach-Intestinal Chromatin States Underlie Human Barrett’s Metaplasia

Singh, H., Ha, K., Hornick, J.L., Madha, S., Cejas, P., Jajoo, K., Singh, P., Polak, P., Lee, h. and Shivdasani, R.A. Gatroenterology, 161, 924-939 (2021)   Background & Aims Tissue metaplasia is uncommon in adults because established cis-element programs resist rewiring. In Barrett’s esophagus, the distal esophageal mucosa acquires a predominantly intestinal character, with notable gastric features, and is predisposed to developing invasive cancers. We sought to understand the chromatin underpinnings of Barrett’s metaplasia and why it commonly displays simultaneous gastric and intestinal properties. Methods We profiled cis-regulatory elements with active histone modifications in primary human biopsy materials using chromatin immunoprecipitation followed by DNA sequencing. Mutations in Barrett’s esophagus were examined in relation to tissue-specific enhancer landscapes using a random forest machine-learning algorithm. We also profiled open chromatin at single-cell resolution in primary Barrett’s biopsy specimens using the assay for transposase-accessible chromatin. We used 1- and 2-color immunohistochemistry to examine protein expression of tissue-restricted genes. Results Barrett’s esophagus bears epigenome fingerprints of human stomach and intestinal columnar, but not esophageal squamous, epithelia. Mutational patterns were best explained as arising on the epigenome background of active gastric cis-elements, supporting the view that adjoining stomach epithelium is a likely tissue source. Individual cells in Barrett’s metaplasia coexpress gastric and intestinal genes, reflecting concomitant chromatin access at enhancers ordinarily restricted to one or the other epithelium. Protein expression of stomach-specific mucins; CLDN18; and a novel gastric marker, ANXA10, showed extensive tissue and subclonal heterogeneity of dual stomach-intestinal cell states. Conclusions These findings reveal mixed and dynamic tissue-restricted chromatin states and phenotypic heterogeneity in Barrett’s esophagus. Pervasive intragland variation argues against stem-cell governance of this phenotype.  

3.4088           ALS/FTD mutations in UBQLN2 are linked to mitochondrial dysfunction through loss-of-function in mitochondrial protein import

Lin, B.C., Phung, T.H., Higgins, N.R., Greenslade, J.E., Prado, M.A. Finley, D., karbowski, M., Polster, B.M. and Monteiro, M.J. Hum. Mol. Genet., 30(13), 1230-1246 (2021)   UBQLN2 mutations cause amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD), but the pathogenic mechanisms by which they cause disease remain unclear. Proteomic profiling identified ‘mitochondrial proteins’ as comprising the largest category of protein changes in the spinal cord (SC) of the P497S UBQLN2 mouse model of ALS/FTD. Immunoblots confirmed P497S animals have global changes in proteins predictive of a severe decline in mitochondrial health, including oxidative phosphorylation (OXPHOS), mitochondrial protein import and network dynamics. Functional studies confirmed mitochondria purified from the SC of P497S animals have age-dependent decline in nearly all steps of OXPHOS. Mitochondria cristae deformities were evident in spinal motor neurons of aged P497S animals. Knockout (KO) of UBQLN2 in HeLa cells resulted in changes in mitochondrial proteins and OXPHOS activity similar to those seen in the SC. KO of UBQLN2 also compromised targeting and processing of the mitochondrial import factor, TIMM44, resulting in accumulation in abnormal foci. The functional OXPHOS deficits and TIMM44-targeting defects were rescued by reexpression of WT UBQLN2 but not by ALS/FTD mutant UBQLN2 proteins. In vitro binding assays revealed ALS/FTD mutant UBQLN2 proteins bind weaker with TIMM44 than WT UBQLN2 protein, suggesting that the loss of UBQLN2 binding may underlie the import and/or delivery defect of TIMM44 to mitochondria. Our studies indicate a potential key pathogenic disturbance in mitochondrial health caused by UBQLN2 mutations.  

3.4089           Dancing with Trojan horses: an interplay between the extracellular vesicles and viruses

Badierah, R.A., Uversky, V.N. and Redwan, E.M.

1. Biomolecular Structure and Dynamics, 39(8), 3034-3060 (2021)

  Extracellular vesicles (EVs) are membrane-encapsulated particles released by eukaryotic and prokaryotic cells into the extracellular environment. Depending on their origin, size, and composition, EVs are grouped in several classes, with one of them being exosomes, which are small EVs (SEVs) generated within the endosomal compartment of eukaryotic cells via the unique multivesicular body pathway. Being able to deliver their content (proteins, lipids, small molecules, and nucleic acids) to other cells, exosomes/SEVs are considered as bioactive vesicles with multiple biological functions. Importantly, the composition of exosomes/SEVs depends on the cell and tissue of origin including a set of specific proteins. However, the pathological conditions may lead to the appearance of diseases-specific exosomes/SEVs containing pathology-specific cargoes utilized in the malicious cell-cell communication and spread of malady. Viruses demonstrate complex ‘dancing’ around the exosome biogenesis system, being able to hijack the host systems responsible for the exosome biogenesis. They use the exosome biogenesis system to promote packaging of their capsids, regulate virion production, and virus secretion. They also utilize a Trojan horse stratagem to place virions inside the SEVs and thereby to spread beyond their normal range of cell hosts using the normal EV uptake process. Another illustration of the virus-based utilization of Trojan horse strategy is given by the ability of human viruses to use exosomes/SEVs as carriers of their exogenous miRNA or viral proteins to the non-infected cells. Taken together, these strategies of dancing with Trojan horses can help viruses to fight with the host defense and to spread the infection.  

3.4090           Cardiovascular Exosomes and MicroRNAs in Cardiovascular Physiology and Pathophysiology

Henning, R.J.

1. Cardiovasc. Transl. Res., 14, 195-212 (2021)

  Cardiac exosomes mediate cell-to-cell communication, stimulate or inhibit the activities of target cells, and affect myocardial hypertrophy, injury and infarction, ventricular remodeling, angiogenesis, and atherosclerosis. The exosomes that are released in the heart from cardiomyocytes, vascular cells, fibroblasts, and resident stem cells are hypoimmunogenic, are physiologically more stable than cardiac cells, can circulate in the body, and are able to cross the blood–brain barrier. Exosomes utilize three mechanisms for cellular communication: (1) internalization by cells, (2) direct fusion to the cell membrane, and (3) receptor-ligand interactions. Cardiac exosomes transmit proteins, mRNA, and microRNAs to other cells during both physiological and pathological process. Cardiac-specific exosome miRNAs can regulate the expression of sarcomeric genes, ion channel genes, autophagy, anti-apoptotic and anti-fibrotic activity, and angiogenesis. This review discusses the role of exosomes and microRNAs in normal myocardium, myocardial injury and infarction, atherosclerosis, and the importance of circulating microRNAs as biomarkers of cardiac disease.  

3.4091           Identification of small extracellular vesicle subtypes in follicular fluid: Insights into the function and miRNA profiles

Wang, X., Meng, k., Wang, H., Wang, Y., Zhao, Y., kang, J., Zhang, Y. and Quan, F.

1. Cell. Physiol., 236, 5633-5645 (2021)

  The study of small extracellular vesicles (sEVs) heterogeneity is one of the main problems that must be solved, and the different sEV subtypes in follicular fluid are still unclear, limiting our understanding of their function. This study first separated sEV subtypes from follicular fluid using differential ultracentrifugation combined with iodixanol density gradient flotation and then evaluated their miRNA profile and effects on the proliferation and apoptosis of granulosa cells (GCs). We also performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of potential target genes of differentially expressed miRNAs (DEMs) and KEGG analysis of potential target genes of non-DEMs. Low-density sEVs (sEV_F6) were enriched in TSG101, while high-density sEVs (sEV_F8) were enriched in CD63. The miRNA profiles of sEV_F6 and sEV_F8 were heterogeneous, and the differential signaling pathways were mainly related to the adhesion and hypoxic stress pathways, while the same signaling pathways were mainly related to cell proliferation, apoptosis, cell cycle, and autophagy pathways. In addition, the highly expressed miRNAs in both subtypes were mainly related to cell proliferation and apoptosis. Both subtypes transferred their miRNAs into GCs and promoted the proliferation ability of the GCs and inhibited their apoptosis. The results showed for the first time that there are different subtypes of sEVs in follicular fluid and that the miRNA profiles of subtypes are heterogeneous.  

3.4092           Exosomes from donor-derived adipose mesenchymal stem cells prolong the survival of vascularized composite allografts

Chen, Z., Xue, S., Zhang, S., Cheng, K. and Ye, Q.

1. Cell. Physiol., 236, 5895-5905 (2021)

  Donor-derived adipose-derived mesenchymal stem cells (ADMSCs) dampen the alloimmune response and exosomes are reported to have biological activity similar to their parent cells. Here, we investigated the roles of exosomes from donor-derived ADMSCs (ADMSC-exo) in vascularized composite allotransplantation (VCA). Brown Norway-to-Lewis rat hindlimb transplantations were intravenously treated with either exosome from donor-derived ADMSCs or phosphate-buffered saline, combined with a short course of immunosuppression. We established that the treatment with ADMSC-exo prolongs the survival time of VCA grafts. Skin and muscle samples from ADMSC-exo-treated animals showed no histological signs of rejection, but samples from controls showed rejection of degree III. Comparing to the control group, a significant increase of donor cell chimerism, Tr1 and Treg, while a decrease of CD4⁺ T and Th1 cells were observed in the ADMSC-exo-treated group. Our findings imply that ADMSC-exo may be a valuable and safe treatment for extending VCA graft survival.  

3.4093           Single-Nucleus RNA-Seq Reveals Dysregulation of Striatal Cell Identity Due to Huntington's Disease Mutations

Malaiya, S., Cortes-Gutierrez, M., Herb, B.R., Coffey, S.R., Legg, S.R.W., Cantle, J.P., Colantuoni, C., Carroll, J.B. and Ament, S.A.

1. Neurosci., 41(25), 5534-5552 (2021)

  Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a trinucleotide expansion in exon 1 of the huntingtin (HTT) gene. Cell death in HD occurs primarily in striatal medium spiny neurons (MSNs), but the involvement of specific MSN subtypes and of other striatal cell types remains poorly understood. To gain insight into cell type-specific disease processes, we studied the nuclear transcriptomes of 4524 cells from the striatum of a genetically precise knock-in mouse model of the HD mutation, HttQ175^/+, and from wild-type controls. We used 14- to 15-month-old male mice, a time point at which multiple behavioral, neuroanatomical, and neurophysiological changes are present but at which there is no known cell death. Thousands of differentially expressed genes (DEGs) were distributed across most striatal cell types, including transcriptional changes in glial populations that are not apparent from RNA-seq of bulk tissue. Reconstruction of cell type-specific transcriptional networks revealed a striking pattern of bidirectional dysregulation for many cell type-specific genes. Typically, these genes were repressed in their primary cell type, yet de-repressed in other striatal cell types. Integration with existing epigenomic and transcriptomic data suggest that partial loss-of-function of the polycomb repressive complex 2 (PRC2) may underlie many of these transcriptional changes, leading to deficits in the maintenance of cell identity across virtually all cell types in the adult striatum.  

3.4094           Cross-Species Proteomic Comparison of Outer Membrane Vesicles and Membranes of Francisella tularensis subsp. tularensis versus subsp. Holarctica

Klimentova, J., Rehulka, P., Pavkova, I., Kubelkova, K., Bavlovis, J. and Stulik, J.

1. Proteome Res., 20, 1716-1732 (2021)

  Release of outer membrane vesicles (OMV) is an important phenomenon in Gram-negative bacteria playing multiple roles in their lifestyle, including in relation to virulence and host–pathogen interaction. Francisella tularensis, unlike other bacteria, releases unusually shaped, tubular OMV. We present a proteomic comparison of OMV and membrane fractions from two F. tularensis strains: moderately virulent subsp. holarctica strain FSC200 and highly virulent subsp. tularensis strain SchuS4. Proteomic comparison studies routinely evaluate samples from the same proteome, but sometimes we must compare samples from closely related organisms. This raises quantification issues. We propose a novel approach to cross-species proteomic comparison based on an intersection protein database from the individual single-species databases. This is less prone to quantification errors arising from differences in the sequences. Consecutively comparing subproteomes of OMV and membranes of the two strains allows distinguishing differences in relative protein amounts caused by global expression changes from those caused by preferential protein packing to OMV or membranes. Among the proteins most differently packed into OMV between the two strains, we detected proteins involved in biosynthesis and metabolism of bacterial envelope components like O-antigen, lipid A, phospholipids, and fatty acids, as well as some major structural outer membrane proteins. The data are available via ProteomeXchange with identifier PXD022406.  

3.4095           Cocaine-induced locomotor stimulation involves autophagic degradation of the dopamine transporter

Harraz, M.M., Guha, P., Kang, I.G., Semenza, E.R., Malla, A.P., Song, Y.J., Reilly, L., Treisman, I., Cortes, P., Coggiano, M.A., Veeravelli, V., Rais, R., Tanda, G. and Snyder, S.H. Mol. Psychiatry, 26, 370-382 (2021)   Cocaine exerts its stimulant effect by inhibiting dopamine reuptake leading to increased dopamine signaling. This action is thought to reflect binding of cocaine to the dopamine transporter (DAT) to inhibit its function. However, cocaine is a relatively weak inhibitor of DAT, and many DAT inhibitors do not share the behavioral actions of cocaine. We previously showed that toxic levels of cocaine induce autophagic neuronal cell death. Here, we show that subnanomolar concentrations of cocaine elicit neural autophagy in vitro and in vivo. Autophagy inhibitors reduce the locomotor stimulant effect of cocaine in mice. Cocaine-induced autophagy degrades transporters for dopamine but not serotonin in the nucleus accumbens. Autophagy inhibition impairs cocaine conditioned place preference in mice. Our findings indicate that autophagic degradation of DAT modulates behavioral actions of cocaine.  

3.4096           Extracellular vesicle mediated feto-maternal HMGB1 signaling induces preterm birth

Radnaa, E., Richardson, L.S., Sheller-Miller, S., Baljinnyam, T., de Castro Silva, M., Kammala, A.K., Urrabaz-Garza, R., Kechichian, T., Kim, S., Han, A. and Menon, R. Lab Chip, 21, 1956-1973 (2021)   Preterm birth (PTB; <37 weeks of gestation) impacts ∼11% of all pregnancies and contributes to 1 million neonatal deaths worldwide annually. An understanding of the feto-maternal (F-M) signals that initiate birthing (parturition) at term is critical to design strategies to prevent their premature activation, resulting in PTB. Although endocrine and immune cell signaling are well-reported, fetal-derived paracrine signals capable of transitioning quiescent uterus to an active state of labor are poorly studied. Recent reports have suggested that senescence of the fetal amnion membrane coinciding with fetal growth and maturation generates inflammatory signals capable of triggering parturition. This is by increasing the inflammatory load at the feto-maternal interface (FMi) tissues (i.e., amniochorion-decidua). High mobility group box 1 protein (HMGB1), an alarmin, is one of the inflammatory signals released by senescent amnion cells via extracellular vesicles (exosomes; 40–160 nm). Increased levels of HMGB1 in the amniotic fluid, cord and maternal blood are associated with term and PTB. This study tested the hypothesis that senescent amnion cells release HMGB1, which is fetal signaling capable of increasing FMi inflammation, predisposing them to parturition. To test this hypothesis, exosomes from amnion epithelial cells (AECs) grown under normal conditions were engineered to contain HMGB1 by electroporation (eHMGB1). eHMGB1 was characterized (quantity, size, shape, markers and loading efficiency), and its propagation through FMi was tested using a four-chamber microfluidic organ-on-a-chip device (FMi-OOC) that contained four distinct cell types (amnion and chorion mesenchymal, chorion trophoblast and decidual cells) connected through microchannels. eHMGB1 propagated through the fetal cells and matrix to the maternal decidua and increased inflammation (receptor expression [RAGE and TLR4] and cytokines). Furthermore, intra-amniotic injection of eHMGB1 (containing 10 ng) into pregnant CD-1 mice on embryonic day 17 led to PTB. Injecting carboxyfluorescein succinimidyl ester (CFSE)-labeled eHMGB1, we determined in vivo kinetics and report that eHMGB1 trafficking resulting in PTB was associated with increased FMi inflammation. This study determined that fetal exosome mediated paracrine signaling can generate inflammation and induce parturition. Besides, in vivo functional validation of FMi-OOC experiments strengthens the reliability of such devices to test physiologic and pathologic systems.  

3.4097           Advances in microfluidic extracellular vesicle analysis for cancer diagnostics

Cheng, S., Li, Y., Yand, h., Wen, Y., Zhou, X., Friedman, L. and Zeng, Y. Lab Chip, 21, 3219-3243 (2021)   Extracellular vesicles (EVs) secreted by cells into the bloodstream and other bodily fluids, including exosomes, have been demonstrated to be a class of significant messengers that mediate intercellular communications. Tumor-derived extracellular vesicles are enriched in a selective set of biomolecules from original cells, including proteins, nucleic acids, and lipids, and thus offer a new perspective of liquid biopsy for cancer diagnosis and therapeutic monitoring. Owing to the heterogeneity of their biogenesis, physical properties, and molecular constituents, isolation and molecular characterization of EVs remain highly challenging. Microfluidics provides a disruptive platform for EV isolation and analysis owing to its inherent advantages to promote the development of new molecular and cellular sensing systems with improved sensitivity, specificity, spatial and temporal resolution, and throughput. This review summarizes the state-of-the-art advances in the development of microfluidic principles and devices for EV isolation and biophysical or biochemical characterization, in comparison to the conventional counterparts. We will also survey the progress in adapting the new microfluidic techniques to assess the emerging EV-associated biomarkers, mostly focused on proteins and nucleic acids, for clinical diagnosis and prognosis of cancer. Lastly, we will discuss the current challenges in the field of EV research and our outlook on future development of enabling microfluidic platforms for EV-based liquid biopsy.  

3.4098           A proteomic view on lysosomes

Muthukottiappan, P. and Winter, D. Mol. Omics, 17, 842-859 (2021)   Lysosomes are the main degradative organelles of almost all eukaryotic cells. They fulfil a crucial function in cellular homeostasis, and impairments in lysosomal function are connected to a continuously increasing number of pathological conditions. In recent years, lysosomes are furthermore emerging as control centers of cellular metabolism, and major regulators of cellular signaling were shown to be activated at the lysosomal surface. To date, >300 proteins were demonstrated to be located in/at the lysosome, and the lysosomal proteome and interactome is constantly growing. For the identification of these proteins, and their involvement in cellular mechanisms or disease progression, mass spectrometry (MS)-based proteomics has proven its worth in a large number of studies. In this review, we are recapitulating the application of MS-based approaches for the investigation of the lysosomal proteome, and their application to a diverse set of research questions. Numerous strategies were applied for the enrichment of lysosomes or lysosomal proteins and their identification by MS-based methods. This allowed for the characterization of the lysosomal proteome, the investigation of lysosome-related disorders, the utilization of lysosomal proteins as biomarkers for diseases, and the characterization of lysosome-related cellular mechanisms. While these >60 studies provide a comprehensive picture of the lysosomal proteome across several model organisms and pathological conditions, various proteomics approaches have not been applied to lysosomes yet, and a large number of questions are still left unanswered.  

3.4099           Fascin-induced actin protrusions are suppressed by dendritic networks in giant unilamellar vesicles

Wubshet, N.H., Bashirzadeh, Y. and Liu, A.P. Mol. Biol. Cell, 32(18), 1634-1640 (2021)   The interactions between actin networks and cell membrane are immensely important for eukaryotic cell functions including cell shape changes, motility, polarity establishment, and adhesion. Actin-binding proteins are known to compete and cooperate using a finite amount of actin monomers to form distinct actin networks. How actin-bundling protein fascin and actin-branching protein Arp2/3 complex compete to remodel membranes is not entirely clear. To investigate fascin- and Arp2/3-mediated actin network remodeling, we applied a reconstitution approach encapsulating bundled and dendritic actin networks inside giant unilamellar vesicles (GUVs). Independently reconstituted, membrane-bound Arp2/3 nucleation forms an actin cortex in GUVs, whereas fascin mediates formation of actin bundles that protrude out of GUVs. Coencapsulating both fascin and Arp2/3 complex leads to polarized dendritic aggregates and significantly reduces membrane protrusions, irrespective of whether the dendritic network is membrane bound or not. However, reducing Arp2/3 complex while increasing fascin restores membrane protrusion. Such changes in network assembly and the subsequent interplay with membrane can be attributed to competition between fascin and Arp2/3 complex to utilize a finite pool of actin.  

3.4100           Stromal-Derived Extracellular Vesicles Suppress Proliferation of Bone Metastatic Cancer Cells Mediated by ERK2

Shupp, A.B., Neupane, M., Agostini, L.C., Ning, G., Brody, J.R. and Bussard, K.M. Mol. Cancer Res., 19, 1763-1766 (2021)   Bone is a common site of cancer metastasis, including cancers such as breast, prostate, and multiple myeloma. Disseminated tumor cells (DTC) shed from a primary tumor may travel to bone and can survive undetected for years before proliferating to form overt metastatic lesions. This period of time can be defined as metastatic latency. Once in the metastatic microenvironment, DTCs engage in intercellular communication with surrounding stromal cells, which can influence cancer cell survival, proliferation, and ultimately disease progression. The role of the surrounding tumor microenvironment in regulating DTC fate is becoming increasingly recognized. We have previously shown that in the bone microenvironment, osteoblasts are “educated” by interactions with breast cancer cells, and these “educated” osteoblasts (EO) produce soluble factors that regulate cancer cell proliferation. In this study, we provide evidence indicating that EOs produce small extracellular vesicles (sEV) that suppress breast cancer proliferation, in part through regulation of ERK1/2 signaling. In addition, using EdU-incorporation assays and propidium iodide staining we demonstrate that exposure to EO-derived sEVs decreases breast cancer cell entry to S-phase of cell cycle. We also have evidence that particular microRNAs, including miR-148a-3p, are enriched in EO-derived sEVs, and that miR-148a-3p is capable of regulating breast cancer proliferation.  

3.4101           Unsupervised Machine Learning-Based Clustering of Nanosized Fluorescent Extracellular Vesicles

Kuypers, S., Smisdom, N., Pintelon, I., Timmermans, J-P., Ameloot, M., Michiels, L., Hendrix, J. and Hosseinkhani, B. Small, 17, 2006786 (2021)   Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease.  

3.4102           A Holistic Review of the State-of-the-Art Microfluidics for Exosome Separation: An Overview of the Current Status, Existing Obstacles, and Future Outlook

Ding, L., Yang, X., Gao, Z., Effah, C.Y., Zhang, X., Wu, Y. and Qu, L. Small, 17, 2007174 (2021)   Exosomes, a class of small extracellular vesicles (30–150 nm), are secreted by almost all types of cells into virtually all body fluids. These small vesicles are attracting increasing research attention owing to their potential for disease diagnosis and therapy. However, their inherent heterogeneity and the complexity of bio-fluids pose significant challenges for their isolation. Even the “gold standard,” differential centrifugation, suffers from poor yields and is time-consuming. In this context, recent developments in microfluidic technologies have provided an ideal system for exosome extraction and these devices exhibit some fascinating properties such as high speeds, good portability, and low sample volumes. In this review, the focus is on the state-of-the-art microfluidic technologies for exosome isolation and highlight potential directions for future research and development by analyzing the challenges faced by the current strategies.  

3.4103           Exosomes in Atherosclerosis, a Double-Edged Sword: Their Role in Disease Pathogenesis and Their Potential as Novel Therapeutics

Patel, N., Chin, D.D. and Chung, E.J. AAPS J., 23:95 (2021)   Cardiovascular disease (CAD) due to atherosclerosis is a major cause of death worldwide. The development of atherosclerosis involves intercellular communication facilitated by exosomes secreted from vascular endothelial cells (VECs), vascular smooth muscle cells (VSMCs), immune cells, and platelets. In this review, we summarize the current understanding of exosome biogenesis and uptake, and discuss atherogenic and atheroprotective functions of exosomes secreted from these cell types. In addition, we examine the potential of enhancing the therapeutic and targeting ability of exosomes exhibiting atheroprotective function by drug loading and surface modification with targeting ligands. We conclude with current challenges associated with exosome engineering for therapeutic use.  

3.4104           Extracellular Vesicles and Hematopoietic Stem Cell Aging

Goldberg, L.R. Arterioscler. Thromb. Vasc. Biol., 41(8), e399-e416 (2021)   Graphical AbstractExtracellular vesicles (EVs), important mediators of intercellular communication, play a critical role in modulating hematopoiesis within the bone marrow microenvironment. Although few studies have explicitly examined the connections between EVs and hematopoietic stem cell (HSC) aging, there is a growing body of evidence that implicates EVs in numerous age-related biologic processes and diseases. This, coupled with their tremendous capacity to influence hematopoiesis, suggests EVs may be key mediators of HSC aging. This review provides an overview of the effects of aging on HSCs, the role of EVs in aging in general, and then details key work in EV modulation of normal and malignant hematopoiesis, with a particular focus on how these effects may translate into the ability of EVs to drive HSC aging. Finally, it describes an exciting emerging literature that provides direct evidence for EV modulation of HSC phenotypes during natural aging and highlights their potential in HSC rejuvenation. Taken collectively, this body of research has profound implications for the future of HSC aging studies. More clearly defining how EVs modify HSC function in an age-dependent fashion and determining the molecular mechanisms by which they drive these age-related HSC phenotype changes will undoubtedly yield innovative strategies to delay or even reverse age-related hematologic dysfunction.    

3.4105           Tissue-derived extracellular vesicles in cancers and non-cancer diseases: Present and future

Li, S-R., Man, Q-W., Gao, X., Lin, H., Wang, J., Su, F-C., Wang, H-Q., Bu, L-L., Liu, B. and Chen, G.

1. Extracell. Vesicles, 10(14), e12175 (2021)

  Extracellular vesicles (EVs) are lipid-bilayer membrane structures secreted by most cell types. EVs act as messengers via the horizontal transfer of lipids, proteins, and nucleic acids, and influence various pathophysiological processes in both parent and recipient cells. Compared to EVs obtained from body fluids or cell culture supernatants, EVs isolated directly from tissues possess a number of advantages, including tissue specificity, accurate reflection of tissue microenvironment, etc., thus, attention should be paid to tissue-derived EVs (Ti-EVs). Ti-EVs are present in the interstitium of tissues and play pivotal roles in intercellular communication. Moreover, Ti-EVs provide an excellent snapshot of interactions among various cell types with a common histological background. Thus, Ti-EVs may be used to gain insights into the development and progression of diseases. To date, extensive investigations have focused on the role of body fluid-derived EVs or cell culture-derived EVs; however, the number of studies on Ti-EVs remains insufficient. Herein, we summarize the latest advances in Ti-EVs for cancers and non-cancer diseases. We propose the future application of Ti-EVs in basic research and clinical practice. Workflows for Ti-EV isolation and characterization between cancers and non-cancer diseases are reviewed and compared. Moreover, we discuss current issues associated with Ti-EVs and provide potential directions.  

3.4106           Development of Drug-Resistant Klebsiella pneumoniae Vaccine via Novel Vesicle Production Technology

Li, W., Hu, Y., Zhang, Q., Hua, L., yang, Z., Ren, Z., Zheng, X., Huang, W. and Ma, Y. ACS Appl. Mater. Interfaces, 13(28), 32703-32715 (2021)   Drug resistance of Klebsiella pneumoniae severely threatens human health. Overcoming the mechanisms of K. pneumoniae resistance to develop novel vaccines against drug-resistant K. pneumoniae is highly desired. Here, we report a technology platform that uses high pressure to drive drug-resistant K. pneumoniae to pass through a gap, inducing the formation of stable artificial bacterial biomimetic vesicles (BBVs). These BBVs had little to no bacterial intracellular protein or nucleic acid and had high yields. BBVs were efficiently taken up by dendritic cells to stimulate their maturation. BBVs as K. pneumoniae vaccines had the dual functions of inducing bacteria-specific humoral and cellular immune responses to increase animals’ survival rate and reduce pulmonary inflammation and bacterial loads. We believe that BBVs are new-generation technology for bacterial vesicle preparation. Establishment of this BBV vaccine platform can maximally expand preparation technology for vaccines against drug-resistant K. pneumoniae.  

3.4107           Role of Microparticles in Cardiovascular Disease: Implications for Endothelial Dysfunction, Thrombosis, and Inflammation

Lugo-gavidia, L.M., Burger, D., Matthews, V.B., Nolde, J.M., Kiuchi, M.G., Carnagarin, R., Kannenkeril, D., Chan, K.J., Joyson, A., Herat, L.Y., Azzam, O. and Schlaich, M.P Hypertension, 77, 1825-1844 (2021)   Microparticles are small cell vesicles that are derived from the cell membrane in response to different biological processes. There is growing evidence supporting the association between microparticles and cardiovascular disease, as their pathophysiology commonly includes endothelial damage and chronic inflammation which also promote a prothrombotic state. The direct causal link between the release of the different subtypes of microparticles and their implications on physiological and pathological conditions is still not completely elucidated. However, evidence suggests microparticles released from platelets, leukocytes, and endothelium may help to evaluate vascular health as they have a relevant role in inflammation, endothelial function, and thrombosis. This review aims to provide a short overview of the biogenesis, characteristics, and detection methodology of microparticles with a special focus on their possible implication in cardiovascular settings.  

3.4108           Translational regulation in the brain by TDP-43 phase separation

Gao, J., Wang, L., Ren, X., Dunn, J.R., Peters, A., Myagi, M., Fujioka, H., Zhao, F., Askwith, C., Liang, J. and Wang, X.

1. Cell Biol., 220(10), e202101019 (2021)

  The in vivo physiological function of liquid–liquid phase separation (LLPS) that governs non–membrane-bound structures remains elusive. Among LLPS-prone proteins, TAR DNA-binding protein of 43 kD (TDP-43) is under intense investigation because of its close association with neurological disorders. Here, we generated mice expressing endogenous LLPS-deficient murine TDP-43. LLPS-deficient TDP-43 mice demonstrate impaired neuronal function and behavioral abnormalities specifically related to brain function. Brain neurons of these mice, however, did not show TDP-43 proteinopathy or neurodegeneration. Instead, the global rate of protein synthesis was found to be greatly enhanced by TDP-43 LLPS loss. Mechanistically, TDP-43 LLPS ablation increased its association with PABPC4, RPS6, RPL7, and other translational factors. The physical interactions between TDP-43 and translational factors relies on a motif, the deletion of which abolished the impact of LLPS-deficient TDP-43 on translation. Our findings show a specific physiological role for TDP-43 LLPS in the regulation of brain function and uncover an intriguing novel molecular mechanism of translational control by LLPS.  

3.4109           L1CAM is not associated with extracellular vesicles in human cerebrospinal fluid or plasma

Norman, M., Ter-Ovanesyan, D., Trieu, W., Lazarovits, R., Kowal, E.J.K., Lee, J.H., Chen-Plotkin, A.S., Regev, A., Church, G.M. and Walt, D.R. Nature Methods, 18, 631-634 (2021)   L1CAM is a transmembrane protein expressed on neurons that was presumed to be found on neuron-derived extracellular vesicles (NDEVs) in human biofluids. We developed a panel of single-molecule array assays to evaluate the use of L1CAM for NDEV isolation. We demonstrate that L1CAM is not associated with extracellular vesicles in human plasma or cerebrospinal fluid and therefore recommend against its use as a marker in NDEV isolation protocols.  

3.4110           Graft-derived extracellular vesicles transported across subcapsular sinus macrophages elicit B cell alloimmunity after transplantation

Zeng, F., Chen, Z., Chen, R., Shufesky, W.J. et al Sc. Transl. Med., 13, eabb0122 (2021)   Despite the role of donor-specific antibodies (DSAs) in recognizing major histocompatibility complex (MHC) antigens and mediating transplant rejection, how and where recipient B cells in lymphoid tissues encounter donor MHC antigens remains unclear. Contrary to the dogma, we demonstrated here that migration of donor leukocytes out of skin or heart allografts is not necessary for B or T cell allosensitization in mice. We found that mouse skin and cardiac allografts and human skin grafts release cell-free donor MHC antigens via extracellular vesicles (EVs) that are captured by subcapsular sinus (SCS) macrophages in lymph nodes or analog macrophages in the spleen. Donor EVs were transported across the SCS macrophages, and donor MHC molecules on the EVs were recognized by alloreactive B cells. This triggered B cell activation and DSA production, which were both prevented by SCS macrophage depletion. These results reveal an unexpected role for graft-derived EVs and open venues to interfere with EV biogenesis, trafficking, or function to restrain priming or reactivation of alloreactive B cells.  

3.4111           Small extracellular vesicles containing miR-486-5p promote angiogenesis after myocardial infarction in mice and nonhuman primates

Li, Q., Xu, Y., Lv, K., Wang, Y., Zhong, Z., Xiao, C. et al Sci. Transl. Med., 13, eabb0202 (2021)   Stem cell–derived small extracellular vesicles (sEVs) promote angiogenesis after myocardial infarction (MI). However, the components of sEVs that contribute to these effects and the safety and efficiency of engineered sEV treatment for MI remain unresolved. Here, we observed improved cardiac function, enhanced vascular density, and smaller infarct size in mice treated with the sEVs from hypoxia-preconditioned (HP) mesenchymal stem cells (MSCs) (HP-sEVs) than in mice treated with normoxia-preconditioned (N) MSCs (N-sEVs). MicroRNA profiling revealed a higher abundance of miR-486-5p in HP-sEVs than in N-sEVs, and miR-486-5p inactivation abolished the benefit of HP-sEV treatment, whereas miR-486-5p up-regulation enhanced the benefit of N-sEV treatment. Matrix metalloproteinase 19 (MMP19) abundance was lower in HP-sEV–treated than N-sEV–treated mouse hearts but was enriched in cardiac fibroblasts (CFs), and Mmp19 was identified as one of the target genes of miR-486-5p. Conditioned medium from CFs that overexpressed miR-486-5p or silenced MMP19 increased the angiogenic activity of endothelial cells; however, medium from CFs that simultaneously overexpressed Mmp19 and miR-486-5p abolished this effect. Mmp19 silencing in CFs reduced the cleavage of extracellular vascular endothelial growth factor (VEGF). Furthermore, miR-486-5p–overexpressing N-sEV treatment promoted angiogenesis and cardiac recovery without increasing arrhythmia complications in a nonhuman primate (NHP) MI model. Collectively, this study highlights the key role of sEV miR-486-5p in promoting cardiac angiogenesis via fibroblastic MMP19-VEGFA cleavage signaling. Delivery of miR-486-5p–engineered sEVs safely enhanced angiogenesis and cardiac function in an NHP MI model and may promote cardiac repair.  

3.4112           Phosphorylation and chromatin tethering prevent cGAS activation during mitosis

Li, T., Huang, T., Du, M., Chen, X., Du, F., Ren, J. and Chen, Z.J. Science, 371, 1221 (2021)   Cyclic guanosine monophosphate (GMP)–adenosine monophosphate (AMP) synthase (cGAS) detects microbial DNA in the cytosol to trigger innate immune responses. cGAS also senses self-DNA in the cytoplasm to promote sterile inflammation and cellular senescence. cGAS binds to double-stranded DNA in a sequence-independent manner, and the multivalent interactions between cGAS and DNA lead to their liquid-liquid phase separation and cGAS activation in the resulting liquid droplets. Once activated, cGAS catalyzes the synthesis of cyclic GMP-AMP (cGAMP), a second messenger that activates the adaptor protein stimulator of interferon genes (STING), leading to the induction of type I interferons and other immune mediators. Paradoxically, a large fraction of cGAS is tightly associated with chromatin, especially during mitosis when the nuclear envelope breaks down. How cGAS activity is regulated during mitosis remains poorly understood.  

3.4113           Mitochondrial DNA alterations underlie an irreversible shift to aerobic glycolysis in fumarate hydratase–deficient renal cancer

Crooks, D.R., Maio, N., Lang, M., Ricketts, C.J., Vocke, C.D. et al Sci. Signal., 14, eabc4436 (2021)   Understanding the mechanisms of the Warburg shift to aerobic glycolysis is critical to defining the metabolic basis of cancer. Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an aggressive cancer characterized by biallelic inactivation of the gene encoding the Krebs cycle enzyme fumarate hydratase, an early shift to aerobic glycolysis, and rapid metastasis. We observed impairment of the mitochondrial respiratory chain in tumors from patients with HLRCC. Biochemical and transcriptomic analyses revealed that respiratory chain dysfunction in the tumors was due to loss of expression of mitochondrial DNA (mtDNA)–encoded subunits of respiratory chain complexes, caused by a marked decrease in mtDNA content and increased mtDNA mutations. We demonstrated that accumulation of fumarate in HLRCC tumors inactivated the core factors responsible for replication and proofreading of mtDNA, leading to loss of respiratory chain components, thereby promoting the shift to aerobic glycolysis and disease progression in this prototypic model of glucose-dependent human cancer.  

3.4114           Transcriptomic taxonomy and neurogenic trajectories of adult human, macaque, and pig hippocampal and entorhinal cells

Franjic, D., Skarica, M., Ma, S., Sousa, A.M.M., Rakic, P. and Sestan, N. Neuron, 110, 452-469 (2022)   The hippocampal-entorhinal system supports cognitive functions, has lifelong neurogenic capabilities in many species, and is selectively vulnerable to Alzheimer’s disease. To investigate neurogenic potential and cellular diversity, we profiled single-nucleus transcriptomes in five hippocampal-entorhinal subregions in humans, macaques, and pigs. Integrated cross-species analysis revealed robust transcriptomic and histologic signatures of neurogenesis in the adult mouse, pig, and macaque but not humans. Doublecortin (DCX), a widely accepted marker of newly generated granule cells, was detected in diverse human neurons, but it did not define immature neuron populations. To explore species differences in cellular diversity and implications for disease, we characterized subregion-specific, transcriptomically defined cell types and transitional changes from the three-layered archicortex to the six-layered neocortex. Notably, METTL7B defined subregion-specific excitatory neurons and astrocytes in primates, associated with endoplasmic reticulum and lipid droplet proteins, including Alzheimer’s disease-related proteins. This resource reveals cell-type- and species-specific properties shaping hippocampal-entorhinal neurogenesis and function.  

3.4115           Vision-dependent specification of cell types and function in the developing cortex

Cheng, S., Butrus, S., Tan, l., Trachtenberg, J.T., Shekhar, K. and Zipursly, S.L. Cell, 185, 311-327 (2022)   The role of postnatal experience in sculpting cortical circuitry, while long appreciated, is poorly understood at the level of cell types. We explore this in the mouse primary visual cortex (V1) using single-nucleus RNA sequencing, visual deprivation, genetics, and functional imaging. We find that vision selectively drives the specification of glutamatergic cell types in upper layers (L) (L2/3/4), while deeper-layer glutamatergic, GABAergic, and non-neuronal cell types are established prior to eye opening. L2/3 cell types form an experience-dependent spatial continuum defined by the graded expression of ∼200 genes, including regulators of cell adhesion and synapse formation. One of these genes, Igsf9b, a vision-dependent gene encoding an inhibitory synaptic cell adhesion molecule, is required for the normal development of binocular responses in L2/3. In summary, vision preferentially regulates the development of upper-layer glutamatergic cell types through the regulation of cell-type-specific gene expression programs.  

3.4116           Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches

Guilliams, M., Bonnardel, J., Haest, B., Van Vlierberghe, H., Devisscher, L. and Scott, C.L. Cell, 185, 379-396 (2022)   The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here we present a spatial proteogenomic atlas of the healthy and obese human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics, and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells, including a population of lipid-associated macrophages (LAMs) at the bile ducts. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and LAMs. We also uncover the respective spatially resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that LAMs are induced by local lipid exposure, leading to their induction in steatotic regions of the murine and human liver, while Kupffer cell development crucially depends on their cross-talk with hepatic stellate cells via the evolutionarily conserved ALK1-BMP9/10 axis.  

3.4117           The two-domain architecture of LAMP2A regulates its interaction with Hsc70

Ikami, Y., Terasawa, k., Sakamoto, K., Ohtake, K., Harada, H., Watabe, T., Yokoyama, S. and Hara-Yokoyama, M. Exp. Cell Res., 411, 112986 (2022)   Chaperone-mediated autophagy (CMA) is a unique proteolytic pathway, in which cytoplasmic proteins recognized by heat shock cognate protein 70 (Hsc70/HSPA8) are transported into lysosomes for degradation. The substrate/chaperone complex binds to the cytosolic tail of the lysosomal-associated membrane protein type 2A (LAMP2A), but whether the interaction between Hsc70 and LAMP2A is direct or mediated by other molecules has remained to be elucidated. The structure of LAMP2A comprises a large lumenal domain composed of two domains, both with the β-prism fold, a transmembrane domain and a short cytoplasmic tail. We previously reported the structural basis for the homophilic interaction of the lumenal domains of LAMP2A, using site-specific photo-crosslinking and/or steric hindrance within cells. In the present study, we introduced a photo-crosslinker into the cytoplasmic tail of LAMP2A and successfully detected its crosslinking with Hsc70, revealing this direct interaction for the first time. Furthermore, we demonstrated that the truncation of the membrane-distal domain within the lumenal domain of LAMP2A reduced the amount of Hsc70 that coimmunoprecipitated with LAMP2A. Our present results suggested that the two-domain architecture of the lumenal domains of LAMP2A underlies the interaction with Hsc70 at the cytoplasmic surface of the lysosome.  

3.4118           The molecular role of Sigmar1 in regulating mitochondrial function through mitochondrial localization in cardiomyocytes

Abdullah, C.S., Aishwarya, R., Alam, S., Remex, N.S., Morshed, M., Nitu, S., Miriyala, S.et al Mitochondrion, 62, 159-175 (2022)   Sigmar1 is a widely expressed molecular chaperone protein in mammalian cell systems. Accumulating research demonstrated the cardioprotective roles of pharmacologic Sigmar1 activation by ligands in preclinical rodent models of cardiac injury. Extensive biochemical and immuno-electron microscopic research demonstrated Sigmar1′s sub-cellular localization largely depends on cell and organ types. Despite comprehensive studies, Sigmar1′s direct molecular role in cardiomyocytes remains elusive. In the present study, we determined Sigmar1′s subcellular localization, transmembrane topology, and function using complementary microscopy, biochemical, and functional assays in cardiomyocytes. Quantum dots in transmission electron microscopy showed Sigmar1 labeled quantum dots on the mitochondrial membranes, lysosomes, and sarcoplasmic reticulum-mitochondrial interface. Subcellular fractionation of heart cell lysates confirmed Sigmar1′s localization in purified mitochondria fraction and lysosome fraction. Immunocytochemistry confirmed Sigmar1 colocalization with mitochondrial proteins in isolated adult mouse cardiomyocytes. Sigmar1′s mitochondrial localization was further confirmed by Sigmar1 colocalization with Mito-Tracker in isolated mouse heart mitochondria. A series of biochemical experiments, including alkaline extraction and proteinase K treatment of purified heart mitochondria, demonstrated Sigmar1 as an integral mitochondrial membrane protein. Sigmar1′s structural requirement for mitochondrial localization was determined by expressing FLAG-tagged Sigmar1 fragments in cells. Full-length Sigmar1 and Sigmar1′s C terminal-deletion fragments were able to localize to the mitochondrial membrane, whereas N-terminal deletion fragment was unable to incorporate into the mitochondria. Finally, functional assays using extracellular flux analyzer and high-resolution respirometry showed Sigmar1 siRNA knockdown significantly altered mitochondrial respiration in cardiomyocytes. Overall, we found that Sigmar1 localizes to mitochondrial membranes and is indispensable for maintaining mitochondrial respiratory homeostasis in cardiomyocytes.  

3.4119           Exosomes derived from immunogenically dying tumor cells as a versatile tool for vaccination against pancreatic cancer

Zhou, W., Chen, X., Zhou, Y., Shi, S., Liang, C., Yu, X., Chen, H., et al Biomaterials, 280, 121306 (2022)   Despite tremendous progress achieved in immunotherapy, many critical challenges in treating pancreatic ductal adenocarcinoma (PDAC) persist. Considering the poor vascularization of PDAC, after intramuscular administration exosomes can targeted deliver “cargos” to pancreatic tumors and bypass obstructions of the intrinsic overexpressed stroma through lymphatics. Herein, we propose a strategy to derive exosomes from immunogenically dying tumor cells and exploit their properties for several purposes, including antigen presentation, adjuvant supply, and “cargo” delivery of vaccines against pancreatic cancer via intramuscular injection. To enhance the immunostimulatory effects, the MART-1 peptide is modified to the exosomes to expand T-cell-related responses. Furthermore, CCL22 siRNA is electroporated into the exosomes (referred to as spMEXO) to hinder the CCR4/CCL22 axis between DCs and Tregs, thereby suppressing Treg expansion. Both in vitro and in vivo studies demonstrate that spMEXO can serve as an effective prophylactic vaccine to delay tumor growth, whereas combining spMEXO with PDAC first-line chemotherapeutics (co-administration of gemcitabine with albumin-paclitaxel) demonstrated significantly enhanced therapeutic effects in established PANC-02 tumors. Therefore, the present work provides an effective strategy to employ cancer vaccines through intramuscular injection in PDAC and highlights the potential of exosomes derived from immunogenically dying tumor cells as a versatile tool to develop nanovaccines for immunotherapy.  

3.4120           Bacterial membrane vesicle functions, laboratory methods, and applications

Celik, P.A., Derkus, B., Erdogan, K., Barut, D., Manga, E.B., Yildirim, Y., Pecha, S. and Cabuk, A. Biotechnology Advances, 54, 107869 (2022)   Bacterial membrane vesicles (BMVs) are cupped-shaped structures formed by bacteria in response to environmental stress, genetic alteration, antibiotic exposure, and others. Due to the structural similarities shared with the producer organism, they can retain certain characteristics like stimulating immune responses. They are also able to carry molecules for long distances, without changes in the concentration and integrity of the molecule. Bacteria originally secrete membrane vesicles for gene transfer, excretion, cell to cell interaction, pathogenesis, and protection against phages. These functions are unique and have several innovative applications in the pharmaceutical industry that have attracted both scientific and commercial interest.This led to the development of efficient methods to artificially stimulate vesicle production, purification, and manipulation in the lab at nanoscales. Also, for specific applications, engineering methods to impart pathogen antigens against specific diseases or customization as cargo vehicles to deliver payloads to specific cells have been reported. Many applications of BMVs are in cancer drugs, vaccines, and adjuvant development with several candidates in clinical trials showing promising results. Despite this, applications in therapy and commercialization stay timid probably due to some challenges one of which is the poor understanding of biogenesis mechanisms. Nevertheless, so far, BMVs seem to be a reliable and cost-efficient technology with several therapeutic applications. Research toward characterizing more membrane vesicles, genetic engineering, and nanotechnology will enable the scope of applications to widen. This might include solutions to other currently faced medical and healthcare-related challenges.  

3.4121           17β-estradiol promotes angiogenesis through non-genomic activation of Smad1 signaling in endometriosis

Zhao, X., Li, X., Liu, P., Li, P., Xu, X., Chen, Y., Cheng, Y., Zhu, D. and Fu, X. Vasc. Pharmacol., 142, 106932 (2022)   17β-estradiol (E2) plays a key role in endometriosis through regulation of angiogenesis. Smad1 has been reported to be up-regulated in patients with endometriosis. However, the role of Smad1 in E2-mediated angiogenesis during the development of endometriosis remains to be determined. This study aimed to explore the role of Smad1 in E2-mediated angiogenesis during endometriosis and its underlying mechanisms. Immunofluorescence staining and Western blotting were performed to examine the expression of p-Smad1 in ectopic and control endometrium. Western blotting was used to examine activation of Smad1 signaling in NMECs, EMECs and HUVECs. Tube formation assay was performed to examine the effect of E2 on angiogenesis. Cell proliferation and migration was determined using in real-time by xCELLigence RTCA DP instrument. We found that the expression of p-Smad1 was significantly up-regulated in ectopic endometrium and ectopic intima microvascular endothelial cells. E2 non-genomically stimulated phosphorylation of Smad1 in HUVECs. c-Src and p44/42 MAPK(ERK1/2) signaling pathways are required for E2's induction on Smad1 phosphorylation. Moreover, caveolae is involved in E2-induced Smad1 phosphorylation in vascular endothelial cells. E2 promoted tube formation of vascular endothelial cells through c-Src/ERK1/2/Smad1 signaling pathway. Knockdown of Smad1 expression attenuated E2-induced proliferation and migration of HUVECs. In conclusion, E2 promotes proliferation, migration and tube formation of HUVECs through c-Src/ERK1/2/Smad1 signaling pathway. Our data shed new lights on the mechanisms through which E2 contributes to endometriosis, and may provide novel strategies to treat endometriosis.  

3.4122           Porcine uterine luminal fluid-derived extracellular vesicles improve conceptus-endometrial interaction during implantation

Hu, Q., Zang, X., Ding, Y., Gu, T., Shi, J., Li, Z., Cai, G., Liu, D., Wu, Z. and Hong, L. Theriogenology, 178, 8-17 (2022)   Successful implantation of porcine conceptus requires synergistic interaction with various signal molecules in the maternal uterus. Extracellular vesicles (EVs) in uterine luminal fluid (ULF) of mice play important roles in conceptus development. However, studies have not explored the roles of extracellular vesicles (EV) in ULF of pigs. The aim of this study was to identify characteristics, origin, and roles of ULF-derived EVs on day 9 of the estrous cycle and on day 9,12 and 15 of pregnancy in pigs. Western blot, BCA assay and HE staining analysis showed increase in EVs concentration in ULF began from day 12 of pregnancy. Immunofluorescence staining and transmission electron microscopy analysis showed that EVs were mainly derived from endometrial epithelial cells. Fluorescent labeling, CCK-8 and transwell migration assays showed that these EVs were delivered to the trophoblast or parthenogenetic activation embryos to regulate proliferation and migration of trophoblast cells. A total of 305 miRNAs were identified using small RNA sequencing analysis. Functional enrichment analysis showed that miRNAs in these EVs potentially play vital regulatory functions in EV transportation or conceptus implantation. QRT-PCR analysis was used to further verify the RNA-seq data. The findings of this study provide information on the functions of porcine ULF-derived EVs and provide a reference dataset for future translational studies on porcine ULF-derived EVs.  

3.4123           Chapter Nine - Extracellular vesicles in tumor immunotherapy

Li, J., Stephens, E. and Zhang, Y. Systems Drug Delivery Strategies, 231-256 (2022)   As endogenous nanocarriers and mediators of cell-to-cell communication, extracellular vesicles (EVs) have been emerging as promising therapeutic agents to combat many diseases. Of the different categories of EVs, exosomes have gained particular prowess in therapeutic development due to valuable pharmacological properties. Exosomes are small membranous vesicles secreted from diverse types of cells, and upon their biogenesis, they inherit contents including proteins, DNA fragments, and RNAs from parental cells. The subsequent release of exosomes into the extracellular space plays an important role in intercellular communication by initiating signaling through inherited surface ligands and transferring cargo contents upon uptake. Tumor-derived exosomes have been shown to be critical for tumor metastasis, angiogenesis, and microenvironmental modulation through these processes. Moreover, given their abilities to modulate the immune system, exosomes have also become important candidates for immunotherapy. In this chapter, the biogenesis, physiological functions, and roles in immune modulation for exosomes are first introduced. Exosome-based cancer applications are then highlighted such as cancer vaccines, exosome as delivery vehicles of therapeutic cargos, and current clinical and translational research. Lastly, the future development of exosomes in immunotherapy is discussed.  

3.4124           The Yin and Yang of exosome isolation methods: conventional practice, microfluidics, and commercial kits

Shirejini, S.Z. and Inci, F. Biotechnology Advances, 54, 107814 (2022)   Exosomes are a subset of extracellular vesicles released from various cells, and they can be found in different bodily fluids. Exosomes have been utilized as biomarkers to diagnose many diseases and to monitor therapy efficiency as they represent the status and origin of the cell, which they are released from. Considering that they co-exist in bodily fluids with other types of particles, their isolation still remains challenging since conventional methods are time-consuming, user-dependent, and result in low isolation yield. This review summarizes the conventional strategies and microfluidic-based methods for exosome isolation along with their strengths and limitations. In particular, microfluidic devices emerge as a promising approach to tackle the existing limitations of conventional methods, and they provide unique features, such as operating with minute volume of samples and rapid process, in order to isolate exosomes with the high yield and the high purity, which make them unprecedented tools for molecular biology and clinical applications in exosome research. This review further elaborates on the existing microfluidic-based exosome isolation methods and denotes their benefits and drawbacks. Herein, we also introduce various commercially available platforms and kits for exosome isolation along with their working principles.  

3.4125           Subcellular metabolomics: Isolation, measurement, and applications

Qin, S., zhang, Y., Tian, Y., Xu, F. and Zhang, P.

1. Pharmacaeut. Biomed. Analysis, 210, 114557 (2022)

  Metabolomics, a technique that profiles global small molecules in biological samples, has been a pivotal tool for disease diagnosis and mechanism research. The sample type in metabolomics covers a wide range, including a variety of body fluids, tissues, and cells. However, little attention was paid to the smaller, relatively independent partition systems in cells, namely the organelles. The organelles are specific compartments/places where diverse metabolic activities are happening in an orderly manner. Metabolic disorders of organelles were found to occur in various pathological conditions such as inherited metabolic diseases, diabetes, cancer, and neurodegenerative diseases. However, at the cellular level, the metabolic outcomes of organelles and cytoplasm are superimposed interactively, making it difficult to describe the changes in subcellular compartments. Therefore, characterizing the metabolic pool in the compartmentalized system is of great significance for understanding the role of organelles in physiological functions and diseases. So far, there are very few research articles or reviews related to subcellular metabolomics. In this review, subcellular fractionation and metabolite analysis methods, as well as the application of subcellular metabolomics in the physiological and pathological studies are systematically reviewed, as a practical reference to promote the continued advancement in subcellular metabolomics.  

3.4126           Cell-derived artificial nanovesicle as a drug delivery system for malignant melanoma treatment

Lin, Y-Y., Chen, C-Y., Ma, D-L., Leung, C-H., Chang, C-Y. and Wang, H-M.D. Biomed. Pharmacother., 147, 112586 (2022)   Extracellular vehicles have a natural targeting ability and immune tolerance of being usually applied in drug delivery systems; however, the purification of EVs is complicated and the production yield was quite low. We developed an artificial cellular mimetic nanovesicle (NV) with melanoma fragment membrane for the transportation with curcumin to achieve the anticancer purpose. B16F10 derived NVs were manufactured by the breakdown of cells using a series of extrusions through cut-off size filters (10 and 5 µm), and the whole procedure was easy and time-saving. To terminate the suspicion of cancer metastatic issue, B16F10 cells were treated by 30-min sonication and 1-min UVB exposure to remove genetic materials before the extrusion. B16F10 derived NV loaded with curcumin was called NV(S30U1/Cur), and the anticancer effect was evaluated by cell-based viability, immune, migration, and invasion. The results showed that NVs were manufactured by passing through 10 and 5 µm filters having an enviable production yield, and the mRNA amounts were declined within NVs produced by B16F10 cells treated with UVB in a comparison to the control group. NV(S30U1/Cur) were effectively decreased B1610 cell viability, and migratory and invasive abilities were also reduced significantly. Besides, CD8⁺ expression of murine primary lymphocytes was activated with CD4⁺ reduction by NV(S30U1/Cur) to stimulate the inherent tumor suppressive capacity in the immune system. Taken together, we established bioengineered NVs serving as novel cell mimetic nanocarriers to deliver natural compound for malignant melanoma potential immune chemotherapy.  

3.4127           Proteomic identification of the contents of small extracellular vesicles from in vivo Plasmodium yoelii infection

De Sousa, K.P., Potriquet, J., Mulvenna, J., Sotillo, J., Groves, P.L., Loukas, A., Apte, S.H. and doolan, D.L. Int. J. Parasitol., 52, 35-45 (2022)   Small extracellular vesicles, including exosomes, are formed by the endocytic pathway and contain genetic and protein material which reflect the contents of their cells of origin. These contents have a role in vesicle-mediated information transfer, as well as physiological and pathological functions. Thus, these vesicles are of great interest as therapeutic targets, or as vehicles for immunomodulatory control. In Plasmodium spp. infections, vesicles derived from the parasite or parasite-infected cells have been shown to induce the expression of pro-inflammatory elements, which have been correlated with manifestations of clinical disease. Herein, we characterised the protein cargo of naturally occurring sEVs in the plasma of P. yoelii-infected mice. After in vivo infections, extracellular vesicles in the size range of exosomes were collected by sequential centrifugation/ultracentrifugation followed by isopycnic gradient separation. Analysis of the vesicles was performed by transmission electron microscopy, dynamic light scattering, SDS–PAGE and flow cytometry. LC-MS analysis followed by bioinformatics analysis predicted parasite protein cargo associated with exosomes. Within these small extracellular vesicles, we identified proteins of interest as vaccine candidates, uncharacterized proteins which may be targets of T cell immunoreactivity, and proteins involved in metabolic processes, regulation, homeostasis and immunity. Importantly, the small extracellular vesicles studied in our work were obtained from in vivo infection rather than from the supernatant of in vitro cultures. These findings add to the growing interest in parasite small extracellular vesicles, further our understanding of the interactions between host and parasite, and identify novel proteins which may represent potential targets for vaccination against malaria.  

3.4128           Extracellular vesicles and their role in peripheral nerve regeneration

Hercher, D., Nguyen, M.Q. and Dworak, H. Exp. Neurol., 350, 113968 (2022)   Peripheral nerve injuries often result in sensory and motor dysfunction in respective parts of the body. Regeneration after peripheral nerve injuries is a complex process including the differentiation of Schwann cells, recruiting of macrophages, blood vessel growth and axonal regrowth. Extracellular vesicles (EVs) are considered to play a pivotal role in intercellular communication and transfer of biological information. Specifically, their bioactivity and ability to deliver cargos of various types of nucleic acids and proteins have made them a potential vehicle for neurotherapeutics. However, production, characterization, dosage and targeted delivery of EVs still pose challenges for the clinical translation of EV therapeutics. This review summarizes the current knowledge of EVs in the context of the healthy and injured peripheral nerve and addresses novel concepts for modification of EVs as therapeutic agents for peripheral nerve regeneration.  

3.4129           The BMSC-derived exosomal lncRNA Mir9-3hg suppresses cardiomyocyte ferroptosis in ischemia-reperfusion mice via the Pum2/PRDX6 axis

Zhang, J-K., Zhang, Z., Guo, Z-A., Fu, Y., Chen, X-J., Chen, W-J., Wu, H-F. and Cui, X-J. Nutrition, Metabolism & Cardiovascular Diseases, 32, 515-527 (2022)   Background and aims The exosomal long noncoding RNAs (lncRNAs) have been reported to have cardioprotective effects on ischemia-reperfusion (I/R) injury by hindering ferroptosis, but the role of lncRNA Mir9-3 host gene (Mir9-3hg) in cardiac I/R injury remains unclear. Methods and results Exosomes were extracted from mouse bone marrow mesenchymal stem cells (BMSCs) and identified by detecting the exosome specific marker levels, and the results showed that Mir9-3hg was highly expressed in BMSCs-Exo. Hypoxia/reoxygenation (H/R)-treated HL-1 mouse cardiomyocytes were incubated with exosomes extracted from BMSCs transfected with Mir9-3hg siRNA. BMSCs-Exo incubation observably facilitated cell proliferation, increased glutathione (GSH) content, and reduced iron ion concentration, reactive oxygen species (ROS) level and ferroptosis marker protein levels in H/R-treated cells, while interfering Mir9-3hg reversed these effects. RNA binding protein immunoprecipitation assay was found that Mir9-3hg bound with pumilio RNA binding family member 2 (Pum2) protein and downregulated Pum2 expression. Silence of Pum2 reversed the effects of Mir9-3hg inhibition on cell functions. Chromatin immunoprecipitation assay was revealed that Pum2 bound with peroxiredoxin 6 (PRDX6) promoter and restrained PRDX6 expression. Silence of PRDX6 reversed the improved effects of Pum2 downregulation on cell functions. Additionally, BMSCs-Exo treatment ameliorated cardiac function in I/R-treated mice by inhibiting cardiomyocyte ferroptosis. Conclusions BMSCs-Exo treatment attenuates I/R-induced cardiac injury by inhibiting cardiomyocyte ferroptosis through modulating the Pum2/PRDX6 axis, thereby ameliorating cardiac function.  

3.4130           Methylmercury induces lysosomal membrane permeabilization through JNK-activated Bax lysosomal translocation in neuronal cells

Tianji, L., Dingbang, H., Xiao, C., Xiaojing, M., Fei, Z. and Bin, W. Toxicol. Lett., 357, 73-83 (2022)   MeHg, an environmental toxicant, is highly toxic to the central nervous system. Recent studies have reported that LMP is an important way in the lysosomal damage. However, the role and molecular mechanism of LMP in MeHg-induced neurotoxicity remain unknown. To study MeHg-induced LMP, we used 10μM MeHg to treat SH-SY5Y cells and 2μM MeHg to treat rat cerebral cortical neurons. Acridine orange (AO) staining and analysis of cathepsin B (CTSB) release were used to determine LMP. We found that MeHg reduced red AO fluorescence and induced CTSB release from lysosomes to the cytoplasm in a time-dependent manner. Moreover, pretreatment with the CTSB inhibitor alleviated cytotoxicity in neuronal cells. These results indicate MeHg induces LMP and subsequent CTSB-dependent cytotoxicity in neuronal cells. Bax is a pore-forming protein, which is involved in mitochondrial outer membrane permeabilization. Intriguingly, we demonstrated that MeHg induced Bax to translocate to lysosomes by using immunofluorescence and Western blot analysis of subcellular fractions. Furthermore, downregulating Bax expression suppressed MeHg-induced LMP. Bax subcellular localization is regulated by protein interaction with the cytoplasmic 14-3-3. Our previous study demonstrated that JNK participated in neurotoxicity through regulating protein interaction. In the current study, we showed that JNK dissociated Bax-14-3-3 complex to facilitate Bax lysosomal translocation. Finally, inhibition of the JNK/Bax pathway could alleviate MeHg-induced cytotoxicity in neuronal cells. The present study implies that inhibiting lysosomal damage (LMP)-related signaling might alleviate MeHg neurotoxicity.  

3.4131           Technology insight: Plant-derived vesicles—How far from the clinical biotherapeutics and therapeutic drug carriers?

Cong, M., Tan, S., Li, S., Gao, L., Huang, L., Zhang, H-G. and Qiao, H. Adv. Drug Delivery Reviews, 182, 114108 (2022)   Within the past decades, extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in both prokaryotes and higher eukaryotes to regulate a diverse range of biological processes. Besides EVs, exosome-like nanoparticles (ELNs) derived from plants were also emerging. Comparing to EVs, ELNs are source-widespread, cost-effective and easy to obtain. Their definite activities can be utilized for potential prevention/treatment of an abundance of diseases, including metabolic syndrome, cancer, colitis, alcoholic hepatitis and infectious diseases, which highlights ELNs as promising biotherapeutics. In addition, the potential of ELNs as natural or engineered drug carriers is also attractive. In this review, we tease out the timeline of plant EVs and ELNs, introduce the arising separation, purification and characterization techniques, state the stability and transport manner, discuss the therapeutic opportunities as well as the potential as novel drug carriers. Finally, the challenges and the direction of efforts to realize the clinical transformation of ELNs are also discussed.    

3.4132           Chapter 16 - Therapeutic potential of induced pluripotent stem cell–derived extracellular vesicles: Quo Vadis? Terra incognito

Ho, M.S.H., Ho, M.S.H. and Librach, C.L. Current Tpoics in iPSCs Technology, 393-449 (2022)   Steady-state organ homeostasis along with tissue repair and regeneration after injury are processes intricately orchestrated by stem cells. The dynamic bidirectional communication between endogenous and/or administered stem cells with distant sites of injury is mediated by a sophisticated intercellular communication system comprising of soluble factors and extracellular vesicles (EVs). This chapter describes the pleiotropic roles of stem cell–derived EVs in development and disease, as well as their potential therapeutic applications and utility as prognostic/diagnostic biomarkers. We will describe how the biostability of EVs, coupled with their minute size, allows these compact therapeutic vectors to successfully negotiate physiological barriers (e.g., blood–brain barrier). We will also elaborate on how EVs function as agents of mass dissemination, given that their diverse cargo is reflective of the interplay between culture environment and physiological stressors. The nomenclature, biogenesis origins, and characteristics of EV subtypes (microvesicles, exosomes, and apoptotic bodies) will also be reviewed. Insights into EV characteristics will provide a prelude to the various isolation strategies used to enrich for EVs, as well as how EV properties, including their proteomic and lipid profile, could be exploited in bioengineering endeavors to further enhance their therapeutic efficacy. While the therapeutic utility of adult stem cell–derived EVs has been popularized in both preclinical studies and clinical trials, drawbacks such as age-related senescence may be effectively circumvented by EVs secreted by pluripotent stem cells, such as induced pluripotent stem cells (iPSCs). Finally, we will discuss how iPSC's unique capability for indefinite self-renewal and multilineage differentiation positions iPSC-EVs as superior alternatives to MSC-EVs in terms of their ability to enhance regeneration in a context-dependent manner, with the added benefit of permitting large-scale manufacturing required for clinical translation.  

3.4133           Exosomes and exosome-mimetics as targeted drug carriers: Where we stand and what the future holds?

Filipovic, L., Kojaddinovic, M. and Popovic, M.

1. Drug Delivery Sci. Technol., 68, 103057 (2022)

  Exosomes are a sub-group of extracellular vesicles, playing an important part in a cell-cell communication in many physiological and pathological conditions. Their size and competence for transferring material to recipient cells make them a promising nanocarrier for clinical use. Their non-immunogenic nature, similar to the body's own structure make them far superior transporters compared to liposomes and polymeric nanoparticles. This review, will provide an overview of exosome biogenesis, biological role, and purification methods. The focus of this manuscript will be to summarize specific applications of exosomes and exosome-mimetics as drug delivery systems in pharmaceutical drug development. We will describe drug-loading approaches, in vivo and in vitro exosome tracing methods, specific modifications and examples of the delivery of therapeutic and imaging molecules from a variety of biological origins. Challenges in the translation of exosome-based drug carriers to clinical use will also be discussed in this review.  

3.4134           Proteomic Analysis of Trichomonas vaginalis Phagolysosome, Lysosomal Targeting, and Unconventional Secretion of Cysteine Peptidases

Zimmann, N., Rada, P., Zarsky, V., Smutna, T., Zahonova, K., Dacks, J., Harant, K., Hrdy, I. and Tachezy, J. Mol. Cell. Proteomics, 21(1), 100174 (2022)   The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases.  

3.4135           The Role of Exosomes in Cancer Progression

Soltesz, B., Buglyo, G., Nemeth, N., Szilaguy, M., Pös, O., Szemes, T., Balogh, I. and Nagy, B. Int. J. Mol. Sci., 23:8 (2022)   Early detection, characterization and monitoring of cancer are possible by using extracellular vesicles (EVs) isolated from non-invasively obtained liquid biopsy samples. They play a role in intercellular communication contributing to cell growth, differentiation and survival, thereby affecting the formation of tumor microenvironments and causing metastases. EVs were discovered more than seventy years ago. They have been tested recently as tools of drug delivery to treat cancer. Here we give a brief review on extracellular vesicles, exosomes, microvesicles and apoptotic bodies. Exosomes play an important role by carrying extracellular nucleic acids (DNA, RNA) in cell-to-cell communication causing tumor and metastasis development. We discuss the role of extracellular vesicles in the pathogenesis of cancer and their practical application in the early diagnosis, follow up, and next-generation treatment of cancer patients.  

3.4136           Therapeutic Effects of Hypoxic and Pro-Inflammatory Priming of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Inflammatory Arthritis

Kay, A.G., Treadwell, K., Roach, P., Morgan, R., Lodge, R., Hyland, M., Piccini, A.M., Forsyth, N.R. and Kehoe, O. Int. J. Mol. Sci., 23:126 (2022)   Mesenchymal stem cells (MSCs) immunomodulate inflammatory responses through paracrine signalling, including via secretion of extracellular vesicles (EVs) in the cell secretome. We evaluated the therapeutic potential of MSCs-derived small EVs in an antigen-induced model of arthritis (AIA). EVs isolated from MSCs cultured normoxically (21% O₂, 5% CO₂), hypoxically (2% O₂, 5% CO₂) or with a pro-inflammatory cytokine cocktail were applied into the AIA model. Disease pathology was assessed post-arthritis induction through swelling and histopathological analysis of synovial joint structure. Activated CD4+ T cells from healthy mice were cultured with EVs or MSCs to assess deactivation capabilities prior to application of standard EVs in vivo to assess T cell polarisation within the immune response to AIA. All EVs treatments reduced knee-joint swelling whilst only normoxic and pro-inflammatory primed EVs improved histopathological outcomes. In vitro culture with EVs did not achieve T cell deactivation. Polarisation towards CD4+ helper cells expressing IL17a (Th17) was reduced when normoxic and hypoxic EV treatments were applied in vitro. Normoxic EVs applied into the AIA model reduced Th17 polarisation and improved Regulatory T cell (Treg):Th17 homeostatic balance. Normoxic EVs present the optimal strategy for broad therapeutic benefit. EVs present an effective novel technology with the potential for cell-free therapeutic translation.  

3.4137           CRISPR/Cas9 Genome Editing vs. Over-Expression for Fluorescent Extracellular Vesicle-Labeling: A Quantitative Analysis

Strohmeier, K., Hofmann, M., hauser, F., Sivun, D., Puthukodan, S., Karner, A., Sandner, G., Le Renard, P-E., Jacak, j. and mairhofer, M. Int. J. Mol. Sci., 23:282 (2022)   Over-expression of fluorescently-labeled markers for extracellular vesicles is frequently used to visualize vesicle up-take and transport. EVs that are labeled by over-expression show considerable heterogeneity regarding the number of fluorophores on single particles, which could potentially bias tracking and up-take studies in favor of more strongly-labeled particles. To avoid the potential artefacts that are caused by over-expression, we developed a genome editing approach for the fluorescent labeling of the extracellular vesicle marker CD63 with green fluorescent protein using the CRISPR/Cas9 technology. Using single-molecule sensitive fluorescence microscopy, we quantitatively compared the degree of labeling of secreted small extracellular vesicles from conventional over-expression and the CRISPR/Cas9 approach with true single-particle measurements. With our analysis, we can demonstrate a larger fraction of single-GFP-labeled EVs in the EVs that were isolated from CRISPR/Cas9-modified cells (83%) compared to EVs that were isolated from GFP-CD63 over-expressing cells (36%). Despite only single-GFP-labeling, CRISPR-EVs can be detected and discriminated from auto-fluorescence after their up-take into cells. To demonstrate the flexibility of the CRISPR/Cas9 genome editing method, we fluorescently labeled EVs using the HaloTag^® with lipid membrane permeable dye, JaneliaFluor^® 646, which allowed us to perform 3D-localization microscopy of single EVs taken up by the cultured cells.  

3.4138           Ganglioside GD2 Enhances the Malignant Phenotypes of Melanoma Cells by Cooperating with Integrins

Yesmin, F., Bhuiyan, R.H., Ohmi, Y., Yamamoto, S., Kaneko, K., Ohkawa, Y et al Int. J. Mol. Sci., 23:423 (2022)   Gangliosides have been considered to modulate cell signals in the microdomain of the cell membrane, lipid/rafts, or glycolipid-enriched microdomain/rafts (GEM/rafts). In particular, cancer-associated gangliosides were reported to enhance the malignant properties of cancer cells. In fact, GD2-positive (GD2+) cells showed increased proliferation, invasion, and adhesion, compared with GD2-negative (GD2−) cells. However, the precise mechanisms by which gangliosides regulate cell signaling in GEM/rafts are not well understood. In order to analyze the roles of ganglioside GD2 in the malignant properties of melanoma cells, we searched for GD2-associating molecules on the cell membrane using the enzyme-mediated activation of radical sources combined with mass spectrometry, and integrin β1 was identified as a representative GD2-associating molecule. Then, we showed the physical association of GD2 and integrin β1 by immunoprecipitation/immunoblotting. Close localization was also shown by immuno-cytostaining and the proximity ligation assay. During cell adhesion, GD2+ cells showed multiple phospho-tyrosine bands, i.e., the epithelial growth factor receptor and focal adhesion kinase. The knockdown of integrin β1 revealed that the increased malignant phenotypes in GD2+ cells were clearly cancelled. Furthermore, the phosphor-tyrosine bands detected during the adhesion of GD2+ cells almost completely disappeared after the knockdown of integrin β1. Finally, immunoblotting to examine the intracellular distribution of integrins during cell adhesion revealed that large amounts of integrin β1 were localized in GEM/raft fractions in GD2+ cells before and just after cell adhesion, with the majority being localized in the non-raft fractions in GD2− cells. All these results suggest that GD2 and integrin β1 cooperate in GEM/rafts, leading to enhanced malignant phenotypes of melanomas.  

3.4139           Small Extracellular Vesicles from Human Amniotic Fluid Samples as Promising Theranostics

Costa, A., Quarto, R. and Bollini, S. Int. J. Mol. Sci., 23:590 (2022)   Since the first evidence that stem cells can provide pro-resolving effects via paracrine secretion of soluble factors, growing interest has been addressed to define the most ideal cell source for clinical translation. Leftover or clinical waste samples of human amniotic fluid obtained following prenatal screening, clinical intervention, or during scheduled caesarean section (C-section) delivery at term have been recently considered an appealing source of mesenchymal progenitors with peculiar regenerative capacity. Human amniotic fluid stem cells (hAFSC) have been demonstrated to support tissue recovery in several preclinical models of disease by exerting paracrine proliferative, anti-inflammatory and regenerative influence. Small extracellular vesicles (EVs) concentrated from the hAFSC secretome (the total soluble trophic factors secreted in the cell-conditioned medium, hAFSC-CM) recapitulate most of the beneficial cell effects. Independent studies in preclinical models of either adult disorders or severe diseases in newborns have suggested a regenerative role of hAFSC-EVs. EVs can be eventually concentrated from amniotic fluid (hAF) to offer useful prenatal information, as recently suggested. In this review, we focus on the most significant aspects of EVs obtained from either hAFSC and hAF and consider the current challenges for their clinical translation, including isolation, characterization and quantification methods.  

3.4140           The Importance of Small Extracellular Vesicles in the Cerebral Metastatic Process

Tämas, F., Balasa, R., Manu, D., Gyorki, G., Chinezu, R., Tämas, C. and Balasa, A. Int. J. Mol. Sci., 23:1449 (2022)   Brain metastases represent more than 50% of all cerebral tumors encountered in clinical practice. Recently, there has been increased interest in the study of extracellular vesicles, and the knowledge about exosomes is constantly expanding. Exosomes are drivers for organotropic metastatic spread, playing important roles in the brain metastatic process by increasing the permeability of the blood–brain barrier and preparing the premetastatic niche. The promising results of the latest experimental studies raise the possibility of one day using exosomes for liquid biopsies or as drug carriers, contributing to early diagnosis and improving the efficacy of chemotherapy in patients with brain metastases. In this review, we attempted to summarize the latest knowledge about the role of exosomes in the brain metastatic process and future research directions for the use of exosomes in patients suffering from brain metastatic disease.  

3.4141           Methodologies to Isolate and Purify Clinical Grade Extracellular Vesicles for Medical Applications

Akbar, A., Malekian, F., Baghban, N., Kodam, S.P. and Ullah, M. Cells, 11:186 (2022)   The use of extracellular vesicles (EV) in nano drug delivery has been demonstrated in many previous studies. In this study, we discuss the sources of extracellular vesicles, including plant, salivary and urinary sources which are easily available but less sought after compared with blood and tissue. Extensive research in the past decade has established that the breadth of EV applications is wide. However, the efforts on standardizing the isolation and purification methods have not brought us to a point that can match the potential of extracellular vesicles for clinical use. The standardization can open doors for many researchers and clinicians alike to experiment with the proposed clinical uses with lesser concerns regarding untraceable side effects. It can make it easier to identify the mechanism of therapeutic benefits and to track the mechanism of any unforeseen effects observed.  

3.4142           Improving Isolation of Extracellular Vesicles by Utilizing Nanomaterials

Zhang, H., Zhang, Q., Deng, Y., Chen, M. and Yang, C. Membranes, 12:55 (2022)   Extracellular vesicles (EVs) as the new form of cellular communication have been demonstrated their potential use for disease diagnosis, prognosis and treatment. EVs are vesicles with a lipid bilayer and are present in various biofluids, such as blood, saliva and urine. Therefore, EVs have emerged as one of the most appealing sources for the discovery of clinical biomarkers. However, isolation of the target EVs from different biofluids is required for the use of EVs as diagnostic and therapeutic entities in clinical settings. Owing to their unique properties and versatile functionalities, nanomaterials have been widely investigated for EV isolation with the aim to provide rapid, simple, and efficient EV enrichment. Herein, this review presents the progress of nanomaterial-based isolations for EVs over the past five years (from 2017 to 2021) and discusses the use of nanomaterials for EV isolations based on the underlying mechanism in order to offer insights into the design of nanomaterials for EV isolations.  

3.4143           Development of DNA Aptamers to Visualize Release of Mycobacterial Membrane-Derived Extracellular Vesicles in Infected Macrophages

Das, S., Jain, S., Ilyas, M., Anand, A., Kumar, S., Sharma, N., Singh, K., Mahlawat, R., Sharma, T.K. and Atmakuri, K. Pharmaceuticals, 15:45 (2022)   Extracellular vesicles (EVs) have emerged into a novel vaccine platform, a biomarker and a nano-carrier for approved drugs. Their accurate detection and visualization are central to their utility in varied biomedical fields. Owing to the limitations of fluorescent dyes and antibodies, here, we describe DNA aptamer as a promising tool for visualizing mycobacterial EVs in vitro. Employing SELEX from a large DNA aptamer library, we identified a best-performing aptamer that is highly specific and binds at nanomolar affinity to EVs derived from three diverse mycobacterial strains (pathogenic, attenuated and avirulent). Confocal microscopy revealed that this aptamer was not only bound to in vitro-enriched mycobacterial EVs but also detected EVs that were internalized by THP-1 macrophages and released by infecting mycobacteria. To the best of our knowledge, this is the first study that detects EVs released by mycobacteria during infection in host macrophages. Within 4 h, most released mycobacterial EVs spread to other parts of the host cell. We predict that this tool will soon hold huge potential in not only delineating mycobacterial EVs-driven pathogenic functions but also in harboring immense propensity to act as a non-invasive diagnostic tool against tuberculosis in general, and extra-pulmonary tuberculosis in particular.  

3.4144           Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

El-Shennawy, L., Hoffmann, A.D., Dashzeveg, N.K., McAndrews, K.M., Mehl, P.J. et al Nature Comm., 13:405 (2022)   The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of the coronavirus induced disease 2019 (COVID-19) with evolving variants of concern. It remains urgent to identify novel approaches against broad strains of SARS-CoV-2, which infect host cells via the entry receptor angiotensin-converting enzyme 2 (ACE2). Herein, we report an increase in circulating extracellular vesicles (EVs) that express ACE2 (evACE2) in plasma of COVID-19 patients, which levels are associated with severe pathogenesis. Importantly, evACE2 isolated from human plasma or cells neutralizes SARS-CoV-2 infection by competing with cellular ACE2. Compared to vesicle-free recombinant human ACE2 (rhACE2), evACE2 shows a 135-fold higher potency in blocking the binding of the viral spike protein RBD, and a 60- to 80-fold higher efficacy in preventing infections by both pseudotyped and authentic SARS-CoV-2. Consistently, evACE2 protects the hACE2 transgenic mice from SARS-CoV-2-induced lung injury and mortality. Furthermore, evACE2 inhibits the infection of SARS-CoV-2 variants (α, β, and δ) with equal or higher potency than for the wildtype strain, supporting a broad-spectrum antiviral mechanism of evACE2 for therapeutic development to block the infection of existing and future coronaviruses that use the ACE2 receptor.  

3.4145           Exosomes from adipose-derived mesenchymal stem cells alleviate sepsis-induced lung injury in mice by inhibiting the secretion of IL-27 in macrophages

Wang, X., Liu, D., Zhang, X., Yang, L., Xia, Z. and Zhang, Q. Cell Death Discovery, 8:18 (2022)   Acute lung injury (ALI) represents a frequent sepsis-induced inflammatory disorder. Mesenchymal stromal cells (MSCs) elicit anti-inflammatory effects in sepsis. This study investigated the mechanism of exosomes from adipose-derived MSCs (ADMSCs) in sepsis-induced ALI. The IL-27r^−/− (WSX-1 knockout) or wild-type mouse model of sepsis was established by cecal ligation and puncture (CLP). The model mice and lipopolysaccharide (LPS)-induced macrophages were treated with ADMSC-exosomes. The content of Dil-labeled exosomes in pulmonary macrophages, macrophages CD68⁺ F4/80⁺ in whole lung tissues, and IL-27 content in macrophages were detected. The mRNA expression and protein level of IL27 subunits P28 and EBI3 in lung tissue and the levels of IL-6, TNF-α, and IL-1β were measured. The pulmonary edema, tissue injury, and pulmonary vascular leakage were measured. In vitro, macrophages internalized ADMSC-exosomes, and ADMSC-exosomes inhibited IL-27 secretion in LPS-induced macrophages. In vivo, IL-27 knockout attenuated CLP-induced ALI. ADMSC-exosomes suppressed macrophage aggregation in lung tissues and inhibited IL-27 secretion. ADMSC-exosomes decreased the contents of IL-6, TNF-α, and IL-1β, reduced pulmonary edema and pulmonary vascular leakage, and improved the survival rate of mice. Injection of recombinant IL-27 reversed the protective effect of ADMSC-exosomes on sepsis mice. Collectively, ADMSC-exosomes inhibited IL-27 secretion in macrophages and alleviated sepsis-induced ALI in mice.  

3.4146           Profiling of open chromatin in developing pig (Sus scrofa) muscle to identify regulatory regions

Salavati, M., Woolley, S:A., Araya, Y.C., Halstead, M.M., Stenhouse, C., Johnsson, M., Asworth, C.J., Archibald, A.L., Donadeu, F.X., Hassan, M.A. and Clark, E.L. Genes Genomes Genetics, 12(2), jkab424 (2022)   There is very little information about how the genome is regulated in domestic pigs (Sus scrofa). This lack of knowledge hinders efforts to define and predict the effects of genetic variants in pig breeding programs. To address this knowledge gap, we need to identify regulatory sequences in the pig genome starting with regions of open chromatin. We used the “Improved Protocol for the Assay for Transposase-Accessible Chromatin (Omni-ATAC-Seq)” to identify putative regulatory regions in flash-frozen semitendinosus muscle from 24 male piglets. We collected samples from the smallest-, average-, and largest-sized male piglets from each litter through five developmental time points. Of the 4661 ATAC-Seq peaks identified that represent regions of open chromatin, >50% were within 1 kb of known transcription start sites. Differential read count analysis revealed 377 ATAC-Seq defined genomic regions where chromatin accessibility differed significantly across developmental time points. We found regions of open chromatin associated with downregulation of genes involved in muscle development that were present in small-sized fetal piglets but absent in large-sized fetal piglets at day 90 of gestation. The dataset that we have generated provides a resource for studies of genome regulation in pigs and contributes valuable functional annotation information to filter genetic variants for use in genomic selection in pig breeding programs.  

3.4147           Extracellular vesicle PD-L1 in reshaping tumor immune microenvironment: biological function and potential therapy strategies

Liu, J., Peng, X., Yang, S., Li, X., Huang, M., Wei, S., Zhang, S., He, G., Zheng, H., Fan, Q., Yang, L. and Li, H. Cell Comm. Signaling, 20:14 (2022)   Programmed cell death 1 ligand 1 (PD-L1) is the ligand for programmed death protein-1 (PD-1), is associated with immunosuppression. Signaling via PD-1/PD-L1 will transmits negative regulatory signals to T cells, inducing T-cell inhibition, reducing CD8⁺ T-cell proliferation, or promoting T-cell apoptosis, which effectively reduces the immune response and leads to large-scale tumor growth. Accordingly, many antibody preparations targeting PD-1 or PD-L1 have been designed to block the binding of these two proteins and restore T-cell proliferation and cytotoxicity of T cells. However, these drugs are ineffective in clinical practice. Recently, numerous of studies have shown that, in addition to the surface of tumor cells, PD-L1 is also found on the surface of extracellular vesicles secreted by these cells. Extracellular vesicle PD-L1 can also interact with PD-1 on the surface of T cells, leading to immunosuppression, and has been proposed as a potential mechanism underlying PD-1/PD-L1-targeted drug resistance. Therefore, it is important to explore the production, regulation and tumor immunosuppression of PD-L1 on the surface of tumor cells and extracellular vesicles, as well as the potential clinical application of extracellular vesicle PD-L1 as tumor biomarkers and therapeutic targets.  

3.4148           Diverse human astrocyte and microglial transcriptional responses to Alzheimer’s pathology

Smith, A.M., Davey, K., Tsartsalis, S., Khozoie, C., Fancy, N., Tang, S.S., Liaptsi, E., Weinert, M., Mcgarry, A., Muirhead, R.C., Gentleman, S., Owen, D.R. and Matthews, P.M: Acta Neurophatologica, 143, 75-91 (2022)   To better define roles that astrocytes and microglia play in Alzheimer’s disease (AD), we used single-nuclei RNA-sequencing to comprehensively characterise transcriptomes in astrocyte and microglia nuclei selectively enriched during isolation post-mortem from neuropathologically defined AD and control brains with a range of amyloid-beta and phospho-tau (pTau) pathology. Significant differences in glial gene expression (including AD risk genes expressed in both the astrocytes [CLU, MEF2C, IQCK] and microglia [APOE, MS4A6A, PILRA]) were correlated with tissue amyloid or pTau expression. The differentially expressed genes were distinct between with the two cell types and pathologies, although common (but cell-type specific) gene sets were enriched with both pathologies in each cell type. Astrocytes showed enrichment for proteostatic, inflammatory and metal ion homeostasis pathways. Pathways for phagocytosis, inflammation and proteostasis were enriched in microglia and perivascular macrophages with greater tissue amyloid, but IL1-related pathway enrichment was found specifically in association with pTau. We also found distinguishable sub-clusters in the astrocytes and microglia characterised by transcriptional signatures related to either homeostatic functions or disease pathology. Gene co-expression analyses revealed potential functional associations of soluble biomarkers of AD in astrocytes (CLU) and microglia (GPNMB). Our work highlights responses of both astrocytes and microglia for pathological protein clearance and inflammation, as well as glial transcriptional diversity in AD.  

3.4149           The role of TolA, TolB, and TolR in cell morphology, OMVs production, and virulence of Salmonella Choleraesuis

Li, Q., Li, Z., Fei, X., Tian, Y., Zhou, G., Hu, Y., Wang, S. and Shi, H. AMP Express, 12:5 (2022)   The Tol–Pal system of Gram-negative bacteria is necessary for maintaining outer membrane integrity. It is a multiprotein complex of five envelope proteins, TolQ, TolR, TolA, TolB, and Pal. These proteins were first investigated in E. coli, and subsequently been identified in many other bacterial genera. However, the function of the Tol–Pal system in Salmonella Choleraesuis pathogenesis is still unclear. Here, we reported the role of three of these proteins in the phenotype and biology of S. Choleraesuis. We found that mutations in tolA, tolB, and tolR caused severe damage to the cell wall, which was supported by observing the microstructure of spherical forms, long chains, flagella defects, and membrane blebbing. We confirmed that all the mutants significantly decreased S. Choleraesuis survival when exposed to sodium deoxycholate and exhibited a high sensitivity to vancomycin, which may be explained by the disruption of envelope integrity. In addition, tolA, tolB, and tolR mutants displayed attenuated virulence in a mouse infection model. This could be interpreted as a series of defective phenotypes in the mutants, such as severe defects in envelope integrity, growth, and motility. Further investigation showed that all the genes participate in outer membrane vesicles (OMVs) biogenesis. Interestingly, immunization with OMVs from ΔtolB efficiently enhanced murine viability in contrast to OMVs from the wild-type S. Choleraesuis, suggesting its potential use in vaccination strategies. Collectively, this study provides an insight into the biological role of the S. Choleraesuis Tol–Pal system.      

3.4150           Streptomyces coelicolor Vesicles: Many Molecules To Be Delivered

Faddetta, T., Renzone, G., Vassallo, A., Rimini, E., Nasillo, G., Buscarino, G., Agnello, S., Licciardi, M., Botta, L., Scaloni, A., Piccionello, A.P., Puglia, A.M. and Gallo, G. App. Environ. Microbiol., 88(1), e01881-21 (2022)   treptomyces coelicolor is a model organism for the study of Streptomyces, a genus of Gram-positive bacteria that undergoes a complex life cycle and produces a broad repertoire of bioactive metabolites and extracellular enzymes. This study investigated the production and characterization of membrane vesicles (MVs) in liquid cultures of S. coelicolor M145 from a structural and biochemical point of view; this was achieved by combining microscopic, physical and -omics analyses. Two main populations of MVs, with different sizes and cargos, were isolated and purified. S. coelicolor MV cargo was determined to be complex, containing different kinds of proteins and metabolites. In particular, a total of 166 proteins involved in cell metabolism/differentiation, molecular processing/transport, and stress response were identified in MVs, the latter functional class also being important for bacterial morpho-physiological differentiation. A subset of these proteins was protected from degradation following treatment of MVs with proteinase K, indicating their localization inside the vesicles. Moreover, S. coelicolor MVs contained an array of metabolites, such as antibiotics, vitamins, amino acids, and components of carbon metabolism. In conclusion, this analysis provides detailed information on S. coelicolor MVs under basal conditions and on their corresponding content, which may be useful in the near future to elucidate vesicle biogenesis and functions.  

3.4151           Landscape and dynamics of accessible chromatin during pigmentation process in green, white and purple sea cucumber Apostichopus japonicus

Xing, L., Liu, S., Zhang, L. and Yang, H. Aquaculture Reports, 23, 101040 (2022)   Cis-regulatory sequences, such as enhancers, control development and physiology by regulating gene expression. The identification and functional characterization of these distal regulatory elements remains challenging, especially in non-model aquatic organisms like sea cucumbers. Apostichopus japonicus, an important echinoderm species, exhibits diverse body colour. Pigmentation is an important stage of growth and development in this species, but the regulatory mechanism remains poorly understood, especially the extent to which epigenetic changes regulate pigmentation. Herein, we globally profiled chromatin accessibility using the assay for transposase-accessible chromatin sequencing technique during pigmentation in green, white and purple sea cucumbers. We have got 27, 817 peaks in total and discovered 1427 peaks showing differentially accessed DNA sites across the genome, which were categorized into four distinct clusters via unsupervised hierarchical clustering. Among three colour morphs, white sea cucumber exhibited the most extensive changes and the greatest differences from the other two colour morphs. Global increases in chromatin accessibility occur in the late pigmentation stage suggesting that active function in the late pigmentation stage drives body colour formation. The screened key cis-regulatory elements KIT, RPS6KB2 and IRF8 may contributed to the albinism, slow growth and poor disease resistance of white sea cucumber. In conclusion, our study provides a framework for understanding the accessible chromatin dynamics of body colour diversity during echinoderm pigmentation process and a new avenue to unravel the transcriptional regulatory mechanisms that facilitate the occurrence of relevant molecular events.  

3.4152           DNAJB8 in small extracellular vesicles promotes Oxaliplatin resistance through TP53/MDR1 pathway in colon cancer

Wang, Z., Li, Y., Mao, R., Zhang, Y., Wen, J., Liu, Q., Liu, Y. and Zhang, T. Cell Death and Disease, 13:151 (2022)   Chemotherapy is one of the most frequently used therapies for the treatment of colon cancer (COAD). However, Oxaliplatin (L-OHP) resistance is a major obstacle to the effective treatment of COAD. Here, we investigated whether DNAJB8, a heat shock protein 40 (HSP40) family protein, could be used for the prognosis and therapy of L-OHP resistance in COAD. Treatment with small interfering RNA targeting DNAJB8 could restore the response to L-OHP in vitro and in vivo. On the mechanism, we demonstrated that DNAJB8 could interact with TP53 and inhibit the ubiquitination degradation of TP53, leading to MDR1 upregulation which promotes colon cancer L-OHP resistance. We found that small extracellular vesicle (sEV)-mediated transfer of DNAJB8 from L-OHP-resistant COAD cells to sensitive cells contributed to L-OHP resistance. A prognostic signature based on the DNAJB8 levels in both tissue and serum showed that COAD patients with high-risk scores exhibited significantly worse overall survival and disease-free survival than patients with low-risk scores. These results indicate that DNAJB8 levels in serum sEVs may serve as a biomarker for COAD. DNAJB8 from sEVs might be a promising therapeutic target for L-OHP resistance and a prognostic predictor of clinical response.  

3.4153           Infrared and Raman spectroscopy for purity assessment of extracellular vesicles

Zini, J., Saari, H., Ciana, p., Viitala, T., Löhmus, A., Saarinen, J. and Yliperttula, M. Eur. J. Pharmaceut. Sci., 172, 106135 (2022)   Extracellular vesicles (EVs) are a complex and heterogeneous population of nanoparticles involved in cell-to-cell communication. Recently, numerous studies have indicated the potential of EVs as therapeutic agents, drug carriers and diagnostic tools. However, the results of these studies are often difficult to evaluate, since different characterization methods are used to assess the purity, physical and biochemical characteristics of the EV samples. In this study, we compared four methods for the EV sample characterization and purity assessment: i) the particle-to-protein ratio based on particle analyses with nanoparticle tracking and protein concentration by bicinchoninic acid assay, ii) Western Blot analysis for specific EV biomarkers, iii) two spectroscopic lipid-to-protein ratios by either the attenuated total reflection Fourier transform infrared (ATR-FTIR) or Raman spectroscopy. The results confirm the value of Raman and ATR-FTIR spectroscopy as robust, fast and operator independent tools that require only a few microliters of EV sample. We propose that the spectroscopic lipid-to-protein (Li/Pr) ratios are reliable parameters for the purity assessment of EV preparations. Moreover, apart from determining protein concentrations, we show that ATR-FTIR spectroscopy can also be used for indirect measurements of EV concentrations. Nevertheless, the Li/Pr ratios do not represent full characterization of the EV preparations. For a complete characterization of selected EV preparations, we recommend also additional use of particle size distribution and EV biomarker analysis.  

3.4154           Lipid flippase dysfunction as a therapeutic target for endosomal anomalies in Alzheimer’s disease

Kaneshiro, N., Komai, M., Imaoka, R., Sakurai, T., Uehara, T. and Takasugi, N. iScience, 25;103869 (2022)   Endosomal anomalies because of vesicular traffic impairment have been indicated as an early pathology of Alzheimer’| disease (AD). However, the mechanisms and therapeutic targets remain unclear. We previously reported that βCTF, one of the pathogenic metabolites of APP, interacts with TMEM30A. TMEM30A constitutes a lipid flippase with P4-ATPase and regulates vesicular trafficking through the asymmetric distribution of phospholipids. Therefore, the alteration of lipid flippase activity in AD pathology has got attention. Herein, we showed that the interaction between βCTF and TMEM30A suppresses the physiological formation and activity of lipid flippase in AD model cells, A7, and App^{NL−G-F/NL−G-F} model mice. Furthermore, the T-RAP peptide derived from the βCTF binding site of TMEM30A improved endosomal anomalies, which could be a result of the restored lipid flippase activity. Our results provide insights into the mechanisms of vesicular traffic impairment and suggest a therapeutic target for AD.  

3.4155           The mammalian trafficking chaperone protein UNC93B1 maintains the ER calcium sensor STIM1 in a dimeric state primed for translocation to the ER cortex

Wang, W-A. and Demaurex, N.

1. Cell. Biochem., 298(3), 101607 (2022)

  The stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum (ER) Ca²⁺ sensor that regulates the activity of Orai plasma membrane Ca²⁺ channels to mediate the store-operated Ca²⁺ entry pathway essential for immunity. Uncoordinated 93 homolog B1 (UNC93B1) is a multiple membrane-spanning ER protein that acts as a trafficking chaperone by guiding nucleic-acid sensing toll-like receptors to their respective endosomal signaling compartments. We previously showed that UNC93B1 interacts with STIM1 to promote antigen cross-presentation in dendritic cells, but the STIM1 binding site(s) and activation step(s) impacted by this interaction remained unknown. In this study, we show that UNC93B1 interacts with STIM1 in the ER lumen by binding to residues in close proximity to the transmembrane domain. Cysteine crosslinking in vivo showed that UNC93B1 binding promotes the zipping of transmembrane and proximal cytosolic helices within resting STIM1 dimers, priming STIM1 for translocation. In addition, we show that UNC93B1 deficiency reduces store-operated Ca²⁺ entry and STIM1–Orai1 interactions and targets STIM1 to lighter ER domains, whereas UNC93B1 expression accelerates the recruitment of STIM1 to cortical ER domains. We conclude that UNC93B1 therefore acts as a trafficking chaperone by maintaining the pool of resting STIM1 proteins in a state primed for activation, enabling their rapid translocation in an extended conformation to cortical ER signaling compartments.  

3.4156           The transmembrane endoplasmic reticulum–associated E3 ubiquitin ligase TRIM13 restrains the pathogenic-DNA–triggered inflammatory response

Li, X., Yu, Z., Fang, Q., Yang, M., Huang, J., Li, Z., Wang, J. and Chen, T. Sci. Adv., 8, eabh0496 (2022)   The endoplasmic reticulum (ER)–localized stimulator of interferon genes (STING) is the core adaptor for the pathogenic-DNA–triggered innate response. Aberrant activation of STING causes autoinflammatory and autoimmune diseases, raising the concern about how STING is finely tuned during innate response to pathogenic DNAs. Here, we report that the transmembrane domain (TM)–containing ER-localized E3 ubiquitin ligase TRIM13 (tripartite motif containing 13) is required for restraining inflammatory response to pathogenic DNAs. TRIM13 deficiency enhances pathogenic-DNA–triggered inflammatory cytokine production, inhibits DNA virus replication, and causes age-related autoinflammation. Mechanistically, TRIM13 interacts with STING via the TM and catalyzes Lys⁶-linked polyubiquitination of STING, leading to decelerated ER exit and accelerated ER-initiated degradation of STING. STING deficiency reverses the enhanced innate anti-DNA virus response in TRIM13 knockout mice. Our study delineates a potential strategy for controlling the homeostasis of STING by transmembrane ER-associated TRIM13 during the pathogenic-DNA–triggered inflammatory response.  

3.4157           A functional cellular framework for sex and estrous cycle-dependent gene expression and behavior

Knoedler, J.R., Inoue, S., Bayless, D.W., Ramakrishnan, C., Deisseroth, K. and Shah, N.M. Cell, 185, 654-671 (2022)   Sex hormones exert a profound influence on gendered behaviors. How individual sex hormone-responsive neuronal populations regulate diverse sex-typical behaviors is unclear. We performed orthogonal, genetically targeted sequencing of four estrogen receptor 1-expressing (Esr1⁺) populations and identified 1,415 genes expressed differentially between sexes or estrous states. Unique subsets of these genes were distributed across all 137 transcriptomically defined Esr1⁺ cell types, including estrous stage-specific ones, that comprise the four populations. We used differentially expressed genes labeling single Esr1⁺ cell types as entry points to functionally characterize two such cell types, BNSTpr^Tac1/Esr1 and VMHvl^Cckar/Esr1. We observed that these two cell types, but not the other Esr1⁺ cell types in these populations, are essential for sex recognition in males and mating in females, respectively. Furthermore, VMHvl^Cckar/Esr1 cell type projections are distinct from those of other VMHvl^Esr1 cell types. Together, projection and functional specialization of dimorphic cell types enables sex hormone-responsive populations to regulate diverse social behaviors.  

3.4158           Exosomes produced by melanoma cells significantly influence the biological properties of normal and cancer-associated fibroblasts

Strnadova, K., Pfeiferova, L., Prikryl, P., Dvofamkova. B., Vlcak, E. et al Histochem. Cell Biol., 157, 153-172 (2022)   The incidence of cutaneous malignant melanoma is increasing worldwide. While the treatment of initial stages of the disease is simple, the advanced disease frequently remains fatal despite novel therapeutic options . This requires identification of novel therapeutic targets in melanoma. Similarly to other types of tumours, the cancer microenvironment plays a prominent role and determines the biological properties of melanoma. Importantly, melanoma cell-produced exosomes represent an important tool of intercellular communication within this cancer ecosystem. We have focused on potential differences in the activity of exosomes produced by melanoma cells towards melanoma-associated fibroblasts and normal dermal fibroblasts. Cancer-associated fibroblasts were activated by the melanoma cell-produced exosomes significantly more than their normal counterparts, as assessed by increased transcription of genes for inflammation-supporting cytokines and chemokines, namely IL-6 or IL-8. We have observed that the response is dependent on the duration of the stimulus via exosomes and also on the quantity of exosomes. Our study demonstrates that melanoma-produced exosomes significantly stimulate the tumour-promoting proinflammatory activity of cancer-associated fibroblasts. This may represent a potential new target of oncologic therapy.    

3.4159           Schwann cell-derived exosomes: Janus-faced mediators of regeneration and disease

Wong, F.C., Ye, L., Demir, I.E. and kahlert, C. Glia, 70, 20-34 (2022)   The phenotypic plasticity of Schwann cells (SCs) has contributed to the regenerative potential of the peripheral nervous system (PNS), but also pathological processes. This double-sided effect has led to an increasing attention to the role of extracellular vesicles (EVs) or exosomes in SCs to examine the intercellular communication between SCs and their surroundings. Here, we first describe the current knowledge of SC and EV biology, which forms the basis for the updates on advances in SC-derived exosomes research. We seek to explore in-depth the exosome-mediated molecular mechanisms involved in the regulation of SCs and their microenvironment. This review concludes with potential applications of SC-derived exosomes as delivery vehicles for therapeutics and biomarkers. The goal of this review is to emphasize the crucial role of SC-derived exosomes in the functional integration of the PNS, highlighting an emerging area in which there is much to explore and re-explore.  

3.4160           Proteome analysis of the Gram-positive fish pathogen Renibacterium salmoninarum reveals putative role of membrane vesicles in virulence

Kroniger, T., Flender, D., Schlüter, R., Köllner, B., Trautweing-Schult, A. and Becher, D. Scientific Reports, 12:3003 (2022)   Bacterial kidney disease (BKD) is a chronic bacterial disease affecting both wild and farmed salmonids. The causative agent for BKD is the Gram-positive fish pathogen Renibacterium salmoninarum. As treatment and prevention of BKD have proven to be difficult, it is important to know and identify the key bacterial proteins that interact with the host. We used subcellular fractionation to report semi-quantitative data for the cytosolic, membrane, extracellular, and membrane vesicle (MV) proteome of R. salmoninarum. These data can aid as a backbone for more targeted experiments regarding the development of new drugs for the treatment of BKD. Further analysis was focused on the MV proteome, where both major immunosuppressive proteins P57/Msa and P22 and proteins involved in bacterial adhesion were found in high abundance. Interestingly, the P22 protein was relatively enriched only in the extracellular and MV fraction, implicating that MVs may play a role in host–pathogen interaction. Compared to the other subcellular fractions, the MVs were also relatively enriched in lipoproteins and all four cell wall hydrolases belonging to the New Lipoprotein C/Protein of 60 kDa (NlpC/P60) family were detected, suggesting an involvement in the formation of the MVs.  

3.4161           Wnt signaling is boosted during intestinal regeneration by a CD44-positive feedback loop

Walter, R.J., Sönnentag, S.J., Munoz-Sagredo, L., Merkel, M., Richert, L. et al Cell Death and disease, 13:168 (2022)   Enhancement of Wnt signaling is fundamental for stem cell function during intestinal regeneration. Molecular modules control Wnt activity by regulating signal transduction. CD44 is such a positive regulator and a Wnt target gene. While highly expressed in intestinal crypts and used as a stem cell marker, its role during intestinal homeostasis and regeneration remains unknown. Here we propose a CD44 positive-feedback loop that boosts Wnt signal transduction, thus impacting intestinal regeneration. Excision of Cd44 in Cd44^fl/fl;VillinCreER^T2 mice reduced Wnt target gene expression in intestinal crypts and affected stem cell functionality in organoids. Although the integrity of the intestinal epithelium was conserved in mice lacking CD44, they were hypersensitive to dextran sulfate sodium, and showed more severe inflammation and delayed regeneration. We localized the molecular function of CD44 at the Wnt signalosome, and identified novel DVL/CD44 and AXIN/CD44 complexes. CD44 thus promotes optimal Wnt signaling during intestinal regeneration.  

3.4162           Small extracellular vesicle-mediated ITGB6 siRNA delivery downregulates the αVβ6 integrin and inhibits adhesion and migration of recipient prostate cancer cells

Krishn, S.R., Garcia, V., Naranjo, N.M., Quaglia, F., Shields, C.D., harris, M.A., Kossenkov, A.V., Liu, Q., Corey, E., Altieri, D.C. and linguino, L.R: Cancer Biol. Ther., 23(1), 173-185 (2022)   The αVβ6 integrin, an epithelial-specific cell surface receptor absent in normal prostate and expressed during prostate cancer (PrCa) progression, is a therapeutic target in many cancers. Here, we report that transcript levels of ITGB6 (encoding the β6 integrin subunit) are significantly increased in metastatic castrate-resistant androgen receptor-negative prostate tumors compared to androgen receptor-positive prostate tumors. In addition, the αVβ6 integrin protein levels are significantly elevated in androgen receptor-negative PrCa patient derived xenografts (PDXs) compared to androgen receptor-positive PDXs. In vitro, the androgen receptor-negative PrCa cells express high levels of the αVβ6 integrin compared to androgen receptor-positive PrCa cells. Additionally, expression of androgen receptor (wild type or variant 7) in androgen receptor-negative PrCa cells downregulates the expression of the β6 but not αV subunit compared to control cells. We demonstrate an efficient strategy to therapeutically target the αVβ6 integrin during PrCa progression by using short interfering RNA (siRNA) loaded into PrCa cell-derived small extracellular vesicles (sEVs). We first demonstrate that fluorescently-labeled siRNAs can be efficiently loaded into PrCa cell-derived sEVs by electroporation. By confocal microscopy, we show efficient internalization of these siRNA-loaded sEVs into PrCa cells. We show that sEV-mediated delivery of ITGB6-targeting siRNAs into PC3 cells specifically downregulates expression of the β6 subunit. Furthermore, treatment with sEVs encapsulating ITGB6 siRNA significantly reduces cell adhesion and migration of PrCa cells on an αVβ6-specific substrate, LAP-TGFβ1. Our results demonstrate an approach for specific targeting of the αVβ6 integrin in PrCa cells using sEVs encapsulating ITGB6-specific siRNAs.  

3.4163           Alzheimer's disease protease-containing plasma extracellular vesicles transfer to the hippocampus via the choroid plexus

Lee, J-H., Ostalecki, C., Oberstein, T., Schierer, S., Zinser, E., Eberhardt, M., Blume, K.Plosnita, B., Stich, L., Bruns, H., Coras, R., Vera-Gonzales, J., Maler, M and Baur, A.S. EBioMedicine, 77, 103903 (2022)   Background Plasma extracellular vesicles (pEV) can harbor a diverse array of factors including active proteases and the amyloid-precursor-protein (APP) cleavage product Aβ, involved in plaque formation in Alzheimer`s diseases (AD). A potential role of such vesicles in AD pathology is unexplored. Methods In a case-control study of randomly selected patients with AD and other neurological diseases (n = 14), and healthy controls (n = 7), we systematically analyzed the content of pEV, using different assay systems. In addition, we determined their entry path into brain tissue, employing animal (mice) injection experiments with ex vivo generated EV that were similar to AD-pEV, followed by multi antigen analysis (MAA) of brain tissue (n = 4 per condition). The results were compared with an IHC staining of human brain tissue in a small cohort of AD patients (n = 3) and controls with no neurodegenerative diseases (n = 3). Findings We show that pEV levels are considerably upregulated in AD patients. Besides numerous inflammatory effectors, AD-pEV contained α-, β- and γ-secretases, able to cleave APP in in target cells. In vitro generated EV with similar characteristics as AD-pEV accumulated in the choroid plexus (CP) of injected animals and reached primarily hippocampal neurons. Corroborating findings were made in human brain samples. An inhibitor of hyaluronic-acid-synthetase (HAS) blocked uploading of proteases and Hyaluronan onto EV in vitro and abolished CP targeting in animal injection experiments. Interpretation We conclude that protease-containing pEV could be part of a communication axis between the periphery and the brain that could be become detrimental depending on pEV concentration and duration of target cell impact.  

3.4164           Selective targeting of chronic social stress-induced activated neurons identifies neurogenesis-related genes to be associated with resilience in female mice

Dos Sontos Guilherme, M., Tsoutsouli, T., Chongtham, M.C., Winter, J., Gerber, S., Müller, M.B. and Endres, K. Psychoneuroendocrinol., 139, 105700 (2022)   Prolonged social stress is a major cause for depression in humans and is associated with a wide range of subsequent pathophysiological changes such as elevated blood pressure. A routinely used model for investigating this kind of stress in mice is the chronic social defeat paradigm where a smaller intruder is exposed to an aggressive inhabitant of a home cage. This model is restricted to males and includes a high proportion of physical stress that might e.g., interfere with immunological aspects of the stress. The prevalence of depression in humans is even higher in women than in men. Therefore, expanding models to female individuals is desirable. We here tested the social instability model as a tool for administering chronic social stress to female C57BL/6J mice and analyzed short-term as well as long-lasting effects. Animals were housed in groups of four and were shuffled two times a week, resulting in a permanent re-structuration of their social hierarchy. While directly after the stress exposure, serum corticosterone was elevated, increased body weight and fat deposits were observed in stressed mice even one year after discontinuation of the stress. At the behavioral level, animals could be stratified into resilient and susceptible animals directly post-stress, but those subgroups were not distinguishable any more in the long-term analysis. To identify molecular contributors to resilience in the here presented social instability induced stress model, Arc-activity dependent trapping of neurons was conducted in Arc-creERT2/sun1sfGFP mice. RNA samples derived from activated nuclei from the ventral hippocampus, a brain region involved in stress-regulation during attacks or explorative behavior of mice, were subjected to a neurogenesis pathway array. While several genes were differentially regulated by stress, in particular, artemin, a neurotrophic factor was upregulated in resilient versus susceptible individuals.  

3.4165           Exosomal RNAs: Novel Potential Biomarkers for Diseases—A Review

Wang, J., Yue, B-L., Huang, Y-Z., Lan, X-Y., Liu, W-J. and Chen, H. Int. J. Mol. Sci., 23:2461 (82022)   Exosomes are a subset of nano-sized extracellular vesicles originating from endosomes. Exosomes mediate cell-to-cell communication with their cargos, which includes mRNAs, miRNAs, lncRNAs, and circRNAs. Exosomal RNAs have cell specificity and reflect the conditions of their donor cells. Notably, their detection in biofluids can be used as a diagnostic marker for various diseases. Exosomal RNAs are ideal biomarkers because their surrounding membranes confer stability and they are detectable in almost all biofluids, which helps to reduce trauma and avoid invasive examinations. However, knowledge of exosomal biomarkers remains scarce. The present review summarizes the biogenesis, secretion, and uptake of exosomes, the current researches exploring exosomal mRNAs, miRNAs, lncRNAs, and circRNAs as potential biomarkers for the diagnosis of human diseases, as well as recent techniques of exosome isolation.  

3.4166           Recruitment of DNA to tumor-derived microvesicles

Clancy, J.W., Sheehan, C.S., Boomgarden, A.C. and D’Souza-Schorey, C. Cell Reports, 38, 110443 (2022)   The shedding of extracellular vesicles (EVs) represents an important but understudied means of cell-cell communication in cancer. Among the currently described classes of EVs, tumor-derived microvesicles (TMVs) comprise a class of vesicles released directly from the cell surface. TMVs contain abundant cargo, including functional proteins and miRNA, which can be transferred to and alter the behavior of recipient cells. Here, we document that a fraction of extracellular double-stranded DNA (dsDNA) is enclosed within TMVs and protected from nuclease degradation. dsDNA inclusion in TMVs is regulated by ARF6 cycling and occurs with the cytosolic DNA sensor, cGAS, but independent of amphisome or micronuclei components. Our studies suggest that dsDNA is trafficked to TMVs via a mechanism distinct from the multivesicular body-dependent secretion reported for the extracellular release of cytosolic DNA. Furthermore, TMV dsDNA can be transferred to recipient cells with consequences to recipient cell behavior, reinforcing its relevance in mediating cell-cell communication.  

3.4167           Assessing hypoxic damage to placental trophoblasts by measuring membrane viscosity of extracellular vesicles

Huang, C., Li, H., Powell, J.S., Ouyang, Y., Wendell, S.G., Suresh, S., Hsia, K.J., Sadovsky, Y. and Quinn, D. Placenta, 121, 14-22 (2022)   Introduction As highly sophisticated intercellular communication vehicles in biological systems, extracellular vesicles (EVs) have been investigated as both promising liquid biopsy-based disease biomarkers and drug delivery carriers. Despite tremendous progress in understanding their biological and physiological functions, mechanical characterization of these nanoscale entities remains challenging due to the limited availability of proper techniques. Especially, whether damage to parental cells can be reflected by the mechanical properties of their EVs remains unknown. Methods In this study, we characterized membrane viscosities of different types of EVs collected from primary human trophoblasts (PHTs), including apoptotic bodies, microvesicles and small extracellular vesicles, using fluorescence lifetime imaging microscopy (FLIM). The biochemical origin of EV membrane viscosity was examined by analyzing their phospholipid composition, using mass spectrometry. Results We found that different EV types derived from the same cell type exhibit different membrane viscosities. The measured membrane viscosity values are well supported by the lipidomic analysis of the phospholipid compositions. We further demonstrate that the membrane viscosity of microvesicles can faithfully reveal hypoxic injury of the human trophoblasts. More specifically, the membrane of PHT microvesicles released under hypoxic condition is less viscous than its counterpart under standard culture condition, which is supported by the reduction in the phosphatidylethanolamine-to-phosphatidylcholine ratio in PHT microvesicles. Discussion Our study suggests that biophysical properties of released trophoblastic microvesicles can reflect cell health. Characterizing EV's membrane viscosity may pave the way for the development of new EV-based clinical applications.  

3.4168           Small extracellular vesicles from plasma of women with preeclampsia increase myogenic tone and decrease endothelium-dependent relaxation of mouse mesenteric arteries

Powell, J.S., Gandley, R.E., Lackner, e., Dolish, A., Ouyang, Y., Powers, R.W., Morelli, A.E., Hubel, C.A. and Sadovsky, Y. Pregnancy Hypertension: An Int. J. Women’s Cardiovasc. Health, 28, 66-73 (2022)   Preeclampsia (PE) is a common syndrome of pregnancy, characterized by new-onset hypertension and proteinuria after gestational week 20, or new onset of hypertension and significant end-organ dysfunction. In the worst cases, it can threaten the survival of both mother and baby. Extracellular vesicles (EVs) are lipid-bilayer nanoparticles released from cells. They are involved in cell–cell communication and transport of diverse cargo molecules. Small extracellular vesicles (sEVs, exosomes) are defined by their size and biogenesis within the endocytic compartment of the cell or reverse budding of the plasma membrane. The function of circulating gestational EVs, released from maternal organs or the placenta, remains to be explored. Here, we focused on sEVs that circulate in the maternal blood in the third trimester of human pregnancy and hypothesized that sEVs from pregnant women with PE play a role in regulation of vessel tone. When compared to sEVs from women with uncomplicated pregnancies, ex vivo exposure of isolated mouse mesenteric arteries to sEVs purified from the plasma of pregnant women with PE led to constriction in response to intraluminal pressure. This effect was not observed using microvesicles from the plasma of women with PE or using PE plasma that was depleted of EVs. Blood vessels exposed to sEVs from women with PE were also more resistant to methacholine-stimulated relaxation. Immunofluorescence microscopy confirmed the presence of sEVs within the vessel wall. Together, these data support the notion that circulating sEVs from pregnant women play a role in the regulation of arterial tone.  

3.4169           Bacterial extracellular vesicles as bioactive nanocarriers for drug delivery: Advances and perspectives

Liu, H., Zhang, Q., Wang, S., Weng, W., Jing, Y. and Su, J. Bioactive Materials, 14, 169-181 (2022)   Nanosized extracellular vesicles derived from bacteria contain diverse cargo and transfer intercellular bioactive molecules to cells. Due to their favorable intercellular interactions, cell membrane-derived bacterial extracellular vesicles (BEVs) have great potential to become novel drug delivery platforms. In this review, we summarize the biogenesis mechanism and compositions of various BEVs. In addition, an overview of effective isolation and purification techniques of BEVs is provided. In particular, we focus on the application of BEVs as bioactive nanocarriers for drug delivery. Finally, we summarize the advances and challenges of BEVs after providing a comprehensive discussion in each section. We believe that a deeper understanding of BEVs will open new avenues for their exploitation in drug delivery applications.  

3.4170           The Neurotoxicity of Vesicles Secreted by ALS Patient Myotubes Is Specific to Exosome-Like and Not Larger Subtypes

Anakor, E., Milla, V., Conolly, O., martinat, C., Pradat, P.F., Dumonceaux, J., Duddy, W. and Duguez, S. Cells, 11:845 (2022)   Extracellular vesicles can mediate communication between tissues, affecting the physiological conditions of recipient cells. They are increasingly investigated in Amyotrophic Lateral Sclerosis, the most common form of Motor Neurone Disease, as transporters of misfolded proteins including SOD1, FUS, TDP43, or other neurotoxic elements, such as the dipeptide repeats resulting from C9orf72 expansions. EVs are classified based on their biogenesis and size and can be separated by differential centrifugation. They include exosomes, released by the fusion of multivesicular bodies with the plasma membrane, and ectosomes, also known as microvesicles or microparticles, resulting from budding or pinching of the plasma membrane. In the current study, EVs were obtained from the myotube cell culture medium of ALS patients or healthy controls. EVs of two different sizes, separating at 20,000 or 100,000 g, were then compared in terms of their effects on recipient motor neurons, astrocytes, and myotubes. Compared to untreated cells, the smaller, exosome-like vesicles of ALS patients reduced the survival of motor neurons by 31% and of myotubes by 18%, decreased neurite length and branching, and increased the proportion of stellate astrocytes, whereas neither those of healthy subjects, nor larger EVs of ALS or healthy subjects, had such effects.  

3.4171           Low-dose metformin targets the lysosomal AMPK pathway through PEN2

Ma, T., Tian, X., Zhang, B., Li, M., Wang, Y., yang, C. et al Nature, 603, 159-165 (2022)   Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1^,2^,3^,4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4^,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.  

3.4172           Controllable secretion of multilayer vesicles driven by microbial polymer accumulation

Koh, S., Sato, M., Yamashina, k., Usukura, Y., Toyofuku, M., Nomura, N. and Taguchi, S. Scientific Reports, 12:3393 (2022)   Membrane vesicles (MVs) are formed in various microorganisms triggered by physiological and environmental phenomena. In this study, we have discovered that the biogenesis of MV took place in the recombinant cell of Escherichia coli BW25113 strain that intracellularly accumulates microbial polyester, polyhydroxybutyrate (PHB). This discovery was achieved as a trigger of foam formation during the microbial PHB fermentation. The purified MVs were existed as a mixture of outer MVs and outer/inner MVs, revealed by transmission electron microscopy. It should be noted that there was a good correlation between MV formation and PHB production level that can be finely controlled by varying glucose concentrations, suggesting the causal relationship in both supramolecules artificially produced in the microbial platform. Notably, the controllable secretion of MV was governed spatiotemporally through the morphological change of the E. coli cells caused by the PHB intracellular accumulation. Based on a hypothesis of PHB internal-pressure dependent envelope-disorder induced MV biogenesis, here we propose a new Polymer Intracellular Accumulation-triggered system for MV Production (designated “PIA-MVP”) with presenting a mechanistic model for MV biogenesis. The PIA-MVP is a promising microbial platform that will provides us with a significance for further study focusing on biopolymer capsulation and cross-membrane transportation for different application purposes.  

3.4173           Single nucleus transcriptome and chromatin accessibility of postmortem human pituitaries reveal diverse stem cell regulatory mechanisms

Zhang, Z., Zamojski, M., Smith, G.R., Andoniadou, C.L., Sealfon, S.C. and Ruf-Zamojski, F. Cell Reports, 38, 110467 (2022)   Despite their importance in tissue homeostasis and renewal, human pituitary stem cells (PSCs) are incompletely characterized. We describe a human single nucleus RNA-seq and ATAC-seq resource from pediatric, adult, and aged postmortem pituitaries (snpituitaryatlas.princeton.edu) and characterize cell-type-specific gene expression and chromatin accessibility programs for all major pituitary cell lineages. We identify uncommitted PSCs, committing progenitor cells, and sex differences. Pseudotime trajectory analysis indicates that early-life PSCs are distinct from the other age groups. Linear modeling of same-cell multiome data identifies regulatory domain accessibility sites and transcription factors that are significantly associated with gene expression in PSCs compared with other cell types and within PSCs. We identify distinct deterministic mechanisms that contribute to heterogeneous marker expression within PSCs. These findings characterize human stem cell lineages and reveal diverse mechanisms regulating key PSC genes and cell type identity.  

3.4174           Single-Nucleus RNA Sequencing Reveals that Decorin Expression in the Amygdala Regulates Perineuronal Nets Expression and Fear Conditioning Response after Traumatic Brain Injury

Shi, Y., Wu, X., Zhou, J., Cui, W., Wang, J., Hu, Q. et al Adv. Sci., 9, 2104112 (2022)   Traumatic brain injury (TBI) is a risk factor for posttraumatic stress disorder (PTSD). Augmented fear is a defining characteristic of PTSD, and the amygdala is considered the main brain region to process fear. The mechanism by which the amygdala is involved in fear conditioning after TBI is still unclear. Using single-nucleus RNA sequencing (snRNA-seq), transcriptional changes in cells in the amygdala after TBI are investigated. In total, 72 328 nuclei are obtained from the sham and TBI groups. 7 cell types, and analysis of differentially expressed genes (DEGs) reveals widespread transcriptional changes in each cell type after TBI are identified. In in vivo experiments, it is demonstrated that Decorin (Dcn) expression in the excitatory neurons of the amygdala significantly increased after TBI, and Dcn knockout in the amygdala mitigates TBI-associated fear conditioning. Of note, this effect is caused by a Dcn-mediated decrease in the expression of perineuronal nets (PNNs), which affect the glutamate-γ-aminobutyric acid balance in the amygdala. Finally, the results suggest that Dcn functions by interacting with collagen VI α3 (Col6a3). Consequently, the findings reveal transcriptional changes in different cell types of the amygdala after TBI and provide direct evidence that Dcn relieves fear conditioning by regulating PNNs.  

3.4175           Extracellular Vesicles Mediate the Intercellular Exchange of Nanoparticles

Wu, X., Tang, T., Wei, Y., Cummins, K.A., Wood, D.K. and Pang, H-B. Adv. Sci., 9, 2102441 (2022)   To exert their therapeutic effects, nanoparticles (NPs) often need to travel into the tissues composed of multilayered cells. Accumulative evidence has revealed the crucial role of transcellular transport route (entry into one cell, exocytosis, and re-entry into another) in this process. While NP endocytosis and subcellular transport are intensively characterized, the exocytosis and re-entry steps are poorly understood, which becomes a barrier for NP delivery into complex tissues. Here, the authors term the exocytosis and re-entry steps together as intercellular exchange. A collagen-based three-dimension assay is developed to specifically quantify the intercellular exchange of NPs, and distinguish the contributions of several potential mechanisms. The authors show that NPs can be exocytosed freely or enclosed inside extracellular vesicles (EVs) for re-entry, while direct cell–cell contact is hardly involved. EVs account for a significant fraction of NP intercellular exchange, and its importance in NP transport is demonstrated in vitro and in vivo. While freely released NPs engage with the same receptors for re-entry, EV-enclosed ones bypass this dependence. These studies provide an easy and precise system to investigate the intercellular exchange stage of NP delivery, and shed the first light in the importance of EVs in NP transport between cells and into complex tissues.  

3.4176           Single nucleus multi-omics identifies human cortical cell regulatory genome diversity

Lu, C., Liu, H., Xie, F., Ren, B., Mukamel, E.A. and Ecker, J.R. Cell Genomics, 2, 100107 (2022)   Single-cell technologies measure unique cellular signatures but are typically limited to a single modality. Computational approaches allow the fusion of diverse single-cell data types, but their efficacy is difficult to validate in the absence of authentic multi-omic measurements. To comprehensively assess the molecular phenotypes of single cells, we devised single-nucleus methylcytosine, chromatin accessibility, and transcriptome sequencing (snmCAT-seq) and applied it to postmortem human frontal cortex tissue. We developed a cross-validation approach using multi-modal information to validate fine-grained cell types and assessed the effectiveness of computational data fusion methods. Correlation analysis in individual cells revealed distinct relations between methylation and gene expression. Our integrative approach enabled joint analyses of the methylome, transcriptome, chromatin accessibility, and conformation for 63 human cortical cell types. We reconstructed regulatory lineages for cortical cell populations and found specific enrichment of genetic risk for neuropsychiatric traits, enabling the prediction of cell types that are associated with diseases.  

3.4177           Cell type–specific mechanism of Setd1a heterozygosity in schizophrenia pathogenesis

Chen,R., Liu, Y., Djekidel, M.N., Chen, W., Bhattacherjee, A., Chen, Z., Scolnick, E. and Zhang, Y. Sci.Adv., 8, eabm1077 (2022)   Schizophrenia (SCZ) is a chronic, serious mental disorder. Although more than 200 SCZ-associated genes have been identified, the underlying molecular and cellular mechanisms remain largely unknown. Here, we generated a Setd1a (SET domain containing 1A) haploinsufficiency mouse model to understand how this SCZ-associated epigenetic factor affects gene expression in brain regions highly relevant to SCZ. Single-cell RNA sequencing revealed that Setd1a heterozygosity causes highly variable transcriptional adaptations across different cell types in prefrontal cortex (PFC) and striatum. The Foxp2⁺ neurons exhibit the most prominent gene expression changes among the different neuron subtypes in PFC, which correlate with changes in histone H3 lysine 4 trimethylation. Many of the genes dysregulated in Setd1a^+/− mice are involved in neuron morphogenesis and synaptic function. Consistently, Setd1a^+/− mice exhibit certain behavioral features of patients with SCZ. Collectively, our study establishes Setd1a^+/− mice as a model for understanding SCZ and uncovers a complex brain region– and cell type–specific dysregulation that potentially underlies SCZ pathogenesis.  

3.4178           Phage Genes Induce Quorum Sensing Signal Release through Membrane Vesicle Formation

Yasuda, M., Yamamoto, T., Nagamura, T., Morinaga, K., Obana, N., Nomura, N. and Toyofuku, M. Microbes Environ., 37(1), ME21067 (2022)   Membrane vesicles (MVs) released from the bacterium Paracoccus denitrificans Pd1222 are enriched with the quorum sensing (QS) signaling molecule N-hexadecanoyl-l-homoserine lactone (C16-HSL). However, the biogenesis of MVs in Pd1222 remains unclear. Investigations on MV formation are crucial for obtaining a more detailed understanding of the dynamics of MV-assisted signaling. In the present study, live-cell imaging showed that P. denitrificans Pd1222 produced MVs through cell lysis under DNA-damaging conditions. DNA sequencing of MVs and a transcriptome ana­lysis of cells indicated that the expression of a prophage region was up-regulated at the onset of MV formation under DNA-damaging conditions. A further sequence ana­lysis identified a putative endolysin (Pden_0381) and holin (Pden_0382) in the prophage region. The expression of these genes was regulated by RecA. Using gene knockout mutants, we showed that prophage-encoded endolysin was critical for MV formation by P. denitrificans Pd1222 under DNA-damaging conditions. MV triggering by endolysin was dependent on the putative holin, which presumably transported endolysin to the periplasmic space. C16-HSL quantification revealed that more signals were released into the milieu as a consequence of the effects of endolysin. Using a QS reporter strain, we found that the QS response in P. denitrificans was stimulated by inducing the expression of endolysin. Collectively, these results provide novel insights into the mechanisms by which a bacterial cell-to-cell communication system is manipulated by phage genes.  

3.4179           Addressing challenges in the removal of unbound dye from passively labelled extracellular vesicles

Rautaniemi, K., Zini, J., Löfman, E., Saari, H., Haapalehto, I., Laukka, J., Vesemäki, S., Efimov, A., Yliperttula, M., Laaksonen, T., Vuorimaa-Laukkanen, E. and Lisitsyna, E.S. Nanoscale Adv., 4, 226-240 (2022)   Studies of extracellular vesicles (EVs), their trafficking and characterization often employ fluorescent labelling. Unfortunately, little attention has been paid thus far to a thorough evaluation of the purification of EVs after labelling, although the presence of an unbound dye may severely compromise the results or even lead to wrong conclusions on EV functionality. Here, we systematically studied five dyes for passive EV labelling and meticulously compared five typical purification methods: ultracentrifugation (UC), ultracentrifugation with discontinuous density gradient (UCG), ultrafiltration (UF), size exclusion chromatography (SEC), and anion exchange chromatography (AEC). A general methodology for evaluation of EV purification efficiency after the labelling was developed and tested to select the purification methods for the chosen dyes. Firstly, we found that some methods initially lead to high EV losses even in the absence of the dye. Secondly, the suitable purification method needs to be found for each particular dye and depends on the physical and chemical properties of the dye. Thirdly, we demonstrated that the developed parameter E_rp (relative purification efficiency) is a useful tool for the pre-screening of the suitable dye-purification method combinations. Additionally, it was also shown that the labelled EVs properly purified from the unbound dye may show significantly reduced contrast and visibility in the target application, e.g. in the live cell fluorescence lifetime imaging.  

3.4180           The G92 NS2B mutant of Tembusu virus is involved in severe defects in progeny virus assembly

Wu, Z., Hu, T., Chen, W., Cheng, Y., Wang, M., Jia, R. et al Vet. Microbiol., 267, 109396 (2022)   Although the role of the flavivirus non-structural 2B (NS2B) as a viral protease cofactor has been investigated, its role in viral proliferation has rarely been studied. Moreover, important details on the involvement of the flavivirus NS2B in the virus replication cycle, including attachment, entry, RNA replication, assembly and release, remain unknown. Here, an alignment of flavivirus NS2B was performed to select conserved amino acids for the site-directed mutagenesis study. G92, which is not totally conserved to mutation, was also used. Certain mutations (P32A, G36A and G37A) completely blocked viral RNA synthesis, four mutations (D25A, A43Y, V44A, G92A and P112A) showed a slight defect in virus replication, and only the G92A mutant affected virus proliferation by blocking virus release but not RNA replication. By taking advantage of the tembusu virus (TMUV) replicon that represents viral genome RNA replication, we showed that the G92A mutation did not obviously impair viral replication. Naturally, the NS2B3 G92A mutation protein did not affect the self-cleavage ability or NS2B3 protein-mediated cleavage of the NS2AB G92A mutant. These results indicated that the G92A mutation did not affect viral protease activity. By using the TMUV infection clone and the replicon package system, the effect of G92 on the virus lifecycle was narrowed to viral resemblance or release. Subcellular fractionation further confirmed that G92A attenuated TMUV by reducing virus assembly. Collectively, these studies extend the list of NS2B functions beyond protease activity as a cofactor, show that transmembrane region amino acids play an important role in viral replication and indicate that G92 is involved in the assembly of infectious TMUV particles.  

3.4181           Non-cell-autonomous disruption of nuclear architecture as a potential cause of COVID-19-induced anosmia

Zazhytska, M., Kodra, A., Hoagland, D.A., tenOever, D.A., Overdevest, J.B. and Lomvardas, S. Cell, 185, 1052-1064 (2022)   SARS-CoV-2 infects less than 1% of cells in the human body, yet it can cause severe damage in a variety of organs. Thus, deciphering the non-cell-autonomous effects of SARS-CoV-2 infection is imperative for understanding the cellular and molecular disruption it elicits. Neurological and cognitive defects are among the least understood symptoms of COVID-19 patients, with olfactory dysfunction being their most common sensory deficit. Here, we show that both in humans and hamsters, SARS-CoV-2 infection causes widespread downregulation of olfactory receptors (ORs) and of their signaling components. This non-cell-autonomous effect is preceded by a dramatic reorganization of the neuronal nuclear architecture, which results in dissipation of genomic compartments harboring OR genes. Our data provide a potential mechanism by which SARS-CoV-2 infection alters the cellular morphology and the transcriptome of cells it cannot infect, offering insight to its systemic effects in olfaction and beyond.  

3.4182           Brain-derived autophagosome profiling reveals the engulfment of nucleoid-enriched mitochondrial fragments by basal autophagy in neurons

Goldsmith, J., Ordureau, A., Harper, J. W. and Holzbaur, E.L.F. Neuron, 110, 967-976 (2022)   Neurons depend on autophagy to maintain cellular homeostasis, and defects in autophagy are pathological hallmarks of neurodegenerative disease. To probe the role of basal autophagy in the maintenance of neuronal health, we isolated autophagic vesicles from mouse brain tissue and used proteomics to identify the major cargos engulfed within autophagosomes, validating our findings in rodent primary and human iPSC-derived neurons. Mitochondrial proteins were identified as a major cargo in the absence of mitophagy adaptors such as OPTN. We found that nucleoid-associated proteins are enriched compared with other mitochondrial components. In the axon, autophagic engulfment of nucleoid-enriched mitochondrial fragments requires the mitochondrial fission machinery Drp1. We proposed that localized Drp1-dependent fission of nucleoid-enriched fragments in proximity to the sites of autophagosome biogenesis enhances their capture. The resulting efficient autophagic turnover of nucleoids may prevent accumulation of mitochondrial DNA in the neuron, thus mitigating activation of proinflammatory pathways that contribute to neurodegeneration.  

3.4183           Chapter 12 - Cell culture metabolomics and lipidomics

Alecu, I., Sosa-Miranda, C.D., Sandhu, J.K., Bennett, S.A.L. and Cuperlava-Culf, M. Metabolomics Perspectives, From Theory to Practical Application, 415-456 (2022)   Cell cultures are one of the most useful tools in life science research and biotechnology, irreplaceable for applications as diverse as testing of drugs or toxins, development of gene and cell therapies, investigation of the function of biomolecules, production of biologics or vaccine particles. Metabolomics and lipidomics with their closest link to cell phenotype and as indicators of functional effects and changes in biological systems provide a highly valuable window into many applications of cell cultures. These omics tools can be used for the real time monitoring of cell culture growth and provide detailed, nonbiased analysis of changes within cells or in secreted media or extracellular vesicles. Metabolomics can be used to optimize cell growth for enhanced production in bioreactor applications or for monitoring cell health, for example, in cell therapy development. In this chapter, we outline some examples and methods used in metabolomics and lipidomics of cell cultures including analysis of cells, media, and extracellular vesicles. Experimental method presentation is followed by introduction to analysis and modeling tools including statistical, mechanistic, and machine learning methods with particular relevance in the applications of metabolomics and lipidomics in cell cultures.  

3.4184           RUFY3 links Arl8b and JIP4-Dynein complex to regulate lysosome size and positioning

Kumar, G., Chawla, P., Dhiman, N., Chadha, S., Sharma, S., Sethi, K., Sharma, M. and Tuli, A. Nature Communications, 13:1540 (2022)   The bidirectional movement of lysosomes on microtubule tracks regulates their whole-cell spatial arrangement. Arl8b, a small GTP-binding (G) protein, promotes lysosome anterograde trafficking mediated by kinesin-1. Herein, we report an Arl8b effector, RUFY3, which regulates the retrograde transport of lysosomes. We show that RUFY3 interacts with the JIP4-dynein-dynactin complex and facilitates Arl8b association with the retrograde motor complex. Accordingly, RUFY3 knockdown disrupts the positioning of Arl8b-positive endosomes and reduces Arl8b colocalization with Rab7-marked late endosomal compartments. Moreover, we find that RUFY3 regulates nutrient-dependent lysosome distribution, although autophagosome-lysosome fusion and autophagic cargo degradation are not impaired upon RUFY3 depletion. Interestingly, lysosome size is significantly reduced in RUFY3 depleted cells, which could be rescued by inhibition of the lysosome reformation regulatory factor PIKFYVE. These findings suggest a model in which the perinuclear cloud arrangement of lysosomes regulates both the positioning and size of these proteolytic compartments.  

3.4185           Aged bone matrix-derived extracellular vesicles as a messenger for calcification paradox

Wang, Z-X., Luo, Z-W., Li, F-X-Z., Cao, J., Rao, S-S. et al Nature Communications, 13:1453 (2022)   Adipocyte differentiation of bone marrow mesenchymal stem/stromal cells (BMSCs) instead of osteoblast formation contributes to age- and menopause-related marrow adiposity and osteoporosis. Vascular calcification often occurs with osteoporosis, a contradictory association called “calcification paradox”. Here we show that extracellular vesicles derived from aged bone matrix (AB-EVs) during bone resorption favor BMSC adipogenesis rather than osteogenesis and augment calcification of vascular smooth muscle cells. Intravenous or intramedullary injection of AB-EVs promotes bone-fat imbalance and exacerbates Vitamin D3 (VD3)-induced vascular calcification in young or old mice. Alendronate (ALE), a bone resorption inhibitor, down-regulates AB-EVs release and attenuates aging- and ovariectomy-induced bone-fat imbalance. In the VD3-treated aged mice, ALE suppresses the ovariectomy-induced aggravation of vascular calcification. MiR-483-5p and miR-2861 are enriched in AB-EVs and essential for the AB-EVs-induced bone-fat imbalance and exacerbation of vascular calcification. Our study uncovers the role of AB-EVs as a messenger for calcification paradox by transferring miR-483-5p and miR-2861.  

3.4186           Role of selenoprotein P expression in the function of pancreatic β cells: Prevention of ferroptosis-like cell death and stress-induced nascent granule degradation

Kitabayashi, N., Nakao, S., Mita, Y., Arisawa, K., Hoshi, T., Touama, T., Ishii, K-A., Takamura, T., Noguchi, N. and Saito, Y. Free Rad. Biol. Med., 183, 89-103 (2022)   Selenoprotein P (SELENOP) is a major selenium (Se)-containing protein (selenoprotein) in human plasma that is mainly synthesized in the liver. SELENOP transports Se to the cells, while SELENOP synthesized in peripheral tissues is incorporated in a paracrine/autocrine manner to maintain the levels of cellular selenoproteins, called the SELENOP cycle. Pancreatic β cells, responsible for the synthesis and secretion of insulin, are known to express SELENOP. Here, using MIN6 cells as a mouse model for pancreatic β cells and Selenop small interfering (si)RNA, we found that Selenop gene knockdown (KD) resulted in decreased cell viability, cellular pro/insulin levels, insulin secretion, and levels of several cellular selenoproteins, including glutathione peroxidase 4 (Gpx4) and selenoprotein K (Selenok). These dysfunctions induced by Selenop siRNA were recovered by the addition of Se. Ferroptosis-like cell death, regulated by Gpx4, was involved in the decrease of cell viability by Selenop KD, while stress-induced nascent granule degradation (SINGD), regulated by Selenok, was responsible for the decrease in proinsulin. SINGD was also observed in the pancreatic β cells of Selenop knockout mice. These findings indicate a significant role of SELENOP expression for the function of pancreatic β cells by maintaining the levels of cellular selenoproteins such as GPX4 and SELENOK.  

3.4187           SWI/SNF chromatin remodeler complex within the reward pathway is required for behavioral adaptations to stress

Zayed, A., Baranowski, C., Compagnion, A-C., Vernochet, C., Karaki, S., et al Nature Comm., 13:1807 (2022)   Enduring behavioral changes upon stress exposure involve changes in gene expression sustained by epigenetic modifications in brain circuits, including the mesocorticolimbic pathway. Brahma (BRM) and Brahma Related Gene 1 (BRG1) are ATPase subunits of the SWI/SNF complexes involved in chromatin remodeling, a process essential to enduring plastic changes in gene expression. Here, we show that in mice, social defeat induces changes in BRG1 nuclear distribution. The inactivation of the Brg1/Smarca4 gene within dopamine-innervated regions or the constitutive inactivation of the Brm/Smarca2 gene leads to resilience to repeated social defeat and decreases the behavioral responses to cocaine without impacting midbrain dopamine neurons activity. Within striatal medium spiny neurons, Brg1 gene inactivation reduces the expression of stress- and cocaine-induced immediate early genes, increases levels of heterochromatin and at a global scale decreases chromatin accessibility. Altogether these data demonstrate the pivotal function of SWI/SNF complexes in behavioral and transcriptional adaptations to salient environmental challenges.  

3.4188           Commonly used methods for extracellular vesicles’ enrichment: Implications in downstream analyses and use

Clos-Sansalvador, M., Monguio-Torjada, M., Roura, S., Franquesa, M. and Borras, F.E. Eur. J. Cell Biol., 101, 151227 (2022)   Extracellular vesicles (EVs) are becoming promising tools for clinical application, either as sources of disease-specific molecular signatures for the unraveling of disease pathophysiology and establishment of novel biomarkers, or as platforms for cell-free nanotherapy. Yet, an unsolved issue is to define standardized techniques for EV isolation allowing data comparison across laboratories worldwide. Considering the difficulties to find this necessary consensus, it has to be stressed out that the outcome of the downstream analysis might be deeply biased by the isolation method, among other variables. Thus, it is crucial that the researcher is aware of the strengths and weaknesses of each method keeping their intended use in mind, and to sufficiently report the methodology details for the results to be comparable and solid. This review aims to present the most widely used EV isolation methods, from the initial differential ultracentrifugation (dUC) to newest approaches.  

3.4189           Bacterial membrane vesicles for vaccine applications

Krishnan, N., Kubiatowicz, L.J., Holay, M., Zhou, J., Fang, R.H. and Zhang, L. Adv. Drug Delivery Reviews, 185, 114294 (2022)   Vaccines have been highly successful in the management of many diseases. However, there are still numerous illnesses, both infectious and noncommunicable, for which there are no clinically approved vaccine formulations. While there are unique difficulties that must be overcome in the case of each specific disease, there are also a number of common challenges that have to be addressed for effective vaccine development. In recent years, bacterial membrane vesicles (BMVs) have received increased attention as a potent and versatile vaccine platform. BMVs are inherently immunostimulatory and are able to activate both innate and adaptive immune responses. Additionally, BMVs can be readily taken up and processed by immune cells due to their nanoscale size. Finally, BMVs can be modified in a variety of ways, including by genetic engineering, cargo loading, and nanoparticle coating, in order to create multifunctional platforms that can be leveraged against different diseases. Here, an overview of the interactions between BMVs and immune cells is provided, followed by discussion on the applications of BMV vaccine nanotechnology against bacterial infections, viral infections, and cancers.  

3.4190           Lipid-mediated phase separation of AGO proteins on the ER controls nascent-peptide ubiquitination

Gao, Y., Zhu, Y., Wang, H., Cheng, Y., Zhao, D., Sun, Q. and Chen, D. Molecular Cell, 82, 1313-1328 (2022)   AGO/miRNA-mediated gene silencing and ubiquitin-mediated protein quality control represent two fundamental mechanisms that control proper gene expression. Here, we unexpectedly discover that fly and human AGO proteins, which are key components in the miRNA pathway, undergo lipid-mediated phase separation and condense into RNP granules on the endoplasmic reticulum (ER) membrane to control protein production. Phase separation on the ER is mediated by electrostatic interactions between a conserved lipid-binding motif within the AGOs and the lipid PI(4,5)P₂. The ER-localized AGO condensates recruit the E3 ubiquitin ligase Ltn1 to catalyze nascent-peptide ubiquitination and coordinate with the VCP-Ufd1-Npl4 complex to process unwanted protein products for proteasomal degradation. Collectively, our study provides insight into the understanding of post-transcription-translation coupling controlled by AGOs via lipid-mediated phase separation.  

3.4191           Hepatocyte-specific activity of TSC22D4 triggers progressive NAFLD by impairing mitochondrial function

Wolff, G., Sakurai, M., Mhamane, A., Troullinaki, M., Maida, A., Dellgiannis, I.K. et al Mol. Metabolism, 60, 101487 (2022)   Objective Fibrotic organ responses have recently been identified as long-term complications in diabetes. Indeed, insulin resistance and aberrant hepatic lipid accumulation represent driving features of progressive non-alcoholic fatty liver disease (NAFLD), ranging from simple steatosis and non-alcoholic steatohepatitis (NASH) to fibrosis. Effective pharmacological regimens to stop progressive liver disease are still lacking to-date. Methods Based on our previous discovery of transforming growth factor beta-like stimulated clone (TSC)22D4 as a key driver of insulin resistance and glucose intolerance in obesity and type 2 diabetes, we generated a TSC22D4-hepatocyte specific knockout line (TSC22D4-HepaKO) and exposed mice to control or NASH diet models. Mechanistic insights were generated by metabolic phenotyping and single-nuclei RNA sequencing. Results Hepatic TSC22D4 expression was significantly correlated with markers of liver disease progression and fibrosis in both murine and human livers. Indeed, hepatic TSC22D4 levels were elevated in human NASH patients as well as in several murine NASH models. Specific genetic deletion of TSC22D4 in hepatocytes led to reduced liver lipid accumulation, improvements in steatosis and inflammation scores and decreased apoptosis in mice fed a lipogenic MCD diet. Single-nuclei RNA sequencing revealed a distinct TSC22D4-dependent gene signature identifying an upregulation of mitochondrial-related processes in hepatocytes upon loss of TSC22D4. An enrichment of genes involved in the TCA cycle, mitochondrial organization, and triglyceride metabolism underscored the hepatocyte-protective phenotype and overall decreased liver damage as seen in mouse models of hepatocyte-selective TSC22D4 loss-of-function. Conclusions Together, our data uncover a new connection between targeted depletion of TSC22D4 and intrinsic metabolic processes in progressive liver disease. Hepatocyte-specific reduction of TSC22D4 improves hepatic steatosis and promotes hepatocyte survival via mitochondrial-related mechanisms thus paving the way for targeted therapies.  

3.4192           CCT2 is an aggrephagy receptor for clearance of solid protein aggregates

Ma, X., Lu, C., Chen, Y., Zhang, M., Yi, C. and Ge, L. Cell, 185, 1325-1345 (2022)   Protein aggregation is a hallmark of multiple human pathologies. Autophagy selectively degrades protein aggregates via aggrephagy. How selectivity is achieved has been elusive. Here, we identify the chaperonin subunit CCT2 as an autophagy receptor regulating the clearance of aggregation-prone proteins in the cell and the mouse brain. CCT2 associates with aggregation-prone proteins independent of cargo ubiquitination and interacts with autophagosome marker ATG8s through a non-classical VLIR motif. In addition, CCT2 regulates aggrephagy independently of the ubiquitin-binding receptors (P62, NBR1, and TAX1BP1) or chaperone-mediated autophagy. Unlike P62, NBR1, and TAX1BP1, which facilitate the clearance of protein condensates with liquidity, CCT2 specifically promotes the autophagic degradation of protein aggregates with little liquidity (solid aggregates). Furthermore, aggregation-prone protein accumulation induces the functional switch of CCT2 from a chaperone subunit to an autophagy receptor by promoting CCT2 monomer formation, which exposes the VLIR to ATG8s interaction and, therefore, enables the autophagic function.  

3.4193           VAP-A and its binding partner CERT drive biogenesis of RNA-containing extracellular vesicles at ER membrane contact sites

Barman, B., Sung, B.H., Krystofiak, E., Patton, J.G., Liu, Q. and Weaver, A.M. Developmental Cell, 57, 974-994 (2022)   RNA transfer via extracellular vesicles (EVs) influences cell phenotypes; however, lack of information regarding biogenesis of RNA-containing EVs has limited progress in the field. Here, we identify endoplasmic reticulum membrane contact sites (ER MCSs) as platforms for the generation of RNA-containing EVs. We identify a subpopulation of small EVs that is highly enriched in RNA and regulated by the ER MCS linker protein VAP-A. Functionally, VAP-A-regulated EVs are critical for miR-100 transfer between cells and in vivo tumor formation. Lipid analysis of VAP-A-knockdown EVs revealed reductions in the EV biogenesis lipid ceramide. Knockdown of the VAP-A-binding ceramide transfer protein CERT led to similar defects in EV RNA content. Imaging experiments revealed that VAP-A promotes luminal filling of multivesicular bodies (MVBs), CERT localizes to MVBs, and the ceramide-generating enzyme neutral sphingomyelinase 2 colocalizes with VAP-A-positive ER. We propose that ceramide transfer via VAP-A-CERT linkages drives the biogenesis of a select RNA-containing EV population.  

3.4194           Encapsulated actomyosin patterns drive cell-like membrane shape changes

Bashrzadeh, Y., Moghimianavval, H.and Liu, A.P. iScience, 25, 104236 (2022)   Cell shape changes from locomotion to cytokinesis are, to a large extent, driven by myosin-driven remodeling of cortical actin patterns. Passive crosslinkers such as α-actinin and fascin as well as actin nucleator Arp2/3 complex largely determine actin network architecture and, consequently, membrane shape changes. Here we reconstitute actomyosin networks inside cell-sized lipid bilayer vesicles and show that depending on vesicle size and concentrations of α-actinin and fascin actomyosin networks assemble into ring and aster-like patterns. Anchoring actin to the membrane does not change actin network architecture yet exerts forces and deforms the membrane when assembled in the form of a contractile ring. In the presence of α-actinin and fascin, an Arp2/3 complex-mediated actomyosin cortex is shown to assemble a ring-like pattern at the equatorial cortex followed by myosin-driven clustering and consequently blebbing. An active gel theory unifies a model for the observed membrane shape changes induced by the contractile cortex.  

3.4195           Deletion of platelet CLEC-2 decreases GPIbα-mediated integrin αIIbβ3 activation and decreases thrombosis in TTP

Shao, B., Hoover, C., Shi, H., Kondo, Y., Lee, R.H., Chen, J., Shan, X., Song, J. et al Blood, 139(16), 2523-2533 (2022)   Microvascular thrombosis in patients with thrombotic thrombocytopenic purpura (TTP) is initiated by GPIbα-mediated platelet binding to von Willebrand factor (VWF). Binding of VWF to GPIbα causes activation of the platelet surface integrin αIIbβ3. However, the mechanism of GPIbα-initiated activation of αIIbβ3 and its clinical importance for microvascular thrombosis remain elusive. Deletion of platelet C-type lectin-like receptor 2 (CLEC-2) did not prevent VWF binding to platelets but specifically inhibited platelet aggregation induced by VWF binding in mice. Deletion of platelet CLEC-2 also inhibited αIIbβ3 activation induced by the binding of VWF to GPIbα. Using a mouse model of TTP, which was created by infusion of anti-mouse ADAMTS13 monoclonal antibodies followed by infusion of VWF, we found that deletion of platelet CLEC-2 decreased pulmonary arterial thrombosis and the severity of thrombocytopenia. Importantly, prophylactic oral administration of aspirin, an inhibitor of platelet activation, and therapeutic treatment of the TTP mice with eptifibatide, an integrin αIIbβ3 antagonist, reduced pulmonary arterial thrombosis in the TTP mouse model. Our observations demonstrate that GPIbα-mediated activation of integrin αIIbβ3 plays an important role in the formation of thrombosis in TTP. These observations suggest that prevention of platelet activation with aspirin may reduce the risk for thrombosis in patients with TTP.  

3.4196           Engineered bacterial membrane vesicles are promising carriers for vaccine design and tumor immunotherapy

Long, Q., Zheng, P., Zhsng, X., Li, W., Hua, L., Yang, Z., Huang, W. and Ma, Y. Adv. Drug Delivery Reviews, 186, 114121 (2022)   Bacterial membrane vesicles (BMVs) have emerged as novel and promising platforms for the development of vaccines and immunotherapeutic strategies against infectious and noninfectious diseases. The rich microbe-associated molecular patterns (MAMPs) and nanoscale membrane vesicle structure of BMVs make them highly immunogenic. In addition, BMVs can be endowed with more functions via genetic and chemical modifications. This article reviews the immunological characteristics and effects of BMVs, techniques for BMV production and modification, and the applications of BMVs as vaccines or vaccine carriers. In summary, given their versatile characteristics and immunomodulatory properties, BMVs can be used for clinical vaccine or immunotherapy applications.  

3.4197           Comparative proteomics analysis of Pichia pastoris cultivating in glucose and methanol

Hou, R., Gao, L., Liu, J., Liang, Z., Zhou, Y.J., Zhang, L. and Zhang, Y. Synthetic and Systems Biotechnol., 7, 862-868 (2022)   The methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) has been extensively engineered for protein production, and is attracting attention as a chassis cell for methanol biotransformation toward production of small molecules. However, the relatively unclear methanol metabolism hampers the metabolic rewiring to improve the biosynthetic efficiency. We here performed a label-free quantitative proteomic analysis of Pichia pastoris when cultivated in minimal media containing methanol and glucose, respectively. There were 243, 158 up-regulated proteins and 244, 304 down-regulated proteins in log and stationary phase, respectively, when cultivated in methanol medium compared with that of glucose medium. Peroxisome enrichment further improved the characterization of more differentially expressed proteins (481 proteins in log phase and 524 proteins in stationary phase). We demonstrated the transaldolase isoenzyme (Tal2, Protein ID: C4R244) was highly up-regulated in methanol medium cultivation, which plays an important role in methanol utilization. Our work provides important information for understanding methanol metabolism in methyltrophic yeast and will help to engineer methanol biotransformation in P. pastoris.  

3.4198           Lysophosphatidylcholine acyltransferase 1 controls mitochondrial reactive oxygen species generation and survival of retinal photoreceptor cells

Nagata, K., Hishikawa, D,m Sagara, H., Saito, M., Watanabe, S., Shimizu, T and Shindou, H.

1. Biol. Chem., 298(6), 101958 (2022)

  Due to their high energy demands and characteristic morphology, retinal photoreceptor cells require a specialized lipid metabolism for survival and function. Accordingly, dysregulation of lipid metabolism leads to the photoreceptor cell death and retinal degeneration. Mice bearing a frameshift mutation in the gene encoding lysophosphatidylcholine acyltransferase 1 (Lpcat1), which produces saturated phosphatidylcholine (PC) composed of two saturated fatty acids, has been reported to cause spontaneous retinal degeneration in mice; however, the mechanism by which this mutation affects degeneration is unclear. In this study, we performed a detailed characterization of LPCAT1 in the retina and found that genetic deletion of Lpcat1 induces light-independent and photoreceptor-specific apoptosis in mice. Lipidomic analyses of the retina and isolated photoreceptor outer segment (OS) suggested that loss of Lpcat1 not only decreased saturated PC production but also affected membrane lipid composition, presumably by altering saturated fatty acyl-CoA availability. Furthermore, we demonstrated that Lpcat1 deletion led to increased mitochondrial reactive oxygen species levels in photoreceptor cells, but not in other retinal cells, and did not affect the OS structure or trafficking of OS-localized proteins. These results suggest that the LPCAT1-dependent production of saturated PC plays critical roles in photoreceptor maturation. Our findings highlight the therapeutic potential of saturated fatty acid metabolism in photoreceptor cell degeneration–related retinal diseases.  

3.4199           Isolation, profiling, and tracking of extracellular vesicle cargo in Caenorhabditis elegans

Nikonorova, I.A., Wang, J., Cope, A.L., Krauschunas, A.R., Shah, P. and Barr, M.M. Current Biology, 32, 1924-1936 (2022)   Extracellular vesicles (EVs) may mediate intercellular communication by carrying protein and RNA cargo. The composition, biology, and roles of EVs in physiology and pathology have been primarily studied in the context of biofluids and in cultured mammalian cells. The experimental tractability of C. elegans makes for a powerful in vivo animal system to identify and study EV cargo from its cellular source. We developed an innovative method to label, track, and profile EVs using genetically encoded, fluorescent-tagged EV cargo and conducted a large-scale isolation and proteomic profiling. Nucleic acid binding proteins (∼200) are overrepresented in our dataset. By integrating our EV proteomic dataset with single-cell transcriptomic data, we identified and validated ciliary EV cargo: CD9-like tetraspanin (TSP-6), ectonucleotide pyrophosphatase/phosphodiesterase (ENPP-1), minichromosome maintenance protein (MCM-3), and double-stranded RNA transporter SID-2. C. elegans EVs also harbor RNA, suggesting that EVs may play a role in extracellular RNA-based communication.  

3.4200           Blood derived extracellular vesicles as regenerative medicine therapeutics

De Boer, C. and Davies, N.H. Biochemie, 196, 203-215 (2022)   The regenerative promise of nanosized extracellular vesicles (EVs) secreted by cells is widely explored. Recently, the capacity of EVs purified from blood to elicit regenerative effect has begun to be evaluated. Blood might be a readily available source of EVs, avoiding need for extensive cell culturing, but there are specific issues that complicate use of the biofluid in this area. We assess the evidence for blood containing regenerative material, progress made towards delivering blood derived EVs as regenerative therapeutics, difficulties that relate to the complexity of blood and the promise of hydrogel-based delivery of EVs.  

3.4201           Selective isolation of mouse glial nuclei optimized for reliable downstream omics analyses

Pena-Ortiz, M., Shafiq, S., Rowland, M.E. and Berube, N.G.

1. Neurosci. Methods, 369, 109480 (2022)

  Background Isolation of cell types of interest from the brain for molecular applications presents several challenges, including cellular damage during tissue dissociation or enrichment procedures, and low cell number in the tissue in some cases. Techniques have been developed to enrich distinct cell populations using immunopanning or fluorescence activated cell/nuclei sorting. However, these techniques often involve fixation, immunolabeling and DNA staining steps, which could potentially influence downstream omics applications. New method Taking advantage of readily available genetically modified mice with fluorescent-tagged nuclei, we describe a technique for the purification of cell-type specific brain nuclei, optimized to decrease sample preparation time and to limit potential artefacts for downstream omics applications. We demonstrate the applicability of this approach for the purification of glial cell nuclei and show that the resulting cell-type specific nuclei obtained can be used effectively for omics applications, including ATAC-seq and RNA-seq. Results We demonstrate excellent enrichment of fluorescently-tagged glial nuclei, yielding high quality RNA and chromatin. We identify several critical steps during nuclei isolation that help limit nuclei rupture and clumping, including quick homogenization, dilution before filtration and loosening of the pellet before resuspension, thus improving yield. Sorting of fluorescent nuclei can be achieved without fixation, antibody labelling, or DAPI staining, reducing potential artifactual results in RNA-seq and ATAC-seq analyses. We show that reproducible glial cell type-specific profiles can be obtained in transcriptomic and chromatin accessibility assays using this rapid protocol. Comparison with existing methods Our method allows for rapid enrichment of glial nuclei populations from the mouse brain with minimal processing steps, while still providing high quality RNA and chromatin required for reliable omics analyses. Conclusions We provide a reproducible method to obtain nucleic material from glial cells in the mouse brain with a quick and limited sample preparation.  

3.4202           The long noncoding RNA ADIPINT regulates human adipocyte metabolism via pyruvate carboxylase

Kerr, A.G., Wang, Z., Wang, N., Kwok, K.H.M., Jalkannen, J., Ludzki, A., Lecoutre, S., Langin, D., Bergo, M.O., Dahlman, I., Mim, C., Arner, P. and Gao, H. Nature Comm., 13:2958 (82022)   The pleiotropic function of long noncoding RNAs is well recognized, but their direct role in governing metabolic homeostasis is less understood. Here, we describe a human adipocyte-specific lncRNA, ADIPINT, that regulates pyruvate carboxylase, a pivotal enzyme in energy metabolism. We developed an approach, Targeted RNA-protein identification using Orthogonal Organic Phase Separation, which identifies that ADIPINT binds to pyruvate carboxylase and validated the interaction with electron microscopy. ADIPINT knockdown alters the interactome and decreases the abundance and enzymatic activity of pyruvate carboxylase in the mitochondria. Reduced ADIPINT or pyruvate carboxylase expression lowers adipocyte lipid synthesis, breakdown, and lipid content. In human white adipose tissue, ADIPINT expression is increased in obesity and linked to fat cell size, adipose insulin resistance, and pyruvate carboxylase activity. Thus, we identify ADIPINT as a regulator of lipid metabolism in human white adipocytes, which at least in part is mediated through its interaction with pyruvate carboxylase.  

3.4203           Tumor stem cell-derived exosomal microRNA-17-5p inhibits anti-tumor immunity in colorectal cancer via targeting SPOP and overexpressing PD-L1

Sun, W., Cui, J., Ge, Y., Wang, J., Yu, Y., Han, B. and Liu, B. Cell Death Discovery, 8:223 (2022)   Exosomes are known to transmit microRNAs (miRNAs) to affect human cancer progression, and miR-17-5p has been manifested to exert facilitated effects on colorectal cancer (CRC) progression, while the role of tumor stem cells-derived exosomal miR-17-5p in CRC remains unknown. We aim to explore the effect of CRC stem cells-derived exosomes (CRCSC-exos) conveying miR-17-5p on CRC. The exosomes were isolated from CRC stem cells and identified. HCT116 cells were transfected with speckle-type POZ protein (SPOP) interfering vector or co-cultured with exosomes carrying miR-17-5p mimic/inhibitor. Then, the proliferation, migration, invasion, and apoptosis of the cells were determined. The xenograft mouse model was constructed using BALB/C mice and the serum levels of T cell cytokines were assessed. Expression of miR-17-5p, SPOP, CD4, CD8 and programmed death ligand 1 (PD-L1) was detected. The targeting relationship between miR-17-5p and SPOP was verified. MiR-17-5p was upregulated and SPOP was downregulated in CRC tissues. CRCSC-exos transmitted miR-17-5p to HCT116 cells to promote malignant behaviors and suppress anti-tumor immunity of HCT116 cells. The overexpressed SPOP exerted opposite effects. SPOP was confirmed as a target gene of miR-17-5p. Upregulated CRCSC-exosomal miR-17-5p inhibits SPOP to promote tumor cell growth and dampen anti-tumor immunity in CRC through promoting PD-L1.  

3.4204           Gene regulation by gonadal hormone receptors underlies brain sex differences

Gegenhuber, B., Wu, M.V., Bronstein, R. and Tollkuhn, J. Nature, 606, 153-159 (2022)   Oestradiol establishes neural sex differences in many vertebrates1^,2^,3 and modulates mood, behaviour and energy balance in adulthood4^,5^,6^,7^,8. In the canonical pathway, oestradiol exerts its effects through the transcription factor oestrogen receptor-α (ERα)9. Although ERα has been extensively characterized in breast cancer, the neuronal targets of ERα, and their involvement in brain sex differences, remain largely unknown. Here we generate a comprehensive map of genomic ERα-binding sites in a sexually dimorphic neural circuit that mediates social behaviours. We conclude that ERα orchestrates sexual differentiation of the mouse brain through two mechanisms: establishing two male-biased neuron types and activating a sustained male-biased gene expression program. Collectively, our findings reveal that sex differences in gene expression are defined by hormonal activation of neuronal steroid receptors. The molecular targets we identify may underlie the effects of oestradiol on brain development, behaviour and disease.  

3.4205           Human and mouse trigeminal ganglia cell atlas implicates multiple cell types in migraine

Yang, L., Xu, M., Bhuiyan, S.A., levy, D., Lennerz, J.K. and Renthal W. Neuron, 110, 1806-1821 (2022)   Sensitization of trigeminal ganglion neurons contributes to primary headache disorders such as migraine, but the specific neuronal and non-neuronal trigeminal subtypes that are involved remain unclear. We thus developed a cell atlas in which human and mouse trigeminal ganglia are transcriptionally and epigenomically profiled at single-cell resolution. These data describe evolutionarily conserved and human-specific gene expression patterns within each trigeminal ganglion cell type, as well as the transcription factors and gene regulatory elements that contribute to cell-type-specific gene expression. We then leveraged these data to identify trigeminal ganglion cell types that are implicated both by human genetic variation associated with migraine and two mouse models of headache. This trigeminal ganglion cell atlas improves our understanding of the cell types, genes, and epigenomic features involved in headache pathophysiology and establishes a rich resource of cell-type-specific molecular features to guide the development of more selective treatments for headache and facial pain.  

3.4206           Huntingtin Co-Isolates with Small Extracellular Vesicles from Blood Plasma of TgHD and KI-HD Pig Models of Huntington’s Disease and Human Blood Plasma

Anabeh, H., Novak, J., Juhas, S., juhasova, j., Klempir, J., Doleckova, K., Rysankova, I., Turnovcova, K., Hanus, J., Hansikova, H., Vodicka, P. and Skkalikova, K. Int. J. Mol. Sci., 23:5598 (2022)   Background: Huntington’s disease (HD) is rare incurable hereditary neurodegenerative disorder caused by CAG repeat expansion in the gene coding for the protein huntingtin (HTT). Mutated huntingtin (mHTT) undergoes fragmentation and accumulation, affecting cellular functions and leading to neuronal cell death. Porcine models of HD are used in preclinical testing of currently emerging disease modifying therapies. Such therapies are aimed at reducing mHTT expression, postpone the disease onset, slow down the progression, and point out the need of biomarkers to monitor disease development and therapy efficacy. Recently, extracellular vesicles (EVs), particularly exosomes, gained attention as possible carriers of disease biomarkers. We aimed to characterize HTT and mHTT forms/fragments in blood plasma derived EVs in transgenic (TgHD) and knock-in (KI-HD) porcine models, as well as in HD patients’ plasma. (2) Methods: Small EVs were isolated by ultracentrifugation and HTT forms were visualized by western blotting. (3) Results: The full length 360 kDa HTT co-isolated with EVs from both the pig model and HD patient plasma. In addition, a ~70 kDa mutant HTT fragment was specific for TgHD pigs. Elevated total huntingtin levels in EVs from plasma of HD groups compared to controls were observed in both pig models and HD patients, however only in TgHD were they significant (p = 0.02). (4) Conclusions: Our study represents a valuable initial step towards the characterization of EV content in the search for HD biomarkers.  

3.4207           Is P-Glycoprotein Functionally Expressed in the Limiting Membrane of Endolysosomes? A Biochemical and Ultrastructural Study in the Rat Liver

Gericke, B., Wienböker, I., Brandes, G. and Löscher, W. Cells, 11:1556 (2022)   The drug efflux transporter P-glycoprotein (Pgp; ABCB1) plays an important role in drug absorption, disposition, and elimination. There is an ongoing debate whether, in addition to its localization at the plasma membrane, Pgp may also be expressed at the limiting membrane of endolysosomes (ELs), mediating active EL drug sequestration. If true, this would be an important mechanism to prevent drugs from reaching their intracellular targets. However, direct evidence demonstrating the functional expression of Pgp at the limiting membrane of ELs is lacking. This prompted us to perform a biochemical and ultrastructural study on the intracellular localization of Pgp in native rat liver. For this purpose, we established an improved subcellular fractionation procedure for the enrichment of ELs and employed different biochemical and ultrastructural methods to characterize the Pgp localization and function in the enriched EL fractions. Whereas the biochemical methods seemed to indicate that Pgp is functionally expressed at EL limiting membranes, transmission electron microscopy (TEM) indicated that this only occurs rarely, if at all. Instead, Pgp was found in the limiting membrane of early endosomes and intraluminal vesicles. In additional TEM experiments, using a Pgp-overexpressing brain microvessel endothelial cell line (hCMEC/D3-MDR1-EGFP), we examined whether Pgp is expressed at the limiting membrane of ELs when cells are exposed to high levels of the Pgp substrate doxorubicin. Pgp was seen in early endosomes but only rarely in endolysosomes, whereas Pgp immunogold labeling was detected in large autophagosomes. In summary, our data demonstrate the importance of combining biochemical and ultrastructural methods to investigate the relationship between Pgp localization and function.  

3.4208           Extracellular Vesicles in the Progression and Therapeutic Resistance of Nasopharyngeal Carcinoma

Shan, Y., Zhou, P., Zhou, Q. and yang, L. Cancers, 14:2289 (2022)   Nasopharyngeal carcinoma (NPC) is an epithelial malignancy largely associated with Epstein–Barr virus (EBV) infection, which is frequently reported in east and southeast Asia. Extracellular vesicles (EVs) originate from the endosome or plasma membrane, which plays a critical role in tumor pathogenesis for their character of cell-cell communication and its cargos, including proteins, RNA, and other molecules that can target recipient cells and affect their progression. To date, numerous studies have indicated that EVs have crucial significance in the progression, metastasis, and therapeutic resistance of NPC. In this review, we not only summarize the interaction of NPC cells and the tumor microenvironment (TME) through EVs, but also explain the role of EVs in radiation and drug resistance of NPC, which poses a severe threat to cancer therapy. Therefore, EVs may show great potential as biomarkers in the early diagnosis of interfered targets of NPC therapy.  

3.4209           Efficient Small Extracellular Vesicles (EV) Isolation Method and Evaluation of EV-Associated DNA Role in Cell–Cell Communication in Cancer

Chetty, V.K., Ghanam, J., Anchan, S., Reinhardt, K., Brenzel, A., Gelleri, M., Cremer, C., Grueso-Navarro, E., Schneider, M., von Neuhoff, n., Reinhardt, D., Jablonska, J., Nazarenko, I. and Thakur, B.K. Cancers, 14:2068 (2022)   Small extracellular vesicles (sEVs) play essential roles in intercellular signaling both in normal and pathophysiological conditions. Comprehensive studies of dsDNA associated with sEVs are hampered by a lack of methods, allowing efficient separation of sEVs from free-circulating DNA and apoptotic bodies. In this work, using controlled culture conditions, we enriched the reproducible separation of sEVs from free-circulated components by combining tangential flow filtration, size-exclusion chromatography, and ultrafiltration (TSU). EV-enriched fractions (F2 and F3) obtained using TSU also contained more dsDNA derived from the host genome and mitochondria, predominantly localized inside the vesicles. Three-dimensional reconstruction of high-resolution imaging showed that the recipient cell membrane barrier restricts a portion of EV-DNA. Simultaneously, the remaining EV-DNA overcomes it and enters the cytoplasm and nucleus. In the cytoplasm, EV-DNA associates with dsDNA-inflammatory sensors (cGAS/STING) and endosomal proteins (Rab5/Rab7). Relevant to cancer, we found that EV-DNA isolated from leukemia cell lines communicates with mesenchymal stromal cells (MSCs), a critical component in the BM microenvironment. Furthermore, we illustrated the arrangement of sEVs and EV-DNA at a single vesicle level using super-resolution microscopy. Altogether, employing TSU isolation, we demonstrated EV-DNA distribution and a tool to evaluate the exact EV-DNA role of cell–cell communication in cancer.  

3.4210           Quality Control of Bacterial Extracellular Vesicles with Total Protein Content Assay, Nanoparticles Tracking Analysis, and Capillary Electrophoresis

Stec, A., Jonca, j., Waleron, K., Waleron, M., Ploska, A., Kalinowski, L., Wielgomas, B. and Dziomba, S. Int. J. Mol. Sci., 23:4347 (2022)   Extracellular vesicles (EVs) were isolated from Pectobacterium zantedeschiae culturing media using direct ultracentrifugation (UC), iodixanol cushion ultracentrifugation (ICUC), and iodixanol density gradient ultracentrifugation (IDGUC) techniques. The isolates were characterized with total protein content assay (bicinchoninic acid assay, BCA), nanoparticles tracking analysis (NTA), and capillary electrophoresis (CE). A satisfactory correlation (R² > 0.94) between quantitative results obtained with BCA, NTA and CE was achieved only for isolates obtained with the IDGUC. The correlation between protein content and CE was proved to be related to the isolates’ purity. The NTA was found unable to provide reliable information on EVs quantity in samples isolated with UC and ICUC, due to the co-isolated particulate impurities. Moreover, the work reports polysaccharides, used as culturing media components, as a potential source of bias of quantitation with total protein content assay and NTA. The study demonstrates the advantageous selectivity of CE in quality control of EVs and its ability to differentiate subpopulations of EVs of Pectobacterium.  

3.4211           Cancer-Derived Extracellular Vesicles: Their Role in Sarcoma

Adib, A., Sahu, R., Mohta, S., Pollock, R.E. and Casadei, L. Life, 12:481 (2022)   Soft tissue sarcomas (STS) are rare malignancies with limited responses to anticancer therapy. Extracellular vesicles (EVs) are a heterogeneous group of bi-lipid layer sacs secreted by cells into extracellular space. Investigations of tumor-derived EVs have revealed their functional capabilities, including cell-to-cell communication and their impact on tumorigenesis, progression, and metastasis; however information on the roles of EVs in sarcoma is currently limited. In this review we investigate the role of various EV cargos in sarcoma and the mechanisms by which those cargos can affect the recipient cell phenotype and the aggressivity of the tumor itself. The study of EVs in sarcoma may help establish novel therapeutic approaches that target specific sarcoma subtypes or biologies, thereby improving sarcoma therapeutics in the future.  

3.4212           The Role of N-Glycosylation in the Intracellular Trafficking and Functionality of Neuronal Growth Regulator 1

Sim, GX., Jeong, M., Seo, H., Kim, J. and Lee, S. Cells, 11:1242 (2022)   Neuronal growth regulator 1 (NEGR1) is a brain-enriched membrane protein that is involved in neural cell communication and synapse formation. Accumulating evidence indicates that NEGR1 is a generic risk factor for various psychiatric diseases including autism and depression. Endoglycosidase digestion of single NEGR1 mutants revealed that the wild type NEGR1 has six putative N-glycosylation sites partly organized in a Golgi-dependent manner. To understand the role of each putative N-glycan residue, we generated a series of multi-site mutants (2MT–6MT) with additive mutations. Cell surface staining and biotinylation revealed that NEGR1 mutants 1MT to 4MT were localized on the cell surface at different levels, whereas 5MT and 6MT were retained in the endoplasmic reticulum to form highly stable multimer complexes. This indicated 5MT and 6MT are less likely to fold correctly. Furthermore, the removal of two N-terminal sites N75 and N155 was sufficient to completely abrogate membrane targeting. An in vivo binding assay using the soluble NEGR1 protein demonstrated that glycans N286, N294 and N307 on the C-terminal immunoglobulin-like domain play important roles in homophilic interactions. Taken together, these results suggest that the N-glycan moieties of NEGR1 are closely involved in the folding, trafficking, and homodimer formation of NEGR1 protein in a site-specific manner.  

3.4213           Small but Mighty—Exosomes, Novel Intercellular Messengers in Neurodegeneration

Kumari, M. and Anji, A. Biology, 11:413 (2022)   Exosomes of endosomal origin are one class of extracellular vesicles that are important in intercellular communication. Exosomes are released by all cells in our body and their cargo consisting of lipids, proteins and nucleic acids has a footprint reflective of their parental origin. The exosomal cargo has the power to modulate the physiology of recipient cells in the vicinity of the releasing cells or cells at a distance. Harnessing the potential of exosomes relies upon the purity of exosome preparation. Hence, many methods for isolation have been developed and we provide a succinct summary of several methods. In spite of the seclusion imposed by the blood–brain barrier, cells in the CNS are not immune from exosomal intrusive influences. Both neurons and glia release exosomes, often in an activity-dependent manner. A brief description of exosomes released by different cells in the brain and their role in maintaining CNS homeostasis is provided. The hallmark of several neurodegenerative diseases is the accumulation of protein aggregates. Recent studies implicate exosomes’ intercellular communicator role in the spread of misfolded proteins aiding the propagation of pathology. In this review, we discuss the potential contributions made by exosomes in progression of Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Understanding contributions made by exosomes in pathogenesis of neurodegeneration opens the field for employing exosomes as therapeutic agents for drug delivery to brain since exosomes do cross the blood–brain barrier.  

3.4214           The Outer Membrane Vesicles of Salmonella enterica Serovar Typhimurium Activate Chicken Immune Cells through Lipopolysaccharides and Membrane Proteins

Cui, H., Sun, Y., Lin, H., Zhao, Y. and Zhao, X. Pathogens, 11:339 (2022)   Salmonella is a common pathogen which can secrete outer membrane vesicles (OMVs). However, the effect of OMVs from Salmonella enterica Serovar Typhimurium (S. Typhimurium) of poultry origin on cells of the chicken innate immune system is not well known. In this study, S. Typhimurium OMVs were first isolated from three different poultry strains of Salmonella, Salmonella CVCC542, SALA, and SALB. In order to investigate the effect of OMVs on the maturation of monocytes into macrophages, both bone marrow-derived (BMD) monocytes and macrophage cell line HD11 cells were used. OMVs promoted the formation of monocyte dendrites in both types of cells, enabled BMD cells to become larger, and stimulated expression of LPS-induced TNF-αfactor (LITAF), IL-6, and inducible nitric oxide synthase (iNOS) genes in HD11 cells. These results demonstrated the capability of OMVs to promote the development of chicken monocytes into macrophages and the maturation of macrophages. In order to study the effect of OMVs on the phagocytosis of macrophages, chicken spleen-derived monocytes and HD11 cells were used. Phagocytosis of FITC-Salmonella and FITC-dextran by these two types of cells was enhanced after stimulation with OMVs. To determine which components in OMVs were responsible for the above observed results, OMVs were treated with proteinase K(PK) or polymyxin B (PMB). Both treatments reduced the phagocytosis of FITC-Salmonella by HD11 cells and chicken spleen mononuclear cells and reduced the secretion of IL-1β, LITAF, and IL-6 cytokines. These results demonstrated that Salmonella OMVs activated chicken macrophages and spleen mononuclear cells and the activation was achieved mainly through lipopolysaccharides and membrane proteins.  

3.4215           Organelle Engineering in Yeast: Enhanced Production of Protopanaxadiol through Manipulation of Peroxisome Proliferation in Saccharomyces cerevisiae

Choi, B.H., Kang, H.J., Kim, S.C. and Lee, P.C. Microorganisms, 10:650 (2022)   Isoprenoids, which are natural compounds with diverse structures, possess several biological activities that are beneficial to humans. A major consideration in isoprenoid production in microbial hosts is that the accumulation of biosynthesized isoprenoid within intracellular membranes may impede balanced cell growth, which may consequently reduce the desired yield of the target isoprenoid. As a strategy to overcome this suggested limitation, we selected peroxisome membranes as depots for the additional storage of biosynthesized isoprenoids to facilitate increased isoprenoid production in Saccharomyces cerevisiae. To maximize the peroxisome membrane storage capacity of S.cerevisiae, the copy number and size of peroxisomes were increased through genetic engineering of the expression of three peroxisome biogenesis-related peroxins (Pex11p, Pex34p, and Atg36p). The genetically enlarged and high copied peroxisomes in S.cerevisiae were stably maintained under a bioreactor fermentation condition. The peroxisome-engineered S.cerevisiae strains were then utilized as host strains for metabolic engineering of heterologous protopanaxadiol pathway. The yields of protopanaxadiol from the engineered peroxisome strains were ca 78% higher than those of the parent strain, which strongly supports the rationale for harnessing the storage capacity of the peroxisome membrane to accommodate the biosynthesized compounds. Consequently, this study presents in-depth knowledge on peroxisome biogenesis engineering in S.cerevisiae and could serve as basic information for improvement in ginsenosides production and as a potential platform to be utilized for other isoprenoids.  

3.4216           LINE-1 Cargo and Reverse Transcriptase Activity Profiles in Extracellular Vesicles from Lung Cancer Cells and Human Plasma

Bowers, E., Motta, A., Knox, K., McKay, B.S. and Ramos, K.S. Int. J. Mol. Sci., 23:3461 (2022)   Long Interspersed Element-1 (LINE-1) is an oncogenic human retrotransposon that ‘copies and pastes’ DNA into new locations via reverse transcription. Given that enzymatically active LINE-1 can be exported in extracellular vesicles (EVs), and that LINE-1 mRNA and its two encoded proteins, ORF1p and ORF2p, are required for retrotransposition, the present study examined LINE-1 EV loading patterns relative to reverse transcriptase (RT) activity in vivo and in vitro. Density gradient ultracentrifugation identified conserved patterns of LINE-1 mRNA and protein distribution in EVs, with RT activity readily detected in EV fractions containing both LINE-1 mRNA and protein. Unlike whole cell and tissue lysates, the ORF1p in EVs was detected as a dimer. EVs from ostensibly healthy plasma donors showed variable but consistent ORF1p profiles, with residual levels of LINE-1 mRNA measured in some but not all samples. EVs from cancer cell lines had elevated mean LINE-1 levels and 5–85 times greater RT activity than EVs from normal cells or healthy plasma. EV RT activity was associated with EV LINE-1 mRNA content and was highest in cell lines that also expressed an elevated expression of ORF1p and ORF2p. Given that LINE-1 activation is a hallmark of many cancer types, our findings suggest that an EV LINE-1 ‘liquid biopsy’ may be developed to monitor LINE-1 activity during the course of malignant progression  

4           Cells

4.1           Separation of T and B lymphocytes from human peripheral blood mononuclear cells using density perturbation methods

Patel, D., Rubbi, C.P. and Rickwood, D. Clin. Chim. Acta, 240, 187-193 (1995)   The fractionation of sub-populations of peripheral blood mononuclear cells has become an essential routine procedure and some of the main fractionation methods used today are immunomagnetic separations. We describe a less expensive method for the separation of subpopulations of mononuclear cells using density perturbation, which uses the binding of antibody-coated dense polystyrene beads to increase the density of specific sub-populations of cells. By incubating a total mononuclear fraction from human peripheral blood together with antibody-coated beads, in a commercially available lymphocyte separation medium (Nycoprep 1.077), a depletion of 94.9 ± 1.68% of the T cells could be obtained by this procedure; a depletion of 69.7 ± 1.78% of the B cells was also achieved. These results indicate the potential for the separation of different sub-populations of peripheral blood mononuclear cells on the basis of the immunological identity of the surface of cells using density perturbation methods involving antibody-coated dense polystyrene beads.  

4.2           Optimization of conditions for specific binding of antibody-coated beads to cells

Patel, D.and Rickwood, D.

1. Immunol. Methods, 184, 71-80 (1995)

  It has previously been demonstrated that cells can bind antibody-coated beads: this effect can be used to enhance the fractionation of cells using magnetic fields or by centrifugation on isopycnic, isotonic density gradients. As a general rule, the higher the expression of surface antigens the more beads to bind to cells. However, we have also noted that other factors also affect the number of beads found bound to cells. Experiments have been carried out to determine what factors affect binding of antibody-coated beads to cells. The optimum conditions for binding of antibody-coated beads to MOLT-4 T cells were found to be, namely, a 20:1 bead to cell ratio in a 1 ml incubation volume, with continuous end-over-end mixing for 1 h at 25°C. Furthermore, the optimum centrifugation conditions at which the samples were separated on isopycnic, isotonic density gradients were determined as 220xg_max for 90 min, at 20°C. The results indicate the preferred conditions that are necessary to achieve optimum bead binding by cells and their subsequent fractionation. Different antibody-coated beads were examined including Dynabeads M-450, used as a known standard. In addition we describe, as a possible alternative to Dynabeads, dense polystyrene beads, for the separation of cells on the basis of the immunological identity of the surface of cells using density perturbation methods.  

4.3           Use of iodixanol as a density gradient material for the isolation of motile, morphologically normal human sperm from semen.

Smith, T.T., Turner, D. and Whitford, W.

1. Andrology, Supplement 1996

  Although highly desirable, the efficient isolation of motile, morphologically normal spermatozoa from semen has been limited due to the unavailability of an iso-osmotic gradient material with a density higher than human sperm. A new gradient material, iodixanol (60% w/v, OptiPrep, Nycomed), with its relatively high osmolality (260 mOsm) and a density (1.32 g/ml), may be suitable for this purpose. Iodixanol solutions, 1.154 g/ml and 1.054 g/ml (pH 7.3), were prepared using modified Human Tubal Fluid (m-HTF) medium. Osmolarity was adjusted to 300 mOsm with NaCl. Semen specimens (n=15) were obtained from 10 normozoospermic donors. Semen volume, sperm concentration (C), motility (%M), normal forms (%N; Tygerberg strict criteria), total motile (TM), and total normal (TN) sperm were determined. Semen was diluted 4:6.5 with OptiPrep and layered under the 1.154 and 1.054 g/ml solutions (3 ml each) in 4 tubes. The discontinuous gradient was centrifuged for 40 min at 1,500g during which time sperm rose to the 1.154/1.054 g/ml interface. The sperm were collected from the interface and diluted 1:5 in m-HTF, centrifuged for 15 min. at 500g and then resuspended in 1.0 ml of m-HTF. Final volume, C, %M, forward progressive velocity (V_f), TM, %N and TN were determined. To identify any possible cytotoxic effect of iodixanol, an aliquot of prepared sperm suspension was diluted to 10x10⁶/ml in 1 ml of m-HTF supplemented with 0.4% human serum albumin and incubated under mineral oil at 37°C in air for 24 h after which %M and V_f were determined again. Values are expressed "SE. Following centrifugation, a mean of 32.0 " 3.8 x10⁶ motile sperm were recovered from the 1.154/1.054 g/ml interface, representing 21 "1.8% of the motile and 19.5 " 2.5% of the morphologically normal sperm. Following 24 h of incubation at 37°C, %M and V_f had increased 18.1 " 2.4% and 14.3 " 1.7 m/s respectively.   The results of this preliminary study show that iodixanol may be used to obtain motile, morphologically normal sperm for IUI, IVF and ICSI. The relatively small decline in %M and V_f at 24 h after recovery indicates iodixanol was not toxic to the sperm. Since the use of iodixanol as a gradient material appears promising, future studies will involve fine tuning the gradient densities to enhance the yield of motile, morphologically normal sperm.  

4.4           Phenotypic and functional characterization of CD11c⁺ dendritic cell population in mouse Peyer’s patches

Ruedl, C., Rieser, C., Bock, G., Wick, G. and Wolf, H. Eur. J. Immunol., 26; 1801-1806 (1996)   The antigen-presenting cell system in the gastrointestinal tract, one of three main sites (skin and lung being the others) of primary antigen contact, is poorly understood. Our study focused on dendritic cells (DC) as possible candidates for antigen uptake, processing and presentation in mucosal inductive sites, such as Peyer’s patches (PP). To investigate the morphology, immunophenotype and stimulatory activity of intestinal DC, a procedure was developed to obtain a cell population by using collagenase digestion of PP, density centrifugation and cell sorting on the basis of CD11c expression. The resultant low-density cell fraction consisted of a nonadherent cell population expressing different intensities of CD11c that could at least be characterized by typical DC morphology (e.g. abundant cytoplasma with veil-like cytoplasmatic dendrites, irregularly shaped nuclei, multivesicular and multilamellar bodies), constitutive levels of surface MHC class II, the presence of macrophage-specific markers, such as F4/80, Mac-I and Fc receptors, respectively, on subpopulations of CD11c⁺ sorted cells and expression of adhesion and co-stimulatory receptors like ICAM-1 and CD44. The capability of this low-density CDl1c⁺ fraction to stimulate T cell responses was demonstrated in primary allergenic mixed-lymphocyte reactions (MLR). Herein, we show that the freshly isolated CD11c⁺ cells showed weak accessory function, but develop this capacity following short-term culture in vitro in the presence of granulocyte/macrophage colony-stimulating factor. Although the nature and functional capacity of the isolated CD11c⁺ needs fur­ther clarification, these preliminary results describing phenotype and accessory function provide some evidence that these cells isolated from the PP may be immature forms of DC and play a crucial role as antigen-presenting cells with important implications for understanding the complex network regulating intes­tinal antigen uptake, processing and presentation.  

4.5           Rejection of MHC Class II-transfected tumor cells requires induction of tumor-encoded B7-1 and/or B7-2 co-stimulatory molecules

Baskar, S et al.

1. Immunol., 156, 3821-3827 (1996)

  Many tumor cells that have been transfected with genes encoding B7 co-stimulatory molecules become effective cellular vac­cines against wild-type tumor. The improved immunity is dependent on newly induced tumor-specific CD8⁺ and/or CD4⁺ T cells and presumably occurs because the B7 transfectants provide the requisite second signal for activation of T cells in conjunction with tumor cell-presented MHC class I/tumor peptide and/or MHC class II/tumor peptide complexes, respectively. Since B7 expression is such a potent enhancer of tumor immunity, and yet some tumors are immunogenic in the absence of B7 transfection, we have used class I⁺ class II-transfected tumors to investigate whether co-stimulatory molecules are also involved in rejection of immunogenic, non-B7-transfected tumor. Blocking studies with B7 mAbs demonstrate that induction of tumor immunity in naive mice requires B7-1 and/or B7-2 expression, while experiments with tumor-primed mice indicate that once antitumor immunity is established, expression of B7 is not necessary. Flow cytometry analyses demonstrate that co-stimulatory molecules are expressed by the tumor cells via an in vivo induction process. Experiments with class II genes with truncated cytoplasmic tails indicate that the cytoplasmic region of the tumor-expressed class II heterodimer is involved in induction of B7. We therefore conclude that for this class I⁺ class II-transfected tumor, generation of tumor immunity requires induction of tumor cell-encoded B7 molecules that are mediated by the cytoplasmic region of the transfected class II heterodimer.          

4.6           Iodixanol as a density gradient medium for the isolation of motile spermatozoa.

Harrison, K.

1. Assist. Reprod. and Gen., 14(7), 385-387 (1997)

  Purpose: The purpose of this study was to establish concentrations of Iodixanol which could be used in a similar manner to the widely used Mini-Percoll technique for the separation of motile sperm. Methods: Various density gradient combinations of Iodixanol were compared for the isolation of motile spermatozoa from cryopreserved donor semen. The toxicity of Iodixanol was tested by its effect on the growth of two-cell mouse embryos and fresh sperm survival. Results: The best sperm recovery (32%) came from the pellet of sperm passing through a discontinuous gradient of 25% over 40% OptiPrep after 20 min of centrifugation at 400g. There was no evidence of toxicity to mouse embryo growth or sperm survival. Conclusions: Iodixanol (OptiPrep; Nycomed) provides a satisfactory alternative for the efficient separation of motile spermatozoa for ART procedures.  

4.7           The use of iodixanol as a density gradient material for separating human sperm from semen.

Smith, T.T. , Byers, M., Kaftani, D. and Whitford, W. Arch. Androl., 38, 223-230 (1997)   Iodixanol, a new nonionic density gradient material with relatively low osmolality and high density, was evaluated to determine its suitability for the separation of human sperm from semen for their subsequent therapeutic use. Using a three-layer iodixanol gradient (1.17/1.15/1.05 g/ml), sperm were centrifuged at 1000g for 30 min and collected from the 1.05/1.15 interface. Using this method, a mean of 78% of the motile and 99% of the morphologically normal sperm originally present in the semen were recovered at the interface. There was no significant increase in the percentage of motile or morphologically normal sperm in the final preparation compared to the original semen. Sperm survived iodixanol density gradient centrifugation well, showing only modest declines in motility (18%) and velocity (35%) during a subsequent 24 h incubation period. Iodixanol provides a suitable, nontoxic alternative to Percoll for the preparation of human sperm for therapeutic use.  

4.8           A quick, easy and inexpensive method for the isolation of human peripheral blood monocytes.

Graziani-Bowering, G.M., Graham, J. and Filion, L.G.

1. Immunol. Methods, 207, 157-168 (1997)

  A commercial monocyte isolation technique based on OptiPrep density-gradient medium was up-scaled with respect to sample and reagent volumes. The results of 7 isolations are reported in which the average purity ranged from 87.9 to 96.4%. In all but the initial isolation, monocytes were defined as CD15⁺ dim CD4⁺ dim as assessed by flow cytometric analysis; in the first isolation, monocytes were defined by the traditional CD14⁺ CD4⁺ dim combination. The mean yield (the number of isolated monocytes relative to the number present in the buffy coat) of all isolations was 26.1%, with the individual yields ranging from 10.8 to 41.4%. The mean number of isolated monocytes per experiment was 3.6x10⁶ monocytes for those isolations performed using 14 ml of buffy coat/OptiPrep mixture (n=4). The isolated cells were viable (>95%) and were not activated, according to HLA-DR expression. This technique is a convenient, fast (less than 2 h), relatively simple, and inexpensive alternative to traditional monocyte isolation techniques). The up-scaled version of this method also results in significantly higher numbers of monocytes per isolation than some traditional techniques. Furthermore this is the first literature report of the use of OptiPrep density gradient medium for the isolation of monocytes.  

4.9           A new method for the purification of human motile spermatozoa applying density-gradient centrifugation; Polysucrose media compared to Percoll media.

Andersen, C.Y. and Grinsted, J.

1. Assist. Reprod. and Gen., 14(10), 624-628 (1997)

  Purpose: A newly developed method for the isolation of human motile spermatozoa using density-gradient centrifugation was compared with the traditionally used Percoll technique. Method: Sperm samples were divided into two equal aliquots, which were purified with either the traditionally performed Percoll technique or a new alternative based on polysucrose/ OptiPrep media. For each sample the isolation was performed during the same run of the centrifuge. Results: The average recovery of progressively motile spermatozoa with the Polysucrose/ OptiPrep method was significantly higher (48"7%) than with the Percoll method (38"6%) (n=18). The average percentage of motile spermatozoa and the motility score were similar in the purified preparations. Conclusion: The new polysucrose/OptiPrep-based density-gradient centrifugation technique for the isolation of motile human spermatozoa is as good as the traditionally used Percoll method and may replace it in connection with assisted reproduction techniques.  

4.10           The use of OptiPrep to prepare human sperm for the assisted reproductive technologies.

Kaftani, D., Byers, M. and Smith, T.T. ASRM Abstracts 1997   Objectives: Concern regarding the safety of silica-based density gradient materials (e.g. Percoll) for human sperm processing has resulted in the need to find safe, cost-effective alternatives. The purpose of this study was to determine the suitability of OptiPrep, a non-toxic density gradient material, for the preparation of human sperm for subsequent therapeutic use.   Design: Two-layer discontinuous density gradients of OptiPrep and Percoll were compared for their ability to separate motile, morphologically normal sperm from semen. The functional status of processed sperm was evaluated by assessing percent motility (%M) and straight line velocity (m/s, VSL) at 0, 24 and 48 h post-isolation.   Material and Methods: Isotonic Optiprep gradients (35 and 17.5%) were prepared by diluting stock OptiPrep (60% iodixanol) with modified Human Tubal Fluid (mHTF). Likewise, Percoll gradients (90 and 45%) were prepared by diluting isotonic Percoll with mHTF. Two layer (1 ml each) discontinuous density gradients were prepared in 15 ml conical centrifuge tubes. Ten semen samples were obtained from 3 normozoospermic donors. Seminal sperm concentration C, %M and percent normal morphology (%N, strict criteria) were determined. Semen aliquots (0.5 ml) from the same ejaculate were layered over OptiPrep and Percoll gradients and centrifuged at 340 g for 20 min. Sperm pellets were washed once (OptiPrep) or twice (Percoll), resuspended in 0.5 ml of HTF and analyzed for C, %M and %N. The percent recovery of motile and morphologically normal sperm was calculated. At 0, 24 and 48 h, %M and VSL were determined using computer assisted sperm analysis. The percent decline over time in motility and VSL was determined. Data, expressed below as mean ± SEM, were compared using a paired t-test.   Results: There was no significant difference between OptiPrep and Percoll density gradients in the % recovery of motile sperm (106 ± 10.6 vs. 112.6 ± 6.7) or morphologically normal sperm (90.2 ± 9.5 vs. 87.9 ± 8.5). Likewise, there was no significant difference in the % decline in motility at 24 h (4.9 ± 2.5 vs. 5.1 ± 2.3) or 48 h (36.9 ± 6.5 vs. 33.0 ± 6.2), or the decline in VSL at 24 h (11.0 ± 5.7 vs. 8.6 ± 6.8) or 48 h (48.9 ± 0.7 vs. 45.2 ± 6.1).   Conclusion: These data indicate that OptiPrep provides a suitable, cost-effective, non-toxic alternative to silica-based density gradient materials for the isolation of motile, morphologically normal sperm for subsequent therapeutic use.  

4.11           Selection of motile spermatozoa of normal morphology from bovine ejaculates by centrifugation in an iodixanol gradient.

Revell, S.G. et al Liverpool John Moores University, ”Control of Human Fertility”, Seminar Report Nov. 1997   Bovine ejaculates vary in the quality and numbers of the sperm present and if the number of motile sperm in an ejaculate falls below a particular standard percentage, the ejaculate will be discarded. Although the percentage of motile sperm may be too low, the actual numbers can be very high, and it would be economically useful if the motile sperm could be separated from the non-motile and thus be used for Artificial Insemination (A.I.). A new centrifugation medium, OptiPrepÔ, offers a method for the separation of dead and deformed sperm from motile sperm of normal morphology. The method involves low-speed centrifugation of an ejaculate on a discontinuous density gradient which leaves the dead sperm as a pellet, live sperm of normal morphology at one interface of the gradient and deformed sperm, i.e. those with bent and twisted tails or with cytoplasmic droplets, rise to the top of the gradient. The band of motile sperm with normal morphology always contained at least 90% of sperm with normal morphologies. The pelleted sperm always have been shown to be at least 95% dead. After freezing and thawing sperm harvested from the ”normal” band, recovered sperm were 75% live with excellent motility.  

4.12           Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo

Brocker, T., Riedinger, M. and Karjalainen, K.

1. Exp. Med., 185(3), 541-550 (1997)

  It is well established that lymphoid dendritic cells (DC) play an important role in the immune system.  Beside their role as potent inducers of primary T cell responses, DC seem to play a crucial part as major histocompatibility complex (MHC) class II⁺ "interdigitating cells" in the thymus during thymocyte development.  Thymic DC have been implicated in tolerance induction and also by some authors in inducing major histocompatibility complex restriction of thymocytes. Most of our knowledge about thymic DC was obtained using highly invasive and manipulatory experimental protocols such as thymus reaggregation cultures, suspension cultures, thymus grafting, and bone marrow reconstitution experiments.  The DC used in those studies had to go through extensive isolation procedures or were cultured with recombinant growth factors.  Since the functions of DC after these in vitro manipulations have been reported to be not identical to those of DC in vivo, we intended to establish a system that would allow us to investigate DC function avoiding artificial interferences due to handling.  Here we present a transgenic mouse model in which we targeted gene expression specifically to DC.  Using the CD 11c promoter we expressed MHC class II I-E molecules specifically on DC of all tissues, but not on other cell types.  We report that I-E expression on thymic DC is sufficient to negatively select I-E reactive CD4⁺ T cells, and to a less complete extent, CD8⁺ T cells.  In contrast, if only DC expressed I-E in a class II-deficient background, positive selection of CD4⁺ T cells could not be observed.  Thus negative, but not positive, selection events can be induced by DC in vivo.  

4.13           Survival of mature CD4 T lymphocytes is dependent on major histocompatibility complex class II-expression dendritic cells

Brocker, T

1. Exp. Med., 186(8), 1223-1232 (1997)

  Thymic T cell development is controlled by T cell receptor (TCR)-major histocompatibility complex (MHC) interactions, whereas a further dependence of peripheral mature T cells on TCR-MHC contact has not been described so far.  To study this question, CD4 T cell survival was surveyed in mice lacking MHC class II expression and in mice expressing MHC class II exclusively on dendritic cells.  Since neither of these mice positively select CD4 T cells in the thymus, they were grafted with MHC class II-positive embryonic thymic tissue, which had been depleted of bone marrow derived cells.  Although the thymus grafts in both hosts were repopulated with host origin thymocytes of identical phenotype and numbers, an accumulation of CD4⁺ T cells in peripheral lymphoid organs could only be observed in mice expressing MHC class II on dendritic cells, but not in mice that were completely MHC class II deficient.  As assessed by histology, the accumulating peripheral CD4 T cells were found to be in close contact with MHC class II⁺ dendritic cells, suggesting that CD4 T cells need peripheral MHC class II expression for survival and that class II⁺ dendritic cells might play an important role for the longevity of CD4 T cells.  

4.14           Manual and automated methods for the determination of leukocyte counts at extreme low levels: comparative evaluation of the Nageotte chamber

Müller, T.H., Döscher, A., Schunter, F. and Scott, C.S. Transfus. Sci., 18(4), 505-515 (1997)   Leukodepleted or leukocyte-poor blood products (fresh-frozen plasma, packed red cell and platelet concentrates in particular) are widely used in current clinical practice. However, because the monitoring of leukodepletion efficiency is generally carried out (if at all) using the labour-intensive and relatively inaccurate manual Nageotte chamber technique, it is clear that any increased demand for leukodepletion monitoring would be difficult, if not impossible, to meet. As the need to identify an automated alternative to the Nageotte technique is important, this study was undertaken to evaluate such a possibility. White blood cells were enumerated in a representative series of filtered and non-filtered human blood components by both microscopic counting in the Nageotte chamber, and with the Abbott CD3500 automated haematology analyser. For the Nageotte estimate, a single analysis was made in accordance with standard procedures, whereas the automated analysis was achieved by making six replicate counts and determining the mean of four replicates after excluding the highest and lowest estimates. To determine linearity limits of the manual and automated procedures, freshly isolated leukocytes were admixed with cell-free plasma-pheresis plasma. Reasonable reproducibility (mean CV 10% for cell counts exceeding 100 cells/microL) and good linearity (r > 0.9) were observed for CD3500 determinations in four separate experiments. The manual and automated measurements also correlated well (r > 0.9) with no obvious inter-method bias for cell counts up to 40 cells/microL although there was some suggestion of lower absolute CD3500 counts in the range 40-130 cells/microL. For the comparative studies with filtered and non-filtered blood products, no significant method bias was seen with 70 individual red cell concentrates, but systematically higher CD3500 white blood cell counts were observed in the series of 68 platelet concentrates (probably due to the presence of platelet clumps). This study concludes that automation of white cell counts in blood products with the CD3500 analyser is feasible for quality control in the preparation of fresh-frozen plasma and red cell concentrates but is limited for the analysis of filtered platelet concentrates.  

4.15           Endotoxin contamination of reagents used during isolation and purification of human pancreatic islets.

Linetsky, E., Inverardi, L., Kenyon, N.S., Alejandro, R. and Ricordi, C. Transplantation Proc., 30, 345-346 (1998)   A substantial number of islet allografts never function or lose function in the first several weeks after transplantation. This early allograft failure has been attributed to several mechanisms, such as increased imunogenicity of the islet preparation, acute allograft rejection, and recurrence of autoimmunity in recipients with insulin dependent diabetes mellitus. More recently, increasing attention has been directed also to microenvironmental factors at the site of the islet implantation (e.g. intrahepatic). A specific tissue injury, inflammation, and ischemia/reperfusion injury can affect the microenvironment at the transplant site, resulting in the activation of macrophages and other antigen-presenting cells. This can result not only in the local generation of cytokines and radicals known to be deleterious to islet cells, but also in the activation of allospecific immune response as well as b cell-specific autoimmunity. Results from experimental studies indicated that macrophages may play a determining role in the occurrence of early loss of transplanted islets. Endotoxins are well known inducers of macrophage activation and related cytokine and radical generation. The presence of endotoxins in the islet preparation could, by inducing macrophage activation, amplify an early inflammatory response. Endotoxins have been previously associated with failure of intrahepatic islet transplants, and it has always been important to identify reagents for human isolation that are endotoxin free. However, only recently new products have become available to fulfill this need. The aim of this study was to evaluate the endotoxin content of reagents commonly used in the isolation and purification of human pancreatic islets.  

4.16           Porcine islet preservation during isolation in University of Wisconsin solution.

Van der Burg, M.P.M., Basir, I., Zwaan, R.P. and Bouwman, E. Transplantation Proc., 30, 360-361 (1998)   Substitution of the University of Wisconsin Solution (UWS) for the conventional Hanks= Balanced Salt Solution (HBSS) during collagenase digestion of the porcine pancreas has been reported to increase the yield of isolated porcine islets. Little is known, however, on the effect of the collagenase solution and different solutions during subsequent steps of the isolation and purification procedure on islet viability. Since the UWS probably also best preserves the islet tissue during the cold steps of the procedure, we compared the use of UWS for all steps of the isolation procedure and also during purification on our novel UWS-based OptiPrep gradient versus the use of HBSS for digestion and dispersion and similar OptiPrep purification by in parallel processing of two paired segments of the pancreatic body of market-age slaughterhouse pigs.  

4.17           No porcine islet loss during density gradient purification in a novel iodixanol in University of Wisconsin solution.

Van der Burg, M.P.M., Basir, I. and Bouwman, E. Transplantation Proc., 30, 362-363 (1998)   The marked fragility and rapid dissociation and loss of (juvenile) porcine islets during the isolation and especially the purification process are considered a major barrier on the way to pig-to-human islet transplantation. We developed a novel simple density gradient of iodixanol (OptiPrep) in University of Wisconsin solution (UWS) that allows the purification of juvenile islets with no loss or fragmentation and an improved purity and viability as compared to conventional ficoll-sodium-diatrizoate (Histopaque, Sigma, St. Louis, Mo) gradients.    

4.18           Markedly improved outcome of adult porcine islet isolation, purification, and culture using Liberase-P1 versus Collagenase-P, and a novel gradient of OptiPrep in University of Wisconsin solution.

Van der Burg, MPM, Zwaan, RP. and Bouwman, E. Horm. Metab. Res., 30, A23 (1998)   Isolated porcine islet yield, integrity, and viability is endangered by the use of crude enzyme mixtures for digestion, and the changing conditions generally introduced by the use of several different solutions during digestion and further isolation and purification steps. We, therefore, aimed at improving the preservation of pig islets by keeping the islets in the University of Wisconsin solution (UWS) both during isolation and the subsequent density purification in a novel iodixanol (OptiPrep, Nycomed Pharma) in UWS; and we compared a novel Liberase-P1 enzyme blend vs. collagenase-P for islet isolation by in parallel processing of two, paired, segments of the pancreas (whole organ weight 254"15 gram) of large sows in 6 consecutive experiments. After 22 min warm- and 100 min cold ischemia, the segments were digested using 0.5-0.75 mg/ml Liberase for the one- and 2 mg/ml collagenase-P for the other segment, by the stationary single-endpoint method for 32 min at 36°C, and dispersed by shaking and sieving (400 mm) for -30 min in UWS on ice. The digest suspension was mixed with half the volume of a 30% w/v iodixanol in UWS (final density 1.10 g/ml, and osmolality 380 mOsm) and topped with a 1.090 g/ml OptiPrep-UWS, and plain UWS. After centrifugation at 500g and 4°C for 5 min, purified islets were obtained from the top layer, and cultured for 24 h in RPMI-1640 supplemented with 10% adult porcine serum. Islet yield was expressed in islet equivalents (IEQs; mass converted to the equivalent number of islets with an 150 mm diameter). Viability was assessed by fluorometry using acridine orange and propidium iodide. Islet yield (overall mean 2444±446 IEQs/g pancreas) and size (volume-average diameter 155±11 mm) did not differ after Liberase and collagenase-P digestion. After purification, comparison of the Liberase vs. collagenase-P isolated islets, demonstrated a similar >95% purity, but higher yields (2448±392 vs. 1319±224 IEQs; P<.05), larger islets (185±16 vs. 140±8; P=.08), and an improved viability (90±2 vs. 79±4%; P<.05) of the Liberase-isolated islets. Postculture recovery was 24±9% of the IEQs in the Liberase digest vs. 14±8% collagenase-P islets, P<.05). Viability was corroborated in vivo by postprandial normoglycemia (<200 mg%) from day- 1 after grafting of 2500 IEQs under the kidney capsule in streptozotocin diabetic nude mice. We conclude that islet integrity and viability are markedly improved by Liberase digestion, and preservation in UWS throughout isolation and purification in our novel OptiPrep-UWS gradient.  

4.19           Efficacy of the novel iodixanol – UWS density gradient for human islet purification

Van der Burg, M.P.M., Ranuncoli, A., Molano, R., Kirlew, T., Ringers, J., Bouwman, E. and Ricordi, C. Acta Diabetol., 35, 247 (1998)   Consistent human islet isolation and purification success is hampered by the large variability of donor and procurement related factors. Substantial progress in purification has nevertheless been made over the last few years by simple changes in (pre-) purification solutions, suggesting that considerable scope still exists for further improvement in the density gradient purification of human islets. We recently developed a ~100% efficient (approx. complete islet recovery and purity) density gradient of iodixanol (OptiPrep) in University of Wisconsin solution (UWS) in the difficult pig model. This success prompted us to test this gradient during 5 consecutive human islet isolations. Pancreases distantly procured from multiorgan cadaveric donors (27-57 y) and cold preserved with UWS for approx. 12 h were digested with 1.4 mg/ml Liberase-HI in HBSS by the automated method. The digest was collected with cold RPMI, and a small sample of the digest was taken for these pilot experiments – the remainder was used in non-related experiments. Next, the tissue was incubated 60 min in UWS on ice. After taking an aliquot for assessment, half of the prep was loaded in a 1.086 – 1.075 –UWS gradient in 50 ml conical tubes, the other half was saved on ice for optional testing of other density layers. The 1.086 bottom was prepared by mixing 30 ml digest (in UWS) with 10 ml Working OptiPrep (WOP; an 1:1 mixture of OptiPrep and double-strength UWS). The 1.075 (or 1.070) barrier layer was prepared by mixing 5 ml WOP with 22.6 ml (or 28.3) UWS. After 5 min 500g centrifugation at 4°C tissue was collected from the top (UWS – 1.075 interface) and second layer. The 1.086-1.075-UWS gradient was successful (> 80% purity and recovery) during all 4 experiments. In the last experiment, purity was 35%, but again > 80% purity and recovery was obtained by using an 1.070 barrier with the other half of the digest prep. On average the digest loaded in the gradient contained 15287 ± 3712 IEQs and the volume average islet diameter was 238 ± 22 mm. After purification at the top 12766 ± 3129 IEQs were recovered (83 ± 2 % recovery), islet diameter was 204 ± 13 mm (NS) and purity was 89 ± 3 % Viability of the islets was corroborated histologically 1 to 3 weeks after transplantation under the kidney capsule in 3 nude mice. Thus, the consistent high efficacy of this simple OptiPrep-UWS gradient under mild hyperosmotic conditions (approx. 360 mOsm) in this pilot, and other favourable characteristics such as a low endotoxin content, suggest that the gradient may become a new powerful tool for human islet purification.  

4.20           Large scale isopycnic islet purification utilizing non-toxic, endotoxin-free media facilitates immediate single-donor pig islet allograft function.

Matsumoto, S, Zhang, H.J., Gilmore, T., van der Burg, M.P., Sutherland, D.E.R. and Hering, B.J. Transplantation, 66(8), S30 (1998)   Early graft function has been difficult to accomplish in clinical islet allotransplantation. It is becoming apparent that reagents used for islet preparation may stimulate the inflammatory response to islet grafts and may thereby interfere with early islet function and engraftment. The purpose of this study therefore was to develop islet purification density gradient media restricted to non-toxic, endotoxin-tested components and to subsequently test whether islets purified on the refined gradients immediately reverse diabetes in the relevant preclinical single-donor pig allograft model.   Density gradients were constructed based on iodixanol x-ray contrast media and UW solution which are both approved for clinical use. In the first set of experiments, the osmolality most suitable for islet separation was identified. The effect of four different osmolalities (320, 350, 375 and 400 mosm/kg) on the density profile of islet and acinar tissue was studied by means of continuous test gradients. In the second part of the study, iodixanol/UW gradients were applied for large scale continuous islet density gradient purification on a Cobe 2991 and the percentage recovery of islet equivalents (IE) in fractions with purities > 90% from iodixanol/UW was compared to Ficoll Na-diatrizoate gradients (previous standard). To test whether iodixanol/UW gradients interfere with immediate reversal of hyperglycemia posttransplant, 7134 ± 1830 purified IE/kg body weight were intraportally transplanted into five streptozotocin-diabetic pigs. The percentage of exocrine tissue contaminating 95% yield of islets were 0.45 ± 0.96%, 0.0 ± 0.0%, 0.45 ± 0.69%, and 0.80 ± 1.00% for 320, 350, 375 and 400 mosm/kg iodixanol/UW gradients, respectively. The percentage recovery of pure islet equivalents from 350 mosm/kg iodixanol/UW (n=6) gradients was significantly higher compared to the standard Ficoll Na-diatrizoate (n=4) gradient (86.2 ± 11.8% vs. 67.0 ± 9.3%, p=0.013). Four out of five consecutive diabetic single-donor pig islet allograft recipients became normoglycemic and insulin-independent within 24 hrs following transplantation.   The data indicate that single donor pig islet allografts purified on non-toxic, endotoxin-free gradients establish independence immediately. Osmolality adjustments should make iodixanol/UW gradients also suitable for human islet purification. Failure to achieve immediate insulin independence in the clinical setting would then point to non-technical obstacles.  

4.21           Identification, culture, and characterization of pancreatic stellate cells in rat and humans

Bachem, M.G. et al Gastroenterology, 115, 421-432 (1998)   Until now, the basic matrix-producing cell type responsible for pancreas fibrosis has not been identified.  In this report, retinoid-containing pancreatic stellate cells (PSCs) in rat and human pancreas are described, and morphological and biochemical similarities to hepatic stellate cells are shown. Methods:  Electron and immunofluorescence microscopy (collagen types I and III, fibronectin, faminin, a-actin, and desmin) was performed using pancreatic tissue and cultured PSCs.  Extracellular matrix synthesis was shown using quantitative immunoassay and Northern blot analysis.  Results: PSCs are located in interlobular areas and in interacinar regions.  Early primary cultured PSCs contain retinol and fatty acid retinyl-esters.  Addition of retinol to passaged cells resulted in retinol uptake and esterification.  During primary culture, the cells changed from a quiescent fat-storing phenotype to a highly synthetic myofibroblast-like cell expressing iso-a-smooth muscle actin (> 90%) and desmin (20%-40%) and showing strong positive staining with antibodies to collagen types I and III, fibronectin, and laminin.  As determined on protein and messenger RNA level serum growth factors stimulated the synthesis of collagen type I and fibronectin. Conclusions: The identification of PSCs, particularly in fibrotic areas, and the similarities of these cells to hepatic stellate cells suggest that PSCs participate in the development of pancreas fibrosis.  

4.22           Analysis of human immunodeficiency virus in semen: indications of a genetically distinct virus reservoir

Byrn, R.A. and Kiessling, A.A.

1. Reprod. Immunol., 41, 161-176 (1998)

  It is well established that HIV is found in semen, either as cell-free or cell associated virus, yet many questions remain about the source of the virus. A number of factors, including anatomic features of the male reproductive tract, the restricted access of the immune system to the germ cell compartment, and the results from sexually transmitted virus studies, suggest that the source of HIV in semen may be different from that in the peripheral blood. In this study, we examine the HIV in the infected cells of semen as indicators of the virus-producing reservoir. The frequency of HIV positive leukocytes in semen is compared to that of concurrent blood samples from eight donors and these values are found to be highly variable and frequently discordant. The protease gene sequences of HIV strains isolated from semen cells and blood cells were determined and phylogenetic analyses were performed which indicate the virus populations in the two sources are genetically distinct. In one patient receiving anti-HIV protease inhibitor therapy, gene sequences indicative of protease inhibitor resistance were found in the blood, but not the semen cell compartment. These results suggest that HIV in the semen and blood compartments are distinct, and further, may respond differently to antiviral therapy.  

4.23           Residual MHC class II expression on mature dendritic cells and activated B cells in RFX5-deficient mice

Clausen, B.E. et al Immunity, 8, 143-155 (1998)   Patients with major histocompatibility complex class II (MHC-II) deficiency are known to carry mutations in either the RFX complex or the trans-activator CIITA. While the pivotal role of CIITA for MHC-II gene transcription is supported by the essential absence of MHC-II molecules in CIITA-deficient mice, we demonstrate here that RFX5^-/- mice retain expression of MHC-II in thymic medulla, mature dendritic cells, and activated B cells. Nevertheless, RFX5^-/- mice develop a severe immunodeficiency due to the lack of MHC-II in thymic cortex, failure of positive selection of CD4⁺ T cells, and absence of MHC-II on resting B cells and resident or IFNg-activated macrophages. This differential requirement for CIITA and RFX5 in subsets of antigen-presenting cells may be specific for the mouse: it may, however, also exist in humans without having been noticed so far.  

4.24           Drug-metabolizing enzymes in rat liver myofibroblasts

Peterson, T.C. and Rowden, G. Biochem. Pharmacol., 55, 703-708 (1998)   The myofibroblast is considered to be a key component in the pathogenesis of hepatic fibrosis. There is a need for therapeutic intervention in hepatic fibrosis, and, to date, the number of efficacious anti-fibrotic drugs is negligible. At best, the current therapeutic modalities reduce liver enzymes, an indicator of liver damage, but cannot reduce or prevent fibrosis. We have described the anti-fibrotic effect of pentoxifylline in an experimental model of hepatic fibrosis. Evidence suggests that, in addition to pentoxifylline itself, at least two of the metabolites of pentoxifylline are of therapeutic interest. We have reported that one of these metabolites (M-1) has a biological activity similar to that of its parent drug. The second metabolite (M-1R) has been reported to be more potent than the parent drug. Recent evidence suggests that inhibition of cytochrome P450 1A2 (CYP1A2) results in higher levels of pentoxifylline and M-1 and may be responsible for the production of the novel, potent metabolite (M-1R). We therefore investigated whether the myofibroblast, the cell with a crucial role in fibrosis, contains drug-metabolizing enzymes and thus may play a critical role in the anti-fibrotic actions of pentoxifylline. Our results showed that myofibroblasts contain aryl hydrocarbon hydroxylase activity, ethoxyresorufin O-deethylase activity, and methoxyresorufin O-demethylase activity. The results presented here also indicate that aryl hydrocarbon hydroxylase and methoxyresorufin O-demethylase activities can be increased by treatment of cells with dibenzanthracene, an inducer of CYP1A activities.  

4.25           Spleen cells of non-obese diabetic mice fed with pig splenocytes display modified proliferation and reduced aggressiveness in vitro against pig islet cells

You, S., Gouin, E. and Saï, P. Diabetologia, 41, 955-962 (1998)   A new means of modifying xenogeneic reaction to pig islet cells, which involves pre-feeding with pig spleen cells, was investigated for the first time in the non-obese diabetic (NOD) mouse. Compared with controls, mice fed with pig spleen cells displayed much higher splenocyte proliferation in response to pig spleen and islet cells (p < 0.0001). This enhanced proliferation was specific for the species providing the fed cells. Positive relationships (p < 0.01) were found between increased splenocyte proliferation in response to pig spleen or islet cells and the number of cells per feeding or the number of daily feedings. Concomitantly, while co-incubation with splenocytes from control mice led to inhibition of both basal and stimulated insulin releases from pig islet cells (p < 0.001), this aggressiveness was abolished (p < 0.001) after co-culture with splenocytes from mice fed with pig spleen cells. The proliferative responses of splenocytes from fed or control mice to pig islet or spleen cells were abolished after removal of plastic-adherent cells, indicating that the major indirect pathway of T-cell activation was unchanged by pig spleen cell feeding. The main T-splenocyte subsets involved were restricted to MHC class II as they did not proliferate in the presence of monoclonal antibodies (mAbs) directed at I-A molecules. In mice fed with pig spleen cells, as well as in control mice, the blocking of CD4 + T cells with mAbs led to abolition of proliferation (p < 0.002), while the blocking of CD8 + led to a less marked effect. However, an increase in the blocking effect of anti-CD8 mAbs was noted in mice fed with pig spleen cells (p < 0.02). In control mice, the main splenocyte subset involved during proliferation in response to pig islet cells was Th1, since interferon <gamma> (IFN<gamma>) production increased significantly (p < 0.01) while that of interleukin-10 (IL-10) increased only slightly. The main change observed in mice fed with pig spleen cells was a marked increase in basal IL-10 production (p < 0.01) and the basal IL-10/IFN<gamma> ratio (p < 0.001). It seems likely that feeding with pig spleen cells shifted the Th1/Th2 balance towards a dominance of Th2-type class II-restricted CD4 + T cells, which may have been conducive to activating CD8 + suppressor T cells. In any event, oral administration of pig cells modified xenogeneic cellular response, which may have implications for xenografts of pig islets. In a more general sense, physiological feeding of cells from xenogeneic species would appear to have certain effects on the immune system.  

4.26           Viability of fresh vs. cultured pig islets for transplant

Rijkelijkhuizen, J.K.R.A., Bouwman, E. and van der Burg, M.P.M. Cell Transplantation, 8(2), abstract 6 (1999)   Probably because pig islets are difficult to isolate and culture, the impact of pre-transplant culture on graft viability has not been studied yet. We isolated islets from large sows (n=12) by Liberase digestion and OptiPrep-UWS purification. The yield of freshly prepared islets was 1924 ± 346 IEQs/g with a diameter of 162 ± 13 um, purity of 96 ± 2%, and viability of 82 ± 2% by acridine orange – propidium iodide (AOPI) staining. During culture at 37°C in RPMI + 10% porcine serum, at day 1 islet recovery was 21 ± 4%, size was 124 ± 6 um, and viability was 87 ± 2%; at 1 week recovery was 11 ± 2%, size was 113 ± 4 um, and viability was 90 ± 2%. Islet quality was further tested by grafting under the kidney capsule in STZ-diabetic nude mice of ~1500 (n=14) or 3200 (n=5) fresh islets as compared with ~1500 (n=18) or 800 (n=12) cultured islets. Only 1/14 recipients of 1500 fresh islets and 0/5 recipients of 3200 fresh islets became normoglycemic (< 10.7 mM non-fasting)). The other mice had a > 28 mM glycemia before being killed for severe diabetic complications at ~3 wk. Histology of the kidneys demonstrated substantial scarring and near-absence of islets. By contrast, 1500 cultured islets rendered 18/18 recipients normoglycemic within 2 days (1-wk cultured) or ~2 wk (1-day cultured) for > 100 days, with a mean glycemia of 5.5 mM. The 800 IEQs-dose was likewise successful in 5/8 recipients of 1-day and 4/4 recipients of 1-wk cultured islets. Histology of these grafts so far showed a substantial mass of well-preserved islets with little scarring at the graft’s site. Thus, pre-transplant culture markedly improved the viability of the graft, and disintegration of part of the fresh islets may not only reduce the effective islet dose, but also hamper the engraftment of viable tissue, because of scarring and because the cellular debris probably will attract macrophages and induce the release of harmful cytokines.  

4.27           OptiPrep for human islet purification.

Van der Burg, M.P.M., Ranuncoli, A., Molano, R., Kirlew, T., Ringers, J., Bouwman, E., Terpstra, O.T. and Ricordi, C. Cell Transplantation, 8(2), abstract 57 (1999)   Considerable scope exists for improvement in the purification of human islets. We recently developed an approx. 100% efficient density gradient of iodixanol (OptiPrep) in University of Wisconsin solution (UWS) in the difficult pig model. This success prompted us to test this gradient during 5 consecutive human islet isolations. Pancreases distantly procured from multiorgan cadaveric donors (27-57 y) and cold preserved with UWS for ~12 h were digested with Liberase in HBSS by the automated method. The digest was collected with cold RPMI, and a small sample of the digest was taken for these pilot experiments. Next, after pre-incubation for 60 min in UWS on ice, the tissue was bottom-loaded in a 1.086-1.075-1.070-UWS gradient in 50 ml conical tubes. The bottom was prepared by mixing 30 ml digest  (in UWS) with 10 ml OptiUWS (an 1:1 mixture of OptiPrep and double-strength UWS). The 1.075 and 1.070 layers were prepared by mixing 5 ml OptiUWS with 22.6 and 28.3 ml UWS, resp. After 5 min 500g centrifugation at 4°C purified islets were collected from the top or two uppermost layers. The digest loaded in the gradients contained 15287 ± 3712 IEQs and the volume-average islet diameter was 238 ± 22 um. After purification at the top 12766 ± 3129 IEQs were recovered (83 ± 2% recovery), islet diameter was 204 ± 13 um (NS) and purity was 89 ± 3%. Viability of the islets was corroborated histologically 1 to 3 wk after transplantation under the kidney capsule in 3 nude mice. Thus, the consistent high efficacy of this simple OptiPrep-UWS gradient under mild hypertonic conditions (~360 mOsm) in this pilot, and other favorable characteristics such as low endotoxin content, suggest that the gradient may become a new powerful tool for human islet purification.  

4.28           Adult pig islet recovery during Liberase isolation, OptiPrep purification and culture for transplantation in nude mice

Van der Burg, M.P.M., Rijkelijkhuizen, J.K.R.A., Zwaan, R.P. and Bouwman, E. Cell Transplantation, 8(2), abstract 58 (1999)   We aimed at improving the yield of viable intact pig islets by keeping the islets in the University of Wisconsin solution (UWS) both during isolation and during purification in a novel gradient of OptiPrep (Nycomed) in UWS, and by testing the new Liberase-PI blend (0.5 mg/ml) head to head with collagenase-P (2 mg/ml) for digestion of two paired segments of the pancreas (WIT 22 min) of large sows (n=6). The segments were digested for ~ 30 min at 36°C, and dispersed by shaking and sieving in UWS on ice. For purification the bottom (density 1.10 g/ml, 380 mOsm) was prepared by mixing the digest 2:1 with OptiUWS (an 1:1 mixture of OptiPrep and double-strength UWS), layered with 1.090 g/ml OptiPrep-UWS, and topped with UWS. After centrifugation at 500 g and 4°C for 5 min, purified islets were obtained from the top. Islet yield (mean 2444 ± 446 IEQs/g pancreas) and size (volume-average diameter 155 ± 11 um) did not differ after Liberase and collagenase-P digestion. After purification, comparison of the Liberase vs. collagenase-P isolated islets demonstrated a similar > 95% purity, but higher yields (2448 ± 392 vs. 1319 ± 224 IEQs; P<0.05), larger islets (185 ± 16 vs. 140 ± 8 um, P=0.08), and superior viability (90 ± 2 vs. 79 ± 4%; P<0.05; acridine orange – propidium iodide staining) of the Liberase-isolated islets. Overnight culture IEQ recovery was 24 ± 9% for Liberase vs. 14 ± 8% for the collagenase-P islets (P<0.05). After grafting of cultured Liberase islets under the kidney capsule in STZ-diabetic nude mice normoglycemia was obtained in 5/8 recipients of 800 IEQs within 1 month and in 9/9 recipients of 1500 IEQs within ~ 2 wk. We conclude that islet preservation is markedly improved by Liberase digestion, and the use of UWS throughout isolation and purification in our novel OptiPrep-UWS gradient.  

4.29           Autocontrolled, randomized comparison between a tri-layer density gradient (OptiPrep) and the migration-sedimentation-gravity method

Van den Bergh, M., Emiliani, S., Biramane, J., Vannin, A.A.S. and Englert, Y. Human Reprod. Suppl., 14, P211, 246 (1999)   Introduction: OptiPrep is a non-ionic, iodinated derivative from compounds initially developed for X-ray contrast which were by nature of their original purpose rigorously tested to ensure their safety. OptiPrep appears therefore to be a possible alternative for semen density gradient preparation.   Materials and methods: Each of 30 semen samples from men attending a classical in-vitro fertilization procedure was divided in two equal volumes. One part was prepared by the migration-sedimentation-gravity (MSG) technique and the other part by a tri-layer density gradient (50%-25%-12.5% v/v) OptiPrep (Op3). The oocytes were randomized to be inseminated either with the MSG either with the Op3 preparation of the same semen sample. The same concentration of spermatozoa was added to the two series. Initial volume, motility and concentration, as well as final volume, motility and concentration were recorded. Morphology according to strict criteria was assessed on both final preparations after Spermacâ staining. Fertilization, cleavage and embryo quality obtained by both preparations was compared. Data were analyzed by Crosstabs and the Altman Bland (AB) method.   Results: Initial concentration varied between 25x10⁶/ml with a median of 100x10⁶/ml. Initial volume was between 1.2 and 22 ml with a median of 4.2 ml. The motility ranged between 12 and 76% with a median of 53%. The percentage recovery of initial motile spermatozoa used in each preparation was calculated. The recovery for the MSG method ranged between 0.5 and 68% with a median of 3.85% and for the Op3 from 0.17 to 24% with a median of 1.19%. The AB analysis resulted in a linear regression curve P < 0.01 with R = 0.8 for the recovery, indicating that the observed differences were significantly method dependent. The final concentration after preparation ranged from 0.85x10⁶/ml to 320x10⁶/ml with a median of 8.25x10⁶/ml for MSG and from 1.1x10⁶/ml to 250x10⁶/ml with a median of 7.5x10⁶/ml for Op3. The AB analysis resulted in linear regression curve P< 0.01 with R = 0.65 for final concentration, indicating that the observed differences were significantly method dependent. The percentage normal forms varied from 9 to 33% with a median of 15.5% for MSG and between 1 and 23% with a median of 10% for Op3. The AB analysis did not result in any correlation, indicating that the observed differences were not method dependent. The motility after preparation ranged between 50 and 100% with a median of 96% for MSG and from 3 to 97% with a median of 43% for Op3. The AB analysis resulted in a linear regression curve P < 0.01 with R = -0.93 for the final motility, indicating that the observed differences were significantly method dependent. From 163 oocytes inseminated with MSG-prepared semen, 65 (39%) fertilized, whereas 74 (40%) fertilized out of 188 inseminated with Op3-prepared semen (not significant). The embryo score based on a scale between 0 and 6 taking in account the cleavage speed at 45 h post insemination, ranged for both preparations between 0 and 5 with an identical median of 3, reflecting that there was no difference in embryo quality. Thirteen pregnancies were obtained in these 30 patients (= cycles) with two abortions. As the best embryos were selected for embryo transfer, embryos obtained by the two semen preparations were mixed in the transfer and no conclusion can be made concerning the implantation rates.   Conclusion: The differences in final percentage motile spermatozoa, in the final concentration and in the percentage recovery were method dependent. The difference in final percentage spermatozoa with normal morphology was not dependent on method. The results obtained in terms of fertilization and embryo cleavage and quality was not influenced by the method of preparation.  

4.30           Immediate reversal of diabetes in primates following intraportal transplantation of porcine islets on a new histidine-lactobionate-iodixanol gradient

Matsumoto, S et al Transplantation, 67(7), abstract 856 (1999)   Information on islet xenotransplantation in relevant preclinical models is limited. One of the first issues to be addressed is whether porcine islets transplanted into primates are subject to hyperacute rejection. Previous work by others has emphasized the importance of density gradient media. To assure that technical factors do not compromise interpretation of early graft function in this model we sought to refine density gradient media such that immediate function of purified and cultured islets is consistently achieved in diabetic nude mouse recipients. Recently we have shown large-scale isopycnic islet purification utilizing University of Wisconsin solution with iodixanol (UWI) facilitates immediate single-donor pig islet allograft function. However, histidine-lactobionate (HL) solution was shown to be superior to UW for cold storage of purified islets. Therefore, we developed new density gradient media based on HL and iodixanol (HLI). The purpose of this study is to compare HLI to UWI-gradient media and examine whether porcine islets purified with new designed gradient media reverse diabetes promptly in nude mice and rhesus monkeys. Methods: Porcine islets were isolated by the automated method. Free islets were separated from non-islet tissue utilizing continuous UWI or HLI gradients on a Cobe 2991 cell separator. After a 48 hr culture period, islet viability was assessed by a trypan blue uptake test and functional integrity in vivo was evaluated by renal subcapsular transplantation of 2,000islet equivalents (IEQ) into diabetic nude mice. HLI purified and 48 hr cultured islets (20,000 IEQ/kg) were transplanted intraportally into streptozotocin-diabetic rhesus monkeys. Results: The percent recovery and purity of HLI purified islets were significantly higher compared to UWI gradient (100.0 ± 5.8% vs. 81.8 ± 5.0% p<0.05 and 93.0 ± 0.6 vs. 89.3 ± 2.8% p<0.03), respectively. Using HLI, culture recovery was significantly higher (79.3 ± 4.1% and 57.3 ± 2.5%, respectively (p<0.01)) and viability using trypan blue uptake test was also significantly higher (99.4 ± 0.1% vs. 96.8 ± 0.5% respectively (p<0.001)). All nude mice recipients of HLI-purified islets became normoglycemic within one day (n=8) in contrast to recipients of UWI islets (2.3 ± 0.5 days, p<0.02). Diabetes was reversed in 5 of 6 rhesus monkeys within 24 hrs after transplantation of HLI islets. Conclusion: HLI gradients recovered viable porcine islets perfectly. Islets purified by HLI reverse diabetes promptly both in nude mice and rhesus monkeys and seem therefore adequate to be used in ongoing studies designed to clarify the mechanism underlying islet xenograft rejection in primates.

4.31           Definition of dendritic cell subpopulations present in the spleen, Peyer’s patches, lymph nodes, and skin of the mouse

AnjuPre, F. et al Blood, 93(2), 590-598 (1999)   Dendritic cells (DC) are highly efficient antigen-presenting cells (APC) that have an essential function in the development of immune responses against microbial pathogens and tumors.  Although during the past few years our understanding of DC biology has remarkably increased, a precise characterization of the different DC subpopulations remains to be achieved with regard to their phenotype and lineage relationships.  In this report, we have extensively studied the DC subpopulations present in the thymus, spleen, Peyer's patches, lymph nodes (LN) and skin of the mouse.  Thymus DC and 60% spleen DC have a lymphoid DC phenotype, i.e., CD8⁺ DEC-205^high Mac-1^low, whereas 40% spleen DC have a myeloid DC phenotype, i.e., CD8^- DEC-205^low Mac-l^high.  Both CD8⁺ and CD8^- DC are leukocyte function-associated antigen-1 (LFA-l)^high and highly adherent.  Within Peyer's patches the majority of DC correspond to the CD8⁺ DEC-205^high Mac-l^low lymphoid category.  In the LN, together with CD8⁺ and CD8^- DC, an additional nonadherent CD8^int LFA-l^int subpopulation with lymphoid DC characteristics is described.  Finally, in the skin both epidermal Langerhans cells (LC) and dermal DC are CD8^- DEC-205^high Mac-1^high, and do not express LFA-1.  Interestingly, LC migrations experiments indicate that LC underwent the upregulation of CD8 and LFA-1 upon migration to the LN, supporting the hypothesis that LC belong to the CD8⁺ lymphoid lineage.  

4.32           CD8⁺ T cells mediate CD40-independent maturation of dendritic cells in vivo

Ruedl, C., Kopf, M. and Bachmann, M.F.

1. Exp. Med., 189(12), 1875-1883 (1999)

  Induction of cytotoxic T lymphocyte (CTL) responses against minor histocompatibility antigens is dependent upon the presence of T cell help and requires the interaction of CD40 on dendritic cells (DCs) with CD40 ligand on activated T helper cells (Th).  This study demonstrates that CD40 is neither involved in Th-dependent nor Th-independent antiviral CTL responses.  Moreover, the data show that DC maturation occurs in vivo after viral infection in the absence of CD40 and Th.  This maturation did not require viral infection of DCs but was mediated by peptide-specific CD8⁺ T cells.  Surprisingly, naive CD8⁺ T cells were able to trigger DC maturation within 24 h after activation in vivo and in vitro.  Moreover, peptide-activated CD8⁺ T cells were able to induce maturation in trans, as DCs that failed to present the relevant antigen in vivo also underwent maturation.  Upon isolation, the in vivo-stimulated DCs were able to convert a classically Th-dependent CTL response (anti-HY) into a Th-independent response in vitro.  Thus, antiviral CD8⁺ T cells are sufficient for the maturation of DCs in the absence of CD40.  

4.33           Thrombopoietin-induced activation of the mitogen-activated protein kinase (MAPK) pathway in normal megakaryocytes: role in endomitosis

Rojnuckarin, P., Drachman, J.G. and Kaushansky, K. Blood, 94(4), 1273-1282 (1999)   Thrombopoietin (TPO) plays a critical role in megakaryocyte proliferation and differentiation.  Using various cultured cell lines, several recent studies have implicated the mitogen-activated protein kinase (MAPK) pathway in megakaryocyte differentiation.  In the study reported here, we examined the role played by thrombopoietin-induced MAPK activity in a cytokine-dependent cell line (BAF3/Mpl) and in primary murine megakaryocytes.  In both systems, extracellular signal regulated protein kinase (ERK) 1 and 2 MAPK phosphorylation was rapidly induced by TPO stimulation.  To identify the Mpl domain responsible for MAPK activation, BAF3 cells expressing truncated forms of the Mpl receptor were studied.  Phosphorylation of ERKs did not require elements of the cytoplasmic signaling domain distal to Box 2 and was not dependent on phosphorylation of the adapter protein Shc. ERK activation in murine megakaryocytes was maximal at 10 minutes and was markedly decreased over the subsequent 3 hours.  Next, the physiologic consequences of MAPK inhibition were studied.  Using the MAPK kinase (MEK) inhibitor, PD 98059, blockade of MAPK activity substantially reduced TPO-dependent proliferation in BAF3/Mpl cells and markedly decreased mean megakaryocyte ploidy in cultures.  To exclude an indirect effect of MAPK inhibition on stromal cells in whole bone marrow, CD41⁺ cells were selected and then cultured in TPO.  The number of polyploid megakaryocytes derived from the CD41-selected cells was also significantly reduced by MEK inhibition, as was their geometric mean ploidy.  These studies show an important role for MAPK in TPO-induced endomitosis and underscore the value of primary cells when studying the physiologic effects of signaling pathways.  

4.34           A novel b₁ integrin-dependent mechanism of leukocyte adherence to apoptotic cells

Schwartz, B.R., Karsan, A., Bombeli, T. and Harlan, J.M.

1. Immunol., 162, 4842-4848 (1999)

  Adherence of leukocytes to cells undergoing apoptosis has been reported to be dependent on a variety of recognition pathways.  These include a_vb₃, (CD51/CD61, vitronectin receptor), CD36 (thrombospondin receptor), macrophage class A scavenger receptor, phosphatidylserine translocated to the outer leaflet of apoptotic cell membranes, and CD14 (LPS-binding protein).  We investigated the mechanism by which leukocytes adhere to apoptotic endothelial cells (EC).  Peripheral blood mononuclear leukocytes and U937 monocytic cells adhered to human or bovine aortic EC induced to undergo apoptosis by withdrawal of growth factors, treatment with the promiscuous protein kinase inhibitor staurosporine, with the protein synthesis inhibitor and protein kinase activator anisomycin, or with the combination of cycloheximide and TNFa.  Expression of endothelial adherence molecules such as CD62E (E-selectin), CD54 (ICAM-1), and CD106 (VCAM-1) was not induced or increased by these treatments.  A mAb to a_vb₃, exogenous thrombospondin, or blockade of phosphatidylserine by annexin V did not inhibit leukocyte adherence.  Further, leukocyte binding to apoptotic EC was completely blocked by treatment of leukocytes but not EC with mAb tb₁, integrin.  These results define a novel pathway for the recognition of apoptotic cells.  

4.35           Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion

Mohan, S., Mohan, N., Valente, A.J. and Sprague, E.A. Am. J. Physiol., 276, C1100-C1107 (1999)   We recently reported that prolonged exposure of human aortic endothelial cells (HAEC) to low shear stress flow patterns is associated with a sustained increase in the activated form of the transcriptional regulator nuclear factor-kB (NF-kB).  Here we investigate the hypothesis that low shear-induced activation of NF-kB is responsible for enhanced expression of vascular cell adhesion molecule (VCAM-1) resulting in augmented endothelial cell-monocyte (EC-Mn) adhesion and that this activation is dependent on intracellular oxidant activity.  Before exposure to low shear (2 dyn/cm²) for 6 h, HAEC were preincubated with or without the antioxidants pyrrolidine dithiocarbamate (PDTC) or N-acetyl-L-cysteine (NAC).  PDTC strongly inhibited low shear-induced activation of NF-kB, expression of VCAM-1, and EC-Mn adhesion.  Paradoxically, NAC exerted a positive effect on low shear-induced VCAM-1 expression and EC-Mn adhesion and only slightly downregulated NF-kB activation.  However, cytokine-induced NF-kB activation and VCAM-1 expression are blocked by both PDTC and NAC.  These data suggest that NF-kB plays a key role in low shear-induced VCAM-1 expression and that pathways mediating low shear- and cytokine-induced EC-Mn adhesion may be differentially regulated.  

4.36           Lipopolysaccharide-activated macrophages stimulate the synthesis of collagen type 1 and C-fibronectin in cultured pancreatic stellate cells

Schmid-Kotsaas, A. Et al Am. J. Pathol., 155, 1749-1758 (1999)   We have recently identified and characterized pancreatic stellate cells (PSC) in rats and humans (Gastroenterology 1998, 15:421-435).  PSC are suggested to represent the main cellular source of extracellular matrix in chronic pancreatitis.  Now we describe a paracrine stimulatory loop between human macrophages and PSC (rat and human) that results in an increased extracellular matrix synthesis.  Native and transiently acidified supernatants of cultured macrophages were added to cultured PSC in the presence of 0.1% fetal calf serum.  Native supernatants of lipopolysaccharide-activated macrophages stimulated the synthesis of collagen type 1 1.38 ± 0.09-fold of control and c-fibronectin 1.89 ± 0.18-fold of control.  Transiently acidified supernatants stimulated collagen type I and c-fibronectin 2.10 ± 0.2-fold and 2.80 ± 0.05-fold of control, respectively.  Northern blot demonstrated an increased expression of the collagen-I-(a-l)-mRNA and fibronectin-mRNA in PSC 10 hours after addition of the acidified macrophage supernatants.  Cell proliferation measured by bromodeoxyuridine incorporation was not influenced by the macrophage supernatants.  Unstimulated macrophages released 1.97 pg TGFb1/mg of DNA over 24 hours and lipopolysaccharide-activated macrophages released 6.61pg TGFb/mg of DNA over 24 hours.  These data together with the results that, in particular, transiently acidified macrophage supernatants increased matrix synthesis, identify TGFb as the responsible mediator.  In conclusion, our data demonstrate a paracrine stimulation of matrix synthesis of pancreatic stellate cells via TGFb1 released by activated macrophages.  We suggest that macrophages might play a pivotal role in the development of pancreas fibrosis.  

4.37           Synaptophysin: a novel marker for human and rat hepatic stellate cells

Cassiman, D. et al Am. J. Pathol., 155(6), 1831-1839 (1999)   Synaptophysin is a protein involved in neurotransmitter exocytosis and is a neuroendocrine marker.  We studied synaptophysin immunohistochemical expression in 35 human liver specimens (normal and different pathological conditions), in rat models of galactosamine hepatitis and carbon tetrachloride-induced cirrhosis, and in freshly isolated rat stellate cells. Synaptophysin reactivity was present in perisinusoidal stellate cells in both human and rat normal liver biopsies.  The number of synaptophysin-reactive perisinusoidal cells increased in pathological conditions.  Double staining for a-smooth muscle actin and synaptophysin, detected by confocal laser scanning microscopy, unequivocally demonstrated colocalization of both markers in lobular stellate cells.  In addition, freshly isolated rat stellate cells expressed synaptophysin mRNA (detected by polymerase chain reaction) and protein.  Finally, electron microscopy showed the presence of small electron translucent vesicles, comparable to the synaptophysin-reactive synaptic vesicles in neurons, in stellate cell projections.  We conclude that synaptophysin is a novel marker for quiescent as well as activated hepatic stellate cells.  Together with the stellate cell's expression of neural cell adhesion molecule, glial fibrillary acidic protein, and nestin, this finding raises questions about its embryonic origin and its differentiation.  In addition, the presence of synaptic vesicles in stellate cell processes suggests a hitherto unknown mechanism of interaction with neighboring cells.  

4.38           Persistent activation of nuclear factor - kB in cultured rat hepatic stellate cells involves the induction of potentially novel Rel-like factors and prolonged changes in the expression of IkB family proteins

Elsharkawy, A.M. et al Hepatology, 30, 761-769 (1999)   Rat hepatic stellate cells (HSC) cultured in serum-containing medium underwent a rapid (3-hour) classical induction of p5O:p65 and p65:p65 nuclear factor-kB (NFkB) dimers.  Subsequent culturing was associated with prolonged expression of active p50:p65 and persistent induction of a high-mobility NF-kB DNA binding complex consisting of potentially novel Rel-like protein(s). Formation of the latter complex was competed for by specific double-stranded oligonucleotides, was upregulated by treatment of HSCs with tumor necrosis factor a (TNF-a), and was maintained at basal levels of expression by a soluble HSC-derived factor.  An NF-kB-responsive CAT reporter gene was highly active in early cultured HSCs but was also trans-activated at a lower but significant level in longer-term cultured cells and could be completely suppressed by expression of dominant negative IkB-a.  Physiological significance of the lower persistent NF-kB activities was also demonstrated by the ability of long-term cultured HSCs to support the activity of the NF-kB-dependent human intercellular adhesion molecule-1 (ICAM-1) promoter.  Freshly isolated HSCs expressed high levels of IkB-a and IkB-b.  Culture activation was accompanied by a long-term reduction in levels of IkB-a with no detectable expression in the nuclear fraction of cells, under these conditions p50:p65 was detected in the nucleus.  IkB-b expression was transiently reduced and, upon replenishment, was associated with appearance of a lower-mobility IkB-b antibody-reactive species.  Bcl3 expression was absent in freshly isolated HSC but was induced during culturing and became a persistent feature of the activated HSC.  Inhibition of NF-kB DNA binding activity by gliotoxin was associated with increased numbers of apoptotic cells. We suggest that activation of NF-kB in cultured HSC is required for expression of specific genes associated with the activated phenotype such as ICAM-1 and may be antiapoptotic for rat HSCs.  

4.39           Dynamic regulation of expression and phosphorylation of Tau by fibroblast growth  factor-2 in neural progenitor cells from adult rat hippocampus

Tatebayashi, Y., Iqbal, K. and Grundke-Iqbal, I.

1. Neurosci., 19(13), 5245-5254 (1999)

  The nature of the extracellular signals that regulate the expression and the phosphorylation of the microtubule-associated protein tau, which is aberrantly hyperphosphorylated in Alzheimer disease and other adult-onset neurodegenerative diseases, is not known.  We have found that neural progenitor cells from adult rat hippocampus express adult isoforms of tau and that the expression and the phosphorylation of tau are regulated by fibroblast growth factor-2 (FGF-2).  Astrocytes that are differentiated from these cells by stimulation with ciliary neurotrophic factor express phosphorylated tau similarly when cultured in the presence of FGF-2.  In fetal progenitor cells that express only the fetal tau isotope expression, but not the phosphorylation, of this protein is regulated by FGF-2 in cultures of higher passages.  The FGF-2-mediated tau hyper-phosphorylation is inhibited by lithium an inhibitor of glycogen synthase kinase-3 (GSK-3), but not by inhibitors of mitogen-activated protein kinase or the cyclin-dependent kinases. Furthermore, both GSK-3 activity and the phosphorylation of tau increase when the concentration of FGF-2 is increased up to 40 ng/ml.  These results demonstrate that proliferating adult rat hippocampal progenitor cells express adult isoforms of tau stably and that FGF-2 upregulates the expression and, by upregulating GSK-3 activity, the phosphorylation of tau.  

4.40           Effect of the nonpeptide neurotrophic compound SR57746A on the phenotypic survival of purified mouse motoneurons.

Duong, F.H.T., Warter, J.M., Poindron, P. and Passilly, P. Br. J. Pharmacol., 128, 1385-1392 (1999)   1 Neurotrophic factors have been used for the treatment of several neurodegenerative diseases. However, their use is limited by their inability to cross the blood-brain barrier, their short half-life and their side effects. SR 57746A is a new orally active compound that exhibits in vivo and in vitro neurotrophic effects in several experimental models. 2 We show here that SR 57746A (1mM) increases the phenotypic survival of embryonic purified mouse motoneurons in vitro to the same extent as brain-derived neurotrophic factor (100 ng ml^-1) and increases the outgrowth and number of their neurites. It acts in a dose-dependent manner up to 1 mM which is the optimal concentration. Above this concentration, its neurotrophic effect decreases. 3 Genistein (10 mM), a protein tyrosine kinase inhibitor, also increases the phenotypic survival and differentiation of mouse motoneurons. It does not act in a synergistic or additive manner with SR 57746A. However, at concentrations equal or superior to 25 mM, it decreases the survival of motoneurons. This suggests that the neurotrophic effect of genistein is due to a favourable alteration of equilibrium between phosphorylated and dephosphorylated states of proteins involved in survival and differentiation of motoneurons. 4 Like genistein. SR 57746A should be used at a critical concentration (1 mM) to exert its optimal effects. Since SR 57746A does not act synergistically with genistein. It is likely that its mechanism of action involves a pathway similar to that affected by this tyrosine kinase inhibitor. 5 At the present time, SR 57746A is the only orally active compound and the only synthetic compound shown to be active on motoneurons in vitro. It should thus be considered as a good candidate for the treatment of motoneuron diseases.  

4.41           Activation and expression of ERK, JNK, and p38 MAP-kinases in isolated islets of Langerhans: implications for cultured islet survival

Paraskevas, S. et al FEBS Letters, 455, 203-208 (1999)   Isolation and purification of islet cells exposes them to ischemic, osmotic and mechanical stress. The objective of this study was to determine the roles of MAP-kinases in islets immediately following isolation. During the first 48 h, activity of JNK1 and JNK2 declined markedly. Activity of p38 increased steadily with time in culture while extracellular signal regulated kinase (ERK) activity declined dramatically within 24 h post-isolation. High p38 activation relative to ERK activation immediately following isolation correlated with a decrease in islet survival after 36 h in culture. Absence and/or transiency of ERK signaling in conjunction with sustained activation of p38 pathway could be an important regulator of cell death in islets during and following their isolation by commonly employed procedures.  

4.42           Comparison of two colloidal silica-based sperm separation media with a non-silica-based medium

Makkar, G., Ng, H-Y-. Yeung, S-B. and Ho, P-C. Fert. Steril., 72(5), 796-802 (1999)   Objective: To study and compare the effects of three sperm separation media, two silica-based (Percoll; Pharmacia Biotech AB, Uppsala, Sweden, and Isolate; Irvine Scientific, Santa Ana, CA) and one non–silica-based (Ixaprep; Medicult, Copenhagen, Denmark), on the recovery of progressive motile sperm, the percentage of sperm with normal morphology, various sperm motion characteristics determined by computer-aided sperm analysis, and the percentage of acrosome-reacted sperm. Design: Prospective study. Setting: A university-based assisted reproductive technology center. Patient(s): Male partners of couples attending our infertility clinic. Intervention(s): None. Main Outcome Measure(s): Various semen parameters. Result(s): Both Isolate and Ixaprep resulted in enhanced recovery of motile spermatozoa compared with Percoll. The percentage of sperm with forward progressive motility, the percentage of sperm with normal morphology, and various sperm motion characteristics were similar after the use of Percoll and Isolate and were significantly better than after the use of Ixaprep. The same percentage of acrosome-reacted spermatozoa was observed with all three media. Similar results were observed in both normal and subnormal semen samples. Conclusion(s): The use of Isolate and Ixaprep resulted in better recovery of motile spermatozoa. Other semen parameters were similar with the use of Isolate and Percoll, whereas the use of Ixaprep was associated with lower sperm velocities and fewer morphologically normal spermatozoa.  

4.43           Intensity and mechanisms of in vitro xenorecognition of adult pig pancreatic islet cells by CD₄⁺ and CD₈⁺ lymphocytes from type I diabetic or healthy subjects

Lalain, S., Chaillous, L., Gouin, E. and Saï, P. Diabetologia, 42, 330-335 (1999)   The intensity and mechanisms of cell-mediated rejection of pig islet cells were studied in 49 Type I diabetic and 34 healthy subjects. Human peripheral mononuclear cells proliferated strongly in response to pig islet cells (p < 0.001), though with notable interindividual variations (stimulation index 2 to 215). The variance of stimulation index was higher in diabetic than healthy subjects (p < 0.0001). The response to islet cells was stronger (p < 0.01) than that to pig splenocytes. Proliferation in response to islet cells was strongly decreased (p < 0.01) when CD₄⁺ T cells were blocked with monoclonal antibodies, whereas the blocking of CD₈⁺ cells or NK cells gave less pronounced effects. The response to islet cells was decreased (p < 0.01), but not abolished, after antigen-presenting cells were removed. Purified CD₄⁺ cells alone did not proliferate in response to islet cells but recovered their proliferative ability when mixed with antigen-presenting cells, whereas CD₈⁺ cells alone proliferated in the presence of interleukin-2 in response to islet cells. Proliferation was blocked (p < 0.01) by anti-DR monoclonal antibodies. During proliferation in response to islet cells, interleukin-10 increased 43-fold (p < 0.01) but interferon-<gamma> increased only slightly. No statistical differences were detected between diabetic and control subjects with respect to lymphocyte subsets and the recognition mechanisms or to interferon-<gamma> / interleukin-10 production in response to islet cells. These results provide the first detailed information on human cell-mediated xenoreaction to pig islet cells. This situation involves a dominant CD₄ class II-restricted Th2 response, with an indirect recognition pathway, as well as a CD₈ T-cell response resulting from direct recognition. This strong reaction constitutes a serious obstacle which may vary in degree among subjects.  

4.44           Increased levels of soluble Fcg receptor III in gingival fluid from periodontal lesions

Yuan, Z-N, Tolo, K., Schenck, K. and Helgeland, K. Oral Microbiol. Immunol., 14, 172-175 (1999)   Enzyme-linked immunosorbent assay was used for determination of the concen­tration of soluble Fcy receptor III (FcyRIII) in 40 samples of gingival fluid obtained from periodontal pockets in 30 patients with periodontitis. The assay was based on a monoclonal immobilized antibody binding FcgRIII and a poly­clonal FcgIII rabbit antibody for its quantification. The results indicate a sub­stantially increased concentration of soluble FcgRIII in gingival fluid as compared to the serum level. This increased concentration of soluble FcgRIII may interfere with phagocytosis and immune homeostasis in the periodontal lesions.  

4.45           Calcium and ATP regulation of ion transport in larval frog skin

Cox, T.C.

1. Comp. Physiol. B 169, 344-350 (1999)

  Ion transport measured as short circuit cur rent (Isc) across the skin of larva! frogs is activated by amiloride, acetylcholine, and ATP. In many epithelia, ATP stimulation of Isc involves an increase in intracellular calcium. To define the role of changes in intracellular calcium in ATP stimulation of Isc in larval frog skin, epithelial cells were loaded with calcium by adding 5mM ionomycin to a 2 mM calcium apical Ringer’s solution. Calcium loading had no observable effect on baseline Isc or on stimulation by ATP. Minimizing changes in intracellular calcium by loading the cell with the calcium chelator BAPTA also had no measurable effect on ATP stimulation of Isc. When the apical side was bathed with Ca²⁺ -free Ringer’s solution, ionomycin increased Isc up to 15mA. This increase was partially blocked by 2 mM Ca²⁺, 2 mM Mg²⁺, and 10mM W-7. Other experiments showed that baseline-stimulated and ATP-stimulated Isc were always larger in 2 mM Mg²⁺ Ringer’s compared to 2 mM Ca²⁺. In dissociated cells bathed in 2 mM Ca²⁺ Ringer’s, ATP had no effect on intracellular calcium as measured by Fluo-LR fluorescence changes. In conclusion, ATP apparently stimulates Isc without concomitant changes in intracellular calcium. This is consistent with a directly ligand-gated receptor at the apical membrane with P2X-like characteristics.  

4.46           Circulating vascular endothelial growth factor is not increased during relapses of steroid-sensitive nephritic syndrome

Webb, N.J.A. et al Kidney Int., 55, 1063-1071 (1999)   Background. An uncharacterized circulating factor that in­creases vascular permeability has previously been described in childhood steroid-sensitive nephrotic syndrome (SSNS). The aim of this study was to determine whether this factor is vascu­lar endothelial growth factor (VEGF), the recently described endothelial cell mitogen and enhancer of vascular permeability. Methods. Plasma and urine VEGF levels were measured in children with SSNS in both relapse and remission and in nor­mal age- and sex-matched controls. Semiquantitative reverse transcriptase-polymerase chain reaction studies investigating VEGF mRNA expression were performed on peripheral blood mononuclear cells isolated from children with SSNS in relapse and controls. In two experimental models (one-hour and three-day follow-up postinfusion), Sprague-Dawley rats were intra­venously administered 50 mg rVEGF to determine whether this induced either proteinuria or glomerular histologic change. Results. Plasma VEGF levels and urine VEGF/creatinine ratios were not elevated in SSNS relapse compared with remis­sion and control samples. Peripheral blood mononuclear cell VEGF mRNA expression was no different in SSNS patients compared with controls. The administration of VEGF to rats induced an acute reversible fall in systemic blood pressure but did not result in the development of either proteinuria or glomerular histologic change. Conclusion, Increased circulating VEGF levels are not re­sponsible for the proteinuria observed during relapses of SSNS. Further studies are warranted to investigate intrarenal VEGF expression.  

4.47           Functional and phenotypic analysis of thymic B cells: role in the induction of T cell negative selection

Ferrero, I., Anjuere, F., Martin, P., Martinez del Hoyo, G., Lopez Fraga, M., Wright, N., Varona, R., Marquez, G. and Ardavin, C. Eur. J. Immunol., 29(5), 1598-1609 (1999)   The phenotype of mouse thymic B cells and their capacity to induce T cell negative selection in vitro were analyzed. Thymic B cells expressed B cell markers such as IgM, Fc receptor, CD44, heat-stable antigen, LFA-1 and CD40. In addition, they were positive for the activation molecule CD69 and displayed high levels of B7-2. Although thymic B cells expressed CD5 on their surface, no CD5-specific mRNA was detected. Moreover, thymic B cells induced a stronger deletion of TCR-transgenic (TG) thymocytes than splenic B cells, which had low CD69 and B7-2 levels. Interestingly, CD40-activated splenic B cells up-regulated CD69 and B7-2 and acquired a capacity to induce T cell deletion comparable to that of thymic B cells. Moreover, thymic B cells from CD40-deficient mice displayed lower CD69 and B7-2 levels than control thymic B cells, and lower capacity to induce the deletion of TCR TG thymocytes. These results support the hypothesis that CD40-mediated activation of thymic B cells determines a high efficiency of antigen presentation, suggesting that within the thymus B cells may play an important role in the elimination of autoreactive thymocytes.  

4.48           Fractionation of differentiating cells using density perturbation

Bildirici, L. and Rickwood, D.

1. Immunol. Methods, 240 (1-2), 93-99 (2000)

  This paper describes the development of a new method for the fractionation of purified subpopulations of partially differentiated cells on continuous isopycnic gradients, using a density perturbation method based on the ability of cells to bind dense antibody-coated beads. Until now none of the available fractionation techniques, such as magnetic cell fractionation has been efficient for separating subpopulations of partially differentiated cells. The fractionation experiments described in this report used promyeolytic HL-60 and DMSO-induced granulocytic HL-60 cells as a model system. Populations of cells, modified by the binding of dense beads were fractionated on isotonic, isopycnic OptiPrep gradients by centrifugation at 220xg for 90 min at 20 degrees C. Examination of the different gradient fractions showed that, as cells bind increasing numbers of beads, they are found in the denser regions of the isopycnic gradients. Indirect immunofluorescence was combined with flow cytometric techniques to characterize the fractionation of partially differentiated cells. Flow cytometric results confirmed that as antigenic determinants appear on the surface at higher levels of expression, the number of beads binding to each cell increased. Furthermore, after fractionation, when the bead-bound and non-bead-bound cells were cultured in the presence of DMSO, those cells that had bound more beads targeted to differentiated cells were found to achieve terminal differentiation faster than those cells that had not been associated with any beads.  

4.49           Stromal cell-derived factor-1 (SDF-1) acts together with thrombopoietin to enhance the development of megakaryocytic progenitor cells (CFU-MK)

Hodohara, K., Fujii, N., Yamamoto, N. and Kaushansky, K Blood, 95(3), 769-775 (2000)

 

Stromal cell-derived factor-I (SDF-1) is a CXC chemokine that acts as a stimulator of pre-B lymphocyte cell growth and as a chemoattractant for T cells, monocytes, and hematopoietic stem cells.  More recent studies also suggest that megakaryocytes migrate in response to SDF-1. Because genetic elimination of SDF-1 or its receptor lead to marrow aplasia, we investigated the effect of SDF-1 on megakaryocyte progenitors (colony-forming units-megakaryocyte [CFU-MK]).  We report that SDF-1 augments the growth of CFU-MK from whole murine bone marrow cells when combined with thrombopoietin (TPO). The addition of SDF-1 to interleukin-3 (IL-3) or stem cell factor (SCF) had no effect.  Specific antagonists for CXCR4 (the sole receptor for SDF-1), T22, and 1-9 (P2G) SDF-1 reduced megakaryocyte colony growth induced by TPO alone, suggesting that many culture systems contain endogenous levels of the chemokine that contributes to the TPO effect. To examine whether SDF-1 has direct effects on CFU-MK, we developed a new protocol to purify megakaryocyte progenitors. CFU-MK were highly enriched in CD41^high c-kit^high cells generated from lineage-depleted TPO-primed marrow cells. Because the growth-promoting effects of SDF-1 were also observed when highly purified populations of CFU-MK were tested in serum-free cultures, these results suggest that SDF-1 directly promotes the proliferation of megakaryocytic progenitors in the presence of TPO, and in this way contributes to the favorable effects of the bone marrow microenvironment on megakaryocyte development.  

4.50           Tumor necrosis factor alpha and interleukin 1b up-regulate gastric mucosal Fas antigen expression in Helicobacter pylori infection

Houghton, J., Macera-Bloch, L.S., Harrison, L., Kim, K.H. and Korah, R.M. Infection and Immunity, 68(3), 1189-1195 (2000)   Fas-mediated gastric mucosal apoptosis is gaining attention as a cause of tissue damage due to Helicobacter pylori infection.  We explored the effects of H. pylori directly, and the effects of the inflammatory environment established subsequent to H. pylori infection, on Fas-mediated apoptosis in a nontransformed gastric mucosal cell line (RGM-1).  Exposure to H. pylori-activated peripheral blood mononuclear cells (PBMCs), but not H. pylori itself, induced Fas antigen (Fas Ag) expression, indicating a Fas-regulatory role for inflammatory cytokines in this system.  Of various inflammatory cytokines tested, only interleukin lb and tumor necrosis factor alpha induced Fas Ag expression, and removal of either of these from the conditioned medium abrogated the response.  When exposed to Fas ligand, RGM- 1 cells treated with PBMC-conditioned medium underwent massive and rapid cell death, interestingly, with a minimal effect on total cell numbers early on.  Cell cycle analysis revealed a substantial increase in S phase cells among cells exposed to Fas ligand, suggesting an increase in their proliferative response.  Taken together, these data indicate that the immune environment secondary to H. pylori infection plays a critical role in priming gastric mucosal cells to undergo apoptosis or to proliferate based upon their Fas Ag status.  

4.51           Upstream tissue inhibitor of metalloproteinases-1 (TIMP-1) element-1, a novel and  essential regulatory DNA motif in the human TIMP-1 gene promoter, directly  interacts with a 30-kDa nuclear protein

Trim, J.E. et al

1. Biol. Chem., 275(9), 6657-6663 (2000)

  Elevated expression of the tissue inhibitor of metalloproteinases-1 (TIMP-1) protein and mRNA has been reported in human diseases including cancers and tissue fibrosis.  Regulation of TIMP-1 gene expression is mainly mediated at the level of gene transcription and involves the activation of several well-known transcription factors including those belonging to the AP-1, STAT, and Pea3/Ets families.  In the current study, we have used DNase-1 footprinting to identify a new regulatory element (5'-TGTGGTTTCCG-3') present in the human TIMP-1 gene promoter.  Mutagenesis and transfection studies in culture-activated rat hepatic stellate cells and the human Jurkat T cell line demonstrated that the new element named upstream TIMP-1 element-1 (UTE-1) is essential for transcriptional activity of the human TIMP-1 promoter.  Electrophoretic mobility shift assay studies revealed that UTE-1 can form protein-DNA complexes of distinct mobilities with nuclear extracts from a variety of mammalian cell types and showed that induction of a high mobility UTE-1 complex is associated with culture activation of freshly isolated rat hepatic stellate cells.  A combination of UV-cross-linking and Southwestern blotting techniques demonstrated that UTE-1 directly interacts with a 30-kDa nuclear protein that appears to be present in all cell types tested.  We conclude that UTE-1 is a novel regulatory element that in combination with its cellular binding proteins may be an important component of the mechanisms controlling TIMP-1 expression in normal and pathological states.  

4.52           Notch1 deficiency dissociates the intrathymic development of dendritic cells and T cells

Radtke, F. et al

1. Exp. Med., 191(7), 1085-1093 (2000)

  Thymic dendritic cells (DCs) form a discrete subset of bone marrow (BM)-derived cells, the function of which is to mediate negative selection of autoreactive thymocytes.  The developmental origin of thymic DCs remains controversial.  Although cell transfer studies support a model in which T cells and thymic DCs develop from the same intrathymic pluripotential precursor, it remains possible that these two types of cells develop from independent intrathymic precursors.  Notch proteins are cell surface receptors involved in the regulation of cell fate specification.  We have recently reported that T cell development in inducible Notchl-deficient mice is severely impaired at an early stage, before the expression of T cell lineage markers.  To investigate whether development of thymic DCs also depends on Notchl, we have constructed mixed BM chimeric mice.  We report here that thymic DC development from Notchl^-/- BM precursors is absolutely normal (in terms of absolute number and phenotype) in this competitive situation, despite the absence of Notchl^-/-T cells.  Furthermore, we find that peripheral DCs and Langerhans cells are also not affected by Notchl deficiency.  Our results demonstrate that the development of DCs is totally independent of Notchl function, and strongly suggest a dissociation between intrathymic T cell and DC precursors.  

4.53           Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascades  in EL4 thymoma cells

Ku, H. and Meier, K.E.

1. Biol. Chem., 275(15), 11333-11340 (2000)

  Intracellular signals can regulate cell adhesion via several mechanisms in a process referred to as "inside-out" signaling.  In phorbol ester-sensitive EL4 thymoma cells, phorbol-12-myristate 13-acetate (PMA) induces activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases and promotes cell adhesion.  In this study, clonal EL4 cell lines with varying abilities to activate ERKs in response to PMA were used to examine signaling events occurring downstream of ERK activation.  Paxillin, a multifunctional docking protein involved in cell adhesion, was phosphorylated on serine/ threonine residues in response to PMA treatment.  This response was correlated with the extent and time course of ERK activation.  PMA-induced phosphorylation of paxillin was inhibited by compounds that block the ERK activation pathway in EL4 cells, primary murine thymocytes, and primary murine splenocytes. Paxillin was phosphorylated in vitro by purified active ERK2.  Two-dimensional electro-phoresis revealed that PMA treatment generated a complex pattern of phosphorylated paxillin species in intact cells, some of which were generated by ERK-mediated phosphorylation in vitro. An ERK pathway inhibitor interfered with PMA induced adhesion of sensitive EL4 cells to substrate. These findings describe a novel inside-out signaling pathway by which the ERK cascade may regulate events involved in adhesion.  

4.54           Cooperation among Stat1, glucocorticoid receptor, and PU.1 in transcriptional  activation of the high-affinity Fcg receptor I in monocytes

Aittomaki, S. et al.

1. Immunol., 164, 5689-5697 (2000)

  IFN-g and glucocorticoids regulate inflammatory and immune responses through Stat1 and glucocorticoid receptor (GR) transcription factors, respectively.  The biological responses to these polypeptides are determined by integration of various signaling pathways in a cell-type and promoter-dependent manner.  In this study we have characterized the molecular basis for the functional cooperation between IFN-g and dexamethasone (Dex) in the induction of the high-affinity Fcg receptor I (FcgRI) in monocytes.  Dex did not affect IFN-g induced Statl DNA binding activity or induce novel DNA-binding complexes to the FCgRI promoter.  By using cell systems lacking functional GR or Stat1, we showed that GR stimulated Statl-dependent transcription in a ligand-dependent manner, while Stat1 did not influence GR-dependent transcription.  The cooperation required phosphorylation of Tyr⁷⁰¹, DNA binding, and the trans-activation domain of Stat1, but did not involve Ser⁷²⁷ phosphorylation of Stat1 or physical interaction between GR and Statl.  The costimulatory effect of Dex was not dependent on a consensus glucocorticoid response element in the Stat1-responsive promoters, but required the DNA-binding and trans-activation functions of GR and Dex-induced protein synthesis.  GR activated the natural FcgRI promoter construct, and this response required both Statl and the Ets family transcription factor PU.I. Previously, physical association between GR and Stat5 has been shown to enhance Stat5-dependent and suppress GR-dependent transcription.  The results shown here demonstrate a distinct, indirect mechanism of cross-modulation between cytokine and steroid receptor signaling that integrates Statl and GR pathways with cell type-specific PU.1 transcription factor in the regulation of FcgRI gene transcription.  

4.55           Rebound from nitric oxide inhibition triggers enhanced monocyte activation and  chemotaxis

Magazine, H.I., Chang, J., Goumon, Y. and Stefano, G.B.

1. Immunol., 165, 102-107 (2000)

  Exposure of human peripheral blood monocytes to the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) resulted in a rapid shift in cellular conformation of spontaneously activated cells from amoeboid to round.  The population of activated cells, ~7.1 ± 1.2%, was reduced 7-fold to 1.1 ± 0.4% following 0.5 h exposure to SNAP.  Observation of monocytes for 6 h demonstrated a gradual release from NO inhibition initiating at 2.5 h following SNAP treatment and a period of hyperactivity that was maximal at ~5 h following SNAP exposure.  During the rebound from the NO inhibition phase, there was a significant increase in the population of activated monocytes and an increased responsiveness to chemotactic agents such as IL-1, IL-8, and fMLP relative to that of cells treated with the chemotactic agents alone.  Conformational changes induced by SNAP were associated with a reduction in F-actin and loss of filopodial extension.  The loss and recovery of F-actin staining paralleled changes in cell activity, suggesting that NO may alter cellular activity by modulation of cytoskeletal actin.  These data taken together suggest that inhibition of monocyte activity by NO results in an excitatory phase observed subsequent to release from NO inhibition and increased sensitivity to chemotactic agents.  We propose that this rebound from NO inhibition may provide increased immunosurveillance to rectify immunological problems that have been encountered during the period of inhibition.  

4.56           Sterol 27-hydroxylase acts on 7-ketocholesterol in human atherosclerotic lesions and macrophages in culture

Brown, A.J., Watts, G.F., Burnett, J.R., Dean, R.T. and Jessup, W

1. Biol. Chem., 275(36), 27627-27633 (2000)

  27-Hydroxycholesterol (27OH) is the major oxysterol in human atherosclerotic lesions, followed by 7-ketocholesterol (7K).  Whereas 7K probably originates nonenzymically, 27OH arises by the action of sterol 27-hydroxylase, a cytochrome P450 enzyme expressed at particularly high levels in the macrophage and proposed to represent an important pathway by which macrophages eliminate excess cholesterol.  We hypothesized and here show that 27-hydroxylated 7-ketocholesterol (27OH-7K) is present in human lesions, probably generated by the action of sterol 27-hydroxylase on 7K. Moreover, [³H]27OH-7K was produced by human monocyte-derived macrophages (HMDMs) supplied with [³H]7K but not in HMDMs from a patient with cerebrotendinous xanthomatosis (CTX) shown to have a splice-junction mutation of sterol 27-hydroxylase.  Whereas [³H]27OH-7K was predominantly secreted into the medium, [³H]-27OH formed from [³H]-cholesterol was mostly cell-associated.  The majority of supplied [³H]7K was metabolized beyond 27OH-7K to aqueous-soluble products (apparently bile acids derived from the sterol 27-hydroxylase pathway).  Metabolism to aqueous-soluble products was ablated by a sterol 27-hydroxylase inhibitor and absent in CTX cells.  Sterol 27-hydroxylase therefore appears to represent an important pathway by which macrophages eliminate not only cholesterol but also oxysterols such as 7K.  The fact that 7K (and cholesterol) still accumulates in lesions and foam cells indicates that this pathway may be perturbed in atherosclerosis and affords a new opportunity for the development of therapeutic strategies to regress atherosclerotic lesions.  

4.57           Differential infectivity and division of Toxoplasma gondii in human peripheral blood leukocytes

Channon, J.Y., Seguin, R.M. and Kasper, L. Infection and Immunity, 68(8), 4822-4826 (2000)   When tachyzoites were incubated with human peripheral blood leukocytes in vitro, more monocytes and dendritic cells than neutrophils were infected. Although tachyzoites were able to divide in each of these cell types, monocytes and dendritic cells were more permissive to rapid tachyzoite division than neutrophils or lymphocytes.  

4.58           Langerhans cells develop from a lymphoid-committed precursor

AnjuPre, F., Martinez del Hoyo, G., Martin, P. and Ardavin, C Blood, 96, 1633-1637 (2000)   Langerhans cells (LCs) are specialized dendritic cells (DCs) strategically located in stratified epithelia, such as those of the skin, oral cavity, pharynx, esophagus, upper airways, urethra, and female reproductive tract, which are exposed to a wide variety of microbial pathogens. LCs play an essential role in the induction of T-lymphocyte responses against viruses, bacteria, and parasites that gain access to those epithelial surfaces, due to their high antigen capture and processing potential and their capacity to present antigen peptides to T cells on migration to the lymph nodes. Although LCs have been classically considered of myeloid origin, recent reports which demonstrate that existence of lymphoid DCs derived from multipotent lymphoid precursors devoid of myeloid differentiation potential, raise the question of the lymphoid or myeloid origin of LCs. The present study shows that mouse lymphoid-committed CD^low precursors, with the capacity to generate T cells, B cells, CD8⁺ lymphoid DCs, and natural killer cells, also generate epidermal LCs on intravenous transfer, supporting the view that LC belong to the lymphoid lineage.  

4.59           Interferon-a directly represses megakaryopoiesis by inhibiting thrombopoietin- induced signaling through induction of SOCS-1

Wang, Q., Miyakawa, Y., Fox, N. and Kaushansky, K. Blood, 96, 2093-2099 (2000)   Interferon (IFN)-a has proven useful for treating several clinical conditions, including chronic viral hepatitis and chronic myeloproliferative and lymphoproliferative disorders.  In addition to its well-known antiviral effects, the cytokine exerts antiproliferative effects on many cell types, helping to explain its therapeutic usefulness in these latter conditions. However, this same property accounts for several undesirable effects, including thrombocytopenia, which can interfere with the successful clinical application of IFN-a.  Unfortunately, the mechanisms responsible for the myelosuppressive effects of the cytokine are incompletely understood.  The effects of IFN-a on megakaryocyte (MK) development were studied.  Using several marrow cell purification techniques and quantitative culture methods, it was found that IFN-a directly inhibits thrombopoietin (TPO)-induced MK growth.  Previous studies indicated that Janus kinase (JAK) and its substrates mediate the effects of TPO on cellular proliferation and survival. It was found that IFN-a directly suppresses TPO-induced phosphorylation of the JAK2 substrates c-Mpl and STAT 5 in a TPO-dependent hematopoietic cell line and of Mpl and STAT3 in primary murine MK.  Moreover, IFN-a induces SOCS-1 production in these cells, which has been shown to inhibit TPO-induced cell growth.  Because SOCS protein expression is induced by many cytokines and has been reported to extinguish signaling from several hematopoietic cytokine receptors, these results identify a molecular mechanism responsible for cytokine receptor cross-talk.  

4.60           Activated monocytes in sickle cell disease: potential role in the activation of vascular endothelium and vaso-occlusion

Belcher, J.D., Marker, P.H., Weber, J.P., Hebbel, R.P. and Vercellotti, G.M. Blood, 96, 2451-2459 (2000)   Sickle cell anemia is characterized by painful vaso-occlusive crises.  It is hypothesized that monocytes are activated in sickle cell disease and can enhance vaso-occlusion by activating endothelium.  To test this hypothesis, human umbilical vain endothelial cells (HUVEC) and human microvascular endothelial cells (MVEC) with sickle and normal mononuclear leukocytes were incubated, and endothelial activation was measured.  Endothelial cells incubated with sickle mononuclear leukocytes were more activated than those incubated with normal mononuclear leukocytes, as judged by the increased endothelial expression of adhesion molecules and tissue factor and the adhesion of polymorphonuclear leukocytes (PMNL). Monocytes, not lymphocytes or platelets, were the mononuclear cells responsible for activating endothelial cells.  Sickle monocytes triggered endothelial nuclear factor-kappa B (NF-kB) nuclear translocation.  Cell-to-cell contact of monocytes and endothelium enhanced, but was not required for, activation.  Antibodies to tumor necrosis factor-alpha (TNF-a) and interleukin-l-beta (IL-1b) blocked activation of the endothelium by monocytes. Peripheral blood monocytes from patients with sickle cell disease had 34% more IL-1b (P = .002) and 139% more TNF-a (P = .002) per cell than normal monocytes.  Sixty percent of sickle monocytes expressed the adhesion molecule ligand CD11b on their surfaces compared with only 20% of normal monocytes (P = .002). Serum C-reactive protein, a marker of systemic inflammation, was increased 12-fold in sickle serum than in normal serum (P = .003). These results demonstrate that sickle monocytes are activated and can, in turn, activate endothelial cells.  It is speculated that vascular inflammation, marked by activated monocytes and endothelium, plays a significant role in the pathophysiology of vaso-occlusion in sickle cell anemia.  

4.61           Concept of lymphoid versus myeloid dendritic cell lineages revisited: both CD8a^- and CD8a⁺ dendritic cells are generated from CD4^low lymphoid-committed precursors

Martin, P. et al Blood, 96, 2511-2519 (2000)   Two dendritic cell (DC) subsets have been identified in the murine system on the basis of their differential CD8a expression.  CDa⁺ DCs and CD8a^- DCs are considered as lymphoid- and myeloid-derived, respectively, because CD8a⁺ but not CD8a^- splenic DCs were generated from lymphoid CD4^low precursors, devoid of myeloid reconstitution potential.  Although CD8a^- DCs were first described as negative for CD4, our results demonstrate that approximately 70% of them are CD4⁺.  Besides CD4^- CD8a^- and CD4⁺ CD8a^- DCs displayed a similar phenotype and T-call stimulatory potential in mixed lymphocyte reaction (MLR), although among CD8a^- DCs, the CD4⁺ subset appears to have a higher endocytic capacity. Finally, experiments of DC reconstitution after irradiation in which, in contrast to previous studies, donor-type DCs were analyzed without depleting CD4⁺ cells, revealed that both CD8a⁺ DCs and CD8a^- were generated after transfer of CD4^low precursors. These data suggest that both CD8a⁺ and CD8a^- DCs derive from a common precursor and, hence, do not support the concept of the CD8a⁺ lymphoid-derived and CD8a^- myeloid-derived DC lineages. However, because this hypothesis has to be confirmed at the clonal level, it remains possible that CD8a^- DCs arise from a myeloid precursor within the CD4^low precursor population or, alternatively, that both CD8a⁺ and CD8a^- DCs derive from an independent nonlymphoid, nonmyeloid DC precursor. In conclusion, although we favor the hypothesis that both CD8a⁺ and CD8a^- DCs derive from a lymphoid-committed precursor, a precise study of the differentiation process of CD8a⁺ and CD8a^- DCs is required to define conclusively their origin.  

4.62           Protective immunity in macaques vaccinated with a modified vaccinia virus Ankara-based measles virus vaccine in the presence of passively acquired antibodies

Stittelaar, K. et al

1. Virol., 74(9), 4236-4243 (2000)

  Recombinant modified vaccinia virus Ankara (MVA), encoding the measles virus (MV fusion (F) and hemagglutinin (H) (MVA-FH) glycoproteins, was evaluated in an MV vaccination-challenge model with macaques.  Animals were vaccinated twice in the absence or presence of passively transferred MV-neutralizing macaque antibodies and challenged 1 year later intratracheally with wild-type MV. After the second vaccination with MVA-FH, all the animals developed MV-neutralizing antibodies and MV-specific T-cell responses. Although MVA-FH was slightly less effective in inducing MV-neutralizing antibodies in the absence of passively transferred antibodies than the currently used live attenuated vaccine, it proved to be more effective in the presence of such antibodies.  All vaccinated animals were effectively protected from the challenge infection. These data suggest that MVA-FH should be further tested as an alternative to the current vaccine for infants with maternally acquired MV-neutralizing antibodies and for adults with waning vaccine-induced immunity.  

4.63           Anatomical origin of dendritic cells determines their life span in peripheral lymph nodes

Ruedl, C., Koebel, P., Bachmann, M., Hess, M. And Karjalainen, K.

1. Immunol., 165, 4910-4916 (2000)

  Dendritic cells (DCs) exhibit considerable heterogeneity in their anatomical location, surface phenotype, and functional properties. In this study, we demonstrate that peripheral lymph nodes contain at least four major, functionally separable, and independently derived, DC subsets, which can be clearly demarcated by their CD11c, CD40, and CD8 expression pattern. Surprisingly, all DCs derived directly from the bone marrow, the myeloid- and the lymphoid-related subsets, turned over fast with t_1/2 of a couple of days. In contrast, DCs exported from the skin, both dermal and epidermal, accumulated 3- to 4-fold slower, turnover that is dramatically increased by cutaneous inflammation.  

4.64           Enteric infection acts as an adjuvant for the response to a model food antigen

Shi, H.N., Liu, Y. and Nagler-Anderson, C.

1. Immunol., 165, 6174-6182 (2000)

  Oral administration of soluble protein Ags typically induces Ag-specific systemic nonresponsiveness. However, we have found that feeding a model food protein, OVA, to helminth-infected mice primes for a systemic OVA-specific Th2 response. In this report we show that, in addition to creating a Th2-priming cytokine environment, helminth infection up-regulates costimulatory molecule expression on mucosal, but not peripheral, APCs. To examine the consequences of mucosal infection for the T cell response to orally administered Ag, we adoptively transferred, OVA-specific, T cells into normal mice. We found that helminth infection enhances the expansion and survival of transgenic T cells induced by Ag feeding. Transfer of 5,6-carboxyfluorescein diacetate succinimidyl ester-labeled donor cells showed that T cell proliferation in response to Ag feeding takes place primarily in the mesenteric lymph nodes. Upon subsequent peripheral exposure to Ag in adjuvant, the proliferative capacity of the transferred transgenic T cells was reduced in noninfected mice that had been fed OVA. Helminth infection abrogated this reduction in proliferative capacity. Our data suggests that enteric infection can act as an adjuvant for the response to dietary Ags and has implications for allergic responses to food and the efficacy of oral vaccination.  

4.65           Successful suppression of the early rejection of pig islets in monkeys

Rijkelijkhuizen, JK. et al Cell Transplant., 9(6), 909-912 (2000)   Primary nonfunction (PNF) is seen very frequently after xenogeneic transplantation of islets of Langerhans.  In a pig-to-rat model we recently observed that no PNF occurs when the islets are kept in culture at 37 degrees C for 1-2 weeks prior to transplantation.  In order to investigate the rejection mechanisms in a preclinical model, we transplanted cultured porcine islets under the capsule of both kidneys in four cynomolgous monkeys.  Islets were isolated from adult sows by means of digestion with Liberase in University of Wisconsin solution (UWS).  The digest was purified by a density gradient of OptiPrep in UWS.  Highly purified (>95%) islets were cultured 1-2 weeks in RPMI.  All monkeys showed significant titers of preformed anti-pig antibodies.  The immunosuppression of the monkeys consisted of cyclophosphamide (Cy) (2 days), cyclosporin A (CsA), and prednisolone.  Anticipating a fast rejection we carried out nephrectomies at different time points within 2 weeks after transplantation.  Following unilateral nephrectomy, well-preserved islets with no signs of rejection were observed between 3 and 7 days posttransplant. Later, between days 11 and 15 posttransplant, histology in the first three animals demonstrated no islets. In the fourth monkey histology on day 11 showed islets with excellent morphology and some small focal infiltrates. The highest CsA blood levels (around 1000 ng/ml) were found in animals with the best graft survival. We conclude that cultured porcine islets can be grafted without hyperacute rejection in monkeys with preformed anti-pig antibodies. In the presence of high levels of CsA only marginal signs of a cellular immune response were observed 11 days after transplantation.  

4.66           The LIM and SH3 domain-containing protein, lasp-1, may link the cAMP signaling pathway with dynamic membrane restructuring activities in ion transporting epithelia

Chew, C.S., Parente, J.A., Chen, X., Chaponnier, C. and Cameron, R.S.

1. Cell Science, 113(11), 2035-2045 (2000)

  Lasp-1 is a unique LIM and src homology 3 (SH3) domain containing protein that was initially identified as a 40 kDa cAMP-dependent phosphoprotein in the HCI-secreting gastric parietal cell. Because cAMP is a potent stimulator of parietal cell acid secretion, we have hypothesized that changes in lasp1 phosphorylation might be involved in the regulation of ion transport-related activities, perhaps by modulating interactions among cytoskeletal and/or vesicle-associated proteins.  In this study, we demonstrate that the cAMP dependent acid secretary agonist, histamine, induces a rapid, sustained rise in parietal cell lasp-1 phosphorylation and this increase in phosphorylation is closely correlated with the acid secretary response.  In addition, elevation of intracellular cAMP concentrations appear to induce a partial redistribution of lasp-1 from the cell cortex, where it predominates along with the g-isoform of actin in unstimulated cells, to the b-actin enriched, apically-directed intracellular canalicular region, which is the site of active proton transport in the parietal cell.  Additional studies demonstrate that although lasp-1 mRNA and protein are expressed in a wide range of tissues, the expression is specific for certain actin-rich cell types present within these tissues.  For example, gastric chief cells, which contain relatively little F-actin and secrete the enzyme, pepsinogen, by regulated exocytosis, do not appear to express lasp- 1. Similarly, lasp- 1 was not detected in pancreatic acinar cells, which secrete enzymes by similar mechanisms and also contain relatively low levels of F-actin.  Lasp- 1 also was not detectable in proximal tubules in the kidney, in gastrointestinal smooth muscle, heart or skeletal muscle.  In contrast, expression was prominent in the cortical regions of ion-transporting duct cells in the pancreas and in the salivary parotid gland as well as in certain F-actin-rich cells in the distal tubule/collecting duct.  Interestingly, moderate levels of expression were also detected in podocytes present in renal glomeruli and in vascular endothelium.  In primary cultures of gastric fibroblasts, lasp-1 was present mainly within the tips of lamellipodia and at the leading edges of membrane ruffles.  Taken together these results support the hypothesis that the lasp-1 plays an important role in the regulation of dynamic actin-based, cytoskeletal activities.  Agonist-dependent changes in lasp-1 phosphorylation may also serve to regulate actin-associated ion transport activities, not only in the parietal cell but also in certain other F-actin-rich secretory epithelial cell types.  

4.67           SpC3, the complement homologue from the purple sea urchin, Strongylocentrotus purpuratus, is expressed in two subpopulations of the phagocytic coelomocytes

Gross, P.S., Clow, L.A., Courtney Smith, L. Immunogenetics, 51, 1034-1044 (2000)   The lower deuterostomes, including the echinoderms, possess an innate immune system that includes a subsystem with similarities to the vertebrate complement system. A homologue of the central component of this system, C3, has recently been identified in the purple sea urchin, Strongylocentrotus purpuratus, and is called SpC3. We determined previously that coelomocytes specifically express the SpC3 gene (Sp064); however, the sea urchin has at least four different types of coelomocytes: amoeboid phagocytes, red spherule cells, colorless spherule cells, and vibratile cells. To determine which of these subpopulations expresses Sp064 and produces SpC3, coelomocytes were separated by discontinuous gradient density centrifugation. Relatively homogenous fractions were obtained consisting of the four major cell types in addition to two types of amoeboid phagocytes with different densities and distinct morphologies. Analysis of proteins from separated cell subpopulations by Western blot and analysis of gene expression by RT-PCR revealed that phagocytes express the gene and contain the protein. Immunolocalization showed that SpC3⁺ phagocytes are present as subsets of both the low- and high-density subpopulations of phagocytes; however, the subcellular localization of SpC3 is different in these two subpopulations.  

4.68           Treatment of rat pancreatic islets with reactive PEG

Panza, J.L. et al Biomaterials, 21, 1155-1164 (2000)   Covalent attachment of polymers to cells and tissues could be used to solve a variety of problems associated with cellular therapies. Insulin-dependent diabetes mellitus is a disease resulting from the autoimmune destruction of the beta cells of the islets of Langerhans in the pancreas. Transplantation of islets into diabetic patients would be an attractive form of treatment, provided that the islets could be protected from the host's immune system in order to prevent graft rejection. If reaction of polyethylene glycol (PEG) segments with the islet surface did not damage function, the immunogenicity and cell binding characteristics of the islet could be altered. To determine if this process damages islets, rat islets have been isolated and treated with protein-reactive PEG-isocyanate (MW 5000) under mild reaction conditions. An assessment of cell viability using a colorimetric mitochondrial activity assay showed that treatment of the islets with PEG-isocyanate did not reduce cell viability. Insulin release in response to secretagogue challenge was used to evaluate islet function after treatment with the polymer. The insulin response of the PEG-treated islets was not significantly different than untreated islets in a static incubation secretagogue challenge. In addition, PEG-isocyanate-treated islets responded in the same manner as untreated islets in a glucose perifusion assay. Finally, the presence of PEG on the surface of the islets after treatment with the amine-reactive N-hydroxysuccinimide-PEG-biotin (not PEG-isocyanate) was confirmed by indirect fluorescence staining. These results demonstrate the feasibility of treating pancreatic islets with reactive polymeric segments and provide the foundation for further investigation of this novel means of potential immunoisolation.  

4.69           Multiple drug resistance mutations in human immunodeficiency virus in semen but not blood of a man on antiretroviral therapy

Eyre, R.C., Zheng, G. And Kiessling, A.A. Urology, 55, 591xvii-591xx (2000)   The concept that the male reproductive tract harbors isolated reservoirs of human immunodeficiency virus (HIV) infection has now been widely accepted. The significance of semen viral burden to sexual transmission of HIV is obvious; however, its contribution to disease progression is unknown. We report a case study that demonstrates the emergence of resistance-conferring mutations to antiviral therapy in infected seminal leukocytes from a man with asymptomatic prostatitis associated with leukospermia. This finding demonstrates the potential importance of male reproductive tract organs to the development of therapy resistance in HIV-infected men.  

4.70           Investigation of the suitability of a new purification medium in comparison with Percoll to separate bone marrow

Ruijs, W.P.M., Preijers, F. and Schattenberg, A. ABS2000, Poster presentation (2000)   Percoll is commonly used to enrich mononuclear cells from bone marrow (BM) prior to Counterflow Centrifugation Elutriation. Since Percoll contains high levels of endotoxins and regulations are increasingly imposing us to use pharmaceutical approved media, we were forced to search for alternatives. Recently Medi-Cult (Denmark) formulated for us a new clinical grade Purification Medium (PM). In contrast to the silica-based Percoll, PM is based on a specific molecular fraction of polysuccrose and iodixanol. We compared Percoll and PM in floatation gradients in order to establish its separation capacity on bone marrow cells. Therefore 20 ml of buffer was pumped in a 250 ml centrifugation bottle followed by 80 ml of 1.073 g/ml Percoll or PM, respectively. After plasma extraction BM was mixed with 90% Percoll or PM, respectively, to obtain a density of 1.085 g/ml. The mixture was pumped beneath the 1.073 g/ml layer and the 250 ml bottles were centrifuged. Different gradient fractions were collected and cell numbers were counted, analyzed in flow cytometry and clonogenic assays (CFU-GM, BFU-E and GFU-GEMM). The differences in numbers of CD34+ cells, T cells (CD4 and CD8), B cells, NK cells (CD3-/CD56), monocytes and granulocytes (CD66e) were determined as well as the number of dead cells (7AAD) in all these populations. Most CD34 cells, lymphocytes and monocytes were found in the 1.073 interfase layers whereas granulocytes were located in the 1.085 interphase layers. Erythrocytes were found in the bottom layers. Although no significant differences were found in the separation profiles of nucleated cells, the best separation of erythrocytes was found with PM. We conclude that the new Purification Medium can be used as a good alternative for the current gradient separation media in order to isolate mononuclear cells from bone marrow.  

4.71           Expression of the HML-1 epitope on human monocytes is independent of aE integrin mRNA

Miller, L.A., Li, C and Hyde, D.M. Inflammation, 24(3), 195-205 (2000) Interferon gamma (IFN )-mediated activation of the myelomonocytic cell line HL-60 and peripheral blood monocytes will induce expression of an epitope for the monoclonal antibody which recognizes human E 7 integrin, HML-1. Here, we found that the anti-human E 7 monoclonal antibody Ber-ACT8 did not recognize IFN -stimulated, HML-1 positive HL-60 cells. Culture of blood monocytes with IFN also induced expression of HML-1 but not Ber-ACT8 epitopes. Moreover, migration of monocytes across a monolayer of human airway epithelial cells rapidly induced expression of HML-1, with no detectable Ber-ACT8 staining. Using two different sets of primers specific for the human E integrin gene, we were unable to detect E mRNA within HL-60 cells or isolated monocytes by reverse transcriptase polymerase chain reaction methods. We conclude that reactivity of the HML-1 antibody for HL-60 cells and monocytes does not correspond with the expression of the E integrin subunit, and instead detects a marker for cellular activation.  

4.72           Monocyte chemotactic protein-1 stimulates the killing of Leishmania major by human monocytes, acts synergistically with IFN-   and is antagonized by IL-4

Ritter, U. and Moll, H. Eur. J. Immunol., 30(11), 3111-3120 (2000)   We recently demonstrated that monocyte chemotactic protein-1 (MCP-1) is strongly expressed in lesions of patients with self-healing localized cutaneous leishmaniasis (LCL) whereas it is scarce in those of chronic diffuse cutaneous leishmaniasis (DCL). This finding indicated that MCP-1 may contribute to the healing process. In the present study, we analyzed the capacity of MCP-1 to trigger leishmanicidal activities. The results show that MCP-1 directly stimulates the elimination of intracellular Leishmania parasites by human monocytes, a potential that correlates with the induction of reactive oxygen intermediates. Release of NO was not detected. To understand the cross-talk between the chemokine and T cell-associated cytokines, we studied the influence of the Th1 cytokine IFN- and the Th2 cytokine IL-4 on MCP-1-mediated activation of human monocytes. The data demonstrate that IFN- and MCP-1 synergistically activate monocytes to clear intracellular parasites, whereas IL-4 abrogates the effect of MCP-1. Furthermore, IL-4 inhibits MCP-1 expression by infected monocytes, a finding that may explain the lack of MCP-1 in chronic lesions. The data suggest a novel model for macrophage activation in cutaneous leishmaniasis. In lesions of LCL, the synergistic action of MCP-1 and IFN- may stimulate the killing of parasites by macrophages and promote healing, whereas the presence of IL-4 in DCL lesions may favor the suppression of MCP-1 and, together with the lack of IFN- , the progression of disease.  

4.73           Maturation of vulnerability to excitotoxicity: intracellular mechanisms in cultured postnatal hippocampal neurons

Marks, J.D., Bindokas, V.P. and Zhang, X-M. Develop. Brain Res., 124, 101-116 (2001)   Neuronal vulnerability to excitotoxicity changes dramatically during postnatal maturation. To study the intracellular mechanisms by which maturation alters vulnerability in single neurons, we developed techniques to maintain hippocampal neurons from postnatal rats in vitro. After establishing their neuronal phenotype with immunohistochemistry and electrophysiology, we determined that these neurons exhibit developmentally regulated vulnerability to excitotoxicity. At 5 days in vitro, NMDA-induced cell death at 23 h increased from 3.6% in 3-day-old rats to > 90% in rats older than 21 days. Time-laps imaging of neuronal morphology following NMDA demonstrated differences in peak NMDA-induced [Ca²⁺] increases between neurons from younger and older animals. However, neurons from older animals were uniformly distinguished from those from younger animals by their subsequent loss of [Ca²⁺]_i homeostasis. Because of the role of  mitochondrial Ca²⁺ buffering in [Ca²⁺ ]_i homeostasis, we measured NMDA-induced changes in mitochondrial membrane potential (DY) as a function of postnatal age. NMDA markedly dissipated DY in neurons from mature rats, but minimally in those from younger rats. These data demonstrate that, in cultures of postnatal hippocampal neurons (a) vulnerability to excitotoxicity increases as a function of the postnatal age of the animal from which they were harvested, and (b) developmental regulation of vulnerability to NMDA occurs at the level of the mitochondria.  

4.74           Is islet transplantation a realistic therapy for the treatment of type 1 diabetes in the near future?

Stevens, R.B., Matsumoto, S. and Marsh, C. Clinical Diabetes, 19(2), 51-60 (2001)   Shapiro and colleagues recently reported a 100% cure rate for type 1 diabetes with their “Edmonton protocol” for islet transplantation. This unprecedented success has caused a groundswell of enthusiasm and an unparalleled effort to replicate their experience. It has also raised questions about the clinical reality of this therapy and sparked a dialog about which patients should benefit from receiving this scarce allocated resource. This article reviews the factors contributing to the Edmonton success and obstacles to immediate and long-term expansion of islet transplantation. The authors argue that use of the two-layered method of pancreas preservation will enable the Edmonton protocol to cure diabetes from single and marginal cadaveric donors. A concerted effort will be required to expedite routing of pancreases to islet processing centers and transplant programs. The long-term success and expansion of islet transplantation will depend on not only safer forms of immunosuppression, but also new sources of islet tissue.    

4.75           In vivo-matured Langerhans cells continue to take up and process native proteins unlike in vitro-matured counterparts

Ruedl, C., Koebel, P. and Karjalainen, K.

1. Immunol., 166(12), 7178-7182 (2001)

  We have been able to identify the cell subset derived from Langerhans cells in the total dendritic cell population of the peripheral lymph node and hence to follow their trafficking under normal physiological conditions as well as upon skin irritation. As expected, the rapid mobilization of Langerhans cells triggered by inflammatory signals into the draining lymph node correlated with an up-regulation of costimulatory molecules and with an enhanced immunostimulatory capacity. Surprisingly, however, these cells, instead of shutting down, maintain the capacity to capture and process protein. Ags during the couple of days they stay alive in stark contrasts to in vitro-matured dendritic cells.  

4.76           The effect of leukocyte-reduction method on the amount of human cytomegalovirus in blood products: a comparison of apheresis and filtration methods

Dumont, L.J. et al Blood, 97(11), 3640-3647 (2001)   This study examined the effectiveness of 3 leukocyte-reduction (LR) methods in depleting the residual level of cytomegalovirus (CMV) in blood products measured by quantitative polymerase chain reaction (QA-PCR). At 2 locations over 3 allergy seasons, apheresis platelets and whole blood were collected from 52 healthy CMV seropositive subjects having an elevated titer of CMV DNA (median = 2400 genome equivalents [GE]/mL) resulting in 32 evaluable LR apheresis platelets, 31 filtered platelets from whole blood, and 31 filtered red blood cells (RBCs) from whole blood. Leukoreduction by apheresis and filtration resulted in substantial reduction of detectable CMV DNA levels with 99.9% of the LR products expected to have less than 500 GE/mL of CMV DNA. No difference was found between methods (P = .52). CMV genomic leukocyte subset localization was determined by QA-PCR of fluorescence-activated cell sorter (FACS)-sorted peripheral blood from 20 seropositive subjects (n = 10 > 100 GE/mL, n = 10 QA-PCR negative). CMV was detected in monocyte (13 of 20) and granulocyte (3 of 20) fractions. Presence of competent virus in QA-PCR positive (> 100 GE/mL) peripheral blood samples was verified with 4 of 19 subjects positive in shell vial assay, and 8 of 18 positive for CMV gene products (messenger RNA). We observed a seasonal DNAemia variation in seropositive subjects. CMV seropositive subjects (n = 45) entered into longitudinal monitoring in March/April 1999 were QA-PCR negative at baseline. Subjects converted to a positive QA-PCR coincident with increased seasonal allergen levels (Norfolk 15 of 18 evaluable in 43.4 ± 9.48 days. Denver, 16 of 23 evaluable in 96 ± 26.3 days). These data demonstrate effective reduction of CMV load by LR during periods of DNAemia in CMV seropositive subjects.  

4.77           JunD regulates transcription of the tissue inhibitor of metalloproteinases-1 and interleukin-6 genes in activated hepatic stellate cells.

Smart, D. et al

1. Biol. Chem., 276(26), 24414-24421 (2001)

  Activation of hepatic stellate cells to a myofibroblast-like phenotype is the pivotal event in hepatic wound healing and fibrosis. Rat hepatic stellate cells activated in vitro express JunD, and FosB as the predominant AP-1 DNA binding proteins and all three associate with an AP-1 sequence that is essential for activity of the tissue inhibitor of metalloproteinases-1 (TIMP-1) promoter. In this study we used expression vectors for wild type, dominant negative and forced homodimeric (Jun/eb1 chimeric factors) forms of JunD and other Fos and Jun proteins to determine the requirement for JunD in the transcriptional regulation of the TIMP-1 and interleukin-6 (IL-6) genes. JunD activity was required for TIMP-1 gene promoter activity, while over-expression of Fra2 or FosB caused a repression of promoter activity. The ability of homodimeric JunD/eb1 to elevate TIMP-1 promoter activity supports a role for JunD homodimers as the major AP1 dependant trans-activators of the TIMP-1 gene. IL-6 promoter activity was induced with activation of hepatic stellate cells and also required JunD activity, however, expression of JunD/eb1 homodimers resulted in transcriptional repression. Mutagenesis of the IL-6 promoter showed that an AP-1 DNA binding site previously reported to be an activator of transcription in fibroblasts functions as a suppressor of promoter activity in hepatic stellate cells. We conclude that JunD activates IL-6 gene transcription as a heterodimer and operates at an alternative DNA binding site in the promoter. The relevance of these findings to events occurring in the injured liver was addressed by showing that AP-1 DNA binding complexes are induced during HSC activation and contain JunD as the predominant Jun family protein. JunD is therefore an important transcriptional regulator of genes responsive to Jun homo- and heterodimers in activated hepatic stellate cells.  

4.78           Phosphatidylinositol 3-kinase is necessary but not sufficient for thrombopoietin- induced proliferation in engineered Mpl-bearing cell lines as well as in primary megakaryocytic progenitors

Geddis, A.E., Fox, N.E. and Kaushansky, K.

1. Biol. Chem. 276(37), 34473-34479 (2001)

  Thrombopoietin and its receptor (Mpl) support survival and proliferation in megakaryocyte progenitors and in BaF3 cells engineered to stably express Mpl (BaF3/Mpl) (1). The binding of thrombopoietin to Mpl activates multiple kinase pathways, including the Jak/STAT, Ras/Raf/MAPK, and phosphatidylinositol 3-kinase, but it is not clear how these kinases promote cell cycling. Here we show that thrombopoietin induces phosphatidylinositol 3-kinase and that phosphatidylinositol 3-kinase is required for thrombopoietin-induced cell cycling in BaF33/Mpl cells and in primary megakaryocyte progenitors. Treatment of BaF3/Mpl cells and megakaryocytes with the phosphatidylinositol 3-kinase inhibitor LY294002 inhibited mitotic and endomitotic cell cycling. BaF3/Mpl cells treated with thrombopoietin and LY294002 showed both a G1 and a G2 cell cycle block. Expression of constitutively active Akt in BaF3/Mpl cells restored the ability of thrombopoietin to promote cell cycling in the presence of LY294002. Constitutively active Akt was not sufficient to drive proliferation of BaF3/Mpl cells in the absence of thrombopoietin. We conclude that in BaF3/Mpl cells and megakaryocyte progenitors, thrombopoietin-induced phosphatidylinositol 3-kinase activity is necessary but not sufficient for thrombopoietin-induced cell cycle progression. Phosphatidylinositol 3-kinase activity is likely involved in regulating the G1/S transition.  

4.79           Human antibodies isolated from plasma by affinity chromatography increase the coxsackievirus B4-induced synthesis of interferon-a by human peripheral blood mononuclear cells in vitro

Chehadeh, W., Bouzidi, A., Alm, G., WattrJ, P.and Hober, D.

1. Gen. Virol., 82, 1899-1907 (2001)

  Coxsackievirus B4 (CVB4) can be found in circulating blood of patients; however, the interaction of CVB4 with peripheral blood mononuclear cells (PBMCs) is poorly understood. CVB4 induced low levels of IFN-a synthesis in PBMCs from healthy donors. In contrast, preincubation of infectious CVB4 with plasma from these donors containing anti-CVB4 antibodies strongly enhanced the synthesis of IFN-a. IgG obtained from plasma by chromatography formed immune complexes with CVB4 and increased significantly the CVB4-induced production of IFN-a by PBMCs. These antibodies did not have a neutralizing effect on CVB4 infection of Hep-2 cells. The role of CVB and adenovirus receptor (CAR), FcgRII and FcgIII in the increased synthesis of IFN-a induced by CVB4 preincubated with IgG was shown by inhibition with specific antibodies. The major interferon-a-producing cells in response to CVB4-IgG complexes were CD14 cells and monocyte-enriched PBMCs. With the latter, detection of IFN-a by immunostaining was positive whereas in monocyte-depleted PBMCs it was not. This study shows that CVB4-induced synthesis of IFN-a by PBMCs can be enhanced by an antibody-dependent mechanism through interactions between the virus, non-neutralizing antivirus antibodies, FcgII and III and CAR.  

4.80           Neurohormonal regulation of secretion from isolated rat stomach ECL cells: a critical reappraisal

Lindstr`m, E. and Håkanson, R. Regulatory Peptides, 97, 169-180 (2001)   ECL cells are endocrine/paracrine cells in the oxyntic mucosa. They produce, store and secrete histamine and chromogranin A-derived peptides such as pancreastatin. The regulation of ECL-cell secretion has been studied by several groups using purified ECL cells, isolated from rat stomachs. Reports from different laboratories often disagree. The purpose of the present study was to re-evaluate the discrepancies by studying histamine (or pancreastatin) secretion from standardized preparations of pure, well-functioning ECL cells. Cells from rat oxyntic mucosa were dispersed by pronase digestion, purified by repeated counter-flow elutriation and subjected to density gradient centrifugation. The final preparation consisted of more than 90% ECL cells (verified by histamine and/or histidine decarboxylase immunochemistry). They were maintained in primary culture for 48 h before they were exposed to candidate stimulants and inhibitors for 30 min after which the medium was collected for determination of mobilized histamine (or pancreastatin). Gastrin-17 and sulphated cholecystokinin octapeptide (CCK-8s) raised histamine secretion 4-fold, the EC₅₀ for both peptides being around 100 pM. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP-27) (5-fold increase) and the related neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) (3-fold increase) mobilized histamine with similar potency (EC₅₀ ranging from 80 to 140 pM). Adrenaline, isoprenaline and terbutaline stimulated secretion by activating b₂ - receptor subtype, while acetylcholine and carbachol were without effect. Secretion experiments were invariably run in parallel with a gastrin standard curve. Somastatin, prostaglandin E₂ (PGE₂) and the PGE₁ congener misoprostol inhibited PACAP- and gastrin-stimulated secretion by more than 90%, with IC₅₀ values ranging from 90-720 (somatostatin) to 40-200 (misoprostol) pM. The neuropeptide galanin inhibited secretion by 60-70% with a potency similar to that of somatostatin. Proposed inhibitors such as peptide YY, neuropeptide Y and the cytokines interleukinb1- and tumor necrosis factora induced at best a moderate inhibition of gastrin- or PACAP-stimulated secretion at high concentration, while calcitonin gene-related peptide, pancreatic polypeptide and histamine itself were without effect. Inhibition of gastrin- or PACAP-stimulated secretion was routinely compared to a somatostatin standard curve. In conclusion, gastrin, PACAP, VIP/PHI and adrenaline stimulated secretion. Somatostatin and PGE₂ were powerful inhibitors of both gastrin- and PACAP-stimulated secretion; although equally potent, galanin was less effective that somatostatin and PGE₂.  

4.81           An investigation into the suitability of silica beads for cell separations based on density perturbation

Bildirici, L. and Rickwood, D.

1. Immunol. Methods, 252, 57-62 (2001)

  This study has investigated possible alternative types of beads for fractionating cells on the basis of density perturbation. It is well known that uniform magnetic beads can be extremely important tools for separating cells by both magnetic separation techniques and density perturbation. However, because of the inherent expense associated with the use of magnetic beads, it was decided to study the possible use of inexpensive silica beads for density perturbation in terms of their attachment and modification of density of cells and to compare them with uniform Dynabeads. Silica beads were analyzed to determine their size and effect on the density of cells. Differentiated HL60 cells were used as model system. As differentiation occurs, different levels of antigens are expressed on the cell surface and this results in different numbers of beads binding to cells. DMSO-differentiated HL60 cells were mixed with anti-CD11b-coated beads at a ratio of 20:1 (beads/cell), and gentle mixing was carried out at 20°C on the end-over-end mixer. The binding of antibody-coated silica beads and Dynabeads to partially differentiated HL60 cells were compared. The conclusions reached on the basis of these experiments are that antibody-coated silica beads (Ab-coated silica) can be used as alternative beads for some cell fractionations. However, compared with Dynabeads, there are more beads that are only transiently associated with cells, possibly indicating that higher levels of detachment of beads from cells occur when silica beads are used. In addition, silica beads are usually heterogeneous in size and this would make it difficult to use these beads for the isolation of purified subpopulations of differentiated cells.  

4.82           Culture and regeneration of human neurons after brain surgery

Brewer, G.J. et al J, Neurosci Meth., 107, 15-23 (2001)   Cortical human brain tissue was obtained from 11 craniotomies for intractable epilepsy or tumor resection. Neuregen transport medium preserved viability at 4⁰C during transfer to the culture laboratory. Cells were isolated and cultured by methods previously developed for adult rat neurons (Brewer G.J. Isolation and culture of adult rat hippocampal neurons. J. Neurosci. Meth. 1997: 71: 143-155). In about 40% of the cases, cultures regenerated with a majority of neuron-like cells that stained for neurofilament and not GFAP. After 3 weeks of culture from a 70-year-old meningioma case, synapse-like structures were revealed by electron microscopy. Trophic support from basic human recombinant fibroblast growth was synergistically improved with the steroid hormone dehydroepiandrosterone 3-sulfate. Another 40% of the cases resulted in cultures that were predominantly GFAP positive astroglia. The remaining 20% of the cases did not regenerate cells with neuron-like or glial processes. Three postmortem cases did not regenerate neurites. These methods may aid development of human culture models of epilepsy as well as human pharmacology, toxicology and development of improved methods for brain grafts.  

4.83           In vitro culture and synchronous release of Sarcocystis neurona merozoites from host cells

Ellison, S.P., Greiner, E. and Dame, J.B. Vet. Parasitol., 95, 251-261 (2001)   The growth of Sarcocystic neurona, isolate UCD1, in continuous culture was examined in 10 cell lines to identify growth conditions and methods for the preparation of parasites free of gross host cell contamination for molecular studies. The unpredictable, slow release of merozoites in most cell lines prompted development of a method to synchronously release the parasites from infected host cells. The calcium ionophore A23187 at a concentration of 1 mM was found to release intracellular merozoites with a 40 min treatment at 37°C. The release of merozoites en masse from attached host cells allowed for the rapid collection of relatively pure parasites from the culture supernatant. This release of merozoites occurred in five different host cell lines. The ionophore-released parasites were highly infectious for host cells and appeared to be morphologically identical to naturally released merozoites, except that the treated merozoites had an increased number of micronemes when examined by electron microscopy. The ionophore did not enhance the release of sporozoites from sporocysts, but freezing in the presence of 5% DMSO released sporozoites that were infectious to bovine monocytes in in vitro culture.  

4.84           In vitro co-incubation of pig islet cells with xenogeneic human blood mononuclear cells causes loss of insulin release during perifusion: involvement of non-T-cell- and T-cell-mediated mechanism

Lalain, S., ClJmenceau, B., Gouin, E. and Sai, P. Human Immunol., 62, 607-614 (2001)   Because the different steps of the human cellular immune rejection of pig islets are still poorly understood, our previous work concerned the intensity and mechanisms of the proliferation of human peripheral mononuclear cells (PBMC) to adult pig islet cells (PIC). As lymphocyte proliferation is not indicative of alteration of PIC, the present in vitro study evaluated cell-mediated immune effectors possible involved in impairment of adult PIC. A test was thus developed, based on perfusion analysis of the alteration of insulin release from PIC incubated with different human cells. Compared to PIC incubated alone or with autologous pig splenocytes, seven-day co-incubation with whole human peripheral blood mononuclear cells (PBMC) (n= 18) led to almost complete abolition of basal and stimulated insulin releases (p < 0.0001). This effect could not be reversed by extensive sequential washes before perifusion of PIC, and the number of PIC was decreased by 78% after seven-day co-incubation with PBMC. PBMC are a complex mixture of cells involved in different xenogeneic mechanisms, and two components of this PIC impairment were then detected separately. First, the effect of PBMC against PIC was decreased (p< 0.0001) after removal of either MHC class II+ or CD14+ cells from PBMC. On the contrary, decreasing effect (p < 0.001) on insulin secretion was observed when only plastic-adherent or CD14+ cells were co-incubated with PIC. Additionally, alteration of insulin release from PIC cultured with PBMC or plastic-adherent cells was abolished dose-dependently (p < 0.0001 and p < 0.04, respectively) by gadolinium chloride (which inhibits macrophages), but not modified by cyclosporin A or mycophenolate mofetil which did not alter insulin release from PIC but blocked the proliferation of PBMC against PIC. A second mechanism was also detected, since co-incubation of PIC with purified human T-cells remixed with antigen-presenting cells led to a decrease (p < 0.0001) of insulin release. This model based on the alteration of dynamic basal and stimulated insulin secretion provides detailed account of in vitro human cell-mediated impairment of PIC. It shows that the xenogeneic effect of whole mononuclear cells were strong and rapid. A crucial role was played by MHC class II+, CD14+, and plastic-adherent cells. Two mechanisms appear to be responsible for the role of these cells: 1) early direct effect, potentially involved in vivo in primary nonfunction of islets aggressed by monocytes/macrophages; and 2) the presentation of PIC xenoantigens leading to impairment by T lymphocytes, which may be involved in in vivo specific cellular rejection.  

4.85           Isolated rat stomach ECL cells generate prostaglandin E₂ in response to interleukin-1b, tumor necrosis factor-a and bradykinin

Lindström, E., Lerner, U.H. and Håkanson, R. Eur. J. Pharmacol., 416, 255-263 (2001)   The ECL cells control parietal cells by releasing histamine in their immediate vicinity. Gastrin and pituitary adenylate-cyclase-activating peptide (PACAP) stimulate histamine secretion from isolated ECL cells, while somatostatin and galanin inhibit stimulated secretion. Prostaglandin E₂ and related prostaglandins likewise suppress ECL-cell histamine secretion. Conceivably, that is how they inhibit acid secretion. In the present study, we examined if prostaglandin E₂ can be generated by isolated ECL cells. Rat stomach ECL cells were purified (> 90% purity) by counterflow elutriation and gradient centrifugation and cultured for 48 h. ECL cell stimulants  (gastrin and PACAP) and inflammatory agents (interleukin-1b, tumor necrosis factor-a and bradykinin) were tested for their ability to induce prostaglandin E₂ accumulation (24-h incubation), measured by radioimmunoassay. Gastrin and PACAP did not affect prostaglandin E₂ accumulation but interleukin-1b (300 pg/ml), tumor necrosis factor-a (10 ng/ml) and bradykinin (1 mM) induced a 2-fold to 3-fold increase in the amount of prostaglandin E₂ accumulated. While the combination of interleukin-1b and bradykinin induced a 9-fold increase, the combination interleukin-1b + tumor necrosis factor-a and bradykinin + tumor necrosis factor-a induced additive effects only. The combination of interleukin-1b + tumor necrosis factor-a + bradykinin did not induce a greater effect than interleukin-1b + bradykinin. The effect of interleukin-1b + bradykinin was abolished by adding 10 nM hydrocortisone (suppressing phospholipase A₂ and cyclooxygenase) or 1 mM indomethacin (inhibiting cyclooxygenase). Incubating ECL cells in the presence of interleukin-1b + bradykinin for 24 h reduced their ability to secrete histamine in response to gastrin. The inhibitory effect was reversed by 1 mM indomethacin. Also, increasing the concentration of hydrocortisone in the medium resulted in an enhanced gastrin-stimulated histamine secretion. Hence, the previously described acid-inhibiting effect of inflamm-atory agents may be explained by inhibition of ECL-cell histamine mobilization, consequent to enhanced formation of prostaglandin E₂ by cells in the oxyntic mucosa, including the ECL cells themselves.  

4.86           Neuregulins increase a7 nicotinic acetylcholine receptors and enhance excitatory synaptic transmission in GABAergic interneurons of the hippocampus

Liu, Y., Ford, B., Mann, M.A. and Fischbach, G.D.

1. Neurosci., 21, 5660-5669 (2001)

  Neuregulins are highly expressed in the CNS, especially in the cholinergic neurons. We have examined the effect of neuregulin on nicotinic acetylcholine receptors (nAChRs) in neurons dissociated from the rat hippocampus. Rapid application of acetylcholine (ACh) induced a rapidly rising and decaying inward current in some of the neurons, which was completely blocked by methyllycaconitine, a specific antagonist of the a7 subunit of the nAChR. When the cells were treated with 5 nM neuregulin (NRG1-b1) for 2-4 d, a twofold increase in amplitude of the peak ACh-induced current was observed, and there was a comparable increase in ¹²⁵I-a-bungarotoxin binding. The fast ACh-induced peak current was prominent in large neurons that also contained GABA immunoreactivity. These presumptive GABAergic neurons constituted ~ 10% of neurons present in 7-to-9-d-old cultures. In addition to the large inward peak current, ACh also evoked transmitter release from presynaptic nerve terminals. Pharmacologic experiments indicated that the shower of PSCs was mediated by glutamate, with a small minority caused by the action of GABA. Chronic exposure to NRG1-b1 increased the amplitude of ACh-evoked PSCs but not the minimum “quantal” PSC. NRG1-b1 also increased the percentage of neurons that exhibited ACh-evoked PSCs.  

4.87           The peroxisome proliferator-activated receptor delta promotes lipid accumulation in human macrophages

Vosper, H et al

1. Biol. Chem. 276, 44258-44265 (2001)

  The Peroxisome Proliferator Activated Receptors (PPARs) are a family of fatty acid-activated transcription factors which control lipid homeostasis and cellular differentiation. PPARa (NR1C1) controls lipid oxidation and clearance in hepatocytes and PPARg (NR1C3) promotes preadipocyte differentiation and lipogenesis. Drugs that activate PPARa are effective in lowering plasma levels of lipids and have been used in the management of hyperlipidaemia. PPARg agonists increase insulin sensitivity and are used in the management of type 2 diabetes. In contrast, there are no marketed drugs that selectively target PPARd (NR1C2) and the physiological roles of PPARd are unclear. In this report we demonstrate that the expression of PPARd is increased during the differentiation of human macrophages in vitro. In addition, a highly selective agonist of PPARd (compound F) promotes lipid accumulation in primary human macrophages and in macrophages derived from the human monocytic cell line, THP-1. Compound F increases the expression of genes involved in lipid uptake and storage such as the class A and B scavenger receptors (SRA, CD36) and adipophilin. PPARd activation also represses key genes involved in lipid metabolism and efflux i.e. cholesterol 27-hydroxylase and apolipoprotein E. We have generated THP-1 sub-lines that overexpress PPARd and have confirmed that PPARd is a powerful promoter of macrophage lipid accumulation. These data suggest that PPARd may play a role in the pathology of diseases associated with lipid-filled macrophages, such as atherosclerosis, arthritis, and neurodegeneration.  

4.88           Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene

Waldburger, J-M., Suter, T., Fontana, A., Acha-Orbea, H. and Reith, W.

1. Exp. Med., 194(4), 393-406 (2001)

  MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-g-induced MHCII expression on a wide variety of non-bone-marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4+  T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-g-activated cells of the macrophage lineage. PIV-/- mice thus allowed precise definition of CTIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.  

4.89           Maturation of dendritic cells is accompanied by rapid transcriptional silencing of class II transactivator (CIITA) expression

Landmann, S. et al

1. Exp. Med., 194(4), 379-391 (2001)

  Cell surface expression of major histocompatibility complex class II (MHCII) molecules is increased during the maturation of dendritic cells (DCs). This enhances their ability to present antigen and activate naive CD4⁺ T cells. In contrast to increased cell surface MHCII expression, de novo biosynthesis of MHCII mRNA is turned off during DC maturation. We show here that this is due to a remarkably rapid reduction in the synthesis of class II transactivator (CIITA) mRNA and protein. This reduction in CIITA expression occurs in human monocyte-derived DCs and mouse bone marrow-derived DCs, and is triggered by a variety of different maturation stimuli, including lipopolysaccharide, tumor necrosis factor a, CD40 ligand, interferon a, and infection with Salmonella typhimurium or Sendai virus. It is also observed in vivo in splenic DCs in acute myelin oligodendrocytes glycoprotein induced experimental autoimmune encephalitis. The arrest in CIITA expression is the result of a transcriptional inactivation of the MHC2TA gene. This is mediated by a global repression mechanism implicating histone deacylation over a large domain spanning the entire MHC2TA regulatory region.  

4.90           Migratory activity and functional changes of green fluorescent effector cells before and during experimental autoimmune encephalomyelitis

Flugel, A. et al Immunity, 14, 547-560 (2001)   Homing behavior and function of autoimmune CD4⁺ T cells in vivo was analyzed before and during EAE, using MBP-specific T cells retrovirally engineered to express the gene of green fluorescent protein. The cells migrate from parathymic lymph nodes to blood and to the spleen. Preceding disease onset, large numbers of effector cells invade the CNS, with only negligible numbers left in the periphery. In early EAE, most (>90%) infiltrating CD4⁺ cells were effector cells. Migratory effector cells downregulate activation markers (CD25, OX-40) but upregulate several chemokine receptors and absorb MHC class II on their membranes. Within the CNS, the effector cells are reactivated, with upregulated proinflammatory cytokines and downmodulated T cell receptor-associated structures, presumably reflecting autoantigen recognition in situ.  

4.91           Characterization of virus-mediated inhibition of mixed chimerism and allospecific tolerance

Williams, M.A. et al

1. Immunol., 167, 4987-4995 (2001)

  Simultaneous blockade of the CD28 and CD40 T cell costimulatory pathways has been shown to effectively promote skin allograft survival in mice. Furthermore, blockade of one or both of these pathways has played a central role in the development of strategies to induce mixed hematopoietic chimerism and allospecific tolerance. It has recently been observed that the beneficial effects of CD40 blockade and donor splenocytes in prolonging skin graft survival can be abrogated by some viral infection, including lymphocytic choriomeningitis virus (LCMV). In this study, we shown that LCMV infection prevents allograft survival following CD28/CD40 combined blockade. We further show that LCMV prevents the induction of allospecific tolerance mixed hematopoietic chimerism, while delay of infection for 3-4 wk posttransplant has no effect on tolerance induction. Because of reports of anti-H2^d activity following LCMV infection, we assayed the ability of LCMV-specific T cells to respond to alloantigen at a single cell level. Although we confirm that LCMV infection induces the generation of alloreactive cells, we also demonstrate that LCMV-specific T cells do not divide in response to alloantigen. The alloresponse suppressed by costimulation blockade is restored by LCMV infection and correlates with increased dendritic cell maturation. We hypothesize that the costimulation blockade-resistant rejection mediated by LCMV could be partly attributable to the up-regulation of alternative co-stimulatory pathways subsequent to LCMV-induced dendritic cell maturation.  

4.92           Regulation of E-box DNA binding during in vivo and in vitro activation of rat and human hepatic stellate cells

Vincent, K.J. et al Gut, 49, 713-719 (2001)   Background – Activation of hepatic stellate cells (HSCs) to a myofibroblastic phenotype is a key event in liver fibrosis. Identification of transcription factors with activities that are modulated during HSC activation will improve our understanding of the molecular events controlling HSC activation. Aims – To determine if changes in E-box DNA binding activity occur during in vitro and in vivo activation of rat and human HSCs and to investigate mechanisms underlying any observed changes. Methods – Nuclear extracts were prepared from rat HSCs isolated and cultured from normal and carbon tetrachloride injured rat livers and from HSCs isolated from human liver. EMSA analysis of E-box DNA binding activity was performed on nuclear extracts to determine changes during HSC activation. Western and northern blot analysis of MyoD and Id1 basic helix-loop-helix (bHLH) protein was performed to confirm expression in HSC. Results – HSC activation was associated with inducible expression of two low mobility E-box binding complexes that were immunoreactive with an anti-MyoD antibody. MyoD and mRNA expression was found at similar levels in freshly isolated and activated HSCs. Activation of rat HSCs was accompanied by reduced expression of the inhibitory bHLH protein Id1. Conclusions – In vitro and in vivo activation of rat and human HSCs is accompanied by induction of MyoD binding to E-box DNA sequences which appears to be mechanistically associated with elevated MyoD protein expression and reduced expression of the inhibitory Id1 protein. Clarification of the role of MyoD and Id1 proteins in HSC activation and liver fibrogenesis is now required.  

4.93           Human and rat hepatic stellate cells express neurotrophins and neurotrophin receptors

Cassiman, D., Denef, C., Desmet, V.J. and Roskams, T. Hepatology, 33, 148-158 (2001)   The expression of neurotrophins and neurotrophin receptors in non-neural tissue is related to tissue remodeling, differentiation, proliferation and migration of target cells. The literature yields contradictory results on neurotrophin and neurotrophin receptor expression in the liver. We show immunoreactivity to antibodies to nerve growth factor (NGF), brain-derived neurotrophin (BDNF), neurotrophin 3 (NT-3), neurotrophin 4/5 (NT-4/5), the low-affinity nerve growth factor receptor p75 and the high-affinity tyrosine kinase receptors (Trk) B and C in hepatic stellate cells and weak reactivity for BDNF, NT-3, and NT-4/5 in hepatocytes, in cryosections of human and rat liver, in normal and varying pathologic conditions. Immunoreactivity is unequivocally localized to hepatic stellate cells by double staining with a-smooth muscle actin (a-SMA) and desmin, studied by confocal laser scanning microscopy. Finally, the presence of mRNA transcripts for the different neurotrophins and neurotrophin receptors, with the exception of Trk-B, is shown by reverse transcription polymerase chain reaction (RT-PCR) on RNA extracted from freshly isolated rat hepatic stellate cells, compared with hepatocyte RNA. Hepatocyte RNA was found to contain BDNF, NT-3, NT-4/5 mRNA (which is compatible with the immunohistochemical findings) and Trk-A mRNA. In conclusion, hepatic stellate cells are a source of several neurotrophins in the liver and they express neurotrophin receptors. These findings correspond with the known involvement of hepatic stellate cells in tissue remodeling, their production of extracellular matrix components and their proliferation in acute necrotizing liver pathology. In analogy with findings in other organs and systems, neurotrophins are hypothesized to play a role in the pathophysiology of liver disease.  

4.94           Differential regulation of transendothelial migration of THP-1 cells by ICAM-1/LFA-1 and VCAM-1/VLA-4

Ronald, J.A., Ionescu, C.V., Rogers, K.A. and Sandig, M.

1. Leukoc. Biol., 70, 601-609 (2001)

  The adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expressed in atherogenic lesions are thought to regulate monocyte diapedesis. To better understand their specific roles we used function-blocking antibodies and examined in a culture model the morphology, motility, and diapedesis of THP-1 cells interacting with human coronary artery endothelial cells. The number of motile THP-1 cells was reduced only when VCAM-1 or both ICAM-1 and VCAM-1 were blocked. Blockade of ICAM-1 and VCAM-1, either separately or together, reduced to the same degree the distance that THP-1 cells traveled. Diapedesis was reduced only during the simultaneous blockade of both adhesion molecules. Blockade of either ICAM-1 or VCAM-1 inhibited pseudopodia formation, but ICAM-1 blockade induced the formation of filopodial. We suggest that the interactions of endothelial ICAM-1 and VCAM-1 with their ligands differentially regulate distinct steps of diapedesis by modulating the ratio of active and inactive forms of small GTPases such as Rho, Rac, and Cdc42.  

4.95           Effect of 6-hydroxydopamine on host resistance against Listeria monocytogenes infection

Miura, T et al Infection and Immunity, 69(12), 7234-7241 (2001)   Recent studies have shown that immunocompetent cells bear receptors of neuropeptides and neurotransmitters and that these ligands play roles in the immune response. In this study, the role of the sympathetic nervous system in host resistance against Listeria monocytogenes infection was investigated in mice pretreated with 6-hydroxydopamine (6-OHDA), which destroys sympathetic nerve termini. The norepinephrine contents of the plasma and spleens were significantly lower in 6-OHDA-treated mice than in vehicle-treated mice. The 50% lethal dose of L. monocytogenes was about 20 times higher for 6-OHDA-treated mice than for vehicle-treated mice. Chemical sympathectomy by 6-OHDA upregulated interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-a) production in enriched dendritic cell cultures and gamma interferon (IFNg) and TNF-a production in spleen cell cultures, whereas chemical sympathectomy had no apparent effect on phagocytic activities, listericidal activities, and nitric oxide production in peritoneal exudates cells and splenic macrophages. Augmentation of host resistance against L. monocytogenes infection by 6-OHDA was abrogated in IFN-g^-/- or TNF-a^-/- mice suggesting that upregulation of IFNg, IL-12, and TNF-a production may be involved in 6-OHDA-mediated augmentation of antilisterial resistance. Furthermore, adoptive transfer of spleen cells immune to L. monocytogenes from 6-OHDA-treated mice resulted in untreated naive recipients that had a high level of resistance against L. monocytogenes infection. These results suggest that the sympathetic nervous system may modulate host resistance against L. monocytogenes infection through regulation of production of IFN-g, IL-12, and TNF-a, which are critical in antilisterial resistance.  

4.96           Differential regulation of calcitonin secretion in normal and neoplastic pulmonary neuroendocrine cells in vitro

Pu, F.R., Manning, F.C.R., Brannigan, A.E. and Crosby, S.R. Exp. Lung Res., 27, 689-703 (2001)   Within the mammalian lung, cells with a neuroendocrine phenotype are few in number and are sparsely distributed. In contrast, neuroendocrine neoplasms represent a major group of lung cancers. The aim of this study was to develop a model of mammalian PNECs and to compare glucocorticoid regulation of calcitonin secretion in normal and neoplastic cells with neuroendocrine differentiation. Cell cultures of PNECs were initiated after the disaggregation of neonatal hamster lungs with 0.1% collagenase and fractionation of the resultant cell suspension on a gradient of iodixanol (1.320 g/ml). Cell fractions enriched in PNECs were identified by positive staining for 5-hydroxytryptamine and the presence of calcitonin. Calcitonin secretion was investigated after exposure to hydrocortisone (0 to 1000 nM). A dose-dependent inhibition of calcitonin secretion was seen after 7 days between 10 nM (55% of control), and 1000 nM (29%) hydrocortisone. Cell cultures grown in the presence of hydrocortisone also contained significantly fewer PNECs between 10 nM (90% of control), and 1000 nM (45%). Human bronchial carcinoid cells (NCIH727) cultured under identical conditions showed a similar inhibition of calcitonin secretion between 10 nM (53%) and 1000 nM (52%), although at these concentrations, no reduction in cell number was seen. In contrast, 2 human small cell lung cancer cell lines (DMS-79 and COR-L24 cells) showed no dose-dependent inhibition of calcitonin secretion and no effect on cell proliferation in response to hydrocortisone. These results show that enriched cultures of mammalian PNECs can be used to investigate functional aspects of their biology, including peptide secretion in response to potential regulators. Furthermore, calcitonin secretion is inhibited in normal PNECs and bronchial carcinoid cells at physiological concentrations of glucocorticoids, but this feature appears not to be present in the 2 more invasive neuroendocrine neoplasms (small cell lung cancer cells) investigated in this study.  

4.97           A population of PC12 cells that is initiating apoptosis can be rescued by nerve growth factor

Francois, F., Godinho, M.J., Dragunow, M. and Grimes, M.L. Mol. Cell. Neurosci., 18, 347-362 (2001)   Programmed cell death, or apoptosis, occurs asynchronously in neuronal cells. To overcome this asynchrony, rat pheochromocytoma (PC12) cells were separated at different stages of apoptosis on the basis of cell density. Live cells that exhibited no apoptotic features floated to the top of density gradients. The most dense cells showed extensive loss of cytochrome C from mitochondria, caspase activation, chromatin condensation, and DNA fragmentation. These cells were committed to apoptosis and could not be rescued by reculturing in with nerve growth factor (NGF). Cells of intermediate density displayed no DNA fragmentation, but had begun to show cytochrome C loss, caspase activation, and chromatin condensation. This population displayed upregulation of the prodeath factor, c-Jun, and downregulation of prosurvival kinase, Akt. Importantly, apoptosis was reversible by NGF in this population. These studies suggest that increased cell density correlates with an initial step in the apoptosis mechanism that precedes irreversible commitment to suicide.  

4.98           Age-related differences in NMDA responses in cultured rat hippocampal neurons

Cady, C., Evans, M.S. and Brewer, G.J. Brain Res., 921, 1-11 (2001)  

The N-methyl-D-aspartate receptor (NMDAR) is expressed in the cerebral cortex and hippocampus and is important in learning and memory. NMDARs are influenced by aging and implicated in neurodegenerative disorders. We investigated age-related differences in NMDAR ionic currents and intracellular calcium in embryonic (E18), middle-age (9-10 months) and old (26 months) rat hippocampal neurons cultured in serum-free medium for 7-12 days. Responses to 200 uM NMDA with 50 uM glycine were measured using whole cell voltage clamp and fura-2 fluorescence. Embryonic neurons exhibited significantly larger and faster NMDA responses than adults. Old rats had 1.5 fold greater normalized NMDA peak current compared to middle-age rats, while intracellular calcium rose 1.3 fold higher. Differences in regression slopes generated from the integral of NMDA current versus normalized NMDA current indicate age-related differences are not exclusively due to changes in receptor density but likely influenced by changes in receptor function. Corresponding age-related measures of intracellular calcium by fura-2 fluorescence in response to NMDA showed a strong correlation with peak current (r₂ = 0.996). Our data support the hypothesis that NMDAR responsiveness is altered during aging with an enhanced NMDA peak current in both old and embryonic neurons compared to middle-age neurons.

 

4.99           Highly efficient isolation of porcine islets of Langerhans for xenotransplantation: numbers, purity, yield and in vitro function

Krickhahn, M., Meyer, T., Buchler, C., Thiede, A. and Ulrichs, K. Ann. Transplant., 6(3), 48-54 (2001)   Xenogeneic transplantation of porcine islets of Langerhans is regarded as a future treatment for diabetes mellitus. Despite considerable biotechnological progress, however, it is still very difficult and often unreliable to isolate sufficient numbers of highly purified, intact islets from the porcine pancreas with good in vitro function. OBJECTIVE: Of this study was to describe an efficient and reliable method to isolate sufficient numbers of highly purified islets of Langerhans with good in vitro function from adult as well as from young hybrid pigs. METHODS: Islets were isolated from the pancreas of young (4-6 months) hybrid pigs and old (2-3 years) retired breeders using Liberase PI and digestion-filtration. Average islet size was detected by dithizone staining of tissue sections prior to isolation; only organs with an average islet size > or = 200 micron were used. Density gradient purification with OptiPrep was performed in a COBE 2991 cell processor. Viability was investigated using fluorescence staining. Perifusion studies were carried out to assess in vitro function of isolated islets. RESULTS: Islets were successfully isolated from young hybrid pigs (3,671 ± 598 IEQ/g) and old retired breeders (5,182 ± 545 IEQ/g). After purification islet purity was 92% for retired breeders and 87% for young hybrid pigs. Yield after purification was still not satisfactory: 64% for retired breeders  (3,209 ± 444 IEQ/g) and 44% for young hybrid pigs (1,669 ± 386 IEQ/g). Viability of isolated islets was 80-95%. Perifusion studies of porcine islets showed sufficient insulin release upon glucose challenge; however, the level of insulin release depended on the density of islets within the perifusion chamber. Low temperature culture (24°C) prior to perifusion studies had no detrimental effect on insulin release. Long-term culture over 11 days was followed by a dramatic loss of islet function. CONCLUSION: If xenograft rejection can be overcome and the risk of xenosis can be minimised, sufficient numbers of purified porcine islets with good in vitro function can be isolated to serve as a potential source for islet transplantation in diabetic patients.  

4.100           Septic shock and acute lung injury in rabbits with peritonitis

Matute-Bello, G. et al Am. J. Respir. Crit. Care Med., 163, 234-243 (2001)   The major goal of this study was to investigate the mechanisms that link the host response to a local infection in the peritoneal cavity with the development of sepsis and lung injury. Rabbits were infected by intraperitoneal inoculation of fibrin clots containing E.coli cells at 10⁸, 10⁹, or 10¹⁰ cfu/clot. Physiologic, bacteriologic, and inflammatory responses were monitored, and the lungs were examined postmortem. At a dose of 10⁸ cfu/clot the animals had resolving infection, and a dose of 10⁹ cfu/clot resulted in persistent infection at 24h, with minimal systemic manifestations. In contrast, inoculation at 10¹⁰ cfu/clot resulted in rapidly lethal local infection, with septic shock and lung injury. The onset of septic shock was associated with a paradoxical lack of identifiable polymorphonuclear leukocytes (PMN; neutrophils) in the peritoneal cavity. The absence of PMN in the peritoneum was due in part to lysis of intraperitoneal PMN, because the peritoneal fluids contained free myeloperoxidase and induced rapid death of normal rabbit PMN in vitro. Although most animals became bacteremic, only those with a severe systemic inflammation response developed lung injury. These data show that control of an infection in the first compartment in which bacteria enter the host is a critical determinant of the systemic response. Above a threshold dose of bacteria, failure of the local neutrophil response is a key mechanism associated with deleterious systemic responses. Bacteremia alone is not sufficient to cause lung injury. Lung injury occurs only in the setting of a severe systemic inflammatory response and an inadequate leukocyte response at the primary site of infection.  

4.101           Gravity sensing in moss protonemata

Sack, F.D., Schwuchow, J.M., Wagner, T. and Kern, V. Adv. Space Res., 27(5), 871-876 (2001)   Moss protonemata are a valuable system for studying gravitropism because both sensing and upward curvature (oriented tip growth) take place in the same cell. We review existing evidence, especially for Ceratodon purpureus, that addresses whether the mass that functions in sensing is that of amyloplasts that sediment. Recent experiments show that gravitropism can take place in media that are denser than the apical cell. This indicates that gravity sensing relies on an intracellular mass rather than that of the entire cell and provides further support for the starch-statolith hypothesis of sensing. Possible mechanisms for how amyloplast mass functions in sensing and transduction are discussed.  

4.102           Origin and differentiation of dendritic cells

Ardivin, C. et al. TRENDS Immunol., 22(12), 691-700 (2001)   Despite extensive, recent research on the development of dendritic cells (DCs), their origin is a controversial issue in immunology, with important implications regarding their use in cancer immunotherapy Although, under defined experimental conditions, DCs can be generated from myeloid or lymphoid precursors, the differentiation pathways that generate DCs in vivo remain unknown largely. Indeed, experimental results suggest that the in vivo differentiation of a particular DC subpopulation could be unrelated to its possible experimental generation. Nevertheless, the analysis of DC differentiation by in vivo and in vitro experimental systems could provide important insights into the control of the physiological development of DCs and constitutes the basis of a model of common DC differentiation that we propose.  

4.103           Preptin derived from proinsulin growth factor II (preIGF-II) is secreted from pancreatic islet b-cells and enhances insulin secretion

Buchanan, C.M., Phillips, A.R.J. and Cooper, G.J.S. Biochem. J., 360, 431-439 (2001)   Pancreatic islet b-cells secrete the hormones insulin, amylin and pancreastatin. To search for further b-cell hormones, we purified peptides from secretory granules isolated from cultured murine bTC6-F7 b-cells. We identified a 34-amino-acid peptide (3948 Da), corresponding to Asp⁶⁹ –Leu¹⁰² of the proinsulin-like growth factor II E-peptide, which we have termed ‘preptin’. Preptin is present in islet b-cells and undergoes glucose-mediated co-secretion with insulin. Synthetic preptin increases insulin secretion from glucose-stimulated bTC6-F7 cells in a concentration-dependent and saturable manner. Preptin infusion into the isolated, perfused rat pancreas increases the second phase of glucose-mediated insulin secretion by 30% while anti­preptin immunoglobulin infusion decreases the first and second phases of insulin secretion by 29 and 26% respectively. These findings suggest that preptin is a physiological amplifier of glucose-mediated insulin secretion.  

4.104           Low-speed isopycnic islet separation is effeictive and yields islets with superior quantity and quality

Sageshima, S.S. et al IPITA 2001 abstracts P3-06 (2001)   Background: The inconsistency of islet processing remains the major obstacle to the application of islet transplantation. This study addresses the impact of centrifugal force (CF) applied during isopycnic islet separation. Method: Islets were purified in an alternate fashion using low (100G) and standard (800 G) CF on a Cobe2991 (n=18). Continuous iodixanol gradients with low viscosity were used in both groups. The low and standard CF-purified islet were compared with respect to islet enumeration, purity, islet equivalent/islet count (IE/IC), and glucose-stimulated insulin release in vitro. The posttransplant function of low CF-purified islets was evaluated in diabetic pigs using the small bowel intramuscular implantation site. Results: The %recovery and purity of low CF-purified islets were significantly higher compared to islets separated at standard CF (118 vs. 100%, 90.3 vs. 88.8%). The insulin secretory response to glucose was higher for islets separated at low vs. standard CF (2.2 vs. 1.2). At standard CF, the IE/IC after purification was significantly lower compared to that before purification (0.73 vs. 0.84), which is suggestive of islet fragmentation. In contrast, there was no significant difference in IE/IC before and after islet separation at low CF (0.73 vs. 0.73). Low CF-purified islets consistently reversed diabetes in the pig allotransplant model with a follow-up greater than 30days in all immunosuppressed pigs. Conclusions: Low CF-purified islet separation yields islets with superior results compared to standard method with respect to both islet recovery and quality. Low CF islet separation represents an improvement and warrants evaluation for human islet separation.  

4.105           Human islet cell transplantation – future prospects

White, S.A., James, R.F.L., Swift, S.M., Kimber, R.M. and Nicholson, M.L. Diabetic Med., 18, 78-103 (2001)   Background Islet transplantation has the potential to cure diabetes mellitus. Nevertheless despite successful reversal of diabetes in many small animal models, the clinical situation has been far more challenging. The aim of this review is to discuss why insulin-independence after islet allotransplantation has been so difficult to achieve.   Methods A literature review was undertaken using Medline from 1975 to July 2000. Results reported to the International Islet Transplant Registry (ITR) up to December 1998 were also analysed.   Results Up to December 1998, 405 islet allotransplants have been reported the ITR. Of those accurately documented between 1990 and 1998 (n = 267) only 12% have achieved insulin-independence (greater than 7 days). However with refined pen-transplant protocols insulin independence at 1 year can reach 20%.   Conclusions There are many factors which can explain the failure of achieving insulin-independence after islet allotransplantation. These include the use of diabetogenic immunosuppressive agents to abrogate both islet allo-immunity and auto-immunity, the critical islet mass to achieve insulin-independence and the detrimental effects of transplanting islets in an ectopic site. However recent evidence most notably from the Edmonton group demonstrates that islet allotransplantation still has great potential to become an established treatment option for diabetic patients.    

4.106           Innate IFN-g production in cattle in response to MPP14, a secreted protein from Mycobacterium avium subsp. paratuberculosis

Olsen, I. and Storset, A.K. Scand. J. Immunol., 54, 305-313 (2001)   Calves experimentally infected with Mycobacterium avium subsp. paratuberculosis and uninfected calves were tested for interferon(1FN)-g production after stimulation with purified protein derivative from M. avium subsp. paratuberculosis (PPDp) or a secreted 14 kDa protein (MPP14) specific for the M. avium-intracellulare­scrofulaceum (MAIS) complex. Several calves in both groups responded strongly up to about 5 months to both antigens. Two uninfected calves responded repeatedly, but not always, to MPPI4 and PPDp throughout the study. The responses in the uninfected animals seemed to be independent of cell contact between the antigen presenting cells (APC) and the responding population. The supernatant from adherent cells stimulated with MPPI4 induced similar levels of IFN-g production in CDl4⁺/B-ce11 depleted peripheral blood mononuclear cells (PBMC) as when the antigen was used directly on PBMC. In contrast, APCiT-cell contact was necessary to induce the IFN-g production in infected animals, suggesting that both innate and adaptive IFN-g production in response to MPP14 could occur. CD8⁺ cells contributed to some of the 1FN-g production in response to MPP14, but the rest could not be explained, while CD4⁺ cells were responsible for the adaptive response to PPDp. This study showed that secreted proteins could induce innate IFN-g production that interferes with diagnostic testing using the IFN-g-test.  

4.107           A microfluidic device for measuring cellular membrane potential

Farinas, J., Chow, A.W. and Wada, H.G. Anal. Biochem., 295, 138-142 (2001)   Recent developments in microfluidics have enabled the design of a lab-on-a-chip system capable of mea­suring cellular membrane potential. The chip accesses liquid samples sequentially by sipping from a micro-plate through a capillary, mixes the samples with cells flowing through a microchannel, contacts the cells with potential-sensitive dyes, and reads out cellular responses using fluorescence detection. The rate of cellular uptake of membrane-permeable, ionic fluoro­phores by THP-1 cells was found to depend strongly on membrane potential. The ratio of the fluorescence of the anionic dye DiBAC₄(3) and the cationic dye Syto 62 taken up by cells was found to double for every 33 mV change in membrane potential. The utility of this ap­proach was demonstrated by assaying ion channel ac­tivity in human T lymphocytes. Because of the high sensitivity, low cellular and reagent consumption, and high data quality obtained with the microfluidic de­vice, the lab-on-a-chip system should be widely appli­cable in high-throughput screening and functional genomics studies.    

4.108           Co-incubation of pig-islet cells with spleen cells from non-obese mice causes decreased insulin release by non-T-cell- and T-cell-mediated mechanisms

You, S., Rivereau, A-S., Gouin, E. and Sai, P. Clin. Exptl. Immunol., 125, 25-31 (2001)    In vitro studies were conducted in the non-obese diabetic (NOD) mouse, prone to Type 1 autoimmune diabetes, to investigate the mechanisms involved in cell-mediated rejection of pig islet xenografts. Our previous work concerning the mechanisms of proliferation of xenogeneic lymphocytes to pig islet cells (PIC) was not indicative of PIC impairment. Consequently, a test was developed based on perfusion analysis of the alteration of basal and stimulated insulin release from adult PIC incubated with mouse splenocytes or subsets. Compared with PIC incubation alone or with syngeneic pig splenocytes, co-­incubation with mouse whole spleen cells resulted in a decrease of basal and stimulated insulin release (P < 0.001). Two components of this alteration were detected separately: PIC impairment was decreased (P < 0.01) after removal of plastic-adherent cells from spleen cells, but maintained (P < 0.01) when plastic-adherent cells alone were co-incubated with PIC. The increase of murine interleukin-lb when mouse plastic-adherent spleen cells were cultured with PIC (P < 0.04) was indicative of macrophage activation. Soluble factors produced during co-incubation of mouse splenocytes or plastic-adherent cells with PIC were involved in the impairment process, since supernatant fluids collected during previous PIC—mouse cell co-incubations directly altered (P < 0.01) insulin release from PIC. Moreover, impairment of PIC by mouse spleen cells was abolished (P < 0.01) by gadolinium chloride (which inhibits macrophages), but not by cyclosporin A. Another mechanism was apparent, since co-incubation of PIC with purified mouse T cells or CD4⁺ T cells, re-mixed with antigen-presenting cells, led to a decrease (P < 0.01) of insulin release. This model, based on the alteration of dynamic basal and stimulated insulin release, is indicative of in vitro cell-mediated alteration of PIC in the NOD mouse, The effect of whole spleen cells was rapid, and a crucial role was played by plastic-adherent cells. Two mechanisms were responsible for the behaviour of these cells: an early direct effect (at least in part via soluble products): and the indirect presentation of PIC xenoantigens (leading to impairment by CD4⁺ T lymphocytes).  

4.109           Efficient cryopreservation of dendritic cells transfected with cDNA of a tumour antigen for clinical application

Pecher, G., Schirrmann, T., Kaiser, L. and Schenk, J.A. Biotechnol. Appl. Biochem., 34, 161-166 (2001)   Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system and are cur­rently being investigated in clinical applications as cancer vaccines. An efficient cryopreservation method would greatly contribute to their use in clinical trials. We have established a method for freezing of DCs derived from peripheral blood mononuclear cells using the plasma expander Gelifundolâ. This enabled us to reduce the concentration of the toxic DMSO to 5%. The method could be performed without the addition of fetal calf serum or any other serum. After freezing, the viability of the DCs was 90%. The cells exhibited all the phenotypic characteristics (CD11c⁺, HLA-DR⁺, CD8O⁺, CD83⁺, CD86⁺) of DCs, as tested by flow cytometry. Cells transfected with cDNA for the tumour antigen mucin expressed this protein on their surfaces in the same manner as before freezing. The stimulating capacity of a mixed lymphocyte culture was also preserved. These findings offer an efficient method for the cryopreservation of DCs for use in clinical trials.    

4.110           Improved in vivo pancreatic iselt function after prolonged in vitro islet culture

Gaber, A. O. et al Transplant., 72, 1730-1736 (2001)   Background. Difficulties with recovering and preserv­ing pancreatic islets have hampered progress in islet transplantation. In previous in vitro studies, our labora­tory successfully demonstrated that using serum-free medium for prolonged pancreatic Islet culture allows post-culture recovery ratios greater than those obtained with standard media with sustained In vitro Islet func­tion. The goal of this study was to determine whether culturing of islets in a modified serum-free medium (M­SFM) would sustain function in vivo. Methods. Islets were isolated from pancreata pro­cured from 12 cadaveric organ donors and cultured in the M-SFM for up to 2 months, cryopreserved at -70°C within 1-3 days of isolation for 2 months, or placed in short-term culture (3-5 days) before their transplanta­tion under the kidney capsule of non-obese diabetic-severe combined immunodeficient mice (n=4-7 per group/time point). In vivo islet function was assessed by measuring the production of human insulin and C-peptide over a period of 3-15 months. Results. After extended culture of Islets in M-SFM for 1 or 2 months, transplanted islets maintained their viability, and in some instances in vivo function im­proved when compared with short-term cultured is­lets transplanted from the same preparation (P<0.01). Improvement was particularly evident for islets cul­tured for 1 month. Furthermore, when compared with cryopreserved preparations, early function (postoper­ative day 7) of Islets from 1-month culture prepara­tions was statistically better (P<0.05). Prolonged cul­ture in M-SFM bad no significant impact on long-term function, inasmuch as cultured islets functioned for more than 120 days. Conclusion. These data demonstrate that prolonged islet culture in M-SFM sustained viability and func­tion, and in some instances had a positive effect on in vivo islet function, particularly in the 1-month cul­tures. No negative effect on long-term in vivo function was demonstrated in this study. Confirmation in clin­ical models utilizing extended (1-2 months) islet culture in M-SFM could significantly enhance islet trans­plantation by allowing the identification of best­ matched recipients, pre-transplantation recipient conditioning, and possible pre-transplantation islet modifications to promote engraftment and prolonged graft function.  

4.111           In vitro recognition and impairment of pig islet cells by baboon immune cells

Lalain, S., Gianello, P., Gouin, E. and Sai, P. Transplant., 72, 1541-1548 (2001)   Background. Grafting pig islets into patients with type 1 diabetes requires control of the strong cellular ­xenogeneic rejection. This in vitro study compared the cellular reaction of baboons and humans to pig Islet cells (PICs) to confirm the validity of using these animals for further in vivo preclinical trials. Methods. Baboon or human peripheral blood mono-nuclear cells (PBMCs) or subsets were co-incubated with PICs from specific pathogen-free adult pigs for 7 days to determine the mechanisms and intensity of PBMC proliferation. Interleukin (IL) 10 and interferon (IFN) g secretion were assessed by enzyme-linked immunosorbent assay. Because proliferation was not indicative of aggression, a test based on perifusion analysis of the alteration of basal and stimulated insulin releases from PIC incubated with different baboon and human cells was developed. Results. Baboon PBMCs strongly proliferated in response to PICs (stimulation Index [SI]=24.8±6.9 [n=8] vs. 23.9±3.4 [n=34] for human PBMCs), showing considerable variation in intensity among animals (2.3<SI<63) and humans (1.8<SI<97). PBMC proliferation was inhibited in baboons and humans by anti-CD4 (% inhibition of  SI: 71±10% and 75±7%, respectively) and anti-DR (75±35% and 80±6%) monoclonal antibodies (MoAbs) or by depletion of MHC class II+ cells (99±1% and 90±6%).Blocking by anti-CD8 or anti-CD16 MoAbs was weaker and variable among both animals and humans. IL-l0 production by baboon and human PBMCS in response to PICs increased more than IFN-g production after 2 days of co-culture, but the IL-10/IFN-g ratio was inverted after 5 days of co-culture. After 7 days (and even after only 2days) of co-culture with baboon (n=8) or human (n=18) PBMCs, basal and glucose-stimulated insulin secretions from PICs were almost completely abolished (P<0.0001). The drop in insulin release could have mainly resulted from lysis of PICs, because the number of PICs de­creased by 78% after 7 days of co-incubation with PB­MCs. A decrease of insulin release by PBMCs was repro­duced with plastic-adherent cells and was abolished by depletion of MHC class II+ cells or by addition of 100 mg/ml gadolinium (which inhibits macrophages), but not by cyclosporine. In baboons, as in humans, insulin re­lease was also decreased after co-culture of PICs with enriched T lymphocytes remixed with antigen-present­ing cells (APCs). Conclusions. This study provides the first data on in vitro comparison of baboon and human cell-mediated recognition and impairment of PICs. Proliferation of PBMCs against PICs involves mainly CD4 T cells, with indirect recognition mediated by baboon or human MHC class II+ APCs. The Th2/Th1 profile of cytokines secreted in response to PICs was similar in baboon and human PBMCs. The model based on alteration of insulin secretion indicates that PIC impairment by whole mononuclear cells was strong and rapid and that a crucial role was played by MHC class II+ and plastic-adherent cells. Two mechanisms appear to be responsible for the role of these cells: (1) early and strong direct effect, which is potentially involved in vivo in primary non-function of islets aggressed by monocytes and macrophages; and (2) presentation of PIC xenoantigens, which leads to impairment by T lymphocytes possibly involved in in vivo-specific cel­lular rejection. The mechanisms and intensity of ba­boon cellular reactions to PICs in vitro were similar to those observed in humans, which suggests that the baboon is a suitable model for the study of cellular mechanisms during preclinical trials of pig islet xenografts.  

4.112           Long-term follow-up failed to detect in vitro transmission of full-length porcine endogenous retroviruses from specific pathogen-free pig islets to human cells

Clemenceau, B., Jegou, D., Martignat, L. and Sai, P. Diabetologia, 44, 2044-2055 (2001)   Aims/hypothesis: Islets from specific pathogen-free (SPF) pigs could prevent the transmission of conventional zoonosis, but not endogenous retroviruses (PERV), from pigs to diabetic patients. We previously reported that the pancreas showed the lowest expression of PERV mRNA among pig tissues intended for grafting. This study aimed to determine whether PERV from pig islets infect human cells during co-incubation. Methods: Human cells (including highly PERV-sensitive 293 cells) were incubated with SPF pig islet cells under conditions designed to increase contact (a high islet to human cell ratio, extended period of co-culture, and repeated contacts). PK15 and G2 retrovirus-producing pig cells were used in place of islet cells as "positive infection controls". Infection of human cells was monitored on cellular extracts and supernatants by PCR or long PCR, and RT-PCR or long RT-PCR, to detect PERV DNA and mRNA, respectively. Reverse-transcriptase activity was monitored by PERT. Results: Despite the presence of all PERV sequences in pig islet cells, including full-length inserts, no DNA or RNA for gag, pol, and the 3 env sub-types were detected in any human cell line or blood mononuclear cells incubated with pig islet cells, during an 18-week follow-up period. No PERV sequences or RT activity were detected in supernatants. PERV signals were negative even when the pig islet to human cell ratio was increased to 100:1, the time of co-culture was extended to 5 days and two sequential co-incubations were done. By contrast, all PERV DNA and mRNA were detected in all human cells co-incubated with PK15 or G2 cells. Depending on human cell types, productive or non-productive infections were obtained: full-length PERV RNA and RT activity in supernatants were detected or not; and PERV sequences to previously unexposed human cells by PERV-infected human cells were transmitted or not. Some human cells were not productively infected by PK15 cells but became productively infected after co-incubation with PERV-infected 293 cells. Conclusion/interpretation: SPF pig islet cells, even with PERV inserts and transcripts, have very little probability of transmitting PERV to human cells during co-incubation. The sensitivity of human cells to stable and productive infection by PERV depends on the cell type. Human adaptation of PERV was observed.  

4.113           Safety of modified vaccinia virus Ankara (MVA) in immune-suppressed macaaaques

Stittelaar, K.J. et al Vaccine, 19, 3700-3709 (2001)   Modified vaccinia virus Ankara (MVA)-based recombinant viruses have been shown to be potent vaccine candidates for several infectious and neoplastic diseases. Since a major application of these live, replication-deficient vectors would be their use in immunocompromised or potentially immunocompromised individuals, a preclinical safety study was carried out. Macaques were inoculated with high doses of MVA (10⁹) via various routes, after immune-suppression by total-body irradiation, anti-thymocyte globulin treatment, or measles virus (MV) infection. No clinical, haematological or pathological abnormalities related to MVA inoculation were observed during a 13-day follow-up period. The presence of MVA genomes was demonstrated by nested PCR during the course of the experiment in all macaques, but from none of these animals replication competent MVA could be reisolated. These data suggest that MVA can safely be used as a basis for recombinant human vaccines, and that it is also safe for use in immunocompromised individuals.  

Gastrin and the neuropeptide PACAP evoke secretion from rat stomach histamine-containing (ECL) cells by stimulating influx of Ca²⁺ through different Ca²⁺ channels

Lindström, E., Eliasson, L., Björkqvist, M. and Håkonson, R.

1. Physiol., 535(3), 663-677 (2001)

  Gastrin and PACAP stimulate secretion of histamine and pancreastatin from isolated rat stomach ECL cells. We have examined whether or not secretion depends on the free cytosolic Ca2+ concentration ([Ca2+]i) and the pathways by which gastrin and PACAP elevate [Ca2+]i. Secretion was monitored by radioimmunoassay of pancreastatin and changes in [Ca2+]i by video imaging. The patch clamp technique was used to record whole-cell currents and membrane capacitance (reflecting exocytosis). In the presence of 2 mm extracellular Ca²⁺, gastrin and PACAP induced secretion and raised [Ca²⁺]_i. Without extracellular Ca²⁺ (or in the presence of La³⁺) no secretion occurred. The extracellular Ca²⁺ concentration required to stimulate secretion was 10 times higher for gastrin than for PACAP. Depletion of intracellular Ca²⁺ pools by thapsigargin had no effect on the capacity of gastrin and PACAP to stimulate secretion. Gastrin-evoked secretion was inhibited 60-80 % by L-type channel blockers and 40 % by the N-type channel blocker ω-conotoxin GVIA. Combining L-type and N-type channel blockers did not result in greater inhibition than L-type channel blockers alone. Whole-cell patch clamp measurements confirmed that the ECL cells are equipped with voltage-dependent inward Ca²⁺ currents. A 500 ms depolarising pulse from -60 mV to +10 mV which maximally opened these channels resulted in an increase in membrane capacitance of 100 fF reflecting exocytosis of secretory vesicles. PACAP-evoked secretion was reduced 40 % by L-type channel blockers but was not influenced by inhibition of N-type channels. SKF 96365, a blocker of both L-type and receptor-operated Ca²⁺ channels, inhibited PACAP-evoked secretion by 85 %. Combining L-type channel blockade with SKF 96365 abolished PACAP-evoked secretion. The results indicate that gastrin- and PACAP-evoked secretion depends on Ca²⁺ entry and not on mobilisation of intracellular Ca²⁺. While gastrin stimulates secretion via voltage-dependent L-type and N-type Ca²⁺ channels, PACAP acts via L-type and receptor-operated Ca²⁺ channels.   Serum-Free Media for Neural Cell Cultures Price, P.J. and Brewer, G.J. Protocols for Neural Cell Culture, Ed. Fedoroff, s. and Richardson, A., Humana Press 255-264 (2001)   The ability to grow primary neurons under serum-free conditions is facilitating better control in studies of neuronal development, mechanisms of neuronal signaling, electrophysiology, pharmacology, plasticity, in vitro growth requirements, gene expression, and neurotoxicity. Some of the new commercially available media combinations allow for the growth of sparse populations of neurons, which in turn allow for the study of individual neurons and synapses. This has not been possible using serum-supplemented media without a feeder layer of glial cells. In serum-supplemented media, glial cells continue to multiply, necessitating the use of cytotoxic mitotic inhibitors (Wallace and Johnson, 1989). Serum also contains unknown and variable levels of growth factors, hormones, vitamins, and proteins.  

4.114           Kinetics of endomitosis in primary murine megakaryocytes

Carow, C.E., Fox, N.E. and Kaushansky, K.

1. Cell. Physiol., 188(3), 291-303 (2001)

  Megakaryocytes (MKs) develop from diploid progenitor cells via successive rounds of DNA synthesis in the absence of cell division, a process termed endomitosis (EnM). While the mechanism underlying EnM is not known, studies in yeast and leukemic cell lines have suggested that it may be due to reduced levels of cyclin B1 or cdc2, leading to a decrease in mitotic kinase activity. Using flow cytometry to study EnM highly purified marrow-derived MK precursors, we found that: (1) on average, 36% of 8N-32N MKs expressed abundant cyclin B during G2/M. The percentage of cells in G2/M decreased in >64N MKs, suggesting the limit of EnM, (2) the level of cyclin B per G2/M MK increased linearly with ploidy, (3) cyclin B expression oscillated normally in polyploid MKs, (4) MPM-2, a phosphoepitope created by the action of mitotic kinases and specific to M-phase cells, was expressed in a significant fraction of polyploid MKs, and (5) there was an apparent increase of cyclin B in G1-phase in polyploid MKs. This study provides the first qualitative kinetic data regarding the cell cycle status of MKs within individual ploidy classes. It also demonstrates the feasibility of using anti-cyclin B antibody and flow cytometry to resolve G1 from G2/M populations in polyploid MKs. Finally, these findings establish that neither a relative nor absolute deficiency of mitotic kinase components is responsible for EnM, suggesting that the departure from normal cell division kinetics seen in polyploid MKs is likely due to alterations in other cell cycle regulators.  

4.115           Temperature and time interval for culture of postmortem neurons from adult rat cortex

Viel, J.J., McManus, D.Q., Cady, C., Evans, M.S. and Brewer, G.J.

1. Neurosci. Res., 64(4), 311-321 (2001)

  For a model of neurological disease and ischemia, we extended recent work to culture adult postmortem rat brain neurons. Frontal cortex sections were removed from adult rats immediately following sacrifice and at different postmortem intervals and with the brain at either 22°C or 4°C. Brain could be stored four times longer at 4°C between sacrifice and neuronal disaggregation to achieve the same 20% recovery of live cells from those plated compared to 22°C. Each milligram of rat frontal cortex was estimated by the optical disector method to contain 160,000 neurons. When cells were isolated as rapidly as possible, 9% of the neurons originally present in the brain were viable. Various postmortem intervals from 2 to 24 hr resulted in a reduction from 6% to 3% of the cells originally present. After 5 days in culture, viable neurons were 23–42% of those isolated. Neuron-like cells that survived represented 40–75% of the viable cells, or 0.5–2.75% of those originally estimated to be present in the brain. Electrophysiology experiments show that cells isolated 0 and 24 hr postmortem had neuronal electrical properties, including an average resting membrane potential of –48 mV, voltage-sensitive currents, and action potentials. Neuron-like cells were immunoreactive for neuron-specific enolase, neurofilament 200, glutamate, MAP2, and tau after 2 weeks in culture. These experiments show that neuron-like cells can be reliably cultured from adult rat cortex up to 6 hr postmortem when stored at 22°C and up to 24 hr postmortem when stored at 4°C. These findings should encourage donation of human postmortem brain neurons for studies on ischemia, adult pharmacology, and neurological disease.  

4.116           Isolation of Mouse Thymic Dendritic Cells

Anjuere, F. and Ardavin, C. Methods in Mol. Med., 64, 23-28 (2001)   No abstract available  

4.117           Fractalkine is expressed by smooth muscle cells in response to IFN-g and TFN-a and is modulated by metalloproteinase activity

Ludwig, A., Berkhout, T., Moores, K., Groot, P. and Chapman G.

1. Immunol., 168, 604-612 (2002)

  Fractalkine/CX3C-chemokine ligand1 is expressed as a membrane-spanning adhesion molecule that can be cleaved from the cell surface to produce a soluble chemoattractant. Within the vasculature, fractalkine is known to be generated by endothelial cells, but to date there are no reports describing its expression by smooth muscle cells (SMC). IN this study we demonstrate that IFNg and TNF-a, but not IL-1b, cooperate synergistically to induce fractalkine mRNA and protein expression in cultured aortic SMC. We also report the release of functional, soluble fractalkine from the membranes of stimulated SMC. This release is inhibited by the zinc metalloproteinase inhibitor batimastat, resulting in the accumulation of membrane-associated fractalkine on the SMC surface. Therefore, an SMC-derived metalloproteinase activity is involved in fractalkine shedding. While soluble fractalkine present in SMC-conditioned medium is capable of inducing calcium transients in cells expressing the fractalkine receptor (CX3CR1), blocking experiments using neutralizing Abs reveal that it can be inactivated without affecting the chemotactic activity of SMC-conditioned media on monocytes. However, membrane-bound fractalkine plays a major role in promoting adhesion of monocytic cells to activated SMC. This fractalkine-mediated adhesion is further enhanced in the presence of batimastat, indicating that shedding of fractalkine from the cell surface down-regulates the adhesive properties of SMC. Hence, during vascular inflammation, the synergistic induction of fractalkine by IFN-g and TNF-a together with its metalloproteinase-mediated cleavage may finely control the recruitment of monocytes to SMC within the blood vessel wall.  

4.118           CD8a⁺ dendritic cells originate from CD8a^- dendritic cell subset by a maturation process involving CD8a, DEC-205, and CD24 up-regulation

Del Hoyo, G.M., Martin, P., Arias, F.C., Marin, A.R. and Ardavin, C. Blood, 99(3), 999-1004 (2002)   CD8a⁺ and CD8a^- dendritic cells (DCs) have been considered as an independent DC subpopulation both ontogenetically and functionally during recent years. However, it has been demonstrated that both DC subsets can be generated from a single precursor population, supporting the concept that they do not represent separate DC lineages. By using highly purified splenic CD8a^- DCs, which were injected intravenously and traced by means of an Ly5.1/Ly5.2 transfer system, this study shows that CD8a^- DCs acquired the phenotypic characteristics of CD8a⁺ DCs by a differentiation process involving CD8a, DEC-205, and CD24 up-regulation, paralleled by the down-regulation of CD11b, F4/80, and CD4. These data demonstrate that CD8a⁺ DCs derive from CD8a^- DCs, and strongly support that CD8a^- and CD8a⁺ represent different maturation or differentiation stages of the same DC population. Therefore, CD8a⁺ DCs would represent the last stage of DC differentiation, playing an essential role in the induction of T-cell responses, due to their antigen-presenting potential, cross-priming ability, and capacity to secrete large amounts of key cytokines such as interferon g and interleukin-12.  

4.119           Characterization of mitotic neurons derived from adult rat hypothalamus and brain stem

Evans, J. et al

1. Neurophysiol., 87, 1076-1085 (2002)

  Embryonic or neonatal rat neurons retain plasticity and are readily grown in tissue culture, but neurons of the adult brain were thought to be terminally differentiated and therefore difficult to culture. Recent studies, however, suggest that it may be possible to culture differentiated neurons from the hippocampus of adult rats. We modified these procedures to grow differentiated neurons from adult rat hypothalamus and brain stem. At day 7 in tissue culture and beyond, the predominant cell types in hypothalamic and brain stem cultures had a stellate morphology and could be subdivided into two distinct groups, one of which stained with antibodies to the immature neuron marker a-internexin, while the other stained with the astrocytes neuron marker GFAP. The a-internexin positive cells were mitotic and grew to form a characteristic two-dimensional cellular network. These a-internexin positive cells coimmunostained for the neuronal markers MAP2, type III b-tubulin, and tau, and also bound tetanus toxin, but were negative for the oligodendrocytes marker Ga1C and also for the neurofilament triplet proteins NF-L, NF-M, and NF-H, markers of more mature neurons. Patch-clamp analysis of these a-internexin positive cells revealed small Ca²⁺ currents with a peak current of –0.5 ± 0.1 pA/pF at a membrane potential of –20 mV (n = 5) and half-maximal activation at –30 mV (n= 5). Na⁺ currents with a peak current density of –154.5 ± 49.8 pA/pF at a membrane potential of –15 mV (n= 5) were also present. We also show that these cells can be frozen and regrown in tissue culture and that they can be efficiently infected by viral vectors. These cells therefore have the immunological and electrophysiological properties of immature mitotic neurons and should be useful in a variety of future studies of neuronal differentiation and function.  

4.120           Dramatic increase in lymph node dendritic cell number during infection by the mouse mammary tumor virus occurs by a CD62L-dependent blood-borne DC recruitment

Martin, P. et al Blood, 99(4), 1282-1288 (2002)   Despite the information dealing with the differential phenotype and function of the main mouse dendritic cell (DC) subpopulations, namely, CD8a^- and CD8a⁺ DCs, their origin and involvement in antiviral immune responses in vivo are still largely unknown. To address these issues, this study used the changes occurring in DC subpopulations during the experimental infection by the Swiss (SW) strain of the mouse mammary tumor virus (MMTV). MMTV(SW) induced an 18-fold increase in lymph node DCs, which can be blocked by anti-CD62L treatment, concomitant with the presence of high numbers of DCs in the outer cortex, in close association with high endothelial venules. These data suggest that the DC increase caused by MMTV(SW) infection results from the recruitment of blood-borne DCs via high endothelial venules, by a CD62L-dependent mechanism. In addition, skin sensitization assays indicate that MMTV(SW) infection inhibits epidermal Langerhans cell migration to the draining lymph node. Moreover, data on the kinetics of MMTV(SW)-induced expansion of the different DC subsets support the hypothesis that CD8^- and CD8⁺ DCs represent different maturation stages of the same DC population, rather than myeloid-and lymphoid-derived DCs, respectively, as previously proposed. Finally, the fact that DCs were infected by MMTV(SW) suggests their participation in the early phases of infection.    

4.121           Alveolar epithelial ion and fluid transport Na transport proteins are expressed by rat alveolar epithelial type I cells

Borok, Z. et al Am. J. Physiol. Lung Mol. Physiol., 282, L599-608 (2002)   Despite a presumptive role for type I (AT1) cells in alveolar epithelial transport, specific Na transporters have not previously been localized to these cells. To evaluate expression of Na transporters in AT1 cells, double labeling immunofluorescence microscopy was utilized in whole lung and in cytocentrifuged preparations of partially purified alveolar epithelial cells (AEC). Expression of Na pump subunit isoforms and the a-subunit of the rat (r) epithelial Na channel (a-EnaC) was evaluated in isolated AT1 cells identified by their immunoreactivity with AT1 cell-specific antibody markers (VIIIB2 and/or anti-aquaporin-5) and lack of reactivity with antibodies specific for AT22 cells (anti-surfactant protein A) or leukocytes (anti-leukocyte common antigen). Expression of the Na pump a₁-subunit in AEC was assessed in situ. Na pump subunit isoform and a-rENaC expression was also evaluated by RT-PCR in highly purified (~ 95%) AT1 cell preparations. Labeling of isolated AT1 cells with anti-a₁ and anti-b₁ Na pump subunit and anti-a-rENaC antibodies was detected, while reactivity with anti-a₂ Na pump subunit antibody was absent. AT1 cells in situ were reactive with anti-a₁ Na pump subunit antibody. Na pump a₁ – and b₁ – (but not a₂-) subunits and a-rENaC were detected in highly purified AT1 cells by RT-PCR. These data demonstrate that AT1 cells express Na pump and Na channel proteins, supporting a role for AT1 cells in active transalveolar epithelial Na transport.  

4.122           Apolipoprotein E4 inhibits, and apolipoprotein E3 promotes neurite outgrowth in cultured adult mouse cortical neurons through the low-density lipoprotein receptor-related protein

Nathan, B. et al Brain Res., 928, 96-105 (2002)   The apolipoprotein E4 (apoE4) genotype is a major risk factor for Alzheimer's disease (AD); however, the mechanism is unknown. We previously demonstrated that apoE isoforms differentially modulated neurite outgrowth in embryonic neurons and in neuronal cell lines. ApoE3 increased neurite outgrowth whereas apoE4 decreased outgrowth, suggesting that apoE4 may directly affect neurons in the brain. In the present study we examined the effects of apoE on neurite outgrowth from cultured adult mouse cortical neurons to examine if adult neurons respond the same way that embryonic cells do. The results from this study demonstrated that (1) cortical neurons derived from adult apoE-gene knockout (apoE KO) mice have significantly shorter neurites than neurons from adult wild-type (WT) mice; (2) incubation of cortical neurons from adult apoE KO mice with human apoE3 increased neurite outgrowth, whereas human apoE4 decreased outgrowth in a dose-dependent fashion; (3) the isoform specific effects were abolished by incubation of the neurons with either receptor associated protein (RAP) or lactoferrin, both of which block the interaction of apoE-containing lipoproteins with the low-density lipoprotein receptor-related protein (LRP). These data suggest a potential mechanism whereby apoE4 may play a role in regenerative failure and accelerate the development of AD.  

4.123           Expression of Notch ligands, Jagged1, and Delta1 in antigen presenting cells in mice

Yamaguchi, E. et al Immunol. Lett., 81, 59-64 (2002)   Notch1 is indispensable for T cell development. It is anticipated that Notch1 and other Notch receptors expressed on the surface of thymic T cell precursors are activated by ligands present on environmental cells, including antigen presenting cells (APCs), and involved in positive and negative selections. Notch receptors on peripheral T cells may also be activated by ligands on APCs. Here, we examined the expression pattern of three Notch ligands, Jagged1, 2 and Delta1 in APCs by an immunofluorescence cell staining method and a reverse transcriptase-polymerase chain reaction (RT-PCR) method. Peritoneal macrophages were strongly positive for Jagged1 staining. In contrast, macrophages separated from spleen and dendritic cells (DCs) separated from spleen and thymus showed positive staining for all the three ligands at a similar intensity. An analysis by RT-PCR revealed that peritoneal and splenic macrophages and splenic and thymic DCs, show a distinct pattern in Notch ligand expression. These findings may represent that expression of various Notch ligands in APCs has a physiological relevance in each organ.    

4.124           Increased production of nitric oxide stimulated by interferon-g from peripheral blood monocytes in patients with complex regional pain syndrome

Hartrick, C.T. Neurosci. Lett., 323, 75-77 (2002)   This study examines immediate nitric oxide (NO) release from monocytes following interleukin-1beta (IL-1beta), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) challenge in patients with complex regional pain syndrome (CRPS). Study patients exhibited the following: (1), mechanical allodynia; (2), evidence of either vasomotor or sudomotor disturbance; and (3), concordant painful allodynia documented with quantitative sensory testing that was temporarily abolished with sympathetic block. Ten subjects (CRPS, N=5; control, N=5) were enrolled. Peripheral blood monocytes were challenged with 100 ml of IL-1b (1 ng), IFN-g (1 ng), TNF-a (0.01 ng), and normal saline (NS) and the resultant immediate NO release measured. Subjects with CRPS exhibited a statistically significant increase in NO release in response to IFN-g (P<0.012) compared with controls. The NO responses to IFN-g in excess of NS (P<0.025) and as the ratio IFN-g/NS (P<0.022) were also significantly increased.

4.125           Tissue-specific mechanisms control the retention of IL-8 in lungs and skin

Frevert, C.W. et al

1. Immunol., 168, 3550-3556 (2002)

  Chemokines are a group of structurally related peptides that promote the directed migration of leukocytes in tissue. Mechanisms controlling the retention of chemokines in tissue are not well understood. In this study we present evidence that two different mechanisms control the persistence of the CXC chemokine, IL-8, in lungs and skin. ¹²⁵I-labeled IL-8 was injected into the airspaces of the lungs and the dermis of the skin and the amount of ¹²⁵I-labeled IL-8 that remained at specified times was measured by scintillation counting. The ¹²⁵I-labeled IL-8 was cleared much more rapidly from skin than lungs, as only 2% of the ¹²⁵I-labeled IL-8 remained in skin at 4 h whereas 50% of the ¹²⁵I-labeled IL-8 remained in lungs at 4 h. Studies in neutropenic rabbits showed that neutrophils shortened the retention of ¹²⁵I-labeled IL-8 in skin but not lungs. A monomeric form of IL-8, N-methyl-leucine 25 IL-8, was not retained as long in lungs as recombinant human IL-8, indicating that dimerization of IL-8 is a mechanism that increases the local concentration and prolongs the retention of ¹²⁵I-labeled IL-8 in lungs. These observations show that the mechanisms that control the retention of IL-8 in tissue include neutrophil migration and dimerization, and that the importance of these varies in different tissues.  

4.126           Expression and function of 4-1BB and 4-1BB ligand on murine dendritic cells

Futagawa, T. et al Int. Immunol., 14(3), 275-286 (2002)   4-1BB (CDw137) and its ligand (4-1BBL) have been implicated in cellular immune responses. To further characterize the expression and function of 4-1BBL, we newly generated an anti-mouse 4-1BBL mAb (TKS-1), which can inhibit the interaction of 4-1BBL with 4-1BB. Flow cytometric analyses using TKS-1 and an anti-mouse 4-1BB mAb indicated that 4-1BB was inducible on both CD4⁺ and CD8⁺ splenic T cells by stimulation with immobilized anti-CD3 mAb, but 4-1BBL was not expressed on resting or activated T cells. 4-1BBL expression was inducible on splenic B cells by stimulation with anti-IgM antibody plus anti-CD40 mAb, on peritoneal macrophages by stimulation with lipopolysaccharide (LPS) and on splenic dendritic cells (DC) by stimulation with anti-CD40 mAb or LPS. Interestingly, splenic DC expressed 4-1BB constitutively, which was down-regulated by anti-CD40 stimulation. Co-culture of splenic DC with 4-1BBL-transfected cells or 4-1BBL-expressing tumor cell lines led to cytokine (IL-6 and IL-12) production and co-stimulatory molecule up-regulation by splenic DC, indicating that 4-1BBL can directly activate DC. Moreover, IL-12 production by anti-CD40-stimulated DC was partially inhibited by TKS-1. These results suggest that 4-1BB expressed on DC may be involved in DC activation through DC–tumor interaction and DC–DC interaction.    

4.127           CD40L blockade prevents autoimmune diabetes by induction of bitypic NK/DC regulatory cells

Homann, D. et al Immunity, 16, 403-415 (2002)   Systemic treatment with antibody to CD40 ligand (aCD40L) can prevent autoimmunity and transplant rejection in several animal models and is currently under evaluation in clinical trials. While it is known that aCD40L administration inhibits expansion and effector functions of aggressive T cells, it is still unclear whether additional regulatory mechanisms are operative. Here we demonstrate that a single episode of CD40L blockade during development of the autoaggressive immune response completely prevented autoimmune disease in the RIP-LCMV mouse model for virally induced type 1 diabetes. Interestingly, protection could be transferred by a highly potent, bitypic cell population sharing phenotypic and functional properties of both natural killer (NK) and dendritic cells (DC). Furthermore, protection of prediabetic recipients was autoantigen specific and did not result in generalized immunosuppression. The origin, function, and therapeutic potential of these bitypic NK/DC regulatory cells are discussed.  

4.128           Differentiated HL-60 cells are a valid model system for the analysis of human neutrophil migration and chemotaxis

Hauert, A.B., Martinelli, S., Marone, C. And Niggli, V. Int. J. Biochem. Cell Biol., 34, 838-854 (2002)   We have carried out a detailed comparison of the motile properties of differentiated HL-60 cells and human peripheral blood neutrophils. We compared the effects of chemotactic stimuli and of inhibitors of signaling proteins on morphology, chemokinesis and chemotaxis of neutrophils and differentiated HL-60 cells using videomicroscopy and a filter assay for chemotaxis. We also assessed expression of signaling and cytoskeletal proteins using Western blotting. Chemotactic peptide induced a front-tail polarity in HL-60 cells comparable to that of neutrophils. Chemokinetic and chemotactic responses to chemotactic peptide were also very similar for both cell types, concerning mean speed of migration, the fraction of migrated cells and the concentration of stimulus optimal for activation. The cytokine interleukin-8 was in contrast clearly less effective in activating motile responses of differentiated HL-60 cells as compared to neutrophils. An important functional role of Rho-activated kinases and phosphatidylinositol 3-kinase in motile responses of HL-60 cells, consistent with their upregulation during differentiation, could be confirmed using inhibitors with specificity for the corresponding enzymes. The only difference observed here between HL-60 cells and neutrophils concerned the differential effects of a protein kinase C inhibitor. In summary, the results presented here show that differentiated HL-60 cells, stimulated with chemotactic peptide, are a valid model system to study molecular mechanisms of neutrophil emigration.  

4.129           Expression of transforming growth factor-b1 by pancreatic stellate cells and its implications for matrix secretion and turnover in chronic pancreatitis

Wai-Tsing Shek, F. et al Am. J. Pathol., 160(5),1787-1798 (2002)   Pancreatic stellate cells mediate fibrosis in chronic pancreatitis. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs)-1 and -2 are crucial modulators of fibrosis. Transforming growth factor-b (TGF-b) is a key regulator of extracellular matrix production and myofibroblast proliferation. We have examined MMP and TIMP synthesis by transformed cultured pancreatic stellate cells and their regulation by TGF-b1. By Northern analysis they expressed mRNAs for procollagen 1, TIMP-1, TIMP-2, and MMP-2. Expression of membrane type-1 MMP was confirmed by Western blotting. By immunohistochemistry these enzymes localized to fibrotic areas in human chronic pancreatitis. Active TGF-b1 constitutes 2 to 5% of total TGF-b1 secreted by pancreatic stellate cells; they express TGF-b receptors I and II. Exogenous TGF-b1 (10 ng/ml) significantly increased procollagen-1 mRNA by 69% and collagen protein synthesis by 34%. Similarly TGF-b1 at 0.1, 1, and 10 ng/ml significantly reduced cellular proliferation rate by 37%, 44%, and 44%, respectively, whereas pan-TGF-b-neutralizing antibody increased proliferation by 40%. TGF-b1 (10 ng/ml) down-regulated MMP-9 by 54% and MMP-3 by 34% whereas TGF-b1-neutralizing antibody increased MMP-9 expression by 39%. Pancreatic stellate cells express both mediators of matrix remodeling and the regulatory cytokine TGF-b1 that, by autocrine inhibition of MMP-3 and MMP-9, may enhance fibrogenesis by reducing collagen degradation.    

4.130           Expression of the vesicular inhibitory amino acid transporter in pancreatic islet cells

Chessler, S.D., Simonson, W.T., Sweet, I.R. and Hammerle, L.P. Diabetes, 51, 1763-1771 (2002)   g-Aminobutyric acid (GABA) is stored in microvesicles in pancreatic islet cells. Because GAD65 and GAD67, which catalyze the formation of GABA, are cytoplasmic, the existence of an islet vesicular GABA transporter has been postulated. Here, we test the hypothesis that the putative transporter is the vesicular inhibitory amino acid transporter (VIAAT), a neuronal transmembrane transporter of GABA and glycine. We sequenced the human VIAAT gene and determined that the human and rat proteins share over 98% sequence identity. In vitro expression of VIAAT and immunoblotting of brain and islet lysates revealed two forms of the protein: an ~52-kDa and an ~57-kDa form. By immunoblotting and immuno-histochemistry, we detected VIAAT in rat but not human islets. Immunohistochemical staining showed that in rat islets, the distribution of VIAAT expression parallels that of GAD67, with increased expression in the mantle. GABA, too, was found to be present in islet non-b-cells. We conclude that VIAAT is expressed in rat islets and is more abundant in the mantle and that expression in human islets is very low or nil. The rat islet mantle differs from rat and human b-cells in that it contains only GAD67 and relatively increased levels of VIAAT. Cells that express only GAD67 may require higher levels of VIAAT expression.  

4.131           Characterization of a new subpopulation of mouse CD8⁺ B220⁺ dendritic cells endowed with type 1 interferon production capasity and tolerogenic potential

Martin, P. et al Blood, 100(2), 383-390 (2002)   We describe a new B220⁺ subpopulation of immaturelike dendritic cells (B220⁺ DCs) with low levels of expression of major histocompatibility complex (MHC) and costimulatory molecules and markedly reduced T-cell stimulatory potential, located in the thymus, bone marrow, spleen, and lymph nodes. B220⁺ DCs display ultrastructural characteristics resembling those of human plasmacytoid cells and accordingly produce interferon- after virus stimulation. B220⁺ DCs acquired a strong antigen-presenting cell capacity on incubation with CpG oligodeoxynucleotides, concomitant with a remarkable up-regulation of MHC and costimulatory molecules and the production of interleukin-12 (IL-12) and IL-10. Importantly, our data suggest that nonstimulated B220⁺ DCs represent a subset of physiological tolerogenic DCs endowed with the capacity to induce a nonanergic state of T-cell unresponsiveness, involving the differentiation of T regulatory cells capable of suppressing antigen-specific T-cell proliferation. In conclusion, our data support the hypothesis that B220⁺ DCs represent a lymphoid organ subset of immature DCs with a dual role in the immune systemexerting a tolerogenic function in steady state but differentiating on microbial stimulation into potent antigen-presenting cells with type 1 interferon production capacity.    

4.132           The volume set point of KCl cotransport in normal and sickle reticulocytes: effects of cell age and sulfhydryl reduction

Joiner, C.H., Rettig, R.K. and Franco, R.S. Abstracts of papers at the 56th annual meeting of the Society of General Physiologists (2002)   KCl cotransport (KCC) is excessively active in sickle red blood cells (SSRBC) and contributes to pathologi­cal dehydration of sickle reticulocytes. To explore the physiological basis for this pathological behavior, meth­odologies are needed that: (a) reflect KCC activity in the entire reticulocyte population (as opposed to a sub-population selected on the basis of cell density or re­sponse to osmotic stimuli) and (b) permit comparison of aged-matched populations of reticulocytes (SS vs. normal, AA). To this end, we have examined the changes in RBC density profile (as a surrogate marker for flux measurements) upon activation of KCC, cou­pled with flow cytometric detection of reticulocytes to track the density changes of this distinct population. Whole blood (SS or AA) was washed in isotonic HEPES-buffered saline (HBS) and treated with nystatin to adjust hemoglobin concentration (MCHC) to 30 g/dl. After incubation at 37°C under specified conditions and times, cells were washed in isotonic HBS pH 7.4 and subjected to density analysis on stepwise gradients prepared from OptiPrep®. The percentage of RBC at each density level was calculated from cell counts to give the RBC density profile. From the percentage of reticulocytes in each density fraction, the reticulocyte density profile was generated. A density score (DS) was calculated for RBC and reticulocytes as: DS = S (N X P_N) where N = gradient fraction number, P_N = per­centage of cells in fraction N. There was a linear corre­lation between DS and MCHC (r = 0.97) when gradi­ents were calibrated using cells treated with nystatin to yield various MCHC. Thus, the DS of a cell population could be used to calculate its MCHC. When KCC was activated by acidification at 37°C to pH 7.0 in HBS, retic density increased rapidly, and then stabilized between 1 and 2 h. Minimal changes occurred in nitrate me­dia. Thus, the rapid increase in MCHC is a manifesta­tion of KCC activity, and the MCHC obtained at 2 h (MCHC_final) reflects the “volume set point” (VSP) for KCC activity under those conditions. When adjusted to MCHC 30 g/dl, neither SS nor AA reticulocytes changed volume significantly when incubated at pH 7.4. However, when acidified to pH 7.0, both types of RBC became more dense; SS retics had a more rapid change in MCHC than AA retics, and MCHC_final was significantly higher in SS than in AA retics (SS, 35.6 ± 1.0 vs. AA 31.2 ± 1.0, n = 6, P < 0.01 by unpaired t test). Thus, the VSP of SS cells appears to be different from that of AA retics. When the maturity of SS reticulocytes was assessed by fluorescence intensity of reticulum staining by thiazole orange, older SS retics were found to have higher MCHC_final than younger cells (35.0 ± 0.2 vs. 34.0 ± 0.4, n = 6, P < 0.04 by paired t test), suggesting that VSP changes as reticulocytes mature. Finally, treatment of SS cells with the sulfhydryl reducing agent, dithiothrei­tol, lowered MCHC_final (control, 34.4 ± 1.3 vs. treated, 33.4 ± 1.2, n = 6, P < 0.002, paired t test), suggesting that sulfhydryl oxidation contributes to the abnormally high MCHC_final of SS retics. This is plausible in view of the known activation of KCC by sulfhydryl oxidation, and the increased oxidant stress and membrane oxida­tion seen in SS RBC.  

4.133           Marine invertebrate cell lines in the study of coral physiology and pathology

Johnston, C., Larkin, K., Woodley, C. and Morris, P.J. Marine Biomedicine and Environmental Sciences Annual Research Open House, Medical University of South Carolina. Poster Abstracts (2002)   Coral reefs are among the most productive and diverse ecosystems on earth. The scleractinians, or reef-building corals, like all living organisms, are prone to a variety of different diseases and stressors. Diseases of corals are having significant, negative impacts on the structure and appearance of coral reefs throughout the world. Bacteria, fungi, and cyanobacteria are known to cause diseases in corals, and changing environmental conditions and human impacts are suspected contributors to disease. However, the pathogens responsible for most diseases affecting reef organisms and the underlying mechanisms of pathogenesis are elusive and remain unknown. A coral cell line would be an invaluable tool for the study of coral physiology and disease pathology. Previous attempts at creating coral cell cultures have met with obstacles, and no marine invertebrate cell line exists. In this preliminary study, we assessed the viability of different cell types in culture from a marine invertebrate closely related to scleractinian corals, the sea anemone Aiptasia pallida. Several different cell types were dissociated from the anemone tissue using mechanical and chemical dissociation methods. The cells were separated on an Optiprep density gradient and cultured in Dulbecco’s modified Eagle media with heat-inactivated fetal bovine serum, antibiotics, and sterile seawater. Various concentrations of media and heat-inactivated fetal bovine serum were used to optimize primary culture conditions. Primary cultures remained viable up to six days without media change. Future studies will optimize cell viability in media supplemented with trace elements, light, growth factors, and growth substrates.

4.134           Motoneuron death triggered by a specific pathway downstream of Fas: Potentation by ALS-linked SOD1 mutations

Raoul, C. et al Neuron, 35, 1067-1083 (2002)   Death pathways restricted to specific neuronal classes could potentially allow for precise control of developmental neuronal death and also underlie the selectivity of neuronal loss in neurodegenerative disease. We show that Fas-triggered death of normal embryonic motoneurons requires transcriptional upregulation of neuronal NOS and involves Daxx, ASK1, and p38 together with the classical FADD/caspase-8 cascade. No evidence for involvement of this pathway was found in cells other than motoneurons. Motoneurons from transgenic mice overexpressing ALS-linked SOD1 mutants (G37R, G85R, or G93A) displayed increased susceptibility to activation of this pathway: they were more sensitive to Fas- or NO-triggered cell death but not to trophic deprivation or excitotoxic stimulation. Thus, triggering of a motoneuron-restricted cell death pathway by neighboring cells might contribute to motoneuron loss in ALS.  

4.135           Mouse CD11c⁺ B220⁺ Gr1⁺ plasmacytoid dendritic cells develop independently of the T-cell lineage

Ferrero, I., Held, W., Wilson, A., Tacchini-Cottier, F., Radtke, F. and MacDonald, H.R. Blood, 100, 2852-2857 (2002)   The developmental origin of dendritic cells (DCs) is controversial. In the mouse CD8a⁺ and CD8a^- DC subsets are often considered to be of lymphoid and myeloid origin respectively, although evidence on this point is conflicting. Very recently a novel CD11c⁺ B220⁺ DC subset has been identified that appears to be the murine counterpart to interferon alpha (IFNa)-producing human plasmacytoid DCs (PDCs). We show here that CD11c⁺ B220⁺ mouse PDCs, like human PDCs, are present in the thymus and express T lineage markers such as CD8a and CD4. However, the intrathymic development of PDCs can be completely dissociated from immature T lineage cells in mixed chimeras established with bone marrow cells from mice deficient for either Notch-1 or T-cell factor 1, two independent mutations that severely block early T-cell development. Our data indicate that thymic PDCs do not arise from a bipotential T/DCprecursor.  

4.136           Rehydration of high-density sickle erythrocytes in vitro

Holtzclaw, J.D., Jiang, M., Yasin, Z., Joiner, C.H. and Franco, R.S. Blood, 100, 3017-3025 (2002)   Recent studies have identified older low-density sickle red blood cells (SSRBCs) that were resistant to dehydration by valinomycin, a K⁺ ionophore. These cells, thought to derive from dense SSRBCs that have rehydrated, may represent a terminal cellular phase. To study rehydration, we subjected dense SSRBCs (r >1.107 g/cc) to either oxygenated incubation or rapid oxygenated/deoxygenated (oxy/deoxy) cycling (70 seconds per cycle). Light cells (r < 1.087 g/cc) were generated during both oxy incubation (2.9% ± 2.1%; n =42) and oxy/deoxy cycling (5.3%± 2.4%; n = 42). The rehydrated cells were K⁺-depleted (K⁺ = 20 ± 14 mmol/kg hemoglobin [Hb]) and Na⁺ -loaded (Na⁺ = 394 ± 106 mmol/kg Hb), and had high levels of external phosphatidylserine. In the presence of external calcium, the generation of rehydrated SSRBCs was inhibited during oxy/deoxy cycling, but the percentage with external phosphatidyl-serine increased. The calcium-mediated inhibition of rehydration was reversed by charybdotoxin, implying that rehydration was delayed in some cells by the Ca⁺⁺ -activated K⁺ channel. Pre-incubation of dense SSRBCs with DIDS (4,4’-di-isothiocyanato-2,2’-disulfostilbene)inhibited the generation of light cells during fast oxy/deoxy cycling, but not during oxy incubation. These results suggest that the sickling-induced pathway, previously implicated in SSRBC dehydration, may be involved in the deoxy-dependent component of rehydration for dense, K⁺-depleted cells. Light-cell generation was inhibited by 1mM bumetanide during both oxy incubation and oxy/deoxy cycling, providing evidence that a bumetanide-sensitive, deoxy-independent pathway, previously described in circulating light SSRBCs, also contributes to the rehydration of high-density SSRBCs.    

4.137           Isolation of purified oocyst walls and sporocysts from Toxoplasma gondii

Everson, W.V., Ware, M.W., Dubey, J.P. and Lindquist, H.D.

1. Eukaryot. Microbiol., 49(4), 344-349 (2002)

  Toxoplasma gondii oocysts are environmentally resistant and can infect virtually all warm-blooded hosts, including humans and livestock. Little is known about the biochemical basis for this resistance of oocysts, and mechanism for excystation of T. gondii sporozoites. The objective of the present study was to evaluate different methods (mechanical fragmentation, gradients, flow cytometry) to separate and purify T. gondii oocyst walls and sporocysts. Oocyst walls were successfully separated and purified using iodixanol gradients. Sporocysts were successfully separated and purified using iodixanol and Percoll gradients. Purification was also achieved by flow cytometry. Flow cytometry with fluorescence-activated cell sorting (FACS) yielded analytical quantities of oocyst walls and intact sporocysts. Flow cytometry with FACS also proved useful for quantitation of purity obtained following iodixanol gradient fractionation. Methods reported in this paper will be useful for analytical purposes, such as proteomic analysis of components unique to this life cycle stage, development of detection methods, or excystation studies.  

4.138           Correlation between RBC deformability and CR1 activity

Kamiyama, M., Inada, Y., Berezina, T., Zaets, S., Condon, M., Spolarics, Z., Kim, J., Deitch, E.A. and Machiedo, G. Proceedings of 25^th Annual Conference on Shock, June 2002.   We have previously presented data showing that binding of immune complex (IC) to CR1 on RBC membrane induced a decreased RBC deformability (RBCD). Objectives: In this study we investigated correlation between CR1 activity and RBCD by separating younger RBC with higher CR1 activity from older cells with lower CR1 activity. Further, we studied reversibility of RBCD after removal of IC from RBC CR1. Methods: RBC were fractionated according to their density using OptiPrep (iodixanol); RBC suspension was layered on the top of discontinuous 4-layered gradient and centrifuged for 30 mm. at 1,000 x g. Four fractions were harvested and EI (elongation index) of each fraction was measured after incubation in the presence and absence of IC (heat-aggregated human IgG) and complement. EI of RBC after removal of IC was also determined. Results: CR1 activity and EI at 3 Pa of each fraction are shown in the table:

Fraction	CR1	EI at 3 Pa -IC                         +IC
A:1.097 g/ml	++++	0.334±0.008*         0.276±0.016
B:1.099 g/ml	++++	0.289±0.043           0.244±0.042
C:1.101 g/ml	++	0.256±0.029           0.241±0.036
D:1.104 g/ml	-	0.238±0.044           0.230±0.046

*p<005 vs. +IC After removal of IC, EIs of all fractions were not significantly different from control levels (-IC). Electron microscopy revealed an increase in number of abnormally shaped RBC after incubation with IC and partial recovery of shape after removal of IC from RBC membrane. Conclusion: The results confirm that CR1 activity and deformability of RBC are closely related; the higher CR1 activity gives higher basal RBCD but makes the cell more susceptible to damage by IC. Our results also suggest that IC-induced change in RBC deformability is reversible.    

4.139           Sperm separation techniques: comparison and evaluation of gradient products

Tucker, K.E. and Jansen, C.A.M. In: Proceedings 2^nd International workshop for Embryologists: Troubleshooting activities in the ART lab. Ed. R. Basuray and D. Mortimer. (2002)   The human ejaculate is comprised of a mixture of seminal plasma, mature and immature spermatozoa, non-reproductive cells, various micro-organisms and non-specific debris. In preparation for intrauterine insemination (IUI) or in vitro fertilization (IVF), the motile, and hopefully, the most fertilizable population of sperm must be separated from the surrounding milieu. Many studies have been performed comparing direct semen processing procedures (i.e. simple wash, swim-up, etc.) with gradient separation techniques. Sperm separation using, a polyvinylpyrrolidone (PVP)-coated, silica-based density gradient (Percollâ), has been shown by numerous investigators to be an effective and relatively simple way to produce a viable, highly motile, morphologically normal and fertilizable population of sperm for use in both IUI and IVF (Mohan and Lindsay, 1995). The demands on sperm separation techniques have increased with our expanding knowledge of sperm physiology and on their contribution to the embryo. Because of this, there has been rising concern over the safety of any sperm separation procedure with respect to not only the viability of the sperm, but to the long-term effects of any resulting pregnancy. Although extremely effective in sperm separation and apparently safe for clinical use, Percoll (Pharmacia; Sigma Pharmaceutical) has been made unavailable for therapeutic use in human infertility. It has, therefore, become necessary to evaluate and select an alternative product or procedure that will compare favorably in light of all the studies that have advocated the use of Percoll for sperm separation. To address the need for Percoll-substitutes, a new line of density gradient products have been manufactured, specifically, the silane-coated, colloidal silica particle-based density gradients (i.e. PureSpermâ, Isolateâ, Enhance S+â). This paper will attempt to compare the efficacy of these new products with each other and with what has become the first choice for sperm separation, Percoll.  

4.140           Outbreak of cyclosporiasis associated with imported raspberries, Philadelphia, Pennsylvania, 2000

Ho, A.Y. et al Emerging Infectious Diseases, 8(8), 783-788 (2002)   An outbreak of cyclosporiasis occurred in attendees of the wedding reception held in Philadelphia, Pennsylvania, on June 10,2000. In a retrospective cohort study, 54 (68.4%) of the 79 interviewed guests and members of the wedding party met the case definition. The wedding cake, which had a cream filling that included raspberries, was the food item most strongly associated with illness (multivariate relative risk, 5.9; 95% confidence interval, 3.6 to 10.5). Leftover cake was positive for Cyclospora DNA by polymerase chain reaction analyses. Sequencing of the amplified fragments confirmed that the organism was Cyclospora cayetanensis. The year 2000 was the fifth year since 1995 that outbreaks of cyclosporiasis definitely or probably associated with Guatemalan raspberries have occurred in the spring in North America. Additionally, this is the second documented U.S. outbreak, and the first associated with raspberries, for which Cyclospora has been detected in the epidemiologically implicated food item.  

4.141           Long-lived immature dendritic cells mediated by TRANCE-RANK interaction

Cremer, I. et al Blood, 100(10), 3646-3655 (2002)   Immature dendritic cells (DCs) reside in interstitial tissues (int-DC) or in the epidermis, where they capture antigen and, thereafter, mature and migrate to draining lymph nodes (LNs), where they present processed antigen to T cells. We have identified int-DCs that express both TRANCE (tumor necrosis factor-related activation-induced cytokine) and RANK (receptor activator of NF-kB) and have generated these cells from CD34⁺ human progenitor cells using macrophage colony-stimulating factor (M-CSF). These CD34⁺-derived int-DCs, which are related to macrophages, are long-lived, but addition of soluble RANK leads to significant reduction of cell viability and Bcl-2 expression. This suggests that constitutive TRANCE-RANK interaction is responsible for CD34⁺-derived int-DC longevity. Conversely, CD1a⁺ DCs express only RANK and are short-lived. However, they can be rescued from cell death either by recombinant soluble TRANCE or by CD34⁺-derived int-DCs. CD34⁺-derived int-DCs mature in response to lipopolysaccharide (LPS) plus CD40 ligand (L) and become capable of CCL21/CCL19-mediated chemotaxis and naive T-cell activation. Upon maturation, they lose TRANCE, making them, like CD1a⁺ DCs, dependent on exogenous TRANCE for survival. These findings provide evidence that TRANCE and RANK play important roles in the homeostasis of DCs.

4.142           Toxoplasma gondii induces granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor secretion by human fibroblasts: implications for neutrophil apoptosis

Channon, J.Y., Miselis, K.A:, Minns, L.A, Dutta, C. and Kasper, L.H. Infection and Immunity, 70(11), 6048-6057 (2002)   Human neutrophils are rescued from apoptosis following incubation with once-washed, fibroblast-derived Toxoplasma gondii tachyzoites. Both infected and uninfected neutrophils are rescued, implicating a soluble mediator. In this study we investigated the origin and identity of this soluble mediator. Neutrophils were incubated either with purified tachyzoites or with conditioned medium derived from T. gondii-infected human fibroblasts. Conditioned medium was found to be a potent stimulus that delayed neutrophil apoptosis up to 72 h, whereas purified and extensively washed tachyzoites had no effect. Delayed apoptosis correlated with up-regulation of the neutrophil antiapoptotic protein, Mcl-1, and the neutrophil interleukin 3 receptor asubunit (IL-3Ra), suggesting a role for granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF and granulocyte colony-stimulating factor (G-CSF) were measurable in conditioned medium by enzyme-linked immunosorbent assay. Neutralizing antibodies to GM-CSF and G-CSF were additive in abrogating delayed neutrophil apoptosis induced by conditioned medium. Inhibitors of Src family tyrosine kinases, G_i proteins, phosphatidylinositol 3-kinase, p44^erk1 and p42^erk2 mitogen-activated protein kinases, and Jak2 kinases partially attenuated the effect of conditioned medium, consistent with a role for G-CSF and/or GM-CSF. Hence, delayed neutrophil apoptosis is mediated by GM-CSF and G-CSF secreted by T. gondii-infected human fibroblasts. This enhanced neutrophil survival may contribute to the robust proinflammatory response elicited in the T. gondii-infectedhost.  

4.143           Dynamic perifusion to maintain and assess isolated pancreatic islets

Sweet, I.R. et al Diabet. Techn. & Therapeut., 4(1), 67-76 (2002)   Advances in human islet transplant techniques are hampered by the inability to assess the quality of isolated islets. A flow culture system was developed to perifuse isolated pancreatic islets or cultured beta-cell lines in order to continuously and noninvasively assess cell function and viability with high kinetic resolution. Continuous perifusion of large amounts of islet tissue as isolated from human pancreata enables the use of noninvasive measurement technologies not previously applied to islets. To compare dynamic perifusion of tissue at high density with conventional static cultures, we measured glucose-stimulated insulin secretion and O₂ consumption of large amounts of INS-1 cells (45-65 × 10⁶) to confirm that perifused cells were adequately supplied with oxygen and nutrients and remained functionally responsive. Isolated human and monkey islets that were perifused for 18 h showed robust biphasic insulin secretion in response to a step increase in glucose, demonstrating the ability to maintain islets and the high kinetic resolution of the system. As an example of the system's ability to resolve multiple indicator dilution experiments, the retention of [³H]-glibenclamide was kinetically distinguished from that of an extracellular marker. In summary, the perifusion system is able to maintain healthy cells, assess insulin secretion and metabolite fluxes such as oxygen consumption and lactate production, and characterize the kinetics of the interaction between radiopharmaceuticals and islet cells. The ability to systematically assess the metabolic and functional viability of islets will facilitate the optimization of islet isolation procedures, islet transplantation studies, and islet storage methodologies.    

4.144           Isolation of living neurons from human elderly brains using the immunomagnetic sorting DNA-linker system

Konishi, Y., Lindhilm, K., Yang, L-B., Li, R. and Shen, Y. Am. J. Pathol., 161(5), 1567-1576 (2002)   Isolation and culture of mature neurons from affected brain regions during diseased states provide a well-suited in vitro model system to study age-related neurodegeneration under dynamic conditions at cellular levels. We have developed a novel technique to isolate living neurons from rapidly autopsied human elderly brains, and have succeeded in keeping them alive in vitro. Specifically, the parietal cortex blocks were fractionated by density gradients and further enriched for neurons by an immunomagnetic sorting DNA-linker technique. The postmortem interval averaged 2.6 hours. After isolation and purification of neurons using this technology, the cells were maintained in vitro for 2 weeks. Our evaluation revealed that 80% of the isolated cells were neurons and they exhibited neurotransmitter phenotypes (glutamate and g-aminobutyric acid) as well as glutamate receptors. Studies on cell viability and calcium influx suggest that these isolated living cortical neurons still retain their typical neuronal functions. Our present study demonstrates that neurons isolated from human elderly brain autopsies can survive in vitro and maintain their functional properties. Our study has opened an opportunity to apply such neurons to dynamic pharmacological studies of neurological disorders at the single-cell level.  

4.145           Expression of programmed death 1 ligands by murine T cells and APC

Yamazaki, T. et al

1. Immunol., 169, 5538-5545 (2002)

  Programmed death 1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral tolerance. Two ligands for PD-1, namely, B7-H1 (PD-L1) and B7-DC (PD-L2), have recently been identified as new members of the B7 family but their expression at the protein level remains largely unknown. To characterize the expression of B7-H1 and B7-DC, we newly generated an anti-mouse B7-H1 mAb (MIH6) and an anti-mouse B7-DC mAb (TY25). MIH6 and TY25 immunoprecipitated a single molecule of 43 and 42 kDa from the lysate of B7-H1 and B7-DC transfectants, respectively. Flow cytometric analysis revealed that B7-H1 was broadly expressed on the surface of mouse tumor cell lines while the expression of B7-DC was rather restricted. PD-1 was expressed on anti-CD3-stimulated T cells and anti-IgM plus anti-CD40-stimulated B cells at high levels but was undetectable on activated macrophages or DCs. B7-H1 was constitutively expressed on freshly isolated splenic T cells, B cells, macrophages, and dendritic cells (DCs), and up-regulated on T cells by anti-CD3 stimulation on macrophages by LPS, IFN-g, GM-CSF, or IL-4, and on DCs by IFN-g, GM-CSF, or IL-4. In contrast, B7-DC expression was only inducible on macrophages and DCs upon stimulation with IFN-g, GM-CSF, or IL-4. The inducible expression of PD-1 ligands on both T cells and APCs may suggest new paradigms of PD-1-mediated immune regulation.  

4.146           Oligodendrocyte progenitor cells in the adult rat CNS express myelin oligodendrocyte glycoprotein (MOG)

Li, G., Crang, A.J., Rundle, J.L. and Blakemore, W.F. Brain Pathol., 12, 463-471 (2002)   While the effects of high dose X-irradiation on mitotically active progenitor cells and remyelination are well-documented, its effects on myelinating oligodendrocytes are less clear, due in part to divergent views on their mitotic capacity. To examine the effect of X-irradiation on oligodendrocytes, the spinal cord of rats was exposed to 40 Gy of X-irradiation and the number of oligodendrocytes and oligodendrocyte progenitors in the dorsal funiculi at T12 and L1 was determined by in situ hybridization using cRNA-probes for platelet derived growth factor a receptor (PDGFRa) (to identify oligodendrocyte progenitors), exon 3b of proteolipid protein (PLP) (to identify mature oligodendrocytes) and myelin oligodendrocyte glycoprotein (MOG). X-irradiation resulted in no change in the number of PLP positive cells and no loss of myelin internodes, but caused an almost complete loss of PDGFRa-expressing cells, and a reduction in the number of MOG positive cells to a number similar to that found using the PLP exon 3b probe. Importantly, the number of radiation-sensitive MOG-expressing cells was similar to the number of PDGFRa positive cells. To determine if the radiation-sensitive MOG positive cells were the same population as the radiation sensitive PDGFRa-expressing cells, MOG and PDGFRa-expressing cells were isolated from the adult CNS using antibody coated magnetic beads. Twelve to thirteen percent of MOG positive cells were PDGFRa positive and nearly all the PDGFRa isolated cells were MOG and galactocerebroside positive. Double immunofluorescence revealed colocalization of NG2 and MOG on cells in the normal adult rat spinal cord. These results show that in situ in the adult rat spinal cord white matter oligodendrocyte progenitors are MOG positive and indicates that expression of MOG cannot be regarded a marker that only identifies mature myelin-supporting oligodendrocytes in tissue.  

4.147           Proteasome-dependent regulation of Syk tyrosine kinase levels in human basophils

Youssef, L.A., Pharm, B., Wilson, B.S. and Oliver, J.M.  Allergy Clin. Immunol., 110, 366-373 (2002)   Background: In human basophils, FceRI signal initiation, leading to histamine release, relies on activation of Syk protein tyrosine kinase. Basophils from approximately 10% of unselected donors do not degranulate in response to FceRI cross-linking. Their unresponsiveness has been linked to the absence of Syk protein despite apparently normal levels of Syk mRNA. Objective: The aim of this study was to explore pathways of Syk protein degradation as a possible posttranslational mechanism for downregulating Syk protein levels in human basophils and other leukocytes. Methods: Highly purified basophils, lymphocytes, and monocytes were incubated in the presence or absence of a panel of cell-permeable inhibitors of proteolytic degradation pathway(s). Subsequently, the protein level of Syk tyrosine kinase was determined by means of Western blotting. In vitro assays were conducted through use of immunoprecipitated basophil Syk and a rabbit reticulocyte lysate system. Results: Three inhibitors of proteasome-mediated degradation—PSI, lactacystin, and ALLN—substantially increased Syk levels in releaser basophils and restored Syk expression in nonreleaser basophils. Caspase inhibitors were less effective, and inhibitors of calpain-mediated proteolysis had no effect. Among other leukocytes tested, only naive CD4⁺ T cells had more Syk after proteasome inhibitor treatment. In vitro ubiquitination assays demonstrated that Syk is readily ubiquitinated in vitro and also that Syk ubiquitination is associated with a substantial decrease in total levels of Syk protein. Conclusion: These data provide evidence for a ubiquitin/proteasome-dependent mechanism that contributes to Syk regulation in human basophils and might also be relevant to naive T cells. Understanding this regulatory pathway might lead to strategies for suppressing allergic inflammation while preserving essential Syk-mediated functions in other hematopoietic cells.  

4.148           Tip-growing cells of the moss Ceratodon purpureus are gravitrophic in high-density media

Schwuchow, J.M., Kern, V.D. and  Sack, F.D. Plant Physiol., 130, 2095-2100 (2002)   Gravity sensing in plants and algae is hypothesized to rely upon either the mass of the entire cell or that of sedimenting organelles (statoliths). Protonemata of the moss Ceratodon purpureus show upward gravitropism and contain amyloplasts that sediment. If moss sensing were whole-cell based, then media denser than the cell should prevent gravitropism or reverse its direction. Cells that were inverted or reoriented to the horizontal displayed distinct negative gravitropism in solutions of iodixanol with densities of 1.052 to 1.320 as well as in bovine serum albumin solutions with densities of 1.037 to 1.184 g cm^-3. Studies using tagged molecules of different sizes and calculations of diffusion times suggest that both types of media penetrate through the apical cell wall. Estimates of the density of the apical cell range from 1.004 to 1.085. Because protonemata grow upward when the cells have a density that is lower than the surrounding medium, gravitropic sensing probably utilizes an intracellular mass in moss protonemata. These data provide additional support for the idea that sedimenting amyloplasts function as statoliths in gravitropism.  

4.149           Continuous measurement of oxygen consumption by pancreatic islets

Sweet, I.R. et al Diabet. Techn. & Therapeut., 4(5), 661-672 (2002)   The rate of oxygen consumption is an important measure of mitochondrial function in all aerobic cells. In pancreatic beta cells, it is linked to the transduction mechanism that mediates glucose-stimulated insulin secretion. However, measurement of oxygen consumption over long periods of time is technically difficult owing to the error resulting from baseline drift and the challenge of measuring small changes in oxygen tension. We have adapted an ultrastable oxygen sensor based on the detection of the decay of the phosphorescent emission from an oxygen-sensitive dye to a previously developed islet flow culture system. The drift of the sensor is approximately 0.3%/24 h, allowing for the continuous measurement of oxygen consumption by 300 islets (or about 6 x 10⁵ cells) for hours or days. Rat islets placed in the perifusion chamber for 24 h were well maintained as reflected by membrane integrity, insulin secretion, and oxygen consumption. Both acute changes in oxygen consumption as induced by glucose and chronic changes as induced by sequential pulses of azide were resolved. The features of the flow culture system-aseptic conditions, fine temporal control of the composition of the media, and the collection of outflow fractions for measurement of insulin, and other products-facilitate a systematic approach to assessing metabolic and functional viability in responses to a variety of stimuli. Applications to the measurement of effects of hypoxia on insulin secretion, membrane integrity, and the redox state of cytochromes are demonstrated. The system has particular application to the field of human islet transplantation, where assessment and the study of islet viability have been hampered by a lack of experimental methods.  

4.150           Nef protein of human immunodeficiency virus and lipopolysaccharide induce expression of CD14 on human monocytes through differential utilization of interleukin-10

Creery, D. et al Clin. Diagnost. Lab. Immunol., 9(6), 1212-1221 (2002)   We investigated the expression of membrane-bound CD14 (mCD14) on monocytes and soluble CD14 (sCD14) released into the culture supernatants of peripheral blood lymphocytes (PBMC) from human immunodeficiency virus (HIV)-infected individuals. Monocytes from HIV-positive individuals exhibited both enhanced mCD14 expression and sCD14 production in the PBMC culture supernatants compared to the levels of mCD14 and sCD14 in HIV-negative individuals. This enhanced mCD14 expression and sCD14 production in HIV-infected individuals may be due to the effects of cytokines, the bacterial product lipopolysaccharide (LPS), and/or the HIV regulatory antigens Tat and Nef. Interleukin-10 (IL-10), an immunoregulatory cytokine, as well as LPS enhanced mCD14 expression and the release of sCD14 in the culture supernatants. HIV-Nef, unlike Tat, enhanced mCD14 expression on monocytes but did not induce the release of sCD14 into the culture supernatants. Studies conducted to investigate the mechanism underlying HIV-Nef-induced mCD14 expression revealed that HIV-Nef upregulated mCD14 expression via a mechanism that does not involve endogenously produced IL-10. In contrast, LPS upregulated the expression of mCD14 and increased the release of sCD14 via a mechanism that involves, at least in part, endogenously produced IL-10. Furthermore, dexamethasone, an anti-inflammatory and immunosuppressive agent, inhibited HIV-Nef-induced CD14 expression in an IL-10-independent manner. In contrast, dexamethasone inhibited IL-10-dependent LPS-induced CD14 expression by interfering with IL-10-induced signals but not by blocking IL-10 production. These results suggest that HIV-Nef and IL-10 constitute biologically important modulators of CD14 expression which may influence immunobiological responses to bacterial infections in HIV disease.  

4.151           Insulin treatment of mice recipients preserves b-cell function in porcine islet transplantation

Pakhomov, O. et al Cell Transplantation, 11(7), 721-728 (2002)   Encapsulation of islets of Langerhans confers protection against cell-mediated immune destruction and so should allow the transplantation of islets without immunosuppression. Xenotransplantation of encapsulated islets of Langerhans might therefore help overcome problems of human organ donor shortage. Given that islets exposed to sustained hyperglycemia show impaired b-cell function, we set out to determine whether recipient treatment with insulin could improve transplantation success rate. Islets of Langerhans were obtained from Specific Germ-Free (SPF) pig pancreas and cultured overnight. Islets were encapsulated in AN69 fibers and implanted into the peritoneal cavity of diabetic mice. A group of implanted mice was treated with exogenous insulin from day 3 to day 7 after grafting. Islet implantation depressed plasma glucose in all the mice, both insulin treated and untreated. Glycemia slowly increased in the non-insulin-treated mice, whereas the decrease observed in the insulin-treated mice was maintained until day 29 of follow-up. We found significant differences between the two groups (p < 0.05 at day 18 and day 20, p < 0.001 at day 23 and day 29). No improvement of hyperglycemia was observed in diabetic mice implanted with empty fibers. When islet-containing fibers were removed from the peritoneal cavity of mice 1 month after the graft plasma glucose increased markedly. We demonstrate that treatment of recipients with exogenous insulin in the immediate posttransplantation period has a positive effect on b-cell function in transplanted macroencapsulated porcine islets.  

4.152           Efficacy of the oxygen-charged static two-layer method for short-term pancreas preservation and islet isolation from nonhuman primate and human pancreata

Matsumoto, S. et al Cell Transplantation, 11(8), 769-777 (2002)   Previous reports indicate that the two-layer method (TLM) of human pancreas preservation is superior to University of Wisconsin solution (UW) when pancreata are preserved for extended periods (i.e., >24 h) prior to islet isolation. In this study, the efficacy of using the TLM for preserving pancreata for short periods (i.e., <13 h) was evaluated using both nonhuman primate and human pancreata preserved with a TLM kit precharged with oxygen. An oxygen precharged TLM (static TLM) was established and compared with the original TLM with continuous oxygen supply. For the static TLM, the perfluorochemical was fully oxygenated and the oxygen supply removed prior to pancreas preservation. In the primate model, pancreata were preserved by the static TLM, the original TLM, and UW for 5 h prior to islet isolation. In the human model, pancreata were preserved with the static TLM or the original TLM or UW for 4–13 h. Both primate and human pancreata were processed by intraductal collagenase injection and digestion followed by continuous density gradient purification to isolate islets. Islets were assessed for islet yield, purity, viability, and in vitro functionality. In the primate model, islet yield, viability, and in vitro functionality were significantly improved by both the static TLM and the original TLM with similar results. Postculture islet yields were 23,877 ± 3619 IE/g in the static TLM, 21,895 ± 3742 IE/g in the original TLM, and 6773 ± 735 IE/g in UW. In the human model, both the static TLM and the original TLM significantly increased islet yield compared with UW with postculture islet yields of 2659 ± 549 IE/g in the static TLM, 2244 ± 557 IE/g in the original TLM, and 1293 ± 451 IE/g in UW. Nonhuman primate and human pancreata stored in the static TLM, immediately upon procurement, yield isolated islets of a substantially higher quantity than when pancreata are stored in UW. Thus, the use of the static TLM should replace the use of UW for storage of pancreata during transport prior to islet isolation.  

4.153           The morphology of islets within the porcine donor pancreas determines the isolation result: Successful isolation of pancreatic islets can now be achieved from young market pigs

Krickhahn, M., Bühler, C., Meyer, T., Thiede, A. and Ulrichs, K. Cell Transplantation, 11(8), 827-838 (2002)   Clinical islet allotransplantation has become an increasingly efficient “routine” therapy in recent years. Shortage of human donor organs leads to porcine pancreatic islets as a potential source for islet xenotransplantation. Yet it is still very difficult to isolate sufficient numbers of intact porcine islets, particularly from young market pigs. In the following study islets were successfully isolated from retired breeders [4806 ± 720 islet equivalents per gram organ (IEQ/g); n = 25; 2–3 years old; RB] and also from young hybrid pigs [2868 ± 260 IEQ/g; n = 65; 4–6 months old; HY] using LiberasePI and a modified version of Ricordi’s digestion-filtration technique. As expected, isolations from RB showed significantly better results (p < 0.002). A retrospective histological analysis of almost all donor pancreases showed that the majority of organs from RB (80%) contained mainly large islets (diameter >200 mm), in contrast to only 35% of all pancreases from HY. Remarkably, the islet size in situ, regardless whether detected in RB or HY, strongly determined the isolation result. A donor organ with predominantly large islets resulted in significantly higher numbers of IEQs compared with a donor organ with predominantly small islets [RB^{Large Islets}: 5680 ± 3,318 IEQ/g (n = 20); RB^{Small Islets}: 1353 ± 427 IEQ/g (n = 5); p < 0.02]. In addition, isolation results were strongly influenced by the quality of the LiberasePI batch, and therefore single batch testing is invariably required. Purification was performed using Ficoll or OptiPrep^TM density gradient centrifugation manually or in the COBE cell processor. Although islet purity was highest when OptiPrep^TM was used, final islet yields did not differ between the different purification methods. Our study demonstrates that islet size in situ is an extremely critical parameter for highly successful islet isolation; consequently, we are now performing a morphological screening of each donor organ prior to the isolation process. Under these conditions highly successful isolations can reliably be performed even from young market pigs.  

4.154           Elevated C-met in thymic dendritic cells of New Zealand black mice

Okada, T. et al Develop. Immunol., 9(1), 29-34 (2002)   New Zealand Black (NZB) mice are a well-known animal model of human autoimmune disease. Although the mechanism for development of autoimmunity is unclear, NZB mice are well known for severe thymic microarchitecture abnormalities. It is thought that thymic dendritic cells (DC) may play a role in thymic education and contribute to the autoimmune process. To address this issue and, in particular, that qualitative and/or quantitative differences exist in thymic DC, we took advantage of a novel restriction analysis system that allow definition of differences in the expression of tyrosine kinases using highly enriched populations of thymic DC from NZB compared to BALB/c and C57BL/6 mice. The method chosen, restriction analysis of gene expression, allowed the determination of protein tyrosine kinase transcription profiles. We report herein that NZB mice have a significant upregulation of C-met compared to the control strains. The abnormality of the C-met transcription was confined to thymic DC. We believe that its abnormal expression reflects the resistance of thymic cells to apoptosis, which will ultimately lead to defects and/or abnormal signaling by the interaction of thymic DC and thymocytes. Further studies involving such interactions are under way.  

4.155           Comparison of nitric oxide production and motion characteristics after 3-layer Percoll and IxaPrep preparation methods of human sperm

Ding, D-C., Huang, Y-C-, Liu, J-Y and Wu, G-J Arch. Gynecol. Obstet., 266, 210-213 (2002)   Objektive: To compare two different spermatozoa preparation mediums, the three-layer Percoll (Sigma, St. Louis, MO; salica-based) and the IxaPrep (Medicult, Copenhagen, Denmark; non-salica based, polysucrose medium) method, with respect to recovery of pregressive motile sperm and various sperm motion characteristics. Analysis was determined by computer-aided sperm analysis and nitric oxide (NO) production of the supernatant after centrifugation. Method: Thirty-nine semen specimens were obtained from men who presented for semen analysis and each of them was divided into two aliquots for preparation with the two mediums mentioned above. The motile sperm recovery, motility percentage and motion parameters were measured for each semen specimen (n=39) before and after preparation using one of the two above methods. The NO was measured using the chemiluminscence method after centrifugation. Results: Recovery rate was higher in the IxaPrep group (Ixaprep: 45.4±28.7% versus Percoll: 32.3±22.7%; p<0.05). The other motion characteristics such as average path velocity (VAP) and straight line velocity (VSL) were better than those of fresh semen samples [VAP: 72±17.2 µm/s (Percoll), 62.8±18.2 µm/s (Ixaprep) vs 52.2±9.5 µm/s (fresh); p<0.05; VSL: 51.8±13.4 µm/s (Percoll), 44.8±12.9 µm/s (Ixaprep) vs 38.6±7.9 µm/s (fresh); p<0.05]. The motility between fresh and post-preparation semen samples had no significant difference. Hyperactivation of the sperm was improved in the IxaPrep group compared with fresh sperm (Percoll: 24.7±16.9% and IxaPrep: 20.5±10.5% versus fresh: 9.2±9.2%; p<0.05). NO produced in the IxaPrep method was significantly lower than that in the Percoll method (Ixaprep: 0.24±0.3 µM versus Percoll: 0.54±0.91 µM; p<0.05). Conclusion: Our data suggests that the IxaPrep method provides a better recovery rate, but that other motion characteristics did not demonstrate any significant difference. The lower level of NO produced in the IxaPrep preparation method may suggest that better sperm quality achieved is due to the decreased NO production.  

4.156           Mucin gene (MUC1) transfected dendritic cells as vaccine: results of a phase I/II clinical trial

Pecher, G., Häring, A., Kaiser, L. and Thiel, E. Cancer Immunol. Immunother., 51, 669-673 (2002)   Dendritic cells (DC) derived from peripheral blood monocytes are currently being investigated in clinical trials for their role in stimulating the immune system. We performed a phase I/II clinical trial using human autologous DC transfected with cDNA of the human tumor antigen mucin (MUC1) as a vaccine in 10 patients with advanced breast, pancreatic or papillary cancer. After liposomal transfection, flow cytometry testing showed that 2% to 53% of the DC expressed mucin epitopes. Patients were immunized two or three times with 1 million transfected DC injected subcutaneously (s.c.). A vaccine-specific delayed-type hypersensitivity (DTH) reaction was observed in 3 out of 10 patients. After vaccination, 4 patients showed a 2- to 10-fold increase in the frequency of mucin-specific interferon-gamma (IFN-g)-secreting CD8⁺ T cells. We demonstrated the feasibility and safety of a vaccine consisting of autologous gene-transfected DC, and that immunologic responses could be induced even in patients with pretreated and advanced disease.  

4.157           Microchimerism and transmission of porcine endogenous retrovirus from a pig cell line or specific pathogen-free pig islets to mouse tissues and human cells during xenografts in nude mice

Clemenceau, B., Jegou, D., Martignat, L, and Saï, P. Diabetologia, 45, 914-923 (2002)   Aims/hypothesis. Pig islets could transmit porcine endogenous retroviruses (PERV) to diabetic patients. Our previous work showed that pig islets expressed low levels of PERV mRNA and were not likely to transmit PERV to human cells in vitro. The real risk of infection during pig tissue xenografts can only be evaluated by in vivo experiments. Methods. Nude mice bearing tumours containing human 293 cells were grafted with specific pathogen-free pig islets or PERV-producing pig PK15 cells to determine whether pig cells could transmit PERV to mouse and human cells in vivo. Infection was monitored by PCR, long PCR, RT-PCR and long RT-PCR. As detection of PERV sequences could be due to the presence of residual pig cells, we looked for pig mitochondrial (mt) DNA. Quantitative PCR for PERV and pig mt DNA was done to compare the PERV-to-pig mt (P-to-M) ratio of each sample with the reference ratio for grafted pig cells. Results. Among 78 mouse tissues from PK15-grafted mice, 54 and 72 were positive for gag and pig mt DNA, respectively. Human tumours developed in these mice were positive for PERV (78%) and pig mt (89%). The P-to-M ratios for mouse tissues and PERV-positive human tumours from PK15-grafted mice were higher than the ratio in PK15 cells. Among 41 tissues from pig islet cell-grafted mice, 7 were positive for PERV (3 lymph nodes, 1 kidney, 2 salivary glands, 1 ovary), and 14 were positive for pig mt DNA. Three of these samples (1 lymph node, 1 kidney and 1 salivary gland) were positive for gag DNA, but negative for pig mt DNA. One human tumour in these mice was positive for PERV DNA. P-to-M reference ratio in grafted islet cells was 0.05+-0.03. The three PERV-positive lymph nodes contained 78 gag/3 mt copies (P-to-M: 26), 101 gag/3 mt copies (P-to-M: 34), and 4 gag/0 mt copies. The two PERV-positive salivary glands contained 14 gag/1 mt copies, and 28 gag/0 mt copies. The ovary and the kidney contained 46 gag/3 mt and 69 gag/0 mt copies, respectively. The PERV-positive human tumour contained 47 gag/3 mt copies. Conclusions/interpretation. Microchimerism and PERV transmission were frequently observed in both mouse and human tissues during grafting of pig PK15 cells into nude mice bearing human tumours, and sometimes during pig islet xenograft in this model. This strengthens the notion that there is a risk of transmitting PERV during pig islet xenograft.  

4.158           Microglia promote glioma migration

Bettinger, I., Thanos, S. and Paulus, W. Acta Neuropathol., 103, 351-355 (2002)   Diffuse astrocytic gliomas extensively infiltrate brain tissue and contain numerous microglial cells, but it is unknown whether these two characteristic features are pathogenetically related. We therefore studied the effects of murine microglial cells on motility of GL261 mouse glioma cells using Boyden chamber assays. In the presence of microglia, glioma cell migration occurred earlier, and after 48 h it was threefold higher as compared to incubations without microglia. This effect was mediated by substances released from microglia, because similar effects were observed by microglia-conditioned medium, and it was specific to microglia, because oligodendroglia and endothelial cells only weakly stimulated glioma cell migration. Microglia activating substances (GM-CSF, LPS) led to a further increase of motility. These data support the notion that microglia accumulation in diffuse glial tumors does not merely represent a nonspecific reaction to tissue injury, but reflects participation of these cells in supporting and promoting the invasive phenotype of astrocytoma cells.  

4.159           The expression of scavenger receptor class B, type I (SR-BI) and caveolin-1 in parenchymal and nonparenchymal liver cells

Malerød, L., Juvet, L.K., Gjøen, T. and Berg, T, Cell Tissue Res., 307, 173-180 (2002)   The liver is the major site of cholesterol synthesis and metabolism, and the only substantive route for eliminating blood cholesterol. Scavenger receptor class B, type I (SR-BI) has been reported to be responsible for mediating the selective uptake of high-density lipoprotein cholesteryl esters (HDL-CE) in liver parenchymal cells (PC). We analysed the expression of SR-BI in isolated rat liver cells, and found the receptor to be highly expressed in liver PC at both the mRNA and protein levels. We also found SR-BI to be expressed in liver endothelial cells (LEC) and Kupffer cells (KC). SR-BI has not previously been reported to be present in LEC. CD36 mRNA was expressed in all three liver cell types. Since caveolin-1 appears to colocalize with SR-BI and CD36 in caveolae of several cell lines, the distribution and expression of caveolin-1 in the liver cells were investigated. Caveolin-1 was not detected in PC but was found in both LEC and KC. This led to the suggestion that caveolin-1 may be more important in the efflux of cholesterol than in the selective uptake of cholesterol in the liver.  

4.160           Feeding NOD mice with pig splenocytes induces transferable mechanisms that modulate cellular and humoral xenogeneic reactions aginst pig spleen or islet cells

You, S., Gouin, E. and Sai, P. Clin. Exptl. Immunol., 127, 412-422 (2002)   We have reported previously that oral administration of pig cells to NOD mice modified xenogeneic cellular response against pig islet cells (PICs), and hypothesized that it may have induced active sup­pression. This preliminary report evaluated only the effect of feeding pig cells by ‘primary’ prolifera­tion, i.e. when splenocytes from fed mice are confronted with pig cells in vitro. The present study also considered ‘secondary’ proliferation and cytokine production after feeding and subsequent in vivo graft of pig cells. Additionally, serum IgM and IgG isotypes were quantified by ELISA using pig target cells. Induction of active mechanism by feeding was hypothetical, which led us here to transfer splenocytes from mice fed pig spleen cells (PSC) and evaluate ‘primary’ (after transfer) and ‘secondary’ (after trans­fer and subsequent graft of pig cells) proliferations and cytokine secretions in recipient mice. We also determined whether the effects of feeding pig cells persisted after depression of suppressor mechanisms by cyclophosphamide. Mice fed with PSC displayed increased ‘primary’ splenocyte proliferation to PSC or PIC (P < 0.0001), while ‘secondary’ responses were decreased (P < 003) in those fed PSC and subsequently grafted with PSC. The increased ‘primary’ and decreased ‘secondary’ proliferations were reduced (P < 0.04) by pretreatment with cyclophosphamide. The IL-l0/ and IL-4/lFNg ratios produced in response to PSC increased (P < 004) in mice fed and grafted with PSC compared to those grafted only with PSC. IgM and IgG levels against pig cells were, respectively, increased (P < 0.04) and decreased (P < 0.04) in mice fed and grafted with PSC. IgG2a and IgG2b, but not IgG1, levels were lower (P < 0.01). These effects of feeding PSC on ‘secondary’ proliferation, cytokine and antibody productions, were not detected when mice were fed PSC only after graft with PSC. Transfer with splenocytes from mice fed PSC increased ‘primary’ proliferation of splenocytes from recipient mice in response to PSC (P < 0.02) or PIC (P < 0.05). After transfer with splenocytes from PSC-fed mice and graft with PSC, ‘secondary’ proliferation to pig cells were reduced (P < 0.04), and the IL-l0/lFNg ratio produced in response to PSC was increased fourfold. Thus, oral administration of PSC induces active transferable mechanisms, characterized by a biphasic pattern with early increased ‘primary’ xenogeneic cellular reactions to both PSC and PIC, followed by decreased ‘secondary’ responsiveness and a con­comitant shift of the Thl/Th2 balance towards greater Th2 influence. Decreased responsiveness may be due to active suppression, even though induction of anergy or deletion cannot be excluded.    

4.161           High efficiency gene transfer into cultured primary rat and human hepatic stellate cells using baculovirus vectors

Gao, R. et al Liver 22, 15-22 (2002)   Background/aims: Gene transfer into hepatic stellate cells (HSC) is inefficient when using plasmid-based transfection methods; viral-based systems are therefore being developed. A baculovirus system has recently been shown to be useful for expressing genes in mammalian cells. The aim of this study was to determine if baculovirus vectors can infect and express target genes in rat and human HSC and to assess potential cytotoxic and modulatory effects of infection. Methods: A recombinant baculovirus vector (AcCALaZ) carrying the LacZ gene was used to infect HSC. b-Ga1actosidase assays and electron microscopy were used to determine efficiency of infection and gene expression. Counting of trypan blue negative cells was used to assess cytotoxic/cytostatic effects of infection. Measurement of protein content of cells and a-smooth muscle actin expression were performed to assess the effects of baculovirus on cell function/phenotype. Results: Baculovirus infection of activated HSC was highly efficient (>90%) and provided long-term LacZ gene expression (15 days) in the absence of cytotoxic, cytostatic or modulatory effects. Infection of freshly isolated cells was also observed but at lower levels (20%). Conc/usjons: Baculovirus vectors can therefore be used to deliver target genes to cultured rat and human HSC with high efficiency and longevity in the absence of detrimental effects on cell function.  

4.162           Proteome analysis of rat polymorphonuclear leukocytes: A two-dimensional electrophoresis/ mass spectrometry approach

Piubelli, C., Galvani, M., Hamdan, M., Domenici, E. and Righetti, P.G. Electrophoresis, 23(2), 298-310 (2002)   The development of a two-dimensional (2-D) map of rat polymorphonuclear (PMN) leukocytes is here reported for the first time. The map is built up by utilizing a wide immobilized pH gradient (IPG), pH 3–10, in the first dimension and also a narrower IPG pH 4.5–8.5 gradient. In addition, the map is constructed by adopting the most recent protocols in 2-D mapping, which call for reduction and alkylation of the sample prior to the start of any electrophoretic step, including the IPG dimension. Fifty-two major protein spots have been so far identified by utilizing both matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray quadrupole (Q)-TOF mass spectrometry. A large number of house-keeping and cytoskeleton proteins were detected, together with proteins which are specific to PMN organelles or related to PMN functions such as phagocytosis and chemotaxis. The results obtained demonstrate the possibility of obtaining a single 2-D gel based proteomic map of PMN with representative proteins from different cellular compartments, also including membrane components, allowing the study of PMN protein expression on a proteome-wide scale. The aim of this project is to build an extensive database of such proteins, to be utilized for future studies where the expression of PMN proteins is used as a disease- or drug treatment marker.  

4.163           Cross-presentation of virus-like particles by skin-derived CD8^– dendritic cells: a dispensable role for TAP

Ruedl, C., Storni, T., Lechner, F., Bächl, T. and Bachmann, M.F. Eur. J. Immunol., 32(3), 818-825 (2002)   Virus-like particles (VLP) induce efficient CTL responses although they do not carry any genetic information. Here, we analyzed MHC class I associated presentation of VLP-derived CTL-epitopes in vivo. After intradermal injection of VLP containing the immunodominant epitope (p33) of lymphocytic choriomeningitis virus (p33-VLP), presentation of peptide p33 in draining lymph nodes was largely restricted to CD8^– skin-derived dendritic cells (DC). Surprisingly, and in contrast to findings with tumor cells, TAP1-deficient DC and macrophages mediated efficient cross-presentation of VLP-derived p33 in vivo and in vitro. However, the ability of TAP1-deficient DC to cross-present p33-VLP was reduced compared to wild-type DC, indicating that in DC, both TAP-dependent and TAP-independent pathways were operative. In contrast, macrophages cross-presented p33-VLP normally in the absence of TAP. The TAP-dependent pathway of cross-presentation is therefore confined to DC while both macrophages and DC harbor the TAP-independent pathway. In summary, the results show that VLP-derived epitopes are cross-presented by CD8^– DC in vivo in a partial TAP-independent fashion and highlight important differences in the processing machinery of DC versus macrophages.      

4.164           Effective induction of acquired resistance to Listeria monocytogenes by immunizing mice with in vivo-infected dendritic cells

Sashinami, H., Nakane, A., Iwakura, Y. And Sasaki, M. Infect.and. Immun.,71(2), 117-125 (2003)   Splenic dendritic cells (DCs) obtained from mice at 48 h after Listeria monocytogenes infection exhibited up-regulation of CD80 and produced higher titers of gamma interferon (IFN-g) and interleukin-12 (IL-12) than did DCs obtained from uninfected mice. Mice immunized with DCs obtained from mice that had been infected with L. monocytogenes 48 h before acquired host resistance to lethal infection with L. monocytogenes at 4 and 8 weeks. Immunization with DCs from heat-killed L. monocytogenes failed to induce resistance. Acquired antilisterial resistance is specific, since the immunized mice could not be protected from Salmonella enterica serovar Typhimurium infection. Infected DCs stimulated proliferation of naive CD4⁺ and CD8⁺ cells in vitro, suggesting that in vivo-infected DCs activate CD8⁺ T cells, which are critical in acquired antilisterial resistance, as well as CD4⁺ T cells. When wild-type mice were immunized with DCs from IFN-g-deficient mice, they were protected against a lethal L. monocytogenes challenge. In contrast, when mice were immunized with DCs from anti-IL-12 p40 monoclonal antibody-injected mice, they failed to gain acquired antilisterial resistance. These results suggest that DC-derived IL-12, but not IFN-g, may play a critical role in induction of acquired antilisterial resistance. Our present results suggest that splenic DCs obtained from mice infected with L. monocytogenes in vivo may be an effective immunogenwith which to induce antigen-specific immunity.  

4.165           Acetylcholine promotes the proliferation and collagen gene expression of myofibroblastic hepatic stellate cells

Oben, J.A., Yang, S., Lin, H., Ono, M. and Diehl. A.M. Biochem. Biophys. Res. Comm., 300, 172-177 (2003)   The mechanisms that initiate and perpetuate the fibrogenic response, during liver injury, are unclear. Animal studies, however, strongly support a role for the autonomic nervous system (ANS) in wound healing. Therefore, the ANS may also mediate the development of cirrhosis. Hepatic stellate cells (HSC), the liver's major matrix-producing cells, are activated by injury to become proliferative, fibrogenic myofibroblasts. HSC respond to sympathetic neurotransmitters by changing phenotype, suggesting that HSC may be the cellular effectors of ANS signals that modulate hepatic fibrogenesis during recovery from liver damage. We show here that the parasympathetic neurotransmitter acetylcholine markedly stimulates the proliferation of myofibroblastic HSC and induces HSC collagen gene expression in these cells. By extending evidence that HSC are direct targets of the ANS, these results support the proposed neuroglial role of HSC in the liver and suggest that interrupting ANS signalling may be useful in constraining the fibrogenic response to liver injury.  

4.166           CD4 T cell priming in dendritic cell-deficient mice

Castiglioni, P. et al Int. Immunol., 15(1), 127-136 (2003)   Bone marrow (BM) chimeras (BMC) generated from mice carrying a null (–/–) mutation in the relB gene of the NF-kB family represent an ideal model for in vivo studies on the role of dendritic cells (DC) in the adaptive immune response. The spleen and lymph nodes (LN) of relB^–/– BMC contain a small number of residual DC, mainly CD8a⁺, that fail to up-regulate MHC class II and co-stimulatory molecules after stimulation in vitro. Moreover, residual spleen DC of relB^–/– BMC have a 4-fold decrease in the ability to uptake and process soluble model antigen, ovalbumin (OVA), and failed to prime CD4 and CD8 T cells in vitro and in vivo. In addition, they also failed to present OVA peptide to OT-II transgenic T lymphocytes at a normal 1:10 (stimulator:responder) cell ratio. In spite of these multiple DC defects, relB^–/– BMC immunized with plasmid DNA targeted to the spleen as the site of immune induction develop a specific CD4⁺ T cell response comparable to that of relB competent mice. These data demonstrate that CD4 ⁺ T cells can be primed in the absence of functional DC and suggest that relB may gauge the T cell response in vivo.  

4.167           Interleukin-6 protects anterior horn neurons from lethal virus-induced injury

Pavelko, K. et al

1. Neurosci., 23(2), 481-492 (2003)

  We evaluated the role of interleukin-6 (IL-6) in neuronal injury after CNS infection. IL-6^-/- and IL-6^+/+ mice of resistant major histocompatibility complex (MHC) H-2^b haplotype intracerebrally infected with Theiler's virus cleared the infection normally without development of viral persistence, lethal neuronal infection, or late phase demyelination. In contrast, infection of IL-6^-/- mice on a susceptible H-2^q haplotype resulted in frequent deaths and severe neurologic deficits within 2 weeks of infection as compared with infected IL-6^+/+ H-2^q littermate controls. Morphologic analysis demonstrated dramatic injury to anterior horn neurons of IL-6^-/- H-2^q mice at 12 d after infection. Infectious viral titers in the CNS (brain and spinal cord combined) were equivalent between IL-6^-/- H-2^q and IL-6^+/+ H-2^q mice. In contrast, more viral RNA was detected in the spinal cord of IL-6^-/- mice compared with IL-6^+/+ H-2^q mice. Virus antigen was localized predominantly to anterior horn cells in infected IL-6^-/- H-2^q mice. IL-6 deletion did not affect the humoral response directed against virus, nor did it affect the expression of CD4, CD8, MHC class I, or MHC class II in the CNS. Importantly, IL-6 was expressed by astrocytes of infected IL-6^+/+ mice but not in astrocytes of IL-6^-/- mice or uninfected IL-6^+/+ mice. Furthermore, expression of various chemokines was robust at 12 d after infection in both H-2^b and H-2^q IL-6^-/- mice, indicating that intrinsic CNS inflammatory responses did not depend on the presence of IL-6. Finally, in vitro analysis of virus-induced death in neuroblastoma-spinal cord-34 motor neurons and primary anterior horn cell neurons showed that IL-6 exerted a neuroprotective effect. These data support the hypothesis that IL-6 plays a critical role in protecting specific populations of neurons from irreversible injury.    

4.168           Adrenaline inhibits lipopolysaccharide-induced macrophage inflammatory protein-1a in human monocytes: the role of b-receptors

Li, C-Y. et al Anesth. Analg., 96, 518-523 (2003)   Macrophage inflammatory protein-1a (MIP-1a) has an important role in the development of inflammatory responses during infection by regulating leukocyte trafficking and function. Our study was conducted to investigate the effect of adrenaline on lipopolysaccharide (LPS)-induced MIP-1a production by human peripheral blood monocytes and human monocytic THP-1 cells. Monocytes were incubated in vitro with LPS for 4 h at 37°C in the presence and absence of adrenaline and/or specific a- and ß-adrenergic receptor antagonists and agonists. The effects of adrenaline on MIP-1a synthesis were studied at the protein level by using enzyme-linked immunosorbent assays and at the messenger RNA level by using reverse transcriptase-polymerase chain reaction. Adrenaline inhibited LPS-induced MIP-1a production in a dose-dependent manner. The suppressive effect could be completely prevented by propranolol, but not by phentolamine. The specific ß-adrenergic agonist isoproterenol produced the same inhibitory effect on LPS-induced MIP-1a production, whereas the a-adrenergic agonist phenylephrine had a minimal effect. In addition, suppression of MIP-1a production was associated with an increase of intracellular cyclic adenosine monophosphate (cAMP) by the cell membrane-permeable cAMP analog dibutyryl-cAMP. Furthermore, we found that adrenaline inhibited LPS-induced MIP-1a messenger RNA expression. These findings suggest that adrenaline can modulate MIP-1a production in inflammatory diseases and sepsis.  

4.169           Transcutaneous immunization with cholera toxin B subunit adjuvant suppresses IgE antibody responses via selective induction of Th1 immune responses

Anjuere, F. et al

1. Immunol., 170, 1586-1592 (2003)

  Topical application of cholera toxin (CT) onto mouse skin can induce a humoral immune response to CT as well as to coadministered Ags. In this study, we examined the nontoxic cell-binding B subunit of CT (CTB) as a potential adjuvant for cutaneous immune responses when coadministered with the prototype protein Ag, OVA. CTB applied onto skin induced serum Ab responses to itself with magnitudes comparable to those evoked by CT but was poorly efficient at promoting systemic Ab responses to coadministered OVA. However, transcutaneous immunization (TCI) with either CT or CTB and OVA led to vigorous OVA-specific T cell proliferative responses. Furthermore, CTB potentiated Th1-driven responses (IFN-g production) whereas CT induced both Th1 and Th2 cytokine production. Coadministration of the toxic subunit CTA, together with CTB and OVA Ag, led to enhanced Th1 and Th2 responses. Moreover, whereas TCI with CT enhanced serum IgE responses to coadministered OVA, CTB suppressed these responses. TCI with either CT or CTB led to an increased accumulation of dendritic cells in the exposed epidermis and the underlying dermis. Thus, in contrast to CT, CTB appears to behave very differently when given by the transcutaneous as opposed to a mucosal route and the results suggest that the adjuvanticity of CT on Th1- and Th2-dependent responses induced by TCI involves two distinct moieties, the B and the A subunits, respectively.  

4.170           Microtubule-disruption-induced and chemotactic-peptide-induced migration of human neutrophils: implications for differential sets of signaling pathways

Niggli, V.

1. Cell Sci., 116, 813-822 (2003)

  Neutrophil granulocytes rely on a functional actin network for directed migration. Microtubule disassembly does not impair receptor-linked chemotaxis, instead it induces development of polarity and chemokinesis in neutrophils concomitant with polarized distribution of a-actinin and F-actin. Cells stimulated with colchicine, which disassembles microtubules, migrate with a speed comparable to cells exposed to chemotactic peptide. We investigated signaling pathways involved in colchicine-induced neutrophil polarization and migration. Colchicine-induced development of polarity was insensitive to treatment with pertussis toxin, in contrast to chemotactic-peptide-induced shape changes, which were completely abolished by this treatment. Thus, colchicine does not appear to act via activating heterotrimeric G_i proteins. Colchicine does also not seem to act via phosphatidylinositol 3-kinase, as it failed to induce phosphorylation of its downstream  target Akt and the potent phosphatidylinositol 3-kinase inhibitor wortmannin failed to inhibit colchicine-induced shape changes. By contrast, wortmannin significantly reduced chemotactic-peptide-induced shape changes. However, the Rho-kinase inhibitor Y-27632 (10 mM) inhibited colchicineinduced development of polarity by 95±3% (n=5) and chemokinesis by 76±9% (n=3), which suggests that the Rho-Rho-kinase pathway has a crucial role in polarity and migration. Indeed, treatment of cells with colchicine induced a significant increase in membrane-bound Rho-kinase II, which is indicative of activation of this protein. This membrane translocation could be prevented by taxol, which stabilizes microtubules. Colchicine also induced a marked increase in myosin light chain phosphorylation, which could be suppressed by Y-27632 and by taxol. In summary, we provide evidence that microtubule disassembly induces in neutrophils a selective activation of Rho-kinase, bypassing activation of heterotrimeric Gi proteins and phosphatidyl-inositol 3-kinase. This process is sufficient for induction of chemokinesis and mediates increased  phosphorylation of myosin light chain and accumulation of F-actin and a-actinin in the leading edge.  

4.171           Cationic lipid and polymer based gene delivery to human pancreatic cells

Mahato, R.I. et al Mol. Therapy, 7, 89-100 (2003)   Transplantation of pancreatic islets has great potential for treating Type I diabetes. Ex vivo gene therapy may promote re-vascularization or inhibit apoptosis of the islets and promote graft. In this study, we investigated the feasibility of non-viral gene delivery using Enhanced Green Fluorescent Protein (EGFP) and human Vascular Endothelial Growth Factor (hVEGF₁₆₅) expression plasmids as model reporter and therapeutic genes. LipofectAMINE/pDNA and Superfect/pDNA complexes showed high transfection efficiency in rapidly dividing Jurkat cells, but low transfection in non-dividing human islets. LipofectAMINE/pCAGGS-hVEGF transfected islets showed relatively higher levels of hVEGF than in those transfected with LipofectAMINE/pCMS-EGFP complexes or 5% glucose. To exclude endogenously secreted hVEGF, real time RT-PCR experiment was repeated using pCAGGS vector-specific forward primer and hVEGF gene-specific reverse primer. In this case, both non-transfected islets and the islets transfected with LipofectAMINE/pCMS-EGFP complexes showed negligible amplification of hVEGF. On glucose challenge, insulin release from LipofectAMINE/pCAGGS-hVEGF transfected human islets increased from 10.78±4.56 to 65±5 ng/ml, suggesting little adverse effect on islet b cell response to glucose challenge. The low transfection efficiency is due to the islets being a cluster of approximately 1000 non-dividing cells. This underscores the importance of experimentation with the actual human islets  

4.172           Granulocyte-macrophage colony stimulating factor is an anti-apoptotic cytokine for thymic dendritic cells and a significant modelator of their accessory function

Vasilijic, S., Cilic, M. and Vucevic, D. Immunology letters, 86, 99-112 (2003)   Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a growth-promoting factor for myeloid-derived dendritic cells (DC) but not for lymphoid DC. The data about its effect on thymic DC (TDC), which are both of lymphoid and myeloid origin, are very scarce. Using an in vitro model, we demonstrated in this work that GM-CSF significantly increased the survival of rat TDC in culture by inhibiting their apoptosis and the effect correlated with up-regulation of Bcl-2 expression. GM-CSF also stimulated differentiation and maturation of TDC as judged by higher expression of MHC class I and II molecules, CD54, CD80 and CD86. These changes correlated with stronger stimulatory activity of GM-CSF-pulsed TDC in syngeneic thymocyte proliferation assay and MLR. The stimulatory potential of TDC was further increased when thymocytes were cultivated with an anti-ab TCR (R73) monoclonal antibody (mAb). The influence of unstimulated TDC on proliferation of thymocytes was inhibited by anti-CD86 but not anti-CD80 mAb, whereas in cultures with GM-CSF-treated TDC both mAbs exerted an additive blocking effect. After separation of TDC on CD11b⁺ and CD11b^- we demonstrated that GM-CSF inhibited apoptosis and potentiated accesory activity of both TDC subsets independently of the myeloid marker expression. Cummulatively, our results suggest that GM-CSF is one of the regulatory cytokine involved in survival, maturation, differentiation and accessory function of TDC.  

4.173           IkBa-dependent regulation of low-shear flow-induced NF-kB activity: role of nitric oxide

Mohan, S. et al Am. J.Cell Physiol., 284, C1039-C1047 (2003)   We have investigated the role of inhibitor kBa (IkBa) in the activation of nuclear factor kB (NF-kB) observed in human aortic endothelial cells (HAEC) undergoing a low shear stress of 2 dynes/cm². Low shear for 6 h resulted in a reduction of IkBa levels, an activation of NF-kB, and an increase in kB-dependent vascular cell adhesion molecule 1 (VCAM-1) mRNA expression and endothelial-monocyte adhesion. Overexpression of IkBa in HAEC attenuated all of these shear-induced responses. These results suggest that downregulation of IkBa is the major factor in the low shear-induced activation of NF-kB in HAEC. We then investigated the role of nitric oxide (NO) in the regulation of IkBaNF-kB. Overexpression of endothelial nitric oxide synthase (eNOS) inhibited NF-kB activation in HAEC exposed to 6 h of low shear stress. Addition of the structurally unrelated NO donors S-nitrosoglutathione (300 µM) or sodium nitroprusside (1 mM) before low shear stress significantly increased cytoplasmic IB and concomitantly reduced NF-kB binding activity and kB-dependent VCAM-1 promoter activity. Together, these data suggest that NO may play a major role in the regulation of IkBa levels in HAEC and that the application of low shear flow increases NF-kB activity by attenuating NO generation and thus IkBa levels.  

4.174           Norepinephrine and neuropeptide Y promote proliferation and collagen gene expression of hepatic myofibroblastic stellate cells

Oben, J.A., Yang, S., Lin, H., Ono, M. and  Diehl, A.M. Biochem. Biophys. Res. Comm., 302, 685-690 (2003)   The mechanisms initiating and perpetuating the fibrogenic response in the injured liver are not well understood. Hepatic stellate cells are activated by liver injury to become proliferative and fibrogenic myofibroblasts. Emerging evidence suggests that the sympathetic nervous system may play a role in the development of cirrhosis. It is not known, however, whether this requires a direct interaction between sympathetic neurotransmitters and stellate cell receptors, or results indirectly, from sympathetic effects on the vasculature. Using cultured hepatic stellate cells, we show that the sympathetic neurotransmitters, norepinephrine and neuropeptide Y, markedly stimulate the proliferation of activated, myofibroblastic, hepatic stellate cells. Norepinephrine, but not neuropeptide Y, also induces collagen gene expression. In conclusion, physiologically relevant concentrations of sympathetic neurotransmitters directly modulate the phenotype of hepatic stellate cells. This suggests that targeted interruption of sympathetic nervous system signaling in hepatic stellate cells may be useful in constraining the fibrogenic response to liver injury.    

4.175           A novel I-branching b-1,6-N-acetylglucosaminyltransferase involved in human blood group I antigen expression

Inaba, N. et al Blood, 101, 2870-2876 (2003)   The human blood group i and I antigens are determined by linear and branched poly-N-acetyllactosamine structures, respectively. In erythrocytes, the fetal i antigen is converted to the adult I antigen by I-branching b-1,6-N-acetylglucosaminyltransferase (IGnT) during development. Dysfunction of the I-branching enzyme may result in the adult i phenotype in erythrocytes. However, the I gene responsible for blood group I antigen has not been fully confirmed. We report here a novel human I-branching enzyme, designated IGnT3. The genes for IGnT1 (reported in 1993), IGnT2 (also presented in this study), and IGnT3 consist of 3 exons and share the second and third exons. Bone marrow cells preferentially expressed IGnT3 transcript. During erythroid differentiation using CD34⁺ cells, IGnT3 was markedly up-regulated with concomitant decrease in IGnT1/2. Moreover, reticulocytes expressed the IGnT3 transcript, but IGnT1/2 was below detectable levels. By molecular genetic analyses of an adult i pedigree, individuals with the adult i phenotype were revealed to have heterozygous alleles with mutations in exon 2 (1006G>A; Gly336Arg) and exon 3 (1049G>A; Gly350Glu), respectively, of the IGnT3 gene. Chinese hamster ovary (CHO) cells transfected with each mutated IGnT3 cDNA failed to express I antigen. These findings indicate that the expression of the blood group I antigen in erythrocytes is determined by a novel IGnT3, not by IGnT1 or IGnT2.  

4.176           A chemokine, interferon (IFN)-g-inducible protein 10 kDa, is stimulated by IFN-t and recruits immune cells in the ovine endometrium

Hagaoka, K. et al Biol. Reprod., 68, 1413-1421 (2003)   Proper distribution of immune cells in the uterus is a prerequisite for successful implantation and subsequent placentation, but biochemical signals that govern such events have not been well characterized. In the present study, the cDNA of a chemokine, interferon (IFN)-g -inducible protein 10 kDa (IP-10), was identified from a cDNA subtraction study between uterine endometrial tissues from Day 17 pregnant and Day 15 cyclic ewes. The effect of IFN- on IP-10 expression and the involvement of IP-10 in the recruitment of immune cells were then investigated. Northern blot analysis revealed that large amounts of IP-10 mRNA were present during conceptus attachment to maternal endometrium and early placentation. IP-10 mRNA was localized to monocytes distributed in the subepithelial stroma of pregnant but not cyclic uteri. This finding was supported by the discovery of IP-10 mRNA expression in monocytes but not in lymphocytes, uterine epithelial cells, or stromal cells. Moreover, the expression of IP-10 mRNA by the monocytes was stimulated by IFN-a, IFN-g, and IFN-t in a dose-dependent manner, but the expression of IP-10 mRNA by the endometrial explants was most stimulated by IFN-t. In a chemotaxis assay, migration of peripheral blood mononuclear cells was stimulated by the addition of IFN-t stimulated-endometrial culture medium, and the effect was significantly reduced by neutralization with an anti-IP-10 antibody. These results suggest that endometrial IP-10 regulated by conceptus IFN-t regulates recruitment and/or distribution of immune cells seen in the early pregnant uterus.  

4.177           PPARg-dependent anti-flammatory action of rosiglitazone in human monocytes: suppression of TNFa secretion is not mediated by PTEN regulation

Hong, G., Davis, B., Khatoon, N., Baker, S.F. and Brown J. Biochem. Biophys. Res. Comm., 303, 782-787 (2003)   Thiazolidinediones (TZDs) are insulin-sensitising drugs that are ligands for the nuclear receptor PPARg. They have been shown to inhibit PMA-stimulated secretion of TNFa from human monocytes, although only at concentrations well in excess of circulating levels observed during TZD therapy, suggesting a mechanism of action independent of PPARg activation. Here we show that insulin-sensitising concentr-ations of the TZD rosiglitazone partially inhibit serum- or LPS- (but not PMA-) stimulated TNFa secretion from primary human monocytes, with an IC₅₀ of around 50 nM. We also show that the observed effects are independent of PPARg-mediated regulation of the lipid phosphatase PTEN. Reversed stimulus specificity, IC₅₀ in the insulin-sensitising range, and the fact that partial inhibition of TNFa secretion is also observed with a structurally unrelated PPARg agonist, GW7845, demonstrate a mechanism of action distinct from that observed with higher TZD concentrations. These findings thus represent the first report of a PPARg-dependent and therapeutically relevant anti-inflammatory action of TZDs in isolated human monocytes.

4.178           Bovine dendritic cells generated from monocytes and bone marrow progenitors regulate immunoglobulin production in peripheral blood B cells

Pinchuk, L.M., Boyd, B.L., Kruger, E.F., Roditi, I. and Furger, A. Comp. Immuun. Microbiol. Infect. Dis., 26 233-249 (2003)   We examined whether bovine monocyte-derived and bone marrow (BM) dendritic cells (DCs) regulate antibody production in activated peripheral blood B cells. DCs were generated from monocytes and BM progenitors in the presence of bovine recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). Monocyte-derived DCs promoted B cells activated by the anti-CD3 triggered CD4⁺ T cells or through immunoglobulin M (IgM) receptor to increase the level of IgG secretion. Furthermore, the addition of DCs triggered B cells activated through IgM receptors to produce IgG2 and IgA, thus inducing an isotype switch. BM-derived DCs increased the production of IgG in B cells activated by the anti-CD3 triggered CD4⁺ T cells, but unlike monocyte-derived DCs did not have any effect on B cells activated through surface IgM. These data suggest that the regulation of humoral immune responses in cattle depends on the origin of DCs and the mode of B cell activation.  

4.179           Monocyte-derived IL12, CD86 (B7-2) and CD40L expression in relapsing and progressive multiple sclerosis

Filion, L.G., Matusevicius, D., Graziani-Bowering, G.M., Kumar, A. and Freedman, M. Clin. Immunol., 106 127-138 (2003)   Multiple sclerosis has been postulated to be an autoimmune disease in which Th1 immune responses predominate. This response is associated with an increased production of IFNg and IL12 produced by T cells and by cells of the monocyte (MO) lineage, respectively. An increased expression of costimulatory molecules by T cells and antigen-presenting cells is also observed. We hypothesized that in relapsing–remitting MS (RRMS) (with or without of IFNb treatment) and in secondary progressive patients (SPMS) IL12 and costimulatory molecules (CD80 [B7-1], CD86 [B7-2], CD28, CD40, CD40L) would be differentially produced or expressed by MO or T cells. We performed cross-sectional and longitudinal flow cytometric studies (at monthly intervals) on peripheral blood mononuclear cells (PBMC) or on MO from SPMS or untreated and IFNb-treated patients with RRMS. We determined that CD86 and CD40L expression was highest on MO derived from SPMS patients compared to those from RRMS or from healthy controls (HC). In vitro culture of PBMC with recombinant human IL10, a cytokine that may be increased in response to treatment with IFNb and that down-regulates CD86 expression, reduced the expression of CD86 on MO derived from RRMS patients to a much higher degree compared to cells derived from SPMS or HC. In vitro secreted IL12 levels from freshly isolated MO from SPMS patients were more than 10-fold higher than either the treated or the untreated RRMS or HC. RRMS patients treated with IFNb demonstrated slightly lower levels of MO IL12 secretion. Our data suggest that a key mechanism in the pathogenesis of MS is the increased expression of CD86 and CD40L and the increased production of IL12 during disease progression. Part of the mechanism of action of IFNb may be to reduce MO CD86 and CD40L expression and IL12 secretion; failure to do so might signify either a lack of response or a transition to a more progressive phase of illness.    

4.180           Monocyte-derived cytokines in multiple sclerosis

Filion, L.G., Graziani-Bowering, G., Matusevicius, D. and Freedman, M.S. Clin. Exp. Immunol., 131, 324-334 (2003)   MS is an inflammatory, presumably autoimmune, disease mediated by the activation of T cells, B cells and monocytes (MO). Inflammation is thought to occur early during the relapsing-remitting phase of MS (RRMS), whereas in the later phases of MS such as secondary progressive MS (SPMS), inflammation tends to diminish. Our objective was to compare the types and amounts of proinflammatory and regulatory cytokines produced by MO from relapsing-remitting patients with or without treatment with IFN-b (RRMS⁺ therapy, RRMS^- therapy), respectively, from secondary progressive patients (SPMS) and from healthy controls (HC). MO were isolated by a density-gradient technique and three different techniques (RNase protection assay, ELISA and intracellular cytokine staining) were used to assess cytokine levels. An increase in IL6, IL12 and TNF-a was observed by all three methods for RRMS^- therapy and for SPMS patients compared to HC and RRMS⁺ therapy patients. We conclude that proinflammatory and regulatory monokines can be derived from MO of MS patients and that these levels are modulated by IFN-b therapy. Although it is believed that inflammation tends to diminish in SPMS patients, our data show that inflammatory cytokines continue to be released at high levels, suggesting that IFN-b or IL10 treatment may be beneficial for this group.  

4.181           Improved islet yields from Macaca Nemestrina and marginal human pancreata after two-layer method preservation and endogenous trypsin inhibition

Matsumoto, S., Rigley, T.H., Reems, J.A., Kuroda, Y. and Stevens, R.B. Am. J. Transplant., 3, 53-63 (2003)   We tested whether two-layer method (TLM) pancreas preservation and trypsin inhibition (Pefabloc) during processing allows longer preservation while retaining or improving viable islet recovery. Non-marginal primate (Macaca nemestrina) and marginal human (ischemic or preservation-injured) pancreata were processed with a research-oriented pan technique (Seattle method). Organs were processed upon arrival (± Pefabloc), or after TLM or University of Wisconsin solution (UW) preservation (+ Pefabloc). Islet yield, viability, and function were assessed. Pefabloc increased M. nemestrina islet yields from 9696 ± 1749 IE/g to 15 822 ± 1332 IE/g (p < 0.01). Two-layer method preservation (< 6 h) further increased yields, to 23 769 ± 2773 IE/g (vs. + Pefabloc; p < 0.01). Similarly, Pefabloc increased marginal human islet yields from 2473 ± 472 IE/g to 4723 ± 1006 IE/g (p < 0.04). This increase was maintained after lengthy TLM preservation (> 30 h; 4801 ± 1066 IE/g). We also tested the applicability of TLM preservation (23.5 ± 3.2 h) to the processing of marginal human pancreata by the Edmonton/Immune Tolerance Network clinical protocol. Islet yield and function approached published results of pancreata processed 4.8 ± 0.8 h after organ recovery (p = 0.06). Pefabloc, and TLM vs. UW preservation, prolonged the tolerable interval between organ recovery and islet isolation. Islet yield, viability, and functionality improved from both marginal and nonmarginal pancreata.    

4.182           Liver sinusoidal endothelial cells represent an important blood clearance system in pigs

Nedredal, G.I. et al Comp. Hepatol., 2, 1-14 (2003)   Background: Numerous studies in rats and a few other mammalian species, including man, have shown that the sinusoidal cells constitute an important part of liver function. In the pig, however, which is frequently used in studies on liver transplantation and liver failure models, our knowledge about the function of hepatic sinusoidal cells is scarce. We have explored the scavenger function of pig liver sinusoidal endothelial cells (LSEC), a cell type that in other mammals performs vital elimination of an array of waste macromolecules from the circulation. Results: ²⁵I-macromolecules known to be cleared in the rat via the scavenger and mannose receptors were rapidly removed from the pig circulation, 50% of the injected dose being removed within the first 2-5 mm following injection. Fluorescently labeled microbeads (2 mm in diameter) used to probe phagocytosis accumulated in Kupffer cells only, whereas fluorescently labeled soluble macromolecular ligands for the mannose and scavenger receptors were sequestered only by LSEC. Desmin-positive stellate cells accumulated no probes. Isolation of liver cells using collagenase perfusion through the portal vein, followed by various centrifugation protocols to separate the different liver cell populations yielded 280 x 10⁷ (range 50-890 x 10⁷) sinusoidal cells per liver (weight of liver 237. I g (sd 43.6)). Use of specific anti-Kupffer cell- and anti-desmin antibodies, combined with endocytosis of fluorescently labeled macromolecular soluble ligands indicated that the LSEC fraction contained 62 x l0⁷ (sd 12 x 10⁷) purified LSEC. Cultured LSEC avidly endocytosed ligands for the mannose and scavenger receptors. Conclusions: We show here for the first time that pig LSEC, similar to what has been found earlier in rat LSEC, represent an effective scavenger system for removal of macromolecular waste products from the circulation.  

4.183           Biological sample collection and processing for molecular epidemiological studies

Holland, N.T., Smith, M.T., Eskenazi, B. and Bastaki, M. Mutation Res., 543, 217-234 (2003)   Molecular epidemiology uses biomarkers and advanced technology to refine the investigation of the relationship between environmental exposures and diseases in humans. It requires careful handling and storage of precious biological samples with the goals of obtaining a large amount of information from limited samples, and minimizing future research costs by use of banked samples. Many factors, such as tissue type, time of collection, containers used, preservatives and other additives, transport means and length of transit time, affect the quality of the samples and the stability of biomarkers and must be considered at the initial collection stage. An efficient study design includes provisions for further processing of the original samples, such as cryopreservation of isolated cells, purification of DNA and RNA, and preparation of specimens for cytogenetic, immunological and biochemical analyses. Given the multiple uses of the samples in molecular epidemiology studies, appropriate informed consent must be obtained from the study subjects prior to sample collection. Use of barcoding and electronic databases allow more efficient management of large sample banks. Development of standard operating procedures and quality control plans is a safeguard of the samples’ quality and of the validity of the analyses results. Finally, specific state, federal and international regulations are in place regarding research with human samples, governing areas including custody, safety of handling, and transport of human samples, as well as communication of study results. Here, we focus on the factors affecting the quality and the potential future use of biological samples and some of the provisions that must be made during collection, processing, and storage of samples, based on our experience in the Superfund Basic Research Program and Children’s Environmental Health Center, at the University of California, Berkeley.    

4.184           Basal expression of IkBa is controlled by the mammalian transcriptional repressor RBP-J (CBF1) and its activator Notch1

Oakley F. et al

1. Biol. Chem., 278, 24359-24370 (2003)

  By using the hepatic stellate cell (HSC) as a paradigm for cells that undergo long term re-programming of NF-kB dependent transcription, we have determined a novel mechanism by which mammalian cells establish their basal  NF-kB activity. Elevation of NF-kB activity during HSC activation is accompanied by induction of CBF1 expression and DNA binding activity. We show that the transcriptional repressor CBF1 interacts with a dual NF-kB/ CBF1-binding site (kB2) in the IkBa pro­moter. Nucleotide substitutions that disrupt CBF1 bind­ing to the kB2 site result in an elevation of IkBa pro­moter activity and loss of responsiveness of the promoter to a transfected CBF1 reporter vector. Over-expression of CBF1 in COS1 cells was associated with markedly reduced IkBa protein expression and elevated NF-kB DNA binding activity. CBF1-induced repression of IkBa promoter activity was reversed in HSC trans­fected with the Notch1 intracellular domain (NICD). The ability of NICD to enhance IkBa gene transcription was confirmed in COS1 cells and was found to be dependent on an intact RAM domain of NICD that has been shown previously to help mediate the interaction of NICD with CBF1. One of the mechanisms by which NICD is thought to convert CBF1 into an activator of transcription is via the recruitment of transcriptional co-activators/histone acetylases to gene promoters. Co-transfection of HSC with NICD and p53 caused a diminution of IkBa pro­moter activity, by contrast overexpression of p300 en­hanced IkBa promoter function. Taken together, these data suggest that basal IkBa expression (and as a con­sequence NF-kB activity) is under the control of the various components of the CBF1/Notch signal transduc­tion pathway.  

4.185           Characterization of C5aR expression on murine myeloid and lymphoid cells by the use of a novel monoclonal antibody

Soruri, A., Kim, S., Kiafard, Z. and Zwirner, J. Immunol. Lett., 88, 47-52 (2003)   The anaphylatoxin C5a is a potent proinflammatory stimulus with immunomodulatory activities. Expression of its receptor C5aR (CD88) has been detected on cells of myeloid origin such as granulocytes and monocytes/ macrophages. However, controversial results exist on the expression of C5aR on T and B lymphocytes as well as on mature dendritic cells (DC). The aim of the present study was to characterize expression of C5aR protein on myeloid and lymphoid cells in the mouse. For this purpose, rat monoclonal antibodies with specificity against the murine C5aR were generated. Using these reagents a distinct amount of C5aR antigen was observed on neutrophils and macrophages. In contrast, C5aR protein was not detectable on resting or stimulated murine T or B lymphocytes. Furthermore, no C5aR protein could be observed on splenic CD11c positive DC which have been classified in the literature as relatively mature. Taken together, our results suggest that in the mouse expression of C5aR protein may be restricted to leukocytes of myeloid origin whereas previous evidence for C5aR expression on lymphoid cells may be reevaluated.  

4.186           Neuropeptide Y is neuroproliferative for hippocampal presursor cells

Howell, O.W. et al.

1. Neurochem., 86, 646-659 (2003)

  New neurones are produced in the adult hippocampus throughout life and are necessary for certain types of hippo­campal learning. Little, however, is known about the control of hippocampal neurogenesis. We used primary hippocampal cultures from early post-natal rats and neuropeptide Y Y1 receptor knockout mice as well as selective neuropeptide Y receptor antagonists and agonists to demonstrate that neu­ropeptide Y is proliferative for nestin-positive, sphere-forming hippocampal precursor cells and b-tubulin-positive neuro­blasts and that the neuroproliferative effect of neuropeptide Y is mediated via its YI receptor. Immunohistochemistry confirmed Y1 receptor staining on both nestin-positive cells and b-tubulin-positive cells in culture and short pulse 5-bromo-2-deoxyuridine studies demonstrated that neuropeptide Y has a proliferative effect on both cell types. These studies suggest that the proliferation of hippocampal neuroblasts and precur­sor cells is increased by neuropeptide Y and, therefore, that hippocampal learning and memory may be modulated by neuropeptide Y-releasing interneurones.  

4.187           Islet transplants and impact on secondary diabetic complications: does C-peptide protect the kidney

Shapiro, A. M. J.

1. Am. Soc. Nephrol., 14, 2214-2216 (2003)

  Extract Hering et al. have integrated a number of important steps including: PFC pancreas transportation; careful donor and re­cipient selection; a new iodixanol non-ficoll based purification gradient; islet culture; thymoglobulin and anti-TNFa (etaner­cept) induction; less diabetogenic, calcineurin inhibitor-free maintenance immune suppression; and intensive insulin and intravenous heparin in the peri-transplant period. Atten­tion to detail every step of the way led to insulin independence after single donor islet infusions in the first eight recipients, with five of eight maintaining insulin independence in the longer term. With such remarkable progress over the past three years, it is clear that islet transplantation can provide a level of glyce­mic control that far exceeds intensive insulin or pump therapy. Provided rejection and autoimmune recurrence can be prevented by non-diabetogenic but potent antirejection drugs, glycated HbA1C can remain within the normal range, and emerging outcomes in islet transplantation may soon parallel the results in whole pancreas transplantation, but with less potential risk of pen-transplant morbidity.  

4.188           Chemotactically-induced redistribution of CD43 as related to polarity and locomotion of human polymorphonuclear leucocytes

Dehghani Zadeh, A., Seveau, S., Halbwachs-Mecarelli, L. and Keller, H.U. Biol. Cell, 95, 265-273 (2003)   Leukocyte motility involves pseudopods extension at the leading edge and uropod contraction at the cell rear. Previous studies have shown that the glycoprotein CD43 redistributes to the uropod, when the cells develop polarity and locomotion. The present study addresses the question whether the accumulation of specific membrane molecules, such as CD43 at the contracted uropod precedes or follows development of polarity and locomotion. PMNs were labeled with fluorescent anti-CD43 antibodies and guided to polarize in the direction of a chemoattractant-containing micropipette or, once polarized, they were forced to reverse polarity and movement direction by placing the micropipette behind the uropod. This chemotactically-induced reversal of polarity was used as an efficient tool to analyse the sequence of events. CD43, but not another abundant surface glycoprotein CD45, was concentrated at the uropod. This documents that CD43 redistribution is a selective phenomenon. During reversal of polarity and of locomotion direction, the geometric center of the cell clearly changed direction earlier than the center of anti-CD43 fluorescence intensity. Thus, CD43 redistribution to the new uropod follows rather than precedes reversal of polarity, suggesting that CD43 redistribution is a consequence rather than a prerequisite for polarity and locomotion. PMNs making a U-turn maintained the pre-existing polarity and CD43 remained concentrated at the uropod, even when the front was moving in the opposite direction. Our data show that anterior pseudopod formation, rather than capping of CD43 at the uropod or the position of the uropod determines the direction of locomotion.  

4.189           A reliable technique for rodent pancreatic stellate cells isolation

Shek, F.W. et al Pancreatology, 3, 209-269 (2003)   Introduction: Pancreatic stellate cells (PSC) isolation varies between laboratories. There are increasing pressures to limit animal procedures. Our method which uses unwanted pancreata from other experiments and avoids cannulation (Bachem, 1998), was developed from the Bedayan technique acinar cell isolation and local expertise in HSC isolation. Methods: Rat pancreata were minced in Hanks Buffer Salt Solution (HBSS) containing calcium and digested in pronase (1mg/ml) and collagenase P (0.5 mg/ml) and shaken 30 minutes at 37⁰C. Undigested tissue was filtered using nylon mesh. The filtrate was pelleted and resuspended in OptiPrep density gradient – 12% v/v in HBSS with HBSS buffering layer and centrifuged for 20 minutes at 1,000g at 4⁰C. PSC were collected in the OptiPrep/HBSS interface and counted and assessed for viability. Results: For each preparation, we can reliably obtain between 2-4 million cells with at least 85% viability and 100% purity at passage 1. PSC immunostained positively for desmin (mesenchymal marker) and a-smooth muscle actin (activated stellate cells). This was also confirmed by western blot. Discussion: Our PSC supplies come from pancreas not required by other researchers. No surgical skills are required. The time-limitations of tissue outgrowth methods are avoided. Local variation of gradient density and centrifugal force may be required to optimize recovery.    

4.190           Fas/tumor necrosis factor receptor death signaling is required for axotomy-induced death of motoneurons in vivo

Ugolini, G. et al

1. Neurosci.,23(24), 8526-8531 (2003)

  Activation of the Fas death receptor leads to the death of motoneurons in culture. To investigate the role of Fas in programmed cell death and pathological situations, we used several mutant mice deficient for Fas signaling and made a novel transgenic FADD-DN (FAS-associated death domain-dominant-negative) strain. In vitro, motoneurons from all of these mice were found to be resistant to Fas activation and to show a delay in trophic deprivation-induced death. During normal development in vivo, no changes in motoneuron survival were observed. However, the number of surviving motoneurons was twofold higher in animals deficient for Fas signaling after facial nerve transection in neonatal mice. These results reveal a novel role for Fas as a trigger of axotomy-induced death and suggest that the Fas pathway may be activated in pathological degeneration of motoneurons.  

4.191           a-defensins  can have anti-HIV activity but are not CD8 cell anti_HIV factors

Mackewicz, C.E. et al AIDS, 17, F23-F32 (2003)   Background: CD8 T cells from healthy HIV-infected individuals inhibit HIV replication in infected CD4 T cells by a non-cytotoxic mechanism mediated by a soluble CD8 cell antiviral factor, CAF. Recently, the antimicrobial peptides, α-defensins, were reported to constitute CAF. Objective: To examine the antiviral activity of α-defensins and address their potential role in CD8 cell non-cytotoxic antiviral responses. Design and methods: A purified mixture of human neutrophil proteins (HNP) 1-3 (α-defensins) was used to examine the effect of α-defensins on HIV virions and on HIV replication in CD4 cells treated prior to or post infection. α-Defensin expression was analyzed at the RNA and protein level in CD8 cells as well as in various other cell types. Antibodies to the defensins were tested for their ability to inhibit CAF activity in CD8 cell culture fluids. Results: The α-defensins exhibited anti-HIV activity on at least two levels: directly inactivating virus particles; and affecting the ability of target CD4 cells to replicate the virus. However, while we could demonstrate α-defensins in neutrophils and monocytes, we found no evidence for the production of these peptides by CD8 T cells. No messenger RNA encoding these proteins was detected in purified CD8 T cells, nor did these cells produce intracellular or extracellular α-defensin peptides. Moreover, antibodies specific for human α-defensins 1, 2, and 3 did not block the antiviral activity of CAF-active CD8 cell culture fluids. Conclusions: The α-defensins are not produced by CD8 cells but unexpectedly were found to be expressed in monocytes. α-Defensins can have anti-HIV activity but are not CD8 cell antiviral factors.  

4.192           Kinetics of increased deformability of deoxygenated sickle cells upon oxygenation

Huang, Z. et al Biophys. J., 85, 2374-2383 (2003)   We have examined the kinetics of changes in the deformability of deoxygenated sickle red blood cells when they are exposed to oxygen (O₂) or carbon monoxide. A flow-channel laser diffraction technique, similar to ektacytometry, was used to assess sickle cell deformability after mixing deoxygenated cells with buffer that was partially or fully saturated with either O₂ or carbon monoxide. We found that the deformability of deoxygenated sickle cells did not regain its optimal value for several seconds after mixing. Among density-fractionated cells, the deformability of the densest fraction was poor and didn't change as a function of O₂ pressure. The deformability of cells from the light and middle fraction increased when exposed to O₂ but only reached maximum deformability when equilibrated with supraphysiological O₂ concentrations. Cells from the middle and lightest fraction took several seconds to regain maximum deformability. These data imply that persistence of sickle cell hemoglobin polymers during circulation in vivo is likely, due to slow and incomplete polymer melting, contributing to the pathophysiology of sickle cell disease.  

4.193           Prevention of leukocyte migration to inflamed skin with a novel fluorosugar modifier of cutaneous lymphocyte-associated antigen

Dimitroff, C.J., Kupper, T.S. and Sackstein, R.

1. Clin. Invest., 112(7), 1008-1018 (2003)

  E-selectin and P-selectin on dermal postcapillary venules play critical roles in the migration of effector T cells into inflamed skin. P-selectin glycoprotein ligand-1 (PSGL-1) modified by 1,3-fucosyltransferase is the principal selectin ligand on skin-homing T cells and is required for effector T cell entry into inflamed skin. We have previously shown that a fluorinated analog of N-acetylglucosamine peracetylated-4-fluorinated-D-glucosamine (4-F-GlcNAc), inhibits selectin ligand expression on human T cell PSGL-1. To analyze 4-F-GlcNAc efficacy in dampening effector T cell migration to inflamed skin, we elicited allergic contact hypersensitivity (CHS) reactions in mice treated with 4-F-GlcNAc. We also investigated 4-F-GlcNAc efficacy on lymphocyte E-selectin ligand expression in LNs draining antigen-sensitized skin and on other immunological processes requisite for CHS responses. Our results showed that 4-F-GlcNAc treatment attenuated lymphocyte E-selectin ligand expression in skin-draining LNs and prevented CHS reactions. Significant reductions in inflammatory lymphocytic infiltrate were observed, while pathways related to antigenic processing and presentation and naive T cell recognition within skin-draining LNs were unaffected. These data indicate that 4-F-GlcNAc prevents CHS by inhibiting selectin ligand activity and the capacity of effector T cells to enter antigen-challenged skin without affecting the afferent phase of CHS.  

4.194           Lysophospholipids synergistically promote primitive hematopoitic cell chemotaxis via a mechanism involving Vav1

Whetton, A.D., Lu, Y., Pierce, A., Carney, L. and Spooncer, E. Blood, 102, 2798-2802 (2003)   Hematopoiesis is sustained by the proliferation and development of an extremely low number of hematopoietic stem cells resident in the bone marrow. These stem cells can migrate from their bone marrow microenvironment and can be found at low levels in the peripheral blood. The factors that regulate egress or ingress of the stem cells from the marrow include cytokines and chemokines. This process of stem cell trafficking is fundamental to both stem cell biology and stem cell transplantation. We show that primitive hematopoietic cells with cobblestone area–forming cell activity express receptors for and display enhanced motility in response to a new class of stem cell agonists, namely lysophospholipids. These agents synergistically promote chemokinestimulated cell chemotaxis, a process that is crucial in stem cell homing. The response to lysophospholipids is mediated by Rac, Rho, and Cdc42 G proteins and the hematopoietic-specific guanyl nucleotide exchange factor Vav 1. Inhibitor studies also show a critical role for phosphatidylinositol 3 kinase (PI3K). Lipid mediators, therefore, regulate the critical process of primitive hematopoietic cell motility via a PI3K- and Vav-dependent mechanism and may govern stem cell movement in vivo. These results are of relevance to understanding stem cell trafficking during bone marrow transplantation.  

4.195           An alternative efficient procedure for purification of the obligate intracellular fish bacterial pathogen Piscirickettsia salmonis

Henriquez, V., Rojas, M.V., and Marshall, S.H. Appl, Environ. Microbiol., 69(10), 6268-6271 (2003)   Piscirickettsia salmonis is an obligate intracellular bacterial pathogen of salmonid fish and the etiological agent of the aggressive disease salmonid rickettsial syndrome. Today, this disease, also known as piscirickettsiosis, is the cause of high mortality in net pen-reared salmonids in southern Chile. Although the bacteria can be grown in tissue culture cells, genetic analysis of the organism has been hindered because of the difficulty in obtaining P. salmonis DNA free from contaminating host cell DNA. In this report, we describe a novel procedure to purify in vitro-grown bacteria with iodixanol as the substrate to run differential centrifugation gradients which, combined with DNase I digestion, yield enough pure bacteria to do DNA analysis. The efficiency of the purification procedure relies on two main issues: semiquantitative synchrony of the P. salmonis-infected Chinook salmon embryo (CHSE-214) tissue culture cells and low osmolarity of iodixanol to better resolve bacteria from the membranous structures of the host cell. This method resulted in the isolation of intact piscirickettsia organisms and removed salmon and mitochondrial DNA effectively, with only 1.0% contamination with the latter.    

4.196           Advances in islet cell biology. From stem cell differentiation to clinical transplantation: conference report

Kandeel, F., Smith, C.V., Tpdorov, I. And Mullen, Y Pancreas, 27(3), e63-e78 (2003)   The 3rd Annual Rachmiel Levine Symposium entitled "Advances in Islet Cell Biology-From Stem Cell Differentiation to Clinical Transplantation" was organized by the Department of Diabetes, Endocrinology and Metabolism at the City of Hope National Medical Center, with the support of the Southern California Islet Cell Resources Center, American Diabetes Association-David Shapiro Research Fund, Ross Foundation, the National Center for Research Resources (NCRR), and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health. The symposium was held at the Hilton Anaheim Hotel in Anaheim, CA, in October 2002, and was attended by nearly 400 participants from 23 countries and 30 U.S. states. The symposium consisted of 11 sessions focusing on 3 areas: (1) pancreas and islet cell differentiation and islet generation, (2) β cell biology and insulin synthesis and/or secretion, and (3) pancreatic islet transplantation in patients with type I diabetes. Thirty-nine world experts lectured on the most current information in each field. Fifty-three abstracts were selected for presentation and discussed at the poster session. The first author of each of the top 10 posters received a Young Investigator Travel Award provided by the National Center for Research Resources and the Southern California Islet Cell Resources Center. The symposium also offered special Meet the Professor sessions, which gave the attendees an opportunity to closely interact with the participating speakers of the day.  

4.197           T-cell specific immunosuppresion results in more than 53 days survival of porcine islets of Langerhans in the monkey

Rijkelijkhuizen, J.K.R.A. et al Transplantation, 76(9), 1359-1368 82003)   Background. Transplantation of islets of Langerhans can restore insulin production in diabetic patients. Because of the shortage of human donor organs, transplantation of porcine islets may be an alternative solution. The present study was aimed at the characterization of rejection mechanisms of porcine islets transplanted into eight nondiabetic monkeys under the kidney capsule. Methods. Cultured adult pig islets were used, which showed no expression of the galactose(α1,3)galactose epitope, major histocompatibility complex class II, or CD45, and no binding of antibodies or complement after exposure to monkey serum. Immunosuppression consisted of cyclophosphamide, cyclosporine A (CsA), and steroids (group 1); or antithymocyte globulin, anti-interleukin-2 receptor antibody, CsA, and steroids (group 2). In three animals of group 2, islets were also transplanted in the portal vein. Results. Although all monkeys had preformed anti-pig antibodies, no correlation was found between antibody titers and rejection and no deposition of antibodies or complement was observed in the grafts. Group 1 showed islets up to day 11, followed by T-cell infiltration and rejection at approximately day 14. In group 2, two monkeys showed infiltrates consisting predominantly of T cells starting at approximately day 29, whereas two monkeys showed well-preserved islets without infiltration up to day 53. In the livers of the three monkeys that also received islets intraportally and were resectioned on days 21, 33, and 49, no islets could be detected. Conclusions. This study demonstrates that cultured adult pig islets can survive in the monkey for more than 53 days without signs of rejection under standard immunosuppression.                

4.198           CD4 expression decrease by antisense oligonucleotides: inhibition of rat CD4⁺ cell reactivity

Rabanal, N.R. et al Oligonucleotides, 13 217-228 (2003)   In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4⁺ T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4⁺ cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4⁺ cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4⁺ cell reactivity.  

4.199           How to detect Toxoplasma gondii oocysts in environmental samples

Dumètre, A. and Dardè, M-L. FEMS Microbiol. Rev., 27, 651-661 (2003)   Detection of Toxoplasma gondii oocysts in environmental samples is a great challenge for researchers as this coccidian parasite can be responsible for severe infections in humans and in animals via ingestion of a single oocyst from contaminated water, soil, fruits or vegetables. Despite field investigations, oocysts have been rarely recovered from the environment due to the lack of sensitive methods. Immunomagnetic separation, fluorescence-activated cell sorting, and polymerase chain reaction have recently shown promising use in detection of protozoa from complex matrices. Such procedures could be applied to T. gondii detection, if studies on the antigenic and biochemical composition of the oocyst wall are completed. Using such methods, it will be possible to assess the occurrence, prevalence, viability and virulence of T. gondii oocysts in environmental matrices and specify sources of human and animal contamination.  

4.200           Clinical islet transplant: current and future directions towards tolerance

Shapiro, A.M.J., Nanij, S. and Lakey, J.R.T. Immunological Reviews, 96(2), 219-236 (2003)   The ultimate goal of islet transplantation is to completely correct the diabetic state from an unlimited donor source, without the need for chronic immunosuppressive drug therapy. Although islet transplantation provides an opportunity to develop innovative strategies for tolerance in the clinic, both alloimmune and autoimmune barriers must be controlled, if stable graft function is to be maintained long-term. After islet extraction from the pancreas, the cellular graft may be stored in tissue culture or cryopreserved for banking, providing an opportunity not only to optimally condition the recipient but also to allow in vitro immunologic manipulation of the graft before transplantation, unlike solid organ grafts. As such, islets may be considered a 'special case.' Remarkable progress has occurred in the last three years, with dramatic improvements in outcomes after clinical islet transplantation. The introduction of a steroid-free, sirolimus-based, anti-rejection protocol and islets prepared from two (or rarely three) donors led to high rates of insulin independence. The 'Edmonton Protocol' has been successfully replicated by other centers in an international multicenter trial. A number of key refinements in pancreas transportation, processing, purification on non-ficoll-based media, storage of islets in culture for two days and newer immunological conditioning and induction therapies have led to continued advancement through extensive collaboration between key centers. This review outlines the historical development of islet transplantation over the past 30 years, provides an update on current clinical outcomes, and summarizes a series of unique opportunities for development and early testing of tolerance protocols in patients.          

4.201           Successful single donor islet transplantation in type 1 diabetes

Hering, B.J. et al IPITA 2003 abstracts 012 (2003)   Islet transplantation is emerging as a viable treatment option for selected patients with type 1diabetes. This pilot trial sought to determine whether optimization of pancreas preservation, islet processing, induction and maintenance immunosuppression would facilitate reversal of type 1 diabetes after single-donor islet transplantation. Pancreases were obtained from stable donors age <50 years and preserved for <8 hrs using the two-layer method. Islets were isolated using the automated method after perfilsion of the pancreas with cold LiberaseTM. Islets were purified on continuous iodixanol gradients, and cultured for 2 days. Islet preparations with viabilities >70%, total tissue volumes <10 cc, negative Gram stain, and endotoxin content <5 FU/kg were considered for transplant. 6,000 to 8,725 cultured islet equivalents/kg prepared from single donors were transplanted intraportally on day 0 following minilaparotomy or percutaneous transhepatic access into 8 C-peptide negative, non-uremic, type 1 diabetic patients with hypoglycemia unawareness. Immunosuppression was initiated 2 days before transplant and included rabbit antithymocyte globulin (through day +2, total dose 6 mg/kg), methylprednisolone (day -2 only), daclizumab, etanercept (through day +10), sirolimus, and reduced-dose tacrolimus (started on day +1). Tacrolimus was gradually replaced with mycophenolate mofetil starting one month posttransplant. All 8 transplant recipients achieved insulin independence with normal HbA 1 c levels and freedom from hypoglycemia. The time to insulin independence varied from 23 to 122 days. 5 of 8 patients have remained insulin-independent, with follow-up exceeding 1 yr in 2 patients. 3 of 8 patients have resumed exogenous insulin therapy preceded by subtherapeutic sirolimus trough levels (<10 ng/ml) in 3 and subtherapeutic MMF doses (1000 mg/day) in 2 of the 3 patients. Procedural complications, serious infections, or serious, unexpected, and islet- or immunosuppression-related adverse events were not encountered. The data suggests that a combination of maximized viable islet yield, islet culturing, preemptive induction immunosuppression including agents with anti-inflammatory properties, and nondiabetogenic maintenance immunosuppression can result in successfiil single-donor islet transplantation.  

4.202           Iodixanol density gradient preparation in University of Wisconsin solution for porcine islet purification

Van der Burg, M.P.M. and Graham, J.M. The Scientific World J., 3, 1154-1159 (2003)   Previously published as Graham, J.M. (2002) Purification of Islets of Langerhans from porcine pancreas. TheScientificWorld JOURNAL 2, 1657-1661. ISSN 1537-744X; DOI 10.1100/tsw.2002.847. Generally, prior to the purification of isolated pancreatic islets, the collagenase-digested tissue is incubated in the University of Wisconsin solution (UWS; ~320 mOsm) for osmotic stabilization to preserve or improve the density differences between islets and acinar fragments. The adverse effects arising from the subsequent pelleting and resuspension of the islets in a second, different (often highly hyperosmotic) purification solution are avoided in the protocol described here; preparation of the purification medium is simply achieved by mixing the UWS preincubated islets with a second UWS containing the inert impermeant iodixanol. Flotation of the islets isolated from juvenile porcine pancreases through this mildly hypertonic (~380 mOsm) gradient of iodixanol-UWS achieves a much higher recovery of islets of an improved viability than the customary method using a Ficoll gradient. The method has been extended to human islet purification.  

4.203           Improved islet yield and function with ductal injection of university of Wisconsin solution before pancreas preservation

Sawada, T. et al Transplantation, 75(12), 1965-1969 (2003)   Background. Ensuring sufficient islet yield from preserved pancreases is a critical step in clinical islet transplantation. The aim of this study was to investigate whether pancreatic ductal injection, performed at procurement, using a small volume of preservation solution before cold storage (ductal preservation method) would improve islet yield and function from rat pancreases preserved for 6 and 24 hr. Materials and Methods. Islets were isolated from Lewis rats. Pancreases were classified into five groups: fresh (group 1); preserved for 6 hr in University of Wisconsin solution without and with ductal preservation (groups 2 and 3); and preserved for 24 hr in University of Wisconsin solution without and with ductal preservation (groups 4 and 5). We assessed islet yield, function, and viability of pancreatic ductal cells. Results. Islet yields per pancreas in groups 1 to 5 were 2010±774, 674±450, 1418±528, 527±263, and 1655±618 (islet equivalent) (±SD), respectively. Stimulation indices in groups 1 to 5 were 11.97±3.17, 6.48±4.04, 12.44±5.65, 2.56±2.03, and 5.55±2.71. Functional success rates in groups 1 to 5 were 100%, 0%, 100%, 0%, and 66.7%. Percentages of nonviable pancreatic duct cells in groups 1 to 5 were 3.8±2.7%, 59.7±4.4%, 19.5±7.3%, 64.7±4.5%, and 17.2±2.6%. In all experiments, the differences were significant between the groups without versus the groups with ductal preservation (P <0.05, group 2 vs. group 3 and group 4 vs. group 5). Conclusions. Ductal preservation improved islet yield and function after 6 and 24 hr of preservation. Well-preserved pancreatic ducts maintained good distribution of collagenase solution. For human islet transplantation to become more widely available and applicable, it will be important to obtain a viable islet mass adequate for diabetes reversal from one donor pancreas. The Edmonton group reported seven successful islet transplants into patients with type 1 diabetes (1). These unprecedented results have since been confirmed in a larger series of patients in Edmonton and, importantly, at other institutions. The islets in these trials were isolated from two or more donors for each recipient. To reverse type 1 diabetes, one donor pancreas is adequate in the setting of vascularized pancreas transplantation (2) but not in the setting of islet transplantation (1). It has been reported, both in a rat and dog model, that islet yield improved from a stored pancreas after intraductal flush of collagenase resuspended in University of Wisconsin (UW) solution at the time of pancreas procurement (3). Pre-cold storage injection of collagenase resuspended in buffer solution into the pancreatic duct of human pancreases from donors less than 30 years old also resulted in increased islet yields (4). We have hypothesized that islet isolation from a preserved pancreas is hampered by the exceptional susceptibility of the pancreatic ductal system to cold ischemic injury and the resulting inability to achieve satisfactory distribution of collagenase by intraductal distention even after short periods of cold storage. We have further hypothesized that improved preservation of the ductal epithelium would produce a better islet yield after pancreas preservation. To test this hypothesis, we have created a new method in which a small volume of UW preservation solution (without adding collagenase) is injected into the pancreatic duct at the time of procurement (i.e., ductal preservation), thereby preserving the pancreatic duct. We studied the yield and the in vitro and in vivo function of islets isolated from rat pancreases preserved for 6 and 24 hr with standard UW solution immersion and those with ductal preservation combined with pancreas immersion in UW solution. We also assessed the preservation of the pancreatic duct by evaluating the percentage of live cells in it, using trypan blue staining. Ductal preservation improved the yield and function of rat islets after 6 and 24 hr of preservation. Well-preserved pancreatic ducts facilitated good distribution of the collagenase solution.  

4.204           Decline in rate of colonization of oligodendrocyte progenitor cell (OPC)-depleted tissue by adult OPCs with age

Chari, D.M., Phil, A., Crang, A.J. and Blakemore, W.F.

1. Neuropathol. Exp. Neurol., 62(9), 908-816 (2003)

  Rates of remyelination decline with age and this has been attributed to slower recruitment of oligodendrocyte progenitor cells (OPCs) into areas of demyelination and slower differentiation of OPCs into remyelinating oligodendrocytes. When considering causes for reduced recruitment rates, intrinsic causes (alterations in biological properties of OPCs) need to be separated from extrinsic causes (age-related differences in the lesion environment). Using 40 Gy of X-irradiation to deplete tissue of its endogenous OPC-population, we examined the effects of age on the rate at which adult rat OPCs colonize OPC-depleted tissue. We found a significant reduction in the rate of colonization between 2 and 10 months of age (0.6 mm/week versus 0.38 mm/week). To determine if this represented an intrinsic property of OPCs or was due to changes in the environment that the cells were recolonizing, OPCs from 10-month-old animals were transplanted into 2-month-old hosts and OPCs from 2-month-old animals were transplanted into 10-month-old hosts. These experiments showed that the transplanted OPCs retained their age-related rate of colonization, indicating that the decline in colonizing rates of OPCs with age reflects an intrinsic property of OPCs. This age-related decline in the ability of OPCs to repopulate OPC-depleted tissue has implications for understanding remyelination failure in multiple sclerosis (MS) and developing therapies for remyelination failure.                

4.205           Monocyte chemoattractant protein-1 and CC-chemokine receptor-2 in severe hypercholesterolaemia

Blomquist, H.M. and Olsson, A.G. Scand. J. Clin. Lab. Invest., 63, 513-520 (2003)   Objectives: To investigate whether plasma concentrations of monocyte chemoattractant protein-1 (MCP-1) and the gene expression of its receptor on the monocyte cell surface CCR-2 were elevated above normal in subjects with asymptomatic, isolated hypercholesterolaemia and if statin treatment could influence this cytokine. Methods: The investigation was designed as a cross sectional study followed by a single, blind, treatment study of patients receiving pravastatin 80 mg/day for 8 weeks. The study included 23 patients with severe hypercholesterolaemia (LDL>5.2 mmol/L) and 39 normocholesterolaemic controls. Blood samples were obtained from patients and controls at baseline and from patients at end of the study and analysed for lipoproteins and inflammatory mediators: MCP-1, high-sensitivity C-reactive protein (HS-CRP). Isolated peripheral mononuclear cells were analysed for CCR-2 gene expression. Results: Mean plasma LDL-C was significantly higher in patients than in controls. No difference in plasma MCP-1 levels or CCR-2 gene expression was seen between the groups at baseline, nor were there any differences in plasma concentrations of CRP. After treatment with pravastatin, LDL-C decreased by 31%. Treatment did not significantly affect the levels of MCP-1 or CCR-2 gene expression, nor was CRP affected by treatment with pravastatin. Conclusions: Our study does not support the view that MCP-1 plasma levels and CCR-2 gene expression in circulating monocytes are directly responsible for the monocyte recruitment into the arterial intima in patients with severe asymptomatic hypercholesterolaemia. In addition, the inflammatory response of a high concentration of LDL-C in isolated asymptomatic hypercholesterolaemia is minute. Objectives: To investigate whether plasma concentrations of monocyte chemoattractant protein-1 (MCP-1) and the gene expression of its receptor on the monocyte cell surface CCR-2 were elevated above normal in subjects with asymptomatic, isolated hypercholesterolaemia and if statin treatment could influence this cytokine. Methods: The investigation was designed as a cross sectional study followed by a single, blind, treatment study of patients receiving pravastatin 80 mg/day for 8 weeks. The study included 23 patients with severe hypercholesterolaemia (LDL>5.2 mmol/L) and 39 normocholesterolaemic controls. Blood samples were obtained from patients and controls at baseline and from patients at end of the study and analysed for lipoproteins and inflammatory mediators: MCP-1, high-sensitivity C-reactive protein (HS-CRP). Isolated peripheral mononuclear cells were analysed for CCR-2 gene expression. Results: Mean plasma LDL-C was significantly higher in patients than in controls. No difference in plasma MCP-1 levels or CCR-2 gene expression was seen between the groups at baseline, nor were there any differences in plasma concentrations of CRP. After treatment with pravastatin, LDL-C decreased by 31%. Treatment did not significantly affect the levels of MCP-1 or CCR-2 gene expression, nor was CRP affected by treatment with pravastatin. Conclusions: Our study does not support the view that MCP-1 plasma levels and CCR-2 gene expression in circulating monocytes are directly responsible for the monocyte recruitment into the arterial intima in patients with severe asymptomatic hypercholesterolaemia. In addition, the inflammatory response of a high concentration of LDL-C in isolated asymptomatic hypercholesterolaemia is minute.  

4.206           Isolation of rat Kupffer cells: a combined methodology for highly purified primary cultures

Valatas, V., Xidakis, C., Roumpaki, H., Kolios, G. And Kouroumalis, E.A. Cell Biol Int., 27, 67-73 (2003)   We report a four-step procedure that optimizes the methodology for isolation of highly purified rat Kupffer cells (KC). We combined the previously reported techniques of enzymatic tissue treatment, density gradient centrifugation, centrifugal elutriation and selective adherence. ED-2 immunophenotyping and non-specific esterase histochemistry were used for cell identification. This combination resulted in a satisfactorily high yield of 80–100×10⁶KCs per liver, over 95% positive for ED-2 and 98% viable cells. Cultures of isolated KCs were functionally intact and exhibited a concentration and time-dependent LPS-induced TNF- and nitric oxide production.  

4.207           Heterogeneity of dendritic cells in the mouse liver: identification and characterization of four distinct populations

Lian, Z-X. et al

1. Immunol., 170, 2323-2330 (2003)

  Liver dendritic cells (DC) are believed to play important roles in liver immunity, autoimmunity, and in the regulation of hepatic allograft acceptance. However, limited information is available on the phenotypes and functions of DC in the liver. To address this issue, we isolated DC from murine liver using procedures that do not involve collagenase, and characterized the freshly isolated DC population that had not been subjected to in vitro expansion. Thence, based on the expression of CD4, B220, and CD11b, four subsets or groups of hepatic NK1.1^-CD11c⁺ DC were identified with the following phenotypes: B220⁺CD4⁺, B220⁺CD4^-, B220^-CD11b⁺, and B220^-CD11b^-. Each subset was further characterized both phenotypically and functionally. In addition to unique phenotypic expression, each subset displayed different allostimulation capability in mixed lymphocyte reaction assays. All four groups developed DC morphology following in vitro culture with activation agents and synthesized distinct patterns of cytokines in response to different stimuli. Taken together, our results suggest that groups I and II are IFN- -producing plasmacytoid DC, group III cells are myeloid-related DC, while group IV is a heterogenous population containing both myeloid- and lymphoid-related DC. Our results demonstrate the highly heterogeneous nature of hepatic DC, which is in agreement with the unique requirements for APC in the complex liver environment.  

4.208           Murine thymic plasmacytoid dendritic cells

Okada, T., Lian, Z-X., Naiki, M., Ansari, A.A., Ikehara, S. and Gershwin, M.E. Eur. J: Immunol., 33(4), 1012-1019 (2003)   We report herein heterogeneous murine thymic cell subsets expressing CD11c and B220 (CD45R). The CD11c⁺B220⁺ subset expresses Ly6C^high and MHC class II^low in contrast with previously described thymic DC (CD11c⁺B220^– cells). Freshly isolated thymic CD11c⁺B220⁺ cells show typical plasmacytoid morphology which differentiates to mature DC, in vitro with CpG oligodeoxynucleotides (ODN) 2216; we term this subset thymic plasmacytoid DC (pDC). These thymic pDC are highly sensitive to spontaneous apoptosis in vitro and induce low T cell allo-proliferation activity. Thymic pDC express low TLR2, TLR3 and TLR4 mRNA, normally found on human immature DC, and high TLR7 and TLR9 mRNA, normally found on human pDC. Thymic pDC also produce high amounts of IFN- following culture with CpG ODN 2216 (TLR9 ligands) as compared with the previously defined thymic DC lineage which expresses low TLR9 mRNA and produce high IL-12 (p40) with CpG ODN 2216. These results indicate that thymic pDC are similar to IFN-producing cells as well as human pDC. The TLR and cytokine production profiles are consistent with a nomenclature of pDC. The repertoire of this cell lineage to TLR9 ligands demonstrate that such responses are determined not only by the quantity of expression, but also cell lineage.  

4.209           Role of Toll-like receptors in costimulating cytotoxic T cell responses

Schwarz, K., Storni, T., Manolova, V., Didierlaurent, A., Sirard, J-C., Röthlisberger, P. and Bachmann, M.F. Eur. J: Immunol., 33(6), 1465-1470 (2003)   Stimulation of Toll-like receptors (TLR) by pathogen-derived compounds leads to activation of APC, facilitating the induction of protective immunity. This phenomenon is the basis of most adjuvant formulations currently in development. Here, we tested the ability of TLR2, 3, 4, 5, 7 and 9 signaling to enhance CTL responses upon vaccination with virus-like particles. Stimulation of TLR2 and 4 failed to increase CTL responses, whereas ligands for TLR3, 5 and 7 exhibited moderate adjuvant function. In contrast, stimulation of TLR9 dramatically increased CTL responses, indicating that ligands for TLR9 are likely to be the most promising candidates for the development of novel adjuvant formulations for stimulating CTL responses.  

4.210           Age-related changes in neuronal glucose uptake in response to glutamate and β-amyloid

Patel, J.R. and Brewer, G.J.

1. Neurosci. Res., 72(4), 527-536 (2003)

  Energy supplies that may decline with age are crucial for cells to maintain ionic homeostasis and prevent neuron death. We examined baseline glucose transporter expression and rate of glucose uptake in cultured hippocampal neurons from embryonic, middle-age (12-month-old), and old (24-month-old) rats and exposed the neurons to glutamate, β-amyloid, and mitochondrial inhibitors. Without stress, the rate of glucose uptake was similar in middle-age and old neurons, and the rate of glucose uptake in embryonic neurons was threefold greater than that in middle-age and old neurons. Glucose uptake increased in the presence of mitochondrial inhibitors (FCCP and oligomycin) for embryonic and middle-age neurons. The old neurons failed to increase glucose uptake. In the presence of glutamate, FCCP, and oligomycin, embryonic neurons showed a decrease in glucose uptake and the middle-age and old neurons showed no change in glucose uptake. Middle-age neurons took up significantly more glucose than old neurons when under mitochondrial and glutamate stress. In the presence of β-amyloid, only embryonic neurons increased glucose uptake; middle-age and old neurons did not. Fluorescence imaging of immunoreactive glut3 in response to β-amyloid demonstrated a 16–49% increase in glut3 immunoreactivity at the plasma membrane for the three ages. The results suggest that old neurons were not able to upregulate glucose uptake to ensure cell survival. Neuron aging does not indicate a defect in normal glut3 function; rather, our results suggest that mechanisms regulating glucose uptake under stress fail to react in time to ensure cell survival.  

4.211           Platelets and Granulocytes, in Particular the Neutrophils, Form Important Compartments for Circulating Vascular Endothelial Growth Factor

Kusumanto, Y.H., Dam, W.A., Hospers, G.A.P., Meijer, C. and Mulder, N.H. Angiogenesis, 6(4), 283-287 (2003)   The measurement of circulating vascular endothelial growth factor (VEGF) levels as a prognostic factor will gain increasing relevance in the diagnosis and evaluation of treatment in cancer patients. Angiogenesis is an absolute requirement in tumour growth and metastatic disease. In the present study data are presented which indicate that circulating VEGF mainly resides in peripheral blood cells. In 15 healthy volunteers we demonstrated that approximately 34% of the circulating VEGF resides in platelets and approximately 11% in patients with cancer (n= 4). An important part namely 58% in healthy volunteers and 69% in patients with cancer of the total circulating VEGF is contained in granulocytes, particular in the neutrophils, as confirmed by fluorescence-activated cell sorting (FACS). Also an increased VEGF level per granulocyte is found in patients with cancer (77 μg VEGF/l) compared with the healthy volunteers (164 μg VEGF/l). In contrast only 2% was present in plasma. The biological significance of platelet- or granulocyte-derived VEGF is not yet known. Liberation of VEGF from these compartments could well be of importance for tumour angiogenesis. Therefore, future studies on the clinical value of circulating VEGF as a prognostic factor in cancer patients should include measurements of VEGF in peripheral blood cells.  

4.212           Para/autocrine regulation of estrogen receptors in hippocambal neurons

Prange-Kiel, J., Wehrenberg, U., Jarry, H. and Rune, G.M. Hippocampus, 13(2), 226-234 (2003)   Previous studies have shown that estrogens, originating from ovaries, have a wide variety of estrogen receptor (ER)-mediated effects in the hippocampus. In the present study, we have investigated whether estrogens, which are synthesized in the hippocampus, could induce these effects as well. As a parameter, we used ER expression in response to estrogen synthesis, because estrogen receptors are ligandinducible transcription factors. The experiments were carried out with cultures of isolated adult rat hippocampal cells, which contained about 95% neurons and about 5% oligodendrocytes in serum-free and steroidfree medium. Hippocampal neurons express both estrogen receptor isoforms (ER_ and ER_), as shown by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. The release of estrogens by hippocampal neurons was quantified by radioimmunoassay (RIA). The ER isoforms (_ and _) were studied by semiquantitative immunocytochemical image analysis. Hippocampal cells precultured for 4 days were found to synthesize 17_-estradiol for the next 8 days. This synthesis was completely inhibited by letrozol, an aromatase inhibitor. Inhibition of estrogen synthesis by letrozol induced a significant decrease in ER_ expression, but an increase in ER_. As a control, supplementation of the medium with 17_-estradiol resulted in a significant increase of ER_ expression, whereas ER_ was downregulated. Our findings provide evidence for a de novo synthesis of estrogens in the hippocampus, differential regulation of estrogen receptor isoforms by estrogen and consequently for a para/autocrine loop of estrogen action in the hippocampus.  

4.213           Cord blood from collection to expansion : Feasibility in a regional blood bank

Elias, M., Choudhury, N. and Smit Sibinga, CTh. Indian J. Padiatr., 70(4), 327-336 (2003)   This article reviews the various aspect of the experimental phase proceeding the establishment of an umbilical cord blood (UCB) bank within a regular blood bank, a situation totally different from that of de novo establishing a cord blood bank having human and financial resources. An ethically approved two-year study has been conducted to determine the technical feasibility, and the practical problems that might be encountered such as public compliance, the additional workload, introduction of new activities ranging from collection and processing to progenitor expansion, infectious disease testing, development of a quality control system, record keeping and documentation, development of specific procedures and definitions of requirements. The cost benefit aspect, which will ultimately depend on the frequency of units release, was not considered in this study.  

4.214           Erythropoietin: a candidate compound for neuroprotection in schizophrenia

Ehrenreich, H. et al Mol. Psychiatry., 9, 42-54 (2004)   Erythropoietin (EPO) is a candidate compound for neuroprotection in human brain disease capable of combating a spectrum of pathophysiological processes operational during the progression of schizophrenic psychosis. The purpose of the present study was to prepare the ground for its application in a first neuroprotective add-on strategy in schizophrenia, aiming at improvement of cognitive brain function as well as prevention/slowing of degenerative processes. Using rodent studies, primary hippocampal neurons in culture, immunohistochemical analysis of human post-mortem brain tissue and nuclear imaging technology in man, we demonstrate that: (1) peripherally applied recombinant human (rh) EPO penetrates into the brain efficiently both in rat and humans, (2) rhEPO is enriched intracranially in healthy men and more distinctly in schizophrenic patients, (3) EPO receptors are densely expressed in hippocampus and cortex of schizophrenic subjects but distinctly less in controls, (4) rhEPO attenuates the haloperidol-induced neuronal death in vitro, and (4) peripherally administered rhEPO enhances cognitive functioning in mice in the context of an aversion task involving cortical and subcortical pathways presumably affected in schizophrenia. These observations, together with the known safety of rhEPO, render it an interesting compound for neuroprotective add-on strategies in schizophrenia and other human diseases characterized by a progressive decline in cognitive performance.  

4.215           Differential recruitment of Kv1.4 and Kv4.2 to lipid rafts by PSD-95

Wong, W. and Schlichter, L.C.

1. Biol. Chem., 279(1), 444-452 (2004)

  The activity of voltage-gated potassium (Kv) channels, and consequently their influence on cellular functions, can be substantially altered by phosphorylation. Several protein kinases that modulate Kv channel activity are found in membrane subdomains known as lipid rafts, which are thought to organize signaling complexes in the cell. Thus, we asked whether Kv1.4 and Kv4.2, two channels with critical roles in excitable cells, are found in lipid rafts. Acylation can target proteins to raft regions; however, Kv channels are not acylated, and therefore, a different mechanism must exist to bring them into these membrane subdomains. Because both Kv1.4 and Kv4.2 interact with postsynaptic density protein 95 (PSD-95), which is acylated (specifically, palmitoylated), we examined whether PSD-95 can recruit these channels to lipid rafts. We found that a portion of Kv1.4 and Kv4.2 protein in rat brain membranes is raft-associated. Lipid raft patching and immunostaining confirmed that some Kv4.2 is in Thy-1-containing rafts in rat hippocampal neurons. Using a heterologous expression system, we determined that palmitoylation of PSD-95 was crucial to its localization to lipid rafts. We then assessed the contribution of PSD-95 to the raft association of these channels. Co-expression of PSD-95 increased the amount of Kv1.4, but not Kv4.2, in lipid rafts. Deleting the PSD-95 binding motif of Kv1.4 eliminated this recruitment, as did substituting a palmitoylation-deficient PSD-95 mutant. This work represents the first evidence that PSD-95 binding can recruit Kv channels into lipid rafts, a process that could facilitate interactions with the protein kinases that affect channel activity.  

4.216           AMG531 stimulates megakaryopoiesis in vitro by binding to Mpl

Broudy, V.C. and Lin, N.L. Cytokine, 25, 52-60 (2004)   Thrombopoietin (TPO) plays a pivotal role in megakaryopoiesis. TPO initiates its biological effects by binding to its receptor Mpl. A recombinant protein consisting of a carrier Fc domain linked to multiple Mpl-binding domains was constructed, and is called AMG531. To define the biological activity of AMG531, we examined the ability of AMG531 to support CFU-Meg growth and to promote megakaryocyte maturation in vitro. AMG531 stimulates CFU-Meg growth in a dose-dependent manner, and acts in concert with erythropoietin, stem cell factor, interleukin-3, and interleukin-6 to enhance CFU-Meg growth, similar to parallel experiments with TPO. AMG531-stimulated serum-free liquid cultures support the development of mature polyploid megakaryocytes with a predominant DNA content of 32 N and 64 N, identical to that of parallel TPO-stimulated cultures. Competitive binding experiments show that AMG531 effectively competes with ¹²⁵I-TPO for binding to BaF3-Mpl cells or normal platelets. Treatment of BaF3-Mpl cells with AMG531 or with TPO resulted in rapid tyrosine phosphorylation of Mpl, JAK2, and STAT5. These results indicate that AMG531 is a potent stimulant of megakarypoiesis in vitro, and provide support for its further characterization in vivo.  

4.217           Systematic screening of potential b-cell imaging agents

Sweet, I.R. et al Biochem. Biophys. Res. Comm., 314, 976-983 (2004)   The -cell loss seen in diabetes mellitus could be monitored clinically by positron emission tomography (PET) if imaging agents were sufficiently specific for -cells to overcome the high ratio of non- -cell to -cell tissue in pancreas. In this report, we present a screening assay for identifying -cell-specific compounds that is based on the relative accumulation and retention by islet, INS-1, and exocrine (PANC-1) cells of candidate molecules. Molecules thought to have a high affinity for -cells were tested and included glibenclamide, tolbutamide, serotonin, -DOPA, dopamine, nicotinamide, fluorodeoxyglucose, and fluorodithizone. Glibenclamide and fluorodithizone were the most specific, but the specificity ratios fell well below those needed to attain robust signal to background ratio as a PET imaging agent for quantifying -cell mass. In vivo tests of the biodistribution of glibenclamide and fluorodithizone in rats indicated that the compounds were not specifically associated with pancreas, bearing out the predictions of the in vitro screen.  

4.218           Regulation of ATP/ADP in pancreatic islets

Sweet, I.R. et al Diabetes, 53, 401-409 (2004)   ATP and ADP levels are critical regulators of glucose-stimulated insulin secretion. In many aerobic cell types, the phosphorylation potential (ATP/ADP/P_i) is controlled by sensing mechanisms inherent in mitochondrial metabolism that feed back and induce compensatory changes in electron transport. To determine whether such regulation may contribute to stimulus-secretion coupling in islet cells, we used a recently developed flow culture system to continuously and noninvasively measure cytochrome c redox state and oxygen consumption as indexes of electron transport in perifused isolated rat islets. Increasing substrate availability by increasing glucose increased cytochrome c reduction and oxygen consumption, whereas increasing metabolic demand with glibenclamide increased oxygen consumption but not cytochrome c reduction. The data were analyzed using a kinetic model of the dual control of electron transport and oxygen consumption by substrate availability and energy demand, and ATP/ADP/P_i was estimated as a function of time. ATP/ADP/P_i increased in response to glucose and decreased in response to glibenclamide, consistent with what is known about the effects of these agents on energy state. Therefore, a simple model representing the hypothesized role of mitochondrial coupling in governing phosphorylation potential correctly predicted the directional changes in ATP/ADP/P_i. Thus, the data support the notion that mitochondrial-coupling mechanisms, by virtue of their role in establishing ATP and ADP levels, may play a role in mediating nutrient-stimulated insulin secretion. Our results also offer a new method for continuous noninvasive measures of islet cell phosphorylation potential, a critical metabolic variable that controls insulin secretion by ATP-sensitive K⁺–dependent and –independent mechanisms.  

4.219           Interleukin-10 secretion differentiates dendritic cells from human liver and skin

Goddard, S., Youster, J., Morgan, E. and Adams, D.H. Am. J. Pathol., 164(2), 511-519 (2004)   Liver dendritic cells (DCs), which may orchestrate the liver’s unique immunoregulatory functions, remain poorly characterized. We used a technique of overnight migration from pieces of normal human liver and skin to obtain tissue-derived DCs with minimal culture and no additional cytokine treatment. Liver and skin DCs had a monocyte-like morphology and a partially mature phenotype, expressing myeloid markers, MHCII, and co-stimulatory molecules; but only the skin DCs contained a population of CD1a+ cells. Overnight-migrated liver DCs activated naïve cord blood T cells efficiently. Liver DCs produced interleukin (IL)-10 whereas skin DCs failed to secrete IL-10 even after stimulation and neither skin nor liver-derived DCs secreted significant amounts of IL-12p70. Compared with skin DCs, liver DCs were less effective at stimulating T-cell proliferation and stimulated T cells to produce IL-10 and IL-4 whereas skin DCs were more potent stimulators of interferon- and IL-4. Monocyte-derived DCs were down-regulated after culture with liver-conditioned media, suggesting that local microenvironmental factors may be important. Thus we show for the first time clear tissue-specific differences in nonlymphoid DCs. Although it is not possible to conclude from our data whether liver DCs are more regulatory, or skin DCs more proimmunogenic, the ability of liver DCs to secrete IL-10 may be important for regulating local immune responses within the liver in the face of constant exposure to gut antigens.  

4.220           Unraveling the secrets of single donor success in islet transplantation

Shapiro, A.M.J. and Ricordi, C. Am. J. Transplant., 4, 295-298 (2004)   Islet transplantation is emerging as an attractive alternative to solitary pancreas transplantation for a highly select group of patients with severe, labile forms of Type 1 diabetes that had previously tried and failed on intensive insulin therapy. The year 2000 marked a dramatic shift in clinical success with the introduction of the so-called 'Edmonton Protocol', which built upon many years of intensive research and extensive collaborations between islet groups worldwide (1,2).  

4.221           Transplantation of cultured islets from two-layer preserved pancreases in type 1 diabetes with anti-CD3 antibody

Hering, B.J. et al Am. J. Transplant., 4, 390-401 (2004) We sought to determine whether or not optimizing pancreas preservation, islet processing, and induction immunosuppression would facilitate sustained diabetes reversal after single-donor islet transplants. Islets were isolated from two-layer preserved pancreata, purified, cultured for 2 days; and transplanted into six C-peptide-negative, nonuremic, type 1 diabetic patients with hypoglycemia unawareness. Induction immunosuppression, which began 2 days pretransplant, included the Fc receptor nonbinding humanized anti-CD3 monoclonal antibody hOKT3 1 (Ala-Ala) and sirolimus. Immunosuppression was maintained with sirolimus and reduced-dose tacrolimus. Of our six recipients, four achieved and maintained insulin independence with normal HbA1c levels and freedom from hypoglycemia; one had partial islet graft function; and one lost islet graft function 2 weeks post-transplant. The four insulin-independent patients showed prolonged CD4⁺ T-cell lymphocytopenia; inverted CD4:CD8 ratios; and increases in the percentage of CD4⁺CD25⁺ T cells. These cells suppressed the in-vitro proliferative response to donor cells and, to a lesser extent, to third-party cells. Severe adverse events were limited to a transient rash in one recipient and to temporary neutropenia in three. Our preliminary results thus suggest that a combination of maximized viable islet yield, pretransplant islet culture, and preemptive immunosuppression can result in successful single-donor islet transplants.  

4.222           Transplanting encephalomyocarditis virus-infected porcine islet cells reverses diabetes in recipient mice but also transmits the virus

Brewer, L., LaRue, R., Hering, B., Brown, C. and Njenga, M.K. Xenotransplantation, 11, 160-170 (2004)   Previous studies demonstrated that porcine encephalomyocarditis virus (EMCV) caused acute and persistent infection in the myocardium, central nervous system, and spleen of non-human primates (cynomolgus macaques); and it productively infected primary human cardiomyocytes, suggesting that the virus may pose a risk in pig-to-human transplantation. Recently, transplantation of myocardial and pancreatic tissues from acutely infected pigs transmitted the virus to recipient mice, resulting in acute fatal EMCV disease. Here, we examined whether porcine islet cells (PICs), which are under clinical trial for treatment of type I diabetes in humans, are susceptible to porcine EMCV, and whether EMCV-infected PICs could function in vivo to reverse diabetes. PICs were infected with EMCV in vitro for 5 h, and resulting insulin production compared with that produced by uninfected PICs. Subsequently, infected PICs were transplanted intra-abdominally or under the kidney capsule of C57BL/6 mice, and both virus transmission and PIC function analyzed. PICs were highly susceptible to porcine EMCV, resulting in a 1500-fold increase in production of infectious virus within 5 h of inoculation and cytolysis that destroyed up to 50% of cells within 96 h. However, as long as they were viable, infected PICs produced insulin at levels comparable with uninfected PICs. Intra-abdominal transplantation of 2000 PICs, infected with one plaque forming unit (pfu) per cell of porcine EMCV, into C57BL/6 mice transmitted the virus resulting in acute fatal EMCV disease characterized by hind limb paresis and paralysis and acute respiratory distress in 40% of recipient mice. More importantly, transplantation of 2500 EMCV-infected PICs under the kidney capsule of diabetic C57BL/6 mice (glucose level 350 mg/dl) reversed diabetes in 83% of recipient mice (glucose level 170 mg/dl); however these mice succumbed to acute EMCV disease transmitted by the xenograft 5 days after transplantation. EMCV infection does not appear to affect insulin production by PICs, but infected xenografts can transmit the virus to recipient animals, resulting in severe disease.  

4.223           Increased CD36 protein as a response to defective insulin signaling in macrophages

Liang, C-P. Et al

1. Clin. Invest., 113(5), 764-773

  Accelerated atherosclerosis is a major cause of morbidity and death in insulin-resistant states such as obesity and the metabolic syndrome, but the underlying mechanisms are poorly understood. We show that macrophages from obese (ob/ob) mice have increased binding and uptake of oxidized LDL, in part due to a post-transcriptional increase in CD36 protein. Macrophages from ob/ob mice are also insulin resistant, as shown by reduced expression and signaling of insulin receptors. Three lines of evidence indicate that the increase in CD36 is caused by defective insulin signaling: (a) Treatment of wild-type macrophages with LY294002, an inhibitor of insulin signaling via PI3K, results in an increase in CD36; (b) insulin receptor knockout macrophages show a post-transcriptional increase in CD36 protein; and (c) administration of thiazolidinediones to intact ob/ob mice and ob/ob, LDL receptor–deficient mice results in a reversal of macrophage insulin receptor defects and decreases CD36 protein. The last finding contrasts with the increase in CD36 that results from treatment of macrophages with these drugs ex vivo. The results suggest that defective macrophage insulin signaling predisposes to foam cell formation and atherosclerosis in insulin-resistant states and that this is reversed in vivo by treatment with PPAR- activators.  

4.224           Role of tau phosphorylation by glycogen synthase kinase-3b in the regulation of organelle transport

Tatebayashi, Y., Haque, N., Tung, Y-C., Iqbal, K. and Grundke-Iqbal, I.

1. Cell Sci., 117, 1653-1663 (2004)

  Anterograde organelle transport is known to be inhibited by overexpression of the microtubule-associated protein tau in cultured cells. However, the molecular mechanism regulating this function of tau protein has not previously been understood. We found that in PC12 cells treated with NGF or fibroblast growth factor-2, glycogen synthase kinase-3ß and tau were upregulated simultaneously from around day 2 of differentiation, with increasing glycogen synthase kinase-3-mediated tau phosphorylation. This phosphorylation did not alter tau's ability to bind to microtubules but appeared to be required for the maintenance of the anterograde organelle transport in differentiated cells. Lithium, alsterpaullone or valproate, three independent glycogen synthase kinase-3 inhibitors, but not butyrolactone 1, an inhibitor of cyclin-dependent protein kinases, induced mitochondrial clustering in association with tau dephosphorylation. In CHO cells transfected with human tau₄₄₁, mitochondrial clustering was found in cells in which tau was unphosphorylated. These findings raise the possibility that the phosphorylation of tau by glycogen synthase kinase-3 might be involved in the regulation of organelle transport.  

4.225           NALP3 forms an IL-1b-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder

Agostini, L. et al Immunity, 20, 319-325 (2004)   Mutations within the NALP3/cryopyrin/CIAS1 gene are responsible for three autoinflammatory disorders: Muckle-Wells syndrome, familial cold autoinflammatory syndrome, and CINCA. The NALP3 protein is homologous to NALP1, which is a component of the inflammasome, a molecular platform that activates the proinflammatory caspases-1 and -5. NALP3 (and other members of the NALP family) lacks the C-terminal, CARD-containing sequence of NALP1, and its role in caspase activation is unclear. Here, we report that NALP2 and NALP3 associate with ASC, the CARD-containing protein Cardinal, and caspase-1 (but not caspase-5), thereby forming an inflammasome with high proIL-1β-processing activity. Macrophages from Muckle-Wells patients spontaneously secrete active IL-1β. Increased inflammasome activity is therefore likely to be the molecular basis of the symptoms associated with NALP3-dependent autoinflammatory disorders.  

4.226           Octreotide regulates CC but not CXC LPS-induced chemokine secretion in rat Kupffer cells

Valatas, V. et al Br. J. Pharm., 141, 477-487 (2004)   1 Kupffer cells (KC) and lipopolysaccharide (LPS) interactionis the initial event leading to hepatic inflammation and fibrosisin many types of liver injury. We studied chemokine secretionby KC activated with LPS and the possible effect of the somatostatinanalogue octreotide, in the regulation of this process. 2 KC isolated from Sprague–Dawley rats were culturedin the presence of LPS added alone or with different concentrationsof octreotide for 24 and 48 h, and chemokine production wasassessed in culture supernatants by ELISA. CC chemokine mRNAexpression was assessed by semiquantitative RT–PCR. 3 Vehicle-stimulated KC produced a basal amount of CC and CXC chemokines. LPS-stimulated KC secreted significantly increased amounts of IL-8 (GRO/CINC-1) (P<0.001), MIP-2 (P<0.001), MCP-1 (P<0.001), and RANTES (P<0.01). 4 Octreotide inhibited LPS-induced secretion of the CC chemokines MCP-1 (P<0.05) and RANTES (P<0.05), but notthe CXC chemokines IL-8 (GRO/CINC-1) and MIP-2, in a concentration-dependentmanner. Downregulation of basal and LPS-induced mRNA expressionof the CC chemokines was also observed in the presence of octreotide. 5 Pretreatment with phosphatidylinositol 3 (PI3)-kinaseinhibitors reduced chemokine production by LPS-treated KC inboth the mRNA and protein level. Furthermore, it prevented theoctreotide inhibitory effect on LPS-induced chemokine secretion,indicating a possible involvement of the PI3-kinase pathway. 6 In conclusion, these data demonstrate that chemokinesecretion by KC can be differentially regulated by octreotide,and suggest that this somatostatin analogue may have immunoregulatoryeffects on resident liver macrophages.  

4.227           Interleukin-6 deficiency affects bone marrow stromal precursors, resulting in defective hematopoietic support

Del Carmen, M., Bernad, A. And Aracil, M. Blood, 103(9), 3349-3354 (2004)   Interleukin-6 (IL-6) is a critical factor in the regulation of stromal function and hematopoiesis. In vivo bromodeoxyuridine incorporation analysis indicates that the percentage of Lin^-Sca-1⁺ hematopoietic progenitors undergoing DNA synthesis is diminished in IL-6-deficient (IL-6^-/-) bone marrow (BM) compared with wild-type BM. Reduced proliferation of IL-6^-/- BM progenitors is also observed in IL-6^-/- long-term BM cultures, which show defective hematopoietic support as measured by production of total cells, granulocyte macrophage-colony-forming units (CFU-GMs), and erythroid burst-forming units (BFU-Es). Seeding experiments of wild-type and IL-6^-/- BM cells on irradiated wild-type or IL-6-deficient stroma indicate that the hematopoietic defect can be attributed to the stromal and not to the hematopoietic component. In IL-6^-/- BM, stromal mesenchymal precursors, fibroblast CFUs (CFU-Fs), and stroma-initiating cells (SICs) are reduced to almost 50% of the wild-type BM value. Moreover, IL-6^-/- stromata show increased CD34 and CD49e expression and reduced expression of the membrane antigens vascular cell adhesion molecule-1 (VCAM-1), Sca-1, CD49f, and Thy1. These data strongly suggest that IL-6 is an in vivo growth factor for mesenchymal precursors, which are in part implicated in the reduced longevity of the long-term repopulating stem cell compartment of IL-6^-/- mice. (Blood. 2004;103:3349-3354)  

4.228           A syntaxin 1, Ga_o, and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization

Li, Q. et al

1. Neurosci., 24(16), 4070-4081 (2004)

  Presynaptic Ca_V2.2 (N-type) calcium channels are subject to modulation by interaction with syntaxin 1 and by a syntaxin 1-sensitive G _O G-protein pathway. We used biochemical analysis of neuronal tissue lysates and a new quantitative test of colocalization by intensity correlation analysis at the giant calyx-type presynaptic terminal of the chick ciliary ganglion to explore the association of Ca_V2.2 with syntaxin 1 and G _O. Ca_V2.2 could be localized by immunocytochemistry (antibody Ab571) in puncta on the release site aspect of the presynaptic terminal and close to synaptic vesicle clouds. Syntaxin 1 coimmunoprecipitated with Ca_V2.2 from chick brain and chick ciliary ganglia and was widely distributed on the presynaptic terminal membrane. A fraction of the total syntaxin 1 colocalized with the Ca_V2.2 puncta, whereas the bulk colocalized with MUNC18-1. G _O, whether in its trimeric or monomeric state, did not coimmunoprecipitate with Ca_V2.2, MUNC18-1, or syntaxin 1. However, the G-protein exhibited a punctate staining on the calyx membrane with an intensity that varied in synchrony with that for both Ca channels and syntaxin 1 but only weakly with MUNC18-1. Thus, syntaxin 1 appears to be a component of two separate complexes at the presynaptic terminal, a minor one at the transmitter release site with Ca_V2.2 and G _O, as well as in large clusters remote from the release site with MUNC18-1. These syntaxin 1 protein complexes may play distinct roles in presynaptic biology.  

4.229           Postmortem effect of pentobarbital anesthetic on survival of adult coctical neurons in primary culture

Viel, J.J., McManus, D.Q., Brewer, G.J. BrainRres.,1009, 219-222 (2004)   We determined whether pentobarbital anesthetic is required to culture postmortem adult rat neurons. Pentobarbital treatment resulted in two-fold increases in neuron survival in culture after 2 and 4 h postmortem compared to non-anesthetic controls, but was not as effective as simple postmortem treatment on ice and therefore not essential.  

4.230           Preservation of human pancreatic islet in vivo function after 6-month culture in serum-free media

Rush, B. et al Transplantation, 77, 1147-1154 (2004)   Background. Culturing human islets in Memphis serum-free media (M-SFM) is associated with excellent postculture recovery, in vitro function, and in vivo survival. The authors investigate the possibility of preserving islet function for extended periods (6 months) in culture and describe the in vitro and in vivo functional outcomes associated with these extended culture times. Methods. Human islets isolated from three cadaveric donor organs were cultured in M-SFM for 1, 3, or 6 months before transplantation under the kidney capsule of nonobese diabetic (NOD)-severe combined immunodeficiency (SCID) mice. In vitro function was measured by static incubation at the time of transplantation. In vivo function was assessed by measuring human insulin and C-peptide production, and by the ability of 6-month cultured islets to cure streptozotocin-induced diabetes in this mouse model. Results. Islet recovery ratios after 1 month in culture ranged from 85% to 88% and declined to 28% to 53% after 6 months of culture (P <0.01). Insulin stimulation indices did not differ among the fresh or the 6-month cultured preparations. All preparations cultured for 1 to 3 months functioned in the NOD-SCID mice. After 6 months of culture, two of the three preparations demonstrated in vivo function and were able to cure streptozotocin-induced diabetes. Conclusions. These data demonstrate that human islets can be cultured in M-SFM for extended periods and still retain in vitro and in vivo function and the ability to cure experimental diabetes. The ability to maintain islets in culture for prolonged periods is an important step toward the development of islet tissue repositories and distribution centers.  

4.231           Comparative proteomics of primitive hematopoietic cell populations reveals differences in expression of proteins regulating motility

Evans, C.A. et al Blood, 103(10), 3751-3759 (2004)   Lineage-marker depleted (Lin^–) murine bone marrow cells expressing stem cell antigen 1 (Sca-1) were sorted on the basis of stem cell factor receptor (c-kit) expression to obtain Lin^–Sca⁺Kit⁺ or Lin^–Sca⁺Kit^– cells. Lin^–Sca⁺Kit^– cells have a markedly greater chemotactic response to stromal derived factor-1 (SDF-1). Using a novel fluorescent stain, we show that both populations generate similar levels of a key messenger, phosphatidylinositol 3,4,5 trisphosphate (PIP₃), in response to SDF-1. Differences in motile behavior may therefore lie downstream of phosphatidylinositol 3-kinase (PI3-kinase) activation at the level of cytoskeleton regulation. The 2 cell populations were compared using 2-dimensional difference gel electrophoresis (2D-DIGE), with a maleimide CyDye fluorescent protein labeling technique that has enhanced sensitivity for low abundance samples. Comparative proteomic analysis of Cy3- and Cy5-labeled protein samples allows relative quantification of protein spots present in both cell populations; of these, 73% were common. Key protein differences were adseverin and gelsolin, actin micro-filament splicing proteins, regulated by Rac, downstream of PI3-kinase activation. Adseverin was shown to be acetylated, a novel modification for this protein. Differences in major regulators of cell shape and motility between the 2 populations can explain the differential response to SDF-1.  

4.232           Evidence that dietary supplementation with carotenoids and carotenoid-rich foods modulates the DNA damage: repair balance in human lymphocytes

Astley, S.B., Elliott, R.M., Archer, D.B. and Southon, S. Br. J. Nutrition, 91, 63-72 (2004)   Epidemiological evidence has shown that the habitual consumption of diets high in fruits and vegetables is associated with reduced risk of cancers. The challenge is to identify causal mechanisms of effect. The aim of the current study was to determine whether an increase in rate of removal of DNA single-strand breaks (SSB) following oxidative challenge could be provoked ex vivo in peripheral blood lymphocytes (PBL). The PBL were isolated from apparently healthy volunteers following dietary intervention with: (1) a mixed carotene capsule; (2) a daily portion of cooked minced carrots; (3) a matched placebo; (4) a portion of mandarin oranges; (5) vitamin C tablets. Single-cell gel electrophoresis was employed to measure baseline levels of SSB and DNA susceptibility to oxidative damage, and to monitor the number of SSB over 4 h, in both unchallenged and H2O2-treated PBL. The enzymatic capacity for repair of different types of DNA oxidative lesions was also measured using two related cell-free assays. There was no evidence that any of the dietary supplementation regimens altered baseline levels of SSB, provided any direct antioxidant protection or altered DNA repair capacity, with two exceptions: the number of SSB following exposure to H2O2 decreased more rapidly in PBL from volunteers given the mixed carotene capsules and repair patch synthesis activity in PBL increased from volunteers given the cooked carrots. These results suggest that carotenoids and carotenoid-rich foods can influence DNA damage:repair by modulation of discrete stages in the DNA repair mechanisms.  

4.233           Metabotropic glutamate receptors are expressed in adult human glial progenitor cells

Luyt, K., Varadi, A., Halfpenny, C.A., Scolding, N.J. and Molnar, E. Biochem. Biophys. Res. Comm., 319, 120-129 (2004)   Glial precursor cells (GPCs) are present in the adult human central nervous system (CNS) and they can be isolated and maintained in culture for in vitro studies. This study analysed expression of mGluR3 and mGluR5 metabotropic glutamate receptor (mGluR) mRNAs in GPCs. A2B5 surface antigen positive GPCs were isolated using immunomagnetic selection from dissociated temporal lobe subcortical white matter cells. The separated GPCs were maintained in cultures and characterised by immunoreactivity for the differentiation markers A2B5 and human platelet-derived growth factor- receptor (PDGF R). Reverse transcription followed by multiplex PCR analysis showed that the GPCs expressed both mGluR3 and mGluR5a mRNAs. Double immunostaining for glial progenitor markers and mGluR5 proteins demonstrated that all A2B5 and PDGF R-positive cells were also positive for mGluR5. The results indicate that GPCs present in the adult human CNS express mGluR3 and mGluR5a. These neurotransmitter receptors may be involved in the proliferation and differentiation of glial cells.  

4.234           Flagellin promotes myeloid differentiation factor 88-dependent development of Th2-type response

Didierlaurent, A. et al

1. Immunol., 172, 6922-6930 (2004)

  Activation of dendritic cells (DC) by microbial products via Toll-like receptors (TLR) is instrumental in the induction of immunity. In particular, TLR signaling plays a major role in the instruction of Th1 responses. The development of Th2 responses has been proposed to be independent of the adapter molecule myeloid differentiation factor 88 (MyD88) involved in signal transduction by TLRs. In this study we show that flagellin, the bacterial stimulus for TLR5, drives MyD88-dependent Th2-type immunity in mice. Flagellin promotes the secretion of IL-4 and IL-13 by Ag-specific CD4⁺ T cells as well as IgG1 responses. The Th2-biased responses are associated with the maturation of DCs, which are shown to express TLR5. Flagellin-mediated DC activation requires MyD88 and induces NF- B-dependent transcription and the production of low levels of proinflammatory cytokines. In addition, the flagellin-specific response is characterized by the lack of secretion of the Th1-promoting cytokine IL-12 p70. In conclusion, this study suggests that flagellin and, more generally, TLR ligands can control Th2 responses in a MyD88-dependent manner.  

4.235           Regulation of tissue inhibitor of metalloproteinase 1 gene transcription by RUNX1 and RUNX2

Bertrand-Philippe, M. Et al

1. Biol. Chem., 279(23), 24530-24539 (2004)

  Tissue inhibitor of metalloproteinase 1 (TIMP1) is a contributory factor to fibrosis of a variety of organs including the liver. UTE-1 is a regulatory DNA motif essential for TIMP1 promoter activity in a variety of cell types including hepatic stellate cells (HSC), the key profibrogenic cells of the liver. In this study we identify RUNX1 and RUNX2 as UTE-1-binding proteins that are induced at the post-transcriptional level during activation of HSC. RUNX1 is expressed in at least two major isoforms, RUNX1B and RUNX1A. Overexpression of full-length RUNX1B isoform in HSC repressed TIMP1 promoter activity, whereas the truncated RUNX1A isoform and RUNX2 functioned as stimulators. To gain further understanding of the way in which RUNX1 isoforms differentially regulate TIMP1 transcription, we investigated the relationship between the UTE-1 site and its adjacent upstream serum-response element (SRE) in the promoter. The UTE-1 and SRE sites cooperate in a synergistic fashion to stimulate transcription of a heterologous minimal active promoter providing that they are in close proximity. The key regulatory sequence within the SRE is an AP-1 site that in HSC directs high level transcription via its interaction with JunD. RUNX1A was shown to interact directly with JunD, and by contrast RUNX1B failed to interact with JunD. Co-expression studies showed that RUNX1B can repress JunD-stimulated TIMP1 promoter activity. From these observations we propose that JunD and RUNX factors assemble at the adjacent SRE and UTE-1 sites in the TIMP1 promoter and form functional interactions that stimulate transcription. However, RUNX1B is unable to interact with JunD, and as such its occupancy at the UTE-1 site disrupts the optimal assembly of transcriptional activators required for directing high level TIMP1 promoter function.  

4.236           Secretion of inflammatory mediators by isolated rat Kupffer cells; the effect of octreotide

Valatas, V. et al Regulatory Peptides, 120, 215-225 (2004)   Aims: We studied the production of inflammatory mediators by rat KC and the possible in vitro effect of the somatostatin analogue octreotide. Methods: Primary KC cultures were incubated with LPS added alone or with different concentrations of octreotide. The production of TNF , IL-6, IL-10, IL-12 and IL-13 was assessed in culture supernatants by ELISA and that of nitric oxide (NO) by a modification of the Griess reaction. Results: Isolated KC produced a basal amount of TNF , IL-6, IL-12, IL-13, and NO but not IL-10. LPS-stimulated KC secreted significantly increased amounts of TNF (P<0.001), IL-6 (P<0.01), IL-10 (P<0.001), IL-12 (P<0.01), and NO (P<0.001) whereas IL-13 production remained constant. Octreotide reduced IL-12 (P<0.05) and increased IL-13 (P<0.05) production by unstimulated KC. Furthermore, octreotide suppressed TNF production (P<0.05), without modifying TNF mRNA expression and decreased iNOS expression and NO (P≈0.05) production by LPS-activated KC. These effects were reversed with Wortmannin pre-treatment suggesting that octreotide may act via interference with phosphatidylinositol 3-kinase pathways. Conclusions: These data demonstrate that KC is a source of multiple inflammatory mediators, indicating a critical role in liver inflammatory disorders. Octreotide modulates inflammatory mediator production by isolated KC, suggesting that it might have immunoregulatory and anti-inflammatory effects in liver diseases.  

4.237           Establishment of a pure culture of the hitherto uncultured unicellular cyanobacterium Aphanothece sacrum, and phylogenetic position of the organism

Fujishiro, H. et al Appl, Enviroment. Microbiol., 70(6), 3338-3345 (2004)   Aphanothece sacrum, an edible freshwater unicellular cyanobacterium, was isolated by using novel synthetic media (designated AST and AST-5xNP). The media were designed on the basis of the ratio of inorganic elements contained in A. sacrum cells cultured in a natural pond. The isolated strain exhibits unicellular rod-shaped cells 6 µm in length that are scattered in an exopolysaccharide matrix, a feature similar to that of natural A. sacrum. DNA analysis of the isolated strain revealed that it carried two ferredoxin genes whose deduced amino acid sequences were almost identical to previously published sequences of ferredoxins from natural A. sacrum. Analysis of the 16S rRNA gene and ferredoxin genes revealed that A. sacrum occupies a phylogenetically unique position among the cyanobacteria.  

4.238           Apolipoprotein A-I stimulated apolipoprotein E secretion from human macrophages is independent of cholesterol efflux

Kockx, M. et al’

1. Biol. Chem., 279(25), 25966-25977 (2004)

  Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using ³⁵S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E_m) and stable (E_s) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer -helical peptides representing amphipathic -helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal -helix (domains 220–241) stimulates cholesterol efflux. Other -helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220–5222delTCT; and mutations A1046D and c.4629–4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by -helix-containing molecules including apoA-I and apoE.  

4.239           Induction of MIP-1a in Kupffer cell by portal venous transfusion

Park, J.K., Cho, K., Johnson, J. and Perez, R.V. Transplant Immunol., 13, 33-38 (2004)   Introduction: Previous studies have shown that portal venous transfusion (PVT) induces a state of immunosuppression, and Kupffer cells may be involved in the mechanism. Objective: This study was aimed to investigate the effect of PVT on Kupffer cell gene expression. Materials and Methods: Each BALB/C mouse was subjected to laparotomy and received one of five treatments: PVT, portal venous saline injection (PVS), inferior vena caval transfusion (IVCT), inferior vena caval saline injection (IVCS) or sham operation (S). The blood for PVT and IVCT was sampled from C57BL/6J mice. Kupffer cells were then isolated 1 or 24 h after each of the 5 treatments, for a total of 10 experimental groups (1-h PVT, PVS, IVCT, IVCS and S, and 24-h PVT, PVS, IVCT, IVCS and S) from BALB/C mice. To examine the effect of PVT on Kupffer cell gene expression, RT-PCR differential display was performed. Results: Increase in the expression of MIP-1 mRNA post PVT and IVCT was identified by differential display. PVT groups revealed higher levels of serum MIP-1 than any other groups. Conclusion: These results suggest that MIP-1 may be involved in a cascade of signaling events associated with the PVT-mediated immunologic modulation in Kupffer cells.  

4.240           Estrogen facilitates neurite extension via apolipoprotein E in cultured adult mouse cortical neurons

Nathan, B.P., Barsukova, A.G., Shen, F., McAsey, M. and Struble, R.G. Endocrinol., 145(7), 3065-3073 (2004)   Literature review suggests a close relationship between estrogen and apolipoprotein E (ApoE) in the central nervous system. Epidemiology studies show that estrogen replacement therapy (ERT) decreases the morbidity from several chronic neurological diseases. Alleles of ApoE modify the risk for and progression of the same diseases. ApoE levels in the rodent brain vary during the estrous cycle and increase after 17ß-estradiol administration. Both estradiol and ApoE3, the most common isoform of human ApoE, increase the extent of neurite outgrowth in culture. Combined, these observations suggest a common mechanism whereby estrogen may increase ApoE levels to facilitate neurite growth. We tested this hypothesis by characterizing the effects of estradiol and ApoE isoforms on neurite outgrowth in cultured adult mouse cortical neurons. Estradiol increased ApoE levels and neurite outgrowth. ApoE2 increased neurite length more so than ApoE3 in the presence of estradiol. Estradiol had no effect on neurite outgrowth from mice lacking the ApoE gene or when only ApoE4, the isoform of ApoE that is associated with increased risk of neurological disease, was exogenously supplied. Cultures from mice transgenic for human ApoE3 or ApoE4 showed the same isoform-specific effect. Neuronal internalization of recombinant human ApoE3 was greater than ApoE4, and ApoE3 was more effective than ApoE4 in facilitating neuronal uptake of a fatty acid. We conclude that estradiol facilitates neurite growth through an ApoE-dependent mechanism. The effects of ERT on chronic neurological diseases may vary with ApoE genotype. The clinical use of ERT may require ApoE genotyping for optimal efficacy.  

4.241           Liver sinusoidal endothelial cells are insufficient to activate T cells

Katz, S.C., Pillarisetty, V.G., Bleier, J.I., Shah, A.B. and DeMatteo, R.P.

1. Immunol., 173, 230-235 (2004)

  Liver sinusoidal endothelial cells (LSEC) have been reported to express MHC class II, CD80, CD86, and CD11c and effectively stimulate naive T cells. Because dendritic cells (DC) are known to possess these characteristics, we sought to directly compare the phenotype and function of murine LSEC and DC. Nonparenchymal cells from C57BL/6 mice were obtained by collagenase digestion of the liver followed by density gradient centrifugation. From the enriched nonparenchymal cell fraction, LSEC (CD45^–) were then isolated to 99% purity using immunomagnetic beads. Flow cytometric analysis of LSEC demonstrated high expression of CD31, von Willebrand factor, and Fc Rs. However, unlike DC, LSEC had low or absent expression of MHC class II, CD86, and CD11c. LSEC demonstrated a high capacity for Ag uptake in vitro and in vivo. Although acetylated low-density lipoprotein uptake has been purported to be a specific function of LSEC, we found DC captured acetylated low-density lipoprotein to a similar extent in vivo. Consistent with their phenotype, LSEC were poor stimulators of allogeneic T cells. Furthermore, in the absence of exogenous costimulation, LSEC induced negligible proliferation of CD4⁺ or CD8⁺ TCR-transgenic T cells. Thus, contrary to previous reports, our data indicate that LSEC alone are insufficient to activate naive T cells.    

4.242           Hippocampal synapses depend on hippocampal estrogen synthesis

Kretz, O. et al

1. Neurosci., 24(26), 5913-5921 (2004)

  Estrogens have been described to induce synaptogenesis in principal neurons of the hippocampus and have been shown to be synthesized and released by exactly these neurons. Here, we have focused on the significance of local estrogen synthesis on spine synapse formation and the synthesis of synaptic proteins. To this end, we reduced hippocampal estrogen synthesis in vitro with letrozole, a reversible nonsteroidal aromatase inhibitor. In hippocampal slice cultures, letrozole treatment resulted in a dose-dependent decrease of 17 -estradiol as quantified by RIA. This was accompanied by a significant decrease in the density of spine synapses and in the number of presynaptic boutons. Quantitative immunohistochemistry revealed a downregulation of spinophilin, a marker of dendritic spines, and synaptophysin, a protein of presynaptic vesicles, in response to letrozole. Surprisingly, no increase in the density of spines, boutons, and synapses and in spinophilin expression was seen after application of estradiol to the medium of cultures that had not been treated with letrozole. However, synaptophysin expression was upregulated under these conditions. Our results point to an essential role of endogenous hippocampal estrogen synthesis in the maintenance of hippocampal spine synapses.  

4.243           Monocyte activation in patients with age-related macular degeneration

Cousins, S.W., Espinosa-Heidelmann, D.G. and Csaky, K.G. Arch. Ophthalmol., 122, 1013-1018 (2004)   Objective  To evaluate the activation state of macrophagefunction in patients with age-related macular degeneration (AMD)by quantifying the production of the proinflammatory and angiogenicfactor tumor necrosis factor (TNF-) and by correlating itsexpression with dry and wet AMD. Methods  Circulating monocytes were obtained from the bloodof patients with AMD or age-matched control subjects by gradientcentrifugation. The monocytes were then analyzed for eitherTNF- release from cultured macrophages in response to retinalpigment epithelium–derived blebs and cytokines or TNF-messenger RNA content by reverse transcriptase–polymerasechain reaction. Results  In human monocytes obtained from controls andAMD patients, TNF- was expressed by freshly isolated monocytesand produced by macrophages in culture after stimulation withretinal pigment epithelium–derived blebs. However, widevariability in TNF- expression was observed among differentpatients. Patients with monocytes that expressed the greatestamount of TNF- demonstrated higher prevalence of choroidal neovascularization. Conclusions  Both controls and AMD patients vary in theactivation state (defined as TNF- expression) of circulatingmonocytes. Partially active monocytes, defined as high TNF-expression, may be a biomarker to identify patients at riskfor formation of choroidal neovascularization. Clinical Relevance  Early diagnostic testing may proveuseful to detect those patients who will progress to the moresevere complications of the disease.  

4.244           Inhibition of experimental asthma by indoleamine 2,3-dioxygenase

Hayashi, T. et al

1. Clin. Invest., 114(2), 270-279 (2004)

  Epidemiological evidence points to the inverse relationship between microbial exposure and the prevalence of allergic asthma and autoimmune diseases in Westernized countries. The molecular basis for this observation has not yet been completely delineated. Here we report that the administration of certain toll-like receptor (TLR) ligands, via the activation of innate immunity, induces high levels of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme of tryptophan catabolism in various organs. TLR9 ligand–induced pulmonary IDO activity inhibits Th2-driven experimental asthma. IDO activity expressed by resident lung cells rather than by pulmonary DCs suppressed lung inflammation and airway hyperreactivity. Our results provide a mechanistic insight into the various formulations of the hygiene hypothesis and underscore the notion that activation of innate immunity can inhibit adaptive Th cell responses.  

4.245           Adenovirus-based vascular endothelial growth factor gene delivery to human pancreatic islets

Cheng, K. et al Gene Ther., 11, 1105-1116 (2004)   Islet transplantation is limited by islet graft failure due to poor revascularization, host immune rejection and nonspecific inflammatory response. Delivery of human vascular endothelial growth factor (hVEGF) gene to the islets is likely to promote islet revascularization and survival. We used a bicistronic adenoviral vector encoding hVEGF and CpG-free allele of green fluorescent protein (Adv-GFP-hVEGF) and introduced into human pancreatic islets by transfection. We found that transfection efficiency and apoptosis were dependent on the multiplicity of infection (MOI). Compared to Adv-GFP transfected and nontransfected islets, the levels of hVEGF secreted from Adv-GFP-hVEGF transfected islets were higher and exhibit a linear relationship between hVEGF expression and MOI (10-5000). Persistent, but low level expression of hVEGF from nontransfected islets was also observed. This may be due to expression of the endogenous hVEGF gene under hypoxic conditions. The levels of DNA fragmentation determined by ELISA of islet lysates were dependent on the MOI of Adv-GFP-hVEGF. On glucose challenge, insulin release from transfected islets was comparable to nontransfected islets. Immunohistochemical staining for hVEGF was very high in Adv-GFP-hVEGF transfected islets. Weak staining was also observed for hCD31 in both transfected and nontransfected islets. These findings suggest that Adv-GFP-hVEGF is a potential candidate for promoting islet revascularization.  

4.246           Potential involvement of gelatinases and their inhibitors in Mannheimia haemolytica pneumonia in cattle

Starr, A.E., Dan, T., Minhas, K., Shewen, P.E. and Coomber, B.L. Infection and Immunity., 72(8), 4393-4400 (2004)   Mannheimia haemolytica infection of the lower respiratory tract of cattle results in a bronchofibrinous pneumonia characterized by massive cellular influx and lung tissue remodeling and scarring. Since altered levels of gelatinases and their inhibitors have been detected in a variety of inflammatory conditions and are associated with tissue remodeling, we examined the presence of gelatinases in lesional and nonlesional lung tissue obtained from calves experimentally infected with M. haemolytica. Lesional tissue had elevated levels of progelatinase A and B and active gelatinase A and B when compared with nonlesional tissue obtained from the same lung lobe. In vitro, M. haemolytica products stimulated production of gelatinase B, but not its activation, by bovine monocytes. Alveolar macrophages showed constitutive production of gelatinase B but no change in response to M. haemolytica products. Bovine neutrophils exposed to M. haemolytica products also released gelatinase B, and there was a significant increase in the activated form of this enzyme. These effects were virtually identical when recombinant O-sialoglycoprotease was used to stimulate these cells. M. haemolytica products also enhanced the expression by bovine monocytes and alveolar macrophages of the tissue inhibitor of metalloproteinase 1. Our results provide evidence that matrix metalloproteinases are activated in lung lesions from cattle with shipping fever and that M. haemolytica virulence products induce production, release, and especially activation of gelatinase B by bovine inflammatory cells in vitro.  

4.247           Effect of functionalization of multilayered polyelectrolyte films on motoneuron growth

Vodouche, C. et al Biomaterials, 26, 545-554 (2005)   We studied in vitro cell–substrate interaction of motoneurons with functionalized polylectrolyte films. Thin polylectrolyte films were built on glass by alternating polycations, poly(ethylene-imine) PEI, poly( -lysine) PLL, or poly(allylamine hydrochloride) PAH, and polyanions, poly(sodium-4-styrenesulfonate) PSS or poly( -glutamic acid) (PGA). These architectures were functionalized with Brain Derived Neurotrophic Factor (BDNF) or Semaphorin 3A (Sema3A). We used Optical Waveguide Lightmode Spectroscopy (OWLS) and Atomic Force Microscopy (AFM) to characterize the architectures. The viability of motoneurons was estimated by the acid phosphatase method, and morphometrical measures were performed to analyse the influence of different architectures on cell morphology. Motoneurons appeared to adhere and spread on all the architectures tested and preferentially on PSS ending films. The viability of motoneurons on polyelectrolyte multilayers was higher compared to polyelectrolyte monolayers. BDNF and Sema3A embedded in the films remained active and thereby create functionalized nanofilms.  

4.248           In vitro opioid induced proliferation of peripheral blood immune cells correlates with in vivo cold pressor pain tolerance in humans: a biological marker of pain tolerance

Hutchinson, M.R., La Vincente, S.F. and Somogyi, A.A. Pain, 110, 751-755 (2004)   There is substantial evidence for bidirectional communication between the immune system and the central nervous system, as the cells and signalling molecules of the immune system influence many central nervous system functions, for instance nociception. Opioids, such as morphine, produce analgesia and numerous other central and peripheral effects including sedation and euphoria, while their effects on the immune system are wide-ranging. There is considerable interindividual variability in basal nociception and response to opioids, however, the physiological and biological mechanisms underlying this are unclear. Therefore, we investigated the relationship between the immune system and basal nociceptive thresholds, using the proliferative response of isolated peripheral blood mononuclear cells and cold pressor pain tolerance. Here we show that the percent increase in proliferation of peripheral immune cells from 13 healthy subjects incubated with morphine ex vivo is highly correlated with the subjects' tolerance to noxious cold stimuli (Pearson r=0.92, P<0.0001). These pilot data provide evidence of a novel objective biological marker of pain tolerance in humans, which also links the immune and opioid systems with basal pain tolerance.  

4.249           Experimental infection of horses with culture-derived Sarcocystis neurona merozoites as a model for equine protozoal myeloencephalitis

Ellison, S.P., Greiner, E., Brown, K.W. and Kennedy, T. Intern. J. Appl. Res. Vet. Med., 2(2), 79-89 (2004)   A study was designed to develop an experimental model to produce Sarcocystis neurona encephalitis in horses. Sarcocystis neurona, isolated from the spinal cord of an ataxic horse, was placed in continuous culture using bovine turbinate cells, and the cultured S. neurona merozoites were used to infect lymphocytes. A horse that was subsequently experimentally infected with 100,000 of the S. neurona-infected lymphocytes developed encephalitis and ataxia. Sarcocystis neurona was isolated from the spinal tissues of the infected horse by in vitro culture. Three horses, each infected with a different number of merozoite-infected lymphocytes, were used to estimate an infective dose of parasites needed to induce clinical signs of equine protozoal myeloencephalitis. This is the first report of histologically confirmed experimental infection of horses with culture-derived merozoites of S. neurona. Both a hematogenous method of distribution in the host and an intracellular location by the parasite in a lymphocyte is sufficient to produce clinical equine protozoal myeloencephalitis.  

4.250           Short tandem repeat analysis to monitor chimerism in Macaca Fasicularis

Lau, M. et al Am. J. Transplant., 4, 1543-1548 (2004)   Chimerism assessment following bone marrow transplantation (BMT) in cynomolgus monkeys (cynos) has been hampered by the lack of good engraftment markers. In human BMT, such markers have been provided by short tandem repeat (STR) loci. We tested the idea that techniques effective for detecting human STR could be readily adapted to cynos. Genomic DNA was extracted from cyno unseparated blood or peripheral cell subsets. With only slight modifications, reagents for detecting human STR alleles were used to amplify and detect cyno STRs and to quantitate allelic mixtures on an automated sequencer. Of the 15 STR loci tested, only CSF1PO, D18S51, and FGA successfully amplified, with seven, seven and two alleles, respectively. CSF1PO and D18S51 heterozygosity (80% and 55%, respectively) allowed use of these two loci for chimerism quantitation after BMT. The successful adaptation of human STR reagents to monitor chimerism in transplanted cynos will facilitate the use of this species in preclinical tolerance studies.  

4.251           An improved method for isolation of mononuclear cells from peripheral blood

Ahmed, Y., Walton, L.J. and Graham, J. ICO/FOCIS 2004 abstract no. 1758 (2004)   The ability to isolate mononuclear cells (MNC) from human peripheral blood is important in immunology research and diagnosis. A long-established one step method for MNC isolation (Bøyum, 1968) is in routine use in laboratories worldwide. MNC are sedimented on to a 1.077 g/ml density barrier of sodium diatrizoate and Ficoll from whole blood that has been diluted 1:1 with saline. Good yields of MNC are obtained, however the cells are always contaminated by platelets, which lie on top of the MNC band. Although platelets can be remoced by washing, this may seriously compromise the ultimate use of the MNC, particularly for the culture of monocytes. In the current study, OptiPrep (a sterile solution of 60% iodixanol) was mixed with EDTA-anticoagulated blood in order to raise the density of the plasma to approx. 1.1 g/ml. The 1.078 g/ml barrier 8produced by diluting OptiPrep with cell culture medium) was layered on top, together with a small volume of culture medium. During the centrifugation at 700g for 30 min at 4⁰C, the MNC float through the barrier to form a distinct band at the interface with the culture medium. May-Grünwald-Giemsa staining of the harvested MNC band shows the presence of few, if any contaminating platelets, which remain predominantly in the plasma layer. The MNC band is also separated from the plasma proteins by the 1.078 g/ml barrier. Moreover, a comparison between the two isolation methods indicates that the recovery o MNC can be as much as 20% higher using the flotation technique and the 1-2% contamination from erythrocytes which is occasionally observed with some blood samples using the sedimentation format, is not seen with the new flotation technique. Monocytes from the MNC band, isolated by flotation, have been successfully maintained in culture for 21 days.  

4.252           Reversal of diabetes in non-immunosuppressed rhesus macaques by intraportal porcine islet xenografts precedes acute cellular rejection

Kirchhof, N. et al Xenotransplantation, 11, 396-407 (2004)   Background: The functional response and immunobiology of primarily non-vascularized islet cell xenografts remain poorly defined in non-human primates. Methods: We transplanted 20 000 adult porcine islet equivalents/kg (purified and cultured for 48-h) intraportally into six streptozotocin-diabetic and two non-diabetic rhesus macaques. Two recipients were killed at various intervals post-transplant for histologic examination of livers bearing xenografts. Results: Plasma glucose levels in diabetic recipients averaged 94 mg/dl at 12 h, 92 mg/dl at 24 h, 147 mg/dl at 48 h, and 157 mg/dl at 72 h post-transplant. Serum porcine C-peptide was present in eight of eight recipients at 12 h, in five of six at 24 h, in four of four at 48 h, and in one of two at 72 h post-transplant. C3a and SC5b-9 plasma levels increased at 12 h post-transplant and returned to pre-transplant levels by 24 h. IgG, IgM anti-pig and anti-Gal IgG serum antibody levels did not increase post-transplant. Rejection was initiated by IgM and complement deposition on islets. Neutrophils dominated the cellular infiltrate at 12 h; CD4⁺ and CD8⁺ T cells were the main infiltrating cells at 24, 48, and 72 h; and macrophages increasingly infiltrated xenografts starting at 24 h post-transplant. Numerous xenoislets were present at all time points; their proportion without intraislet infiltrates decreased from 65% at 24 h to 17% at 72 h post-transplant. Conclusions: Pig-to-primate intraportal islet xenografts reverse diabetes and the majority of intraportally transplanted xenogeneic islets are not subject to hyperacute rejection. They undergo acute cellular rejection mediated by CD4⁺- and CD8⁺ T cells and macrophages.  

4.253           Reduced cellular expression and activity of the P129T mutant of human fatty acid amide hydrolase: evidence for a link between defects in the endocannabinoid sysem and problem drug use

Chiang, K.P., Gerber, A.L., Sipe, J.C. and Cravatt, B.F. Human Mol. Gen., 13(18), 2113-2119 (2004)   Fatty acid amide hydrolase (FAAH) inactivates the endogenous cannabinoid (endocannabinoid) anandamide and related lipid transmitters in vivo. A single nucleotide polymorphism (SNP) in the human FAAH gene (385C to A) has recently been described that, in homozygous form, is over-represented in subjects with problem drug use. This SNP, which converts a conserved proline residue in FAAH to threonine (P129T), suggests a potential role for the FAAH–endocannabinoid system in regulating addictive behavior. Nonetheless, the impact of the 385A mutation on the biochemical and cellular function of FAAH remains unknown. Here, we report that T-lymphocytes isolated from patients homozygous for the P129T-FAAH variant express less than half of the FAAH protein and activity observed in wild-type (WT) lymphocytes. Transfected COS-7 cells also expressed significantly lower levels of P129T-FAAH compared with WT-FAAH, indicating that the aberrant expression of the former protein is not a cell type-specific phenomenon. A comparison of the transcription/translation efficiencies and cellular stabilities of WT- and P129T-FAAH proteins revealed that the reduced expression of the mutant enzyme is due to a post-translational mechanism that precedes productive folding. These findings indicate that the natural 385A SNP in the human FAAH gene produces a mutant enzyme with reduced cellular stability, thus fortifying a potential link between functional abnormalities in the endocannabinoid system and drug abuse and dependence.  

4.254           Scavenger properties of cultivated pig liver endothelial cells

Elvevold, K.H., Nedredal, G.I., Revhaug, A. and Smedsrød, B. Comp. Mepatol., 3(4), 1-11 (2004)   The liver sinusoidal endothelial cells (LSEC) and Kupffer cells constitute the most powerful scavenger system in the body. Various waste macromolecules, continuously released from tissues in large quantities as a consequence of normal catabolic processes are cleared by the LSEC. In spite of the fact that pig livers are used in a wide range of experimental settings, the scavenger properties of pig LSEC has not been investigated until now. Therefore, we studied the endocytosis and intracellular transport of ligands for the five categories of endocytic receptors in LSEC. Endocytosis of five ¹²⁵I-labelled molecules: collagen α-chains, FITC-biotin-hyaluronan, mannan, formaldehyde-treated serum albumin (FSA), and aggregated gamma globulin (AGG) was substantial in cultured LSEC. The endocytosis was mediated via the collagen-, hyaluronan-, mannose-, scavenger-, or IgG Fc-receptors, respectively, as judged by the ability of unlabelled ligands to compete with labelled ligands for uptake. Intracellular transport was studied employing a morphological pulse-chase technique. Ninety minutes following administration of red TRITC-FSA via the jugular vein of pigs to tag LSEC lysosomes, cultures of the cells were established, and pulsed with green FITC-labelled collagen, -mannan, and -FSA. By 10 min, the FITC-ligands was located in small vesicles scattered throughout the cytoplasm, with no co-localization with the red lysosomes. By 2 h, the FITC-ligands co-localized with red lysosomes. When LSEC were pulsed with FITC-AGG and TRITC-FSA together, co-localization of the two ligands was observed following a 10 min chase. By 2 h, only partial co-localization was observed; TRITC-FSA was transported to lysosomes, whereas FITC-AGG only slowly left the endosomes. Enzyme assays showed that LSEC and Kupffer cells contained equal specific activities of hexosaminidase, aryl sulphates, acid phosphatase and acid lipase, whereas the specific activities of α-mannosidase, and glucuronidase were higher in LSEC. All enzymes measured showed considerably higher specific activities in LSEC compared to parenchymal cells. Pig LSEC express the five following categories of high capacity endocytic receptors: scavenger-, mannose-, hyaluronan-, collagen-, and IgG Fc-receptors. In the liver, soluble ligands for these five receptors are endocytosed exclusively by LSEC. Furthermore, LSEC contains high specific activity of lysosomal enzymes needed for degradation of endocytosed material. Our observations suggest that pig LSEC have the same clearance activity as earlier described in rat LSEC.  

4.255           MHC class II expression is differentially regulated in plasmacytoid and conventional dendritic cells

LeibundGut-Landemann, S., Waldburger, J-M., Reis e Sousa, C. Acha-Orbea, H. And Reith, W. Nature Immunol., 5(9), 899-908 (2004)   Major histocompatibility complex (MHC) class II–restricted antigen presentation is essential for the function of dendritic cells (DCs). We show here that plasmacytoid DCs (pDCs) differ from all other DC subsets with respect to expression of CIITA, the 'master regulator' of MHC class II genes. The gene encoding CIITA is controlled by three cell type–specific promoters: pI, pIII and pIV. With gene targeting in mice, we demonstrate that pDCs rely strictly on the B cell promoter pIII, whereas macrophages and all other DCs depend on pI. The molecular mechanisms driving MHC class II expression in pDCs are thus akin to those operating in lymphoid rather than myeloid cells.  

4.256           The remyelinating potential and in vitro differentiation of MOG-expressing oligodendrocyte precursors isolated from the adult rat CNS

Crang, A.J., Gilson, J.M., Li, W.-W. and Blakemore, W.F. Eur. J. Neurosci., 20, 1445-1460 (2004)   There is a long-standing controversy as to whether oligodendrocytes may be capable of cell division and thus contribute to remyelination. We recently published evidence that a subpopulation of myelin oligodendrocyte glycoprotein (MOG)-expressing cells in the adult rat spinal cord co-expressed molecules previously considered to be restricted to oligodendrocyte progenitors [G. Li et al. (2002) Brain Pathol., 12, 463-471]. To further investigate the properties of MOG-expressing cells, anti-MOG-immunosorted cells were grown in culture and transplanted into acute demyelinating lesions. The immunosorting protocol yielded a cell preparation in which over 98% of the viable cells showed anti-MOG- and O1-immunoreactivity; 12-15% of the anti-MOG-immunosorted cells co-expressed platelet-derived growth factor alpha receptor (PDGFR ) or the A2B5-epitope. When cultured in serum-free medium containing EGF and FGF-2, 15-18% of the anti-MOG-immunosorted cells lost anti-MOG- and O1-immunoreactivity and underwent cell division. On removal of these growth factors, cells differentiated into oligodendrocytes, or astrocytes and Schwann cells when the differentiation medium contained BMPs. Transplantation of anti-MOG-immunosorted cells into areas of acute demyelination immediately after isolation resulted in the generation of remyelinating oligodendrocytes and Schwann cells. Our studies indicate that the adult rat CNS contains a significant number of oligodendrocyte precursors that express MOG and galactocerebroside, molecules previously considered restricted to mature oligodendrocytes. This may explain why myelin-bearing oligodendrocytes were considered capable of generating remyelinating cells. Our study also provides evidence that the adult oligodendrocyte progenitor can be considered as a source of the Schwann cells that remyelinate demyelinated CNS axons following concurrent destruction of oligodendrocytes and astrocytes.  

4.257           Zinc tolerance, uptake, accumulation and distribution in plants and protoplasts of five European populations of the wertland grass Glyceria fluitans

Matthews, D.J., Moran, B.M., McCabe, P.F. and Otte, M.L. Auatic Botany, 80, 39-52 (2004)   Five populations of Glyceria fluitans (L.) R. Br. from metal-contaminated and non-contaminated sites across Europe were investigated for innate zinc tolerance. The plants were grown hydroponically in zinc-amended nutrient solutions. Growth and survival of plants from all five populations occurred at all levels of elevated zinc treatments (2, 300, 600 and 1000 μmol L⁻¹ ZnSO₄·7H₂O). There were only slight differences in growth between the populations from contaminated and non-contaminated sites. Uptake of zinc did differ between populations, but this did not affect tolerance. The findings support the theory that wetland angiosperm species tend to be tolerant to exposure to high levels of metals, regardless of their origin.  

4.258           Monocyte adhesion to decidual endothelial cells is increased in pregnancies complicated by type 1 diabetes but not by gestational diabetes

Galettis, A. et al Diabetes Care, 27(10), 2514-2515 82004)   Type 1 diabetes complicates 1 of every 200 pregnancies and gestational diabetes a further 2–3% of pregnancies (1). Systemic atheromatous vascular disease can develop or accelerate during diabetic pregnancy. We and others (2) have observed similar vascular lesions in placental bed vessels (Fig. 1A) associated with impaired placental function and fetal growth. Genesis of atheroma involves adherence of peripheral blood monocytes to endothelium (3–5). To determine whether a similar process underlies the placental bed vasculopathy of diabetes, we examined cell adhesion in an in vitro coculture system using decidual endothelial cells from normal pregnancies and monocytes from both normal and diabetic pregnancies. Genesis of atheroma involves adherence of peripheral blood monocytes to endothelium (3–5). To determine whether a similar process underlies the placental bed vasculopathy of diabetes, we examined cell adhesion in an in vitro coculture system using decidual endothelial cells from normal pregnancies and monocytes from both normal and diabetic pregnancies.  

4.259           Fibrin stimulates platelets to increase factor VIIIa binding site expression

Phillips, J.E., Lord, S.T. and Gilbert, G.E,

1. Thromb. Haemost., 2, 1806-1815 (2004)

  Factor (F)VIII functions as an enzymatic cofactor on the membranes of stimulated platelets. However, thrombin stimulates platelets to express only a small number of binding sites for FVIII. We wished to determine whether molecules that are likely to be present in a developing thrombus stimulate platelets to up-regulate FVIII binding site expression. Flow cytometry was utilized to measure binding of fluorescein-labeled FVIIIa to activated platelets and a FXase assay was utilized to measure platelet-dependent function. Various agonists as well as normal and mutant fibrinogens and fibrin were evaluated as co-stimuli. Thrombin-stimulated platelets expressed 214 ± 67 binding sites for thrombin-activated FVIII (FVIIIa) and none of the established soluble agonists enhanced binding site exposure. However, the presence of 5 µg mL ¹ fibrin increased the number of FVIIIa binding sites/platelet three- to eight-fold (1470 ± 130, range 600-1800) with a parallel increase in platelet-based FXase assay. Binding site up-regulation was not stimulated by fibrinogen and was blocked by inhibitors of GPIIbIIIa. Mutant fibrin lacking the -chain C-terminal four residues was ineffective while fibrin with altered RGD sequences did stimulate expression of FVIIIa binding sites indicating that co-stimulation is mediated by the fibrin -chain termini. Fibrin-enhanced expression of FVIIIa binding sites was not supported by D364H fibrin, which does not aggregate normally, and was blocked by the GPRP peptide, which inhibits fibrin polymerization. Polymerized fibrin can function as a platelet co-stimulus, up-regulating expression of binding sites for FVIIIa.  

4.260           Improvement in islet yield from obese donors for human islet transplants

Matsumoto, I. Et al Transplantation, 78(6), 880-885 (2004)   Background. The feasibility of human islet transplantations has been firmly established. To increase the number of islet transplants, the suitability of pancreases from organ donors considered inappropriate for pancreas transplantations must be evaluated. Methods. We isolated islets from 114 human cadaver donor pancreases by the automated Ricordi method, followed by purification using continuous-density gradients. We divided the pancreases into two groups by donor body mass index (BMI)-group 1: n=51, BMI of 30 or more; group 2: n=63, BMI of less than 30. We compared the results of human islet isolation, in vitro potency assays, and a nude mouse bioassay. Results. In group 1 (vs. group 2), we found a significantly higher mean pancreas weight (109.5+/-30.7 vs. 90.6+/-24.0 g; P=0.0002); higher mean islet equivalents/pancreas, after digestion (442,565+/-238,741 vs. 289,860+/-158,995; P<0.0001) and after purification (319,129+/-164,002 vs. 215,753+/-126,089; P=0.0002); and a higher islet isolation success rate-defined as isolations yielding more than 300,000 islet equivalents/pancreas, with purities of more than 50% (37.3% [19 of 51 pancreases] vs. 15.9% [10 of 63]; P=0.009). Our in vitro potency assays and bioassay uncovered no differences between the two groups. Notably, all except one of the donor BMIs for the successful isolations in group 2 exceeded 26; the mean donor BMI for the successful isolations (27.3+/-3.0, n=10) was significantly higher than for the unsuccessful isolations (24.8+/-3.3, n=53) (P=0.03). Conclusions. Pancreases from both overweight (BMI >=26 but <30) and obese (BMI >=30) cadaver donors are suitable for islet isolation and transplantations. Their use could increase the size of the islet donor pool.  

4.261           In vivo adjuvant-induced mobilization and maturation of gut dendritic cells after oral administration of cholera toxin

Anjuere, F. et al

1. Immunol., 173, 5103-5111 (2004)

  Although dendritic cells (DCs) regulate immune responses, they exhibit functional heterogeneity depending on their anatomical location. We examined the functional properties of intestinal DCs after oral administration of cholera toxin (CT), the most potent mucosal adjuvant. Two CD11c⁺ DC subsets were identified both in Peyer’s patches and mesenteric lymph nodes (MLN) based on the expression of CD8 (CD8⁺ and CD8^– DCs, respectively). A third subset of CD11c⁺CD8^int was found exclusively in MLN. Feeding mice with CT induced a rapid and transient mobilization of a new CD11c⁺CD8^– DC subset near the intestinal epithelium. This recruitment was associated with an increased production of the chemokine CCL20 in the small intestine and was followed by a massive accumulation of CD8^int DCs in MLN. MLN DCs from CT-treated mice were more potent activators of naive T cells than DCs from control mice and induced a Th2 response. This increase in immunostimulating properties was accounted for by CD8^int and CD8^– DCs, whereas CD8⁺ DCs remained insensitive to CT treatment. Consistently, the CD8^int and CD8^– subsets expressed higher levels of costimulatory molecules than CD8⁺ and corresponding control DCs. Adoptive transfer experiments showed that these two DC subsets, unlike CD8⁺ DCs, were ableto present Ags orally coadministered with CT in an immunostimulatingmanner. The ability of CT to mobilize immature DCs in the intestinalepithelium and to promote their emigration and differentiationin draining lymph nodes may explain the exceptional adjuvantproperties of this toxin on mucosal immune responses.  

4.262           Prevalent human coxsaxkie B-5 virus infects porcine islet cells primarily using the coxsackie-adenovirus receptor

Myers, S.E. et al Xenotransplantation, 11, 536-546 (2004)   Background: We have previously demonstrated that transplanting porcine encephalomyocarditis virus (EMCV)-infected porcine islet cells (PICs) results in transmission of the virus to recipient mice, which is manifested by acute fatal infection within 5 to 8 days. Here, we determined PIC susceptibility to a related and highly prevalent human picornavirus, coxsackie B-5 virus (CVB-5). Methods: PICs were inoculated with CVB-5 in vitro for up to 96 hours and infectivity, level of virus replication, and cellular function determined. Subsequently, monoclonal and polyclonal antibody blocking experiments were used to investigate the receptor CVB-5 uses to enter PICs, and the ability of CVB-5-infected islets to reverse diabetes analyzed in mice. Results: Adult pig islets inoculated with CVB-5 in vitro showed a typical picornaviral replication cycle with a 2-h lag phase followed by a 4-h exponential phase during which the virus titer increased by 4 logs. However, CVB-5 was less cytolytic to PICs than EMCV, resulting in a persistent productive infection lasting for up to 96 h, with minimal evidence of cell lysis. Double immunostaining confirmed the presence of CVB-5 antigens in insulin-producing islets. Infection of PICs in the presence of antibodies against human coxsackie-adenovirus receptor (CAR) resulted in near complete blockage in production of infectious virus particles whereas blocking with anti-porcine decay-accelerating factor (DAF, also called CD55) or anti-porcine membrane cofactor protein (MCP, also called CD46) only slightly decreased the number of infectious CVB-5 particles produced. Immunofluoresence staining showed CAR and MCP expression on the islet surface, but not DAF. Transplanting CVB-5-infected PICs into diabetic C57BL/6 mice resulted in reversal of diabetes. Conclusion: Although PICs are susceptible to human CVB-5, the infection does not appear to affect xenograft function in vitro or in vivo in the short term.  

4.263           Comparison of six density gradient media for selection of cryopreserved donor spermatozoa

Mousset-Simeon, N., Rives, N., Masse, L., Chevallier, F. and Mace, B.

1. Androl., 25(6), 881-884 (2004)

  The aim of our study was to evaluate the efficiency of 4 density gradient media for motile cryopreserved spermatozoa selection to Percoll (Kabi Pharmacia, Uppsala, Sweden) and to Puresperm (J.C.D. International Laboratory, L'Aigle, France). Puresperm was the new medium chosen in our laboratory in 1996 as the substitute for Percoll. The solutions tested were 3 colloidal silane-coated silica particle media (Isolate, SpermGrad-100, Sil-Select Plus) and iodixanol (Optiprep). Semen parameters analyzed after selection were concentration, motility, and morphology. Semen parameters after Puresperm gradient had similar values compared to Percoll. Optiprep was less efficient with a poor concentration. Isolate had a comparatively better concentration, but the capacity of selection was not satisfactory. SpermGrad-100 and Sil-Select Plus were less effective than Puresperm. In conclusion, Puresperm could be considered a better alternative to Percoll for cryopreserved spermatozoa migration.  

4.264           KCl cotransport mediates abnormal sulfhydryl-dependent volume regulation in sickle reticulocytes

Joiner, C.H., Rettig, R.K., Jiang, M. And Franco, R.S. Blood, 104, 2954-2960 (2004)   KCl cotransport (KCC) activation by cell swelling and pH was compared in sickle (SS) and normal (AA) red blood cells (RBCs). KCC fluxes had the same relationship to mean corpuscular hemoglobin concentration (MCHC) in SS and AA RBCs when normalized to the maximal volume-stimulated (VS_max) flux (MCHC < 270 g/L [27 g/dL]). Acid-stimulated (pH 6.9) KCC flux in SS RBCs was 60% to 70% of VS_max KCC versus 20% in AA RBCs. Density gradients were used to track changes in reticulocyte MCHC during KCC-mediated regulatory volume decrease (RVD). Swelling to MCHC of 260 g/L (26 g/dL) produced Cl-dependent RVD that resulted in higher MCHC in SS than AA reticulocytes. In acid pH, RVD was also greater in SS than AA reticulocytes. Sulfhydryl reduction by dithiothreitol (DTT) lowered VS_max KCC flux in AA and SS RBCs by one third but did not alter swelling-induced RVD. DTT lowered acid-activated KCC in SS RBCs by 50% and diminished acid-induced RVD in SS reticulocytes. Thus, swelling activation of KCC is normal in SS RBCs but KCC-mediated RVD produces higher MCHC in SS than AA reticulocytes. Acid activation of KCC is exaggerated in SS RBCs and causes dehydration in SS reticulocytes. KCC response to acid stimulation was mitigated by DTT, suggesting that it arises from sulfhydryl oxidation.  

4.265           Factor XIIIA transglutaminase crosslinks AT₁ receptor dimers of monocytes at the onset of atherosclerosis

AbdAlla, S., Lother, H., Langer, A., el Faramaway, Y. and Quitterer, U. Cell, 119, 343-354 (2004)   Many G protein-coupled receptors form dimers in cells. However, underlying mechanisms are barely understood. We report here that intracellular factor XIIIA transglutaminase crosslinks agonist-induced AT₁ receptor homodimers via glutamine³¹⁵ in the carboxyl-terminal tail of the AT₁ receptor. The crosslinked dimers displayed enhanced signaling and desensitization in vitro and in vivo. Inhibition of angiotensin II release or of factor XIIIA activity prevented formation of crosslinked AT₁ receptor dimers. In agreement with this finding, factor XIIIA-deficient individuals lacked crosslinked AT₁ dimers. Elevated levels of crosslinked AT₁ dimers were present on monocytes of patients with the common atherogenic risk factor hypertension and correlated with an enhanced angiotensin II-dependent monocyte adhesion to endothelial cells. Elevated levels of crosslinked AT₁ receptor dimers on monocytes could sustain the process of atherogenesis, because inhibition of angiotensin II generation or of intracellular factor XIIIA activity suppressed the appearance of crosslinked AT₁ receptors and symptoms of atherosclerosis in ApoE-deficient mice.  

4.266           Elastic light scattering from single cells: orientational dynamics in optical trap

Watson, D. et al Biophys. J, 87, 1298-1306 (2004)   Light-scattering diagrams (phase functions) from single living cells and beads suspended in an optical trap were recorded with 30-ms time resolution. The intensity of the scattered light was recorded over an angular range of 0.5–179.5° using an optical setup based on an elliptical mirror and rotating aperture. Experiments revealed that light-scattering diagrams from biological cells exhibit significant and complex time dependence. We have attributed this dependence to the cell's orientational dynamics within the trap. We have also used experimentally measured phase function information to calculate the time dependence of the optical radiation pressure force on the trapped particle and show how it changes depending on the orientation of the particle. Relevance of these experiments to potential improvement in the sensitivity of label-free flow cytometry is discussed.  

4.267           Type 1 collagen promotes the malignant phenotype of pancreatic ductal adenocarcinoma

Armstrong, T. et al Clin. Can. Res., 10, 7427-7437 (2004)   Purpose: The purpose of this study was to determine the roleof functional interactions between pancreatic cancer cells andpancreatic stellate cells (PSCs) in the formation of the desmoplasticreaction (DR) in pancreatic cancer and to characterize the effectof type I collagen (the predominant component of the DR) onpancreatic cancer cell phenotype. Experimental Design: PSCs and type I collagen were identifiedin sections of pancreatic cancer using immunohistochemistry,and their anatomic relationship was studied. Interactions amongpancreatic cancer cell lines (MIA PaCa-2, Panc-1, and AsPC-1),primary cultures of human PSCs, and type I collagen were investigatedin a series of tissue culture models. Results: In vivo, the DR causes gross distortion of normal pancreas, bringing cancer cells into close contact with numerous PSCs and abundant type I collagen. In tissue culture models of pancreatic cancer, conditioned media from each cell line increased PSC [³H]thymidine incorporation up to 6.3-fold that of controls, and AsPC-1 cells also increased PSC collagen synthesis 1.3-fold. Type I collagen was observed to increase long-term survival of pancreatic cancer cells treated with 5-fluorouracil, by up to 62% in clonogenic assays. This was because type I collagen increased the proliferation of cancer cells ([³H]thymidine incorporationwas up to 2.8-fold that of cells cultured on tissue cultureplastic) and reduced apoptosis of AsPC-1 cells in response to5-fluorouracil (by regulating mcl-1). Conclusions: These experiments elucidate a mechanism by whichthe DR in pancreatic cancer may form and, via the collagen withinit, promote the malignant phenotype of pancreatic cancer cells,suggesting significant detriment to the host.  

4.268           Synergy between IL-8 and GM-CSF in reproductive tract epithelial cell secretions promotes enhanced neutrophil chemotaxis

Shen, L. et al Cellular Immunol., 230, 23-32 (2004)   Neutrophils occur in tissues of the female reproductive tract (FRT) under non-infected conditions. These cells generally enter tissues under the influence of chemoattractants called chemokines. Primary epithelial cells (EC) from FRT were a potent source of chemokines, IL-8 being the chief neutrophil chemoattractant secreted. Blocking with neutralizing anti-IL-8 showed that IL-8 did not account for all of the chemoattraction observed. A mixture of 25 ng/mL rIL-8 and 1 ng/mL rGM-CSF mediated 2.7-fold more chemotaxis than that expected if the two agents were additive. We then found that GM–CSF was produced by EC in amounts that synergised strongly with IL-8 to enhance chemotaxis. Treatment of uterine EC conditioned medium with saturating doses of anti-IL-8 plus anti-GM–CSF antibodies produced an 84% inhibition of chemotaxis, These findings demonstrate that the majority of neutrophil chemoattractant activity produced by FRT EC results from the synergistic effects of IL-8 and GM–CSF.  

4.269           Combined disruption of both the MEK/ERK and the IL-6R/STAT3 pathways is required to induce apoptosis of multiple myeloma cells in the presence of bone marrow stromal cells

Chatterjee, M. et al Blood, 104(12), 3712-3721 (2004)   The interleukin-6 receptor (IL-6R)/signal transducer and activator of transcription 3 (STAT3) pathway contributes to the pathogenesis of multiple myeloma (MM) and protects MM cells from apoptosis. However, MM cells survive the IL-6R blockade if they are cocultured with bone marrow stromal cells (BMSCs), suggesting that the BM microenvironment stimulates IL-6–independent pathways that exert a pro-survival effect. The goal of this study was to investigate the underlying mechanism. Detailed pathway analysis revealed that BMSCs stimulate STAT3 via the IL-6R, and mitogen-activated protein (MAP) kinases via IL-6R–independent mechanisms. Abolition of MEK1,2 activity with PD98059, or ERK1,2 small interfering RNA knockdown, was insufficient to induce apoptosis. However, the combined disruption of the IL-6R/STAT3 and MEK1,2/ERK1,2 pathways led to strong induction of apoptosis even in the presence of BMSCs. This effect was observed with MM cell lines and with primary MM cells, suggesting that the BMSC-induced activation of MEK1,2/ERK1,2 renders MM cells IL-6R/STAT3 independent. Therefore, in the presence of cells from the BM micro-environment, combined targeting of different (and independently activated) pathways is required to efficiently induce apoptosis of MM cells. This might have direct implications for the development of future therapeutic strategies for MM.  

4.270           Isolating pure populations of monocytes from the blood of pregnant women: comparison of flotation in iodixanol with elutriation

Nutt, J.C., Willis, C.C., Morris, J.M. and Gallery, E.D.M.

1. Immunol. Methods, 293, 215-218 (2004)

  Observations that the innate arm of the immune system is upregulated in pregnancy have highlighted the need for methods of isolating pure populations of monocytes for studies into pregnancy and pre-eclampsia without activating them during the isolation process. Density gradient centrifugation using iodixanol is a useful method for isolating relatively pure populations of unactivated monocytes from human blood but has not been validated in pregnant subjects. We compared the ability of monocytes isolated from pregnant women by density gradient centrifugation using iodixanol (n=6) with monocytes isolated by countercurrent centrifugal elutriation (n=6) in terms of their ability to produce interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) under basal conditions and after stimulation with bacterial lipopolysaccharide (LPS). Under basal conditions, monocytes isolated by density gradient centrifugation produced low amounts of IL-6 and MCP-1. Production of IL-6 and MCP-1 after stimulation of the monocytes with LPS was much greater (p<0.01). There was no statistically significant difference between the two methods in terms of stimulated levels of either cytokine.  

4.271           SIVdrl detection in captive mandrills: are mandrill infected with a third strain of simian immunodeficiency virus?

Gruters, R.A., Osterhaus, A.D.M.E. and Berhout, B. Retrovirology, 1(36), 1-5 (2004)   A pol-fragment of simian immunodeficiency virus (SIV) that is highly related to SIVdrl-pol from drill monkeys (Mandrillus leucophaeus) was detected in two mandrills (Mandrillus sphinx) from Amsterdam Zoo. These captivity-born mandrills had never been in contact with drill monkeys, and were unlikely to be hybrids. Their mitochondrial haplotype suggested that they descended from founder animals in Cameroon or northern Gabon, close to the habitat of the drill. SIVdrl has once before been found in a wild-caught mandrill from the same region, indicating that mandrills are naturally infected with a SIVdrl-like virus. This suggests that mandrills are the first primate species to be infected with three strains of SIV: SIVmnd1, SIVmnd2, and SIVdrl.  

4.272           Role of myosin VIIa and Rab27a in the motility and localization of RPE melanosomes

Gibbs, D. et al

1. Cell Sci., 117, 6473-6483 (2004)

  Myosin VIIa functions in the outer retina, and loss of this function causes human blindness in Usher syndrome type 1B (USH1B). In mice with mutant Myo7a, melanosomes in the retinal pigmented epithelium (RPE) are distributed abnormally. In this investigation we detected many proteins in RPE cells that could potentially participate in melanosome transport, but of those tested, only myosin VIIa and Rab27a were found to be required for normal distribution. Two other expressed proteins, melanophilin and myosin Va, both of which are required for normal melanosome distribution in melanocytes, were not required in RPE, despite the association of myosin Va with the RPE melanosome fraction. Both myosin VIIa and myosin Va were immunodetected broadly in sections of the RPE, overlapping with a region of apical filamentous actin. Some 70-80% of the myosin VIIa in RPE cells was detected on melanosome membranes by both subcellular fractionation of RPE cells and quantitative immunoelectron microscopy, consistent with a role for myosin VIIa in melanosome motility. Time-lapse microscopy of melanosomes in primary cultures of mouse RPE cells demonstrated that the melanosomes move in a saltatory manner, interrupting slow movements with short bursts of rapid movement (>1 µm/second). In RPE cells from Myo7a-null mice, both the slow and rapid movements still occurred, except that more melanosomes underwent rapid movements, and each movement extended approximately five times longer (and further). Hence, our studies demonstrate the presence of many potential effectors of melanosome motility and localization in the RPE, with a specific requirement for Rab27a and myosin VIIa, which function by transporting and constraining melanosomes within a region of filamentous actin. The presence of two distinct melanosome velocities in both control and Myo7a-null RPE cells suggests the involvement of at least two motors other than myosin VIIa in melanosome motility, most probably, a microtubule motor and myosin Va.  

4.273           Telomere dynamics in Fancg-deficient mouse and human cells

Franco, S. et al Blood, 104, 3927-3935 (2004)   A number of DNA repair proteins also play roles in telomere metabolism. To investigate whether the accelerated telomere shortening reported in Fanconi anemia (FA) hematopoietic cells relates to a direct role of the FA pathway in telomere maintenance, we have analyzed telomere dynamics in Fancg-deficient mouse and human cells. We show here that both hematopoietic (stem and differentiated bone marrow cells, B and T lymphocytes) and nonhematopoietic (germ cells, mouse embryonic fibroblasts [MEFs]) Fancg^-/- mouse cells display normal telomere length, normal telomerase activity, and normal chromosome end-capping, even in the presence of extensive clastogen-induced cytogenetic instability (mitomycin C [MMC], gamma-radiation). In addition, telomerase-deficient MEFs with humanlike telomere length and decreased Fancg expression (G5 Terc^-/-/Fancg shRNA3 MEFs) display normal telomere maintenance. Finally, early-passage primary fibroblasts from patients with FA of complementation group G as well as primary human cells with reduced FANCG expression (FANCG shRNA IMR90 cells) show no signs of telomere dysfunction. Our observations indicate that accelerated telomere shortening in patients with FA is not due to a role of FANCG at telomeres but instead may be secondary to the disease. These findings suggest that telomerase-based therapies could be useful prophylactic agents in FA aplastic anemia by preserving their telomere reserve in the context of the disease.  

4.274           Immune function is impaired with a mini nutritional assessment score indicative of malnutrition in nursing home elders with pressure ulcers

Hudgens, J. et al

1. Parenteral and Enteral Nutrition, 28(6), 416-422 (2004)

  Background: Malnutrition is prevalent in elders with pressure ulcers and is associated with increased morbidity and mortality. This study compared nutritional status, assessed by the Mini Nutrition Assessment (MNA), to immune function in nursing home elders with pressure ulcers. Methods: Nutritional status was assessed in nursing home residents (>65 years) with a stage II or more severe pressure ulcer. Subjects were classified as well nourished, at risk of malnutrition, or malnourished according to MNA score. Blood was drawn to assess whole blood mitogen-induced lymphocyte proliferation and neutrophil respiratory burst. Delayed-type hypersensitivity to 3 antigens was measured. MNA status was compared with immune parameters using the Kruskall-Wallis test. Results: Of the 24 subjects (23 men, 1 woman) who completed the study protocol, only 4 (17%) were classified as well nourished, whereas 7 (29%) were at risk and 13 (54%) were malnourished according to MNA score. Whole blood lymphocyte proliferation was significantly lower in the malnourished vs at risk subjects with both pokeweed (median [25^th,75^th percentile], 0.6 [0.3, 0.9] vs 1.8 [1.2, 2.1] disintegrations per minute [dpm]/cell, p < .05); and concanavalin A (1.7 [0.9, 2.0] vs 2.8 [2.6, 3.9] dpm/cell, p < .05) mitogens. Neutrophil respiratory burst normalized to a young control was significantly lower in malnourished subjects vs well-nourished subjects (0.8 [0.5, 0.9] vs 1.4 [1.0, 1.7], p < .05). Total induration to 3 skin-test antigens was 13.4 ± 4.6, 3.5 ± 2.6, and 3.8 ± 1.8 (mean ± SEM) for well-nourished, at risk, and malnourished, respectively (p = .059). Conclusions: Immune function is impaired with an MNA score indicative of malnutrition in nursing home elders with pressure ulcers.  

4.275           Systemic treatment of cerebral cortex lesions in rats with a new secreted phospholipase A2 inhibitor

Cunningham, T.J., Souayah, N., Jameson, B., Mitchell, J. and Yao, L.

1. Neurotrauma, 21(11), 1683-1691 (2004)

  An internal fragment of the human neuroprotective polypeptide DSEP (Diffusible Survival Evasion Peptide) was delivered at 0.4 mg/kg (subcutaneously) 20-30 min after stab wound lesions in the parietal cortex of anesthetized rats. The peptide, CHEASAAQC or CHEC-9, inhibited the inflammatory response to the lesion and the degeneration of neurons adjacent to the wound. Four days after surgery, peptide-treated animals (n = 6) had 75% fewer reactive ameboid microglia/brain macrophages in the cortical parenchyma surrounding the lesion compared to vehicle-injected control rats (n = 6, p = 0.004). The cortical laminae in area 2 adjacent to the lesion were completely obscured in controls because of the increase in inflammatory cells and frank degeneration of neurons, while there was preservation of the neurons and cytoarchitecture after peptide treatment. In parallel experiments, CHEC-9 was found to inhibit the enzymatic activity of secreted phospholipase A2 (sPLA2), including activity present in the serum of peptide-injected rats. Kinetic analysis revealed the peptide increased the average Km for serum by 318% when tested 45 min after treatment (peptide-treated, n = 6; control-treated, n = 6; p = 0.0087), suggesting the principal effect of the peptide was to lower the affinity of serum sPLA2 for substrate. The sPLA2 inhibition by this particular peptide sequence appeared to be highly specific since inversion of a single pair of amino acids eliminated the inhibitory effect. Phorbol-12-myristate-13-acetate stimulated platelet aggregation, a PLA2-regulated activity, was also inhibited by the peptide. The discovery of CHEC-9 makes it possible to study in vivo the long appreciated contribution made by PLA2-directed inflammation to both acute and chronic neurodegeneration and may be helpful in designing therapies to limit neuron death in these conditions.  

4.276           Heterogeneity among DN1 prothymocytes reveals multiple progenitors with different capacities to generate T cell and non-T cell lineages

Porritt, H.E. et al Immunity, 20, 735-745 (2004)   The nature of early T lineage progenitors in the thymus or bone marrow remains controversial. Here we assess lineage capacity and proliferative potential among five distinct components of the earliest intrathymic stage (DN1, CD25⁻44⁺). All of these express one or more hemato-lymphoid lineage markers. All can produce T lineage cells, but only two of them display kinetics of differentiation, proliferative capacity, and other traits consistent with being canonical T progenitors. The latter also appeared limited to producing cells of the T or NK lineages, while B lineage potential derived mainly from the other, less typical T progenitors. In addition to precisely defining canonical early progenitors in the thymus, this work reconciles conflicting results from numerous groups by showing that multiple progenitors with a DN1 phenotype home to the thymus and make T cells, but possess different proliferative potentials and lineage capacities.  

4.277           Constitutive expression and alternative splicing of the exons encoding SCRs in Sp152, the sea urchin homologue of complement factor B. Implications on the evolution of the Bf/C2 gene family

Terwilliger, D.P., Clow, L.A., Gross, P.S. and Smith, L.C. Immunogenetics, 56, 531-543 (2004)   The purple sea urchin, Strongylocentrotus purpuratus, possesses a non-adaptive immune system including elements homologous to C3 and factor B (Bf) of the vertebrate complement system. SpBf is composed of motifs typical of the Bf/C2 protein family. Expression of Sp152 (encodes SpBf) was identified in the phagocyte type of coelomocyte in addition to gut, pharynx and esophagus, which may have been due to the presence of these coelomocytes in and on all tissues of the animal. Sp152 expression in coelomocytes was constitutive and non-inducible based on comparisons between pre- and post-injection with lipopolysaccharide or sterile seawater. The pattern of five short consensus repeats (SCRs) in SpBf has been considered ancestral compared to other deuterostome Bf/C2 proteins that contain either three or four SCRs. Three alternatively spliced messages were identified for Sp152 and designated Sp152 1, Sp152 4, and Sp152 1+ 4, based on which of the five SCRs were deleted. Sp152 4 had an in-frame deletion of SCR4, which would encode a putative SpBf 4 protein with four SCRs rather than five. On the other hand, both Sp152 1 and Sp152 1+ 4 had a frame-shift that introduced a stop codon six amino acids downstream of the splice site for SCR1, and would encode putative proteins composed only of the leader. Comparisons between the full-length SpBf and its several splice variants with other Bf/C2 proteins suggested that the early evolution of this gene family may have involved a combination of gene duplications and deletions of exons encoding SCRs.  

4.278           Both soluble and membrane-bound forms of Flt3 ligand enhance tumor immunity following “suicide” gene therapy in a murine colon carcinoma model

Alsheihly, A-R., Zweiri, J., Walmesley, A.J., Watson, A.J.M. and Christmas, S.E. Cancer Immunol. Immunother, 53, 946-954 (2004)   In prodrug-activated ( suicide ) gene therapy, tumor cells are transfected with the gene for an enzyme that converts an inactive prodrug, such as ganciclovir (GCV), to a toxic compound. Transfected cells are killed on administration of GCV, as also are untransfected bystander cells. The ability of the dendritic cell stimulatory cytokine Flt3 ligand (Flt3-L) to modulate prodrug-activated gene therapy has been investigated. Transfectants of the murine colon carcinoma MC26 were generated expressing soluble (FLS) and membrane-bound forms of Flt3-L. They were inoculated together with wild-type MC26 cells and cells expressing herpes simplex virus-1 (HSV1) thymidine kinase into BALB/c mice, which were then administered GCV. Expression of Flt3-L or FLS prevented regrowth of tumor in most mice, which was comparable to the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), while tumors recurred in all mice receiving suicide gene therapy alone. Recurring tumor cells were resistant to direct killing by GCV but sensitive to bystander killing in vitro. Mice without tumor recurrence were rechallenged with unmodified MC26 cells. Of those mice given transfectants expressing GM-CSF, Flt3-L, or FLS, approximately 50% were immune to rechallenge. These mice also showed cytotoxic and proliferative responses to MC26 cells. These experiments show that both soluble and membrane-bound forms of Flt3-L were able to induce a protective immune response to colon carcinoma cells in a fashion similar to GM-CSF.  

4.279           Vascular endothelial growth factor gene delivery for revascularization in transplanted human islets

Narang, A.S., et al Pharmaceut. Res., 21(1), 15-25 (2004)   Purpose. Islet transplantation is limited by islet graft failure because of poor revascularization, host immune rejection, and nonspecific inflammatory response. Human vascular endothelial growth factor (hVEGF) gene delivery is likely to promote islet revascularization and survival. Methods. We evaluated gene expression from a bicistronic plasmid encoding hVEGF and enhanced green fluorescent protein (EGFP) (pCMS-EGFP-hVEGF). Glucose responsiveness of islets was evaluated both in vitro and in vivo, and revascularization in islet graft was evaluated by immunohistochemistry. Results. After transfection, hVEGF and EGFP expression levels were comparable with original monocistronic plasmids in Jurkat cells but higher and prolonged hVEGF expression in islets transfected with the bicistronic plasmid was observed, possibly as the result of differences in promoter strength and hypoxia response. The 3:1 w/w complexes showed little toxicity to islets at a dose of 5 g DNA per 2000 islets. On glucose challenge, insulin release from transfected islets as well as secretion from islets after transplantation under the mouse kidney capsules in response to glucose stimulation, increased with time. Immunohistochemical staining of transplanted islets using mouse anti-human insulin, mouse anti-human von Willebrand factor, and rat anti-mouse CD31 antibodies suggests that islets are functional and there is new blood vessel formation. Conclusions. These findings suggest that transient hVEGF gene expression by the islets may promote islet revascularization and prolong islet survival after transplantation.    

4.280           Sodium channels b1 subunits promote neurite outgrowth in cerebellar granule neurons

Davis, T.H., Chen, C. and Isom, L.L.

1. Biol. Chem., 279(49), 51424-51432 (2004)

  Many immunoglobulin superfamily members are integral in development through regulation of processes such as growth cone guidance, cell migration, and neurite outgrowth. We demonstrate that homophilic interactions between voltage-gated sodium channel 1 subunits promote neurite extension in cerebellar granule neurons. Neurons isolated from wild-type or 1(-/-) mice were plated on top of parental, mock-, or 1-transfected fibroblasts. Wild-type neurons consistently showed increased neurite length when grown on 1-transfected monolayers, whereas 1(-/-) neurons showed no increase compared with control conditions. 1-Mediated neurite extension was mimicked using a soluble 1 extracellular domain and was blocked by antibodies directed against the 1 extracellular domain. Immunohistochemical analysis suggests that the 1 and 4 subunits, but not 2 and 3, are expressed in cerebellar Bergmann glia as well as granule neurons. These results suggest a novel role for 1 during neuronal development and are the first demonstration of a functional role for sodium channel subunit-mediated cell adhesive interactions.  

4.281           Oleic and docosahexaenoic acid differentially phase separate from lipid raft molecules: a comparative NMR, DSC, AFM, and detergent extraction study

Shaikh, S.R. et al Biophys. J., 87, 1752-1766 (2004)   We have previously suggested that the -3 polyunsaturated fatty acid, docosahexaenoic acid (DHA) may in part function by enhancing membrane lipid phase separation into lipid rafts. Here we further tested for differences in the molecular interactions of an oleic (OA) versus DHA-containing phospholipid with sphingomyelin (SM) and cholesterol (CHOL) utilizing ²H NMR spectroscopy, differential scanning calorimetry, atomic force microscopy, and detergent extractions in model bilayer membranes. ²H NMR and DSC (differential scanning calorimetry) established the phase behavior of the OA-containing 1-[²H₃₁]palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1PE-d₃₁)/SM (1:1) and the DHA-containing 1-[²H₃₁]palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6PE-d₃₁)/SM (1:1) in the absence and presence of equimolar CHOL. CHOL was observed to affect the OA-containing phosphatidylethanolamine (PE) more than the DHA-containing PE, as exemplified by >2x greater increase in order measured for the perdeuterated palmitic chain in 16:0-18:1PE-d₃₁/SM (1:1) compared to 16:0-22:6PE-d₃₁/SM (1:1) bilayers in the liquid crystalline phase. Atomic force microscopy (AFM) experiments showed less lateral phase separation between 16:0-18:1PE-rich and SM/CHOL-rich raft domains in 16:0-18:1PE/SM/CHOL (1:1:1) bilayers than was observed when 16:0-22:6PE replaced 16:0-18:1PE. Differences in the molecular interaction of 16:0-18:1PE and 16:0-22:6PE with SM/CHOL were also found using biochemical detergent extractions. In the presence of equimolar SM/CHOL, 16:0-18:1PE showed decreased solubilization in comparison to 16:0-22:6PE, indicating greater phase separation with the DHA-PE. Detergent experiments were also conducted with cardiomyocytes fed radiolabeled OA or DHA. Although both OA and DHA were found to be largely detergent solubilized, the amount of OA that was found to be associated with raft-rich detergent-resistant membranes exceeded DHA by almost a factor of 2. We conclude that the OA-PE phase separates from rafts far less than DHA-PE, which may have implications for cellular signaling.    

4.282           Analysis of transcription factor expression during discrete stages of postnatal thymocyte differentiation

Tabrizifard, S. et al

1. Immunol., 173, 1094-1102 (2004)

  Postnatal T lymphocyte differentiation in the thymus is a multistage process involving serial waves of lineage specification, proliferative expansion, and survival/cell death decisions. Although these are believed to originate from signals derived from various thymic stromal cells, the ultimate consequence of these signals is to induce the transcriptional changes that are definitive of each step. To help to characterize this process, high density microarrays were used to analyze transcription factor gene expression in RNA derived from progenitors at each stage of T lymphopoietic differentiation, and the results were validated by a number of appropriate methods. We find a large number of transcription factors to be expressed in developing T lymphocytes, including many with known roles in the control of differentiation, proliferation, or cell survival/death decisions in other cell types. Some of these are expressed throughout the developmental process, whereas others change substantially at specific developmental transitions. The latter are particularly interesting, because stage-specific changes make it increasingly likely that the corresponding transcription factors may be involved in stage-specific processes. Overall, the data presented here represent a large resource for gene discovery and for confirmation of results obtained through other methods.  

4.283           Circulating cytokine profile in anti-neutrophilic cytoplasmatic autoantibody-associated vasculitis: prediction of outcome?

Ohlsson, S., Wieslander, J. and Segelmark, M. Mediators of inflammation,13(4), 275-283 (2004)   AIMS: The anti-neutrophilic cytoplasmatic autoantibody-associated vasculitides (AASV) are diseases of relapsing-remitting inflammation. Here we explore the cytokine profile in different phases of disease, looking for pathogenic clues of possible prognostic value. Results: Interleukin (IL)-6, IL-8 and IL-10 were significantly elevated in plasma. Patients in the stable phase who subsequently developed adverse events had higher IL-8 values. Patients in the stable phase who relapsed within 3 months had lower IL-10 values and higher IL-6 levels. Conclusions: Patients with AASV have raised circulating cytokine levels compared with healthy controls, even during remission. Raised IL-8 seems associated with poor prognosis. Lower levels of IL-10 and higher levels of IL-6 herald a greater risk of relapse. Patients with systemic vasculitis in clinical remission have persistent disease activity, kept under control by inhibitory cytokines.  

4.284           Inhibition of phenylephrine-induced cardiac hypertrophy by docosahexaenoic acid

Siddiqui, R.A., Shaikh, S.R., Kovacs, R., Stillwell, W. and Zaloga, G.

1. Cell. Biochem., 92(6), 1141-1159 (2004)

  Many of the cardiovascular benefits of fish oil result from the antiarrhythmic actions of the n-3 polyunsaturated lipids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The beneficial effects of DHA/EPA in patients with coronary artery disease and myocardial infarction may also result from modulation of the myocardial hypertrophic response. Hypertrophy was assessed in neonatal cardiomyocytes exposed to phenylephrine (PE) by measuring cell surface area, total protein synthesis (¹⁴C leucine incorporation), and the organization of sarcomeric -actinin and by monitoring expression of atrial natriuretic factor (ANF). We report that PE induced a twofold increase in cell surface area and protein synthesis in cardiomyocytes. The hypertrophied cardiomyocytes also exhibited increased expression of ANF in perinuclear regions and organization of sarcomeric -actinin into classical z-bands. Treatment of cardiomyocytes with 5 μM DHA effectively prevented PE-induced hypertrophy as shown by inhibition of surface area expansion and protein synthesis, inhibition of ANF expression, and prevention of -actinin organization into z-bands. DHA treatment prevented PE-induced activation of Ras and Raf-1 kinase. The upstream inhibition of Ras → Raf-1 effectively prevented translocation and nuclear localization of phosphorylated extracellularly regulated kinase 1 and 2 (Erk1/2). These effects consequently led to inhibition of nuclear translocation, and hence, activation of the downstream signaling enzyme p90 ribosomal S6 kinase (p90^rsk). These results indicate that PE-induced cardiac hypertrophy can be minimized by DHA. Our results suggest that inhibition of Ras → Raf-1 → Erk1/2 → p90^rsk → hypertrophy is one possible pathway by which DHA can inhibit cardiac hypertrophy. In vivo studies are needed to confirm these in vitro effects of DHA.  

4.285           Primary Adipocyte Culture: Adipocyte Purification Methods May Lead to a New Understanding of Adipose Tissue Growth and Development

Fernyhough, M.E., Vierck, J:L., Hausman, G.J., Mir, P.S., Okine, E.K. and Dodson, M.V. Cytotechnology, 46(2-3), 163-172 (2004) In the present manuscript, the methods required to generate purified cultures of mature adipocytes, as well as stromal vascular cells, from the same isolation are detailed. Also, we describe the in vitro conditions for the dedifferentiation of the isolated mature adipocytes. These two types of cells may be used to reevaluate differences between presently available cellular models for lipogenesis/lipolysis and might provide a new cellular physiological system for studies utilizing the proliferative progeny from mature adipocyte dedifferentiation. Alternative possibilities to the dedifferentiation phenomenon are proposed, as this new area of research is novel.  

4.286           The recognition of adsorbed and denatured proteins of different topographics by b₂ integrins and effects on leukocyte adhesion and activation

Brevig, T. et al Biomaterials, 26, 3039-3053 (2005)   Leukocyte β₂ integrins Mac-1 and p150,95 are promiscuous cell-surface receptors that recognise and mediate cell adhesion to a variety of adsorbed and denatured proteins. We used albumin as a model protein to study whether leukocyte adhesion and activation depended on the nm-scale topography of a protein adlayer. Albumin adsorbed from the native conformation gave rise to different adlayer topographies and different amounts of adsorbed protein on hydrophobic and relatively hydrophilic polystyrene and silanised silicon-wafer surfaces, whereas adsorption of pre-denatured Alb resulted in similar adlayer topographies and similar amounts of adsorbed protein on these surfaces. All three distinct protein-adlayer topographies supported adhesion of in vitro differentiated, macrophage-like U937 and THP-1 cells, but did not support adhesion of their promonocytic precursors. Human monocytes freshly isolated from peripheral blood did not adhere to adsorbed albumin, not even in the presence of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1α chemokines. Adhesion of the macrophage-like cells to albumin in any of the three topographies was inhibited by antibodies against β₂ integrins, but not by antibodies against β₁ integrins, and did not induce secretion of the proinflammatory cytokine tumour necrosis factor-α.  

4.287           Virus-like particles as carriers for T-cell epitopes: limited inhibition of T-cell priming by carrier-specific antibodies

Ruedl. C. et al

1. Virol., 79(2), 717-724 (2005)

  Virus-like particles (VLPs) are able to induce cytotoxic T-cell responses in the absence of infection or replication. This makes VLPs promising candidates for the development of recombinant vaccines. However, VLPs are also potent inducers of B-cell responses, and it is generally assumed that such VLP-specific antibodies interfere with the induction of protective immune responses, a phenomenon summarized as carrier suppression. In this study, we investigated the impact of preexisting VLP-specific antibodies on the induction of specific cytotoxic T-cell and Th-cell responses in mice. The data show that VLP-specific antibodies did not measurably reduce antigen presentation in vitro or in vivo. Nevertheless, T-cell priming was slightly reduced by antigen-specific antibodies; however, the overall reduction was limited and vaccination with VLPs in the presence of VLP-specific antibodies still resulted in protective T-cell responses. Thus, carrier suppression is unlikely to be a limiting factor for VLP-based T-cell vaccines.  

4.288           Brain-derived neurotrophic factor in platelets and airflow limitation in asthma

Lommatzsch, M. et al Am. J. Respir. Care Med., 171, 115-120 (2005)   Brain-derived neurotrophic factor (BDNF), a key mediator of neuronal plasticity, contributes to airway obstruction and hyperresponsiveness in a model of allergic asthma. BDNF is stored in human platelets and circulates in human plasma, but the significance of BDNF in this compartment is poorly understood. We investigated the relationship between platelet and plasma BDNF levels and pulmonary function in a cohort of 26 adult patients with recently diagnosed allergic asthma. BDNF levels in serum, platelets, and plasma were significantly increased in participants with asthma, as compared with 26 age- and sex-matched control subjects. In steroid-naive patients, but not in patients using inhaled corticosteroids, enhanced platelet BDNF levels correlated with parameters of airway obstruction and airway hyperresponsiveness to histamine. Experiments with activated peripheral blood mononuclear cells revealed that corticosteroids such as fluticasone effectively suppress BDNF secretion. In conclusion, we demonstrate that enhanced platelet BDNF is associated with airflow limitation and airway hyperresponsiveness in asthma. In addition, we provide evidence that corticosteroids suppress BDNF production by activated immune cells.  

4.289           IQGAP are differentially expressed and regulated in polarized gastric epithelial cells

Chew, C.S., Okamoto, C.T., Chen, X. and Qin, H.Y. Am. J. Physiol., 288, G376-G387 (2005)   IQGAPs, GTPase-activating proteins with an IQ motif, are thought to regulate many actin cytoskeleton-based activities through interactions with Cdc42 and Rac. Recently, Cdc42 was implicated in regulation of gastric parietal cell HCl secretion, and IQGAP2 was immunolocalized with Cdc42 to F-actin-rich intracellular canalicular membranes of isolated gastric parietal cells in primary culture. Here we sought to define distribution and localization of IQGAP1 and IQGAP2 in major oxyntic (acid-secreting) gastric mucosal cell types and to determine whether secretory agonists modulate these proteins. Differential staining protocols were used to identify different cell populations (parietal, chief, surface/pit, and mucous neck cells) in semi-intact glands isolated from rabbit gastric mucosae and to characterize these same cells after dispersion and fractionation on isopycnic density gradients with simultaneous staining for F-actin, H⁺-K⁺-ATPase, and GSII lectin-binding sites. There was a pronounced increase in intracellular F-actin staining in dispersed chief cells, apparently from internalization of F-actin-rich apical membranes that normally abut the gland lumen. Therefore, other membrane-associated proteins might also be redistributed by disruption of cell-cell contacts. Western blot analyses were used to quantitate relative concentrations of IQGAPs in defined mucosal cell fractions, and gastric glands were used for in situ localizations. We detected uniform levels of IQGAP2 expression in oxyntic mucosal cells with predominant targeting to regions of cell-cell contact and nuclei of all cell types. IQGAP2 was not detected in parietal cell intracellular canaliculi. IQGAP1 expression was variable and targeted predominantly to the cortex of chief and mucous neck cells. Parietal cells expressed little or no IQGAP1 vs. other mucosal cell types. Phosphoprotein affinity chromatography, isoelectric focusing, and phosphorylation site analyses indicated that both IQGAP1 and IQGAP2 are phosphoproteins potentially regulated by [Ca²⁺]_i/PKC and cAMP signaling pathways, respectively. Stimulation of glands with carbachol, which elevates [Ca²⁺]_i and activates PKC, induced apparent translocation of IQGAP1, but not IQGAP2, to apical poles of chief (zymogen) and mucous neck cells. This response was mimicked by PMA but not by ionomycin or by elevation of [cAMP]_i with forskolin. Our observations support a novel, PKC-dependent role for IQGAP1 in regulated exocytosis and suggest that IQGAP2 may play a more general role in regulating cell-cell interactions and possibly migration within the gastric mucosa.  

4.290           Microfluid sorting of mammalian cells by optical force switching

Wang, M.M. et al Nature Biotech., 23(1), 83-87 (2005)   Microfluidic-based devices have allowed miniaturization and increased parallelism of many common functions in biological assays; however, development of a practical technology for microfluidic-based fluorescence-activated cell sorting has proved challenging. Although a variety of different physical on-chip switch mechanisms have been proposed1, 2, 3, 4, 5, 6, none has satisfied simultaneously the requirements of high throughput, purity, and recovery of live, unstressed mammalian cells. Here we show that optical forces can be used for the rapid (2−4 ms), active control of cell routing on a microfluidic chip. Optical switch controls reduce the complexity of the chip and simplify connectivity. Using all-optical switching, we have implemented a fluorescence-activated microfluidic cell sorter and evaluated its performance on live, stably transfected HeLa cells expressing a fused histone−green fluorescent protein. Recovered populations were verified to be both viable and unstressed by evaluation of the transcriptional expression of two genes, HSPA6 and FOS, known indicators of cellular stress.  

4.291           The multifunctional DNA repair/redox enzyme Apel/Ref-1 promotes survival of neurons after oxidative stress

Vasko, M.R., Guo, C. and Kelley, M.R. DNA Repair, 4(3), 367-379 (2005)   Although correlative studies demonstrate a reduction in the expression of apurinic/apyrimidinic endonuclease/redox effector factor (Ape1/Ref-1 or Ape1) in neural tissues after neuronal insult, the role of Ape1 in regulating neurotoxicity remains to be elucidated. To address this issue, we examined the effects of reducing Ape1 expression in primary cultures of hippocampal and sensory neurons on several endpoints of neurotoxicity induced by H₂O₂. Ape1 is highly expressed in hippocampal and sensory neurons grown in culture as indicated by immunohistochemistry, immunoblotting and activity. Exposing hippocampal or sensory neuronal cultures to 25 or 50 nM small interfering RNA to Ape1 (Ape1siRNA), respectively, for 48 h, causes a reduction in immunoreactive Ape1 by approximately 65 and 54%, and an equivalent loss in endonuclease activity. The reduced expression of Ape1 is maintained for up to 5 days after the siRNA in the medium is removed, whereas exposing cultures to scrambled sequence siRNA (SCsiRNA) has no effect of Ape1 protein levels. The reduction in Ape1 significantly reduces cell viability in cultures 24 h after a 1-h exposure to 25–300 μM H₂O₂, compared to SCsiRNA treated controls. In cells treated with SCsiRNA, exposure to 300 μM H₂O₂ reduced cell viability by 40 and 30% in hippocampal and sensory neuronal cultures, respectively, whereas cultures treated with Ape1siRNA lost 93 and 80% of cells after the peroxide. Reduced Ape1 levels also increase caspase-3 activity in the cells, 2–3-fold, 60 min after a 1-h exposure to 100 μM H₂O₂ in the cultures. Exposing neuronal cultures with reduced expression of Ape1 to 65 μM H₂O₂ (hippocampal) or 300 μM H₂O₂ (sensory) for 1 h results in a 3-fold and 1.5-fold increase in the phosphorylation of histone H2A.X compared to cells exposed to SCsiRNA. Overexpressing wild-type Ape1 in hippocampal and sensory cells using adenoviral expression constructs results in significant increase in cell viability after exposure to various concentrations of H₂O₂. The C65A repair competent/redox incompetent Ape1 when expressed in the hippocampal and sensory cells conferred only partial protection on the cells. These data support the notion that both of functions of Ape1, redox and repair are necessary for optimal levels of neuronal cell survival.  

4.292           Changes in immune cell distribution and IL-10 production are regulated through endometrial IP-10 expression in the goat uterus

Imakawa, K., Nagaoka, K., Nojima, H., Hara, Y. and Christensen, R.K. Am. J. Reprod. Immunol., 53, 54-64 (2005)   Problem: Changes in distribution or redistribution of immune cells are required for the establishment and maintenance of pregnancy, but these changes during early pregnancy have been poorly understood in the ruminant ungulates. Expression of a chemokine, interferon- (IFN- )-inducible protein 10 kDa (IP-10, CXCL10), was identified in the endometrium of pregnant goats. Population and/or distribution of endometrial immune cells and their cytokine productions could be regulated by IP-10 during the period of pregnancy establishment. Method of study: Using reverse transcriptase-polymerase chain reaction (RT-PCR), expression of IP-10, IFN- , tumor necrosis factor- , interleukin-10 (IL-10), CXCR3 mRNA and leukocyte cell surface markers, CD4, CD8, CD11b and CD45 mRNA during the caprine early pregnancy was investigated. The ability of IP-10 to stimulate peripheral blood mononuclear cells (PBMCs) migration was demonstrated using a chemotaxis assay. Changes in migration of PBMCs' immune cell population and cytokine expressions with IP-10 stimulation were investigated using flow cytometry and RT-PCR respectively. Results: Levels of IP-10, IL-10, CD4 and CD11b mRNA, and the number of CD4 and CD11b positive cells in pregnant goat endometrium were higher than those of cyclic goat endometrium. Migration of PBMCs was stimulated by recombinant caprine IP-10, and the effect was significantly reduced by neutralization with the use of an anti-IP-10 antibody. In the flow cytometric and RT-PCR analyses, migrated cells stimulated by IP-10 increased the expression of IL-10 and CD11b mRNA. Furthermore, IP-10 could stimulate the expression of IL-10 mRNA from PBMCs. Conclusion: Endometrial chemokine IP-10 could regulate IL-10 production by resident and possibly migrated cells expressing CD11b, probably natural killer cells, and these changes may result in immune environments of the uterus suitable for conceptus implantation in ruminants.  

4.293           Rat embryonic motoneurons in long-term co-culture with Schwann cells – a system to investigate motoneuron diseases on a cellular level in vitro

Haastert, K. et al

1. Neurosci. Methods, 142(2), 275-284 (2005)

  Investigations of motoneuron diseases on a cellular and molecular level require long-term cultivation of primary cells. Here we present a new culture system in which matured motoneurons interact with their physiological partners like interneurons, astroglia and peripheral glia cells. This enables motoneuron-maturation for up to 3 weeks, while motoneurons consistently reached large diameters of their somata of 30–45 μm, occasionally more than 80 μm. Dissociated rat embryonic ventral spinal cord cells were enriched for motoneurons by density gradient centrifugation and seeded on a non-confluent mono-layer of highly enriched neonatal rat Schwann cells. Immunocytochemical visualization of neuron specific βIII-tubulin in all neurons and of motoneuron specific non-phosphorylated neurofilament H/M, respectively, revealed that after 3 days in vitro >70% of all neurons were motoneurons. After 20 days in vitro, a motoneuron fraction of 12% was maintained. Motoneurons were susceptible to transient transfection with green fluorescent protein cDNA when liposomal transfection and an enhancer substance were combined. Synaptic connections enabled formation of spontaneously active neuronal networks which provide a culture model to study glutamate excitotoxicity and calcium deregulation on a molecular level. Both mechanisms are implied in the pathophysiology of amyotrophic lateral sclerosis, a neurodegenerative motoneuron disorder.  

4.294           Identification of a novel tumor necrosis factor a-responsive region in the NCF2 promoter

Gauss, K.A. et al

1. Leukoc. Biol., 77, 267-278 (2005)

  The phagocyte reduced nicotinamide adenine dinucleotide phosphate oxidase is a multiprotein enzyme that catalyzes the production of microbicidal oxidants. Although oxidase assembly involves association of several membrane and cytosolic oxidase proteins, one of the cytosolic cofactors, p67^phox, appears to play a more prominent role in final activation of the enzyme complex. Based on the importance of p67^phox, we investigated transcriptional regulation of the p67^phox gene [neutrophil cytosolic factor 2 (NCF2)] and demonstrated previously that activator protein-1 (AP-1) was essential for basal transcriptional activity. As p67^phox can be up-regulated by tumor necrosis factor (TNF- ), which activates AP-1, we hypothesized that TNF- might regulate NCF2transcription via AP-1. In support of this hypothesis, we show here that NCF2 promoter-reporter constructs are up-regulated by TNF- but only when AP-1 factors were coexpressed. Consistent with this observation, we also demonstrate that NCF2 mRNA and p67^phox protein are up-regulated by TNF- in various myeloid cell lines as well as in human monocytes. It was surprising that mutagenesis of the AP-1 site in NCF2 promoter constructs did not eliminate TNF- induction, suggesting additional elements were involved in this response and that AP-1 might play a more indirect role. Indeed, we used NCF2 promoter-deletion constructs to map a novel TNF- -responsive region (TRR) located between –56 and –16 bp upstream of the translational start site and demonstrated its importance in vivo using transcription factor decoy analysis. Furthermore, DNase footprinting verified specific binding of factor(s) to the TRR with AP-1 binding indirectly to this region. Thus, we have identified a novel NCF2 promoter/enhancer domain, which is essential for TNF- -induced up-regulation of p67^phox.  

4.295           Characterization of microglia induced from mouse embryonic stem cells and their migration into brain parenchyma

Tsuchiya, T. et al

1. Neuroimmunol., 160, 210-218 (2005)

  We derived microglia from mouse embryonic stem cells (ES cells) at very high density. Using the markers Mac1⁺/CD45^low and Mac1⁺/CD45^high to define microglia and macrophages, respectively, we show that Mac1⁺ cells are induced by GM-CSF stimulation following neuronal differentiation of mouse ES cells using a five-step method. CD45^low expression was high and CD45^high expression was low on induced cells. We used a density gradient method to obtain a large amount of microglia-like cells, approximately 90% of Mac1⁺ cells. Microglia-like cells expressed MHC class I, class II, CD40, CD80, CD86, and IFN-γR. The expression level of these molecules on microglia-like cells was barely enhanced by IFN-γ. Intravenously transferred GFP⁺ microglia derived from GFP⁺ ES cells selectively accumulated in brain but not in peripheral tissues such as spleen and lymph node. GFP⁺ cells were detected mainly in corpus callosum and hippocampus but were rarely seen in cerebral cortex, where Iba1, another marker of microglia, is primarily expressed. Furthermore, both GFP⁺ and Iba1⁺ cells exhibited a ramified morphology characteristic of mature microglia. These studies suggest that ES cell-derived microglia-like cells obtained using our protocol are functional and migrate selectively into the brain but not into peripheral tissues after intravenous transplantation.  

4.296           Clinical response in Japanese metastatic melanoma patients treated with peptide cocktail-pulsed dendritic cells

Akiyama, Y. et al

1. Transplant. Med., 3(4), 1-10 (2005)

  Background Metastatic, chemotherapy-resistant melanoma is an intractable cancer with a very poor prognosis. As to immunotherapy targeting metastatic melanoma, HLA-A2⁺ patients were mainly enrolled in the study in Western countries. However, HLA-A24⁺ melanoma patients-oriented immunotherapy has not been fully investigated. In the present study, we investigated the effect of dendritic cell (DC)-based immunotherapy on metastatic melanoma patients with HLA-A2 or A24 genotype. Methods Nine cases of metastatic melanoma were enrolled into a phase I study of monocyte-derived dendritic cell (DC)-based immunotherapy. HLA-genotype analysis revealed 4 cases of HLA-A*0201, 1 of A*0206 and 4 of A*2402. Enriched monocytes were obtained using OptiPrep™ from leukapheresis products, and then incubated with GM-CSF and IL-4 in a closed serum-free system. After pulsing with a cocktail of 5 melanoma-associated synthetic peptides (gp100, tyrosinase, MAGE-2, MAGE-3 and MART-1 or MAGE-1) restricted to HLA-A2 or A24 and KLH, cells were cryopreserved until used. Finally, thawed DCs were washed and injected subcutaneously (s.c.) into the inguinal region in a dose-escalation manner. Results The mean percentage of DCs rated as lin^-HLA-DR⁺ in melanoma patients was 46.4 ± 15.6 %. Most of DCs expressed high level of co-stimulatory molecules and type1 phenotype (CD11c⁺HLA-DR⁺), while a moderate number of mature DCs with CD83 and CCR7 positive were contained in DC products. DC injections were well tolerated except for transient liver dysfunction (elevation of transaminases, Grade I-II). All 6 evaluable cases except for early PD showed positive immunological responses to more than 2 melanoma peptides in an ELISPOT assay. Two representative responders demonstrated strong HLA-class I protein expression in the tumor and very high scores of ELISPOT that might correlate to the regression of metastatic tumors. Clinical response through DC injections was as follows : 1CR, 1 PR, 1SD and 6 PD. All 59 DC injections in the phase I study were tolerable in terms of safety, however, the maximal tolerable dose of DCs was not determined. Conclusions These results suggested that peptide cocktail-treated DC-based immunotherapy had the potential for utilizing as one of therapeutic tools against metastatic melanoma in Japan.  

4.297           Immunomagnetic separation of Toxoplasma gondii oocysts using a monoclonal antibody directed against the oocyst wall

Dumetre, A. and Darde, M-L.

1. Microbiol. Methods, 61, 209-217 (2005)

  Recent outbreaks of waterborne toxoplasmosis have stimulated the development of sensitive methods to detect Toxoplasma gondii oocysts in samples suspected to be contaminated. The immunomagnetic separation (IMS) have been standardised to detect waterborne protozoa, but it did not exist for Toxoplasma oocysts. In this study, we describe two monoclonal antibodies (mAbs 3G4 and 4B6) produced against the oocyst wall, and the incorporation of mAb 3G4 in an IMS procedure. We found that an indirect IMS method gave better mean recoveries than a direct one (69.4% and 25.2%, respectively). Dissociation of oocyst_magnetic bead complexes was greatly improved by using a 2% aqueous H₂SO₄ solution instead of a 0.1 N HCl solution (82.8% and 17.4%, respectively). With these parameters, mean recoveries of less than 1000 oocysts ranged from 44.6% to 82.9%, depending on incubating temperature and buffe r. Age of oocysts (1 or 12 months old) does not influence IMS performances. Results of this study indicate that the described IMS is an efficient technique to recover Toxoplasma oocysts.  

4.298           Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes

Hering, B.J: et al JAMA, 293(7), 830-835 (2005)   Context  Islet allografts from 2 to 4 donors can reversetype 1 diabetes. However, for islet transplants to become awidespread clinical reality, diabetes reversal must be achievedwith a single donor to reduce risks and costs and increase theavailability of transplantation. Objective  To assess the safety of a single-donor, marginal-doseislet transplant protocol using potent induction immunotherapyand less diabetogenic maintenance immunosuppression in recipientswith type 1 diabetes. A secondary objective was to assess theproportion of islet transplant recipients who achieve insulinindependence in the first year after single-donor islet transplantation. Design, Setting, and Participants  Prospective, 1-yearfollow-up trial conducted July 2001 to August 2003 at a singleUS center and enrolling 8 women with type 1 diabetes accompaniedby recurrent hypoglycemia unawareness or advanced secondarycomplications. Interventions  Study participants underwent a primary isletallotransplant with 7271 (SD, 1035) islet equivalents/kg preparedfrom a single cadaver donor pancreas. Induction immunosuppressionwas with antithymocyte globulin, daclizumab, and etanercept.Maintenance immunosuppression consisted of mycophenolate mofetil,sirolimus, and no or low-dose tacrolimus. Main Outcome Measures  Safety (assessed by monitoring theseverity and duration of adverse events) and efficacy (assessedby studying the recipients’ insulin requirements, C-peptidelevels, oral and intravenous glucose tolerance results, intravenousarginine stimulation responses, glycosylated hemoglobin levels,and hypoglycemic episodes) associated with the study transplantprotocol. Results  There were no serious, unexpected, or procedure-or immunosuppression-related adverse events. All 8 recipientsachieved insulin independence and freedom from hypoglycemia.Five remained insulin-independent for longer than 1 year. Graftfailure in 3 recipients was preceded by subtherapeutic sirolimusexposure in the absence of measurable tacrolimus trough levels. Conclusions  The tested transplant protocol restored insulinindependence and protected against hypoglycemia after single-donor,marginal-dose islet transplantation in 8 of 8 recipients. Theseresults may be related to improved islet engraftment secondaryto peritransplant administration of antithymocyte globulin andetanercept. These findings may have implications for the ongoingtransition of islet transplantation from clinical investigationto routine clinical care.  

4.299           Are platelets activated after a rapid, one-step density gradient centrifugation? Evidence from flow cytometric analysis

Bagamery, K., Kvell, K., Barnet, M., Landau, R. and Graham, J. Clin. Lab. Haem., 27(1), 75-77 (2005)   This procedure describes the preparation of platelets from whole blood of healthy donors and pregnancy-induced hypertensive (PIH) patients by a rapid, one-step density gradient centrifugation, and the direct immunofluorescence staining of obtained platelets (CD63). Platelets are relatively fragile structures. Consequently, for the investigation of their biochemical properties it is recommended to isolate them by a simple method that does not damage their functional parameters and induce their activation. During platelet activation, several changes occur at the platelet surface. CD63 is the receptor for a lysosomal glycoprotein expressed in activated platelets. Currently, flow cytometry (fluorescence-activated cell sorting) is the most sensitive method to detect increased surface exposure of activation antigens on the platelet surface. The present technical note describes that compared with other whole blood flow cytometric techniques, our one-step density-gradient centrifugation method using OptiPrep(TM) can also prevent artificial, sample manipulation-related platelet activation.  

4.300           CD4^- plasmacytoid dendritic cells (pDCs) migrate in lymph nodes by CpG inoculation and represent a potent functional subset of pDCs

Yang, G-X. Et al

1. Immunol., 174, 3197-3203 (2005)

  We have recently identified two groups of plasmacytoid dendritic cells (pDCs) isolated from murine liver based on the expression of CD4 and other cell surface markers uniquely expressed by pDCs. Herein, we describe the identification of both CD4⁺ and CD4^– pDCs that clearly exist in lymph nodes (LNs), spleen, liver, thymus, bone marrow, and lung. Normally, CD4⁺ pDCs are enriched in LNs. However, after in vivo systemic injection with bacterial CpG, a larger number of CD4^– pDCs are recruited to the LNs and local inoculation by CpG drives CD4^– pDCs migrating into local sentinel LNs, suggesting that CD4^– pDCs are the main subpopulation migrating to the peripheral LNs. Furthermore, although both freshly isolated CD4⁺ pDCs and CD4^– pDCs appear as an immature plasmacytoid cell and develop into a DC morphology following activation, the two subsets have strikingly different immune features, including differences in the production pattern of cytokines stimulated with CpG and in T cell activation.  

4.301           Influence of ESAT-6 secretion system 1 (RD1) of Mycobacterium tuberculosis on the interaction between mycobacteria and the host immune system

Majlessi, L. et al

1. Immunol., 174, 3570-3579 (2005)

  The chromosomal locus encoding the early secreted antigenic target, 6 kDa (ESAT-6) secretion system 1 of Mycobacterium tuberculosis, also referred to as "region of difference 1 (RD1)," is absent from Mycobacterium bovis bacillus Calmette-Guérin (BCG). In this study, using low-dose aerosol infection in mice, we demonstrate that BCG complemented with RD1 (BCG::RD1) displays markedly increased virulence which albeit does not attain that of M. tuberculosis H37Rv. Nevertheless, phenotypic and functional analyses of immune cells at the site of infection show that the capacity of BCG::RD1 to initiate recruitment/activation of immune cells is comparable to that of fully virulent H37Rv. Indeed, in contrast to the parental BCG, BCG::RD1 mimics H37Rv and induces substantial influx of activated (CD44^highCD45RB^–CD62L^–) or effector (CD45RB^–CD27^–) T cells and of activated CD11c⁺CD11b^high cells to the lungs of aerosol-infected mice. For the first time, using in vivo analysis of transcriptome of inflammatory cytokines and chemokines of lung interstitial CD11c⁺ cells, we show that in a low-dose aerosol infection model, BCG::RD1 triggered an activation/inflammation program comparable to that induced by H37Rv while parental BCG, due to its overattenuation, did not initiate the activation program in lung interstitial CD11c⁺ cells. Thus, products encoded by the ESAT-6 secretion system 1 of M. tuberculosis profoundly modify the interaction between mycobacteria and the host innate and adaptive immune system. These modifications can explain the previously described improved protective capacity of BCG::RD1 vaccine candidate against M. tuberculosis challenge.  

4.302           A phase I study of a-galactosylceramide (KRN7000) – pulsed dendritic cells in patients with advanced and recurrent non – small cell lung cancer

Ishikawa, A. et al Clin. Can. Res., 11, 1910-1917 (2005)   Purpose: Human V24 natural killer T (NKT) cells bearing an invariantV24JQ antigen receptor, the counterpart of murine V14 NKT cells,are activated by a specific ligand, -galactosylceramide (GalCer,KRN7000), in a CD1d-dependent manner. I.v. administration ofGalCer-pulsed dendritic cells (DC) induces significant activationand expansion of V14 NKT cells in the lung and resulting potentantitumor activities in mouse tumor metastatic models. We dida phase I dose escalation study with GalCer-pulsed DCs in lungcancer patients. Experimental Design: Patients with advanced non–small cell lung cancer or recurrent lung cancer received i.v. injections of GalCer-pulsed DCs (level 1: 5 x 10⁷/m²; level 2: 2.5 x 10⁸/m²; and level 3: 1 x 10⁹/m²) to test the safety, feasibility, andclinical response. Immunomonitoring was also done in all completedcases. Results: Eleven patients were enrolled in this study. No severeadverse events were observed during this study in any patient.After the first and second injection of GalCer-pulsed DCs, dramaticincrease in peripheral blood V24 NKT cells was observed in onecase and significant responses were seen in two cases receivingthe level 3 dose. No patient was found to meet the criteriafor partial or complete responses, whereas two cases in thelevel 3 group remained unchanged for more than a year with goodquality of life. Conclusions: In this clinical trial, GalCer-pulsed DC administrationwas well tolerated and could be safely done even in patientswith advanced disease.  

4.303           Nuclear factor-kB1 (p50) limits the inflammatory and fibrogenic responses to chronic injury

Oakley, F. et al Am. J. Pathol., 166(3), 695-708 (2005)   In this study we addressed the role of the nuclear factor (NF)- B1/p50 subunit in chronic injury of the liver by determining the inflammatory and fibrotic responses of nf b1-null mice in an experimental model that mimics chronic liver disease. Mice received repeated hepatic injuries throughout 12 weeks by intraperitoneal injection of the hepatotoxin carbon tetrachloride. In response nf b1^–/– mice developed more severe neutrophilic inflammation and fibrosis compared to nf b1^+/+ mice. This phenotype was associated with elevated hepatic expression of tumor necrosis factor (TNF)- , which was localized to regions of the liver associated with inflammation and fibrosis. Hepatic stellate cells are important regulators of hepatic inflammatory and fibrogenic events but normally do not express TNF- . Hepatic stellate cells derived from nf b1^–/– mice expressed TNF- promoter activity, mRNA, and protein. By contrast the expression of other NF- B-responsive genes (ICAM1 and interleukin-6) was similar between nf b1^–/– and nf b1^+/+ cells. We provide experimental evidence that the inappropriate expression of TNF- by nf b1^–/– cells is because of lack of a p50-dependent histone deacetylase 1 (HDAC1)-mediated repression of TNF- gene transcription. Taken together these data indicate that the p50 NF- B subunit plays a critical protective role in the injured liver by limiting the expression of TNF- and its recruitment of inflammatory cells.  

4.304           Arginine supplementation does not e enhance serum nitric oxide levels in elderly nursing home residents with pressure ulcers

Techmiller, J.K. et al Biol. Res. for Nursing, 6(4), 289-299 (2005)   The purpose of this study was to determine whether arginine supplementation enhances in vitro (neutrophil burst and mitogen-induced lymphocyte proliferation) and in vivo (delayed-type hypersensitivity [DTH] and serum nitric oxide) measures of immune function in nursing home elders with pressure ulcers. Twenty-six elders, 65 years of age or older, with one or more pressure ulcers, were randomized to receive 8.5 g of arginine or an isonitrogenous supplement for 4 weeks. Immune function studies and serum arginine, ornithine, citrulline, and nitric oxide were measured at baseline, 4 weeks postsupplementation (Week 4) and after a 6-week washout (Week 10). At Week 4, serum ornithine increased (p = .01) and arginine trended to increase (p = .055), but there was no increasein citrulline or nitric oxide with arginine supplementation.There were no differences in neutrophil burst or DTH responsesbetween groups. Whole blood mitogen–induced proliferationdecreased significantly atWeek 10 in the isonitrogenous butnot in the arginine-supplemented group. There is mounting concernthat arginine supplementation during an inflammatory state couldbe detrimental due to overwhelming nitric oxide production.A key finding of this study is that arginine supplementationdid not increase serum nitric oxide levels over that observedin elders with pressure ulcers given an isonitrogenous supplement.  

4.305           Pancreatic b-cell failure and diabetes in mice with a delition mutation of the endoplastic reticulum molecular chaperone gene P58^IPK

Ladiges, W. et al Diabetes, 54, 1074-1081 (2005)   The endoplasmic reticulum (ER) transmits apoptotic signals in the pancreas during ER stress, implicating ER stress–mediated apoptosis in the development of diabetes. P58^IPK (DNAJC3) is induced during ER stress and functions as a negative feedback component to inhibit eIF-2 signaling and attenuate the later phases of the ER stress response. To gain insight into a more comprehensive role of P58^IPK function, we generated deletion mutant mice that showed a gradual onset of glucosuria and hyperglycemia associated with increasing apoptosis of pancreatic islet cells. Lack of P58^IPK had no apparent effect on the functional integrity of viable ß-cells. A set of genes associated with apoptosis showed altered expression in pancreatic islets from P58^IPK-null mice, further substantiating the apoptosis phenotype. The data provide in vivo evidence to support the concept that P58^IPK functions as a signal for the downregulation of ER-associated proteins involved in the initial ER stress response, thus preventing excessive cell loss by degradation pathways. Insulin deficiency associated with the absence of P58^IPK mimics ß-cell failure associated with type 1 and late-stage type 2 diabetes. P58^IPK function and activity may therefore provide a novel area of investigation into ER-mediated mechanistic and therapeutic approaches for diabetes.  

4.306           Neuropeptide Y stimulates neuronal precursor proliferation in the post-natal and adult dentate gyrus

Howell, O.W. et al

1. Neurochem., 93, 560-570 (2005)

  Adult dentate neurogenesis is important for certain types of hippocampal-dependent learning and also appears to be important for the maintenance of normal mood and the behavioural effects of antidepressants. Neuropeptide Y (NPY), a peptide neurotransmitter released by interneurons in the dentate gyrus, has important effects on mood, anxiety-related behaviour and learning and memory. We report that adult NPY receptor knock-out mice have significantly reduced cell proliferation and significantly fewer immature doublecortin-positive neurons in the dentate gyrus. We also show that the neuroproliferative effect of NPY is dentate specific, is Y₁-receptor mediated and involves extracellular signal-regulated kinase (ERK)1/2 activation. NPY did not exhibit any effect on cell survival in vitro but constitutive loss of the Y₁ receptor in vivo resulted in greater survival of newly generated neurons and an unchanged total number of dentate granule cells. These results show that NPY stimulates neuronal precursor proliferation in the dentate gyrus and suggest that NPY-releasing interneurons may modulate dentate neurogenesis.  

4.307           Flow cytometric analysis of CD41-labeled platelets isolated by the rapid, one-step OptiPrep method from human blood

Bagamery, K., Kvell, K., Landau, R. and Graham, J. Cytometry Part A, 65A, 84-87 (2005)   Although platelet-rich plasma is relatively easy to produce by centrifugation of whole blood, yields of platelets may be variable because many of them are trapped within the erythrocyte layer. Although they can be recovered by washing these cells, it is a general rule that the number of centrifugations should be kept to a minimum to avoid activation of platelets. This work describes the rapid, one-step OptiPrep method for the isolation of highly purified platelets from human blood (buffy coat). To provide a functionally intact and uncontaminated platelet fraction, a density gradient centrifugation was performed by using a density barrier prepared from OptiPrep. CD41 antibody staining was performed to assess the purity of the obtained platelet population by means of a FACScan flow cytometer. Platelets were identified by a morphologic gate in which events were further studied for CD41 expression. Data were analyzed by CellQuest (Becton Dickinson). Platelet-specific CD41 antibody staining showed that the purity of the platelet population recovered from this density barrier method was greater than 90%. The platelets showed an excellent morphologic state. The rapid, one-step OptiPrep density gradient centrifugation is a reliable method for obtaining highly purified platelets from human blood that are ready for further pharmacologic investigations.  

4.308           Reversal of long-term sepsis-induced immunosuppression by dendritic cells

Benjamin, C.F., Lundy, S.K., Lukacs, N.W., Hogaboam, C.M. and Kunkel, S.L. Blood, 105, 3588-3595 (2005)   Severe sepsis leads to long-term systemic and local immunosuppression, which is the cause of a number of complications, including pulmonary infection. A therapeutic strategy that reverses this immunosuppression is required, given the ongoing high mortality rate of patients who have survived a severe sepsis. The present study demonstrates that experimental severe sepsis renders the lung susceptible to a normally innocuous Aspergillus fumigatus fungus challenge, due to a dominant lung type 2 cytokine profile. Dendritic cells (DCs) obtained from the lungs of mice subjected to cecal ligation and puncture (CLP) model were skewed toward type 2 cytokine profile, which occurred with exaggerated expression of Toll-like receptor 2 (TLR2). The intrapulmonary transfer of bone marrow–derived DCs (BMDCs) in postseptic mice prevented fatal Aspergillus infection. This therapy reduced the overall inflammatory response and fungal growth in the lung, and promoted the balance of proinflammatory and suppressive cytokines in the lung. Thus, intrapulmonary DC supplementation appears to restore the pulmonary host response in the postseptic lung in our animal model. These data strongly suggest that lung DCs are profoundly affected as a consequence of the systemic impact of severe sepsis, and the identification of mechanisms that restore their function may serve as a key strategy to reverse sepsis-induced immunosuppression.  

4.309           TGF-b signaling regulates CD8+ T cell responses to high- and low-affinity TCR interactions

Mehal, W., Sheikh, S.Z., Gorelik, L. and Flavell, R.A. Int. Immunol., 17(5), 531-538 (2005)   Absence of transforming growth factor-ß (TGF-ß) signaling to T cells in mice results in an increase in T cell numbers, an activated CD44 high, CD69–, CD25– T cell phenotype and a T cell-mediated injury to many organs. It is not known if such T cell activation in the absence of TGF-ß signaling is spontaneous or due to aberrant T cell responses to a physiological stimulus. We used adoptive transfer of CD8+ T cells from mice double transgenic for the OT-1 TCR and the TGF-ß1-dominant negative transgene [OT-dominant-negative receptor (DNR)] to investigate the role of TGF-ß in regulating CD8+ T cell activation in vivo. The activation and expansion of single-transgenic OT and double-transgenic OT-DNR cells to oral antigens, high-affinity and low-affinity peptides were indistinguishable. Activation with high-affinity peptide and CFA however resulted in greater expansion of OT-DNR cells in comparison to OT cells. Low-affinity peptide and adjuvant did not result in OT cell activation or expansion but results in up-regulation of CD44 on OT-DNR cells. These data show that TGF-ß functions in vivo to limit the scale of CD8+ T cell expansion after high-affinity peptide–MHC interactions. TGF-ß also limits T cell activation to the highest affinity peptide–MHC interactions. The increase in T cell number and activation present in TGF-ß-deficient and TGF-ß DNR-expressing mice may be due to the loss of these two phenomena.  

4.310           Effect of P-selectin on phosphatidylserine exposure and surface-dependent thrombin generation on monocytes

Del Conde, I. Et al Arterioscler. Thromb. Vasc. Biol., 25, 1065-1070  (2005)   Objective— Stimulation of monocytes with P-selectin inducesthe synthesis of an array of mediators of inflammation, as wellas the expression of tissue factor (TF), the main initiatorof coagulation. Because the membrane-bound reactions of coagulationare profoundly influenced by the presence of phosphatidylserineon the membranes of cells, factors that increase its expressionmay have an impact on coagulation. Methods and Results— Using flow cytometry, we studiedthe effect of P-selectin on phosphatidylserine expression inblood monocytes and in the monocytic cells, THP-1. Soluble P-selectinat biologically relevant concentrations (0.31 to 2.5 µg/mL)induced a time-dependent increase in phosphatidylserine expression,an effect that could be inhibited with an anti–PSGL-1blocking antibody, and by genistein, a tyrosine kinase inhibitor.Binding of activated platelets to THP-1 cells also resultedin a significant increase in phosphatidylserine expression thatwas dependent on PSGL-1. Consistent with the role of phosphatidylserineon surface-dependent reactions of coagulation, treatment ofmonocytic cells with soluble P-selectin led to increased thrombingeneration. We excluded P-selectin induced apoptosis of monocyteas a mechanism for the increased phosphatidylserine exposure. Conclusion— In summary, we show that P-selectin, eithersoluble or in its membrane-bound form, induces phosphatidylserineexposure in monocytes through a mechanism dependent on PSGL-1. Phosphatidylserine expression on monocytes is an important cofactorin the enzymatic reactions of coagulation. Stimulation of monocyteswith P-selectin leads to cell activation and the expressionof tissue factor, the main initiator of coagulation. Here weshow that P-selectin induces phosphatidylserine expression onmonocytes through a mechanism dependent on PSGL-1 on the monocyte.  

4.311           Quantitation and characterization of myosin peptide-specific CD4⁺ T cells in autoimmune myocarditis

Maier, R. et al

1. Immunol. Methods, 304, 117-125 (2005)

  Characterization of autoantigen-specific CD4⁺ T cells at the single cell level is crucial for understanding the immunopathological mechanisms underlying autoimmune diseases. Cardiac myosin heavy chain (myhca) is the major autoantigen associated with autoimmune myocarditis both in humans and in experimental autoimmune myocarditis (EAM) in mice. In the current study, we evaluated two methods for the enumeration and phenotypic characterization of myhca-specific CD4⁺ T cells during the course of EAM. Both enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) assays were suitable for the detection and characterization of myhca-specific Th cells during acute myocardial inflammation and the late healing phase of the disease. Cytokine production of myhca-specific Th cells was restricted to interferon-γ (IFNγ). Only trace amounts of the Th2 cytokines IL-4 and IL-5 could be detected. Concomitant surface marker analysis in the CFC assay revealed the prototypical effector phenotype of myhca-specific Th1 cells during the acute phase of the disease. Taken together, the combination of both methods appears to be most appropriate for a comprehensive ex vivo single cell analysis of Th cells in heart-specific autoimmune disorders.    

4.312           Primary defect in UVB-induced systemic immunomodulation does not relate to immature or functionally impaired APCs in regional lymph nodes

Gorman, S. et al

1. Immunol., 174, 6677-6685 (2005)

  UVB irradiation of the shaved dorsal skin of mice can cause both local and systemic suppression of contact hypersensitivity responses; the former demonstrated by administration of the sensitizing Ag/hapten to the irradiated site and the latter by its administration at least 72 h later to distal unirradiated sites. The immunological basis of systemic immunomodulation is not clear. When haptens (trinitrochlorobenzene, FITC) were administered to the shaved ventral skin 4 days after irradiation (8 kJ/m²) to the shaved dorsum of BALB/c mice, CD11c⁺/FITC⁺ cells in the skin-draining lymph nodes from control and irradiated mice produced on a per cell basis similar levels of IL-12 and PGE₂ were phenotypically mature and efficient at presenting FITC to lymphocytes from FITC-sensitized mice. Ag presentation by FACS-sorted CD11c⁺ lymph node cells isolated 4 days after UVB irradiation was as efficient as were cells from unirradiated mice at presentation in vitro of an OVA peptide (OVA_323–339) to CD4⁺ cells from OVA-TCR-transgenic DO11.10 mice. Further, IFN- levels were increased in the cultures containing CD11c⁺ cells from UVB-irradiated mice, suggesting that inflammation may precede downstream immunosuppression. These results suggest that the primary cause of reduced contact hypersensitivity responses in mice in which UV irradiation and the sensitizing Ag are applied to different sites several days apart must originate from cells other than CD11c⁺ APCs that directly or by production of soluble mediators (IL-12, PGE₂) affect cellular responses in the nodes of UVB-irradiated mice.  

4.313           Identification of a cell population that produces alpha/beta interferon in vitro and in vivo in response to noncytopathic bovine viral diarrhea virus

Brackenbury, L.S. et al J.Virol., 79(12), 7738-7744 (2005)   In vitro infection of bovine cells of many origins with the cytopathogenic bovine viral diarrhea virus (cpBVDV) results in the induction of alpha/beta interferon (IFN- /ß), whereas noncytopathogenic BVDV (ncpBVDV) isolates have been shown not to induce IFN- /ß in vitro. Similarly, cpBVDV induces IFN- /ß in the early bovine fetus, but ncpBVDV does not. However, acute infection of naïve cattle with ncpBVDV results in IFN- /ß production. In this study, we identified and characterized a minor population of cells, present in lymph nodes that produce IFN- in response to ncpBVDV. These cells expressed the myeloid markers CD14, CD11b, and CD172a but did not express CD4 and CD45RB. We also established that these cells produced IFN- in the absence of detectable productive infection.  

4.314           Islet transplantation: progress and challenge

Gaglia, J.L., Shapiro, A.M.J. and Weir, G.C. Arch. Med. Res., 36, 273-280 (2005)   For over 30 years, investigators have explored islet transplantation as a logical approach to restoring glucose homeostasis in persons with diabetes. Islet transplantation can currently provide improved glycemic control, relief from recurrent severe hypoglycemia, and potentially insulin independence. In this review, we describe details of the evolution of modern islet transplantation and provide insight into ongoing clinical and basic research efforts to overcome current obstacles for this promising therapy.  

4.315           Uptake and neuritic transport of scrapie prion protein coincident with infection of neuronal cells

Magalhaes, A. C. Et al

1. Neurosci., 25(21), 5207-5216 (2005)

  Invasion of the nervous system and neuronal spread of infection are critical, but poorly understood, steps in the pathogenesis of transmissible spongiform encephalopathies or prion diseases. To characterize pathways for the uptake and intraneuronal trafficking of infectious, protease-resistant prion protein (PrP-res), fluorescent-labeled PrP-res was used to infect a neuronally derived murine cell line (SN56) and adult hamster cortical neurons in primary culture. Concurrent with the establishment of persistent scrapie infection, SN56 cells internalized PrP-res aggregates into vesicles positive for markers for late endosomes and/or lysosomes but not synaptic, early endocytic, or raft-derived vesicles. Internalized PrP-res was then transported along neurites to points of contact with other cells. Similar trafficking was observed with dextran, Alzheimer's A 1-42 fibrils and noninfectious recombinant PrP fibrils, suggesting that PrP-res is internalized by a relatively nonspecific pinocytosis or transcytosis mechanism. Hamster cortical neurons were also capable of internalizing and disseminating exogenous PrP-res. Similar trafficking of exogenous PrP-res by cortical neurons cultured from the brains of PrP knock-out mice showed that uptake and neuritic transport did not require the presence of endogenous cellular PrP. These experiments visualize and characterize the initial steps associated with prion infection and transport within neuronal cells.  

4.316           Increased monocyte transcription of the proteinase 3 gene in small vessel vasculitis

Ohlsson, S. et al Clin. Exp. Immunol., 141, 174-182 (2005)   Proteinase 3 (PR3) is a pleiotropic and destructive serine protease and it is also a major target for autoantibodies in systemic small vessel vasculitis. We have shown recently that patients in stable remission have increased circulating levels of PR3, independent of autoantibody titre, inflammation, neutrophil degranulation and renal function. Here we explore the possibility of increased PR3 gene transcription. RNA was purified from peripheral blood monocytes from vasculitis patients and controls. Specific mRNA was measured by TaqMan real-time polymerase chain reaction (PCR). The monocyte-like cell lines THP-1 and U937 and human peripheral blod monocytes from healthy controls were stimulated with cytokines and lipopolysaccharide (LPS) for different time periods. PR3 protein was measured in plasma with enzyme-linked immunosorbent assay (ELISA). The median result for PR3 mRNA was 9·6 (1·8 680) for 22 patients, compared to 1 (0·1 2·8) for the 15 healthy controls. Elastase expression was also significantly increased, whereas myeloperoxidase and interleukin-8 were not. Stimulation of monocytes with tumour necrosis factor (TNF)- , interferon (IFN)- or LPS did not result in any increase of PR3 or elastase transcription, whereas interleukin (IL)-8 transcription was increased 10-fold. Circulating monocytes from patients with systemic vasculitis display increased PR3 gene transcription compared to healthy controls and patients with sytemic lupus erythematosus (SLE). This may be important for the development of vasculitis. Our results do not favour a role for cytokines, antineutrophil cytoplasmic antibodies (ANCA) or immunosuppressive medication in the upregulation of PR3 transcription in vasculitis.  

4.317           Improved success of myoblast transplantation in mdx mice by blocking the myostatin signal

Benabdallah, B.F., Bouchentouf, M. and Tremblay, J.P. Transplantation, 79(12), 1696-1702 (2005)   Background. Duchenne muscular dystrophy (DMD) is caused by a dystrophin gene mutation. Transplantation of normal myoblasts results in long-term restoration of dystrophin. However, the success of this approach is compromised by the limited time of regeneration following muscle damage. Myostatin is known to be responsible for limiting skeletal muscle regeneration. Our purpose is to verify whether blocking the myostatin signal in mdx host mice or in normal myoblasts transplanted in mdx host mice would increase the extent of muscle repair and thus allow the formation of more dystrophin-positive fibers. Methods. Transgenic mdx mice carrying a dominant negative form of myostatin receptor (dnActRIIB) were used to test the fiber resistance to damage and to act as a host for normal myoblast transplantation. Myoblasts obtained from nondystrophic transgenic mice carrying the dominant negative myostatin receptor were also transplanted in nontransgenic mdx mice. Results. Transgenic mdx mice carrying the dnActRIIB gene have bigger muscles than mdx mice with the normal gene of ActRIIB. Their fiber resistance to exercise-induced damage was also greatly improved. Moreover, the success of normal myoblast transplantation was significantly enhanced in mdx/dnActRIIB mice. Finally, nondystrophic dnActRIIB myoblasts formed more abundant and bigger dystrophin positive fibers when transplanted in mdx mice. Conclusions. Blocking the myostatin signal in mdx mice allowed the size of muscle fibers to increase, the fiber resistance to damage induced by exercise to increase, and the success of normal myoblast transplantation to improve. The transplantation in mdx mice of dnActRIIB myoblasts formed more abundant and larger dystrophin positive fibers.  

4.318           Different platelet activation levels in non-pregnant, normotensive pregnant, pregnancy-induced hypertensive and pre-eclamptic women. A pilot study of flow cytometric analysis

Bagamery, K. and Landau, R. Eur. J. Obstet. Gynecol. Reprod. Biol., 121, 117-123 (2005)   No abstract available  

4.319           Molecular epidemiology biomarkers – sample collection and processing considerations

Holland, N.T., Pfleger, L., Berger, E., Ho, A. and Bastaki, M. Tox. Appl. Pharmacol., 206, 261-268 (2005)   Biomarker studies require processing and storage of numerous biological samples with the goals of obtaining a large amount of information and minimizing future research costs. An efficient study design includes provisions for processing of the original samples, such as cryopreservation, DNA isolation, and preparation of specimens for exposure assessment. Use of standard, two-dimensional and nanobarcodes and customized electronic databases assure efficient management of large sample collections and tracking results of data analyses. Standard operating procedures and quality control plans help to protect sample quality and to assure validity of the biomarker data. Specific state, federal and international regulations are in place regarding research with human samples, governing areas including custody, safety of handling, and transport of human samples. Appropriate informed consent must be obtained from the study subjects prior to sample collection and confidentiality of results maintained. Finally, examples of three biorepositories of different scale (European Cancer Study, National Cancer Institute and School of Public Health Biorepository, University of California, Berkeley) are used to illustrate challenges faced by investigators and the ways to overcome them. New software and biorepository technologies are being developed by many companies that will help to bring biological banking to a new level required by molecular epidemiology of the 21st century.  

4.320           Immunological role of neuronal receptor vanilloid receptor 1 expressed on dendritic cells

Basu, S. and Srivastava, P. PNAS, 102(14), 5120-5125 (2005)   Capsaicin (CP), the pungent component of chili pepper, acts on sensory neurons to convey the sensation of pain. The CP receptor, vanilloid receptor 1 (VR1), has been shown to be highly expressed by nociceptive neurons in dorsal root and trigeminal ganglia. We demonstrate here that the dendritic cell (DC), a key cell type of the vertebrate immune system, expresses VR1. Engagement of VR1 on immature DCs such as by treatment with CP leads to maturation of DCs as measured by up-regulation of antigen-presenting and costimulatory molecules. This effect is present in DCs of VR1^+/+ but not VR1^–/– mice. In VR1^+/+ mice, this effect is inhibited by the VR1 antagonist capsazepine. Further, intradermal administration of CP leads to migration of DCs to the draining lymph nodes in VR1^+/+ but not VR1^–/– mice. These data demonstrate a powerful influence of a neuroactive ligand on a central aspect of immune function and a commonality of mechanistic pathways between neural and immune functions.  

4.321           Debrin E2 is differentially expressed and phosphorylated in paretal cells in the gastric mucosa

Chew, C.S., Okamoto, C.T., Chen, X. and Thomas, R. Am. J. Physiol., 289, G320-G331 (2005)   Developmentally regulated brain proteins (drebrins) are highly expressed in brain where they may regulate actin filament formation in dendritic spines. Recently, the drebrin E2 isoform was detected in certain epithelial cell types including the gastric parietal cell. In gastric parietal cells, activation of HCl secretion is correlated with actin filament formation and elongation within intracellular canaliculi, which are the sites of acid secretion. The aim of this study was to define the pattern of drebrin expression in gland units in the intact rabbit oxyntic gastric mucosa and to initiate approaches to define the functions of this protein in parietal cells. Drebrin E2 expression was limited entirely or almost entirely to parietal cells and depended upon the localization of parietal cells along the gland axis. Rabbit drebrin E2 was cloned and found to share 86% identity with human drebrin 1a and to possess a number of cross-species conserved protein-protein interaction and phosphorylation consensus sites. Two-dimensional Western blot and phosphoaffinity column analyses confirmed that drebrin is phosphorylated in parietal cells, and several candidate phosphorylation sites were identified by mass spectrometry. Overexpression of epitope-tagged drebrin E2 led to the formation of microspikes and F-actin-rich ring-like structures in cultured parietal cells and suppressed cAMP-dependent acid secretory responses. In Madin-Darby canine kidney cells, coexpression of epitope-tagged drebrin and the Rho family GTPase Cdc42, which induces filopodial extension, produced an additive increase in the length of microspike projections. Coexpression of dominant negative Cdc42 with drebrin E2 did not prevent drebrin-induced microspike formation. These findings suggest that 1) drebrin can induce the formation of F-actin-rich membrane projections by Cdc42-dependent and -independent mechanisms; and that 2) drebrin plays an active role in directing the secretagogue-dependent formation of F-actin-rich filaments on the parietal cell canalicular membrane. Finally, the differential distribution of drebrin in parietal cells along the gland axis suggests that drebrin E2 may be an important marker of parietal cell differentiation and functionality.  

4.322           Mitochondrial nitric oxide mediates decreased vulnerability of hippocampal neurons from immature animals to NMDA

Marks, J.D., Boriboun, C. and Wang, J.

1. Neurosci., 25(28), 6561-6575 (2005)

  Mitochondrial membrane potential ( _m)-dependent Ca²⁺ uptake plays a central role in neurodegeneration after NMDA receptor activation. NMDA-induced _m dissipation increases during postnatal development, coincident with increasing vulnerability to NMDA. NMDA receptor activation also produces nitric oxide (NO), which can inhibit mitochondrial respiration, dissipating _m. Because _m dissipation reduces mitochondrial Ca²⁺ uptake, we hypothesized that NO mediates the NMDA-induced _m dissipation in immature neurons, underlying their decreased vulnerability to excitotoxicity. Using hippocampal neurons cultured from 5- and 19-d-old rats, we measured NMDA-induced changes in [Ca²⁺]_cytosol, _m, NO, and [Ca²⁺]_mito. In postnatal day 5 (P5) neurons, NMDA mildly dissipated _m in a NO synthase (NOS)-dependent manner and increased NO. The NMDA-induced NO increase was abolished with carbonyl cyanide 4-(trifluoromethoxy)phenyl-hydrazone and regulated by [Ca²⁺]_mito. Mitochondrial Ca²⁺ uptake inhibition prevented the NO increase, whereas inhibition of mitochondrial Ca²⁺ extrusion increased it. Consistent with this mitochondrial regulation, NOS and cytochrome oxidase immunoreactivity demonstrated mitochondrial localization of NOS. Furthermore, NOS blockade increased mitochondrial Ca²⁺ uptake during NMDA. Finally, at physiologic O₂ tensions (3% O₂), NMDA had little effect on survival of P5 neurons, but NOS blockade during NMDA markedly worsened survival, demonstrating marked neuroprotection by mitochondrial NO. In P19 neurons, NMDA dissipated _m in an NO-insensitive manner. NMDA-induced NO production was not regulated by _m, and NOS immunoreactivity was cytosolic, without mitochondrial localization. NOS blockade also protected P19 neurons from NMDA. These data demonstrate that mitochondrial NOS mediates much of the decreased vulnerability to NMDA in immature hippocampal neurons and that cytosolic NOS contributes to NMDA toxicity in mature neurons.  

4.323           Novel sulfasalazine analogues with enhanced NF-kB inhibitory and apoptosis promoting activity

Habens, F. Et al Apoptosis, 10, 481-491 (2005)   The NF-kB transcription factor plays a key role in the regulation of apoptosis by modulating expression of a wide range of cell death control molecules. NF-kB also plays an important role in human diseases by promoting inappropriate cell survival. Small molecule inhibitors of NF-kB are therefore likely to provide novel therapeutic opportunities. Sulfasalazine (SFZ) is a synthetic anti-inflammatory comprising an aminosalicylate, 5-amino salicylic acid (5-ASA), linked to an antibiotic, sulfapyridine (SPY). SFZ, but not 5-ASA or SPY, inhibits activation of NF-kB. We synthesised a small number of SFZ analogues and determined their ability to inhibit NF-kB activity and promote apoptosis in chronic lymphocytic leukaemia and hepatic stellate cells, where NF-kB plays an important role in cell survival. Remarkably, 3 of the 6 analogues synthesised were significantly more effective (up to 8-fold) inhibitors of NF-kB dependent transcription and this increased activity was associated with enhanced apoptosis. Therefore, it is possible to readily improve the NF-kB inhibiting activity of SFZ and analogues of SFZ may be attractive therapeutic agents for malignancies and chronic liver disease where NF-kB is thought to play a significant role.  

4.324           Blockade of B7-H1 on macrophages suppresses CD4⁺ T cell proliferation by augmenting IFN-g-induced nitric oxide production

Yamazaki, T. et al

1. Immunol., 175, 1586-1592 (2005)

  PD-1 is an immunoinhibitory receptor that belongs to the CD28/CTLA-4 family. B7-H1 (PD-L1) and B7-DC (PD-L2), which belong to the B7 family, have been identified as ligands for PD-1. Paradoxically, it has been reported that both B7-H1 and B7-DC costimulate or inhibit T cell proliferation and cytokine production. To determine the role of B7-H1 and B7-DC in T cell-APC interactions, we examined the contribution of B7-H1 and B7-DC to CD4⁺ T cell activation by B cells, dendritic cells, and macrophages using anti-B7-H1, anti-B7-DC, and anti-PD-1 blocking mAbs. Anti-B7-H1 mAb and its Fab markedly inhibited the proliferation of anti-CD3-stimulated naive CD4⁺ T cells, but enhanced IL-2 and IFN- production in the presence of macrophages. The inhibition of T cell proliferation by anti-B7-H1 mAb was abolished by neutralizing anti-IFN- mAb. Coculture of CD4⁺ T cells and macrophages from IFN- -deficient or wild-type mice showed that CD4⁺ T cell-derived IFN- was mainly responsible for the inhibition of CD4⁺ T cell proliferation. Anti-B7-H1 mAb induced IFN- -mediated production of NO by macrophages, and inducible NO synthase inhibitors abrogated the inhibition of CD4⁺ T cell proliferation by anti-B7-H1 mAb. These results indicated that the inhibition of T cell proliferation by anti-B7-H1 mAb was due to enhanced IFN- production, which augmented NO production by macrophages, suggesting a critical role for B7-H1 on macrophages in regulating IFN- production by naive CD4⁺ T cells and, hence, NO production by macrophages.  

4.325           TLR-4 regulates CD8⁺ T cell trapping in the liver

John, B. and Crispe, I.N.

1. Immunol., 175, 1643-1650 (2005)

  Mammalian TLRs are understood primarily as an activating system for innate and adaptive immunity, but have also been implicated in sensing cellular damage and in promoting intestinal integrity. In this study we show that TLR-4 also controls the in vivo distribution of activated CD8⁺ T cells. The liver is a site for trapping and apoptosis of activated CD8⁺ T cells during systemic immune responses, but the reason for this is unknown. In this study we tested the hypothesis that the liver’s constant exposure to endotoxin, derived from commensal bacteria in the gut, acts via TLR-4 to promote activated T cell adhesion. In the absence of TLR-4, the liver was compromised in its ability to sequester activated CD8⁺ T cells, and there was an inverse correlation between the frequency of activated CD8⁺ T cells trapped in the liver and their frequency in the circulating pool. Thus, in the absence of any inflammation, TLR-4 ligands play a significant role in the ability of the liver to trap activated CD8⁺ T cells. This provides a new perspective on the regulation of immune responses by TLR-4 under basal conditions.  

4.326           Inhibition of inhibitor of kB kinases stimulates hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis

Oakley, F. et al Gastroenterol., 128, 108-120 (2005)   Background & Aims: Resolution of liver fibrosis is associated with clearance of hepatic myofibroblasts by apoptosis; development of strategies that promote this process in a selective way is therefore important. The aim of this study was to determine whether the inhibitor of κB kinase suppresser sulfasalazine stimulates hepatic myofibroblast apoptosis and recovery from fibrosis. Methods: Hepatic myofibroblasts were generated by culture activation of rat and human hepatic stellate cells. Fibrosis was established in rat livers by chronic injury with carbon tetrachloride followed by recovery with or without sulfasalazine (150 mg/kg) treatment. Results: Treatment of hepatic stellate cells with sulfasalazine (0.5–2.0 mmol/L) induced apoptosis of activated rat and human hepatic stellate cells. A single in vivo administration of sulfasalazine promoted accelerated recovery from fibrosis as assessed by improved fibrosis score, selective clearance of smooth muscle α-actin-positive myofibroblasts, reduced hepatic procollagen I and tissue inhibitor of metalloproteinase 1 messenger RNA expression, and increased matrix metalloproteinase 2 activity. Mechanistic studies showed that sulfasalazine selectively blocks nuclear factor-κB-dependent gene transcription, inhibits hepatic stellate cell expression of Gadd45β, stimulates phosphorylation of Jun N-terminal kinase 2, and promotes apoptosis by a mechanism that is prevented by the Jun N-terminal kinase inhibitor SP600125. As further evidence for a survival role for the inhibitor of κB kinase/nuclear factor-κB pathway in activated hepatic stellate cells, a highly selective cell-permeable peptide inhibitor of κB kinase activation also stimulated hepatic stellate cell apoptosis via a Jun N-terminal kinase-dependent mechanism. Conclusions: Inhibition of the inhibitor of κB kinase/nuclear factor-κB pathway is sufficient to increase the rate at which activated hepatic stellate cells undergo apoptosis both in vitro and in vivo, and drugs that selectively target inhibitor of κB kinase have potential as antifibrotics.  

4.327           Dendritic cells acquire tolerogenic properties at the site of sterile granulomatous inflammation

Vasilijic, S. et al Cell. Immunol., 233, 148-157 (2005)   Subcutaneous implantation of polyvinyl sponges represents a suitable model for studying the mechanisms of acute and chronic inflammation, granulomatous foreign-body reaction, as well as wound healing. Using such a model in rats, we studied the phenotypic and functional characteristics of dendritic cells (DC). DC were purified from the sponge exudate using a combination of separation gradients, adherence to plastics, and immunomagnetic sorting. We have shown that the number of DC progressively increased in the sponges, reaching maximal values at day 10 after implantation, followed by their decrease thereafter. Inflammatory DC expressed MHC class II molecules and myeloid markers CD11b, CD11c, and CD68. A subset of DC expressed CD4, R-MC46, DEC-205, R-MC17, and CCR1. Compared to DC isolated in the early phase of inflammation (day 6 DC), DC in the late stage of inflammation (day 14 DC) had a lower capability to stimulate the proliferation of allogeneic lymphocytes and CD4⁺ T cells. This finding correlated with the downregulation of CD80, CD86, and CD54 expression and the increased proportion of plasmacytoid MHC class II⁺ His 24⁺ His 48⁺ DC. The suppression of allogeneic lymphocyte proliferation was abrogated by the treatment of DC with lipopolysaccharide. In addition, day 14 DC exerted tolerogenic capability in co-culture with allogenic CD4⁺ T cells. These results correlated with the increased levels of IL-10 and TGF-β in culture supernatants and the sponge exudate.  

4.328           Characteristics of poly-L-ornithine-coated alginate microcapsules

Darrabie, M.D., Kendall, W.F. and Opara, E.C. Biomaterials, 26, 6846-6852 (2005)   Poly-l-Lysine (PLL) is the most widely used biomaterial for providing perm-selectivity in alginate microcapsules for islet transplantation. We had previously reported that Poly-l-Ornithine (PLO) is less immunogenic than PLL, and in the present study, we have compared the physical characteristics of PLO- and PLL-coated hollow alginate microcapsules. Microspheres made with 1.5% alginate were divided into 2 groups that were first coated with either 0.1% PLO or PLL, followed by a second coating with 0.25% alginate. After liquefaction of the inner alginate core with sodium citrate, the microcapsules were washed with saline and used for experiments. Pore size exclusion studies were performed with FITC-labeled lectins incubated with encapsulated pig islets followed by examination for fluorescence activity. Mechanical strength was assessed by an osmotic pressure test and by 36 h of mechanical agitation of microcapsules with inert soda lime beads. The pore size exclusion limit of microcapsules after 20 min of coating was significantly smaller with PLO. While the mean±SEM diameter of PLL-coated microcapsules increased from 718±17 to 821±17 μm (p<0.05) during 14 days incubation at 37 °C, the PLO group did not change in size. Also, PLL group had a higher percentage of broken capsules (52.7±4.9%) compared to 3.1±2.05% for PLO capsules (p<0.0001,n=6). We conclude that PLO-coated alginate microcapsules are mechanically stronger and provide better perm-selectivity than PLL-coated microcapsules.  

4.329           Microglia Kv1.3 channels contribute to their ability to kill neurons

Fordyce, C.B., Jagasia, R., Zhu, X. and Schlichter, L.C.

1. Neurosci., 25(31), 7139-7149 (2005)

  Many CNS disorders involve an inflammatory response that is orchestrated by cells of the innate immune system: macrophages, neutrophils, and microglia (the endogenous CNS immune cell). Hence, there is considerable interest in anti-inflammatory strategies that target these cells. Microglia express Kv1.3 (KCNA3) channels, which we showed previously are important for their proliferation and the NADPH-mediated respiratory burst. Here, we demonstrate the potential for targeting Kv1.3 channels to control CNS inflammation. Rat microglia express Kv1.2, Kv1.3, and Kv1.5 transcripts and protein, but only a Kv1.3 current was detected. When microglia were activated with lipopolysaccharide or a phorbol ester, only the Kv1.3 transcript (but not protein) expression changed. Using a Transwell cell-culture system that allows separate drug treatment of microglia or neurons, we found that activated microglia killed postnatal hippocampal neurons through a process that requires Kv1.3 channel activity in microglia but not in neurons. A major neurotoxic molecule in this model was peroxynitrite, which is formed from superoxide and nitric oxide; thus, it is significant that Kv1.3 channel blockers reduced the respiratory burst, but not nitric oxide production, by the activated microglia. In addressing the biochemical pathway affected by Kv1.3 channel activity, we found that Kv1.3 acts via a different cellular mechanism from the broad-spectrum drug minocycline, which is often used in animal models of neuroinflammation. That is, the dose-dependent reduction in neuron killing by minocycline corresponded with a reduction in p38 mitogen-activated protein kinase activation in microglia; however, none of the Kv1.3 blockers affected p38 activation.  

4.330           Agrin promotes synaptic differentiation by counteracting an inhibitory effect of neurotransmitter

Misgeld, T., Kummer, T.T., Lichtman, J.W. and Sanes, J.R. PNAS, 102(31), 11088-11093 (2005)   Synaptic organizing molecules and neurotransmission regulate synapse development. Here, we use the skeletal neuromuscular junction to assess the interdependence of effects evoked by an essential synaptic organizing protein, agrin, and the neuromuscular transmitter, acetylcholine (ACh). Mice lacking agrin fail to maintain neuromuscular junctions, whereas neuromuscular synapses differentiate extensively in the absence of ACh. We now demonstrate that agrin's action in vivo depends critically on cholinergic neurotransmission. Using double-mutant mice, we show that synapses do form in the absence of agrin provided that ACh is also absent. We provide evidence that ACh destabilizes nascent postsynaptic sites, and that one major physiological role of agrin is to counteract this "antisynaptogenic" influence. Similar interactions between neurotransmitters and synaptic organizing molecules may operate at synapses in the central nervous system.  

4.331           Ephrin-B3 is a myelin-based inhibitor of neurite outgrowth

Benson, M.D. et al PNAS, 102(30), 10694-10699 (2005)   The inability of CNS axons to regenerate after traumatic spinal cord injury is due, in part, to the inhibitory effects of myelin. The three major previously identified constituents of this activity (Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein) were isolated based on their potent inhibition of axon outgrowth in vitro. All three myelin components transduce their inhibitory signals through the same Nogo receptor/p75 neurotrophin receptor/LINGO-1 (NgR1/p75/LINGO-1) complex. In this study, we considered that molecules known to act as repellants in vertebrate embryonic axonal pathfinding may also inhibit regeneration. In mice, ephrin-B3 functions during development as a midline repellant for axons of the corticospinal tract. We therefore investigated whether this repellant was expressed in the adult spinal cord and retained inhibitory activity. We demonstrate that ephrin-B3 is expressed in postnatal myelinating oligodendrocytes and, by using primary CNS neurons, show that ephrin-B3 accounts for an inhibitory activity equivalent to that of the other three myelin-based inhibitors, acting through p75, combined. Our data describe a known vertebrate axon guidance molecule as a myelin-based inhibitor of neurite outgrowth.  

4.332           Production and characterization of monoclonal antibodies against Enterocytozoon bieneusi purified from rhesus macaques

Zhang, Q. et al Infect. Immun., 73(8), 5166-5172 (2005)   Enterocytozoon bieneusi spores derived from rhesus macaque feces were purified by serial salt-Percoll-sucrose-iodixanol centrifugation, resulting in two bands with different specific densities of 95.6% and 99.5% purity and with a recovery efficiency of 10.8%. An ultrastructural examination revealed typical E. bieneusi spores. Twenty-six stable hybridomas were derived from BALB/c mice immunized with spores and were cloned twice by limiting dilution or growth on semisolid medium. Four monoclonal antibodies (MAbs), reacting exclusively with spores, were further characterized. These MAbs specifically reacted with spores present in stools of humans and macaques, as visualized by immunofluorescence, and with spore walls, as visualized by immunoelectron microscopy. A blocking enzyme-linked immunosorbent assay and Western blotting revealed that the epitope recognized by 8E2 was different from those recognized by 7G2, 7H2, and 12G8, which identified the same 40-kDa protein. These MAbs will be valuable tools for diagnostics, for epidemiological investigations, for host-pathogen interaction studies, and for comparative genomics and proteomics.  

4.333           Production of pro- and anti-fibrotic agents by rat kupffer cells; the effect of octreotide

Xidakis, C. et al Digest. Dis. Sci., 50(5), 935-941 (2005)   Kupffer cells may be involved in liver fibrogenesis through production of TGF-1. Their role in fibrinolysis is less clear. Octreotide, a synthetic analogue of somatostatin, is often used in cirrhotic patients. Its effect on Kupffer cells was studied. Isolated rat Kupffer cells were cultured in the presence of lipopolysaccharide and/or octreotide. TGF-1, leptin, collagenase (MMP-1), and urokinase-type plasminogen activator (uPA) were assessed in supernatants by ELISA, and MMP-2 and MMP-9 by zymography. Kupffer cells produced large amounts of MMP-1 and lipopolysaccharide induced a significant (P < 0.02) early increase. Octreotide and lipopolysaccharide caused a synergistic effect on MMP-1 secretion. By contrast, MMP-9 production stimulated by lipopolysaccharide was suppressed by octreotide. Kupffer cells produced a basal amount of uPA, significantly increased after lipopolysaccharide or octreotide incubation (P < 0.001). Large amounts of TGF-1 were produced in a time-dependent manner by unstimulated Kupffer cells. Lipopolysaccharide and octreotide, alone or in combination, induced a significant inhibition of this production (P < 0.01). Kupffer cells did not produce leptin, a recently identified mediator of liver fibrosis, or MMP-2. Kupffer cells may play a significant role in liver fibrinolysis. Octreotide, acting on TGF-1, uPA, and MMP-1 production, may be a useful agent for fibrosis resolution.  

4.334           Bone marrow is a major reservoir and site of recruitment for central memory CD8⁺ T cells

Mazo, I.B: et al Immunity, 22, 259-270 (2005)   Normal bone marrow (BM) contains T cells whose function and origin are poorly understood. We observed that CD8⁺ T cells in BM consist chiefly of CCR7⁺ L-selectin⁺ central memory cells (T_CMs). Adoptively transferred T_CMs accumulated more efficiently in the BM than naive and effector T cells. Intravital microscopy (IVM) showed that T_CMs roll efficiently in BM microvessels via L-, P-, and E-selectin, whereas firm arrest required the VCAM-1/α4β1 pathway. α4β1 integrin activation did not depend on pertussis toxin (PTX)-sensitive Gαi proteins but was reduced by anti-CXCL12. In contrast, T_CM diapedesis did not require CXCL12 but was blocked by PTX. After extravasation, T_CMs displayed agile movement within BM cavities, remained viable, and mounted potent antigen-specific recall responses for at least two months. Thus, the BM functions as a major reservoir for T_CMs by providing specific recruitment signals that act in sequence to mediate the constitutive recruitment of T_CMs from the blood.  

4.335           Pancreatic islet transplantation using non-heart-beating donors (NHBDs)

Matsumoto, S. and Tanaka, K.

1. Hepatobiliary Pancreat. Surg., 12, 227-230 (2005)

  Recent dramatic improvements in clinical islet cell transplantation demonstrated by the Edmonton group have increased the demand for this treatment, and donor shortage could become a major problem. Utilization of marginal donors could alleviate the donor shortage, and non-heart-beating donors (NHBDs) might be good resources. The University of Pennsylvania group demonstrated that it was possible to isolate islets from NHBDs, and the group actually transplanted islets from NHBDs, for the first time. The patient became insulin-independent; however, there had been no more cases using NHBDs until our group initiated islet transplantations from NHBDs in Japan. In order to utilize NHBDs effectively, we modified the standard islet isolation method. These modifications included minimizing the warm ischemic time, the use of trypsin inhibition during isolation, carrying out density measurement before purification and the use of a less toxic islet purification solution. With these modifications we were able to transplant nine of ten islet preparations from ten NHBDs (90%), into five type-1 diabetic patients. The first transplantation was performed on April 7, 2004 (the first time in Japan), and this patient became insulin-independent after the second islet transplantation (first time in Japan). All patients showed improved glycemic control and reduced insulin requirements, without hypoglycemic events. We also performed living-donor islet transplantation, with our modified islet isolation protocol, on January 19, 2005. The improved islet isolation protocol enabled us to perform effective islet transplantations from NHBDs, and it also enabled us to perform the living-donor islet transplantation.  

4.336           Multiple isoforms of the KC1 cotransporter are expressed in sickle and normal erythroid cells

Crable, S.C. et al Exp. Hematol., 33, 624-631 (2005)   Objective The KCl cotransporter (KCC) plays an important role in cellular cation and volume regulation and contributes to the process of volume reduction that accompanies reticulocyte maturation. In human red cells containing sickle hemoglobin, KCl cotransporter activity is high compared to normal cells, and contributes to the deleterious dehydration of sickle reticulocytes. To date, genes for four KCC isoforms have been identified. As a step toward determining which isoform(s) is responsible for the Cl-dependent K fluxes in reticulocytes, human erythroid cells were examined for the presence of various KCC isoform transcripts. Methods In vitro differentiated erythroid precursors, and reticulocytes isolated from normal individuals and sickle patients, were examined by reverse-transcriptase PCR for the expression of KCC isoforms. Transient transfection experiments were subsequently performed to characterize a novel KCC1 promoter. Results Expression of multiple isoforms was detected, with transcripts for KCC1, 3, and 4 detected in all samples of erythroid cells. Two N-terminal splicing variants were detected for both KCC1 and 3. Sickle hemoglobin containing reticulocytes demonstrated KCC isoform expression patterns similar to wild-type cells, except for a consistent difference in the relative abundance of one KCC1 splice variant. This N-terminal variant initiates from a newly described promoter in the KCC1 gene. Conclusion Three KCC genes are expressed in human red cells. Splicing variants arising from the KCC1 and 3 genes are also evident. Structure/function studies of mouse KCC1 suggest that these natural variants could profoundly affect overall cotransporter activity in the red cell.  

4.337           Decreases in phosphoinositide-3-kinase/Akt and extracellular signal-regulated kinase ½ signaling activate components of spinal motoneuron death

Newbern, J., Taylor, A., Robinson, M., Li, L. and Milligan, C.E.

1. Neurochem., 94, 1652-1665 (2005)

  Motoneuron dependence on target-derived trophic factors during development is well established, with loss of trophic support leading to the death of these cells. A complete understanding of the intracellular signal transduction machinery associated with extracellular survival signals requires the examination of individual pathways in various cellular and environmental contexts. In cells deprived of trophic support, and hence compromised for survival, phosphoinositide-3-kinase (PI3K) is decreased when compared with healthy cells supplied with trophic support. Extracellular signal-regulated kinase 1/2 (ERK1/2) signaling is dramatically decreased in deprived cells. We have examined the role of these two pathways to understand how changes in their activity regulate motoneuron survival and death. Pharmacological inhibition of PI3K attenuated motoneuron survival and was important in the regulation of Bcl-2 serine phosphorylation, limited release of cytochrome c into the cytoplasm and caspase activation. Bax translocation from cytoplasm to mitochondria was not altered when PI3K was inhibited. High levels of ERK1/2 inhibition robustly attenuated motoneuron survival in cells supplied with trophic support, whereas moderate inhibition of ERK1/2 activation had little effect. ERK1/2 inhibition in these cells decreased Bcl-2 phosphorylation and resulted in release of cytochrome c from the mitochondria. Bax translocation and caspase activation were not affected by ERK1/2 inhibition. These data reveal that changes in PI3K and ERK1/2 signaling lead to individual and overlapping effects on the cell-death machinery. Characterizing the role of these pathways is critical for a fundamental understanding of the development and degeneration of specific neuronal populations.  

4.338           Long-term preservation of high endocytic activity in primary cultures of pig liver sinusoidal endothelial cells

Elvevold, K., Nedredal, G.I., Revhaug, A., Bertheussen, K. And Smedsrød, B. Eur. J. Cell Biol., 84(9), 749-764 (2005)   Together with Kupffer cells, liver sinusoidal endothelial cells (LSECs) constitute the most powerful scavenger system in the body. However, studies on LSEC function are hampered by the fact that the cells lose their scavenger ability and start deteriorating after a few days in culture. The purpose of the present study was to improve the conditions of cultivation to prolong the survival of pig LSECs in vitro. We used the high capacity receptor-mediated endocytosis of soluble waste molecules as a marker for functionally intact cells in the cultures. Compared with two commercially-, and two other media specifically designed for use with either SECs or hepatocytes from rat, our newly developed serum-free medium, DM 110/SS, devoid of any components of animal origin, was superior in maintaining the endocytic activity. Of six growth factors studied for their effect on endocytosis, basic fibroblast, and recombinant epidermal, but not vascular endothelial growth factor, were found to be most beneficial. After 8 days in DM 110/SS, LSECs maintained endocytosis via the scavenger receptor, mannose receptor, collagen α-chain receptor and the Fc-γ receptor. All endocytosed ligands, except for aggregated IgG were degraded in 8-day-old cultures. Using the new medium, the cells endocytosed ligands for up to 20 days, and survived for at least an additional 10 days, albeit without the high endocytic activity typical of intact LSECs. Importantly, DNA synthesis in prolonged cultures of LSECs was observed only when maintained in DM 110/SS medium. In conclusion, we describe a protocol for the maintenance of LSECs in culture for the longest period yet reported.  

4.339           GluR2 deficiency accelerates motor neuron degeneration in a mouse model of amyothropic lateral sclerosis

Van Damme, P., Braeken, D., Callewaert, G., Robberecht, W. and Van Den Bosch, L.

1. Neuropathol. Exp. Neurol., 64(7), 605-612 (2005)

  AMPA receptor-mediated excitotoxicity has been implicated in the selective degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). Motor neurons in vitro are particularly vulnerable to excessive AMPA receptor stimulation and one of the factors underlying this selective vulnerability is the presence of a large proportion of Ca2+-permeable (i.e. GluR2-lacking) AMPA receptors. However, the precise role of GluR2-lacking AMPA receptors in motor neuron degeneration remains to be defined. We therefore studied the impact of GluR2 deficiency on motor neuron death in vitro and in vivo. Cultured motor neurons from GluR2-deficient embryos displayed an increased Ca2+ influx through AMPA receptors and an increased vulnerability to AMPA receptor-mediated excitotoxicity. We deleted the GluR2 gene in mutant SOD1G93A mice by crossbreeding them with GluR2 knockout mice. GluR2 deficiency clearly accelerated the motor neuron degeneration and shortened the life span of mutant SOD1G93A mice. These findings indicate that GluR2 plays a pivotal role in the vulnerability of motor neurons in vitro and in vivo, and that therapies that limit Ca2+ entry through AMPA receptors might be beneficial in ALS patients.  

4.340           Connective tissue growth factor (CCN2) in rat pancreatic stellate cell function: integrin a₅b₁ as a novel CCN2 receptor

Gao, R. and Brigstock, D.R. Gastroenterology, 129, 1019-1030 (2005)   Background & Aims: Pancreatic stellate cells (PSCs) are proposed to play a key role in the development of pancreatic fibrosis. The aim of this study was to evaluate the production by rat activated PSCs of the fibrogenic protein, connective tissue growth factor (CCN2), and to determine the effects of CCN2 on PSC function. Methods: CCN2 production was evaluated by immunoprecipitation and promoter activity assays. Expression of integrin α₅β₁ was examined by immunoprecipitation and Western blot. Binding between CCN2 and integrin α₅β₁ was determined in cell-free systems. CCN2 was assessed for its stimulation of PSC adhesion, migration, proliferation, DNA synthesis, and collagen I synthesis. Results: CCN2 was produced by activated PSCs, and its levels were enhanced by transforming growth factor β1 treatment. CCN2 promoter activity was stimulated by transforming growth factor β1, platelet-derived growth factor, alcohol, or acetaldehyde. CCN2 stimulated integrin α₅β₁–dependent adhesion, migration, and collagen I synthesis in PSCs. Integrin α₅β₁ production by PSCs was verified by immunoprecipitation, while direct binding between integrin α₅β₁ and CCN2 was confirmed in cell-free binding assays. Cell surface heparan sulfate proteoglycans functioned as a partner of integrin α₅β₁ in regulating adhesion of PSCs to CCN2. PSC proliferation and DNA synthesis were enhanced by CCN2. Conclusions: PSCs synthesize CCN2 during activation and after stimulation by profibrogenic molecules. CCN2 regulates PSC function via cell surface integrin α₅β₁ and heparan sulfate proteoglycan receptors. These data support a role for CCN2 in PSC-mediated fibrogenesis and highlight CCN2 and its receptors as potential novel therapeutic targets.  

4.341           Developmental pluripotency of the nuclei of neurons in the cerebral cortex of juvenile mice

Osada, K. et al

1. Neurosci., 25, 8368-8374 (2005)

  Nuclei isolated from green fluorescent protein-marked neurons in the cerebral cortex of juvenile mice (14–21 d after birth) were injected into enucleated oocytes that were allowed to develop into blastocysts. Embryonic stem (ES) cell lines were established from the inner cell mass of 76 cloned blastocysts after injecting 2026 neuronal nuclei. Some ES cells were injected individually into enucleated oocytes (nuclear transfer). Other ES cells were transferred into the blastocoeles of tetraploid blastocysts (tetraploid complementation). Two-cell embryos after nuclear transfer were transferred to the oviducts of surrogate mothers. Four (1.5%) of 272 nuclear-transferred two-cell embryos developed to term, and two (0.7%) developed into fertile adults. Nineteen (1.9%) of 992 tetraploid blastocysts receiving ES cells reached term, and 10 (1.0%) developed into adults. These findings demonstrate that some of the nuclei of differentiated neurons in the cerebral cortex of juvenile mice maintain developmental pluripotency.  

4.342           Exercise-induced oxidative stress leads hemolysis in sedentary but not trained humans

Sentürk, Ü.K. et al

1. Appl. Physiol., 99, 1434-1441 (2005)

  Intravascular hemolysis is one of the most emphasized mechanisms for destruction of erythrocytes during and after physical activity. Exercise-induced oxidative stress has been proposed among the different factors for explaining exercise-induced hemolysis. The validity of oxidative stress following exhaustive cycling exercise on erythrocyte damage was investigated in sedentary and trained subjects before and after antioxidant vitamin treatment (A, C, and E) for 2 mo. Exercise induced a significant increase in thiobarbituric acid-reactive substance and protein carbonyl content levels in sedentary subjects and resulted in an increase of osmotic fragility and decrease in deformability of erythrocytes, accompanied by signs for intravascular hemolysis (increase in plasma hemoglobin concentration and decrease in haptoglobulin levels). Administration of antioxidant vitamins for 2 mo prevented exercise-induced oxidative stress (thiobarbituric acid-reactive substance, protein carbonyl content) and deleterious effects of exhaustive exercise on erythrocytes in sedentary subjects. Trained subjects' erythrocyte responses to exercise were different from those of sedentary subjects before antioxidant vitamin treatment. Osmotic fragility and deformability of erythrocytes, plasma hemoglobin concentration, and haptoglobulin levels were not changed after exercise, although the increased oxidative stress was observed in trained subjects. After antioxidant vitamin treatment, functional and structural parameters of erythrocytes were not altered in the trained group, but exercise-induced oxidative stress was prevented. Increased percentage of young erythrocyte populations was determined in trained subjects by density separation of erythrocytes. These findings suggest that the exercise-induced oxidative stress may contribute to exercise-induced hemolysis in sedentary humans.  

4.343           Helicobacter pylori outer membrane protein 18 (Hp1125) induces dendritic cell maturation and function

Rathinavelu, S., Kao, J.Y., Zavros, Y. and Merchant, J.L. Helicobacter, 10(5), 424-432 (2005)   Background. Dendritic cells (DCs) are potent antigen-presenting cells that initiate T-cell responses. A robust adaptive Th1 immune response is crucial to an adaptive (Th2) immune response necessary for vaccine-induced protective immunity against Helicobacter pylori. It has been shown that several outer membrane proteins (Omps) induce a robust antibody response. However, it is also known that the antibodies generated are not protective. Moreover there is great variation in the recognition of high molecular weight H. pylori proteins by sera from infected patients. In contrast to the high molecular weight proteins, serologic responses to small molecular weight proteins provide assessment of current infection with H. pylori and also of its eradication. Aim. The goal of the study was to analyze the activation of the immune response by a specific low molecular weight Omp that is universally expressed by all H. pylori strains. Therefore, we studied interaction of H. pylori Omp18 with DCs. Methods. Activation of murine bone marrow-derived DCs and production of cytokines by Omp18 was assessed by fluorescence-activated cell sorter (FACS) for costimulatory markers and ELISA, respectively. The ability of Omp18 stimulated DCs to induce lymphocyte proliferation was measured in a mixed leukocyte reaction. Results. Omp18 induced higher expression of the B7 (CD80 and CD86) costimulatory molecule after 18 hours indicating processing and presentation of the antigen on the surface by bone marrow-derived DCs. The maturing DCs also secreted significant levels of IL-12, but was 4-fold less than that stimulated by whole bacteria. Omp18-primed DCs induced proliferation and release of IFN by syngeneic splenocytes. Conclusion. We concluded that Omp18 is capable of activating DCs initiating a Th1 immune response.  

4.344           A novel role of hepatocyte growth factor as an immune regulator through suppressing dendritic cell function

Okunishi, K. et al

1. Immunol., 175, 4745-4753 (2005)

  Hepatocyte growth factor (HGF) plays an important role in many biological events such as angiogenesis, cell proliferation, anti-fibrosis and antiapoptosis. It is well known that HGF promotes tumor progression and suppresses development of fibrosis after tissue injury. In contrast, its role in immune-mediated disorders has not been fully clarified. In the present study, we examined the role of HGF in Ag-specific immune response using in vitro studies and an experimental model of allergic airway inflammation. We first confirmed that dendritic cells (DCs) expressed the receptor for HGF, c-met, which was not expressed in T cells. Treatment with HGF both in vitro and in vivo potently suppressed DC functions such as Ag-presenting capacity, thus down-regulating Ag-induced Th1- and Th2-type immune responses. Exogenous administration of the HGF expression plasmid into Ag-primed mice markedly suppressed the development of airway eosinophilia and airway hyperresponsiveness, which was induced by Ag inhalation, with suppression of the Ag-presenting capacity of DCs in the lung. HGF exhibited these immunosuppressive effects without up-regulation of IL-10 or TGF- . We also found that expression of endogenous HGF in the lung significantly increased following Ag sensitization and inhalation challenges. Finally, neutralization of endogenous HGF in vivo significantly increased airway eosinophilia and airway hyperresponsiveness with up-regulation of the Ag-presenting capacity of DCs in the lung. These results demonstrated a novel, significant, and possibly therapeutic role of HGF as a potent regulator in immune-mediated disorders such as asthma.  

4.345           Herpesvirus saimiri-based vector biodistribution using noninvasive optical imaging

Smith, P.G. et al Gen. Ther., 12, 1465-1476 (2005)   Herpesvirus saimiri (HVS) is capable of infecting a range of human cell types with high efficiency and the viral genome persists as high copy number, circular, nonintegrated episomes which segregate to progeny upon cell division. This allows the HVS-based vector to stably transduce a dividing cell population and provide sustained transgene expression for an extended period of time both in vitro and in vivo. Here we assess the dissemination of HVS-based vectors in vivo following intravenous and intraperitoneal administration. Bioluminescence imaging of an HVS-based vector expressing luciferase demonstrates that the virus can infect and establish a persistent latent infection in a variety of mouse tissues. Moreover, the long-term in vivo maintenance of the HVS genome as a nonintegrated circular episome provided sustained expression of luciferase over a 10-week period. A particularly high level of transgene expression in the liver and the ability of HVS to infect and persist in hepatic stellate cells suggest that HVS-based vectors may have potential for the treatment of inherited and acquired liver diseases.  

4.346           Superior efficacy of dendritic cell-tumor fusion vaccine compared with tumor lysate-pulsed dendritic cell vaccine in colon cancer

Kao, J.Y., Zhang, M., Chen, C-M. and Chen, J-J. Immunology Letters, 101(2), 154-159 (2005)   Dendritic cell (DC)-based tumor vaccine is a promising therapy for malignancies. Recent studies showed greater potency with DC/tumor fusion vaccines against acute myeloid leukemia and melanoma compared with lysate-pulsed DC vaccines. We compared these two vaccine strategies against murine colon cancer and investigated whether DC/tumor fusion cells continue to produce tumor antigens following fusion as a possible explanation for their increased potency. Using a mouse colon cancer model, CT26, we first showed that the DC/CT26 fusion vaccine is more effective in preventing tumor implantation than CT26 lysate-pulsed DC vaccine. Next, CT26 made to constitutively produce bioactive TGF-β, a surrogate of tumor-derived products, was fused to DCs and found to produce bioactive TGF-β 72 h after fusion. Our results suggest the DC/tumor fusion vaccine is more potent against colon cancer than the lysate-pulsed DC vaccine. These fusion cells have the distinct advantage of prolonged interaction with tumor antigens in vivo.  

4.347           Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells

Cavanagh, L.L. et al Nature Immunol., 6(10), 1029-1037 (2005)   Dendritic cells (DCs) carry antigen from peripheral tissues via lymphatics to lymph nodes. We report here that differentiated DCs can also travel from the periphery into the blood. Circulating DCs migrated to the spleen, liver and lung but not lymph nodes. They also homed to the bone marrow, where they were retained better than in most other tissues. Homing of DCs to the bone marrow depended on constitutively expressed vascular cell adhesion molecule 1 and endothelial selectins in bone marrow microvessels. Two-photon intravital microscopy in bone marrow cavities showed that DCs formed stable antigen-dependent contacts with bone marrow−resident central memory T cells. Moreover, using this previously unknown migratory pathway, antigen-pulsed DCs were able to trigger central memory T cell−mediated recall responses in the bone marrow.  

4.348           Defects in secretory pathway trafficking during sperm development in Adam2 knockout mice

Stein, K.K., Go, J.C., Primakoff, P. and Myles, D.G. Biol. Reprod., 73, 1032-1038 (2005)   Adam2-null and Adam3-null male mice exhibit reduced levels of one or more ADAM proteins on mature sperm, in addition to the loss of the genetically targeted protein. ADAM protein loss was believed to occur posttranslationally, although the timing of loss and the mechanism by which the loss occurred were not explored. In this study we have found that in Adam3-null mice, fertilin beta (also known as ADAM2) is lost during the formation of testicular sperm. In Adam2-null males, most cyritestin (ADAM3) protein is also lost at this stage, but 25% of cyritestin is lost later, during sperm passage through the epididymis. Although normal levels of cyritestin are synthesized and acquire Endoglycosidase H resistance, indicating transit through the Golgi, the protein does not reach the cell surface. We also discovered that the majority of both fertilin beta and cyritestin are found in a Triton X-100 insoluble compartment on testicular sperm, when most of the cyritestin was observed on the cell surface. This insoluble compartment may represent a sorting platform, because in Adam2-knockout cells, only a small fraction of the cyritestin becomes Triton X-100 insoluble. Thus, it appears that cyritestin loss in Adam2-knockout mice may result, at least in part, from a disruption in protein trafficking.  

4.349           Murine plasmacytoid dendritic cells produce IFN-g upon IL-4 stimulation

Suto, A. et al

1. Immunol., 175, 5681-5689 (2005)

  IL-4 plays a key role in inducing IL-4 production in CD4⁺ T cells, functioning as an important determinant for Th2 cell differentiation. We show here that IL-4 induces IFN- production in B220⁺ plasmacytoid dendritic cells (PDCs). By searching for cell populations that produce IFN- upon IL-4 stimulation, we found that PDCs were a major IFN- -producing cell upon IL-4 stimulation in wild-type and Rag-2^–/– splenocytes. Isolated PDCs, but not CD11b⁺ DCs or CD8⁺ DCs, produced IFN- upon IL-4 stimulation. In vivo, the depletion of PDCs by anti-Ly6G/C Ab prevented IFN- production induced by IL-4 administration. We also found that IL-4 induced IFN- production, but not IL-12 or IFN- production, in PDCs and also strongly enhanced CpG oligodeoxynucleotide-induced IFN- production, but not CpG oligodeoxynucleotide-induced IL-12 or IFN- production. However, IL-4 did not induce IFN- production in Stat6^–/– PDCs. Moreover, IL-4 induced Stat4 expression in PDCs through a Stat6-dependent mechanism, and only the Stat4-expressing PDCs produced IFN- . Furthermore, IL-4 did not induce IFN- production in Stat4^–/– PDCs. These results indicate that PDCs preferentially produce IFN- upon IL-4 stimulation by Stat6- and Stat4-dependent mechanisms.  

4.350           A dual role for TGF-b1 in the control and persistence of fungal pneumonia

Shao, X., Rivera, J., Niang, R., Casadevall, A. and Goldman, D.L.

1. Immunol., 175, 6757-6763 (2005)

  TGF- 1 (TGF) has been implicated in the pathogenesis of several chronic infections and is thought to promote microbial persistence by interfering with macrophage function. In rats with experimental pulmonary cryptococcosis, increased lung levels of TGF were present at 12 mo of infection. Within the lung, expression of TGF localized to epithelioid cells and foamy macrophages in areas of inflammation. Increased TGF expression was also observed in the lungs of experimentally infected mice and a patient with pulmonary cryptococcosis. TGF reduced Ab and serum-mediated phagocytosis of Cryptococcus neoformans by rat alveolar macrophages (AM) and peripheral blood monocytes, and this was associated with decreased chemokine production and oxidative burst. Interestingly, TGF-treated rat AM limited both intracellular and extracellular growth of C. neoformans. Control of C. neoformans growth by TGF-treated rat AM was due to increased secretion of lysozyme, a protein with potent antifungal activity. The effects of TGF on the course of infection were dependent on the timing of TGF administration relative to the time of infection. TGF treatment of chronically infected rats resulted in reduced lung fungal burden, while treatment early in the course of infection resulted in increased fungal burden. In summary, our studies suggest a dual role for TGF in persistent fungal pneumonia whereby it contributes to the local control of infection by enhancing macrophage antifungal efficacy through increased lysozyme secretion, while limiting inflammation by inhibiting macrophage/monocyte phagocytosis and reducing associated chemokine production and oxidative burst.  

4.351           Kyoto islet isolation method: the optimized one for non-heart-beating donors with highly efficient islet retrieval

Okitsu, T. et al Transplant. Proc., 37, 3391-3392 (2005)   The availability of pancreata for clinical cadaveric islet transplantation is restricted to non-heart-beating donors (NHBDs) in Japan. This forced us to modify the current standard islet isolation protocol that was made up for brain-dead donors and make it suitable for NHBDs. The Kyoto islet isolation method is the one with induction of several steps based on the ideas both already reported literally and invented originally by ourselves. Using this islet isolation method, we isolated islets from 13 human pancreata of NHBDs and transplanted 11 preparations to six type-1 diabetic patients. The rate to meet release criteria of Edmonton protocol was 84.6%. Establishment of this method allowed us to begin a clinical islet transplantation program in Japan and to continue to perform the preparation of islets from NHBDs with high rate to meet the release criteria of the Edmonton protocol.

4.352           Mullerian inhibiting substance acts as a motor neuron survival factor in vitro

Wang, P-Y. et al PNAS, 102(45), 16421-16425 (2005)   The survival of motor neurons is controlled by multiple factors that regulate different aspects of their physiology. The identification of these factors is important because of their relationship to motor neuron disease. We investigate here whether Mullerian Inhibiting Substance (MIS) is a motor neuron survival factor. We find that motor neurons from adult mice synthesize MIS and express its receptors, suggesting that mature motor neurons use MIS in an autocrine fashion or as a way to communicate with each other. MIS was observed to support the survival and differentiation of embryonic motor neurons in vitro. During development, male-specificMIS may have a hormone effect because the blood-brain barrierhas yet to form, raising the possibility that MIS participatesin generating sex-specific differences in motor neurons.        

4.353           Nitric oxide scavenging by red blood cells as a function of hematocrit and oxygenation

Azarov, I. et al

1. Biol. Chem., 280(47), 39024-39032 (2005)

  The reaction rate between nitric oxide and intraerythrocytic hemoglobin plays a major role in nitric oxide bioavailability and modulates homeostatic vascular function. It has previously been demonstrated that the encapsulation of hemoglobin in red blood cells restricts its ability to scavenge nitric oxide. This effect has been attributed to either factors intrinsic to the red blood cell such as a physical membrane barrier or factors external to the red blood cell such as the formation of an unstirred layer around the cell. We have performed measurements of the uptake rate of nitric oxide by red blood cells under oxygenated and deoxygenated conditions at different hematocrit percentages. Our studies include stopped-flow measurements where both the unstirred layer and physical barrier potentially participate, as well as competition experiments where the potential contribution of the unstirred layer is limited. We find that deoxygenated erythrocytes scavenge nitric oxide faster than oxygenated cells and that the rate of nitric oxide scavenging for oxygenated red blood cells increases as the hematocrit is raised from 15% to 50%. Our results 1) confirm the critical biological phenomenon that hemoglobin compartmentalization within the erythrocyte reduces reaction rates with nitric oxide, 2) show that extra-erythocytic diffusional barriers mediate most of this effect, and 3) provide novel evidence that an oxygen-dependent intrinsic property of the red blood cell contributes to this barrier activity, albeit to a lesser extent. These observations may have important physiological implications within the microvasculature and for pathophysiological disruption of nitric oxide homeostasis in diseases.  

4.354           Glucose stimulation of cytochrome C reduction and oxygen consumption as assessment of human islet quality

Sweet, I.R. et al Transplantation, 80(8), 1003-1011 (2005)   Background. An in vitro method to assess human islets could prevent transplantation of nonviable islets and facilitate the optimization of islet preparation. We hypothesize that glucose-stimulated cytochrome c reduction and oxygen consumption by human islets can be used as predictors of transplant success. Methods. Isolated human islets were obtained from research-grade pancreata. Using a previously developed islet flow culture system, the response of cytochrome c reduction and oxygen consumption to glucose was compared to the ability of islets transplanted into nondiabetic NOD-SCID mice to secrete C-peptide in response to a glucose tolerance test conducted 7 days following transplant (n=10). Results. In vitro responses by human islets were qualitatively similar to those seen in rat islets: glucose increased both oxygen consumption and cytochrome c reduction. However, the responses were smaller in magnitude and quite variable. Scatter plots of C-peptide and quantiles for ln(C-peptide) indicated that 12 ng/ml could be used as threshold of transplant success with which to evaluate the diagnostic potential of cytochrome c and oxygen consumption. Data was analyzed by generating receiver operating curves and the area under the curve was 0.889 (95% CI: 0.645-1.000) and 0.738 (95% CI: 0.413-1.000) for cytochrome c reuction and oxygen consumption respectively (1 indicates absolute predictive capability and 0.5 indicates no predictive capability). Conclusions. The detection of glucose-stimulated cytochrome c reduction and oxygen consumption may have utility as criteria for the assessment of human islet quality.  

4.355           Targeted expression of human CD1d in transgenic mice reveals independent roles for thymocytes and thymic APCs in positive and negative selection of Va14i NKT cells

Schäumann, J. et al

1. Immunol., 175, 7303-7310 (2005)

  CD1d-dependent invariant V 14 (V 14i) NKT cells are innate T lymphocytes expressing a conserved semi-invariant TCR, consisting, in mice, of the invariant V 14-J 18 TCR -chain paired mostly with V 8.2 and V 7. The cellular requirements for thymic positive and negative selection of V 14i NKT cells are only partially understood. Therefore, we generated transgenic mice expressing human CD1d (hCD1d) either on thymocytes, mainly CD4⁺ CD8⁺ double positive, or on APCs, the cells implicated in the selection of V 14i NKT cells. In the absence of the endogenous mouse CD1d (mCD1d), the expression of hCD1d on thymocytes, but not on APCs, was sufficient to select V 14i NKT cells that proved functional when activated ex vivo with the Ag -galactosyl ceramide. V 14i NKT cells selected by hCD1d on thymocytes, however, attained lower numbers than in control mice and expressed essentially V 8.2. The low number of V 8.2⁺ V 14i NKT cells selected by hCD1d on thymocytes was not reversed by the concomitant expression of mCD1d, which, instead, restored the development of V 7⁺ V 14i NKT cells. V 8.2⁺, but not V 7⁺, NKT cell development was impaired in mice expressing both hCD1d on APCs and mCD1d. Taken together, our data reveal that selective CD1d expression by thymocytes is sufficient for positive selection of functional V 14i NKT cells and that both thymocytes and APCs may independently mediate negative selection.  

4.356           CCL5-CCR5 interaction provides antiapoptotic signals for macrophage survival viral infection

Tyner, J.W. et al Nature Med., 11(11), 1180-1187 (2005)   Host defense against viruses probably depends on targeted death of infected host cells and then clearance of cellular corpses by macrophages. For this process to be effective, the macrophage must presumably avoid its own virus-induced death. Here we identify one such mechanism. We show that mice lacking the chemokine Ccl5 are immune compromised to the point of delayed viral clearance, excessive airway inflammation and respiratory death after mouse parainfluenza or human influenza virus infection. Virus-inducible levels of Ccl5 are required to prevent apoptosis of virus-infected mouse macrophages in vivo and mouse and human macrophages ex vivo. The protective effect of Ccl5 requires activation of the Ccr5 chemokine receptor and consequent bilateral activation of G _i-PI3K-AKT and G _i-MEK-ERK signaling pathways. The antiapoptotic action of chemokine signaling may therefore allow scavengers to finally stop the host cell-to-cell infectious process.  

4.357           Nongenotoxic activation of the p53 pathway as a therapeutic strategy for multiple myeloma

Stühmer, T. et al Blood, 106(10), 3609-3617 (2005)   Mutation of p53 is a rare event in multiple myeloma, but it is unknown if p53 signaling is functional in myeloma cells, and if targeted nongenotoxic activation of the p53 pathway is sufficient to kill tumor cells. Here, we demonstrate that treatment of primary tumor samples with a small-molecule inhibitor of the p53–murine double minute 2 (MDM2) interaction increases the level of p53 and induces p53 targets and apoptotic cell death. Significantly, given the importance of the bone marrow microenvironment for the support and drug resistance of myeloma cells, tumor cells undergo effective apoptosis also in the presence of stromal cells, which themselves appear to tolerate exposure to nutlin-3. The in vitro toxicity of nutlin-3 was similar to that of the genotoxic drug melphalan. Because nutlin-mediated p53 activation is not dependent on DNA damage, MDM2 antagonists may help to avoid or reduce the severe genotoxic side effects of chemotherapeutic agents currently used to treat multiple myeloma. Therefore, MDM2 antagonists may offer a new treatment option for this disease.  

4.358           Elevated plasma arginase levels in hemoglobinopathies

Larkin, S.K., Morris, C.R., Styles, L.A. and kuypers, F.A. Blood, 106(11), abstract 2346 (2005)   The decreased bioavailability of arginine (Arg) and the resultinglower nitric oxide (NO) production has been shown to be an importantfactor in the pathology of sickle cell disease (SCD) and thalassemia.Vascular alterations leading to pulmonary hypertension are importantfactors of heart failure and death in these hemoglobinopathies.Red blood cell (RBC) hemolysis in these patients will releasearginase into the circulation and contribute to the reductionof plasma Arg levels, change the Arg-to-ornithine ratio, andincrease other downstream amino acid metabolites. Such compounds,including proline and polyamines, may contribute to vascularand airway remodeling. To study the contribution of arginasereleased from RBC, we measured the arginase activity and arginaseprotein concentration in the plasma and RBC lysates of normalcontrols, SCD patients, and thalassemia patients. Arginase activity was determined by the conversion of ¹⁴C-labeled guanidine-L-arginine to ¹⁴C-labeled urea, then to ¹⁴CO₂ by urease and trapped as Na₂ ¹⁴CO₃ for scintillation counting. Arginase and hemoglobin concentration were measured by ELISA (BioVendor, Candler, NC and Bethyl Labs Inc., Montgomery, TX respectively). Mean cellular hemoglobin concentration was measured with a Coulter Counter (Beckman Coulter, Fullerton, CA) or Technicon H3 analyzer (Tarrytown, NY). RBC were separated by density gradient centrifugation with OptiPrep (Axis Shield, Dundee, UK). Arginase specific activity(SA) was calculated by dividing the activity by the concentrationto yield mole of Arg metabolized per g of arginase per hour.We found a higher level of arginase in the plasma and RBC ofSCD and thalassemia patients as compared to normal controls.These levels correlated with cell-free hemoglobin content (plasma)or reticulocyte count (RBC). The light density fraction (<1.077g/ml) of RBC, enriched in reticulocytes, showed higher arginaselevels confirming increased arginase levels in young RBC. Interestingly,as the plasma arginase protein and activity increased with cell-freehemoglobin, the SA decreased. Similarly in RBC lysates, theSA decreases with hemoglobin concentration. This suggests thata factor exists within the RBC that negatively modulates arginaseactivity. Due to the high SA of arginase at low hemoglobin concentrations,low levels of intravascular hemolysis can generate relativelyhigh levels of Arg breakdown. We conclude that low levels of intravascular hemolysis of RBC with increased arginase content in SCD and thalassemia, play a role in the altered Arg metabolism and decreased NO production. While the reason for high levels of arginase in the RBC as well as complete clinical impact of elevated arginase activity in plasma remain to be determined, dysregulated Arg metabolism and a shift toward Arg catabolic products, may be associated with the development of pulmonary hypertension and mortality in SCD and thalassemia.  

4.359           Characterization of progesterone receptor isoform expression in fetal membrane

Mills, A., Yonish, B., Feng, L. and Murtha, A. Am. J. Obstet. Gynecol., 193(6), Suppl. 1, abstract 164, (2005)   Objective To quantify the levels of expression of progesterone receptor (PR) isoforms A and B in amnion, chorion, and decidua, and to determine whether expression changes in cell culture. Study design After IRB approval, placentas from term gestations delivered by cesarean section without labor were collected. Layers of amnion, chorion, and decidua were separated manually and then digested. Cell layers were further separated using Opti-prep (Sigma Aldrich) density gradient. Purity of cell separation was confirmed using immunocytochemistry. RNA was immediately extracted from an aliquot of cells from each cell layer and the remainder was cultured for 48 hours. RNA was then extracted for quantitative RT-PCR using primers specific for PR isoforms A and B, as well as for β-2 microglobulin (β2M), a constituitively expressed gene used to normalize quantification across tissue samples. Standard curves were generated from known concentrations of each transcript. Data were analyzed using t-test and Mann Whitney U. Results PR isoforms A and B were identified in the chorion and decidua but were below the lower limits of detection of our assay in the amnion. For both isoforms, decidual expression was higher than expression in the chorion (PR-A/β2M 0.006 vs. 0.001, p = 0.02; PR-B/β2M 0.005 vs. 0.0004, p = 0.02). Ratios of PR-A to PR-B were similar in both decidua and chorion. When fresh cells were compared to cultured cells there was no significant difference in PR expression. Conclusion PR isoforms A and B are present in chorion and decidua, with the highest concentrations found in the maternally-derived decidua. PR isoforms appear to be equally expressed in cultured and fresh cells. These findings will aid in our understanding of how progesterone contributes to the pathogenesis of preterm delivery.  

4.360           Decrease in Langerhans cells and increase in lymph node dendritic cells following chronic exposure of mice to suberythemal doses of solar simulated radiation

McLoone, P., Woods, G.M. and Norval, M. Photochem. Photobiol., 81(5), 1168-1173 (2005)   Exposure of certain strains of mice to ultraviolet radiation (UVR) causes suppression of some innate and adaptive immune responses. One such consequence of acute UVB exposure is a reduction in the number of Langerhans cells (LC) in the epidermis and an increase in dendritic cells (DC) in lymph nodes draining the irradiated skin sites. Exposure to chronic UVB irradiation also has effects on the immune system, but it is unknown what effects are caused by repeated doses of solar simulated radiation (SSR). Consequently, the main aims of the present study were to determine whether repeated exposure to low doses of SSR would lead to similar changes in these cell populations and whether chronic doses of SSR activate a protective photoadaptation mechanism. Groups of C3H/HeN mice were irradiated daily with 3.7 J/cm² SSR from Cleo Natural lamps for 2, 10, 20, 30 or 60 days. Further groups of mice received an additional dose of 7.4 J/cm² SSR on days 2, 10, 30 or 60 to test for photoadaptation. The numbers of LC in the epidermis and DC in the lymph nodes draining irradiated skin sites were counted 24 h after the final irradiation. With the exception of mice irradiated for only 2 days, LC were significantly reduced throughout the chronic irradiation protocol, and no recovery occurred. DC numbers were significantly increased in the draining lymph nodes of mice irradiated for 20 days and 60 days.  

4.361           Blocking intrahepatic deletion of activated CD8⁺ T cells by an altered peptide ligand

Kuniyasu, Y. et al Cell. Immunol., 238, 31-37 (2005)   Background Activated CD8⁺ T cells are retained by the healthy liver where the majority undergo apoptosis. The intrahepatic apoptosis of activated CD8⁺ T cells is enhanced by the presence of SIINFEKL peptide. It is of great interest to identify strategies for maintaining intrahepatic T cell number and function in the presence of SIINFEKL peptides. Aim Our aim was to test if low affinity peptides can block SIINFEKL peptide induced T cell deletion. Methods We used an in vivo model of intrahepatic CD8⁺ T cell deletion with peptides of different affinities. Results and discussion We show that the intrahepatic deletion of CD8⁺ T cells by SIINFEKL peptide results in loss of in vivo cytotoxic T lymphocyte function. In contrast we show that a low affinity peptide (G4) does not result in intrahepatic deletion of CD8⁺ T cells. High concentrations G4 peptide can however block intrahepatic deletion of activated CD8⁺ T cells, and prevent loss of in vivo cytotoxicity due to SIINFEKL peptide. This is the first demonstration of blocking of SIINFEKL peptide induced CD8⁺ T cell deletion in the liver, with enhancement of in vivo cytotoxicity.  

4.362           Improved Methods for Culturing Rat and Mouse Motor Neurons

Guettier-Sigrist, S et al New Methods for Cultering Cells from Nervous Tissues, 1, 96-108 (2005)   How synapses are formed and which molecules contribute to this process are essential, but not yet fully understood, aspects of neuronal development. However, identifying the steps of formation of neuromuscular junction would be very important to understand the physiopathology of neuromuscular disorders. Those who want to study this process in vitro need to prepare purified motor neuron suspensions, together with other cells contributing to the formation of neuromuscular junction [1]. Similarly, in order to study neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), it is essential to have these cells at our disposal. With the development of transgenic animal strains displaying such diseases [2], it is now possible to study the behavior of diseased motor neurons in culture and especially follow the biochemical events occurring in these cells [3]. It is also possible to study the effects of putative neurotrophic or neuroprotective drugs [4]. However, it should be underlined that an isolated motor neuron does not have an entire physiological or pathophysiological significance, since, in vivo, it is always connected to muscle fibers. The pertinent unit for studying more precisely motor neuron diseases or neuromuscular disorders is the motor unit, which is defined as the anatomo-functional entity consisting of a motor neuron and the set of muscle fibers innervated by the ramifications of its axon. We describe here the preparation of the rat and mouse purified motor neuron suspensions.  

4.363           Cellular Uptake of Vitamin B₁₂ in Patients with Chronic Renal Failure

Obeid, R., Kuhlmann, M., Kirsch, C-M. And Hermann, W. Nephron. Clin. Pract., 99, c42-c48 (2005)   Background/Aims: Elevated concentration of plasma homocysteine (tHcy) is common in renal patients, however, the reason behind the resistance to vitamin B₁₂ and folate therapy are poorly understood. Methods: We investigated vitamin B₁₂ uptake by mononuclear cells (MC) from predialysis patients (n = 19) as compared to healthy controls (n = 15). Serum levels of tHcy, methylmalonic acid and cystathionine, holotranscobalamin (holoTC), total vitamin B₁₂ and folate were also measured. Results: The uptake of vitamin B₁₂ by MC from renal patients was lower than that by MC from controls (9.3 vs. 12.5 pg/3 × 10⁶ cells; p = 0.001). Nonetheless, the receptor-binding capacity was comparable between patients and controls (6.1 vs. 6.5 pg/3 × 10⁶ cells; p = 0.627). Average reduction of vitamin B₁₂ uptake in patients as compared to the controls was 18.1%. Conclusions: Our results show that vitamin B₁₂ uptake is impaired in MC from renal patients, with no evidence that the surface receptor is down-regulated. High serum concentrations of holoTC are common in renal patients and might be related to a generalized resistance to this vitamin. Serum concentrations of vitamin B₁₂ within the reference range are not likely to ensure vitamin delivery into the cells. Supraphysiological doses of vitamin B₁₂ may be necessary to deliver a sufficient amount of the vitamins to the cells via mechanisms largely independent of holoTC receptor.  

4.364           Amelioration of rat adjuvant-induced arthritis by Met-RANTES

Shahrara, S., Proudfoot, A.E.I., Woods, J.M., Ruth, J.H., Amin, M.A., Park, C.C., Haas, C.S., Pope, R.M., Haines, G.K., Zha, Y.Y. and Koch, A.E. Arthritis & Rheumatism, 52(6), 1907-1919 (2005)   Objective CC chemokines and their receptors play a fundamental role in trafficking and activation of leukocytes at sites of inflammation, contributing to joint damage in rheumatoid arthritis. Met-RANTES, an amino-terminal–modified methionylated form of RANTES (CCL5), antagonizes the binding of the chemokines RANTES and macrophage inflammatory protein 1 (MIP-1 ; CCL3) to their receptors CCR1 and CCR5, respectively. The aim of this study was to investigate whether Met-RANTES could ameliorate adjuvant-induced arthritis (AIA) in the rat. Methods Using immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription–polymerase chain reaction, Western blot analysis, adoptive transfer, and chemotaxis, we defined joint inflammation, bony destruction, neutrophil and macrophage migration, Met-RANTES binding affinity to rat receptors, proinflammatory cytokine and bone marker levels, CCR1 and CCR5 expression and activation, and macrophage homing into joints with AIA. Results Administration of Met-RANTES as a preventative reduced the severity of joint inflammation. Administration of Met-RANTES to ankles with AIA showed decreases in inflammation, radiographic soft tissue swelling, and bone erosion. Met-RANTES significantly reduced the number of neutrophils and macrophages at the peak of arthritis compared with saline-injected controls. Competitive chemotaxis in peripheral blood mononuclear cells demonstrated that Met-RANTES inhibited MIP-1 and MIP-1β at 50% inhibition concentrations of 5 nM and 2 nM, respectively. Furthermore, levels of tumor necrosis factor , interleukin-1β, macrophage colony-stimulating factor, and RANKL were decreased in joints with AIA in the Met-RANTES group compared with the control group. Interestingly, the expression and activation of CCR1 and CCR5 in the joint were down-regulated in the Met-RANTES group compared with the control group. Functionally, Met-RANTES administration decreased adoptively transferred peritoneal macrophage homing into the joint. Conclusion The data suggest that the targeting of Th1-associated chemokine receptors reduce joint inflammation, bone destruction, and cell recruitment into joints with AIA.  

4.365           CD46 on glial cells can function as a receptor for viral glycoprotein-mediated cell–cell fusion

Cassiani-Ingoni, R., Greenstone, H.L., Donati, D., Fogdell-Hahn, A., Martinell, E., Refai, D., Martin, R., Berger, E.A. and Jacobson, S. GLIA, 52(3), 252-258 (2005)   Membrane cofactor protein (CD46) is a regulator of complement activation that also serves as the entry receptor for human herpes virus 6 (HHV-6) and measles virus (MV) into human cells. While it is clear that oligodendrocytes and astrocytes are cell types commonly infected by these viruses, it is unclear whether oligodendrocytes express CD46, or which are the cellular mechanisms underlying the infection. We show that adult oligodendrocytes, as well as astrocytes and microglial cells, express CD46 on the cellular surface. Moreover, we employed a quantitative fusion assay to demonstrate that HHV-6A infection of T lymphocytes enables cell–cell fusion of these cells to astrocytes or to oligodendroglial cells. This fusion is mediated by the interaction between viral glycoproteins expressed on the membrane of the infected cells and CD46 on the glial targets, and is also observed using cells expressing recombinant MV glycoproteins. These data suggest a mechanism that involves cell–cell fusion by which certain viruses could spread the infection from the periphery to the cells in the nervous system.  

4.366           Lineage-negative bone marrow cells travel bidirectionally in the olfactory migratory stream but maintain hematopoietic phenotype

Moore, B.E., Colvin, G.A., Dooner, M.S. and Quesenberry, P.J.

1. Cell. Physiol., 202(1), 147-152 (2005)

  The mammalian olfactory system is a physiologically plastic region of the brain with the potential to support implanted stem cells. We performed direct injection of lineage-negative (lin-neg), green fluorescent protein-positive (GFP+) bone marrow cells into the olfactory bulb to assess cell survival and motility within the central nervous system (CNS). Before direct injection of 100,000 lin-neg cells, some of the C57/Bl mice received 1,000 cGy brain irradiation with the aim of disabling the endogenous reservoir of periventricular neural progenitor cells. Brain harvest took place up to 2 weeks after cell implantation. Brains were evaluated for presence of GFP positivity via fluorescence microscopy. Many GFP+ cells were identified within the turbinate neuroepithelium, olfactory bulb, and frontal lobe. Most of the cells that had traveled from the implantation site adopted an elongated, arborizing morphology consistent with cellular extensions arrayed in the direction of the rostral migratory stream (RMS). No difference was seen in brain-irradiated versus non-irradiated mice. Antibody staining revealed that these cells did not take on a neural, glial, or endothelial phenotype, while largely retaining their hematopoietic lineage as demonstrated by CD45 positivity.    

4.367           rAAV-mediated stable expression of heme oxygenase-1 in stellate cells: A new approach to attenuate liver fibrosis in rats

Tsui, T-Y., Lau, C-K., Ma, J., Wu, X., Wang, Y-Q., Farkas, S., Xu, R., Schlitt, H.J. and Fan, S-T. Hepatology, 42(2), 335-342 (2005)   Liver fibrosis is the consequence of activation of hepatic stellate cells mediated by persistent or recurrent liver injury, where oxidative stress or inflammatory response resulting from immune cells and cytokines are involved. Targeting of hepatic stellate cells could be an important strategy for the therapy of liver fibrosis. In this study, we showed a tropism of recombinant adeno-associated virus (rAAV, serotype 2) with high efficiency in transduction of a homeostatic gene, heme oxygenase-1 (HO-1), to activated stellate cells. The binding of rAAVs to stellate cells increased significantly after serum-stimulated activation compared with quiescent status. Portal injection of rAAVs to normal or carbon tetrachloride (CCl4)-induced liver fibrosis showed a distinct distribution of rAAV binding. The majority of injected rAAVs bound to the cells in fibrotic areas that were associated with higher expression levels of fibroblast growth factor receptor-1 at 2 hours after administration. Isolation of different types of cells from CCl4-induced fibrotic livers showed predominant expression of transgene in stellate cells after rAAV/HO-1 administration on day 3 and remained stable for 12 weeks. In addition, HO-1–transduced stellate cells showed reduced transcript levels of type 1 collagen and impaired proliferative ability compared with controls. With this approach, the severity of established micronodular cirrhosis was markedly reduced. In conclusion, these findings suggest a new approach for the treatment of liver fibrosis using adeno-associated virus-mediated gene transfer.  

4.368           Expression of a dominant negative form of Daxx in vivo rescues motoneurons from Fas (CD95)-induced cell death

Raoul, C., Barthelemy, C., Couzinet, A., Hancock, D., Pettmann, B. and Hueber, A-O.

1. Neurobiol., 62(2), 178-188 (2005)

  Fas-induced death of motoneurons in vitro has been shown to involve two signaling cascades that act together to execute the death program: a Fas-Daxx-ASK-1-p38 kinase-nNOS branch, which controls transcriptional and post-translational events, and the second classical Fas-FADD-caspase-8 branch. To analyze the role of Daxx in the developmental motoneuron cell death, we studied Fas-dependent cell death in motoneurons from transgenic mice that overexpress a dominant-negative form of Daxx. Motoneurons purified from these transgenic mice are resistant to Fas-induced death. This protective effect is specific to Fas because ultraviolet irradiation-triggered death is not affected by the transgene. The Daxx and the FADD pathways work in parallel because only Daxx, but not FADD, is involved in the transcriptional control of neuronal nitric oxide synthase and nitric oxide production. Nevertheless, we do not observe involvement of Daxx in developmental motoneuronal cell death, as the pattern of naturally occurring programmed cell death in vivo is normal in transgenic mice overexpressing the dominant negative form of Daxx, suggesting that Daxx-independent pathways are used during development.  

4.369           CD86 molecule is a specific marker for canine monocyte-derived dendritic cells

Bonnefont-Rebeix, C. et al Vet. Immunol. Immunopathol., 109, 167-176 (2006)   In this study, canine monocyte-derived dendritic cells (cMo-DC) were produced in presence of canine GM-CSF (cGM-CSF) and canine IL-4 (cIL-4), and they were characterized by their dendritic morphology, MLR functionality and phenotype. We noticed that cMo-DC were labelled with three anti-human CD86 (FUN-1, BU63 and IT2.2 clones), whereas resting and activated lymphocytes or monocytes were not stained. CD86 expression was induced by cIL-4 and was up-regulated during the differentiation of the cMo-DC, with a maximum at day 7. Furthermore, cMo-DC were very potent even in low numbers as stimulator cells in allogeneic MLR, and BU63 mAb was able to completely block the cMo-DC-induced proliferation in MLR. We also observed that cMo-DC highly expressed MHC Class II and CD32, but we failed to determine their maturation state since the lack of commercially available canine markers. Moreover, cMo-DC contained cytoplasmic periodic microstructures, potentially new ultrastructural markers of canine DC recently described. In conclusion, this work demonstrates that the CD86 costimulatory marker is now usable for a better characterization of in vitro canine DC.  

4.370           Helminth-primed dendritic cells: alter the host response to enteric bacterial infection

Chen, C-C., Louie, S., McCormick, B.A., Walker, W.A. and Shi, H.N.

1. Immunol., 176, 472-483 (2006)

  To examine whether intestinal helminth infection may be a risk factor for enteric bacterial infection, a murine model was established using the intestinal helminth Heligomosomoides polygyrus and a murine pathogen Citrobacter rodentium, which causes infectious colitis. Using this model we recently have shown that coinfection with the Th2-inducing H. polygyrus and C. rodentium promotes bacterial-associated disease and colitis. In this study, we expand our previous observations and examine the hypothesis that dendritic cells (DC) stimulated by helminth infection may play an important role in the regulation of the intestinal immune response to concurrent C. rodentium infection as well as in the modulation of the bacterial pathogenesis. We show that H. polygyrus infection induces DC activation and IL-10 expression, and that adoptive transfer of parasite-primed DC significantly impairs host protection to C. rodentium infection, resulting in an enhanced bacterial infection and in the development of a more severe colonic injury. Furthermore, we demonstrate that adoptive transfer of parasite-primed IL-10-deficient DCs fails to result in the development of a significantly enhanced C. rodentium-mediated colitis. Similarly, when the DC IL-10 response was neutralized by anti-IL-10 mAb treatment in mice that received parasite-primed DC, no deleterious effect of the parasite-primed DC on the host intestinal response to C. rodentium was detected. Thus, our results provide evidence to indicate that the H. polygyrus-dependent modulation of the host response to concurrent C. rodentium infection involves IL-10-producing DCs.  

4.371           Antitumor efficiacy of a combination of CMC-544 (inotuzumab ozogamicin), a CD22-targeted cytotoxic immunoconjugate of calicheamicin, and rituximab against non-Hodgkin’s B-cell lymphoma

DiJoseph, J.F. et al Clin. Cancer Res., 12(1), 242-249 (2006)   Purpose: CMC-544 is a CD22-targeted cytotoxic immunoconjugate,currently being evaluated in B-cell non-Hodgkin's lymphoma (B-NHL)patients. Rituximab is a CD20-targeted antibody commonly usedin B-NHL therapy. Here, we describe antitumor efficacy of acombination of CMC-544 and rituximab against B-cell lymphoma(BCL) in preclinical models. Experimental Design: BCLs were cultured in vitro with CMC-544,rituximab, or their combination. BCLs were injected either s.c.or i.v. to establish localized s.c. BCL in nude mice or disseminatedBCL in severe combined immunodeficient mice, respectively. I.p.treatment with CMC-544 or rituximab was initiated at varioustimes either alone or in combination and its effect on s.c.BCL growth or survival of mice with disseminated BCL was monitored. Results: In vitro growth-inhibitory activity of CMC-544 combinedwith rituximab was additive. Rituximab but not CMC-544 exhibitedeffector functions, such as antibody-dependent cellular cytotoxicityand complement-dependent cytotoxicity. Rituximab was less effectivein inhibiting growth of established BCL xenografts than developingxenografts. In contrast, CMC-544 was equally effective againstboth developing and established BCL xenografts. Although CMC-544and rituximab individually caused partial inhibition of thegrowth of BCL xenografts at suboptimal doses examined, theircombination suppressed xenograft growth by >90%. In a disseminatedBCL model, 60% of CMC-544-treated mice and 20% of rituximab-treatedmice survived for 125 days. In contrast, 90% of mice treatedwith the combination of CMC-544 and rituximab survived for longerthan 125 days. Conclusion: The demonstration of superior antitumor activityof a combination of CMC-544 and rituximab described here providesthe preclinical basis for its clinical evaluation as a treatmentoption for B-NHL.  

4.372           Motor neurone targeting of IGF-1 prevents specific force decline in ageing mouse muscle

Payne, A.M. et al

1. Physiol., 570(2), 283-294 (2006)

  IGF-1 is a potent growth factor for both motor neurones and skeletal muscle. Muscle IGF-1 is known to provide target-derived trophic effects on motor neurones. Therefore, IGF-1 overexpression in muscle is effective in delaying or preventing deleterious effects of ageing in both tissues. Since age-related decline in muscle function stems partly from motor neurone loss, a tetanus toxin fragment-C (TTC) fusion protein was created to target IGF-1 to motor neurones. IGF-1–TTC retains IGF-1 activity as indicated by [³H]thymidine incorporation into L6 myoblasts. Spinal cord motor neurones effectively bound and internalized the IGF-1–TTC in vitro. Similarly, IGF-1–TTC injected into skeletal muscles was taken up and retrogradely transported to the spinal cord in vivo, a process prevented by denervation of injected muscles. Three monthly IGF-1–TTC injections into muscles of ageing mice did not increase muscle weight or muscle fibre size, but significantly increased single fibre specific force over aged controls injected with saline, IGF-1, or TTC. None of the injections changed muscle fibre type composition, but neuromuscular junction post-terminals were larger and more complex in muscle fibres injected with IGF-1–TTC, compared to the other groups, suggesting preservation of muscle fibre innervation. This work demonstrates that induced overexpression of IGF-1 in spinal cord motor neurones of ageing mice prevents muscle fibre specific force decline, a hallmark of ageing skeletal muscle.  

4.373           Differential regulation of neutrophil chemotaxis to IL-8 and fMLP by GM-CSF: lack of direct effect of oestradiol

Shen, L. et al Immunology, 117(2), 205-212 (2006)   Neutrophils are a normal constituent of the female reproductive tract and their numbers increase in the late secretory phase of the menstrual cycle prior to menses. Several cytokines are produced in female reproductive tract tissue. In particular granulocyte–macrophage colony-stimulating factor (GM-CSF), a potent activator of neutrophils, is secreted in high concentrations by female reproductive tract epithelia. We previously observed that GM-CSF synergizes strongly with interleukin-8 (IL-8) in enhancing chemotaxis of neutrophils. Thus we investigated whether pretreatment of neutrophils with GM-CSF would prime subsequent chemotaxis to IL-8 in the absence of GM-CSF. Surprisingly, a 3-hr pulse of GM-CSF severely diminished chemotaxis to IL-8, whereas N-formyl-methyl-leucyl-phenylalanine (fMLP)-mediated chemotaxis was retained. Conversely, when cells were incubated without GM-CSF they retained IL-8-mediated migration but lost fMLP chemotaxis. These changes in chemotaxis did not correlate with expression of CXCR1, CXCR2 or formyl peptide receptor. However, IL-8-mediated phosphorylation of p44/42 mitogen-activated protein kinase was greatly reduced in neutrophils that no longer migrated to IL-8, and was diminished in cells that no longer migrated to fMLP. Oestradiol, which is reported by some to exert an anti-inflammatory effect on neutrophils, did not change the effects of GM-CSF. These data suggest that neutrophil function may be altered by cytokines such as GM-CSF through modulation of signalling and independently of surface receptor expression.  

4.374           Alveolar epithelial cells secrete chemokines in response to IL-1b and lipopolysaccharide but not to ozone

Manzer, R., Wang, J., Nishina, K., McConville, G. and Mason, R.J. Am. J. Respir. Cell Mol., 34, 158-166 (2006)   Ozone exposure produces acute inflammation and neutrophil influx in the distal lung. Alveolar epithelial cells cover a large surface area, secrete chemokines, and may initiate or modify the inflammatory response. The effect of ozone on chemokine production by these cells has not been defined. Isolated rat type II cells were cultured in different conditions to express the morphologic appearance and biochemical markers for the type I and the type II cell phenotypes. These cells were exposed to ozone at an air/liquid interface. The type I–like cells were more susceptible to injury than the type II cells and showed signs of injury at exposure levels of 100 ppb ozone for 60 min. Both phenotypes showed evidence of lipid peroxidation after ozone exposure as measured by 8-isoprostane production, but neither phenotype secreted increased amounts of MIP-2 (CXCL3), CINC-1 (CXCL1), or MCP-1 (CCL2) in response to ozone. Both cell phenotypes secreted MIP-2 and MCP-1 in response to IL-1 or lipopolysaccharide, but there was no priming or synergy with ozone. It is likely that the inflammatory response to ozone in the alveolar compartment is not due to the direct effect of ozone on epithelial cells.  

4.375           Oxygen tension regulates the in vitro maturation of GM-CSF expanded murine bone marrow dendritic cells by modulating class II MHC expression

Goth, S.R., Chu, R.A. and Pessah, I.N.

1. Immunol. Methods, 308, 179-191 (2006)

  Conventional culture conditions for GM-CSF expanded murine bone marrow derived dendritic cells (BMDCs) uses ambient (hyperoxic) oxygen pressure (20% v/v, 152 Torr) and medium supplemented with the thiol 2-mercaptoethanol (2-Me). Given the redox activities of O₂ and 2-Me, the effects of 2%, 5%, 10%, and 20% v/v O₂ atmospheres and omitting 2-Me from the medium were tested upon the generation of GM-CSF expanded BMDCs. DC yield, phenotype and function were compared to BMDCs grown using conventional conditions. All cultures yielded DC subsets with CD11c⁺ MHC II^NEG, CD11c⁺ MHC II^INT, CD11c⁺ MHC II^HI expression phenotypes, classed as precursor, immature, and mature DCs (IDC, MDC). Low O₂ tensions generated significantly fewer precursor DCs, and more IDCs and MDCs. Cytometer sorted precursor DCs expressed surface class II MHC after transfer to low, but not high O₂ atmospheres. Expression of myeloid markers was similar between BMDC cultures generated in 5% O₂ or conventional conditions, and MDCs from low O₂ cultures had the morphology typical of mature myeloid DCs. IDCs and MDCs from low O₂ and conventional culture conditions were similarly potent allostimulatory APCs. The O₂ tension (but not 2-Me addition) in vitro significantly influences overall DC subset frequencies and yield, and governs DC maturation by regulating the surface class II MHC expression of GM-CSF expanded BMDC cultures.  

4.376           Adjuvant IL-15 does not enhance the efficacy of tumor cell lysate-pulsed dendritic cell vaccines for active immunotherapy of T cell lymphoma

Gatza, E. and Okada, C.Y. Cancer Immunol. Immunother., 55, 420-432 (2006)   There has been a recent interest in using IL-15 to enhance antitumor activity in several models because of its ability to stimulate CD8⁺ T cell expansion, inhibit apoptosis and promote memory T cell survival and maintenance. Previously, we reported that C6VL tumor lysate-pulsed dendritic cell vaccines significantly enhanced the survival of tumor-bearing mice by stimulating a potent tumor-specific CD8⁺ T cell response. In this study, we determined whether IL-15 used as immunologic adjuvant would augment vaccine-primed CD8⁺ T cell immunity against C6VL and further improve the survival of tumor-bearing mice. We report that IL-15 given after C6VL lysate-pulsed dendritic cell vaccines stimulated local and systemic expansion of NK, NKT and CD8⁺ CD44^hi T cells. IL-15 did not, however, augment innate or cellular responses against the tumor. T cells from mice infused with IL-15 following vaccination did not secrete increased levels of tumor-specific TNF-α or IFN-γ or have enhanced C6VL-specific CTL activity compared to T cells from recipients of the vaccine alone. Lastly, IL-15 did not enhance the survival of tumor-bearing vaccinated mice. Thus, while activated- and memory-phenotype CD8⁺ T cells were dramatically expanded by IL-15 infusion, vaccine-primed CD8⁺ T cell specific for C6VL were not significantly expanded. This is the first account of using IL-15 as an adjuvant in a therapeutic model of active immunotherapy where there was not a preexisting pool of tumor-specific CD8⁺ T cells. Our results contrast the recent studies where IL-15 was successfully used to augment tumor-reactivity of adoptively transferred transgenic CD8⁺ T cells. This suggests that the adjuvant potential of IL-15 may be greatest in settings where it can augment the number and activity of preexisting tumor-specific CD8⁺ T cells.  

4.377           Migratory monocytes and granulocytes are major lymphatic carriers of Salmonella from tissue to draining lymph node

Bonneau, M. et al

1. Leukoc. Biol., 79, 268-276 (2006)

  Dendritic cells (DC) are recognized as sentinels, which capture antigens in tissue and migrate to the lymph node, where they initiate immune responses. However, when a vaccine strain of green fluorescent protein-expressing Salmonella abortusovis (SAO) was inoculated into sheep oral mucosa, it induced accumulation of myeloid non-DC in the subcapsular sinus and paracortex of the draining lymph node, and SAO was mainly found associated with these cells (granulocytes and macrophages) but rarely with DC. To analyze how bacteria reached lymph nodes, we used cervical pseudo-afferent lymph duct catheterization. We showed that Salmonella administered in the oral mucosa were traveling free in lymph or associated with cells, largely with lymph monocytes and granulocytes but less with DC. SAO also induced a strong influx of these phagocytic cells in afferent lymph. Migrating DC presented a semi-mature phenotype, and SAO administration did not alter their expression of major histocompatibility complex type 2 and coactivation molecules. Compared with blood counterparts, lymph monocytes expressed lower levels of CD40, and granulocytes expressed higher levels of CD80. The data suggest that immunity to bacteria may result from the complex interplay between a mixture of phagocytic cell types, which transport antigens and are massively recruited via lymph to decisional lymph nodes.  

4.378           Identification and characterization of a novel isoform of the vesicular g-aminobutyric acid transporter with glucose-regulated expression in rat islets

Suckow, A.T. et al

1. Mol. Endocrinol., 36, 187-199 (2006)

  Pancreatic islets are unique outside the nervous system in that they contain high levels of the inhibitory neurotransmitter -aminobutyric acid (GABA), synthesized by the enzyme glutamic acid decarboxylase (GAD). Since the role that GABA plays in the islet and the mechanisms whereby the two major GAD isoforms (GAD65 and GAD67) function as diabetes-associated autoantigens are unknown, continued characterization of the islet GAD–GABA system is important. We previously demonstrated that the GABA and glycine transporter vesicular inhibitory amino acid transporter (VIAAT also known as VGAT) is present in rat islets. Here we identify a novel 52 kDa variant of VIAAT in rat islets: VIAAT-52 (V52). V52 is an amino-terminally truncated form of VIAAT (V57) that likely results from utilization of a downstream start site of translation. V57 and V52 display different patterns of post-translational modification and cellular expression. Our results have indicated that islet content of V52, but not V57, is responsive to changes in glucose concentration and other extracellular conditions. VIAAT is expressed in the islet cells, but there have been conflicting findings regarding the presence of VIAAT in the ß cells. Here we have also provided additional evidence for the presence of VIAAT in islet ß cells and show that the ß cell line INS-1 expresses V57. V52 may be better adapted than V57 to the unique rat cell GAD–GABA system, which lacks GAD65 and in which VIAAT traffics to secretory granules rather than just to synaptic microvesicles.  

4.379           Modified two-layer preservation method (M-Kyoto/PFC) improves islet yields in islet isolation

Noguchi, H. et al Am. J. Transplant., 6, 496-504 (2006)   Islet allotransplantation can achieve insulin independence in patients with type I diabetes. Recent reports show that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and UW solution, is superior to simple cold storage in UW for pancreas preservation in islet transplantation. However, UW solution has several disadvantages, including the inhibition of Liberase activity. In this study, we investigated the features of a new solution, designated M-Kyoto solution. M-Kyoto solution contains trehalose and ulinastatin as distinct components. Trehalose has a cytoprotective effect against stress, and ulinastatin inhibits trypsin. In porcine islet isolation, islet yield was significantly higher in the M-Kyoto/PFC group compared with the UW/PFC group. There was no significant difference in ATP content in the pancreas between the two groups, suggesting that different islet yields are not due to their differences as energy sources. Compared with UW solution, M-Kyoto solution significantly inhibited trypsin activity in the digestion step; moreover, M-Kyoto solution inhibited collagenase digestion less than UW solution. In conclusion, the advantages of M-Kyoto solution are trypsin inhibition and less collagenase inhibition. Based on these data, we now use M-Kyoto solution for clinical islet transplantation from nonheart-beating donor pancreata.  

4.380           Variants of the 5’-untranslated region of human NCF2: expression and translational efficiency

Gauss, K.A. et al Gene, 366, 169-179 (2006)   The NCF2 gene encodes p67^phox, an essential component of the multi-protein NADPH oxidase enzyme in phagocytic leukocytes, as well as in certain non-phagocytic cells. In humans, the NCF2 gene is expressed as multiple NCF2 variants that differ in the 5′-untranslated region (5′-UTR). Previously, we reported the presence of four NCF2 5′-UTR mRNA variants (designated as NCF2 exon 1, intron 1a, intron 1b and intron 1c). As each of the gene variants encodes an identical p67^phox protein, the functional significance of these message variants was not apparent. In this study, we investigated the relative expression levels and tissue-specificity of NCF2 5′-UTR variant mRNAs and their translation efficiency and stability. NCF2 5′-UTR variant transcripts were differentially expressed in various cell lines and human tissues. In vitro translation assays indicated that the NCF2 5′-UTR variants also differed in their effects on the translation of a luciferase reporter mRNA and NCF2 mRNA. Notably, NCF2 intron 1 5′-UTR variants, which are the predominantly expressed variants found in vivo, strongly inhibited translation when compared to the NCF2 exon 1 5′-UTR variant. In contrast, RNA decay assays demonstrated that there was no significant difference between stability of NCF2 intron 1 transcripts and the exon 1 5′-UTR variant in HL-60, MonoMac 6, and U937 cells. Moreover, expression of the variant transcripts remained unchanged after neutrophil phagocytosis, and was similar in normal neutrophils and neutrophils from a patient with X-linked chronic granulomatous disease. These studies suggest that expression of p67^phox is regulated through mechanisms that include modulation of transcription and translation.  

4.381           Natural killer cells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent manners

Radaeva, S. et al Gastroenterology, 130(2), 434-452 (2006)   Background & Aims: Viral hepatitis infection, which is a major cause of liver fibrosis, is associated with activation of innate immunity. However, the role of innate immunity in liver fibrosis remains obscure. Methods: Liver fibrosis was induced either by feeding mice with the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet or by injecting them with carbon tetrachloride. The Toll-like receptor 3 ligand, polyinosinic-polycytidylic acid, was used to activate innate immunity cells and mediators, including natural killer cells and interferon γ. Results: In the mouse model of DDC-induced liver fibrosis, natural killer cell activation by polyinosinic-polycytidylic acid induced cell death to activated hepatic stellate cells and attenuated the severity of liver fibrosis. Polyinosinic-polycytidylic acid treatment also ameliorated liver fibrosis induced by carbon tetrachloride. The observed protective effect of polyinosinic-polycytidylic acid on liver fibrosis was diminished through either depletion of natural killer cells or by disruption of the interferon γ gene. Expression of retinoic acid early inducible 1, the NKG2D ligand, was undetectable on quiescent hepatic stellate cells, whereas high levels were found on activated hepatic stellate cells, which correlated with the resistance and susceptibility of quiescent hepatic stellate cells and activated hepatic stellate cells to natural killer cell lysis, respectively. Moreover, treatment with polyinosinic-polycytidylic acid or interferon γ enhanced the cytotoxicity of natural killer cells against activated hepatic stellate cells and increased the expression of NKG2D and tumor necrosis factor–related apoptosis-inducing ligand on liver natural killer cells. Blocking NKG2D or tumor necrosis factor–related apoptosis-inducing ligand with neutralizing antibodies markedly diminished the cytotoxicity of polyinosinic-polycytidylic acid–activated natural killer cells against activated hepatic stellate cells. Conclusions: Our findings suggest that natural killer cells kill activated hepatic stellate cells via retinoic acid early inducible 1/NKG2D-dependent and tumor necrosis factor–related apoptosis-inducing ligand–dependent mechanisms, thereby ameliorating liver fibrosis.                      

4.382           Somatostatin inhibits dendritic cell responsiveness to Helicobacter pylori

Kao, J.Y. et al Regulatory Peptides, 134, 23-29 (2006)   Somatostatin is a regulatory peptide found in abundance in the stomach. We have previously shown that somatostatin is required for IL-4-mediated resolution of Helicobacter pylori gastritis. In the current study, we hypothesize that somatostatin acts directly on antigen-presenting cells in the stomach to lessen the severity of gastritis. To test this hypothesis, we first show that CD11c+ dendritic cells are present in the infected tissue of mice with H. pylori-induced gastritis. Pretreatment of bone marrow-derived dendritic cells with somatostatin results in decreased IL-12 production, and lower splenocyte proliferation induced by H. pylori-stimulated dendritic cells. Furthermore, octreotide, a somatostatin analogue, is more potent than somatostatin in suppressing IL-12 release by H. pylori-stimulated dendritic cells through an NF-kappaB-independent pathway. In addition, IL-4 stimulates somatostatin secretion from dendritic cells. In conclusion, somatostatin inhibits dendritic cell activation by H. pylori; a possible mechanism by which IL-4 mediates resolution of gastritis. We suggest that octreotide may be effective in treating immune-mediated diseases of the stomach.  

4.383           H2-O expression in primary dendritic cells

Chen, X., Reed-Loisel, L.M., Karlsson, L. and Jensen, P.E.

1. Immunol., 176, 3548-3556 (2006)

  H2-O is a nonpolymorphic class II molecule whose biological role remains to be determined. H2-O modulates H2-M function, and it has been generally believed to be expressed only in B lymphocytes and thymic medullary epithelial cells, but not in dendritic cells (DCs). In this study, we report identification of H2-O expression in primary murine DCs. Similar to B cells, H2-O is associated with H2-M in DCs, and its expression is differentially regulated in DC subsets as well as during cell maturation and activation. Primary bone marrow DCs and plasmacytoid DCs in the spleen and lymph nodes express MHC class II and H2-M, but not the inhibitor H2-O. In contrast, myeloid DCs in secondary lymphoid organs express both H2-M and H2-O. In CD8 ⁺ DCs, the ratio of H2-O to H2-M is higher than in CD8 ^– DCs. In DCs generated from GM-CSF- and IL-4-conditioned bone marrow cultures, H2-O expression is not detected regardless of the maturation status of the cells. Administration of LPS induces in vivo activation of myeloid DCs, and this activation is associated with down-regulation of H2-O expression. Primary splenic DCs from H2-O^–/– and H2-O^+/+ mice present exogenous protein Ags to T cell hybridomas similarly well, but H2-O^–/– DCs induce stronger allogeneic CD4 T cell response than the H2-O^+/+ DCs in mixed leukocyte reactions. Our results suggest that H2-O has a broader role than previously appreciated in regulating Ag presentation.      

4.384           Complete differentiation of CD8⁺ T cells activated locally within the transplanted liver

Klein, I. and Crispe, I.N.

1. Exp. Med., 203(2), 437-447 (2006)

  The transplanted liver elicits systemic tolerance, and the underlying mechanism may also account for the persistence of liver infections, such as malaria and viral hepatitis. These phenomena have led to the hypothesis that antigen presentation within the liver is abortive, leading to T cell tolerance or apoptosis. Here we test this hypothesis in an optimized orthotopic liver transplantation model. In direct contradiction to this model, the liver itself induces full CD8⁺ T cell activation and differentiation. The effects of microchimerism were neutralized by bone marrow transplantation in the liver donor, and the lack of liver-derived antigen-presenting cells was documented by eight-color flow cytometry and by sensitive functional assays. We conclude that local antigen presentation cannot explain liver tolerance. On the contrary, the liver may be an excellent priming site for naive CD8⁺ T cells.  

4.385           Effector T cell differentiation and memory T cell maintenance outside secondary lymphoid organs

Obharai, J. et al

1. Immunol., 176, 4051-4058 (2006)

  Naive T cell circulation is restricted to secondary lymphoid organs. Effector and memory T cells, in contrast, acquire the ability to migrate to nonlymphoid tissues. In this study we examined whether nonlymphoid tissues contribute to the differentiation of effector T cells to memory cells and the long-term maintenance of memory T cells. We found that CD4, but not CD8, effector T cell differentiation to memory cells is impaired in adoptive hosts that lack secondary lymphoid organs. In contrast, established CD4 and CD8 memory T cells underwent basal homeostatic proliferation in the liver, lungs, and bone marrow, were maintained long-term, and functioned in the absence of secondary lymphoid organs. CD8 memory T cells found in nonlymphoid tissues expressed both central and effector memory phenotypes, whereas CD4 memory T cells displayed predominantly an effector memory phenotype. These findings indicate that secondary lymphoid organs are not necessary for the maintenance and function of memory T cell populations, whereas the optimal differentiation of CD4 effectors to memory T cells is dependent on these organs. The ability of memory T cells to persist and respond to foreign Ag independently of secondary lymphoid tissues supports the existence of nonlymphoid memory T cell pools that provide essential immune surveillance in the periphery.  

4.386           Hedgehog signaling maintains resident hepatic progenitors throughout life

Sicklick, J.K. et al Am. J. Gastrointest. Liver Physiol., 290, G859-G870 (2006)   Hedgehog signaling through its receptor, Patched, activates transcription of genes, including Patched, that regulate the fate of various progenitors. Although Hedgehog signaling is required for endodermal commitment and hepatogenesis, the possibility that it regulates liver turnover in adults had not been considered because mature liver epithelial cells lack Hedgehog signaling. Herein, we show that this pathway is essential throughout life for maintaining hepatic progenitors. Patched-expressing cells have been identified among endodermally lineage-restricted, murine embryonic stem cells as well as in livers of fetal and adult Ptc-lacZ mice. An adult-derived, murine hepatic progenitor cell line expresses Patched, and Hedgehog-responsive cells exist in stem cell compartments of fetal and adult human livers. In both species, manipulation of Hedgehog activity influences hepatic progenitor cell survival. Therefore, Hedgehog signaling is conserved in hepatic progenitors from fetal development through adulthood and may be a new therapeutic target in patients with liver damage.  

4.387           Effect of donor age on function of isolated human islets

Ihm, S-H. et al Diabetes, 55, 1361-1368 (2006)   This study intended to evaluate the impact of donor age on the function of isolated islets. Analysis of human islets from cadaveric donors (age 16–70 years) was performed using glucose-stimulated insulin release (GSIR) (n = 93), islet ATP content (n = 27), diabetic nude mouse bioassay (n = 72), and the insulin secretory function after single-donor clinical islet allotransplantation (n = 7). The GSIR index was significantly higher in younger donors (age 40 years) than in older donors and negatively correlated with the donor age (r = –0.535). Islet ATP was higher in younger donors (115.7 ± 17.7 vs. 75.7 ± 6.6 pmol/µg DNA). The diabetes reversal rate of mice with 2,000 IE was significantly higher in younger donors (96 vs. 68%). C-peptide increment to glucose during intravenous glucose tolerance test at days 90–120 after clinical transplantation showed negative correlation with donor age (r = –0.872) and positive correlation with the islet mass (r = 0.832). On the other hand, acute insulin response to arginine only showed correlation with the islet mass and not with donor age. These results show that insulin secretory response to glucose deteriorates with increasing age and that it may be related to changes in ATP generation in ß-cells.  

4.388           Pretransplant culture selects for high-quality porcine islets

Rijkelijkhuizen, J.K.R.A., van der Burg, M.P.M., Töns, A., Terpstra, O.T. and Bouwman, E. Transplantation, 32(4), 403-407 (2006)   Objectives: The pig is generally considered a suitable alternative donor for clinical islet transplantation. However, adult pig islets are difficult to isolate and culture, often behave variably in in vitro assays, and do not consistently cure diabetic nude mice. In this study, we compared the in vivo function of freshly isolated and cultured adult porcine islets by transplantation in diabetic nude mice. Methods: Freshly isolated and cultured islets were transplanted in different doses to diabetic nude mice (N = 48). Results: Average islet yield was 1924 islet-equivalents per gram of pancreas, purity 96%, and the viability that was measured by acridine orange and propidium iodide was greater than 80% in all freshly isolated islet preparations. Grafts of freshly isolated islets failed to reduce hyperglycemia in 17 of 18 recipients. Although after 1 day of culture islet recovery was only 21%, grafts of these islets cured 12 of 17 mice. After 7 to 14 days of culture, the recovery had decreased to 11%; however, these islets reversed hyperglycemia in all mice (13/13) and showed shorter time-to-normoglycemia and more tightly regulated blood glucose. Conclusions: Although freshly isolated adult porcine islets survive culture and transplantation poorly, islets selected by prolonged culture are of high potential.  

4.389           Aberrant T helper cell response in tumor-bearing mice limits the efficacy of dendritic cell vaccine

Kao, J.Y., Zhang, M., Chen, C-M., Pierzchala, A. and Chen, J-J. Immunol. Lett., 105, 16-25 (2006)   Dendritic cell (DC) vaccine is a promising immunotherapy for malignancies, but its clinical efficacy has been questioned. Here we examined the mechanisms of treatment failure with DC vaccine in a murine colon cancer model. DC vaccination of naive mice prevents tumor implantation, but it is ineffective in tumor-bearing hosts despite the induction of tumor-specific CTL activity. Analyses of tumor-specific T helper cell type 1 (Th1)/T helper cell type 2 (Th2) responses showed that DC vaccine induced a mixed Th1/Th2 response in naive mice. Interestingly, CD4⁺ T cells from tumor-bearing mice showed a Th1-predominant response before DC vaccination but Th2 after DC vaccination. Furthermore, interleukin-10 production was higher in CD4⁺ T cells from vaccinated tumor-bearing mice than in CD4⁺ T cells from unvaccinated tumor-bearing mice. CD4⁺ T cells from mice treated with lipopolysaccharide (LPS)-matured DC fusion vaccine had lower production of interleukin-10 than CD4⁺ T cells from mice treated with non-LPS-treated DC vaccine. However, similar to the non-LPS-treated DC vaccine, the LPS-matured DC vaccine failed to suppress tumor growth and induced a Th2 predominant tumor-specific response in tumor-bearing mice. These results suggest that the presence of tumor in the host induces an aberrant CD4⁺ T cell response to DC vaccine, which may contribute to the failure of the vaccine to eradicate established tumors.  

4.390           CCR6-mediated dendritic cell activation of pathogen-specific T cells in Peyer’s patches

Salazar-Gonzales, R.M. et al Immunity, 24, 623-632 (2006)   T cell activation by dendritic cells (DCs) is critical to the initiation of adaptive immune responses and protection against pathogens. Here, we demonstrate that a specialized DC subset in Peyer's patches (PPs) mediates the rapid activation of pathogen specific T cells. This DC subset is characterized by the expression of the chemokine receptor CCR6 and is found only in PPs. CCR6⁺ DCs were recruited into the dome regions of PPs upon invasion of the follicle associated epithelium (FAE) by an enteric pathogen and were responsible for the rapid local activation of pathogen-specific T cells. CCR6-deficient DCs were unable to respond to bacterial invasion of PPs and failed to initiate T cell activation, resulting in reduced defense against oral infection. Thus, CCR6-dependent regulation of DCs is responsible for localized T cell dependent defense against entero-invasive pathogens.    

4.391           A closed system for the preparation of islets of Langerhans using the COBE2991 cell processor

Lembert, N., Biesemeier, A., Klaffschenkel, R. And Königsrainer, A. Cytotherapy,8, Suppl. 2 (2006) Abstracts of the 2^nd International Conference “Strategies in Tissue Engineering” May 31–June 2, 2006 Würzburg, Germany   Introduction: During the isolation of human islets of Langerhans, the digest is repeatedly and directly exposed to the ambient atmosphere. Therefore, the entire cell isolation must be performed in a clean room facility to fulfill the GMP requirements of German authorities. We used the pig model for a modification of the isolation and purification process by performing all islet preparation steps in a closed system consisting of a Ricordi chamber, a cooling device, and the COBE2991 cell processor. This study evaluated whether this entirely closed system to avoid the need of clean room facilities can deliver functional and purified islets. Materials & Methods: Pancreata from 6-month-old market-weight pigs were procured in the local slaughterhouse. After continuous digestion-filtration (Liberase PI, 1.5 mg/g pancreas) the digest was flushed through the cooling device and pumped directly into the COBE2991. Centrifugation (1500 U/min, 1 min), automatic supernatant pump-out, and Ricordi-chamber washing and refilling (HBSS) were repeated five times until a total of 3 l was flushed through the COBE2991, leaving the concentrated digest inside the COBE bag. A discontinuous gradient was applied consisting of a bottom layer (UW/Optiprep, density 1.12), a middle layer (UW/Optiprep, density 1.09), and a top layer (UW solution, density 1.05). Centrifugation (1000 U/min) was stopped after 5 min, and 20-ml fractions were collected. Islet equivalents (IEQ , areal density), insulin (ELISA), amylase activity (kinetic assay), and cell composition (FACS) were determined. Results: The islet yield in six preparations was 116875±/17759 IEQ or 1290±/118 IEQ/gP (mean9/SEM). Compared with the native organ, the insulin content of the islet fraction increased from 0.5±/0.2 to 95±/52 mg/mg protein. The insulin content in the islet fraction was 92±/32 U. The amylase activity decreased from 2857±/1001 to 63±/28 U/mg protein. Glucose stimulated insulin output in 4/6 preparations. FACS identified 70% living cells, and 40% living beta cells. The entire preparation was completed within 4 h by two people, which doubled the routine preparation speed. Discussion: The presented technique allows a reproducible and efficient preparation of pure and vital porcine islets in a closed system. The preparation is much faster and less expensive than the traditional islet preparation. This technique may be applicable for human islet preparations without the need of preparations inside clean room facilities.    

4.392           Comparison of Methods of Extracting Salmonella enterica Serovar Enteritidis DNA from Environmental Substrates and Quantification of Organisms by Using a General Internal Procedural Control

Klerks, M.M., van Bruggen, A.H.C., Zijlstra, C. And Donnikov, M. Appl. Envir. Micorbiol., 72(6), 3879-3886 (2006)   This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of S. enterica serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 x 10³ to 1.8 x 10³ CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent S. enterica-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.  

4.393           Quantitative proteomics reveals posttranslational control as a regulatory factor in primary hematopoietic stem cells

Unwin, R.D. et al Blood, 107(12), 4687-4694 (2006)   The proteome is determined by rates of transcription, translation, and protein turnover. Definition of stem cell populations therefore requires a stem cell proteome signature. However, the limit to the number of primary cells available has restricted extensive proteomic analysis. We present a mass spectrometric method using an isobaric covalent modification of peptides for relative quantification (iTRAQ), which was employed to compare the proteomes of approximately 1 million long-term reconstituting hematopoietic stem cells (Lin^–Sca⁺Kit⁺; LSK⁺) and non–long-term reconstituting progenitor cells (Lin^–Sca⁺Kit^–; LSK^–), respectively. Extensive 2-dimensional liquid chromatography (LC) peptide separation prior to mass spectrometry (MS) enabled enhanced proteome coverage with relative quantification of 948 proteins. Of the 145 changes in the proteome, 54% were not seen in the transcriptome. Hypoxia-related changes in proteins controlling metabolism and oxidative protection were observed, indicating that LSK⁺ cells are adapted for anaerobic environments. This approach can define proteomic changes in primary samples, thereby characterizing the molecular signature of stem cells and their progeny.  

4.394           Helicobacter pylori-secreted factors inhibit dendritic cell IL-12 secretion: a mechanism of ineffective host defense

Kao, J.Y. et al Am. J. Physiol Gastrointest. Liver Physiol., 291, G73-G81 (2006)   Helicobacter pylori evades host immune defenses and causes chronic gastritis. Immunity against intestinal pathogens is largely mediated by dendritic cells, yet the role of dendritic cells in acute H. pylori infection is largely unknown. We observed the recruitment of dendritic cells to the gastric mucosa of H. pylori-infected mice. Bone marrow-derived dendritic cells from mice responded to live H. pylori by upregulating the expression of proinflammatory cytokine mRNA (i.e., IL-1 , IL-1 , and IL-6). The supernatant from dendritic cells stimulated with H. pylori for 18 h contained twofold higher levels of IL-12p70 than IL-10 and induced the proliferation of syngeneic splenocytes and type 1 T helper cell cytokine release (IFN- and TNF- ). These responses were significantly lower compared with those induced by Acinetobacter lwoffi, another gastritis-causing pathogen more susceptible to host defenses. Analysis of whole H. pylori sonicate revealed the presence of a heat-stable factor secreted from H. pylori that specifically inhibited IL-12 but not IL-10 release from dendritic cells activated by A. lwoffi. Our findings suggest that dendritic cells participate in the host immune response against H. pylori and that their suppression by H. pylori may explain why infected hosts fail to prevent bacterial colonization.  

4.395           Cyclin dependent kinase inhibitors prevent apoptosis of postmitotic mouse motoneurons

Appert-Collin, A. et al Life Sciences, 79(5), 484-490 (2006)   Recent evidence suggests that apoptosis in post-mitotic neurons involves an aborted attempt of cells to re-enter the cell cycle which is characterized by increased expression of cyclins, such as cyclin D1, prior to death. However, such cyclins activation prior to apoptotic cell death remains controversial. Many neurological disorders are characterized by neuronal loss, particularly amyotrophic lateral sclerosis (ALS). ALS is a motoneuronal degenerative condition in which motoneuron loss could be due to an inappropriate return of these cells in the cell cycle. In the present study, we observed that deprivation of neurotrophic factor in purified motoneuron cultures induces an apoptotic pathway. After neurotrophic factor withdrawal, DAPI (4,6-diamidin-2-phenylindol dichlorohydrate) staining revealed the presence of nuclear condensation, DNA fragmentation, and perinuclear apoptotic body. Similarly, release of apoptotic microparticles and activation of caspases-3 and -9 were observed within the first hours following neurotrophic factor withdrawal. Next, we tested whether inhibition of cell cycle-related cyclin-dependent kinases (cdks) can prevent motoneuronal cell death. We showed that three cdk inhibitors, olomoucine, roscovitine and flavopiridol, suppress the death of motoneurons. Finally, we observed early increases in cyclin D1 and cyclin E expression after withdrawal of neurotrophic factors. These findings support the hypothesis that after removal of trophic support, post-mitotic neuronal cells die due to an attempt to re-enter the cell cycle in an uncoordinated and inappropriate manner.  

4.396           The phyto-chemical (−)-epigallocatechin gallate suppresses gene expression of epidermal growth factor receptor in rat hepatic stellate cells in vitro by reducing the activity of Egr-1

Fu, Y. and Chen, A. Biochem. Pharmacol., 72(2), 227-238   Hepatic stellate cells (HSC) are the major effectors in hepatic fibrogenesis. During liver injury, HSC become activated and proliferative. Platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) are the potent mitogens for many cell types. We previously demonstrated that (−)-epigallocatechin gallate (EGCG), the major and active component in green tea extracts, inhibited HSC growth, including reducing cell proliferation, and inducing apoptosis. We have reported that EGCG interrupts PDGF signaling by reducing receptor tyrosine phosphorylation and gene expression of PDGF-β receptor. Additional experiments are necessary to elucidate the effect of EGCG on EGF signaling in activated HSC. The aims of this study are to evaluate the effect of EGCG on the expression of EGFR and to elucidate the underlying molecular mechanisms in activated HSC. We hypothesize that EGCG might interrupt EGF signaling by suppressing gene expression of EGF receptor (EGFR) in activated HSC, which, together with the interruption of PDGF signaling, might collectively result in the inhibition of HSC growth. The present report demonstrates that the phyto-chemical dose-dependently suppresses gene expression of EGFR in activated HSC in vitro. The Egr-1 binding site located in the egfr promoter is found to be cis-activating element in regulating the promoter activity of the gene. EGCG inhibits the trans-activation activity of Egr-1 in activated HSC by suppressing gene expression of the transcription factor. The interruption of the ERK signaling pathway by EGCG reduces the trans-activation activity of Egr-1 and the promoter activity of EGFR gene in HSC. Taken together, our results demonstrate that EGCG suppresses gene expression of EGFR in rat activated HSC in vitro mediated by reducing the trans-activation activity of Egr-1.  

4.397           A comparison of ovine monocyte-derived macrophage function following infection with Mycobacterium avium ssp. avium and Mycobacterium avium ssp. paratuberculosis

Berger, S.T. and Griffin, F.T. Immunol. Cell Biol., 84(4), 349-356 (2006)   Mycobacterium avium ssp. paratuberculosis causes Johne's disease in ruminants, whereas the antigenically and genetically similar subspecies Mycobacterium avium ssp. avium is less virulent. In this study, we compared one strain of each subspecies for its ability to survive, induce cytokines, suppress MHC class I and II expression and induce apoptosis or necrosis in ovine monocyte-derived macrophages. Both subspecies survived intracellularly and induced the secretion of IL-10. Low levels of TNF-α were detected after infection with both subspecies at 4 h. IL-12 was not upregulated after infection. Downregulation of MHC class I and II was evident in response to infection with both M. avium ssp. avium and M. avium ssp. paratuberculosis. No significant cytotoxicity was detectable in ovine macrophages after the addition of bacteria. M. avium ssp. paratuberculosis induced slightly more apoptosis than M. avium ssp. avium. Still the overall rate of apoptosis was very low and both subspecies suppressed LPS-induced macrophage apoptosis.  

4.398           Phenotypic and Functional Characterization of Vaginal Dendritic Cells in a Rat Model of Candida albicans Vaginitis

De Bernardis, F. Et al Infect. Immun., 74(7), 4282-4294 (2006)   This study analyzes the phenotype of vaginal dendritic cells (VDCs), their antigenic presentation and activation of T-cell cytokine secretion, and their protective role in a rat model of Candida vaginitis. Histological observation demonstrated a significant accumulation of OX62⁺ VDCs in the mucosal epithelium of Candida albicans-infected rats at the third round of infection. We identified two subsets of OX62⁺ VDCs differing in the expression of CD4 molecule in both noninfected and Candida-infected rats. The OX62⁺ CD4⁺ subset of VDCs displayed a lymphoid cell-like morphology and expressed the T-cell antigen CD5, whereas the OX62⁺ CD4^– VDC subset exhibited a myeloid morphology and was CD5 negative. Candida infection resulted in VDC maturation with enhanced expression of CD80 and CD134L on both CD4⁺ and CD4^– VDC subsets at 2 and 6 weeks after Candida infection. CD5^– CD4^– CD86^– CD80^– CD134L⁺ VDCs from infected, but not noninfected, rats spontaneously released large amounts of interleukin-12 (IL-12) and tumor necrosis factor alpha, whereas all VDC subsets released comparable levels of IL-10 and IL-2 cytokines. Furthermore, OX62⁺ VDCs from infected rats primed naïve CD4⁺ T-cell proliferation and release of cytokines, including gamma interferon, IL-2, IL-6, and IL-10, in response to staphylococcal enterotoxin B stimulation in vitro. Adoptive transfer of highly purified OX62⁺ VDCs from infected rats induced a significant acceleration of fungal clearance compared with that in rats receiving naive VDCs, suggesting a protective role of VDCs in the anti-Candida mucosal immunity. Finally, VDC-mediated protection was associated with their ability to rapidly migrate to the vaginal mucosa and lymph nodes, as assessed by adoptive transfer of OX62⁺ VDCs labeled with 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester.  

4.399           The antifibrogenic effect of (-)-epigallocatechin gallate results from the induction of de novo synthesis of glutathione in passaged rat hepatic stellate cells

Yumei, F., Zhou, Y., Zheng, S. and Chen, A. Lab. Invest., 86, 697-709 (2006)   Hepatic stellate cells (HSC) are the major players during hepatic fibrogenesis. Overproduction of extracellular matrix (ECM) is a characteristic of activated HSC. Transforming growth factor-beta (TGF- ) is the most potent fibrogenic cytokine while connective tissue growth factor (CTGF) mediates the production of TGF- -induced ECM in activated HSC. HSC activation and hepatic fibrogenesis are stimulated by oxidative stress. Glutathione (GSH) is the most important intracellular antioxidant. The aim of this study is to explore the mechanisms of (-)-epigallocatechin-3-gallate (EGCG), the major and most active component in green tea extracts, in the inhibition of ECM gene expression in activated HSC. It is hypothesized that EGCG inhibits ECM gene expression in activated HSC by interrupting TGF- signaling through attenuating oxidative stress. It is found that EGCG interrupts TGF- signaling in activated HSC by suppressing gene expression of type I and II TGF- receptors. EGCG inhibits CTGF gene expression, leading to the reduction in the abundance of ECM, including I(I) procollagen. Exogenous CTGF dose dependently eliminates the antifibrogenic effect. EGCG attenuates oxidative stress in passaged HSC by scavenging reactive oxygen species and reducing lipid peroxidation. De novo synthesis of GSH is a prerequisite for EGCG to interrupt TGF- signaling and to reduce the abundance of I(I) procollagen in activated HSC in vitro. Taken together, our results demonstrate that the interruption of TGF- signaling by EGCG results in the suppression of gene expression of CTGF and ECM in activated HSC in vitro. In addition, our results, for the first time, demonstrate that the antioxidant property of EGCG derived from de novo synthesis of intracellular GSH plays a critical role in its antifibrogenic effect. These results provide novel insights into the mechanisms of EGCG as an antifibrogenic candidate in the prevention and treatment of liver fibrosis.      

4.400           Local Intrahepatic CD8+ T Cell Activation by a Non-Self- Antigen Results in Full Functional Differentiation

Wuensch, S.A., Pierce, R.H. and Crispe, I.N.

1. Immunol., 177, 1689-1697 (2006)

  The response of T cells to liver Ags sometimes results in immune tolerance. This has been proposed to result from local, intrahepatic priming, while the expression of the same Ag in liver-draining lymph nodes is believed to result in effective immunity. We tested this model, using an exogenous model Ag expressed only in hepatocytes, due to infection with an adeno-associated virus vector. T cell activation was exclusively intrahepatic, yet in contrast to the predictions of the current model, this resulted in clonal expansion, IFN- synthesis, and cytotoxic effector function. Local activation of naive CD8⁺ T cells can therefore cause full CD8⁺ T cell activation, and hepatocellular presentation cannot be used to explain the failure of CTL effector function against some liver pathogens such as hepatitis C.  

4.401           The oncofetal protein glypican-3 is a novel marker of hepatic progenitor/oval cells

Grozdanov, P.N., Yovchev, M.I. and Dabeva, D. Lab. Invest., 86, 1272-1284 (2006)   Glypican-3 (Gpc3), a cell surface-linked heparan sulfate proteoglycan is highly expressed during embryogenesis and is involved in organogenesis. Its exact biological function remains unknown. We have studied the expression of Gpc3 in fetal and adult liver, in liver injury models of activation of liver progenitor cells: D-galactosamine and 2-acetylaminofluorene (2-AAF) administration followed by partial hepatectomy (PH) (2-AAF/PH); and in the Solt-Farber carcinogenic model: by initiation with a single dose of diethylnitrosamine and promotion with 2-AAF followed by PH treatment. Gpc3 expression was studied using complementary DNA microarrays, reverse transcriptase-polymerase chain reaction, in situ hybridization (ISH); ISH combined with immunohistochemistry (IHC) and immunofluorescent microscopy. We found that Gpc3 is highly expressed in fetal hepatoblasts from embryonic days 13 through 16 and its expression gradually decreases towards birth. Dual ISH with Gpc3 and -fetoprotein (AFP) probes confirmed that only hepatoblasts and no other fetal liver cells express Gpc3. At 3 weeks after birth the expression of Gpc3 mRNA and protein was hardly detected in the liver. Gpc3 expression was highly induced in oval cell of D-gal and 2-AAF/PH treated animals. Dual ISH/IHC with Gpc3 riboprobe and cytokeratin-19 (CK-19) antibody revealed that Gpc3 is expressed in activated liver progenitor cells. ISH for Gpc3 and AFP performed on serial liver sections also showed coexpression of the two-oncofetal proteins. FACS isolated oval cells with anti-rat Thy1 revealed expression of Gpc3. Gpc3 expression persists in atypical duct-like structures and liver lesions of animals subjected to the Solt-Farber model of initiation and promotion of liver cancer expressing CK-19. In this work we report for the first time that the oncofetal protein Gpc3 is a marker of hepatic progenitor cells and of early liver lesions. Our findings show further that hepatic progenitor/oval cells are the target for malignant transformation in the Solt-Farber model of hepatic carcinogenesis.  

4.402           Intramuscular immunization with DNA construct containing Der p 2 and signal peptide sequences primed strong IgE production

Tan, L.K., Huang, C-H., Kuo, I-C., Liew, L.M. and Chua, K.Y. Vaccine, 24(29-30), 5762-5771 (2006)   Background Previous studies demonstrated that allergen gene vaccination induced TH1-skewed responses and inhibited IgE production. This study evaluated and characterized the immune responses induced by three DNA constructs encoding different forms of Der p 2 for safe and efficacious vaccination against mite allergy. Methods Mice were immunized intramuscularly with DNA constructs encoding a major mite allergen, Der p 2, without a signal peptide (p2), with a signal peptide (p52), and with a signal peptide plus lysosomal-targeting sequence (p52-LA), respectively, followed by TH2-skewed protein challenge. Antibody and T-cell cytokine responses were assessed by ELISA. Primed dendritic cells (DCs) were adoptively transferred to naïve mice and humoral responses were examined after protein challenge. The circulating Der p 2 protein was detected by sandwich ELISA. Results Mice immunized with p52-LA showed strong and clear-cut TH1-type response, as evident by high IFN-γ production and elevated levels of Der p 2-specific IgG2a production whereas construct p2 induced only moderate levels of TH1 response. In contrast, mice immunized with construct p52 showed a mixed TH1/TH2 phenotype and produced substantial circulating Der p 2 protein. Mice adoptively transferred with DCs primed by p52 construct, but not by the p2 or p52-LA constructs, were sensitized to produce high levels of Der p 2-specific IgE. Conclusions Immunization with DNA construct encoding a signal peptide could potentially prime TH2-skewed responses and IgE production. The additional inclusion of lysosomal-targeting sequences to such construct could improve the safety and efficacy of DNA vaccination against allergy.    

4.403           Effects of cushioned centrifugation on sperm quality in stallion semen stored cooled at 5°C for 24h, and stored cooled for 2h or 24h and then frozen

Sieme, H., Knop, K. And Rath, D. Animal Reprod. Sci., 94(1-2), 99-103 (2006)   Removing seminal plasma from stallion spermatozoa before use in a cooled semen or frozenthawed semen breeding program has been reported to be beneficial. The centrifugation involved is not without detrimental effects on the motility and morphology of spermatozoa, and may lead to loss of spermatozoa. To reduce this, semen can be underlaid with a dense, liquid cushion, on which the spermatozoa float during the centrifugation process (Revell et al., 1997; Ecot et al., 2005). Transportation of stallion spermatozoa for freezing at appropriate facilities would allow stallions to remain at home. Most reports recommend that semen be centrifuged at room temperature to remove seminal plasma, cooled slowly to 5 ◦C, and shipped at 5 ◦C prior to freezing (Crockett et al., 2001; Backman et al., 2004). However, the effects of cushioned centrifugation techniques on stallion spermatozoa after cooled-storage at 5 ◦C for 24 h, and after subsequent cryopreservation are unknown. This study evaluates the effects of high-speed centrifugation (20 min×1000×g) with various centrifugation extenders (Eqcellsire®, INRA-82, HBS) with or without a cushion (Cushion-Fluid®, Minit¨ub, Landshut, Germany; Eqcellsire®, IMV, L ´ Aigle, France) in stallions with good and poor semen freezability.  

4.404           The Phenotypes of Pluripotent Human Hepatic Progenitors

Schmelzer, E., Wauther, E. and Reid, L.M. Stem Cells, 24, 1852-1858 (2006)   Human livers contain two pluripotent hepatic progenitors, hepatic stem cells and hepatoblasts, with size, morphology, and gene expression profiles distinct from that of mature hepatocytes. Hepatic stem cells, the precursors to hepatoblasts, persist in stable numbers throughout life, and those isolated from the livers of all age donors from fetal to adult are essentially identical in their gene and protein expression profiles. The gene expression profile of hepatic stem cells throughout life consists of high levels of expression of cytokeratin 19 (CK19), neuronal cell adhesion molecule (NCAM), epithelial cell adhesion molecule (EpCAM), and claudin-3 (CLDN-3); low levels of albumin; and a complete absence of expression of -fetoprotein (AFP) and adult liver-specific proteins. By contrast, hepatoblasts, the dominant cell population in fetal and neonatal livers, decline in numbers with age and are found as <0.1% of normal adult livers. They express high levels of AFP, elevated levels of albumin, low levels of expression of adult liver-specific proteins, low levels of CK19, and a loss of NCAM and CLDN-3. Mature hepatocytes lack expression altogether of EpCAM, NCAM, AFP, CLDN-3, cytokeratin 19, and have acquired the well-known adult-specific profile that includes expression of high levels of albumin, cytochrome P4503A4, connexins, phosphoenolpyruvate carboxykinase, and transferrin. Thus, hepatic stem cells have a unique stem cell phenotype, whereas hepatoblasts have low levels of expression of both stem cell genes and genes expressed in high levels in mature hepatocytes.  

4.405           Cytokine-induced monocyte adhesion to endothelial cells involves platelet-activating factor: Suppression by conjugated linoleic acid

Sneddon, A.A., McLeod, E., Wahle, K.W.J. and Arthur, J.R. Biochim. Biophys. Acta, 1761(7), 793-801 (2006)   Monocyte–endothelium interaction is key to many acute and chronic inflammatory diseases. We have investigated the factors regulating monocyte attachment to cytokine-activated human umbilical vein endothelial cells (HUVEC) and the modulatory effect of the polyunsaturated fatty acid (PUFA), conjugated linoleic acid (CLA) in this process. Both TNF-α and IL-1β induced HUVEC platelet-activating factor (PAF) production and PAF was required for subsequent firm THP-1 monocyte adhesion since it was inhibited by both PAF receptor antagonists (BN-52021 or CV-6209) and a PAF synthesis inhibitor (sanguinarine). CLA inhibited the binding of both THP-1 and isolated human peripheral blood monocytes to HUVEC by up to 40% with the CLA t10,c12 isomer suppressing adhesion dose-dependently. Investigation into the mechanism involved demonstrated that with IL-1β, VCAM-1 and ICAM-1 levels and pro-inflammatory cytokine expression were largely unaffected by CLA. Through the use of PAF receptor antagonists and PAF synthesis inhibitors, CLA was shown to inhibit cytokine-induced binding by suppressing PAF production. Direct assay of PAF levels confirmed this result. We conclude that endothelial-generated PAF plays a central role in cytokine-induced monocyte adherence to endothelium and that the anti-inflammatory action of PUFAs such as CLA in suppressing monocyte–endothelial interaction is mediated through attenuation of pro-inflammatory phospholipids such as PAF.  

4.406           An -Glucan of Pseudallescheria boydii Is Involved in Fungal Phagocytosis and Toll-like Receptor Activation

Bittencourt, V.C.B. et al

1. Biol. Chem., 281(32), 22614-22623 (2006)

  The host response to fungi is in part dependent on activation of evolutionarily conserved receptors, including toll-like receptors and phagocytic receptors. However, the molecular nature of fungal ligands responsible for this activation is largely unknown. Herein, we describe the isolation and structural characterization of an -glucan from Pseudallescheria boydii cell wall and evaluate its role in the induction of innate immune response. These analyses indicate that -glucan of P. boydii is a glycogen-like polysaccharide consisting of linear 4-linked -D-Glcp residues substituted at position 6 with -D-Glcp branches. Soluble -glucan, but not -glucan, led to a dose-dependent inhibition of conidia phagocytosis. Furthermore, a significant decrease in the phagocytic index occurred when -glucan from conidial surface was removed by enzymatic treatment with -amyloglucosidase, thus indicating an essential role of -glucan in P. boydii internalization by macrophages. -Glucan stimulates the secretion of inflammatory cytokines by macrophages and dendritic cells; again this effect is abolished by treatment with -amyloglucosidase. Finally, -glucan induces cytokine secretion by cells of the innate immune system in a mechanism involving toll-like receptor 2, CD14, and MyD88. These results might have relevance in the context of infections with P. boydii and other fungi, and -glucan could be a target for intervention during fungal infections.  

4.407           The p110δ Isoform of PI3K Differentially Regulates β1 and β2 Integrin-Mediated Monocyte Adhesion and Spreading and Modulates Diapedesis

Ferreira, A.M., Isaacs, H., Hayflick, J.S., Rogers, K.A. and Sandig, M. Microcirculation, 13, 439-456 (2006)   Objective: Leukocyte diapedesis is misregulated in inflammatory disease and depends on the binding of monocytic LFA-1 and VLA-4 to endothelial ICAM-1 and VCAM-1, respectively. The authors hypothesized that these different molecular interactions elicit specific signaling cascades within monocytes regulating specific steps in adhesion, motility, and diapedesis. Methods: The authors employed the PI3K p110δ catalytic subunit specific inhibitor IC87114 (2 μ M) and the broad-spectrum PI3K inhibitory agents LY294002 (50 μ M) and wortmannin (100 nM), to examine the role of PI3Kδ in monocyte diapedesis through endothelial monolayers and its role in monocyte adhesion and spreading upon carpets of ICAM-1 or VCAM-1. They further explored the effects of PI3Kδ inhibition on the activation state of β 1 and β 2 integrins with immunocytochemistry and flow cytometry. Results: In human peripheral blood monocytes IC87114 was as effective as wortmannin and LY294002 at inhibiting diapedesis, however, in THP-1 cells LY294002 and wortmannin caused a 5-fold reduction in diapedesis, while IC87114 only decreased diapedesis 2-fold. PI3Kδ activity was specifically required for THP-1 cell adhesion and spreading on VCAM-1, but not on ICAM-1 protein substrates. Flow cytometric analysis demonstrated that PI3Kδ inhibition decreased the amount of conformationally active β 1-integrins, while having no effect on the prevalence of conformationally active β 2-integrins expressed on the cell surface. In addition, PI3Kδ inhibition resulted in a 4-fold decrease in the activation state of Rac-1 and Cdc42. Conclusions: These results demonstrate the specific necessity of PI3Kδ in regulating monocytic integrin activation and the general role of PI3K signaling during diapedesis, implicating PI3K as a target for therapeutic intervention.  

4.408           The efficient isolation of murine splenic dendritic cells and their cytochemical features

Zarnani, A.H. et al Histochem. Cell Biol., 126, 275-282 (2006)   Despite their importance in professional antigen presentation and their ubiquitous presence, dendritic cells (DCs) are usually found in such trace amounts in tissues that their isolation with high purity is a difficult task. Because of their scarcity, accurate determination of the purity of isolated dendritic cells is very important. In this study, we purified murine splenic dendritic cells by a three-step enrichment method and evaluated their morphological, cytochemical and functional characteristics. Purity of the isolated cells was determined by established methods such as flow cytometry (FC) and immunocytochemistry (ICC) using anti-CD11c monoclonal antibody. In order to test purified DC functional properties, we used in vivo antigen presentation assay. Our results showed that antigen-pulsed DCs are potent stimulators of antigen-specific lymphocyte proliferation. We studied myeloperoxidase (MPO) and non-specific esterase (NSE) activity in isolated cells to determine the purity of dendritic cells compared to more conventional methods. Our results showed that murine splenic dendritic cells were deficient in both MPO and NSE activity and the percentage of purity obtained by NSE staining on isolated cells was comparable to the results obtained by either FC or ICC. To our knowledge, this is the first report on using NSE activity for determination of the purity of isolated murine splenic dendritic cells. We, therefore, recommend that NSE activity be employed as a simple, inexpensive and yet accurate method for evaluation of the purity of isolated murine splenic dendritic cells.  

4.409           Post-mortem semen cryopreservation and characterization in two different endangered gazelle species (Gazella gazella and Gazella dorcas) and one subspecies (Gazella gazelle acaiae)

Saragusty, J., Gacitua, H., King, R. and Arav, A. Theriogenology, 66(4), 775-784 (2006)   Both Gazella gazella and Gazella dorcas are endangered species with continually dwindling population size, yet basic knowledge on their spermatozoa is missing. Semen collected post-mortem (PM) from the cauda epididymis of five adult gazelles (three Gazella gazella gazella, one Gazella gazella acaiae and one G. dorcas) was cryopreserved using directional freezing of large volumes (8 mL) with egg-yolk-free extender. Sperm size measurements and SYBR-14/propodium iodide (PI) viability stain validation for use in gazelles were conducted. Post-thaw characterization included motility, viability, acrosome damage evaluation, computerized motility characterization and morphology and sperm motility index (SMI) was calculated. Extracted sperm motility was 71.67 ± 11.67% (mean ± S.E.M.). Post-thaw motility ranged between 15% and 63%, viability was 57.49 ± 3.24%, intact acrosome was detected in 63.74 ± 2.6% (median 64.8%, upper/lower quartiles 71.79%, 61.82%), and normal morphology ranged between 41% and 63%. Motility characterization showed two sub-groups—highly active and progressively motile spermatozoa with SMI of 62.75 ± 0.38 and low activity and poorly progressive with SMI of 46.16 ± 1.53. Our results indicate that PM preservation of gazelle spermatozoa with satisfactory post-thaw viability is possible and cryobanking is achievable.  

4.410           Arginase 1 Regulation of Nitric Oxide Production Is Key to Survival of Trophic Factor-Deprived Motor Neurons

Estevez, A.G. et al

1. Neurosci., 26(33), 8512-8516 (2006)

  When deprived of trophic factors, the majority of cultured motor neurons undergo nitric oxide-dependent apoptosis. However, for reasons that have remained unclear, 30–50% of the motor neurons survive for several days without trophic factors. Here we hypothesize that the resistance of this motor neuron subpopulation to trophic factor deprivation can be attributed to diminished nitric oxide production resulting from the activity of the arginine-degrading enzyme arginase. When incubated with nor-N^G-hydroxy-nor-L-arginine (NOHA), the normally resistant trophic factor-deprived motor neurons showed a drop in survival rates, whereas trophic factor-treated neurons did not. NOHA-induced motor neuron death was inhibited by blocking nitric oxide synthesis and the scavenging of superoxide and peroxynitrite, suggesting that peroxynitrite mediates NOHA toxicity. When we transfected arginase 1 into motor neurons to see whether it alone could abrogate trophic factor deprivation-induced death, we found that its forced expression did indeed do so. The protection afforded by arginase 1 expression is reversed when cells are incubated with NOHA or with low concentrations of nitric oxide. These results reveal that arginase acts as a central regulator of trophic factor-deprived motor neuron survival by suppressing nitric oxide production and the consequent peroxynitrite toxicity. They also suggest that the resistance of motor neuron subpopulations to trophic factor deprivation may result from increased arginase activity.  

4.411           Multiprotein Complexes of the Survival of Motor Neuron Protein SMN with Gemins Traffic to Neuronal Processes and Growth Cones of Motor Neurons

Zhang, H. et al

1. Neurosci., 26(33), 8622-8632 (2006)

  Spinal muscular atrophy (SMA), a progressive neurodegenerative disease affecting motor neurons, is caused by mutations or deletions of the SMN1 gene encoding the survival of motor neuron (SMN) protein. In immortalized non-neuronal cell lines, SMN has been shown to form a ribonucleoprotein (RNP) complex with Gemin proteins, which is essential for the assembly of small nuclear RNPs (snRNPs). An additional function of SMN in neurons has been hypothesized to facilitate assembly of localized messenger RNP complexes. We have shown that SMN is localized in granules that are actively transported into neuronal processes and growth cones. In cultured motor neurons, SMN granules colocalized with ribonucleoprotein Gemin proteins but not spliceosomal Sm proteins needed for snRNP assembly. Quantitative analysis of endogenous protein colocalization in growth cones after three-dimensional reconstructions revealed a statistically nonrandom association of SMN with Gemin2 (40%) and Gemin3 (48%). SMN and Gemin containing granules distributed to both axons and dendrites of differentiated motor neurons. A direct interaction between SMN and Gemin2 within single granules was indicated by fluorescence resonance energy transfer analysis of fluorescently tagged and overexpressed proteins. High-speed dual-channel imaging of live neurons depicted the rapid and bidirectional transport of the SMN–Gemin complex. The N terminus of SMN was required for the recruitment of Gemin2 into cytoplasmic granules and enhanced Gemin2 stability. These findings provide new insight into the molecular composition of distinct SMN multiprotein complexes in neurons and motivation to investigate deficiencies of localized RNPs in SMA.  

4.412           Role of Transcription Factor T-bet Expression by CD4+ Cells in Gastritis Due to Helicobacter pylori in Mice

Eaton, K.A., Benson, L.H., Haeger, J. and Gray, B.M. Infect. Immun., 74(8), 4673-4684 (2006)   Gastritis due to Helicobacter pylori is induced by a Th1-mediated response that is CD4 cell and gamma interferon (IFN- ) dependent. T-bet is a transcription factor that directs differentiation of and IFN- secretion by CD4⁺ Th1 T cells. The goal of this study was to use two mouse models to elucidate the role of T-bet in gastritis due to H. pylori. C57BL/6J mice, congenic T-bet knockout (KO) mutants, or congenic SCID (severe, combined immunodeficient) mutants were given live H. pylori by oral inoculation. SCID mice were given CD4⁺ splenocytes from C57BL/6J or T-bet KO mice by intraperitoneal injection. Twelve or 24 weeks after bacterial inoculation, C57BL/6J mice developed moderate gastritis but T-bet KO mice and SCID mice did not. In contrast, SCID recipients of either C57BL/6J T cells or T-bet KO T cells developed gastritis 4 or 8 weeks after adoptive transfer. In recipients of C57BL/6J CD4⁺ cells but not recipients of T-bet KO cells, gastritis was associated with a delayed-type hypersensitivity response to H. pylori antigen and elevated gastric and serum IFN- , interleukin 6, and tumor necrosis factor alpha. In spite of the absence of IFN- expression, indicating failure of Th1 differentiation, CD4⁺ T cells from T-bet KO mice induce gastritis in H. pylori-infected recipient SCID mice. This indicates that Th1-independent mechanisms can cause gastric inflammation and disease due to H. pylori.  

4.413           Procurement of the Human Pancreas for Pancreatic Islet Transplantation from Marginal Cadaver Donors

Nagata, H. et al Transplantation, 82(3), 327-331 (2006)   Background. Recent advances in pancreatic islet transplantation (PIT) have contributed significantly to the treatment of patients with type 1 diabetes. The specific aim of this study was to develop an effective technique for the procurement of pancreas for PIT from nonheart-beating-donor (NHBDs). Methods. Between January 2004 and August 2004, eight human pancreata were procured and processed for isolation of islets at a cell processing center. After confirmation of brain death status, a double balloon catheter was inserted to prevent warm ischemic damage to the donor pancreas by using an in situ regional organ cooling system that was originally developed for procurement of kidneys. The catheter position of the cooling system was modified specifically for the pancreas and kidney. Furthermore, we worked in cooperation with a kidney procurement team to protect the pancreas during kidney procurement. Results. Warm ischemic time could be controlled with the modified in situ regional cooling system at 3.0+/-0.8 min (mean+/-SE). The operations for procurement of the kidneys and pancreata lasted 45.6+/-3.6 min and 10.6+/-1.8 min, respectively. Islet yield per isolation was 444,426+/-35,172 IE (islet equivalent). All eight cases met the criteria for PIT based on the Edmonton protocol. Conclusion. We developed a novel procurement technique in cooperation with our kidney procurement team. This protocol for the procurement of pancreas and kidney from a NHBD enabled us to transplant islets into a type 1 diabetic patient and kidney into a renal failure patient.        

4.414           T cell surface redox levels determine T cell reactivity and arthritis susceptibility

Gelderman, K.A., Hultquist, M., Holmberg, J., Olafsson, P. and Holmdahl, R. PNAS, 103(34), 12831-12836 (2006)   Rats and mice with a lower capacity to produce reactive oxygen species (ROS) because of allelic polymorphisms in the Ncf1 gene (which encodes neutrophil cytosolic factor 1) are more susceptible to develop severe arthritis. These data suggest that ROS are involved in regulating the immune response. We now show that the lower capacity to produce ROS is associated with an increased number of reduced thiol groups (–SH) on T cell membrane surfaces. Artificially increasing the number of reduced thiols on T cells from animals with arthritis-protective Ncf1 alleles by glutathione treatment lowered the threshold for T cell reactivity and enhanced proliferative responses in vitro and in vivo. Importantly, T cells from immunized congenic rats with an E3-derived Ncf1 allele (DA.Ncf1^E3 rats) that cannot transfer arthritis to rats with an arthritis-associated Dark Agouti (DA)-derived mutated Ncf1 allele (DA.Ncf1^DA rats) became arthritogenic after increasing cell surface thiol levels. This finding was confirmed by the reverse experiment, in which oxidized T cells from DA.Ncf1^DA rats induced less severe arthritis compared with controls. Therefore, we conclude that ROS production as controlled by Ncf1 is important in regulating surface redox levels of T cells and thereby suppresses autoreactivity and arthritis development.  

4.415           Human Fallopian Tube Neutrophils – A Distinct Phenotype from Blood Neutrophils

Smith, J.M., Wira, C.R., Fanger, M.W. and Shen, L. Am. J. Reprod. Immunol., 56, 218-229 (2006)   Problem The role of neutrophils in the human Fallopian tube (FT) is unknown. In order to provide insights into their functions in the FT, we systematically compared neutrophils from normal FT and peripheral blood (PB). Method of study Flow cytometric analysis of surface receptors, granule proteins, and intracellular cytokines expressed by neutrophils from enzymatically dispersed FT and PB was performed. Results Fallopian tube neutrophils expressed significantly higher levels of CD64, human class II histocompatibility antigen DR (HLA-DR), γ-interferon, and vascular endothelial growth factor than those from PB. Fewer FT neutrophils expressed IL-8 receptors compared to PB, while more expressed the receptor for the bacterial-derived chemoattractant formyl-Met-Leu-Phe (fMLP). The number of FT neutrophils containing the granule proteins matrix metalloproteinase-9, lactoferrin, and myeloperoxidase was decreased versus PB. Conclusion Fallopian tube neutrophils exhibit a phenotype distinct from PB neutrophils, suggesting functional activation of innate immune defense in the female reproductive tract as well as a potential role in maintaining normal FT physiology.  

4.416           Successful Islet Transplantation from Nonheartbeating Donor Pancreata Using Modified Ricordi Islet Isolation Method

Matsumoto, S. et al Transplantation, 82(4), 460-465 (2006)   Background. Current success of islet transplantation has led to donor shortage and the need for marginal donor utilization to alleviate this shortage. The goal of this study was to improve the efficacy of islet transplantation using nonheartbeating donors (NHBDs). Methods. First, we used porcine pancreata for the implementation of several strategies and applied to human pancreata. These strategies included ductal injection with trypsin inhibitor for protection of pancreatic ducts, ET-Kyoto solution for pancreas preservation, and Iodixanol for islet purification. Results. These strategies significantly improved both porcine and human islet isolation efficacy. Average 399,469+/-36,411 IE human islets were obtained from NHBDs (n=13). All islet preparations met transplantation criteria and 11 out of 13 cases (85%) were transplanted into six type 1 diabetic patients for the first time in Japan. All islets started to secrete insulin and all patients showed better blood glucose control without hypoglycemic loss of consciousness. The average HbA1c levels of the six recipients significantly improved from 7.5+/-0.4% at transplant to 5.1+/-0.2% currently (P<0.0003). The average insulin amounts of the six recipients significantly reduced from 49.2+/-3.3 units at transplant to 11+/-4.4 units (P<0.0005) and five out of six patients reduced to less than half dose. The first patient is now insulin free, the first such case in Japan. Conclusion. This demonstrates that our current protocol makes it feasible to use NHBDs for islet transplant into type 1 diabetic patients efficiently.  

4.417           Evidence for epithelial-mesenchymal transitions in adult liver cells

Sicklick, J.K: et al

1. J. Physiol. Liver Physiol., 291, G575-G583 (2006)

  Both myofibroblastic hepatic stellate cells (HSC) and hepatic epithelial progenitors accumulate in damaged livers. In some injured organs, the ability to distinguish between fibroblastic and epithelial cells is sometimes difficult because cells undergo epithelial-mesenchymal transitions (EMT). During EMT, cells coexpress epithelial and mesenchymal cell markers. To determine whether EMT occurs in adult liver cells, we analyzed the expression profile of primary HSC, two HSC lines, and hepatic epithelial progenitors. As expected, all HSC expressed HSC markers. Surprisingly, these markers were also expressed by epithelial progenitors. In addition, one HSC line expressed typical epithelial progenitor mRNAs, and these epithelial markers were inducible in the second HSC line. In normal and damaged livers, small ductular-type cells stained positive for an HSC marker. In conclusion, HSC and hepatic epithelial progenitors both coexpress epithelial and mesenchymal markers, providing evidence that EMT occurs in adult liver cells.  

4.418           Evaluation of Islet Transplantation from Non-Heart Beating Donors

Noguchi, H. et al Am. J. transplant., 6, 2476-2482 (2006)   We evaluated islet transplantation from non-heart beating donors (NHBDs) with our Kyoto Islet Isolation Method. All patients had positive C-peptide after transplantation. The average HbA_1C levels of the five recipients significantly improved from 7.8 ± 0.4% at transplant to 5.2 ± 0.2% currently (p < 0.01). Three patients with no or a single autoantibody became insulin independent while the other two patients with double autoantibodies reduced their insulin requirement but did not become insulin independent. C-peptide in patients who became insulin-independent gradually increased after each transplantation whereas C-peptide in patients who did not become insulin-independent from 3 months after the first transplantation to the next transplantation dramatically decreased. The β-score of the three patients who became insulin independent was the best of eight. In conclusion, our method makes it feasible to use NHBDs for islet transplant into type 1 diabetic patients efficiently.  

4.419           Immunophenotype and functions of fetal baboon bone-marrow derived dendritic cells

Awasthi, S. and Cropper, J. Cell. Immunol., 240(1), 31-40 (2006)   Dendritic cells (DCs) are unique antigen-presenting cells that can take up pathogens, pathogens-derived and stress-antigens and stimulate antigen-specific immune response. Here we investigated the immunobiology of fetal DCs and compared their phenotype and activation status against infectious stimuli with those of young and adult baboons. The DCs were obtained from femoral bone-marrow (BMDCs) of fetus (140 and 175 days of gestation), young (4–5 years old) and mature adult (10–35 years old) baboons. The cells were cultured in the presence of GM-CSF and IL-4. To study phagocytic ability of BMDCs, the cells were harvested on 6th day and incubated with fluorescent-labeled Escherichia coli bioparticles. The BMDCs were also treated with E. coli O111:B4 lipopolysaccharide (LPS) for 24 h and changes in expression of cell-surface markers and IL-12 were studied using distinct immunoassays. We found that the phenotype and morphology of BMDCs from fetal, young and adult baboons were similar and showed increased expression of HLA-DP, DQ, DR and T cell co-stimulatory molecules upon LPS treatment. However, significant differences were observed in phagocytic activity and IL-12 secretion among BMDCs from these sources. The ability of fetal baboon BMDCs to phagocytose E. coli bioparticles was significantly lower and they secreted lower level of LPS-stimulated IL-12 as compared to the BMDCs from adult baboon. These results suggest that compared to adult BMDCs, fetal baboon BMDCs are less efficient in mounting immune response against Gram-negative bacterial stimuli.  

4.420           NF- B/Rel Regulates Inhibitory and Excitatory Neuronal Function and Synaptic Plasticity

O’Mahony, A. et al Mol. Biol. Cell., 26(19), 7283-7298 (2006)   Changes in synaptic plasticity required for memory formation are dynamically regulated through opposing excitatory and inhibitory neurotransmissions. To explore the potential contribution of NF- B/Rel to these processes, we generated transgenic mice conditionally expressing a potent NF- B/Rel inhibitor termed I B superrepressor (I B -SR). Using the prion promoter-enhancer, I B -SR is robustly expressed in inhibitory GABAergic interneurons and, at lower levels, in excitatory neurons but not in glia. This neuronal pattern of I B -SR expression leads to decreased expression of glutamate decarboxylase 65 (GAD65), the enzyme required for synthesis of the major inhibitory neurotransmitter, -aminobutyric acid (GABA) in GABAergic interneurons. I B -SR expression also results in diminished basal GluR1 levels and impaired synaptic strength (input/output function), both of which are fully restored following activity-based task learning. Consistent with diminished GAD65-derived inhibitory tone and enhanced excitatory firing, I B -SR⁺ mice exhibit increased late-phase long-term potentiation, hyperactivity, seizures, increased exploratory activity, and enhanced spatial learning and memory. I B -SR⁺ neurons also express higher levels of the activity-regulated, cytoskeleton-associated (Arc) protein, consistent with neuronal hyperexcitability. These findings suggest that NF- B/Rel transcription factors act as pivotal regulators of activity-dependent inhibitory and excitatory neuronal function regulating synaptic plasticity and memory.  

4.421           Clonal deletion of thymocytes by circulating dendritic cells homing to the thymus

Bonasio, R. et al Nature Immunol., 7(10), 1092-1100 (2006)   Dendritic cell (DC) presentation of self antigen to thymocytes is essential to the establishment of central tolerance. We show here that circulating DCs were recruited to the thymic medulla through a three-step adhesion cascade involving P-selectin, interactions of the integrin VLA-4 with its ligand VCAM-1, and pertussis toxin–sensitive chemoattractant signaling. Ovalbumin-specific OT-II thymocytes were selectively deleted after intravenous injection of antigen-loaded exogenous DCs. We documented migration of endogenous DCs to the thymus in parabiotic mice and after painting mouse skin with fluorescein isothiocyanate. Antibody to VLA-4 blocked the accumulation of peripheral tissue–derived DCs in the thymus and also inhibited the deletion of OT-II thymocytes in mice expressing membrane-bound ovalbumin in cardiac myocytes. These findings identify a migratory route by which peripheral DCs may contribute to central tolerance.  

4.422           Mutational Analyses of Taiwanese Kindred With X-linked Adrenoleukodystrophy

Chiu, H-C., Liang, J-S., Wang, J-S. and Feng, J-F. Pediatric Neurol., 35(4), 250-256 (2006)   X-linked adrenoleukodystrophy is a neurodegenerative disorder with highly variable clinical presentation, including the childhood cerebral form, adult form adrenomyeloneuropathy, and Addison disease. The biochemical hallmark of the disorder is the accumulation of saturated very long chain fatty acids in all tissues and body fluids. This accumulation results from mutations in the ABCD1 gene localized to Xq28. Using polymerase chain reaction and direct sequencing of deoxyribonucleic acid, we identified five novel mutations, including a microdeletion (1624 del ATC), a splicing site mutation (intervening sequence 1 [IVS1] -2a>c), and three missense mutations (1172 T>C, 1520 G>A, and 1754 T>C), from Taiwanese kindred with X-linked adrenoleukodystrophy. A polymorphism involving a single nucleotide deletion in the intervening sequence 5 (IVS5 -6 del c) of the ABCD1 gene, previously misattributed as a mutation in the Chinese population, was also identified. The dinucleotide deletion (1415 del AG) mutation common in Japan and Western countries was not found as frequently in the Chinese and Taiwanese populations. Instead, a higher mutation frequency was observed in exon 6 of the ABCD1 gene among Japanese, Chinese, and Taiwanese kindred with X-linked adrenoleukodystrophy, representing a potential mutational hotspot for future mutational screening among these Asian populations.  

4.423           Islet neogenesis associated protein transgenic mice are resistant to hyperglycemia induced by streptozotocin

Taylor-Fishwick, D.A. et al

1. Endocrinol., 190, 729-737 (2006)

  Islet neogenesis associated protein (INGAP) is a protein factor that can stimulate new islet mass from adult pancreatic progenitor cells. In models of islet neogenesis, INGAP expression is elevated in pancreatic acinar cells. Using a transgenic model to drive a sustained expression of INGAP in pancreatic acinar cells, we have identified a protection to chemical-induced hyperglycemia. A sustained expression of INGAP during development did not perturb islet development or basal blood glucose homeostasis, although ß-cell mass and pancreatic insulin content were significantly increased in the INGAP transgenic mice. When challenged with a diabetogenic dose of streptozotocin (STZ), mice carrying the INGAP transgene did not become hyperglycemic. In contrast, wild-type mice became and remained hyperglycemic, blood glucose > 550 mg/dl. The serum insulin levels and islet morphology were preserved in the transgenic mice after STZ treatment. These data suggest that the sustained expression of INGAP in the acinar pancreas confers resistance to a diabetogenic insult. The INGAP transgenic mouse provides a new model to uncover factors that are protective to diabetes onset and biomarkers to track ß-cell pathology.  

4.424           Characterization of progesterone receptor isoform expression in fetal membranes

Mills, A.A. et al Am. J. Obstet. Gynecol., 195, 998-1003 (2006)   Objective To quantify expression of progesterone receptor (PR) messenger RNA (mRNA) isoforms in fetal membranes, and to determine whether these levels change in culture. Study design Placentas from women undergoing term cesarean delivery before labor were collected. Layers of amnion, chorion, and decidua were separated manually, enzymatically digested, and separated further with the use of a density gradient. RNA was extracted immediately and after culture for 48 hours, then analyzed by quantitative reverse transcription polymerase chain reaction for PR-A, PR-B, and β-2 microglobulin mRNA expression. Separation of cell types was confirmed by immunohistochemistry. Results PR isoform expression was identified in fetal membranes, with levels highest in decidua and below the limits of detection in amnion. The ratio of PR-A/PR-B mRNA was not significantly different between cell layers. PR mRNA isoform levels did not differ significantly in fresh versus cultured cells. Conclusion Quantitative reverse transcription polymerase chain reaction was used to quantitate expression of PR mRNA isoforms in cells of fetal membranes and to validate systems for further study of PR with respect to inflammation, infection, and preterm delivery.    

4.425           A detailed study of time-dependent changes in human red blood cells: from reticulocyte maturation to erythrocyte senescence

Gifford, S.C., Derganc, J., Shevkoplyas, S.S., Yoshida, T. and Bitensky, M.W. Br. J. Hematol., 135, 395-404 (2006)   The use of microfabrication technology in the study of biological systems continues to grow rapidly in both prevalence and ascendancy. Customised microdevices that provide superior results than traditional macroscopic methods can be designed in order to investigate specific cell types and cellular processes. This study showed the benefit of this approach in precisely characterising the progressive losses of surface area and haemoglobin (Hb) content by the human red blood cell (RBC), from newborn reticulocyte to senescent erythrocyte. The high-throughput, multiparametric measurements made on individual cells with a specialised microdevice enabled, for the first time, delineation and quantification of the losses that occur during the two stages of the human RBC lifespan. Data acquired on tens of thousands of red cells showed that nearly as much membrane area is lost during the 1–2 d of reticulocyte maturation (c. 10–14%) as in the subsequent 4 months of erythrocyte ageing (c. 16–17%). The total decrease in Hb over the red cell lifespan is also estimated (c. 15%) and a model describing the complete time-course of diminishing mean RBC area and Hb is proposed. The relationship between the losses of Hb and area, and their possible influence on red cell lifespan, are discussed.  

4.426           The Src Homology 2 Domain-Containing Leukocyte Protein of 76-kDa Adaptor Links Integrin Ligation with p44/42 MAPK Phosphorylation and Podosome Distribution in Murine Dendritic Cells

Luckashenak, N.A., Ryszkiewizs, R..L., Ramsey, K.D. and Clements, J.L.

1. Immunol., 177, 5177-5185 (2006)

  The Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) is an important molecular intermediate in multiple signaling pathways governing immune cell function. In this study, we report that SLP-76 is expressed in CD11c⁺B220^– dendritic cells (DCs) isolated from murine thymus or spleen, and that SLP-76 is rapidly phosphorylated on tyrosine residues upon plating of bone marrow-derived DCs (BMDCs) on integrin agonists. SLP-76 is not required for the in vitro or in vivo generation of DCs, but SLP-76-deficient BMDCs adhere poorly to fibronectin, suggesting impaired integrin function. Consistent with impaired adhesion, cutaneous SLP-76-deficient DCs leave ear tissue at an elevated frequency compared with wild-type DCs. In addition, the pattern and distribution of actin-based podosome formation are visibly altered in BMDCs lacking SLP-76 following integrin engagement. SLP-76-deficient BMDCs manifest multiple signaling defects following integrin ligation, including reduced global tyrosine phosphorylation and markedly impaired phosphorylation of p44/42 MAPK (ERK1/2). These data implicate SLP-76 as an important molecular intermediate in the signaling pathways regulating multiple integrin-dependent DC functions, and add to the growing body of evidence that hemopoietic cells may use unique molecular intermediates and mechanisms for regulating integrin signaling.  

4.427           Oestrogen Synthesis in the Hippocampus: Role in Axon Outgrowth

Von Schassen, C. et al

1. Neuroendocrinol., 18, 847-856 (2006)

  Ovarian oestrogens have been postulated to be neuroprotective. It has also been shown that considerable amounts of oestrogens are synthesised in hippocampal neurones. In the present study, we focused on a potential role of hippocampus-derived oestradiol compared to gonad-derived oestradiol on axon outgrowth of hippocampal neurones. To address the role of hippocampus-derived oestradiol, we inhibited oestrogen synthesis by treatment of neonatal hippocampal cell cultures with letrozole, a specific aromatase inhibitor. As an alternative, we used siRNA against steroidogenic acute regulatory protein (StAR). Axon outgrowth and GAP-43 expression were significantly down-regulated in response to letrozole and in siRNA-StAR transfected cells. The effects after inhibition of oestrogen synthesis in response to letrozole and in siRNA-StAR transfected cells were reversed by oestrogen supplementation. No difference was found between ovariectomised animals, cycling animals at pro-oestrus and ovariectomised and subsequently oestradiol-treated animals. However, high pharmacological doses of oestradiol promoted axon outgrowth, which was possible to abolish by the oestrogen receptor antagonist ICI 182,780. Our results show that oestradiol-induced neurite outgrowth is very likely mediated by genomic oestrogen receptors and requires higher doses of oestradiol than physiological serum concentrations derived from the gonads.      

4.428           Prion Protein Expression by Mouse Dendritic Cells Is Restricted to the Nonplasmacytoid Subsets and Correlates with the Maturation State

Martinez de Hoya, G., Lopez-Bravo, M., Metharom, P., Ardavin, C. and Aucounterier, P.

1. Immunol., 177, 6137-6142 (2006)

  Expression of the physiological cellular prion protein (PrP^C) is remarkably regulated during differentiation and activation of cells of the immune system. Among these, dendritic cells (DCs) display particularly high levels of membrane PrP^C, which increase upon maturation, in parallel with that of molecules involved in Ag presentation to T cells. Freshly isolated mouse Langerhans cells, dermal DCs, and DCs from thymus, spleen, and mesenteric lymph nodes expressed low to intermediate levels of PrP^C. Highest levels of both PrP^C and MHC class II molecules were displayed by lymph node CD8 ^int DCs, which represent fully mature cells having migrated from peripheral tissues. Maturation induced by overnight culture resulted in increased levels of surface PrP^C, as did in vivo DC activation by bacterial LPS. Studies on Fms-like tyrosine kinase 3 ligand bone marrow-differentiated B220^– DCs confirmed that PrP^C expression followed that of MHC class II and costimulatory molecules, and correlated with IL-12 production in response to TLR-9 engagement by CpG. However, at variance with conventional DCs, B220⁺ plasmacytoid DCs isolated from the spleen, or in vitro differentiated, did not significantly express PrP^C, both before and after activation by TLR-9 engagement. PrP knockout mice displayed higher numbers of spleen CD8 ⁺ DCs, but no significant differences in their maturation response to stimulation through TLR-4 and TLR-9 were noticed. Results are discussed in relation to the functional relevance of PrP^C expression by DCs in the induction of T cell responses, and to the pathophysiology of prion diseases.  

4.429           Lentiviral vector expressing retinoic acid receptor ß2 promotes recovery of function after corticospinal tract injury in the adult rat spinal cord

Yip, P.K. et al Hum. Mol. Genet., 15(21), 3107-3118 (2006)   Spinal cord injury often results in permanent and devastating neurological deficits and disability. This is due to the limited regenerative capacity of neurones in the central nervous system (CNS). We recently demonstrated that a transcription factor retinoic acid receptor ß2 (RARß2) promoted axonal regeneration in adult sensory neurones located peripherally. However, it is not known if RARß2 can promote axonal regeneration in cortical neurones of the CNS. Here, we demonstrate that delivery of RARß2 via a lentiviral vector to adult dissociated cortical neurones significantly enhances neurite outgrowth on adult cortical cryosections, which normally provide an unfavourable substrate for growth. We also show that lentiviral-mediated transduction of corticospinal neurones resulted in robust transgene expression in layer V corticospinal neurones and their axonal projections in the corticospinal tract (CST) of the spinal cord. Expression of RARß2 in these neurones enhanced regeneration of the descending CST fibres after injury to these axons in the mid-cervical spinal cord. Furthermore, we observed functional recovery in sensory and locomotor behavioural tests in RARß2-treated animals. These results suggest that a direct and selective delivery of RARß2 to the corticospinal neurones promotes long-distance functional regeneration of axons in the spinal cord and may thus offer new therapeutic gene strategy for the treatment of human spinal cord injuries.  

4.430           Preadipocytes Mediate Lipopolysaccharide-Induced Inflammation and Insulin Resistance in Primary Cultures of Newly Differentiated Human Adipocytes

Chung, S. et al Endocrinology, 147(11), 5340-5351 (2006)   Recent data suggest that proinflammatory cytokines secreted from adipose tissue contribute to the morbidity associated with obesity. However, characterization of the cell types involved in inflammation and how these cells promote insulin resistance in human adipocytes are unclear. We simulated acute inflammation using the endotoxin lipopolysaccharide (LPS) to define the roles of nonadipocytes in primary cultures of human adipocytes. LPS induction of the mRNA levels of proinflammatory cytokines (e.g. IL-6, TNF- , and IL-1ß) and chemokines (e.g. IL-8, monocyte chemoattractant protein-1) occurred primarily in the nonadipocyte fraction of newly differentiated human adipocytes. Nonadipocytes were characterized as preadipocytes based on their abundant mRNA levels of preadipocyte markers preadipocyte factor-1 and adipocyte enhancer protein-1 and only trace levels of markers for macrophages and myocytes. The essential role of preadipocytes in inflammation was confirmed by modulating the degree of differentiation in the cultures from approximately 0–90%. LPS-induced proinflammatory cytokine/chemokine expression and nuclear factor- B and MAPK signaling decreased as differentiation increased. LPS-induced cytokine/chemokine expression in preadipocytes was associated with: 1) decreased adipogenic gene expression, 2) decreased ligand-induced activation of a peroxisome proliferator activated receptor (PPAR)- reporter construct and increased phosphorylation of PPAR , and 3) decreased insulin-stimulated glucose uptake. Collectively, these data demonstrate that LPS induces nuclear factor- B- and MAPK-dependent proinflammatory cytokine/chemokine expression primarily in preadipocytes, which triggers the suppression of PPAR activity and insulin responsiveness in human adipocytes.  

4.431           Naltrexone, an opioid receptor antagonist, attenuates liver fibrosis in bile duct ligated rats

Ebrahimkhani, M.R. et al Gut, 55, 1606-1616 (2006)   Aim: The aim of this study was to investigate the hypothesisthat the opioid system is involved in the development of hepaticfibrosis. Methods: The effect of naltrexone (an opioid receptor antagonist)on hepatic fibrosis in bile duct ligated (BDL) or sham ratswas assessed by histology and hepatic hydroxyproline levels.Liver matrix metalloproteinase 2 (MMP-2) was measured by zymography,and smooth muscle actin (-SMA) and CD45 (leucocyte common antigen)by immunohistochemistry. The redox state of the liver was assessedby hepatic glutathione (GSH)/oxidised glutathione (GSSG) andS-nitrosothiol levels. Subtypes of opioid receptors in culturedhepatic stellate cells (HSCs) were characterised by reversetranscriptase-polymerase chain reaction, and the effects ofselective opioid receptor agonists on cellular proliferation,tissue inhibitor of metalloproteinase 1 (TIMP-1), and procollagenI expression in HSCs determined. Results: Naltrexone markedly attenuated the development of hepatic fibrosis as well as MMP-2 activity (p<0.01), and decreased the number of activated HSCs in BDL rats (p<0.05). The development of biliary cirrhosis altered the redox state with a decreased hepatic GSH/GSSG ratio and increased concentrations of hepatic S-nitrosothiols, which were partially or completely normalised by treatment with naltrexone, respectively. Activated rat HSCs exhibited expression of 1 receptors, with increased procollagen I expression, and increased TIMP-1 expression in response to ₁ and ₂ agonists, respectively. Conclusions: This is the first study to demonstrate that administrationof an opioid antagonist prevents the development of hepaticfibrosis in cirrhosis. Opioids can influence liver fibrogenesisdirectly via the effect on HSCs and regulation of the redoxsensitive mechanisms in the liver.  

4.432           CLC-3 Channels Modulate Excitatory Synaptic Transmission in Hippocampal Neurons

Wang, X.Q. et al Neuron, 52, 321-333 (2006)   It is well established that ligand-gated chloride flux across the plasma membrane modulates neuronal excitability. We find that a voltage-dependent Cl⁻ conductance increases neuronal excitability in immature rodents as well, enhancing the time course of NMDA receptor-mediated miniature excitatory postsynaptic potentials (mEPSPs). This Cl⁻ conductance is activated by CaMKII, is electrophysiologically identical to the CaMKII-activated CLC-3 conductance in nonneuronal cells, and is absent in clc-3^−/− mice. Systematically decreasing [Cl⁻]_i to mimic postnatal [Cl⁻]_i regulation progressively decreases the amplitude and decay time constant of spontaneous mEPSPs. This Cl⁻-dependent change in synaptic strength is absent in clc-3^−/− mice. Using surface biotinylation, immunohistochemistry, electron microscopy, and coimmunoprecipitation studies, we find that CLC-3 channels are localized on the plasma membrane, at postsynaptic sites, and in association with NMDA receptors. This is the first demonstration that a voltage-dependent chloride conductance modulates neuronal excitability. By increasing postsynaptic potentials in a Cl⁻ dependent fashion, CLC-3 channels regulate neuronal excitability postsynaptically in immature neurons.  

4.433           A Phase I Study of In vitro Expanded Natural Killer T Cells in Patients with Advanced and Recurrent Non–Small Cell Lung Cancer

Motohashi, S. et al Clin. Cancer Res., 12(20), 6079-6086 (2006)   Purpose: Human V24 natural killer T (V24 NKT) cells bearingan invariant V24JQ antigen receptor are activated by a glicolipidligand -galactosylceramide (GalCer; KRN7000) in a CD1d-dependentmanner. The human V24 NKT cells activated with GalCer and interleukin-2have been shown to produce large amounts of cytokines, suchas IFN-, and also exerting a potent killing activity againstvarious tumor cell lines. We did a phase I study with autologousactivated V24 NKT cell therapy. Experimental Design: Patients with advanced or recurrent non–small cell lung cancer received i.v. injections of activated V 24 NKT cells (level 1: 1 x 10⁷/m² and level 2: 5 x 10⁷/m²) to testthe safety, feasibility, and clinical response of this therapeuticstrategy. Immunomonitoring was also done in all cases. Results: Six patients were enrolled in this study. No severeadverse events were observed during this study in any patients.After the first and second injection of activated V24 NKT cells,an increased number of peripheral blood V24 NKT cells was observedin two of three cases receiving a level 2 dose of activatedV24 NKT cells. The number of IFN--producing cells in peripheralblood mononuclear cells increased after the administration ofactivated V24 NKT cells in all three cases receiving the level2 dose. No patient was found to meet the criteria for eithera partial or a complete response. Conclusions: The clinical trial with activated V24 NKT celladministration was well tolerated and carried out safely withminor adverse events even in patients with advanced diseases.  

4.434           Temporospatial coupling of networked synaptic activation of AMPA-type glutamate receptor channels and calcium transients in cultured motoneurons

Jahn, K. et al Neuroscience, 142, 1019-1029 (2006)   AMPA-type glutamate receptor (GluR) channels provide fast excitatory synaptic transmission in the CNS, but mediate also cytotoxic insults. It could be shown that AMPA-type GluR channel–mediated chronic excitotoxicity leads to an increased intracellular calcium concentration and plays an important role in neurodegenerative diseases like for example amyotrophic lateral sclerosis (ALS). As calcium is an important mediator of various processes in the cell and calcium signals have to be very precise in the temporospatial resolution, excessive intracellular calcium increases can seriously impair cell function. It is still unclear if AMPA-type receptors can directly interact with the intracellular calcium homeostasis or if other mechanisms are involved in this process. The objective of this study was therefore to investigate the calcium homeostasis in rat motoneurons under physiological stimulation of AMPA-type GluR channels using calcium imaging techniques and patch-clamp recordings simultaneously. It was found that spontaneous excitatory postsynaptic currents of cultured motoneurons did not elicit significant intracellular calcium transients. Large intracellular calcium transients occurred only when preceding fast sodium currents were observed. Pharmacological experiments showed that activation of AMPA-type GluR channels during synaptic transmission has a great functional impact on the calcium homeostasis in motoneurons as all kinds of activity was completely blocked by application of the selective kainate- and AMPA-type GluR channel blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Furthermore we suggest from our experiments that calcium transients of several hundred milliseconds’ duration result from release of calcium from the endoplasmic reticulum via activation of ryanodine receptors (calcium-induced calcium release, CICR). Our results help to understand the regulatory function of AMPA-type GluR channels in the intracellular calcium homeostasis which is known to be disturbed in neurodegenerative diseases.  

4.435           Isolation of an adult blood-derived progenitor cell population capable of differentiation into angiogenic, myocardial and neural lineages

Porat, Y. et al Br. J. Hematol., 135, 703-714 (2006)   Blood-derived adult stem cells were previously considered impractical for therapeutic use because of their small numbers. This report describes the isolation of a novel human cell population derived from the peripheral blood, termed synergetic cell population (SCP), and defined by the expression of CD31^Bright, CD34⁺, CD45 ^/Dim and CD34^Bright, but not lineage-specific features. The SCP was capable of differentiating into a variety of cell lineages upon exposure to defined culture conditions. The resulting cells exhibited morphological, immunocytochemical and functional characteristics of angiogenic, neural or myocardial lineages. Angiogenic cell precursors (ACPs) expressed CD34, CD133, KDR, Tie-2, CD144, von Willebrand factor, CD31^Bright, concomitant binding of Ulex-Lectin and uptake of acetylated low density lipoprotein (Ac-LDL), secreted interleukin-8, vascular endothelial growth factor and angiogenin and formed tube-like structures in vitro. The majority of CD31^Bright ACP cells demonstrated Ac-LDL uptake. Neural cell precursors (NCPs) expressed the neuronal markers Nestin, βIII-Tubulin, and Neu-N, the glial markers GFAP and O4, and responded to neurotransmitter stimulation. Myocardial cell precursors (MCPs) expressed Desmin, cardiac Troponin and Connexin 43. In conclusion, the simple and rapid method of SCP generation and the resulting considerable quantities of lineage-specific precursor cells makes it a potential source of autologous treatment for a variety of diseases.    

4.436           Proinflammatory cytokine secretion from preadipocytes decreases PPARg expression and activity in primary cultures of human adipocytes

Chung, S., LaPoint, K., Kennedy, A., Troy, A. and McIntosh, M. FASEB J., 20, A163 (2006)   Recent data suggest that proinflammatory cytokines secreted from adipose tissue contribute to the morbidity associated with obesity. However, characterization of the cell types involved in inflammation in adipose tissue is unclear. To delineate the role that non-adipocytes play in inflammation, primary cultures of newly-differentiated human stromal vascular cells containing ~50% adipocytes and ~50% non-adipocytes were stimulated with LPS for 3 h. Subsequently, non-adipocytes were fractionated from adipocytes by centrifugation in 6% iodixanol (1.03g/ml) and gene expression in each fraction was quantified by qPCR. LPS induction of proinflammatory cytokine mRNA (IL-6, IL-8, TNF , IL-1ß, COX-2) occurred primarily in non-adipocytes. Non-adipocytes were characterized as preadipocytes based on high mRNA levels of preadipocyte markers Pref-1 and adipocytes enhancer protein-1(AEBP-1) and only trace levels of macrophage (CD68 and Mac-1) and myocyte (MyoD) markers. LPS-induced cytokine expression in preadipocytes was associated with suppression of peroxisome proliferator activated receptor (PPAR) and adiponectin gene expression in adipocytes. In parallel, LPS treatment increased PPAR phosphorylation and decreased the activity of a luciferase reporter construct containing a PPAR response element, indicating that LPS attenuates PPAR activity. Collectively, these data demonstrate that LPS induces proinflammatory gene expression in preadipocytes, leading to suppression of PPAR activity in adipocytes, thereby impairing insulin sensitivity.  

4.437           Rat liver endothelial cells isolated by anti-CD31 immunomagnetic separation lack fenestrae and sieve plates

DeLeve, L.D., Wang, X., McCuskey, M.K. and McCuskey, R.S. Am. J. Physiol. Gastrointest. Liver Physiol., 291, G1187-G1189 (2006)   The gold standard for the identification of sinusoidal endothelial cells (SEC) is the presence of fenestrae organized in sieve plates, which is characteristic of SEC in vivo. One of the methods currently in use to isolate SEC is immunomagnetic sorting for CD31. However, there is evidence to suggest that CD31 is not present on the surface of differentiated SEC. The present study used scanning electron microscopy to image rat hepatic endothelial cells isolated by anti-CD31 and immunomagnetic sorting and cells isolated by gradient centrifugation and centrifugal elutriation. Cells isolated by elutriation had well-developed fenestrae and sieve plates, whereas cells isolated by anti-CD31 and immunomagnetic sorting had significantly fewer fenestrae organized in sieve plates. In conclusion, cells isolated by anti-CD31 and immunomagnetic sorting lacked the hallmark features of SEC.  

4.438           Distinctive role of donor strain immature dendritic cells in the creation of allograft tolerance

Kim, Y.S. et al Int. Immunol., 18(12), 1771-1777 (2006)   Dendritic cells (DCs) are pivotal antigen-presenting cells and serve a unique role in initiating immunity. To test the hypothesis that pre-immunization of recipient with certain DC subsets of donor origin can influence graft outcome, we have studied the effects of immunization with allogeneic CD4⁺CD8^–CD11c⁺ dendritic cell (CD4⁺DC) and CD4^–CD8⁺CD11c⁺ dendritic cell (CD8⁺DC) on the allograft response. Although both immature CD4⁺DC and CD8⁺DC subsets from DBA/2 were able to prime naive allogeneic C57BL/6 (B6) T cells in mixed lymphocyte reaction (MLR), CD8⁺DC exerted more vigorous alloimmune responses than CD4⁺DC did. Also, CD4⁺DC-driven allogeneic T cell response was attenuated more significantly by anti-CD154 mAb than CD8⁺DC-driven response. Consistent with the MLR results, combined pre-treatment with CD4⁺DC, but not CD8⁺DC, plus anti-CD154 mAb produced donor strain-specific long-term graft survival and induced tolerance while treatment with CD8⁺DC plus anti-CD154 mAb created minimal prolongation of allograft survival in a pancreas islet transplant model (DBA/2 B6). The beneficial effects exerted by CD4⁺DC and anti-CD154 mAb pre-treatment were correlated with T_h1 to T_h2 immune deviation and with the amplified donor-specific suppressive capacity by recipient CD4⁺CD25⁺ T cells. These findings highlight the capacity of CD4⁺DC to modulate alloimmune responses, and suggest therapeutic approaches for the induction of donor-specific tolerance.  

4.439           Contribution of Calcium Influx in Mediating Glucose-Stimulated Oxygen Consumption in Pancreatic Islets

Sweet, I.R. and Gilbert, M. Diabetes, 55(12), 3509-3519 (2006)   In brain, muscle, and pancreatic islets, depolarization induces an increase in respiration, which is dependent on calcium influx. The goal of this study was to assess the quantitative significance of this effect in islets relative to glucose-stimulated ATP turnover, to examine the molecular mechanism mediating the changes, and to investigate the functional implications with respect to insulin secretion. Glucose (3–20 mmol/l) increased steady-state levels of cytochrome c reduction (32–66%) in isolated rat islets, reflecting an increased production of NADH, and oxygen consumption rate (OCR) by 0.32 nmol/min/100 islets. Glucose-stimulated OCR was inhibited 30% by inhibitors of calcium influx (diazoxide or nimodipine), whereas a protein synthesis inhibitor (emetine) decreased it by only 24%. None of the inhibitors affected cytochrome c reduction, suggesting that calcium’s effect on steady-state OCR is mediated by changes in ATP usage rather than the rate of NADH generation. 3-isobutyl-1-methylxanthine increased insulin secretion but had little effect on OCR, indicating that the processes of movement and exocytosis of secretory granules do not significantly contribute to ATP turnover. At 20 mmol/l glucose, a blocker of sarcoendoplasmic reticulum calcium ATPase (SERCA) had little effect on OCR despite a large increase in cytosolic calcium, further supporting the notion that influx of calcium, not bulk cytosolic calcium, is associated with the increase in ATP turnover. The glucose dose response of calcium influx–dependent OCR showed a remarkable correlation with insulin secretion, suggesting that the process mediating the effect of calcium on ATP turnover has a role in the amplification pathway of insulin secretion.  

4.440           Age-related changes in monocyte and platelet cyclooxygenase expression in healthy male humans and rats

Kang, K.B., Van Der Zypp, A., Iannazzo, L. and Majewski, H. Translational Res., 148, 289-294 (2006)   Cyclooxygenase (COX) catalyses the formation of prostanoids that are crucial in maintaining hemostasis and important in inflammation. Animal studies reveal that COX-1 and COX-2 expression increase in some cell types during aging. This study determined age-related changes in COX expression in platelets and monocytes. Platelets and mononuclear cells were isolated from healthy male human volunteers from 18 to 28 and from 55 to 65 years of age, as well as male rats 8 and 54 weeks old for comparison. Western blot analysis was performed using selective antibodies against COX-1 and COX-2, followed by densitometrical analysis. In humans, an age-related increase in COX-2 expression in mononuclear cells was observed, with a 70% increase in the older age group. In rat studies, a 50% increase of COX-2 protein occurred in mononuclear cells of 54-week-old rats, compared with 8-week-old rats. For COX-1, an age-related increase of 50% occurred in rat platelets, but no difference occurred in the platelets’ COX-1 levels between young and elderly human age groups. The increased COX-2 in monocytes of older humans, which is mirrored in rats, may have downstream implications in atherosclerosis and cardiovascular risk as mononuclear prostanoids are implicated in atherosclerotic plaque stability.  

4.441           Aminophospholipid Translocase Activity and Phosphatidylserine Externalization in Sickle Red Blood Cell Subpopulations

Arnold, L.E., Palascak, M.B., Ciraolo, P., Joiner, C.H. and Franco, R.S. Blood, 108, Abstract 1241 (2006)   Red blood cell (RBC) membrane phosphatidylserine (PS) is normally confined to the inner leaflet. In sickle RBC, however, PS externalization has been observed and may contribute to thrombogenesis, endothelial adhesion, and shortened RBC lifespan. Increased calcium leads to PS externalization through inhibition of aminophospholipid translocase (APLT), which normally returns external PS to the inner leaflet, and activation of phospholipid scramblase, which allows nonspecific phospholipid equilibration between the leaflets. Sickle RBC in the light and dense (dehydrated) fractions exhibit increased PS externalization compared to sickle RBC in the normal density fractions. Sickle RBC with modest PS exposure (Type I PS+) occur in both light and dense fractions and, especially in the light fractions, include many reticulocytes. Sickle Cells with high levels of PS exposure (Type II PS+) occur predominantly in the dense fraction. Type II PS+ sickle cells have low levels of HbF, are more adherent than Type I PS+ cells, and tend to increase in number as sickle cells age in the circulation. Dense sickle cells have decreased APLT activity and this has been considered necessary (but probably not sufficient) for PS externalization. In the current studies we have examined the relationship between PS externalization and translocase activity in sickle cell density fractions. APLT activity and PS externalization were determined flow cytometrically using NBD-PS internalization and annexin V-phycoerythrin binding respectively. Sickle RBC density fractions were isolated using a discontinuous Optiprep® gradient,and two fractions were selected for further study, Fx 4 withessentially normal hydration (1.093< g/cc <1.100) andthe dehydrated Fx 6 (> 1.120 g/cc). As expected, Fx 6 sickleRBC had a decreased percentage of APLT positive cells (29.9±10.8,N=4) compared to normal RBC (94.5±3.6, N=3) and Fx 4sickle RBC (92.9±2.3, N=4). Fx 4 contained 5.7±2.8%Type I and 0.3±0.2% Type II PS+ cells. Fx 6 had 9.1±2.5%Type I and 3.2±2.3% Type II PS+ cells. Figure 1 showsthe percentage of APLT positive cells in the PS negative (PS-),Type I PS+, and Type II PS+ groups for Fx 4 and Fx 6. Type IPS+ cells in Fx 4 are positive for APLT, whereas those in Fx6 are mostly negative. In Fx 4, the Type I PS+ group most likelycontains many reticulocytes, and in a separate experiment TfR+reticulocytes had a uniform, slightly elevated APLT activity.The Type II PS+ cells in both Fx 4 and Fx 6 had decreased percentagesof APLT positive cells. It thus appears that high levels ofPS externalization, regardless of hydration state, are associatedwith low APLT activity. We therefore conclude: 1.In normally hydrated sickle cells, Type II PS+ cells, but notType I PS+ cells, are associated with decreased APLT; 2.In densesickle RBC, both Type I and Type II PS+ cells are associatedwith decreased APLT. These results further emphasize the pathologicnature of the Type II PS+ sickle cells.  

4.442           Advanced methods for Handling and Preparation of Stallion Semen

Loomis, P.R. Vet. Clin. Equine, 22, 663-676 (2006)   Since the advent of artificial insemination, practitioners and researchers have been concerned with semen handling techniques. Practices that minimize damage and maximize viability, survival, and fertility of spermatozoa are required for assisted reproductive technologies (ARTs). Many procedures for processing semen and ARTs require the separation of spermatozoa from seminal components. Semen is composed of a heterogeneous population of viable and nonviable spermatozoa suspended in secretions from various accessory sex glands. Semen may also contain other cells (eg. Leukocytes, epithelial cells, erythrocytes, immature germ cells) and contaminants (eg. bacteria, viruses, urine).  Sperm separation techniques are used by practitioners and researchers for several reasons. One reason is to concentrate spermatozoa and remove seminal plasma before cooling or freezing semen. Another reason is to separate spermatozoa from seminal plasma and to select an enriched population of viable spermatozoa from the ejaculate. The purpose of this article is to outline reasons and procedures for separation and selection of equine spermatozoa.  

4.443           Cervical duct cannulation in sheep for collection of afferent lymph dendritic cells from head tissues

Schwartz-Cornil, I., Epardaud, M. and Bonneau, M. Nature Protocols, 1(2), 874-879 (2006)   Pseudo-afferent cervical lymph-duct cannulation in a sheep model allows large amounts of lymph cells to be collected under physiological conditions, carrying immune signaling information from the head tissues, including oro-nasal mucosae. Importantly, large quantities of dendritic cells (DCs) of several subtypes are obtained (up to 8 million per overnight collection), as well as many other trafficking leukocytes. The technique includes three steps: removal of all head lymph nodes on one side (2 h), catheterization of cervical lymph ducts after 2 months (2–3 h) and collection/purification of lymph-cell subsets (4 h). The approach is challenging (1 in 3 success rate) but fruitful, and can be used to study DC subsets under immunomodulation, in order to assess lymph-cell subset dynamic changes and antigen transportation from oro-nasal tissues. This protocol is directed to experienced postdoctoral researchers.  

4.444           Neuropeptide Urocortin and Its Receptors Are Expressed in Rat Kupffer Cells

Charalampopoulos, I. et al Neuroendocrinol., 84, 49-57 (2006)  

The stress neuropeptides, corticotropin-releasing hormone (CRH) and urocortin (UCN), modulate the inflammatory response via the hypothalamus-pituitary-adrenal axis and locally, in a paracrine manner, act on mast and macrophage cells. Kupffer cells (KCs) are the resident macrophages of the liver. They represent the bulk of tissue macrophages in the body and they are the first to face invading noxious agents reaching the body via the portal circulation. The aim of the present report was to study the expression of the CRH system in rat KC and test its functionality. Our findings are as follows: (1) In highly purified KCs the transcripts of UCN, of its receptors CRHR1, CRHR2 and that of the pseudoreceptor CRH-binding protein (CRHBP) were present while that of CRH was not detectable. (2) Similarly, immunoreactive UCN, CRHR1, CRHR2 and CRHBP were easily detectable by immunohistochemistry and immunofluorescence in sections of whole rat liver (localized in KC) as well as in purified KC while CRH was again not detectable. (3) Exposure of purified KC to CRH or UCN suppressed lipopolysaccharide-induced tumor necrosis factor alpha production, an effect completely prevented by the CRHR1 and CRHR2 receptor antagonist astressin. Our data demonstrate the presence of UCN and its receptors in rat KC, the absence of CRH, and the functionality of these receptors. We propose that a UCN-based system may affect local inflammatory phenomena in the liver acting in a paracrine manner.

 

4.445           Lack of UCP2 reduces fas-mediated liver injury in ob/ob mice and reveals importance of cell-specific UCP2 expression

Fülöp, P., Derdak, Z., Sheets, A., Sabo, E., Berthiaume, E.P., Resnick, M.B., Wands, J.R., Pragh, G. and baffy, G. Hepatology, 44(3), 592-601 (2006)   Fatty liver is vulnerable to conditions that challenge hepatocellular energy homeostasis. Lipid-laden hepatocytes highly express uncoupling protein-2 (UCP2), a mitochondrial carrier that competes with adenosine triphosphate (ATP) synthesis by mediating proton leak. However, evidence for a link between UCP2 expression and susceptibility of liver to acute injury is lacking. We asked whether absence of UCP2 protects ob/ob mice from Fas-mediated acute liver damage. UCP2-deficient ob/ob mice (ob/ob:ucp2−/−) and UCP2-competent littermates (ob/ob:ucp2+/+) received a single dose of agonistic anti-Fas antibody (Jo2). Low-dose Jo2 (0.15 mg/kg intraperitoneally) caused less serum alanine aminotransferase (ALT) elevation and lower apoptosis rates in ob/ob:ucp2−/− mice. High-dose Jo2 (0.40 mg/kg intraperitoneally) proved uniformly fatal; however, ob/ob:ucp2−/− mice survived longer with less depletion of liver ATP stores, indicating that fatty hepatocytes may benefit from lack of UCP2 during Jo2 challenge. Although UCP2 reportedly controls mitochondrial oxidant production, its absence had no apparent effect on fatty liver tissue malondialdehyde levels augmented by Jo2. This finding prompted us to determine UCP2 expression in Kupffer cells, a major source of intrahepatic oxidative stress. UCP2 expression was found diminished in Kupffer cells of untreated ob/ob:ucp2+/+ mice, conceivably contributing to increased oxidative stress in fatty liver and limiting the impact of UCP2 ablation. In conclusion, whereas UCP2 abundance in fatty hepatocytes exacerbates Fas-mediated injury by compromising ATP stores, downregulation of UCP2 in Kupffer cells may account for persistent oxidative stress in fatty liver. Our data support a cell-specific approach when considering the therapeutic effects of mitochondrial uncoupling in fatty liver disease.  

4.446           Proteomic analysis of sperm regions that mediate sperm-egg interactions

Stein, K.K., Go, J.C., Lane, W.S., Primakoff, P. and Myles, D.G. Proteomics, 6(12), 3533-3543 (2006)   The sperm interacts with three oocyte-associated structures during fertilization: the cumulus cell layer surrounding the oocyte, the egg extracellular matrix (the zona pellucida), and the oocyte plasma membrane. Each of these interactions is mediated by the sperm head, probably through proteins both on the sperm surface and within the acrosome, a specialized secretory granule. In this study, we have used subcellular fractionation in order to generate a proteome of the sperm head subcellular compartments that interact with oocytes. Of the proteins we identified for which a gene knockout has been tested, a third have been shown to be essential for efficient reproduction in vivo. Many of the other presently untested proteins are likely to have a similarly important role. Twenty-five percent of the cell surface fraction proteins are previously uncharacterized. We have shown that at least two of these novel proteins are localized to the sperm head. In summary, we have identified over 100 proteins that are expressed on mature sperm at the site of sperm-oocyte interactions.    

4.447           STAT1 inhibits liver fibrosis in mice by inhibiting stellate cell proliferation and stimulating NK cell cytotoxicity

Jeong, W-I., Park, O., Radaeva, S. and Gao, B. Hepatology, 44(6), 1441-1451 (2006)   Liver fibrosis, a common scarring response to chronic liver injury, is a precursor to cirrhosis and liver cancer. Here, we identified signal transducer and activator of transcription 1 (STAT1) as an important negative regulator in liver fibrosis. Our findings show that disruption of the STAT1 gene accelerated liver fibrosis and hepatic stellate cell (HSC) proliferation in an in vivo model of carbon tetrachloride (CCl₄)-induced liver fibrosis. In vitro treatment with IFN- inhibited proliferation and activation of wild-type HSCs, but not STAT1^−/− HSCs. Moreover, compared to wild-type cells, cellular proliferation stimulated by serum or platelet-derived growth factor (PDGF) was enhanced and accelerated in STAT1^−/− HSCs, which was partially mediated via elevated PDGF receptor β expression on such cells. Polyinosinic-polycytidylic acid (poly I:C) or IFN- treatment inhibited liver fibrosis in wild-type mice but not in STAT1^−/− mice. Induction of NK cell killing of activated HSCs by poly I:C was attenuated in STAT1^−/− mice compared to wild-type mice, which was likely due to reduced NKG2D and TRAIL expression on STAT1^−/− NK cells. Finally, activation of TGF-β/Smad3 signaling pathway was accelerated, whereas induction of Smad7 was diminished in the liver of STAT1^−/− mice after CCl₄ administration compared to wild-type mice. In conclusion, activation of STAT1 attenuates liver fibrosis through inhibition of HSC proliferation, attenuation of TGF-β signaling, and stimulation of NK cell killing of activated HSCs. STAT1 could be a new therapeutic target for treating liver fibrosis.  

4.448           Single Cell Analysis on Microfluidic Devices

Culbertson, C. Methods in Mol. Biol., 339, 203-216 (2006)   There is significant variability among cells of the same type at the single cell level. This variability may be because of external stimuli that vary temporally or spatially among a population of cells. It may also be owing to the nonsynchronized responses of cells to various stimuli. In addition, differences in otherwise similar cells may be generated by genetic mutations acquired by one or more of the cells. Often times multiple biochemical pathways and molecules are involved in such differences. In order to better understand these differences and to detect those rare cells in a large population that may be indicative of early disease states, methods that are capable of rapidly quantifying multiple molecular species in single cells are desired. Microfluidic devices may provide the optimal platform upon which to develop such methods. Microfluidics has the capability of combining the high-throughput manipulation and transport of cells with rapid, high-efficiency separations and high-sensitivity detection. This chapter describes how to fabricate microfluidic devices for the high-throughput manipulation and rapid electrical lysis of single, nonadherent (suspension) cells followed by the injection and separation of the fluorescently labeled cell contents.    

4.449           Stem cell continuum: Directed differentiation hotspots

Colvin, G.A. et al Exp. Hematol., 35(1), 96-107 (2007)   Objective The purpose of this study was to evaluate the technique of stem cell-directed differentiation in the context of cell-cycle position. The hypothesis was that stem cells would have different sensitivities to an identical inductive signal through cell-cycle transit and that this would affect the outcome of its progeny. Materials and Methods Differentiation of murine marrow lineage^negativerhodamine-123^low-Hoechst-33342^low (LRH) stem cells was determined at different points in cell cycle under stimulation by thrombopoietin, flt3 ligand, and steel factor. LRH stem cells were subcultured in granulocyte macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and steel factor at different points in cell cycle and differentiation determined 14 days later. Results There was a significant, reproducible, and pronounced reversible increase in differentiation to megakaryocytes in early S-phase and to nonproliferative granulocytes in mid S-phase. Megakaryocyte hotspots also were seen on a clonal basis. Elevations of the transcription factor FOG-1 were seen at the hotspot along with increases in Nfe2 and Fli1. Conclusions We show that the potential of marrow stem cells to differentiate changes reversibly with cytokine-induced cell-cycle transit, suggesting that stem cell regulation is not based on the classic hierarchical model, but instead on a functional continuum. We propose that there is a tight linkage of commitment to a lineage and a particular phase of cell cycle. Thus, windows of vulnerability for commitment can open and close depending on the phase of cell cycle. These data indicate that stem cell differentiation occurs on a cell-cycle–related continuum with fluctuating windows of transcriptional opportunity.  

4.450           Detection of Mattesia oryzaephili (Neogregarinorida: Lipotrophidae) in grain beetle laboratory colonies with an enzyme-linked immunosorbent assay

Lord, J.C.

1. Invert. Pathol., 94(1), 74-76 (2007)

  An indirect sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of the neogregarine Mattesia oryzaephili was developed with monoclonal antibodies. It was used to screen laboratory colonies of Oryzaephilus surinamensis, Cryptolestes ferrugineus, C. pusillus, and C. turcicus from the United States, Canada, and Australia. All of the colonies except C. turcicus had larvae that tested positive with the percent of positives ranging from 0.2 to 83.9, but only colonies that tested positive had reported population declines. This assay will make possible epizootiological studies to assess the impact of M. oryzaephili on pest populations.  

4.451           Expression of mRNAs related to connective tissue metabolism in rat hepatic stellate cells and myofibroblasts

Jiroutova, A. et al Exp. Toxicol. Pathol., 58(4), 263-273 (2007)   Hepatic stellate cells (HSC) and liver myofibroblasts (MFB) are two cell populations most likely responsible for the synthesis of most connective tissue components in fibrotic liver. They differ in their origin and location, and possibly in patterns of gene expression. Normal and carbon tetrachloride-cirrhotic livers from rats were used to isolate HSC. Liver was perfused with pronase and collagenase solutions, followed by centrifugation of the cell suspension on a density gradient. HSC were quiescent 2 days after plating on plastic but they became activated after another 5 days in culture. When the culture was passaged 5 times, its character changed profoundly as HSC were replaced by MFB. Microarray analysis was used to determine gene expression in quiescent HSC, activated HSC and MFB. The expression of 49 genes coding for connective tissue proteins, proteoglycans, metalloproteinases and their inhibitors, growth factors and cellular markers was determined. The pattern of gene expression changed during HSC activation and there were distinct differences between HSC and MFB. Little difference between normal cells and cells isolated from cirrhotic liver was found.  

4.452           Neuroprotection by estradiol: A role of aromatase against spine synapse loss after blockade of GABAA receptors

Zhou, L. et al Exp. Neurol., 203(1), 72-81 (2007)   Estrogen has been suggested to be pro-epileptic by reducing GABA synthesis, resulting in increased spine density and a decreased threshold for seizures in the hippocampus, which, once they occur, are characterized by a dramatic spine loss in the affected brain areas. As considerable amounts of estradiol are synthesized in the hippocampus, in this study we focused on aromatase, the rate-limiting enzyme in estrogen synthesis in order to examine the role of locally synthesized estrogens in epilepsy. To this end, we first examined the effects of letrozole, a potent aromatase inhibitor, on GABA metabolism in single interneurons of hippocampal dispersion cultures. Letrozole downregulated estradiol release into the medium, as well as glutamate decarboxylase (GAD) expression and GABA synthesis, and decreased the number of GAD positive cells in the cultures. Next, we counted spine synapses and measured estradiol release of hippocampal slice cultures, in which GABA_A receptors had been blocked by bicuculline, in order to mimic epileptic activity. Treatment of slice cultures with bicuculline resulted in a dramatic decrease in the number of spine synapses and in a significant suppression of estrogen synthesis. The decrease in synapse number in response to bicuculline was restored by combined application of estradiol and bicuculline. Surprisingly, estradiol alone had no effect on either spine synapse number or on GAD expression and GABA synthesis. “Rescue” of synapse number in “epileptic slices” by estradiol and maintenance of GABA metabolism by hippocampus-derived estradiol points to a neuroprotective role of aromatase in epilepsy. Re-filling of estradiol stores after their depletion due to overexcitation may therefore add to therapeutical strategies in epilepsy.  

4.453           Cloning and characterization of guinea pig CXCR1

Takahashi, M., Jeevan, A., Sawant, K., McMurray, D.N. and Yoshimuro, T. Mol. Immunol., 44(5), 878-888 (2007)   IL-8/CXCL8 plays a critical role in the trafficking and activation of neutrophils via its receptors, CXCR1 and CXCR2, in humans. CXCR1 is highly selective for IL-8, whereas CXCR2 is activated by all CXC chemokines with an ELR motif. In mice and rats, neither IL-8 nor CXCR1 is present, making it difficult to evaluate the in vivo roles of the IL-8/CXCR1 interactions. We previously demonstrated the presence of IL-8 in the guinea pig (gp), suggesting that its specific receptor CXCR1 is also present in this species. Here, we obtained two gp genomic DNA clones, clones 8 and 10, coding for the potential orthologues of CXCR1 and CXCR2, respectively. Transcripts for these genes were expressed in neutrophils, but not in macrophages. Functionally, both gp and human (h) IL-8 induced cell migration and ERK phosphorylation in HEK 293 cells expressing either receptor, whereas hGRO activated only cells expressing the clone 10 protein, confirming that clone 8 indeed coded for gpCXCR1. ¹²⁵I-labeled hIL-8 bound to gpCXCR1 and addition of unlabeled hIL-8 completely abolished the binding; however, unlabeled gpIL-8 failed to compete against ¹²⁵I-labeled hIL-8, strongly suggesting that the avidity of hIL-8 to gpCXCR1 is higher than that of gpIL-8. Identification and characterization of CXCR1 in the guinea pig will allow us to use this small animal model to evaluate the role of the IL-8/CXCR1 interactions and to examine the efficacy of CXCR1 antagonists in vivo.  

4.454           Ivermectin inhibits AMPA receptor-mediated excitotoxicity in cultured motor neurons and extends the life span of a transgenic mouse model of amyotrophic lateral sclerosis

Andries, M., Van Damme, P., Robberecht, W. and Van Den Bosch, L. Neurobiol. Disease, 25(1), 8-16 (2007)   α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-mediated excitotoxicity contributes to the selective motor neuron death in amyotrophic lateral sclerosis (ALS). In this study, we investigated the effect of P2 receptor-influencing substances on kainate-induced motor neuron death in an in vitro model for AMPA receptor-mediated excitotoxicity. Complete protection was found after preincubation of the motor neurons with ivermectin or Cibacron Blue 3G-A. Preincubation with both P2X₄ modulators did not influence the number or Ca²⁺ permeability of the AMPA receptors and addition during kainate stimulation alone had no effect. Preincubation with a low concentration of ATP, the natural agonist of the P2X₄ receptor, also protected the motor neurons against a subsequent excitotoxic stimulation, while high concentrations of ATP were toxic. Moreover, ivermectin increased the toxicity of low ATP concentrations, indicating that ivermectin can potentiate the effect of ATP on its receptor. Ivermectin and ATP also protected against hypoxia/hypoglycemia. To further investigate the relevance of these findings for ALS, we treated SOD1(G93A)-mice, a transgenic animal model for familial ALS, with ivermectin. This resulted in an extension of the life span of these mice with almost 10%. We conclude that ivermectin induces a mechanism in motor neurons, in vivo and in vitro, that protects against subsequent excitotoxic insults. Our in vitro data indicate that this protective mechanism is due to the potentiation by ivermectin of an effect of ATP mediated by the P2X₄ receptor.  

4.455           Surfactant Protein D Augments Bacterial Association but Attenuates Major Histocompatibility Complex Class II Presentation of Bacterial Antigens

Hansen, S. et al Am. J. Respir. Cell Mol. Biol.36, 94-102 (2007)   Surfactant protein D (SP-D) is a secreted pattern recognition molecule associated with lung surfactant and mediates the clearance of pathogens in multiple ways. SP-D is an established part of the innate immune system, but it also modulates the adaptive immune response by interacting with both antigen-presenting cells and T cells. In a previous study, antigen presentation by bone marrow–derived dendritic cells was enhanced by SP-D. As dendritic cell function varies depending on the tissue of origin, we extended these studies to antigen-presenting cells isolated from mouse lung. Flow cytometric studies showed that SP-D binds calcium dependently and specifically to lung CD11c-positive cells. Opsonization of fluorescently labeled Escherichia coli by SP-D enhanced uptake by lung dendritic cells. SP-D facilitated the association of E. coli and antigen-presenting cells by increasing the frequency of CD11+ cells associated with E. coli by up to 10-fold. In contrast to the effect on bone marrow–derived dendritic cells, SP-D decreased the antigen presentation of ovalbumin, expressed in E. coli, to ovalbumin-specific major histocompatibility complex class II–specific T-cell hybridomas by 30–50%. The reduction of antigen presentation did not depend on whether the dendritic cells were isolated from the lungs of nonstimulated mice or mice that had been exposed to LPS aerosols. Our results show that SP-D increases the opsonization of pathogens, but decreases the antigen presentation by lung dendritic cells, and thereby, potentially dampens the activation of T cells and an adaptive immune response against bacterial antigens—during both steady-state conditions and inflammation.  

4.456           Regulation of myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for wound healing and fibrogenesis

Mann, J. et al Cell Death and Differentiation, 14, 275-285 (2007)   Myofibroblasts are critical cellular elements of wound healing generated at sites of injury by transdifferentiation of resident cells. A paradigm for this process is conversion of hepatic stellate cells (HSC) into hepatic myofibroblasts. Treatment of HSC with DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-azadC) blocked transdifferentiation. 5-azadC also prevented loss of I B and PPAR expression that occurs during transdifferentiation to allow acquisition of proinflammatory and profibrogenic characteristics. ChIP analysis revealed I B promoter is associated with transcriptionally repressed chromatin that converts to an active state with 5-azadC treatment. The methyl-CpG-binding protein MeCP2 which promotes repressed chromatin structure is selectively detected in myofibroblasts of diseased liver. siRNA knockdown of MeCP2 elevated I B promoter activity, mRNA and protein expression in myofibroblasts. MeCP2 interacts with I B promoter via a methyl-CpG-dependent mechanism and recruitment into a CBF1 corepression complex. We conclude that MeCP2 and DNA methylation exert epigenetic control over hepatic wound healing and fibrogenesis.  

4.457           Discoidin domain receptor 2 is involved in the activation of bone marrow-derived dendritic cells caused by type I collagen

Lee, J-E. et al Biochem. Biophys. Res. Comm., 352(1), 244-250 (2007)   Discoidin domain receptors (DDRs), DDR1 and DDR2, are non-integrin receptor tyrosine kinases for collagen in many cell types. In this study, we investigated the contributions of DDRs to the activation of mouse bone marrow-derived dendritic cells (DCs) by type I collagen (ColI). Our data showed that transcript and protein of DDR2 were expressed constitutively in immature DCs and upregulated in TNF-α-stimulated mature DCs. ColI treatment induced DDR2 phosphorylation and subsequently induced the upregulation of IL-12 production, CD86 expression, and antigen uptake activity by immature DCs. Depletion of DDR2 by specific siRNA attenuated significantly an increase in expression of IL-12 and CD86 in ColI-treated DCs. Additionally, DDR2–ColI interaction upregulated the ability of mature DCs to activate allogeneic T cells. These findings suggest that DDR2 is a critical collagen receptor for DC activation and that DDR2–collagen interaction plays an important role in the functional capacity of DCs regulating immune responses.  

4.458           CNS myeloid DCs presenting endogenous myelin peptides 'preferentially' polarize CD4+ TH-17 cells in relapsing EAE

Bailey, S.I., Schreiner, B., McMahon, E.J. and Miller, S.D. Nature Immunol., 8(2), 172-180 (2007)   Peripherally derived CD11b⁺ myeloid dendritic cells (mDCs), plasmacytoid DCs, CD8 ⁺ DCs and macrophages accumulate in the central nervous system during relapsing experimental autoimmune encephalomyelitis (EAE). During acute relapsing EAE induced by a proteolipid protein peptide of amino acids 178–191, transgenic T cells (139TCR cells) specific for the relapse epitope consisting of proteolipid protein peptide amino acids 139–151 clustered with mDCs in the central nervous system, were activated and differentiated into T helper cells producing interleukin 17 (T_H-17 cells). CNS mDCs presented endogenously acquired peptide, driving the proliferation of and production of interleukin 17 by naive 139TCR cells in vitro and in vivo. The mDCs uniquely biased T_H-17 and not T_H1 differentiation, correlating with their enhanced expression of transforming growth factor- 1 and interleukins 6 and 23. Plasmacytoid DCs and CD8 ⁺ DCs were superior to macrophages but were much less efficient than mDCs in presenting endogenous peptide to induce T_H-17 cells. Our findings indicate a critical function for CNS mDCs in driving relapses in relapsing EAE.    

4.459           Influence of strain and age differences on the yields of porcine islet isolation: extremely high islet yields from SPF CMS miniature pigs

Kim, J.H. et al Xenotransplantation, 14, 60-66 (2007)   Background: Porcine pancreas is a potential source of material for islet xenotransplantation. However, the difficulty in isolating islets, because of their fragility and the variability of isolation outcome in donor age and breed, represents a major obstacle to porcine islet xenotransplantation. In this study, we compared the islet isolation yield of specific pathogen-free (SPF) Chicago Medical School (CMS) miniature pigs with that of another miniature pig breed and market pigs from a local slaughterhouse. Methods: Nine adult CMS miniature (ACM) pigs (>12 months), six young CMS miniature (YCM) pigs (6–7 months), four adult Prestige World Genetics (PWG) miniature (APM) pigs (>12 months), and 13 adult market (AM) pigs from a local slaughterhouse were used for islet isolation. Results: The islet yield per gram of pancreas from ACM pigs (9589 ± 2823 IEQ/g) was significantly higher than that from APM pigs (1752 ± 874 IEQ/g, P < 0.05), AM pigs (1931 ± 947 IEQ/g, P < 0.05), or YCM pigs (3460 ± 1985 IEQ/g, P < 0.05). Isolated islets from ACM pigs were significantly larger than those from AM pigs or YCM pigs. The in vitro and in vivo function of isolated islets showed no difference among experimental groups. The pancreases of ACM pigs contained higher mean islet volume density percentages and larger size of islets than those of AM or APM pigs. Conclusions: We isolated extremely high yields of well-functioning islets from ACM pigs bred under SPF conditions. SPF CMS miniature pigs should be one of the best porcine islet donors for clinical porcine islet xenotransplantation.  

4.460           Control of coronavirus infection through plasmacytoid dendritic-cell–derived type I interferon

Cervantes-Barragan, L. et al Blood, 109(3), 1131-1137 (2007)   This study demonstrates a unique and crucial role of plasmacytoid dendritic cells (pDCs) and pDC-derived type I interferons (IFNs) in the pathogenesis of mouse coronavirus infection. pDCs controlled the fast replicating mouse hepatitis virus (MHV) through the immediate production of type I IFNs. Recognition of MHV by pDCs was mediated via TLR7 ensuring a swift IFN- production following encounter with this cytopathic RNA virus. Furthermore, the particular type I IFN response pattern was not restricted to the murine coronavirus, but was also found in infection with the highly cytopathic human severe acute respiratory syndrome (SARS) coronavirus. Taken together, our results suggest that rapid production of type I IFNs by pDCs is essential for the control of potentially lethal coronavirus infections.  

4.461           Selective abrogation of Th1 response by STA-5326, a potent IL-12/IL-23 inhibitor

Wada, Y. et al Blood, 109(3), 1156-1164 (2007)   The interleukin-12 (IL-12) cytokine induces the differentiation of naive T cells to the T helper cell type 1 (Th1) phenotype and is integral to the pathogenesis of Th1-mediated immunologic disorders. A more recently discovered IL-12 family member, IL-23, shares the p40 protein subunit with IL-12 and plays a critical role in the generation of effector memory T cells and IL-17–producing T cells. We introduce a novel compound, STA-5326, that down-regulates both IL-12 p35 and IL-12/IL-23 p40 at the transcriptional level, and inhibits the production of both IL-12 and IL-23 cytokines. Oral administration of STA-5326 led to a suppression of the Th1 but not Th2 immune response in mice. In vivo studies using a CD4⁺CD45Rb^high T-cell transfer severe combined immunodeficiency (SCID) mouse inflammatory bowel disease model demonstrated that oral administration of STA-5326 markedly reduced inflammatory histopathologic changes in the colon. A striking decrease in interferon- (IFN- ) production was observed in ex vivo culture of lamina propria cells harvested from animals treated with STA-5326, indicating a down-regulation of the Th1 response by STA-5326. These results suggest that STA-5326 has potential for use in the treatment of Th1-related autoimmune or immunologic disorders. STA-5326 currently is being evaluated in phase 2 clinical trials in patients with Crohn disease and rheumatoid arthritis.  

4.462           Astrocyte and Muscle-Derived Secreted Factors Differentially Regulate Motoneuron Survival

Taylor, A.R. et al

1. Neurosci., 27(3), 634-644 (2007)

  During development, motoneurons (MNs) undergo a highly stereotyped, temporally and spatially defined period of programmed cell death (PCD), the result of which is the loss of 40–50% of the original neuronal population. Those MNs that survive are thought to reflect the successful acquisition of limiting amounts of trophic factors from the target. In contrast, maturation of MNs limits the need for target-derived trophic factors, because axotomy of these neurons in adulthood results in minimal neuronal loss. It is unclear whether MNs lose their need for trophic factors altogether or whether, instead, they come to rely on other cell types for nourishment. Astrocytes are known to supply trophic factors to a variety of neuronal populations and thus may nourish MNs in the absence of target-derived factors. We investigated the survival-promoting activities of muscle- and astrocyte-derived secreted factors and found that astrocyte-conditioned media (ACM) was able to save substantially more motoneurons in vitro than muscle-conditioned media (MCM). Our results indicate that both ACM and MCM are significant sources of MN trophic support in vitro and in ovo, but only ACM can rescue MNs after unilateral limb bud removal. Furthermore, we provide evidence suggesting that MCM facilitates the death of a subpopulation of MNs in a p75^NTR - and caspase-dependent manner; however, maturation in ACM results in MN trophic independence and reduced vulnerability to this negative, pro-apoptotic influence from the target.  

4.463           Pro-NGF secreted by astrocytes promotes motor neuron cell death

Domeniconi, M., Hempstead, B.L. and Chao, M.V. Mol. Cell. Neurosci., 34(2), 271-279 (2007)   It is well established that motor neurons depend for their survival on many trophic factors. In this study, we show that the precursor form of NGF (pro-NGF) can induce the death of motor neurons via engagement of the p75 neurotrophin receptor. The pro-apoptotic activity was dependent upon the presence of sortilin, a p75 co-receptor expressed on motor neurons. One potential source of pro-NGF is reactive astrocytes, which up-regulate the levels of pro-NGF in response to peroxynitrite, an oxidant and producer of free radicals. Indeed, motor neuron viability was sensitive to conditioned media from cultured astrocytes treated with peroxynitrite and this effect could be reversed using a specific antibody against the pro-domain of pro-NGF. These results are consistent with a role for activated astrocytes and pro-NGF in the induction of motor neuron death and suggest a possible therapeutic target for the treatment of motor neuron disease.  

4.464           Both Ca2+-dependent and -independent pathways are involved in rat hepatic stellate cell contraction and intrahepatic hyperresponsiveness to methoxamine

Laleman, W. et al Am. J. Physiol. Gastrointest. Liver Physiol., 292, G556-G564 (2007)   In chronic liver injury, hepatic stellate cells (HSCs) have been implicated as regulators of sinusoidal vascular tone. We studied the relative role of Ca²⁺-dependent and Ca²⁺-independent contraction pathways in rat HSCs and correlated these findings to in situ perfused cirrhotic rat livers. Contraction of primary rat HSCs was studied by a stress-relaxed collagen lattice model. Dose-response curves to the Ca²⁺ ionophore A-23187 and to the calmodulin/myosin light chain kinase inhibitor W-7 served to study Ca²⁺-dependent pathways. Y-27632, staurosporin, and calyculin (inhibitors of Rho kinase, protein kinase C, and myosin light chain phosphatase, respectively) were used to investigate Ca²⁺-independent pathways. The actomyosin interaction, the common end target, was inhibited by 2,3-butanedione monoxime. Additionally, the effects of W-7, Y-27632, and staurosporin on intrahepatic vascular resistance were evaluated by in situ perfusion of normal and thioacetamide-treated cirrhotic rat livers stimulated with methoxamine (n = 25 each). In vitro, HSC contraction was shown to be actomyosin based with a regulating role for both Ca²⁺-dependent and -independent pathways. Although the former seem important, an important auxiliary role for the latter was illustrated through their involvement in the phenomenon of "Ca²⁺ sensitization." In vivo, preincubation of cirrhotic livers with Y-27632 (10^–4 M) and staurosporin (25 nM), more than with W-7 (10^–4 M), significantly reduced the hyperresponsiveness to methoxamine (10^–4 M) by –66.8 ± 1.3%, –52.4 ± 2.7%, and –28.7 ± 2.8%, respectively, whereas in normal livers this was significantly less: –43.1 ± 4.2%, –40.2 ± 4.2%, and –3.8 ± 6.3%, respectively. Taken together, these results suggest that HSC contraction is based on both Ca²⁺-dependent and -independent pathways, which were shown to be upregulated in the perfused cirrhotic liver, with a predominance of Ca²⁺-independent pathways.  

4.465           Immunological characterization of a bacterial protein isolated from salmonid fish naturally infected with Piscirickettsia salmonis

Marshall, S.H. et al Vaccine, 25, 2095-2102 (2007)   The Salmon Rickettsia syndrome (SRS) remains a major infectious disease in the Chilean aquaculture. A limited number of Piscirickettsia salmonis proteins have been characterized so far for their use as potential candidates for vaccines studies. In this study, we identified and expressed a highly immunogenic protein of P. salmonis extracted by selective hydrophobicity from crude-cell macerates of naturally infected salmonid fish. One and two-D PAGE gels followed by Western blot analysis with a battery of polyclonal anti-P. salmonis antibodies have allowed the isolation of the target protein. Basic local alignment search (BLAST) done after partial sequencing of the pure protein identified it as a member of the heat-shock protein (HSP) family of prokaryotes. The protein, named ChaPs, was cloned as a single open reading frame encoding 545 amino acid residues with a predicted molecular mass of 57.3 kDa. The amplicon representing the entire novel gene was expressed in vitro in different heterologous systems: the PurePro Caulobacter crescentus expression system from where most of the characterization was attained, and also in the Escherichia coli BL-21 CodonPlus model for commercially potential purposes. The immunologic potential of ChaPs was determined with serum from naturally infected fish.  

4.466           DCs metabolize sunlight-induced vitamin D3 to 'program' T cell attraction to the epidermal chemokine CCL27

Sigmundsdottir, H. et al Nature Immunol., 8(3), 285-293 (2007)   During adaptive immune responses, dendritic cells activate T cells and endow them with specific homing properties. Mechanisms that 'imprint' specific tropisms, however, are not well defined. We show here that 1,25(OH)2D3, the active form of vitamin D3, signaled T cells to express CC chemokine receptor 10, which enabled them to migrate to the skin-specific chemokine CCL27 secreted by keratinocytes of the epidermis. In contrast, 1,25(OH)2D3 suppressed the gut-homing receptors 4 7 and CCR9. Vitamin D3, the inactive prohormone naturally generated in the skin by exposure to the sun, was processed by dendritic cells and T cells to the active metabolite, providing a mechanism for the local regulation of T cell 'epidermotropism'. Our findings support a model in which dendritic cells process and 'interpret' locally produced metabolites to 'program' T cell homing and microenvironmental positioning.  

4.467           Gene Gun-delivered pGM-CSF Adjuvant Induces Enhanced Emigration of two Dendritic Cell Subsets from the Skin

Matthews, K et al Scand. J. Immunol., 65, 231-239 (2007)   Two subsets of sheep afferent lymph dendritic cells (DC) are defined by the differential expression of CD172a and CD45RA. The majority ( 70%) of CD172a⁺ subset is CD45RA/CD11c⁺/CD207⁺/TLR4⁺. The CD172a DC are CD45RA⁺/CD207 and express low levels of CD11c and CD86. Real-time RT-PCR showed that CD172⁺ DC produce IL-1β and IL-10 and high levels of IL-18 but almost no IL-12p40; CD172a DC express IL-12p40 but no IL-10 and low levels of IL-1β and IL-18. Gene gun-delivered granulocyte-macrophage colony-stimulating factor (pGM-CSF) caused an early rise in the output of CD172a⁺ DC, changes to DC phenotype and significant increases in the levels of expression cytokine transcripts. However, pGM-CSF did not affect any qualitative changes to cytokine expression, CD172a⁺ DC remained IL-10⁺/IL-12p40 and the CD172 DC remained IL-10 /IL-12p40⁺.  

4.468           Comparative immunobiology of thymic DC mRNA in autoimmune-prone mice

Okada, T. et al

1. Autoimmunity, 28, 41-45 (2007)

  New Zealand Black (NZB) mice have multiple defects in both innate and acquired immunity. A fundamental defect, described more than 25years ago, is premature thymic involution. Subsequent studies have disclosed multiple defects in thymic epithelial cells, and it has been proposed that thymic dendritic cells (DCs) play an important role not only in thymic involution but also in the appearance of immunopathology. However, the number of available thymic DCs makes this population extremely difficult to study. We have taken advantage of our ability to isolate pure populations of thymic DCs and have examined several key mRNA levels of enzymes involved in signal transduction. Our data on NZB mice was compared to that of NZB x NZW F1 (B/WF1), BXSB-Yaa, MRL/lpr, NOD and control mice. Importantly, we demonstrate herein that a common feature in autoimmune-prone mice is an increase of thymic DC c-met mRNA. Indeed, the increase in c-met mRNA levels appeared specific to the thymus and was not noted in the spleen. Additionally, we demonstrate that E-cadherin, a downstream molecule of c-met, is also reduced. Finally, we note that the levels of HGF mRNA are normal in the autoimmune strains examined herein, confirming that the abnormality of c-met mRNA is not due to primary defects in thymic stromal cells. We submit that these results highlight the possibility of a selective defect in thymic DCs which will be a pivotal step in loss of tolerance, and suggest that future studies focus on adoptive cell transfer involving this population.  

4.469           Evaluation of a cushioned method for centrifugation and processing for freezing boar semen

Matas, C., Decuadro, G., Martinez-Miro, S. and Gadea, J. Theriogenology, 67, 1087-1091 (2007)   The purpose of this investigation was to evaluate the use of an iodixanol cushion during centrifugation on sperm recovery and yield after centrifugation (sperm recovery, sperm motility, viability, membrane lipid disorder, acrosome reaction and ROS generation); and to investigate how this procedure affects sperm function after freezing–thawing (sperm motility, membrane lipid disorder, acrosomal status and homologous in vitro penetration test). The sperm-rich fractions from fertile boars were centrifuged under two centrifugation régimes: 800×g for 10min (standard method) and 1000×g for 20min with an iodixanol (60% w/v) cushion at the bottom of the centrifuge tubes (Cushion method). The highest recovery was achieved using the cushion method (sperm loss for cushion method was 0.50%±0.18 versus 2.97%±0.43 for standard method, P<0.01) and sperm quality was not significantly affected by the centrifugation régime. The motion parameters (% progressive motility, % motility, VCL, VSL, VAP, ALH, BCF, P<0.05) of frozen–thawed samples showed higher values using the standard method. However, a higher number of viable spermatozoa with lower lipid disorders were found in spermatozoa processed with the cushion method. The in vitro penetration assay showed that the individual boar influenced the parameters studied but there were no differences between the two centrifugation régimes used. Our results support the hypothesis that the proportion of sperm loss in frozen–thawed semen was significantly influenced by the centrifugation régime. Therefore, the iodixanol cushion method is a suitable tool for cryopreservation of boar semen in order to reduce sperm loss without affecting sperm quality.  

4.470           Nitroflurbiprofen, a Nitric Oxide-Releasing Cyclooxygenase Inhibitor, Improves Cirrhotic Portal Hypertension in Rats

Laleman, W. et al Gastroenterol., 132(2), 709-719 (2007)   Background & Aims: We studied whether administration of nitroflurbiprofen (HCT-1026), a cyclooxygenase inhibitor with nitric oxide (NO)-donating properties, modulates the increased intrahepatic vascular tone in portal hypertensive cirrhotic rats. Methods: In vivo hemodynamic measurements (n = 8/condition) and evaluation of the increased intrahepatic resistance by in situ perfusion (n = 5/condition) were performed in rats with thioacetamide-induced cirrhosis that received either nitroflurbiprofen (45 mg/kg), flurbiprofen (30 mg/kg, equimolar concentration to nitroflurbiprofen), or vehicle by intraperitoneal injection 24 hours and 1 hour prior to the measurements. Additionally, we evaluated the effect of acute administration of both drugs (250 μmol/L) on the intrahepatic vascular tone in the in situ perfused cirrhotic rat liver (endothelial dysfunction and hyperresponsiveness to methoxamine) and on hepatic stellate cell contraction in vitro. Typical systemic adverse effects of nonsteroidal anti-inflammatory drugs, such as gastrointestinal ulceration, renal insufficiency, and hepatotoxicity, were actively explored. Results: In vivo, nitroflurbiprofen and flurbiprofen equally decreased portal pressure (8 ± 0.8 and 8.4 ± 0.1 mm Hg, respectively, vs 11.8 ± 0.6 mm Hg) and reduced the total intrahepatic vascular resistance. Systemic hypotension was not aggravated in the different treatment groups (P = .291). In the perfused cirrhotic liver, both drugs improved endothelial dysfunction and hyperresponsiveness. This was associated with a decreased hepatic thromboxane A₂-production and an increased intrahepatic nitrate/nitrite level. In vitro, nitroflurbiprofen, more than flurbiprofen, decreased hepatic stellate cells contraction. Flurbiprofen-treated rats showed severe gastrointestinal ulcerations (bleeding in 3/8 rats) and nefrotoxicity, which was not observed in nitroflurbiprofen-treated cirrhotic rats. Conclusions: Treatment with nitroflurbiprofen, an NO-releasing cyclooxygenase inhibitor, improves portal hypertension without major adverse effects in thioacetamide-induced cirrhotic rats by attenuating intrahepatic vascular resistance, endothelial dysfunction, and hepatic hyperreactivity to vasoconstrictors.  

4.471           Progesterone protects fetal chorion and maternal decidua cells from calcium-induced death

Murtha, A.P., Feng, L., Yonish, B., Leppert, P.C. and Schomberg, D.W. Am. J. Obstet. Gynecol., 196(3), 257.e1-257.e5   Objective The purpose of this study was to determine whether progesterone exerts a protective effect in chorion and decidua cells when exposed to calcimycin. Study design Fetal membrane samples were collected from term elective repeat cesarean deliveries and chorion and decidua cells that are separated and cultured. Cells were pretreated with progesterone and exposed to calcimycin. Cell viability was determined, and percent cell viability was calculated. Results Exposure to calcimycin resulted in a reduction of cell viability in both chorion and decidua cells in a dose-dependent fashion. In chorion and decidua cells, progesterone pretreatment followed by calcimycin increased cell viability compared with calcimycin treatment alone (chorion, 67%, vs controls, 24%; P < .001; decidua, 58%, vs controls, 35%; P < .001). The progesterone receptor antagonist, RTI 6413-49a, blocked the protective effect of progesterone in both chorion and decidua cells. Conclusion These preliminary results suggest that progesterone may provide a protective effect in fetal membrane cells and that this effect may be mediated through the progesterone receptor.  

4.472           Innate Immune Response to Adenoviral Vectors Is Mediated by both Toll-Like Receptor-Dependent and -Independent Pathways

Zhu, J., Huang, X. and Yang, Y.

1. Virol., 81(7), 3170-3180 (2007)

  Recombinant adenoviral vectors have been widely used for gene therapy applications and as vaccine vehicles for treating infectious diseases such as human immunodeficiency virus disease. The innate immune response to adenoviruses represents the most significant hurdle in clinical application of adenoviral vectors for gene therapy, but it is an attractive feature for vaccine development. How adenovirus activates innate immunity remains largely unknown. Here we showed that adenovirus elicited innate immune response through the induction of high levels of type I interferons (IFNs) by both plasmacytoid dendritic cells (pDCs) and non-pDCs such as conventional DCs and macrophages. The innate immune recognition of adenovirus by pDCs was mediated by Toll-like receptor 9 (TLR9) and was dependent on MyD88, whereas that by non-pDCs was TLR independent through cytosolic sensing of adenoviral DNA. Furthermore, type I IFNs were pivotal in innate and adaptive immune responses to adenovirus in vivo, and type I IFN blockade diminished immune responses, resulting in more stable transgene expression and reduction of inflammation. These findings indicate that adenovirus activates innate immunity by its DNA through TLR-dependent and -independent pathways in a cell type-specific fashion, and they highlight a critical role for type I IFNs in innate and adaptive immune responses to adenoviral vectors. Our results that suggest strategies to interfere with type I IFN pathway may improve the outcome of adenovirus-mediated gene therapy, whereas approaches to activate the type I IFN pathway may enhance vaccine potency.  

4.473           The Use of Iodixanol for the Purification of Rat Pancreatic Islets

Delle, H., Saito, M.H., Yoshimoto, P.M. and Noronha, I.L. Transplant. Proceed., 39(2), 467-469 (2007)   Transplantation of pancreatic islets is a promising therapeutic treatment for type 1 diabetes mellitus. For clinical and experimental transplantation, a large number of pure pancreatic islets are required for transplantation. Thus, the improvement of islet isolation and purification techniques are crucial. In this context, iodixanol-based solution, successfully used for the purification of porcine islets, seems to be a possible alternative to Ficoll for purification of islets. The aim of this study was to test the efficacy of iodixanol compared with Ficoll density gradients for the purification of rat pancreatic islets. Twelve Wistar rats were used for isolation and purification of pancreatic islets. Pancreata were digested with Liberase R1 and islets purified by two gradients: Ficoll or iodixanol gradient. The number and the purity of the pancreatic islets were assessed. To analyze the response of isolated pancreatic islet to glucose challenge, in vitro experiments were performed by measuring the insulin concentration in the Supernatant. The results demonstrated that the iodixanol gradient provided a higher purity of pancreatic islets compared to the Ficoll gradient. In addition, the rat islet yield by iodixanol gradient was significantly higher compared to a Ficoll gradient (751 ± 16 versus 464 ± 19 pancreatic islets, respectively; P < .001). The viability of pancreatic islets isolated by an iodixanol gradient was confirmed by high glucose challenge, with more than twofold higher increase in insulin secretion. The present study demonstrated that iodixanol density gradient overcomes Ficoll density gradient, providing a greater number of pure and functional rat pancreatic islets.  

4.474           Establishment and characterization of porcine Sertoli cell line for the study of xenotransplantation

Lee, H-M. et al Xenotransplantation, 14, 112-118 (2007)   Background: An understanding of the main mechanism that determines the ability of immune privilege related to Sertoli cells (SC) will provide clues for promoting a local tolerogenic environment. In this report, we established neonatal porcine SC line and evaluated their characteristics. Methods: SC line was established following the transfection of primary SC (NPSC) from the testis of neonatal pig with plasmid pRNS-1 carrying genes for neomycin resistance and the SV40 large T antigen. Immunohistochemistry and RT-PCR were performed to evaluate the character of immortalized SC lines. Results: Our immortalized SC line (iPS) proliferated stably and had a phenotype similar to NPSC, as indicated by the immunoexpression of follicle stimulating hormone receptor (FSHR), and mRNA expression of androgen receptor (AR), and Wilms’ tumor antigen (WT1). Interestingly, NPSC and iPS expressed mRNA of complement regulatory proteins (CRP) such as membrane cofactor protein (CD46), decay accelerating factor (DAF or CD55), and protectin (CD59), but CD59 mRNA expression was negligible in iPS. Conclusion: These results suggest that iPS, immortalized by the introduction of SV40 T, retain their original characteristics, except for the relatively low expression of CD59, and that they may be useful for future in vitro and in vivo studies of immune privilege mechanisms related to SC.  

4.475           BMP6 is axonally transported by motoneurons and supports their survival in vitro

Wang, P-Y., Koishi, K. and McLennan, I.S. Mol. Cell. Neurosci., 34(4), 653-661 (2007)   The regulation of motoneuron survival is only partially elucidated. We have sought new survival factors for motoneuron by analyzing which receptors they produce. We report here that the type II bone morphogenetic receptor (BMPRII) mRNA is one of the most abundant receptor mRNAs in laser microdissected motoneurons. Motoneurons were intensely stained by an anti-BMPRII antibody, indicating the presence of BMPRII protein. One of its ligands (BMP6) supported the survival of motoneurons in vitro. BMP6 was produced by myotubes and mature Schwann cells and was retrogradely transported in mature motor axons. BMP6 thus joins a list of known Schwann-cell-derived regulators of motoneurons, which includes GDNF, CNTF, LIF and TGF-β2. The control of the production of these factors by Schwann cells and the direction of their movement in motor axons is diverse. This suggests that the multiplicity of motoneuron factors is because cells use different factors to regulate different aspects of motoneuron function.  

4.476           Costimulatory ligand CD70 is delivered to the immunological synapse by shared intracellular trafficking with MHC class II molecules

Keller, A.M. et al PNAS, 104(14), 5989-5994 (2007)   TNF family member CD70 is the ligand of CD27, a costimulatory receptor that shapes effector and memory T cell pools. Tight control of CD70 expression is required to prevent lethal immunodeficiency. By selective transcription, CD70 is largely confined to activated lymphocytes and dendritic cells (DC). We show here that, in addition, specific intracellular routing controls its plasma membrane deposition. In professional antigen-presenting cells, such as DC, CD70 is sorted to late endocytic vesicles, defined as MHC class II compartments (MIIC). In cells lacking the machinery for antigen presentation by MHC class II, CD70 travels by default to the plasma membrane. Introduction of class II transactivator sufficed to reroute CD70 to MIIC. Vesicular trafficking of CD70 and MHC class II is coordinately regulated by the microtubule-associated dynein motor complex. We show that when maturing DC make contact with T cells in a cognate fashion, newly synthesized CD70 is specifically delivered via MIIC to the immunological synapse. Therefore, we propose that routing of CD70 to MIIC serves to coordinate delivery of the T cell costimulatory signal in time and space with antigen recognition.  

4.477           IS-741 Attenuates Local Migration of Monocytes and Subsequent Pancreatic Fibrosis in Experimental Chronic Pancreatitis Induced by Dibutyltin Dichloride in Rats

Kaku, T. et al Pancreas, 34(3), 299-309 (2007)   Objectives: Chronic pancreatitis consists of excessive leukocyte infiltration and fibrosis. IS-741 has been reported to be an antiinflammatory drug through an inhibitory action on cell adhesion. In this study, we investigated whether IS-741 could inhibit the progression of pancreatic fibrosis through monocyte infiltration. Moreover, we investigated the effect of IS-741 on rat pancreatic stellate cells (PSCs). Methods: Chronic pancreatitis was induced by dibutyltin dichloride in rats. From days 7 to 28 after dibutyltin dichloride application, IS-741 or distilled water was administered. At days 14 and 28, histological [hematoxylin-eosin stain and immunostain for ED1 and [alpha] smooth muscle actin ([alpha]-SMA)] and biochemical evaluations (intrapancreatic amylase, protein, cytokines, chemokines, and [alpha]-SMA) were performed. In vitro, rat PSCs were incubated with cytokine, chemokine, and growth factor simultaneously with IS-741, and their proliferation and activation were examined. Results: Histologically, IS-741 inhibited pancreatic fibrosis and decreased the number of ED1- and [alpha]-SMA-positive cells. The intrapancreatic expression of cytokines, chemokine, and [alpha]-SMA were also decreased. In vitro, IS-741 has no direct effect on the proliferation, [alpha]-SMA expression, and collagen synthesis of PSCs. Conclusions: These results suggest that IS-741 suppressed macrophage infiltration and subsequent pancreatic fibrosis and that the infiltration of monocytes into pancreas is essential for pancreatic fibrosis.  

4.478           Gamma-Glutamyltransferase Activity and Total Antioxidant Status in Serum and Platelets of Patients with Community-acquired Pneumonia

Laskaj, R., Slavica, D., Cepelak, I. and Kuzman, I. Arc. Med. Res., 38(4), 424-431 (2007)   Background We undertook this study to analyze serum and platelet γ-glutamyltransferase (GGT) activity and total antioxidant status (TAS) concentration during the course of pneumonia and to compare them between patients with normal platelet count and those who developed reactive thrombocytosis. Methods Platelet count, GGT activity and TAS concentration in serum (S) and platelet (Plt) isolates were measured in 60 patients with community-acquired pneumonia (CAP) on admission and at discharge. Results At the end of treatment, platelet count increased significantly from the value recorded on admission. By the end of treatment, 42% of patients developed reactive thrombocytosis. Serum and platelet GGT activity was higher, whereas (S)TAS was significantly lower in CAP patients than in control subjects. On admission, (Plt)TAS was significantly higher in CAP patients as compared with control subjects; at discharge, (Plt)TAS was lower in comparison with either patient admission and control subjects. GGT activity and TAS concentration in serum and platelet isolate on admission did not differ significantly between patients with and without thrombocytosis. At discharge, (S)GGT activity showed no significant changes, whereas (Plt)GGT decreased significantly in patients with thrombocytosis as compared with those without thrombocytosis. In patients with thrombocytosis, (S)TAS concentration showed no significant difference, whereas (Plt)TAS concentration measured at discharge was significantly lower in patients with thrombocytosis as compared to those with normal platelet count. Conclusions The pattern of changes in (Plt)GGT catalytic activity and TAS concentration might be indicative of a certain role of thrombocytosis during treatment in patients with CAP. Further investigations are necessary to clarify these changes.  

4.479           Tertiary Lymphoid Tissues Generate Effector and Memory T Cells That Lead to Allograft Rejection

Nasr, I.W. et al Am. J. Transplant., 7, 1071-1079 (2007)   Tertiary lymphoid tissues are lymph node-like cell aggregates that arise at sites of chronic inflammation. They have been observed in transplanted organs undergoing chronic rejection, but it is not known whether they contribute to the rejection process by supporting local activation of naïve lymphocytes. To answer this question, we established a murine transplantation model in which the donor skin contains tertiary lymphoid tissues due to transgenic expression of lymphotoxin-α(RIP-LTα), whereas the recipient lacks all secondary lymphoid organs and does not mount primary alloimmune responses. We demonstrate in this model that RIP-LTα allografts that harbor tertiary lymphoid tissues are rejected, while wild-type allografts that lack tertiary lymphoid tissues are accepted. Wild-type allografts transplanted at the same time as RIP-LTα skin or 60 days later were also rejected, suggesting that tertiary lymphoid tissues, similar to secondary lymphoid organs, generate both effector and memory immune responses. Consistent with this observation, naive T cells transferred to RIP-LTα skin allograft but not syngeneic graft recipients proliferated and differentiated into effector and memory T cells. These findings provide direct evidence that tertiary lymphoid structures perpetuate the rejection process by supporting naïve T-cell activation.  

4.480           Cutting Edge: Conventional Dendritic Cells Are the Critical APC Required for the Induction of Experimental Cerebral Malaria

DeWalick, S. et al

1. Immunol., 178, 6033-6037 (2007)

  Cerebral malaria (CM) is a serious complication of Plasmodium falciparum infection, causing significant morbidity and mortality among young children and nonimmune adults in the developing world. Although previous work on experimental CM has identified T cells as key mediators of pathology, the APCs and subsets therein required to initiate immunopathology remain unknown. In this study, we show that conventional dendritic cells but not plasmacytoid dendritic cells are required for the induction of malaria parasite-specific CD4⁺ T cell responses and subsequent experimental CM. These data have important implications for the development of malaria vaccines and the therapeutic management of CM.  

4.481           Regulation by Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Substrate-1 of -Galactosylceramide-Induced Antimetastatic Activity and Th1 and Th2 Responses of NKT Cells

Okajo, J. et al

1. Immunol., 178, 6164-6172 (2007)

  Interaction of -galactosylceramide ( -GalCer) presented by CD1d on dendritic cells (DCs) with the invariant TCR of NKT cells activates NKT cells. We have now investigated the role of Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), a transmembrane protein abundantly expressed on DCs, in regulation of NKT cells with the use of mice that express a mutant form of SHPS-1. The suppression by -GalCer of experimental lung metastasis was markedly attenuated in SHPS-1 mutant mice compared with that apparent in wild-type (WT) mice. The antimetastatic effect induced by adoptive transfer of -GalCer-pulsed DCs from SHPS-1 mutant mice was also reduced compared with that apparent with WT DCs. Both the production of IFN- and IL-4 as well as cell proliferation in response to -GalCer in vitro were greatly attenuated in splenocytes or hepatic mononuclear cells from SHPS-1 mutant mice compared with the responses of WT cells. Moreover, CD4⁺ mononuclear cells incubated with -GalCer and CD11c⁺ DCs from SHPS-1 mutant mice produced markedly smaller amounts of IFN- and IL-4 than did those incubated with -GalCer and CD11c⁺ DCs from WT mice. SHPS-1 on DCs thus appears to be essential for -GalCer-induced antimetastatic activity and Th1 and Th2 responses of NKT cells. Moreover, our recent findings suggest that SHPS-1 on DCs is also essential for the priming of CD4⁺ T cells by DCs.  

4.482           Inaugural Article: Toxicity from different SOD1 mutants dysregulates the complement system and the neuronal regenerative response in ALS motor neurons

Lobsiger, C.S., Boilee, S. and Cleveland, D.W. PNAS, 104(18), 7319-7326 (2007)   Global, age-dependent changes in gene expression from rodent models of inherited ALS caused by dominant mutations in superoxide-dismutase 1 (SOD1) were identified by using gene arrays and RNAs isolated from purified embryonic and adult motor neurons. Comparison of embryonic motor neurons expressing a dismutase active ALS-linked mutant SOD1 with those expressing comparable levels of wild-type SOD1 revealed the absence of mutant-induced mRNA changes. An age-dependent mRNA change that developed presymptomatically in adult motor neurons collected by laser microdissection from mice expressing dismutase active ALS-linked mutants was dysregulation of the D/L-serine biosynthetic pathway, previously linked to both excitotoxic and neurotrophic effects. An unexpected dysregulation common to motor neurons expressing either dismutase active or inactive mutants was induction of neuronally derived components of the classic complement system and the regenerative/injury response. Alteration of these mutant SOD1-induced pathways identified a set of targets for therapies for inherited ALS.  

4.483           A Host Lipase Detoxifies Bacterial Lipopolysaccharides in the Liver and Spleen

Shao, B. et al

1. Biol. Chem., 282(18), 13726-13735 (2007)

  Much of the inflammatory response of the body to bloodborne Gram-negative bacteria occurs in the liver and spleen, the major organs that remove these bacteria and their lipopolysaccharide (LPS, endotoxin) from the bloodstream. We show here that LPS undergoes deacylation in the liver and spleen by acyloxyacyl hydrolase (AOAH), an endogenous lipase that selectively removes the secondary fatty acyl chains that are required for LPS recognition by its mammalian signaling receptor, MD-2-TLR4. We further show that Kupffer cells produce AOAH and are required for hepatic LPS deacylation in vivo. AOAH-deficient mice did not deacylate LPS and, whereas their inflammatory responses to low doses of LPS were similar to those of wild type mice for 3 days after LPS challenge, they subsequently developed pronounced hepatosplenomegaly. Providing recombinant AOAH restored LPS deacylating ability to Aoah^-/- mice and prevented LPS-induced hepatomegaly. AOAH-mediated deacylation is a previously unappreciated mechanism that prevents prolonged inflammatory reactions to Gram-negative bacteria and LPS in the liver and spleen.  

4.484           Modulation of virulence factors in Francisella tularensis determines human macrophage responses

Carlson Jr., P.E., Carroll, J.A., O’Dee, D.M. and Nau, G.J. Microbial Pathogenesis, 42(5-6), 204-214 (2007)   Francisella tularensis, the causative agent of tularemia and Category A biodefense agent, is known to replicate within host macrophages, though the pathogenesis of this organism is incompletely understood. We have isolated a variant of F. tularensis live vaccine strain (LVS) based on colony morphology and its effect on macrophages. Human monocyte-derived macrophages produced more tumor necrosis factor α (TNFα), interleukin (IL)-1β, IL-6, and IL-12 p40 following exposure to the variant, designated the activating variant (ACV). The immunoreactivity of the lipopolysaccharide (LPS) from both LVS and ACV was comparable to the previously described blue variant and was distinct from the gray variant of LVS. We found, however, the soluble protein fractions of LVS and ACV differed. Further investigation using two-dimensional gel electrophoresis demonstrated higher levels of several proteins in the parental LVS isolate. The differentially expressed proteins featured several associated with virulence in F. tularensis and other pathogens, including intracellular growth locus C (IglC), a σ⁵⁴-modulation protein family member (YhbH), and aconitase. ACV reverted to the LVS phenotype, indicated by low cytokine induction and high IglC expression, after growth in a chemically defined medium. These data provide evidence that the levels of virulence factors in F. tularensis are modulated based on culture conditions and that this modulation impacts host responses. This work provides a basis for investigation of Francisella virulence factor regulation and the identification of additional factors, co-regulated with IglC, that affect macrophage responses.  

4.485           Suppression of reactive oxygen species production enhances neuronal survival in vitro and in vivo in the anoxia-tolerant turtle Trachemys scripta

Milton, S.L., Nayak, G., Kesaraju, S., Kara, L. and Prentice, H.M.

1. Neurochem., 101, 993-1001 (2007)

Hypoxia-ischemia with reperfusion is known to cause reactive oxygen species-related damage in mammalian systems, yet, the anoxia tolerant freshwater turtle is able to survive repeated bouts of anoxia/reoxygenation without apparent damage. Although the physiology of anoxia tolerance has been much studied, the adaptations that permit survival of reoxygenation stress have been largely ignored. In this study, we examine ROS production in the turtle striatum and in primary neuronal cultures, and examine the effects of adenosine (AD) on cell survival and ROS. Hydroxyl radical formation was measured by the conversion of salicylate to 2,3-dihydroxybenzoic acid (2,3-DHBA) using microdialysis; reoxygenation after 1 or 4 h anoxia did not result in increased ROS production compared with basal normoxic levels, nor did H₂O₂ increase after anoxia/reoxygenation in neuronally enriched cell cultures. Blockade of AD receptors increased both ROS production and cell death in vitro, while AD agonists decreased cell death and ROS. As turtle neurons proved surprisingly susceptible to externally imposed ROS stress (H₂O₂), we propose that the suppression of ROS formation, coupled to high antioxidant levels, is necessary for reoxygenation survival. As an evolutionarily selected adaptation, the ability to suppress ROS formation could prove an interesting path to investigate new therapeutic targets in mammals.  

4.486           Bacterially Derived 400 nm Particles for Encapsulation and Cancer Cell Targeting of Chemotherapeutics

MacDiamid, J.A. et al Cancer Cell, 11(5), 431-445 (2007)   Systemic administration of chemotherapeutic agents results in indiscriminate drug distribution and severe toxicity. Here we report a technology potentially overcoming these shortcomings through encapsulation and cancer cell-specific targeting of chemotherapeutics in bacterially derived 400 nm minicells. We discovered that minicells can be packaged with therapeutically significant concentrations of chemotherapeutics of differing charge, hydrophobicity, and solubility. Targeting of minicells via bispecific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release. This affects highly significant tumor growth inhibition and regression in mouse xenografts and case studies of lymphoma in dogs despite administration of minute amounts of drug and antibody; a factor critical for limiting systemic toxicity that should allow the use of complex regimens of combination chemotherapy.  

4.487           Cell cooperation in coelomocyte cytotoxic activity of Paracentrotus lividus coelomocytes

Arizza, V., Giaramita, F.T., Parinello, D., Cammarata, M. and Parinello, N. Comp. Biochem. Physiol. Part A, 147(2), 389-394 (2007)   The coelomic fluid from the sea urchin Paracentrotus lividus contains several coelomocyte types including amoebocytes and uncoloured spherulocytes involved in immune defences. In the present paper, we show a Ca²⁺-dependent cytotoxic activity for the unfractionated coelomocytes assayed in vitro, with rabbit erythrocytes and the K562 tumour cell line. In a plaque-forming assay, whole coelomocyte preparations as well as density gradient separated coelomocyte populations revealed that cell populations enriched in uncoloured spherulocytes, exerted high cytotoxic activity by releasing lysins in the presence of amoebocytes. This cooperative effect could be dependent on soluble factors released by amoebocytes. With regard to this, we show that an enhanced cytotoxic activity was found by adding the supernatant from sonicated amoebocytes or hemocyte culture medium into spherulocyte preparations.  

4.488           Differentiated Human Alveolar Epithelial Cells and Reversibility of their Phenotype In Vitro

Wang, J. et al Am. J. Respir. Cell Mol. Biol., 36, 661-668 (2007)   Cultures of differentiating fetal human type II cells have been available for many years. However, studies with differentiated adult human type II cells are limited. We used a published method for type II cell isolation and developed primary culture systems for maintenance of differentiated adult human alveolar epithelial cells for in vitro studies. Human type II cells cultured on Matrigel (basolateral access) or a mixture of Matrigel and rat tail collagen (apical access) in the presence of keratinocyte growth factor, isobutylmethylxanthine, 8-bromo-cyclicAMP, and dexamethasone (KIAD) expressed the differentiated type II cell phenotype as measured by the expression of surfactant protein (SP)-A, SP-B, SP-C, and fatty acid synthase and their morphologic appearance. These cells contain lamellar inclusion bodies and have apical microvilli. In both systems the cells appear well differentiated. In the apical access system, type II cell differentiation markers initially decreased and then recovered over 6 d in culture. Lipid synthesis was also increased by the addition of KIAD. In contrast, type II cells cultured on rat tail collagen (or tissue culture plastic) slowly lose their lamellar inclusions and expression of the surfactant proteins and increase the expression of type I cell markers. The expression of the phenotypes is regulated by the culture conditions and is, in part, reversible in vitro.  

4.489           Role of mitochondria in kainate-induced fast Ca²⁺ transients in cultured spinal motor neurons

Grosskreutz, J. et al Cell Calcium, 42, 59-69 (2007)   Motor neuron death in amyotrophic lateral sclerosis (ALS) has been linked to selective vulnerability towards AMPA receptor-mediated excitotoxicity. We investigated intracellular mechanisms leading to impairment of motor neuron Ca²⁺ homeostasis with near physiological AMPA receptor activation. Using fast solution exchange on patch-clamped cultured neurons, kainate (KA) was applied for 2 s. This induced a transient increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) for seconds. Inhibition of the mitochondrial uniporter by RU-360 abolished the decay of the Ca²⁺ transient and caused immediate [Ca²⁺]_c overload. Repetitive short KA stimulation caused a slowing of the decay of the Ca²⁺ transient and a gradual increase in peak and baseline [Ca²⁺]_c in motor neurons, but not in other neurons, indicating saturation of the mitochondrial buffer. Furthermore, mitochondrial density was lower in motor neurons and, in a network of neurons with physiological synaptic AMPA receptor input, RU-360 acutely induced an increase in Ca²⁺ transients. We conclude that motor neurons have an insufficient mitochondrial capacity to buffer large Ca²⁺ elevations which is partly due to a reduced mitochondrial density per volume compared to non-motor neurons. This may exert deleterious effects in motor neuron disease where mitochondrial function is thought to be compromised.  

4.490           Effector and regulatory T-cell function is differentially regulated by RelB within antigen-presenting cells during GVHD

MacDonald, K.P.A. et al Blood, 109, 5049-5057 (2007)   Antigen-presenting cells (APCs) are critical for the initiation of graft-versus-host disease (GVHD), although the responsible APC subset and molecular mechanisms remain unclear. Because dendritic cells (DCs) are the most potent APCs and the NF-kB/Rel family member RelB is associated with DC maturation and potent APC function, we examined their role in GVHD. Within 4 hours of total body irradiation, RelB nuclear translocation was increased and restricted to CD11c^hi DCs within the host APC compartment. Furthermore, the transient depletion of CD11c^hi donor DCs that reconstitute in the second week after transplantation resulted in a transient decrease in GVHD severity. By using RelB^–/– bone marrow chimeras as transplant recipients or RelB^–/– donor bone marrow, we demonstrate that the induction and maintenance of GVHD is critically dependent on this transcription factor within both host and donor APCs. Critically, RelB within APCs was required for the expansion of donor helper T cell type 1 (Th1) effectors and subsequent alloreactivity, but not the peripheral expansion or function of donor FoxP3⁺ regulatory T cells. These data suggest that the targeted inhibition of nuclear RelB translocation within APCs represents an attractive therapeutic strategy to dissociate effector and regulatory T-cell function in settings of Th1-mediated tissue injury.  

4.491           Osteopontin prevents monocyte recirculation and apoptosis

Burdo, T.H., Wood, M.R. and Fox, H.S.

1. Leukoc. Biol., 81, 1504-1511 (2007)

  Cells of the monocyte/macrophage lineage have been shown to be the principal targets for productive HIV-1 replication within the CNS. In addition, HIV-1-associated dementia (HAD) has been shown to correlate with macrophage abundance in the brain. Although increased entry of monocytes into the brain is thought to initiate this process, mechanisms that prevent macrophage egress from the brain and means that prevent macrophage death may also contribute to cell accumulation. We hypothesized that osteopontin (OPN) was involved in the accumulation of macrophages in the brain in neuroAIDS. Using in vitro model systems, we have demonstrated the role of OPN in two distinct aspects of macrophage accumulation: prevention from recirculation and protection from apoptosis. In these unique mechanisms, OPN would aid in macrophage survival and accumulation in the brain, the pathological substrate of  HAD.  

4.492           Trophic factors counteract elevated FGF-2-induced inhibition of adult neurogenesis

Chen, H., Tung, Y-C., Li, B., Iqbal, K. and Grundke-Iqbal, I. Neurobioliology of Aging, 28, 1148-1162 (2007)   The dentate gyrus of adult mammalian brain contains neural progenitor cells with self-renewal and multi-lineage potential. The lineage and maturation of the neural progenitors are determined by the composition and levels of the trophic factors in their microenvironment. In Alzheimer disease (AD) brain, especially the hippocampus, the level of basic fibroblast growth factor (FGF-2) is markedly elevated. Here we show that elevated FGF-2 enhances the division and nestin levels of cultured adult rat hippocampal progenitors but impairs neuronal lineage determination and maturation of these cells in culture. The trophic factors ciliary neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF), and insulin-like growth factors-1 and -2 (IGF-1, IGF-2) as well as an Alzheimer peptidergic drug, Cerebrolysin^® (CL), in which we found these neurotrophic activities, counteract the effect of FGF-2 in inducing neuronal lineage (early neurogenesis). Whereas CNTF is the most active of the neurotrophic factors studied in promoting neurogenesis, CL, probably because of a combined effect of these factors, induces similar changes but without inhibiting cell proliferation. These findings suggest that CNTF, GDNF, IGF-1, and IGF-2 are promising therapeutic targets for AD and other diseases in which neurogenesis is probably inhibited.  

4.493           Identification of an ADAM2-ADAM3 Complex on the Surface of Mouse Testicular Germ Cells and Cauda Epididymal Sperm

Nishimura, H., Myles, D.G. and Primakoff, P.

1. Biol. Chem., 282(24), 17900-17907 (2007)

  Male mice lacking ADAM2 (fertilin ) or ADAM3 (cyritestin) are infertile; cauda epididymal sperm (mature sperm) from these mutant mice cannot bind to the egg zona pellucida. ADAM3 is barely present in Adam2-null sperm, despite normal levels of this protein in Adam2-null testicular germ cells (TGCs; sperm precursor cells). Here, we have explored the molecular basis for the loss of ADAM3 in Adam2-null TGCs to clarify the biosynthetic and functional linkage of ADAM2 and ADAM3. A small portion of total ADAM3 was found present on the surface of wild-type and Adam2^-/- TGCs at similar levels. In the Adam2-null TGCs, however, surface-localized ADAM3 exhibited an increased amount of an endoglycosidase H-resistant form that may be related to instability of ADAM3. Moreover, we found a complex between ADAM2 and ADAM3 on the surface of TGCs and sperm. The intracellular chaperone calnexin was a component of the testicular ADAM2-ADAM3 complex. Our findings suggest that the association with ADAM2 is a key element for stability of ADAM3 in epididymal sperm. The presence of the ADAM2-ADAM3 complex in sperm also suggests a potential role of ADAM2 with ADAM3 in sperm binding to the egg zona pellucida.  

4.494           Binding of Pleomorphic Adenoma Gene-like 2 to the Tumor Necrosis Factor (TNF)- -responsive Region of the NCF2 Promoter Regulates p67phox Expression and NADPH Oxidase Activity

Ammons, M.C., Siemsen, D.W., Nelson-Overton, L.K., Quinn, M.T. and Gauss, K.A.

1. Biol. Chem., 282(24), 17941-17952 (2007)

  NCF2, the gene encoding the NADPH oxidase cytosolic component p67^phox, is up-regulated by TNF- , and we recently mapped a region in the NCF2 promoter that was required for this TNF- -dependent response. Because this TNF- -responsive region (TRR) lacked recognizable transcription factor binding elements, we performed studies to identify factors involved in regulating NCF2 via the TRR. Using the TRR sequence as bait in a yeast one-hybrid screen, we identified the zinc finger transcription factor Pleomorphic Adenoma Gene-Like 2 (PLAGL2) as a candidate regulator of NCF2 expression. PLAGL2-specific antibodies were generated that detected the native and SUMO1-modified forms of endogenous PLAGL2. EMSA and DNA-binding protein affinity purification analyses demonstrated specific binding of in vitro-translated as well as endogenously expressed PLAGL2 to the TRR, and chromatin immunoprecipitation assays demonstrated enhanced binding of endogenous PLAGL2 to the TRR in vivo with TNF- treatment. Knockdown of PLAGL2 protein inhibited up-regulation of NCF2 transcript, p67^phox protein expression, and subsequent superoxide production in response to TNF- . Furthermore, relative levels of native and SUMO1-modified endogenous PLAGL2 protein were modulated in a time-dependant manner in response to TNF- treatment. These data clearly identify PLAGL2 as a novel regulator of NCF2 gene expression as well as NADPH oxidase activity and contribute to a greater understanding of the transcriptional regulation of NCF2.  

4.495           Inhibition of human neutrophil degranulation by transforming growth factor-β1

Shen, L. et al Clin. Exp. Immunol., 149, 155-161 (2007)   Neutrophils enter tissues including the uterus and are found in the endometrium in increased numbers prior to menses. In this environment, they are exposed to transforming growth factor (TGF)-β1 produced by endometrial stromal and epithelial cells. We observed that incubation of neutrophils in vitro with TGF-β1 at 1 pg/ml significantly reduced their secretion of lactoferrin in response to lipopolysaccharide (LPS). This effect was achieved with as little as 15 min of pretreatment with TGF-β1. Inhibition of lactoferrin release by TGF-β1 was observed irrespective of whether neutrophils were stimulated by ligands for Toll-like receptor (TLR)-2, TLR-4 or FPR, the G protein-coupled receptor for formylated peptides. Inhibition by TGF-β1 was negated by SB-431542, a small molecule inhibitor that specifically blocks the kinase activity of the type I TGF-β receptor (ALK5) In contrast to lactoferrin release, another important neutrophil function, interleukin (IL)-8 driven chemotaxis, was not affected by TGF-β1 at 1 pg/ml or 100 pg/ml. We conclude that in tissues of the female reproductive tract, TGF-β1 inhibition of neutrophil degranulation may prevent these cells from initiating an inflammatory response or releasing degradative enzymes that could potentially damage the oocyte or fetus.  

4.496           Isolation and culture of adult neurons and neurospheres

Brewer, G,J. and Torricelli, J.R. Nature Protocols, 2(6), 1490-1498 (2007)   Here we present a protocol for extraction and culture of neurons from adult rat or mouse CNS. The method proscribes an optimized protease digestion of slices, control of osmolarity and pH outside the incubator with Hibernate and density gradient separation of neurons from debris. This protocol produces yields of millions of cortical, hippocampal neurons or neurosphere progenitors from each brain. The entire process of neuron isolation and culture takes less than 4 h. With suitable growth factors, adult neuron regeneration of axons and dendrites in culture proceeds over 1–3 weeks to allow controlled studies in pharmacology, electrophysiology, development, regeneration and neurotoxicology. Adult neurospheres can be collected in 1 week as a source of neuroprogenitors ethically preferred over embryonic or fetal sources. This protocol emphasizes two differences between neuron differentiation and neurosphere proliferation: adhesion dependence and the differentiating power of retinyl acetate.  

4.497           In vitro methods to prepare astrocyte and motoneuron cultures for the investigation of potential in vivo interactions

Taylor, A.R., Robinson, M.B. and Milligan, C.E. Nature protocols, 2(6), 1499-1507 (2007)   This protocol details methods to isolate and purify astrocytes and motoneurons (MNs) from the chick lumbar spinal cord. In addition, an approach to study the influences of astrocyte secreted factors on MNs is provided. Astrocytes are isolated between embryonic days 10 and 12 (E10–12), propagated in serum (2–3 h) and differentiated in chemically defined medium (3–4 h). When prepared according to this protocol, astrocyte cultures are more than 98% pure when assessed using the astrocyte-specific markers glial fibrillary acidic protein (GFAP) and S100 . MNs are isolated between E5.5 and 6.0 (3–4 h) using a procedure that takes selective advantage of the large size of these cells. These cultures can be maintained using individual trophic factors, target-derived factors or astrocyte-derived factors, the preparation of which is also described (5–6 h). All or part of these techniques can be used to investigate a variety of processes that occur during nervous system development and disease or after injury.  

4.498           Tocotrienols Induce Apoptosis and Autophagy in Rat Pancreatic Stellate Cells Through the Mitochondrial Death Pathway

Rickmann, M., Vaquero, E.C., Ramon, J., Malagelada, J.R. and Molero, X. Gastroenterology, 132, 2518-2532 (2007)   Background & Aims: Selective removal of activated pancreatic stellate cells (PSCs) through induction of their own programmed death is a goal of therapeutic interest in patients with chronic pancreatitis. Here, we investigated the effects of tocotrienols on PSC death outcomes. Methods: Activated and quiescent PSCs and acinar cells from rat pancreas were treated with vitamin E derivatives α-tocopherol; individual α-, β-, γ-, and δ-tocotrienols; and a tocotrienol rich fraction (TRF) from palm oil. Results: TRF, but not α-tocopherol, reduced viability of activated PSC by setting up a full death program, independent of cell cycle regulation. Activated PSCs died both through apoptosis, as indicated by increased DNA fragmentation and caspase activation, and through autophagy, as denoted by the formation of autophagic vacuoles and LC3-II accumulation. In contrast to α-tocopherol, TRF caused an intense and sustained mitochondrial membrane depolarization and extensive cytochrome c release. Caspase inhibition with zVAD-fmk suppressed TRF-induced apoptosis but enhanced autophagy. However, mitochondrial permeability transition pore blockade with cyclosporin A completely abolished the deadly effects of TRF. β-, γ-, and δ-tocotrienol, but not α-tocotrienol nor α-tocopherol, reproduced TRF actions on activated PSCs. TRF death induction was restricted to activated PSCs because it did not cause apoptosis either in quiescent PSCs or in acinar cells. Conclusions: Tocotrienols selectively trigger activated pancreatic stellate cell death by targeting the mitochondrial permeability transition pore. Our findings unveil a novel potential for tocotrienols to ameliorate the fibrogenesis associated with chronic pancreatitis.  

4.499           A Key Role for Itk in Both IFN and IL-4 Production by NKT Cells

Byron, B., Au-Yeung, and Fowell, D.J.

1. Immunol., 179, 111-119 (2007)

  NKT cells rapidly secrete cytokines upon TCR stimulation and thus may modulate the acquired immune response. Recent studies suggest that signaling for development and effector function in NKT cells may differ from conventional T cells. The tyrosine kinase Itk is activated downstream of the TCR, and its absence in CD4⁺ T cells results in impaired Th2, but not Th1 responses. In this study, we investigated NKT cell function in the absence of Itk as impaired type 2 responses in vivo could be manifest through IL-4 defects in a number of cell types. We show that Itk-deficient NKT cells up-regulate IL-4 mRNA in the thymus and express constitutive IL-4 and IFN- transcripts in peripheral organs. Thus, Itk is not required for the developmental activation of cytokine loci in NKT cells. Nevertheless, Itk-deficient NKT cells are severely impaired in IL-4 protein production. Strikingly, unlike conventional CD4⁺ T cells, Itk-deficient NKT cells also have profound defects in IFN- production. Furthermore, both IL-4 and IFN- production were markedly impaired following in vivo challenge with -galactosyl ceramide. Function can be restored in Itk-deficient NKT cells by provision of calcium signals using ionomycin. These results suggest that NKT cells are highly dependent on Itk for IL-4- and IFN- -mediated effector function. Thus, the pattern of cytokine genes that are affected by Itk deficiency appears to be cell lineage-specific, likely reflecting differences in activation threshold between immune effectors. The severe defect in NKT cell function may underlie a number of the Th1 and Th2 immune defects in Itk-deficient mice.  

4.500           Early Intrahepatic Accumulation of CD8+ T Cells Provides a Source of Effectors for Nonhepatic Immune Responses

Poløakos, N.K. et al

1. Immunol., 179, 201-210 (2007)

  Interactions between the liver and CD8⁺ T cells can lead to tolerance, due in part to CD8⁺ T cell death. To test whether this was the case in an extrahepatic infection, we investigated the fate and effector capacity of intrahepatic CD8⁺ T cells during lung-restricted influenza infection in mice. Virus-specific T cells accumulated in livers without detectable intrahepatic presentation of viral Ags, and this accumulation was not restricted to the contraction phase, but was apparent as early as day 5. Intrahepatic influenza-specific cells were functionally similar to those recovered from the bronchioalveolar lavage, based on ex vivo cytokine production and specific target lysis. Both adoptive transfer of liver lymphocytes and orthotopic liver transplant of organs containing accumulated effector T cells revealed that activated CD8s from the liver were viable, expanded during reinfection, and generated a memory population that trafficked to lymphoid organs. Thus, intrahepatic CD8⁺ T cells re-enter circulation and generate functional memory, indicating that the liver does not uniformly incapacitate activated CD8⁺ T cells. Instead, it constitutes a substantial reservoir of usable Ag-specific effector CD8⁺ T cells involved in both acute and recall immune responses.  

4.501           Resistance to Experimental Autoimmune Encephalomyelitis and Impaired T Cell Priming by Dendritic Cells in Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Substrate-1 Mutant Mice

Tomizawa, T. et al

1. Immunol., 179, 869-877 (2007)

  Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is expressed on the surface of CD11c⁺ dendritic cells (DCs) and macrophages. In this study, we show that mice that express a mutant form of SHPS-1 lacking most of the cytoplasmic region are resistant to experimental autoimmune encephalomyelitis (EAE) in response to immunization with a peptide derived from myelin oligodendrocyte glycoprotein (MOG (35–55)). The MOG (35–55)-induced proliferation of, and production of IFN- , IL-2, and IL-17, by T cells from immunized SHPS-1 mutant mice were reduced compared with those apparent for wild-type cells. The abilities of splenic DCs from mutant mice to stimulate an allogenic MLR and to prime Ag-specific T cells were reduced. Both IL-12-stimulated and TLR-dependent cytokine production by DCs of mutant mice were also impaired. Finally, SHPS-1 mutant mice were resistant to induction of EAE by adoptive transfer of MOG (35–55)-specific T cells. These results show that SHPS-1 on DCs is essential for priming of naive T cells and the development of EAE. SHPS-1 is thus a potential therapeutic target in inflammatory disorders of the CNS and other autoimmune diseases.  

4.502           Expression of Tyk2 in dendritic cells is required for IL-12, IL-23, and IFN- production and the induction of Th1 cell differentiation

Tokumasa, N. et al Blood, 110(2), 553-560 (2007)   It is well documented that dendritic cells (DCs), representative antigen-presenting cells, are important sources of Th1-promoting cytokines and are actively involved in the regulation of T-helper–cell differentiation. However, the intracellular event that regulates this process is still largely unknown. In this study, we examined the role of Tyk2, a JAK kinase that is involved in the signaling pathway under IL-12 and IL-23, in DC functions. While the differentiation and maturation of DCs was normal in Tyk2-deficient (Tyk2^–/–) mice, IL-12–induced Stat4 phosphorylation was diminished in Tyk2^–/– DCs. IL-12–induced IFN- production was also significantly diminished in Tyk2^–/– DCs to levels similar to those in Stat4^–/– DCs. Interestingly, Tyk2^–/– DCs were defective in IL-12 and IL-23 production upon stimulation with CpG ODN. Furthermore, Tyk2^–/– DCs were impaired in their ability to induce Th1-cell differentiation but not Th2-cell differentiation. Taken together, these results indicate that the expression of Tyk2 in DCs is crucial for the production of Th1-promoting cytokines such as IL-12 and IFN- from DCs and thereby for the induction of antigen-specific Th1-cell differentiation.  

4.503           Characterization of Heme as Activator of Toll-like Receptor 4

Figueiredo, R.T. et al

1. Biol. Chem., 282(28), 20221-20229 (2007)

  Heme is an ancient and ubiquitous molecule present in organisms of all kingdoms, composed of an atom of iron linked to four ligand groups of porphyrin. A high amount of free heme, a potential amplifier of the inflammatory response, is a characteristic feature of diseases with increased hemolysis or extensive cell damage. Here we demonstrate that heme, but not its analogs/precursors, induced tumor necrosis factor- (TNF- ) secretion by macrophages dependently on MyD88, TLR4, and CD14. The activation of TLR4 by heme is exquisitely strict, requiring its coordinated iron and the vinyl groups of the porphyrin ring. Signaling of heme through TLR4 depended on an interaction distinct from the one established between TLR4 and lipopolysaccharide (LPS) since anti-TLR4/MD2 antibody or a lipid A antagonist inhibited LPS-induced TNF- secretion but not heme activity. Conversely, protoporphyrin IX antagonized heme without affecting LPS-induced activation. Moreover, heme induced TNF- and keratinocyte chemokine but was ineffective to induce interleukin-6, interleukin-12, and interferon-inducible protein-10 secretion or co-stimulatory molecule expression. These findings support the concept that the broad ligand specificity of TLR4 and the different activation profiles might in part reside in its ability to recognize different ligands in different binding sites. Finally, heme induced oxidative burst, neutrophil recruitment, and heme oxygenase-1 expression independently of TLR4. Thus, our results presented here reveal a previous unrecognized role of heme as an extracellular signaling molecule that affects the innate immune response through a receptor-mediated mechanism.  

4.504           c-Jun N-terminal kinase signaling regulates events associated with both health and degeneration in motoneurons

Newbern, J., Taylor, A., Robinson, M., Livel, M.O. and Milligan, C.E. Neuroscience, 147(3), 680-692 (2007)   The c-Jun N-terminal kinases (JNKs) are activated by various stimuli and are critical for neuronal development as well as for death following a stressful stimulus. Here, we have evaluated JNK activity in both healthy and dying motoneurons from developing chick embryos and found no apparent difference in overall JNK activity between the conditions, suggesting that this pathway maybe critical in both circumstances. Pharmacological inhibition of JNK in healthy motoneurons supplied with trophic support resulted in decreased mitochondrial membrane potential, neurite outgrowth, and phosphorylation of microtubule-associated protein 1B. On the other hand, in motoneurons deprived of trophic support, inhibition of JNK attenuated caspase activation, and nuclear condensation. We also examined the role of JNK’s downstream substrate c-Jun in mediating these events. While c-Jun expression and phosphorylation were greater in cells supplied with trophic support as compared with those deprived, inhibition of c-Jun had no effect on nuclear condensation in dying cells or neurite outgrowth in healthy cells, suggesting that JNK’s role in these events is independent of c-Jun. Together, our data underscore the dualistic nature of JNK signaling that is critical for both survival and degenerative changes in motoneurons.    

4.505           Effects of Menstrual Cycle Status and Gender on Human Neutrophil Phenotype

Smith, J.M., Shen, Z., Wira, C.R., Fanger, M.W. and Shen, L. Am. J. Reprod. Immunol., 58, 111-119 (2007)   The effects of gender and fluctuating ovarian hormones on neutrophil phenotype have yet to be characterized. Method of study Neutrophils from females at days 7, 14, 21, and 28 of the menstrual cycle were analyzed by flow cytometry for surface receptor, granule protein, and intracellular cytokine expression. Comparisons were made to neutrophils from males isolated at 7-day intervals during 1 month. Results Decreased MMP-9 and TNF-α expression by neutrophils from females was observed during the periovulatory period. Comparing the genders, cells from females during the periovulatory period expressed less CD11b and CD18 than those from males. CXCR1 surface levels were higher on neutrophils from female donors. Conclusions Neutrophil phenotype varies minimally during the menstrual cycle and between the genders. Our data provide support for a potential anti-inflammatory effect of ovarian hormones on neutrophils.  

4.506           Fatty acids isolated from royal jelly modulate dendritic cell-mediated immune response in vitro

Vucevic, D. et al Int. Immunopharmacol., 7, 1211-1220 (2007)   Royal jelly (RJ), especially its protein components, has been shown to possess immunomodulatory activity. However, almost nothing is known about the influence of RJ fatty acids on the immune system. In this work we studied the effect of 10-hydroxy-2-decanoic acid (10-HDA) and 3,10-dihydroxy-decanoic acid (3,10-DDA), isolated from RJ, on the immune response using a model of rat dendritic cell (DC)–T-cell cocultures. Both fatty acids, at higher concentrations, inhibited the proliferation of allogeneic T cells. The effect of 10-HDA was stronger and was followed by a decrease in interleukin-2 (IL-2) production and down-regulation of IL-2 receptor expression. Spleen DC, cultivated with 10 μg/ml of fatty acids down-regulated the expression of CD86 and the production of IL-12, but up-regulated the production of IL-10. In contrast, DC, pretreated with 100 μg/ml of 3,10-DDA, up-regulated the expression of CD86 and augmented the proliferation of allogeneic T cells. The highest dose (200 μg/ml) of both fatty acids which was non-apoptotic for both T cells and DC, down-regulated the expression of MHC class II and CD86, decreased the production of IL-12 and made these DC less allostimulatory. The immunosuppressive activity of 3,10-DDA was also confirmed in vivo, using a model of Keyhole lymphet hemocyanine immunization of rats. In conclusion, our results showed the immunomodulatory activity of RJ fatty acids and suggest that DC are a significant target of their action.  

4.507           Evaluation of nonleukoreduced red blood cell transfusion units collected at delivery from the placenta

Widing, L., Bechensteen, A.G., Mirlashari, M.R., Vetlesen, A. and Kjeldsen-Kragh, J. Transfusion, 47, 1481-1487 (2007)   BACKGROUND: The objective of this study was to evaluate the suitability of cord blood (CB) as a source of red blood cells (RBCs) for autologous transfusion. STUDY DESIGN AND METHODS: CB was collected in 150-mL storage containers with citrate phosphate dextrose (CPD) as anticoagulant and stored in either saline, adenine, glucose, and mannitol (SAG-M; n = 18) or phosphate, adenine, glucose, guanosine, saline, and mannitol (PAGGS-M; n = 18) for 35 days at 4°C. Hematologic status and hemolysis were studied. The lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β1 from CB monocytes was analyzed after incubation with addition of weekly sampled supernatants from the CB RBC units. Five additional units (PAGGS-M) were leukoreduced and thereafter analyzed as indicated above. RESULTS: Hemolysis increased significantly over time, in SAG-M more than in PAGGS-M. During storage in both media, the number of white blood cells (WBCs) decreased, and the LPS-induced production of TNF-α and TGF-β1 decreased and increased, respectively. There were no significant changes in the LPS-induced production of TNF-α and TGF-β1 in the leukoreduced CB RBC units. CONCLUSION: Hemolysis in CB RBC units increased significantly over time, and PAGGS-M appears to be superior to SAG-M as a preservation solution for CB RBC. The changes in LPS-induced TNF-α and TGF-β1 production over time were probably caused by substances released from apoptotic and/or necrotic WBCs. Further studies are needed to identify both which substances are responsible for the changes in LPS-induced cytokine release and the clinical significance hereof.  

4.508           RT-PCR and immunocytochemistry studies support the presence of somatostatin, cortistatin and somatostatin receptor subtypes in rat Kupffer cells

Xidakis, C. et al RegulatoryPeptides, 143(1-3), 76-82 (2007)   The present study investigated the presence of somatostatin receptor subtypes (ssts) and the endogenous peptides somatostatin and cortistatin in rat Kupffer cells, since modulation of these cells by somatostatin may be important for the beneficial effect of somatostatin analogues in a selected group of hepatocellular carcinoma patients. Kupffer cells were isolated from rat liver in agreement with national and EU guidelines. RT-PCR was employed to assess the expression of somatostatin, cortistatin and ssts in Kupffer cells. Western blot analysis and immunocytochemistry were employed to assess the expression and the localization of the receptors, respectively. Quiescent Kupffer cells were found to express sst_1–4 mRNA, while immunocytochemical studies supported the presence of only the sst₃ and sst₄ receptors, which were found to be internalized. However, sst₁ and sst_2A receptors were detected by western blotting. RT-PCR and RIA measurements support the presence of both somatostatin and cortistatin. Stimulation of the cells with LPS activated the expression of the sst₂, sst₃ and sst₄ receptors. The present data provide evidence to support the presence of ssts and the endogenous neuropeptides somatostatin and CST in rat Kupffer cells. Both peptides may act in an autocrine manner to regulate sst receptor distribution. Studies are in progress in order to further characterize the role of ssts in Kupffer cells and in hepatic therapeutics.  

4.509           Human hepatic stem cells from fetal and postnatal donors

Schmelzer, E. et al

1. Exp. Med., 204(8), 1973-1987 (2007)

  Human hepatic stem cells (hHpSCs), which are pluripotent precursors of hepatoblasts and thence of hepatocytic and biliary epithelia, are located in ductal plates in fetal livers and in Canals of Hering in adult livers. They can be isolated by immunoselection for epithelial cell adhesion molecule–positive (EpCAM+) cells, and they constitute 0.5–2.5% of liver parenchyma of all donor ages. The self-renewal capacity of hHpSCs is indicated by phenotypic stability after expansion for >150 population doublings in a serum-free, defined medium and with a doubling time of 36 h. Survival and proliferation of hHpSCs require paracrine signaling by hepatic stellate cells and/or angioblasts that coisolate with them. The hHpSCs are 9 µm in diameter, express cytokeratins 8, 18, and 19, CD133/1, telomerase, CD44H, claudin 3, and albumin (weakly). They are negative for -fetoprotein (AFP), intercellular adhesion molecule (ICAM) 1, and for markers of adult liver cells (cytochrome P450s), hemopoietic cells (CD45), and mesenchymal cells (vascular endothelial growth factor receptor and desmin). If transferred to STO feeders, hHpSCs give rise to hepatoblasts, which are recognizable by cordlike colony morphology and up-regulation of AFP, P4503A7, and ICAM1. Transplantation of freshly isolated EpCAM+ cells or of hHpSCs expanded in culture into NOD/SCID mice results in mature liver tissue expressing human-specific proteins. The hHpSCs are candidates for liver cell therapies.  

4.510           Cytoskeletal Rearrangements via Vasodilator-Stimulated Phosphoprotein (VASP) Phosphorylation and Redistribution in Human Retinal Endothelial Cells (HREC), Endothelial Precursor Cells(EPC) and Platelets: Implications for Retinal Vessel Repair

Calzi, S.L. et al Invest. Ophthalmol. Vis. Sci., 48, E-abstract 4096 (2007)   Purpose:The actin motor protein VASP powers cell migration, vasodilatation,and inhibition of platelet aggregation- essential processesfor maintaining retinal vascular health. VASP has three phosphorylationsites (Ser-157, Ser-239, and Thr-278) that are regulated bycGMP and cAMP protein kinases. Therefore, we examined the effectof the vasoactive angiogenic agents CO, NO and stromal derivedfactor-1 (SDF-1) on cell migration, VASP phosphorylation, andVASP localization. Methods:CD34+ EPCs were isolated from human peripheral blood using magnetic microbeads and platelets using Opti-Prep^TM. HREC were prepared as previously described (Grant MB 1991). After 1, 5 and 15 min exposure to the NO donor DETA/NO, the CO donor Ru(II)Cl₂(CO)₃dimer or SDF-1, VASP phosphorylation in platelets and endothelialcells was evaluated by Western analysis. FACS analysis was usedto evaluate EPCs. Phosphospecific anti-pSer-157 antibody oranti-pSer-239 antibody was used for analysis. Immunohistochemistrywas performed to evaluate VASP redistribution in HREC and EPCsusing anti-VASP antibody following exposure to CO, NO or SDF-1.Migration to CO, NO and SDF-1 was performed using the modifiedBoyden chamber assay in HREC and EPC. Results:Upon CO stimulation in human platelets, VASP was phosphorylatedon Ser-157, but not Ser-239, while NO exposure resulted in phosphorylationmainly of Ser-239. In endothelial cells, VASP is phosphorylatedon Ser-239 in response to both CO and NO exposure. In EPC andHREC, VASP was redistributed to filopodia after incubation witheither CO, NO or SDF-1. At the concentrations tested, all threeagents induced EPC and endothelial cell migration in a dose-dependentmanner. Conclusion: In the retinal vasculature, NO, CO and SDF-1 may compensate for one another or work together to promote cytoskeletal changes through site-specific phosphorylation of VASP. Vasoactive agents may promote retinal vascular repair and improved tissue perfusion by increasing EPC and endothelial cell migration and by preventing platelet aggregation.  

4.511           Guinea Pig Neutrophils Infected with Mycobacterium tuberculosis Produce Cytokines Which Activate Alveolar Macrophages in Noncontact Cultures

Sawant, K.T. and McMurray, D.N. Infect. Immun., 75(4), 1870-1877 (2007)   The early influx of neutrophils to the site of infection may be an important step in host resistance against Mycobacterium tuberculosis. In this study, we investigated the effect of M. tuberculosis infection on the ability of guinea pig neutrophils to produce interleukin-8 (IL-8; CXCL8) and tumor necrosis factor alpha (TNF- ) and to activate alveolar macrophages. Neutrophils and alveolar macrophages were isolated from naïve guinea pigs, cultured together or alone, and infected with virulent M. tuberculosis for 3, 12, and 24 h. IL-8 protein production in cocultures, as measured by using an enzyme-linked immunosorbent assay, was found to be additive at 24 h and significantly greater in M. tuberculosis-infected cocultures than in uninfected cocultures and in cultures of the infected neutrophils or macrophages alone. The IL-8 mRNA levels, determined by real-time reverse transcription-PCR, were elevated at 24 h in infected cocultures and infected cells cultured alone. In order to elucidate the contributions of neutrophils and their soluble mediators to the activation of alveolar macrophages, neutrophils and alveolar macrophages were cultured in a contact-independent manner by using a Transwell insert system. Neutrophils were infected with virulent M. tuberculosis in the upper wells, and alveolar macrophages were cultured in the lower wells. The release of hydrogen peroxide from alveolar macrophages exposed to soluble products from infected neutrophils was significantly increased compared to that from unexposed alveolar macrophages. Significant up-regulation of IL-1ß and TNF- mRNA levels in alveolar macrophages was observed at 24 and 30 h, respectively, compared to those in cells not exposed to soluble neutrophil products. Treatment with anti-guinea pig TNF- polyclonal antibody completely abolished the response of alveolar macrophages to neutrophil products. This finding suggests that TNF- produced by infected neutrophils may be involved in the activation of alveolar macrophages and hence may contribute to the containment of M. tuberculosisinfection during the early period of infection.  

4.512           The Blood Contains Multiple Distinct Progenitor Populations with Clonogenic B and T Lineage Potential

Umland, O., Mwangi, W.N., Anderson, B.M., Walker, J.C. and Petrie, H.T.

1. Immunol., 178, 4147-4152 (2007)

  The thymus is seeded by bone marrow-derived progenitors that circulate in the blood. Multiple cell types can be found in the thymus early after i.v. administration or in steady state, but most fail to satisfy the known characteristics of true T progenitors. Cells that do conform to classical definitions retain multilineage potential, but surprisingly, cannot make B cells. Because acquisition of the T lineage fate among noncommitted progenitors is a lengthy process, the absence of B cell potential in early thymocytes suggests that B and T lineages diverge prethymically. To test this suggestion, we screened numerous presumptive progenitor populations for T cell growth and differentiation potential, as well as for clonogenic T or B cell development. We find that blood and marrow each contain multiple distinct subsets that display growth and differentiation potential consistent with being canonical T progenitors. Assessment of clonogenic potential further shows that although all blood and marrow populations have high T cell cloning potential, no T/non-B cells are apparent. These data suggest that either true thymic reconstitution potential derives from a small T/non-B cell subset of one of these populations, or that most of the cells defined as canonical progenitors within the thymus do not, in fact, reside in the mainstream of T progenitor differentiation.    

4.513           Effect of fetuin, a TGFβ antagonist and pentoxifylline, a cytokine antagonist on hepatic stellate cell function and fibrotic parameters in fibrosis

Verma-Gandhu, M., Peterson, M.R. and Peterson, T.C. Eur. J. Pharmacol., 572, 220-227 (2007)   We have previously shown that monocyte conditioned medium (MCM) from patients with liver fibrosis stimulated proliferation of hepatic stellate cells (HSCs), the major cell involved in hepatic fibrosis. To investigate the potential role of fetuin and pentoxifylline in fibrosis we used MCM samples obtained from patients with biopsy proven hepatic fibrosis related to Hepatitis C (HCV). Our results indicate that the MCM obtained from patients with HCV-related liver fibrosis significantly stimulated collagen synthesis in HSCs as assessed by tritiated proline incorporation into a collagenase sensitive trichloroacetic acid (TCA) precipitate. Collagen synthesis was also stimulated in HSCs using transforming growth factor beta (TGFβ) and this effect was neutralized using TGFβ antibody. Incubation of HSCs with fetuin (but not TGFβ antibody) significantly inhibited collagen synthesis in HSCs that were stimulated by HCV MCM samples. Patient MCM samples would also stimulate proliferation of HSCs as assessed by tritiated thymidine uptake but this effect was not attenuated by fetuin. Likewise the significant stimulatory effect of platelet derived growth factor (PDGF) on HSC proliferation and collagen synthesis was not inhibited by fetuin but could be significantly reduced by 70% and 40% respectively, when treated with pentoxifylline. We also investigated the ability of samples obtained from patients with hepatic fibrosis to inhibit HSC apoptosis, as determined by okadaic acid-induced 4-hydroxynonenal immunocytochemistry in HSCs. We have previously reported that okadaic acid induces apoptosis in HSCs as assessed by Hoescht and TUNEL. Okadaic acid treatment produced a positive 4-hydroxynonenal (4-HNE) immunoreactivity in HSCs and treatment with HCV patient MCM or TGFβ decreased the 4-HNE positive immunoreactivity in HSCs treated with okadaic acid. Our results suggest that fetuin may be beneficial in hepatic fibrosis and suggest that combination of fetuin and pentoxifylline may target the two key events in hepatic fibrosis by modifying the effects of TGFβ and PDGF, the two major growth factors in fibrosis.  

4.514           Pancreatic Islet Cell Transplantation: Update and New Developments

Onaca, N. et al Nutr. Clin. Pract., 22, 485-493 (2007)   Pancreatic islet cell transplantation is a treatment alternative for patients with type 1 diabetes who experience hypoglycemic unawareness despite maximal care. The good results obtained by the group from Edmonton and other centers, with 80% insulin independence at 1 year posttransplant, are not sustainable over time, with 5-year insulin independence achieved in only 10% of patients. However, persistent graft function, even without insulin independence, results in improved glucose control and avoidance of hypoglycemic events. Changes in organ preservation, islet processing technique, and immunosuppression regimens can result in improvement of results in the future. Islet autotransplantation is an option for patients who undergo total pancreatectomy for chronic pancreatitis with debilitating pain, in which reinfusion of the islets from the resected pancreas can result in avoidance of postsurgical diabetes or enhanced glucose control.  

4.515           Plasmodium falciparum glycosylphosphatidylinositol induces limited apoptosis in liver and spleen mouse tissue

Wichmann, D. et al Apoptosis, 12, 1037-1041 (2007)   Plasmodium falciparum malaria affects about 500 million people worldwide and is responsible for approximately 2.5 million deaths per year. Glycosylphosphatidylinositol (GPI) is the major anchor for membrane-associated proteins of P. falciparum and GPI plays a major role as a toxin in the pathology of malaria. Therefore, we tested the hypothesis that GPI, like LPS, induces apoptosis in vitro and in vital organs of mice. Our data does not provide evidence for direct cardiomyocyte apoptosis induced by GPI in vitro. However, in vivo injection of GPI induced limited apoptosis in mouse liver and spleen tissue. Apoptosis may be due to a direct GPI apoptotic effect or to an indirect effect via the induction of TNFα and nitric oxide production.  

4.516           Retinoic acid signaling sensitizes hepatic stellate cells to NK cell killing via upregulation of NK cell activating ligand RAE1

Radaeva, S. et al Am. J. Physiol. Gastrointest. Liver Physiol., 293, G809-G816 (2007)   Hepatic stellate cells (HSCs) store 75% of the body's supply of vitamin A (retinol) and play a key role in liver fibrogenesis. During liver injury, HSCs become activated and susceptible to natural killer (NK) cell killing due to increased expression of the NK cell activating ligand retinoic acid early inducible gene 1 (RAE-1). To study the mechanism by which RAE-1 is upregulated in HSCs during activation, an in vitro model of cultured mouse HSCs was employed. RAE-1 was detected at low levels in quiescent HSCs but upregulated in 4- and 7-day cultured HSCs (early activated HSCs), whereas 21-day cultured HSCs (fully activated HSCs) lost RAE-1 expression. High levels of RAE-1 in 4- and 7-day cultured HSCs correlated with their susceptibility to NK cell killing, which was diminished by treatment with RAE-1 neutralizing antibody. Furthermore, retinoic acid (RA) and retinal dehydrogenase (Raldh) levels were upregulated in early activated HSCs compared with quiescent or fully activated HSCs. Blocking RA synthesis by the Raldh inhibitor or blocking RA signaling by the retinoic acid receptor antagonist abolished upregulation of RAE-1 whereas treatment with RA induced RAE-1 expression in HSCs. In conclusion, during activation, HSCs lose retinol, which is either secreted out or oxidized into RA; the latter stimulates RAE-1 expression and sensitizes early activated HSCs to NK cell killing. In contrast, fully activated HSCs become resistant to NK cell killing because of lack of RAE1 expression, leading to chronic liver fibrosis and disease.  

4.517           Methods in Cell Separations

Dainiak, M.B., Kumar, A., Galaev, I.Y. and Matthiasson, B. Adv. Biochem. Engin/Biotechnol., Springer Berlin/Heidelberg (2007)   Research in the field of cell biology and biomedicine relies on technologies that fractionate cell populations and isolate rare cell types to high purity. A brief overview of methods and commercially available products currently used in cell separations is presented. Cell fractionation by size and density and highly selective affinity-based technologies such as affinity chromatography, fluorescence-activated cell sorting (FACS) and magnetic cell sorting are discussed in terms of throughput, yield, and purity.  

4.518           The Long Form of Fas Apoptotic Inhibitory Molecule Is Expressed Specifically in Neurons and Protects Them against Death Receptor-Triggered Apoptosis

Segura, M.F. et al

1. Neurosci., 27(42), 11228-11241 (2007)

  Death receptors (DRs) and their ligands are expressed in developing nervous system. However, neurons are generally resistant to death induction through DRs and rather their activation promotes neuronal outgrowth and branching. These results suppose the existence of DRs antagonists expressed in the nervous system. Fas apoptosis inhibitory molecule (FAIM_S) was first identified as a Fas antagonist in B-cells. Soon after, a longer alternative spliced isoform with unknown function was identified and named FAIM_L. FAIM_S is widely expressed, including the nervous system, and we have shown previously that it promotes neuronal differentiation but it is not an anti-apoptotic molecule in this system. Here, we demonstrate that FAIM_L is expressed specifically in neurons, and its expression is regulated during the development. Expression could be induced by NGF through the extracellular regulated kinase pathway in PC12 (pheochromocytoma cell line) cells. Contrary to FAIM_S, FAIM_L does not increase the neurite outgrowth induced by neurotrophins and does not interfere with nuclear factor B pathway activation as FAIM_S does. Cells overexpressing FAIM_L are resistant to apoptotic cell death induced by DRs such as Fas or tumor necrosis factor R1. Reduction of endogenous expression by small interfering RNA shows that endogenous FAIM_L protects primary neurons from DR-induced cell death. The detailed analysis of this antagonism shows that FAIM_L can bind to Fas receptor and prevent the activation of the initiator caspase-8 induced by Fas. In conclusion, our results indicate that FAIM_L could be responsible for maintaining initiator caspases inactive after receptor engagement protecting neurons from the cytotoxic action of death ligands.  

4.519           The Role of Dendritic Cells in the Development of Acute Dextran Sulfate Sodium Colitis

Berndt, B.E., Zhang, M., Chen, G-H., Huffnagle, G.B. and Kao, J.Y.

1. Immunol., 179, 6255-6262 (2007)

  Dendritic cells (DCs) are essential mediators of the host immune response to surrounding microbes. In this study, we investigate the role of DCs in the pathogenesis of a widely used colitis model, dextran sulfate sodium-induced colitis. The effect of dextran sulfate sodium on the production of proinflammatory cytokines and chemokines by bone marrow-derived DCs (BM-DCs) was analyzed. BM-DCs were adoptively transferred into C57BL/6 mice or DCs were ablated using transgenic CD11c-DTR/GFP mice before treatment with 5% dextran sulfate sodium in drinking water. We found that dextran sulfate sodium induced production of proinflammatory cytokines (IL-12 and TNF- ) and chemokines (KC, MIP-1 , MIP-2, and MCP-1) by DCs. Adoptive transfer of BM-DCs exacerbated dextran sulfate sodium colitis while ablation of DCs attenuated the colitis. We conclude that DCs are critical in the development of acute dextran sulfate sodium colitis and may serve a key role in immune balance of the gut mucosa.  

4.520           Estradiol Attenuates Lipopolysaccharide-Induced CXC Chemokine Ligand 8 Production by Human Peripheral Blood Monocytes

Pioli, P.A. et al

1. Immunol., 179, 6284-6290 (2007)

  Regulation of the inflammatory response is imperative to the maintenance of immune homeostasis. Activated monocytes elaborate a broad variety of proinflammatory cytokines that mediate inflammation, including CXCL8. Release of this chemokine attracts neutrophils to sites of bacterial invasion and inflammation; however, high levels of CXCL8 may result in excessive neutrophil infiltration and subsequent tissue damage. In this study, we demonstrate that 17 -estradiol (E2) attenuates LPS-induced expression of CXCL8 in human peripheral blood monocytes. Treatment of monocytes with estradiol before administration of LPS reduces CXCL8 message and protein production through an estrogen receptor-dependent mechanism, and luciferase reporter assays demonstrate that this inhibition is mediated transcriptionally. Importantly, the ability of estradiol-pretreated LPS-activated monocytes to mobilize neutrophils is impaired. These results implicate a role for estradiol in the modulation of the immune response, and may lead to an enhanced understanding of gender-based differences in inflammatory control mechanisms.  

4.521           Thymosin 4 Upregulates the Expression of Hepatocyte Growth Factor and Downregulates the Expression of PDGF- Receptor in Human Hepatic Stellate Cells

Barnaeva, E., Nadezhda, A., Hannappell, E., Sjogren, M.H: and Rojkind, M. Ann. N.Y. Acad. Sci., 1112, 154-160 (2007)   Hepatic stellate cells (HSCs) are the main producers of type I collagen in the liver, and therefore are responsible, in part, for the fibrous scar observed in cirrhotic livers. Although there is no approved treatment for this deadly disease, drugs inducing HSC apoptosis in animals (gliotoxin) and hepatocyte regeneration in man (hepatocyte growth factor [HGF]), have been used successfully in ameliorating liver fibrosis. In this communication we investigated whether thymosin ₄ (T ₄), an actin-sequestering peptide that prevents scarring of the heart after a myocardial infarction and that prevents kidney fibrosis in animals, has the potential to be used to treat liver fibrosis. To this end we studied whether the administration of T ₄ to HSCs could alter the expression of genes encoding for extracellular matrix components, as well as those required for differentiation of HSCs. Our preliminary findings show that T ₄ had no effect on the expression of 2 (I) collagen, tissue inhibitor of metalloproteinases-1, and matrix metalloproteinase-2 mRNAs. However, it upregulated the expression of HGF and downregulated the expression of platelet-derived growth factor- receptor mRNAs in these cells. Overall, these findings suggest that T ₄ has antifibrogenic potential.  

4.522           Conventional dendritic cells regulate the outcome of colonic inflammation independently of T cells

Abe, K. et al PNAS, 104(43), 17022-17027 (2007)   We explored the physiological role of conventional dendritic cells (cDCs) in acute colitis induced by a single cycle of dextran sodium sulfate administration. Depending on their mode of activation and independently of T cells, cDCs can enhance or attenuate the severity of dextran sodium sulfate-induced colitis. The latter beneficial effect was achieved, in part, by IFN-1 induced by Toll-like receptor 9-activated cDCs. IFN-1 inhibits colonic inflammation by regulating neutrophil and monocyte trafficking to the inflamed colon and restraining the inflammatory products of tissue macrophages. These data highlight a novel role of cDCs in the regulation of other innate immune cells and position them as major players in acute colonic inflammation.    

4.523           A closed system for islet isolation and purification using the COBE2991 cell processor may reduce the need of clean room facilities

Klaffschenkel, R.A. et al Cell Transplant., 16(6), 587-594 (2007)   During the isolation of human islets of Langerhans the digest has repeated direct contact with the ambient atmosphere. In order to fulfill GMP requirements in clinical applications, the entire cell preparation must be performed in clean room facilities. We hypothesized that the use of a closed system, which avoids the direct exposure of tissue to the atmosphere, would significantly ease the preparation procedure. To avoid the direct atmosphere exposure we tested a modification of the isolation and purification process by performing all islet preparation steps in a closed system. In this study we compared the isolation outcome of the traditional open preparation technique with the new closed system. Pancreata from 6-month-old hybrid pigs were procured in the local slaughterhouse. After digestion/filtration the digest was cooled, collected, and concentrated in centrifugation containers and purified thereafter in the COBE2991 by top loading (control). In the control group 502±253 IEQ per gram pancreas were purified. The total preparation time amounted to 12 h. In the closed system the digest were cooled and directly pumped into the COBE2991 for centrifugation followed by supernatant expelling. Bag filling, centrifugation and expelling were repeated several times. Islets in pellet form were than purified by adding a gradient (bottom loading). Using this closed system 1098±489 IEQ per gram pancreas were purified with a total cell viability of 67±10% and a b-cell viability of 41±13%. The total preparation time reduced to 6 h. After 24 h of cell culture the viability of b-cells was still 56±10% and was only reduced after the addition of proapoptotic IL-1 and TNF-a to 40±4%, indicating that freshly isolated islets are not apoptotic. In conclusion, the closed system preparation is much faster, more effective, and less expensive than the traditional islet preparation. The closed system may be applicable for human islets preparations to restrict the need of clean room facilities for islets preparations to a minimum and may open the way for islet preparations without clean room demand.  

4.524           Interleukin-22 but Not Interleukin-17 Provides Protection to Hepatocytes during Acute Liver Inflammation

Zenewicz, L.A. et al Immunity, 27(4), 647-659 (2007)   The cytokine interleukin-22 (IL-22) is primarily expressed by T helper 17 (Th17) CD4⁺ T cells and is highly upregulated during chronic inflammatory diseases. IL-22 receptor expression is absent on immune cells, but is instead restricted to the tissues, providing signaling directionality from the immune system to the tissues. However, the role of IL-22 in inflammatory responses has been confounded by data suggesting both pro- and anti-inflammatory functions. Herein, we provide evidence that during inflammation, IL-22 played a protective role in preventing tissue injury. Hepatocytes from mice deficient in IL-22 were highly sensitive to the detrimental immune response associated with hepatitis. Additionally, IL-22-expressing Th17 cells provided protection during hepatitis in IL-22-deficient mice. On the other hand, interleukin-17 (IL-17), which is coexpressed with IL-22 and can induce similar cellular responses, had no observable role in liver inflammation. Our data suggest that IL-22 serves as a protective molecule to counteract the destructive nature of the immune response to limit tissue damage.  

4.525           Generation of islets from stem cells

Soria, B., Hmadcha, A., Bedoya, F.J. and Tejedo, J.R. Tissue Eengineering, 3^rd Ed., 605-618 (2007)   No abstract available  

4.526           Directional Freezing of Equine Semen in Large Volumes

Saragusty, J., Gacitua, H., Petit, M.T. and Arav, A. Reprod. Dom. Anim., 42, 610-615 (2007)   Despite its potential impact on the horse industry, sperm cryopreservation is not an established technology throughout the industry, for a number of reasons that include a reduction in pregnancy rate and increased cost per pregnancy. We have evaluated a novel directional freezing technique, based on a multi-thermal gradient (MTG), by comparing it with the conventional, controlled-rate cryopreservation method (CRCM). Ninety-seven ejaculates with ≥50% motility, collected from 31 stallions were each divided into two parts and subsequently frozen by either MTG or CRCM. Frozen samples were then stored in liquid nitrogen until thawing. The two treatments were evaluated by three methods: progressive linear motility (PLM), viability stain and hypoosmotic swelling (HOS) test. High correlation was found between the three evaluation methods for all post-thaw samples. Eighty-eight per cent of the ejaculates frozen by MTG had post-thaw PLM ≥35%, whereas only 59% of the ejaculates frozen by CRCM had such motility. Post-thaw evaluations of samples frozen by MTG and CRCM were: PLM – 50.2 ± 1.5% and 37.4 ± 1.5%, respectively; viability – 53.6 ± 1.5% and 39.5 ± 1.4%, respectively; membrane integrity, as evaluated by HOS – 36.2 ± 1.3% and 26.5 ± 1.1%, respectively. The differences according to all the evaluation methods were highly significant (p < 0.001), and the results indicate that freezing stallion semen by MTG is superior to CRCM.  

4.527           Confocal light absorption and scattering spectroscopic microscopy monitors organelles in live cells with no exogenous labels

Itzkan, I. et al PNAS, 104(44), 17255-17260 (2007)   This article reports the development of an optical imaging technique, confocal light absorption and scattering spectroscopic (CLASS) microscopy, capable of noninvasively determining the dimensions and other physical properties of single subcellular organelles. CLASS microscopy combines the principles of light-scattering spectroscopy (LSS) with confocal microscopy. LSS is an optical technique that relates the spectroscopic properties of light elastically scattered by small particles to their size, refractive index, and shape. The multispectral nature of LSS enables it to measure internal cell structures much smaller than the diffraction limit without damaging the cell or requiring exogenous markers, which could affect cell function. Scanning the confocal volume across the sample creates an image. CLASS microscopy approaches the accuracy of electron microscopy but is nondestructive and does not require the contrast agents common to optical microscopy. It provides unique capabilities to study functions of viable cells, which are beyond the capabilities of other techniques.  

4.528           Neurotrophins Redirect p75^NTR from a Clathrin-Independent to a Clathrin-Dependent Endocytic Pathway Coupled to Axonal Transport

Deinhardt, K., Reversi, A., Berninghausen, O., Hopkins, C.R. and Schiavo, G. Traffic, 8, 1736-1749 (2007)   The p75 neurotrophin receptor (p75^NTR) plays multiple roles in neuronal physiology through interactions with many ligands and coreceptors. However, its intracellular neuronal trafficking prior to and after neurotrophin activation is still poorly characterized. We have previously shown that in response to nerve growth factor (NGF), p75^NTR is retrogradely transported along the axons of motor neurons (MNs) in carriers shared with NGF, brain-derived neurotrophic factor and the tyrosine kinase receptor TrkB. Here, we report that NGF does not enhance the internalization or degradation of p75^NTR, which undergoes a rapid dynamin-dependent and clathrin-independent recycling process in MNs. Instead, incubation of cells with NGF leads to the redirection of a pool of plasma membrane p75^NTR into clathrin-coated pits. The subsequent internalization of p75^NTR via clathrin-mediated endocytosis, as well as the activity of Rab5, are essential for the sorting of the p75^NTR-containing endosomes to the axonal retrograde transport pathway and for the delivery of p75^NTR to the soma. Our findings suggest that the spatial regulation of p75^NTR signalling is controlled by these ligand-driven routes of endocytosis.  

4.529           Fasting induces changes in peripheral blood mononuclear cell gene expression profiles related to increases in fatty acid ß-oxidation: functional role of peroxisome proliferator–activated receptor in human peripheral blood mononuclear cells

Bouwens, M., Afman, L.A. and Müller, M. Am. J. Clin. Nutr., 86, 1515-1523 (2007)   Background: Peripheral blood mononuclear cells (PBMCs) are theonly readily available cells in healthy humans. Various studiesshowed disease-characteristic gene expression patterns in PBMCs.However, little is known of nutritional effects on PBMC geneexpression patterns. Fatty acids are nutrients that regulategene expression by activating the nuclear receptor peroxisomeproliferator–activated receptor (PPAR). PBMCs expressPPAR, making these cells interesting to study FA-dependent geneexpression. Objective: The aim of this study was to elucidate whether PBMCgene expression profiles also reflect nutrition-related metabolicchanges. Furthermore, we focused on the specific role of PPARin regulation of PBMC gene expression during fasting, when plasmafree fatty acids are elevated. Design: Four healthy male volunteers fasted for 48 h. PBMC RNAwas hybridized on Affymetrix whole genome microarrays. To elucidatethe role of PPAR, PBMCs of 9 blood donors were incubated withthe specific PPAR ligand Wy14643. Results: After 24 and 48 h of fasting, 1200 and 1386 genes werechanged >1.4-fold, respectively. Many of those genes wereinvolved in fatty acid ß-oxidation and are known PPARtarget genes. Incubation of PBMCs with Wy14643 resulted in up-regulationof genes that were also up-regulated during fasting. Conclusions: We conclude that PBMC gene expression profiles reflect nutrition-related metabolic changes such as fasting and that part of the fasting-induced changes are likely regulated by PPAR .  

4.530           Endothelial protein C receptor is overexpressed in rheumatoid arthritic (RA) synovium and mediates the anti-inflammatory effects of activated protein C in RA monocytes

Xue, M., March, L., Sambrook, P.N., Fukudome, K. and Jackson, C.J. Ann. Rheum. Dis., 66, 1574-1580 (2007)   Objectives: (1) To investigate whether inflammatory synovial tissues frompatients with rheumatoid arthritis (RA) express endothelialprotein C receptor (EPCR) and (2) to determine the major celltype(s) that EPCR is associated with and whether EPCR functionsto mediate the effects of activated protein C (APC) on thesecells. Methods: EPCR, CD68 and PC/APC in synovial tissues were detected by immunostainingand in situ PCR. Monocytes were isolated from peripheral bloodof patients with RA and treated with APC, lipopolysaccharide(LPS), and/or EPCR blocking antibody RCR252. Cells and supernatantswere collected for RT-PCR, western blotting, enzyme-linked immuosorbentassay and chemotaxis assay. Results: EPCR was expressed by both OA and RA synovial tissuesbut was markedly increased in RA synovium. EPCR was colocalisedwith PC/APC mostly on CD68 positive cells in synovium. In RAmonocytes, APC upregulated EPCR expression and reduced monocytechemoattractant protein-1-induced chemotaxis of monocytes byapproximately 50%. APC also completely suppressed LPS-stimulatedNF-B activation and attenuated TNF- protein by more than 40%in RA monocytes. The inhibitory effects of APC were reversedby RCR252, indicating that EPCR is required. Conclusions: Our results demonstrate for the first time that EPCR is expressedby synovial tissues, particularly in RA, where it co-localiseswith PC/APC on monocytes/macrophages. In addition, APC inhibitsthe migration and activation of RA monocytes via EPCR. Theseinhibitory effects on RA monocytes suggest that PC pathway mayhave a beneficial therapeutic effect in RA.  

4.531           Rat coronaviruses infect rat alveolar type I epithelial cells and induce expression of CXC chemokines

Miura, T.A., Wang, J., Holmes, K.V. and Mason, R.J. Virology, 369(2), 288-298 (2007)   We analyzed the ability of two rat coronavirus (RCoV) strains, sialodacryoadenitis virus (SDAV) and Parker's RCoV (RCoV-P), to infect rat alveolar type I cells and induce chemokine expression. Primary rat alveolar type II cells were transdifferentiated into the type I cell phenotype. Type I cells were productively infected with SDAV and RCoV-P, and both live virus and UV-inactivated virus induced mRNA and protein expression of three CXC chemokines: CINC-2, CINC-3, and LIX, which are neutrophil chemoattractants. Dual immunolabeling of type I cells for viral antigen and CXC chemokines showed that chemokines were expressed primarily by uninfected cells. Virus-induced chemokine expression was reduced by the IL-1 receptor antagonist, suggesting that IL-1 produced by infected cells induces uninfected cells to express chemokines. Primary cultures of alveolar epithelial cells are an important model for the early events in viral infection that lead to pulmonary inflammation.  

4.532           The use of test gradient s to determine the bottom layer density for subsequent continuous isopycnic purification of human islets on a COBE 2991 cell processor

Yonekawa, Y. et al Xenotransplantation, 14(5), poster PJ1007, 449-549 (2007)   Objectives: The standard human islet purification method utilizes fixed densities of 1.075 to 1.095 g/cm3 for continuous density gradients and 1.095 g/cm3 for bottom layers. We hypothesized that acinar tissue densities vary among donor organs and that test gradients (TGs) measuring acinar tissue density improve our understanding of the dynamics of tissue density during pancreas processing and thereby separation of islet from acinar tissue during subsequent continuous density gradient separation. Materials and Methods: We determined the acinar tissue density of tissue digests prepared from pancreata from 50 deceased donors and 4 donors who underwent total pancreatectomy and islet autotransplantation. 5-ml iodixanol gradients with densities of 1.085, 1.090, 1.095, 1.100, 1.105, and 1.110 g/cm3 were transferred to six 15-ml conical tubes. Each gradient was overlaid with 1 ml of cap solution (1.035). Pancreatic digest (200 ll), obtained at the start of phase 2, was top-loaded onto each of the 6 gradients. These TGs were centrifuged at 430xg for 3 min at 8 _C. The density chosen as the density of the bottom layer for the subsequent COBE run was 0.005 g/cm3 higher than the highest density that still allowed sedimentation of digest to the bottom of the tube. Results: The mean ± SD density selected as the bottom layer for autoislet purification was 1.106 ± 0.005 (range: 1.100-1.110). This density was significantly higher (p<0.001) than the density selected for deceased donor islet purification, which was 1.091 ± 0.005 (range: 1.070-1.100). For autoislet purification, fractions excluding the COBE bag and denser than 1.095g/cm3 contained 27.0 ± 23.8% of the total islet volume and only 1.7 ± 2.3 ml (range: 0.5-6.0) tissue volume. Conclusions: TGs appear useful for facilitating the separation of acinar tissue with densities >1.095 g/cm3, as found typically in preparations intended for islet autotransplantation. Using TGs, we were able to return to the pancreatectomized patient a large proportion of autoislets that would have otherwise been lost in the COBE bag. Interventions that maintain the high density of acinar tissue throughout pancreas preservation and processing are likely to improve density gradient purification of deceased donor islets.  

4.533           Excitotoxicity mediated by non-NMDA receptors causes distal axonopathy in long-term cultured spinal motor neurons

King, A.E. et al Eur. J. Neurosci., 26, 2151-2159 (2007)   Excitotoxicity has been implicated as a potential cause of neuronal degeneration in amyotrophic lateral sclerosis (ALS). It has not been clear how excitotoxic injury leads to the hallmark pathological changes of ALS, such as the abnormal accumulation of filamentous proteins in axons. We have investigated the effects of overactivation of excitatory receptors in rodent neurons maintained in long-term culture. Excitotoxicity, mediated principally via non-N-methyl-D-aspartate (NMDA) receptors, caused axonal swelling and accumulation of cytoskeletal proteins in the distal segments of the axons of cultured spinal, but not cortical, neurons. Axonopathy only occurred in spinal neurons maintained for 3 weeks in vitro, indicating that susceptibility to axonal pathology may be related to relative maturity of the neuron. Excitotoxic axonopathy was associated with the aberrant colocalization of phosphorylated and dephosphorylated neurofilament proteins, indicating that disruption to the regulation of phosphorylation of neurofilaments may lead to their abnormal accumulation. These data provide a strong link between excitotoxicity and the selective pattern of axonopathy of lower motor neurons that underlies neuronal dysfunction in ALS.  

4.534           Transition from enhanced T cell infiltration to inflammation in the myelin-degenerative central nervous system

Grundtner, R. et al Neurobiology of Disease, 28(3), 261-275 82007)   Myelin degeneration in the central nervous system (CNS) is often associated with elevated numbers of T cells in brain and spinal cord (SC). In some degenerative diseases, this T cell immigration has no clinical relevance, in others, it may precede severe inflammation and tissue damage. We studied T cells in the myelin-degenerative SC of transgenic (tg) Lewis rats overexpressing the proteolipid protein (PLP). These lymphocytes are T_H1/T_C1 cells and represent different T cell clones unique to individual animals. The SC-infiltrating CD8⁺ T cell pool is more restricted than its CD4⁺ counterpart, possibly due to constrictions in the peripheral CD8⁺ T cell repertoire. Some SC-infiltrating T cells are highly motile and cover large distances within their target tissue, others are tethered to MHC class II⁺ microglia cells. The activation of the tethered cells may trigger the formation of inflammatory foci and could pave the way for inflammation in degenerative CNS disease.  

4.535           Fibrillar beta-amyloid (Aβ) (1–42) elevates extracellular Aβ in cultured hippocampal neurons of adult rats

Maja, S., Rastegar, K., Zarifkar, A. and Takhshid, M.A. Brain Res., 1185, 321-327 (2007)   Alzheimer's disease (AD) is a chronic disorder with progressive neurodegeneration associated with aging and is characterized by fibrillar beta-amyloid (Aβ) deposits in the brain. Although the increased production of Aβ seems to play a noticeable role in AD pathogenesis and its progression, all the mechanisms which are involved in this extracellular Aβ elevation are not known completely. In the present study, we used adult hippocampal neuronal culture as an in vitro model which is favorable for adult neurodegenerative diseases' studies. We introduced a toxic concentration for fibrillar Aβ1–42 in adult neurons which was much lower from the toxic concentration in embryonic neurons. To determine the effect of fibrillar Aβ1–42 which is the most toxic part of amyloid plaques, on extracellular Aβ1–40, as the main part of βAPP proteolysis products, we treated the neurons with fibrillar Aβ1–42 at nontoxic concentrations of 2 × 10^− 6, 2 × 10^− 5 and 2 × 10^− 4 μM and measured extracellular Aβ1–40. Our findings show that even very low levels of fibrillar Aβ1–42 can contribute to subsequent extracellular Aβ elevation in a dose dependent manner. These results suggest that even low levels of fibrillar Aβ may have deleterious actions if it remains in extracellular space for a period of time.  

4.536           Defective T Helper Response of Hepatocyte-Stimulated CD4 T Cells Impairs Antiviral CD8 Response and Viral Clearance

Wiegard, C. et al Gasteroenterology, 133, 2010-2018 (2007)   Background & Aims: In hepatitis, hepatocytes gain the ability to express major histocompatibility complex (MHC) class II molecules and to present antigen to CD4 T cells. Here, we investigated whether MHC class II-expressing hepatocytes influence in vitro the differentiation of CD4 T cells and in vivo the T-cell response to and control of viral infection. Methods: Class II transactivator-transgenic hepatocytes that constitutively express MHC class II molecules were used to stimulate CD4 T cells in vitro, and the effector response type of the stimulated CD4 T cells was determined. The in vivo relevance of the obtained findings was confirmed by infecting nontransgenic or class II transactivator-transgenic mice with lymphocytic choriomeningitis virus. Results: MHC II-expressing hepatocytes induced T helper cell (Th) 2 differentiation of uncommitted CD4 T cells and abrogated the ability of previously differentiated Th1 to secrete interferon-γ, even in the presence of proinflammatory microbial signals. The suppression of Th1 responses by hepatocytes was associated with poor expression levels of Th1-promoting Delta-like Notch ligands. In vivo, MHC II expression by hepatocytes impaired the interferon-γ production by lymphocytic choriomeningitis virus-specific CD4 and CD8 T cells and prolonged viral persistence. Conclusions: By instructing infiltrating CD4 T cells to differentiate into a less inflammatory phenotype, MHC II-expressing hepatocytes seem to impair antiviral CD8 T-cell responses and viral clearance. Thus, hepatocytes may contribute to the chronicity of hepatitis virus infection.  

4.537           Expression of transforming growth factor-b by human islets: Impact on inslets viability and function

Sabek, O.M. et al Cell Transplant., 16, 775-785 (2007)   Transforming growth factor-b1 (TGF-b1) is a pleotrophic cytokine that promotes angiogenesis and extracellular matrix protein synthesis in addition to its immunosuppressive effects. The purpose of this study is to identify optimal conditions for in vivo expression of TGF-b1 by human islets to exploit the possible beneficial effects and minimize undesirable side effects. We transduced human islets with adenoviral vectors encoding the active form of Ad-TGF-b1 or Ad-LacZ to test the effects of TGF-b1 gene expression on islet in vivo function following their transplantation into a NOD-SCID mouse model. Islets were transduced with multipliticy of infection (MOI) of 20, 10, 5 and 2.5 per islet cell. At a MOI ranging from 2.5 to 20, expression of TGF-b1 in islet supernatant persisted for 1-2 months and ranged from 153 ± 5 to 2574 ± 1299 pg/ml, respectively. Transduction with the lowest MOI (2.5) did not comprise the in vivo production of human C-peptide. We conclude that TGF-b1 expression in transplanted islets does not compromise viability and that adenoviral transduction with the TGF-b1 gene has a dose-dependent effect, with larger MOIs being used successfully to evaluate the nonimmune effects of gene transduction.  

4.538           Kupffer cell heterogeneity: functional properties of bone marrow–derived and sessile hepatic macrophages

Klein, I. et al Blood, 110, 4077-4085 (2007)   Kupffer cells form a large intravascular macrophage bed in the liver sinusoids. The differentiation history and diversity of Kupffer cells is disputed; some studies argue that they are derived from blood monocytes, whereas others support a local origin from intrahepatic precursor cells. In the present study, we used both flow cytometry and immunohistochemistry to distinguish 2 subsets of Kupffer cells that were revealed in the context both of bone marrow transplantation and of orthotopic liver transplantation. One subset was radiosensitive and rapidly replaced from hematogenous precursors, whereas the other was relatively radioresistant and long-lived. Both were phagocytic but only the former population was recruited into inflammatory foci in response to CD8⁺ T-cell activation. We propose the name "sessile" for the radioresistant Kupffer cells that do not participate in immunoinflammatory reactions. However, we found no evidence that these sessile Kupffer cells arise from immature intrahepatic precursors. Our conclusions resolve a long-standing controversy and explain how different experimental approaches may reveal one or both of these subsets.  

4.539           Surface Modification of RGD-Liposomes for Selective Drug Delivery to Monocytes/Neutrophils in Brain

Qin, J. et al Chem. Pharm. Bul., 55(8), 1192-1197 (2007)   In the present study, RGD peptide was coupled with ferulic acid (FA) liposomes for binding to monocytes and neutrophils in peripheral blood for brain targeting in response to leukocyte recruitment. Cholesterol (Ch) was esterified with succinic anhydride to introduce a carboxylic end group (Ch-COOH). Soybean phosphatidylcholine, cholesterol and Ch-COOH were in a molar ratio of 1 : 0.23 : 0.05. FA was loaded into liposomes with 80.2±5.2% entrapment efficiency (EE) using a calcium acetate gradient method since it was difficult to load FA by other methods. RGD peptide was a novel compound coupled with Ch-COOH via carbodiimide and N-hydroxysulfosuccinimide. The results of the in vitro flow cytometric study showed that RGD conjugation liposomes (RGD-liposomes) could bind to monocytes/neutrophils efficiently. The rats were subjected to intrastriatal microinjections of 100 μl of human recombinant IL-1β to produce brain inflammation and subsequently sacrificed after 15, 30, 60 and 120 min of administration of three formulations (FA solution, FA liposome, RGD-coated FA liposome). The body distribution results showed that RGD-liposomes could be directed to the target site, i.e. the brain, by cell selectivity in case of an inflammatory response. For RGD coated liposomes, the concentration of FA in brain was 6-fold higher than that of FA solution and 3-fold higher than that of uncoated liposomes. MTT assay and flow cytometry were used in the pharmacodynamic studies where it was found that FA liposomes exhibited greater antioxidant activity to FA solution on U937 cell.  

4.540           Body Distributioin of RGD-mediated Liposome in Brain-targeting Drug Delivery

Qin, J. et al Yakugaku Zasshi, 127(9), 1497-1501 (2007)   RGD conjugation liposomes (RGD-liposomes) were evaluated for brain-targeting drug delivery. The flow cytometric in vitro study demonstrated that RGD-liposomes could bind to monocytes and neutrophils effectively. Ferulic acid (4-hydroxy-3-methoxycinnamic, FA) was loaded into liposomes. Rats were subjected to intrastriatal microinjections of 100 units of human recombinant IL-1β to produce brain inflammation and caudal vein injection of three formulations (FA solution, FA liposome and RGD-coated FA liposome). Animals were sacrificed 15, 30, 60 and 120 min after administration to study the body distribution of the FA in the three formulations. HPLC was used to determine the concentration of FA in vivo with salicylic acid as internal standard. The results of body distribution indicated that RGD-coated liposomes could be mediated into the brain with a 6-fold FA concentration compared to FA solution and 3-fold in comparison to uncoated liposome. Brain targeted delivery was achieved and a reduction in dosage might be allowed.  

4.541           Coronavirus Non-Structural Protein 1 Is a Major Pathogenicity Factor: Implications for the Rational Design of Coronavirus Vaccines

Züst, R. et al PloS Pathogens, 3(8), 1062-1072 (2007)   Attenuated viral vaccines can be generated by targeting essential pathogenicity factors. We report here the rational design of an attenuated recombinant coronavirus vaccine based on a deletion in the coding sequence of the non-structural protein 1 (nsp1). In cell culture, nsp1 of mouse hepatitis virus (MHV), like its SARS-coronavirus homolog, strongly reduced cellular gene expression. The effect of nsp1 on MHV replication in vitro and in vivo was analyzed using a recombinant MHV encoding a deletion in the nsp1-coding sequence. The recombinant MHV nsp1 mutant grew normally in tissue culture, but was severely attenuated in vivo. Replication and spread of the nsp1 mutant virus was restored almost to wild-type levels in type I interferon (IFN) receptor-deficient mice, indicating that nsp1 interferes efficiently with the type I IFN system. Importantly, replication of nsp1 mutant virus in professional antigen-presenting cells such as conventional dendritic cells and macrophages, and induction of type I IFN in plasmacytoid dendritic cells, was not impaired. Furthermore, even low doses of nsp1 mutant MHV elicited potent cytotoxic T cell responses and protected mice against homologous and heterologous virus challenge. Taken together, the presented attenuation strategy provides a paradigm for the development of highly efficient coronavirus vaccines.  

4.542           Novel hepatic progenitor cell surface markers in the adult rat liver

Yovchev, M.I., Groszdanov, P.N., Hoseph, B., Gupta, S.and Daabeva, M.D. Hepatology, 45(1), 139-149 (2007)   Hepatic progenitor/oval cells appear in injured livers when hepatocyte proliferation is impaired. These cells can differentiate into hepatocytes and cholangiocytes and could be useful for cell and gene therapy applications. In this work, we studied progenitor/oval cell surface markers in the liver of rats subjected to 2-acetylaminofluorene treatment followed by partial hepatectomy (2-AAF/PH) by using rat genome 230 2.0 Array chips and subsequent RT-PCR, immunofluorescent (IF), immunohistochemical (IHC) and in situ hybridization (ISH) analyses. We also studied expression of the identified novel cell surface markers in fetal rat liver progenitor cells and FAO-1 hepatoma cells. Novel cell surface markers in adult progenitor cells included tight junction proteins, integrins, cadherins, cell adhesion molecules, receptors, membrane channels and other transmembrane proteins. From the panel of 21 cell surface markers, 9 were overexpressed in fetal progenitor cells, 6 in FAO-1 cells and 6 are unique for the adult progenitors (CD133, claudin-7, cadherin 22, mucin-1, ros-1, Gabrp). The specificity of progenitor/oval cell surface markers was confirmed by ISH and double IF analyses. Moreover, study of progenitor cells purified with Ep-CAM antibodies from D-galactosamine injured rat liver, a noncarcinogenic model of progenitor cell activation, verified that progenitor cells expressed these markers. Conclusion: We identified novel cell surface markers specific for hepatic progenitor/oval cells, which offers powerful tool for their identification, isolation and studies of their physiology and pathophysiology. Our studies also reveal the mesenchymal/epithelial phenotype of these cells and the existence of species diversity in the hepatic progenitor cell identity.  

4.543           Immune role of hepatic TLR-4 revealed by orthotopic mouse liver transplantation

John, B., Klein, I. and Crispe, I.N. Hepatology, 45(1), 178-186 (2007)   Activated CD8+ T cells migrate to the liver at the end of an immune response and go through apoptosis there, but this mechanism is impaired in mice lacking Toll-like receptor-4. This allowed us to test the importance of liver trapping in an ongoing immune response. In the absence of Toll-like receptor-4, reduced liver accumulation was associated with an increase in the circulating CD8+ T cell pool, more long-lived memory T cells and increased CD8+ T cell memory responses. Using experimental orthotopic liver transplantation, we showed that the effect of Toll-like receptor-4 on the formation of the CD8+ T cell memory resides in the liver. Conclusion: These studies reveal a new function for the liver, which is to regulate the magnitude of T cell memory responses through a Toll-like receptor-4–dependent mechanism.  

4.544           Murine liver plasmacytoid dendritic cells become potent immunostimulatory cells after Flt-3 ligand expansion

Kingham, T.P., Chaudhry, U.I., Plitas, G., Katz, S.C., Raab, J. and DeMatteo, R.P. Hepatology, 45(2), 445-454 (2007)   The liver has unique immunological properties. Although dendritic cells (DCs) are central mediators of immune regulation, little is known about liver DCs. Plasmacytoid DCs (pDCs) are a recently identified subtype of murine liver DC. We sought to define the function of freshly isolated murine liver pDCs. We found that normal liver pDCs were weak in stimulating T cells, yet they possessed a proinflammatory cytokine profile with high tumor necrosis factor- and low IL-10 secretion. To facilitate the investigation of murine liver pDCs, we expanded them in vivo with fms-like tyrosine kinase 3 ligand (Flt3L). After Toll-like receptor-9 ligation, expanded liver pDCs secreted high levels of IFN- and were able to stimulate NK cells, NKT cells, and antigen-specific CD8+ T cells in vitro. In addition, Flt3L expansion alone generated pDCs capable of activating antigen-specific CD8+ T cells in vivo. Conclusion: Unstimulated liver pDCs exist in a latent state with the potential to become potent activators of the innate and adaptive immune systems through their interactions with other immune effectors. Our findings have implications for understanding the role of the liver in tolerance and immunity.  

4.545           Transdifferentiation of hepatocyte-like cells from the human hepatoma HepaRG cell line through bipotent progenitor

Cerec, V., glaise, D., Garnier, D., Morosan, S., Turlin, B., Drenou, B., Gripon, P., Kremsdorf, D., Guguen-Guillouzo, C. and Corlu, A. Hepatology, 45(4), 957-967 (2007)   Hepatic tumors, exhibiting mature hepatocytes and undifferentiated cells merging with cholangiocyte and hepatocyte phenotypes, are frequently described. The mechanisms by which they occur remain unclear. We report differentiation and transdifferentiation behaviors of human HepaRG cells isolated from a differentiated tumor developed consecutively to chronic HCV infection. We demonstrate that, in vitro, proliferating HepaRG cells differentiate toward hepatocyte-like and biliary-like cells at confluence. If hepatocyte-like cells are selectively isolated and cultured at high cell density, they proliferate and preserve their differentiation status. However, when plated at low density, they transdifferentiate into hepatocytic and biliary lineages through a bipotent progenitor. In accordance, transplantation of either undifferentiated or differentiated HepaRG cells in uPA/SCID mouse damaged liver gives rise mainly to functional human hepatocytes infiltrating mouse parenchyma. Analysis of the differentiation/transdifferentiation process reveals that: (1) the reversible differentiation fate of HepaRG cells is related to the absence of p21CIP1 and p53 accumulation in differentiated cells; (2) HepaRG bipotent progenitors express the main markers of in vivo hepatic progenitors, and that cell differentiation process is linked to loss of their expression; (3) early and transient changes of β-catenin localization and HNF3β expression are correlated to Notch3 upregulation during hepatobiliary commitment of HepaRG cells. Conclusion: Our results demonstrate the great plasticity of transformed hepatic progenitor cells and suggest that the transdifferentiation process could supply the pool of hepatic progenitor cells. Moreover, they highlight possible mechanisms by which transdifferentiation and proliferation of unipotent hepatocytes might cooperate in the development of mixed and differentiated tumors.  

4.546           Atorvastatin lowers portal pressure in cirrhotic rats by inhibition of RhoA/Rho-kinase and activation of endothelial nitric oxide synthase

Trebicka, J., Hennenberg, M., Laleman, W., Shelest, W., Biecker, E., Schepke, M., Nevens, F., Sauerbruch, T. and Heller, J. Hepatology, 46(1), 242-253 (2007)   In cirrhosis, increased RhoA/Rho-kinase signaling and decreased nitric oxide (NO) availability contribute to increased intrahepatic resistance and portal hypertension. Hepatic stellate cells (HSCs) regulate intrahepatic resistance. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) inhibit synthesis of isoprenoids, which are necessary for membrane translocation and activation of small GTPases like RhoA and Ras. Activated RhoA leads to Rho-kinase activation and NO synthase inhibition. We therefore investigated the effects of atorvastatin in cirrhotic rats and isolated HSCs. Rats with secondary biliary cirrhosis (bile duct ligation, BDL) were treated with atorvastatin (15 mg/kg per day for 7 days) or remained untreated. Hemodynamic parameters were determined in vivo (colored microspheres). Intrahepatic resistance was investigated in in situ perfused livers. Expression and phosphorylation of proteins were analyzed by RT-PCR and immunoblots. Three-dimensional stress-relaxed collagen lattice contractions of HSCs were performed after incubation with atorvastatin. Atorvastatin reduced portal pressure without affecting mean arterial pressure in vivo. This was associated with a reduction in intrahepatic resistance and reduced responsiveness of in situ–perfused cirrhotic livers to methoxamine. Furthermore, atorvastatin reduced the contraction of activated HSCs in a 3-dimensional stress-relaxed collagen lattice. In cirrhotic livers, atorvastatin significantly decreased Rho-kinase activity (moesin phosphorylation) without affecting expression of RhoA, Rho-kinase and Ras. In activated HSCs, atorvastatin inhibited the membrane association of RhoA and Ras. Furthermore, in BDL rats, atorvastatin significantly increased hepatic endothelial nitric oxide synthase (eNOS) mRNA and protein levels, phospho-eNOS, nitrite/nitrate, and the activity of the NO effector protein kinase G (PKG). Conclusion: In cirrhotic rats, atorvastatin inhibits hepatic RhoA/Rho-kinase signaling and activates the NO/PKG-pathway. This lowers intrahepatic resistance, resulting in decreased portal pressure. Statins might represent a therapeutic option for portal hypertension in cirrhosis.  

4.547           Vaccination with plasmacytoid dendritic cells induces protection against infection with Leishmania major in mice

Remer, K., Apetrei, C., Schwarz, T., Linden, C. and Moll, H. Eur. J. Immunol., 37(9), 2463-2473 (2007)   DC-based vaccination against Leishmania major induces a parasite-specific Th1 response and long-lasting protective immunity in susceptible mice. Since distinct DC subsets have been proposed to direct the predominant development of either Th1 or Th2 cells, we analyzed the capability of plasmacytoid DC (pDC) to induce protection and elicit a Th1 response against L. major. Pulsing with L. major lysate induced the activation and maturation of semi-mature murine pDC that had been isolated from the spleen, as indicated by up-regulation of the co-stimulatory molecules CD86 and CD80, but did not enhance the level of IFN- secretion by pDC. Vaccination of susceptible mice with L. major lysate-pulsed pDC induced highly effective T cell-mediated immunity against subsequent infection with L. major parasites. Surprisingly, the protection was not accompanied by a polarized Th1 cytokine profile. Co-activation of pDC with CpG-containing oligodeoxynucleotides, which has been shown to be critical for activating the protective potential of myeloid DC, was not required for the protective effect of L. major antigen-pulsed pDC. These findings demonstrate that antigen-loaded pDC are able to induce T cell-mediated protection against a parasite disease and that experimental leishmaniasis is a suitable model to elucidate the mechanisms underlying DC-based vaccination against infections.  

4.548           Simultaneous age-related depolarization of mitochondrial membrane potential and increased mitochondrial reactive oxygen species production correlate with age-related glutamate excitotoxicity in rat hippocampal neurons

Parihar, M.S. and Brewer, G.J.

1. Neurosci. Res., 85(5), 1018-1032 (2007)

  Mitochondria are implicated in glutamate excitotoxicity by causing bioenergetic collapse, loss of Ca2+ homeostasis, and generation of reactive oxygen species (ROS), all of which become increasingly important clinically with age. Little is known about how aging affects the relative importance of mitochondrial membrane potential (ΔΨm) and ROS production. To determine aging affects on ΔΨm and ROS production in individual somal and axonal/dendritic mitochondria, we compared ROS production while simultaneously monitoring ΔΨm before and after glutamate treatment of live neurons from embryonic (day 18), middle-aged (9–12 months), and old (24 months) rats. At rest, old neuronal mitochondria 1) showed a higher rate of ROS production that was particularly strong in axonal/dendritic mitochondria relative to that in middle-age neurons, 2) were more depolarized in comparison with neurons of other ages, and 3) showed no differences in ROS or ΔΨm as a function of distance from the nucleus. All ΔΨm grouped into three classes of high (less than –120 mV), medium (–85 to –120 mV), and low (greater than –85 mV) polarization that shifted toward the lower classes with age at rest. Glutamate exposure dramatically depolarized the ΔΨm in parallel with greatly increased ROS production, with a surprising absence of an effect of age or distance from the nucleus on these mitochondrial parameters. These data suggest that old neurons are more susceptible to glutamate excitotoxicity because of an insidious depolarization of ΔΨm and rate of ROS generation at rest that lead to catastrophic failure of phosphorylative and reductive energy supplies under stress.  

4.549           Differential regulation of matrix metalloproteinase 2 and matrix metalloproteinase 9 by activated protein C: Relevance to inflammation in rheumatoid arthritis

Xue, M., March, L., Sambrook, P.N. and Jackson, C.J. Arthritis & Rheumatism, 56(9), 2864-2874 (2007)   Objective To investigate the in vitro effect of activated protein C (APC), a natural anticoagulant and novel antiinflammatory agent, on the regulation of the gelatinases matrix metalloproteinase 2 (MMP-2) and MMP-9. Methods Synovial fibroblasts and peripheral blood monocytes isolated from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) and Mono Mac6 cells were used in this study. After treatment, cells and culture supernatants were collected for zymography, enzyme-linked immunosorbent assay, reverse transcription–polymerase chain reaction, and Western blot analysis. Results Fibroblasts and monocytes from RA patients produced substantially more MMP-9 than did those from OA patients; however, there was no difference in MMP-2 production. The addition of recombinant APC markedly reduced MMP-9 at the gene and protein levels. In contrast, APC up-regulated and activated MMP-2. Using a blocking antibody to the endothelial protein C receptor (EPCR), we showed that the inhibition of MMP-9 by APC was EPCR-dependent. Furthermore, APC directly suppressed the production of tumor necrosis factor (TNF) and the activation of NF-κB and MAP kinase p38, and inhibitors of NF-κB or p38 reduced the production of MMP-9, suggesting that APC inhibits MMP-9 by blocking TNF, NF-κB, and p38. Thus, APC acts on MMP-9 by binding to EPCRs on the cell surface and, subsequently, inhibiting the intracellular activation of the proinflammatory signaling molecules NF-κB and p38. Conclusion APC appears to be the first physiologic agent to inhibit the production of proinflammatory MMP-9, yet increase antiinflammatory MMP-2 activity. Our results provide the initial evidence that APC may be beneficial in the prevention of inflammation and joint destruction in RA.  

4.550           Platelet imidazoline receptors as state marker of depressive symptomatology

Piletz, J., Baker, R. and Halaris, A.

1. Psychiatric Res., 42, 41-49 (2008)

  Objective Previous studies have shown that imidazoline receptors (IR-1) are increased in platelets and frontal cortex of depressed patients, and this up-regulation is normalized (down-regulated) after antidepressant drug treatments. It has been hypothesized that IR-1 up-regulation during the depressive episode may be a state marker for depressive symptomatology. The goal of the present study was to address the state versus trait question. Method Twelve healthy subjects (six males and six females) met stringent inclusion and exclusion criteria for physical and mental health. They received desipramine for 6 weeks in order to simulate the length of time and dosing used previously to obtain an IR-1 down-regulation and a therapeutic response in depressed patients. Outcome and safety measures included clinical, psychological, and cardiovascular assessments obtained throughout the study. Plasma concentrations of desipramine were measured throughout the 6 weeks of treatment and again after 2 weeks following tapered discontinuation of desipramine. Platelet receptors were assessed by Western blotting and radioligand binding assays. Results Healthy subjects taking desipramine experienced mild dysphoric effects but there were no adverse events. The binding of 8 nM p-[¹²⁵I]clonidine to IR-1 and α₂-adrenoceptors in healthy subjects did not change during desipramine treatment. The immunodensity of the 33 kDa band associated with IR-1 gradually increased to a maximum, by week-6, of 26% higher than baseline (p < 0.01 compared to baseline). Two weeks after desipramine discontinuation, there was a decline in α₂-adrenoceptor binding and 33 kDa band’s immunodensity (p = 0.04). Conclusions The findings support the hypothesis that platelet IR-1 binding sites are a marker of mood state rather than of antidepressant-induced pharmacological regulation. By comparison, platelet α₂-adrenoceptors appear to be regulated by desipramine as a pharmacological effect independent of mood state.  

4.551           Abrogation of the Antifibrotic Effects of Natural Killer Cells/Interferon-γ Contributes to Alcohol Acceleration of Liver Fibrosis

Jeong, W-L., Park, O. and Gao, B. Gastroenterology, 134, 248-258 (2008)   Background & Aims: Chronic alcohol drinking accelerates liver fibrosis in patients with viral hepatitis that cannot be fully explained by ethanol-enhanced liver damage. Here, we identified a novel mechanism by which alcohol accelerates liver fibrosis: inhibition of the antifibrotic effects of natural killer (NK) cells and interferon-γ (IFN-γ). Methods: Alcohol administration was achieved by feeding mice with a liquid diet containing 5% ethanol for 8 weeks. Liver fibrosis was induced by administration of carbon tetrachloride (CCl₄) for 2 weeks. Hepatic stellate cells (HSCs) were also isolated and cultured for in vitro studies. Results: CCl₄ treatment induced greater fibrosis and less apoptosis of HSCs in ethanol-fed mice compared with pair-fed mice. Polyinosinic-polycytidylic acid (Poly I:C) or IFN-γ treatment inhibited liver fibrosis in pair-fed but not in ethanol-fed mice. Poly I:C activation of NK cell cytotoxicity against HSCs was attenuated in ethanol-fed mice compared with pair-fed mice, which was due to reduced natural killer group 2 member D (NKG2D), tumor necrosis factor-related apoptosis-inducing ligand, and IFN-γ expression on NK cells from ethanol-fed mice. In vitro, HSCs from ethanol-fed mice were resistant to IFN-γ–induced cell cycle arrest and apoptosis compared with pair-fed mice. Such resistance was due to diminished IFN-γ activation of signal transducer and activator of transcription 1 (STAT1) in HSCs from ethanol-fed mice caused by the induction of suppressors of cytokine signaling proteins and the production of oxidative stress. Finally, HSCs from ethanol-fed mice were resistant to NK cell killing, which can be reversed by transforming growth factor-β1 (TGF-β1) neutralizing antibody. Conclusions: Chronic ethanol consumption attenuates the antifibrotic effects of NK/IFN-γ/STAT1 in the liver, representing new and different therapeutic targets with which to treat alcoholic liver fibrosis.  

4.552           T Cell Development from Kit-Negative Progenitors in the Foxn1 / Mutant Thymus

Xiao, S., Su, D-m. and Manley, N.R.

1. Immunol., 180, 914-921 (2008)

  Foxn1 is a hypomorphic allele of the nude gene that causes arrested thymic epithelial cell differentiation and abnormal thymic architecture lacking cortical and medullary domains. T cells develop in the Foxn1 ^/ adult thymus to the double- and single-positive stages, but in the apparent absence of double-negative 3 (DN3) cells; however, DN3 cells are present in the fetal thymus. To investigate the origin of this seemingly contradictory phenotype, we performed an analysis of fetal and adult DN cells in these mutants. Neither adult bone marrow-derived cells nor fetal liver cells from wild-type or Rag1^–/– mice were able to differentiate to the DN2 or DN3 stage in the Foxn1 ^/ thymus. Our data suggest that thymopoiesis in the Foxn1 ^/ adult thymus proceeds from CD117^– atypical progenitors, while CD117⁺ DN1a cells are absent or blocked in their ability to differentiate to the T lineage. Wild-type cells generated by this pathway in the postnatal thymus were exported to the periphery, demonstrating that these atypical cells contributed to the peripheral T cell pool. The Foxn1 ^/ adult (but not fetal) thymus also preferentially supports B cell development, specifically of the B-1 type, and this phenotype correlated with reduced Notch ligand expression in the adult stroma.  

4.553           Red Blood Cells Are the Major Source of Alpha-Synuclein in Blood

Barbour, R. et al Neurodegenerative Dis., 5, 55-59 (2008)   Background: -Synuclein has been directly linked to Parkinson's disease etiology by mutations in and multiplication of its gene that result in a familial form of Parkinson's disease. -Synuclein has been detected in blood, and was found to be elevated in the blood of those individuals with the -synuclein gene multiplication. Objective: A complete analysis of the level of -synuclein in blood has not been performed. In this report, we determine the quantitative distribution of -synuclein in the plasma and different cellular fractions of human blood. The levels of -synuclein in human and mouse blood are compared. Methods: -Synuclein levels in the different fractions of blood were quantified by a sandwich ELISA with purified recombinant -synuclein as an assay standard. Samples were further characterized by Western immunoblot analysis. Results: More than 99% of the -synuclein resides in the red blood cells (RBCs) with less than 1% of the total detected in the plasma, platelets and peripheral blood mononuclear cells. Conclusions: More than 99% of the -synuclein in human blood is present in the peripheral blood cells, with the remainder in plasma. Fractionation of peripheral blood cells from human blood and quantification of -synuclein revealed that only a very small amount of the total -synuclein is present in peripheral blood mononuclear cells, and platelets, with the majority of -synuclein in blood being present in RBCs. Considering the abundance and fragility of RBCs, -synuclein levels in these other blood fractions or other bodily fluids such as cerebrospinal fluid may be artificially elevated by contamination with intact or lysed RBCs.  

4.554           Effects of 30 min of aerobic exercise on gene expression in human neutrophils

Radom-Aizik, S., Zalvidar, Jr., F., Leu, S-Y., Galasetti, P. and Cooper, D.M.

1. Appl. Physiol., 104, 236-243 (2008)

  Relatively brief bouts of exercise alter gene expression in peripheral blood mononuclear cells (PBMCs), but whether exercise changes gene expression in circulating neutrophils (whose numbers, like PBMCs, increase) is not known. We hypothesized that exercise would activate neutrophil genes involved in apoptosis, inflammation, and cell growth and repair, since these functions in leukocytes are known to be influenced by exercise. Blood was sampled before and immediately after 30 min of constant, heavy ( 80% peak O₂ uptake) cycle ergometer exercise in 12 healthy men (19–29 yr old) of average fitness. Neutrophils were isolated using density gradients; RNA was hybridized to Affymetrix U133+2 Genechip arrays. With false discovery rate (FDR) <0.05 with 95% confidence, a total of 526 genes were differentially expressed between before and after exercise. Three hundred and sixteen genes had higher expression after exercise. The Jak/STAT pathway, known to inhibit apoptosis, was significantly activated (EASE score, P < 0.005), but 14 genes were altered in a way likely to accelerate apoptosis as well. Similarly, both proinflammatory (e.g., IL-32, TNFSF8, and CCR5) and anti-inflammatory (e.g., ANXA1) were affected. Growth and repair genes like AREG and FGF2 receptor genes (involved in angiogenesis) were also activated. Finally, a number of neutrophil genes known to be involved in pathological conditions like asthma and arthritis were altered by exercise, suggesting novel links between physical activity and disease or its prevention. In summary, brief heavy exercise leads to a previously unknown substantial and significant alteration in neutrophil gene expression.    

4.555           Pancreatic Islet Immunoreactivity to the Reg Protein INGAP

Taylor-Fishwick, D.A., Bowman, A., Korngiebel-Rosique. M.C. and Vinik, A.I.

1. Histochem. Cytochem., 56(2), 183-191 (2008)

  The Reg-related protein family member INGAP (islet neogenesis-associated protein) is a pleiotropic factor enhancing islet neogenesis, neurite growth, β-cell protection, and β-cell function. Using an antibody to the N-termini of INGAP, we have identified that immunoreactivity to INGAP localized to the pancreatic endocrine cells in mouse. INGAP- and insulin-immunoreactive cells are mutually exclusive, with INGAP-immunoreactive cells being preserved after streptozotocin-mediated destruction of β-cells. Glucagon- and INGAP-immunoreactive cells colocalize, although respective antigen expression occurs in different intracellular locations. These data suggest that INGAP-immunoreactive cells include -cells; however, detection of single INGAP-immunoreactive/glucagon-negative cells indicates that this may not be exclusive. In addition to mouse, detection of islet endocrine cells that were INGAP immunoreactive/glucagon immunoreactive/insulin negative was also observed in islets from human, monkey, and rat. These findings reveal that INGAP and/or related group 3 Reg proteins have a conserved expression in the pancreatic islet.  

4.556           Salmonella infection of afferent lymph dendritic cells

Chan, S.S.M., Mastroeni, P., McConnell, I. and Blacklaws, B.A.

1. Leukoc. Biol., 88, 272-279 (2008)

  The interactions of Salmonella enterica subspecies I serotype Abortusovis (S. Abortusovis) with ovine afferent lymph dendritic cells (ALDCs) were investigated for their ability to deliver Maedi visna virus (MVV) GAG p25 antigens to ALDCs purified from afferent lymph. Salmonellae were found to enter ALDC populations by a process of cell invasion, as confirmed by electron and confocal microscopy. This led to phenotypical changes in ALDC populations, as defined by CD1b and CD14 expression. No differences in the clearance kinetics of intracellular aroA-negative Salmonella from CD1b⁺ CD14^lo and CD1b⁺ CD14^– ALDC populations were noted over 72 h. ALDCs were also shown to present MVV GAG p25 expressed by aroA-negative S. Abortusovis to CD4⁺ T lymphocytes. Thus, the poor immune responses that Salmonella vaccines elicited in large animal models compared with mice are neither a result of an inability of Salmonella to infect large animal DCs nor an inability of these DCs to present delivered antigens. However, the low efficiency of infection of ALDC compared with macrophages or monocyte-derived DCs may account for the poor immune responses induced in large animal models.  

4.557           iPLA2β: front and center in human monocyte chemotaxis to MCP-1

Mishra, R.S., Carnevale, K.A. and Cathcart, M.K.

1. Exp. Med., 205(2), 347-359 (2008)

  Monocyte chemoattractant protein-1 (MCP-1) directs migration of blood monocytes to inflamed tissues. Despite the central role of chemotaxis in immune responses, the regulation of chemotaxis by signal transduction pathways and their in vivo significance remain to be thoroughly deciphered. In this study, we examined the intracellular location and functions of two recently identified regulators of chemotaxis, Ca²⁺-independent phospholipase (iPLA₂β) and cytosolic phospholipase (cPLA₂ ), and substantiate their in vivo importance. These enzymes are cytoplasmic in unstimulated monocytes. Upon MCP-1 stimulation, iPLA₂β is recruited to the membrane-enriched pseudopod. In contrast, cPLA₂ is recruited to the endoplasmic reticulum. Although iPLA₂β or cPLA₂ antisense oligodeoxyribonucleotide (ODN)–treated monocytes display reduced speed, iPLA₂β also regulates directionality and actin polymerization. iPLA₂β or cPLA₂ antisense ODN–treated adoptively transferred mouse monocytes display a profound defect in migration to the peritoneum in vivo. These converging observations reveal that iPLA₂β and cPLA₂ regulate monocyte migration from different intracellular locations, with iPLA₂β acting as a critical regulator of the cellular compass, and identify them as potential targets for antiinflammatory strategies.  

4.558           SARS-CoV replicates in primary human alveolar type II cell cultures but not in type I-like cells

Mossel, E.C. et al Virology, 372, 127-135 (2008)   Severe acute respiratory syndrome (SARS) is a disease characterized by diffuse alveolar damage. We isolated human alveolar type II cells and maintained them in a highly differentiated state. Type II cell cultures supported SARS-CoV replication as evidenced by RT-PCR detection of viral subgenomic RNA and an increase in virus titer. Virus titers were maximal by 24 h and peaked at approximately 10⁵ pfu/mL. Two cell types within the cultures were infected. One cell type was type II cells, which were positive for SP-A, SP-C, cytokeratin, a type II cell-specific monoclonal antibody, and Ep-CAM. The other cell type was composed of spindle-shaped cells that were positive for vimentin and collagen III and likely fibroblasts. Viral replication was not detected in type I-like cells or macrophages. Hence, differentiated adult human alveolar type II cells were infectible but alveolar type I-like cells and alveolar macrophages did not support productive infection.  

4.559           Phase I study of α-galactosylceramide-pulsed antigen presenting cells administration to the nasal submucosa in unresectable or recurrent head and neck cancer

Uchida, T. et al Cancer Immunol. Immunother., 57, 337-345 (2008)   Background  Human Vα24 natural killer T (NKT) cells are activated by the specific ligand, α-galactosylceramide (α-GalCer), in a CD1d-dependent manner. Potent anti-tumor activity of activated NKT cells has been previously demonstrated. Methods   We conducted a phase I study with α-GalCer-pulsed antigen presenting cells (APCs) administered in the nasal submucosa of patients with head and neck cancer, and evaluated the safety and feasibility of such a treatment. Nine patients with unresectable or recurrent head and neck cancer received two treatments 1 week apart, of 1 × 10⁸ of α-GalCer-pulsed autologous APCs into the nasal submucosa. Results   During the clinical study period, no serious adverse events (Common Terminology Criteria for Adverse Events version 3.0 greater than grade 3) were observed. After the first and the second administration of α-GalCer-pulsed APCs, an increased number of NKT cells was observed in four patients and enhanced natural killer activity was detected in the peripheral blood of eight patients. Conclusion   The administration of α-GalCer-pulsed APCs into the nasal submucosa was found to be safe and induce anti-tumor activity in some patients.  

4.560           Islet Cell Transplantation.  How Effective Is It?

Liu, E.H. and Harlan, D.M. Controversies in Treating Diabetes, 11-32 (2008), Humana Press   Islet cell transplantation as a treatment for diabetes has shown great promise, but has significant limitations. Since early animal studies in rats demonstrated its ability to restore euglycemia, islet transplantation has not proven to be a durable or practical therapy for type 1 diabetes. Here we review some of the history, technique, clinical outcomes, and potential alternatives of islet transplantation.  

4.561           Galectin-3 Is an Amplifier of Inflammation in Atherosclerotic Plaque Progression Through Macrophage Activation And Monocyte Chemoattraction

Papaspyridonos, M. et al Arterioscler. Thromb. Vasc. Biol., 28, 433-440 (2008)   Objective— Galectin-3 (Gal-3) is a 26-kDa lectin knownto regulate many aspects of inflammatory cell behavior. We assessedthe hypothesis that increased levels of Gal-3 contribute toatherosclerotic plaque progression by enhancing monocyte chemoattractionthrough macrophage activation. Methods and Results— Gal-3 was found to be upregulated in unstable plaque regions of carotid endarterectomy (CEA) specimens compared with stable regions from the same patient (3.2-fold, P<0.05) at the mRNA (n=12) and (2.3-fold, P<0.01) at the protein level (n=9). Analysis of aortic tissue from ApoE^–/– mice on a high fat diet (n=14) and wild-type controls (n=9) showed that Gal-3 mRNA and protein levels are elevated by 16.3-fold (P<0.001) and 12.2-fold (P<0.01) and that Gal-3 staining colocalizes with macrophages. In vitro, conditioned media from Gal-3–treated human macrophages induced an up to 6-fold increase in human monocyte chemotaxis (P<0.01, ANOVA), aneffect that was reduced by 66 and 60% by Pertussis Toxin (PTX)and the Vaccinia virus protein 35K, respectively. Microarrayanalysis of human macrophages and subsequent qPCR validationconfirmed the upregulation of CC chemokines in response to Gal-3treatment. Conclusions— Our data suggest that Gal-3 is both a markerof atherosclerotic plaque progression and a central contributorto the pathology by amplification of key proinflammatory molecules. Galectin-3 was upregulated in advanced human and murine ApoE^–/– atherosclerotic plaques. Mediators released in response to Gal-3treatment in macrophages increased monocyte chemotaxis, andmicroarray analysis confirmed the upregulation of several keychemoattractant molecules. Gal-3 is an amplifier of inflammationthat could be used as a marker of atherosclerotic plaque progressionor target for atherosclerosis.

4.562           PECAM-1 Polymorphism Affects Monocyte Adhesion to Endothelial Cells

Goodman, R.S. et al Transplantation, 85(3), 471-477 (2008)   Background. Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) plays an important role in leukocyte-endothelial cell adhesion and transmigration. Single nucleotide polymorphisms of PECAM-1 encoding amino acid substitutions at positions 98 leucine/valine (L/V), 536 serine/asparagine (S/N), and 643 arginine/glycine (R/G) occur in strong genetic linkage resulting in two common haplotypes (LSR and VNG). These PECAM-1 polymorphisms are associated with graft-versus-host disease after hematopoietic stem cell transplantation and with cardiovascular disease, but whether they influence PECAM-1 function is unknown. Methods. We examined the effect of homozygous and heterozygous expression of the PECAM-1 LSR and VNG genotypes on the adhesive interactions of peripheral blood monocytes and activated endothelial cell monolayers under shear stress in a flow-based cell adhesion assay. Results. There was no difference in monocyte adhesion between the two homozygous genotypes of PECAM-1 but when monocytes expressed both alleles in heterozygous form, firm adhesion of monocytes to endothelial cells was markedly increased. PECAM-1 polymorphism expressed in homozygous or heterozygous form by endothelial cells did not influence monocyte adhesion. Conclusions. This is, to our knowledge, the first demonstration that PECAM-1 genotype can alter the level of monocyte binding to endothelial cells and a demonstration that heterozygous expression of a polymorphic protein may lead to altered function.  

4.563           Acetylcholinesterase Expression in Muscle Is Specifically Controlled by a Promoter-Selective Enhancesome in the First Intron

Camp, S. et al

1. Neurosci., 28(10), 2459-2470 (2008)

  Mammalian acetylcholinesterase (AChE) gene expression is exquisitely regulated in target tissues and cells during differentiation. An intron located between the first and second exons governs a 100-fold increase in AChE expression during myoblast to myotube differentiation in C2C12 cells. Regulation is confined to 255 bp of evolutionarily conserved sequence containing functional transcription factor consensus motifs that indirectly interact with the endogenous promoter. To examine control in vivo, this region was deleted by homologous recombination. The knock-out mouse is virtually devoid of AChE activity and its encoding mRNA in skeletal muscle, yet activities in brain and spinal cord innervating skeletal muscle are unaltered. The transcription factors MyoD and myocyte enhancer factor-2 appear to be responsible for muscle regulation. Selective control of AChE expression by this region is also found in hematopoietic lineages. Expression patterns in muscle and CNS neurons establish that virtually all AChE activity at the mammalian neuromuscular junction arises from skeletal muscle rather than from biosynthesis in the motoneuron cell body and axoplasmic transport.  

4.564           Paracrine Activation of Hepatic CB₁ Receptors by Stellate Cell-Derived Endocannabinoids Mediates Alcoholic Fatty Liver

Jeong, W-i. et al Cell Metabolism, 7, 227-235 (2008)   Alcohol-induced fatty liver, a major cause of morbidity, has been attributed to enhanced hepatic lipogenesis and decreased fat clearance of unknown mechanism. Here we report that the steatosis induced in mice by a low-fat, liquid ethanol diet is attenuated by concurrent blockade of cannabinoid CB₁ receptors. Global or hepatocyte-specific CB₁ knockout mice are resistant to ethanol-induced steatosis and increases in lipogenic gene expression and have increased carnitine palmitoyltransferase 1 activity, which, unlike in controls, is not reduced by ethanol treatment. Ethanol feeding increases the hepatic expression of CB₁ receptors and upregulates the endocannabinoid 2-arachidonoylglycerol (2-AG) and its biosynthetic enzyme diacylglycerol lipase β selectively in hepatic stellate cells. In control but not CB₁ receptor-deficient hepatocytes, coculture with stellate cells from ethanol-fed mice results in upregulation of CB₁ receptors and lipogenic gene expression. We conclude that paracrine activation of hepatic CB₁ receptors by stellate cell-derived 2-AG mediates ethanol-induced steatosis through increasing lipogenesis and decreasing fatty acid oxidation.      

4.565           Increased intraneuronal resting [Ca²⁺] in adult Alzheimer’s disease mice

Lopez, J.R. et al

1. Neurochem., 105, 262-271 (2008)

  Neurodegeneration in Alzheimer’s disease (AD) has been linked to intracellular accumulation of misfolded proteins and dysregulation of intracellular Ca²⁺. In the current work, we determined the contribution of specific Ca²⁺ pathways to an alteration in Ca²⁺ homeostasis in primary cortical neurons from an adult triple transgenic (3xTg-AD) mouse model of AD that exhibits intraneuronal accumulation of β-amyloid proteins. Resting free Ca²⁺ concentration ([Ca²⁺]_i), as measured with Ca²⁺-selective microelectrodes, was greatly elevated in neurons from 3xTg-AD and APP_SWE mouse strains when compared with their respective non-transgenic neurons, while there was no alteration in the resting membrane potential. In the absence of the extracellular Ca²⁺, the [Ca²⁺]_i returned to near normal levels in 3xTg-AD neurons, demonstrating that extracellular Ca²⁺contributed to elevated [Ca²⁺]_i. Application of nifedipine, or a non-L-type channel blocker, SKF-96365, partially reduced [Ca²⁺]_i. Blocking the ryanodine receptors, with ryanodine or FLA-365 had no effect, suggesting that these channels do not contribute to the elevated [Ca²⁺]_i. Conversely, inhibition of inositol trisphosphate receptors with xestospongin C produced a partial reduction in [Ca²⁺]_i. These results demonstrate that an elevation in resting [Ca²⁺]_i, contributed by aberrant Ca²⁺entry and release pathways, should be considered a major component of the abnormal Ca²⁺ homeostasis associated with AD.  

4.566           Characterization and Application of a Glucose-Repressible Promoter in Francisella tularensis

Horzempa, J., Tarwacki, D.M., Carlson Jr., P.E., Robinson, C.M. and Nau, G.J. Appl. Envir. Microbiol., 74(7), 2161-2170 (2008)   Francisella tularensis, the causative agent of tularemia, is a category A biodefense agent. The examination of gene function in this organism is limited due to the lack of available controllable promoters. Here, we identify a promoter element of F. tularensis LVS that is repressed by glucose (termed the Francisella glucose-repressible promoter, or FGRp), allowing the management of downstream gene expression. In bacteria cultured in medium lacking glucose, this promoter induced the expression of a red fluorescent protein allele, tdtomato. FGRp activity was used to produce antisense RNA of iglC, an important virulence factor, which severely reduced IglC protein levels. Cultivation in glucose-containing medium restored IglC levels, indicating the usefulness of this promoter for controlling both exogenous and chromosomal gene expression. Moreover, FGRp was shown to be active during the infection of human macrophages by using the fluorescence reporter. In this environment, the FGRp-mediated expression of antisense iglC by F. tularensis LVS resulted in reduced bacterial fitness, demonstrating the applicability of this promoter. An analysis of the genomic sequence indicated that this promoter region controls a gene, FTL_0580, encoding a hypothetical protein. A deletion analysis determined the critical sites essential for FGRp activity to be located within a 44-bp region. This is the first report of a conditional promoter and the use of antisense constructs in F. tularensis, valuable genetic tools for studying gene function both in vitro and in vivo.  

4.567           Morphological features and responses to AMPA receptor-mediated excitotoxicity of mouse motor neurons: comparison in purified, mixed anterior horn or motor neuron/glia cocultures

De Paola, M., Diana, V., Bigini, P. And Mennini, T.

1. Neurosci. Methods, 170, 85-95 (2008)

  Primary motor neuron cultures are widely used as in vitro model to study the early mechanisms involved in the aetiology of amyotrophic lateral sclerosis. In this study, we directly compared the morphological features and the responses to AMPA receptor (AMPAR) activation of mouse spinal cord motor neurons under different culture conditions (OptiPrep -purified, mixed anterior horn or motor neuron/glia cocultures). Motor neurons cocultured with a confluent glial layer had significant improvements in axonal length and in somata perimeter and area, compared both to mixed anterior horn cultures and to purified cultures, suggesting that the presence of more “mature” glial cells was determinant to obtain healthier motor neurons. By immuno-cytochemical assays we found that both in mixed anterior horn cultures and in cocultures, lower AMPA (0.3 μM) or kainate (5 μM) concentrations, but not the higher (1 or 15 μM, respectively), induced classical apoptotic events such as the nuclear fragmentation, the membrane externalization of phosphatidylserine residues and the activation of caspases-9 and -3. The morphological features and the different degenerative pathways induced by AMPAR agonist concentrations suggest that the experimental conditions used for in vitro studies are key factors that should be deeply considered to obtain more valid and reproducible results.    

4.568           Progranulin functions as a neurotrophic factor to regulate neurite outgrowth and enhance neuronal survival

Van Damme, P. et al

1. Cell Biolo., 181(1), 37-41 (2008)

  Recently, mutations in the progranulin (PGRN) gene were found to cause familial and apparently sporadic frontotemporal lobe dementia (FTLD). Moreover, missense changes in PGRN were identified in patients with motor neuron degeneration, a condition that is related to FTLD. Most mutations identified in patients with FTLD until now have been null mutations. However, it remains unknown whether PGRN protein levels are reduced in the central nervous system from such patients. The effects of PGRN on neurons also remain to be established. We report that PGRN levels are reduced in the cerebrospinal fluid from FTLD patients carrying a PGRN mutation. We observe that PGRN and GRN E (one of the proteolytic fragments of PGRN) promote neuronal survival and enhance neurite outgrowth in cultured neurons. These results demonstrate that PGRN/GRN is a neurotrophic factor with activities that may be involved in the development of the nervous system and in neurodegeneration.  

4.569           Costimulation of Dectin-1 and DC-SIGN Triggers the Arachidonic Acid Cascade in Human Monocyte-Derived Dendritic Cells

Valera, I. et al

1. Immunol., 180, 5727-5736 (2008)

  Inflammatory mediators derived from arachidonic acid (AA) alter the function of dendritic cells (DC), but data regarding their biosynthesis resulting from stimulation of opsonic and nonopsonic receptors are scarce. To address this issue, the production of eicosanoids by human monocyte-derived DC stimulated via receptors involved in Ag recognition was assessed. Activation of Fc R induced AA release, short-term, low-grade PG biosynthesis, and IL-10 production, whereas zymosan, which contains ligands of both the mannose receptor and the human β-glucan receptor dectin-1, induced a wider set of responses including cyclooxygenase 2 induction and biosynthesis of leukotriene C₄ and IL-12p70. The cytosolic phospholipase A₂ inhibitor pyrrolidine 1 completely inhibited AA release stimulated via all receptors, whereas the spleen tyrosine kinase (Syk) inhibitors piceatannol and R406 fully blocked AA release in response to immune complexes, but only partially blocked the effect of zymosan. Furthermore, anti-dectin-1 mAb partially inhibited the response to zymosan, and this inhibition was enhanced by mAb against DC-specific ICAM-3-grabbing nonintegrin (SIGN). Immunoprecipitation of DC lysates showed coimmunoprecipitation of DC-SIGN and dectin-1, which was confirmed using Myc-dectin-1 and DC-SIGN constructs in HEK293 cells. These data reveal a robust metabolism of AA in human DC stimulated through both opsonic and nonopsonic receptors. The Fc R route depends on the ITAM/Syk/cytosolic phospholipase A₂ axis, whereas the response to zymosan involves the interaction with the C-type lectin receptors dectin-1 and DC-SIGN. These findings help explain the distinct functional properties of DC matured by immune complexes vs those matured by β-glucans.  

4.570           Adenoviral-Mediated Overexpression of Either Membrane-Bound Human FasL or Human Decoy Fas Can Prolong Pig Islet Xenograft Survival in a Rat Transplant Model

Kawamoto, K. et al Transplantation Proceedings, 40, 477-479 (2008)   The success of pancreatic islet transplantation is limited because of the severe shortage of allogeneic pancreas donors. Accordingly, pig islets are considered to be an attractive, promising alternative. However, cell-mediated immunity, especially CD8⁺ cytotoxic T lymphocyte (CTL)-mediated cytotoxicity, remains a formidable barrier to prevent long-term islet survival in xenograft recipients. Therefore, it is particularly important to explore methods to specifically prevent cell-mediated immunity against pig islets. Our group previously demonstrated that the overexpression of either membrane-bound human FasL or human decoy Fas antigen in pig endothelial cells prevented CTL xenocytotoxicity. In this study, we assessed the cytoprotective effects of adenoviral-mediated overexpression of either membrane-bound human FasL or human decoy Fas antigen in pig islets to inhibit CTL xenocytotoxicity. The CTL-mediated killing of pig islets infected with an adenoviral vector carrying either membrane-bound human FasL or human decoy Fas was significantly reduced compares with that of control pig islets transfected with adenoviral vector encoding enhanced green fluorescent protein (EGFP). Moreover, we transfected pig islets with these molecules to confirm their cytoprotective effects in in vivo studies. The significant long-term survival of pig islets expressing these molecules was elicited through days 3 to 5 posttransplantation. Thus, these results demonstrated that the remodeling of either death receptor or death ligand on pig islets by adenoviral gene transfer prevented innate cellular immunity against xeno-islet grafts facilitating long-term xenograft survival.  

4.571           Comparative methodologies of regulatory T cell depletion in a murine melanoma model

Matsushita, N., Pilon-Thomas, S.A., Martin, L.M. and Riker, A.I.

1. Immunol. Methods, 333, 167-179 (2008)

  There has been recent interest in the depletion of regulatory T cells (Tregs) as part of a multi-faceted approach to the immunotherapy of melanoma patients. This is in part due recent findings that convincingly show that Tregs are an integral part of regulating and even suppressing an immune response to growing tumor cells. We therefore compared three methods of Treg depletion and/or elimination, utilizing low dose cyclophosphamide (CY), a specific antibody directed against the IL-2 receptor found on Tregs (PC61) and the use of denileukin diftitox (DD), which is a fusion protein designed to have a direct cytocidal action on cells which express the IL-2 receptor. We show that CY administration resulted in the highest reduction in Tregs among the three reagents. However, the reduction in Tregs with CY was also associated with the concomitant reduction of CD8(+) T cells and a lack of tumor antigen priming. Utilization of DD resulted in a > 50% Treg cell reduction without parallel cytocidal effects upon other T cell subsets but did not enhance anti-tumor immunity against B16 melanoma. Lastly, the PC61 showed a moderate reduction of Tregs that lasted longer than the other reagents, without a reduction in the total number of CD8(+) T cells. Furthermore, PC61 treatment did not abrogate tumor antigen-specific immunity elicited by dendritic cells (DC). We therefore conclude that PC61 administration was the most effective method of reducing Tregs in a murine melanoma model in addition to providing evidence of a synergistic effect when combined with DC-based immunotherapy.  

4.572           Tonsilar NK Cells Restrict B Cell Transformation by the Epstein-Barr Virus via IFN-γ

Strowig, T. et al PLOS Pathogens, 4(2), e27 (2008)   Cells of the innate immune system act in synergy to provide a first line of defense against pathogens. Here we describe that dendritic cells (DCs), matured with viral products or mimics thereof, including Epstein-Barr virus (EBV), activated natural killer (NK) cells more efficiently than other mature DC preparations. CD56^brightCD16⁻ NK cells, which are enriched in human secondary lymphoid tissues, responded primarily to this DC activation. DCs elicited 50-fold stronger interferon-γ (IFN-γ) secretion from tonsilar NK cells than from peripheral blood NK cells, reaching levels that inhibited B cell transformation by EBV. In fact, 100- to 1,000-fold less tonsilar than peripheral blood NK cells were required to achieve the same protection in vitro, indicating that innate immune control of EBV by NK cells is most efficient at this primary site of EBV infection. The high IFN-γ concentrations, produced by tonsilar NK cells, delayed latent EBV antigen expression, resulting in decreased B cell proliferation during the first week after EBV infection in vitro. These results suggest that NK cell activation by DCs can limit primary EBV infection in tonsils until adaptive immunity establishes immune control of this persistent and oncogenic human pathogen.  

4.573           Epigallocatechin-3-gallate Inhibits Growth of Activated Hepatic Stellate Cells by Enhancing the Capacity of Glutathione Synthesis

Fu, Y., Zheng, S., Lu, S.C. and Chen, A. Mol. Pharmacol., 73(5), 1465-1473 (2008)   Activation of hepatic stellate cells (HSC), the key effectors in hepatic fibrogenesis, is characterized by enhanced cell proliferation and overproduction of extracellular matrix. Oxidative stress promotes HSC activation. Glutathione (GSH) is the most important intracellular antioxidant, whose synthesis is mainly regulated by glutamate-cysteine ligase (GCL). We reported previously that (-)-epigallocatechin-3-gallate (EGCG), the major and most active component in green tea extracts, inhibited HSC activation. The aim of this study is to elucidate the underlying mechanisms. We hypothesize that this inhibitory effect of EGCG might mainly result from its antioxidant capability by increasing de novo synthesis of GSH. In this report, we observe that EGCG enhances the levels of cytoplasmic and mitochondrial GSH and increases GCL activity by inducing gene expression of the catalytic subunit GCLc, leading to de novo synthesis of GSH. Real-time polymerase chain reaction and Western blotting analyses show that de novo synthesis of GSH is required for EGCG to regulate the expression of genes relevant to apoptosis and to cell proliferation. Additional experiments demonstrate that exogenous transforming growth factor (TGF)-β1 suppresses GCLc gene expression and reduces the level of GSH in cultured HSC. Transient transfection assays and Western blotting analyses further display that EGCG interrupts TGF-β signaling by reducing gene expression of TGF-β receptors and Smad4, leading to increased expression of GCLc. These results support our hypothesis and collectively demonstrate that EGCG increases the level of cellular GSH in HSC by stimulating gene expression of GCLc, leading to the inhibition of cell proliferation of activated HSC in vitro.  

4.574           Isolation of chicken follicular dendritic cells

Del Cacho, E., Gallego, M., Lopez-Bernard, D.F., Sanchez-Acedo, C. and Liillehoj, H.S.

1. Immunol. Methods, 334, 59-69 (2008)

  The aim of the present study was to isolate chicken follicular dendritic cells (FDC). A combination of methods involving panning, iodixanol density gradient centrifugation, and magnetic cell separation technology made it possible to obtain functional FDC from the cecal tonsils from chickens, which had been infected with Eimeria tenella. CD45⁻ dendritic cells were selected using the specific monoclonal antibody against chicken CD45, which is a marker for chicken leukocytes, but is not expressed on chicken FDC. Isolated FDC were characterized morphologically, phenotypically and functionally. The phenotype of the selected cells was consistent with FDC in that they expressed IgG, IgM, complement factors C3 and B, ICAM-1, and VCAM-1, but lacked cell surface markers characteristic of macrophages, T-, and B cells. Transmission electron microscopy confirmed their characteristic dendritic morphology. In addition, the identity of the FDC was further confirmed by their ability to trap chicken immune complexes (ICs) on their surface, whereas they did not trap naive antigen (ovalbumin) or ICs generated with mammalian immunoglobulins. Co-culturing allogeneic or autologous isolated FDC with B cells resulted in enhanced B cell proliferation and immunoglobulin production. The lack of MHC restriction, a functional characteristic feature of FDC, further reinforces the identity of the isolated cells as chicken FDC.  

4.575           Activation of tonsil dendritic cells with immuno-adjuvants

Polak, M.E., Borthwick, N.J., Gabriel, F.G., Jager, M.J. and Cree, I.A. BMC Immunolog, 9, 10-21 (2008)   Background Dendritic cells (DC) play the key role in directing antigen-specific immune responses and manipulating their function may be a useful tool for immunotherapy. The balance between immune stimulation and tolerance is particularly important at mucosal interfaces, where discrimination between dangerous pathogens and innocuous antigens takes place. In humans, although much is known about the responses of monocyte derived DC, relatively little is known about effect of immuno-stimulatory adjuvants on DC found in tonsil. Results To examine this, tonsil DC were isolated and cultured with potent DC activators; IFNγ, anti-CD40 antibody, LPS and Poly I:C either singly or in combination. To measure maturation and activation, DC were examined for changes in the expression of HLA-DR, HLA- class I, CD83, CD40, CD80 and CD86 and the release of IL12p70. The DC isolated from tonsil were a mixed population containing both myeloid and plasmacytoid DC, but all showed similar responses. Tonsil DC released IL12p70 upon stimulation with IFNγ , anti-CD40 antibody, and LPS, but unlike monocyte-derived DC, they did not increase the expression of cell surface activation molecules above those induced by culture alone. Poly I:C, a potent stimulator of laboratory generated DC inhibited the activation of tonsil DC by other adjuvants. Conclusion As the response of this mixed population of DC does not mirror that of DC generated in vitro, this may have implications for other tissue residing DC and might be an important consideration for immunotherapy.  

4.576           Plasmacytoid Dendritic Cells Migrate in Afferent Skin Lymph

Pascale, F. et al

1. Immunol., 180, 5963-5972 (2008)

  Conventional dendritic cells enter lymph nodes by migrating from peripheral tissues via the lymphatic route, whereas plasmacytoid dendritic cells (pDC), also called IFN-producing cells (IPC), are described to gain nodes from blood via the high endothelial venules. We demonstrate here that IPC/pDC migrate in the afferent lymph of two large mammals. In sheep, injection of type A CpG oligodinucleotide (ODN) induced lymph cells to produce type I IFN. Furthermore, low-density lymph cells collected at steady state produced type I IFN after stimulation with type A CpG ODN and enveloped viruses. Sheep lymph IPC were found within a minor B^negCD11c^neg subset expressing CD45RB. They presented a plasmacytoid morphology, expressed high levels of TLR-7, TLR-9, and IFN regulatory factor 7 mRNA, induced IFN- production in allogeneic CD4^pos T cells, and differentiated into dendritic cell-like cells under viral stimulation, thus fulfilling criteria of bona fide pDC. In mini-pig, a CD4^posSIRP^pos subset in afferent lymph cells, corresponding to pDC homologs, produced type I IFN after type A CpG-ODN triggering. Thus, pDC can link innate and acquired immunity by migrating from tissue to draining node via lymph, similarly to conventional dendritic cells.  

4.577           Activation of hepatic natural killer cells and control of liver-adapted lymphoma in the murine model of cytomegalovirus infection

Erlach, K.C. et al Med. Microbiol. Immunol., 197, 167-178 (2008)   Hematopoietic stem cell transplantation (HSCT) is a promising therapeutic option against hematopoietic malignancies. Infection with cytomegalovirus (CMV) and tumor relapse are complications that limit the success of HSCT. In theory, CMV infection can facilitate tumor relapse and growth by inhibiting “graft take” and reconstitution of the immune system or by inducing the secretion of tumor cell growth-promoting cytokines. Conversely, one can also envisage an anti-tumoral effect of CMV by cytopathic/oncolytic infection of tumor cells, by inducing the secretion of death ligands for tumor cell apoptosis, and by the activation of systemic innate and adaptive immunity. Here we will briefly review the current knowledge about tumor control in a murine model of CMV infection and liver-adapted B cell lymphoma, with a focus on a putative implication of CD49⁺NKG2D⁺ hepatic natural killer cells.  

4.578           Ductal Injection of Preservation Solution Increases Islet Yields in Islet Isolation and Improves Islet Graft Function

Noguchi, H. et al Cell Transplantation, 17, 69-81 (2008)   For islet transplantation, it is important to obtain an available islet mass adequate for diabetes reversal from a single donor pancreas. A recent report demonstrated that the use of M-Kyoto solution instead of UW solution improved islet yields in the two-layer method for pancreas preservation. The present study investigated whether the ductal injection of a large volume of preservation solution (UW and M-Kyoto solution) before pancreas storage improves islet yields. Islet yield both before and after purification was significantly higher in the ductal injection (+) group compared with the ductal injection (−) group. TUNEL-positive cells in the ductal injection (+) group were significantly decreased in comparison to the ductal injection (−) group. The ductal injection of preservation solution increased the ATP level in the pancreas tissue and reduced trypsin activity during the digestion step. Annexin V and PI assays showed that the ductal injection prevents islet apoptosis. In a transplant model, the ductal injection improved islet graft function. These findings suggest that the ductal injection of preservation solution, especially the M-Kyoto solution, leads to improved outcomes for pancreatic islet transplantation. Based on these data, this technique is now used for clinical islet transplantation from non-heart-beating donor pancreata or living donor pancreas.  

4.579           Secretory Unit of Islet in Transplantation (SUIT) and Engrafted Islet Rate (EIR) Indexes Are Useful for Evaluating Single Islet Transplantation

Noguchi, H. et al Cell Transplantation, 17, 121-128 (2008)   The evaluation of engraftment is important to assess the success of islet transplantation, but it is complex because islet transplantation usually requires two or more donors to achieve euglycemia. Islet transplantation from NHBDs was evaluated using new assessment forms for the secretory unit of islet in transplantation (SUIT) and engrafted islet rate (EIR) indexes. Insulin independence was obtained when the SUIT index was more than 28, which might indicate that 28% of the β-cell mass of a normal subject is required for insulin independence. Because the average EIR for a single transplantation is about 30, the percentage of engrafted islets following one transplantation is about 30%, assuming that a normal subject has 1 million islet equivalents. Although few cultured islet transplants have been performed, the increase of the SUIT and EIR indexes in patients who received cultured islets was significantly lower than in patients who received fresh islets, suggesting that fresh islets may be more effective than cultured islets. The SUIT and EIR indexes are thus considered to be useful values for evaluating islet transplantation, especially for single islet transplantation.  

4.580           Nonmyeloablative Chemotherapy Followed by T-cell Adoptive Transfer and Dendritic Cell-based Vaccination Results in Rejection of Established Melanoma

Koike, N., Pilon-Thomas, S. and Mule, J.J.

1. Immunother., 31(4), 402-412 (2008)

  We demonstrated previously that dendritic cell (DC)-based vaccines could mediate a specific and long-lasting antitumor immune response during early lymphoid reconstitution after lethal irradiation and bone marrow transplant. The purpose of this current study was to examine the potential therapeutic efficacy of DC-based vaccines in combination with sublethal lymphodepletion and T-cell transfer. In an aggressive model of melanoma, treatment with the combination of 200 mg/kg cyclophosphamide (Cy) and 100 mg/kg fludarabine (Flu) led to a lymphopenic state lasting approximately 14 days, but had no effect on the growth of an established M05 melanoma. Addition of ovalbumin (OVA) peptide-pulsed DC-based immunization resulted in a delay in tumor growth but did not enhance overall survival in this model. To improve treatment, adoptively transferred naive T cells were added. After induction of lymphopenia with Cy and Flu, transferred T cells demonstrated an activated memory phenotype including high expression of CD44 and low expression of CD62L. Induction of lymphopenia with Cy and Flu in combination with adoptive transfer of naive T cells and OVA peptide-pulsed DCs immunization led to an enhancement in the number of OVA specific, CD8+ T cells that demonstrated specific cytotoxic activity, proliferation, and interferon-[gamma] production in response to the OVA expressing M05 melanoma. This combination therapy also led to tumor regression and enhanced survival in mice bearing M05 melanoma.  

4.581           Uridine diphosphate (UDP) stimulates insulin secretion by activation of P2Y6 receptors

Parandeh, F., Abaraviciene, S.M., Amisten, S., erlinge, D. and Salehi, A. Biochem. Biophys. Res. Comm., 370, 499-503 (2008)   We examined the transcriptional expression and functional effects of receptors for the extracellular pyrimidines uridine triphosphate (UTP) and uridine diphosphate (UDP), on insulin and glucagon secretion in isolated mouse pancreatic islets and purified β-cells. Using real-time PCR, the UDP receptor P2Y₆ was found to be highly expressed in both whole islets and β-cells purified by repeated counter-flow elutriation, whereas no mRNA expression for UTP receptors P2Y₄ and P2Y₂ could be detected. Functional in vitro experiments revealed that the P2Y₆ agonist UDPβS dose-dependently enhanced insulin and glucagon release during short-term incubation (1 h), while P2Y₆ activation during a longer period (24 h), selectively increased insulin release, especially at high glucose levels. The corresponding EC₅₀ value for UDPβS ranged from 3.2 × 10⁻⁸ M to 1.6 × 10⁻⁸ M for both glucose concentrations. The P2Y₆ antagonist MRS2578 inhibited the effects of UDPβS, supporting a P2Y₆ specific effect. In addition to negative RT-PCR results, the lack of response to UTPγS a selective P2Y_2/4 agonist further rule out the involvement of P2Y_2/4 receptors in the islet hormone release. Our results suggest a modulatory role for UDP via a functional active P2Y₆ receptor in the regulation of islet hormone release.  

4.582           Helicobacter pylori-Pulsed Dendritic Cells Induce H. pylori-Specific Immunity in Mice

Zhang, M. et al Helicobacter, 13, 200-208 (2008)   Background: The growing concern over the emergence of antibiotic-resistant Helicobacter pylori infection is propelling the development of an efficacious vaccine to control this highly adaptive organism. Aim: We studied the use of a dendritic cell (DC)-based vaccine against H. pylori infection in mice. Methods: The cellular immune responses to murine bone marrow-derived DCs pulsed with phosphate-buffered saline (PBS-DC) or live H. pylori SS1 (HP-DC) were assessed in vitro and in vivo. The protective immunity against H. pylori SS1 oral challenge was compared between HP-DC or PBS-DC immunized mice. The effect of regulatory T-cell (Treg) depletion by anti-CD25 antibody on HP-DC vaccine efficacy was also evaluated. Results: HP-DC induced a Th1-dominant response in vitro. In vivo, HP-DC immunized mice were characterized by a mixed Th1/Th2 peripheral immune response. However, in the stomach, HP-DC immunized mice expressed a higher level of IFN-γ compared to PBS-DC immunized mice; no difference was found for interleukin-5 expressions in the stomach. A lower bacterial colonization post-H. pylori challenge was observed in HP-DC immunized mice compared to PBS-DC immunized mice with no significant difference in gastritis severity. H. pylori-specific Th1 response and protective immunity were further enhanced in vivo by depletion of Treg with anti-CD25 antibody. Conclusion: DC-based anti-H. pylori vaccine induced H. pylori-specific helper T-cell responses capable of limiting bacterial colonization. Our data support the critical role of effector cellular immune response in the development of H. pylori vaccine.    

4.583           Conversion Potential of Marrow Cells into Lung Cells Fluctuates with Cytokine-Induced Cell Cycle

Dooner, M.S. et al Stem Cells and Development, 17, 207-219 (2008)   Green fluorescent protein (GFP)-labeled marrow cells transplanted into lethally irradiated mice can be detected in the lungs of transplanted mice and have been shown to express lung-specific proteins while lacking the expression of hematopoietic markers. We have studied marrow cells induced to transit the cell cycle by exposure to interleukin-3 (IL-3), IL-6, IL-11, and Steel factor at different times of culture corresponding to different phases of cell cycle. We have found that marrow cells at the G₁ /S interface of the cell cycle have a three-fold increase in cells that assume a nonhematopoietic or pulmonary epithelial cell phenotype and that this increase is no longer seen in late S/G ₂. These cells have been characterized as GFP⁺ CD45 and GFP⁺ cytokeratin⁺. Thus, marrow cells with the capacity to convert into cells with a lung phenotype after transplantation show a reversible increase with cytokine-induced cell cycle transit. Previous studies have shown that the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse the cell cycle, leading to a continuum model of stem cell regulation. The present study indicates that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.  

4.584           Fewer active motors per vesicle may explain slowed vesicle transport in chick motoneurons after three days in vitro

Macosko, J.C. et al Brain Res., 1211, 6-12 (2008)   Vesicle transport in cultured chick motoneurons was studied over a period of 3 days using motion-enhanced differential interference contrast (MEDIC) microscopy, an improved version of video-enhanced DIC. After 3 days in vitro (DIV), the average vesicle velocity was about 30% less than after 1 DIV. In observations at 1, 2 and 3 DIV, larger vesicles moved more slowly than small vesicles, and retrograde vesicles were larger than anterograde vesicles. The number of retrograde vesicles increased relative to anterograde vesicles after 3 DIV, but this fact alone could not explain the decrease in velocity, since the slowing of vesicle transport in maturing motoneurons was observed independently for both anterograde and retrograde vesicles. In order to better understand the slowing trend, the distance vs. time trajectories of individual vesicles were examined at a frame rate of 8.3/s. Qualitatively, these trajectories consisted of short (1–2 s) segments of constant velocity, and the changes in velocity between segments were abrupt (< 0.2 s). The trajectories were therefore fit to a series of connected straight lines. Surprisingly, the slopes of theses lines, i.e. the vesicle velocities, were often found to be multiples of ~ 0.6 μm/s. The velocity histogram showed multiple peaks, which, when fit with Gaussians using a least squares minimization, yielded an average spacing of 0.57 μm/s (taken as the slope of a fit to peak position vs. peak number, R² = 0.994). We propose that the abrupt velocity changes occur when 1 or 2 motors suddenly begin or cease actively participating in vesicle transport. Under this hypothesis, the decrease in average vesicle velocity observed for maturing motoneurons is due to a decrease in the average number of active motors per vesicle.  

4.585           Translating innate response into long-lasting antibody response by the intrinsic antigen-adjuvant properties of papaya mosaic virus

Acosta-Ramirez, E. et al Immunology, 124, 186-197 (2008)   Identifying the properties of a molecule involved in the efficient activation of the innate and adaptive immune responses that lead to long-lasting immunity is crucial for vaccine and adjuvant development. Here we show that the papaya mosaic virus (PapMV) is recognized by the immune system as a pathogen-associated molecular pattern (PAMP) and as an antigen in mice (Pamptigen). A single immunization of PapMV without added adjuvant efficiently induced both cellular and specific long-lasting antibody responses. PapMV also efficiently activated innate immune responses, as shown by the induction of lipid raft aggregation, secretion of pro-inflammatory cytokines, up-regulation of co-stimulatory molecules on dendritic cells and macrophages, and long-lasting adjuvant effects upon the specific antibody responses to model antigens. PapMV mixed with Salmonella enterica serovar Typhi (S. typhi) outer membrane protein C increased its protective capacity against challenge with S. typhi, revealing the intrinsic adjuvant properties of PapMV in the induction of immunity. Antigen-presenting cells loaded with PapMV efficiently induced antibody responses in vivo, which may link the innate and adaptive responses observed. PapMV recognition as a Pamptigen might be translated into long-lasting antibody responses and protection observed. These properties could be used in the development of new vaccine platforms.  

4.586           CD24a Expression Levels Discriminate Langerhans Cells from Dermal Dendritic Cells in Murine Skin and Lymph Nodes

Stutte, S., Jux, B., esser, C. and Förster, I.

1. Invest. Dermatol., 128, 1470-1475 (2008)

  Langerhans cells (LCs) and dermal dendritic cells (dDCs) are the professional antigen-presenting cells of the skin. Recently, their immunogenic versus tolerogenic role has come under re-investigation. LCs are distinguished from dDCs by Langerin (CD207) staining or by detection of Birbeck granules. However, for in vitro experiments it is desirable to have a simple and robust flow cytometric demarcation of both cell types. We show here that CD24a is expressed on LCs but not on dDCs isolated directly from the skin. Moreover, in combination with major histocompatibility complex class II (MHCII), CD24a expression levels distinguish LCs from dDCs in skin-draining lymph nodes after antigen activation and migration. High expression of CD24a correlated strictly with CD207 expression. MHCIIhigh cells were unique for skin-draining lymph nodes and were shown to be the only cells carrying antigen after FITC painting of the skin. CD24a expression levels further differentiated LCs and dDCs in the MHCIIhigh population. As staining for CD24a does not require fixation of cells, CD24a-stained cells can be used for in vitro experiments to analyze and compare the functional roles and properties of dDCs and LCs.  

4.587           The receptor tyrosine kinase Flt3 is required for dendritic cell development in peripheral lymphoid tissues

Waskow, C. et al Nature Immunol., 9(6), 676-683 (2008)   Dendritic cell (DC) development begins in the bone marrow but is not completed until after immature progenitors reach their sites of residence in lymphoid organs. The hematopoietic growth factors regulating these processes are poorly understood. Here we examined the effects of signaling by the receptor tyrosine kinase Flt3 on macrophage DC progenitors in the bone marrow and on peripheral DCs. We found that the macrophage DC progenitor compartment was responsive to superphysiological amounts of Flt3 ligand but was not dependent on Flt3 for its homeostatic maintenance in vivo. In contrast, Flt3 was essential to the regulation of homeostatic DC development in the spleen, where it was needed to maintain normal numbers of DCs by controlling their division in the periphery.  

4.588           Assessing meiofaunal variation among individuals utilising morphological and molecular approaches: an example using the Tardigrada Sands, C.J., Convey, P., Linse, K. and McInnes, S.J.

BMC Ecology, 8(7), 1-11 (2008)   Background: Meiofauna – multicellular animals captured between sieve size 45 μm and 1000 μm – are a fundamental component of terrestrial, and marine benthic ecosystems, forming an integral element of food webs, and playing a critical roll in nutrient recycling. Most phyla have meiofaunal representatives and studies of these taxa impact on a wide variety of sub-disciplines as well as having social and economic implications. However, studies of variation in meiofauna are presented with several important challenges. Isolating individuals from a sample substrate is a time consuming process, and identification requires increasingly scarce taxonomic expertise. Finding suitable morphological characters in many of these organisms is often difficult even for experts. Molecular markers are extremely useful for identifying variation in morphologically conserved organisms. However, for many species markers need to be developed de novo, while DNA can often only be extracted from pooled samples in order to obtain sufficient quantity and quality. Importantly, multiple independent markers are required to reconcile gene evolution with species evolution. In this primarily methodological paper we provide a proof of principle of a novel and effective protocol for the isolation of meiofauna from an environmental sample. We also go on to illustrate examples of the implications arising from subsequent screening for genetic variation at the level of the individual using ribosomal, mitochondrial and single copy nuclear markers. Results: To isolate individual tardigrades from their habitat substrate we used a non-toxic density gradient media that did not interfere with downstream biochemical processes. Using a simple DNA release technique and nested polymerase chain reaction with universal primers we were able amplify multi-copy and, to some extent, single copy genes from individual tardigrades. Maximum likelihood trees from ribosomal 18S, mitochondrial cytochrome oxidase subunit 1, and the single copy nuclear gene Wingless support a recent study indicating that the family Hypsibiidae is a non-monophyletic group. From these sequences we were able to detect variation between individuals at each locus that allowed us to identify the presence of cryptic taxa that would otherwise have been overlooked. Conclusion: Molecular results obtained from individuals, rather than pooled samples, are a prerequisite to enable levels of variation to be placed into context. In this study we have provided a proof of principle of this approach for meiofaunal tardigrades, an important group of soil biota previously not considered amenable to such studies, thereby paving the way for more comprehensive phylogenetic studies using multiple nuclear markers, and population genetic studies.  

4.589           Dendritic cell vaccine with mRNA targeted to the proteasome by polyubiquitination

Hosoi, A. et al Biochem. Biophys. Res. Comm., 371, 242-246 (2008)   Dendritic cells (DCs) transfected with mRNA encoding tumor-associated antigens (TAAs) can induce tumor-specific T-cell responses. To potentiate this, we transfected mature DCs (mDCs) with mRNA encoding TAA targeted to the proteasome. DCs were generated from bone marrow cells by culture with 20 ng/ml GM-CSF and maturation with 1 μg/ml LPS. These mDCs were then electroporated with 10 μg of mRNA. Antigen presentation after electroporation with in vitro transcribed mRNA was compared with mRNA from a construct of the TAA preceded by ubiquitin. Proteasomal targeting of mRNA encoding cotranslationally ubiquitinated antigen was found to enhance intracellular degradation of target protein, and result in more efficient priming and expansion of TAA-specific CD8⁺ T-cells. We therefore suggest that RNA-transfected DC vaccine efficacy could be improved by the use of mRNA targeted to the proteasome.  

4.590           The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons

Corey, J.M. et al Acta Biomaterialia, 4, 863-875 (2008)   Aligned electrospun nanofibers direct neurite growth and may prove effective for repair throughout the nervous system. Applying nanofiber scaffolds to different nervous system regions will require prior in vitro testing of scaffold designs with specific neuronal and glial cell types. This would be best accomplished using primary neurons in serum-free media; however, such growth on nanofiber substrates has not yet been achieved. Here we report the development of poly(l-lactic acid) (PLLA) nanofiber substrates that support serum-free growth of primary motor and sensory neurons at low plating densities. In our study, we first compared materials used to anchor fibers to glass to keep cells submerged and maintain fiber alignment. We found that poly(lactic-co-glycolic acid) (PLGA) anchors fibers to glass and is less toxic to primary neurons than bandage and glue used in other studies. We then designed a substrate produced by electrospinning PLLA nanofibers directly on cover slips pre-coated with PLGA. This substrate retains fiber alignment even when the fiber bundle detaches from the cover slip and keeps cells in the same focal plane. To see if increasing wettability improves motor neuron survival, some fibers were plasma etched before cell plating. Survival on etched fibers was reduced at the lower plating density. Finally, the alignment of neurons grown on this substrate was equal to nanofiber alignment and surpassed the alignment of neurites from explants tested in a previous study. This substrate should facilitate investigating the behavior of many neuronal types on electrospun fibers in serum-free conditions.  

4.591           Culturing Adult Rat Hippocampal Neurons with Long-Interval Changing Media

Majd, S., Zarifkar, A., Rastegar, K. And Takhshid, M.A. Iranian Biomed. J., 12(2), 101-107 (2008)   Background: Primary cultures of embryonic neurons have been used to introduce a model of neurons in physiological and pathological conditions. However, age-related cellular events limit this method as an optimal model in adult neurodegenerative diseases studies. Besides, short-interval changing media in previous cultures decreases the effectiveness of this model. As an example of this matter, we can refer to the study on some special neuronal secreted factors or the influence of some experimental materials on neurons. Meanwhile, short-interval changing media could remove the effects of some released factors from the environment. In this study, the method for isolation and culturing adult rat hippocampal neurons with longintervals medium changing has been described. Methods: The hippocampal neurons of adult male rats were cultured. We used Neurobasal A/B27 culture medium, papain (2 mg/ml), trypsin 0.25% and collagenase (1mg/ml) for neuronal isolation, OptiPrep density gradient for separation of neurons from other cell types and also debris and FGF2 (10 ng/ml) for increasing neuronal survival and regeneration. Results: The neuronal sprouting and viability were increased by using papain and mild triturating (P<0.05). Adult neuronal culturing and their regeneration were impossible without FGF2. It was shown that adding new fresh medium every 4 days and exchanging half of it every 8 days had no detrimental effect on neuronal viability. Conclusion: This investigation shows the possibility of culturing adult neuronal cells and their maintaining in long-interval media. It could be happened because of adult neurons rely significantly on the neighboring cells secreted factors for living and making synaptic connections. This model is very useful in physiological and pathological studies which need stable conditions of neuronal culture in a long period of time.  

4.592           Adenosine Triphosphate Production by Bovine Spermatozoa and Its Relationship to Semen Fertilizing Ability

Garrett, L.J.A., Revell, S.G. and Leese, H.J.

1. Androl., 29, 449-458 (2008)

  This article's objectives are to investigate the relationship between adenosine triphosphate (ATP) production (oxidative phosphorylation and glycolysis) and fertility of bovine spermatozoa, determine the proportion of oxygen consumption devoted to proton leak and that due to nonmitochondrial processes, and discover whether freeze/thawing affects sperm oxygen consumption. Oxygen consumption of bovine spermatozoa was measured using a standard Clark electrode and, for the first time, in an Oxygen Biosensor System (OBS). Total ATP formation by bovine spermatozoa was calculated from the oxygen consumption and lactate production (glycolysis) by the same spermatozoa sample. ATP production varied from 1.99 to 8.09 µmol ATP per 10⁸ spermatozoa per hour; glycolysis accounted for 16% to 38% of ATP. Nonmitochondrial oxygen consumption could not be detected in bovine spermatozoa using these methods. A significant proportion (16%–43%) of oxygen consumption was insensitive to oligomycin and was due to "proton leak." There was no significant difference between oxygen consumption of frozen/thawed and fresh spermatozoa for 2 of the 3 bulls tested. However, oxygen consumption of frozen/thawed spermatozoa was significantly higher (P < .05) than fresh spermatozoa for the third bull. When ZO₂ of frozen/thawed spermatozoa from 20 bulls was compared with their 49 day nonreturn rates (NRRs), oxygen consumption was correlated positively with NRR (ie, fresh spermatozoa with a higher ZO₂ were more fertile). Moreover, total ATP production correlated with NNR better than ZO₂. Bulls with a lower NRR produce spermatozoa that are susceptible to damage during the freeze/thawing process, causing an increase in ZO₂, possibly due to mitochondrial membrane damage resulting in more energy being expended in maintaining the proton gradient, or capacitation-like changes causing hyperactivation. Oxygen consumption measured in the OBS may be useful in assessing bovine sperm fertility.  

4.593           Persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease

Kim, E.Y. et al Nature Med., 14(6), 633-640 (2008)   To understand the pathogenesis of chronic inflammatory disease, we analyzed an experimental mouse model of chronic lung disease with pathology that resembles asthma and chronic obstructive pulmonary disease (COPD) in humans. In this model, chronic lung disease develops after an infection with a common type of respiratory virus is cleared to only trace levels of noninfectious virus. Chronic inflammatory disease is generally thought to depend on an altered adaptive immune response. However, here we find that this type of disease arises independently of an adaptive immune response and is driven instead by interleukin-13 produced by macrophages that have been stimulated by CD1d-dependent T cell receptor–invariant natural killer T (NKT) cells. This innate immune axis is also activated in the lungs of humans with chronic airway disease due to asthma or COPD. These findings provide new insight into the pathogenesis of chronic inflammatory disease with the discovery that the transition from respiratory viral infection into chronic lung disease requires persistent activation of a previously undescribed NKT cell–macrophage innate immune axis.  

4.594           The Proinflammatory Mediator CD40 Ligand Is Increased in the Metabolic Syndrome and Modulated by Adiponectin

Natal, C. et al

1. Clin. Endocrin. Metab., 93(6), 2319-2327 (2008)

  Objectives: We hypothesized that the CD40/CD40 ligand (CD40L)system is up-regulated in the metabolic syndrome (MS) and modulatedby adiponectin (AN). The objectives were: 1) to compare plasmaand monocyte CD40L in patients with MS and controls and itsassociation with clinical and biochemical parameters, 2) toinvestigate platelets as a source of soluble CD40L (sCD40L),and 3) to analyze the effects of AN on CD40/CD40L. Methods: Plasma sCD40L and AN were measured in 246 controlsand 128 patients with MS by ELISA. Monocyte CD40/CD40L expressionand platelet CD40L content and release were compared in patientswith MS and controls. Monocytes and endothelial cells were culturedwith AN and CD40/CD40L expression determined by real-time RT-PCRand Western blotting. Results: Patients with MS had higher sCD40L and lower AN levels than controls (0.89 ± 0.1 vs. 0.76 ± 0.07 ng/ml and 10.10 ± 0.65 vs. 12.99 ± 0.80 µg/ml, P < 0.05). Monocyte CD40/CD40L expression was higher (P < 0.05) in patients than controls (CD40: 1.31 ± 0.31 vs. 0.80 ± 0.14 arbitrary units; CD40L: 1.24 ± 0.85 vs. 0.43 ± 0.14 pg/µg protein). No differences were observed on CD40L content between resting platelets from patients with MS and controls (7.7 ± 3.5 vs. 7.2 ± 2.2 pg/µg protein). Stimulated platelets from patients with the MS released more (P < 0.05) sCD40L than controls (582 ± 141 vs. 334 ± 60% change vs. nonstimulatedplatelets). AN reduced CD40L mRNA and protein expression inmonocytes from MS patients and endothelial cells. Conclusions: The enhanced sCD40L and cellular CD40L expressionin the MS suggests that CD40L is of pathophysiological relevancein MS. Also, a new antiinflammatory effect of AN is describedthrough the modulation of the CD40/CD40L system.  

4.595           Islet transplantation at the Diabetes Research Institute Japan

Noguchi, H. and Matsumoto, S.

1. Hepatobiliary Pancreat. Surg., 15, 278-283 (2008)

  Since the Edmonton Protocol was announced, more than 600 patients with type 1 diabetes at more than 50 institutions have received islet transplantation to treat their disease. We recently established a new islet isolation protocol, called the Kyoto Islet Isolation Method, based on the Ricordi method. It includes an in-situ cooling system for pancreas procurement, pancreatic ductal protection, a modified two-layer (M-Kyoto /perfluorochemical [PFC]) method of pancreas preservation, and a new islet purification solution (Iodixanol-based solution). Using this islet isolation method, we isolated islets from 19 human pancreata of non-heart-beating donors and transplanted 16 preparations into seven patients with type 1 diabetes between April 7, 2004 and November 18, 2005. The percentage of those meeting the release criteria of the Edmonton Protocol was more than 80%. We also performed living-donor transplantation of islets for unstable diabetes on January 19, 2005. Establishment of this method enables us to make diabetic patients insulin-independent, using islets not only from two or three pancreata of non-heart-beating donors but also using islets from half a pancreas from a living donor.  

4.596           Glycosylphosphatidylinositol-induced cardiac myocyte death might contribute to the fatal outcome of Plasmodium falciparum malaria

Wennicke, K. et al Apoptosis, 13, 857-866 (2008)   Background  Glycosylphosphatidylinositol (GPI) purified from Plasmodium falciparum has been shown to play an important role as a toxin in the pathology of malaria. Previous studies demonstrated cardiac involvement in patients suffering from severe malaria due to P. falciparum. Therefore, we tested the hypothesis that GPI induces apoptosis in cardiomyocytes. Methods and results  By using TUNEL and caspase activity assays, we provided evidence for apoptosis induction in cardiomyocytes by P. falciparum GPI after 48 h of incubation. A similar result was obtained in heart cells of mice 48 h after in vivo injection of GPI. Gene expression analyses in GPI-treated cardiomyocytes showed an up-regulation of apoptotic genes (apaf-1, bax) and of a myocardial damage marker bnp (brain natriuretic peptide), while a down-regulation was observed for the anti-apoptotic gene bcl-2 and for the heat shock protein hsp70. In spite of inflammatory cytokine gene up-regulation by GPI, co-culture with peripheral mononuclear cells (PMNCs) did not change the results obtained with cardiomyocytes alone, indicating a direct effect of GPI on cardiac myocytes. Co-culture with non-myocytic cardiac cells (NMCCs) resulted in up-regulation of Hsp70 and Bcl-2 genes in GPI-treated cardiomyocytes but without repercussion on the apoptosis level. A malaria-infected patient, presenting fulminant heart failure showed typical signs of cardiac myocyte apoptosis demonstrating the clinical relevance of toxin induced heart damage for the lethality of malaria. Our studies performed in vitro and in mice suggest that the GPI could be responsible for cardiomyocyte apoptosis that occurred in this patient. Conclusion   Plasmodium falciparum GPI-induced apoptosis might participate in the lethality of malaria.    

4.597           Different fibrillar Aβ 1–42 concentrations induce adult hippocampal neurons to reenter various phases of the cell cycle

Majd, S., Zarifkar, A., Rastegar, K. And Takhshid, M.A. Brain Res., 1218, 224-229 (2008)   In Alzheimer's disease (AD) cell cycle reentry precedes neuronal death, which could be induced by many cytotoxic factors. It is believed that beta amyloid (Aβ), the major component of extracellular plaques in AD, is potent in inducing neurons to reenter cell cycle. In AD brains, neurons expressing cell cycle markers are reported in many brain regions without any plaque formation, although very low levels of Aβ may still be detected. In the other side, because cell cycle reentry is not an immediate cause of apoptosis, neurons may remain in cell cycle phases for some time prior to their final death. In this study we examined if very low concentrations of Aβ 1–42 (picomolar) can trigger the adult neurons to reenter the cell cycle, and the effect of different Aβ concentrations on neuronal progression through different cell cycle phases. Primary adult neurons were treated with Aβ 1–42 at 2 × 10^− 6, 2 × 10^− 5, 2 × 10^− 4, 0.5 and 2.5 µM concentrations. Cyclin D1 and cyclin B1 (the markers for G1 and G2 phases of the cell cycle, respectively) and apoptosis were assessed. Treatment with Aβ at 2.5 µM induced apoptosis. At lower levels however, Aβ promoted neurons entering G1 and G2 phases without apoptosis, with 0.5 µM of Aβ inducing neurons into G2, and 2 × 10^− 5, 2 × 10^− 4 into G1 phases. Our results suggested that lower concentrations of Aβ induced neurons to reenter the cell cycle, and different concentrations had differential abilities to promote neurons into various cell cycle phases or trigger their death.  

4.598           Quantitative nitric oxide production by rat, bovine and porcine macrophages

Zelnickowa, P. et al Nitric Oxide, 19, 36-41 (2008)   The aim of this work was to compare in vitro nitric oxide (NO) production by rat, bovine and porcine macrophages. NO production was induced by lipopolysaccharide (LPS) or by phorbol 12-myristate 13-acetate (PMA) with ionomycin or recombinant interferon gamma (rIFN-γ) and was assessed by Griess reaction. NO synthase type II (NOS II) expression was quantified by immunocytochemistry, Western blot and real-time polymerase chain reaction (RT-PCR). There were differences in NO production by pulmonary alveolar macrophages (PAM) in all species tested. The largest amounts of NO were produced by rat PAM. Less NO was produced by bovine PAM. Moreover, PAM in rats and cows differed in their abilities to respond to various stimulators. Neither porcine PAM nor Kupffer cells produced NO. Stimulation of porcine PAM with alternative concentrations of LPS did not lead to inducing NO production. Stimulation of porcine PAM with rIFN-γ together with LPS led to a significant increase in the expression of NOS II mRNA, albeit without detectable NO production or NOS II expression on the protein level.  

4.599           Characterization of early and terminal complement proteins associated with polymorphonuclear leukocytes in vitro and in vivo after spinal cord injury

Nguyen, H.X., Galvan, M.D. and Anderson, A.J.

1. Neuroinflammation, 5, 26-38 (2008)

  Background The complement system has been suggested to affect injury or disease of the central nervous system (CNS) by regulating numerous physiological events and pathways. The activation of complement following traumatic CNS injury can also result in the formation and deposition of C5b-9 membrane attack complex (C5b-9/MAC), causing cell lysis or sublytic effects on vital CNS cells. Although complement proteins derived from serum/blood-brain barrier breakdown can contribute to injury or disease, infiltrating immune cells may represent an important local source of complement after injury. As the first immune cells to infiltrate the CNS within hours post-injury, polymorphonuclear leukocytes (PMNs) may affect injury through mechanisms associated with complement-mediated events. However, the expression/association of both early and terminal complement proteins by PMNs has not been fully characterized in vitro, and has not observed previously in vivo after traumatic spinal cord injury (SCI). Method We investigated the expression of complement mRNAs using rt-PCR and the presence of complement proteins associated with PMNs using immunofluroescence and quantitative flow cytometry. Results Stimulated or unstimulated PMNs expressed mRNAs encoding for C1q, C3, and C4, but not C5, C6, C7 or C9 in culture. Complement protein C1q or C3 was also detected in less than 30% of cultured PMNs. In contrast, over 70% of PMNs that infiltrated the injured spinal cord were associated with C1q, C3, C7 and C5b-9/MAC 3 days post-SCI. The localization/association of C7 or C5b-9/MAC with infiltrating PMNs in the injured spinal cord suggests the incorporation or internalization of C7 or C5b-9/MAC bound cellular debris by infiltrating PMNs because C7 and C5b-9/MAC were mostly localized to granular vesicles within PMNs at the spinal cord epicenter region. Furthermore, PMN presence in the injured spinal cord was observed for many weeks post-SCI, suggesting that this infiltrating cell population could chronically affect complement-mediated events and SCI pathogenesis after trauma. Conclusion Data presented here provide the first characterization of early and terminal complement proteins associated with PMNs in vitro and in vivo after SCI. Data also suggest a role for PMNs in the local internalization or deliverance of complement and complement activation in the post-SCI environment.  

4.600           The Absence of Lymphoid CD8+ Dendritic Cell Maturation in L-Selectin–/– Respiratory Compartment Attenuates Antiviral Immunity

Pascual, D.W., Wang, X., Kochetkova, I., Callis, G. and Riccardi, C.

1. Immunol., 181, 1345-1356 (2008)

  Intratracheal instillation of L-selectin-deficient (L-Sel^–/–) mice with an adenovirus 2 (Ad2) vector resulted in the lack of respiratory Ad2- or β-galactosidase-specific CTLs with concomitant long-lived β-galactosidase transgene expression in the lungs. The absence of Ag-specific CTLs was attributed to a deficiency in lymphoid CD11c⁺CD8⁺ dendritic cells (DCs) in the lower respiratory lymph nodes (LRLNs). To enable L-Sel^–/– CTL activity, cell-sorted L-Sel^–/–CD8⁺ T cells were cocultured with cell-sorted L-Sel^+/+CD8⁺ or CD8^– DCs or L-Sel^–/–CD8^– DCs. Only the CD8⁺ DCs restored CTL activity; L-Sel^–/–CD8^– DCs failed to support L-Sel^+/+ CTLs because these remained immature, lacking the ability to express costimulatory molecules CD40, CD80, or CD86. Although no lung CD8⁺ DCs were detected, the DC environment remained suppressive in L-Sel^–/– mice evident by the lack of CTL responses following adenoviral challenge with OVA in recipient L-Sel^–/– adoptively transferred with OT-1 CD8⁺ T cells. To assess whether the L-Sel^–/–CD8^– DCs could be induced into maturity, microbial stimulation studies were performed showing the failure of L-Sel^–/– LRLN to make matured DCs. When L-Sel^–/– mice were subjected in vivo to microbial activation before Ad2 vector dosing, CTL activity was restored stimulating the renewed presence of LRLN CD8⁺ DCs in L-Sel^–/– mice. These studies show that impairment of L-Sel^–/– DC maturation results in insufficient mature DCs that require microbial activation to restore increases in respiratory CD8⁺ DCs to support CTL responses.  

4.601           Proteomic Methodological Recommendations for Studies Involving Human Plasma, Platelets, and Peripheral Blood Mononuclear Cells

De Roos, B. et al

1. Proteome Res., 7, 2280-2290 (2008)

  This study was designed to develop, optimize and validate protocols for blood processing prior to proteomic analysis of plasma, platelets and peripheral blood mononuclear cells (PBMC) and to determine analytical variation of a single sample of depleted plasma, platelet and PBMC proteins within and between four laboratories each using their own standard operating protocols for 2D gel electrophoresis. Plasma depleted either using the Beckman Coulter IgY-12 proteome partitioning kit or the Amersham albumin and IgG depletion columns gave good quality gels, but reproducibility appeared better with the single-use immuno-affinity column. The use of the Millipore Filter Device for protein concentration gave a 16% (p < 0.005) higher recovery of protein in flow-through sample compared with acetone precipitation. The use of OptiPrep gave the lowest level of platelet contamination (1:0.8) during the isolation of PBMC from blood. Several proteins (among which are α-tropomyosin, fibrinogen and coagulation factor XIII A) were identified that may be used as biomarkers of platelet contamination in future studies. When identifying preselected spots, at least three out of the four centers found similar identities for 10 out of the 10 plasma proteins, 8 out of the 10 platelet proteins and 8 out of the 10 PBMC proteins. The discrepancy in spot identifications has been described before and may be explained by the mis-selection of spots due to laboratory-to-laboratory variation in gel formats, low scores on the peptide analysis leading to no or only tentative identifications, or incomplete resolution of different proteins in what appears as a single abundant spot. The average within-laboratory coefficient of variation (CV) for each of the matched spots after automatic matching using either PDQuest or ProteomWeaver software ranged between 18 and 69% for depleted plasma proteins, between 21 and 55% for platelet proteins, and between 22 and 38% for PBMC proteins. Subsequent manual matching improved the CV with on average between 1 and 16%. The average between laboratory CV for each of the matched spots after automatic matching ranged between 4 and 54% for depleted plasma proteins, between 5 and 60% for platelet proteins, and between 18 and 70% for PBMC proteins. This variation must be considered when designing sufficiently powered studies that use proteomics tools for biomarker discovery. The use of tricine in the running buffer for the second dimension appears to enhance the resolution of proteins especially in the high molecular weight range.  

4.602           Cerebellar granule cells cultured from adolescent rats express functional NMDA receptors: an in vitro model for studying the developing cerebellum 

Popp, R.L., Reneau, J.C. and Dertien, J.S.

1. Neurochem., 106, 900-911 (2008)

  In the developing rat cerebellum functional NMDA receptors (NMDARs) expressing the NR2C subunit have been identified on or after postnatal day 19. We obtained primary cultured cells from 19- to 35-day-old rat cerebellum that expressed few oligodendrocytes or astrocytes. Cultured cells were immunoreactive for neuron-specific proteins thus indicating a neuronal population. The primary neuron present was the granule cell as indicated by immunofluorescence for the GABA_A alpha 6 subunit. Whole-cell patch-clamp experiments indicated that functional NMDARs were present. Functional characteristics of NMDARs expressed in cerebellar granule cells (CGCs) obtained from adolescent animals were similar to those previously reported for NMDARs expressed in CGCs obtained from neonatal rats. Cultured CGCs obtained from older animals contained NMDARs that were inhibited by EtOH and were less sensitive to the NR2B subunit-specific antagonist Ro 25-6981. Furthermore, NMDA-induced currents were smaller than those observed in CGCs. Western blot analysis indicated the presence of the NMDA NR2A and NR2C subunits, but not the NR2B in cultures obtained from the adolescent rats. CGCs obtained from adolescent rats express functional NMDARs consistent with a developmental profile observed in vivo.  

4.603           NO-synthase-/NO-independent regulation of human and murine platelet soluble guanylyl cyclase activity

Gambaryan, S. et al

1. Thromb. Haemostasis, 6, 1376-1384 (2008)

  Summary.  Objectives: Platelets, specialized adhesive cells, play key roles in normal and pathological hemostasis through their ability to rapidly adhere to subendothelial matrix proteins (adhesion) and to other activated platelets (aggregation), functions which are inhibited by nitric oxide (NO). Platelets have been reported to be regulated not only by exogenous endothelium-derived NO, but also by two isoforms of NO synthase, endothelial (eNOS) and inducible (iNOS), endogenously expressed in platelets. However, data concerning expression, regulation and function of eNOS and iNOS in platelets remain controversial. Methods and results: Using important positive (endothelial cells, stimulated macrophages) and negative (eNOS/iNOS knock-out mouse) controls, as well as human platelets highly purified by a newly developed protocol, we now demonstrate that human and mouse platelets do not contain eNOS/iNOS proteins or mRNA. NOS substrate (l-arginine), NOS inhibitors (L-NAME, L-NMMA), and eNOS/iNOS deficiency did not produce detectable functional effects on human and mouse platelets. von Willebrand factor (VWF)/ristocetin treatment of platelets increased cGMP by NO-independent activation of soluble guanylyl cyclase (sGC) which correlated with Src kinase-dependent phosphorylation of sGC β₁-subunit-Tyr¹⁹². Conclusions: Human and mouse platelets do not express eNOS/iNOS. VWF/ristocetin-mediated activation of the sGC/cGMP signaling pathway may contribute to feedback platelet inhibition.  

4.604           Use of functional highly purified human platelets for the identification of new proteins of the IPP signaling pathway

Birschmann, I. et al Thromb. Res., 122, 59-68 (2008)   Introduction Identification of the full content of platelet proteins and their mRNAs would be helpful for further studies of human platelet function. For this purpose, proteomic as well as transcriptomic methods (SAGE and qRT-PCR) can be utilized, but the purity of the platelet samples studied is crucial. Here we report the development of a new, effective, and efficient technique for purification of human platelets from washed apheresis platelet concentrates and whole blood. Materials and methods Methods used are a combination of differential and gradient centrifugation steps. The level of purification was determined by nephelometry, FACS, and PCR. Results We could show that even the P2Y purinoceptor 12 (P2Y₁₂) receptor, which undergoes rapid homologous desensitization, was still functional after the purification procedure. The presence of PINCH (particularly interesting new Cys–His protein) and -parvin, which constitute the IPP (ILK–PINCH–parvin) complex together with the integrin-linked kinase (ILK), has been predicted in platelets by proteomic analysis. We could confirm this observation with our purified platelets. Detection of these proteins is an example of the application of this purification protocol that can be used for the verification of proteins postulated by high-throughput studies. Conclusions The procedure for obtaining purified platelets described here provides an essential, much-needed tool for the comprehensive investigation of platelet proteins and functions.  

4.605           Factors impacting equine sperm recovery rate and quality following cushioned centrifugation

Waite, J.A. et al Theriogenology, 70(4), 704-714 (2008)   Two experiments were conducted to investigate modifications in cushioned centrifugation of stallion semen. Specifically, the effects of tube type, centrifugation medium, cushion type, and centrifugation force on post-centrifugation sperm recovery rate and quality were evaluated. In Experiment 1, sperm recovery rate was higher (P < 0.05) in conventional plastic conical-bottom tubes (103%) than in newly developed glass nipple-bottom tubes (96%) following cushioned centrifugation; however, several measures of semen quality (i.e., % total motility [MOT], % progressive motility [PMOT], curvilinear velocity, and average-path velocity) yielded higher values following centrifugation in nipple-bottom tubes (P < 0.05). Sperm recovery rate following cushioned centrifugation was similar between semen previously diluted in optically clear centrifugation extender (100%) and semen diluted in opaque centrifugation extender (100%); however, MOT and PMOT were higher in semen subjected to cushioned centrifugation in opaque extender (P < 0.05). An extender by tube-type interaction was not detected for recovery rate or post-centrifugation semen quality. In Experiment 2, sperm recovery rate following cushioned centrifugation in nipple-bottom tubes was similar when forces of 400 × g or 600 × g were applied (90 and 90%, respectively; P > 0.05), and no resulting differences in semen quality were detected between these treatment groups (P > 0.05). The type of iodixanol cushion medium used (i.e., OptiPrep™, Eqcellsire^® Component B, or Cushion Fluid™) did not impact post-centrifugation semen quality, based on the laboratory values measured (P > 0.05). In conclusion, cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes yielded a high sperm harvest, while maintaining sperm function. An optically opaque extender, commonly used in the equine breeding industry, can be used to achieve this goal.  

4.606           Expression of a Soluble TGF-β Receptor by Tumor Cells Enhances Dendritic Cell/Tumor Fusion Vaccine Efficacy

Zhang, M., Berndt, B.E., Chen, J-J. And Kao, J.Y.

1. Immunol., 181, 3690-3697 (2008)

  Dendritic cell (DC)-based antitumor immunotherapy is a promising cancer therapy. We have previously shown that tumor-derived TGF-β limits the efficacy of the DC/tumor fusion vaccine in mice. In the current study we investigated the effect of neutralizing tumor-derived TGF-β on the efficacy of the DC/tumor fusion vaccine. An adenovirus encoding human TGF-β receptor type II fused to the Fc region of human IgM (Adv-TGF-β-R) or a control adenovirus encoding LacZ (Adv-LacZ) was used to express a soluble form of the neutralizing TGF-β receptor (TGF-β-R). Murine breast carcinoma cells, 4T1, but not bone marrow-derived DCs, were successfully transfected with Adv-TGF-β-R (4T1+Adv-TGF-β-R) using a multiplicity of infection of 300. Immunization with irradiated 4T1+Adv-TGF-β-R tumor cells conferred enhanced antitumor immunity compared with immunization with irradiated 4T1+Adv-LacZ tumor cells. The DC/4T1+Adv-TGF-β-R fusion vaccine offered enhanced protective and therapeutic efficacy compared with the DC/4T1-Adv-LacZ fusion vaccine. Because TGF-β is known to induce regulatory T cells (Tregs), we further showed that the DC/4T1+Adv-TGF-β-R fusion vaccine induced fewer CD4⁺CD25⁺Foxp3⁺ Tregs than the DC/4T1+Adv-LacZ fusion vaccine in vitro and in vivo. The suppressive role of splenic CD4⁺CD25⁺ Tregs isolated from mice immunized with DC/4T1+Adv-LacZ was demonstrated using a CTL killing assay. Similar enhanced therapeutic efficacy was observed in murine renal cell carcinoma, RenCa, which expresses a high level of TGF-β. We conclude that the blockade of tumor-derived TGF-β reduces Treg induction by the DC/tumor fusion vaccine and enhances antitumor immunity. This may be an effective strategy to enhance human DC-based antitumor vaccines.  

4.607           Age-related differences in NFκB translocation and Bcl-2/Bax ratio caused by TNFα and Abeta42 promote survival in middle-age neurons and death in old neurons

Patel, J.R. and Brewer, G.J. Exp. Neurol., 213, 93-100 (2008)   Alzheimer's disease is associated with an age-related accumulation of Abeta and inflammation. The inflammatory mediator, TNFα activates a signaling cascade involving NFκB translocation to the nucleus and a beneficial or detrimental transcriptional response, depending on the age of the neurons and the type of stress applied. Relative to treatment with Abeta42 alone, previously we found that TNFα plus Abeta42, applied to old rat neurons (24 month) is toxic, while the same treatment of middle-age neurons (10 month) is protective. In contrast to improved survival of middle-age rat cortical neurons, neurons from old rats are killed by TNFα plus Abeta42 despite greater p50 nuclear translocation. In middle-age neurons, blocking TNFR1 does not affect NFκB translocation, whereas blocking TNFR2 results in an increase in NFκB translocation. For old neurons, blocking either receptor, does not change NFκB translocation, but improves cell survival. To account for these effects on cell viability in response to TNF + Abeta, measures of the Bcl-2/Bax ratio positively correlate with survival. In the setting of old neurons, these results suggest that overactivated nuclear translocation of NFκB and lower Bcl-2 levels promote death that is reduced by inhibition of either TNFR1 or R2.  

4.608           THE PURIFICATION METHOD USING IODIXANOL (OPTIPREP)-BASED DENSITY GRADIENT SIGNIFICANTLY REDUCE CYTOKINE/CHEMOKINE PRODUCTION FROM HUMAN ISLET PREPARATIONS, LEADING TO PROLONG ~ A-CELL SURVIVAL DURING CULTURE

Mita, A. et al Transplantation. 86(2S) Supplement:570 ( 2008)   Background: Although Ficoll-based density gradient has been widely used for human islet purifi cation in most islet processing centers, OptiPrep-based density gradient was recently used in the limited centers. It has been reported that Islet preparations purifi ed using OptiPrep-based density gradient provided better clinical outcomes. It is well known that cytokine/chemokine production from islet preparations widely varies. Reducing cytokine/chemokine production from islet preparations may be a key to improve islet transplantation outcomes. The aim of current study is to investigate the variability of pro-infl ammatory cytokine/chemokine production from human islet preparations purifi ed using different density gradients. Methods: Human islet isolations were performed using automated method. After digestion phase, pre-purification digests were divided into two groups andpurified using semi-automated cell processor with Ficoll-based or OptiPrepbased density gradient. Human Islet preparations were cultured for 2 days, and assessed regarding glucose stimulated insulin release, islet cell viability (FDA/PI), fractional ƒÀ-cell viability and ƒÀ-cell content. Cytokine/chemokine production from islet preparations was also examined. Results: Between Ficoll-based and OptiPrep-based density gradient groups, islet purity (90.0 ¦ } 4.1% and 91.3 ¦ } 3.2%, respectively, p=0.718), postpurification IEQ (128,504.0 ¦ } 28,893.4 IEQ and 140,881.0 ¦ } 16,644.0 IEQ, respectively, p=0.726) and islet recovery rate (46.4 ¦ } 4.2% and 69.7 ¦ } 22.8%, respectively, p=0.390) were comparable. Although stimulated insulin release (stimulation index 0.5 ¦ } 0.2 and 0.8 ¦ } 0.3, respectively, p=0.123), FDA/PI (92.4 ¦ } 2.5% and 90.6 ¦ } 3.6%, respectively, p=0.550) and fractional ƒÀ-cell viability showed no signifi cant differences (88.2 ¦ } 7.7% relative to OptiPrep-based density gradient group, p=0.178), ƒÀ-cell survival during culture signifi cantly improved in OptiPrep-based density gradient group when compared to Ficoll-based density gradient group (130.6 ¦ } 1.8% relative, p<0.05). TNF-ƒ¿, IL-1ƒÀ, IFN-ƒÁ, IL-6 and MIP-1ƒÀ production from OptiPrep-based density gradient group signifi cantly decreased when compared to Ficoll-based density gradient group (22.8 ¦ } 11.8% relative, p=0.003, 27.7 ¦ } 14.0% relative, p=0.002, 37.3 ¦ } 11.2% relative, p=0.001, 45.3 ¦ } 16.2% relative, p=0.015, and 38.5 ¦ } 18.6% relative, p=0.016, respectively). Conclusions: The purifi cation method using OptiPrep-based density gradient can signifi cantly reduce cytokine/chemokine production from islet preparationswhen compared to Ficoll-based density gradient, leading to the improvement of quantity of ƒÀ-cell mass. Our results suggest that the purifi cation method using OptiPrep-based density gradient may be of assistance in improving clinical outcomes, and that Cytokine/chemokine profi ling from islet preparations could be helpful to develop better isolation methods.  

4.609           Microfluidic-Based Cell Sorting of Francisella tularensis Infected Macrophages Using Optical Forces

Perroud, T.D. et al Anal. Chem., 80, 6365-6372 (2008)   We have extended the principle of optical tweezers as a noninvasive technique to actively sort hydrodynamically focused cells based on their fluorescence signal in a microfluidic device. This micro fluorescence-activated cell sorter (µFACS) uses an infrared laser to laterally deflect cells into a collection channel. Green-labeled macrophages were sorted from a 40/60 ratio mixture at a throughput of 22 cells/s over 30 min achieving a 93% sorting purity and a 60% recovery yield. To rule out potential photoinduced cell damage during optical deflection, we investigated the response of mouse macrophage to brief exposures (<4 ms) of focused 1064-nm laser light (9.6 W at the sample). We found no significant difference in viability, cell proliferation, activation state, and functionality between infrared-exposed and unexposed cells. Activation state was measured by the phosphorylation of ERK and nuclear translocation of NF-κB, while functionality was assessed in a similar manner, but after a lipopolysaccharide challenge. To demonstrate the selective nature of optical sorting, we isolated a subpopulation of macrophages highly infected with the fluorescently labeled pathogen Francisella tularensis subsp. novicida. A total of 10 738 infected cells were sorted at a throughput of 11 cells/s with 93% purity and 39% recovery.  

4.610           An improved method for the extraction of nematodes using iodixanol (OptiPrep Ô)

Deng, D. et al African J. Microbiol. Res., 2, 167-170 (2008)   A new nematode extraction technique was established, which is based on an iso-osmotic densitygradient medium (OptiPrepTM). This technique resulted in significantly higher numbers of clean eggs and vermiform nematodes that retain higher viability (48.6%) than samples processed with the sucrose method (28.7%). Nematodes survived exposure to OptiPrepTM for 22 hours without significant mortality whereas all nematodes died in the sucrose medium. OptiPrepTM provided a suitable, non-toxic alternative to the traditional density gradient material for the isolation of nematodes. This technique is convenient and relatively simple, with the added benefit of yielding cleaner samples compared to traditional isolation techniques.  

4.611           Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans

Jaensson, E. et al

1. Exp. Med., 205(9), 2139-2149 (2008)

  A functionally distinct subset of CD103⁺ dendritic cells (DCs) has recently been identified in murine mesenteric lymph nodes (MLN) that induces enhanced FoxP3⁺ T cell differentiation, retinoic acid receptor signaling, and gut-homing receptor (CCR9 and 4β7) expression in responding T cells. We show that this function is specific to small intestinal lamina propria (SI-LP) and MLN CD103⁺ DCs. CD103⁺ SI-LP DCs appeared to derive from circulating DC precursors that continually seed the SI-LP. BrdU pulse-chase experiments suggested that most CD103⁺ DCs do not derive from a CD103^– SI-LP DC intermediate. The majority of CD103⁺ MLN DCs appear to represent a tissue-derived migratory population that plays a central role in presenting orally derived soluble antigen to CD8⁺ and CD4⁺ T cells. In contrast, most CD103^– MLN DCs appear to derive from blood precursors, and these cells could proliferate within the MLN and present systemic soluble antigen. Critically, CD103⁺ DCs with similar phenotype and functional properties were present in human MLN, and their selective ability to induce CCR9 was maintained by CD103⁺ MLN DCs isolated from SB Crohn's patients. Thus, small intestinal CD103⁺ DCs represent a potential novel target for regulating human intestinal inflammatory responses.        

4.612           Chromatin-bound mitogen-activated protein kinases transmit dynamic signals in transcription complexes in β-cells

Lawrence, M.C. et al PNAS, 105(36), 13315-13320 (2008)   MAPK pathways regulate transcription through phosphorylation of transcription factors and other DNA-binding proteins. In pancreatic β-cells, ERK1/2 are required for transcription of the insulin gene and several other genes in response to glucose. We show that binding of glucose-sensitive transcription activators and repressors to the insulin gene promoter depends on ERK1/2 activity. We also find that glucose and NGF stimulate the binding of ERK1/2 to the insulin gene and other promoters. An ERK1/2 cascade module, including MEK1/2 and Rsk, are found in complexes bound to these promoters. These findings imply that MAPK-containing signaling complexes are positioned on sensitive promoters with their protein substrates to modulate transcription in situ in response to incoming signals.  

4.613           Adenovirus-Mediated VEGF Gene Therapy Enhances Venous Thrombus Recanalization and Resolution

Modarai, B. et al Arterioscler. Thromb. Vasc. Biol., 28, 1753-1759 (2008)   Objective— Rapid thrombus recanalization reduces the incidenceof post–thrombotic complications. This study aimed todiscover whether adenovirus-mediated transfection of the vascularendothelial growth factor gene (ad.VEGF) enhanced thrombus recanalizationand resolution. Methods and Results— In rats, thrombi were directly injected with either ad.VEGF (n=40) or ad.GFP (n=37). Thrombi in SCID mice (n=12) were injected with human macrophages transfected with ad.VEGF or ad.GFP. Thrombi were analyzed at 1 to 14 days. GFP was found mainly in the vein wall and adventitia by 3 days, but was predominantly found in cells within the body of thrombus by day 7. VEGF levels peaked at 4 days (376±299 pg/mg protein). Ad.VEGF treatment reduced thrombus size by >50% (47.7±5.1 mm² to 22.0±4.0 mm², P=0.0003) and increased recanalization by >3-fold (3.9±0.69% to 13.6±4.1%, P=0.024) compared with controls. Ad.VEGF treatment increased macrophage recruitment into the thrombus by more than 50% (P=0.002). Ad.VEGF-transfected macrophages reduced thrombus size by 30% compared with controls (12.3±0.89 mm² to 8.7±1.4 mm², P=0.04) and enhanced vein lumen recanalization (3.39±0.34% to 5.07±0.57%, P=0.02). Conclusion— Treatment with ad.VEGF enhanced thrombus recanalizationand resolution, probably as a consequence of an increase inmacrophage recruitment.  

4.614           Microenvironment of the feto–maternal interface protects the semiallogenic fetus through its immunomodulatory activity on dendritic cells

Zarnani, A.H. et al Fertility and Stwerility, 90(3), 781-788 (2008)   Objective To investigate the immunomodulatory activity of decidual culture supernatant on dendritic cell (DC) functions. Design In vivo and in vitro experimental study using mice. Setting Academic research laboratory. Animal(s) C57BL/6-mated female Balb/c mice. Intervention(s) Culture supernatants of decidual cells obtained from the uteri of allogenic pregnant mice (Balb/c × C57BL/6) were collected. Dendritic cells were purified from Balb/c mice spleens and pulsed with antigen during overnight culture. In some cultures, decidual supernatant was added at 5%, 10%, or 20% final concentration. Endometrial culture supernatant-treated DCs served as a control. Antigen-pulsed DCs were injected into the front footpads of syngeneic mice. Main Outcome Measure(s) Lymph nodes of primed mice were removed 5 days after DC injection. Antigen-specific proliferation and interleukin-10 and interferon gamma production by lymphocytes were measured by ³H-Thymidine incorporation and ELISA, respectively. Result(s) The results showed that decidual culture supernatant markedly blocked in vivo antigen presentation by DCs and inhibited their capacity to induce interferon gamma (but not interleukin-10) production by primed lymphocytes. Conclusion(s) It seems that soluble factors produced by decidual cells are important mediators of immunoregulation at the feto–maternal interface, which provide the two fundamental requirements for protection of the semiallogenic fetus, namely immunologic tolerance and predominance of T helper 2 immunity, through modulation of DCs function.  

4.615           Carbon Monoxide and Nitric Oxide Mediate Cytoskeletal Reorganization in Microvascular Cells via Vasodilator-Stimulated Phosphoprotein Phosphorylation: Evidence for Blunted Responsiveness in Diabetes

Calzi, S.U. et al Diabetes, 57, 2488-2494 (2008)   OBJECTIVE— We examined the effect of the vasoactive agentscarbon monoxide (CO) and nitric oxide (NO) on the phosphorylationand intracellular redistribution of vasodilator-stimulated phosphoprotein(VASP), a critical actin motor protein required for cell migrationthat also controls vasodilation and platelet aggregation. RESEARCH DESIGN AND METHODS— We examined the effect ofdonor-released CO and NO in endothelial progenitor cells (EPCs)and platelets from nondiabetic and diabetic subjects and inhuman microvascular endothelial cells (HMECs) cultured underlow (5.5 mmol/l) or high (25 mmol/l) glucose conditions. VASPphosphorylation was evaluated using phosphorylation site-specificantibodies. RESULTS— In control platelets, CO selectively promotesphosphorylation at VASP Ser-157, whereas NO promotes phosphorylationprimarily at Ser-157 and also at Ser-239, with maximal responsesat 1 min with both agents on Ser-157 and at 15 min on Ser-239with NO treatment. In diabetic platelets, neither agent resultedin VASP phosphorylation. In nondiabetic EPCs, NO and CO increasedphosphorylation at Ser-239 and Ser-157, respectively, but thisresponse was markedly reduced in diabetic EPCs. In endothelialcells cultured under low glucose conditions, both CO and NOinduced phosphorylation at Ser-157 and Ser-239; however, thisresponse was completely lost when cells were cultured underhigh glucose conditions. In control EPCs and in HMECs exposedto low glucose, VASP was redistributed to filopodia-like structuresfollowing CO or NO exposure; however, redistribution was dramaticallyattenuated under high glucose conditions. CONCLUSIONS— Vasoactive gases CO and NO promote cytoskeletalchanges through site- and cell type–specific VASP phosphorylation,and in diabetes, blunted responses to these agents may leadto reduced vascular repair and tissue perfusion.  

4.616           Activation of peroxisome proliferator-activated receptor-γ by curcumin blocks the signaling pathways for PDGF and EGF in hepatic stellate cells

Lin, J. and Chen, A. Lab. Invest., 88, 529-540 (2008)   During hepatic fibrogenesis, reduction in the abundance of peroxisome proliferator-activated receptor- (PPAR ) is accompanied by activation of mitogenic signaling for platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) in hepatic stellate cells (HSCs), the major effector cells. We previously reported that curcumin, the yellow pigment in curry, interrupted PDGF and EGF signaling, stimulated PPAR gene expression, and enhanced its activity, leading to inhibition of cell proliferation of activated HSC in vitro and in vivo. The aim of this study was to elucidate the underlying mechanisms. We hypothesized that the enhancement of PPAR activity by curcumin might result in the interruption of PDGF and EGF signaling. Our experiments demonstrated that curcumin, with different treatment strategies, showed different efficiencies in the inhibition of PDGF- or EGF-stimulated HSC proliferation. Further experiments observed that curcumin dose dependently reduced gene expression of PDGF and EGF receptors (ie, PDGF- R and EGFR), which required PPAR activation. The activation of PPAR by its agonist suppressed pdgf- r and egfr expression in HSC. In addition, curcumin reduced the phosphorylation levels of PDGF- R and EGFR, as well as their downstream signaling cascades, including ERK1/2 and JNK1/2. Moreover, activation of PPAR induced gene expression of glutamate–cysteine ligase, the rate-limiting enzyme in de novo synthesis of the major intracellular antioxidant, glutathione. De novo synthesis of glutathione was required for curcumin to suppress pdgf- r and egfr expression in activated HSCs. Our results collectively demonstrated that enhancement of PPAR activity by curcumin interrupted PDGF and EGF signaling in activated HSCs by reducing the phosphorylation levels of PDGF- R and EGFR, and by suppressing the receptor gene expression. These results provide novel insights into the mechanisms of curcumin in the inhibition of HSC activation and the suppression of hepatic fibrogenesis.  

4.617           GM-CSF mediates autoimmunity by enhancing IL-6–dependent Th17 cell development and survival

Sonderegger, I. et al

1. Exp. Med., 205(10), 2281-2294 (2008)

  Granulocyte macrophage–colony stimulating factor (GM-CSF) is critically involved in development of organ-related autoimmune inflammatory diseases including experimental allergic encephalitis and collagen-induced arthritis. Roles of GM-CSF in the initiation and in the effector phase of the autoimmune response have been proposed. Our study was designed to investigate the mechanisms of GM-CSF in autoimmunity using a model of autoimmune heart inflammatory disease (myocarditis). The pathological sequel after immunization with heart myosin has been shown previously to depend on IL-1, IL-6, IL-23, and IL-17. We found that innate GM-CSF was critical for IL-6 and IL-23 responses by dendritic cells and generation of pathological Th17 cells in vivo. Moreover, GM-CSF promoted autoimmunity by enhancing IL-6–dependent survival of antigen specific CD4⁺ T cells. These results suggest a novel role for GM-CSF in promoting generation and maintenance of Th17 cells by regulation of IL-6 and IL-23 in vivo.  

4.618           IFN- -Dependent Recruitment of Mature CD27high NK Cells to Lymph Nodes Primed by Dendritic Cells

Watt, S.V., Andrews, D.M., Takeda, K., Smyth, M.J. and Hayakawa, Y.

1. Immunol., 181, 5323-5330 (2008)

  NK cells have been proposed to be an initial source of IFN- that supports either Th1 or CTL priming. Although NK cells reside in naive lymph nodes (LN) at a very low frequency, they can be recruited into LN draining sites of infection, inflammation, or immunization where they potentially influence adaptive immunity. In this study, we report that mature CD27^high NK cells are predominantly recruited into the draining LN following dendritic cell (DC) challenge. Importantly, the recruitment of the CD27^high NK cell subset in the draining LN was dependent on host IFN- and the activation status of NK cells. Endogenous epidermal DC migration induced by hapten challenge also triggers NK cell recruitment to the draining LN in an IFN- -dependent mechanism. Thus, our results identify that CD27^high NK cells are the dominant population recruited to the draining LN and NK cell recruitment requires endogenous IFN- in coordinating with DC migration.  

4.619           CD11c identifies a subset of murine liver natural killer cells that responds to adenoviral hepatitis

Burt, B.M. et al

1. Leukoc. Biol., 84(4), 1039-1046 (2008)

  The liver contains a unique repertoire of immune cells and a particular abundance of NK cells. We have found that CD11c defines a distinct subset of NK cells (NK1.1⁺CD3^–) in the murine liver whose function was currently unknown. In naïve animals, CD11c⁺ liver NK cells displayed an activated phenotype and possessed enhanced effector functions when compared with CD11c^– liver NK cells. During the innate response to adenovirus infection, CD11c⁺ NK cells were the more common IFN- -producing NK cells in the liver, demonstrated enhanced lytic capability, and gained a modest degree of APC function. The mechanism of IFN- production in vivo depended on TLR9 ligation as well as IL-12 and -18. Taken together, our findings demonstrate that CD11c⁺ NK cellsare a unique subset of NK cells in the murine liver that contributeto the defense against adenoviral hepatitis.  

4.620           Combined functional and molecular analysis of tumor cell signaling defines 2 distinct myeloma subgroups: Akt-dependent and Akt-independent multiple myeloma

Zöllinger, A. et al Blood, 112(8), 3403-3411 (2008)   Although the phosphatidylinositide 3-kinase (PI3K)/Akt pathway has been reported to contribute to the malignant growth of multiple myeloma (MM), the true relevance of Akt kinases for this disease is still unclear. In particular, functional analyses in primary tumor cells and genetic target validation experiments are missing. Here, we used combined functional and molecular analyses to determine the importance of Akt activity in a large panel of primary MM samples and in MM cell lines. Akt down-regulation with isoform-specific siRNA constructs or with an Akt1/2-specific pharmacologic inhibitor strongly induced apoptosis in approximately half of the primary MM samples analyzed. Sensitivity to Akt inhibition strongly correlated with the activation status of Akt as determined by immunohistochemistry, phospho-Akt–specific flow cytometry, and Western analysis. Additional blockade of the MAPK and the IL-6R/STAT3 pathways was often not sufficient to decrease the viability of MM cells resilient to Akt inhibition. Taken together, these experiments led to the identification of 2 myeloma subgroups: Akt-dependent and Akt-independent MM.  

4.621           Novel Role for Vascular Endothelial Growth Factor (VEGF) Receptor-1 and Its Ligand VEGF-B in Motor Neuron Degeneration

Poesen, K. et al

1. Neurosci., 28(42), 10451-10459 (2008)

  Although vascular endothelial growth factor-B (VEGF-B) is a homolog of the angiogenic factor VEGF, it has only minimal angiogenic activity, raising the question of whether this factor has other (more relevant) biological properties. Intrigued by the possibility that VEGF family members affect neuronal cells, we explored whether VEGF-B might have a role in the nervous system. Here, we document that the 60 kDa VEGF-B isoform, VEGF-B¹⁸⁶, is a neuroprotective factor. VEGF-B¹⁸⁶ protected cultured primary motor neurons against degeneration. Mice lacking VEGF-B also developed a more severe form of motor neuron degeneration when intercrossed with mutant SOD1 mice. The in vitro and in vivo effects of VEGF-B¹⁸⁶ were dependent on the tyrosine kinase activities of its receptor, Flt1, in motor neurons. When delivered intracerebroventricularly, VEGF-B¹⁸⁶ prolonged the survival of mutant SOD1 rats. Compared with a similar dose of VEGF, VEGF-B¹⁸⁶ was safer and did not cause vessel growth or blood–brain barrier leakiness. The neuroprotective activity of VEGF-B, in combination with its negligible angiogenic/permeability activity, offers attractive opportunities for the treatment of neurodegenerative diseases.  

4.622           Analysis of Donor- and Isolation-Related Variables From Non-Heart-Beating Donors (NHBDs) Using the Kyoto Islet Isolation Method

Liu, X. et al Cel Transplantation, 17, 649-656 (2008)   Recently, we demonstrated that islet transplantation from non-heart-beating donors (NHBDs) using the Kyoto islet isolation method (KIIM) successfully reversed patients' diabetes state. In this study, we evaluated the effects of donor- and isolation-related variables on islet isolation results from NHBDs by KIIM. Twenty-one islet preparations from the pancreata of NHBDs were isolated by KIIM. Islet preparations that met transplantation criteria and achieved improved patient diabetes control after transplantation were defined as successful isolations. Potential risk factors deemed to affect islet isolation results, such as age, gender, body mass index, hospital stay, donors' blood biochemical tests, a modified pancreata procurement method, and isolation and purification procedure-related variables, were analyzed. Seventeen out of 21 islet isolations (81%) were successful isolations. Postpurification islet yield was 447,639 ± 39,902 islet equivalents (IE) in the successful isolation group and 108,007 ± 31,532 IE in the failure group. Donor age was significantly younger in the success group (41.9 ± 4.0 years old in the success group vs. 57.5 ± 2.2 years old in the failure group, p = 0.003). Chronic pancreatitis significantly decreased islet yields (p = 0.006). Phase I time was significantly shorter (p = 0.010) and undigested tissue volume was significantly smaller (p = 0.020) in the success group. Purity was in positive correlation to postpurification islet yield, while donor age was in reverse correlation to postpurification islet yield. KIIM enables us to perform islet transplantation from NHBDs; however, the decision to use pancreata from older donors or those with chronic pancreatitis requires careful consideration.  

4.623           Ikaros Regulates Notch Target Gene Expression in Developing Thymocytes

Chari, S. and Winandy, S.

1. Immunol., 181, 6265-6274 (2008)

  Both Ikaros and Notch are essential for normal T cell development. Collaborative mutations causing a reduction in Ikaros activity and an increase in Notch activation promote T cell leukemogenesis. Although the molecular mechanisms of this cooperation have been studied, its consequences in thymocyte development remain unexplored. In this study, we show that Ikaros regulates expression of a subset of Notch target genes, including Hes1, Deltex1, pTa, Gata3, and Runx1, in both Ikaros null T cell leukemia lines and Ikaros null primary thymocytes. In Ikaros null leukemia cells, Notch deregulation occurs at both the level of Notch receptor cleavage and expression of Notch target genes, because re-expression of Ikaros in these cells down-regulates Notch target gene expression without affecting levels of intracellular cleaved Notch. In addition, abnormal expression of Notch target genes is observed in Ikaros null double-positive thymocytes, in the absence of detectable intracellular cleaved Notch. Finally, we show that this role of Ikaros is specific to double-positive and single-positive thymocytes because derepression of Notch target gene expression is not observed in Ikaros null double-negative thymocytes or lineage-depleted bone marrow. Thus, in this study, we provide evidence that Ikaros and Notch play opposing roles in regulation of a subset of Notch target genes and that this role is restricted to developing thymocytes where Ikaros is required to appropriately regulate the Notch program as they progress through T cell development.  

4.624           Effect of Interferon-Alpha, Ribavirin, Pentoxifylline, and Interleukin-18 Antibody on Hepatitis C Sera-Stimulated Hepatic Stellate Cell Proliferation

Khan, F., Peltekian, K.M. and Peterson, T.C.

1. Interferon & Cytokine Res., 28, 643-652 (2008)

  Chronic hepatitis C virus (HCV) infection is a major cause of liver fibrosis ultimately leading to cirrhosis. Hepatic stellate cell (HSC) proliferation is crucial in fibrosis development. Current antiviral treatment for HCV involves interferon-alpha (IFN-α) and Ribavirin combination therapy. IL-18, a novel cytokine of the IL-1 family of cytokines, is involved in inflammation and may be important in HCV-related inflammation. We hypothesize that block of one of the crucial events will block fibrosis due to HCV. The effect of HCV patient sera with and without IFN-α, ribavirin, and IL-18 antibody on HSC proliferation was assessed by [³H]-thymidine incorporation assays. Western analysis was used to assess the effect of pentoxifylline (PTX) on c-Jun immediate early gene phosphorylation (p-c-Jun formation). We demonstrate that HCV patient sera-stimulated HSC proliferation. Ribavirin with or without IFN-α significantly decreased HCV sera-stimulated HSC proliferation by 50%. Western analysis revealed that HCV serum increased p-c-Jun levels, which were decreased with Ribavirin and PTX. ELISA results showed an elevation of IL-18 levels in HCV sera when compared to normal sera. IL-18 did not stimulate HSC proliferation. However, IL-18 antibody significantly decreased patient sera-stimulated HSC proliferation. In conclusion, Ribavirin decreased HSC proliferation and may act by decreasing p-c-Jun levels in HSCs. IL-18 alone did not stimulate HSC proliferation but IL-18 antibody decreased stimulation, suggesting that IL-18 may work in conjunction with some other factor to increase HSC proliferation.  

4.625           Global transcriptional response to mammalian temperature provides new insight into Francisella tularensis pathogenesis

Horzempa, J., Carlson, P.E., O’Dee, D.N., Shank, R.M.Q. and Nau, G.J. BMC Microbiol., 8, 172-188 (2008)   Background After infecting a mammalian host, the facultative intracellular bacterium, Francisella tularensis, encounters an elevated environmental temperature. We hypothesized that this temperature change may regulate genes essential for infection. Results Microarray analysis of F. tularensis LVS shifted from 26°C (environmental) to 37°C (mammalian) showed ~11% of this bacterium's genes were differentially-regulated. Importantly, 40% of the protein-coding genes that were induced at 37°C have been previously implicated in virulence or intracellular growth of Francisella in other studies, associating the bacterial response to this temperature shift with pathogenesis. Forty-four percent of the genes induced at 37°C encode proteins of unknown function, suggesting novel Francisella virulence traits are regulated by mammalian temperature. To explore this possibility, we generated two mutants of loci induced at 37°C [FTL_1581 and FTL_1664 (deoB)]. The FTL_1581 mutant was attenuated in a chicken embryo infection model, which was likely attributable to a defect in survival within macrophages. FTL_1581 encodes a novel hypothetical protein that we suggest naming temperature-induced, virulence-associated locus A, tivA. Interestingly, the deoB mutant showed diminished entry into mammalian cells compared to wild-type LVS, including primary human macrophages and dendritic cells, the macrophage-like RAW 264.7 line, and non-phagocytic HEK-293 cells. This is the first study identifying a Francisella gene that contributes to uptake into both phagocytic and non-phagocytic host cells. Conclusion Our results provide new insight into mechanisms of Francisella virulence regulation and pathogenesis. F. tularensis LVS undergoes considerable gene expression changes in response to mammalian body temperature. This temperature shift is important for the regulation of genes that are critical for the pathogenesis of Francisella. Importantly, the compilation of temperature-regulated genes also defines a rich collection of novel candidate virulence determinants, including tivA (FTL_1581). An analysis of tivA and deoB (FTL_1664) revealed that these genes contribute to intracellular survival and entry into mammalian cells, respectively.  

4.626           Improved Islet Yields After Purification Following the Novel Endogenous Trypsin Inhibitor and Histidine-Tryptophan-Ketoglutarate Treatment in Pigs

Wee, Y.M. et al Transplant. Proceedings, 40, 2585-2587 (2008)   Background Adult porcine islet xenotransplantation into humans is greatly diminished by the difficulty to isolate islets because of their fragility. The goal of this study was to improve the efficacy of islet yields using endogenous trypsin inhibitor and histidine-tryptophan-ketoglutarate (HTK) perfusate. Method We compared two porcine islet isolation protocols: Eurocollins solution for in situ pancreas perfusion without use of an endogenous trypsin inhibitor versus HTK solution including endogenous trypsin inhibitor for pancreas perfusion. Results Endogenous trypsin inhibitor and HTK strategies significantly improved total islet yield, recovery, and islet index after purification (P < .05), whereas unpurified islet yield did not increase. An average of 228,000 ± 95,000 islet equivalents (IEQ) (n = 20) purified islets were obtained in the first group compared with 115,000 ± 56,000 IEQ (n = 18) in the second group. The average islet index was significantly increased in the first group compared with the second group before and after purification: before: 0.28 versus 0.49 versus after: 0.25 versus 0.4 (P < .05). At this time, islet purity, viability, and stimulation index did not show a significant difference between groups. Conclusion Our study showed that endogenous trypsin inhibitor and HTK strategies significantly improved purified islet isolation efficacy because of reduction of islet fragility.  

4.627           Essential roles of SHPS-1 in induction of contact hypersensitivity of skin

Motegi, S-I. et al Immunol. Lett., 121, 52-60 (2008)   SHPS-1 is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 and is abundant on the surface of CD11c⁺ dendritic cells (DCs). We recently showed that SHPS-1 is essential for priming by DCs of CD4⁺ T cells and for development of Th17 cell-mediated experimental autoimmunity. We have now further evaluated the importance of SHPS-1 and that of its ligand CD47 in contact hypersensitivity (CHS) to 2,4-dinitro-1-fluorobenzene (DNFB). Whereas the DNFB-induced CHS response was impaired in mice that express a mutant form of SHPS-1 lacking most of the cytoplasmic region, it was unaffected in CD47-deficient mice. Moreover, treatment of wild-type mice with mAbs to SHPS-1 that either block or do not block the binding of SHPS-1 to CD47 inhibited the CHS response. A mAb to CD47 had no such effect. The 2,4-dinitro-benzenesulfonic acid-induced proliferation of, and production of IFN-γ or IL-17 by, T cells from DNFB-sensitized wild-type mice were inhibited by either mAb to SHPS-1 but not by that to CD47. In contrast, the blocking mAbs to SHPS-1, but not that to CD47, inhibited an allogeneic mixed leukocyte reaction. Both mAbs to SHPS-1, but not that to CD47, also inhibited the lipopolysaccharide- or polyinosinic–polycytidylic acid-induced production of TNF-α by DCs. These results suggest that SHPS-1 is essential for development of CHS, likely as a result of its positive regulation of the priming by DCs of CD4⁺ T cells. However, such regulation by SHPS-1 does not appear to require its interaction with CD47.  

4.628           Nitric oxide and MCP-1 regulation in LPS activated rat Kupffer cells

Kolios, G. et al Mol. Cell. Biochem., 319, 91-98 (2008)   Nitric oxide (NO) and Monocyte Chemoattractant Protein (MCP)-1 co-regulation has been found in endotoxin-activated macrophages. Kupffer cells (KC) are a main source of soluble-mediators production in liver abnormalities. We investigated in vitro similar co-regulation of NO and MCP-1 production in rat activated KC. Isolated rat KC were cultured in the presence of 1 μg/ml LPS and various concentrations of Wortmannin (0–300 nM), L-NAME (0–500 μM) or MCP-1 (0–100 ng/ml). Production of MCP-1 and NO were measured in supernatants, by ELISA and a modification of the Griess reaction, respectively. Growth arrested KC, stimulated with vehicle, produced a basal amount of NO and MCP-1. In the presence of LPS, cultured KC secreted significantly (P < 0.01) increased amounts of MCP-1 and NO. Pre-treatment of KC with various concentrations of L-NAME significantly (P < 0.05) reduced the LPS-induced secretion of NO in a concentration dependent manner, but the MCP-1 production remained unaffected. Pre-treatment with Wortmannin significantly (P < 0.05) inhibited LPS-induced secretion of MCP-1 and NO in a concentration dependent manner. Linear regression analysis revealed a positive correlation between MCP-1 and NO in the LPS (r = 0.59171, P < 0.0001) and Wortmannin (r = 0.9215, P = 0.009) treated groups, but not in the L-NAME (r = −0.08513, P = 0.873). Incubation of KC with various concentrations of MCP-1 did not increase the NO production. These results indicate that KC might be the main source of NO and MCP-1 production in liver disorders, probably through the induction of PI3-kinase(s) and without any co-regulation between these molecules, which might represent two independent immunoregulatory pathways in the role of KC in hepatic disorders.  

4.629           Regulation of CCN2 mRNA expression and promoter activity in activated hepatic stellate cells

Leask, A., Chen, S., Pala, D and Brigstock, D.R.

1. Cell Commun. Signal., 2, 49-56 (2008)

  The matricellular protein connective tissue growth factor (CCN2) is considered a faithful marker of fibroblast activation in wound healing and in fibrosis. CCN2 is induced during activation of hepatic stellate cells (HSC). Here, we investigate the molecular basis of CCN2 gene expression in HSC. Fluoroscence activated cell sorting was used to investigate CCN2 expression in HSC in vivo in mice treated with CCl₄. CCN2 and TGF-β mRNA expression were assessed by polymerase chain reaction as a function of culture-induced activation of HSC. CCN2 promoter/reporter constructs were used to map cis-acting elements required for basal and TGFβ-induced CCN2 promoter activity. Real-time polymerase chain reaction analysis was used to further clarify signaling pathways required for CCN2 expression in HSC. CCl₄ administration in vivo increased CCN2 production by HSC. In vitro, expression of CCN2 and TGF-β mRNA were concommitantly increased in mouse HSC between days 0 and 14 of culture. TGFβ-induced CCN2 promoter activity required the Smad and Ets-1 elements in the CCN2 promoter and was reduced by TGFβ type I receptor (ALK4/5/7) inhibition. CCN2 overexpression in activated HSC was ALK4/5/7-dependent. As CCN2 overexpression is a faithful marker of fibrogenesis, our data are consistent with the notion that signaling through TGFβ type I receptors such as ALK5 contributes to the activation of HSC and hence ALK4/5/7 inhibition would be expected to be an appropriate treatment for liver fibrosis.  

4.630           Hydrogen Peroxide-Induced VCAM-1 Expression in Pancreatic Islets and [beta]-Cells Through Extracellular Ca2+ Influx

Lee, S. et al Transplantation, 86(9), 1257-1266 (2008)   Background. The use of porcine islets as alternatives to transplantable human islets is hampered by xenotransplant rejection. To identify molecular mechanisms that would allow subversion of xenoislet rejection, we investigated the role of H2O2 in vascular cell adhesion molecule-1 (VCAM-1) expression by porcine and mouse islets and [beta]-cell lines. Methods. Porcine islets were treated with H2O2, tumor necrosis factor alpha, interferon-[gamma], interleukin-1[beta], and lipopolysaccharide, to assess the effects of inflammatory stimulators on VCAM-1 expression using flow cytometry. The role of Ca2+ in H2O2-induced VCAM-1 expression was investigated in [beta]-cell lines using an extracellular Ca2+ chelator and Ca2+-depleted media. Furthermore, H2O2-induced VCAM-1 expression was measured in [beta]-cells, pretreated with inhibitors of protein kinase C, phospholipase D, and phosphatidylinositol-3 kinase/Akt. Finally, H2O2-induced VCAM-1 expression was evaluated in porcine islets and rodent [beta]-cell lines infected with an adenovirus encoding catalase, a H2O2-removing enzyme. Results. H2O2 was most potent inflammatory stimulator of VCAM-1 expression in porcine islets and had the greatest effect on VCAM-1 expression by [beta]-cells. Signaling pathway analysis demonstrated that extracellular Ca2+ influx was critical to H2O2-mediated VCAM-1 expression; however, protein kinase C, phospholipase D, and phosphatidylinositol-3 kinase/Akt activation were not required for VCAM-1 expression. Finally, catalase overexpression inhibited H2O2-induced VCAM-1 expression by islets and [beta]-cell lines. Conclusion. An extracellular calcium-dependent H2O2 pathway is the critical mediator of VCAM-1 expression by pancreatic islets and [beta]-cells. Inhibition of this pathway by catalase overexpression in donor islets can be exploited to protect against xenoislet immune responses.  

4.631           Ex vivo priming of CD4 T cells converts immunological tolerance into effective antitumor immunity in a murine model of acute lymphoblastic leukemia

Hegazy, A.N. and Klein, C. Leukemia, 22, 2070-2079 (2008)   Tumor escape mechanisms in leukemia are not well defined. To dissect immunological mechanisms responsible for immune tolerance toward leukemia, we established a murine model system allowing clonotypic analysis of leukemia-specific CD4 T cells recognizing ovalbumin (OVA). Upon i.v. injection of genetically engineered leukemia cells, dendritic cells (DCs) engulfed, processed and presented OVA to OVA-specific CD4 T cells. Consequently, leukemia-specific T cells were primed in vivo as shown by expression of activation markers and proliferative responses. However, in spite of detectable CD4 T cell responses in vitro and in vivo, no effective anti-leukemia immunity was established. In contrast, adoptively transferred DO11.10 T cells that were primed ex vivo mediated effective antitumor immunity. Furthermore, ex vivo primed DO11.10 T cells showed high expression of Th1 cytokines (interferon- , tumor necrosis factor- and interleukin-2) whereas in vivo primed OVA-specific CD4 T cells showed incomplete differentiation (proliferation without cytokine production). We conclude that activated T cells lacking effector function develop through incomplete differentiation in leukemia-bearing mice. Thus, priming conditions of leukemia-specific CD4 T cells critically determines the balance between immunity or tolerance toward leukemia.  

4.632           Expression of Neurexin, Neuroligin, and Their Cytoplasmic Binding Partners in the Pancreatic β-Cells and the Involvement of Neuroligin in Insulin Secretion

Suckow, A.T. et al Endocrinology, 149(12), 6006-6017 (2008)   The composition of the β-cell exocytic machinery is very similar to that of neuronal synapses, and the developmental pathway of β-cells and neurons substantially overlap. β-Cells secrete -aminobutyric acid and express proteins that, in the brain, are specific markers of inhibitory synapses. Recently, neuronal coculture experiments have identified three families of synaptic cell-surface molecules (neurexins, neuroligins, and SynCAM) that drive synapse formation in vitro and that control the differentiation of nascent synapses into either excitatory or inhibitory fully mature nerve terminals. The inhibitory synapse-like character of the β-cells led us to hypothesize that members of these families of synapse-inducing adhesion molecules would be expressed in β-cells and that the pattern of expression would resemble that associated with neuronal inhibitory synaptogenesis. Here, we describe β-cell expression of the neuroligins, neurexins, and SynCAM, and show that neuroligin expression affects insulin secretion in INS-1 β-cells and rat islet cells. Our findings demonstrate that neuroligins and neurexins are expressed outside the central nervous system and help confer an inhibitory synaptic-like phenotype onto the β-cell surface. Analogous to their role in synaptic neurotransmission, neurexin-neuroligin interactions may play a role in the formation of the submembrane insulin secretory apparatus.  

4.633           Semen Processing for the Subfertile Stallion

Varner, D.D. et al

1. Equine Vet. Sci., 28(11), 677-685 (2008)

  Stallions become sires based on three qualities: pedigree, performance record, and conformation. Conspicuously absent from this formula for sire status is that of reproductive health. Stallions represent 50% of the breeding equation, and the horse industry is replete with stallions whose level of fertility is undesirable. In natural-cover programs, physical, mental, or environmental aberrations can result in disruption of efficient semen transfer from the stallion to the reproductive tract of the mare. Some forms of subfertility may have a genetic basis, but subfertility is often associated with aging in stallions, and the attendant effects of age on testicular function. Effects of long-term medications, such as progestogens or anabolic steroids, on testicular health and fertility of younger sires that have recently retired from a performance career, must also be considered. Other environmental effects, such as hot environmental temperature, fever, and genital trauma can also induce a subfertile state in an otherwise fertile stallion. Taken together, these scenarios rationalize the need for veterinary intervention as a means to maximize the fertility of subfertile stallions. This communication contains an assortment of stallion cases in which various semen-processing methods were used in an effort to improve the fertility of subfertile stallions.      

4.634           The Effect of Isolation Methods and the Use of Different Enzymes on Islet Yield and In Vivo Function

Sabek, O.M., Cowan, P., Fraga, D.W. and Gaber, A.O. Cell Transplantation, 17, 785-792 (2008)   The ability to isolate high-yield pure and viable islets from human cadaver pancreas donors is dependent on donor factor as well as isolation factors. The aim of this study was to examine factors influencing islets recovery and in vivo function with an emphasis on donor and isolation methods as well as to compare the effectiveness of Liberase, widely used in clinical islet isolation, with Serva for the isolation of pure functional islets. The results of 123 islet isolations using Liberase for digestion were compared with those of 113 isolations with Serva. Islet equivalents per gram of tissue were similar between Liberase and Serva (3620 ± 1858 vs. 4132 ± 2104, p < 0.2) as well as the percent purity (75 ± 16 vs. 74 ± 15, p < 0.9). In vivo function of islets from 71 isolations (Liberase = 45, Serva = 26) were further tested by transplantation into NOD-SCID mice following short-term culture (<6 days, n = 71). Our data show that both Liberase- and Serva-isolated islets showed similar function results following short-term culture. These data demonstrate that there is no difference in islet yield, purity, and function between the two enzymes. However, when these 71 isolations were analyzed for in vivo function with emphasis on donor factors, cold ischemia time (12.0 ± 5.3 vs. 15.0 ± 5.7, p < 0.04), islet integrity (1.6 ± 0.7 vs. 1.3 ± 0.5, p < 0.05), and female gender were the only factors that correlated with in vivo function. We also compared the mechanical-shaking method for islets isolation with hand-shaking methods. Our results show that although there is no different in islet yield, purity, and integrity between different enzymes using the same method, hand-shaking method yields more islets with better integrity than mechanical-shaking method.  

4.635           Non-Cell-Autonomous Effect of Human SOD1G37R Astrocytes on Motor Neurons Derived from Human Embryonic Stem Cells

Marchetto, M.C.N. et al Cell Stem Cell, 3, 649-657 (2008)   Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron death. ALS can be induced by mutations in the superoxide dismutase 1 gene (SOD1). Evidence for the non-cell-autonomous nature of ALS emerged from the observation that wild-type glial cells extended the survival of SOD1 mutant motor neurons in chimeric mice. To uncover the contribution of astrocytes to human motor neuron degeneration, we cocultured hESC-derived motor neurons with human primary astrocytes expressing mutated SOD1. We detected a selective motor neuron toxicity that was correlated with increased inflammatory response in SOD1-mutated astrocytes. Furthermore, we present evidence that astrocytes can activate NOX2 to produce superoxide and that effect can be reversed by antioxidants. We show that NOX2 inhibitor, apocynin, can prevent the loss of motor neurons caused by SOD1-mutated astrocytes. These results provide an assay for drug screening using a human ALS in vitro astrocyte-based cell model.  

4.636           FUSION OF THE TETANUS TOXIN C FRAGMENT BINDING DOMAIN AND BCL-XL FOR PROTECTION OF PERIPHERAL NERVE NEURONS

Carlton, E. et al Neurosurgery, 63(6), 1175-1184 (2008)   OBJECTIVE: Apoptosis has been shown to play an important role in motor neuron (MN) degeneration in both neurodegenerative disease and peripheral neuropathy. Bcl-xL, an antiapoptotic protein, is down-regulated in these origins. The carboxyl-terminal domain of the tetanus toxin heavy chain (Hc) has high affinity for axon terminal binding and uptake into motor and dorsal root ganglion (DRG) neurons. We report the development of a fusion protein between Hc and Bcl-xL to enhance uptake of Bcl-xL by MNs as a strategy for inhibiting peripheral neuronal apoptosis. METHODS: The genes for Hc, Bcl-xL, and green fluorescent protein were cloned into an Escherichia coli expression system in 2 different arrangements. Fusion proteins were purified through chromatography. Cultured E15 rat spinal cord MNs and DRG cells were used to demonstrate neuron-specific uptake and retrograde transport of the fusion proteins mediated by Hc. Finally, glutamate-induced apoptosis was used as an in vitro model to measure the antiapoptotic effects of the fusion proteins. RESULTS: Bcl-xL fusion proteins were found to bind specifically and undergo uptake into cultured rat spinal MNs. The fusion proteins were also taken up by DRG axonal terminals and transported back to the cell bodies in Campenot compartmentalized chambers (Tyler Research Corp., Edmonton, Canada). Finally, fusion protein application improved cell survival and decreased apoptosis in glutamate-mediated excitotoxicity of the SH-SY5Y neuronal cells. CONCLUSION: Hc can be applied as a universal carrier for therapeutic cargo delivery specifically to MNs or DRGs. The fusion proteins between Bcl-xL and Hc constructed in this study might bear applications to the treatment of MN disease, neuropathy, or nerve injury through nerve or intramuscular injection.  

4.637           Elastin peptide receptor-directed monocyte chemotactic polysaccharides derived from seaweed sporophyll and from infectious fungus

Li,Y. et al Microbial Pathogenesis, 45, 423-434 (2008)   We discovered that a seaweed sporophyll-derived polysaccharide of brown alga, Wakame (Undaria pinnatifida) bound to monocytes and attracted them in vitro and in vivo. Physicochemical properties, affinity to a lectin-bead column and sugar composition of the chemotactic polysaccharide indicated this molecule to be a highly sulfated fucogalactan. We then identified the monocyte receptor of the sulfated fucogalactan as the elastin peptide receptor by prophylactic inhibition of the binding and the chemoattraction with lactose and the synthetic elastin peptide, Val-Gly-Val-Ala-Pro-Gly. We assume that the galactose-binding lectin, which is a component of the elastin peptide receptor complex, would recognize a Gal residue of the sulfated fucogalactan. We also observed a similar chemoattracting polysaccharide in a pathogenic fungus, Candida albicans, although the content of it was much lower than in the case of seaweed sporophyll. We speculate that the chemotactic response of monocytes to the sulfated fucogalactan is part of the innate immune system to fungal infection.  

4.638           Nrf2 Activation in Astrocytes Protects against Neurodegeneration in Mouse Models of Familial Amyotrophic Lateral Sclerosis

Vargas, M.R., Johnson, D.A., Sirkis, D.W., Messing, A. and Johnson, J.A.

1. Neurosci., 28(50), 13574-13581 (2008)

  Activation of the transcription factor Nrf2 in astrocytes coordinates the upregulation of antioxidant defenses and confers protection to neighboring neurons. Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. Non-neuronal cells, including astrocytes, shape motor neuron survival in ALS and are a potential target to prevent motor neuron degeneration. The protective effect of Nrf2 activation in astrocytes has never been examined in a chronic model of neurodegeneration. We generated transgenic mice over-expressing Nrf2 selectively in astrocytes using the glial fibrillary acidic protein (GFAP) promoter. The toxicity of astrocytes expressing ALS-linked mutant hSOD1 to cocultured motor neurons was reversed by Nrf2 over-expression. Motor neuron protection depended on increased glutathione secretion from astrocytes. This protective effect was also observed by crossing the GFAP-Nrf2 mice with two ALS-mouse models. Over-expression of Nrf2 in astrocytes significantly delayed onset and extended survival. These findings demonstrate that Nrf2 activation in astrocytes is a viable therapeutic target to prevent chronic neurodegeneration.  

4.639           Deficiency in Complement C1q Improves Histological and Functional Locomotor Outcome after Spinal Cord Injury

Galvan, M.D., Luchetti, S., Burgos, A.M., Nguyen, H.X., Hooshmand, M.J., Hamers, F.P.T. and Anderson, A.J.

1. Neurosci., 28(51), 13876-13888 (2008)

  Although studies have suggested a role for the complement system in the pathophysiology of spinal cord injury (SCI), that role remains poorly defined. Additionally, the relative contribution of individual complement pathways in SCI is unknown. Our initial studies revealed that systemic complement activation was strongly influenced by genetic background and gender. Thus, to investigate the role of the classical complement pathway in contusion-induced SCI, male C1q knock-out (KO) and wild-type (WT) mice on a complement sufficient background (BUB) received a mild-moderate T9 contusion injury with the Infinite Horizon impactor. BUB C1q KO mice exhibited greater locomotor recovery compared with BUB WT mice (p < 0.05). Improved recovery observed in BUB C1q KO mice was also associated with decreased threshold for withdrawal from a mild stimulus using von Frey filament testing. Surprisingly, quantification of microglia/macrophages (F4/80) by FACS analysis showed that BUB C1q KO mice exhibited a significantly greater percentage of macrophages in the spinal cord compared with BUB WT mice 3 d post-injury (p < 0.05). However, this increased macrophage response appeared to be transient as stereological assessment of spinal cord tissue obtained 28 d post-injury revealed no difference in F4/80-positive cells between groups. Stereological assessment of spinal cord tissue showed that BUB C1q KO mice had reduced lesion volume and an increase in tissue sparing compared with BUB WT mice (p < 0.05). Together, these data suggest that initiation of the classical complement pathway via C1q is detrimental to recovery after SCI.  

4.640           Innate and Adaptive Interleukin-22 Protects Mice from Inflammatory Bowel Disease

Zenewicz, L.A., Yancopoulos, G.D., Valenzuela, D.M., Murphy, A.J., Stevens, S. and Flavell, R.A. Immunity, 29, 947-957 (2008)   Inflammatory bowel disease (IBD) is a chronic inflammatory disease thought to be mediated by dysfunctional innate and/or adaptive immunity. This aberrant immune response leads to the secretion of harmful cytokines that destroy the epithelium of the gastrointestinal tract and thus cause further inflammation. Interleukin-22 (IL-22) is a T helper 17 (Th17) T cell-associated cytokine that is bifunctional in that it has both proinflammatory and protective effects on tissues depending on the inflammatory context. We show herein that IL-22 protected mice from IBD. Interestingly, not only was this protection mediated by CD4⁺ T cells, but IL-22-expressing natural killer (NK) cells also conferred protection. In addition, IL-22 expression was differentially regulated between NK cell subsets. Thus, both the innate and adaptive immune responses have developed protective mechanisms to counteract the damaging effects of inflammation on tissues.  

4.641           Efficient tumour formation by single human melanoma cells

Quintana, E., Shackleton, M., Sabel, M.S., Fullen, D.R., Johnson, T.M. and Morrison, S.J. Nature, 456, 593-599 (2008)   A fundamental question in cancer biology is whether cells with tumorigenic potential are common or rare within human cancers. Studies on diverse cancers, including melanoma, have indicated that only rare human cancer cells (0.1–0.0001%) form tumours when transplanted into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. However, the extent to which NOD/SCID mice underestimate the frequency of tumorigenic human cancer cells has been uncertain. Here we show that modified xenotransplantation assay conditions, including the use of more highly immunocompromised NOD/SCID interleukin-2 receptor gamma chain null (Il2rg ^-/-) mice, can increase the detection of tumorigenic melanoma cells by several orders of magnitude. In limiting dilution assays, approximately 25% of unselected melanoma cells from 12 different patients, including cells from primary and metastatic melanomas obtained directly from patients, formed tumours under these more permissive conditions. In single-cell transplants, an average of 27% of unselected melanoma cells from four different patients formed tumours. Modifications to xenotransplantation assays can therefore dramatically increase the detectable frequency of tumorigenic cells, demonstrating that they are common in some human cancers.    

4.642           Identification of adult hepatic progenitor cells capable of repopulating injured rat liver

Yovchev, M:I., Grozdanov, P.N., Zhou, H., Racherla, H., Guha, C. and Dabeva, M.D. Hepatology, 47(2), 636-647 (2008)   Oval cells appear and expand in the liver when hepatocyte proliferation is compromised. Many different markers have been attributed to these cells, but their nature still remains obscure. This study is a detailed gene expression analysis aimed at revealing their identity and repopulating in vivo capacity. Oval cells were activated in 2-acetylaminofluorene–treated rats subjected to partial hepatectomy or in D-galactosamine–treated rats. Two surface markers [epithelial cell adhesion molecule (EpCAM) and thymus cell antigen 1 (Thy-1)] were used for purification of freshly isolated cells. Their gene expression analysis was studied with Affymetrix Rat Expression Array 230 2.0, reverse-transcriptase polymerase chain reaction, and immunofluorescent microscopy. We found that EpCAM⁺ and Thy-1⁺ cells represent two different populations of cells in the oval cell niche. EpCAM⁺ cells express the classical oval cell markers (alpha-fetoprotein, cytokeratin-19, OV-1 antigen, a6 integrin, and connexin 43), cell surface markers recently identified by us (CD44, CD24, EpCAM, aquaporin 5, claudin-4, secretin receptor, claudin-7, V-ros sarcoma virus oncogene homolog 1, cadherin 22, mucin-1, and CD133), and liver-enriched transcription factors (forkhead box q, forkhead box a2, onecut 1, and transcription factor 2). Oval cells do not express previously reported hematopoietic stem cell markers Thy-1, c-kit, and CD34 or the neuroepithelial marker neural cell adhesion molecule 1. However, oval cells express a number of mesenchymal markers including vimentin, mesothelin, bone morphogenetic protein 7, and Tweak receptor (tumor necrosis factor receptor superfamily, member 12A). A group of novel differentially expressed oval cell genes is also presented. It is shown that Thy-1⁺ cells are mesenchymal cells with characteristics of myofibroblasts/activated stellate cells. Transplantation experiments reveal that EpCAM⁺ cells are true progenitors capable of repopulating injured rat liver. Conclusion: We have shown that EpCAM⁺ oval cells are bipotential adult hepatic epithelial progenitors. These cells display a mixed epithelial/mesenchymal phenotype that has not been recognized previously. They are valuable candidates for liver cell therapy.  

4.643           Dendritic cells are required for effective cross-presentation in the murine liver

Plitas, G., Burt, B.M., Stableford, J.A., Nguyen, H.M., Welles, A.P. and DeMatteo, R.P. Hepatology, 47(4), 1343-1351 (2008)   The liver harbors a diversity of cell types that have been reported to stimulate T cells. Although most hepatic dendritic cells are immature, a small population of CD11chigh conventional dendritic cells (cDCs) exists that expresses high levels of costimulatory molecules. We sought to determine the relative contribution of cDCs to cross-presentation by the liver. In vitro, liver nonparenchymal cells (NPCs) depleted of cDCs induced only minimal proliferation and activation of antigen-specific CD8+ T cells when loaded with soluble protein antigen. Using a transgenic mouse with the CD11c promoter driving expression of the human diphtheria toxin receptor, we found that selective depletion of cDCs in vivo reduced the number and activation of antigen-specific CD8+ T cells in the liver after intravenous administration of soluble protein antigen. Adoptive transfer of DCs, but not CD40 stimulation, restored the hepatic T-cell response. Conclusion: Our findings indicate that the ability of the liver to effectively cross-present soluble protein to antigen-specific CD8+ T cells depends primarily on cDCs. Despite costimulation, other resident liver antigen-presenting cells cannot compensate for the absence of cDCs.  

4.644           Systemic transmigration of allosensitizing donor dendritic cells to host secondary lymphoid organs after rat liver transplantation

Ueta, H., Shi, C., Miyanari, N., Xu, X-D., Zhou, S., Yamashita, M., Ezaki, T. and Matsuno, K. Hepatology, 47(4), 1352-1362 (2008)   Donor dendritic cell (DC) migration and allosensitization in host secondary lymphoid organs after liver transplantation are ill defined. We used rat models to investigate graft-derived cells and intrahost allosensitization. Liver transplantation induced diffuse blood-borne migration of donor major histocompatibility class II antigen–positive (MHCII+) cells and MHCI+ cells from the graft to host secondary lymphoid organs, not only the spleen, but also lymph nodes and Peyer's patches. The migrated MHCII+ cells included DCs and some T cells and B cells. The DCs formed clusters with host BrdU+ cells where they up-regulated CD86+, and a CD8+ T cell proliferative response originated within 24 hours after liver transplantation, demonstrating that these DCs can quickly mature and trigger direct allosensitization in host lymphoid organs. Transfer of allogeneic bone marrow cells also induced DC transmigration and a similar host response. In contrast, allogeneic thoracic duct lymph cells contained many fewer transmigrating DCs, and their transfer induced a comparable T cell response but significantly weaker CD8+ T cell proliferation. Thus, there is a different outcome via the indirect pathway by host DCs that have captured donor alloantigens. Conclusion: The rat liver as well as bone marrow contains an immature DC population that can systemically transmigrate through blood vessel walls of the host secondary lymphoid organs, quickly mature, and induce diffuse intrahost CD8+ T cell responses, which may promote graft rejection.  

4.645           Sinusoidal endothelial cells prevent rat stellate cell activation and promote reversion to quiescence

DeLeve, L.D., Wang, X. and Guo, Y. Hepatology, 48(3), 920-930 (2008)   Capillarization precedes hepatic fibrosis. We hypothesize that capillarization of sinusoidal endothelial cells (SEC) is permissive for hepatic stellate cell (HSC) activation and therefore permissive for fibrosis. We examined whether freshly isolated SECs prevent activation of HSCs and promote reversion to quiescence, and whether this effect was lost in capillarization. HSCs were cultured alone or co-cultured with differentiated or capillarized SECs. Results: Co-culture with freshly isolated SECs markedly decreased HSC activation after 3 days in culture, but co-culture with capillarized SEC had no effect. Inhibition of nitric oxide (NO) synthesis abolished SEC suppression of HSC activation. Activated HSCs reverted to quiescence when co-cultured with SEC plus vascular endothelial growth factor (VEGF) (that is, with SECs that maintained differentiation), but co-culture with capillarized SECs did not. Reversion of activated HSCs to quiescence in the presence of SECs plus VEGF was abolished by inhibition of NO synthesis. To establish whether there was indeed reversion, activated and quiescent HSCs were counted before and 3 days after adding freshly isolated SECs plus VEGF to activated HSCs, and proliferation was quantified in quiescent HSCs; the stoichiometry demonstrated reversion. Conclusion: Differentiated SECs prevent HSC activation and promote reversion of activated HSCs to quiescence through VEGF-stimulated NO production. Capillarized SECs do not promote HSC quiescence, because of loss of VEGF-stimulated NO production.  

4.646           Age-related changes to tumor necrosis factor receptors affect neuron survival in the presence of beta-amyloid

Patel, J.R. and Brewer, G.J.

1. Neurosci. Res., 86(10), 2303-2313 (2008)

  Inflammation including local accumulations of tumor necrosis factor alpha (TNF- ) is a part of Alzheimer's disease pathology and may exacerbate age-related neurodegeneration. Most studies on TNF- and TNF neuronal receptors are conducted by using embryonic neurons. Few studies consider age-related deficits that may occur in neurons. Age-related changes in susceptibility to TNF- through TNF receptor 1 (TNFR1) and receptor 2 (TNFR2) expression could increase susceptibility to β-amyloid (1–42, Aβ42). Evidence is conflicting about which receptor mediates survival and/or apoptosis. We determined how aging affects receptor expression in cultured adult rat cortical neurons. Old neurons were more susceptible to Aβ42 toxicity than middle-aged neurons, and the addition of TNF- was neuroprotective in middle-aged neurons, but exacerbated the toxicity from Aβ42 in old neurons. These pathologic and protective responses in old and middle-aged neurons, respectively, correlated with higher starting TNFR1 and TNFR2 mRNA levels in old vs. middle-aged neurons. Middle-aged neurons treated with TNF- plus Aβ42 did not show an increase in either TNFR1 or TNFR2 mRNA, but old neurons showed an up-regulation in TNFR2 mRNA and not TNFR1 mRNA. Despite these mRNA changes, surface immunoreactivity of both TNFR1 and TNFR2 increased with the dose of TNF- in middle-aged neurons. However, middle-aged neurons treated with TNF- plus Aβ42 showed an up-regulation in both TNFR1 and TNFR2 surface expression, whereas old neurons failed to up-regulate surface expression of either receptor. These findings support the hypothesis that age-related changes in TNF- surface receptor expression contribute to the neuronal loss associated with inflammation in Alzheimer's disease.  

4.647           Age-related decreases in NAD(P)H and glutathione cause redox declines before ATP loss during glutamate treatment of hippocampal neurons

Parihar, M.S., Kunz, E.A. and Brewer, G.J.

1. Neurosci. Res., 86(10), 2339-2352 (2008)

  Age-related glutamate excitotoxicity depends in an unknown manner on active mitochondria, which are key determinants of the cellular redox potential. Compared with embryonic and middle-aged neurons, old-aged rat hippocampal neurons have a lower resting reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and a lower redox ratio (NAD(P)H/flavin adenine nucleotide). Glutamate treatment resulted in an initial increase in NAD(P)H concentrations in all ages, followed by a profound calcium-dependent, age-related decline in NAD(P)H concentration and redox ratio. With complex I of the electron transport chain inhibited by rotenone, treatment with glutamate or ionomycin only resulted in the increase in NAD(P)H fluorescence. High-performance liquid chromatography analysis of adenine nucleotides in brain extracts showed 50% less nicotinamide adenine dinucleotide (NADH) and almost twice as much oxidized nicotinamide adenine dinucleotide, demonstrating a more oxidized ratio in old than middle-aged brain. Resting glutathione content also declined with age and further decreased with glutamate treatment without accompanying changes in adenosine triphosphate levels. We conclude that age does not affect production of NADH by dehydrogenases but that old-aged neurons consume more NADH and glutathione, leading to a catastrophic decline in redox ratio.  

4.648           Phylum Tardigrada: an “individual” approach

Sands, C.J., McInnes, S.J., Marley, N.J., Goodall-Copestake, W.P., Convey, P. and Linse, K. Cladistics, 24(6), 861-871 (2008)   Phylum Tardigrada consists of ∼1000 tiny, hardy metazoan species distributed throughout terrestrial, limno-terrestrial and oceanic habitats. Their phylogenetic status has been debated, with current evidence placing them in the Ecdysozoa. Although there have been efforts to explore tardigrade phylogeny using both morphological and molecular data, limitations such as their few morphological characters and low genomic DNA concentrations have resulted in restricted taxonomic coverage. Using a protocol that allows us to identify and extract DNA from individuals, we have sequenced 18S rDNA from 343 tardigrades from across the globe. Using maximum parsimony and Bayesian analyses we have found support for dividing Order Parachela into three super-families and further evidence that indicates the traditional taxonomic perspective of families in the class Eutardigrada are nonmonophyletic and require re-working. It appears that conserved morphology within Tardigrada has resulted in conservative taxonomy as we have found cases of several discrete lineages grouped into single genera. Although this work substantially adds to the understanding of the evolution and taxonomy of the phylum, we highlight that inferences gained from this work are likely to be refined with the inclusion of further taxa—specifically representatives of the nine families yet to be sampled.  

4.649           Exogenous Hsc70, but not thermal preconditioning, confers protection to motoneurons subjected to oxidative stress

Robinson, M.B., Taylor, A.R., Gifondorwa, D.J., Tytell, M. and Milligan, C.E. Develop. Neurobiol., 68(1), 1-17 (2008)   Proper sensing of stress and the initiation of the stress response are critical to maintaining cell viability in response to noxious stimuli. Induction of the stress response prior to the exposure of a lethal stress (preconditioning) can be protective. Heat shock proteins (Hsps), the main products of the stress response, are considered to be responsible for this protective effect. Most cells readily initiate a stress response, but some neuronal phenotypes, including motoneurons (MNs), have a diminished capacity to do so. We have found that, given a proper stimulus, MNs can execute a heat stress response; but, it does not protect them from death caused by hydrogen peroxide (H2O2) induced oxidative stress, despite inhibiting H2O2-induced caspase activation. Conversely, we demonstrate that incubation with the heat shock cognate 70 (Hsc70) protein prior to oxidative insult can protect MNs from oxidative stress. This survival promoting effect may be mediated through the substrate binding domain (SBD) of Hsc70. Our data suggest that stress preconditioning may not be beneficial to MNs, but that pharmacological interventions and alternative means of acquiring components of the stress response are an effective means of ameliorating lethal stress in MNs and may be potentially useful therapeutically in preventing pathological MN loss.  

4.650           Gene expression fluctuations in murine hematopoietic stem cells with cell cycle progression

Dooner, G.J., Colvin, G.A., Dooner, M.S., Johnson, K.W. and Quesenberry, P.J.

1. Cell. Physiol., 214(3), 786-795 (2008)

  Evolving data suggest that marrow hematopoietic stem cells show reversible changes in homing, engraftment, and differentiation phenotype with cell cycle progression. Furthermore, marrow stem cells are a cycling population. Traditional concepts hold that the system is hierarchical, but the information on the lability of phenotype with cycle progression suggests a model in which stem cells are on a reversible continuum. Here we have investigated mRNA expression in murine lineage negative stem cell antigen-1 positive stem cells of a variety of cell surface epitopes and transcription regulators associated with stem cell identity or regulation. At isolation these stem cells expressed almost all cell surface markers, and transcription factors studied, including receptors for G-CSF, GM-CSF, and IL-7. When these stem cells were induced to transit cell cycle in vitro by exposure to interleukin-3 (IL-3), Il-6, IL-11, and steel factor some (CD34, CD45R c-kit, Gata-1, Gata-2, Ikaros, and Fog) showed stable expression over time, despite previously documented alterations in phenotype, while others showed variation of expression between and within experiments. These latter included Sca-1, Mac-1, c-fms, and c-mpl. Tal-1, endoglin, and CD4. These studies indicate that defined marrow stem cells express a wide variety of genes at isolation and with cytokine induced cell cycle transit show marked and reversible phenotype lability. Altogether, the phenotypic plasticity of gene expression for murine stem cells indicates a continuum model of stem cell regulation and extends the model to reversible expression with cell cycle transit of mRNA for cytokine receptors and stem cell markers.  

4.651           Prolonged Insulin Independence After Islet Allotransplants in Recipients with Type 1 Diabetes

Bellin, M.D., Kandaswamy, R., Parkey, J., Zhang, H-J., Liu, B., Ihm, S.H., Ansite, J.D., Witson, J., Bansal-Pakala, P., Balamurugan, A.N., Papas, K., Sutherland, D.E.R., Moran, A. and Hering, B.J. Am. J. Transplant., 8(11), 2463-2470 (2008)   We sought to determine the long-term outcomes in type 1 diabetic recipients of intraportal alloislet transplants on a modified immunosuppressive protocol. Six recipients with hypoglycemia unawareness received one to two islet infusions. Induction therapy was with antithymocyte globulin (ATG) plus etanercept for tumor necrosis factor- blockade. Recipients received cyclosporine and everolimus for maintenance immunosuppression for the first year posttransplant, with mycophenolic acid or mycophenolate mofetil subsequently substituted for everolimus. Recipients have been followed for 1173 ± 270 days since their last infusion for islet graft function (insulin independence, hemoglobin A_1c levels and C-peptide production) and for adverse events associated with the study protocol. Of the six recipients, five were insulin-independent at 1 year, and four continue to be insulin-independent at a mean of 3.4 ± 0.4 years posttransplant. None of the six recipients experienced recurrence of severe hypoglycemia. Measured glomerular filtration rate decreased from 110.5 ± 21.2 mL/min/1.73 m² pretransplant to 82.6 ±19.1 mL/min/1.73 m² at 1 year posttransplant. In conclusion, islet transplants restored insulin independence for a mean of >3 years in four of six recipients treated with ATG and etanercept induction therapy and with cyclosporine and, initially, everolimus for maintenance. Our results suggest this immunosuppressive protocol may allow long-term graft survival.  

4.652           CD40L-expressing CD8 T cells prime CD8 ⁺ DC for IL-12p70 production

Wong, K.L., Lew, F.C., MacAry, P.A. and Kemeny, D.M. Eur. J. Immunol., 38(8), 2251-2262 (2008)   CD8 ⁺ DC are implicated as the principle DC subset for cross-presentation and cross-priming of cytotoxic CD8 T cell responses. In this study, we demonstrate another unique facet of the CD8 ⁺ DC and CD8 T cell relationship, by showing that CD8 T cells reciprocally activate CD8 ⁺ DC, but not CD8 ^– DC, for IL-12p70 production, the key Th1-promoting cytokine. This effect was observed during an antigen-specific interaction between DC and activated CD8 T cells, along with secondary TLR stimulation of DC by LPS. Activated CD8 T cells use a combination of IFN- and CD40L, which is rapidly up-regulated post-stimulation, to prime DC for IL-12p70 production during an antigen-specific response. Our results suggest that the interaction between CD8 ⁺ DC and antigen-primed CD8 T cells may form an important component of Th1-mediated immunity through the induction of IL-12p70.  

4.653           A new in vitro model of the glial scar inhibits axon growth

Wanner, I.B., Deik, A., Torres, M., Rosendahl, A., Neary, J.T., Lemmon, V.P. and Bixby, J.L. GLIA, 56(15), 1691-1709 (2008)   Astrocytes respond to central nervous system (CNS) injury with reactive astrogliosis and participate in the formation of the glial scar, an inhibitory barrier for axonal regeneration. Little is known about the injury-induced mechanisms underlying astrocyte reactivity and subsequent development of an axon-inhibitory scar. We combined two key aspects of CNS injury, mechanical trauma and co-culture with meningeal cells, to produce an in vitro model of the scar from cultures of highly differentiated astrocytes. Our model displayed widespread morphological signs of astrocyte reactivity, increases in expression of glial fibrillary acidic protein (GFAP), and accumulation of GFAP in astrocytic processes. Expression levels of scar-associated markers, phosphacan, neurocan, and tenascins, were also increased. Importantly, neurite growth from various CNS neuronal populations was significantly reduced when neurons were seeded on the scar-like cultures, compared with growth on cultures of mature astrocytes. Quantification of neurite growth parameters on the scar model demonstrated significant reductions in neuronal adhesion and neurite lengths. Interestingly, neurite outgrowth of postnatal neurons was reduced to a greater extent than that of embryonic neurons, and outgrowth inhibition varied among neuronal populations. Scar-like reactive sites and neurite-inhibitory patches were found throughout these cultures, creating a patchwork of growth-inhibitory areas mimicking a CNS injury site. Thus, our model showed relevant aspects of scar formation and produced widespread inhibition of axonal regeneration; it should be useful both for examining mechanisms underlying scar formation and to assess various treatments for their potential to improve regeneration after CNS injury  

4.654           Differential regulation of sphingomyelin synthesis and catabolism in oligodendrocytes and neurons

Kilkus, J.P., Goswami, R., Dawson, S.A., Testai, F.D., Berdyshev, E.V., Han, X. and dawson, G.

1. Neurochem., 106(4), 1745-1757 (2008)

  Neurons (both primary cultures of 3-day rat hippocampal neurons and embryonic chick neurons) rapidly converted exogenous NBD-sphingomyelin (SM) to NBD-Cer but only slowly converted NBD-Cer to NBD-SM. This was confirmed by demonstrating low in vitro sphingomyelin synthase (SMS) and high sphingomyelinase (SMase) activity in neurons. Similar results were observed in a human neuroblastoma cell line (LA-N-5). In contrast, primary cultures of 3-day-old rat oligodendrocytes only slowly converted NBD-SM to NBD-Cer but rapidly converted NBD-Cer to NBD-SM. This difference was confirmed by high in vitro SMS and low SMase activity in neonatal rat oligodendrocytes. Similar results were observed in a human oligodendroglioma cell line. Mass-Spectrometric analyses confirmed that neurons had a low SM/Cer ratio of (1.5 : 1) whereas oligodendroglia had a high SM/Cer ratio (9 : 1). Differences were also confirmed by [³H]palmitate-labeling of ceramide, which was higher in neurons compared with oligodendrocytes. Stable transfection of human oligodendroglioma cells with neutral SMase, which enhanced the conversion of NBD-SM to NBD-Cer and increased cell death, whereas transfection with SMS1 or SMS2 enhanced conversion of NBD-Cer to NBD-SM and was somewhat protective against cell death. Thus, SMS rather than SMases may be more important for sphingolipid homeostasis in oligodendrocytes, whereas the reverse may be true for neurons.  

4.655           Graft-versus-Host Disease Prevents the Maturation of Plasmacytoid Dendritic Cells

Banovic, T., Markey, K.A., Kuns, R.D., Olver, S.D., Raffelt, N.C., Don, A.L., Degli-Esposti, M.A., Engwerda, C.R., MacDonald, K.P.A. and Hill, G.R.

1. Immunol., 182, 912-920 (2009)

  The role of Ag presenting cell subsets in graft-versus-host disease (GVHD) remains unclear. We have thus examined the ability of plasmacytoid dendritic cells (pDC) to modulate transplant outcome. Surprisingly, host pDC were exquisitely sensitive to total body irradiation and were depleted before transplantation, thus allowing us to focus on donor pDC. The depletion of all pDC from bone marrow grafts resulted in an acceleration of GVHD mortality while the depletion of mature pDC from G-CSF mobilized splenic grafts had no effect. Thus, donor bone marrow pDC, but not mature pDC contained within stem cell grafts attenuate acute GVHD. In the presence of GVHD, donor pDC completely failed to reconstitute although a CD11c^low120G8⁺ precursor DC reconstituted in an exaggerated and transient manner. These cells expressed Flt-3, the macrophage colony stimulating factor receptor and, consistent with a common dendritic cell (DC) precursor, were capable of differentiation into pDC and conventional DC in vivo in the absence of GVHD. These precursors were MHC class II⁺ and CD80/86⁺ but lacked CD40, were actively presenting host Ag and inhibited GVHD and T cell proliferation in a contact-dependent fashion. These data demonstrate that GVHD prevents the maturation of pDC and instead promotes the generation of a suppressive precursor DC, further contributing to the state of immune paralysis after transplantation.  

4.656           Type I IFN-Mediated Protection of Macrophages and Dendritic Cells Secures Control of Murine Coronavirus Infection

Cervantes-Barragan, L., Kalinke, U., Züst, R., König, M., Reizis, B., Lopez-Macias, C., Thiel, V. And Ludewig, B.

1. Immunol., 182, 1099-1106 (2009)

  The swift production of type I IFNs is one of the fundamental aspects of innate immune responses against viruses. Plasmacytoid dendritic cell-derived type I IFNs are of prime importance for the initial control of highly cytopathic viruses such as the mouse hepatitis virus (MHV). The aim of this study was to determine the major target cell populations of this first wave of type I IFNs. Generation of bone marrow-chimeric mice expressing the type I IFN receptor (IFNAR) on either hemopoietic or non-bone marrow-derived cells revealed that the early control of MHV depended mainly on IFNAR expression on hemopoietic cells. To establish which cell population responds most efficiently to type I IFNs, mice conditionally deficient for the IFNAR on different leukocyte subsets were infected with MHV. This genetic analysis revealed that IFNAR expression on LysM⁺ macrophages and CD11c⁺ dendritic cells was most important for the early containment of MHV within secondary lymphoid organs and to prevent lethal liver disease. This study identifies type I IFN-mediated cross-talk between plasmacytoid dendritic cells on one side and macrophages and conventional dendritic cells on the other, as an essential cellular pathway for the control of fatal cytopathic virus infection.  

4.657           A fluorescence-based assay for measuring the viable cell concentration of mixed microbial communities in soil

Pascaud, A., Amellal, S., Soulas, M-L. and Soulas, G.

1. Microbiol. Methods, 76(1), 81-87 (2009)

  Microbial cell concentration is a particularly important bioindicator of soil health and a yardstick for determining biological quotients which are likely to gain in ecological significance if they are calculated in relation to the viable, rather than total, microbial density. A dual-staining technique with fluorescent dyes was used for the spectrofluorimetric quantitative determination of the concentration of viable microbial cells present in three different soil types. This is a novel and substantially modified application of the dual-staining procedure implemented in the LIVE/DEAD™ BacLight® viability kit which has never been successfully applied to the quantification of naturally occurring soil microbial communities. Indigenous microbial cell concentrations were quantified using an internal standard, i.e. spiking environmental samples with suspensions containing different concentrations of live E. coli cells, and external calibration, by comparing fluorescence emission by indigenous bacteria and known concentrations of E. coli in nutrient saline. Two types of environmental samples were tested: bacterial preparations obtained by density gradient centrifugation and soil suspensions. In both cases, prior dilution of the sample was necessary to minimise fluorescence quenching by soil particulate matter. Spectrofluorimetric measurements of indigenous cell concentration in bacterial preparations were in close agreement with those found using epifluorescence microscopy. Limits of detection of 5 × 10⁶ for the soil bacterial preparations and 8 × 10⁷ for the soil suspensions were estimated. Deviations observed when soil suspensions are dealt with are likely due to the selection of a unique bacterial strain for standardisation and calibration. Thorough testing of a variety of reference bacteria and fungi is suggested to determine a more accurate average fluorescence enhancement per microbial cell or mass unit.  

4.658           Differential expression of Prnp and Sprn in scrapie infected sheep also reveals Prnp genotype specific differences

Gossner, A.G., Bennet, N., Hunter, N. And Hopkins, J. Biochem. Biophys. Res. Comm., 378(4), 862-866 (2009)   The central role for PrP in the pathogenesis of the transmissible spongiform encephalopathies (TSEs) is illustrated by the resistance of Prnp^0/0 mice to disease and by the inverse association of Prnp gene dosage with incubation period. Understanding the role of PrP^C in TSEs necessitates knowledge of expression levels of the Prnp gene during the development of disease. SSBP/1 scrapie shows a defined pattern of disease progression and here we show that Prnp and shadow of PrP (Sprn) are differentially expressed in different brain areas and lymphoid tissues. Counter-intuitively we found that there is no positive correlation between expression of Prnp or Sprn and patterns of disease progression. Prnp and Sprn expression levels are both influenced by Prnp genotype; although the scrapie-sensitive VRQ/VRQ sheep did not express the highest level of either. In addition, infection with SSBP/1 scrapie seems to have little effect on either PrP or Shadoo expression levels.  

4.659           Regeneration and characterization of adult mouse hippocampal neurons in a defined in vitro system

Varghese, K., Das, M., Bhargava, N., Stancescu, M., Molnar, P., Kindy, M.S. and Hickman, J.J.

1. Neurosci. Methods, 177, 51-59 (2009)

  Although the majority of human illnesses occur during adulthood, most of the available in vitro disease models are based upon cells obtained from embryonic/fetal tissues because of the difficulties involved with culturing adult cells. Development of adult mouse neuronal cultures has a special significance because of the abundance of transgenic disease models that use this species. In this study a novel cell culture method has been developed that supports the long-term survival and physiological regeneration of adult mouse hippocampal cells in a serum-free defined environment. In this well-defined, controlled system, adult mouse hippocampal cells survived for up to 21 days in culture. The cultured cells exhibited typical hippocampal neuronal morphology and electrophysiological properties after recovery from the trauma of dissociation, and stained positive for the expected neuronal markers. This system has great potential as an investigative tool for in vitro studies of adult diseases, the aging brain or transgenic models of age-associated disorders.    

4.660           Calumenin but not reticulocalbin forms a Ca2+-dependent complex with thrombospondin-1. A potential role in haemostasis and thrombosis

Westegaard Hansen, G.A., Vorum, H., Jacobsen, C. and Honore, B. Mol. Cell Biochem., 320, 25-33 (2009)   Thrombocytes express thrombospondin-1 (TSP1), as well as the CREC proteins, calumenin and reticulocalbin. TSP1 and calumenin are released upon stimulation with thrombin. Calumenin has recently been shown to influence the synthesis of certain coagulation factors. Calumenin is present in atherosclerotic lesions but not in normal vasculature [Coppinger et al. (Blood 103:2096–2104, 2004)] and is able to modulate the protein expression pattern as well as the cell cycle of fibroblasts [Østergaard et al. (Proteomics 6:3509–3519, 2006)]. We here show that calumenin in the presence of Ca²⁺ binds to TSP1 with a dissociation constant K _d around 0.4 μM. This interaction is specific with respect to the secreted calumenin as the closest relative among the CREC family members, the non-secreted reticulocalbin, does not form a similar complex. This further indicates that calumenin may be broadly involved in haemostasis and in the pathophysiology of thrombosis.  

4.661           Critical age-related loss of cofactors of neuron cytochrome C oxidase reversed by estrogen

Jones, T.T. and Brewer, G.J. Exp. Neurol., 215, 212-219 (2009)   The mechanistic basis for the correlation between mitochondrial dysfunction and neurodegenerative disease is unclear, but evidence supports involvement of cytochrome C oxidase (CCO) deficits with age. Neurons isolated from the brains of 24 month and 9 month rats and cultured in common conditions provide a model of intrinsic neuronal aging. In situ CCO activity was decreased in 24 month neurons relative to 9 month neurons. Possible CCO-related deficits include holoenzyme activity, cofactor, and substrate. No difference was found between neurons from 24 month and 9 month rats in mitochondrial counts per neuron, CCO activity in submitochondrial particles, or basal respiration. Immunostaining for cytochrome C in individual mitochondria revealed an age-related deficit of this electron donor. 24 month neurons did not have adequate respiratory capacity to upregulate respiration after a glutamate stimulus, in spite of a two-fold upregulation of respiration seen in 9 month neurons. Respiration in 24 month neurons was inhibited by lower concentrations of potassium cyanide, suggesting a 50% deficit in functional enzyme in 24 month compared to 9 month neurons. In addition to cytochrome C, CCO requires cardiolipin to function. Staining with nonylacridine orange revealed an age-related deficit in cardiolipin. Treatment of 24 month neurons with 17-β-estradiol restored cardiolipin levels (10 ng/mL) and upregulated respiration under glutamate stress (1 pg/mL). Attempts to induce mitochondrial turnover by neuronal multiplication also rejuvenated CCO activity in 24 month neurons. These data suggest cytochrome C and cardiolipin levels are deficient in 24 month neurons, preventing normal upregulation of respiration needed for oxidative phosphorylation in response to stress. Furthermore, the data suggest this deficit can be corrected with estrogen treatment.  

4.662           Impaired calcium homeostasis in aged hippocampal neurons

Hajieva, P., Kuhlmann, C., Luhmann, H.J. and Behl, C. Neurosci. Lett., 451, 119-123 (2009)   Development of neurodegenerative diseases such as Alzheimer's and Parkinson's disease is strongly age-associated. The impairment of calcium homeostasis is considered to be a key pathological event leading to neuronal dysfunction and cell death. However, the exact impact of aging on calcium homeostasis in neurons remains largely unknown. In the present work we have investigated intracellular calcium levels in cultured primary hippocampal neurons from young (2 months) and aged (24 months) rat brains. Upon stimulation with glutamate or hydrogen peroxide aged neurons in comparison to young neurons demonstrated an increased vulnerability to these disease-related toxins. Measurement of calpain activity using Western blot analysis showed a significant increase in basal activity of calpains in aged neurons. The observed increase of calpain activity was correlated with elevated protein levels of μ-calpain. Ca²⁺-imaging experiments performed on living individual neurons using the dye calcium green demonstrated a twofold increase in intracellular calcium concentration in aged neurons as compared to young neurons. The observed changes of intracellular calcium in aged neurons might play a role in their increased vulnerability to neurodegeneration.      

4.663           Genomic Survey of the Non-Cultivatable Opportunistic Human Pathogen, Enterocytozoon bieneusi

Akiyoshi, D.E., Morrison, H.G., Kei, S., Feng, X., Zhang, Q., Corradi, N., Mayanja, H., Tumwine, J.K., Keeling, P.J., Weiss, L.M. and Tzipori,.S. PloSPathogens, 5(1), e100261 (2009)   Enterocytozoon bieneusi is the most common microsporidian associated with human disease, particularly in the immunocompromised population. In the setting of HIV infection, it is associated with diarrhea and wasting syndrome. Like all microsporidia, E. bieneusi is an obligate, intracellular parasite, but unlike others, it is in direct contact with the host cell cytoplasm. Studies of E. bieneusi have been greatly limited due to the absence of genomic data and lack of a robust cultivation system. Here, we present the first large-scale genomic dataset for E. bieneusi. Approximately 3.86 Mb of unique sequence was generated by paired end Sanger sequencing, representing about 64% of the estimated 6 Mb genome. A total of 3,804 genes were identified in E. bieneusi, of which 1,702 encode proteins with assigned functions. Of these, 653 are homologs of Encephalitozoon cuniculi proteins. Only one E. bieneusi protein with assigned function had no E. cuniculi homolog. The shared proteins were, in general, evenly distributed among the functional categories, with the exception of a dearth of genes encoding proteins associated with pathways for fatty acid and core carbon metabolism. Short intergenic regions, high gene density, and shortened protein-coding sequences were observed in the E. bieneusi genome, all traits consistent with genomic compaction. Our findings suggest that E. bieneusi is a likely model for extreme genome reduction and host dependence.  

4.664           Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation

Veljkovic, D.K., Rivard, G.E., Diamandis, M., Blavignac, J., Cramer-Borde, E.M. and Hayward, C.P.M. Blood, 113(7), 1535-1542 (2009)   Quebec platelet disorder (QPD) is an inherited bleeding disorder associated with increased urokinase plasminogen activator (uPA) in platelets but not in plasma, intraplatelet plasmin generation, and -granule protein degradation. These abnormalities led us to investigate uPA expression by QPD CD34⁺ progenitors, cultured megakaryocytes, and platelets, and whether uPA was stored in QPD -granules. Although QPD CD34⁺ progenitors expressed normal amounts of uPA, their differentiation into megakaryocytes abnormally increased expression of the uPA gene but not the flanking genes for vinculin or calcium/calmodulin-dependent protein kinase II on chromosome 10. The increased uPA production by cultured QPD megakaryocytes mirrored their production of -granule proteins, which was normal. uPA was localized to QPD -granules and it showed extensive colocalization with -granule proteins in both cultured QPD megakaryocytes and platelets, and with plasminogen in QPD platelets. In QPD megakaryocytes, cultured without or with plasma as a source of plasminogen, -granule proteins were stored undegraded and this was associated with much less uPA-plasminogen colocalization than in QPD platelets. Our studies indicate that the overexpression of uPA in QPD emerges with megakaryocyte differentiation, without altering the expression of flanking genes, and that uPA is costored with -granule proteins prior to their proteolysis in QPD.  

4.665           Discovery of a functional protein complex of netrin-4, laminin 1 chain, and integrin 6β1 in mouse neural stem cells

Staquicini, F.I., Dias-neto, E., Li, J., Snyder, E.Y., Sidman, R.L., Pasqualini, R. and Arap, W. PNAS, 106(8), 2903-2908 (2009)   Molecular and cellular interactions coordinating the origin and fate of neural stem cells (NSCs) in the adult brain are far from being understood. We present a protein complex that controls proliferation and migration of adult NSCs destined for the mouse olfactory bulb (OB). Combinatorial selection based on phage display technology revealed a previously unrecognized complex between the soluble protein netrin-4 and laminin γ1 subunit that in turn activates an α6β1 integrin-mediated signaling pathway in NSCs. Differentiation of NSCs is accompanied by a decrease in netrin-4 receptors, indicating that netrin-4 participates in the continual propagation of this stem cell population. Notably, the stem cells themselves do not synthesize netrin-4. Further, we show that netrin-4 is produced by selected GFAP-positive astrocytes positioned close to newborn neurons migrating in the anterior part of the rostral migratory stream (RMS) and within the OB. Our findings present a unique molecular mechanism mediating astrocytic/neuronal crosstalk that regulates ongoing neurogenesis in the adult olfactory system.      

4.666           Distinct Subtypes of Cholecystokinin (CCK)-Containing Interneurons of the Basolateral Amygdala Identified Using a CCK Promoter-Specific Lentivirus

Jasnow, A.M., Ressler, K.J., Hammack, S.E., Chhatwal, J.P. and Rainnie, D.G.

1. Neurophysiol., 101, 1494-1506 (2009)

  The basolateral amygdala (BLA) is critical for the formation of emotional memories. Little is known about the physiological properties of BLA interneurons, which can be divided into four subtypes based on their immunocytochemical profiles. Cholecystokinin (CCK) interneurons play critical roles in feedforward inhibition and behavioral fear responses. Evidence suggests that interneurons within a subgroup can display heterogeneous physiological properties. However, little is known about the physiological properties of CCK interneurons in the BLA and/or whether they represent a homogeneous or heterogeneous population. To address this question, we generated a lentivirus-expressing GFP under the control of the CCK promoter to identify CCK neurons in vivo. We combined this with whole cell patch-clamp recording techniques to examine the physiological properties of CCK-containing interneurons of the rat BLA. Here, we describe the physiological properties of 57 cells recorded in current-clamp mode; we used hierarchical cluster and discriminant function analysis to demonstrate that CCK interneurons can be segregated into three distinct subtypes (I, II, III) based on their passive and active membrane properties. Additionally, Type II neurons could be further separated into adapting and nonadapting types based on their rates of spike frequency adaptation. These data suggest that CCK interneurons of the BLA are a heterogeneous population and may be functionally distinct subpopulations that differentially contribute to the processing of emotionally salient stimuli.  

4.667           A Phase I-II Study of -Galactosylceramide-Pulsed IL-2/GM-CSF-Cultured Peripheral Blood Mononuclear Cells in Patients with Advanced and Recurrent Non-Small Cell Lung Cancer

Motohashi, S., Nagato, K., Kunii, N., Yamamoto, H., Yamasaki, K., Okita, K., Hanaoka, H., Shimizu, N., Suzuki, M., Yoshino, I., Taniguchi, M., Fujisawa, T. and Nakayama, T.,

1. Immunol., 182, 2492-2501 (2009)

  To evaluate the safety, immune responses, and antitumor responses after the administration of -galactosylceramide ( GalCer) KRN7000-pulsed PBMC cultured with IL-2 and GM-CSF (IL-2/GM-CSF-cultured PBMCs), a phase I-II study in patients with non-small cell lung cancer was conducted. Patients with advanced non-small cell lung cancer or recurrent lung cancer refractory to the standard therapy were eligible. GalCer-pulsed IL-2/GM-CSF-cultured PBMCs (1 x 10⁹/m²) were i.v. administered four times. Immune responses were monitored weekly. Twenty-three patients were enrolled in this study and 17 cases (73.9%) completed. No severe adverse event related to the treatment was observed. After the injection of GalCer-pulsed IL-2/GM-CSF-cultured PBMCs, an increased number of IFN- -producing cells in the peripheral blood were detected in 10 patients (58.8%). Five cases remained as stable disease, and the remaining 12 cases were evaluated as progressive disease. The estimated median survival time (MST) of the 17 cases was 18.6 mo (range, 3.8 to 36.3 mo). Ten patients who displayed increased IFN- -producing cells ( 2-fold) showed prolonged MST (31.9 mo; range, 14.5 to 36.3 mo) as compared with poor-responder patients (n = 7) MST (9.7 mo; range, 3.8 to 25.0 mo) (log-rank test, p = 0.0015). The administration of GalCer-pulsed IL-2/GM-CSF-cultured PBMCs was well tolerated and was accompanied by the successful induction of NKT cell-dependent immune responses. The increased IFN- -producing cells that result from GalCer stimulation in PBMCs were significantly associated with prolonged MST. These results are encouraging and warrant further evaluation for survival benefit of this immunotherapy.  

4.668           Blood Glucose Levels Regulate Pancreatic β-Cell Proliferation during Experimentally-Induced and Spontaneous Autoimmune Diabetes in Mice

Pechhold, K., Koczwara, K., Zhu, X., Harrison, V.S., Walker, G., Lee, J.and Harlan, D.M.   Background Type 1 diabetes mellitus is caused by immune-mediated destruction of pancreatic β-cells leading to insulin deficiency, impaired intermediary metabolism, and elevated blood glucose concentrations. While at autoimmune diabetes onset a limited number of β-cells persist, the cells' regenerative potential and its regulation have remained largely unexplored. Using two mouse autoimmune diabetes models, this study examined the proliferation of pancreatic islet ß-cells and other endocrine and non-endocrine subsets, and the factors regulating that proliferation. Methodology and Principal Findings We adapted multi-parameter flow cytometry techniques (including DNA-content measurements and 5′-bromo-2′-deoxyuridine [BrdU] incorporation) to study pancreatic islet single cell suspensions. These studies demonstrate that β-cell proliferation rapidly increases at diabetes onset, and that this proliferation is closely correlated with the diabetic animals' elevated blood glucose levels. For instance, we show that when normoglycemia is restored by exogenous insulin or islet transplantation, the β-cell proliferation rate returns towards low levels found in control animals, yet surges when hyperglycemia recurs. In contrast, other-than-ß endocrine islet cells did not exhibit the same glucose-dependent proliferative responses. Rather, disease-associated alterations of BrdU-incorporation rates of δ-cells (minor decrease), and non-endocrine islet cells (slight increase) were not affected by blood glucose levels, or were inversely related to glycemia control after diabetes onset (α-cells). Conclusion We conclude that murine β-cells' ability to proliferate in response to metabolic need (i.e. rising blood glucose concentrations) is remarkably well preserved during severe, chronic β-cell autoimmunity. These data suggest that timely control of the destructive immune response after disease manifestation could allow spontaneous regeneration of sufficient β-cell mass to restore normal glucose homeostasis.  

4.669           Superoxide dismutase, copper and zinc concentrations in platelet-rich plasma of pneumonia patients

Laskaj, R., Dodig, S., Cepelak, I. and Kuzman, I. Ann. Clin. Biochem., 46, 123-128 (2009)   Background: The aim of this study was to analyse platelet superoxide dismutase(SOD) activities (total SOD, manganese SOD and copper zinc SOD)and copper (Cu) and zinc (Zn) concentrations during the courseof community-acquired pneumonia (CAP), and to compare them betweenpatients with normal platelet count and those who have developedreactive thrombocytosis (RT). Methods: Platelet count, SOD activities and Cu and Zn concentrationsin platelet-rich plasma were measured in patients with CAP onadmission and at discharge. Results: Post-therapeutic platelet count increased significantly fromthe value recorded on admission. By the end of treatment, 42%of patients developed RT. All platelet SOD activities as wellas Cu concentration were significantly lower in CAP patientsthan in control subjects. The initial Zn concentration was greaterin CAP patients compared with controls and showed a decreaseat discharge. On admission, there was no difference in all SODactivities between either subgroup with normal platelet countor subgroup with RT. At discharge all SOD activities were significantlylower in patients with RT. Also, catalytic activities of thoseenzymes were significantly lower in both subgroups in comparisonwith the initial values. Post-therapeutic Cu value was lowerin patients with RT in comparison with patients having normalplatelet count. Zn concentration decreased significantly atdischarge when compared with the initial values only in patientswith RT. Conclusion: The pattern of changes might be indicative of a certain roleof platelets in antioxidant response during treatment in CAPpatients.  

4.670           Differentiated Human Alveolar Type II Cells Secrete Antiviral IL-29 (IFN- 1) in Response to Influenza A Infection

Wang, J., Oberley-deegan, R., Wang, S., Nikrad, M., Funk, C.J., Hartshorn, K.L. and Mason, R.J.

1. Immunol., 182, 1296-1304 (2009)

  Alveolar type II epithelial cells (ATIIs) are one of the primary targets for influenza A pneumonia. The lack of a culture system for maintaining differentiated ATIIs hinders our understanding of pulmonary innate immunity during viral infection. We studied influenza A virus (IAV)-induced innate immune responses in differentiated primary human ATIIs and alveolar macrophages (AMs). Our results indicate that ATIIs, but not AMs, support productive IAV infection. Viral infection elicited strong inflammatory chemokine and cytokine responses in ATIIs, including secretion of IL-8, IL-6, MCP-1, RANTES, and MIP-1β, but not TNF- , whereas AMs secreted TNF- as well as other cytokines in response to infection. Wild-type virus A/PR/8/34 induced a greater cytokine response than reassortant PR/8 virus, A/Phil/82, despite similar levels of replication. IAV infection increased mRNA expression of IFN genes IFN-β, IL-29 (IFN- 1), and IL-28A (IFN- 2). The major IFN protein secreted by type II cells was IL-29 and ATIIs appear to be a major resource for production of IL-29. Administration of IL-29 and IFN-β before infection significantly reduced the release of infectious viral particles and CXC and CC chemokines. IL-29 treatment of type II cells induced mRNA expression of antiviral genes MX1, OAS, and ISG56 but not IFN-β. IL-29 induced a dose-dependent decrease of viral nucleoprotein and an increase of antiviral genes but not IFN-β. These results suggest that IL-29 exerts IFN-β-independent protection in type II cells through direct activation of antiviral genes during IAV infection.  

4.671           Protective role of angiotensin II type 2 receptor signaling in a mouse model of pancreatic fibrosis

Ulmasov, B., Xu, Z., Tetri, L.H., Inagami, T. and Neuschwander-tetri, B.A. Am. J. Physiol. Gastrointest. Liver Physiol., 296, G282-G294 (2009)   The renin-angiotensin system contributes to pathological processes in a variety of organs. In the pancreas, blocking the angiotensin II (AII) type 1 receptor (AT1) attenuates pancreatic fibrogenesis in animal models of pancreatitis. Because the role of the AII type 2 receptor (AT2) in modulating pancreatic injury is unknown we investigated the role of AT2 in pancreatic injury and fibrosis. Pancreatic fibrosis was induced by repetitive cerulein administration in C57BL/6 wild-type (WT) or AT2-deficient (AT2–/–) mice and assessed by morphology and gene expression at 10 days. There was no difference between WT and AT2–/– mice in the degree of acute pancreatic injury as assessed by amylase release at 9 and 12 h and by histological examination of the pancreas at 12 h. In contrast, parenchymal atrophy and fibrosis were more pronounced in AT2–/– mice compared with WT mice at 10 days. Fibrosis was accompanied by activation of pancreatic stellate cells (PSC) evaluated by Western blot analysis for -smooth muscle actin and by immunocytochemistry; PSC activation was further increased in AT2–/– mice compared with WT mice. The level of pancreatic transforming growth factor-β1 mRNA and protein after repetitive cerulein treatment was higher in AT2–/– mice than in WT mice. Our results demonstrate that, in contrast to AT1 receptor signaling, AT2 receptor signaling modulates protective antifibrogenic effects in a mouse model of cerulein-induced pancreatic fibrogenesis. We propose that the effects of AII on injury-induced pancreatic fibrosis may be determined by the balance between AT1 and AT2 receptor signaling.  

4.672           The ataxia3 Mutation in the N-Terminal Cytoplasmic Domain of Sodium Channel Nav1.6 Disrupts Intracellular Trafficking

Sharkey, L.M., Cheng, X., Drews, V., Buchner, D.A., Jones, J.M., Justice, M.J., Waxman, S.G., Dib-Hajj, S.D. and Meisler, M.H.

1. Neurosci., 29(9), 2733-2741 (2009)

  ENU-induced neurological mutant ataxia3 was mapped to distal mouse chromosome 15. Sequencing of the positional candidate gene Scn8a encoding the sodium channel Na_v1.6 identified a T>C transition in exon 1 resulting in the amino acid substitution p.S21P near the N terminus of the channel. The cytoplasmic N-terminal region is evolutionarily conserved but its function has not been well characterized. ataxia3 homozygotes exhibit a severe disorder that includes ataxia, tremor, and juvenile lethality. Unlike Scn8a null mice, they retain partial hindlimb function. The mutant transcript is stable but protein abundance is reduced and the mutant channel is not detected in its usual site of concentration at nodes of Ranvier. In whole-cell patch-clamp studies of transfected ND7/23 cells that were maintained at 37°C, the mutant channel did not produce sodium current, and function was not restored by coexpression of β1 and β2 subunits. However, when transfected cells were maintained at 30°C, the mutant channel generated voltage-dependent inward sodium currents with an average peak current density comparable with wild type, demonstrating recovery of channel activity. Immunohistochemistry of primary cerebellar granule cells from ataxia3 mice demonstrated that the mutant protein is retained in the cis-Golgi. This trafficking defect can account for the low level of Na_v1.6-S21P at nodes of Ranvier in vivo and at the surface of transfected cells. The data demonstrate that the cytoplasmic N-terminal domain of the sodium channel is required for anterograde transport from the Golgi complex to the plasma membrane.  

4.673           Effect of Probiotics Lactobacillus acidophilus on Citrobacter rodentium Colitis: The Role of Dendritic Cells

Chen, C-C., Chiu, C-H., Lin, T-Y., Shi, H.N. and Walker, W. Pediatric Res., 65(2), 169-175 (2009)   Modulation of the intestinal immune response early in life by administration of probiotic bacteria may be an effective strategy for preventing or attenuating infectious diarrhea. We preinoculated the mice early in life with the probiotic bacteria Lactobacillus acidophilus NCFM (La) at age 2 wk. Dendritic cells (DCs) were collected and purified from mesenteric lymph nodes (MLN) and spleens of the BalbC/ByJ mice. DC isolation and adoptive transfer was used to examine the function of probiotics. We demonstrated that when mice were adoptively transferred with La-primed DCs (t-LaDC) instead of oral consumption with La, there was a similar effect on fecal bacteria counts, IgA levels, and colonic histopathology, as well as cytokine levels in MLN when there was intestinal bacterial infection. The above findings suggest that DCs play a key role in probiotics attenuating Citrobacter rodentium (Cr) colitis. Moreover, the location of La-primed DC hints that there is interaction of DCs and T cells in the digestive system of the host. Up-regulated expression of a surface marker on DCs indicated that inoculation with probiotics will stimulate the function of DCs, thereby further increasing immune response triggered by DC.  

4.674           ATP Measurement Predicts Porcine Islet Transplantation Outcome in Nude Mice

Kim, J.H., Park, S.G., Lee, H.N., Lee, Y.Y., Park, H.S., Kim, H-I., Yu, J.E., Kim, S.H., Park, C-G., Ha, J., Kim, S.J. and Park, K.S. Transplantation, 87(2), 166-169 (2009)   Current nude mice islet transplantation studies cannot be used prospectively. Therefore, to predict transplantation outcomes, reliable and rapid assays for islet quality assessment are warranted. This study evaluated the predictive power of the porcine islet ATP content on the outcomes of islet transplantation in nude mice. Here, we report that the ATP measurement using a small number of handpicked islets with a diameter of 100 to 150 μm is a good predictor of islet graft efficacy in nude mice. Using receiver-operator characteristic analysis, the area under the curve of the ATP content using a small number of handpicked islets was 0.867 (95% confidence interval 0.744-0.989, P<0.001). The sensitivity and the specificity measured were 83.3% and 73.3%, respectively. In conclusion, a simple and a rapid measurement of intraislet ATP content could be a promising substitute for current nude mice islet transplantation studies.  

4.675           Mitigation of peroxynitrite-mediated nitric oxide (NO) toxicity as a mechanism of induced adaptive NO resistance in the CNS

Bishop, A., Gooch, R., Eguchi, A., Jeffrey, S., Smallwood, L., Anderson, J. and Estevez, A.G.

1. Neurochem., 109, 74-84 (2009)

  During CNS injury and diseases, nitric oxide (NO) is released at a high flux rate leading to formation of peroxynitrite (ONOO^•) and other reactive nitrogenous species, which nitrate tyrosines of proteins to form 3-nitrotyrosine (3NY), leading to cell death. Previously, we have found that motor neurons exposed to low levels of NO become resistant to subsequent cytotoxic NO challenge; an effect dubbed induced adaptive resistance (IAR). Here, we report IAR mitigates, not only cell death, but 3NY formation in response to cytotoxic NO. Addition of an NO scavenger before NO challenge duplicates IAR, implicating reactive nitrogenous species in cell death. Addition of uric acid (a peroxynitrite scavenger) before cytotoxic NO challenge, duplicates IAR, implicating peroxynitrite, with subsequent 3NY formation, in cell death, and abrogation of this pathway as a mechanism of IAR. IAR is dependent on the heme-metabolizing enzyme, heme oxygenase-1 (HO1), as indicated by the elimination of IAR by a specific HO1 inhibitor, and by the finding that neurons isolated from HO1 null mice have increased NO sensitivity with concomitant increased 3NY formation. This data indicate that IAR is an HO1-dependent mechanism that prevents peroxynitrite-mediated NO toxicity in motor neurons, thereby elucidating therapeutic targets for the mitigation of CNS disease and injury.  

4.676           Differential sensitivity of oligodendrocytes and motor neurons to reactive nitrogen species: implications for multiple sclerosis

Bishop, A., Green Hobbs, K., Eguchi, A., Jeffrey, S., Smallwood, L., Pennie, C., Anderson, J. and estevez, A.G.

1. Neurochem., 109, 93-104 (2009)

  Depending on its concentration, nitric oxide (NO) has beneficial or toxic effects. In pathological conditions, NO reacts with superoxide to form peroxynitrite, which nitrates proteins forming nitrotyrosine residues (3NY), leading to loss of protein function, perturbation of signal transduction, and cell death. 3NY immunoreactivity is present in many CNS diseases, particularly multiple sclerosis. Here, using the high flux NO donor, spermine-NONOate, we report that oligodendrocytes are resistant to NO, while motor neurons are NO sensitive. Motor neuron sensitivity correlates with the NO-dependent formation of 3NY, which is significantly more pronounced in motor neurons when compared with oligodendrocytes, suggesting peroxynitrite as the toxic molecule. The heme-metabolizing enzyme, heme-oxygenase-1 (HO1), is necessary for oligodendrocyte NO resistance, as demonstrated by loss of resistance after HO1 inhibition. Resistance is reinstated by peroxynitrite scavenging with uric acid further implicating peroxynitrite as responsible for NO sensitivity. Most importantly, differential sensitivity to NO is also present in cultures of primary oligodendrocytes and motor neurons. Finally, motor neurons cocultured with oligodendrocytes, or oligodendrocyte-conditioned media, become resistant to NO toxicity. Preliminary studies suggest oligodendrocytes release a soluble factor that protects motor neurons. Our findings challenge the current paradigm that oligodendrocytes are the exclusive target of multiple sclerosis pathology.    

4.677           Thymus-homing peripheral dendritic cells constitute two of the three major subsets of dendritic cells in the steady-state thymus

Li, J., Park, J., Foss, D. and Goldschneider, I.

1. Exp. Med., 206(3), 607-622 (2009)

  Many dendritic cells (DCs) in the normal mouse thymus are generated intrathymically from common T cell/DC progenitors. However, our previous work suggested that at least 50% of thymic DCs originate independently of these progenitors. We now formally demonstrate by parabiotic, adoptive transfer, and developmental studies that two of the three major subsets of thymic DCs originate extrathymically and continually migrate to the thymus, where they occupy a finite number of microenvironmental niches. The thymus-homing DCs consisted of immature plasmacytoid DCs (pDCs) and the signal regulatory protein –positive (Sirp ⁺) CD11b⁺ CD8 ^– subset of conventional DCs (cDCs), both of which could take up and transport circulating antigen to the thymus. The cDCs of intrathymic origin were mostly Sirp ^– CD11b^– CD8 ^hi cells. Upon arrival in the thymus, the migrant pDCs enlarged and up-regulated CD11c, major histocompatibility complex II (MHC II), and CD8 , but maintained their plasmacytoid morphology. In contrast, the migrant cDCs proliferated extensively, up-regulated CD11c, MHC II, and CD86, and expressed dendritic processes. The possible functional implications of these findings are discussed.  

4.678           Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Inhibits Experimental Autoimmune Thyroiditis by the Expansion of CD4+CD25+ Regulatory T Cells

Wang, S.H., Chen. G-H., Fan, Y., Van Antwerp, M. and Baker Jr., J.R. Endocrinology, 150(4), 2000-2007 (2009)   There have been several reports that TNF-related apoptosis-inducing ligand (TRAIL) has the ability to suppress the development of experimental autoimmune diseases, including a mouse model of experimental autoimmune encephalomyelitis, a rabbit model of rheumatoid arthritis, type 1 diabetes mellitus, in mice and experimental autoimmune thyroiditis (EAT) in mice. However, the mechanism underlying TRAIL effect is not well defined. In the present study, we specifically examined TRAIL effects on CD4⁺CD25⁺ regulatory T cells. CD4⁺CD25⁺ T cells prepared from mouse thyroglobulin (mTg)-immunized CBA/J mice proliferate in the presence of TRAIL and dendritic cells in vitro. These CD4⁺CD25⁺ T cells included both CD4⁺CD25⁺CD45RB^Low (regulatory) and CD4⁺CD25⁺CD45RB^High (effector) T cells. Our results demonstrated that mTg-immunized mice treated with TRAIL showed significant increases in the number of CD4⁺CD25⁺CD45RB^Low T cells compared with mice immunized with mTg alone. CD4⁺CD25⁺CD45RB^Low T cells expressed much higher levels of the forkhead family transcription factor, IL-10, and TGFβ1 than CD4⁺CD25⁺CD45RB^High T cells, and these cells can completely suppress the proliferation of the mTg-primed splenocytes in lower concentrations than the unfractionated CD4⁺CD25⁺ T cells. Furthermore, transfer of these cells into CBA/J mice prior to mTg-primed splenocyte injection could markedly reduce the frequency and severity of EAT development. CD4⁺CD25⁺CD45RB^Low T cells were more effective at suppressing histological thyroiditis than unfractionated cells. These results indicated that TRAIL can increase the number of mTg-specific CD4⁺CD25⁺CD45RB^Low T cells, inhibiting autoimmune responses and preventing the progression of EAT. These findings reveal a novel mechanism by which TRAIL could inhibit autoimmune disease.  

4.679           Brief Bout of Exercise Alters Gene Expression in Peripheral Blood Mononuclear Cells of Early- and Late-Pubertal Males

Radom-Aizik, S., Zaldivar Jr., F., Leu, S-Y. and Cooper, D.M. Pediatric Res., 65(4), 447-452 (2009)   Peripheral blood mononuclear cells (PBMCs) are stimulated by exercise and contribute not only to host defense, but also to growth, repair, and disease pathogenesis. Whether PBMC gene expression is altered by exercise in children is not known. Ten early pubertal boys (8-12 y) and 10 late pubertal boys (15-18 y) performed ten 2-min bouts of strenuous, constant work rate exercise with 1-min rest intervals. PBMCs were isolated before and after exercise and microarray (Affymetrix U133 + 2 chips) analyzed. Statistical criterion to identify gene expression changes was less than 5% false discovery rate (FDR) with 95% confidence interval. One thousand two hundred forty-six genes were altered in older boys (517 up, 729 down), but only 109 were altered in the younger group (79 up, 30 down). In older boys, 13 gene pathways (using Expression Analysis Systematic Explorer, p < 0.05) were found (e.g. natural killer cell cytotoxicity, apoptosis). Epiregulin gene expression (EREG, a growth factor involved in wound healing) increased in older boys. In older boys exercise altered genes such as TBX21, GZMA, PGTDR, and CCL5 also play roles in pediatric inflammatory diseases like asthma. Sixty-six genes were changed significantly in both groups. The pattern of PBMC gene expression suggests the initiation of an immunologic danger signal associated with a sudden change in energy expenditure.  

4.680           Elevated plasma prostaglandins and acetylated histone in monocytes in Type 1 diabetes patients

Chen, S.S.H., jenkins, A.J. and Majewski, H. Diabet. Med., 26, 182-186 (2009)   Aims/hypothesis: Inflammation is implicated in diabetes and cyclooxygenase (COX) is involved in vascular inflammatory processes, participating in both atherosclerosis and thrombosis. The aims were to determine whether levels of monocyte COX and plasma COX metabolites are increased in Type 1 diabetic patients and to determine whether these could be linked to histone hyperacetylation. Materials and methods: Monocytes from 19 Type 1 diabetic and 39 non-diabetic control subjects were probed for COX and acetylated histone H4 proteins by immunoblotting. Plasma COX metabolite levels [thromboxane B₂ (TXB₂) and prostaglandin E₂ (PGE₂)] were determined by enzyme immunoassay. Results: Monocyte COX-2 expression was significantly up-regulated (1.3-fold) in diabetic relative to the non-diabetic control subjects and plasma PGE₂ was markedly elevated (2.7-fold). In diabetic subjects, monocyte acetylated histone H4 levels were significantly elevated; sub-group analysis indicated that the increased histone acetylation was found only in the complication-free group. Conclusions: Results support increased inflammatory activity in Type 1 diabetes that involves COX-2 and increased prostaglandin production, which may predispose patients to cardiovascular events. The observation of elevated histone acetylation only in complication-free diabetic subjects suggests that this may be a protective mechanism. This merits further investigation as histone hyperacetylation has been associated with reduced expression of factors involved in vascular injury and remodelling.  

4.681           Ethanol-mediated expression of connective tissue growth factor (CCN2) in mouse pancreatic stellate cells

Lawrencia, C., Charrier, A., Huang, g. and Brigstock, D.R. Growth Factors, 27(2), 91-99 (2009)   Activated pancreatic stellate cells (PSC) play a central role in the pathogenesis of pancreatic fibrosis, a common feature of chronic pancreatitis which is often caused by excessive alcohol consumption. In view of the central role of connective tissue growth factor (CCN2) in fibrosis, we investigated the mechanisms by which CCN2 is regulated in PSC following their exposure to ethanol or acetaldehyde. Primary cultures of PSC from Balb/c mice were treated with 0-50 mM ethanol or 0-200 μM acetaldehyde in the presence or absence of 4-methylpyrazole (4MP; an inhibitor of alcohol dehydrogenase), diallyl sulfide (DAS; an inhibitor of cytochrome P4502E1) or anti-oxidant catalase or vitamin D. CCN2 production, assessed by reverse-transcriptase polymerase chain reaction to measure CCN2 mRNA levels or by fluorescence activated cell sorting to assess CCN2 protein, was enhanced in a dose-dependent manner by ethanol or acetaldehyde. In the presence of 4MP, DAS, or the anti-oxidants vitamin D or catalase, there was a substantial decrease in the ability of ethanol to stimulate CCN2 mRNA expression and a concomitant decrease in CCN2-positive PSC. Accumulation of reactive oxygen species in PSC after exposure to ethanol was verified by loading the cells with dichlorofluorescin diacetate and showing that there was a stimulation of its oxidized fluorescent product, the latter of which was diminished in the presence of catalase or vitamin D. These results show the production of acetaldehyde and oxidant stress in mouse PSC are the cause of increased CCN2 mRNA and protein production after exposure of the cells to ethanol. The potential therapeutic effects of inhibitors of ethanol metabolism or anti-oxidants in alcoholic pancreatitis may arise in part through their ability to attenuate CCN2 production by PSC.  

4.682           Deciphering von Hippel-Lindau (VHL/Vhl)-Associated Pancreatic Manifestations by Inactivating Vhl in Specific Pancreatic Cell Populations

Shen, H-C.J., Adem, A., Ylaya, K., Wilson, A., He, M., Lorang, D., Hewitt, S.M., Pechhold, K., harlan, D.M., Lubensky, I.A., Schmidt, L.S., Linehan, W.M. and Libutti, S.K. PloSOne, 4(4), e4897 (2009)   The von Hippel-Lindau (VHL) syndrome is a pleomorphic familial disease characterized by the development of highly vascularized tumors, such as hemangioblastomas of the central nervous system, pheochromocytomas, renal cell carcinomas, cysts and neuroendocrine tumors of the pancreas. Up to 75% of VHL patients are affected by VHL-associated pancreatic lesions; however, very few reports in the published literature have described the cellular origins and biological roles of VHL in the pancreas. Since homozygous loss of Vhl in mice resulted in embryonic lethality, this study aimed to characterize the functional significance of VHL in the pancreas by conditionally inactivating Vhl utilizing the Cre/LoxP system. Specifically, Vhl was inactivated in different pancreatic cell populations distinguished by their roles during embryonic organ development and their endocrine lineage commitment. With Cre recombinase expression directed by a glucagon promoter in α-cells or an insulin promoter in β-cells, we showed that deletion of Vhl is dispensable for normal functions of the endocrine pancreas. In addition, deficiency of VHL protein (pVHL) in terminally differentiated α-cells or β-cells is insufficient to induce pancreatic neuroendocrine tumorigenesis. Most significantly, we presented the first mouse model of VHL-associated pancreatic disease in mice lacking pVHL utilizing Pdx1-Cre transgenic mice to inactivate Vhl in pancreatic progenitor cells. The highly vascularized microcystic adenomas and hyperplastic islets that developed in Pdx1-Cre;Vhl f/f homozygous mice exhibited clinical features similar to VHL patients. Establishment of three different, cell-specific Vhl knockouts in the pancreas have allowed us to provide evidence suggesting that VHL is functionally important for postnatal ductal and exocrine pancreas, and that VHL-associated pancreatic lesions are likely to originate from progenitor cells, not mature endocrine cells. The novel model systems reported here will provide the basis for further functional and genetic studies to define molecular mechanisms involved in VHL-associated pancreatic diseases.  

4.683           The Toll-Like Receptor Signaling Molecule Myd88 Contributes to Pancreatic Beta-Cell Homeostasis in Response to Injury

Bollyky, P.L., Bice, J.B., Sweet, I.R., Falk, B.A., Gebe, J.A., Clark, A.E., Gersuk, V.H., Aderem, A., Hawn, T.R. and Nepom, G.T. PloSOne, 4(4), e5063 (2009)   Commensal flora and pathogenic microbes influence the incidence of diabetes in animal models yet little is known about the mechanistic basis of these interactions. We hypothesized that Myd88, an adaptor molecule in the Toll-like-receptor (TLR) pathway, regulates pancreatic β-cell function and homeostasis. We first examined β-cells histologically and found that Myd88−/− mice have smaller islets in comparison to C57Bl/6 controls. Myd88−/− mice were nonetheless normoglycemic both at rest and after an intra-peritoneal glucose tolerance test (IPGTT). In contrast, after low-dose streptozotocin (STZ) challenge, Myd88−/−mice had an abnormal IPGTT relative to WT controls. Furthermore, Myd88−/− mice suffer enhanced β-cell apoptosis and have enhanced hepatic damage with delayed recovery upon low-dose STZ treatment. Finally, we treated WT mice with broad-spectrum oral antibiotics to deplete their commensal flora. In WT mice, low dose oral lipopolysaccharide, but not lipotichoic acid or antibiotics alone, strongly promoted enhanced glycemic control. These data suggest that Myd88 signaling and certain TLR ligands mediate a homeostatic effect on β-cells primarily in the setting of injury.  

4.684           Islet cell transplantation for the treatment of type 1 diabetes in the USA

Ikemoto, T., Noguchi, H., Shimoda, M., Naziruddin, B., Jackson, A., Tamura, Y., Fujita, Y., Onaca, N., Levy, M.F. and Matsumoto, S.

1. Hepatobiliary Pancreat. Surg., 16, 118-123 (2009)

  Islet cell transplantation (ICTx) is one of the most effective treatments for type 1 diabetes and is less invasive compared to whole organ transplantation. The US has been the leader in the research and clinical applications of ICTx for the last 40 years. ICTx requires complex procedures, including pancreas procurement and preservation; pancreas digestion; islet purification; and transplantation. Even with the dramatic progresses in each of the procedures listed above, there are still challenges to make ICTx the standard therapy. These challenges are: (1) obtaining enough islets from a single donor and (2) preventing graft loss due to allogenic rejection and recurrence of autoimmune islet destruction. A new preservation strategy for pancreata and pancreatic ducts using ET-Kyoto solution as well as a new islet purification method using iodixanol has substantially improved islet yields. Continuous research to improve the efficacy of islet isolation will solve the issue of obtaining enough islets from a single donor. Immunological tolerance is an ideal solution for the issue of rejection and autoimmune recurrence and a regulatory T cell strategy seems promising. Moreover, the SUITO index is a simple and powerful tool to assess engrafted islet mass and is, therefore, useful for evaluating the efficacy of new immunosuppressant strategies. Once ICTx becomes a standard treatment, the donor shortage will become the next challenge. Marginal or living donor islet transplantations could help alleviate this issue; however, bio-artificial islet transplantation with animal islets could be the ultimate solution.  

4.685           An in vitro screening cascade to identify neuroprotective antioxidants in ALS

Barber, S.C., Higginbottom, A., Mead, R.J., Barber, S. and Shaw, P.J. Free Radical Biology & Medicine, 46, 1127-1138 (2009)   Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease, characterized by progressive dysfunction and death of motor neurons. Although evidence for oxidative stress in ALS pathogenesis is well described, antioxidants have generally shown poor efficacy in animal models and human clinical trials. We have developed an in vitro screening cascade to identify antioxidant molecules capable of rescuing NSC34 motor neuron cells expressing an ALS-associated mutation of superoxide dismutase 1. We have tested known antioxidants and screened a library of 2000 small molecules. The library screen identified 164 antioxidant molecules, which were refined to the 9 most promising molecules in subsequent experiments. Analysis of the in silico properties of hit compounds and a review of published literature on their in vivo effectiveness have enabled us to systematically identify molecules with antioxidant activity combined with chemical properties necessary to penetrate the central nervous system. The top-performing molecules identified include caffeic acid phenethyl ester, esculetin, and resveratrol. These compounds were tested for their ability to rescue primary motor neuron cultures after trophic factor withdrawal, and the mechanisms of action of their antioxidant effects were investigated. Subsequent in vivo studies can be targeted using molecules with the greatest probability of success.  

4.686           Sperm sex-sorting in the Asian elephant (Elephas maximus)

Hermes, R., Behr, B., Hildebrandt, T.B., Blottner, S., Sieg, B., Frenzel, A., Knieriem, A., Saragusty, J. and Rath, D. Animal Reprod. Sci., 112, 390-396 (2009)   In captive Asian elephants, there is a strong need for production of female offspring to enhance reproduction, counter premature aging processes in female animals and reduce challenging management situations derived from husbandry of several bulls in one institution. Artificial insemination of flow cytometrically sex-sorted spermatozoa offers the possibility to predetermine the sex of offspring with high accuracy. The aims of this study were to determine a suitable semen extender and basic parameters for flow cytometrical sex-sorting of Asian elephant spermatozoa. In total 18 semen samples were collected by manual rectal stimulation from one bull. Sperm quality parameters and sex sortability of spermatozoa were evaluated after dilution in three semen extenders (MES-HEPES-skim milk, MES-HEPES, TRIS–citric acid) and DNA staining. MES-HEPES-skim milk was the only semen extender found suitable to sex Asian elephant spermatozoa. From 18 ejaculates collected, 12 were successfully sorted with a purity of 94.5 ± 0.7% at an average sort rate of 1945.5 ± 187.5 spermatozoa per second. Sperm integrity, progressive and total motility were 42.6 ± 3.9%, 48.1 ± 3.3%, 59.4 ± 3.8% after DNA labelling, and 64.8 ± 3.2%, 58.0 ± 5.0%, 70.8 ± 4.4% after sorting, respectively. After liquid storage of sorted spermatozoa for 12 h at 4 °C, sperm integrity, progressive and total motility were 46.4 ± 5.2%, 32.2 ± 4.2% and 58.2 ± 3.9%, respectively. The obtained results provide a promising base to inseminate Asian elephants with sexed semen.  

4.687           Flotillins Interact with PSGL-1 in Neutrophils and, upon Stimulation, Rapidly Organize into Membrane Domains Subsequently Accumulating in the Uropod

Rossy, J., Schlicht, D., engelhardt, B. and Niggli, V. PloSOne, 4, e5403 (2009)   Background Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that are ubiquitously enriched in detergent resistant membranes and are thought to shape membrane microdomains by forming homo- and hetero-oligomers. It was the goal of this study to investigate dynamic membrane microdomain reorganization during neutrophil activation. Methodology/Principal Findings We show now, using immunofluorescence staining and co-immunoprecipitation, that endogenous flotillin-1 and -2 colocalize and associate in resting spherical and polarized primary neutrophils. Flotillins redistribute very early after chemoattractant stimulation, and form distinct caps in more than 90% of the neutrophils. At later time points flotillins accumulate in the uropod of polarized cells. Chemotactic peptide-induced redistribution and capping of flotillins requires integrity and dynamics of the actin cytoskeleton, but does not involve Rho-kinase dependent signaling related to formation of the uropod. Both flotillin isoforms are involved in the formation of this membrane domain, as uropod location of exogenously expressed flotillins is dramatically enhanced by co-overexpression of tagged flotillin-1 and -2 in differentiated HL-60 cells as compared to cells expressing only one tagged isoform. Flotillin-1 and -2 associate with P-selectin glycoprotein ligand 1 (PSGL-1) in resting and in stimulated neutrophils as shown by colocalization and co-immunoprecipitation. Neutrophils isolated from PSGL-1-deficient mice exhibit flotillin caps to the same extent as cells isolated from wild type animals, implying that PSGL-1 is not required for the formation of the flotillin caps. Finally we show that stimulus-dependent redistribution of other uropod-located proteins, CD43 and ezrin/radixin/moesin, occurs much slower than that of flotillins and PSGL-1. Conclusions/Significance These results suggest that flotillin-rich actin-dependent membrane microdomains are importantly involved in neutrophil uropod formation and/or stabilization and organize uropod localization of PSGL-1.  

4.688           Tim-3 mediates phagocytosis of apoptotic cells and cross-presentation

Nakayama, M., Akiba, H., Takeda, K., Kojima, Y., Hashiguchi, M., Azuma, M., Yagita, H. and Okumura, K. Blood, 113(16), 3821-3830 (2009)   Phagocytes such as macrophages and dendritic cells (DCs) engulf apoptotic cells to maintain peripheral immune tolerance. However, the mechanism for the recognition of dying cells by phagocytes is not fully understood. Here, we demonstrate that T-cell immunoglobulin mucin-3 (Tim-3) recognizes apoptotic cells through the FG loop in the IgV domain, and is crucial for clearance of apoptotic cells by phagocytes. Whereas Tim-4 is highly expressed on peritoneal resident macrophages, Tim-3 is expressed on peritoneal exudate macrophages, monocytes, and splenic DCs, indicating distinct Tim-mediated phagocytic pathways used by different phagocytes. Furthermore, phagocytosis of apoptotic cells by CD8⁺ DCs is inhibited by anti–Tim-3 mAb, resulting in a reduced cross-presentation of dying cell-associated antigens in vitro and in vivo. Administration of anti–Tim-3 as well as anti–Tim-4 mAb induces autoantibody production. These results indicate a crucial role for Tim-3 in phagocytosis of apoptotic cells and cross-presentation, which may be linked to peripheral tolerance.  

4.689           T-cell responses associated with neonatal alloimmune thrombocytopenia: isolation of HPA-1a–specific, HLA-DRB3*0101–restricted CD4+ T cells

Ahlen, M.T., Husebekk, A., Killie, M.K., Skogen, B. and Stuge, T.B. Blood, 113(16), 3838-3844 (2009)   T-cell responses have been implicated in the development of HPA-1a–induced neonatal alloimmune thrombocytopenia (NAIT). However, HPA-1a–specific T cells have neither been isolated nor characterized. Here, we aimed to determine whether HPA-1a–specific T cells could be isolated from HPA-1a–immunized women. In the present study, peripheral blood mononuclear cells (PBMCs) from an HPA-1a–alloimmunized woman were cultured for weeks in the presence of HPA-1a peptide, labeled with CFSE, and assayed for antigen-specific proliferation. Individual proliferating cells were isolated by fluorescence-activated cell sorting and expanded in culture. Antigen specificity and HLA restriction were determined by cytokine secretion (enzyme-linked immunospot [ELISPOT]) and proliferation assays. Several CD3⁺CD4⁺ T-cell clones were isolated that proliferated and secreted cytokines in response to HPA-1a peptide. Two of these clones have been established in long-term culture in our laboratory. Both of these recognize synthetic as well as naturally processed HPA-1a antigen, and the recognition is restricted by the MHC molecule HLA-DRB3*0101 that is strongly associated with NAIT. These HPA-1a–specific T-cell clones represent unambiguous evidence for the association of T-cell responses with NAIT, and they will serve as unique tools to elucidate the cellular immune response that may result in NAIT.  

4.690           Differential contribution of the Na+-K+-2Cl– cotransporter NKCC1 to chloride handling in rat embryonic dorsal root ganglion neurons and motor neurons

Chabwine, J.N., Talavwera, K., Verbert, L., Eggermont, J., Vanderwinden, J-M., De Smedt, H., Van Den Bosch, L., Robberecht, W. and Callewaert, G. FASEB J., 23, 1168-1176 (2009)   Plasma membrane chloride (Cl^–) pathways play an important role in neuronal physiology. Here, we investigated the role of NKCC1 cotransporters (a secondary active Cl^– uptake mechanism) in Cl^– handling in cultured rat dorsal root ganglion neurons (DRGNs) and motor neurons (MNs) derived from fetal stage embryonic day 14. Gramicidin-perforated patch-clamp recordings revealed that DRGNs accumulate intracellular Cl^– through a bumetanide- and Na⁺-sensitive mechanism, indicative of the functional expression of NKCC1. Western blotting confirmed the expression of NKCC1 in both DRGNs and MNs, but immunocytochemistry experiments showed a restricted expression in dendrites of MNs, which contrasts with a homogeneous expression in DRGNs. Both MNs and DRGNs could be readily loaded with or depleted of Cl^– during GABA_A receptor activation at depolarizing or hyperpolarizing membrane potentials. After loading, the rate of recovery to the resting Cl^– concentration (i.e., [Cl^–]_i decrease) was similar in both cell types and was unaffected by lowering the extracellular Na⁺ concentration. In contrast, the recovery on depletion (i.e., [Cl^–]_i increase) was significantly faster in DRGNs in control conditions but not in low extracellular Na⁺. The experimental observations could be reproduced by a mathematical model for intracellular Cl^– kinetics, in which DRGNs show higher NKCC1 activity and smaller Cl^–-handling volume than MNs. On the basis of these results, we conclude that embryonic DRGNs show a higher somatic functional expression of NKCC1 than embryonic MNs. The high NKCC1 activity in DRGNs is important for maintaining high [Cl^–]_i, whereas lower NKCC1 activity in MNs allows large [Cl^–]_i variations during neuronal activity.—Chabwine, J. N., Talavera, K., Verbert, L., Eggermont, J., Vanderwinden, J.-M., De Smedt, H., Van Den Bosch, L., Robberecht, W., Callewaert, G. Differential contribution of the Na⁺-K⁺-2Cl^– cotransporter NKCC1 to chloride handling in rat embryonic dorsal root ganglion neurons and motor neurons.  

4.691           Immunostimulatory cancer chemotherapy using local ingenol-3-angelate and synergy with immunotherapies

Le, T.T.T., Gardner, J., Hoang-Le, D., Schmidt, C.W., MacDonald, K.P., Lambley, e., Schroder, W.A., Ogbourne, S.M. and Suhrbier, A. Vaccine, 27, 3053-3062 (2009)   Ingenol-3-angelate is a new local chemotherapeutic agent in clinical trails that induces primary necrosis of tumour cells and transient local inflammation. Here we show that cure of subcutaneous tumours with ingenol-3-angelate (PEP005) resulted in the generation of anti-cancer CD8 T cells that could regress metastases. Furthermore, PEP005-mediated cure synergized with several CD8 T cell-based immunotherapies to regress further distant metastases. PEP005 was shown to have adjuvant properties, being able to upregulate CD80 and CD86 expression on dendritic cells in vivo, and to promote CD8 T cell induction when co-delivered with a protein antigen. PEP005 thus emerges as a unique local chemotherapeutic immunostimulatory debulking agent that could be used in conjunction with immunotherapies to promote regression of metastases.  

4.692           Mutual Helper Effect in Copulsing of Dendritic Cells With 2 Antigens: A Novel Approach for Improvement of Dendritic-based Vaccine Efficacy Against Tumors and Infectious Diseases Simultaneously

Shojaeian, J. et al

1. Immunother., 32(4), 325-332 (2009)

  To develop an efficient dendritic cell (DC)-based immunotherapy protocol, we examined whether simultaneous pulsing of DCs with a given antigen and a third-party antigen could enhance their antigen presentation capacity. Purified splenic DCs of Balb/c mice were pulsed separately with immunoglobulin G, ovalbumin, conalbumin, P15 peptide of Mycobacterium tuberculosis, and prostate-specific antigen or double combinations of the aforementioned antigens. In some settings, DCs pulsed with 1 antigen were mixed equally with those pulsed with another antigen. Antigen-pulsed DCs were injected into the footpad of syngeneic mice and proliferation of whole, CD4- and CD8- depleted lymph node cells was measured after restimulation with cognate antigen. Antigen-specific production of interferon-gamma (IFN[gamma]) was tested in culture supernatants. Frequency of responding lymph node cells was determined by IFN[gamma] enzyme-linked immunosorbent spot assay. Our results showed that copulsing of DCs with 2 unrelated antigens increased the capacity of DCs to induce antigen-specific T-cell proliferation against both antigens up to 16-fold. Injection of 2 populations of DCs each pulsed with a different antigen, increased proliferation of primed T cells significantly as well. Both CD4- and CD8- depleted populations showed vigorous proliferative response in copulsing system. In addition, copulsing of DCs with 2 antigens resulted in higher frequency of antigen-specific responding cells and significantly more IFN[gamma] production. Our results clearly showed that unrelated peptides and proteins could be used to enhance efficacy of DC-based vaccines    

4.693           Superiority of Iodixanol (OptiPrep) over Ficoll in Human Islet Purification

Mita, A. et al Am. J. Transplant., 9, Suppl.2, 406 (2009)   Continuous Ficoll-based density gradient purifi cation with top-loading using semiautomated computerized COBE-2991 cell processor is considered the gold-standard method in islet transplantation at the present time. The purifi cation methods using OptiPrep-based density gradient have been recently reported to provide more successful clinical outcomes. The aim of current study was to investigate the effects of purifi cation method using OptiPrep-based density gradients. Human islet isolations were performed using a modifi ed automated method. After digestion phase, pancreatic digests were equally separated into two groups and purifi ed using OptiPrep-based density gradient (OptiPrep group) or Ficollbased density gradient (Ficoll group). The quantity, purity, viability and cellular composition of islet preparations from each group were assessed. Cytokine/ chemokine and tissue factor production from islet preparations after 48h culture were also measured. Although islet purity, post-purifi cation IEQ, islet recovery rate, FDA/PI and fractional â-cell viability were comparable, absolute â-cell mass after 48h culture signifi cantly improved in OptiPrep group when compared to Ficoll group. TNF-á, IL-1â, IFN-ã, IL-6, IL-8, MIP-1â, MCP-1 and RANTES production except for tissue factor in OptiPrep group were signifi cantly lower. Each preparation contained the similar number of ductal cell and macrophage, which are one of cytokine/chemokine producers in human islet preparation. Endotoxin level in both gradient medium was also comparable. The purifi cation method using OptiPrep gradient media signifi cantly reduced cytokine/chemokine production but not tissue factor from human islet preparations and improved beta cell survival during pre-transplant culture. Our results suggest that the purifi cation method using OptiPrep gradient media may be of assistance in increasing successful islet transplantation.  

4.694           Identification of Critical Factors Leading to Successful Islet Isolations and Transplantation

Avila, J.G. et al Am. J. Transplant., 9, Suppl. 2, 403-404 (2009)   Islet transplantation remains a feasible therapy for the treatment of a specifi c group of patients with type I diabetes. However, islet isolation remains inconsistent due to many factors that infl uence fi nal islet yield. New products are leading to improved outcome, even in younger donor pancreata. The purpose of this study was to identify a group of variables with a critical role in obtaining successful outcomes in islet isolations, leading to single donor islet transplants with insulin independence in our clinical trial. Methods: Thirty-nine human islet isolations where studied. Final islet yields in islet equivalents (IEQ) were compared from pancreata procured from our surgical group versus or surgeons from non-islet centers. In addition, age and body mass index (BMI) of the donor, enzyme type and purifi cation methods were compared between successful (transplanted) isolations and non-transplanted ones. Results: Final islet yields were signifi cantly higher when an islet experienced surgeon performed the procurement compared to a surgeon from another institution (401,787±177,894 IEQ vs. 237,825±124,987, mean±SD, p<0.01) respectively. A new generation of collagenase (NB1) and neutral protease (NB) available enabled us to obtain higher islet yields from younger pancreata. The mean age of donor pancreata resulting in an islet transplant was signifi cantly younger than in unsuccessful isolations using the same enzyme (32±4 years vs. 44±10 respectively, p=0.04). Regression analysis shows a negative correlation between age and islet yield using this enzyme (Spearman R=-0.58, p=0.039). As in previous studies, isolations leading to a transplant resulted from donors with signifi cantly higher body mass index (BMI) than the unsuccessful isolations (38.4±4.9 vs. 30±6.4 respectively, p=0.04). An improved method for islet purifi cation using Penta starch and Iodixanol resulted in a significantly higher percent recovery of islets than the Ficoll-based method (86%±17 vs. 64%±25 respectively, p=0.01). Conclusion: Results suggest that surgeons with interest in islets may have a positive impact in the outcome of the isolation. In addition, islets from younger and heavier donors can be successfully isolated with the new enzyme combination NB1-NB. Moreover, the combination of a careful procurement, an appropriate enzyme and an effi cient purifi cation method can result in improved outcomes of islet isolations.        

4.695           Protective effects of iodixanol during bovine sperm cryopreservation

Saragusty, J., Gacitua, H., Rozenboim, I. and Arav, A. Theriogenology, 71, 1425-1432 (2009)   The aim of cryopreservation is to maintain cellular integrity, thereby enabling resumption of proper biological functioning after thawing. Here we propose OptiPrep ™ (60% iodixanol in water) as a protectant during sperm cryopreservation using pooled bull semen as the model. We evaluated OptiPrep concentration effect and its relation to cryopreservation by comparing frozen-thawed and chilled samples. Semen, extended in Andromed^® with 0 (control), 1.25%, 2.5%, and 5% OptiPrep ™, was compared after either chilling or freezing in large volume by directional freezing. Sample evaluation included sperm motility upon thawing and after 3 h incubation at 37 °C for frozen-thawed samples and after 3 h and 6 h of chilling for chilled samples; viability, acrosomal integrity, and hypoosmotic swelling were also tested for frozen-thawed and chilled samples. Chilled samples with 5% OptiPrep ™ showed inferior viability (P = 0.047) and 3 h motility (P = 0.017) relative to that for chilled samples with 2.5% OptiPrep and inferior viability (P = 0.042), acrosomal integrity (P = 0.045), and 0 h motility (P = 0.024) relative to that for chilled samples with 1.25% OptiPrep. The 1.25%, 2.5%, and control samples did not differ. In frozen-thawed samples, 2.5% OptiPrep was superior to all other concentrations for 3 h motility (control, P = 0.007; 5% OptiPrep, P = 0.005; 1.25% OptiPrep, P = 0.004) and to 1.25% OptiPrep for acrosomal integrity (P = 0.001). In a search for a protection mechanism, we measured glass transition temperature (T_g) of Andromed^® and of Andromed^® with 1.25%, 2.5%, and 5% OptiPrep ™. Andromed^® (–58.78 °C) and 1.25% OptiPrep ™ (–58.75 °C) groups had lower mean T_g than that of the 2.5% (–57.67 °C) and the 5% (–57.10 °C) groups. Directional cryomicroscopy revealed that the presence of iodixanol alters ice crystal formation into an intricate net of dendrites. Thus, iodixanol appears to possess cryoprotective properties by helping spermatozoa maintain motility and membrane integrity, possibly through altering ice crystals formation into a more hospitable environment and increasing the glass transition temperature.  

4.696           Foxn1 is required to maintain the postnatal thymic microenvironment in a dosage-sensitive manner

Chen, L., Xiao, S. and Manley, N.R. Blood, 113(3), 567-574 (2009)   The postnatal thymus is the primary source of T cells in vertebrates, and many if not all stages of thymocyte development require interactions with thymic epithelial cells (TECs). The Foxn1 gene is a key regulator of TEC differentiation, and is required for multiple aspects of fetal TEC differentiation. Foxn1 is also expressed in the postnatal thymus, but its function after birth is unknown. We generated a Foxn1 allele with normal fetal expression and thymus development, but decreased expression in the postnatal thymus. This down-regulation causes rapid thymic compartment degeneration and reduced T-cell production. TEC subsets that express higher Foxn1 levels are most sensitive to its down-regulation, in particular MHCII^hiUEA-1^hi medullary TECs. The requirement for Foxn1 is extremely dosage sensitive, with small changes in Foxn1 levels having large effects on thymus phenotypes. Our results provide the first evidence that Foxn1 is required to maintain the postnatal thymus. Furthermore, the similarities of this phenotype to accelerated aging-related thymic involution support the possibility that changes in Foxn1 expression in TECs during aging contribute to the mechanism of involution.  

4.697           Motor neuronal protection by l-arginine prolongs survival of mutant SOD1 (G93A) ALS mice

Lee, J., Ryu, H. and Kowall, N.W. Biochem. Biophys. Res. Comm., 384, 524-529 (2009)   Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to motor neuron degeneration. Despite the fact that many different therapeutic strategies have been applied to prevent disease progression, no cure or effective therapy is currently available for ALS. We found that l-arginine protects cultured motor neurons from excitotoxic injury. We also found that l-arginine supplementation both prior to and after the onset of motor neuron degeneration in mtSOD1 (G93A) transgenic ALS mice significantly slowed the progression of neuropathology in lumbar spinal cord, delayed onset of motor dysfunction, and prolonged life span. Moreover, l-arginine treatment was associated with preservation of arginase I activity and neuroprotective polyamines in spinal cord motor neurons. Our findings show that l-arginine has potent in vitro and in vivo neuroprotective properties and may be a candidate for therapeutic trials in ALS.    

4.698           Up-regulation of uPARAP/Endo180 during culture activation of rat hepatic stellate cells and its presence in hepatic stellate cell lines from different species

Mousavi, S.A., Fønhus, M.S. and berg, T. BMC Cell Biol., 10, 39-49 (2009)   Background The urokinase plasminogen activator receptor associated protein (uPARAP)/Endo180 is a novel endocytic receptor that mediates collagen uptake and is implicated to play a role in physiological and pathological tissue-remodelling processes by mediating intracellular collagen degradation. Result This study investigates the expression of uPARAP/Endo180 protein and messenger RNA in primary rat hepatic stellate cell (HSC) cultures. The results show that uPARAP/Endo180 protein is not expressed in freshly isolated HSCs or during the first few days of culture while the cells still display quiescent features. In contrast, uPARAP/Endo180 protein is expressed early during HSC activation when cells are transdifferentiated into myofibroblast-like cells. Very low levels of uPARAP/Endo180 mRNA are detectable during the first days of culture but uPARAP/Endo180 mRNA is strongly up-regulated with increasing time in culture. Moreover, endocytic uptake of denatured collagen increases as transdifferentiation proceeds over time and correlates with increased expression of uPARAP/Endo180. Finally, analysis of uPARAP/Endo180 expression in four hepatic stellate cell lines from three different species showed that all these cell lines express uPARAP/Endo180 and are able to take up denatured collagen efficiently. Conclusion These results demonstrate that uPARAP/Endo180 expression by rat HSCs is strongly up-regulated during culture activation and identify this receptor as a feature common to culture-activated HSCs.  

4.699           Langerhans Cell Maturation and Contact Hypersensitivity Are Impaired in Aryl Hydrocarbon Receptor-Null Mice

Jux, B., Kadow, S. and Esser, C.

1. Immunol., 182, 6709-6717 (2009)

  Langerhans cells (LC) are professional APCs of the epidermis. Recently, it was suggested that they are tolerogenic and control adverse immune reactions, including against low molecular mass chemicals. The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, is involved in low molecular mass chemical metabolism and cell differentiation. Growing evidence suggests a role for the AhR in the immune system, for example, by influencing dendritic cell and T cell differentiation. We found that the AhR and its repressor AhRR are expressed in LC of C57BL/6 mice. LC, unexpectedly, did not respond to a strong AhR agonist with induction of transcripts of xenobiotic metabolizing enzymes. To test for a physiological role of the AhR in LC, we investigated how AhR deficiency affects LC. We found that AhR-deficient LC were impaired in maturation; they remained smaller and less granular, did not up-regulate expression of costimulatory molecules CD40, CD80, and CD24a during in vitro maturation, and their phagocytic capacity was higher. Interestingly, the mRNA expression of tolerogenic Ido was severely decreased in AhR-deficient LC, and enzyme activity could not be induced in AhR-deficient bone marrow-derived dendritic cells. GM-CSF, needed for LC maturation, was secreted in significantly lower amounts by AhR-deficient epidermal cells. Congruent with this impaired maturity and capacity to mature, mice mounted significantly weaker contact hypersensitivity against FITC. Our data suggest that the AhR is involved in LC maturation, both cell autonomously and through bystander cells. At the same time, the AhR might be part of the risk strategy of LC against unwanted immune activation by potential skin allergens.  

4.700           SP-A Preserves Airway Homeostasis During Mycoplasma pneumoniae Infection in Mice

Ledford, J.G., Goto, H., Potts, E.N., Degan, S., Chu, H.W., Voelker, D.R., Sunday, M.E., Cianciolo, G.J., Foster, W.M., Kraft, M. and Wright, J.R.

1. Immunol., 182, 7818-7827 (2009)

  The lung is constantly challenged during normal breathing by a myriad of environmental irritants and infectious insults. Pulmonary host defense mechanisms maintain homeostasis between inhibition/clearance of pathogens and regulation of inflammatory responses that could injure the airway epithelium. One component of this defense mechanism, surfactant protein-A (SP-A), exerts multifunctional roles in mediating host responses to inflammatory and infectious agents. SP-A has a bacteriostatic effect on Mycoplasma pneumoniae (Mp), which occurs by binding surface disaturated phosphatidylglycerols. SP-A can also bind the Mp membrane protein, MPN372. In this study, we investigated the role of SP-A during acute phase pulmonary infection with Mp using mice deficient in SP-A. Biologic responses, inflammation, and cellular infiltration, were much greater in Mp infected SP-A^–/– mice than wild-type mice. Likewise, physiologic responses (airway hyperresponsiveness and lung compliance) to Mp infection were more severely affected in SP-A^–/– mice. Both Mp-induced biologic and physiologicchanges were attenuated by pharmacologic inhibition of TNF-.Our findings demonstrate that SP-A is vital to preserving lunghomeostasis and host defense to this clinically relevant strainof Mp by curtailing inflammatory cell recruitment and limitingan overzealous TNF- response.  

4.701           Cyclooxygenase-2 Induced by Zymosan in Human Monocyte-Derived Dendritic Cells Shows High Stability, and Its Expression Is Enhanced by Atorvastatin

Alvarez, Y., Municio, C., Alonso, S., Roman, J.A.S., Sanchez Crespo, M and Fernandez, N.Infect. Immun.,

1. Pharmacol. Exp. Ther., 329(3), 987-994 (2009)

  Cyclooxygenase (COX)-2 is a central enzyme of arachidonic acid metabolism, and its modulation by statins may explain some of the myocardial protective effects of these drugs. Dendritic cells (DCs) play a central role in microbial defense and in atherogenesis, and COX-2 expression in DCs is important for their migration to lymph nodes and antibody response, thus explaining why prostaglandin E₂ is a main component of the cocktails used to prepare DCs for clinical applications. On this basis, we addressed the effect of atorvastatin (ATV) on the release of arachidonic acid and on the expression of COX-2 in human monocyte-derived DCs. Although ATV on its own lacked any effect on COX-2 protein induction expression, it enhanced the release of arachidonic acid, the expression of COX-2 protein, and the production of prostaglandin E₂ induced by the fungal wall extract zymosan, and to a lower extent the effect of peptidoglycan. The effect on COX-2 protein was observed mainly 24 h after stimulation by zymosan and was not reverted by mevalonate, thus pointing to an effect unrelated to cholesterol metabolism. It is noteworthy that COX-2 protein showed a great stability, with a t of approximately 12 h, which was enhanced in the presence of ATV. In view of the important role played by COX-2 on DC function, these data indicate that ATV, by enhancing COX-2 stability, may increase DC function after infectious bouts and also counteract some of the risks associated with sustained inhibition of COX-2.  

4.702           Interruption of β-Catenin Signaling Reduces Neurogenesis in Alzheimer's Disease

He, P. and Shen, Y.

1. Neurosci., 29(20), 6545-6557 (2009)

  Although recent studies have shown that new neurons can be generated from progenitor cells in the neocortices of healthy adults, the neurogenic potential of the stem/progenitor cells of AD patients is not known. To answer this question, we compared the properties of glial progenitor cells (GPCs) from the cortices of healthy control (HC) and AD subjects. The GPCs from AD brain samples displayed reduced renewal capability and reduced neurogenesis compared with GPCs from HC brains. To investigate the mechanisms underlying this difference, we compared β-catenin signaling proteins in GPCs from AD versus HC subjects and studied the effect of amyloid β peptide (Aβ, a hallmark of AD pathology) on GPCs. Interestingly, GPCs from AD patients exhibited elevated levels of glycogen synthase kinase 3β (GSK-3β, an enzyme known to phosphorylate β-catenin), accompanied by an increase in phosphorylated β-catenin and a decrease in nonphosphorylated β-catenin compared with HC counterparts. Furthermore. we found that Aβ treatment impaired the ability of GPCs from HC subjects to generate new neurons and caused changes in β-catenin signaling proteins similar to those observed in GPCs from AD patients. Similar results were observed in GPCs isolated from AD transgenic mice. These results suggest that Aβ-induced interruption of β-catenin signaling may contribute to the impairment of neurogenesis in AD progenitor cells.  

4.703           Nonhematopoietic Cells Control the Outcome of Infection with Listeria monocytogenes in a Nucleotide Oligomerization Domain 1-Dependent Manner

Mosa, A., Trumstedt, C., Eriksson, E., Soehnlein, O., Heuts, F., Janik, K., Klos, A., Dittrich-Breiholz, O., Kracht, M., Hidmark, Å., Wigzell, H. and Rottenberg, M.E. Infect. Immun., 77(7), 2908-2918 (2009)   We analyzed the defensive role of the cytosolic innate recognition receptor nucleotide oligomerization domain 1 (NOD1) during infection with Listeria monocytogenes. Mice lacking NOD1 showed increased susceptibility to systemic intraperitoneal and intravenous infection with high or low doses of L. monocytogenes, as measured by the bacterial load and survival. NOD1 also controlled dissemination of L. monocytogenes into the brain. The increased susceptibility to reinfection of NOD1^–/– mice was not associated with impaired triggering of listeria-specific T cells, and similar levels of costimulatory molecules or activation of dendritic cells was observed. Higher numbers of F480⁺ Gr1⁺ inflammatory monocytes and lower numbers of F480^– Gr1⁺ neutrophils were recruited into the peritoneum of infected WT mice than into the peritoneum of infected NOD1^–/– mice. We determined that nonhematopoietic cells accounted for NOD1-mediated resistance to L. monocytogenes in bone marrow radiation chimeras. The levels of NOD1 mRNA in fibroblasts and bone marrow-derived macrophages (BMM) were upregulated after infection with L. monocytogenes or stimulation with different Toll-like receptor ligands. NOD1^–/– BMM, astrocytes, and fibroblasts all showed enhanced intracellular growth of L monocytogenes compared to WT controls. Gamma interferon-mediated nitric oxide production and inhibition of L. monocytogenes growth were hampered in NOD1^–/– BMM. Thus, NOD1 confers nonhematopoietic cell-mediated resistance to infection with L. monocytogenes and controls intracellular bacterial growth in different cell populations in vitro.  

4.704           Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3

Gamerdinger, M., Hajieva, P., Kaya, A.M., Wolfrum, U., Hart, F.U. and Behl, C. EMBO J., 28, 889-901 (2009)   The Hsc/Hsp70 co-chaperones of the BAG (Bcl-2-associated athanogene) protein family are modulators of protein quality control. We examined the specific roles of BAG1 and BAG3 in protein degradation during the aging process. We show that BAG1 and BAG3 regulate proteasomal and macroautophagic pathways, respectively, for the degradation of polyubiquitinated proteins. Moreover, using models of cellular aging, we find that a switch from BAG1 to BAG3 determines that aged cells use more intensively the macroautophagic system for turnover of polyubiquitinated proteins. This increased macroautophagic flux is regulated by BAG3 in concert with the ubiquitin-binding protein p62/SQSTM1. The BAG3/BAG1 ratio is also elevated in neurons during aging of the rodent brain, where, consistent with a higher macroautophagy activity, we find increased levels of the autophagosomal marker LC3-II as well as a higher cathepsin activity. We conclude that the BAG3-mediated recruitment of the macroautophagy pathway is an important adaptation of the protein quality control system to maintain protein homeostasis in the presence of an enhanced pro-oxidant and aggregation-prone milieu characteristic of aging.  

4.705           Ontogeny and phagocytic function of baboon lung dendritic cells

Awasthi, S., Wolf, R. and White, G. Immunol. Cell Biol., 87, 419-427 (2009)   Dendritic cells (DCs) are the most potent antigen-presenting cells, but the ontogeny and functions of lung DCs are not known during prenatal period. Here, we isolated lung DC population from fetal (125–175 days of gestation age) and adult baboons. The cells were stained with fluorochrome-conjugated-HLA-DP, DQ, DR, CD1a, CD11c, CD14, CD40, CD80, CD86, CD209, CMKLR1, ILT7-specific antibodies, and staining was analyzed by flow cytometry. The phagocytic function was investigated by incubating the cells with fluorescent-labeled Escherichia coli bioparticles and analyzed by flow cytometry and fluorescence microscopy. The fetal baboon lung DCs expressed low levels of HLA-DP, DQ, DR, CD11c and CD86 as compared to adult baboon lung DCs and showed distinct DC morphology. The fetal lung DCs were also less capable of phagocytosing E. coli as compared to the adult lung DCs (P<0.05). In conclusion, the fetal lung DCs are not only phenotypically immature, but also less efficient in phagocytosing E. coli.  

4.706           Dynamic Changes in Pancreatic Endocrine Cell Abundance, Distribution, and Function in Antigen-Induced and Spontaneous Autoimmune Diabetes

Pechhold, K., Zhu, X., Harison, V.S., Lee, J., Chakabarty, S., Kocwara, K., Gavrilova, O. and Harlan, D.M. Diabetes, 58(5), 1175-1184 (2009)   OBJECTIVE Insulin deficiency in type 1 diabetes and in rodent autoimmune diabetes models is caused by β-cell–specific killing by autoreactive T-cells. Less is known about β-cell numbers and phenotype remaining at diabetes onset and the fate of other pancreatic endocrine cellular constituents. RESEARCH DESIGN AND METHODS We applied multicolor flow cytometry, confocal microscopy, and immunohistochemistry, supported by quantitative RT-PCR, to simultaneously track pancreatic endocrine cell frequencies and phenotypes during a T-cell–mediated β-cell–destructive process using two independent autoimmune diabetes models, an inducible autoantigen-specific model and the spontaneously diabetic NOD mouse. RESULTS The proportion of pancreatic insulin-positive β-cells to glucagon-positive α-cells was about 4:1 in nondiabetic mice. Islets isolated from newly diabetic mice exhibited the expected severe β-cell depletion accompanied by phenotypic β-cell changes (i.e., hypertrophy and degranulation), but they also revealed a substantial loss of α-cells, which was further confirmed by quantitative immunohistochemisty. While maintaining normal randomly timed serum glucagon levels, newly diabetic mice displayed an impaired glucagon secretory response to non–insulin-induced hypoglycemia. CONCLUSIONS Systematically applying multicolor flow cytometry and immunohistochemistry to track declining β-cell numbers in recently diabetic mice revealed an altered endocrine cell composition that is consistent with a prominent and unexpected islet α-cell loss. These alterations were observed in induced and spontaneous autoimmune diabetes models, became apparent at diabetes onset, and differed markedly within islets compared with sub–islet-sized endocrine cell clusters and among pancreatic lobes. We propose that these changes are adaptive in nature, possibly fueled by worsening glycemia and regenerative processes.      

4.707           Iodixanol-Controlled Density Gradient During Islet Purification Improves Recovery Rate in Human Islet Isolation

Noguchi, H., Ikemoto, T., Naziruddin, B., Jackson, A., Shimoda, M., Fujita, Y., Chujo, D., Takita, M., Kobayashi, N., Onaca, N., Levy, M.F. and Matsumoto, S. Transplantation, 87(11), 1629-1635 (2009)   Background. For pancreatic islet transplantation, islet purification minimizes the risks associated with islet infusion through the portal vein by reducing the amount of transplanted tissue. However, the purification step may result in decreased numbers of islets recovered from digested tissue and be traumatic to the islets. In this study, we evaluated the effectiveness of iodixanol-controlled density gradients on the islet purification step. Methods. For 14.3% of the isolations, the density was 1.085 g/cm3, 32.1% were 1.090 g/cm3, 46.4% were 1.095 g/cm3, 3.6% were 1.100 g/cm3, and 3.6% were 1.105 g/cm3, indicating that the density varies with each isolation. This has profound implications for the difficulty of islet purification. According to the density of digested tissue before purification, the density of the purification solutions was controlled by changing the volumetric ratio of iodixanol and the purification solutions (iodixanol-Kyoto [IK] solutions). Results. Islet yield after purification and rate of postpurification recovery were significantly higher in the IK group than with standard continuous gradient purification by Ficoll solutions (islet yield=Ficoll group: 377,230+/-50,207 islet equivalents, IK group: 594,136+/-50,570 islet equivalents, P less than 0.01; percentage of recovery=Ficoll group: 55.6%+/-5.8%, IK group: 84.9%+/-4.2%, P less than 0.01). In vitro and in vivo assays suggest that the quality of islets was similar between the two groups. Conclusion. Our data suggest that using an iodixanol-controlled density gradient improves the islet recovery rate in human islet isolation. On the basis of these data, we now use this purification method for clinical islet transplantation.  

4.708           A brief bout of exercise alters gene expression and distinct gene pathways in peripheral blood mononuclear cells of early- and late-pubertal females

Radom-Aizik, S., Zaldivar Jr, F., Leu, S-Y. and Cooper, D.M.

1. Appl. Physiol., 107, 168-175 (2009)

  Recent studies show that brief exercise alters circulating neutrophil and peripheral blood mononuclear cell (PBMC) gene expression, ranging from cell growth to both pro-and anti-inflammatory processes. These initial observations were made solely in males, but whether PBMC gene expression is altered by exercise in females is not known. Ten early-pubertal girls (8–11 yr old) and 10 late-pubertal girls (15–17 yr old) performed ten 2-min bouts of cycle ergometry ( 90% peak heart rate) interspersed with 1-min rest intervals. Blood was obtained at rest and after exercise, and microarrays were performed in each individual subject. RNA was hybridized to Affymetrix U133+2.0 Arrays. Exercise induced significant changes in PBMC gene expression in early (1,320 genes)- and late (877 genes)-pubertal girls. The expression of 622 genes changed similarly in both groups. Exercise influenced a variety of established gene pathways (EASE < 0.04) in both older (6 pathways) and younger girls (11 pathways). Five pathways were the same in both groups and were functionally related to inflammation, stress, and apoptosis, such as natural killer cell-mediated cytotoxicity, antigen processing and presentation, B cell receptor signaling, and apoptosis. In summary, brief exercise alters PBMC gene expression in early- and late-pubertal girls. The pattern of change involves diverse genetic pathways, consistent with a global danger-type response, perhaps readying PBMCs for a range of physiological functions from inflammation to tissue repair that would be useful following a bout of physical activity.  

4.709           Gut Homing Receptors on CD8 T Cells Are Retinoic Acid Dependent and Not Maintained by Liver Dendritic or Stellate Cells

Eksteen, B., Rodrigo Mora, J., Haughton, E.L., henderson, N.C., Lee-Turner, L., Villablanca, E.J., Curbishley, S.M., Aspinall, A.I., von Andrian, U.H. and Adams, D.H. Gastroenterology, 137(1), 320-329 (2009)   Background & Aims Lymphocytes primed by intestinal dendritic cells (DC) express the gut-homing receptors CCR9 and α4β7, which recognize CCL25 and mucosal addressin cell-adhesion molecule-1 in the intestine promoting the development of regional immunity. In mice, imprinting of CCR9 and α4β7 is dependent on retinoic acid during T-cell activation. Tissue specificity is lost in primary sclerosing cholangitis (PSC), an extraintestinal manifestation of inflammatory bowel disease, when ectopic expression of mucosal addressin cell-adhesion molecule-1 and CCL25 in the liver promotes recruitment of CCR9+α4β7+ T cells to the liver. We investigated the processes that control enterohepatic T-cell migration and whether the ability to imprint CCR9 and α4β7 is restricted to intestinal DCs or can under some circumstances be acquired by hepatic DCs in diseases such as PSC. Methods Human and murine DCs from gut, liver, or portal lymph nodes and hepatic stellate cells were used to activate CD8 T cells. Imprinting of CCR9 and α4β7 and functional migration responses were determined. Crossover activation protocols assessed plasticity of gut homing. Results Activation by gut DCs imprinted high levels of functional CCR9 and α4β7 on naïve CD8 T cells, whereas hepatic DCs and stellate cells proved inferior. Imprinting was RA dependent and demonstrated plasticity. Conclusions Imprinting and plasticity of gut-homing human CD8 T cells requires primary activation or reactivation by gut DCs and is retinoic acid dependent. The inability of liver DCs to imprint gut tropism implies that α4β7+CCR9+ T cell that infiltrate the liver in PSC are primed in the gut.  

4.710           CD44high Memory CD8 T Cells Synergize with CpG DNA to Activate Dendritic Cell IL-12p70 Production

Wong, K.L., Tang, L.F.M., Lew, F.C., Wong, H.S.K., Chua, Y.L., MacAry, P.A. and Kemeny, D.M.

1. Immunol., 183, 41-50 (2009)

  Protective memory CD8 T cell responses are generally associated with the rapid and efficient acquisition of CTL function. However, the ability of memory CD8 T cells to modulate immune responses through interactions with dendritic cells (DCs) during the early states of secondary Ag exposure is poorly understood. In this study, we show that murine Ag-specific CD44^high CD8 T cells, representing CD8 T cells of the memory phenotype, potently activate DCs to produce high levels of IL-12p70 in conjunction with stimulation of DCs with the TLR 9 ligand, unmethylated CpG DNA. IL-12p70 production was produced predominantly by CD8 ⁺ DCs and plasmacytoid DCs, and mediated by CD8 T cell-derived cytokines IFN- , GM-CSF, TNF- , and surface CD40L. We also find that CD44^high memory phenotype CD8 T cells were better DC IL-12p70 stimulators than CD44^low naive phenotype CD8 T cells, and this was attributed to higher levels of IFN- and GM-CSF produced by CD44^high memory phenotype CD8 T cells during their Ag specific interaction with DCs. Our study identifies CpG DNA as the most effective TLR ligand that cooperates with CD8 T cells for DC IL-12p70 production, and suggests that effectiveness of memory CD8 T cells could be attributed to their ability to rapidly and effectively induce protective Th1 immunity during early stages of pathogen reinfection.  

4.711           The Induction of IL-10 by Zymosan in Dendritic Cells Depends on CREB Activation by the Coactivators CREB-Binding Protein and TORC2 and Autocrine PGE2

Alvarez,  Y., Municio, C., Alonso, S., Sanchez Crespo, M. and Fernandez, N.

1. Immunol., 183, 1471-1479 (2009)

  Stimulation of human monocyte-derived dendritic cells with the yeast extract zymosan is characterized by a predominant production of IL-10 and a strong induction of cyclooxygenase-2, but the molecular mechanisms underlying this response are only partially understood. To address this issue, the activation of transcription factors that may bind to the il10 proximal promoter was studied. Binding activity to Sp1, Sp3, NF-Y, and cAMP response element (CRE) sites was detected in the nuclear extracts of dendritic cells; however these binding activities were not influenced by zymosan. No binding activity to Stat1, Stat3, and c/EBP sites was detected. Notably, zymosan activated B-binding activity, but inhibition of NF- B was associated with enhanced IL-10 production. In sharp contrast, treatments acting on CREB (CRE binding protein), including 8-Br-cAMP, PGE₂, and inhibitors of PKA, COX, and glycogen-synthase kinase-3β showed a direct correlation between CREB activation and IL-10 production. Zymosan induced binding of both P-CREB and CREB-binding protein (CBP) to the il10 promoter as judged from chromatin immunoprecipitation assays, whereas negative results were obtained with Ab reactive to Sp1, Sp3, c-Maf, and NF-Y. Zymosan also induced nuclear translocation of the CREB coactivator transducer of regulated CREB activity 2 (TORC2) and interaction of TORC2 with P-CREB coincidental with the association of CREB to the il10 promoter. Altogether, our data show that zymosan induces il10 transcription by a CRE-dependent mechanism that involves autocrine secretion of PGE₂ and a network of interactions of PKA, MAP/ERK, glycogen-synthase kinase-3β, and calcineurin, which regulate CREB transcriptional activity by binding the coactivators CBP and TORC2 and inhibiting CBP interaction with other transcription factors.  

4.712           Carbon Monoxide Rescues Heme Oxygenase-1-Deficient Mice from Arterial Thrombosis in Allogeneic Aortic Transplantation

Chen, B., Guo, L., Fan, C., Bolisetty, S., Joseph, R., Wright, M.M., Agarwal, A. and George, J.F. Am. J. Pathol., 175(1), 422-429 (2009)   Heme oxygenase-1 (HO-1) catalyzes the conversion of heme into carbon monoxide (CO), iron, and biliverdin. In preliminary studies, we observed that the absence of HO-1 in aortic allograft recipients resulted in 100% mortality within 4 days due to arterial thrombosis. In contrast, recipients normally expressing HO-1 showed 100% graft patency and survival for more than 56 days. Abdominal aortic transplants were performed using Balb/cJ mice as donors and either HO-1^+/+ or HO-1^–/– (C57BL/6xFVB) mice as recipients. Light and electron microscopy revealed extensive platelet-rich thrombi along the entire length of the graft in HO-1^–/– recipients at 24 hours. Treatment of recipients with CORM-2, a CO-releasing molecule (10 mg/kg of body weight intravenously), 1 hour prior and 1, 3, and 6 days after transplantation, significantly improved survival (62% at >56 days, P < 0.001) compared with HO-1^–/– recipients treated with inactive CORM-2 (median survival 1 day). Histological analyses revealed that CO treatment markedly reduced platelet aggregation within the graft. Adoptive transfer of wild-type platelets to HO-1^–/– recipients also conferred protection and increased survival. Aortic transplants from either HO-1^–/– or HO-1^+/+ C57BL/6 donors into HO-1^+/+ (Balb/cJ) mice did not develop arterial thrombosis, surviving more than 56 days. These studies demonstrate an important role for systemic HO-1/CO for protection against vascular arterial thrombosis in murine aortic allotransplantation.  

4.713           Role of neuroglobin in regulating reactive oxygen species in the brain of the anoxia-tolerant turtle Trachemys scripta 

Nayak, G., Prentice, H.M. and Milton, S.L.

1. Neurochem., 110(2), 603-612 (2009)

  Neuroglobin (Ngb) is an oxygen binding heme protein found in nervous tissue with a yet unclear physiological and protective role in the hypoxia-sensitive mammalian brain. Here we utilized in vivo and in vitro studies to examine the role of Ngb in anoxic and post-anoxic neuronal survival in the freshwater turtle. We employed semiquantitative RT-PCR and western blotting to analyze Ngb mRNA and protein levels in turtle brain and neuronally enriched cultures. Ngb expression is strongly up-regulated by hypoxia and post-anoxia reoxygenation but increases only modestly in anoxia. The potential neuroprotective role of Ngb in this species was analyzed by knocking down Ngb using specific small interfering RNA. Ngb knockdown in neuronally enriched cell cultures resulted in significant increases in H₂O₂ release compared to controls but no change in cell death. Cell survival may be linked to activation of other protective responses such as the extracellular regulated kinase transduction pathway, as phosphorylated extracellular regulated kinase levels in anoxia were significantly higher in Ngb knockdown cultures compared to controls. The greater expression of Ngb when reactive oxygen species are likely to be high, and the increased susceptibility of neurons to H₂O₂ release and external oxidative stress in knockdown cultures, suggests a role for Ngb in reducing reactive oxygen species production or in detoxification, though it does not appear to be of primary importance in the anoxia tolerant turtle in the presence of compensatory survival mechanisms.        

4.714           Successful cryopreservation of Asian elephant (Elephas maximus) spermatozoa

Saragusty, J., Hildebrandt, T.B., Behr, B., Knieriem, A., Kruse, J. and Hermes, R. Animal Reprod. Sci., 115, 255-266 (2009)   Reproduction in captive elephants is low and infant mortality is high, collectively leading to possible population extinction. Artificial insemination was developed a decade ago; however, it relies on fresh-chilled semen from just a handful of bulls with inconsistent sperm quality. Artificial insemination with frozen–thawed sperm has never been described, probably, in part, due to low semen quality after cryopreservation. The present study was designed with the aim of finding a reliable semen freezing protocol. Screening tests included freezing semen with varying concentrations of ethylene glycol, propylene glycol, trehalose, dimethyl sulfoxide and glycerol as cryoprotectants and assessing cushioned centrifugation, rapid chilling to suprazero temperatures, freezing extender osmolarity, egg yolk concentration, post-thaw dilution with cryoprotectant-free BC solution and the addition of 10% (v/v) of autologous seminal plasma. The resulting optimal freezing protocol uses cushioned centrifugation, two-step dilution with isothermal 285 m Osm/kg Berliner Cryomedium (BC) with final glycerol concentration of 7% and 16% egg yolk, and freezing in large volume by the directional freezing technique. After thawing, samples are diluted 1:1 with BC solution. Using this protocol, post-thaw evaluations results were: motility upon thawing: 57.2 ± 5.4%, motility following 30 min incubation at 37 °C: 58.5 ± 6.0% and following 3 h incubation: 21.7 ± 7.6%, intact acrosome: 57.1 ± 5.2%, normal morphology: 52.0 ± 5.8% and viability: 67.3 ± 6.1%. With this protocol, good quality semen can be accumulated for future use in artificial inseminations when and where needed.  

4.715           Melatonin protects against alcoholic liver injury by attenuating oxidative stress, inflammatory response, and apoptosis

Hu, S., Yin, S., Jiang, X., Huang, D. and Shen, G. Eur. J. Pharmacol., 616, 287-292 (2009)   Melatonin is reported to exhibit a wide variety of biological effects, including antioxidant and anti-inflammatory. Previous studies show that melatonin has a protective role in different types of liver injury and fibrosis. But its role in the pathogenesis of alcoholic liver injury remains obscure. The present investigation was designed to determine the effects of melatonin on alcohol-induced hepatic injury in mice. The degree of alcoholic liver injury was evaluated by measuring serum markers and pathological examination. Treatment with melatonin significantly attenuated the increased level of serum aminotransferase, reduced the severe extent of hepatic cell damage, steatosis and the immigration of inflammatory cells, but had no effects on hepatic expression of lipogenic genes. Furthermore, melatonin decreased serum and tissue inflammatory cytokines levels, tissue lipid peroxidation, neutrophil infiltration and inhibited the apoptosis of hepatocytes. Kupffer cells isolated from ethanol-fed mice produced high amounts of reactive oxygen species and tumor necrosis factor alpha, whereas Kupffer cells from melatonin treatment mice produced less reactive oxygen species and tumor necrosis factor alpha compared with model alcohol-feeding mice. These findings suggest that melatonin may represent a novel, protective strategy against alcoholic liver injury by attenuating oxidative stress, inflammatory response and apoptosis.  

4.716           Tumor necrosis factor alpha and interferon gamma cooperatively induce oxidative stress and motoneuron death in rat spinal cord embryonic explants

Mir, M., Asensio, V.J., Tolosa, L., Gou-Fabregas, M., Soler, R.M., llado, J. And Olmos, G. Neuroscience, 162, 959-971 (2009)   The accumulation of reactive microglia in the degenerating areas of amyotrophic lateral sclerosis (ALS) tissue is a key cellular event creating a chronic inflammatory environment that results in motoneuron death. We have developed a new culture system that consists in rat spinal cord embryonic explants in which motoneurons migrate outside the explant, growing as a monolayer in the presence of glial cells. The proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) have been proposed to be involved in ALS-linked microglial activation. In our explants, the combined exposure to these cytokines resulted in an increased expression of the pro-oxidative enzymes inducible nitric oxide synthase (iNOS), the catalytic subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, gp91^phox and cyclooxygenase-2 (COX-2), as compared to each cytokine alone. This effect was related to their cooperation in the activation of the transcription factor nuclear factor kappa B (NF-κB). TNF-α and IFN-γ also cooperated to promote protein oxidation and nitration, thus increasing the percentage of motoneurons immunoreactive for nitrotyrosine. Apoptotic motoneuron death, measured through annexin V-Cy3 and active caspase-3 immunoreactivities, was also found cooperatively induced by TNF-α and IFN-γ. Interestingly, these cytokines did not affect the viability of purified spinal cord motoneurons in the absence of glial cells. It is proposed that the proinflammatory cytokines TNF-α and IFN-γ have cooperative/complementary roles in inflammation-induced motoneuron death.  

4.717           Bluetongue Virus Targets Conventional Dendritic Cells in Skin Lymph

Hemati, B., Contreras, V., Urien, C., Bonneau, M., Takamatsu, H-H., Mertens, P.P., Breard, E., Sailleau, C., Zientara, S. and Schwartz-Cornil, I.

1. Virol., 83(17), 8789-8799 (2009)

  Bluetongue virus (BTV) is the etiological agent of bluetongue, a hemorrhagic disease of ruminants (particularly sheep), which causes important economic losses around the world. BTV is transmitted primarily via the bites of infected midges, which inject the virus into the ruminant's skin during blood feeding. The virus initially replicates in the draining lymph node and then disseminates to secondary organs where it induces edema, hemorrhages, and necrosis. In this study, we show that ovine conventional dendritic cells (cDCs) are the primary targets of BTV that contribute to the primary dissemination of BTV from the skin to draining lymph nodes. Lymph cDCs support BTV RNA and protein synthesis, as well as the production of infectious virus belonging to several different BTV serotypes, regardless of their level of attenuation. Afferent lymph cell subsets, other than cDCs, showed only marginal levels of BTV protein expression. BTV infection provoked a massive recruitment of cDCs to the sheep skin and afferent lymph, providing cellular targets for infection. Although BTV productively infects cDCs, no negative impact on their physiology was detected. Indeed, BTV infection and protein expression in cDCs enhanced their survival rate. Several serotypes of BTV stimulated the surface expression of the CD80 and CD86 costimulatory molecules on cDCs as well as the mRNA synthesis of cytokines involved in inflammation and immunity, i.e., interleukin-12 (IL-12), IL-1β, and IL-6. BTV-infected cDCs stimulated antigen-specific CD4 and CD8 proliferation as well as gamma interferon production. BTV initially targets cDCs while preserving their functional properties, reflecting the optimal adaptation of the virus to its host cells for its first spread.  

4.718           Liver Sinusoidal Endothelial Cells Are a Site of Murine Cytomegalovirus Latency and Reactivation

Seckert, C.K., Renzaho, A., Tervo, H-M., Krause, C., Deegen, P., Kühnapfel, B.,. Reddehase, M.J. and Grzimek, K.A.

1. Virol., 83(17), 8869-8884 (2009)

  Latent cytomegalovirus (CMV) is frequently transmitted by organ transplantation, and its reactivation under conditions of immunosuppressive prophylaxis against graft rejection by host-versus-graft disease bears a risk of graft failure due to viral pathogenesis. CMV is the most common cause of infection following liver transplantation. Although hematopoietic cells of the myeloid lineage are a recognized source of latent CMV, the cellular sites of latency in the liver are not comprehensively typed. Here we have used the BALB/c mouse model of murine CMV infection to identify latently infected hepatic cell types. We performed sex-mismatched bone marrow transplantation with male donors and female recipients to generate latently infected sex chromosome chimeras, allowing us to distinguish between Y-chromosome (gene sry or tdy)-positive donor-derived hematopoietic descendants and Y-chromosome-negative cells of recipients' tissues. The viral genome was found to localize primarily to sry-negative CD11b^– CD11c^– CD31⁺ CD146⁺ cells lacking major histocompatibility complex class II antigen (MHC-II) but expressing murine L-SIGN. This cell surface phenotype is typical of liver sinusoidal endothelial cells (LSECs). Notably, sry-positive CD146⁺ cells were distinguished by the expression of MHC-II and did not harbor latent viral DNA. In this model, the frequency of latently infected cells was found to be 1 to 2 per 10⁴ LSECs, with an average copy number of 9 (range, 4 to 17) viral genomes. Ex vivo-isolated, latently infected LSECs expressed the viral genes m123/ie1 and M122/ie3 but not M112-M113/e1, M55/gB, or M86/MCP. Importantly, in an LSEC transfer model, infectious virus reactivated from recipients' tissue explants with an incidence of one reactivation per 1,000 viral-genome-carrying LSECs. These findings identified LSECs as the main cellular site of murine CMV latency and reactivation in the liver.  

4.719           An In Vitro Model of Cell Transplantation for Evaluation of Cell Engraftment Enhancers

Alfaro, F.J., Grau, M., Ramirez, E., Cevey, M., Mellado, M., Castro, M.J., Meneu, J.C., Abradelo, M., Camanas, C., Moreno, E., Morales, P., Paz-Artal, E. and Serrano, A. Transplant. Proceedings, 41, 2487-2490 (2009)   The limited availability of organs for liver transplantation has focused interest on the use of cell transplants to restore hepatic function. Advances have been made in rodent models, but efficacy is limited in humans due to low engraftment efficiency. In rodents, pretransplantation treatment of the liver with engraftment enhancers (EE) shows that repopulation is feasible, although the toxicity of the substances impedes their application in humans. Evaluation of low-toxicity engraftment enhancers for human use requires testing in animal models, a time-consuming, expensive process that also raises ethical issues. To reduce animal use in the preliminary evaluation of a new EE, we designed an easily quantitated in vitro method that mimics an intraportal cell transplant. It is based on EE-mediated disruption of intercellular adhesion in confluent endothelial cell cultures.  

4.720           Aminophospholipid translocase and phospholipid scramblase activities in sickle erythrocyte subpopulations

Barber, L.A., Palascak, M.B., Joiner, C.H. and Franco, R.S. Br. J. Hematol., 146, 447-455 (2009)   Phosphatidylserine (PS) externalization may contribute to Sickle Cell Disease (SCD) characteristics including thrombogenesis, endothelial adhesion and shortened red blood cell (RBC) lifespan. Aminophospholipid translocase (APLT) returns externalized PS to the inner membrane, and phospholipid scramblase (PLSCR) equilibrates phospholipids (PL) across the membrane. APLT inhibition and PLSCR activation appear to be important for PS externalization. We examined relationships between APLT, PLSCR and external PS in mature sickle RBC and reticulocytes. Normally-hydrated sickle RBC without external PS had active APLT and inactive PLSCR. PS-exposing sickle RBC had inhibited APLT and active PLSCR. Sickle reticulocytes had active APLT and active PLSCR independent of external PS. Sickle RBC dehydrated in vivo had the highest proportion of PS-exposing RBC and markedly inhibited APLT. Normal and sickle RBC dehydrated in vitro had moderately decreased APLT. Rehydration resulted in significant recovery of APLT in RBC previously dehydrated in vitro, but not in sickle RBC dehydrated in vivo. These findings indicate that (i) PS externalization in mature sickle RBC depends on the balance between APLT and PLSCR activities, (ii) PS externalization in sickle reticulocytes depends primarily on PLSCR activation and (iii) APLT inhibition in sickle RBC dehydrated in vivo is due to dehydration itself and other factors.  

4.721           A central role for monocytes in Toll-like receptor-mediated activation of the vasculature

Ward, J.R., Francis, S.E., Marsden, L., Sudddddddddason, T., Lord, G.M., Dower, S.K., Crossman, D.C. and Sabroe, I. Immunology, 128, 58-68 (2009)   There is increasing evidence that activation of inflammatory responses in a variety of tissues is mediated co-operatively by the actions of more than one cell type. In particular, the monocyte has been implicated as a potentially important cell in the initiation of inflammatory responses to Toll-like receptor (TLR)-activating signals. To determine the potential for monocyte-regulated activation of tissue cells to underpin inflammatory responses in the vasculature, we established cocultures of primary human endothelial cells and monocytes and dissected the inflammatory responses of these systems following activation with TLR agonists. We observed that effective activation of inflammatory responses required bidirectional signalling between the monocyte and the tissue cell. Activation of cocultures was dependent on interleukin-1 (IL-1). Although monocyte-mediated IL-1[beta] production was crucial to the activation of cocultures, TLR specificity to these responses was also provided by the endothelial cells, which served to regulate the signalling of the monocytes. TLR4-induced IL-1[beta] production by monocytes was increased by TLR4-dependent endothelial activation in coculture, and was associated with increased monocyte CD14 expression. Activation of this inflammatory network also supported the potential for downstream monocyte-dependent T helper type 17 activation. These data define co-operative networks regulating inflammatory responses to TLR agonists, identify points amenable to targeting for the amelioration of vascular inflammation, and offer the potential to modify atherosclerotic plaque instability after a severe infection.  

4.722           Coadministration of the fungal immunomodulatory protein FIP-Fve and a tumour-associated antigen enhanced antitumour immunity

Ding, Y., Seow, S.V., Huang, C.H., Liew, L.M., Lim, T.C., Kuo, J.C. and Chua, K.Y. Immunology, 128, e881-e894 (2009)   Fve is a fungal protein isolated from the golden needle mushroom Flammulina velutipes and has previously been reported to trigger immunological responses in both mouse and human lymphocytes. In this study, we evaluated the potential application of Fve as an adjuvant for tumour immunotherapy and examined the underlying mechanism(s). When the human papillomavirus (HPV)-16 E7 oncoprotein was used as a model antigen, mice coimmunized with HPV-16 E7 and Fve showed enhanced production of HPV-16 E7-specific antibodies as well as expansion of HPV-16 E7-specific interferon (IFN)-[gamma]-producing CD4+ and CD8+ T cells as compared with mice immunized with HPV-16 E7 alone. Tumour protection assays showed that 60% of mice coimmunized with HPV-16 E7 plus Fve, as compared with 20% of those immunized only with HPV-16 E7, remained tumour-free for up to 167 days after challenge with the tumour cells. Tumour therapeutic assays showed that HPV-16 E7 plus Fve treatment significantly prolonged the survival of tumour-bearing mice as compared with those treated only with HPV-16 E7. In vivo cell depletion and adoptive T-cell transfer assays showed that CD4+ and CD8+ T cells and IFN-[gamma] played critical roles in conferring the antitumour effects. Interestingly, Fve could stimulate the maturation of splenic dendritic cells in vivo and induce antigen-specific CD8+ T-cell immune responses. In summary, Fve has potent adjuvant properties that enhance T helper type 1 antigen-specific humoral and cellular immune responses which confer strong antitumour effects. The use of Fve as an adjuvant could be an attractive alternative to the current vaccination strategy for cancer immunotherapy.  

4.723           Probiotic Preparation VSL#3 Alters the Distribution and Phenotypes of Dendritic Cells within the Intestinal Mucosa in C57BL/10J Mice

Wang, X., O’Gorman, M.R.G., Bu, H-F., Koti, V., Zuo, X-L. and Tan, X-D.

1. Nutr., 139, 1595-1602 (2009)

  Probiotic nutrients have shown promise in therapy for the treatment of gastrointestinal inflammation, infection, and atopic disease. Intestinal dendritic cells (DC) play a critical role in shaping the intestinal immune response. In this study, we tested the effect of a probiotic preparation (VSL#3) on DC distribution and phenotypes within the intestinal mucosa using a lineage depletion-based flow cytometric analysis. In naïve C57BL/10J mice, intestinal mucosal DC were composed of plasmacytoid DC (pDC) and myeloid DC (mDC). The pDC were the dominant form in lamina propria and Peyer's patches, whereas mDC were the prevailing type in the mesenteric lymph nodes. Additional characterization of pDC and mDC with flow cytometry revealed that they expressed heterogeneous phenotypes in the intestinal mucosa. In mice gavaged with the probiotic VSL#3 for 7 d, the proportion of pDC within the lamina propria was >60% lower, whereas the pDC subset in the mesenteric lymph nodes was more than 200% greater than in sham-treated controls (P < 0.01). Within pDC, the proportion of functionally unique CX3CR1⁺ DC was greater than in controls in both the lamina propria and the Peyer's patches (P < 0.01). In contrast to pDC, the mDC number was greater than in controls in all intestinal lymphoid tissue compartments in VSL#3-treated mice (P < 0.01). In conclusion, this study suggests that phenotypically and functionally distinct DC subsets are localized to specific lymphoid tissues within the intestinal mucosa and that the VSL#3 probiotic nutritional supplement alters the distribution of the DC subsets within the intestinal mucosa. These changes may be important in the alteration of mucosal immunity following probiotic VSL#3 therapy.  

4.724           A highly energetic process couples calcium influx through L-type calcium channels to insulin secretion in pancreatic β-cells

Jung, S-R., Reed, B.J. and Sweet, I.R. Am. J. Physiol. Endocrinol. Metab., 297, E717-E727 (2009)   Calcium (Ca²⁺) influx is required for the sustained secretion of insulin and is accompanied by a large rate of energy usage. We hypothesize that the energy usage reflects a process [Ca²⁺/metabolic coupling process (CMCP)] that couples Ca²⁺ to insulin secretion by pancreatic islets. The aim of the study was to test this hypothesis by testing the effect of inhibiting candidate Ca²⁺-sensitive proteins proposed to play a critical role in the CMCP. The effects of the inhibitors on oxygen consumption rate (OCR), a reflection of ATP usage, and insulin secretion rate (ISR) were compared with those seen when L-type Ca²⁺ channels were blocked with nimodipine. We reasoned that if a downstream Ca²⁺-regulated site was responsible for the OCR associated with the CMCP, then its inhibition should mimic the effect of nimodipine. Consistent with previous findings, nimodipine decreased glucose-stimulated OCR by 36% and cytosolic Ca²⁺ by 46% and completely suppressed ISR in rat pancreatic islets. Inhibitors of three calmodulin-sensitive proteins (myosin light-chain kinase, calcineurin, and Ca²⁺/calmodulin-dependent protein kinase II) did not meet the criteria. In contrast, KN-62 severed the connection between Ca²⁺ influx, OCR, and ISR without interfering with Ca²⁺ influx. In the presence of nimodipine or KN-62, potentiators of ISR, acetylcholine, GLP-1, and arginine had little effect on insulin secretion, suggesting that the CMCP is also essential for the amplification of ISR. In conclusion, a KN-62-sensitive process directly mediates the effects of Ca²⁺ influx via L-type Ca²⁺ channels on OCR and ISR, supporting the essential role of the CMCP in mediating ISR.  

4.725           Salmonella Induces Flagellin- and MyD88-Dependent Migration of Bacteria-Capturing Dendritic Cells Into the Gut Lumen

Arques, J.L., Hautefort, I., Ivory, K., Bertelli, E., Regoli, M., Clare, S., Hinton, J.C.D. and Nicoletti, C. Gastroenterology, 137, 579-587 (2009)   Background & Aims Intestinal dendritic cells (DCs) sample bacteria, such as Salmonella, by extending cellular processes into the lumen to capture bacteria and shuttle them across the epithelium; however, direct evidence of bacteria-loaded DCs travelling back into the tissue is lacking. We hypothesized that sampling is paralleled by migration of DCs into the lumen prior to or following the internalization of Salmonella. Methods The small intestine and the colon of BALB/c and C57BL/6 mice were challenged with noninvasive Salmonella enterica serovar Typhimurium SL1344-ΔSalmonella pathogenicity island (SPI) 1 or Escherichia coli DH5α by using isolated loops or oral administration by gavage. Transepithelial migration of DCs was documented by immunohistochemistry, microscopy, and flow cytometry. The role of flagellin was determined by using flagellin (ΔfliC ΔfljB)- and SPI1-SPI2 (ΔSPI1 ΔssrA)-deficient Salmonella, flagellated E coli K12, and MyD88 mice. Results Salmonella ΔSPI1 induced migration of CD11c⁺CX₃CR1⁺MHCII⁺CD11b⁻CD8α⁻ DCs into the small intestine, whereas flagellin- and SPI1-SPI2-deficient Salmonella, soluble flagellin, and E coli DH5α or flagellated K12, failed to do so. DC migration did not occur in the colon; it was not observed in MyD88 mice, and intraluminal DCs internalized Salmonella but did not cross the epithelium to return into tissues. Finally, DC migration was not linked to Salmonella-induced damage of the epithelium. Conclusions DC-mediated sampling of Salmonella is accompanied by flagellin- and MyD88-dependent migration of Salmonella-capturing DCs into the intestinal lumen. We suggest that the rapid intraluminal migration of Salmonella-capturing DCs may play a role in the protection of the intestinal mucosa against bacterial infection.  

4.726           Opposing Effects of TGF-β and IL-15 Cytokines Control the Number of Short-Lived Effector CD8⁺ T Cells

Sanjabi, S., Mosaheb, M.M. and Flavell, R.A. Immunity, 31, 131-144 (2009)   An effective immune response against infectious agents involves massive expansion of CD8⁺ T cells. Once the infection is cleared, the majority of these effector cells die through unknown mechanisms. How is expansion controlled to maximize pathogen clearance and minimize immunopathology? We found, after Listeria infection, plasma transforming growth factor β (TGF-β) titers increased concomitant with the expansion of effector CD8⁺ T cells. Blocking TGF-β signaling did not affect effector function of CD8⁺ T cells. However, TGF-β controlled effector cell number by lowering Bcl-2 amounts and selectively promoting the apoptosis of short-lived effector cells. TGF-β-mediated apoptosis of this effector subpopulation occurred during clonal expansion and contraction, whereas interleukin-15 (IL-15) promoted their survival only during contraction. We demonstrate that the number of effector CD8⁺ T cells is tightly controlled by multiple extrinsic signals throughout effector differentiation; this plasticity should be exploited during vaccine design and immunotherapy against tumors and autoimmune diseases.  

4.727           Differential regulation of neuronal and inducible nitric oxide synthase (NOS) in the spinal cord of mutant SOD1 (G93A) ALS mice

Lee, J., Ryu, H. and Kowall, N.W. Biochem. Biophys. Res. Comm., 387, 202-206 (2009)   Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by degeneration of motor neurons throughout the central nervous system. Mutations of the free radical scavenging enzyme superoxide dismutase-1 (SOD1) are a cause of familial ALS but it is not known how mutations lead to cell death. Free radicals such as nitric oxide (NO) are thought to play a key pathogenic role. NO is synthesized by NO synthases (NOSs) from arginine, which is a rate-limiting factor for NO production. We found that neuronal NOS (nNOS)-positive motor neurons were depleted while inducible NOS (iNOS)-positive activated glial cells were increased in transgenic mtSOD1 (G93A) ALS mice. iNOS expression was up regulated consistent with the increases of motor neuron loss and glial activation and citrulline and NO levels while nNOS expression was decreased in G93A ALS mice. Administration of l-arginine to G93A mice reduced the severity of motor neuron depletion and glial activation. In treated animals, nNOS expression was preserved while citrulline and NO were reduced, possibly due to reduced activation of glia expressing iNOS. Our findings show that high concentrations of NO correlate with iNOS expression rather than nNOS expression in G93A ALS mice. This suggests that therapy focused on iNOS inhibition might be a fruitful direction for future ALS therapeutic trials.  

4.728           Purification Method Using Iodixanol (OptiPrep)-Based Density Gradient Significantly Reduces Cytokine Chemokine Production From Human Islet Preparations, Leading to Prolonged β-Cell Survival During Pretransplantation Culture

Mita, A., Ricordi, C., Miki, A. Barker, S., Khan, A., Alvarez, A., Hashikura, Y., Miyagawa, S. and Ichii, H. Transplant. Proc., 41, 314-315 (2009)   Purification is one of the most important steps in human islet isolation. Although Ficoll-based density gradients are widely used, OptiPrep-based density gradients are used in few centers. Cytokine/chemokine production from human islet preparations varies widely. Some cytokines/chemokines have been reported to have adverse effects on human islet preparations. Control of cytokine/chemokine production may be a key to improve islet quality and quantity, leading to better transplantation outcomes. The aim of the present study was to investigate the effects on islet preparations of purification methods using various density gradients on viability, cellular composition, and proinflammatory cytokine/chemokine production. After the digestion phase, the extracts were divided into 2 groups for purification using a semiautomated cell processor with Ficoll-based or OptiPrep-based density gradients. Islet preparations cultured for 2 days were assessed regarding islet cell viability (fluorescein diacetate/propidium iodide [FDA/PI]), fractional β-cell viability by FACS, and β-cell content using iCys. Cytokine/chemokine production from islet preparations was also measured by Bio-plex. After purification, the purity, islet equivalents (IEQ), and islet recovery rates were comparable between the 2 groups. Although FDA/PI and fractional β-cell viability showed no significant difference, survival of β cells during culture was significantly higher in the OptiPrep compared with the Ficoll-based density gradient group. There were significantly lower tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (IFN)-γ, IL-6, and MIP-1β productions from the OptiPrep-based density gradient group. OptiPrep-based density gradients reduced cytokine/chemokine production by islet preparations. In addition, OptiPrep-based density gradient purification significantly reduced the loss of β-cell mass during pretransplantation culture.  

4.729           Node of Ranvier formation on motoneurons in vitro

Rumsey, J.W., Das, M., Stancescu, M., Bott, M., Fernandez-Valle, C. And Hickman, J.J. Biomaterials, 30, 3567-3572 (2009)   One of the most significant interactions between Schwann cells and neurons is myelin sheath formation. Myelination is a vertebrate adaptation that enables rapid conduction of action potentials without a commensurate increase in axon diameter. In vitro neuronal systems provide a unique modality to study both factors influencing myelination and diseases associated with myelination. Currently, no in vitro system for motoneuron myelination by Schwann cells has been demonstrated. This work details the myelination of motoneuron axons by Schwann cells, with complete Node of Ranvier formation, in a defined in vitro culture system. This defined system utilizes a novel serum-free medium in combination with the non-biological substrate, N-1[3 (trimethoxysilyl) propyl] diethylenetriamine (DETA). The myelinated segments and nodal proteins were visualized and quantified using confocal microscopy. This defined system provides a highly controlled, reproducible model for studying Schwann cell interactions with motoneurons as well as the myelination process and its effect on neuronal plasticity. Furthermore, an in vitro system that would allow studies of motoneuron myelination would be beneficial for understanding peripheral demyelinating neuropathies such as diabetes induced peripheral neuropathy and could lead to a better understanding of CNS demyelinating diseases like multiple sclerosis, as well as neuromuscular junction maturation and maintenance.  

4.730           Effect of Centrifugation Technique on Post-storage Characteristics of Stallion Spermatozoa

Webb, G.W. and Dean, M.M.

1. Equine Vet. Sci., 29(9), 675-680 (2009)

  Three ejaculates were collected from four stallions and used to compare the effects of three centrifugation methods on post-storage motility and recovery of available sperm. Two aliquots per ejaculate were diluted with skim milk-glucose (SKMG) extender to 50×10⁶ sperm/mL, placed in 50-mL conical bottom tubes, and centrifuged at either 700g for 15 minutes (700g) or 600g for 12 minutes (600g). A third aliquot was diluted 1:1 with SKMG, placed in 15-mL conical tubes, and centrifuged at 400g for 7 minutes (400g). Subsamples from each pre-treated diluted ejaculate were held at room temperature and evaluated for motility at the same time as the post-centrifugation pre-storage motility evaluation was made for treated aliquots. After centrifugation, samples from each aliquot were stored at 5°C for evaluation after 24 and 48 hours or frozen in liquid nitrogen. Percentage of available sperm harvested was higher (P ≤ .05) for aliquots centrifuged in 15-mL tubes at 400g versus 600g in 50-mL tubes. After centrifugation, total but not progressive motility of aliquots centrifuged at 700g was lower than that for noncentrifuged controls and sperm from aliquots centrifuged at 400g in 15-mL tubes. After cold storage, values for total but not progressive motility or velocity were higher (P ≤ .05) for aliquots centrifuged in 15-mL tubes at 400g compared with those centrifuged in 50-mL tubes at both 600g and 700g. Postthaw motility of frozen sperm was not different between centrifugation treatments. Poststorage percentages of intact acrosomes and detached heads did not differ because of centrifugation treatment.  

4.731           Determinants of Leukocyte Margination in Rectangular Microchannels

Jain, A. and Munn, L.L. PloSOne, 4(9), e7104 (2009)   Microfabrication of polydimethylsiloxane (PDMS) devices has provided a new set of tools for studying fluid dynamics of blood at the scale of real microvessels. However, we are only starting to understand the power and limitations of this technology. To determine the applicability of PDMS microchannels for blood flow analysis, we studied white blood cell (WBC) margination in channels of various geometries and blood compositions. We found that WBCs prefer to marginate downstream of sudden expansions, and that red blood cell (RBC) aggregation facilitates the process. In contrast to tubes, WBC margination was restricted to the sidewalls in our low aspect ratio, pseudo-2D rectangular channels and consequently, margination efficiencies of more than 95% were achieved in a variety of channel geometries. In these pseudo-2D channels blood rheology and cell integrity were preserved over a range of flow rates, with the upper range limited by the shear in the vertical direction. We conclude that, with certain limitations, rectangular PDMS microfluidic channels are useful tools for quantitative studies of blood rheology.  

4.732           Comparison of Trypsin Inhibitors in Preservation Solution for Islet Isolation

Noguchi, H., Ueda, M., Hayashi, S., Kobayashi, N., Okitsu, T., Iwanaga, Y., Nagata, Y., Nagata, H., Liu, X., Kamiya, H., Levy, M.F. and Matsumoto, S. Cell Transplant., 18(5-6), 541-547 (2009)   Islet transplantation has recently emerged as an effective therapy and potential cure for type 1 diabetes mellitus. Recent reports show that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and University of Wisconsin (UW) solution, is superior to simple cold storage in UW for pancreas preservation in islet transplantation. Moreover, we recently reported that islet yield was significantly higher in the ET-Kyoto solution with ulinastatin (MK)/PFC preservation solution compared with the UW/PFC preservation solution in the porcine model and that the advantages of MK solution are trypsin inhibition and less collagenase inhibition. In this study, we compared ulinastatin with another trypsin inhibitor, Pefabloc, in preservation solution for islet isolation. Islet yield before purification was higher in the MK/PFC group compared with the ET-Kyoto with Pefabloc (PK)/PFC group. The stimulation index was higher for the MK/PFC group than for the PK/PFC group. These data suggest that ET-Kyoto with ulinastatin was the better combination for pancreas preservation than ET-Kyoto with Pefabloc. Based on these data, we now use ET-Kyoto solution with ulinastatin for clinical islet transplantation.  

4.733           Estimation of Donor Usability for Islet Transplantation in the United States With the Kyoto Islet Isolation Method

Matsumoto, S., Noguchi, H., Hatanaka, N., Shimoda, M., Kobayashi, N., jackson, A., Onaca, N., Naziruddin, B. and Levy, M.F. Cell Transplant, 18, 549-556 (2009)   The quality of donor pancreata is important for successful islet isolation. However, in some countries like Japan, the number of donor pancreata is very low; therefore, marginal donors have been used with less restrictive donor criteria. In order to use marginal donor pancreata, we established the Kyoto islet isolation method (KIIM). According to United Network for Organ Sharing (UNOS) in 2005, more than 6,000 pancreata were not clinically used in the US. In this study, we applied the KIIM for brain-dead donors and reevaluated donor usability based on the Japanese islet donor criteria. Islets were isolated with the Ricordi method using pancreata stored in University of Wisconsin (UW) solution (UW group) or by the two-layer method (TLM group) or the TLM combined with ductal injection (DI group). We implemented the KIIM (KIIM group) to confirm the effect of the KIIM on brain-dead donors. Donor charts in Texas from 2005 to 2006 were reviewed. If pancreata were not used clinically, the reason was reviewed and donors were reevaluated based on Japanese criteria. There were no significant differences of islet yield, viability, and purity between the UW and TLM groups. The DI group significantly improved islet yields and isolations were further improved in the KIIM group [UW: 251,663 ± 60,217 islet equivalent (IE); TLM: 243,738 ± 54,170 IE; DI: 498,639 ± 28,853 IE; KIIM: 678,286 ± 55,853]. The KIIM provided high-quality islets in high numbers from islet isolations from brain-dead donors. A total of 236 donor charts were reviewed and 194 pancreata (82%) were not used. Of these, 185 cases identified the reasons that the pancreata were not used. When we applied the Japanese criteria, an additional 82 cases out of 185 (44%) seem to be suitable for islet isolations. With the KIIM, more than 2,500 additional donor pancreata can be used for islet isolation in the US every year when the Japanese criteria are applied.  

4.734           Human Islet Isolation for Autologous Transplantation: Comparison of Yield and Function Using SERVA/Nordmark Versus Roche Enzymes

Anazawa, T., Balamurugan, A.N., Bellin, M., Zhang, H.J., Matsumoto,, S., Yonekawa, Y., Tanaka, T., Loganathan, G., Papas, K.K., Beilman, G:J., hering, B.J. and Sutherland, E.R. Am. J. Transplant., 9, 2383-2391 (2009)   Islet autotransplantation (IAT) is used to preserve as much insulin-secretory capacity as possible in patients undergoing total pancreatectomy for painful chronic pancreatitis. The enzyme used to dissociate the pancreas is a critical determinant of islet yield, which is correlated with posttransplant function. Here, we present our experience with IAT procedures to compare islet product data using the new enzyme SERVA/Nordmark (SN group; n = 46) with the standard enzyme Liberase-HI (LH group; n = 40). Total islet yields (mean +/- standard deviation; 216 417 +/- 79 278 islet equivalent [IEQ] in the LH group; 227 958 +/- 58 544 IEQ in the SN group; p = 0.67) were similar. However, the percentage of embedded islets is higher in the SN group compared to the LH group. Significant differences were found in pancreas digestion time, dilution time, and digested pancreas weight between the two groups. Multivariate linear regression analysis showed the two groups differed in portal venous pressure changes. The incidence of graft function and insulin independence was not different between the two groups. The SN and LH enzymes are associated with similar outcomes for IAT. Further optimization of the collagenase/neutral protease ratio is necessary to reduce the number of embedded islets obtained when using the SN enzyme.  

4.735           Successful Clinical Islet Isolation Using a GMP-Manufactured Collagenase and Neutral Protease

Szot, G.L., Lee, M.R., Tavakol., M.M., Lang, J., Derkovic, F., Kerlan, R.K., Stock, P.G. and Posselt, A.M. Transplantation, 88(6), 753-756 (2009)   In 2007, the islet community was notified that the collagenase product most commonly used for human islet isolations contained bovine neural tissue contaminants. To minimize this potential hazard, we adapted our human islet processing procedure to use a GMP-manufactured, bovine neural tissue-free collagenase blend. Here, we describe the factors that we consider most important for achieving reproducible and clinically useable islet isolations using this product.  

4.736           Specific vulnerability of mouse spinal cord motoneurons to membrane depolarization

Grou-Fabregas, M., Garcera, A., Mincheva, S., Perez-Garcia, M.J., Comella, J.X. and Soler, R.M.

1. Neurochem., 110(6), 1842-1854 (2009)

  Intracellular calcium (Ca²⁺) concentration determines neuronal dependence on neurotrophic factors (NTFs) and susceptibility to cell death. Ca²⁺ overload induces neuronal death and the consequences are thought to be a probable cause of motoneuron (MN) degeneration in neurodegenerative diseases. In the present study, we show that membrane depolarization with elevated extracellular potassium (K⁺) was toxic to cultured embryonic mouse spinal cord MNs even in the presence of NTFs. Membrane depolarization induced an intracellular Ca²⁺ increase. Depolarization-induced toxicity and increased intracellular Ca²⁺ were blocked by treatment with antagonists to some of the voltage-gated Ca²⁺ channels (VGCCs), indicating that Ca²⁺ influx through these channels contributed to the toxic effect of depolarization. Ca²⁺ activates the calpains, cysteine proteases that degrade a variety of substrates, causing cell death. We investigated the functional involvement of calpain using a calpain inhibitor and calpain gene silencing. Pre-treatment of MNs with calpeptin (a cell-permeable calpain inhibitor) rescued MNs survival; calpain RNA interference had the same protective effect, indicating that endogenous calpain contributes to the cell death caused by membrane depolarization. These findings suggest that MNs are especially vulnerable to extracellular K⁺ concentration, which induces cell death by causing both intracellular Ca²⁺ increase and calpain activation.  

4.737           Human telomerase activity, telomerase and telomeric template expression in hepatic stem cells and in livers from fetal and postnatal donors

Schmelzer, E. and Reid, L.M. Eur. J. Gastroenterol. Hepatol., 21(10), 1191-1198 (2009)   Background: Although telomerase activity has been analyzed in various normal and malignant tissues, including liver, it is still unknown to what extent telomerase can be associated with specific maturational lineage stages. Methods: We assessed human telomerase activity, protein and gene expression for the telomerase reverse transcriptase, as well as expression of the telomeric template RNA hTER in hepatic stem cells and in various developmental stages of the liver from fetal to adult. In addition, the effect of growth factors on telomerase activity was analyzed in hepatic stem cells in vitro. Results: Telomerase was found to be highly active in fetal liver cells and was significantly higher than in hepatic stem cells, correlating with gene and protein expression levels. Activity in postnatal livers from all donor ages varied considerably and did not correlate with age or gene expression levels. The hter expression could be detected throughout the development. A short stimulation by growth factors of cultured hepatic stem cells did not increase telomerase activity. Conclusion: Telomerase is considerably active in fetal liver and variably in postnatal livers. Although telomerase protein is present at varying levels in liver cells of all donor ages, gene expression is solely associated with fetal liver cells.  

4.738           Molecular and immunocytochemical characterization of primary neuronal cultures from adult rat brain: Differential expression of neuronal and glial protein markers

Ray, B., Bailey, J.A., Sarkar, S. and Lahiri, D.K.

1. Neurosci. Methods, 184, 294-302 (2009)

  Neurobiological studies using primary neuronal cultures commonly employ fetal-derived neurons, but much less often adult brain-derived neurons. Our goal is to perform morphological and molecular characterization of primary neuronal cultures from adult rat brain, including the relative expression of neuronal and glial cell markers at different time points. We tested the hypothesis that long-term neuronal viability is compatible with glial proliferation in adult neuron culture. We examined neuron culture from adult rat brain, which was maintained at steady state up to 24 days, and characterized them on the basis of cellular, molecular and biochemical properties at different time points of the culture. We identified neuronal and glial cells by both immunocytochemical and western immunoblotting techniques using NSE and Tau as neuronal markers and GFAP as glial protein marker, which revealed the presence of predominantly neuronal cells in the initial phase of the culture and a rise in glial cells from day 12 onwards. Notably, neuronal cells were preserved in the culture along with the glial cells even at day 24. Transfection of the cultured cells with a GFP expression vector and plasmids containing a luciferase reporter gene under the control of two different gene promoters demonstrated DNA transfectability. Taken together, these results suggest a differential expression of neuronal and glial cells at different time points and long-term neuronal viability in the presence of glial proliferation. Such adult neurons serve as a suitable system for the application of neurodegeneration models and for drug target discovery in various brain disorders including Alzheimer's disease.  

4.739           Proinflammatory and immunoregulatory mechanisms in periapical lesions

Colic, M., Gazivoda, D., Vucevic, D., Vasilijic, S., Rudolf, R. and Lukic, A. Mol. Immunol., 47, 101-113 (2009)   Proinflammatory and immunoregulatory cytokines are important for the pathogenesis of periapical lesions. However, little is known about how their functions are balanced and controlled at different phases of lesion development. The aim of this study was to examine the relationship between the production of Th1, Th2, Th17 and T regulatory cell (T reg) cytokines by human periapical lesion mononuclear cells (PL-MNC) in culture and their correlation with cellular composition and clinical presentation of the lesions. We show that symptomatic lesions are characterized by the infiltration of neutrophils, high production of IL-17, positive correlation between IL-17 and IFN-γ, but not between IL-17 and IL-23 production. Most IL-17⁺ cells coexpressed IFN-γ. Asymptomatic lesions were phenotypically heterogeneous. The lesions with the predominance of T cells over B cells/plasma cells expressed higher levels of IFN-γ which correlated with higher production of IL-12 and the frequency of macrophages. In contrast, in most B-type lesions higher levels of IL-5 and TGF-β were observed, as well as positive correlation between the production of TGF-β and IL-10. The addition of Th cytokines in PL-MNC cultures confirmed that Th1, Th2 and Th17 cytokines are mutually antagonistic, except that IL-17, unexpectedly, augmented the production of IFN-γ. IL-10 and TGF-β inhibited the production of both Th1 and Th17 cytokines. Dendritic cells (DCs) from periapical lesions, composed of immature (CD83⁻), and mature (CD83⁺) myeloid type DCs and plasmacytoid (BDCA2⁺) DCs produced higher levels of IL-12 and IL-23 but lower levels of IL-10 and TNF-α than monocyte (Mo) -derived DCs. IL-23 stimulated the production of IL-17 by PL-MNC, whereas the secretion of IFN-γ was enhanced by both IL-12 and IL-23. Cumulatively, these results suggest that: (1) Th1 immune response is most probably important for all stages of periapical lesion development; (2) Th2 and immunoregulatory cytokines are more significant for advanced types of lesions with the predominance of B cells/plasma cells; (3) Th17 immune response seems to play a dominant role in exacerbating inflammation.  

4.740           Mitochondrial DNA content in peripheral blood monocytes: relationship with age of diabetes onsetand diabetic complications

Wong, J., McLennan, S.V., Molyneaux, L., Min, D., Twigg, S.M. and Yue, D.K. Diabetologia, 52, 1953-1961 (2009)   Aims/hypothesis  We examined whether age of type 2 diabetes onset is related to mitochondrial DNA content in peripheral blood monocytes (PBMCs). Methods  PBMCs were isolated from 65 patients with type 2 diabetes. To minimise age as a confounder, only patients aged ≥50 years were studied. Sample mitochondrial DNA (mtDNA) content was determined by amplification of the mitochondrial gene CYT-B (also known as MT-CYB) and adjusted for single-copy nuclear control genes (36B4 [also known as RPLPO] and GAPDH). Results  Age of diabetes onset ranged from 25 to 69 years. There was a significant positive relationship between age of diabetes onset in quartiles and mtDNA content for the whole group (p = 0.02 for trend). When stratified by the presence of diabetes complications, a strong positive relationship was observed between age of diagnosis and mtDNA content for participants without diabetic complications (r = 0.7; p = 0.0002), but not for those with complications (r = −0.04; p = 0.8). Multivariate analysis confirmed age of onset and complication status as independent determinants. There was co-linearity between age of onset and disease duration, with similar relationships also seen between duration and mtDNA content. Conclusions/interpretation  An earlier age of type 2 diabetes onset is associated with a lower PBMC mtDNA content, but only in patients without diabetes complications. This may reflect a differing biology of PBMC mtDNA in those with early-onset diabetes and those who are prone to complications. PBMC mtDNA depletion may accelerate diabetes onset; however the independent effect of diabetes duration remains to be evaluated.  

4.741           Analysis of Neuroprotective Effects of Valproic Acid on Primary Motor Neurons in Monoculture or Co-cultures with Astrocytes or Schwann Cells

Ragancokova, D., Jahn, K., Kotsiari, A., Schlesinger, F., Haastert, K., Stangel, M., petri, S. and Krampfl, K. Cell. Mol. Neurobiol., 29, 1037-1043 (2009)   Chronic dysregulation of the intracellular Ca²⁺ homeostasis (excitotoxicity) is thought to contribute to the development of motor neuron diseases. Valproic acid (VPA) is widely used as an antiepileptic drug and acts mainly by inhibition of sodium channels and by enhancing the level of the inhibitory neurotransmitter γ-aminobutyric acid. Neuroprotective capacities of VPA are supposed to arise also from the inhibition of histone deacetylases. We investigated the viability of highly purified rat embryonic motor neurons cultured on glial feeder layers, composed of either astrocytes or Schwann cells, or in the absence of glia, monoculture in presence of VPA and/or kainate (KA) using immunocytochemistry and calcium imaging. A significant effect of the culture and co-culture conditions on the viability of motor neurons in our in vitro model of excitotoxicity was detected. The neuroprotective effect of VPA on primary embryonic motor neuron cultures was not proven. A functional interaction between VPA and KA occurred during the first 10 days in culture.  

4.742           Transcriptional analysis of intracytoplasmically stained, FACS-purified cells by high-throughput, quantitative nuclease protection

Pechhold, S., Stouffer, M., Walker, G., Martel, R., Seligmann, B., Hang, Y., Stein, R., harlan, D.M. and Pechhold, K. Nature Biotechnol., 27(11), 1038-1042 (2009)   Analyzing specialized cells in heterogeneous tissues is crucial for understanding organ function in health and disease. Thus far, however, there has been no convenient method for studying gene expression in cells purified by fluorescence-activated cell sorting (FACS) using intracellular markers. Here we show that the quantitative nuclease protection assay (qNPA) enables transcriptional analysis of intracytoplasmically stained cells sorted by FACS. Applying the method to mouse pancreatic islet–cell subsets, we detected both expected and unknown lineage-specific gene expression patterns. Some beta cells from pregnant animals were found to express Mafb, previously observed only in immature beta cells during embryonic development. The four 'housekeeping' genes tested were expressed in purified islet-cell subpopulations with a notable variability, dependent on both cell lineage and developmental stage. Application of qNPA to intracellularly stained, FACS-sorted cells should be broadly applicable to the analysis of gene expression in subpopulations of any heterogeneous tissue, including tumors.  

4.743           Galactosylated LDL Nanoparticles: A Novel Targeting Delivery System To Deliver Antigen to Macrophages and Enhance Antigen Specific T Cell Responses

Wu, F., Wuensch, S.A., Azadniv, M., Ebrahimkhani, M.R. and Crispe, I.N. Mol. Pharmaceutics, 6(5), 1506-1517 (2009)   We aim to define the role of Kupffer cells in intrahepatic antigen presentation, using the selective delivery of antigen to Kupffer cells rather than other populations of liver antigen-presenting cells. To achieve this we developed a novel antigen delivery system that can target antigens to macrophages, based on a galactosylated low-density lipoprotein nanoscale platform. Antigen was delivered via the galactose particle receptor (GPr), internalized, degraded and presented to T cells. The conjugation of fluoresceinated ovalbumin (FLUO-OVA) and lactobionic acid with LDL resulted in a substantially increased uptake of FLUO-OVA by murine macrophage-like ANA1 cells in preference to NIH3T3 cells, and by primary peritoneal macrophages in preference to primary hepatic stellate cells. Such preferential uptake led to enhanced proliferation of OVA specific T cells, showing that the galactosylated LDL nanoscale platform is a successful antigen carrier, targeting antigen to macrophages but not to all categories of antigen presenting cells. This system will allow targeted delivery of antigen to macrophages in the liver and elsewhere, addressing the question of the role of Kupffer cells in liver immunology. It may also be an effective way of delivering drugs or vaccines directly at macrophages.  

4.744           Th1/Th17 Immune Response Is Induced by Mesenteric Lymph Node Dendritic Cells in Crohn's Disease

Sakuraba, A., Sato, T., Kamada, N., Kitazume, M., Sugita, A. And Hibi, T. Gastroenterol., 137, 1736-1745 (2009)   Background & Aims Dendritic cells (DCs) possess the most potent ability to induce acquired immunity. However, their involvement in the pathogenesis of Crohn's disease (CD) has not yet been determined. We aimed to establish the immune status of mesenteric lymph nodes, the major gut-associated lymphoid tissue, and isolated DCs and determine their involvement in the pathogenesis of CD. Methods CD4⁺ T cells and DCs were isolated from mesenteric lymph nodes of CD, ulcerative colitis, and normal control. The immune status of CD4⁺ T cells was analyzed by cytokine production and transcriptional profile. Surface phenotype of DCs was analyzed by flow cytometry. Cytokine production by myeloid DCs was analyzed by real-time polymerase chain reaction and exogenous bacterial stimulation. Immune stimulating activity of DCs was determined by mixed lymphocyte reaction. Results In CD, mesenteric lymph node CD4⁺ T cells produced higher amounts of interferon-γ and interleukin (IL)-17 compared with ulcerative colitis and normal control, and this was dictated by increased T-bet and retinoic acid-related orphan receptor-γ expression. Three subtypes of DCs, myeloid DC, plasmacytoid DC, and mature DC, were identified in all groups. When stimulated with exogenous bacterial derivative, myeloid DCs from CD produced a higher amount of IL-23 and a lower amount of IL-10. Myeloid DCs from CD induced stronger T helper cell (Th)1 immune response in mixed lymphocyte reaction compared with those from ulcerative colitis and normal control. Conclusions Our findings revealed that mesenteric lymph node is the key pathogenic location of CD elicited by the unique cytokine milieu produced by DCs leading to a dysregulated Th1/Th17 immune response.  

4.745           Factor VIII C1 domain residues Lys 2092 and Phe 2093 contribute to membrane binding and cofactor activity

Meems, H., Meijer, A.B., Cullinan, D.B., Mertens, K. and Gilbert, G.E. Blood, 114(18), 3938-3946 (2009)   Binding of factor VIII to membranes containing phosphatidyl-L-serine (Ptd-L-Ser) is mediated, in part, by a motif localized to the C2 domain. We evaluated a putative membrane-binding role of the C1 domain using an anti-C1 antibody fragment, KM33_scFv, and factor VIII mutants with an altered KM33 epitope. We prepared a dual mutant Lys2092/Phe2093 Ala/Ala (fVIII_{YFP 2092/93}) and 2 single mutants Lys2092 Ala and Phe2093 Ala. KM33_scFv inhibited binding of fluorescein-labeled factor VIII to synthetic membranes and inhibited at least 95% of factor Xase activity. fVIII_{YFP 2092/93} had 3-fold lower affinity for membranes containing 15% Ptd-L-Ser but more than 10-fold reduction in affinity for membranes with 4% Ptd-L-Ser. In a microtiter plate, KM33_scFv was additive with an anti-C2 antibody for blocking binding to vesicles of 15% Ptd-L-Ser, whereas either antibody blocked binding to vesicles of 4% Ptd-L-Ser. KM33_scFv inhibited binding to platelets and fVIII_{YFP 2092/93} had reduced binding to A23187 [GenBank] -stimulated platelets. fVIII_{YFP 2092} exhibited normal activity at various Ptd-L-Ser concentrations, whereas fVIII_{YFP 2093} showed a reduction of activity with Ptd-L-Ser less than 12%. fVIII_{YFP 2092/93} had a greater reduction of activity than either single mutant. These results indicate that Lys 2092 and Phe 2093 are elements of a membrane-binding motif on the factor VIII C1 domain.  

4.746           Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver

Schreiber, R., Taschler, U., Wolinski, H., Seper, A., Tamegger, S.N., Graf, M., Kohlwein, S.D., Haemmerle, G., Zimmermann, R., Zechner, R. and Lass, A.

1. Lipid Res., 50, 2514-2523 (2009)

  Excess dietary vitamin A is esterified with fatty acids and stored in the form of retinyl ester (RE) predominantly in the liver. According to the requirements of the body, liver RE stores are hydrolyzed and retinol is delivered to peripheral tissues. The controlled mobilization of retinol ensures a constant supply of the body with the vitamin. Currently, the enzymes catalyzing liver RE hydrolysis are unknown. In this study, we identified mouse esterase 22 (Es22) as potent RE hydrolase highly expressed in the liver, particularly in hepatocytes. The enzyme is located exclusively at the endoplasmic reticulum (ER), implying that it is not involved in the mobilization of RE present in cytosolic lipid droplets. Nevertheless, cell culture experiments revealed that overexpression of Es22 attenuated the formation of cellular RE stores, presumably by counteracting retinol esterification at the ER. Es22 was previously shown to form a complex with β-glucuronidase (Gus). Our studies revealed that Gus colocalizes with Es22 at the ER but does not affect its RE hydrolase activity. Interestingly, however, Gus was capable of hydrolyzing the naturally occurring vitamin A metabolite retinoyl β-glucuronide. In conclusion, our observations implicate that both Es22 and Gus play a role in liver retinoid metabolism.  

4.747           AIRE activated tissue specific genes have histone modifications associated with inactive chromatin

Org, T., Rebane, A., Kisand, K., Laan, M., Haljasorg, U., Andreson, R. And Peterson, P. Hum. Mol. Genet., 18(24), 4699-4710 (2009)   The Autoimmune Regulator (AIRE) protein is expressed in thymic medullary epithelial cells, where it promotes the ectopic expression of tissue-restricted antigens needed for efficient negative selection of developing thymocytes. Mutations in AIRE cause APECED syndrome, which is characterized by a breakdown of self-tolerance. The molecular mechanism by which AIRE increases the expression of a variety of different genes remains unknown. Here, we studied AIRE-regulated genes using whole genome expression analysis and chromatin immunoprecipitation. We show that AIRE preferentially activates genes that are tissue-specific and characterized by low levels of initial expression in stably transfected HEK293 cell model and mouse thymic medullary epithelial cells. In addition, the AIRE-regulated genes lack active chromatin marks, such as histone H3 trimethylation (H3K4me3) and acetylation (AcH3), on their promoters. We also show that during activation by AIRE, the target genes acquire histone H3 modifications associated with transcription and RNA polymerase II. In conclusion, our data show that AIRE is able to promote ectopic gene expression from chromatin associated with histone modifications characteristic to inactive genes.  

4.748           Monocyte urokinase-type plasminogen activator up-regulation reduces thrombus size in a model of venous thrombosis

Humphries, J., Gossage, J.A., Modarai, B., Burnand, K.G., Sisson, T.H., Murdoch, C. and Smith, A.

1. Vasc. Surg., 50, 1127-1134 (2009)

  Background Our previous studies showed that the direct injection of an adenovirus construct expressing urokinase-type plasminogen activator (uPA) into experimental venous thrombi significantly reduces thrombus weight. The systemic use of adenovirus vectors is limited by inherent hepatic tropism and inflammatory response. As macrophages are recruited into venous thrombi, it is reasonable to speculate that these cells could be used to target the adenovirus uPA (ad-uPA) gene construct to the thrombus. The aims of this study were to determine whether macrophages transduced with ad-uPA have increased fibrinolytic activity and whether systemic injection of transduced cells could be used to target uPA expression to the thrombus and reduce its size. Methods The effect of up-regulating uPA was examined in an immortalized macrophage cell line (MM6) and macrophages differentiated from human blood monocyte-derived macrophages (HBMMs). Cells were infected with ad-uPA or blank control virus (ad-blank). Fibrinolytic mediator expression, cell viability, and cytokine expression were measured by activity assays and enzyme-linked immunosorbent assays. Monocyte migration was measured using a modified Boyden chamber assay. A model of venous thrombosis was developed and characterized in mice with severe combined immunodeficiency (SCID). This model was used to study whether systemically administered macrophages over-expressing uPA reduced thrombus size. Uptake of HBMMs into the thrombus induced in these mice was confirmed by a combination of PKH2-labeled cell tracking and colocalization with human leukocyte antigen (HLA) by immunohistology. Results Compared with ad-blank, treated HBMMs transduction with ad-uPA increased uPA production by >1000-fold (P = .003), uPA activity by 150-fold (P = .0001), and soluble uPA receptor (uPAR) by almost twofold (P = .043). Expression of plasminogen activator inhibitor (PAI-1) and PAI-2 was decreased by about twofold (P = .011) and threefold (P = .005), respectively. Up-regulation of uPA had no effect on cell viability or inflammatory cytokine production compared with ad-blank or untreated cells. Ad-uPA transduction increased the migration rate of HBMMs (about 20%, P = .03) and MM6 cells (>twofold, P = .005) compared with ad-blank treated controls. Human macrophage recruitment into the mouse thrombus was confirmed by the colocalization of HLA with the PKH2-marked cells. Systemic injection of uPA-up-regulated HBMMs reduced thrombus weight by approximately 20% compared with ad-blank (P = .038) or sham-treated controls (P = .0028). Conclusion Transduction of HBBM with ad-uPA increases their fibrinolytic activity. Systemic administration of uPA up-regulated HBBMs reduced thrombus size in an experimental model of venous thrombosis. Alternative methods of delivering fibrinolytic agents are worth exploring. Clinical Relevance The use of thrombolysis in the treatment of acute iliofemoral deep vein thrombosis is not suitable for all patients. Our previous studies have shown that direct injection of an adenovirus construct expressing urokinase plasminogen activator (uPA) into experimental venous thrombi significantly reduced thrombus weight. The systemic use of adenovirus vectors is, however, limited by both their inherent hepatic tropism, which precludes targeted delivery to disease sites, and by the associated host inflammatory response. As macrophages are recruited into venous thrombi, these cells could be used to target uPA gene constructs to the thrombus after systemic administration.  

4.749           Muscle-conditioned media and cAMP promote survival and neurite outgrowth of adult spinal cord motor neurons

Montoya, J.V., Sutachan, J.J., Chan, W.S., Sideris, A., Blanck, T.J.J. and Recio-Pinto, E. Exp. Neurol., 220, 303-315 (2009)   Embryonic spinal cord motor neurons (MNs) can be maintained in vitro for weeks with a cocktail of trophic factors and muscle-derived factors under serum-containing conditions. Here we investigated the beneficial effects of muscle-derived factors in the form of muscle-conditioned medium (MCM) on the survival and neurite outgrowth of adult rat spinal cord MNs under serum-free conditions. Ventral horn dissociated cell cultures from the cervical enlargement were maintained in the presence of one or more of the following factors: brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), a cell permeant cyclic adenosine-3′,5′-monophosphate (cAMP) analog and MCM. The cell cultures were immunostained with several antibodies recognizing a general neuronal marker the microtubule-associated protein 2 (MAP2) and either one or more motor neuronal markers: the non-phosphorylated neurofilament heavy isoform (SMI32), the transcription factors HB9 and Islet-1 and the choline acetyl transferase. We found that treatment with MCM together with the cAMP analog was sufficient to promote selective survival and neurite outgrowth of adult spinal cord MNs. These conditions can be used to maintain adult spinal cord MNs in dissociated cultures for several weeks and may have therapeutic potential following spinal cord injury or motor neuropathies. More studies are necessary to evaluate how MCM and the cAMP analog act in synergy to promote the survival and neurite outgrowth of adult MNs.  

4.750           Phosphodiesterase 3 Inhibition Reduces Platelet Activation and Monocyte Tissue Factor Expression in Knee Arthroplasty Patients

Beppu, S., Nakajima, Y., Shibasaki, M., Kageyama, K., Mizobe, T., Shime, N. and Matsuda, N. Anesthesiology, 111, 1227-1237 (2009)   Background: Tissue damage during surgery activates platelets and provokes a prothrombic state. The current study attempted to determine the impact of phosphodiesterase 3 inhibitors on platelet activation, platelet-leukocyte aggregate formation, and monocyte tissue factor expression during and after total knee arthroplasty. Methods: Thirty-four patients undergoing scheduled total knee arthroplasty were randomly assigned to receive either the phosphodiesterase 3 inhibitor milrinone or the same amount of saline perioperatively. The effects of milrinone on platelet and leukocyte function in vitro were then assessed in healthy volunteers. Results: Perioperative infusion of milrinone significantly attenuated platelet activation; phosphorylation of intraplatelet p38 mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2, and Akt; and platelet-leukocyte aggregation. Furthermore, perioperative tissue factor expression on monocytes and fibrin monomer complex production were reduced by milrinone infusion in patients undergoing total knee arthroplasty. In vitro studies using adenosine diphosphate- and collagen-stimulated blood samples from healthy volunteers confirmed the antiplatelet effects and reduced monocyte tissue factor expression by milrinone. These studies further showed that platelet aggregation and integrin [alpha]IIb[beta]3 activation were modified by intraplatelet phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways, and that P-selectin expression on platelets and platelet-leukocyte aggregation were modulated by intraplatelet p38 mitogen-activated protein kinase pathway. Conclusion: Continuous milrinone infusion has the potential to reduce platelet activation and monocyte tissue factor expression during the perioperative period in total knee arthroplasty. These events may be mediated in part by the ability of milrinone to reduce activation of intraplatelet mitogen-activated protein kinases and phosphatidylinositol 3-kinase. The clinical impact of phosphodiesterase 3 inhibition on perioperative hemostasis remains to be elucidated.  

4.751           Improved, Low-Cost Methods for Pancreatic Islet Purification in Rats

Zhang, X-L., Yang, J-K., Yu, M. And Xue, G-F. Transplant. Proceedings, 41, 4297-4301 (2009)   Objective Density gradient separation of islets from exocrine tissue is usually performed using Ficoll. However, this reagent significantly increases the cost of isolation. The aim of the present study was to investigate the effects on islet preparations of purification methods using Lymphoprep and Iodixanol (OptiPrep) density gradients. Methods Pancreata were procured from 46 Wistar rats, loaded with collagenase V (Sigma), and mechanically dissociated using standard procedures. After the digestion phase, the islets purified by 3 methods—Ficoll, Lymphoprep, and Iodixanol (OptiPrep) —were assessed for yields, purity, morphology, and in vitro function. Result We expressed the yields as islet equivalents (IEQ, diameter standardizing to 150 μm), showing no significant differences. Compared with the Ficoll group, the purity was significantly higher in the Lymphoprep (P = .005) and Iodixanol (OptiPrep) groups (P = .011). While the viability was >90% in all 3 groups, the viability in the Lymphoprep Group and OptiPrep groups was significantly higher than that of the Ficoll group (P < .001). In vitro islet function did not differ among the 3 experimental groups. Conclusion Lymphoprep and Iodixanol were as effective as Ficoll in terms of islet yield and in vitro function. High-purity and high-viability islet cells were obtained using improved Lymphoprep-based or Iodixanol (OptiPrep) -based density gradient methods, potential low-cost substitutes for Ficoll.  

4.752           Stimulation of stellate cells by injured acinar cells: a model of acute pancreatitis induced by alcohol and fat (VLDL)

Siech, M., Zhou, Z., Zhou, S., Bair, B., Alt, A., Hammm, S., Gross, H., Mayer, J., Beger, H.G., Tian, X., Kornmann, M. and Bachem, M.G. Am. J. Physiol. Gastrintest. Liver Physiol., 297, G1163-G1171 (2009)   Mechanisms leading to acute pancreatitis after a fat-enriched meal combined with excess alcohol are incompletely understood. We have studied the effects of alcohol and fat (VLDL) on pancreatic acinar cell (PAC) function, oxidative stress, and repair mechanisms by pancreatic stellate cells (PSC) leading to fibrogenesis. To do so, PAC (rat) were isolated and cultured up to 24 h. Ethanol and/or VLDL were added to PAC. We measured PAC function (amylase, lipase), injury (lactic dehydrogenase), apoptosis (TUNEL, Apo2.7, annexin V binding), oxidative stress, and lipid peroxidation (conjugated dienes, malondialdehyde, chemoluminescence); we also measured PSC proliferation (bromodeoxyuridine incorporation), matrix synthesis (immunofluorescence of collagens and fibronectin, fibronectin immunoassay), and fatty acids in PAC supernatants (gas chromatography). Within 6 h, cultured PAC degraded and hydrolyzed VLDL completely. VLDL alone (50 µg/ml) and in combination with alcohol (0.2, 0.5, and 1% vol/vol) induced PAC injury (LDL, amylase, and lipase release) within 2 h through generation of oxidative stress. Depending on the dose of VLDL and alcohol, apoptosis and/or necrosis were induced. Antioxidants (Trolox, Probucol) reduced the cytotoxic effect of alcohol and VLDL. Supernatants of alcohol/VLDL-treated PAC stimulated stellate cell proliferation and extracellular matrix synthesis. We concluded that, in the presence of lipoproteins, alcohol induces acinar cell injury. Our results provide a biochemical pathway for the clinical observation that a fat-enriched meal combined with excess alcohol consumption can induce acinar cell injury (acute pancreatitis) followed by repair mechanisms (proliferation and increased matrix synthesis in PSC).  

4.753           Toll-Like Receptor Triggering and T-Cell Costimulation Induce Potent Antitumor Immunity in Mice

Westwood, J.A., Haynes, N.M., Sharkey, J., McLaughlin, N., Pegram, H.J., Schwendener, R.A., Smyth, M.J., Darcy, P.K. and Kershaw, M.H. Clin. Cancer Res., 15(24), 7624-7633 (2009)   Purpose: To determine the antitumor activity of a novel combination of two immunomodulatory agents that simultaneously direct multiple components of immunity against cancer. Experimental Design: We combined the Toll-like receptor agonist CpG 1826 with a T-cell costimulatory antibody specific for CD137 in an optimal treatment route and dosing schedule against established tumors in two mouse models. Mechanistic insight was gained using gene-deficient mice and cell-depleting antibodies. Results: The combination was shown to eradicate tumors in a large proportion of mice. Crucial roles for CD8⁺ T cells, natural killer cells, and IFNs were shown. CpG and anti-CD137 injection led to activation of dendritic cells and optimal expansion of activated T cells in the blood. Macrophages were not necessary for therapeutic effect, and indeed depletion of macrophages in vivo enhanced therapy leading to tumor rejection in 100% of mice, which has not been previously reported in the immunotherapeutic setting. Long-term surviving mice were resistant to tumor rechallenge, demonstrating immunologic memory. In addition, we show, for the first time, that mice lacking B cells have a total loss of a recall response against tumor, suggesting a role for B cells in the induction of antitumor immunologic memory. Conclusion: This study provides support for the use of a novel combination of immunomodulatory agents stimulating multiple facets of immunity for the effective immunotherapy of cancer.  

4.754           Neuron survival in vitro is more influenced by the developmental age of the cells than by glucose condition

Sepehr, A., Ruud, J. and Mohseni, S. Cytotechnology, 61, 73-79 (2009)   The objective of this study was to determine whether the sensitivity to varying glucose conditions differs for the peripheral and central nervous system neurons at different developmental stages. Ventral horn neurons (VHN) and dorsal root ganglion neurons (DRG) from rats of different postnatal ages were exposed to glucose-free or glucose-rich culture conditions. Following 24 h at those conditions, the number of protein gene product 9.5 positive (PGP+) DRG neurons and choline acetyltransferase positive (ChAT+) VHN were counted and their neurite lengths and soma diameters were measured. For both DRG and VHN, the highest number of cells with and without neurite outgrowth was seen when cells from postnatal day 4 donors were cultured, while the lowest cell numbers were when neurons were from donors early after birth and grown under glucose-free conditions. The length of the neurites and the soma diameter for VHN were not affected by either glucose level or age. DRG neurons, however, exhibited the shortest neurites and smallest soma diameter when neurons were obtained and cultured early after birth. Our results indicate that survival of neurons in vitro is more influenced by the developmental stage than by glucose concentrations.  

4.755           Hoechst 33342 Side Population Identification Is a Conserved and Unified Mechanism in Urological Cancers

Oates, J.E., Grey, B.R., Addla, S.K., Samuel, J.D., Hart, C.A., Ramani, V.A.C., Brown, M.D. and Clarke, N.W. Stem Cells and Development, 18(10), 1515-1521 (2009) Mutation within the adult human stem cell (SC) compartment has been proposed as a factor in the initiation and promotion of carcinogenesis. Isolation of these cancer stem cells (CSCs) has proven difficult, limiting their subsequent phenotypic, functional, and genetic characterization. We have used the Hoechst 33342 dye efflux technique to isolate an epithelial side population (SP) from genitourinary (GU) cancers, which is enriched for cells with SC traits. With informed consent, samples were taken from patients with primary tumors and undergoing surgery for prostatic (CaP), invasive bladder transitional cell (TCC), and renal cell carcinomas (RCC). Single cell epithelial suspensions were extracted from these and incubated with Hoechst 33342. Hoechst SP/non-SP profiles were then generated by flow cytometry using standardized protocols. SP/non-SP cell cycle status was established by Hoechst 33342 and Pyronin Y staining. Immunocytochemistry staining was performed for markers suggested as stem markers as well as lineage-specific markers. Functionality was determined using colony-forming assays and long-term monolayer culture. A characteristic verapamil-sensitive SP was isolated from all 3 urological malignancies and represented 0.57% ± 0.11% (CaP), 0.52% ± 0.49% (TCC), and 5.9% ± 0.9% (RCC) of the total epithelial population. Cell cycle analysis showed that the SP had enhanced numbers of cells in G₀ as compared to the total cell population (CaP 12.4% ± 3.2 vs. 3.8% ± 1.0, RCC 23.2% ± 3.4 vs. 1.8% ± 0.9, and TCC 28.5% ± 4.9 vs. 4% ± 1.3). Immunocytochemistry demonstrated an increased expression of proliferative and putative stem markers within the SP fraction. Cultures confirmed significant enhancement of colony-forming ability and proliferative capacity of the SP fraction. A characteristic SP enriched for stem-like cells has been isolated from the 3 most common urological malignancies. This provides strong evidence that Hoechst 33342 efflux is a conserved and unified mechanism in GU cancer.  

4.756           Indirect action of tumor necrosis factor-alpha in liver injury during the CD8+ T cell response to an adeno-associated virus vector in mice

Ginnandrea, M., Pierce, R.H. and Crispe, I.N. Hepatology, 49(6), 2010-2020 (2009)   CD8+ T cells can cause hepatocellular injury by two distinct mechanisms. In addition to their direct cytotoxic effect, there is also collateral liver injury, which occurs when cells are killed in an antigen-independent manner. Whereas immune effector cytokines interferon-gamma (IFN ) and tumor necrosis factor-alpha (TNF ) have both been implicated in various forms of hepatitis, their respective roles in direct and/or collateral liver damage remains unclear. In order to investigate these elements of liver injury, we developed a new experimental model of CD8+ T-cell–mediated hepatitis based on an adeno-associated virus-based gene therapy vector. This vector is used to deliver antigen to hepatocytes, and CD8+ T cells specific for the vector-encoded transgene are adoptively transferred to produce liver immunopathology. In this experimental model, CD8+ T-cell IFN acts on Kupffer cells, inducing TNF secretion and liver injury. Both IFN and TNF are important in this injury process, but TNF acts as an autocrine amplifier of Kupffer cell function, rather than as a direct effector of hepatocellular damage. Conclusions: TNF indirectly promotes liver damage and is not a direct hepatotoxic agent. IFN also indirectly contributes to liver injury through Kupffer cell activation while, in parallel, directly promoting hepatitis through induction of hepatocyte major histocompatability complex class I. In principle, it may be possible to ameliorate this immunopathologic indirect mechanism by developing therapies that target Kupffer cells, without impairing CD8+ T-cell-mediated antiviral immunity. This would have great therapeutic potential in chronic viral hepatitis.  

4.757           Thymus cell antigen-1-expressing cells in the oval cell compartment

Yovchev, M.I., Zhang, J., Neufeld, D.S., Grozdanov, P.N. and Dabeva, M.D. Hepatology, 50(2), 601-611 (2009)   Thymus cell antigen-1 (Thy-1)-expressing cells proliferate in the liver during oval cell (OC)-mediated liver regeneration. We characterized these cells in normal liver, in carbon tetrachloride-injured liver, and in several models of OC activation. The gene expression analyses were performed using reverse-transcriptase polymerase chain reaction (RT-PCR), quantitative RT-PCR (Q-RT-PCR) of cells isolated by fluorescence-activated cell sorting (FACS), and by immunofluorescent microscopy of tissue sections and isolated cells. In normal liver, Thy-1+ cells are a heterogeneous population: those located in the periportal region do not coexpress desmin or alpha smooth muscle actin ( -SMA). The majority of Thy-1+ cells located at the lobular interface and in the parenchyma coexpress desmin but not -SMA, i.e., they are not resident myofibroblasts. Although Thy-1+ cells proliferate moderately after carbon tetrachloride injury, in all models of OC-mediated liver regeneration they proliferate quickly and expand significantly and disappear from the liver when the OC response subsides. Activated Thy-1+ cells do not express OC genes but they express genes known to be expressed in mesenchymal stem cells (CD105, CD73, CD29), genes considered specific for activated stellate cells (desmin, collagen I-a2, Mmp2, Mmp14) and myofibroblasts ( -SMA, fibulin-2), as well as growth factors and cytokines (Hgf, Tweak, IL-1b, IL-6, IL-15) that can affect OC growth. Activated in vitro stellate cells do not express Thy-1. Subcloning of Thy-1+ cells from OC-activated livers yield Thy-1+ fibroblastic cells and a population of E-cadherin+ mesenchymal cells that gradually discontinue expression of Thy-1 and begin to express cytokeratins. However, upon transplantation these cells do not differentiate into hepatocytes or cholangiocytes. Activated Thy-1+ cells produce predominantly latent transforming growth factor beta. Conclusion: Thy-1+ cells in the OC niche are activated mesenchymal-epithelial cells that are distinct from resident stellate cells, myofibroblasts, and oval cells.  

4.758           Porcine liver sinusoidal endothelial cells contribute significantly to intrahepatic ammonia metabolism

Nedredal, G.I., Elvevold, K., Ytrebø, L.M., Fuskevåg, O-M., Pettersen, I., McCourt, P.A., Bertheussen, K., Smedsrød, b. and Revhaug, A. Hepatology, 50(3), 900-908 (2009)   Ammonia metabolism in the liver has been largely credited to hepatocytes (HCs). We have shown that liver nonparenchymal cells that include liver sinusoidal endothelial cells (LSECs) produce ammonia. To address the limited knowledge regarding a role for LSECs in ammonia metabolism, we investigated the ammonia metabolism of isolated LSECs and HCs under three different conditions: (1) bioreactors containing LSECs (LSEC-bioreactors), (2) bioreactors containing HCs (HC-bioreactors), and (3) separate bioreactors containing LSECs and HCs connected in sequence (Seq-bioreactors). Our results showed that LSEC-bioreactors released six-fold more ammonia (22.2 nM/hour/106 cells) into the growth media than HC-bioreactors (3.3 nM/hour/106 cells) and Seq-bioreactors (3.8 nM/hour/106 cells). The glutamate released by LSEC-bioreactors (32.0 nM/hour/106 cells) was over four-fold larger than that released by HC-bioreactors and Seq-bioreactors (<7 nM/hour/106 cells). LSEC-bioreactors and HC-bioreactors consumed large amounts of glutamine (>25 nM/hour/106 cells). Glutaminase is known for catalyzing glutamine into glutamate and ammonia. To determine if this mechanism may be responsible for the large levels of glutamate and ammonia found in LSEC-bioreactors, immunolabeling of glutaminase and messenger RNA expression were tested. Our results demonstrated that glutaminase was present with colocalization of an LSEC-specific functional probe in lysosomes of LSECs. Furthermore, using a nucleotide sequence specific for kidney-type glutaminase, reverse-transcription polymerase chain reaction revealed that this isoform of glutaminase was expressed in porcine LSECs. Conclusion: LSECs released large amounts of ammonia, perhaps due to the presence of glutaminase in lysosomes. The ammonia and glutamate released by LSECs in Seq-bioreactors were used by hepatocytes, suggesting an intrahepatic collaboration between these two cell types.  

4.759           Qualitative and quantitative comparison of N-glycans between pig endothelial and islet cells by high-performance liquid chromatography and mass spectrometry-based strategy

Kim, Y-G., Gil, G-C., Jang, K-S., Lee, S., Kim, H-i., Kim, J-S., Chung, J., Park, C-G., Harvey, D.J. and Kim, B-G.

1. Mass. Spectrom., 44(7), 1087-1104 (2009)

  N-glycan structures released from miniature pig endothelial and islet cells were determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), negative ion electrospray ionization (ESI) MS/MS and normal-phase high performance liquid chromatography (NP-HPLC) combined with exoglycosidase digestion. Totally, the identified structures were 181 N-glycans including 129 sialylated and 18 -galactosylated glycans from pig endothelial cells and 80 N-glycans including 41 sialylated and one -galactosylated glycans from pig islet cells. The quantity of the -galactosylated glycans from pig islet cells was certainly neglectable compared to pig endothelial cells. A number of NeuGc-terminated N-glycans (80 from pig endothelial cells and 13 from pig islet cells) are newly detected by our mass spectrometric strategies. The detailed structural information will be a matter of great interest in organ or cell xenotransplantation using 1,3-galactosyltransferase gene-knockout (GalT-KO) pig.  

4.760           Mass spectrometric analysis of the glycosphingolipid-derived glycans from miniature pig endothelial cells and islets: identification of NeuGc epitope in pig islets

Kim, Y-G., Harvey, D.J., Yang, Y-H., Park, C-G. and Kim, B-G.

1. Mass. Spectrom., 44(10), 1489-1499 (2009)

  Glycosphingolipid (GSL) is a major component of the plasma membrane in eukaryotic cells that is involved directly in a variety of immunological events via cell-to-cell or cell-to-protein interactions. In this study, qualitative and quantitative analyses of GSL-derived glycans on endothelial cells and islets from a miniature pig were performed and their glycosylation patterns were compared. A total of 60 and 47 sialylated and neutral GSL-derived glycans from the endothelial cells and islets, respectively, were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and collision-induced fragmentation using positive-ion electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). In accordance with previous immunohistochemistry studies, the -Gal-terminated GSL was not detected but NeuGc-terminated GSLs were newly detected from miniature pig islets. In addition, the neutral GSL-derived glycans were relatively quantified by derivatization with carboxymethyl trimethylammonium hydrazide (so called Girard's T reagent) and MALDI-TOF MS. The structural information of the GSL-derived glycans from pig endothelial cells and islets suggests that special attention should be paid to all types of glycoconjugates expressed on pig tissues or cells for successful clinical xenotransplantation.  

4.761           Rapamycin inhibits hepatic fibrosis in rats by attenuating multiple profibrogenic pathways

Bridle, K.R., Popa, C., Morgan, M.L., Sobbe, A.L., Clouston, A.D., Fletcher, L.M. aand Crawford, D.H.G. Liver Transplant, 15(10), 1315-1324 (2009)   Hepatic stellate cell transdifferentiation, epithelial-mesenchymal cell transition, and the ductular reaction each contribute to the development of hepatic fibrosis in cholestatic liver diseases. Inhibitors of mammalian target of rapamycin have antifibrotic properties. We evaluated the hypothesis that the antifibrotic action of rapamycin is due to attenuated myofibroblast proliferation in addition to an inhibitory effect on epithelial-mesenchymal transition and the ductular reaction. Hepatic fibrosis was induced by bile duct ligation, and rodents received 1.5 mg/kg/day rapamycin by subcutaneous infusion for 21 days. The expression of various markers of hepatic fibrosis, stellate cell transactivation, epithelial-mesenchymal transition, and the ductular reaction was compared between treated and untreated animals. Hepatic fibrosis, hepatic procollagen type 1 messenger RNA, and alpha-smooth muscle actin expression were significantly reduced in treated animals. Hepatic stellate cell procollagen expression and proliferation were also reduced by rapamycin. The following markers of epithelial-mesenchymal transition—vimentin protein expression, S100 calcium binding protein A4 and transforming growth factor beta 1 messenger RNA, and the mothers against decapentaplegic homolog signaling pathway—were all reduced after rapamycin treatment. The intensity of the ductular reaction was reduced by rapamycin as assessed by histopathological scoring and by reduced cytokeratin 19 expression. Rapamycin caused a reduction in hepatic progenitor cell proliferation. Together, these data show that multiple profibrogenic pathways are activated in an animal model of cholestasis and that rapamycin attenuates epithelial-mesenchymal transition and the ductular reaction as well as hepatic stellate cell activation.  

4.762           Cholesterol-promoted synaptogenesis requires the conversion of chelosterol to estradiol in the Hippocampus

Fester, L., Zhou, L., Bütow, A., Huber, C., von Lossow, R., Prange-Kiel, J., Jarry, H. and Rune, G.M. Hippocampus, 19(8), 692-705 (2009)   Cholesterol of glial origin promotes synaptogenesis (Mauch et al., (2001) Science 294:1354–1357). Because in the hippocampus local estradiol synthesis is essential for synaptogenesis, we addressed the question of whether cholesterol-promoted synapse formation results from the function of cholesterol as a precursor of estradiol synthesis in this brain area. To this end, we treated hippocampal cultures with cholesterol, estradiol, or with letrozole, a potent aromatase inhibitor. Cholesterol increased neuronal estradiol release into the medium, the number of spine synapses in hippocampal slice cultures, and immunoreactivity of synaptic proteins in dispersed cultures. Simultaneous application of cholesterol and letrozole or blockade of estrogen receptors by ICI 182 780 abolished cholesterol-induced synapse formation. As a further approach, we inhibited the access of cholesterol to the first enzyme of steroidogenesis by knock-down of steroidogenic acute regulatory protein, the rate-limiting step in steroidogenesis. A rescue of reduced synaptic protein expression in transfected cells was achieved by estradiol but not by cholesterol. Our data indicate that in the hippocampus cholesterol-promoted synapse formation requires the conversion of cholesterol to estradiol.  

4.763           Androgen ablation augments prostate cancer vaccine immunogenicity only when applied after immunization

Koh, Y.T., gray, A., Higgins, S.A., Hubby, B. and Kast, W.M. The Prostate, 69(6), 571-584 (2009)   BACKGROUND Androgen ablation (AA) causes apoptosis of normal and neoplastic prostate cells. It is a standard treatment for advanced prostate cancer. Androgen ablation-mediated immunological effects include bone marrow hyperplasia, thymic regeneration, T and B cell lymphopoeisis and restoration of age-related peripheral T cell dysfunction. Androgens also regulate the transcription of several cytokines. Dendritic cells (DC) are the most potent antigen presenting cells that can activate antigen-specific naïve T cells. Despite myriad clinical trials involving DC-based prostate cancer immunotherapies, the effects of AA on DC function remain largely uncharacterized. Therefore, we investigated the effects of AA on DC and whether it could improve the efficacy of prostate cancer immunotherapy. METHODS Cytokine expression changes due to AA were quantified by multiplex ELISA. Flow cytometry was used to assess AA-mediated effects on DC maturation and expression of costimulatory markers. Mixed leukocyte reactions and cell-mediated lysis assays elucidated the role of androgens in DC function. The effect of AA on the efficacy of vaccination against a prostate tumor-associated antigen was tested using Elispot assays. RESULTS Androgen ablation increased dendritic cell maturation and costimulatory marker expression, but had no effect on DC costimulatory function. However, DC isolated from castrated mice increased the expression of key cytokines by antigen-experienced T cells while decreasing their expression in naïve cells. Finally, androgen ablation improved immune responses to vaccination only when applied after immunization. CONCLUSION Androgen ablation causes differential effects of DC on primary and secondary T cell responses, thus augmenting vaccine immunogenicity only when applied after immunization.  

4.764           Parameters for successful pig islet isolation as determined using 68 specific-pathogen-free miniature pigs

Kim, H-I., Lee, S-Y., Jin, S.M., Kim, K.S., YU, J.E., Yeom, S-C., Yoon, T.W., Kim, J.H., Ha, J., Park, C-G. and Kim, S-J. Xenotransplant.,16(1), 11-18 (2009)   Abstract: Background: Islet cell transplantation is a novel therapeutic modality for the cure of diabetes. Pig islet cells are an attractive substitute for human islet cells; however, they are known to be particularly difficult to isolate because of a weak islet capsule and a tendency to be fragmented during enzymatic digestion. Therefore, parameters favoring successful pig islet isolation were investigated using specific-pathogen-free (SPF) miniature pigs. Methods: Sixty-eight SPF miniature pigs were used for islet isolation. Birth weight, body weight, age, sex, pregnancy history, and the fasting blood glucose levels of each pig were determined. Each pig’s general condition was assessed with regard to feeding status and physical activity. Pancreas procurement was performed by one surgical team. Anesthesia duration, operation duration, procedure quality, and perfusate type were recorded. After pancreatectomy, a biopsy was performed for islet density analysis. Decapsulation, cannulation duration, degree of distension, and cold ischemic time were assessed. During islet isolation, pancreas weight, digestion time, and digested tissue proportion were recorded. Isolation results were evaluated by total islet equivalents (IEQ), islet equivalents per gram of pancreas (IEQ/g), isolation index, islet recovery rate, purity, and visual grade. To identify the predictors of higher islet isolation yield, we performed binary logistic regression analysis with significant (P < 0.05) variables from the univariate analysis. Results: The pigs were categorized into high (n = 34) and low yield (n = 34) groups according to the median IEQ/g or total IEQ values. Body weight and age were significantly different between the two groups. Being male or a positive history of pregnancy in females was factors favoring successful islet isolation. General condition assessments failed to estimate islet isolation results. Long anesthesia duration, which might have caused ischemic injury to the pancreas, negatively affected islet isolation results. Decapsulation, cannulation duration, and subsequent pancreas distension were significantly important in successful islet isolation. Inter-lot variability of Liberase was not observed because of screening processes performed before purchase. Isolation index and islet recovery rate correlated well with islet yields. Conclusions: Multivariate analysis using total IEQ and IEQ/g as outcome variables indicated that age older than 2, being male and moderate distension by Liberase injection are major determinants influencing successful islet isolation.  

4.765           Tissue targeting of anti-RNP autoimmunity: Effects of T cells and myeloid dendritic cells in a murine model

Greidinger, E.L., Zang, Y., Fernandez, I., Berho, M., Nassiri, M., Martinez, L. and Hoffmann, R.W. Arthritis & Rheumatism, 60(2), 534-542 (2009)   Objective To explore the role of immune cells in anti-RNP autoimmunity in a murine model of pneumonitis or glomerulonephritis, using adoptive transfer techniques. Methods Donor mice were immunized with 50 μg of U1–70-kd small nuclear RNP fusion protein and 50 μg of U1 RNA adjuvant. Whole splenocytes as well as CD4+ cell and dendritic cell (DC) subsets from the immunized mice were infused into naive syngeneic recipients. Anti-RNP and T cell responses were assessed by immunoblotting, enzyme-linked immunosorbent assay, and flow cytometry. Development of renal or lung disease was assessed by histology and urinalysis. Results Unfractionated splenocytes from donor mice without proteinuria induced predominantly lung disease in recipients (8 [57%] of 14 versus 2 [14%] of 14 developing renal disease; P = 0.046). However, infusion of CD4+ cells from donors without proteinuria induced renal disease more frequently than lung disease (7 [70%] of 10 versus 2 [20%] of 10; P = 0.01); adoptive transfer of RNP+CD4+ T cells from short-term culture yielded similar results (renal disease in 8 [73%] of 11 recipients versus lung disease in 3 [27%] of 11). Cotransfer of splenic myeloid DCs and CD4+ T cells from immunized donors prevented induction of renal disease in all 5 recipients (P = 0.026 versus recipients of fresh CD4+ cells alone), although lung disease was still observed in 1 of 5 mice. Transfer of myeloid DCs alone from immunized donors induced lung disease in 3 (60%) of 5 recipients, without evidence of nephritis. Cotransfer of splenocytes from mice with and those without nephritis led to renal disease in 4 of 5 recipients, without evidence of lung disease. Conclusion These findings indicate that RNP+CD4+ T cells are sufficient to induce anti-RNP autoimmunity, tissue targeting in anti-RNP autoimmunity can be deviated to either a renal or pulmonary phenotype depending on the presence of accessory cells such as myeloid DCs, and DC subsets can play a role in both propagation of autoimmunity and end-organ targeting.  

4.766           Effect of short-term culture on functional and stress-related parameters in isolated human islets

Ihm, S-H., Matsumoto, I., Zhang, H.J., Ansite, J.D. and hering, B.J. Transplant Int., 22(2), 207-216 (2009)   The Edmonton protocol for islet transplantation utilizes fresh islet grafts but other protocols increasingly transplant short-term cultured grafts mainly for practical reasons. To improve our understanding of the impact of culture pretreatment of human islets, we assessed post-transplant function by nude mouse bioassay, islet ATP, activity of stress-activated MAP kinases, and expression of stress-related genes by focused cDNA array in freshly isolated and cultured islets. Mean blood glucose levels over 4 weeks after transplantation (2000 IE) of (i) freshly isolated, (ii) cultured and preculture counted (recovery rate; 78 ± 6%), and (iii) cultured and postculture counted islets in diabetic mice were 330 ± 40, 277 ± 65, and 256 ± 52 mg/dl (i versus ii, P = 0.004; i versus iii, P = 0.002). During culture, islet ATP/DNA and ATP/ADP increased; JNK and p38 MAPK activities decreased. Among 96 genes studied, mRNA expression of heat shock protein 70 genes decreased >twofold during culture in all four pairs; expression of cyclooxygenase-2, superoxide dismutase-2, interleukin-6 and cytochromes P450 1A1 genes increased. Our results show that culturing human islets before transplantation is not disadvantageous in regard of functional recovery from changes induced by nonphysiologic stimuli during islet isolation. The increase in expression of several stress-related genes during culture also shows that improving culture conditions may further enhance post-transplant islet function.  

4.767           Vascular endothelial growth factor prevents G93A-SOD1-induced motor neuron degeneration

Lunn, J.S., Sakowski, S.A., Kim, B., Rosenberg, A.A. and Feldman, E.L. Develop. Neurobiol., 69(13), 871-884 (2009)   Amyotrophic lateral sclerosis (ALS)  is an adult-onset neurodegenerative disorder characterized by selective loss of motor neurons (MNs). Twenty percent of familial ALS cases are associated with mutations in Cu2+/Zn2+ superoxide dismutase (SOD1). To specifically understand the cellular mechanisms underlying mutant SOD1 toxicity, we have established an in vitro model of ALS using rat primary MN cultures transfected with an adenoviral vector encoding a mutant SOD1, G93A-SOD1. Transfected cells undergo axonal degeneration and alterations in biochemical responses characteristic of cell death such as activation of caspase-3. Vascular endothelial growth factor (VEGF) is an angiogenic and neuroprotective growth factor that can increase axonal outgrowth, block neuronal apoptosis, and promote neurogenesis. DecreasedVEGF gene expression in mice results in a phenotype similar to that seen in patients with ALS, thus linking loss of VEGF to the pathogenesis of MN degeneration. Decreased neurotrophic signals prior to and during disease progression may increase MN susceptibility to mutant SOD1-induced toxicity. In this study, we demonstrate a decrease in VEGF and VEGFR2 levels in the spinal cord of G93A-SOD1 ALS mice. Furthermore, in isolated MN cultures, VEGF alleviates the effects of G93A-SOD1 toxicity and neuroprotection involves phosphatidylinositol 3-kinase/protein kinase B (PI3K/ Akt) signaling. Overall, these studies validate the usefulness of VEGF as a potential therapeutic factor for the treatment of ALS and give valuable insight into the responsible signaling pathways and mechanisms involved.  

4.768           A Re-evaluation of the Mechanisms Leading to Dengue Hemorrhagic Fever

Noisakran, S., Chokephaibulkit, K., Songprakhon, P., Onlamoon, N., Hsiao, H-M., Villinger, F., Ansari, A. and Perng, G.C. Ann. N.Y. Acad. Sc., 1171, E24-E35 (2009)   Viremia is one of the features of dengue virus infection among the flaviviruses. Dengue virus infection results in a spectrum of clinical symptoms, ranging from undifferentiated flu-like illness, mild dengue fever, to dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS), a life-threatening illness. Several mechanisms have been hypothesized based primarily on data collected from post-acute clinical phase to account for DHF/DSS. Lack of a suitable animal model for DHF/DSS has hindered progress in defining the etiology of DHF/DSS. Levels of circulating dengue virus have been well-correlated to severe dengue disease. However, the cell lineage(s) serving as a primary target for the source of viremia are largely unknown. Results from in vivo and in vitro pilot studies using molecular and more advanced technologies reveal that dengue virus appears to be associated with platelets and the megakaryocytic lineage. The observation may partially explain the dysfunction of platelets observed in dengue affected patients.  

4.769           Short-term exposure to human cytomegalovirus–infected fibroblasts induces a proportional increase of active CD94/NKG2A⁺ natural killer cells

Petersen, L., Petersen, C.C., Møller-Larsen, A. and Hokland, M.E. Human Immunol., 71, 29-35 (2010)   Natural killer (NK) cells are essential components of the immune response against human cytomegalovirus (HCMV). As NK cells are part of the innate immune system providing an immediate defense against pathogens, short-term exposure to HCMV-infected cells may induce changes in the phenotype and function of these cells. To identify immediate reactions of NK cells to HCMV, we co-cultured peripheral blood mononuclear cells with HCMV-infected fibroblasts for 24 and 72 hours. A distinct, HCMV-mediated, proportional enlargement of a subset of NK cells expressing CD94/NKG2A was sustained throughout the period of incubation. As preceding studies have shown that HCMV can cause an increase in CD94/NKG2C⁺ NK cells, our results were surprising. The NK cells showed intense upregulation of the early activation marker CD69 in response to HCMV. The CD94/NKG2A⁺ NK cells demonstrated the highest expression of CD69. Studies of HCMV-induced interferon-γ expression after 24 hours of co-culture showed that this cytokine was almost exclusively produced by the CD94/NKG2A⁺ subset of NK cells. In summary, our data demonstrate that HCMV induces an immediate proportional enlargement of a functionally active CD94/NKG2A expressing subset of NK cells.  

4.770           The quest for liver progenitor cells: A practical point of view

Dolle, L., Best, J., Mei, J., Al Battah, F., Reynaert, H., van Grunsven, L.A. and Geerts, A.

1. Hepatol., 52, 117-129 (2010)

  Many chronic liver diseases can lead to hepatic dysfunction with organ failure. At present, orthotopic liver transplantation represents the benchmark therapy of terminal liver disease. However this practice is limited by shortage of donor grafts, the need for lifelong immunosuppression and very demanding state-of-the-art surgery. For this reason, new therapies have been developed to restore liver function, primarily in the form of hepatocyte transplantation and artificial liver support devices. While already offered in very specialized centers, both of these modalities still remain experimental. Recently, liver progenitor cells have shown great promise for cell therapy, and consequently they have attracted a lot of attention as an alternative or supportive tool for liver transplantation. These liver progenitor cells are quiescent in the healthy liver and become activated in certain liver diseases in which the regenerative capacity of mature hepatocytes and/or cholangiocytes is impaired. Although reports describing liver progenitor cells are numerous, they have not led to a consensus on the identity of the liver progenitor cell. In this review, we will discuss some of the characteristics of these cells and the different ways that have been used to obtain these from rodents. We will also highlight the challenges that researchers are facing in their quest to identify and use liver progenitor cells.    

4.771           Cytokines Involved in Interferon- Production by Human Macrophages

Robinson, C.M., O’Dee, D., Hamilton, T. and Nau, G.J.

1. Innat. Immun., 2, 56-65 (2010)

  Interferon (IFN)- is important to the immune defense against intracellular pathogens and specifically the ability of macrophages to control Mycobacterium tuberculosis (MTB). Increasing evidence has accumulated to support the idea that macrophages produce IFN- . We describe here the cytokine interactions that determine IFN- expression and secretion during MTB infection of human macrophages. Detection of biologically important IFN- levels in culture supernatants of MTB-infected human macrophages requires the addition of interleukin (IL)-12. IL-18 augmented IFN- production from human macrophages in response to the combination of MTB and supplemental IL-12. Although IL-18 gene expression was generally unchanged, IL-18 protein secretion was enhanced by the combination of MTB and IL-12, and functioned primarily to stimulate IFN- release. Importantly, IL-27 induced by MTB infection opposed IFN- production by antagonizing IL-18 activity in human macrophages. Neutralization of IL-27 increased the expression of the IL-18 receptor -chain. Additionally, IL-27 blocked NF- B activation in response to IL-18. These results define the signals required for IFN- production by human macrophages and highlight the interactions between cytokines produced during MTB infection. Together, they identify a novel role for IL-27 in regulating macrophage function by disrupting IL-18 activity.  

4.772           Age-related deficiencies in complex I endogenous substrate availability and reserve capacity of complex IV in cortical neuron electron transport

Jones, T.T. and Brewer, G.J. Biochim. Biophys. Acta, 1797, 167-176 (2010)   Respiratory enzyme complex dysfunction is mechanistically involved in mitochondrial failure leading to neurodegenerative disease, but the pathway is unclear. Here, age-related differences in mitochondrial respiration were measured in both whole and permeabilized neurons from 9-month and 24-month adult rat cortex cultured in common conditions. After permeabilization, respiration increased in both ages of neurons with excess substrates. To dissect specific deficiencies in the respiratory chain, inhibitors for each respiratory chain complex were used to isolate their contributions. Relative to neurons from 9-month rats, in neurons isolated from 24-month rats, complexes I, III, and IV were more sensitive to selective inhibition. Flux control point analysis identified complex I in neurons isolated from 24-month rats as the most sensitive to endogenous substrate availability. The greatest age-related deficit in flux capacity occurred at complex IV with a 29% decrease in neurons isolated from 24-month rats relative to those from 9-month rats. The deficits in complexes I and III may contribute to a redox shift in the quinone pool within the electron transport chain, further extending these age-related deficits. Together these changes could lead to an age-related catastrophic decline in energy production and neuronal death.  

4.773           A Large-Scale Chemical Screen for Regulators of the Arginase 1 Promoter Identifies the Soy Isoflavone Daidzeinas a Clinically Approved Small Molecule That Can Promote Neuronal Protection or Regeneration via a cAMP-Independent Pathway

Ma, T.C., Campana, A., Lange, P.S., Lee, H-H., Banerjee, K., Bryson, J.B., Mahishi, L., Alam, S., Giger, R.J., Barnes, S., Morris jr, S.M., Willis, D.E., Rwiss, J.L., Filbin, M.T. and Ratan, R.R.

1. Neurosci., 30(2), 739-748 (2010)

  An ideal therapeutic for stroke or spinal cord injury should promote survival and regeneration in the CNS. Arginase 1 (Arg1) has been shown to protect motor neurons from trophic factor deprivation and allow sensory neurons to overcome neurite outgrowth inhibition by myelin proteins. To identify small molecules that capture Arg1's protective and regenerative properties, we screened a hippocampal cell line stably expressing the proximal promoter region of the arginase 1 gene fused to a reporter gene against a library of compounds containing clinically approved drugs. This screen identified daidzein as a transcriptional inducer of Arg1. Both CNS and PNS neurons primed in vitro with daidzein overcame neurite outgrowth inhibition from myelin-associated glycoprotein, which was mirrored by acutely dissociated and cultured sensory neurons primed in vivo by intrathecal or subcutaneous daidzein infusion. Further, daidzein was effective in promoting axonal regeneration in vivo in an optic nerve crush model when given intraocularly without lens damage, or most importantly, when given subcutaneously after injury. Mechanistically, daidzein requires transcription and induction of Arg1 activity for its ability to overcome myelin inhibition. In contrast to canonical Arg1 activators, daidzein increases Arg1 without increasing CREB phosphorylation, suggesting its effects are cAMP-independent. Accordingly, it may circumvent known CNS side effects of some cAMP modulators. Indeed, daidzein appears to be safe as it has been widely consumed in soy products, crosses the blood–brain barrier, and is effective without pretreatment, making it an ideal candidate for development as a therapeutic for spinal cord injury or stroke.  

4.774           Rotavirus Infection Activates Dendritic Cells from Peyer's Patches in Adult Mice

Lopez-Guerrero, D.V., Meza-Perez, S., Ramirez-Pliego, O., Santana-Calderon, M.A., Espino-Solis, P., Gutierrez-Xicotencatl, L., Flores-Romo, L. and Eszuivel-Guadarrama, F.R.

1. Virol., 84(4), 1856-1866 (2010)

  This study used an in vivo mouse model to analyze the response of dendritic cells (DCs) in Peyer's patches (PPs) within the first 48 h of infection with the wild-type murine rotavirus EDIM (EDIMwt). After the infection, the absolute number of DCs was increased by 2-fold in the PPs without a modification of their relative percentage of the total cell number. Also, the DCs from PPs of infected mice showed a time-dependent migration to the subepithelial dome (SED) and an increase of the surface activation markers CD40, CD80, and CD86. This response was more evident at 48 h postinfection (p.i.) and depended on viral replication, since DCs from PPs of mice inoculated with UV-treated virus did not show this phenotype. As a result of the activation, the DCs showed an increase in the expression of mRNA for the proinflammatory cytokines interleukin-12/23p40 (IL-12/23p40), tumor necrosis factor alpha (TNF- ), and beta interferon (IFN-β), as well as for the regulatory cytokine IL-10. These results suggest that, a short time after rotavirus infection, the DCs from PPs play a critical role in controlling the infection and, at the same time, avoiding an excessive inflammatory immune response.  

4.775           Low-Temperature Preservation of Isolated Islets is Superior to Conventional Islet Culture Before Islet Transplantation

Noguchi, H., Naziruddin, B., Jackson, A., Shimoda, M., Ikemoto, T., Fujita, Y., Chujo, D., Takota, M., Kobayashi, N., Onaca, N., Levy, M.F. and Matsumoto, S. Transplantation, 89(1), 47-54 (2010)   Background. Although culturing islets before transplantation provides flexibility for evaluation of isolated islets and pretreatment of patients, it is well-known that isolated islets deteriorate rapidly in culture. In this study, we evaluated optimal temperature for culture/preservation of isolated human islets before transplantation. Methods. Isolated islets were cultured or preserved for 48 hr in the following culture/preservation conditions: preservation at 4[degrees]C in University of Wisconsin solution and culture at 22[degrees]C or 37[degrees]C in culture medium. Results. Islet morphology after 4[degrees]C preservation was similar to that of fresh islets, whereas islet diameter after 37[degrees]C or 22[degrees]C culture was smaller than that of fresh islets. Islet yield significantly decreased at higher temperatures (24% loss in 37[degrees]C culture and 19% loss in 22[degrees]C culture, but <5% loss in 4[degrees]C preservation). Cultured/preserved islets were transplanted into diabetic nude mice. The attainability of posttransplantation normoglycemia was significantly higher in the 4[degrees]C preservation group than in 22[degrees]C and 37[degrees]C culture groups. Conclusion. Preservation of isolated islets at 4[degrees]C improves the outcome of islet transplantation more efficiently than preservation at 22[degrees]C or 37[degrees]C. Based on these data, we have performed short-time cold storage of isolated islets instead of culturing for current clinical islet transplantation.  

4.776           Paeonol, the main active principles of Paeonia moutan, ameliorates alcoholic steatohepatitis in mice

Hu, A., Shwn, G., Zhao, W., Wang, F., Jiang, X. and Huang, D.

1. Ethnopharmacol., 128, 100-106 (2010)

  Aim of study Paeonol, a major phenolic component of Moutan Cortex, is traditionally used as a Chinese herbal medicine in various diseases including hepatitis. Evidence shows that paeonol has anti-inflammatory, anti-tumor, and anti-atherosclerosis effects. However, the effect of paeonol on alcoholic liver injury remains obscure. The present investigation was designed to determine the effects of paeonol on alcohol-induced hepatic injury in mice. Materials and methods The degree of alcoholic liver injury was evaluated biochemically by measuring serum markers and pathological examination. Real-time PCR and ELISA methods were used to check the expression of cytokines. Western blotting was used to check CYP 450 expression. Results Treatment with paeonol significantly attenuated the level of serum aminotransferase, reduced the severe extent of hepatic cell damage, steatosis, and the infiltration of inflammatory cells in a model of alcoholic liver injury (P < 0.05). Interestingly, paeonol markedly decreased hepatic mRNA expression of lipogenic genes (P < 0.05) while had no effect on protein expression of hepatic CYP2E1. Furthermore, paeonol significantly decreased serum and tissue inflammatory cytokine levels, tissue lipid peroxidation, neutrophil infiltration and inhibited the apoptosis of hepatocytes (P < 0.05). Kupffer cells isolated from ethanol-fed mice produced high amounts of tumor necrosis factor alpha, whereas Kupffer cells from paeonol treatment ethanol-fed mice produced less tumor necrosis factor alpha (P < 0.05). Conclusions These findings suggest that paeonol may represent a novel, protective strategy against alcoholic liver injury by attenuating hepatic steatosis, inflammatory response and apoptosis.  

4.777           Quantitative analysis of cellular inflammation after traumatic spinal cord injury: evidence for a multiphasic inflammatory response in the acute to chronic environment

Beck, K.D., Nguyen, H.X., Galvan, M.D., Salazar, D.L., Woodruff, T.M. and Anderson, A.J. Brain, 133(2), 433-447 (2010)   Traumatic injury to the central nervous system results in the disruption of the blood brain/spinal barrier, followed by the invasion of cells and other components of the immune system that can aggravate injury and affect subsequent repair and regeneration. Although studies of chronic neuroinflammation in the injured spinal cord of animals are clinically relevant to most patients living with traumatic injury to the brain or spinal cord, very little is known about chronic neuroinflammation, though several studies have tested the role of neuroinflammation in the acute period after injury. The present study characterizes a novel cell preparation method that assesses, quickly and effectively, the changes in the principal immune cell types by flow cytometry in the injured spinal cord, daily for the first 10 days and periodically up to 180 days after spinal cord injury. These data quantitatively demonstrate a novel time-dependent multiphasic response of cellular inflammation in the spinal cord after spinal cord injury and are verified by quantitative stereology of immunolabelled spinal cord sections at selected time points. The early phase of cellular inflammation is comprised principally of neutrophils (peaking 1 day post-injury), macrophages/microglia (peaking 7 days post-injury) and T cells (peaking 9 days post-injury). The late phase of cellular inflammation was detected after 14 days post-injury, peaked after 60 days post-injury and remained detectable throughout 180 days post-injury for all three cell types. Furthermore, the late phase of cellular inflammation (14–180 days post-injury) did not coincide with either further improvements, or new decrements, in open-field locomotor function after spinal cord injury. However, blockade of chemoattractant C5a-mediated inflammation after 14 days post-injury reduced locomotor recovery and myelination in the injured spinal cord, suggesting that the late inflammatory response serves a reparative function. Together, these data provide new insight into cellular inflammation of spinal cord injury and identify a surprising and extended multiphasic response of cellular inflammation. Understanding the role of this multiphasic response in the pathophysiology of spinal cord injury could be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions.  

4.778           Adjuvant Activity on Murine and Human Macrophages

Quesniaux, V., Erard, F. and Ryffel, B. Methods in Mol. Biol., 626, 117-130 (2010)   Activation of cells of the innate immunity such as macrophages and dendritic cells is critical to mount an adaptive immune response. Recent advances on the understanding of innate immune receptors such as the Toll-like receptors (TLR) and NOD-like receptors (NLR) and the demonstration that microbial products activate specific receptors. This discovery represented a major advance and provided tools to test novel adjuvants in vitro to investigate activation on innate immune cells. Here the isolation and culture of murine macrophages is described, and the use of macrophages derived from gene-deficient mice is proposed to define receptor usage. Novel adjuvants may be tested for their capacity to induce cytokines, chemokines and the expression of costimulatory molecules. The basic methods to assess macrophage activation are given, which may predict an in vivo activity of a novel adjuvant.  

4.779           Pathogenic cysteine mutations affect progranulin function and production of mature granulins

Wang, J., Van Damme, P., Cruchaga, C., Gitcho, M.A., Vidal, J.M., Seijo-Martinez, M., Wang, L., Wu, J.Y., Robberecht, W. and Goate, A.

1. Neurochem., 112, 1305-1315 (2010)

  Frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U) can be caused by mutations in the progranulin gene (GRN). Progranulin (PGRN) is a cysteine-rich growth factor, which is proteolytically cleaved by elastase to produce several granulins (GRNs). All FTLD-U mutations in GRN characterized to date result in reduced secreted PGRN protein. We recently reported a Spanish family with progressive non-fluent aphasia and dementia in which a novel C521Y mutation segregates with disease. A second cysteine mutation (C139R) has also been reported to be disease specific. Allele-specific mRNA expression assays in brain reveal that the C521Y mutant allele is expressed at similar levels to the wild-type allele. Furthermore, plasma PGRN levels in C521Y carriers are comparable with non-carrier family relatives, suggesting that the mutation does not affect PGRN protein expression and secretion in vivo. Despite normal PGRN levels C521Y and C139R mutant GRNs show reduced neurite growth-stimulating activity in vitro. Further study revealed that these mutations also cause impaired cleavage of PGRN by elastase. Our data suggest that these mutations affect the function of full-length PGRN as well as elastase cleavage of PGRN into GRNs, leading to neurodegeneration.  

4.780           Microvesicle entry into marrow cells mediates tissue-specific changes in mRNA by direct delivery of mRNA and induction of transcription

Aliotta, J.M., Pereira, M., Johnson, K.W., de Paz, N., Dooner, M.S., Puente, N., Ayala, C., Brilliant, K., Berz, D., Lee, D., Ramratman, B., McMillan, P.N., Hixson, D.C., Josic, D. and Quesenberry, P.J. Exp. Hematol., 38, 233-245 (2010)   Objective Microvesicles have been shown to mediate intercellular communication. Previously, we have correlated entry of murine lung-derived microvesicles into murine bone marrow cells with expression of pulmonary epithelial cell-specific messenger RNA (mRNA) in these marrow cells. The present studies establish that entry of lung-derived microvesicles into marrow cells is a prerequisite for marrow expression of pulmonary epithelial cell-derived mRNA. Materials and Methods Murine bone marrow cells cocultured with rat lung, but separated from them using a cell-impermeable membrane (0.4-μm pore size), were analyzed using species-specific primers (for rat or mouse). Results These studies revealed that surfactant B and C mRNA produced by murine marrow cells were of both rat and mouse origin. Similar results were obtained using murine lung cocultured with rat bone marrow cells or when bone marrow cells were analyzed for the presence of species-specific albumin mRNA after coculture with rat or murine liver. These studies show that microvesicles both deliver mRNA to marrow cells and mediate marrow cell transcription of tissue-specific mRNA. The latter likely underlies the longer-term stable change in genetic phenotype that has been observed. We have also observed microRNA in lung-derived microvesicles, and studies with RNase-treated microvesicles indicate that microRNA negatively modulates pulmonary epithelial cell-specific mRNA levels in cocultured marrow cells. In addition, we have also observed tissue-specific expression of brain, heart, and liver mRNA in cocultured marrow cells, suggesting that microvesicle-mediated cellular phenotype change is a universal phenomena. Conclusion These studies suggest that cellular systems are more phenotypically labile than previously considered.  

4.781           Lethal Antibody Enhancement of Dengue Disease in Mice Is Prevented by Fc Modification

Balsitis, S.J., Williams, K.L., Lachica, R., Flores, D., Kyle, J.L., Mehlhop, E., Johnson, S., Diamond, M.S., Beatty, P.R. and Harris, E. PloSPathogens, 6(2), e1000790 (2010)   Immunity to one of the four dengue virus (DV) serotypes can increase disease severity in humans upon subsequent infection with another DV serotype. Serotype cross-reactive antibodies facilitate DV infection of myeloid cells in vitro by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody-dependent enhancement (ADE). However, despite decades of investigation, no in vivo model for antibody enhancement of dengue disease severity has been described. Analogous to human infants who receive anti-DV antibodies by transplacental transfer and develop severe dengue disease during primary infection, we show here that passive administration of anti-DV antibodies is sufficient to enhance DV infection and disease in mice using both mouse-adapted and clinical DV isolates. Antibody-enhanced lethal disease featured many of the hallmarks of severe dengue disease in humans, including thrombocytopenia, vascular leakage, elevated serum cytokine levels, and increased systemic viral burden in serum and tissue phagocytes. Passive transfer of a high dose of serotype-specific antibodies eliminated viremia, but lower doses of these antibodies or cross-reactive polyclonal or monoclonal antibodies all enhanced disease in vivo even when antibody levels were neutralizing in vitro. In contrast, a genetically engineered antibody variant (E60-N297Q) that cannot bind FcγR exhibited prophylactic and therapeutic efficacy against ADE-induced lethal challenge. These observations provide insight into the pathogenesis of antibody-enhanced dengue disease and identify a novel strategy for the design of therapeutic antibodies against dengue.  

4.782           TDP-43 transgenic mice develop spastic paralysis and neuronal inclusions characteristic of ALS and frontotemporal lobar degeneration

Wils, H., Kleinberger, G., Janssens, J., Pereson, S., Joris, G., Cuijt, I., Smits, V., Ceuterick-de Groote, C., Van Broechoven, C and Kumar-Singh, S. PNAS, 107(8), 3858-3863 (2010)   Neuronal cytoplasmic and intranuclear aggregates of RNA-binding protein TDP-43 are a hallmark feature of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). ALS and FTLD show a considerable clinical and pathological overlap and occur as both familial and sporadic forms. Though missense mutations in TDP-43 cause rare forms of familial ALS, it is not yet known whether this is due to loss of TDP-43 function or gain of aberrant function. Moreover, the role of wild-type (WT) TDP-43, associated with the majority of familial and sporadic ALS/FTLD patients, is also currently unknown. Generating homozygous and hemizygous WT human TDP-43 transgenic mouse lines, we show here a dose-dependent degeneration of cortical and spinal motor neurons and development of spastic quadriplegia reminiscent of ALS. A dose-dependent degeneration of nonmotor cortical and subcortical neurons characteristic of FTLD was also observed. Neurons in the affected spinal cord and brain regions showed accumulation of TDP-43 nuclear and cytoplasmic aggregates that were both ubiquitinated and phosphorylated as observed in ALS/FTLD patients. Moreover, the characteristic ≈25-kDa C-terminal fragments (CTFs) were also recovered from nuclear fractions and correlated with disease development and progression in WT TDP-43 mice. These findings suggest that ≈25-kDa TDP-43 CTFs are noxious to neurons by a gain of aberrant nuclear function.  

4.783           Deletion of the Mucin-Like Molecule Muc1 Enhances Dendritic Cell Activation in Response to Toll-Like Receptor Ligands

Williams, M.A., Bauer, S., Lu, W., Guo, J., Walter, S., Brushnell, T.P., Lillehoj, E.P. and Georas, S.N.

1. Innate Immun., 2(2), 123-143 (2010)

  Dendritic cells (DC) are potent professional antigen-presenting cells that drive primary immune responses to infections or other agonists perceived as ‘dangerous’. Muc1 is the only cell surface mucin or MUC gene product that is expressed in DC. Unlike other members of this glycoprotein family, Muc1 possesses a unique cytosolic region capable of signal transduction and attenuating toll-like receptor (TLR) activation. The expression and function of Muc1 has been intensively investigated on epithelial and tumor cells, but relatively little is known about its function on DC. We hypothesized that Muc1 would influence in vitro generation and primary DC activation in response to the TLR4 and TLR5 ligands lipopolysaccharide and flagellin. Compared with Muc1^+/+ DC, we found that Muc1^–/– DC were constitutively activated, as determined by higher expression of co-stimulatory molecules (CD40, CD80 and CD86), greater secretion of immunoregulatory cytokines (TNF-α and VEGF), and better stimulation of allogeneic naïve CD4+ T cell proliferation. After activation by either LPS or flagellin and co-culture with allogeneic CD4+ T cells, Muc1^–/– DC also induced greater secretion of TNF-α and IFN-γ compared to similarly activated Muc1^+/+ DC. Taken together, our results indicate that deletion of Muc1 promotes a heightened functional response of DC in response to TLR4 and TLR5 signaling pathways, and suggests a previously under-appreciated role for Muc1 in regulating innate immune responses of DC.  

4.784           Enhanced Infection of Liver Sinusoidal Endothelial Cells in a Mouse Model of Antibody-Induced Severe Dengue Disease

Zellweger, R.M., Prestwood, T.R. and Shresta, S. Cell Host & Microbe, 7(2), 128-139 (2010)   Dengue virus (DENV) causes disease ranging from dengue fever (DF), a self-limited febrile illness, to the potentially lethal dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). DHF/DSS usually occurs in patients who have acquired DENV-reactive antibodies prior to infection, either from a previous infection with a heterologous DENV serotype or from an immune mother. Hence, it has been hypothesized that subneutralizing levels of antibodies exacerbate disease, a phenomenon termed antibody-dependent enhancement (ADE). However, given the lack of suitable animal models for DENV infection, the mechanism of ADE and its contribution to pathology remain elusive. Here we demonstrate in mice that DENV-specific antibodies can sufficiently increase severity of disease so that a mostly nonlethal illness becomes a fatal disease resembling human DHF/DSS. Antibodies promote massive infection of liver sinusoidal endothelial cells (LSECs), resulting in increased systemic levels of virus. Thus, a subprotective humoral response may, under some circumstances, have pathological consequences.  

4.785           Use of Iodixanol Self-Generated Density Gradients to Enrich for Viable Urothelial Cells from Nonneurogenic and Neurogenic Bladder Tissue

Bruce, A.T., Sangha, N., Richmond, A., Johnson, K., Jones, S., Spencer, T. and Ludlow, J.W. Tissue Engineering: Part C, 16(1), 33-40 (2010)   Suspensions of viable urothelial cells (UC) isolated from patient bladder biopsies often contain considerable amounts of extraneous materials comprised of cellular debris, dead and dying UC, and red blood cells. We have consistently observed an inversely proportional relationship between UC attachment efficiency and the amount of extraneous materials in the suspension; viable UC cell attachment efficiency decreases as the amount of extraneous materials in the cell suspension increases. Processing the initial cell isolate to reduce the amount of extraneous materials can enrich for viable UC capable of attaching and proliferating in ex vivo cultures. In this report, we describe the isolation of an enriched population of viable UC from nonneurogenic and neurogenic bladder tissue biopsies using iodixanol self-generated density gradients (OptiPrep™), and characterization by trypan blue exclusion, fluorescence-activated cell sorting, immunofluorescence, and growth kinetics.  

4.786           A Physiological Function of Inflammation-Associated SerpinB2 Is Regulation of Adaptive Immunity

Schroder, W.A., Le, T.T.T., Major, L., Street, S., Gardner, J., Lambley, E., Markey, K., MacDonald, K.P., Fish, R.J., Thomas, R. and Suhrbier, A.

1. Immunol., 184, 2663-2670 (2010)

  SerpinB2 (plasminogen activator inhibitor-2) is widely described as an inhibitor of urokinase plasminogen activator; however, SerpinB2^–/– mice show no detectable increase in urokinase plasminogen activator activity. In this study, we describe an unexpected immune phenotype in SerpinB2^–/– mice. After immunization with OVA in CFA, SerpinB2^–/– mice made 6-fold more IgG2c and generated 2.5-fold more OVA-specific IFN- –secreting T cells than SerpinB2^+/+ littermate controls. In SerpinB2^+/+ mice, high inducible SerpinB2 expression was seen at the injection site and in macrophages low levels in draining lymph nodes and conventional dendritic cells, and no expression was seen in plasmacytoid dendritic, B, T, or NK cells. SerpinB2^–/– macrophages promoted greater IFN- secretion from wild-type T cells in vivo and in vitro and, when stimulated with anti-CD40/IFN- or cultured with wild-type T cells in vitro, secreted more Th1-promoting cytokines than macrophages from littermate controls. Draining lymph node SerpinB2^–/– myeloid APCs similarly secreted more Th1-promoting cytokines when cocultured with wild-type T cells. Regulation of Th1 responses thus appears to be a physiological function of inflammation-associated SerpinB2; an observation that may shed light on human inflammatory diseases like pre-eclampsia, lupus, asthma, scleroderma, and periodontitis, which are associated with SerpinB2 polymorphisms or dysregulated SerpinB2 expression.  

4.787           MeCP2 Controls an Epigenetic Pathway That Promotes Myofibroblast Transdifferentiation and Fibrosis

Mann, J., Chu, D.C.K., Maxwell, A., Oakley, F., Zhu, N-L., Tsukamoto, H. and Mann, D.A. Gastroenterology, 138, 705-714 (2010)   Background & Aims Myofibroblast transdifferentiation generates hepatic myofibroblasts, which promote liver fibrogenesis. The peroxisome proliferator-activated receptor γ (PPARγ) is a negative regulator of this process. We investigated epigenetic regulation of PPARγ and myofibroblast transdifferentiation. Methods Chromatin immunoprecipitation (ChIP) assays assessed the binding of methyl-CpG binding protein 2 (MeCP2) to PPARγ and chromatin modifications that silence this gene. MeCP2^−/y mice and an inhibitor (DZNep) of the epigenetic regulatory protein EZH2 were used in the carbon tetrachloride model of liver fibrosis. Liver tissues from mice were assessed by histologic analysis; markers of fibrosis were measured by quantitative polymerase chain reaction (qPCR). Reverse transcription PCR detected changes in expression of the microRNA miR132 and its target, elongated transcripts of MeCP2. Myofibroblasts were transfected with miR132; PPARγ and MeCP2 expressions were analyzed by qPCR or immunoblotting. Results Myofibroblast transdifferentiation of hepatic stellate cells is controlled by a combination of MeCP2, EZH2, and miR132 in a relay pathway. The pathway is activated by down-regulation of miR132, releasing the translational block on MeCP2. MeCP2 is recruited to the 5′ end of PPARγ, where it promotes methylation by H3K9 and recruits the transcription repressor HP1α. MeCP2 also stimulates expression of EZH2 and methylation of H3K27 to form a repressive chromatin structure in the 3′ exons of PPARγ. Genetic and pharmacologic disruptions of MeCP2 or EZH2 reduced the fibrogenic characteristics of myofibroblasts and attenuated fibrogenesis. Conclusions Liver fibrosis is regulated by an epigenetic relay pathway that includes MeCP2, EZH2, and miR132. Reagents that interfere with this pathway might be developed to reduce fibrogenesis in chronic liver disease.  

4.788           CD36 Is a Novel Serum Amyloid A (SAA) Receptor Mediating SAA Binding and SAA-induced Signaling in Human and Rodent Cells

Baranova, I.N., Bochatrov, A.V., Vishnyakova, T.G., Kurlander, R., Chen, Z., Fu, D., Arias, I.M., Csako, G., Patterson, A.P. and Eggerman, T.L.

1. Biol. Chem., 285(11), 8942-8506 (2010)

  Serum amyloid A (SAA) is a major acute phase protein involved in multiple physiological and pathological processes. This study provides experimental evidence that CD36, a phagocyte class B scavenger receptor, functions as a novel SAA receptor mediating SAA proinflammatory activity. The uptake of Alexa Fluor® 488 SAA as well as of other well established CD36 ligands was increased 5–10-fold in HeLa cells stably transfected with CD36 when compared with mock-transfected cells. Unlike other apolipoproteins that bind to CD36, only SAA induced a 10–50-fold increase of interleukin-8 secretion in CD36-overexpressing HEK293 cells when compared with control cells. SAA-mediated effects were thermolabile, inhibitable by anti-SAA antibody, and also neutralized by association with high density lipoprotein but not by association with bovine serum albumin. SAA-induced cell activation was inhibited by a CD36 peptide based on the CD36 hexarelin-binding site but not by a peptide based on the thrombospondin-1-binding site. A pronounced reduction (up to 60–75%) of SAA-induced pro-inflammatory cytokine secretion was observed in cd36^−/− rat macrophages and Kupffer cells when compared with wild type rat cells. The results of the MAPK phosphorylation assay as well as of the studies with NF-κB and MAPK inhibitors revealed that two MAPKs, JNK and to a lesser extent ERK1/2, primarily contribute to elevated cytokine production in CD36-overexpressing HEK293 cells. In macrophages, four signaling pathways involving NF-κB and three MAPKs all appeared to contribute to SAA-induced cytokine release. These observations indicate that CD36 is a receptor mediating SAA binding and SAA-induced pro-inflammatory cytokine secretion predominantly through JNK- and ERK1/2-mediated signaling.  

4.789           Distinct Roles for CCR4 and CXCR3 in the Recruitment and Positioning of Regulatory T Cells in the Inflamed Human Liver

Oo, Y., Weston, C.J., Lalor, P.F., Curbishley, S.M., Withers, D.R., Reynolds, G.M., Shetty, S., Harki, J., Shaw, J.C., Eksteen, B., Hubscher, S.G., Walker, L.S. and Adams, D.H.

1. Immunol., 184, 2886-2898 (2010)

  Regulatory T cells (T_regs) are found at sites of chronic inflammation where they mediate bystander and Ag-specific suppression of local immune responses. However, little is known about the molecular control of T_reg recruitment into inflamed human tissues. We report that up to 18% of T cells in areas of inflammation in human liver disease are forkhead family transcriptional regulator box P3 (FoxP3)⁺ T_regs. We isolated CD4⁺CD25⁺CD127^lowFoxP3⁺ T_regs from chronically inflamed human liver removed at transplantation; compared with blood-derived T_regs, liver-derived T_regs express high levels of the chemokine receptors CXCR3 and CCR4. In flow-based adhesion assays using human hepatic sinusoidal endothelium, T_regs used CXCR3 and 4β1 to bind and transmigrate, whereas CCR4 played no role. The CCR4 ligands CCL17 and CCL22 were absent from healthy liver, but they were detected in chronically inflamed liver where their expression was restricted to dendritic cells (DCs) within inflammatory infiltrates. These DCs were closely associated with CD8 T cells and CCR4⁺ T_regs in the parenchyma and septal areas. Ex vivo, liver-derived T_regs migrated to CCR4 ligands secreted by intrahepatic DCs. We propose that CXCR3 mediates the recruitment of T_regs via hepatic sinusoidal endothelium and that CCR4 ligands secreted by DCs recruit T_regs to sites of inflammation in patients with chronic hepatitis. Thus, different chemokine receptors play distinct roles in the recruitment and positioning of T_regs at sites of hepatitis in chronic liver disease.  

4.790           Blockade of Programmed Death Ligand 1 Enhances the Therapeutic Efficacy of Combination Immunotherapy against Melanoma

Pilon-Thomas, S., Mackay, A., Vohra, N. and Mule, J.J.

1. Immunol., 184, 3442-3449 (2010)

  Inhibition of antitumor T cell responses can be mediated by the productive interaction between the programmed death-1 (PD-1) receptor on T cells and its ligand PD-L1. PD-L1 is highly expressed on both murine bone marrow-derived dendritic cells (DCs) and B16 melanoma. In this study, in vitro blockade of PD-L1 interaction on DCs led to enhanced IFN- production and cytotoxicity by Ag-specific T cells. In vivo, the systemic administration of anti–PD-L1 Ab plus melanoma peptide-pulsed DCs resulted in a higher number of melanoma peptide-specific CD8⁺ T cells, but this combination was insufficient to delay the growth of established B16 melanoma. Although the addition of 600 rad of total body irradiation delayed tumor growth, further adoptive transfer of Ag-specific CD8⁺ T cells was needed to achieve tumor regression and long-term survival of the treated mice. Lymphopenic mice treated with anti–PD-L1 Ab demonstrated increased activation and persistence of adoptively transferred T cells, including a higher number of CD8⁺ T cells infiltrating the tumor mass. Together, these studies support the blocking of PD-L1 signaling as a means to enhance combined immunotherapy approaches against melanoma.  

4.791           Human Peripheral Lymphoid Tissues Contain Autoimmune Regulator-Expressing Dendritic Cells

Poliani, P.L., Kisand, K., Marrella, V., Ravanini, M., Notarangelo, L.D., Villa, A., Peterson, P. and Facchetti, F. Am. J. Pathol., 176(3), 1104-1112 (2010)   Autoimmune regulator (AIRE) modulates the expression of tissue-restricted antigens (TSAs) and promotes central tolerance in the thymus. However, few autoreactive T cells escape negative selection and reach the periphery, where peripheral tolerance is required to avoid autoimmunity. Murine lymph nodes (LNs) have been shown to contain "stromal" cells expressing AIRE and TSAs. Here we report the occurrence of AIRE-expressing cells in human peripheral lymphoid tissues, including LNs, tonsils, and gut-associated lymphoid tissue, with the exception of the spleen. Notably, AIRE⁺ cells are absent in fetal LNs and, in postnatal life, they are more numerous in abdominal than in superficial LNs, thus suggesting that their development in periphery may depend on instructive signals from microenvironment and antigen challenge. Extrathymic AIRE⁺ cells show a dendritic morphology, consistently express human leukocyte antigen-DR (HLADR) and fascin, and are largely positive for CD11c and S100 and for the dendritic cell-activation markers CD40, CD83, DC-LAMP/CD208, and CCR7. Lymphoid, myelomonocytic, mesenchymal, and epithelial cell lineage markers are negative. The HLADR^high/AIRE⁺ cell fraction isolated from mesenteric LNs expressed TSAs (insulin, CYP17A1, and CYP21A2), as well as molecules associated with tolerogenic functions, such as interleukin-10 and indoleamine 2,3-dioxygenase. Data indicate that AIRE⁺ cells in human peripheral lymphoid tissues correspond to a subset of activated interdigitating dendritic cells expressing TSAs and the tolerogenic molecules indoleamine 2,3-dioxygenase and interleukin-10, suggestive of a potential tolerogenic function.  

4.792           Islet Isolation for Clinical Transplantation

Kin, T. Adv. Exp. Med. and Biol., 654, 683-710 (2010)   Islet transplantation is emerging as a viable treatment option for selected patients with type 1 diabetes. Following the initial report in 2000 from Edmonton of insulin independence in seven out of seven consecutive recipients, there has been a huge expansion in clinical islet transplantation. The challenge we now face is the apparent decline in graft function over time. Isolating high-quality human islets which survive and function for a longer period will no doubt contribute to further improvement in long-term clinical outcome. This chapter reviews the selection of appropriate donors for islet isolation and transplantation, describes each step during islet isolation, and discusses the scope for further improvements.    

4.793           Tumour necrosis factor alpha induces rapid reduction in AMPA receptor-mediated calcium entry in motor neurones by increasing cell surface expression of the GluR2 subunit: relevance to neurodegeneration

Rainey-Smith, S.R., Andersson, D.A., Williams, R.J. and Rattray, M.

1. Neurochem., 113, 692-703 (2010)

  The [alpha]-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNF[alpha]) have both been implicated in motor neurone vulnerability in amyotrophic lateral sclerosis/motor neurone disease. TNF[alpha] has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNF[alpha] receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNF[alpha] (10 ng/mL, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using fura-2-acetoxymethyl ester microfluorimetry, we showed that exposure to TNF[alpha] caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggest that TNF[alpha] acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in amyotrophic lateral sclerosis.  

4.794           Survival effect of PDGF-CC rescues neurons from apoptosis in both brain and retina by regulating GSK3β phosphorylation

Tang, Z., Arjunan, P., Lee, C., Li, Y., Kumar, A., Hou, X., Wang, B., Wardega, P., Zhang, F., Ddong, L., Zhang, Y., Zhang, S-Z., Ding, H., Fariss, R.N., Becker, K.G., Lennartsson, J., Nagai, N., Cao, Y. and Li, X. J: Exp. Med., 207(4), 867-880 (2010)   Platelet-derived growth factor CC (PDGF-CC) is the third member of the PDGF family discovered after more than two decades of studies on the original members of the family, PDGF-AA and PDGF-BB. The biological function of PDGF-CC remains largely to be explored. We report a novel finding that PDGF-CC is a potent neuroprotective factor that acts by modulating glycogen synthase kinase 3β (GSK3β) activity. In several different animal models of neuronal injury, such as axotomy-induced neuronal death, neurotoxin-induced neuronal injury, 6-hydroxydopamine–induced Parkinson’s dopaminergic neuronal death, and ischemia-induced stroke, PDGF-CC protein or gene delivery protected different types of neurons from apoptosis in both the retina and brain. On the other hand, loss-of-function assays using PDGF-C null mice, neutralizing antibody, or short hairpin RNA showed that PDGF-CC deficiency/inhibition exacerbated neuronal death in different neuronal tissues in vivo. Mechanistically, we revealed that the neuroprotective effect of PDGF-CC was achieved by regulating GSK3β phosphorylation and expression. Our data demonstrate that PDGF-CC is critically required for neuronal survival and may potentially be used to treat neurodegenerative diseases. Inhibition of the PDGF-CC–PDGF receptor pathway for different clinical purposes should be conducted with caution to preserve normal neuronal functions.  

4.795           A defined long-term in vitro tissue engineered model of neuromuscular junctions

Das, M., Rumsey, J.W., Bhaargava, N., Stancescu, M. and Hickman, J.J. Biomaterials, 31, 4880-4888 (2010)   Neuromuscular junction (NMJ) formation, occurring between motoneurons and skeletal muscle, is a complex multistep process involving a variety of signaling molecules and pathways. In vitro motoneuron-muscle co-cultures are powerful tools to study the role of different growth factors, hormones and cellular structures involved in NMJ formation. In this study, a serum-free culture system utilizing defined temporal growth factor application and a non-biological substrate resulted in the formation of robust NMJs. The system resulted in long-term survival of the co-culture and selective expression of neonatal myosin heavy chain, a marker of myotube maturation. NMJ formation was verified by colocalization of dense clusters of acetylcholine receptors visualized using alpha-bungarotoxin and synaptophysin containing vesicles present in motoneuron axonal terminals. This model will find applications in basic NMJ research and tissue engineering applications such as bio-hybrid device development for limb prosthesis and regenerative medicine as well as for high-throughput drug and toxin screening applications.  

4.796           Apoptosis induced by ozone and oxysterols in human alveolar epithelial cells

Kosmider, B., Loader, J.E., Murphy, R.C. and Mason, R.J. Free Radical Biol. & Med., 48, 1513-1524 (2010)   The mechanism of ozone-induced lung cell injury is poorly understood. One hypothesis is that ozone induces lipid peroxidation and that these peroxidated lipids produce oxidative stress and DNA damage. Oxysterols are lipid peroxides formed by the direct effects of ozone on pulmonary surfactant and cell membranes. We studied the effects of ozone and the oxysterol 5β,6β-epoxycholesterol (β-epoxide) and its metabolite cholestan-6-oxo-3,5-diol (6-oxo-3,5-diol) on human alveolar epithelial type I-like cells (ATI-like cells) and type II cells (ATII cells). Ozone and oxysterols induced apoptosis and cytotoxicity in ATI-like cells. They also generated reactive oxygen species and DNA damage. Ozone and β-epoxide were strong inducers of nuclear factor erythroid 2-related factor 2, heat shock protein 70, and Fos-related antigen 1 protein expression. Furthermore, we found higher sensitivity of ATI-like cells compared to ATII cells exposed to ozone or treated with β-epoxide or 6-oxo-3,5-diol. In general the response to the cholesterol epoxides was similar to the effect of ozone. Understanding the response of human ATI-like cells and ATII cells to oxysterols may be useful for further studies, because these compounds may represent useful biomarkers in other diseases.  

4.797           Neurons and Oligodendrocytes Recycle Sphingosine 1-Phosphate to Ceramide: SIGNIFICANCE FOR APOPTOSIS AND MULTIPLE SCLEROSIS

Qin, J., Berdyshev, E., Goya, J., Natarajan, V. and Dawson, G.

1. Biol. Chem., 285(19), 14134-14143 (2010)

  Both cultured neonatal rat hippocampal neurons and differentiated oligodendrocytes rapidly metabolized exogenous C₂- and C₆-ceramides to sphingosine (Sph) and sphingosine 1-phosphate (S1P) but only minimally to C_16–24-ceramides. Dihydrosphinolipids were unaffected but were increased by exogenous C₆-dihydroceramide. Conversely, quantitative liquid chromatography-tandem mass spectrometry technology showed that exogenous S1P (0.25–10 μm) was rapidly metabolized to both Sph (a >200-fold increase) and predominantly C₁₈-ceramide (a >2-fold increase). Longer treatments with either C₂-ceramide (>2.5 μm) or S1P (10 μm) led to apoptotic cell death. Thus, there is an active sphingolipid salvage pathway in both neurons and oligodendrocytes. Staurosporine-induced cell death was shown to be associated with decreased S1P and increased Sph and C_16/18-ceramide levels. The physiological significance of this observation was confirmed by the analysis of affected white matter and plaques from brains of multiple sclerosis patients in which reduced S1P and increased Sph and C_16/18-ceramides were observed.  

4.798           Adult human brain cell culture for neuroscience research

Gibbons, H.M. and Dragunow, M. Int. J. Biochem. & Cell Biol., 42, 844-856 (2010)   Studies of the brain have progressed enormously through the use of in vivo and in vitro non-human models. However, it is unlikely such studies alone will unravel the complexities of the human brain and so far no neuroprotective treatment developed in animals has worked in humans. In this review we discuss the use of adult human brain cell culture methods in brain research to unravel the biology of the normal and diseased human brain. The advantages of using adult human brain cells as tools to study human brain function from both historical and future perspectives are discussed. In particular, studies using dissociated cultures of adult human microglia, astrocytes, oligodendrocytes and neurons are described and the applications of these types of study are evaluated. Alternative sources of human brain cells such as adult neural stem cells, induced pluripotent stem cells and slice cultures of adult human brain tissue are also reviewed. These adult human brain cell culture methods could benefit basic research and more importantly, facilitate the translation of basic neuroscience research to the clinic for the treatment of brain disorders.  

4.799           Distinct populations of metastases-enabling myeloid cells expand in the liver of mice harboring invasive and preinvasive intra-abdominal tumor

Conolly, M.K., Mallen-St. Clair, J., Bedrosian, A.S., Malhotra, A., Vera, V., Ibrahim, J., Henning, J., Pachter, H.L., Bar-Sagi, D., Frey, A.B. and Miller, G.

1. Leukoc. Biol., 87, 713-725 (2010)

  The liver is the most common site of adenocarcinoma metastases, even in patients who initially present with early disease. We postulated that immune-suppressive cells in the liver of tumor-bearing hosts inhibit anti-tumor T cells, thereby accelerating the growth of liver metastases. Using models of early preinvasive pancreatic neoplasia and advanced colorectal cancer, aims of this study were to determine immune phenotype, stimulus for recruitment, inhibitory effects, and tumor-enabling function of immune-suppressive cells in the liver of tumor-bearing hosts. We found that in mice with intra-abdominal malignancies, two distinct CD11b⁺Gr1⁺ populations with divergent phenotypic and functional properties accumulate in the liver, becoming the dominant hepatic leukocytes. Their expansion is contingent on tumor expression of KC. These cells are distinct from CD11b⁺Gr1⁺ populations in other tissues of tumor-bearing hosts in terms of cellular phenotype and cytokine and chemokine profile. Liver CD11b⁺Gr1⁺ cells are highly suppressiveof T cell activation, proliferation, and cytotoxicity and inducethe development of Tregs. Moreover, liver myeloid-derived suppressorcells accelerate the development of hepatic metastases by inactivationof cytotoxic T cells. These findings may explain the propensityof patients with intra-abdominal cancers to develop liver metastasesand suggest a promising target for experimental therapeutics.  

4.800           Glucose Intolerance and Reduced Proliferation of Pancreatic β-Cells in Transgenic Pigs With Impaired Glucose-Dependent Insulinotropic Polypeptide Function

Renner, S., Fehlings, C., Herbach, N., Hofmann, A., von Waldthausen, D.C., Kessler, B., Ulrichs, K., Chodnevskaja, I., Moskalenko, V., Amselgruber, W., Göke, B., Pfeifer, A., wanke, R. and Wolf, E. Diabetes, 59, 1228-1238 (2010)   OBJECTIVE The insulinotropic action of the incretin glucose-dependent insulinotropic polypeptide (GIP) is impaired in type 2 diabetes, while the effect of glucagon-like peptide-1 (GLP-1) is preserved. To evaluate the role of impaired GIP function in glucose homeostasis and development of the endocrine pancreas in a large animal model, we generated transgenic pigs expressing a dominant-negative GIP receptor (GIPR^dn) in pancreatic islets. RESEARCH DESIGN AND METHODS GIPR^dn transgenic pigs were generated using lentiviral transgenesis. Metabolic tests and quantitative stereological analyses of the different endocrine islet cell populations were performed, and β-cell proliferation and apoptosis were quantified to characterize this novel animal model. RESULTS Eleven-week-old GIPR^dn transgenic pigs exhibited significantly reduced oral glucose tolerance due to delayed insulin secretion, whereas intravenous glucose tolerance and pancreatic β-cell mass were not different from controls. The insulinotropic effect of GIP was significantly reduced, whereas insulin secretion in response to the GLP-1 receptor agonist exendin-4 was enhanced in GIPR^dn transgenic versus control pigs. With increasing age, glucose control deteriorated in GIPR^dn transgenic pigs, as shown by reduced oral and intravenous glucose tolerance due to impaired insulin secretion. Importantly, β-cell proliferation was reduced by 60% in 11-week-old GIPR^dn transgenic pigs, leading to a reduction of β-cell mass by 35% and 58% in 5-month-old and 1- to 1.4-year-old transgenic pigs compared with age-matched controls, respectively. CONCLUSIONS The first large animal model with impaired incretin function demonstrates an essential role of GIP for insulin secretion, proliferation of β-cells, and physiological expansion of β-cell mass.  

4.801           Langerhans Cells Prime IL-17–Producing T Cells and Dampen Genital Cytotoxic Responses following Mucosal Immunization

Hervouet, C., Luci, C., Rol., N., Rlousseau, D., Kissenpfennig, A., Malissen, B., Czerkinsky, C. and Anjuere, F.

1. Immunol., 184, 4842-4851 (2010)

  Langerhans cells (LCs) are dendritic cells (DCs) localized in stratified epithelia, such as those overlaying skin, buccal mucosa, and vagina. The contribution of LCs to the promotion or control of immunity initiated at epithelial sites remains debated. We report in this paper that an immunogen comprising OVA linked to the B subunit of cholera toxin, used as delivery vector, was efficient to generate CTLs after vaginal immunization. Using Lang-EGFP mice, we evaluated the contribution of distinct DC subsets to the generation of CD4 and CD8 T cell responses. We demonstrate that the vaginal epithelium, unlike the skin epidermis, includes a minor population of LCs and a major subset of langerin^– DCs. Intravaginally administered Ag is taken up by LCs and langerin^– DCs and carried up to draining lymph nodes, where both subsets prime CD8 T cells, unlike blood-derived DCs, although with distinct capabilities. LCs prime CD8 T cells with a cytokine profile dominated by IL-17, whereas Lang^– DCs induce IFN- –producing T cells. Using Lang-DTR-EGFP mice to ensure a transient ablation of LCs, we found that these cells not only are dispensable for the generation of genital CTL responses but also downregulate these responses, by a mechanism that may involve IL-10 and IL-17 cytokines. This finding has implications for the development of mucosal vaccines and immunotherapeutic strategies designed for the targeting of DCs.  

4.802           High-efficiency transfection of cultured primary motor neurons to study protein localization, trafficking, and function

Fallini, C., Bassell, G.J. and Rossoll, W. Mol. Neurodegeneration, 5, 17-26 (2010)   Background Cultured spinal motor neurons are a valuable tool to study basic mechanisms of development, axon growth and pathfinding, and, importantly, to analyze the pathomechanisms underlying motor neuron diseases. However, the application of this cell culture model is limited by the lack of efficient gene transfer techniques which are available for other neurons. To address this problem, we have established magnetofection as a novel method for the simple and efficient transfection of mouse embryonic motor neurons. This technique allows for the study of the effects of gene expression and silencing on the development and survival of motor neurons. Results We found that magnetofection, a novel transfection technology based on the delivery of DNA-coated magnetic nanobeads, can be used to transfect primary motor neurons. Therefore, in order to use this method as a new tool for studying the localization and transport of axonal proteins, we optimized conditions and determined parameters for efficient transfection rates of >45% while minimizing toxic effects on survival and morphology. To demonstrate the potential of this method, we have used transfection with plasmids encoding fluorescent fusion-proteins to show for the first time that the spinal muscular atrophy-disease protein Smn is actively transported along axons of live primary motor neurons, supporting an axon-specific role for Smn that is different from its canonical function in mRNA splicing. We were also able to show the suitability of magnetofection for gene knockdown with shRNA-based constructs by significantly reducing Smn levels in both cell bodies and axons, opening new opportunities for the study of the function of axonal proteins in motor neurons. Conclusions In this study we have established an optimized magnetofection protocol as a novel transfection method for primary motor neurons that is simple, efficient and non-toxic. We anticipate that this novel approach will have a broad applicability in the study of motor neuron development, axonal trafficking, and molecular mechanisms of motor neuron diseases.  

4.803           Successful Human Islet Isolation and Transplantation Indicating the Importance of Class 1 Collagenase and Collagen Degradation Activity Assay

Balamurugan, A.N., Breite, A.G., Anazawa, T., Loganathan, G., Wilhelm, J.J., Papas, K.K., Dwulet, F.E., McCarthy, R.C. and Hering, B.J. Transplantation, 89, 954-961 (2010)   Background. Purified tissue dissociation enzymes (TDEs) are critical to successful human islet isolation required for clinical transplantation, but little is known about the characteristics of the key enzymes-class I (C1) and class II (C2) collagenase from Clostridium histolyticum-used in these procedures. Here, we show the differences between the C1 collagenase found in purified collagenase products manufactured by three suppliers and the impact of differences in C1 between two suppliers on human islet yield. Methods. Collagenase from Roche, Serva/Nordmark (Uetersen, Germany), and VitaCyte (Indianapolis, IN) were analyzed by analytical high-performance liquid chromatography and collagen degradation activity (CDA), an assay that preferentially detects intact C1 collagenase. Human islet isolations were performed using current standard practices. Results. These studies showed that the highest amount of intact C1 that correlated with a high specific CDA (CDA unit per milligram of protein). The highest specific CDA was found in VitaCyte product followed by the Roche and Serva/Nordmark products. The products of VitaCyte were used successfully for human islet isolation (n=14) with an average final islet yield obtained was 419,100+/-150,900 islet equivalent number (IEQ) (4147+/-1759 IEQ/g pancreas). Four of these preparations were used successfully in clinical transplantation procedures. These TDEs gave significantly better results when compared with earlier data where 27 isolations were performed using Serva NB1 collagenase and NB neutral protease where the final islet yield was 217,500+/-152,400 IEQ (2134+/-1524 IEQ/g pancreas). Conclusions. These data indicate the importance of intact C1 and the use of the appropriate analytical assays to correlate biochemical characteristics of TDEs to islet quality and yield.    

4.804           Expression of Cell Cycle–Related Genes With Cytokine-Induced Cell Cycle Progression of Primitive Hematopoietic Stem Cells

Quesenberry, P.J., Dooner, G.J., Del Tatto, M., Colvin, G.A., Johnson, K. and Dooner, M.S. Stem Cells and Development, 19(4), 453-460 (2010)   Primitive marrow lineage-negative rhodamine low and Hoechst low (LRH) stem cells isolated on the basis of quiescence respond to the cytokines thrombopoietin, FLT3L, and steel factor by synchronously progressing through cell cycle. We have now profiled the mRNA expression, as determined by real-time RT-PCR, of 47 hematopoietic or cell cycle-related genes, focusing on the variations in the cell cycle regulators with cycle transit. LRH stem cells, at isolation, showed expression of all interrogated genes, but at relatively low levels. In our studies, there was a good deal of consistency with regard to cell cycle regulatory genes involved in the G1/S progression point of LRH murine stem cells. The observed pattern of expression of cyclin A2 is consistent with actions at these phases of cell cycle. Minimal elevations were seen at 16 h with higher elevations at 24, 32, 40, and 48 h times encompassing S, G2, and M phases. CDK2 expression pattern was also consistent with a role in G1/S transition with a modest elevation at 24 h and more substantial elevation at 32 h. The observed pattern of expression of cyclin F mRNA with marked elevations at 16–40 h was also consistent with actions in S and G2 phases. Cyclin D1 expression pattern was less consistent with its known role in G1 progression. The alterations in multiple other cell cycle regulators were consistent with previous information obtained in other cell systems. The cycle regulatory mechanics appears to be preserved across broad ranges of cell types.  

4.805           Cultured hypothalamic neurons are resistant to inflammation and insulin resistance induced by saturated fatty acids

Choi, S.J., Kim, F., Schwartz, M.W. and Wisse, B.E. Am. J. Physiol. Endocrinol. Metab., 298, E1122-E1130 (2010)   Hypothalamic inflammation induced by high-fat feeding causes insulin and leptin resistance and contributes to the pathogenesis of obesity. Since in vitro exposure to saturated fatty acids causes inflammation and insulin resistance in many cultured cell types, we determined how cultured hypothalamic neurons respond to this stimulus. Two murine hypothalamic neuronal cell cultures, N43/5 and GT1–7, were exposed to escalating concentrations of saturated fatty acids for up to 24 h. Harvested cells were evaluated for activation of inflammation by gene expression and protein content. Insulin-treated cells were evaluated for induction of markers of insulin receptor signaling (p-IRS, p-Akt). In both hypothalamic cell lines, inflammation was induced by prototypical inflammatory mediators LPS and TNF , as judged by induction of I B (3- to 5-fold) and IL-6 (3- to 7-fold) mRNA and p-I B protein, and TNF pretreatment reduced insulin-mediated p-Akt activation by 30% (P < 0.05). By comparison, neither mixed saturated fatty acid (100, 250, or 500 µM for 6 h) nor palmitate exposure alone (200 µM for 24 h) caused inflammatory activation or insulin resistance in cultured hypothalamic neurons, whereas they did in control muscle and endothelial cell lines. Despite the lack of evidence of inflammatory signaling, saturated fatty acid exposure in cultured hypothalamic neurons causes endoplasmic reticulum stress, induces mitogen-activated protein kinase, and causes apoptotic cell death with prolonged exposure. We conclude that saturated fatty acid exposure does not induce inflammatory signaling or insulin resistance in cultured hypothalamic neurons. Therefore, hypothalamic neuronal inflammation in the setting of DIO may involve an indirect mechanism mediated by saturated fatty acids on nonneuronal cells.  

4.806           Transport of Bacillus anthracis from the lungs to the draining lymph nodes is a rapid process facilitated by CD11c+ cells

Shetron-Rama, L.M., Herring-Palmer, A.C., Huffnagle, G.B. and Hanna, P. Microbial Pathogenesis, 49, 38-46 (2010)   Inhalational anthrax is established after inhaled Bacillus anthracis spores are transported to the lung associated lymph nodes. Dendritic cells (CD11c+ cells) located in the lungs are phagocytes that maintain many capabilities consistent with transport. This study investigates the role of dendritic cells as conduits of spores from the lung to the draining lymph nodes. The intratracheally spore-challenged mouse model of inhalational anthrax was utilized to investigate in vivo activities of CD11c+ cells. FITC labeled spores were delivered to the lungs of mice. Subsequently lung associated lymph nodes were isolated after infection and CD11c+ cells were found in association with the labeled spores. Further investigation of CD11c+ cells in early anthrax events was facilitated by use of the CD11c-diphtheria toxin (DT) receptor-green fluorescent protein transgenic mice in which CD11c+ cells can be transiently depleted by treatment with DT. We found that the presence of CD11c+ cells was necessary for efficient traffic of the spore to lung associated lymph nodes at early times after infection. Cultured dendritic cells were used to determine that these cells are capable of B. anthracis spore phagocytosis, and support germination and outgrowth. This data demonstrates that CD11c+ cells are likely carriers of B. anthracis spores from the point of inhalation in the lung to the lung associated lymph nodes. The cultured dendritic cell allows for spore germination and outgrowth supporting the concept that the CD11c+ cell responsible for this function can be a dendritic cell.  

4.807           The CagA protein of Helicobacter pylori suppresses the functions of dendritic cell in mice

Tanaka, H., Yoshida, M., Nishiumi, S., Ohnishi, N., Kobayashi, K., Yamamoto, K., Fujita, T., Hatakeyama, M. and Azuma, T. Arch. Biochem. Biophys., 498, 35-42 (2010)   CagA protein is the most assessed effecter molecule of Helicobacter pylori. In this report, we demonstrate how CagA protein regulates the functions of dendritic cells (DC) against H. pylori infection. In addition, we found that CagA protein was tyrosine-phosphorylated in DC. The responses to cagA-positive H. pylori in DC were reduced in comparison to those induced by cagA-negative H. pylori. CagA-overexpressing DC also exhibited a decline in the responses against LPS stimulation and the differentiation of CD4⁺ T cells toward Th1 type cells compared to wild type DC. In addition, the level of phosphorylated IRF3 decreased in CagA-overexpressing DC stimulated with LPS, indicating that activated SHP-2 suppressed the enzymatic activity of TBK1 and consequently IRF3 phosphorylation. These data suggest that CagA protein negatively regulates the functions of DC via CagA phosphorylation and that cagA-positive H. pylori strains suppress host immune responses resulting in their chronic colonization of the stomach.  

4.808           The PXR is a drug target for chronic inflammatory liver disease

Wallace, K., Cowie, D.E., Konstantinou, D.K., Hill, S.J., Tjelle, T.E., Axon, A., Koruth, M., White, S.A., Carlsen, H., Mann, D.A. and Wright, M.C.

1. Steroid Biochem. Mol. Biol., 120, 137-148 (2010)

  PXR activators are used to treat pruritus in chronic inflammatory liver diseases such as primary biliary cirrhosis (PBC). The aims of this study were to determine whether PXR activators could have an additional benefit of inhibiting inflammation in the liver, and determine whether cyclosporin A – which more effectively prevents PBC recurrence in transplanted patients than FK506 – is a PXR activator. In SJL/J mice (which have constitutively high levels of hepatic portal tract inflammatory cell recruitment), feeding a PXR activator inhibited inflammation, TNFα and Il-1α mRNA expression in SJL/J-PXR^+/+, but not SJL/J-PXR^−/−. Monocytic cells – a major source of inflammatory mediators such as TNFα – expressed the PXR and PXR activators inhibited endotoxin-induced NF-κB activation and TNFα expression. PXR activation also inhibited endotoxin-stimulated TNFα secretion from liver monocytes/macrophages isolated from PXR^+/+ mice, but not from cells isolated from PXR^−/− mice. To confirm that PXR activation inhibits NF-κB in vivo, 3x-κB-luc fibrotic mice (which express a luciferase gene regulated by NF-κB) were imaged after treatment with the hepatotoxin CCl₄. PXR activator inhibited the induction of hepatic NF-κB activity without affecting CCl₄ toxicity/hepatic damage. Using a PXR reporter gene assay, cyclosporin A – but not FK506 – was shown to be a direct PXR activator, and also to induce expression of the classic PXR-regulated CYP3A4 gene in human hepatocytes and in a cell line null for the FXR, a nuclear receptor with similar properties to the PXR. Conclusion: PXR activation is anti-inflammatory in the liver and the effects of cyclosporin A in PBC disease recurrence may be mediated in part via the PXR. Since PXR activation promotes hepatocyte growth and is also anti-fibrogenic, the PXR may be an excellent drug target for the treatment of chronic inflammatory liver disease.  

4.809           trans-10,cis-12-Conjugated Linoleic Acid Instigates Inflammation in Human Adipocytes Compared with Preadipocytes

Martinez, K., Kennedy, A., West, T., Milatovic, D., Aschner, M. and McIntosh, M.

1. Biol. Chem., 285(23), 17701-17721 (2010)

  We showed previously in cultures of primary human adipocytes and preadipocytes that lipopolysaccharide and trans-10,cis-12-conjugated linoleic acid (10,12-CLA) activate the inflammatory signaling that promotes insulin resistance. Because our published data demonstrated that preadipocytes are the primary instigators of inflammatory signaling in lipopolysaccharide-treated cultures, we hypothesized that they played the same role in 10,12-CLA-mediated inflammation. To test this hypothesis, we employed four distinct models. In model 1, a differentiation model, CLA activation of MAPK and induction of interleukin-8 (IL-8), IL-6, IL-1β, and cyclo-oxygenase-2 (COX-2) were greatest in differentiated compared with undifferentiated cultures. In model 2, a cell separation model, the mRNA levels of these inflammatory proteins were increased by 10,12-CLA compared with bovine serum albumin vehicle in the adipocyte fraction and the preadipocyte fraction. In model 3, a co-culture insert model, inserts containing ∼50% adipocytes (AD50) or ∼100% preadipocytes (AD0) were suspended over wells containing AD50 or AD0 cultures. 10,12-CLA-induced IL-8, IL-6, IL-1β, and COX-2 mRNA levels were highest in AD50 cultures when co-cultured with AD0 inserts. In model 4, a conditioned medium (CM) model, CM collected from CLA-treated AD50 but not AD0 cultures induced IL-8 and IL-6 mRNA levels and activated phosphorylation of MAPK in naive AD0 and AD50 cultures. Consistent with these data, 10,12-CLA-mediated secretions of IL-8 and IL-6 from AD50 cultures were higher than from AD0 cultures. Notably, blocking adipocytokine secretion prevented the inflammatory capacity of CM from 10,12-CLA-treated cultures. These data suggest that CLA instigates the release of inflammatory signals from adipocytes that subsequently activate adjacent preadipocytes.  

4.810           CCR5 Signaling Suppresses Inflammation and Reduces Adverse Remodeling of the Infarcted Heart, Mediating Recruitment of Regulatory T Cells

Dobaczewski, M., Xia, Y., Bujak, M., Gonzales-Quesada, C. and Frangogiannis, N.G. Am. J. Pathol., 176(5), 2177-2187 (2010)   Myocardial infarction triggers an inflammatory reaction that is involved in cardiac remodeling. Cardiac repair is dependent on regulatory mechanisms that suppress inflammation and prevent excessive matrix degradation. Chemokine induction in the infarcted heart mediates recruitment of leukocyte subsets with distinct properties. We demonstrate that signaling through the CC chemokine receptor 5 (CCR5) prevents uncontrolled postinfarction inflammation and protects from adverse remodeling by recruiting suppressive mononuclear cells. CCR5 and its ligands macrophage inflammatory protein (MIP)–1 and MIP-1β were markedly induced in the infarcted mouse myocardium. In addition, almost 40% of the mononuclear cells infiltrating the infarct expressed CCR5. CCR5^–/– mice exhibited marked upregulation of proinflammatory cytokine and chemokine expression in the infarct. In wild-type infarcts CCR5⁺ mononuclear cells had anti-inflammatory properties, expressing higher levels of IL-10 than CCR5^– cells. In contrast, mononuclear cells isolated from CCR5^–/– infarcts had reduced IL-10 expression. Moreover, enhanced inflammation in the absence of CCR5 was associated with impaired recruitment of CD4⁺/foxp3⁺ regulatory T cells (Tregs). The CCR5⁺ Treg subset exhibited increased IL-10 expression, reflecting potent anti-inflammatory activity. Accentuated inflammation in CCR5^–/– infarcts was associated with increased matrix metalloproteinase (MMP) expression, reduced TIMP levels, and enhanced MMP-2 and MMP-9 activity, resulting in worse cardiac dilation. These results suggest that CCR5-mediated Treg recruitment may restrain postinfarction inflammation, preventing excessive matrix degradation and attenuating adverse remodeling.  

4.811           Comparison of density gradient and simple centrifugation of equine spermatozoa: effect on fertility of an oligospermic-subfertile stallion

Mari, G., Castagnetti, C., Morganti, M., Rizzato, G., Mislei, B., Iacono, E. and Merlo, B. Animal Reprod., 121S, S153-S154 (2010)   Low dose insemination methods have recently evolved to satisfy the desire to exploit newly available technology such as sex pre-selection of spermatozoa. Additional benefits, such as improvements in the efficiency of the use of frozen semen and the potential to enhance the fertility of subfertile stallions, have become apparent during the development and evaluation of these techniques (Morris and Allen, 2002). Vazquez et al. (1998) investigated the use of hysteroscopic insemination in an attempt to bypass the uterine transport phase of sperm migration and to increase the fertility of subfertile stallions. Inseminating only the highest quality sperm by selecting for quality prior to insemination may further improve the outcome of techniques delivering low numbers of sperm. Macpherson et al. (2002) demonstrated that EquiPureTM, a proprietary silan-coated silica particle density gradient, can improve semen quality in stallions. In two studies, fresh spermatozoa from fertile stallions were processed through density gradients to enhance selection of those with intact plasma membranes (Morris et al., 2000; Lindsey et al., 2002) and successful low dose hysteroscopic inseminations were achieved. Similarly, frozen-thawed ejaculated  (Alvarenga and Leão, 2002) and epididymal (Morris and Allen, 2002) spermatozoa have been centrifuged through density gradients to remove those damaged by the cryopreservation process. However, no beneficial effect of this treatment was observed on the fertility of low numbers    

4.812           Cortical Overexpression of Neuronal Calcium Sensor-1 Induces Functional Plasticity in Spinal Cord Following Unilateral Pyramidal Tract Injury in Rat

Yip, P.K., Wong, L-F., Sears, T.A., Yanez-Munoz, R.J. and McMahon, S.B. PloSBiology, 8(6), e1000399 (2010)   Following trauma of the adult brain or spinal cord the injured axons of central neurons fail to regenerate or if intact display only limited anatomical plasticity through sprouting. Adult cortical neurons forming the corticospinal tract (CST) normally have low levels of the neuronal calcium sensor-1 (NCS1) protein. In primary cultured adult cortical neurons, the lentivector-induced overexpression of NCS1 induces neurite sprouting associated with increased phospho-Akt levels. When the PI3K/Akt signalling pathway was pharmacologically inhibited the NCS1-induced neurite sprouting was abolished. The overexpression of NCS1 in uninjured corticospinal neurons exhibited axonal sprouting across the midline into the CST-denervated side of the spinal cord following unilateral pyramidotomy. Improved forelimb function was demonstrated behaviourally and electrophysiologically. In injured corticospinal neurons, overexpression of NCS1 induced axonal sprouting and regeneration and also neuroprotection. These findings demonstrate that increasing the levels of intracellular NCS1 in injured and uninjured central neurons enhances their intrinsic anatomical plasticity within the injured adult central nervous system.  

4.813           Dicer-Dependent MicroRNAs Control Maturation, Function, and Maintenance of Langerhans Cells In Vivo

Kuipers, H., Schnorfeil, F.M., Fehling, H-J., Bartels, H. and Brocker, T.

1. Immunol., 185, 400-409 (2010)

  Dendritic cells (DCs) are central for the induction of T cell immunity and tolerance. Fundamental for DCs to control the immune system is their differentiation from precursors into various DC subsets with distinct functions and locations in lymphoid organs and tissues. In contrast to the differentiation of epidermal Langerhans cells (LCs) and their seeding into the epidermis, LC maturation, turnover, and MHC class II Ag presentation capacities are strictly dependent on the presence of Dicer, which generates mature microRNAs (miRNAs). Absence of miRNAs caused a strongly disturbed steady-state homeostasis of LCs by increasing their turnover and apoptosis rate, leading to progressive ablation of LCs with age. The failure to maintain LCs populating the epidermis was accompanied by a proapoptotic gene expression signature. Dicer-deficient LCs showed largely increased cell sizes and reduced expression levels of the C-type lectin receptor Langerin, resulting in the lack of Birbeck granules. In addition, LCs failed to properly upregulate MHC class II, CD40, and CD86 surface molecules upon stimulation, which are critical hallmarks of functional DC maturation. This resulted in inefficient induction of CD4 T cell proliferation, whereas Dicer-deficient LCs could properly stimulate CD8 T cells. Taken together, Dicer-dependent generation of miRNAs affects homeostasis and function of epidermal LCs.  

4.814           Innate immunity defines the capacity of antiviral T cells to limit persistent infection

Andrews, D.M., Estcourt, M.J., Andoniou, C.E., Wikstrom, M.E., Khong, A., Voigt, V., Fleming, P., Tabarias, H., Hill, G.R., vander Most, R.G., Scalzo, A.A., Smyth, M.J. and Degli-Esposti, M.A.

1. Exp. Med., 207(6), 1333-1343 (2010)

  Effective immunity requires the coordinated activation of innate and adaptive immune responses. Natural killer (NK) cells are central innate immune effectors, but can also affect the generation of acquired immune responses to viruses and malignancies. How NK cells influence the efficacy of adaptive immunity, however, is poorly understood. Here, we show that NK cells negatively regulate the duration and effectiveness of virus-specific CD4+ and CD8+ T cell responses by limiting exposure of T cells to infected antigen-presenting cells. This impacts the quality of T cell responses and the ability to limit viral persistence. Our studies provide unexpected insights into novel interplays between innate and adaptive immune effectors, and define the critical requirements for efficient control of viral persistence.  

4.815           Characterization of human DNGR-1+ BDCA3+ leukocytes as putative equivalents of mouse CD8 + dendritic cells

Poulin, L.F., Salio, M., Griessinger, E., Anjos-Afonso, F., Craciun, L., Chen, J-L., Keller, A.M., Joffre, O., Zelenay, S., Nye, E., Moine, A.L., Faure, F., Donckier, V., Sancho, D., Cerundolo, V., Bonnet, D. and Reis e Sousa, C.

1. Exp. Med., 207(6), 1261-1271 (2010)

  In mouse, a subset of dendritic cells (DCs) known as CD8α⁺ DCs has emerged as an important player in the regulation of T cell responses and a promising target in vaccination strategies. However, translation into clinical protocols has been hampered by the failure to identify CD8α⁺ DCs in humans. Here, we characterize a population of human DCs that expresses DNGR-1 (CLEC9A) and high levels of BDCA3 and resembles mouse CD8α⁺ DCs in phenotype and function. We describe the presence of such cells in the spleens of humans and humanized mice and report on a protocol to generate them in vitro. Like mouse CD8α⁺ DCs, human DNGR-1⁺ BDCA3^hi DCs express Necl2, CD207, BATF3, IRF8, and TLR3, but not CD11b, IRF4, TLR7, or (unlike CD8α⁺ DCs) TLR9. DNGR-1⁺ BDCA3^hi DCs respond to poly I:C and agonists of TLR8, but not of TLR7, and produce interleukin (IL)-12 when given innate and T cell–derived signals. Notably, DNGR-1⁺ BDCA3⁺ DCs from in vitro cultures efficiently internalize material from dead cells and can cross-present exogenous antigens to CD8⁺ T cells upon treatment with poly I:C. The characterization of human DNGR-1⁺ BDCA3^hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy.  

4.816           Predicting islet yield in pediatric patients undergoing pancreatectomy and autoislet transplantation for chronic pancreatitis

Bellin, M.D., Blondet, J.J., Beilman, G.J., Dunn, Ty.B., Balamurugan, A.N., Thomas, W., Sutherland, D.E.R. and Moran, A. Pedriatric Diabetes, 11, 227-234 (2010)   Background/Objective: Chronic pancreatitis (CP) in children is associated with significant morbidity and can lead to narcotic dependence. Total pancreatectomy (TP) may be indicated in refractory CP to relieve pain; simultaneous islet autotransplant (IAT) may prevent postsurgical diabetes. About half of pediatric patients are insulin independent 1 yr after IAT. Insulin independence correlates best with the number of islets available for transplantation (islet yield). Currently there is no known method to predict islet yield in a given patient. We assessed the ability of preoperative metabolic tests to predict islet yields in 10 children undergoing TP/IAT. Design/Methods: Hemoglobin A1c (HbA1c) and mixed meal tolerance tests (MMTT) were obtained prior to surgery in 10 patients age <= 18 yr. Fasting glucose, C-peptide, and creatinine were used to calculate the C-peptide to glucose* creatinine ratio (CPGCR). C-peptide peak and area under the curve (AUC) were determined from 2 h MMTT. Linear regressions were performed to predict islet yield from baseline test results. Results: Islet yield ranged from 7000 to 434 000 islet equivalents (IE) (mean 222 452 +/- 148 697 IE). Islet yield was well predicted from body weight and fasting plasma glucose (R 2 = 57%, adjusted for overfitting by bootstrap). Islet yield was positively associated with CPGCR, peak C-peptide, and AUC C-peptide and negatively associated with HbA1c. Conclusions: Pilot data from 10 pediatric patients suggest that simple preoperative measurement of fasting plasma glucose may give a useful prediction of islet yield. Islet yield correlates with HbA1c and C-peptide levels. This information allows individual candidates to weigh the specific risk of becoming diabetic against the benefit of pain relief should they undergo TP-IAT.  

4.817           Transplantation of Long-term Cultured Porcine Islets in the Rat: Prolonged Graft Survival and Recipient Growth on Reduced Immunosuppression

Rijkelijkhuizen, J.K.R.A., Töns, A., Terpstra, O.T. and Bouwman, E. Cell Transplantation, 19, 387-398 (2010)   To evaluate whether further improvement in porcine islet xenotransplantation is feasible, a number of questions were addressed. Earlier we showed significant improvement in the nude mouse of the porcine islets by selection through long-term culture. Now these islets were tested in the stringent pig-to-rat model. Islets were isolated from adult pigs, cultured for 1.5-3 weeks and transplanted to rats. Possible rejection mechanisms were assessed by interference of the cellular response with cyclosporine A (CsA), blocking macrophages with gadolinium chloride (GdCl), and suppressing the humoral response with cyclophosphamide. Modifications in graft size and condition were analyzed. Untreated control recipients showed primary nonfunction (PNF). CsA treatment could fully overcome PNF and resulted in graft survival from 10 to over 134 days. Rejection was the main cause of function loss. Although rejection could not be prevented by intensifying the induction therapy, increased maintenance immunosuppression effectively blocked rejection, albeit at the expense of toxicity. Blocking the humoral response was ineffective; all grafts showed PNF. In contrast, depletion of macrophages fully prevented PNF. Combination of GdCl and CsA gave no additional effect, and grafts were rejected between 57 and 162 days. Generally, graft survivals were similar to those reported in the literature; however, long-term cultured islets required much less maintenance immunosuppression. Cessation of graft function was not always due to rejection; in some cases “islet exhaustion” was found, possibly caused by discrepancy between the graft size and the rapidly growing recipient. Neither the presence of damaged islet tissue in the graft nor the size of the graft exerted any influence on graft survival. On rejection, no real infiltration of the graft was seen; destruction gradually processed from the outside. The good functional capability of the cultured islets was illustrated by disappearance of the clinical symptoms and increase in body weight, which almost doubled in the long-term survivors.  

4.818           Evidence for microRNA involvement in exercise-associated neutrophil gene expression changes

Radom-Aizik, S., Zaldivar Jr., F., oliver, S., Galassetti, P. and Cooper, D.M.

1. Appl. Physiol., 109(1), 252-261 (2010)

  Exercise leads to a rapid change in the profile of gene expression in circulating neutrophils. MicroRNAs (miRNAs) have been discovered to play important roles in immune function and often act to attenuate or silence gene translation. We hypothesized that miRNA expression in circulating neutrophils would be affected by brief exercise. Eleven healthy men (19–30 yr old) performed 10, 2-min bouts of cycle ergometer exercise interspersed with 1-min rest at a constant work equivalent to 76% of maximal oxygen uptake ( O_2max). We used the Agilent Human miRNA V2 Microarray. A conservative statistical approach was used to determine that exercise significantly altered 38 miRNAs (20 had lower expression). Using RT-PCR, we verified the expression level changes from before to after exercise of seven miRNAs. In silico analysis showed that collectively 36 miRNAs potentially targeted 4,724 genes (2 of the miRNAs had no apparent gene targets). Moreover, when we compared the gene expression changes (n = 458) in neutrophils that have been altered by exercise, as previously reported, with the miRNAs altered by exercise, we identified three pathways, Ubiquitin-mediated proteolysis, Jak-STAT signaling pathway, and Hedgehog signaling pathway, in which an interaction of miRNA and gene expression was plausible. Each of these pathways is known to play a role in key mechanisms of inflammation. Brief exercise alters miRNA profile in circulating neutrophils in humans. These data support the hypothesis that exercise-associated changes in neutrophil miRNA expression play a role in neutrophil gene expression in response to physical activity.  

4.819           Addition of glutamate to serum-free culture promotes recovery of electrical activity in adult hippocampal neurons in vitro

Edwards, D., Das, M., Molnar, P. and Hickman, J.J.

1. Neurosci. Methods, 190, 155-163 (2010)

  A long-term cell culture system utilizing normal adult hippocampal neurons would represent an important tool that could be useful in research on the mature brain, neurological disorders and age-related neurological diseases. Historically, in vitro neuronal systems are derived from embryonic rather than mature brain tissue, a practice predicated upon difficulties in supporting regeneration, functional recovery and long-term survival of adult neurons in vitro. A few studies have shown that neurons derived from the hippocampal tissue of adult rats can survive and regenerate in vitro under serum-free conditions. However, while the adult neurons regenerated morphologically under these conditions, both the electrical activity characteristic of in vivo neurons as well as long-term neuronal survival was not consistently recovered in vitro. In this study, we report on the development of a defined culture system with the ability to support functional recovery and long-term survival of adult rat hippocampal neurons. In this system, the cell-adhesive substrate, N-1 [3-(trimethoxysilyl) propyl]-diethylenetriamine, supported neuronal attachment, regeneration, and long-term survival of adult neurons for more than 80 days in vitro. Additionally, the excitatory neurotransmitter glutamate, applied at 25 μM for 1–7 days after morphological neuronal regeneration in vitro, enabled full recovery of neuronal electrical activity. This low concentration of glutamate promoted the recovery of neuronal electrical activity but with minimal excitotoxicity. These improvements allowed electrically active adult neurons to survive in vitro for several months, providing a stable test-bed for the long-term study of regeneration in adult-derived neuronal systems, especially for traumatic brain injury (TBI).  

4.820           Enhanced Prediction of Porcine Islet Yield and Posttransplant Outcome Using a Combination of Quantitative Histomorphometric Parameters and Flow Cytometry

Jin, S-M., Kim, K.S., Lee, S-Y., Gong, C-H., Park, S.K., Yu, J.E., Yeom, S-C., Yoon, T.W., Ha, J., Park, C-G. and Kim, S-J. Cell Transplant., 19, 299-311 (2010)   Prediction of islet yield and posttransplant outcome is essential for clinical porcine islet xenotransplantation. Although several histomorphometric parameters of biopsied porcine pancreases are predictive of islet yield, their role in the prediction of in vivo islet potency is unknown. We investigated which histomorphometrical parameter best predicts islet yield and function, and determined whether it enhanced the predictive value of in vitro islet function tests for the prediction of posttransplant outcome. We analyzed the histomorphometry of pancreases from which 60 adult pig islet isolations were obtained. Islet function was assessed using the β-cell viability index based on flow cytometry analysis, oxygen consumption rate, ADP/ATP ratio, and/or concurrent transplantation into NOD/SCID mice. Receiver operating characteristic (ROC) analysis revealed that only islet equivalent (IEQ)/cm² and the number of islets >200 μm in diameter significantly predicted an islet yield of >2000 IEQ/g (p < 0.001 for both) and in vivo islet potency (p = 0.024 and p = 0.019, respectively). Although not predictive of islet yield, a high proportion of large islets (>100 μm in diameter) best predicted diabetes reversal (p = 0.001). Multiple regression analysis revealed that the β-cell viability index (p = 0.003) and the proportion of islets >100 μm in diameter (p = 0.048) independently predicted mean posttransplant blood glucose level (BGL). When BGL was estimated using both these parameters [area under the ROC curve (AUC), 0.868; 95% confidence interval (CI), 0.730-1.006], it predicted posttransplant outcome more accurately than the β-cell viability index alone (AUC, 0.742; 95% CI, 0.544-0.939). In conclusion, we identified the best histomorphometric predictors of islet yield and posttransplant outcome. This further enhanced the predictive value of the flow cytometry analysis. These parameters should be useful for predicting islet yield and in vivo potency before clinical adult porcine islet xenotransplantation.  

4.821           Seven Consecutive Successful Clinical Islet Isolations With Pancreatic Ductal Injection

Matsumoto, S., Noguichi, H., Shimoda, M., Ikemoto, T., Nazirruddin, B., Jackson, A., Tamura, Y., Olson, G., Fujita, Y., Chujo, D., Takita, M., Kobayashi, N., Onaca, N. and Levy, M. Cell Transplant., 19, 291-297 (2010)   Inconsistent islet isolation is one of the issues of clinical islet transplantation. In the current study, we applied ductal injection to improve the consistency of islet isolation. Seven islet isolations were performed with the ductal injection of ET-Kyoto solution (DI group) and eight islet isolations were performed without the ductal injection (standard group) using brain-dead donor pancreata. Isolated islets were evaluated based on the Edmonton protocol for transplantation. The DI group had significantly higher islet yields (588,566 ± 64,319 vs. 354,836 ± 89,649 IE, p < 0.01) and viability (97.3 ± 1.2% vs. 92.6 ± 1.2%, p < 0.02) compared with the standard group. All seven isolated islet preparations in the DI group (100%), versus only three out of eight isolated islet preparations (38%) in the standard group met transplantation criteria. The islets from the DI group were transplanted into three type 1 diabetic patients and all three patients became insulin independent. Ductal injection significantly improved quantity and quality of isolated islets and resulted in high success rate of clinical islet transplantation. This simple modification will reduce the risk of failure of clinical islet isolation.  

4.822           Mutant HSPB8 causes motor neuron-specific neurite degeneration

Irobi, J., Almeida-Souza, L., Asselbergh, B., De Winter, V., Goethais, S., Dierick, I., Krishnan, J., Timmermans, J-P., Robberecht, W., De Johnghe, P., van den Bosch, L., Janssens, S. and Timmerman, V. Hum. Mol. Genet., 19(16), 3254-3265 (2010)   Missense mutations (K141N and K141E) in the -crystallin domainof the small heat shock protein HSPB8 (HSP22) cause distal hereditarymotor neuropathy (distal HMN) or Charcot-Marie-Tooth neuropathytype 2L (CMT2L). The mechanism through which mutant HSPB8 leadsto a specific motor neuron disease phenotype is currently unknown.To address this question, we compared the effect of mutant HSPB8in primary neuronal and glial cell cultures. In motor neurons,expression of both HSPB8 K141N and K141E mutations clearly resultedin neurite degeneration, as manifested by a reduction in numberof neurites per cell, as well as in a reduction in average lengthof the neurites. Furthermore, expression of the K141E (and toa lesser extent, K141N) mutation also induced spheroids in theneurites. We did not detect any signs of apoptosis in motorneurons, showing that mutant HSPB8 resulted in neurite degenerationwithout inducing neuronal death. While overt in motor neurons,these phenotypes were only very mildly present in sensory neuronsand completely absent in cortical neurons. Also glial cellsdid not show an altered phenotype upon expression of mutantHSPB8. These findings show that despite the ubiquitous presenceof HSPB8, only motor neurons appear to be affected by the K141Nand K141E mutations which explain the predominant motor neuronphenotype in distal HMN and CMT2L.  

4.823           Islet Transplantation: Lessons Learned Since the Edmonton Breakthrough

Langer, R.M. Transplant. Proceedings, 42, 1421-1424 (2010) This work sought to summarize the main issues of the last decade in the field of clinical islet transplantation. Ten years ago in Edmonton, a new protocol initiated for islet transplantation brought a breakthrough to the field. The earlier, rather poor results were in a sharp contrast to the first published results of 100% insulin freedom at 1 year. However, later it became clear that the promising initial results decline with time; at around 5 years, only about 10% of the patients maintain freedom from external insulin. Despite that fact, a milestone was set and intensive research started worldwide. New hopes were raised for patients. Modifications of the original protocol have been implemented to improve clinical results; however, islet transplantation remains an experimental procedure to date.  

4.824           Tauroursodeoxycholate (TUDCA), chemical chaperone, enhances function of islets by reducing ER stress

Lee, Y.Y., Hong, S.H., Lee, Y.J., Chung, S.S., Jung, H.S., Park, S.G. and Park, K.S. Biochem. Biophys. Res. Comm., 397, 735-739 (2010)   The exposure to acute or chronic endoplasmic reticulum (ER) stress has been known to induce dysfunction of islets, leading to apoptosis. The reduction of ER stress in islet isolation for transplantation is critical for islet protection. In this study, we investigated whether tauroursodeoxycholate (TUDCA) could inhibit ER stress induced by thapsigargin, and restore the decreased glucose stimulation index of islets. In pig islets, thapsigargin decreased the insulin secretion by high glucose stimulation in a time-dependent manner (1 h, 1.35 ± 0.16; 2 h, 1.21 ± 0.13; 4 h, 1.17 ± 0.16 vs. 0 h, 1.81 ± 0.15, n = 4, p < 0.05, respectively). However, the treatment of TUDCA restored the decreased insulin secretion index induced by thapsigargin (thapsigargin, 1.25 ± 0.12 vs. thapsigargin + TUDCA, 2.13 ± 0.19, n = 5, p < 0.05). Furthermore, the culture of isolated islets for 24 h with TUDCA significantly reduced the rate of islet regression (37.4 ± 5.8% vs. 14.5 ± 6.4%, n = 12, p < 0.05). The treatment of TUDCA enhanced ATP contents in islets (27.2 ± 3.2 pmol/20IEQs vs. 21.7 ± 2.8 pmol/20IEQs, n = 9, p < 0.05). The insulin secretion index by high glucose stimulation is also increased by treatment of TUDCA (2.42 ± 0.15 vs. 1.92 ± 0.12, n = 12, p < 0.05). Taken together, we suggest that TUDCA could be a useful agent for islet protection in islet isolation for transplantation.  

4.825           Live attenuated Salmonella enterica serovar Typhimurium expressing swine interferon-α has antiviral activity and alleviates clinical signs induced by infection with transmissible gastroenteritis virus in piglets

Kim, S.J., Han, Y.W., Rahman, M.M., Kim, S.B., Uyangaa, E., Lee, B.M., Kim, J.H., Roh, Y.S., Kang, S.H., Kim, K., Lee, J.H., Kim, B., Park, K.I. and Eo, S.K. Vaccine, 28, 5031-5037 (2010)   Enhancing innate and acquired immunity by cytokines such as IFN-α appears to be useful as a first line of defense against viral infection. However, the practical use of cytokines in livestock is not evident due to cost and production issues associated with mass administration. In this study, we tested the efficacy of live attenuated Salmonella enterica serovar Typhimurium designed to secrete swine IFN-α (swIFN-α) protein for preventing the clinical signs caused by infection with transmissible gastroenteritis virus (TGEV), one of the diarrhea-causing viruses in the swine industry. Attenuated Salmonella vaccine (χ8501) containing swIFN-α-encoding pYA3560 vector (χ8501/swIFN-α) successfully induced the secretion of swIFN-α protein into the culture supernatants, as confirmed by SDS-PAGE and Western blot. The culture supernatants of χ8501/swIFN-α had antiviral activity against TGEV with 50% effective dose (ED₅₀) of 320 per mg of supernatant protein. In addition, oral administration of χ8501/swIFN-α reduced the severity of clinical signs caused by TGEV infection with the effect more apparent at 6–8 days post-infection, and reduced excretion of TGEV in feces. Similarly, the amount of TGEV in intestinal tissues and mesenteric lymph node of χ8501/swIFN-α-administered piglets was lower than in piglets that were treated with control bacteria. These results indicate the value of attenuated Salmonella vaccines as delivery systems of cytokines that can be used for mass administration, thereby overcoming cost and production issues.  

4.826           Angiotensin-converting Enzyme Inhibition Down-regulates the Pro-atherogenic Chemokine Receptor 9 (CCR9)-Chemokine Ligand 25 (CCL25) Axis

Alla, J.A., Langer, A., Elzahwy, S.S., Arman-Kalcek, G., Streichert, T. and Quitterer, U.

1. Biol. Chem., 285(30), 23496-23505 (2010)

  Many experimental and clinical studies suggest a relationship between enhanced angiotensin II release by the angiotensin-converting enzyme (ACE) and the pathophysiology of atherosclerosis. The atherosclerosis-enhancing effects of angiotensin II are complex and incompletely understood. To identify anti-atherogenic target genes, we performed microarray gene expression profiling of the aorta during atherosclerosis prevention with the ACE inhibitor, captopril. Atherosclerosis-prone apolipoprotein E (apoE)-deficient mice were used as a model to decipher susceptible genes regulated during atherosclerosis prevention with captopril. Microarray gene expression profiling and immunohistology revealed that captopril treatment for 7 months strongly decreased the recruitment of pro-atherogenic immune cells into the aorta. Captopril-mediated inhibition of plaque-infiltrating immune cells involved down-regulation of the C-C chemokine receptor 9 (CCR9). Reduced cell migration correlated with decreased numbers of aorta-resident cells expressing the CCR9-specific chemoattractant factor, chemokine ligand 25 (CCL25). The CCL25-CCR9 axis was pro-atherogenic, because inhibition of CCR9 by RNA interference in hematopoietic progenitors of apoE-deficient mice significantly retarded the development of atherosclerosis. Analysis of coronary artery biopsy specimens of patients with coronary artery atherosclerosis undergoing bypass surgery also showed strong infiltrates of CCR9-positive cells in atherosclerotic lesions. Thus, the C-C chemokine receptor, CCR9, exerts a significant role in atherosclerosis.  

4.827           Contributions of selective knockout studies to understanding cholinesterase disposition and function

Camp, S., Zhang, L., Krejci, E., Dobbertin, A., Bernanrd, V., Girard, E., Duysen, E., Lockridge, O., De Jaco, A. and Taylor, P. Chemico-Biological Interantions, 187, 72-77 (2010)   The complete knockout of the acetylcholinesterase gene (AChE) in the mouse yielded a surprising phenotype that could not have been predicted from deletion of the cholinesterase genes in Drosophila, that of a living, but functionally compromised animal. The phenotype of this animal showed a sufficient compromise in motor function that precluded precise characterization of central and peripheral nervous functional deficits. Since AChE in mammals is encoded by a single gene with alternative splicing, additional understanding of gene expression might be garnered from selected deletions of the alternatively spliced exons. To this end, transgenic strains were generated that deleted exon 5, exon 6, and the combination of exons 5 and 6. Deletion of exon 6 reduces brain AChE by 93% and muscle AChE by 72%. Deletion of exon 5 eliminates AChE from red cells and the platelet surface. These strains, as well as knockout strains that selectively eliminate the AChE anchoring protein subunits PRiMA or ColQ (which bind to sequences specified by exon 6) enabled us to examine the role of the alternatively spliced exons responsible for the tissue disposition and function of the enzyme. In addition, a knockout mouse was made with a deletion in an upstream intron that had been identified in differentiating cultures of muscle cells to control AChE expression. We found that deletion of the intronic regulatory region in the mouse essentially eliminated AChE in muscle and surprisingly from the surface of platelets. The studies generated by these knockout mouse strains have yielded valuable insights into the function and localization of AChE in mammalian systems that cannot be approached in cell culture or in vitro.  

4.828           Superiority of Visipaque (Iodixanol) -Controlled Density Gradient Over Ficoll-400 in Adult Porcine Islet Purification

Min, T., Yi, L., Chao, Z., Haitoa, Z., Wei, W., Liang, Y and  Bo, W. Transplant. Proccedings, 42(5), 1825-1829 (2010)   Objective Sufficient and favorable biological functions of islets are major problems hindering xenotransplantation. The aim of the present study was to evaluate the effects on harvesting, purity, viability, and function of using improved Visipaque ( iodixanol) and Ficoll-400 for adult porcine islet purification. Methods Twelve adult porcine pancreata were randomly divided into an Iodixanol -University of Wisconsin (UW) group and a Ficoll-400-UW group according to the purification method. Porcine pancreata were isolated by collagenase digestion. After isolation and purification, the islet yield and purity were evaluated by dithizone staining, and islet function assessed by in vitro insulin release assays and in vivo islet xenotransplantation. Results There were no marked differences in the islet yield before purification (5254.67 ± 189.44 IEQ/g vs 5092.67 ± 178.94 IEQ/g, P > .05). After purification, there were significantly more islets harvested in Iodixanol -UW group than in the Ficoll-400-UW group: 4222.00 ± 228.84 IEQ/g vs 3036.83 ± 79.60 IEQ/g (P < .05). Islets from the two groups showed satisfactory insulin secretory ability. There were no significant differences in islet survival times between the two groups in diabetic rats: 8.2 ± 1.619 days vs 6.9 ± 1.197 days (P > .05). Conclusion The improved iodixanol -UW density gradient method was superior to Ficoll-400 method to improve the number, viability, and insulin secret of purified adult porcine islets although the benefits did not improve in vivo survival.  

4.829           Improved Method of Porcine Pancreas Procurement With Arterial Flush and Ductal Injection Enhances Islet Isolation Outcome

  Anazawa, T., Balamuragan, A.N., Papas, K.K., Avgoustiniatos, E.S., Ferrer, J., Matsumoto, S., Sutherland, D.E.R. and Hering, B.J. Transplant. Proceedings, 42(6), 2032-2035 (2010)   Background Several pancreas procurement procedures have been used for porcine islet isolation; however, their impact on outcomes has not been extensively studied. We evaluated an advanced procurement technique for porcine islet isolation designed to reduce warm ischemia, to remove blood content, and enhance cooling of the pancreas by implementing a vascular flush and ductal preservation. Method Pancreata procured from adult Landrace pigs were divided into 3 different surgical protocols: Pancreatectomy utilizing only surface cooling (group 1; n = 24); surface cooling and ductal injection with cold preservation solution before pancreatectomy (group 2; n = 12); or surface cooling, ductal injection, and an approach by selectively flushing through the celiac trunk and the superior mesenteric artery (group 3; n = 14). We assessed the islet isolation results and quality using in vitro and in vivo assays. Results Significantly higher overall yield and islet yield per gram pancreas were obtained from group 3 pigs compared with the other groups. Measurements of islet viability after 7 days of culture, as assessed by oxygen consumption rate per DNA, showed that group 3 islets displayed the highest values. Sustained normoglycemia was observed in diabetic nude mice transplanted with 2000 islet equivalents from all 3 groups. Discussion This study demonstrated that an advanced pancreas procurement technique including ductal preservation and selective arterial flush with cold preservation solution provided significant improvements in porcine islet isolation outcomes.  

4.830           Rapid Quantitative Assessment of the Pig Pancreas Biopsy Predicts Islet Yield

Anazawa, T., Balamurugan, A.N., Matsumoto, S., LaFreniere, S.A., O’Brien, T.D., Sutherland, D.E.R. and Hering, B.J. Transplant. Proceedings, 42(6), 2036-2039 (2010)   Background The cost of islet procurement from donor pigs is increased by the use of organs that produce low yields. We developed an assessment system using dithizone-stained pig pancreas biopsies to enable the preselection of donor organs. Methods Pig pancreas biopsy slices were soaked in dithizone solution. The islets were evaluated before islet isolation by converting the islet counts (IC) to islet equivalents (IE), and then determining the IE/cm², IE/IC, % islets >150 μm, and % islets >200 μm. These parameters were evaluated in 3 different areas of the pancreas (duodenal, splenic, and connecting lobe; n = 42 each). Stepwise multivariate linear regression analysis was performed to assess for correlations with islet yield and decide which area of the pancreas had the most predictive value. To identify other predictors, including donor and islet isolation variables, we performed binary logistic regression analysis with significant variables from the univariate analysis (n = 67). For this analysis, the pigs were categorized into high (n = 23) and low (n = 44) yield groups. Results Stepwise multivariate linear regression analysis revealed that IE/cm² of the splenic lobe significantly predicted islet yield. Binary logistic regression analysis indicated that the IE/mm² of the splenic lobe was the only parameter that significantly correlated with successful pig islet isolations (P = .01; odds ratio 3.605). Variables associated with donor and islet isolation, such as age, gender, ischemic time, or enzyme lot, were not significantly correlated with islet yield. Conclusion Our study suggests that the islet distribution of splenic lobe biopsies can be a reliable predictor of islet yield from pig pancreata.  

4.831           Assessment of Human Islet Isolation With Four Different Collagenases

Shimoda, M., Noguchi, H., Naziruddin, BG., Fujita, Y., Chujo, D., Takita, M., Peng, H., Tamura, Y., Olsen, G.S., Sugimoto, K., Itoh, T., Onaca, N., Levy, M.F., Grayburn, P.A. and Matsumoto, S. Transplant. Proceedings, 42(6), 2049-2051 (2010)   Background The isolation of islets from the human pancreas critically depends on the efficiency of the digestive enzymes. Liberase HI had been used as a standard preparation until the issues concerning bovine spongiform encephalopathy. Thus, we must now use other collagenases for clinical islet transplantation, four of which we have evaluated herein. Methods The digestion of each of 17 pancreata from brain-dead donors was performed using the following collagenases: Liberase HI (HI; Roche, n = 9); Liberase MTF C/T (MTF; Roche, n = 4); Collagenase NB1 Premium Grade (NB1; Serva, n = 7); or Clzyme Collagenase HA (CI, VitaCyte, n = 4). Islet isolations were based on the Edmonton protocol for HI, whereas our modified islet isolation method was used for the three new enzymes (MTF, NB1, and CI). Results There were no significant differences in donor age, body mass index, pancreas size, and cold ischemic time among the four groups. The phase I time in the NB1 group was significantly shorter than in the CI group (P = .0014). The prepurification IEQ/g in the HI group was significantly lower than the others (P = .0003 vs MTF, .0007 vs NB1, and .0009 vs CI, respectively). The postpurification IEQ/g in the MTF group was significantly higher than in the HI group (P = .006). The viability in the NB1 group was significantly greater than the HI group (P = .003). Conclusion Three new enzymes (MTF, NB1, and CI) may enable us to obtain higher islet yields than with HI.  

4.832           ET-Kyoto Ductal Injection and Density-Adjusted Purification Combined With Potent Anti-Inflammatory Strategy Facilitated Single-Donor Islet Transplantation: Case Reports

Matsumoto, S., Noguchi, H., Takita, M., Shimoda, M., Tamura, Y., Olsen, G., Naziruddin, B., Onaca, N. and Levy, M.F. Transplant. Proceedings, 42(6), 2159-2161 (2010)   Background The necessity to use multiple donors for achieving insulin independence in clinical islet transplantation is still a major issue. We have developed a modified islet isolation method for non–heart-beating donors (Kyoto method) to significantly increase islet yield. In this study, we further modified the method for brain-dead donors and in addition, introduced a potent anti-inflammatory strategy aiming for single-donor islet transplantation. Materials and methods Two islet isolations used pancreatic ductal preservation with the modified Kyoto solution and a density-adjusted purification method. Anti-interleukin-1-beta antibody (Anakinra) and anti-tumor necrosis factor-alpha (Eternacept) were administered during and after transplantation. The efficacy of the islet transplantation was assessed by the insulin requirement and SUITO (Secretory Unit of Islet Transplant Objects) index, wherein a value of more than 26.0 seems to be associated with insulin independence. Results Both isolated islet preparations met the criteria for transplantation. They were transplanted into two type 1 diabetic patients, both of whom became insulin independent with stable glycemic control. The average SUITO index within 1 month was 29.2 and 45.3. Conclusion The islet isolation method combined with a potent anti-inflammation strategy made it possible to achieve single-donor islet transplantation achieving a high SUITO index.  

4.833           Neural precursor-derived astrocytes of wobbler mice induce apoptotic death of motor neurons through reduced glutamate uptake

Diana, V., Ottalina, A., Botti, F., Fumagalli, E., Calcagno, E., De Paola, M., Cagnotto, A., Invernici, G., Parati, E., Curti, D. and Mennini, T. Exp. Neurol., 225, 163-172 (2010)   In the present study, we investigated whether cultured astrocytes derived from adult neural precursor cells (NPCs) obtained from the subventricular zone (SVZ) of wobbler mice display metabolic traits of the wobbler astrocytes in situ and in primary culture. We also utilized NPC-derived astrocytes as a tool to investigate the involvement of astrocytes in the molecular mechanism of MND focusing on the possible alteration of glutamate reuptake since excitotoxicity glutamate-mediated may be a contributory pathway. NPC-derived wobbler astrocytes are characterized by high immunoreactivity for GFAP, significant decrease of glutamate uptake and reduced immunoreactivity for glutamate transporters GLT1 and GLAST. Spinal cord motor neurons obtained from healthy mouse embryos, when co-cultured with wobbler NPC-derived astrocytes, show reduced viability and morphologic alterations. These suffering motor neurons are caspase-7 positive, and treatment with anti-apoptotic drug V5 increases cell survival. Physical contact with wobbler astrocytes is not essential because purified motor neurons display reduced survival also when treated with the medium conditioned by wobbler NPC-derived astrocytes. Toxic levels of glutamate were revealed by HPLC assay in the extracellular medium of wobbler NPC-derived astrocytes, whereas the level of intracellular glutamate is reduced if compared with controls. Moreover, glutamate receptor antagonists are able to enhance motor neuron survival. Therefore, our results demonstrate that astrocytes derived from wobbler neural precursor cells display impaired glutamate homeostasis that may play a crucial role in motor neuron degeneration. Finally, the cultured astrocytes derived from NPCs of adult mice may offer a useful alternative in vitro model to study the molecular mechanisms involved in neurodegeneration.  

4.834           The NF-κB p50:p50:HDAC-1 repressor complex orchestrates transcriptional inhibition of multiple pro-inflammatory genes

Elsharkawy, A.M., Oakley, F., Lin, F., Packham, G., Mann, D.A. and Mann, J.

1. Hepatol., 53, 519-527 (2010)

  Background & Aims The pro-inflammatory functions of NF-κB must be tightly regulated to prevent inappropriate tissue damage and remodelling caused by activated inflammatory and wound-healing cells. The p50 subunit of NF-κB is emerging as an important repressor of immune and inflammatory responses, but by mechanisms that are poorly defined. This study aims to delineate p50 target genes in activated hepatic stellate cells and to outline mechanisms utilised in their repression. Methods Hepatic stellate cells were isolated from nfkb1(p50)-deficient or Wt mice and gene expression compared using microarray. Target genes were verified by qRT-PCR and p50-mediated HDAC-1 recruitment to the target genes demonstrated using chromatin immunoprecipitation. Results We identify p50 as transcriptional repressor of multiple pro-inflammatory genes including Ccl2, Cxcl10, Gm-csf, and Mmp-13. These genes are over-expressed in nfkb1(p50)-deficient mice suffering from chronic hepatitis and in fibrogenic/inflammatory hepatic stellate cells isolated from nfkb1^−/− liver. We identify Mmp-13 as a bona-fide target gene for p50 and demonstrate that p50 is required for recruitment of the transcriptional repressor histone deacetylase (HDAC)-1 to κB sites in the Mmp-13 promoter. Chromatin immunoprecipitations identified binding of HDAC-1 to specific regulatory regions of the Ccl2, Cxcl10, Gm-csf genes that contain predicted κB binding motifs. Recruitment of HDAC-1 to these genes was not observed in nfkb1^−/− cells suggesting a requirement for p50 in a manner similar to that described for Mmp-13. Conclusions Recruitment of HDAC-1 to inflammatory genes provides a widespread mechanism to explain the immunosuppressive properties of p50.  

4.835           Diesel exhaust particles override natural injury-limiting pathways in the lung

Chaudhuri, N., Paiva, C., Donaldsen, K., Duffin, r., Parker, L.C. and Sabroe, I. Am. J. Physiol. Lung Cell. Mol. Phsyiol., 299, L263-L271 (2010)   Induction of effective inflammation in the lung in responseto environmental and microbial stimuli is dependent on cooperativesignaling between leukocytes and lung tissue cells. We exploredhow these inflammatory networks are modulated by diesel exhaustparticles (DEP) using cocultures of human monocytes with epithelialcells. Cocultures, or monoculture controls, were treated withDEP in the presence or absence of LPS or flagellin. Productionof cytokines was explored by Western blotting and ELISA; cellsignaling was analyzed by Western blotting. Here, we show thatresponses of epithelial cells to DEP are amplified by the presenceof monocytes. DEP amplified the responses of cellular coculturesto very low doses of TLR agonists. In addition, in the presenceof DEP, the responses induced by LPS or flagellin were lessamenable to antagonism by the physiological IL-1 antagonist,IL-1ra. This was paralleled by the uncoupling of IL-1 productionand release from monocytes, potentially attributable to an abilityof DEP to sequester or degrade extracellular ATP. These datadescribe a model of inflammation where DEP amplifies responsesto low concentrations of microbial agonists and alters the natureof the inflammatory milieu induced by TLR agonists.  

4.836           In Hepatic Fibrosis, Liver Sinusoidal Endothelial Cells Acquire Enhanced Immunogenicity

Conolly, M.K., Bedrosian, A.S., Malhotra, A., Henning, J.R., Ibrahim, J., Vera, V., Cieza-Rubio, N.E., Hassan, B.U., Pachter, H.L., Cohen, S., Frey, A.B. and Miller, G.

1. Immunol., 185, 2200-2208 (2010)

  The normal liver is characterized by immunologic tolerance. Primary mediators of hepatic immune tolerance are liver sinusoidal endothelial cells (LSECs). LSECs block adaptive immunogenic responses to Ag and induce the generation of T regulatory cells. Hepatic fibrosis is characterized by both intense intrahepatic inflammation and altered hepatic immunity. We postulated that, in liver fibrosis, a reversal of LSEC function from tolerogenic to proinflammatory and immunogenic may contribute to both the heightened inflammatory milieu and altered intrahepatic immunity. We found that, after fibrotic liver injury from hepatotoxins, LSECs become highly proinflammatory and secrete an array of cytokines and chemokines. In addition, LSECs gain enhanced capacity to capture Ag and induce T cell proliferation. Similarly, unlike LSECs in normal livers, in fibrosis, LSECs do not veto dendritic cell priming of T cells. Furthermore, whereas in normal livers, LSECs are active in the generation of T regulatory cells, in hepatic fibrosis LSECs induce an immunogenic T cell phenotype capable of enhancing endogenous CTLs and generating potent de novo CTL responses. Moreover, depletion of LSECs from fibrotic liver cultures mitigates the proinflammatory milieu characteristic of hepatic fibrosis. Our findings offer a critical understanding of the role of LSECs in modulating intrahepatic immunity and inflammation in fibro-inflammatory liver disease.  

4.837           AAV-mediated expression of wild-type and ALS-linked mutant VAPB selectively triggers death of motoneurons through a Ca2+-dependent ER-associated pathway

Langou, K., Moumen, A., Pellegrino, C., Aebischer, J., Medina, I., Aebischer, P. and Raoul, C.

1. Neurochem., 114, 795-809 (2010)

  A dominant mutation in the gene coding for the vesicle-associated membrane protein-associated protein B (VAPB) was associated with amyotrophic lateral sclerosis, a fatal paralytic disorder characterized by the selective loss of motoneurons in the brain and spinal cord. Adeno-associated viral vectors that we show to transduce up to 90% of motoneurons in vitro were used to model VAPB-associated neurodegenerative process. We observed that Adeno-associated viral-mediated over-expression of both wild-type and mutated form of human VAPB selectively induces death of primary motoneurons, albeit with different kinetics. We provide evidence that ER stress and impaired homeostatic regulation of calcium (Ca2+) are implicated in the death process. Finally, we found that completion of the motoneuron death program triggered by the over-expression of wild-type and mutant VAPB implicates calpains, caspase 12 and 3. Our viral-based in vitro model, which recapitulates the selective vulnerability of motoneurons to the presence of mutant VAPB and also to VAPB gene dosage effect, identifies aberrant Ca2+ signals and ER-derived death pathways as important events in the motoneuron degenerative process.  

4.838           Induction of Indoleamine 2,3-Dioxygenase by Gene Delivery in Allogeneic Islets Prolongs Allograft Survival

Delle, H. and Noronha, L. Am. J: Transplant., 10, 1918-1924 (2010)   Indoleamine 2,3-dioxygenase (IDO), an enzyme that plays a critical role in fetomaternal tolerance, exerts immunoregulatory functions suppressing T-cell responses. The aims of this study were to promote IDO expression in rat islets using a nonviral gene transfer approach, and to analyze the effect of the in vivo induction of IDO in a model of allogeneic islet transplantation. The IDO cDNA was isolated from rat placenta, subcloned into a plasmid and transfected into rat islets using Lipofectamine. The efficiency of transfection was confirmed by qRT-PCR and functional analysis. The in vivo effect of IDO expression was analyzed in streptozotocin-induced diabetic Lewis rats transplanted with allogeneic islets under the renal capsule. Transplantation of IDO-allogeneic islets reversed diabetes and maintained metabolic control, in contrast to transplantation of allogeneic nontransfected islets, which failed shortly after transplantation in all animals. Graft survival of allograft islets transfected with IDO transplanted without any immunosuppression was superior to that observed in diabetic rats receiving nontransfected islets. These data demonstrated that IDO expression induced in islets by lipofection improved metabolic control of streptozotocin-diabetic rats and prolonged allograft survival.  

4.839           Caffeine protects against alcoholic liver injury by attenuating inflammatory response and oxidative stress

Lv, X., Chen, Z., Li, J., Zhang, L., Liu, H., Huang, C. and Zhu, P. Inflamm. Res., 59, 635-645 (2010)   Objective and design The present investigation was designed to determine the effects of caffeine on alcohol-induced hepatic injury in mice. Material Five groups of mice (8 each) were used. Treatment The mice treated with different doses of caffeine (5, 10, and 20 mg/kg, respectively). Methods The degree of alcoholic liver injury was evaluated biochemically by measuring serum markers and pathological examination. Real time PCR and ELISA methods were used to check the expression of cytokines and CYP 450. Results Treatment with caffeine significantly attenuated the elevated serum aminotransferase enzymes and reduced the severe extent of hepatic cell damage, steatosis and the immigration of inflammatory cells. Interestingly, caffeine decreased hepatic mRNA expression of lipogenic genes, while it had no effect on protein expression of hepatic CYP2E1. Furthermore, caffeine decreased serum and tissue inflammatory cytokines levels, tissue lipid peroxidation and inhibited the necrosis of hepatocytes. Kupffer cells isolated from ethanol-fed mice produced high amounts of reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-α), whereas Kupffer cells from caffeine treatment mice produced less ROS and TNF-α. Conclusions These findings suggest that caffeine may represent a novel, protective strategy against alcoholic liver injury by attenuating oxidative stress and inflammatory response.  

4.840           PD-1 on Immature and PD-1 Ligands on Migratory Human Langerhans Cells Regulate Antigen-Presenting Cell Activity

Pena-Cruz, V., McDonough, S.M., Diaz-Grieffero, F., Crum, C.P., Carrasco, R.D. and Freeman, G.J.

1. Invest. Dermatol., 130, 2222-2230 (2010)

  Langerhans cells (LCs) are known as “sentinels” of the immune system that function as professional antigen-presenting cells (APCs) after migration to draining lymph node. LCs are proposed to have a role in tolerance and the resolution of cutaneous immune responses. The Programmed Death-1 (PD-1) receptor and its ligands, PD-L1 and PD-L2, are a co-inhibitory pathway that contributes to the negative regulation of T-lymphocyte activation and peripheral tolerance. Surprisingly, we found PD-1 to be expressed on immature LCs (iLCs) in situ. PD-1 engagement on iLCs reduced IL-6 and macrophage inflammatory protein (MIP)-1α cytokine production in response to TLR2 signals but had no effect on LC maturation. PD-L1 and PD-L2 were expressed at very low levels on iLCs. Maturation of LCs upon migration from epidermis led to loss of PD-l expression and gain of high expression of PD-L1 and PD-L2 as well as co-stimulatory molecules. Blockade of PD-L1 and/or PD-L2 on migratory LCs (mLCs) and DDCs enhanced T-cell activation, as has been reported for other APCs. Thus the PD-1 pathway is active in iLCs and inhibits iLC activities, but expression of receptor and ligands reverses upon maturation and PD-L1 and PD-L2 on mLC function to inhibit T-cell responses.  

4.841           Existence of CD8 -Like Dendritic Cells with a Conserved Functional Specialization and a Common Molecular Signature in Distant Mammalian Species

Contreras, V., Urien, C., Guiton, R., Alexandre, Y., Vu Manh, T-P., Andrieu, T., Crozat, K., Jouneau, L., Bertho, N., Epardaud, M., Hope, J., Savina, A., Amigorena, S., Bonneau, M., Dalod, M. and Schwartz-Cornil, I.

1. Immunol., 185, 3313-3325 (2010)

  The mouse lymphoid organ-resident CD8 ⁺ dendritic cell (DC) subset is specialized in Ag presentation to CD8⁺ T cells. Recent evidence shows that mouse nonlymphoid tissue CD103⁺ DCs and human blood DC Ag 3⁺ DCs share similarities with CD8 ⁺ DCs. We address here whether the organization of DC subsets is conserved across mammals in terms of gene expression signatures, phenotypic characteristics, and functional specialization, independently of the tissue of origin. We study the DC subsets that migrate from the skin in the ovine species that, like all domestic animals, belongs to the Laurasiatheria, a distinct phylogenetic clade from the supraprimates (human/mouse). We demonstrate that the minor sheep CD26⁺ skin lymph DC subset shares significant transcriptomic similarities with mouse CD8 ⁺ and human blood DC Ag 3⁺ DCs. This allowed the identification of a common set of phenotypic characteristics for CD8 -like DCs in the three mammalian species (i.e., SIRP^lo, CADM1^hi, CLEC9A^hi, CD205^hi, XCR1^hi). Compared to CD26^– DCs, the sheep CD26⁺ DCs show 1) potent stimulation of allogeneic naive CD8⁺ T cells with high selective induction of the Ifn and Il22 genes; 2) dominant efficacy in activating specific CD8⁺ T cells against exogenous soluble Ag; and 3) selective expression of functional pathways associated with high capacity for Ag cross-presentation. Our results unravel a unifying definition of the CD8 ⁺-like DCs across mammalian species and identify molecularcandidates that could be used for the design of vaccines applyingto mammals in general.  

4.842           In Vitro Cultured Cardiomyocytes for Evaluating Cardiotoxicity

Liu, S.J. and Melchert, R.B. Comprehensive Toxicology, 6, 113-131 (2010)   Cardiotoxicity has often been observed with the advent of pharmaceuticals for conditions such as cancer and characterized by abnormality of cardiac electrical activity and contractile dysfunction, ultimately leading to heart failure. Finding and understanding the primary cause of cardiotoxicity would improve pharmaceutical development for effective treatment without cardiac side effects. However, complexities of muscle structure and the heterogeneity of cell populations in the heart make in vivo studies and in vitro studies of the whole-heart preparations problematic to identify the primary cause and mechanisms underlying cardiotoxicity at the cellular level. Thus, isolated cardiomyocytes have been powerful tools to discover functional changes of individual cardiac myocytes in response to stimuli. With careful design and control of cell growth, isolated cardiomyocytes can be maintained for a long time in culture and can provide stable model systems for both short-term and long-term studies of genetic physiology, evaluation of cardiotoxicity, and reparative medicine. Understanding limitations of these cell cultures and utilizing multidisciplinary technologies in refined experimental conditions would enable one to take best advantage of unique varieties of these in vitro model systems. Then, cultured cardiomyocytes become the best systems and the choice for physiological, pharmacological, and toxicological studies to evaluate direct effects and underlying mechanisms of xenobiotics on the heart at cellular, subcellular, and molecular levels. Knowledge obtained from using in vitro cultured cardiomyocytes is essential and critical for further in vivo studies in whole animals and drug development to reduce cardiotoxicity.    

4.843           The Morphology of Poly(3,4-Ethylenedioxythiophene)

Martin, D.C., Wu, J., Shaw, C.M., King, Z., Spanninga, S.A., Richardson-Burns, S., Hendricks, J. and Yang, J. Polymer Reviews, 50, 340-384 (2010)   Poly(3,4-ethylene dioxythiophene) (PEDOT) is a chemically stable, conjugated polymer that is of considerable interest for a variety of applications including coatings for interfacing electronic biomedical devices with living tissue. Here, we describe recent work from our laboratory and elsewhere to investigate the morphology of PEDOT in the solid state. We discuss the importance of oxidative chemical and electrochemical polymerization, as well as the critical role of the counterion used during synthesis and film deposition. We have obtained information about the morphology of PEDOT from a number of different complimentary techniques including X-ray diffraction, optical microscopy, scanning electron microscopy, transmission high-resolution electron microscopy, and low-voltage electron microscopy. We also discuss results from ultraviolet-visible light spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). PEDOT is a relatively rigid polymer that packs in the solid state at a characteristic face-to-face distance (010) of ∼0.34 nm, similar to graphite. These sheets of oriented PEDOT molecules are separated from one another by ∼1.4 nm laterally, with the (100) distance between layers quite sensitive to the choice of counterion used during sample preparation. The order in the films is typically modest, although this also depends on the counterion used and the method of film deposition. The films can be organized into useful structures with a variety of nanoscale dissolvable templates (including fibers, particles, and lyotropic mesophases). When PEDOT is electrochemically deposited in the presence of bromine counterions, highly ordered crystalline phases are observed. It is also possible to deposit PEDOT around living cells, both in vitro and in vivo.  

4.844           Islet cell transplantation for Type 1 diabetes

Matsumoto, S.

1. Diabetes, 2, 16-22 (2010)

  Islet transplantation is an attractive concept for the treatment of Type 1 diabetes because of its potential high efficacy and minimal invasion to patients. The treatment may effectively control blood glucose for brittle Type 1 diabetes, resulting in a marked reduction in hypoglycemic episodes and improvements in HbA1c. In addition, approximately 70% of transplanted Type 1 diabetic patients have achieved insulin independence. However, there are still important issues to be addressed before this treatment is widely applicable, including difficulty in maintaining insulin independence, low islet isolation success rate, multiple donor requirements, and side effects associated with the use of immunosuppressants. Donor shortage is another dilemma. To address the issue of donor shortage, living donor islet transplantation and bioartificial islet transplantation using pig islets are being evaluated. Bioartificial islet transplantation could be the ultimate solution of the donor shortage. Currently, overcoming immunological hurdles, establishing reliable islet isolation methods, and controlling porcine endogenous retrovirus are the primary obstacles to the implementation of this treatment. If bioartificial islet transplant becomes a clinical reality, it may even be applicable in the treatment of select Type 2 diabetic patients. β-Cell regeneration from naïve pancreas and β-cell generation from embryonic stem cells or induced pluripotent stem cells are the next-generation treatments for Type 1 diabetes.  

4.845           Identifying Homing Interactions in T-Cell Traffic in Human Disease

Lalor, P.F., Curbishley, S.M. and Adams, D.H. Methods in Mol. Biol., 616(4), 231-252 82010)   Description of the molecular mechanisms which regulate the traffic of lymphocyte populations over recent years [for useful reviews see (1, 2)] has significantly enhanced our understanding of the processes underlying acquired immunity and also permitted the development of therapies targeted at specific leukocyte subpopulations. Such therapies are dependent upon a detailed knowledge of the molecular regulation of lymphocyte adhesion to and migration through endothelium in specific tissues. Whereas animal models have been central to understanding the underlying mechanisms, it is crucial to confirm and extend observations in man by using analysis of tissues and in vitro cell-based models. In this chapter, we discuss expertise developed in our laboratory for the isolation of specific lymphocyte and endothelial populations from explanted human liver tissue specimens. We then move on to provide specific examples of assays such as the Stamper–Woodruff assay, the transmigration assay and the tissue-specific endothelial static and flow-based adhesion assays, which can be used to interrogate the tissue-specific adhesion and migration of lymphocyte subsets. Although our own experience is with human liver tissue, the general principles apply to analysing any organ of interest.  

4.846           Isolation and Culture of Adult Human Liver Progenitor Cells: In Vitro Differentiation to Hepatocyte-Like Cells

Gerbal-Chaloin, S., Duret, C., Raulet, E., Navarro, F., Blanc, P., Ramos, J., Maurel, P. and Daujat-Chavanieu, M. Methods in Mol. Biol., 640, 247-260 (2010)   Highly differentiated normal human hepatocytes represent the gold standard cellular model for basic and applied research in liver physiopathology, pharmacology, toxicology, virology, and liver biotherapy. Nowadays, although livers from organ donors or medically required resections represent the current sources of hepatocytes, the possibility to generate hepatocytes from the differentiation of adult and embryonic stem cells represents a promising opportunity. The aim of this chapter is to describe our experience with the isolation from adult human liver and culture of non-parenchymal epithelial cells. Under appropriate conditions, these cells differentiate in vitro in hepatocyte-like cells and therefore appear to behave as liver progenitor cells.  

4.847           Pediatric Islet Autotransplantation: Indication, Technique, and Outcome

Bellin, M.D. and Sutherland, D.E.R. Cur. Diab. Rep., 10, 326-331 (2010)   Chronic pancreatitis is a rare disease in childhood. However, when severe, a total pancreatectomy may be the only option to relieve pain and restore quality of life. An islet autotransplant performed at the time of pancreatectomy can prevent or minimize the postsurgical diabetes that would otherwise result from pancreatectomy alone. In this procedure, the resected pancreas is mechanically disrupted and enzymatically digested to separate the islets from the surrounding exocrine tissue, and the isolated islets are infused into the portal vein and engraft in the liver. Because patients are receiving their own tissue, no immunosuppression is required. Islet autotransplant is successful in two thirds of children—these patients are insulin independent or require little insulin to maintain euglycemia. Factors associated with a more successful outcome include a younger age at transplant (<13 years), more islets transplanted, and lack of prior surgical procedures on the pancreas (partial pancreatectomy or surgical drainage procedures).  

4.848           ADP mediates inhibition of insulin secretion by activation of P2Y13 receptors in mice

Amisten, S., Meidute-Abaraviciene, S., Tan, C., Olde, B., Lundquist, I., Salehi, A. and Erlinge, D. Diabetologia, 53, 1927-1934 (2010)   Aims/hypotheses To investigate the effects of extracellular purines on insulin secretion from mouse pancreatic islets. Methods Mouse islets and beta cells were isolated and examined with mRNA real-time quantification, cAMP quantification and insulin and glucagon secretion. ATP release was measured in MIN6c4 cells. Insulin and glucagon secretion were measured in vivo after glucose injection. Results Enzymatic removal of extracellular ATP at low glucose levels increased the secretion of both insulin and glucagon, while at high glucose levels insulin secretion was reduced and glucagon secretion was stimulated, indicating an autocrine effect of purines. In MIN6c4 cells it was shown that glucose does induce release of ATP into the extracellular space. Quantitative real-time PCR demonstrated the expression of the ADP receptors P2Y1 and P2Y13 in both intact mouse pancreatic islets and isolated beta cells. The stable ADP analogue 2-MeSADP had no effect on insulin secretion. However, co-incubation with the P2Y1 antagonist MRS2179 inhibited insulin secretion, while co-incubation with the P2Y13 antagonist MRS2211 stimulated insulin secretion, indicating that ADP acting via P2Y1 stimulates insulin secretion, while signalling via P2Y13 inhibits the secretion of insulin. P2Y13 antagonism through MRS2211 per se increased the secretion of both insulin and glucagon at intermediate (8.3 mmol/l) and high (20 mmol/l) glucose levels, confirming an autocrine role for ADP. Administration of MRS2211 during glucose injection in vivo resulted in both increased secretion of insulin and reduced glucose levels. Conclusions/interpretation In conclusion, ADP acting on the P2Y13 receptors inhibits insulin release. An antagonist to P2Y13 increases insulin release and could be evaluated for the treatment of diabetes.  

4.849           Human primary cultured hepatic stellate cells can be cryopreserved

Nakamura, A., Ueno, T., Yagi, Y., Okuda, K., Ogata, T., Nakamura, T., Torimura, T., Iwamoto, H., Ramadoss, S., Sata, M., Tsutsumi, V., Yasuda, K., Tomiyashi, K., Tashiro, K. and Kuhara, S. Med. Mol. Morphol., 43, 107-115 (2010)   We compared the morphological and functional characteristics of cultured unfrozen hepatic stellate cells (HSCs) and cryopreserved HSCs obtained from human livers. We used liver tissues obtained by surgical resection from patients with metastatic liver cancer or with hepatocellular carcinoma. HSCs were isolated and allowed to spread in culture. Comparison of morphological and functional features between the unfrozen HSCs and cryopreserved HSCs was performed at each passage using the following techniques: light microscopy, immunohistochemistry, cell growth curve, metallothionein (MTT) assay, and PI staining, Western blot, real-time polymerase chain reaction (PCR), and gene expression analysis using microarrays. The purity of HSCs was more than 90% in all passages. α-Smooth muscle actin (SMA-)positive HSCs gradually increased in successive passages, and the positive cell rate and rate of increase in cell number were similar in both groups. Expression of platelet-derived growth factor (PDGF) receptor, transforming growth factor (TGF)-β receptor, and α-SMA mRNAs and protein was similar during each passage in the two groups. Gene expression was nearly identical at each passage in unfrozen and frozen/thawed samples obtained from the same patient. In conclusion, an adequate protocol for the cryopreservation of human primary cultured HSCs could be established.  

4.850           Modulation of Synaptic Transmission and Analysis of Neuroprotective Effects of Valproic Acid and Derivates in Rat Embryonic Motoneurons

Ragancokova, D., Song, Y., Nau, H., Dengler, R., Krammpfl, K. and Petri, S. Cell. Mol. Neurobiol., 30, 891-900 (2010)   Amyotrophic lateral sclerosis is a devastating motoneuron disorder for which no effective treatment exists. There is some evidence for neuroprotective effects of valproic acid (VPA). The beneficial effects, however, are limited due to the adverse effects of VPA. To overcome this problem, a number of VPA derivates with fewer side effects have been synthesized. In the present study, we investigated the viability of highly purified embryonic motoneurons cultured on glial feeder layers, composed of either astrocytes or Schwann cells, or in monoculture, in presence of VPA and its three derivates 3-propyl-heptanoic acid (3-PHA), PE-4-yn enantiomers (R- and S-PE-4-yn). An excitotoxic stimulus, kainate (KA), was added at day in vitro 9 (DIV9) and the neuroprotective effect of either simultaneous incubation (DIV9) or pre-incubation (DIV1) of VPA and its derivates was tested. The survival of motoneurons under simultaneous application of KA and VPA derivates was not remarkably increased. Pre-incubation with VPA and even more with the derivates before the addition of KA, however, significantly reduced their vulnerability against the KA-induced neurotoxic effect. Our data suggest that the neuroprotective capacities of VPA and its three derivates tested here drastically increase when they are added several days before KA. Most prominent neuroprotective effects were seen for the PE-4-yn enantiomers. Patch-clamp experiments revealed an antiexcitotoxic effect of the S-PE-4-yn enantiomer that reduces the frequency of postsynaptic currents and enhances the inhibitory postsynaptic transmission dependent on the co-culture condition.  

4.851           CX3CR1 and vascular adhesion protein-1-dependent recruitment of CD16+ monocytes across human liver sinusoidal endothelium

Aspinall, A., Curbishley, S.M., Lalor, P:F., Weston, C.J., Blahova, M., Liaskou, E., Adams, R.M., Holt, A.P. ansd Adams, D.H. Hepatology, 51, 2030-2039 (2010)   The liver contains macrophages and myeloid dendritic cells (mDCs) that are critical for the regulation of hepatic inflammation. Most hepatic macrophages and mDCs are derived from monocytes recruited from the blood through poorly understood interactions with hepatic sinusoidal endothelial cells (HSECs). Human CD16+ monocytes are thought to contain the precursor populations for tissue macrophages and mDCs. We report that CD16+ cells localize to areas of active inflammation and fibrosis in chronic inflammatory liver disease and that a unique combination of cell surface receptors promotes the transendothelial migration of CD16+ monocytes through human HSECs under physiological flow. CX3CR1 activation was the dominant pertussis-sensitive mechanism controlling transendothelial migration under flow, and expression of the CX3CR1 ligand CX3CL1 is increased on hepatic sinusoids in chronic inflammatory liver disease. Exposure of CD16+ monocytes to immobilized purified CX3CL1 triggered β1-integrin-mediated adhesion to vascular cell adhesion molecule-1 and induced the development of a migratory phenotype. Following transmigration or exposure to soluble CX3CL1, CD16+ monocytes rapidly but transiently lost expression of CX3CR1. Adhesion and transmigration across HSECs under flow was also dependent on vascular adhesion protein-1 (VAP-1) on the HSECs. Conclusion: Our data suggest that CD16+ monocytes are recruited by a combination of adhesive signals involving VAP-1 and CX3CR1 mediated integrin-activation. Thus a novel combination of surface molecules, including VAP-1 and CX3CL1 promotes the recruitment of CD16+ monocytes to the liver, allowing them to localize at sites of chronic inflammation and fibrosis.  

4.852           The hepatic vagus nerve stimulates hepatic stellate cell proliferation in rat acute hepatitis via muscarinic receptor type 2

Bockx, I., Vander elst, I., Roskams, T. and Cassiman, D. Liver Int., 30(5), 693-702 (2010)   Background & aims: We have previously shown that the hepatic vagus nerve stimulates the activation of hepatic progenitor cells (HPC), via muscarinic acetylcholine receptor type 3. Given the coproliferation of HPC and hepatic stellate cells (HSC) in acute hepatitis, we determined whether HSC proliferation is also modulated by vagal activity. Methods: We induced acute hepatitis in Wistar rats by injection of galactosamine and lipopolysaccharides. Hepatitis was preceded by hepatic branch vagotomy or sham vagotomy, by electrical stimulation or sham stimulation and by muscarinic receptor antagonist atropine, nicotinic receptor antagonist mecamylamine or saline injection. Rats were sacrificed after 12 and 48 h and HSC numbers were quantified on immunohistochemical stainings. Furthermore, we performed reverse transcriptase-polymerase chain reaction with receptor-specific primers on total RNA from isolated HSC and determined the in vitro proliferation of HSC in response to acetylcholine, atropine and mecamylamine. Results: HSC numbers were significantly lower after vagotomy than after sham vagotomy. Conversely, more HSC were seen after electrical stimulation than after sham stimulation. Atropine resulted in less HSC than saline at both time points, while mecamylamine treatment only diminished HSC after 12 h, suggesting a predominant involvement of muscarinic receptors. Moreover, HSC express muscarinic receptor type 2 mRNA and protein, as well as nicotinic receptor 1, 5, β1 and vasoactive intestinal peptide receptor 1 mRNA. Furthermore, acetylcholine enhanced the in vitro proliferation of HSC, which was inhibited by atropine, but not by mecamylamine. Conclusions: We show here that the hepatic vagus nerve stimulates HSC proliferation, most likely through binding of acetylcholine on muscarinic receptor type 2.  

4.853           Toll-like receptor expression in the peripheral nerve

Goethals, S., Ydens, E., Timmerman, V. and Janssens, S. Glia, 58, 1701-1709 (2010)   Toll-like receptors comprise a family of evolutionary conserved pattern recognition receptors that act as a first defense line in the innate immune system. Upon stimulation with microbial ligands, they orchestrate the induction of a host defense response by activating different signaling cascades. Interestingly, they appear to detect the presence of endogenous signals of danger as well and as such, neurodegeneration is thought to trigger an immune response through ligation of TLRs. Though recent data report the expression of various TLRs in the central nervous system, TLR expression patterns in the peripheral nervous system have not been determined yet. We observed that Schwann cells express relatively high levels of TLRs, with especially TLR3 and TLR4 being prominent. Sensory and motor neurons hardly express TLRs at all. Through the use of NF-κB signaling as read-out, we could show that all TLRs are functional in Schwann cells and that bacterial lipoprotein, a ligand for TLR1/TLR2 receptors yields the strongest response. In sciatic nerve, basal levels of TLRs closely reflect the expression patterns as determined in Schwann cells. TLR3, TLR4, and TLR7 are majorly expressed, pointing to their possible role in immune surveillance. Upon axotomy, TLR1 becomes strongly induced, while most other TLR expression levels remain unaffected. Altogether, our data suggest that similar to microglia in the brain, Schwann cells might act as sentinel cells in the PNS. Furthermore, acute neurodegeneration induces a shift in TLR expression pattern, most likely illustrating specialized functions of TLRs in basal versus activated conditions of the peripheral nerve.  

4.854           Multicenter Analysis of Novel and Established Variables Associated with Successful Human Islet Isolation Outcomes

Kaddis, J.S., Danobeitia, J.S., Niland, J.C., Stiller, T. and Fernandez, L.A. Am. J. Transplant., 10, 646-656 (2010)   Islet transplantation is a promising therapy used to achieve glycometabolic control in a select subgroup of individuals with type I diabetes. However, features that characterize human islet isolation success prior to transplantation are not standardized and lack validation. We conducted a retrospective analysis of 806 isolation records from 14 pancreas-processing laboratories, considering variables from relevant studies in the last 15 years. The outcome was defined as postpurification islet equivalent count, dichotomized into yields ≥315 000 or ≤220 000. Univariate analysis showed that donor cause of death and use of hormonal medications negatively influenced outcome. Conversely, pancreata from heavier donors and those containing elevated levels of surface fat positively influence outcome, as did heavier pancreata and donors with normal amylase levels. Multivariable logistic regression analysis identified the positive impact on outcome of surgically intact pancreata and donors with normal liver function, and confirmed that younger donors, increased body mass index, shorter cold ischemia times, no administration of fluid/electrolyte medications, absence of organ edema, use of University of Wisconsin preservation solution and a fatty pancreas improves outcome. In conclusion, this multicenter analysis highlights the importance of carefully reviewing all donor, pancreas and processing parameters prior to isolation and transplantation.  

4.855           Primary Sensory and Motor Neuron Cultures

Vincent, A.M. and Feldman, E.L. Protocols for Neural Cell Culture, 161-173 (2010) Springer Protocols Handbook   The ability to culture primary neurons is critical to the study of neuronal biochemistry and molecular biology. Because committed neurons do not proliferate, neuronal cell lines are fundamentally altered in all areas of activity. Transformation of neuronal lines necessarily alters their survival, signaling, and metabolism, so for these and many other studies primary cells are essential. The issue of cell identity is also an important advantage of primary neuron cultures. An investigator tends to have high confidence in the identity of neurons reproducibly harvested from a specific site from an animal. Issues of senescence and enrichment of more robust population subtypes are eliminated in primary cell culture. We have particularly focused on enriched neuronal cultures so that we can study neuronal biology in isolation. These cultures are amenable to extraction of protein, lipid, and nucleic acid for assessment of expression and post-translational modifications with high confidence that the measurements are neuronal and not occurring in accessory cells. We have established protocols for many embryonic neuronal cultures and also a limited number of adult neurons. In some studies, co-culture with glial cells is important, so we also culture various glial cells. These cultures are used for immunohistochemistry (1), biochemical enzyme assays (2), gene expression at mRNA and protein level (3, 4), live cell fluorescence-based metabolic assays (5, 6), survival (5, 6), and signaling measurements (1, 7). We have found that the most effective method for gene transfer to primary neurons is viral infection. Using adeno- and adeno-associated viruses, we achieve high expression levels and greater than 90% infection rates within 24–48 h (3, 8). This fits well within our 3-day culture period. These gene transfer experiments prove powerful for the rescue of a knockout phenotype or the study of gene overexpression (2, 8). We also can use lower multiplicities of infection to achieve 10% or 50% infection. With a bicistronic vector that expresses GFP as well as the gene of interest, we can microscopically compare neurons in the same dish with and without the transgene and separate them by green fluorescence. We also have devised serum-free defined media for our neuronal cultures. This development confers many advantages over other protocols. The media permits the culture of neurons at very low density in the absence of a glial feeder layer. This is important for our studies of neurite growth on different extracellular matrix proteins and aligned nanofibers (9) and for immunohistochemistry (6, 7). The absence of serum also is important for our studies into the effects of growth factors on signaling and survival (1, 3). We and others have found that embryonic and neonatal neurons require basal 25 mM glucose in order to remain viable in culture, and this is contained in the formulation of neurobasal media (Gibco, #21103). This is consistent with many neuronal cells, but contrasts with most other primary cell types (5, 10). Adult DRG neurons may be grown in either low- or high-glucose media, although very large neurons are decreased in high glucose and fluctuations in glucose are detrimental to the neurons (11). Representative examples of the cells isolated in the following protocols are shown in Fig. 9.1.  

4.856           Preparation of Normal and Reactive Astrocyte Cultures

De Villes, J., Ghiani, C.A., Wanner, I.B. and Cole, R. Protocols for Neural Cell Culture, 193-215 (2010) SpringerProtocols Handbooks   The study of glial cell development and function has been considerably enhanced by the development of methods to culture oligodendrocytes, astrocytes, and microglia from central nervous system tissue. A primary mixed glial culture, composed of astrocytes, oligodendrocytes, and microglia, is obtained when newborn disaggregated cerebral brain cells from rat are plated at high cell density (2 × 10⁵/cm²) in serum-supplemented medium (1). Neurons fail to develop or survive in this culture model. At low cell density (e.g., 5 × 10⁴ cells/cm²), few oligodendrocytes develop, and the culture consists mostly of astrocytes. At high cell density, phase-dark, process-bearing spindle or spider-shaped cells appear by 4 days and stratify into clusters and individual cells above the bed-layer of cells. The bed layer consists of astrocytes rich in glial filaments. This observation led to the development of the shaking procedure, which results in selective removal of the process-bearing cells from the underlying astrocytes (1).Thus, highly purified cultures of astrocytes and oligodendrocytes, as wells as microglia, can be obtained from the same piece of brain tissue. Microglia cells (2) can be harvested from the stationary cultures by harvesting the medium on days 6 and 7, when they can be microscopically observed to be suspended in the medium. The remaining microglia and loosely adhering astrocytes are then removed from the mixed culture by a 6-h pre-shake, before the oligodendrocyte lineage cells are removed. The microglia cultures are about 95% pure, as characterized with immunocytochemistry using the microglia marker, ED 1 (3). At the time of harvesting the process-bearing cells from the 7- to 9-day-old cultures, the cell population is mostly composed of oligodendrocyte progenitor cells and immature oligodendrocytes (4). If the process-bearing cells are placed in a chemically defined serum-free medium, <4% of the cells express astrocyte markers, such as glial fibrillary acidic protein (GFAP) (5). In fetal bovine serum (FBS)-supplemented medium, 25–35% of the cells express GFAP, originally called astrocyte type II. It has now been determined that astrocytes type II are an in vitro phenomenon with no in vivo counterpart (6). The bed-layer cells can be maintained as pure astrocyte cultures by keeping the flasks shaking slowly, to keep removing dividing oligodendrocyte progenitors and microglia. When cultured in the presence of horse serum, astrocytes grow flat processes and acquire a highly differentiated quiescent phenotype while continuing to form a lawn (7). This primary culture of astrocytes can be used to set up pure secondary astrocyte cultures in serum or serum-free medium (8, 9). Astroglial and oligodendroglial cell lines have been developed from the cultures described above (10, 11). The usage of astrocyte cultures derived from neonatal brains has the advantage of using a population of astroglial cells with a purity of 99% (1). Primary cultures of astrocytes offer an invaluable tool to characterize the changes that may occur in these cells following, for instance, a brain injury. Astrocytes respond to injury by becoming hypertrophic, hyperplastic, and increasing GFAP. This process, named reactive gliosis or astrogliosis, occurs in the brain and spinal cord in response to different types of injury including ischemia and inflammatory diseases. Its hallmark is an increase in GFAP (12, 13). Astrogliosis leads to the formation of a glial scar, a boundary necessary to contain the injury, but also an inhibitory obstacle to successful regeneration of damaged neuronal networks and axon tracts. The differences in the response to a harmful event, either chemical or physical, have been examined in astrocytes cultured from either the neonatal or the adult brain (12). It is important to point out that neonatal astrocytes in culture as well as in vivo display higher levels of GFAP compared to adult astrocytes. Yet, cultured astrocytes derived from adult brain or spinal cord were shown to arise from glial progenitor cells, similarly to those prepared from newborn brains (14–16). Hence, a protocol to obtain purified and highly differentiated astrocytes in culture is desirable to study mechanisms of astrogliosis and scar formation. Here, we provide a set of detailed protocols preparing and using purified astrocytes that were kept in culture for up to 8 weeks, as well as of reactive astrocytes (1, 7 , 12, 17).  

4.857           Production of IL-10 and IL-12 by antigen-presenting cells in periapical lesions

Colic, M., Gazivoda, D., Vasilijic, S., Vucevic, D. and Lukic, A.

1. Oral Pathol. Med., 39(9), 690-696 (2010)

  Background: Interferon- (IFN- ) plays an important role in the pathogenesis of periapical lesions. Its expression is up-regulated by interleukin (IL)-12) and down-regulated by IL-10. The aim of this work was to study the cellular source of these cytokines and their mutual interactions in human periapical lesions. Methods: Mononuclear cells, macrophages and dendritic cells were isolated from periapical lesions using plastic adherence and osmotic gradients. Cytokines were measured in culture supernatants by a microbeads fluorescence assay. Phenotypic characteristics of cells were studied by immunocytochemistry, whereas allostimulatory activity of antigen-presenting cells was tested using a mixed leukocyte reaction. Results: We observed the positive correlations between the levels of IL-12 and IFN- as well as IL-12 and IL-10 in cultures of mononuclear cells. As IL-10 and IL-12 are produced by dendritic cells and activated macrophages, we examined their contribution to the production of these cytokines. Macrophages, CD14⁺ adherent cells, produced high levels of IL-10 and very low levels of IL-12. In contrast, non-adherent, strongly HLA-DR⁺ dendritic cells, potent stimulators of the alloreactive T-cell response, produced low levels of IL-10 and moderate levels of IL-12. Dendritic cells stimulated the production of IFN- by allogeneic CD4⁺ T cells. In contrast, the level of IFN- was significantly decreased and the production of IL-10 was enhanced by addition of macrophages to the culture system. Conclusion: Our results suggest that a fine balance between the production of IL-10 and IL-12 by different antigen-presenting cells, through IFN- , may control the course of chronic inflammation in periapical lesions.  

4.858           Surfactant Protein-A Inhibits Mycoplasma-Induced Dendritic Cell Maturation through Regulation of HMGB-1 Cytokine Activity

Ledford, J.G., Lo, B., Kislan, M.M., Thomas, J.M., Evans, K., Cain, D.W., Kraaft, M., Williams, K.L. and Wright, J.R.

1. Immunol., 185, 3884-3894 (2010)

  During pulmonary infections, a careful balance between activation of protective host defense mechanisms and potentially injurious inflammatory processes must be maintained. Surfactant protein A (SP-A) is an immune modulator that increases pathogen uptake and clearance by phagocytes while minimizing lung inflammation by limiting dendritic cell (DC) and T cell activation. Recent publications have shown that SP-A binds to and is bacteriostatic for Mycoplasma pneumoniae in vitro. In vivo, SP-A aids in maintenance of airway homeostasis during M. pneumoniae pulmonary infection by preventing an overzealous proinflammatory response mediated by TNF- . Although SP-A was shown to inhibit maturation of DCs in vitro, the consequence of DC/SP-A interactions in vivo has not been elucidated. In this article, we show that the absence of SP-A during M. pneumoniae infection leads to increased numbers of mature DCs in the lung and draining lymph nodes during the acute phase of infection and, consequently, increased numbers of activated T and B cells during the course of infection. The findings that glycyrrhizin, a specific inhibitor of extracellular high-mobility group box-1 (HMGB-1) abrogated this effect and that SP-A inhibits HMGB-1 release from immune cells suggest that SP-A inhibits M. pneumoniae-induced DC maturation by regulating HMGB-1 cytokine activity.  

4.859           Cu,Zn-Superoxide Dismutase Increases Toxicity of Mutant and Zinc-deficient Superoxide Dismutase by Enhancing Protein Stability

Sahawneh, M.A., Ricart, K.C., Roberts, B.R., Bomben, V.C., Basso, M., Ye, Y., Sahawneh, J., Franco, M.C., Beckman, J.S. and Estevez, A.G.

1. Biol. Chem., 285(44), 33885-33897 (2010)

  When replete with zinc and copper, amyotrophic lateral sclerosis (ALS)-associated mutant SOD proteins can protect motor neurons in culture from trophic factor deprivation as efficiently as wild-type SOD. However, the removal of zinc from either mutant or wild-type SOD results in apoptosis of motor neurons through a copper- and peroxynitrite-dependent mechanism. It has also been shown that motor neurons isolated from transgenic mice expressing mutant SODs survive well in culture but undergo apoptosis when exposed to nitric oxide via a Fas-dependent mechanism. We combined these two parallel approaches for understanding SOD toxicity in ALS and found that zinc-deficient SOD-induced motor neuron death required Fas activation, whereas the nitric oxide-dependent death of G93A SOD-expressing motor neurons required copper and involved peroxynitrite formation. Surprisingly, motor neuron death doubled when Cu,Zn-SOD protein was either delivered intracellularly to G93A SOD-expressing motor neurons or co-delivered with zinc-deficient SOD to nontransgenic motor neurons. These results could be rationalized by biophysical data showing that heterodimer formation of Cu,Zn-SOD with zinc-deficient SOD prevented the monomerization and subsequent aggregation of zinc-deficient SOD under thiol-reducing conditions. ALS mutant SOD was also stabilized by mutating cysteine 111 to serine, which greatly increased the toxicity of zinc-deficient SOD. Thus, stabilization of ALS mutant SOD by two different approaches augmented its toxicity to motor neurons. Taken together, these results are consistent with copper-containing zinc-deficient SOD being the elusive “partially unfolded intermediate” responsible for the toxic gain of function conferred by ALS mutant SOD.  

4.860           Monocyte CD147 is induced by advanced glycation end products and high glucose concentration: possible role in diabetic complications

Bao, W., Min, D., Twigg, S.M., Shackel, N.A., Warner, F.J., Yue, D.K. and McLennan, S.V. Am. J. Physiol. Cell Physiol., 299, C1212-C1219 (2010)   CD147 is a highly glycosylated transmembrane protein that is known to play a role in regulation of many protein families. It has the unique ability to maintain functional activity in both the membrane bound state and in the soluble form. CD147 is known to play a role in regulation of matrix metalloproteinase (MMP) expression, but whether its expression is affected by the diabetic milieu is not known, and its role in regulation of monocyte MMPs in this environment has not been investigated. Therefore, in this study we investigated the effect of advanced glycation end products (AGEs) and high glucose (HG; 25 mM), on monocyte CD147 expression. Culture of THP-1 monocytes in the presence of AGEs or HG significantly increased CD147 at the gene and protein level. THP-1 cell results were confirmed using freshly isolated monocytes from human volunteers. The effect of AGEs and HG on CD147 expression was also mimicked by addition of proinflammatory cytokines. Addition of AGEs or HG also increased expression of monocyte MMP-1 and MMP-9 but not MMP-2. This increase in MMPs was significantly attenuated by inhibition of CD147 using either a small interfering RNA or an anti-CD147 antibody. Inhibition of NF- B or addition of antibodies to either TNF- or the receptor for AGE (RAGE) each significantly prevented in a dose-dependent manner the induction of CD147 gene and protein by AGE and also decreased MMP-1 and MMP-9. This novel result shows that AGEs can induce monocyte CD147 expression, an effect mediated by inflammatory pathways and RAGE. Because MMPs play a role in monocyte migration, inhibition of their regulator CD147 may assist in the prevention of diabetic complications, particularly those where monocyte infiltration is an early initiating event.  

4.861           Isolation of periportal, midlobular, and centrilobular rat liver sinusoidal endothelial cells enables study of zonated drug toxicity

Xie, G., Wang, L., Wang, X., Wang, L. and DeLeve, L.D. Am. J. Physiol. Gastrointest. Liver Physiol., 299, G1204-G1210 (2010)   Many liver sinusoidal endothelial cell (LSEC)-dependent processes, including drug-induced liver injury, ischemia-reperfusion injury, acute and chronic rejection, fibrosis, and the HELLP (hemolytic anemia, elevated liver enzymes, low platelet count) syndrome, may have a lobular distribution. Studies of the mechanism of this distribution would benefit from a reliable method to isolate LSEC populations from different regions. We established and verified a simple method to isolate periportal, midlobular, and centrilobular LSEC. Three subpopulations of LSEC were isolated by immunomagnetic separation on the basis of CD45 expression. Flow cytometry showed that 78.2 ± 2.3% of LSEC were CD45 positive and that LSEC could be divided into CD45 bright (28.6 ± 2.7% of total population), dim (49.6 ± 1.0%), and negative populations (21.8 ± 2.3%). Immunohistochemistry confirmed that in vivo expression of CD45 in LSEC had a lobular distribution with enhanced CD45 staining in periportal LSEC. Cell diameter, fenestral diameter, number of fenestrae per sieve plate and per cell, porosity, and lectin uptake were significantly different in the subpopulations, consistent with the literature. Endocytosis of low concentrations of the LSEC-specific substrate, formaldehyde-treated serum albumin, was restricted to CD45 bright and dim LSEC. Acetaminophen was more toxic to the CD45 dim and negative populations than to the CD45 bright population. In conclusion, CD45 is highly expressed in periportal LSEC, low in midlobular LSEC, and negative in centrilobular LSEC, and this provides an easy separation method to isolate LSEC from the three different hepatic regions. The LSEC subpopulations obtained by this method are adequate for functional studies and drug toxicity testing.  

4.862           Tubular cell-enriched subpopulation of primary renal cells improves survival and augments kidney function in rodent model of chronic kidney disease

Kelley, R., Werdin, E.S., Bruce, A.T., Choudhury, S., Wallace, S.M., Ilagan, R.M., Cox, B.R., Tatsumi-Ficht, P., Rivera, E.A., Spencer, T., Rapoport, H., Wagner, B.J., Guthrie, K., Jayo, M.J., Bertram, T.A. and Presnell, S.C. Am. J. Physiol. Renal Physiol., 299, F1026-F1039 (2010)   Established chronic kidney disease (CKD) may be identified by severely impaired renal filtration that ultimately leads to the need for dialysis or kidney transplant. Dialysis addresses only some of the sequelae of CKD, and a significant gap persists between patients needing transplant and available organs, providing impetus for development of new CKD treatment modalities. Some postulate that CKD develops from a progressive imbalance between tissue damage and the kidney's intrinsic repair and regeneration processes. In this study we evaluated the effect of kidney cells, delivered orthotopically by intraparenchymal injection to rodents 4–7 wk after CKD was established by two-step 5/6 renal mass reduction (NX), on the regeneration of kidney function and architecture as assessed by physiological, tissue, and molecular markers. A proof of concept for the model, cell delivery, and systemic effect was demonstrated with a heterogeneous population of renal cells (UNFX) that contained cells from all major compartments of the kidney. Tubular cells are known contributors to kidney regeneration in situ following acute injury. Initially tested as a control, a tubular cell-enriched subpopulation of UNFX (B2) surprisingly outperformed UNFX. Two independent studies (3 and 6 mo in duration) with B2 confirmed that B2 significantly extended survival and improved renal filtration (serum creatinine and blood urea nitrogen). The specificity of B2 effects was verified by direct comparison to cell-free vehicle controls and an equivalent dose of non-B2 cells. Quantitative histological evaluation of kidneys at 6 mo after treatment confirmed that B2 treatment reduced severity of kidney tissue pathology. Treatment-associated reduction of transforming growth factor (TGF)-β1, plasminogen activator inhibitor (PAI)-1, and fibronectin (FN) provided evidence that B2 cells attenuated canonical pathways of profibrotic extracellular matrix production.  

4.863           Cdc42-mediated MTOC polarization in dendritic cells controls targeted delivery of cytokines at the immune synapse

Pulecio, J., Petrovic, J., Prete, F., Chiaruttini, G., Lennon-Dumenil, A-M., Desdouets, C., Gasman, S., Burrone, O.R. and Benvenuti, F.

1. Exp. Med., 207(12), 2719-2732 (2010)

  The immune synapse (IS) forms as dendritic cells (DCs) and T cells interact in lymph nodes during initiation of adaptive immunity. Factors that contribute to the formation and maintenance of IS stability and function have been mostly studied in T cells, whereas little is known about events occurring during synapse formation in DCs. Here, we show that DCs activated by Toll-like receptor (TLR) agonists reorient the microtubule-organizing center (MTOC) toward the interacting T cell during antigen-specific synapse formation through a mechanism that depends on the Rho GTPase Cdc42. IL-12, a pivotal cytokine produced by DCs, is found enriched around the MTOC at early time points after TLR ligation and is dragged to the DC–T cell interface in antigen-specific synapses. Synaptic delivery of IL-12 induces activation of pSTAT4 and IFN-γ neosynthesis in CD8⁺ naive T cells engaged in antigen-specific conjugates and promotes the survival of antigen-primed T cells. We propose that DC polarization increases the local concentration of proinflammatory mediators at the IS and that this represents a new mechanism by which T cell priming is controlled.  

4.864           Regulation by SIRP of dendritic cell homeostasis in lymphoid tissues

Saito, Y., Iwamura, H., Kaneko, T., Ohnishi, H., Murata, Y., Okazawa, H., Kanazawa, Y., Sato-Hashimoto, M., Kobayashi, H., Oldenborg, P-A., Naito, M., Kaneko, Y., Nojima, Y. and Matozaki, T. Blood, 116(18), 3517-3525 (2010)   The molecular basis for regulation of dendritic cell (DC) development and homeostasis remains unclear. Signal regulatory protein (SIRP ), an immunoglobulin superfamily protein that is predominantly expressed in DCs, mediates cell-cell signaling by interacting with CD47, another immunoglobulin superfamily protein. We now show that the number of CD11c^high DCs (conventional DCs, or cDCs), in particular, that of CD8^–CD4⁺ (CD4⁺) cDCs, is selectively reduced in secondary lymphoid tissues of mice expressing a mutant form of SIRP that lacks the cytoplasmic region. We also found that SIRP is required intrinsically within cDCs or DC precursors for the homeostasis of splenic CD4⁺ cDCs. Differentiation of bone marrow cells from SIRP mutant mice into DCs induced by either macrophage-granulocyte colony-stimulating factor or Flt3 ligand in vitro was not impaired. Although the accumulation of the immediate precursors of cDCs in the spleen was also not impaired, the half-life of newly generated splenic CD4⁺ cDCs was markedly reduced in SIRP mutant mice. Both hematopoietic and nonhematopoietic CD47 was found to be required for the homeostasis of CD4⁺ cDCs and CD8^–CD4^–(double negative) cDCs in the spleen. SIRP as well as its ligand, CD47, are thus important for the homeostasis of CD4⁺ cDCs or double negative cDCs in lymphoid tissues. The molecular basis for regulation of dendritic cell (DC) development and homeostasis remains unclear. Signal regulatory protein (SIRP ), an immunoglobulin superfamily protein that is predominantly expressed in DCs, mediates cell-cell signaling by interacting with CD47, another immunoglobulin superfamily protein. We now show that the number of CD11c^high DCs (conventional DCs, or cDCs), in particular, that of CD8^–CD4⁺ (CD4⁺) cDCs, is selectively reduced in secondary lymphoid tissues of mice expressing a mutant form of SIRP that lacks the cytoplasmic region. We also found that SIRP is required intrinsically within cDCs or DC precursors for the homeostasis of splenic CD4⁺ cDCs. Differentiation of bone marrow cells from SIRP mutant mice into DCs induced by either macrophage-granulocyte colony-stimulating factor or Flt3 ligand in vitro was not impaired. Although the accumulation of the immediate precursors of cDCs in the spleen was also not impaired, the half-life of newly generated splenic CD4⁺ cDCs was markedly reduced in SIRP mutant mice. Both hematopoietic and nonhematopoietic CD47 was found to be required for the homeostasis of CD4⁺ cDCs and CD8^–CD4^–(double negative) cDCs in the spleen. SIRP as well as its ligand, CD47, are thus important for the homeostasis of CD4⁺ cDCs or double negative cDCs in lymphoid tissues.  

4.865           Trim17, a novel E3 ubiquitin-ligase, initiates neuronal apoptosis

Lassot, I., Robbins, I., Kristiansen, M., Rahmeh, R., Jaudon, F., Magiera, M.M., Mora, S., Vanhille, L., Lidkin, A., Pettmann, B., Ham, J. and Desagher, S. Cell Death and Differentiation, 17(12), 1928-1941 (2010)   Accumulating data indicate that the ubiquitin–proteasome system controls apoptosis by regulating the level and the function of key regulatory proteins. In this study, we identified Trim17, a member of the TRIM/RBCC protein family, as one of the critical E3 ubiquitin ligases involved in the control of neuronal apoptosis upstream of mitochondria. We show that expression of Trim17 is increased both at the mRNA and protein level in several in vitro models of transcription-dependent neuronal apoptosis. Expression of Trim17 is controlled by the PI3K/Akt/GSK3 pathway in cerebellar granule neurons (CGN). Moreover, the Trim17 protein is expressed in vivo, in apoptotic neurons that naturally die during post-natal cerebellar development. Overexpression of active Trim17 in primary CGN was sufficient to induce the intrinsic pathway of apoptosis in survival conditions. This pro-apoptotic effect was abolished in Bax^−/− neurons and depended on the E3 activity of Trim17 conferred by its RING domain. Furthermore, knock-down of endogenous Trim17 and overexpression of dominant-negative mutants of Trim17 blocked trophic factor withdrawal-induced apoptosis both in CGN and in sympathetic neurons. Collectively, our data are the first to assign a cellular function to Trim17 by showing that its E3 activity is both necessary and sufficient for the initiation of neuronal apoptosis.    

4.866           Phenotypic Heterogeneity among Tumorigenic Melanoma Cells from Patients that Is Reversible and Not Hierarchically Organized

Quintana, E., Shackleton, M., Foster, H.R., Fullen, D.R., Sabel, M.S., Johnson, T,M. and Morrison, S.J. Cancer Cell, 18(5), 510-523 (2010)   We investigated whether melanoma is hierarchically organized into phenotypically distinct subpopulations of tumorigenic and nontumorigenic cells or whether most melanoma cells retain tumorigenic capacity, irrespective of their phenotype. We found 28% of single melanoma cells obtained directly from patients formed tumors in NOD/SCID IL2R ^null mice. All stage II, III, and IV melanomas obtained directly from patients had common tumorigenic cells. All tumorigenic cells appeared to have unlimited tumorigenic capacity on serial transplantation. We were unable to find any large subpopulation of melanoma cells that lacked tumorigenic potential. None of 22 heterogeneously expressed markers, including CD271 and ABCB5, enriched tumorigenic cells. Some melanomas metastasized in mice, irrespective of whether they arose from CD271 or CD271⁺ cells. Many markers appeared to be reversibly expressed by tumorigenic melanoma cells.  

4.867           Accelerated neuritogenesis and maturation of primary spinal motor neurons in response to nanofibers

Gertz, C., Leach, M.K., Birrell, L.K., Martin, D.C., Feldman, E.L. and Corey, J.M. Develop. Neurobiol., 70(8), 589-603 (2010)   Neuritogenesis, neuronal polarity formation, and maturation of axons and dendrites are strongly influenced by both biochemical and topographical extracellular components. The aim of this study was to elucidate the effects of polylactic acid electrospun fiber topography on primary motor neuron development, because regeneration of motor axons is extremely limited in the central nervous system and could potentially benefit from the implementation of a synthetic scaffold to encourage regrowth. In this analysis, we found that both aligned and randomly oriented submicron fibers significantly accelerated the processes of neuritogenesis and polarity formation of individual cultured motor neurons compared to flat polymer films and glass controls, likely due to restricted lamellipodia formation observed on fibers. In contrast, dendritic maturation and soma spreading were inhibited on fiber substrates after 2 days in vitro. This study is the first to examine the effects of electrospun fiber topography on motor neuron neuritogenesis and polarity formation. Aligned nanofibers were shown to affect the directionality and timing of motor neuron development, providing further evidence for the effective use of electrospun scaffolds in neural regeneration applications.  

4.868           The effect of interleukin-13 (IL-13) and interferon-γ (IFN-γ) on expression of surfactant proteins in adult human alveolar type II cells in vitro

Ito, Y. and Mason, R.J. Respiratory Res., 11, 157-169 (2010)   Background Surfactant proteins are produced predominantly by alveolar type II (ATII) cells, and the expression of these proteins can be altered by cytokines and growth factors. Th1/Th2 cytokine imbalance is suggested to be important in the pathogenesis of several adult lung diseases. Recently, we developed a culture system for maintaining differentiated adult human ATII cells. Therefore, we sought to determine the effects of IL-13 and IFN-γ on the expression of surfactant proteins in adult human ATII cells in vitro. Additional studies were done with rat ATII cells. Methods Adult human ATII cells were isolated from deidentified organ donors whose lungs were not suitable for transplantation and donated for medical research. The cells were cultured on a mixture of Matrigel and rat-tail collagen for 8 d with differentiation factors and human recombinant IL-13 or IFN-γ. Results IL-13 reduced the mRNA and protein levels of surfactant protein (SP)-C, whereas IFN-γ increased the mRNA level of SP-C and proSP-C protein but not mature SP-C. Neither cytokine changed the mRNA level of SP-B but IFN-γ slightly decreased mature SP-B. IFN-γ reduced the level of the active form of cathepsin H. IL-13 also reduced the mRNA and protein levels of SP-D, whereas IFN-γ increased both mRNA and protein levels of SP-D. IL-13 did not alter SP-A, but IFN-γ slightly increased the mRNA levels of SP-A. Conclusions We demonstrated that IL-13 and IFN-γ altered the expression of surfactant proteins in human adult ATII cells in vitro. IL-13 decreased SP-C and SP-D in human ATII cells, whereas IFN-γ had the opposite effect. The protein levels of mature SP-B were decreased by IFN-γ treatment, likely due to the reduction in active form cathpesin H. Similarly, the active form of cathepsin H was relatively insufficient to fully process proSP-C as IFN-γ increased the mRNA levels for SP-C and proSP-C protein, but there was no increase in mature SP-C. These observations suggest that in disease states with an overexpression of IL-13, there would be some deficiency in mature SP-C and SP-D. In disease states with an excess of IFN-γ or therapy with IFN-γ, these data suggest that there might be incomplete processing of SP-B and SP-C.  

4.869           Islet Transplantation Using Donors After Cardiac Death: Report of the Japan Islet Transplantation Registry

Saito, T., Gotoh, M., Satomi, S., Uemoto, S., Kenmochi, T., Itoh, T., Kuroda, Y., Yasunami, Y., Matsumoto, S. and Teraoka, S. Transplantation, 90(7), 740-747 (2010)   Background. This report summarizes outcomes of islet transplantation employing donors after cardiac death (DCD) between 2004 and 2007 as reported to the Japan Islet Transplantation Registry. Method. Sixty-five islet isolations were performed for 34 transplantations in 18 patients with insulin-dependent diabetes mellitus, including two patients who had prior kidney transplantation. All but one donor (64/65) was DCD at the time of harvesting. Results. Factors influencing criteria for islet release included duration of low blood pressure of the donor, cold ischemic time, and usage of Kyoto solution for preservation. Multivariate analysis selected usage of Kyoto solution as most important. Of the 18 recipients, 8, 4, and 6 recipients received 1, 2, and 3 islet infusions, respectively. Overall graft survival defined as C-peptide level more than or equal to 0.3 ng/mL was 76.5%, 47.1%, and 33.6% at 1, 2, and 3 years, respectively, whereas corresponding graft survival after multiple transplantations was 100%, 80.0%, and 57.1%, respectively. All recipients remained free of severe hypoglycemia while three achieved insulin independence for 14, 79, and 215 days. HbA1c levels and requirement of exogenous insulin were significantly improved in all patients. Conclusion. Islet transplantation employing DCD can ameliorate severe hypoglycemic episodes, significantly improve HbA1c levels, sustain significant levels of C-peptide, and achieve insulin independence after multiple transplantations. Thus, DCD can be an important resource for islet transplantation if used under strict releasing criteria and in multiple transplantations, particularly in countries where heart-beating donors are not readily available.  

4.870           miR-194 is a marker of hepatic epithelial cells and suppresses metastasis of liver cancer cells in mice

Meng, Z., Fu, X., Chen, X., Zeng, S., Tian, Y., Jove, R., Xu, R. and Huang, W. Hepatology, 52(6), 2148-2157 (2010)   MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by interacting with the 3′ untranslated region (3′-UTR) of multiple mRNAs. Recent studies have linked miRNAs to the development of cancer metastasis. In this study, we show that miR-194 is specifically expressed in the human gastrointestinal tract and kidney. Moreover, miR-194 is highly expressed in hepatic epithelial cells, but not in Kupffer cells or hepatic stellate cells, two types of mesenchymal cells in the liver. miR-194 expression was decreased in hepatocytes cultured in vitro, which had undergone a dedifferentiation process. Furthermore, expression of miR-194 was low in liver mesenchymal-like cancer cell lines. The overexpression of miR-194 in liver mesenchymal-like cancer cells reduced the expression of the mesenchymal cell marker N-cadherin and suppressed invasion and migration of the mesenchymal-like cancer cells both in vitro and in vivo. We further demonstrated that miR-194 targeted the 3′-UTRs of several genes that were involved in epithelial-mesenchymal transition and cancer metastasis. Conclusion: These results support a role of miR-194, which is specifically expressed in liver parenchymal cells, in preventing liver cancer cell metastasis.  

4.871           B7-H4 mediates inhibition of T cell responses by activated murine hepatic stellate cells

Chinnadurai, R. and Grakoui, A. Hepatology, 52(6), 2177-2185 (2010)   Liver fibrosis is mediated by the transformation of hepatic stellate cells (HSC) from a quiescent to an activated state. To understand the role of HSC in liver immunity, we investigated the effect of this transition on T cell stimulation in vitro. Unlike quiescent HSC, activated HSC did not induce proliferation of antigen-specific T cells. Phenotypic analysis of quiescent and activated HSC revealed that activated HSC expressed the coinhibitory molecule B7-H4. Silencing B7-H4 by small interfering RNA (siRNA) in activated HSC restored the ability of T cells to proliferate, differentiate, and regain effector recall responses. Furthermore, expression of B7-H4 on HSC inhibits early T cell activation and addition of exogenous interleukin (IL)-2 reversed the T cell anergy induced by activated HSC. Conclusion: These studies reveal a novel role for activated HSC in the attenuation of intrahepatic T cell responses by way of expression of the coinhibitory molecule B7-H4, and may provide fundamental insight into intrahepatic immunity during liver fibrogenesis.  

4.872           Calcium and Calmodulin-dependent Kinase Kinase Beta Signalling Controls Human Platelet Aggregation

Onselaer, M-B., eeckoudt, S., de Meester, C., Hue, L., Vanoverschelde, J-L., Bertrand, L., Oury, C., Beauloye, C., and Norman, S. Circulation, 122, Abstract A17868 (2010)   Background: Several agonists, thrombin (T), thromboxane (TXA2) and collagen (C) induce platelet activation and aggregation. Their effects are mediated by increase in intracellular calcium (Ca2+) and remodelling of actin cytoskeleton. Ca2+/calmodulin-dependent protein kinase kinase beta (CaMKKbeta) controls actin cytoskeleton organization through AMP-activated protein kinase (AMPK) in epithelial cells, extending the role of AMPK beyond metabolism. Therefore, our hypothesis was that activation of CaMKKbeta-AMPK pathway by these agonists mediated platelet aggregation by regulating cytoskeletal targets. Methods: Blood freshly drawn was collected from healthy donors. Platelet aggregation was analyzed on the platelet rich fraction (PRP) using a lumi-aggregometer (Chrono-log). For in vitro treatments and western blot analysis, PRP was purified by the OptiPrep method. Results: 10μM STO609, a selective inhibitor of CaMKKbeta, inhibited thrombin-induced platelet aggregation (0.025U/ml T: 69±3; T + STO609: 11±4 % of aggregation, P<0.001). STO609 also reduced platelet aggregation induced by TXA2 and collagen (1 μM TXA2: 79±5; TXA2+STO609, 29±11, 2μg/ml C: 50±4; C+STO609, 19±2 % o aggregation, P<0.01). Thrombin induced an increase in acetyl CoA carboxylase (ACC) phosphorylation, a substrate of AMPK (control: 15±10; 0.1 U/ml T:81±17 A.U., P<0.05). TXA2 and collagen also increased ACC phosphorylation to a lower extent. STO609 prevented thrombin effect on ACC phosphorylatio (Control: 15±10 ; Control+STO609: 16±11; 0.1U/ml T: 81±17; T+STO609: 16±6 A.U., P<0.05). Finally, inhibition of CaMKKbeta was associated with a decrease in the phosphorylation of two AMPK cytoskeletal targets, namely myosin light chains (MLC) and vasodilatator-phosphoprotein (VASP), known to be modulated during platelet aggregation. Conclusion: Inhibition of CaMKKbeta by STO609 blocked platelet aggregation induced by thrombin, TXA2 and collagen. The molecular mechanism by which CaMKKbeta control aggregation could be a direct activation of AMPK and subsequent phosphorylation of MLC and VASP. CaMKKbeta is a potential target for new anti-platelet therapies.  

4.873           Combined Transplantation of Pancreatic Islets and Adipose Tissue-Derived Stem Cells Enhances the Survival and Insulin Function of Islet Grafts in Diabetic Mice

Ohmura, Y., tanemura, M., Kawaguchi, N., Machida, T., Tanida, T., Deguchi, T., Wada, H., Kobayashi, S., Marubashi, S., Eguchi, H., Takeda, Y., Matsuura, N., Ito, T., Nagano, H., Doki, Y. and Mori, M. Transplantation, 90(12), 1366-1373 (2010)   Background. Overcoming significant loss of transplanted islet mass is important for successful islet transplantation. Adipose tissue-derived stem cells (ADSCs) seem to have angiogenic potential and antiinflammatory properties. We hypothesized that the inclusion of ADSCs with islet transplantation should enhance the survival and insulin function of the islet graft. Methods. Syngeneic ADSCs and allogeneic islets were transplanted simultaneously under the kidney capsules of diabetic C57BL/6J mice. Rejection of the graft was examined by measurement of blood glucose level. Revascularization and inflammatory cell infiltration were examined by immunohistochemistry. Results. Transplantation of 400 islets only achieved normoglycemia with graft survival of 13.6±1.67 days (mean±standard deviation), whereas that of 100 or 200 allogeneic islets never reversed diabetes. Transplantation of 200 islets with 2×10⁵ ADSCs reversed diabetes and significantly prolonged graft survival (13.0±5.48 days). Results of glucose tolerance tests performed on day 7 were significantly better in islets-ADSCs than islets-alone recipients. Immunohistochemical analysis confirmed the presence of insulin-stained islet grafts with well-preserved structure in islets-ADSCs transplant group. Significant revascularization (larger number of von Willebrand factor-positive cells) and marked inhibition of inflammatory cell infiltration, including CD4⁺ and CD8⁺ T cells and macrophages, were noted in the islets-ADSCs transplant group than islets-alone transplant group. Conclusions. Our results indicated that cotransplantation of ADSCs with islet graft promoted survival and insulin function of the graft and reduced the islet mass required for reversal of diabetes. This innovative protocol may allow “one donor to one recipient” islet transplantation.  

4.874           In vivo trafficking and immunostimulatory potential of an intranasally-administered primary dendritic cell-based vaccine

Vilekar, P., Awasthi, V., Lagisetty, P., King, C., Shankar, N. and Awasthi, S. BMC Immunol., 11, 60-76 (2010)   Background Coccidioidomycosis or Valley fever is caused by a highly virulent fungal pathogen: Coccidioides posadasii or immitis. Vaccine development against Coccidioides is of contemporary interest because a large number of relapses and clinical failures are reported with antifungal agents. An efficient Th1 response engenders protection. Thus, we have focused on developing a dendritic cell (DC)-based vaccine for coccidioidomycosis. In this study, we investigated the immunostimulatory characteristics of an intranasal primary DC-vaccine in BALB/c mouse strain that is most susceptible to coccidioidomycosis. The DCs were transfected nonvirally with Coccidioides-Ag2/PRA-cDNA. Expression of DC-markers, Ag2/PRA and cytokines were studied by flow cytometry, dot-immunoblotting and cytometric bead array methods, respectively. The T cell activation was studied by assessing the upregulation of activation markers in a DC-T cell co-culture assay. For trafficking, the DCs were co-transfected with a plasmid DNA encoding HSV1 thymidine kinase (TK) and administered intranasally into syngeneic mice. The trafficking and homing of TK-expressing DCs were monitored with positron emission tomography (PET) using ¹⁸F-FIAU probe. Based on the PET-probe accumulation in vaccinated mice, selected tissues were studied for antigen-specific response and T cell phenotypes using ELISPOT and flow cytometry, respectively. Results We found that the primary DCs transfected with Coccidioides-Ag2/PRA-cDNA were of immature immunophenotype, expressed Ag2/PRA and activated naïve T cells. In PET images and subsequent biodistribution, intranasally-administered DCs were found to migrate in blood, lung and thymus; lymphocytes showed generation of T effector memory cell population (T_EM) and IFN-γ release. Conclusions In conclusion, our results demonstrate that the intranasally-administered primary DC vaccine is capable of inducing Ag2/PRA-specific T cell response. Unique approaches utilized in our study represent an attractive and novel means of producing and evaluating an autologous DC-based vaccine.  

4.875           Antiproinflammatory Effects of Iodixanol (OptiPrep)-Based Density Gradient Purification on Human Islet Preparations

Mita, A., Ricordi, C., Messinger, S., Miki, A., Misawa, R., Barker, S., Molano, R.D., Haertter, R., Khan, A., Miyagawa, S., PIleggi, A., Inverardi, L., Ellejandro, R., Hering, B.J. and Ichii, H. Cell Transplant., 19(12), 1537-1546 (2010)   Islet isolation and purification using a continuous density gradient may reduce the volume of tissue necessary for implantation into patients, therefore minimizing the risks associated with intraportal infusion in islet transplantation. On the other hand, the purification procedure might result in a decreased number of islets recovered due to various stresses such as exposure to cytokine/chemokine. While a Ficoll-based density gradient has been widely used in purification for clinical trials, purification with iodixanol (OptiPrep) has been recently reported in islet transplant series with successful clinical outcomes. The aim of the current study was to compare the effects of the purification method using OptiPrep-based and Ficoll-based density gradients. Human islet isolations were performed using a modified automated method. After the digestion phase, prepurification digests were divided into two groups and purified using a semiautomated cell processor with either a continuous Ficoll- or OptiPrep-based density gradient. The quantity, purity, viability, and cellular composition of islet preparations from each group were assessed. Cytokine/chemokine and tissue factor production from islet preparations after 48-h culture were also measured. Although islet purity, postpurification IEQ, islet recovery rate, FDA/PI, and fractional β-cell viability were comparable, β-cell mass after 48-h culture significantly improved in the OptiPrep group when compared to the Ficoll group. The production of cytokine/chemokine including IL-1β, TNF-α, IFN-γ, IL-6, IL-8, MIP-1β, MCP-1, and RANTES but not tissue factor from the OptiPrep group was significantly lower during 48-h culture after isolation. Each preparation contained the similar number of ductal cells and macrophages. Endotoxin level in both gradient medium was also comparable. The purification method using OptiPrep gradient media significantly reduced cytokine/chemokine production but not tissue factor from human islet preparations and improved β-cell survival during pretransplant culture. Our results suggest that the purification method using OptiPrep gradient media may be of assistance in increasing successful islet transplantation.    

4.876           Growth temperature-dependent expression of structural variants of Listeria monocytogenes lipoteichoic acid

Dehus, O., Pfitzenmaier, M., Stuebs, G., Fischer, N., Schwaeble, W., Morath, S., Hartung, T., Geyer, A. and Hermann, C. Immunobiol., 216, 23-31 (2011)   Investigating the expression of lipoteichoic acid (LTA) from Listeria monocytogenes, we found two distinct structural variants of LTA (LTA1 and LTA2) using NMR and MS technology. While both LTA consisted of a poly-glycerophosphate backbone (differing in length) bound via a disaccharide to a diacyl-glycerol moiety, one LTA type (LTA2) possessed a second diacyl-glycerol moiety linked to the disaccharide via a phosphodiester. As examined in vitro, LTA2 in contrast to LTA1 failed to activate the l-ficolin dependent pathway of complement. Most interestingly, growth temperature had a strong influence on the expression levels of LTA1 and LTA2 in the cell wall: while the amount of LTA1 was comparable, the expression of LTA2 was low when Listeria had grown at room temperature (ratio of LTA1 to LTA2 was 1:0.06), but increased when Listeria had been cultivated at 37 °C (ratio of LTA1 to LTA2 was 1:0.68). The observed shift in LTA expression, probably accompanying the switch from the saprophytic to the virulent entity, indicates an important adaptation to the different structural requirements inside the host cells.  

4.877           Indole-3-carbinol inhibits hepatic stellate cells proliferation by blocking NADPH oxidase/reactive oxygen species/p38 MAPK pathway

Ping, J., Li, J-t., Liao, Z-x., Shang, L. and Wang, H. Eur. J. Pharmocol., 650, 656-662 (2011)   During the course of liver fibrogenesis, hepatic stellate cell (HSC) proliferation as well as subsequent synthesis of excessive extracellular matrix components is known to be the central events. Thus, factors that could limit HSC proliferation are potential anti-fibrotic agents. The aim of this study was to investigate the effects of indole-3-carbinol (I3C), a nutritional component derived from Brassica family vegetables, on the proliferation of cultured HSC and to clarify the underline molecular mechanism. HSC-T6, an activated rat HSC line, was treated with I3C (50, 100 and 200 μM) for 24 h. The results indicated that I3C can significantly inhibit HSC proliferation in a concentration-dependent manner with or without platelet-derived growth factor-BB (PDGF-BB) stimulation (P < 0.01). I3C could also block HSC in the G₀/G₁ phase from entering the S phase. The expressions of α-smooth muscle actin in HSC treated with I3C, were significantly decreased at levels of protein and mRNA (P < 0.01). In addition, the type I collagen level, cyclin D₁ and cyclin-dependent kinase 4 mRNA expressions, intracellular nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and reactive oxygen species generation were significantly decreased by I3C (P < 0.05). We also observed that the phosphorylated p38 MAPK in HSC-T6 was inhibited by I3C in a concentration-dependent manner; however, the phosphorylated ERK1/2 was unaltered. In conclusion, I3C could inhibit the proliferation of HSC by blocking the NADPH oxidase/reactive oxygen species/p38 MAPK signal pathway. These findings suggest that dietary I3C might play a novel role in prevention and treatment of chronic liver diseases.  

4.878           Hyperbranched polyglycerols on the nanometer and micrometer scale

Steinhilber, D., Seiffert, S., Heyman, J.A., Paulus, F., Weitz, D.A. and Haag, r. Biomaterials, 32, 1311-1316 (2011)   We report the preparation of polyglycerol particles on different length scales by extending the size of hyperbranched polyglycerols (3 nm) to nanogels (32 nm) and microgels (140 and 220 μm). We use miniemulsion templating for the preparation of nanogels and microfluidic templating for the preparation of microgels, which we obtain through a free-radical polymerization of hyperbranched polyglycerol decaacrylate and polyethylene glycol-diacrylate. The use of mild polymerization conditions allows yeast cells to be encapsulated into the resultant microgels with cell viabilities of approximately 30%.    

4.879           Circulating cytokines and growth factors in professional soccer players: correlation with in vitro-induced motor neuron death

De Paola, M., Visconti, L., Vianello, E., Mattana, F., Banfi, G., Corsi, M.M., Beghi, E. and Mennini, T. Eur. J. Neurol., 18, 85-92 (2011)   Background: Professional soccer players are susceptible to amyotrophic lateral sclerosis. Strenuous physical activity has been associated with persistent inflammatory conditions and elevation of systemic cytokine levels, which could contribute to the vulnerability of these athletes. To investigate changes induced by playing soccer in the systemic profiles of growth factors and of the principal cytokines involved in the inflammatory response, we compared the serum concentrations of these factors in Italian professional soccer players and sedentary subjects. We also investigated the effects of the sera on primary cultured motor neurons in relation to their cytokine and growth factor content. Methods: Serum concentrations of cytokines and growth factors were measured by a biochip array analyzer. Neurotoxicity of sera was assessed by immunocytochemical assays in primary motor neuron cultures from mouse embryos. Results: Circulating levels of interleukin-8, tumor necrosis factor-alpha and interleukin-4 were lower in soccer players than controls. However, the viability of primary cultured mouse motor neurons treated with sera from the two groups did not differ significantly. Vascular endothelial growth factor (VEGF) independently emerged as a systemic protective factor for motor neurons. Conclusions: We found significant alterations in circulating pro-inflammatory cytokines in Italian professional soccer players, showing an unbalanced inflammatory condition in these subjects. VEGF was a protective serum factor affecting motor neuron survival.  

4.880           Mechanisms by Which Chronic Ethanol Feeding Limits the Ability of Dendritic Cells to Stimulate T-Cell Proliferation

Fan, J., Edsen-Moore, R., Turner, L.E., Cook, R.T., Legge, K.L., Waldschmidt, T.J. and Schkuter, A.J. Alcoholism: Clin. Exp. Res., 35(1), 47-59 (2011)   Background: As initiators of immune responses, dendritic cells (DCs) are required for antigen (Ag)-specific activation of naïve T cells in the defense against infectious agents. The increased susceptibility to and severity of infection seen in chronic alcoholics could be because of impaired DCs initiation of naïve T-cell responses. Specifically, these DCs may not provide adequate Signals 1 (Ag presentation), 2 (costimulation), or 3 (cytokine production) to these T cells. Methods: Using the Meadows-Cook murine model of chronic alcohol abuse, the ability of ethanol (EtOH)-exposed DCs to stimulate T-cell proliferation, acquire and process Ag, express costimulatory molecules, and produce inflammatory cytokines was assessed. Results: Normal naïve T cells primed by EtOH-exposed DCs showed decreased proliferation in vitro and in vivo, compared to water-fed control mice. These EtOH-exposed DCs, after activation by CpG or tumor necrosis factor alpha (TNF ), were less able to upregulate costimulatory molecules CD40, CD80, or CD86, and produced less IL-12 p40, TNF , and IFN than DCs from water-fed mice. TLR9 and TNF receptor expression were also reduced in/on EtOH-exposed DCs. No evidence of defective Ag acquisition or processing as a result of EtOH feeding was identified. Conclusions: Inadequate proliferation of normal T cells following stimulation by EtOH-exposed DCs is likely a result of diminished Signal 2 and Signal 3. Lack of adequate inflammatory stimulation of EtOH-exposed DCs because of diminished receptors for inflammatory mediators appears to be at least partially responsible for their dysfunction. These findings provide a mechanism to explain increased morbidity and mortality from infectious diseases in alcoholics and suggest targets for therapeutic intervention.  

4.881           SOD1 targeted to the mitochondrial intermembrane space prevents motor neuropathy in the Sod1 knockout mouse

Fischer, L.R., Igoudjil, A., Magrane, J., Li, Y., Hansen, J.M., Manfredi, g. and Glass, J.D. Brain, 134, 196-209 (2011)   Motor axon degeneration is a critical but poorly understood event leading to weakness and muscle atrophy in motor neuron diseases. Here, we investigated oxidative stress-mediated axonal degeneration in mice lacking the antioxidant enzyme, Cu,Zn superoxide dismutase (SOD1). We demonstrate a progressive motor axonopathy in these mice and show that Sod1−/− primary motor neurons extend short axons in vitro with reduced mitochondrial density. Sod1−/− neurons also show oxidation of mitochondrial—but not cytosolic—thioredoxin, suggesting that loss of SOD1 causes preferential oxidative stress in mitochondria, a primary source of superoxide in cells. SOD1 is widely regarded as the cytosolic isoform of superoxide dismutase, but is also found in the mitochondrial intermembrane space. The functional significance of SOD1 in the intermembrane space is unknown. We used a transgenic approach to express SOD1 exclusively in the intermembrane space and found that mitochondrial SOD1 is sufficient to prevent biochemical and morphological defects in the Sod1−/− model, and to rescue the motor phenotype of these mice when followed to 12 months of age. These results suggest that SOD1 in the mitochondrial intermembrane space is fundamental for motor axon maintenance, and implicate oxidative damage initiated at mitochondrial sites in the pathogenesis of motor axon degeneration.  

4.882           Marek's Disease Virus Type 1 MicroRNA miR-M3 Suppresses Cisplatin-Induced Apoptosis by Targeting SMAD2 of the Transforming Growth Factor Beta Signal Pathway

Xu, S., Xue, C., Li, J., Bi, Y. and Cao, Y.

1. Virol., 85(1), 276-285 (2011)

  Viruses cause about 15% of the cancers that are still the leading causes of human mortality. The discovery of viral oncogenes has enhanced our understanding of viral oncogenesis. However, the underlying molecular mechanisms of virus-induced cancers are complex and require further investigation. The present study has attempted to investigate the effects of the microRNAs (miRNAs) encoded by Marek's disease virus 1 (MDV1), a chicken herpesvirus causing acute T-cell lymphomas and solid visceral tumors in chickens, on anti-cancer drug-induced apoptosis and identify the targets of the miRNAs. The results showed that of the total 14 miRNAs encoded by MDV1, MDV1-miR-M3 significantly promoted cell survival under treatment with cisplatin, a widely used chemotherapy drug. MDV1-miR-M3 suppressed cisplatin-induced apoptosis by directly downregulating expression at the protein but not the mRNA level of Smad2, a critical component in the transforming growth factor β signal pathway. Our data suggest that latent/oncogenic viruses may encode miRNAs to directly target cellular factors involved in antiviral processes including apoptosis, thus proactively creating a cellular environment beneficial to viral latency and oncogenesis. Furthermore, the knowledge of the apoptosis resistance conferred by viral miRNAs has great practical implications for improving the efficacy of chemotherapies for treating cancers, especially those induced by oncogenic viruses.  

4.883           Effective Caspase Inhibition Blocks Neutrophil Apoptosis and Reveals Mcl-1 as Both a Regulator and a Target of Neutrophil Caspase ActivationHuman tissue inflammation is terminated, at least in part, by the death of inflammatory neutrophils by apoptosis. The regulation of this process is therefore key to understanding and manipulating inflammation resolution. Previous data have suggested that the short-lived pro-survival Bcl-2 family protein, Mcl-1, is instrumental in determining neutrophil lifespan. However, Mcl-1 can be cleaved following caspase activity, and the possibility therefore remains that the observed fall in Mcl-1 levels is due to caspase activity downstream of caspase activation, rather than being a key event initiating apoptosis in human neutrophilsWe demonstrate that apoptosis in highly purified neutrophils can be almost completely abrogated by caspase inhibition with the highly effective di-peptide caspase inhibitor, Q-VD.OPh, confirming the caspase dependence of neutrophil apoptosis. Effective caspase inhibition does not prevent the observed fall in Mcl-1 levels early in ultrapure neutrophil culture, suggesting that this fall in Mcl-1 levels is not a consequence of neutrophil apoptosis. However, at later timepoints, declines in Mcl-1 can be reversed with effective caspase inhibition, suggesting that Mcl-1 is both an upstream regulator and a downstream target of caspase activity in human neutrophils.

Wardle, D.J., Burgon, J., Sabroe, I., Bingle, C.D., Whyte, M.K.B. and Renshaw, S.A. PloSOne, 6(1), e15768 (2011)   Human tissue inflammation is terminated, at least in part, by the death of inflammatory neutrophils by apoptosis. The regulation of this process is therefore key to understanding and manipulating inflammation resolution. Previous data have suggested that the short-lived pro-survival Bcl-2 family protein, Mcl-1, is instrumental in determining neutrophil lifespan. However, Mcl-1 can be cleaved following caspase activity, and the possibility therefore remains that the observed fall in Mcl-1 levels is due to caspase activity downstream of caspase activation, rather than being a key event initiating apoptosis in human neutrophils. We demonstrate that apoptosis in highly purified neutrophils can be almost completely abrogated by caspase inhibition with the highly effective di-peptide caspase inhibitor, Q-VD.OPh, confirming the caspase dependence of neutrophil apoptosis. Effective caspase inhibition does not prevent the observed fall in Mcl-1 levels early in ultrapure neutrophil culture, suggesting that this fall in Mcl-1 levels is not a consequence of neutrophil apoptosis. However, at later timepoints, declines in Mcl-1 can be reversed with effective caspase inhibition, suggesting that Mcl-1 is both an upstream regulator and a downstream target of caspase activity in human neutrophils.  

4.884           Regulation of Neutrophil Senescence by MicroRNAs

Ward, J.R., Heath, P.R., Catto, J.W., Whyte, M.K.B., Milo, M. and renshaw, S.A. PlosOne, 6(1), e15810 (2011)   Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease.  

4.885           CpG Promotes Cross-Presentation of Dead Cell-Associated Antigens by Pre-CD8α⁺ Dendritic Dells

De Brito, C., Tomkowiak, M., Ghittoni, R., Caux, C., Leverrier, Y. and Marvel, J.

1. Immunol., 186, 1503-1511 (2011)

  Cross-presentation of cell-associated Ags by dendritic cells (DC) plays an important role in immunity. DC in lymphoid tissues are short lived, being continuously replaced by precursors that proliferate and differentiate locally. Paradoxically, although TLR ligands promote immune responses and stimulate DC replenishment, they impair the cross-priming capacity of terminally differentiated splenic CD8α⁺ DC, the major subset involved in cross-priming. In this study, we have investigated the cross-presentation capacity of newly generated murine DC and especially immediate precursors of CD8α⁺ DC. We show that these DC do not cross-present Ag from dead cells unless stimulated by TLR ligands before Ag capture. TLR ligand CpG induced the expression of costimulatory molecules required for CD8 T cell activation but also regulated the intracellular mechanisms of cross-presentation such as Ag degradation rates without regulating Ag uptake. GM-CSF, an inflammatory cytokine associated with infections, also promoted cross-presentation acquisition by pre-CD8α⁺ DC and synergized with TLR9 ligand. The concept that TLR ligands as well as inflammatory cytokines promote the acquisition of cross-presenting properties by pre-CD8α⁺ DC has important implications during immune responses and when considering the use of these cells for vaccination.  

4.886           Rhesus macaque θ-defensin isoforms: expression, antimicrobial activities, and demonstration of a prominent role in neutrophil granule microbicidal activities

Tongaonkar, P., Tran, P., Roberts, K., Schaal, J., Ösapay, G., Tran, D., Ouellette, A.J. and Selsted, M.E.

1. Leukoc. Biol., 89(2), 283-290 (2011)

  Mammalian defensins are cationic, antimicrobial peptides that play a central role in innate immunity. The peptides are composed of three structural subfamilies: α-, β-, and θ-defensins. θ-Defensins are macrocyclic octadecapeptides expressed only in Old World monkeys and Orangutans and are produced by the pair-wise, head-to-tail splicing of nonapeptides derived from their respective precursors. The existence of three active θ-defensin genes predicts that six different RTDs (1–6) are produced in this species. In this study, we isolated and quantified RTDs 1–6 from the neutrophils of 10 rhesus monkeys. RTD-1 was the most abundant θ-defensin, constituting ∼50% of the RTD content; total RTD content varied by as much as threefold between animals. All peptides tested were microbicidal at ∼1 μM concentrations. The contribution of θ-defensins to macaque neutrophil antimicrobial activity was assessed by analyzing the microbicidal properties of neutrophil granule extracts after neutralizing θ-defensin content with a specific antibody. θ-Defensin neutralization markedly reduced microbicidal activities of the corresponding extracts. Macaque neutrophil granule extracts had significantly greater microbicidal activity than those of human neutrophils, which lack θ-defensins. Supplementation of human granule extracts with RTD-1 markedly increased the microbicidal activity of these preparations, further demonstrating a prominent microbicidal role for θ-defensins.  

4.887           Neuroprotective signaling pathways are modulated by adenosine in the anoxia tolerant turtle

Nayak, G.H., Prentice, H.M. and Milton, S.L.

1. Cerebral Blood Flow & Metabolism, 31(2), 467-475 (2011)

  Cumulative evidence shows a protective role for adenosine A1 receptors (A1R) in hypoxia/ischemia; A1R stimulation reduces neuronal damage, whereas blockade exacerbates damage. The signal transduction pathways may involve the mitogen-activated protein kinase (MAPK) pathways and serine/threonine kinase (AKT), with cell survival depending on the timing and degree of upregulation of these cascades as well as the balance between pro-survival and pro-death pathways. Here, we show in vitro that extracellular signal-regulated kinase (ERK1/2) and phosphatidylinositol 3-kinase (PI3-K/AKT) activation is dependent on A1R stimulation, with further downstream effects that promote neuronal survival. Phosphorylated ERK1/2 (p-ERK) and AKT (p-AKT) as well as Bcl-2 are upregulated in anoxic neuronally enriched primary cultures from turtle brain. This native upregulation is further increased by the selective A1R agonist 2-chloro-N-cyclopentyladenosine (CCPA), whereas the selective antagonist 8-cyclopentyl-1,3-dihydropylxanthine (DPCPX) decreases p-ERK and p-AKT expression. Conversely, A1R antagonism resulted in increases in phosphorylated JNK (p-JNK), p38 (p-p38), and Bax. As pathological and adaptive changes occur simultaneously during anoxia/ischemia in mammalian neurons, the turtle provides an alternative model to analyze protective mechanisms in the absence of evident pathologies.  

4.888           Rapid brain-derived neurotrophic factor-dependent sequestration of amygdala and hippocampal GABA_A receptors via different tyrosine receptor kinase B-mediated phosphorylation pathways

Mou, L., heldt, S.A. and Ressler, K.J. Neuroscience, 176, 72-85 (2011)   During the consolidation of fear memory, it has been shown that GABA_A receptors (GABA_AR) are rapidly downregulated in amygdala. This rapid decrease in GABA_AR functioning may permit transient hyperexcitablity, contributing to cellular mechanisms of memory consolidation. Memory consolidation also requires brain-derived neurotrophic factor (BDNF) activation of tyrosine receptor kinase B (TrkB) receptors in the amygdala and hippocampus. We hypothesized that rapid internalization of GABA_ARα1 is mediated via TrkB activation of PKA and PKC-dependent processes. Primary neuronal cell cultures, from postnatal day 14–21 mouse amygdala and hippocampus, were analyzed with immunofluorescence using cell-surface, whole-cell permeabilization, and antibody internalization techniques, as well as with ³H-muscimol binding assays. In both hippocampal and amygdala cultures, we found a >60% reduction in surface GABA_ARα1 within 5 min of BDNF treatment. Notably, the rapid decrease in surface GABA_ARα1 was confirmed biochemically using surface biotinylation assays followed by western blotting. This rapid effect was accompanied by TrkB phosphorylation and increased internal GABA_ARα1 immunofluorescence, and was blocked by k252a, a broad-spectrum tyrosine kinase antagonist. To further demonstrate TrkB specificity, we used previously characterized TrkB^F616A mice, in which the highly selective TrkB-mutant specific antagonist, 1NMPP1, prevented the BDNF-dependent GABA_ARα1 internalization. In hippocampus, we found both PKA and PKC inhibition, using Rp-8-Br-cAMP and Calphostin C, respectively, blocked GABA_ARα1 internalization, whereas inhibition of MAPK (U0126) and PI3K (LY294002) did not prevent rapid internalization. By contrast in amygdala cultures, Rp-8-Br-cAMP had no effect. Together, these data suggest that rapid GABA_AR internalization during memory consolidation is BDNF-TrkB dependent. Further, it appears that hippocampal GABA_AR internalization is PKA and PKC dependent, while it may be primarily PKC dependent in amygdala, implying differential roles for TrkB-dependent kinase activation in BDNF-dependent memory formation.  

4.889           Characterization of Dendritic Cells Subpopulations in Skin and Afferent Lymph in the Swine Model

Marquet, F., Bonneau, M., Pascale, F., Urien, C., Kang, C., Schwartz-Cornil, I. and Bertho, N. PloSOne, 6(1), e16320 (2011)   Transcutaneous delivery of vaccines to specific skin dendritic cells (DC) subsets is foreseen as a promising strategy to induce strong and specific types of immune responses such as tolerance, cytotoxicity or humoral immunity. Because of striking histological similarities between human and pig skin, pig is recognized as the most suitable model to study the cutaneous delivery of medicine. Therefore improving the knowledge on swine skin DC subsets would be highly valuable to the skin vaccine field. In this study, we showed that pig skin DC comprise the classical epidermal langerhans cells (LC) and dermal DC (DDC) that could be divided in 3 subsets according to their phenotypes: (1) the CD163^neg/CD172a^neg, (2) the CD163^highCD172a^pos and (3) the CD163^lowCD172a^pos DDC. These subtypes have the capacity to migrate from skin to lymph node since we detected them in pseudo-afferent lymph. Extensive phenotyping with a set of markers suggested that the CD163^high DDC resemble the antibody response-inducing human skin DC/macrophages whereas the CD163^negCD172^low DDC share properties with the CD8⁺ T cell response-inducing murine skin CD103^pos DC. This work, by showing similarities between human, mouse and swine skin DC, establishes pig as a model of choice for the development of transcutaneous immunisation strategies targeting DC.  

4.890           p75NTR Regulates Aβ Deposition by Increasing Aβ Production But Inhibiting Aβ Aggregation with Its Extracellular Domain

Wang, Y-J., Wang, X., Lu, J-J., Li, Q-X., Gao, C-Y., Liu, X-H., Sun, Y., Yang, M., Lim, Y., Evin, G., Zhong, J-H., Masters, C. and Zhou, X-F.

1. Neurosci., 31(6), 2292-2304 (2011)

  Accumulation of toxic amyloid-β (Aβ) in the cerebral cortex and hippocampus is a major pathological feature of Alzheimer's disease (AD). The neurotrophin receptor p75NTR has been proposed to mediate Aβ-induced neurotoxicity; however, its role in the development of AD remains to be clarified. The p75NTR/ExonIII–/– mice and APPSwe/PS1dE9 mice were crossed to generate transgenic AD mice with deletion of p75NTR gene. In APPSwe/PS1dE9 transgenic mice, p75NTR expression was localized in the basal forebrain neurons and degenerative neurites in neocortex, increased with aging, and further activated by Aβ accumulation. Deletion of the p75NTR gene in APPSwe/PS1dE9 mice reduced soluble Aβ levels in the brain and serum, but increased the accumulation of insoluble Aβ and Aβ plaque formation. There was no change in the levels of amyloid precursor protein (APP) and its proteolytic derivatives, or -, β-, and -secretase activities, or in levels of BACE1, neprilysin (NEP), and insulin-degrading enzyme (IDE) proteins. Aβ production by cortical neurons of APPSwe/PS1dE9 mice was reduced by deletion of p75NTR gene in vitro. Recombinant extracellular domain of p75NTR attenuated the oligomerization and fibrillation of synthetic Aβ₄₂ peptide in vitro, and reduced local Aβ plaques after hippocampus injection in vivo. In addition, deletion of p75NTR attenuated microgliosis but increased the microhemorrhage profiles in the brain. The deletion of p75NTR did not significantly change the cognitive function of the mice up to the age of 9 months. Our data suggest that p75NTR plays a critical role in regulating Aβ levels by both increasing Aβ production and attenuating its aggregation, and they caution that a therapeutic intervention simply reducing p75NTR may exacerbate AD pathology.  

4.891           Lung Effector Memory and Activated CD4+ T Cells Display Enhanced Proliferation in Surfactant Protein A-Deficient Mice during Allergen-Mediated Inflammation

Pastva, A.M., Mukherjee, S., Giamberardino, C., Hsia, B., Lo, B., Sempowski, G.D. and Wright, J.R.

1. Immunol., 186, 2842-2849 (2011)

  Although many studies have shown that pulmonary surfactant protein (SP)-A functions in innate immunity, fewer studies have addressed its role in adaptive immunity and allergic hypersensitivity. We hypothesized that SP-A modulates the phenotype and prevalence of dendritic cells (DCs) and CD4⁺ T cells to inhibit Th2-associated inflammatory indices associated with allergen-induced inflammation. In an OVA model of allergic hypersensitivity, SP-A^−/− mice had greater eosinophilia, Th2-associated cytokine levels, and IgE levels compared with wild-type counterparts. Although both OVA-exposed groups had similar proportions of CD86⁺ DCs and Foxp3⁺ T regulatory cells, the SP-A^−/− mice had elevated proportions of CD4⁺ activated and effector memory T cells in their lungs compared with wild-type mice. Ex vivo recall stimulation of CD4⁺ T cell pools demonstrated that cells from the SP-A^−/− OVA mice had the greatest proliferative and IL-4–producing capacity, and this capability was attenuated with exogenous SP-A treatment. Additionally, tracking proliferation in vivo demonstrated that CD4⁺ activated and effector memory T cells expanded to the greatest extent in the lungs of SP-A^−/− OVA mice. Taken together, our data suggested that SP-A influences the prevalence, types, and functions of CD4⁺ T cells in the lungs during allergic inflammation and that SP deficiency modifies the severity of inflammation in allergic hypersensitivity conditions like asthma.  

4.892           Similar Islet Function in Islet Allotransplant and Autotransplant Recipients, Despite Lower Islet Mass in Autotransplants

Bellin, M.D., Sutherland, D.E.R., Beilman, G:J., Hong-Mcatee, I., Balamurugan, A.N., Hering, B.J. and Moran, A. Transplantation, 91(3), 367-372 (2011)   Background. Despite high initial rates of insulin independence after islet allotransplant for type 1 diabetes, long-term islet function is suboptimal. Possible contributing factors include autoimmune recurrence, alloimmune rejection, or immunosuppressant medication toxicity. In contrast, islet autografts, infused at the time of pancreatectomy for chronic pancreatitis, are not subject to these variables. Islet function was compared in autograft and allograft recipients. Methods. Eight autograft and eight allograft recipients, insulin independent or requiring minimal insulin, were matched for similar duration posttransplant (mean 2.1±1.2 years). Eleven healthy control subjects were also enrolled. Subjects underwent oral and intravenous glucose tolerance testing and arginine stimulation testing. Results. Age, gender, body mass index, duration posttransplant, and hemoglobin A_1c levels were similar between groups. Glucose tolerance was worse in transplant recipients compared with controls. Alloislet recipients received significantly more islet equivalents per kg body weight (IE/kg) than autograft recipients (9958±6229 IE/kg vs. 4589±1232 IE/kg, P=0.03). However, the glycemic response to oral glucose tolerance testing, the acute insulin response to glucose, and the acute insulin response to arginine did not differ significantly between islet allograft and autograft recipients. Conclusions. Insulin secretion and glucose excursion were similar in allograft and autograft recipients, despite the latter group receiving less than half as many islets. Better preservation of islet mass in the autograft setting is likely related to the lack of autoimmunity, alloimmunity, and immunosuppressive drug toxicity, highlighting the potential for better outcomes in islet allotransplant for type 1 diabetes mellitus with refinements in immunosuppression.  

4.893           Migration and immunological reaction after the administration of αGalCer-pulsed antigen-presenting cells into the submucosa of patients with head and neck cancer

Kurosaki, M., Horiguchi, S., Yamasaki, K., Uchida, Y., Motohashi, S., Nakayama, T., Sugimoto, A. and Okamoto, Y. Cancer Immunol. Immunother., 60(2), 207-215 (2011)   Background Antigen-presenting cells (APCs) play a crucial role in the induction of immune responses. However, the optimal administration route of tumor-specific APCs for inducing effective immunological responses via cancer immunotherapy remains to be elucidated. Human NKT cells are known to have strong anti-tumor activities and are activated by the specific ligand, namely, α-galactosylceramide (αGalCer). Methods Seventeen patients with head and neck squamous cell carcinoma (HNSCC) were enrolled in this study. Patients received an injection of αGalCer-pulsed APCs into the nasal, or the oral floor submucosa. Then total body image and single photon emission computed tomography (SPECT) images were examined. The immunological responses including the number of peripheral blood NKT cells, anti-tumor activities and the CD4⁺ CD25^high Foxp3⁺ T cells (Tregs) induced following APCs were also compared. Results APCs injected into the nasal submucosa quickly migrated to the lateral lymph nodes and those injected into the oral floor submucosa dominantly migrated to the submandibular nodes rather than the lateral lymph nodes. An increase in the absolute number of NKT cells and the IFN-γ producing cells was observed in peripheral blood after injection of the APCs into the nasal submucosa, however, these anti-tumor activities were not detected and the increased frequency of Treg cells were observed after administration into oral floor. Conclusions These results indicate that a different administration route of APCs has the potential to bring a different immunological reaction. The submucosal administration of αGalCer into the oral submucosa tends to induce immunological suppression.  

4.894           COX-2 Inhibition and Inhibition of Cytosolic Phospholipase A2 Increase CD36 Expression and Foam Cell Formation in THP-1 Cells

Anwar, K., Voloshyna, I., Littlefield, M.J., Carsons, S.E., Wirkowski, P.A., jaber, N.L., Sohn, A., Eapen, S. and Reiss, A.B. Lipids, 46(2), 131-142 (2011)   Cardiovascular safety of cyclooxygenase (COX)-2-selective inhibitors and nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) is of worldwide concern. COX-2 inhibitors and NSAIDs act by inhibiting arachidonic acid metabolism to prostaglandins. They confer a cardiovascular hazard manifested as an elevated risk of myocardial infarction. Mechanisms underlying these cardiovascular effects are uncertain. Here we determine whether interference with cytosolic phospholipase A2 (cPLA-2) or COX-2 through pharmacologic blockade or silencing RNA impacts expression of scavenger receptor CD36 and scavenger receptor A, both involved in cholesterol uptake in monocytes and macrophages. THP-1 human monocytes and human peripheral blood mononuclear cells were exposed to celecoxib, a COX-2 selective inhibitor currently in clinical use, and to arachidonyl trifluoromethyl ketone (AACOCF3), an arachidonic acid analog that selectively inhibits cPLA-2. Celecoxib and AACOCF3 each upregulated expression of CD36, but not scavenger receptor A, as determined by quantitative PCR and immunoblotting. Silencing of cPLA-2 or COX-2 had comparable effects to pharmacologic treatments. Oil red O staining revealed a profound increase in foam cell transformation of THP-1 macrophages exposed to either celecoxib or AACOCF3 (both 25 μM), supporting a role for the COX pathway in maintaining macrophage cholesterol homeostasis. Demonstration of disrupted cholesterol balance by AACOCF3 and celecoxib provides further evidence of the possible mechanism by which COX inhibition may promote lipid overload leading to atheromatous lesion formation and increased cardiovascular events.  

4.895           Loss of Caspase-2-dependent Apoptosis Induces Autophagy after Mitochondrial Oxidative Stress in Primary Cultures of Young Adult Cortical Neurons

Tiwari, M., Lopez-Cruzaqn, M., Morgan, W.W. and Herman, B.

1. Biol. Chem., 286(10), 8493-8506 (2011)

  Mitochondrial dysfunctions have been associated with neuronal apoptosis and are characteristic of neurodegenerative conditions. Caspases play a central role in apoptosis; however, their involvement in mitochondrial dysfunction-induced neuronal apoptosis remains elusive. In the present report using rotenone, a complex I inhibitor that causes mitochondrial dysfunction, we determined the initiator caspase and its role in cell death in primary cultures of cortical neurons from young adult mice (1–2 months old). By pretreating the cells with a cell-permeable, biotinylated pan-caspase inhibitor that irreversibly binds to and traps the active caspase, we identified caspase-2 as an initiator caspase activated in rotenone-treated primary neurons. Loss of caspase-2 inhibited rotenone-induced apoptosis; however, these neurons underwent a delayed cell death by necrosis. We further found that caspase-2 acts upstream of mitochondria to mediate rotenone-induced apoptosis in neurons. The loss of caspase-2 significantly inhibited rotenone-induced activation of Bid and Bax and the release of cytochrome c and apoptosis inducing factor from mitochondria. Rotenone-induced downstream activation of caspase-3 and caspase-9 were also inhibited in the neurons lacking caspase-2. Autophagy was enhanced in caspase-2 knock-out neurons after rotenone treatment, and this response was important in prolonging neuronal survival. In summary, the present study identifies a novel function of caspase-2 in mitochondrial oxidative stress-induced apoptosis in neurons cultured from young adult mice.  

4.896           The N-methyl-D-aspartate-evoked cytoplasmic calcium increase in adult rat dorsal root ganglion neuronal somata was potentiated by substance P pretreatment in a protein kinase C-dependent manner

Castillo, C., Norcini, M., Baquero-Buitrago, J., levavic, D., Medina, R., Montoya-Gacharna. J.V., Blanck, T.J.J., Dubois, M. and Recio-Pinto, E. Neuroscience, 177, 308-320 (2011)   The involvement of substance P (SP) in neuronal sensitization through the activation of the neurokinin-1-receptor (NK1r) in postsynaptic dorsal horn neurons has been well established. In contrast, the role of SP and NK1r in primary sensory dorsal root ganglion (DRG) neurons, in particular in the soma, is not well understood. In this study, we evaluated whether SP modulated the NMDA-evoked transient increase in cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) in the soma of dissociated adult DRG neurons. Cultures were treated with nerve growth factor (NGF), prostaglandin E₂ (PGE₂) or both NGF+PGE₂. Treatment with NGF+PGE₂ increased the percentage of N-methyl-d-aspartate (NMDA) responsive neurons. There was no correlation between the percentage of NMDA responsive neurons and the level of expression of the NR1 and NR2B subunits of the NMDA receptor or of the NK1r. Pretreatment with SP did not alter the percentage of NMDA responsive neurons; while it potentiated the NMDA-evoked [Ca²⁺]_cyt transient by increasing its magnitude and by prolonging the period during which small- and some medium-sized neurons remained NMDA responsive. The SP-mediated potentiation was blocked by the SP-antagonist ([D-Pro⁴, D-Trp^7,9]-SP (4–11)) and by the protein kinase C (PKC) blocker bisindolylmaleimide I (BIM); and correlated with the phosphorylation of PKCε. The Nk1r agonist [Sar⁹, Met(O₂)¹¹]-SP (SarMet-SP) also potentiated the NMDA-evoked [Ca²⁺]_cyt transient. Exposure to SP or SarMet-SP produced a rapid increase in the labeling of phosphorylated-PKCε. In none of the conditions we detected phosphorylation of the NR2B subunit at Ser-1303. Phosphorylation of the NR2B subunit at Tyr1472 was enhanced to a similar extent in cells exposed to NMDA, SP or NMDA+SP, and that enhancement was blocked by BIM. Our findings suggest that NGF and PGE₂ may contribute to the injury-evoked sensitization of DRG neurons in part by enhancing their NMDA-evoked [Ca²⁺]_cyt transient in all sized DRG neurons; and that SP may further contribute to the DRG sensitization by enhancing and prolonging the NMDA-evoked increase in [Ca²⁺]_cyt in small- and medium-sized DRG neurons.  

4.897           Nrf2 regulates ferroportin 1-mediated iron efflux and counteracts lipopolysaccharide-induced ferroportin 1 mRNA suppression in macrophages

Harada, N., Kanayama, M., Maruyama, A., Yoshida, A., Tazumi, K., Hosoya, T., Mimura, J., Toki, T., Maher, J.M., Yamamoto, M. and Itoh, K. Arch. Biochem. Biophys., 508, 101-109 (2011)   Iron is an essential element of hemoglobin, and efficient iron recycling from senescent erythrocytes by splenic macrophages is required for erythrocyte hemoglobin synthesis during erythropoiesis. Ferroportin 1 (Fpn1) is the sole iron exporter in mammals, and it also regulates iron reutilization. In this study, we demonstrated genetically that a redox-sensitive transcription factor, Nrf2, regulates Fpn1 mRNA expression in macrophages. Nrf2 activation by several electrophilic compounds commonly resulted in the upregulation of Fpn1 mRNA in bone marrow-derived and peritoneal macrophages obtained from wild-type mice but not from Nrf2 knockout mice. Further, Nrf2 activation enhanced iron release from the J774.1 murine macrophage cell line. Previous studies showed that inflammatory stimuli, such as LPS, downregulates macrophage Fpn1 by transcriptional and hepcidin-mediated post-translational mechanisms leading to iron sequestration by macrophages. We showed that two Nrf2 activators, diethyl maleate and sulforaphane (SFN; a natural Nrf2 activator found in broccoli), restored the LPS-induced suppression of Fpn1 mRNA in human and mouse macrophages, respectively. Furthermore, SFN counteracted the LPS-induced increase of Hepcidin mRNA by an Nrf2-independent mechanism in mouse peritoneal macrophages. These results demonstrate that Nrf2 regulates iron efflux from macrophages through Fpn1 gene transcription and suggest that Nrf2 may control iron metabolism during inflammation.  

4.898           Scavenger receptor CD36 mediates uptake of high density lipoproteins in mice and by cultured cells

Brundert, M., Heeren, J., Merkel, M., Carambia, A., herkel, J., Groitl, P., Dobner, T., Ramakrishnan, R., Moore, K.J. and Rinninger, F.

1. Lipid Res., 52, 745-758 (2011)

  The mechanisms of HDL-mediated cholesterol transport from peripheral tissues to the liver are incompletely defined. Here the function of scavenger receptor cluster of differentiation 36 (CD36) for HDL uptake by the liver was investigated. CD36 knockout (KO) mice, which were the model, have a 37% increase (P = 0.008) of plasma HDL cholesterol compared with wild-type (WT) littermates. To explore the mechanism of this increase, HDL metabolism was investigated with HDL radiolabeled in the apolipoprotein (¹²⁵I) and cholesteryl ester (CE, [³H]) moiety. Liver uptake of [³H] and ¹²⁵I from HDL decreased in CD36 KO mice and the difference, i. e. hepatic selective CE uptake ([³H]¹²⁵I), declined (–33%, P = 0.0003) in CD36 KO compared with WT mice. Hepatic HDL holo-particle uptake (¹²⁵I) decreased (–29%, P = 0.0038) in CD36 KO mice. In vitro, uptake of ¹²⁵I-/[³H]HDL by primary liver cells from WT or CD36 KO mice revealed a diminished HDL uptake in CD36-deficient hepatocytes. Adenovirus-mediated expression of CD36 in cells induced an increase in selective CE uptake from HDL and a stimulation of holo-particle internalization. In conclusion, CD36 plays a role in HDL uptake in mice and by cultured cells. A physiologic function of CD36 in HDL metabolism in vivo is suggested.  

4.899           Prolonged survival of E coli but not Staphylococcus aureus in monocytes from patients with Crohn's disease

Elliott, T.R., Hudspith, b., Karaiskos, C., Rayment, N. and Sanderson, J.D. Gut, 60, A61-A62 (2011)   Introduction A primary macrophage defect has been proposed to play a role in Crohn's disease (CD) pathogenesis.1 In CD, Escherichia coli persist within lamina propria macrophages and peripheral blood monocytes are unable to effectively kill E coli in vitro.2 3 It is uncertain whether this abnormal bacterial handling by monocytes and macrophages in CD is limited to certain strains of E coli or whether a broader bacterial killing defect exists. The aim of this study was to compare intracellular survival of E coli with Staphylococcus aureus (not a usual resident of gut flora) within CD-derived and control monocytes. Methods Peripheral blood was taken from eight healthy controls and nine CD patients. CD distribution was ileal (n=1) and ileocolonic (n=8). Three CD patients were on thiopurines. Monocyte isolation was performed by iodixanol barrier flotation. Monocytes were challenged with a CD-derived strain of E coli and methicillin sensitive Staphylococcus aureus (MSSA). After 1 h incubation, the gentamicin protection assay was performed. Monocytes were lysed to release internalized bacteria after further incubation for 1 and 4 h. Cell lysates were plated on agar and incubated for 24 h at 37°C. Colony forming units (CFU) were counted after both 1 and 4h incubation. The CFU counts at these time points allowed relative replication to be determined. Results Viable CD-derived E coli were cultured from cell lysates at 1 and 4 h from 9/9 CD patients, and in 3/8 HC's (mean increase CFUs from 1 to 4 h: CD +394%, HC −7%). Viable S aureus was cultured in cell lysates at 1 and 4 h respectively from 1/9 and 0/9 CD patients and 0/8 healthy controls at both time points. The difference in the number of CFU's present for each subject at 1 and 4 h was calculated and there was a statistically significant difference in mean rank of (CFU (4 h) – CFU (1 h)) between CD and HC's for E coli but not for staph aureus (Mann–Whitney U test). Conclusion Within CD-derived peripheral monocytes, intracellular survival is prolonged for E coli but not for S aureus, illustrating that impairment of killing by monocytes in CD is restricted to particular bacteria. This is consistent with the clinical observation that CD primarily affects the gut which has an extensive but specific microbiome, rather than causing systemic immunodeficiency characterised by infections in other systems.  

4.900           Prediction of Pancreatic Tissue Densities by an Analytical Test Gradient System Before Purification Maximizes Human Islet Recovery for Islet Autotransplantation/Allotransplantation

Anazawa, T., Matsumoto, S., Yonekawa, Y., Loganathan, G., Wilhelm, J.J., Soltani, S.M., Papas, K.K., Sutherland, D.E.R., hering, B.J. and Balamurugan, A.N. Transplantation, 91(5), 508-514 (2011)   Background. Using standard density gradient (SDG) ranges for human islet purification frequently results in islet loss and transplantation of lower islet mass. Measuring the densities of islet and acinar tissue beforehand to customize the gradient range for the actual COBE 2991 cell processor (COBE) purification is likely to maximize the recovery of islets. We developed an analytical test gradient system (ATGS) for predicting pancreatic tissue densities before COBE purification to minimize islet loss during purification. Methods. Human islets were isolated from deceased donor (n=30) and chronic pancreatitis pancreata (n=30). Pancreatic tissue densities were measured before purification by the ATGS, and the density gradient range for islet purification in a COBE was customized based on density profiles determined by the ATGS. The efficiency of custom density gradients (CDGs) to recover high islet yield was compared with predefined SDGs. Results. Pancreatic tissue densities from autografts were significantly higher than in allograft preparations. In allograft purifications, a higher proportion of islets were recovered using ATGS-guided CDGs (85.9%±18.0%) compared with the SDG method (69.2%±27.0%; P=0.048). Acinar contamination at 60%, 70%, and 80% cumulative islet yield for allografts was significantly lower in the CDG group. In autograft purifications, more islets were recovered with CDGs (81.9%±28.0%) than SDGs (55.8%±22.8%; P=0.03). CDGs effectively reduced islet loss by minimizing islet sedimentation in the COBE bag. Conclusions. Using ATGS-guided CDGs maximizes the islet recovery for successful transplantations by reducing acinar contamination in allograft preparations and by reducing sedimentation of islets in the COBE bag in autograft preparations.  

4.901           Enhanced proliferation of primary rat type II pneumocytes by Jaagsiekte sheep retrovirus envelope protein

Johnson, C., Jahid, S., Voelker, D.R. and Fan, H. Virology, 412, 349-356 (2011)   Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep. The envelope protein (Env) is the oncogene, as it can transform cell lines in culture and induce tumors in animals, although the mechanisms for transformation are not yet clear because a system to perform transformation assays in differentiated type II pneumocytes does not exist. In this study we report culture of primary rat type II pneumocytes in conditions that favor prolonged expression of markers for type II pneumocytes. Env-expressing cultures formed more colonies that were larger in size and were viable for longer periods of time compared to vector control samples. The cells that remained in culture longer were confirmed to be derived from type II pneumocytes because they expressed surfactant protein C, cytokeratin, displayed alkaline phosphatase activity and were positive for Nile red. This system will be useful to study JSRV Env in the targets of transformation.  

4.902           Characterization of mouse sperm TMEM190, a small transmembrane protein with the trefoil domain: evidence for co-localization with IZUMO1 and complex formation with other sperm proteins

Nishimura, H., Gupta, S., Myles, D.G. and Primakoff, P. Reproduction, 141, 437-451 (2011)   TMEM190, a small transmembrane protein containing the trefoil domain, was previously identified by our proteomic analysis of mouse sperm. Two structural features of TMEM190, ‘trefoil domain’ and ‘small transmembrane protein’, led us to hypothesize that this protein forms a protein–protein complex required during fertilization, and we characterized TMEM190 by biochemical, cytological, and genetic approaches. We showed in this study that the mouse Tmem190 gene exhibits testis-specific mRNA expression and that the encoded RNA is translated into a 19-kDa protein found in both testicular germ cells and cauda epididymal sperm. Treatment of the cell surface with proteinase K, subcellular fractionation, and immunofluorescence assay all revealed that mouse TMEM190 is an inner-acrosomal membrane protein of cauda epididymal sperm. During the acrosome reaction, TMEM190 partly relocated onto the surface of the equatorial segment, on which sperm–oocyte fusion occurs. Moreover, TMEM190 and IZUMO1, which is an immunoglobulin-like protein required for gamete fusion, co-localized in mouse sperm both before and after the acrosome reaction. However, immunoprecipitates of TMEM190 contained several sperm proteins, but did not include IZUMO1. These findings suggest that a mouse sperm protein complex(es) including TMEM190 plays an indirect role(s) in sperm–oocyte fusion. The role(s), if any, is probably dispensable since Tmem190-null male mice were normally fertile.  

4.903           Co-adjuvant effects of retinoic acid and IL-15 induce inflammatory immunity to dietary antigens

DePaolo, R.W., Abadie, V., Tang, F., Fehlner-Peach, H., Hall, J.A., Wang, W., Marietta, E.V., Kaserda, D.D., Waldmann, T.A., Murray, J.A., Semrad, C., Kupfer, S.S., Belkaid, Y., Guandalini, S. and Jabri, B. Nature, 471, 220-225 (2011)   Under physiological conditions the gut-associated lymphoid tissues not only prevent the induction of a local inflammatory immune response, but also induce systemic tolerance to fed antigens^{1, 2}. A notable exception is coeliac disease, where genetically susceptible individuals expressing human leukocyte antigen (HLA) HLA-DQ2 or HLA-DQ8 molecules develop inflammatory T-cell and antibody responses against dietary gluten, a protein present in wheat³. The mechanisms underlying this dysregulated mucosal immune response to a soluble antigen have not been identified. Retinoic acid, a metabolite of vitamin A, has been shown to have a critical role in the induction of intestinal regulatory responses^{4, 5, 6}. Here we find in mice that in conjunction with IL-15, a cytokine greatly upregulated in the gut of coeliac disease patients^{3, 7}, retinoic acid rapidly activates dendritic cells to induce JNK (also known as MAPK8) phosphorylation and release the proinflammatory cytokines IL-12p70 and IL-23. As a result, in a stressed intestinal environment, retinoic acid acted as an adjuvant that promoted rather than prevented inflammatory cellular and humoral responses to fed antigen. Altogether, these findings reveal an unexpected role for retinoic acid and IL-15 in the abrogation of tolerance to dietary antigens.  

4.904           MyD88-dependent pathway is essential for the innate immunity to Enterocytozoon bieneusi

Zhinag, Q., Feng, X., Nie, W., Golenbock, D.T., Mayanja-Kizza, H., Tzipori, S. and Feng, H. Parasite Immunol., 33, 217-225 (2011)   Enterocytozoon bieneusi is clinically the most significant microsporidian parasite associated with persistent diarrhoea, wasting and cholangitis in 30–50% of individuals with HIV/AIDS, as well as in malnutritional children and in the recipients of immunosuppressive therapy. However, the host immune responses to E. bieneusi have not been investigated until recently because of lack of sources of spores, cell culture system and animal models. In this study, we purified spores from heavily infected human or monkey faeces by serial salt-Percoll-sucrose-iodixanol centrifugation, and the purity of spores was confirmed by FACS and scanning electron microscopy. Exposure of dendritic cells to E. bieneusi spores induced the upregulation of the surface markers and production of pro-inflammatory cytokines. The cytokine production was independent of toll-like receptor 4, but MyD88 dependent, because dendritic cells from MyD88 knockout mice failed to secrete these pro-inflammatory cytokines, whereas dendritic cells from C3H/HeJ (a toll-like receptor 4 mutant) were activated by E. bieneusi and secreted these cytokines. Furthermore, MyD88-deficient mice were susceptible to E. bieneusi infection, in contrast to wild-type mice that resisted the infection. Collectively, the data demonstrate innate recognition of E. bieneusi by dendritic cells and the importance of MyD88-dependent signalling in resisting infection in a murine challenge model.  

4.905           Activation of the mitochondrial permeability transition pore modulates Ca²⁺ responses to physiological stimuli in adult neurons

Barsukova, A., Komarov, A., Hajnoczky, G., Bernardi, P,m Bourdette, D. and Forte, M. Eur. J. Neurosci., 33, 831-842 (2011)   The participation of mitochondria in cellular and neuronal Ca²⁺ homeostatic networks is now well accepted. Yet, critical tests of specific mitochondrial pathways in neuronal Ca²⁺ responses have been hampered because the identity of mitochondrial proteins that must be integrated within this dynamic system remain uncertain. One putative pathway for Ca²⁺ efflux from mitochondria exists through the formation of the permeability transition pore (PTP) that is often associated with cellular and neuronal death. Here, we have evaluated neuronal Ca²⁺ dynamics and the PTP in single adult neurons in wild-type mice and those missing cyclophilin D (CyPD), a key regulator of the PTP. Using high-resolution time-lapse imaging, we demonstrate that PTP opening only follows simultaneous activation with two physiological stimuli that generate critical threshold levels of cytosolic and mitochondrial Ca²⁺. Our results are the first to demonstrate CyPD-dependent PTP opening in normal neuronal Ca²⁺ homeostatic mechanisms not leading to activation of cell death pathways. As neurons in mice lacking CyPD are protected in a number of neurodegenerative disease models, the results suggest that improved viability of CyPD-knockout animals in these pathological states may be due to the transient, rather than persistent, activation of the PTP in mutant mitochondria, thereby shielding neurons from cytoplasmic Ca²⁺ overload.  

4.906           Isolation, Characterization, and Expansion Methods for Defined Primary Renal Cell Populations from Rodent, Canine, and Human Normal and Diseased Kidneys

Presnell, S.C., Bruce, A.T., Wallace, S.M., Choudury, S., Genheimer, C.W., Cox, B., Guthrie, K., Werdin, E.S., Tatsumi-Ficht, P., Ilagan, R.M., Kelley, R.W., Rivera, E.A., ludlow, J.W., Wagner, B.J., Jayo, M.J. and Bertram, T.A. Tissue Engineering: Part C, 17(3), 261-273 (2011)   Chronic kidney disease (CKD) is a global health problem; the growing gap between the number of patients awaiting transplant and organs actually transplanted highlights the need for new treatments to restore renal function. Regenerative medicine is a promising approach from which treatments for organ-level disorders (e.g., neurogenic bladder) have emerged and translated to clinics. Regenerative templates, composed of biodegradable material and autologous cells, isolated and expanded ex vivo, stimulate native-like organ tissue regeneration after implantation. A critical step for extending this strategy from bladder to kidney is the ability to isolate, characterize, and expand functional renal cells with therapeutic potential from diseased tissue. In this study, we developed methods that yield distinct subpopulations of primary kidney cells that are compatible with process development and scale-up. These methods were translated to rodent, large mammal, and human kidneys, and then to rodent and human tissues with advanced CKD. Comparative in vitro studies demonstrated that phenotype and key functional attributes were retained consistently in ex vivo cultures regardless of species or disease state, suggesting that autologous sourcing of cells that contribute to in situ kidney regeneration after injury is feasible, even with biopsies from patients with advanced CKD.  

4.907           Porcine CTLA4-Ig prolong islet xenografts in rats by downregulating the direct pathway of T-cell activation

Zhai, C., Yu, L., Zhu, H., Tian, M., Xiaogang, Z. and Bo, W. Xenotransplantation, 18, 40-45 (2011)   Abstract: Aim: Porcine pancreatic islets fused with pCTLA4-Ig were transplanted into diabetic rats. Xenografts survival was observed, and the underlying immunological rejection mechanisms were investigated. Methods: Control porcine islets, empty vector (Adv-GFP)–transfected, and gene-modified porcine islets were transplanted into the renal capsule of diabetic rats. The survival rates of the xenografts were observed. Changes in serum levels of IL-4 and -IFN in the recipients were assessed. Results: The survival time of xenografts in the gene-modified porcine islets group was 34.50 ± 4.14 days, which was longer than those in the control group (34.50 ± 4.14 days vs. 7.43 ± 1.72 days and 7.22 ± 1.72 days; P < 0.01). Changes in the serum levels of IL-4 and -IFN between the groups of rats post-transplantation indicated the differentiation bias of T helper cells. Conclusions: The donor-originated pCTLA-IgG4 fusion protein inhibits the direct pathway of recipient T-cell priming, which might prolong xenograft survival.  

4.908           Tristetraprolin-dependent Post-transcriptional Regulation of Inflammatory Cytokine mRNA Expression by Apolipoprotein A-I: ROLE OF ATP-BINDING MEMBRANE CASSETTE TRANSPORTER A1 AND SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 3

Yin, K., Deng, X., Mo, Z-C., Zhao, G-J., Jiang, J., Cui, L-B., Tan, C-Z., Wen, G-B., Fu, Y. and Tang, C-K.

1. Biol. Chem., 286(16), 13834-13845 (2011)

Atherosclerosis is an inflammatory disease characterized by the accumulation of macrophages in the arterial intima. The activated macrophages secreted more pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, which promote the development of the disease. Apolipoprotein A-I (apoA-I), the major component of high density lipoprotein, is involved in reverse cholesterol transport of lipid metabolism. Recently, it has been found that apoA-I suppresses inflammation via repression of inflammatory cytokine expression; the mechanisms of the apoA-I-suppressive action, however, are not yet well characterized. In this study, we have for the first time found that apoA-I suppresses the expression of some inflammatory cytokines induced by lipopolysaccharide via a specific post-transcriptional regulation process, namely mRNA destabilization, in macrophages. Our further studies have also shown that AU-rich elements in the 3′-untranslated region of TNF-α mRNA are responsive to the apoA-I-mediated mRNA destabilization. The apoA-I-induced inflammatory cytokine mRNA destabilization was associated with increased expression of mRNA-destabilizing protein tristetraprolin through a JAK2/STAT3 signaling pathway-dependent manner. When blocking interaction of apoA-I with ATP-binding membrane cassette transporter A1 (ABCA1), a major receptor for apoA-I in macrophages, it would almost totally abolish the effect of apoA-I on tristetraprolin expression. These results present not only a novel mechanism for the apoA-I-mediated inflammation suppression in macrophages but also provide new insights for developing strategies for modulating vascular inflammation and atherosclerosis.  

4.909           Bovine Plasmacytoid Dendritic Cells Are the Major Source of Type I Interferon in Response to Foot-and-Mouth Disease Virus In Vitro and In Vivo

Reid, E., Juleff, N., Gubbins, S., Prentice, H., Seago, J and Charleston, B.

1. Virol., 85(9), 4297-4308 (2011)

  Type I interferons (alpha/beta interferons [IFN-α/β]) are the main innate cytokines that are able to induce a cellular antiviral state, thereby limiting viral replication and disease pathology. Plasmacytoid dendritic cells (pDCs) play a crucial role in the control of viral infections, especially in response to viruses that have evolved mechanisms to block the type I IFN signal transduction pathway. Using density gradient separation and cell sorting, we have highly enriched a population of bovine cells capable of producing high levels of biologically active type I IFN. These cells represented less than 0.1% of the total lymphocyte population in blood, pseudoafferent lymph, and lymph nodes. Phenotypic analysis identified these cells as bovine pDCs (CD3– CD14– CD21– CD11c– NK– TCR – CD4+ MHC II+ CD45RB+ CD172a+ CD32+). High levels of type I IFN were generated by these cells in vitro in response to Toll-like receptor 9 (TLR-9) agonist CpG and foot-and-mouth disease virus (FMDV) immune complexes. In contrast, immune complexes formed with UV-inactivated FMDV or FMDV empty capsids failed to elicit a type I IFN response. Depletion of CD4 cells in vivo resulted in levels of type I IFN in serum early during FMDV infection that were significantly lower than those for control animals. In conclusion, pDCs interacting with immune-complexed virus are the major source of type I interferon production during acute FMDV infection in cattle.  

4.910           Surfactant protein-A and toll-like receptor-4 modulate immune functions of preterm baboon lung dendritic cell precursor cells

Awasthi, S., Madhusoodhanan, R. and Wolf, R. Cell. Immunol., 268, 87-96 (2011)   Lung infections are important risk factors for an increased morbidity and mortality in prematurely-delivered babies. Immaturity of the innate immune components makes them extremely susceptible to infection. Recently, we isolated lung dendritic cell (DC)-precursor cells from preterm fetal baboons. The isolated cells were found to be defective in phagocytosing Escherichia coli under basal conditions. In this study, we investigated the effects of exogenously-added purified native lung surfactant protein (SP)-A and recombinant toll-like receptor (TLR)-4-MD2 proteins on phagocytic uptake and cytokine secreting ability of fetal baboon lung DC-precursor cells. The cells were pulsed with SP-A and/or TLR4-MD2 proteins and the phagocytic function was investigated by incubating the cells with fluorescent-labeled E. coli bioparticles and analyzed by spectrofluorometry. The amounts of TNF-α secreted in cell-free supernatants were measured by ELISA. Our results demonstrate that SP-A and TLR4-MD2 proteins, whether added alone or together, induce phagocytosis of E. coli (p < 0.05). The SP-A does not affect TNF-α secretion, while the TLR4-MD2 protein induces TNF-α. However, simultaneous addition of SP-A with TLR4-MD2 protein reduces the TLR4-MD2-protein induced TNF-α to basal level. In conclusion, our results indicate that an exogenous administration of SP-A can potentially induce phagocytic activity and anti-inflammatory effect in preterm babies, and help control infection and inflammation. Keywords: Dendritic cells; Surfactant protein-A; Toll-like receptor-4; Preterm babies; Innate immunity Abbreviations: DC(s), dendritic cell(s); dGA, days of gestation age; D-PBS, Dulbecco’s phosphate buffered saline; FACS, fluorescence activated cell sorting; FBS, fetal bovine serum; FITC, fluorescien isothiocyanate; HBSS, Hanks balanced salt solution; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; MFI, mean fluorescent intensity; PE, phycoerythrinin; SEM, standard error of measurement  

4.911           A new model to study spinal muscular atrophy: Neurite degeneration and cell death is counteracted by BCL-X_L Overexpression in motoneurons

Garcera, A., Mincheva, S., Gou-Fabregas, M., Caraballo-Miralles, V., Llado, J., Comella, J.X. and Soler, R.M. Neurobiology of Disease, 42, 415-426 (2011)   Spinal muscular atrophy (SMA) is a motoneuron disorder characterized by deletions or specific mutations in the Survival Motor Neuron gene (SMN). SMN is ubiquitously expressed and has a general role in the assembly of small nuclear ribonucleoprotein (snRNP) and pre-mRNA splicing requirements. However, in motoneuron axons SMN deficiency results in inappropriate levels of certain transcripts in the distal axon, suggesting that the specific susceptibility of motoneurons to SMN deficiency is related to a specialized function in these cells. Although mouse models of SMA have been generated and are useful for in vivo and in vitro studies, the limited number of isolated MNs that could be obtained from them makes it difficult to perform biochemical, genetic and pharmacological approaches. We describe here an in vitro model of isolated embryonic mouse motoneurons in which the cellular levels of endogenous SMN are reduced. These cells show neurite degeneration and cell death after several days of SMN knockdown. We found that the over-expression of the anti-apoptotic protein Bcl-x_L into motoneurons rescues these cells from the phenotypic changes observed. This result demonstrates that Bcl-x_L signaling could be a possible pharmacological target of SMA therapeutics.  

4.912           Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death

Mukhopadhyay, P., Rajesh, M., Horvath, B., Batkai, S., Park, O., Tanchian, G., Gao, R.Y., Patel, V., Wink, D.A., Liaudet, L., Hasko, G., Mechoulam, R. and Pachet, P. Free Radical Biology & Medicine, 50, 1368-1381 (2011)   Ischemia/reperfusion (I/R) is a pivotal mechanism of liver damage after liver transplantation or hepatic surgery. We have investigated the effects of cannabidiol (CBD), the nonpsychotropic constituent of marijuana, in a mouse model of hepatic I/R injury. I/R triggered time-dependent increases/changes in markers of liver injury (serum transaminases), hepatic oxidative/nitrative stress (4-hydroxy-2-nonenal, nitrotyrosine content/staining, and gp91phox and inducible nitric oxide synthase mRNA), mitochondrial dysfunction (decreased complex I activity), inflammation (tumor necrosis factor α (TNF-α), cyclooxygenase 2, macrophage inflammatory protein-1α/2, intercellular adhesion molecule 1 mRNA levels; tissue neutrophil infiltration; nuclear factor κB (NF-κB) activation), stress signaling (p38MAPK and JNK), and cell death (DNA fragmentation, PARP activity, and TUNEL). CBD significantly reduced the extent of liver inflammation, oxidative/nitrative stress, and cell death and also attenuated the bacterial endotoxin-triggered NF-κB activation and TNF-α production in isolated Kupffer cells, likewise the adhesion molecule expression in primary human liver sinusoidal endothelial cells stimulated with TNF-α and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB₂ knockout mice and were not prevented by CB_1/2 antagonists in vitro. Thus, CBD may represent a novel, protective strategy against I/R injury by attenuating key inflammatory pathways and oxidative/nitrative tissue injury, independent of classical CB_1/2 receptors.  

4.913           Silencing tissue inhibitors of metalloproteinases (TIMPs) with short interfering RNA reveals a role for TIMP-1 in hepatic stellate cell proliferation

Fowell, A.J., Collins, J.E., Duncombe, D.R., Pickering, J.A., Rosenberg, W.M.C. and Benyon, R.C. Biochem. Biophys. Res. Comm., 407, 277-282 (2011)   Myofibroblastic, activated hepatic stellate cells (HSC) play a pivotal role in the development of liver fibrosis through the secretion of fibrillar collagens and the tissue inhibitors of metalloproteinase (TIMP)-1 and -2. TIMPs are believed to promote hepatic fibrosis by inhibiting both matrix degradation and apoptosis of HSC. In other cell types, there is evidence that TIMP-1 has effects on proliferation, however the role of TIMPs in the regulation of HSC proliferation remains unexplored. Therefore, we have used short interfering RNA (siRNA) to investigate the effects of autocrine TIMP-1 and -2 on HSC proliferation. TIMP-1 and -2 siRNA were highly effective, producing peak target protein knockdown compared to negative control siRNA of 92% and 63%, respectively. Specific silencing of TIMP-1, using siRNA, significantly reduced HSC proliferation. TIMP-1 was localised in part to the HSC nucleus and TIMP-1 siRNA resulted in loss of both cytoplasmic and nuclear TIMP-1. Attenuated proliferation was associated with reduced Akt phosphorylation and was partially rescued by addition of recombinant TIMP-1. We have revealed a novel autocrine mitogenic effect of TIMP-1 on HSC, which may involve Akt-dependent and specific nuclear mechanisms of action. We suggest that TIMP-1 might promote liver fibrosis by means other than its previously described anti-apoptotic effect on HSC. Moreover, these findings, together with our previous reports and the emerging data from in vivo studies of TIMP inhibition, provide strong evidence that TIMP-1 is mechanistically central to liver fibrosis and an important potential therapeutic target.  

4.914           GalR2/3 mediates proliferative and trophic effects of galanin on postnatal hippocampal precursors

Abbosh, C., Lawkowski, A., Zaben, M. and Gray, W.

1. Neurochem., 117(3), 425-436 (2011)

  Understanding how neural activity is functionally linked to the stem cell niche, is assuming ever increasing importance as hippocampal neurogenesis is shown to be important for modulating the behavioural responses to stress and for certain forms of learning and memory. Neuropeptides such as neuropeptide Y and vasoactive intestinal peptide have emerged as important mediators for signalling local interneuron activity to subgranular zone precursors, however, little is known regarding the effects of neuropeptides that are extrinsic modulators of hippocampal information processing. Here, we show that the galanin GalR2/3 agonist Gal2–11 is both trophic and proliferative for postnatal subgranular precursors and proliferating neuroblasts at 10 nM and is purely trophic at doses as low as 100 pM. We found no effect mediated via GalR1. As galanin is co-released from noradrenergic and serotonergic projection neurons to the dentate gyrus, these findings support a direct effect of galanin on hippocampal neurogenesis, which may partly mediate its antidepressant effect via GalR2/3 receptors.  

4.915           IFNγ triggers a LIGHT-dependent selective death of motoneurons contributing to the non-cell-autonomous effects of mutant SOD1

Aebischer, J., Cassina, P., Otsmane, B., Moumen, A., Seilhean, D., Meininger, V., Barbeito, L., Pettmann, B. and Raoul, C. Cell Death and Differentiation, 18, 754-768 (2011)   Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that primarily affects motoneurons in the brain and spinal cord. Dominant mutations in superoxide dismutase-1 (SOD1) cause a familial form of ALS. Mutant SOD1-damaged glial cells contribute to ALS pathogenesis by releasing neurotoxic factors, but the mechanistic basis of the motoneuron-specific elimination is poorly understood. Here, we describe a motoneuron-selective death pathway triggered by activation of lymphotoxin-β receptor (LT-βR) by LIGHT, and operating by a novel signaling scheme. We show that astrocytes expressing mutant SOD1 mediate the selective death of motoneurons through the proinflammatory cytokine interferon-γ (IFNγ), which activates the LIGHT-LT-βR death pathway. The expression of LIGHT and LT-βR by motoneurons in vivo correlates with the preferential expression of IFNγ by motoneurons and astrocytes at disease onset and symptomatic stage in ALS mice. Importantly, the genetic ablation of Light in an ALS mouse model retards progression, but not onset, of the disease and increases lifespan. We propose that IFNγ contributes to a cross-talk between motoneurons and astrocytes causing the selective loss of some motoneurons following activation of the LIGHT-induced death pathway.  

4.916           The Canonical Nuclear Factor- B Pathway Regulates Cell Survival in a Developmental Model of Spinal Cord Motoneurons

Mincheva, S., Garcera, A., Gou-Fabregas, M., Encinas, M., Dolcet, X. and Soler, R.M.

1. Neurosci., 31(17), 6493-6503 (2011)

  In vivo and in vitro motoneuron survival depends on the support of neurotrophic factors. These factors activate signaling pathways related to cell survival or inactivate proteins involved in neuronal death. In the present work, we analyzed the involvement of the nuclear factor-κB (NF-κB) pathway in mediating mouse spinal cord motoneuron survival promoted by neurotrophic factors. This pathway comprises ubiquitously expressed transcription factors that could be activated by two different routes: the canonical pathway, associated with IKKα/IKKβ kinase phosphorylation and nuclear translocation RelA (p65)/p50 transcription factors; and the noncanonical pathway, related to IKKα kinase homodimer phosphorylation and RelB/p52 transcription factor activation. In our system, we show that neurotrophic factors treatment induced IKKα and IKKβ phosphorylation and RelA nuclear translocation, suggesting NF-κB pathway activation. Protein levels of different members of the canonical or noncanonical pathways were reduced in a primary culture of isolated embryonic motoneurons using an interference RNA approach. Even in the presence of neurotrophic factors, selective reduction of IKKα, IKKβ, or RelA proteins induced cell death. In contrast, RelB protein reduction did not have a negative effect on motoneuron survival. Together these results demonstrated that the canonical NF-κB pathway mediates motoneuron survival induced by neurotrophic factors, and the noncanonical pathway is not related to this survival effect. Canonical NF-κB blockade induced an increase of Bim protein level and apoptotic cell death. Bcl-x_L overexpression or Bax reduction counteracted this apoptotic effect. Finally, RelA knockdown causes changes of CREB and Smn protein levels.  

4.917           The Inhibition of Neutrophil Elastase Ameliorates Islet Yield and Islet Graft Survival

Tanemura, M., Machida, T., Nagano, H., Wada, S., Kobayashi, S., Murabashi, S., Eguchi, H., Ito, T., Mori, M. and Doki, Y. Am. J. Transplant., 11, 249 (2011)   Introduction: One important key to achieve successful insulin-independence after islet transplantation (ITx) is acquiring a suffi cient donor islet mass. Activated neutrophils play an important role for acute tissue injury in transplant organs. Neutrophil elastase (NE), released from activated neutrophils can directly cause tissue injury. We focused on the crucial role of NE in the cytotoxic effects against islets during islet isolation and after ITx. Objectives: The objectives are to determine whether the addition of the specifi c NE inhibitor, sivelestat (Si) into the islet isolation solution could improve islet yield and, to investigate the cytoprotective effects of the administration of Si in islet recipients. Materials and Methods: Islet isolation: Anesthetized male C57BL/6 mice underwent bile duct cannulation with pancreatic inflation using either UW, UW containing 20 μM of Si (ie: S-UW), ET-Kyoto or ET-Kyoto containing 20 μM of Si (ie: S-Kyoto). The infl ated pancreas was digested with collagenase, followed by purifi cation using a discontinuous Iodixanol gradient. Assessment of islet viability: Fluorescence labeling with TMRE was performed. Glucose-Stimulated Insulin Release: To assess in vitro potency of isolated islets, static glucose change was performed. Measurement of NE activity: NE activity during isolation was measured by the incubation with N-methoxysuccinyl-Ala-Ala- Pro-Val-p-nitroanilide as the substrate. IT experiments: The diabetic recipient, male Balb c/A mice received 500 of allogeneic islets isolated from C57BL/6J mice under kidney capsule. To assess the benefi cial effects of Si, intraperitoneal administration of 100 mg/kg of Si per one day was performed at 1 day before ITx and every day until 14 day after ITx. Results: Data are summarized in the Table. Conclusion: NE inhibitor (ie: sivelestat) treatment should be considered a potential strategy for recovering transplantable islets and long-term islet allograft survival, and patients may be cured with islets from one donor.  

4.918           Attenuation of microglial and IL-1 signaling protects mice from acute alcohol-induced sedation and/or motor impairment

Wu, Y., Lousberg, E.L., Moldenhauer, L.M., Hayball, J.D., Robertson, S.A., Coller, J.K., Watkins, L.R., Somogyi, A.A. and Hutchinson, M.R. Brain, Behavior, and immunity, 25, 5155-5164 (2011)   Alcohol-induced proinflammatory central immune signaling has been implicated in the chronic neurotoxic actions of alcohol, although little work has examined if these non-neuronal actions contribute to the acute behavioral responses elicited by alcohol administration. The present study examined if acute alcohol-induced sedation (loss of righting reflex, sleep time test) and motor impairment (rotarod test) were influenced by acute alcohol-induced microglial-dependent central immune signaling. Inhibition of acute alcohol-induced central immune signaling, through the reduction of proinflammatory microglial activation with minocycline, or by blocking interleukin-1 (IL-1) receptor signaling using IL-1 receptor antagonist (IL-1ra), reduced acute alcohol-induced sedation in mice. Mice treated with IL-1ra recovered faster from acute alcohol-induced motor impairment than control animals. However, minocycline led to greater motor impairment induced by alcohol, implicating different mechanisms in alcohol-induced sedation and motor impairment. At a cellular level, IκBα protein levels in mixed hippocampal cells responded rapidly to alcohol in a time-dependent manner, and both minocycline and IL-1ra attenuated the elevated levels of IκBα protein by alcohol. Collectively these data suggest that alcohol is capable of rapid modification of proinflammatory immune signaling in the brain and this contributes significantly to the pharmacology of alcohol.  

4.919           Immunological tolerance in a mouse model of immune-mediated liver injury induced by 16,16 dimethyl PGE2 and PGE2-containing nanoscale hydrogels

Okamoto, T., Saito, T., Tabata, Y. and Uemoto, S. Biomaterials, 32, 4925-4935 (2011)   Although immunosuppressive agents play a pivotal role in the success of organ transplantation, chronic toxicity has been a major issue for long-term treatment. The development of therapies that induce donor-specific immunological tolerance remains an important clinical challenge. In the present study, we investigated the underlying mechanisms and applications of prostaglandin (PG) E2 for the induction of immunological tolerance in mice with concanavalin A(Con A)-induced immune-mediated liver injury. The immunological tolerogenic effect of 16,16 dimethyl PGE2 (dmPGE2) pretreatment in C57B/6 male mice with Con A-induced liver injury was observed, and it was revealed that its response was partially associated with the expression of interleukin (IL)-10, an anti-inflammatory cytokine, in Kupffer cells. To apply native eicosanoids of PGE2 for tolerance induction in vivo, PGE2 was incorporated into l-lactic acid oligomer-grafted pullulan of an amphiphilic polymer to form a nano-sized hydrogel (PGE2-nanogel). Pharmacokinetics studies revealed that nanogel incorporation enabled PGE2 to have a prolonged life-time in circulating blood, and a tolerogenic effect was also observed in Con A-induced liver injury, the same as with dmPGE2 pretreatment. Nanogel-based prostaglandin administration might be developed as a therapeutic agent to induce immunological tolerance, which is necessary in allogenic organ and cell transplantation.  

4.920           New Insight into the Antifibrotic Effects of Praziquantel on Mice in Infection with Schistosoma japonicum

Liang, Y-J., Luo, J., Yuan, Q., Zheng, D., Liu, Y-P., Shi, L., Zhou, Y., Chen, A-L., Ren, Y-Y., Sun, K-Y., Sun, Y., Wang, T. and Zhang, Z-S. PloS One, 6(5), e20247 (2011)   Background Schistosomiasis is a parasitic disease infecting more than 200 million people in the world. Although chemotherapy targeting on killing schistosomes is one of the main strategies in the disease control, there are few effective ways of dealing with liver fibrosis caused by the parasite infection in the chronic and advanced stages of schistosomiasis. For this reason, new strategies and prospective drugs, which exert antifibrotic effects, are urgently required. Methods and Findings The antifibrotic effects of praziquantel were assessed in the murine models of schistosomiasis japonica. Murine fibrosis models were established by cutaneous infection with 14±2 Schistosoma japonicum cercariae. Then, the mice of both chronic (8 weeks post-infection) and advanced (15 weeks post-infection) schistosomiasis were treated by gavage of praziquantel (250 mg/kg, once daily for 3 days) to eliminate worms, and followed by praziquantel anti-fibrosis treatment (300 mg/kg, twice daily for 30 days). The fibrosis-related parameters assessed were areas of collagen deposition, content of hydroxyproline and mRNA expressions of Col1α1, Col3α1, α-SMA, TGF-β, MMP9, TIMP1, IL-4, IL-10, IL-13 and IFN-γ of liver. Spleen weight index, alanine aminotransferase activity and liver portal venous pressure were also measured. The results showed that anti-fibrosis treatment improved liver fibrosis, splenomegaly, hepatic function, as well as liver portal hypertension. In order to confirm the anti-fibrotic properties of praziquantel, we established a CCL4-induced model and revealed that CCL4-induced liver fibrosis was inhibited by PZQ treatment for 30 days. Furthermore, we analyzed the effects of praziquantel on mouse primary hepatic stellate cells (HSCs). It is indicated that mRNA expressions of Col1α1, Col3α1, α-SMA, TGF-β, MMP9 and TIMP1 of HSCs were all inhibited after praziquantel anti-parasite treatments. Conclusions The significant amelioration of hepatic fibrosis by praziquantel treatment validates it as a promising drug of anti-fibrosis and offers potential of a new chemotherapy for hepatic fibrosis resulting from schistosomiasis.  

4.921           Cellular and intracellular distribution of growth hormone in the adult chicken testis

Martinez-Moreno, C.G., Palma, L., Carranza, M., Harvey, S., Aramburo, C. and Luna, M. Gen. Comp. Endocrinol., 172, 344-357 (2011)   Endocrine actions of growth hormone (GH) have been implicated during the development of adult testicular function in several mammalian species, and recently intracrine, autocrine, and paracrine effects have been proposed for locally expressed GH. Previous reports have shown the distribution of GH mRNA and the molecular heterogeneity of GH protein in both adult chicken testes and vas deferens. This study provides evidence of the presence and distribution of GH and its receptor (GHR) during all stages of spermatogenesis in adult chicken testes. This hormone and its receptor are not restricted to the cytoplasm; they are also found in the nuclei of spermatogonia, spermatocytes, and spermatids. The pattern of GH isoforms was characterized in the different, isolated germ cell subpopulations, and the major molecular variant in all subpopulations was 17 kDa GH, as reported in other chicken extra-pituitary tissues. Another molecular variant, the 29 kDa moiety, was found mainly in the enriched spermatocyte population, suggesting that it acts at specific developmental stages. The co-localization of GH with the proliferative cell nuclear antigen PCNA (a DNA replication marker present in spermatogonial cells) was demonstrated by immunohistochemistry. These results show for the first time that GH and GHR are present in the nuclei of adult chicken germinal cells, and suggest that GH could participate in proliferation and differentiation during the complex process of spermatogenesis.  

4.922           Neuroprotective effect of Nrf2/ARE activators, CDDO ethylamide and CDDO trifluoroethylamide, in a mouse model of amyotrophic lateral sclerosis

Neymotin, A., Calingasan, N:Y., Wille, E., Naseri, N., Petri, S., Damiano, M., Liby, K.T., Risingsong, R., Sporn, M., Beal, M.F. and Kiaei, M. Free Radical Biology & Medicine, 51, 88-96 (2011)   Oxidative damage, neuroinflammation, and mitochondrial dysfunction contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS), and these pathologic processes are tightly regulated by the Nrf2/ARE (NF-E2-related factor 2/antioxidant response element) signaling program. Therefore, modulation of the Nrf2/ARE pathway is an attractive therapeutic target for neurodegenerative diseases such as ALS. We examined two triterpenoids, CDDO (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid) ethylamide and CDDO trifluoroethylamide (CDDO-TFEA), that potently activate Nrf2/ARE in a cell culture model of ALS and in the G93A SOD1 mouse model of ALS. Treatment of NSC-34 cells stably expressing mutant G93A SOD1 with CDDO-TFEA upregulated Nrf2 expression and resulted in translocation of Nrf2 into the nucleus. Western blot analysis showed an increase in the expression of Nrf2/ARE-regulated proteins. When treatment started at a “presymptomatic age” of 30 days, both of these compounds significantly attenuated weight loss, enhanced motor performance, and extended the survival of G93A SOD1 mice. Treatment started at a “symptomatic age,” as assessed by impaired motor performance, was neuroprotective and slowed disease progression. These findings provide further evidence that compounds that activate the Nrf2/ARE signaling pathway may be useful in the treatment of ALS.  

4.923           A5-Positive Primary Sensory Neurons Are Nonpermissive for Productive Infection with Herpes Simplex Virus 1 In Vitro

Bertke, A.S., Swanson, S.M., Chen, J., Imai, Y., Kinchington, P.R. and Margolis, T.P.

1. Virol., 85(13), 6699-6677 (2011)

  Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) establish latency and express the latency-associated transcript (LAT) preferentially in different murine sensory neuron populations, with most HSV-1 LAT expression in A5+ neurons and most HSV-2 LAT expression in KH10+ neurons. To study the mechanisms regulating the establishment of HSV latency in specific subtypes of neurons, cultured dissociated adult murine trigeminal ganglion (TG) neurons were assessed for relative permissiveness for productive infection. In contrast to that for neonatal TG, the relative distribution of A5+ and KH10+ neurons in cultured adult TG was similar to that seen in vivo. Productive infection with HSV was restricted, and only 45% of cultured neurons could be productively infected with either HSV-1 or HSV-2. A5+ neurons supported productive infection with HSV-2 but were selectively nonpermissive for productive infection with HSV-1, a phenomenon that was not due to restricted viral entry or DNA uncoating, since HSV-1 expressing β-galactosidase under the control of the neurofilament promoter was detected in 90% of cultured neurons, with no preference for any neuronal subtype. Infection with HSV-1 reporter viruses expressing enhanced green fluorescent protein (EGFP) from immediate early (IE), early, and late gene promoters indicated that the block to productive infection occurred before IE gene expression. Trichostatin A treatment of quiescently infected neurons induced productive infection preferentially from non-A5+ neurons, demonstrating that the nonpermissive neuronal subtype is also nonpermissive for reactivation. Thus, HSV-1 is capable of entering the majority of sensory neurons in vitro; productive infection occurs within a subset of these neurons; and this differential distribution of productive infection is determined at or before the expression of the viral IE genes.  

4.924           Isolation of Inflammatory Cells from Human Tumours

Polak, M.E. Methods in Mol. Biol., 731, 201-208 (2011)   Inflammatory cells are present in many tumours, and understanding their function is of increasing importance, particularly to studies of tumour immunology. The tumour-infiltrating leukocytes encompass a variety of cell types, e.g. T lymphocytes, macrophages, dendritic cells, NK cells, and mast cells. Choice of the isolation method greatly depends on the tumour type and the leukocyte subset of interest, but the protocol usually includes tissue disaggregation and cell enrichment. We recommend density centrifugation for initial enrichment, followed by specific magnetic bead negative or positive panning with leukocyte and tumour cell selective antibodies.  

4.925           Increased Expression of CD69 on T Cells as an Early Immune Marker for Human Cytomegalovirus Reactivation in Chronic Lymphocytic Leukemia Patients

Petersen, C.C., Nederby, L., Roug, A.S., Skovbo, A., Peterslund, N.A., Hokland, P., Nielsen, B. and Hokland, M. Viral Immunol., 24(2), 165-169 (2011)   Reactivation of human cytomegalovirus (HCMV) remains a serious problem in immunosuppressed individuals. To investigate whether a change in the immune status can be used as an earlier marker for HCMV reactivation than the traditional PCR analysis, eight chronic lymphocytic leukemia (CLL) patients at risk for reactivation due to commencement of alemtuzumab (anti-CD52) treatment were longitudinally followed. Five series of consecutive weekly blood samples were immunophenotyped by flow cytometry to cover both the innate and adaptive immune responses. Concurrently, patients were monitored by PCR for HCMV reactivation. We found a minor upregulation of the early activation marker CD69 on NK cells immediately before HCMV was detected in circulation by PCR. Interestingly, for the specific immune response, CD69 was highly upregulated on CD3⁺ T cells, especially for the CD8⁺ subset, in the two patients experiencing an HCMV reactivation between 6 and 20 d before HCMV viremia was measured by PCR. Moreover, a CD4⁺:CD8⁺ ratio lower than 0.6 may indicate a trend toward an increased risk for viral reactivation. In conclusion, an increase in CD69 expression is a promising candidate as an early predictor of HCMV reactivation.  

4.926           Viral Replication and Innate Host Responses in Primary Human Alveolar Epithelial Cells and Alveolar Macrophages Infected with Influenza H5N1 and H1N1 Viruses

Yu, W.C.L., Chan, R.W.Y., Wang, J., Travanty, E.A., Nicholls, J.M., Peiris, J.S.M., Mason, R.J. and Chan, M.C.W.

1. Virol., 85(14), 6844-6855 (2011)

  Highly pathogenic influenza H5N1 virus continues to pose a threat to public health. Although the mechanisms underlying the pathogenesis of the H5N1 virus have not been fully defined, it has been suggested that cytokine dysregulation plays an important role. As the human respiratory epithelium is the primary target cell for influenza viruses, elucidating the viral tropism and innate immune responses of influenza H5N1 virus in the alveolar epithelium may help us to understand the pathogenesis of the severe pneumonia associated with H5N1 disease. Here we used primary cultures of differentiated human alveolar type II cells, alveolar type I-like cells, and alveolar macrophages isolated from the same individual to investigate viral replication competence and host innate immune responses to influenza H5N1 (A/HK/483/97) and H1N1 (A/HK/54/98) virus infection. The viral replication kinetics and cytokine and chemokine responses were compared by quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). We demonstrated that influenza H1N1 and H5N1 viruses replicated productively in type II cells and type I-like cells although with different kinetics. The H5N1 virus replicated productively in alveolar macrophages, whereas the H1N1 virus led to an abortive infection. The H5N1 virus was a more potent inducer of proinflammatory cytokines and chemokines than the H1N1 virus in all cell types. However, higher levels of cytokine expression were observed for peripheral blood monocyte-derived macrophages than for alveolar macrophages in response to H5N1 virus infection. Our findings provide important insights into the viral tropisms and host responses of different cell types found in the lung and are relevant to an understanding of the pathogenesis of severe human influenza disease.  

4.927           Expression and Function of Fibroblast Growth Factor (FGF) 7 during Liver Regeneration

Tsai, S-M. and Wang, W-P. Cell. Physiol. Biochem., 27(6), 641-652 (2011)   Background/Aim: Previous studies have shown that fibroblast growth factors (FGFs) are involved in the process of liver injury repair. Liver regeneration after partial hepatectomy (PH) is impaired in transgenic mice expressing dominant-negative FGFR2b in hepatocytes. Although FGF7, a ligand specifically bound to FGFR2b, is expressed by activated hepatic stellate cells (HSCs) in fibrotic livers, the expressions and functions of FGF7 and FGFR2b after PH remain unexplored. Therefore, this study sought to examine the potential role of FGF7 signaling during liver regeneration. Methods: We examined the expression of FGF7 and FGFR2b in normal and regenerating livers. Effects of FGF7 on hepatocytes were examined in vitro using primary hepatocyte culture with FGF7 recombinant protein and in vivo by hydrodynamic-based gene transfer method. Results: We found that FGF7 expression was increased according to the activation status of HSCs after PH. The receptor, FGFR2b, was also increased in hepatocytes during liver regeneration. In vitro treatment with FGF7 protein activated ERK1/2 and promoted proliferation of hepatocytes isolated from regenerating livers. In vivo overexpression of exogenous FGF7 could notably promote hepatic proliferation and activate MAPKs after PH. Conclusion: This study suggests a role for activated HSC-expressed FGF7 in stimulating FGF signaling pathways in hepatocytes and regulating liver regeneration.  

4.928           Thymosin-β4 (Tβ4) Blunts PDGF-Dependent Phosphorylation and Binding of AKT to Actin in Hepatic Stellate Cells

Reyes-Gordillo, K., Shah, R., Popratiloff, A., Fu, S., Hindle, A., Brody, F. and Rojkind, M. Am. J. Pathol., 178(5), 2100-2108 (2011)   Hepatic stellate cell transdifferentiation is a key event in the fibrogenic cascade. Therefore, attempts to prevent and/or revert the myofibroblastic phenotype could result in novel therapeutic approaches to treat liver cirrhosis. The expression of platelet-derived growth factor (PDGF)-β receptor and the proliferative response to platelet-derived growth factor-ββ (PDGF-ββ) are hallmarks of the transdifferentiation of hepatic stellate cells (HSC). In this communication, we investigated whether thymosin-β4 (Tβ4), a chemokine expressed by HSC could prevent PDGF-BB-mediated proliferation and migration of cultured HSC. Using early passages of human HSC, we showed that Tβ4 inhibited cell proliferation and migration and prevented the expression of PDGF-β receptor (PDGF-βr), α-smooth muscle actin and α1(I) collagen mRNAs. Tβ4 also inhibited the reappearance of PDGF-βr after its PDGF-BB-dependent degradation. These PDGF-dependent events were associated with the inhibition of AKT phosphorylation at both T308 and S473 amino acid residues. The lack of AKT phosphorylation was not due to the inhibition of PDGF-βr phosphorylation, the activation of phosphoinositide 3-kinase (PI3K), pyruvate dehydrogenase kinase isozyme 1 (PDK1), and mammalian target of rapamycin (mTOR). We found that PDGF-BB induced AKT binding to actin, and that Tβ4 prevented this effect. Tβ4 also prevented the activation of freshly isolated HSC cultured in the presence of Dulbecco's modified Eagle's medium or Dulbecco's minimal essential medium containing 10% fetal bovine serum. In conclusion, overall, our findings suggest that Tβ4 by sequestering actin prevents binding of AKT, thus inhibiting its phosphorylation. Therefore, Tβ4 has the potential to be an antifibrogenic agent.  

4.929           Enhancement of dentate gyrus neurogenesis, dendritic and synaptic plasticity and memory by a neurotrophic peptide

Chohan, M.O., Li, B., Blanchard, J., Tung, Y-C., Heaney, A.T., Rabe, A., Izbal, K. and Grundke-Iabal, I.

Neurobiology of Aging, 32, 1420-1434 (2011)   Pharmacological enhancement of hippocampal neurogenesis is a therapeutic approach for improvement of cognition in learning and memory disorders such as Alzheimer's disease. Here we report the development of an 11-mer peptide that we designed based on a biologically active region of the ciliary neurotrophic factor. This peptide, Peptide 6, induced proliferation and increased survival and maturation of neural progenitor cells into neurons in the dentate gyrus of normal adult C57BL6 mice. Furthermore, Peptide 6 increased the MAP2 and synaptophysin immunoreactivity in the dentate gyrus. Thirty-day treatment of the mice with a slow release bolus of the peptide implanted subcutaneously improved reference memory of the mice in Morris water maze. Peptide 6 has a plasma half life of over 6 h, is blood–brain barrier permeable, and acts by competitively inhibiting the leukemia inhibitory factor signaling. The fact that Peptide 6 is both neurogenic and neurotrophic and that this peptide is effective when given peripherally, demonstrates its potential for prevention and treatment of learning and memory disorders.  

4.930           Hepatic Stellate Cells Function as Regulatory Bystanders

Ichikawa, S., Mucida, D., Tyznik, A.J., Kronenberg, M. and Cheroutre, H.

J. Immunol., 186, 5549-5555 (2011)

Regulatory T cells (Tregs) contribute significantly to the tolerogenic nature of the liver. The mechanisms, however, underlying liver-associated Treg induction are still elusive. We recently identified the vitamin A metabolite, retinoic acid (RA), as a key controller that promotes TGF-β–dependent Foxp3+ Treg induction but inhibits TGF-β–driven Th17 differentiation. To investigate whether the RA producing hepatic stellate cells (HSC) are part of the liver tolerance mechanism, we investigated the ability of HSC to function as regulatory APC. Different from previous reports, we found that highly purified HSC did not express costimulatory molecules and only upregulated MHC class II after in vitro culture in the presence of exogenous IFN-γ. Consistent with an insufficient APC function, HSC failed to stimulate naive OT-II TCR transgenic CD4+T cells and only moderately stimulated α-galactosylceramide–primed invariant NKT cells. In contrast, HSC functioned as regulatory bystanders and promoted enhanced Foxp3 induction by OT-II TCR transgenic T cells primed by spleen dendritic cells, whereas they greatly inhibited the Th17 differentiation. Furthermore, the regulatory bystander capacity of the HSC was completely dependent on their ability to produce RA. Our data thus suggest that HSC can function as regulatory bystanders, and therefore, by promoting Tregs and suppressing Th17 differentiation, they might represent key players in the mechanism that drives liver-induced tolerance.

 

4.931           Development of an In Vitro Model to Evaluate the Regenerative Capacity of Adult Brain-Derived Tyrosine Hydroxylase-Expressing Dopaminergic Neurons

Majd, S., Smardencas, A., Parish, C.L. and Drago, J.

Neurochem. Res., 36(6), 967-977 (2011)

 

The loss of nigral dopaminergic (DA) neurons is the disease-defining pathological change responsible for progressive motor dysfunction in Parkinson’s disease. In this study, we sought to establish a culture method for adult rat tyrosine hydroxylase (TH)-immunoreactive DA neurons. In this context, we investigated the role of fibroblast growth factor 2 (FGF2), brain-derived neurotrophic factor (BDNF), transforming growth factor-β3 (TGF-β3), glial-derived neurotrophic factor (GDNF) and dibutyryl-cyclic AMP (dbcAMP) in these cultures. Culturing in the presence of FGF2, BDNF and GDNF enhanced the survival of DA neurons by 15-fold and promoted neurite growth. In contrast, dbcAMP promoted neurite growth in all neurons but did not enhance DA cell survival. This study demonstrates that long-term cultures of DA neurons can be established from the mature rat brain and that survival and regeneration of DA neurons can be manipulated by epigenetic factors such as growth factors and intracellular cAMP pathways.

 

4.932           Assessment of the protective effects of oral tocotrienols in arginine chronic-like pancreatitis

Gonzales, A.M., Garcia, T., Samper, E., Rickmann, M., Vaquero, E.C. and Molero, X. Am. J. Physiol. Gastrointest. Liver Physiol., 301, G846-G855 (2011)

 

Tocotrienols exhibit anti-inflammatory properties over macrophages and promote cytotoxicity in activated pancreatic stellate cells, suggesting that they may limit chronic pancreatitis progression. We aimed to quantitate the effect of oral tocotrienols on a rat model of chronic pancreatic injury. Chronic-like pancreatitis was induced by repeated arginine pancreatitis. Palm oil tocotrienol-rich fraction (TRF) was given by gavage before and after pancreatitis inductions. Amylase and hydroxyproline were determined in pancreatic homogenates; collagen, fibronectin, α-smooth muscle actin (SMA), glial fibrillary acidic protein (GFAP), and phosphorylated Smad3 were assessed by Western blotting. Transforming growth factor (TGF)-β1 was measured in plasma. Morphological assessment included light microscopy, fibrosis area fraction, and collagen network fractal analysis. Arginine pancreatitis induced pancreatic atrophy and increased hydroxyproline that ameliorated after TRF. Arginine increased TGF-β1 (185 ± 40 vs. 15 ± 2 ng/ml; P <0.01) that was blunted by TRF (53 ± 19; P < 0.01). TRF reduced protease and Smad3 activation, collagen, and fibronectin. α-SMA increased and GFAP diminished in arginine pancreatitis, consistent with long-term stellate cell activation, and TRF reverted these changes to basal. Arginine pancreatitis increased fibrosis area fraction (4.5 ± 0.3% vs. 0.2 ± 0.2%), collagen network complexity (fractal dimension 1.52 ± 0.03 vs. 1.42 ± 0.01; P < 0.001), and inhomogeneity (lacunarity 0.63 ± 0.03 vs. 0.40 ± 0.02; P < 0.001), which were all reduced by TRF (1.3 ± 0.4%, 1.43 ± 0.02%, and 0.51 ± 0.03%, respectively; P < 0.01). Best correlation coefficients were obtained when comparing fibrosis area fraction with lacunarity (r = 0.88) and both parameters with pancreatic weight (r = −0.91 and −0.79, respectively). TRF administered only before pancreatitis best, but not fully, recapitulated the beneficial effects of TRF. Tocotrienols improve quantitative measures of chronic pancreatic damage. They may be of benefit in human chronic pancreatitis.  

4.933           Rodent blood-stage Plasmodium survive in dendritic cells that infect naive mice

Wykes, M.N. et al PNAS, 108(27), 11205-11210 (2011)   Plasmodium spp. parasites cause malaria in 300 to 500 million individuals each year. Disease occurs during the blood-stage of the parasite's life cycle, where the parasite is thought to replicate exclusively within erythrocytes. Infected individuals can also suffer relapses after several years, from Plasmodium vivax and Plasmodium ovale surviving in hepatocytes. Plasmodium falciparum and Plasmodium malariae can also persist after the original bout of infection has apparently cleared in the blood, suggesting that host cells other than erythrocytes (but not hepatocytes) may harbor these blood-stage parasites, thereby assisting their escape from host immunity. Using blood stage transgenic Plasmodium berghei-expressing GFP (PbGFP) to track parasites in host cells, we found that the parasite had a tropism for CD317⁺ dendritic cells. Other studies using confocal microscopy, in vitro cultures, and cell transfer studies showed that blood-stage parasites could infect, survive, and replicate within CD317⁺ dendritic cells, and that small numbers of these cells released parasites infectious for erythrocytes in vivo. These data have identified a unique survival strategy for blood-stage Plasmodium, which has significant implications for understanding the escape of Plasmodium spp. from immune-surveillance and for vaccine development.  

4.934           Role of Interleukin-1 and MyD88-Dependent Signaling in Rhinovirus Infection

Stokes, C.A., Ismail, S., Dick, E.P., Bennett, J.A., Johnston, S.L., Edwards, M.R., Sabroe, I. and Parker, L.C.

1. Virol., 85(15), 7912-7921 (2011)

  Rhinoviral infection is an important trigger of acute inflammatory exacerbations in patients with underlying airway disease. We have previously established that interleukin-1β (IL-1β) is central in the communication between epithelial cells and monocytes during the initiation of inflammation. In this study we explored the roles of IL-1β and its signaling pathways in the responses of airway cells to rhinovirus-1B (RV-1B) and further determined how responses to RV-1B were modified in a model of bacterial coinfection. Our results revealed that IL-1β dramatically potentiated RV-1B-induced proinflammatory responses, and while monocytes did not directly amplify responses to RV-1B alone, they played an important role in the responses observed with our coinfection model. MyD88 is the essential signaling adapter for IL-1β and most Toll-like receptors. To examine the role of MyD88 in more detail, we created stable MyD88 knockdown epithelial cells using short hairpin RNA (shRNA) targeted to MyD88. We determined that IL-1β/MyD88 plays a role in regulating RV-1B replication and the inflammatory response to viral infection of airway cells. These results identify central roles for IL-1β and its signaling pathways in the production of CXCL8, a potent neutrophil chemoattractant, in viral infection. Thus, IL-1β is a viable target for controlling the neutrophilia that is often found in inflammatory airway disease and is exacerbated by viral infection of the airways.  

4.935           Classical Swine Fever Virus Npro Limits Type I Interferon Induction in Plasmacytoid Dendritic Cells by Interacting with Interferon Regulatory Factor 7

Fiebach, A.R., Guzylack-Piriou. L., Python, S., Summerfield, A. and Ruggli, N.

1. Virol., 85(16), 8002-8011 (2011)

  Viruses are detected by different classes of pattern recognition receptors that lead to the activation of interferon regulatory factors (IRF) and consequently to the induction of alpha/beta interferon (IFN-α/β). In turn, efficient viral strategies to escape the type I IFN-induced antiviral mechanisms have evolved. Previous studies established that pestivirus N^pro antagonizes the early innate immune response by targeting the transcription factor IRF3 for proteasomal degradation. Here, we report that N^pro of classical swine fever virus (CSFV) interacts also with IRF7, another mediator of type I IFN induction. We demonstrate that the Zn-binding domain of N^pro is essential for the interaction of N^pro with IRF7. For IRF3 and IRF7, the DNA-binding domain, the central region, and most of the regulatory domain are required for the interaction with N^pro. Importantly, the induction of IRF7-dependent type I IFN responses in plasmacytoid dendritic cells (pDC) is reduced after wild-type CSFV infection compared with infection with virus mutants unable to interact with IRF7. This is associated with lower levels of IRF7 in pDC. Consequently, wild-type but not N^pro mutant CSFV-infected pDC show reduced responses to other stimuli. Taken together, the results of this study show that CSFV N^pro is capable of manipulating the function of IRF7 in pDC and provides the virus with an additional strategy to circumvent the innate defense.  

4.936           Enhanced protection against infection with transmissible gastroenteritis virus in piglets by oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing swine interferon-α and interleukin-18

Lee, B.M. et al Comp. Immunol. Microbiol. Infect. Dis., 34, 369-380 (2011)   The enhanced effect of cytokine combinations has been assessed empirically, based on their immunobiological mechanisms. However, far less is known of the enhanced protection of practical cytokine combinations against viral infection in the livestock industry, due to cost and production issues associated with mass administration. This study demonstrates the enhanced protection of oral co-administration of swine interferon-α (swIFN-α) and interleukin-18 (swIL-18) against infection with transmissible gastroenteritis virus (TGEV) in piglets using attenuated Salmonella enterica serovar Typhimurium as carrier of cytokine proteins. A single oral co-administration of S. enterica serovar Typhimurium expressing swIFN-α and swIL-18 induced enhanced alleviation of the severity of diarrhea caused by TGEV infection, compared to piglets administered S. enterica serovar Typhimurium expressing swIFN-α or swIL-18 alone. This enhancement was further observed by the reduction of TGEV shedding and replication, and the expression of IFN-stimulated gene products in the intestinal tract. The results suggest that the combined administration of the swIFN-α and swIL-18 cytokines using attenuated S. enterica serovar Typhimurium as an oral carrier provides enhanced protection against intestinal tract infection with TGEV.  

4.937           iNOS potentiates mouse Ig isotype switching through AID expression

Lee, M-R., Seo, G-Y., Kim, Y-M. and Kim, P-H. Biochem. Biophys. Res. Comm., 410, 602-607 82011)   The IgA antibody plays an important role in protecting mucosal surfaces against pathogens. It has recently been shown that nitric oxide (NO) plays a critical role in mouse IgA synthesis. In the present study, we further characterized inducible-nitric oxide synthase-deficient (iNOS^−/−) mice in the context of Ig expression. The amount of IgA in fecal pellets was substantially diminished in iNOS^−/− mice and was paralleled by a decrease in IgA production by Peyer’s patch cells. Interestingly, the amount of all IgG subisotypes, as well as IgA, was substantially diminished in sera and in cultured spleen B cells from iNOS^−/− mice. Moreover, the synthesis of TGF-β1-inducible IgA and IgG2b in iNOS^−/− mice was also lower than that in WT mice. However, levels of Ig germ-line transcripts, and expression of TGF-β receptor type II (TβRII) and BAFF/APRIL, were comparable between iNOS^−/− and WT mice. Expression of activation-induced cytidine deaminase (AID) was diminished in iNOS^−/− B cells, but restored by a NO donor, SNAP. These results indicate that iNOS regulates Ig isotype switching events at the level of AID gene expression.  

4.938           Decreased glutathione accelerates neurological deficit and mitochondrial pathology in familial ALS-linked hSOD1^G93A mice model

Vargas, M.R., Johnson, D.A. and Johnson, J.A. Neurobiol. of Disease, 43, 543-551 (2011)   Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. To investigate the role of antioxidant defenses in ALS we used knockout mice for the glutamate-cysteine ligase modifier subunit (GCLM−/−), which have a 70–80% reduction in total glutathione. Although GCLM(−/−) mice are viable and fertile, the life span of GCLM(−/−)/hSOD1^G93A mice decreased in 55% when compared to GCLM(+/+)/hSOD1^G93A mice. Decreased life span in GCLM(−/−)/hSOD1^G93A mice was associated to increased oxidative stress, aggravated mitochondrial pathology and increased association of hSOD1 with the mitochondria. Interestingly, when the GCLM(−/−) animals were mated with a different ALS-model which overexpress the experimental mutation hSOD1^H46R/H48Q, no effect was observed in survival of GCLM(−/−)/hSOD1^H46R/H48Q mice; and little or no mitochondrial pathology was observed. Since a specific disease modifier, such as glutathione deficiency, may affect only certain hSOD1 mutants, these findings contribute to our understanding of the potential difference in the molecular pathways by which different hSOD1 mutants generate disease.  

4.939           Effect of cryopreservation protocol on postthaw characteristics of stallion sperm

Salazar Jr., J.L., Teague, S.R., Love, C.C., Brinsko, S.P., Blanchard, T.L. and Varner, D.D. Theriogenology, 76, 409-418 (2011)   Three ejaculates from each of eight stallions were subjected to cryopreservation in a milk/egg yolk-based freezing extender or an egg yolk-based freezing extender. Semen was exposed to a fast prefreeze cooling rate (FAST; semen immediately subjected to cryopreservation) or a slow prefreeze cooling rate (SLOW; semen pre-cooled at a controlled rate for 80 min prior to cryopreservation). Postthaw semen was diluted in initial freezing medium (FM) or INRA 96 (IMV Technologies, L'Aigle, France) prior to analysis of 10 experimental end points: total motility (MOT; %), progressive motility (PMOT; %), curvilinear velocity (VCL; μm/s), linearity (LIN; %), intact acrosomal and plasma membranes (AIMI; %), intact acrosomal membranes (AI; %), intact plasma membranes (MI; %), and DNA quality. Eight of 10 experimental endpoints (MOT, PMOT, average-path velocity [VAP], mean straight-line velocity [VSL], LIN AIMI, AI, and MI) were affected by extender type, with egg yolk-based extender yielding higher values than milk/egg yolk-based extender (P < 0.05). Exposure of extended semen to a slow prefreeze cooling period resulted in increased values for six of eight endpoints (MOT, PMOT, VCL, AIMI, AI, and MI), as compared with a fast prefreeze cooling period (P < 0.05). As a postthaw diluent, INRA 96 yielded higher mean values than FM for MOT, PMOT, VCL, average-path velocity, and mean straight-line velocity (P < 0.05). Treatment group FM yielded slightly higher values than INRA 96 for LIN and MI (P < 0.05). In conclusion, a slow prefreeze cooling rate was superior to a fast prefreeze cooling rate, regardless of freezing extender used, and INRA 96 served as a satisfactory postthaw diluent prior to semen analysis.  

4.940           Obstructive Jaundice Expands Intrahepatic Regulatory T Cells, Which Impair Liver T Lymphocyte Function but Modulate Liver Cholestasis and Fibrosis

Katz, S., Ryan, K., Ahmed, N., Plitas, G., Chaudhry, U.I., Kingham, T.P., Naheed, S., Nguyen, C., Somasundar, P., espat, N.J., Junghans, R.P. and DeMatteo, r.P

1. Immunol., 187, 1150-1156 (2011)

  Although obstructive jaundice has been associated with a predisposition toward infections, the effects of bile duct ligation (BDL) on bulk intrahepatic T cells have not been clearly defined. The aim of this study was to determine the consequences of BDL on liver T cell phenotype and function. After BDL in mice, we found that bulk liver T cells were less responsive to allogeneic or syngeneic Ag-loaded dendritic cells. Spleen T cell function was not affected, and the viability of liver T cells was preserved. BDL expanded the number of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg), which were anergic to direct CD3 stimulation and mediated T cell suppression in vitro. Adoptively transferred CD4⁺CD25⁻ T cells were converted into Treg within the liver after BDL. In vivo depletion of Treg after BDL restored bulk liver T cell function but exacerbated the degrees of inflammatory cytokine production, cholestasis, and hepatic fibrosis. Thus, BDL expands liver Treg, which reduce the function of bulk intrahepatic T cells yet limit liver injury.  

4.941           Evidence for proteolysis of a recombinant prion protein in a lamb brain-amended loamy soil

Rapp, D., Richaume, A., Jame, P., Rigou, P., Rezaei, H., Alcouffe, P., Chapel, J-P., Quiquampoix, H. and POtier, P. Eur. J. Soil Sci., 62(4), 607-616 (2011)   Soils contaminated by prions, the infectious agents responsible for transmissible spongiform encephalopathy diseases, remain infectious to grazing animals for many years. In this study, the ability of enzymes produced by soil microbes to degrade a recombinant prion protein (recPrP) was investigated in a loamy soil. A ¹⁵N-labelled recPrP was added to soil in which microbial biomass and soil proteolytic activity had been increased by either simultaneous or prior amendment with lamb brain, and distribution of ¹⁵N among soil solid particles, soluble molecules and bacterial biomass was determined. After 1 day the proportions of recovered recPrP-N associated with microbial biomass and soluble molecules were 6–9 and 15–19%, respectively, which is consistent with the hypothesis of degradation. A greater incorporation of ¹⁵N-derived β-sheeted recPrP into the microbial biomass pool occurred when the soil proteolytic activity was pre-stimulated by a lamb brain amendment, suggesting that the recPrP degradation in soil is mediated by the activity level of proteolytic enzymes produced by the microbial biomass. The majority (35–87%) of the recovered recPrP-N was associated with the soil particles. An observed partial degradation of recPrP deposited on a mica surface by soil soluble enzymes indicated a sorption-related resistance to proteolysis. In conclusion, integration of the stimulation and turnover of the soil microbial component, after an input of a large amount of animal organic matter with the sorption properties of prion protein, is required to model and predict prion survivability, transformation and transmissibility in soil.  

4.942           The earliest intrathymic precursors of CD8α+ thymic dendritic cells correspond to myeloid-type double-negative 1c cells

Luche, H., Ardouin, L., Teo, P., See, P., henri, S., Merad, M., Ginhoux, F and Malissen, B. Eur. J. Immunol., 41(8), 21645-2175 (2011)   The dendritic cells (DCs) present in lymphoid and non-lymphoid organs are generated from progenitors with myeloid-restricted potential. However, in the thymus a major subset of DCs expressing CD8α and langerin (CD207) appears to stand apart from all other DCs in that it is thought to derive from progenitors with lymphoid potential. Using mice expressing a fluorescent reporter and a diphtheria toxin receptor under the control of the cd207 gene, we demonstrated that CD207⁺CD8α⁺ thymic DCs do not share a common origin with T cells but originate from intrathymic precursors that express markers that are normally present on all (CD11c⁺ and MHCII molecules) or on some (CD207, CD135, CD8α, CX3CR1) DC subsets. Those intrathymic myeloid-type precursors correspond to CD44⁺CD25^– double-negative 1c (DN1c) cells and are continuously renewed from bone marrow-derived canonical DC precursors. In conclusion, our results demonstrate that the earliest intrathymic precursors of CD8α⁺ thymic DCs correspond to myeloid-type DN1c cells and support the view that under physiological conditions myeloid-restricted progenitors generate the whole constellation of DCs present in the body including the thymus.  

4.943           Effects of Mycobacterium bovis on monocyte-derived macrophages from bovine tuberculosis infection and healthy cattle

Wang, Y., Zhou, X., Lin, J., Yin, F., Xu, L., Huang, Y., Ding, T. and Zhao, D. FEMS Microbiol. Lett., 321(1), 30-36 (2011)   Bovine tuberculosis (BTB) is a chronic infectious disease caused by the pathogen Mycobacterium bovis and poses a long-standing threat to livestock worldwide. To further elucidate the poorly defined BTB immune response in cattle, we utilized monocyte-derived macrophages (MDMs) to assess the gene expression related to M. bovis Beijing strain stimulation. Here, we demonstrate the existence of distinctive gene expression patterns between macrophages of healthy cattle and those exposed to BTB. In comparing MDMs cells from healthy cattle (n=5) and cattle with tuberculosis (n=5) 3 h after M. bovis stimulation, the differential expressions of seven genes (IL1β, IL1R1, IL1A, TNF-α, IL10, TLR2 and TLR4) implicated in M. bovis response were examined. The expressions of these seven genes were increased in both the tuberculosis-infected and the healthy cattle to M. bovis stimulation, and two of them (TLR2 and IL10) were significantly different in the tuberculosis and the healthy control groups (P≤0.05). The increase in the expression of the TLR2 gene is more significant in healthy cattle response to stimulation, and the change of IL10 gene expression is more significant in tuberculosis cattle. Additionally, we investigated the cytopathic effect caused by M. bovis stimulation and the relationship between M. bovis and MDMs cells to obtain a general profile of pathogen–host interaction.  

4.944           A Francisella tularensis Locus Required for Spermine Responsiveness Is Necessary for Virulence

Russo, B.C., Horzempa, J., O’Dee, D.M., Schmitt, D.M., Brown, M.J., Carlson Jr., P.E., Xavier, R.J. and Nau, G.J. Infect. Immun., 79(9), 3665-3676 (2011)   Tularemia is a debilitating febrile illness caused by the category A biodefense agent Francisella tularensis. This pathogen infects over 250 different hosts, has a low infectious dose, and causes high morbidity and mortality. Our understanding of the mechanisms by which F. tularensis senses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions of F. tularensis with host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis of F. tularensis subsp. holarctica live vaccine strain (LVS), we identified FTL_0883 as a gene important for spermine responsiveness. In-frame deletion mutants of FTL_0883 and FTT_0615c, the homologue of FTL_0883 in F. tularensis subsp. tularensis Schu S4 (Schu S4), elicited higher levels of cytokines from human and murine macrophages compared to wild-type strains. Although deletion of FTL_0883 attenuated LVS replication within macrophages in vitro, the Schu S4 mutant with a deletion in FTT_0615c replicated similarly to wild-type Schu S4. Nevertheless, both the LVS and the Schu S4 mutants were significantly attenuated in vivo. Growth and dissemination of the Schu S4 mutant was severely reduced in the murine model of pneumonic tularemia. This attenuation depended on host responses to elevated levels of proinflammatory cytokines. These data associate responsiveness to polyamines with tularemia pathogenesis and define FTL_0883/FTT_0615c as an F. tularensis gene important for virulence and evasion of the host immune response.  

4.945           The repair function of the multifunctional DNA repair/redox protein APE1 is neuroprotective after ionizing radiation

Vasko, M.R., Guo, C., Thompson, E.L. and Kelley, M.R. DNA Repair, 10, 942-952 (2011)   Although exposure to ionizing radiation (IR) can produce significant neurotoxicity, the mechanisms mediating this toxicity remain to be determined. Previous studies using neurons isolated from the central nervous system show that IR produces reactive oxygen species and oxidative DNA damage in those cells. Because the base excision DNA repair pathway repairs single-base modifications caused by ROS, we asked whether manipulating this pathway by altering APE1 expression would affect radiation-induced neurotoxicity. In cultures of adult hippocampal and sensory neurons, IR produces DNA damage as measured by phosphorylation of histone H2A.X and results in dose-dependent cell death. In isolated sensory neurons, we demonstrate for the first time that radiation decreases the capsaicin-evoked release of the neuropeptide CGRP. Reducing APE1 expression in cultured cells augments IR-induced neurotoxicity, whereas overexpressing APE1 is neuroprotective. Using lentiviral constructs with a neuronal specific promoter that selectively expresses APE1s different functions in neurons, we show that selective expression of the DNA repair competent (redox inactive) APE1 constructs in sensory neurons resurrects cell survival and neuronal function, whereas use of DNA-repair deficient (redox active) constructs is not protective. Use of an APE1 redox-specific inhibitor, APX3330, also facilitates neuronal protection against IR-induced toxicity. These results demonstrate for the first time that the repair function of APE1 is required to protect both hippocampal and DRG neuronal cultures—specifically neuronal cells—from IR-induced damage, while the redox activity of APE1 does not appear to be involved.        

4.946           Sample preparation method for isolation of single-cell types from mouse liver for proteomic studies

Liu, W., Hou, Y., Chen, H., Wei, H., Lin, W., Li, J., Zhang, M., He, F. and Jiang, Y. Proteomics, 11(17), 3556-3564 (2011)   It becomes increasingly clear that separation of pure cell populations provides a uniquely sensitive and accurate approach to protein profiling in biological systems and opens up a new area for proteomic analysis. The method we described could simultaneously isolate population of hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) by a combination of collagenase-based density gradient centrifugation and magnetic activated cell sorting with high purity and yield for the first time. More than 98% of the isolated HCs were positive for cytokeratin 18, with a viability of 91%. Approximately 97% of the isolated HSCs expressed glial fibrillary acidic protein with a viability of 95%. Nearly 98% of isolated KCs expressed F4/80 with a viability of 94%. And the purity of LSECs reached up to 91% with a viability of 94%. And yield for HCs, HSCs, LSECs and KCs were 6.3, 1.3, 2.6 and 5.0 million per mouse. This systematic isolation method enables us to study the proteome profiling of different types of liver cells with high purity and yield, which is especially useful for sample preparation of Human Liver Proteome Project.  

4.947           NOX1/nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase promotes proliferation of stellate cells and aggravates liver fibrosis induced by bile duct ligation

Cui, W., Matsuno, K., Iwata, K., Ibi, M., Matsumoto, M., Zhang, J., Zhu, K., Katsuyama, M., Torok, N.J. and Yabe-Nishimura, C. Hepatology, 54(3), 949-958 (2011)   Among multiple isoforms of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase expressed in the liver, the phagocytic NOX2 isoform in hepatic stellate cells (HSCs) has been demonstrated to play a key role in liver fibrogenesis. The aim of this study was to clarify the role of NOX1, a nonphagocytic form of NADPH oxidase, in the development of fibrosis using Nox1-deficient mice (Nox1KO). Liver injury and fibrosis were induced by bile duct ligation (BDL) and carbon tetrachloride in Nox1KO and wildtype littermate mice (WT). Primary HSCs were isolated to characterize the NOX1-induced signaling cascade involved in liver fibrogenesis. Following BDL, a time-dependent increase in NOX1 messenger RNA (mRNA) was demonstrated in WT liver. Compared with those in WT, levels of collagen-1α mRNA and hydroxyproline were significantly suppressed in Nox1KO with a reduced number of activated HSCs and less severe fibrotic lesions. The expression levels of α-smooth muscle actin, a marker of HSCs activation, were similar in cultured HSCs isolated from both genotypes. However, cell proliferation was significantly attenuated in HSCs isolated from Nox1KO. In these cells, the expression of p27^kip1, a cell cycle suppressor, was significantly up-regulated. Concomitantly, a significant reduction in phosphorylated forms of Akt and forkhead box O (FOXO) 4, a downstream effector of Akt that regulates the transcription of p27^kip1 gene, was demonstrated in Nox1KO. Finally, the level of the oxidized inactivated form of phosphatase and tensin homolog (PTEN), a negative regulator of PI3K/Akt pathway, was significantly attenuated in HSCs of Nox1KO.  

4.948           Dendritic cell depletion exacerbates acetaminophen hepatotoxicity

Conolly, M.K. et al Hepatology, 54(3), 959-968 (2011)   Acetaminophen (APAP) overdose is one of the most frequent causes of acute liver failure in the United States and is primarily mediated by toxic metabolites that accumulate in the liver upon depletion of glutathione stores. However, cells of the innate immune system, including natural killer (NK) cells, neutrophils, and Kupffer cells, have also been implicated in the centrilobular liver necrosis associated with APAP. We have recently shown that dendritic cells (DCs) regulate intrahepatic inflammation in chronic liver disease and, therefore, postulated that DC may also modulate the hepatotoxic effects of APAP. We found that DC immune-phenotype was markedly altered after APAP challenge. In particular, liver DC expressed higher MHC II, costimulatory molecules, and Toll-like receptors, and produced higher interleukin (IL)-6, macrophage chemoattractant protein-1 (MCP-1), and tumor necrosis factor alpha (TNF-α). Conversely, spleen DC were unaltered. However, APAP-induced centrilobular necrosis, and its associated mortality, was markedly exacerbated upon DC depletion. Conversely, endogenous DC expansion using FMS-like tyrosine kinase 3 ligand (Flt3L) protected mice from APAP injury. Our mechanistic studies showed that APAP liver DC had the particular capacity to prevent NK cell activation and induced neutrophil apoptosis. Nevertheless, the exacerbated hepatic injury in DC-depleted mice challenged with APAP was independent of NK cells and neutrophils or numerous immune modulatory cytokines and chemokines. Conclusion: Taken together, these data indicate that liver DC protect against APAP toxicity, whereas their depletion is associated with exacerbated hepatotoxicity  

4.949           Neuroprotective Signaling Mechanisms of Telomerase Are Regulated by Brain-Derived Neurotrophic Factor in Rat Spinal Cord Motor Neurons

Niu, C. and Yip, H.K.

1. Neuropathol. Exp. Neurol., 70(7), 634-652 (2011)

  Telomerase can promote neuron survival and can be regulated by growth factors such as brain-derived neurotrophic factor (BDNF). Increases of BDNF expression and telomerase activity after brain injury suggest that telomerase may be involved in BDNF-mediated neuroprotection. We investigated BDNF regulation of telomerase in rat spinal cord motor neurons (SMNs). Our results indicate that BDNF increases telomerase expression and activity levels in SMNs and activates mitogen-activated protein kinase/extracellular signal-regulated kinases 1 and 2 and phosphatidylinositol-3-OH kinase/protein kinase B signals, and their downstream transcription factors nuclear factor-κB, c-Myc, and Sp1. Administration of the tyrosine kinase receptor B inhibitor K-252a, the mitogen-activated protein kinase 1 inhibitor PD98059, and the phosphatidylinositol-3-OH kinase inhibitor LY294002 abolished BDNF-induced upregulation of these transcription factors and telomerase expression. The nuclear factor-κB inhibitor Bay11-7082 also attenuated c-Myc and Sp1 expression and increased telomerase promoter activity. Spinal cord motor neurons with higher telomerase levels induced by BDNF became more resistant to apoptosis; survival of SMNs that overexpressed the catalytic protein component of telomerase with reverse transcriptase activity was also enhanced against apoptosis. The neuronal survival-promoting effect of telomerase was mediated through the regulation of Bcl-2, Bax, p53, and maintenance of mitochondrial membrane potential. Taken together, these data suggest that the neuroprotective effect of BDNF via telomerase is mediated by inhibition of apoptotic pathways.  

4.950           5′ Triphosphorylated Small Interfering RNAs Control Replication of Hepatitis B Virus and Induce an Interferon Response in Human Liver Cells and Mice

Ebert, G., Poeck, H., Lucifora, J., Baschuk, N., Esser, K., Essposito, I., Hartmann, G. and Protzer, U. Gastroenterology, 141(2), 696-706 (2011)   Background & Aims Approved therapies for chronic hepatitis B include systemic administration of interferon (IFN)-alfa and inhibitors of hepatitis B virus (HBV) reverse-transcription. Systemic application of IFN-alfa is limited by side effects. Reverse-transcriptase inhibitors effectively control HBV replication, but rarely eliminate the virus and can select drug-resistant variants. We aimed to develop an alternative therapeutic approach that combines gene silencing with induction of IFN in the liver. Methods To stimulate an immune response while inhibiting HBV activity, we designed 3 small interfering (si)RNAs that target highly conserved sequences and multiple HBV transcripts of all genotypes. A 5′-triphosphate (3p) was added to the siRNAs, turning them into a ligand for the cytosolic helicase retinoic acid–inducible protein I, which becomes activated and induces expression of type-I IFNs. Antiviral activity was investigated in cell lines that replicate HBV, in HBV-infected primary human hepatocytes, and in HBV transgenic mice. Results 3p-double-stranded RNA (3p-RNA) activated retinoic acid–inducible protein I, induced a strong type I IFN response (expression of IFN-β) in liver cells and showed transient but strong antiviral activity. Bifunctional, HBV-specific, 3p-siRNAs controlled replication of HBV more efficiently and for longer periods of time than 3p-RNAs without silencing capacity or siRNAs that targeted identical sequences but did not contain 3p. Conclusions HBV-specific 3p-siRNAs are bifunctional antiviral molecules that induce production of type I IFNs in the liver and target HBV RNAs to inhibit viral replication.  

4.951           Thymic but not splenic CD8+ DCs can efficiently cross-prime T cells in the absence of licensing factors

Dresch, C., Ackermann, M., Vogt, B., de Andrada Pereira, B., Shortman, K. and Fraefel, C. Eur. J. Immunol., 41(9), 2544-2555 (2011)   Cross-presentation is an important mechanism to elicit both immune defenses and tolerance. Although only a few DC subsets possess the machinery required for cross-presentation, little is known about differences in cross-presenting capabilities of DCs belonging to the same subpopulation but localized in different lymphoid organs. In this study, we demonstrate that steady-state thymic CD8⁺ DCs can efficiently cross-prime naïve CD8⁺ T cells in the absence of costimulation. Surprisingly, cross-priming by splenic CD8⁺ DCs was dependent on licensing factors such as GM-CSF. In the absence of GM-CSF, antigen–MHC-class-I complexes were detected on thymic but not on splenic CD8⁺ DCs, indicating that the cross-presentation capacity of the thymic subpopulation was higher. The observed cross-priming differences between thymic and splenic CD8⁺ DCs did not correlate with differential antigen capture or costimulatory molecules found on the surface of DCs. Moreover, we did not detect overall impairment of antigen presentation, as peptide-loaded splenic CD8⁺ DCs were able to induce CD8⁺ T-cell proliferation. The observation that thymic CD8⁺ DCs are more efficient than splenic CD8⁺ DCs in T-cell cross-priming in the absence of licensing factors indicates that the requirements for efficient antigen presentation differ between these cells.  

4.952           Immunization with dendritic cells transfected in vivo with HIV-1 plasmid DNA induces HIV-1-specific immune responses

Malm, M., Krohn, K. and Blazevic, V. Arch. Virol., 156(9), 1607-1610 (2011)   We evaluated the importance of dendritic cells (DCs) in the induction of the immune response after immunization of mice with DNA plasmid Auxo-GTU^®-MultiHIV. First, GTU^®-encoded protein was shown to be expressed by DCs of the draining lymph nodes (LNs) following intradermal (i.d.) immunization. Next, donor mice were immunized with the MultiHIV DNA plasmid, and DCs were enriched and further used to immunize naïve recipient mice. For the first time, the results show that i.d. immunization with Auxo-GTU^®-MultiHIV transfects DCs in vivo, enabling them to present antigens and induce HIV-specific immune responses in recipient mice.  

4.953           The role of dystroglycan in PDGF-BB-dependent migration of activated hepatic stellate cells/myofibroblasts

Kastanis, G.J., Hernandez-Nazara, Z., Nieto, N., Rincon-Sanchez, A.R., Popratiloff, A., Dominguez-Rosales, J.A., Lechuga, C.G. and Rojkind, M. Am. J. Physiol. Gastrintest. Liver Physiol.,  301,G464-G474 (2011)   Hepatic stellate cells are embedded in the loose connective tissue matrix within the space of Disse. This extracellular matrix contains several basement membrane components including laminin, but its composition changes during liver injury because of the production of extracellular matrix components found in scar tissue. These changes in extracellular matrix composition and in cell-extracellular matrix interactions may play a key role in hepatic stellate cell transdifferentiation. In this communication we used early passages of mouse hepatic stellate cells (activated HSC/myofibroblasts) to study the platelet-derived growth factor BB (PDGF-BB)-dependent expression and regulation of β-dystroglycan and its role in activated HSC/myofibroblast migration. We used Northern and Western analysis to study dystroglycan expression and confocal microscopy to investigate changes in subcellular distribution of the protein. Activated HSC migration was investigated using an in vitro wound-healing assay. PDGF-BB induced significant changes in dystroglycan regulation and subcellular distribution of the protein. Whereas steady-state levels of dystroglycan mRNA remained constant, PDGF-BB increased dystroglycan transcription but shortened the t_1/2 by 50%. Moreover, PDGF-BB changed dystroglycan and α5-integrin cellular distribution. Cell migration experiments revealed that PDGF-BB-dependent migration of activated HSC/myofibroblasts was completely blocked by neutralizing antibodies to fibronectin, α5-integrin, laminin, and β-dystroglycan. Overall, these findings suggest that both laminin and fibronectin and their receptors play a key role in PDGF-BB-induced activated HSC migration.  

4.954           Quality of Life Improves for Pediatric Patients After Total Pancreatectomy and Islet Autotransplant for Chronic Pancreatitis

Bellin, M.D., Freeman, M.L., Schwarzenberg, S.J., Dunn, T.B., Beilman, G.J., Vickers, S.M., Chinnakotla, S., Balamurugan, A.N., Hering, B.J., Radosevich, D.M., Moran, A. and Sutherland, D.E.R. Clin. Gastroenterol. Hepatol., 9, 793-799 (2011)   Background & Aims Total pancreatectomy (TP) and islet autotransplant (IAT) have been used to treat patients with painful chronic pancreatitis. Initial studies indicated that most patients experienced significant pain relief, but there were few validated measures of quality of life. We investigated whether health-related quality of life improved among pediatric patients undergoing TP/IAT. Methods Nineteen consecutive children (aged 5–18 years) undergoing TP/IAT from December 2006 to December 2009 at the University of Minnesota completed the Medical Outcomes Study 36-item Short Form (SF-36) health questionnaire before and after surgery. Insulin requirements were recorded. Results Before TP/IAT, patients had below average health-related quality of life, based on data from the Medical Outcomes Study SF-36; they had a mean physical component summary (PCS) score of 30 and mental component summary (MCS) score of 34 (2 and 1.5 standard deviations, respectively, below the mean for the US population). By 1 year after surgery, PCS and MCS scores improved to 50 and 46, respectively (global effect, PCS P < .001, MCS P = .06). Mean scores improved for all 8 component subscales. More than 60% of IAT recipients were insulin independent or required minimal insulin. Patients with prior surgical drainage procedures (Puestow) had lower yields of islets (P = .01) and greater incidence of insulin dependence (P = .04). Conclusions Quality of life (physical and emotional components) significantly improve after TP/IAT in subsets of pediatric patients with severe chronic pancreatitis. Minimal or no insulin was required for most patients, although islet yield was reduced in patients with previous surgical drainage operations. Keywords: Pancreas; Inflammation; Therapy; Clinical Trial Abbreviations: CP, chronic pancreatitis; HbA, hemoglobin A; HRQOL, health-related quality of life; IAT, islet autotransplant; IE, islet equivalent; MCS, mental component summary; PCS, physical component summary; SF-36, 36-Item Short Form Medical Outcomes Survey; TP, total pancreatectomy  

4.955           A knockout of the caspase 2 gene produces increased resistance of the nigrostriatal dopaminergic pathway to MPTP-induced toxicity

Tiwari, M., Herman, B. and Morgan, W.W. Exp. Neurol., 229, 421-428 (2011)   This study investigated the effect of a knockout of the caspase 2 gene on the sensitivity of murine nigral dopaminergic neurons to 1-methyl-4-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity. Female wild type (WT), heterozygous caspase 2 NL (HET) and homozygous caspase 2 null (NL) mice were treated with cumulative dosages of 0, 10, 15 or 20 mg/kg MPTP free base. Without MPTP treatment, one week later dopamine (DA) levels were not significantly different in HET or NL versus WT mice. Twenty mg/kg MPTP reduced striatal DA in WT and HET (p < 0.01) but not NL mice. This same MPTP dosage regimen also induced a significantly greater decrease in tyrosine hydroxylase immunopositive (TH+) protein in striata of WT compared to NL mice (p < 0.001). Subsequently, WT and NL mice were treated daily with 20 mg/kg MPTP for 3 days and 25 mg/kg MPTP for 2 additional days, and TH+ neurons in the substantia nigra (SN) were estimated using unbiased stereology. When compared to untreated WT, the numbers of TH+ neurons were significantly lower in the SN of untreated NL mice (p < 0.05). Treatment with the MPTP regimen significantly reduced TH+ neurons in WT mice but not NL mice. In primary mesencephalic cultures both the cell bodies and the neuronal processes of TH immunopositive (TH+) neurons from NL embryos were significantly (p < 0.001) more resistant to 10 μM MPP+ compared to WT. Following MPP+ treatment, features of apoptotic cell death were also significantly (p < 0.001) more prevalent in nuclei of TH+ neurons in cultures prepared from WT versus NL mouse pups. These results suggest that caspase 2 may play a role in modulating the MPTP-induced damage to the nigrostriatal dopaminergic system.  

4.956           Integrin expression and function in the response of primary culture hepatic stellate cells to connective tissue growth factor (CCN2)

Huang, G. and Brigstock, D.R.

1. Cell. Mol. Med., 15(5), 1087-1095 (2011)

  Production of connective tissue growth factor (CCN2, also known as CTGF) is a hallmark of hepatic fibrosis. This study examined early primary cultures of hepatic stellate cells (HSC) for (i) CCN2 regulation of its cognate receptor integrin subunits; and (ii) interactions between CCN2 and integrin α₅β₁, heparan sulphate proteoglycans (HSPG) or fibronectin (FN) in supporting cell adhesion. HSC were isolated from healthy male Balb/c mice. mRNA levels of CCN2 or α₅, β₁, αv or β₃ integrin subunits were measured in days 1–7 primary culture HSC, and day 3 or day 7 cells treated with recombinant CCN2 or CCN2 small interfering RNA. Interactions between CCN2 and integrin α₅β₁, HSPG or FN were investigated using an in vitro cell adhesion assay. Co-incident with autonomous activation over the first 7 days, primary culture HSC increasingly expressed mRNA for CCN2 or integrin subunits. Addition of exogenous CCN2 or knockdown of endogenous CCN2 differentially regulated integrin gene expression in day 3 versus day 7 cells. Either full length CCN2 (‘CCN2_1–4’) or residues 247–349 containing module 4 alone (‘CCN2₄’) supported day 3 cell adhesion in an integrin α₅β₁- and HSPG-dependent fashion. Adhesion of day 3 cells to FN was promoted in an integrin α₅β₁-dependent manner by CCN2_1–4 or CCN2₄, whereas FN promoted HSPG-dependent HSC adhesion to CCN2_1–4 or CCN2₄. These findings suggest CCN2 regulates integrin expression in primary culture HSC and supports HSC adhesion via its binding of cell surface integrin α₅β₁, a novel CCN2 receptor in primary culture HSC which interacts co-operatively with HSPG or FN.  

4.957           Necdin Protects Embryonic Motoneurons from Programmed Cell Death

Aebisher, J., Sturny, R., Andrieu, D., Rieusset, A., Schaller, F., Geib, S., Raoul, C. and Muscatelli, F. PloS One, 6(9), e23764 (2011)   NECDIN belongs to the type II Melanoma Associated Antigen Gene Expression gene family and is located in the Prader-Willi Syndrome (PWS) critical region. Necdin-deficient mice develop symptoms of PWS, including a sensory and motor deficit. However, the mechanisms underlying the motor deficit remain elusive. Here, we show that the genetic ablation of Necdin, whose expression is restricted to post-mitotic neurons in the spinal cord during development, leads to a loss of 31% of specified motoneurons. The increased neuronal loss occurs during the period of naturally-occurring cell death and is not confined to specific pools of motoneurons. To better understand the role of Necdin during the period of programmed cell death of motoneurons we used embryonic spinal cord explants and primary motoneuron cultures from Necdin-deficient mice. Interestingly, while Necdin-deficient motoneurons present the same survival response to neurotrophic factors, we demonstrate that deletion of Necdin leads to an increased susceptibility of motoneurons to neurotrophic factor deprivation. We show that by neutralizing TNFα this increased susceptibility of Necdin-deficient motoneurons to trophic factor deprivation can be reduced to the normal level. We propose that Necdin is implicated through the TNF-receptor 1 pathway in the developmental death of motoneurons.  

4.958           Nonoverlapping functions for Notch1 and Notch3 during murine steady-state thymic lymphopoiesis

Shi, J., Fallahi, M., Luo, J-L. and Petrie, H.T. Blood, 118(9), 2511-2519 (2011)   Notch1 signaling is absolutely essential for steady-state thymic lymphopoiesis, but the role of other Notch receptors, and their potential overlap with the function of Notch1, remains unclear. Here we show that like Notch1, Notch3 is differentially expressed by progenitor thymocytes, peaking at the DN3 progenitor stage. Using mice carrying a gene-trapped allele, we show that thymic cellularity is slightly reduced in the absence of Notch3, although progression through the defined sequence of TCR-αβ development is normal, as are NKT and TCRγδ cell production. The absence of a profound effect from Notch3 deletion is not explained by residual function of the gene-trapped allele because insertion mapping suggests that the targeted allele would not encode functional signaling domains. We also show that although Notch1 and Notch3 are coexpressed on some early intrathymic progenitors, the relatively mild phenotype seen after Notch3 deletion does not result from the compensatory function of Notch1, nor does Notch3 function explain the likewise mild phenotype seen after conditional (intrathymic) deletion of Notch1. Our studies indicate that Notch1 and Notch3 carry out nonoverlapping functions during thymocyte differentiation, and that while Notch1 is absolutely required early in the lymphopoietic process, neither receptor is essential at later stages.  

4.959           Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry: a Novel Use for a Cell Purification Method

Nguyen, H.X., Beck, K.D. and Anderson, A.J.

1. Vis. Exp., 50, (2011) http://www.jove.com/details.php?id=2698

  Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck & Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.  

4.960           The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Leach, M.K., Feng, Z-Q., Gertz, C.C., Tuck, S.J., Regan, T.M., Naim, Y., Vincent, A.M. and Corey, J.M.

1. Vis. Exp., 48, (2011), http://www.jove.com/details.php?id=2389

  Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.  

4.961           Transcriptional profiling of peripheral lymphoid tissue reveals genes and networks linked to SSBP/1 scrapie pathology in sheep

Gossner, A., Roupaka, S., Foster, J., Hunter, N. and Hopkins, J. Vet. Microbiol., 153, 218-228 (2011)   Transmissible spongiform encephalopathies (TSEs) are slow and progressive neurodegenerative diseases of humans and animals. The major target organ for all TSEs is the brain but some TSE agents are associated with prior accumulation within the peripheral lymphoid system. Many studies have examined the effects of scrapie infection on the expression of central nervous system (CNS) genes, but this study examines the progression of scrapie pathology in the peripheral lymphoid system and how scrapie infection affects the transcriptome of the lymph nodes and spleen. Infection of sheep with SSBP/1 scrapie resulted in PrP^Sc deposition in the draining prescapular lymph node (PSLN) by 25 days post infection (dpi) in VRQ/VRQ genotype sheep and 75 dpi in tonsils and spleen. Progression of PrP^Sc deposition in VRQ/ARR animals was 25 dpi later in the PSLN and 250 dpi later in spleen. Microarray analysis of 75 dpi tissues from VRQ/VRQ sheep identified 52 genes in PSLN and 37 genes in spleen cells that showed significant difference (P ≤ 0.05) between scrapie-infected and mock-infected animals. Transcriptional pathway analysis highlighted immunological disease, cell death and neurological disease as the biological pathways associated with scrapie pathogenesis in the peripheral lymphoid system. PrP^Sc accumulation of lymphoid tissue resulted in the repression of genes linked to inflammation and oxidative stress, and the up-regulation of genes related to apoptosis.  

4.962           Dysregulation of astrocyte–motoneuron cross-talk in mutant superoxide dismutase 1-related amyotrophic lateral sclerosis

Ferraiuolo, L., Higginbottom, A., Hath, P.R., Barber, S., Greenald, D., Kirby, J. and Shaw, P.J. Brain, 134, 2627-2641 (2011)   Amyotrophic lateral sclerosis is a neurodegenerative disease in which death of motoneurons leads to progressive failure of the neuromuscular system resulting in death frequently within 2–3 years of symptom onset. Focal onset and propagation of the disease symptoms to contiguous motoneuron groups is a striking feature of the human disease progression. Recent work, using mutant superoxide dismutase 1 murine models and in vitro culture systems has indicated that astrocytes are likely to contribute to the propagation of motoneuron injury and disease progression. However, the basis of this astrocyte toxicity and/or failure of motoneuron support has remained uncertain. Using a combination of in vivo and in vitro model systems of superoxide dismutase 1-related amyotrophic lateral sclerosis, linked back to human biosamples, we set out to elucidate how astrocyte properties change in the presence of mutant superoxide dismutase 1 to contribute to motoneuron injury. Gene expression profiling of spinal cord astrocytes from presymptomatic transgenic mice expressing mutant superoxide dismutase 1 revealed two striking changes. First, there was evidence of metabolic dysregulation and, in particular, impairment of the astrocyte lactate efflux transporter, with resultant decrease of spinal cord lactate levels. Second, there was evidence of increased nerve growth factor production and dysregulation of the ratio of pro-nerve growth factor to mature nerve growth factor, favouring p75 receptor expression and activation by neighbouring motoneurons. Functional in vitro studies showed that astrocytes expressing mutant superoxide dismutase 1 are toxic to normal motoneurons. We provide evidence that reduced metabolic support from lactate release and activation of pro-nerve growth factor-p75 receptor signalling are key components of this toxicity. Preservation of motoneuron viability could be achieved by increasing lactate provision to motoneurons, depletion of increased pro-nerve growth factor levels or p75 receptor blockade. These findings are likely to be relevant to human amyotrophic lateral sclerosis, where we have demonstrated increased levels of pro-nerve growth factor in cerebrospinal fluid and increased expression of the p75 receptor by spinal motoneurons. Taken together, these data confirm that altered properties of astrocytes are likely to play a crucial role in the propagation of motoneuron injury in superoxide dismutase 1-related amyotrophic lateral sclerosis and indicate that manipulation of the energy supply to motoneurons as well as inhibition of p75 receptor signalling may represent valuable neuroprotective strategies.  

4.963           Motor neuron impairment mediated by a sumoylated fragment of the glial glutamate transporter EAAT2

Foran, E., Bogush, A., Goffredo, M., Roncaglia, P., Gustincich, S., Pasinell, P. and Trotti, D. Glia, 59(11), 1719-1731 (2011)   Dysregulation of glutamate handling ensuing downregulation of expression and activity levels of the astroglial glutamate transporter EAAT2 is implicated in excitotoxic degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). We previously reported that EAAT2 (a.k.a. GLT-1) is cleaved by caspase-3 at its cytosolic carboxy-terminus domain. This cleavage results in impaired glutamate transport activity and generates a proteolytic fragment (CTE) that we found to be post-translationally conjugated by SUMO1. We show here that this sumoylated CTE fragment accumulates in the nucleus of spinal cord astrocytes of the SOD1-G93A mouse model of ALS at symptomatic stages of disease. Astrocytic expression of CTE, artificially tagged with SUMO1 (CTE-SUMO1) to mimic the native sumoylated fragment, recapitulates the nuclear accumulation pattern of the endogenous EAAT2-derived proteolytic fragment. Moreover, in a co-culture binary system, expression of CTE-SUMO1 in spinal cord astrocytes initiates extrinsic toxicity by inducing caspase-3 activation in motor neuron-derived NSC-34 cells or axonal growth impairment in primary motor neurons. Interestingly, prolonged nuclear accumulation of CTE-SUMO1 is intrinsically toxic to spinal cord astrocytes, although this gliotoxic effect of CTE-SUMO1 occurs later than the indirect, noncell autonomous toxic effect on motor neurons. As more evidence on the implication of SUMO substrates in neurodegenerative diseases emerges, our observations strongly suggest that the nuclear accumulation in spinal cord astrocytes of a sumoylated proteolytic fragment of the astroglial glutamate transporter EAAT2 could participate to the pathogenesis of ALS and suggest a novel, unconventional role for EAAT2 in motor neuron degeneration.  

4.964           Isolation and Culture of Postnatal Spinal Motoneurons

Milligan, C. and Difondorwa, D. Methods in Mol. Biol., 793, 77-85 (2011)   Neuronal cultures, including motoneuron (MN) cultures, are established from embryonic animals. These approaches have provided novel insights into developmental and possibly disease mechanisms mediating cell survival or death. Motoneurons isolated from mouse models of disease, such as the SOD1G93A mouse, demonstrate subtle abnormalities that may contribute to pathology. Nonetheless, in the animal model, pathological events become more prominent as the animal matures, but the ability to isolate individual cells to investigate these events is limited. Here, we describe a protocol derived and modified from previously published protocols to isolate motoneurons from mature animals. While the yield of cells is low, the ability to examine mature motoneurons provides a new platform to investigate pathological changes associated with motoneuron disease.  

4.965           Proteomic Analysis of Anaplasma phagocytophilum during Infection of Human Myeloid Cells Identifies a Protein That Is Pronouncedly Upregulated on the Infectious Dense-Cored Cell

Troese, M.J., Kahlon, A., Ragland, S.A., Ottens, A.K., Ojogun, N., Nelson, K.T., Walker, N.J., Borjesson, D.L. and Carlyon, J.A. Infect. Immun., 79(11), 4696-4707 (2011)   Anaplasma phagocytophilum is an obligate intracellular bacterium that invades neutrophils to cause the emerging infectious disease human granulocytic anaplasmosis. A. phagocytophilum undergoes a biphasic developmental cycle, transitioning between an infectious dense-cored cell (DC) and a noninfectious reticulate cell (RC). To gain insights into the organism's biology and pathogenesis during human myeloid cell infection, we conducted proteomic analyses on A. phagocytophilum organisms purified from HL-60 cells. A total of 324 proteins were unambiguously identified, thereby verifying 23.7% of the predicted A. phagocytophilum proteome. Fifty-three identified proteins had been previously annotated as hypothetical or conserved hypothetical. The second most abundant gene product, after the well-studied major surface protein 2 (P44), was the hitherto hypothetical protein APH_1235. APH_1235 homologs are found in other Anaplasma and Ehrlichia species but not in other bacteria. The aph_1235 RNA level is increased 70-fold in the DC form relative to that in the RC form. Transcriptional upregulation of and our ability to detect APH_1235 correlate with RC to DC transition, DC exit from host cells, and subsequent DC binding and entry during the next round of infection. Immunoelectron microscopy pronouncedly detects APH_1235 on DC organisms, while detection on RC bacteria minimally, at best, exceeds background. This work represents an extensive study of the A. phagocytophilum proteome, discerns the complement of proteins that is generated during survival within human myeloid cells, and identifies APH_1235 as the first known protein that is pronouncedly upregulated on the infectious DC form.  

4.966           Vasoactive intestinal peptide reduces oxidative stress in pancreatic acinar cells through the inhibition of NADPH oxidase

Fujimori, N., Oono, T., Igarashi, H., Ito, T., Nakamura, T., Uchida, M., Coy, D.H., Jensen, R.T. and Takayanagi, R. Peptides, 32, 2067-2076 (2011)   Vasoactive intestinal peptide (VIP) attenuates experimental acute pancreatitis (AP) by inhibition of cytokine production from inflammatory cells. It has been suggested that reactive oxygen species (ROS) as well as cytokines play pivotal roles in the early pathophysiology of AP. This study aimed to clarify the effect of VIP on the oxidative condition in pancreas, especially pancreatic acinar cells (acini). Hydrogen peroxide (H₂O₂)-induced intracellular ROS, assessed with CM-H₂DCFDA, increased time- and dose-dependently in acini isolated from rats. Cell viability due to ROS-induced cellular damage, evaluated by MTS assay, was decreased with ≥100 μmol/L H₂O₂. VIP significantly inhibited ROS production from acini and increased cell viability in a dose-dependent manner. Expression of antioxidants including catalase, glutathione reductase, superoxide dismutase (SOD) 1 and glutathione peroxidase was not altered by VIP except for SOD2. Furthermore, Nox1 and Nox2, major components of NADPH oxidase, were expressed in pancreatic acini, and significantly increased after H₂O₂ treatment. Also, NADPH oxidase activity was provoked by H₂O₂. VIP decreased NADPH oxidase activity, which was abolished by PKA inhibitor H89. These results suggested that VIP affected the mechanism of ROS production including NADPH oxidase through induction of a cAMP/PKA pathway. In conclusion, VIP reduces oxidative stress in acini through the inhibition of NADPH oxidase. These results combined with findings of our previous study suggest that VIP exerts its protective effect in pancreatic damage, not only through an inhibition of cytokine production, but also through a reduction of the injury caused by oxidative stress.  

4.967           A Double-Inactivated Severe Acute Respiratory Syndrome Coronavirus Vaccine Provides Incomplete Protection in Mice and Induces Increased Eosinophilic Proinflammatory Pulmonary Response upon Challenge

Bolles, M., Deming, D., Long, K., Agnihothram, S., Whitmore, A., Ferris, M., Funkhouser, W., Gralinski, L., Totura, A., Heise, M. and Baric, R.S.

1. Virol., 85(23), 12201-12215 (2011)

  Severe acute respiratory syndrome coronavirus (SARS-CoV) is an important emerging virus that is highly pathogenic in aged populations and is maintained with great diversity in zoonotic reservoirs. While a variety of vaccine platforms have shown efficacy in young-animal models and against homologous viral strains, vaccine efficacy has not been thoroughly evaluated using highly pathogenic variants that replicate the acute end stage lung disease phenotypes seen during the human epidemic. Using an adjuvanted and an unadjuvanted double-inactivated SARS-CoV (DIV) vaccine, we demonstrate an eosinophilic immunopathology in aged mice comparable to that seen in mice immunized with the SARS nucleocapsid protein, and poor protection against a nonlethal heterologous challenge. In young and 1-year-old animals, we demonstrate that adjuvanted DIV vaccine provides protection against lethal disease in young animals following homologous and heterologous challenge, although enhanced immune pathology and eosinophilia are evident following heterologous challenge. In the absence of alum, DIV vaccine performed poorly in young animals challenged with lethal homologous or heterologous strains. In contrast, DIV vaccines (both adjuvanted and unadjuvanted) performed poorly in aged-animal models. Importantly, aged animals displayed increased eosinophilic immune pathology in the lungs and were not protected against significant virus replication. These data raise significant concerns regarding DIV vaccine safety and highlight the need for additional studies of the molecular mechanisms governing DIV-induced eosinophilia and vaccine failure, especially in the more vulnerable aged-animal models of human disease.  

4.968           Immune Adjuvant Efficacy of CpG Oligonucleotide in Cancer Treatment Is Founded Specifically upon TLR9 Function in Plasmacytoid Dendritic Cells

Nierkens, S., den Brok, M.H., Garcia, Z. et al Cancer Res., 71(20), 6428-6437 (2011)   The differences in function, location, and migratory pattern of conventional dendritic cells (cDC) and plasmacytoid DCs (pDC) not only point to specialized roles in immune responses but also signify additive and interdependent relationships required to clear pathogens. We studied the in vivo requirement of cross-talk between cDCs and pDCs for eliciting antitumor immunity against in situ released tumor antigens in the absence or presence of the Toll-like receptor (TLR) 9 agonist CpG. Previous data indicated that CpG boosted tumor-specific T-cell responses after in vivo tumor destruction and increased survival after tumor rechallenges. The present study shows that cDCs are indispensable for cross-presentation of ablation-released tumor antigens and for the induction of long-term antitumor immunity. Depletion of pDCs or applying this model in type I IFN receptor–deficient mice abrogated CpG-mediated responses. CD8α⁺ cDCs and the recently identified merocytic cDCs were dependent on pDCs for CpG-induced upregulation of CD80. Moreover, DC transfer studies revealed that merocytic cDCs and CD8α⁺ cDCs were most susceptible to pDC help and subsequently promoted tumor-free survival in a therapeutic setting. By transferring wild-type pDCs into TLR9-deficient mice, we finally showed that TLR9 expression in pDCs is sufficient to benefit from CpG as an adjuvant. These studies indicate that the efficacy of CpG in cancer immunotherapy is dependent on cross-talk between pDCs and specific subsets of cDCs.  

4.969           Low-avidity anti-HPA-1a alloantibodies are capable of antigen-positive platelet destruction in the NOD/SCID mouse model of alloimmune thrombocytopenia

Backchoul, T., Kubiak, S., Krautwurst, A., Roderfeld, M., Siebert, H.C., Bein, G., Sachs, U.J. and Santosa, S. Transfusion, 51(11), 2455-2461 82011)   BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is mostly caused by maternal antibodies against human platelet antigen 1a (HPA-1a) expressed on glycoprotein (GP) IIb/IIIa. Accumulated evidence indicated that anti-HPA-1a could be overlooked by standard methods due to low avidity. Low-avidity HPA-1a antibodies were shown to be detectable by surface plasmon resonance (SPR). We sought to investigate the frequency and in vivo relevance of low-avidity anti-HPA-1a. STUDY DESIGN AND METHODS: A retrospective cohort consisting of 82 HPA-1bb mothers of HPA-1ab newborns with thrombocytopenia was analyzed using standard serologic methods. Maternal immunoglobulin (Ig)G fractions were investigated for low-avidity antibodies in SPR using purified GPIIb/IIIa (HPA-1a or -1b). The capability of HPA-1a antibodies to clear platelets (PLTs) in vivo was analyzed using the NOD/SCID mouse model of alloimmune thrombocytopenia. RESULTS: HPA antibodies were detectable in sera from 68 of 82 (83%) mothers using standard serologic methods and undetectable in 14 of 82 sera. In SPR, IgG fractions of sera reacting positive in monoclonal antibody immobilization of PLT antigen (MAIPA) assay showed specific binding to an HPA-1a flow cell (mean, 87 ± 21 resonance units [RU]). When MAIPA-negative sera were tested in SPR, binding with low avidity was observed in 7 of 14 to HPA-1a (mean, 31 ± 5 RU), but not to HPA-1b flow cell (mean, 5 ± 2 RU). In vivo, low-avidity antibodies were capable of clearing HPA-1ab PLTs but not HPA-1bb PLTs in a NOD/SCID mouse model. Elimination kinetics were slower than observed with MAIPA-positive antibodies. CONCLUSIONS: Low-avidity HPA-1a antibodies are present in a significant number of NAIT cases and, although they can escape detection by standard serology, they harbor the capability of PLT destruction in vivo.  

4.970           Ginger Phenylpropanoids Inhibit IL-1β and Prostanoid Secretion and Disrupt Arachidonate-Phospholipid Remodeling by Targeting Phospholipases A2

Nievergelt, A., Marazzi, J., Schoop, r., Altmann, K-H. and Gertsch, J.

1. Immunol., 187(8), 4140-4150 (2011)

  The rhizome of ginger (Zingiber officinale) is employed in Asian traditional medicine to treat mild forms of rheumatoid arthritis and fever. We have profiled ginger constituents for robust effects on proinflammatory signaling and cytokine expression in a validated assay using human whole blood. Independent of the stimulus used (LPS, PMA, anti-CD28 Ab, anti-CD3 Ab, and thapsigargin), ginger constituents potently and specifically inhibited IL-1β expression in monocytes/macrophages. Both the calcium-independent phospholipase A₂ (iPLA₂)-triggered maturation and the cytosolic phospholipase A₂ (cPLA₂)-dependent secretion of IL-1β from isolated human monocytes were inhibited. In a fluorescence-coupled PLA₂ assay, most major ginger phenylpropanoids directly inhibited i/cPLA₂ from U937 macrophages, but not hog pancreas secretory phospholipase A₂. The effects of the ginger constituents were additive and the potency comparable to the mechanism-based inhibitor bromoenol lactone for iPLA₂ and methyl arachidonyl fluorophosphonate for cPLA₂, with 10-gingerol/-shogaol being most effective. Furthermore, a ginger extract (2 μg/ml) and 10-shogaol (2 μM) potently inhibited the release of PGE₂ and thromboxane B2 (>50%) and partially also leukotriene B₄ in LPS-stimulated macrophages. Intriguingly, the total cellular arachidonic acid was increased 2- to 3-fold in U937 cells under all experimental conditions. Our data show that the concurrent inhibition of iPLA₂ and prostanoid production causes an accumulation of free intracellular arachidonic acid by disrupting the phospholipid deacylation-reacylation cycle. The inhibition of i/cPLA₂, the resulting attenuation of IL-1β secretion, and the simultaneous inhibition of prostanoid production by common ginger phenylpropanoids uncover a new anti-inflammatory molecular mechanism of dietary ginger that may be exploited therapeutically.  

4.971           Helicobacter pylori DNA decreases pro-inflammatory cytokine production by dendritic cells and attenuates dextran sodium sulphate-induced colitis

Luther, J., Owyang, S.Y., takeuchi, T., Cole, T.S., Zhang, M., Liu, M., Erb-Downward, J., Rubenstein, J.H., Chen, C-C., Pierzchala, A.V., Paul, J.A. and Kao, J.Y. Gut, 60, 1479-1486 (2011)   Background and aims Epidemiological data have recently emerged to suggest Helicobacter pylori may protect against certain chronic inflammatory diseases such as inflammatory bowel disease (IBD). However, the mechanism for the observed inverse association between H pylori and IBD has not been described. Methods The frequency of immunoregulatory (IRS) to immunostimulatory (ISS) sequences within the genome of various bacteria was calculated using MacVector software. The induction of type I IFN and IL-12 responses by DNA-pulsed murine bone marrow-derived dendritic cells (BMDC) and human plasmacytoid dendritic cells (DC) was analysed by cytokine production. The effect of H pylori DNA on Escherichia coli DNA production of type I IFN and IL-12 was assessed. The in-vivo significance of H pylori DNA suppression was assessed in a dextran sodium sulphate (DSS) model of colitis. The systemic levels of type I IFN were assessed in H pylori-colonised and non-colonised patients. Results H pylori DNA has a significantly elevated IRS:ISS ratio. In-vitro experiments revealed the inability of H pylori DNA to stimulate type I IFN or IL-12 production from mouse BMDC or human plasmacytoid DC. H pylori DNA was also able to suppress E coli DNA production of type I IFN and IL-12. The administration of H pylori DNA before the induction of DSS colitis significantly ameliorated the severity of colitis compared with E coli DNA or vehicle control in both an acute and chronic model. Finally, the systemic levels of type I IFN were found to be lower in H pylori-colonised patients than non-colonised controls. Conclusions This study indicates that H pylori DNA has the ability to downregulate pro-inflammatory responses from DC and this may partly explain the inverse association between H pylori and IBD.        

4.972           Mitochondrial calcium and its regulation in neurodegeneration induced by oxidative stress

Barsukova, A.G., Bourdette, D. and Forte, M. Eur. J. Neurosci., 34(3), 437-447 (2011)   A proposed mechanism of neuronal death associated with a variety of neurodegenerative diseases is the response of neurons to oxidative stress and consequent cytosolic Ca²⁺ overload. One hypothesis is that cytosolic Ca²⁺ overload leads to mitochondrial Ca²⁺ overload and prolonged opening of the permeability transition pore (PTP), resulting in mitochondrial dysfunction. Elimination of cyclophilin D (CyPD), a key regulator of the PTP, results in neuroprotection in a number of murine models of neurodegeneration in which oxidative stress and high cytosolic Ca²⁺ have been implicated. However, the effects of oxidative stress on the interplay between cytosolic and mitochondrial Ca²⁺ in adult neurons and the role of the CyPD-dependent PTP in these dynamic processes have not been examined. Here, using primary cultured cerebral cortical neurons from adult wild-type (WT) mice and mice missing cyclophilin D (CyPD-KO), we directly assess cytosolic and mitochondrial Ca²⁺, as well as ATP levels, during oxidative stress. Our data demonstrate that during acute oxidative stress mitochondria contribute to neuronal Ca²⁺ overload by release of their Ca²⁺ stores. This result contrasts with the prevailing view of mitochondria as a buffer of cytosolic Ca²⁺ under stress conditions. In addition, we show that CyPD deficiency reverses the release of mitochondrial Ca²⁺, leading to lower of cytosolic Ca²⁺ levels, attenuation of the decrease in cytosolic and mitochondrial ATP, and a significantly higher viability of adult CyPD-knockout neurons following exposure of neurons oxidative stress. The study offers a first insight into the mechanism underlying CyPD-dependent neuroprotection during oxidative stress.  

4.973           Mechanism for antioxidative effects of thiazolidinediones in pancreatic β-cells

Chung, S.S., Kim, M., Lee, J.S., Ahn, B.Y., Jung, H.S., Lee, H.M. and Park, K.S. Am. J. Physiol. Endocrinol. Metab., 301, E912-E921 (2011)   Thiazolidinediones (TZDs) are synthetic ligands of peroxisome proliferator-activated receptor-γ (PPARγ), a member of the nuclear receptor superfamily. TZDs are known to increase insulin sensitivity and also to have an antioxidative effect. In this study, we tested whether TZDs protect pancreatic β-cells from oxidative stress, and we investigated the mechanism involved in this process. To generate oxidative stress in pancreatic β-cells (INS-1 and βTC3) or isolated islets, glucose oxidase was added to the media. The extracellular and intracellular reactive oxygen species (ROS) were measured to directly determine the antioxidant effect of TZDs. The phosphorylation of JNK/MAPK after oxidative stress was detected by Western blot analysis, and glucose-stimulated insulin secretion and cell viability were also measured. TZDs significantly reduced the ROS levels that were increased by glucose oxidase, and they effectively prevented β-cell dysfunction. The antioxidative effect of TZDs was abolished in the presence of a PPARγ antagonist, GW9662. Real-time PCR was used to investigate the expression levels of antioxidant genes. The expression of catalase, an antioxidant enzyme, was increased by TZDs in pancreatic β-cells, and the knockdown of catalase significantly inhibited the antioxidant effect of TZDs. These results suggest that TZDs effectively protect pancreatic β-cells from oxidative stress, and this effect is dependent largely on PPARγ. In addition, the expression of catalase is increased by TZDs, and catalase, at least in part, mediates the antioxidant effect of TZDs in pancreatic β-cells.  

4.974           Loss of CFTR Affects Biliary Epithelium Innate Immunity and Causes TLR4–NF-κB—Mediated Inflammatory Response in Mice

Fiorotto, R., Scirpo, R., Trauner, M., Fabris, L., Hoque, R., Spirli, C. and Strazzabosco, M. Gastroenterology, 141(4), 1498-1508 (2011)   Background & Aims Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) in the biliary epithelium reduces bile flow and alkalinization in patients with cystic fibrosis (CF). Liver damage is believed to result from ductal cholestasis, but only 30% of patients with CF develop liver defects, indicating that another factor is involved. We studied the effects of CFTR deficiency on Toll-like receptor 4 (TLR4)-mediated responses of the biliary epithelium to endotoxins. Methods Dextran sodium sulfate (DSS) was used to induce colitis in C57BL/6J-Cftr^tm1Unc (Cftr-KO) mice and their wild-type littermates. Ductular reaction and portal inflammation were quantified by keratin-19 and CD45 immunolabeling. Cholangiocytes isolated from wild-type and Cftr-KO mice were challenged with lipopolysaccharide (LPS); cytokine secretion was quantified. Activation of nuclear factor κB (NF-κB), phosphorylation of TLR4, and activity of Src were determined. HEK-293 that expressed the secreted alkaline phosphatase reporter and human TLR4 were transfected with CFTR complementary DNAs. Results DSS-induced colitis caused biliary damage and portal inflammation only in Cftr-KO mice. Biliary damage and inflammation were not attenuated by restoring biliary secretion with 24-nor-ursodeoxycholic acid but were significantly reduced by oral neomycin and polymyxin B, indicating a pathogenetic role of gut-derived bacterial products. Cftr-KO cholangiocytes incubated with LPS secreted significantly higher levels of cytokines regulated by TLR4 and NF-κB. LPS-mediated activation of NF-κB was blocked by the TLR4 inhibitor TAK-242. TLR4 phosphorylation by Src was significantly increased in Cftr-KO cholangiocytes. Expression of wild-type CFTR in the HEK293 cells stimulated with LPS reduced activation of NF-κB. Conclusions CFTR deficiency alters the innate immunity of the biliary epithelium and reduces its tolerance to endotoxin, resulting in an Src-dependent inflammatory response mediated by TLR4 and NF-κB. These findings might be used to develop therapies for CF-associated cholangiopathy.  

4.975           Preferential Expression of Integrin αvβ8 Promotes Generation of Regulatory T Cells by Mouse CD103+ Dendritic Cells

Paidassi, H., Acharya, M., Zhang, A., Mukhopadhyay, S., Kwon, M., Chow, C., Stuart, L.M., Savill, J. and Lacy-Hulbert, A. Gastroenterology, 141(5), 1813-1820 (2011)   Background & Aims Immune responses in the intestine are controlled by regulatory T cells (Tregs), which prevent inflammation in response to commensal bacteria. A specific population of intestinal dendritic cells (DCs), marked by expression of CD103, generate Tregs more efficiently than other DC populations through mechanisms that involve retinoic acid and transforming growth factor (TGF)-β. However, it is not clear how CD103⁺ DCs are specialized for this function. We investigated the ability of CD103⁺ DCs to promote Treg generation through activation of TGF-β and the role of integrins with the αv subunit in this process. Methods Naïve T cells were cultured with purified DCs from mesenteric lymph nodes (MLNs) or intestines of wild-type and αv conditional knockout mice to assess generation of Tregs. Antigens were administered orally to mice, and antigen-specific generation of Tregs was measured in intestinal tissues. Expression of the integrin αv subunit was measured in purified subpopulations of DCs by quantitative polymerase chain reaction and immunoblot analyses. Results In vitro, CD103⁺ DCs generated more Tregs in the presence of latent TGF-β than other MLN DCs. Efficient generation of Tregs required expression of the integrin αv subunit by DCs; mice that lacked αv in immune cells did not convert naïve T cells to intestinal Tregs in response to oral antigen. CD103⁺ DCs derived from the MLNs selectively expressed high levels of integrin αvβ8 compared with other populations of DCs. Conclusions Expression of αvβ8 is required for CD103⁺ DCs to become specialized and activate latent TGF-β and generate Tregs during the induction of tolerance to intestinal antigens in mice.  

4.976           Increased Expression of the Transient Receptor Potential Melastatin 7 Channel Is Critically Involved in Lipopolysaccharide-Induced Reactive Oxygen Species-Mediated Neuronal Death

Nunez-Villena, F., Becerra, A., Echeverria, C., Briceno, N., Porras, O., Armisen, R., Varela, D., Montorfano, I., Sarmiento, D. and Simon, F. Antioxidants & Redox Signaling, 15(9), 2425-2438 (2011)   Aims: To assess the mechanisms involved in lipopolysaccharide (LPS)-induced neuronal cell death, we examined the cellular consequences of LPS exposure in differentiated PC12 neurons and primary hippocampal neurons. Results: Our data show that LPS is able to induce PC12 neuronal cell death without the participation of glial cells. Neuronal cell death was mediated by an increase in cellular reactive oxygen species (ROS) levels. Considering the prevalent role of specific ion channels in mediating the deleterious effect of ROS, we assessed their contribution to this process. Neurons exposed to LPS showed a significant intracellular Ca²⁺ overload, and nonselective cationic channel blockers inhibited LPS-induced neuronal death. In particular, we observed that both LPS and hydrogen peroxide exposure strongly increased the expression of the transient receptor protein melastatin 7 (TRPM7), which is an ion channel directly implicated in neuronal cell death. Further, both LPS-induced TRPM7 overexpression and LPS-induced neuronal cell death were decreased with dithiothreitol, dipheniliodonium, and apocynin. Finally, knockdown of TRPM7 expression using small interference RNA technology protected primary hippocampal neurons and differentiated PC12 neurons from the LPS challenge. Innovation: This is the first report showing that TRPM7 is a key protein involved in neuronal death after LPS challenge. Conclusion: We conclude that LPS promotes an abnormal ROS-dependent TRPM7 overexpression, which plays a crucial role in pathologic events, thus leading to neuronal dysfunction and death.  

4.977           Ineffective CD8+ T-Cell Immunity to Adeno-Associated Virus Can Result in Prolonged Liver Injury and Fibrogenesis

Spahn, J., Pierce, R.H. and Crispe, I.N. Am. J. Pathol., 179(5), 2370-2381 (2011)   Chronic viral hepatitis depends on the inability of the T-cell immune response to eradicate antigen. This results in a sustained immune response accompanied by tissue injury and fibrogenesis. We have created a mouse model that reproduces these effects, based on the response of CD8⁺ T cells to hepatocellular antigen delivered by an adeno-associated virus (AAV) vector. Ten thousand antigen-specific CD8⁺ T cells undergo slow expansion in the liver and can precipitate a subacute inflammatory hepatitis with stellate cell activation and fibrosis. Over time, antigen-specific CD8⁺ T cells show signs of exhaustion, including high expression of PD-1, and eventually both inflammation and fibrosis resolve. This model allows the investigation of both chronic liver immunopathology and its resolution.  

4.978           Steady state pig dendritic cells migrating in skin draining pseudo-afferent lymph are semi-mature

Bertho, N., marquet, F., Pascale, F., Kang, C., Bonneau, M. and Schwartz-Cornil, I. Vet. Immunol. Immunopathol., 144, 430-436 (2011)   Dendritic cells (DC) in peripheral tissues are considered as immature cells that mature and migrate towards lymph nodes upon stimulation with pathogens. This commonly accepted paradigm is challenged by the fact that tolerance to peripheral self antigen is controlled by mature DC and that DC collected from afferent lymph draining different tissues from several species, in the absence of pathogen signaling, were inconsistently found to be either at a mature or semi-mature state. In order to better define the maturation state of DC that migrate in lymph in absence of pathogen stimulation, we compared skin lymph DC to resident and LPS (lipopolysaccharide)-activated skin DC thanks to the establishment of a mini-pig model of lymph duct cannulation. Based on their co-stimulatory molecules expression and endocytotic capacities, pig lymph skin DC were found at an intermediate state of maturation between resident and LPS-activated skin DC and were fully capable of allogeneic T cell stimulation. Furthermore, lymph skin DC could be further matured by LPS or influenza stimulation. Thus, using the pig skin model which is relevant to human, we show that skin-derived DC constantly migrate at an intermediate state of maturation that can be further enhanced upon appropriate stimulation.  

4.979           Sperm selection using single layer centrifugation prior to cryopreservation can increase thawed sperm quality in stallions

Hoogewijs, M., Morrell, J., Van Soom, A., Govaere, J., Johannisson, A., Piepers, S., De Schauwer, C., De Kruif, A. and De Vliegher, S. Equine Vet. J., 43 8Suppl 40), 35-41 (2011)   Reasons for performing study: The increasing use of modern reproductive techniques in human medicine has led to a higher demand for isolation of motile sperm. Several of these isolation techniques have been adapted for veterinary use and can be applied for the selection of a superior sperm sample from stallion semen. Until recently a major disadvantage of such isolation techniques was the limitation in sperm volume that could be handled. Androcoll-E had been shown to be successful for processing large volumes of equine semen but there are few data to substantiate the potential beneficial effect of freezing an Androcoll-E selected equine sperm sample to obtain higher quality following thawing.   Objectives and methods: In this study, the effect of Androcoll-E treatment of sperm prior to cryopreservation was compared with cushioned centrifugation using ejaculates from 8 different stallions selected because they were known to have semen of differing quality following freezing.   Results: Androcoll-E treatment increased measures of semen quality prior to freezing. However, Androcoll-E treatment reduced the yield of sperm following centrifugation when compared with the cushion centrifuged control group (50.9 ± 14.2% vs. 97.1 ± 9.0%, respectively). Quality analysis following thawing showed an overall improved sperm quality for Androcoll-E treated samples and average post thaw progressive motility (PM) was 41.6% compared with 30.5% for the cushion centrifuged group.   Conclusions and potential relevance: Androcoll-E can be used with good results to select a superior sperm population prior to cryopreservation, in order to produce good-quality frozen thawed semen.    

4.980           Islet isolation from adult designated pathogen-free pigs: use of the newer bovine nervous tissue–free enzymes and a revised donor selection strategy would improve the islet graft function

Jin, S-M., Shin, J.S., Kim, K.S., Gong, C-H., park, S.K., Kim, J-S., Yeom, S-C., Hwang, E.S., Lee, C.T., Kim, S-J., and Park, C-G. Xenotransplantation, 18, 369-379 (2011)   Background:  In clinical trials using adult porcine islet products, islets should be isolated from the designated pathogen-free (DPF) pigs under the current good manufacturing practice (GMP) regulations. Our previous studies suggested that male DPF pigs are better donors than retired breeder pigs and histomorphometrical parameters of donor pancreas predict the porcine islet quality. We aimed to investigate whether the use of the newer bovine nervous tissue–free enzymes and a revised donor selection strategy could improve the islet graft function in the context of islet isolation with DPF pigs. Methods:  Using 30 DPF pigs within a closed herd, we compared the islet yield of porcine islets isolated with Liberase PI (n = 11, as a historical control group), Liberase MTF C/T, which is a GMP-grade enzyme (n = 12), and CIzyme collagenase MA/BP protease (n = 7). We analyzed the relationship between the diabetes reversal rate of recipient NOD/SCID mice (n = 75) and histomorphometric parameters of each donor pancreas as well as donor characteristics. Results:  Proportion of islets larger than 200 μm from the biopsied donor pancreas (P = 0.006) better predicted islet yield than age (P = 0.760) or body weight (P = 0.371) of donor. The proportion of islets larger than 200 μm from the biopsied donor pancreas was not related to the sex of the donor miniature pig (P = 0.358). The islet yield obtained with the three enzymes did not differ, even after stratification of the donor with the histomorphometric parameters of the biopsied donor pancreas and the sex of donor. The use of the newer bovine nervous tissue–free enzymes (P < 0.001), a higher proportion of large islets in donor pancreas (P = 0.006), and a male sex of the donor (P = 0.025) were independent predictors of earlier diabetes reversal. Conclusions:  Use of the newer bovine nervous tissue–free enzymes including a GMP-grade enzyme resulted in better islet quality than that of islet isolated using Liberase PI. To obtain high-quality islet from DPF pigs, the donor should be male pig and histomorphometrical parameters from donor pancreas should be considered.

4.981           Lung CD103+ Dendritic Cells Efficiently Transport Influenza Virus to the Lymph Node and Load Viral Antigen onto MHC Class I for Presentation to CD8 T Cells

Ho, A.W.S., Prabhu, N., Betts, R.J., Ge, M.Q., Dai, X., Hutchinson, P.E., Lew, F.C., Wong, K.L., Hanson, B.J., macary, P.A. and Kemeny, D.M.

1. Immunol., 187(11), 6011-6021 (2011)

  The uptake, transport, and presentation of Ags by lung dendritic cells (DCs) are central to the initiation of CD8 T cell responses against respiratory viruses. Although several studies have demonstrated a critical role of CD11b^low/negCD103⁺ DCs for the initiation of cytotoxic T cell responses against the influenza virus, the underlying mechanisms for its potent ability to prime CD8 T cells remain poorly understood. Using a novel approach of fluorescent lipophilic dye-labeled influenza virus, we demonstrate that CD11b^low/negCD103⁺ DCs are the dominant lung DC population transporting influenza virus to the posterior mediastinal lymph node as early as 20 h postinfection. By contrast, CD11b^highCD103^neg DCs, although more efficient for taking up the virus within the lung, migrate poorly to the lymph node and remain in the lung to produce proinflammatory cytokines instead. CD11b^low/negCD103⁺ DCs efficiently load viral peptide onto MHC class I complexes and therefore uniquely possess the capacity to potently induce proliferation of naive CD8 T cells. In addition, the peptide transporters TAP1 and TAP2 are constitutively expressed at higher levels in CD11b^low/negCD103⁺ DCs, providing, to our knowledge, the first evidence of a distinct regulation of the Ag-processing pathway in these cells. Collectively, these results show that CD11b^low/negCD103⁺ DCs are functionally specialized for the transport of Ag from the lung to the lymph node and also for efficient processing and presentation of viral Ags to CD8 T cells.  

4.982           Small molecule tyrosine kinase inhibitors for the treatment of intestinal inflammation

Sidhu, M., Cotoner, C.A., Guleng, B., Arihiro, S., Chang, S., Duncan, K.W., Ajami, A.M., Chau, M. and Reinecker, H-C. Inflamm. Bowel Dis., 17(12), 2416-2426 (2011)   Background: We developed a series of dendritic cell autoimmune modulators (DCAMs) based on small molecule Flt3 receptor tyrosine kinase inhibitors (TKIs) for the inhibition of intestinal inflammation and oral delivery. Methods: DCAMs were administered orally during and after induction of dextran sodium sulfate (DSS)-induced colitis. Dendritic cell recruitment and inflammatory responses were determined in the mucosal immune system during acute intestinal inflammatory responses and mucosal recovery. Bone marrow-derived macrophages were utilized to define the mechanisms by which DCAMs can modify responses to microbial signals. Results: Oral doses of DCAMs prevented severe weight loss and mucosal inflammation associated with DSS colitis in mice. The presence of DCAMs increased the number of CD11c⁺PDCA1⁺ dendritic cells, induced interleukin (IL)-10 expression, and reduced inflammatory cytokine expression in the mucosal immune system. Surprisingly, DCAMs regulated innate immune responses in macrophages resulting in the inhibition of tumor necrosis factor alpha (TNF-α) production and the induction of IL-10 expression during Toll-like receptor-mediated signaling. Conclusions: We identified two new imidazoacridinone derivatives that protect mice from severe colitis and promote mucosal recovery by enhancing protective cytokine production while inhibiting proinflammatory stimuli during microbial recognition. These compounds are promising candidates for further development into potent orally available drugs for the prevention of colitis and promotion of mucosal recovery.  

4.983           Successful Isolation and Transplantation of Nonhuman Primate Islets Using a Novel Purified Enzyme Blend

Abouaish, J., Graham, M., Bansal-Pakala, P., Loganathan, G., Soltani, S.M., Tiwari, M., Yusa, T., papas, K.K., Sutherland, D.E.R., Mccarthy, R.C., Hering, B.J. andBalamurugan, A.N. Transplantation, 92(8), e40-e42 (2011)   No abstract available.  

4.984           Natural killer (NK)–dendritic cell interactions generate MHC class II-dressed NK cells that regulate CD4+ T cells

Nakayama, M., Takeda, K., Kawano, M., takai, T., Ishii, N. and Ogasawara, K. PNAS, 108(45), 18360-18365 (2011)   Natural killer (NK) cells contribute to not only innate but also to adaptive immunity by interacting with dendritic cells (DCs) and T cells. All activated human NK cells express HLA-DR and can initiate MHCII-dependent CD4⁺ T-cell proliferation; however, the expression of MHCII by mouse NK cells and its functional significance are controversial. In this study, we show that NK–DC interactions result in the emergence of MHCII-positive NK cells. Upon in vitro or in vivo activation, mouse conventional NK cells did not induce MHCII transcripts, but rapidly acquired MHCII protein from DCs. MHCII H2-Ab1–deficient NK cells turned I-A^b-positive when adoptively transferred into wild-type mice or when cultured with WT splenic DCs. NK acquisition of MHCII was mediated by intercellular membrane transfer called “trogocytosis,” but not upon DAP10/12- and MHCI-binding NK cell receptor signaling. MHCII-dressed NK cells concurrently acquired costimulatory molecules such as CD80 and CD86 from DCs; however, their expression did not reach functional levels. Therefore, MHCII-dressed NK cells inhibited DC-induced CD4⁺ T-cell responses rather than activated CD4⁺ T cells by competitive antigen presentation. In a mouse model for delayed-type hypersensitivity, adoptive transfer of MHCII-dressed NK cells attenuated footpad swelling. These results suggest that MHCII-dressed NK cells generated through NK–DC interactions regulate T cell-mediated immune responses.  

4.985           In situ induction of dendritic cell–based T cell tolerance in humanized mice and nonhuman primates

Jung, K.C. et al

1. Exp. Med., 208(12), 2477-2488 (2011)

  Induction of antigen-specific T cell tolerance would aid treatment of diverse immunological disorders and help prevent allograft rejection and graft versus host disease. In this study, we establish a method of inducing antigen-specific T cell tolerance in situ in diabetic humanized mice and Rhesus monkeys receiving porcine islet xenografts. Antigen-specific T cell tolerance is induced by administration of an antibody ligating a particular epitope on ICAM-1 (intercellular adhesion molecule 1). Antibody-mediated ligation of ICAM-1 on dendritic cells (DCs) led to the arrest of DCs in a semimature stage in vitro and in vivo. Ablation of DCs from mice completely abrogated anti–ICAM-1–induced antigen-specific T cell tolerance. T cell responses to unrelated antigens remained unaffected. In situ induction of DC-mediated T cell tolerance using this method may represent a potent therapeutic tool for preventing graft rejection.  

4.986           An Effective Method to Release Human Islets From Surrounding Acinar Cells With Agitation in High Osmolality Solution

Shimoda, M., Itoh, T., Sugimoto, K., Takita, M., Chujo, D., Iwahashi, S., SoRelle, J.A., Naziruddin, B., Levy, M.F., Grayburn, P.A. and Matsumoto, S. Transplantation Proceedings, 43, 3161-3166 (2011)   Introduction Islet purification is mainly performed by the density gradient method. However, purification of the embedded islets that are surrounded by exocrine tissue should be difficult, because their density is similar to exocrine tissue. In this study, we performed chart review to assess the relationship between the ratio of embedded islets and efficacy of purification. Then, we tested several conditions of a new method to free the islets from surrounded exocrine tissues using high osmolality solution with gentle agitation. Materials and Methods First, we performed chart review of our human islet isolation. Second, embedded islet-enriched human islet fractions (embedded islets >50%) were suspended in University of Wisconsin (UW) solution (UW group, 320 mOsm/kg/H₂0) or osmolality-adjusted UW solution (400, 500, and 600 mOsm/kg/H₂0; 400 group, 500 group, and 600 group, respectively). Each tube was gently shaken at 4°C. The tissue samples were taken before shaking and after 15, 30, and 60 minutes. Islet yield, percentage of embedded islets, and viabilities were assessed. Results The chart review revealed that high ratio of embedded islets deteriorated the efficacy of islet purification. The islet yield in all groups except for the 600 group did not change at 15 minutes, but it decreased in all groups at 60 minutes. The average percentage of embedded islets before shaking was 62.6%. Although percentage of embedded islets were decreasing in all groups, it was < 20% at 15 minutes in the 500 and 600 groups whereas it was >44% in the UW group, which indicated that higher osmolality would have a greater effect. Viability was >95% in all groups at 30 minutes. Conclusions The embedded islets deteriorated the efficacy of islet purification. Gentle agitation of embedded islets in high osmolality (500 mOsm/kg/H₂O, 15 minutes) could release islets from surrounded exocrine tissue.  

4.987           Usefulness of the Secretory Unit of Islet Transplant Objects (SUITO) Index for Evaluation of Clinical Autologous Islet Transplantation

Matsumoto, S., Takita, M., Shimoda, M., Itoh, T., Iwahashi, S., Chujo, D., SoRelle, J.A., Tamura, Y., Tahman, A., Purcell, K., Onaca, N., Naziruddin, B. and Levy, M.F. Transplantation Proceedings, 43, 3246-3249 (2011)   Background Assessing the engrafted islet mass is important in evaluating the efficacy of islet transplantation. We previously demonstrated that the average secretory unit of islet transplant objects (SUITO) index within 1 month of allogeneic islet transplantation was an excellent predictor of insulin independence. However, the usefulness of the SUITO index for evaluating autologous islet transplantation has not been explored. The purpose of the present study was to assess the relationship between the SUITO index and clinical outcomes after total pancreatectomy followed by autologous islet transplantation. Methods We performed 27 total pancreatectomies followed by autologous islet transplantation from October 2006 to January 2011. Cases were divided into an insulin-independent group (IIG; n = 12) and an insulin-dependent group (lDG; n = 15). The SUITO index was calculated by the formula [fasting C-peptide (ng/mL)/fasting glucose (mg/dL) −63] × 1,500. The average SUITO index within the first month of transplantation except for days 0, 1, and 2, maximum SUITO index, and most recent SUITO index were calculated in each case, and values were compared between the IIG and the IDG. Results The average SUITO index within 1 month was significantly higher in the IIG than in the IDG (24.6 ± 3.4 vs 14.9 ± 2.0, respectively; P < .02). The maximum SUITO indices were 45.7 ± 7.7 in the IIG and 30.1 ± 8.1 in the IDG (not significant), and the recent SUITO indices were 36.9 ± 6.7 in the IIG and 22.8 ± 6.1 in the IDG (not significant). Conclusions The average SUITO index within 1 month was an excellent predictor of insulin independence after total pancreatectomy followed by autologous islet transplantation.  

4.988           Human Alveolar Epithelial Cell Injury Induced by Cigarette Smoke

Kosmider, B., Messier, E.M., Chu, H.W. and Mason, R.J. PloS One, 6(12), e26059 (2011)   Background Cigarette smoke (CS) is a highly complex mixture and many of its components are known carcinogens, mutagens, and other toxic substances. CS induces oxidative stress and cell death, and this cell toxicity plays a key role in the pathogenesis of several pulmonary diseases. Methodology/Principal Findings We studied the effect of cigarette smoke extract (CSE) in human alveolar epithelial type I-like (ATI-like) cells. These are isolated type II cells that are differentiating toward the type I cell phenotype in vitro and have lost many type II cell markers and express type I cell markers. ATI-like cells were more sensitive to CSE than alveolar type II cells, which maintained their differentiated phenotype in vitro. We observed disruption of mitochondrial membrane potential, apoptosis and necrosis that were detected by double staining with acridine orange and ethidium bromide or Hoechst 33342 and propidium iodide and TUNEL assay after treatment with CSE. We also detected caspase 3 and caspase 7 activities and lipid peroxidation. CSE induced nuclear translocation of Nrf2 and increased expression of Nrf2, HO-1, Hsp70 and Fra1. Moreover, we found that Nrf2 knockdown sensitized ATI-like cells to CSE and Nrf2 overexpression provided protection against CSE-induced cell death. We also observed that two antioxidant compounds N-acetylcysteine and trolox protected ATI-like cells against injury by CSE. Conclusions Our study indicates that Nrf2 activation is a major factor in cellular defense of the human alveolar epithelium against CSE-induced toxicity and oxidative stress. Therefore, antioxidant agents that modulate Nrf2 would be expected to restore antioxidant and detoxifying enzymes and to prevent CS-related lung injury and perhaps lessen the development of emphysema.  

4.989           Association Between the Secretory Unit of Islet Transplant Objects Index and Satisfaction With Insulin Therapy Among Insulin-Dependent Islet Recipients

Takita, M., Matsumoto, S., Shimoda, M., Chujo, D.,Itoh, T., Iwaaaahashi, S., SoRelle, J.A., Onaca, N., Nazxiruddin, B. and Levy, M.F. Transplantation Proceedings, 43, 3250-3255 (2011)   Introduction When patients do not become insulin independent after islet cell transplantation (ICT), another aim is to eliminate severe hypoglycemia. Previously we reported that a secretory unit of islet transplant objects (SUITO) index score >10 was associated with a reduction of severe hypoglycemia. In this study, we assessed patients' satisfaction with their insulin therapy based on the SUITO index. Methods The study involved 11 islet recipients with type 1 diabetes who underwent ICT but still used insulin. From those patients, 41 Insulin Therapy Satisfaction Questionnaires (ITSQ) were collected. The SUITO index (fasting C-peptide [ng/mL] × 1500/blood glucose [mg/dL] − 63) was calculated at the same outpatient visits that the survey was administered. ITSQ scores were summarized using subscales and compared among 3 groups: the pre-ICT group, the low-SUITO group (SUITO index score <10 post-ICT), and the high-SUITO group (SUITO index score ≥10). Higher survey scores indicated better satisfaction. Results Significant trend relationships across the 3 groups were observed in the ITSQ total score (P = .02 with Jonckheere-Terpstra test) and subscale scores of glycemic control (P < .001), hypoglycemic control (P = .01), and inconvenience of regimen (P = .004). The pairwise comparisons between the 3 groups found significant differences: high SUITO versus both pre-ICT and low SUITO for the total ITSQ score (P = .03 and .005, respectively) and glycemic control score (P = .008 and .001, respectively), and high SUITO versus low SUITO for hypoglycemic control score (P = .04) and inconvenience of regimen score (P = .008). Conclusion Islet recipients with a SUITO index ≥10 experienced higher satisfaction with insulin injection therapy compared with the pre-ICT group, even though they were insulin dependent. A SUITO index ≥10 is a reasonable benchmark for successful ICT.  

4.990           Autologous islet cell transplantation to prevent surgical diabetes

Matsumoto, S.

1. Diabetes, 3(4), 328-336 (2011)

  Autologous islet transplantation (AIT) is performed to prevent surgical diabetes after total or semi-total pancreatectomy for the treatment of chronic pancreatitis with severe abdominal pain. In addition, AIT is used in cases of benign pancreatic tumors and pancreatic trauma. It has been shown that AIT results in better outcomes in terms of glycemic control compared with allogeneic islet transplantation. The reasons for the favorable outcomes of AIT are thought to be: (i) patients have no autoimmune diseases; (ii) the transplanted islets do not suffer allogeneic rejection; (iii) diabetogenic antirejection drugs are not required; (iv) pancreata do not undergo a cytokine storm as a result of periods of brain death; (v) the period of cold preservation of retrieved pancreata is short; (vi) the isolated islets are immediately transplanted without culture; and (vii) pancreata with pancreatitis may contain more progenitor cells. Further research into AIT would help improve the results of allogeneic islet transplantation. Conversely, the technical difficulties associated with islet isolation appear to be the largest hurdle for AIT; therefore, remote center islet isolation may prove to be key in the promotion of this treatment.  

4.991           Insulin independence by supplemental islet transplantation 5 years after initial islet transplantation

Matsumoto, S., Takita, M., Shimoda, M., Chujo, D., Itoh, T., Iwaaaaaaaaaahashi, S., Sorelle, J.A., Tamura, Y., Rahman, A., Purcell, K., Naziruddin, B., Onaca, N. and Levy, M.F.

1. Diabetes, 3(4), 353-355 (2011)

  The Edmonton protocol, which proved islet cell transplantation could make Type 1 diabetic patients insulin independent, was introduced in 2000.1 However, in 2005, the same Edmonton group demonstrated that <10% of recipients could maintain insulin independence. 2 Therefore, >90% of patients who received islet transplantation experienced decreased islet function and resumed exogenous insulin injections. Most patients could maintain better glycemic control after resuming insulin injections,2 but still hoped to again be insulin independent. Here we report a case of supplemental islet transplantation in a patient wh received initial islet transplantation >5 years earlier. The patient had achieved temporary insulin independence and maintained some islet function before the supplemental islet infusion. Now, the patient has again become insulin independent.  

4.992           Velocity Effect on Aptamer-Based Circulating Tumor Cell Isolation in Microfluidic Devices

Wan, Y., Tan, J., Asghar, W.,. Kim, Y-t., Liu, Y. and Iqbal, S.M.

1. Phys. Chem. B., 115(47), 13891-13896 (2011)

  The isolation and detection of rare circulating tumor cells (CTCs) has been one of the focuses of intense research recently. In a microfluidic device, a number of factors can influence the enrichment capability of surface-bound probe molecules. This article analyzes the important factor of flow velocity in a microfluidic channel. The competition of surface-grafted anti-EGFR aptamers to bind the overexpressed EGFR on cell membranes against the drag force from the fluid flow is an important efficiency determining factor. The flow rate variations are applied both in experiments and in simulation models to study their effects on CTC capture efficiency. A mixture of mononuclear cells and human Glioblastoma cells is used to isolate cancer cells from the cellular flow. The results show interdependence between the adhesion probability, isolation efficiency, and flow rate. This work can help in designing flow-through lab-on-chip devices that use surface-bound probe affinities against overexpressed biomarkers for cell isolation. This work demonstrates that microfluidic based approaches have strong potential applications in CTC detection and isolation.  

4.993           Mechanisms of Slower Nitric Oxide Uptake by Red Blood Cells and Other Hemoglobin-containing Vesicles

Azarov, I., Liu, C., Reynolds, H., Tsekouras, Z., Lee, J.S., Gladwin, M.T. and Kim-Shapiro, D.B.

1. Biol. Chem., 286(39), 33567-33579 (2011)

  Nitric oxide (NO) acts as a smooth muscle relaxation factor and plays a crucial role in maintaining vascular homeostasis. NO is scavenged rapidly by hemoglobin (Hb). However, under normal physiological conditions, the encapsulation of Hb inside red blood cells (RBCs) significantly retards NO scavenging, permitting NO to reach the smooth muscle. The rate-limiting factors (diffusion of NO to the RBC surface, through the RBC membrane or inside of the RBC) responsible for this retardation have been the subject of much debate. Knowing the relative contribution of each of these factors is important for several reasons including optimization of the development of blood substitutes where Hb is contained within phospholipid vesicles. We have thus performed experiments of NO uptake by erythrocytes and microparticles derived from erythrocytes and conducted simulations of these data as well as that of others. We have included extracellular diffusion (that is, diffusion of the NO to the membrane) and membrane permeability, in addition to intracellular diffusion of NO, in our computational models. We find that all these mechanisms may modulate NO uptake by membrane-encapsulated Hb and that extracellular diffusion is the main rate-limiting factor for phospholipid vesicles and erythrocytes. In the case of red cell microparticles, we find a major role for membrane permeability. These results are consistent with prior studies indicating that extracellular diffusion of several gas ligands is also rate-limiting for erythrocytes, with some contribution of a low membrane permeability.  

4.994           Histopaque provides optimal mouse islet purification kinetics: Comparison study with Ficoll, iodixanol and dextran

McCall, M.D., Maciver, A.H., Pawlick, R., Edgar, R. and Shapiro, A,M.J. Islets, 3(4), 144-149 (2011)   Islet transplantation has become a very promising treatment for type 1 diabetes. To facilitate further clinical improvements in this exciting field, rodent islets are used to evaluate new strategies and modifications. One method to purify islets is on a density gradient, although the optimal gradient component can be debated. N=6 separate mouse islet isolations were used and the resulting islets were separated and purified on either a Ficoll, Histopaque, Dextran or Iodixanol gradient. Islets were assessed for recovery, viability, purity and in vitro functionality. Aliquots were transplanted into diabetic mice to assess in vivo functionality and survival. There was no difference in the number of islets recovered across groups nor in the size of recovered islets. Use of a Ficoll or Histopaque gradient led to the most pure and viable islets in comparison to Dextran and Iodixanol. Functionally, islets isolated on a Ficoll gradient had the highest glucose-stimulated insulin release in vitro while performing equally to Histopaque and Dextran gradients in vivo. Using a Ficoll gradient, however, comes at a higher monetary cost. We recommend using a Histopaque gradient, which led to the isolation of viable and functional islets with a reduced cost as compared to a Ficoll gradient.      

4.995           Improving Efficacy of Clinical Islet Transplantation with Iodixanol Based Islet Purification, Thymoglobulin Induction and Blockage of IL-1-beta and TNF-alpha

Matsumoto, S., Takita, M., Chaussabel, D., Noguchi, H., Shimoda, M., Sugimoto, K., Itoh, T., Chujo, D., Sorelle, J., Onaca, N., Naziruddin, B. and Levy, M.F. Cell Transplant, 20(10), 1641-1647 (2011)   Background: Poor efficacy is one of the issues for clinical islet transplantation. Recently, we demonstrated that pancreatic ductal preservation significantly improved the success rate of islet isolation; however, two transplants were necessary to achieve insulin independence. In this study, we introduced iodixanol based purification, thymoglobulin induction and double blockage of IL-1 beta and TNF-alpha as well as sirolimus free immunosuppression to improve the efficacy of clinical islet transplantation. Methods: Nine clinical grade human pancreata were procured. Pancreatic ductal preservation was performed using ET-Kyoto solution in all cases. When the isolated islets met the clinical criteria, they were transplanted. We utilized two methods of immunosuppression and anti-inflammation. The first protocol prescribed daclizumab for induction, then sirolimus and tacrolimus to maintain immunosuppression. The second protocol used thymoglobulin for induction and tacrolimus and mycophenolate mofetil to maintain immunosuppression. Eternacept and anakinra were administered as anti-inflammatory drugs. The total amount of insulin required, HbA1c, and the SUITO index were determined to analyze and compare the results of transplantation. Results: All isolated islet preparations (9/9) met the criteria for clinical transplantation, and they were transplanted into 6 type 1 diabetic patients. All patients achieved insulin independence with normal HbA1c levels; however, the first protocol required two islet infusions (N=3) and the second protocol only required a single infusion (N=3).The average SUITO index, at one month after a single-donor islet transplantation, was significantly higher in the second protocol (49.6±8.3 vs. 19.3±6.3, p<0.05). Conclusions: Pancreatic ductal preservation, iodixanol based purification combined with thymoglobulin induction and blockage of IL-1 beta and TNF-alpha as well as sirolimus free immunosuppression dramatically improved the efficacy of clinical islet transplantations. This protocol enabled us to perform successful single-donor islet transplantations. Further large scale studies are necessary to confirm these results and clarify the mechanism of each component.   4.995a A Rapid and Simple Method to Obtain Canine Peripheral Blood-Derived Macrophages Goto-Koshino, Y., Ohno, K., Nakajima, M., Mochizuki, H., Kanemoto, H. and Tsujimoto, H.

1. Vet. Med. Sci., 73(6), 773-778 (2011)

  Macrophages play an important role in a variety of situations, including pathogen elimination, inflammation, and tissue repair. However, these cells are not fully studied in dogs, in part, due to the difficulty of efficiently isolating and culturing them in vitro. In this study, we cultured canine peripheral blood mononuclear cells (PBMCs) with 10 ng/ml of phorbol 12-myristate-13-acetate (PMA) for 5 days to obtain macrophages. A high number of round-adherent cells were obtained without the addition of any cytokine. These cells showed active phagocytic activity and a cell surface antigen profile different from dendritic cells. Our method facilitates a high yield of macrophages in a short cultivation period compared with previous studies. This method might be a powerful tool to study macrophage functions in dogs.  

4.996           Diapocynin and apocynin administration fails to significantly extend survival in G93A SOD1 ALS mice

Trumbull, K.A., McAllister, D.,Gandelman, M.M., Fung, W.Y., lew, T., Brennan, L., Lopez, N., Morre, J., Kalyanaraman, B. and Beckman, J.S. Neurobiol. of Dis., 45, 137-144 (2012)   NADPH oxidase has recently been identified as a promising new therapeutic target in ALS. Genetic deletion of NADPH oxidase (Nox2) in the transgenic SOD1^G93A mutant mouse model of ALS was reported to increase survival remarkably by 97 days. Furthermore, apocynin, a widely used inhibitor of NADPH oxidase, was observed to dramatically extend the survival of the SOD1^G93A ALS mice even longer to 113 days (Harraz et al. J Clin Invest 118: 474, 2008). Diapocynin, the covalent dimer of apocynin, has been reported to be a more potent inhibitor of NADPH oxidase. We compared the protection of diapocynin to apocynin in primary cultures of SOD1^G93A-expressing motor neurons against nitric oxide-mediated death. Diapocynin, 10 μM, provided significantly greater protection compared to apocynin, 200 μM, at the lowest statistically significant concentrations. However, administration of diapocynin starting at 21 days of age in the SOD1^G93A-ALS mouse model did not extend lifespan. Repeated parallel experiments with apocynin failed to yield protection greater than a 5-day life extension in multiple trials conducted at two separate institutions. The maximum protection observed was an 8-day extension in survival when diapocynin was administered at 100 days of age at disease onset. HPLC with selective ion monitoring by mass spectrometry revealed that both apocynin and diapocynin accumulated in the brain and spinal cord tissue to low micromolar concentrations. Diapocynin was also detected in the CNS of apocynin-treated mice. The failure to achieve significant protection with either apocynin or diapocynin raises questions about the utility for treating ALS patients.  

4.997           Case Study of Processing and Insemination Techniques: Attempts to Improve Fertility of an Aged Stallion with Dilute Semen of Poor Quality

Blanchard, T.L., Brinsko, S.P., Love, C.C., Vest, D.D., Berezowski, C.B., Wendt, K.M., Stich, K. and Varner, D.D.

1. Equine Vet. Sci., 32, 5-11 (2012)

  The objective of this case study was to investigate whether semen centrifugation and low-dose insemination techniques would improve fertility of an aged subfertile Quarter Horse stallion with low sperm concentration, motility, and morphology in ejaculates. Forty-five mares were bred by one of five treatments (n = 9 per group) using the entire ejaculate as follows: (1) Group Body: body insemination with ejaculate diluted 1:1 in TAMU extender; (2) Group Body-Cent: body insemination after centrifugation and re-suspension of sperm pellet to 1 mL in TAMU extender; (3) Group Horn-Cent: deep horn insemination after centrifugation and re-suspension of sperm pellet to 1 mL in TAMU extender; (4) Group Cent-Hys: hysteroscopic insemination onto the uterotubal papilla after centrifugation and re-suspension of sperm pellet to 200 μL in Kenney-Modified Tyrode’s extender; and (5) Group Dens-Hys: hysteroscopic insemination onto the uterotubal papilla after discontinuous density gradient centrifugation and re-suspension of the sperm pellet in 200-μL Kenney-Modified Tyrode’s extender. Pregnancy rates did not differ among treatment groups (P = .77). Semen centrifugation for low dose insemination did not appear to improve fertility of this subfertile stallion, despite use of entire ejaculates for each individual insemination dose.  

4.998           Toll-like receptor 2-mediated innate immune response in human nonparenchymal liver cells toward adeno-associated viral vectors

Hösel, M., Broxtermann, M., Janicki, H., Esser, K., Arzberger, S., Hartmann, P., Gillen, S., Kleeff, J., Stabenow, D., Odenthal, M., Knolle, P., Hallek, M., Protzer, U. and Büning, H. Hepatology, 55(1), 287-297 (2012)   Adeno-associated viral vectors (rAAV) are frequently used in gene therapy trials. Although rAAV vectors are of low immunogenicity, humoral as well as T cell responses may be induced. While the former limits vector reapplication, the expansion of cytotoxic T cells correlates with liver inflammation and loss of transduced hepatocytes. Because adaptive immune responses are a consequence of recognition by the innate immune system, we aimed to characterize cell autonomous immune responses elicited by rAAV in primary human hepatocytes and nonparenchymal liver cells. Surprisingly, Kupffer cells, but also liver sinusoidal endothelial cells, mounted responses to rAAV, whereas neither rAAV2 nor rAAV8 were recognized by hepatocytes. Viral capsids were sensed at the cell surface as pathogen-associated molecular patterns by Toll-like receptor 2. In contrast to the Toll-like receptor 9–mediated recognition observed in plasmacytoid dendritic cells, immune recognition of rAAV in primary human liver cells did not induce a type I interferon response, but up-regulated inflammatory cytokines through activation of nuclear factor κB. Conclusion: Using primary human liver cells, we identified a novel mechanism of rAAV recognition in the liver, demonstrating that alternative means of sensing rAAV particles have evolved. Minimizing this recognition will be key to improving rAAV-mediated gene transfer and reducing side effects in clinical trials due to immune responses against rAAV.  

4.999           Unraveling features of the natural MHC class II peptidome of skin-migrated dendritic cells

Muixi, L., Contreras, V., Collado, J.A., Alexandre, Y., Ballingall, K., Bonneau, M., Jaraquemada, D. and Schwartz-Cornil, I. Int. Immunol., 24(1), 59-69 (2012)   Dendritic cells (DCs) migrating from peripheral tissues at steady state are considered the most efficient antigen-presenting cells (APCs) involved in the induction of peripheral T-cell tolerance via self-antigen presentation on MHC class II molecules. However, difficulties in obtaining sufficient numbers of such DCs have precluded previous analyses of their natural MHC class II peptidome in laboratory animals or humans. Here, we overcome this difficulty by collecting the large quantities of sheep DCs that migrate from the skin via the afferent lymphatics at steady state to the draining lymph node. We compared the repertoire of MHC class II-bound peptides from afferent lymph DCs with autologous APCs derived from peripheral blood. A large fraction of the MHC class II peptidome from skin DCs was derived from membrane-recycling proteins (59%) and from proteins of the antigen presentation machinery (50%), whereas these types of peptides constituted a more limited fraction in blood APCs (21 and 11%, respectively). One sheep cytokeratin peptide was identified in the skin DC peptidome indicating active processing of epithelium-derived antigens. Conversely, peptides derived from cytosolic and soluble antigens of the extracellular milieu were more represented in blood APCs than skin DCs. The biased peptidome of skin-migrated DCs indicates that these cells express a peptide repertoire for the generation of self-reactive and/or regulatory T cells mainly directed toward DC molecules from internal and external membranes and to a lesser extent toward antigens of the extracellular milieu, including some tissue-specific peptides.  

4.1000           Mitochondrial Dynamics and Bioenergetic Dysfunction Is Associated with Synaptic Alterations in Mutant SOD1 Motor Neurons

Magrane, J., Sahaweh, M.A., Przedborski, S., Estevez, A.G. and Manfredi, G.

1. Neurosci., 32(1), 229-242 (2012)

  Mutations in Cu,Zn superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis (FALS), a rapidly fatal motor neuron disease. Mutant SOD1 has pleiotropic toxic effects on motor neurons, among which mitochondrial dysfunction has been proposed as one of the contributing factors in motor neuron demise. Mitochondria are highly dynamic in neurons; they are constantly reshaped by fusion and move along neurites to localize at sites of high-energy utilization, such as synapses. The finding of abnormal mitochondria accumulation in neuromuscular junctions, where the SOD1-FALS degenerative process is though to initiate, suggests that impaired mitochondrial dynamics in motor neurons may be involved in pathogenesis. We addressed this hypothesis by live imaging microscopy of photo-switchable fluorescent mitoDendra in transgenic rat motor neurons expressing mutant or wild-type human SOD1. We demonstrate that mutant SOD1 motor neurons have impaired mitochondrial fusion in axons and cell bodies. Mitochondria also display selective impairment of retrograde axonal transport, with reduced frequency and velocity of movements. Fusion and transport defects are associated with smaller mitochondrial size, decreased mitochondrial density, and defective mitochondrial membrane potential. Furthermore, mislocalization of mitochondria at synapses among motor neurons, in vitro, correlates with abnormal synaptic number, structure, and function. Dynamics abnormalities are specific to mutant SOD1 motor neuron mitochondria, since they are absent in wild-type SOD1 motor neurons, they do not involve other organelles, and they are not found in cortical neurons. Together, these results suggest that impaired mitochondrial dynamics may contribute to the selective degeneration of motor neurons in SOD1-FALS  

4.1001           Stella TUM: current consensus and discussion on pancreatic stellate cell research

Erkan, M., Adler, GX. Apte, M.V. et al Gut, 61, 172-178 (2012)   The field of pancreatic stellate cell (PSC) biology is very young, as the essential in-vitro tools to study these cells (ie, methods to isolate and culture PSC) were only developed as recently as in 1998. Nonetheless, there has been an exponential increase in research output in this field over the past decade, with numerous research groups around the world focusing their energies into elucidating the biology and function of these cells. It is now well established that PSC are responsible for producing the stromal reaction (fibrosis) of two major diseases of the pancreas—chronic pancreatitis and pancreatic cancer. Despite exponentially increasing data, the methods for studying PSC remain variable. Although within individual laboratories methods are consistent, different methodologies used by various research groups make it difficult to compare results and conclusions. This article is not a review article on the functions of PSC. Instead, members of the Pancreatic Star Alliance (http://www.pancreaticstaralliance.com) discuss here and consolidate current knowledge, to outline and delineate areas of consensus or otherwise (eg, with regard to methodological approaches) and, more importantly, to identify essential directions for future research.    

4.1002           Isolation and Culture of Spinal Cord Astrocytes

Kerstetter, A. and Miller, R.H. Methods in Mol. Biol., 814, 93-104 (2012)   Astrocytes are possibly the most numerous cells of the vertebrate central nervous system, yet a detailed characterization of their functions is still missing. One potential reason for the obscurity of astrocytic function is that they represent a diverse population of cells that all share some critical characteristics. In the CNS, astrocytes have been proposed to perform many functions. For example, they are supportive cells that provide guidance to newly formed migrating neurons and axons. They regulate the functions of endothelial cells at the blood brain barrier, provide nutrients, and maintain homeostasis including ionic balance within the CNS. More recently, dissecting the central role of astrocytes in mediating injury responses in the CNS, particularly the spinal cord, has become an area of considerable importance. The ability to culture-enriched populations of astrocytes has facilitated a detailed dissection of their potential roles in the developing and adult, normal, and injured brain and spinal cord. Most importantly, in vitro models have defi ned molecular signals that may mediate or regulate astrocyte functions and the capacity to modulate these signals may provide new opportunities for therapeutic intervention after spinal cord injury and other neural insults.  

4.1003           Recipient nonhematopoietic antigen-presenting cells are sufficient to induce lethal acute graft-versus-host disease

Koyama, M. et al Nature Med., 18(1), 135-142 (2012)   The presentation pathways by which allogeneic peptides induce graft-versus-host disease (GVHD) are unclear. We developed a bone marrow transplant (BMT) system in mice whereby presentation of a processed recipient peptide within major histocompatibility complex (MHC) class II molecules could be spatially and temporally quantified. Whereas donor antigen presenting cells (APCs) could induce lethal acute GVHD via MHC class II, recipient APCs were 100–1,000 times more potent in this regard. After myeloablative irradiation, T cell activation and memory differentiation occurred in lymphoid organs independently of alloantigen. Unexpectedly, professional hematopoietic-derived recipient APCs within lymphoid organs had only a limited capacity to induce GVHD, and dendritic cells were not required. In contrast, nonhematopoietic recipient APCs within target organs induced universal GVHD mortality and promoted marked alloreactive donor T cell expansion within the gastrointestinal tract and inflammatory cytokine generation. These data challenge current paradigms, suggesting that experimental lethal acute GVHD can be induced by nonhematopoietic recipient APCs.  

4.1004           Insulin Protects Pancreatic Acinar Cells from Cytosolic Calcium Overload and Inhibition of Plasma Membrane Calcium Pump

Mmandad, P., James, A., Siriwardena, A.K., Elliott, A.C. and Bruce, J.I.E.

1. Biol. Chem., 287(3), 1823-1836 (2012)

  Acute pancreatitis is a serious and sometimes fatal inflammatory disease of the pancreas without any reliable treatment or imminent cure. In recent years, impaired metabolism and cytosolic Ca²⁺ ([Ca²⁺]_i) overload in pancreatic acinar cells have been implicated as the cardinal pathological events common to most forms of pancreatitis, regardless of the precise causative factor. Therefore, restoration of metabolism and protection against cytosolic Ca²⁺ overload likely represent key therapeutic untapped strategies for the treatment of this disease. The plasma membrane Ca²⁺-ATPase (PMCA) provides a final common path for cells to “defend” [Ca²⁺]_i during cellular injury. In this paper, we use fluorescence imaging to show for the first time that insulin treatment, which is protective in animal models and clinical studies of human pancreatitis, directly protects pancreatic acinar cells from oxidant-induced cytosolic Ca²⁺ overload and inhibition of the PMCA. This protection was independent of oxidative stress or mitochondrial membrane potential but appeared to involve the activation of Akt and an acute metabolic switch from mitochondrial to predominantly glycolytic metabolism. This switch to glycolysis appeared to be sufficient to maintain cellular ATP and thus PMCA activity, thereby preventing Ca²⁺ overload, even in the face of impaired mitochondrial function.  

4.1005           Proteomic Analysis of Fractionated Toxoplasma Oocysts Reveals Clues to Their Environmental Resistance

Fritz, H.M., Bowyer, P.W., Bogyo, M., Conrad, P.A. and Boothroyd, J.C.

PloS One, 7(1), e29955 (2012)

Toxoplasma gondii is an obligate intracellular parasite that is unique in its ability to infect a broad range of birds and mammals, including humans, leading to an extremely high worldwide prevalence and distribution. This work focuses on the environmentally resistant oocyst, which is the product of sexual replication in felids and an important source of human infection. Due to the difficulty in producing and working with oocysts, relatively little is known about how this stage is able to resist extreme environmental stresses and how they initiate a new infection, once ingested. To fill this gap, the proteome of the wall and sporocyst/sporozoite fractions of mature, sporulated oocysts were characterized using one-dimensional gel electrophoresis followed by LC-MS/MS on trypsin-digested peptides. A combined total of 1021 non-redundant T. gondii proteins were identified in the sporocyst/sporozoite fraction and 226 were identified in the oocyst wall fraction. Significantly, 172 of the identified proteins have not previously been identified in Toxoplasma proteomic studies. Among these are several of interest for their likely role in conferring environmental resistance including a family of small, tyrosine-rich proteins present in the oocyst wall fractions and late embryogenesis abundant domain-containing (LEA) proteins in the cytosolic fractions. The latter are known from other systems to be key to enabling survival against desiccation.

   

4.1006           Biochemical properties of bull spermatozoa separated in iodixanol density solution

Beer-Ljubic, B., Aladrovic, J., Marenjak, T.S:, Majic-Balic. I., Laskaj, R. and Milinkovic-Tur, S. Res.Vet. Sci., 92, 292-294 (2012)   Bull spermatozoa samples contain variable portion of motile and normal morphology spermatozoa along with spermatozoa incapable of fertilization due to their pathologic changes. As semen quality is influenced by biochemical and morphological characteristics of all spermatozoa, the aim of the study was to separate spermatozoa in discontinuous iodixanol density gradient solution and to determine their cholesterol, phospholipid, triacylglycerol and lipid peroxide concentrations and creatine kinase activity. The study was performed in winter and included seven Simmental bulls aged 1.5–3.5 years. Semen samples were collected by use of artificial vagina. Upon evaluation of semen quality (volume, concentration and progressive sperm motility), the samples were centrifuged in iodixanol density solution to obtain two sperm fractions. The two fractions included sperms with progressive motility greater than 90% and less than 20%, respectively. A statistically significantly higher lipid peroxide concentration was determined in sperm fraction with <20% progressive motility. Different sperm subpopulations can be obtained by separating bull spermatozoa in different iodixanol density gradient solutions, while monitoring their biochemical properties can help assess the sperm quality.  

4.1007           Homing and Long-Term Engraftment of Long- and Short-Term Renewal Hematopoietic Stem Cells

Liu, L., Papa, E.F., Dooner, M.S., Machan, J.T., Johnson, K.W., Goldberg, L.R., Qusenberry, P.J. and Covin, G.A. PloS One, 7(2), e31300 (2012)   Long-term hematopoietic stem cells (LT-HSC) and short-term hematopoietic stem cells (ST-HSC) have been characterized as having markedly different in vivo repopulation, but similar in vitro growth in liquid culture. These differences could be due to differences in marrow homing. We evaluated this by comparing results when purified ST-HSC and LT-HSC were administered to irradiated mice by three different routes: intravenous, intraperitoneal, and directly into the femur. Purified stem cells derived from B6.SJL mice were competed with marrow cells from C57BL/6J mice into lethally irradiated C57BL/6J mice. Serial transplants into secondary recipients were also carried out. We found no advantage for ST-HSC engraftment when the cells were administered intraperitoneally or directly into femur. However, to our surprise, we found that the purified ST-HSC were not short-term in nature but rather gave long-term multilineage engraftment out to 387 days, albeit at a lower level than the LT-HSC. The ST-HSC also gave secondary engraftment. These observations challenge current models of the stem cell hierarchy and suggest that stem cells are in a continuum of change.  

4.1008           Infection of bone marrow cells by dengue virus in vivo

Noisakran, S., Onlamoon, N., Hsiao, H-M., Clark, K.B., Villinger, F., Ansari, A.A. and Perng, G.C. Exp. Hematol., 240, 250-259 (2012)   Abnormal bone marrow (BM) suppression is one of the hallmarks of dengue virus (DENV) infection in patients. Although the etiology remains unclear, direct viral targeting of the BM has been reasoned to be a contributing factor. The present studies were carried out in an effort to determine the potential effect of DENV infection on the cellularity of BM using a previously established nonhuman primate model of DENV-induced coagulopathy. BM aspirates were collected at various times from the infected nonhuman primate and cells were phenotypically defined and isolated using standard flow cytometry (fluorescence-activated cell sorting). These isolated cells were subjected to detection of DENV utilizing quantitative real-time reverse transcription polymerase chain reaction, electron microscopy, and immunostaining techniques. DENV RNA was detectable by quantitative real-time reverse transcription polymerase chain reaction in BM specimens and the presence of DENV-like particles within platelet was confirmed by electron microscopy. Enumeration of BM cells revealed a transient surge in cellularity at day 1, followed by a gradual decline from days 2 to 10 post infection. Detailed phenotypic studies showed similar kinetics in the frequencies of CD41⁺CD61⁺ cells, regardless of CD34 and CD45 expression. The CD61⁺ cells were not only the predominant cells that stained for DENV antigen but fluorescence-activated cell sorting–assisted isolation of CD61⁺ cells from the BM were shown to contain infectious DENV by coculture with Vero cells. These data support the view that intravenous infection of nonhuman primate with DENV leads to direct infection of the BM, which is likely to be a contributing factor for transient cell suppression in the peripheral blood characteristic of acute DENV infection.    

4.1009           Oral administration of Salmonella enterica serovar Typhimurium expressing swine interleukin-18 induces Th1-biased protective immunity against inactivated vaccine of pseudorabies virus

Kim, S.B., Kim, S.J., Lee, B.M., Han, Y.W., Rahman, M.M., Uyangaa, E., Kim, J.H., Choi, J.Y., Yoo, D.J., Kim, K. and Eo, S.K. Vet. Microbiol., 155, 172-182 (2012)   Enhancing and/or modulating innate and adaptive immunity by cytokines appears to be greatly useful to provide effective protective immunity against infectious diseases. However, an effective delivery system for mass administration in livestock industry is needed because of limitations such as cost, labor, time, and protein stability. Here the immunomodulatory functions of swine interleukine-18 (swIL-18), known as IFN-γ-inducing factor (IGIF), were evaluated in a vaccination model of pseudorabies virus (PrV) using attenuated Salmonella enterica serovar Typhimurium as the oral delivery system. The oral administration of S. enterica serovar Typhimurium expressing swIL-18 prior to vaccination with inactivated PrV vaccine induced enhanced levels of serum PrV-specific IgG and its IgG2 isotype, compared to administration of S. enterica serovar Typhimurium harboring the empty vector. Furthermore, S. enterica serovar Typhimurium expressing swIL-18 mounted Th1-biased cellular immune responses against PrV antigen, as evaluated by the production of IFN-γ and IL-4 from peripheral blood mononuclear cells of piglets. Subsequently, Th1-biased immunity induced by S. enterica serovar Typhimurium expressing swIL-18 showed rapid response and rendered piglets displayed more alleviated clinical signs following the virulent PrV challenge. Also, this alleviation of clinical signs was further confirmed by the reduction of nasal excretion of PrV after challenge. The present study demonstrates the extended use of immunomodulatory functions of swIL-18 orally delivered by attenuated S. enterica serovar Typhimurium.  

4.1010           A Francisella tularensis Live Vaccine Strain That Improves Stimulation of Antigen-Presenting Cells Does Not Enhance Vaccine Efficacy

Schmitt, D.M., O’Dee, D.M., Horzempa, J., Carlson, P.E., Russo, B.C., Bales, J.M., Brown, M.J. and Nau, G.J. PloS One, 7(2), e31172 (2012)   Vaccination is a proven strategy to mitigate morbidity and mortality of infectious diseases. The methodology of identifying and testing new vaccine candidates could be improved with rational design and in vitro testing prior to animal experimentation. The tularemia vaccine, Francisella tularensis live vaccine strain (LVS), does not elicit complete protection against lethal challenge with a virulent type A Francisella strain. One factor that may contribute to this poor performance is limited stimulation of antigen-presenting cells. In this study, we examined whether the interaction of genetically modified LVS strains with human antigen-presenting cells correlated with effectiveness as tularemia vaccine candidates. Human dendritic cells infected with wild-type LVS secrete low levels of proinflammatory cytokines, fail to upregulate costimulatory molecules, and activate human T cells poorly in vitro. One LVS mutant, strain 13B47, stimulated higher levels of proinflammatory cytokines from dendritic cells and macrophages and increased costimulatory molecule expression on dendritic cells compared to wild type. Additionally, 13B47-infected dendritic cells activated T cells more efficiently than LVS-infected cells. A deletion allele of the same gene in LVS displayed similar in vitro characteristics, but vaccination with this strain did not improve survival after challenge with a virulent Francisella strain. In vivo, this mutant was attenuated for growth and did not stimulate T cell responses in the lung comparable to wild type. Therefore, stimulation of antigen-presenting cells in vitro was improved by genetic modification of LVS, but did not correlate with efficacy against challenge in vivo within this model system.  

4.1011           Surfactant Protein A Integrates Activation Signal Strength To Differentially Modulate T Cell Proliferation

Mukherjee, S., Giamberardino, C., Thomas, J., Evans, K., Goto, H., Ledford, J.G., Hsia, B., Pastva, A.M. and Wright, J.R.

1. Immunol., 188(3), 957-967 (2012)

  Pulmonary surfactant lipoproteins lower the surface tension at the alveolar–airway interface of the lung and participate in host defense. Previous studies reported that surfactant protein A (SP-A) inhibits lymphocyte proliferation. We hypothesized that SP-A–mediated modulation of T cell activation depends upon the strength, duration, and type of lymphocyte activating signals. Modulation of T cell signal strength imparted by different activating agents ex vivo and in vivo in different mouse models and in vitro with human T cells shows a strong correlation between strength of signal (SoS) and functional effects of SP-A interactions. T cell proliferation is enhanced in the presence of SP-A at low SoS imparted by exogenous mitogens, specific Abs, APCs, or in homeostatic proliferation. Proliferation is inhibited at higher SoS imparted by different doses of the same T cell mitogens or indirect stimuli such as LPS. Importantly, reconstitution with exogenous SP-A into the lungs of SP-A^−/− mice stimulated with a strong signal also resulted in suppression of T cell proliferation while elevating baseline proliferation in unstimulated T cells. These signal strength and SP-A–dependent effects are mediated by changes in intracellular Ca²⁺ levels over time, involving extrinsic Ca²⁺-activated channels late during activation. These effects are intrinsic to the global T cell population and are manifested in vivo in naive as well as memory phenotype T cells. Thus, SP-A appears to integrate signal thresholds to control T cell proliferation.  

4.1012           RANKL Induces Organized Lymph Node Growth by Stromal Cell Proliferation

Hess, E., Duheron, V., Decossas, M., Lezot, F., Berdal, A., Chea, S., Golub, R., Bosisio, M.R., Bridal, S.L., Choi, Y., Yagita, H. and Mueller, C.G.

1. Immunol., 188(3), 1245-1254 (2012)

  RANK and its ligand RANKL play important roles in the development and regulation of the immune system. We show that mice transgenic for Rank in hair follicles display massive postnatal growth of skin-draining lymph nodes. The proportions of hematopoietic and nonhematopoietic stromal cells and their organization are maintained, with the exception of an increase in B cell follicles. The hematopoietic cells are not activated and respond to immunization by foreign Ag and adjuvant. We demonstrate that soluble RANKL is overproduced from the transgenic hair follicles and that its neutralization normalizes lymph node size, inclusive area, and numbers of B cell follicles. Reticular fibroblastic and vascular stromal cells, important for secondary lymphoid organ formation and organization, express RANK and undergo hyperproliferation, which is abrogated by RANKL neutralization. In addition, they express higher levels of CXCL13 and CCL19 chemokines, as well as MAdCAM-1 and VCAM-1 cell-adhesion molecules. These findings highlight the importance of tissue-derived cues for secondary lymphoid organ homeostasis and identify RANKL as a key molecule for controlling the plasticity of the immune system.  

4.1013           CD64 Expression Distinguishes Monocyte-Derived and Conventional Dendritic Cells and Reveals Their Distinct Role during Intramuscular Immunization

Llanglet, C., Tamoutounour, S., Henri, S., Luche, H., Ardouin, L., Gregoire, C., Malissen, B. and Guilliams, M.

1. Immunol., 188(4), 1751-1760 (2012)

  Although most vaccines are administered i.m., little is known about the dendritic cells (DCs) that are present within skeletal muscles. In this article, we show that expression of CD64, the high-affinity IgG receptor FcγRI, distinguishes conventional DCs from monocyte-derived DCs (Mo-DCs). By using such a discriminatory marker, we defined the distinct DC subsets that reside in skeletal muscles and identified their migratory counterparts in draining lymph nodes (LNs). We further used this capability to analyze the functional specialization that exists among muscle DCs. After i.m. administration of Ag adsorbed to alum, we showed that alum-injected muscles contained large numbers of conventional DCs that belong to the CD8α⁺- and CD11b⁺-type DCs. Both conventional DC types were capable of capturing Ag and of migrating to draining LNs, where they efficiently activated naive T cells. In alum-injected muscles, Mo-DCs were as numerous as conventional DCs, but only a small fraction migrated to draining LNs. Therefore, alum by itself poorly induces Mo-DCs to migrate to draining LNs. We showed that addition of small amounts of LPS to alum enhanced Mo-DC migration. Considering that migratory Mo-DCs had, on a per cell basis, a higher capacity to induce IFN-γ–producing T cells than conventional DCs, the addition of LPS to alum enhanced the overall immunogenicity of Ags presented by muscle-derived DCs. Therefore, a full understanding of the role of adjuvants during i.m. vaccination needs to take into account the heterogeneous migratory and functional behavior of muscle DCs and Mo-DCs revealed in this study.  

4.1014           A novel neuroprotective therapy for Parkinson’s disease using a viral noncoding RNA that protects mitochondrial Complex I activity

Kuan, W-L., Poole, E., Fletcher, M., KArniely, S., Tyers, P., Wills, M., Barker, R.A. and Sinclair, J.H.

1. Exp. Med., 209(1), 1-10 (2012)

  Parkinson’s disease (PD) is a neurodegenerative disorder that results in the loss of nigrostriatal dopamine neurons. The etiology of this cell loss is unknown, but it involves abnormalities in mitochondrial function. In this study, we have demonstrated that the administration of a novel noncoding p137 RNA, derived from the human cytomegaloviral β2.7 transcript, can prevent and rescue dopaminergic cell death in vitro and in animal models of PD by protecting mitochondrial Complex I activity. Furthermore, as this p137 RNA is fused to a rabies virus glycoprotein peptide that facilitates delivery of RNA across the blood–brain barrier, such protection can be achieved through a peripheral intravenous administration of this agent after the initiation of a dopaminergic lesion. This approach has major implications for the potential treatment of PD, especially given that this novel agent could have the same protective effect on all diseased neurons affected as part of this disease process, not just the dopaminergic nigrostriatal pathway.  

4.1015           Effect of centrifugal fractionation protocols on quality and recovery rate of equine sperm

Edmond, A.J., Brinsko, S.P., Love, C.C., Blanchard, T.L., Teague, S.R. and Varner, D.D. Theriogenology, 77, 959-966 (2012)   Centrifugal fractionation of semen is commonly done to improve quality of human semen in assisted-reproduction laboratories, allowing sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, promoting their retention above defined density media, with structurally normal sperm passing through the media following centrifugation. Three experiments were conducted to evaluate the effects of density-medium type, centrifuge-tube size, sperm number, and density-medium volume (column height) on stallion sperm quality and recovery rate in sperm pellets following centrifugation. In all three experiments, equine semen was initially centrifuged to increase sperm concentration. In Experiment 1, semen was layered over continuous or discontinuous gradients. For Experiment 2, semen was layered over three column heights of continuous gradients in 15- or 50-ml conical-bottom tubes. For Experiment 3, increasing sperm numbers were layered over continuous gradient in 15- or 50-ml conical-bottom tubes. Following centrifugation, sperm pellets were evaluated for sperm morphologic quality, motility, DNA integrity, and recovery rate. Centrifugal fractionation improved (P < 0.05) sperm morphology, motility, and DNA integrity, as compared to controls. The continuous gradient increased (P < 0.05) sperm recovery rate relative to the discontinuous gradient, whereas sperm processed in 15-ml tubes yielded higher velocity and higher recovery rates (P < 0.05 for each) than that processed in 50-ml tubes. Sperm recovery rate was not affected (P > 0.05) by column height of gradient. Increasing sperm number subjected to gradient centrifugation decreased (P < 0.05) sperm recovery rate when 15-ml tubes were used.  

4.1016           Evaluation of the qualitative and quantitative effectiveness of three media of centrifugation (Maxifreeze, Cushion Fluid Equine, and PureSperm 100) in preparation of fresh or frozen-thawed brown bear spermatozoa

Nicolas, M., Alvarez, M., Borragan, S., Martinez-Pastor, F., Chamorro, C.A., Alvarez-Rodriguez, M., de Paz, P. and Anel, L. Theriogenology, 77, 1119-1128 (2012)   Centrifugation is a crucial procedure in sperm cryopreservation protocols of brown bear (Ursus arctos), because the semen must be processed to increase sperm concentration and/or clean urine-contaminated samples. The efficacy of three media for centrifugation (Maxifreeze [IMV technologies, L'Aigle, France], Cushion Fluid Equine (Minitübe, Tiefenbach, Germany), and PureSperm [Nidacon, Gothenburg, Sweden]) on the quality of bear spermatozoa was evaluated. In experiment one, two cushioned media used for protecting against mechanical stress during centrifugation were analyzed. In experiment two, a density gradient based on PureSperm was assessed in relation to the maximum retrieval and the quality of fresh spermatozoa, and the freezability of the spermatozoa selected in this density gradient was studied in experiment three. Finally, the selection of frozen-thawed sperm using PureSperm was analyzed in experiment four. Our results indicate that the use of dense isotonic cushion solutions (Maxifreeze, Cushion Fluid Equine) in centrifugation did not improve the quality of recovered spermatozoa compared with standard centrifugation. However, a density gradient prepared with PureSperm improved the quality of spermatozoa in fresh semen and frozen-thawed semen, but the spermatozoa selected from the fresh sample with this density gradient did not show a better resistance to freezing with this density gradient in comparison with the control sample.  

4.1017           The unfolded protein response in models of human mutant G93A amyotrophic lateral sclerosis

Prell, T., Lautenschlläger, J., Witte, O.W., Carri, M.T. and Grosskreutz, J. Eur. J. Neurosci., 35, 652-660 82012)   Recent studies indicate that endoplasmic reticulum (ER) stress is involved in the pathogenesis of familial and sporadic amyotrophic lateral sclerosis (ALS). ER stress occurs when the ER–mitochondria calcium cycle (ERMCC) is disturbed and misfolded proteins accumulate in the ER. To cope with ER stress, the cell engages the unfolded protein response (UPR). While activation of the UPR has been shown in some ALS models and tissues, ER stress elements have not been studied directly in motor neurons. Here we investigated the expression of XBP1 and ATF6α and phosphorylation of eIF2α, and their modulation, in mutated SOD1^G93A NSC34 and animal model of ALS. Expression of XBP1 and ATF6α mRNA and protein was enhanced in SOD1^G93A NSC34 cells. Activation of ATF6α and XBP1 and phosphorylation of eIF2α were detectable in mutated SOD1^G93A motor but not in wild-type motor neurons. Treatment with the ER stressor thapsigargin enhanced phosphorylation of eIF2α and activated proteolysis of ATF6α and splicing of XBP1 in NSC34 and motor neurons in a time-dependent manner. The present study thus provides direct evidence of activated UPR in motor neurons which overexpress human pathogenic mutant SOD1^G93A, providing evidence that ER stress plays a major role in ALS.  

4.1018           Effects of Exercise on microRNA Expression in Young Males Peripheral Blood Mononuclear Cells

Radom-Aizik, S., Zaldivar, F., Leu, S-Y., Adams, G.R., Oliver, S. and Cooper, D.M. Clin. Trans. Sci., 5, 32-38 (2012)   MicroRNAs are increasingly seen as targets of drug discovery because they influence gene function acting both to silence and subtly modulate protein translation. Little is known about effects of dynamic physiological states on microRNA regulation in humans. We hypothesized that microRNA expression in peripheral blood mononuclear cells (PBMCs) would be affected by brief exercise. Twelve young men performed brief bouts of heavy exercise. PBMC microRNA was analyzed before and immediately after exercise using the Agilent Human microRNA V2 Microarray. Exercise altered expression level of 34 microRNAs (FDR < 0.05). Many of them play roles in inflammatory processes (e.g., miR-125b[↓], down-regulated by proinflammatory factor LPS; and miR-132[↑], 125b[↓] and let-7e[↓] involved inTLR4 signaling). Using previous exercise data in PBMCs, we linked the microRNA changes to specific gene pathways. This analysis identified 12 pathways including the TGF-β and MAPK signaling. We also compared exercise-associated microRNA changes in PBMCs with the exercise-associated microRNAs previously identified in neutrophils. Nine microRNAs were affected in both PBMCs and neutrophils, but only six changed in the same direction. A commonly occurring physiologic perturbation, brief heavy exercise, changes microRNA profiles in PBMCs, many of which are related to inflammatory processes. The pattern of change suggests that exercise differentially influences microRNAs in leukocyte subtypes  

4.1019           ER stress activates the NLRP3 inflammasome via an UPR-independent pathway

Menu, P., Mayor, A., Zhou, R., Tardivel, A., Ichijo, H., Mori, K. and Tschopp, J. Cell Death and Differentiation, 3, e261, (2012)   Uncontrolled endoplasmic reticulum (ER) stress responses are proposed to contribute to the pathology of chronic inflammatory diseases such as type 2 diabetes or atherosclerosis. However, the connection between ER stress and inflammation remains largely unexplored. Here, we show that ER stress causes activation of the NLRP3 inflammasome, with subsequent release of the pro-inflammatory cytokine interleukin-1β. This ER-triggered proinflammatory signal shares the same requirement for reactive oxygen species production and potassium efflux compared with other known NLRP3 inflammasome activators, but is independent of the classical unfolded protein response (UPR). We thus propose that the NLRP3 inflammasome senses and responds to ER stress downstream of a previously uncharacterized ER stress response signaling pathway distinct from the UPR, thus providing mechanistic insight to the link between ER stress and chronic inflammatory diseases.  

4.1020           Stromal TIMP3 Regulates Liver Lymphocyte Populations and Provides Protection against Th1 T Cell-Driven Autoimmune Hepatitis

Murthy, A., Washington Shao, Y., Defamie, V., Wedeles, C., Smookler, D. and Khokha, R.

1. Immunol., 188, 2876-2883 (2012)

  Lymphocyte infiltration into epithelial tissues and proinflammatory cytokine release are key steps in autoimmune disease. Although cell-autonomous roles of lymphocytes are well studied in autoimmunity, much less is understood about the stromal factors that dictate immune cell function. Tissue inhibitor of metalloproteinases 3 (TIMP3) controls systemic cytokine bioavailability and signaling by inhibiting the ectodomain shedding of cytokines and their receptors. The role of TIMP3 in cytokine biology is emerging; however, its contribution to cellular immunology remains unknown. In this study, we show that TIMP3 produced by the hepatic stroma regulates the basal lymphocyte populations in the liver and prevents autoimmune hepatitis. TIMP3 deficiency in mice led to spontaneous accumulation and activation of hepatic CD4⁺, CD8⁺, and NKT cells. Treatment with Con A in a model of polyclonal T lymphocyte activation resulted in a greatly enhanced Th1 cytokine response and acute liver failure, which mechanistically depended on TNF signaling. Bone marrow chimeras demonstrated that TIMP3 derived from the stromal rather than hematopoietic compartment provided protection against autoimmunity. Finally, we identified hepatocytes as the major source of Timp3 in a resting liver, whereas significant Timp3 gene transcription was induced by hepatic stellate cells in the inflamed liver. These results uncover metalloproteinase inhibitors as critical stromal factors in regulating cellular immunity during autoimmune hepatitis.  

4.1021           Gastroprotective effect of anti-cancer compound rohitukine: possible role of gastrin antagonism and H+ K+-ATPase inhibition

Singh, N., Singh, P., Shrivastva, S., Mishra, S.K., Lakshmi, V., Sharma, R. and Palit, G. Naunyn-Schmiederberg’s Arch. Pharmacol., 385(3), 277-286 (2012)   The present study was designed to evaluate the anti-ulcerogenic properties of an alkaloid chromane, rohitukine from Dysoxylum binectariferum. Anti-ulcer potential of rohitukine was assessed in cold restrained, pyloric ligated and ethanol induced ulcers in rats. In addition, rohitukine was tested in vitro for H⁺ K⁺-ATPase inhibitory activity in gastric microsomes. Moreover, we studied the role of rohitukine on the cytosolic concentration of Ca²⁺ in parietal cell-enriched cell suspension in order to ascertain its mechanism of action. Cytoprotective activity was evaluated through PGE₂ level. Rohitukine significantly attenuated the ulcers in cold restraint ulcer (CRU) model in a dose-related manner. Moreover, it significantly lowered the free acidity and pepsin activity in pyloric ligated rats while improved the depleted level of mucin. Furthermore, rohitukine significantly reversed the cold restrained-induced increase in gastrin level. Our in vitro study revealed that rohitukine moderately inhibited the microsomal H⁺ K⁺-ATPase activity with respect to positive control omeprazole. Furthermore, rohitukine potently antagonized the gastrin-elicited increase in cytosolic Ca²⁺ level in parietal cell-enriched suspension. In ethanol-induced gastric lesions in rats, rohitukine significantly inhibited the formation of erosions and increased PGE₂ content showing more potency than reference drug sucralfate. Our results thus suggest that rohitukine possess significant anti-ulcer and anti-gastrinic activity in rats. It is likely that gastro-protective influences of rohitukine are dependent partly on its acid-lowering potential and partly on cytoprotective property. The acid-reducing effect of rohitukine might be attributed to its lowering effect on gastrin production and/or antagonism of gastrin-evoked functional responses of parietal cells. Thus, rohitukine represent a useful agent in the treatment of peptic ulcer disease.  

4.1022           Microfluidic Cytometer for the Characterization of Cell Lysis

SooHoo, J.R., Herr, J.K., Ramsey, J.M. and Walker, G.M. Anal. Chem., 84(5), 2195-2201 (2012)   Blood cytometry and intercellular analysis typically requires lysis as a preparatory step, which can alter the results of downstream analyses. We fabricated a microfluidic cytometer to characterize erythrocyte lysis kinetics. Forward light scatter from erythrocytes was used for enumeration at specific locations on a microfluidic chip. Diffusive transport coupled with laminar flow was used to control the concentration and exposure time of the lysis reagent Zap-OGLOBIN II to erythrocytes. Standard clinical practice is to expose erythrocytes to lysis reagent for 10 min. Under optimum conditions, we achieved complete erythrocyte lysis of a blood sample in 0.7 s. A maximum lysis reaction rate of 1.55 s^–1 was extrapolated from the data. Lysis began after 0.2 s and could be initiated with a lysis reagent concentration of 1.0% (68.5 mM). An equation that related lysis reagent concentration, [A], to erythrocyte lysis, [B], was determined to be [B] = −0.77[A]^0.29t.  

4.1023           The impact of cushioned centrifugation protocols on semen quality of stallions

Bliss, S.B., Voge, J.L., Hayden, S.S., Teague, S.R., Brinsko, S.P., Love, C.C., Blanchard, T.L. and varner, D.D. Theriogenology, 77, 1232-1239 (2012)   The objective was to determine if decreased cushion-fluid volume and increased sperm number during centrifugation, or if sperm concentration of extended semen following centrifugation, affected stallion sperm quality. Three ejaculates from each of three stallions were subjected to cushioned centrifugation (1,000g for 20 min). Cushion-fluid volume was set at 1 or 3.5 ml, and sperm number per centrifuge tube was set 1 billion or 3 billion. Following centrifugation, sperm pellets were resuspended in semen extender containing 20% seminal plasma (v/v) with sperm concentrations of 25 or 250 million/mL. Sperm recovery rate among centrifugation treatment groups was compared. Motion characteristics, plasma membrane intactness (SMI), and DNA quality (COMPαt) of sperm were compared among treatment groups and uncentrifuged controls immediately following centrifugation (Time 0 h) and following 24 h of cooled storage (Time 24 h). Centrifugation treatment did not affect sperm recovery rate (P > 0.05). At Time 0 h, no differences in experimental end points were detected between cushion-fluid volumes tested (P > 0.05). Values for percent total sperm motility, percent progressive sperm motility, and track straightness were similar between sperm-number treatments subjected to centrifugation (P > 0.05). At Time 24 h, values for all experimental endpoints were similar between centrifugation treatments for cushion volume per tube, and between centrifugation treatments for sperm number per tube (P > 0.05). Centrifugation treatments and control treatments were similar for five of six variables tested (P > 0.05). Sperm storage concentrations of 25 × 10⁶ and 250 × 10⁶/mL yielded similar values for percent total sperm motility, percent progressive sperm motility, percent SMI, and percent COMPαt (P > 0.05). A storage concentration of 250 × 10⁶ sperm/ml yielded higher values for curvilinear velocity, and lower values for straightness, than all other groups (P < 0.05). In conclusion, centrifugation with as little as 1 ml of cushion fluid and a sperm number of up to 3 × 10⁹ sperm in 50-ml conical-bottom centrifuge tubes had no detrimental effect on initial or cool-stored sperm quality. Additionally, storage of centrifuged sperm at a concentration of 250 × 10⁶/mL with 20% seminal plasma (v/v) did not have a detrimental effect on percentages of motile or progressively motile sperm, or sperm DNA quality.  

4.1024           High-Throughput Examination of Fluorescence Resonance Energy Transfer-Detected Metal-Ion Response in Mammalian Cells

Ma, H., Gibson, E.A., Dittmeer, P.J., Jimenez, R. and Palmer, A.E.

1. Am. Chem. Soc., 134(5), 2488-2491 (2012)

  Fluorescence resonance energy transfer (FRET)-based genetically encoded metal-ion sensors are important tools for studying metal-ion dynamics in live cells. We present a time-resolved microfluidic flow cytometer capable of characterizing the FRET-based dynamic response of metal-ion sensors in mammalian cells at a throughput of 15 cells/s with a time window encompassing a few milliseconds to a few seconds after mixing of cells with exogenous ligands. We have used the instrument to examine the cellular heterogeneity of Zn²⁺ and Ca²⁺ sensor FRET response amplitudes and demonstrated that the cluster maps of the Zn²⁺ sensor FRET changes resolve multiple subpopulations. We have also measured the in vivo sensor response kinetics induced by changes in Zn²⁺ and Ca²⁺ concentrations. We observed an 30 fold difference between the extracellular and intracellular sensors.  

4.1025           Anti-Inflammatory Effects of Nicotinic Acid in Human Monocytes Are Mediated by GPR109A Dependent Mechanisms

Digby, J.E., Martinez, F., Jefferson, A., Ruparelia, N., Chai, J., Wamil, M., Greaves, D.RS. and Choudhury, R.P. Arterioscler. Thromb. Vasc. Biol., 32, 669-676 (2012)   Objective—Nicotinic acid (NA) treatment has been associated with benefits in atherosclerosis that are usually attributed to effects on plasma lipoproteins. The NA receptor GPR109A is expressed in monocytes and macrophages, suggesting a possible additional role for NA in modulating function of these immune cells. We hypothesize that NA has the potential to act directly on monocytes to alter mediators of inflammation that may contribute to its antiatherogenic effects in vivo. Methods and Results—In human monocytes activated by Toll-like receptor (TLR)-4 agonist lipopolysaccharide, NA reduced secretion of proinflammatory mediators: TNF-α (by 49.2±4.5%); interleukin-6 (by 56.2±2.8%), and monocyte chemoattractant protein-1 (by 43.2±3.1%) (P<0.01). In TLR2 agonist, heat-killed Listeria monocytogenes-activated human monocytes, NA reduced secretion of TNF-α (by 48.6±7.1%), interleukin-6 (by 60.9±1.6%), and monocyte chemoattractant protein-1 (by 59.3±5.3%) (P<0.01; n=7). Knockdown of GPR109A by siRNA resulted in a loss of this anti-inflammatory effect in THP-1 monocytes. However, inhibition of prostaglandin D₂ receptor by MK0524 or COX2 by NS398 did not alter the anti-inflammatory effects of NA observed in activated human monocytes. Preincubation of THP-1 monocytes with NA 0.1 mmol/L reduced phosphorylated IKKβ by 42±2% (P<0.001) IKB-α by 54±14% (P<0.01). Accumulation of nuclear p65 NF-κB in response to lipopolysaccharide treatment was also profoundly inhibited, by 89±1.3% (n=4; P<0.01). NA potently inhibited monocyte adhesion to activated HUVEC, and VCAM, mediated by the integrin, very late antigen 4. Monocyte chemotaxis was also significantly reduced (by 45.7±1.2%; P<0.001). Conclusion—NA displays a range of effects that are lipoprotein-independent and potentially antiatherogenic. These effects are mediated by GPR109A and are independent of prostaglandin pathways. They suggest a rationale for treatment with NA that is not dependent on levels of plasma cholesterol and possible applications beyond the treatment of dyslipidemia.

4.1026           Induction of Biogenic Magnetization and Redox Control by a Component of the Target of Rapamycin Complex 1 Signaling Pathway

Nishida, K. and Silver, P.A. PloS Biology, 10(2), e1001269 (2012)   Author Summary Most organisms do not respond to magnetic fields. However, “magnetotactic” bacteria and migratory animals can sense geomagnetic fields and alter their behavior accordingly. These organisms often contain small magnetic particles that may be responsible for sensing magnetic fields. In magnetotactic bacteria, specific genes are crucial for the formation of these magnetic particles, but no such genes have yet been characterized in migratory animals. In humans, formation of magnetic particles can be observed in the neuronal tissue in neurodegenerative diseases. One explanation for the appearance of these magnetic particles is that they are the result of alterations in metabolism, which occur in neurodegenerative diseases. Here, we explore this hypothesis by inducing magnetism in yeast cells, which are not naturally magnetic and examine how changes in metabolism contribute to particle formation and magnetism. We find that yeast cells expressing a set of human proteins that sequester iron contain iron particles and become attracted by a magnet when grown with ferric citrate. Through physiological and genetic studies we show that target of rapamycin complex 1 (TORC1) signaling, which responds to nutritional signals, is important for the magnetization of these cells by altering the intracellular oxidation (or redox) state. We also show that genes involved in carbon metabolism affect magnetization. We propose that local redox control mediated by carbon metabolism and iron homeostasis, processes that exist in normal unmagnetized cells, are key for iron particle formation and magnetization. We conclude that magnetization of normal cells will be possible with these existing gene sets.  

4.1027           Preparation of Adult Spinal Cord Motor Neuron Cultures Under Serum-Free Conditions

Montoya-Gacharna, J.V., Sutachan, J.J., Chan, W.S., Sideris, A., Blanck, T.J.J. and Recio-Pinto, E. Methods in Mol. Biol., 846, 103-116 (2012)   Spinal cord motor neuron cultures are an important tool for the study of mechanisms involved in motor neuron survival, degeneration and regeneration, volatile anesthetic-induced immobility, motor neuron disorders such as amyotrophic lateral sclerosis or spinal muscular atrophy as well as in spinal cord injury. Embryonic spinal cord motor neurons derived from rats have been successfully cultured; unfortunately, the culture of adult motor neurons has been problematic due to their short-term survival. Recently, by using a cocktail of target-derived factors, neurotrophins (brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor) and a permeable cyclic adenosine monophosphate analog, we have established a reproducible protocol for long-term cultures of healthy and functional adult motor neurons (Exp Neurol 220:303–315, 2009). Here, we now describe in detail the steps that we used for the optimization of the process of isolation and maintenance of adult rat ventral horn motor neurons in vitro.  

4.1028           Effects of subacute oral warfarin administration on peripheral blood granulocytes in rats

Belij, S., Miljkovic, D., Popov, A., Subota, V., Timotijevic, G., Slavic, M., Mirkov, I., KAtaranovski, D. and Kataranovski, M. Food Chem. Tox., 50, 1499-1507 (2012)   Warfarin affects mainly vitamin K dependent (VKD) processes, but the effects on some non-VKD-related activities such as tumor growth inhibition and mononuclear cell-mediated immune reactions were shown as well. In this study, the effect of subchronic (30 days) oral warfarin (0.35 mg/l and 3.5 mg/l) intake on peripheral blood granulocytes in rats was investigated. Increase in prothrombin and partial thromboplastin time at high warfarin dose reflected its basic activity. Priming effect for respiratory burst was noted at both warfarin doses, while only high warfarin dose resulted in priming for adhesion, the rise in intracellular myeloperoxidase content/release and stimulation of nitric oxide production. Differential effects of high warfarin dose were noted on granulocyte cytokines IL-6 (lack of the effect), TNF-α (decreased release and mRNA expression) and IL-12 (increase in mRNA for IL-12 subunits p35 and p40). Changes in granulocytes seems not to rely on mitogen activated kinases p38 and ERK. Warfarin intake was associated with an increase in circulating IL-6, fibrinogen and haptoglobin and with changes in the activity of erythrocyte antioxidant enzymes superoxide dismutase and catalase. The effects of oral warfarin intake on peripheral blood granulocytes demonstrated in this study might be relevant for oral anticoagulant therapy strategies in humans.    

4.1029           An integrated analytical approach for assessing the biological status of the soil microbial community

Pascaud, A., Soulas, M-L., Amellal, S. and Soulas, G. Eur. J. Soil Biol., 49, 98-106 (2012)   An integrated multicriteria analytical procedure for rapid, cost-effective characterisation of the biological status of soil bacterial community was developped. Commercially-available, light emission-based bioassays were selected for measuring cell density, activity, and diversity. All but Terminal Restriction Fragment Length Polymorphism (T-RFLP) were designed for multiwell-plate formats and high-throughput screening potential. Adenosine Tri Phosphate (ATP) was measured using a bioluminescence assay. Dehydrogenase activity (DHA) was measured on growing cells. Kinetic measurements of the formation of a coloured formazan derivative was used after nutrient broth addition to estimate initial cell concentrations by reference to Escherichia coli added as internal standard. Compared to conventional ATP and DHA determinations in soils, the procedures described here do not require extraction of ATP or formazan derivative from the soil matrix. Metabolic diversity was characterised using the Biolog™ system. T-RFLP was chosen for assessing bacterial community structure. The bioassays were performed on microbial preparations obtained after either direct dilution of soil suspensions or prior density-gradient separation of microbial cells from the soil matrix. Dilution maintains the original structure of native dominant microbial communities. Density-gradient separation of microbial cells is highly selective, drastically modifying metabolic (CLP Profiles) and species (T-RFLP patterns) diversity, as well as activity parameters.  

4.1030           Differential changes in gene expression in rainbow trout hepatocytes exposed to extracts of oil sands process-affected water and the Athabasca River

Gagne, F., Douville, M., Andre, C., Debenest, T., talbot, A., Sherry, J., Hewitt, L.M., Frank, R.A., McMaster, M.E., Parrott, J. and Bickerton, G. Comp. Biochem. Physiol. B, 155, 551-559 (2012)   The oil sands region of northern Alberta represents the world's largest reserves of bitumen, and the accelerated pace of industrial extraction activity has raised concern about the possible impacts on the Athabasca River and its tributaries. An ecotoxicogenomic study was undertaken on Oncorhynchus mykiss trout hepatocytes exposed to extracts of water samples near the oil sand development area, as well as to oil sands process-affected water (OSPW) extracts using the quantitative reverse transcriptase polymerase chain reaction technique. The expression of the following genes (mRNA) was monitored to track changes in xenobiotic biotransformation (CYP1A1, CYP3A4, glutathione S-transferase, multi-drug resistance transporter), estrogenicity (estrogen receptor and vitellogenin), oxidative stress (superoxide dismutase and metallothionein) and DNA repair activity (DNA ligase). The extent of DNA-aromatic hydrocarbon adducts was also determined in cells by immuno-staining. A comparative analysis of gene expression between the river/lake and OSPW samples revealed that CYP3A4, metallothioneins, DNA ligase and GST genes, were specifically expressed by OSPW. Cells exposed to OSPW, commercial naphthenic acids, and benzo(a)pyrene showed increased polyaromatic hydrocarbon DNA-adducts, as determined by cell immunofluorescence analysis. Other genes were induced by all types of water samples, although the induction potential was stronger in OSPW most of the time (e.g., VTG gene was expressed nearly 15-fold by surface waters from the lake and river samples but increased to a maximum of 31-fold in OSPW). A multivariate discriminant function analysis revealed that the lake and river water samples were well discriminated from the OSPW. The CYP3A4 gene was the most highly expressed gene in cells exposed to OSPW and responded less to the lake or river water in the Athabasca River area. This study identified a suite of gene targets that responded specifically to OSPW extracts, which could serve as toxicogenomic fingerprints of OSPW contamination.  

4.1031           Olesoxime delays muscle denervation, astrogliosis, microglial activation and motoneuron death in an ALS mouse model

Sunyach, C., Michaud, M., Arnoux, T., Bernard-Marissal, N., Aebisvher, J., Latyszenok, V., Gouarne, C., Raoul, C., Pruss, R.M., Bordet, st. and Pettmann, B. Neuropharmacol., 62, 2345-2352 (2012)   Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. The pathology is mimicked to a striking degree in transgenic mice carrying familial ALS-linked SOD1 gene mutations. Olesoxime (TRO19622), a novel neuroprotective and reparative compound identified in a high-throughput screen based on motoneuron (MN) survival, delays disease onset and improves survival in mutant SOD1G93A mice, a model for ALS. The present study further analyses the cellular basis for the protection provided by olesoxime at the neuromuscular junctions (NMJ) and the spinal cord. Studies were carried out at two disease stages, 60 days, presymptomatic and 104 days, symptomatic. Cohorts of wild type and SOD1G93A mice were randomized to receive olesoxime-charged food pellets or normal diet from day 21 onward. Analysis showed that olesoxime initially reduced denervation from 60 to 30% compared to SOD1G93A mice fed with control food pellets while at the symptomatic stage only a few NMJs were still preserved. Immunostaining of cryostat sections of the lumbar spinal cord with VAChT to visualize MNs, GFAP for astrocytes and Iba1 for microglial cells showed that olesoxime strongly reduced astrogliosis and microglial activation and prevented MN loss. These studies suggest that olesoxime exerts its protective effect on multiple cell types implicated in the disease process in SOD1G93A mice, slowing down muscle denervation, astrogliosis, microglial activation and MN death. A Phase 3 clinical study in ALS patients will determine whether olesoxime could be beneficial for the treatment of ALS.  

4.1032           A new cannabinoid CB2 receptor agonist HU-910 attenuates oxidative stress, inflammation and cell death associated with hepatic ischaemia/reperfusion injury

Horvath, B., Magid, L., Mukhopadhyay, P., Batkai, S., Rajesh, M., Park, O., Tanchian, G., Gao, R.Y., Goodfellow, C.E., Glass, M., Mechoulam, R. and Pacher, P. Br. J. Pharmacol., 165(8), 2462-2478 (2012)   BACKGROUND AND PURPOSE Cannabinoid CB₂ receptor activation has been reported to attenuate myocardial, cerebral and hepatic ischaemia-reperfusion (I/R) injury. EXPERIMENTAL APPROACH We have investigated the effects of a novel CB₂ receptor agonist ((1S,4R)-2-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-7,7-dimethylbicyclo[2.2.1]hept-2-en-1-yl)methanol (HU-910) on liver injury induced by 1 h of ischaemia followed by 2, 6 or 24 h of reperfusion, using a well-established mouse model of segmental hepatic I/R. KEY RESULTS Displacement of [³H]CP55940 by HU-910 from specific binding sites in CHO cell membranes transfected with human CB₂ or CB₁ receptors (hCB_1/2) yielded K_i values of 6 nM and 1.4 µM respectively. HU-910 inhibited forskolin-stimulated cyclic AMP production by hCB₂ CHO cells (EC₅₀= 162 nM) and yielded EC₅₀ of 26.4 nM in [³⁵S]GTPγS binding assays using hCB₂ expressing CHO membranes. HU-910 given before ischaemia significantly attenuated levels of I/R-induced hepatic pro-inflammatory chemokines (CCL3 and CXCL2), TNF-α, inter-cellular adhesion molecule-1, neutrophil infiltration, oxidative stress and cell death. Some of the beneficial effect of HU-910 also persisted when given at the beginning of the reperfusion or 1 h after the ischaemic episode. Furthermore, HU-910 attenuated the bacterial endotoxin-triggered TNF-α production in isolated Kupffer cells and expression of adhesion molecules in primary human liver sinusoidal endothelial cells stimulated with TNF-α. Pretreatment with a CB₂ receptor antagonist attenuated the protective effects of HU-910, while pretreatment with a CB₁ antagonist tended to enhance them. CONCLUSION AND IMPLICATIONS HU-910 is a potent CB₂ receptor agonist which may exert protective effects in various diseases associated with inflammation and tissue injury.  

4.1033           Modulation of Astrocytic Mitochondrial Function by Dichloroacetate Improves Survival and Motor Performance in Inherited Amyotrophic Lateral Sclerosis

Miquel, E., Cassina, A., Martinez-Palma, L., Bolatto, C., Trias, e., Gandelman, M., Radi, R., Barbeito, L. and Cassina, P. PloS One, 7(4), e34776 (2012)   Mitochondrial dysfunction is one of the pathogenic mechanisms that lead to neurodegeneration in Amyotrophic Lateral Sclerosis (ALS). Astrocytes expressing the ALS-linked SOD1^G93A mutation display a decreased mitochondrial respiratory capacity associated to phenotypic changes that cause them to induce motor neuron death. Astrocyte-mediated toxicity can be prevented by mitochondria-targeted antioxidants, indicating a critical role of mitochondria in the neurotoxic phenotype. However, it is presently unknown whether drugs currently used to stimulate mitochondrial metabolism can also modulate ALS progression. Here, we tested the disease-modifying effect of dichloroacetate (DCA), an orphan drug that improves the functional status of mitochondria through the stimulation of the pyruvate dehydrogenase complex activity (PDH). Applied to astrocyte cultures isolated from rats expressing the SOD1^G93A mutation, DCA reduced phosphorylation of PDH and improved mitochondrial coupling as expressed by the respiratory control ratio (RCR). Notably, DCA completely prevented the toxicity of SOD1^G93A astrocytes to motor neurons in coculture conditions. Chronic administration of DCA (500 mg/L) in the drinking water of mice expressing the SOD1^G93A mutation increased survival by 2 weeks compared to untreated mice. Systemic DCA also normalized the reduced RCR value measured in lumbar spinal cord tissue of diseased SOD1^G93A mice. A remarkable effect of DCA was the improvement of grip strength performance at the end stage of the disease, which correlated with a recovery of the neuromuscular junction area in extensor digitorum longus muscles. Systemic DCA also decreased astrocyte reactivity and prevented motor neuron loss in SOD1^G93A mice. Taken together, our results indicate that improvement of the mitochondrial redox status by DCA leads to a disease-modifying effect, further supporting the therapeutic potential of mitochondria-targeted drugs in ALS.  

4.1034           Prevention of Trinitrobenzene Sulfonic Acid-Induced Experimental Colitis by Oral Administration of a Poly(lactic-coglycolic Acid) Microsphere Containing Prostaglandin E2 Receptor Subtype 4 Agonist

Okamoto, T., Uemoto, S. and Tabata, Y.

1. Pharmacol. Exp. Ther., 341(2), 340-349 (2012)

  Prostaglandin E₂ receptor subtype 4 (EP4) agonists are known to reduce intestinal inflammation and enhance epithelium regeneration. We explored the possibility of colonic delivery of an EP4 agonist, 2-[(4-{[2-((1R,2R,3R)-3-hydroxy-2-{(1E,3S)-3-hydroxy-4-[3-(methoxymethyl)phenyl]but-1-enyl}-5-oxocyclopentyl)ethyl]sulfanyl}butanoyl)oxy]ethyl nonanoate (ONO-AE2-724), using poly(lactic-coglycolic acid) (PLGA) microspheres. Colitis was induced in mice by the intrarectal administration of trinitrobenzene sulfonic acid (TNBS). ONO-AE2-724-PLGA microspheres (EP4-MS) were prepared by the standard technique. Drug distributions after oral administration of EP4-MS were determined by liquid chromatography-tandem mass spectrometry analysis. To evaluate the protective effect of EP4-MS, animals were orally treated by gavage with single doses of EP4-MS 24 h before TNBS instillation. The changes in body weight, histopathology, immunohistochemistry, and expression of inflammatory cytokines were evaluated. Oral administration of EP4-MS enhanced colonic tissue drug concentration without any increase in the serum concentration during the 48 h after intake. EP4-MS pretreatment, but not unloaded ONO-AE2-724, significantly attenuated TNBS-induced colitis and diminished colonic mRNA expression levels of proinflammatory cytokines. In addition, a significant increase in the expression of CD25 and FoxP3 was found in isolated lamina propria CD4⁺ T cells of EP4-MS-treated mice. Immunohistochemical analysis of Ki-67 and single-stranded DNA revealed that EP4-MS pretreatment significantly suppressed apoptosis of colonic cells and promoted epithelial cell proliferation. These results suggest that EP4-MS protect mice from TNBS-induced colitis by intestinal local ONO-AE2-724 delivery. The EP4-MS may offer a promising new therapeutic strategy to treat inflammatory bowel diseases.  

4.1035           Epigenetic Modulation of Homer1a Transcription Regulation in Amygdala and Hippocampus with Pavlovian Fear Conditioning

Mahan, A.L., Mou, L., Shah, N., Hu, J-H., Worley, P.F. and Ressler, K.J.

1. Neurosci., 32(13), 4651-4659 (2012)

  The consolidation of conditioned fear involves upregulation of genes necessary for long-term memory formation. An important question remains as to whether this results in part from epigenetic regulation and chromatin modulation. We examined whether Homer1a, which is required for memory formation, is necessary for Pavlovian cued fear conditioning, whether it is downstream of BDNF-TrkB activation, and whether this pathway utilizes histone modifications for activity-dependent transcriptional regulation. We initially found that Homer1a knock-out mice exhibited deficits in cued fear conditioning (5 tone–shock presentations with 70 dB, 6 kHz tones and 0.5 s, 0.6 mA footshocks). We then demonstrated that: (1) Homer1a mRNA increases after fear conditioning in vivo within both amygdala and hippocampus of wild-type mice; (2) it increases after BDNF application to primary hippocampal and amygdala cultures in vitro; and (3) these increases are dependent on transcription and MAPK signaling. Furthermore, using chromatin immunoprecipitation we found that both in vitro and in vivo manipulations result in decreases in Homer1 promoter H3K9 methylation in amygdala cells but increases in Homer1 promoter H3 acetylation in hippocampal cells. However, no changes were observed in H4 acetylation or H3K27 dimethylation. Inhibition of histone deacetylation by sodium butyrate enhanced contextual but not cued fear conditioning and enhanced Homer1 H3 acetylation in the hippocampus. These data provide evidence for dynamic epigenetic regulation of Homer1a following BDNF-induced plasticity and during a BDNF-dependent learning process. Furthermore, upregulation of this gene may be regulated through distinct epigenetic modifications in the hippocampus and amygdala.  

4.1036           Actin polymerization stabilizes α4β1 integrin anchors that mediate monocyte adhesion

Rullo, J., Becker, H., Hyduk, S.J., Wong, J.C., Digby, G., Arora, P.D., Cano, A.P., HArtwig, J., McCulloch, C.A. ansd Cybulsky, M.I.

1. Cell Biol., 197(1), 115-129 (2012)

  Leukocytes arrested on inflamed endothelium via integrins are subjected to force imparted by flowing blood. How leukocytes respond to this force and resist detachment is poorly understood. Live-cell imaging with Lifeact-transfected U937 cells revealed that force triggers actin polymerization at upstream α4β1 integrin adhesion sites and the adjacent cortical cytoskeleton. Scanning electron microscopy revealed that this culminates in the formation of structures that anchor monocyte adhesion. Inhibition of actin polymerization resulted in cell deformation, displacement, and detachment. Transfection of dominant-negative constructs and inhibition of function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. These included activation of Rap1, phosphoinositide 3-kinase γ isoform, and Rac but not Cdc42. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces.  

4.1037           Reduced Calreticulin Levels Link Endoplasmic Reticulum Stress and Fas-Triggered Cell Death in Motoneurons Vulnerable to ALS

Bernard-Marissal, N., Moumen, A., Sunyach, C., Pellegrino, C., Dudley, K., Henderson, C.E., Raoul, C. and Pettmann, B.

1. Neurosci., 32(14), 4901-4912 (2012)

  Cellular responses to protein misfolding are thought to play key roles in triggering neurodegeneration. In the mutant superoxide dismutase (mSOD1) model of amyotrophic lateral sclerosis (ALS), subsets of motoneurons are selectively vulnerable to degeneration. Fast fatigable motoneurons selectively activate an endoplasmic reticulum (ER) stress response that drives their early degeneration while a subset of mSOD1 motoneurons show exacerbated sensitivity to activation of the motoneuron-specific Fas/NO pathway. However, the links between the two mechanisms and the molecular basis of their cellular specificity remained unclear. We show that Fas activation leads, specifically in mSOD1 motoneurons, to reductions in levels of calreticulin (CRT), a calcium-binding ER chaperone. Decreased expression of CRT is both necessary and sufficient to trigger SOD1^G93A motoneuron death through the Fas/NO pathway. In SOD1^G93A mice in vivo, reductions in CRT precede muscle denervation and are restricted to vulnerable motor pools. In vitro, both reduced CRT and Fas activation trigger an ER stress response that is restricted to, and required for death of, vulnerable SOD1^G93A motoneurons. Our data reveal CRT as a critical link between a motoneuron-specific death pathway and the ER stress response and point to a role of CRT levels in modulating motoneuron vulnerability to ALS.  

4.1038           Endogenous GFAP-Positive Neural Stem/Progenitor Cells in the Postnatal Mouse Cortex Are Activated following Traumatic Brain Injury

Ahmed, A.I., Shtaya, A.B., Zaben, M.J., Owens, E.V., Kiecker, C. and Gray, W.P.

1. Neurotrauma, 29(5), 828-842 (2012)

  Interest in promoting regeneration of the injured nervous system has recently turned toward the use of endogenous stem cells. Elucidating cues involved in driving these precursor cells out of quiescence following injury, and the signals that drive them toward neuronal and glial lineages, will help to harness these cells for repair. Using a biomechanically validated in vitro organotypic stretch injury model, cortico-hippocampal slices from postnatal mice were cultured and a stretch injury equivalent to a severe traumatic brain injury (TBI) applied. In uninjured cortex, proliferative potential under in vitro conditions is virtually absent in older slices (equivalent postnatal day 15 compared to 8). However, following a severe stretch injury, this potential is restored in injured outer cortex. Using slices from mice expressing a fluorescent reporter on the human glial fibrillary acidic protein (GFAP) promoter, we show that GFAP+ cells account for the majority of proliferating neurospheres formed, and that these cells are likely to arise from the cortical parenchyma and not from the subventricular zone. Moreover, we provide evidence for a correlation between upregulation of sonic hedgehog signaling, a pathway known to regulate stem cell proliferation, and this restoration of regenerative potential following TBI. Our results indicate that a source of quiescent endogenous stem cells residing in the cortex and subcortical tissue proliferate in vitro following TBI. Moreover, these proliferating cells are multipotent and are derived mostly from GFAP-expressing cells. This raises the possibility of using this endogenous source of stem cells for repair following TBI.  

4.1039           A New Enzyme Mixture to Increase the Yield and Transplant Rate of Autologous and Allogeneic Human Islet Products

Balamurugan, A.N., Loganathan, G., Bellin, M.D., Wilhelm, J.J., Harmon, J., Anazawa, t., Soltani, S.M., Radosevich, D.M., Yuasa, T., Tiwari, M., Papas, K.K., McCarthy, sr., Sutherland, D.E.R. and Hering, B.J. Transplantation, 93(7), 693-702 (2012) Background. The optimal enzyme blend that maximizes human islet yield for transplantation remains to be determined. In this study, we evaluated eight different enzyme combinations (ECs) in an attempt to improve islet yield. The ECs consisted of purified, intact or truncated class 1 (C1) and class 2 (C2) collagenases from Clostridium histolyticum (Ch), and neutral protease (NP) from Bacillus thermoproteolyticus rokko (thermolysin) or Ch (ChNP). Methods. We report the results of 249 human islet isolations, including 99 deceased donors (research n=57, clinical n=42) and 150 chronic pancreatitis pancreases. We prepared a new enzyme mixture (NEM) composed of intact C1 and C2 collagenases and ChNP in place of thermolysin. The NEM was first tested in split pancreas (n=5) experiments and then used for islet autologous (n=21) and allogeneic transplantation (n=10). Islet isolation outcomes from eight different ECs were statistically compared using multivariate analysis. Results. The NEM consistently achieved higher islet yields from pancreatitis (P<0.003) and deceased donor pancreases (P<0.001) than other standard ECs. Using the NEM, islet products met release criteria for transplantation from 8 of 10 consecutive pancreases, averaging 6510±2150 islet equivalent number/gram (IEQ/g) pancreas and 694,681±147,356 total IEQ/transplantation. In autologous isolation, the NEM yielded more than 200,000 IEQ from 19 of 21 pancreases (averaging 422,893±181,329 total IEQ and 5979±1469 IEQ/kg recipient body weight) regardless of the severity of fibrosis. Conclusions. A NEM composed of ChNP with CIzyme high intact C1 collagenase recovers higher islet yield from deceased and pancreatitis pancreases while retaining islet quality and function.  

4.1040           Effect of daily semen centrifugation and resuspension on the longevity of equine sperm quality following cooled storage

Love, C.C., Blanchard, T.L., varner, D.D., Brinsko, S.P., Voge, J., Sudderth, K., Teague, S. and LaCaze, K. Theriogenology, 77, 1911-1917 (2012)   An experiment was conducted to determine whether cooled semen quality could be maintained for a longer interval by conducting daily centrifugation of extended semen, with resuspension of the sperm pellet in fresh extender. Semen treatments included SP10NC and SP50NC which contained 10 and 50% seminal plasma, respectively, were not centrifuged (NC), and were stored at 4 to 7 °C for 96 h. Treatments SP10C and SP50C contained 10 and 50% seminal plasma, respectively, but were centrifuged (C) after 24, 48, and 72 h of cooled storage, with daily resuspension in fresh extender containing 10% seminal plasma. Percent total sperm motility (TMOT) and progressively motile (PMOT) was reduced (P < 0.05) in the SP50NC treatment after 24, 48, 72, and 96 h of storage, and TMOT did not differ (P > 0.05) in the SP10C, SP50C, SP10NC groups after the same storage periods. The % COMP-_αt did not differ (P > 0.05) among treatments at any time period. Percent membrane intact sperm (SMI) was reduced in SP50NC, as compared to SP10C at 48, 72, and 96 h (P < 0.05). Daily centrifugation and resuspension of sperm exposed to 50% seminal plasma for the first 24 h (SP50C) yielded similar TMOT, PMOT, VCL, SMI, % COMP-_αt (P > 0.05) to Groups SP10NC and SP10C after 96 h of storage. Daily centrifugation and resuspension of cool-stored equine semen in fresh extender may be a method to increase sperm longevity.  

4.1041           Loss of Sialic Acid Binding Domain Redirects Protein σ1 to Enhance M Cell-Directed Vaccination

Zlotkowska, D., Maddaloni, M., Riccardi, C., Walters, N., Holderness, K., CaLLIS, g., Rynda-Apple, A. and Pascual, D.W. PloS One, 7(4), e36182 (2012)   Ovalbumin (OVA) genetically fused to protein sigma 1 (pσ1) results in tolerance to both OVA and pσ1. Pσ1 binds in a multi-step fashion, involving both protein- and carbohydrate-based receptors. To assess the relative pσ1 components responsible for inducing tolerance and the importance of its sialic binding domain (SABD) for immunization, modified OVA-pσ1, termed OVA-pσ1(short), was deleted of its SABD, but with its M cell targeting moiety intact, and was found to be immunostimulatory and enhanced CD4⁺ and CD8⁺ T cell proliferation. When used to nasally immunize mice given with and without cholera toxin (CT) adjuvant, elevated SIgA and serum IgG responses were induced, and OVA-pσ1(s) was more efficient for immunization than native OVA+CT. The immune antibodies (Abs) were derived from elevated Ab-forming cells in the upper respiratory tissues and submaxillary glands and were supported by mixed Th cell responses. Thus, these studies show that pσ1(s) can be fused to vaccines to effectively elicit improved SIgA responses.      

4.1042           P2X7 receptor-mediated purinergic signaling promotes liver injury in acetaminophen hepatotoxicity in mice

Hoque, R., Sohail, M.A., Salhanick, S.,, Malik, A.F., Ghani, A., Robson, S.C. and Mehal, W.Z. Am. J. Physiol. Gastrointest. Liver Physiol., 302, G1171-G1179 (2012)   Inflammation contributes to liver injury in acetaminophen (APAP) hepatotoxicity in mice and is triggered by stimulation of immune cells. The purinergic receptor P2X7 is upstream of the nod-like receptor family, pryin domain containing-3 (NLRP3) inflammasome in immune cells and is activated by ATP and NAD that serve as damage-associated molecular patterns. APAP hepatotoxicity was assessed in mice genetically deficient in P2X7, the key inflammatory receptor for nucleotides (P2X7−/−), and in wild-type mice. P2X7−/− mice had significantly decreased APAP-induced liver necrosis. In addition, APAP-poisoned mice were treated with the specific P2X7 antagonist A438079 or etheno-NAD, a competitive antagonist of NAD. Pre- or posttreatment with A438079 significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury in wild-type but not P2X7−/− mice. Pretreatment with etheno-NAD also significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury. In addition, APAP toxicity in mice lacking the plasma membrane ecto-NTPDase CD39 (CD39−/−) that metabolizes ATP was examined in parallel with the use of soluble apyrase to deplete extracellular ATP in wild-type mice. CD39−/− mice had increased APAP-induced hemorrhage and mortality, whereas apyrase also decreased APAP-induced mortality. Kupffer cells were treated with extracellular ATP to assess P2X7-dependent inflammasome activation. P2X7 was required for ATP-stimulated IL-1β release. In conclusion, P2X7 and exposure to the ligands ATP and NAD are required for manifestations of APAP-induced hepatotoxicity.  

4.1043           Outbreak of Equine Herpesvirus Myeloencephalopathy in France: a Clinical and Molecular Investigation

Pronost, S., Legrand, L., Pitel, P-H., Wegge, B., Lissens, J., Freymuth, F., Richard, E. and Fortier, G. Transboundary and Emerging Dis., 59(3), 256-263 (2012)   Equid herpesvirus 1 (EHV-1)-associated myeloencephalopathy (EHM) is a disease affecting the central nervous system of horses. Despite the constantly increasing interest about this syndrome, epidemiological data are limited especially when related to the description of large outbreaks. The aim of this article is to describe clinical, virological and molecular data obtained throughout a severe outbreak of EHM, with emphasis on laboratory diagnostic methods. The epidemic disease concerned a riding school in France where 7/66 horses aged 12–22 years developed signs of neurological disease in July 2009. Diagnosis of EHM was supported by EHV-1 detection using both real-time PCR and virus culture, and SNP-PCR test for viral strain characterization. EHM morbidity was 10.6% (7/66), mortality was 7.5% (5/66) and case fatality rate was 71.4% (5/7). Clinical presentation of the disease was characterized by the fact that fever was systematically present within 2 days before the severe neurological signs were noted. EHV-1 was detected by PCR in each available blood and nasal swab samples. Neuropathogenic strain only (G₂₂₅₄) was isolated during the current outbreak; C_t values, used as an indicative level of the viral load, ranged 26.0–37.0 among the six sampled horses. The amount of virus in biological samples was not systematically related to the intensity of the clinical signs being observed. In conclusion, this article described a severe outbreak of EHM while limited in time and restricted to one premise. Molecular data strongly suggested taking into account any low viral load as being a potential risk factor for neurological manifestations.  

4.1044           Isolation of Porcine Pancreatic Islets for Xenotransplantation

Ulrichs, K., Eber, S., Schneiker, b., Gahn, S., Strauss, A., Moskalenko, V.a dn Chodnevskaja, I. Methods in Mol. Biol., 885, 213-232 (2012)   This chapter deals with a technique for isolating intact islets of Langerhans from the pig pancreas based on our experience performing approximately 750 isolations. The procedure we describe involves identi fi cation of an optimal donor pancreas, puri fi cation and in vitro culture of islets, diabetes induction in recipients, and transplantation of islets and their immunomodulation. Besides the sophistication of the technical equipment employed, the major factors in fl uencing the isolation outcome are the pig breed, the number and morphology of the islets in the donor pancreas, the quality of the collagenase/neutral protease, and the skill of the team members.          

4.1045           Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice

Huang, G., Besner, G.E. and Brigstock, D.R. Lab. Invest., 92(5), 703-712 (2012)   Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cytoprotective agent in several organ systems but its roles in liver fibrosis are unclear. We studied the roles of HB-EGF in experimental liver fibrosis in mice and during hepatic stellate cell (HSC) activation. Thioacetamide (TAA; 100 mg/kg) was administered by intraperitoneal injection three times a week for 4 weeks to wild-type HB-EGF^+/+ or HB-EGF-null (HB-EGF^−/−) male mice. Livers were examined for histology and expression of key fibrotic markers. Primary cultured HSCs isolated from untreated HB-EGF^+/+ or HB-EGF^−/− mice were examined for fibrotic markers and/or cell migration either during culture-induced activation or after exogenous HB-EGF (100 ng/ml) treatment. TAA induced liver fibrosis in both HB-EGF^+/+ and HB-EGF^−/− mice. Hepatic HB-EGF expression was decreased in TAA-treated HB-EGF^+/+ mice by 37.6% (P<0.05) as compared with animals receiving saline alone. HB-EGF^−/− mice treated with TAA showed increased hepatic α-smooth muscle actin-positive cells and collagen deposition, and, as compared with HB-EGF^+/+ mice, TAA-stimulated hepatic mRNA levels in HB-EGF^−/− mice were, respectively, 2.1-, 1.7-, 1.8-, 2.2-, 1.2- or 3.3-fold greater for α-smooth muscle actin, α1 chain of collagen I or III (COL1A1 or COL3A1), transforming growth factor-β1, connective tissue growth factor or tissue inhibitor of metalloproteinase-1 (P<0.05). HB-EGF expression was detectable in primary cultured HSCs from HB-EGF^+/+ mice. Both endogenous and exogenous HB-EGF inhibited HSC activation in primary culture, and HB-EGF enhanced HSC migration. These findings suggest that HB-EGF gene knockout in mice increases susceptibility to chronic TAA-induced hepatic fibrosis and that HB-EGF expression or action is associated with suppression of fibrogenic pathways in HSCs.  

4.1046           The Double-Stranded RNA Bluetongue Virus Induces Type I Interferon in Plasmacytoid Dendritic Cells via a MYD88-Dependent TLR7/8-Independent Signaling Pathway

Ruscanu, S. et al

1. Virol., 86(10), 5817-5828 (2012)

  Dendritic cells (DCs), especially plasmacytoid DCs (pDCs), produce large amounts of alpha/beta interferon (IFN-α/β) upon infection with DNA or RNA viruses, which has impacts on the physiopathology of the viral infections and on the quality of the adaptive immunity. However, little is known about the IFN-α/β production by DCs during infections by double-stranded RNA (dsRNA) viruses. We present here novel information about the production of IFN-α/β induced by bluetongue virus (BTV), a vector-borne dsRNA Orbivirus of ruminants, in sheep primary DCs. We found that BTV induced IFN-α/β in skin lymph and in blood in vivo. Although BTV replicated in a substantial fraction of the conventional DCs (cDCs) and pDCs in vitro, only pDCs responded to BTV by producing a significant amount of IFN-α/β. BTV replication in pDCs was not mandatory for IFN-α/β production since it was still induced by UV-inactivated BTV (UV-BTV). Other inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-12p40, were also induced by UV-BTV in primary pDCs. The induction of IFN-α/β required endo-/lysosomal acidification and maturation. However, despite being an RNA virus, UV-BTV did not signal through Toll-like receptor 7 (TLR7) for IFN-α/β induction. In contrast, pathways involving the MyD88 adaptor and kinases dsRNA-activated protein kinase (PKR) and stress-activated protein kinase (SAPK)/Jun N-terminal protein kinase (JNK) were implicated. This work highlights the importance of pDCs for the production of innate immunity cytokines induced by a dsRNA virus, and it shows that a dsRNA virus can induce IFN-α/β in pDCs via a novel TLR-independent and Myd88-dependent pathway. These findings have implications for the design of efficient vaccines against dsRNA viruses.  

4.1047           Controlled Synthesis of Cell-Laden Microgels by Radical-Free Gelation in Droplet Microfluidics

Rossow, T., Heyman, J.A., Ehrlicher, A.J., Langhoff, A., Weitz, D.A., Haag, R. and Seiffert, S.

1. Am. Chem. Soc., 134(10), 4983-4989 (2012)

  Micrometer-sized hydrogel particles that contain living cells can be fabricated with exquisite control through the use of droplet-based microfluidics and bioinert polymers such as polyethyleneglycol (PEG) and hyperbranched polyglycerol (hPG). However, in existing techniques, the microgel gelation is often achieved through harmful reactions with free radicals. This is detrimental for the viability of the encapsulated cells. To overcome this limitation, we present a technique that combines droplet microfluidic templating with bio-orthogonal thiol–ene click reactions to fabricate monodisperse, cell-laden microgel particles. The gelation of these microgels is achieved via the nucleophilic Michael addition of dithiolated PEG macro-cross-linkers to acrylated hPG building blocks and does not require any initiator. We systematically vary the microgel properties through the use of PEG linkers with different molecular weights along with different concentrations of macromonomers to investigate the influence of these parameters on the viability and proliferation of encapsulated yeast cells. We also demonstrate the encapsulation of mammalian cells including fibroblasts and lymphoblasts.  

4.1048           A Reversible Early Oxidized Redox State That Precedes Macromolecular ROS Damage in Aging Nontransgenic and 3xTg-AD Mouse Neurons

Ghosh,, D., LeVault, K.R., Barnett, A.J. and Brewer, G.J.

1. Neurosci., 32(17), 5821-5832 (2012)

  The brain depends on redox electrons from nicotinamide adenine dinucleotide (reduced form; NADH) to produce ATP and oxyradicals (reactive oxygen species [ROS]). Because ROS damage and mitochondrial dysregulation are prominent in aging and Alzheimer's disease (AD) and their relationship to the redox state is unclear, we wanted to know whether an oxidative redox shift precedes these markers and leads to macromolecular damage in a mouse model of AD. We used the 3xTg-AD mouse model, which displays cognitive deficits beginning at 4 months. Hippocampal/cortical neurons were isolated across the age span and cultured in common nutrients to control for possible hormonal and vascular differences. We found an increase of NAD(P)H levels and redox state in nontransgenic (non-Tg) neurons until middle age, followed by a decline in old age. The 3xTg-AD neurons maintained much lower resting NAD(P)H and redox states after 4 months, but the NADH regenerating capacity continuously declined with age beginning at 2 months. These redox characteristics were partially reversible with nicotinamide, a biosynthetic precursor of NAD⁺. Nicotinamide also protected against glutamate excitotoxicity. Compared with non-Tg neurons, 3xTg-AD neurons had more mitochondria/neuron and lower glutathione (GSH) levels that preceded age-related increases in ROS levels. These GSH deficits were again reversible with nicotinamide in 3xTg-AD neurons. Surprisingly, low macromolecular ROS damage was only elevated after 4 months in the 3xTg-AD neurons if antioxidants were removed. The present data suggest that a more oxidized redox state and a lower antioxidant GSH defense can be dissociated from neuronal ROS damage, changes that precede the onset of cognitive deficits in the 3xTg-AD model.  

4.1049           Consumption of Rice Bran Increases Mucosal Immunoglobulin A Concentrations and Numbers of Intestinal Lactobacillus spp.

Henderson, A.J., Kumar, A., Barnett, B., Dow, S.W. and Ryan, E.P.

1. Med. Food, 15(5), 469-475 (2012)

  Gut-associated lymphoid tissue maintains mucosal homeostasis by combating pathogens and inducing a state of hyporesponsiveness to food antigens and commensal bacteria. Dietary modulation of the intestinal immune environment represents a novel approach for enhancing protective responses against pathogens and inflammatory diseases. Dietary rice bran consists of bioactive components with disease-fighting properties. Therefore, we conducted a study to determine the effects of whole dietary rice bran intake on mucosal immune responses and beneficial gut microbes. Mice were fed a 10% rice bran diet for 28 days. Serum and fecal samples were collected throughout the study to assess total immunoglobulin A (IgA) concentrations. Tissue samples were collected for cellular immune phenotype analysis, and concentrations of native gut Lactobacillus spp. were enumerated in the fecal samples. We found that dietary rice bran induced an increase in total IgA locally and systemically. In addition, B lymphocytes in the Peyer's patches of mice fed rice bran displayed increased surface IgA expression compared with lymphocytes from control mice. Antigen-presenting cells were also influenced by rice bran, with a significant increase in myeloid dendritic cells residing in the lamina propria and mesenteric lymph nodes. Increased colonization of native Lactobacillus was observed in rice bran–fed mice compared with control mice. These findings suggest that rice bran–induced microbial changes may contribute to enhanced mucosal IgA responses, and we conclude that increased rice bran consumption represents a promising dietary intervention to modulate mucosal immunity for protection against enteric infections and induction of beneficial gut bacteria.  

4.1050           Role of Lentivirus-Mediated Overexpression of Programmed Death-Ligand 1 on Corneal Allograft Survival

Nosov, M., Wilk, M., Morcos, M., Cregg, M., O’Flynn, L., Treacy, O. and Ritter, T. Am. J. Transplant., 12(5), 1313-1322 (2012)   To investigate the role of lentivirus-mediated overexpression of programmed death-ligand 1 (PD-L1) on rat corneal allograft survival. A fully allogeneic rat cornea transplant model was used for in vivo studies. Lentiviral (LV) vectors are efficient tools for ex vivo genetic modification of cultured corneas. LV vector encoding for PD-L1 (LV.PD-L1) and LV vector encoding for eGFP (LV.eGFP, as control) were constructed and tested. PD-L1 or eGFP expression was increased on corneal cells upon LV.PD-L1 and LV.eGFP transduction, respectively. Both allogeneic controls and allogeneic LV.eGFP transduced corneas were uniformly rejected (MST: 13.8 ± 1.7 days and 12.3 ± 1.9 days, respectively). In contrast, allogeneic LV.PD-L1 transduced corneas showed a high percentage (83%) of graft survival (MST > 30 days, n = 5, 15 days, n = 1). Graft opacity of PD-L1 transduced corneas was present but was significantly reduced compared to control or eGFP expressing corneas. Flow cytometric analysis revealed that percentages of CD3⁺CD8⁺CD161⁺ and CD3⁺CD8⁺CD161^– lymphocytes were decreased in animals receiving LV.PD-L1 transduced corneas compared to animals grafted with LV.eGFP transduced corneas. Moreover, reduced expression of proinflammatory cytokines (IFN-γ and IL-6) in PD-L1 transduced corneas compared to allogeneic controls was also observed. Local PD-L1 gene transfer in cultured corneas is a promising approach for the prolongation of corneal allograft survival and attenuation of graft rejection.  

4.1051           Motoneuron programmed cell death in response to proBDNF

Taylor, A.R., Gifondorwa, D.J., Robinson, M.B., Strupe, J.L., Prevette, D., Johnson, J.E., Hemstead, B., Oppenheim, R.W. and Milligan, C.E. Develop. Neurobiol., 72(5), 699-712 (2012)   Motoneurons (MN) as well as most neuronal populations undergo a temporally and spatially specific period of programmed cell death (PCD). Several factors have been considered to regulate the survival of MNs during this period, including availability of muscle-derived trophic support and activity. The possibility that target-derived factors may also negatively regulate MN survival has been considered, but not pursued. Neurotrophin precursors, through their interaction with p75^NTR and sortilin receptors have been shown to induce cell death during development and following injury in the CNS. In this study, we find that muscle cells produce and secrete proBDNF. ProBDNF through its interaction with p75^NTR and sortilin, promotes a caspase-dependent death of MNs in culture. We also provide data to suggest that proBDNF regulates MN PCD during development in vivo.  

4.1052           High-frequency vagus nerve stimulation improves portal hypertension in cirrhotic rats

Bockx, I., Verdrengh, K., Vander Elst, I. et al Gut, 61, 604-612 (2012)   Objective The liver is innervated by the vagus nerve. Its efferent neurotransmitters acetylcholine (ACh) and vasoactive intestinal peptide (VIP) are both well-known vasodilators. A study was undertaken to determine whether electrical vagus nerve stimulation (STIM) influences portal vein pressure. Methods The left vagus nerve upstream of the hepatic branch was stimulated at 5 Hz (ACh release) and 10 Hz (VIP release) in normal and cirrhotic rats. Results STIM at both frequencies decreased portal pressure in normal rats while, in cirrhotic rats, only 10 Hz STIM resulted in long-lasting reduction of portal pressure. Hepatic branch vagotomy prevented the STIM-induced decrease in pressure, proving that the effect is a direct hepatic effect. Deafferentation of the left vagus nerve by pretreatment with capsaicin did not change the effect of STIM, showing that the vagus efferents and not the afferents are responsible for the decrease in portal pressure. Injecting microspheres before and after STIM showed that STIM did not lead to redistribution of systemic blood flow but decreased portal pressure by lowering intrahepatic resistance. Using in situ liver perfusion to evaluate the intrahepatic effect of ACh and VIP, both neurotransmitters significantly decreased the perfusion pressure in normal rats. VIP also decreased portal pressure in cirrhotic rats, confirming the results of STIM. This VIP-induced decrease in pressure could be prevented by a VIP receptor 2 antagonist. L-NAME did not inhibit the VIP effect in cirrhotic rats, indicating that VIP does not act via nitric oxide. Conclusion High-frequency electrical vagus stimulation improves portal hypertension in cirrhotic rats, most likely through release of VIP, binding to VIP receptor 2. As the technology is already in use for other applications, vagus nerve stimulation might be an important new strategy in the treatment of portal hypertension.  

4.1053           Tissue engineering the monosynaptic circuit of the stretch reflex arc with co-culture of embryonic motoneurons and proprioceptive sensory neurons

Guo, X., Ayala, J.E., Gonzales, M., Stancescu, M., Lambert, S. and Hickman, J.J. Biomaterials, 33, 5723-5731 (2012)   The sensory circuit of the stretch reflex arc is composed of intrafusal muscle fibers and their innervating proprioceptive neurons that convert mechanical information regarding muscle length and tension into action potentials that synapse onto the homonymous motoneurons in the ventral spinal cord which innervate the extrafusal fibers of the same muscle. To date, the in vitro synaptic connection between proprioceptive sensory neurons and spinal motoneurons has not been demonstrated. A functional in vitro system demonstrating this connection would enable the understanding of feedback by the integration of sensory input into the spinal reflex arc. Here we report a co-culture of rat embryonic motoneurons and proprioceptive sensory neurons from dorsal root ganglia (DRG) in a defined serum-free medium on a synthetic silane substrate (DETA). Furthermore, we have demonstrated functional synapse formation in the co-culture by immunocytochemistry and electrophysiological analysis. This work will be valuable for enabling in vitro model systems for the study of spinal motor control and related pathologies such as spinal cord injury, muscular dystrophy and spasticity by improving our understanding of the integration of the mechanosensitive feedback mechanism.  

4.1054           Endothelial Progenitor Cell Cotransplantation Enhances Islet Engraftment by Rapid Revascularization

Kang, S., Park, H.S., Jo, A., Hong, S.H., Lee, H.N., Lee, Y.Y., Park, J.S., Jung, H.S., Chung, S.S. and Park, K.S. Diabetes, 61, 866-876 (2012)   Impaired revascularization of transplanted islets is a critical problem that leads to progressive islet loss. Since endothelial progenitor cells (EPCs) are known to aid neovascularization, we aimed to enhance islet engraftment by cotransplanting EPCs with islets. Porcine islets, with (islet-EPC group) or without (islet-only group) human cord blood–derived EPCs, were transplanted into diabetic nude mice. The islet-EPC group reached euglycemia by ∼11 days posttransplantation, whereas the islet-only group did not. Also, the islet-EPC group had a higher serum porcine insulin level than the islet-only group. Islets from the islet-EPC group were more rapidly revascularized at the early period of transplantation without increment of final capillary density at the fully revascularized graft. Enhanced revascularization rate in the islet-EPC group was mainly attributed to stimulating vascular endothelial growth factor-A production from the graft. The rapid revascularization by EPC cotransplantation led to better graft perfusion and recovery from hypoxia. EPC cotransplantation was also associated with greater β-cell proliferation, probably by more basement membrane production and hepatocyte growth factor secretion. In conclusion, cotransplantation of EPCs and islets induces better islet engraftment by enhancing the rate of graft revascularization. These findings might provide a directly applicable tool to enhance the efficacy of islet transplantation in clinical practice.  

4.1055           PMO-126 The role of vascular-adhesion-protein 1 (vap-1) in mediating monocyte migration across inflamed hepatic sinusoidal endothelium

Zimmermann, H., Weston, C.J., Curbishley, S.M. and Adams, D.H. Gut, 61, A124 (2012)   Introduction There is compelling evidence that accumulating monocyte-derived macrophages are pivotally involved in driving liver fibrogenesis. It remains unclear which molecules mediate transmigration of these cells across hepatic sinusoidal endothelial cells (HSEC). VAP-1 is an atypical adhesion molecule with enzymatic monoamine oxidase activity that is predominantly expressed in the liver microvasculature. It possesses key function in the recruitment of various lymphocyte subsets. The aim of this study was to decipher VAP-1 contribution to monocytic transendothelial transmigration. Methods Primary human HSEC were isolated from explanted and grown to confluence in flow chambers. After activation with TNF-α/IFN-γ for 24h HSEC were treated with VAP-1 antibody and enzyme inhibitors. Monocytes were enriched from peripheral blood by using OptiPrep gradient. Monocyte subsets (CD14⁺⁺CD16⁻, CD14⁺⁺CD16⁺, CD14⁺CD16⁺⁺) were isolated by FACS-sorting. Isolated monocytes were perfused over HSEC monolayers under constant flow simulating physiological shear stress (0.05 Pa). Adhesion and transmigration was studied using phase contrast microscopy. Transwell assays were used to study the phenotype of transmigrated monocytes by flowcytometry. Results HSEC pretreatment with VAP-1 antibody (TK8-14) or enzyme inhibitor Semicarbizide equally reduced monocyte transmigration by ∼50%. VCAM-1 blockade had a similar but redundant effect whereas CLEVER-1-antibody or LOX-inhibitor (β-APN) did not alter monocyte transmigration. VAP-1 antibody acted in a time-dependent manner with influence on monocyte adhesion only after short-term application (15 min). Inhibiting VAP-1 led to profound reduction of proinflammatory nonclassical CD14⁺CD16⁺⁺ monocyte transmigration but also affected classical CD14⁺⁺CD16⁻ whereas the intermediate CD14⁺⁺CD16⁺ subtype was not affected. Under static conditions VAP-1 enzymatic or antibody inhibition was significantly blunted suggesting flow to be a mandatory prerequisite for the biological function of VAP-1 on monocytes. Increased expression of HLA-DR and the M2 macrophage marker CD206 on monocyte subsets after endothelial transmigration was not altered by VAP-1 inhibition. Conclusion Endothelial VAP-1 differentially modulates monocyte recruitment under flow conditions in a time-dependent fashion and .favours transmigration of a proinflammatory monocyte subset. The critical role of VAP-1 enzyme function renders small molecules as a promising therapeutic approach in combating liver inflammation and subsequent fibrosis.  

4.1056           Antigen-specific effector CD8 T cells regulate allergic responses via IFN-γ and dendritic cell function

Tang, Y., Guan, S.P., Chua, B.Y.L., Zhou, Q., Ho, A.W.S., Wong, K.H.S., Wong, K.L., Wong, W.S.F. and Kemeny, D.M.

1. Allergy Clin. Immunol., 129, 1611-1620 (2012)

  Background Previous studies have shown that CD8 T cells can both prevent and cause allergic responses. However, the underlying mechanisms remain to be elucidated. Objective We aim to investigate the potential of CD8 T cells with different IFN-γ expressions to modulate the elicitation of allergic inflammation following ovalbumin (OVA) challenge and investigate the underlying mechanisms. Methods To study the role of IFN-γ in the effect of CD8 T cells, effector CD8 T cells from CD8 OVA transgenic (OT-I) mice and IFN-γ^−/−OT-I mice were transferred to OVA-sensitized mice the day before 3 challenges with OVA. The effect on lung dendritic cells (DCs) exerted by CD8 T cells was studied with ex vivo culture of sorted DCs from treatment mice with CD4 T cells. Results Effector OT-I, but not IFN-γ^−/−OT-I CD8 T cells, attenuated eosinophilia and mucus secretion in the lungs of sensitized mice in an antigen-specific manner. Effector IFN-γ^−/−OT-I CD8 T cells displayed a Tc2-/Tc17-biased phenotype with weaker cytotoxicity and were able to both induce and exacerbate eosinophilia as well as neutrophilia. OT-I CD8 T cells increased the ability of lung CD11b⁺CD103⁻ DCs to both prime the differentiation of naive OVA-specific CD4 T cells toward a T_H1 phenotype and enhance IFN-γ production by antigen-experienced lung CD4 T cells. Conclusion Effector CD8 T cells attenuate pulmonary inflammation and alter the ability of DCs within the allergic lung to polarize T cells to a T_H1 phenotype during a T_H2 response. In the absence of IFN-γ, CD8 T cells assume a Tc2-/Tc17-biased phenotype and potentiate inflammation.  

4.1057           Expression of Transient Receptor Potential Ankyrin 1 (TRPA1) and Its Role in Insulin Release from Rat Pancreatic Beta Cells

Cao, D-S., Zhong, L., Hsieh, T-h., Abooj, M., Bishnoi, M., Hughes, L. and Premkumar, L.S. PloS One, 7(5), e38005 (2012)   Objective Several transient receptor potential (TRP) channels are expressed in pancreatic beta cells and have been proposed to be involved in insulin secretion. However, the endogenous ligands for these channels are far from clear. Here, we demonstrate the expression of the transient receptor potential ankyrin 1 (TRPA1) ion channel in the pancreatic beta cells and its role in insulin release. TRPA1 is an attractive candidate for inducing insulin release because it is calcium permeable and is activated by molecules that are produced during oxidative glycolysis. Methods Immunohistochemistry, RT-PCR, and Western blot techniques were used to determine the expression of TRPA1 channel. Ca²⁺ fluorescence imaging and electrophysiology (voltage- and current-clamp) techniques were used to study the channel properties. TRPA1-mediated insulin release was determined using ELISA. Results TRPA1 is abundantly expressed in a rat pancreatic beta cell line and freshly isolated rat pancreatic beta cells, but not in pancreatic alpha cells. Activation of TRPA1 by allyl isothiocyanate (AITC), hydrogen peroxide (H₂O₂), 4-hydroxynonenal (4-HNE), and cyclopentenone prostaglandins (PGJ₂) and a novel agonist methylglyoxal (MG) induces membrane current, depolarization, and Ca²⁺ influx leading to generation of action potentials in a pancreatic beta cell line and primary cultured pancreatic beta cells. Activation of TRPA1 by agonists stimulates insulin release in pancreatic beta cells that can be inhibited by TRPA1 antagonists such as HC030031 or AP-18 and by RNA interference. TRPA1-mediated insulin release is also observed in conditions of voltage-gated Na⁺ and Ca²⁺ channel blockade as well as ATP sensitive potassium (K_ATP) channel activation. Conclusions We propose that endogenous and exogenous ligands of TRPA1 cause Ca²⁺ influx and induce basal insulin release and that TRPA1-mediated depolarization acts synergistically with K_ATP channel blockade to facilitate insulin release.  

4.1058           miR-132 Enhances Dendritic Morphogenesis, Spine Density, Synaptic Integration, and Survival of Newborn Olfactory Bulb Neurons

Pathania, M., Torres-Reveron, J., Yan, L., Kimura, T., Lin, T.V., Gordon, V., Teng, Z-Q., Zhao, X., Fulga, T.A., Van Vactor, D. and Borsdey, A. PloS One, 7(5), e38174 (2012)   An array of signals regulating the early stages of postnatal subventricular zone (SVZ) neurogenesis has been identified, but much less is known regarding the molecules controlling late stages. Here, we investigated the function of the activity-dependent and morphogenic microRNA miR-132 on the synaptic integration and survival of olfactory bulb (OB) neurons born in the neonatal SVZ. In situ hybridization revealed that miR-132 expression occurs at the onset of synaptic integration in the OB. Using in vivo electroporation we found that sequestration of miR-132 using a sponge-based strategy led to a reduced dendritic complexity and spine density while overexpression had the opposite effects. These effects were mirrored with respective changes in the frequency of GABAergic and glutamatergic synaptic inputs reflecting altered synaptic integration. In addition, timely directed overexpression of miR-132 at the onset of synaptic integration using an inducible approach led to a significant increase in the survival of newborn neurons. These data suggest that miR-132 forms the basis of a structural plasticity program seen in SVZ-OB postnatal neurogenesis. miR-132 overexpression in transplanted neurons may thus hold promise for enhancing neuronal survival and improving the outcome of transplant therapies.  

4.1059           CD70–CD27 Interaction Augments CD8+ T-Cell Activation by Human Epidermal Langerhans Cells

Polak, M.E., Newell, L., Taraban, V.Y., Pickard, C., Healy, E., Friedmann, P.S., Al-Shamkhani, A. and Ardern-Jones, M.R.

1. Invest. Dermatol., 132(6), 1636-1644 (2012)

  Human cutaneous dendritic cells (DCs) from epidermal and dermal compartments exhibit functional differences in their induction of CD4+ T-cell and humoral immune responses; however, differences in the regulation of memory CD8+ T-cell responses by human skin DCs remain poorly characterized. We tested the capacity of human Langerhans cells (LCs) and dermal dendritic cells (DDCs) to induce antigen-specific cytokine production and proliferation of memory CD8+ cells. Although tumor necrosis factor-α-matured human DCs from both epidermal and dermal compartments showed efficient potential to activate CD8+ cells, LCs were constitutively more efficient than DDCs in cross-presenting CD8+ epitopes, as well as direct presentation of viral antigen to Epstein–Barr virus-specific CD8+ T cells. LCs showed greater expression of CD70, and blockade of CD70–CD27 signaling demonstrated that superiority of CD8+ activation by epidermal LC is CD70 dependent. This CD70-related activation of CD8+ cells by LCs denotes a central role of LCs in CD8+ immunity in skin, and suggests that regulation of LC CD70 expression is important in enhancing immunity against cutaneous epithelial pathogens and cancer.  

4.1060           Elucidating the Mechanism By Which Monocytes Can Inhibit Hypoxic PA-SMC Proliferation

Parmar, S., Thompson, A.A.R., Higgins, K.R., Sabroe, I., Parker, L.C., Lawrie, A., Arnold, J., Walker, S., Elliot, C.A., Conddliffe, R., Kiely, D.G., Whyte, M.K. and Walmsley, S.R. Am. J. Respir. Crit. Care Med., 185, A5660 (2012)   Introduction Pulmonary arterial hypertension (PAH) is a devastating disease characterised by the narrowing and occlusion of the small pulmonary arteries leading to increased right ventricular pressures and eventual failure. The triggers for the vascular remodelling events are still poorly understood. Understanding the pathogenesis of PAH is key to developing novel therapies. Interestingly, inflammation and local tissue hypoxia have been linked to the process. Using co-culture models we investigated the role of hypoxia monocyte PASMC interactions. Methods Peripheral blood mononuclear cells from healthy volunteers, patients with idiopathic PAH (IPAH) and patients with systemic sclerosis associated PAH (SScPAH) were isolated by Optiprep and CD14+ monocytes purified by negative selection. Monocytes were then cultured with hPA-SMCs (1:5) in normoxia (19 kPa) and hypoxia (3 kPa) for 24 hours +/- 10 ng/ml LPS and cell counts were. Results Monocytes undergo significantly less apoptosis in 24 hr hypoxia (3kPa) (32.88% +/- 3.09) than in normoxia (13kPa) (63.51% +/- 14.44) (*P< 0.05, n=4). Total PA-SMC counts increased significantly in hypoxia to normoxia (36420 +/- 6792 to 21167 +/- 1113, **P<0.01, n=8).This increase was abrogated with addition of monocytes (Normoxia: 26936 +/- 4269, hypoxia 24787 +/- 1859, n=10). Supernatant transfer reduced total cell numbers to co-culture values (Normoxia: 28276.67 +/- 1508.645 Hypoxia: 26578.0 +/- 908.847 n=4). Monocytes could not inhibit platelet derived growth factor induced proliferation (3 ng/ml PDGF monoculture: 31616.4 +/- 1395.0, co-culture: 30732.7 +/- 1276.1, ***P<0.001, n=6). Exdogenous IL-1ra could inhibit hypoxia induced proliferation to the extent of co-cultures (3 ng/ml IL-1ra hypoxic monoculture: 29846.7 +/- 1039.3, normoxic monoculture: 25830.0 +/- 994.9, n=6). We saw no inhibition of hypoxic PASMC proliferation by IPAH monocytes in contrast to monocytes from healthy controls or from patients with SSc-PAH (IPAH: 64307.5 +/- 12566.4, controls: 27935 +/- 672.5, SScPAH: 36517 +/- 3571 n=3, n=6 and n=6 respectively). Monocytes from IPAH patients are unable to suppress hypoxic PA-SMC proliferation Smooth muscle cells were seeded at 25,000 cells /well and cultured in normoxia (room air) (open bars) and hypoxia (3 kPa) (closed bars) for 24 hours before serum starvation. Plates were cultured for further 24 hours +/- monocytes. Total cell counts were carried out by Coulter Counter. (bars represent mean +/- SEM , n=6 control, n=4 SScPAH, n=3 IPAH *P<0.05, **P<0.01, ***P<0.001) Results are also expressed in terms of percentage change from normoxic monoculture Conclusions Both monocytes and hPASMCs demonstrate a pro-survival and proliferative phenotype in hypoxia. Monocytes inhibit the hypoxia induced increase in hPA-SMC cell counts. Supernatant transfers suggest this is independent of monocyte hypoxia and direct cell contact and is selective for hypoxia. Interestingly, IL-1ra was able to inhibit hypoxic proliferation, although we were unable to detect IL-1ra, IL-1 beta or IL-1 alpha in our culture supernatants. Importantly we show that monocytes from patients with IPAH cannot inhibit hypoxic proliferation compared to monocytes from SSc-PAH or controls. Acknowledgements SP: NIHR CVBRU funded PhD studentship SRW: Wellcome Intermediate fellowship.  

4.1061           Cytotoxic activity to acute myeloid leukemia cells by Antp-TPR hybrid peptide targeting Hsp90

Horibe, T., Kawamoto, M., Kohno, M. and Kawakami, K.

1. Bioscience and Bioengineering, 114(1), 96-103 (2012)

  We previously reported that Antp-TPR hybrid peptide inhibited the interaction of Hsp90 with TPR2A and had selective cytotoxic activity discriminating between normal and cancer cells to induce cancer cell death. In this study, we investigated the cytotoxic activity of Antp-TPR peptide toward acute myeloid leukemia (AML) cells. It was demonstrated that Antp-TPR peptide induced AML cell death in cell lines such as U937, K562, THP-1, and HL-60 via activation of caspases 3 and 7, and disruption of mitochondrial membrane potential. Conversely, Antp-TPR peptide did not reduce the viability of normal cells including peripheral blood mononuclear cells (PBMCs), although both geldanamycin and 17-AAG, small-molecule inhibitors of Hsp90, mediated cytotoxicity to these normal cells at low concentrations. In addition, mutation analysis of TPR peptide demonstrated that the highly conserved amino acids Lys and Arg were critical to the cytotoxic activity. These results indicated that Antp-TPR hybrid peptide would provide potent and selective therapeutic options in the treatment of AML.  

4.1062           PD-1 Blockage Reverses Immune Dysfunction and Hepatitis B Viral Persistence in a Mouse Animal Model

Tzeng, H-T.,Tsai, H-F., Liao, H-J., Lin, Y-J., Chen, L., Chen, P-J.and Hsu, P-N. PloS One, 7(6), e39179 (2012)   Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Recent studies in animal models of viral infection indicate that the interaction between the inhibitory receptor, programmed death (PD)-1, on lymphocytes and its ligand (PD-L1) play a critical role in T-cell exhaustion by inducing T-cell inactivation. High PD-1 expression levels by peripheral T-lymphocytes and the possibility of improving T-cell function by blocking PD-1-mediated signaling confirm the importance of this inhibitory pathway in inducing T-cell exhaustion. We studied T-cell exhaustion and the effects of PD-1 and PD-L1 blockade on intrahepatic infiltrating T-cells in our recently developed mouse model of HBV persistence. In this mouse animal model, we demonstrated that there were increased intrahepatic PD-1-expressing CD8+ and CD4+ T cells in mice with HBV persistence, but PD-1 upregulation was resolved in mice which had cleared HBV. The Intrahepatic CD8+ T-cells expressed higher levels of PD-1 and lower levels of CD127 in mice with HBV persistence. Blockade of PD-1/PD-L1 interactions increased HBcAg-specific interferon (IFN)-γ production in intrahepatic T lymphocytes. Furthermore, blocking the interaction of PD-1 with PD-L1 by an anti-PD-1 monoclonal antibody (mAb) reversed the exhausted phenotype in intrahepatic T lymphocytes and viral persistence to clearance of HBV in vivo. Our results indicated that PD-1 blockage reverses immune dysfunction and viral persistence of HBV infection in a mouse animal model, suggesting that the anti-PD-1 mAb might be a good therapeutic candidate for chronic HBV infection.  

4.1063           Role of Differentiation of Liver Sinusoidal Endothelial Cells in Progression and Regression of Hepatic Fibrosis in Rats

Xie, G., Wang, X., Wang, L., Wang, L., Atkinson, R.D., Kanel, G.C., Gaarde, W.A. and Deleve, L.D. Gastroenterology, 142(4), 918-927 (2012)   Background & Aims Capillarization, characterized by loss of differentiation of liver sinusoidal endothelial cells (LSECs), precedes the onset of hepatic fibrosis. We investigated whether restoration of LSEC differentiation would normalize crosstalk with activated hepatic stellate cells (HSC) and thereby promote quiescence of HSC and regression of fibrosis. Methods Rat LSECs were cultured with inhibitors and/or agonists and examined by scanning electron microscopy for fenestrae in sieve plates. Cirrhosis was induced in rats using thioacetamide, followed by administration of BAY 60-2770, an activator of soluble guanylate cyclase (sGC). Fibrosis was assessed by Sirius red staining; expression of α-smooth muscle actin was measured by immunoblot analysis. Results Maintenance of LSEC differentiation requires vascular endothelial growth factor-A stimulation of nitric oxide–dependent signaling (via sGC and cyclic guanosine monophosphate) and nitric oxide–independent signaling. In rats with thioacetamide-induced cirrhosis, BAY 60-2770 accelerated the complete reversal of capillarization (restored differentiation of LSECs) without directly affecting activation of HSCs or fibrosis. Restoration of differentiation to LSECs led to quiescence of HSCs and regression of fibrosis in the absence of further exposure to BAY 60-2770. Activation of sGC with BAY 60-2770 prevented progression of cirrhosis, despite continued administration of thioacetamide. Conclusions The state of LSEC differentiation plays a pivotal role in HSC activation and the fibrotic process.  

4.1064           Application of antioxidants and centrifugation for cryopreservation of boar spermatozoa

Zhang, W., Yi, K., Chen, C., Hou, X. and Zhou, X. Animal Reprod.  Sci., 132, 123-128 (2012)   Although cryopreserved boar semen has been available since 1975, a major breakthrough in commercial application has not yet occurred due to the high susceptibility of boar spermatozoa to damage during cryopreservation and the complicated process required for deep freezing. In recent years, the application of antioxidants during the cryopreservation of boar semen has been the subject of considerable research aimed at improving the quality of post-thaw semen. Centrifugation is necessary before using cryopreservation protocols for freezing boar spermatozoa. Studies of the effect of different centrifugation regimens on boar sperm recovery, yield and cryosurvival have made significant contributions. Therefore this review elucidates results of recent applications of various antioxidants and centrifugation regimens used in efforts to improve cryopreservation of boar spermatozoa. This review is intended to enhance understanding of the roles of these antioxidants and centrifugation regimens with respect to mechanisms that increase resistance to cryodamage of boar spermatozoa. In addition, the discussion addresses the need for developing an objective evaluation of effectiveness and estimating the prospect of application of new techniques for the cryopreservation of boar semen and its use in artificial insemination.  

4.1065           Delayed cryopreservation of stallion sperm: effect of iodixanol density gradient centrifugation

Heutelbeck, A., Oldenhof, H., Henke, S., Martinsson, G. and Sieme, H.

1. Equine Vet. Sci., 32, 488-489 (2012)

  The infrastructure needed for cryopreservation of stallion sperm is not always available. In these cases, diluted semen needs to be shipped to a facility where cryopreservation can be done typically after one day storage at 4^oC. Centrifugation processing of semen, including density centrifugation, is generally used in the selection of high quality sperm for artificial insemination. The aim of this study was to evaluate if selection of sperm via two-layer iodixanol density centrifugation improves cryosurvival after delayed cryopreservation, as compared to the standard centrifugation processing. Moreover, centrifugation processing directly after semen collection and storage of selected sperm was compared with centrifugation of stored sperm just prior to cryopreservation. For each processing method, twelve ejaculates were tested from stallions of the Hannoverian warmblood breed (two ejaculates from each of six stallions). Sperm motility was evaluated using computer assisted sperm analysis. Sperm stained with both propidium iodide and Sybr-14 was analyzed by flow cytometry to discriminate between membrane damaged and intact cells, respectively. Fractions of normal double stranded and denatured single-stranded DNA were determined after acid treatment and acridine orange staining, as a measure for sperm chromatin stability and integrity. Two-layer iodixanol density centrifugation resulted in a 5% increase in the percentage of plasma membrane intact sperm, as compared to diluted sperm or sperm processed using ordinary centrifugation. In addition, chromatin stability was found to be increased for sperm obtained after iodixanol density centrifugation. Centrifugation processing at the day of collection or after storage resulted in similar percentages of plasma membrane intact sperm after one day storage at 4^oC, whereas progressive motility of sperm processed after storage was slightly decreased. Iodixanol density centrifugation resulted in increased cryosurvival rates as compared to ordinary centrifugation. Highest survival rates after freezing and thawing were obtained when centrifugation processing and cryopreservation were performed at the day of collection. In case of delayed cryopreservation, survival rates could not be increased by centrifugation processing. Post-freeze plasma membrane integrity tended to be higher when sperm was selected via centrifugation processing directly after semen collection as compared to sperm processed just prior to cryopreservation, both for ordinary as well as for iodixanol density centrifugation. Taken together, survival after delayed cryopreservation can be increased when two-layer iodixanol density centrifugation is applied directly after semen collection.  

4.1066           Arachidonic acid stimulates TNFα production in Kupffer cells via a reactive oxygen species-pERK1/2-Egr1-dependent mechanism

Cubero, F.J. and Nieto, N. Am. J. Physiol. Gastrointest. Liver Physiol., 303, G228-G239 (2012)   Kupffer cells are a key source of mediators of alcohol-induced liver damage such as reactive oxygen species, chemokines, growth factors, and eicosanoids. Since diets rich in polyunsaturated fatty acids are a requirement for the development of alcoholic liver disease, we hypothesized that polyunsaturated fatty acids could synergize with ethanol to promote Kupffer cell activation and TNFα production, hence, contributing to liver injury. Primary Kupffer cells from control and from ethanol-fed rats incubated with arachidonic acid showed similar proliferation rates than nontreated cells; however, arachidonic acid induced phenotypic changes, lipid peroxidation, hydroperoxides, and superoxide radical generation. Similar effects occurred in human Kupffer cells. These events were greater in Kupffer cells from ethanol-fed rats, and antioxidants and inhibitors of arachidonic acid metabolism prevented them. Arachidonic acid treatment increased NADPH oxidase activity. Inhibitors of NADPH oxidase and of arachidonic acid metabolism partially prevented the increase in oxidant stress. Upon arachidonic acid stimulation, there was a rapid and sustained increase in TNFα, which was greater in Kupffer cells from ethanol-fed rats than in Kupffer cells from control rats. Arachidonic acid induced ERK1/2 phosphorylation and nuclear translocation of early growth response-1 (Egr1), and ethanol synergized with arachidonic acid to promote this effect. PD98059, a mitogen extracellular kinase 1/2 inhibitor, and curcumin, an Egr1 inhibitor, blocked the arachidonic acid-mediated upregulation of TNFα in Kupffer cells. This study unveils the mechanism whereby arachidonic acid and ethanol increase TNFα production in Kupffer cells, thus contributing to alcoholic liver disease.  

4.1067           Surface Proteome Analysis and Characterization of Surface Cell Antigen (Sca) or Autotransporter Family of Rickettsia typhi

Sears, K.T., Ceraul, S.M., Gillespie, J.J., Allen Jr., E.D., Popov, V.L., Ammerman, N.C., Rahman, M.S. and Azad, A.F. PloS Pathogens, 8(8), e1002856 (2012)   Surface proteins of the obligate intracellular bacterium Rickettsia typhi, the agent of murine or endemic typhus fever, comprise an important interface for host-pathogen interactions including adherence, invasion and survival in the host cytoplasm. In this report, we present analyses of the surface exposed proteins of R. typhi based on a suite of predictive algorithms complemented by experimental surface-labeling with thiol-cleavable sulfo-NHS-SS-biotin and identification of labeled peptides by LC MS/MS. Further, we focus on proteins belonging to the surface cell antigen (Sca) autotransporter (AT) family which are known to be involved in rickettsial infection of mammalian cells. Each species of Rickettsia has a different complement of sca genes in various states; R. typhi, has genes sca1 thru sca5. In silico analyses indicate divergence of the Sca paralogs across the four Rickettsia groups and concur with previous evidence of positive selection. Transcripts for each sca were detected during infection of L929 cells and four of the five Sca proteins were detected in the surface proteome analysis. We observed that each R. typhi Sca protein is expressed during in vitro infections and selected Sca proteins were expressed during in vivo infections. Using biotin-affinity pull down assays, negative staining electron microscopy, and flow cytometry, we demonstrate that the Sca proteins in R. typhi are localized to the surface of the bacteria. All Scas were detected during infection of L929 cells by immunogold electron microscopy. Immunofluorescence assays demonstrate that Scas 1–3 and 5 are expressed in the spleens of infected Sprague-Dawley rats and Scas 3, 4 and 5 are expressed in cat fleas (Ctenocephalides felis). Sca proteins may be crucial in the recognition and invasion of different host cell types. In short, continuous expression of all Scas may ensure that rickettsiae are primed i) to infect mammalian cells should the flea bite a host, ii) to remain infectious when extracellular and iii) to infect the flea midgut when ingested with a blood meal. Each Sca protein may be important for survival of R. typhi and the lack of host restricted expression may indicate a strategy of preparedness for infection of a new host.  

4.1068           Gene Profile of Chemokines on Hepatic Stellate Cells of Schistosome-Infected Mice and Antifibrotic Roles of CXCL9/10 on Liver Non-Parenchymal Cells

Liang, Y-j., Luo, J., Lu, Q., Zhou, Y., Wu, H-w., Zheng, D., Ren, Y-y., Sun, K-y., Wang, Y. and Zhang, Z-s. PloS One, 7(8), e42490 (2012)   Hepatic stellate cells (HSCs) play a key role in the development of liver fibrosis caused by schistosomiasis. Chemokines were widely expressed and involved in cellular activation, proliferation and migration in inflammatory and infectious diseases. However, little is known about the expressions of chemokines on HSCs in the schistosoma infection. In addition, the roles of chemokines in pathogenesis of liver fibrosis are not totally clear. In our study, we used microarray to analyze the temporal gene expressions of primary HSCs isolated from mice with both acute and chronic schistosomiasis. Our microarray data showed that most of the chemokines expressed on HSCs were upregulated at 3 weeks post-infection (p.i) when the egg granulomatous response was not obviously evoked in the liver. However, some of them like CXCL9, CXCL10 and CXCL11 were subsequently decreased at 6 weeks p.i when the granulomatous response reached the peak. In the chronic stage, most of the differentially expressed chemokines maintained persistent high-abundances. Furthermore, several chemokines including CCR2, CCR5, CCR7, CXCR3, CXCR4, CCL2, CCL5, CCL21, CXCL9 and CXCL10 were expressed by HCSs and the abundances of them were changed following the praziquantel treatment in the chronic stage, indicating that chemokines were possibly necessary for the persistence of the chronic stage. In vitro experiments, hepatic non-parenchymal cells, primary HSCs and human HSCs line LX-2 were stimulated by chemokines. The results showed that CXCL9 and CXCL10, but not CXCL11 or CXCL4, significantly inhibited the gene expressions of Col1α1, Col3α1 and α-SMA, indicating the potential anti-fibrosis effect of CXCL9 and CXCL10 in schistosomiasis. More interestingly, soluble egg antigen (SEA) of Schistosoma japonicum was able to inhibit transcriptional expressions of some chemokines by LX-2 cells, suggesting that SEA was capable of regulating the expression pattern of chemokine family and modulating the hepatic immune microenvironment in schistosomiasis.  

4.1069           EF24 suppresses maturation and inflammatory response in dendritic cells

Vilekar, P., Awasthi, S., Natarajan, A., Anant, S. and Awasthi, V. Int. Immunol., 24(7), 455-464 (2012)   Synthetic curcuminoid EF24 was studied for its effect on the maturation and inflammatory response in murine bone marrow derived immortalized JAWS II dendritic cells (DCs). EF24 reduced the expression of LPS-induced MHC class II, CD80 and CD86 molecules. It also abrogated the appearance of dendrites, a typical characteristic of mature DCs. These effects were accompanied by the inhibition of LPS-induced activation of transcription factor nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB). Simultaneous reduction of pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, IL-6] both at the mRNA and secreted levels was also observed. To investigate the dependency of LPS effects on MyD88 adaptor protein, we transfected JAWS II DCs with dominant negative MyD88 plasmid construct (MyD88-DN). EF24 reduced NF-κB activity and TNF-α secretion in a MyD88-dependent manner. These results suggest that EF24 modulates DCs by suppressing their maturation and reducing the secretion of inflammatory cytokines. Further, it appears that EF24 acts at or upstream of MyD88 in the LPS-TLR4/MyD88/NF-κB pathway.

4.1070           Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10−/− mice

Devkota, S., Wang, Y., Musch, M.W., Leone, V., Fehlner-Peach, H., Nadimpalli, A., Antonopoulos, D.A., Jabri, B. and Chang, E.B. Nature, 487, 104-109 (2012)   The composite human microbiome of Western populations has probably changed over the past century, brought on by new environmental triggers that often have a negative impact on human health¹. Here we show that consumption of a diet high in saturated (milk-derived) fat, but not polyunsaturated (safflower oil) fat, changes the conditions for microbial assemblage and promotes the expansion of a low-abundance, sulphite-reducing pathobiont, Bilophila wadsworthia². This was associated with a pro-inflammatory T helper type 1 (T_H1) immune response and increased incidence of colitis in genetically susceptible Il10^−/−, but not wild-type mice. These effects are mediated by milk-derived-fat-promoted taurine conjugation of hepatic bile acids, which increases the availability of organic sulphur used by sulphite-reducing microorganisms like B. wadsworthia. When mice were fed a low-fat diet supplemented with taurocholic acid, but not with glycocholic acid, for example, a bloom of B. wadsworthia and development of colitis were observed in Il10^−/− mice. Together these data show that dietary fats, by promoting changes in host bile acid composition, can markedly alter conditions for gut microbial assemblage, resulting in dysbiosis that can perturb immune homeostasis. The data provide a plausible mechanistic basis by which Western-type diets high in certain saturated fats might increase the prevalence of complex immune-mediated diseases like inflammatory bowel disease in genetically susceptible hosts.  

4.1071           Co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing swine interleukin-18 and interferon-α provides enhanced Th1-biased protective immunity against inactivated vaccine of pseudorabies virus

Kim, J.S., Kim, S.B., Han, Y.W., uyangaa, E., Kim, J.H., Choi, J.Y., Kim, K. and Eo, S.K. Microbiol.  Immunol., 56(8), 529-540 (2012)   The co-administration of two or more cytokines may generate additive or synergistic effects for controlling infectious diseases. However, the practical use of cytokine combinations for the modulation of immune responses against inactivated vaccine has not been demonstrated in livestock yet, primarily due to protein stability, production, and costs associated with mass administration. In light of the current situation, we evaluated the immunomodulatory functions of the combined administration of swine interleukin-18 (swIL-18) and interferon-α (swIFN-α) against an inactivated PrV vaccine using attenuated Salmonella enterica serovar Typhimurium as a cytokine delivery system. Co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α produced enhanced Th1-biased humoral and cellular immune responses against the inactivated PrV vaccine, when compared to single administration of S. enterica serovar Typhimurium expressing either swIL-18 or swIFN-α. Also, enhanced immune responses in co-administered piglets occurred rapidly after virulent PrV challenge, and piglets that received co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α displayed a greater alleviation of clinical severity following the virulent PrV challenge, as determined by clinical scores and cumulative daily weight gain. Furthermore, this enhancement was confirmed by reduced nasal shedding of PrV following viral challenge. Therefore, these results suggest that oral co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α provide enhanced Th1-biased immunity against inactivated PrV vaccine to alleviate clinical signs caused by PrV challenge.  

4.1072           Interferon-γ, tumor necrosis factor, and interleukin-18 cooperate to control growth of Mycobacterium tuberculosis in human macrophages

Robinson, C.M., Jung, J-Y. and Nau, G.J. Cytokine, 60, 233-241 (2012)   Mycobacterium tuberculosis (MTB) remains a leading infectious threat to human health. Macrophages are the cells targeted for infection by the bacterium as well as key effector cells for clearance of the pathogen. Interleukin (IL)-27 opposes macrophage-mediated control of MTB because supplying IL-12 and blocking the activity of IL-27 limits bacterial growth in primary human macrophages. The purpose of this study was to determine the immunological regulators of this macrophage mechanism to restrict MTB growth. Interferon (IFN)-γ, TNF-α, and IL-18 were all demonstrated to be important to the environment that limits bacterial growth when IL-12 is supplied and IL-27 is neutralized. We find IL-18 works in conjunction with IL-12 to achieve optimal IFN-γ production in this system. We also demonstrate novel interactions between these cytokines to influence the expression or responsiveness to one another. Quantitative assays show that IFN-γ enhances expression of the IL-18 receptor signaling chain, as well as TNF expression and secretion. In turn, TNF-α augments expression of the receptor for IFN-γ, the amount at the cell surface, and the extent of IFN-γ -induced signaling. We further define how the cytokine environment supports an enhanced state of classical macrophage activation. Collectively, these results describe novel immunological mechanisms that provide additional insights into the effects of IL-12 and IL-27 on macrophage regulation during MTB infection.  

4.1073           Iodixanol density gradient centrifugation for selecting stallion sperm for cold storage and cryopreservation

Stuhtmann, G., Oldenhof, H., peters, P., Klewitz, J., Martinsson, G. and Sieme, H. Animal Reprod. Sci., 133(3-4), 184-190 (2012)   Density gradient centrifugation can be used for selection of sperm of superior quality and removal of seminal plasma for use in artificial insemination. In this study, the use of two-layer iodixanol density gradient centrifugation was evaluated for processing of stallion semen. The protocol includes centrifugation through a 16% iodixanol top layer of 1.090 g mL⁻¹ and collection of motile and intact sperm on a 30% iodixanol bottom layer of 1.165 g mL⁻¹. Sperm recovery and effects on sperm quality were determined during cold storage as well as after cryopreservation and compared with ordinary dilution and centrifugation. Two-layer iodixanol density gradient centrifugation allows for selection of greater percentages of morphologically normal and progressively motile sperm compared to ordinary centrifugation. This likely results from collecting sperm on the bottom layer that functions as cushion fluid, which prevents mechanical forces as occur when sperm are packed in a pellet. In addition, percentages of membrane and chromatin integrity are increased when cells are selected based on their density via centrifugation through the top and bottom layers. Removal of seminal plasma and increased initial percentages of motile and membrane intact sperm after iodixanol density gradient centrifugation also result in greater percentages of progressively motile and membrane intact sperm during cold storage as well as after freezing and thawing. In conclusion, the two-layer iodixanol density gradient centrifugation protocol described in this manuscript allows for selection of stallion sperm with greater survival rates for cold storage and cryopreservation.  

4.1074           Increased Responsiveness of Peripheral Blood Mononuclear Cells to In Vitro TLR 2, 4 and 7 Ligand Stimulation in Chronic Pain Patients

Kwok, Y.H., Hutchinson, M.R., Gentgall, M.G. and Rolan, P.E. PloS One, 7(8), e44232 (2012)   Glial activation via Toll-like receptor (TLR) signaling has been shown in animals to play an important role in the initiation and establishment of chronic pain. However, our ability to assess this central immune reactivity in clinical pain populations is currently lacking. Peripheral blood mononuclear cells (PBMCs) are an accessible source of TLR expressing cells that may mirror similarities in TLR responsiveness of the central nervous system. The aim of this study was to characterize the IL-1β response to various TLR agonists in isolated PBMCs from chronic pain sufferers (on and not on opioids) and pain-free controls. Venous blood was collected from 11 chronic pain sufferers on opioids (≥ 20 mg of morphine / day), 8 chronic pain sufferers not on opioids and 11 pain-free controls. PBMCs were isolated and stimulated in vitro with a TLR2 (Pam3CSK4), TLR4 (LPS) or TLR7 (imiquimod) agonist. IL-1β released into the supernatant was measured with ELISA. Significantly increased IL-1β expression was found in PBMCs from chronic pain sufferers (on and not on opioids) compared with pain-free controls for TLR2 (F _{(6, 277)} = 15, P<0.0001), TLR4 (F _{(8, 263)} = 3, P = 0.002) and TLR7 (F _(2,201) = 5, P = 0.005) agonists. These data demonstrate that PBMCs from chronic pain sufferers were more responsive to TLR agonists compared with controls, suggesting peripheral cells may have the potential to become a source of biomarkers for chronic pain.  

4.1075           Enriching the captive elephant population genetic pool through artificial insemination with frozen-thawed semen collected in the wild

Hildebrandt, T.B., Hermes, R., Saragusty, J., Potier, R., Schwammer, H.M., Balfanz, F., Vielgrader, H., Baker, B., Bartels, P. and Göritz, F. Theriogenology, 78, 1398-1404 (2012)   The first successful AI in an elephant was reported in 1998, using fresh semen. Since then almost 40 calves have been produced through AI in both Asian and African elephants worldwide. Following these successes, with the objective of enriching the captive population with genetic material from the wild, we evaluated the possibility of using frozen-thawed semen collected from wild bulls for AI in captivity. Semen, collected from a 36-yr-old wild African savanna elephant (Loxodonta africana) in South Africa was frozen using the directional freezing technique. This frozen-thawed semen was used for four inseminations over two consecutive days, two before and two after ovulation, in a 26-yr-old female African savanna elephant in Austria. Insemination dose of 1200 × 10⁶ cells per AI with 61% motility resulted in pregnancy, which was confirmed through ultrasound examination 75, 110 and 141 days after the AI procedure. This represents the first successful AI using wild bull frozen-thawed semen in elephants. The incorporation of AI with frozen-thawed semen into the assisted reproduction toolbox opens the way to preserve and transport semen between distant individuals in captivity or, as was done in this study, between wild and captive populations, without the need to transport stressed or potentially disease-carrying animals or to remove animals from the wild. In addition, cryopreserved spermatozoa, in combination with AI, are useful methods to extend the reproductive lifespan of individuals beyond their biological lifespan and an important tool for genetic diversity management and phenotype selection in these endangered mammals.  

4.1076           3′poly-G-Tailed ODNs Inhibit F-spondin to Induce Cell Death and Neurite Retraction in Rat Embryonic Neurons

Cheng, Y-C., Chen, T-A., Chen, C-Y., Liang, C-M. and Liang, S-M. Mol. Neurobiol., 45(3), 536-549 (2012)   The effects and mechanism of action of oligodeoxyribonucleotides containing CpG motif (CpG-ODNs) on neuron cells are largely unexamined. Here, we found that CpG-A ODNs but not other types of CpG-ODNs induced neurite retraction and cell apoptosis of rat embryonic neurons in a TLR9-independent manner. These effects of CpG-A ODNs were primarily due to the poly-guanosine at the 3′ terminus (3′G-ODNs). Pull-down analysis showed that 3′G-ODNs associated with transcription factor Y-BOX1 (YB-1) to facilitate the translocation of YB-1 into the nucleus via the nuclear localizing sequence of YB-1. YB-1 then interacted with the promoter of F-spondin directly at −45 and −1,375 sites as demonstrated by chromatin immunoprecipitation (ChIP) analysis. Binding of YB-1 to F-spondin promoter resulted in downregulation of F-spondin expression. Overexpression of F-spondin rescued the cell death and neurite retraction induced by 3′G-ODNs in embryonic neuron cells. Taken together, these findings suggest that 3′G-ODNs enhance nucleus YB-1 to inhibit F-spondin leading to cell death and neurite retraction of embryonic neuron cells  

4.1077           Focal Increases of Axoplasmic Ca2+, Aggregation of Sodium–Calcium Exchanger, N-type Ca2+ Channel, and Actin Define the Sites of Spheroids in Axons Undergoing Oxidative Stress

Barsukova, A.G., Forte, M. and Bourdette, D.

1. Neurosci., 32(35), 12028-12037 (2012)

  The excitotoxic effects of kainic acid (KA) in the mouse hippocampus is strain dependent. Following KA administration, the large majority of hippocampal pyramidal cells die in the FVB/N (FVB) mouse, while the pyramidal cells of the C57BL/6 (B6) strain are largely spared. We generated aggregation chimeras between the sensitive FVB and the resistant B6 strains to investigate whether intrinsic or extrinsic features of a neuron confer cell vulnerability or resistance to KA. The constitutive expression of transgenic green fluorescence protein (GFP) or β-galactosidase expressed from the ROSA26 locus was used to mark cells in FVB or B6 mice, respectively. These makers enable the identification of cells from each parental genotype while TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling)-staining labeled dying cells. The analysis of the percentage of dying cells in FVB-GFP ↔ B6-ROSA chimeras yielded an intriguing mix of both intrinsic and extrinsic factors in the readout of cell phenotype. Thus, normally resistant B6-ROSA pyramidal neurons demonstrated an increasing sensitivity to KA, in a linear fashion, when the percentage of FVB-GFP cells was increased, either across chimeras or in different regions of the same chimera. However, the death of B6-ROSA pyramidal cells never exceeded ∼70% of the total amount of B6 neurons regardless of the amount of FVB cells in the chimeric hippocampus. In a similar manner, FVB-GFP cells show lower amounts of cell death in chimeras that are colonized by B6-ROSA cells, but again, are never fully rescued. These data indicate that both intrinsic and extrinsic factors modulate the sensitivity of hippocampal pyramidal cells to kainic acid.  

4.1078           Neuroprotective effects of phenolic antioxidant tBHQ associate with inhibition of FoxO3a nuclear translocation and activity

Bahia, P.K., Pugh, V., Hoyland, K., Hensley, V., Rattray, M. and Williams, R.J.

1. Neurochem., 123(1), 182-191(2012)

  The Forkhead transcription factor, FoxO3a induces genomic death responses in neurones following translocation from the cytosol to the nucleus. Nuclear translocation of FoxO3a is triggered by trophic factor withdrawal, oxidative stress and the stimulation of extrasynaptic NMDA receptors. Receptor activation of phosphatidylinositol 3-kinase (PI3K)–Akt signalling pathways retains FoxO3a in the cytoplasm, thereby inhibiting the transcriptional activation of death-promoting genes. We hypothesized that phenolic antioxidants such as tert-Butylhydroquinone (tBHQ), which is known to stimulate PI3K–Akt signalling, would inhibit FoxO3a translocation and activity. Treatment of cultured cortical neurones with NMDA increased the nuclear localization of FoxO3a, reduced the phosphorylation of FoxO3a, increased caspase activity and up-regulated Fas ligand expression. In contrast the phenolic antioxidant, tBHQ, caused retention of FoxO3a in the cytosol coincident with enhanced PI3K- dependent phosphorylation of FoxO3a. tBHQ-induced nuclear exclusion of FoxO3a was associated with reduced FoxO-mediated transcriptional activity. Exposure of neurones to tBHQ inhibited NMDA-induced nuclear translocation of FoxO3a, prevented NMDA-induced up-regulation of FoxO-mediated transcriptional activity, blocked caspase activation and protected neurones from NMDA-induced excitotoxic death. Collectively, these data suggest that phenolic antioxidants such as tBHQ oppose stress-induced activation of FoxO3a and therefore have potential neuroprotective utility in neurodegeneration.  

4.1079           Histone methyltransferase ASH1 orchestrates fibrogenic gene transcription during myofibroblast transdifferentiation

Perugorria, M.J., Wilson, C.L., Zeybel, M., Walsh, M., Amin, S., Robinson, S., White, S.A., Burt, A.D., Oakley, F., Tsukamoto, H., Mann, D.A. and Mann, J. Hepatology, 56(3), 1129-1139 (2012)   Transdifferentiation of hepatic stellate cells (HSCs) to a myofibroblast-like phenotype is the pivotal event in liver fibrosis. The dramatic change in phenotype associated with transdifferentiation is underpinned by a global change in gene expression. Orchestrated changes in gene expression take place at the level of chromatin packaging which is regulated by enzymatic activity of epigenetic regulators that in turn affect histone modifications. Using expression profiling of epigenetic regulators in quiescent and activated primary HSCs we found a number of histone methyltranferases including MLL1, MLL5, Set1 and ASH1 to be highly up-regulated during transdifferentiation of HSCs. All of these histone methyltranferases regulate methylation of lysine 4 of histone H3, which is a signature of actively transcribed genes. We therefore postulated that one or more of these enzymes may be involved in positively influencing expression of profibrogenic genes. Conclusion: We find that ASH1 directly binds to the regulatory regions of alpha smooth muscle actin (αSMA), collagen I, tissue inhibitor of metalloproteinase-1 (TIMP1) and transforming growth factor beta1 (TGFβ1) in activated HSCs while depletion of ASH1 caused broad suppression of fibrogenic gene expression. We also discovered that MeCP2 positively regulates ASH1 expression and therefore identify ASH1 as a key transcriptional activator component of the MeCP2 epigenetic relay pathway that orchestrates coordinated induction of multiple profibrogenic genes.  

4.1080           Wiskott–Aldrich Syndrome Protein Deficiency in Innate Immune Cells Leads to Mucosal Immune Dysregulation and Colitis in Mice

Ngyen, D.D. et al Gastroenterology, 143(3), 719-729 (2012)   Background & Aims Immunodeficiency and autoimmune sequelae, including colitis, develop in patients and mice deficient in Wiskott–Aldrich syndrome protein (WASP), a hematopoietic cell-specific intracellular signaling molecule that regulates the actin cytoskeleton. Development of colitis in WASP-deficient mice requires lymphocytes; transfer of T cells is sufficient to induce colitis in immunodeficient mice. We investigated the interactions between innate and adaptive immune cells in mucosal regulation during development of T cell–mediated colitis in mice with WASP-deficient cells of the innate immune system. Methods Naïve and/or regulatory CD4⁺ T cells were transferred from 129 SvEv mice into RAG-2–deficient (RAG-2 KO) mice or mice lacking WASP and RAG-2 (WRDKO). Animals were observed for the development of colitis; effector and regulatory functions of innate immune and T cells were analyzed with in vivo and in vitro assays. Results Transfer of unfractionated CD4⁺ T cells induced severe colitis in WRDKO, but not RAG-2 KO, mice. Naïve wild-type T cells had higher levels of effector activity and regulatory T cells had reduced suppressive function when transferred into WRDKO mice compared with RAG-2 KO mice. Regulatory T-cell proliferation, generation, and maintenance of FoxP3 expression were reduced in WRDKO recipients and associated with reduced numbers of CD103⁺ tolerogenic dendritic cells and levels of interleukin-10. Administration of interleukin-10 prevented induction of colitis following transfer of T cells into WRDKO mice. Conclusions Defective interactions between WASP-deficient innate immune cells and normal T cells disrupt mucosal regulation, potentially by altering the functions of tolerogenic dendritic cells, production of interleukin-10, and homeostasis of regulatory T cells.  

4.1081           Isolation and Identification of Red Cell Progenitor Subsets from Whole Blood, Mobilized Peripheral Blood and Cord Blood

He, J., Wang, F. and  Zhu, F. Transfusion, 52(s3), 185A (2012)   Background/Case Studies: Red blood cell (RBC) transfusion is an essential therapeutic act and the need for blood is thereby widespread. However there is a major imbalance between demand and supply of donors, so that there is mounting research to develop suitable surrogates for human donated blood. Functional RBCs have already been generated from a variety of cellular progenitors. To investigate a similar population of cells that can be also isolated from the whole blood, mobilized peripheral blood and cord blood, we purified and identified a population of ‘immature’ red cells that are phenotypically similar to red cell progenitor subsets. Study Design/Methods: The whole blood, mobilized peripheral blood and cord blood were mixed with 6% polysucrose400 respectively, incubated at 37 C for 1 hour. Leukocyte rich supernatant above the sedimented red cells was collected and washed. Suspended with PBS cells were layered onto the erythrocyte and leukocyte Ficoll-Optiprep gradient (the three densities in one tube up to down were 1.091, 1.109, 1.117). After centrifugation the isolated fraction were harvested to analyze the phenotype using cell surface markers CD45+/ CD34+/CD133+, CD45+/CD71+/ CD235a+, CD45+/C14+/CD66b+ by FACS. CD34+133+ cells were isolated from interfaces A, CD71+cells and CD71−cells were separated from the depletion of CD34+133+ cells population from interfaces A by MACS. Five fractions included above three separated population, interfaces B and C were expanded to assess BFU-E, CFU-E, BFU-GM, BFU-GEMM in methylcellulose medium. The results of phenotype and colony assay from three kinds of samples were compared. Results/Findings: After centrifugation the sample showed A, B and C bands up to down. Large number of red cells and white cells held at interface A giving poorly separated bands; thin bands of red cells were isolated from interface B and C. Greater enrichment for CD45+/CD34+/CD133+ and CD45+/CD14+/CD66b+ could be found in interface A and greater enrichment for CD45+/CD71+/CD235a+ could be found in interfaces B and C. CD45+/CD71+/CD235a+ population from interfaces B and C of UCB cells are signifi cantly rich compared to interfaces B and C of the whole blood and mobilized peripheral blood. The result of red cell colony-forming unit of CD34+133+, CD71+, CD71−, B and C cell populations from three kinds of samples were 0.35 ± 0.15%, 66.16 ± 5.19%, 20 ± 3.87%, 83.33 ± 1.24%, 85.45 ± 2.31% respectively. Almost all the red cell colony-forming potential was within the B and C fractions from three kinds of samples. Conclusion: Using 6% polysucrose400 sedimentation and erthrocte-Ficoll-Optiprep gradient can isolate immature red cells from the leukocyte fraction. Cord blood is the best source for red cell progenitor subsets which are suitable for the generation of mature and functional RBCs.  

4.1082           Nilotinib protects the murine liver from ischemia/reperfusion injury

Ocuin, L.M., Cavnar, M.J., Sorenson, E.C., Bamboat, Z.M., Greer, J.B., Kim, T.S., Popow, R. and DeMatteo, R.P.

1. Hepatol., 57, 766-773 (2012)

  Background & aims The mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase (JNK), and p38, mediate liver ischemia/reperfusion (I/R) injury via cell death and inflammatory cytokine expression, respectively. Nilotinib is an orally available receptor tyrosine kinase inhibitor used for chronic myelogenous leukemia that also has in vitro activity against JNK and p38. In this study, we examine its therapeutic potential against hepatic I/R injury. Methods The effects of nilotinib on liver I/R injury were tested using a murine model of warm, segmental liver I/R. Serum ALT was measured and livers were analyzed by histology, RT-PCR, Western blot, and flow cytometry. The in vitro effects of nilotinib on hepatocyte and non-parenchymal cell (NPC) MAPK activation and cytokine production were also tested. Results Mice receiving nilotinib had markedly lower serum ALT levels and less histologic injury and apoptosis following liver I/R. Nilotinib did not inhibit its known receptor tyrosine kinases. Nilotinib lowered intrahepatic expression of IL-1β, IL-6, MCP-1, and MIP-2 and systemic levels of IL-6, MCP-1, and TNF. Nilotinib reduced NPC activation of p38 MAPK signaling and decreased the recruitment of inflammatory monocytes and their production of TNF. Nilotinib attenuated JNK phosphorylation and hepatocellular apoptosis. In vitro, nilotinib demonstrated direct inhibition of JNK activation in isolated hepatocytes cultured under hypoxic conditions, and blocked activation of p38 MAPK and cytokine production by stimulated NPCs. Conclusions Nilotinib lowers both liver JNK activation and NPC p38 MAPK activation and may be useful for ameliorating liver I/R injury in humans.  

4.1083           Progenitor/Stem Cell Fate Determination: Interactive Dynamics of Cell Cycle and Microvesicles

Aliotta, J.M., Lee, D., Puente, N., Faradyan, S., Sears, E.H., Amaral, A., Goldberg, L., Dooner, M.S., Pereira, M. and Quesenberry, P.J. Stem Cells and Development, 21(10), 1627-1638 (2012)   We have shown that hematopoietic stem/progenitor cell phenotype and differentiative potential change throughout cell cycle. Lung-derived microvesicles (LDMVs) also change marrow cell phenotype by inducing them to express pulmonary epithelial cell-specific mRNA and protein. These changes are accentuated when microvesicles isolated from injured lung. We wish to determine if microvesicle-treated stem/progenitor cell phenotype is linked to cell cycle and to the injury status of the lung providing microvesicles. Lineage depleted, Sca-1+ (Lin-/Sca-1+) marrow isolated from mice were cultured with interleukin 3 (IL-3), IL-6, IL-11, and stem cell factor (cytokine-cultured cells), removed at hours zero (cell cycle phase G0/G1), 24 (late G1/early S), and 48 (late S/early G2/M), and cocultured with lung tissue, lung conditioned media (LCM), or LDMV from irradiated or nonirradiated mice. Alternatively, Lin-/Sca-1+ cells not exposed to exogenous cytokines were separated into G0/G1 and S/G2/M cell cycle phase populations by fluorescence-activated cell sorting (FACS) and used in coculture. Separately, LDMV from irradiated and nonirradiated mice were analyzed for the presence of adhesion proteins. Peak pulmonary epithelial cell-specific mRNA expression was seen in G0/G1 cytokine-cultured cells cocultured with irradiated lung and in late G1/early S cells cocultured with nonirradiated lung. The same pattern was seen in cytokine-cultured Lin-/Sca-1 cells cocultured with LCM and LDMV and when FACS-separated Lin-/Sca-1 cells unexposed to exogenous cytokines were used in coculture. Cells and LDMV expressed adhesion proteins whose levels differed based on cycle status (cells) or radiation injury (LDMV), suggesting a mechanism for microvesicle entry. These data demonstrate that microvesicle modification of progenitor/stem cells is influenced by cell cycle and the treatment of the originator lung tissue.  

4.1084           In vivo reprogramming of Sox9+ cells in the liver to insulin-secreting ducts

Banga, A., Akinci, E., Greder, L.V., Dutton, J.R. and Slack, J.M.W. PNAS, 109(38), 15336-15341 (2012)   In embryonic development, the pancreas and liver share developmental history up to the stage of bud formation. Therefore, we postulated that direct reprogramming of liver to pancreatic cells can occur when suitable transcription factors are overexpressed. Using a polycistronic vector we misexpress Pdx1, Ngn3, and MafA in the livers of NOD-SCID mice rendered diabetic by treatment with streptozotocin (STZ). The diabetes is relieved long term. Many ectopic duct-like structures appear that express a variety of β-cell markers, including dense core granules visible by electron microscopy (EM). Use of a vector also expressing GFP shows that the ducts persist long after the viral gene expression has ceased, indicating that this is a true irreversible cell reprogramming event. We have recovered the insulin⁺ cells by cell sorting and shown that they display glucose-sensitive insulin secretion. The early formed insulin⁺ cells can be seen to coexpress SOX9 and are also labeled in mice lineage labeled for Sox9 expression. SOX9⁺ cells are normally found associated with small bile ducts in the periportal region, indicating that the duct-like structures arise from this source. This work confirms that developmentally related cells can be reprogrammed by suitable transcription factors and also suggests a unique therapy for diabetes.

4.1085           Circumventing Antivector Immunity by Using Adenovirus-Infected Blood Cells for Repeated Application of Adenovirus-Vectored Vaccines: Proof of Concept in Rhesus Macaques

Sun, C., Feng, L., Zhang, Y., Xiao, L., Pan, W., Li, C., Zhang, L. and Chen, L.

1. Virol., 86(20), 11031-11042 (2012)

  Adenovirus has been extensively exploited as a vector platform for delivering vaccines. However, preexisting antiadenovirus immunity is the major stumbling block for application of adenovirus-vectored vaccines. In this study, we found that freshly isolated peripheral blood mononuclear cells (PBMCs), mostly CD14⁺ cells, from adenovirus serotype 5 (Ad5)-seropositive primates (humans and rhesus macaques) can be efficiently infected with Ad5 in vitro. On the basis of this observation, a novel strategy based on adenoviral vector-infected PBMC (AVIP) immunization was explored to circumvent antivector immunity. Autologous infusion of Ad5-SIVgag-infected PBMCs elicited a strong Gag-specific cellular immune response but induced weaker Ad5-neutralizing antibody (NAb) in Ad5-seronegative macaques than in macaques intramuscularly injected with Ad5-SIVgag. Moreover, Ad5-seropositive macaques receiving multiple AVIP immunizations with Ad5-SIVenv, Ad5-SIVgag, and Ad5-SIVpol vaccines elicited escalated Env-, Gag-, and Pol-specific immune responses after each immunization that were significantly greater than those in macaques intramuscularly injected with these Ad5-SIV vaccines. After challenged intravenously with a highly pathogenic SIVmac239 virus, macaques receiving AVIP immunization demonstrated a significant reduction in viral load at both the peak time and set-point period compared with macaques without Ad5-SIV vaccines. Our study warranted further research and development of the AVIP immunization as a platform for repeated applications of adenovirus-vectored vaccines.  

4.1086           Dendritic Cells Are Central Coordinators of the Host Immune Response to Staphylococcus aureus Bloodstream Infection

Schindler, D., Gutierrez, M.O., Beineke, A., Rauter, Y., Rohde, M., Foster, S., Goldmann, O. and Medina, E. Am. J. Pathol., 181(4), 1327-1337 (2012)   Dendritic cells (DCs) play an important role in integration of the immune responses induced by pathogens. The purpose of this study was to determine the importance of DCs in host defense against Staphylococcus aureus bacteremia. Using a murine infection model, we demonstrated that DCs are rapidly recruited into infected tissue after intravenous inoculation with S. aureus. The recruited DCs were fully functional and in a more advanced stage of maturation than those isolated from uninfected mice. Depletion of DCs in CD11c-DTR transgenic mice resulted in substantial worsening of infection, as indicated by increased bacterial loads in kidneys and lungs, accelerated mortality, and more severe pathology. Furthermore, DC depletion completely abolished IL-12 production in response to infection. The beneficial effect afforded by DCs during S. aureus infection was not mediated by their contribution to direct bacterial killing, nor by increased neutrophil recruitment. Instead, neutrophil influx (along with expression of CXC chemokines) was significantly enhanced in infected tissue after depletion of DCs. We also found that the bactericidal capacity of the recruited neutrophils was significantly impaired in DC-depleted mice. More importantly, the detrimental effect of DC depletion was practically reversed by treatment with exogenous recombinant mouse IL-12. Our results demonstrated that DCs, probably through their production of IL-12, play an important role in coordinating the inflammatory response during S. aureus infection.  

4.1087           Elevated levels of serum-soluble triggering receptor expressed on myeloid cells-1 in patients with IBD do not correlate with intestinal TREM-1 mRNA expression and endoscopic disease activity

Saurer, L., Rihs, S., Birrer, M., Saxer-Seculic, N., RAdsak, M. and Mueller, C.

1. Chron’s and Colitis, 6, 913-923 (2012)

  Background & aims Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory responses. We have previously demonstrated a substantial increase in TREM-1-expressing macrophages in the inflamed intestinal mucosa of patients with inflammatory bowel diseases (IBD). TREM-1 is also produced as a soluble receptor (sTREM-1). Here, we aimed to determine whether serum sTREM-1 could be used as a surrogate marker of disease activity in patients with IBD. Methods Intestinal biopsies and concurrently collected sera from patients with Crohn's disease (CD) and Ulcerative colitis (UC) enrolled in the Swiss IBD cohort study were analyzed for intestinal TREM-1 mRNA and serum sTREM-1 expression. TREM-1 mRNA and sTREM-1 were correlated with the endoscopically determined disease activity. Serum sTREM-1 and TREM-1 mRNA expression levels were further determined in sera and colonic tissues collected at various time-points post disease induction in an experimental mouse model of colitis and correlated with disease activity. Results Expression of TREM-1 mRNA was upregulated in intestinal biopsies from patients with active disease but not in patients with quiescent disease. Serum sTREM-1 was elevated in IBD patients compared to normal controls. No substantial differences in sTREM-1 expression levels were found in patients with active versus quiescent disease. In colitic mice, colonic TREM-1 mRNA and serum sTREM-1 were also upregulated. While colonic TREM-1 mRNA expression levels correlated with disease activity, augmented serum sTREM-1 in fact associated with a milder course of disease. Conclusions Analysis of sTREM-1 as a surrogate marker of disease activity in patients with IBD warrants caution.  

4.1088           Improvement of Porcine Islet Isolation by Inhibition of Trypsin Activity During Pancreas Preservation and Digestion Using α1-Antitrypsin

Shimoda, M., Noguchi, H., Fujita, Y., Takita, M., Ikemoto, TS., Chujo, D., Naziruddin, B., Levy, M.F., Kobayashi, N., Grayburn, P.A. and Matsumoto, S. Cell Transplant., 21(2-3), 465-471 (2012)   Porcine islets are considered to be a promising resource for xenotransplantation. However, it is difficult to isolate porcine islets because of the marked fragility and rapid dissociation. Endogenous trypsin is one of the main factors to damage islets during the isolation procedure. Recent studies have suggested that trypsin inhibitors during the preservation of pancreas or the collagenase digestion can improve the result of islet isolation. In this study, we examined whether α1-antitrypsin (Aralast^TM), which inhibits several endogenous proteases and has immunomodulatory properties, can protect islets from the proteases and improve the results of porcine islet isolation. Twelve porcine pancreata were divided into three groups: without Aralast group (standard, n = 5), preserved with Aralast using the ductal injection (DI) method (DI, n = 3), and with Aralast using the DI method and in the collagenase solution (DI+C, n = 4). Efficacy of islet isolation was assessed by islet yields, purity, and viability. The trypsin activity of the preservation and the digestion solution during the isolation procedure was measured. During islet isolation, the trypsin activity in DI+C group was significantly inhibited compared to the standard group, whereas DI group showed less effect than DI+C group. The average of postpurification islet equivalents (IEQ) per pancreas weight in the DI+C group was significantly higher than the standard group (standard: 3516 ± 497 IEQ/g, DI: 4607 ± 1090 IEQ/g, DI+C: 7097 ± 995 IEQ/g; p = 0.017 between standard and DI+C). In the DI+C group, stimulation index was higher than in other groups, although there was no significant difference. The presence of Aralast in both DI solution and collagenase solution markedly inhibited trypsin activity during pancreas digestion procedure and improved the porcine islet isolation. Inhibition of trypsin activity by Aralast could improve porcine islet isolation.  

4.1089           Evaluation of Osmolality of Density Gradient for Human Islet Purification

Noguchi, H., Naziruddin, B., Shimmoda, M., Fujita, Y., Chujo, D., Takita, M., Peng, H., Sugimoto, K., Itoh, T., Kobayashi, N., Onaca, N., levy, M.F. and Matsumoto, S.

Cell Transplant., 21(2-3), 493-500 (2012)

For pancreatic islet transplantation, the most common method of islet purification is density gradient centrifugation because of the differences in density between islets and acinar tissue. The density of islets/acinar tissue depends on several conditions, such as osmolality of purification solution. In this study, we evaluated the osmolality of iodixanol-controlled density gradients (400, 450, and 500 mOsm/kg) on the islet purification step. The density of the purification solutions was controlled by changing the volumetric ratio of iodixanol and the purification solutions (iodixanol-Kyoto solutions; IK solutions). The osmolality of density gradients was controlled by addition of 10× Hanks balanced salt solution (HBSS) solution. Density of both islets and acinar tissue increased relative to increase of the osmolality of purification solutions. There were no significant differences among the three groups on islet yield after density-adjusted purification and the rate of postpurification recovery. In vitro and in vivo assays suggest that the quality of islets was similar among the three groups. Our data suggest that efficacy of purification and quality of isolated islets is similar when the osmolality of purification solutions is between 400 and 500 mOsm/kg and density adjustment is applied. Since the density of islet and acinar tissue is changed according to osmolality, the density adjustment is important when using several osmolality solutions.

   

4.1090           Islet Purification Method Using Large Bottles Effectively Achieves High Islet Yield from Pig Pancreas

Shimoda, M., Noguchi, H., Fujita, Y., Takita, M., Ikemoto, T., Chujo, D., Naziruddin, B., Levy, M.F., Kobayashi, N., Grayburn, P.A. and Matsumoto, S. Cell Transplant, 21 (2-3), 501-508 (2012)   Porcine islets are a promising resource for xenotransplantation. However, low efficacy of islet isolation because of their marked fragility remains a problem. Recently we found that the standard purification method using COBE 2991 cell processor (COBE) with Ficoll density gradient solution damaged islets mechanically by high shearing force. In this study, we evaluated our new purification method using large plastic bottles for the efficacy of islet purification. Ten porcine pancreata were used. The average warm ischemic time was over 40 min; therefore, these pancreata were considered to be in a marginal condition. After digestion, the digested tissue was divided into three groups. Each group was purified using either top loading method with bottle (top group) or bottom loading method with bottle (bottom group) or standard COBE method (COBE group). Islet yield per pancreas weight (IEQ/g) and the rate of postpurification recovery in the top group were significantly higher than the COBE group (top: 8060 ± 1652 IEQ/g, bottom: 4572 ± 614 IE/g, COBE: 3900 ± 734 IE/g. p < 0.02 in top vs. COBE; top percentage of recovery: 99.3 ± 12.3%, bottom: 62.6 ± 8.8%, COBE: 49.5 ± 6.7%, p < 0.02 in top vs. bottom and COBE). The average sizes of purified islets in the top and bottom groups were significantly larger than COBE group (Average diameter top: 156 ± 8 μm, bottom: 147 ± 6 μm, COBE: 119 ± 6 μm, p < 0.01 in top vs. COBE and in bottom vs. COBE), which indicated that bottle method can reduce shear force during purification. Our new purification using top loading bottle method enabled us to obtain a high yield of porcine islets from marginal pancreata.  

4.1091           Comparison of Ulinastatin, Gabexate Mesilate, and Nafamostat Mesilate in Preservation Solution for Islet Isolation

Noguchi, H., Naziruddin, B., Jackson, A., Shimoda, M., Fujita, Y., Chujo, D., Takita, M., Peng, H., Sugimoto, K., Itoh, T., Kobayashi, N., Ueda, M., Okitsu, T., Iwanaga, Y., Nagata, H., Liu, X., Kamiya, H., Onaca, N., Levy, M.F. and Matsumoto, S. Cell Transplant., 21(2-3), 509-516 (2012)   For islet transplantation, maintaining organ viability after pancreas procurement is critically important for optimal graft function and survival. We recently reported that islet yield was significantly higher in the modified ET-Kyoto (MK) solution, which includes a trypsin inhibitor (ulinastatin), compared with the UW solution, and that the advantages of MK solution are trypsin inhibition and less collagenase inhibition. In this study, we compared ulinastatin with other trypsin inhibitors, gabexate mesilate, and nafamostat mesilate, in preservation solution for islet isolation. Ulinastatin was easily dissolved in ET-Kyoto solution, while ET-Kyoto with gabexate mesilate and nafamostat mesilate became cloudy immediately after addition. Although there were no significant differences in islet yield among the three groups, viability was significantly higher for the MK group than for the GK group or the NK group. The stimulation index was significantly higher for the MK group than for the GK group. In summary, there are no other trypsin inhibitors that are more effective than ulinastatin. Based on these data, we now use ET-Kyoto solution with ulinastatin for clinical islet transplantation.  

4.1092           Fresh Islets Are More Effective for Islet Transplantation Than Cultured Islets

Noguchi, H., Naziruddin, B., Jackson, A., Shimoda, M., Ikemoto, T., Fujita, Y., Chujo, D., Takita, M., Peng, H., Sugimoto, K., Itoh, T., Kobayashi, N., Onaca, N., Levy, M.L. and Matsumoto, S. Cell Transplant., 21(2-3), 517-523 (2012)   For clinical islet transplantation, isolated islets deteriorate rapidly in culture, although culturing islets prior to transplantation provides flexibility for evaluation of isolated islets and pretreatment of patients. In the present study, we compared human fresh islets to cultured islets with in vitro and in vivo assays. After culture for 24, 48, and 72 h, islet yield significantly decreased from 2,000 to 1,738 ± 26 (13% loss), 1,525 ± 30 (24% loss), or 1,298 ± 18 IEQ (35% loss), respectively. The ATP contents were significantly higher in the 6-h cultured group (near fresh group) than in 48-h culture groups. The stimulation index was relatively higher in the 6-h cultured group than in 48-h cultured group. Human islets with or without culture were transplanted into diabetic nude mice. The attainability of posttransplantation normoglycemia was significantly higher in fresh group than in the culture groups. Intraperitoneal glucose tolerance testing (IPGTT) showed that the blood glucose levels of mice transplanted with fresh islets were significantly lower than with cultured islets at 30, 60, 90, and 120 min after injection. These data suggest that human islet transplantation without culture could avoid the deterioration of islets during culture and improve the outcome of islet transplantation. Based on these data, we have transplanted fresh islets without culture for our current clinical islet transplantation protocol.  

4.1093           Adverse Events in Clinical Islet Transplantation: One Institutional Experience

Takita, M., Matsumoto, S., Noguchi, H., Shimoda, M., Ikemoto, T., Chujo, D., Tamura, Y., Olsen, G.S., Naziruddin, B., Purcell, K., Onaca, N. and Levy, M.F. Cell Transplant., 21(2-3), 547-551 (2012)   Islet transplantation is one of the most promising treatments for an unstable form of type 1 diabetes. However, islet transplantation still has some obstacles, such as low success rate of islet isolation, difficulty to obtain long-term insulin freedom, and adverse events related to transplant protocol. We describe the adverse events of current clinical islet transplantation at our institute in this report. Nine type 1 diabetic patients received 17 islet infusions from March 2005 to October 2008. The islet infusion procedure and immunosuppression regimen were based on a modified Edmonton protocol. Severe adverse events (SAEs) were defined as events that were more than grade 3 according to the Terminology Criteria for Adverse Events in Trials of Adult Pancreatic Islet Transplantation, version 4.1 (Collaborative Islet Transplant Registry, CITR). Sixteen events were reported as SAEs and among them 12 events were probably or definitely related to transplant protocols; all occurred within 1 year after infusion except for one. Five adverse events (31%) occurred within 10 days after transplantation and were related to infusion procedures. Seven events (44%) occurred after 50 days and were related to immunosuppressive therapy. SAEs related to the protocol included three events of elevated liver enzymes, two of hemorrhage into gall bladder or peritoneal cavity, two of neutropenia, two of infection, one of vomiting, one of diarrhea, and one of renal dysfunction. All events were grade 3, except for one case that was grade 4 of neutropenia. All SAEs resolved with no sequelae. Neoplasms and deaths were not observed in our study. The present study suggests need to improve both infusion procedure and immunosuppressive strategy from the view of preventing SAEs.  

4.1094           Atorvastatin inhibits proliferation and apoptosis, but induces senescence in hepatic myofibroblasts and thereby attenuates hepatic fibrosis in rats

Klein, S., Klösel, J., Schierwagen, R., Körner, C., Granzow, M., Huss, S., Gretchen, I., Mazar, R., Weber, S., van den Ven, P.F.M., Pieper-Fürst, U., Fürst, D.O., Nattermann, J., Lammert, F., Sauerbruch, T. and Trebicka, J. Lab. Invest., 92(10), 1440-1450 (2012)   Hepatic myofibroblasts (MFB) show increased proliferation, migration and collagen production, which are crucial for hepatic fibrogenesis. Atorvastatin treatment inhibits proliferation, apoptosis and cytokine production of MFB in bile duct-ligated (BDL) rats in vivo. Here, we have further investigated the underlying mechanisms. Primary rat hepatic stellate cells (HSC) were isolated and culture-activated to hepatic MFB. Following 3 days of incubation with atorvastatin (10⁻⁴, 10⁻⁵ and 10⁻⁶ M), transcription levels of profibrotic cytokines (transforming growth factor-β1, connective tissue growth factor and TIMP1) and procollagen Ia were analyzed by real time PCR. Proliferation was investigated by 5'-bromo-2'-deoxyuridine assays. α-Smooth muscle actin protein expression was examined by western blotting. Fluorescence-activated cell sorting analysis of Annexin V and propidium iodide were used to measure apoptosis. Furthermore, p21 western blotting and β-galactosidase staining were investigated in MFB as senescence markers. Subsequently, hepatic expression of desmin and senescence markers were analyzed in the livers of rats receiving atorvastatin (15 mg/kg*d) for 1 week starting 3 and 5 weeks after BDL. Atorvastatin inhibited the activation of HSC to MFB and decreased cytokine and collagen production in MFB in vitro. In addition, proliferation, cytokine and collagen production of MFB were reduced by atorvastatin. Atorvastatin initiated apoptosis at 10⁻⁴ M and attenuated it at 10⁻⁵ M. Atorvastatin induced p21 protein expression and β-galactosidase staining of MFB in vitro and in vivo. Atorvastatin elicits similiar effects on MFB as previously seen in vivo: it decreases MFB turnover and fibrogenesis. We suggest that a further mechanism explaining these effects is senescence of cells.  

4.1095           TRIM50 Protein Regulates Vesicular Trafficking for Acid Secretion in Gastric Parietal Cells

Nishi, M., Aoyama, F., Kisa, F., Zhu, H., Sun, M., Lin, P., Ohta, H., Van, B., Yamamoto, S., Kakizawa, S., Sakai, H., Ma, J., Sawaguchi, A. and Takeshima, H.

1. Biol. Chem., 287(40), 333523-33532 (2012)

  Of the TRIM/RBCC family proteins taking part in a variety of cellular processes, TRIM50 is a stomach-specific member with no defined biological function. Our biochemical data demonstrated that TRIM50 is specifically expressed in gastric parietal cells and is predominantly localized in the tubulovesicular and canalicular membranes. In cultured cells ectopically expressing GFP-TRIM50, confocal microscopic imaging revealed dynamic movement of TRIM50-associated vesicles in a phosphoinositide 3-kinase-dependent manner. A protein overlay assay detected preferential binding of the PRY-SPRY domain from the TRIM50 C-terminal region to phosphatidylinositol species, suggesting that TRIM50 is involved in vesicular dynamics by sensing the phosphorylated state of phosphoinositol lipids. Trim50 knock-out mice retained normal histology in the gastric mucosa but exhibited impaired secretion of gastric acid. In response to histamine, Trim50 knock-out parietal cells generated deranged canaliculi, swollen microvilli lacking actin filaments, and excess multilamellar membrane complexes. Therefore, TRIM50 seems to play an essential role in tubulovesicular dynamics, promoting the formation of sophisticated canaliculi and microvilli during acid secretion in parietal cells.  

4.1096           CD8 T cell - Dendritic Cell crosstalk up-regulates CD40L expression and IL-12p70 production

Tay, Q. W., Ho, L.C., Low, P.Y. and Kemeny, D.M. Immunology, 137 (Supp 1), P0874, 185-772 (2012)   Purpose/Objective: CD8 T cells have been shown to skew the immune response by stimulating dendritic cells (DC) to produce IL-12p70. The purpose of our study was to investigate the effect on CD8 T cell expression of CD40L. Materials and methods: Ovalbumin (OVA)-specific T cell receptor transgenic mouse CD8 T cells (OT-I) were isolated by positive selection using anti-CD8 coated MACS beads and co-cultured splenic DC isolated using Optiprep and by positive selection with anti-CD11c coated MACS beads, and cultured in complete medium (RPMI 1640, 10% Fetal calf serum (FCS), antibiotics and non-essential amino acids and 0.5% 2 mercaptoethanol). CD40L expression was measured by Flow cytometry, IL-12p70 was determined by ELISA (R&D systems). CD8 T cells were activated with PMA (10 ng/ml) and Ionomycin (400 ng/ml) to induce CD44high effector memory cells. CD11c+ splenic DCs were pulsed with 1 lg/ml of peptide (typically SIINFEKL) for 1 h before co-culture with pre-activated CD8 T cells at a ratio of 1 DC to 3 T cells. Altered SIINFEKL peptides (SAINFEKL, EIINFEKL, SIIRFEKL, SIINYEKL) were purchased from AnaSpec Inc (USA). Results: Co-culture of effector memory CD8 T cells with DC up regulated CD8 T cell expression of CD40L that reached a maximum after 6 h. IL-12p70 levels in the culture reached 80% of its maximum value after 8 h. Altered peptide ligands SAINFEKL and SIINYEKL induced intermediate levels of CD40L while EINFEKYL and SIIRFEKL failed to induce CD40L on CD8 T cells. There was a corresponding reduction in IL-12p70 in the medium. Using CD8 T cells stimulated with anti-CD3 and CD28 addition of IL-12p70 enhanced CD40L expression on CD8s and addition of IL-12p70 neutralising antibody reduced CD40L expression. Conclusions: CD8 T cell receptor dependent signals stimulate DCs to secrete IL-12p70 that up-regulates CD40L expression on CD8 T cells.  

4.1097           Developmental rates of IVP bovine embryos using minigradient of Percoll, Isolate and Optiprep

Vianna, L.L., pradiee, J., Santos, E.C., Gonc, A., Pfeifer, L.F.M., Dode, M.A., Vieira, A.D., De Lima, V.F.H., Correal, M.N. and pegoraro, L.M.C. Reproducion in Domestic Animals, 47(s4), 416-613 (2012)   The sperm selection method is one important step of embryo IVP systems and may influence the embryonic development rates. The objective of this study was to compare the efficiency of different sperm selection methods used in bovine IVP systems in terms of embryo development. A total of 2455 oocytes obtained from ovaries of Bos taurus cattle collected in a slaughterhouse were used in this experiment (n = 11 replicates). After in vitro maturation oocytes were distributed and inseminated according to four treatments: (i) Conventional Percoll group (90 and 45%) – 4 ml, centrifuged at 700· g for 20 min, (ii) Minipercoll group (90 and 45%) – 800 lL, centrifuged at 700· g for 5 min, (iii) Miniisolate group (90 and 45%) – 800 lL, centrifuged at 700· g for 5 min; and (iv) Minioptiprep group (30.28 and 26%) – 1.2 ml, centrifuged at 900· g for 15 min. Developmental rates at D2 (cleaved/oocytes inseminated) and at D8 (blastocyst/ oocytes inseminated) of culture were compared among treatments. Data were analysed by using chi-squre test. The cleavage rates were similar among the Conventional Percoll_ and Miniisolate_ groups (70.4% vs. 67.1%; p > 0.05). Minipercoll_ and Minioptiprep_ groups had lower cleavage rate than Conventional Percoll_ (65.8%, 57.9% vs. 70.4%; p < 0.05). At Day 8 blastocyst rate for Minipercoll, Minioptiprep and Conventional Percoll were 16.1%, 16.9%, and 18.4%, respectively (p > 0.05). Among the minigradientes, the Miniisolate had higher blastocyst yield than Minipercoll and Minioptiprep (21.1% vs. 16.1% and 16.9%, respectively; p < 0.05). In conclusion, the minigradients, in the concentration and volume used in our experiment, reached similar results to the Percoll Conventional group. Therefore, they can be use in the routine of IVP bovine embryos as alternatives with same viability and lower time consuming and production cost.  

4.1098

 

4.1099           The Peroxisome Proliferator-activated Receptor {gamma} (PPAR{gamma}) Controls Natural Protective Mechanisms against Lipid Peroxidation in Amyotrophic Lateral Sclerosis

Benedusi, V., Martorana, F., Brambilla, L., Maggi, A. and Rossi, D.

1. Biol. Chem., 287(43), 35899-35911 (2012)

  Recent evidence highlights the peroxisome proliferator-activated receptors (PPARs) as critical neuroprotective factors in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). To gain new mechanistic insights into the role of these receptors in the context of ALS, here we investigated how PPAR transcriptional activity varies in hSOD1^G93A ALS transgenic mice. We demonstrate that PPARγ-driven transcription selectively increases in the spinal cord of symptomatic hSOD1^G93A mice. This phenomenon correlates with the up-regulation of target genes, such as lipoprotein lipase and glutathione S-transferase α-2, which are implicated in scavenging lipid peroxidation by-products. Such events are associated with enhanced PPARγ immunoreactivity within motor neuronal nuclei. This observation, and the fact that PPARγ displays increased responsiveness in cultured hSOD1^G93A motor neurons, points to a role for this receptor in neutralizing deleterious lipoperoxidation derivatives within the motor cells. Consistently, in both motor neuron-like cultures and animal models, we report that PPARγ is activated by lipid peroxidation end products, such as 4-hydroxynonenal, whose levels are elevated in the cerebrospinal fluid and spinal cord from ALS patients. We propose that the accumulation of critical concentrations of lipid peroxidation adducts during ALS progression leads to the activation of PPARγ in motor neurons. This in turn triggers self-protective mechanisms that involve the up-regulation of lipid detoxification enzymes, such as lipoprotein lipase and glutathione S-transferase α-2. Our findings indicate that anticipating natural protective reactions by pharmacologically modulating PPARγ transcriptional activity may attenuate neurodegeneration by limiting the damage induced by lipid peroxidation derivatives.  

4.1100           Impact of Tissue Volume and Purification on Clinical Autologous Islet Transplantation for the Treatment of Chronic Pancreatitis

Matsumoto, S., Takita, M., AShimoda, M., Sugimoto, K., Itoh, T., Chujo, D., SoRelle, J.A., Tamura, Y., Rahman, A.M., Onaca, N., Naziruddin, B. and Levy, M.F. Cell Transplantation, 21(4), 625-632 (2012)   Autologous islet transplantation after total pancreatectomy is an excellent treatment for painful chronic pancreatitis. Traditionally, islets have been isolated without purification; however, purification is applied when the tissue volume is large. Nevertheless, the impact of tissue volume and islet purification on clinical outcomes of autologous islet transplantation has not been well examined. We analyzed 27 cases of autologous islet transplantation performed from October 2006 to January 2011. After examining the relationship between tissue volume and portal pressure at various time points, we compared islet characteristics and clinical outcomes between cases with complications (complication group) and without (noncomplication group), as well as cases with purification (purification group) and without (nonpurification group). Tissue volume significantly correlated with maximum (R = 0.61), final (R = 0.53), and delta (i.e., difference between base and maximum; R = 0.71) portal pressure. The complication group had a significantly higher body mass index, tissue volume, islet yield, and portal pressure (maximum, final, delta), suggesting that complications were associated with high tissue volume and high portal pressure. Only one of four patients (25%) in the complication group became insulin free, whereas 11 of 23 patients (49%) in the noncomplication group became insulin free with smaller islet yields. The purification group had a higher islet yield and insulin independence rate but had similar final tissue volume, portal pressure, and complication rates compared with the nonpurification group. In conclusion, high tissue volume was associated with high portal pressure and complications in autologous islet transplantation. Islet purification effectively reduced tissue volume and had no negative impact on islet characteristics. Therefore, islet purification can reduce the risk of complications and may improve clinical outcome for autologous islet transplantation when tissue volume is large.  

4.1101           Correlation of Released HMGB1 Levels with the Degree of Islet Damage in Mice and Humans and With the Outcomes of Islet Transplantation in Mice

Itoh, T., Takita, M., SoRelle, J.A., Shimoda, M., Sugimoto, K., Chujo, D., Qin, H., Naziruddin, B., Levy, M.F. and Matsumoto, S. Cell Transplantation, 21(7), 1371-1381 (2012)   Establishing reliable islet potency assay is a critical and unmet issue for clinical islet transplantation. Recently, we reported that islets contained high levels of high mobility group box 1 (HMGB1) and damaged islets released HMGB1 in a mouse model. In this study, we hypothesized that the amount of released HMGB1 could reflect the degree of islet damage, and could predict the outcome of islet transplantation. Four groups of damaged mouse islets and three groups of damaged human islets were generated by hypoxic conditions. These islets were assessed by in vivo (transplantation) and in vitro (released HMGB1 levels, released C-peptide levels, PI staining, TUNEL staining, ATP/DNA, and glucose-stimulated insulin release test) assays. In addition, the ability of each assay to distinguish between noncured (n = 13) and cured (n = 7) mice was assessed. The curative rates of STZ-diabetic mice after receiving control, hypoxia-3h, hypoxia-6h, and hypoxia-24h mouse islets were 100%, 40%, 0%, and 0%, respectively. Only amounts of released HMGB1 and ratio of PI staining significant increased according to the degree of damages in both human and mouse islets. In terms of predictability of curing diabetic mice, amounts of released HMGB1 showed the best sensitivity (100%), specificity (100%), positive (100%), and negative predictive values (100%) among all the assays. The amount of released HMGB1 reflected the degree of islet damage and correlated with the outcome of islet transplantation in mice. Hence, released HMGB1 levels from islets should be a useful marker to evaluate the potency of isolated islets.  

4.1102           Antagonism of sphingosine 1-phosphate receptor 2 causes a selective reduction of portal vein pressure in bile duct-ligated rodents

Kageyama, Y. et al Hepatology, 56(4), 1427-1438 (2012)   Sinusoidal vasoconstriction, in which hepatic stellate cells operate as contractile machinery, has been suggested to play a pivotal role in the pathophysiology of portal hypertension. We investigated whether sphingosine 1-phosphate (S1P) stimulates contractility of those cells and enhances portal vein pressure in isolated perfused rat livers with Rho activation by way of S1P receptor 2 (S1P₂). Rho and its effector, Rho kinase, reportedly contribute to the pathophysiology of portal hypertension. Thus, a potential effect of S1P₂ antagonism on portal hypertension was examined. Intravenous infusion of the S1P₂ antagonist, JTE-013, at 1 mg/kg body weight reduced portal vein pressure by 24% without affecting mean arterial pressure in cirrhotic rats induced by bile duct ligation at 4 weeks after the operation, whereas the same amount of S1P₂ antagonist did not alter portal vein pressure and mean arterial pressure in control sham-operated rats. Rho kinase activity in the livers was enhanced in bile duct-ligated rats compared to sham-operated rats, and this enhanced Rho kinase activity in bile duct-ligated livers was reduced after infusion of the S1P₂ antagonist. S1P₂ messenger RNA (mRNA) expression, but not S1P₁ or S1P₃, was increased in bile duct-ligated livers of rats and mice and also in culture-activated rat hepatic stellate cells. S1P₂ expression, determined in S1P mice, was highly increased in hepatic stellate cells of bile duct-ligated livers. Furthermore, the increase of Rho kinase activity in bile duct-ligated livers was observed as early as 7 days after the operation in wildtype mice, but was less in S1P mice. Conclusion: S1P may play an important role in the pathophysiology of portal hypertension with Rho kinase activation by way of S1P₂. The S1P₂ antagonist merits consideration as a novel therapeutic agent for portal hypertension.  

4.1103           Velocimetry of red blood cells in microvessels by the dual-slit method: Effect of velocity gradients

Roman, S., Lorthois, S., Duru, P. and Risso, F. Microvascular Res., 84, 249-261 (2012)   The dual-slit is a photometric technique used for the measurement of red blood cell (RBC) velocity in microvessels. Two photometric windows (slits) are positioned along the vessel. Because the light is modulated by the RBCs flowing through the microvessel, a time dependent signal is captured for each window. A time delay between the two signals is obtained by temporal cross correlation, and is used to deduce a velocity, knowing the distance between the two slits. Despite its wide use in the field of microvascular research, the velocity actually measured by this technique has not yet been unambiguously related to a relevant velocity scale of the flow (e.g. mean or maximal velocity) or to the blood flow rate. This is due to a lack of fundamental understanding of the measurement and also because such a relationship is crucially dependent on the non-uniform velocity distribution of RBCs in the direction parallel to the light beam, which is generally unknown. The aim of the present work is to clarify the physical significance of the velocity measured by the dual-slit technique. For that purpose, dual-slit measurements were performed on computer-generated image sequences of RBCs flowing in microvessels, which allowed all the parameters related to this technique to be precisely controlled. A parametric study determined the range of optimal parameters for the implementation of the dual-slit technique. In this range, it was shown that, whatever the parameters governing the flow, the measured velocity was the maximal RBC velocity found in the direction parallel to the light beam. This finding was then verified by working with image sequences of flowing RBCs acquired in PDMS micro-systems in vitro. Besides confirming the results and physical understanding gained from the study with computer generated images, this in vitro study showed that the profile of RBC maximal velocity across the channel was blunter than a parabolic profile, and exhibited a non-zero sliding velocity at the channel walls. Overall, the present work demonstrates the robustness and high accuracy of the optimized dual-slit technique in various flow conditions, especially at high hematocrit, and discusses its potential for applications in vivo.  

4.1104           Upregulation of the Tim-3/Galectin-9 Pathway of T Cell Exhaustion in Chronic Hepatitis B Virus Infection

Nebbia, G., Peppa, D., Schurich, A., Khanna, P., Singh, H.D., Cheng, Y., Rosenberg, W., Dusheiko, G., Gilson, R., ChinAleong, J., Kennedy, P. and Maini, M.K. PloS One, 7(10), e7648 (2012)   The S-type lectin galectin-9 binds to the negative regulatory molecule Tim-3 on T cells and induces their apoptotic deletion or functional inactivation. We investigated whether galectin-9/Tim-3 interactions contribute to the deletion and exhaustion of the antiviral T cell response in chronic hepatitis B virus infection (CHB). We found Tim-3 to be expressed on a higher percentage of CD4 and CD8 T cells from patients with CHB than healthy controls (p<0.0001) and to be enriched on activated T cells and those infiltrating the HBV-infected liver. Direct ex vivo examination of virus-specific CD8 T cells binding HLA-A2/peptide multimers revealed that Tim-3 was more highly upregulated on HBV-specific CD8 T cells than CMV-specific CD8 T cells or the global CD8 T cell population in patients with CHB (p<0.001) or than on HBV-specific CD8 after resolution of infection. T cells expressing Tim-3 had an impaired ability to produce IFN-γ and TNF-α upon recognition of HBV-peptides and were susceptible to galectin-9-triggered cell death in vitro. Galectin-9 was detectable at increased concentrations in the sera of patients with active CHB-related liver inflammation (p = 0.02) and was strongly expressed by Kupffer cells within the liver sinusoidal network. Tim-3 blockade resulted in enhanced expansion of HBV-specific CD8 T cells able to produce cytokines and mediate cytotoxicity in vitro. Blocking PD-1 in combination with Tim-3 enhanced the number of patients from whom functional antiviral responses could be recovered and/or the strength of responses, indicating that these co-inhibitory molecules play a non-redundant role in driving T cell exhaustion in CHB. Patients taking antivirals able to potently suppress HBV viraemia continued to express Tim-3 on their T cells and respond to Tim-3 blockade. In summary, both Tim-3 and galectin-9 are increased in CHB and may contribute to the inhibition and deletion of T cells as they infiltrate the HBV-infected liver.  

4.1105           Neurotrophic factor-secreting autologous muscle stem cell therapy for the treatment of laryngeal denervation injury

Halum, S.L., McRae, B., Bijangi-Vishehsaraei, K. and Hiatt, K. The Laryngoscope, 122(11), 2482-2496 (2012)   Objectives/Hypothesis: To determine if the spontaneous reinnervation that characteristically ensues after recurrent laryngeal nerve (RLN) injury could be selectively promoted and directed to certain laryngeal muscles with the use of neurotrophic factor (NF)-secreting muscle stem cell (MSC) vectors while antagonistic reinnervation is inhibited with vincristine (VNC). Study Design: Basic science investigation involving primary cell cultures, gene cloning/transfer, and animal experiments. Methods: MSC survival assays were used to test multiple individual NFs in vitro. Motoneuron outgrowth assays assessed the trophic effects of identified NF on cranial nerve X (CNX)-derived motoneurons in vitro. Therapeutic NF was cloned into a lentiviral vector, and MSCs were tranduced to secrete NF. Sixty rats underwent left RLN transection injury, and at 3 weeks received injections of either MSCs (n = 24), MSCs secreting NF (n = 24), or saline (n = 12) into the left thyroarytenoid muscle complex; half of the animals in the MSC groups simultaneously received left posterior cricoarytenoid injections of VNC, whereas half of the animals received saline. Results: Ciliary neurotrophic factor (CNTF) had the greatest survival-promoting effect on MSCs in culture. The addition of CNTF (50 ng/mL) to CNX motoneuron cultures resulted in enhanced neurite outgrowth and branching. In the animal model, the injected MSCs fused with the denervated myofibers, immunohistochemistry demonstrated enhanced reinnervation based on motor endplate to nerve contact, and reverse transcriptase-polymerase chain reaction confirmed stable CNTF expression at longest follow-up (4 months) in the CNTF-secreting MSC treated groups. Conclusions: MSC therapy may have a future role in selectively promoting and directing laryngeal reinnervation after RLN injury.  

4.1106           Interplay of foot-and-mouth disease virus, antibodies and plasmacytoid dendritic cells: virus opsonization under non-neutralizing conditions results in enhanced interferon-alpha responses

Lannes, N., Python, S. and Summerfield, A. Vet. Res., 43, 64-72 (2012)   Foot-and-mouth disease virus (FMDV) is a highly infectious member of the Picornaviridae inducing an acute disease of cloven-hoofed species. Vaccine-induced immune protection correlates with the presence of high levels of neutralizing antibodies but also opsonising antibodies have been proposed as an important mechanism of the immune response contributing to virus clearance by macrophages and leading to the production of type-I interferon (IFN) by plasmacytoid dendritic cells (pDC). The present study demonstrates that the opsonising antibody titres mediating enhanced IFN-α responses in pDC were similar to neutralizing titres, when antigenically related viruses from the same serotype were employed. However, sera cross-reacted also with non-neutralized isolates of multiple serotypes, when tested in this assay. Both uncomplexed virus and immune complexed virus stimulated pDC via Toll-like receptor 7. An additional finding of potential importance for strain-specific differences in virulence and/or immunogenicity was that pDC activation by FMDV strongly differed between viral isolates. Altogether, our results indicate that opsonising antibodies can have a broader reactivity than neutralizing antibodies and may contribute to antiviral responses induced against antigenically distant viruses.  

4.1107           Human Melanoma Metastasis in NSG Mice Correlates with Clinical Outcome in Patients

Quintana, E., Piskounova, E., Shackleton, M., Weinberg, D., Eskiocak, U., Fullen, D.R., Johnson, T.M. and Morrison, S.J. Science Translational Med., 4(159), 159ra149 (2012)   Studies of human cancer metastasis have been limited by a lack of experimental assays in which cancer cells from patients metastasize in vivo in a way that correlates with clinical outcome. This makes it impossible to study intrinsic differences in the metastatic properties of cancers from different patients. We recently developed an assay in which human melanomas readily engraft in nonobese diabetic/severe combined immunodeficient interleukin-2 receptor-γ chain null (NSG) mice. We show that melanomas from 25 patients exhibited reproducible differences in the rate of spontaneous metastasis after transplantation into NSG mice and that these differences correlated with clinical outcome in the patients. Stage IIIB/C melanomas that formed distant metastases within 22 months in patients also formed tumors that metastasized widely in NSG mice, whereas stage IIIB/C melanomas that did not form distant metastases within 22 to 50 months in patients metastasized more slowly in NSG mice. These differences in the efficiency of metastasis correlated with the presence of circulating melanoma cells in the blood of NSG mice, suggesting that the rate of entry into the blood is one factor that limits the rate of metastasis. The study of NSG mice can therefore yield information about the metastasis of human melanomas in vivo, in this case revealing intrinsic differences among stage III melanomas in their ability to circulate/survive in the blood and to metastasize.  

4.1108           Miscellaneous Pathogens

Austin, B. and Austin, D.A. Bacterial Fish Pathogens, 413-441 (2012)   Pseudoalteromonas piscicida, Pseudoalteromonas undina, Shewanella putrefaciens, Arcobacter cryaerophilus, Halomonas (=Deleya) cupida, Acinetobacter sp., Moraxella sp., Moritella marina, Moritella viscosa, Mycoplasma mobile, Myxococcus piscicola, Aquaspirillum sp., Janthinobacterium lividum, Pasteurella skyensis, Piscirickettsia salmonis, Rickettsia-like organisms, Streptobacillus, ‘Candidatus Arthromitus’, ‘Candidatus Branchiomonas cysticola’, ‘Candidatus Clavochlamydia salmonicola’, ‘Candidatus Piscichlamydia salmonis’ and ‘Candidatus Renichlamydia lutjani’ have been associated with fish diseases. Moritella viscosa has been recovered from winter ulcer disease (= skin lesions) in Atlantic salmon with pathogenicity mechanisms reflecting the presence of extracellular products. Protection has been achieved with an adjuvanted formalin inactivated whole cell vaccine. Piscirickettsia salmonis is an obligate parasite, which has been associated with coho salmon syndrome, Huito disease and salmonid rickettsial septicaemia. Good protection was recorded by use of a formalised whole cell suspension. Candidatus are uncultured organisms, which may be visualised in pathological material.  

4.1109           3.03 – Cell Separation, Perfusion from Tissue, Organelle Fractionation: A Comparison of the Methods Used for Porcine Islet Isolation for Transplantation as a Treatment for Type 1 Diabetes Mellitus

Rafati, S., Le, C., Rajotte, R.V. and Rayat, G.R. Comp. Sampling and Sample Preparation, 3, 33-51 (2012)   Diabetes mellitus is a chronic condition of disordered glucose metabolism characterized by high blood glucose levels. Its prevalence has rapidly increased in recent years and has been predicted to continue to increase in the years to come. Type 1 diabetes mellitus is commonly treated by insulin therapy; however, secondary complications of diabetes still develop. The replacement of damaged beta cells in the islets could be accomplished by whole pancreas or islet transplantation. However, transplantation is currently challenged by the increasing demand for organs. The transplantation of porcine islets could potentially overcome the shortage of human organ donors and is being considered as one of the most feasible alternative treatments for type 1 diabetes mellitus. The methods for adult porcine islet isolation have dramatically improved since the 1970s and have lead to the enhancement of the quality of adult porcine islet preparations. Neonatal porcine islets with simpler isolation technique and differentiation capacity are becoming more attractive for preclinical large animal studies. The optimization of enzymatic digestion methods, culture conditions, quality control, and improvement of immunosuppressive therapies to overcome rejection are among the challenges that need to be met before porcine islet xenotransplantation can be applied to patients with type 1 diabetes mellitus.  

4.1110           Safety and tolerability of the T-cell depletion protocol coupled with anakinra and etanercept for clinical islet cell transplantation

Takita, M., Matsumoto, S., Shimoda, M., Chujo, D., Itoh, T., SoRelle, J.A., Purcell, K., Onaca, N., Naziruddin, B. and Levy, M.F. Clin. Transplant., 26, E471-E484 (2012)   Background Islet cell transplantation (ICT) is a promising approach to cure patients with type 1 diabetes. We have implemented a new immunosuppression protocol with antithymoglobulin plus anti-inflammatory agents of anakinra and eternacept for induction and tacrolimus plus mycophenolate mofetil for maintenance [T-cell depletion with anti-inflammatory (TCD-AI) protocol], resulting in successful single-donor ICT. Methods Eight islet recipients with type 1 diabetes reported adverse events (AEs) monthly. AEs were compared between three groups: first infusion with the TCD-AI protocol (TCD-AI-1st) and first and second infusion with the Edmonton-type protocol (Edmonton-1st and Edmonton-2nd). Results The incidence of symptomatic AEs within the initial three months in the TCD-AI-1st group was less than in the Edmonton-1st and Edmonton-2nd groups, with a marginally significant difference (mean ± SE: 5.5 ± 0.3, 7.5 ± 0.5, and 8.3 ± 1.3, respectively; p = 0.07). A significant reduction in liver enzyme elevation after ICT was found in the TCD-AI-1st group compared with the Edmonton-1st and Edmonton-2nd groups (p < 0.05). Because of AEs, all patients in the Edmonton protocol eventually converted to the TCD-AI protocol, whereas all patients tolerated the TCD-AI protocol. Conclusions TCD-AI protocol can be tolerated for successful ICT, although this study includes small cohort, and large population trial should be taken.  

4.1111           Gamma Interferon (IFN-γ) Receptor Restricts Systemic Dengue Virus Replication and Prevents Paralysis in IFN-α/β Receptor-Deficient Mice

Prestwood, T.R., Morar, M.M., Zellweger, R.M., Miller, R., May, M.M., Yauch, L.E., Lada, S.M. and Shresta, S.

1. Virol., 86(23), 12561-12570 (2012)

  We previously reported that mice lacking alpha/beta and gamma interferon receptors (IFN-α/βR and -γR) uniformly exhibit paralysis following infection with the dengue virus (DENV) clinical isolate PL046, while only a subset of mice lacking the IFN-γR alone and virtually no mice lacking the IFN-α/βR alone develop paralysis. Here, using a mouse-passaged variant of PL046, strain S221, we show that in the absence of the IFN-α/βR, signaling through the IFN-γR confers approximately 140-fold greater resistance against systemic vascular leakage-associated dengue disease and virtually complete protection from dengue-induced paralysis. Viral replication in the spleen was assessed by immunohistochemistry and flow cytometry, which revealed a reduction in the number of infected cells due to IFN-γR signaling by 2 days after infection, coincident with elevated levels of IFN-γ in the spleen and serum. By 4 days after infection, IFN-γR signaling was found to restrict DENV replication systemically. Clearance of DENV, on the other hand, occurred in the absence of IFN-γR, except in the central nervous system (CNS) (brain and spinal cord), where clearance relied on IFN-γ from CD8⁺ T cells. These results demonstrate the roles of IFN-γR signaling in protection from initial systemic and subsequent CNS disease following DENV infection and demonstrate the importance of CD8⁺ T cells in preventing DENV-induced CNS disease.  

4.1112           Nucleoside salvage pathway kinases regulate hematopoiesis by linking nucleotide metabolism with replication stress

Austin, W.R., Armijo, A.L., Campbell, D.O., Singh, A.S., Hsieh, T., Nathanson, D., Herschman, H.R., Phelps, M.E., Witte, O.N., Czermin, J. and Radu, C.G.

1. Exp. Med., 209(12), 2215-2228 (2012)

  Nucleotide deficiency causes replication stress (RS) and DNA damage in dividing cells. How nucleotide metabolism is regulated in vivo to prevent these deleterious effects remains unknown. In this study, we investigate a functional link between nucleotide deficiency, RS, and the nucleoside salvage pathway (NSP) enzymes deoxycytidine kinase (dCK) and thymidine kinase (TK1). We show that inactivation of dCK in mice depletes deoxycytidine triphosphate (dCTP) pools and induces RS, early S-phase arrest, and DNA damage in erythroid, B lymphoid, and T lymphoid lineages. TK1^−/− erythroid and B lymphoid lineages also experience nucleotide deficiency but, unlike their dCK^−/− counterparts, they still sustain DNA replication. Intriguingly, dCTP pool depletion, RS, and hematopoietic defects induced by dCK inactivation are almost completely reversed in a newly generated dCK/TK1 double-knockout (DKO) mouse model. Using NSP-deficient DKO hematopoietic cells, we identify a previously unrecognized biological activity of endogenous thymidine as a strong inducer of RS in vivo through TK1-mediated dCTP pool depletion. We propose a model that explains how TK1 and dCK “tune” dCTP pools to both trigger and resolve RS in vivo. This new model may be exploited therapeutically to induce synthetic sickness/lethality in hematological malignancies, and possibly in other cancers.  

4.1113           Accumulation of Activated Invariant Natural Killer T Cells in the Tumor Microenvironment after α-Galactosylceramide-Pulsed Antigen Presenting Cells

Nagato, K., Motohashi, S., Ishibashi, F., Okita, K., Yamasaki, K., Moriya, Y., Hoshino, H., Yoshida, S., Hanaoka, H., Fujii, S-i., Taniguchi, M., Yoshino, I. and Nakayama, T.

1. Clin. Immunol., 32(5), 1071-1081 (2012)

  Purpose The intravenous administration of α-Galactosylceramide (α-GalCer)-pulsed antigen presenting cells (APCs) is well tolerated and the increased IFN-γ producing cells in the peripheral blood after the treatment appeared to be associated with prolonged survival. An exploratory study protocol was designed with the preoperative administration of α-GalCer-pulsed APCs to clarify the mechanisms of these findings, while especially focusing on the precise tumor site. Methods Patients with operable advanced lung cancer received an intravenous injection of α-GalCer-pulsed APCs before surgery. The resected lung and tumor infiltrating lymphocytes (TILs) as well as peripheral blood mononuclear cells were collected and the invariant NKT (iNKT) cell-specific immune responses were analyzed. Results Four patients completed the study protocol. We observed a significant increase in iNKT cell numbers in the TILs and augmented IFN-γ production by the α-GalCer-stimulated TILs. Conclusion The administration of α-GalCer-pulsed APCs successfully induced the dramatic infiltration and activation of iNKT cells in the tumor microenvironment.  

4.1114           Blockade of Myeloid-Derived Suppressor Cells after Induction of Lymphopenia Improves Adoptive T Cell Therapy in a Murine Model of Melanoma

Kodumudi, K.N., Weber, A., Sarnaik, A.A. and Pilon-Thomas, S.

1. Immunol., 189(11), 5147-5154 (2012)

  Administration of nonmyeloablative chemotherapeutic agents or total body irradiation (TBI) prior to adoptive transfer of tumor-specific T cells may reduce or eliminate immunosuppressive populations such as T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSC). Little is known about these populations during immune reconstitution. This study was designed to understand the reconstitution rate and function of these populations post TBI in melanoma tumor‑bearing mice. Reconstitution rate and suppressive activity of CD4⁺CD25⁺Foxp3⁺ Tregs and CD11b⁺Gr1⁺ MDSC following TBI-induced lymphopenia was measured in B16 melanoma tumor‑bearing mice. To ablate the rapid reconstitution of suppressive populations, we treated mice with docetaxel, a known chemotherapeutic agent that targets MDSC, in combination with adoptive T cell transfer and dendritic cell immunotherapy. Both Treg and MDSC populations exhibited rapid reconstitution after TBI-induced lymphopenia. Although reconstituted Tregs were just as suppressive as Tregs from untreated mice, MDSC demonstrated enhanced suppressive activity of CD8⁺ T cell proliferation compared with endogenous MDSC from tumor-bearing mice. TBI-induced lymphopenia followed by docetaxel treatment improved the efficacy of adoptive T cell transfer and dendritic cell immunotherapy in melanoma-bearing mice, inducing a significant reduction in tumor growth and enhancing survival. Tumor regression correlated with increased CTL activity and persistence of adoptively transferred T cells. Overall, these findings suggest that TBI-induced MDSC are highly immunosuppressive and blocking their rapid reconstitution may improve the efficacy of vaccination strategies and adoptive immunotherapy.  

4.1115           Hepatic Vascular Endothelial Growth Factor Regulates Recruitment of Rat Liver Sinusoidal Endothelial Cell Progenitor Cells

Wang, L., Wang, X., Wang, L., Chiu, J.D., Van De Ven, G., Gaarde, W.A. and Deleve, L.D: Gastroenterology, 143(6), 1555-1563 (2012)   Background & Aims After liver injury, bone marrow–derived liver sinusoidal endothelial cell progenitor cells (BM SPCs) repopulate the sinusoid as liver sinusoidal endothelial cells (LSECs). After partial hepatectomy, BM SPCs provide hepatocyte growth factor, promote hepatocyte proliferation, and are necessary for normal liver regeneration. We examined how hepatic vascular endothelial growth factor (VEGF) regulates recruitment of BM SPCs and their effects on liver injury. Methods Rats were given injections of dimethylnitrosamine to induce liver injury, which was assessed by histology and transaminase assays. Recruitment of SPCs was analyzed by examining BM SPC proliferation, mobilization to the circulation, engraftment in liver, and development of fenestration (differentiation). Results Dimethylnitrosamine caused extensive denudation of LSECs at 24 hours, followed by centrilobular hemorrhagic necrosis at 48 hours. Proliferation of BM SPCs, the number of SPCs in the bone marrow, and mobilization of BM SPCs to the circulation increased 2- to 4-fold by 24 hours after injection of dimethylnitrosamine; within 5 days, 40% of all LSECs came from engrafted BM SPCs. Allogeneic resident SPCs, infused 24 hours after injection of dimethylnitrosamine, repopulated the sinusoid as LSECs and reduced liver injury. Expression of hepatic VEGF messenger RNA and protein increased 5-fold by 24 hours after dimethylnitrosamine injection. Knockdown of hepatic VEGF with antisense oligonucleotides completely prevented dimethylnitrosamine-induced proliferation of BM SPCs and their mobilization to the circulation, reduced their engraftment by 46%, completely prevented formation of fenestration after engraftment as LSECs, and exacerbated dimethylnitrosamine injury. Conclusions BM SPC recruitment is a repair response to dimethylnitrosamine liver injury in rats. Hepatic VEGF regulates recruitment of BM SPCs to liver and reduces this form of liver injury.  

4.1116           The Role of Interferon-γ Inducible Protein-10 in a Mouse Model of Acute Liver Injury Post Induced Pluripotent Stem Cells Transplantation

Chan, C-C., Cheng, L-Y., Lu, J., Huang, Y-H., Chiou, S-H., Tsai, P-H., Huo, T-l., Lin, H-C. and Lee, F-Y. PloS One, 7(12), e50577 (2012)  

BackgroundLiver injuries are important medical problems that require effective therapy. Stem cell or hepatocyte transplantation has the potential to restore function of the damaged liver and ameliorate injury. However, the regulatory factors crucial for the repair and regeneration after cell transplantation have not been fully characterized. Our study investigated the effects and the expression of the regulatory factors in mouse models of acute liver injury either transplanted with the induced pluripotent stem cells (iPS) or the hepatocytes that differentiated from iPS cells (iHL).Methods/Principal FindingsMice received CCl4 injection and were randomized to receive vehicle, iPS, or iHL transfusions vial tail veins and were observed for 24, 48 or 72 hours. The group of mice with iPS transplantation performed better than the group of mice receiving iHL in reducing the serum alanine aminotransferase, aspartate aminotransferase, and liver necrosis areas at 24 hours after CCl4 injury. Moreover, iPS significantly increased the numbers of proliferating hepatocytes at 48 hours. Cytokine array identified that chemokine IP-10 could be the potential regulatory factor that ameliorates liver injury. Further studies revealed that iPS secreted IP-10 in vitro and transfusion of iPS increased IP-10 protein and mRNA expressions in the injured livers in vivo. The primary hepatocytes and non-parenchyma cells were isolated from normal and injured livers. Hepatocytes from injured livers that received iPS treatment expressed more IP-10 mRNA than their non-hepatocyte counter-parts. In addition, animal studies revealed that administration of recombinant IP-10 (rIP-10) effectively reduced liver injuries while IP-10-neutralizing antibody attenuated the protective effects of iPS and decreased hepatocyte proliferation. Both iPS and rIP-10 significantly reduced the 72-hour mortality rate in mice that received multiple CCl4-injuries.Conclusions/SignificanceThese findings suggested that IP-10 may have an important regulatory role in facilitating the repair and regeneration of injured liver after iPS transplantation.

 

4.1117           β-Glucan Curdlan Induces IL-10–Producing CD4+ T Cells and Inhibits Allergic Airway Inflammation

Kawashima, S., Hirose, K., Iwata, A., Takahashi, K., Ohkubo, A., Tamachi, T., Ikeda, K., Kagami, S-i. and Nakajima, H.

1. Immunol., 189(12), 5713-5721 (2012)

  A number of studies have suggested a correlation between a decreased incidence in infectious diseases and an increased incidence of allergic diseases, including asthma. Although several pathogen-derived products have been shown to possess therapeutic potential for allergic diseases, it remains largely unknown whether β-glucan, a cell wall component of a variety of fungi, yeasts, and bacteria, has a regulatory potential for allergic diseases. In this study, we examined the effect of curdlan, a linear β-(1-3)-glucan, on the development of allergic airway inflammation. We found that i.p. injection of curdlan significantly inhibited Ag-induced eosinophil recruitment and Th2 cytokine production in the airways. The activation of CD4⁺ T cells in the presence of curdlan induced IL-10–producing CD4⁺ T cells with high levels of c-Maf expression. Curdlan-induced development of IL-10–producing CD4⁺ T cells required the presence of APCs and ICOS/ICOS ligand interaction. Curdlan-induced development of IL-10–producing CD4⁺ T cells also required intrinsic expression of STAT6. Furthermore, the transfer of Ag-specific CD4⁺ T cells that were stimulated in the presence of curdlan inhibited Ag-induced eosinophil recruitment into the airways. Taken together, these results suggest that curdlan is capable of inducing IL-10–producing CD4⁺ T cells and inhibiting the development of eosinohilic airway inflammation, underscoring the therapeutic potential of curdlan for allergic diseases.  

4.1118           Role of Galectin-3 in Classical and Alternative Macrophage Activation in the Liver following Acetaminophen Intoxication

Docan Dragomir, A-C., Sun, R., Choi, H., Laskin, J.D. and Laskin, D.L.

1. Immunol., 189(12), 5934-5941 (2012)

  Inflammatory macrophages have been implicated in hepatotoxicity induced by the analgesic acetaminophen (APAP). In these studies, we characterized the phenotype of macrophages accumulating in the liver following APAP intoxication and evaluated the role of galectin-3 (Gal-3) in macrophage activation. Administration of APAP (300 mg/kg, i.p.) to wild-type mice resulted in the appearance of two distinct subpopulations of CD11b⁺ cells in the liver, which expressed high or low levels of the monocyte/macrophage activation marker Ly6C. Whereas CD11b⁺/Ly6C^hi macrophages exhibited a classically activated proinflammatory phenotype characterized by increased expression of TNF-α, inducible NO synthase, and CCR2, CD11b⁺/Ly6C^lo macrophages were alternatively activated, expressing high levels of the anti-inflammatory cytokine IL-10. APAP intoxication was also associated with an accumulation of Gal-3⁺ macrophages in the liver; the majority of these cells were Ly6C^hi. APAP-induced increases in CD11b⁺/Ly6C^hi macrophages were significantly reduced in Gal-3^−/− mice. This reduction was evident 72 h post APAP and was correlated with decreased expression of the classical macrophage activation markers, inducible NO synthase, IL-12, and TNF-α, as well as the proinflammatory chemokines CCL2 and CCL3, and chemokine receptors CCR1 and CCR2. Conversely, numbers of CD11b⁺/Ly6C^lo macrophages increased in livers of APAP-treated Gal-3^−/− mice; this was associated with increased expression of the alternative macrophage activation markers Ym1 and Fizz1, increased liver repair, and reduced hepatotoxicity. These data demonstrate that both classically and alternatively activated macrophages accumulate in the liver following APAP intoxication; moreover, Gal-3 plays a role in promoting a persistent proinflammatory macrophage phenotype.  

4.1119           Diesel Exhaust Particle Exposure In Vitro Alters Monocyte Differentiation and Function

Chaudhuri, N., Jary, H., Lea, S., Khan, N., Piddock, K.C., Dockrell, D.H., Donaldson, K., Duffin, R., Singh, D., Parker, L.C. and Sabroe, I. PloS One, 7(12), e51107 (2012)   Air pollution by diesel exhaust particles is associated with elevated mortality and increased hospital admissions in individuals with respiratory diseases such as asthma and chronic obstructive pulmonary disease. During active inflammation monocytes are recruited to the airways and can replace resident alveolar macrophages. We therefore investigated whether chronic fourteen day exposure to low concentrations of diesel exhaust particles can alter the phenotype and function of monocytes from healthy individuals and those with chronic obstructive pulmonary disease. Monocytes were purified from the blood of healthy individuals and people with a diagnosis of chronic obstructive pulmonary disease. Monocyte-derived macrophages were generated in the presence or absence of diesel exhaust particles and their phenotypes studied through investigation of their lifespan, cytokine generation in response to Toll like receptor agonists and heat killed bacteria, and expression of surface markers. Chronic fourteen day exposure of monocyte-derived macrophages to concentrations of diesel exhaust particles >10 µg/ml caused mitochondrial and lysosomal dysfunction, and a gradual loss of cells over time both in healthy and chronic obstructive pulmonary disease individuals. Chronic exposure to lower concentrations of diesel exhaust particles impaired CXCL8 cytokine responses to lipopolysaccharide and heat killed E. coli, and this phenotype was associated with a reduction in CD14 and CD11b expression. Chronic diesel exhaust particle exposure may therefore alter both numbers and function of lung macrophages differentiating from locally recruited monocytes in the lungs of healthy people and patients with chronic obstructive pulmonary disease.  

4.1120           Canine Recombinant Adenovirus Vector Induces an Immunogenicity-Related Gene Expression Profile in Skin-Migrated CD11b+ -Type DCs

Contreras, V., Urien, C., Jouneau, L., Bourge, M., Bouet-Cararo, C., Bonneau, M., Zientara, S., Klonjkowski, B., Schwartz-Cornil, I. PloS One, 7(12), e52513 (2012)   Gene expression profiling of the blood cell response induced early after vaccination has previously been demonstrated to predict the immunogenicity of vaccines. In this study, we evaluated whether the analysis of the gene expression profile of skin-migrated dendritic cells (DCs) could be informative for the in vitro prediction of immunogenicity of vaccine, using canine adenovirus serotype 2 (CAV2) as vaccine vector. CAV2 has been shown to induce immunity to transgenes in several species including sheep and is an interesting alternative to human adenovirus-based vectors, based on the safety records of the parental strain in dogs and the lack of pre-existing immunity in non-host species. Skin-migrated DCs were collected from pseudo-afferent lymph in sheep. Both the CD11b⁺ -type and CD103⁺ -type skin-migrated DCs were transduced by CAV2. An analysis of the global gene response to CAV2 in the two skin DC subsets showed that the gene response in CD11b⁺ -type DCs was far higher and broader than in the CD103⁺ -type DCs. A newly released integrative analytic tool from Ingenuity systems revealed that the CAV2-modulated genes in the CD11b⁺ -type DCs clustered in several activated immunogenicity-related functions, such as immune response, immune cell trafficking and inflammation. Thus gene profiling in skin-migrated DC in vitro indicates that the CD11b⁺ DC type is more responsive to CAV2 than the CD103⁺ DC type, and provides valuable information to help in evaluating and possibly improving viral vector vaccine effectiveness.  

4.1121           LAT Region Factors Mediating Differential Neuronal Tropism of HSV-1 and HSV-2 Do Not Act in Trans

Bertke, A.S., Apakupakul, K., Ma, A., Imai, Y., Gussow, A.M., Wang, K., Cohen, J.I., Bloom, D.C. and Margolis, T.P. PloS One, 7(12), e53281 (2012)   After HSV infection, some trigeminal ganglion neurons support productive cycle gene expression, while in other neurons the virus establishes a latent infection. We previously demonstrated that HSV-1 and HSV-2 preferentially establish latent infection in A5+ and KH10+ sensory neurons, respectively, and that exchanging the latency-associated transcript (LAT) between HSV-1 and HSV-2 also exchanges the neuronal preference. Since many viral genes besides the LAT are functionally interchangeable between HSV-1 and HSV-2, we co-infected HSV-1 and HSV-2, both in vivo and in vitro, to determine if trans-acting viral factors regulate whether HSV infection follows a productive or latent pattern of gene expression in sensory neurons. The pattern of HSV-1 and HSV-2 latent infection in trigeminal neurons was no different following co-infection than with either virus alone, consistent with the hypothesis that a trans-acting viral factor is not responsible for the different patterns of latent infection of HSV-1 and HSV-2 in A5+ and KH10+ neurons. Since exchanging the LAT regions between the viruses also exchanges neuronal preferences, we infected transgenic mice that constitutively express 2.8 kb of the LAT region with the heterologous viral serotype. Endogenous expression of LAT did not alter the pattern of latent infection after inoculation with the heterologous serotype virus, demonstrating that the LAT region does not act in trans to direct preferential establishment of latency of HSV-1 and HSV-2. Using HSV1-RFP and HSV2-GFP in adult trigeminal ganglion neurons in vitro, we determined that HSV-1 and HSV-2 do not exert trans-acting effects during acute infection to regulate neuron specificity. Although some neurons were productively infected with both HSV-1 and HSV-2, no A5+ or KH10+ neurons were productively infected with both viruses. Thus, trans-acting viral factors do not regulate preferential permissiveness of A5+ and KH10+ neurons for productive HSV infection and preferential establishment of latent infection.  

4.1122           Molecular Characterization of the Regenerative Response Induced by Intrarenal Transplantation of Selected Renal Cells in a Rodent Model of Chronic Kidney Disease

Genheimer, C.W., Ilagan, R.M., Spencer, T., Kelley, R.W., Werdin, E., Choudhury, S., Jain, D., Ludlow, J.W. and Basu, J. Cell Tissues Organs, 196(4), 374-384 (2012)   Dedifferentiation and proliferation of resident tubular epithelial cells is a mechanism of action potentially contributing to repair and regeneration in kidneys presenting with ischemic or chronic disease. To more efficiently develop cell and tissue engineering technologies for the kidney, we have developed molecular assays to evaluate the acquisition of a pluripotent state associated with stem/progenitor cell phenotype during induction of a regenerative response within the kidneys of rats with chronic kidney disease (CKD) following therapeutic intervention. Intrarenal delivery of selected bioactive renal cells leads to significant upregulation of pluripotency-associated SOX2 mRNA within the diseased kidney tissue from 1 to 24 weeks after treatment. The overall regenerative response index was assessed by quantitative composite expression of CD24, NODAL and LEFTY1 proteins, which were induced within 1 week of cell treatment and peaked at 12 weeks after treatment, reaching statistical significance (p < 0.05) compared to untreated CKD controls. Molecular assays that incorporate the assessment of SOX2 and the regenerative response index may prove to be valuable tools for the detection and monitoring of the tissue response after the delivery of regenerative treatments for CKD, thereby significantly shortening the developmental timelines associated with such therapies.  

4.1123           Butyrate increases IL-23 production by stimulated dendritic cells

Berndt, B.E., Zhang, M., Owyang, S.Y., Cole, T.S., Wang, T.W., Luther, J., Veniaminova, N.A., Merchant, J.L., Chen, C-C., Huffnagle, G.B. and Kao, J.Y. Am. J. Physiol. Gastrointest. Liver Physiol., 303, G1384-G1392 (2012)   The gut microbiota is essential for the maintenance of intestinal immune homeostasis and is responsible for breaking down dietary fiber into short-chain fatty acids (SCFAs). Butyrate, the most abundant bioactive SCFA in the gut, is a histone deacetylase inhibitor (HDACi), a class of drug that has potent immunomodulatory properties. This characteristic of butyrate, along with our previous discovery that conventional dendritic cells (DCs) are required for the development of experimental colitis, led us to speculate that butyrate may modulate DC function to regulate gut mucosal homeostasis. We found that butyrate, in addition to suppressing LPS-induced bone marrow-derived DC maturation and inhibiting DC IL-12 production, significantly induced IL-23 expression. The upregulation of mRNA subunit IL-23p19 at the pretranslational level was consistent with the role of HDACi on the epigenetic modification of gene expression. Furthermore, the mechanism of IL-23p19 upregulation was independent of Stat3 and ZBP89. Coculture of splenocytes with LPS-stimulated DCs pretreated with or without butyrate was performed and showed a significant induction of IL-17 and IL-10. We demonstrated further the effect of butyrate in vivo using dextran sulfate sodium (DSS)-induced colitis and found that the addition of butyrate in the drinking water of mice worsened DSS-colitis. This is in contrast to the daily intraperitoneal butyrate injection of DSS-treated mice, which mildly improved disease severity. Our study highlights a novel effect of butyrate in upregulating IL-23 production of activated DCs and demonstrates a difference in the host response to the oral vs. systemic route of butyrate administration.  

4.1124           Characterization of coagulation factor synthesis in nine human primary cell types

Dashty, M:, Akbarkhanzadeh, V., Zeebregts, C.J., Spek, C.A., Sijbrands, E.J., Pepplenbosch, M.P. and Rezaee, F. Scientific Reports, 2:787, DOI: 10.1038/srep00787 (2012)   The coagulation/fibrinolysis system is essential for wound healing after vascular injury. According to the standard paradigm, the synthesis of most coagulation factors is restricted to liver, platelets and endothelium. We challenged this interpretation by measuring coagulation factors in nine human primary cell types. FX mRNA was expressed by fibroblasts, visceral preadipocytes/adipocytes and hepatocytes, but not in macrophages or other cells. All cells expressed FVIII except endothelial cells. Fibroblasts, endothelial cells and macrophages produced thrombomodulin but not FV. Interestingly, vascular-related cells (platelets/monocytes) that expressed FV did not express FX and vice versa. Monocytes expressed FV, FVIII and FXIIIA, which are positive regulators of clot formation, but these cells also contained thrombomodulin, a negative regulator of coagulation. Our data show that the expression of coagulation factors is much more complex than previously thought, and we speculate that this intricate regulation of coagulation factor expression is necessary for correct fine-tuning of fibrinogenesis versus fibrinolysis.  

4.1125           Suppressors of Cytokine Signaling Promote Fas-Induced Apoptosis through Downregulation of NF-κB and Mitochondrial Bfl-1 in Leukemic T Cells

Oh, J., Kim, S-H., Ahn, S. and Lee, C-E.

1. Immunol., 189(12), 5561-5571 (2012)

  Suppressors of cytokine signaling (SOCS) are known as negative regulators of cytokine- and growth factor–induced signal transduction. Recently they have emerged as multifunctional proteins with regulatory roles in inflammation, autoimmunity, and cancer. We have recently reported that SOCS1 has antiapoptotic functions against the TNF-α– and the hydrogen peroxide–induced T cell apoptosis through the induction of thioredoxin, which protects protein tyrosine phosphatases and attenuates Jaks. In this study, we report that SOCS, on the contrary, promote death receptor Fas-mediated T cell apoptosis. The proapoptotic effect of SOCS1 was manifested with increases in Fas-induced caspase-8 activation, truncated Bid production, and mitochondrial dysfunctions. Both caspase-8 inhibitor c-Flip and mitochondrial antiapoptotic factor Bfl-1 were significantly reduced by SOCS1. These proapoptotic responses were not associated with changes in Jak or p38/Jnk activities but were accompanied with downregulation of NF-κB and NF-κB–dependent reporter gene expression. Indeed, p65 degradation via ubiquitination was accelerated in SOCS1 overexpressing cells, whereas it was attenuated in SOCS1 knockdown cells. With high NF-κB levels, the SOCS1-ablated cells displayed resistance against Fas-induced apoptosis, which was abrogated upon siBfl-1 transfection. The results indicate that the suppression of NF-κB–dependent induction of prosurvival factors, such as Bfl-1 and c-Flip, may serve as a mechanism for SOCS action to promote Fas-mediated T cell apoptosis. SOCS3 exhibited a similar proapoptotic function. Because both SOCS1 and SOCS3 are induced upon TCR stimulation, SOCS would play a role in activation-induced cell death by sensitizing activated T cells toward Fas-mediated apoptosis to maintain T cell homeostasis.   An effective purification method using large bottles for human pancreatic islet isolation Shimoda, M., Itoh, T., Iwahashi, S., Takita, M., Sugimoto, K., Kanak, M.A., Chujo, D., Naziruddin, B., Levy, M.F., Grayburn, P.A. and Matsumoto, S. Islets, 4(6), 1-7 (2012)   The purification process is one of the most difficult procedures in pancreatic islet isolation. It was demonstrated that the standard purification method using a COBE 2991 cell processor with Ficoll density gradient solution harmed islets mechanically by high shear force. We reported that purification using large bottles with a lower viscosity gradient solution could improve the efficacy of porcine islet purification. In this study, we examined whether the new bottle purification method could improve the purification of human islets. Nine human pancreata from brain-dead donors were used. After pancreas digestion, the digested tissue was divided into three groups. Each group was purified by continuous density gradient using ET-Kyoto and iodixanol gradient solution with either the standard COBE method (COBE group) or the top loading (top group) or bottom loading (bottom group) bottle purification methods. Islet yield, purity, recovery rate after purification, and in vitro and in vivo viability were compared. Islet yield per pancreas weight (IE/g) and the recovery rate in the top group were significantly higher than in the COBE and bottom groups. Furthermore, the average size of purified islets in the top group was significantly larger than in the COBE group, which indicated that the bottle method could reduce the shear force to the islets. In vivo viability was also significantly higher in the top group compared with the COBE group. In conclusion, the top-loading bottle method could improve the quality and quantity of human islets after purification.   α-type-1 polarized dendritic cell-based vaccination in recurrent high-grade glioma: a phase I clinical trial Akiyama, Y., Oshita, C., Kume, A., Lizuka, A., Miyata, H., Komiyama, M., Ashizawa, T., Yagoto, M., Abe, Y., Mitsuya, K., Watanabe, R., Sugino, T., Yamaguchi, K. and Nakasu, Y. BMC Cancer, 12:623 (2012)   Background High-grade gliomas including glioblastoma multiforme (GBM) are among the most malignant and aggressive of tumors, and have a very poor prognosis despite a temozolomide-based intensive treatment. Therefore, a novel therapeutic approach to controlling recurrence is needed. In the present study, we investigated the effect of activated dendritic cell (DC) (α-type-1 polarized DC)-based immunotherapy on high-grade glioma patients with the HLA-A2 or A24 genotype. Methods Nine patients with recurrent high-grade gliomas including 7 with GBMs who fulfilled eligibility criteria were enrolled into a phase I study of monocyte-derived DC-based immunotherapy. HLA-genotyping revealed 1 case of HLA-A*0201 and 8 cases of A*2402. Enriched monocytes obtained using OptiPrep^TM from leukapheresis products on day1, were incubated with GM-CSF and IL-4 in a closed serum-free system, and activated on day6 with TNF-α, IL-1β, IFN-α, IFN-γ, and poly I/C. After pulsing with a cocktail of 5 synthetic peptides (WT-1, HER2, MAGE-A3, and MAGE-A1 or gp100) restricted to HLA-A2 or A24 and KLH, cells were cryopreserved until used. Thawed DCs were injected intradermally in the posterior neck at a dose per cohort of 1.0, 2.0 and 5.0× 10⁷/body. Results The frequency of CD14⁺ monocytes increased to 44.6% from 11.9% after gradient centrifugation. After a 7-day-incubation with cytokines, the mean percentage of DCs rated as lin^-HLA-DR⁺ in patients was 56.2 ± 19.1%. Most DCs expressed high levels of maturation markers, co-stimulatory molecules and type-1 phenotype (CD11c⁺HLA-DR⁺) with a DC1/2 ratio of 35.6. The amount of IL-12 produced from activated DCs was 1025 ± 443 pg/ml per 10⁵ cells. All 76 DC injections were well tolerated except for transient liver dysfunction with grade II. Six patients showed positive immunological responses to peptides in an ELISPOT assay, and positive skin tests to peptide-pulsed DC and KLH were recognized in 4 cases. The clinical response to DC injections was as follows :1 SD and 8 PD. Interestingly, the SD patient, given 24 DC injections, showed a long-term recurrence-free and immunological positive response period. Conclusions These results indicate peptide cocktail-treated activated α-type-1 DC-based immunotherapy to be a potential therapeutic tool against recurrent high-grade glioma with mainly HLA-A*2402. Trial registration Current non-randomized investigational trial UMIN-CTR UMIN ID: 000000914.   Isolation of Chlamydia trachomatis and membrane vesicles derived from host and bacteria Frohlich, K., Hua, Z., Wang, J. and Shen, L.

1. Microbiol. Methods, 91, 222-230 (2012)

  The study of intracellular bacteria and nanometer-size membrane vesicles within infected host cells poses an important challenge as it is difficult to identify each distinct population in the context of the complex populations generated from active host-pathogen interactions. Here, suspension cultures of L929 cells infected with the prevalent obligate intracellular bacterium Chlamydia trachomatis strain F/Cal-IC-13 are utilized for the large scale preparation and isolation of natural membrane vesicles and bacterial forms. Cell lysis with nitrogen cavitation in combination with differential centrifugation, OptiPrep™ density gradient separation, and immunoenrichment using anti-chlamydial lipopolysaccharide antibodies and MagnaBind beads allows for the isolation of both productive and persistent bacterial forms, as well as membrane vesicles derived from the host and pathogen. We have evaluated these populations by electron microscopy and Western blot analysis for identification of biomarkers. In addition, purified persistent forms of C. trachomatis induced by ampicillin display adenosine-5′-triphosphate (ATP) transport activity, suggesting that ampicillin-induced persistent C. trachomatis organisms, at least in part, rely upon host ATP as an energy source. Importantly, several chlamydial cytotoxic and/or secreted proteins are demonstrated to be associated with these vesicles, supporting the idea that membrane vesicles are generated by Chlamydia as a means of carrying and delivering virulence factors necessary for pathogenesis. The ability to produce large-scale infections and generate distinct bacteria and host-derived populations for biochemical analysis, while reducing the burdens of time and cost have implications in all areas of chlamydiology. These protocols can be applied to other strains of C. trachomatis or other intracellular bacteria.    

4.1126           Resveratrol mediates anti-atherogenic effects on cholesterol flux in human macrophages and endothelium via PPARγ and adenosine

Voloshyna, I., Hai, O., Littlefield, M:J., Carsons, S. and Reiss, A. Eur. J. Pharmacol., 698, 299-309 (2013)   Resveratrol is a bioactive molecule used in dietary supplements and herbal medicines and consumed worldwide. Known cardioprotective and anti-inflammatory properties of resveratrol have spurred investigation of the mechanisms involved. The present study explored potential atheroprotective actions of resveratrol on cholesterol metabolism in cells of the arterial wall, including human macrophages and arterial endothelium. Using QRT-PCR and Western blotting techniques, we measured expression of the proteins involved in reverse cholesterol transport (ABCA1, ABCG1 and SR-B1) and the scavenger receptors responsible for uptake of modified cholesterol (CD36, SR-A1 and LOX-1). We analyzed the effect of resveratrol on apoA-1-and HDL-mediated cholesterol efflux in human THP-1 macrophages. The effect of resveratrol on oxLDL internalization and foam cell formation were evaluated using confocal and light microscopy. Our data indicate that resveratrol regulates expression of major proteins involved in cholesterol transport, promotes apoA-1 and HDL-mediated efflux, downregulates oxLDL uptake and diminishes foam cell formation. Mechanistically, resveratrol effects were dependent upon PPAR-γ and adenosine 2A receptor pathways. For the first time we demonstrate that resveratrol regulates expression of the cholesterol metabolizing enzyme cytochrome P450 27-hydroxylase, providing efficient cholesterol elimination via formation of oxysterols. This study establishes that resveratrol attenuates lipid accumulation in cultured human macrophages via effects on cholesterol transport. Further in vivo studies are needed to determine whether resveratrol may be an additional resource available to reduce lipid deposition and atherosclerosis in humans.  

4.1127           Inhibition of inflammatory CD4 T cell activity by murine liver sinusoidal endothelial cells

Carambia, A., Frenzel, C., Bruns, O.T., Schwinge, D., Reimer, R., Hohenberg, H., Huber, S., Tiegs, G., Schramm, C., Lohse, A.W. and Herkel, J.

1. Hepatol., 58, 112-118 (2013)

  Background & Aims The liver can mitigate the inflammatory activity of infiltrating T cells by mechanisms that are not entirely clear. Here we investigated the role of liver sinusoidal endothelial cells (LSECs) in regulating the activity of inflammatory CD4 T cells. Methods Interactions between T helper (Th) 1 or Th17 cells and LSEC were studied by intravital microscopy and by in vitro stimulation assays. Results Circulating CD4 T cells established lasting and repeated interactions with liver endothelium in vivo. Stimulation of Th1 and Th17 cells by LSEC greatly inhibited their capacity to secrete interferon-γ or interleukin-17 in vitro; in contrast, stimulation by dendritic cells (DCs) resulted in considerable secretion of both cytokines. Cytokine release by Th1 or Th17 cells seemed to be actively suppressed by LSEC, as indicated by the inhibition of cytokine secretion even in the presence of Th1- and Th17-promoting DC. This inhibition of CD4 T cell effector function seemed to depend on the dominance of inhibitory over activating co-stimulatory signals on LSEC, since (1) cytokine secretion could be restored by increased CD28 co-activation; (2) LSEC from interleukin-10^−/− mice, which manifest increased activating signals, such as MHC II, and decreased inhibitory signals, such as PD-L1, failed to suppress cytokine secretion; and (3) cytokine secretion by Th1 or Th17 cells that lacked PD-1, the ligand for inhibitory PD-L1, could not be suppressed by LSEC. Conclusions LSEC inhibit inflammatory cytokine secretion of Th1 and Th17 effector CD4 T cells in dependence of interleukin-10 and PD-1.  

4.1128           Macrophage-derived hedgehog ligands promotes fibrogenic and angiogenic responses in human schistosomiasis mansoni

Pereira, T.A., Xie, G., Choi, S.S., Syn, W-K., Voieta, I., Lu, J., Chan, I.S., Swiderska, M., Amaral, K.B., Antunes, C.M., Secor, W.E., Witek, R.P., Lambertucci, J.R., Pereira, F.L. and Diehl, A.M. Liver Int., 33(1), 149-161 (2013)   Background Schistosomiasis mansoni is a major cause of portal fibrosis and portal hypertension. The Hedgehog pathway regulates fibrogenic repair in some types of liver injury. Aims Determine if Hedgehog pathway activation occurs during fibrosis progression in schistosomiasis and to determine if macrophage-related mechanisms are involved. Methods Immunohistochemistry was used to characterize the cells that generate and respond to Hedgehog ligands in 28 liver biopsies from patients with different grades of schistosomiasis fibrosis staged by ultrasound. Cultured macrophages (RAW264.7 and primary rat Kupffer cells) and primary rat liver sinusoidal endothelial cells (LSEC) were treated with schistosome egg antigen (SEA) and evaluated using qRT-PCR. Inhibition of the Hedgehog pathway was used to investigate its role in alternative activation of macrophages (M2) and vascular tube formation. Results Patients with schistosomiasis expressed more ligands (Shh and Ihh) and target genes (Patched and Gli2) than healthy individuals. Activated LSEC and myofibroblasts were Hedgehog responsive [Gli2(+)] and accumulated in parallel with fibrosis stage (P < 0.05). Double IHC for Ihh/CD68 showed that Ihh(+) cells were macrophages. In vitro studies demonstrated that SEA-stimulated macrophages to express Ihh and Shh mRNA (P < 0.05). Conditioned media from such macrophages induced luciferase production by Shh-LightII cells (P < 0.001) and Hedgehog inhibitors blocked this effect (P < 0.001). SEA-treated macrophages also up-regulated their own expression of M2 markers, and Hh pathway inhibitors abrogated this response (P < 0.01). Inhibition of the Hedgehog pathway in LSEC blocked SEA-induced migration and tube formation. Conclusion SEA stimulates liver macrophages to produce Hh ligands, which promote alternative activation of macrophages, fibrogenesis and vascular remodelling in schistosomiasis.  

4.1129           Changes in the properties of normal human red blood cells during in vivo aging

Franco, R.S., Puchulu-Campanella, M.E., Barber, L.A., Palascak,  M.B., Joiner, C.H., Low, P.S. and Cohen, R.M. Am. J. Hematol., 88(1), 44-51 (2013)   The changes in red blood cells (RBC) as they age and the mechanisms for their eventual removal have been of interest for many years. Proposed age-related changes include dehydration with increased density and decreased size, increased membrane IgG, loss of membrane phospholipid asymmetry, and decreased activity of KCl cotransport. The biotin RBC label allows unambiguous identification of older cells and exploration of their properties as they age. Autologous normal human RBC were labeled ex vivo and, after reinfusion, compared with unlabeled RBC throughout their lifespan. RBC density increased with age, with most of the change in the first weeks. Near the end of their lifespan, RBC had increased surface IgG. However, there was no evidence for elevated external phosphatidylserine (PS) even though older RBC had significantly lower activity of aminophospholipid translocase (APLT). KCl cotransport activity persisted well past the reticulocyte stage, but eventually decreased as the RBC became older. These studies place limitations on the use of density fractionation for the study of older human RBC, and do not support loss of phospholipid asymmetry as a mechanism for human RBC senescence. However, increased levels of IgG were associated with older RBC, and may contribute to their removal from the circulation.  

4.1130           Hedgehog signalling regulates liver sinusoidal endothelial cell capillarisation

Xie, G., Choi, S.S., Syn, W-K. et al Gut, 62, 299-309 (2013)   Objective Vascular remodelling during liver damage involves loss of healthy liver sinusoidal endothelial cell (LSEC) phenotype via capillarisation. Hedgehog (Hh) signalling regulates vascular development and increases during liver injury. This study therefore examined its role in capillarisation. Design Primary LSEC were cultured for 5 days to induce capillarisation. Pharmacological, antibody-mediated and genetic approaches were used to manipulate Hh signalling. Effects on mRNA and protein expression of Hh-regulated genes and capillarisation markers were evaluated by quantitative reverse transcription PCR and immunoblot. Changes in LSEC function were assessed by migration and tube forming assay, and gain/loss of fenestrae was examined by electron microscopy. Mice with acute or chronic liver injury were treated with Hh inhibitors; effects on capillarisation were assessed by immunohistochemistry. Results Freshly isolated LSEC expressed Hh ligands, Hh receptors and Hh ligand antagonist Hhip. Capillarisation was accompanied by repression of Hhip and increased expression of Hh-regulated genes. Treatment with Hh agonist further induced expression of Hh ligands and Hh-regulated genes, and upregulated capillarisation-associated genes; whereas Hh signalling antagonist or Hh ligand neutralising antibody each repressed expression of Hh target genes and capillarisation markers. LSEC isolated from Smo^loxP/loxP transgenic mice that had been infected with adenovirus expressing Cre-recombinase to delete Smoothened showed over 75% knockdown of Smoothened. During culture, Smoothened-deficient LSEC had inhibited Hh signalling, less induction of capillarisation-associated genes and retention of fenestrae. In mice with injured livers, inhibiting Hh signalling prevented capillarisation. Conclusions LSEC produce and respond to Hh ligands, and use Hh signalling to regulate complex phenotypic changes that occur during capillarisation.  

4.1131           Activation of toll-like receptor 3 attenuates alcoholic liver injury by stimulating Kupffer cells and stellate cells to produce interleukin-10 in mice

Byun, J-S., Suh, Y-G., Yi, H-S., Lee, Y-S. and Jeong, W-I.

1. Hepatol., 58, 342-349 (2013)

  Background & Aims The important function of toll-like receptor (TLR) 4 in Kupffer cells and hepatic stellate cells (HSCs) has been well documented in alcoholic liver injury. However, little is known about the role of TLR3. Thus, we tested whether TLR3 activation in HSCs and Kupffer cells could attenuate alcoholic liver injury in vivo, and investigated its possible mechanism in vitro. Methods Alcoholic liver injury was achieved by feeding wild type (WT), TLR3 knockout (TLR3^−/−) and interleukin (IL)-10^−/− mice with high-fat diet plus binge ethanol drinking for 2 weeks. To activate TLR3, polyinosinic–polycytidylic acid (poly I:C) was injected into mice. For in vitro studies, HSCs and Kupffer cells were isolated and treated with poly I:C. Results In WT mice, poly I:C treatment reduced alcoholic liver injury and fat accumulation by suppressing nuclear factor-κB activation and sterol response element-binding protein 1c expression in the liver. In addition, freshly isolated HSCs and Kupffer cells from poly I:C-treated mice showed enhanced expression of IL-10 compared to controls. Infiltrated macrophage numbers and the expression of tumor necrosis factor-α, monocyte chemoattractant protein-1 and IL-6 on these cells were decreased after poly I:C treatment. In vitro, poly I:C treatment enhanced the expression of IL-10 via a TLR3-dependent mechanism in HSCs and Kupffer cells. Finally, the protective effects of poly I:C on alcoholic liver injury were diminished in TLR3^−/− and IL-10^−/− mice. Conclusions TLR3 activation ameliorates alcoholic liver injury via the stimulation of IL-10 production in HSCs and Kupffer cells. TLR3 could be a novel therapeutic target for the treatment of alcoholic liver injury.  

4.1132           Mutant SOD1G93A triggers mitochondrial fragmentation in spinal cord motor neurons: Neuroprotection by SIRT3 and PGC-1α

Song, W., Song, Y., Kincaid, B., Bossy, B. and Bossy-Wetzel, E. Neurobiology of Disease, 51, 72-81 (2013)   Mutations in the Cu/Zn Superoxide Dismutase (SOD1) gene cause an inherited form of ALS with upper and lower motor neuron loss. The mechanism underlying mutant SOD1-mediated motor neuron degeneration remains unclear. While defects in mitochondrial dynamics contribute to neurodegeneration, including ALS, previous reports remain conflicted. Here, we report an improved technique to isolate, transfect, and culture rat spinal cord motor neurons. Using this improved system, we demonstrate that mutant SOD1^G93A triggers a significant decrease in mitochondrial length and an accumulation of round fragmented mitochondria. The increase of fragmented mitochondria coincides with an arrest in both anterograde and retrograde axonal transport and increased cell death. In addition, mutant SOD1^G93A induces a reduction in neurite length and branching that is accompanied with an abnormal accumulation of round mitochondria in growth cones. Furthermore, restoration of the mitochondrial fission and fusion balance by dominant-negative dynamin-related protein 1 (DRP1) expression rescues the mutant SOD1^G93A-induced defects in mitochondrial morphology, dynamics, and cell viability. Interestingly, both SIRT3 and PGC-1α protect against mitochondrial fragmentation and neuronal cell death by mutant SOD1^G93A. This data suggests that impairment in mitochondrial dynamics participates in ALS and restoring this defect might provide protection against mutant SOD1^G93A-induced neuronal injury.  

4.1133           IKKβ in Myeloid Cells Controls the Host Response to Lethal and Sublethal Francisella tularensis LVS Infection

Samaniego, S. and  Marcu, K.B. PloS One, 8(1), e54124 (2013)   Background The NF-κB activating kinases, IKKα and IKKβ, are key regulators of inflammation and immunity in response to infection by a variety of pathogens. Both IKKα and IKKβ have been reported to modulate either pro- or anti- inflammatory programs, which may be specific to the infectious organism or the target tissue. Here, we analyzed the requirements for the IKKs in myeloid cells in vivo in response to Francisella tularensis Live Vaccine Strain (Ft. LVS) infection. Methods and Principal Findings In contrast to prior reports in which conditional deletion of IKKβ in the myeloid lineage promoted survival and conferred resistance to an in vivo group B streptococcus infection, we show that mice with a comparable conditional deletion (IKKβ cKO) succumb more rapidly to lethal Ft. LVS infection and are unable to control bacterial growth at sublethal doses. Flow cytometry analysis of hepatic non-parenchymal cells from infected mice reveals that IKKβ inhibits M1 classical macrophage activation two days post infection, which has the collateral effect of suppressing IFN-γ⁺ CD8⁺ T cells. Despite this early enhanced inflammation, IKKβ cKO mice are unable to control infection; and this coincides with a shift toward M2a polarized macrophages. In comparison, we find that myeloid IKKα is dispensable for survival and bacterial control. However, both IKKα and IKKβ have effects on hepatic granuloma development. IKKα cKO mice develop fewer, but well-contained granulomas that accumulate excess necrotic cells after 9 days of infection; while IKKβ cKO mice develop numerous micro-granulomas that are less well contained. Conclusions Taken together our findings reveal that unlike IKKα, IKKβ has multiple, contrasting roles in this bacterial infection model by acting in an anti-inflammatory capacity at early times towards sublethal Ft. LVS infection; but in spite of this, macrophage IKKβ is also a critical effector for host survival and efficient pathogen clearance.  

4.1134           Significant Improvement in Islet Yield and Survival With Modified ET-Kyoto Solution: ET-Kyoto/Neutrophil Elastase Inhibitor

Machida, T., Tanemura, M., Ohmura, Y., tanida, T., Wada, H., Kobayashi, S., Marubashi, S., Eguchi, H., Ito, T., Nagano, H., Mori, M., Doki, Y. and Sawas, Y. Cell Transplantation, 22, 159-173 (2013)   Although islet transplantation can achieve insulin independence in patients with type 1 diabetes, sufficient number of islets derived from two or more donors is usually required to achieve normoglycemia. Activated neutrophils and neutrophil elastase (NE), which is released from these neutrophils, can directly cause injury in islet grafts. We hypothesized that inhibition of NE improves islet isolation and islet allograft survival. We tested our hypothesis by examining the effects of modified ET-Kyoto solution supplemented with sivelestat, a NE inhibitor (S-Kyoto solution), on islet yield and viability in islet isolation and the effect of intraperitoneally injected sivelestat on islet graft survival in a mouse allotransplant model. NE and proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 increased markedly at the end of warm digestion during islet isolation and exhibited direct cytotoxic activity against the islets causing their apoptosis. The use of S-Kyoto solution significantly improved islet yield and viability. Furthermore, treatment with sivelestat resulted in significant prolongation of islet allograft survival in recipient mice. Furthermore, serum levels of IL-6 and TNF-α at 1 and 2 weeks posttransplantation were significantly higher in islet recipients than before transplantation. Our results indicated that NE released from activated neutrophils negatively affects islet survival and that its suppression both in vitro and in vivo improved islet yield and prolonged islet graft survival. The results suggest that inhibition of NE activity could be potentially useful in islet transplantation for patients with type 1 diabetes mellitus.  

4.1135           Circulatory Antigen Processing by Mucosal Dendritic Cells Controls CD8+ T Cell Activation

Chang, S-Y., Song, J-H., Guleng, B., Alonso, C., Arihiro, S., Zhao, Y., Chiang, H-S., O’Keeffe, M., Liao, G., Karp, C.L., Kweon, M-N., Sharpe, A.H., Bhan, A., Terhorst, C. and Reinecker, H-C. Immunity, 38(1), 153-165 (2013)   Circulatory antigens transit through the small intestine via the fenestrated capillaries in the lamina propria prior to entering into the draining lymphatics. But whether or how this process controls mucosal immune responses remains unknown. Here we demonstrate that dendritic cells (DCs) of the lamina propria can sample and process both circulatory and luminal antigens. Surprisingly, antigen cross-presentation by resident CX3CR1⁺ DCs induced differentiation of precursor cells into CD8⁺ T cells that expressed interleukin-10 (IL-10), IL-13, and IL-9 and could migrate into adjacent compartments. We conclude that lamina propria CX3CR1⁺ DCs facilitate the surveillance of circulatory antigens and act as a conduit for the processing of self- and intestinally absorbed antigens, leading to the induction of CD8⁺ T cells, that partake in the control of T cell activation during mucosal immune responses.  

4.1136           Hepatic Stellate Cells Preferentially Induce Foxp3⁺ Regulatory T Cells by Production of Retinoic Acid

Dunham, R.M., Thapa, M., Velazquez, V.M., Elrod, E.J., Denning, T.L., Pulendran, B. and Grakoui, A.

1. Immunol., 190, 2009-2016 (2013)

  The liver has long been described as immunosuppressive, although the mechanisms underlying this phenomenon are incompletely understood. Hepatic stellate cells (HSCs), a population of liver nonparenchymal cells, are potent producers of the regulatory T cell (Treg)–polarizing molecules TGF-β1 and all-trans retinoic acid, particularly during states of inflammation. HSCs are activated during hepatitis C virus infection and may therefore play a role in the enrichment of Tregs during infection. We hypothesized that Ag presentation in the context of HSC activation will induce naive T cells to differentiate into Foxp3⁺ Tregs. To test this hypothesis, we investigated the molecular interactions between murine HSCs, dendritic cells, and naive CD4⁺ T cells. We found that HSCs alone do not present Ag to naive CD4⁺ T cells, but in the presence of dendritic cells and TGF-β1, preferentially induce functional Tregs. This Treg induction was associated with retinoid metabolism by HSCs and was dependent on all-trans retinoic acid. Thus, we conclude that HSCs preferentially generate Foxp3⁺ Tregs and, therefore, may play a role in the tolerogenic nature of the liver.  

4.1137           Phagocytosis Is the Main CR3-Mediated Function Affected by the Lupus-Associated Variant of CD11b in Human Myeloid Cells

Fossati-Jimack, L., Ling, G.S., Cortini, A., Szajnaa, M., Malik, T.H., McDonald, J.U., Pickering, M.C., Cook, H.T., Taylor, P.R. and Botto, M. PloS One, 8(2), e57082 (2013)   The CD11b/CD18 integrin (complement receptor 3, CR3) is a surface receptor on monocytes, neutrophils, macrophages and dendritic cells that plays a crucial role in several immunological processes including leukocyte extravasation and phagocytosis. The minor allele of a non-synonymous CR3 polymorphism (rs1143679, conversation of arginine to histidine at position 77: R77H) represents one of the strongest genetic risk factor in human systemic lupus erythematosus, with heterozygosity (77R/H) being the most common disease associated genotype. Homozygosity for the 77H allele has been reported to reduce adhesion and phagocytosis in human monocytes and monocyte-derived macrophages, respectively, without affecting surface expression of CD11b. Herein we comprehensively assessed the influence of R77H on different CR3-mediated activities in monocytes, neutrophils, macrophages and dendritic cells. R77H did not alter surface expression of CD11b including its active form in any of these cell types. Using two different iC3b-coated targets we found that the uptake by heterozygous 77R/H macrophages, monocytes and neutrophils was significantly reduced compared to 77R/R cells. Allele-specific transduced immortalized macrophage cell lines demonstrated that the minor allele, 77H, was responsible for the impaired phagocytosis. R77H did not affect neutrophil adhesion, neutrophil transmigration in vivo or Toll-like receptor 7/8-mediated cytokine release by monocytes or dendritic cells with or without CR3 pre-engagement by iC3b-coated targets. Our findings demonstrate that the reduction in CR3-mediated phagocytosis associated with the 77H CD11b variant is not macrophage-restricted but demonstrable in other CR3-expressing professional phagocytic cells. The association between 77H and susceptibility to systemic lupus erythematosus most likely relates to impaired waste disposal, a key component of lupus pathogenesis  

4.1138           Mesothelial cells give rise to hepatic stellate cells and myofibroblasts via mesothelial–mesenchymal transition in liver injury

Li, Y., Wang, J. and Asahina, K. PNAS, 110(6), 2324-2329 (2013)   In many organs, myofibroblasts play a major role in the scarring process in response to injury. In liver fibrogenesis, hepatic stellate cells (HSCs) are thought to transdifferentiate into myofibroblasts, but the origins of both HSCs and myofibroblasts remain elusive. In the developing liver, lung, and intestine, mesothelial cells (MCs) differentiate into specific mesenchymal cell types; however, the contribution of this differentiation to organ injury is unknown. In the present study, using mouse models, conditional cell lineage analysis has demonstrated that MCs expressing Wilms tumor 1 give rise to HSCs and myofibroblasts during liver fibrogenesis. Primary MCs, isolated from adult mouse liver using antibodies against glycoprotein M6a, undergo myofibroblastic transdifferentiation. Antagonism of TGF-β signaling suppresses transition of MCs to mesenchymal cells both in vitro and in vivo. These results indicate that MCs undergo mesothelial–mesenchymal transition and participate in liver injury via differentiation to HSCs and myofibroblasts.  

4.1139           Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays

Bachy, V., Hervouet, C., Becker, P.D., Chorro, L., Carlin, L.M., Herath, S., Papagatsias, T., Barbaroux, J-B., Oh, S-J., Benlahrech, A., Athanasopolous, T., Dickson, G., Patterson, S., Kwon, S-Y., Geissmann, F. and Klavinskis, L.S. PNAS, 110(8), 3041-3046 (2013)   Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8⁺ T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c⁺ dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c⁺ MHCII^hi CD8α^neg epithelial cell adhesion molecule (EpCAM^neg) CD11b⁺ langerin (Lang; CD207)^neg DCs, but neither Langerhans cells nor Lang⁺ DCs were required for CD8⁺ T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8⁺ T-cell priming by live rAdHu5 MAs.  

4.1140           BRG1-mediated immune tolerance: facilitation of Treg activation and partial independence of chromatin remodeling

Chaiyachati, B.H., Jani, A., Wan, Y., Huang, H., Flavell, R. and chi, T. EMBO J., 32(3), 395-408 (2013)   Treg activation in response to environmental cues is necessary for regulatory T cells (Tregs) to suppress inflammation, but little is known about the transcription mechanisms controlling Treg activation. We report that despite the known proinflammatory role of the chromatin-remodelling factor BRG1 in CD4 cells, deleting Brg1 in all αβ T cell lineages led to fatal inflammation, which reflected essential roles of BRG1 in Tregs. Brg1 deletion impaired Treg activation, concomitant with the onset of the inflammation. Remarkably, as the inflammation progressed, Tregs became increasingly activated, but the activation levels could not catch up with the severity of inflammation. In vitro assays indicate that BRG1 regulates a subset of TCR target genes including multiple chemokine receptor genes. Finally, using a method that can create littermates bearing either a tissue-specific point mutation or deletion, we found the BRG1 ATPase activity partially dispensable for BRG1 function. Collectively, these data suggest that BRG1 acts in part via remodelling-independent functions to sensitize Tregs to inflammatory cues, thus allowing Tregs to promptly and effectively suppress autoimmunity.  

4.1141           Freezing African Elephant Semen as a New Population Management Tool

Hermes, R., Saragusty, J., Göritz, F., Bartels, P., Potier, R.., Baker, B., Streich, W.J. and Hildebrandt, T.B. PloS One, 8(3), e57616 (2013)   Background The captive elephant population is not self-sustaining and with a limited number of breeding bulls, its genetic diversity is in decline. One way to overcome this is to import young and healthy animals from the wild. We introduce here a more sustainable alternative method - importation of semen from wild bulls without removing them from their natural habitat. Due to the logistics involved, the only practical option would be to transport cryopreserved sperm. Despite some early reports on African elephant semen cryopreservation, the utility of this new population management tool has not been evaluated. Methodology/Principal Findings Semen was collected by electroejaculation from 14 wild African savanna elephant (Loxodonta africana) bulls and cryopreserved using the directional freezing technique. Sperm treatments evaluated included the need for centrifugation, the use of hen or quail yolk, the concentration of glycerol (3%, 5% or 7%) in the extender, and maintenance of motility over time after thawing. Our results suggest that dilution in an extender containing hen yolk and 7% glycerol after centrifugation best preserved post-thaw sperm motility when compared to all other treatments (P≤0.012 for all). Using this approach we were able to achieve after thawing (mean ± SD) 54.6±3.9% motility, 85.3±2.4% acrosome integrity, and 86.8±4.6% normal morphology with no decrease in motility over 1 h incubation at 37°C. Sperm cryopreserved during this study has already lead to a pregnancy of a captive female elephant following artificial insemination. Conclusions/Significance With working techniques for artificial insemination and sperm cryopreservation of both African and Asian elephants in hand, population managers can now enrich captive or isolated wild elephant populations without removing valuable individuals from their natural habitat.  

4.1142           Erythrocyte NADPH oxidase activity modulated by Rac GTPases, PKC, and plasma cytokines contributes to oxidative stress in sickle cell disease

George, A., Pushkaran, S., Konstantinidis, D.G., Koochaki, S., Malik, P., Mohandas, N., Zheng, Y., Joiner, C.H. and Kalfa, T.A. Blood, 121(11), 2099-2107 (2013)   Chronic inflammation has emerged as an important pathogenic mechanism in sickle cell disease (SCD). One component of this inflammatory response is oxidant stress mediated by reactive oxygen species (ROS) generated by leukocytes, endothelial cells, plasma enzymes, and sickle red blood cells (RBC). Sickle RBC ROS generation has been attributed to sickle hemoglobin auto-oxidation and Fenton chemistry reactions catalyzed by denatured heme moieties bound to the RBC membrane. In this study, we demonstrate that a significant part of ROS production in sickle cells is mediated enzymatically by NADPH oxidase, which is regulated by protein kinase C, Rac GTPase, and intracellular Ca²⁺ signaling within the sickle RBC. Moreover, plasma from patients with SCD and isolated cytokines, such as transforming growth factor β1 and endothelin-1, enhance RBC NADPH oxidase activity and increase ROS generation. ROS-mediated damage to RBC membrane components is known to contribute to erythrocyte rigidity and fragility in SCD. Erythrocyte ROS generation, hemolysis, vaso-occlusion, and the inflammatory response to tissue damage may therefore act in a positive-feedback loop to drive the pathophysiology of sickle cell disease. These findings suggest a novel pathogenic mechanism in SCD and may offer new therapeutic targets to counteract inflammation and RBC rigidity and fragility in SCD.  

4.1143           Monoacylglycerol Lipase Controls Endocannabinoid and Eicosanoid Signaling and Hepatic Injury in Mice

Cao, Z., Mulvihill, M.M., Mukhopadhyay, P., Xu, H., Erdelyi, K., hao, E., Holovac, E., Hasko, G., Cravatt, B.F., Nomura, D.K. and Pacher, P. Gastroenterology, 144, 808-817 (2013)   Background & Aims The endocannabinoid and eicosanoid lipid signaling pathways have important roles in inflammatory syndromes. Monoacylglycerol lipase (MAGL) links these pathways, hydrolyzing the endocannabinoid 2-arachidonoylglycerol to generate the arachidonic acid precursor pool for prostaglandin production. We investigated whether blocking MAGL protects against inflammation and damage from hepatic ischemia/reperfusion (I/R) and other insults. Methods We analyzed the effects of hepatic I/R in mice given the selective MAGL inhibitor JZL184, in Mgll^-/- mice, fatty acid amide hydrolase^-/- mice, and in cannabinoid receptor type 1^−/− (CB₁-/-) and cannabinoid receptor type 2^−/− (CB₂-/-). Liver tissues were collected and analyzed, along with cultured hepatocytes and Kupffer cells. We measured endocannabinoids, eicosanoids, and markers of inflammation, oxidative stress, and cell death using molecular biology, biochemistry, and mass spectrometry analyses. Results Wild-type mice given JZL184 and Mgll^-/- mice were protected from hepatic I/R injury by a mechanism that involved increased endocannabinoid signaling via CB₂ and reduced production of eicosanoids in the liver. JZL184 suppressed the inflammation and oxidative stress that mediate hepatic I/R injury. Hepatocytes were the major source of hepatic MAGL activity and endocannabinoid and eicosanoid production. JZL184 also protected from induction of liver injury by D-(+)-galactosamine and lipopolysaccharides or CCl₄. Conclusions MAGL modulates hepatic injury via endocannabinoid and eicosanoid signaling; blockade of this pathway protects mice from liver injury. MAGL inhibitors might be developed to treat conditions that expose the liver to oxidative stress and inflammatory damage.      

4.1144           Secretory Leukocyte Protease Inhibitor Reverses Inhibition by CNS Myelin, Promotes Regeneration in the Optic Nerve, and Suppresses Expression of the Transforming Growth Factor-β Signaling Protein Smad2

Hannila, S.S., Siddiq, M.M., Carmel, J.B., Hou, J., Chaudhry, N., Bradley, P.M.J., Hillaire, M., Richman, E.L., Hart, R.P. and Filbin, M.T.

1. Neurosci., 33(12), 5138-5151 (2013)

  After CNS injury, axonal regeneration is limited by myelin-associated inhibitors; however, this can be overcome through elevation of intracellular cyclic AMP (cAMP), as occurs with conditioning lesions of the sciatic nerve. This study reports that expression of secretory leukocyte protease inhibitor (SLPI) is strongly upregulated in response to elevation of cAMP. We also show that SLPI can overcome inhibition by CNS myelin and significantly enhance regeneration of transected retinal ganglion cell axons in rats. Furthermore, regeneration of dorsal column axons does not occur after a conditioning lesion in SLPI null mutant mice, indicating that expression of SLPI is required for the conditioning lesion effect. Mechanistically, we demonstrate that SLPI localizes to the nuclei of neurons, binds to the Smad2 promoter, and reduces levels of Smad2 protein. Adenoviral overexpression of Smad2 also blocked SLPI-induced axonal regeneration. SLPI and Smad2 may therefore represent new targets for therapeutic intervention in CNS injury.  

4.1145           Regulation of ERα Protein Expression by 17β-Estradiol in Cultured Neurons of Hypothalamic Ventromedial Nucleus

Mallikov, V. and Madeira, M.D. Neurochem. Res., 38, 82-89 (2013)   The activation of the subtype α of estrogen receptors (ERα) in the hypothalamic ventromedial nucleus (VMNvl) is required to stimulate female sexual receptivity. Moreover, the hormone was found to govern the expression of the receptor. Its removal due to ovariectomy and subsequent substitution suggest that the hormone down-regulates the expression of ERα. In contrast, in normally cycling animals the expression of the receptor peaks at proestrus, the phase of highest concentration of 17β-estradiol in estrous cycle. Therefore, in this study we examined the influence of the hormone on ERα expression in primary dissociated cultures of neurons isolated from the VMNvl of young adult female rats. Measurements of ERα immunofluorescence revealed that both supraphysiological and physiological concentrations of 17β-estradiol increase the expression of ERα. Analyses with selective agonists showed that both nuclear ERs are able to mediate the action of the hormone. However, the activation of ERα had a stronger effect on the expression of its own receptor than the activation of ERβ. Simultaneous activation of both receptors attenuated the influence of ERα alone. Physiological concentrations of progesterone were found to revoke the effect of 17β-estradiol, whereas the expression of ERα is up-regulated by progesterone alone. These data indicate that the expression of ERα in VMNvl neurons is under the control of both types of nuclear ERs and, in addition, progesterone receptors (PRs). The particular contribution of the receptors is dependent on their level of expression and the hormonal context. In neurons expressing high quantity of ERα, ERβ attenuates the overall expression of the receptor, whereas in cells containing mostly ERβ it contributes to the up-regulation of ERα synthesis. Simultaneous activation of ERs and PRs reverses the influences of the receptors due to inter-inhibition of their transcriptional activities.  

4.1146           Reciprocal Induction Between α-Synuclein and β-Amyloid in Adult Rat Neurons

Majd, S., Chegini, F., Chataway, T., Zhou, X-F. and Gai, W. Neurotox. Res., 23, 69-78 (2013)   In spite of definite roles for β-amyloid (Aβ) in familial Alzheimer’s disease (AD), the cause of sporadic AD remains unknown. Amyloid senile plaques and Lewy body pathology frequently coexist in neocortical and hippocampal regions of AD and Parkinson’s diseases. However, the relationship between Aβ and α-synuclein (α-Syn), the principle components in the pathological structures, in neuronal toxicity and the mechanisms of their interaction are not well studied. As Aβ and α-Syn accumulate in aging patients, the biological functions and toxicity of these polypeptides in the aging brain may be different from those in young brain. We examined the neurotoxicity influences of Aβ1-42 or α-Syn on mature neurons and the effects of Aβ1-42 or α-Syn on the production of endogenous α-Syn or Aβ1-40 reciprocally using a model of culture enriched with primary neurons from the hippocampus of adult rats. Treatment of neurons with high concentrations of Aβ1-42 or α-Syn caused significant apoptosis of neurons. Following Aβ1-42 treatment at sub apoptotic concentrations, both intra- and extra-cellular α-Syn levels were significantly increased. Reciprocally, the non-toxic levels of α-Syn treatment also increased intra- and extra-cellular Aβ1-40 levels. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, suppressed α-Syn-induced Aβ1-40 elevation, as well as Aβ1-42-induced α-Syn elevation. Thus, high concentrations of Aβ1-42 and α-Syn exert toxic effects on mature neurons; however, non-toxic concentration treatment of these polypeptides induced the production of each other reciprocally with possible involvement of PI3K pathway.  

4.1147           Isolation, Characterization, and Transplantation of Adult Liver Progenitor Cells

Yovchev, M.I., Dabeva, M.D. and Oertel, M. Methods in Mol. Biol., 976, 37-51 (2013)   Many chronic liver diseases are life-threatening. When the liver loses the ability to repair itself the only treatment currently available is liver transplant. However, there are not enough donors to treat all the patients. This requires the search of alternative therapies utilizing stem and progenitor cells for treatment of these patients and restoration of their normal liver function. Hepatic progenitor cells can be isolated from livers at different developmental stages including adult liver. In the adult rat liver, there is clear evidence that progenitor cells (also called “oval cells”) derive from precursors in the canals of Herring that are capable to differentiate into hepatocytes and bile duct cells. In experimental models, hepatic progenitor cells can be isolated and propagated in vitro and used for restoration of the diseased liver. The first step in utilization of progenitor cells is their identification in the liver, isolation of purified progenitor cell fractions, which are subsequently transplanted in the diseased liver for evaluation of liver repopulation by transplanted cells, and evaluation their potentials for clinical application. The present protocol describes the isolation of non-parenchymal cells (NPCs) from wt DPPIV⁺ F344 rats, followed by purification of “oval cells”, immunohistochemical staining techniques to characterize these cells, their transplantation into retrorsine-treated mutant DPPIV⁻ rats, as well as the enzyme histochemical staining for DPPIV to detect transplanted cells in the host liver.  

4.1148           Isolation of Urothelial Cells from Bladder Tissue

Sangha, N. Methods in Mol. Biol., 1001, 21-33 (2013)   Presented below is a methodology for the isolation, expansion, and maintenance of urothelial cells derived from human bladder. Such bladder-derived urothelial cells, taken together with bladder or alternately sourced smooth muscle cells, may be complexed with an appropriately shaped biodegradable scaffold to create regenerative constructs capable of seeding formation of new bladder or bladder-like neo-organs upon implantation in human cystectomy patients.  

4.1149           Ex Vivo Culture and Separation of Functional Renal Cells

Bruce, A.T., Guthrie, K.I. and Kelley, R. Methods in Mol. Biol., 1001, 53-64 (2013)   The following methods outline the procedures for isolating primary renal cells from kidney tissue via enzymatic digestion, followed by their culture, harvest, and then fractionation of renal subpopulations from primary culture. The current methods describe procedures to sub-fractionate biologically active cells that have been used to treat and stabilize renal function in models of chronic kidney disease (Kelley et al. Am J Physiol Renal Physiol 299(5):F1026–F1039, 2010).  

4.1150           Formulation of Selected Renal Cells for Implantation into a Kidney

Halberstadt, C., Robbins, N., McCoy, D.W., Guthrie, K.I., Bruce, A.T., Knight, T.A. and Payne, R.G. Methods in Mol. Biol., 1001, 279-287 (2013)   Delivery of cells to organs has primarily relied on formulating the cells in a nonviscous liquid carrier. We have developed a methodology to isolate selected renal cells (SRC) that have provided functional stability to damaged kidneys in preclinical models (Kelley et al. Poster presentation at 71st scientific sessions of American diabetes association, 2011; Kelley et al. Oral presentation given at Tissue Engineering and Regenerative Medicine International Society (TERMIS)—North America annual conference, 2010; Presnell et al. Tissue Eng Part C Methods 17:261–273, 2011; Kelley et al. Am J Physiol Renal Physiol 299:F1026–F1039, 2010). In order to facilitate SRC injection into the kidney of patients who have chronic kidney disease, we have developed a strategy to immobilize the cells in a hydrogel matrix. This hydrogel (gelatin) supports cells by maintaining them in a three-dimensional state during storage and shipment (both at cold temperatures) while facilitating the delivery of cells by liquefying when engrafting into the kidney. This chapter will define a method for the formulation of the kidney epithelial cells within a hydrogel.  

4.1151           All-trans-retinoic acid ameliorates experimental allergic encephalomyelitis by affecting dendritic cell and monocyte development

Zhan, X-X., Liu, Y., Yang, J-F., Wang, G-Y., Mu, L., Zhang, T-S., Xie, X-L., Wang, J-H., Liu, Y-M., Kong, Q-F., Li, H-L. and Sun, B. Immunology, 138(4), 333-345 (2013)   Experimental allergic encephalomyelitis (EAE) can be induced in animal models by injecting the MOG_35–55 peptide subcutaneously. Dendritic cells (DCs) that are located at the immunization site phagocytose the MOG_35–55 peptide. These DCs mature and migrate into the nearest draining lymph nodes (dLNs), then present antigen, resulting in the activation of naive T cells. T helper type 1 (Th1) and Th17 cells are the primary cells involved in EAE progression. All-trans-retinoic acid (AT-RA) has been shown to have beneficial effects on EAE progression; however, whether AT-RA influences DC maturation or mediates other functions is unclear. In the present study, we showed that AT-RA led to the down-regulation of MHC class II, CD80 (B7-1) and CD86 (B7-2) expressed on the surface of DCs that were isolated from dLNs or spleen 3 days post-immunization in an EAE model. Changes to DC function influenced Th1/Th17 subset polarization. Furthermore, the number of CD44⁺ monocytes (which might trigger EAE progression) was also significantly decreased in dLNs, spleen, subarachnoid space and the spinal cord parenchyma after AT-RA treatment. These findings are the first to demonstrate that AT-RA impairs the antigen-presenting capacity of DCs, leading to down-regulation of pathogenic Th1 and Th17 inflammatory cell responses and reducing EAE severity.  

4.1152           Droplets as Reaction Compartments for Protein Nanotechnology

Devenish, S.R.A., Kaltenbach, M., Fishlechner, M. and Hollfelder, F. Methods in Mol. Biol., 996, 269-286 (2013)   Extreme miniaturization of biological and chemical reactions in pico- to nanoliter microdroplets is emerging as an experimental paradigm that enables more experiments to be carried out with much lower sample consumption, paving the way for high-throughput experiments. This review provides the protein scientist with an experimental framework for (a) formation of polydisperse droplets by emulsification or, alternatively, of monodisperse droplets using microfluidic devices; (b) construction of experimental rigs and microfluidic chips for this purpose; and (c) handling and analysis of droplets.  

4.1153           Platelets Recognize Brain-Specific Glycolipid Structures, Respond to Neurovascular Damage and Promote Neuroinflammation

Sotnikov, I., Veremeyko, T., Starossom, S.C., Barteneva, N., Weiner, H.L. and Ponomarev, E.D. PloS One, 8(3), e58979 (2013)   Platelets respond to vascular damage and contribute to inflammation, but their role in the neurodegenerative diseases is unknown. We found that the systemic administration of brain lipid rafts induced a massive platelet activation and degranulation resulting in a life-threatening anaphylactic-like response in mice. Platelets were engaged by the sialated glycosphingolipids (gangliosides) integrated in the rigid structures of astroglial and neuronal lipid rafts. The brain-abundant gangliosides GT1b and GQ1b were specifically recognized by the platelets and this recognition involved multiple receptors with P-selectin (CD62P) playing the central role. During the neuroinflammation, platelets accumulated in the central nervous system parenchyma, acquired an activated phenotype and secreted proinflammatory factors, thereby triggering immune response cascades. This study determines a new role of platelets which directly recognize a neuronal damage and communicate with the cells of the immune system in the pathogenesis of neurodegenerative diseases  

4.1154           Trolox contributes to Nrf2-mediated protection of human and murine primary alveolar type II cells from injury by cigarette smoke

Messier, E.M., Bahmed, K., Tuder, R.M., Chu, H.W., Bowler, R.P. and Kosmider, B. Cell Death and Disease, 4, e573 (2013)   Cigarette smoke (CS) is a main risk factor for chronic obstructive pulmonary disease (COPD). Oxidative stress induced by CS causes DNA and lung damage. Oxidant/antioxidant imbalance occurs in the distal air spaces of smokers and in patients with COPD. We studied the effect of oxidative stress generated by CS both in vivo and in vitro on murine primary alveolar type II (ATII) cells isolated from nuclear erythroid 2-related factor-2 (Nrf2)^−/− mice. We determined human primary ATII cell injury by CS in vitroand analyzed ATII cells isolated from smoker and non-smoker lung donorsex vivo. We also studied whether trolox (water-soluble derivative of vitamin E) could protect murine and human ATII cells against CS-induced DNA damage and/or decrease injury. We analyzed oxidative stress by 4-hydroxynonenal expression, reactive oxygen species (ROS) generation by Amplex Red Hydrogen Peroxide Assay, Nrf2, heme oxygenase 1, p53 and P53-binding protein 1 (53BP1) expression by immonoblotting, Nrf2 nuclear translocation, Nrf2 and p53 DNA-binding activities, apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and cytokine production by ELISA. We found that ATII cells isolated from Nrf2^−/−mice are more susceptible to CS-induced oxidative DNA damage mediated by p53/53BP1 both in vivo and in vitro compared with wild-type mice. Therefore, Nrf2 activation is a key factor to protect ATII cells against injury by CS. Moreover, trolox abolished human ATII cell injury and decreased DNA damage induced by CS in vitro. Furthermore, we found higher inflammation and p53 mRNA expression by RT-PCR in ATII cells isolated from smoker lung donors in comparison with non-smokers ex vivo. Our results indicate that the Nrf2 and p53 cross talk in ATII cells affect the susceptibility of these cells to injury by CS. Trolox can protect against oxidative stress, genotoxicity and inflammation induced by CS through ROS scavenging mechanism, and serve as a potential antioxidant prevention strategy against oxidative injury of ATII cells in CS-related lung diseases.  

4.1155           Preparation of Kupffer cell enriched non-parenchymal liver cells with high yield and reduced damage of surface markers by a modified method for flow cytometry

Xu, F., Zhen, P., Zheng, Y., Lljuan, F., Aiting, Y., Min, C., Hong, Y. and Jidong, J. Cell Biol. Int., 37, 284-291 (2013)   The aim of this study was to optimise a collagenase perfusion protocol for the isolation of a liver non-parenchymal cell (NPC) suspension enriched for Kupffer cells that reduced damage to F4/80 antigen cell surface expression to allow analysis by flow cytometry. Kupffer cell-enriched liver NPCs were isolated from C57BL/6 mice using different protocols. Flow cytometry was used to examine the effect of collagenase digestion on F4/80 expression on Kupffer cells, and results were represented by the percentage of F4/80 positive cells and by the F4/80 mean fluorescence intensity (MFI). The perfusion temperature, concentration of collagenase solution and total dosage of collagenase for liver perfusion influenced the effect of collagenase perfusion on the expression of F4/80 antigen on Kupffer cells. Collagenase perfusion at 28°C resulted in an increased percentage of F4/80 positive cells (P = 0.001) and MFI (P = 0.005) compared with 37°C. Perfusion with a total dose of 1.0 g/kg BW collagenase (using a 0.75 mg/mL solution) resulted in the highest percentage of F4/80 positive cells (P = 0.001) compared with 0.8 g/kg BW and 1.2 g/kg BW collagenase. Isolation of cells using the modified protocol resulted in a higher percentage of Kupffer cells (P < 0.001) and a higher MFI of F4/80 antigen (P < 0.001) compared with the common protocol.  

4.1156           Characterization of the Two Intra-Individual Sequence Variants in the 18S rRNA Gene in the Plant Parasitic Nematode, Rotylenchulus reniformis

Nyaku, S.T., Sripathi, V.R., Kantety, R.V., Gu, Y.Q., Lawrence, K. and Sharma, G.C. PloS One, 8(4), e60891(2013)   The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene.    

4.1157           Overexpression of human mutated G93A SOD1 changes dynamics of the ER mitochondria calcium cycle specifically in mouse embryonic motor neurons

Lautenschläger, J., Prell, T., Ruhmer, J., Weidemann, L., Witte, O.W. and Grosskreutz, J. Exp. Neurol., 247, 91-100 (2013)   Motor neurons vulnerable to the rapidly progressive deadly neurodegenerative disease amyotrophic lateral sclerosis (ALS) inherently express low amounts of calcium binding proteins (CaBP), likely to allow physiological motor neuron firing frequency modulation. At the same time motor neurons are susceptible to AMPA receptor mediated excitotoxicity and internal calcium deregulation which is not fully understood. We analysed ER mitochondria calcium cycle (ERMCC) dynamics with subsecond resolution in G93A hSOD1 overexpressing motor neurons as a model of ALS using fluorescent calcium imaging. When comparing vulnerable motor neurons and non-motor neurons from G93A hSOD1 mice and their non-transgenic littermates, we found a decelerated cytosolic calcium clearance in the presence of G93A hSOD1. While both non-transgenic as well as G93A hSOD1 motor neurons displayed large mitochondrial calcium uptake by the mitochondrial uniporter (mUP), the mitochondrial calcium extrusion system was altered in the presence of G93A hSOD1. In addition, ER calcium uptake by the sarco-/endoplasmic reticulum ATPase (SERCA) was increased in G93A hSOD1 motor neurons. In survival assays, blocking the mitochondrial sodium calcium exchanger (mNCE) by CGP37157 as well as inhibiting SERCA by cyclopiazonic acid showed protective effects against kainate induced excitotoxicity. Thus, our study shows for the first time that the functional consequence of G93A hSOD1 overexpression in intact motor neurons is indeed a disturbance of the ER mitochondria calcium cycle, and identified two promising targets for therapeutic intervention in the pathology of ALS.  

4.1158           Up-regulation of immunoglobulin G gene expression in the hippocampus of rats subjected to acute immobilization stress

Wang, S., Huang, G., Wang, Y., Huang, Y., Lin, S. and Gu, J.

1. Neuroimmunol.,258, 1-9 (2013)

  Immunoglobulin G (IgG) is thought to be produced by matured B lymphocytes, however, it was recently found to be synthesized in neurons of the brain, especially showing higher expression level in the hippocampus. To study the possible effects of IgG in the hippocampus, we examined IgG protein and mRNA expressions in rat hippocampal neurons with immunohistochemistry, immunofluorescence, in situ hybridization and laser microdissection-assisted RT-PCR. Increased IgG expressions at both protein and mRNA levels were detected in the hippocampus of an acute immobilization stress model of rat. No change was observed in the cortex or the thalamus. Furthermore, the microtubule-associated protein 2 (MAP2) and β III tubulin proteins did not show significant changes. Based on these findings, we hypothesize that hippocampal IgG may play a key role in adverse circumstances such as stress. The finding of increased IgG expression in the hippocampus following stress may also provide possibilities for developing antidepressant medication.  

4.1159           Histone Deacetylase Inhibitors (HDACis) That Release the Positive Transcription Elongation Factor b (P-TEFb) from Its Inhibitory Complex Also Activate HIV Transcription

Bartholomeeusen, K., Fujinaga, K., Xiang, Y. and Peterlin, B.M.

1. Biol. Chem., 288(20), 14400-14407 (2013)

  Numerous studies have looked at the effects of histone deacetylase inhibitors (HDACis) on HIV reactivation in established transformed cell lines and primary CD4⁺ T cells. However, their findings remain confusing, and differences between effects of class I- and class II-specific HDACis persist. Because no clear picture emerged, we decided to determine how HDACis reactivate HIV in transformed cell lines and primary cells. We found that neither histone H3 nor tubulin acetylation correlated with HIV reactivation in Jurkat and HeLa cells. Rather, HDACis that could reactivate HIV in chromatin or on episomal plasmids also released free positive transcription elongation factor b (P-TEFb) from its inhibitory 7SK snRNP. In resting primary CD4⁺ T cells, where levels of P-TEFb are vanishingly low, the most potent HDACi, suberoylanilide hydroxyamic acid (SAHA), had minimal effects. In contrast, when these cells were treated with a PKC agonist, bryostatin 1, which increased levels of P-TEFb, then SAHA once again reactivated HIV. We conclude that HDACis, which can reactivate HIV, work via the release of free P-TEFb from the 7SK snRNP.    

4.1160           Comparison of cryoprotective effects of iodixanol, trehalose and cysteamine on ram semen

Cirit, U., Bagis, H., Demir, K., Agca, C., Pabuccuoglu, S., Varisli, Ö., Clifford-Rathert, C. and Agca, Y. Animal Reprod. Science, 139, 38-44 (2013)   This study was conducted to improve cryosurvival of electroejaculated (EE) ram semen in the presence of iodixanol (OptiPrep™), trehalose or cysteamine. A tris-based extender was used to prepare 12 extenders containing OptiPrep™ (Op), trehalose (Tr) or cysteamine (Cy) alone, or different combinations of these compounds. Extenders were designated as follows: Tris (control), Op1.25 (1.25% Op, v/v), Op2.5 (2.5% Op, v/v), Op5 (5% Op, v/v), Tr50 (50 mM Tr), Tr100 (100 mM Tr), Cy (5 mM Cy), OpTr (2.5% Op and 100 mM Tr), OpCy (2.5% Op and 5 mM Cy), TrCy (100 mM Tr and 5 mM Cy), OpTrCy1 (2.5% Op, 100 mM Tr and 5 mM Cy) and OpTrCy2 (1.25% Op, 50 mM Tr and 2.5 mM Cy). A two-step dilution was used and glycerol was added at 5 °C in the second step. Diluted samples were equilibrated for 1 h, loaded in 0.25 mL straws and frozen in a programmable freezing machine. Supplementation of 5% OptiPrep™ significantly protected post-thaw progressive motility, membrane integrity, acrosomal integrity and morphological damages. Trehalose supplementation protected membrane integrity of ram sperm; however, it did not help post-thaw motility and morphology. Supplementation of 5 mM cysteamine had detrimental effect on cryosurvival of EE ram semen. These results demonstrate that the supplementation of iodixanol increases the cryosurvival of EE ram semen in a dose-dependent manner.  

4.1161           Nilotinib induces apoptosis and autophagic cell death of activated hepatic stellate cells via inhibition of histone deacetylases

Shaker, M.E., Ghani, A., Shiha, G.E., Ibrahim, T.M. and Mehal, W.Z. Biochim. Biophys. Acta, 1833, 1992-2003 (2013)   Increasing hepatic stellate cell (HSC) death is a very attractive approach for limiting liver fibrosis. Tyrosine kinase inhibitors have been shown to have anti-fibrotic properties, but the mechanisms are poorly understood. Here, we identified the mechanism of action of the second-generation tyrosine kinase inhibitor nilotinib in inducing HSC death. Human HSC line (LX-2) and rat HSCs were treated with nilotinib and its predecessor, imatinib, in the absence or presence of various blockers, known to interfere with death signaling pathways. Nilotinib, but not imatinib, induced progressive cell death of activated, but not quiescent, HSCs in a dose-dependent manner. Activated HSCs died through apoptosis, as denoted by increased DNA fragmentation and caspase activation, and through autophagy, as indicated by the accumulation of autophagic markers, light chain (LC)3A-II and LC3B-II. Although inhibition of caspases with Z-VAD-FMK suppressed nilotinib-induced HSCs' apoptosis, there was no increase in HSCs' survival, because autophagy was exacerbated. However, blocking the mitochondrial permeability transition pore (mPTP) opening with cyclosporin A completely abolished both apoptosis and autophagy due to nilotinib. Moreover, nilotinib treatment decreased the protein expression of histone deacetylases 1, 2 and 4. Interestingly, pretreament with C646, a selective p300/CBP histone acetyl transferase inhibitor, resulted in diverting nilotinib-induced apoptosis and autophagy towards necrosis. In conclusion, the identification of mPTP as a target of nilotinib in activated HSCs suggests coordination with histone deacetylases inhibition to induce apoptosis and autophagy. Thus, our study provides novel insights into the anti-fibrotic effects of nilotinib.  

4.1162           Aging Exacerbates Microvascular Endothelial Damage Induced by Circulating Factors Present in the Serum of Septic Patients

Tucsek, Z., Gautam, T., Sonntag, W.E., Toth, P., Saito, H., Salomao, R., Szabo, C., Csiszar, A. and Ungvari, Z.

1. Geronol. A Biol. Sci. Med. Sci., 68(6), 652-660 (2013)

  The elderly patients show a significantly elevated mortality rate during sepsis than younger patients, due to their higher propensity to microvascular dysfunction and consequential multiorgan failure. We tested whether aging renders vascular endothelial cells more susceptible to damage induced by inflammatory factors present in the circulation during sepsis. Primary microvascular endothelial cells derived from young (3 months) and aged (24 months) Fischer 344 × Brown Norway rats were treated with sera obtained from sepsis patients and healthy controls. Oxidative stress (MitoSox fluorescence), death receptor activation (caspase 8 activity), and apoptotic cell death (caspase 3 activity) induced by treatment with septic sera were exacerbated in aged endothelial cells as compared with responses obtained in young cells. Induction of heme oxygenase-1 and thrombomodulin in response to treatment with septic sera was impaired in aged endothelial cells. Treatment with septic sera elicited greater increases in tumor necrosis factor-α expression in aged endothelial cells, as compared with young cells, whereas induction of inducible nitric oxide synthase, intercellular adhesion molecule-1, and vascular cell adhesion molecule did not differ between the two groups. Collectively, aging increases sensitivity of microvascular endothelial cells (MVECs) to oxidative stress and cellular damage induced by inflammatory factors present in the circulation during septicemia. We hypothesize that these responses may contribute to the increased vulnerability of elderly patients to multiorgan failure associated with sepsis.  

4.1163           Treatment with the cytochrome P450 ω-hydroxylase inhibitor HET0016 attenuates cerebrovascular inflammation, oxidative stress and improves vasomotor function in spontaneously hypertensive rats

Toth, P., Csiszar, A., Sosnowska, D., Tucsek, Z., Cseplo, P., Springo, Z., Tarantini, S., Sonntag, W.E., Ungvari, Z. and Koller, A. Br. J. Pharmacol., 168(8), 1878-1888 (2013)   Background and Purpose Hypertension increases cerebrovascular oxidative stress and inflammation and impairs vasomotor function. These pathological alterations lead to dysregulation of cerebral blood flow and exacerbate atherogenesis, increasing the morbidity of ischaemic cerebrovascular diseases and promoting vascular cognitive impairment. We aimed to test the hypothesis that increased production of the arachidonic acid metabolite 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) contributes to hypertension-induced cerebrovascular alterations. Experimental Approach We treated male spontaneously hypertensive rats (SHR) with HET0016 (N-hydroxy-N′-(4-butyl-2-methylphenyl)-formamidine), an inhibitor of 20-HETE synthesis. In middle cerebral arteries (MCAs) of SHRs, we focused on vasomotor responses and end points that are highly relevant for cellular reactive oxygen species (ROS) production, inflammatory cytokine expression and NF-κB activation. Key Results SHRs treated with HET0016 remained hypertensive (SHR + HET0016: 149 ± 8 mmHg, Wistar-Kyoto rat: 115 ± 4 mmHg; P < 0.05.), although their systolic blood pressure was decreased compared to untreated SHRs (191 ± 6 mmHg). In MCAs of SHRs, flow-induced constriction was increased, whereas ACh- and ATP-induced dilations were impaired. This functional impairment was reversed by treatment with HET0016. Treatment with HET0016 also significantly decreased oxidative stress in MCAs of SHRs (as shown by dihydroethidium staining and analysis of vascular 5-nitrotyrosine, 4-hydroxynonenal and carbonyl content) and inhibited cerebrovascular inflammation (shown by the reduced mRNA expression of TNF , IL-1β and IL-6). Treatment of SHRs with HET0016 also attenuated vascular NF-κB activation. In vitro treatment with 20-HETE significantly increased vascular production of ROS and promoted NF-κB activation in cultured cerebromicrovascular endothelial cells. Conclusions and Implications Taken together, treatment with HET0016 confers anti-oxidative and anti-inflammatory effects in the cerebral arteries of SHRs by disrupting 20-HETE-mediated autocrine/paracrine signalling pathways in the vascular wall. It is likely that HET0016-induced decreases in blood pressure also potentiate the cerebrovascular protective effects of the drug.  

4.1164           Trypomastigotes and amastigotes of Trypanosoma cruzi induce apoptosis and STAT3 activation in cardiomyocytes in vitro

Stahl, P., Ruppert, V., Meyer, T., Schmidt, J., Campos, M.A., Gazzinelli, R.T., Maisch, B., Schwarz, R.T. and Debierre-Grockiego, F. Apoptosis, 18(6), 653-663 (2013)   The haemoflagellate Trypanosoma cruzi is the causative agent of Chagas’ disease that occurs in approximately 8 million people in Latin America. Patients infected with T. cruzi frequently suffer of cardiomegaly and may die of myocardial failure. Here we show that T. cruzi trypomastigotes (extracellular form) increased in vitro apoptosis of rat cardiomyocytes. Additionally, we demonstrated that amastigotes (intracellular form), for which a method for purification was established, were also able to induce cardiomyocyte apoptosis. Increase of apoptosis was associated with up-regulation of the apoptotic gene bax by trypomastigotes, while expression of the anti-apoptotic gene bcl-2 was down-regulated by amastigotes. The transcription factor STAT3 but not STAT1 was activated in cardiomyocytes by trypomastigotes. In addition, tlr7 gene expression was up-regulated in cardiomyocytes incubated with trypomastigotes, suggesting that this Toll-like receptor is involved in the intracellular recognition after host cell invasion by T. cruzi. Glycosylphosphatidylinositols purified from trypomastigotes did not induce cardiomyocyte apoptosis and STAT activation but down-regulated tlr7 gene expression. In conclusion, cardiomyopathy observed in Chagas’ disease might be in part due to apoptosis of cardiomyocytes induced directly by the parasite.  

4.1165           Antifibrotic Effects of a Recombinant Adeno-Associated Virus Carrying Small Interfering RNA Targeting TIMP-1 in Rat Liver Fibrosis

Cong, M., Liu, T., Wang, P., Fan, X., yang, A., Bai, Y., Peng, Z., Wu, P., Tong, X., Chen, J., Li, H., Cong, R., Tang, S., Wang, B., Jia, J. and You, H. Am. J. Pathol., 182(5), 1607-1616 (2013)   Elevated tissue inhibitor of metalloproteinase 1 (TIMP-1) expression contributes to excess production of extracellular matrix in liver fibrosis. Herein, we constructed a recombinant adeno-associated virus (rAAV) carrying siRNA of the TIMP-1 gene (rAAV/siRNA–TIMP-1) and investigated its effects on liver fibrosis in rats. Two models of rat liver fibrosis, the carbon tetrachloride and bile duct ligation models, were treated with rAAV/siRNA–TIMP-1. In the carbon tetrachloride model, rAAV/siRNA–TIMP-1 administration attenuated fibrosis severity, as determined by histologic analysis of hepatic collagen accumulation, hydroxyproline content, and concentrations of types I and III collagen in livers and sera. Levels of mRNA and active matrix metalloproteinase (MMP) 13 were elevated, whereas levels of mRNA and active MMP-2 were decreased. Moreover, a marked decrease was noted in the expression of α-smooth muscle actin, a biomarker of activated hepatic stellate cells (HSCs), and transforming growth factor-β1, critical for the development of liver fibrosis. Similarly, rAAV/siRNA–TIMP-1 treatment significantly alleviated bile duct ligation–induced liver fibrosis. Furthermore, this treatment dramatically suppressed TIMP-1 expression in HSCs from both model rats. These data indicate that the administration of rAAV/siRNA–TIMP-1 attenuated liver fibrosis by directly elevating the function of MMP-13 and diminishing activated HSCs. It also resulted in indirect decreased expression of type I collagen, MMP-2, and transforming growth factor-β1. In conclusion, rAAV/siRNA–TIMP-1 may be an effective antifibrotic gene therapy agent.  

4.1166           FACS Array Profiling Identifies Ecto-5′ Nucleotidase as a Striatopallidal Neuron-Specific Gene Involved in Striatal-Dependent Learning

Ena, S.L., De Backer, J-F., Schiffmann, S.N., and de Kerchove d’Exaerde, A.

1. Neurosci., 33(20), 8794-8809 (2013)

  The striatopallidal (STP) and striatonigral (STN) neurons constitute the main neuronal populations of the striatum. Despite the increasing knowledge concerning their involvement in multiple tasks associated with the striatum, it is still challenging to understand the precise differential functions of these two neuronal populations and to identify and study new genes involved in these functions. Here, we describe a reliable approach, applied on adult mouse brain, to generate specific STP and STN neuron gene profiles. STP and STN neurons were identified in the same animal using the transgenic Adora2A-Cre × Z/EG mouse model combined with retrograde labeling, respectively. Gene profiling was generated from FACS-purified neurons leading to the identification of new STP and STN neuron-specific genes. Knock-down models based on Cre-dependent lentiviral vector were developed to investigate their function either in striatal or in STP neurons. Thereby, we demonstrate that ecto-5′-nucleotidase (NT5e) is specifically expressed in STP neurons and is at the origin of most of the extracellular adenosine produced in the striatum. Behavioral analysis of striatal and STP neuron knock-down mouse models as well as NT5e knock-out mice demonstrates the implication of this STP neuron enzyme in motor learning.  

4.1167           Role of Fatty-Acid Synthesis in Dendritic Cell Generation and Function

Rehman, A., Hemmert, K.C., Ochi, A., Jamal, M., Henning, J.R., Barilla, R., Quesada, J.P., Zambirinis, C.P., Tang, K., Ego-Osuala, M., Rao, R.S., Greco, S., Deutsch, M., Narayan, S., Pachter, H.L., Graffeo, C.S., Acehan, D. and Miller, G.

1. Immunol., 190(9), 4640-4649 (2013)

  Dendritic cells (DC) are professional APCs that regulate innate and adaptive immunity. The role of fatty-acid synthesis in DC development and function is uncertain. We found that blockade of fatty-acid synthesis markedly decreases dendropoiesis in the liver and in primary and secondary lymphoid organs in mice. Human DC development from PBMC precursors was also diminished by blockade of fatty-acid synthesis. This was associated with higher rates of apoptosis in precursor cells and increased expression of cleaved caspase-3 and BCL-xL and downregulation of cyclin B1. Further, blockade of fatty-acid synthesis decreased DC expression of MHC class II, ICAM-1, B7-1, and B7-2 but increased their production of selected proinflammatory cytokines including IL-12 and MCP-1. Accordingly, inhibition of fatty-acid synthesis enhanced DC capacity to activate allogeneic as well as Ag-restricted CD4⁺ and CD8⁺ T cells and induce CTL responses. Further, blockade of fatty-acid synthesis increased DC expression of Notch ligands and enhanced their ability to activate NK cell immune phenotype and IFN-γ production. Because endoplasmic reticulum (ER) stress can augment the immunogenic function of APC, we postulated that this may account for the higher DC immunogenicity. We found that inhibition of fatty-acid synthesis resulted in elevated expression of numerous markers of ER stress in humans and mice and was associated with increased MAPK and Akt signaling. Further, lowering ER stress by 4-phenylbutyrate mitigated the enhanced immune stimulation associated with fatty-acid synthesis blockade. Our findings elucidate the role of fatty-acid synthesis in DC development and function and have implications to the design of DC vaccines for immunotherapy.  

4.1168           Single-cell analysis and sorting using droplet-based microfluidics

Mazutis, L., Gilbert, J., Ung, W.L., Weitz, D.A., Griffiths, A.D. and Heyman, J.A. Nature Protocols, 8(5), 870-891 (2013)   We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2–6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5–7 d.  

4.1169           Role of host cell traversal by the malaria sporozoite during liver infection

Tavares, J., Formaglio, P., Thiberge, S., Mordelet, E., Van Rooijen, N., Medvinsky, A., Menard, R. and Amino, R.

1. Exp. Med., 210(5), 905-915 (2013)

  Malaria infection starts when the sporozoite stage of the Plasmodium parasite is injected into the skin by a mosquito. Sporozoites are known to traverse host cells before finally invading a hepatocyte and multiplying into erythrocyte-infecting forms, but how sporozoites reach hepatocytes in the liver and the role of host cell traversal (CT) remain unclear. We report the first quantitative imaging study of sporozoite liver infection in rodents. We show that sporozoites can cross the liver sinusoidal barrier by multiple mechanisms, targeting Kupffer cells (KC) or endothelial cells and associated or not with the parasite CT activity. We also show that the primary role of CT is to inhibit sporozoite clearance by KC during locomotion inside the sinusoid lumen, before crossing the barrier. By being involved in multiple steps of the sporozoite journey from the skin to the final hepatocyte, the parasite proteins mediating host CT emerge as ideal antibody targets for vaccination against the parasite.  

4.1170           Protoplast isolation optimization and regeneration of cell wall in Gracilaria gracilis (Gracilariales, Rhodophyta)

Huddy, S.M., Meyers, A.E. and Coyne, V.E.

1. Appl. Phycol., 25(2), 433-443 (2013)

  This paper reports the first successful isolation and cell wall regeneration of Gracilaria gracilis (Stackhouse) Steentoft, Irvine et Farnham protoplasts. These results form an important foundation for the development of a successful tissue culture system for G. gracilis. Initially, an isolation protocol was optimized by investigation of the effects of the enzyme constituents and concentrations, the pre-treatment of thalli, the incubation period and temperature, and the pH of the enzymatic medium on protoplast yields. A pre-treatment of G. gracilis thalli with 1 % (w/v) papain for 30 min followed by a 3-h enzymatic digestion of thalli with an enzymatic mixture containing 2 % (w/v) cellulase Onozuka R-10, 1 % (w/v) macerozyme R-10, and 10 U mL−1 agarase at pH 6.15 was found to produce the highest yield of protoplasts at 22 °C. Reliably high yields (20–30 × 105 protoplasts g−1 f.wt) of protoplasts could be obtained from G. gracilis thalli when this optimized protocol was used. Cell wall re-synthesis by G. gracilis protoplasts, which constitutes the first step towards whole plant regeneration, was followed using calcoflour staining and scanning electron microscopy. Protoplasts were shown to complete the initial stages of cell wall re-synthesis within the first 24 h of culturing.  

4.1171           Toll Like Receptor 3 Plays a Critical Role in the Progression and Severity of Acetaminophen-Induced Hepatotoxicity

Cavassani, K.A., Moreira, A.P., Habiel, D., Ito, T., Coelho, A.L., Allen, R.M., Hu, B., Raphelson, J., Carson IV, W.F., Schaller, M.A., Lukacs, N.W., Omary, M.B., Hogaboam, C.M. and Kunkel, S.L: PloS One, 8(6), e65899 (2013)   Toll-like receptor (TLR) activation has been implicated in acetaminophen (APAP)-induced hepatotoxicity. Herein, we hypothesize that TLR3 activation significantly contributed to APAP-induced liver injury. In fasted wildtype (WT) mice, APAP caused significant cellular necrosis, edema, and inflammation in the liver, and the de novo expression and activation of TLR3 was found to be necessary for APAP-induced liver failure. Specifically, liver tissues from similarly fasted TLR3-deficient (tlr3^−/−) mice exhibited significantly less histological and biochemical evidence of injury after APAP challenge. Similar protective effects were observed in WT mice in which TLR3 was targeted through immunoneutralization at 3 h post-APAP challenge. Among three important death ligands (i.e. TNFα, TRAIL, and FASL) known to promote hepatocyte death after APAP challenge, TNFα was the only ligand that was significantly reduced in APAP-challenged tlr3^−/− mice compared with APAP-challenged WT controls. In vivo studies demonstrated that TLR3 activation contributed to TNFα production in the liver presumably via F4/80⁺ and CD11c⁺ immune cells. In vitro studies indicated that there was cooperation between TNFα and TLR3 in the activation of JNK signaling in isolated and cultured liver epithelial cells (i.e. nMuLi). Moreover, TLR3 activation enhanced the expression of phosphorylated JNK in APAP injured livers. Thus, the current study demonstrates that TLR3 activation contributes to APAP-induced hepatotoxicity.  

4.1172           Efficient gene expression from integration-deficient lentiviral vectors in the spinal cord

Peluffo, H., Foster, E., Ahmed, S.G., lago, N., Hutson, T.H., Moon, L., Wanisch, K., Caraballo-miralles, V., Olmos, G., Llado, J., McMahon, S.B. and Yanez-Munoz, R.J. Gene Therapy, 20, 645-657 (2013)   Gene transfer to spinal cord cells may be crucial for therapy in spinal muscular atrophy, amyotrophic lateral sclerosis and spinal cord injury. Lentiviral vectors are efficient for transduction of a variety of cells, but like all integrating vectors they pose a risk of insertional mutagenesis. Integration-deficient lentiviral vectors (IDLVs) remain episomal but retain the transduction efficiency of standard integrating lentiviral vectors, particularly when the episomes are not diluted out through repeated cell division. We have now applied IDLVs for transduction of spinal cord in vitro, in explants and in vivo. Our results demonstrate similar efficiency of eGFP expression from integrating lentiviral vectors and IDLVs in most cell types analyzed, including motor neurons, interneurons, dorsal root ganglia (DRG) neurons and astroglia. IDLV-mediated expression of pro-glial-cell-derived neurotrophic factor (Gdnf) rescues motor neuron cultures from death caused by removal of exogenous trophic support. IDLVs also mediate efficient RNA interference in DRG neuron cultures. After intraparenchymal injection in the rat and mouse cervical and lumbar regions in vivo, transduction is mainly neuronal, with both motor neurons and interneurons being efficiently targeted. These results suggest that IDLVs could be efficient and safer tools for spinal cord transduction in future therapeutic strategies.  

4.1173           Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases

Meng, W., Pan, W., Zhang, A.J.X., Li, Z., Wei, G., Feng, L., Dong, Z., Li, C., Hu, X., Sun, C., Luo, Q., Yuen, K-Y., Zhong, N. and Chen, L: PloS One, 8(6), e66278 (2013)   Background The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs). Methodology/Principal Findings The genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA. Conclusions/Significance Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases.  

4.1174           Separation of Penaeus vannamei haemocyte subpopulations by iodixanol density gradient centrifugation

Dantas-Lima, J.J., Tuan, V.V., Corteel, M., Grauwet, K., An, N.T.T., Sorgeloos, P. and Nauwynck, H.J. Aquaculture, 408-409, 128-135 (2013)   Methodologies for separation of immune cell subpopulations are essential tools in immunology studies. Up to date, only one methodology for separating crustacean haemocyte subpopulations using Percoll density gradient centrifugation has been described. In the present work, a new methodology to separate Penaeus vannamei haemocyte subpopulations was developed, using a two-step iodixanol density gradient centrifugation. P. vannamei haemolymph was collected with anticoagulant and centrifuged through a first gradient (densities from 1.063 to 1.109 g/ml) for 10 min at 2000 g. Three bands were formed: two bands with lower density close together, and a third band with higher density. The first two were collected together whilst the third band was collected separately. The volume fraction in-between these bands contained dispersed cells and was also collected. The suspension containing the mixture of the first two bands was centrifuged through a second gradient (densities from 1.047 to1.087 g/ml) for 15 min at 2000 g. Two bands were formed and collected individually. All the cell suspensions were used for in vitro culture (cell survival evaluation) and for evaluation of cell morphology by flow cytometry and light microscopy. Each of the three bands contained a major cell type with distinct morphology and behaviour. The dispersed cell fraction contained a mixture of two different cell types, which were distinct from the cell types in the bands. By order of appearance from the top of the gradient, the cell types were named: subpopulations (Sub) 1 (band 1), Sub 2 (band 2), Sub 3 + 4 (dispersed cells) and Sub 5 (band 3). The purity level (percentage of the major cell type) of Sub 1, 2 and 5 was 95.0 ± 1.0%, 97.7 ± 1.2% and 99.4 ± 0.8%, respectively. Cells of Sub 2 showed the best survival time in vitro (up to 96 h) followed by cells from Sub 1, Sub 3 + 4 and Sub 5. Phagocytic activity was detected in Sub 1 and 4. This methodology allowed the separation and characterization of five morphologically distinct and physiologically active P. vannamei haemocyte subpopulations, from which three were isolated with a very high degree of purity. Therefore, we consider this methodology a valuable alternative for the traditional crustacean haemocyte separation procedure in Percoll.  

4.1175           Microfluidic primary culture model of the lower motor neuron–neuromuscular junction circuit

Southan, K.A., King, A.E., Blizzard, C.A., McCormack, G.H. and Dickson, T.C.

1. Neurosci. Methods, 218, 164-169 (2013)

  Modelling the complex process of neuromuscular signalling is key to understanding not only normal circuit function but also importantly the mechanisms underpinning a range of degenerative diseases. We describe a novel in vitro model of the lower motor neuron–neuromuscular junction circuit, incorporating primary spinal motor neurons, supporting glia and skeletal muscle. This culture model is designed to spatially mimic the unique anatomical and cellular interactions of this circuit in compartmented microfluidic devices, such that the glial cells are located with motor neuron cell bodies in the cell body chamber and motor neuron axons extend to a distal chamber containing skeletal muscle cells whilst simultaneously allowing targeted intervention. This model is suitable for use in conjunction with a range of downstream experimental approaches and could also be modified to utilise other cellular sources including appropriate immortal cell lines, cells derived from transgenic models of disease and also patient derived stem cells.  

4.1176           Examination of MARCO Activity on Dendritic Cell Phenotype and Function Using a Gene Knockout Mouse

Komine, H., Kuhn, L., Matsushita, N., Mule, J.J. and Pilon-Thomas, S. PloS One, 8(7), e67795 (2013)   We have reported the upregulation of MARCO, a member of the class A scavenger receptor family, on the surface of murine and human dendritic cells (DCs) pulsed with tumor lysates. Exposure of murine tumor lysate-pulsed DCs to an anti-MARCO antibody led to loss of dendritic-like processes and enhanced migratory capacity. In this study, we have further examined the biological and therapeutic implications of MARCO expression by DCs. DCs generated from the bone marrow (bm) of MARCO knockout (MARCO^-/-) mice were phenotypically similar to DCs generated from the bm of wild-type mice and produced normal levels of IL-12 and TNF-α when exposed to LPS. MARCO^-/- DCs demonstrated enhanced migratory capacity in response to CCL-21 in vitro. After subcutaneous injection into mice, MARCO^-/- TP-DCs migrated more efficiently to the draining lymph node leading to enhanced generation of tumor-specific IFN-γ producing T cells and improved tumor regression and survival in B16 melanoma-bearing mice. These results support targeting MARCO on the surface of DCs to improve trafficking and induction of anti-tumor immunity.  

4.1177           Low Immunogenicity of Neural Progenitor Cells Differentiated from Induced Pluripotent Stem Cells Derived from Less Immunogenic Somatic Cells

Liu, P., Chen, S., Li, X., Qin, L., Huang, K., Wang, L., Huang, W., Li, S., Jia, B., Zhong, M., Pan, G., Cai, J. and Pei, D. PloS One, 8(7), e69617 (2013)   The groundbreaking discovery of induced pluripotent stem cells (iPS cells) provides a new source for cell therapy. However, whether the iPS derived functional lineages from different cell origins have different immunogenicity remains unknown. It had been known that the cells isolated from extra-embryonic tissues, such as umbilical cord mesenchymal cells (UMCs), are less immunogenic than other adult lineages such as skin fibroblasts (SFs). In this report, we differentiated iPS cells from human UMCs and SFs into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with allologous peripheral blood mononuclear cells (PBMCs), we showed that UMCs were indeed less immunogenic than skin cells to simulate proliferation of PBMCs. Surprisingly, we found that the NPCs differentiated from UMC-iPS cells retained low immunogenicity as the parental UMCs based on the PBMC proliferation assay. In cytotoxic expression assay, reactions in most kinds of immune effector cells showed more perforin and granzyme B expression with SF-NPCs stimulation than that with UMC-NPCs stimulation in PBMC co-culture system, in T cell co-culture system as well. Furthermore, through whole genome expression microarray analysis, we showed that over 70 immune genes, including all members of HLA-I, were expressed at lower levels in NPCs derived from UMC-iPS cells than that from SF-iPS cells. Our results demonstrated a phenomenon that the low immunogenicity of the less immunogenic cells could be retained after cell reprogramming and further differentiation, thus provide a new concept to generate functional lineages with lower immunogenicity for regenerative medicine.  

4.1178           Alternative Immunomodulatory Strategies for Xenotransplantation: CD80/CD86-CTLA4 Pathway-Modified Immature Dendritic Cells Promote Xenograft Survival

Tian, M., Lv, Y., Zhai, C., Zhu, H., Yu, L. and Wang, B. PloS One, 8(7), e69640 (2013)   Background Xenotransplantation is a promising approach to circumventing the current organ shortage. However, T-cell-dependent anti-xenoresponses are a major challenge to successful xenografts. Given the advantages of the use of CTLA4-Ig in the survival of allografts, the purpose of the study was to investigate the therapeutic potential of CTLA4-IgG4 modified immature dendritic cells (imDCs) in the prevention of islets xenograft rejection. Methods CTLA4-IgG4 was constructed by the fusion of the extracellular regions of porcine CTLA4 to human the hIgG4 Fc region. The imDCs were induced and cultured from porcine peripheral blood mononuclear cells (PBMC). The CTLA4-IgG4 modified imDCs were delivered via the portal vein to the liver of diabetic mice (insulin-dependent diabetes mellitus) before islet xenografting, and mCTLA4-Ig was administered intravenously after xenotransplantation. Results The xenograft survival of mice receiving unmodified imDCs was approximately 30 days. However, following administration of CTLA4-IgG4 modified imDCs before grafting and mCTLA4-Ig after grafting, xenografts survived for more than 100 days. Flow cytometric analysis showed that the CD4⁺CD25⁺Foxp3⁺ Treg population was increased in spleens. The efficacy of donor CTLA4-IgG4 modified imDCs correlated partially with the amplification of Tregs. Conclusions These results confirm that selective inhibition of the direct and indirect pathways of T-cell activation by donor CTLA4-IgG4 modified imDCs and receptor CTLA4-Ig is a highly effective strategy to promote survival of xenografts.  

4.1179           High-quality RNA extraction from rat pancreatic islet

Kiba, T., Tanemura, M. and Yagyu, K. Cell Biol. Int. Reports, 20(1), 1-4 (2013)   In recent years, increasing interest surrounding islet replacement therapies in human has provided the drive for advances in the methods used to isolate from humans as well as a host of animal research models. However, there has been no reports describing a technique that reliably improves the quality of RNA extracted from rat pancreatic islet. Male Sprague–Dawley rats, 10- to 12-week-old, were housed in a certified animal care facility. The rats were underwent bile duct cannulation with pancreatic inflation after clamping the distal common bile duct. The pancreas was excised and digested with ETK/Liberase TL solution at 37°C for 30 min without shaking. Cold ETK was added to stop digestion. The islets were purified by discontinuous iodixanol density gradients of 25, 23, 20 and 11% in a modified ETK/OptiPrep® solution. After a 15 min centrifugation at 1,000g, islets were collected from the interface between the 20 and 11% layer. Immediately after purification, islets were used for RNA extraction. RNA was extracted from isolated rat pancreatic islet cells, using the commercially available kit. In the present study, we have described a technique that reliably improves the quality of RNA extracted from rat pancreatic islet using the perfusion technique in the bile duct. The islet cells that we isolated using this technique were suitable for high quality RNA extraction.  

4.1180           Ultra-pure platelet isolation from canine whole blood

Trichler, S.A., Bulla, S.C., Thomason, J., Lunsford, K.V. and Bulla, C. BMC Vet. Res., 9:144 (2013)   Background Several research applications involving platelets, such as proteomic and transcriptomic analysis, require samples with very low numbers of contaminating leukocytes, which have considerably higher RNA and protein content than platelets. We sought to develop a platelet purification protocol that would minimize contamination, involve minimal centrifugation steps, and yield highly pure platelet samples derived from low volume whole blood samples from healthy dogs. Results Using an optimized OptiPrep density gradient technique, platelet recovery was 51.56% with 99.99% platelet purity and leukocyte contamination of 100 leukocytes per 10⁸ platelets, on average. Platelet samples were subjected to additional purification with CD45-labeled Dynabeads after density barrier centrifugation resulting in a 95-fold depletion of residual leukocytes. Platelets purified using these methods remained inactivated as assessed by Annexin V and P-selectin labeling with flow cytometry. Conclusions The use of OptiPrep density gradient is a quick method for obtaining highly purified platelet samples from low volumes of canine whole blood with minimal contamination. Additional depletion of residual leukocytes can be achieved using CD45-labeled beads. These platelet samples can then be used for many downstream applications that require ultra-pure platelet samples such as RNA and protein analysis.  

4.1181           Dendritic Cell Subtypes from Lymph Nodes and Blood Show Contrasted Gene Expression Programs upon Bluetongue Virus Infection

Ruscanu, S., Jouneau, L., Urien, C., Bourge, M., Lecardonnel, J., Moroldo, M., Loup, B., Dalod, M., Elhmouzi-Younes, J., Bevilacqua, C. and Schwartz-Cornil, I.

1. Virol., 87(16), 9333-9343 (2013)

  Human and animal hemorrhagic viruses initially target dendritic cells (DCs). It has been proposed, but not documented, that both plasmacytoid DCs (pDCs) and conventional DCs (cDCs) may participate in the cytokine storm encountered in these infections. In order to evaluate the contribution of DCs in hemorrhagic virus pathogenesis, we performed a genome-wide expression analysis during infection by Bluetongue virus (BTV), a double-stranded RNA virus that induces hemorrhagic fever in sheep and initially infects cDCs. Both pDCs and cDCs accumulated in regional lymph nodes and spleen during BTV infection. The gene response profiles were performed at the onset of the disease and markedly differed with the DC subtypes and their lymphoid organ location. An integrative knowledge-based analysis revealed that blood pDCs displayed a gene signature related to activation of systemic inflammation and permeability of vasculature. In contrast, the gene profile of pDCs and cDCs in lymph nodes was oriented to inhibition of inflammation, whereas spleen cDCs did not show a clear functional orientation. These analyses indicate that tissue location and DC subtype affect the functional gene expression program induced by BTV and suggest the involvement of blood pDCs in the inflammation and plasma leakage/hemorrhage during BTV infection in the real natural host of the virus. These findings open the avenue to target DCs for therapeutic interventions in viral hemorrhagic diseases.  

4.1182           Aging-Induced Dysregulation of Dicer1-Dependent MicroRNA Expression Impairs Angiogenic Capacity of Rat Cerebromicrovascular Endothelial Cells

Ungvari, Z., Tucsek, Z., Sosnowska, D., Toth, P., Gautam, T., Podlutsky, A., Csiszar, A., Losonczy, G., Valcarcel-Ares, M.N., Sonntag, W.E. and Csiszar, A.

1. Gerontol. A Biol. Sci. Med. Sci., 68(8), 877-891 (2013)

  Age-related impairment of angiogenesis is likely to play a central role in cerebromicrovascular rarefaction and development of vascular cognitive impairment, but the underlying mechanisms remain elusive. To test the hypothesis that dysregulation of Dicer1 (ribonuclease III, a key enzyme of the microRNA [miRNA] machinery) impairs endothelial angiogenic capacity in aging, primary cerebromicrovascular endothelial cells (CMVECs) were isolated from young (3 months old) and aged (24 months old) Fischer 344 × Brown Norway rats. We found an age-related downregulation of Dicer1 expression both in CMVECs and in small cerebral vessels isolated from aged rats. In aged CMVECs, Dicer1 expression was increased by treatment with polyethylene glycol–catalase. Compared with young cells, aged CMVECs exhibited altered miRNA expression profile, which was associated with impaired proliferation, adhesion to vitronectin, collagen and fibronectin, cellular migration (measured by a wound-healing assay using electric cell–substrate impedance sensing technology), and impaired ability to form capillary-like structures. Overexpression of Dicer1 in aged CMVECs partially restored miRNA expression profile and significantly improved angiogenic processes. In young CMVECs, downregulation of Dicer1 (siRNA) resulted in altered miRNA expression profile associated with impaired proliferation, adhesion, migration, and tube formation, mimicking the aging phenotype. Collectively, we found that Dicer1 is essential for normal endothelial angiogenic processes, suggesting that age-related dysregulation of Dicer1-dependent miRNA expression may be a potential mechanism underlying impaired angiogenesis and cerebromicrovascular rarefaction in aging.  

4.1183           The neurotrophic properties of progranulin depend on the granulin E domain but do not require sortilin binding

De Muynck, L., Herdewyn, S., Beel, S., Scheveneels, W., Van Den Bosch, L., Robberecht, W. and Van Damme P. Neurobiol. Of Aging, 34, 2541-2547 (2013)   Progranulin (PGRN) is a growth factor involved in wound healing, inflammation, tumor growth, and neurodegeneration. Mutations in the gene encoding PGRN give rise to shortage of PGRN and cause familial frontotemporal lobar degeneration. PGRN exerts neurotrophic functions and binding of PGRN to the membrane receptor sortilin (SORT1) mediates the endocytosis of PGRN. SORT1-mediated uptake plays an important role in the regulation of extracellular PGRN levels. We studied the role of SORT1 in PGRN-mediated neuroprotection in vitro and in vivo. The survival-enhancing effect of PGRN seemed to be dependent on the granulin E (GRN E) domain. Pharmacologic inhibition of the GRN E–SORT1 interaction or deletion of the SORT1 binding site of GRN E did not abolish its neurotrophic function. In addition, the in vivo phenotype of PGRN knockdown in zebrafish embryos was not phenocopied by SORT1 knockdown. These results suggest that GRN E mediates the neurotrophic properties of PGRN and that binding to SORT1 is not required for this effect.    

4.1184           A DISINTEGRIN AND METALLOPROTEINASE 17 REGULATES TNF AND TNFR1 LEVELS IN INFLAMMATION AND LIVER REGENERATION IN MICE

McMahan, R.S., Riehle, K.J., Fausto, N. and Campbell, J.S. Am. J: Physiol. Gastrointest. Liver Physiol., 305, G25-G34 (2013)   A Disintegrin And Metalloproteinase 17 (ADAM17), or TNF-alpha Converting Enzyme (TACE), is a key metalloproteinase and physiological convertase for a number of putative targets that play critical roles in cytokine and growth factor signaling. These interdependent pathways are essential components of the signaling network that links liver function with the compensatory growth that occurs during liver regeneration following 2/3 partial hepatectomy (PH) or chemically induced hepatotoxicity. Despite identification of many soluble factors needed for efficient liver regeneration, very little is known about how such ligands are regulated in the liver. To directly study the role of ADAM17 in the liver, we employed two cell-specific ADAM17 KO mouse models. Using LPS as a robust stimulus for TNF release, we found attenuated levels of circulating TNF in myeloid-specific ADAM17 KO mice (ADAM17 m-KO) and unexpectedly, in mice with hepatocyte-specific ADAM17 deletion (ADAM17 h-KO), indicating that ADAM17 expression in both cell types plays a role in TNF shedding. After 2/3 PH, induction of TNF, TNFR1, and amphiregulin (AR) was significantly attenuated in ADAM17 h-KO mice, implicating ADAM17 as the primary sheddase for these factors in the liver. Surprisingly, the extent and timing of hepatocyte proliferation were not affected after PH or carbon tetrachloride (CCl₄) injection in ADAM17 h-KO or ADAM17 m-KO mice. We conclude that ADAM17 regulates TNF, TNFR1, and AR in the liver, and its expression in both hepatocytes and myeloid cells is important for TNF regulation after LPS injury or 2/3 PH, but is not required for liver regeneration.  

4.1185           Dendritic cell immunotherapy combined with gemcitabine chemotherapy enhances survival in a murine model of pancreatic carcinoma

Ghansah, T., Vohra, N., Kinney, K., Weber, A., Kodumudi, K., Springett, G., Sarnaik, A.A. and Pilon-Thomas, S. Cancer Immunol. Immunother.,62(6), 1083-1091 (2013)   Pancreatic cancer is an extremely aggressive malignancy with a dismal prognosis. Cancer patients and tumor-bearing mice have multiple immunoregulatory subsets including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC) that may limit the effectiveness of anti-tumor immunotherapies for pancreatic cancer. It is possible that modulating these subsets will enhance anti-tumor immunity. The goal of this study was to explore depletion of immunoregulatory cells to enhance dendritic cell (DC)-based cancer immunotherapy in a murine model of pancreatic cancer. Flow cytometry results showed an increase in both Tregs and MDSC in untreated pancreatic cancer–bearing mice compared with control. Elimination of Tregs alone or in combination with DC-based vaccination had no effect on pancreatic tumor growth or survival. Gemcitabine (Gem) is a chemotherapeutic drug routinely used for the treatment for pancreatic cancer patients. Treatment with Gem led to a significant decrease in MDSC percentages in the spleens of tumor-bearing mice, but did not enhance overall survival. However, combination therapy with DC vaccination followed by Gem treatment led to a significant delay in tumor growth and improved survival in pancreatic cancer–bearing mice. Increased MDSC were measured in the peripheral blood of patients with pancreatic cancer. Treatment with Gem also led to a decrease of this population in pancreatic cancer patients, suggesting that combination therapy with DC-based cancer vaccination and Gem may lead to improved treatments for patients with pancreatic cancer.  

4.1186           A Comparative Toxicogenomic Investigation of Oil Sand Water and Processed Water in Rainbow Trout Hepatocytes

Gagne, F., Andre, C., Turcotte, P., Gagnon, C., Sherry, J. and Talbot, A. Arch. Environ Contam. Toxicol., 65(2), 309-323 (2013)   The purpose of this study was to compare the expression of gene transcripts involved in toxic stress in rainbow trout hepatocytes exposed to oil sand water (OSW), lixiviate (OSLW), and processed water (OSPW). We pose the hypothesis that the changes in gene expression responses in cells exposed to a simulated oil sand extraction procedure (OSPW) differ from the gene expression responses of OSLW and OS. Rainbow trout hepatocytes were exposed to increasing concentrations of OSW, OSLW, and OSPW for 48 h at 15 °C. Cell viability was assessed by measuring membrane permeability, total RNA levels, and gene expression using an array of 16 genes involved in xenobiotic biotransformation (GST, CYP1A1, CYP3A4, MDR), metal homeostasis and oxidative stress (MT, SOD, and CAT), estrogenicity (VTG, ERβ), DNA repair (LIG, APEX, UNG, and OGG), cell growth (GADD45 and PCNA), and glycolysis (GAPDH). The results showed that the toxicogenomic properties of OSPW differed from those of OSLW and OSW. Gene transcripts that were influenced by OSW and OSLW, and strongly expressed in OSPW, were MT, CAT, GST (induction), CYP1A1, VTG, UNG/OGG, and PCNA. These genes are therefore considered not entirely specific to OSPW but to water in contact with OS. We also found gene transcripts that responded only with OSPW: SOD, GST (inhibition), MDR (inhibition), CYP3A4, GAPDH, GADD45, and APEX. Of these gene transcripts, the ones strongly associated with toxicity (loss of cell viability and RNA levels) were CYP3A4, GST, and GAPDH. Genes involved in DNA repair were also strongly related to the loss of cell viability but responded to both OSLW and OSPW. The observed changes in cell toxicity and gene expression therefore support the hypothesis that OSPW has a distinct toxic fingerprint from OSLW and OSW.  

4.1187           Expression Pattern of Interferon-Inducible Transcriptional Genes in Neutrophils During Bovine Tuberculosis Infection no access

Wang, J., Zhou, X., Pan, B., Wang, H., Shi, F., Gan, W., Yang, L., Yin, X., Xu, B. and Zhao, D. DNA and Cell Biol., 32(8), 480-486 (2013)   Mycobacterium bovis, the classical causative agent of bovine tuberculosis (BTB), infects animals of agricultural importance and other mammals, including humans. Neutrophils are one of the first lines of defense against all microbes and produce a diverse collection of antimicrobial molecules, which play an important role in the early control of tuberculosis progression. An interferon (IFN)-inducible neutrophil-driven blood transcriptional signature that consisted of both IFN-γ and type I IFN-α/β signaling has been identified in human tuberculosis, supporting a role for neutrophils in the pathogenesis of tuberculosis disease. However, it is unknown whether bovine neutrophils play a similar role during M. bovis infection. Thus, we assessed the expression levels of ten IFN-inducible transcriptional genes in neutrophils from healthy cattle stimulated by M. bovis and neutrophils isolated from three groups of cattle of different infection status, and in addition, examined the changes in the expression of myeloperoxidase (MPO) and pentraxin-related protein pentraxin-inducible protein (PTX3) genes during bovine tuberculosis infection. Our results demonstrated a specific expression pattern of IFN-inducible transcriptional genes and MPO and PTX3 genes in neutrophils during bovine tuberculosis infection. The observed expression pattern provides a potential diagnostic tool, which may have implications for vaccine and therapeutic development to combat the bovine tuberculosis epidemic.  

4.1188           Truncated Form of TGF-βRII, But Not Its Absence, Induces Memory CD8+ T Cell Expansion and Lymphoproliferative Disorder in Mice

Ishigame, H., Mosaheb, M.M., Sanjabi, S. and Flavell, R.A.

1. Immunol., 190(12), 6340-6350 (2013)

  Inflammatory and anti-inflammatory cytokines play an important role in the generation of effector and memory CD8⁺ T cells. We used two different models, transgenic expression of truncated (dominant negative) form of TGF-βRII (dnTGFβRII) and Cre-mediated deletion of the floxed TGF-βRII to examine the role of TGF-β signaling in the formation, function, and homeostatic proliferation of memory CD8⁺ T cells. Blocking TGF-β signaling in effector CD8⁺ T cells using both of these models demonstrated a role for TGF-β in regulating the number of short-lived effector cells but did not alter memory CD8⁺ T cell formation and their function upon Listeria monocytogenes infection in mice. Interestingly, however, a massive lymphoproliferative disorder and cellular transformation were observed in Ag-experienced and homeostatically generated memory CD8⁺ T cells only in cells that express the dnTGFβRII and not in cells with a complete deletion of TGF-βRII. Furthermore, the development of transformed memory CD8⁺ T cells expressing dnTGFβRII was IL-7– and IL-15–independent, and MHC class I was not required for their proliferation. We show that transgenic expression of the dnTGFβRII, rather than the absence of TGF-βRII–mediated signaling, is responsible for dysregulated expansion of memory CD8⁺ T cells. This study uncovers a previously unrecognized dominant function of the dnTGFβRII in CD8⁺ T cell proliferation and cellular transformation, which is caused by a mechanism that is different from the absence of TGF-β signaling. These results should be considered during both basic and translational studies where there is a desire to block TGF-β signaling in CD8⁺ T cells.  

4.1189           Macrophage Migration Inhibitory Factor Potentiates Autoimmune-Mediated Neuroinflammation

Cox, G.M., Kithcart, A.P., Pitt, D., Guan, Z., Alexander, J., Williams, J.L., Shawler, T., Dagia, N.M., Popovich, P.G., Satoskar, A.R. and Whittacre, C.C.

1. Immunol., 191(3), 1043-1054 82013)

  Macrophage migration inhibitory factor (MIF) is a multipotent cytokine that is associated with clinical worsening and relapses in multiple sclerosis (MS) patients. The mechanism through which MIF promotes MS progression remains undefined. In this study, we identify a critical role for MIF in regulating CNS effector mechanisms necessary for the development of inflammatory pathology in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Despite the ability to generate pathogenic myelin-specific immune responses peripherally, MIF-deficient mice have reduced EAE severity and exhibit less CNS inflammatory pathology, with a greater percentage of resting microglia and fewer infiltrating inflammatory macrophages. We demonstrate that MIF is essential for promoting microglial activation and production of the innate soluble mediators IL-1β, IL-6, TNF-α, and inducible NO synthase. We propose a novel role for MIF in inducing microglial C/EBP-β, a transcription factor shown to regulate myeloid cell function and play an important role in neuroinflammation. Intraspinal stereotaxic microinjection of MIF resulted in upregulation of inflammatory mediators in microglia, which was sufficient to restore EAE-mediated inflammatory pathology in MIF-deficient mice. To further implicate a role for MIF, we show that MIF is highly expressed in human active MS lesions. Thus, these results illustrate the ability of MIF to influence the CNS cellular and molecular inflammatory milieu during EAE and point to the therapeutic potential of targeting MIF in MS.  

4.1190           IL-17A Plays a Critical Role in the Pathogenesis of Liver Fibrosis through Hepatic Stellate Cell Activation

Tan, Z., Xiaofeng, Q., Jiang, R., Liu, Q., Wang, Y., Chen. C.,Wang, X., Ryffel, B. and Sun, B.

1. Immunol., 191(4), 1835-1844 (2013)

  Liver fibrosis is a severe, life-threatening clinical condition resulting from nonresolving hepatitis of different origins. IL-17A is critical in inflammation, but its relation to liver fibrosis remains elusive. We find increased IL-17A expression in fibrotic livers from HBV-infected patients undergoing partial hepatectomy because of cirrhosis-related early-stage hepatocellular carcinoma in comparison with control nonfibrotic livers from uninfected patients with hepatic hemangioma. In fibrotic livers, IL-17A immunoreactivity localizes to the inflammatory infiltrate. In experimental carbon tetrachloride–induced liver fibrosis of IL-17RA–deficient mice, we observe reduced neutrophil influx, proinflammatory cytokines, hepatocellular necrosis, inflammation, and fibrosis as compared with control C57BL/6 mice. IL-17A is produced by neutrophils and T lymphocytes expressing the Th17 lineage–specific transcription factor Retinoic acid receptor–related orphan receptor γt. Furthermore, hepatic stellate cells (HSCs) isolated from naive C57BL/6 mice respond to IL-17A with increased IL-6, α-smooth muscle actin, collagen, and TGF-β mRNA expression, suggesting an IL-17A–driven fibrotic process. Pharmacologic ERK1/2 or p38 inhibition significantly attenuated IL-17A–induced HSC activation and collagen expression. In conclusion, IL-17A⁺ Retinoic acid receptor–related orphan receptor γt⁺ neutrophils and T cells are recruited into the injured liver driving a chronic, fibrotic hepatitis. IL-17A–dependent HSC activation may be critical for liver fibrosis. Thus, blockade of IL-17A could potentially benefit patients with chronic hepatitis and liver fibrosis.  

4.1191           P2X7 receptor-induced death of motor neurons by a peroxynitrite/FAS-dependent pathway

Gandelman, M., Levy, M., Cassina, P., Barbeito, L. and Beckman, J.S.

1. Neurochem., 126(3), 382-388 (2013)

  The P2X7 receptor/channel responds to extracellular ATP and is associated with neuronal death and neuroinflammation in spinal cord injury and amyotrophic lateral sclerosis. Whether activation of P2X7 directly causes motor neuron death is unknown. We found that cultured motor neurons isolated from embryonic rat spinal cord express P2X7 and underwent caspase-dependent apoptosis when exposed to exceptionally low concentrations of the P2X7 agonist 2′(3′)-O-(4-Benzoylbenzoyl)-ATP. The P2X7 inhibitors BBG, oATP, and KN-62 prevented 2′(3′)-O-(4-Benzoylbenzoyl)-ATP-induced motor neuron death. The endogenous P2X7 agonist ATP induced motor neuron death at low concentrations (1-100 μM). High concentrations of ATP (1 mM) paradoxically became protective due to degradation in the culture media to produce adenosine and activate adenosine receptors. P2X7-induced motor neuron death was dependent on neuronal nitric oxide synthase-mediated production of peroxynitrite, p38 activation, and autocrine FAS signaling. Taken together, our results indicate that motor neurons are highly sensitive to P2X7 activation, which triggers apoptosis by activation of the well-established peroxynitrite/FAS death pathway in motor neurons.    

4.1192           Neonatal macrophages express elevated levels of interleukin-27 that oppose immune responses

Kraft, J.D., Horzempa, J., Davis, C., Jung, J-Y., Pena, M.M. and Robinson, C.M. Immunology, 139(4), 484-493 (2013)   Microbial infections are a major cause of infant mortality worldwide because of impaired immune defences in this population. The nature of this work was to further understand the mechanistic limitations of the neonatal and infant immune response. Interleukin-27 (IL-27) is a heterodimeric cytokine of the IL-12 family that is produced primarily by antigen-presenting cells and is immunosuppressive toward a variety of immune cell types. We show that IL-27 gene expression is elevated in cord blood-derived macrophages relative to macrophages originating from healthy adults. We also evaluated the duration over which elevated IL-27 gene expression may impact immune responses in mice. Age-dependent analysis of IL-27 gene expression indicated that levels of IL-27 remained significantly elevated throughout infancy and then declined in adult mice. Flow cytometric analysis of intracellular cytokine-stained splenocytes further confirmed these results. Interleukin-27 may be induced during pregnancy to contribute to the immunosuppressive environment at the fetal–maternal interface because we demonstrate dose-responsive gene expression to progesterone in macrophages. Neutralization of IL-27 in neonatal macrophages improved the ability of these cells to limit bacterial replication. Moreover, neutralization of IL-27 during incubation with the Mycobacterium bovis bacillus Calmette–Guérin vaccine augmented the level of interferon- elicited from allogeneic CD4⁺ T lymphocytes. This suggests that blocking IL-27 during vaccination and infection may improve immune responses in newborn and infant populations. Furthermore, mice will be a suitable model system to further address these possibilities.  

4.1193           Venezuelan equine encephalitis virus glycoprotein pseudotyping confers neurotropism to lentiviral vectors

Trabalza, A., Geortgiadis, C., Eleftheriadou, I., Hislop, J.N., Karavassilis, M.E. and Mazarakis, N.D. Gene Therapy, 20(7), 723-732 (2013)   We have produced high-titre HIV-1 green fluorescent protein-expressing lentiviral (LV) vectors pseudotyped with strain 3908 Venezuelan equine encephalitis virus glycoprotein (VEEV-G) and used them to study transduction of: (1) rat embryonic motor neuron (MN) and striatal neuron primary cultures, (2) differentiated MN cell line NSC-34 and (3) adult rat striatum. In primary neuronal cultures, transduction with VEEV-G-pseudotyped LV was more efficient and more neuronal than with vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped LV. In NSC-34 cells clear retrograde transport of VEEV-G vector particles was observed. In the striatum at the injection site, transduction with the VEEV-G vectors driven by cytomegalovirus or phosphoglycerate kinase promoters exhibited a distinct neuronal tropism with no microglial and only a minor astroglial component, superior to that obtained with VSV-G-pseudotyped LV, irrespective of the promoter used. Neuronal transduction efficiency increased over time. Distal to the injection site transduction of mitral cells in the olfactory bulb, thalamic neurons and dopaminergic neurons in the substantia nigra pars compacta was detected. This, together with observations of retrograde axonal trafficking in vitro indicates that these vectors also possess low level of retrograde neuronal transduction capability in vivo. In this study, we demonstrate both strong neurotropism as well as sustainability of expression and minimal host immune response in vivo, making the VEEV-G-pseudotyped LV vectors potentially useful for gene therapy of neurodegenerative diseases.  

4.1194           Paracrine Wnt signaling both promotes and inhibits human breast tumor growth

Green, J.L., La, J., Yum, K.W., Desai, P., Redewald, L-W., Zhang, X., leblanc, M., Nusse, R., lewis, M.T. and Wahl, G.M. PNAS, 110(17), 6991-6996 (2013)   Wnt signaling in mouse mammary development and tumorigenesis has been heavily studied and characterized, but its role in human breast cancer remains elusive. Although Wnt inhibitors are in early clinical development, it is unclear whether they will be of therapeutic benefit to breast cancer patients, and subsequently, to which ones. To address this, we generated a panel of Wnt reporting human breast cancer cell lines and identified a previously unrecognized enrichment for the ability to respond to Wnt in the basal B or claudin-low subtype, which has a poor prognosis and no available targeted therapies. By co-injecting Wnt3A expressing human mammary fibroblasts with human breast cancer cell lines into mouse mammary fat pads, we showed that elevated paracrine Wnt signaling was correlated with accelerated tumor growth. Using this heterotypic system and a dual lentiviral reporter system that enables simultaneous real-time measurement of both Wnt-responsive cells and bulk tumor cells, we analyzed the outcome of elevated Wnt signaling in patient-derived xenograft (PDX) models. Interestingly, the PDX models exhibited responses not observed in the cell lines analyzed. Exogenous WNT3A promoted tumor growth in one human epidermal growth factor receptor 2-overexpressing PDX line but inhibited growth in a second PDX line obtained from a patient with triple-negative breast cancer. Tumor suppression was associated with squamous differentiation in the latter. Thus, our work suggests that paracrine Wnt signaling can either fuel or repress the growth of human breast cancers depending on yet to be determined aspects of the molecular pathways they express.  

4.1195           MST1 functions as a key modulator of neurodegeneration in a mouse model of ALS

Lee, J.K:, Shin, J.H., Hwang, S.G., Gwag, B.J., McKee, A.C., Lee, J., Kowall, N.W., Ryu, H., Lim, D.S. and Choi, E-J. PNAS, 110(29), 12066-12071 (2013)   Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder characterized by loss of motor neurons. Dominant mutations in the gene for superoxide dismutase 1 (SOD1) give rise to familial ALS by an unknown mechanism. Here we show that genetic deficiency of mammalian sterile 20-like kinase 1 (MST1) delays disease onset and extends survival in mice expressing the ALS-associated G93A mutant of human SOD1. SOD1(G93A) induces dissociation of MST1 from a redox protein thioredoxin-1 and promotes MST1 activation in spinal cord neurons in a reactive oxygen species–dependent manner. Moreover, MST1 was found to mediate SOD1(G93A)-induced activation of p38 mitogen-activated protein kinase and caspases as well as impairment of autophagy in spinal cord motoneurons of SOD1(G93A) mice. Our findings implicate MST1 as a key determinant of neurodegeneration in ALS.  

4.1196           The Intracellular Environment of Human Macrophages That Produce Nitric Oxide Promotes Growth of Mycobacteria

Jung, J-Y., Madan-Lala, R., georgieva, M., Rengarajan, J., Sohaskey, C.D., Bange, F-C. and Robinson, C.M. Infect. Immun., 81(9), 3198-3209 (2013)   Nitric oxide (NO) is a diffusible radical gas produced from the activity of nitric oxide synthase (NOS). NOS activity in murine macrophages has a protective role against mycobacteria through generation of reactive nitrogen intermediates (RNIs). However, the production of NO by human macrophages has remained unclear due to the lack of sensitive reagents to detect NO directly. The purpose of this study was to investigate NO production and the consequence to mycobacteria in primary human macrophages. We found that Mycobacterium bovis BCG or Mycobacterium tuberculosis infection of human macrophages induced expression of NOS2 and NOS3 that resulted in detectable production of NO. Treatment with gamma interferon (IFN-γ), l-arginine, and tetrahydrobiopterin enhanced expression of NOS2 and NOS3 isoforms, as well as NO production. Both of these enzymes were shown to contribute to NO production. The maximal level of NO produced by human macrophages was not bactericidal or bacteriostatic to M. tuberculosis or BCG. The number of viable mycobacteria was increased in macrophages that produced NO, and this requires expression of nitrate reductase. An narG mutant of M. tuberculosis persisted but was unable to grow in human macrophages. Taken together, these data (i) enhance our understanding of primary human macrophage potential to produce NO, (ii) demonstrate that the level of RNIs produced in response to IFN-γ in vitro is not sufficient to limit intracellular mycobacterial growth, and (iii) suggest that mycobacteria may use RNIs to enhance their survival in human macrophages.  

4.1197           In vivo CD8+ T Cell Dynamics in the Liver of Plasmodium yoelii Immunized and Infected Mice

Cabrera, M., Pewe, L.L., Harty, J.T. and Frevert, U. PloS One, 8(8), e70842 (2013)   Plasmodium falciparum malaria remains one of the most serious health problems globally and a protective malaria vaccine is desperately needed. Vaccination with attenuated parasites elicits multiple cellular effector mechanisms that lead to Plasmodium liver stage elimination. While granule-mediated cytotoxicity requires contact between CD8+ effector T cells and infected hepatocytes, cytokine secretion should allow parasite killing over longer distances. To better understand the mechanism of parasite elimination in vivo, we monitored the dynamics of CD8+ T cells in the livers of naïve, immunized and sporozoite-infected mice by intravital microscopy. We found that immunization of BALB/c mice with attenuated P. yoelii 17XNL sporozoites significantly increases the velocity of CD8+ T cells patrolling the hepatic microvasculature from 2.69±0.34 μm/min in naïve mice to 5.74±0.66 μm/min, 9.26±0.92 μm/min, and 7.11±0.73 μm/min in mice immunized with irradiated, early genetically attenuated (Pyuis4-deficient), and late genetically attenuated (Pyfabb/f-deficient) parasites, respectively. Sporozoite infection of immunized mice revealed a 97% and 63% reduction in liver stage density and volume, respectively, compared to naïve controls. To examine cellular mechanisms of immunity in situ, naïve mice were passively immunized with hepatic or splenic CD8+ T cells. Unexpectedly, adoptive transfer rendered the motile CD8+ T cells from immunized mice immotile in the liver of P. yoelii infected mice. Similarly, when mice were simultaneously inoculated with viable sporozoites and CD8+ T cells, velocities 18 h later were also significantly reduced to 0.68±0.10 μm/min, 1.53±0.22 μm/min, and 1.06±0.26 μm/min for CD8+ T cells from mice immunized with irradiated wild type sporozoites, Pyfabb/f-deficient parasites, and P. yoelii CS_280–288 peptide, respectively. Because immobilized CD8+ T cells are unable to make contact with infected hepatocytes, soluble mediators could potentially play a key role in parasite elimination under these experimental conditions.  

4.1198           Sorafenib perpetuates cellular anticancer effector functions by modulating the crosstalk between macrophages and natural killer cells

Sprinzl, M.F., Reisinger, F., Puschnik, A., Ringelhan, M., Ackermann, K., Hartmann, D., Schiemann, M., Weinmann, A., Galle, P.R., Schuchmann, M., Friess, H., Otto, G., Heikenwalder, M. and Protzer, U. Hepatology, 57(6), 2358-2368 (2013)   Alternatively polarized macrophages (Mϕ) shape the microenvironment of hepatocellular carcinoma (HCC) and temper anticancer immune responses. We investigated if sorafenib alters the HCC microenvironment by restoring classical macrophage polarization and triggering tumor-directed natural killer (NK) cell responses. In vivo experiments were conducted with sorafenib (25 mg/kg)-treated C57BL/6 wildtype as well as hepatitis B virus (HBV) and lymphotoxin transgenic mice with and without HCC. Monocyte-derived Mϕ or tumor-associated macrophages (TAM) isolated from HCC tissue were treated with sorafenib (0.07-5.0 μg/mL) and cocultured with autologous NK cells. Mϕ and NK cell activation was analyzed by flow cytometry and killing assays, respectively. Cytokine and growth factor release was measured by enzyme-linked immunosorbent assay. Short-term administration of sorafenib triggered activation of hepatic NK cells in wildtype and tumor-bearing mice. In vitro, sorafenib sensitized Mϕ to lipopolysaccharide, reverted alternative Mϕ polarization and enhanced IL12 secretion (P = 0.0133). NK cells activated by sorafenib-treated Mϕ showed increased degranulation (15.3 ± 0.2% versus 32.0 ± 0.9%, P < 0.0001) and interferon-gamma (IFN- ) secretion (2.1 ± 0.2% versus 8.0 ± 0.2%, P < 0.0001) upon target cell contact. Sorafenib-triggered NK cell activation was verified by coculture experiments using TAM. Sorafenib-treated Mϕ increased cytolytic NK cell function against K562, Raji, and HepG2 target cells in a dose-dependent manner. Neutralization of interleukin (IL)12 or IL18 as well as inhibition of the nuclear factor kappa B (NF-κB) pathway reversed NK cell activation in Mϕ/NK cocultures. Conclusion: Sorafenib triggers proinflammatory activity of TAM and subsequently induces antitumor NK cell responses in a cytokine- and NF-κB-dependent fashion. This observation is relevant for HCC therapy, as sorafenib is a compound in clinical use that reverts alternative polarization of TAM in HCC  

4.1199           Maternal obesity programs offspring nonalcoholic fatty liver disease by innate immune dysfunction in mice

Mouralidarane, A., Soeda, J., Visconti-Pugmire, C., Samuelsson, A-M., Pombo, J., maragkoudaki, X., Butt, A., Saraswati, R., Novelli, M., Fusai, G., Poston, L., Taylor, P.D. and oben, J.A. Hepatology, 58(1), 128-138 (2013)   The global prevalence of obesity-induced liver disease (nonalcoholic fatty liver disease; NAFLD) is rising. Suggested causes include a role for in utero influences of maternal obesity compounded by the availability of energy-dense foods throughout postnatal life. Using a physiologically relevant model, we investigated the role of the innate immune system in liver injury induced by maternal obesity followed by a postnatal obesogenic diet. Female C57BL/6J mice were fed a standard or obesogenic diet before and throughout pregnancy and during lactation. Female offspring were weaned onto a standard or obesogenic diet at 3 weeks postpartum. Biochemical and histological indicators of dysmetabolism, NAFLD and fibrosis, analysis of profibrotic pathways, liver innate immune cells, and reactive oxygen species (ROS) were investigated at 3, 6, and 12 months. Female offspring exposed to a postweaning obesogenic diet (OffCon-OD) demonstrated evidence of liver injury, which was exacerbated by previous exposure to maternal obesity (OffOb-OD), as demonstrated by raised alanine aminotransferase, hepatic triglycerides, and hepatic expression of interleukin (IL)-6, tumor necrosis factor alpha, transforming growth factor beta, alpha smooth muscle actin, and collagen (P < 0.01). Histological evidence of hepatosteatosis and a more-robust NAFLD phenotype with hepatic fibrosis was observed at 12 months in OffOb-OD. A role for the innate immune system was indicated by increased Kupffer cell numbers with impaired phagocytic function and raised ROS synthesis (P < 0.01), together with reduced natural killer T cells and raised interleukin (IL)-12 and IL-18. Conclusion: Maternal obesity in the context of a postnatal hypercalorific obesogenic diet aggressively programs offspring NAFLD associated with innate immune dysfunction, resulting in a comprehensive phenotype that accurately reflects the human disease.  

4.1200           Dendritic cells limit fibroinflammatory injury in nonalcoholic steatohepatitis in mice

Henning, J.R., Graffeo, C.S., Rehman, A., fallon, N.C., Zambirinis, C.P., Ochi, A., Barilla, R., Jamal, M., Deutsch, M., Greco, S., Ego-Osuala, M., Bin-Saeed, U., Rao, R.S., Badar, S., Quesada, J.P., Acehan, D. and Miller, G. Hepatology, 58(2), 589-602 (2013)   Nonalcoholic steatohepatitis (NASH) is the most common etiology of chronic liver dysfunction in the United States and can progress to cirrhosis and liver failure. Inflammatory insult resulting from fatty infiltration of the liver is central to disease pathogenesis. Dendritic cells (DCs) are antigen-presenting cells with an emerging role in hepatic inflammation. We postulated that DCs are important in the progression of NASH. We found that intrahepatic DCs expand and mature in NASH liver and assume an activated immune phenotype. However, rather than mitigating the severity of NASH, DC depletion markedly exacerbated intrahepatic fibroinflammation. Our mechanistic studies support a regulatory role for DCs in NASH by limiting sterile inflammation through their role in the clearance of apoptotic cells and necrotic debris. We found that DCs limit CD8⁺ T-cell expansion and restrict Toll-like receptor expression and cytokine production in innate immune effector cells in NASH, including Kupffer cells, neutrophils, and inflammatory monocytes. Consistent with their regulatory role in NASH, during the recovery phase of disease, ablation of DC populations results in delayed resolution of intrahepatic inflammation and fibroplasia. Conclusion: Our findings support a role for DCs in modulating NASH. Targeting DC functional properties may hold promise for therapeutic intervention in NASH.  

4.1201           Mutational Analyses on X-Linked Adrenoleukodystrophy Reveal a Novel Cryptic Splicing and Three Missense Mutations in the ABCD1 Gene

Hung, K-L., Wang, J-S., Keng, W.T., Chen, H-J., Liang, J-S., Ngu, L.H. and Lu, J-F. Pediatric Neurol., 49, 185-190 (2013)   Background X-linked adrenoleukodystrophy is caused by a defective peroxisomal membrane transporter, ABCD1, responsible for transporting very-long-chain fatty acid substrate into peroxisomes for degradation. The main biochemical defect, which is also one of the major diagnostic hallmarks, of X-linked adrenoleukodystrophy is the accumulation of saturated very-long-chain fatty acids in all tissues and body fluids. Methods Direct and reverse-transcribed polymerase chain reactions followed by DNA sequencing-based mutational analyses were performed on one Taiwanese and three Malaysian X-linked adrenoleukodystrophy families. Results A novel splicing donor site mutation (c.1272+1g>a) was identified in a Taiwanese X-linked adrenoleukodystrophy patient, resulting in a deletion of 121 bp and a premature stop codon (p.Val425fs*92) in messenger-RNA transcript. This deletion is caused by the activation of a cryptic splicing donor site in exon 4 of the ABCD1 gene, which is consistent with the prediction by several online algorithms. In addition, three previously described missense mutations (c.965T>C, c.1978C>T, and c.2006A>G), leading to aberrant ABCD1 of p.Leu322Pro, p.Arg660Trp, and p.His669Arg, were also identified in Malaysian probands. Conclusions This is the first report to unveil unequivocally that cryptic splicing-induced aberrant messenger-RNA carrying an internal frameshift deletion results from an intronic mutation in the ABCD1 gene. Furthermore, a polymorphism in intron 9 (c.1992-32c/t; refSNP: rs4898368) of the ABCD1 gene was commonly observed in both Taiwanese and Malaysian populations.  

4.1202           Interleukin-33-Dependent Innate Lymphoid Cells Mediate Hepatic Fibrosis

Mchedlidze, T., Waldner, M., Zopf, S., Walker, J., Rankin, A.L., Schuchmann, M., Voehringer, D., McKenzie, A.N.J., Neurarath, M.F., Pflanz, S. and Wirtz, S. Immunity, 39, 357-371 (2013)   Liver fibrosis is a consequence of chronic liver diseases and thus a major cause of mortality and morbidity. Clinical evidence and animal studies suggest that local tissue homeostasis is disturbed due to immunological responses to chronic hepatocellular stress. Poorly defined stress-associated inflammatory networks are thought to mediate gradual accumulation of extracellular-matrix components, ultimately leading to fibrosis and liver failure. Here we have reported that hepatic expression of interleukin-33 (IL-33) was both required and sufficient for severe hepatic fibrosis in vivo. We have demonstrated that IL-33’s profibrotic effects related to activation and expansion of liver resident innate lymphoid cells (ILC2). We identified ILC2-derived IL-13, acting through type-II IL-4 receptor-dependent signaling via the transcription factor STAT6 and hepatic stellate-cell activation, as a critical downstream cytokine of IL-33-dependent pathologic tissue remodeling and fibrosis. Our data reveal key immunological networks implicated in hepatic fibrosis and support the concept of modulation of IL-33 bioactivity for therapeutic purposes.  

4.1203           Differentiation of glutamatergic neurons from mouse embryonic stem cells requires raptor S6K signaling

Chuang, J-H., Tung, L-C., Yin, Y. and Lin, Y. Stem Cell Res., 11, 1117-1128 (2013)   Although the mammalian target of rapamycin complex 1 (mTORC1) functions as an important signaling complex in many cellular processes, the role of mTORC1 in neurons derived from embryonic stem cells (ESCs) has been less explored. Here, using a modified protocol to differentiate mouse ESCs (mESCs) into almost uniform glutamatergic neurons, we explored the importance of raptor/mTORC1 in the differentiation of mESCs. Raptor gene-trap mESCs, and raptor-knockdown mESCs formed smaller-sized embryonic bodies than the wild type and failed to undergo neuronal differentiation. Treatment with 1 μM rapamycin starting at the point when neuronal precursors began to differentiate from mESCs caused the gradual loss of neurites, shrinkage of soma, and a decreased ratio of neurite length to cell number over 48 to 72 h of treatment. This change was accompanied by activation of caspase-3 and S6 kinase (S6K), but not 4E-binding protein 1 (4EBP1). Knockdown of raptor during neuronal differentiation from mESCs also resulted in gradual loss of neurites and shrinkage of cell bodies. Loss of neurite density resulting from rapamycin treatment could be reversed by overexpression of S6K T389E. Taken together, these data demonstrate that raptor/mTORC1/S6K plays a critical role in the differentiation and survival of neurons derived from mESCs.  

4.1204           Cushioned versus noncushioned centrifugation: Sperm recovery rate and integrity

Len, J.A., Beehan, D.P., Lyle, S.K. and Eilts, B.E. Theriogenology, 80, 648-653 (2013)   It was hypothesized that optimal sperm recovery rate (RR) without damage to the sperm would be obtained after centrifugation without a cushion solution. Semen collected three times from six light breed stallions was extended to 25 × 10⁶ sperm/mL and centrifuged at CON (noncentrifuged), 900NC (no-cushion), 900C (cushion), 1800NC, and 1800C × g for 10 minutes. Sperm concentration, motility (TM and PM), and intact plasma membranes (PLM) and acrosomes (ACR) pre- and postcentrifugation (D0) and after 24 hours (D1) of cooling were evaluated. The RR in the CON (100 ± 0.0), 900NC (93.7 ± 2.9), and 1800NC (96.7 ± 2.6) groups was significantly higher than the 900C (68.7 ± 4.6) and 1800C (79.6 ± 3.5) groups. The D0 TM and PM were not different between the CON, 900NC, 900C, and 1800C, but were lower for the 1800NC group. The D1 TM and PM of the 900NC (75.2 ± 3.8 and 71.1 ± 4.1) and 900C (76.2 ± 3.7 and 72.4 ± 4.0) groups were significantly higher than the 1800NC (71.7 ± 4.1 and 67.3 ± 4.4) and 1800C (71.6 ± 4.1 and 67.2 ± 4.4) groups, and the CON (66.2 ± 4.5 and 60.0 ± 4.8) group was significantly lower than the other groups. The D1 PLM of the CON, 900NC, 900C, 1800NC, and 1800C groups were not different. The ACR on D1 was significantly lower for the CON (93.0 ± 2.4) group compared with all other groups. Optimal RR preserving sperm integrity was obtained in the 900NC group.  

4.1205           Influences of cerebral stent implantation on CD4+CD25+FOXP3+Treg, Th1 and Th17 cells

Wang, S., Ni, B., Chen, K. and Shi, S. Int. Immunopharmacol., 17, 519-525 (2013)   Stent implantation is primarily used for the treatment of artery stenosis. However, the application and use of stent struts induces local and systemic inflammation, leading to intractable neointimal hyperplasia. CD4⁺ T cells are involved in artery stenosis diseases, but little is known about the influence of the CD4⁺ T cells on the inflammation reaction after stent implantation. In this study, we analyzed the frequency of signature transcription factors and proinflammatory cytokine expression from each subtype of CD4⁺ T cells in 50 patients receiving intracranial or cervical stent implantations from December 2011 to June 2012. The results showed that the frequency of signature transcription factor/cytokine production in Treg cells was reduced in the first week and returned to control levels at 3 months after stent implantation. However, we observed opposite trends for Th17 cells, showing increased signature transcription factor/cytokine production during the acute phase, which returned to control levels after 3 months. No significant difference in the frequency of signature transcription factor/cytokine expression was observed in Th1 cells from patients before and after stent implantation. We speculate that the maintenance of the frequency and function of Tregs controls the inflammatory response, which otherwise induces acute inflammation after stent implantation.  

4.1206           Small Molecule Suppressors of Drosophila Kinesin Deficiency Rescue Motor Axon Development in a Zebrafish Model of Spinal Muscular Atrophy

Gassmann, A., Hao, L.T., Bhoite, L., Braadford, C.L., Chien, C-B., Beattie, C.E. and Manfredi, J.P. PloS One, 8(9), e74325 (2013)   Proximal spinal muscular atrophy (SMA) is the most common inherited motor neuropathy and the leading hereditary cause of infant mortality. Currently there is no effective treatment for the disease, reflecting a need for pharmacologic interventions that restore performance of dysfunctional motor neurons or suppress the consequences of their dysfunction. In a series of assays relevant to motor neuron biology, we explored the activities of a collection of tetrahydroindoles that were reported to alter the metabolism of amyloid precursor protein (APP). In Drosophila larvae the compounds suppressed aberrant larval locomotion due to mutations in the Khc and Klc genes, which respectively encode the heavy and light chains of kinesin-1. A representative compound of this class also suppressed the appearance of axonal swellings (alternatively termed axonal spheroids or neuritic beads) in the segmental nerves of the kinesin-deficient Drosophila larvae. Given the importance of kinesin-dependent transport for extension and maintenance of axons and their growth cones, three members of the class were tested for neurotrophic effects on isolated rat spinal motor neurons. Each compound stimulated neurite outgrowth. In addition, consistent with SMA being an axonopathy of motor neurons, the three axonotrophic compounds rescued motor axon development in a zebrafish model of SMA. The results introduce a collection of small molecules as pharmacologic suppressors of SMA-associated phenotypes and nominate specific members of the collection for development as candidate SMA therapeutics. More generally, the results reinforce the perception of SMA as an axonopathy and suggest novel approaches to treating the disease  

4.1207           Apoptotic cell administration enhances pancreatic islet engraftment by induction of regulatory T cells and tolerogenic dendritic cells

Wu, C., Zhang, Y., Jiang, Y., Wang, Q., Long, Y., Wang, C., Cao, X. and Chen, G. Cell. Mol. Immunol., 10, 393-402 (2013)   Apoptotic cell transfer has been found to be able to facilitate engraftment of allograft. However, the underlying mechanisms remain to be fully understood. Here we demonstrate that intravenous administration of donor apoptotic splenocytes can promote pancreatic islet engraftment by inducing generation of tolerogenic dendritic cells (Tol-DCs) and expansion of CD4⁺Foxp3⁺ regulatory T cells (Tregs). In vivo clearance of either dendritic cells (DCs) or Tregs prevented the induction of immune tolerance by apoptotic cell administration. Transient elimination of Tregs using anti-CD25, monoclonal antibody (mAb) abrogated the generation of Tol-DCs after administration of apoptotic splenocytes. Reciprocally, depletion of DCs within CD11c-DTR mice using diphtheria toxin (DT) prevented the generation of Tregs in the recipients with administration of apoptotic splenocytes. Induction of Tregs by Tol-DCs required direct cell contact between the two cell types, and programmed death 1 ligand (PD-L1) played important role in the Tregs expansion. Apoptotic cell administration failed to induce Tol-DCs in IL-10-deficient and Smad3-deficient mice, suggesting that IL-10 and transforming growth factor-β (TGF-β) are needed to maintain DCs in the tolerogenic state. Therefore, we demonstrate that Tol-DCs promote the expansion of Tregs via PD-L1 on their surface and reciprocally Tregs facilitate Tol-DCs to maintain transplantation tolerance induced by apoptotic cells via secreting IL-10 and TGF-β.  

4.1208           TLR2 Mediates Helicobacter pylori–Induced Tolerogenic Immune Response in Mice

Sun, X., Zhang, M., El-Zataari, M., Qwyang, S.Y., Eaton, K.A., Liu, M., Chang, Y-M., Zou, W. and Kao, J.Y. PloS One, 8(9), e74595 (2013)   We have shown that Helicobacter pylori induces tolerogenic programming of dendritic cells and inhibits the host immune response. Toll-like receptors (TLRs) represent a class of transmembrane pattern recognition receptors essential for microbial recognition and control of the innate immune response. In this study, we examined the role of TLRs in mediating H. pylori tolerogenic programming of dendritic cells and their impact on anti–H. pylori immunity using C57BL/6 wild-type and TLR2-knockout (TLR2KO) mice. We analyzed the response of TLR2KO bone marrow-derived dendritic cells (BMDCs) to H. pylori SS1 stimulation and the outcome of chronic H. pylori infection in TLR2KO mice. We showed that H. pylori–stimulated BMDCs upregulated the expression of TLR2, but not TLR4, TLR5, or TLR9. H. pylori-stimulated BMDCs from TLRKO mice induced lower Treg and Th17 responses, but a higher IFN-γ response compared to H. pylori-stimulated BMDCs from wild-type mice. In vivo analyses following an H. pylori infection of 2 months duration showed a lower degree of gastric H. pylori colonization in TLR2KO mice and more severe gastric immunopathology compared to WT mice. The gastric mucosa of the infected TLR2KO mice showed a lower mRNA expression of Foxp3, IL-10, and IL-17A, but higher expression of IFN-γ compared to the gastric mRNA expression in infected wild-type mice. Moreover, the H. pylori–specific Th1 response was higher and the Treg and Th17 responses were lower in the spleens of infected TLR2KO mice compared to infected WT mice. Our data indicate that H. pylori mediates immune tolerance through TLR2-derived signals and inhibits Th1 immunity, thus evading host defense. TLR2 may be an important target in the modulation of the host response to H. pylori.  

4.1209           Dynamics of the Major Histocompatibility Complex Class I Processing and Presentation Pathway in the Course of Malaria Parasite Development in Human Hepatocytes: Implications for Vaccine Development

Ma, J., Trop, S., Baer, S., Rakjmanaliev, E., Arany, Z., Dumoulin, P., Zhang, H., Romano, J., Coppens, I., levitsky, V. and Levitskaya, J. PloS One, 8(9), e75321 (2013)   Control of parasite replication exerted by MHC class I restricted CD8+ T-cells in the liver is critical for vaccination-induced protection against malaria. While many intracellular pathogens subvert the MHC class I presentation machinery, its functionality in the course of malaria replication in hepatocytes has not been characterized. Using experimental systems based on specific identification, isolation and analysis of human hepatocytes infected with P. berghei ANKA GFP or P. falciparum 3D7 GFP sporozoites we demonstrated that molecular components of the MHC class I pathway exhibit largely unaltered expression in malaria-infected hepatocytes until very late stages of parasite development. Furthermore, infected cells showed no obvious defects in their capacity to upregulate expression of different molecular components of the MHC class I machinery in response to pro-inflammatory lymphokines or trigger direct activation of allo-specific or peptide-specific human CD8+ T-cells. We further demonstrate that ectopic expression of circumsporozoite protein does not alter expression of critical genes of the MHC class I pathway and its response to pro-inflammatory cytokines. In addition, we identified supra-cellular structures, which arose at late stages of parasite replication, possessed the characteristic morphology of merosomes and exhibited nearly complete loss of surface MHC class I expression. These data have multiple implications for our understanding of natural T-cell immunity against malaria and may promote development of novel, efficient anti-malaria vaccines overcoming immune escape of the parasite in the liver.  

4.1210           Definition of a third VLR gene in hagfish

Li, J., Das, S., Herrin, B.R., Hirano, M. and Cooper, M.D. PNAS, 110(37), 15013-15018 (2013)   Jawless vertebrates (cyclostomes) have an alternative adaptive immune system in which lymphocytes somatically diversify their variable lymphocyte receptors (VLR) through recombinatorial use of leucine-rich repeat cassettes during VLR gene assembly. Three types of these anticipatory receptors in lampreys (VLRA, VLRB, and VLRC) are expressed by separate lymphocyte lineages. However, only two VLR genes (VLRA and VLRB) have been found in hagfish. Here we have identified a third hagfish VLR, which undergoes somatic assembly to generate sufficient diversity to encode a large repertoire of anticipatory receptors. Sequence analysis, structural comparison, and phylogenetic analysis indicate that the unique hagfish VLR is the counterpart of lamprey VLRA and the previously identified hagfish “VLRA” is the lamprey VLRC counterpart. The demonstration of three orthologous VLR genes in both lampreys and hagfish suggests that this anticipatory receptor system evolved in a common ancestor of the two cyclostome lineages around 480 Mya.  

4.1211           Histones Activate the NLRP3 Inflammasome in Kupffer Cells during Sterile Inflammatory Liver Injury

Huang, H., Chen, H-W., Evankovich, J., Yan, W., Rosborough, B.R., Nace, G.W., Ding, Q., Loughran, P., Beer-Stolz, D., Billiar, T.R., Esmon, C.T. and Tsung, A.

1. Immunol., 191, 2665-2679 (2013)

  Cellular processes that drive sterile inflammatory injury after hepatic ischemia/reperfusion (I/R) injury are not completely understood. Activation of the inflammasome plays a key role in response to invading intracellular pathogens, but mounting evidence suggests that it also plays a role in inflammation driven by endogenous danger-associate molecular pattern molecules released after ischemic injury. The nucleotide-binding domain, leucine-rich repeat containing protein 3 (NLRP3) inflammasome is one such process, and the mechanism by which its activation results in damage and inflammatory responses following liver I/R is unknown. In this article, we report that both NLRP3 and its downstream target caspase-1 are activated during I/R and are essential for hepatic I/R injury, because both NLRP3 and caspase-1 knockout mice are protected from injury. Furthermore, inflammasome-mediated injury is dependent on caspase-1 expression in liver nonparenchymal cells. Although upstream signals that activate the inflammasome during ischemic injury are not well characterized, we show that endogenous extracellular histones activate the NLRP3 inflammasome during liver I/R through TLR9. This occurs through TLR9-dependent generation of reactive oxygen species. This mechanism is operant in resident liver Kupffer cells, which drive innate immune responses after I/R injury by recruiting additional cell types, including neutrophils and inflammatory monocytes. These novel findings illustrate a new mechanism by which extracellular histones and activation of NLRP3 inflammasome contribute to liver damage and the activation of innate immunity during sterile inflammation.  

4.1212           OPEN

   

4.1213           Ultrahigh-Throughput Mammalian Single-Cell Reverse-Transcriptase Polymerase Chain Reaction in Microfluidic Drops

Eastburn, D.J., Sciambi, A. and Abate, A.R. Anal. Chem., 85(16), 8016-8021 (2013)   The behaviors of complex biological systems are often dictated by the properties of their heterogeneous and sometimes rare cellular constituents. Correspondingly, the analysis of individual cells from a heterogeneous population can reveal information not obtainable by ensemble measurements. Reverse-transcriptase polymerase chain reaction (RT-PCR) is a widely used method that enables transcriptional profiling and sequencing analysis on bulk populations of cells. Major barriers to successfully implementing this technique for mammalian single-cell studies are the labor, cost, and low-throughput associated with current approaches. In this report, we describe a novel droplet-based microfluidic system for performing 50000 single-cell RT-PCR reactions in a single experiment while consuming a minimal amount of reagent. Using cell type-specific staining and TaqMan RT-PCR probes, we demonstrate the identification of specific cells from a mixed human cell population. The throughput, robust detection rate and specificity of this method makes it well-suited for characterizing large, heterogeneous populations of cells at the transcriptional level.  

4.1214           Absence of Siglec-H in MCMV Infection Elevates Interferon Alpha Production but Does Not Enhance Viral Clearance

Puttur, F., Arnold-Schrauf, C., Lahl, K., Solmaz, G., Lindenberg, M., Mayer, C.T., Gohmert, M., Swallow, M., van Helt, C., Schmitt, H., Nitschke, L., Lambrecht, B.N., Lang, R., Messerle, M. and Sparwasser, T. PloS One, 9(9), e1003648 (2013)   Plasmacytoid dendritic cells (pDCs) express the I-type lectin receptor Siglec-H and produce interferon α (IFNα), a critical anti-viral cytokine during the acute phase of murine cytomegalovirus (MCMV) infection. The ligands and biological functions of Siglec-H still remain incompletely defined in vivo. Thus, we generated a novel bacterial artificial chromosome (BAC)-transgenic “pDCre” mouse which expresses Cre recombinase under the control of the Siglec-H promoter. By crossing these mice with a Rosa26 reporter strain, a representative fraction of Siglec-H⁺ pDCs is terminally labeled with red fluorescent protein (RFP). Interestingly, systemic MCMV infection of these mice causes the downregulation of Siglec-H surface expression. This decline occurs in a TLR9- and MyD88-dependent manner. To elucidate the functional role of Siglec-H during MCMV infection, we utilized a novel Siglec-H deficient mouse strain. In the absence of Siglec-H, the low infection rate of pDCs with MCMV remained unchanged, and pDC activation was still intact. Strikingly, Siglec-H deficiency induced a significant increase in serum IFNα levels following systemic MCMV infection. Although Siglec-H modulates anti-viral IFNα production, the control of viral replication was unchanged in vivo. The novel mouse models will be valuable to shed further light on pDC biology in future studies.  

4.1215           Aging affects AO rat splenic conventional dendritic cell subset composition, cytokine synthesis and T-helper polarizing capacity

Stojic-Vukanic, Z., Bufan, B., Arsenovic-Eanin. N., Kosec, D., Pilipovic, I., Nanut, M.P. and Leposavic, G. Biogerontology, 14, 443-459 (2013)   It is well-established that almost all cellular components of innate and adaptive immunity undergo age-related remodelling. The findings on age-related changes in both human and mouse dendritic cells (DCs) are conflicting, whereas there are no data on the influence of aging on rat DCs. In an attempt to fill this gap, freshly isolated splenic DCs expressing CD103 (α_OX-62 integrin), a DC specific marker recognized by MRC OX62 monoclonal antibody, from 3- (young) and 26-month-old (aged) Albino Oxford rats were examined for subset composition, expression of activation/differentiation markers (CD80, CD86 and CD40 and MHC II molecules) and endocytic capacity using flow cytometric analysis (FCA). In addition, splenic OX62+ DCs cultured in the presence or absence of LPS were analysed for the activation marker and TNF-α, IL-6, IL-12, IL-23, TGF-β1, IL-10 expression using FCA, RT-PCR and ELISA, respectively. Moreover, the allostimulatory capacity of OX62+ DCs and IFN-γ, IL-4 and IL-17 production by CD4+ T cells in mixed leukocyte reaction was quantified using FCA and ELISA, respectively. It was found that aging: i) shifts the CD4+:CD4− subset ratio in the OX62+ DCs population towards the CD4− subset and ii) influences DCs maturation (judging by activation marker expression and efficiency of endocytosis) by affecting the expression of intrinsic (TNF-α and IL-10) and extrinsic maturation regulators. Furthermore, in LPS-matured OX62+ DCs from aged rats expression of TNF-α, IL-12, IL-23 and IL-6 was increased, whereas that of IL-10 was diminished compared with the corresponding cells from young rats. Moreover, in MLR, OX62+ DCs from aged rats exhibited enhanced Th1/Th17 driving force and diminished allostimulatory capacity compared with those from young rats.  

4.1216           Down-regulation of microglial activity attenuates axotomized nigral dopaminergic neuronal cell loss

Song, D-Y., Yu, H-N., Park, C-R., Lee, J-S., Lee, J-Y., park, B-G., Woo, R-S., Han, J-T., Cho, B-P. and Baik, T-K. BMC Neurosci., 14:112 (2013)   Background There is growing evidence that inflammatory processes of activated microglia could play an important role in the progression of nerve cell damage in neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease which harbor features of chronic microglial activation, though the precise mechanism is unknown. In this study, we presented in vivo and ex vivo experimental evidences indicating that activated microglia could exacerbate the survival of axotomized dopaminergic neurons and that appropriate inactivation of microglia could be neuroprotective. Results The transection of medial forebrain bundle (MFB) of a rat induced loss of dopaminergic neurons in a time-dependent manner and accompanied with microglial activation. Along with microglial activation, production of reactive oxygen species (ROS) was upregulated and TH/OX6/hydroethidine triple-immunofluorescence showed that the microglia mainly produced ROS. When the activated microglial cells that were isolated from the substantia nigra of the MFB axotomized animal, were transplanted into the substantia nigra of which MFB had been transected at 7 days ago, the survival rate of axotomized dopaminergic neurons was significantly reduced as compared with sham control. Meanwhile, when the microglial activation was attenuated by administration of tuftsin fragment 1-3 (microglia inhibitory factor) into the lateral ventricle using mini-osmotic pump, the survival rate of axotomized dopaminergic neurons was increased. Conclusion The present study suggests that activated microglia could actively produce and secrete unfavorable toxic substances, such as ROS, which could accelerate dopaminergic neuronal cell loss. So, well-controlled blockade of microglial activation might be neuroprotective in some neuropathological conditions.  

4.1217           Characterization of functional capacity of adult ventricular myocytes in long-term culture

Liu, S.J. Int. J. Cardiol., 168, 1923-1936 (2013)   Background Functional properties of freshly isolated adult ventricular myocytes (AVMs) or those of AVMs during first few weeks in culture were well described. However, the functional capacity of these AVMs such as regenerative potential remains unknown, in part, due to the short lifespan of AVMs in culture. This study modified culture conditions that extended the lifespan of AVMs, isolated from adult rat hearts, longer than 6 months. Methods Temporal changes in the morphology of individual AVMs, cell–cell interaction, formation of myofibers, self-repair capacity after injury, expression of senescence biomarkers, and contractile function of AVMs over 5 weeks (defined as long-term culture) were chronologically characterized and quantified with live-cell video and fluorescence microscopy, and immunocytochemistry. Results Cell growth in size reached a plateau after 4 weeks in culture concomitantly with continuous increase in structural remodeling in long-term culture. Dynamic remodeling of AVMs promoted self-contact of filopodia and cell–cell contact where these contained abundant myofilaments, connexin 43 proteins, and high density and high integrity of mitochondria. Such high capacity also enabled self-repair of AVMs after injury, cytokinesis, and formation of myofibers. AVMs in long-term culture displayed spontaneous contraction and importantly were responsive to electrical stimulation. Moreover, AVMs expressed senescence-associated β-galactosidase, p16, and stress-associated atrial natriuretic peptides that resulted likely from cellular modeling. Conclusions Prolonged longevity of AVMs in culture with characteristics of high functional capacity of organelle regeneration and contraction makes them invaluable for further longitudinal mechanistic studies in cardiac (patho)physiology (e.g., hypertrophy and aging), single-cell analysis (e.g., function of hetero-phenotypes) and drug discovery.  

4.1218           Tolerance develops to the antiallodynic effects of the peripherally acting opioid loperamide hydrochloride in nerve-injured rats

He, S-Q., Yang, F., Perez, F.M., Xu, Q., Shechter, R., Cheong, Y-K., Carteret, A.F., Dong, X., Sweitzer, S.M., Raja, S.N. and Guan, Y. Pain, 154, 2477-2486 (2013)   Peripherally acting opioids are potentially attractive drugs for the clinical management of certain chronic pain states due to the lack of centrally mediated adverse effects. However, it remains unclear whether tolerance develops to peripheral opioid analgesic effects under neuropathic pain conditions. We subjected rats to L5 spinal nerve ligation (SNL) and examined the analgesic effects of repetitive systemic and local administration of loperamide hydrochloride, a peripherally acting opioid agonist. We found that the inhibition of mechanical hypersensitivity, an important manifestation of neuropathic pain, by systemic loperamide (1.5 mg/kg subcutaneously) decreased after repetitive drug treatment (tolerance-inducing dose: 0.75 to 6.0 mg/kg subcutaneously). Similarly, repeated intraplantar injection of loperamide (150 μg/50 μL intraplantarly) and D-Ala²-MePhe⁴-Glyol⁵ enkephalin (300 μg/50 μL), a highly selective mu-opioid receptor (MOR) agonist, also resulted in decreased inhibition of mechanical hypersensitivity. Pretreatment with naltrexone hydrochloride (5 mg/kg intraperitoneally) and MK-801 (0.2 mg/kg intraperitoneally) attenuated systemic loperamide tolerance. Western blot analysis showed that repetitive systemic administration of morphine (3 mg/kg subcutaneously), but not loperamide (3 mg/kg subcutaneously) or saline, significantly increased MOR phosphorylation in the spinal cord of SNL rats. In cultured rat dorsal root ganglion neurons, loperamide dose-dependently inhibited KCl-induced increases in [Ca²⁺]_i. However, this drug effect significantly decreased in cells pretreated with loperamide (3 μM, 72 hours). Intriguingly, in loperamide-tolerant cells, the delta-opioid receptor antagonist naltrindole restored loperamide’s inhibition of KCl-elicited [Ca²⁺]_i increase. Our findings indicate that animals with neuropathic pain may develop acute tolerance to the antiallodynic effects of peripherally acting opioids after repetitive systemic and local drug administration.  

4.1219           A Specific and Facile Method for Isolating Murine Liver Sinusoidal Endothelial Cells using a CD32B Monoclonal Antibody

Donofrio, B., Kasumov, T., Dasarathy, S. and McCullough, A.J. Hepatology, 58(4), 455A-459A (2013)   Background: Liver sinusoidal endothelial cells (LSEC) comprise 20% of all liver cells and are now recognized to be both a target and mediator of injury in a number of liver diseases. Despite their emerging importance, current methods of LSEC isolation (centrificugal elutriation or percoll adherence) are cumbersome, produce variable yields and have limited utility as a practical laboratory method. The proposed technique is based on the differential staining characteristics between CD31 (a marker of all endothelial cells) and CD32B (a marker specific for LSEC). Aim: To develop an isolation method for LSEC that would reliably obtain high yield with a specificity and reproducibility that would be easily available for wide spread use. Methods: We used an immunomagnetic method with the monoclonal antibodies: Rat Anti-Mouse F4/80 and Rat Anti-Mouse Fc gamma RIIB/CD32B. Non-Parenchymal cells were obtained by 0.05% Collagenase liver digestion and a 17% OptiPrep (iodixanol) density gradient. Cells were then subjected to Rat Anti-Mouse F4/80-conjugated Dynabeads to remove Kupffer cells and blood Monocytes. The remaining cells were subjected to magnetic bead cell isolation using Rat Anti-Mouse Fc gamma RIIB/CD32B. The attached LSEC were released from the beads with a specific decoupling buffer. Cells were then plated on a fibronectin matrix. Ninety percent of bead isolated cells were recovered with an average yield of 5.5 x10exp6 ±2 LSEC per mouse liver. Both mouse liver tissue and CD32B isolated cells were stained with immune labeled LSEC markers; CD31,CD32B and Stabilin-2 (Stab-2). Isotype controls were run for CD31, CD32B and a blocking peptide control was run for Stab-2. Results: All three markers stained positive in both mouse liver tissue and CD32B isolated cells. A co-stain with CD32B and Stab-2 showed co-localization in isolated LSEC and in liver tissue in a sinusoidal distribution. The CD32B isolated cells also showed typical LSEC fenestrae and sieve plates on scanning electron microscopy. Importantly, 99% of the isolated cells took up formaldehyde-treated serum albumin and oxidized low-density lipoprotein (oxLDL), which are functions and markers specific for LSEC. Conclusion: This straight forward isolation method allows for the culture of LSEC with high purity and reproducibility with a potential for more wide spread use than currently available methods.  

4.1220           Manual adult porcine islet isolation technique and optimal condition for adult

pig islets

Okitsu, T. Xenotransplantation, 20(5), 349 (2013)   In the case of requiring a high islet yield obtained efficiently through a single series of pancreas procurement and islet isolation procedure, adult pigs, especially retired breeders, are considered suitable to the islet donors. Besides the age of the pig, its race and particular feeding protocols could also influence the number and the morphology of the islets in the pancreas. Because islet yield depends on the na€õve number and how well morphology is preserved in the isolated islets, a series of pancreas procurement and islet isolation is recognized to be an important factor to determine islet yield. This presentation will introduce a simple islet isolation procedure that is modified based upon the static digestion method originally described by O’Neil et al [2001 Cell Transplantation]. In the procedure, at slaughterhouse, a whole pancreas from a retired breeder pig is procured within 30 min of warm ischemic time. A cannula is inserted into the main pancreatic duct, which had been connected to the duodenum. Through the cannula, 200–300 ml of M-Kyoto solution is infused into the pancreatic duct. Then the pancreas is preserved in oxygenized perfluorocarbon and transported to the laboratory. At the isolation laboratory, pancreas is distended using 300–400ml of Hank’s balanced salt solution containing 0.5 mg/ml of collagenase. The pancreas is cut into seven to nine pieces, put into a one liter Nalgene jar, and left in water bath at 37^a„ƒ for approximately 1 h until almost all the pancreas is digested. After the serous membrane of the pancreas is teared off using two pairs of forceps to allow the dissociated tissue to be released into the solution, all the tissue is filtrated through 500 lm mesh in a large filtration chamber and collected in 250 ml conical tubes. For islet purification, four 500 ml plastic containers with flat bottom are used to load discontinuous density gradients. This system allows us to purify up to 120 ml of dissociated pancreas tissue at a time. The discontinuous gradients consist of 100 ml each of M-Kyoto solution containing iodixanol at the density of 1.060, 1.096 and 1.110 g/ml; the bottom gradient of 1.110 g/ml solution is mixed with the dissociated pancreas tissue in advance. This system is centrifuged at 1000 rpm (240 9 g) for 5 min at 4^a„ƒ. The purified islets form a layer between the top and the second gradients and they are carefully collected and washed three times. Using this islet isolation method, we obtained a mean of 560,000 islet equivalent after purification with islet purity of greater than 60% (n = 6 isolations) from one adult pig. This method would be useful to isolate porcine islets of quite a few numbers in a stable and efficient manner through a single series of pancreas procurement and islet isolation using adult pigs.  

4.1221           Optimization of a porcine islet isolation and purification procedure that

utilizes recombinant collagenase

Green, M., Beechler, C., Breite, D., Dwulet, F. and McCarthy,R. Xenotransplantation, 20(5), 334 (2013)   Porcine pancreata represent an alternative xenogeneic source of islets for the clinical treatment of type 1 diabetes. However, the isolation and subsequent purification of porcine islets presents some challenging technical hurdles. Donor characteristics, procurement steps, type and level of enzymes used for tissue dissociation, digestion method, and purification protocol all significantly influence the isolation outcome. Using a standardized Ricordi procedure, which had been previously optimized for use with natural collagenases purified from C. histolyticum fermentation broth, we evaluated the performance of recombinant collagenases expressed in E. coli for use in porcine islet isolation. Pancreata from retired Landrace breeders (18–36 months of age) were divided into the splenic and duodenal/connecting lobes such that direct comparisons between test formulations could be made. Equivalent enzymatic target activities per gram of tissue were maintained between test formulations using specific activities determined from in-house substrate-specific assays. Recombinantly-expressed class II collagenase (rC2) targeted at 7.5 Wunsch units/g pancreas caused a 33% reduction (P < 0.001) in the mass of undigested tissue, an approximate 5 min quicker (P < 0.001) switch time and a 55% increase (P < 0.01) in packed tissue volume relative to naturally-derived C2 at the same target levels. Moreover, a further improvement (P = 0.03) in digestion was observed when recombinant C1 (rC1) rather than natural C1 was used in combination with rC2. Molecular form [i.e. intact (rC1) vs. truncated protein (rC1c)]of class I collagenase had no effect on any digestion parameter measured when equivalent collagen degrading activity was targeted, however, achieving such targets required approximately 19-fold higher enzyme masses for rC1c. Clostripain, a tryptic-like protease co-expressed in C. histolyticum fermentations, reduced (P < 0.01 and P < 0.001) the amount of undigested tissue by 21.6 and 35.5% when combined with naturally-derived and recombinant collagenase, respectively. Addition of a serine protease inhibitor (AEBSF; 0.4 mM) to the Ricordi circuit dramatically reduced the diameter of the liberated exocrine tissue pieces, and, consequently, improved islet release. Discontinuous polysucrose density gradients were inadequate in separating porcine islets from the contaminating exocrine tissue when recombinant collagenase was used for tissue dissociation. However, purification via a continuous iodixanol density gradient (1.069–1.108 g/cm3) resulted in high purity (>90%) preparations of porcine islets with high viability (>85%) as determined by SYTO13/propidium iodide staining. In summary, recombinantlyexpressed collagenases possess some inherent advantages over their naturallyderived counterparts that improve some digestion parameters, but also mandate modifications to the porcine islet isolation and purification protocol. The optimal formulation of rC2, rC1 and neutral protease for maximizing porcine islet yield and function is currently bein determined in a 23 factorial design.    

4.1222           Biliary obstruction results in PD-1-dependent liver T cell dysfunction and acute inflammation mediated by Th17 cells and neutrophils

Licata, L.A., Ngyuen, C.T., Burga, R.A., Falanga, V., Espat, N.J., Ayala, A., Thorn, M., Junghans, R.P. and katz, S.C.

1. Leukoc. Biol., 94(4), 813-823 (2013)

  Biliary obstruction is a common clinical problem that is associated with intrahepatic inflammation and impaired immunity. PD-1 is well known to mediate T cell dysfunction but has been reported to promote and attenuate acute inflammation in various injury models. With the use of a well-established murine model of BDL, we studied the effects of intrahepatic PD-1 expression on LTC function, inflammation, and cholestasis. Following BDL, PD-1 expression increased significantly among LTCs. Increased PD-1 expression following BDL was associated with decreased LTC proliferation and less IFN-γ production. Elimination of PD-1 expression resulted in significantly improved proliferative capacity among LTC following BDL, in addition to a more immunostimulatory cytokine profile. Not only was LTC function rescued in PD-1^−/− mice, but also, the degrees of biliary cell injury, cholestasis, and inflammation were diminished significantly compared with WT animals following BDL. PD-1-mediated acute inflammation following BDL was associated with expansions of intrahepatic neutrophil and Th17 cell populations, with the latter dependent on IL-6. PD-1 blockade represents an attractive strategy for reversing intrahepatic immunosuppression while limiting inflammatory liver damage.  

4.1223           Th9 Cells Drive Host Immunity against Gastrointestinal Worm Infection

Licona-Limon, P., Henao-Mejia, J., Temann, A.U., Gagliani, N., Licona.Limon, I., Ishigame, H., Hao, L., Herbert, D.B.R. and Flavell, A. Immunity, 39(4), 744-757 (2013)   Type 2 inflammatory cytokines, including interleukin-4 (IL-4), IL-5, IL-9, and IL-13, drive the characteristic features of immunity against parasitic worms and allergens. Whether IL-9 serves an essential role in the initiation of host-protective responses is controversial, and the importance of IL-9- versus IL-4-producing CD4⁺ effector T cells in type 2 immunity is incompletely defined. Herein, we generated IL-9-deficient and IL-9-fluorescent reporter mice that demonstrated an essential role for this cytokine in the early type 2 immunity against Nippostrongylus brasiliensis. Whereas T helper 9 (Th9) cells and type 2 innate lymphoid cells (ILC2s) were major sources of infection-induced IL-9 production, the adoptive transfer of Th9 cells, but not Th2 cells, caused rapid worm expulsion, marked basophilia, and increased mast cell numbers in Rag2-deficient hosts. Taken together, our data show a critical and nonredundant role for Th9 cells and IL-9 in host-protective type 2 immunity against parasitic worm infection.  

4.1224           3,3′-Diindolylmethane ameliorates experimental hepatic fibrosis via inhibiting miR-21 expression

Zhang, Z., Gao, Z., Hu, W., Yin, S., Wang, C., Zang, Y., Chen, J., Zhang, J. and Dong, L. Br. J. Pharmacol., 170(3), 649-660 (2013)   Background and Purpose Hepatic fibrosis is a type of liver disease characterized by excessive collagen deposition produced by activated hepatic stellate cells (HSCs), and no appropriate drug treatment is available clinically. The microRNA, miR-21 exhibits an important role in the pathogenesis and progression of hepatic fibrosis. 3,3′-Diindolylmethane (DIM) is a natural autolytic product in plants and can down-regulate miR-21 expression. Here we have assessed the therapeutic effects of DIM against hepatic fibrosis and investigated the underlying mechanisms. Experimental Approach The effects of DIM on HSC activation were measured by analysing the expression of α-smooth muscle actin and collagen I in both HSC-T6 cell line and primary HSCs. Expression of miR-21 was also measured after DIM treatment and the therapeutic effect of DIM was further studied in vivo, using the model of hepatic fibrosis induced by thioacetamide in mice. The antagonist oligonucleotide, antagomir-21, was also used to suppress the effects of miR-21. Key Results DIM suppressed the central TGF-β signalling pathway underlying HSC activation by down-regulating the expression of miR-21. The decreased miR-21 expression was achieved by inhibiting the activity of the transcription factor, AP-1. Moreover, DIM blunted the activation phenotype of primary HSCs. Administration of DIM in vivo attenuated liver fibrosis induced by thioacetamide, as assessed by collagen deposition and profiles of profibrogenic markers. Conclusions and Implications DIM shows potential as a therapeutic agent for the treatment of hepatic fibrosis.  

4.1225           Metabolic Assessment Prior to Total Pancreatectomy and Islet Autotransplant: Utility, Limitations and Potential

Lundberg, R., Beilman, G.J., Dunn, T.B., Pruett, T.L., Chinnakotla, S.C., Radosevich, D.M., Robertson, R.P., Ptacek, P., Balamurugan, A.N., Wilhelm, J.J., Hering, B.J., Sutherland, D.E.R., Moran, A. and Bellin, M.D. Am. J. Transplant., 13(10), 2664-2671 (2013)   Islet autotransplant (IAT) may ameliorate postsurgical diabetes following total pancreatectomy (TP), but outcomes are dependent upon islet mass, which is unknown prior to pancreatectomy. We evaluated whether preoperative metabolic testing could predict islet isolation outcomes and thus improve assessment of TPIAT candidates. We examined the relationship between measures from frequent sample IV glucose tolerance tests (FSIVGTT) and mixed meal tolerance tests (MMTT) and islet mass in 60 adult patients, with multivariate logistic regression modeling to identify predictors of islet mass ≥2500 IEQ/kg. The acute C-peptide response to glucose (ACRglu) and disposition index from FSIVGTT correlated modestly with the islet equivalents per kilogram body weight (IEQ/kg). Fasting and MMTT glucose levels and HbA1c correlated inversely with IEQ/kg (r values −0.33 to −0.40, p ≤ 0.05). In multivariate logistic regression modeling, normal fasting glucose (<100 mg/dL) and stimulated C-peptide on MMTT ≥4 ng/mL were associated with greater odds of receiving an islet mass ≥2500 IEQ/kg (OR 0.93 for fasting glucose, CI 0.87–1.0; OR 7.9 for C-peptide, CI 1.75–35.6). In conclusion, parameters obtained from FSIVGTT correlate modestly with islet isolation outcomes. Stimulated C-peptide ≥4 ng/mL on MMTT conveyed eight times the odds of receiving ≥2500 IEQ/kg, a threshold associated with reasonable metabolic control postoperatively.  

4.1226           TLR 2 and 4 Responsiveness from Isolated Peripheral Blood Mononuclear Cells from Rats and Humans as Potential Chronic Pain Biomarkers

Kwok, Y.H., Tuke, J., Nicotra, L.L., Grace, P.M., Rolan, R.E. and Hutchinson, M.R. PloS One, 8(10), e77799 (2013)   Background Chronic pain patients have increased peripheral blood mononuclear cell Interkeukin-1β production following TLR2 and TLR4 simulation. Here we have used a human-to-rat and rat-to-human approach to further investigate whether peripheral blood immune responses to TLR agonists might be suitable for development as possible systems biomarkers of chronic pain in humans. Methods and Results Study 1: using a graded model of chronic constriction injury in rats, behavioral allodynia was assessed followed by in vitro quantification of TLR2 and TLR4 agonist-induced stimulation of IL-1β release by PBMCs and spinal cord tissues (n = 42; 6 rats per group). Statistical models were subsequently developed using the IL-1β responses, which distinguished the pain/no pain states and predicted the degree of allodynia. Study 2: the rat-derived statistical models were tested to assess their predictive utility in determining the pain status of a published human cohort that consists of a heterogeneous clinical pain population (n = 19) and a pain-free population (n = 11). The predictive ability of one of the rat models was able to distinguish pain patients from controls with a ROC AUC of 0.94. The rat model was used to predict the presence of pain in a new chronic pain cohort and was able to accurately predict the presence of pain in 28 out of the 34 chronic pain participants. Conclusions These clinical findings confirm our previous discoveries of the involvement of the peripheral immune system in chronic pain. Given that these findings are reflected in the prospective graded rat data, it suggests that the TLR response from peripheral blood and spinal cord were related to pain and these clinical findings do indeed act as system biomarkers for the chronic pain state. Hence, they provide additional impetus to the neuroimmune interaction to be a drug target for chronic pain.  

4.1227           Gephyrin plays a key role in BDNF-dependent regulation of amygdala surface GABAARs

Mou, L., Dias, B.G., Gosnell, H. and ressler, K.J. Neuroscience, 255, 33-44 (2013)   Brain-derived neurotrophic factor (BDNF) is critically involved in synaptic plasticity and neurotransmission. Our lab has previously found that BDNF activation of neurotrophic tyrosine kinase, receptor, type 2 (TrkB) is required for fear memory formation and that GABA_A receptor (GABA_AR) subunits and the GABA_A clustering protein gephyrin are dynamically regulated during fear memory consolidation. We hypothesize that TrkB-dependent internalization of GABA_ARs may partially underlie a transient period of amygdala hyperactivation during fear memory consolidation. We have previously reported that BDNF modulates GABA_AR α1 subunit sequestration in cultured hippocampal and amygdala neurons by differential phosphorylation pathways. At present, no studies have investigated the regulation of gephyrin and GABA_AR α1 subunits following BDNF activation in the amygdala. In this study, we confirm the association of GABA_AR α1 and γ2 subunits with gephyrin on mouse amygdala neurons by coimmunoprecipitation and immunocytochemistry. We then demonstrate that rapid BDNF treatment, as well as suppression of gephyrin protein levels on amygdala neurons, induced sequestration of surface α1 subunits. Further, we find that rapid exposure of BDNF to primary amygdala cultures produced decreases in gephyrin levels, whereas longer exposure resulted in an eventual increase. While total α1 subunit levels remained unchanged, gephyrin was downregulated in whole cell homogenates, but enhanced in complexes with GABA_ARs. Our data with anisomycin suggest that BDNF may rapidly induce gephyrin protein degradation, with subsequent gephyrin synthesis occurring. Together, these findings suggest that gephyrin may be a key factor in BDNF-dependent GABA_AR regulation in the amygdala. This work may inform future studies aimed at elucidating the pathways connecting BDNF, GABA_A systems, gephyrin, and their role in underlying amygdala-dependent learning.  

4.1228           Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity

Ungvari, Z., Podlutski, A., Sosnowska, D., Tucsek, Z., Toth, P., Deak, F., Gautman, T., Csiszar, A. and Sonntag, W.E.

1. Gerontol. A Biol. Sci. Med. Sci., 68(12), 1443-1457 (2013)

  Cerebromicrovascular rarefaction is believed to play a central role in cognitive impairment in patients receiving whole-brain irradiation therapy. To elucidate the mechanism underlying the deleterious effects of γ-irradiation on the cerebral microcirculation, rat primary cerebromicrovascular endothelial cells (CMVECs) were irradiated in vitro. We found that in CMVECs, γ-irradiation (2–8 Gy) elicited increased DNA damage, which was repaired less efficiently in CMVECs compared with neurons, microglia, and astrocytes. Increased genomic injury in CMVECs associated with increased apoptotic cell death. In the surviving cells, γ-irradiation promotes premature senescence (indicated by SA-β-galactosidase positivity and upregulation of p16^INK4a), which was associated with impaired angiogenic capacity (decreased proliferation and tube-forming capacity). γ-Irradiated CMVECs acquired a senescence-associated secretory phenotype, characterized by upregulation of proinflammatory cytokines and chemokines (including IL-6, IL-1α, and MCP-1). Collectively, increased vulnerability of γ-irradiated CMVECs and their impaired angiogenic capacity likely contribute to cerebromicrovascular rarefaction and prevent regeneration of the microvasculature postirradiation. The acquisition of a senescence-associated secretory phenotype in irradiated CMVECs is biologically highly significant as changes in the cytokine microenvironment in the hippocampus may affect diverse biological processes relevant for normal neuronal function (including regulation of neurogenesis and the maintenance of the blood brain barrier).  

4.1229           Toll-Like Receptor 4–Dependent Microglial Activation Mediates Spinal Cord Ischemia–Reperfusion Injury

Bell, M.T., Puskas, F., Agoston, V.A., Cleveland Jr., J.C., Freeman, K.A., Gamboni, F., Herson, P.S., Meng, X., Smith, P.D., Weyant, M.J., Fullerton, D.A. and Reece, T.B. Circulation, 128, S152-S156 (2013)   Background—Paraplegia continues to complicate thoracoabdominal aortic interventions. The elusive mechanism of spinal cord ischemia–reperfusion injury has delayed the development of pharmacological adjuncts. Microglia, the resident macrophages of the central nervous system, can have pathological responses after a variety of insults. This can occur through toll-like receptor 4 (TLR-4) in stroke models. We hypothesize that spinal cord ischemia–reperfusion injury after aortic occlusion results from TLR-4–mediated microglial activation in mice. Methods and Results—TLR-4 mutant and wild-type mice underwent aortic occlusion for 5 minutes, followed by 60 hours of reperfusion when spinal cords were removed for analysis. Spinal cord cytokine production and microglial activation were assessed at 6 and 36 hours after surgery. Isolated microglia from mutant and wild-type mice were subjected to oxygen and glucose deprivation for 24 hours, after which the expression of TLR-4 and proinflammatory cytokines was analyzed. Mice without functional TLR-4 demonstrated decreased microglial activation and cytokine production and had preserved functional outcomes and neuronal viability after thoracic aortic occlusion. After oxygen and glucose deprivation, wild-type microglia had increased TLR-4 expression and production of proinflammatory cytokines. Conclusions—The absence of functional TLR-4 attenuated neuronal injury and microglial activation after thoracic aortic occlusion in mice. Furthermore, microglial upregulation of TLR-4 occurred after oxygen and glucose deprivation, and the absence of functional TLR-4 significantly attenuated the production of proinflammatory cytokines. In conclusion, TLR-4–mediated microglia activation in the spinal cord after aortic occlusion is critical in the mechanism of paraplegia after aortic cross-clamping and may provide targets for pharmacological intervention.  

4.1230           Purification and Culture of Spinal Motor Neurons from Rat Embryos

Graber, D.J. and Harris, B.T. Cold Spring Harb. Protoc., pdb.top070920, 310-311 (2013)   We describe an immunopanning protocol to isolate, enrich, and culture spinal motor neurons from rat embryonic spinal cords. The method takes advantage of several distinct properties of rat lower motor neurons to isolate them from neighboring cells. First, an ideal stage in development after motor neurons are born (embryonic day 14 during rat gestation), but prior to extensive axonal extension or developmental apoptosis, is exploited. Lower motor neurons cannot be viably isolated using this method after birth. After dissociating embryonic spinal cord tissue, which contains lower motor neurons among many other cell types, the uniquely large motor neurons are enriched using density gradient centrifugation. Finally, the collected cell population is further purified based on selective immunopanning for motor neurons, which express the low-affinity nerve growth factor (NGF) receptor often referred to as p75. The near-pure lower motor neuron cultures are plated and seeded in defined conditions optimal for survival and can be maintained for several weeks. The expected yield is approximately 70,000 cells per embryonic spinal cord.  

4.1231           Induction of pulmonary hypertensive changes by extracellular vesicles from monocrotaline-treated mice

Aliotta, J.M., Pereira, M., Amaral, A., Sorokina, A., Igbinoba, Z., Hasslinger, A., El-Bizri, R., Rounds, S.I., Quesenberry, P.J. and Klinger, J.R. Cardiovasc. Res.,100, 354-362 (2013)   Aims Circulating endothelium-derived extracellular vesicles (EV) levels are altered in pulmonary arterial hypertension (PAH) but whether they are biomarkers of cellular injury or participants in disease pathogenesis is unknown. Previously, we found that lung-derived EVs (LEVs) induce bone marrow-derived progenitor cells to express lung-specific mRNA and protein. In this study, we sought to determine whether LEV or plasma-derived EV (PEV) alter pulmonary vascular endothelial or marrow progenitor cell phenotype to induce pulmonary vascular remodelling. Methods and results LEV, PEV isolated from monocrotaline (MCT-EV)- or vehicle-treated mice (vehicle-EV) were injected into healthy mice. Right ventricular (RV) hypertrophy and pulmonary vascular remodelling were assessed by RV-to-body weight (RV/BW) and blood vessel wall thickness-to-diameter (WT/D) ratios. RV/BW, WT/D ratios were elevated in MCT- vs. vehicle-injected mice (1.99 ± 0.09 vs. 1.04 ± 0.09 mg/g; 0.159 ± 0.002 vs. 0.062 ± 0.009%). RV/BW, WT/D ratios were higher in mice injected with MCT-EV vs. mice injected with vehicle-EV (1.63 ± 0.09 vs. 1.08 ± 0.09 mg/g; 0.113 ± 0.02 vs. 0.056 ± 0.01%). Lineage-depleted bone marrow cells incubated with MCT-EV and marrow cells isolated from mice infused with MCT-EV had greater expression of endothelial progenitor cell mRNAs and mRNAs abnormally expressed in PAH than cells incubated with vehicle-EV or isolated from vehicle-EV infused mice. MCT-EV induced an apoptosis-resistant phenotype in murine pulmonary endothelial cells and lineage-depleted bone marrow cells incubated with MCT-EV induced pulmonary hypertension when injected into healthy mice. Conclusions EV from MCT-injured mice contribute to the development of MCT-induced pulmonary hypertension. This effect may be mediated directly by EV on the pulmonary vasculature or by differentiation of bone marrow cells to endothelial progenitor cells that induce pulmonary vascular remodelling.  

4.1232            A method for biomarker measurements in peripheral blood mononuclear cells isolated from anxious and depressed mice: β-arrestin 1 protein levels in depression and treatment

Mendez-David, I., El-Ali, Z., Hen, R., Falissard, B., Corruble, E., gardier, A.M., Kerdine-Römer, S. and David, D.J. Frontiers in Pharmacol., 4:124 (2013)   A limited number of biomarkers in the central and peripheral systems which are known may be useful for diagnosing major depressive disorders and predicting the effectiveness of antidepressant (AD) treatments. Since 60% of depressed patients do not respond adequately to medication or are resistant to ADs, it is imperative to delineate more accurate biomarkers. Recent clinical studies suggest that β-arrestin 1 levels in human mononuclear leukocytes may be an efficient biomarker. If potential biomarkers such as β-arrestin 1 could be assessed from a source such as peripheral blood cells, then they could be easily monitored and used to predict therapeutic responses. However, no previous studies have measured β-arrestin 1 levels in peripheral blood mononuclear cells (PBMCs) in anxious/depressive rodents. This study aimed to develop a method to detect β-arrestin protein levels through immunoblot analyses of mouse PBMCs isolated from whole blood. In order to validate the approach, β-arrestin levels were then compared in naïve, anxious/depressed mice, and anxious/depressed mice treated with a selective serotonin reuptake inhibitor (fluoxetine, 18 mg/kg/day in the drinking water). The results demonstrated that mouse whole blood collected by submandibular bleeding permitted isolation of enough PBMCs to assess circulating proteins such as β-arrestin 1. β-Arrestin 1 levels were successfully measured in healthy human subject and naïve mouse PBMCs. Interestingly, PBMCs from anxious/depressed mice showed significantly reduced β-arrestin 1 levels. These decreased β-arrestin 1 expression levels were restored to normal levels with chronic fluoxetine treatment. The results suggest that isolation of PBMCs from mice by submandibular bleeding is a useful technique to screen putative biomarkers of the pathophysiology of mood disorders and the response to ADs. In addition, these results confirm that β-arrestin 1 is a potential biomarker for depression.  

4.1233           Investigation of the effect of Mycobacterium bovis infection on bovine neutrophils functions

Wang, J., Zhou, X., Pan, B., Yang, L., Yin, X., Xu, B. and Zhao, D. Tuberculosis, 93, 675-687 (2013)   Bovine tuberculosis is a disease in cattle caused by infection with Mycobacterium bovis. The disease has posed significant economic losses and remains a public health hazard worldwide. Interactions between M. bovis and bovine macrophages have been extensively characterized in various studies, while similar analyses in neutrophils, which are one of the other types of white blood cells in mammals, were often overlooked. Neutrophils provide defense against all microbes and can present a diverse collection of antimicrobial molecules, which play an important role in the control of tuberculosis progression. Much of the available data about the involvement of neutrophils in the killing M. bovis is controversial. In this study, we assessed the effect of in vitro infection with M. bovis on some parameters of neutrophils functions including phenotypic changes, apoptosis rate and inflammatory cytokines production. Our results demonstrated that phagocytosis of M. bovis activated and enhanced bovine neutrophils functions as well as initialed their defense mechanism, but failed to eliminate the mycobacteria. Moreover, autophagy might get involved in the defense infection process functioning as a protective mechanism, and inducible-autophagy by lipopolysaccharides stimulation and starvation treatment could efficiently reverse the inability of neutrophils for killing M. bovis, suggesting a potential target for anti-mycobacterial drug-therapy.  

4.1234           Stage specific reprogramming of mouse embryo liver cells to a beta cell-like phenotype

Yang, Y., Akinci, E., Dutton, J.R., banga, A. and Slack, J.M.W. Mechanisms of Development, 130, 602-612 (2013)   We show that cultures of mouse embryo liver generate insulin-positive cells when transduced with an adenoviral vector encoding the three genes: Pdx1, Ngn3 and MafA (Ad-PNM). Only a proportion of transduced cells become insulin-positive and the highest yield occurs in the period E14–16, declining at later stages. Insulin-positive cells do not divide further although they can persist for several weeks. RT-PCR analysis of their gene expression shows the upregulation of a whole battery of genes characteristic of beta cells including upregulation of the endogenous counterparts of the input genes. Other features, including a relatively low insulin content, the expression of genes for other pancreatic hormones, and the fact that insulin secretion is not glucose-sensitive, indicate that the insulin-positive cells remain immature. The origin of the insulin-positive cells is established both by co-immunostaining for α-fetoprotein and albumin, and by lineage tracing for Sox9, which is expressed in the ductal plate cells giving rise to biliary epithelium. This shows that the majority of insulin-positive cells arise from hepatoblasts with a minority from the ductal plate cells.  

4.1235           121 Effects of iodixanol during rat epididymal sperm cryopreservation

Kim, S., Agca, C.a dn Agca, Y. Cryobiology, 67(3), 432 (2013)   Sperm cryopreservation is an effective method of maintaining valuable strains for biomedical research. However, successful cryopreservation of rat spermatozoa remains a challenge. The objective of this study was to determine if OptiPrepTM (60% iodixanol in water) acts as a protectant during Sprague Dawley (SD) rat sperm cryopreservation. We first evaluated OptiPrepTM concentration effect on sperm motility 10 min and 3 h after freezing-thawing. Acrosomal integrity and mitochondrial membrane potential (MMP) were also evaluated for frozen-thawed spermatozoa. In addition, the toxic effects of OptiPrepTM were tested via motility after 1 h or 3 h incubation with OptiPrepTM in fresh and chilled rat spermatozoa. The concentrations of OptiPrepTM used for these studies were 0% (control), 1%, 2%, 3%, and 4% (v/v). There were no significant differences on motility among OptiPrepTM treatment groups in fresh and chilled rat spermatozoa. OptiPrepTM did not have toxic effect on SD rat spermatozoa. In frozen-thawed samples, 2% OptiPrepTM improved total motility at 10 min after thawing and progressive motility and average path velocity at 3 h after thawing (P < 0.05) and MMP (P < 0.05) while OptiPrepTM did not show difference for acrosomal integrity compared to control. Intra uterine insemination (IUI) was performed for OptiPrepTM -contained cryopreserved spermatozoa showing best results on sperm evaluation parameters. While 9 out of 10 (90%) rats become pregnant after intrauterine insemination (IUI), 4 out of 11 (36%) rats become pregnant after IUI using frozen-thaw SD sperm. These data suggest that iodixanol may have cryoprotective effect during rat sperm freezing without any toxic effects.  

4.1236           CD4 blockade directly inhibits mouse and human CD4+ T cell functions independent of Foxp3+ Tregs

Mayer, C.T., Huntenberg, J., Nandan, A., Schmitt, E., Czeloth, N. and Sparwasser, T.

1. Autoimunity, 47, 73-82 (2013)

  CD4⁺ helper T cells orchestrate protective immunity against pathogens, yet can also induce undesired pathologies including allergies, transplant rejection and autoimmunity. Non-depleting CD4-specific antibodies such as clone YTS177.9 were found to promote long-lasting T cell tolerance in animal models. Thus, CD4 blockade could represent a promising therapeutic approach for human autoimmune diseases. However, the mechanisms underlying anti-CD4-induced tolerance are incompletely resolved. Particularly, multiple immune cells express CD4 including Foxp3⁺ regulatory T cells (Tregs) and dendritic cells (DCs), both controlling the activation of CD4⁺Foxp3⁻ helper T cells. Utilizing mixed leukocyte reactions (MLRs) reflecting physiological interactions between T cells and DCs, we report that anti-CD4 treatment inhibits CD4⁺Foxp3⁻ T cell proliferation in an IL-2-independent fashion. Notably, YTS177.9 binding induces a rapid internalization of CD4 on both CD4⁺Foxp3⁻ T cells and Foxp3⁺ Tregs. However, no expansion or activation of immunosuppressive CD4⁺Foxp3⁺ Tregs was observed following anti-CD4 treatment. Additionally, cytokine production, maturation and T cell priming capacity of DCs are not affected by anti-CD4 exposure. In line with these data, the selective ablation of Foxp3⁺ Tregs from MLRs by the use of diphtheria toxin (DT)-treated bacterial artificial chromosome (BAC)-transgenic DEREG mice completely fails to abrogate the suppressive activity of multiple anti-CD4 antibodies. Instead, tolerization is associated with the defective expression of various co-stimulatory receptors including OX40 and CD30, suggesting altered signaling through the TCR complex. Consistent with our findings in mice, anti-CD4 treatment renders human CD4⁺ T cells tolerant in the absence of Tregs. Thus, our results establish that anti-CD4 antibodies can directly tolerize pathogenic CD4⁺Foxp3⁻ helper T cells. This has important implications for the treatment of human inflammatory diseases.  

4.1237           GABA Protects Human Islet Cells Against the Deleterious Effects of Immunosuppressive Drugs and Exerts Immunoinhibitory Effects Alone

Prud’homme, G.J., Glinka, Y., Hasilo, C., Paraskevas, S., Li, X. and Wang, Q. Transplantation, 98(7), 616-623 (2013)   Background: We recently found that γ-aminobutyric acid (GABA) protects mouse islet β cells. It prevented autoimmune type 1 diabetes in mice, induced islet β-cell regeneration, and exerted immunoinhibitory effects. However, it is not known whether GABA would be equally active on human islet and immune cells. Methods: In vitro culture of human islets and immune cells with or without GABA and immunosuppressive drugs. In vitro analysis of apoptosis, proliferation, nuclear factor (NF)-κB activation, calcium signaling, and insulin secretion. Results: GABA reduced human islet cell apoptosis in culture, such that the yield of live cells was approximately tripled after 1 week, and it stimulated insulin secretion. It protected against the deleterious effects of rapamycin, tacrolimus, and mycophenolate mofetil. In human immune cells, GABA had inhibitory effects similar to mouse cells, such as suppressed anti-CD3–stimulated T-cell proliferation, in a GABA type A receptor–dependent fashion. The immunosuppressive mechanisms have been unclear, but we found that GABA blocked calcium influx, which is a key activation signal. GABA also suppressed NF-κB activation in both human islet cells and immune cells. We found that it could be combined with rapamycin to increase its suppressive effects. Conclusions: GABA improved human islet cell survival and had suppressive effects on human immune cells. It inhibited canonical NF-κB activation in both islet and immune cells. This is important because activation of this pathway is detrimental to islet cells and likely promotes damaging autoimmunity and alloreactivity against transplanted islets. These findings suggest that GABA might find applications in clinical islet transplantation.  

4.1238           Long-Term Functions of Encapsulated Islets Grafted in Nonhuman Primates Without Immunosuppression

Sasikala, M., Rao, G.V., Vijayalakshmi, V., Pradeep, R., Pothani, S., Kumar, P.P., Gaddipati, R., Sirisha, G., Cheemalakonda, R., Tandan, M., Subramanyam, C., Vasudevan, S. and Reddy, D.N. Transplantation, 96(7), 624-632 (2013) Background: Long-term survival and functions of encapsulated islet grafts need to be evaluated in the absence of immunosuppression. The present study aimed to assess the viability and functions of macroencapsulated islets grafted in nonhuman primates without immunosuppression for 1 year. Methods: Islet transplantations were performed in partially pancreatectomized rhesus monkeys (two autologous and four allogenic) without immunosuppression using immunoisolatory devices. Macroencapsulated islets were implanted subcutaneously (5000–8000 IEQ/device) at two sites (left thigh and interscapular region) and were explanted at 2, 6, and 12 months after implantation. Staining for viability and apoptosis, in vivo and in vitro glucose-stimulated insulin release, expression of insulin and glucagon genes, and histopathologic examination of the device were used to assess engraftment potential, viability, and functions of islets. Animals were regularly monitored for dietary intake, body weight, and fasting blood glucose levels after islet transplantation. Results: Devices explanted showed vascularization at the end of 2, 6, and 12 months with occasional lymphocytes and minimal fibrosis outside the device. Flow cytometric analysis revealed 97.9%±1.5% and 94.3%±5.71% viable β cells in interscapular site and thigh in autologous recipients and 85.6%±4.01% (interscapular site) and 74.1%±12.05% (thigh) viable β cells in allogenic islet recipients. In vivo glucose challenge test revealed significantly increased glucose-stimulated insulin release (P=0.028) in the left thigh with implant (17.58±3.13 mU/L) compared with the thigh without implant (9.86±1.063 mU/L). Insulin and glucagon gene expression was evident in islets recovered from explanted device. Conclusions: These results indicate that subcutaneous implantation of macroencapsulated islets is minimally invasive and has potential for transplantation without immunosuppression.  

4.1239           Electrical Stimulation of Embryonic Neurons for 1 Hour Improves Axon Regeneration and the Number of Reinnervated Muscles That Function

Liu, Y., Grumbles, R.M. and Thomas, C.K.

1. Neuropathol. Exp. Neurol., 72(7), 697-707 (2013)

  Motoneuron death after spinal cord injury or disease results in muscle denervation, atrophy, and paralysis. We have previously transplanted embryonic ventral spinal cord cells into the peripheral nerve to reinnervate denervated muscles and to reduce muscle atrophy, but reinnervation was incomplete. Here, our aim was to determine whether brief electrical stimulation of embryonic neurons in the peripheralnerve changes motoneuron survival, axon regeneration, and muscle reinnervation and function because neural depolarization is crucial for embryonic neuron survival and may promote activity-dependent axon growth. At 1 week after denervation by sciatic nerve section, embryonic day 14 to 15 cells were purified for motoneurons, injected into the tibial nerve of adult Fischer rats, and stimulated immediatelyfor up to 1 hour. More myelinated axons were present in tibial nerves 10 weeks after transplantation when transplants had been stimulated acutely at 1 Hz for 1 hour. More muscles were reinnervated if the stimulation treatment lasted for 1 hour. Reinnervation reduced muscle atrophy, with or without the stimulation treatment. These data suggest that brief stimulation of embryonic neurons promotes axon growth, which has a long-term impact on muscle reinnervation and function. Muscle reinnervation is important because it may enable the use of functional electrical stimulation to restore limb movements.  

4.1240           The ALS disease-associated mutant TDP-43 impairs mitochondrial dynamics and function in motor neurons

Wang, W., Li, L., Lin, W-L., Dickson, D.W., Petrucelli, L., Zhang, T. and Wang, X. Human Mol. Genet., 22(23), 4706-4719 (2013)   Mutations in TDP-43 lead to familial ALS. Expanding evidence suggests that impaired mitochondrial dynamics likely contribute to the selective degeneration of motor neurons in SOD1-associated ALS. In this study, we investigated whether and how TDP-43 mutations might impact mitochondrial dynamics and function. We demonstrated that overexpression of wild-type TDP-43 resulted in reduced mitochondrial length and density in neurites of primary motor neurons, features further exacerbated by ALS-associated TDP-43 mutants Q331K and M337V. In contrast, suppression of TDP-43 resulted in significantly increased mitochondrial length and density in neurites, suggesting a specific role of TDP-43 in regulating mitochondrial dynamics. Surprisingly, both TDP-43 overexpression and suppression impaired mitochondrial movement. We further showed that abnormal localization of TDP-43 in cytoplasm induced substantial and widespread abnormal mitochondrial dynamics. TDP-43 co-localized with mitochondria in motor neurons and their colocalization was enhanced by ALS associated mutant. Importantly, co-expression of mitochondrial fusion protein mitofusin 2 (Mfn2) could abolish TDP-43 induced mitochondrial dynamics abnormalities and mitochondrial dysfunction. Taken together, these data suggest that mutant TDP-43 impairs mitochondrial dynamics through enhanced localization on mitochondria, which causes mitochondrial dysfunction. Therefore, abnormal mitochondrial dynamics is likely a common feature of ALS which could be potential new therapeutic targets to treat ALS.  

4.1241           Fortilin reduces apoptosis in macrophages and promotes atherosclerosis

Pinkaew, D., Le, R.J., Chen, Y., Eltorky, M., teng, B-B. and Fujise, K. Am. J. Physiol. Heart Circ. Physiol., 305, H1519-H1529 (2013)   Atherosclerosis, a deadly disease insufficiently addressed by cholesterol-lowering drugs, needs new therapeutic strategies. Fortilin, a 172-amino acid multifunctional polypeptide, binds p53 and blocks its transcriptional activation of Bax, thereby exerting potent antiapoptotic activity. Although fortilin-overexpressing mice reportedly exhibit hypertension and accelerated atherosclerosis, it remains unknown if fortilin, not hypertension, facilitates atherosclerosis. Our objective was to test the hypothesis that fortilin in and of itself facilitates atherosclerosis by protecting macrophages against apoptosis. We generated fortilin-deficient (fortilin^+/−) mice and wild-type counterparts (fortilin^+/+) on a LDL receptor (Ldlr)^−/− apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (Apobec1)^−/− hypercholesterolemic genetic background, incubated them for 10 mo on a normal chow diet, and assessed the degree and extent of atherosclerosis. Despite similar blood pressure and lipid profiles, fortilin^+/− mice exhibited significantly less atherosclerosis in their aortae than their fortilin+/+ littermate controls. Quantitative immunostaining and flow cytometry analyses showed that the atherosclerotic lesions of fortilin^+/− mice contained fewer macrophages than those of fortilin^+/+ mice. In addition, there were more apoptotic cells in the intima of fortilin^+/− mice than in the intima of fortilin^+/+ mice. Furthermore, peritoneal macrophages from fortilin^+/− mice expressed more Bax and underwent increased apoptosis, both at the baseline level and in response to oxidized LDL. Finally, hypercholesterolemic sera from Ldlr^−/−Apobec1^−/− mice induced fortilin in peritoneal macrophages more robustly than sera from control mice. In conclusion, fortilin, induced in the proatherosclerotic microenvironment in macrophages, protects macrophages against Bax-induced apoptosis, allows them to propagate, and accelerates atherosclerosis. Anti-fortilin therapy thus may represent a promising next generation antiatherosclerotic therapeutic strategy.  

4.1242           Dendritic Cell-Specific Delivery of Flt3L by Coronavirus Vectors Secures Induction of Therapeutic Antitumor Immunity

Perez-Shibayama, C., Gil-Cruz, C., Nussbacher, M., Allgäuer, E., Cervantes-Barragan, L., Züst, R. and Ludewig, B. PloS One, 8(11), e81442 (2013)   Efficacy of antitumor vaccination depends to a large extent on antigen targeting to dendritic cells (DCs). Here, we assessed antitumor immunity induced by attenuated coronavirus vectors which exclusively target DCs in vivo and express either lymphocyte- or DC-activating cytokines in combination with a GFP-tagged model antigen. Tracking of in vivo transduced DCs revealed that vectors encoding for Fms-like tyrosine kinase 3 ligand (Flt3L) exhibited a higher capacity to induce DC maturation compared to vectors delivering IL-2 or IL-15. Moreover, Flt3L vectors more efficiently induced tumor-specific CD8⁺ T cells, expanded the epitope repertoire, and provided both prophylactic and therapeutic tumor immunity. In contrast, IL-2- or IL-15-encoding vectors showed a substantially lower efficacy in CD8⁺ T cell priming and failed to protect the host once tumors had been established. Thus, specific in vivo targeting of DCs with coronavirus vectors in conjunction with appropriate conditioning of the microenvironment through Flt3L represents an efficient strategy for the generation of therapeutic antitumor immunity.  

4.1243           Ezh1 and Ezh2 differentially regulate PSD-95 gene transcription in developing hippocampal neurons

Henriquez, B., Bustos, F., Aguilar, R., Becerra, A., Simon, F., Montecino, M. and van Zundert, B. Mol. Cell. Neurosci., 57, 130-143 (2013)   Polycomb Repressive Complex 2 (PRC2) mediates transcriptional silencing by catalyzing histone H3 lysine 27 trimethylation (H3K27me3), but its role in the maturation of postmitotic mammalian neurons remains largely unknown. We report that the PRC2 paralogs Ezh1 and Ezh2 are differentially expressed during hippocampal development. We show that depletion of Ezh2 leads to increased expression of PSD-95, a critical plasticity gene, and that reduced PSD-95 gene transcription is correlated with enrichment of Ezh2 at the PSD-95 gene promoter; however, the H3K27me3 epigenetic mark is not present at the PSD-95 gene promoter, likely due to the antagonizing effects of the H3S28P and H3K27Ac marks and the activity of the H3K27 demethylases JMJD3 and UTX. In contrast, increased PSD-95 gene transcription is accompanied by the presence of Ezh1 and elongation-engaged RNA Polymerase II complexes at the PSD-95 gene promoter, while knock-down of Ezh1 reduces PSD-95 transcription. These results indicate that Ezh1 and Ezh2 have antagonistic roles in regulating PSD-95 transcription.  

4.1244           Engineering the type III secretion system in non-replicating bacterial minicells for antigen delivery

Carleton, H.A., Lara-Tejero, M., Liu, X. and Galan, J.E. Nature Communications, 4:1590 (2013)   Type III protein secretion systems are being considered for vaccine development as virtually any protein antigen can be engineered for delivery by these nanomachines into the class I antigen presentation pathway to stimulate antigen-specific CD8⁺ T cells. A limitation in the use of this system is that it requires live virulence-attenuated bacteria, which may preclude its use in certain populations such as children and the immunocompromised. Here we report the engineering of the Salmonella Typhimurium type III secretion system in achromosomal, non-replicating nanoparticles derived from bacterial minicells. The engineered system is shown to be functional and capable of delivering heterologous antigens to the class I antigen presentation pathway stimulating immune responses both in vitro and in vivo. This antigen delivery platform offers a novel approach for vaccine development and cellular immunotherapy.    

4.1245           The single-nucleotide polymorphism (GPX4c718t) in the glutathione peroxidase 4 gene influences endothelial cell function: Interaction with selenium and fatty acids

Crosley, L.K., Bashir, S., Nicol., F., Arthur,J.R., Hesketh, J.E. and Sneddon, A.A. Mol. Nutr. Food Res., 57(12), 2185-2194 (2013)   Scope Selenium (Se) is incorporated into selenoproteins as selenocysteine, which requires structures in the 3′-untranslated region (3′-UTR) of selenoprotein mRNAs. The functional consequences of a single nucleotide polymorphism (SNP) within the 3′-UTR of the selenoprotein GPX4 gene (GPX4c718t) was assessed in human umbilical vein endothelial cells (HUVECs) and monocytes from human volunteers. Methods and results HUVEC and monocytes homozygous for the T- or C-variant of the GPX4c718t SNP were assessed for monocyte–endothelial cell adhesion, expression of VCAM-1 and sensitivity to oxidative challenge. Interaction of the SNP with Se and different PUFA and effects on selenoprotein expression were also investigated. HUVEC and monocytes homozygous for the T-variant showed elevated adhesion levels compared to cells of the C-variant. This effect was modified by Se and PUFA. HUVEC homozygous for the T-variant showed elevated levels of VCAM-1 protein in the presence of arachidonic acid, were more sensitive to oxidative challenge and showed Se-dependant changes in lipid peroxide levels and expression of additional selenoproteins. Conclusion These findings demonstrate functional effects of the GPX4c718t SNP in endothelial cells and may suggest that individuals with the TT genotype have impaired endothelial function and are at greater risk of vascular disease compared to individuals with the CC genotype.  

4.1246           Anti-Inflammatory Potency of Nano-Formulated Puerarin and Curcumin in Rats Subjected to the Lipopolysaccharide-Induced Inflammation no access

Singh, A.K:, Jiang, Y., Gupta, s., Younus, M. and Ramzan, M.

1. Medicinal Food, 16(10), 899-911 (2013)

  Puerarin (PU) and curcumin (CU), used commonly in traditional Chinese medicine and Ayurveda, have been shown to possess potent anti-inflammatory, anti-oxidation, and neuro-protective properties. Despite the experimental success of CU and PU in in vitro and animal models, their effectiveness has not yet been demonstrated in clinical trials, possibly because of their poor bioavailability. We hypothesized that gold nanoparticle (AuNP)-formulated PU (PU-AuNP), CU (CU-AuNP), or a combination of PU and CU (PU-CU-AuNP) were a more effective and nontoxic alternative to their bulk (nonformulated) counterparts. To test the hypothesis, bioavailability, therapeutic potency, and toxicity of bulk CU and/or PU were compared with those of their nanotized counterparts in rats subjected to the lipopolysaccharide (LPS)-induced inflammation. This study showed that a 20-mg/kg dose of bulk PU or a mixture of PU and CU did not, while their nanotized counterparts, PU-AuNP, CU-AuNP, or PU-CU-AuNP, effectively suppressed the LPS-induced inflammation and cytotoxicity in rats. In addition, PU-CU-AuNP was more potent than PU-AuNP or CU-AuNP alone. The blank AuNP (bAuNP) at ≤40 mg/kg dose did not cause any adverse effects (blood and brain lactic acid concentrations, kidney function, and neuronal apoptosis were measured) in animals. Therefore, the present observations suggest that a bi-functional AuNP loaded with CU and PU may effectively suppress the LPS-induced inflammation and cytotoxicity provided the following conditions are met: (1) The AuNP dose is at or below the no-effect dose; (2) the nanoparticles release a therapeutic dose of CU and PU in vivo; and (3) the active ingredients are released into the intracellular component of the brain.  

4.1247           Upregulation of axon guidance molecules in the adult central nervous system of Nogo-A knockout mice restricts neuronal growth and regeneration

Kempf, A., Montani, L., Petrinovic, M.M., Schroeter, A., Weinmann, O., Patrigani, A. and Schwab, M.E. Eur. J. Neurosci., 38(11), 3567-3579 (2013)   Adult central nervous system axons show restricted growth and regeneration properties after injury. One of the underlying mechanisms is the activation of the Nogo-A/Nogo receptor (NgR1) signaling pathway. Nogo-A knockout (KO) mice show enhanced regenerative growth in vivo, even though it is less pronounced than after acute antibody-mediated neutralization of Nogo-A. Residual inhibition may involve a compensatory component. By mRNA expression profiling and immunoblots we show increased expression of several members of the Ephrin/Eph and Semaphorin/Plexin families of axon guidance molecules, e.g. EphrinA3 and EphA4, in the intact spinal cord of adult Nogo-A KO vs. wild-type (WT) mice. EphrinA3 inhibits neurite outgrowth of EphA4-positive neurons in vitro. In addition, EphrinA3 KO myelin extracts are less growth-inhibitory than WT but more than Nogo-A KO myelin extracts. EphA4 KO cortical neurons show decreased growth inhibition on Nogo-A KO myelin as compared with WT neurons, supporting increased EphA4-mediated growth inhibition in Nogo-A KO mice. Consistently, in vivo, Nogo-A/EphA4 double KO mice show increased axonal sprouting and regeneration after spinal cord injury as compared with EphA4 KO mice. Our results reveal the upregulation of developmental axon guidance cues following constitutive Nogo-A deletion, e.g. the EphrinA3/EphA4 ligand/receptor pair, and support their role in restricting neurite outgrowth in the absence of Nogo-A.  

4.1248           Salvianolic acid B inhibits hepatic stellate cell activation through transforming growth factor beta-1 signal transduction pathway in vivo and in vitro

Tao, Y-Y., Wang, Q-L., Shen, L., Fu, W-W. and Liu, C-H. Exp.Biol. Med., 238(11), 1284-1296 (2013)   Salvianolic acid B (Sal B) is a major water soluble component extracted from Radix Salviae miltiorrhizae, a traditional Chinese herb widely used for treating cardiovascular and hepatic diseases. Sal B has been reported to inhibit transforming growth factor (TGF)-β1-stimulated hepatic stellate cells (HSCs) activation and collagen type I expression. In this study, we further investigated the mechanisms of Sal B on liver fibrosis relating to TGF-β/Smads signalling pathway, especially to TGF-β1 receptors. Liver fibrosis model was induced by intraperitoneal injection of dimethylnitrosamine (DMN) for four weeks. Rats were randomly divided into three groups: normal, model, and Sal B groups. Rats in Sal B group were treated by oral administration of Sal B for four weeks from the first day of DMN exposure. Hydroxyproline (Hyp) content in liver tissue was assayed using Jamall's method and collagen deposition was visualized using Sirius red staining. HSCs were isolated from normal rats, and were cultured primarily in uncoated plastics. At day 4 after isolation, cells were stimulated with 2.5 ng/mL TGF-β1, and treated with 1 and 10 µmol/L Sal B and 10 µmol/L SB-431542 (TβR-I inhibitor) for 24 h, respectively. Cell proliferation was examined with 5-ethynyl-2'-deoxyuridine assay. The expressions of alpha smooth muscle actin (α-SMA) and Smad3 were assayed by immunofluorescent stain and Western blotting. The expression of TβR-I was analysed by Western blotting and real-time polymerase chain reaction. The activity of TβR-I kinase was measured by ADP-Glo kinase assay. The results showed that Sal B could inhibit collagen deposition and reduce Hyp content significantly, and decrease expressions of TGF-β1 and TβR-I in fibrotic liver in vivo. Also, Sal B decreased the expressions of α-SMA and TβR-I, inhibited Smad3 nuclear translocation and down-regulated TβR-I kinase activity in vitro. These findings suggested that Sal B could prevent HSCs activation through TGF-β signalling pathway, i.e. inhibiting TGF-β1 expression, activity of TβR-I kinase and Smads phosphorylation.  

4.1249           Selective Nanovector Mediated Treatment of Activated Proinflammatory Microglia/Macrophages in Spinal Cord Injury

Papa, S.et al ACSNano, 7(11), 9881-9895 (2013)   Much evidence shows that acute and chronic inflammation in spinal cord injury (SCI), characterized by immune cell infiltration and release of inflammatory mediators, is implicated in development of the secondary injury phase that occurs after spinal cord trauma and in the worsening of damage. Activation of microglia/macrophages and the associated inflammatory response appears to be a self-propelling mechanism that leads to progressive neurodegeneration and development of persisting pain state. Recent advances in polymer science have provided a huge amount of innovations leading to increased interest for polymeric nanoparticles (NPs) as drug delivery tools to treat SCI. In this study, we tested and evaluated in vitro and in vivo a new drug delivery nanocarrier: minocycline loaded in NPs composed by a polymer based on poly-ε-caprolactone and polyethylene glycol. These NPs are able to selectively target and modulate, specifically, the activated proinflammatory microglia/macrophages in subacute progression of the secondary injury in SCI mouse model. After minocycline-NPs treatment, we demonstrate a reduced activation and proliferation of microglia/macrophages around the lesion site and a reduction of cells with round shape phagocytic-like phenotype in favor of a more arborized resting-like phenotype with low CD68 staining. Treatment here proposed limits, up to 15 days tested, the proinflammatory stimulus associated with microglia/macrophage activation. This was demonstrated by reduced expression of proinflammatory cytokine IL-6 and persistent reduced expression of CD68 in traumatized site. The nanocarrier drug delivery tool developed here shows potential advantages over the conventionally administered anti-inflammatory therapy, maximizing therapeutic efficiency and reducing side effects.  

4.1250           The Autoregulatory Feedback Loop of MicroRNA-21/Programmed Cell Death Protein 4/Activation Protein-1 (MiR-21/PDCD4/AP-1) as a Driving Force for Hepatic Fibrosis Development

Zhang, Z., Zha, Y., Hu, W., Huang, Z., Gao, Z., Zang, Y., Chen, J. Dong, L. and Zhang, J.

1. Biol. Chem., 288(52), 37082-37093 (2013)

  Sustained activation of hepatic stellate cells (HSCs) leads to hepatic fibrosis, which is characterized by excessive collagen production, and for which there is no available drug clinically. Despite tremendous progress, the cellular activities underlying HSC activation, especially the driving force in the perpetuation stage, are only partially understood. Recently, microRNA-21 (miR-21) has been found to be prevalently up-regulated during fibrogenesis in different tissues, although its detailed role needs to be further elucidated. In the present study, miR-21 expression was examined in human cirrhotic liver samples and in murine fibrotic livers induced by thioacetamide or carbon tetrachloride. A dramatic miR-21 increase was noted in activated HSCs. We further found that miR-21 maintained itself at constant high levels by using a microRNA-21/programmed cell death protein 4/activation protein-1 (miR-21/PDCD4/AP-1) feedback loop. Disrupting this loop with miR-21 antagomir or AP-1 inhibitors significantly suppressed fibrogenic activities in HSCs and ameliorated liver fibrosis. In contrast, reinforcing this loop with small interfering RNA (siRNA) against PDCD4 promoted fibrogenesis in HSCs. Further analysis indicated that the up-regulated miR-21 promoted the central transforming growth factor-β (TGF-β) signaling pathway underlying HSC activation. In summary, we suggest that the miR-21/PDCD4/AP-1 autoregulatory loop is one of the main driving forces for hepatic fibrosis progression. Targeting this aberrantly activated feedback loop may provide a new therapeutic strategy and facilitate drug discovery against hepatic fibrosis.  

4.1251           Activation of the PD-1 Pathway Contributes to Immune Escape in EGFR-Driven Lung Tumors

Akbay, E.A., Koyama, S., Carretero, J. et al Cancer Discovery, 3, 1355-1363 (2013)   The success in lung cancer therapy with programmed death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between EGF receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, CTL antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased CTLs and increased markers of T-cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T-cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non–small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape and mechanistically link treatment response to PD-1 inhibition. Significance: We show that autochthonous EGFR-driven lung tumors inhibit antitumor immunity by activating the PD-1/PD-L1 pathway to suppress T-cell function and increase levels of proinflammatory cytokines. These findings indicate that EGFR functions as an oncogene through non–cell-autonomous mechanisms and raise the possibility that other oncogenes may drive immune escape  

4.1252           Adenosine is required for sustained inflammasome activation via the A2A receptor and the HIF-1α pathway

Ouyang, X., Ghani, A., Malik, A., Wilder, T., Colegio, O.R., Flavell, R.A., Cronstein, B.N. and Mehal, W.Z. Nature Communications, 4:2909 (2013)   Inflammasome pathways are important in chronic diseases; however, it is not known how the signalling is sustained after initiation. Inflammasome activation is dependent on stimuli such as lipopolysaccharide (LPS) and ATP that provide two distinct signals resulting in rapid production of interleukin (IL)-1β, with the lack of response to repeat stimulation. Here we report that adenosine is a key regulator of inflammasome activity, increasing the duration of the inflammatory response via the A_2A receptor. Adenosine does not replace signals provided by stimuli such as LPS or ATP but sustains inflammasome activity via a cAMP/PKA/CREB/HIF-1α pathway. In the setting of the lack of IL-1β responses after previous exposure to LPS, adenosine can supersede this tolerogenic state and drive IL-1β production. These data reveal that inflammasome activity is sustained, after initial activation, by A_2A receptor-mediated signalling.    

4.1253           Particle Focusing in Curved Microfluidic Channels

Martel, J.M. and Toner, M. Scientific Reports, 3:3340 (2013)   The decoupled effects of Reynolds and Dean numbers are examined in inertial focusing flows. In doing so, a complex set of inertial focusing behavioral regimes is discovered within curved microfluidic channels over a range of channel Reynolds numbers, curvature ratios and particle confinement ratios. These regimes are characterized by particle migration either towards or away from the center of curvature as the channel Reynolds number is increased. The transition between these two regimes is shown to be a set of conditions where single-point equilibrium position focusing of particles of different sizes is achieved. A mechanism describing the observed motion of particles in such flows is hypothesized incorporating the redistribution of the main flow velocities caused by Dean flow and its effect on the balance forces on suspended particles.  

4.1254           Quiescent Hepatic Stellate Cells Functionally Contribute to the Hepatic Innate Immune Response via TLR3

Wilson, C.L., Mann, J., Walsh, M., Perrugoria, M.J., Oakley, F., Wright, M.C., Brignole, C., Di Paolo, D., Perri, P., Ponzoni, M., Karin, M. and Mann, D.A. PloS One, 9(1), e83391 (2014)   Toll-like Receptor 3 (TLR3) is a pathogen pattern recognition receptor that plays a key role in innate immunity. TLR3 signalling has numerous functions in liver, both in health and disease. Here we report that TLR3 is expressed by quiescent hepatic stellate cells (HSC) where it functions to induce transcription and secretion of functional interferons as well as a number of other cytokines and chemokines. Upon transdifferentiation into myofibroblasts, HSCs rapidly loose the ability to produce interferon gamma (IFNγ). Mechanistically, this gene silencing may be due to Polycomb complex mediated repression via methylation of histone H3 lysine 27. In contrast to wild type, quiescent HSC isolated from tlr3 knockout mice do not produce IFNγ in response to Poly(I:C) treatment. Therefore, quiescent HSC may contribute to induction of the hepatic innate immune system in response to injury or infection.  

4.1255           Overcoming barriers in clinical islet transplantation: Current limitations and future prospects

Chhabra, P. et al Current Problems in Surgery, 51, 49-86 (2014)   Type 1 diabetes (T1D) has been determined to be an autoimmune disease involving the selective destruction of insulin-producing β-cells in the pancreatic islets of Langerhans that results in the progressive loss of insulin secretion and ultimately in glycometabolic dysregulation.¹ As many as 3 million Americans may have T1D, and according to the most recent population-based estimates of diabetes incidence and prevalence, the number of youth with T1D will increase by more than 20% over the next 40 years.² Complications associated with long-standing T1D include retinopathy, neuropathy, and nephropathy, as well as cerebrovascular and cardiovascular disease.¹ Discovering strategies that could prevent or arrest the onset and progression of autoimmune T1D, reverse β-cell destruction, and permanently restore glycometabolic control preferably without long-term immunosuppression is the ultimate goal of clinical intervention. The current treatment for T1D is primarily focused on the combination of intensive diet management along with lifelong insulin administration, either by multiple daily injections or more recently through pump delivery.^3 and 4 Tight glucose control through insulin therapy has succeeded in preventing and even reversing long-term complications of T1D in some patients,⁵ but the treatment often renders patients susceptible to severe episodes of hypoglycemia and hypoglycemic unawareness.^6 and 7 Recent innovations in insulin therapy have succeeded in achieving more physiological glycometabolic control. For instance, genetically modified recombinant human insulin analogues, aspart and lispro insulin, are rapid acting, with faster onset and offset than regular insulin given subcutaneously, which allows both prandial and corrective boluses.⁸ BIOD-123 and BIOD-125 are ultrarapid-acting recombinant human insulin analogues with acceptable injection site toleration that have an even faster absorption rate and onset of action compared with insulin lispro.⁹ Degludec, an ultralong-acting basal insulin analogue, was shown to improve glycemic control and lower the risk of nocturnal hypoglycemia better than long-acting insulin analogue, glargine, in basal-bolus treatment with mealtime insulin aspart in patients with T1D.¹⁰ Similarly, development of the “artificial pancreas,” an integrated closed-loop control system that combines continuous glucose monitoring with subcutaneous insulin infusion, has been shown to significantly improve glycemic control, although refinement for routine ambulatory use is still in evolution.¹¹ Despite advances such as these, insulin therapy alone has not been successful in reducing the frequency of hypoglycemic episodes and unawareness, highlighting the necessity for developing alternative strategies to permanently restore glycometabolic homeostasis.   To date, pancreas or islet transplantation has been the most reliable clinical approach to cure T1D.^{12, 13 and 14} The success rate of whole-pancreas transplantation alone has continued to improve, with graft survival rates of up to 86% at 1 year and 69% at 3 years with tacrolimus-based maintenance therapy.^15 and 16 Moreover, successful β-cell replacement through transplantation is often accompanied by a reduction in secondary complications of T1D, such as autonomic neuropathy and retinopathy.¹⁷ However, major drawbacks of the pancreas transplant procedure persist, including the risks of major surgery with associated morbidity or mortality and complications, such as graft thrombosis, graft pancreatitis, fistulae, sepsis, and peritonitis.^18 and 19 Importantly, the side effects associated with long-term immunosuppressive therapy (infection and malignancy) are also of concern.^{13, 20 and 21} Pancreas transplantation is often restricted to patients with T1D receiving simultaneous kidney-pancreas transplants or those that have undergone successful kidney transplantation who are already on immunosuppressive therapy,^{19, 20, 21, 22 and 23} and often patient selection excludes high-risk older patients or those with coronary artery disease.^{19, 24 and 25} In contrast to whole-pancreas transplantation, isolated islet transplantation represents a safe, effective, and definitive treatment option, offering substantial benefits in terms of lowering daily insulin requirements, improving levels of glycated hemoglobin (HbA1c), reducing incidences of debilitating hypoglycemic episodes and unawareness, and affording potential insulin independence.^{19, 26, 27, 28 and 29} It is also less invasive than whole-pancreas transplantation and considered hypothetically superior from a metabolic, immunologic, and patient-ease perspective. Islet transplantation restores insulin sensitivity and glucose disposal, as well as improves free fatty acid clearance dynamics.^30 and 31 According to the most recent Collaborative Islet Transplant Registry (CITR) report, the periprocedural complications of islet transplantation have an estimated 20-fold lower morbidity risk when compared with pancreatic transplantation.¹⁹ An additional advantage of islet transplantation includes the utilization of islets isolated from deceased donor pancreas that are considered unsuitable for whole-pancreas transplantation, a decided plus given the shortage of donor organs.³² Ex vivo manipulation of isolated islet tissue also allows a window of opportunity, before transplantation, for attempting various therapeutic manipulations aimed at achieving superior transplant outcomes as well as bypassing the need for long-term immunosuppression.³³ An overview of the procedure is provided in Figure 1.^{29, 34, 35 and 36} However, despite impressive advances in the field in the last decade, widespread application of the procedure is hindered by an inadequate supply of donor pancreata, innate and alloimmune graft rejection, recurrence of autoimmunity,³⁷ diabetogenic and nephrotoxic side effects related to long-term immunosuppression,³⁸ and financial considerations related to the fact that adequate funding streams for the procedure have not advanced through Medicare and private, commercial sources.^38 and 39 This article reviews the strategies that are being employed to overcome some of these hurdles to help transition islet cell transplantation into a widespread, accepted, and fundable clinical option as a permanent cure for T1D (Fig 2).  

4.1256           Dual-energy precursor and nuclear erythroid–related factor 2 activator treatment additively improve redox glutathione levels and neuron survival in aging and Alzheimer mouse neurons upstream of reactive oxygen species

Ghosh, D., LeVault, K.R. and Brewer, G.J. Neurobiology of Aging, 35, 179-190 (2014)   To determine whether glutathione (GSH) loss or increased reactive oxygen species (ROS) are more important to neuron loss, aging, and Alzheimer's disease (AD), we stressed or boosted GSH levels in neurons isolated from aging 3xTg-AD neurons compared with those from age-matched nontransgenic (non-Tg) neurons. Here, using titrating with buthionine sulfoximine, an inhibitor of γ-glutamyl cysteine synthetase (GCL), we observed that GSH depletion increased neuronal death of 3xTg-AD cultured neurons at increasing rates across the age span, whereas non-Tg neurons were resistant to GSH depletion until old age. Remarkably, the rate of neuron loss with ROS did not increase in old age and was the same for both genotypes, which indicates that cognitive deficits in the AD model were not caused by ROS. Therefore, we targeted for neuroprotection activation of the redox sensitive transcription factor, nuclear erythroid–related factor 2 (Nrf2) by 18 alpha glycyrrhetinic acid to stimulate GSH synthesis through GCL. This balanced stimulation of a number of redox enzymes restored the lower levels of Nrf2 and GCL seen in 3xTg-AD neurons compared with those of non-Tg neurons and promoted translocation of Nrf2 to the nucleus. By combining the Nrf2 activator together with the NADH precursor, nicotinamide, we increased neuron survival against amyloid beta stress in an additive manner. These stress tests and neuroprotective treatments suggest that the redox environment is more important for neuron survival than ROS. The dual neuroprotective treatment with nicotinamide and an Nrf2 inducer indicates that these age-related and AD-related changes are reversible.  

4.1257           Differential effects of cadmium administration on peripheral blood granulocytes in rats

Djokic, J., Ninkov, M., Mirkov, I., Popov, A., Aleksandrov, A.P., Zolotarevski, L., Kataranovski, D. and Kataranovski, M. Environ. Toxicol. Pharmacol., 37, 210-219 (2014)   Infiltration of circulatory inflammatory cells is a common histopathological finding in target organs following cadmium administration, but there is paucity of data concerning their activity. In this study, the effects of sublethal (1 mg/kg) cadmium on peripheral blood polymorphonuclear (PMN) cells were examined 48 h following administration in rats, when tissue (liver and lung) infiltration of these cells was observed. Cadmium administration resulted in systemic inflammatory cytokine and acute phase response with an increase in circulatory neutrophil numbers and cells that express CD11b molecules. Rise in basic aspects of oxidative activity including intracellular myeloperoxidase (MPO), reactive oxygen (nitroblue tetrazolium/NBT cytochemical assay) and nitrogen (Griess assay) species production was observed in PMNs from cadmium-administered rats. A decrease in levels of mRNA for IL-1β, TNF-α and IL-6 was noted, but production of these cytokines was affected differentially. Described effects of cadmium on PMNs add further to the understanding of inflammatory potential of this environmental contaminant.  

4.1258           In situ measurement of superoxide and hydroxyl radicals by frequency mixing detection technique

Hong, H., Krause, H.J., Sohn, S.W., Baik, T., Park, J.H., Shin, S., Park, C. and Song, D. Anal. Biochem., 447, 141-145 (2014)   Frequency mixing magnetic detection (FMMD) was used to detect superoxide from hypoxanthine and xanthine reaction and to detect hydroxyl radical from the Fenton reaction. FMMD was also applied to measure the reactive oxygen species (ROS) level released from microglial cells. We could assess the formation and extinction of the free radicals without a spin trap reagent. The FMMD signal amplitude scaled with the concentration of the radicals. It was verified that no signals are obtained from the substrates and reagents. Based on the observations and on previous research, we suggest that the FMMD signals originate from superoxide and hydroxyl radicals, indicating that FMMD can be used to detect O-centered radicals. Subsequent analysis of free radicals generated from living microglial cells showed that there were significant differences between the activated microglial cells and resting ones. The results of this research are promising regarding the applications of FMMD for in situ measurement of free radicals from various sources, including the cell.  

4.1259           Expression of antimicrobial peptides in coelomocytes and embryos of the green sea urchin (Strongylocentrotus droebachiensis)

Li, C., Blencke, H-M., Haug, T., Jørgensen, Ø. And Stensvågt, K. Developmental and Comparative Immunol., 43, 106-113 (2014)   Antimicrobial peptides (AMPs) play a crucial role in innate immunity. We have previously reported the isolation and characterization of the AMPs, strongylocins 1 and 2, and centrocin 1, from coelomocyte extracts of Strongylocentrotus droebachiensis. Here we show that these AMPs were expressed in phagocytes. In addition, transcripts of strongylocin 1 were detected in vibratile cells and/or colorless spherule cells, while transcripts of strongylocin 2 were found in red spherule cells. Results from immunoblotting and immunocytochemistry studies showed that centrocin 1 was produced by phagocytes and stored in granular vesicles. Co-localization of centrocin 1 and phagocytosed bacteria suggests that the granular vesicles containing centrocin 1 may be involved in the formation of phagolysosomes. We also analyzed the temporal and spatial expression of AMPs throughout larval development. Strongylocins were expressed in the early pluteus stage, while centrocin 1 was expressed in the mid pluteus stage. The spatial expression pattern showed that centrocin 1 was mainly located in blastocoelar cells (BCs) around the stomach and the esophagus. In addition, a few patrolling BCs were detected in some larval arms. Together, these results suggest that AMPs are expressed in different types of coelomocytes and that centrocin 1 is involved in response against bacteria. Furthermore, the expression of AMPs in larval pluteus stage, especially in BCs, indicates that AMPs and BCs are engaged in the larval immune system.      

4.1260           Polymeric nanoparticle system to target activated microglia/macrophages in spinal cord injury

Papa, S. et al

1. Controlled Release, 174, 15-26 (2014)

  The possibility to control the fate of the cells responsible for secondary mechanisms following spinal cord injury (SCI) is one of the most relevant challenges to reduce the post traumatic degeneration of the spinal cord. In particular, microglia/macrophages associated inflammation appears to be a self-propelling mechanism which leads to progressive neurodegeneration and development of persisting pain state. In this study we analyzed the interactions between poly(methyl methacrylate) nanoparticles (PMMA-NPs) and microglia/macrophages in vitro and in vivo, characterizing the features that influence their internalization and ability to deliver drugs. The uptake mechanisms of PMMA-NPs were in-depth investigated, together with their possible toxic effects on microglia/macrophages. In addition, the possibility to deliver a mimetic drug within microglia/macrophages was characterized in vitro and in vivo. Drug-loaded polymeric NPs resulted to be a promising tool for the selective administration of pharmacological compounds in activated microglia/macrophages and thus potentially able to counteract relevant secondary inflammatory events in SCI.  

4.1261           Curcumin up-regulates phosphatase and tensin homologue deleted on chromosome 10 through microRNA-mediated control of DNA methylation – a novel mechanism suppressing liver fibrosis

Zheng, J., Wu, C., Lin, Z., Guo, Y., Shi, L., Dong, P., Lu, Z., Gao, S., Liao, Y., Chen, B. and Yu, F. FEBS J., 281, 88-103 (2014)   Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) has been reported to play a role in the suppression of activated hepatic stellate cells (HSCs). Moreover, it has been demonstrated that hypermethylation of the PTEN promoter is responsible for the loss of PTEN expression during HSC activation. Methylation is now established as a fundamental regulator of gene transcription. MicroRNAs (miRNAs), which can control gene expression by binding to their target genes for degradation and/or translational repression, were found to be involved in liver fibrosis. However, the mechanism responsible for miRNA-mediated epigenetic regulation in liver fibrosis still remained unclear. In the present study, curcumin treatment significantly resulted in the inhibition of cell proliferation and an increase in the apoptosis rate through the up-regulation of PTEN associated with a decreased DNA methylation level. Only DNA methyltransferase 3b (DNMT3b) was reduced in vivo and in vitro after curcumin treatment. Further studies were performed aiming to confirm that the knockdown of DNMT3b enhanced the loss of PTEN methylation by curcumin. In addition, miR-29b was involved in the hypomethylation of PTEN by curcumin. MiR-29b not only was increased by curcumin in activated HSCs, but also was confirmed to target DNMT3b by luciferase activity assays. Curcumin-mediated PTEN up-regulation, DNMT3b down-regulation and PTEN hypomethylation were all attenuated by miR-29b inhibitor. Collectively, it is demonstrated that curcumin can up-regulate miR-29b expression, resulting in DNMT3b down-regulation in HSCs and epigenetically-regulated PTEN involved in the suppression of activated HSCs. These results indicate that miRNA-mediated epigenetic regulation may be a novel mechanism suppressing liver fibrosis.  

4.1262           Mycobacterium tuberculosis infection of human dendritic cells decreases integrin expression, adhesion and migration to chemokines

Roberts, L.L. and Robinson, M. Immunology, 141, 39-51 (2014)   Tuberculosis (TB) remains a major global health problem accounting for millions of deaths annually. Approximately one-third of the world's population is infected with the causative agent Mycobacterium tuberculosis. The onset of an adaptive immune response to M. tuberculosis is delayed compared with other microbial infections. This delay permits bacterial growth and dissemination. The precise mechanism(s) responsible for this delay have remained obscure. T-cell activation is preceded by dendritic cell (DC) migration from infected lungs to local lymph nodes and synapsis with T cells. We hypothesized that M. tuberculosis may impede the ability of DCs to reach lymph nodes and initiate an adaptive immune response. We used primary human DCs to determine the effect of M. tuberculosis on expression of heterodimeric integrins involved in cellular adhesion and migration. We also evaluated the ability of infected DCs to adhere to and migrate through lung endothelial cells, which is necessary to reach lymph nodes. We show by flow cytometry and confocal microscopy that M. tuberculosis-infected DCs exhibit a significant reduction in surface expression of the β₂ (CD18) integrin. Distribution of integrin β₂ is also markedly altered in M. tuberculosis-infected DCs. A corresponding reduction in the αL (CD11a) and αM (CD11b) subunits that associate with integrin β₂ was also observed. Consistent with reduced integrin surface expression, we show a significant reduction in adherence to lung endothelial cell monolayers and migration towards lymphatic chemokines when DCs are infected with M. tuberculosis. These findings suggest that M. tuberculosis modulates DC adhesion and migration to increase the time required to initiate an adaptive immune response.  

4.1263           Impact of Surfactant Protein D, Interleukin-5, and Eosinophilia on Cryptococcosis

Holmer, S.M., Evans, K.S., Asfaw, Y.G., Saini, D., Schell, W.A., Ledford, J.G., Frothingham, R., Wright, J.R., Sempowski, G.D.and Perfect, J.R. Infect. Immun., 82(2), 683-693 (2014)   Cryptococcus neoformans is an opportunistic fungal pathogen that initiates infection following inhalation. As a result, the pulmonary immune response provides a first line of defense against C. neoformans. Surfactant protein D (SP-D) is an important regulator of pulmonary immune responses and is typically host protective against bacterial and viral respiratory infections. However, SP-D is not protective against C. neoformans. This is evidenced by previous work from our laboratory demonstrating that SP-D-deficient mice infected with C. neoformans have a lower fungal burden and live longer than wild-type (WT) control animals. We hypothesized that SP-D alters susceptibility to C. neoformans by dysregulating the innate pulmonary immune response following infection. Thus, inflammatory cells and cytokines were compared in the bronchoalveolar lavage fluid from WT and SP-D^−/− mice after C. neoformans infection. Postinfection, mice lacking SP-D have reduced eosinophil infiltration and interleukin-5 (IL-5) in lung lavage fluid. To further explore the interplay of SP-D, eosinophils, and IL-5, mice expressing altered levels of eosinophils and/or IL-5 were infected with C. neoformans to assess the role of these innate immune mediators. IL-5-overexpressing mice have increased pulmonary eosinophilia and are more susceptible to C. neoformans infection than WT mice. Furthermore, susceptibility of SP-D^−/− mice to C. neoformans infection could be restored to the level of WT mice by increasing IL-5 and eosinophils by crossing the IL-5-overexpressing mice with SP-D^−/− mice. Together, these studies support the conclusion that SP-D increases susceptibility to C. neoformans infection by promoting C. neoformans-driven pulmonary IL-5 and eosinophil infiltration.  

4.1264           Paracrine activation of hepatic stellate cells in platelet-derived growth factor C transgenic mice: Evidence for stromal induction of hepatocellular carcinoma

Wright, J.H., Johnson, M.M., Shimizu-Albergine, M., Bauer, R.L., Hayes, B.J., Surapisitchat, J., Hudkins, K.L., Riehle, K.J., Johnson, S.C., Yeh, M.M., Bammler, T.K., Beyer, R.P., Gilbertson, D.G., Alpers, C.E., Fausto, N. and Campell, J.S. Int. J. Cancer, 134(4), 778-788 (2014)   Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet-derived growth factor C (PDGF-C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF-CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast-like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell-derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte-derived PDGF-C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF-CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF-C transgenic mice provide a unique model for the in vivo study of tumor–stromal interactions in the liver.  

4.1265           Study of Adenovirus and CAR Axonal Transport in Primary Neurons

Zussy, C. and Salinas, S. Methods in Mol. Biol., 1089, 71-78 (2014)   Vectors derived from the canine adenovirus serotype 2 (CAV-2) possess a high neurotropism and efficient retrograde transport that lead to widespread neuronal transduction in the central nervous system (CNS) of various animals. These abilities are due to the engagement of virions to the coxsackievirus and adenovirus receptor at the surface of neurons, which is linked to the endocytic and axonal transport machineries. The trafficking of CAV-2 and the coxsackievirus and adenovirus receptor (CAR) can be visualized ex vivo by incubating primary neurons (e.g., motoneurons and hippocampal neurons) with fluorescently labeled virions or recombinant viral proteins. Using this approach, we could recapitulate the mechanisms responsible for long-range transport of adenovirus in neurons.  

4.1266           Neural progenitor cells from human induced pluripotent stem cells generated less autogenous immune response

Huang, K., Liu, P., Li, X., Chen, S., Wang, L., Qin, L., Su, Z., Huang, W., Liu, J., Jia, B., Liu, J., Cai, J., Pei, D. and Pan, G. Science China Life Sciences, 57(2), 162-170 (2014)   The breakthrough development of induced pluripotent stem cells (iPSCs) raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells. However, whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear. In this study, we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with autogenous peripheral blood mononuclear cells (PBMCs), we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation. However, a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs. Furthermore, no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells (CD3⁺CD8⁻ T cells, CD3⁺CD8⁺ T cells or CD3⁻CD56⁺ NK cells) by NPCs in both PBMC and T cell co-culture systems. These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants, and thus set a base for further preclinical evaluation of human iPSCs.  

4.1267           Vitamin D confers protection to motoneurons and is a prognostic factor of amyotrophic lateral sclerosis

Camu, W., Tremblier, B., Plassot, C., Alphandery, S., Salsac, C., Pageot, N., Juntas-Morales, R., Scamps, F., Daures, J-P. and Raoul, C. Neurobiology of Aging, 35, 1198-1205 (2014)   Amyotrophic lateral sclerosis (ALS) is an incurable paralytic disorder primarily typified by the selective and progressive degeneration of motoneurons in the brain and spinal cord. ALS causes muscle wasting and atrophy, resulting eventually in respiratory failure and death within 3–5 years of diagnosis. Vitamin D is a potent secosteroid hormone with diverse biological functions that include protection against neuronal damage. The detrimental consequences of vitamin D dietary deficiency have been documented in other neurodegenerative diseases. However, the protective effect of vitamin D on motoneuron and the influence of its levels on disease course remains elusive. Here we found that the biologically active form of vitamin D significantly potentiated the effect of neurotrophic factors and prevented motoneurons from a Fas-induced death, while electrophysiological properties of motoneurons were not affected. In ALS patients, we report that a severe vitamin D deficiency accelerates by 4 times the rate of decline and were associated with a marked shorter life expectancy. Our findings support a neuroprotective function of vitamin D on motoneurons and propose vitamin D as a reliable prognostic factor of ALS.  

4.1268           No Major Role for Insulin-Degrading Enzyme in Antigen Presentation by MHC Molecules

Culina, S., Mauvais, F-X., Hsu, H-T., Burgevin, A., Guenette, S., Moser, A. and van Endert, P. PloS One, 9(2), e88365 (2014)   Antigen presentation by MHC class I molecules requires degradation of epitope source proteins in the cytosol. Although the preeminent role of the proteasome is clearly established, evidence suggesting a significant role for proteasome-independent generation of class I ligands has been reported repeatedly. However, an enzyme responsible for such a role has not been identified. Recently insulin-degrading enzyme (IDE) was shown to produce an antigenic peptide derived from the tumor antigen MAGE-A3 in an entirely proteasome-independent manner, raising the question of the global impact of IDE in MHC class I antigen processing. Here we report that IDE knockdown in human cell lines, or knockout in two different mouse strains, has no effect on cell surface expression of various MHC class I molecules, including allomorphs such as HLA-A3 and HLA-B27 suggested to be loaded in an at least a partly proteasome-independent manner. Moreover, reduced or absent IDE expression does not affect presentation of five epitopes including epitopes derived from beta amyloid and proinsulin, two preferred IDE substrates. Thus, IDE does not play a major role in MHC class I antigen processing, confirming the dominant and almost exclusive role of the proteasome in cytosolic production of MHC class I ligands.  

4.1269           OX40 ligand regulates splenic CD8− dendritic cell-induced Th2 responses in vivo

Kamachi, F., Harada, N., Usui, Y., Sakanishi, T., Ishii, N., Okumura, K., Miyake, S. and Akiba, H. Biochem. Biophys. Res. Comm., 444, 235-240 (2014)   In mice, splenic conventional dendritic cells (cDCs) can be separated, based on their expression of CD8α into CD8⁻ and CD8⁺ cDCs. Although previous experiments demonstrated that injection of antigen (Ag)-pulsed CD8⁻ cDCs into mice induced CD4 T cell differentiation toward Th2 cells, the mechanism involved is unclear. In the current study, we investigated whether OX40 ligand (OX40L) on CD8⁻ cDCs contributes to the induction of Th2 responses by Ag-pulsed CD8⁻ cDCs in vivo, because OX40–OX40L interactions may play a preferential role in Th2 cell development. When unseparated Ag-pulsed OX40L-deficient cDCs were injected into syngeneic BALB/c mice, Th2 cytokine (IL-4, IL-5, and IL-10) production in lymph node cells was significantly reduced. Splenic cDCs were separated to CD8⁻ and CD8⁺ cDCs. OX40L expression was not observed on freshly isolated CD8⁻ cDCs, but was induced by anti-CD40 mAb stimulation for 24 h. Administration of neutralizing anti-OX40L mAb significantly inhibited IL-4, IL-5, and IL-10 production induced by Ag-pulsed CD8⁻ cDC injection. Moreover, administration of anti-OX40L mAb with Ag-pulsed CD8⁻ cDCs during a secondary response also significantly inhibited Th2 cytokine production. Thus, OX40L on CD8⁻ cDCs physiologically contributes to the development of Th2 cells and secondary Th2 responses induced by Ag-pulsed CD8⁻ cDCs in vivo.  

4.1270           Abstract 192: Bone Marrow Mononuclear Cells May Enhance Recovery After Stroke By Modulating the Microglial Response

Yang, B., Parsha, K., Migliati, E. and Savitz, S. Stroke, 45, A192 (2014)   Background: Autologous bone marrow mononuclear cells (MNCs) have been shown in multiple labs to improve stroke recovery in animal models but the mechanisms remain unclear. We assessed whether MNCs modulate macrophage-microglia responses in acute stroke. Methods: C57/BL mice were subjected to middle cerebral artery occlusion (MCAo) for 60 minutes or sham surgery. 24 hours later, they were randomized to receive saline infusion IV or 1x 10⁶ autologous MNCs IV. At various time points after stroke, 1ml peripheral blood was collected and brains were harvested up to day14. Peripheral blood was labeled with anti-mouse CD45, CD115, F4/80 or Gr-1 antibodies and then assessed by flow cytometry. Microglia were isolated from brains by Optiprep gradient and stained with anti-mouse CD11b, CD45, and CD86 antibodies for evaluation by flow cytometry. For gene expression analyses by RT-PCR, microglia-macrophages were further sorted by magnetic activated-cells sorting using CD11b antibody. Results: 1) At day 2 after stroke, the percentage of CD115⁺ monocytes and CD115⁺/Gr-1^lowcells were significantly decreased in whole peripheral blood but F4/80⁺ macrophages were increased. MNCs increased the abundance of CD115⁺/Gr-1^low cells and decreased the abundance of F4/80⁺ macrophages in peripheral blood compared to saline control at day 2 after stroke (n=5, p<0.05); 2)Compared to saline, MNCs significantly reduced the total number of CD11b⁺ microglia-macrophages in the stroke-affected hemisphere at day 2 to day 6. MNCs significantly decreased the proportion of CD11b⁺/CD45^high among CD11b⁺ cells at day 2 and day 4 and CD86⁺ cells among CD11b⁺ cells at day 2. Among CD11b⁺ microglia-macrophages isolated from the ipsilateral hemisphere of MNC treated mice, compared to saline treated controls, iNOS and IL-1β genes expression were down-regulated at day 2. At day 6, iNOS remained down-regulated while now IL-10, Arganase-1 and CD206 genes expression (markers for the “healing” macrophage phenotype) were up-regulated (n=3 to 5, p<0.05). Conclusions: MNCs may enhance recovery after stroke by altering monocyte trafficking and changing microglial polarization into a healing phenotype.            

4.1271           Dendritic Cells Coordinate Innate Immunity via MyD88 Signaling to Control Listeria monocytogenes Infection

Arnold-Schrauf, C., Dudek, M., Dielmann, A., Pace, L., Swallow, M., Kruse, F., Kühl, A.A., Holzmann, B., Berod, L. and Sparwasser, T. Cell Reports, 6(4), 698-708 (2014)   Listeria monocytogenes (LM), a facultative intracellular Gram-positive pathogen, can cause life-threatening infections in humans. In mice, the signaling cascade downstream of the myeloid differentiation factor 88 (MyD88) is essential for proper innate immune activation against LM, as MyD88-deficient mice succumb early to infection. Here, we show that MyD88 signaling in dendritic cells (DCs) is sufficient to mediate the protective innate response, including the production of proinflammatory cytokines, neutrophil infiltration, bacterial clearance, and full protection from lethal infection. We also demonstrate that MyD88 signaling by DCs controls the infection rates of CD8α⁺ cDCs and thus limits the spread of LM to the T cell areas. Furthermore, in mice expressing MyD88 in DCs, inflammatory monocytes, which are required for bacterial clearance, are activated independently of intrinsic MyD88 signaling. In conclusion, CD11c⁺ conventional DCs critically integrate pathogen-derived signals via MyD88 signaling during early infection with LM in vivo.  

4.1272           Schistosoma japonicum soluble egg antigens induce apoptosis and inhibit activation of hepatic stellate cells: a possible molecular mechanism

Duan, Y., Gu, X., Zhu, D., Sun, W., Chen, J., Feng, J., Song, K., Xu, F., He, X. and He, X. Int. J. Parasitol., 44, 217-224 (2014)   Hepatic stellate cells play a key role in the development of hepatic fibrosis. Activated hepatic stellate cells can be reversed to a quiescent-like state or apoptosis can be induced to reverse fibrosis. Some studies have recently shown that Schistosoma mansoni eggs could suppress the activation of hepatic stellate cells and that soluble egg antigens from schistosome eggs could promote immunocyte apoptosis. Hence, in this study, we attempt to assess the direct effects of Schistosoma japonicum soluble egg antigens on hepatic stellate cell apoptosis, and to explore the mechanism by which the apoptosis of activated hepatic stellate cells can be induced by soluble egg antigens, as well as the mechanism by which hepatic stellate cell activation is inhibited by soluble egg antigens. Here, it was shown that S. japonicum-infected mouse livers had increased apoptosis phenomena and a variability of peroxisome proliferator-activated receptor γ expression. Soluble egg antigens induce morphological changes in the hepatic stellate cell LX-2 cell line, inhibit cell proliferation and induce cell-cycle arrest at the G₁ phase. Soluble egg antigens also induce apoptosis in hepatic stellate cells through the TNF-related apoptosis-inducing ligand/death receptor 5 and caspase-dependent pathways. Additionally, soluble egg antigens could inhibit the activation of hepatic stellate cells through peroxisome proliferator-activated receptor γ and the transforming growth factor β signalling pathways. Therefore, our study provides new insights into the anti-fibrotic effects of S. japonicum soluble egg antigens on hepatic stellate cell apoptosis and the underlying mechanism by which the liver fibrosis could be attenuated by soluble egg antigens.  

4.1273           GAP, an aequorin-based fluorescent indicator for imaging Ca2+ in organelles

Rodriguez-Garcia, A., Rojo-Ruiz, J., Navas-Navarro, P., Aulestia, F.J., Gallego-Sendin, S., Garcia-Sancho, J. and Alonso, M.T. PNAS, 111(7), 2584-2589 (2014)   Genetically encoded calcium indicators allow monitoring subcellular Ca²⁺ signals inside organelles. Most genetically encoded calcium indicators are fusions of endogenous calcium-binding proteins whose functionality in vivo may be perturbed by competition with cellular partners. We describe here a novel family of fluorescent Ca²⁺ sensors based on the fusion of two Aequorea victoria proteins, GFP and apo-aequorin (GAP). GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg²⁺, tunable Ca²⁺ affinity, uncomplicated calibration, and targetability to five distinct organelles. Moreover, transgenic mice for endoplasmic reticulum-targeted GAP exhibited a robust long-term expression that correlated well with its reproducible performance in various neural tissues. This biosensor fills a gap in the actual repertoire of Ca²⁺ indicators for organelles and becomes a valuable tool for in vivo Ca²⁺ imaging applications.  

4.1274           Isolation and culture of hepatic stellate cells from mouse liver

Chang, W., Yang, M., Song, L., Shen, K., Wang, H., Gao, X., Li, M., Niu, W. and Qin, X. Acta Biochim. Biophys. Sin., 46(4), 291-298 (2014)   Hepatic stellate cells (HSCs) are the primary extracellular matrix-producing cells within the liver and have numerous vital functions. A robust protocol for the isolation and culture of HSCs is important for further investigations of cell functions and related mechanisms in liver disease. The volume of the mouse liver is much smaller than that of the rat liver, which makes it much more difficult to isolate mouse HSCs (mHSCs) than rat HSCs. At present, isolating mHSCs is still a challenge because there is no efficient, robust method to isolate and culture these cells. In the present study, C57BL/6J mice were intravenously injected with liposome-encapsulated dichloromethylene diphosphate (CL2MDP) to selectively eliminate Kupffer cells from the liver. The mouse livers were then perfused in situ, and the mHSCs were isolated with an optimized density gradient centrifugation technique. In the phosphate buffer solution (PBS)-liposome group, the yield of mHSCs was (1.37 ± 0.23) × 10⁶/g liver, the cell purity was (90.18 ± 1.61)%, and the cell survival rate was (94.51 ± 1.61)%. While in the CL2MDP-liposome group, the yield of mHSCs was (1.62 ± 0.34) × 10⁶/g liver, the cell purity was (94.44 ± 1.89)%, and the cell survival rate was (94.41 ± 1.50)%. Based on the yield and purity of mHSCs, the CL2MDP-liposome treatment was superior to the PBS-liposome treatment (P < 0.05, P < 0.01). This study established successfully a robust and efficient protocol for the separation and purification of mHSCs, and both a high purity and an adequate yield of mHSCs were obtained.  

4.1275           Adoptive cytotoxic T lymphocyte therapy triggers a counter-regulatory immunosuppressive mechanism via recruitment of myeloid-derived suppressor cells

Hosoi, A., Matsushita, H., Shimizu, K., Fujii, S-i.,. Ueha, S., Abe, J., Kurach, M., Maekawa, R., Matsushima, K. and Kakimi, K. Int. J. Cancer, 134(8), 1810-1822 (2014)   Complex interactions among multiple cell types contribute to the immunosuppressive milieu of the tumor microenvironment. Using a murine model of adoptive T-cell immunotherapy (ACT) for B16 melanoma, we investigated the impact of tumor infiltrating cells on this complex regulatory network in the tumor. Transgenic pmel-1-specific cytotoxic T lymphocytes (CTLs) were injected intravenously into tumor-bearing mice and could be detected in the tumor as early as on day 1, peaking on day 3. They produced IFN-γ, exerted anti-tumor activity and inhibited tumor growth. However, CTL infiltration into the tumor was accompanied by the accumulation of large numbers of cells, the majority of which were CD11b⁺Gr1⁺ myeloid-derived suppressor cells (MDSCs). Notably, CD11b⁺Gr1^intLy6G⁻Ly6C⁺ monocytic MDSCs outnumbered the CTLs by day 5. They produced nitric oxide, arginase I and reactive oxygen species, and inhibited the proliferation of antigen-specific CD8⁺ T cells. The anti-tumor activity of the adoptively-transferred CTLs and the accumulation of MDSCs both depended on IFN-γ production on recognition of tumor antigens by the former. In CCR2^−/− mice, monocytic MDSCs did not accumulate in the tumor, and inhibition of tumor growth by ACT was improved. Thus, ACT triggered counter-regulatory immunosuppressive mechanism via recruitment of MDSCs. Our results suggest that strategies to regulate the treatment-induced recruitment of these MDSCs would improve the efficacy of immunotherapy.  

4.1276           Evidence for Instant Blood-Mediated Inflammatory Reaction in Clinical Autologous Islet Transplantation

Naziruddin, B., Iwahashi, S., Kanak, M.A., Takita, M., Itoh, T. and Levy, M.F. Am. J. Transplant., 14(2), 428-437 (2014)   A nonspecific inflammatory and thrombotic reaction termed instant blood-mediated inflammatory reaction (IBMIR) has been reported when allogenic or xenogenic islets come into contact with blood. This reaction is known to cause significant loss of transplanted islets. We hypothesized that IBMIR occurs in patients undergoing total pancreatectomy followed by autologous islet transplantation (TP-AIT) and tested this hypothesis in 24 patients and in an in vitro model. Blood samples drawn during the peritransplant period showed a significant and rapid increase of thrombin–anti-thrombin III complex (TAT) and C-peptide during islet infusion, which persisted for up to 3 h, along with a decreased platelet count. A concomitant increase in levels of inflammatory proteins IL-6, IL-8 and interferon-inducible protein-10 was observed. An in vitro model composed of pure islets plus autologous blood also demonstrated significantly increased levels of TAT (p < 0.05), C-peptide (p < 0.05), tumor necrosis factor-alpha (p < 0.05) and MCP-1 (p < 0.05), as well as strong tissue factor expression in islets. Islet viability decreased significantly but was rescued by the presence of low-molecular-weight dextran sulfate. In conclusion, AIT-induced elevation of TAT and destruction of islets suggests that IBMIR might occur during AIT. Modulating this process may help improve islet engraftment and the insulin independence rate in TP-AIT patients.  

4.1277           Concentration, Activity and Biochemical Characterization of Myeloperoxidase in Fresh and Post-Thaw Equine Semen and their Implication on Freezability

Ponthier, J., Franck, T., Parrilla-Hernandez, S., Niesten, A., de la Rebiere, G., Serteyn, D. and Deleuze, S. Reproduction in Domestic Animals, 49(2), 285-291 (2014)   Myeloperoxidase (MPO) is a pro-oxidant enzyme associated with decreased motility in thawed equine semen. This study aimed to describe MPO concentration, activity and subunits in raw and thawed semen and to correlate these data with motilities in raw and thawed semen. Semen samples from five stallions were collected four times. Motilities were assessed in raw and thawed semen. MPO assays were performed in raw seminal plasma, raw sperm-rich pellet and thawed semen. Total and active MPO concentrations were, respectively, assayed by enzyme-linked immunosorbent assay and specific immunological extraction followed by enzymatic detection. MPO subunits present in semen were characterized by Western blot. Purified active MPO was added in saline solution and freezing extender to control its activity during freezing procedure. Differences between medians were determined using Kruskal–Wallis test, and correlations were determined using Spearman's test for nonparametric data. Active MPO concentration was low in seminal plasma and thawed semen, but high in pellet (p = 0.0058), as the opposite relation was observed for total MPO concentration (p < 0.0001). In seminal plasma and post-thaw semen, inactive 86-kDa MPO precursor was mainly observed. Purified MPO activity was decreased in the extender (p = 0.0286). MPO activity in pellet was highly correlated with thawed progressive motility (r = −0.5576, p = 0.0086). Inactive MPO precursor and unknown low molecular weight inactive MPO precursor subunits explain low MPO activity in semen. Major MPO activity was observed in pellet, and post-thaw loss of activity is partially explained by MPO inactivation in extender. Thawed semen motility was negatively correlated with MPO activity in pellet, becoming a potential freezability predictor.  

4.1278           Cerebrospinal fluid-targeted delivery of neutralizing anti-IFNγ antibody delays motor decline in an ALS mouse model

Otsmane, B., Aebischer, J., Moumen, A. and Raoul, C. Neuroreport, 25(1), 49-54 (2014)   Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the selective and gradual loss of motoneurons in the brain and spinal cord. A persistent inflammation, typified by the activation of astrocytes and microglia, accompanies the progressive degeneration of motoneurons. Interferon gamma (IFNγ), a potent proinflammatory cytokine that is aberrantly present in the spinal cord of ALS mice and patients, has been proposed to contribute to motoneuron death by eliciting the activation of the lymphotoxin-β receptor (LT-βR) through its ligand LIGHT. However, the implication of IFNγ in the pathogenic process remains elusive. Here, we show that an antagonistic anti-IFNγ antibody efficiently rescues motoneurons from IFNγ-induced death. When transiently delivered in the cerebrospinal fluid through a subcutaneously implanted osmotic minipump, the neutralizing anti-IFNγ antibody significantly retarded motor function decline in a mouse model of ALS. However, this transient infusion of anti-IFNγ antibody did not increase the life expectancy of ALS mice. Our results suggest that IFNγ contributes to ALS pathogenesis and represents a potential therapeutic target for ALS.  

4.1279           Chronic Pancreatitis and Primary Sclerosing Cholangitis—First Report of Intrahepatic Autologous Islet Transplantation

Wang, L-j., Young, S., Misawa, r., Azzam, R., Wang, X., Golab, K., Cochet, O., Savari, O., Tibudan, M., Millis, J.M., Matthews, J.B. and Witkowski, P.

1. Gastrointest. Surg., 18(4), 845-850 (2014)

  Background We are reporting first successful intrahepatic autologous islet transplantation after total pancreatectomy in a patient with chronic pancreatitis and primary sclerosing cholangitis. Methods Total pancreatectomy and subsequent islet autotransplantation were performed in a 16-year-old boy with intractable pain due to chronic pancreatitis in the setting of ulcerative colitis and primary sclerosing cholangitis (PSC). Liver biopsy revealed PSC with focal bridging fibrosis. The pancreas was surgically removed and digested, and islets were isolated, highly purified, and infused intraportally. Results Over 18-month follow-up, the patient did not show progression of chronic liver disease or signs of portal hypertension. Magnetic resonance cholangiopancreatography revealed no new changes, and liver biopsy did not show progression of the periportal fibrosis. Pain medication was weaned over 12 months at which time glycemic control was excellent without exogenous insulin supplementation. HbgA1c was 5.9. Fifteen months after the procedure, stimulation with a mixed meal led to a fourfold increase of serum C-peptide and an eightfold increase of insulin level. Conclusion Pancreatic autologous islets can be successfully transplanted into a liver affected by PSC without compromising hepatic or graft function. Durability of the procedure may be limited in the future by the natural course of the liver injury caused by PSC.  

4.1280           Microfluidic encapsulation of cells in alginate particles via an improved internal gelation approach

Akbari, S. and Pirbodaghi, T. Microfluid. Nanofluid., 16(3), 773-777 (2014)   An improved internal gelation approach is developed to encapsulate single mammalian cells in monodisperse alginate microbeads as small as 26 μm in diameter and at rates of up to 1 kHz with high cell viability. The cell damage resulting from contact with calcium carbonate nanoparticles as gelation reagents is eliminated by employing a co-flow microfluidic device, and the cell exposure to low pH is minimized by a chemically balanced off-chip gelation step. These modifications significantly improve the viability of cells encapsulated in gelled alginate particles. Two different mammalian cell types are encapsulated with viability of over 84 %. The cells are functional and continue to grow inside the microparticles.  

4.1281           Resveratrol treatment rescues neurovascular coupling in aged mice: role of improved cerebromicrovascular endothelial function and downregulation of NADPH oxidase

Toth, P., Tarantini, S., Tucsek, Z., Ashpole, N.M., Sosnowska, D., Gautam, T., Ballabh, P., Koller, A., Sonntag, W., Csiszar, a. and Ungvari, Z. Am. J. Physiol. Heart Circ. Physiol., 30683), H299-H308 (2014)   Moment-to-moment adjustment of cerebral blood flow (CBF) to neuronal activity via neurovascular coupling is essential for the maintenance of normal neuronal function. Increased oxidative stress that occurs with aging was shown to impair neurovascular coupling, which likely contributes to a significant age-related decline in higher cortical function, increasing the risk for vascular cognitive impairment. Resveratrol is a polyphenolic compound that exerts significant antiaging protective effects in large vessels, but its effects on the cerebromicrovasculature remain poorly defined. The present study was undertaken to investigate the capacity of resveratrol to improve neurovascular coupling in aging. In aged (24-mo-old) C57BL/6 mice N^ω-nitro-l-arginine methyl ester-sensitive, nitric oxide-mediated CBF responses to whisker stimulation and to the endothelium-dependent dilator acethylcholine (ACh) were impaired compared with those in young (3-mo-old) mice. Treatment of aged mice with resveratrol rescued neurovascular coupling and ACh-induced responses, which was associated with downregulation of cortical expression of NADPH oxidase and decreased levels of biomarkers of oxidative/nitrative stress (3-nitrotyrosine, 8-isoprostanes). Resveratrol also attenuated age-related increases in reactive oxygen species (ROS) production in cultured cerebromicrovascular endothelial cells (DCF fluorescence, flow cytometry). In conclusion, treatment with resveratrol rescues cortical neurovascular coupling responses to increased neuronal activity in aged mice, likely by restoring cerebromicrovascular endothelial function via downregulation of NADPH oxidase-derived ROS production. Beneficial cerebromicrovascular effects of resveratrol may contribute to its protective effects on cognitive function in aging.  

4.1282           Involvement of IGF-II receptors in the antioxidant and neuroprotective effects of IGF-II on adult cortical neuronal cultures

Martin-Montanez, E., pavia, J., Santin, L.J., Boraldi, F., Estivill-Torrus, G., Aguirre, J.A. and Garcia-Fernandez, M. Biochim. Biophys. Acta, 1842, 1041-1051 (2014)   Insulin-like growth factor-II (IGF-II) is a naturally occurring peptide that exerts known pleiotropic effects ranging from metabolic modulation to cellular development, growth and survival. IGF-II triggers its actions by binding to and activating IGF (IGF-I and IGF-II) receptors. In this study, we assessed the neuroprotective effect of IGF-II on corticosterone-induced oxidative damage in adult cortical neuronal cultures and the role of IGF-II receptors in this effect. We provide evidence that treatment with IGF-II alleviates the glucocorticoid-induced toxicity to neuronal cultures, and this neuroprotective effect occurred due to a decrease in reactive oxygen species (ROS) production and a return of the antioxidant status to normal levels. IGF-II acts via not only the regulation of synthesis and/or activity of antioxidant enzymes, especially manganese superoxide dismutase, but also the restoration of mitochondrial cytochrome c oxidase activity and mitochondrial membrane potential. Although the antioxidant effect of IGF-I receptor activation has been widely reported, the involvement of the IGF-II receptor in these processes has not been clearly defined. The present report is the first evidence describing the involvement of IGF-II receptors in redox homeostasis. IGF-II may therefore contribute to the mechanisms of neuroprotection by acting as an antioxidant, reducing the neurodegeneration induced by oxidative insults. These results open the field to new pharmacological approaches to the treatment of diseases involving imbalanced redox homeostasis. In this study, we demonstrated that the antioxidant effect of IGF-II is at least partially mediated by IGF-II receptors.  

4.1283           IL-12 and IL-27 regulate the phagolysosomal pathway in mycobacteria-infected human macrophages

Jung, J-Y. and Robinson, C.M. Cell Communication and Signaling, 12:16 (2014)   Background The cytokine environment at the site of infection is important to the control of mycobacteria by host macrophages. During chronic infection immunosuppressive cytokines are likely to favor mycobacterial growth, persistence, and an avoidance of proper antigen processing and presentation. The activity of interleukin (IL)-27 toward macrophages is anti-inflammatory and this compromises control of mycobacteria. Modulation of the cytokine environment may enhance both protective and vaccine-induced responses. Results In this study we showed that supplying IL-12 and neutralizing IL-27 enhanced acidification and fusion of mycobacterial-containing phagosomes with lysosomes. This was achieved by phagosomal acquisition of vacuolar ATPase (V-ATPase) and CD63. Both V-ATPase and CD63 protein levels were increased by the addition of IL-12 and neutralization of IL-27. In addition, cathepsin D associated with the bacteria and matured to the active form when IL-12 was supplied and IL-27 was neutralized. Lysosomal acidification and cathepsin D activity were associated with control of mycobacteria. The acidification of lysosomes, association with mycobacteria, and maturation of cathepsin D required macrophage production of IFN-γ and signaling through signal transducer and activator of transcription (STAT)-1. In contrast, STAT-3 signaling opposed these events. Conclusions Our results have identified novel influences of IL-12, IL-27, and STAT-3 on lysosomal activity and further demonstrate that modulating the cytokine environment promotes enhanced trafficking of mycobacteria to lysosomes in human macrophages. This has important implications in approaches to control infection and improve vaccination. Overcoming bacterial resistance to lysosomal fusion may expand the repertoire of antigens presented to the adaptive arm of the immune response.  

4.1284           Long-chain Acyl-CoA Dehydrogenase Deficiency as a Cause of Pulmonary Surfactant Dysfunction

Goetzman, E.S. et al

1. Biol. Chem., 289(15), 10668-10679 (2014)

  Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD^−/− mice. LCAD^−/− mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD^−/− mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD^−/− surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD^−/− lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.  

4.1285           A comparison of the main structures of N-glycans of porcine islets with those from humans

Miyagawa, S., Maeda, A., Kawamura, T., Ueno, T., Usui, N., Kondo, S., Matsumoto, S., Okitsu, T., Goto, M. and Nagashima, H. Glycobiology, 24(2), 125-138 (2014)   After producing α1-3-galactosyltransferase knockout (GKO) pigs, most of the organs of these pigs showed less antigenicity to the human body. However, wild-type adult pig islets (API) that originally contained negligible levels of α-galactosidase now showed a clear antigenicity to human serum. In this study, N-glycans were isolated from both APIs and human islets. Their structures were then analyzed by a mapping technique based on their high-performance liquid chromatography elution positions and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric data. Both preparations contained substantial amounts of high-mannose structures. The N-glycans from human islets were separated into 17 neutral, 8 mono-sialyl and 4 di-sialyl glycans, and the API glycans were comprised of 11 neutral, 8 mono-sialyl, 3 di-sialyl, 2 mono-sulfated, 3 mono-sialyl-mono-sulfated and 1 di-sulfated glycans. Among them, the API preparation contained one neutral, five mono-sialyl glycans and six sulfated glycans that were not detected in human islets. The structures of 9 of these 12 could be clearly determined. In addition, a study of the sulfate-depleted API suggests that sulfate residues could be antigenic to humans. The data herein will be helpful for future studies of the antigenicity associated with API.  

4.1286           Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: Role of NADPH oxidase

Wang, M., Abais, J.M., Meng, N., Zhang, Y., Ritter, J.K., Li, P-L. and Tang, W-X. Free Radical Biology abd Medicine, 71, 109-120 (2014)   The endocannabinoid system (CS) has been implicated in the development of hepatic fibrosis such as schistosomiasis-associated liver fibrosis (SSLF). However, the mechanisms mediating the action of the CS in hepatic fibrosis are unclear. The present study hypothesized that Schistosoma J. infection upregulates cannabinoid receptor 1 (CB1) due to activation of NADPH oxidase leading to a fibrotic phenotype in hepatic stellate cells (HSCs). The SSLF model was developed by infecting mice with Schistosoma J. cercariae in the skin, and HSCs from control and infected mice were then isolated, cultured, and confirmed by analysis of HSC markers α-SMA and desmin. CB1 significantly increased in HSCs isolated from mice with SSLF, which was accompanied by a greater expression of fibrotic markers α-SMA, collagen I, and TIMP-1. CB1 upregulation and enhanced fibrotic changes were also observed in normal HSCs treated with soluble egg antigen (SEA) from Schistosoma J. Electron spin resonance (ESR) analysis further demonstrated that superoxide (O₂ ^{<img border="0" alt="radical dot" src="http://origin-cdn.els-cdn.com/sd/entities/rad" class="glyphImg">−}) production was increased in infected HSCs or normal HSCs stimulated with SEA. Both Nox4 and Nox1 siRNA prevented SEA-induced upregulation of CB1, α-SMA, collagen I, and TIMP-1 by inhibition of O₂ ^{<img border="0" alt="radical dot" src="http://origin-cdn.els-cdn.com/sd/entities/rad" class="glyphImg">−} production, while CB1 siRNA blocked SEA-induced fibrotic changes without effect on O₂ ^{<img border="0" alt="radical dot" src="http://origin-cdn.els-cdn.com/sd/entities/rad" class="glyphImg">−} production in these HSCs. Taken together, these data suggest that the fibrotic activation of HSCs on Schistosoma J. infection or SEA stimulation is associated with NADPH oxidase-mediated redox regulation of CB1 expression, which may be a triggering mechanism for SSLF.  

4.1287           Nanoparticle Incorporation of Melittin Reduces Sperm and Vaginal Epithelium Cytotoxicity

Jallouk, A.P., Moley, K.H., Omurtag, K., Hu, G., Lanza, G.M., Wickline, S.A. and Hood, J.L. PloS One, 9(4), e95411 (2014)   Melittin is a cytolytic peptide component of bee venom which rapidly integrates into lipid bilayers and forms pores resulting in osmotic lysis. While the therapeutic utility of free melittin is limited by its cytotoxicity, incorporation of melittin into the lipid shell of a perfluorocarbon nanoparticle has been shown to reduce its toxicity in vivo. Our group has previously demonstrated that perfluorocarbon nanoparticles containing melittin at concentrations <10 µM inhibit HIV infectivity in vitro. In the current study, we assessed the impact of blank and melittin-containing perfluorocarbon nanoparticles on sperm motility and the viability of both sperm and vaginal epithelial cells. We found that free melittin was toxic to sperm and vaginal epithelium at concentrations greater than 2 µM (p<0.001). However, melittin nanoparticles were not cytotoxic to sperm (p = 0.42) or vaginal epithelium (p = 0.48) at an equivalent melittin concentration of 10 µM. Thus, nanoparticle formulation of melittin reduced melittin cytotoxicity fivefold and prevented melittin toxicity at concentrations previously shown to inhibit HIV infectivity. Melittin nanoparticles were toxic to vaginal epithelium at equivalent melittin concentrations ≥20 µM (p<0.001) and were toxic to sperm at equivalent melittin concentrations ≥40 µM (p<0.001). Sperm cytotoxicity was enhanced by targeting of the nanoparticles to the sperm surface antigen sperm adhesion molecule 1. While further testing is needed to determine the extent of cytotoxicity in a more physiologically relevant model system, these results suggest that melittin-containing nanoparticles could form the basis of a virucide that is not toxic to sperm and vaginal epithelium. This virucide would be beneficial for HIV serodiscordant couples seeking to achieve natural pregnancy.  

4.1288           Endoplasmic reticulum stress is accompanied by activation of NF-κB in amyotrophic lateral sclerosis

Prell, T., Lautenschläger, J., Weidemann, L., Ruhmer, J., Witte, O.W.and Grosskreutz, J.

1. Neuroimmunol., 270, 29-36 (2014)

  Background Recent studies have indicated that endoplasmic reticulum (ER) stress is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). ER stress occurs when the ER–mitochondria calcium cycle is disturbed and misfolded proteins accumulate in the ER. To cope with ER stress, cells activate the unfolded protein response (UPR). Accumulating evidence from non-neuronal cell models suggests that there is extensive cross-talk between the UPR and the NF-κB pathway. Methods Here we investigated the expression of NF-κB and the main UPR markers X-box binding protein 1 (XBP1), basic leucine-zipper transcription factor 6 (ATF6) and phosphorylated eukaryotic initiation factor-2α (p-eIF2) in mutated SOD1^G93A cell models of ALS, as well as their modulation by lipopolysaccharide and ER-stressing (tunicamycin) stimuli. Results Expression of NF-κB was enhanced in the presence of SOD1^G93A. Lipopolysaccharide did not induce the UPR in NSC34 cells and motor neurons in a mixed motor neuron–glia coculture system. The induction of the UPR by tunicamycin was accompanied by activation of NF-κB in NSC34 cells and motor neurons. Conclusion Our data linked two important pathogenic mechanisms of ALS, ER stress and NF-κB signalling, in motor neurons.  

4.1289           Optimization of murine small intestine leukocyte isolation for global immune phenotype analysis

Goodyear, A.W., Kumar, A., Dow, S. and Ryan, E.P.

1. Immunol. Methods, 405, 97-108 (2014)

  New efforts to understand complex interactions between diet, gut microbiota, and intestinal immunity emphasize the need for a standardized murine protocol that has been optimized for the isolation of lamina propria immune cells. In this study multiple mouse strains including BALB/c, 129S6/Sv/EvTac and ICR mice were utilized to develop an optimal protocol for global analysis of lamina propria leukocytes. Incubation temperature was found to significantly improve epithelial cell removal, while changes in media formulation had minor effects. Tissue weight was an effective method for normalization of solution volumes and incubation times. Collagenase digestion in combination with thermolysin was identified as the optimal method for release of leukocytes from tissues and global immunophenotyping, based on the criteria of minimizing marker cleavage, improving cell viability, and reagent cost. The effects of collagenase in combination with dispase or thermolysin on individual cell surface markers revealed diverse marker specific effects. Aggressive formulations cleaved CD8α, CD138, and B220 from the cell surface, and resulted in relatively higher expression levels of CD3, γδ TCR, CD5, DX5, Ly6C, CD11b, CD11c, MHC-II and CD45. Improved collagenase digestion significantly improved viability and reduced debris formation, eliminating the need for density gradient purification. Finally, we demonstrate that two different digestion protocols yield significant differences in detection of CD4⁺ and CD8⁺ T cells, NK cells, monocytes and interdigitating DC (iDC) populations, highlighting the importance and impact of cell collection protocols on assay outputs. The optimized protocol described herein will help assure the reproducibility and robustness of global assessment of lamina propria immune responses. Moreover, this technique may be applied to isolation of leukocytes from the entire gastrointestinal tract.  

4.1290           The contribution of the glycine cleavage system to the pathogenesis of Francisella tularensis

Brown, M.J., Russo, B.C., O’Dee, D.M., Schmitt, D.M. and Nau, G.J. Microbes and Infection, 16, 300-309 (2014)   Biosynthesis and acquisition of nutrients during infection are integral to pathogenesis. Members of a metabolic pathway, the glycine cleavage system, have been identified in virulence screens of the intracellular bacterium Francisella tularensis but their role in pathogenesis remains unknown. This system generates 5,10-methylenetetrahydrofolate, a precursor of amino acid and DNA synthesis, from glycine degradation. To characterize this pathway, deletion of the gcvT homolog, an essential member of this system, was performed in attenuated and virulent F. tularensis strains. Deletion mutants were auxotrophic for serine but behaved similar to wild-type strains with respect to host cell invasion, intracellular replication, and stimulation of TNF-α. Unexpectedly, the glycine cleavage system was required for the pathogenesis of virulent F. tularensis in a murine model. Deletion of the gcvT homolog delayed mortality and lowered bacterial burden, particularly in the liver and bloodstream. To reconcile differences between the cell culture model and animal model, minimal tissue culture media was employed to mimic the nutritionally limiting environment of the host. This reevaluation demonstrated that the glycine cleavage system contributes to the intracellular replication of virulent F. tularensis in serine limiting environments. Thus, the glycine cleavage system is the serine biosynthetic pathway of F. tularensis and contributes to pathogenesis in vivo.  

4.1291           Genotoxic potential of several naphthenic acids and a synthetic oil sands process-affected water in rainbow trout (Oncorhynchus mykiss)

Lacaze, E., Devaux, A., Bruneau, A., Bony, S., Sheery, J. and gagne, F. Aquatic Toxicol., 152, 291-299 (2014)   The exploitation of oil sands has raised major environmental concerns, particularly regarding the presence of high concentration in contaminants such as polycyclic aromatic hydrocarbons (PAHs) and naphthenic acids (NAs) in oil sands process-affected water (OSPW). The purpose of this study was, first to evaluate the genotoxic impact of OSPW-related compounds such as NAs and PAHs in a salmonid species and secondly to assess if OSPW exposure leads to genotoxicity. For this purpose, rainbow trout hepatocytes were exposed in vitro to environmentally relevant concentrations of synthetic NAs, naphtalene, benzo(a)pyrene, and extracts of synthetic OSPW (generated by a laboratory bitumen extraction) and of oil sands leaching water (OSLW, mimicking leaching of oil sands in river water). Primary DNA damage was assessed by the formamidopyrimidine-DNA glycolyase (Fpg)-modified comet assay. Genotoxicity was observed in hepatocytes exposed to several NAs, mixture of them, OSPW and OSLW extracts. The chemical structure of NAs influences the genotoxicity potential: among the NAs tested, the most cyclic NA was the most genotoxic. It also appears that genotoxicity was more marked for OSPW than for OSLW. Because exposure to OSPW led to oxidative DNA damage, while after exposure to several NAs, these types of DNA damage were limited, the NAs tested in this study could not be qualified as the only major contaminants responsible for OSPW genotoxicity. Notwithstanding, it should be noteworthy that exposure to NAs resulted in genotoxic impact at concentrations lower than those documented by literature for fresh OSPW. Further research is needed to explore the relationships between the chemical structure of NAs and their genotoxicity in the light of the distribution of NAs in fresh OSPW samples as well as in surface waters.  

4.1292           Mass and Density Measurements of Live and Dead Gram-Negative and Gram-Positive Bacterial Populations

Lewis, C.L., Craig, C.C. and Senecal, A.G. Appl. Environ. Microbiol., 80(12), 3622-3631 (2014)   Monitoring cell growth and measuring physical features of food-borne pathogenic bacteria are important for better understanding the conditions under which these organisms survive and proliferate. To address this challenge, buoyant masses of live and dead Escherichia coli O157:H7 and Listeria innocua were measured using Archimedes, a commercially available suspended microchannel resonator (SMR). Cell growth was monitored with Archimedes by observing increased cell concentration and buoyant mass values of live growing bacteria. These growth data were compared to optical density measurements obtained with a Bioscreen system. We observed buoyant mass measurements with Archimedes at cell concentrations between 10⁵ and 10⁸ cells/ml, while growth was not observed with optical density measurements until the concentration was 10⁷ cells/ml. Buoyant mass measurements of live and dead cells with and without exposure to hydrogen peroxide stress were also compared; live cells generally had a larger buoyant mass than dead cells. Additionally, buoyant mass measurements were used to determine cell density and total mass for both live and dead cells. Dead E. coli cells were found to have a larger density and smaller total mass than live E. coli cells. In contrast, density was the same for both live and dead L. innocua cells, while the total mass was greater for live than for dead cells. These results contribute to the ongoing challenge to further develop existing technologies used to observe cell populations at low concentrations and to measure unique physical features of cells that may be useful for developing future diagnostics.  

4.1293           Saxitoxins induce cytotoxicity, genotoxicity and oxidative stress in teleost neurons in vitro

Da Silva, C.A., de Morais, E.C.P., Costa, M.D.M., Ribas, J.L.C., Guiloski, I.C., Ramsdorf, W.A., Zanata, S.M., Cestari, M.M., Ribeiro, C.A.O., magalhaes, V.F., Trudeau, V.L. and de Assis, H.C.S. Toxicon, 86, 8-15 (2014)   The aim of this study was establish a protocol for isolation and primary culture of neurons from tropical freshwater fish species Hoplias malabaricus for assessment of the effects of neurotoxic substances as saxitoxins (STXs). Cells from brain of H. malabaricus were treated with different concentrations of trypsin, dispase and papain for tissue dissociation. Cells type was separated by cellular gradient and basic fibroblast growth factor (bFGF) supplement nutrition media were added. The dissociated cells were plated with medium and different STXs concentrations and the toxic cellular effects such as oxidative stress, neurotoxicity, and genotoxicity and apoptosis process were evaluated. Cultures treated with bFGF showed the greatest adherence, survival and cellular development. STXs increased specific activity of glutathione peroxidase and lipoperoxidation levels, were cytotoxic and genotoxic indicated by the comet assay. Although the STXs effects due the blockage of sodium channels is reported to be reversible, the time exposure and concentration of STXs suggested cellular injuries which can lead to neuropathology. The establishment of primary neuronal culture protocol enables new applications for neurotoxicological assessments.  

4.1294           Macrophagic and microglial responses after focal traumatic brain injury in the female rat

Turtzo, L.C., Lescher, J., Janes, L., Dean, D.D., Budde, M.D. and Frank, J.A.

1. Neuroinflammation, 11:82 (2014)

  Background After central nervous system injury, inflammatory macrophages (M1) predominate over anti-inflammatory macrophages (M2). The temporal profile of M1/M2 phenotypes in macrophages and microglia after traumatic brain injury (TBI) in rats is unknown. We subjected female rats to severe controlled cortical impact (CCI) and examined the postinjury M1/M2 time course in their brains. Methods The motor cortex (2.5 mm left laterally and 1.0 mm anteriorly from the bregma) of anesthetized female Wistar rats (ages 8 to 10 weeks; N = 72) underwent histologically moderate to severe CCI with a 5-mm impactor tip. Separate cohorts of rats had their brains dissociated into cells for flow cytometry, perfusion-fixed for immunohistochemistry (IHC) and ex vivo magnetic resonance imaging or flash-frozen for RNA and protein analysis. For each analytical method used, separate postinjury times were included for 24 hours; 3 or 5 days; or 1, 2, 4 or 8 weeks. Results By IHC, we found that the macrophagic and microglial responses peaked at 5 to 7 days post-TBI with characteristics of mixed populations of M1 and M2 phenotypes. Upon flow cytometry examination of immunological cells isolated from brain tissue, we observed that peak M2-associated staining occurred at 5 days post-TBI. Chemokine analysis by multiplex assay showed statistically significant increases in macrophage inflammatory protein 1α and keratinocyte chemoattractant/growth-related oncogene on the ipsilateral side within the first 24 hours after injury relative to controls and to the contralateral side. Quantitative RT-PCR analysis demonstrated expression of both M1- and M2-associated markers, which peaked at 5 days post-TBI. Conclusions The responses of macrophagic and microglial cells to histologically severe CCI in the female rat are maximal between days 3 and 7 postinjury. The response to injury is a mixture of M1 and M2 phenotypes.  

4.1295           Differential expression of CD45 isoforms in canine leukocytes

Goto-Koshino, Y., Tomiyasu, H., Suzuki, H., Tamamoto, T., Mitzutani, N., Fujino, Y., Ohno, K. and Tsujimoto, H. Veterinary Immunol. Immunopathol., 160, 118-122 (2014)   CD45 is one of the most abundant molecules expressed on the white blood cell surface in various mammals. In this study, we investigated the differential expression of CD45 isoforms in normal canine white blood cells. It has been shown that all canine nucleated blood cells express CD45. We characterized two major isoforms of canine CD45 derived from alternative splicing: a higher molecular weight isoform, CD45RA, and a lower molecular weight isoform, CD45RO. The nucleotide sequences of the two isoforms were identical, except for the region corresponding to a part in the extracellular domain. Flow cytometry analysis using an antibody that recognizes CD45RA, but not CD45RO, revealed that granulocytes did not express CD45RA, and monocytes express low levels of CD45RA. We further analyzed the expression levels of CD45RA in each lymphocyte subpopulation and found that the expression of CD45RA on CD21+ B cells was uniform. On the other hand, expression of CD45RA on CD3+ T cells was variable. Upon stimulation of lymphocytes with Con A, the CD45RA+ fraction increased, indicating that not only the phenotypes but also the activation status influences the isoform expression pattern of CD45. Our finding provides a basic knowledge of the expression of canine CD45, which could be a tool to study lymphocytes with various phenotypes, developmental stages, and activation status.  

4.1296           Rabies Virus Envelope Glycoprotein Targets Lentiviral Vectors to the Axonal Retrograde Pathway in Motor Neurons

Hislop, J.N., Islam, T.A., eleftheriadou, I., Carpentier, D.C.J., Trabalza, A., Parkinson, M., Schhiavo, G. and Mazarakis, N.D.

1. Biol. Chem., 289(23), 16148-16163 (2014)

  Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labeling techniques and confocal microscopy, we demonstrated that pseudotyping with rabies virus envelope glycoprotein (RV-G) enabled the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process revealed that these vectors trafficked through Rab5-positive endosomes and accumulated within a non-acidic Rab7 compartment. RV-G pseudotyped vectors were co-transported with both the tetanus neurotoxin-binding fragment and the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, and p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultured in compartmentalized chambers, we demonstrated that axonal retrograde transport of these vectors was rapid and efficient; however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, which suggests a novel area for improvement of gene therapy vectors.  

4.1297           Efficient Transient Transfection of Human Multiple Myeloma Cells by Electroporation – An Appraisal

Steinbrunn, T., Chatterjee, M., Bargou, R.C. and Stühmer, T. PloS One, 9(6), e97443 (2014)   Cell lines represent the everyday workhorses for in vitro research on multiple myeloma (MM) and are regularly employed in all aspects of molecular and pharmacological investigations. Although loss-of-function studies using RNA interference in MM cell lines depend on successful knockdown, no well-established and widely applied protocol for efficient transient transfection has so far emerged. Here, we provide an appraisal of electroporation as a means to introduce either short-hairpin RNA expression vectors or synthesised siRNAs into MM cells. We found that electroporation using siRNAs was much more efficient than previously anticipated on the basis of transfection efficiencies deduced from EGFP-expression off protein expression vectors. Such knowledge can even confidently be exploited in “hard-to-transfect” MM cell lines to generate large numbers of transient knockdown phenotype MM cells. In addition, special attention was given to developing a protocol that provides easy implementation, good reproducibility and manageable experimental costs.  

4.1298           Human Tonsil-derived Dendritic Cells are Poor Inducers of T cell Immunity to Mucosally Encountered Pathogens

Hallisey, C.M., Heydeman, R.S. and Williams, N.A.

1. Infectious Disease, 209, 1847-1856 (2014)

  The mucosal immune system must initiate and regulate protective immunity, while balancing this immunity with tolerance to harmless antigens and bacterial commensals. We have explored the hypothesis that mucosal dendritic cells (DC) control the balance between regulation and immunity, by studying the responses of human tonsil-derived DC to Neisseria meningitidis as a model organism. We show that tonsil DC are able to sample their antigenic environment, internalizing Nm and expressing high levels of HLA-DR and CD86. However, in comparison to monocyte-derived DC (moDC), they respond to pathogen encounter with only low level cytokine production, largely dominated by TGFβ. Functionally, tonsil DC also only stimulated low levels of antigen-specific T cell proliferation and cytokine production when compared to moDC. We therefore propose that the default role for DC in the nasopharynx is to maintain tolerance/ignorance of the large volume of harmless antigens and bacterial commensals encountered at the nasopharyngeal mucosa.  

4.1299           Dietary Zinc Supplementation to the Donor Improves Insulin Secretion After Islet Transplantation in Chemically Induced Diabetic Rats

Mishima, T., Kuroki, T., Tajima, Y., Adachi, T., Hirabaru, M., Tanaka, T., Kitasato, A., Takatsuki, M. and Eguchi, S. Pancreas, 43(2), 236-239 (2014)   Objectives Zinc (Zn) is related to insulin synthesis, storage, and secretion. This study demonstrates the effects of Zn supplementation in donor rats on the outcomes of islet transplantation.   Methods Donor rats received 3 different regimens of dietary Zn supplementation for 2 weeks before undergoing pancreas donation: a standard diet containing Zn at 50 ppm (control), 1 ppm (low-Zn group) or 1000 ppm (high-Zn group), respectively. Diabetic recipient rats underwent islet transplantation, and the blood glucose levels and insulin secretion were monitored for 7 days after transplantation.   Results The serum and pancreatic Zn levels at the time of donation were significantly lower in the low-Zn group (48.8 ± 25.5 µg/dL and 11.3 ± 1.9 µg/g) and higher in the high-Zn group (147.3 ± 17.6 µg/dL and 18.7 ± 2.2 µg/g) when compared with those observed in the controls (118.7 ± 7.9 µg/dL and 14.6 ± 2.0 µg/g) (P < 0.05). The blood glucose levels became re-elevated 2 days after transplantation in rats receiving islet grafts from the controls and the low-Zn groups. In contrast, in the rats that received islets from the high-Zn groups, these were maintained within a reference range (P < 0.01).   Conclusions These data indicate that a Zn-rich diet for donor rats improves the function of islet grafts in chemically induced diabetic rats.  

4.1300           Long-Term Function of Islets Encapsulated in a Redesigned Alginate Microcapsule Construct in Omentum Pouches of Immune-Competent Diabetic Rats

Pareta, R., McQuilling, J.P., Sittadjody, S., Jenkins, R., Bowden, S., Orlando, G., Farney, A.C., Brey, E.M. and Opara, E.C. Pancreas, 43(4), 605-613 (2014)   Objective Our study aim was to determine encapsulated islet graft viability in an omentum pouch and the effect of fibroblast growth factor 1 (FGF-1) released from our redesigned alginate microcapsules on the function of the graft. Methods Isolated rat islets were encapsulated in an inner core made with 1.5% low-viscosity–high-mannuronic-acid alginate followed by an external layer made with 1.25% low-viscosity high-guluronic acid alginate with or without FGF-1, in microcapsules measuring 300 to 400 µm in diameter. The 2 alginate layers were separated by a perm-selective membrane made with 0.1% poly-l-ornithine, and the inner low-viscosity–high-mannuronic-acid core was partially chelated using 55 mM sodium citrate for 2 minutes. Results A marginal mass of encapsulated islet allografts (∼2000 islets/kg) in streptozotocin-diabetic Lewis rats caused significant reduction in blood glucose levels similar to the effect observed with encapsulated islet isografts. Transplantation of alloislets coencapsulated with FGF-1 did not result in better glycemic control, but induced greater body weight maintenance in transplant recipients compared with those that received only alloislets. Histological examination of the retrieved tissue demonstrated morphologically and functionally intact islets in the microcapsules, with no signs of fibrosis. Conclusions We conclude that the omentum is a viable site for encapsulated islet transplantation.  

4.1301           Lactate Reduces Liver and Pancreatic Injury in Toll-Like Receptor– and Inflammasome-Mediated Inflammation via GPR81-Mediated Suppression of Innate Immunity

Hoque, R., Farooq, A., Ghani, A., Gorelick, F. and Mehal, W.Z. Gastroenterology, 146, 1763-1774 (2014)   Background & Aims The NACHT, LRR, and pyrin domain–containing protein 3 (NLRP3) inflammasome induces inflammation in response to organ injury, but little is known about its regulation. Toll-like receptors (TLRs) provide the first signal required for activation of the inflammasome and stimulate aerobic glycolysis to generate lactate. We examined whether lactate and the lactate receptor, G_i-protein–coupled receptor 81 (GPR81), regulate TLR induction of signal 1 and limit inflammasome activation and organ injury. Methods Primary mouse macrophages and human monocytes were incubated with TLR4 agonists and lactate and assayed for levels of pro-interleukin (IL)1β, NLRP3, and caspase-1 (CASP1); release of IL1β; and activation of nuclear factor-κB (NF-κB) and caspase-1. Small interfering RNAs were used to reduce levels of GPR81 and arrestin β-2 (ARRB2), and an NF-κB luciferase reporter transgene was transfected in RAW 264.7 cells. Cell lysates were analyzed by immunoprecipitation with an antibody against GPR81. Acute hepatitis was induced in C56BL/6N mice by administration of lipopolysaccharide and D-galactosamine. Acute pancreatitis was induced by administration of lipopolysaccharide and cerulein. Some mice were given intraperitoneal injections of sodium lactate or small interfering RNA against Gpr81. Activation of NF-κB in tissue macrophages was assessed in mice that expressed a reporter transgene. Results In macrophages and monocytes, increasing concentrations of lactate reduced TLR4-mediated induction of Il1B, Nlrp3, and Casp1; activation of NF-κB; release of IL1β; and cleavage of CASP1. GPR81 and ARRB2 physically interacted and were required for these effects. The administration of lactate reduced inflammation and organ injury in mice with immune hepatitis; this reduction required Gpr81 dependence in vivo. Lactate also prevented activation of NF-κB in macrophages of mice, and, when given after injury, reduced the severity of acute pancreatitis and acute liver injury. Conclusions Lactate negatively regulates TLR induction of the NLRP3 inflammasome and production of IL1β, via ARRB2 and GPR81. Lactate could be a promising immunomodulatory therapy for patients with acute organ injury.  

4.1302           Despite Differences in Cytosolic Calcium Regulation, Lidocaine Toxicity Is Similar in Adult and Neonatal Rat Dorsal Root Ganglia In Vitro

Doan, L.V., Eydlin, O., Piskoun, B., Kline, R., Recio-Pinto, E., Rosenberg, A.D., Blanck, T.J.J. and Xu, F. Anesthesiology,120(1), 50-61 (2014)   Background: Neuraxial local anesthetics may have neurological complications thought to be due to neurotoxicity. A primary site of action of local anesthetics is the dorsal root ganglia (DRG) neuron. Physiologic differences have been noted between young and adult DRG neurons; hence, the authors examined whether there were any differences in lidocaine-induced changes in calcium and lidocaine toxicity in neonatal and adult rat DRG neurons. Methods: DRG neurons were cultured from postnatal day 7 (P7) and adult rats. Lidocaine-induced changes in cytosolic calcium were examined with the calcium indicator Fluo-4. Cells were incubated with varying concentrations of lidocaine and examined for viability using calcein AM and ethidium homodimer-1 staining. Live imaging of caspase-3/7 activation was performed after incubation with lidocaine. Results: The mean KCl-induced calcium transient was greater in P7 neurons (P < 0.05), and lidocaine significantly inhibited KCl-induced calcium responses in both ages (P < 0.05). Frequency distribution histograms of KCl-evoked calcium increases were more heterogeneous in P7 than in adult neurons. With lidocaine, KCl-induced calcium transients in both ages became more homogeneous but remained different between the groups. Interestingly, cell viability was decreased by lidocaine in a dose-dependent manner similarly in both ages. Lidocaine treatment also activated caspase-3/7 in a dose- and time-dependent manner similarly in both ages. Conclusions: Despite physiological differences in P7 and adult DRG neurons, lidocaine cytotoxicity is similar in P7 and adult DRG neurons in vitro. Differences in lidocaine- and KCl-evoked calcium responses suggest the similarity in lidocaine cytotoxicity involves other actions in addition to lidocaine-evoked effects on cytosolic calcium responses.  

4.1303           NKT Cells Determine Titer and Subtype Profile of Virus-Specific IgG Antibodies during Herpes Simplex Virus Infection

Rafter, M.J., Wolter, E., Fillatreau, S., Meisel, H., Kaufmann, H.E. and Schönrich, G.

1. Immunol., 192(9), 4294-4302 (2014)

  Invariant NKT cells (iNKT cells) are innate lymphocytes that recognize lipid-derived Ags presented by the MHC class I–related protein CD1d. In this study, we analyzed the role of iNKT cells in the generation of Abs against HSV type 1 (HSV-1). In sera from healthy hman donors, we found a correlation between HSV-1–specific IgG titers and proportions of CD4⁺ iNKT cells. In HSV-1–infected iNKT cell–deficient mice, the amount of specific IgM and IgG Abs were significantly reduced compared with wild-type mice. Moreover, iNKT cell–deficient mice were unable to upregulate CD1d on B cells and failed to establish an IFN-γ–driven subtype profile of HSV-1–specific IgG Abs. In spleens of HSV-1–infected wild-type mice, the percentage of iNKT cells expressing CCR6, a marker for inflammatory iNKT cells secreting IFN-γ, was significantly decreased at 6 mo postinfection, suggesting that these cells were released from the spleen to other tissues. Finally, in vitro experiments showed that in the absence of CD1d-restricted cells, HSV-1 induced markedly lower IFN-γ production in splenocytes from naive mice. Taken together, our results indicate that iNKT cells shape the Ab response to HSV-1 infection and provide a basis for rational development of antiviral vaccines.  

4.1304           Adult Porcine Islet Isolation Using a Ductal Preservation Method and Purification With a Density Gradient Composed of Histidine-Tryptophan-Ketoglutarate Solution and Iodixanol

Jin, S-M.,Lee, H-S., Oh, S-H., Park, H.J., Park, J.B., Kim, J.H. and Kim, S.J Transplantation Proceedings, 46, 1628-1632 (2014)   Background Given the fragility of adult porcine islets, reduction of shearing stress in islet purification using histidine-tryptophan-ketoglutarate (HTK) solution and iodixanol could be an effective strategy. We examined the effect of ductal preservation with HTK solution and an islet purification protocol that utilizes HTK solution and iodixanol in adult porcine islet isolation. Methods Islets were isolated with a modified Ricordi method using adult Prestige World Genetics (PWG) and Yucatan pigs. The discontinuous density gradient was composed of either HTK solution/iodixanol (n = 23, iodixanol group) or Hank's balanced salt solution (HBSS)/Ficoll (n = 17, Ficoll group). In the iodixanol group, ductal injection of HTK solution was performed before purification. Results In PWG pigs, significantly higher islet yield after purification (3480 ± 214.2 islet equivalent [IEQ]/g, P = .003) and higher recovery rate (85.45% ± 3.49%, P = .0043) were obtained from the HTK/iodixanol group as compared to the HBSS/Ficoll group (1905 ± 323.2 IEQ/g, and 67.22% ± 4.77%, respectively). Similar results were obtained in Yucatan pigs with greater body weight. Conclusion Ductal preservation and iodixanol-based islet purification using HTK solution improved the yield of adult porcine islet isolation compared to the conventional method using HBSS and Ficoll. The results of this study support the feasibility of an adult porcine islet isolation protocol using HTK solution and iodixanol, which have the favorable physical properties.  

4.1305           Endoplasmic reticulum stress in spinal and bulbar muscular atrophy: a potential target for therapy

Montague, K., Malik, B., Gray, A.L., La Spada, A.R., Hanna, M.G., Szabadkai, G. and Linda Greensmith Brain, 137, 1894-1906 (2014)   Spinal and bulbar muscular atrophy is an X-linked degenerative motor neuron disease caused by an abnormal expansion in the polyglutamine encoding CAG repeat of the androgen receptor gene. There is evidence implicating endoplasmic reticulum stress in the development and progression of neurodegenerative disease, including polyglutamine disorders such as Huntington’s disease and in motor neuron disease, where cellular stress disrupts functioning of the endoplasmic reticulum, leading to induction of the unfolded protein response. We examined whether endoplasmic reticulum stress is also involved in the pathogenesis of spinal and bulbar muscular atrophy. Spinal and bulbar muscular atrophy mice that carry 100 pathogenic polyglutamine repeats in the androgen receptor, and develop a late-onset neuromuscular phenotype with motor neuron degeneration, were studied. We observed a disturbance in endoplasmic reticulum-associated calcium homeostasis in cultured embryonic motor neurons from spinal and bulbar muscular atrophy mice, which was accompanied by increased endoplasmic reticulum stress. Furthermore, pharmacological inhibition of endoplasmic reticulum stress reduced the endoplasmic reticulum-associated cell death pathway. Examination of spinal cord motor neurons of pathogenic mice at different disease stages revealed elevated expression of markers for endoplasmic reticulum stress, confirming an increase in this stress response in vivo. Importantly, the most significant increase was detected presymptomatically, suggesting that endoplasmic reticulum stress may play an early and possibly causal role in disease pathogenesis. Our results therefore indicate that the endoplasmic reticulum stress pathway could potentially be a therapeutic target for spinal and bulbar muscular atrophy and related polyglutamine diseases.  

4.1306           Microglial VPAC1R mediates a novel mechanism of neuroimmune-modulation of hippocampal precursor cells via IL-4 release

Nunan, R., Sivasathiaseelan, H., Khan, D., Zaben, M. and Gray, W. Glia, 62, 1313-1327 (2014)   Neurogenesis, the production of new neurons from neural stem/progenitor cells (NSPCs), occurs throughout adulthood in the dentate gyrus of the hippocampus, where it supports learning and memory. The innate and adaptive immune systems are increasingly recognized as important modulators of hippocampal neurogenesis under both physiological and pathological conditions. However, the mechanisms by which the immune system regulates hippocampal neurogenesis are incompletely understood. In particular, the role of microglia, the brains resident immune cell is complex, as they have been reported to both positively and negatively regulate neurogenesis. Interestingly, neuronal activity can also regulate the function of the immune system. Here, we show that depleting microglia from hippocampal cultures reduces NSPC survival and proliferation. Furthermore, addition of purified hippocampal microglia, or their conditioned media, is trophic and proliferative to NSPCs. VIP, a neuropeptide released by dentate gyrus interneurons, enhances the proliferative and pro-neurogenic effect of microglia via the VPAC1 receptor. This VIP-induced enhancement is mediated by IL-4 release, which directly targets NSPCs. This demonstrates a potential neuro-immuno-neurogenic pathway, disruption of which may have significant implications in conditions where combined cognitive impairments, interneuron loss, and immune system activation occurs, such as temporal lobe epilepsy and Alzheimer's disease.  

4.1307           Control of Insulin Secretion by Cytochrome c and Calcium Signaling in Islets with Impaired Metabolism

Rountree, A.M., Neal, A.S., Lisowsky, M., Rizzo, M., Radtke, J., White, S., Luciani, D.S., Kim, F., Hampe, C.S. and Sweet, I.R.

1. Biol. Chem., 289(27), 19110-19119 (2014)

  The aim of the study was to assess the relative control of insulin secretion rate (ISR) by calcium influx and signaling from cytochrome c in islets where, as in diabetes, the metabolic pathways are impaired. This was achieved either by culturing isolated islets at low (3 mm) glucose or by fasting rats prior to the isolation of the islets. Culture in low glucose greatly reduced the glucose response of cytochrome c reduction and translocation and ISR, but did not affect the response to the mitochondrial fuel α-ketoisocaproate. Unexpectedly, glucose-stimulated calcium influx was only slightly reduced in low glucose-cultured islets and was not responsible for the impairment in glucose-stimulated ISR. A glucokinase activator acutely restored cytochrome c reduction and translocation and ISR, independent of effects on calcium influx. Islets from fasted rats had reduced ISR and cytochrome c reduction in response to both glucose and α-ketoisocaproate despite normal responses of calcium. Our data are consistent with the scenario where cytochrome c reduction and translocation are essential signals in the stimulation of ISR, the loss of which can result in impaired ISR even when calcium response is normal.  

4.1308           Islet cell transplantation

McCall, M. and Shapiro, A.M.J. Seminars in Pediatric Surgery, 23, 83-90 (2014)   Islet transplantation has become a promising treatment for selected patients with type 1 diabetes. Here we provide an overview of the procedure including its history, the process of donor selection, and the techniques and procedures involved in a successful transplant. A brief overview of the current immunosuppressive regimens, the long-term follow-up and the reported outcomes will also be discussed. While islet transplantation is currently generally reserved for adults with type 1 diabetes with severe hypoglycemia or glycemic lability, we herein consider the possibility of its application to the pediatric population.  

4.1309           Isolation and characterization of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart

Smith, A.J., lewis, F.C., Aquila, I., Waring, C.D., Nocera, A., Agosti, V., Nadal-Ginard, b., Torella, D. and Ellison, G.M. Nature Protocols, 9(7), 1662-1681 (2014)   This protocol describes the isolation of endogenous c-Kit (also known as CD117)-positive (c-Kit⁺), CD45-negative (CD45⁻) cardiac stem cells (eCSCs) from whole adult mouse and rat hearts. The heart is enzymatically digested via retrograde perfusion of the coronary circulation, resulting in rapid and extensive breakdown of the whole heart. Next, the tissue is mechanically dissociated further and cell fractions are separated by centrifugation. The c-Kit⁺CD45⁻ eCSC population is isolated by magnetic-activated cell sorting technology and purity and cell numbers are assessed by flow cytometry. This process takes ∼4 h for mouse eCSCs or 4.5 h for rat eCSCs. We also describe how to characterize c-Kit⁺CD45⁻ eCSCs. The c-Kit⁺CD45⁻ eCSCs exhibit the defining characteristics of stem cells: they are self-renewing, clonogenic and multipotent. This protocol also describes how to differentiate eCSCs into three main cardiac lineages: functional, beating cardiomyocytes, smooth muscle, and endothelial cells. These processes take 17–20 d.  

4.1310           Specific Retrograde Transduction of Spinal Motor Neurons Using Lentiviral Vectors Targeted to Presynaptic NMJ Receptors

Eleftheriadou, I., trabalza, A., Ellison, S.M., Gharun, K. and Mazarakis, N.D. Molecular Therapy, 22(7), 1285-1298 (2014)   To understand how receptors are involved in neuronal trafficking and to be able to utilize them for specific targeting via the peripheral route would be of great benefit. Here, we describe the generation of novel lentiviral vectors with tropism to motor neurons that were made by coexpressing onto the lentiviral surface a fusogenic glycoprotein (mutated sindbis G) and an antibody against a cell-surface receptor (Thy1.1, p75^NTR, or coxsackievirus and adenovirus receptor) on the presynaptic terminal of the neuromuscular junction. These vectors exhibit binding specificity and efficient transduction of receptor positive cell lines and primary motor neurons in vitro. Targeting of each of these receptors conferred to these vectors the capability of being transported retrogradely from the axonal tip, leading to transduction of motor neurons in vitro in compartmented microfluidic cultures. In vivo delivery of coxsackievirus and adenovirus receptor-targeted vectors in leg muscles of mice resulted in predicted patterns of motor neuron labeling in lumbar spinal cord. This opens up the clinical potential of these vectors for minimally invasive administration of central nervous system-targeted therapeutics in motor neuron diseases.  

4.1311           Micropatterned Cell–Cell Interactions Enable Functional Encapsulation of Primary Hepatocytes in Hydrogel Microtissues No Access

Li, C.Y., Stevens, K:R., Schwartz, R.E., Alejandro, B.S., Huang, J.H. and Bhatia, S.N: Tissue Eingineering Part A, 20(15), 2200-2212 (2014)   Drug-induced liver injury is a major cause of drug development failures and postmarket withdrawals. In vitro models that incorporate primary hepatocytes have been shown to be more predictive than model systems which rely on liver microsomes or hepatocellular carcinoma cell lines. Methods to phenotypically stabilize primary hepatocytes ex vivo often rely on mimicry of hepatic microenvironmental cues such as cell–cell interactions and cell–matrix interactions. In this work, we sought to incorporate phenotypically stable hepatocytes into three-dimensional (3D) microtissues, which, in turn, could be deployed in drug-screening platforms such as multiwell plates and diverse organ-on-a-chip devices. We first utilize micropatterning on collagen I to specify cell–cell interactions in two-dimensions, followed by collagenase digestion to produce well-controlled aggregates for 3D encapsulation in polyethylene glycol (PEG) diacrylate. Using this approach, we examined the influence of homotypic hepatocyte interactions and composition of the encapsulating hydrogel, and achieved the maintenance of liver-specific function for over 50 days. Optimally preaggregated structures were subsequently encapsulated using a microfluidic droplet-generator to produce 3D microtissues. Interactions of engineered hepatic microtissues with drugs was characterized by flow cytometry, and yielded both induction of P450 enzymes in response to prototypic small molecules and drug–drug interactions that give rise to hepatotoxicity. Collectively, this study establishes a pipeline for the manufacturing of 3D hepatic microtissues that exhibit stabilized liver-specific functions and can be incorporated into a wide array of emerging drug development platforms.

4.1312           Cutting Edge: Antigen-Specific Thymocyte Feedback Regulates Homeostatic Thymic Conventional Dendritic Cell Maturation

Spidale, N.A., Wang, B. and Tisch, R.

1. Immunol., 193(1), 21-25 (2014)

  Thymic dendritic cells (DC) mediate self-tolerance by presenting self-peptides to and depleting autoreactive thymocytes. Despite a significant role in negative selection, the events regulating thymic DC maturation and function under steady-state conditions are poorly understood. We report that cross-talk with thymocytes regulates thymic conventional DC (cDC) numbers, phenotype, and function. In mice lacking TCR-expressing thymocytes, thymic cDC were reduced and exhibited a less mature phenotype. Furthermore, thymic cDC in TCR-transgenic mice lacking cognate Ag expression in the thymus were also immature; notably, however, thymic cDC maturation was re-established by an Ag-specific cognate interaction with CD4⁺ or CD8⁺ single-positive thymocytes (SP). Blockade of CD40L during Ag-specific interactions with CD4 SP, but not CD8 SP, limited the effect on cDC maturation. Together, these novel findings demonstrate that homeostatic maturation and function of thymic cDC are regulated by feedback delivered by CD4 SP and CD8 SP via distinct mechanisms during a cognate Ag–specific interaction.  

4.1313           GM-CSF–Licensed CD11b+ Lung Dendritic Cells Orchestrate Th2 Immunity to Blomia tropicalis

Zhou, Q., Ho, A.W.S., Schlitzer, A., Tang, Y., Wwong, K.H.S., Wong, F.H.S., Chua, Y.L, Angeli, V., Mortellaro, A., Ginhoux, F. and Kemeny, D.M.

1. Immunol., 193(2), 496-509 (2014)

  The Blomia tropicalis dust mite is prevalent in tropical and subtropical regions of the world. Although it is a leading cause of asthma, little is known how it induces allergy. Using a novel murine asthma model induced by intranasal exposure to B. tropicalis, we observed that a single intranasal sensitization to B. tropicalis extract induces strong Th2 priming in the lung draining lymph node. Resident CD11b⁺ dendritic cells (DCs) preferentially transport Ag from the lung to the draining lymph node and are crucial for the initiation of Th2 CD4⁺ T cell responses. As a consequence, mice selectively deficient in CD11b⁺ DCs exhibited attenuated Th2 responses and more importantly did not develop any allergic inflammation. Conversely, mice deficient in CD103⁺ DCs and CCR2-dependent monocyte-derived DCs exhibited similar allergic inflammation compared with their wild-type counterparts. We also show that CD11b⁺ DCs constitutively express higher levels of GM-CSF receptor compared with CD103⁺ DCs and are thus selectively licensed by lung epithelial-derived GM-CSF to induce Th2 immunity. Taken together, our study identifies GM-CSF–licensed CD11b⁺ lung DCs as a key component for induction of Th2 responses and represents a potential target for therapeutic intervention in allergy.  

4.1314           Multiple pathogenic proteins implicated in neuronopathic Gaucher disease mice

Xu, Y-h., Xu, K., Sun, Y., Liou, B., Quinn, B., Li, R-h., Xue, L., Zhang, W., Setchell, K.D.R., Witte, D. and Grabowski, G.A. Hum. Mol. Genet., 23(15), 3943-3957 (2014)   Gaucher disease, a prevalent lysosomal storage disease (LSD), is caused by insufficient activity of acid β-glucosidase (GCase) and the resultant glucosylceramide (GC)/glucosylsphingosine (GS) accumulation in visceral organs (Type 1) and the central nervous system (Types 2 and 3). Recent clinical and genetic studies implicate a pathogenic link between Gaucher and neurodegenerative diseases. The aggregation and inclusion bodies of α-synuclein with ubiquitin are present in the brains of Gaucher disease patients and mouse models. Indirect evidence of β-amyloid pathology promoting α-synuclein fibrillation supports these pathogenic proteins as a common feature in neurodegenerative diseases. Here, multiple proteins are implicated in the pathogenesis of chronic neuronopathic Gaucher disease (nGD). Immunohistochemical and biochemical analyses showed significant amounts of β-amyloid and amyloid precursor protein (APP) aggregates in the cortex, hippocampus, stratum and substantia nigra of the nGD mice. APP aggregates were in neuronal cells and colocalized with α-synuclein signals. A majority of APP co-localized with the mitochondrial markers TOM40 and Cox IV; a small portion co-localized with the autophagy proteins, P62/LC3, and the lysosomal marker, LAMP1. In cultured wild-type brain cortical neural cells, the GCase-irreversible inhibitor, conduritol B epoxide (CBE), reproduced the APP/α-synuclein aggregation and the accumulation of GC/GS. Ultrastructural studies showed numerous larger-sized and electron-dense mitochondria in nGD cerebral cortical neural cells. Significant reductions of mitochondrial adenosine triphosphate production and oxygen consumption (28–40%) were detected in nGD brains and in CBE-treated neural cells. These studies implicate defective GCase function and GC/GS accumulation as risk factors for mitochondrial dysfunction and the multi-proteinopathies (α-synuclein-, APP- and Aβ-aggregates) in nGD.  

4.1315           Connective tissue growth factor (CCN2) and microRNA-21 are components of a positive feedback loop in pancreatic stellate cells (PSC) during chronic pancreatitis and are exported in PSC-derived Exosomes

Charrier, A., Chen, R., Chen, L., Kemper, S., Hattori, T., Takigawa, M. and Brigstock, D.R.

1. Cell Commun. Signal., 8, 147-156 (2014)

  Pancreatitis is an inflammatory condition of the pancreas which, in its chronic form, involves tissue destruction, exocrine and endocrine insufficiency, increased risk of pancreatic cancer, and an extensive fibrotic pathology which is due to unrelenting collagen deposition by pancreatic stellate cells (PSC). In response to noxious agents such as alcohol—excessive consumption of which is a major cause of pancreatitis in the West—normally quiescent PSC undergo a phenotypic and functional transition to activated myofibroblasts which produce and deposit collagen at high levels. This process is regulated by connective tissue growth factor (CCN2), expression of which is highly up-regulated in activated PSC. We show that CCN2 production by activated PSC is associated with enhanced expression of microRNA-21 (miR-21) which was detected at high levels in activated PSC in a murine model of alcoholic chronic pancreatitis. A positive feedback loop between CCN2 and miR-21 was identified that resulted in enhancement of their respective expression as well as that of collagen α1(I). Both miR-21 and CCN2 mRNA were present in PSC-derived exosomes, which were characterized as 50–150 nm CD9-positive nano-vesicles. Exosomes from CCN2-GFP- or miR-21-GFP-transfected PSC were taken up by other PSC cultures, as shown by direct fluorescence or qRT-PCR for GFP. Collectively these studies establish miR-21 and CCN2 as participants in a positive feedback loop during PSC activation and as components of the molecular payload in PSC-derived exosomes that can be delivered to other PSC. Thus interactions between cellular or exosomal miR-21 and CCN2 represent novel aspects of fibrogenic regulation in PSC. Summary Chronic injury in the pancreas is associated with fibrotic pathology which is driven in large part by CCN2-dependent collagen production in pancreatic stellate cells. This study shows that CCN2 up-regulation in PSC is associated with increased expression of miR-21 which, in turn, is able to stimulate CCN2 expression further via a positive feedback loop. Additionally miR-21 and CCN2 were identified in PSC-derived exosomes which effected their delivery to other PSC. The cellular and exosomal miR-21-CCN2 axis is a novel component in PSC fibrogenic signaling.  

4.1316           Mesodermal mesenchymal cells give rise to myofibroblasts, but not epithelial cells, in mouse liver injury

Lua, I., James, D., Wang, J., Wang, K.S. and Asahina, K. Hepatology, 60(1), 311-322 (2014)   Hepatic stellate cells (HSCs) and portal fibroblasts (PFs) are believed to be the major source of myofibroblasts that participate in fibrogenesis by way of synthesis of proinflammatory cytokines and extracellular matrices. Previous lineage tracing studies using MesP1^Cre and Rosa26lacZ^flox mice demonstrated that MesP1+ mesoderm gives rise to mesothelial cells (MCs), which differentiate into HSCs and PFs during liver development. In contrast, several in vivo and in vitro studies reported that HSCs can differentiate into other cell types, including hepatocytes, cholangiocytes, and progenitor cell types known as oval cells, thereby acting as stem cells in the liver. To test whether HSCs give rise to epithelial cells in adult liver, we determined the hepatic lineages of HSCs and PFs using MesP1^Cre and Rosa26mTmG^flox mice. Genetic cell lineage tracing revealed that the MesP1+ mesoderm gives rise to MCs, HSCs, and PFs, but not to hepatocytes or cholangiocytes, in the adult liver. Upon carbon tetrachloride injection or bile duct ligation surgery-mediated liver injury, mesodermal mesenchymal cells, including HSCs and PFs, differentiate into myofibroblasts but not into hepatocytes or cholangiocytes. Furthermore, differentiation of the mesodermal mesenchymal cells into oval cells was not observed. These results indicate that HSCs are not sufficiently multipotent to produce hepatocytes, cholangiocytes, or oval cells by way of mesenchymal-epithelial transition in vivo. Conclusion: Cell lineage tracing demonstrated that mesodermal mesenchymal cells including HSCs are the major source of myofibroblasts but do not differentiate into epithelial cell types such as hepatocytes, cholangiocytes, and oval cells  

4.1317           Lung fibroblasts accelerate wound closure in human alveolar epithelial cells through hepatocyte growth factor/c-Met signaling

Ito, Y., Correll, K., Schiel, J.A., Finigan, J.H., Prekeris, R. and Mason, R.J. Am. J. Physiol. Lung Cell Mol. Physiol., 307(1), L94-L105 (2014)   There are 190,600 cases of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) each year in the United States, and the incidence and mortality of ALI/ARDS increase dramatically with age. Patients with ALI/ARDS have alveolar epithelial injury, which may be worsened by high-pressure mechanical ventilation. Alveolar type II (ATII) cells are the progenitor cells for the alveolar epithelium and are required to reestablish the alveolar epithelium during the recovery process from ALI/ARDS. Lung fibroblasts (FBs) migrate and proliferate early after lung injury and likely are an important source of growth factors for epithelial repair. However, how lung FBs affect epithelial wound healing in the human adult lung has not been investigated in detail. Hepatocyte growth factor (HGF) is known to be released mainly from FBs and to stimulate both migration and proliferation of primary rat ATII cells. HGF is also increased in lung tissue, bronchoalveolar lavage fluid, and serum in patients with ALI/ARDS. Therefore, we hypothesized that HGF secreted by FBs would enhance wound closure in alveolar epithelial cells (AECs). Wound closure was measured using a scratch wound-healing assay in primary human AEC monolayers and in a coculture system with FBs. We found that wound closure was accelerated by FBs mainly through HGF/c-Met signaling. HGF also restored impaired wound healing in AECs from the elderly subjects and after exposure to cyclic stretch. We conclude that HGF is the critical factor released from FBs to close wounds in human AEC monolayers and suggest that HGF is a potential strategy for hastening alveolar repair in patients with ALI/ARDS.  

4.1318           The Ca2+/calmodulin-dependent kinase kinase β-AMP-activated protein kinase-α1 pathway regulates phosphorylation of cytoskeletal targets in thrombin-stimulated human platelets

Onselaer, M.B., Oury, C., Hunter, R.W., eeckhoudt, S., Barile, N., Lecut, C., Morel, N., Viollet, B., Jacquet, L.M., Bertrand, L., Sakamoto, K., Vanoverschelde, J.L., Beauloye, C. and Horman, S.

1. Thrombosis and Haemostatis, 12, 973-986 (2014)

  Background Platelet activation requires sweeping morphologic changes, supported by contraction and remodeling of the platelet actin cytoskeleton. In various other cell types, AMP-activated protein kinase (AMPK) controls the phosphorylation state of cytoskeletal targets. Objective To determine whether AMPK is activated during platelet aggregation and contributes to the control of cytoskeletal targets. Results We found that AMPK-α1 was mainly activated by thrombin, and not by other platelet agonists, in purified human platelets. Thrombin activated AMPK-α1 ex vivo via a Ca²⁺/calmodulin-dependent kinase kinase β (CaMKKβ)-dependent pathway. Pharmacologic inhibition of CaMKKβ blocked thrombin-induced platelet aggregation and counteracted thrombin-induced phosphorylation of several cytoskeletal proteins, namely, regulatory myosin light chains (MLCs), cofilin, and vasodilator-stimulated phosphoprotein (VASP), three key elements involved in actin cytoskeletal contraction and polymerization. Platelets isolated from mice lacking AMPK-α1 showed reduced aggregation in response to thrombin, and this was associated with defects in MLC, cofilin and VASP phosphorylation and actin polymerization. More importantly, we show, for the first time, that the AMPK pathway is activated in platelets of patients undergoing major cardiac surgery, in a heparin-sensitive manner. Conclusion AMPK-α1 is activated by thrombin in human platelets. It controls the phosphorylation of key cytoskeletal targets and actin cytoskeletal remodeling during platelet aggregation.  

4.1319           Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice

Prakash, T. et al Nucleic Acids Res., 42(13), 8796-8807 (2014)   Triantennary N-acetyl galactosamine (GalNAc, GN3), a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense oligonucleotides (ASOs) 6–10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S-cEt (S-2′-O-Et-2′,4′-bridged nucleic acid) gapmer ASOs, ∼60-fold enhancement in potency relative to the parent MOE (2′-O-methoxyethyl RNA) ASO was observed. GN3-conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3-ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3-ASO conjugate was detected in plasma suggesting that GN3 acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver. GalNAc conjugation also enhanced potency and duration of the effect of two ASOs targeting human apolipoprotein C-III and human transthyretin (TTR) in transgenic mice. The unconjugated ASOs are currently in late stage clinical trials for the treatment of familial chylomicronemia and TTR-mediated polyneuropathy. The ability to translate these observations in humans offers the potential to improve therapeutic index, reduce cost of therapy and support a monthly dosing schedule for therapeutic suppression of gene expression in the liver using ASOs.  

4.1320           Limited Type I Interferons and Plasmacytoid Dendritic Cells during Neonatal Respiratory Syncytial Virus Infection Permit Immunopathogenesis upon Reinfection

Cormier, S.A., Shrestha, B., Saravia, J., Lee, G.I., Shen, L., DeVincenzo, J.P., Kim, Y-i. and You, D.

1. Virol., 88(16), 9350-9360 (2014)

  Respiratory syncytial virus (RSV) infection is the number one cause of bronchiolitis in infants, yet no vaccines are available because of a lack of knowledge of the infant immune system. Using a neonatal mouse model, we previously revealed that mice initially infected with RSV as neonates develop Th2-biased immunopathophysiologies during reinfection, and we demonstrated a role for enhanced interleukin-4 receptor α (IL-4Rα) expression on T helper cells in these responses. Here we show that RSV infection in neonates induced limited type I interferon (IFN) and plasmacytoid dendritic cell (pDC) responses. IFN alpha (IFN-α) treatment or adoptive transfer of adult pDCs capable of inducing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. A reduced viral load and downregulation of IL-4Rα on Th2 cells were observed in IFN-α-treated neonatal mice, suggesting dual mechanisms of action.  

4.1321           Intrinsic Innate Immunity Fails To Control Herpes Simplex Virus and Vesicular Stomatitis Virus Replication in Sensory Neurons and Fibroblasts

Rosato, P.C. and Leib, D.A.

1. Virol., 88(17), 9991-10001 (2014)

  Herpes simplex virus 1 (HSV-1) establishes lifelong latent infections in the sensory neurons of the trigeminal ganglia (TG), wherein it retains the capacity to reactivate. The interferon (IFN)-driven antiviral response is critical for the control of HSV-1 acute replication. We therefore sought to further investigate this response in TG neurons cultured from adult mice deficient in a variety of IFN signaling components. Parallel experiments were also performed in fibroblasts isolated concurrently. We showed that HSV-1 replication was comparable in wild-type (WT) and IFN signaling-deficient neurons and fibroblasts. Unexpectedly, a similar pattern was observed for the IFN-sensitive vesicular stomatitis virus (VSV). Despite these findings, TG neurons responded to IFN-β pretreatment with STAT1 nuclear localization and restricted replication of both VSV and an HSV-1 strain deficient in γ34.5, while wild-type HSV-1 replication was unaffected. This was in contrast to fibroblasts in which all viruses were restricted by the addition of IFN-β. Taken together, these data show that adult TG neurons can mount an effective antiviral response only if provided with an exogenous source of IFN-β, and HSV-1 combats this response through γ34.5. These results further our understanding of the antiviral response of neurons and highlight the importance of paracrine IFN-β signaling in establishing an antiviral state.  

4.1322           A genetic fiber modification to achieve matrix-metalloprotease-activated infectivity of oncolytic adenovirus

Jose, A., Rovira-Rigau, M., Luna, J., Gimenez-Alejandre, M., Vaquero, E., de la Torre, B.G., Andreu, D., Alemany, R. and Fillat, C.

1. Controlled Release, 192, 148-156 (2014)

  Selective tumor targeting of oncolytic adenovirus at the level of cell entry remains a major challenge to improve efficacy and safety. Matrix metalloproteases (MMPs) are overexpressed in a variety of tumors and in particular in pancreatic cancer. In the current work, we have exploited the expression of MMPs together with the penetration capabilities of a TAT-like peptide to engineer tumor selective adenoviruses. We have generated adenoviruses containing CAR-binding ablated fibers further modified with a C-terminus TAT-like peptide linked to a blocking domain by an MMP-cleavable sequence. This linker resulted in a MMP-dependent cell transduction of the reporter MMP-activatable virus AdTATMMP and in efficient transduction of neoplastic cells and cancer-associated fibroblasts. Intravenous and intraductal administration of AdTATMMP into mice showed very low AdTATMMP activity in the normal pancreas, whereas increased transduction was observed in pancreatic tumors of transgenic Ela-myc mice. Intraductal administration of AdTATMMP into mice bearing orthotopic tumors led to a 25-fold increase in tumor targeting compared to the wild type fiber control. A replication competent adenovirus, Ad^RCMMP, with the MMP-activatable fiber showed oncolytic efficacy and increased antitumor activity compared to Adwt in a pancreatic orthotopic model. Reduced local and distant metastases were observed in Ad^RCMMP treated-mice. Moreover, no signs of pancreatic toxicity were detected. We conclude that MMP-activatable adenovirus may be beneficial for pancreatic cancer treatment.    

4.1323           Activation of AMP-activated Protein Kinase Regulates Hippocampal Neuronal pH by Recruiting Na+/H+ Exchanger NHE5 to the Cell Surface

Jinadasa, T.S., Szabo, E.Z., Numata, M. and Orlowski, J.

1. Biol. Chem.,289(30), 20879-20897 (2014)

  Strict regulation of intra- and extracellular pH is an important determinant of nervous system function as many voltage-, ligand-, and H⁺-gated cationic channels are exquisitely sensitive to transient fluctuations in pH elicited by neural activity and pathophysiologic events such as hypoxia-ischemia and seizures. Multiple Na⁺/H⁺ exchangers (NHEs) are implicated in maintenance of neural pH homeostasis. However, aside from the ubiquitous NHE1 isoform, their relative contributions are poorly understood. NHE5 is of particular interest as it is preferentially expressed in brain relative to other tissues. In hippocampal neurons, NHE5 regulates steady-state cytoplasmic pH, but intriguingly the bulk of the transporter is stored in intracellular vesicles. Here, we show that NHE5 is a direct target for phosphorylation by the AMP-activated protein kinase (AMPK), a key sensor and regulator of cellular energy homeostasis in response to metabolic stresses. In NHE5-transfected non-neuronal cells, activation of AMPK by the AMP mimetic AICAR or by antimycin A, which blocks aerobic respiration and causes acidification, increased cell surface accumulation and activity of NHE5, and elevated intracellular pH. These effects were effectively blocked by the AMPK antagonist compound C, the NHE inhibitor HOE694, and mutation of a predicted AMPK recognition motif in the NHE5 C terminus. This regulatory pathway was also functional in primary hippocampal neurons, where AMPK activation of NHE5 protected the cells from sustained antimycin A-induced acidification. These data reveal a unique role for AMPK and NHE5 in regulating the pH homeostasis of hippocampal neurons during metabolic stress.  

4.1324           Activation of Nuclear Factor Kappa B in the Hepatic Stellate Cells of Mice with Schistosomiasis Japonica

He, X., Pu, G., Tang, R., Zhang, D. and Pan, W. PloS One, 9(8), e104323 (2014)   Schistosomiasis japonica is a serious tropical parasitic disease in humans, which causes inflammation and fibrosis of the liver. Hepatic stellate cells (HSCs) are known to play an important role in schistosome-induced fibrosis, but their role in schistosome-induced inflammation is still largely unknown. Here, we use a murine model of schistosomiasis japonica to investigate the role that nuclear factor kappa B (NF-κB), a critical mediator of inflammatory responses, plays in schistosome-induced inflammation. We revealed that NF-κB was significantly activated in HSCs at the early stage of infection, but not at later stages. We also show that the expression levels of several chemokines regulated by NF-κB signaling (Ccl2, Ccl3 and Ccl5) were similarly elevated at early infection. TLR4 signaling, one of the strongest known inducers of NF-κB activation, seemed not activated in HSCs post-infection. Importantly, we found that levels of miR-146 (a known negative regulator of NF-κB signaling) in HSCs opposed those of NF-κB signaling, elevating at later stage of infection. These results indicate that HSCs might play an important role in the progression of hepatic schistosomiasis japonica by linking liver inflammation to fibrosis via NF-κB signaling. Moreover, our work suggests that miR-146 appeared to regulate this process. These findings are significant and imply that manipulating the function of HSCs by targeting either NF-κB signaling or miR-146 expression may provide a novel method of treating hepatic schistosomiasis japonica.  

4.1325           Pretreatment of Donor Pigs With a Diet Rich in Soybean Oil Increases the Yield of Isolated Islets

Loganathan, G., Graham, M.L., Spizzo, T., Tiwari, M., Lockridge, A.D., Soltani, S., Wilhelm, J.J., Balamurugan, A.N. and Hering, B.J. Transplant. Proceedings, 46, 1945-1949 (2014)   Introduction The pig is considered the donor species of choice for islet xenotransplantation. However, isolation of porcine islets is difficult, particularly from young pigs. Early life exposure to a high-fat diet (HFD) reportedly encourages islet β-cell expansion in neonatal rodents and improves islet viability in culture from pretreated weanling pigs. In this study, we examined the influence of young donor pretreatment with a soybean oil–enriched HFD on porcine islet mass and yield after islet isolation. Materials and Methods Postweaning and between days 70 and 250, pigs were fed either a standard diet (control group; n = 5) or an HFD (experimental group; n = 6). Biochemical blood parameters and acute C-peptide response to intravenous glucose were monitored before pancreas procurement. The study was blinded to objectively evaluate the influence of treated diet. After procurement, pancreas biopsy samples were taken from control and pretreated donor pigs to assess islet number by using a dithizone scoring method and histologic islet area fraction determination. Control and HFD donor pig islets were isolated by using our standard isolation protocol to determine islet yield. Islet isolation characteristics and islet quality were assessed in both groups, and the results were compared. Results There were no significant differences in the donor characteristics (age, body weight, glucose disposal rate, acute C-peptide response to intravenous glucose, cholesterol, and aspartate aminotransferase) except fasting blood glucose level between the control and treatment groups (84 ± 6 vs 99 ± 12 mg/dL; P = .0317). The stimulated insulin and C-peptide levels between groups were similar. However, the dithizone score was slightly higher in the treatment group compared with the control group (95.4 ± 38.5 vs 62.6 ± 23.9; P = .1208). Digestion time, digested pancreas weight, pellet volume, and the fragility index were similar in both groups. However, the average islet count (islet equivalent number/g pancreas) at the digest level was significantly higher in the HFD group than in the control group (1578 ± 994 vs 738 ± 202; P = .0344). The functional viability of 2- and 7 day-cultured islets, as assessed by using oxygen consumption rate corrected for DNA, was similar in both groups. Conclusions Pretreatment of pigs with HFD enriched with soybean oil could potentially be used to improve the islet mass in donor pigs. Further studies are needed to confirm and optimize the use of HFD for the purpose of increasing islet yield from young donor pigs.  

4.1326           TGF-β-dependent induction of CD4+CD25+Foxp3+ Tregs by liver sinusoidal endothelial cells

Carambia, A., Freund, B., Schwinge, D., Heine, M., Laschtowitz, A., Huber, S., Wraith, D.C., Korn, T., Schramm, C., Lohse, A.W., Heeren, J. and Herkel, J.

1. Hepatol., 61, 594-599 (2014)

  Background & Aims CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (Tregs) have a profound ability to control immune responses. We have previously shown that the liver is a major source of peripherally induced Tregs. Here, we investigate the liver cell types and molecular mechanisms responsible for hepatic Treg induction. Methods To assess the Treg-inducing potential of liver resident antigen-presenting cell types, we studied the conversion of Foxp3⁻ non-Tregs into Foxp3⁺ Tregs induced by liver dendritic cells (DCs), liver sinusoidal endothelial cells (LSECs), or Kupffer cells (KCs). The dependency of Treg induction on TGF-β was tested in Treg conversion assays using T cells with reduced TGF-β sensitivity. The suppressive potential of liver cell-induced Tregs was assessed by an in vitro suppression assay and in vivo, in the model of experimental autoimmune encephalomyelitis (EAE). Results All tested liver cell types were capable of inducing Foxp3⁺ Tregs; however, LSECs were most efficient in inducing Tregs. Treg-induction was antigen-specific and depended on TGF-β. LSECs featured membrane-bound LAP/TGF-β and the anchor molecule GARP, which is required for tethering LAP/TGF-β to the cell membrane. LSEC-induced Tregs suppressed proliferation and cytokine secretion of effector T cells in vitro. LSEC-induced Tregs were also functional suppressors in vivo, as neuroantigen-specific Tregs induced by LSECs were able to suppress EAE. Conclusions We demonstrate that LSECs are the major liver cell type responsible for TGF-β dependent hepatic Treg induction. The extraordinary capacity of LSECs to induce Tregs was associated with their unique ability to tether TGF-β to their membrane.  

4.1327           Evaluation of the Contribution of Multiple DAMPs and DAMP Receptors in Cell Death-Induced Sterile Inflammatory Responses

Kataoka, H., Kono, H., patel, Z. and Rock, K.L. PloS One, 9(8), e104741 (2014)   When cells die by necrosis in vivo they stimulate an inflammatory response. It is thought that this response is triggered when the injured cells expose proinflammatory molecules, collectively referred to as damage associated molecular patterns (DAMPs), which are recognized by cells or soluble molecules of the innate or adaptive immune system. Several putative DAMPs and/or their receptors have been identified, but whether and how much they participate in responses in vivo is incompletely understood, and they have not previously been compared side-by-side in the same models. This study focuses on evaluating the contribution of multiple mechanisms that have been proposed to or potentially could participate in cell death-induced inflammation: The third component of complement (C3), ATP (and its receptor P2X7), antibodies, the C-type lectin receptor Mincle (Clec4e), and protease-activated receptor 2 (PAR2). We investigate the role of these factors in cell death-induced inflammation to dead cells in the peritoneum and acetaminophen-induced liver damage. We find that mice deficient in antibody, C3 or PAR2 have impaired inflammatory responses to dying cells. In contrast there was no reduction in inflammation to cell death in the peritoneum or liver of mice that genetically lack Mincle, the P2X7 receptor or that were treated with apyrase to deplete ATP. These results indicate that antibody, complement and PAR2 contribute to cell death-induced inflammation but that Mincle and ATP- P2X7 receptor are not required for this response in at least 2 different in vivo models.  

4.1328           131. Centrifugation of Semen: Cushion Technique

Bradecamp, E.A. Equine Reproductive Procedures, 429-432 (2014)   Centrifugation of semen can be performed with or without the aid of a cushion medium. The purpose of the cushion is to allow the semen to be centrifuged at a higher g force to maximize the percentage of sperm harvested post-centrifugation without having a detrimental effect on the viability of the sperm. This chapter tabulates iodixanol-based cushions for the centrifugation of equine semen. It summarizes step by step procedure for centrifugation of semen, and the equipment and supplies needed.  

4.1329           Reduction of ARNT in myeloid cells causes immune suppression and delayed wound healing

Scott, C., Bonner, J., Min, D., Boughton, P., Stokes, R., Cha, K.M., Walters, S.N., Maskowski, K., Sierro, F., Grey, S.T., Twigg, S., McLennan, S. and Gunton, J.E. Am. J. Physiol. Cell Physiol., 307, C349-C357 (2014)   Aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor that binds to partners to mediate responses to environmental signals. To investigate its role in the innate immune system, floxed ARNT mice were bred with lysozyme M-Cre recombinase animals to generate lysozyme M-ARNT (LAR) mice with reduced ARNT expression. Myeloid cells of LAR mice had altered mRNA expression and delayed wound healing. Interestingly, when the animals were rendered diabetic, the difference in wound healing between the LAR mice and their littermate controls was no longer present, suggesting that decreased myeloid cell ARNT function may be an important factor in impaired wound healing in diabetes. Deferoxamine (DFO) improves wound healing by increasing hypoxia-inducible factors, which require ARNT for function. DFO was not effective in wounds of LAR mice, again suggesting that myeloid cells are important for normal wound healing and for the full benefit of DFO. These findings suggest that myeloid ARNT is important for immune function and wound healing. Increasing ARNT and, more specifically, myeloid ARNT may be a therapeutic strategy to improve wound healing  

4.1330           Caloric restriction confers persistent anti-oxidative, pro-angiogenic, and anti-inflammatory effects and promotes anti-aging miRNA expression profile in cerebromicrovascular endothelial cells of aged rats

Csiszar, A., Gautam, T., Sosnowska, D., Tarantini, S., Banki, E., Tucsek, Z., Toth, P., Losonczy, G., Koller, A., Reglodi, D., Giles, C.B., Wren, J.D., Sonntag, W.E. and Ungvari, Z. Am. J. Physiol. Heart Circ. Physiol., 307, H292-H306 (2014)   In rodents, moderate caloric restriction (CR) without malnutrition exerts significant cerebrovascular protective effects, improving cortical microvascular density and endothelium-dependent vasodilation, but the underlying cellular mechanisms remain elusive. To elucidate the persisting effects of CR on cerebromicrovascular endothelial cells (CMVECs), primary CMVECs were isolated from young (3 mo old) and aged (24 mo old) ad libitum-fed and aged CR F344xBN rats. We found an age-related increase in cellular and mitochondrial oxidative stress, which is prevented by CR. Expression and transcriptional activity of Nrf2 are both significantly reduced in aged CMVECs, whereas CR prevents age-related Nrf2 dysfunction. Expression of miR-144 was upregulated in aged CMVECs, and overexpression of miR-144 significantly decreased expression of Nrf2 in cells derived from both young animals and aged CR rats. Overexpression of a miR-144 antagomir in aged CMVECs significantly decreases expression of miR-144 and upregulates Nrf2. We found that CR prevents age-related impairment of angiogenic processes, including cell proliferation, adhesion to collagen, and formation of capillary-like structures and inhibits apoptosis in CMVECs. CR also exerts significant anti-inflammatory effects, preventing age-related increases in the transcriptional activity of NF-κB and age-associated pro-inflammatory shift in the endothelial secretome. Characterization of CR-induced changes in miRNA expression suggests that they likely affect several critical functions in endothelial cell homeostasis. The predicted regulatory effects of CR-related differentially expressed miRNAs in aged CMVECs are consistent with the anti-aging endothelial effects of CR observed in vivo. Collectively, we find that CR confers persisting anti-oxidative, pro-angiogenic, and anti-inflammatory cellular effects, preserving a youthful phenotype in rat cerebromicrovascular endothelial cells, suggesting that through these effects CR may improve cerebrovascular function and prevent vascular cognitive impairment.  

4.1331           Soluble Adenylyl Cyclase Is Necessary and Sufficient to Overcome the Block of Axonal Growth by Myelin-Associated Factors

Martinez, J., Stressin, A., Campana, A., Hou, J., Nikulina, E., Buck, J., Levin, L.R. and Filbin, M.T.

1. Neurosci., 34(28), 9281-9289 (2014)

  Neurons in the CNS do not regenerate following injury; regeneration is blocked by inhibitory proteins in myelin, such as myelin-associated glycoprotein (MAG). Elevating neuronal levels of the second messenger cAMP overcomes this blocked axonal outgrowth. One way to elevate cAMP is pretreating neurons with neurotrophins, such as brain-derived neurotrophic factor (BDNF). However, pleiotropic effects and poor bioavailability make exogenous administration of neurotrophins in vivo problematic; therefore, alternative targets must be considered. In neurons, two families of adenylyl cyclases synthesize cAMP, transmembrane adenylyl cyclases (tmACs), and soluble adenylyl cyclase (sAC). Here, we demonstrate that sAC is the essential source of cAMP for BDNF to overcome MAG-dependent inhibition of neurite outgrowth. Elevating sAC in rat and mouse neurons is sufficient to induce neurite outgrowth on myelin in vitro and promotes regeneration in vivo. These results suggest that stimulators of sAC might represent a novel therapeutic strategy to promote axonal growth and regeneration.  

4.1332           Electrical stimulation of transplanted motoneurons improves motor unit formation

Liu, Y., Grumbles, R.M. and Thomas, C.K.

1. Neurophysiol., 112, 660-670 (2014)

  Motoneurons die following spinal cord trauma and with neurological disease. Intact axons reinnervate nearby muscle fibers to compensate for the death of motoneurons, but when an entire motoneuron pool dies, there is complete denervation. To reduce denervation atrophy, we have reinnervated muscles in Fisher rats from local transplants of embryonic motoneurons in peripheral nerve. Since growth of axons from embryonic neurons is activity dependent, our aim was to test whether brief electrical stimulation of the neurons immediately after transplantation altered motor unit numbers and muscle properties 10 wk later. All surgical procedures and recordings were done in anesthetized animals. The muscle consequences of motoneuron death were mimicked by unilateral sciatic nerve section. One week later, 200,000 embryonic day 14 and 15 ventral spinal cord cells, purified for motoneurons, were injected into the tibial nerve 10–15 mm from the gastrocnemii muscles as the only neuron source for muscle reinnervation. The cells were stimulated immediately after transplantation for up to 1 h using protocols designed to examine differential effects due to pulse number, stimulation frequency, pattern, and duration. Electrical stimulation that included short rests and lasted for 1 h resulted in higher motor unit counts. Muscles with higher motor unit counts had more reinnervated fibers and were stronger. Denervated muscles had to be stimulated directly to evoke contractions. These results show that brief electrical stimulation of embryonic neurons, in vivo, has long-term effects on motor unit formation and muscle force. This muscle reinnervation provides the opportunity to use patterned electrical stimulation to produce functional movements.  

4.1333           TLR2 and TLR4 mediate the TNFα response to Vibrio vulnificus biotype 1

Stamm, L.V. and Drapp, R.L. Pathogens and Disease, 71(3), 357-361 (2014)   Vibrio vulnificus (Vv) is a pathogenic bacterium that can cause life-threatening infections in humans. Most fatal cases are due to septic shock that results from dysregulation of cytokines, particularly TNFα, which plays a critical role in the outcome of Vv infection. The goal of this study was to investigate the Toll-like receptor (TLR)-mediated TNFα response to four Vv biotype 1 strains using mice deficient for TLR2, TLR4, and TLR2/TLR4. Ex vivo assays were performed with blood, splenocytes, and Kupffer cells (KC) from wild-type (WT) and TLR-knockout (KO) mice using formalin-inactivated Vv (f-Vv) as stimulant. All f-Vv biotype 1 strains elicited strong TNFα production by WT mouse blood and cells, which was TLR2 and TLR4 dependent. OxPAPC, an inhibitor of TLR2 and TLR4 signaling, effectively blunted the TLR-mediated TNFα response to f-Vv. Furthermore, TLR2 KO and TLR2/TLR4 KO mice were more resistant to lethal infection with Vv ATCC 27562 than WT mice, perhaps due to attenuation of the TNFα response. These data suggest that it may be possible to devise strategies to specifically target the harmful TLR-mediated TNFα response as an adjunct to antibiotic treatment of severe Vv infection.  

4.1334           Identification of an Atg8-Atg3 Protein–Protein Interaction Inhibitor from the Medicines for Malaria Venture Malaria Box Active in Blood and Liver Stage Plasmodium falciparum Parasites

Hain, A.U.P., bartee, D., Sanders, N.G., Miller, A.S., Sullivan, D.J., Levitskaya, J., Meyers, C.F. and Bosch, J.

1. Med. Chem., 57(11), 4521-4531 (2014)

  Atg8 is a ubiquitin-like autophagy protein in eukaryotes that is covalently attached (lipidated) to the elongating autophagosomal membrane. Autophagy is increasingly appreciated as a target in diverse diseases from cancer to eukaryotic parasitic infections. Some of the autophagy machinery is conserved in the malaria parasite, Plasmodium. Although Atg8’s function in the parasite is not well understood, it is essential for Plasmodium growth and survival and partially localizes to the apicoplast, an indispensable organelle in apicomplexans. Here, we describe the identification of inhibitors from the Malaria Medicine Venture Malaria Box against the interaction of PfAtg8 with its E2-conjugating enzyme, PfAtg3, by surface plasmon resonance. Inhibition of this protein–protein interaction prevents PfAtg8 lipidation with phosphatidylethanolamine. These small molecule inhibitors share a common scaffold and have activity against both blood and liver stages of infection by Plasmodium falciparum. We have derivatized this scaffold into a functional platform for further optimization.  

4.1335           Nrf2 Activation in Astrocytes Contributes to Spinal Cord Ischemic Tolerance Induced by Hyperbaric Oxygen Preconditioning No Access

Xu, J., Huang, G., Zhang, K., Sun, J., Xu, T., Li, R., Tao, H. and Xu, W.

1. Neurotrauma, 31(15), 1343-1353 (2014)

  In this study, we investigated whether nuclear factor erythroid 2-related factor 2 (Nrf2) activation in astrocytes contributes to the neuroprotection induced by a single hyperbaric oxygen preconditioning (HBO-PC) against spinal cord ischemia/reperfusion (SCIR) injury. In vivo: At 24 h after a single HBO-PC at 2.5 atmospheres absolute for 90 min, the male ICR mice underwent SCIR injury by aortic cross-clamping surgery and observed for 48 h. HBO-PC significantly improved hindlimb motor function, reduced secondary spinal cord edema, ameliorated the reactivity of spinal motor-evoked potentials, and slowed down the process of apoptosis to exert neuroprotective effects against SCIR injury. At 12 h or 24 h after HBO-PC without aortic cross-clamping surgery, Western blot, enzyme-linked immunosorbent assay, realtime-polymerase chain reaction and double-immunofluorescence staining were used to detect the Nrf2 activity of spinal cord tissue, such as mRNA level, protein content, DNA binding activity, and the expression of downstream gene, such as glutamate-cysteine ligase, γ-glutamyltransferase, multidrug resistance protein 1, which are key proteins for intracellular glutathione synthesis and transit. The Nrf2 activity and downstream genes expression were all enhanced in normal spinal cord with HBO-PC. Glutathione content of spinal cord tissue with HBO-PC significantly increased at all time points after SCIR injury. Moreover, Nrf2 overexpression mainly occurs in astrocytes. In vitro: At 24 h after HBO-PC, the primary spinal astrocyte-neuron co-cultures from ICR mouse pups were subjected to oxygen-glucose deprivation (OGD) for 90 min to simulate the ischemia-reperfusion injury. HBO-PC significantly increased the survival rate of neurons and the glutathione content in culture medium, which was mainly released from asctrocytes. Moreover, the Nrf2 activity and downstream genes expression induced by HBO-PC were mainly enhanced in astrocytes, but not in neurons. In conclusion, our findings demonstrated that spinal cord ischemic tolerance induced by HBO-PC may be mainly related to Nrf2 activation in astrocytes.  

4.1336           Subchronic olanzapine treatment decreases the expression of pancreatic glucose transporter 2 in rat pancreatic β cells

Shu, S., Liu, H., Wang, M., Su, D., Yao, L. and Wang, G.

1. Endocrinol. Invest., 37, 667-673 (2014)

  Background Olanzapine is a second generation antipsychotic. A common side effect in humans is weight gain, but the mechanisms are mostly unknown. Aim To study the effects of subchronic olanzapine treatment on body weight, fasting plasma glucose (FPG), fasting insulin (FINS), C-peptide, insulin sensitivity index (ISI), and expression of glucose transporter 2 (GLUT2) in rat pancreatic β cells. Materials and methods Female Sprague-Dawley rats were randomly divided into two groups: the olanzapine-treated group and the control group (each n = 8). Rats in the olanzapine-treated group intragastrically received olanzapine 5 mg/kg/day for 28 days; the rats in the control group received the same volume of vehicle. FPG and body weight were measured on the 1st, 7th, 14th and 28th day. FINS and C-peptide were measured using immunoradiometric assays at baseline and on the 28th day. GLUT2 mRNA and protein expressions in pancreatic β cells were analyzed by RT-PCR and western blot. Results Olanzapine-treated rats had higher body weight (227.4 ± 8.9 vs. 211.0 ± 9.9 g), FPG (5.86 ± 0.42 vs. 4.24 ± 0.29 mmol/L), FINS (17.34 ± 3.64 vs. 10.20 ± 1.50 µIU/mL), and C-peptide (0.154 ± 0.027 vs. 0.096 ± 0.009 ng/mL) than those in controls (all P < 0.05) at the 28th day. Pancreatic β cells of the olanzapine-treated group showed lower ISI (−4.60 ± 0.23 vs. −3.76 ± 0.20) and GLUT2 levels (mRNA: 1.12 ± 0.02 vs. 2.00 ± 0.03; protein: 0.884 ± 0.134 vs. 1.118 ± 0.221) than those in controls (all P < 0.05). Conclusions Subchronic olanzapine treatment inhibited expression of GLUT2 in rat pancreatic β cells. Therefore, it may disturb glucose metabolism via the insulin resistance of β cells, but confirmation in humans is needed.  

4.1337           Blood-Derived Mesenchymal Stem Cells Heal Calvarial Defects and Promote Wound Healing

Hu, M., Huang, K-J., Li, S., Wu, J-C., Lo, D.D., Hyun, J.S., Chung, M.T., Hu, M., Longaker, M. and Lorenz, H.P.

1. American College of Surgeons, 219(S3), S85-S86 (2014)

  Introduction Mesenchymal stem cells (MSCs) are promising for their potential in cell-based regenerative therapeutics. However, typically obtained from bone marrow, MSC harvesting techniques are invasive and costly. Here we demonstrate the capacity of peripheral blood-derived MSCs (BD-MSCs) for cell-based calvarial defect and wound healing applications. Methods BD-MSCs were obtained from the peripheral blood of 8-10 week-old CD1 wild type or FVB-Tg(CAG-luc,-GFP)L2G85Chco/J mice, which express firefly luciferase and cytoplasmic eGFP constitutively in all cells, by a novel method utilizing OptiPrep density gradient isolation and hepatocyte stimulation. Cells were characterized by fluorescence-activated cell sorting (FACS). For calvarial defect healing, cells (5.0x105) were seeded onto hydroxyapatite-poly(lactic-co-glycolic acid) (HA-PLGA) scaffolds and placed onto critical-sized (4 mm) calvarial defects on CD1 athymic nude mice. For wound healing, BD-MSCs were seeded onto pullulan-collagen composite dermal hydrogels, and transplanted (2.5×105 cells per wound) onto 6 mm splinted full thickness excisional wounds on the dorsum of FVB/NJ mice. Results Quantification of calvarial defect healing demonstrated increased bone formation in defects treated with HA-PLGA scaffold seeded with BD-MSCs vs HA-PLGA scaffold alone (*p<0.01). BD-MSC-seeded hydrogels demonstrated improved wound healing as compared to un-seeded hydrogel controls on days 8-12 (*p<0.05). The average time for complete wound healing was 12.7 days in the BD-MSC group vs 14 days in the control group (*p<0.05). Conclusions We demonstrate the ability of peripheral blood-derived MSCs to heal calvarial defects and accelerate wound healing in vivo. This novel, less invasive, approach to obtaining MSCs may provide a promising alternative to available techniques for cell-based regenerative applications.  

4.1338           Anti-Inflammatory Activity of Bone Morphogenetic Protein Signaling Pathways in Stomachs of Mice

Takabayashi, H., Shinohara, M., Mao, M., Phaosawasdi, P., El-Zaatari, M., Zhang, M., Ji, T., Eaton, K.A., Dang, D., Kao, J. and Todisco, A. Gastroenterology, 147, 396-406 (2014)   Background & Aims Bone morphogenetic protein (BMP)4 is a mesenchymal peptide that regulates cells of the gastric epithelium. We investigated whether BMP signaling pathways affect gastric inflammation after bacterial infection of mice. Methods We studied transgenic mice that express either the BMP inhibitor noggin or the β- galactosidase gene under the control of a BMP-responsive element and BMP4^βgal/+ mice. Gastric inflammation was induced by infection of mice with either Helicobacter pylori or Helicobacter felis. Eight to 12 weeks after inoculation, gastric tissue samples were collected and immunohistochemical, quantitative, reverse-transcription polymerase chain reaction and immunoblot analyses were performed. We used enzyme-linked immunosorbent assays to measure cytokine levels in supernatants from cultures of mouse splenocytes and dendritic cells, as well as from human gastric epithelial cells (AGS cell line). We also measured the effects of BMP-2, BMP-4, BMP-7, and the BMP inhibitor LDN-193189 on the expression of interleukin (IL)8 messenger RNA by AGS cells and primary cultures of canine parietal and mucus cells. The effect of BMP-4 on NFkB activation in parietal and AGS cells was examined by immunoblot and luciferase assays. Results Transgenic expression of noggin in mice increased H pylori– or H felis–induced inflammation and epithelial cell proliferation, accelerated the development of dysplasia, and increased expression of the signal transducer and activator of transcription 3 and activation-induced cytidine deaminase. BMP-4 was expressed in mesenchymal cells that expressed α-smooth muscle actin and activated BMP signaling pathways in the gastric epithelium. Neither BMP-4 expression nor BMP signaling were detected in immune cells of C57BL/6, BRE–β-galactosidase, or BMP-4^βgal/+ mice. Incubation of dendritic cells or splenocytes with BMP-4 did not affect lipopolysaccharide-stimulated production of cytokines. BMP-4, BMP-2, and BMP-7 inhibited basal and tumor necrosis factor α–stimulated expression of IL8 in canine gastric epithelial cells. LDN-193189 prevented BMP4-mediated inhibition of basal and tumor necrosis factor α–stimulated expression of IL8 in AGS cells. BMP-4 had no effect on TNFα-stimulated phosphorylation and degradation of IκBα, or on TNFα induction of a NFκβ reporter gene. Conclusions BMP signaling reduces inflammation and inhibits dysplastic changes in the gastric mucosa after infection of mice with H pylori or H felis.  

4.1339           Interleukin 17–Producing γδT Cells Promote Hepatic Regeneration in Mice

Rao, R., Graffeo, C.S., Gulati, R., Jamal, M., Narayan, S., Zambirinis, C.R., Barilla, R., Deutsch, M., Greco, S.H., Ochi, A., Tomkötter, L., Blobstein, R., Avanzi, A., Tippens, D.M., Gelstein, Y., Van Heerden, E. and Miller, G. Gastroenterology, 147, 473-484 (2014)   Background & Aims Subsets of leukocytes synergize with regenerative growth factors to promote hepatic regeneration. γδT cells are early responders to inflammation-induced injury in a number of contexts. We investigated the role of γδT cells in hepatic regeneration using mice with disruptions in Tcrd (encodes the T-cell receptor δ chain) and Clec7a (encodes C-type lectin domain family 7 member a, also known as DECTIN1). Methods We performed partial hepatectomies on wild-type C57BL/6, CD45.1, Tcrd^−/−, or Clec7a^−/− mice. Cells were isolated from livers of patients and mice via mechanical and enzymatic digestion. γδT cells were purified by fluorescence-activated cell sorting. Results In mice, partial hepatectomy up-regulated expression of CCL20 and ligands of Dectin-1, which was associated with recruitment and activation of γδT cells and their increased production of interleukin (IL)-17 family cytokines. Recruited γδT cells induced production of IL-6 by antigen-presenting cells and suppressed expression of interferon gamma by natural killer T cells, promoting hepatocyte proliferation. Absence of IL-17–producing γδT cells or deletion of Dectin-1 prevented development of regenerative phenotypes in subsets of innate immune cells. This slowed liver regeneration and was associated with reduced expression of regenerative growth factors and cell cycle regulators. Conversely, exogenous administration of IL-17 family cytokines or Dectin-1 ligands promoted regeneration. More broadly, we found that γδT cells are required for inflammatory responses mediated by IL-17 and Dectin-1. Conclusions γδT cells regulate hepatic regeneration by producing IL-22 and IL-17, which have direct mitogenic effects on hepatocytes and promote a regenerative phenotype in hepatic leukocytes, respectively. Dectin-1 ligation is required for γδT cells to promote hepatic regeneration.  

4.1340           Small-molecule screening identifies inhibition of salt-inducible kinases as a therapeutic strategy to enhance immunoregulatory functions of dendritic cells

Sundberg, T.B. et al PNAS, 111(34), 12468-12473 (2014)   Genetic alterations that reduce the function of the immunoregulatory cytokine IL-10 contribute to colitis in mouse and man. Myeloid cells such as macrophages (MΦs) and dendritic cells (DCs) play an essential role in determining the relative abundance of IL-10 versus inflammatory cytokines in the gut. As such, using small molecules to boost IL-10 production by DCs–MΦs represents a promising approach to increase levels of this cytokine specifically in gut tissues. Toward this end, we screened a library of well-annotated kinase inhibitors for compounds that enhance production of IL-10 by murine bone-marrow–derived DCs stimulated with the yeast cell wall preparation zymosan. This approach identified a number of kinase inhibitors that robustly up-regulate IL-10 production including the Food and Drug Administration (FDA)-approved drugs dasatinib, bosutinib, and saracatinib that target ABL, SRC-family, and numerous other kinases. Correlating the kinase selectivity profiles of the active compounds with their effect on IL-10 production suggests that inhibition of salt-inducible kinases (SIKs) mediates the observed IL-10 increase. This was confirmed using the SIK-targeting inhibitor HG-9-91-01 and a series of structural analogs. The stimulatory effect of SIK inhibition on IL-10 is also associated with decreased production of the proinflammatory cytokines IL-1β, IL-6, IL-12, and TNF-α, and these coordinated effects are observed in human DCs–MΦs and anti-inflammatory CD11c⁺ CX₃CR1^hi cells isolated from murine gut tissue. Collectively, these studies demonstrate that SIK inhibition promotes an anti-inflammatory phenotype in activated myeloid cells marked by robust IL-10 production and establish these effects as a previously unidentified activity associated with several FDA-approved multikinase inhibitors.  

4.1341           Long-Acting Atypical Antipsychotics: Characterization of the Local Tissue Response

Paquette, S.M., Dawit, H., Hickey, M.B., Merisko-Liversidge, E., Almarsson, Ö and Deaver, D.R. Pharm. Res., 31, 2065-2077 (2014)   Purpose Long-acting injectables (LAIs) are increasingly recognized as an effective therapeutic approach for treating chronic conditions. Many LAIs are formulated to create a poorly soluble depot from which the active agent is delivered over time. This long residing depot can cause localized chronic-active inflammation in the tissue, which has not been well defined in the literature. The purpose of this work is to establish an experimental baseline for describing these responses. Methods Non-human primates and rodents were used to examine the response to LAI formulations of two clinically relevant atypical antipsychotics, aripiprazole monohydrate and olanzapine pamoate monohydrate. Results A foreign body response develops with elevations of key cytokines such as IL-1α, IL-1β, TNFα, and IL6 at the site of injection. However, the tissue response for the two atypical antipsychotics compounds diverge as evidenced by quantitative differences observed in cytokine levels at various time points after dosing. Conclusions Our studies show that, while the drugs are in the same therapeutic class, the response to each of these compounds can be distinguished qualitatively and quantitatively, supporting the idea that the injection site reaction involves a multiplicity of factors including the properties of the compound and cellular dynamics at the site of injection.  

4.1342           Distinct Dictation of Japanese Encephalitis Virus-Induced Neuroinflammation and Lethality via Triggering TLR3 and TLR4 Signal Pathways

Han, Y.W., Choi, J.Y., Uyangaa, E., Kim, S.B., Kim, J.H., Kim, B.S., Kim, K. and Eo, S.K. PloS Pathogens, 10(9), e1004319 (2014)   Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3^−/− and TLR4^−/− mice, i.e. TLR3^−/− mice were highly susceptible to JE, whereas TLR4^−/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b⁺Ly-6C^high monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4^−/− mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3^−/− myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4⁺ and CD8⁺ T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b⁺Ly-6C^high monocytes) and CD4⁺Foxp3⁺ Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components during JE progression could be responsible for determining disease outcome through regulating negative and positive factors.  

4.1343           Identification and genetic analysis of cancer cells with PCR-activated cell sorting

Eastburn, D.J., Sciambi, A. and Abate, A.R. Nucleic Acids Res., 42(16), e128 (2014)   Cell sorting is a central tool in life science research for analyzing cellular heterogeneity or enriching rare cells out of large populations. Although methods like FACS and FISH-FC can characterize and isolate cells from heterogeneous populations, they are limited by their reliance on antibodies, or the requirement to chemically fix cells. We introduce a new cell sorting technology that robustly sorts based on sequence-specific analysis of cellular nucleic acids. Our approach, PCR-activated cell sorting (PACS), uses TaqMan PCR to detect nucleic acids within single cells and trigger their sorting. With this method, we identified and sorted prostate cancer cells from a heterogeneous population by performing >132 000 simultaneous single-cell TaqMan RT-PCR reactions targeting vimentin mRNA. Following vimentin-positive droplet sorting and downstream analysis of recovered nucleic acids, we found that cancer-specific genomes and transcripts were significantly enriched. Additionally, we demonstrate that PACS can be used to sort and enrich cells via TaqMan PCR reactions targeting single-copy genomic DNA. PACS provides a general new technical capability that expands the application space of cell sorting by enabling sorting based on cellular information not amenable to existing approaches.  

4.1344           In vitro antiparasitic activity of new thiosemicarbazones in strains of Trypanosoma cruzi

Moreno-Rodriguez, A., Salazar-Schettino, P.M., Bautista, J.K., hernandez-Luis, F., Torrens, H., Guevara-Gomez, Y., Pina-canseco, S., Torres, M.B., Cabrera-bravo, M., Martinez, C.M. and Perez-campos, E. Eur. J. Medicinal Chem., 87, 23-29 (2014)   In this study thiosemicarbazones derivatives of 5-[(trifluoromethyl)phenylthio]-2-furaldehyde were synthesized and evaluated in terms of their efficiency in challenging the growth of epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas' disease. A number of compounds were synthesized from 5-bromo-2-furfuraldehyde using nucleophilic aromatic substitution, with a series of trifluoromethyl thiolates, followed by condensation reactions with thiosemicarbazide. Their molecular structures were determined by ¹H, ¹³C and ¹⁹F NMR, MS and IR spectroscopy. When tested with T.cruzi, they showed a stronger reaction, similar to nifurtimox and benznidazole, with the 5-[nitro-4-(trifluoromethyl)phenyltio]-2-furaldehyde thiosemicarbazone (compound 4) showing the highest antiparasitic activity. This improved activity may be explained due to the nitro group present in the molecule, which potentiates its activity. The thiosemicarbazone derivatives in this study showed no apoptosis in platelets or monocytes, nor did they induce platelet activation. The trypanocidal activity of these substances represents a good starting point for a medicinal chemistry program aimed at therapy for Chagas' disease.  

4.1345           Endothelin-1 induced desensitization in primary afferent neurons

Smith, T.P., Smith, S.N. and Sweitzer, S.M. Neuroscience Letters, 582, 59-64 (2014)   Endothelin-1 (ET-1) is a known algogen that causes acute pain and sensitization in humans and spontaneous nociceptive behaviors when injected into the periphery in rats, and is elevated during vaso-occlusive episodes (VOEs) in sickle cell disease (SCD) patients. Previously, our lab has shown that a priming dose of ET-1 produces sensitization to capsaicin-induce secondary hyperalgesia. The goal of this study was to determine if the sensitization induced by ET-1 priming is occurring at the level of the primary afferent neuron. Calcium imaging in cultured dorsal root ganglion (DRG) neurons was utilized to examine the effects of ET-1 on primary afferent neurons. ET-1 induces [Ca²⁺]_i transients in unprimed cells. ET-1 induced [Ca²⁺]_i transients are attenuated by priming with ET-1. This priming effect occurs whether the priming dose is given 0–4 days prior to the challenge dose. Similarly, ET-1 priming decreases capsaicin-induced [Ca²⁺]_i transients. At the level of the primary afferent neuron, ET-1 priming has a desensitizing effect on challenge exposures to ET-1 and capsaicin.  

4.1346           Identification of the clpB and bipA genes and an evaluation of their expression as related to intracellular survival for the bacterial pathogen Piscirickettsia salmonis

Isla, A., Haussmann, D., Vera, T., Kausel, G. and Figueroa, J. Vet. Microbiol., 173, 390-394 (2014)   Piscirickettsia salmonis is the pathogen responsible for salmonid rickettsial septicemia (SRS), a disease that affects a wide variety of marine cultivated fish species and causes economic losses for the aquaculture industry worldwide. Many in vitro studies have reported on the capacity of this microorganism to replicate in the interior of cytoplasmic vesicles from varied fish cell lines. However, the mechanisms used by this bacteria to survive, replicate, and propagate in cell lines, especially in macrophages and monocytes, are unknown. A number of studies have described the diverse proteins in pathogens such as Legionella pneumophila, Coxiella burnetii, and Francisella tularensis which allow these to evade the cellular immune response and replicate in the interior of macrophages in different hosts. Some of these proteins are the virulence factor BipA/TypA and the heat shock protein ClpB, both of which have been widely characterized. The results of the current study present the complete coding sequence of the genes clpB and bipA from the P. salmonis genome. Moreover, the experimental results suggest that during the infectious process of the SHK-1 cellular line in P. salmonis, the pathogen significantly increases the expression of proteins ClpB and BipA. This would permit the pathogen to adapt to the hostile conditions produced by the macrophage and thus evade mechanisms of cellular degradation while facilitating replication in the interior of this salmon cell line.  

4.1347           Obesity in Aging Exacerbates Blood–Brain Barrier Disruption, Neuroinflammation, and Oxidative Stress in the Mouse Hippocampus: Effects on Expression of Genes Involved in Beta-Amyloid Generation and Alzheimer’s Disease

Tucsek, Z., Toth, P., Sosnowska, D., Gautam, T., Mitschelen, M., Koller, A., Szalai, G., Sonntag, W.E., Ungvari, Z. and Csiszar, A.

1. Gerontol. A Biol. Sci. Med. Sci., 69(10), 1212-1226 (2014)

  There is growing evidence that obesity has deleterious effects on the brain and cognitive function in the elderly population. However, the specific mechanisms through which aging and obesity interact to promote cognitive decline remain unclear. To test the hypothesis that aging exacerbates obesity-induced cerebromicrovascular damage and neuroinflammation, we compared young (7 months) and aged (24 months) high fat diet–fed obese C57BL/6 mice. Aging exacerbated obesity-induced systemic inflammation and blood–brain barrier disruption, as indicated by the increased circulating levels of proinflammatory cytokines and increased presence of extravasated immunoglobulin G in the hippocampus, respectively. Obesity-induced blood–brain barrier damage was associated with microglia activation, upregulation of activating Fc-gamma receptors and proinflammatory cytokines, and increased oxidative stress. Treatment of cultured primary microglia with sera derived from aged obese mice resulted in significantly more pronounced microglia activation and oxidative stress, as compared with treatment with young sera. Serum-induced activation and oxidative stress were also exacerbated in primary microglia derived from aged animals. Hippocampal expression of genes involved in regulation of the cellular amyloid precursor protein–dependent signaling pathways, beta-amyloid generation, and the pathogenesis of tauopathy were largely unaffected by obesity in aged mice. Collectively, obesity in aging is associated with a heightened state of systemic inflammation, which exacerbates blood–brain barrier disruption. The resulting neuroinflammation and oxidative stress in the mouse hippocampus likely contribute to the significant cognitive decline observed in aged obese animals.    

4.1348           Activation of N-methyl-d-aspartate receptor downregulates inflammasome activity and liver inflammation via a β-arrestin-2 pathway

Farooq, A., Hoque, R., Ouyang, X., Farooq, A., Ghani, A., Ahsan, K., Guerra, M. and Mehal, W.Z. Am. J. Physiol. Gastrointest. Liver Physiol., 307, G732-G740 (2014)   Activation of the cytosolic inflammasome machinery is responsible for acute and chronic liver inflammation, but little is known about its regulation. The N-methyl-d-aspartate (NMDA) receptor families are heterotetrameric ligand-gated ion channels that are activated by a range of metabolites, including aspartate, glutamate, and polyunsaturated fatty acids. In the brain NMDA receptors are present on neuronal and nonneuronal cells and regulate a diverse range of functions. We tested the role of the NMDA receptor and aspartate in inflammasome regulation in vitro and in models of acute hepatitis and pancreatitis. We demonstrate that the NMDA receptor is present on Kupffer cells, and their activation on primary mouse and human cells limits inflammasome activation by downregulating NOD-like receptor family, pyrin domain containing 3 and procaspase-1. The NMDA receptor pathway is active in vivo, limits injury in acute hepatitis, and can be therapeutically further activated by aspartate providing protection in acute inflammatory liver injury. Downregulation of inflammasome activation by NMDA occurs via a β-arrestin-2 NF-kβ and JNK pathway and not via Ca²⁺ mobilization. We have identified the NMDA receptor as a regulator of inflammasome activity in vitro and in vivo. This has identified a new area of immune regulation associated by metabolites that may be relevant in a diverse range of conditions, including nonalcoholic steatohepatitis and total parenteral nutrition-induced immune suppression.  

4.1349           Regulator of G-Protein Signaling-5 Is a Marker of Hepatic Stellate Cells and Expression Mediates Response to Liver Injury

Bahrami, A.J., Gunaje, J.J., Hayes, B.J., Riehle, K.J., Kenerson, H.L., Yeung, R.S., Stempien-Otero, A.S., Campbell, J.S. and Mahoney Jr, W.M. PloS One, 9(10), e108505 (2014)   Liver fibrosis is mediated by hepatic stellate cells (HSCs), which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR)-mediated signaling, via endothelin-1 (ET-1) and angiotensin II (AngII), increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5), an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs). Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that Rgs5 expression is regulated during carbon tetrachloride (CCl₄)-induced acute and chronic liver injury in Rgs5^LacZ/LacZ reporter mice. Furthermore, CCl₄ treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl₄ and have increased expression of markers of HSC activation. Knockdown of Rgs5 enhances ET-1-mediated signaling in HSCs in vitro. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury.  

4.1350           Three Distinct Subsets of Thymic Epithelial Cells in Rats and Mice Defined by Novel Antibodies

Sawanobori, Y., Ueta, H., Dijkstra, C.D., park, C.G., Satou, M., Kitazawa, Y. and matsuno, K: PloS One, 9(10), e109995 (2014)   Aim Thymic epithelial cells (TECs) are thought to play an essential role in T cell development and have been detected mainly in mice using lectin binding and antibodies to keratins. Our aim in the present study was to create a precise map of rat TECs using antibodies to putative markers and novel monoclonal antibodies (i.e., ED 18/19/21 and anti-CD205 antibodies) and compare it with a map from mouse counterparts and that of rat thymic dendritic cells. Results Rat TECs were subdivided on the basis of phenotype into three subsets; ED18⁺ED19^+/−keratin 5 (K5)⁺K8⁺CD205⁺ class II MHC (MHCII)⁺ cortical TECs (cTECs), ED18⁺ED21⁻K5⁻K8⁺Ulex europaeus lectin 1 (UEA-1)⁺CD205⁻ medullary TECs (mTEC1s), and ED18⁺ED21⁺K5⁺K8^dullUEA-1⁻CD205⁻ medullary TECs (mTEC2s). Thymic nurse cells were defined in cytosmears as an ED18⁺ED19^+/−K5⁺K8⁺ subset of cTECs. mTEC1s preferentially expressed MHCII, claudin-3, claudin-4, and autoimmune regulator (AIRE). Use of ED18 and ED21 antibodies revealed three subsets of TECs in mice as well. We also detected two distinct TEC-free areas in the subcapsular cortex and in the medulla. Rat dendritic cells in the cortex were MHCII⁺CD103⁺ but negative for TEC markers, including CD205. Those in the medulla were MHCII⁺CD103⁺ and CD205⁺ cells were found only in the TEC-free area. Conclusion Both rats and mice have three TEC subsets with similar phenotypes that can be identified using known markers and new monoclonal antibodies. These findings will facilitate further analysis of TEC subsets and DCs and help to define their roles in thymic selection and in pathological states such as autoimmune disorders.  

4.1351           Morphology and Intrinsic Excitability of Regenerating Sensory and Motor Neurons Grown on a Line Micropattern

Benzina, O., Cloitre, T., martin, M., Raoul, C., gergely, C. and Scamps, F. PloS One, 9(10), e110687 (2014)   Axonal regeneration is one of the greatest challenges in severe injuries of peripheral nerve. To provide the bridge needed for regeneration, biological or synthetic tubular nerve constructs with aligned architecture have been developed. A key point for improving axonal regeneration is assessing the effects of substrate geometry on neuronal behavior. In the present study, we used an extracellular matrix-micropatterned substrate comprising 3 µm wide lines aimed to physically mimic the in vivo longitudinal axonal growth of mice peripheral sensory and motor neurons. Adult sensory neurons or embryonic motoneurons were seeded and processed for morphological and electrical activity analyses after two days in vitro. We show that micropattern-guided sensory neurons grow one or two axons without secondary branching. Motoneurons polarity was kept on micropattern with a long axon and small dendrites. The micro-patterned substrate maintains the growth promoting effects of conditioning injury and demonstrates, for the first time, that neurite initiation and extension could be differentially regulated by conditioning injury among DRG sensory neuron subpopulations. The micro-patterned substrate impacts the excitability of sensory neurons and promotes the apparition of firing action potentials characteristic for a subclass of mechanosensitive neurons. The line pattern is quite relevant for assessing the regenerative and developmental growth of sensory and motoneurons and offers a unique model for the analysis of the impact of geometry on the expression and the activity of mechanosensitive channels in DRG sensory neurons.  

4.1352           Transcriptome analysis of peripheral blood mononuclear cells in human subjects following a 36 h fast provides evidence of effects on genes regulating inflammation, apoptosis and energy metabolism

Elliott, R.M., de Roos, B., Duthie, S.J., Bouwman, F.G., Rubio-Aliaga, I., Crosley, L.K., Mayer, C., Polley, A.C., Heim, C., Coort, S.L., Evelo, C.T., Mulholland, F., Daniel, H., mariman, E.C. and Jonhson, L.T. Genes Nutr., 9:432 (2014)   There is growing interest in the potential health benefits of diets that involve regular periods of fasting. While animal studies have provided compelling evidence that feeding patterns such as alternate-day fasting can increase longevity and reduce incidence of many chronic diseases, the evidence from human studies is much more limited and equivocal. Additionally, although several candidate processes have been proposed to contribute to the health benefits observed in animals, the precise molecular mechanisms responsible remain to be elucidated. The study described here examined the effects of an extended fast on gene transcript profiles in peripheral blood mononuclear cells from ten apparently healthy subjects, comparing transcript profiles after an overnight fast, sampled on four occasions at weekly intervals, with those observed on a single occasion after a further 24 h of fasting. Analysis of the overnight fasted data revealed marked inter-individual differences, some of which were associated with parameters such as gender and subject body mass. For example, a striking positive association between body mass index and the expression of genes regulated by type 1 interferon was observed. Relatively subtle changes were observed following the extended fast. Nonetheless, the pattern of changes was consistent with stimulation of fatty acid oxidation, alterations in cell cycling and apoptosis and decreased expression of key pro-inflammatory genes. Stimulation of fatty acid oxidation is an expected response, most likely in all tissues, to fasting. The other processes highlighted provide indications of potential mechanisms that could contribute to the putative beneficial effects of intermittent fasting in humans.  

4.1353           GABA Promotes Human β-Cell Proliferation and Modulates Glucose Homeostasis

Purwana, I. et al Diabetes, 63, 4197-4205 (2014)   γ-Aminobutyric acid (GABA) exerts protective and regenerative effects on mouse islet β-cells. However, in humans it is unknown whether it can increase β-cell mass and improve glucose homeostasis. To address this question, we transplanted a suboptimal mass of human islets into immunodeficient NOD-scid-γ mice with streptozotocin-induced diabetes. GABA treatment increased grafted β-cell proliferation, while decreasing apoptosis, leading to enhanced β-cell mass. This was associated with increased circulating human insulin and reduced glucagon levels. Importantly, GABA administration lowered blood glucose levels and improved glucose excursion rates. We investigated GABA receptor expression and signaling mechanisms. In human islets, GABA activated a calcium-dependent signaling pathway through both GABA A receptor and GABA B receptor. This activated the phosphatidylinositol 3-kinase–Akt and CREB–IRS-2 signaling pathways that convey GABA signals responsible for β-cell proliferation and survival. Our findings suggest that GABA regulates human β-cell mass and may be beneficial for the treatment of diabetes or improvement of islet transplantation.  

4.1354           Virus-Specific Immune Memory at Peripheral Sites of Herpes Simplex Virus Type 2 (HSV-2) Infection in Guinea Pigs

Xia, J., Veselenak, R.L., Gorder, S.R., Bourne, N. and Milligan, G.N. PloS One, 9(12), e114652 (2014)   Despite its importance in modulating HSV-2 pathogenesis, the nature of tissue-resident immune memory to HSV-2 is not completely understood. We used genital HSV-2 infection of guinea pigs to assess the type and location of HSV-specific memory cells at peripheral sites of HSV-2 infection. HSV-specific antibody-secreting cells were readily detected in the spleen, bone marrow, vagina/cervix, lumbosacral sensory ganglia, and spinal cord of previously-infected animals. Memory B cells were detected primarily in the spleen and to a lesser extent in bone marrow but not in the genital tract or neural tissues suggesting that the HSV-specific antibody-secreting cells present at peripheral sites of HSV-2 infection represented persisting populations of plasma cells. The antibody produced by these cells isolated from neural tissues of infected animals was functionally relevant and included antibodies specific for HSV-2 glycoproteins and HSV-2 neutralizing antibodies. A vigorous IFN-γ-secreting T cell response developed in the spleen as well as the sites of HSV-2 infection in the genital tract, lumbosacral ganglia and spinal cord following acute HSV-2 infection. Additionally, populations of HSV-specific tissue-resident memory T cells were maintained at these sites and were readily detected up to 150 days post HSV-2 infection. Unlike the persisting plasma cells, HSV-specific memory T cells were also detected in uterine tissue and cervicothoracic region of the spinal cord and at low levels in the cervicothoracic ganglia. Both HSV-specific CD4⁺ and CD8⁺ resident memory cell subsets were maintained long-term in the genital tract and sensory ganglia/spinal cord following HSV-2 infection. Together these data demonstrate the long-term maintenance of both humoral and cellular arms of the adaptive immune response at the sites of HSV-2 latency and virus shedding and highlight the utility of the guinea pig infection model to investigate tissue-resident memory in the setting of HSV-2 latency and spontaneous reactivation.  

4.1355           Fuzheng Huayu recipe alleviates hepatic fibrosis via inhibiting TNF-α induced hepatocyte apoptosis

Tao, Y-y., Yan, X-c., Zhou, T., Shen, L., Liu, Z-l. and Liu, C-h. BMC Complementary and Alternative Medicine, 14:449 (2014)   Background What was the relationship of Fuzheng Huayu recipe (FZHY) inhibiting hepatocyte apoptosis and HSC activation at different stage of liver fibrosis? In order to answer this question, the study was carried out to dynamically observe FZHY’s effect on hepatocyte apoptosis and HSC activation and further explored underling mechanism of FZHY against hepatocyte apoptosis. Methods Mice were randomly divided into four groups: normal, model, FZHY, and N-acetylcystein (NAC) groups. Acute hepatic injury and liver fibrosis in mice were induced by CCl₄. Three days before the first CCl₄ injection, treatment with FZHY powder or NAC respectively was started. In vitro, primary hepatocytes were pretreated with FZHY medicated serum or Z-VAD-FMK and then incubated with ActD and TNF-α. Primary HSCs were treated with DNA from apoptotic hepatocytes incubated by Act D/TNF-α or FZHY medicated. Liver sections were analyzed for HE staining and immunohistochemical evaluation of apoptosis. Serum ALT and AST, Alb content and TNF-α expression in liver tissue were detected. Hyp content was assayed and collagen deposition was visualized. Expressions of α-SMA and type I collagen were analyzed by immunofluorescence and immunoblotting. Flow cytometry, immunofluorescence, and DNA ladder for hepatocyte apoptosis and immunoblotting for TNF-R1, Bcl-2 and Bax were also analyzed. Results Mice showed characteristic features of massive hepatocytes apoptosis in early stage of liver injury and developed severe hepatic fibrosis in later phase. FZHY treatment significantly alleviated acute liver injury and hepatocyte apoptosis, and inhibited liver fibrosis by decreasing α-SMA expression and hepatic Hyp content. In vitro, primary hepatocytes were induced by TNF-α and Act D. The anti-apoptotic effect of FZHY was generated by reducing TNFR1 expression and balancing the expressions of Bcl-2 and Bax. Meanwhile, the nuclear DNA from apoptotic hepatocytes stimulated HSC activation in a dose dependent manner, and the DNA from apoptotic hepatocytes treated with FZHY or Z-VAD-FMK reduced HSC activation and type I collagen expression. Conclusion These findings suggested that FZHY suppressed hepatocyte apoptosis through regulating mediators in death receptor and mitochondrial pathways, and the effect of FZHY on hepatocyte apoptosis might play an important role in inhibiting liver fibrosis.  

4.1356           Aag-initiated base excision repair promotes ischemia reperfusion injury in liver, brain, and kidney

Ebrahimkhani, M.R., Daneshmand, A., Mazumder, A., Allocca, M., Calvo, J.A., Abolhassani, N., Jhun, I., Muthupalani, S., Ayata, C. and Samson, L.D. PNAS, 111(43), E4878-E4886 (2014)   Inflammation is accompanied by the release of highly reactive oxygen and nitrogen species (RONS) that damage DNA, among other cellular molecules. Base excision repair (BER) is initiated by DNA glycosylases and is crucial in repairing RONS-induced DNA damage; the alkyladenine DNA glycosylase (Aag/Mpg) excises several DNA base lesions induced by the inflammation-associated RONS release that accompanies ischemia reperfusion (I/R). Using mouse I/R models we demonstrate that Aag^−/− mice are significantly protected against, rather than sensitized to, I/R injury, and that such protection is observed across three different organs. Following I/R in liver, kidney, and brain, Aag^−/− mice display decreased hepatocyte death, cerebral infarction, and renal injury relative to wild-type. We infer that in wild-type mice, Aag excises damaged DNA bases to generate potentially toxic abasic sites that in turn generate highly toxic DNA strand breaks that trigger poly(ADP-ribose) polymerase (Parp) hyperactivation, cellular bioenergetics failure, and necrosis; indeed, steady-state levels of abasic sites and nuclear PAR polymers were significantly more elevated in wild-type vs. Aag^−/− liver after I/R. This increase in PAR polymers was accompanied by depletion of intracellular NAD and ATP levels plus the translocation and extracellular release of the high-mobility group box 1 (Hmgb1) nuclear protein, activating the sterile inflammatory response. We thus demonstrate the detrimental effects of Aag-initiated BER during I/R and sterile inflammation, and present a novel target for controlling I/R-induced injury.  

4.1357           Pancreatic Ductal Perfusion at Organ Procurement Enhances Islet Yield in Human Islet Isolation

Takita, M., Itoh, T., Shimoda, M., Kanak, M.A., Shahbazov, R., Kunnathodi, F., Lawrence, M.C., Naziruddin, B. and Levy, M.F. Pancreas, 43(8), 1249-1255 (2014)   Objective Pancreas preservation is a major factor influencing the results of islet cell transplantation. This study evaluated the effects of 2 different solutions for pancreatic ductal perfusion (PDP) at organ procurement. Methods Eighteen human pancreases were assigned to 3 groups: non-PDP (control), PDP with ET-Kyoto solution, and PDP with cold storage/purification stock solution. Pancreatic islets were isolated according to the modified Ricordi method. Results No significant differences in donor characteristics, including cold ischemia time, were observed between the 3 groups. All islet isolations in the PDP groups had more than 400,000 islet equivalence in total islet yield after purification, a significant increase when compared with the control (P = 0.04 and P < 0.01). The islet quality assessments, including an in vivo diabetic nude mice assay and the response of high-mobility group box protein 1 to cytokine stimulation, also showed no significant differences. The proportion of terminal deoxynucleotidyl transferase dUTP nick-end labeling–positive cells showing apoptosis in islets in the PDP groups was significantly lower than in the control group (P < 0.05). Conclusions Both ET-Kyoto solution and cold storage/purification stock solution are suitable for PDP and consistently resulted in isolation success. Further studies with a larger number of pancreas donors should be done to compare the effects of the PDP solutions.  

4.1358           Neuronal Transgene Expression in Dominant-Negative SNARE Mice

Fujita, T., Chen, M.J., Li, B., Smith, N.A., Peng, W., Sun, W., Toner, M.J., Kress, B.T., Wang, L., Benraiss, A., Takano, T., Wang, S. and Nedergaard, M.

1. Neurosci., 34(50), 16594-16604 (2014)

  Experimental advances in the study of neuroglia signaling have been greatly accelerated by the generation of transgenic mouse models. In particular, an elegant manipulation that interferes with astrocyte vesicular release of gliotransmitters via overexpression of a dominant-negative domain of vesicular SNARE (dnSNARE) has led to documented astrocytic involvement in processes that were traditionally considered strictly neuronal, including the sleep–wake cycle, LTP, cognition, cortical slow waves, depression, and pain. A key premise leading to these conclusions was that expression of the dnSNARE was specific to astrocytes. Inconsistent with this premise, we report here widespread expression of the dnSNARE transgene in cortical neurons. We further demonstrate that the activity of cortical neurons is reversibly suppressed in dnSNARE mice. These findings highlight the need for independent validation of astrocytic functions identified in dnSNARE mice and thus question critical evidence that astrocytes contribute to neurotransmission through SNARE-dependent vesicular release of gliotransmitters.  

4.1359           Micropatterned Coumarin Polyester Thin Films Direct Neurite Orientation

Mccormick, A.M., Maddipatla, M.V.S.N., Shi, S., Chamsaz, E.A., Yokoyama, H., Joy, A. and Leipzig, N.D. ACS Appl. Mater. Interfaces, 6(22), 19655-19667 (2014)   Guidance and migration of cells in the nervous system is imperative for proper development, maturation, and regeneration. In the peripheral nervous system (PNS), it is challenging for axons to bridge critical-sized injury defects to achieve repair and the central nervous system (CNS) has a very limited ability to regenerate after injury because of its innate injury response. The photoreactivity of the coumarin polyester used in this study enables efficient micropatterning using a custom digital micromirror device (DMD) and has been previously shown to be biodegradable, making these thin films ideal for cell guidance substrates with potential for future in vivo applications. With DMD, we fabricated coumarin polyester thin films into 10 × 20 μm and 15 × 50 μm micropatterns with depths ranging from 15 to 20 nm to enhance nervous system cell alignment. Adult primary neurons, oligodendrocytes, and astrocytes were isolated from rat brain tissue and seeded onto the polymer surfaces. After 24 h, cell type and neurite alignment were analyzed using phase contrast and fluorescence imaging. There was a significant difference (p < 0.0001) in cell process distribution for both emergence angle (from the body of the cell) and orientation angle (at the tip of the growth cone) confirming alignment on patterned surfaces compared to control substrates (unpatterned polymer and glass surfaces). The expected frequency distribution for parallel alignment (≤15°) is 14% and the two micropatterned groups ranged from 42 to 49% alignment for emergence and orientation angle measurements, where the control groups range from 12 to 22% for parallel alignment. Despite depths being 15 to 20 nm, cell processes could sense these topographical changes and preferred to align to certain features of the micropatterns like the plateau/channel interface. As a result this initial study in utilizing these new DMD micropatterned coumarin polyester thin films has proven beneficial as an axon guidance platform for future nervous system regenerative strategies.   Molecular aspects, genomic arrangement and immune responsive mRNA expression profiles of two CXC chemokine receptor homologs (CXCR1 and CXCR2) from rock bream, Oplegnathus fasciatus Umasuthan, N., Wan, Q., Revathy, K.S., Whang, I., Noh, J.K., Kim, S., park, M-A. and Lee, J. Fish & Shellfish Immunology, 40(1), 304-318 (2014)   The CXCR1 and CXCR2 are the prototypical receptors and are the only known receptors for mammalian ELR+ (Glu-Leu-Arg) CXC chemokines, including CXCL8 (interleukin 8). These receptors transduce the ELR+ chemokine signals and operate the downstream signaling pathways in inflammation and innate immunity. In this study, we report the identification and characterization of CXCR1 and CXCR2 genes from rock bream fish (OfCXCR1 and OfCXCR2) at the molecular level. The cDNA and genomic DNA sequences of the OfCXCR1 and OfCXCR2 were identified from a transcriptome library and a custom-constructed BAC library, respectively. Both OfCXCR genes consisted of two exons, separated by an intron. The 5′-flanking regions of OfCXCR genes possessed multiple putative transcription factor binding sites related to immune response. The coding sequences of OfCXCR1 and OfCXCR2 encoded putative peptides of 355 and 360 amino acids (aa), respectively. The deduced aa sequences of OfCXCR1 and OfCXCR2 comprised of a G-protein coupled receptors (GPCR) family 1 profile with a GPCR signature and a DRY motif. In addition, seven conserved transmembrane regions were predicted in both OfCXCRs. While our multiple alignment study revealed the functionally significant conserved elements of the OfCXCR1 and OfCXCR2, phylogeny analyses further confirmed their position in teleost sub clade, in which they manifested an evolutionary relatedness with other fish counterparts. Based on comparative analyses, teleost CXC chemokine receptors appear to be distinct from their non-fish orthologs in terms of evolution (both CXCR1 andCXCR2) and genomic organization (CXCR2). Quantitative real-time PCR (qPCR) detected the transcripts of OfCXCR1 and OfCXCR2 in eleven examined tissues, with higher levels in head kidney, kidney and spleen highlighting their crucial importance in immunity. In vitro stimulation of peripheral blood leukocytes (PBLs) with concanavalin A (Con A) resulted in modulation of OfCXCR2 transcription, but not that ofOfCXCR1. In addition, the magnitude of the OfCXCR1 and OfCXCR2 transcripts in head kidney and spleen was differentially increased after the in vivo administration of immune stimulants, LPS and poly I:C and in the infection models injected with rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. These lines of evidence suggest that these receptors may play an important role(s) in immune responsive signaling during pathogenesis of rock bream.   Isolation and characterization of platelet-derived extracellular vesicles Aatonen, M.T., Ôhman, T., Nyman, T.A., laitinen, S., Grönholm, M. and Siljander, P.R.-M.

1. Extracellulaar Vesicles, 3:24692 (2014)

  Background: Platelet-derived extracellular vesicles (EVs) participate, for example, in haemostasis, immunity and development. Most studies of platelet EVs have targeted microparticles, whereas exosomes and EV characterization under various conditions have been less analyzed. Studies have been hampered by the difficulty in obtaining EVs free from contaminating cells and platelet remnants. Therefore, we optimized an EV isolation protocol and compared the quantity and protein content of EVs induced by different agonists. Methods: Platelets isolated with iodixanol gradient were activated by thrombin and collagen, lipopolysaccharide (LPS) or Ca²⁺ ionophore. Microparticles and exosomes were isolated by differential centrifugations. EVs were quantitated by nanoparticle tracking analysis (NTA) and total protein. Size distributions were determined by NTA and electron microscopy. Proteomics was used to characterize the differentially induced EVs. Results: The main EV populations were 100–250 nm and over 90% were <500 nm irrespective of the activation. However, activation pathways differentially regulated the quantity and the quality of EVs, which also formed constitutively. Thrombogenic activation was the most potent physiological EV-generator. LPS was a weak inducer of EVs, which had a selective protein content from the thrombogenic EVs. Ca²⁺ ionophore generated a large population of protein-poor and unselectively packed EVs. By proteomic analysis, EVs were highly heterogeneous after the different activations and between the vesicle subpopulations. Conclusions: Although platelets constitutively release EVs, vesiculation can be increased, and the activation pathway determines the number and the cargo of the formed EVs. These activation-dependent variations render the use of protein content in sample normalization invalid. Since most platelet EVs are 100–250 nm, only a fraction has been analyzed by previously used methods, for example, flow cytometry. As the EV subpopulations could not be distinguished and large vesicle populations may be lost by differential centrifugation, novel methods are required for the isolation and the differentiation of all EVs.  

4.1360           S100A4 promotes liver fibrosis via activation of hepatic stellate cells

Chen, L. et al

1. Hepatol., 62, 156-164 (2015)

  Background & Aims S100A4 has been linked to the fibrosis of several organs due to its role as a fibroblast-specific marker. However, the role of S100A4 itself in the development of fibrosis has not been much investigated. Here, we determined whether S100A4 regulates liver fibrogenesis and examined its mechanism by focusing on the activation of hepatic stellate cells (HSCs). Methods S100A4 deficient mice were used to determine the role of S100A4 in liver fibrogenesis. The effect of S100A4 on HSC activation was estimated by using primary mouse HSCs and the human HSC cell line LX-2. Serum levels of S100A4 in cirrhotic patients were determined by ELISA. Results S100A4 was found to be secreted by a subpopulation of macrophages and to promote the development of liver fibrosis. It accumulated in the liver during the progression of liver fibrosis and activated HSCs in mice. In vitro studies demonstrated that S100A4 induced the overexpression of alpha-smooth muscle actin through c-Myb in HSCs. Both, the selective depletion of S100A4-expressing cells and knockdown of S100A4 in the liver by RNA interference, resulted in a reduction of liver fibrosis following injury. Importantly, increased S100A4 levels in both the liver tissue and serum correlated positively with liver fibrosis in humans. Conclusions S100A4 promotes liver fibrosis by activating HSCs, which may represent a potential target for anti-fibrotic therapies.  

4.1361           The xanthine oxidase inhibitor Febuxostat reduces tissue uric acid content and inhibits injury-induced inflammation in the liver and lung

Kataoka, H., yang, K. and Rock, K.L. Eur. J. Pharmacol., 746, 174-179 (2015)   Necrotic cell death in vivo induces a robust neutrophilic inflammatory response and the resulting inflammation can cause further tissue damage and disease. Dying cells induce this inflammation by releasing pro-inflammatory intracellular components, one of which is uric acid. Cells contain high levels of intracellular uric acid, which is produced when purines are oxidized by the enzyme xanthine oxidase. Here we test whether a non-nucleoside xanthine oxidase inhibitor, Febuxostat (FBX), can reduce intracellular uric acid levels and inhibit cell death-induced inflammation in two different murine tissue injury models; acid-induced acute lung injury and acetaminophen liver injury. Infiltration of inflammatory cells induced by acid injection into lungs or peritoneal administration of acetaminophen was evaluated by quantification with flow cytometry and tissue myeloperoxidase activity in the presence or absence of FBX treatment. Uric acid levels in serum and tissue were measured before giving the stimuli and during inflammation. The impact of FBX treatment on the peritoneal inflammation caused by the microbial stimulus, zymosan, was also analyzed to see whether FBX had a broad anti-inflammatory effect. We found that FBX reduced uric acid levels in acid-injured lung tissue and inhibited acute pulmonary inflammation triggered by lung injury. Similarly, FBX reduced uric acid levels in the liver and inhibited inflammation in response to acetaminophen-induced hepatic injury. In contrast, FBX did not reduce inflammation to zymosan, and therefore is not acting as a general anti-inflammatory agent. These results point to the potential of using agents like FBX to treat cell death-induced inflammation.  

4.1362           Identification of quinoline-chalcone hybrids as potential antiulcer agents

Sashidhara, K.V., Avula, S.R., Mishra, V., Palnati, G.R., Singh, L.R., Singh, N., Chhonker, Y.S., Swami, P., Bhata, R.S. and palit, G. Eur. J. Medicinal Chem., 89, 638-653 (2015)   Antiulcer activity of novel quinoline-chalcone hybrids (13–37) was investigated. Among them, eight compounds (14, 16, 17, 23, 29, 31, 32 and 35) were found to be active in various ulcer models in Sprague–Dawley (SD) rats. To understand the mechanism of action of these hybrids, the effects of the compounds on antisecretory and cytoprotective activities were studied. All these active hybrids improved the depleted levels of mucin and consequently inhibited the formation of erosions in a pyloric ligated ulcer model. In addition, they also significantly increased the gastric PGE₂ content in an aspirin induced ulcer model. The additional experiments including the in vitro metabolic stability and in vivo pharmacokinetics led to the identification of compound 17 as an orally active and safe candidate that is worthy of further investigation to be developed as an antiulcer agent.  

4.1363           Spinal neuroimmune activation is independent of T-cell infiltration and attenuated by A3 adenosine receptor agonists in a model of oxaliplatin-induced peripheral neuropathy

Janes, K., Wahlman, C., Little, J.W., Doyle, T., Tosh, D.K., Jacobson, K.A. and Salvemini, D. Brain, Behavior, and Immunity, 44, 91-99 (2015)   Many commonly used chemotherapeutics including oxaliplatin are associated with the development of a painful chemotherapy-induced peripheral neuropathy (CIPN). This dose-limiting complication can appear long after the completion of therapy causing a significant reduction in quality-of-life and impeding cancer treatment. We recently reported that activation of the G_i/G_q-coupled A₃ adenosine receptor (A₃AR) with selective A₃AR agonists (i.e., IB-MECA) blocked the development of chemotherapy induced-neuropathic pain in models evoked by distinct agents including oxaliplatin without interfering with their anticancer activities. The mechanism(s) of action underlying these beneficial effects has yet to be explored. Our results herein demonstrate that the development of oxaliplatin-induced mechano-hypersensitivity (allodynia and hyperalgesia) in rats is associated with the hyperactivation of astrocytes, but not microglial cells, increased production of pro-inflammatory and neuroexcitatory cytokines (TNF, IL-1β), and reductions in the levels of anti-inflammatory/neuroprotective cytokines (IL-10, IL-4) in the dorsal horn of the spinal cord. These events did not require lymphocytic mobilization since oxaliplatin did not induce CD45⁺/CD3⁺ T-cell infiltration into the spinal cord. A₃AR agonists blocked the development of neuropathic pain with beneficial effects strongly associated with the modulation of spinal neuroinflammatory processes: attenuation of astrocytic hyperactivation, inhibition of TNF and IL-1β production, and an increase in IL-10 and IL-4. These results suggest that inhibition of an astrocyte-associated neuroinflammatory response contributes to the protective actions of A₃AR signaling and continues to support the pharmacological basis for selective A₃AR agonists as adjuncts to chemotherapeutic agents for the management of chronic pain.  

4.1364           Alcohol directly stimulates epigenetic modifications in hepatic stellate cells

Page, A., Paoli, P.P., Hill, S.J., Howarth, R., Wu, R., Kweon, S-M., French, J., White, S., Tsukamoto, H., Mann, D.E. and Mann, J.

1. Hepatol., 62, 388-397 (2015)

  Background & Aims Alcohol is a primary cause of liver disease and an important co-morbidity factor in other causes of liver disease. A common feature of progressive liver disease is fibrosis, which results from the net deposition of fibril-forming extracellular matrix (ECM). The hepatic stellate cell (HSC) is widely considered to be the major cellular source of fibrotic ECM. We determined if HSCs are responsive to direct stimulation by alcohol. Methods HSCs undergoing transdifferentiation were incubated with ethanol and expression of fibrogenic genes and epigenetic regulators was measured. Mechanisms responsible for recorded changes were investigated using ChIP-Seq and bioinformatics analysis. Ethanol induced changes were confirmed using HSCs isolated from a mouse alcohol model and from ALD patient’s liver and through precision cut liver slices. Results HSCs responded to ethanol exposure by increasing profibrogenic and ECM gene expression including elastin. Ethanol induced an altered expression of multiple epigenetic regulators, indicative of a potential to modulate chromatin structure during HSC transdifferentiation. MLL1, a histone 3 lysine 4 (H3K4) methyltransferase, was induced by ethanol and recruited to the elastin gene promoter where it was associated with enriched H3K4me3, a mark of active chromatin. Chromatin immunoprecipitation sequencing (ChIPseq) revealed that ethanol has broad effects on the HSC epigenome and identified 41 gene loci at which both MML1 and its H3K4me3 mark were enriched in response to ethanol. Conclusions Ethanol directly influences HSC transdifferentiation by stimulating global changes in chromatin structure, resulting in the increased expression of ECM proteins. The ability of alcohol to remodel the epigenome during HSC transdifferentiation provides mechanisms for it to act as a co-morbidity factor in liver disease.  

4.1365           Decabrominated diphenyl ether and methylmercury impair fetal nervous system development in mice at documented human exposure levels

Mariani, A., Fanelli, R., Re Depaolini, A. and De Paola, M. Develop. Neurobiol., 75, 23-38 (2015)   The central nervous system (CNS) is extremely vulnerable to the toxic effects of environmental pollutants during development. Polybrominated diphenyl ethers (PBDEs) are persistent contaminants, increasingly present in the environment and in human tissues. Recent investigations identified a correlation between maternal exposure to PBDEs and impairment in fetal neurobehavioral development, suggesting that these contaminants pose a potential risk for children. We investigated on the potential effects of environmental decabrominated diphenyl ether (decaBDE, the fully brominated congener) on key neurodevelopmental molecules (e.g., synaptic proteins and immature neuron markers) in fetal mouse neurons. Methylmercury was used as reference neurotoxic contaminant and to evaluate its possible synergism with decaBDE. The neurotoxic effects of decaBDE and methylmercury were determined in developing cultured neurons from mouse fetal hippocampus and cerebellum. Neuron death, dendritic branching, synaptic protein expression, markers of immature neurons, and microglia activation were evaluated by immunocytochemistry. Brain samples from prenatally treated embryos were also examined for neurotoxicity signs by immunoblotting and histochemistry. DecaBDE significantly affected (down to 0.4 nM) the number of dendritic branches, and the levels of synaptic proteins and doublecortin in cultured neurons. Prenatal exposure to decaBDE decreased the synaptic proteins and increased the expression of the immature neuron and microglial markers in mouse fetuses. In conclusion, prenatal exposure to realistic (relevant for human exposure) concentrations of decaBDE induces impairment of fetal CNS development in mice, suggesting a potential risk of fetotoxicity in humans.  

4.1366           Myeloid-specific disruption of recombination signal binding protein Jκ ameliorates hepatic fibrosis by attenuating inflammation through cylindromatosis in mice

He, F., Guo, F-C., Li, Z., Yu, H-C., Ma, P-F., Zhao, J-L., Feng, L., Li, W-N., Liu, X-W., Qin, H-Y., Dou, K-F. and Han, H. Hepatology, 61, 303-314 (2015)   Macrophages play multidimensional roles in hepatic fibrosis, but their control has not been fully understood. The Notch pathway mediated by recombination signal binding protein Jκ (RBP-J), the transcription factor transactivated by signals from four mammalian Notch receptors, is implicated in macrophage activation and plasticity. In this study, by using mouse hepatic fibrosis models, we show that myeloid-specific disruption of RBP-J resulted in attenuated fibrosis. The activation of hepatic stellate cells and production of profibrotic factors including platelet-derived growth factor (PDGF)-B and transforming growth factor beta1 (TGF-β1) reduced significantly in myeloid-specific RBP-J deficient mice. The infiltration of inflammatory cells and production of proinflammatory factors were reduced in liver of myeloid-specific RBP-J-deficient mice during fibrosis. In RBP-J-deficient macrophages, the nuclear factor kappa B (NF-κB) activation was remarkably attenuated as compared with the control. This could be attributed to the up-regulation of cylindromatosis (CYLD), a negative regulator of NF-κB, in Notch signal-compromised macrophages, because the knockdown of CYLD in RBP-J-deficient macrophages or overexpression of p65 in RBP-J knockdown cells both restored NF-κB activation and the production of proinflammatory and/or profibrotic factors by macrophages. In human hepatic fibrosis biopsies, stronger Notch activation is correlated with more severe fibrosis, which is accompanied by a lower level of CYLD but irrespective of etiological reasons. Conclusion: RBP-J-mediated Notch signaling is required for macrophages to promote hepatic fibrosis by up-regulation of NF-κB activation through CYLD  

4.1367           Inhibition of H3K27me3-Specific Histone Demethylases JMJD3 and UTX Blocks Reactivation of Herpes Simplex Virus 1 in Trigeminal Ganglion Neurons

Messer, H.G.P., Jacobs, D., Dhummakupt, A. and Bloom, D.C.

1. Virol., 89(6), 3417-3420 (2015)

  Herpes simplex virus 1 (HSV-1) genomes are associated with the repressive heterochromatic marks H3K9me2/me3 and H3K27me3 during latency. Previous studies have demonstrated that inhibitors of H3K9me2/me3 histone demethylases reduce the ability of HSV-1 to reactivate from latency. Here we demonstrate that GSK-J4, a specific inhibitor of the H3K27me3 histone demethylases UTX and JMJD3, inhibits HSV-1 reactivation from sensory neurons in vitro. These results indicate that removal of the H3K27me3 mark plays a key role in HSV-1 reactivation.  

4.1368           Efficient Uptake and Dissemination of Scrapie Prion Protein by Astrocytes and Fibroblasts from Adult Hamster Brain

Hollister, J.R., Lee, K.S., Dorward, D.W. and baron, G.S. PloS One, 10(1), e0115351 (2015)   Prion infections target neurons and lead to neuronal loss. However, the role of non-neuronal cells in the initiation and spread of infection throughout the brain remains unclear despite the fact these cells can also propagate prion infectivity. To evaluate how different brain cells process scrapie prion protein (PrPres) during acute infection, we exposed neuron-enriched and non-neuronal cell cultures from adult hamster brain to fluorescently-labeled purified PrPres and followed the cultures by live cell confocal imaging over time. Non-neuronal cells present in both types of cultures, specifically astrocytes and fibroblasts, internalized PrPres more efficiently than neurons. PrPres was trafficked to late endosomal/lysosomal compartments and rapidly transported throughout the cell bodies and processes of all cell types, including contacts between astrocytes and neurons. These observations suggest that astrocytes and meningeal fibroblasts play an as yet unappreciated role in prion infections via efficient uptake and dissemination of PrPres.  

4.1369           Kinetics of sickle cell biorheology and implications for painful vasoocclusive crisis

Du, E., Diez-Silva, M., kato, G.J., Dao, M. and Suresh, S. PNAS, 112(5), 1422-1427 (2015)   We developed a microfluidics-based model to quantify cell-level processes modulating the pathophysiology of sickle cell disease (SCD). This in vitro model enabled quantitative investigations of the kinetics of cell sickling, unsickling, and cell rheology. We created short-term and long-term hypoxic conditions to simulate normal and retarded transit scenarios in microvasculature. Using blood samples from 25 SCD patients with sickle hemoglobin (HbS) levels varying from 64 to 90.1%, we investigated how cell biophysical alterations during blood flow correlated with hematological parameters, HbS level, and hydroxyurea (HU) therapy. From these measurements, we identified two severe cases of SCD that were also independently validated as severe from a genotype-based disease severity classification. These results point to the potential of this method as a diagnostic indicator of disease severity. In addition, we investigated the role of cell density in the kinetics of cell sickling. We observed an effect of HU therapy mainly in relatively dense cell populations, and that the sickled fraction increased with cell density. These results lend support to the possibility that the microfluidic platform developed here offers a unique and quantitative approach to assess the kinetic, rheological, and hematological factors involved in vasoocclusive events associated with SCD and to develop alternative diagnostic tools for disease severity to supplement other methods. Such insights may also lead to a better understanding of the pathogenic basis and mechanism of drug response in SCD.  

4.1370           Regeneration of whole plants from protoplasts of Gracilaria gracilis (Gracilariales, Rhodophyta)

Huddy, S.M., Meyers, A.E. and Coyne, V.E.

1. Appl. Phycol., 27(1), 427-435 (2015)

  This paper reports the first successful regeneration of whole plants from protoplasts of Gracilaria gracilis (Stackhouse) Steentoft, Irvine and Farnham. Protoplasts were isolated and purified using a previously optimized protocol. Protoplasts with regenerated cell walls divided to produce callus-like cell masses which showed the presence of uniseriate, filamentous outgrowths. Bud outgrowth from callus masses was associated with a distinct change in colour intensity at the point of outgrowth. Ultimately, whole plants were regenerated from the callus-like cell masses with overall yields of approximately two to three whole plants per 10⁴ protoplasts seeded. Two distinctive patterns of regeneration were observed. In the first case, protoplasts regenerated slowly to produce plants which resembled the parent plants, exhibiting slender, branched thalli. Growth rates of regenerated seaweed were similar to that of wild-type G. gracilis cultured under the same conditions with 115 g of seaweed cultured from 15 individually regenerated plants over a year. In the second case, protoplasts regenerated rapidly to produce plants which remained small with thalli that were thick and unbranched and had a limited life span. These results provide an important foundation for the development of a successful tissue culture system for G. gracilis.  

4.1371           Upregulation of miR21 and Repression of Grhl3 by Leptin Mediates Sinusoidal Endothelial Injury in Experimental Nonalcoholic Steatohepatitis

Pourhoseini, S., Seth, R.K., Das, S., Dattatoy, D., Kadilska, B., Xie, G., Michelotti, G.A., nagarkatti, M., Diehl, A.M. and Chatterjee, S. PloS One, 10(2), e0116780 (2015)   Sinusoidal endothelial dysfunction (SED) has been found to be an early event in nonalcoholic steatohepatitis (NASH) progression but the molecular mechanisms underlying its causation remains elusive. We hypothesized that adipokine leptin worsens sinusoidal injury by decreasing functionally active nitric oxide synthase 3 (NOS)3 via miR21. Using rodent models of NASH, and transgenic mice lacking leptin and leptin receptor, results showed that hyperleptinemia caused a 4–5 fold upregulation of hepatic miR21 as assessed by qRTPCR. The upregulation of miR21 led to a time-dependent repression of its target protein Grhl3 levels as shown by western blot analyses. NOS3-p/NOS3 ratio which is controlled by Grhl3 was significantly decreased in NASH models. SED markers ICAM-1, VEGFR-2, and E-selectin as assessed by immunofluorescence microscopy were significantly up regulated in the progressive phases of NASH. Lack of leptin or its receptor in vivo, reversed the upregulation of miR21 and restored the levels of Grhl3 and NOS3-p/NOS3 ratio coupled with decreased SED dysfunction markers. Interestingly, leptin supplementation in mice lacking leptin, significantly enhanced miR21 levels, decreased Grhl3 repression and NOS3 phosphorylation. Leptin supplementation in isolated primary endothelial cells, Kupffer cells and stellate cells showed increased mir21 expression in stellate cells while sinusoidal injury was significantly higher in all cell types. Finally miR21 KO mice showed increased NOS3-p/NOS3 ratio and reversed SED markers in the rodent models of NASH. The experimental results described here show a close association of leptin-induced miR21 in aiding sinusoidal injury in NASH.  

4.1372           Cytosolic Access of Mycobacterium tuberculosis: Critical Impact of Phagosomal Acidification Control and Demonstration of Occurrence In Vivo

Simeone, R., Sayes, F., Song, O., Gröschel, M.I., Brodin, P., Brosch, R. and Majlessi, L. PloS Pathogens, 10(2), e1004650 (2015)   Mycobacterium tuberculosis (Mtb) uses efficient strategies to evade the eradication by professional phagocytes, involving—as recently confirmed—escape from phagosomal confinement. While Mtb determinants, such as the ESX-1 type VII secretion system, that contribute to this phenomenon are known, the host cell factors governing this important biological process are yet unexplored. Using a newly developed flow-cytometric approach for Mtb, we show that macrophages expressing the phagosomal bivalent cation transporter Nramp-1, are much less susceptible to phagosomal rupture. Together with results from the use of the phagosome acidification inhibitor bafilomycin, we demonstrate that restriction of phagosomal acidification is a prerequisite for mycobacterial phagosomal rupture and cytosolic contact. Using different in vivo approaches including an enrichment and screen for tracking rare infected phagocytes carrying the CD45.1 hematopoietic allelic marker, we here provide first and unique evidence of M. tuberculosis-mediated phagosomal rupture in mouse spleen and lungs and in numerous phagocyte types. Our results, linking the ability of restriction of phagosome acidification to cytosolic access, provide an important conceptual advance for our knowledge on host processes targeted by Mtb evasion strategies.  

4.1373           CD40 Is Required for Protective Immunity against Liver Stage Plasmodium Infection

Murray, S.A., Mohar, I., Miller, J.L., Brempelis, K.J., Vaughan, A.M., Kappe, S.I. and Crispe, I.N.

1. Immunol., 194(5), 2268-2279 (2015)

  The costimulatory molecule CD40 enhances immunity through several distinct roles in T cell activation and T cell interaction with other immune cells. In a mouse model of immunity to liver stage Plasmodium infection, CD40 was critical for the full maturation of liver dendritic cells, accumulation of CD8⁺ T cells in the liver, and protective immunity induced by immunization with the Plasmodium yoelii fabb/f⁻ genetically attenuated parasite. Using mixed adoptive transfers of polyclonal wild-type and CD40-deficient CD8⁺ T cells into wild-type and CD40-deficient hosts, we evaluated the contributions to CD8⁺ T cell immunity of CD40 expressed on host tissues including APC, compared with CD40 expressed on the CD8⁺ T cells themselves. Most of the effects of CD40 could be accounted for by expression in the T cells’ environment, including the accumulation of large numbers of CD8⁺ T cells in the livers of immunized mice. Thus, protective immunity generated during immunization with fabb/f⁻ was largely dependent on effective APC licensing via CD40 signaling.  

4.1374           Resveratrol Encapsulated in Novel Fusogenic Liposomes Activates Nrf2 and Attenuates Oxidative Stress in Cerebromicrovascular Endothelial Cells From Aged Rats

Csiszar, A., Csiszar, A., Pinto, J.T., Gautaqm, T., Kleusch, C., Hoffmann, B., Tucsek, Z., Toth, P., Sonntag, W. and Ungvari, Z.

1. Gerontol. A Biol. Sci. Med. Sci., 70(3), 303-314 (2015)

  Resveratrol (3,4′,5-trihydroxystilbene) is a plant-derived polyphenolic trans-stilbenoid, which exerts multifaceted antiaging effects. Here, we propose a novel delivery system for resveratrol, which significantly increases its cellular uptake into aged cells. Combination of resveratrol with a positively charged lipid component to “conventional” liposomes converts these lipid vesicles to a robust fusogenic system. To study their cellular uptake and cellular effects, we treated primary cerebromicrovascular endothelial cells isolated from aged F344xBN rats with resveratrol encapsulated in fusogenic liposomes (FL-RSV). To demonstrate effective cellular uptake of FL-RSV, accumulation of the lipophilic tracer dye, DiR, and resveratrol in cerebromicrovascular endothelial cells was confirmed using flow cytometry and confocal microscopy and high-performance liquid chromatography electrochemical detection. Treatment of aged cerebromicrovascular endothelial cells with FL-RSV activated Nrf2 (assessed with a reporter gene assay), significantly decreased cellular production of reactive oxygen species (assessed by a flow cytometry-based H₂DCFDA fluorescence method), and inhibited apoptosis. Taken together, encapsulation of resveratrol into novel fusogenic liposomes significantly enhances the delivery of resveratrol into aged cells, which subsequently results in rapid activation of cellular Nrf2-driven antioxidant defense mechanisms. Our studies provide proof-of-concept for the development of a novel, translationally relevant interventional strategy for prevention and/or control of oxidative stress-related pathophysiological conditions in aging.    

4.1375           Comparison of surface modification chemistries in mouse, porcine, and human islets

SoRelle, J.A., kanak, M.A., Itoh, T., Horton, J.M., Naziruddin, B. and Kane, R.R.

1. Biomed. Mater. Res. part A, 103A(3), 869-877 (2015)

  Beta cell replacement therapy, the transplantation of isolated pancreatic islets by intraportal infusion, offers patients with brittle type 1 diabetes blood glucose regulation with a minimally invasive technique. Chemical modification of islets prior to transplantation, providing a nanothin barrier that potentially includes active protective compounds, has been proposed as a strategy to minimize the inflammatory and immune reactions that often significantly limit graft function and duration. Chemical modification also has the potential to allow the use of alternative sources of islets, such as porcine islets, for transplantation. This investigation compared three orthogonal covalent islet modification techniques across three species (human, porcine, and murine), using multiple measures to determine biocompatibility and effectiveness. All three conjugation chemistries were well tolerated, and the overall efficiency, gross uniformity, and stability of the surface modifications were dependent upon the conjugation chemistry as well as the islet source (human, porcine, or murine). Notably, the reductive modification of surface disulfides was shown to afford intense and long-lasting modification of human islets. This study demonstrates that murine, human, and porcine islets tolerate a variety of covalent modifications, that these modifications are relatively stable, and that the murine islet model may not be predictive for some chemical contexts  

4.1376           Use of Density Centrifugation for Delayed Cryopreservation of Stallion Sperm: Perform Sperm Selection Directly after Collection or after Storage?

Heutelbeck, A., Oldenhof, H., Rohn, K., Martinsson, G., Morrell, J.M. and Sieme, H. Reprod. Dom. Anim., 50(1), 76-83 (2015)   Equipment for cryopreservation of stallion sperm is not always available. In such cases, diluted semen can be shipped to a facility for later cryopreservation. The aim of this study was to evaluate if selection of sperm via density centrifugation yields higher survival rates when cryopreservation is to be delayed (i.e. carried out after 1 day of storage at 5°C). Two-layer iodixanol as well as single-layer Androcoll density centrifugation were tested and compared with samples prepared with standard centrifugation. Special emphasis was placed on comparing centrifugation on the day of semen collection with centrifugation after 1-day refrigerated storage. Sperm morphology and motility as well as membrane and chromatin integrity were evaluated before and after centrifugation. Sperm motility and membrane integrity were also assessed after cryopreservation. It was found that both two- and single-layer density centrifugation processing resulted in higher percentages of morphologically normal and motile sperm with higher membrane and chromatin integrity, as compared to standard centrifugation or diluted samples. Differences were only in the order of magnitude of 5%. Recovery rates after density centrifugation were only approximately 30–40%. When cryopreservation was carried out after 1-day refrigerated storage, centrifugation processing of sperm directly after semen collection resulted in higher percentages of plasma membrane intact sperm post-thaw as compared to performing centrifugation processing of stored sperm just prior to cryopreservation. No significant differences in progressively motile sperm post-thaw were seen. Taken together, for delayed cryopreservation, it is best to perform density centrifugation directly after collection rather than immediately prior to cryopreservation.  

4.1377           Dual characterization of biological cells by optofluidic microscope and resistive pulse sensor

Guo, J., Chen, L., Huang, X., Li, C.M., Ai, Y. and kang, Y. Electrophoresis, 36(3), 420-423 (2015)   Label-free detection technique has emerged as a powerful platform for biomedical applications since it can avoid laborious multi-step sample preparation. In this paper, we demonstrate a dual analysis of biological cells using a single microfluidic system combining optofluidic microscopy and resistive pulse sensing. Both red blood cells (RBCs) and circulating tumor cells (CTCs) have been used to validate the concept of dual analysis and also to test the performance of the microfluidic device. The cell characterization by resistive pulse sensing is in good agreement with the analysis by optofluidic microscopy, further verified by the commercial Beckman-Coulter® FC500 flow cytometry. The present system has attractive merits such as simple fabrication, easy integration, high portability, and low cost. This study has great potentials for the development of innovative on-chip flow cytometry with concurrent imaging sensing and resistive sensing.  

4.1378           Generation, cryopreservation, function and in vivo persistence of ex vivo expanded cynomolgus monkey regulatory T cells

Guo, H., Zhang, H., Lu, L., Ezzelarab, M.B. and Thomson, A.W. Cell. Immunol., 295, 19-28 (2015)   We expanded flow-sorted Foxp3⁺ cynomolgus monkey regulatory T cells (Treg) >1000-fold after three rounds of stimulation with anti-CD3 mAb-loaded artificial antigen-presenting cells, rapamycin (first round only) and IL-2. The expanded Treg maintained their expression of Treg signature markers, CD25, CD27, CD39, Foxp3, Helios, and CTLA-4, as well as CXCR3, which plays an important role in T cell migration to sites of inflammation. In contrast to expanded effector T cells (Teff), expanded Treg produced minimal IFN-γ and IL-17 and no IL-2 and potently suppressed Teff proliferation. Following cryopreservation, thawed Treg were less viable than their freshly-expanded counterparts, although no significant changes in phenotype or suppressive ability were observed. Additional rounds of stimulation/expansion restored maximal viability. Furthermore, adoptively-transferred autologous Treg expanded from cryopreserved second round stocks and labeled with CFSE or VPD450 were detected in blood and secondary lymphoid tissues of normal or immunosuppressed recipients at least two months after their systemic infusion.  

4.1379           Identifying the primary site of pathogenesis in amyotrophic lateral sclerosis – vulnerability of lower motor neurons to proximal excitotoxicity

Blizzard, C.A., Southam, K.A., Dawkins, E., Lewis, K.E., King, A.E., Clark, J.A. and Dickson, ST.C. Diseases Modles & Mechanisms, 8, 215-224 (2015)   There is a desperate need for targeted therapeutic interventions that slow the progression of amyotrophic lateral sclerosis (ALS). ALS is a disorder with heterogeneous onset, which then leads to common final pathways involving multiple neuronal compartments that span both the central and peripheral nervous system. It is believed that excitotoxic mechanisms might play an important role in motor neuron death in ALS. However, little is known about the mechanisms by which excitotoxicity might lead to the neuromuscular junction degeneration that characterizes ALS, or about the site at which this excitotoxic cascade is initiated. Using a novel compartmentalised model of site-specific excitotoxin exposure in lower motor neurons in vitro, we found that spinal motor neurons are vulnerable to somatodendritic, but not axonal, excitotoxin exposure. Thus, we developed a model of somatodendritic excitotoxicity in vivo using osmotic mini pumps in Thy-1-YFP mice. We demonstrated that in vivo cell body excitotoxin exposure leads to significant motor neuron death and neuromuscular junction (NMJ) retraction. Using confocal real-time live imaging of the gastrocnemius muscle, we found that NMJ remodelling preceded excitotoxin-induced NMJ degeneration. These findings suggest that excitotoxicity in the spinal cord of individuals with ALS might result in a die-forward mechanism of motor neuron death from the cell body outward, leading to initial distal plasticity, followed by subsequent pathology and degeneration.  

4.1380           Physiological Electrical Signals Promote Chain Migration of Neuroblasts by Up-Regulating P2Y1 Purinergic Receptors and Enhancing Cell Adhesion

Cao, L., Pu, J., Scott, R.H., CHing, J. and McCaig, C.D. Stem Cell Rev. and Rep., 11, 75-86 (2015)   Neuroblasts migrate as directed chains of cells during development and following brain damage. A fuller understanding of the mechanisms driving this will help define its developmental significance and in the refinement of strategies for brain repair using transplanted stem cells. Recently, we reported that in adult mouse there are ionic gradients within the extracellular spaces that create an electrical field (EF) within the rostral migratory stream (RMS), and that this acts as a guidance cue for neuroblast migration. Here, we demonstrate an endogenous EF in brain slices and show that mimicking this by applying an EF of physiological strength, switches on chain migration in mouse neurospheres and in the SH-SY5Y neuroblastoma cell line. Firstly, we detected a substantial endogenous EF of 31.8 ± 4.5 mV/mm using microelectrode recordings from explants of the subventricular zone (SVZ). Pharmacological inhibition of this EF, effectively blocked chain migration in 3D cultures of SVZ explants. To mimic this EF, we applied a physiological EF and found that this increased the expression of N-cadherin and β-catenin, both of which promote cell-cell adhesion. Intriguingly, we found that the EF up-regulated P2Y purinoceptor 1 (P2Y1) to contribute to chain migration of neuroblasts through regulating the expression of N-cadherin, β-catenin and the activation of PKC. Our results indicate that the naturally occurring EF in brain serves as a novel stimulant and directional guidance cue for neuronal chain migration, via up-regulation of P2Y1.  

4.1381           Blood cell transcriptomic-based early biomarkers of adverse programming effects of gestational calorie restriction and their reversibility by leptin supplementation

Konieczna, J., Sanchez, J., Palou, M., Pico, C. and Palou, A. Scientific Reports, 5:9088 (2015)   The challenge of preventing major chronic diseases requires reliable, early biomarkers. Gestational mild undernutrition in rats is enough to program the offspring to develop later pathologies; the intake of leptin, a breastmilk component, during lactation may reverse these programming effects. We used these models to identify, in peripheral blood mononuclear cells (PBMCs), transcriptomic-based early biomarkers of programmed susceptibility to later disorders, and explored their response to neonatal leptin intake. Microarray analysis was performed in PBMCs from the offspring of control and 20% gestational calorie-restricted dams (CR), and CR-rats supplemented with physiological doses of leptin throughout lactation. Notably, leptin supplementation normalised 218 of the 224 mRNA-levels identified in PBMCs associated to undernutrition during pregnancy. These markers may be useful for early identification and subsequent monitoring of individuals who are at risk of later diseases and would specifically benefit from the intake of appropriate amounts of leptin during lactation.  

4.1382           Lipolysis of Visceral Adipocyte Triglyceride by Pancreatic Lipases Converts Mild Acute Pancreatitis to Severe Pancreatitis Independent of Necrosis and Inflammation

Patel, K., Trivedi, R.N., Durgampudi, C., Noel, P:, Cline, R.A., DeLany, J.P., navina, S. and Singh, V.P: Am. J. Pathol., 185(3), 808-819 (2015)   Visceral fat necrosis has been associated with severe acute pancreatitis (SAP) for over 100 years; however, its pathogenesis and role in SAP outcomes are poorly understood. Based on recent work suggesting that pancreatic fat lipolysis plays an important role in SAP, we evaluated the role of pancreatic lipases in SAP-associated visceral fat necrosis, the inflammatory response, local injury, and outcomes of acute pancreatitis (AP). For this, cerulein pancreatitis was induced in lean and obese mice, alone or with the lipase inhibitor orlistat and parameters of AP induction (serum amylase and lipase), fat necrosis, pancreatic necrosis, and multisystem organ failure, and inflammatory response were assessed. Pancreatic lipases were measured in fat necrosis and were overexpressed in 3T3-L1 cells. We noted obesity to convert mild cerulein AP to SAP with greater cytokines, unsaturated fatty acids (UFAs), and multisystem organ failure, and 100% mortality without affecting AP induction or pancreatic necrosis. Increased pancreatic lipase amounts and activity were noted in the extensive visceral fat necrosis of dying obese mice. Lipase inhibition reduced fat necrosis, UFAs, organ failure, and mortality but not the parameters of AP induction. Pancreatic lipase expression increased lipolysis in 3T3-L1 cells. We conclude that UFAs generated via lipolysis of visceral fat by pancreatic lipases convert mild AP to SAP independent of pancreatic necrosis and the inflammatory response. Supported by grants from the Clinical Translational Science Institute (RO1DK092460 to V.P.S.) and the NIH (UL1RR024153 and UL1TR000005; V.P.S., S.N.). This project used the UPCI Cancer Biomarkers Facility: Luminex Core Laboratory, which is supported in part by an award from NIH (P30CA047904). Funding was also provided by a startup package from the Department of Medicine, University of Pittsburgh (V.P.S.).  

4.1383           Complement Protein C1q Modulates Neurite Outgrowth In Vitro and Spinal Cord Axon Regeneration In Vivo

Peterson, S.L., Nguyen, H.X., Mendez, O.A. and Anderson, A.J:

1. Neurosci., 35(10), 4332-4349 (2015)

  Traumatic injury to CNS fiber tracts is accompanied by failure of severed axons to regenerate and results in lifelong functional deficits. The inflammatory response to CNS trauma is mediated by a diverse set of cells and proteins with varied, overlapping, and opposing effects on histological and behavioral recovery. Importantly, the contribution of individual inflammatory complement proteins to spinal cord injury (SCI) pathology is not well understood. Although the presence of complement components increases after SCI in association with axons and myelin, it is unknown whether complement proteins affect axon growth or regeneration. We report a novel role for complement C1q in neurite outgrowth in vitro and axon regrowth after SCI. In culture, C1q increased neurite length on myelin. Protein and molecular assays revealed that C1q interacts directly with myelin associated glycoprotein (MAG) in myelin, resulting in reduced activation of growth inhibitory signaling in neurons. In agreement with a C1q-outgrowth-enhancing mechanism in which C1q binding to MAG reduces MAG signaling to neurons, complement C1q blocked both the growth inhibitory and repulsive turning effects of MAG in vitro. Furthermore, C1q KO mice demonstrated increased sensory axon turning within the spinal cord lesion after SCI with peripheral conditioning injury, consistent with C1q-mediated neutralization of MAG. Finally, we present data that extend the role for C1q in axon growth and guidance to include the sprouting patterns of descending corticospinal tract axons into spinal gray matter after dorsal column transection SCI.  

4.1384           Protein kinase CK2 enables regulatory T cells to suppress excessive TH2 responses in vivo

Ulges, A. et al Nature Immunol., 16(3), 267-275 (2015)   The quality of the adaptive immune response depends on the differentiation of distinct CD4⁺ helper T cell subsets, and the magnitude of an immune response is controlled by CD4⁺Foxp3⁺ regulatory T cells (T_reg cells). However, how a tissue- and cell type–specific suppressor program of T_reg cells is mechanistically orchestrated has remained largely unexplored. Through the use of T_reg cell–specific gene targeting, we found that the suppression of allergic immune responses in the lungs mediated by T helper type 2 (T_H2) cells was dependent on the activity of the protein kinase CK2. Genetic ablation of the β-subunit of CK2 specifically in T_reg cells resulted in the proliferation of a hitherto-unexplored ILT3⁺ T_reg cell subpopulation that was unable to control the maturation of IRF4⁺PD-L2⁺ dendritic cells required for the development of T_H2 responses in vivo.  

4.1385           Abnormal erythroid maturation leads to microcytic anemia in the TSAP6/Steap3 null mouse model

Blanc, L., Papoin, J., Debnath, G., Vidal, M., Amson, R., Telerman, A., An, X. and Mohandas, N. Am. J. Hematol., 90(3), 235-241 (2015)   Genetic ablation of the ferrireductase STEAP3, also known as TSAP6, leads to severe microcytic and hypochromic red cells with moderate anemia in the mouse. However, the mechanism leading to anemia is poorly understood. Previous results indicate that TSAP6/Steap3 is a regulator of exosome secretion. Using TSAP6/Steap3 knockout mice, we first undertook a comprehensive hematologic characterization of the red cell compartment, and confirmed a dramatic decrease in the volume and hemoglobin content of these erythrocytes. We observed marked anisocytosis as well as the presence of fragmenting erythrocytes. Consistent with these observations, we found by ektacytometry decreased membrane mechanical stability of knockout red cells. However, we were unable to document significant changes in the expression levels of the major skeletal and transmembrane proteins to account for this decrease in the membrane stability. Furthermore, there were no differences in red cell survival between wild type and knockout animals. However, when we monitored erythropoiesis, we found a decreased number of proerythroblasts in the bone marrow of TSAP6/Steap3^−/− animals. In addition, progression from the proerythroblastic to the orthochromatic stage was affected, with accumulation of cells at the polychromatic stage. Altogether, our findings demonstrate that abnormal erythroid maturation is the main cause of anemia in these mice  

4.1386           The presence of interleukin-27 during monocyte-derived dendritic cell differentiation promotes improved antigen processing and stimulation of T cells

Jung, J-Y., Roberts, L.L. and Robinson, C.M. Immunology, 144(4), 649-660 (2015)   Dendritic cells (DCs) are potent antigen-presenting cells necessary to establish effective adaptive immune responses. The cytokine environment that exists at the time of DC differentiation may be an important but often ignored determinant in the phenotypic and functional properties of DCs. Interleukin-27 (IL-27) is a unique cytokine that has both inflammatory and immune suppressive activities. Although it can both promote and oppose activity of different T-cell subsets, mostly anti-inflammatory activity has been described toward macrophages and DCs. However, the specific effect of IL-27 during DC differentiation and how that may change the nature of the antigen-presenting cell has not been investigated. In this report, we show that IL-27 treatment during monocyte-derived DC differentiation enhanced the ability to process antigens and stimulate T-cell activity. DCs differentiated in the presence of IL-27 showed enhanced acidification of latex bead-containing phagosomes that was consistent with elevated expression of vacuolar-ATPases. This resulted in inhibition of intracellular growth of Staphylococcus aureus. In addition, the levels of MHC class II surface expression were higher in DCs differentiated in the presence of IL-27. Production of IL-12 was also significantly increased during S. aureus infection of IL-27-differentiated DCs. The net effect of these activities was enhanced CD4⁺ T-cell proliferation and T helper type 1 cytokine production. These findings are important to a wide number of immunological contexts and should be considered in the development of future vaccines.  

4.1387           Testosterone Suppresses Hepatic Inflammation by the Downregulation of IL-17, CXCL-9, and CXCL-10 in a Mouse Model of Experimental Acute Cholangitis

Schwinge, D., Carambia, A., Quaas, A., Krech, T., Wegscheid, CC., Tiegs, G., Prinz, I., Lohse, A.W., Herkel, J. and Schramm, C.

1. Immunol., 194(6), 2522-2530 (2015)

  Autoimmune liver diseases predominantly affect women. In this study, we aimed to elucidate how sex affects autoimmune hepatic inflammation. Acute experimental cholangitis was induced by adoptive transfer of OVA-specific CD8⁺ T cells into mice, which express the cognate Ag on cholangiocytes. In contrast to previous mouse models of cholangitis, this model displayed a strong sexual dimorphism: female mice developed marked cholangitis, whereas male mice were resistant to cholangitis induction. The recruitment of endogenous CD4⁺ T cells, but not transferred CD8⁺ T cells into female livers was strongly increased. These cells expressed higher amounts of the proinflammatory cytokine IL-17, which was at least in part responsible for the liver inflammation observed. The recruitment of endogenous CD4⁺ T cells was associated with increased expression of the chemokines CXCL-9 and CXCL-10 in female livers. The sex-specific factor responsible for the observed differences was found to be testosterone: male mice could be rendered susceptible to liver inflammation by castration, and testosterone treatment was sufficient to completely suppress liver inflammation in female mice. Accordingly, testosterone treatment of female mice significantly reduced the expression of IL-17A, CXCL-9, and CXCL-10 within the liver. Serum testosterone levels of untreated mice negatively correlated with the IL-17, CXCL-9, and CXCL-10 expression in the liver, further supporting a role for testosterone in hepatic immune homeostasis. In conclusion, testosterone was found to be the major determinant of the observed sexual dimorphism. Further study into the role of testosterone for liver inflammation could lead to novel treatment targets in human autoimmune liver diseases.  

4.1388           Disrupting Protein Expression with Peptide Nucleic Acids Reduces Infection by Obligate Intracellular Rickettsia

Pelc, R., McClure, J.C., kaur, S.J., Sears, K., Rahman, M.S. and Ceraul, S.M. PloS One, 10(3), e119283 (2015)   Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria’s ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria.    

4.1389           High Expression Level of Tra2-β1 Is Responsible for Increased SMN2 Exon 7 Inclusion in the Testis of SMA Mice

Chen, Y-C., Chang, J-G., Jong, Y-J., Liu, T-Y. and You, C-Y. PloS One, 10(3), e120721 (2015)   Spinal muscular atrophy (SMA) is an inherited neuromuscular disease caused by deletion or mutation of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene, which produces low level of SMN protein due to mRNA exon 7 exclusion. Previously, we found that the testis of SMA mice (smn^−/− SMN2) expresses high level of SMN2 full-length mRNA, indicating a testis-specific mechanism for SMN2 exon 7 inclusion. To elucidate the underlying mechanism, we established primary cultures of testis cells from SMA mice and analyzed them for SMN2 exon 7 splicing. We found that primary testis cells after a 2-hour culture still expressed high level of SMN2 full-length mRNA, but the level decreased after longer cultures. We then compared the protein levels of relevant splicing factors, and found that the level of Tra2-β1 also decreased during testis cell culture, correlated with SMN2 full-length mRNA downregulation. In addition, the testis of SMA mice expressed the highest level of Tra2-β1 among the many tissues examined. Furthermore, overexpression of Tra2-β1, but not ASF/SF2, increased SMN2 minigene exon 7 inclusion in primary testis cells and spinal cord neurons, whereas knockdown of Tra2-β1 decreased SMN2 exon 7 inclusion in primary testis cells of SMA mice. Therefore, our results indicate that high expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice. This study also suggests that the expression level of Tra2-β1 may be a modifying factor of SMA disease and a potential target for SMA treatment.  

4.1390           Monocyte mediated brain targeting delivery of macromolecular drug for the therapy of depression

Qin, J., Yang, X., Zhang, R-X., Luo, Y-X., Li, J-L., Hou, J., Zhang, C., Li, Y-J., Shi, J., Lu, L., Wang, J-X. and Xhu, W.L. Nanomedicine: Nanothechnology, Biology, and Medicine, 11, 391-400 (2015)   Leukocytes can cross intact blood-brain barrier under healthy conditions and in many neurological diseases, including psychiatric diseases. In present study, a cyclic RGD (cRGD) peptide with high affinity for integrin receptors of leukocytes was used to modify liposomes. The cRGD-modified liposomes (cRGDL) showed high affinity for monocytes in vitro and in vivo and co-migrated across in vitro BBB model with THP-1. The trefoil factor 3 (TFF3), a macromolecular drug, was rapidly and persistently delivered to brain for at least 12 h when loaded into cRGDL while 2.8-fold increase in drug concentration in basolateral amygdala regions related to depression was observed. A systemic administration of cRGDL-TFF3 mimicked antidepressant-like effect of direct intra-basolateral amygdala administration of TFF3 solution in rats subjected to chronic mild stress. The effective dual-brain targeting delivery resulting from the combination and co-migration of cRGDL with leukocyte cross BBB may be a promising strategy for targeted brain delivery.  

4.1391           Transcriptome changes upon in vitro challenge with Mycobacterium bovis in monocyte-derived macrophages from bovine tuberculosis-infected and healthy cows

Lin, J., Zhao, D., Wang, J., Wang, Y., Li, H., Yin, X., Yang, L. and Zhou, X. Veterinary Immunology and Immunopathology, 163, 146-156 (2015)   As innate immune cells, macrophages are expected to respond to mycobacterial infection equally in both Mycobacterium bovis-infected cows and healthy cows. We previously found that monocyte-derived macrophages (MDMs) from M. bovis-infected cows respond differently than MDMs from healthy cows when exposed to in vitro M. bovis challenge. We have now used the Agilent™ Bovine Gene Expression Microarray to examine transcriptional differences between these MDMs. At a high multiplicity of infection (10), in vitro challenge led to changes in several thousands of genes, with dysregulation at multiple orders of magnitude. For example, significant changes were seen for colony stimulating factor 3 (granulocyte) (CSF3), colony stimulating factor 2 (granulocyte-macrophage) (CSF2), and chemokine (C-C motif) ligand 20 (CCL20). Classical macrophage activation was also observed, although to a lesser degree in interleukin 12 (IL12) expression. For macrophages, kallikrein-related peptidase 12 (KLK12) and protease, serine, 2 (trypsin 2) (PRSS2), as well as a secreted protein, acidic, cysteine-rich (osteonectin) (SPARC)-centered matricellular gene network, were differentially expressed in infected animals. Finally, global transcriptome fold-changes caused by in vitro challenge were higher in healthy cows than in tuberculosis-positive cows, suggesting that healthy macrophages responded marginally better to in vitro infection. Macrophages from healthy and already infected animals can both be fully activated during M. bovis infection, yet there are differences between these macrophages: distinct expression pattern in matricellular proteins, and their different responses to in vitro infection.  

4.1392           5-HT4 Receptor Subtype, β-Arrestin Level, and Rapid-Onset Effects of Antidepressant Drugs

Mendez-David, I., David, ; D.J., Guilloux, J-P., Hen, R. and Gardier, A.M. Neuromethods, 95, 101-121 (2015)   Understanding the pathophysiology of affective disorders and their treatment relies on the availability of experimental models that accurately mimic aspects of the disease. The use of exogenously administered corticosterone (CORT model) can mimic the effects of a chronic stress and has been validated as an animal model to study disease states displaying some hallmark characteristics of anxiety and depression observed in patients. Recently, we have adapted the CORT model protocol to screen for rapid-onset drugs to treat anxiety/depression disorders. In spite of the fact that selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed drugs for the treatment of depression and several anxiety disorders, the onset of action of SSRIs is often delayed by 3–6 weeks. The existence of this delayed action combined with the fact that one-third of patients do not respond to treatment emphasizes the need for faster acting and more effective antidepressants. This chapter gives laboratory protocols including step-by-step recommendations to explain how the CORT model in mice can be used to screen for candidate drugs. For this purpose we examined the behavioral and cellular effects of a 5-HT₄ receptor ligand, RS67333, and compared it with the SSRI, fluoxetine. Likewise, we emphasize that mononuclear cells (PBMCs) isolated from whole blood in corticosterone-treated mice could serve as a marker of treatment response(s) and fast onset of drug action in the mouse CORT model.  

4.1393           Andrographolide inhibits intracellular Chlamydia trachomatis multiplication and reduces secretion of proinflammatory mediators produced by human epithelial cells

Hua, Z., Frolich, K.M., Zhang, Y., Feng, X., Zhang, J. and Shen, L.

Pathog. Dis., 73, 1-11 (2015)

Chlamydia trachomatis is the most common sexually transmitted bacterial disease worldwide. Untreated C. trachomatis infections may cause inflammation and ultimately damage tissues. Here, we evaluated the ability of Andrographolide (Andro), a natural diterpenoid lactone component of Andrographis paniculata, to inhibit C. trachomatis infection in cultured human cervical epithelial cells. We found that Andro exposure inhibited C. trachomatis growth in a dose- and time-dependent manner. The greatest inhibitory effect was observed when exponentially growing C. trachomatis was exposed to Andro. Electron micrographs demonstrated the accumulation of unusual, structurally deficient chlamydial organisms, correlated with a decrease in levels of OmcB expressed at the late stage of infection. Additionally, Andro significantly reduced the secretion of interleukin6, CXCL8 and interferon-γ-induced protein10 produced by host cells infected with C. trachomatis. These results indicate the efficacy of Andro to perturb C. trachomatis transition from the metabolically active reticulate body to the infectious elementary body and concurrently reduce the production of a proinflammatory mediator by epithelial cells in vitro. Further dissection of Andro's anti-Chlamydia action may provide identification of novel therapeutic targets.

 

4.1394           Alpha-2 agonist attenuates ischemic injury in spinal cord neurons

Freeman, K.A., Puskas, F., Bell, M.T., Mares, J.M., Foley, L.S., Weyant, M.J., Cleveland, J.C., Fullerton, D.A., Meng, X., Herson, P.S. and Reece, T.B:

1. Surg. Res., 195, 21-28 (2015)

  Background Paraplegia secondary to spinal cord ischemia–reperfusion injury remains a devastating complication of thoracoabdominal aortic intervention. The complex interactions between injured neurons and activated leukocytes have limited the understanding of neuron-specific injury. We hypothesize that spinal cord neuron cell cultures subjected to oxygen-glucose deprivation (OGD) would simulate ischemia–reperfusion injury, which could be attenuated by specific alpha-2a agonism in an Akt-dependent fashion. Materials and methods Spinal cords from perinatal mice were harvested, and neurons cultured in vitro for 7–10 d. Cells were pretreated with 1 μM dexmedetomidine (Dex) and subjected to OGD in an anoxic chamber. Viability was determined by MTT assay. Deoxyuridine-triphosphate nick-end labeling staining and lactate dehydrogenase (LDH) assay were used for apoptosis and necrosis identification, respectively. Western blot was used for protein analysis. Results Vehicle control cells were only 59% viable after 1 h of OGD. Pretreatment with Dex significantly preserves neuronal viability with 88% viable (P < 0.05). Dex significantly decreased apoptotic cells compared with that of vehicle control cells by 50% (P < 0.05). Necrosis was not significantly different between treatment groups. Mechanistically, Dex treatment significantly increased phosphorylated Akt (P < 0.05), but protective effects of Dex were eliminated by an alpha-2a antagonist or Akt inhibitor (P < 0.05). Conclusions Using a novel spinal cord neuron cell culture, OGD mimics neuronal metabolic derangement responsible for paraplegia after aortic surgery. Dex preserves neuronal viability and decreases apoptosis in an Akt-dependent fashion. Dex demonstrates clinical promise for reducing the risk of paraplegia after high-risk aortic surgery.  

4.1395           Plasma bioavailability and changes in PBMC gene expression after treatment of ovariectomized rats with a commercial soy supplement

Islam, M.A., Hooiveld, G.J.E.J., van der berg, J.H.J., Boekschoten, M.V., van der Velpen, V., Murk, A.J., Rietjens, I.M.C.M. and van Leewen, F.X.R. Toxicology Reports, 2, 308-321 82015)   The health effects of soy supplementation in (post)menopausal women are still a controversial issue. The aim of the present study was to establish the effect of the soy isoflavones (SIF) present in a commercially available supplement on ovariectomized rats and to investigate whether these rats would provide an adequate model to predict effects of SIF in (post)menopausal women. Two dose levels (i.e. 2 and 20 mg/kg b.w.) were used to characterize plasma bioavailability, urinary and fecal concentrations of SIF and changes in gene expression in peripheral blood mononuclear cells (PBMC). Animals were dosed at 0 and 48 h and sacrificed 4 h after the last dose. A clear dose dependent increase of SIF concentrations in plasma, urine and feces was observed, together with a strong correlation in changes in gene expression between the two dose groups. All estrogen responsive genes and related biological pathways (BPs) that were affected by the SIF treatment were regulated in both dose groups in the same direction and indicate beneficial effects. However, in general no correlation was found between the changes in gene expression in rat PBMC with those in PBMC of (post)menopausal women exposed to a comparable dose of the same supplement. The outcome of this short-term study in rats indicates that the rat might not be a suitable model to predict effects of SIF in humans. Although the relative exposure period in this rat study is comparable with that of the human study, longer repetitive administration of rats to SIF may be required to draw a final conclusion on the suitability of the rat a model to predict effects of SIF in humans.  

4.1396           Retinoic Acid Can Exacerbate T Cell Intrinsic TLR2 Activation to Promote Tolerance

Nguyen, V., Pearson, K., Kim, J-H., Kamdar, K. and DePaolo, W. PloS One, 10(3), e0118875 (2015)   The contribution of vitamin A to immune health has been well established. However, recent evidence indicates that its active metabolite, retinoic acid (RA), has the ability to promote both tolerogenic and inflammatory responses. While the outcome of RA-mediated immunity is dependent upon the immunological status of the tissue, the contribution of specific innate signals influencing this response have yet to be delineated. Here, we found that treatment with RA can dampen inflammation during intestinal injury. Importantly, we report a novel and unexpected requirement for TLR2 in RA-mediated suppression. Our data demonstrate that RA treatment enhances TLR2-dependent IL-10 production from T cells and this, in turn, potentiates T regulatory cell (T_REG) generation without the need for activation of antigen presenting cells. These data also suggest that combinatorial therapy using RA and TLR2 ligands may be advantageous in the design of therapies to treat autoimmune or inflammatory disease.  

4.1397           Sprouty2 in the Dorsal Hippocampus Regulates Neurogenesis and Stress Responsiveness in Rats

Dow, A.L., Lin, T.V., Chartoff, E.H., Potter, D., McPhie, D.L., Van’t Veer, A.V., Knoll, A.T., Lee, K.N., Neve, R.L., Patel, T.B., Ongur, D., Cohen, B.M. and Carlezon Jr., W.A. PloS One, 10(3), e0120693 (2015)   Both the development and relief of stress-related psychiatric conditions such as major depression (MD) and post-traumatic stress disorder (PTSD) have been linked to neuroplastic changes in the brain. One such change involves the birth of new neurons (neurogenesis), which occurs throughout adulthood within discrete areas of the mammalian brain, including the dorsal hippocampus (HIP). Stress can trigger MD and PTSD in humans, and there is considerable evidence that it can decrease HIP neurogenesis in laboratory animals. In contrast, antidepressant treatments increase HIP neurogenesis, and their efficacy is eliminated by ablation of this process. These findings have led to the working hypothesis that HIP neurogenesis serves as a biomarker of neuroplasticity and stress resistance. Here we report that local alterations in the expression of Sprouty2 (SPRY2), an intracellular inhibitor of growth factor function, produces profound effects on both HIP neurogenesis and behaviors that reflect sensitivity to stressors. Viral vector-mediated disruption of endogenous Sprouty2 function (via a dominant negative construct) within the dorsal HIP of adult rats stimulates neurogenesis and produces signs of stress resilience including enhanced extinction of conditioned fear. Conversely, viral vector-mediated elevation of SPRY2 expression intensifies the behavioral consequences of stress. Studies of these manipulations in HIP primary cultures indicate that SPRY2 negatively regulates fibroblast growth factor-2 (FGF2), which has been previously shown to produce antidepressant- and anxiolytic-like effects via actions in the HIP. Our findings strengthen the relationship between HIP plasticity and stress responsiveness, and identify a specific intracellular pathway that could be targeted to study and treat stress-related disorders.  

4.1398           Recommendations for the use of the non-obese diabetic/severe combined immunodeficiency mouse model in autoimmune and drug-induced thrombocytopenia: communication from the SSC of the ISTH

Backhoul, T., Fuhrmann, J., Chong, B.H., Boougie, D. and Aster, R.

1. Thrombosis and Haemostasis, 13, 1-4 (2015)

  Human platelet survival studies have been hampered by the lack of a suitable animal model. Transfusion of human platelets into immunocompetent animals leads to the rapid destruction of these platelets by naturally occurring xenoantibodies. The non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse lacks T and B cells, and therefore lack natural antibodies that could destroy infused human platelets [1]. Because of this property, human platelets given to the mouse intravenously circulate for several days, permitting the model to be used for testing the ability of human antibodies to cause platelet destruction in vivo [2-7]. Preliminary studies have demonstrated the usefulness of the NOD/SCID mouse model for monitoring the survival and immune destruction of human platelets. However, differences exist between the research groups regarding the method of platelet injection, the amount and route of antibody injection, and the preparation of blood samples collected from the animal, making the results poorly comparable. Basically, in all laboratories, resting human platelets are injected intravenously into the mouse circulation, where they can, in the absence of platelet-reactive antibodies, circulate for up to 48 h [8]. After estimatation of a baseline value (100%) of human platelets, platelet-reactive antibodies (with or without drug administration) can be infused. The impact of these antibodies on the survival of human platelets can then be analyzed by taking blood samples from the mouse over time [8]. Methodological details that require attention in this model include: platelet preparation and resuspension in plasma or ‘synthetic plasma’; the concentration and volume of applied analytes (platelet, antibody, or drug); the route of platelet injection (retro-orbital injection or tail vein injection); and the route of antibody injection (intravenous or intraperitoneal). The method of data capture, including time points of blood sampling and subsequent sample preparation for analysis, the percentage of circulating human platelets, and software details, should also be reported in detail. Additional steps required to answer the scientific questions, e.g. platelet preincubation with a drug of interest or an antibody in pooled plasma or ‘synthetic plasma’, should also be reported [2, 9]. Surprisingly, application procedures and the amount of injected platelets and antibodies have been only loosely defined, and standardization is necessary in order to improve the reproducibility of the procedures and to enable reliable comparison of the results. This report is not didactic in relation to how to measure the survival of human platelets with the NOD/SCID mouse model. Its purpose is to suggest standardized procedures and to define variables that should be considered when presenting methodology in published reports. The presented procedures were introduced and discussed during the meetings of the Subcommittee of Platelet Immunology of the Scientific and Standardization Committee (SSC) in Liverpool 2012 and Milwaukee 2014. Suggestions were introduced to the SSC members and the presented recommendations had unanimous agreement. Adopting these recommendations will be of advantage for investigators and laboratories to reduce imprecision and harmonize results, and will allow other laboratories to readily reproduce reported methods and findings and interpret results appropriately.      

4.1399           Bmp6 Expression in Murine Liver Non Parenchymal Cells: A Mechanism to Control their High Iron Exporter Activity and Protect Hepatocytes from Iron Overload?

Rausa, M., Pagani, A., Nai, A., campanella, A., Gilberti, M.E., Apostoli, P., Camaschella, C. and Silvestri, L. PloS One, 10(4), e0122696 (2015)   Bmp6 is the main activator of hepcidin, the liver hormone that negatively regulates plasma iron influx by degrading the sole iron exporter ferroportin in enterocytes and macrophages. Bmp6 expression is modulated by iron but the molecular mechanisms are unknown. Although hepcidin is expressed almost exclusively by hepatocytes (HCs), Bmp6 is produced also by non-parenchymal cells (NPCs), mainly sinusoidal endothelial cells (LSECs). To investigate the regulation of Bmp6 in HCs and NPCs, liver cells were isolated from adult wild type mice whose diet was modified in iron content in acute or chronic manner and in disease models of iron deficiency (Tmprss6 KO mouse) and overload (Hjv KO mouse). With manipulation of dietary iron in wild-type mice, Bmp6 and Tfr1 expression in both HCs and NPCs was inversely related, as expected. When hepcidin expression is abnormal in murine models of iron overload (Hjv KO mice) and deficiency (Tmprss6 KO mice), Bmp6 expression in NPCs was not related to Tfr1. Despite the low Bmp6 in NPCs from Tmprss6 KO mice, Tfr1 mRNA was also low. Conversely, despite body iron overload and high expression of Bmp6 in NPCs from Hjv KO mice, Tfr1 mRNA and protein were increased. However, in the same cells ferritin L was only slightly increased, but the iron content was not, suggesting that Bmp6 in these cells reflects the high intracellular iron import and export. We propose that NPCs, sensing the iron flux, not only increase hepcidin through Bmp6 with a paracrine mechanism to control systemic iron homeostasis but, controlling hepcidin, they regulate their own ferroportin, inducing iron retention or release and further modulating Bmp6 production in an autocrine manner. This mechanism, that contributes to protect HC from iron loading or deficiency, is lost in disease models of hepcidin production.  

4.1400           Adipose triglyceride lipase is involved in the mobilization of triglyceride and retinoid stores of hepatic stellate cells

Taschler, U., Schreiber, R., Chitraju, C., Grabner, G.F., Romauch, M., Wolinski, H., Haemmerle, G., Breinbauer, R., Zechner, R., Lass, A. and Zimmermann, R. Biochim. Biophys. Acta, 1851, 937-945 (2015)   Hepatic stellate cells (HSCs) store triglycerides (TGs) and retinyl ester (RE) in cytosolic lipid droplets. RE stores are degraded following retinoid starvation or in response to pathogenic stimuli resulting in HSC activation. At present, the major enzymes catalyzing lipid degradation in HSCs are unknown. In this study, we investigated whether adipose triglyceride lipase (ATGL) is involved in RE catabolism of HSCs. Additionally, we compared the effects of ATGL deficiency and hormone-sensitive lipase (HSL) deficiency, a known RE hydrolase (REH), on RE stores in liver and adipose tissue. We show that ATGL degrades RE even in the presence of TGs, implicating that these substrates compete for ATGL binding. REH activity was stimulated and inhibited by comparative gene identification-58 and G0/G1 switch gene-2, respectively, the physiological regulators of ATGL activity. In cultured primary murine HSCs, pharmacological inhibition of ATGL, but not HSL, increased RE accumulation. In mice globally lacking ATGL or HSL, RE contents in white adipose tissue were decreased or increased, respectively, while plasma retinol and liver RE levels remained unchanged. In conclusion, our study shows that ATGL acts as REH in HSCs promoting the degradation of RE stores in addition to its established function as TG lipase. HSL is the predominant REH in adipocytes but does not affect lipid mobilization in HSCs.  

4.1401           Spinal cord protection via alpha-2 agonist-mediated increase in glial cell-line–derived neurotrophic factor

Freeman, K., Fullerton, D.A., Foley, L.S., Bell, M.T., Cleveland Jr., J.C., Weyant, M.J., Mares, J., Meng, X., Puskas, F. and Reece, T.B.

1. Thorac. Cardiovasc. Surg., 149(2), 578-586 (2015)

  Objectives Delayed paraplegia secondary to ischemia–reperfusion injury is a devastating complication of thoracoabdominal aortic surgery. Alpha-2 agonists have been shown to attenuate ischemia–reperfusion injury, but the mechanism for protection has yet to be elucidated. A growing body of evidence suggests that astrocytes play a critical role in neuroprotection by release of neurotrophins. We hypothesize that alpha-2 agonism with dexmedetomidine increases glial cell-line–derived neurotrophic factor in spinal cord astrocytes to provide spinal cord protection. Methods Spinal cords were isolated en bloc from C57BL/6 mice, and primary spinal cord astrocytes and neurons were selected for and grown separately in culture. Astrocytes were treated with dexmedetomidine, and glial cell-line–derived neurotrophic factor was tested for by enzyme-linked immunosorbent assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess neuronal viability. Results Spinal cord primary astrocytes treated with dexmedetomidine at 1 μmol/L and 10 μmol/L had significantly increased glial cell-line–derived neurotrophic factor production compared with control (P < .05). Neurons subjected to oxygen glucose deprivation had significant preservation (P < .05) of viability with use of dexmedetomidine-treated astrocyte media. Glial cell-line–derived neurotrophic factor neutralizing antibody eliminated the protective effects of the dexmedetomidine-treated astrocyte media (P < .05). Conclusions Astrocytes have been shown to preserve neuronal viability via release of neurotrophic factors. Dexmedetomidine increases glial cell–derived neurotrophic factor from spinal cord astrocytes via the alpha-2 receptor. Treatment with alpha-2 agonist dexmedetomidine may be a clinical tool for use in spinal cord protection in aortic surgery.  

4.1402           Vaccine molecules targeting Xcr1 on cross-presenting DCs induce protective CD8+ T-cell responses against influenza virus

Fossum, E., Grødeland, G., Terhorst, D., Tveita, A.A., Vikse, E., Mjaaland, S., Henri, S., Malissen, B. and Bogen, B. Eur. J. Immunol., 45(2), 624-635 (2015)   Targeting antigens to cross-presenting dendritic cells (DCs) is a promising method for enhancing CD8⁺ T-cell responses. However, expression patterns of surface receptors often vary between species, making it difficult to relate observations in mice to other animals. Recent studies have indicated that the chemokine receptor Xcr1 is selectively expressed on cross-presenting murine CD8α⁺ DCs, and that the expression is conserved on homologous DC subsets in humans (CD141⁺ DCs), sheep (CD26⁺ DCs), and macaques (CADM1⁺ DCs). We therefore tested if targeting antigens to Xcr1 on cross-presenting DCs using antigen fused to Xcl1, the only known ligand for Xcr1, could enhance immune responses. Bivalent Xcl1 fused to model antigens specifically bound CD8α⁺ DCs and increased proliferation of antigen-specific T cells. DNA vaccines encoding dimeric Xcl1-hemagglutinin (HA) fusion proteins induced cytotoxic CD8⁺ T-cell responses, and mediated full protection against a lethal challenge with influenza A virus. In addition to enhanced CD8⁺ T-cell responses, targeting of antigen to Xcr1 induced CD4⁺ Th1 responses and highly selective production of IgG2a antibodies. In conclusion, targeting of dimeric fusion vaccine molecules to CD8α⁺ DCs using Xcl1 represents a novel and promising method for induction of protective CD8⁺ T-cell responses.  

4.1403           Vitamin D counteracts fibrogenic TGF-β signalling in human hepatic stellate cells both receptor-dependently and independently

Beilfuss, A., Sowa, J-P., Sydor, S., Beste, M., Bechmann, L.P., Schlattjan, M., Syn, W-K., Wedemeyer, I., Mathe, Z., Jochum, C., Gerken, G., Gieseler, R.K. and Canbay, A. Gut, 64(5), 791-799 (2015)   Objective Non-alcoholic fatty liver disease (NAFLD) is closely linked to obesity and constitutes part of the metabolic syndrome, which have been associated with low serum vitamin D (VD). Due to known crosstalk between VD and transforming growth factor (TGF)-β signalling, VD has been proposed as an antifibrotic treatment. Design We evaluated the association between VD, the vitamin D receptor (VDR) and liver fibrosis in primary human hepatic stellate cells (phHSC) and 106 morbidly obese patients with NAFLD. Results Treating phHSC with VD ameliorated TGF-β-induced fibrogenesis via both VDR-dependent and VDR-independent mechanisms. Reduction of fibrogenic response was abolished in cells homozygous for GG at the A1012G single nucleotide polymorphisms within the VDR gene. Compared with healthy livers, NAFLD livers expressed higher levels of VDR mRNA and VDR fragments. VDR mRNA was lower in patients homozygous for GG at A1012G and expression of pro-fibrogenic genes was higher in patients carrying the G allele. Conclusions VD may be an antifibrotic treatment option early in the onset of fibrosis in specific genotypes for VDR. Known polymorphisms of the VDR may influence the response to VD treatment.    

4.1404           Dysfunction in endoplasmic reticulum-mitochondria crosstalk underlies SIGMAR1 loss of function mediated motor neuron degeneration

Bernard-Marissal, N., Medard, J-J., Azzedine, H. and Chrast, R. Brain, 138, 875-890 (2015)   Mutations in Sigma 1 receptor (SIGMAR1) have been previously identified in patients with amyotrophic lateral sclerosis and disruption of Sigmar1 in mouse leads to locomotor deficits. However, cellular mechanisms underlying motor phenotypes in human and mouse with disturbed SIGMAR1 function have not been described so far. Here we used a combination of in vivo and in vitro approaches to investigate the role of SIGMAR1 in motor neuron biology. Characterization of Sigmar1^−/− mice revealed that affected animals display locomotor deficits associated with muscle weakness, axonal degeneration and motor neuron loss. Using primary motor neuron cultures, we observed that pharmacological or genetic inactivation of SIGMAR1 led to motor neuron axonal degeneration followed by cell death. Disruption of SIGMAR1 function in motor neurons disturbed endoplasmic reticulum–mitochondria contacts, affected intracellular calcium signalling and was accompanied by activation of endoplasmic reticulum stress and defects in mitochondrial dynamics and transport. These defects were not observed in cultured sensory neurons, highlighting the exacerbated sensitivity of motor neurons to SIGMAR1 function. Interestingly, the inhibition of mitochondrial fission was sufficient to induce mitochondria axonal transport defects as well as axonal degeneration similar to the changes observed after SIGMAR1 inactivation or loss. Intracellular calcium scavenging and endoplasmic reticulum stress inhibition were able to restore mitochondrial function and consequently prevent motor neuron degeneration. These results uncover the cellular mechanisms underlying motor neuron degeneration mediated by loss of SIGMAR1 function and provide therapeutically relevant insight into motor neuronal diseases.  

4.1405           Peripheral Blood-Derived Mesenchymal Stem Cells: Candidate Cells Responsible for Healing Critical-Sized Calvarial Bone Defects

Li, S., Huang, K-L., Wu, J-C., Hu, M.S., Sanyal, M., Hu, M., Longaker, M.T. and Lorenz, H.P. Stem Cells Trans. Med., 4, 359-368 (2015)   Postnatal tissue-specific stem/progenitor cells hold great promise to enhance repair of damaged tissues. Many of these cells are retrieved from bone marrow or adipose tissue via invasive procedures. Peripheral blood is an ideal alternative source for the stem/progenitor cells because of its ease of retrieval. We present a coculture system that routinely produces a group of cells from adult peripheral blood. Treatment with these cells enhanced healing of critical-size bone defects in the mouse calvarium, a proof of principle that peripheral blood-derived cells can be used to heal bone defects. From these cells, we isolated a subset of CD45⁻ cells with a fibroblastic morphology. The CD45⁻ cells were responsible for most of the differentiation-induced calcification activity and were most likely responsible for the enhanced healing process. These CD45⁻ fibroblastic cells are plastic-adherent and exhibit a surface marker profile negative for CD34, CD19, CD11b, lineage, and c-kit and positive for stem cell antigen 1, CD73, CD44, CD90.1, CD29, CD105, CD106, and CD140α. Furthermore, these cells exhibited osteogenesis, chondrogenesis, and adipogenesis capabilities. The CD45⁻ fibroblastic cells are the first peripheral blood-derived cells that fulfill the criteria of mesenchymal stem cells as defined by the International Society for Cellular Therapy. We have named these cells “blood-derived mesenchymal stem cells.”  

4.1406           Olfactory ensheathing cell–neurite alignment enhances neurite outgrowth in scar-like cultures

Khhankan, R.R., Wanner, I.B. and Phelps, P.E. Exp. Neurol., 269, 93-101 (2015)   The regenerative capacity of adult CNS neurons after injury is strongly inhibited by the spinal cord lesion site environment that is composed primarily of the reactive astroglial scar and invading meningeal fibroblasts. Olfactory ensheathing cell (OEC) transplantation facilitates neuronal survival and functional recovery after a complete spinal cord transection, yet the mechanisms by which this recovery occurs remain unclear. We used a unique multicellular scar-like culture model to test if OECs promote neurite outgrowth in growth-inhibitory areas. Astrocytes were mechanically injured and challenged by meningeal fibroblasts to produce key inhibitory elements of a spinal cord lesion. Neurite outgrowth of postnatal cerebral cortical neurons was assessed on three substrates: quiescent astrocyte control cultures, reactive astrocyte scar-like cultures, and scar-like cultures with OECs. Initial results showed that OECs enhanced total neurite outgrowth of cortical neurons in a scar-like environment by 60%. We then asked if the neurite growth-promoting properties of OECs depended on direct alignment between neuronal and OEC processes. Neurites that aligned with OECs were nearly three times longer when they grew on inhibitory meningeal fibroblast areas and twice as long on reactive astrocyte zones compared to neurites not associated with OECs. Our results show that OECs can independently enhance neurite elongation and that direct OEC–neurite cell contact can provide a permissive substrate that overcomes the inhibitory nature of the reactive astrocyte scar border and the fibroblast-rich spinal cord lesion core.  

4.1407           A Novel In Vitro Primary Culture Model of the Lower Motor Neuron–Neuromuscular Junction Circuit

Southam, K.A., King, A.E., Blizzard, C.A., McCormack, G.H. and Dickson, T.C. Neuromethods, 103, 181-193 (2015)   Modelling the complex process of neuromuscular signalling is key to understanding not only normal circuit function but also importantly the mechanisms underpinning a range of degenerative diseases. Here, we describe a compartmented in vitro model of the lower motor neuron–neuromuscular junction circuit, incorporating primary spinal motor neurons, supporting glia and skeletal muscle. This culture model is designed to spatially mimic the unique anatomical and cellular interactions of this circuit in compartmented microfluidic devices, such that the glial cells are located with motor neuron cell bodies in the cell body chamber and motor neuron axons extend to a distal chamber containing skeletal muscle cells whilst simultaneously allowing targeted intervention.  

4.1408           Sperm Cleanup and Centrifugation Processing for Cryopreservation

Sieme, H. and Oldenhof, H. Methods in Mol. Biol., 1257, 343-352 (2015)   Fertility rates with artificial insemination are highest with good-quality sperm samples. Therefore, nonviable sperm, cellular debris, and seminal plasma are preferably removed from semen samples prior to use or for preservation. Such compounds are sources where reactive oxygen species are generated during storage or upon cryopreservation, impairing sperm function. In this chapter we describe methods to remove seminal plasma and cellular debris from sperm samples, and for selecting morphologically normal motile sperm. The methods that are described here include: ordinary centrifugation, sperm swim-up, glass wool and Sephadex filtration/adherence, and single-layer as well as discontinuous two-layer iodixanol density gradient centrifugation.  

4.1409           FcRn Rescues Recombinant Factor VIII Fc Fusion Protein from a VWF Independent FVIII Clearance Pathway in Mouse Hepatocytes

Van der Flier, A., Liu, Z., Tan, S., Chen, K., Grager, D., Liu, T., Patarroyo-White, S., Jiang, H. and Light, D.R. Plos One, 10(4), e0124930 (2015)   We recently developed a longer lasting recombinant factor VIII-Fc fusion protein, rFVIIIFc, to extend the half-life of replacement FVIII for the treatment of people with hemophilia A. In order to elucidate the biological mechanism for the elongated half-life of rFVIIIFc at a cellular level we delineated the roles of VWF and the tissue-specific expression of the neonatal Fc receptor (FcRn) in the biodistribution, clearance and cycling of rFVIIIFc. We find the tissue biodistribution is similar for rFVIIIFc and rFVIII and that liver is the major clearance organ for both molecules. VWF reduces the clearance and the initial liver uptake of rFVIIIFc. Pharmacokinetic studies in FcRn chimeric mice show that FcRn expressed in somatic cells (hepatocytes or liver sinusoidal endothelial cells) mediates the decreased clearance of rFVIIIFc, but FcRn in hematopoietic cells (Kupffer cells) does not affect clearance. Immunohistochemical studies show that when rFVIII or rFVIIIFc is in dynamic equilibrium binding with VWF, they mostly co localize with VWF in Kupffer cells and macrophages, confirming a major role for liver macrophages in the internalization and clearance of the VWF-FVIII complex. In the absence of VWF a clear difference in cellular localization of VWF-free rFVIII and rFVIIIFc is observed and neither molecule is detected in Kupffer cells. Instead, rFVIII is observed in hepatocytes, indicating that free rFVIII is cleared by hepatocytes, while rFVIIIFc is observed as a diffuse liver sinusoidal staining, suggesting recycling of free-rFVIIIFc out of hepatocytes. These studies reveal two parallel linked clearance pathways, with a dominant pathway in which both rFVIIIFc and rFVIII complexed with VWF are cleared mainly by Kupffer cells without FcRn cycling. In contrast, the free fraction of rFVIII or rFVIIIFc unbound by VWF enters hepatocytes, where FcRn reduces the degradation and clearance of rFVIIIFc relative to rFVIII by cycling rFVIIIFc back to the liver sinusoid and into circulation, enabling the elongated half-life of rFVIIIFc.    

4.1410           Lack of GDAP1 Induces Neuronal Calcium and Mitochondrial Defects in a Knockout Mouse Model of Charcot-Marie-Tooth Neuropathy

Barneo-Munoz, M., Juarez, P., Civera-Tregon, A., Yndriago, L., Pla-Martin, D., Zenker, J., Cuevas-Martin, C., Estela, A., Sanchez-Arago, M., Forteza-Villa, J., Cuezva, J.M., Chrast, R. and Palau, F. PloS Genetics, 11(4), e1005115 (2015)   Mutations in GDAP1, which encodes protein located in the mitochondrial outer membrane, cause axonal recessive (AR-CMT2), axonal dominant (CMT2K) and demyelinating recessive (CMT4A) forms of Charcot-Marie-Tooth (CMT) neuropathy. Loss of function recessive mutations in GDAP1 are associated with decreased mitochondrial fission activity, while dominant mutations result in impairment of mitochondrial fusion with increased production of reactive oxygen species and susceptibility to apoptotic stimuli. GDAP1 silencing in vitro reduces Ca²⁺ inflow through store-operated Ca²⁺ entry (SOCE) upon mobilization of endoplasmic reticulum (ER) Ca²⁺, likely in association with an abnormal distribution of the mitochondrial network. To investigate the functional consequences of lack of GDAP1 in vivo, we generated a Gdap1 knockout mouse. The affected animals presented abnormal motor behavior starting at the age of 3 months. Electrophysiological and biochemical studies confirmed the axonal nature of the neuropathy whereas histopathological studies over time showed progressive loss of motor neurons (MNs) in the anterior horn of the spinal cord and defects in neuromuscular junctions. Analyses of cultured embryonic MNs and adult dorsal root ganglia neurons from affected animals demonstrated large and defective mitochondria, changes in the ER cisternae, reduced acetylation of cytoskeletal α-tubulin and increased autophagy vesicles. Importantly, MNs showed reduced cytosolic calcium and SOCE response. The development and characterization of the GDAP1 neuropathy mice model thus revealed that some of the pathophysiological changes present in axonal recessive form of the GDAP1-related CMT might be the consequence of changes in the mitochondrial network biology and mitochondria–endoplasmic reticulum interaction leading to abnormalities in calcium homeostasis.  

4.1411           A CXC chemokine gene, CXCL12, from rock bream, Oplegnathus fasciatus: Molecular characterization and transcriptional profile

Thulasitha, W.S., Umasuthan, N., Whang, I., Lim, B-S., Jung, H-B., Noh, J.K. and Lee, J. Fish & Shellfish Immunology, 45, 560-566 (2015)   Chemokines are small, structurally related chemotactic cytokines characterized by the presence of conserved cysteine residues. In the present study, we identified the cDNA of a CXC chemokine fromOplegnathus fasciatus, designated as OfCXCL12. An open reading frame of 297 bp encoded a 98 amino acid peptide with a putative signal peptide of 23 amino acids. The CXC family-specific small cytokine domain (SCY), which is highly conserved among vertebrates, was located between residues 29 and 87. The characteristic conserved cysteine residues in the CXC motif of OfCXCL12 were separated by tyrosine (Y). Similar to other vertebrate CXCL12 proteins, OfCXCL12 also lacked the ELR motif and hence belongs to ELR⁻ subfamily. Phylogenetic analysis revealed two distinct clades, consisting of fish and tetrapod CXCL12 homologs. Constitutive expression with significantly higher levels of OfCXCL12 mRNA transcription was detected in immune-related organs, including the head kidney, spleen, and kidney. Infection with bacterial and viral agents led to significant upregulation of mRNA expression in both the head kidney and spleen, in a stimulant-specific manner. Stimulation of peripheral blood leukocytes by the mitogen concanavalin-A significantly induced OfCXCL12 transcription. Results from the present study suggest an important role for OfCXCL12 in immune defense against bacterial and viral infection in rock bream.  

4.1412           Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells

Klein, A.M., Mazutis, L., Akartuna, I., Tallapragada, N., Veres, A., Peshkin, L., Weitz, D.A. and Kirschner, M.W. Cell, 161, 1187-1201 (2015)   It has long been the dream of biologists to map gene expression at the single-cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single-cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after leukemia inhibitory factor (LIF) withdrawal. The reproducibility of these high-throughput single-cell data allowed us to deconstruct cell populations and infer gene expression relationships.  

4.1413           Divergent effects of RIP1 or RIP3 blockade in murine models of acute liver injury

Deutsch, M., Graffeo, C.S., Rokosh, R., pansari, M., Ochi, A., Levie, E.M., Van Heerden, E., Tippens, D.M., Greco, S., Barilla, R., Tomkötter, L., Zambrinis, C.P., Avanzi, N., Gulati, R., pachter, H.L., Torres-Hernandez, A., Eisenthal, A., Daley, D. and Miller, G. Cell Death and Disease, 6, e1759 (2015)   Necroptosis is a recently described Caspase 8-independent method of cell death that denotes organized cellular necrosis. The roles of RIP1 and RIP3 in mediating hepatocyte death from acute liver injury are incompletely defined. Effects of necroptosis blockade were studied by separately targeting RIP1 and RIP3 in diverse murine models of acute liver injury. Blockade of necroptosis had disparate effects on disease outcome depending on the precise etiology of liver injury and component of the necrosome targeted. In ConA-induced autoimmune hepatitis, RIP3 deletion was protective, whereas RIP1 inhibition exacerbated disease, accelerated animal death, and was associated with increased hepatocyte apoptosis. Conversely, in acetaminophen-mediated liver injury, blockade of either RIP1 or RIP3 was protective and was associated with lower NLRP3 inflammasome activation. Our work highlights the fact that diverse modes of acute liver injury have differing requirements for RIP1 and RIP3; moreover, within a single injury model, RIP1 and RIP3 blockade can have diametrically opposite effects on tissue damage, suggesting that interference with distinct components of the necrosome must be considered separately.  

4.1414           Endotoxic shock-expanded murine CD11clowCD45RB+ regulatory dendritic cells modulate inflammatory T cell responses through multiple mechanisms

Wang, X., Wang, Q., Zhang, X., Li, Y., Wang, J., Hou, C., Chen, J., Shen, B., Shi, Y. and Zhang, J. Scientific Reports, 5:10653 (2015)   Changes in the number and function of dendritic cells (DCs) have been reported to play an important role in endotoxin tolerance. It has been reported that expansion of splenic CD11c^lowCD45RB⁺ DCs occurs in mice injected with sublethal doses of lipopolysaccharide (LPS). However, the function of endotoxic shock-expanded CD11c^lowCD45RB⁺ DCs has not been examined. In this work, we show that endotoxic shock promotes the expansion of CD11c^lowCD45RB⁺ cells with dendritic morphology and the production of low levels of inflammatory cytokines and costimulatory molecules. The expanded cells induce the generation of regulatory T cells (Tregs), show incapability to stimulate T cells, and induce apoptosis of CD4⁺ T cells in vitro. As compared to CD11c^hiCD45RB⁻ conventional DCs, the expanded cells exert better protection against colitis induction by CD4⁺ CD25⁻ T cells, even though both subpopulations show similar ability to induce Tregs in vivo. The better control of proinflammatory cytokine responses in vivo by the expanded cells is associated with more apoptosis in the Payer’s patches and in colonic tissue-infiltrating cells. Thus, the expanded cells can modulate inflammatory T cell responses through multiple mechanisms. Our study facilitates a better understanding how innate immune responses may shape adaptive immunity and immune suppression following LPS-induced acute inflammation.  

4.1415           Influenza induces IL-8 and GM-CSF secretion by human alveolar epithelial cells through HGF/c-Met and TGF-α/EGFR signaling

Ito, Y., Correll, K., Zemans, R.I., Leslie, C.C., Murphy, R.C. and mason, R.J: Am. J. Physiol. Lung Cell Mol. Physiol., 308, L1178-L1188 (2015)   The most severe complication of influenza is viral pneumonia, which can lead to the acute respiratory distress syndrome. Alveolar epithelial cells (AECs) are the first cells that influenza virus encounters upon entering the alveolus. Infected epithelial cells produce cytokines that attract and activate neutrophils and macrophages, which in turn induce damage to the epithelial-endothelial barrier. Hepatocyte growth factor (HGF)/c-Met and transforming growth factor-α (TGF-α)/epidermal growth factor receptor (EGFR) are well known to regulate repair of damaged alveolar epithelium by stimulating cell migration and proliferation. Recently, TGF-α/EGFR signaling has also been shown to regulate innate immune responses in bronchial epithelial cells. However, little is known about whether HGF/c-Met signaling alters the innate immune responses and whether the innate immune responses in AECs are regulated by HGF/c-Met and TGF-α/EGFR. We hypothesized that HGF/c-Met and TGF-α/EGFR would regulate innate immune responses to influenza A virus infection in human AECs. We found that recombinant human HGF (rhHGF) and rhTGF-α stimulated primary human AECs to secrete IL-8 and granulocyte macrophage colony-stimulating factor (GM-CSF) strongly and IL-6 and monocyte chemotactic protein 1 moderately. Influenza infection stimulated the secretion of IL-8 and GM-CSF by AECs plated on rat-tail collagen through EGFR activation likely by TGF-α released from AECs and through c-Met activated by HGF secreted from lung fibroblasts. HGF secretion by fibroblasts was stimulated by AEC production of prostaglandin E₂ during influenza infection. We conclude that HGF/c-Met and TGF-α/EGFR signaling enhances the innate immune responses by human AECs during influenza infections.  

4.1416           Metabolic regulation of hepatitis B immunopathology by myeloid-derived suppressor cells

Pallett, L.J. et al Nature Med., 21(6), 591-600 (2015)   Infection with hepatitis B virus (HBV) results in disparate degrees of tissue injury: the virus can either replicate without pathological consequences or trigger immune-mediated necroinflammatory liver damage. We investigated the potential for myeloid-derived suppressor cells (MDSCs) to suppress T cell–mediated immunopathology in this setting. Granulocytic MDSCs (gMDSCs) expanded transiently in acute resolving HBV, decreasing in frequency prior to peak hepatic injury. In persistent infection, arginase-expressing gMDSCs (and circulating arginase) increased most in disease phases characterized by HBV replication without immunopathology, whilst L-arginine decreased. gMDSCs expressed liver-homing chemokine receptors and accumulated in the liver, their expansion supported by hepatic stellate cells. We provide in vitro and ex vivo evidence that gMDSCs potently inhibited T cells in a partially arginase-dependent manner. L-arginine–deprived T cells upregulated system L amino acid transporters to increase uptake of essential nutrients and attempt metabolic reprogramming. These data demonstrate the capacity of expanded arginase-expressing gMDSCs to regulate liver immunopathology in HBV infection.  

4.1417           Non-Aggregating Tau Phosphorylation by Cyclin-Dependent Kinase 5 Contributes to Motor Neuron Degeneration in Spinal Muscular Atrophy

Miller, N. et al

1. Neurosci., 35(15), 6038-6050 (2015)

  Mechanisms underlying motor neuron degeneration in spinal muscular atrophy (SMA), the leading inherited cause of infant mortality, remain largely unknown. Many studies have established the importance of hyperphosphorylation of the microtubule-associated protein tau in various neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, tau phosphorylation in SMA pathogenesis has yet to be investigated. Here we show that tau phosphorylation on serine 202 (S202) and threonine 205 (T205) is increased significantly in SMA motor neurons using two SMA mouse models and human SMA patient spinal cord samples. Interestingly, phosphorylated tau does not form aggregates in motor neurons or neuromuscular junctions (NMJs), even at late stages of SMA disease, distinguishing it from other tauopathies. Hyperphosphorylation of tau on S202 and T205 is mediated by cyclin-dependent kinase 5 (Cdk5) in SMA disease condition, because tau phosphorylation at these sites is significantly reduced in Cdk5 knock-out mice; genetic knock-out of Cdk5 activating subunit p35 in an SMA mouse model also leads to reduced tau phosphorylation on S202 and T205 in the SMA;p35^−/− compound mutant mice. In addition, expression of the phosphorylation-deficient tauS202A,T205A mutant alleviates motor neuron defects in a zebrafish SMA model in vivo and mouse motor neuron degeneration in culture, whereas expression of phosphorylation-mimetic tauS202E,T205E promotes motor neuron defects. More importantly, genetic knock-out of tau in SMA mice rescues synapse stripping on motor neurons, NMJ denervation, and motor neuron degeneration in vivo. Altogether, our findings suggest a novel mechanism for SMA pathogenesis in which hyperphosphorylation of non-aggregating tau by Cdk5 contributes to motor neuron degeneration.  

4.1418           CD81 Controls Immunity to Listeria Infection through Rac-Dependent Inhibition of Proinflammatory Mediator Release and Activation of Cytotoxic T Cells

Martinez del Hoya, G., Ramirez-Huesca, M., Levy, S., Boucheix, C., Rubinstein, E., Minguito de la Escalera, M., Gonzalez-Cintado, L., Arvadin, C., Veiga, E., Yanez-Mo, M. and Sanchez-Madrid, F.

1. Immunol., 194(12), 6090-6101 (2015)

  Despite recent evidence on the involvement of CD81 in pathogen binding and Ag presentation by dendritic cells (DCs), the molecular mechanism of how CD81 regulates immunity during infection remains to be elucidated. To investigate the role of CD81 in the regulation of defense mechanisms against microbial infections, we have used the Listeria monocytogenes infection model to explore the impact of CD81 deficiency in the innate and adaptive immune response against this pathogenic bacteria. We show that CD81^−/− mice are less susceptible than wild-type mice to systemic Listeria infection, which correlates with increased numbers of inflammatory monocytes and DCs in CD81^−/− spleens, the main subsets controlling early bacterial burden. Additionally, our data reveal that CD81 inhibits Rac/STAT-1 activation, leading to a negative regulation of the production of TNF-α and NO by inflammatory DCs and the activation of cytotoxic T cells by splenic CD8α⁺ DCs. In conclusion, this study demonstrates that CD81–Rac interaction exerts an important regulatory role on the innate and adaptive immunity against bacterial infection and suggests a role for CD81 in the development of novel therapeutic targets during infectious diseases.  

4.1419           Tweak regulates astrogliosis, microgliosis and skeletal muscle atrophy in a mouse model of amyotrophic lateral sclerosis

Bowerman, M., Salsac, C., Coque, E., Eiselt, E., Deschaumes, R.G., Brodovitch, A., Burkly, L.C., Scamps, F. and Raoul, C. Hum. Mol. Genet., 24(12), 3440-3456 (2015)   Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that primarily affects motoneurons in the brain and spinal cord. Astrocyte and microglia activation as well as skeletal muscle atrophy are also typical hallmarks of the disease. However, the functional relationship between astrocytes, microglia and skeletal muscle in the pathogenic process remains unclear. Here, we report that the tumor necrosis factor-like weak inducer of apoptosis (Tweak) and its receptor Fn14 are aberrantly expressed in spinal astrocytes and skeletal muscle of SOD1^G93A mice. We show that Tweak induces motoneuron death, stimulates astrocytic interleukin-6 release and astrocytic proliferation in vitro. The genetic ablation of Tweak in SOD1^G93A mice significantly reduces astrocytosis, microgliosis and ameliorates skeletal muscle atrophy. The peripheral neutralization of Tweak through antagonistic anti-Tweak antibody ameliorates muscle pathology and notably, decreases microglial activation in SOD1^G93A mice. Unexpectedly, none of these approaches improved motor function, lifespan and motoneuron survival. Our work emphasizes the multi-systemic aspect of ALS, and suggests that a combinatorial therapy targeting multiple cell types will be instrumental to halt the neurodegenerative process.  

4.1420           Basic fibroblast growth factor-treated adipose tissue-derived mesenchymal stem cell infusion to ameliorate liver cirrhosis via paracrine hepatocyte growth factor

Tang, W-P., Akahoshi, T., Piao, J-S., Narahara, S., Murata, M., Kawano, T., Hamano, N., Ikeda, T. and Hashizume, M.

1. Gastroenterol. and Hepatol., 30(6), 1065-1074 (2015)

  Background and Aim Recent studies show that adipose tissue-derived mesenchymal stem cells have potential clinical applications. However, the mechanism has not been fully elucidated yet. Here, we investigated the effect of basic fibroblast growth factor-treated adipose tissue-derived mesenchymal stem cells infusion on a liver fibrosis rat model and elucidated the underlying mechanism. Methods Adipose tissue-derived mesenchymal stem cells were infused into carbon tetrachloride-induced hepatic fibrosis rats through caudal vein. Liver functions and pathological changes were assessed. A co-culture model was used to clarify the potential mechanism. Results Basic fibroblast growth factor treatment markedly improved the proliferation, differentiation, and hepatocyte growth factor expression ability of adipose tissue-derived mesenchymal stem cells. Although adipose tissue-derived mesenchymal stem cells infusion alone slightly ameliorated liver functions and suppressed fibrosis progression, basic fibroblast growth factor-treatment significantly enhanced the therapeutic effect in association with elevated hepatocyte growth factor expression. Moreover, double immunofluorescence staining confirmed that the infused cells located in fibrosis area. Furthermore, co-culture with adipose tissue-derived mesenchymal stem cell led to induction of hepatic stellate cell apoptosis and enhanced hepatocyte proliferation. However, these effects were significantly weakened by knockdown of hepatocyte growth factor. Mechanism investigation revealed that co-culture with adipose tissue-derived mesenchymal stem cells activated c-jun N-terminal kinase-p53 signaling in hepatic stellate cell and promoted apoptosis. Conclusions Basic fibroblast growth factor treatment enhanced the therapeutic effect of adipose tissue-derived mesenchymal stem cells, and secretion of hepatocyte growth factor from adipose tissue-derived mesenchymal stem cells plays a critical role in amelioration of liver injury and regression of fibrosis.  

4.1421           Image-based cell-resolved screening assays in flow

Cheung, M.C., McKenna, B., Wang, S.S., Wolf, D. and Erhlich, D.J. Cytometry Part A, 87(6), 541-548 (2015)   A parallel microfluidic cytometer (PMC) is based on a one-dimensional (1D) scanning detector, a parallel array of flow channels, and new multiparameter analysis algorithms that operate on low-pixel-count 1D images. In this article, we explore a series of image-based live- and fixed-cell screening assays, including two NF-kB nuclear translocations and T-cell capping. We then develop a new multiparametric linear weighted classifier that achieves a Z′ factor sufficient for scaled pharmaceutical discovery with Jurkat cells in suspension. We conclude that the PMC should have the throughput and statistical power to permit a new capability for image-based high-sample-number pharmaceutical screening with suspension samples  

4.1422           Activated endothelial cells limit inflammatory response, but increase chemoattractant potential and bacterial clearance by human monocytes

Mancilla-Herrera, I., Alvarado-Moreno, J.A., Cerbulo-Vazquez, A., Prieto-Chavez. J.L., Ferat-Osorio, E., Lopez-Macias, C., Estrada-Parra, S., Isibasi, A. and Arriaga-Pizano, L. Cell Biol. Int., 39(6), 721-732 (2015)   Inflammation is the normal immune response of vascularized tissues to damage and bacterial products, for which leukocyte transendothelial migration (TEM) is critical. The effects of cell-to-cell contact seen in both leukocyte and endothelial cells include cytoskeleton rearrangement, and dynamic expression of adhesion molecules and metalloproteinases. TEM induces expression of anti-apoptotic molecules, costimulatory molecules associated with antigen presentation, and pattern recognition receptors (PRR), such as TLR-4, in monocytes. However, little is known about how TLR-4 increment operates in monocytes during an inflammatory response. To understand it better, we used an in vitro model in which monocytes crossed a layer of IL-1β stimulated Human Umbilical Vein Endothelial Cells (HUVEC). After TEM, monocytes were tested for the secretion of inflammatory cytokines and chemokines, their phenotype (CD14, CD16, TLR-4 expression), and TLR-4 canonical [Nuclear Factor kappa B, (NF-κB) pathway] and non-canonical [p38, extracellular signal-regulated kinases (ERK) 1/2 pathway] signal transduction induced by lipopolysaccharide (LPS). Phagocytosis and bacterial clearance were also measured. There was diminished secretion of LPS-induced inflammatory cytokines (IL-1β, IL-6, and TNF-α) and higher secretion of chemokines (CXCL8/IL-8 and CCL2/MCP-1) in supernatant of TEM monocytes. These changes were accompanied by increases in TLR-4, CD14 (surfaces expression), p38, and ERK1/2 phosphorylated cytoplasmic forms, without affecting NF-κB activation. It also increased bacterial clearance after TEM by an O₂-independent mechanism. The data suggest that interaction between endothelial cells and monocytes fine-tunes the inflammatory response and promotes bacterial elimination.  

4.1423           Age-Related Decline of Autocrine Pituitary Adenylate Cyclase-Activating Polypeptide Impairs Angiogenic Capacity of Rat Cerebromicrovascular Endothelial Cells

Banki, E., Sosnowska, D., Tucsek, Z., Gautam, T., Toth, P., Tarantini, S., Tamas, A., Helyes, Z., Reglodi, D., Sonntag, W.E., Csiszar, A. and Ungvari, Z.

1. Gerontol. A Biol. Sci. Med. Sci., 70, 665-674 (2015)

  Aging impairs angiogenic capacity of cerebromicrovascular endothelial cells (CMVECs) promoting microvascular rarefaction, but the underlying mechanisms remain elusive. PACAP is an evolutionarily conserved neuropeptide secreted by endothelial cells and neurons, which confers important antiaging effects. To test the hypothesis that age-related changes in autocrine PACAP signaling contributes to dysregulation of endothelial angiogenic capacity, primary CMVECs were isolated from 3-month-old (young) and 24-month-old (aged) Fischer 344 x Brown Norway rats. In aged CMVECs, expression of PACAP was decreased, which was associated with impaired capacity to form capillary-like structures, impaired adhesiveness to collagen (assessed using electric cell-substrate impedance sensing [ECIS] technology), and increased apoptosis (caspase3 activity) when compared with young cells. Overexpression of PACAP in aged CMVECs resulted in increased formation of capillary-like structures, whereas it did not affect cell adhesion. Treatment with recombinant PACAP also significantly increased endothelial tube formation and inhibited apoptosis in aged CMVECs. In young CMVECs shRNA knockdown of autocrine PACAP expression significantly impaired tube formation capacity, mimicking the aging phenotype. Cellular and mitochondrial reactive oxygen species production (dihydroethidium and MitoSox fluorescence, respectively) were increased in aged CMVECs and were unaffected by PACAP. Collectively, PACAP exerts proangiogenic effects and age-related dysregulation of autocrine PACAP signaling may contribute to impaired angiogenic capacity of CMVECs in aging.  

4.1424           Histone H3K9 demethylase JMJD1A modulates hepatic stellate cells activation and liver fibrosis by epigenetically regulating peroxisome proliferator-activated receptor γ

Jiang, Y., Wang, S., Zhao, Y., Lin, C., Zhong, F., Jin, L., He, F. and Wang, H. FASEB J., 29(5), 1830-1841 (2015)   As a central event in liver fibrogenesis, hepatic stellate cell (HSC) transdifferentiation involves loss of regulation by adipogenic transcription factors such as peroxisome proliferator-activated receptor γ; (PPARγ), which is epigenetically silenced during HSC activation. We hypothesized that JMJD1A, an H3K9 demethylase involved in adipogenic metabolism, could regulate PPARγ. In human HSC cell line, rat primary HSCs, and carbontetrachloride-induced mouse liver fibrogenesis model, we down-regulated the expression of JMJD1A using small interfering or short hairpin RNAs, and overexpressed its wild-type and mutant. We analyzed the effects of JMJD1A manipulation on the histone di-methyl-H3K9 (H3k9me2) status of PPARγ gene and the expression of PPARγ and fibrosis markers using chromatin immunoprecipitation, real-time quantitative RT-PCR and Western blot, and also investigated the in vitro and in vivo consequences on liver fibrosis and necrosis by Masson or hematoxylin-eosin staining, respectively. JMJD1A knockdown in HSCs correlated with reinforced H3K9me2 in the PPARγ gene promoter, and its down-regulation in both mRNA and protein led to increased expression of fibrosis markers, which could be consistently rescued by JMJD1A overexpression. Jmjd1a knockdown in situ resulted in significantly increased expression of α-smooth muscle actin (P = 0.005) and Col1a (P = 0.036), strengthened production of collagens (P = 0.028), and remarkably enhanced necrosis (P = 0.007) 4 weeks after treatment. This study suggests JMJD1A as a novel epigenetic regulator that modulates HSC activation and liver fibrosis through targeting PPARγ gene expression.—Jiang, Y., Wang, S., Zhao, Y., Lin, C., Zhong, F., Jin, L., He, F., Wang, H. Histone H3K9 demethylase JMJD1A modulates hepatic stellate cells activation and liver fibrosis by epigenetically regulating peroxisome proliferator-activated receptor γ.  

4.1425           Continuous Flow Microfluidic Bioparticle Concentrator

Martel, J.M., Smith, K.C., Dlamini, M., Pletchert, K., yang, J., karabacak, M., haber, D.A., Kapur, R. and Toner, M. Scientific Reports, 5:11300 (2015)   Innovative microfluidic technology has enabled massively parallelized and extremely efficient biological and clinical assays. Many biological applications developed and executed with traditional bulk processing techniques have been translated and streamlined through microfluidic processing with the notable exception of sample volume reduction or centrifugation, one of the most widely utilized processes in the biological sciences. We utilize the high-speed phenomenon known as inertial focusing combined with hydraulic resistance controlled multiplexed micro-siphoning allowing for the continuous concentration of suspended cells into pre-determined volumes up to more than 400 times smaller than the input with a yield routinely above 95% at a throughput of 240 ml/hour. Highlighted applications are presented for how the technology can be successfully used for live animal imaging studies, in a system to increase the efficient use of small clinical samples, and finally, as a means of macro-to-micro interfacing allowing large samples to be directly coupled to a variety of powerful microfluidic technologies.  

4.1426           PTPRO-Associated Hepatic Stellate Cell Activation Plays a Critical Role in Liver Fibrosis

Zhang, X., Tan, Z., Wang, Y., Tang, J., Jiang, R., Hou, J., Zhuo, H., Wang, X., Ji, J., Qin, X. and Sun, B. Cell. Physiol. Biochem., 35, 885-898 (2015)   Background/Aims: PTPRO (protein tyrosine phosphatase, receptor type O) is implicated in diverse physiological and pathological processes in cancer and hepatic ischemia/reperfusion injury, although little is known about its role in hepatic fibrosis. Methods: Here, by using genetically deficient mice, we reported that PTPRO knockout (PTPRO^-/-) significantly attenuated liver injury, release of inflammatory factors, tissue remodeling, and liver fibrosis in two experimental mouse models of fibrogenesis induced by bile-duct ligation or carbon tetrachloride administration. Results: However, we proved that PTPRO expression was strongly downregulated in clinical and experimental liver fibrosis specimens. Further investigations revealed that stimulation of primary hepatic stellate cells (HSCs) and hepatocytes with specific activator platelet-derived growth factor (PDGF)-BB increased PTPRO transcription in HSCs but had the opposite effect in primary hepatocytes. More importantly, synthetic short hairpin RNA targeting PTPRO significantly neutralized PDGF-BB-induced HSC proliferation and myofibroblast marker expression through downregulated phosphorylation of extracellular signal-regulated kinase (ERK) and AKT. Conclusion: These observations confirm that PTPRO plays a critical role in liver fibrogenesis by affecting PDGF signaling in HSC activation and might be developed into a feasible therapeutic approach for the treatment of chronic fibrotic liver diseases.  

4.1427           High-Speed Discrimination and Sorting of Submicron Particles Using a Microfluidic Device

Rajauria, S., Axline, C., Gottstein, C. and Cleland, A.N. Nano Lett., 15(1), 469-475 (2015)   The size- and fluorescence-based sorting of micro- and nanoscale particles suspended in fluid presents a significant and important challenge for both sample analysis and for manufacturing of nanoparticle-based products. Here, we demonstrate a disposable microfluidic particle sorter that enables high-throughput, on-demand counting and binary sorting of submicron particles and cells using either fluorescence or an electrically based determination of particle size. Size-based sorting uses a resistive pulse sensor integrated on-chip, whereas fluorescence-based discrimination is achieved using on-the-fly optical image capture and analysis. Following detection and analysis, the individual particles are deflected using a pair of piezoelectric actuators, directing the particles into one of two desired output channels; the main flow goes into a third waste channel. The integrated system can achieve sorting fidelities of better than 98%, and the mechanism can successfully count and actuate, on demand, more than 60 000 particles/min.  

4.1428           Assessment of the Performance of Membrane Bioreactors Applied to the Treatment of Industrial Effluents Containing Poly(vinyl alcohol)

Blanco, L., Hermosilla, D., Blanco, A., Swinnen, N., Prieto, D. and Negro, C. Ind. Eng. Chem. Res., 54(20), 5442-5449 (2015)   To assess and to optimize the treatment of effluents containing poly(vinyl alcohol) (PVA) from a poly(vinyl chloride) (PVC) production site, two aerobic membrane bioreactors (MBR) were run in parallel. PVA and total organics degradation, pH, temperature, oxygen consumption, organic loading rate, hydraulic retention time, and macro- and micronutrients contents were measured and optimized. Total removal of PVA, total nitrification, > 90% chemical oxygen demand (COD) removal, and >95% biological oxygen demand (BOD₅) reduction were achieved from a mixed culture of microorganisms after an adequate adaptation period. In addition, different bacteria cultures were assessed and analyzed in order to study PVA biodegradation: γ-proteobacteria and α-proteobacteria were the main groups of PVA-degrading bacteria. Membranes showed a good performance during the trials. Furthermore, permeability was completely recovered by chemical cleaning. After the assessed MBR treatment, the effluent fulfills appropriate water quality requirements for its post-treatment in a desalination unit in order to be reused within the PVC production processes.  

4.1429           Clinical outcomes of a novel therapeutic vaccine with Tax peptide-pulsed dendritic cells for adult T cell leukaemia/lymphoma in a pilot study

Suehiro, Y. et al Br. J. hematol., 169(6), 356-367 (2015)   Adult T cell leukaemia/lymphoma (ATL) is a human T cell leukaemia virus type-I (HTLV-I)-infected T cell malignancy with poor prognosis. We herein developed a novel therapeutic vaccine designed to augment an HTLV-I Tax-specific cytotoxic T lymphocyte (CTL) response that has been implicated in anti-ATL effects, and conducted a pilot study to investigate its safety and efficacy. Three previously treated ATL patients, classified as intermediate- to high-risk, were subcutaneously administered with the vaccine, consisting of autologous dendritic cells (DCs) pulsed with Tax peptides corresponding to the CTL epitopes. In all patients, the performance status improved after vaccination without severe adverse events, and Tax-specific CTL responses were observed with peaks at 16–20 weeks. Two patients achieved partial remission in the first 8 weeks, one of whom later achieved complete remission, maintaining their remission status without any additional chemotherapy 24 and 19 months after vaccination, respectively. The third patient, whose tumour cells lacked the ability to express Tax at biopsy, obtained stable disease in the first 8 weeks and later developed slowly progressive disease although additional therapy was not required for 14 months. The clinical outcomes of this pilot study indicate that the Tax peptide-pulsed DC vaccine is a safe and promising immunotherapy for ATL.  

4.1430           Defects in optineurin- and myosin VI-mediated cellular trafficking in amyotrophic lateral sclerosis

Sundaramoorthy, V., Walker, A.K., Tan, V., Fifita, J.A., McCann, E.P., Williams, K.L., Blair, I.P., Guillemin, G.J., Frag, M.A. and Atkin, J.D. Hum. Mol. Genet., 24(13), 3830-3846 (2015)   Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting motor neurons. Mutations in optineurin cause a small proportion of familial ALS cases, and wild-type (WT) optineurin is misfolded and forms inclusions in sporadic ALS patient motor neurons. However, it is unknown how optineurin mutation or misfolding leads to ALS. Optineurin acts an adaptor protein connecting the molecular motor myosin VI to secretory vesicles and autophagosomes. Here, we demonstrate that ALS-linked mutations p.Q398X and p.E478G disrupt the association of optineurin with myosin VI, leading to an abnormal diffuse cytoplasmic distribution, inhibition of secretory protein trafficking, endoplasmic reticulum (ER) stress and Golgi fragmentation in motor neuron-like NSC-34 cells. We also provide further insight into the role of optineurin as an autophagy receptor. WT optineurin associated with lysosomes and promoted autophagosome fusion to lysosomes in neuronal cells, implying that it mediates trafficking of lysosomes during autophagy in association with myosin VI. However, either expression of ALS mutant optineurin or small interfering RNA-mediated knockdown of endogenous optineurin blocked lysosome fusion to autophagosomes, resulting in autophagosome accumulation. Together these results indicate that ALS-linked mutations in optineurin disrupt myosin VI-mediated intracellular trafficking processes. In addition, in control human patient tissues, optineurin displayed its normal vesicular localization, but in sporadic ALS patient tissues, vesicles were present in a significantly decreased proportion of motor neurons. Optineurin binding to myosin VI was also decreased in tissue lysates from sporadic ALS spinal cords. This study therefore links several previously described pathological mechanisms in ALS, including defects in autophagy, fragmentation of the Golgi and induction of ER stress, to disruption of optineurin function. These findings also indicate that optineurin–myosin VI dysfunction is a common feature of both sporadic and familial ALS.  

4.1431           Liver fibrosis occurs through dysregulation of MyD88-dependent innate B-cell activity

Thapa, M., Chinnadurai, r., Velazquez, V.M., Tedesco, D., Elrod, E., Han, J-H., Sharma, P., Ibegbu, C., Gewirtz, A., Anania, F., Pulendran, b., Suthar, M. and Grakoui, A. Hepatology, 61(6), 2067-2079 (2015)   Chronic liver disease mediated by activation of hepatic stellate cells (HSCs) leads to liver fibrosis. Here, we postulated that the immune regulatory properties of HSCs might promote the profibrogenic activity of B cells. Fibrosis is completely attenuated in carbon tetrachloride–treated, B cell–deficient µMT mice, showing that B cells are required. The retinoic acid produced by HSCs augmented B-cell survival, plasma cell marker CD138 expression, and immunoglobulin G production. These activities were reversed following addition of the retinoic acid inhibitor LE540. Transcriptional profiling of fibrotic liver B cells revealed increased expression of genes related to activation of nuclear factor κ light chain enhancer of activated B cells, proinflammatory cytokine production, and CD40 signaling, suggesting that these B cells are activated and may be acting as inflammatory cells. Biological validation experiments also revealed increased activation (CD44 and CD86 expression), constitutive immunoglobulin G production, and secretion of the proinflammatory cytokines tumor necrosis factor-α, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1α. Likewise, targeted deletion of B-cell-intrinsic myeloid differentiation primary response gene 88 signaling, an innate adaptor with involvement in retinoic acid signaling, resulted in reduced infiltration of migratory CD11c⁺ dendritic cells and Ly6C⁺⁺ monocytes and, hence, reduced liver pathology. Conclusion: Liver fibrosis occurs through a mechanism of HSC-mediated augmentation of innate B-cell activity. These findings highlight B cells as important “first responders” of the intrahepatic immune environment.  

4.1432           Rapid Identification by Surface-Enhanced Raman Spectroscopy of Cancer Cells at Low Concentrations Flowing in a Microfluidic Channel

Pallaoro, A., Hoonejani, M.R., Braun, G.B., Meinhart, C.D. and Moskovits, M. ACS Nano, 9(4), 4328-4336 (2015)   Reliable identification and collection of cells from bodily fluids is of growing interest for monitoring patient response to therapy and for early detection of disease or its recurrence. We describe a detection platform that combines microfluidics with surface-enhanced Raman spectroscopy (SERS) for the identification of individual mammalian cells continuously flowing in a microfluidics channel. A mixture of cancerous and noncancerous prostate cells was incubated with SERS biotags (SBTs) developed and synthesized by us, then injected into a flow-focused microfluidic channel, which forces the cells into a single file. The spectrally rich SBTs are based on a silver nanoparticle dimer core labeled with a Raman-active small reporter molecule paired with an affinity biomolecule, providing a unique barcode whose presence in a composite SERS spectrum can be deconvoluted. Individual cancer cells passing through the focused laser beam were correctly identified among a proportionally larger number of other cells by their Raman signatures. We examine two deconvolution strategies: principal component analysis and classical least-squares. The deconvolution strategies are used to unmix the overall spectrum to determine the relative contributions between two SBT barcodes, where one SBT barcode indicates neuropilin-1 overexpression, while a second SBT barcode is more universal and indicates unspecific binding to a cell’s membrane. Highly reliable results were obtained for all of the cell mixture ratios tested, the lowest being 1 in 100 cells.  

4.1433           Metallothionein-I/II Promotes Axonal Regeneration in the Central Nervous System

Siddiq, M.M., Hannila, S.S., Carmel, J.B., Bryson, J.B., Hou, J., Nikulina, E., Willis, M.R., Mellado, W., Richman, E.L., Hilaire, M., hart, R.P. and Filbin, M.T.

1. Biol. Chem., 290(26), 16343-16356 (2015)

  The adult CNS does not spontaneously regenerate after injury, due in large part to myelin-associated inhibitors such as myelin-associated glycoprotein (MAG), Nogo-A, and oligodendrocyte-myelin glycoprotein. All three inhibitors can interact with either the Nogo receptor complex or paired immunoglobulin-like receptor B. A conditioning lesion of the sciatic nerve allows the central processes of dorsal root ganglion (DRG) neurons to spontaneously regenerate in vivo after a dorsal column lesion. After a conditioning lesion, DRG neurons are no longer inhibited by myelin, and this effect is cyclic AMP (cAMP)- and transcription-dependent. Using a microarray analysis, we identified several genes that are up-regulated both in adult DRGs after a conditioning lesion and in DRG neurons treated with cAMP analogues. One gene that was up-regulated under both conditions is metallothionein (MT)-I. We show here that treatment with two closely related isoforms of MT (MT-I/II) can overcome the inhibitory effects of both myelin and MAG for cortical, hippocampal, and DRG neurons. Intrathecal delivery of MT-I/II to adult DRGs also promotes neurite outgrowth in the presence of MAG. Adult DRGs from MT-I/II-deficient mice extend significantly shorter processes on MAG compared with wild-type DRG neurons, and regeneration of dorsal column axons does not occur after a conditioning lesion in MT-I/II-deficient mice. Furthermore, a single intravitreal injection of MT-I/II after optic nerve crush promotes axonal regeneration. Mechanistically, MT-I/II ability to overcome MAG-mediated inhibition is transcription-dependent, and MT-I/II can block the proteolytic activity of α-secretase and the activation of PKC and Rho in response to soluble MAG.  

4.1434           Regulation of neuronal high-voltage activated CaV2 Ca2+ channels by the small GTPase RhoA

Rousset, M., Cens, T., menard, C., Bowerman, M., Bellis, M., Bruses, J., Raoul, C., Scaamps, F. and Charnet, P. Neuropharmacol., 97, 201-209 (2015)   High-Voltage-Activated (HVA) Ca²⁺ channels are known regulators of synapse formation and transmission and play fundamental roles in neuronal pathophysiology. Small GTPases of Rho and RGK families, via their action on both cytoskeleton and Ca²⁺ channels are key molecules for these processes. While the effects of RGK GTPases on neuronal HVA Ca²⁺ channels have been widely studied, the effects of RhoA on the HVA channels remains however elusive. Using heterologous expression in Xenopus laevis oocytes, we show that RhoA activity reduces Ba²⁺ currents through Ca_V2.1, Ca_V2.2 and Ca_V2.3 Ca²⁺ channels independently of Ca_Vβ subunit. This inhibition occurs independently of RGKs activity and without modification of biophysical properties and global level of expression of the channel subunit. Instead, we observed a marked decrease in the number of active channels at the plasma membrane. Pharmacological and expression studies suggest that channel expression at the plasma membrane is impaired via a ROCK-sensitive pathway. Expression of constitutively active RhoA in primary culture of spinal motoneurons also drastically reduced HVA Ca²⁺ current amplitude. Altogether our data revealed that HVA Ca²⁺ channels regulation by RhoA might govern synaptic transmission during development and potentially contribute to pathophysiological processes when axon regeneration and growth cone kinetics are impaired.  

4.1435           Herpes Simplex Virus 1 Reactivates from Autonomic Ciliary Ganglia Independently from Sensory Trigeminal Ganglia To Cause Recurrent Ocular Disease

Lee, S., Ives, A.M. and Bertke, A.S.

1. Virol., 89(16), 8383-8391 (2015)

  Herpes simplex virus 1 (HSV-1) and HSV-2 establish latency in sensory and autonomic neurons after ocular or genital infection, but their recurrence patterns differ. HSV-1 reactivates from latency to cause recurrent orofacial disease, and while HSV-1 also causes genital lesions, HSV-2 recurs more efficiently in the genital region and rarely causes ocular disease. The mechanisms regulating these anatomical preferences are unclear. To determine whether differences in latent infection and reactivation in autonomic ganglia contribute to differences in HSV-1 and HSV-2 anatomical preferences for recurrent disease, we compared HSV-1 and HSV-2 clinical disease, acute and latent viral loads, and viral gene expression in sensory trigeminal and autonomic superior cervical and ciliary ganglia in a guinea pig ocular infection model. HSV-2 produced more severe acute disease, correlating with higher viral DNA loads in sensory and autonomic ganglia, as well as higher levels of thymidine kinase expression, a marker of productive infection, in autonomic ganglia. HSV-1 reactivated in ciliary ganglia, independently from trigeminal ganglia, to cause more frequent recurrent symptoms, while HSV-2 replicated simultaneously in autonomic and sensory ganglia to cause more persistent disease. While both HSV-1 and HSV-2 expressed the latency-associated transcript (LAT) in the trigeminal and superior cervical ganglia, only HSV-1 expressed LAT in ciliary ganglia, suggesting that HSV-2 is not reactivation competent or does not fully establish latency in ciliary ganglia. Thus, differences in replication and viral gene expression in autonomic ganglia may contribute to differences in HSV-1 and HSV-2 acute and recurrent clinical disease.  

4.1436           Imaging the immunological synapse between dendritic cells and T cells

Markey, K.A., Gartlan, K.H., Kuns, R.D., MacDonald, K.P.A. and Hill, G.R.

1. Immunol. Methods, 423, 40-44 (2015)

  Immunological synapse formation between antigen-specific T cells and antigen presenting cells (APC) involves reorganization of the cellular cytoskeleton (polymerization of filamentous actin) and recruitment of adhesion molecules (e.g. LFA-1, ICAM-1). This engagement is critical for the generation of specific immune responses. Until recently, quantitative, high-throughput measurements of these interactions have not been possible. Instead, previous assessment was reliant on qualitative microscopy of live cells, where typically the APC is adhered to a surface and the suspended T cell is required to migrate to facilitate synapse formation. While this methodology can demonstrate the capacity for synapse formation, it cannot accommodate quantification of large numbers of interacting cell pairs, nor does it allow for statistically robust comparison between test conditions. We have developed a method for assessing immunological synapse formation between purified ex vivo dendritic cells (DCs) and responder antigen-specific CD4⁺ T cells using imaging flow cytometry, allowing us to quantify LFA-1 and f-actin rearrangement at the interface between DC/T cell pairs. This novel application of imaging flow cytometry represents a major advance in dendritic cell function and immunological synapse research as it facilitates quantitative, high throughput analysis of the interaction between live, ex vivo DC and T cells.  

4.1437           Endolysosomal Deficits Augment Mitochondria Pathology in Spinal Motor Neurons of Asymptomatic fALS Mice

Xie, Y., Zhou, B., Lin, M-Y., Wang, S., Foust, K.D. and Sheng, Z-H. Neuron, 87, 355-370 (2015)   One pathological hallmark in ALS motor neurons (MNs) is axonal accumulation of damaged mitochondria. A fundamental question remains: does reduced degradation of those mitochondria by an impaired autophagy-lysosomal system contribute to mitochondrial pathology? We reveal MN-targeted progressive lysosomal deficits accompanied by impaired autophagic degradation beginning at asymptomatic stages in fALS-linked hSOD1^G93A mice. Lysosomal deficits result in accumulation of autophagic vacuoles engulfing damaged mitochondria along MN axons. Live imaging of spinal MNs from the adult disease mice demonstrates impaired dynein-driven retrograde transport of late endosomes (LEs). Expressing dynein-adaptor snapin reverses transport defects by competing with hSOD1^G93A for binding dynein, thus rescuing autophagy-lysosomal deficits, enhancing mitochondrial turnover, improving MN survival, and ameliorating the disease phenotype in hSOD1^G93A mice. Our study provides a new mechanistic link for hSOD1^G93A-mediated impairment of LE transport to autophagy-lysosomal deficits and mitochondrial pathology. Understanding these early pathological events benefits development of new therapeutic interventions for fALS-linked MN degeneration.  

4.1438           Type 1 innate lymphoid cells contribute to the pathogenesis of chronic hepatitis B

Yang, Z., Tang, T., Wei, X., Yang, S. and Tian, Z. Innate Immun., 21(6), 665-673 (2015)   Innate lymphoid cells (ILCs) function in producing effector cytokines in response to pathogenic infections. However, the roles and related mechanisms of the ILC subpopulations, ILC1 and ILC2, which mirror Th1 and Th2 in adaptive immunity, remain unclear. In this study, we found the markedly elevated levels of the ILC1 transcription factor T-bet, the effector cytokine IFN-γ and the IL/receptor signaling molecules IL-12/IL-12R, which are indispensable for ILC1 differentiation, in the helper ILCs of chronic hepatitis B (CHB) patients. The elevated level of the ILC1 population was significantly associated with hepatic damage in CHB patients, and was not related to telbivudine treatment. In contrast, although we also observed elevated levels of ILC2-related factors, including IL-33, ST2, GATA3 and IL-13 in helper ILCs, the extent of elevation shown by each was lower than that shown by the ILC1-related factors. Furthermore, the activity of the ILC2s did not correlate with either HBV copies or liver damage. The findings of this study suggest potential pro-inflammatory roles for ILC1s in CHB pathogenesis, potentiating these cells and their related molecules as targets of diagnostic, prognostic and/or therapeutic strategies for hepatitis B.  

4.1439           Liver myeloid-derived suppressor cells expand in response to liver metastases in mice and inhibit the anti-tumor efficacy of anti-CEA CAR-T

Burga, R.A., Thorn, M., Point, G.R., Guha, P., Nguyen, C.T., Licata, L.A., DeMatteo, R.P., ayala, A., Espat, N.J., Junghans, R.P. and Katz, S.C. Cancer Immunol. Immunother., 64, 817-829 (2015)   Chimeric antigen receptor-modified T cell (CAR-T) technology, a promising immunotherapeutic tool, has not been applied specifically to treat liver metastases (LM). While CAR-T delivery to LM can be optimized by regional intrahepatic infusion, we propose that liver CD11b+Gr-1+ myeloid-derived suppressor cells (L-MDSC) will inhibit the efficacy of CAR-T in the intrahepatic space. We studied anti-CEA CAR-T in a murine model of CEA+ LM and identified mechanisms through which L-MDSC expand and inhibit CAR-T function. We established CEA+ LM in mice and studied purified L-MDSC and responses to treatment with intrahepatic anti-CEA CAR-T infusions. L-MDSC expanded threefold in response to LM, and their expansion was dependent on GM-CSF, which was produced by tumor cells. L-MDSC utilized PD-L1 to suppress anti-tumor responses through engagement of PD-1 on CAR-T. GM-CSF, in cooperation with STAT3, promoted L-MDSC PD-L1 expression. CAR-T efficacy was rescued when mice received CAR-T in combination with MDSC depletion, GM-CSF neutralization to prevent MDSC expansion, or PD-L1 blockade. As L-MDSC suppressed anti-CEA CAR-T, infusion of anti-CEA CAR-T in tandem with agents targeting L-MDSC is a rational strategy for future clinical trials.  

4.1440           Genome-wide analysis of DNA methylation associated with HIV infection based on a pair of monozygotic twins

Zhang, Y., Li, S-K., Tsui, S.K-W. Genomics Data, 6, 12-15 82015)   Alteration of DNA methylation in mammalian cells could be elicited by many factors, including viral infections [1]. HIV has shown the ability to interact with host cellular factors to change the methylation status of some genes [2], [3] and [4]. However, the change of the DNA methylation associated with HIV infection based on the whole genome has not been well illustrated. In this study, a unique pair of monozygotic twins was recruited: one of the twins was infected with HIV without further anti-retroviral therapy while the other one was healthy, which could be considered as a relatively ideal model for profiling the alterations of DNA methylation associated with HIV infection. Therefore, using methylated DNA immunoprecipitation–microarray method (MeDIP–microarray), we found the increased DNA methylation level in peripheral blood mononuclear cells from HIV infected twin compared to her normal sibling. Moreover, several distinguished differential methylation regions (DMRs) in HIV infected twin worth further study. The raw data has been deposited in Gene Expression Omnibus (GEO) datasets with reference number GSE68028.  

4.1441           17β-Estradiol influences in vitro response of aged rat splenic conventional dendritic cells to TLR4 and TLR7/8 agonists in an agonist specific manner

Stojic-Vukanic, Z., Nacka-Aleksic, M., Bufan, b., Pilipovic, I., Arsenovic-Ranin, N., Djikic, J., Kosec, D. and Leposavic, G. Int. Immunopharmacol., 24, 24-35 (2015)   This study was undertaken considering that, despite the broad use of the unopposed estrogen replacement therapy in elderly women, data on estrogen influence on the functional capacity of dendritic cells (DCs), and consequently immune response are limited. We examined the influence of 17β-estradiol on phenotype, cytokine secretory profile, and allostimulatory and polarizing capacity of splenic (OX62+) conventional DCs from 26-month-old (aged) Albino Oxford rats matured in vitro in the presence of LPS, a TLR4 agonist, and R848, a TLR7/8 agonist. In the presence of 17β-estradiol, DCs from aged rats exhibited an impaired ability to mature upon stimulation with LPS, as shown by the lower surface density of MHC II and costimulatory CD80 and CD86 molecules. 17β-Estradiol alone enhanced CD40 expression in OX62+ DCs without affecting the expression of other costimulatory molecules, thereby confirming that the expression of this molecule is regulated independently from the regulation of other costimulatory molecules. However, although R848 upregulated the expression of MHC II and CD80 and CD40 costimulatory molecules on DCs, 17β-estradiol diminished the effect of this TLR agonist only on MHC II expression. In conjunction, the previous findings suggest that LPS and R848 elicit changes in the expression of costimulatory molecules via triggering differential intracellular signaling pathways. Furthermore, 17β-estradiol diminished the stimulatory influence of both LPS- and R848-matured OX62+ DCs on allogeneic CD4+ T lymphocyte proliferation in a mixed lymphocyte reaction (MLR). Moreover, as shown in MLR, the exposure to 17β-estradiol during LPS- and R848-induced maturation diminished Th1- and enhanced Th17-driving capacity and reduced Th1-driving capacity of OX62+ DCs, respectively. This suggests that LPS and R848 affect not only the surface phenotype, but also functional characteristics of OX62+ DCs triggering distinct intracellular signaling pathways. Collectively, the findings indicate that estrogen directly acting on OX62+ DCs, may affect CD4+ lymphocyte-dependent immune response in aged female rats.  

4.1442           Normal Human Lung Epithelial Cells Inhibit Transforming Growth Factor-β Induced Myofibroblast Differentiation via Prostaglandin E2

Epa, A.P:, Thatcher, T.H., Pollock, S.J., Wahl, L.A., Lyda, E., Kottmann, R.M., Phipps, R. and Sime, P.J. PloS One, 10(8), e0135266 (2015)   Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with very few effective treatments. The key effector cells in fibrosis are believed to be fibroblasts, which differentiate to a contractile myofibroblast phenotype with enhanced capacity to proliferate and produce extracellular matrix. The role of the lung epithelium in fibrosis is unclear. While there is evidence that the epithelium is disrupted in IPF, it is not known whether this is a cause or a result of the fibroblast pathology. We hypothesized that healthy epithelial cells are required to maintain normal lung homeostasis and can inhibit the activation and differentiation of lung fibroblasts to the myofibroblast phenotype. To investigate this hypothesis, we employed a novel co-culture model with primary human lung epithelial cells and fibroblasts to investigate whether epithelial cells inhibit myofibroblast differentiation. Measurements and Main Results In the presence of transforming growth factor (TGF)-β, fibroblasts co-cultured with epithelial cells expressed significantly less α-smooth muscle actin and collagen and showed marked reduction in cell migration, collagen gel contraction, and cell proliferation compared to fibroblasts grown without epithelial cells. Epithelial cells from non-matching tissue origins were capable of inhibiting TGF-β induced myofibroblast differentiation in lung, keloid and Graves’ orbital fibroblasts. TGF-β promoted production of prostaglandin (PG) E₂ in lung epithelial cells, and a PGE₂ neutralizing antibody blocked the protective effect of epithelial cell co-culture. Conclusions We provide the first direct experimental evidence that lung epithelial cells inhibit TGF-β induced myofibroblast differentiation and pro-fibrotic phenotypes in fibroblasts. This effect is not restricted by tissue origin, and is mediated, at least in part, by PGE₂. Our data support the hypothesis that the epithelium plays a crucial role in maintaining lung homeostasis, and that damaged and/ or dysfunctional epithelium contributes to the development of fibrosis.  

4.1443           Nlrp6 promotes recovery after peripheral nerve injury independently of inflammasomes

Ydens, E., Demon, D., Lornet, g., De Winter, V., Timmerman, V., Lamkanfi, M. and Janssens, S. Journal of Neuroinflammation, 12:143 (2015)   Background NOD-like receptors (Nlrs) are key regulators of immune responses during infection and autoimmunity. A subset of Nlrs assembles inflammasomes, molecular platforms that are activated in response to endogenous danger and microbial ligands and that control release of interleukin (IL)-1β and IL-18. However, their role in response to injury in the nervous system is less understood. Methods In this study, we investigated the expression profile of major inflammasome components in the peripheral nervous system (PNS) and explored the physiological role of different Nlrs upon acute nerve injury in mice. Results While in basal conditions, predominantly members of NOD-like receptor B (Nlrb) subfamily (NLR family, apoptosis inhibitory proteins (NAIPs)) and Nlrc subfamily (ICE-protease activating factor (IPAF)/NOD) are detected in the sciatic nerve, injury causes a shift towards expression of the Nlrp family. Sterile nerve injury also leads to an increase in expression of the Nlrb subfamily, while bacteria trigger expression of the Nlrc subfamily. Interestingly, loss of Nlrp6 led to strongly impaired nerve function upon nerve crush. Loss of the inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC) and effector caspase-1 and caspase-11 did not affect sciatic nerve function, suggesting that Nlrp6 contributed to recovery after peripheral nerve injury independently of inflammasomes. In line with this, we did not detect release of mature IL-1β upon acute nerve injury despite potent induction of pro-IL-1β and inflammasome components Nlrp3 and Nlrp1. However, Nlrp6 deficiency was associated with increased pro-inflammatory extracellular regulated MAP kinase (ERK) signaling, suggesting that hyperinflammation in the absence of Nlrp6 exacerbated peripheral nerve injury. Conclusions Together, our observations suggest that Nlrp6 contributes to recovery from peripheral nerve injury by dampening inflammatory responses independently of IL-1β and inflammasomes.  

4.1444           A novel multicolor immunostaining method using ethynyl deoxyuridine for analysis of in situ immunoproliferative response

Kitazawa, Y., Ueta, H., Hünig, T., Sawanobori, Y. and Matsuno, K. Histochem. Cell Biol., 144, 195-208 (2015)   Immune responses are generally accompanied by antigen presentation and proliferation and differentiation of antigen-specific lymphocytes (immunoproliferation), but analysis of these events in situ on tissue sections is very difficult. We have developed a new method of simultaneous multicolor immunofluorescence staining for immunohistology and flow cytometry using a thymidine analogue, 5-ethynyl-2′-deoxyuridine (EdU). Because of the small size of azide dye using click chemistry and elimination of DNA denaturation steps, EdU staining allowed for immunofluorescence staining of at least four colors including two different markers on a single-cell surface, which is impossible with the standard 5-bromo-2′-deoxyuridine method. By using two rat models, successfully detected parameters were the cluster of differentiation antigens including phenotypic and functional markers of various immune cells, histocompatibility complex antigens, and even some nuclear transcription factors. Proliferating cells could be further sorted and used for RT-PCR analysis. This method thus enables functional in situ time-kinetic analysis of immunoproliferative responses in a distinct domain of the lymphoid organs, which are quantitatively confirmed by flow cytometry.  

4.1445           Adverse effects of stromal vascular fraction during regenerative treatment of the intervertebral disc: observations in a goat model

Detiger, S.E.L., Helder, M.N., Smit, T.H. and Hoogendoorn, RS.J.W. Eur. Spine J., 24, 1992-2000 (2015)   Stromal vascular fraction (SVF), an adipose tissue-derived heterogeneous cell mixture containing, among others, multipotent adipose stromal cells (ASCs) and erythrocytes, has proved beneficial for a wide range of applications in regenerative medicine. We sought to establish intervertebral disc (IVD) regeneration by injecting SVF intradiscally during a one-step surgical procedure in an enzymatically (Chondroitinase ABC; cABC) induced goat model of disc degeneration. Unexpectedly, we observed a severe inflammatory response that has not been described before, including massive lymphocyte infiltration, neovascularisation and endplate destruction. A second study investigated two main suspects for these adverse effects: cABC and erythrocytes within SVF. The same destructive response was observed in healthy goat discs injected with SVF, thereby eliminating cABC as a cause. Density gradient removal of erythrocytes and ASCs purified by culturing did not lead to adverse effects. Following these observations, we incorporated an extra washing step in the SVF harvesting protocol. In a third study, we applied this protocol in a one-step procedure to a goat herniation model, in which no adverse responses were observed either. However, upon intradiscal injection of an identically processed SVF mixture into our goat IVD degeneration model during a fourth study, the adverse effects surprisingly occurred again. Despite our quest for the responsible agent, we eventually could not identify the mechanism through which the observed destructive responses occurred. Although we cannot exclude that the adverse effects are species-dependent or model-specific, we advertise caution with the clinical application of autologous SVF injections into the IVD until the responsible agent(s) are identified.  

4.1446           Bioengineering mini functional thymic units with EAK16-II/EAKIIH6 self-assembling hydrogel

Tajima, A., Liu, W., Pradhan, I., Bertera, S., bagia, C., Trucco, M., Meng, W.S. and Fan, Y. Clin. Immunol., 160, 82-89 (2015)   Herein, we highlight the technical feasibility of generating a functional mini thymus with a novel hydrogel system, based on a peptide-based self-assembly platform that can induce the formation of 3-D thymic epithelial cell (TEC) clusters. Amphiphilic peptide EAK16-II co-assembled with its histidinylated analogue EAKIIH6 into beta-sheet fibrils. When adaptor complexes (recombinant protein A/G molecules loaded with both anti-His and anti-EpCAM IgGs) were added to the mix, TECs were tethered to the hydrogel and formed 3-D mini clusters. TECs bound to the hydrogel composites retained their molecular properties; and when transplanted into athymic nude mice, they supported the development of functional T-cells. These mini thymic units of TECs can be useful in clinical applications to reconstitute T-cell adaptive immunity.  

4.1447           Recent Advances in Boar Sperm Cryopreservation: State of the Art and Current Perspectives

Yeste, M. Reprod. Dom. Anim., 50, Suppl. 2, 71-79 (2015)   While sperm cryopreservation is the best technology to store boar semen for long-term periods, only 1% of all artificial inseminations (AI) conducted worldwide are made using frozen–thawed boar sperm. With the emergence of long-term extenders for liquid storage, the use of cryopreserved sperm in routine AI is less required. However, banks of boar semen contain cryopreserved sperm and planning inseminations in AI centres may benefit from the use of frozen–thawed semen. Therefore, there is an interest in the use of this technology to preserve boar sperm. In this regard, although the first attempts to cryopreserve boar semen date back to the seventies and this technology is still considered as optimal, some relevant improvements have been made in the last decade. After giving a general picture about boar sperm cryodamage, the present review seeks to shed light on these recent cryopreservation advances. These contributions regard to protein markers for predicting ejaculate freezability, sperm selection prior to start cryopreservation procedures, additives to freezing and thawing extenders, relevance of the AI-technique and insemination-to-ovulation interval. In conclusion, most of these progresses have allowed counteracting better boar sperm cryodamage and are thus considered as forward steps for this storage method. It is also worth noting that, despite being lower than fresh/extended semen, reproductive performance outcomes following AI with frozen-thawed boar sperm are currently acceptable.  

4.1448           CD68 acts as a major gateway for malaria sporozoite liver infection

Cha, S-J., Park, K., Srinivasan, P., Schindler, C.W., van Rooijen, N., Stins, M. and Jacobs-Lorena, M.

1. Exp. Med., 212(9), 1391-1403 (2015)

  After being delivered by the bite from an infected mosquito, Plasmodium sporozoites enter the blood circulation and infect the liver. Previous evidence suggests that Kupffer cells, a macrophage-like component of the liver blood vessel lining, are traversed by sporozoites to initiate liver invasion. However, the molecular determinants of sporozoite–Kupffer cell interactions are unknown. Understanding the molecular basis for this specific recognition may lead to novel therapeutic strategies to control malaria. Using a phage display library screen, we identified a peptide, P39, that strongly binds to the Kupffer cell surface and, importantly, inhibits sporozoite Kupffer cell entry. Furthermore, we determined that P39 binds to CD68, a putative receptor for sporozoite invasion of Kupffer cells that acts as a gateway for malaria infection of the liver.  

4.1449           Selective Expression of the MAPK Phosphatase Dusp9/MKP-4 in Mouse Plasmacytoid Dendritic Cells and Regulation of IFN-β Production

Niedzielska, M. et al

1. Immunol., 195(4), 1753-1762 (2015)

  Plasmacytoid dendritic cells (pDCs) efficiently produce large amounts of type I IFN in response to TLR7 and TLR9 ligands, whereas conventional DCs (cDCs) predominantly secrete high levels of the cytokines IL-10 and IL-12. The molecular basis underlying this distinct phenotype is not well understood. In this study, we identified the MAPK phosphatase Dusp9/MKP-4 by transcriptome analysis as selectively expressed in pDCs, but not cDCs. We confirmed the constitutive expression of Dusp9 at the protein level in pDCs generated in vitro by culture with Flt3 ligand and ex vivo in sorted splenic pDCs. Dusp9 expression was low in B220⁻ bone marrow precursors and was upregulated during pDC differentiation, concomitant with established pDC markers. Higher expression of Dusp9 in pDCs correlated with impaired phosphorylation of the MAPK ERK1/2 upon TLR9 stimulation. Notably, Dusp9 was not expressed at detectable levels in human pDCs, although these displayed similarly impaired activation of ERK1/2 MAPK compared with cDCs. Enforced retroviral expression of Dusp9 in mouse GM-CSF–induced cDCs increased the expression of TLR9-induced IL-12p40 and IFN-β, but not of IL-10. Conditional deletion of Dusp9 in pDCs was effectively achieved in Dusp9^flox/flox; CD11c-Cre mice at the mRNA and protein levels. However, the lack of Dusp9 in pDC did not restore ERK1/2 activation after TLR9 stimulation and only weakly affected IFN-β and IL-12p40 production. Taken together, our results suggest that expression of Dusp9 is sufficient to impair ERK1/2 activation and enhance IFN-β expression. However, despite selective expression in pDCs, Dusp9 is not essential for high-level IFN-β production by these cells.  

4.1450           The Effect of Progestins on Tumor Necrosis Factor α-Induced Matrix Metalloproteinase-9 Activity and Gene Expression in Human Primary Amnion and Chorion Cells In Vitro

Allen, T.K., Feng, L., Nazzal, M., Grotegut, C.A., Buhimschi, I.A. and Murtha, A.P. Anesth. Analg., 120(5), 1085-1094 (2015)   BACKGROUND: Current treatment modalities for preventing preterm premature rupture of membranes are limited, but progestins may play a role. Tumor necrosis factor α (TNFα) enhances matrix metalloproteinase-9 (MMP-9) gene expression and activity in fetal membranes, contributing to membrane weakening and rupture. We previously demonstrated that progestins attenuate TNFα-induced MMP-9 activity in a cytotrophoblast cell line. However, whether they have a similar effect in primary amnion and chorion cells of fetal membranes is unknown. In this study, we evaluated the effect of progestins on basal and TNFα-induced MMP-9 activity and gene expression in primary chorion and amnion cells harvested from the fetal membranes of term nonlaboring patients. METHODS: Primary amnion and chorion cells were isolated from fetal membranes obtained from term uncomplicated nonlaboring patients following elective cesarean delivery (n = 11). Confluent primary amnion and chorion cell cultures were both pretreated with vehicle (control), progesterone (P4), 17α-hydroxyprogesterone caproate (17P), or medroxyprogesterone acetate (MPA) at 10^–6 M concentration for 6 hours followed by stimulation with TNFα at 10 ng/mL for an additional 24 hours. Cell cultures pretreated with the vehicle only served as the unstimulated control and the vehicle stimulated with TNFα served as the stimulated control. Both controls were assigned a value of 100 units. Cell culture medium was harvested for MMP-9 enzymatic activity quantification using gelatin zymography. Total RNA was extracted for quantifying MMP-9 gene expression using real-time quantitative PCR. Basal MMP-9 activity and gene expression data were normalized to the unstimulated control. TNFα-stimulated MMP-9 activity and gene expression were normalized to the stimulated control. The primary outcome was the effect of progestins on TNFα-induced MMP-9 enzymatic activity in term human primary amnion and chorion cells in vitro. Secondary outcomes included the effect of progestin therapy on TNFα-induced MMP-9 gene expression and on basal MMP-9 activity and gene expression in primary amnion and chorion cells in vitro. RESULTS: Primary cells were harvested from 11 patients. Compared with the unstimulated control, TNFα increased MMP-9 activity (P = 0.005 versus control in primary amnion cells and P < 0.001 versus control in primary chorion cells) and MMP-9 gene expression (P = 0.030 versus control in primary amnion cells, P < 0.001 versus control in primary chorion cells). Compared with the unstimulated controls, MPA, but not P4 or 17P, reduced basal MMP-9 activity [mean difference (95% CI) −49.6 (−81.9, −17.3) units, P = 0.001] and gene expression [mean difference (95% CI) −53.4 (−105.9, −0.9) units, P = 0.045] in primary amnion cells. Compared with the stimulated control, MPA also reduced TNFα-induced MMP-9 activity [mean difference (95% CI) −69.0 (−91.8, −46.3) units, P < 0.001] and gene expression [mean difference (95% CI) −86.0 (−120.7, −51.3) units, P < 0.001] in primary amnion cells. Progestin pretreatment had no significant effect on basal or TNFα-induced MMP-9 activity and gene expression in primary chorion cells. CONCLUSIONS: The inhibitory effect of MPA on both basal and TNFα-induced MMP-9 activity and gene expression in primary amnion cells demonstrate a possible mechanism by which progestins may prevent fetal membrane weakening leading to preterm premature rupture of membranes.  

4.1451           Axon stretch growth of adult primary motor neurons

Brinn, M., Zhao, S., Kumuratilake, J., Lu, T-F., Freeman, B., Al-Sarawi, S. and Henneberg, M.

1. Neurochem., Suppl. 1, 333 (2015)

  Growth of embryonic dorsal root ganglion (DRG) axons has been markedly enhanced by controlled in vitro stretching without the involvement of the growth cone. However limited work has been done on enhancement of axonal growth on adult motor neurons. The spinal cord was harvested from adult Sprague–Dawley rats (8– 12 weeks) by bilateral neural arch dissection, demyelinated and separated neurons from the homogenized spinal cord using Papain (36 U/mL in 6 mLs processing media) digestion and trituration. Motor neurons were isolated from the homogenate using an established 4 step Optiprep density gradient centrifugation and plated onto poly-D-lysine (100 mg/mL) coated glass and aclar substrate (at a cell density of 320–500 cells/mm3) within the axon stretch bioreactor. Cells were grown in the bioreactor using an optimized neuron culture media. The bioreactor consisted of three sections, which were assembled, autoclaved, plated with neurons and sealed. Nerve cells were grown under controlled temperature (37°C) and 5% CO2 atmosphere for 8 days prior to commencement of stretching of the axons. The axons were stretched using a motor driven device controlled by PIMikro- Move software, where the axons were subjected to stretching, resting and stretching in sequence at an incremental rate commencing at 0.5 mm per day in 2 lm increments with 500 ms resting between stretching. Stretching of axons was continued for up to 13 days. The amount of stretch was monitored using a microcamera mounted onto the objective of an inverted microscope. Cultured neurons typically developed axons and dendrites by 4 days and remained viable in excess of 21 days. Cells were identified as motor neurons using HB9, Islet-1 and Neurofilament-M antibodies. Early results indicate feasibility in using this tailored adult primary motor neuron protocol for axon stretch growth in vitro experiments.  

4.1452           Myelin-associated glycoprotein modulates apoptosis of motoneurons during early postnatal development via NgR/p75NTR receptor-mediated activation of RhoA signaling pathways

Palandri, A., Salvador, V.R., Wojnacki, J., Vivinetto, A.L., Schnaar, R.L. and Lopez, P.H.H. Cell Death and Disease, 6, e1876 (2015)   Myelin-associated glycoprotein (MAG) is a minor constituent of nervous system myelin, selectively expressed on the periaxonal myelin wrap. By engaging multiple axonal receptors, including Nogo-receptors (NgRs), MAG exerts a nurturing and protective effect the axons it ensheaths. Pharmacological activation of NgRs has a modulatory role on p75^NTR-dependent postnatal apoptosis of motoneurons (MNs). However, it is not clear whether this reflects a physiological role of NgRs in MN development. NgRs are part of a multimeric receptor complex, which includes p75^NTR, Lingo-1 and gangliosides. Upon ligand binding, this multimeric complex activates RhoA/ROCK signaling in a p75^NTR-dependent manner. The aim of this study was to analyze a possible modulatory role of MAG on MN apoptosis during postnatal development. A time course study showed that Mag-null mice suffer a loss of MNs during the first postnatal week. Also, these mice exhibited increased susceptibility in an animal model of p75^NTR-dependent MN apoptosis induced by nerve-crush injury, which was prevented by treatment with a soluble form of MAG (MAG-Fc). The protective role of MAG was confirmed in in vitro models of p75^NTR-dependent MN apoptosis using the MN1 cell line and primary cultures. Lentiviral expression of shRNA sequences targeting NgRs on these cells abolished protection by MAG-Fc. Analysis of RhoA activity using a FRET-based RhoA biosensor showed that MAG-Fc activates RhoA. Pharmacological inhibition of p75^NTR/RhoA/ROCK pathway, or overexpression of a p75^NTR mutant unable to activate RhoA, completely blocked MAG-Fc protection against apoptosis. The role of RhoA/ROCK signaling was further confirmed in the nerve-crush model, where pretreatment with ROCK inhibitor Y-27632 blocked the pro-survival effect of MAG-Fc. These findings identify a new protective role of MAG as a modulator of apoptosis of MNs during postnatal development by a mechanism involving the p75^NTR/RhoA/ROCK signaling pathway. Also, our results highlight the relevance of the nurture/protective effects of myelin on neurons.  

4.1453           Inhibition of pancreatic stellate cell activity by adipose-derived stem cells

Yu, F-X., Su, L-F., Dai, C-L., Wang, DY., teng, Y-Y., Fu, J-H., Zhang, Q-Y. and Tang, Y-H. Hepatobiliary Pancreat. Dis. Inst., 14(2), 215-221 (2015)   Background Pancreatic stellate cells (PSCs) play a critical role in the development of pancreatic fibrosis. In this study we used a novel method to isolate and culture rat PSCs and then investigated the inhibitory effects of adipose-derived stem cells (ADSCs) on activation and proliferation of PSCs. Methods Pancreatic tissue was obtained from Sprague-Dawley rats for PSCs isolation. Transwell cell cultures were adopted for co-culture of ADSCs and PSCs. PSCs proliferation and apoptosis were determined using CCK-8 and flow cytometry, respectively. α-SMA expressions were analyzed using Western blotting. The levels of cytokines [nerve growth factor (NGF), interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1)] in conditioned medium were detected by ELISA. Gene expression (MMP-2, MMP-9 and TIMP-1) was analyzed using qRT-PCR. Results This method produced 17.6±6.5×10³ cells per gram of the body weight with a purity of 90%–95% and a viability of 92%–97%. Co-culture of PSCs with ADSCs significantly inhibited PSCs proliferation and induced PSCs apoptosis. Moreover, α-SMA expression was significantly reduced in PSCs+ADSCs compared with that in PSC-only cultures, while expression of fibrinolytic proteins (e.g., MMP-2 and MMP-9) was up-regulated and anti-fibrinolytic protein (TIMP-1) was down-regulated. In addition, NGF expression was up-regulated, but IL-10 and TGF-β1 expressions were down-regulated in the co-culture conditioned medium compared with those in the PSC-only culture medium. Conclusions This study provided an easy and reliable technique to isolate PSCs. The data demonstrated the inhibitory effects of ADSCs on the activation and proliferation of PSCs in vitro.  

4.1454           Spinal Cord Ischemia-Reperfusion Injury Induces Erythropoietin Receptor Expression

Foley, L.S., Fullerton, D.A., Bennett, D.T., Freeman, K.A., Mares, J., Bell, M.T., Cleveland, J.C., Weyant, M.J., Meng, X., Puskas, F. and Reece, TS.B. Ann. Thorac. Surg., 100, 41-46 (2015)   Background Paraplegia remains a devastating complication of aortic surgery, occurring in up to 20% of complex thoracoabdominal repairs. Erythropoietin (EPO) attenuates this injury in models of spinal cord ischemia. Upregulation of the beta-common receptor (βcR) subunit of the EPO receptor is associated with reduced damage in murine models of neural injury. This receptor activates anti-apoptotic pathways including signaling transducer and activator of transcription 3 (STAT3). We hypothesized that spinal cord ischemia-reperfusion injury upregulates the βcR subunit with a subsequent increase in activated STAT3. Methods Adult male C57/BL6 mice received an intraperitoneal injection of 0.5 mL of EPO (10 U/kg) or 0.9% saline after induction of anesthesia. Spinal cord ischemia was induced through sternotomy and 4-minute thoracic aortic cross-clamp. Sham mice underwent sternotomy without cross-clamp placement. Four groups were studied: ischemic and sham groups, each with and without EPO treatment. After 4 hours of reperfusion, spinal cords were harvested and homogenized. The βcR subunit expression and STAT3 activation were evaluated by immunoblot. Results Ischemia reperfusion increased βcR subunit expression in spinal cords of ischemia + saline and ischemia + EPO mice compared with shams (3.4 ± 1.39 vs 1.31 ± 0.3, p = 0.01 and 3.80 ± 0.58 vs 1.56 ± 0.32, p = 0.01). Additionally, both ischemic groups demonstrated increased STAT3 activation compared with shams (1.35 ± 0.14 vs 1.09 ± 0.07, p = 0.01 and 1.66 ± 0.35 vs 1.08 ± 0.17, p = 0.02). Conclusions Ischemia-reperfusion injury induces EPO receptor βcR subunit expression and early downstream anti-apoptotic signaling through STAT3 activation. Further investigation into the role of the βcR subunit is warranted to determine tissue protective functions of EPO. Elucidation of mechanisms involved in spinal cord protection is essential for reducing delayed paraplegia. Delayed paraplegia resulting from spinal cord ischemia-reperfusion injury remains a devastating complication of complex aortic operations, occurring in up to 20% of thoracoabdominal repairs 1 and 2. Advances in operative technique, use of hypothermia and cerebrospinal fluid drainage have spared some patients from this unfortunate outcome over the last decade; however, these innovations have led to only minor reductions in its overall incidence [3]. Furthermore, there is currently no widely accepted pharmacologic treatment available that is proven to reduce this injury. Neurologic damage after ischemia reperfusion occurs on a spectrum ranging from dense immediate postoperative paraplegia to delayed onset functional deterioration. This bimodal pattern of injury is due to an initial ischemic insult at the time of surgery followed by an inflammatory response during reperfusion, which accelerates neuronal loss and spinal cord dysfunction 4 and 5. Tissue hypoxia and metabolic stress activate the innate-adaptive immune response, characterized by proinflammatory cytokines, which amplify the injury, activate cell death pathways, and ultimately increase damage beyond the initial lesion [6]. Curtailing this pathophysiologic response has proven promising in translational brain and spinal cord injury research [7]. Erythropoietin (EPO) has emerged as a potential therapeutic tool, with anti-inflammatory and neuroprotective effects in murine models of ischemic stroke and contusive spinal cord injury [8]. Local cerebral production of EPO and its ability to cross the blood-brain barrier are important characteristics that highlight its potential as an endogenous neuroprotective cytokine. Erythropoietin has been shown to improve functional outcomes and reduce neuronal loss in multiple animal models of ischemic injury, including in our own laboratory’s murine model of spinal cord ischemia-reperfusion injury using a thoracic aortic cross-clamp 9 and 10. The gross functional benefits of EPO treatment have been described; however, its mechanisms remain unclear. Anti-inflammatory properties and activation of anti-apoptotic pathways, including that of signal transducer and activator of transcription 3 (STAT3) and B-cell lymphoma 2 protein (BCL-2, an inhibitor of cell death) are thought to mediate these effects. Recent investigations into tissue-protective properties of EPO have led to the identification of a unique receptor that specifically mediates these effects, distinct from the EPO receptor that mediates hematopoiesis [11]. This tissue-protective receptor is present in various organs including the brain, heart, and kidney, and is identifiable by a beta-common subunit (βcR or CD131) domain. The βcR subunit expression is characteristically low at baseline, and significantly induced by hypoxia and metabolic stress resulting in improved functional outcomes with EPO administration [12]. Expression of this receptor on injured cells precedes a local increase in tissue production of EPO temporally [13]. The tissue production of EPO lagging behind receptor expression results briefly in a relative deficit of EPO and creates a window of opportunity for therapeutic intervention, as seen in Figure 1[14].  

4.1455           The pro-fibrotic and anti-inflammatory foam cell macrophage paradox

Thomas, A.C., Eijgelaar, W.J., daemen, M.J.A.P. and Newby, A.C. Genomics Data, 6, 136-138 (2015)   The formation of foamy macrophages by sequestering extracellular modified lipids is a key event in atherosclerosis. However, there is controversy about the effects of lipid loading on macrophage phenotype, with in vitro evidence suggesting either pro- or anti-inflammatory consequences. To investigate this in vivo we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in experimental subcutaneous granulomas in fat-fed ApoE null mice or normal chow-fed wild-type mice, respectively. Consistent with previous studies in peritoneal macrophages from LDL receptor null mice (Spann et al., 2012 [1]), we found that anti-inflammatory LXR/RXR pathway genes were over-represented in the foamy macrophages, but there was no change in M1 or M2 phenotypic markers. Quite unexpectedly, however, we found that genes related to the induction of fibrosis had also been up-regulated (Thomas et al., 2015 [2]). The progression of the foamy macrophages along anti-inflammatory and pro-fibrotic pathways was confirmed using immunohistochemistry (described fully in our primary research article (Thomas et al., 2015 [2]). Here we provide additional details on production of the macrophages and their transcriptomic comparison, with the raw and processed microarray data deposited in GEO (accession number GSE70126). Our observations on these cells are indeed paradoxical, because foamy macrophages have long been implicated in promoting inflammation, extracellular matrix degradation and atherosclerotic plaque rupture, which must be provoked by additional local mediators. Our findings probably explain how very early macrophage-rich lesions maintain their structural integrity.  

4.1456           MicroRNA125b-mediated Hedgehog signaling influences liver regeneration by chorionic plate-derived mesenchymal stem cells

Hyun, J., Wang, S., Kim, J., Kim, G.J. and Jung, Y. Scientific Reports, 5:14135 (2015)   Although chorionic plate-derived mesenchymal stem cells (CP-MSCs) were shown to promote liver regeneration, the mechanisms underlying the effect remain unclear. Hedgehog (Hh) signaling orchestrates tissue reconstruction in damaged liver. MSCs release microRNAs mediating various cellular responses. Hence, we hypothesized that microRNAs from CP-MSCs regulated Hh signaling, which influenced liver regeneration. Livers were obtained from carbon tetrachloride (CCl4)-treated rats transplanted with human CP-MSCs (Tx) or saline (non-Tx). Sonic Hh, one of Hh ligands, increased in CCl4-treated liver, whereas it decreased in CP-MSC-treated liver with CCl4. The expression of Hh-target genes was significantly downregulated in the Tx. Reduced expansion of progenitors and regressed fibrosis were observed in the liver of the Tx rats. CP-MSCs suppressed the expression of Hh and profibrotic genes in co-cultured LX2 (human hepatic stellate cell) with CP-MSCs. MicroRNA-125b targeting smo was retained in exosomes of CP-MSCs. CP-MSCs with microRNA-125b inhibitor failed to attenuate the expression of Hh signaling and profibrotic genes in the activated HSCs. Therefore, these results demonstrated that microRNA-125b from CP-MSCs suppressed the activation of Hh signaling, which promoted the reduced fibrosis, suggesting that microRNA-mediated regulation of Hh signaling contributed to liver regeneration by CP-MSCs.  

4.1457           Inhibition of NOX2 reduces locomotor impairment, inflammation, and oxidative stress after spinal cord injury

Khayrullina, G., Bermudez, S and Byrnes, K.R.

1. Neuroinflammation, 12:172 (2015)

  Background Spinal cord injury (SCI) results in the activation of the NADPH oxidase (NOX) enzyme, inducing production of reactive oxygen species (ROS). We hypothesized that the NOX2 isoform plays an integral role in post-SCI inflammation and functional deficits. Methods Moderate spinal cord contusion injury was performed in adult male mice, and flow cytometry, western blot, and immunohistochemistry were used to assess NOX2 activity and expression, inflammation, and M1/M2 microglia/macrophage polarization from 1 to 28 days after injury. The NOX2-specific inhibitor, gp91ds-tat, was injected into the intrathecal space immediately after impact. The Basso Mouse Scale (BMS) was used to assess locomotor function at 24 h post-injury and weekly thereafter. Results Our findings show that gp91ds-tat treatment significantly improved functional recovery through 28 days post-injury and reduced inflammatory cell concentrations in the injured spinal cord at 24 h and 7 days post-injury. In addition, a number of oxidative stress markers were reduced in expression at 24 h after gp91ds-tat treatment, which was accompanied by a reduction in M1 polarization marker expression. Conclusion Based on our findings, we now conclude that inhibition of NOX2 significantly improves outcome after SCI, most likely via acute reductions in oxidative stress and inflammation. NOX2 inhibition may therefore have true potential as a therapy after SCI.  

4.1458           All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells

Werner, M., Driftmann, S., Kleinehr, K., Kaiser, G.M., mathe, Z., treckmann, J-w., paul, A., Sibbe, K., Timm, J., Canbay, A., gerken, G., Schlaak, J.F. and Boering, R. PloS One, 10(9), e0138655 (2015)   Background & Aims Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen. Methods Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity. Results Cell preparation yielded the following cell counts per gram of liver tissue: 2.0±0.4×10⁷ hepatocytes, 1.8±0.5×10⁶ Kupffer cells, 4.3±1.9×10⁵ liver sinusoidal endothelial cells, and 3.2±0.5×10⁵ stellate cells. Hepatocytes were identified by albumin (95.5±1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5±1.2%) and exhibited phagocytic activity, as determined with 1μm latex beads. Endothelial cells were CD146⁺ (97.8±1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1±1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence. Conclusions Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.  

4.1459           Single human sperm cryopreservation method using hollow-core agarose capsules

Araki, Y., Yao, T., Asayama, Y., matsuhisa, A. and Araki, Y. Fertility and Sterility, 104(4), 1004-1009 (2015)   Objective: To develop an efficient cryopreservation method using a single sperm. Design: Experimental study. Setting: Laboratory of a private institute. Patient(s): A fertile donor. Intervention(s):We produced hollow-core capsules with agarose walls. A single human sperm was injected into each capsule as per the conventional intracytoplasmic sperm injection (ICSI) method. The capsules that contained the spermatozoa were cryopreserved on polycarbonate or nylon mesh sheets using nitrogen vapor. Before their use, the capsules were thawed and recovered. The motile spermatozoa in the capsules were counted. Main Outcome Measure(s) The recovery rates of the agarose capsules and the spermatozoa in these capsules after thawing and the mortality and survival rates of the spermatozoa. Result(s) The recovery rates of the capsules were 91.5% (75/82) using polycarbonate sheets (PS) and 98.3% (59/60) using mesh sheets (MS) after thawing. The recovered capsules were not at all damaged. The recovery rates of the spermatozoa were 91.5% (75/82) using PS and 96.7% (58/60) using MS. Sperm motility rates were 85.3% (64/75) and 82.8% (48/58), whereas the survival rates of the immotile spermatozoa by the hypoosmotic swelling test were 81.8% (9/11) and 50.0% (5/10); furthermore, the total survival rates of the spermatozoa were 97.3% (73/75) and 91.4% (53/58) using PS and MS, respectively. There was no significant difference between the results obtained using PS and MS. Conclusion(s) A cryopreservation method for a single sperm using an agarose capsule has been developed. The method is expected to be useful in ICSI treatment in patients with few spermatozoa.  

4.1460           Pilot Study Evaluating Regulatory T Cell–Promoting Immunosuppression and Nonimmunogenic Donor Antigen Delivery in a Nonhuman Primate Islet Allotransplantation Model

Lei, J. et al Am. J. Transplant., 15(10), 2739-2749 (2015)   The full potential of islet transplantation will only be realized through the development of tolerogenic regimens that obviate the need for maintenance immunosuppression. Here, we report an immunotherapy regimen that combines 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide (ECDI)-treated donor lymphoid cell infusion (ECDI-DLI) with thymoglobulin, anti-interleukin-6 receptor antibody and rapamycin to achieve prolonged allogeneic islet graft survival in a nonhuman primate (NHP) model. Prolonged graft survival is associated with Treg expansion, donor-specific T cell hyporesponsiveness and a transient absence of donor-specific alloantibody production during the period of graft survival. This regimen shows promise for clinical translation.  

4.1461           Acute GVHD results in a severe DC defect that prevents T-cell priming and leads to fulminant cytomegalovirus disease in mice

Wikstrom, M., Fleming, P., Kuns, R.D., Schuster, I.S., Voigt, V., Miller, G., Clouston, A.D., Tey, S-K., Andoniou, C.E., Hill, G.R. and Degli-Esposti, M.A. Blood, 126(12), 1503-1514 (2015)   Viral infection is a common, life-threatening complication after allogeneic bone marrow transplantation (BMT), particularly in the presence of graft-versus-host disease (GVHD). Using cytomegalovirus (CMV) as the prototypic pathogen, we have delineated the mechanisms responsible for the inability to mount protective antiviral responses in this setting. Although CMV infection was self-limiting after syngeneic BMT, in the presence of GVHD after allogeneic BMT, CMV induced a striking cytopathy resulting in universal mortality in conjunction with a fulminant necrotizing hepatitis. Critically, GVHD induced a profound dendritic cell (DC) defect that led to a failure in the generation of CMV-specific CD8⁺ T-cell responses. This was accompanied by a defect in antiviral CD8⁺ T cells. In combination, these defects dramatically limited antiviral T-cell responses. The transfer of virus-specific cells circumvented the DC defects and provided protective immunity, despite concurrent GVHD. These data demonstrate the importance of avoiding GVHD when reconstructing antiviral immunity after BMT, and highlight the mechanisms by which the adoptive transfer of virus-specific T cells overcome the endogenous defects in priming invoked by GVHD.  

4.1462           Density-gradient centrifugation enables the purification of cultured corneal endothelial cells for cell therapy by eliminating senescent cells

Okumura, N., Kusakabe, A., Hirano, H., Inoue, R., Okazaki, Y., nakano, S., Kinoshita, S. and Koizumi, N. Scientific Reports, 5:15005 (2015)   The corneal endothelium is essential for maintaining corneal transparency; therefore, corneal endothelial dysfunction causes serious vision loss. Tissue engineering-based therapy is potentially a less invasive and more effective therapeutic modality. We recently started a first-in-man clinical trial of cell-based therapy for treating corneal endothelial dysfunction in Japan. However, the senescence of corneal endothelial cells (CECs) during the serial passage culture needed to obtain massive quantities of cells for clinical use is a serious technical obstacle preventing the push of this regenerative therapy to clinical settings. Here, we show evidence from an animal model confirming that senescent cells are less effective in cell therapy. In addition, we propose that density-gradient centrifugation can eliminate the senescent cells and purify high potency CECs for clinical use. This simple technique might be applicable for other types of cells in the settings of regenerative medicine.  

4.1463           Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

Gilbert, G.E., Novakovic, V.A., Shi, J., Rasmussen, J. and Pipe, S.W.

Blood, 126(10), 1237-1244 (2015)

Thrombin-stimulated platelets expose very little phosphatidylserine (PS) but express binding sites for factor VIII (fVIII), casting doubt on the role of exposed PS as the determinant of binding sites. We previously reported that fVIII binding sites are increased three- to sixfold when soluble fibrin (SF) binds the αIIbβ3 integrin. This study focuses on the hypothesis that platelet-bound SF is the major source of fVIII binding sites. Less than 10% of fVIII was displaced from thrombin-stimulated platelets by lactadherin, a PS-binding protein, and an fVIII mutant defective in PS-dependent binding retained platelet affinity. Therefore, PS is not the determinant of most binding sites. FVIII bound immobilized SF and paralleled platelet binding in affinity, dependence on separation from von Willebrand factor, and mediation by the C2 domain. SF also enhanced activity of fVIII in the factor Xase complex by two- to fourfold. Monoclonal antibody (mAb) ESH8, against the fVIII C2 domain, inhibited binding of fVIII to SF and platelets but not to PS-containing vesicles. Similarly, mAb ESH4 against the C2 domain, inhibited >90% of platelet-dependent fVIII activity vs 35% of vesicle-supported activity. These results imply that platelet-bound SF is a component of functional fVIII binding sites.

       

4.1464           Moderate- and high-intensity exhaustive exercise in the heat induce a similar increase in monocyte Hsp72

Periard, J.D., Ruell, P.A., Thompson, M.W. and Caillaud, C. Cell Stress and Chaperones, 20(6), 1037-1042 (2015)   This study examined the relationship between exhaustive exercise in the heat at moderate and high intensities on the intracellular heat shock protein 72 (iHsp72) response. Twelve male subjects cycled to exhaustion at 60 and 75 % of maximal oxygen uptake in hot conditions (40 °C, 50 % RH). iHsp72 concentration was measured in monocytes before, at exhaustion and 24 h after exercise. Rectal temperature, heart rate and oxygen uptake were recorded during exercise. Volitional exhaustion occurred at 58.9 ± 12.1 and 27.3 ± 9.5 min (P < 0.001) and a rectal temperature of 39.8 ± 0.4 and 39.2 ± 0.6 °C (P = 0.002), respectively, for 60 and 75 %. The area under the curve above a rectal temperature of 38.5 °C was greater at 60 % (17.5 ± 6.6 °C min) than 75 % (3.4 ± 4.8 °C min; P < 0.001), whereas the rate of increase in rectal temperature was greater at 75 % (5.1 ± 1.7 vs. 2.2 ± 1.4 °C h⁻¹; P < 0.001). iHsp72 concentration increased similarly at exhaustion relative to pre-exercise (P = 0.044) and then increased further at 24 h (P < 0.001). Multiple regression analysis revealed no predictor variables associated with iHsp72 expression; however, a correlation was observed between exercise intensities for the increase in iHsp expression at exhaustion and 24 h (P < 0.05). These results suggest that iHsp72 expression increased in relation to the level of hyperthermia attained and sustained at 60 % and the higher metabolic rate and greater rate of increase in core temperature at 75 %, with the further increase in iHsp72 concentration 24 h after exercise reinforcing its role as a chaperone and cytoprotective agent.  

4.1465           Increased Sensitivity to Binge Alcohol-Induced Gut Leakiness and Inflammatory Liver Disease in HIV Transgenic Rats

Banerjee, A., Abdelmegeed, M.A., jang, S. And Song, B-J. PloS One, 10(10), e0140498 (2015)   The mechanisms of alcohol-mediated advanced liver injury in HIV-infected individuals are poorly understood. Thus, this study was aimed to investigate the effect of binge alcohol on the inflammatory liver disease in HIV transgenic rats as a model for simulating human conditions. Female wild-type (WT) or HIV transgenic rats were treated with three consecutive doses of binge ethanol (EtOH) (3.5 g/kg/dose oral gavages at 12-h intervals) or dextrose (Control). Blood and liver tissues were collected at 1 or 6-h following the last dose of ethanol or dextrose for the measurements of serum endotoxin and liver pathology, respectively. Compared to the WT, the HIV rats showed increased sensitivity to alcohol-mediated gut leakiness, hepatic steatosis and inflammation, as evidenced with the significantly elevated levels of serum endotoxin, hepatic triglycerides, histological fat accumulation and F4/80 staining. Real-time PCR analysis revealed that hepatic levels of toll-like receptor-4 (TLR4), leptin and the downstream target monocyte chemoattractant protein-1 (MCP-1) were significantly up-regulated in the HIV-EtOH rats, compared to all other groups. Subsequent experiments with primary cultured cells showed that both hepatocytes and hepatic Kupffer cells were the sources of the elevated MCP-1 in HIV-EtOH rats. Further, TLR4 and MCP-1 were found to be upregulated by leptin. Collectively, these results show that HIV rats, similar to HIV-infected people being treated with the highly active anti-retroviral therapy (HAART), are more susceptible to binge alcohol-induced gut leakiness and inflammatory liver disease than the corresponding WT, possibly due to additive or synergistic interaction between binge alcohol exposure and HIV infection. Based on these results, HIV transgenic rats can be used as a surrogate model to study the molecular mechanisms of many disease states caused by heavy alcohol intake in HIV-infected people on HAART.  

4.1466           Gene expression of peripheral blood mononuclear cells is affected by cold exposure

Reynes, B., Garcia-Ruiz, E., Oliver, P. and Palou, A. Am. J. Physiol. Regul. Comp. Physiol., 309, R824-R834 (2015)   Because of the discovery of brown adipose tissue (BAT) in humans, there is increased interest in the study of induction of this thermogenic tissue as a basis to combat obesity and related complications. Cold exposure is one of the strongest stimuli able to activate BAT and to induce the appearance of brown-like (brite) adipocytes in white fat depots (browning process). We analyzed the potential of peripheral blood mononuclear cells (PBMCs) to reflect BAT and retroperitoneal white adipose tissue (rWAT) response to 1-wk cold acclimation (4°C) at different ages of rat development (1, 2, 4, and 6 mo). As expected, cold exposure increased fatty acid β-oxidation capacity in BAT and rWAT (increased Cpt1a expression), explaining increased circulating nonesterified free fatty acids and decreased adiposity. Cold exposure increased expression of the key thermogenic gene, Ucp1, in BAT and rWAT, but only in 1-mo-old animals. Additionally, other brown/brite markers were affected by cold during the whole developmental period studied in BAT. However, in rWAT, cold exposure increased studied markers mainly at early age. PBMCs did not express Ucp1, but expressed other brown/brite markers, which were cold regulated. Of particular interest, PBMCs reflected adipose tissue-increased Cpt1a mRNA expression in response to cold (in older animals) and browning induction occurring in rWAT of young animals (1 mo) characterized by increased Cidea expression and by the appearance of a high number of multilocular CIDE-A positive adipocytes. These results provide evidence pointing to PBMCs as an easily obtainable biological material to be considered to perform browning studies with minimum invasiveness.  

4.1467           Liver Sinusoidal Endothelial Cells Escape Senescence by Loss of p19ARF

Kouldelkova, P., Weber, G. and Mikulits, W. PloS One, 10(11), e0142134 (2015)   Liver sinusoidal endothelial cells (LSECs) represent a highly differentiated cell type that lines hepatic sinusoids. LSECs form a discontinuous endothelium due to fenestrations under physiological conditions, which are reduced upon chronic liver injury. Cultivation of rodent LSECs associates with a rapid onset of stress-induced senescence a few days post isolation, which limits genetic and biochemical studies ex vivo. Here we show the establishment of LSECs isolated from p19^ARF-/- mice which undergo more than 50 cell doublings in the absence of senescence. Isolated p19^ARF-/- LSECs display a cobblestone-like morphology and show the ability of tube formation. Analysis of DNA content revealed a stable diploid phenotype after long-term passaging without a gain of aneuploidy. Notably, p19^ARF-/- LSECs express the endothelial markers CD31, vascular endothelial growth factor receptor (VEGFR)-2, VE-cadherin, von Willebrand factor, stabilin-2 and CD146 suggesting that these cells harbor and maintain an endothelial phenotype. In line, treatment with small molecule inhibitors against VEGFR-2 caused cell death, demonstrating the sustained ability of p19^ARF-/- LSECs to respond to anti-angiogenic therapeutics. From these data we conclude that loss of p19^ARF overcomes senescence of LSECs, allowing immortalization of cells without losing endothelial characteristics. Thus, p19^ARF-/- LSECs provide a novel cellular model to study endothelial cell biology.  

4.1468           Non-Canonical Wnt Predominates in Activated Rat Hepatic Stellate Cells, Influencing HSC Survival and Paracrine Stimulation of Kupffer Cells

Corbett, L., Mann, J. and Mann,. D.A. PloS One, 10(11), e0142794 (2015)   The Wnt system is highly complex and is comprised of canonical and non-canonical pathways leading to the activation of gene expression. Our aim was to examine changes in the expression of Wnt ligands and regulators during hepatic stellate cell (HSC) transdifferentiation and assess the relative contributions of the canonical and non-canonical Wnt pathways in fibrogenic activated HSC. The expression profile of Wnt ligands and regulators in HSC was not supportive for a major role for β-catenin-dependent canonical Wnt signalling, this verified by inability to induce Topflash reporter activity in HSC even when expressing a constitutive active β-catenin. We detected expression of Wnt5a in activated HSC which can signal via non-canonical mechanisms and showed evidence for non-canonical signalling in these cells involving phosphorylation of Dvl2 and pJNK. Stimulation of HSC or Kupffer cells with Wnt5a regulated HSC apoptosis and expression of TGF-β1 and MCP1 respectively. We were unable to confirm a role for β-catenin-dependent canonical Wnt in HSC and instead propose autocrine and paracrine functions for Wnts expressed by activated HSC via non-canonical pathways. The data warrant detailed investigation of Wnt5a in liver fibrosis.  

4.1469           Type I IFN-mediated synergistic activation of mouse and human DC subsets by TLR agonists

Kreutz, M., Bakdash, G., Dolen, Y., Sköld, A.E., van Hout-Kuijer, M.A., de Vries, I.J.M. and Figdor, C.G. Eur. J. Immunol., 45(10), 2798-2909 (2015)   Novel approaches of dendritic cell (DC) based cancer immunotherapy aim at harnessing the unique attributes of different DC subsets. Classical monocyte-derived DC vaccines are currently being replaced by either applying primary DCs or specifically targeting antigens and adjuvants to these subsets in vivo. Appropriate DC activation in both strategies is essential for optimal effect. For this purpose TLR agonists are favorable adjuvant choices, with TLR7 triggering being essential for inducing strong Th1 responses. However, mouse CD8α⁺ DCs, considered to be the major cross-presenting subset, lack TLR7 expression. Interestingly, this DC subset can respond to TLR7 ligand upon concurrent TLR3 triggering. Nevertheless, the mechanism underlying this synergy remains obscure. We now show that TLR3 ligation results in the production of IFN-α, which rapidly induces the expression of TLR7, resulting in synergistic activation. Moreover, we demonstrate that this mechanism conversely holds for plasmacytoid DCs that respond to TLR3 ligation when TLR7 pathway is mobilized. We further demonstrate that this mechanism of sharpening DC senses is also conserved in human BDCA1⁺ DCs and plasmacytoid DCs. These findings have important implications for future clinical trials as it suggests that combinations of TLR ligands should be applied irrespective of initial TLR expression profiles on natural DC subsets for optimal stimulation.  

4.1470           Dynamics and Transcriptomics of Skin Dendritic Cells and Macrophages in an Imiquimod-Induced, Biphasic Mouse Model of Psoriasis

Terhorst, D., Chelbi, R., Wohn, C., Malosse, C., Tamoutounour, S., Jorquera, A., bajenoff, M., Ddalod, M., Malissen, B. and Henri, S.

1. Immunol., 195(10), 4953-4961 (2015)

  Psoriasis is a chronic inflammatory skin disease of unknown etiology. Previous studies showed that short-term, 5–7 d-long application of imiquimod (IMQ), a TLR7 agonist, to the skin of mice triggers a psoriasis-like inflammation. In the current study, by applying IMQ for 14 consecutive d, we established an improved mouse psoriasis-like model in that it recapitulated many of the clinical and cellular hallmarks observed in human patients during both the early-onset and the late-stable phase of psoriasis. Although macrophages and dendritic cells (DCs) have been proposed to drive the psoriatic cascade, their largely overlapping phenotype hampered studying their respective role. Based on our ability to discriminate Langerhans cells (LCs), conventional DCs, monocytes, monocyte-derived DCs, macrophages, and plasmacytoid DCs in the skin, we addressed their dynamics during both phases of our biphasic psoriasis-like model. Plasmacytoid DCs were not detectable during the whole course of IMQ treatment. During the early phase, neutrophils infiltrated the epidermis, whereas monocytes and monocyte-derived DCs were predominant in the dermis. During the late phase, LCs and macrophage numbers transiently increased in the epidermis and dermis, respectively. LC expansion resulted from local proliferation, a conclusion supported by global transcriptional analysis. Genetic depletion of LCs permitted to evaluate their function during both phases of the biphasic psoriasis-like model and demonstrated that their absence resulted in a late phase that is associated with enhanced neutrophil infiltration. Therefore, our data support an anti-inflammatory role of LCs during the course of psoriasis-like inflammation.  

4.1471           Brief Exercises Affect Gene Expression in Circulating Monocytes

Wang, D., Cai, F., Ge, J. and Yin, L. Scand. J. Immunol., 82(5), 429-235 (2015)   We aimed to give a systematic hypothesis on the functions of exercise on circulating monocytes by identifying a discrete set of genes in circulating monocytes that were altered by exercise. The microarray expression profile of GSE51835 was downloaded from gene expression omnibus (GEO) database for the identification of differentially expressed genes (DEGs) using limma and affy packages in R language. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for DEGs, followed by the construction of co-expression network and protein–protein interaction (PPI) network. The top 10 nodes in PPI network were screened, and subnetwork was constructed for the key genes identification. Totally, 35 DEGs, including 2 upregulated genes and 33 downregulated genes, were identified. The enriched GO terms were mainly linked to immune response and defence response, and the enriched KEGG pathways were mainly associated with natural killer cell-mediated cytotoxicity and graft-versus-host disease. Dual-specificity phosphatase 2 (DUSP2) was identified as a key node in the co-expression network. In the PPI network, CD247 module (CD247), chemokine (C-X-C motif) receptor 4 (CXCR4), granzyme B (GZMB) and perforin 1 (PRF1) were identified as key nodes. An important interaction, GZMB/PRF1, was detected. Five key genes, including DUSP2, CD247, CXCR4, GZMB and PRF1, and an interaction of GZMB/PRF1, were significant factors in the immune processes of circulating monocytes, which might be regulated by brief exercises, leading to the enhancement of immune function.  

4.1472           Expression of Peroxiredoxin 1 After Traumatic Spinal Cord Injury in Rats

Huang, S., Liu, X., Zhang, J., Bao, g., Xu, G., Sun, Y., Shen, Q., Lian, M., Huang, Y. and Cui, Z. Cell. Mol. Neurobiol., 35(8), 1217-1226 (2015)   Reactive astrogliosis and microgliosis after spinal cord injury (SCI) contribute to glial scar formation that impedes axonal regeneration. The mechanisms underlying reactive astrocyte and microglia proliferation upon injury remain partially understood. Peroxiredoxin 1 (PRDX1) is an antioxidant participating in cell proliferation, differentiation, and apoptosis. However, PRDX1 functions in SCI-induced astrocyte and microglia proliferation are unknown. In this study, we established an acute spinal cord contusion injury model in adult rats to investigate the potential role of PRDX1 during the pathological process of SCI. We found the palpable expression increase of PRDX1 after SCI by western blot and immunohistochemistry staining. Double immunofluorescence staining showed that PRDX1 expression mainly increased in astrocytes and microglia. In addition, PRDX1/proliferating cell nuclear antigen (PCNA) colocalized in astrocytes and microglia. Furthermore, PCNA expression also elevated after SCI, as well as was positively correlated with PRDX1 expression. In vitro, PRDX1 expression in primary rat spinal cord astrocytes and microglia changed in a concentration- and time-dependent manner according to LPS treatment. In addition, PRDX1 knockdown in astrocytes and microglia resulted in the decrease of PCNA expression after LPS stimulation, showing that PRDX1 promoted astrocyte and microglia proliferation after inflammation. Our results suggested that PRDX1 might play a crucial role in astrocyte and microglia proliferation after SCI.

4.1473           Characterizing Cellular Biophysical Responses to Stress by Relating Density, Deformability, and Size

Byun, S., Hecht, V.C. and Manalis, S.R. Biophys. J., 109(8)   Cellular physical properties are important indicators of specific cell states. Although changes in individual biophysical parameters, such as cell size, density, and deformability, during cellular processes have been investigated in great detail, relatively little is known about how they are related. Here, we use a suspended microchannel resonator (SMR) to measure single-cell density, volume, and passage time through a narrow constriction of populations of cells subjected to a variety of environmental stresses. Osmotic stress significantly affects density and volume, as previously shown. In contrast to density and volume, the effect of an osmotic challenge on passage time is relatively small. Deformability, as determined by comparing passage times for cells with similar volume, exhibits a strong dependence on osmolarity, indicating that passage time alone does not always provide a meaningful proxy for deformability. Finally, we find that protein synthesis inhibition, cell-cycle arrest, protein kinase inhibition, and cytoskeletal disruption result in unexpected relationships among deformability, density, and volume. Taken together, our results suggest that by measuring multiple biophysical parameters, one can detect unique characteristics that more specifically reflect cellular behaviors.  

4.1474           Ubiquitin C-terminal hydrolase 1: A novel functional marker for liver myofibroblasts and a therapeutic target in chronic liver disease

Wilson, C.L., Murphy, L.B., Leslie, J., Kendrick, S., French, J., Fox, C.R., Sheerin, N.S., Fisher, A., Robinson, J.H., Tiniakos, D.G., Gray, D.A., Oakley, F. and Mann, D.A.

1. Hepatol., 63, 1421-1428 (2015)

  Background & Aims Ubiquitination is a reversible protein modification involved in the major cellular processes that define cell phenotype and behaviour. Ubiquitin modifications are removed by a large family of proteases named deubiquitinases. The role of deubiquitinases in hepatic stellate cell (HSC) activation and their contribution to fibrogenesis are poorly defined. We have identified that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is highly induced following HSC activation, determined its function in activated HSC and its potential as a therapeutic target for fibrosis. Methods Deubiquitinase expression was determined in day 0 and day 10 HSC. Increased UCHL1 expression was confirmed in human HSC and in an alcoholic liver disease (ALD) patient liver. The importance of UCHL1 in hepatic fibrosis was investigated in CCl₄ and bile duct ligation injured mice using a pharmacological inhibitor (LDN 57444). The effects of UCHL1 inhibition on HSC proliferation were confirmed by Western blot and 3H thymidine incorporation. Results Here we report that pharmacological inhibition of UCHL1 blocks progression of established fibrosis in CCl₄ injured mice. UCHL1 siRNA knockdown, LDN 57444 treatment, or HSC isolated from UCHL1^−/− mice show attenuated proliferation in response to the mitogen, platelet-derived growth factor. Additionally, we observed changes in the phosphorylation of the cell cycle regulator retinoblastoma protein (Rb) in the absence of UCHL1 highlighting a potential mechanism for the reduced proliferative response. Conclusions UCHL1 expression is highly upregulated upon HSC activation and is involved in the regulation of HSC proliferation. This study highlights therapeutic opportunities for pharmacological targeting of UCHL1 in chronic liver disease.  

4.1475           Long Non-coding RNA Growth Arrest-specific Transcript 5 (GAS5) Inhibits Liver Fibrogenesis through a Mechanism of Competing Endogenous RNA

Yu, F., Zheng, J., Mao, Y., Dong, P., Lu, Z., Li, G., Guo, C., Liu, Z. and Fan, X.

1. Biol. Chem., 290(47), 28286-28298 (2015)

  Effective control of hepatic stellate cell (HSC) activation and proliferation is critical to the treatment of liver fibrosis. Long non-coding RNAs have been shown to play a pivotal role in the regulation of cellular processes. It has been reported that growth arrest-specific transcript 5 (GAS5) acts as a crucial mediator in the control of cell proliferation and growth. However, little is known about the role and underlying mechanism of GAS5 in liver fibrosis. In this study, our results indicated that GAS5 expression was reduced in mouse, rat, and human fibrotic liver samples and in activated HSCs. Overexpression of GAS5 suppressed the activation of primary HSCs in vitro and alleviated the accumulation of collagen in fibrotic liver tissues in vivo. We identified GAS5 as a target of microRNA-222 (miR-222) and showed that miR-222 could inhibit the expression of GAS5. Interestingly, GAS5 could also repress miR-222 expression. A pulldown assay further validated that GAS5 could directly bind to miR-222. As a competing endogenous RNAs, GAS5 had no effect on primary miR-222 expression. In addition, GAS5 was mainly localized in the cytoplasm. Quantitative RT-PCR further demonstrated that the copy numbers of GAS5 per cell are higher than those of miR-222. GAS5 increased the level of p27 protein by functioning as a competing endogenous RNA for miR-222, thereby inhibiting the activation and proliferation of HSCs. Taken together, a new regulatory circuitry in liver fibrosis has been identified in which RNAs cross-talk by competing for shared microRNAs. Our findings may provide a new therapeutic strategy for liver fibrosis.  

4.1476           Dectin-1 Regulates Hepatic Fibrosis and Hepatocarcinogenesis by Suppressing TLR4 Signaling Pathways

Seifert, L. et al Cell Reports, 13, 1-13 (2015)   Dectin-1 is a C-type lectin receptor critical in anti-fungal immunity, but Dectin-1 has not been linked to regulation of sterile inflammation or oncogenesis. We found that Dectin-1 expression is upregulated in hepatic fibrosis and liver cancer. However, Dectin-1 deletion exacerbates liver fibro-inflammatory disease and accelerates hepatocarcinogenesis. Mechanistically, we found that Dectin-1 protects against chronic liver disease by suppressing TLR4 signaling in hepatic inflammatory and stellate cells. Accordingly, Dectin-1–/– mice exhibited augmented cytokine production and reduced survival in lipopolysaccharide (LPS)-mediated sepsis, whereas Dectin-1 activation was protective. We showed that Dectin-1 inhibits TLR4 signaling by mitigating TLR4 and CD14 expression, which are regulated by Dectin-1-dependent macrophage colony stimulating factor (M-CSF) expression. Our study suggests that Dectin-1 is an attractive target for experimental therapeutics in hepatic fibrosis and neoplastic transformation. More broadly, our work deciphers critical cross-talk between pattern recognition receptors and implicates a role for Dectin-1 in suppression of sterile inflammation, inflammation-induced oncogenesis, and LPS-mediated sepsis.  

4.1477           CD11chi Dendritic Cells Regulate Ly-6Chi Monocyte Differentiation to Preserve Immune-privileged CNS in Lethal Neuroinflammation

Kim, J.H., Choi, J.Y., Kim, S.B., Uyangaa, E., patil, A.M., Han, Y.W., park, S-Y., Lee, J.H., Kim, K. and Eo, S.K. Scientific Reports, 5:17548 (2015)   Although the roles of dendritic cells (DCs) in adaptive defense have been defined well, the contribution of DCs to T cell-independent innate defense and subsequent neuroimmunopathology in immune-privileged CNS upon infection with neurotropic viruses has not been completely defined. Notably, DC roles in regulating innate CD11b+Ly-6Chi monocyte functions during neuroinflammation have not yet been addressed. Using selective ablation of CD11chiPDCA-1int/lo DCs without alteration in CD11cintPDCA-1hi plasmacytoid DC number, we found that CD11chi DCs are essential to control neuroinflammation caused by infection with neurotropic Japanese encephalitis virus, through early and increased infiltration of CD11b+Ly-6Chi monocytes and higher expression of CC chemokines. More interestingly, selective CD11chi DC ablation provided altered differentiation and function of infiltrated CD11b+Ly-6Chi monocytes in the CNS through Flt3-L and GM-CSF, which was closely associated with severely enhanced neuroinflammation. Furthermore, CD11b+Ly-6Chi monocytes generated in CD11chi DC-ablated environment had a deleterious rather than protective role during neuroinflammation, and were more quickly recruited into inflamed CNS, depending on CCR2, thereby exacerbating neuroinflammation via enhanced supply of virus from the periphery. Therefore, our data demonstrate that CD11chi DCs provide a critical and unexpected role to preserve the immune-privileged CNS in lethal neuroinflammation via regulating the differentiation, function, and trafficking of CD11b+Ly-6Chi monocytes.  

4.1478           Amelioration of Japanese encephalitis by blockage of 4-1BB signaling is coupled to divergent enhancement of type I/II IFN responses and Ly-6Chi monocyte differentiation

Kim, S.B., Choi, J.Y., Kim, J.H., Uyangaa, E., patil, A.M., park, S-Y., Lee, J.H., Kim, K., Han, Y.W. and Eo, S.K.

1. Neuroinfammation, 12:216 (2015)

  Background Japanese encephalitis (JE), a neuroinflammation caused by zoonotic JE virus, is the major cause of viral encephalitis worldwide and poses an increasing threat to global health and welfare. To date, however, there has been no report describing the regulation of JE progression using immunomodulatory tools for developing therapeutic strategies. We tested whether blocking the 4-1BB signaling pathway would regulate JE progression using murine JE model. Methods Infected wild-type and 4-1BB-knockout (KO) mice were examined daily for mortality and clinical signs, and neuroinflammation in the CNS was evaluated by infiltration of inflammatory leukocytes and cytokine expression. In addition, viral burden, JEV-specific T cell, and type I/II IFN (IFN-I/II) innate responses were analyzed. Results Blocking the 4-1BB signaling pathway significantly increased resistance to JE and reduced viral burden in extraneural tissues and the CNS, rather than causing a detrimental effect. In addition, treatment with 4-1BB agonistic antibody exacerbated JE. Furthermore, JE amelioration and reduction of viral burden by blocking the 4-1BB signaling pathway were associated with an increased frequency of IFN-II-producing NK and CD4⁺ Th1 cells as well as increased infiltration of mature Ly-6C^hi monocytes in the inflamed CNS. More interestingly, DCs and macrophages derived from 4-1BB KO mice showed potent and rapid IFN-I innate immune responses upon JEV infection, which was coupled to strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7), and antiviral ISG genes (ISG49, ISG54, ISG56). Further, the ablation of 4-1BB signaling enhanced IFN-I innate responses in neuron cells, which likely regulated viral spread in the CNS. Finally, we confirmed that blocking the 4-1BB signaling pathway in myeloid cells derived from hematopoietic stem cells (HSCs) played a dominant role in ameliorating JE. In support of this finding, HSC-derived leukocytes played a dominant role in generating the IFN-I innate responses in the host. Conclusions Blocking the 4-1BB signaling pathway ameliorates JE via divergent enhancement of IFN-II-producing NK and CD4⁺ Th1 cells and mature Ly-6C^hi monocyte infiltration, as well as an IFN-I innate response of myeloid-derived cells. Therefore, regulation of the 4-1BB signaling pathway with antibodies or inhibitors could be a valuable therapeutic strategy for the treatment of JE.  

4.1479           Cation Homeostasis in Red Cells From Patients With Sickle Cell Disease Heterologous for HbS and HbC (HbSC Genotype)

Hannemann, A., Rees, D.C., Tewari, S. and Gibson, J.S. EBioMedicine, 2, 1669-1676 (2015)   Sickle cell disease (SCD) in patients of HbSC genotype is considered similar, albeit milder, to that in homozygous HbSS individuals — but with little justification. In SCD, elevated red cell cation permeability is critical as increased solute loss causes dehydration and encourages sickling. Recently, we showed that the KCl cotransporter (KCC) activity in red cells from HbSC patients correlated significantly with disease severity, but that in HbSS patients did not. Two transporters involved in red cell dehydration, the conductive channels P_sickle and the Gardos channel, behaved similarly in red cells from the two genotypes, but were significantly less active in HbSC patients. By contrast, KCC activity was quantitatively greater in HbSC red cells. Results suggest that KCC is likely to have greater involvement in red cell dehydration in HbSC patients, which could explain its association with disease severity in this genotype. This work supports the hypothesis that SCD in HbSC patients is a distinct disease entity to that in HbSS patients. Results suggest the possibility of designing specific treatments of particular benefit to HbSC patients and a rationale for the development of prognostic markers, to inform early treatment of children likely to develop more severe complications of the disease.  

4.1480           Chronic Nicotine Exposure In Vivo and In Vitro Inhibits Vitamin B1 (Thiamin) Uptake by Pancreatic Acinar Cells

Srinivasan, P., Thrower, E.C., Loganaathan, G., Balamurugan, A.N., Subramanian, V.S., Gorelick, F.S. and Said, H.M. PloS One, 10(12), e0143575 (2015)   Thiamin (vitamin B1), a member of the water-soluble family of vitamins, is essential for normal cellular functions; its deficiency results in oxidative stress and mitochondrial dysfunction. Pancreatic acinar cells (PAC) obtain thiamin from the circulation using a specific carrier-mediated process mediated by both thiamin transporters -1 and -2 (THTR-1 and THTR-2; encoded by the SLC19A2 and SLC19A3 genes, respectively). The aim of the current study was to examine the effect of chronic exposure of mouse PAC in vivo and human PAC in vitro to nicotine (a major component of cigarette smoke that has been implicated in pancreatic diseases) on thiamin uptake and to delineate the mechanism involved. The results showed that chronic exposure of mice to nicotine significantly inhibits thiamin uptake in murine PAC, and that this inhibition is associated with a marked decrease in expression of THTR-1 and THTR-2 at the protein, mRNA and hnRNAs level. Furthermore, expression of the important thiamin-metabolizing enzyme, thiamin pyrophosphokinase (TPKase), was significantly reduced in PAC of mice exposed to nicotine. Similarly, chronic exposure of cultured human PAC to nicotine (0.5 μM, 48 h) significantly inhibited thiamin uptake, which was also associated with a decrease in expression of THTR-1 and THTR-2 proteins and mRNAs. This study demonstrates that chronic exposure of PAC to nicotine impairs the physiology and the molecular biology of the thiamin uptake process. Furthermore, the study suggests that the effect is, in part, mediated through transcriptional mechanism(s) affecting the SLC19A2 and SLC19A3 genes.  

4.1481           Patient-specific blood rheology in sickle-cell anaemia

Li, X., Du, E., lei, H., tang, Y-H., Dao, M., Suresh, S and karniadakis, G.E. Interface Focus, 6, 20150065 (2015)   Sickle-cell anaemia (SCA) is an inherited blood disorder exhibiting heterogeneous cell morphology and abnormal rheology, especially under hypoxic conditions. By using a multiscale red blood cell (RBC) model with parameters derived from patient-specific data, we present a mesoscopic computational study of the haemodynamic and rheological characteristics of blood from SCA patients with hydroxyurea (HU) treatment (on-HU) and those without HU treatment (off-HU). We determine the shear viscosity of blood in health as well as in different states of disease. Our results suggest that treatment with HU improves or worsens the rheological characteristics of blood in SCA depending on the degree of hypoxia. However, on-HU groups always have higher levels of haematocrit-to-viscosity ratio (HVR) than off-HU groups, indicating that HU can indeed improve the oxygen transport potential of blood. Our patient-specific computational simulations suggest that the HVR level, rather than the shear viscosity of sickle RBC suspensions, may be a more reliable indicator in assessing the response to HU treatment.  

4.1482           Microencapsulated Pig Islet Xenotransplantation as an Alternative Treatment of Diabetes

Zhu, H., Yu, L., He, Y., Lyu, Y. and Wang, B. Tissue Engineering: Part B, 21(5), 474-489 (2015)   Islet transplantation is emerging as an attractive option for the treatment of type 1 diabetes mellitus (T1DM). However, some major obstacles are needed to be overcome, including shortage of islet supply and excessive immunosuppressive therapy. Xenotransplantation of bioartificial pancreas (BAP) made of microencapsulated pig islets will effectively solve these problems. Before widespread application of this therapy, several important issues should be addressed to further improve pig islet viability and functionality, such as pig islet source, optimization of microcapsule preparation, cryopreservation of implant, selection of biocompatible material, and implant site, as well as prevention of xenoreaction and biosafety concern. It is hoped that improvements in these critical aspects will lead to wider human application of microencapsulation of pig islets.  

4.1483           Evaluation of small noncoding RNAs in ex vivo stored human mature red blood cells: changes in noncoding RNA levels correlate with storage lesion events

Sarachana, T., Kulkarni, S. and Atreya, C.D. Transfusion, 55(11), 2672-2683 (2015)   BACKGROUND While biomarkers of storage lesions (SLs) for red blood cells (RBCs) abound, the physiologic consequences of SLs and associated important events are poorly understood. Previously we have identified differentially expressed regulatory small noncoding RNAs (ncRNAs) in stored RBCs, suggesting their role in the RBC SL process and their potential as quality biomarkers of stored RBCs. STUDY DESIGN AND METHODS Comprehensive ncRNA expression analysis of RBCs stored for up to 56 days was performed on RNAs collected from enriched mature RBCs on Days 0, 7, 14, 28, 42, and 56. Three known RBC SL processes, that is, mature RBCs’ suicidal death (eryptosis), ATP loss, and changes in RBC indices, were correlated with differentially expressed ncRNAs to gain knowledge on the SL molecular processes. RESULTS The analysis identified four ncRNAs whose changes in the expression levels were correlated with the selected three SL processes. Differential expression on Days 14 and 28 of the four selected ncRNAs was confirmed by TaqMan quantitative reverse transcription–polymerase chain reaction analysis. Bioinformatics analysis identified potential targets and biologic functions of these ncRNAs. Overexpression of one such ncRNA, hsa-miR-196a, in a human erythroblast cell line confirmed its protective effects against the cell death and ATP loss. CONCLUSION Overall, this study demonstrates that changes in the levels of small ncRNAs of stored RBCs correlate with some of the SL events and thus they have the potential to serve as the storage quality markers.  

4.1484           GDF10 is a signal for axonal sprouting and functional recovery after stroke

Li, S., Nie, E.H., Yin, Y., Benowitz, L.I., Tung, S., Vinters, H.V., Bahjat, F.R., Stenzel-Poore, M.P., Kawaguchi, R., Xoppala, G. and Carmichael, T. Nature Neuroscience, 18(12), 1737-1745 (2015)   Stroke produces a limited process of neural repair. Axonal sprouting in cortex adjacent to the infarct is part of this recovery process, but the signal that initiates axonal sprouting is not known. Growth and differentiation factor 10 (GDF10) is induced in peri-infarct neurons in mice, non-human primates and humans. GDF10 promotes axonal outgrowth in vitro in mouse, rat and human neurons through TGFβRI and TGFβRII signaling. Using pharmacogenetic gain- and loss-of-function studies, we found that GDF10 produced axonal sprouting and enhanced functional recovery after stroke; knocking down GDF10 blocked axonal sprouting and reduced recovery. RNA sequencing from peri-infarct cortical neurons revealed that GDF10 downregulated PTEN, upregulated PI3 kinase signaling and induced specific axonal guidance molecules. Using unsupervised genome-wide association analysis of the GDF10 transcriptome, we found that it was not related to neurodevelopment, but may partially overlap with other CNS injury patterns. Thus, GDF10 is a stroke-induced signal for axonal sprouting and functional recovery.  

4.1485           Hepatic CD206-positive macrophages express amphiregulin to promote the immunosuppressive activity of regulatory T cells in HBV infection

Dai, K., Huang, L., Sun, X., yang, L. and Gong, Z.

1. Leukoc. Biol., 98(6), 1071-1080 (2015)

  Hepatitis B virus is a major cause of chronic liver inflammation worldwide. Innate and adaptive immune responses work together to restrain or eliminate hepatitis B virus in the liver. Compromised or failed adaptive immune response results in persistent virus replication and spread. How to promote antiviral immunity is a research focus for hepatitis B virus prevention and therapy. In this study, we investigated the role of macrophages in the regulation of antiviral immunity. We found that F4/80⁺CD206⁺CD80^lo/+ macrophages were a particular hepatic macrophage subset that expressed amphiregulin in our mouse hepatitis B virus infection model. CD206⁺ macrophage-derived amphiregulin promoted the immunosuppressive activity of intrahepatic regulatory T cells, demonstrated by higher expression of CTLA-4, ICOS, and CD39, as well as stronger inhibition of antiviral function of CD8⁺ T cells. Amphiregulin-neutralizing antibody diminished the effect of CD206⁺ macrophages on regulatory T cells. In addition, we found that CD206⁺ macrophage-derived amphiregulin activated mammalian target of rapamycin signaling in regulatory T cells, and this mammalian target of rapamycin activation was essential for promotion of regulatory T cell activity by CD206⁺ macrophages. Adoptive transfer of CD206⁺ macrophages into hepatitis B virus-infected mice increased cytoplasmic hepatitis B virus DNA in hepatocytes and also increased serum hepatitis B surface antigen. The antiviral activity of CD8⁺ T cells was decreased after macrophage transfer. Therefore, our research indicated that amphiregulin produced by CD206⁺ macrophages plays an important role in modulating regulatory T cell function and subsequently restrains the antiviral activity of CD8⁺ T cells. Our study offers new insights into the immunomodulation in hepatitis B virus infection.  

4.1486           lincRNA-p21 inhibits hepatic stellate cell activation and liver fibrogenesis via p21

Zheng, J., Dong, P., Mao, Y., Chen, S., Wu, X., Li, G., Lu, Z. and Yu, F. FEBS  J., 282(24), 4810-4821 (2015)   Long non-coding RNAs are involved in various biological processes and diseases. The biological role of long intergenic non-coding RNA-p21 (lincRNA-p21) in liver fibrosis remains unknown before this study. In this study, we observed marked reduction of lincRNA-p21 expression in mice liver fibrosis models and human cirrhotic liver. Over-expression of lincRNA-p21 suppressed activation of hepatic stellate cells (HSCs) in vitro. Lentivirus-mediated lincRNA-p21 transfer into mice decreased the severity of liver fibrosis in vivo. Additionally, lincRNA-p21 reversed the activation of HSCs to their quiescent phenotype. The mRNA levels of lincRNA-p21 and p21 were positively correlated. Our results show that over-expression of lincRNA-p21 promotes up-regulation of p21 at both the mRNA and protein levels. Furthermore, lincRNA-p21 inhibited cell-cycle progression and proliferation of primary HSCs through enhancement of p21 expression. Compared with healthy subjects, serum lincRNA-p21 levels were significantly lower in patients with liver cirrhosis, especially those with decompensation. These findings collectively indicate that lincRNA-p21 is a mediator of HSC activation, supporting its utility as a novel therapeutic target for liver fibrosis.  

4.1487           Bioremediation strategies for removal of residual atrazine in the boreal groundwater zone

Nousiainen, A.O., Bjöklöf, K., Sagarkar, S., Lund Nielsen, J., Kapley, A. and Jørgensen, K.S. Appl. Microbiol. Biotechnol., 99(23), 10249-10259 (2015)   Strategies for bioremediation of atrazine, a pesticide commonly polluting groundwater in low concentrations, were studied in two boreal nonagricultural soils. Atrazine was not mineralized in soil without bioremediation treatments. In biostimulation treatment with molasses, up to 52 % of atrazine was mineralized at 10 °C, even though the degradation gene copy numbers did not increase. Incubations with radioactively labeled atrazine followed by microautoradiographic analysis revealed that bioremediation strategies increased the relative proportion of active degraders from 0.3 up to 1.9 % of the total bacterial count. These results indicate that atrazine degradation might not solely be facilitated by atzA/trzN–atzB genes. In combined biostimulation treatment using citrate or molasses and augmentation with Pseudomonas citronellolis ADP or Arthrobacter aurescens strain TC1, up to 76 % of atrazine was mineralized at 30 °C, and the atrazine degradation gene numbers increased up to 10⁷ copies g⁻¹ soil. Clone libraries from passive samplers in groundwater monitoring wells revealed the presence of phylogenetic groups formerly shown to include atrazine degraders, and the presence of atrazine degradation genes atzA and atzB. These results show that the mineralization of low concentrations of atrazine in the groundwater zone at low temperatures is possible by bioremediation treatments.  

4.1488           Myeloperoxidase–Hepatocyte–Stellate Cell Cross Talk Promotes Hepatocyte Injury and Fibrosis in Experimental Nonalcoholic Steatohepatitis

Pulli, B., Ali, M., Iwamoto, Y., Zeller, M.W.G., Schob, S., Linnoila, J.J. and Chen, J.W. Antioxidants & Redox Signaling, 23(16), 1255-1269 (2015)   Aims: Myeloperoxidase (MPO), a highly oxidative enzyme secreted by leukocytes has been implicated in human and experimental nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain unknown. In this study, we investigated how MPO contributes to progression from steatosis to NASH. Results: In C57Bl/6J mice fed a diet deficient in methionine and choline to induce NASH, neutrophils and to a lesser extent inflammatory monocytes are markedly increased compared with sham mice and secrete abundant amounts of MPO. Through generation of HOCl, MPO directly causes hepatocyte death in vivo. In vitro experiments demonstrate mitochondrial permeability transition pore induction via activation of SAPK/JNK and PARP. MPO also contributes to activation of hepatic stellate cells (HSCs), the most important source of collagen in the liver. In vitro MPO-activated HSCs have an activation signature (MAPK and PI3K-AKT phosphorylation) and upregulate COL1A1, α-SMA, and CXCL1. MPO-derived oxidative stress also activates transforming growth factor β (TGF-β) in vitro, and TGF-β signaling inhibition with SB-431542 decreased steatosis and fibrosis in vivo. Conversely, congenital absence of MPO results in reduced hepatocyte injury, decreased levels of TGF-β, fewer activated HSCs, and less severe fibrosis in vivo. Innovation and Conclusion: Cumulatively, these findings demonstrate important cross talk between inflammatory myeloid cells, hepatocytes, and HSCs via MPO and establish MPO as part of a proapoptotic and profibrotic pathway of progression in NASH, as well as a potential therapeutic target to ameliorate this disease.   Lung-derived exosome uptake into and epigenetic modulation of marrow progenitor/stem and differentiated cells Aliotta, J.M., Pereira, M., Sears, E.H., Dooner, M.S., Wen, S., Goldberg, L.R. and Quesenberry, P.J.

1. Extracellular Vesicles, 4:26166 (2015)

  Background: Our group has previously demonstrated that murine whole bone marrow cells (WBM) that internalize lung-derived extracellular vesicles (LDEVs) in culture express pulmonary epithelial cell–specific genes for up to 12 weeks. In addition, the lungs of lethally irradiated mice transplanted with lung vesicle–modulated marrow have 5 times more WBM-derived type II pneumocytes compared to mice transplanted with unmanipulated WBM. These findings indicate that extracellular vesicle modification may be an important consideration in the development of marrow cell–based cellular therapies. Current studies were performed to determine the specific marrow cell types that LDEV stably modify. Methods: Murine WBM-derived stem/progenitor cells (Lin-/Sca-1+) and differentiated erythroid cells (Ter119+), granulocytes (Gr-1+) and B cells (CD19+) were cultured with carboxyfluorescein N-succinimidyl ester (CFSE)-labelled LDEV. LDEV+ cells (CFSE+) and LDEV− cells (CFSE−) were separated by flow cytometry and visualized by fluorescence microscopy, analyzed by RT-PCR or placed into long-term secondary culture. In addition, murine Lin-/Sca-1+ cells were cultured with CFSE-labelled LDEV isolated from rats, and RT-PCR analysis was performed on LDEV+ and–cells using species-specific primers for surfactant (rat/mouse hybrid co-cultures). Results: Stem/progenitor cells and all of the differentiated cell types studied internalized LDEV in culture, but heterogeneously. Expression of a panel of pulmonary epithelial cell genes was higher in LDEV+cells compared to LDEV− cells and elevated expression of these genes persisted in long-term culture. Rat/mouse hybrid co-cultures revealed only mouse-specific surfactant B and C expression in LDEV+ Lin-/Sca-1+cells after 4 weeks of culture, indicating stable de novo gene expression. Conclusions: LDEV can be internalized by differentiated and more primitive cells residing in the bone marrow in culture and can induce stable de novo pulmonary epithelial cell gene expression in these cells for several weeks after internalization. The gene expression represents a transcriptional activation of the target marrow cells. These studies serve as the basis for determining marrow cell types that can be used for cell-based therapies for processes that injure the pulmonary epithelial surfaces.  

4.1489           In vitro fertilization in pigs: New molecules and protocols to consider in the forthcoming years

Romar, R., Funahashi, H. and Coy, P. Theriogenology, 85, 125-134 (2016)   Assisted reproduction technology (ART) protocols are used in livestock for the improvement and preservation of their genetics and to enhance reproductive efficiency. In the case of pigs, the potential use of embryos for biomedicine is being followed with great interest by the scientific community. Owing to the physiological similarities with humans, embryos produced in vitro and many of those produced in vivo are used in research laboratories for the procurement of stem cells or the production of transgenic animals, sometimes with the purpose of using their organs for xenotransplantation. Several techniques are required for the production of an in vitro–derived embryo. These include in vitro oocyte maturation, sperm preparation, IVF, and further culture of the putative zygotes. Without doubt, among these technologies, IVF is still a critical limiting factor because of the well-known, but still unsolved, question of polyspermy. Despite the improvements made in the past decade, current IVF systems hardly reach 50% to 60% efficiency and any progression in porcine ARTs requires an unavoidable improvement in the monospermy rate. It is time, then, to learn from what happens under in vivo physiological conditions and to transfer this knowledge into ART. This review describes the latest advances in porcine IVF, from sperm preparation procedures to culture media supplements with special attention paid to molecules with a known or potential role in in vivo fertilization. Oviductal fluid is the natural medium in which fertilization takes place, and, in the near future, could become the definitive supplement for culture media, where it would help to solve many of the problems inherent in ARTs in swine and improve the quality of in vitro–derived porcine embryos.  

4.1490           Synthetic and natural small molecule TLR4 antagonists inhibit motoneuron death in cultures from ALS mouse model

De Paola, M., Sestito, S.E., Mariani,A., Memo, C., Fanelli, R., Freschi, M., Bendotti, C., Calabrese, V. and Peri, F. Pharmacol. Res., 103, 180-187 (2016)   Increasing evidence indicates that inflammatory responses could play a critical role in the pathogenesis of motor neuron injury in amyotrophic lateral sclerosis (ALS). Recent findings have underlined the role of Toll-like receptors (TLRs) and the involvement of both the innate and adaptive immune responses in ALS pathogenesis. In particular, abnormal TLR4 signaling in pro-inflammatory microglia cells has been related to motoneuron degeneration leading to ALS. In this study the effect of small molecule TLR4 antagonists on in vitro ALS models has been investigated. Two different types of synthetic glycolipids and the phenol fraction extracted from commercial extra-virgin olive oil (EVOO) were selected since they efficiently inhibit TLR4 stimulus in HEK cells by interacting with the TLR4·MD-2 complex and CD14 co-receptor. Here, TLR4 antagonists efficiently protected motoneurons from LPS-induced lethality in spinal cord cultures, and inhibited the interleukine-1β production by LPS-stimulated microglia. In motoneurons/glia cocultures obtained from wild type or SOD1 G93A mice, motoneuron death induced by SOD1mut glia was counteracted by TLR4 antagonists. The release of nitric oxide by LPS treatment or SOD1mut glia was also inhibited by EVOO, suggesting that the action of this natural extract could be mainly related to the modulation of this inflammatory mediator.  

4.1491           Reciprocal interaction among gasotransmitters in isolated pancreatic β-cells

Moustafa, A. and Habara, Y. Free Radical Biology and medicine, 90, 47-58 (2016)   We aimed to elucidate the interplay among the three well-known gas molecules, nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H₂S), and their effects on intracellular Ca²⁺ concentration ([Ca²⁺]i) and insulin secretion in rat pancreatic β-cells. Immunofluorescence studies demonstrated the expression of constitutive enzymes that are responsible for the production of NO, CO and H₂S. CO and H₂S increased NO production as indicated by the increase in diaminofluorescein-2 triazole fluorescence. NO and CO induced an elevation in the sulfane sulfur pool and concomitantly H₂S production. The NO- and CO-induced H₂S production was partially inhibited by hypotaurine, an H₂S scavenger. NO and H₂S produced CO production as revealed by a myoglobin assay. A calmodulin antagonist in the absence of extracellular Ca²⁺ significantly attenuated NO and H₂S production. NO and CO induced a [Ca²⁺]_i increase mainly via Ca²⁺ release from internal stores; however, H₂S induced a [Ca²⁺]_i increase via the influx of extracellular Ca²⁺. NO dose-dependently stimulated basal insulin release but CO dose-dependently inhibited it. H₂S showed an insignificant effect on basal insulin secretion from freshly isolated pancreatic islets. Herein, we address for the first time the reciprocal and synergistic relation among gasotransmitters with diverse effects on basal insulin secretion that regulate β-cells functions and homeostasis.  

4.1492           Transdermal toxicity of topically applied anticoagulant rodenticide warfarin in rats

Subota, V., Mirkov, I., Demenesku, J., Aleksandrov, A.P., Ninkov, M., Mileusnic, D., Kataranovski, D. and Kataranovski, M. Environmental Toxicology and Pharmacology, 41, 232-2540 (2016)   Occupational/accidental exposure data have showed hemorrhage as a result of transdermal exposure to warfarin, however, other effects are not known. In the present study, the impact of epicutaneous application of 10 μg or 100 μg of warfarin (three times, once a day) on peripheral blood polymorphonuclear (PMN) and mononuclear cells (PBMC) was examined in rats. Both doses resulted in prolongation of prothrombin time and changes in hematologic parameters. Increases in PMN intracellular myeloperoxidase (MPO) activity were seen at higher warfarin dose and both doses resulted in higher percentages of granular CD11b⁺ cells. In contrast, a decrease in PMN TNF and IL-6 production (ELISA) and gene expression (RT-PCR) was observed. Epicutaneous application of warfarin resulted in decreased numbers of PBMC, higher numbers of mononuclear CD11b⁺ cells, but without effect on PMBC cytokine production. The data obtained showed differential effects of transdermal exposure to warfarin depending on leukocyte type and activity.  

4.1493           Early modulation of pro-inflammatory microglia by minocycline loaded nanoparticles confers long lasting protection after spinal cord injury

Papa, S. et al Biomaterials, 75, 13-24 (2016)   Many efforts have been performed in order to understand the role of recruited macrophages in the progression of spinal cord injury (SCI). Different studies revealed a pleiotropic effect played by these cells associated to distinct phenotypes (M1 and M2), showing a predictable spatial and temporal distribution in the injured site after SCI. Differently, the role of activated microglia in injury progression has been poorly investigated, mainly because of the challenges to target and selectively modulate them in situ. A delivery nanovector tool (poly-ε-caprolactone-based nanoparticles) able to selectively treat/target microglia has been developed and used here to clarify the temporal and spatial involvement of the pro-inflammatory response associated to microglial cells in SCI. We show that a treatment with nanoparticles loaded with minocycline, the latter a well-known anti-inflammatory drug, when administered acutely in a SCI mouse model is able to efficiently modulate the resident microglial cells reducing the pro-inflammatory response, maintaining a pro-regenerative milieu and ameliorating the behavioral outcome up to 63 days post injury. Furthermore, by using this selective delivery tool we demonstrate a mechanistic link between early microglia activation and M1 macrophages recruitment to the injured site via CCL2 chemokine, revealing a detrimental contribution of pro-inflammatory macrophages to injury progression after SCI.  

4.1494           Inversion of hematocrit partition at microfluidic bifurcations

Shen, Z., Coupier, G., Kaoui, B., Polack, B., Harting, J., Misbah, C. and Podgorski, T. Microvascular Res., 105, 40-46 (2016)   Partitioning of red blood cells (RBCs) at the level of bifurcations in the microcirculatory system affects many physiological functions yet it remains poorly understood. We address this problem by using T-shaped microfluidic bifurcations as a model. Our computer simulations and in vitro experiments reveal that the hematocrit (ϕ₀) partition depends strongly on RBC deformability, as long as ϕ₀ < 20% (within the normal range in microcirculation), and can even lead to complete deprivation of RBCs in a child branch. Furthermore, we discover a deviation from the Zweifach–Fung effect which states that the child branch with lower flow rate recruits less RBCs than the higher flow rate child branch. At small enough ϕ₀, we get the inverse scenario, and the hematocrit in the lower flow rate child branch is even higher than in the parent vessel. We explain this result by an intricate up-stream RBC organization and we highlight the extreme dependence of RBC transport on geometrical and cell mechanical properties. These parameters can lead to unexpected behaviors with consequences on the microcirculatory function and oxygen delivery in healthy and pathological conditions.  

4.1495           Molecular characterization and expression analysis of B cell activating factor from rock bream (Oplegnathus fasciatus)

Godahewa, G.I., Perera, N.C.N., Umasuthan, N., Wan, Q., Whang, I. and Lee, J. Development and Comparative Immunology, 55, 1-11 (2016)   B cell activating factor (BAFF) is a member of the tumor necrosis factor (TNF) ligand family. BAFF has been shown to induce survival and proliferation of lymphocytes. We characterized the gene encoding BAFF (RbBAFF) in rock bream (Oplegnathus fasciatus), and attempted to determine its biological functions upon immune responses. In silico analysis of RbBAFF demonstrated the presence of common TNF ligand family features, including a TNF domain, a D-E loop, and three cysteine residues that are crucial for trimer formation. Amino acid sequence alignment confirmed that RbBAFF and its homologs were conserved at secondary and tertiary levels. Transcriptional analysis indicated that RbBAFF mRNAs were ubiquitously expressed in wide array of tissues. The higher levels of constitutive expression were observed in the kidney, head kidney and spleen, suggesting an important physiological relationship with lymphocytes. Under pathological conditions, RbBAFF mRNA levels were significantly elevated. The role of RbBAFF in lymphocyte survival and proliferation was confirmed by MTT assays and flow cytometry. Recombinant RbBAFF protein (10 μg/mL) was able to prolong the survival and/or enhance the proliferation of rock bream lymphocytes by approximately 30%. Transcription of IL-10 and NFκB-1 was significantly stimulated by RbBAFF. Our findings provide further information regarding fish BAFF gene and its role in adaptive immunity.  

4.1496           Activation of Kupffer Cells Is Associated with a Specific Dysbiosis Induced by Fructose or High Fat Diet in Mice

Ferrere, G., Leroux, A., Wrzosek, L., Puchols, V., Gaudin, F., Ciocan, D., Renoud, M-L., Naveau, S., Perlemuter, G. and Cassars, A-M. PloS One, 11(1), e0146177 (2016)   The increase consumption of fructose in diet is associated with liver inflammation. As a specific fructan substrate, fructose may modify the gut microbiota which is involved in obesity-induced liver disease. Here, we aimed to assess whether fructose-induced liver damage was associated with a specific dysbiosis, especially in mice fed a high fat diet (HFD). To this end, four groups of mice were fed with normal and HFD added or not with fructose. Body weight and glucose sensitivity, liver inflammation, dysbiosis and the phenotype of Kupffer cells were determined after 16 weeks of diet. Food intake was increased in the two groups of mice fed with the HFD. Mice fed with HFD and fructose showed a higher infiltration of lymphocytes into the liver and a lower inflammatory profile of Kupffer cells than mice fed with the HFD without fructose. The dysbiosis associated with diets showed that fructose specifically prevented the decrease of Mouse intestinal bacteria in HFD fed mice and increased Erysipelotrichi in mice fed with fructose, independently of the amount of fat. In conclusion, fructose, used as a sweetener, induced a dysbiosis which is different in presence of fat in the diet. Consequently, the activation of Kupffer cells involved in mice model of HFD-induced liver inflammation was not observed in an HFD/fructose combined diet. These data highlight that the complexity of diet composition could highly impact the development of liver lesions during obesity. Specific dysbiosis associated with the diet could explain that the progressions of liver damage are different.  

4.1497           HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species

Kivi, G., Teesalu, K., parik, J., Kontkar, E., Ustav Jr., M. and Männik, A. BMC Biotechnol., 16:2 (2016)   Background The production of recombinant monoclonal antibodies in mammalian cell culture is of high priority in research and medical fields. A critical step in this process is the isolation of the antigen-binding domain sequences of antibodies possessing the desired properties. Many different techniques have been described to achieve this goal, but all have shortcomings; most techniques have problems with robustness, are time-consuming and costly, or have complications in the transfer from isolation to production phase. Here, we report a novel HybriFree technology for the development of monoclonal antibodies from different species that is robust, rapid, inexpensive and flexible and can be used for the subsequent production of antibodies in mammalian cell factories. Results HybriFree technology is illustrated herein via detailed examples of isolating mouse, rabbit and chicken monoclonal antibody sequences from immunized animals. Starting from crude spleen samples, antigen capturing of specific B-cells is performed initially. cDNA of antibody variable domains is amplified from the captured cells and used a source material for simple and rapid restriction/ligation free cloning of expression vector library in order to produce scFv-Fc or intact IgG antibodies. The vectors can be directly used for screening purposes as well as for the subsequent production of the developed monoclonal antibodies in mammalian cell culture. The antibodies isolated by the method have been shown to be functional in different immunoassays, including ELISA, immunofluorescence and Western blot. In addition, we demonstrate that by using a modified method including a negative selection step, we can isolate specific antibodies targeting the desired epitope and eliminate antibodies directed to undesired off-targets. Conclusions HybriFree can be used for the reliable development of monoclonal antibodies and their subsequent production in mammalian cells. This simple protocol requires neither the culturing of B-cells nor single-cell manipulations, and only standard molecular biology laboratory equipment is needed. In principle, the method is applicable to any species for which antibody cDNA sequence information is available.  

4.1498           Telomerase reverse transcriptase acts in a feedback loop with NF-κB pathway to regulate macrophage polarization in alcoholic liver disease

Wu, W-q., Yang, Y., Li, W-x., Cheng, Y-h., Li, X-f., Huang, C.,Meng, X-m., Wu, B-m., Liu, X-h., Zhang, L., Lv, X-w. and Li, J. Scientific Reports, 6:18685 (2016)   Activation of Kupffer cells (KCs) plays a central role in the pathogenesis of alcoholic liver disease (ALD). C57BL/6 mice fed EtOH-containing diet showed a mixed induction of hepatic classical (M1) and alternative (M2) macrophage markers. Since telomerase activation occurs at critical stages of myeloid and lymphoid cell activation, we herein investigated the role of telomerase reverse transcriptase (TERT), the determining factor of telomerase, in macrophage activation during ALD. In our study, TERT expression and telomerase activity (TA) were remarkably increased in liver tissue of EtOH-fed mice. Moreover, EtOH significantly up-regulated TERT in isolated KCs and RAW 264.7 cells and LPS induced TERT production in vitro. These data indicate that up-regulation of TERT may play a critical role in macrophages during ALD. Furthermore, loss- and gain-of-function studies suggested that TERT switched macrophages towards M1 phenotype by regulating NF-κB signaling, but had limited effect on M2 macrophages polarization in vitro. Additionally, PDTC, a chemical inhibitor of NF-κB, could dramatically down-regulate TERT expression and the hallmarks of M1 macrophages. Therefore, our study unveils the role of TERT in macrophage polarization and the cross-talk between TERT and p65, which may provide a possible explanation for the ethanol-mediated hepatic proinflammatory response and M1 macrophage polarization.  

4.1499           Analyses of movement and contact of two nucleated cells using a gas-driven micropipette aspiration technique

Yang, H., Tong, C., Fu, C., Xu, Y., Liu, X., Chen, Q., Zhang, Y., Lü, S., Li, N. and Long, M.

1. Immunol. Methods, 428, 20-29 (2016)

  Adhesion between two nucleated cells undergoes specific significances in immune responses and tumor metastasis since cellular adhesive molecules usually express on two apposed cell membranes. However, quantification of the interactions between two nucleated cells is still challenging in microvasculature. Here distinct cell systems were used, including three types of human cells (Jurkat cell or PMN vs. MDA-MB-231 cell) and two kinds of murine native cells (PMN vs. liver sinusoidal endothelial cell). Cell movement, compression to, and relaxation from the counterpart cell were quantified using an in-house developed gas-driven micropipette aspiration technique (GDMAT). This assay is robust to quantify this process since cell movement and contact inside a pipette are independent of the repeated test cycles. Measured approaching or retraction velocity follows well a normal distribution, which is independent on the cycle period. Contact area or duration also fits a Gaussian distribution and moreover contact duration is linearly correlated with the cycle period. Cell movement is positively related to gas flux but negatively associated to medium viscosity. Cell adhesion tends to reach an equilibrium state with increase of cycle period or contact duration. These results further the understanding in the dynamics of cell movement and contact in microvasculature.    

4.1500           Endogenous adaptation to low oxygen modulates T-cell regulatory pathways in EAE

Esen, N., katyshev, V., Serkin, Z., Kaytsheva, S. and Dore-Duffy, P.

1. Neuroinflammation, 13:13 (2016)

  Background In the brain, chronic inflammatory activity may lead to compromised delivery of oxygen and glucose suggesting that therapeutic approaches aimed at restoring metabolic balance may be useful. In vivo exposure to chronic mild normobaric hypoxia (10 % oxygen) leads to a number of endogenous adaptations that includes vascular remodeling (angioplasticity). Angioplasticity promotes tissue survival. We have previously shown that induction of adaptive angioplasticity modulates the disease pattern in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). In the present study, we define mechanisms by which adaptation to low oxygen functionally ameliorates the signs and symptoms of EAE and for the first time show that tissue hypoxia may fundamentally alter neurodegenerative disease. Methods C57BL/6 mice were immunized with MOG, and some of them were kept in the hypoxia chambers (day 0) and exposed to 10 % oxygen for 3 weeks, while the others were kept at normoxic environment. Sham-immunized controls were included in both hypoxic and normoxic groups. Animals were sacrificed at pre-clinical and peak disease periods for tissue collection and analysis. Results Exposure to mild hypoxia decreased histological evidence of inflammation. Decreased numbers of cluster of differentiation (CD)4+ T cells were found in the hypoxic spinal cords associated with a delayed Th17-specific cytokine response. Hypoxia-induced changes did not alter the sensitization of peripheral T cells to the MOG peptide. Exposure to mild hypoxia induced significant increases in anti-inflammatory IL-10 levels and an increase in the number of spinal cord CD25+FoxP3+ T-regulatory cells. Conclusions Acclimatization to mild hypoxia incites a number of endogenous adaptations that induces an anti-inflammatory milieu. Further understanding of these mechanisms system may pinpoint possible new therapeutic targets to treat neurodegenerative disease.  

4.1501           IgG-Immune Complexes Promote B Cell Memory by Inducing BAFF

Kang, S.A., Keener, A.B., Jones, S.Z., Benschop, R.J., Caro-Maldonado, A., Rathmell, J.C., Clarke, S.H., Matsushima, G.K., Whitmire, J.K. and Vilen, B.J.

1. Immunol., 196(1), 196-206 (2016)

  Memory B cell responses are vital for protection against infections but must also be regulated to prevent autoimmunity. Cognate T cell help, somatic hypermutation, and affinity maturation within germinal centers (GCs) are required for high-affinity memory B cell formation; however, the signals that commit GC B cells to the memory pool remain unclear. In this study, we identify a role for IgG-immune complexes (ICs), FcγRs, and BAFF during the formation of memory B cells in mice. We found that early secretion of IgG in response to immunization with a T-dependent Ag leads to IC–FcγR interactions that induce dendritic cells to secrete BAFF, which acts at or upstream of Bcl-6 in activated B cells. Loss of CD16, hematopoietic cell–derived BAFF, or blocking IC:FcγR regions in vivo diminished the expression of Bcl-6, the frequency of GC and memory B cells, and secondary Ab responses. BAFF also contributed to the maintenance and/or expansion of the follicular helper T cell population, although it was dispensable for their formation. Thus, early Ab responses contribute to the optimal formation of B cell memory through IgG-ICs and BAFF. Our work defines a new role for FcγRs in GC and memory B cell responses.  

4.1502           Semaphorin 7A Promotes Chemokine-Driven Dendritic Cell Migration

Van Rijn, A., paulis, L., te Riet, J., Vasauturo, A., Reinieren-Beeren, I., van der Schaaf, A., Kuipers, A.J., Schulte, L.P., Jongbloets, B.C., Pasterkamp, R.J., Figdor, C.G., van Spriel, A.B. and Buschow, S.I.

1. Immunol., 196, 459-468 (2016)

  Dendritic cell (DC) migration is essential for efficient host defense against pathogens and cancer, as well as for the efficacy of DC-based immunotherapies. However, the molecules that induce the migratory phenotype of DCs are poorly defined. Based on a large-scale proteome analysis of maturing DCs, we identified the GPI-anchored protein semaphorin 7A (Sema7A) as being highly expressed on activated primary myeloid and plasmacytoid DCs in human and mouse. We demonstrate that Sema7A deficiency results in impaired chemokine CCL21-driven DC migration in vivo. Impaired formation of actin-based protrusions, resulting in slower three-dimensional migration, was identified as the mechanism underlying the DC migration defect. Furthermore, we show, by atomic force microscopy, that Sema7A decreases adhesion strength to extracellular matrix while increasing the connectivity of adhesion receptors to the actin cytoskeleton. This study demonstrates that Sema7A controls the assembly of actin-based protrusions that drive DC migration in response to CCL21.  

4.1503           Induction and regulation of murine emphysema by elastin peptides

Sellami, M., meghraoui-Kheddar, A., Terryn, C., Fichel, C., Bouland, N., Diebold, M-D., Guenounou, M., Hery-Huynh, S. and Le Naour, R. Am. J. Physiol. Lung Cell Mol. Physiol., 310, L8-L23 (2016)   Emphysema is the major component of chronic obstructive pulmonary disease (COPD). During emphysema, elastin breakdown in the lung tissue originates from the release of large amounts of elastase by inflammatory cells. Elevated levels of elastin-derived peptides (EP) reflect massive pulmonary elastin breakdown in COPD patients. Only the EP containing the GXXPG conformational motif with a type VIII β-turn are elastin receptor ligands inducing biological activities. In addition, the COOH-terminal glycine residue of the GXXPG motif seems a prerequisite to the biological activity. In this study, we endotracheally instilled C57BL/6J mice with GXXPG EP and/or COOH-terminal glycine deleted-EP whose sequences were designed by molecular dynamics and docking simulations. We investigated their effect on all criteria associated with the progression of murine emphysema. Bronchoalveolar lavages were recovered to analyze cell profiles by flow cytometry and lungs were prepared to allow morphological and histological analysis by immunostaining and confocal microscopy. We observed that exposure of mice to EP elicited hallmark features of emphysema with inflammatory cell accumulation associated with increased matrix metalloproteinases and desmosine expression and of remodeling of parenchymal tissue. We also identified an inactive COOH-terminal glycine deleted-EP that retains its binding-activity to EBP and that is able to inhibit the in vitro and in vivo activities of emphysema-inducing EP. This study demonstrates that EP are key actors in the development of emphysema and that they represent pharmacological targets for an alternative treatment of emphysema based on the identification of EP analogous antagonists by molecular modeling studies.  

4.1504           Biotin-conjugated fusogenic liposomes for high-quality cell purification

Hersch, N., Wolters, B., Ungvari, Z., Gautam, T., Deshpande, D., Merkel, R., Cziszar, A., Hoffmann, B. and Cziszar, A.

1. Biomater. Appl., 30(6), 846-856 (2016)

  Purification of defined cell populations from mixed primary cell sources is essential for many biomedical and biotechnological applications but often very difficult to accomplish due to missing specific surface markers. In this study, we developed a new approach for efficient cell population separation based on the specific membrane fusion characteristics of distinct cell types upon treatment with fusogenic liposomes. When such liposomes are conjugated with biotin, specific cell populations can be efficiently surface functionalized by biotin after liposomal treatment while other populations remain unlabeled. Due to the high affinity of biotin for avidin-like proteins, biotin functionalized cells are ideal targets for conjugation of e.g. avidin tagged magnetic beads, fluorophores or antibodies with bioanalytical relevance. Here, based on the differential biotinylation of distinct cell populations high quality separation of cardiac fibroblasts from myocytes, and cerebromicrovascular endothelial cells from fibroblasts was successfully established.  

4.1505           Post-thaw ATP supplementation enhances cryoprotective effect of iodixanol in rat spermatozoa

Kim, S., Hooper, S., Agca, C. and Agca, Y. Reproductive Biol.and Endocrinol., 14:5 (2016)   Background Successful cryopreservation of rat spermatozoa from various strains still remains a challenge. The objective of this study was to determine if combinations of OptiPrep™ (iodixanol) and adenosine 5′-triphosphate (ATP) can improve rat sperm function during the cryopreservation procedure. Methods Epididymal rat spermatozoa were frozen under different OptiPrep™ concentrations (0, 1, 2, 3 or 4 %) and were diluted with media supplemented with or without 2 mM ATP after thawing. Post-thaw sperm motility, acrosomal membrane integrity (AMI) and mitochondrial membrane potential (MMP) were then evaluated. In addition, the effect of different OptiPrep™ concentrations on fresh and cooled rat spermatozoa was tested via motility. Results There was no effect of OptiPrep™ on motility of fresh and cooled spermatozoa. The supplementation of 1 and 2 % OptiPrep™ increased motility of frozen spermatozoa at 10 min after thawing, while it did not improve motility of spermatozoa at 3 h after thawing in the absence of ATP. During incubation of thawed spermatozoa, the ATP addition protected time-dependent decrease in motility after thawing in OptiPrep™-treated samples. OptiPrep™ had no effect on AMI and MMP in frozen-thawed spermatozoa but combinations of OptiPrep™ and ATP improved MMP in frozen-thawed spermatozoa. Conclusions Iodixanol has cryoprotective effects during rat sperm freezing without any toxic effect. Moreover, the combinations of iodixanol and ATP have a beneficial role in maintaining function of frozen-thawed rat spermatozoa for long period of incubation post-thaw.  

4.1506           Proteomic Analysis of Dynein-Interacting Proteins in Amyotrophic Lateral Sclerosis Synaptosomes Reveals Alterations in the RNA-Binding Protein Staufen1

Gershoni-Emek, N., Mazza, A., Chein, M., Gradus-Pery, T., Xiang, X., Wan, K., Sharan, R. and Perlson, E. Mol. Cell. Proteomics, 15, 506-522 (2016)   Synapse disruption takes place in many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the mechanistic understanding of this process is still limited. We set out to study a possible role for dynein in synapse integrity. Cytoplasmic dynein is a multisubunit intracellular molecule responsible for diverse cellular functions, including long-distance transport of vesicles, organelles, and signaling factors toward the cell center. A less well-characterized role dynein may play is the spatial clustering and anchoring of various factors including mRNAs in distinct cellular domains such as the neuronal synapse. Here, in order to gain insight into dynein functions in synapse integrity and disruption, we performed a screen for novel dynein interactors at the synapse. Dynein immunoprecipitation from synaptic fractions of the ALS model mSOD1^G93A and wild-type controls, followed by mass spectrometry analysis on synaptic fractions of the ALS model mSOD1^G93A and wild-type controls, was performed. Using advanced network analysis, we identified Staufen1, an RNA-binding protein required for the transport and localization of neuronal RNAs, as a major mediator of dynein interactions via its interaction with protein phosphatase 1–beta (PP1B). Both in vitro and in vivo validation assays demonstrate the interactions of Staufen1 and PP1B with dynein, and their colocalization with synaptic markers was altered as a result of two separate ALS-linked mutations: mSOD1^G93A and TDP43^A315T. Taken together, we suggest a model in which dynein's interaction with Staufen1 regulates mRNA localization along the axon and the synapses, and alterations in this process may correlate with synapse disruption and ALS toxicity.  

4.1507           Combination strategy of multi-layered surface camouflage using hyperbranched polyethylene glycol and immunosuppressive drugs for the prevention of immune reactions against transplanted porcine islets

Haque, M.R., Jeong, J-H. and Byun, Y. Biomaterials, 84, 144-156 (2016)   This study suggests a novel method of stabilizing fragile porcine islets to prevent the dissociation after isolation and reducing immune cell invasion in a combination therapy of ‘surface camouflaging’ and immunosuppressive drugs (FK506, Rapamycin, MR-1, anti-CD19 mAb, and Clodrosome^®) to effectively alleviate overall immune reactions against xenotransplanted porcine islets. The surface camouflage of pancreatic islets using biocompatible materials improved stabilization of pancreatic islet and prevented the infiltration of immune cells. Firstly, the surface of porcine islets was camouflaged by SH-6-arm-PEG-lipid and gelatin-catechol (artificial extracellular matrix) in order to stabilize the fragile isolated islets. Secondly, three different PEG layers (6-arm-PEG-SH, 6-arm-PEG-catechol, and linear PEG-SH) were chemically conjugated onto the surface of the stabilized porcine islets. Both artificial extracellular matrix (artificial ECM) and PEGylation effectively covered the surface of porcine islets without increasing the size of the whole islet. In addition, the viability and functionality of the islets were not affected by this multi-layer surface modification. The multi-layer modification significantly reduced the attachment of human serum albumin, fibronectin, and immunoglobulin G in comparison to the control collagen surface. The combination effect of multi-layer PEGylation and cocktailed immunosuppressive drugs on the survival time of the transplanted islets was assessed in a xenogeneic porcine-to-mouse model. The median survival time (MST) of ‘artificial ECM + PEGylation' group was 4-fold increased compared to that of control group. In addition, the MST of ‘artificial ECM + PEGylation + drug' group was 2.16-fold increased, compared to the ‘control + drug’ group. In conclusion, we proposed a novel porcine islet transplantation protocol using surface multi-layer modification and cocktailed immunosuppressive drugs, for stabilization and immunoprotection against xenogeneic immune reactions.  

4.1508           Co-culture of Hepatocytes and Kupffer Cells as an In Vitro Model of Inflammation and Drug-Induced Hepatotoxicity

Rose, K.A., Holman, N.S., Green, A.M., Andersen, M.E. and LeCluyse, E.L.

1. Pharmaceut. Sci., 105, 950-964 (2016)

  Immune-mediated drug-induced hepatotoxicity is often unrecognized as a potential mode of action due to the lack of appropriate in vitro models. We have established an in vitro rat donor-matched hepatocyte and Kupffer cell co-culture (HKCC) model to study immune-related responses to drug exposure. Optimal cell culture conditions were identified for the maintenance of co-cultures based on cell longevity, monolayer integrity, and cytokine response after lipopolysaccharide (LPS) exposure. Hepatocyte monocultures and HKCCs were then used to test a subset of compounds associated with hepatotoxic effects with or without LPS. Cytokine levels and metabolic activity (cytochrome P450 3A [Cyp3A]) were measured after a 48-h exposure to monitor endotoxin-induced changes in acute phase and functional end points. LPS-activated HKCCs, but not hepatocyte monocultures, treated with trovafloxacin or acetaminophen, compounds associated with immune-mediated hepatotoxicity, showed LPS-dependent decreases in interleukin-6 production with concomitant increases in Cyp3A activity. Differential endotoxin- and model-dependent alterations were observed in cytokine profiles and Cyp3A activity levels that corresponded to specific compounds. These results indicate the utility of the HKCC model system to discern compound-specific effects that may lead to enhanced or mitigate hepatocellular injury due to innate or adaptive immune responses.  

4.1509           Pregnancy outcomes using stallion epididymal sperm stored at 5 °C for 24 or 48 hours before harvest

Stawicki, R.J., McDonnell, S.M., Giguere, S. and Turner, R.M. Theriogenology, 85, 698-702 (2016)   The cryopreservation of epididymal sperm can be useful in a variety of circumstances for ensuring genetic preservation of a valued stallion. Although early studies have reported pregnancy rates significantly lower than those achieved with cryopreserved ejaculated sperm, two recent studies report over 60% one-cycle pregnancy rates with epididymal sperm stored for 24 hours at 5 °C before harvest and cryopreservation. The aims of this study were to: (1) attempt to replicate the one-cycle pregnancy rate of over 60% using epididymal sperm cooled and stored within the epididymis for 24 hours before harvest and cryopreservation and (2) evaluate pregnancy outcome with sperm cooled and stored within the epididymis for 48 hours before sperm harvest and cryopreservation. Testicles were obtained from 13 stallions undergoing routine castration. The epididymides were stored at 5 °C for either 24 or 48 hours before sperm harvest and cryopreservation in an egg yolk and dimethylformamide-based freezing extender. Thirteen mares were bred on one cycle with cryopreserved epididymal sperm stored for 24 hours before harvest, and 10 of those 13 mares were also bred on a previous or subsequent cycle with samples from the same stallion that had been stored for 48 hours before harvest. Pregnancy occurred in 7 of the 13 inseminations of sperm stored for 24 hours before harvest, and in 4 of the 10 inseminations of sperm stored for 48 hours before harvest. The pregnancy rate using epididymal sperm stored for 24 hours before harvest is consistent with that of previous reports. In addition, these results provide evidence that pregnancies can be achieved when the epididymides are cooled and stored for 48 hours before sperm harvest and cryopreservation.  

4.1510           Substitution of egg yolk by a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing medium of equine sperm

Blommaert, D., Franck, T., Donnay, I., Lejeune, J-P., Detilleux, J. and Serteyn, D. Cryobiology, 72, 27-32 (2016)   The aim of this work was to completely replace the egg yolk a classical diluent for freezing equine semen by a cyclodextrin-cholesterol complex. At the same time, the reduction in the glycerol content used for cryopreservation and the incubation time between sperm and the freezing media were evaluated. Horse ejaculates were frozen with four different freezing extenders: a frozen reference medium (IF) containing egg yolk and 2.5% glycerol and media without egg yolk but supplemented with 1.5 mg 2-hydroxypropyl-beta-cyclodextrin cholesterol (HPβCD-C) complex and containing either 1% (G1), 2% (G2) or 3% glycerol (G3). Three incubation times (90, 120 and 180 min) at 4 °C between the fresh semen and the different media were tested before freezing. Viability and motility analyses were performed with computer assisted semen analysis (CASA). Results showed that the freezing media containing the HPβCD–C complex with 1%, 2% and 3% glycerol significantly improve the 3 in vitro parameters of post thawing semen quality (viability, progressive and total mobilities) compared to IF. The best improvement of the parameters was obtained with G1 medium and the longest contact time. The substitution of egg yolk by HPβCD–C complex allows the decrease of protein charge of the medium while favouring the cholesterol supply to membrane spermatozoa offering it a better resistance to osmotic imbalance and a better tolerance to the glycerol toxicity. Our results highlight that the egg yolk of an extender for the freezing of horse semen can be completely substituted by HPβCD–C complex.  

4.1511           The Evolutionarily Conserved Tre2/Bub2/Cdc16 (TBC), Lysin Motif (LysM), Domain Catalytic (TLDc) Domain Is Neuroprotective against Oxidative Stress

Finelli, M.J., Sanchez-Pulido, L., Liu, K.X., Davies, K.E. and Oliver, P.L.

1. Biol. Chem., 291(6), 2751-2763 (2016)

  Oxidative stress is a pathological feature of many neurological disorders; therefore, utilizing proteins that are protective against such cellular insults is a potentially valuable therapeutic approach. Oxidation resistance 1 (OXR1) has been shown previously to be critical for oxidative stress resistance in neuronal cells; deletion of this gene causes neurodegeneration in mice, yet conversely, overexpression of OXR1 is protective in cellular and mouse models of amyotrophic lateral sclerosis. However, the molecular mechanisms involved are unclear. OXR1 contains the Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic (TLDc) domain, a motif present in a family of proteins including TBC1 domain family member 24 (TBC1D24), a protein mutated in a range of disorders characterized by seizures, hearing loss, and neurodegeneration. The TLDc domain is highly conserved across species, although the structure-function relationship is unknown. To understand the role of this domain in the stress response, we carried out systematic analysis of all mammalian TLDc domain-containing proteins, investigating their expression and neuroprotective properties in parallel. In addition, we performed a detailed structural and functional study of this domain in which we identified key residues required for its activity. Finally, we present a new mouse insertional mutant of Oxr1, confirming that specific disruption of the TLDc domain in vivo is sufficient to cause neurodegeneration. Our data demonstrate that the integrity of the TLDc domain is essential for conferring neuroprotection, an important step in understanding the functional significance of all TLDc domain-containing proteins in the cellular stress response and disease.  

4.1512           Characterization of an IgG monoclonal antibody targeted to both tissue cyst and sporocyst walls of Toxoplasma gondii

Gondim, L.F.P., Wolf, A., Vrhovec, M.G., pantchev, N., Bauer, C., langennayer, M.C., Bohne, W., Teifke, J.P., Dubey, J.P., Conratahs, F.J. and Schares, G. Exp. Parasitol., 163, 46-56 (2016)   Toxoplasma gondii infects animals habiting terrestrial and aquatic environments. Its oocysts and tissue cysts are important for the horizontal transmission of this parasite. The oocyst and tissue cyst walls are crucial for the ability of the parasite to persist in the environment or in animal tissues, respectively. However, the composition of these walls is not well understood. We report the generation of monoclonal antibodies directed against wall components using mice immunized with oocyst antigens of T. gondii. One monoclonal antibody (mAb) G1/19 reacted solely with T. gondii sporozoites. The respective antigen had a relative molecular weight (Mr) of 30 kDa. MAb G1/19 failed to react with sporozoites of any other coccidian parasite species tested (Hammondia hammondi, Hammondia heydorni, Cystoisospora felis, Eimeria bovis, Sarcocystis sp.). Another mAb, designated K8/15-15, recognized antigens in sporocyst walls of the parasite and in the walls of in vivo or in vitro produced tissue cysts, as demonstrated by immunofluorescence and immunoblot assays. Antigens of 80 to a high molecular weight protein of about 350 kDa Mr were recognized by this antibody using antigen extracts from sporocysts, and from in vitro or in vivo generated tissue cysts of the parasite. Tissue cyst and sporocyst walls of H. hammondi and H. heydorni, and tissue cysts of Neospora caninum were also recognized by mAb K8/15-15. Sporocyst walls of C. felis also reacted to this mAb. The cyst walls of Sarcocystis sp. and Besnoitia besnoiti were not recognized by mAb K8/15-15. Reactivity by a single mAb against T. gondii antigens in tissue cysts and sporocysts had not been reported previously. MAb K8/15-15 may be a practical tool for the identification of both cysts and sporocysts of the parasite, and may also be potentially employed in proteomic studies on the identification of new components of the cyst and sporocyst walls of T. gondii.  

4.1513           Spatial distribution of osteoblast activating peptide in the rat stomach

Noreldin, A.E., Sogabe, M., Yamano, Y., Uehara, M., Mahdy, M.A.A, Elnasharty, M.A., Sayed-Ahmed, A., Warita, K. and Hosaka, Y.Z. Acta Histochemica, 118, 109-117 (2016)   Osteoblast activating peptide (OBAP) was previously reported to be expressed in the rat stomach and to have a vital role in osteogenesis, but its distribution in rat stomach has not been determined. Thus, the aim of the present study was to identify the cell types expressing OBAP in the rat stomach. The stomachs of twelve 10-to-11-week-old male Jc1:SD rats were used. Samples were collected for immunohistochemistry, immunoelectron microscopy and dot blot assay. Immunohistochemical investigation revealed that OBAP was distributed mainly in parietal cells without any expression in chief cells, X/A-like cells or enterochromaffin-like cells. Moreover, OBAP-immunopositive cells were observed mainly in the upper and lower parts of the gastric gland. Significantly high optical density of immunopositive cells was observed in the upper and lower gastric gland regions. The dot blot assay confirmed that OBAP is secreted by parietal cells and that it is present in the gastric gland lumen. Immunoelectron microscopy demonstrated that OBAP was confined to the mitochondrial inner membrane within parietal cells and that the number of mitochondria in the upper and lower parts of the gastric epithelium was significantly larger than the number in the middle part of the gastric epithelium. Based on the results, it was concluded that OBAP is mainly produced by mitochondria of parietal cells in the upper and lower parts of the gastric epithelium. Moreover, the presence of OBAP in the gastric gland lumen suggests an exocrine mechanism of release.  

4.1514           Hepatic stellate cell transdifferentiation involves genome-wide remodeling of the DNA methylation landscape

Page, A., Paoli, P., Salvador, E.M., White, S., French, J. and Mann, J.

1. Hepatol., 64, 661-673 (2016)

  Background & Aims DNA methylation (5-mC) is an epigenetic mark that is an established regulator of transcriptional repression with an important role in liver fibrosis. Currently, there is very little knowledge available as to how DNA methylation controls the phenotype of hepatic stellate cell (HSC), the key cell type responsible for onset and progression of liver fibrosis. Moreover, recently discovered DNA hydroxymethylation (5-hmC) is involved in transcriptional activation and its patterns are often altered in human diseases. The aim of this study is to investigate the role of DNA methylation/hydroxymethylation in liver fibrosis. Methods Levels of 5-mC and 5-hmC were assessed by slot blot in a range of animal liver fibrosis models and human liver diseases. Expression levels of TET and DNMT enzymes were measured by qRT-PCR and Western blotting. Reduced representation bisulfite sequencing (RRBS) method was used to examine 5-mC and 5-hmC patterns in quiescent and in vivo activated rat HSC. Results We demonstrate global alteration in 5-mC and 5-hmC and their regulatory enzymes that accompany liver fibrosis and HSC transdifferentiation. Using RRBS, we show exact genomic positions of changed methylation patterns in quiescent and in vivo activated rat HSC. In addition, we demonstrate that reduction in DNMT3a expression leads to attenuation of pro-fibrogenic phenotype in activated HSC. Conclusions Our data suggest that DNA 5-mC/5-hmC is a crucial step in HSC activation and therefore fibrogenesis. Changes in DNA methylation during HSC activation may bring new insights into the molecular events underpinning fibrogenesis and may provide biomarkers for disease progression as well as potential new drug targets.  

4.1515           Determination and Characterization of Tetraspanin-Associated Phosphoinositide-4 Kinases in Primary and Neoplastic Liver Cells

Rombouts, K. and Carloni, V. Methods in Mol. Biol., 1376, 203-212 (2016)   Accumulating evidence implicates phosphoinositide 4-phosphate as a regulatory molecule in its own right recruiting specific effector proteins to cellular membranes. Here, we describe biochemical and immunocytochemical methods to evaluate tetraspanin-associated phosphoinositide-4 kinases activity in primary human hepatic stellate cells (hHSC) and neoplastic hepatoblastoma cells.    

4.1516           Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints

Koyama, S. et al Nature Communications, 7:10501 (2016)   Despite compelling antitumour activity of antibodies targeting the programmed death 1 (PD-1): programmed death ligand 1 (PD-L1) immune checkpoint in lung cancer, resistance to these therapies has increasingly been observed. In this study, to elucidate mechanisms of adaptive resistance, we analyse the tumour immune microenvironment in the context of anti-PD-1 therapy in two fully immunocompetent mouse models of lung adenocarcinoma. In tumours progressing following response to anti-PD-1 therapy, we observe upregulation of alternative immune checkpoints, notably T-cell immunoglobulin mucin-3 (TIM-3), in PD-1 antibody bound T cells and demonstrate a survival advantage with addition of a TIM-3 blocking antibody following failure of PD-1 blockade. Two patients who developed adaptive resistance to anti-PD-1 treatment also show a similar TIM-3 upregulation in blocking antibody-bound T cells at treatment failure. These data suggest that upregulation of TIM-3 and other immune checkpoints may be targetable biomarkers associated with adaptive resistance to PD-1 blockade.  

4.1517           All-Trans Retinoic Acid Induces Expression of a Novel Intergenic Long Noncoding RNA in Adult rat Primary Hippocampal Neurons

Kour, S. and Rath, P.C.

1. Mol. Sci., 58(2), 266-276 (2016)

  Around 90 % of the mammalian genome undergoes pervasive transcription into various types of small and long regulatory noncoding RNAs, whereas only ∼1.5 % codes for proteins. Long noncoding RNAs (lncRNAs) constitute diverse classes of sense- and antisense transcripts that are abundantly expressed in the mammalian central nervous system (CNS) in cell type- and developmental stage-specific manners. They are implicated in brain development, differentiation, neuronal plasticity, and other cognitive functions. Mammalian brain requires the vitamin A metabolite all-trans retinoic acid (atRA) for its normal development, differentiation, and cell-fate determination. However, its role in adult brain function is less understood. Here, we report atRA-mediated transcriptional upregulation of endogenous expression of a novel long intergenic noncoding RNA-rat brain expressed (LINC-RBE) in cultured primary hippocampal neurons from adult rat. We have previously reported LINC-RBE as an intergenic, simple repeat sequence containing lncRNA highly expressed in the rat brain. This is a first-time report of involvement of atRA in transcriptional upregulation of lncRNA expression in rat hippocampal neurons. Therefore, it may be involved in regulation of brain function and disease.  

4.1518           Biophysical changes reduce energetic demand in growth factor–deprived lymphocytes

Hecht, V.C., Sullivan, L.B., Kimmerling, R.J., Kim, D-H., Hosios, A.M., Stockslager, M.A., Stevens, M.M., Kang, J.H., Wirtz, D., Vander heiden, M.G. and Manalis, S.R.

1. Cell Biol., 212(4), 439-447 (2016)

  Cytokine regulation of lymphocyte growth and proliferation is essential for matching nutrient consumption with cell state. Here, we examine how cellular biophysical changes that occur immediately after growth factor depletion promote adaptation to reduced nutrient uptake. After growth factor withdrawal, nutrient uptake decreases, leading to apoptosis. Bcl-x_L expression prevents cell death, with autophagy facilitating long-term cell survival. However, autophagy induction is slow relative to the reduction of nutrient uptake, suggesting that cells must engage additional adaptive mechanisms to respond initially to growth factor depletion. We describe an acute biophysical response to growth factor withdrawal, characterized by a simultaneous decrease in cell volume and increase in cell density, which occurs before autophagy initiation and is observed in both FL5.12 Bcl-x_L cells depleted of IL-3 and primary CD8⁺ T cells depleted of IL-2 that are differentiating toward memory cells. The response reduces cell surface area to minimize energy expenditure while conserving biomass, suggesting that the biophysical properties of cells can be regulated to promote survival under conditions of nutrient stress.  

4.1519           Hepatic stellate cells regulate liver immunity to visceral leishmaniasis through P110δ-dependent induction and expansion of regulatory T cells in mice

Khadem, F., Gao, X., Mou, Z., Jia, P., Movassagh, H., Onyilagha, C., Gounni, A.S., Wright, M.C. and Uzonna, J.E. Hepatology, 63(2), 620-632 (2016)   Visceral leishmaniasis (VL) is associated with severe immune dysfunction and if untreated leads to death. Because the liver is one of the primary target organs in VL, unraveling the mechanisms governing the local hepatic immune response is important for understanding the immunopathogenesis of VL. We previously reported that mice with inactivating knockin mutation in the p110δ gene (p110δ^D910A) are resistant to VL, due in part to impaired regulatory T-cell (Treg) expansion. In this study, we investigated the mechanism of this resistance by focusing on hepatic stellate cells (HSCs), which are known to regulate Treg induction and expansion. We show that HSCs are infected with Leishmania donovani in vivo and in vitro and that this infection leads to the production of interleukin-2, interleukin-6, and transforming growth factor-β, cytokines known to induce Tregs. We further demonstrate that L. donovani infection leads to expansion of HSCs in a p110δ-dependent manner and that this correlated with proliferation of hepatic Tregs in vivo. In vitro studies clearly show that L. donovani–infected HSCs induce CD4⁺ T cells to become Tregs and expand Tregs in a p110δ-dependent manner. Targeted depletion of HSCs during infection caused a dramatic reduction in liver Treg numbers and proliferation, which was associated with a decrease in interleukin-10 production by hepatic T cells and a more efficient parasite control. Conclusion: These results demonstrate the critical role of HSCs in the pathogenesis of VL and suggest that the enhanced resistance of p110δ^D910A mice to L. donovani infection is due in part to impaired expansion and inability of their HSCs to induce and expand Tregs in the liver.  

4.1520           Role of TGF-β signaling in differentiation of mesothelial cells to vitamin A-poor hepatic stellate cells in liver fibrosis

Li, Y., Lua, I., French, S.W. and Asahina, K. Am. J. Physiol. Gastrointest. Liver Physiol., 310, G262-G272 (2016)   Mesothelial cells (MCs) form a single layer of the mesothelium and cover the liver surface. A previous study demonstrated that, upon liver injury, MCs migrate inward from the liver surface and give rise to hepatic stellate cells (HSCs) in biliary fibrosis induced by bile duct ligation (BDL) or myofibroblasts in CCl₄-induced fibrosis. The present study analyzed the role of transforming growth factor-β (TGF-β) signaling in mesothelial-mesenchymal transition (MMT) and the fate of MCs during liver fibrosis and its regression. Deletion of TGF-β type II receptor (Tgfbr2) gene in cultured MCs suppressed TGF-β-mediated myofibroblastic conversion. Conditional deletion of Tgfbr2 gene in MCs reduced the differentiation of MCs to HSCs and myofibroblasts in the BDL and CCl₄ models, respectively, indicating that the direct TGF-β signaling in MCs is responsible to MMT. After BDL and CCl₄ treatment, MC-derived HSCs and myofibroblasts were distributed near the liver surface and the thickness of collagen was increased in Glisson's capsule beneath the liver surface. Fluorescence-activated cell sorting analysis revealed that MC-derived HSCs and myofibroblasts store little vitamin A lipids and have fibrogenic phenotype in the fibrotic livers. MCs contributed to 1.4 and 2.0% of activated HSCs in the BDL and CCl₄ models, respectively. During regression of CCl₄-induced fibrosis, 20% of MC-derived myofibroblasts survived in the liver and deactivated to vitamin A-poor HSCs. Our data indicate that MCs participate in capsular fibrosis by supplying vitamin A-poor HSCs during a process of liver fibrosis and regression.  

4.1521           Increases in core temperature counterbalance effects of haemoconcentration on blood viscosity during prolonged exercise in the heat

Buono, M.J., Krippes, T., Kolkhorst, F.W., Williams, A.T. and Cabrales, P. Exp. Physiol., 101(2), 332-342 (2016)   Previous studies have reported that blood viscosity is significantly increased following exercise. However, these studies measured both pre- and postexercise blood viscosity at 37°C even though core and blood temperatures would be expected to have increased during the exercise. Consequently, the effect of exercise-induced hyperthermia on mitigating change in blood viscosity may have been missed. The purpose of this study was to isolate the effects of exercise-induced haemoconcentration and hyperthermia and to determine their combined effects on blood viscosity. Nine subjects performed 2 h of moderate-intensity exercise in the heat (37°C, 40% relative humidity), which resulted in significant increases from pre-exercise values for rectal temperature (from 37.11 ± 0.35 to 38.76 ± 0.13°C), haemoconcentration (haematocrit increased from 43.6 ± 3.6 to 45.6 ± 3.5%) and dehydration (change in body weight = −3.6 ± 0.7%). Exercise-induced haemoconcentration significantly (P < 0.05) increased blood viscosity by 9% (from 3.97 to 4.33 cP at 300 s⁻¹), whereas exercise-induced hyperthermia significantly decreased blood viscosity by 7% (from 3.97 to 3.69 cP at 300 s⁻¹). When both factors were considered together, there was no overall change in blood viscosity (from 3.97 to 4.03 cP at 300 s⁻¹). The effects of exercise-induced haemoconcentration, increased plasma viscosity and increased red blood cell aggregation, all of which increased blood viscosity, were counterbalanced by increased red blood cell deformability (e.g. red blood cell membrane shear elastic modulus and elongation index) caused by the hyperthermia. Thus, blood viscosity remained unchanged following prolonged moderate-intensity exercise in the heat.  

4.1522           Sonic hedgehog stimulates neurite outgrowth in a mechanical stretch model of reactive-astrogliosis

Berretta, A., Gowing, E.K., Jasoni, C.L. and Clarkson, A.N. Scientific Reports, 6:21896 (2016)   Although recovery following a stroke is limited, undamaged neurons under the right conditions can establish new connections and take on-board lost functions. Sonic hedgehog (Shh) signaling is integral for developmental axon growth, but its role after injury has not been fully examined. To investigate the effects of Shh on neuronal sprouting after injury, we used an in vitro model of glial scar, whereby cortical astrocytes were mechanically traumatized to mimic reactive astrogliosis observed after stroke. This mechanical trauma impaired neurite outgrowth from post-natal cortical neurons plated on top of reactive astrocytes. Addition of Shh to the media, however, resulted in a concentration-dependent increase in neurite outgrowth. This response was inhibited by cyclopamine and activated by oxysterol 20(S)-hydroxycholesterol, both of which modulate the activity of the Shh co-receptor Smoothened (Smo), demonstrating that Shh-mediated neurite outgrowth is Smo-dependent. In addition, neurite outgrowth was not associated with an increase in Gli-1 transcription, but could be inhibited by PP2, a selective inhibitor of Src family kinases. These results demonstrate that neurons exposed to the neurite growth inhibitory environment associated with a glial scar can be stimulated by Shh, with signaling occurring through a non-canonical pathway, to overcome this suppression and stimulate neurite outgrowth.  

4.1523           8-Oxoguanine accumulation in mitochondrial DNA causes mitochondrial dysfunction and impairs neuritogenesis in cultured adult mouse cortical neurons under oxidative conditions

Leon, J., Sakumi. K., Castillo, E., Sheng, Z., Oka, S. and Makabeppy, Y. Scientific Reports, 6:22086 (2016)   Oxidative stress and mitochondrial dysfunction are implicated in aging-related neurodegenerative disorders. 8-Oxoguanine (8-oxoG), a common oxidised base lesion, is often highly accumulated in brains from patients with neurodegenerative disorders. MTH1 hydrolyses 8-oxo-2′-deoxyguanosine triphosphate (8-oxo-dGTP) to 8-oxo-dGMP and pyrophosphate in nucleotide pools, while OGG1 excises 8-oxoG paired with cytosine in DNA, thereby minimising the accumulation of 8-oxoG in DNA. Mth1/Ogg1-double knockout (TO-DKO) mice are highly susceptible to neurodegeneration under oxidative conditions and show increased accumulation of 8-oxoG in mitochondrial DNA (mtDNA) in neurons, suggesting that 8-oxoG accumulation in mtDNA causes mitochondrial dysfunction. Here, we evaluated the contribution of MTH1 and OGG1 to the prevention of mitochondrial dysfunction during neuritogenesis in vitro. We isolated cortical neurons from adult wild-type and TO-DKO mice and maintained them with or without antioxidants for 2 to 5 days and then examined neuritogenesis. In the presence of antioxidants, both TO-DKO and wild-type neurons exhibited efficient neurite extension and arborisation. However, in the absence of antioxidants, the accumulation of 8-oxoG in mtDNA of TO-DKO neurons was increased resulting in mitochondrial dysfunction. Cells also exhibited poor neurite outgrowth with decreased complexity of neuritic arborisation, indicating that MTH1 and OGG1 are essential for neuritogenesis under oxidative conditions.  

4.1524           Adoptive transferred hepatic stellate cells attenuated drug-induced liver injury by modulating the rate of regulatory T cells/T helper 17 cells

Feng, M., Wang, Q., Jiang, z., Ding, J., Wang, H., Wang, M., Lu, L. and Guan, W. Clin. Immunol., 165, 12-18 (2016)   Our study showed that hepatic stellate cells (HSCs) promote the healing of the liver after drug-induced acute injury. However, the relevant mechanisms by which this is accomplished remain unclear. The objective of this study was to investigate the role of the adoptive transfer of HSCs in acute liver injury and the underlying mechanisms for healing. It was found that adoptive transfer of HSCs resulted in an increase in Tregs and a decrease in Th17 cells. Liver insult was consistently attenuated by HSC treatment. HSC cultured medium induced Tregs from naive T cells and suppressed the differentiation of Th17 cells. This study demonstrated that the adoptive transfer of HSCs protected the liver from drug-induced acute injury. Promoting the differentiation of Tregs and suppressing the development of Th17 cells are possibly involved in the protective effect of adoptive transfer of HSCs.  

4.1525           Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation

Balamurugan, A.N., Green, M.L., Breite, A.G., Loganathan, G., Wilhelm, J.J., Tweed, B., Vargova, L., Lockbridge, A., Kuriti, M., Hughes, M.G., Williams, S-K., Hering, B.J., Dwulet, F.E. and McCarthy, R.C. Transplantation Direct, 2:e54 (2016)   Background: Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation. Methods: We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42). Results: Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas. Conclusions: A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.  

4.1526           Bicyclic-Capped Histone Deacetylase 6 Inhibitors with Improved Activity in a Model of Axonal Charcot–Marie–Tooth Disease

Shen, S., Benoy, V., bergman, J.A., Kalin, J.H., Frojuello, M., Vistoli, G., Haeck, W., Van Den Bosch, L. and Kozikowski, A.P. ACS Chem. Neurosci., 7(2), 240-258 (2016)   Charcot–Marie–Tooth (CMT) disease is a disorder of the peripheral nervous system where progressive degeneration of motor and sensory nerves leads to motor problems and sensory loss and for which no pharmacological treatment is available. Recently, it has been shown in a model for the axonal form of CMT that histone deacetylase 6 (HDAC6) can serve as a target for the development of a pharmacological therapy. Therefore, we aimed at developing new selective and activity-specific HDAC6 inhibitors with improved biochemical properties. By utilizing a bicyclic cap as the structural scaffold from which to build upon, we developed several analogues that showed improved potency compared to tubastatin A while maintaining excellent selectivity compared to HDAC1. Further screening in N2a cells examining both the acetylation of α-tubulin and histones narrowed down the library of compounds to three potent and selective HDAC6 inhibitors. In mutant HSPB1-expressing DRG neurons, serving as an in vitro model for CMT2, these inhibitors were able to restore the mitochondrial axonal transport deficits. Combining structure-based development of HDAC6 inhibitors, screening in N2a cells and in a neuronal model for CMT2F, and preliminary ADMET and pharmacokinetic profiles, resulted in the selection of compound 23d that possesses improved biochemical, functional, and druglike properties compared to tubastatin A.  

4.1527           Long-term Efficacy and Biocompatibility of Encapsulated Islet Transplantation With Chitosan-Coated Alginate Capsules in Mice and Canine Models of Diabetes

Yang, H.K. et al Transplantation, 100(2), 334-343 (2016)   Background: Clinical application of encapsulated islet transplantation is hindered by low biocompatibility of capsules leading to pericapsular fibrosis and decreased islet viability. To improve biocompatibility, we designed a novel chitosan-coated alginate capsules and compared them to uncoated alginate capsules. Methods: Alginate capsules were formed by crosslinking with BaCl₂, then they were suspended in chitosan solution for 10 minutes at pH 4.5. Xenogeneic islet transplantation, using encapsulated porcine islets in 1,3-galactosyltransferase knockout mice, and allogeneic islet transplantation, using encapsulated canine islets in beagles, were performed without immunosuppressants. Results: The chitosan-alginate capsules showed similar pore size, islet viability, and insulin secretory function compared to alginate capsules, in vitro. Xenogeneic transplantation of chitosan-alginate capsules demonstrated a trend toward superior graft survival (P = 0.07) with significantly less pericapsular fibrosis (cell adhesion score: 3.77 ± 0.41 vs 8.08 ± 0.05; P < 0.001) compared to that of alginate capsules up to 1 year after transplantation. Allogeneic transplantation of chitosan-alginate capsules normalized the blood glucose level up to 1 year with little evidence of pericapsular fibrotic overgrowth on graft explantation. Conclusions: The efficacy and biocompatibility of chitosan-alginate capsules were demonstrated in xenogeneic and allogeneic islet transplantations using small and large animal models of diabetes. This capsule might be a potential candidate applicable in the treatment of type 1 diabetes mellitus patients, and further studies in nonhuman primates are required.  

4.1528           Ammonia produces pathological changes in human hepatic stellate cells and is a target for therapy of portal hypertension

Jalan, R., De Chiara, F., Balasubramaniyan, V., Andreola, F., Kheta, V., Malago, M., Pinzani, M., Mookerjee, R.P. and Rombouts, K.

1. Hepatol., 64, 823-833 (2016)

  Background & Aims Hepatic stellate cells (HSCs) are vital to hepatocellular function and the liver response to injury. They share a phenotypic homology with astrocytes that are central in the pathogenesis of hepatic encephalopathy, a condition in which hyperammonemia plays a pathogenic role. This study tested the hypothesis that ammonia modulates human HSC activation in vitro and in vivo, and evaluated whether ammonia lowering, by using l-ornithine phenylacetate (OP), modifies HSC activation in vivo and reduces portal pressure in a bile duct ligation (BDL) model. Methods Primary human HSCs were isolated and cultured. Proliferation (BrdU), metabolic activity (MTS), morphology (transmission electron, light and immunofluorescence microscopy), HSC activation markers, ability to contract, changes in oxidative status (ROS) and endoplasmic reticulum (ER) were evaluated to identify effects of ammonia challenge (50 μM, 100 μM, 300 μM) over 24–72 h. Changes in plasma ammonia levels, markers of HSC activation, portal pressure and hepatic eNOS activity were quantified in hyperammonemic BDL animals, and after OP treatment. Results Pathophysiological ammonia concentrations caused significant and reversible changes in cell proliferation, metabolic activity and activation markers of hHSC in vitro. Ammonia also induced significant alterations in cellular morphology, characterised by cytoplasmic vacuolisation, ER enlargement, ROS production, hHSC contraction and changes in pro-inflammatory gene expression together with HSC-related activation markers such as α-SMA, myosin IIa, IIb, and PDGF-Rβ. Treatment with OP significantly reduced plasma ammonia (BDL 199.1 μmol/L ± 43.65 vs. BDL + OP 149.27 μmol/L ± 51.1, p <0.05) and portal pressure (BDL 14 ± 0.6 vs. BDL + OP 11 ± 0.3 mmHg, p <0.01), which was associated with increased eNOS activity and abrogation of HSC activation markers. Conclusions The results show for the first time that ammonia produces deleterious morphological and functional effects on HSCs in vitro. Targeting ammonia with the ammonia lowering drug OP reduces portal pressure and deactivates hHSC in vivo, highlighting the opportunity for evaluating ammonia lowering as a potential therapy in cirrhotic patients with portal hypertension.  

4.1529           Complement receptor 3 mediates renal protection in experimental C3 glomerulopathy

Barbour, T.D., Ling, G.S., Ruseva, M.M., Fossati-Jimack, L., Cook, H.T., Botto, M. and Pickering, M.C. Kidney Int., 89, 823-832 (2016)   C3 glomerulopathy is a complement-mediated renal disease that is frequently associated with abnormalities in regulation of the complement alternative pathway. Mice with deficiency of factor H (Cfh–/–), a negative alternative pathway regulator, are an established experimental model of C3 glomerulopathy in which complement C3 fragments including iC3b accumulate along the glomerular basement membrane. Here we show that deficiency of complement receptor 3 (CR3), the main receptor for iC3b, enhances the severity of spontaneous renal disease in Cfh–/– mice. This effect was found to be dependent on CR3 expression on bone marrow–derived cells. CR3 also mediated renal protection outside the setting of factor H deficiency, as shown by the development of enhanced renal injury in CR3-deficient mice during accelerated nephrotoxic nephritis. The iC3b–CR3 interaction downregulated the proinflammatory cytokine response of both murine and human macrophages to lipopolysaccharide stimulation in vitro, suggesting that the protective effect of CR3 on glomerular injury was mediated via modulation of macrophage-derived proinflammatory cytokines. Thus, CR3 has a protective role in glomerulonephritis and suggests that pharmacologic potentiation of the macrophage CR3 interaction with iC3b could be therapeutically beneficial.  

4.1530           Controlled ice nucleation—Is it really needed for large-volume sperm cryopreservation?

Saragusty, J., Osmers, J-H. and Hildebrandt, T.B. Theriogenology, 85, 1328-1333 (2016)   Controlled ice nucleation (CIN) is an integral stage of slow freezing process when relatively large volumes (usually 1 mL or larger) of biological samples in suspension are involved. Without it, a sample will supercool to way below its melting point before ice crystals start forming, resulting in multiple damaging processes. In this study, we tested the hypothesis that when freezing large volumes by the directional freezing technique, a CIN stage is not needed. Semen samples collected from ten bulls were frozen in 2.5-mL HollowTubes in a split-sample manner with and without a CIN stage. Thawed samples were evaluated for viability, acrosome integrity, rate of normal morphology, and, using computer-aided sperm analysis system, for a wide range of motility parameters that were also evaluated after 3 hours of incubation at 37 °C. Analysis of the results found no difference between freezing with and without CIN stage in any and all of the 29 parameters compared (P > 0.1 for all). This similarity was maintained through 3 hours of incubation at 37 °C. Possibly, because of its structure, the directional freezing device promotes continuous ice nucleation so a specific CIN stage is no longer needed, thus reducing costs, energy use, and carbon footprint.  

4.1531           Versican: a novel modulator of hepatic fibrosis

Bukong, T.N., Maurice, S.B., Chahai, B., Schaeffer, D.F. and Winwood, P.J. Lab. Invest., 96(3), 361-374 (2016)   Little is known about the deposition and turnover of proteoglycans in liver fibrosis, despite their abundance in the extracellular matrix. Versican plays diverse roles in modulating cell behavior in other fibroproliferative diseases, but remains poorly described in the liver. Hepatic fibrosis was induced by carbon tetrachloride treatment of C57BL/6 mice over 4 weeks followed by recovery over a 28-day period. Primary mouse hepatic stellate cells (HSCs) were activated in culture and versican was transiently knocked down in human (LX2) and mouse HSCs. Expression of versican, A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs (ADAMTS)-1, -4, -5, -8, -9, -15, and -20, and markers of fibrogenesis were studied using immunohistochemistry, real-time quantitative PCR, and western blotting. Immunohistochemistry showed increased expression of versican in cirrhotic human livers and the mouse model of fibrosis. Carbon tetrachloride treatment led to significant increases in versican expression and the proteoglycanases ADAMTS-5, -9, -15, and -20, alongside TNF-α, α-smooth muscle actin (α-SMA), collagen-1, and TGF-β expression. During recovery, expression of many of these genes returned to control levels. However, expression of ADAMTS-5, -8, -9, and -15 showed delayed increases in expression at 28 days of recovery, which corresponded with decreases in versican V0 and V1 cleavage products (G1-DPEAAE¹⁴⁰¹ and G1-DPEAAE⁴⁴¹). Activation of primary HSCs in vitro significantly increased versican, α-SMA, and collagen-1 expression. Transient knockdown of versican in HSCs led to decreases in markers of fibrogenesis and reduced cell proliferation, without inducing apoptosis. Versican expression increases during HSC activation and liver fibrosis, and proteolytic processing occurs during the resolution of fibrosis. Knockdown studies in vitro suggest a possible role of versican in modulating hepatic fibrogenesis.  

4.1532           Mechanical tension applied to substrate films specifies location of neuritogenesis and promotes major neurite growth at the expense of minor neurite development

Feng, Z-Q., Franz, E.W., Leach, M.K., Winterroth, F., White, C.M., Rastogi, A., Gu, Z-Z. and Corey, J.M.

1. Biomed. Res. Part A, 104(4), 966-974 (2016)

  One obstacle in neural repair is facilitating axon growth long enough to reach denervated targets. Recent studies show that axonal growth is accelerated by applying tension to bundles of neurites, and additional studies show that mechanical tension is critical to all neurite growth. However, no studies yet describe how individual neurons respond to tensile forces applied to cell bodies and neurites simultaneously; neither do any test motor neurons, a phenotype critical to neural repair. Here we examine the growth of dissociated motor neurons on stretchable substrates. E15 spinal motor neurons were cultured on poly-lactide-co-glycolide films stretched at 4.8, 9.6, or 14.3 mm day⁻¹. Morphological analysis revealed that substrate stretching has profound effects on developing motor neurons. Stretching increases major neurite length; it also forces neuritogenesis to occur nearest poles of the cell closest to the sources of tension. Stretching also reduces the number of neurites per neuron. These data show that substrate stretching affects neuronal morphology by specifying locations on the cell where neuritogenesis occurs and favoring major neurite growth at the expense of minor neurites. These results serve as a building block for development of new techniques to control and improve the growth of neurons for nerve repair purposes.  

4.1533           Cyclic AMP and Polyamines Overcome Inhibition by Myelin-Associated Glycoprotein through eIF5A-Mediated Increases in p35 Expression and Activation of Cdk5

He, H., Deng, K., Siddiz, M.M., Pyie, A., Mellado, W., Hannila, S.S. and Filbin, M.T.

1. Neurosci., 36(10), 3079-3091 (2016)

  Inhibitory molecules associated with CNS myelin, such as myelin-associated glycoprotein (MAG), represent major obstacles to axonal regeneration following CNS injury. Our laboratory has shown that elevating levels of intracellular cAMP, via application of the nonhydrolyzable analog dibutyryl cAMP (dbcAMP), can block the inhibitory effects of MAG and myelin. We have also shown that elevation of cAMP results in upregulation of arginase I and increased polyamine synthesis. Treatment with putrescine or spermidine blocks myelin-mediated inhibition of neurite outgrowth, but the mechanism underlying this effect has not yet been elucidated. Here we show that cyclin-dependent kinase 5 (Cdk5) is required for dbcAMP and putrescine to overcome MAG-mediated inhibition. The ability of dbcAMP and putrescine to overcome inhibition by MAG is abolished in the presence of roscovitine, a Cdk inhibitor that has greater selectivity for Cdk5, and expression of dominant negative Cdk5 abolishes the ability of dbcAMP or putrescine to enhance neurite outgrowth in the presence of MAG. Importantly, dbcAMP and putrescine increase expression of p35, the neuron-specific activator of Cdk5, and rat DRG neurons transduced with HSV overexpressing p35 can overcome inhibition by MAG. The upregulation of p35 by putrescine is also reflected in increased localization of p35 to neurites and growth cones. Last, we show that putrescine upregulates p35 expression by serving as a substrate for hypusine modification of eIF5A, and that this hypusination is necessary for putrescine's ability to overcome inhibition by MAG. Our findings reveal a previously unknown mechanism by which polyamines may encourage regeneration after CNS injury.  

4.1534           Effector T-cell trafficking between the leptomeninges and the cerebrospinal fluid

Schläger, C. et al Nature, 530, 349-353 (2016)   In multiple sclerosis, brain-reactive T cells invade the central nervous system (CNS) and induce a self-destructive inflammatory process. T-cell infiltrates are not only found within the parenchyma and the meninges, but also in the cerebrospinal fluid (CSF) that bathes the entire CNS tissue^{1, 2}. How the T cells reach the CSF, their functionality, and whether they traffic between the CSF and other CNS compartments remains hypothetical^{3, 4, 5, 6}. Here we show that effector T cells enter the CSF from the leptomeninges during Lewis rat experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. While moving through the three-dimensional leptomeningeal network of collagen fibres in a random Brownian walk, T cells were flushed from the surface by the flow of the CSF. The detached cells displayed significantly lower activation levels compared to T cells from the leptomeninges and CNS parenchyma. However, they did not represent a specialized non-pathogenic cellular sub-fraction, as their gene expression profile strongly resembled that of tissue-derived T cells and they fully retained their encephalitogenic potential. T-cell detachment from the leptomeninges was counteracted by integrins VLA-4 and LFA-1 binding to their respective ligands produced by resident macrophages. Chemokine signalling via CCR5/CXCR3 and antigenic stimulation of T cells in contact with the leptomeningeal macrophages enforced their adhesiveness. T cells floating in the CSF were able to reattach to the leptomeninges through steps reminiscent of vascular adhesion in CNS blood vessels, and invade the parenchyma. The molecular/cellular conditions for T-cell reattachment were the same as the requirements for detachment from the leptomeningeal milieu. Our data indicate that the leptomeninges represent a checkpoint at which activated T cells are licensed to enter the CNS parenchyma and non-activated T cells are preferentially released into the CSF, from where they can reach areas of antigen availability and tissue damage.  

4.1535           Probiotics modulated gut microbiota suppresses hepatocellular carcinoma growth in mice

Li, J., Sung, C.Y.J., Lee, N., Ni, Y., Pihlajamäki, J., Panagiotou, G. and El-Nezami, H. PNAS, 113(9), E1306-E1315 (2016)   The beneficial roles of probiotics in lowering the gastrointestinal inflammation and preventing colorectal cancer have been frequently demonstrated, but their immunomodulatory effects and mechanism in suppressing the growth of extraintestinal tumors remain unexplored. Here, we adopted a mouse model and metagenome sequencing to investigate the efficacy of probiotic feeding in controlling s.c. hepatocellular carcinoma (HCC) and the underlying mechanism suppressing the tumor progression. Our result demonstrated that Prohep, a novel probiotic mixture, slows down the tumor growth significantly and reduces the tumor size and weight by 40% compared with the control. From a mechanistic point of view the down-regulated IL-17 cytokine and its major producer Th17 cells, whose levels decreased drastically, played critical roles in tumor reduction upon probiotics feeding. Cell staining illustrated that the reduced Th17 cells in the tumor of the probiotic-treated group is mainly caused by the reduced frequency of migratory Th17 cells from the intestine and peripheral blood. In addition, shotgun-metagenome sequencing revealed the crosstalk between gut microbial metabolites and the HCC development. Probiotics shifted the gut microbial community toward certain beneficial bacteria, including Prevotella and Oscillibacter, that are known producers of antiinflammatory metabolites, which subsequently reduced the Th17 polarization and promoted the differentiation of antiinflammatory Treg/Tr1 cells in the gut. Overall, our study offers novel insights into the mechanism by which probiotic treatment modulates the microbiota and influences the regulation of the T-cell differentiation in the gut, which in turn alters the level of the proinflammatory cytokines in the extraintestinal tumor microenvironment.  

4.1536           MicroRNA-378 limits activation of hepatic stellate cells and liver fibrosis by suppressing Gli3 expression

Hyun, J., Wang, S., Kim, J., Rao, K.M., park, S.Y., Chung, I., Ha, C-S., Kim, S-W., Yun, Y.H: and Jung, Y. Nature Communications, 7:10993 (2016)   Hedgehog (Hh) signalling regulates hepatic fibrogenesis. MicroRNAs (miRNAs) mediate various cellular processes; however, their role in liver fibrosis is unclear. Here we investigate regulation of miRNAs in chronically damaged fibrotic liver. MiRNA profiling shows that expression of miR-378 family members (miR-378a-3p, miR-378b and miR-378d) declines in carbon tetrachloride (CCl₄)-treated compared with corn-oil-treated mice. Overexpression of miR-378a-3p, directly targeting Gli3 in activated hepatic stellate cells (HSCs), reduces expression of Gli3 and profibrotic genes but induces gfap, the inactivation marker of HSCs, in CCl₄-treated liver. Smo blocks transcriptional expression of miR-378a-3p by activating the p65 subunit of nuclear factor-κB (NF-κB). The hepatic level of miR-378a-3p is inversely correlated with the expression of Gli3 in tumour and non-tumour tissues in human hepatocellular carcinoma. Our results demonstrate that miR-378a-3p suppresses activation of HSCs by targeting Gli3 and its expression is regulated by Smo-dependent NF-κB signalling, suggesting miR-378a-3p has therapeutic potential for liver fibrosis.  

4.1537           Methods for Isolation and Purification of Murine Liver Sinusoidal Endothelial Cells: A Systematic Review

Meyer, J., Gonelle-Gispert. C., Morel, P. and Bühler, L. PloS One, 11(3), E0151945 (2016)   To study the biological functions of liver sinusoidal endothelial cells (LSEC) and to identify their interplay with blood or liver cells, techniques allowing for the isolation and purification of LSEC have been developed over the last decades. The objective of the present review is to summarize and to compare the efficiency of existing methods for isolating murine LSEC. Toward this end, the MEDLINE database was searched for all original articles describing LSEC isolation from rat and mouse livers. Out of the 489 publications identified, 23 reported the main steps and outcomes of the procedure and were included in our review. Here, we report and analyse the technical details of the essential steps of the techniques used for LSEC isolation. The correlations between the prevalence of some steps and the efficiency of LSEC isolation were also identified. We found that centrifugal elutriation, selective adherence and, more recently, magnetic-activated cell sorting were used for LSEC purification. Centrifugal elutriation procured high yields of pure LSEC (for rats 30–141.9 million cells for 85–98% purities; for mice 9–9.25 million cells for >95% purities), but the use of this method remained limited due to its high technical requirements. Selective adherence showed inconsistent results in terms of cell yields and purities in rats (5–100 million cells for 73.7–95% purities). In contrast, magnetic-activated cell sorting allowed for the isolation of highly pure LSEC, but overall lower cell yields were reported (for rats 10.7 million cells with 97.6% purity; for mice 0.5–9 million cells with 90–98% purities). Notably, the controversies regarding the accuracy of several phenotypic markers for LSEC should be considered and their use for both magnetic sorting and characterization remain doubtful. It appears that more effort is needed to refine and standardize the procedure for LSEC isolation, with a focus on the identification of specific antigens. Such a procedure is required to identify the molecular mechanisms regulating the function of LSEC and to improve our understanding of their role in complex cellular processes in the liver.  

4.1538           TLR4-Dependent Secretion by Hepatic Stellate Cells of the Neutrophil-Chemoattractant CXCL1 Mediates Liver Response to Gut Microbiota

Bigorgne, A.E., John, B., ebrahimkhani, M.R., Shimizu-Albergine, M., Campbell, J.S. and Crispe, I.N: PloS One, 11(3), e0151063 (2016)   Background & Aims The gut microbiota significantly influences hepatic immunity. Little is known on the precise mechanism by which liver cells mediate recognition of gut microbes at steady state. Here we tested the hypothesis that a specific liver cell population was the sensor and we aimed at deciphering the mechanism by which the activation of TLR4 pathway would mediate liver response to gut microbiota. Methods Using microarrays, we compared total liver gene expression in WT versus TLR4 deficient mice. We performed in situ localization of the major candidate protein, CXCL1. With an innovative technique based on cell sorting, we harvested enriched fractions of KCs, LSECs and HSCs from the same liver. The cytokine secretion profile was quantified in response to low levels of LPS (1ng/mL). Chemotactic activity of stellate cell-derived CXCL1 was assayed in vitro on neutrophils upon TLR4 activation. Results TLR4 deficient liver had reduced levels of one unique chemokine, CXCL1 and subsequent decreased of neutrophil counts. Depletion of gut microbiota mimicked TLR4 deficient phenotype, i.e., decreased neutrophils counts in the liver. All liver cells were responsive to low levels of LPS, but hepatic stellate cells were the major source of chemotactic levels of CXCL1. Neutrophil migration towards secretory hepatic stellate cells required the TLR4 dependent secretion of CXCL1. Conclusions Showing the specific activation of TLR4 and the secretion of one major functional chemokine—CXCL1, the homolog of human IL-8-, we elucidate a new mechanism in which Hepatic Stellate Cells play a central role in the recognition of gut microbes by the liver at steady state.  

4.1539           Microfluidic fabrication of bioactive microgels for rapid formation and enhanced differentiation of stem cell spheroids

Siltanen, C., Yaghoobi, M., haque, A., You, J., Lowen, J., Soleimani, M. and Revzin, A. Acta Biomaterialia, 34, 125-132 (2016)   A major challenge in tissue engineering is to develop robust protocols for differentiating ES and iPS cells to functional adult tissues at a clinically relevant scale. The goal of this study is to develop a high throughput platform for generating bioactive, stem cell-laden microgels to direct differentiation in a well-defined microenvironment. We describe a droplet microfluidics system for fabricating microgels composed of polyethylene glycol and heparin, with tunable geometric, mechanical, and chemical properties, at kHz rates. Heparin-containing hydrogel particles sequestered growth factors Nodal and FGF-2, which are implicated in specifying pluripotent cells to definitive endoderm. Mouse ESCs were encapsulated into heparin microgels with a single dose of Nodal and FGF-2, and expressed high levels of endoderm markers Sox17 and FoxA2 after 5 days. These results highlight the use of microencapsulation for tailoring the stem cell microenvironment to promote directed differentiation, and may provide a straightforward path to large scale bioprocessing in the future.  

4.1540           Establishment of a Drug-Induced, Bile Acid–Dependent Hepatotoxicity Model Using HepaRG Cells

Susukida, T., Sekine, S., Nozaki, M., Tokizono, M., Oizumi, K., Horie, T. and Ito, K.

1. Pharmaceut. Sci., 105, 1550-1560 (2016)

  Bile acid (BA) retention within hepatocytes is an underlying mechanism of cholestatic drug-induced liver injury (DILI). We previously developed an assay using sandwich-cultured human hepatocytes (SCHHs) to evaluate drug-induced hepatocyte toxicity accompanying intracellular BA accumulation. However, due to shortcomings commonly associated with the use of primary human hepatocytes (e.g., limited availability, lot-to-lot variability, and high cost), we examined if the human hepatic stem cell line, HepaRG, might also be applicable to our assay system. Consequently, mRNA expression levels of human BA efflux and uptake transporters were lower in HepaRG cells than in SCHHs but higher than in HepG2 human hepatoma cells. Nevertheless, HepaRG cells and SCHHs showed similar toxicity responses to 22 selected drugs, including cyclosporine A (CsA). CsA (10 μM) was cytotoxic toward HepaRG cells in the presence of BAs and also reduced the biliary efflux rate of [³H]taurocholic acid from 38.5% to 19.2%. Therefore, HepaRG cells are useful for the evaluation of BA-dependent drug toxicity caused by biliary BA efflux inhibition. Regardless, the prediction accuracy for cholestatic DILI risk was poor for HepaRG cells versus SCHHs, suggesting that our DILI model system requires further improvements to increase the utility of HepaRG cells as a preclinical screening tool.  

4.1541           Long noncoding RNAs expressed in human hepatic stellate cells form networks with extracellular matrix proteins

Zhou, C., York, S.R., Chen, J.Y., Pondick, J.V., Motola, D.L., Chung, R.T. and Mullen, A.C. Genome Medicine, 8:31 (2016)   Background Hepatic fibrosis is the underlying cause of cirrhosis and liver failure in nearly every form of chronic liver disease, and hepatic stellate cells (HSCs) are the primary cell type responsible for fibrosis. Long noncoding RNAs (lncRNAs) are increasingly recognized as regulators of development and disease; however, little is known about their expression in human HSCs and their function in hepatic fibrosis. Methods We performed RNA sequencing and ab initio assembly of RNA transcripts to define the lncRNAs expressed in human HSC myofibroblasts. We analyzed chromatin immunoprecipitation data and expression data to identify lncRNAs that were regulated by transforming growth factor beta (TGF-β) signaling, associated with super-enhancers and restricted in expression to HSCs compared with 43 human tissues and cell types. Co-expression network analyses were performed to discover functional modules of lncRNAs, and principle component analysis and K-mean clustering were used to compare lncRNA expression in HSCs with other myofibroblast cell types. Results We identified over 3600 lncRNAs that are expressed in human HSC myofibroblasts. Many are regulated by TGF-β, a major fibrotic signal, and form networks with genes encoding key components of the extracellular matrix (ECM), which is the substrate of the fibrotic scar. The lncRNAs directly regulated by TGF-β signaling are also enriched at super-enhancers. More than 400 of the lncRNAs identified in HSCs are uniquely expressed in HSCs compared with 43 other human tissues and cell types and HSC myofibroblasts demonstrate different patterns of lncRNA expression compared with myofibroblasts originating from other tissues. Co-expression analyses identified a subset of lncRNAs that are tightly linked to collagen genes and numerous proteins that regulate the ECM during formation of the fibrotic scar. Finally, we identified lncRNAs that are induced during progression of human liver disease. Conclusions lncRNAs are likely key contributors to the formation and progression of fibrosis in human liver disease.  

4.1542           The HSV-1 Latency-Associated Transcript Functions to Repress Latent Phase Lytic Gene Expression and Suppress Virus Reactivation from Latently Infected Neurons

Nicoll, M.P., Hann, W., Shivkumar, M., harmann, L.E.R., Connor, V., Coleman, H.M., Proenca, J.T. and Efstathiou, S. PloS Pathogens, 12(4), e1005539 (2016)   Herpes simplex virus 1 (HSV-1) establishes life-long latent infection within sensory neurons, during which viral lytic gene expression is silenced. The only highly expressed viral gene product during latent infection is the latency-associated transcript (LAT), a non-protein coding RNA that has been strongly implicated in the epigenetic regulation of HSV-1 gene expression. We have investigated LAT-mediated control of latent gene expression using chromatin immunoprecipitation analyses and LAT-negative viruses engineered to express firefly luciferase or β-galactosidase from a heterologous lytic promoter. Whilst we were unable to determine a significant effect of LAT expression upon heterochromatin enrichment on latent HSV-1 genomes, we show that reporter gene expression from latent HSV-1 genomes occurs at a greater frequency in the absence of LAT. Furthermore, using luciferase reporter viruses we have observed that HSV-1 gene expression decreases during long-term latent infection, with a most marked effect during LAT-negative virus infection. Finally, using a fluorescent mouse model of infection to isolate and culture single latently infected neurons, we also show that reactivation occurs at a greater frequency from cultures harbouring LAT-negative HSV-1. Together, our data suggest that the HSV-1 LAT RNA represses HSV-1 gene expression in small populations of neurons within the mouse TG, a phenomenon that directly impacts upon the frequency of reactivation and the maintenance of the transcriptionally active latent reservoir.  

4.1543           Characterization of hepatic stellate cells, portal fibroblasts, and mesothelial cells in normal and fibrotic livers

Lua, I., Li, Y., Zagory, J.A., Wang, K.S., French, S.W., Sevigny, J. and Asahina, K.

1. Hepatol., 64, 1137-1146 (2016)

  Background & Aims Contribution of hepatic stellate cells (HSCs), portal fibroblasts (PFs), and mesothelial cells (MCs) to myofibroblasts is not fully understood due to insufficient availability of markers and isolation methods. The present study aimed to isolate these cells, characterize their phenotypes, and examine their contribution to myofibroblasts in liver fibrosis. Methods Liver fibrosis was induced in Collagen1a1-green fluorescent protein (Col1a1^GFP) mice by bile duct ligation (BDL), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet, or CCl₄ injections. Combining vitamin A (VitA) lipid autofluorescence and expression of GFP and glycoprotein M6a (GPM6A), we separated HSCs, PFs, and MCs from normal and fibrotic livers by fluorescence-activated cell sorting (FACS). Results Normal Col1a1^GFP livers broadly expressed GFP in HSCs, PFs, and MCs. Isolated VitA+ HSCs expressed reelin, whereas VitA−GFP+GPM6A− PFs expressed ectonucleoside triphosphate diphosphohydrolase-2 and elastin. VitA−GFP+GPM6A+ MCs expressed keratin 19, mesothelin, and uroplakin 1b. Transforming growth factor (TGF)-β1 treatment induced the transformation of HSCs, PFs, and MCs into myofibroblasts in culture. TGF-β1 suppressed cyclin D1 mRNA expression in PFs but not in HSCs and MCs. In biliary fibrosis, PFs adjacent to the bile duct expressed α-smooth muscle actin. FACS analysis revealed that HSCs are the major source of GFP+ myofibroblasts in the injured Col1a1^GFP mice after DDC or CCl₄ treatment. Although PFs partly contributed to GFP+ myofibroblasts in the BDL model, HSCs were still dominant source of myofibroblasts. Conclusion HSCs, PFs, and MCs have distinct phenotypes, and PFs partly contribute to myofibroblasts in the portal triad in biliary fibrosis.  

4.1544           CCL2–CCR2 signaling promotes hepatic ischemia/reperfusion injury

Zhang, J., Xu, P., Song, P., Wang, H., Zhang, Y., Hu, Q., Wang, G., Zhang, S., Yu, Q., Billiar, T.R., Wang, C. and Zhang, J. J.Surg. Res., 15, 352-362 (2016)   Background Liver ischemia/reperfusion (I/R) injury is a type of uncontrolled inflammatory cascade in which neutrophils, an early infiltrating immune cell population, elicit significant tissue damage. However, the precise mechanism for neutrophil recruitment and infiltration remains to be fully characterized. Methods A hepatic partial I/R model was reproduced in wild-type, CCL2^−/− and CCR2^−/− mice. Tissue damage was evaluated by serum enzyme analysis, hematoxylin–eosin staining, and cytokine production measurement. Mobilization of neutrophils from the bone marrow and subsequent infiltration into the liver were measured by flow cytometry. C-C motif chemokine receptor 2 (CCR2) expression on neutrophils and C-C motif chemokine ligand 2 (CCL2) chemotaxis were measured using flow cytometry. The cellular source of CCL2 in the liver was determined by deleting specific cell groups and performing intracellular staining. Results Liver damage was ameliorated, and neutrophil recruitment and accumulation were decreased in both CCL2^−/− and CCR2^−/− mice compared with wild-type mice. Neutrophils displayed upregulated expression of CCR2 during I/R, and these cells were required for CCL2-induced chemotaxis. Depletion of Kupffer cells protected the liver from I/R injury. Furthermore, genetic ablation of CCL2 reduced liver injury, as demonstrated by decreases in the levels of alanine aminotransferase and aspartate aminotransferase and subsequent reductions in neutrophil recruitment and accumulation. Conclusions Kupffer cells secrete CCL2 to promote CCR2-expressing neutrophil recruitment from the bone marrow and subsequent infiltration into the liver during I/R. These findings reveal a novel pro-inflammatory role of cell-mediated CCL2–CCR2 interactions during this sterile insult.  

4.1545           Caspase 6 has a protective role in SOD1G93A transgenic mice

Hogg, M.C., Mitchem., M.R., König, H-G. and Prehn, J.H.M. Biochim. Biophys. Acta, 1862, 1063-1073 (2016)   In amyotrophic lateral sclerosis (ALS), it has been suggested that the process of neurodegeneration starts at the neuromuscular junction and is propagated back along axons towards motor neurons. Caspase-dependent pathways are well established as a cause of motor neuron death, and recent work in other disease models indicated a role for caspase 6 in axonal degeneration. Therefore we hypothesised that caspase 6 may be involved in motor neuron death in ALS. To investigate the role of caspase 6 in ALS we profiled protein levels of caspase-6 throughout disease progression in the ALS mouse model SOD1^G93A; this did not reveal differences in caspase 6 levels during disease. To investigate the role of caspase 6 further we generated a colony with SOD1^G93A transgenic mice lacking caspase 6. Analysis of the transgenic SOD1^G93A; Casp6^−/− revealed an exacerbated phenotype with motor dysfunction occurring earlier and a significantly shortened lifespan when compared to transgenic SOD1^G93A; Casp6^+/+ mice. Immunofluorescence analysis of the neuromuscular junction revealed no obvious difference between caspase 6^+/+ and caspase 6^−/− in non-transgenic mice, while the SOD1^G93A transgenic mice showed severe degeneration compared to non-transgenic mice in both genotypes. Our data indicate that caspase-6 does not exacerbate ALS pathogenesis, but may have a protective role.  

4.1546           Olfactory Ensheathing Cells Express α7 Integrin to Mediate Their Migration on Laminin

Ingram, N.T., Khankan, R.R. and Phelps, P.E. PloS One, 11(4), e0153394 (2016)   The unique glia located in the olfactory system, called olfactory ensheathing cells (OECs), are implicated as an attractive choice for transplantation therapy following spinal cord injury because of their pro-regenerative characteristics. Adult OECs are thought to improve functional recovery and regeneration after injury by secreting neurotrophic factors and making cell-to-cell contacts with regenerating processes, but the mechanisms are not well understood. We show first that α7 integrin, a laminin receptor, is highly expressed at the protein level by OECs throughout the olfactory system, i.e., in the olfactory mucosa, olfactory nerve, and olfactory nerve layer of the olfactory bulb. Then we asked if OECs use the α7 integrin receptor directly to promote neurite outgrowth on permissive and neutral substrates, in vitro. We co-cultured α7+/+ and α7lacZ/lacZ postnatal cerebral cortical neurons with α7+/+ or α7lacZ/lacZ OECs and found that genotype did not effect the ability of OECs to enhance neurite outgrowth by direct contact. Loss of α7 integrin did however significantly decrease the motility of adult OECs in transwell experiments. Twice as many α7+/+ OECs migrated through laminin-coated transwells compared to α7+/+ OECs on poly-L-lysine (PLL). This is in contrast to α7lacZ/lacZ OECs, which showed no migratory preference for laminin substrate over PLL. These results demonstrate that OECs express α7 integrin, and that laminin and its α7 integrin receptor contribute to adult OEC migration in vitro and perhaps also in vivo.  

4.1547           Blockage of indoleamine 2,3-dioxygenase regulates Japanese encephalitis via enhancement of type I/II IFN innate and adaptive T-cell responses

Kim, S.B. Choi, J.Y., Uyangaa, E., patil, A.M., Hossain, F.M.A., Hur, J., park, S-Y., Lee, J-H., Kim, K. and Eo, S.K. Journal of Neuroinflammation, 13:79 (2016)   Background Japanese encephalitis (JE), a leading cause of viral encephalitis, is characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV). Indoleamine 2,3-dioxygenase (IDO) has been identified as an enzyme associated with immunoregulatory function. Although the regulatory role of IDO in viral replication has been postulated, the in vivo role of IDO activity has not been fully addressed in neurotropic virus-caused encephalitis. Methods Mice in which IDO activity was inhibited by genetic ablation or using a specific inhibitor were examined for mortality and clinical signs after infection. Neuroinflammation was evaluated by central nervous system (CNS) infiltration of leukocytes and cytokine expression. IDO expression, viral burden, JEV-specific T-cell, and type I/II interferon (IFN-I/II) innate responses were also analyzed. Results Elevated expression of IDO activity in myeloid and neuron cells of the lymphoid and CNS tissues was closely associated with clinical signs of JE. Furthermore, inhibition of IDO activity enhanced resistance to JE, reduced the viral burden in lymphoid and CNS tissues, and resulted in early and increased CNS infiltration by Ly-6C^hi monocytes, NK, CD4⁺, and CD8⁺ T-cells. JE amelioration in IDO-ablated mice was also associated with enhanced NK and JEV-specific T-cell responses. More interestingly, IDO ablation induced rapid enhancement of type I IFN (IFN-I) innate responses in CD11c⁺ dendritic cells (DCs), including conventional and plasmacytoid DCs, following JEV infection. This enhanced IFN-I innate response in IDO-ablated CD11c⁺ DCs was coupled with strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7, STAT1), and antiviral ISG genes (Mx1, Mx2, ISG49, ISG54, ISG56). IDO ablation also enhanced the IFN-I innate response in neuron cells, which may delay the spread of virus in the CNS. Finally, we identified that IDO ablation in myeloid cells derived from hematopoietic stem cells (HSCs) dominantly contributed to JE amelioration and that HSC-derived leukocytes played a key role in the enhanced IFN-I innate responses in the IDO-ablated environment. Conclusions Inhibition of IDO activity ameliorated JE via enhancement of antiviral IFN-I/II innate and adaptive T-cell responses and increased CNS infiltration of peripheral leukocytes. Therefore, our data provide valuable insight into the use of IDO inhibition by specific inhibitors as a promising tool for therapeutic and prophylactic strategies against viral encephalitis caused by neurotropic viruses.  

4.1548           Autoantibody-boosted T-cell reactivation in the target organ triggers manifestation of autoimmune CNS disease

Flach, A-C., Litke, T., Strauss, J., Haberl, M., Gomez, C.C., reindl, M., Saiz, A., Fehling, H-J., Wienands, J., Odoardi, F., Lühder, F. and Flügel, A. PNAS, 113(12), 3323-3328 (2016)   Multiple sclerosis (MS) is caused by T cells that are reactive for brain antigens. In experimental autoimmune encephalomyelitis, the animal model for MS, myelin-reactive T cells initiate the autoimmune process when entering the nervous tissue and become reactivated upon local encounter of their cognate CNS antigen. Thereby, the strength of the T-cellular reactivation process within the CNS tissue is crucial for the manifestation and the severity of the clinical disease. Recently, B cells were found to participate in the pathogenesis of CNS autoimmunity, with several diverse underlying mechanisms being under discussion. We here report that B cells play an important role in promoting the initiation process of CNS autoimmunity. Myelin-specific antibodies produced by autoreactive B cells after activation in the periphery diffused into the CNS together with the first invading pathogenic T cells. The antibodies accumulated in resident antigen-presenting phagocytes and significantly enhanced the activation of the incoming effector T cells. The ensuing strong blood–brain barrier disruption and immune cell recruitment resulted in rapid manifestation of clinical disease. Therefore, myelin oligodendrocyte glycoprotein (MOG)-specific autoantibodies can initiate disease bouts by cooperating with the autoreactive T cells in helping them to recognize their autoantigen and become efficiently reactivated within the immune-deprived nervous tissue.  

4.1549           Herpes Simplex Virus and Interferon Signaling Induce Novel Autophagic Clusters in Sensory Neurons

Katzenell, A. and Leib, D.A.

1. Virol., 90(9), 4706-4719 (2016)

  Herpes simplex virus 1 (HSV-1) establishes lifelong infection in the neurons of trigeminal ganglia (TG), cycling between productive infection and latency. Neuronal antiviral responses are driven by type I interferon (IFN) and are crucial to controlling HSV-1 virulence. Autophagy also plays a role in this neuronal antiviral response, but the mechanism remains obscure. In this study, HSV-1 infection of murine TG neurons triggered unusual clusters of autophagosomes, predominantly in neurons lacking detectable HSV-1 antigen. Treatment of neurons with IFN-β induced a similar response, and cluster formation by infection or IFN treatment was dependent upon an intact IFN-signaling pathway. The autophagic clusters were decorated with both ISG15, an essential effecter of the antiviral response, and p62, a selective autophagy receptor. The autophagic clusters were not induced by rapamycin or starvation, consistent with a process of selective autophagy. While clusters were triggered by other neurotropic herpesviruses, infection with unrelated viruses failed to induce this response. Following ocular infection in vivo, clusters formed exclusively in the infected ophthalmic branch of the TG. Taken together, our results show that infection with HSV and antiviral signaling in TG neurons produce an unorthodox autophagic response. This autophagic clustering is associated with antiviral signaling, the presence of viral genome, and the absence of HSV protein expression and may therefore represent an important neuronal response to HSV infection and the establishment of latency.    

4.1550           MyD88 Mediates Instructive Signaling in Dendritic Cells and Protective Inflammatory Response during Rickettsial Infection

Bechelli, J., Smalley, C., Zhao, X., Judy, B., Valdes, P., Walker, D.H. and Fang, R. Infect. Immun., 84(4), 883-893 (2016)   Spotted fever group rickettsiae cause potentially life-threatening infections throughout the world. Several members of the Toll-like receptor (TLR) family are involved in host response to rickettsiae, and yet the mechanisms by which these TLRs mediate host immunity remain incompletely understood. In the present study, we found that host susceptibility of MyD88^−/− mice to infection with Rickettsia conorii or Rickettsia australis was significantly greater than in wild-type (WT) mice, in association with severely impaired bacterial clearance in vivo. R. australis-infected MyD88^−/− mice showed significantly lower expression levels of gamma interferon (IFN-γ), interleukin-6 (IL-6), and IL-1β, accompanied by significantly fewer inflammatory infiltrates of macrophages and neutrophils in infected tissues, than WT mice. The serum levels of IFN-γ, IL-12, IL-6, and granulocyte colony-stimulating factor were significantly reduced, while monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, and RANTES were significantly increased in infected MyD88^−/− mice compared to WT mice. Strikingly, R. australis infection was incapable of promoting increased expression of MHC-II^high and production of IL-12p40 in MyD88^−/− bone marrow-derived dendritic cells (BMDCs) compared to WT BMDCs, although costimulatory molecules were upregulated in both types of BMDCs. Furthermore, the secretion levels of IL-1β by Rickettsia-infected BMDCs and in the sera of infected mice were significantly reduced in MyD88^−/− mice compared to WT controls, suggesting that in vitro and in vivo production of IL-1β is MyD88 dependent. Taken together, our results suggest that MyD88 signaling mediates instructive signals in DCs and secretion of IL-1β and type 1 immune cytokines, which may account for the protective inflammatory response during rickettsial infection.  

4.1551           Isolation of Conventional Dendritic Cells from Mouse Lungs

Van de Laar, L., Guilliams, M. and Tavernier, S. Methods in Mol. Biol., 1423, 139-152 (2016)   The lungs are in direct contact with the environment. Separated only by a thin layer of mucosa, the lung immune system is being exposed to dangers like pathogens, allergens, or pollutants. The lung dendritic cells form an elaborate network at the basolateral side of the epithelium and continuously sample antigens from the airway lumen. The conventional dendritic cells (cDCs) in the lung can be subdivided into two distinct subsets based on their ontogeny and are described to have distinct immunological functions. High-quality ex vivo isolation of these cells is required for experiments such as functional assays, transfer experiments, or transcriptomics and is crucial to further our knowledge concerning these subpopulations. In this chapter we describe a protocol for the isolation of both CD103⁺ and CD11b⁺ cDCs. In our protocol we compare different methods of cell isolation. We propose that the optimal isolation technique is based on the number of cells needed and the type of experiment that will be performed. If low cell numbers are required, simple flow cytometry-assisted cell sorting (FACS) is sufficient. In the case of high cell numbers that will be lysed or fixed upon sorting, positive selection of CD11c⁺ cells followed by FACS can be utilized. Purification of cDCs through gradient selection and subsequent sorting is found to be optimal for experiments that require large amount of cells for functional assays.  

4.1552           Negative regulation of the hepatic fibrogenic response by suppressor of cytokine signaling 1

Kandhi, R., Bobbala, D., Yeganeh, M., Mayhue, M., Menendez, A. and Ilangumaran, S. Cytokine, 82, 58-69 (2016)   Suppressor of cytokine signaling 1 (SOCS1) is an indispensable regulator of IFNγ signaling and has been implicated in the regulation of liver fibrosis. However, it is not known whether SOCS1 mediates its anti-fibrotic functions in the liver directly, or via modulating IFNγ, which has been implicated in attenuating hepatic fibrosis. Additionally, it is possible that SOCS1 controls liver fibrosis by regulating hepatic stellate cells (HSC), a key player in fibrogenic response. While the activation pathways of HSCs have been well characterized, the regulatory mechanisms are not yet clear. The goals of this study were to dissociate IFNγ-dependent and SOCS1-mediated regulation of hepatic fibrogenic response, and to elucidate the regulatory functions of SOCS1 in HSC activation. Liver fibrosis was induced in Socs1−/−Ifng−/− mice with dimethylnitrosamine or carbon tetrachloride. Ifng−/− and C57BL/6 mice served as controls. Following fibrogenic treatments, Socs1−/−Ifng−/− mice showed elevated serum ALT levels and increased liver fibrosis compared to Ifng−/− mice. The latter group showed higher ALT levels and fibrosis than C57BL/6 controls. The livers of SOCS1-deficient mice showed bridging fibrosis, which was associated with increased accumulation of myofibroblasts and abundant collagen deposition. SOCS1-deficient livers showed increased expression of genes coding for smooth muscle actin, collagen, and enzymes involved in remodeling the extracellular matrix, namely matrix metalloproteinases and tissue inhibitor of metalloproteinases. Primary HSCs from SOCS1-deficient mice showed increased proliferation in response to growth factors such as HGF, EGF and PDGF, and the fibrotic livers of SOCS1-deficient mice showed increased expression of the Pdgfb gene. Taken together, these data indicate that SOCS1 controls liver fibrosis independently of IFNγ and that part of this regulation may occur via regulating HSC proliferation and limiting growth factor availability.  

4.1553           Single and double layer centrifugation improve the quality of cryopreserved bovine sperm from poor quality ejaculates

Gloria, A., Carluccio, A., Wegher, L., Robbe, D., Befacchia, G. and Contri, A.

1. Animal Science and Biotechnology, 7:30 (2016)

  Background Density gradient centrifugation was reported as a technique of semen preparation in assisted reproductive techniques in humans and animals. This technique was found to be efficient in improving semen quality after harmful techniques such as cryopreservation. Recently a modified technique, single layer centrifugation, was proposed as a technique providing a large amount of high quality spermatozoa, and this treatment was performed before conservation. Single layer centrifugation has been studied prevalently in stallions and in boars, but limited data were available for bulls. Occasionally bulls are known to experience a transient reduction in semen quality, thus techniques that allow improvement in semen quality could be applied in this context. The aim of this study was the evaluation of single layer and double layer centrifugation by the use of iodixanol, compared with conventional centrifugation and non-centrifuged semen, on the sperm characteristics during the cryopreservation process in bulls with normal and poor semen quality.  

4.1554           Whole Blood RNA as a Source of Transcript-Based Nutrition- and Metabolic Health-Related Biomarkers

Petrov, P.D., Bonet, M.L., Reynes, B., Oliver, P., Palou, A. and Ribot, J. PloS One, 11(5), e0155361 (2016)   Blood cells are receiving an increasing attention as an easily accessible source of transcript-based biomarkers. We studied the feasibility of using mouse whole blood RNA in this context. Several paradigms were studied: (i) metabolism-related transcripts known to be affected in rat tissues and peripheral blood mononuclear cells (PBMC) by fasting and upon the development of high fat diet (HFD)-induced overweight were assessed in whole blood RNA of fasted rats and mice and of HFD-fed mice; (ii) retinoic acid (RA)-responsive genes in tissues were assessed in whole blood RNA of control and RA-treated mice; (iii) lipid metabolism-related transcripts previously identified in PBMC as potential biomarkers of metabolic health in a rat model were assessed in whole blood in an independent model, namely retinoblastoma haploinsufficient (Rb+/-) mice. Blood was collected and stored in RNAlater® at -80°C until analysis of selected transcripts by real-time RT-PCR. Comparable changes with fasting were detected in the expression of lipid metabolism-related genes when RNA from either PBMC or whole blood of rats or mice was used. HFD-induced excess body weight and fat mass associated with expected changes in the expression of metabolism-related genes in whole blood of mice. Changes in gene expression in whole blood of RA-treated mice reproduced known transcriptional actions of RA in hepatocytes and adipocytes. Reduced expression of Fasn, Lrp1, Rxrb and Sorl1 could be validated as early biomarkers of metabolic health in young Rb+/- mice using whole blood RNA. Altogether, these results support the use of whole blood RNA in studies aimed at identifying blood transcript-based biomarkers of nutritional/metabolic status or metabolic health. Results also support reduced expression of Fasn, Lrp1, Rxrb and Sorl1 in blood cells at young age as potential biomarkers of metabolic robustness.  

4.1555           Iso-acoustic focusing of cells for size-insensitive acousto-mechanical phenotyping

Augustsson, P., Karlsen, J.T., Su, H-W., Bruus, H. and Voldman, J. Nature Communications, 7:11556 (2016)   Mechanical phenotyping of single cells is an emerging tool for cell classification, enabling assessment of effective parameters relating to cells’ interior molecular content and structure. Here, we present iso-acoustic focusing, an equilibrium method to analyze the effective acoustic impedance of single cells in continuous flow. While flowing through a microchannel, cells migrate sideways, influenced by an acoustic field, into streams of increasing acoustic impedance, until reaching their cell-type specific point of zero acoustic contrast. We establish an experimental procedure and provide theoretical justifications and models for iso-acoustic focusing. We describe a method for providing a suitable acoustic contrast gradient in a cell-friendly medium, and use acoustic forces to maintain that gradient in the presence of destabilizing forces. Applying this method we demonstrate iso-acoustic focusing of cell lines and leukocytes, showing that acoustic properties provide phenotypic information independent of size.  

4.1556           Unique microbial-derived volatile organic compounds in portal venous circulation in murine non-alcoholic fatty liver disease

Reid, D.T., McDonald, B., Khalid, T., Vo, T., Schenck, L.P., Surette, M.G., Beck, P.L., Reimer, R.A., Robert, C.S., Rioux, K.P. and Eksteen, B. Biochim. Biophys. Acta, 1862, 1337-1344 (2016)   Background and aims Non-alcoholic fatty liver disease is now the leading liver disease in North America. The progression of non-alcoholic fatty liver disease to the inflammatory condition, non-alcoholic steatohepatitis is complex and currently not well understood. Intestinal microbial dysbiosis has been implicated in the development of non-alcoholic fatty liver disease and progression of non-alcoholic steatohepatitis. Volatile organic compounds are byproducts of microbial metabolism in the gut that may enter portal circulation and have hepatotoxic effects contributing to the pathogenesis of non-alcoholic steatohepatitis. To test this hypothesis, we measured volatile organic compounds in cecal luminal contents and portal venous blood in a mouse model of non-alcoholic steatohepatitis. Methods Gas chromatography–mass spectrometry analysis was conducted on cecal content and portal vein blood for volatile organic compound detection from mice fed a methionine and choline deficient diet, which induces non-alcoholic steatohepatitis. The colonic microbiome was studied by 16S rRNA gene amplification using the Illumina MiSeq platform. Results Sixty-eight volatile organic compounds were detected in cecal luminal content, a subset of which was also present in portal venous blood. Importantly, differences in portal venous volatile organic compounds were associated with diet-induced steatohepatitis establishing a biochemical link between gut microbiota-derived volatile organic compounds and increased susceptibility to non-alcoholic steatohepatitis. Conclusion Our model creates a novel tool to further study the role of gut-derived volatile organic compounds in the pathogenesis of non-alcoholic steatohepatitis.  

4.1557           RNA-Seq following PCR-based sorting reveals rare cell transcriptional signatures

Pellegrino, M., Sciambi, A., Yates, J.L., Mast, J.D., Silver, C. and Eastburn, D.J. BMC Genomics, 17:361 (2016)   Background Rare cell subtypes can profoundly impact the course of human health and disease, yet their presence within a sample is often missed with bulk molecular analysis. Single-cell analysis tools such as FACS, FISH-FC and single-cell barcode-based sequencing can investigate cellular heterogeneity; however, they have significant limitations that impede their ability to identify and transcriptionally characterize many rare cell subpopulations. Results PCR-activated cell sorting (PACS) is a novel cytometry method that uses single-cell TaqMan PCR reactions performed in microfluidic droplets to identify and isolate cell subtypes with high-throughput. Here, we extend this method and demonstrate that PACS enables high-dimensional molecular profiling on TaqMan-targeted cells. Using a random priming RNA-Seq strategy, we obtained high-fidelity transcriptome measurements following PACS sorting of prostate cancer cells from a heterogeneous population. The sequencing data revealed prostate cancer gene expression profiles that were obscured in the unsorted populations. Single-cell expression analysis with PACS was subsequently used to confirm a number of the differentially expressed genes identified with RNA sequencing. Conclusions PACS requires minimal sample processing, uses readily available TaqMan assays and can isolate cell subtypes with high sensitivity. We have now validated a method for performing next-generation sequencing on mRNA obtained from PACS isolated cells. This capability makes PACS well suited for transcriptional profiling of rare cells from complex populations to obtain maximal biological insight into cell states and behaviors.  

4.1558           Expansion and Hepatic Differentiation of Adult Blood-Derived CD34+ Progenitor Cells and Promotion of Liver Regeneration After Acute Injury

Hu, M., Li, S., Menon, S., Liu, B., Hu, M.S., Longaker, M.T. and Lorentz, H.P. Stem Cells Translational Medicine, 5, 723-732 (2016)   The low availability of functional hepatocytes has been an unmet demand for basic scientific research, new drug development, and cell-based clinical applications for decades. Because of the inability to expand hepatocytes in vitro, alternative sources of hepatocytes are a focus of liver regenerative medicine. We report a new group of blood-derived CD34⁺ progenitor cells (BDPCs) that have the ability to expand and differentiate into functional hepatocyte-like cells and promote liver regeneration. BDPCs were obtained from the peripheral blood of an adult mouse with expression of surface markers CD34, CD45, Sca-1, c-kit, and Thy1.1. BDPCs can proliferate in vitro and differentiate into hepatocyte-like cells expressing hepatocyte markers, including CK8, CK18, CK19, α-fetoprotein, integrin-β1, and A6. The differentiated BDPCs (dBDPCs) also display liver-specific functional activities, such as glycogen storage, urea production, and albumin secretion. dBDPCs have cytochrome P450 activity and express specific hepatic transcription factors, such as hepatic nuclear factor 1α. To demonstrate liver regenerative activity, dBDPCs were injected into mice with severe acute liver damage caused by a high-dose injection of carbon tetrachloride (CCl₄). dBDPC treatment rescued the mice from severe acute liver injury, increased survival, and induced liver regeneration. Because of their ease of access and application through peripheral blood and their capability of rapid expansion and hepatic differentiation, BDPCs have great potential as a cell-based therapy for liver disease.  

4.1559           Analysis of Dendritic Cell Function Using Clec9A-DTR Transgenic Mice

Tetlak, P. and Ruedl, C. Methods in Mol. Biol., 1423, 275-289 (2016)   The Clec9A-diphtheria toxin receptor (DTR) transgenic mouse strain provides a robust animal model to study the function of lymphoid organ-resident CD8⁺ dendritic cells (DCs) and nonlymphoid organ-specific CD103⁺ DCs in infectioous diseases and inflammation. Here we describe some basic protocols for CD8⁺/CD103⁺ DC isolation, for their in vivo depletion, and for their characterization by multi-color flow cytometry analysis. As an example for in vivo functional characterization of this DC subset, we present here the experimental cerebral malaria model. Furthermore, we illustrate advantages and pitfalls of the Clec9A-DTR system.  

4.1560           Producing megakaryocytes from a human peripheral blood source

Ivetic, N., Nazi, I., Karim, N., Clare, R., Smith, J.W., Moore, J.C., Hope, K.J., Kelton, J.G. and Arnold, D.M. Transfusion, 56(5), 1066-1074 (2016)   BACKGROUND Cultured megakaryocytes could prove useful in the study of human diseases, but it is difficult to produce sufficient numbers for study. We describe and evaluate the use of an expansion process to develop mature megakaryocytes from peripheral blood–derived human hematopoietic stem and progenitor cells (HSPCs). STUDY DESIGN AND METHODS HSPCs (CD34+) were isolated from peripheral blood by positive selection and expanded using an optimal CD34+ expansion supplement. We evaluated megakaryocyte growth, maturation, and morphology in response to thrombopoietin (TPO) stimulation using flow cytometry and electron microscopy. TPO demonstrated a dose-dependent stimulatory effect on both megakaryocyte number and maturation. RESULTS From 90 to 120 mL of unmanipulated peripheral blood, we isolated a mean of 1.5 × 10⁵ HSPCs (1.5 × 10³ cells/mL of whole blood). HSPCs expanded nine-fold after a 4-day culture using an expansion supplement. Expanded cells were cultured for an additional 8 days with TPO (20 ng/mL), which resulted in a 2.9-fold increase in megakaryocytic cells where 83% of live cells expressed CD41a+, a marker of megakaryocyte commitment, and 50% expressed CD42b+, a marker for megakaryocyte maturation. The expanded HSPCs responded to TPO stimulation to yield more than 1.0 × 10⁶ megakaryocytes. This cell number was sufficient for morphologic studies that demonstrated these expanded HSPCs produced mature polyploid megakaryocytes capable of forming proplatelet extensions. CONCLUSIONS Peripheral blood HSPCs can be expanded and differentiated into functional, mature megakaryocytes, a finding that supports the use of this process to study inherent platelet (PLT) production disorders as well as study factors that impair normal PLT production.  

4.1561           ResolvinD1 reduces apoptosis and inflammation in primary human alveolar epithelial type 2 cells

Xie, W., Wang, H., Liu, Q., Li, Y., Wang, J., Yao, S. and Wu, Q. Lab. Invest., 96(5), 526-536 (2016)   Lung epithelial apoptosis and inflammatory responses are important pathological processes in many pulmonary disorders. ResolvinD1 (RvD1), generated in inflammatory resolution processes, reduces inflammatory responses in animal models of lung diseases. The aim of this study was to investigate whether RvD1 attenuates apoptosis and proinflammatory responses in primary human alveolar epithelial type 2 cells (AEC2 cells) that are exposed to lipopolysaccharide (LPS) in vitro. We examined the percentage of apoptotic AEC2 cells by flow cytometry. The expression levels of cytokines and chemokines were determined by ELISA and microarray. The expression levels of molecular signaling modulators were evaluated by western blot. LPS-stimulated AEC2 cells pretreated with RvD1 exhibited a statistically significant reduction in apoptosis. The pretreatment of LPS-stimulated cells with RvD1 stimulated the phosphorylation of AKT and prevented the cleavage of caspase-3, the upregulation of Bax, and the downregulation of Bcl-2. The antiapoptotic effects of RvD1 were abrogated upon pretreatment with a PI3K inhibitor. In addition, RvD1 reduced the release of cytokines and chemokines, and inhibited the degradation and phosphorylation of IκB-α in LPS-stimulated AEC2 cells. RvD1 reduces apoptosis of LPS-exposed AEC2 cells by inducing the phosphorylation of AKT and attenuates the inflammatory response by suppressing the degradation and phosphorylation of IκB-α.  

4.1562           Striatopallidal Neuron NMDA Receptors Control Synaptic Connectivity, Locomotor, and Goal-Directed Behaviors

Lambot, L., Rodriguez, E.C., Houtteman, D., Li, Y., Schiffmann, S.N., Gall, D. and de Kerchove d’Exaerde, A.

1. Neurosci., 36(18), 4976-4992 (2016)

  The basal ganglia (BG) control action selection, motor programs, habits, and goal-directed learning. The striatum, the principal input structure of BG, is predominantly composed of medium-sized spiny neurons (MSNs). Arising from these spatially intermixed MSNs, two inhibitory outputs form two main efferent pathways, the direct and indirect pathways. Striatonigral MSNs give rise to the activating, direct pathway MSNs and striatopallidal MSNs to the inhibitory, indirect pathway (iMSNs). BG output nuclei integrate information from both pathways to fine-tune motor procedures and to acquire complex habits and skills. Therefore, balanced activity between both pathways is crucial for harmonious functions of the BG. Despite the increase in knowledge concerning the role of glutamate NMDA receptors (NMDA-Rs) in the striatum, understanding of the specific functions of NMDA-R iMSNs is still lacking. For this purpose, we generated a conditional knock-out mouse to address the functions of the NMDA-R in the indirect pathway. At the cellular level, deletion of GluN1 in iMSNs leads to a reduction in the number and strength of the excitatory corticostriatopallidal synapses. The subsequent scaling down in input integration leads to dysfunctional changes in BG output, which is seen as reduced habituation, delay in goal-directed learning, lack of associative behavior, and impairment in action selection or skill learning. The NMDA-R deletion in iMSNs causes a decrease in the synaptic strength of striatopallidal neurons, which in turn might lead to a imbalanced integration between direct and indirect MSN pathways, making mice less sensitive to environmental change. Therefore, their ability to learn and adapt to the environment-based experience was significantly affected.  

4.1563           Type I and III IFNs Produced by Plasmacytoid Dendritic Cells in Response to a Member of the Flaviviridae Suppress Cellular Immune Responses

Reid, E., Juleff, N., Windsor, M., Gubbins, S., Roberts, L., Morgan, s., Meyers, G., Perez-Martin, E., Tchilian, E., Charleston, B. and Seago, J.

1. Immunol., 196(10), 4214-4226 (2016)

  The pestivirus noncytopathic bovine viral diarrhea virus (BVDV) can suppress IFN production in the majority of cell types in vitro. However, IFN is detectable in serum during acute infection in vivo for ∼5–7 d, which correlates with a period of leucopoenia and immunosuppression. In this study, we demonstrate that a highly enriched population of bovine plasmacytoid dendritic cells (DCs) produced IFN in response to BVDV in vitro. We further show that the majority of the IFN produced in response to infection both in vitro and in vivo is type III IFN and acid labile. Further, we show IL-28B (IFN-λ3) mRNA is induced in this cell population in vitro. Supernatant from plasmacytoid DCs harvested postinfection with BVDV or recombinant bovine IFN-α or human IL-28B significantly reduced CD4⁺ T cell proliferation induced by tubercle bacillus Ag 85–stimulated monocyte-derived DCs. Furthermore, these IFNs induced IFN-stimulated gene expression predominantly in monocyte-derived DCs. IFN-treated immature DCs derived from murine bone marrow also had a reduced capacity to stimulate T cell proliferative responses to tubercle bacillus Ag 85. Immature DCs derived from either source had a reduced capacity for Ag uptake following IFN treatment that is dose dependent. Immunosuppression is a feature of a number of pestivirus infections; our studies suggest type III IFN production plays a key role in the pathogenesis of this family of viruses. Overall, in a natural host, we have demonstrated a link between the induction of type I and III IFN after acute viral infection and transient immunosuppression.  

4.1564           VEGF-sdf1 recruitment of CXCR7+ bone marrow progenitors of liver sinusoidal endothelial cells promotes rat liver regeneration

Delever, L.D., Wang, X. and Wang, L. Am. J. Physiol. Gastrointest. Liver Physiol., 310(9), G739-G746 (2016)   In liver injury, recruitment of bone marrow (BM) progenitors of liver sinusoidal endothelial cells (sprocs) is necessary for normal liver regeneration. Hepatic vascular endothelial growth factor (VEGF) is a central regulator of the recruitment process. We examine whether stromal cell-derived factor 1 [sdf1, or CXC ligand 12 (CXCL12)] acts downstream from VEGF to mediate recruitment of BM sprocs, what the sdf1 receptor type [CXC receptor (CXCR)-4 or CXCR7] is on sprocs, and whether sdf1 signaling is required for normal liver regeneration. Studies were performed in the rat partial hepatectomy model. Tracking studies of BM sprocs were performed in wild-type Lewis rats that had undergone BM transplantation from transgenic enhanced green fluorescent protein-positive Lewis rats. Knockdown studies were performed using antisense oligonucleotides (ASOs). Expression of sdf1 doubles in liver and liver sinusoidal endothelial cells (LSECs) after partial hepatectomy. Upregulation of sdf1 expression increases proliferation of sprocs in the BM, mobilization of CXCR7⁺ BM sprocs to the circulation, and engraftment of CXCR7⁺ BM sprocs in the liver and promotes liver regeneration. Knockdown of hepatic VEGF with ASOs decreases hepatic sdf1 expression and plasma sdf1 levels. When the effect of VEGF knockdown on sdf1 is offset by infusion of sdf1, VEGF knockdown-induced impairment of BM sproc recruitment after partial hepatectomy is completely attenuated and liver regeneration is normalized. These data demonstrate that the VEGF-sdf1 pathway regulates recruitment of CXCR7⁺ BM sprocs to the hepatic sinusoid after partial hepatectomy and is required for normal liver regeneration.  

4.1565           Olfactory Ensheathing Cell Transplantation after a Complete Spinal Cord Transection Mediates Neuroprotective and Immunomodulatory Mechanisms to Facilitate Regeneration

Khankan, R.R., Griffis, K.G., Haggerty-Skeans, J.R., Zhong, H., Roy, R.R., Edgerton, V.R. and Phelps, P.E.

1. Neurosci., 36(23), 6269-6286 (2016)

  Multiple neural and peripheral cell types rapidly respond to tissue damage after spinal cord injury to form a structurally and chemically inhibitory scar that limits axon regeneration. Astrocytes form an astroglial scar and produce chondroitin sulfate proteoglycans (CSPGs), activate microglia, and recruit blood-derived immune cells to the lesion for debris removal. One beneficial therapy, olfactory ensheathing cell (OEC) transplantation, results in functional improvements and promotes axon regeneration after spinal cord injury. The lack of an OEC-specific marker, however, has limited the investigation of mechanisms underlying their proregenerative effects. We compared the effects of enhanced green fluorescent protein-labeled fibroblast (FB) and OEC transplants acutely after a complete low-thoracic spinal cord transection in adult rats. We assessed the preservation of neurons and serotonergic axons, the levels of inhibitory CSPGs and myelin debris, and the extent of immune cell activation between 1 and 8 weeks postinjury. Our findings indicate that OECs survive longer than FBs post-transplantation, preserve axons and neurons, and reduce inhibitory molecules in the lesion core. Additionally, we show that OECs limit immune-cell activation and infiltration, whereas FBs alter astroglial scar formation and increase immune-cell infiltration and concomitant secondary tissue damage. Administration of cyclosporine-A to enhance graft survival demonstrated that immune suppression can augment OEC contact-mediated protection of axons and neurons during the first 2 weeks postinjury. Collectively, these data suggest that OECs have neuroprotective and immunomodulatory mechanisms that create a supportive environment for neuronal survival and axon regeneration after spinal cord injury.  

4.1566           β1-C121W Is Down But Not Out: Epilepsy-Associated Scn1b-C121W Results in a Deleterious Gain-of-Function

Kruger, L.C., O’Malley, H.A., Hull, J.M., Kleeman, A., Patino, G.A.and Isom, L.L.

1. Neurosci., 36(23), 6213-6224 (2016)

  Voltage-gated sodium channel (VGSC) β subunits signal through multiple pathways on multiple time scales. In addition to modulating sodium and potassium currents, β subunits play nonconducting roles as cell adhesion molecules, which allow them to function in cell–cell communication, neuronal migration, neurite outgrowth, neuronal pathfinding, and axonal fasciculation. Mutations in SCN1B, encoding VGSC β1 and β1B, are associated with epilepsy. Autosomal-dominant SCN1B-C121W, the first epilepsy-associated VGSC mutation identified, results in genetic epilepsy with febrile seizures plus (GEFS+). This mutation has been shown to disrupt both the sodium-current-modulatory and cell-adhesive functions of β1 subunits expressed in heterologous systems. The goal of this study was to compare mice heterozygous for Scn1b-C121W (Scn1b^+/W) with mice heterozygous for the Scn1b-null allele (Scn1b^+/−) to determine whether the C121W mutation results in loss-of-function in vivo. We found that Scn1b^+/W mice were more susceptible than Scn1b^+/− and Scn1b^+/+ mice to hyperthermia-induced convulsions, a model of pediatric febrile seizures. β1-C121W subunits are expressed at the neuronal cell surface in vivo. However, despite this, β1-C121W polypeptides are incompletely glycosylated and do not associate with VGSC α subunits in the brain. β1-C121W subcellular localization is restricted to neuronal cell bodies and is not detected at axon initial segments in the cortex or cerebellum or at optic nerve nodes of Ranvier of Scn1b^W/W mice. These data, together with our previous results showing that β1-C121W cannot participate in trans-homophilic cell adhesion, lead to the hypothesis that SCN1B-C121W confers a deleterious gain-of-function in human GEFS+ patients.  

4.1567           Activation of hepatic stellate cell in Pten null liver injury model

He, L., Gubbins, J., Peng, Z., Medina, V., Fei, F., Ashina, K., Wang, J., Kahn, M., Rountree, C.B. and Stiles, B.L. Fibrogenesis & Tissue Repair, 9:8 (2016)   Background Hepatic fibrosis is a prominent pathological feature associated with chronic liver disease including non-alcoholic hepatosteatosis (NASH), and a precursor for liver cancer development. We previously reported that PTEN loss in the liver, which leads to hyperactivated liver insulin signaling results in NASH development. Here we used the same mouse model to study the progression from steatosis to fibrosis. Results The Pten null livers develop progressive liver fibrosis as indicated by Sirius Red staining and increased expression of collagen I, Timp 1, SMAα, and p75NTR. Consistently, hepatic stellate cells (HSCs) isolated from Pten null livers are readily activated when compared with that from mice with intact PTEN. Deletion of AKT2, the downstream target of PTEN signal, blocked NASH development, and alleviated fibrosis. HSCs from the Pten/Akt2 double null mice are quiescent like those isolated from the control livers. Our analysis shows that the activation of HSCs does not depend on the intrinsic signals regulated by PI3K/AKT, the target of PTEN, but does depend on steatosis and injury to the liver. During the progression of liver fibrosis in the Pten null model, Wnt ligands and signaling receptor are induced, concurrent with the reduction of sFRP5, a Wnt antagonist. We showed that treatment of HSCs with Wnt receptor antagonist blocks the observed morphological changes when HSCs undergo activation in culture. This signal appears to be mediated by β-catenin, as manipulating β-catenin signaling alters marker gene expressions of HSC activation. Conclusions Wnt/β-catenin activation serves as an important mediator for fibrosis development resulting from NASH using a mouse model where NASH is mimicked by PTEN loss.  

4.1568           Assessment of human platelet survival in the NOD/SCID mouse model: technical considerations

Fuhrmann, J., Jouni, R., Alex, J., Zöllner, H., Wesche, J., Greinacher, A. and Bakchoul, T. Transfusion, 56, 1370-1376 (82016)   BACKGROUND The NOD/SCID mouse model is a unique and sophisticated method to study the survival of human platelets (PLTs) in vivo. Meanwhile, several research groups adopted this model to analyze a wide range of PLT antibodies. Differences exist between the research groups regarding the method of PLT injection, the amount and route of antibody injection, and the preparation of blood samples collected from the animal, making it difficult to compare results between studies. STUDY DESIGN AND METHODS We compared the survival of human PLTs infused into NOD/SCID mice via the tail vein or the retro-orbital plexus. The percentage of circulating human PLTs in the mouse circulation was determined by flow cytometry. Murine blood samples were prepared using two different methods: 1) direct fixation of whole blood samples and 2) isolation of PLTs by density gradient centrifugation. RESULTS Recovery of human PLTs after tail vein injection was comparable to retro-orbital injection (13% vs. 11% of all circulating PLTs, p = 0.401). However, the survival rate of tail vein–infused PLTs was higher than that of retro-orbitally injected PLTs (median PLT survival after 5 hr 84% vs. 56%, p = 0.025). Moreover, we observed that determination of circulating human PLTs in directly fixed murine whole blood samples shows better reproducibility compared to the density gradient centrifugation method. CONCLUSIONS Tail vein injection of human PLTs into the NOD/SCID mice is superior to retro-orbital injection in terms of human PLT survival. Direct fixation of whole blood samples allows better reproducibility of results compared to the density gradient centrifugation method.  

4.1569           The Human Pancreas as a Source of Protolerogenic Extracellular Matrix Scaffold for a New-generation Bioartificial Endocrine Pancreas

Peloso, A. et al Annals of Surgery, 264(1), 169-179 (2016)   Objectives: Our study aims at producing acellular extracellular matrix scaffolds from the human pancreas (hpaECMs) as a first critical step toward the production of a new-generation, fully human-derived bioartificial endocrine pancreas. In this bioartificial endocrine pancreas, the hardware will be represented by hpaECMs, whereas the software will consist in the cellular compartment generated from patient's own cells. Background: Extracellular matrix (ECM)-based scaffolds obtained through the decellularization of native organs have become the favored platform in the field of complex organ bioengineering. However, the paradigm is now switching from the porcine to the human model. Methods: To achieve our goal, human pancreata were decellularized with Triton-based solution and thoroughly characterized. Primary endpoints were complete cell and DNA clearance, preservation of ECM components, growth factors and stiffness, ability to induce angiogenesis, conservation of the framework of the innate vasculature, and immunogenicity. Secondary endpoint was hpaECMs’ ability to sustain growth and function of human islet and human primary pancreatic endothelial cells. Results: Results show that hpaECMs can be successfully and consistently produced from human pancreata and maintain their innate molecular and spatial framework and stiffness, and vital growth factors. Importantly, hpaECMs inhibit human naïve CD4⁺ T-cell expansion in response to polyclonal stimuli by inducing their apoptosis and promoting their conversion into regulatory T cells. hpaECMs are cytocompatible and supportive of representative pancreatic cell types. Discussion: We, therefore, conclude that hpaECMs has the potential to become an ideal platform for investigations aiming at the manufacturing of a regenerative medicine-inspired bioartificial endocrine pancreas.  

4.1570           Isolating High Islet Mass Even from Alcoholic Pancreatitis Pancreases Intended for Clinical Islet Auto-Transplantation: Improved Strategies to Human Islet Isolation Technique

Balamurugan, A., Loganathan, G., Tweed, B., Tucker, W., Mokshagundam, S., Williams, S.and Hughes, M, Am. J. Transplant., 16(S3), abstract 81, 405-798 (2016)   It has been reported in the literature that after pancreatic resection with planned islet auto-transplantation (IAT), islets isolated from alcoholic pancreatitis (AP) pancreases resulted in lower islet yields and in some cases, failed isolations (J Am Coll Surg. 2013 Apr;216:59: Should pancreatectomy with islet cell autotransplantation in patients with chronic alcoholic pancreatitis be abandoned?). Isolating islets from AP pancreases poses multiple challenges (alterations in duct structure and accumulation of fibrotic bundles) which make it difficult to obtain high islet yield. We have developed new approaches to maximize the islet yield from pancreases with AP. The modified islet isolation process includes 1) Dosing the collagenase enzyme based on severity of fibrosis instead of using the standard (brain-dead donor pancreas) one full vial approach. 2) Adequate delivery of tissue dissociation enzyme throughout the pancreas by using both intraductal and parenchymal injection methods 3) Judging the quality of distention after sectioning the pancreases and perform additional enzyme distention 4) Warm enzyme recirculation if digestion time is prolonged for >30minutes. Etiology of chronic pancreatitis (CP) was alcoholism (n=4), and idiopathic (n=3). Islets were isolated according to the clinical transplantation protocols. AP cases were compared with idiopathic patients. The new enzyme mixture (VitaCyte CIzyme HA+ Serva Neutral Protease) was used for pancreas digestion. Islet purification was done with iodixanol density gradients. The final islet product was tested for sterility and viability. Transplant outcome was monitored by c-peptide measurements. The average trimmed pancreas weight was 75 ± 9 grams. The average transplanted islet mass was 4,593 ± 728 IEQ/kg (nonalcoholic cases: 5,728 ± 278) and the average IEQ/gram pancreas was 4,487 ± 1,158 (nonalcoholic: 5,788 ± 1,055). The average infused tissue volume was 9.3 ± 6.2 cc. The average islet viability was >88%. Positive c-peptide secretion was monitored in all patients one month after transplantation. Our results indicated that it was possible to obtain >4,000 IEQ/gram from AP pancreases and transplant >4500 IEQ/kg of patient body weight. Modified islet isolation approaches were effective in maximizing islet yield.  

4.1571           Attenuated viral hepatitis in Trem1−/− mice is associated with reduced inflammatory activity of neutrophils

Kozik, J-H., Trautmann, T., Carambia, A., Preti, M., Lütgehetmann, M., Krech, T., Wiegard, C., Heeren, J. and Herkel, J: Scientific Reports, 6:28556 (2016)   TREM1 (Triggering Receptor Expressed on Myeloid Cells 1) is a pro-inflammatory receptor expressed by phagocytes, which can also be released as a soluble molecule (sTREM1). The roles of TREM1 and sTREM1 in liver infection and inflammation are not clear. Here we show that patients with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection manifest elevated serum levels of sTREM1. In mice, experimental viral hepatitis induced by infection with Lymphocytic Choriomeningitis Virus (LCMV)-WE was likewise associated with increased sTREM1 in serum and urine, and with increased TREM1 and its associated adapter molecule DAP12 in the liver. Trem1−/− mice showed accelerated clearance of LCMV-WE and manifested attenuated liver inflammation and injury. TREM1 expression in the liver of wild-type mice was mostly confined to infiltrating neutrophils, which responded to LCMV by secretion of CCL2 and TNF-α, and release of sTREM1. Accordingly, the production of CCL2 and TNF-α was decreased in the livers of LCMV-infected Trem1−/− mice, as compared to LCMV-infected wildtype mice. These findings indicate that TREM1 plays a role in viral hepatitis, in which it seems to aggravate the immunopathology associated with viral clearance, mainly by increasing the inflammatory activity of neutrophils.  

4.1572           EPIGENETIC FACTORS IN THE EFFECTS OF NEONATAL ALCOHOL EXPOSURE ON

HYPOTHALAMIC MICROGLIA IN RATS

Chastain, L., Shrivastava, P., Cabrera, M. and  Sarkar, D.K. Alcoholism: Clin. Exp. Res., 40(S1), abstract 256, 16A-247A   Microglia are the brain’s resident immune cells, and experimental evidence shows that microglia over-activation may play a role in the detrimental effects of alcohol on the hypothalamus, but the mechanism for this is unknown. These studies sought to (i) characterize the effects of developmental alcohol exposure onmicroglia activation and (ii) investigate the effects of alcohol on epigenetic factors in microglia using a rat model for fetal alcohol exposure. Neonatal rat pups (third trimester human equivalent) were fed by oral gavage a milk formula containing 11.34%ethanol (vol/vol), yielding a total daily ethanol dose of 2.5 g/kg (AF), or isocaloric control (PF), or they were left in the litter with themother (AD) for 5 days (postnatal days 2–6). Two hours after the last feeding, the pups were sacrificed and hypothalamus was dissected.Whole hypothalamic tissue was used for quantification of RNA transcripts involved in inflammation using quantitative real time PCR. In another set of animals, microglia was purified from hypothalamic tissue by differential gradient centrifugation using OptiPrep gradient.Microglia characterization and comparison between AF, PF, and AD pups was performed by flow cytometry. In the gene expression study, AF pups showed a significant increase in pro-inflammatory transcripts including monocyte chemoattractant protein-1 (MCP-1) and colony stimulating factor 1 receptor (CSFR1) compared to AD controls, indicating increased pro-inflammatory gene expression as a result of neonatal alcohol exposure. Flow cytometry characterization of purified microglia showed a significant increase in microglia-specific calcium-binding protein IBA1 and chemokine interferon gamma (IFN-c) in AF pups compared to AD pups. As these results confirmed an increase in microglia activation and pro-inflammatory gene expression in the hypothalamus after neonatal alcohol exposure, the effects of neonatal alcohol on epigenetic regulators of gene expression were also investigated. AF pups showed a significant decrease in transcriptional repressors methyl CpG binding protein 2 (MeCP2) and histone deacetylase 1 (HDAC1) proteins compared to PF and AD pups. These results suggest that neonatal alcohol activates microglia possibly by decreasing transcriptional repressors, revealing a possible epigenetic mechanism for the adverse effects of alcohol on hypothalamic inflammation. (Supported by NIH grants F32AA023434 and R37AA008757).  

4.1573           Rickettsia australis Activates Inflammasome in Human and Murine Macrophages

Smalley, C., Bechelli, J., Rockx-Brouwer, D., Saito, T., Azar, S.R., Ismali, N., Walker, D.H. and Fang, R. PloS One, 11(6), e0157231 (2016)   Rickettsiae actively escape from vacuoles and replicate free in the cytoplasm of host cells, where inflammasomes survey the invading pathogens. In the present study, we investigated the interactions of Rickettsia australis with the inflammasome in both mouse and human macrophages. R. australis induced a significant level of IL-1β secretion by human macrophages, which was significantly reduced upon treatment with an inhibitor of caspase-1 compared to untreated controls, suggesting caspase-1-dependent inflammasome activation. Rickettsia induced significant secretion of IL-1β and IL-18 in vitro by infected mouse bone marrow-derived macrophages (BMMs) as early as 8–12 h post infection (p.i.) in a dose-dependent manner. Secretion of these cytokines was accompanied by cleavage of caspase-1 and was completely abrogated in BMMs deficient in caspase-1/caspase-11 or apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), suggesting that R. australis activate the ASC-dependent inflammasome. Interestingly, in response to the same quantity of rickettsiae, NLRP3-/- BMMs significantly reduced the secretion level of IL-1β compared to wild type (WT) controls, suggesting that NLRP3 inflammasome contributes to cytosolic recognition of R. australis in vitro. Rickettsial load in spleen, but not liver and lung, of R. australis-infected NLRP3-/- mice was significantly greater compared to WT mice. These data suggest that NLRP3 inflammasome plays a role in host control of bacteria in vivo in a tissue-specific manner. Taken together, our data, for the first time, illustrate the activation of ASC-dependent inflammasome by R. australis in macrophages in which NLRP3 is involved.  

4.1574           ITGAV and ITGA5 diversely regulate proliferation and adipogenic differentiation of human adipose derived stem cells

Morandi, E.M., Verstappen, R., Zwierzina, M.E., Geley, S., Pierer, G. and Ploner, C. Scientific Reports, 6, 28889 (2016)   The fate of human adipose tissue stem cells (ASCs) is largely determined by biochemical and mechanical cues from the extracellular matrix (ECM), which are sensed and transmitted by integrins. It is well known that specific ECM constituents influence ASC proliferation and differentiation. Nevertheless, knowledge on how individual integrins regulate distinct processes is still limited. We performed gene profiling of 18 alpha integrins in sorted ASCs and adipocytes, identifying downregulations of RGD-motif binding integrins integrin-alpha-V (ITGAV) and integrin-alpha-5 (ITGA5), upregulation of laminin binding and leukocyte-specific integrins and individual regulations of collagen and LDV-receptors in differentiated adipocytes in-vivo. Gene function analyses in in-vitro cultured ASCs unraveled differential functions of ITGA5 and ITGAV. Knockdown of ITGAV, but not ITGA5 reduced proliferation, caused p21Cip1 induction, repression of survivin and specific regulation of Hippo pathway mediator TAZ. Gene knockdown of both integrins promoted adipogenic differentiation, while transgenic expression impaired adipogenesis. Inhibition of ITGAV using cilengitide resulted in a similar phenotype, mimicking loss of pan-ITGAV expression using RNAi. Herein we show ASC specific integrin expression patterns and demonstrate distinct regulating roles of both integrins in human ASCs and adipocyte physiology suggesting a negative impact of RDG-motif signaling on adipogenic differentiation of ASCs via ITGA5 and ITGAV.  

4.1575           Expanding the tools for identifying mononuclear phagocyte subsets in swine: Reagents to porcine CD11c and XCR1

Deloizy, C., Bouguyon, E., Fossum, E., Sebo, P., Osicka, R., Bole, A., Pierres, M., Biacchesi, S., Salod, M., Bogen, B., Bertho, N. and Schwartz-Cornil, I. Development. Comp. Immunol., 65, 31-40 (2016)   Pig is a domestic species of major importance in the agro-economy and in biomedical research. Mononuclear phagocytes (MNP) are organized in subsets with specialized roles in the orchestration of the immune response and new tools are awaited to improve MNP subset identification in the pig. We cloned pig CD11c cDNA and generated a monoclonal antibody to pig CD11c which showed a pattern of expression by blood and skin MNP subsets similar to humans. We also developed a porcine XCL1-mCherry dimer which specifically reacted with the XCR1-expressing dendritic cell subset of the type 1 lineage in blood and skin. These original reagents will allow the efficient identification of pig MNP subsets to study their role in physiological and pathological processes and also to target these cells in novel intervention and vaccine strategies for veterinary applications and preclinical evaluations.  

4.1576           Free Fatty Acids Differentially Downregulate Chemokines in Liver Sinusoidal Endothelial Cells: Insights into Non-Alcoholic Fatty Liver Disease

McMahan, R.H., Porsche, C.E., Edwards, M.G. and Rosen, H.R. PloS One, 11(7), e015917 (2016)   Non-alcoholic fatty liver disease is a prevalent problem throughout the western world. Liver sinusoidal endothelial cells (LSEC) have been shown to play important roles in liver injury and repair, but their role in the underlying pathogenetic mechanisms of non-alcoholic fatty liver disease remains undefined. Here, we evaluated the effects of steatosis on LSEC gene expression in a murine model of non-alcoholic fatty liver disease and an immortalized LSEC line. Using microarray we identified distinct gene expression profiles following exposure to free fatty acids. Gene pathway analysis showed a number of differentially expressed genes including those involved in lipid metabolism and signaling and inflammation. Interestingly, in contrast to hepatocytes, fatty acids led to decreased expression of pro-inflammatory chemokines including CCL2 (MCP-1), CXCL10 and CXCL16 in both primary and LSEC cell lines. Chemokine downregulation translated into a significant inhibition of monocyte migration and LSECs isolated from steatotic livers demonstrated a similar shift towards an anti-inflammatory phenotype. Overall, these pathways may represent a compensatory mechanism to reverse the liver damage associated with non-alcoholic fatty liver disease.  

4.1577           Nasal delivery of chitosan-coated poly(lactide-co-glycolide)-encapsulated honeybee (Apis mellifera) venom promotes Th 1-specific systemic and local intestinal immune responses in weaned pigs

Lee, J-A., Kim, Y-M., Kim, T-H., Lee, S-H., Lee, C-A., Cho, C-W., Jeon, J-w., Park, J-k., Kim, S-K., Jung, B-G. and Lee, B-J. Vet. Immunol. Immunopathol., 178, 99-106 (2016)   Nasal delivery is a convenient and acceptable route for drug administration, and has been shown to elicit a much more potent local and systemic response compared with other drug delivery routes. We previously demonstrated that rectal administration of poly(lactide-co-glycolide)-encapsulated honeybee venom (P-HBV) could enhance systemic Th 1-specific immune responses. We therefore synthesized chitosan-coated P-HBV (CP-HBV) and then evaluated the immune-boosting efficacy of nasally administered CP-HBV on systemic and local intestinal immunity compared with non-chitosan-coated P-HBV. The nasally delivered CP-HBV effectively enhanced Th 1-specific responses, eliciting a significant increase in the CD3⁺CD4⁺CD8⁻ Th cell population, lymphocyte proliferation capacity, and expression of Th 1 cytokines (IFN-γ, IL-12, and IL-2) in peripheral blood mononuclear cells. Furthermore, these immune-boosting effects persisted up to 21 days post CP-HBV administration. Nasal administration of CP-HBV also led to an increase of not only the CD4⁺ Th 1 and IFN-γ secreting CD4⁺ Th 1 cell population but also Th 1-specific cytokines and transcription factors, including IL-12, IFN-γ, STAT4, and T-bet, in isolated mononuclear cells from the spleen and ileum.  

4.1578           Exosome-mediated activation of toll-like receptor 3 in stellate cells stimulates interleukin-17 production by γδ T cells in liver fibrosis

Seo, W., Eun, H., Kim, S.Y., Yi, H-S., Lee, Y-S., park, S-H., jang, M-J., Jo, E., Kim, S.C., Han, Y-M., Park, K-G. and Jeong, W-I. Hepatology, 64(2), 616-631 (2016)   During liver injury, hepatocytes secrete exosomes that include diverse types of self-RNAs. Recently, self-noncoding RNA has been recognized as an activator of Toll-like receptor 3 (TLR3). However, the roles of hepatic exosomes and TLR3 in liver fibrosis are not yet fully understood. Following acute liver injury and early-stage liver fibrosis induced by a single or 2-week injection of carbon tetrachloride (CCl₄), increased interleukin (IL)-17A production was detected primarily in hepatic γδ T cells in wild-type (WT) mice. However, liver fibrosis and IL-17A production by γδ T cells were both significantly attenuated in TLR3 knockout (KO) mice compared with WT mice. More interestingly, IL-17A-producing γδ T cells were in close contact with activated hepatic stellate cells (HSCs), suggesting a role for HSCs in IL-17A production by γδ T cells. In vitro treatments with exosomes derived from CCl₄-treated hepatocytes significantly increased the expression of IL-17A, IL-1β, and IL-23 in WT HSCs but not in TLR3 KO HSCs. Furthermore, IL-17A production by γδ T cells was substantially increased upon coculturing with exosome-treated WT HSCs or conditioned medium from TLR3-activated WT HSCs. However, similar increases were not detected when γδ T cells were cocultured with exosome-treated HSCs from IL-17A KO or TLR3 KO mice. Using reciprocal bone marrow transplantation between WT and TLR3 KO mice, we found that TLR3 deficiency in HSCs contributed to decreased IL-17A production by γδ T cells, as well as liver fibrosis. Conclusion: In liver injury, the exosome-mediated activation of TLR3 in HSCs exacerbates liver fibrosis by enhancing IL-17A production by γδ T cells, which might be associated with HSC stimulation by unknown self-TLR3 ligands from damaged hepatocytes. Therefore, TLR3 might be a novel therapeutic target for liver fibrosis.  

4.1579           The respiratory DC/macrophage network at steady-state and upon influenza infection in the swine biomedical model

Maisonnasse, P., Bouguyon, E., Piton, G., Ezquerra, A., Urien, C., Deloizy, C., Bourge, M., leplat, J-J., Simon, G., Chevalier, C., Vincent-Naulleau, S., Crisci, E., Montoya, M., Schwartz-Cornil, I. and bertho, N. Mucosal Immunol., 9(4), 835-849 (2016)   Human and mouse respiratory tracts show anatomical and physiological differences, which will benefit from alternative experimental models for studying many respiratory diseases. Pig has been recognized as a valuable biomedical model, in particular for lung transplantation or pathologies such as cystic fibrosis and influenza infection. However, there is a lack of knowledge about the porcine respiratory immune system. Here we segregated and studied six populations of pig lung dendritic cells (DCs)/macrophages (Mθs) as follows: conventional DCs (cDC) 1 and cDC2, inflammatory monocyte-derived DCs (moDCs), monocyte-derived Mθs, and interstitial and alveolar Mθs. The three DC subsets present migratory and naive T-cell stimulation capacities. As observed in human and mice, porcine cDC1 and cDC2 were able to induce T-helper (Th)1 and Th2 responses, respectively. Interestingly, porcine moDCs increased in the lung upon influenza infection, as observed in the mouse model. Pig cDC2 shared some characteristics observed in human but not in mice, such as the expression of FCεRIα and Langerin, and an intra-epithelial localization. This work, by unraveling the extended similarities of the porcine and human lung DC/Mθ networks, highlights the relevance of pig, both as an exploratory model of DC/Mθ functions and as a model for human inflammatory lung pathologies.  

4.1580           Posttranscriptional control of NLRP3 inflammasome activation in colonic macrophages

Filardy, A.A., He, J., bennink, J., Yewdell, J. and Kelsall, B.L. Mucosal Immunol., 9(4), 850-858 (2016)   Colonic macrophages (cMPs) are important for intestinal homeostasis as they kill microbes and yet produce regulatory cytokines. Activity of the NLRP3 (nucleotide-binding leucine-rich repeat-containing pyrin receptor 3) inflammasome, a major sensor of stress and microorganisms that results in pro-inflammatory cytokine production and cell death, must be tightly controlled in the intestine. We demonstrate that resident cMPs are hyporesponsive to NLRP3 inflammasome activation owing to a remarkable level of posttranscriptional control of NLRP3 and pro-interleukin-1β (proIL-1β) protein expression, which was also seen for tumor necrosis factor-α and IL-6, but lost during experimental colitis. Resident cMPs rapidly degraded NLRP3 and proIL-1β proteins by the ubiquitin/proteasome system. Finally, blocking IL-10R-signaling in vivo enhanced NLRP3 and proIL-1β protein but not mRNA levels in resident cMPs, implicating a role for IL-10 in environmental conditioning of cMPs. These data are the first to show dramatic posttranscriptional control of inflammatory cytokine production by a relevant tissue-derived macrophage population and proteasomal degradation of proIL-1β and NLRP3 as a mechanism to control inflammasome activation, findings which have broad implications for our understanding of intestinal and systemic inflammatory diseases.  

4.1581           Characterization of the Expression and Function of the C-Type Lectin Receptor CD302 in Mice and Humans Reveals a Role in Dendritic Cell Migration

Lo, T-H., Silveira, P.A., Fromm, P.D., verma, N.D., Vu, P.A., Kupresanin, F., Adam, R., Kato, M., Cogger, V.C., Clark, G.J. and hart, D.N.J.

1. Immunol., 197(3), 885-898 (2016)

  C-type lectin receptors play important roles in immune cell interactions with the environment. We described CD302 as the simplest, single domain, type I C-type lectin receptor and showed it was expressed mainly on the myeloid phagocytes in human blood. CD302 colocalized with podosomes and lamellopodia structures, so we hypothesized that it played a role in cell adhesion or migration. In this study, we used mouse models to obtain further insights into CD302 expression and its potential immunological function. Mouse CD302 transcripts were, as in humans, highest in the liver, followed by lungs, lymph nodes (LN), spleen, and bone marrow. In liver, CD302 was expressed by hepatocytes, liver sinusoidal endothelial cells, and Kupffer cells. A detailed analysis of CD302 transcription in mouse immune cells revealed highest expression by myeloid cells, particularly macrophages, granulocytes, and myeloid dendritic cells (mDC). Interestingly, 2.5-fold more CD302 was found in migratory compared with resident mDC populations and higher CD302 expression in mouse M1 versus M2 macrophages was also noteworthy. CD302 knockout (CD302KO) mice were generated. Studies on the relevant immune cell populations revealed a decrease in the frequency and numbers of migratory mDC within CD302KO LN compared with wild-type LN. In vitro studies showed CD302KO and wild-type DC had an equivalent capacity to undergo maturation, prime T cells, uptake Ags, and migrate toward the CCL19/CCL21 chemokines. Nevertheless, CD302KO migratory DC exhibited reduced in vivo migration into LN, confirming a functional role for CD302 in mDC migration.  

4.1582           IL4-10 Fusion Protein Is a Novel Drug to Treat Persistent Inflammatory Pain

Eijkelkamp, N., Steen-Louws, C., Hartgring, S.A.Y., Willemen, H.L.D.M., Prado, J., Lafeber, F.P.J.G., Heijnen, C.J., Hack, C.E., van Roon, J.A.G., and Kavelaars, A.

1. Neurosci., 36(28), 7353-7363 (2016)

  Chronic pain is a major clinical problem that is difficult to treat and requires novel therapies. Although most pain therapies primarily target neurons, neuroinflammatory processes characterized by spinal cord and dorsal root ganglion production of proinflammatory cytokines play an important role in persistent pain states and represent potential therapeutic targets. Anti-inflammatory cytokines are attractive candidates to regulate aberrant neuroinflammatory processes, but the therapeutic potential of these cytokines as stand-alone drugs is limited. Their optimal function requires concerted actions with other regulatory cytokines, and their relatively small size causes rapid clearance. To overcome these limitations, we developed a fusion protein of the anti-inflammatory cytokines interleukin 4 (IL4) and IL10. The IL4-10 fusion protein is a 70 kDa glycosylated dimeric protein that retains the functional activity of both cytokine moieties. Intrathecal administration of IL4-10 dose-dependently inhibited persistent inflammatory pain in mice: three IL4-10 injections induced full resolution of inflammatory pain in two different mouse models of persistent inflammatory pain. Both cytokine moieties were required for optimal effects. The IL4-10 fusion protein was more effective than the individual cytokines or IL4 plus IL10 combination therapy and also inhibited allodynia in a mouse model of neuropathic pain. Mechanistically, IL4-10 inhibited the activity of glial cells and reduced spinal cord and dorsal root ganglion cytokine levels without affecting paw inflammation. In conclusion, we developed a novel fusion protein with improved efficacy to treat pain, compared with wild-type anti-inflammatory cytokines. The IL4-10 fusion protein has potential as a treatment for persistent inflammatory pain.  

4.1583           Antitumor effect of antiplatelet agents in gastric cancer cells: an in vivo and in vitro study

Mikami, J., Kurokawa, Y., Takahashi, T., Miyazaki, Y., Yamasaki, M., Miyata, H., nakajima, K., Takiguchi, S., Mori, M. and Doki, Y. Gastric Cancer, 19, 817-826 (2016)   Background The antitumor effects of antiplatelet agents in gastric cancer cells are not well known. In this study, the possibility of gastric cancer treatment with an antiplatelet agent, mainly aspirin, was examined both in vivo and in vitro. Methods For in vivo experiments, tumor-bearing mice were treated by an antiplatelet antibody or aspirin, and the tumor growth was compared. For in vitro experiments, human gastric cancer cell lines were used to confirm the cancer cell growth and inhibition by reducing the platelet count or using aspirin. We also examined several cytokines by using an ELISA assay and conducted microRNA microarray analysis of MKN-45 tumor cells to determine the influence of platelets or aspirin. Results In vivo experiments showed that tumor growth was inhibited by halving the circulating platelet count by using an antiplatelet antibody or peroral daily aspirin. In vitro experiments showed that the proliferation rates of gastric cancer cell lines were increased after coincubation with platelets and that the effect was inhibited by aspirin. Although the expression of interleukin-6, platelet-derived growth factor, transforming growth factor-β, and prostaglandin E2 did not correlate with tumor growth inhibition by aspirin, seven microRNAs showed altered expression in cancer cells in response to coincubation with platelets or addition of aspirin. Cells transfected with mir-4670-5p showed a significant increase in proliferation compared to negative control cells. Conclusions Our study showed that platelets increased the proliferation of gastric cancer cells and that this increase was inhibited by antiplatelet antibody or aspirin. Mir-4670-5p may play an important role in these responses.  

4.1584           Sperm is epigenetically programmed to regulate gene transcription in embryos

Teperek, M., Simeone, A., Gaggioli, V., Miyamoto, K., Allen, G.E., Erkek, S., Kwon, T., marcotte, E.M., Zegerman, P., Bradshaw, C.R., Peters, A.H.M., Gurdon, J.B. and Jullien, J. Genome Res., 26, 1034-1046 (2016)   For a long time, it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo. However, conflicting reports have challenged the existence of epigenetic marks on sperm genes, and there are no functional tests supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of sperm- and spermatid-derived frog embryos, we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marks. Experimental removal of these epigenetic marks at fertilization de-regulates gene expression in the resulting embryos in a paternal chromatin-dependent manner. This demonstrates that epigenetic instructions delivered by the sperm at fertilization are required for correct regulation of gene expression in the future embryos. The epigenetic mechanisms of developmental programming revealed here are likely to relate to the mechanisms involved in transgenerational transmission of acquired traits. Understanding how parental experience can influence development of the progeny has broad potential for improving human health.  

4.1585           Muscle specific nucleus ambiguus neurons isolation and culturing

Hernandez-Morato, I., Pitman, M.J. and Sharma, S.

1. Neuroscience Methods, 273, 33-39 (2016)

  Background Peripheral nerve injury leads to a regenerative state. However, the reinnervation process is highly non-selective. Growing axons are often misrouted and establish aberrant synapsis to abductor or adductor muscles. Determining the complex properties of abductor and adductor motoneurons in a neuron culture, may lay the groundwork for future studies on axon guidance, leading to a clinical treatment for a selective reinnervation. New method In the present study we develop a neuron culture protocol to isolate recurrent laryngeal nerve abductor and adductor motoneurons in order to study their unique properties. Comparison with existing methods the best period to perform the present protocol for postnatal rat cranial motoneurons isolation was determined. In addition, the method allows identification of specific motoneurons from other primary motoneurons and interneurons within brainstem. Conclusion The present protocol will allow investigators to perform targeted and novel studies of the mechanisms of peripheral nerve regeneration.  

4.1586           The effects of urine concentration, and cushion centrifugation to remove urine, on the quality of cool-stored stallion sperm

Voge, J., Varner, D.D., Blanchard, T.L., Meschini, M., Turner, C., Teague, S.R., Brinsko, S.P. and Love, C.C. Theriogenology, 86, 1294-1298 (2016)   Urine-contaminated stallion semen is a clinical problem due to a variety of causes. The effect of the level of urine contamination on the longevity of sperm quality has not been evaluated. The aim of this study was to determine the effects of urine concentration level (0%, 10%, 20%, 30%, and 40%) and cushioned centrifugation and resuspension of the sperm pellet in fresh extender, on measures of sperm quality, immediately after semen collection (T₀), after 1 hour of storage at room temperature (T₁), and after 24 hours of cooled storage (T₂₄). In general, most sperm quality measures declined with increasing urine concentration starting at T₀. Cushioned centrifugation (CC), but not simple dilution, generally maintained sperm quality at T₂₄ as compared with T₁. At T₂₄, total sperm motility was higher in all urine-contaminated CC samples compared with uncentrifuged samples (P < 0.05); sperm viability was lower in CC than uncentrifuged at a urine concentration of 20%, but higher at 30% and 40% (P < 0.05); and DNA quality was decreased (higher % cells outside the main population) in all urine concentrations (P < 0.05). Immediate extension in semen extender, followed by cushioned centrifugation and resuspension of the sperm pellet in fresh extender, provided the best option for preserving sperm quality of urospermic semen.  

4.1587           Hepatic immunophenotyping for streptozotocin-induced hyperglycemia in mice

Lee, Y-S., Eun, H.S., Kim, S.Y., Jeong, J-M., Seo, W., Byun, J-S., Jeong, W-i. and Yi, H-S. Scientific Reports, 6:30656 (2016)   Emerging evidence revealed that diabetes induces abnormal immune responses that result in serious complications in organs. However, the effect of hyperglycemia on hepatic immunity remains obscure. We evaluated the population and function of hepatic immune cells in streptozotocin (STZ)-induced hyperglycemic mice. CC chemokine receptor 2 (CCR2)-knockout mice and mice with a depletion of regulatory T cells (DEREG) were used to investigate the migration and role of regulatory T cells (Tregs) in hyperglycemic mice. The inflammatory cytokines and hepatic transaminase levels were significantly increased in the hyperglycemic mice. The population and number of infiltrating monocytes, granulocytes, and Tregs were enhanced in the livers of the hyperglycemic mice. Hepatic monocytes other than macrophages showed the increased expression of inflammatory cytokines and chemokines in the hyperglycemic mice. The CCR2 knockout and DEREG chimeric mice exhibited increased populations of activated T cells and neutrophils compared to the WT chimeric mice, which promoted hepatic inflammation in the hyperglycemic mice. The migration of CCR2 knockout Tregs into the liver was significantly reduced compared to the WT Tregs. We demonstrated that hyperglycemia contributes to increase in infiltrating monocytes and Tregs, which are associated with hepatic immune dysfunction in mice. CCR2-mediated migration of Tregs regulates hyperglycemia-induced hepatic inflammation.  

4.1588           Leucine Zipper-bearing Kinase promotes axon growth in mammalian central nervous system neurons

Chen, M., Geoffroy, C.G., Wong, H.N., Tress, O., Nguyen, M.T., Holzman, L.B., Jin, Y. and Zheng, B. Scientific Reports, 6:31482 (2016)   Leucine Zipper-bearing Kinase (LZK/MAP3K13) is a member of the mixed lineage kinase family with high sequence identity to Dual Leucine Zipper Kinase (DLK/MAP3K12). While DLK is established as a key regulator of axonal responses to injury, the role of LZK in mammalian neurons is poorly understood. By gain- and loss-of-function analyses in neuronal cultures, we identify LZK as a novel positive regulator of axon growth. LZK signals specifically through MKK4 and JNKs among MAP2Ks and MAPKs respectively in neuronal cells, with JNK activity positively regulating LZK protein levels. Neuronal maturation or activity deprivation activates the LZK-MKK4-JNK pathway. LZK and DLK share commonalities in signaling, regulation, and effects on axon extension. Furthermore, LZK-dependent regulation of DLK protein expression and the lack of additive effects on axon growth upon co-manipulation suggest complex functional interaction and cross-regulation between these two kinases. Together, our data support the possibility for two structurally related MAP3Ks to work in concert to mediate axonal responses to external insult or injury in mammalian CNS neurons.  

4.1589           Splenectomy enhances the therapeutic effect of adipose tissue-derived mesenchymal stem cell infusion on cirrhosis rats

Tang, W-P., Akahoshi, T., Piao, J-S., narahara, S., Murata, M., Kawano, T., Hamano, N., Ikeda, T. and Hashizume, M. Liver Int., 36(8), 1151-1159 (2016)   Background & Aims Clinical studies suggest that splenectomy improves liver function in cirrhotic patients, but the influence of splenectomy on stem cell transplantation is poorly understood. This study investigated the effect of splenectomy on stem cell infusion and elucidated its mechanism. Methods Rat adipose tissue-derived mesenchymal stem cells were infused into cirrhosis rats with or without splenectomy, followed by the assessment of the in vivo distribution of stem cells and pathological changes. Stromal cell-derived factor-1 and hepatocyte growth factor expression were also investigated in splenectomized cirrhosis patients and rats. Results Splenectomy, prior to cell infusion, improved liver function and suppressed fibrosis progression more efficiently than cell infusion alone in the experimental cirrhosis model. Stromal cell-derived factor-1 and hepatocyte growth factor levels after splenectomy were increased in patients and rats. These upregulated cytokines significantly facilitated stem cell motility, migration and proliferation in vitro. C-X-C chemokine receptor type 4 neutralization weakened the promotion of cell migration by these cytokines. The infused cells integrated into liver fibrosis septa and participated in regeneration more efficiently in splenectomized rats. Direct coculture with stem cells led to inhibition of hepatic stellate cell proliferation. In addition, hepatocyte growth factor induced hepatic stellate cell apoptosis via the c-jun N-terminal kinase-p53 pathway. Conclusions Splenectomy prior to cell infusion enhanced the therapeutic effect of stem cells on cirrhosis, which involved upregulation of stromal cell-derived factor-1 and hepatocyte growth factor after splenectomy.  

4.1590           TPL-2 Regulates Macrophage Lipid Metabolism and M2 Differentiation to Control TH2-Mediated Immunopathology

Kannan, Y., Perez-Lloret, J., Li, Y., Entwistle, L.J., Khoury, H., Papoutsopoulou, S., Mahmood, R., Mansour, N.R., Huang, S.C-C., Pearce, E.J., de Carvalho, L.P.S., Ley, S.C. and Wilson, M.S. PloS Pathogens, 12(8), e1005783 (2016)   Persistent TH2 cytokine responses following chronic helminth infections can often lead to the development of tissue pathology and fibrotic scarring. Despite a good understanding of the cellular mechanisms involved in fibrogenesis, there are very few therapeutic options available, highlighting a significant medical need and gap in our understanding of the molecular mechanisms of TH2-mediated immunopathology. In this study, we found that the Map3 kinase, TPL-2 (Map3k8; Cot) regulated TH2-mediated intestinal, hepatic and pulmonary immunopathology following Schistosoma mansoni infection or S. mansoni egg injection. Elevated inflammation, TH2 cell responses and exacerbated fibrosis in Map3k8–/–mice was observed in mice with myeloid cell-specific (LysM) deletion of Map3k8, but not CD4 cell-specific deletion of Map3k8, indicating that TPL-2 regulated myeloid cell function to limit TH2-mediated immunopathology. Transcriptional and metabolic assays of Map3k8–/–M2 macrophages identified that TPL-2 was required for lipolysis, M2 macrophage activation and the expression of a variety of genes involved in immuno-regulatory and pro-fibrotic pathways. Taken together this study identified that TPL-2 regulated TH2-mediated inflammation by supporting lipolysis and M2 macrophage activation, preventing TH2 cell expansion and downstream immunopathology and fibrosis.  

4.1591           PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis

Martin, K., Pritchett, J., Llewellym, J., Mullan, A.F., Athwal, V.S., Dobie, R., Harvey, E., Zeef, L., Farrow, S., Streuli, C., Henderson, N.C., Friedman, S.L., Hanley, N.A. and Hanley, K.P. Nature Communications, 7:12502 (2016)   Fibrosis due to extracellular matrix (ECM) secretion from myofibroblasts complicates many chronic liver diseases causing scarring and organ failure. Integrin-dependent interaction with scar ECM promotes pro-fibrotic features. However, the pathological intracellular mechanism in liver myofibroblasts is not completely understood, and further insight could enable therapeutic efforts to reverse fibrosis. Here, we show that integrin beta-1, capable of binding integrin alpha-11, regulates the pro-fibrotic phenotype of myofibroblasts. Integrin beta-1 expression is upregulated in pro-fibrotic myofibroblasts in vivo and is required in vitro for production of fibrotic ECM components, myofibroblast proliferation, migration and contraction. Serine/threonine-protein kinase proteins, also known as P21-activated kinase (PAK), and the mechanosensitive factor, Yes-associated protein 1 (YAP-1) are core mediators of pro-fibrotic integrin beta-1 signalling, with YAP-1 capable of perpetuating integrin beta-1 expression. Pharmacological inhibition of either pathway in vivo attenuates liver fibrosis. PAK protein inhibition, in particular, markedly inactivates the pro-fibrotic myofibroblast phenotype, limits scarring from different hepatic insults and represents a new tractable therapeutic target for treating liver fibrosis.  

4.1592           Elevated interleukin-27 levels in human neonatal macrophages regulate indoleamine dioxygenase in a STAT-1 and STAT-3-dependent manner

Jung, J-Y., Parson, M. G., Kraft, J.D., Lyda, L., Kobe, B., Davis, C., Robinson, J., Marjorette, M., Pena, O. and Robinson, C.M. Immunology, 149(1), 35-47 (2016)   Microbial infections are a major cause of infant mortality as a result of limitations in immune defences. Interleukin-27 (IL-27) is a heterodimeric cytokine produced primarily by leucocytes and is immunosuppressive toward lymphocytes and leucocytes. Our laboratory demonstrated that human neonatal macrophages express IL-27 more abundantly than adult macrophages. Similarly in mice, IL-27 expression is elevated early in life and maintained through infancy. To determine IL-27-regulated mechanisms that may limit immunity, we evaluated the expression of a number of genes in response to this cytokine in primary human neonatal macrophages. Indoleamine 2,3-dioxygenase (IDO) gene expression was increased dose-responsively by IL-27. We have previously demonstrated inhibition of T-cell proliferation and cytokine production by neonatal macrophage-generated IL-27, and IDO is often implicated in this negative regulation. An increase in IDO protein was demonstrated by immunofluorescence microscopy and was consistent with increased enzyme activity following treatment with IL-27. Inclusion of a soluble receptor to neutralize endogenous IL-27, decreased IDO expression and activity compared with untreated macrophages. In response to IL-27, neonatal macrophages phosphorylate signal transdcuer and activator of transcription 1 (STAT-1) and STAT-3. Both transcription factors are recruited to the IDO regulatory region. STAT-3 dominates during steady-state regulation by lower levels of endogenous IL-27 production. A shift to enhanced STAT-1 recruitment occurs during increased levels of exogenously supplied IL-27. These data suggest an interesting interplay of STAT-1 and STAT-3 to regulate IDO activity and immunosuppression in response to different levels of IL-27 in the microenvironment of the immune response that may further our understanding of this interesting cytokine.  

4.1593           OX40+ Regulatory T Cells in Cutaneous Squamous Cell Carcinoma Suppress Effector T-Cell Responses and Associate with Metastatic Potential

Lai, C., August, S., Albibas, A., Behar, R., Cho, S-Y., Polak, M.E., Theaker, J., MacLeod, A.S., French, R.R., Glennie, M.J., Al-Shamkani, A. and Healy, E. Clin. Cancer Res., 22(16), 4236-4248 (2016)   Purpose: Cutaneous squamous cell carcinoma (cSCC) is the most common human cancer with metastatic potential. Despite T cells accumulating around cSCCs, these tumors continue to grow and persist. To investigate reasons for failure of T cells to mount a protective response in cSCC, we focused on regulatory T cells (Tregs) as this suppressive population is well represented among the infiltrating lymphocytes. Experimental Design: Flow cytometry was conducted on cSCC lymphocytes and in vitro functional assays were performed using sorted tumoral T cells. Lymphocyte subsets in primary cSCCs were quantified immunohistochemically. Results: FOXP3⁺ Tregs were more frequent in cSCCs than in peripheral blood (P < 0.0001, n = 86 tumors). Tumoral Tregs suppressed proliferation of tumoral effector CD4⁺ (P = 0.005, n = 10 tumors) and CD8⁺ T cells (P = 0.043, n = 9 tumors) and inhibited IFNγ secretion by tumoral effector T cells (P = 0.0186, n = 11 tumors). The costimulatory molecule OX40 was expressed predominantly on tumoral Tregs (P < 0.0001, n = 15 tumors) and triggering OX40 with an agonist anti-OX40 antibody overcame the suppression exerted by Tregs, leading to increased tumoral effector CD4⁺ lymphocyte proliferation (P = 0.0098, n = 10 tumors). Tregs and OX40⁺ lymphocytes were more abundant in primary cSCCs that metastasized than in primary cSCCs that had not metastasized (n = 48 and n = 49 tumors, respectively). Conclusions: Tregs in cSCCs suppress effector T-cell responses and are associated with subsequent metastasis, suggesting a key role for Tregs in cSCC development and progression. OX40 agonism reversed the suppressive effects of Tregs in vitro, suggesting that targeting OX40 could benefit the subset of cSCC patients at high risk of metastasis.    

4.1594           Oxygen-Purged Microfluidic Device to Enhance Cell Viability in Photopolymerized PEG Hydrogel Microparticles

Xia, B., Krutkramelis, K and Oakey, J. Biomacromolecules, 17(7), 2459-2465 (2016)   Encapsulating cells within biocompatible materials is a widely used strategy for cell delivery and tissue engineering. While cells are commonly suspended within bulk hydrogel-forming solutions during gelation, substantial interest in the microfluidic fabrication of miniaturized cell encapsulation vehicles has more recently emerged. Here, we utilize multiphase microfluidics to encapsulate cells within photopolymerized picoliter-volume water-in-oil droplets at high production rates. The photoinitiated polymerization of polyethylene glycol diacrylate (PEGDA) is used to continuously produce solid particles from aqueous liquid drops containing cells and hydrogel forming solution. It is well understood that this photoinitiated addition reaction is inhibited by oxygen. In contrast to bulk polymerization in which ambient oxygen is rapidly and harmlessly consumed, allowing the polymerization reaction to proceed, photopolymerization within air permeable polydimethylsiloxane (PDMS) microfluidic devices allows oxygen to be replenished by diffusion as it is depleted. This sustained presence of oxygen and the consequential accumulation of peroxy radicals produce a dramatic effect upon both droplet polymerization and post-encapsulation cell viability. In this work we employ a nitrogen microjacketed microfluidic device to purge oxygen from flowing fluids during photopolymerization. By increasing the purging nitrogen pressure, oxygen concentration was attenuated, and increased post-encapsulation cell viability was achieved. A reaction-diffusion model was used to predict the cumulative intradroplet concentration of peroxy radicals, which corresponded directly to post-encapsulation cell viability. The nitrogen-jacketed microfluidic device presented here allows the droplet oxygen concentration to be finely tuned during cell encapsulation, leading to high post-encapsulation cell viability.  

4.1595           Identification of proliferative and mature β-cells in the islets of Langerhans

Bader, E. et al Nature, 535, 430-434 (2016)   Insulin-dependent diabetes is a complex multifactorial disorder characterized by loss or dysfunction of β-cells. Pancreatic β-cells differ in size, glucose responsiveness, insulin secretion and precursor cell potential^{1, 2, 3, 4, 5}; understanding the mechanisms that underlie this functional heterogeneity might make it possible to develop new regenerative approaches. Here we show that Fltp (also known as Flattop and Cfap126), a Wnt/planar cell polarity (PCP) effector and reporter gene⁶, acts as a marker gene that subdivides endocrine cells into two subpopulations and distinguishes proliferation-competent from mature β-cells with distinct molecular, physiological and ultrastructural features. Genetic lineage tracing revealed that endocrine subpopulations from Fltp-negative and -positive lineages react differently to physiological and pathological changes. The expression of Fltp increases when endocrine cells cluster together to form polarized and mature 3D islet mini-organs^{7, 8, 9}. We show that 3D architecture and Wnt/PCP ligands are sufficient to trigger β-cell maturation. By contrast, the Wnt/PCP effector Fltp is not necessary for β-cell development, proliferation or maturation. We conclude that 3D architecture and Wnt/PCP signalling underlie functional β-cell heterogeneity and induce β-cell maturation. The identification of Fltp as a marker for endocrine subpopulations sheds light on the molecular underpinnings of islet cell heterogeneity and plasticity and might enable targeting of endocrine subpopulations for the regeneration of functional β-cell mass in diabetic patients.  

4.1596           Progesterone, Inflammatory Cytokine (TNF-α), and Oxidative Stress (H2O2) Regulate Progesterone Receptor Membrane Component 1 Expression in Fetal Membrane Cells

Meng, Y., Murtha, A.P. and Feng, L. Reproductive Sciences, 23(9), 1168-1178 (2016)   Progesterone receptor membrane component 1 (PGRMC1) is an important novel mediator of progesterone (P4) function in fetal membrane cells. We demonstrated previously that PGRMC1 is differentially expressed in fetal membranes among pregnancy subjects and diminished in preterm premature rupture of membrane subjects. In the current study, we aim to elucidate whether PGRMC1 expression is regulated by P4, tumor necrosis factor α (TNF-α), and H₂O₂ in fetal membrane cells. Primary cultured membrane cells were serum starved for 24 hours followed by treatments of P4, 17 hydroxyprogesterone caproate, and medroxyprogesterone 17 acetate (MPA) at 10⁻⁷ mol/L with ethanol as vehicle control; TNF-α at 10, 20, and 50 ng/mL with phosphate-buffered saline (PBS) as control; and H₂O₂ at 10 and 100 μmol/L with culture media as control for 24, 48, and 72 hours. The messenger RNA (mRNA) and protein expression of PGRMC1 was quantified using polymerase chain reaction and Western blotting, respectively. We found that PGRMC1 protein expression was regulated by MPA, TNF-α, and H₂O₂ in a dose-dependent manner. This regulation is also specific to the type of cell (amnion, chorion, or decidua). The upregulation of PGRMC1 by MPA might be mediated through glucocorticoid receptor (GR) demonstrated using amnion and chorion cells model with GR knockdown by specific small interfering RNA transfection. The mRNA expression of PGRMC1 was decreased by H₂O₂ (100 μmol/L) treatment in amnion cells, which might ultimately result in downregulation of PGRMC1 protein as our data demonstrated. None of other treatments changed PGRMC1 mRNA level in these cells. We conclude that these stimuli act as regulatory factors of PGRMC1 in a cell-specific manner.  

4.1597           Early steatohepatitis in hyperlipidemic mice with endothelial-specific gain of TRPC3 function precedes changes in aortic atherosclerosis

Smedlund, K., Dube, P. and Vazquez, G. Physiol. Genomics, 48, 644-649 (2016)   Nonalcoholic fatty liver disease (NAFLD) and its more advanced form nonalcoholic steatohepatitis (NASH) are the most common chronic liver diseases in developed countries. Moreover, NAFLD and NASH are considerable risk factors for atherosclerosis, the most frequent vascular pathology in these and other metabolic diseases. Despite this strong connection, current knowledge of the relationship between NAFLD/NASH and atherosclerosis is scarce. Recently, we studied hyperlipidemic Apoe knockout mice with endothelial-specific gain of transient receptor potential canonical 3 channel function (TgESTRPC3/ApoeKO) and found that these animals had increased burden of advanced aortic atherosclerosis (16 wk on high-fat diet) compared with nontransgenic ApoeKO littermate controls (non-Tg/ApoeKO), whereas early lesions (10 wk on high-fat diet) were not different. Here, we report that at the early stage when differences in aortic atherosclerosis are not yet manifest, the livers of TgESTRPC3/ApoeKO mice show steatosis, fibrosis, and altered hepatic enzymes compared with non-Tg/ApoeKO animals. Because differences in liver pathology were noticeable long before differences in atherosclerosis were evident, our studies suggest that TRPC3-related endothelial mechanisms that promote steatohepatitis may also contribute to atherosclerosis progression. In vitro, downregulation of TRPC3 in liver sinusoid endothelial cells reduces their susceptibility to endoplasmic reticulum stress-induced apoptosis, suggesting that a proapoptotic effect of TRPC3 may add to other fibrogenic factors in vivo. These novel findings show a positive association between augmented expression of an endothelial TRPC channel, development of early steatohepatitis, and atherosclerotic burden in a hyperlipidemic mouse model of NAFLD fed conventional Western-type diet.  

4.1598           Cellular normoxic biophysical markers of hydroxyurea treatment in sickle cell disease

Hosseini, P., Abidi, S.Z., Du, E., papageorgiou, D.P., Choi, Y., Park, Y., Higgins, J.M., Kato, G.J., Suresh, S., Dao, M., Yaqoob, Z. and So, P.T.C. PNAS, 113(34), 9527-9532 (2016)   Hydroxyurea (HU) has been used clinically to reduce the frequency of painful crisis and the need for blood transfusion in sickle cell disease (SCD) patients. However, the mechanisms underlying such beneficial effects of HU treatment are still not fully understood. Studies have indicated a weak correlation between clinical outcome and molecular markers, and the scientific quest to develop companion biophysical markers have mostly targeted studies of blood properties under hypoxia. Using a common-path interferometric technique, we measure biomechanical and morphological properties of individual red blood cells in SCD patients as a function of cell density, and investigate the correlation of these biophysical properties with drug intake as well as other clinically measured parameters. Our results show that patient-specific HU effects on the cellular biophysical properties are detectable at normoxia, and that these properties are strongly correlated with the clinically measured mean cellular volume rather than fetal hemoglobin level.  

4.1599           CCR5 ameliorates Japanese encephalitis via dictating the equilibrium of regulatory CD4+Foxp3+ T and IL-17+CD4+ Th17 cells

Kim, J.H., Patil, A.M., Choi, J.Y., Kim, S.B., Uyangaa, E., Hossain, F.M.A., Park, S-Y., Lee, J.H. and Eo, S.K.

1. Neuroinflammation, 13:223 (2016)

  Background CCR5 is a CC chemokine receptor involved in the migration of effector leukocytes including macrophages, NK, and T cells into inflamed tissues. Also, the role of CCR5 in CD4⁺Foxp3⁺ regulatory T cell (Treg) homing has recently begun to grab attention. Japanese encephalitis (JE) is defined as severe neuroinflammation of the central nervous system (CNS) following infection with mosquito-borne flavivirus JE virus. However, the potential contribution of CCR5 to JE progression via mediating CD4⁺Foxp3⁺ Treg homing has not been investigated. Methods Infected wild-type (Ccr5^+/+) and CCR5-deficient (Ccr5^−/−) mice were examined daily for mortality and clinical signs, and neuroinflammation in the CNS was evaluated by infiltration of inflammatory leukocytes and cytokine expression. In addition, viral burden, NK- and JEV-specific T cell responses were analyzed. Adoptive transfer of CCR5⁺CD4⁺Foxp3⁺ Tregs was used to evaluate the role of Tregs in JE progression. Results CCR5 ablation exacerbated JE without altering viral burden in the extraneural and CNS tissues, as manifested by increased CNS infiltration of Ly-6C^hi monocytes and Ly-6G^hi granulocytes. Compared to Ccr5^+/+ mice, Ccr5^−/− mice unexpectedly showed increased responses of IFN-γ⁺NK and CD8⁺ T cells in the spleen, but not CD4⁺ T cells. More interestingly, CCR5-ablation resulted in a skewed response to IL-17⁺CD4⁺ Th17 cells and correspondingly reduced CD4⁺Foxp3⁺ Tregs in the spleen and brain, which was closely associated with exacerbated JE. Our results also revealed that adoptive transfer of sorted CCR5⁺CD4⁺Foxp3⁺ Tregs into Ccr5^−/− mice could ameliorate JE progression without apparently altering the viral burden and CNS infiltration of IL-17⁺CD4⁺ Th17 cells, myeloid-derived Ly-6C^hi monocytes and Ly-6G^hi granulocytes. Instead, adoptive transfer of CCR5⁺CD4⁺Foxp3⁺ Tregs into Ccr5^−/− mice resulted in increased expression of anti-inflammatory cytokines (IL-10 and TGF-β) in the spleen and brain, and transferred CCR5⁺ Tregs were found to produce IL-10. Conclusions CCR5 regulates JE progression via governing timely and appropriate CNS infiltration of CD4⁺Foxp3⁺ Tregs, thereby facilitating host survival. Therefore, this critical and extended role of CCR5 in JE raises possible safety concerns regarding the use of CCR5 antagonists in human immunodeficiency virus (HIV)-infected individuals who inhabit regions in which both HIV and flaviviruses, such as JEV and West Nile virus, are endemic.  

4.1600           The Deficiency of Indoleamine 2,3-Dioxygenase Aggravates the CCl4-Induced Liver Fibrosis in Mice

Ogiso, H., Ito, H., Ando, T., Arioka, Y., Kanbe, A., Ando, K., Ishikawa, T., Saito, K., Hara, A., Moriwaki, H., Shimizu, M. and Seishima, M. PloS One, 11(9), e0162183 (2016)   In the present study, we examined the role of indoleamine 2,3-dioxygenase (IDO) in the development of CCl4-induced hepatic fibrosis. The liver fibrosis induced by repetitive administration with CCl4 was aggravated in IDO-KO mice compared to WT mice. In IDO-KO mice treated with CCl4, the number of several inflammatory cells and the expression of pro-inflammatory cytokines increased in the liver. In the results, activated hepatic stellate cells (HSCs) and fibrogenic factors on HSCs increased after repetitive CCl4 administration in IDO-KO mice compared to WT mice. Moreover, the treatment with l-tryptophan aggravated the CCl4-induced hepatic fibrosis in WT mice. Our findings demonstrated that the IDO deficiency enhanced the inflammation in the liver and aggravated liver fibrosis in repetitive CCl4-treated mice.  

4.1601           Rabbit M1 and M2 macrophages can be induced by human recombinant GM-CSF and M-CSF

Yamane, K. and Leung, K-P. FEBS Open Bio, 6(9), 945-953 (2016)   Macrophages can change their phenotype in response to environmental cues. Polarized macrophages are broadly classified into two groups: classical activated M1 and alternative activated M2. Characterization of human macrophages has been widely studied, but polarized macrophages in rabbits have not been characterized. We characterized rabbit macrophages that were polarized using human recombinant GM-CSF and M-CSF. GM-CSF-treated macrophages had higher mRNA expression of proinflammatory cytokines (M1 phenotype) than did the M-CSF-treated counterpart. By contrast, high levels of TGF-β and IL-10 expression (M2 phenotype) were found in M-CSF-treated macrophages. The present study may be useful to understand roles of polarized macrophages in rabbit disease models.  

4.1602           An optimized method for obtaining adult rat spinal cord motor neurons to be used for tissue culture

Brinn, M., O’Neill, K., Musgrave, I., Freeman, B.J.C., Henneberg, M. and Kumaratilake, J.

1. Neurosci. Methods, 273, 128-137 (2016)

  Background There is a paucity of detailed methods describing how to harvest and process motor neurons obtained from the adult rat spinal cord. New method Removal of intra-cardiac perfusion step. The spinal cord is extruded intact from the rat in under 60 s post-decapitation then processed without differentiation of ventral and dorsal regions. The temperature during processing was maintained at room temperature (22 °C) except during the Papain processing step where the temperature was increased to 30 °C. Results Cell debris interfered with the counting of cells at the time of plating. Also, cell types could not be identified since they appear rounded structures with no projections. Cell viability counts reduced to 91% and 63% from day 7 to day 14 and days 7–28 respectively. Red blood cell counts in stepped density gradient layers 2 and 3 were low. Comparison with existing method(s) No requirement for intra-cardiac perfusion. No requirement to cool to 4 °C post harvesting, No requirement for specialized substrates. Reduces processing time by at least 2 h and reduces the potential for processing errors through a reduction in complexity. Procedures are also explained suitable for those new to the culture of primary adult motor neurons. Conclusions Cell viability counts indicate that removal of the perfusion step has a minimal effect on the viability of the cultured nerve cells, which may be due to the reduction in the spinal cord harvesting time and the inclusion of Hibernate based media during extrusion and processing.  

4.1603           Latency Entry of Herpes Simplex Virus 1 Is Determined by the Interaction of Its Genome with the Nuclear Environment

Ali Maroui, M., Calle, A., Cohen, C., Streichenberger, N., Texier, P., Takissian, J., Rousseau, A., Poccardi, N., Welsch, J., Corpet, A., Schaeffer, L., labetoulle, M. and Lomonte, P. PloS Pathogens, 12(9), e1005834 (2016)   Herpes simplex virus 1 (HSV-1) establishes latency in trigeminal ganglia (TG) sensory neurons of infected individuals. The commitment of infected neurons toward the viral lytic or latent transcriptional program is likely to depend on both viral and cellular factors, and to differ among individual neurons. In this study, we used a mouse model of HSV-1 infection to investigate the relationship between viral genomes and the nuclear environment in terms of the establishment of latency. During acute infection, viral genomes show two major patterns: replication compartments or multiple spots distributed in the nucleoplasm (namely “multiple-acute”). Viral genomes in the “multiple-acute” pattern are systematically associated with the promyelocytic leukemia (PML) protein in structures designated viral DNA-containing PML nuclear bodies (vDCP-NBs). To investigate the viral and cellular features that favor the acquisition of the latency-associated viral genome patterns, we infected mouse primary TG neurons from wild type (wt) mice or knock-out mice for type 1 interferon (IFN) receptor with wt or a mutant HSV-1, which is unable to replicate due to the synthesis of a non-functional ICP4, the major virus transactivator. We found that the inability of the virus to initiate the lytic program combined to its inability to synthesize a functional ICP0, are the two viral features leading to the formation of vDCP-NBs. The formation of the “multiple-latency” pattern is favored by the type 1 IFN signaling pathway in the context of neurons infected by a virus able to replicate through the expression of a functional ICP4 but unable to express functional VP16 and ICP0. Analyses of TGs harvested from HSV-1 latently infected humans showed that viral genomes and PML occupy similar nuclear areas in infected neurons, eventually forming vDCP-NB-like structures. Overall our study designates PML protein and PML-NBs to be major cellular components involved in the control of HSV-1 latency, probably during the entire life of an individual.  

4.1604           Estradiol enhances capacity of TLR-matured splenic dendritic cells to polarize CD4+ lymphocytes into IL-17/GM-CSF-producing cells in vitro

Stojic-Vukanic, Z., Bufan, B., Pilipovic, I., Vujnovic, I., Nacka-Aleksic, M., Petrovic, R., Arsenovic-Ranin, N. and Leposavic, G. Int. Immunopharmacol., 40, 244-253 (2016)   There are little data on modulatory effects of estrogens on rat dendritic cell (DC) responses to inflammatory stimuli, and consequently their ability to activate and polarize CD4+ T lymphocyte-mediated immune responses. Splenic conventional DCs from young female Albino Oxford rats were activated in vitro with LPS (TLR4 agonist) or R848 (TLR7/8 agonist) in the presence and absence of 17β-estradiol (E2), and their allostimulatory and CD4+ lymphocyte polarizing ability in mixed leukocyte culture (MLC) were studied. Irrespective of the E2 presence, LPS and R848 up-regulated the expression of MHC II on DCs, so they exhibited enhanced allostimulatory capacity in co-culture with CD4+ lymphocytes. On the other hand, E2 promoted stimulatory action of both TLRs on OX62+ DC IL-23 production, augmented their stimulatory effects on IL-6 and IL-1β production, but diminished their enhancing effects on the expression IL-10 and IL-27 by DCs. Consequently, in MLC, OX62+ DCs activated/matured in the co-presence of E2 and either LPS or R848 increased the levels of IL-17, the signature Th17 cell cytokine, when compared with those activated/matured in the absence of E2. GM-CSF levels were also increased in these MLC. Given that the expression of IL-7 mRNA was diminished in DCs activated/matured in the co-presence of E2 and TLR, this increase most likely did not reflect enhanced differentiation of Th cells producing GM-CSF only (Th-GM).  

4.1605           Hepatocyte Toll-Like Receptor 5 Promotes Bacterial Clearance and Protects Mice Against High-Fat Diet–Induced Liver Disease

Etienne-Mesmin, L., Vijay-Kumar, M., Gewirtz, A.T. and Chassaing, B. Cell. Mol. Gastroenterol. Hepatol., 2, 584-604 (2016)   Background & Aims Innate immune dysfunction can promote chronic inflammatory diseases of the liver. For example, mice lacking the flagellin receptor Toll-like receptor 5 (TLR5) show microbial dysbiosis and predisposition to high-fat diet (HFD)-induced hepatic steatosis. The extent to which hepatocytes play a direct role in detecting bacterial products in general, or flagellin in particular, is poorly understood. In the present study, we investigated the role of hepatocyte TLR5 in recognizing flagellin, policing bacteria, and protecting against liver disease. Methods Mice were engineered to lack TLR5 specifically in hepatocytes (TLR5^ΔHep) and analyzed relative to sibling controls (TLR5^fl/fl). TLR5 messenger RNA levels, responses to exogenous flagellin, elimination of circulating motile bacteria, and susceptibility of liver injury (concanavalin A, carbon tetrachloride, methionine- and choline-deficient diet, and HFD) were measured. Results TLR5^ΔHep expressed similar levels of TLR5 as TLR5^fl/fl in all organs examined, except in the liver, which showed a 90% reduction in TLR5 levels, indicating that hepatocytes accounted for the major portion of TLR5 expression in this organ. TLR5^ΔHep showed impairment in responding to purified flagellin and clearing flagellated bacteria from the liver. Although TLR5^ΔHep mice did not differ markedly from sibling controls in concanavalin A or carbon tetrachloride–induced liver injury models, they showed exacerbated disease in response to a methionine- and choline-deficient diet and HFD. Such predisposition of TLR5^ΔHep to diet-induced liver pathology was associated with increased expression of proinflammatory cytokines, which was dependent on the Nod-like-receptor C4 inflammasome and rescued by microbiota ablation. Conclusions Hepatocyte TLR5 plays a critical role in protecting liver against circulating gut bacteria and against diet-induced liver disease.  

4.1606           Blocking Notch signal in myeloid cells alleviates hepatic ischemia reperfusion injury by repressing the activation of NF-κB through CYLD

Yu, H-C., Bai, L., Yang, Z-X., Qin, H-Y., Tao, K-S., Han, H. and Dou, K-F. Scientific Reports, 6:32226 (2016)   Ischemia-reperfusion (I/R) is a major reason of hepatocyte injury during liver surgery and transplantation. Myeloid cells including macrophages and neutrophils play important roles in sustained tissue inflammation and damage, but the mechanisms regulating myeloid cells activity have been elusive. In this study, we investigate the role of Notch signaling in myeloid cells during hepatic I/R injury by using a mouse model of myeloid specific conditional knockout of RBP-J. Myeloid-specific RBP-J deletion alleviated hepatic I/R injury. RBP-J deletion in myeloid cells decreased hepatocytes apoptosis after hepatic I/R injury. Furthermore, myeloid-specific RBP-J deletion led to attenuated inflammation response in liver after I/R injury. Consistently, Notch blockade reduced the production of inflammatory cytokines by macrophages in vitro. We also found that blocking Notch signaling reduced NF-κB activation and increased cylindromatosis (CYLD) expression and knockdown of CYLD rescued reduction of inflammatory cytokines induced by Notch blockade in macrophages during I/R injury in vitro. On the other hand, activation of Notch signaling in macrophages led to increased inflammatory cytokine production and NF-κB activation and decreased CYLD expression in vitro. These data suggest that activation of Notch signaling in myeloid cells aggravates I/R injury, by enhancing the inflammation response by NF-κB through down regulation of CYLD.  

4.1607           ICER is requisite for Th17 differentiation

Yoshida, N., Comte, D., Mizui, M., Otomo, K., Rosetti, F., Mayadas, T.N., Crispin, J.C., Bradley, S.J., Koga, T., Kono, M., karampetsou, M.P., Kyttaris, V.C., Tenbrock, K. and Ysokos, G.C: Nature Communications, 7:12993 (2016)   Inducible cAMP early repressor (ICER) has been described as a transcriptional repressor isoform of the cAMP response element modulator (CREM). Here we report that ICER is predominantly expressed in Th17 cells through the IL-6–STAT3 pathway and binds to the Il17a promoter, where it facilitates the accumulation of the canonical enhancer RORγt. In vitro differentiation from naive ICER/CREM-deficient CD4+ T cells to Th17 cells is impaired but can be rescued by forced overexpression of ICER. Consistent with a role of Th17 cells in autoimmune and inflammatory diseases, ICER/CREM-deficient B6.lpr mice are protected from developing autoimmunity. Similarly, both anti-glomerular basement membrane-induced glomerulonephritis and experimental encephalomyelitis are attenuated in ICER/CREM-deficient mice compared with their ICER/CREM-sufficient littermates. Importantly, we find ICER overexpressed in CD4+ T cells from patients with systemic lupus erythematosus. Collectively, our findings identify a unique role for ICER, which affects both organ-specific and systemic autoimmunity in a Th17-dependent manner.  

4.1608           Identification of early gene expression changes in primary cultured neurons treated with topoisomerase I poisons

Rossi, S.L., Lumpkin, C.J., Harris, A.W., Holbrook, J., Gentillon, C., McVahan, S.M., Wang, W. and Butchbach, M.E.R. Biochem. Biophys. Res. Comm., 479, 319-324 (2016)   Topoisomerase 1 (TOP1) poisons like camptothecin (CPT) are currently used in cancer chemotherapy but these compounds can have damaging, off-target effects on neurons leading to cognitive, sensory and motor deficits. To understand the molecular basis for the enhanced sensitivity of neurons to CPT, we examined the effects of compounds that inhibit TOP1—CPT, actinomycin D (ActD) and β-lapachone (β-Lap)—on primary cultured rat motor (MN) and cortical (CN) neurons as well as fibroblasts. Neuronal cells expressed higher levels of Top1 mRNA than fibroblasts but transcript levels are reduced in all cell types after treatment with CPT. Microarray analysis was performed to identify differentially regulated transcripts in MNs in response to a brief exposure to CPT. Pathway analysis of the differentially expressed transcripts revealed activation of ERK and JNK signaling cascades in CPT-treated MNs. Immediate-early genes like Fos, Egr-1 and Gadd45b were upregulated in CPT-treated MNs. Fos mRNA levels were elevated in all cell types treated with CPT; Egr-1, Gadd45b and Dyrk3 transcript levels, however, increased in CPT-treated MNs and CNs but decreased in CPT-treated fibroblasts. These transcripts may represent new targets for the development of therapeutic agents that mitigate the off-target effects of chemotherapy on the nervous system.  

4.1609           Characterization of brevetoxin (PbTx-3) exposure in neurons of the anoxia-tolerant freshwater turtle (Trachemys scripta)

Cocilova, C.C. and Milton, S.L. Quatic Toxicology, 180, 115-122 (2016)   Harmful algal blooms are increasing in frequency and extent worldwide and occur nearly annually off the west coast of Florida where they affect both humans and wildlife. The dinoflagellate Karenia brevis is a key organism in Florida red tides that produces a suite of potent neurotoxins collectively referred to as the brevetoxins (PbTx). Brevetoxins bind to and open voltage gated sodium channels (VGSC), increasing cell permeability in excitable cells and depolarizing nerve and muscle tissue. Exposed animals may thus show muscular and neurological symptoms including head bobbing, muscle twitching, paralysis, and coma; large HABs can result in significant morbidity and mortality of marine life, including fish, birds, marine mammals, and sea turtles. Brevetoxicosis however is difficult to treat in endangered sea turtles as the physiological impacts have not been investigated and the magnitude and duration of brevetoxin exposure are generally unknown. In this study we used the freshwater turtle Trachemys scripta as a model organism to investigate the effects of the specific brevetoxin PbTx-3 in the turtle brain. Primary turtle neuronal cell cultures were exposed to a range of PbTx-3 concentrations to determine excitotoxicity. Agonists and antagonists of voltage-gated sodium channels and downstream targets were utilized to confirm the toxin’s mode of action. We found that turtle neurons are highly resistant to PbTx-3; while cell viability decreased in a dose dependent manner across PbTx-3 concentrations of 100–2000 nM, the EC₅₀ was significantly higher than has been reported in mammalian neurons. PbTx-3 exposure resulted in significant Ca²⁺ influx, which could be fully abrogated by the VGSC antagonist tetrodotoxin, NMDA receptor blocker MK-801, and tetanus toxin, indicating that the mode of action in turtle neurons is the same as in mammalian cells. As both turtle and mammalian VGSCs have a high affinity for PbTx-3, we suggest that the high resistance of the turtle neuron to PbTx-3 may be related to its ability to withstand anoxic depolarization. The ultimate goal of this work is to design treatment protocols for sea turtles exposed to red tides worldwide.  

4.1610           Acoustic Cell Manipulation

Lenshof, A., Johannesson, C., Evander, M., Nilsson, J. and Laurel, T. Microtechnology for Cell Manipulation and Sorting, 129-173 (2016)   This chapter reviews recent developments in the field of acoustic manipulation and processing of cells in microfluidic systems and gives an overview of different acoustofluidic operating modalities. Continuous flow-based acoustophoresis and acoustic trapping are key areas of interest. In view of the topic of this publication we have limited this chapter to mainly cover acoustofluidic work that concerns cell handling and cell-based studies. A focus is therefore maintained on developments that demonstrate how microscale acoustofluidic systems can be designed to solve unmet needs in the everyday work of life science laboratories related to cell biology or clinically relevant research.  

4.1611           End Sequence Analysis Toolkit (ESAT) expands the extractable information from single-cell RNA-seq data

Alan Derr, Chaoxing Yang, Rapolas Zilionis, Alexey Sergushichev, David M. Blodgett, Sambra Redick, Rita Bortell, Jeremy Luban, David M. Harlan, Sebastian Kadener, Dale L. Greiner, Allon Klein, Maxim N. Artyomov, and Manuel Garber Genome Res., 26, 1397-1410 (2016)   RNA-seq protocols that focus on transcript termini are well suited for applications in which template quantity is limiting. Here we show that, when applied to end-sequencing data, analytical methods designed for global RNA-seq produce computational artifacts. To remedy this, we created the End Sequence Analysis Toolkit (ESAT). As a test, we first compared end-sequencing and bulk RNA-seq using RNA from dendritic cells stimulated with lipopolysaccharide (LPS). As predicted by the telescripting model for transcriptional bursts, ESAT detected an LPS-stimulated shift to shorter 3′-isoforms that was not evident by conventional computational methods. Then, droplet-based microfluidics was used to generate 1000 cDNA libraries, each from an individual pancreatic islet cell. ESAT identified nine distinct cell types, three distinct β-cell types, and a complex interplay between hormone secretion and vascularization. ESAT, then, offers a much-needed and generally applicable computational pipeline for either bulk or single-cell RNA end-sequencing.  

4.1612           Clinical Islet Isolation

Hawthorne, W.J., Williams, L. and Chew, Y.V. Advances in Exp. Med., 938, 89-122 (2016)   The overarching success of islet transplantation relies on the success in the laboratory to isolate the islets. This chapter focuses on the processes of human islet cell isolation and the ways to optimally provide islet cells for transplantation. The major improvements in regards to the choice of enzyme type, way the digested pancreas tissue is handled to best separate islets from the acinar and surrounding tissues, the various methods of purification of the islets, their subsequent culture and quality assurance to improve outcomes to culminate in safe and effective islet transplantation will be discussed. After decades of improvements, islet cell isolation and transplantation now clearly offer a safe, effective and feasible therapeutic treatment option for an increasing number of patients suffering from type 1 diabetes specifically for those with severe hypoglycaemic unawareness.  

4.1613           Miscellaneous Pathogens

Austin, B. and Austin, D.A. Bacterial Fish Pathogens, 603-642 (2016)   Pseudoalteromonas piscicida, Pseudoalteromonas undina, Shewanella putrefaciens, Arcobacter cryaerophilus, Halomonas (=Deleya) cupida, Acinetobacter sp., Moraxella sp., Moritella marina, Moritella viscosa, Mycoplasma mobile, Myxococcus piscicola, Aquaspirillum sp., Janthinobacterium lividum, Pasteurella skyensis, Piscirickettsia salmonis, Rickettsia-like organisms, Streptobacillus, ‘Candidatus Arthromitus’, ‘Candidatus Branchiomonas cysticola’, ‘Candidatus Clavochlamydia salmonicola’, ‘Candidatus Piscichlamydia salmonis’ and ‘Candidatus Renichlamydia lutjani’ have been associated with fish diseases. Moritella viscosa has been recovered from winter ulcer disease (= skin lesions) in Atlantic salmon with pathogenicity mechanisms reflecting the presence of extracellular products. Protection has been achieved with an adjuvanted formalin inactivated whole cell vaccine. Piscirickettsia salmonis is an obligate parasite, which has been associated with coho salmon syndrome, Huito disease and salmonid rickettsial septicaemia. Good protection was recorded by use of a formalised whole cell suspension. ‘Candidatus’ are uncultured organisms, which may be visualised in pathological material.  

4.1614           HMGB1 Activates Proinflammatory Signaling via TLR5 Leading to Allodynia

Das, N., Dewan, V., Grace, P. et al Cell Reports, 17, 1128-1140 (2016)   Infectious and sterile inflammatory diseases are correlated with increased levels of high mobility group box 1 (HMGB1) in tissues and serum. Extracellular HMGB1 is known to activate Toll-like receptors (TLRs) 2 and 4 and RAGE (receptor for advanced glycation endproducts) in inflammatory conditions. Here, we find that TLR5 is also an HMGB1 receptor that was previously overlooked due to lack of functional expression in the cell lines usually used for studying TLR signaling. HMGB1 binding to TLR5 initiates the activation of NF-κB signaling pathway in a MyD88-dependent manner, resulting in proinflammatory cytokine production and pain enhancement in vivo. Biophysical and in vitro results highlight an essential role for the C-terminal tail region of HMGB1 in facilitating interactions with TLR5. These results suggest that HMGB1-modulated TLR5 signaling is responsible for pain hypersensitivity.  

4.1615           Helicobacter suis affects the health and function of porcine gastric parietal cells

Zhang, G., Ducatelle, R., Mihi, B., Smet, A., Flahou, B. and Haesebrouk, F. Vet. Res., 47:10 (2016)   The stomach of pigs at slaughter age is often colonized by Helicobacter (H.) suis, which is also the most prevalent gastric non-H. pylori Helicobacter (NHPH) species in humans. It is associated with chronic gastritis, gastric ulceration and other gastric pathological changes in both hosts. Parietal cells are highly specialized, terminally differentiated epithelial cells responsible for gastric acid secretion and regulation. Dysfunction of these cells is closely associated with gastric pathology and disease. Here we describe a method for isolation and culture of viable and responsive parietal cells from slaughterhouse pigs. In addition, we investigated the interactions between H. suis and gastric parietal cells both in H. suis-infected six-month-old slaughter pigs, as well as in our in vitro parietal cell model. A close interaction of H. suis and parietal cells was observed in the fundic region of stomachs from H. suis positive pigs. The bacterium was shown to be able to directly interfere with cultured porcine parietal cells, causing a significant impairment of cell viability. Transcriptional levels of Atp4a, essential for gastric acid secretion, showed a trend towards an up-regulation in H. suis positive pigs compared to H. suis-negative pigs. In addition, sonic hedgehog, an important factor involved in gastric epithelial differentiation, gastric mucosal repair, and stomach homeostasis, was also significantly up-regulated in H. suis positive pigs. In conclusion, this study describes a successful approach for the isolation and culture of porcine gastric parietal cells. The results indicate that H. suis affects the viability and function of this cell type.  

4.1616           Non-invasive epicutaneous vaccine against Respiratory Syncytial Virus: Preclinical proof of concept

Herve, P-L., Descamps, D., Deloizy, C., Dhelft, V., Laubreton, D., Bouguyon, E., Boukadiri, A., Dubuquoy, C., Larcher, T., Benhamou, P-H., Benhamou, P-H., Eleouët, J.F., bertho, N., Mondoulet, L. and Riffault, S.

1. Controlled Release, 243, 146-159 (2016)

  To put a Respiratory Syncytial Virus (RSV) vaccine onto the market, new vaccination strategies combining scientific and technical innovations need to be explored. Such a vaccine would also need to be adapted to the vaccination of young children that are the principal victims of acute RSV infection. In the present project, we describe the development and the preclinical evaluation of an original epicutaneous RSV vaccine that combines two technologies: Viaskin® epicutaneous patches as a delivery platform and RSV N-nanorings (N) as a subunit antigen. Such a needle-free vaccine may have a better acceptability for the vaccination of sensible population such as infants since it does not require any skin preparation. Moreover, this self-applicative vaccine would overcome some issues associated to injectable vaccines such as the requirement of sterile medical devices, the need of skilled health-care professionals and the necessity of stringent store conditions. Here, we demonstrate that Viaskin® patches loaded with a formulation containing N-nanorings (Viaskin®-N) are highly immunogenic in mice and promotes a Th1/Th17 oriented immune response. More importantly, Viaskin®-N epicutaneous vaccine confers a high level of protection against viral replication upon RSV challenge in mice, without exacerbating clinical symptoms. In swine, which provides the best experimental model for the transcutaneous passage of drug/antigen in human skin, we have shown that GFP fluorescent N-nanorings, delivered epicutaneously with Viaskin® patches, are taken up by epidermal Langerhans cells. We have also demonstrated that Viaskin®-N induced a significant RSV N-specific T-cell response in pig. In conclusion, Viaskin®-N epicutaneous vaccine seems efficient to protect against RSV infection in animal model.  

4.1617           Histamine H3 receptor activation stimulates calcium mobilization in a subpopulation of rat striatal neurons in primary culture, but not in synaptosomes

Rivera-Ramirez, N., Montejo-Lopez, W., Lopez-Mendez. M-C., Guerrero-Hernandez, A., Molina-Hernandez, A., Garcia-Hernandez-U. and Arias-Montano, J-A. Neurochem. Int., 101, 38-47 (2016)   The histamine H₃ receptor (H₃R) is abundantly expressed in the Central Nervous System where it regulates several functions pre and postsynaptically. H₃Rs couple to Gα_i/o proteins and trigger or modulate several intracellular signaling pathways, including the cAMP/PKA pathway and the opening of N- and P/Q-type voltage-gated Ca²⁺ channels. In transfected cells, activation of the human H₃R of 445 amino acids (hH₃R₄₄₅) results in phospholipase C (PLC) stimulation and release of Ca²⁺ from intracellular stores. In this work we have studied whether H₃R activation induces Ca²⁺ mobilization from intracellular stores in native systems, either isolated nerve terminals (synaptosomes) or neurons in primary culture. In rat striatal synaptosomes H₃R activation induced inositol 1,4,5-trisphosphate (IP₃) formation but failed to increase the intracellular calcium concentration ([Ca²⁺]_i). In striatal primary cultures H₃R activation resulted in IP₃ formation and increased the [Ca²⁺]_i in 18 out of 70 cells that responded with an elevation in the [Ca²⁺]_i to membrane depolarization with KCl (100 mM) as evaluated by microfluorometry. Confocal microscopy studies corroborated the increase in [Ca²⁺]i induced by H₃R activation in a fraction of those cells that were responsive to membrane depolarization. These results indicate that H₃R activation stimulates the PLC/IP₃/Ca²⁺ pathway but only in a subpopulation of striatal neurons.  

4.1618           National Institutes of Health–Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities

Ricordi, C., Goldstein, J.S., Balamurugan, A.N. et al Diabetes, 65(11), 3418-3428 (2016)   Eight manufacturing facilities participating in the National Institutes of Health–sponsored Clinical Islet Transplantation (CIT) Consortium jointly developed and implemented a harmonized process for the manufacture of allogeneic purified human pancreatic islet (PHPI) product evaluated in a phase 3 trial in subjects with type 1 diabetes. Manufacturing was controlled by a common master production batch record, standard operating procedures that included acceptance criteria for deceased donor organ pancreata and critical raw materials, PHPI product specifications, certificate of analysis, and test methods. The process was compliant with Current Good Manufacturing Practices and Current Good Tissue Practices. This report describes the manufacturing process for 75 PHPI clinical lots and summarizes the results, including lot release. The results demonstrate the feasibility of implementing a harmonized process at multiple facilities for the manufacture of a complex cellular product. The quality systems and regulatory and operational strategies developed by the CIT Consortium yielded product lots that met the prespecified characteristics of safety, purity, potency, and identity and were successfully transplanted into 48 subjects. No adverse events attributable to the product and no cases of primary nonfunction were observed.  

4.1619           Plasticity and Aggregation of Juvenile Porcine Islets in Modified Culture: Preliminary Observations

Weegman, B.P., Taylor, M.J., Baicu, S.C., Mueller, K., O’Brien, T.D., Wilson, J. and Papas, K.K. Cell Transplant., 25, 1763-1775 (2016)   Diabetes is a major health problem worldwide, and there is substantial interest in developing xenogeneic islet transplantation as a potential treatment. The potential to relieve the demand on an inadequate supply of human pancreata is dependent upon the efficiency of techniques for isolating and culturing islets from the source pancreata. Porcine islets are favored for xenotransplantation, but mature pigs (>2 years) present logistic and economic challenges, and young pigs (3‐6 months) have not yet proven to be an adequate source. In this study, islets were isolated from 20 juvenile porcine pancreata (∼3 months; 25 kg Yorkshire pigs) immediately following procurement or after 24 h of hypothermic machine perfusion (HMP) preservation. The resulting islet preparations were characterized using a battery of tests during culture in silicone rubber membrane flasks. Islet biology assessment included oxygen consumption, insulin secretion, histopathology, and in vivo function. Islet yields were highest from HMP-preserved pancreata (2,242 ± 449 IEQ/g). All preparations comprised a high proportion (>90%) of small islets (<100 μm), and purity was on average 63 ± 6%. Morphologically, islets appeared as clusters on day 0, loosely disaggregated structures at day 1, and transitioned to aggregated structures comprising both exocrine and endocrine cells by day 6. Histopathology confirmed both insulin and glucagon staining in cultures and grafts excised after transplantation in mice. Nuclear staining (Ki-67) confirmed mitotic activity consistent with the observed plasticity of these structures. Metabolic integrity was demonstrated by oxygen consumption rates = 175 ± 16 nmol/min/mg DNA, and physiological function was intact by glucose stimulation after 6‐8 days in culture. In vivo function was confirmed with blood glucose control achieved in nearly 50% (8/17) of transplants. Preparation and culture of juvenile porcine islets as a source for islet transplantation require specialized conditions. These immature islets undergo plasticity in culture and form fully functional multicellular structures. Further development of this method for culturing immature porcine islets is expected to generate small pancreatic tissue-derived organoids termed “pancreatites,” as a therapeutic product from juvenile pigs for xenotransplantation and diabetes research.  

4.1620           Prior voluntary wheel running attenuates neuropathic pain

Grace, P.M., Fabisiak, T.J., Green-Fulgham, S.M., Anderson, N.D., Strand, K.A., Kwilasz, A.J., Galer, E.L., Walker, F.R., Greenwood, B.N., Maier, S.F., Fleshner, M. and Watkins, L.R. Pain, 157, 2012-2023 (2016)   Exercise is known to exert a systemic anti-inflammatory influence, but whether its effects are sufficient to protect against subsequent neuropathic pain is underinvestigated. We report that 6 weeks of voluntary wheel running terminating before chronic constriction injury (CCI) prevented the full development of allodynia for the ∼3-month duration of the injury. Neuroimmune signaling was assessed at 3 and 14 days after CCI. Prior exercise normalized ipsilateral dorsal spinal cord expression of neuroexcitatory interleukin (IL)-1β production and the attendant glutamate transporter GLT-1 decrease, as well as expression of the disinhibitory P2X4R-BDNF axis. The expression of the macrophage marker Iba1 and the chemokine CCL2 (MCP-1), and a neuronal injury marker (activating transcription factor 3), was attenuated by prior running in the ipsilateral lumbar dorsal root ganglia. Prior exercise suppressed macrophage infiltration and/or injury site proliferation, given decreased presence of macrophage markers Iba1, iNOS (M1), and Arg-1 (M2; expression was time dependent). Chronic constriction injury–driven increases in serum proinflammatory chemokines were suppressed by prior running, whereas IL-10 was increased. Peripheral blood mononuclear cells were also stimulated with lipopolysaccharide ex vivo, wherein CCI-induced increases in IL-1β, nitrite, and IL-10 were suppressed by prior exercise. Last, unrestricted voluntary wheel running, beginning either the day of, or 2 weeks after, CCI, progressively reversed neuropathic pain. This study is the first to investigate the behavioral and neuroimmune consequences of regular exercise terminating before nerve injury. This study suggests that chronic pain should be considered a component of “the diseasome of physical inactivity,” and that an active lifestyle may prevent neuropathic pain.  

4.1621           The long non-coding RNA Morrbid regulates Bim and short-lived myeloid cell lifespan

Kotzin, J.J. et al Nature, 537(7619), 239-243 (2016)   Neutrophils, eosinophils and ‘classical’ monocytes collectively account for about 70% of human blood leukocytes and are among the shortest-lived cells in the body^{1, 2}. Precise regulation of the lifespan of these myeloid cells is critical to maintain protective immune responses and minimize the deleterious consequences of prolonged inflammation^{1, 2}. However, how the lifespan of these cells is strictly controlled remains largely unknown. Here we identify a long non-coding RNA that we termed Morrbid, which tightly controls the survival of neutrophils, eosinophils and classical monocytes in response to pro-survival cytokines in mice. To control the lifespan of these cells, Morrbid regulates the transcription of the neighbouring pro-apoptotic gene, Bcl2l11 (also known as Bim), by promoting the enrichment of the PRC2 complex at the Bcl2l11 promoter to maintain this gene in a poised state. Notably, Morrbid regulates this process in cis, enabling allele-specific control of Bcl2l11 transcription. Thus, in these highly inflammatory cells, changes in Morrbid levels provide a locus-specific regulatory mechanism that allows rapid control of apoptosis in response to extracellular pro-survival signals. As MORRBID is present in humans and dysregulated in individuals with hypereosinophilic syndrome, this long non-coding RNA may represent a potential therapeutic target for inflammatory disorders characterized by aberrant short-lived myeloid cell lifespan.      

4.1622           Fetal liver endothelium regulates the seeding of tissue-resident macrophages

Rantakari, P., Jäppinen, N., Lokka, E., Mokkala, E., Gerke, H., Peuhu, E., Ivaska, J., Elima, K., Auvinen, K. and Salmi, M. Nature, 538(7625), 392-396 (2016)   Macrophages are required for normal embryogenesis, tissue homeostasis and immunity against microorganisms and tumours^{1, 2, 3, 4}. Adult tissue-resident macrophages largely originate from long-lived, self-renewing embryonic precursors and not from haematopoietic stem-cell activity in the bone marrow^{4, 5}. Although fate-mapping studies have uncovered a great amount of detail on the origin and kinetics of fetal macrophage development in the yolk sac and liver^{6, 7, 8, 9, 10, 11}, the molecules that govern the tissue-specific migration of these cells remain completely unknown. Here we show that an endothelium-specific molecule, plasmalemma vesicle-associated protein (PLVAP), regulates the seeding of fetal monocyte-derived macrophages to tissues in mice. We found that PLVAP-deficient mice have completely normal levels of both yolk-sac- and bone-marrow-derived macrophages, but that fetal liver monocyte-derived macrophage populations were practically missing from tissues. Adult PLVAP-deficient mice show major alterations in macrophage-dependent iron recycling and mammary branching morphogenesis. PLVAP forms diaphragms in the fenestrae of liver sinusoidal endothelium during embryogenesis, interacts with chemoattractants and adhesion molecules and regulates the egress of fetal liver monocytes to the systemic vasculature. Thus, PLVAP selectively controls the exit of macrophage precursors from the fetal liver and, to our knowledge, is the first molecule identified in any organ as regulating the migratory events during embryonic macrophage ontogeny.  

4.1623           Visceral motor neuron diversity delineates a cellular basis for nipple- and pilo-erection muscle control

Furlan, A., La Manno, G., Lübke, M., Häring, M., Abdo, H., Hochgerner, H., Kupari, J., Usoskin, D., Airaksinen, M.S., Oliver, G., Linnarsson, S. and Ernfors, P. Nature Neurosci., 19(10), 1331-1340 (2016)   Despite the variety of physiological and target-related functions, little is known regarding the cellular complexity in the sympathetic ganglion. We explored the heterogeneity of mouse stellate and thoracic ganglia and found an unexpected variety of cell types. We identified specialized populations of nipple- and pilo-erector muscle neurons. These neurons extended axonal projections and were born among other neurons during embryogenesis, but remained unspecialized until target organogenesis occurred postnatally. Target innervation and cell-type specification was coordinated by an intricate acquisition of unique combinations of growth factor receptors and the initiation of expression of concomitant ligands by the nascent erector muscles. Overall, our results provide compelling evidence for a highly sophisticated organization of the sympathetic nervous system into discrete outflow channels that project to well-defined target tissues and offer mechanistic insight into how diversity and connectivity are established during development.  

4.1624           CCL2, but not its receptor, is essential to restrict immune privileged central nervous system-invasion of Japanese encephalitis virus via regulating accumulation of CD11b+ Ly-6Chi monocytes

Kim, J.H., Patil, A.M., Choi, J.Y., Kim, S.B., Uyangaa, E., Hossain, F.M.A., Park, S-Y., Lee, J.H., Kim, K. and Eo, S.K. Immunology, 149(2), 186-203 (2016)   Japanese encephalitis virus (JEV) is a re-emerging zoonotic flavivirus that poses an increasing threat to global health and welfare due to rapid changes in climate and demography. Although the CCR2–CCL2 axis plays an important role in trafficking CD11b⁺ Ly-6C^hi monocytes to regulate immunopathological diseases, little is known about their role in monocyte trafficking during viral encephalitis caused by JEV infection. Here, we explored the role of CCR2 and its ligand CCL2 in JE caused by JEV infection using CCR2- and CCL2-ablated murine models. Somewhat surprisingly, the ablation of CCR2 and CCL2 resulted in starkly contrasting susceptibility to JE. CCR2 ablation induced enhanced resistance to JE, whereas CCL2 ablation highly increased susceptibility to JE. This contrasting regulation of JE progression by CCR2 and CCL2 was coupled to central nervous system (CNS) infiltration of Ly-6C^hi monocytes and Ly-6G^hi granulocytes. There was also enhanced expression of CC and CXC chemokines in the CNS of CCL2-ablated mice, which appeared to induce CNS infiltration of these cell populations. However, our data revealed that contrasting regulation of JE in CCR2- and CCL2-ablated mice was unlikely to be mediated by innate natural killer and adaptive T-cell responses. Furthermore, CCL2 produced by haematopoietic stem cell-derived leucocytes played a dominant role in CNS accumulation of Ly-6C^hi monocytes in infected bone marrow chimeric models, thereby exacerbating JE progression. Collectively, our data indicate that CCL2 plays an essential role in conferring protection against JE caused by JEV infection. In addition, blockage of CCR2, but not CCL2, will aid in the development of strategies for prophylactics and therapeutics of JE.  

4.1625           VOLIN and KJON—Two novel hyperdiploid myeloma cell lines

Våtsveen, T., Børset, M., Dikic, A., Tian, E., Micci, F., Lid, A.H.B., Meza-Zepeda, L.A., Coward, E., Waage, A., Sundan, A., Kuehl, W.M. and Holien, T. Genes, Chromosomes & Cancer, 55(11), 890-901 (2016)   Multiple myeloma can be divided into two distinct genetic subgroups: hyperdiploid (HRD) or nonhyperdiploid (NHRD) myeloma. Myeloma cell lines are important tools to study myeloma cell biology and are commonly used for preclinical screening and testing of new drugs. With few exceptions human myeloma cell lines are derived from NHRD patients, even though about half of the patients have HRD myeloma. Thus, there is a need for cell lines of HRD origin to enable more representative preclinical studies. Here, we present two novel myeloma cell lines, VOLIN and KJON. Both of them were derived from patients with HRD disease and shared the same genotype as their corresponding primary tumors. The cell lines' chromosomal content, genetic aberrations, gene expression, immunophenotype as well as some of their growth characteristics are described. Neither of the cell lines was found to harbor immunoglobulin heavy chain translocations. The VOLIN cell line was established from a bone marrow aspirate and KJON from peripheral blood. We propose that these unique cell lines may be used as tools to increase our understanding of myeloma cell biology.  

4.1626           Cell sources for in vitro human liver cell culture models

Zeilinger, K., Freyer, N., Damm, G., Seehofer, D. and Knöspel, F. Exp. Biol. Med., 241(15), 1684-1698 (2016)   In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro. However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro. Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described.  

4.1627           Sevelamer Improves Steatohepatitis, Inhibits Liver and Intestinal Farnesoid X Receptor (FXR), and Reverses Innate Immune Dysregulation in a Mouse Model of Non-alcoholic Fatty Liver Disease

McGettigan, B.M., McMahan, R.H., Luo, Y., Wang, X.X., Orlicky, D.J., Porsche, C., Levi, M. and Rosen, H.R.

1. Biol. Chem., 291(44), 23058-23067 (2016)

  Bile acid sequestrants are synthetic polymers that bind bile acids in the gut and are used to treat dyslipidemia and hyperphosphatemia. Recently, these agents have been reported to lower blood glucose and increase insulin sensitivity by altering bile acid signaling pathways. In this study, we assessed the efficacy of sevelamer in treating mice with non-alcoholic fatty liver disease (NAFLD). We also analyzed how sevelamer alters inflammation and bile acid signaling in NAFLD livers. Mice were fed a low-fat or Western diet for 12 weeks followed by a diet-plus-sevelamer regimen for 2 or 12 weeks. At the end of treatment, disease severity was assessed, hepatic leukocyte populations were examined, and expression of genes involved in farnesoid X receptor (FXR) signaling in the liver and intestine was analyzed. Sevelamer treatment significantly reduced liver steatosis and lobular inflammation. Sevelamer-treated NAFLD livers had notably fewer pro-inflammatory infiltrating macrophages and a significantly greater fraction of alternatively activated Kupffer cells compared with controls. Expression of genes involved in FXR signaling in the liver and intestine was significantly altered in mice with NAFLD as well as in those treated with sevelamer. In a mouse model of NAFLD, sevelamer improved disease and counteracted innate immune cell dysregulation in the liver. This study also revealed a dysregulation of FXR signaling in the liver and intestine of NAFLD mice that was counteracted by sevelamer treatment.  

4.1628           A Single-Cell Transcriptomic Map of the Human and Mouse Pancreas Reveals Inter- and Intra-cell Population Structure

Baron, M., Veres, A., Wolock, S.L. et al Cell Systems, 3(4), 346-360 (2016)   Although the function of the mammalian pancreas hinges on complex interactions of distinct cell types, gene expression profiles have primarily been described with bulk mixtures. Here we implemented a droplet-based, single-cell RNA-seq method to determine the transcriptomes of over 12,000 individual pancreatic cells from four human donors and two mouse strains. Cells could be divided into 15 clusters that matched previously characterized cell types: all endocrine cell types, including rare epsilon-cells; exocrine cell types; vascular cells; Schwann cells; quiescent and activated stellate cells; and four types of immune cells. We detected subpopulations of ductal cells with distinct expression profiles and validated their existence with immuno-histochemistry stains. Moreover, among human beta- cells, we detected heterogeneity in the regulation of genes relating to functional maturation and levels of ER stress. Finally, we deconvolved bulk gene expression samples using the single-cell data to detect disease-associated differential expression. Our dataset provides a resource for the discovery of novel cell type-specific transcription factors, signaling receptors, and medically relevant genes.  

4.1629           Adult human pancreas-derived cells expressing stage-specific embryonic antigen 4 differentiate into Sox9-expressing and Ngn3-expressing pancreatic ducts in vivo

Lee, S., Lee, C.M. and Kim, S.C. Stem Cell Res. & Ther., 7:162 (2016)   Background Tissue-specific stem/progenitor cells are found in various adult tissues and may have the capacity for lineage-specific differentiation, facilitating applications in autologous transplantation. Stage-specific embryonic antigen 4 (SSEA-4), an early embryonic glycolipid antigen, is expressed in cells derived from adult human pancreas exocrine tissue. Here, we examined the characteristics and lineage-specific differentiation capacity of SSEA-4⁺ cells. Methods Human adult partial pancreas tissues were obtained from different donors and cultured in vitro. SSEA-4⁺ and CA19-9⁺ cells were isolated from adult human pancreas exocrine cells using magnetic-activated cell sorting, and gene expression was validated by quantitative polymerase chain reaction. To confirm in-vivo differentiation, SSEA-4⁺ and CA19-9⁺ cells were transplanted into the dorsal subcutaneous region of mice. Finally, morphological features of differentiated areas were confirmed by immunostaining and morphometric analysis. Results SSEA-4-expressing cells were detected in isolated pancreas exocrine cells from adult humans. These SSEA-4⁺ cells exhibited coexpression of CA19-9, a marker of pancreatic duct cells, but not amylase expression, as shown by immunostaining and flow cytometry. SSEA-4⁺ cells exhibited higher relative expression of Oct4, Nanog, Klf4, Sox2, and c-Myc mRNAs than CA19-9⁺ cells. Pancreatic intralobular ducts (PIDs) were generated from SSEA-4⁺ or CA19-9⁺ cells in vivo at 5 weeks after transplantation. However, newly formed PIDs from CA19-9⁺ cells were less abundant and showed an incomplete PID morphology. In contrast, newly formed PIDs from SSEA-4⁺ cells were abundant in the transplanted area and showed a crowded morphology, typical of PIDs. Sox9 and Ngn3, key transcription factors associated with pancreatic development and regeneration, were expressed in PIDs from SSEA-4⁺ cells. Conclusions SSEA-4-expressing cells in the adult human pancreas may have the potential for regeneration of the pancreas and may be used as a source of stem/progenitor cells for pancreatic cell lineage-specific differentiation.  

4.1630           Red blood cell phase separation in symmetric and asymmetric microchannel networks: effect of capillary dilation and inflow velocity

Clavica, F., Homsy, A., jeandupeux, L. and Obrist, D. Scientific Reports, 6:36763 (2016)   The non-uniform partitioning or phase separation of red blood cells (RBCs) at a diverging bifurcation of a microvascular network is responsible for RBC heterogeneity within the network. The mechanisms controlling RBC heterogeneity are not yet fully understood and there is a need to improve the basic understanding of the phase separation phenomenon. In this context, in vitro experiments can fill the gap between existing in vivo and in silico models as they provide better controllability than in vivo experiments without mathematical idealizations or simplifications inherent to in silico models. In this study, we fabricated simple models of symmetric/asymmetric microvascular networks; we provided quantitative data on the RBC velocity, line density and flux in the daughter branches. In general our results confirmed the tendency of RBCs to enter the daughter branch with higher flow rate (Zweifach-Fung effect); in some cases even inversion of the Zweifach-Fung effect was observed. We showed for the first time a reduction of the Zweifach-Fung effect with increasing flow rate. Moreover capillary dilation was shown to cause an increase of RBC line density and RBC residence time within the dilated capillary underlining the possible role of pericytes in regulating the oxygen supply.  

4.1631           An optimized method for mouse liver sinusoidal endothelial cell isolation

Meyer, J., Lacotte, S., Morel, P., Gonelle-Gispert, C. and Bühler, L. Exp. Cell Res., 349, 291-301 (2016)   The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions.  

4.1632           Development of a toolbox to dissect host-endosymbiont interactions and protein trafficking in the trypanosomatid Angomonas deanei

Morales, J., Kokkori, S., Weidauer, D., Chapman, J., Goltsman, E., Rokhsar, D., Grossman, A.R. and Nowack, E.C.M. BMC Evolutionary Biol., 16:247 (2016)   Background Bacterial endosymbionts are found across the eukaryotic kingdom and profoundly impacted eukaryote evolution. In many endosymbiotic associations with vertically inherited symbionts, highly complementary metabolic functions encoded by host and endosymbiont genomes indicate integration of metabolic processes between the partner organisms. While endosymbionts were initially expected to exchange only metabolites with their hosts, recent evidence has demonstrated that also host-encoded proteins can be targeted to the bacterial symbionts in various endosymbiotic systems. These proteins seem to participate in regulating symbiont growth and physiology. However, mechanisms required for protein targeting and the specific endosymbiont targets of these trafficked proteins are currently unexplored owing to a lack of molecular tools that enable functional studies of endosymbiotic systems. Results Here we show that the trypanosomatid Angomonas deanei, which harbors a β-proteobacterial endosymbiont, is readily amenable to genetic manipulation. Its rapid growth, availability of full genome and transcriptome sequences, ease of transfection, and high frequency of homologous recombination have allowed us to stably integrate transgenes into the A. deanei nuclear genome, efficiently generate null mutants, and elucidate protein localization by heterologous expression of a fluorescent protein fused to various putative targeting signals. Combining these novel tools with proteomic analysis was key for demonstrating the routing of a host-encoded protein to the endosymbiont, suggesting the existence of a specific endosymbiont-sorting machinery in A. deanei. Conclusions After previous reports from plants, insects, and a cercozoan amoeba we found here that also in A. deanei, i.e. a member of a fourth eukaryotic supergroup, host-encoded proteins can be routed to the bacterial endosymbiont. This finding adds further evidence to our view that the targeting of host proteins is a general strategy of eukaryotes to gain control over and interact with a bacterial endosymbiont. The molecular resources reported here establish A. deanei as a time and cost efficient reference system that allows for a rigorous dissection of host-symbiont interactions that have been, and are still being shaped over evolutionary time. We expect this system to greatly enhance our understanding of the biology of endosymbiosis.  

4.1633           Engineering Genetically-Encoded Mineralization and Magnetism via Directed Evolution

Liu, X., Lopez, P.A., Giessen, T.W., Gilers, M., Way, J.C. and Silver, P.A. Scientific Reports, 6:38019 (2016)   Genetically encoding the synthesis of functional nanomaterials such as magnetic nanoparticles enables sensitive and non-invasive biological sensing and control. Via directed evolution of the natural iron-sequestering ferritin protein, we discovered key mutations that lead to significantly enhanced cellular magnetism, resulting in increased physical attraction of ferritin-expressing cells to magnets and increased contrast for cellular magnetic resonance imaging (MRI). The magnetic mutants further demonstrate increased iron biomineralization measured by a novel fluorescent genetic sensor for intracellular free iron. In addition, we engineered Escherichia coli cells with multiple genomic knockouts to increase cellular accumulation of various metals. Lastly to explore further protein candidates for biomagnetism, we characterized members of the DUF892 family using the iron sensor and magnetic columns, confirming their intracellular iron sequestration that results in increased cellular magnetization.  

4.1634           The Role of Progesterone and a Novel Progesterone Receptor, Progesterone Receptor Membrane Component 1, in the Inflammatory Response of Fetal Membranes to Ureaplasma parvum Infection

Feng, L., Ransom, C.E., Nazzai, M., Allen, T.K., Li, Y-J., Truong, T., Potts, L.C., Seed, P.-C. and Murtha, A.P. PloS One, 11(12), e0168102 (2016)   Ureaplasma parvum (U. parvum) is gaining recognition as an important pathogen for chorioamnionitis and preterm premature rupture of membranes. We aimed to investigate the roles of progesterone (P4) and a novel progesterone receptor, progesterone receptor membrane component 1 (PGRMC1), in the response of fetal membranes to U. parvum. Fetal membrane cells (amnion, chorion and decidua) were isolated and confirmed to be free of Mycoplasmataceae. Cells were treated with U. parvum (5x10⁶ CFU), and adherence was quantified by qPCR. Amnion and chorion cells were transfected with scrambled siRNA or validated PGRMC1 siRNA for 72h. Cells were then treated with U. parvum for 4h with or without pretreatment with P4 (10⁻⁷ M) or ethanol for 1h. Interleukin-8 (IL-8), matrix metalloproteinase 9 (MMP9) and cyclooxygenase (COX-2) mRNA expression were quantified by qRT-PCR. Culture medium was harvested and analyzed for IL-8 and prostaglandin (PGE₂) secretion by ELISA and MMP9 activity by zymography. U. parvum had a mean adherence of 15.0±0.6%, 16.9± 3.7% and 4.7±0.3% in cultured amnion, chorion and decidua cells, respectively. Exposure to U. parvum elicited significant inflammatory responses including induction of IL-8, COX-2, PGE2 and MMP9. A possible role of PGRMC1 was identified in the inhibition of U. parvum-stimulated COX-2 and MMP9 mRNA expression in chorion cells and MMP9 activity in amnion cells. On the other hand, it might enhance the U. parvum-stimulated IL-8 protein secretion in amnion cells. P4, mediated through PGRMC1, significantly inhibited U. Parvum-induced MMP9 mRNA and COX-2 mRNA expression in chorion cells. P4 appeared to attenuate U. parvum induced IL-8 mRNA expression in chorion cells, but this P4 effect might not mediated through PGRMC1. In summary, U. parvum preferentially adheres to and induces inflammatory responses in chorion and amnion cells. P4 and PGRMC1 appear to differentially modulate the inflammatory responses induced by U. parvum among amnion and chorion cells.  

4.1635           Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation

Mortimer, M., Petersen, E.J., Buchholz, B.A. and Holden, P.A: Nanomaterials, 6:181 (2016)   Sustainable production and use of carbon nanotube (CNT)-enabled materials require efficient assessment of CNT environmental hazards, including the potential for CNT bioaccumulation and biomagnification in environmental receptors. Microbes, as abundant organisms responsible for nutrient cycling in soil and water, are important ecological receptors for studying the effects of CNTs. Quantification of CNT association with microbial cells requires efficient separation of CNT-associated cells from individually dispersed CNTs and CNT agglomerates. Here, we designed, optimized, and demonstrated procedures for separating bacteria (Pseudomonas aeruginosa) from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT agglomerates using sucrose density gradient centrifugation. We demonstrate separation of protozoa (Tetrahymena thermophila) from MWCNTs, bacterial agglomerates, and protozoan fecal pellets by centrifugation in an iodixanol solution. The presence of MWCNTs in the density gradients after centrifugation was determined by quantification of ¹⁴C-labeled MWCNTs; the recovery of microbes from the density gradient media was confirmed by optical microscopy. Protozoan intracellular contents of MWCNTs and of bacteria were also unaffected by the designed separation process. The optimized methods contribute to improved efficiency and accuracy in quantifying MWCNT association with bacteria and MWCNT accumulation in protozoan cells, thus supporting improved assessment of CNT bioaccumulation.  

4.1636           Broncho Alveolar Dendritic Cells and Macrophages Are Highly Similar to Their Interstitial Counterparts

Maisonnasse, P., Bordet, E., Bouuguyon, E. and Bertho, N. PloS One, 11(12), e0167315 (2016)   In human medicine, bronchoalveolar lavage is the main non-traumatic procedure allowing an insight into the respiratory Dendritic Cells (DC) and Macrophages populations. However, it has never been demonstrated in a relevant model that alveolar DC subpopulations were comparable to their interstitial counterparts. In a precedent work we observed that respiratory pig DC and Macrophages were more similar to the human ones than to the mouse ones. In the present work, thanks to our animal model, we were able to collect the rare bronchoalveolar DC and compare them to their interstitial counterparts. We observed that DC presented very similar gene-expression patterns in the alveolar and interstitial compartments, validating the study of human bronchoalveolar DC as surrogate of their interstitium counterparts.  

4.1637           Mincle Signaling Promotes Con A Hepatitis

Greco, S.H. et al

1. Immunol., 197(7), 2816-2827 (2016)

  Con A hepatitis is regarded as a T cell–mediated model of acute liver injury. Mincle is a C-type lectin receptor that is critical in the immune response to mycobacteria and fungi but does not have a well-defined role in preclinical models of non-pathogen–mediated inflammation. Because Mincle can ligate the cell death ligand SAP130, we postulated that Mincle signaling drives intrahepatic inflammation and liver injury in Con A hepatitis. Acute liver injury was assessed in the murine Con A hepatitis model using C57BL/6, Mincle^−/−, and Dectin-1^−/− mice. The role of C/EBPβ and hypoxia-inducible factor-1α (HIF-1α) signaling was assessed using selective inhibitors. We found that Mincle was highly expressed in hepatic innate inflammatory cells and endothelial cells in both mice and humans. Furthermore, sterile Mincle ligands and Mincle signaling intermediates were increased in the murine liver in Con A hepatitis. Most significantly, Mincle deletion or blockade protected against Con A hepatitis, whereas Mincle ligation exacerbated disease. Bone marrow chimeric and adoptive transfer experiments suggested that Mincle signaling in infiltrating myeloid cells dictates disease phenotype. Conversely, signaling via other C-type lectin receptors did not alter disease course. Mechanistically, we found that Mincle blockade decreased the NF-κβ–related signaling intermediates C/EBPβ and HIF-1α, both of which are necessary in macrophage-mediated inflammatory responses. Accordingly, Mincle deletion lowered production of nitrites in Con A hepatitis and inhibition of both C/EBPβ and HIF-1α reduced the severity of liver disease. Our work implicates a novel innate immune driver of Con A hepatitis and, more broadly, suggests a potential role for Mincle in diseases governed by sterile inflammation.  

4.1638           A Critical Role for P2X7 Receptor–Induced VCAM-1 Shedding and Neutrophil Infiltration during Acute Lung Injury

Mishra, A., Guo, Y., Zhang, L., More, S., Weng, T., Chintagari, R., Huang, C., Liang, Y., Pushparaj, S., Gou, D., Breshears, M. and Liu, L.

1. Immunol., 197(7), 2828-2837 (2016)

  Pulmonary neutrophils are the initial inflammatory cells that are recruited during lung injury and are crucial for innate immunity. However, pathological recruitment of neutrophils results in lung injury. The objective of this study is to determine whether the novel neutrophil chemoattractant, soluble VCAM-1 (sVCAM-1), recruits pathological levels of neutrophils to injury sites and amplifies lung inflammation during acute lung injury. The mice with P2X₇ receptor deficiency, or treated with a P2X₇ receptor inhibitor or anti–VCAM-1 Abs, were subjected to a clinically relevant two-hit LPS and mechanical ventilation–induced acute lung injury. Neutrophil infiltration and lung inflammation were measured. Neutrophil chemotactic activities were determined by a chemotaxis assay. VCAM-1 shedding and signaling pathways were assessed in isolated lung epithelial cells. Ab neutralization of sVCAM-1 or deficiency or antagonism of P2X₇R reduced neutrophil infiltration and proinflammatory cytokine levels. The ligands for sVCAM-1 were increased during acute lung injury. sVCAM-1 had neutrophil chemotactic activities and activated alveolar macrophages. VCAM-1 is released into the alveolar airspace from alveolar epithelial type I cells through P2X₇ receptor–mediated activation of the metalloproteinase ADAM-17. In conclusion, sVCAM-1 is a novel chemoattractant for neutrophils and an activator for alveolar macrophages. Targeting sVCAM-1 provides a therapeutic intervention that could block pathological neutrophil recruitment, without interfering with the physiological recruitment of neutrophils, thus avoiding the impairment of host defenses.  

4.1639           Blomia tropicalis–Specific TCR Transgenic Th2 Cells Induce Inducible BALT and Severe Asthma in Mice by an IL-4/IL-13–Dependent Mechanism

Chua, Y.L. et al

1. Immunol., 197(10), 3771-3781 (2016)

  Previous studies have highlighted the importance of lung-draining lymph nodes in the respiratory allergic immune response, whereas the lung parenchymal immune system has been largely neglected. We describe a new in vivo model of respiratory sensitization to Blomia tropicalis, the principal asthma allergen in the tropics, in which the immune response is focused on the lung parenchyma by transfer of Th2 cells from a novel TCR transgenic mouse, specific for the major B. tropicalis allergen Blo t 5, that targets the lung rather than the draining lymph nodes. Transfer of highly polarized transgenic CD4 effector Th2 cells, termed BT-II, followed by repeated inhalation of Blo t 5 expands these cells in the lung >100-fold, and subsequent Blo t 5 challenge induced decreased body temperature, reduction in movement, and a fall in specific lung compliance unseen in conventional mouse asthma models following a physiological allergen challenge. These mice exhibit lung eosinophilia; smooth muscle cell, collagen, and goblet cell hyperplasia; hyper IgE syndrome; mucus plugging; and extensive inducible BALT. In addition, there is a fall in total lung volume and forced expiratory volume at 100 ms. These pathophysiological changes were substantially reduced and, in some cases, completely abolished by administration of neutralizing mAbs specific for IL-4 and IL-13 on weeks 1, 2, and 3. This IL-4/IL-13–dependent inducible BALT model will be useful for investigating the pathophysiological mechanisms that underlie asthma and the development of more effective drugs for treating severe asthma.  

4.1640           Kinetic analysis of internalization of white spot syndrome virus by haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei (Boone), and the outcome for virus and cell

Tuan, V.V., De Gryse, G.M.A., Thuong, K.V., Bossier, P. and Nauwynck, H.J.

1. Fish Diseases, 39(12), 1477-1493 (2016)

  Little is known about the innate antiviral defence of shrimp haemocytes. In this context, the haemocytes of penaeid shrimp Litopenaeus vannamei (Boone) were separated by iodixanol density gradient centrifugation into five subpopulations (sub): sub 1 (hyalinocytes), sub 2 and 3 (prohyalinocytes), sub 4 (semigranulocytes) and sub 5 (granulocytes) and exposed to beads, white spot syndrome virus (WSSV) and ultraviolet (UV)-killed WSSV. In a first experiment, the uptake of beads, white spot syndrome virus (WSSV) and UV-killed WSSV by these different haemocyte subpopulations was investigated using confocal microscopy. Only haemocytes of sub 1, 4 and 5 were internalizing beads, WSSV and UV-killed WSSV. Beads were engulfed by a much larger percentage of cells (91.2 in sub 1; 84.1 in sub 4 and 58.1 in sub 5) compared to WSSV (9.6 in sub 1; 10.5 in sub 4 and 7.9 in sub 5) and UV-killed WSSV (12.9 in sub 1; 13.3 in sub 4; and 11.8 in sub 5). In a second experiment, it was shown that upon internalization, WSS virions lost their envelope most probably by fusion with the cellular membrane of the endosome (starting between 30 and 60 min post-inoculation) and that afterwards the capsid started to become disintegrated (from 360 min post-inoculation). Expression of new viral proteins was not observed. Incubation of haemocyte subpopulations with WSSV but not with UV-killed WSSV and polystyrene beads resulted in a significant drop in haemocyte viability. To find the underlying mechanism, a third experiment was performed in which haemocyte subpopulations were exposed to a short WSSV DNA fragment (VP19) and CpG ODNs. These small DNA fragments induced cell death. In conclusion, WSSV is efficiently internalized by hyalinocytes, semigranulocytes and granulocytes, after which the virus loses its envelope; as soon as the capsids start to disintegrate, cell death is activated, which in part may be explained by the exposure of viral DNA to cellular-sensing molecules.  

4.1641           Mesenchymal stromal cell-derived extracellular vesicles rescue radiation damage to murine marrow hematopoietic cells

Wen, S., Dooner, M., Cheng, Y., Papa, E., Del Tatto, M., Pereira, M., Deng, Y., Goldberg, L., Aliotta, J., Chatterjee, D., Stewart, C., Carpanetto, A., Collino, F., Bruno, S., Camussi, G. and Quesenberry, P. Leukemia, 30(12), 2221-2231 (2016)   Mesenchymal stromal cells (MSCs) have been shown to reverse radiation damage to marrow stem cells. We have evaluated the capacity of MSC-derived extracellular vesicles (MSC-EVs) to mitigate radiation injury to marrow stem cells at 4 h to 7 days after irradiation. Significant restoration of marrow stem cell engraftment at 4, 24 and 168 h post irradiation by exposure to MSC-EVs was observed at 3 weeks to 9 months after transplant and further confirmed by secondary engraftment. Intravenous injection of MSC-EVs to 500cGy exposed mice led to partial recovery of peripheral blood counts and restoration of the engraftment of marrow. The murine hematopoietic cell line, FDC-P1 exposed to 500cGy, showed reversal of growth inhibition, DNA damage and apoptosis on exposure to murine or human MSC-EVs. Both murine and human MSC-EVs reverse radiation damage to murine marrow cells and stimulate normal murine marrow stem cell/progenitors to proliferate. A preparation with both exosomes and microvesicles was found to be superior to either microvesicles or exosomes alone. Biologic activity was seen in freshly isolated vesicles and in vesicles stored for up to 6 months in 10% dimethyl sulfoxide at −80 °C. These studies indicate that MSC-EVs can reverse radiation damage to bone marrow stem cells.  

4.1642           Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions

Martins, R. et al Nature Immunol., 17(12), 1361-1372 (2016)   Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.  

4.1643           Evolution of Osteocrin as an activity-regulated factor in the primate brain

Ataman, B. et al Nature, 539(7628), 242-247 (2016)   Sensory stimuli drive the maturation and function of the mammalian nervous system in part through the activation of gene expression networks that regulate synapse development and plasticity. These networks have primarily been studied in mice, and it is not known whether there are species- or clade-specific activity-regulated genes that control features of brain development and function. Here we use transcriptional profiling of human fetal brain cultures to identify an activity-dependent secreted factor, Osteocrin (OSTN), that is induced by membrane depolarization of human but not mouse neurons. We find that OSTN has been repurposed in primates through the evolutionary acquisition of DNA regulatory elements that bind the activity-regulated transcription factor MEF2. In addition, we demonstrate that OSTN is expressed in primate neocortex and restricts activity-dependent dendritic growth in human neurons. These findings suggest that, in response to sensory input, OSTN regulates features of neuronal structure and function that are unique to primates.  

4.1644           Different tissue phagocytes sample apoptotic cells to direct distinct homeostasis programs

Cummings, R.J., Barbet, G., Bongers, G., hartmann, B.M., Gettler, K., Muniz, L., Furtado, G.C., Cho, J., Lira, S.A. and Blander, J.M. Nature, 539(7630), 565-569 (2016)   Recognition and removal of apoptotic cells by professional phagocytes, including dendritic cells and macrophages, preserves immune self-tolerance and prevents chronic inflammation and autoimmune pathologies^{1, 2}. The diverse array of phagocytes that reside within different tissues, combined with the necessarily prompt nature of apoptotic cell clearance, makes it difficult to study this process in situ. The full spectrum of functions executed by tissue-resident phagocytes in response to homeostatic apoptosis, therefore, remains unclear. Here we show that mouse apoptotic intestinal epithelial cells (IECs), which undergo continuous renewal to maintain optimal barrier and absorptive functions³, are not merely extruded to maintain homeostatic cell numbers⁴, but are also sampled by a single subset of dendritic cells and two macrophage subsets within a well-characterized network of phagocytes in the small intestinal lamina propria^{5, 6}. Characterization of the transcriptome within each subset before and after in situ sampling of apoptotic IECs revealed gene expression signatures unique to each phagocyte, including macrophage-specific lipid metabolism and amino acid catabolism, and a dendritic-cell-specific program of regulatory CD4⁺ T-cell activation. A common ‘suppression of inflammation’ signature was noted, although the specific genes and pathways involved varied amongst dendritic cells and macrophages, reflecting specialized functions. Apoptotic IECs were trafficked to mesenteric lymph nodes exclusively by the dendritic cell subset and served as critical determinants for the induction of tolerogenic regulatory CD4⁺ T-cell differentiation. Several of the genes that were differentially expressed by phagocytes bearing apoptotic IECs overlapped with susceptibility genes for inflammatory bowel disease⁷. Collectively, these findings provide new insights into the consequences of apoptotic cell sampling, advance our understanding of how homeostasis is maintained within the mucosa and set the stage for development of novel therapeutics to alleviate chronic inflammatory diseases such as inflammatory bowel disease.  

4.1645           Strategies for Processing Semen from Subfertile Stallions for Cooled Transport

Varner, D.D. Vet. Clin. Equine, 32(3), 547-560 (2016)   Simple dilution of semen in extender is generally satisfactory for cooled transport of semen if certain guidelines are applied. Subfertility following insemination with cool-transported semen can be associated with different inciting factors. Concentration of sperm in semen can be achieved by filtration or centrifugation procedures. Currently, centrifugation is most commonly applied. Centrifugal fractionation of semen (also termed density gradient centrifugation) can be used to enhance sperm quality but recovery rates can be low, thereby necessitating low-dose insemination techniques for breeding purposes.  

4.1646           Molecular immunology profiles of monkeys following xenografting with the islets and heart of α-1,3-galactosyltransferase knockout pigs

Ock, S.A., Lee, J., Oh, K.B., Hwang, S., Yun, I.J., Ahn, C.,, Chee, H.k., Kim, H., park, J.B., Kim, S.J., Im, G-S.and Park, E.W. Xenotransplantation, 23(5), 357-369 (2016)   Effective immunosuppression strategies and genetically modified animals have been used to prevent hyperacute and acute xenograft rejection; however, the underlying mechanisms remain unknown. In this study, we evaluated the expression of a comprehensive set of immune system-related genes (89 genes, including five housekeeping genes) in the blood of cynomolgus monkeys (~5 yr old) used as graft recipients, before and after the xenografting of the islets and heart from single and double α-1,3-galactosyltransferase (GalT) knockout (KO) pigs (<6 weeks old). The immunosuppressive regimen included administration of cobra venom factor, anti-thymocyte globulin, rituximab, and anti-CD154 monoclonal antibodies to recipients before and after grafting. Islets were xenografted into the portal vein in type 1 diabetic monkeys, and the heart was xenografted by heterotopic abdominal heart transplantation. Genes from recipient blood were analyzed using RT² profiler PCR arrays and the web-based RT² profiler PCR array software v.3.5. Recipients treated with immunosuppressive agents without grafting showed significant downregulation of CCL5, CCR4, CCR6, CD4, CD40LG, CXCR3, FASLG, CXCR3, FOXP3, GATA3, IGNG, L10, IL23A, TRAF6, MAPK8, MIF, STAT4, TBX21, TLR3, TLR7, and TYK2 and upregulation of IFNGR1; thus, genes involved in protection against viral and bacterial infection were downregulated, confirming the risk of infection. Notably, C3-level control resulted in xenograft failure within 2 days because of a 7- to 11-fold increase in all xenotransplanted models. Islet grafting using single GalT-KO pigs resulted in upregulation of CXCL10 and MX1, early inflammation, and acute rejection-associated signals at 2 days after xenografting. We observed at least 5-fold upregulation in recipients transplanted with islets grafts from single (MX1) or double (C3, CCR8, IL6, IL13, IRF6, CXCL10, and MX1) GalT-KO pigs after 77 days; single GalT-KO incurred early losses owing to immune attacks. Our results suggest that this novel, simple, non-invasive, and time-efficient procedure (requiring only 1.5 ml blood) for evaluating graft success, minimizing immune rejection, and blocking infection.  

4.1647           Ethanol Exposures During the Developmental Period Increase Microglia Sensitivity to a Stress Challenge During Adulthood: A Possible Cause for the Stress Hyperresponse in Fetal Alcohol Exposed Offspring

Sarkar, D., Chastain, L., Cabrera, M. and Shrivastava , P.

Neurosychopharmacology, 41, S136, abstract M33 (2016)   Background: In the healthy central nervous system (CNS), microglia, the immune cells of the CNS, quickly detect a stimulus, become activated and elicit the proper response. Once the stimulus is removed and homeostasis is restored, microglia revert to their inactive ramified state and returns to their surveillance duties. Recently it has been shown that a single exposure to Escherichia coli (E. Coli) activates microglia in rat neonates, and causes long-term alterations in microglia response and behavior in adulthood. No previous investigation on the long-term effects of ethanol induced microglia activation during the neonatal period in rats, a period equivalent to the third trimester of human pregnancy, has been reported. In this study, we investigate the effects of neonatal ethanol on microglia and their influence in regulation of the hypothalamic-pituitary-adrenal (HPA) axis response to an immune stress challenge during adulthood. Methods: Neonatal rat pups were fed by oral gavage a milk formula containing 11.34% ethanol (vol/vol), yielding a total daily ethanol dose of 2.5 g/kg (AF), or isocaloric control (PF), or they were left in the litter with the mother (AD) for 5 days (postnatal days 2-6). Some of the AF rats additionally received a minocycline pre-treatment one hour prior to the first feeding on each day by subcutaneous injection of minocycline solution (45μg/kg bodyweight). Two hours after the last feeding, some of the pups were sacrificed and hypothalamus was dissected for microglia separation by differential gradient centrifugation using OptiPrep gradient and characterization by measuring production of various activation markers and cytokines. The rest of the pups were maintained in the controlled condition until 90 days of age when they were used for immune stress challenge (Lipopolysaccharide, LPS; 100ug/kg bodyweight i.p.) and microglia characterization and HPA axis responses by measuring plasma levels of corticosterone and adrenocorticotrophin (ACTH). Animal surgery and care were performed in accordance with institutional guidelines and complied with the National Institutes of Health policy. Results: We found binge-like ethanol exposures during the postnatal period increased microglial activation markers (e.g., IBA1, CX3CR1), inflammatory cytokine (e.g., TNF-α), an inflammatory signal receptor (TLR-4), and inflammatory cell signaling molecules (IKBA) in microglia isolated from the hypothalamus. Comparable results were also obtained following a known activator of microglia, LPS. In association with the microglial activation, we found increased proopiomelanocortin (POMC) neuronal (a neuronal population known to regulate the HPA axis function) apoptosis in the arcuate nucleus of the hypothalamus. Treatment with minocycline, an inhibitor of microglia activation, blocked ethanol effect on POMC neuronal apoptosis. Neonatal ethanol also promotes the adult response of the microglia to an immune challenge. In addition, neonatal ethanol increased the response of the HPA axis, plasma corticosterone and ACTH, following LPS challenge, while neonatal minocycline prevented ethanol action on the stress hormone responses. Neonatal ethanol altered the expression of various proteins related to Dnmts, HDACs and MeCp2 and increased histone acetylation and decreased DNA methylation in microglia. Conclusions: These data suggest that neonatal exposure to a high dose of ethanol activates microglia by producing various inflammatory cytokines leading to killing of POMC neurons and a deficiency in feedback regulatory control over the stress axis function. Additionally, neonatal ethanol exposures increase microglia sensitivity to the subsequent stimuli by possibly altering epigenetic mechanisms. In conclusion, this work provides important insights into the effects of both immediate and long-term effects of ethanol induced microglia activation during third trimester gestation. In our model, ethanol induced microglia activation leads to long-term alterations in microglia responses to subsequent stimuli that contribute to changes in HPA response in adulthood.  

4.1648           FREE

 

4.1649           Reducing neuroinflammation by delivery of IL-10 encoding lentivirus from multiple-channel bridges

Margul, D.J. et al Bioeng. Translational Med., 1, 136-148 (2016)   The spinal cord is unable to regenerate after injury largely due to growth-inhibition by an inflammatory response to the injury that fails to resolve, resulting in secondary damage and cell death. An approach that prevents inhibition by attenuating the inflammatory response and promoting its resolution through the transition of macrophages to anti-inflammatory phenotypes is essential for the creation of a growth permissive microenvironment. Viral gene delivery to induce the expression of anti-inflammatory factors provides the potential to provide localized delivery to alter the host inflammatory response. Initially, we investigated the effect of the biomaterial and viral components of the delivery system to influence the extent of cell infiltration and the phenotype of these cells. Bridge implantation reduces antigen-presenting cell infiltration at day 7, and lentivirus addition to the bridge induces a transient increase in neutrophils in the spinal cord at day 7 and macrophages at day 14. Delivery of a lentivirus encoding IL-10, an anti-inflammatory factor that inhibits immune cell activation and polarizes the macrophage population towards anti-inflammatory phenotypes, reduced neutrophil infiltration at both day 7 and day 28. Though IL-10 lentivirus did not affect macrophages number, it skewed the macrophage population toward an anti-inflammatory M2 phenotype and altered macrophage morphology. Additionally, IL-10 delivery resulted in improved motor function, suggesting reduced secondary damage and increased sparing. Taken together, these results indicate that localized expression of anti-inflammatory factors, such as IL-10, can modulate the inflammatory response following spinal cord injury, and may be a key component of a combinatorial approach that targets the multiple barriers to regeneration and functional recovery.  

4.1650           Effect of GDNF on Morphology, Proliferation, and Phagocytic Activity of Rat Neonatal Cortex Isolated Microglia

Zhuravleva, M., Rizvanov, A. and Mukhamedshina, Y. BioNanoSci., 6, 379-383 (2016)   Microglia are the main defenders of the central nervous system and at the same time are involved in the pathogenesis of various neurological disorders. Microglia hyperactivity or phagocytic impairment exacerbates degenerative processes in nervous tissue leading to further loss of function. A variety of factors and cytokines may modify microglia function. In our study, it was shown that glial cell line-derived neurotrophic factor (GDNF), a well-known neuroprotective molecule, decreases phagocytic activity of microglia in vitro model of spinal cord injury. Recombinant adenovirus encoding GDNF (Ad5-GDNF) transfected microglia have shown the same effect and can be potentially used as a therapeutic agent in case of neurotrauma due to its debris phagocytic and GDNF-associated neuroprotective role.  

4.1651           Dendritic cell sphingosine-1-phosphate lyase regulates thymic egress

Zamora-Pineda, J., Kumar, A., Suh, J.H., Zhang, M. and Saba, J.D.

1. Exp. Med., 213(12), 2773-2791 (2016)

  T cell egress from the thymus is essential for adaptive immunity and involves chemotaxis along a sphingosine-1-phosphate (S1P) gradient. Pericytes at the corticomedullary junction produce the S1P egress signal, whereas thymic parenchymal S1P levels are kept low through S1P lyase (SPL)–mediated metabolism. Although SPL is robustly expressed in thymic epithelial cells (TECs), in this study, we show that deleting SPL in CD11c⁺ dendritic cells (DCs), rather than TECs or other stromal cells, disrupts the S1P gradient, preventing egress. Adoptive transfer of peripheral wild-type DCs rescued the egress phenotype of DC-specific SPL knockout mice. These studies identify DCs as metabolic gatekeepers of thymic egress. Combined with their role as mediators of central tolerance, DCs are thus poised to provide homeostatic regulation of thymic export.  

4.1652           Motor neuron mitochondrial dysfunction in spinal muscular atrophy

Miller, N., Shi, H., Zelikovich, A.S. and Ma, Y-C. Hum. Mol. Genet., 25(16), 3395-3406 (2016)   Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, predominantly affects high metabolic tissues including motor neurons, skeletal muscles and the heart. Although the genetic cause of SMA has been identified, mechanisms underlying tissue-specific vulnerability are not well understood. To study these mechanisms, we carried out a deep sequencing analysis of the transcriptome of spinal motor neurons in an SMA mouse model, in which we unexpectedly found changes in many genes associated with mitochondrial bioenergetics. Importantly, functional measurement of mitochondrial activities showed decreased basal and maximal mitochondrial respiration in motor neurons from SMA mice. Using a reduction-oxidation sensitive GFP and fluorescence sensors specifically targeted to mitochondria, we found increased oxidative stress level and impaired mitochondrial membrane potential in motor neurons affected by SMA. In addition, mitochondrial mobility was impaired in SMA disease conditions, with decreased retrograde transport but no effect on anterograde transport. We also found significantly increased fragmentation of the mitochondrial network in primary motor neurons from SMA mice, with no change in mitochondria density. Electron microscopy study of SMA mouse spinal cord revealed mitochondria fragmentation, edema and concentric lamellar inclusions in motor neurons affected by the disease. Intriguingly, these functional and structural deficiencies in the SMA mouse model occur during the presymptomatic stage of disease, suggesting a role in initiating SMA. Altogether, our findings reveal a critical role for mitochondrial defects in SMA pathogenesis and suggest a novel target for improving tissue health in the disease.  

4.1653           Platelet-borne complement proteins and their role in platelet–bacteria interactions

Arbesu, I., Bucsaiova, M., Fisher, M.B. and mannhalter, C.

1. Thrombosis and Haemostasis, 14, 2241-2252 (2016)

  Background The role of platelets in immune defense is increasingly being recognized. Platelets bind complement proteins from plasma, initiate complement activation, and interact with bacteria. However, the contribution of platelets to complement-mediated defense against bacterial infections is not known in detail. Objectives To assess platelet interactions with Escherichia coli strains, and evaluate the contributions of platelet complement proteins to host defense. Methods We studied the cell–cell interactions of a pathogenic and a non-pathogenic E. coli strain with platelet concentrates, washed platelets and manually isolated platelets by flow cytometry and ELISA. The presence of complement proteins and complement RNA in megakaryocytes and platelets was analyzed by PCR, RT-PCR, confocal microscopy, and western blotting. Results Incubation with E. coli leads to platelet activation, as indicated by the expression of CD62P and CD63 on the platelet surface. RNA and protein analyses show that megakaryocytes and platelets contain complement C3, and that platelet C3 migrates differently on polyacrylamide gels than plasmatic C3. Activation of platelets by bacteria leads to translocation of C3 to the cell surface. This translocation is not induced by thrombin receptor activating peptide or lipopolysaccharide. Interaction of platelets with E. coli occurs even in the absence of plasma proteins, and is independent of platelet toll-like receptor 4 and α_2bβ₃ (glycoprotein IIbIIIa). Conclusion Platelets contain a specific form of C3. Importantly, they can modulate immune defense against bacteria by enhancing plasmatic complement activation.   The Warburg Effect Mediator Pyruvate Kinase M2 Expression and Regulation in the Retina Rajala, R.V.S., Rajala, A., Kooker, C., Wang, Y. and Anderson, R.E. Scientific Reports, 6:37727 (2016)   The tumor form of pyruvate kinase M2 (PKM2) undergoes tyrosine phosphorylation and gives rise to the Warburg effect. The Warburg effect defines a pro-oncogenic metabolism switch such that cancer cells take up more glucose than normal tissue and favor incomplete oxidation of glucose, even in the presence of oxygen. Retinal photoreceptors are highly metabolic and their energy consumption is equivalent to that of a multiplying tumor cell. In the present study, we found that PKM2 is the predominant isoform in both rod- and cone-dominant retina, and that it undergoes a light-dependent tyrosine phosphorylation. We also discovered that PKM2 phosphorylation is signaled through photobleaching of rhodopsin. Our findings suggest that phosphoinositide 3-kinase activation promotes PKM2 phosphorylation. Light and tyrosine phosphorylation appear to regulate PKM2 to provide a metabolic advantage to photoreceptor cells, thereby promoting cell survival.  

4.1654           Isolation of Platelet-Derived Extracellular Vesicles

Aatonen, M., Valkonen, S., Böing, A., Yuana, Y., Nieuwland, R. and Siljander, P. Methods in Mol. Biol., 1545, 177188 (2017)   Platelets participate in several physiological functions, including hemostasis, immunity, and development. Additionally, platelets play key roles in arterial thrombosis and cancer progression. Given this plethora of functions, there is a strong interest of the role of platelet-derived (extracellular) vesicles (PDEVs) as functional mediators and biomarkers. Moreover, the majority of the blood-borne EVs are thought to originate from either platelets or directly from the platelet precursor cells, the megakaryocytes, which reside in the bone marrow. To circumvent confusion, we use the term PDEVs for both platelet-derived and/or megakaryocyte-derived EVs. PDEVs can be isolated from blood or from isolated platelets after activation. In this chapter, we describe all commonly used PDEV isolation methods from blood and prepurified platelets.  

4.1655           Association of a novel GABRG2 splicing variation and a PTGS2/COX-2 single nucleotide polymorphism with Taiwanese febrile seizures

Hung, K-L., Liang, J-S., Wang, J-S., Chen, H-J., Lin, J-J. and Lu, J-F. Epilepsy Res., 129, 1-7 (2017)   Abstract: Febrile seizure (FS) is the most common type of convulsion in infants and young children. The occurrence of FS in a subset of children with febrile illness suggested genetic factors may have an important effect on the predisposition of the disease. Using targeted next generation sequencing (NGS), a novel splicing variation (NM_198903.2:c.1249-1G>T) was identified in the γ-aminobutyric acid type A (GABA-A) receptor γ2 subunit (GABRG2) gene of a FS patient. To investigate possible association of FS with single nucleotide polymorphisms (SNPs) in prostaglandin-endoperoxide synthase-2 (prostaglandin G/H synthase-2; PTGS2/cyclooxygenase-2; COX2) gene involving in thermoregulatory pathway, eight SNPs, rs689465, rs689466, rs20417, rs13306038, rs201931599, rs689470, rs4648306 and rs4648308, along with 2 previously reported variations in IL1RN (86-bp VNTR) and IL10 (rs1900872) were genotyped and utilized for case-control association studies on 35 FS and 31 non-FS controls. A single SNP (rs689466) localized at 5′-1192 of the PTGS2 gene exhibited significant association with FS (p = 0.045) based on case-control allelic association analyses. A significant decrease in the frequency of the G allele in FS (0.357) was observed compared to that in controls (0.536) with an estimated odds ratio (OR) of 0.48 (95% CI, 0.23–0.99) for the G versus A allele. Using case-control genotypic association analysis, the −1192 A allele is most likely to confer susceptibility to FS by a recessive action model (p = 0.045, pointwise empirical p value (EMP1) = 0.049). The association of SNPs in PTGS2, in addition to IL6, IL-6 receptor (IL6R) and prostaglandin E receptor 3 (PTGER3) in prior reports, with FS suggests their possible action in concert to modulate phenotypes in FS as well as the involvement of thermoregulatory pathway in pathogenesis of FS.  

4.1656           Reactive gamma-ketoaldehydes as novel activators of hepatic stellate cells in vitro

Longato, L., Andreola, F., Davies, S.S., Roberts, J.L., Fusai, G., Pinzani, M., Moore, K. and Rombouts, K. Free Radical Biol. Med., 102, 162-173 (2017)   Aims Products of lipid oxidation, such as 4-hydroxynonenal (4-HNE), are key activators of hepatic stellate cells (HSC) to a pro-fibrogenic phenotype. Isolevuglandins (IsoLG) are a family of acyclic γ-ketoaldehydes formed through oxidation of arachidonic acid or as by-products of the cyclooxygenase pathway. IsoLGs are highly reactive aldehydes which are efficient at forming protein adducts and cross-links at concentrations 100-fold lower than 4-hydroxynonenal. Since the contribution of IsoLGs to liver injury has not been studied, we synthesized 15-E₂-IsoLG and used it to investigate whether IsoLG could induce activation of HSC. Results Primary human HSC were exposed to 15-E₂-IsoLG for up to 48 h. Exposure to 5 μM 15-E₂-IsoLG in HSCs promoted cytotoxicity and apoptosis. At non-cytotoxic doses (50 pM-500 nM) 15-E₂-IsoLG promoted HSC activation, indicated by increased expression of α-SMA, sustained activation of ERK and JNK signaling pathways, and increased mRNA and/or protein expression of cytokines and chemokines, which was blocked by inhibitors of JNK and NF-kB. In addition, IsoLG promoted formation of reactive oxygen species, and induced an early activation of ER stress, followed by autophagy. Inhibition of autophagy partially reduced the pro-inflammatory effects of IsoLG, suggesting that it might serve as a cytoprotective response. Innovation This study is the first to describe the biological effects of IsoLG in primary HSC, the main drivers of hepatic fibrosis. Conclusions IsoLGs represent a newly identified class of activators of HSC in vitro, which are biologically active at concentrations as low as 500 pM, and are particularly effective at promoting a pro-inflammatory response and autophagy.  

4.1657           Identification of axon growth promoters in the secretome of the deer antler velvet

Pita-Thomas, W., Barroso-Garcia, G., Moral, V., Hackett, A.R., Cavalli, V. and Nieto-Diaz, M. Neuroscience, 340, 333-344 (2017)   Every spring, deer cast their old antlers and initiate a regeneration process, which yields a new set of antlers of up to 1 m in length. Over the course of three months, branches of the trigeminal nerve, originating from the frontal skull, innervate velvet, a modified skin that covers the regenerating antler. The rate of growth of these axons reaches up to 2 cm per day making them the fastest regenerating axons in adult mammals. Here, we aim to identify the factors secreted by velvet that promote such high speed axon growth. Our experiments with cultures of adult rat trigeminal neurons demonstrate that conditioned medium harvested from velvet organotypic cultures has greater axon growth-promoting properties than a medium conditioned by normal skin. The axon growth-promoting effects of velvet act synergistically with the extracellular matrix (ECM) protein laminin, a component of the basal lamina present in the deer antler. Our proteomic analyses identified several axon growth promoters in the velvet-conditioned medium (VCM), including soluble proteins such as nerve growth factor (NGF) and apolipoprotein A-1, as well as matrix extracellular proteins, such as periostin and SPARC. Additional in vitro analyses allowed us to determine that a synergic relationship between periostin and NGF may contribute to neurite growth-promoting effects of velvet secretome. A combinatorial approach using these factors may promote regeneration at high speeds in patients with peripheral neuropathies.  

4.1658           Death of Monocytes through Oxidative Burst of Macrophages and Neutrophils: Killing in Trans

Ponath, V. and Kaina, B. PloS One, 12(1), e0170347 (2017)   Monocytes and their descendants, macrophages, play a key role in the defence against pathogens. They also contribute to the pathogenesis of inflammatory diseases. Therefore, a mechanism maintaining a balance in the monocyte/macrophage population must be postulated. Our previous studies have shown that monocytes are impaired in DNA repair, rendering them vulnerable to genotoxic stress while monocyte-derived macrophages are DNA repair competent and genotoxic stress-resistant. Based on these findings, we hypothesized that monocytes can be selectively killed by reactive oxygen species (ROS) produced by activated macrophages. We also wished to know whether monocytes and macrophages are protected against their own ROS produced following activation. To this end, we studied the effect of the ROS burst on DNA integrity, cell death and differentiation potential of monocytes. We show that monocytes, but not macrophages, stimulated for ROS production by phorbol-12-myristate-13-acetate (PMA) undergo apoptosis, despite similar levels of initial DNA damage. Following co-cultivation with ROS producing macrophages, monocytes displayed oxidative DNA damage, accumulating DNA single-strand breaks and a high incidence of apoptosis, reducing their ability to give rise to new macrophages. Killing of monocytes by activated macrophages, termed killing in trans, was abolished by ROS scavenging and was also observed in monocytes co-cultivated with ROS producing activated granulocytes. The data revealed that monocytes, which are impaired in the repair of oxidised DNA lesions, are vulnerable to their own ROS and ROS produced by macrophages and granulocytes and support the hypothesis that this is a mechanism regulating the amount of monocytes and macrophages in a ROS-enriched inflammatory environment.  

4.1659           Steatosis induced CCL5 contributes to early-stage liver fibrosis in nonalcoholic fatty liver disease progress

Li, B-H., He, F-P., Yang, X., Chen, Y-W. and Fan, J-G. Translational Res., 180, 103-117 (2017)   The rapidly increasing prevalence of nonalcoholic fatty liver disease (NAFLD) has become one of the major public health threats in China and worldwide. However, during the development of NAFLD, the key mechanism underlying the progression of related fibrosis remains unclear, which greatly impedes the development of optimal NAFLD therapy. In the current study, we were endeavored to characterize a proinflammatory cytokine, CCL5, as a major contributor for fibrosis in NAFLD. The results showed that CCL5 was highly expressed in fatty liver and NASH patients. In NAFLD rats induced by 8-week-HFD, CCL5 and its receptor, CCR5, were significantly up-regulated and liver fibrosis exclusively occurred in this group. In addition, we showed that hepatocytes are the major source contributing to this CCL5 elevation. Interestingly, a CCL5 inhibitor Met-CCL5, significantly decreased liver fibrosis but not hepatic steatosis. Using a cell model of hepatic steatosis, we found that the conditioned medium of lipid-overloaded hepatocytes (Fa2N-4 cells) which produced excessive CCL5 stimulated the profibrotic activities of hepatic stellate cells (LX-2) as manifested by increased migration rate, proliferation and collagen production of LX-2 cells. CCL5 knockdown in Fa2N-4 cells, Met-CCL5 or CCR5 antibody treatment on LX-2 cells all significantly inhibited the conditioned medium of FFA-treated Fa2N-4 cells to exert stimulatory effects on LX-2 cells. Consistently, the conditioned medium of Fa2N-4 cells with CCL5 over-expression significantly enhanced migration rate, cell proliferation and collagen production of LX-2 cells. All these results support that CCL5 produced by steatotic hepatocytes plays an essential role in fibrotic signaling machinery of NAFLD. In addition, we were able to identify C/EBP-β as the up-stream regulator of CCL5 gene transcription in hepatocytes treated with free fatty acid (FFA). Our data strongly supported that CCL5 plays a pivotal regulatory role in hepatic fibrosis during NAFLD, which constitutes a novel and exciting observation that may call for potential future development of specific CCL5-targeted NAFLD therapy.  

4.1660           A Proof-of-Concept for Epigenetic Therapy of Tissue Fibrosis: Inhibition of Liver Fibrosis Progression by 3-Deazaneplanocin A

Zeybel, M. et al Molecular Therapy, 25(1), 218-231 (2017)   The progression of fibrosis in chronic liver disease is dependent upon hepatic stellate cells (HSCs) transdifferentiating to a myofibroblast-like phenotype. This pivotal process is controlled by enzymes that regulate histone methylation and chromatin structure, which may be targets for developing anti-fibrotics. There is limited pre-clinical experimental support for the potential to therapeutically manipulate epigenetic regulators in fibrosis. In order to learn if epigenetic treatment can halt the progression of pre-established liver fibrosis, we treated mice with the histone methyltransferase inhibitor 3-deazaneplanocin A (DZNep) in a naked form or by selectively targeting HSC-derived myofibroblasts via an antibody-liposome-DZNep targeting vehicle. We discovered that DZNep treatment inhibited multiple histone methylation modifications, indicative of a broader specificity than previously reported. This broad epigenetic repression was associated with the suppression of fibrosis progression as assessed both histologically and biochemically. The anti-fibrotic effect of DZNep was reproduced when the drug was selectively targeted to HSC-derived myofibroblasts. Therefore, the in vivo modulation of HSC histone methylation is sufficient to halt progression of fibrosis in the context of continuous liver damage. This discovery and our novel HSC-targeting vehicle, which avoids the unwanted effects of epigenetic drugs on parenchymal liver cells, represents an important proof-of-concept for epigenetic treatment of liver fibrosis.  

4.1661           Single-cell barcoding and sequencing using droplet microfluidics

Zilionis, R., Nainys, J., Veres, A., Savova, v., Zemmeour, D., Klein, A.M. and Mazautis, L. Nature Protocol., 12(1), 44-73 (2017)   Single-cell RNA sequencing has recently emerged as a powerful tool for mapping cellular heterogeneity in diseased and healthy tissues, yet high-throughput methods are needed for capturing the unbiased diversity of cells. Droplet microfluidics is among the most promising candidates for capturing and processing thousands of individual cells for whole-transcriptome or genomic analysis in a massively parallel manner with minimal reagent use. We recently established a method called inDrops, which has the capability to index >15,000 cells in an hour. A suspension of cells is first encapsulated into nanoliter droplets with hydrogel beads (HBs) bearing barcoding DNA primers. Cells are then lysed and mRNA is barcoded (indexed) by a reverse transcription (RT) reaction. Here we provide details for (i) establishing an inDrops platform (1 d); (ii) performing hydrogel bead synthesis (4 d); (iii) encapsulating and barcoding cells (1 d); and (iv) RNA-seq library preparation (2 d). inDrops is a robust and scalable platform, and it is unique in its ability to capture and profile >75% of cells in even very small samples, on a scale of thousands or tens of thousands of cells.    

4.1662           Sirtuin 2 aggravates postischemic liver injury by deacetylating mitogen-activated protein kinase phosphatase-1

Wang, J., Koh, H-W., Zhou, L., Bae, U-J., Lee, H-S., Bang, I.H., Ka, S-O., Oh, S-H., Bae, E.J. and Park, B-H. Hepatology, 65(1), 225-236 (2017)   Sirtuin 2 (Sirt2) is known to negatively regulate anoxia-reoxygenation injury in myoblasts. Because protein levels of Sirt2 are increased in ischemia-reperfusion (I/R)-injured liver tissues, we examined whether Sirt2 is protective or detrimental against hepatic I/R injury. We overexpressed Sirt2 in the liver of C57BL/6 mice using a Sirt2 adenovirus. Wild-type and Sirt2 knockout mice were subjected to a partial (70%) hepatic ischemia for 45 minutes, followed by various periods of reperfusion. In another set of experiments, wild-type mice were pretreated intraperitoneally with AGK2, a Sirt2 inhibitor. Isolated hepatocytes and Kupffer cells from wild-type and Sirt2 knockout mice were subjected to hypoxia-reoxygenation injury to determine the in vitro effects of Sirt2. Mice subjected to I/R injury showed typical patterns of hepatocellular damage. Prior injection with Sirt2 adenovirus aggravated liver injury, as demonstrated by increases in serum aminotransferases, prothrombin time, proinflammatory cytokines, hepatocellular necrosis and apoptosis, and neutrophil infiltration relative to control virus-injected mice. Pretreatment with AGK2 resulted in significant improvements in serum aminotransferase levels and histopathologic findings. Similarly, experiments with Sirt2 knockout mice also revealed reduced hepatocellular injury. The molecular mechanism of Sirt2's involvement in this aggravation of hepatic I/R injury includes the deacetylation and inhibition of mitogen-activated protein kinase phosphatase-1 and consequent activation of mitogen-activated protein kinases. Conclusion: Sirt2 is an aggravating factor during hepatic I/R injury.  

4.1663           Synaptopodin is regulated by aromatase activity

Fester, L., Zhou, L., Ossig, C., labitzke, J., Bläute, C., Bader, M., Vollmer, G., jarry, H. and Rune, G.M..

1. Neurochem., 140, 126-139 (2017)

  Locally synthesized estradiol plays an important role in synaptic plasticity in the hippocampus. We have previously shown that in hippocampal neurons, activity of the enzyme aromatase, which converts testosterone into estradiol, is reduced via Ca²⁺-dependent phosphorylation. Synaptopodin is a highly estrogen responsive protein, and it has been shown that it is an important regulator of synaptic plasticity, mediated by its close association with internal calcium stores. In this study, we show that the expression of synaptopodin is stronger in the hippocampus of female animals than in that of male animals. Phosphorylation of aromatase, using letrozole, however, down-regulates synaptopodin immunohistochemistry in the hippocampus of both male and females. Similarly, in aromatase knock-out mice synaptopodin expression in the hippocampus is reduced sex independently. Using primary-dissociated hippocampal neurons, we found that evoked release of Ca²⁺ from internal stores down-regulates aromatase activity, which is paralleled by reduced expression of synaptopodin. Opposite effects were achieved after inhibition of the release. Calcium-dependent regulation of synaptopodin expression was abolished when the control of aromatase activity by the Ca²⁺ transients was disrupted. Our data suggest that the regulation of aromatase activity by Ca²⁺ transients in neurons contributes to synaptic plasticity in the hippocampus of male and female animals as an on-site regulatory mechanism.  

4.1664           The upregulation of annexin A2 after spinal cord injury in rats may have implication for astrocyte proliferation

Chen, J., Cui, Z., yang, S., Wu, C., Li, W., Bao, G., Xu, G., Sun, Y., Wang, L. and Zhang, J. Neuropeptides, 61, 67-76 (2017)   Annexin A2 (ANXA2), is a member of the annexin family of proteins that exhibit Ca^2 +-dependent binding to phospholipids. One attractive biological function of ANXA2 is participating in DNA synthesis and cell proliferation. Previous studies have shown that ANXA2 play a role in the development of the central nervous system. However, the biological function of ANXA2 after spinal cord injury (SCI) is still with limited acquaintance. In the present study, we performed a SCI model in adult rats and investigated the dynamic changes of ANXA2 expression in the spinal cord. Western blot analysis indicated a striking expression upregulation of ANXA2 after SCI. Immunohistochemistry further confirmed that ANXA2 immunoactivity was expressed at low levels in normal condition and increased at 5 day after SCI. Double immunofluorescence staining prompted that ANXA2 immunoreactivity was found in astrocytes and neurons. Interestingly, ANXA2 expression was increased predominantly in astrocytes. We also examined the expression profiles of proliferating cell nuclear antigen (PCNA), Cyclin D1 and active caspase-3 in the injured spinal cords by western blot. Co-expression of ANXA2/PCNA, ANXA2/Cyclin D1 was detected in glial fibrillary acidic protein. Importantly, double immunofluorescence staining revealed that cell proliferation evaluated by PCNA appeared in many ANXA2-expressing cells and rare caspase-3 was observed in ANXA2-expressing cells after SCI. In addition, ANXA2 knockdown in astrocytes resulted in the increase of PCNA expression after LPS stimulation, showing that ANXA2 inhibited astrocyte proliferation after inflammation. Our data suggested that ANXA2 might play important roles in CNS pathophysiology after SCI.  

4.1665           Understanding quasi-apoptosis of the most numerous enucleated components of blood needs detailed molecular autopsy

Petrovich Gusev, G., Govekar, R., Gadewal, N. and Ivanovna Agalakova, N. Ageing Res. Reviews, 35, 46-62 (2017)   Erythrocytes are the most numerous cells in human body and their function of oxygen transport is pivotal to human physiology. However, being enucleated, they are often referred to as a sac of molecules and their cellularity is challenged. Interestingly, their programmed death stands a testimony to their cell-hood. They are capable of self-execution after a defined life span by both cell-specific mechanism and that resembling the cytoplasmic events in apoptosis of nucleated cells. Since the execution process lacks the nuclear and mitochondrial events in apoptosis, it has been referred to as quasi-apoptosis or eryptosis. Several studies on molecular mechanisms underlying death of erythrocytes have been reported. The data has generated a non-cohesive sketch of the process. The lacunae in the present knowledge need to be filled to gain deeper insight into the mechanism of physiological ageing and death of erythrocytes, as well as the effect of age of organism on RBCs survival. This would entail how the most numerous cells in the human body die and enable a better understanding of signaling mechanisms of their senescence and premature eryptosis observed in individuals of advanced age.  

4.1666           Platelets Inhibit Migration of Canine Osteosarcoma Cells

Bulla, S.C., Badial, P.R., Silva, R.C., Lunsford, K. and Bulla, C.

1. Comp. Path., 156, 3-13 (2017)

  The interaction between platelets and tumour cells is important for tumour growth and metastasis. Thrombocytopenia or antiplatelet treatment negatively impact on cancer metastasis, demonstrating potentially important roles for platelets in tumour progression. To our knowledge, there is no information regarding the role of platelets in cancer progression in dogs. This study was designed to test whether canine platelets affected the migratory behaviour of three canine osteosarcoma cell lines and to give insights of molecular mechanisms. Intact platelets, platelet lysate and platelet releasate inhibited the migration of canine osteosarcoma cell lines. Addition of blood leucocytes to the platelet samples did not alter the inhibitory effect on migration. Platelet treatment also significantly downregulated the transcriptional levels of SNAI2 and TWIST1 genes. The interaction between canine platelets or molecules released during platelet activation and these tumour cell lines inhibits their migration, which suggests that canine platelets might antagonize metastasis of canine osteosarcoma. This effect is probably due to, at least in part, downregulation of genes related to epithelial–mesenchymal transition.  

4.1667           Tumor progression locus 2 reduces severe allergic airway inflammation by inhibiting Ccl24 production in dendritic cells

Kannan, Y., Li, Y., Coomes, S.M., Okoye, I.S., Pelly, V.S., Sriskantharajah, S., Gückel, E., Webb, L., Czieso, S., Nikolov, N., MacDonald, A.S., Ley, S. and Wilson, M.S.

1. Allergy Clin. Immunol., 139(2), 655-666e7 (2017)

  Background The molecular and cellular pathways driving the pathogenesis of severe asthma are poorly defined. Tumor progression locus 2 (TPL-2) (COT, MAP3K8) kinase activates the MEK1/2-extracellular-signal regulated kinase 1/2 MAP kinase signaling pathway following Toll-like receptor, TNFR1, and IL-1R stimulation. Objective TPL-2 has been widely described as a critical regulator of inflammation, and we sought to investigate the role of TPL-2 in house dust mite (HDM)-mediated allergic airway inflammation. Methods A comparative analysis of wild-type and Map3k8−^/− mice was conducted. Mixed bone marrow chimeras, conditional knockout mice, and adoptive transfer models were also used. Differential cell counts were performed on the bronchoalveolar lavage fluid, followed by histological analysis of lung sections. Flow cytometry and quantitative PCR was used to measure type 2 cytokines. ELISA was used to assess the production of IgE, type 2 cytokines, and Ccl24. RNA sequencing was used to characterize dendritic cell (DC) transcripts. Results TPL-2 deficiency led to exacerbated HDM-induced airway allergy, with increased airway and tissue eosinophilia, lung inflammation, and IL-4, IL-5, IL-13, and IgE production. Increased airway allergic responses in Map3k8−^/− mice were not due to a cell-intrinsic role for TPL-2 in T cells, B cells, or LysM⁺ cells but due to a regulatory role for TPL-2 in DCs. TPL-2 inhibited Ccl24 expression in lung DCs, and blockade of Ccl24 prevented the exaggerated airway eosinophilia and lung inflammation in mice given HDM-pulsed Map3k8−^/− DCs. Conclusions TPL-2 regulates DC-derived Ccl24 production to prevent severe type 2 airway allergy in mice.  

4.1668           489 – Modulation of platelet levels by an anti-IL-1α antibody (MABp1) in advanced colorectal cancer patients

Hickish, T., Mohanty, P., Shivaswamy, M.S., Sunley, K., Varshney, A., Martin, R. and Simard, J. Eur. J. Cancer, 72, Suppl. 1, S70 (2017)   Background: In a Phase III study, treatment with MABp1, an anti-IL-1a antibody, has demonstrated 76% relative increase in clinical response rate versus placebo in end-stage colorectal cancer patients. In addition to the primary end point (improved health status as measured by lean body mass and pain, fatigue and appetite), secondary measures included monitoring of pharmacodynamic parameters such as serum IL-6 levels and platelet counts. With respect to these secondary endpoints, patient receiving MABp1 treatment showed decreased serum IL6 levels as well as platelet counts. IL-1a is known to upregulate IL-6, a known inducer of megakaryocytopoesis. The reduction in IL-6 and platelet counts in patients treated with IL-1a suggests that platelet-derived IL-1a may be both a target of antibody therapy and play an important role in regulation of megakaryocytopoiesis. While the role of platelet-derived IL-1a has been established in animal models for vascular endothelial cell activation and the pathogenesis of cerebrovascular inflammation, few studies have even confirmed the expression of IL-1a on human platelets. Here we present findings to confirm theexpression of platelet IL-1a on platelets within a healthy human population. Material and Methods: Platelets from human blood were isolated on a discontinuous iodixanol density gradient. Platelets were stained with MABp1 or isotype control before and after activation with thrombin and lipopolysaccharide, and observed by confocal microscopy and flow cytometry. In addition, the isolated platelets were lysed and the membranes were isolated by ultracentrifugation. The IL-1a on the membrane was immunoprecipitated using a pro-IL-1a antibody. The membrane proteins were resolved on a SDS-PAGE and human IL-1a was detected using various antibodies. The protein was digested with trypsin, and the isolatedpeptides were subjected to peptide mass fingerprinting. Results: Our work has confirmed the presence of IL-1a on the surface of platelets. Confocal microscopy and Flow Cytometry has shown an activation dependent increase in membrane IL-1a levels. Western blotting and immunoprecipitation confirmed that the IL-1a is present on the membrane in its propeptide form. In addition, the propeptide is sensitive to cleavage into its mature form by calpain-like protease also present on the surface of platelets. Peptide mass fingerprinting of the immunoprecipitant using a QTof Micro Mass Spectrometer identified five unique peptides specific to human IL-1a at high confidence levels. Conclusions: Based on our findings and results from previous studies, platelets appear to play an important role in the development of important conditions, including cancer. We confirm that IL-1a is present on human platelets and that this may represent an important factor in regulatingplatelet thrombocytosis and consequent pathology in cancer.  

4.1669           Doxycycline attenuates breast cancer related inflammation by decreasing plasma lysophosphatidate concentrations and inhibiting NF-κB activation

Tang, X., Wang, X., Zhao, Y.Y., Curtis, J.M. and Brindley, D.N. Mol. Cancer, 16:36 (2017)   Background We previously discovered that tetracyclines increase the expression of lipid phosphate phosphatases at the surface of cells. These enzymes degrade circulating lysophosphatidate and therefore doxycycline increases the turnover of plasma lysophosphatidate and decreases its concentration. Extracellular lysophosphatidate signals through six G protein-coupled receptors and it is a potent promoter of tumor growth, metastasis and chemo-resistance. These effects depend partly on the stimulation of inflammation that lysophosphatidate produces. Methods In this work, we used a syngeneic orthotopic mouse model of breast cancer to determine the impact of doxycycline on circulating lysophosphatidate concentrations and tumor growth. Cytokine/chemokine concentrations in tumor tissue and plasma were measured by multiplexing laser bead technology. Leukocyte infiltration in tumors was analyzed by immunohistochemistry. The expression of IL-6 in breast cancer cell lines was determined by RT-PCR. Cell growth was measured in Matrigel™ 3D culture. The effects of doxycycline on NF-κB-dependent signaling were analyzed by Western blotting. Results Doxycycline decreased plasma lysophosphatidate concentrations, delayed tumor growth and decreased the concentrations of several cytokines/chemokines (IL-1β, IL-6, IL-9, CCL2, CCL11, CXCL1, CXCL2, CXCL9, G-CSF, LIF, VEGF) in the tumor. These results were compatible with the effects of doxycycline in decreasing the numbers of F4/80⁺ macrophages and CD31⁺ blood vessel endothelial cells in the tumor. Doxycycline also decreased the lysophosphatidate-induced growth of breast cancer cells in three-dimensional culture. Lysophosphatidate-induced Ki-67 expression was inhibited by doxycycline. NF-κB activity in HEK293 cells transiently expressing a NF-κB-luciferase reporter vectors was also inhibited by doxycycline. Treatment of breast cancer cells with doxycycline also decreased the translocation of NF-κB to the nucleus and the mRNA levels for IL-6 in the presence or absence of lysophosphatidate. Conclusion These results contribute a new dimension for understanding the anti-inflammatory effects of tetracyclines, which make them potential candidates for adjuvant therapy of cancers and other inflammatory diseases.  

4.1670           Selective Osmotic Shock (SOS)-Based Islet Isolation for Microencapsulation

Enck, K., McQuilling, J.P., Orlando, G., Tamburrini, R., Sivanandane, S. and Opara, E.C. Methods in Mol. Biol., 1479, 191-198 (2017)   Islet transplantation (IT) has recently been shown to be a promising alternative to pancreas transplantation for reversing diabetes. IT requires the isolation of the islets from the pancreas, and these islets can be used to fabricate a bio-artificial pancreas. Enzymatic digestion is the current gold standard procedure for islet isolation but has lingering concerns. One such concern is that it has been shown to damage the islets due to nonselective tissue digestion. This chapter provides a detailed description of a nonenzymatic method that we are exploring in our lab as an alternative to current enzymatic digestion procedures for islet isolation from human and nonhuman pancreatic tissues. This method is based on selective destruction and protection of specific cell types and has been shown to leave the extracellular matrix (ECM) of islets intact, which may thus enhance islet viability and functionality. We also show that these SOS-isolated islets can be microencapsulated for transplantation.  

4.1671           Neutrophil adhesion and crawling dynamics on liver sinusoidal endothelial cells under shear flow

Yang, H., Li, N., Du, Y., Tong, C., Lü, S., Hu, J., Zhang, Y. and Long, M. Exp. Cell Res., 351, 91-00 (2017)   Neutrophil (polymorphonuclear leukocyte, PMN) recruitment in the liver sinusoid takes place in almost all liver diseases and contributes to pathogen clearance or tissue damage. While PMN rolling unlikely appears in liver sinusoids and Mac-1 or CD44 is assumed to play respective roles during in vivo local or systematic inflammatory stimulation, the regulating mechanisms of PMN adhesion and crawling dynamics are still unclear from those in vivo studies. Here we developed a two-dimensional in vitro sinusoidal model with primary liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) to investigate TNF-α-induced PMN recruitment under shear flow. Our data demonstrated that LFA-1 dominates the static or shear resistant adhesion of PMNs while Mac-1 decelerates PMN crawling on LSEC monolayer. Any one of LFA-1, Mac-1, and CD44 molecules is not able to work effectively for mediating PMN transmigration across LSEC monolayer. The presence of KCs only affects the randomness of PMN crawling. These findings further the understandings of PMN recruitment under shear flow in liver sinusoids.  

4.1672           Zinc finger protein ZPR9 functions as an activator of AMPK-related serine/threonine kinase MPK38/MELK involved in ASK1/TGF-β/p53 signaling pathways

Seong, H-A., Manoharan, R. and Ha, H. Scientific Reports, 7:42502 (2017)   Murine protein serine-threonine kinase 38 (MPK38), an AMP‐activated protein kinase (AMPK)-related kinase, has been implicated in the induction of apoptosis signal-regulating kinase 1 (ASK1)-, transforming growth factor-β (TGF‐β)-, and p53-mediated activity involved in metabolic homeostasis. Here, zinc finger protein ZPR9 was found to be an activator of MPK38. The association of MPK38 and ZPR9 was mediated by cysteine residues present in each of these two proteins, Cys269 and Cys286 of MPK38 and Cys305 and Cys308 of ZPR9. MPK38 phosphorylated ZPR9 at Thr252. Wild‐type ZPR9, but not the ZPR9 mutant T252A, enhanced ASK1, TGF‐β, and p53 function by stabilizing MPK38. The requirement of ZPR9 Thr252 phosphorylation was validated using CRISPR/Cas9-mediated ZPR9 (T252A) knockin cell lines. The knockdown of endogenous ZPR9 showed an opposite trend, resulting in the inhibition of MPK38‐dependent ASK1, TGF‐β, and p53 function. This effect was also demonstrated in mouse embryonic fibroblast (MEF) cells that were haploinsufficient (+/−) for ZPR9, NIH 3T3 cells with inducible knockdown of ZPR9, and CRISPR/Cas9-mediated ZPR9 knockout cells. Furthermore, high-fat diet (HFD)-fed mice displayed reduced MPK38 kinase activity and ZPR9 expression compared to that in mice on control chow, suggesting that ZPR9 acts as a physiological activator of MPK38 that may participate in obesity.  

4.1673           PARP inhibition protects against alcoholic and non-alcoholic steatohepatitis

Mukhopadhyay, P. et al

1. Hepatol., 66, 589-600 (2017)

  Background & Aims Mitochondrial dysfunction, oxidative stress, inflammation, and metabolic reprograming are crucial contributors to hepatic injury and subsequent liver fibrosis. Poly(ADP-ribose) polymerases (PARP) and their interactions with sirtuins play an important role in regulating intermediary metabolism in this process. However, there is little research into whether PARP inhibition affects alcoholic and non-alcoholic steatohepatitis (ASH/NASH). Methods We investigated the effects of genetic deletion of PARP1 and pharmacological inhibition of PARP in models of early alcoholic steatohepatitis, as well as on Kupffer cell activation in vitro using biochemical assays, real-time PCR, and histological analyses. The effects of PARP inhibition were also evaluated in high fat or methionine and choline deficient diet-induced steatohepatitis models in mice. Results PARP activity was increased in livers due to excessive alcohol intake, which was associated with decreased NAD⁺ content and SIRT1 activity. Pharmacological inhibition of PARP restored the hepatic NAD⁺ content, attenuated the decrease in SIRT1 activation and beneficially affected the metabolic-, inflammatory-, and oxidative stress-related alterations due to alcohol feeding in the liver. PARP1^−/− animals were protected against alcoholic steatohepatitis and pharmacological inhibition of PARP or genetic deletion of PARP1 also attenuated Kupffer cell activation in vitro. Furthermore, PARP inhibition decreased hepatic triglyceride accumulation, metabolic dysregulation, or inflammation and/or fibrosis in models of NASH. Conclusion Our results suggests that PARP inhibition is a promising therapeutic strategy in steatohepatitis with high translational potential, considering the availability of PARP inhibitors for clinical treatment of cancer.  

4.1674           One step fabrication of hydrogel microcapsules with hollow core for assembly and cultivation of hepatocyte spheroids

Siltanen, C., Diakataou, M., Lowen, J., Haque, A., Rahimian, A., Stybayeva, G. and Revzin, A. Acta Biomaterialia, 50, 428-436 (2017)   3D hepatic microtissues can serve as valuable liver analogues for cell-based therapies and for hepatotoxicity screening during preclinical drug development. However, hepatocytes rapidly dedifferentiate in vitro, and typically require 3D culture systems or co-cultures for phenotype rescue. In this work we present a novel microencapsulation strategy, utilizing coaxial flow-focusing droplet microfluidics to fabricate microcapsules with liquid core and poly(ethylene glycol) (PEG) gel shell. When entrapped inside these capsules, primary hepatocytes rapidly formed cell-cell contacts and assembled into compact spheroids. High levels of hepatic function were maintained inside the capsules for over ten days. The microencapsulation approach described here is compatible with difficult-to-culture primary epithelial cells, allows for tuning gel mechanical properties and diffusivity, and may be used in the future for high density suspension cell cultures.    

4.1675           Laquinimod enhances central nervous system barrier functions

Lühder, F., Kebir, H., Odoardi, F., Litke, T., Sonneck, M., Alvarez, J.I., Winchenbrch, J., Eckert, N., Hayardeny, L., Sorani, E., Lodygin, D., Flügel, A. and Prat, A. Neurobiology of Disease, 102, 60-69 (2017)   Laquinimod is currently being tested as a therapeutic drug in multiple sclerosis. However, its exact mechanism of action is still under investigation. Tracking of fluorescently-tagged encephalitogenic T cells during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, revealed that laquinimod significantly reduces the invasion of pathogenic effector T cells into the CNS tissue. T-cell activation, differentiation and amplification within secondary lymphoid organs after immunization with myelin antigen, their migratory capacity and re-activation within the nervous tissue were either only mildly affected or remained unchanged. Instead, laquinimod directly impacted the functionality of the CNS vasculature. The expression of tight junction proteins p120 and ZO-1 in human brain endothelial cells was up-regulated upon laquinimod treatment, resulting in a significant increase in the transendothelial electrical resistance of confluent monolayers of brain endothelial cells. Similarly, expression of the adhesion molecule activated leukocyte cell adhesion molecule (ALCAM) and inflammatory chemokines CCL2 and IP-10 was suppressed, leading to a significant reduction in the migration of memory T_H1 and T_H17 lymphocytes across the blood brain barrier (BBB). Our data indicate that laquinimod exerts its therapeutic effects by tightening the BBB and limiting parenchymal invasion of effector T cells, thereby reducing CNS damage.  

4.1676           The cryoprotective effect of iodixanol in buffalo semen cryopreservation

Singh Swami, D., Kumar, P., Malik, R.K., Saini, M., Kumar, d. and Jan, M.H. Animal Reprod. Sci., 179, 20-26 (2017)   This is the first report to examine the effect of iodixanol (OptiPrep^TM) on cryosurvival of buffalo spermatozoa. A total of thirty ejaculates (five ejaculates from each bull) from six buffalo bulls were used for this experiment. Each ejaculate was divided into four aliquots and diluted in freezing extender supplemented with different concentrations of OptiPrep^TM (0, 1.25, 2.5 and 5%) and then cryopreserved. The semen quality variables were evaluated before and after freezing of the semen. There were no effects of OptiPrep^TM (P > 0.05) on sperm kinetics, motility, abnormality and membrane integrity of fresh extended spermatozoa. However, after freeze-thaw, sperm motility, plasma membrane integrity and distance travelled in cervical mucus of 2.5% OptiPrep^TM treated samples showed significantly higher (P < 0.05) compared to other treated and control samples. No significant differences (P > 0.05) were seen in sperm abnormality and acrosomal integrity of treated and control frozen-thawed samples. The total antioxidant capacity of 2.5 and 5% OptiPrep^TM treated frozen-thawed sperm were found to be higher (P < 0.05) as compared to other groups; whereas the MDA level in OptiPrep^TM treated sperm was significantly lower than the control (P < 0.05). In incubation test, 2.5% OptiPrep^TM proved to be better in preservation of sperm motility as compared to other treated and control samples. In conclusion, the present study has shown that iodixanol has the ability protect spermatozoa against oxidative stress and resulting overall improvement in the post-thaw semen quality.  

4.1677           Identification of novel autoantigens via mass spectroscopy-based antibody-mediated identification of autoantigens (MS-AMIDA) using immune thrombocytopenic purpura (ITP) as a model disease

Kamhieh-Milz, J., Sterzer, V., Celik, H., Khorramshahi, O., Hassan Noftah, R.F. and Salama, A.

1. Proteomics, 157, 59-70 (2017)

  Immune thrombocytopenic purpura (ITP) is one of the best characterized autoimmune diseases. Autoantibodies (AABs) against platelet antigens are considered as the diagnostic hallmark of ITP, but are detectable in only 50% of patients. We designed and applied a novel proteomic approach termed Mass Spectroscopy-based Antibody-Mediated Identification of Autoantigens (MS-AMIDA) for platelet antigens. Patients were separated into patients with classical AABs [ITP(+)] and patients without AABs [ITP(−)]. Altogether, 181 potential AAGs were found in ITP(+) and 135 AAGs in ITP(−), with 34 and 23 AAGs reproducibly found in two runs of MS-AMIDA. After subtracting identifiers from the controls, 57 AAGs in ITP(+) and 29 AAGs in ITP(+) remained, with 16 AAGs commonly found in ITP(+) and ITP(−) patients. Label-free quantification (LFQ) revealed 15 potential AAGs that are quantitatively stronger in ITP. Dot blot validation was performed on hexokinase 1 (HK1), E1 pyruvate dehydrogenase (E1-PDH), coagulation factor XIII, filamin A (FLNA), non-muscle myosin 9. Eleven patients were found to have anti-HK1 AABs, one patient had anti-E1-PDH AABs, and two patients had anti-FLNA AABs. Most antigens were of intracellular origin with significant association with actin-cytoskeleton and regulation of programmed cell death. In conclusion, novel AAGs for ITP were identified using MS-AMIDA.  

4.1678           The redox environment triggers conformational changes and aggregation of hIAPP in Type II Diabetes

Rodriguez Camargo, D.C. et al Scientific Reports, 7:44041 (2017)   Type II diabetes (T2D) is characterized by diminished insulin production and resistance of cells to insulin. Among others, endoplasmic reticulum (ER) stress is a principal factor contributing to T2D and induces a shift towards a more reducing cellular environment. At the same time, peripheral insulin resistance triggers the over-production of regulatory hormones such as insulin and human islet amyloid polypeptide (hIAPP). We show that the differential aggregation of reduced and oxidized hIAPP assists to maintain the redox equilibrium by restoring redox equivalents. Aggregation thus induces redox balancing which can assist initially to counteract ER stress. Failure of the protein degradation machinery might finally result in β-cell disruption and cell death. We further present a structural characterization of hIAPP in solution, demonstrating that the N-terminus of the oxidized peptide has a high propensity to form an α-helical structure which is lacking in the reduced state of hIAPP. In healthy cells, this residual structure prevents the conversion into amyloidogenic aggregates.  

4.1679           Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

Shahi, P., Kim, S.C., Haliburton, J.R., Gartner, Z.J. and Abate, A.R. Scientific Reports, 7:44447 (2017)   Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.  

4.1680           An efficient method for isolation of representative and contamination-free population of blood platelets for proteomic studies

Wrzyszcz, A., Urbaniak, J., Sapa, A. and Wozniak, M. Platelets, 28(1), 43-53 (2017)   To date, there has been no ideal method for blood platelet isolation which allows one to obtain a preparation devoid of contaminations, reflecting the activation status and morphological features of circulating platelets. To address these requirements, we have developed a method which combines the continuous density gradient centrifugation with washing from PGI₂-supplemented platelet-rich plasma (PRP). We have assessed the degree of erythrocyte and leukocyte contamination, recovery of platelets, morphological features, activation status, and reactivity of isolated platelets. Using our protocol, we were able to get a preparation free from contaminations, representing well the platelet population prior to the isolation in terms of size and activity. Besides this, we have obtained approximately 2 times more platelets from the same volume of blood compared to the most widely used method. From 10 ml of whole citrated blood we were able to get on average 2.7 mg of platelet-derived protein. The method of platelet isolation presented in this paper can be successfully applied to tests requiring very pure platelets, reflecting the circulating platelet state, from a small volume of blood.  

4.1681           Deterministic encapsulation of single cells in thin tunable microgels for niche modelling and therapeutic delivery

Mao, A.S., Shin, J-W., utech, S., Wang, H., Uzun, O., Li, W., Cooper, M., Hu, Y., Zhang, L., Weitz, D.A. and Mooney, D.J. Nature Materials, 16(2), 236-243 (2017)   Existing techniques to encapsulate cells into microscale hydrogels generally yield high polymer-to-cell ratios and lack control over the hydrogel’s mechanical properties1. Here, we report a microfluidic-based method for encapsulating single cells in an approximately six-micrometre layer of alginate that increases the proportion of cell-containing microgels by a factor of ten, with encapsulation efficiencies over 90%. We show that in vitro cell viability was maintained over a three-day period, that the microgels are mechanically tractable, and that, for microscale cell assemblages of encapsulated marrow stromal cells cultured in microwells, osteogenic differentiation of encapsulated cells depends on gel stiffness and cell density. We also show that intravenous injection of singly encapsulated marrow stromal cells into mice delays clearance kinetics and sustains donor-derived soluble factors in vivo. The encapsulation of single cells in tunable hydrogels should find use in a variety of tissue engineering and regenerative medicine applications.  

4.1682           Age-Related Expression of a Repeat-Rich Intergenic Long Noncoding RNA in the Rat Brain

Kour, S. and Rath, P.C. Mol. Neurobiol., 54, 639-660 (2017)   Genome-wide transcriptome analysis has shown that ∼90 % of the mammalian genome undergoes pervasive transcription into various small and long noncoding RNAs with diverse biological functions and only ∼1.5 % is protein coding. Recent literature suggests that various structurally diverse sense and antisense long noncoding RNAs (lncRNAs) (>200 nt) are expressed from the intronic, intergenic and repeat-rich regions in the mammalian central nervous system (CNS). Till date, many of them have been found to be regulated in developmental, spatio-temporal and cell type-specific manners and are involved in various neurological processes. However, still much is left to be understood regarding their functional relevance in mammalian brain development, maturation and ageing. Furthermore, various signalling factors and metabolites such as all-trans retinoic acid (atRA) have been known to regulate brain functions during development, though their role in adult brain function is much less known. Here, we report differential and age-related expression of a novel repeat sequence-rich, long intergenic nonprotein coding RNA (lincRNA), named as LINC-RSAS (repeat-rich sense-antisense transcript) in different neuroanatomical regions of the rat brain. The LINC-RSAS was found to be moderately conserved and contained regulatory elements of various cell growth- and development-specific transcriptional factors in its up/downstream flanking sequences in the genome. Through RNA expression by reverse transcription polymerase chain reaction (RT-PCR) and localization by in situ RNA hybridization, we found that both sense and antisense transcripts of LINC-RSAS were expressed in the cortex, hippocampus and cerebellum regions of the rat brain in cell type-specific and age-related manner. Furthermore, both the expression level and subcellular localization of the antisense LINC-RSAS transcript were significantly induced in the cultured primary hippocampal neurons after treatment with atRA. Overall, our study provides insights into the possible involvement of an atRA-inducible, intergenic lncRNA in different functional regions of mammalian brain and its association with brain maturation and ageing.  

4.1683           Microfluidic fluorescence-activated cell sorting (μFACS) chip with integrated piezoelectric actuators for low-cost mammalian cell enrichment

Cheng, Z., Wu, X., Cheng, J. and Liu, P. Microfluid. Nanofluid., 21:9 (2017)   A low-cost, microfluidic fluorescence-activated cell sorting (μFACS) microchip integrated with two piezoelectric lead–zirconate–titanate actuators was demonstrated for automated, high-performance mammalian cell analysis and enrichment. In this PDMS–glass device, cells were hydrodynamically focused into a single file line in the lateral direction by two sheath flows, and then interrogated with a forward scattering and confocal fluorescent detection system. The selected cells were displaced transversely into a collection channel by two piezoelectric actuators that worked in a pull–push relay manner with a minimal switching time of ~0.8 ms. High detection throughput (~2500 cells/s), high sorting rate (~1250 cells/s), and high sorting efficiency (~98%) were successfully achieved on the μFACS system. Six cell mixture samples containing 22.87% of GFP-expressing HeLa cells were consecutively analyzed and sorted on the chip, revealing a stable sorting efficiency of 97.7 ± 0.93%. In addition, cell mixtures containing 37.65 and 3.36% GFP HeLa cells were effectively enriched up to 83.82 and 78.51%, respectively, on the microchip, and an enrichment factor of 105 for the low-purity (3.36%) sample was successfully obtained. This fully enclosed, disposable microfluidic chip provides an automated platform for low-cost fluorescence-based cell detection and enrichment, and is attractive to applications where cross-contamination between runs and aerosol hazard are the primary concerns.  

4.1684           Rapid Encapsulation of Cell and Polymer Solutions with Bubble-Triggered Droplet Generation

Yan, Z., Clark, I.C. and Abate, A.R. Macromolecul. Chem. Physics, 218(2), 1600297 (2017)   The generation of monodisperse droplets with microfluidics is valuable for applications ranging from material science to single cell analysis. However, conventional methods for forming droplets are limited in throughput, particularly when the fluids have low interfacial tension or high viscosity, like biological or polymer fluids. Rapid emulsification of biological and polymer fluids using bubble-triggered droplet generation is demonstrated. In addition to making droplets over tenfold faster than conventional drop makers with equivalent monodispersity, bubble-triggering can form droplets smaller than the nozzle, allowing droplets of the desired size to be generated in large channels that are robust against clogging.  

4.1685           Single cell-laden protease-sensitive microniches for long-term culture in 3D

Lienemann, P.S., Rossow, T., Mao, A.S., Vallmajo-Martin, Q., Ehrbar, M. and Mooney, D.J. Lab on a Chip, 17, 727-737 (2017)   Single cell-laden three-dimensional (3D) microgels that can serve to mimic stem cell niches in vitro, and are therefore termed microniches, can be efficiently fabricated by droplet-based microfluidics. In this technique an aqueous polymer solution along with a highly diluted cell solution is injected into a microfluidic device to create monodisperse pre-microgel droplets that are then solidified by a polymer crosslinking reaction to obtain monodisperse single cell-laden microniches. However, problems limiting this approach studying the fate of single cells include Poisson encapsulation statistics that result in mostly empty microniches, and cells egressing from the microniches during subsequent cell culture. Here, we present a strategy to bypass Poisson encapsulation statistics in synthetic microniches by selective crosslinking of only cell-laden pre-microgel droplets. Furthermore, we show that we can position cells in the center of the microniches, and that even in protease-sensitive microniches this greatly reduces cell egress. Collectively, we present the development of a versatile protocol that allows for unprecedented efficiency in creation of synthetic protease-sensitive microniches for probing single stem cell fate in 3D.  

4.1686           Thymic Dendritic Cell Subsets Display Distinct Efficiencies and Mechanisms of Intercellular MHC Transfer

Kroger, C.J., Spidale, N.A., Wang, B. and Tisch, R.

1. Immunol., 198(1), 249-256 (2017)

  Thymic dendritic cells (DC) delete self-antigen–specific thymocytes, and drive development of Foxp3-expressing immunoregulatory T cells. Unlike medullary thymic epithelial cells, which express and present peripheral self-antigen, DC must acquire self-antigen to mediate thymic negative selection. One such mechanism entails the transfer of surface MHC–self peptide complexes from medullary thymic epithelial cells to thymic DC. Despite the importance of thymic DC cross-dressing in negative selection, the factors that regulate the process and the capacity of different thymic DC subsets to acquire MHC and stimulate thymocytes are poorly understood. In this study intercellular MHC transfer by thymic DC subsets was investigated using an MHC-mismatch–based in vitro system. Thymic conventional DC (cDC) subsets signal regulatory protein α (SIRPα⁺) and CD8α⁺ readily acquired MHC class I and II from thymic epithelial cells but plasmacytoid DC were less efficient. Intercellular MHC transfer was donor-cell specific; thymic DC readily acquired MHC from TEC plus thymic or splenic DC, whereas thymic or splenic B cells were poor donors. Furthermore DC origin influenced cross-dressing; thymic versus splenic DC exhibited an increased capacity to capture TEC-derived MHC, which correlated with direct expression of EpCAM by DC. Despite similar capacities to acquire MHC–peptide complexes, thymic CD8α⁺ cDC elicited increased T cell stimulation relative to SIRPα⁺ cDC. DC cross-dressing was cell-contact dependent and unaffected by lipid raft disruption of donor TEC. Furthermore, blocking PI3K signaling reduced MHC acquisition by thymic CD8α⁺ cDC and plasmacytoid DC but not SIRPα⁺ cDC. These findings demonstrate that multiple parameters influence the efficiency of and distinct mechanisms drive intercellular MHC transfer by thymic DC subsets.  

4.1687           Extracellular vesicles released by hepatocytes from gastric infusion model of alcoholic liver disease contain a MicroRNA barcode that can be detected in blood

Eguchi, A., Lazaro, R.G., Wang, J., Kim, J., Povero, D., Williams, B., Ho, S.B., Stärkel, P., Schnabl, B., Ohno-Machado, L., Tsukamoto, H. and Feldstein, A.E. Hepatology, 65(2), 475-490 (2017)   Extracellular vesicles (EVs) released during cell stress, or demise, can contain a barcode of the cell origin, including specific microRNAs (miRNAs). Here, we tested the hypothesis that during early alcoholic steatohepatitis (ASH) development, hepatocytes (HCs) release EVs with an miRNA signature that can be measured in circulation. A time-course experiment showed that after 2 weeks of intragastric infusion, a time point that results in isolated steatosis, there was no increase of blood EVs. After 4 weeks of infusion, mice developed features of early ASH accompanied by a marked increase in the level of EVs in blood (P < 0.05), as well as in culture media of isolated HCs (P < 0.001) and hepatic macrophages (P < 0.001), with HCs being the predominant source of EVs. The transcriptome analysis of HC-EVs from ASH mice detected differentially expressed miRNAs, including nine significantly up-regulated and four significantly down-regulated miRNAs. Target prediction and pathway analyses of the up-regulated miRNAs identified 121 potential target genes involved in inflammatory and cancer pathways, such as nuclear factor kappa B, EGF, Wnt, and B-cell lymphoma 2. Three miRNAs, let7f, miR-29a, and miR-340, were increased in blood EVs from ASH mice (P < 0.05), but not in blood EVs from three other models of chronic liver injury, including bile duct ligation, nonalcoholic steatohepatitis, and obese mice, as well as EVs released from hepatocytes exposed to ethanol. Blood EV level (P < 0.01) and three miRNAs (P < 0.05) were significantly increased in patients with ambulatory mild ALD as compared to nonalcoholics. Conclusion: Damaged hepatocytes from ASH mice are a key EV source with a specific miRNA cargo, which are specific for ASH-related liver injury. These findings uncover EVs as a potentially novel diagnostic for ASH.  

4.1688           Wildtype motoneurons, ALS-Linked SOD1 mutation and glutamate profoundly modify astrocyte metabolism and lactate shuttling

Hounoum, B.M., mavel, S., Coque, E., patin, F., Vourc’h, P., marouillat, S., nadaal-Desbarats, L., Emond, P., Corcia, P., Andres, C.R., Raoul, C. and Blasco, H. Glia, 65(4), 592-605 (2017)   The selective degeneration of motoneuron that typifies amyotrophic lateral sclerosis (ALS) implicates non-cell-autonomous effects of astrocytes. However, mechanisms underlying astrocyte-mediated neurotoxicity remain largely unknown. According to the determinant role of astrocyte metabolism in supporting neuronal function, we propose to explore the metabolic status of astrocytes exposed to ALS-associated conditions. We found a significant metabolic dysregulation including purine, pyrimidine, lysine, and glycerophospholipid metabolism pathways in astrocytes expressing an ALS-causing mutated superoxide dismutase-1 (SOD1) when co-cultured with motoneurons. SOD1 astrocytes exposed to glutamate revealed a significant modification of the astrocyte metabolic fingerprint. More importantly, we observed that SOD1 mutation and glutamate impact the cellular shuttling of lactate between astrocytes and motoneurons with a decreased in extra- and intra-cellular lactate levels in astrocytes. Based on the emergent strategy of metabolomics, this work provides novel insight for understanding metabolic dysfunction of astrocytes in ALS conditions and opens the perspective of therapeutics targets through focusing on these metabolic pathways.  

4.1689           Droplet size influences division of mammalian cell factories in droplet microfluidic cultivation

Rajaeswari, P.K.P., Joensson, H.N. and Andersson-Svahn, H. Electrophoresis, 38(2), 305-310 (2017)   The potential of using droplet microfluidics for screening mammalian cell factories has been limited by the difficulty in achieving continuous cell division during cultivation in droplets. Here, we report the influence of droplet size on mammalian cell division and viability during cultivation in droplets. Chinese Hamster Ovary (CHO) cells, the most widely used mammalian host cells for biopharmaceuticals production were encapsulated and cultivated in 33, 180 and 320 pL droplets for 3 days. Periodic monitoring of the droplets during incubation showed that the cell divisions in 33 pL droplets stopped after 24 h, whereas continuous cell division was observed in 180 and 320 pL droplets for 72 h. The viability of the cells cultivated in the 33 pL droplets also dropped to about 50% in 72 h. In contrast, the viability of the cells in the larger droplets was above 90% even after 72 h of cultivation, making them a more suitable droplet size for 72-h cultivation. This study shows a direct correlation of microfluidic droplet size to the division and viability of mammalian cells. This highlights the importance of selecting suitable droplet size for mammalian cell factory screening assays.    

4.1690           Upregulation of hydroxysteroid sulfotransferase 2B1b promotes hepatic oval cell proliferation by modulating oxysterol-induced LXR activation in a mouse model of liver injury

Wang, Z., yang, X., Chen, L., Zhi, X., Lu, H., Ning, Y., Yeong, J., Chen, S., Yin, L., Wang, X. and Li, X. Arch. Toxicol., 91, 271-287 (2017)   Hydroxysteroid sulfotransferase 2B1b (SULT2B1b) sulfates cholesterol and oxysterols. Hepatic oval cells (HOCs), thought to be progenitor cells, can be triggered in chemically injured livers. The present study focused on the role of SULT2B1b in HOC proliferation after liver injury. Our experiments revealed that the expression of SULT2B1b was increased dramatically in a chemical-induced liver injury model, mainly in HOCs. Upon challenge with a hepatotoxic diet containing 0.1 % 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), SULT2B1^−/− mice presented alleviated liver injury and less HOC proliferation compared with wild-type (WT) mice, and these findings were verified by serum analysis, histopathology, immunofluorescence staining, RNA-seq, and Western blotting. HOCs derived from SULT2B1^−/− mice showed lower proliferative capability than those from WT mice. SULT2B1b overexpression promoted growth of the WB-F344 hepatic oval cell line, whereas SULT2B1b knockdown inhibited growth of these cells. The IL-6/STAT3 signaling pathway also was promoted by SULT2B1b. Liquid chromatography and mass spectrometry indicated that the levels of 22-hydroxycholesterol, 25-hydroxycholesterol, and 24,25-epoxycholesterol were higher in the DDC-injured livers of SULT2B1^−/− mice than in livers of WT mice. The above oxysterols are physiological ligands of liver X receptors (LXRs), and SULT2B1b suppressed oxysterol-induced LXR activation. Additional in vivo and in vitro experiments demonstrated that LXR activation could inhibit HOC proliferation and the IL-6/STAT3 signaling pathway, and these effects could be reversed by SULT2B1b. Our data indicate that upregulation of SULT2B1b might promote HOC proliferation and aggravate liver injury via the suppression of oxysterol-induced LXR activation in chemically induced mouse liver injury.  

4.1691           Droplet Microfluidic Flow Cytometer For Sorting On Transient Cellular Responses Of Genetically-Encoded Sensors

Fiedler, B.L., Van Buskirk, S., Carter, K.P., Qin, Y., Carpenter, M.C., Palmer, A.E. and Jimenez, R. Anal. Chem., 89(1), 711-719 (2017)   Fluorescent biosensors are important measurement tools for in vivo quantification of pH, concentrations of metal ions and other analytes, and physical parameters such as membrane potential. Both the development of these sensors and their implementation in examining cellular heterogeneity requires technology for measuring and sorting cells based on the fluorescence levels before and after chemical or physical perturbations. We developed a droplet microfluidic platform for the screening and separation of cell populations on the basis of the in vivo response of expressed fluorescence-based biosensors after addition of an exogenous analyte. We demonstrate the capability to resolve the responses of two genetically encoded Zn²⁺ sensors at a range of time points spanning several seconds and subsequently sort a mixed-cell population of varying ratios with high accuracy.  

4.1692           Anakinra Protects Against Serum Deprivation-Induced Inflammation and Functional Derangement in Islets Isolated From Nonhuman Primates

Jin, S-M., Shim, W., Oh, B.J., Oh, S-H., Yu, S.J., Choi, J.M., Park, H.J., Park, J.B. and Kim, J.H. Am. J. Transplant., 17(2), 365-376 (2017)   We investigated whether serum deprivation induces islet amyloid polypeptide (IAPP) oligomer accumulation and/or a proinflammatory response and, if so, whether the addition of interleukin (IL)-1 receptor antagonist to the culture medium can relieve the proinflammatory response during serum-deprived culture of nonhuman primate (NHP) islets. After culture in medium with and without Ana under serum-deprived culture conditions, IAPP oligomer/amyloid accumulation, in vitro viability, islet function, cytokine secretion, and posttransplantation outcome in streptozotocin-induced diabetic nude mice were determined in islets isolated from heterozygote human IAPP transgenic (hIAPP^+/−) mice and/or NHP islets. Serum deprivation induced accumulation of IAPP oligomer, but not amyloid, in NHP islets. Anakinra (Ana) protected islets from the serum deprivation–induced impairment of in vitro viability and glucose-stimulated insulin secretion and attenuated serum deprivation–induced caspase-1 activation, transcription, and secretion of IL-1β, IL-6, and tumor necrosis factor-α in hIAPP^+/− mice and NHP islets. Supplementation of medium with Ana during serum-deprived culture also improved posttransplantation in vivo outcomes of NHP islets. In conclusion, serum deprivation induced accumulation of IAPP oligomers and proinflammatory responses in cultured isolated islets. Supplementation of the culture medium with Ana attenuated the functional impairment and proinflammatory responses induced by serum deprivation in ex vivo culture of NHP islets.  

4.1693           Transient Monocytosis Subjugates Low Platelet Count in Adult Dengue Patients

Tsai, J-J., Chang, J-S., Chang, K., Chen, P-C., Liu, L-T., Ho, T-C., Tan, S-S., Chien, Y-W., Lo, Y-C. and Perng, G.C. Biomedicine Hub, 2:457785 (2017)   Background: Dengue is one of the most important vector-borne human viral diseases globally. The kinetic changes of hematological parameters of dengue in adult Taiwanese patients have seldomly been systematically investigated and characterized. Methodology/Principal Findings: Serial laboratory data of 1,015 adult patients who were diagnosed with dengue virus serotype 2 (DENV2) and 3 (DENV3) infections in southern Taiwan were retrospectively examined. Prominent parameters were verified with specimens from a 2015 dengue outbreak. Higher absolute monocyte counts on day 5 in severe patients than mild fever subjects after the onset of fever was seen. The absolute number of monocytes was significantly greater in those with DENV2 than DENV3 infections in spite of subtle differences in laboratory tests. Platelet counts were lowest and activated partial thromboplastin time was highest on day 5 in patients with severe conditions. In addition, sudden downward platelet counts corresponding to a transient surge of monocytes on day 4 onward was observed. Fluorescence-activated cell sorting analysis of peripheral blood mononuclear cells obtained from acute dengue patients and experimental investigations revealed that phagocytic effects of innate immune cells contribute to thrombocytopenia in dengue patients. Conclusion: Innate phagocytic cells play an essential role in low platelet counts in adult patients with dengue virus infections.  

4.1694           ALS Along the Axons – Expression of Coding and Noncoding RNA Differs in Axons of ALS models

Rotem, N., magen, I., Ionescu, A., Gershoni-Emek, N., Altman, T., Costa, C.J., Gradus, T., Pasmanik-Chor, M., Willis, D.E., Ben-Dov, I.Z., Hornstein, E. and Perlson, E. Scientific Reports, 7:44500 (2017)   Amyotrophic lateral sclerosis (ALS) is a multifactorial lethal motor neuron disease with no known treatment. Although the basic mechanism of its degenerative pathogenesis remains poorly understood, a subcellular spatial alteration in RNA metabolism is thought to play a key role. The nature of these RNAs remains elusive, and a comprehensive characterization of the axonal RNAs involved in maintaining neuronal health has yet to be described. Here, using cultured spinal cord (SC) neurons grown using a compartmented platform followed by next-generation sequencing (NGS) technology, we find that RNA expression differs between the somatic and axonal compartments of the neuron, for both mRNA and microRNA (miRNA). Further, the introduction of SOD1G93A and TDP43A315T, established ALS-related mutations, changed the subcellular expression and localization of RNAs within the neurons, showing a spatial specificity to either the soma or the axon. Altogether, we provide here the first combined inclusive profile of mRNA and miRNA expression in two ALS models at the subcellular level. These data provide an important resource for studies on the roles of local protein synthesis and axon degeneration in ALS and can serve as a possible target pool for ALS treatment.  

4.1695           Genome-wide mapping of histone H3K9me2 in acute myeloid leukemia reveals large chromosomal domains associated with massive gene silencing and sites of genome instability

Salzberg, A.C., Harris-Becker, A., Popova, E.Y., Keasey, N., Loughran, T.P., Claxton, D.F. and Grigoryev, S.A. PloS One, 12(3), e0173723 (2017)   A facultative heterochromatin mark, histone H3 lysine 9 dimethylation (H3K9me2), which is mediated by histone methyltransferases G9a/GLP (EHMT2/1), undergoes dramatic rearrangements during myeloid cell differentiation as observed by chromatin imaging. To determine whether these structural transitions also involve genomic repositioning of H3K9me2, we used ChIP-sequencing to map genome-wide topography of H3K9me2 in normal human granulocytes, normal CD34+ hematopoietic progenitors, primary myeloblasts from acute myeloid leukemia (AML) patients, and a model leukemia cell line K562. We observe that H3K9me2 naturally repositions from the previously designated “repressed” chromatin state in hematopoietic progenitors to predominant association with heterochromatin regions in granulocytes. In contrast, AML cells accumulate H3K9me2 on previously undefined large (> 100 Kb) genomic blocks that are enriched with AML-specific single nucleotide variants, sites of chromosomal translocations, and genes downregulated in AML. Specifically, the AML-specific H3K9me2 blocks are enriched with genes regulated by the proto-oncogene ERG that promotes stem cell characteristics. The AML-enriched H3K9me2 blocks (in contrast to the heterochromatin-associated H3K9me2 blocks enriched in granulocytes) are reduced by pharmacological inhibition of the histone methyltransferase G9a/GLP in K562 cells concomitantly with transcriptional activation of ERG and ETS1 oncogenes. Our data suggest that G9a/GLP mediate formation of transient H3K9me2 blocks that are preserved in AML myeloblasts and may lead to an increased rate of AML-specific mutagenesis and chromosomal translocations.  

4.1696           PEGylated insulin-like growth factor-I affords protection and facilitates recovery of lost functions post-focal ischemia

Parker, K., Berretta, A., Saenger, S., Sivaramakrishnan, M., Shirley, S.A., Metzger, F. and Clarkson, A.N. Scientific Reports, 7:241 (2017)   Insulin-like growth factor-I (IGF-I) is involved in the maturation and maintenance of neurons, and impaired IGF-I signaling has been shown to play a role in various neurological diseases including stroke. The aim of the present study was to investigate the efficacy of an optimized IGF-I variant by adding a 40 kDa polyethylene glycol (PEG) chain to IGF-I to form PEG-IGF-I. We show that PEG-IGF-I has a slower clearance which allows for twice-weekly dosing to maintain steady-state serum levels in mice. Using a photothrombotic model of focal stroke, dosing from 3 hrs post-stroke dose-dependently (0.3–1 mg/kg) decreases the volume of infarction and improves motor behavioural function in both young 3-month and aged 22–24 month old mice. Further, PEG-IGF-I treatment increases GFAP expression when given early (3 hrs post-stroke), increases Synaptophysin expression and increases neurogenesis in young and aged. Finally, neurons (P5–6) cultured in vitro on reactive astrocytes in the presence of PEG-IGF-I showed an increase in neurite length, indicating that PEG-IGF-I can aid in sprouting of new connections. This data suggests a modulatory role of IGF-I in both protective and regenerative processes, and indicates that therapeutic approaches using PEG-IGF-I should be given early and where the endogenous regenerative potential is still high.  

4.1697           Tricyclic Antidepressants Promote Ceramide Accumulation to Regulate Collagen Production in Human Hepatic Stellate Cells

Chen, J.Y. et al Scientific Reports, 7:44867 (2017)   Activation of hepatic stellate cells (HSCs) in response to injury is a key step in hepatic fibrosis, and is characterized by trans-differentiation of quiescent HSCs to HSC myofibroblasts, which secrete extracellular matrix proteins responsible for the fibrotic scar. There are currently no therapies to directly inhibit hepatic fibrosis. We developed a small molecule screen to identify compounds that inactivate human HSC myofibroblasts through the quantification of lipid droplets. We screened 1600 compounds and identified 21 small molecules that induce HSC inactivation. Four hits were tricyclic antidepressants (TCAs), and they repressed expression of pro-fibrotic factors Alpha-Actin-2 (ACTA2) and Alpha-1 Type I Collagen (COL1A1) in HSCs. RNA sequencing implicated the sphingolipid pathway as a target of the TCAs. Indeed, TCA treatment of HSCs promoted accumulation of ceramide through inhibition of acid ceramidase (aCDase). Depletion of aCDase also promoted accumulation of ceramide and was associated with reduced COL1A1 expression. Treatment with B13, an inhibitor of aCDase, reproduced the antifibrotic phenotype as did the addition of exogenous ceramide. Our results show that detection of lipid droplets provides a robust readout to screen for regulators of hepatic fibrosis and have identified a novel antifibrotic role for ceramide.  

4.1698           Petri Net computational modelling of Langerhans cell Interferon Regulatory Factor Network predicts their role in T cell activation

Polak, M.E., Ung, C.Y., Masapust, J., Freeman, T.C. and Ardern-Jones, M.R. Scientific Reports, 7:668 (2017)   Langerhans cells (LCs) are able to orchestrate adaptive immune responses in the skin by interpreting the microenvironmental context in which they encounter foreign substances, but the regulatory basis for this has not been established. Utilising systems immunology approaches combining in silico modelling of a reconstructed gene regulatory network (GRN) with in vitro validation of the predictions, we sought to determine the mechanisms of regulation of immune responses in human primary LCs. The key role of Interferon regulatory factors (IRFs) as controllers of the human Langerhans cell response to epidermal cytokines was revealed by whole transcriptome analysis. Applying Boolean logic we assembled a Petri net-based model of the IRF-GRN which provides molecular pathway predictions for the induction of different transcriptional programmes in LCs. In silico simulations performed after model parameterisation with transcription factor expression values predicted that human LC activation of antigen-specific CD8 T cells would be differentially regulated by epidermal cytokine induction of specific IRF-controlled pathways. This was confirmed by in vitro measurement of IFN-γ production by activated T cells. As a proof of concept, this approach shows that stochastic modelling of a specific immune networks renders transcriptome data valuable for the prediction of functional outcomes of immune responses.  

4.1699           Papilloma-pseudovirus eradicates intestinal tumours and triples the lifespan of ApcMin/+ mice

Zhong, Z., Zhai, Y., Bu, P., Shah, S. and Qiao, L. Nature Communications, 8:15004 (2017)   Inducing tumour-specific adaptive immunity, such as cytotoxic T lymphocyte (CTL) response, can result in promising antitumour effect against several human malignancies, especially in combination with immune checkpoint blockade strategies. However, little is known whether activation of innate immunity can lead to direct tumoricidal effect. Here, we develop a papilloma pseudovirus-based oral immunotherapeutic approach that shows strong tumoricidal effects in the gut, resulting in an almost tripled lifespan of ApcMin/+ mice (an animal model of human intestinal tumorigenesis). Mechanistically, these pseudoviruses activate the NLRP3 and AIM2 inflammasomes, leading to caspase-1-mediated tumour regression that is dependent on neither cytotoxic T lymphocytes nor humoral immune response. Blocking caspase-1 activation abrogated the therapeutic effects of the pseudoviruses. Thus, targeting innate immune sensors in tumours by the pseudoviruses might represent a strategy to treat intestinal tumours.  

4.1700           Mimicking liver sinusoidal structures and functions using a 3D-configured microfluidic chip

Long, M. et al Lab. Chip, 17(5), 782-794 (2017)   Physiologically, four major types of hepatic cells – the liver sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, and hepatocytes – reside inside liver sinusoids and interact with flowing peripheral cells under blood flow. It is hard to mimic an in vivo liver sinusoid due to its complex multiple cell–cell interactions, spatiotemporal construction, and mechanical microenvironment. Here we developed an in vitro liver sinusoid chip by integrating the four types of primary murine hepatic cells into two adjacent fluid channels separated by a porous permeable membrane, replicating liver's key structures and configurations. Each type of cells was identified with its respective markers, and the assembled chip presented the liver-specific unique morphology of fenestration. The flow field in the liver chip was quantitatively analyzed by computational fluid dynamics simulations and particle tracking visualization tests. Intriguingly, co-culture and shear flow enhance albumin secretion independently or cooperatively, while shear flow alone enhances HGF production and CYP450 metabolism. Under lipopolysaccharide (LPS) stimulations, the hepatic cell co-culture facilitated neutrophil recruitment in the liver chip. Thus, this 3D-configured in vitro liver chip integrates the two key factors of shear flow and the four types of primary hepatic cells to replicate key structures, hepatic functions, and primary immune responses and provides a new in vitro model to investigate the short-duration hepatic cellular interactions under a microenvironment mimicking the physiology of a liver.  

4.1701           CCR2 mediates Helicobacter pylori-induced immune tolerance and contributes to mucosal homeostasis

Sun, X., Zhang, M., El-Zaatari, M., Huffnagle, G.B. and Kao, J.Y. Helicobacter, 22(2), e12366 (2017)   Background We previously demonstrated that H. pylori infection leads to increased induction of regulatory T cells in local and systemic immune compartments. Here, we investigate the role of CCR2 in the tolerogenic programing of dendritic cells in a mouse model of H. pylori infection. Materials and Methods CCR2 deficient (CCR2KO) mice and wild-type (Wt) mice infected with H. pylori SS1 strain were analyzed by qPCR and FACS analysis. In vitro, bone marrow-derived DC on day 6 from CCR2KO and Wt mice cocultured with or without H. pylori were examined to determine the impact of CCR2 signaling on dendritic cells function by qPCR, ELISA, and FACS analyses. Results Acute H. pylori infection was associated with a threefold increase in CCR2 mRNA expression in the gastric mucosa. H. pylori-infected CCR2KO mice exhibited a higher degree of mucosal inflammation, that is, increased gastritis scores and pro-inflammatory cytokine mRNA levels, but lower degree of H. pylori gastric colonization compared to infected Wt mice. Peripheral H. pylori-specific immune response measured in the CCR2KO spleen was characterized by a higher Th17 response and a lower Treg response. In vitro, CCR2KO bone marrow-derived DC was less mature and shown a lower Treg/Th17 ratio. Moreover, blockade of CCR2 signaling by MCP-1 neutralizing antibody inhibited H. pylori-stimulated bone marrow-derived DC maturation. Conclusions Our results indicate that CCR2 plays an essential role in H. pylori-induced immune tolerance and shed light on a novel mechanism of CCR2-dependent DC Treg induction, which appears to be important in maintaining mucosal homeostasis during H. pylori infection.    

4.1702           Adenovirus vector-mediated macrophage erythroblast attacher (MAEA) overexpression in primary mouse hepatocytes attenuates hepatic gluconeogenesis

Shimizu, K., Okamoto, M., Terada, T., Sakurai, F., Mizuguchi, H., Tomita, K. and Nishinaka, T. Biochem. Biophys. Reports, 10, 192-197 (2017)   Japanese patients with type 2 diabetes mellitus present a different responsiveness in terms of insulin secretion to glucose and body mass index (BMI) from other populations. The genetic background that predisposes Japanese individuals to type 2 diabetes mellitus is under study. Recent genetic studies demonstrated that the locus mapped in macrophage erythroblast attacher (MAEA) increases the susceptibility to type 2 diabetes mellitus in East Asians, including Japanese individuals. MAEA encodes a protein that plays a role in erythroblast enucleation and in the normal differentiation of erythroid cells and macrophages. However, the contribution of MAEA to type 2 diabetes mellitus remains unknown. In this study, to overexpress MAEA in the mouse liver and primary mouse hepatocytes, we generated a MAEA-expressing adenovirus (Ad) vector using a novel Ad vector exhibiting significantly lower hepatotoxicity (Ad-MAEA). Blood glucose and insulin levels in Ad-MAEA-treated mice were comparable to those in control Ad-treated mice. Primary mouse hepatocytes transduced with Ad-MAEA showed lower levels of expression of gluconeogenesis genes than those transduced with the control Ad vector. Hepatocyte nuclear factor-4α (HNF-4α) mRNA expression in primary mouse hepatocytes was also suppressed by MAEA overexpression. These results suggest that MAEA overexpression attenuates hepatic gluconeogenesis, which could potentially lead to improvement of type 2 diabetes mellitus.  

4.1703           Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol

Ahrivastava, P., Cabrera, M.A., Chastain, L.G., Boyadjieva, N.I., Jabbar, S., Franklin, T. and Sarkar, D.K.

1. Neuroinflammatioan, 14:83, (2017)

  Background Opioid receptors are known to control neurotransmission of various peptidergic neurons, but their potential role in regulation of microglia and neuronal cell communications is unknown. We investigated the role of mu-opioid receptors (MOR) and delta-opioid receptors (DOR) on microglia in the regulation of apoptosis in proopiomelanocortin (POMC) neurons induced by neonatal ethanol in the hypothalamus. Methods Neonatal rat pups were fed a milk formula containing ethanol or control diets between postnatal days 2–6. Some of the alcohol-fed rats additionally received pretreatment of a microglia activation blocker minocycline. Two hours after the last feeding, some of the pups were sacrificed and processed for histochemical detection of microglial cell functions or confocal microscopy for detection of cellular physical interaction or used for gene and protein expression analysis. The rest of the pups were dissected for microglia separation by differential gradient centrifugation and characterization by measuring production of various activation markers and cytokines. In addition, primary cultures of microglial cells were prepared using hypothalamic tissues of neonatal rats and used for determination of cytokine production/secretion and apoptotic activity of neurons. Results In the hypothalamus, neonatal alcohol feeding elevated cytokine receptor levels, increased the number of microglial cells with amoeboid-type circularity, enhanced POMC and microglial cell physical interaction, and decreased POMC cell numbers. Minocycline reversed these cellular effects of alcohol. Alcohol feeding also increased levels of microglia MOR protein and pro-inflammatory signaling molecules in the hypothalamus, and MOR receptor antagonist naltrexone prevented these effects of alcohol. In primary cultures of hypothalamic microglia, both MOR agonist [D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin (DAMGO) and ethanol increased microglial cellular levels and secretion of pro-inflammatory cell signaling proteins. However, a DOR agonist [D-Pen2,5]enkephalin (DPDPE) increased microglial secretion of anti-inflammatory cytokines and suppressed ethanol’s ability to increase microglial production of inflammatory signaling proteins and secretion of pro-inflammatory cytokines. In addition, MOR-activated inflammation promoted while DOR-suppressed inflammation inhibited the apoptotic effect of ethanol on POMC neurons. Conclusions These results suggest that ethanol’s neurotoxic action on POMC neurons results from MOR-activated neuroinflammatory signaling. Additionally, these results identify a protective effect of a DOR agonist against the pro-inflammatory and neurotoxic action of ethanol.  

4.1704           Development of Improved HDAC6 Inhibitors as Pharmacological Therapy for Axonal Charcot–Marie–Tooth Disease

Benoy, V., Vanden berghe, P., jarpe, M., Van Damme, P., Robberecht, W. and Van Den Bosch, L. Neurotherapeutics, 14, 417-428 (2017)   Charcot–Marie–Tooth disease (CMT) is the most common inherited peripheral neuropathy, with an estimated prevalence of 1 in 2500. The degeneration of motor and sensory nerve axons leads to motor and sensory symptoms that progress over time and have an important impact on the daily life of these patients. Currently, there is no curative treatment available. Recently, we identified histone deacetylase 6 (HDAC6), which deacetylates α-tubulin, as a potential therapeutic target in axonal CMT (CMT2). Pharmacological inhibition of the deacetylating function of HDAC6 reversed the motor and sensory deficits in a mouse model for mutant “small heat shock protein B1” (HSPB1)-induced CMT2 at the behavioral and electrophysiological level. In order to translate this potential therapeutic strategy into a clinical application, small drug-like molecules that are potent and selective HDAC6 inhibitors are essential. To screen for these, we developed a method that consisted of 3 distinct phases and that was based on the pathological findings in the mutant HSPB1-induced CMT2 mouse model. Three different inhibitors (ACY-738, ACY-775, and ACY-1215) were tested and demonstrated to be both potent and selective HDAC6 inhibitors. Moreover, these inhibitors increased the innervation of the neuromuscular junctions in the gastrocnemius muscle and improved the motor and sensory nerve conduction, confirming that HDAC6 inhibition is a potential therapeutic strategy in CMT2. Furthermore, ACY-1215 is an interesting lead molecule as it is currently tested in clinical trials for cancer. Taken together, these results may speed up the translation of pharmacological inhibition of HDAC6 into a therapy against CMT2.  

4.1705           Hesperetin derivative-14 alleviates inflammation by activating PPAR-γ in mice with CCl4-induced acute liver injury and LPS-treated RAW264.7 cells

Chen, X., Ding, H-W., Li, H-D., Huang, H-M., Li, X-F., Yang, Y., Zhang, Y-L., Pan, X-Y., Huang, C. and Meng, X-M. Toxicol. Lett., 274, 51-63 (2017)   Hesperetin is a flavanone glycoside compound naturally occurring in the fruit peel of Citrusaurantium L. (Rutaceae). Previous studies revealed that hesperetin possesses various pharmacological effects, including anti-inflammation, anti-tumor, anti-oxidant and neuroprotective properties. Hesperetin derivative-14 (HD-14) is a derivative of hesperetin improved in water solubility and bioavailability. In this study, we indicated that HD-14 (2 μM) significantly attenuated inflammation in LPS-treated RAW264.7 cells, besides, HD-14 (100 mg/kg) exhibited hepato-protective effects and anti-inflammatory effects on C57BL/6J mice with CCl₄-induced acute liver injury. In addition, it was demonstrated that HD-14 dramatically up-regulated the expression of PPAR-γ in vivo and in vitro. Interestingly, over-expression of PPAR-γ had anti-inflammatory effects on the expressions of TNF-α, IL-6, and IL-1β, whereas, knockdown of PPAR-γ with small interfering RNA had pro-inflammatory effects in LPS-treated RAW264.7 cells. Thus, our findings demonstrated that HD-14 alleviated inflammation by activating PPAR-γ expression at least in part. Further studies founded that HD-14 remarkably inhibited the expression of p-JAK1 and p-STAT1 through up-regulating PPAR-γ. Together, these results suggested that HD-14 served as an activator of PPAR-γ and the JAK1/STAT1 signaling pathway may be involved in the progress of inflammation. Collectively, HD-14 may be utilized as a potential anti-inflammation monomeric compound in the treatment of acute liver injury.  

4.1706           Both MAPK and STAT3 signal transduction pathways are necessary for IL-6-dependent hepatic stellate cells activation

Kagan, P., Sultan, M., Tachlytski, I., Safran, M. and Ben-Ari, Z. PloS One, 12(5), e0176173 (2017)   Background During liver injury, hepatic stellate cells (HSCs) can undergo activation and transform into alpha-smooth muscle actin (αSMA)-expressing contractile myofibroblast-like cells, leading to deposition of excessive scar matrix. We have recently demonstrated that depletion of adenosine deaminase acting on double-stranded RNA (ADAR1) from mouse hepatocytes leads to HSC activation and induction of inflammation and hepatic fibrosis that is mediated by interleukin 6 (IL-6). Our aim was to identify and characterize the molecular pathways involved in the direct, inflammation-independent activation of HSCs by IL-6. Methods Primary HSCs were isolated from mouse livers. mRNA levels of αSMA and Col1a were analyzed using qRT-PCR. Protein levels of αSMA, MAPK, p-MAPK, p38, p-p38, STAT3 and p-STAT3 were assessed by Western Blot analysis. The effect of specific signal transduction pathway inhibitors (i.e., SB203580 (P-38 inhibitor), U0126 (MAPK inhibitor), S3I-201 (STAT3 inhibitor) and Ruxolitinib (Jak1/2 inhibitor)) was also studied. Results Primary HSCs treated with IL-6 demonstrated upregulation of αSMA and Col1a mRNA levels as well as increased αSMA protein levels. Moreover, the phenotypic transition of quiescent HSCs toward myofibroblast-like cells was noted upon administration of IL-6 and not in untreated samples. In addition, the phosphorylation levels of p38, MAPK and STAT3 increased 30 minutes after treatment, and was followed by a decline in the phosphorylation levels 2–4 hours post-treatment. However, addition of specific signal transduction pathway inhibitors curbed this effect, and resulted in αSMA and Col1a expression levels similar to those measured in untreated control samples. Conclusion IL-6 can directly induce the transition of HSCs toward myofibroblast-like cells. The effect is mediated by the activation of both MAPK and JAK/STAT signaling pathways. Elimination of either MAPK or JAK/STAT signaling pathways inhibits HSC stimulation. These results might pave the road toward the development of potential therapeutic interventions for hepatic fibrosis.  

4.1707           Xenotransplantation of layer-by-layer encapsulated non-human primate islets with a specified immunosuppressive drug protocol

Haque, M.R., Kim, J., Park, H., Lee, H.S., Lee, K.W., Al-Hilal, T.A., Jeong, J-H., Ahn, C-H., Lee, D.S., Kim, S.J. and Byun, Y.

1. Controlled Release, 258, 10-21 (2017)

  Islet transplantation is as effective as but also less immunogenic than pancreas transplantation for the treatment of type 1 diabetes mellitus. However, as the complete elimination of immunogenicity still remains a major obstacle in islet transplantation, layer-by-layer encapsulation (LbL) of pancreatic islets using biocompatible polymers offers a rational approach to reducing host immune response towards transplanted islets. We investigated the effect of LbL of non-human primate (NHP) islets on reducing immunogenicity as a preclinical model since NHPs have close phylogenetic and immunological relationship with humans. LbL with three-layers of polyethylene glycol (PEG) molecules (SH-6-arm-PEG-NHS, 6-arm-PEG-catechol and linear PEG-SH) showed a uniform nano-shielding on islets without the loss of viability or function of islets. An immunosuppressive drug protocol was also combined to improve the survival rate of the transplanted islets in vivo. A xenorecipient (C57BL/6 mice) of LbL islet transplanted along with our immunosuppressive drug protocol showed 100% survival rate for 150 days after transplantation. On the other hand, naked islet recipients showed poor survival time of 5.5 ± 1.4 days without drugs and 77.5 ± 42 days with the drug protocol. Immunohistochemistry of the transplanted grafts and serum cytokine concentration demonstrated less immunogenicity in the LbL islet transplanted recipients compared with the naked islet ones.  

4.1708           Follicular size predicts success in artificial insemination with frozen-thawed sperm in donkeys

Saragusty, J., Lemma, A., Hildebrandt, T.B. and Göritz, F. PloS One, 12(5), e0175637 (2017)   In asses, semen collection, cryopreservation, and artificial insemination (AI) with frozen-thawed semen have been scarcely described and success rate, particularly following AI, is reportedly low. In the absence of reliable protocols, assisted reproductive technologies cannot support the conservation efforts aimed at endangered wild ass species and domestic donkey breeds. Two experiments were conducted in this study. In experiment 1 we evaluated freezing Abyssinian donkey (N = 5, 4 ejaculates each) spermatozoa using three freezing extenders (Berliner Cryomedium + glycerol, BC+G; BotuCrio, BOTU; INRAFreeze, INRA) and two cryopreservation techniques (liquid nitrogen vapour, LNV; directional freezing, DF). Post-thaw evaluation indicated that BOTU and INRA were similar and both superior to BC+G (P ≤ 0.004 for all motility tests), and that DF was superior to LNV (P < 0.002 for all evaluation parameters). In experiment 2, relying on these results, we used Abyssinian donkey sperm frozen in BOTU and INRA by DF for AI (N = 20). Prior to AI, thawed samples were diluted in corresponding centrifugation media or autologous seminal fluids at 1:1 ratio. No difference was found between BOTU and INRA or between the addition of seminal fluids or media, all resulting in ~50% pregnancy, and no differences were noted between males (N = 4). The size of pre-ovulatory follicle was a significant (P = 0.001) predictor for AI success with 9/10 pregnancies occurring when follicular size ranged between 33.1–37.4 mm, no pregnancy when it was smaller, and only one when larger. A number of ass species face the risk of extinction. Knowledge gained in this study on the Abyssinian donkey can be customised and transferred to its closely related endangered species and breeds.  

4.1709           Clinical effectiveness of a pylorus-preserving procedure on total pancreatectomy with islet autotransplantation

Shahbazov, R., Yoshimatsu, G., Haque, W.Z., Khan, O.S., Saracino, G., Lawrence, M.C., Kim, P.T., Onaca, N., Naziruddin, B. and Levy, M.F. Am. J. Surg., 213, 1065-1071 (2017)   Background The impact of pylorus preserving procedures (PP) on total pancreatectomy with islet autotransplantation (TPIAT) has not been examined. This study aimed to investigate the clinical impact of the PP on TPIAT. Methods The Baylor Simmons Transplant Institute database was queried to identify seventy-three patients who underwent TPIAT from 2006 to 2014. All patients were investigated in postoperative complications, long-term nutritional status, and graft function. Results Patients with PP did not face worse outcomes in terms of delayed gastric emptying and length of hospital stay. Also, nutritional status and metabolic outcome, such as body weight, serum albumin level, serum vitamin level, HbA1c level, graft survival rate and insulin independent rate, were similar between both groups. Conclusions Clinical results including the graft function indicated that patients undergoing TPIAT with PP did not amplify surgical complications such as delayed gastric emptying and showed no significant advantage of nutrition and metabolic outcome.  

4.1710           High-throughput generation of hyaluronic acid microgels via microfluidics-assisted enzymatic crosslinking and/or Diels–Alder click chemistry for cell encapsulation and delivery

Ma, T., Gao, X., Dong, H., He, H. and Cao, X. Applied Materials Today, 9, 49-59 (2017)   Cell encapsulation in 3D microgels offers unique advantages to meet the complicated requirements in tissue engineering, regenerative medicine and cell therapy. Herein we report high-throughput microfluidic generation of cell-laden microgels based on a new hyaluronic acid (HA) derivative, i.e. furylamine and tyramine grafted HA molecules, which can be crosslinked via either enzymatic crosslinking, or Diels–Alder click chemistry, or both. Compared with traditional photoinitiated free radical polymerization and Michael conjugate addition reaction, enzymatic crosslinking and click chemistry crosslinking show higher chemical selectivity and milder reaction conditions. More importantly, the switching of crosslinking strategy in the same molecule makes it possible to fabricate microgels with almost the same composition but variable gelation time and elasticity. Using ATDC-5 cells as model cells, three types of cell-laden microgels were generated in high-throughput manner and their potentials as cell carriers were evaluated in detail by comparing their mechanical elasticity, gelation time, microgel size, swelling ratio, cell viability, enzymatic degradation and bioactivity of released cells. Our results reveal that the new HA-derived microgels crosslinked by enzymatic crosslinking and Diels–Alder click chemistry are very promising candidate for cell encapsulation and delivery.  

4.1711           GalR3 mediates galanin proliferative effects on postnatal hippocampal precursors

Khan, D., Khan, M., Runesson, J., Zaben, M. and Gray, W.P. Neuropeptides, 63, 14-17 (2017)   Galanin, a neuropeptide co-released from noradrenergic and serotonergic projection neurons to the dentate gyrus, has recently emerged as an important mediator for signaling neuronal activity to the subgranular neurogenic stem cell niche supporting adult hippocampal neurogenesis. Galanin and its receptors appear to play key roles in depression-like behavior, and effects on hippocampal neurogenesis are relevant to pharmacological strategies for treating depression, which in part appear to rely on restoring altered neurogenesis. We previously demonstrated that the GalR2/3 receptor agonist Gal 2–11 is proliferative and proneurogenic for postnatal hippocampal progenitor cells; however, the specific receptor mediation remained to be identified. With the recent availability of M1145 (a specific GalR2 agonist), and SNAP 37889 (GalR3 specific antagonist), we extend our previous studies and show that while M1145 has no proliferative effect, the co-treatment of postnatal rat hippocampal progenitors with Gal 2–11 and SNAP 37889 completely abolished the Gal 2–11 proliferative effects. Taken together, these results clearly demonstrate that GalR3 and not GalR2 is the specific receptor subtype that mediates the proliferative effects of galanin on hippocampal progenitor cells. These results implicate GALR3 in the mediation of galanin neurogenic effects and, potentially, its neurogenic anti-depressant effects.  

4.1712           Foxa2 and Pdx1 cooperatively regulate postnatal maturation of pancreatic β-cells

Bastidas-Ponce, A., Roscioni, S.S., Burtscher, I., Bader, E., Sterr, M., Bakhti, M. and Lickert, H. Mol. Metabolism, 6, 524-534 (2017)   Objective The transcription factors (TF) Foxa2 and Pdx1 are key regulators of beta-cell (β-cell) development and function. Mutations of these TFs or their respective cis-regulatory consensus binding sites have been linked to maturity diabetes of the young (MODY), pancreas agenesis, or diabetes susceptibility in human. Although Foxa2 has been shown to directly regulate Pdx1 expression during mouse embryonic development, the impact of this gene regulatory interaction on postnatal β-cell maturation remains obscure. Methods In order to easily monitor the expression domains of Foxa2 and Pdx1 and analyze their functional interconnection, we generated a novel double knock-in homozygous (FVFPBF^DHom) fluorescent reporter mouse model by crossing the previously described Foxa2-Venus fusion (FVF) with the newly generated Pdx1-BFP (blue fluorescent protein) fusion (PBF) mice. Results Although adult PBF homozygous animals exhibited a reduction in expression levels of Pdx1, they are normoglycemic. On the contrary, despite normal pancreas and endocrine development, the FVFPBF^DHom reporter male animals developed hyperglycemia at weaning age and displayed a reduction in Pdx1 levels in islets, which coincided with alterations in β-cell number and islet architecture. The failure to establish mature β-cells resulted in loss of β-cell identity and trans-differentiation towards other endocrine cell fates. Further analysis suggested that Foxa2 and Pdx1 genetically and functionally cooperate to regulate maturation of adult β-cells. Conclusions Our data show that the maturation of pancreatic β-cells requires the cooperative function of Foxa2 and Pdx1. Understanding the postnatal gene regulatory network of β-cell maturation will help to decipher pathomechanisms of diabetes and identify triggers to regenerate dedifferentiated β-cell mass.  

4.1713           IGF-II promotes neuroprotection and neuroplasticity recovery in a long-lasting model of oxidative damage induced by glucocorticoids

Martin-Montanez, E., Millon, C., Boraldi, F., Garcia-Guirades, F., Pedroza, C., Lara, e., Santin, L.J., Pavia, J. and Garcia-Fernandez, M. Redex Biol., 13, 69-81 (2017)   Insulin-like growth factor-II (IGF-II) is a naturally occurring hormone that exerts neurotrophic and neuroprotective properties in a wide range of neurodegenerative diseases and ageing. Accumulating evidence suggests that the effects of IGF-II in the brain may be explained by its binding to the specific transmembrane receptor, IGFII/M6P receptor (IGF-IIR). However, relatively little is known regarding the role of IGF-II through IGF-IIR in neuroprotection. Here, using adult cortical neuronal cultures, we investigated whether IGF-II exhibits long-term antioxidant effects and neuroprotection at the synaptic level after oxidative damage induced by high and transient levels of corticosterone (CORT). Furthermore, the involvement of the IGF-IIR was also studied to elucidate its role in the neuroprotective actions of IGF-II. We found that neurons treated with IGF-II after CORT incubation showed reduced oxidative stress damage and recovered antioxidant status (normalized total antioxidant status, lipid hydroperoxides and NAD(P) H:quinone oxidoreductase activity). Similar results were obtained when mitochondria function was analysed (cytochrome c oxidase activity, mitochondrial membrane potential and subcellular mitochondrial distribution). Furthermore, neuronal impairment and degeneration were also assessed (synaptophysin and PSD-95 expression, presynaptic function and FluoroJade B® stain). IGF-II was also able to recover the long-lasting neuronal cell damage. Finally, the effects of IGF-II were not blocked by an IGF-IR antagonist, suggesting the involvement of IGF-IIR. Altogether these results suggest that, in or model, IGF-II through IGF-IIR is able to revert the oxidative damage induced by CORT. In accordance with the neuroprotective role of the IGF-II/IGF-IIR reported in our study, pharmacotherapy approaches targeting this pathway may be useful for the treatment of diseases associated with cognitive deficits (i.e., neurodegenerative disorders, depression, etc.).  

4.1714           Simultaneous Subtotal Pancreatectomy and Streptozotocin Injection for Diabetes Modeling in Cynomolgus Monkeys

Park, H., Park, J.B., Kim, J.H., Lee, K.W., Lee, H.S., Kim, G.S., Shin, D-Y., Oh, S.H., Jin, S-M. and Kim, S.J. Transplant. Proceedings, 49, 1142-1149 (2017)   Background In an experimental animal model of islet transplantation, stable induction of insulin-dependent diabetes mellitus (IDDM) and islet isolation from donor pancreas are essential. Total pancreatectomy for IDDM induction and islet procurement in nonhuman primates leads to unwanted loss of exocrine function and may lead to morbidities associated with IDDM. Methods IDDM induction with streptozotocin (STZ) is associated with drug toxicity of STZ and necessitates the killing of another animal for islet procurement. In this study, we performed a subtotal pancreatectomy combined with reduced STZ injection to induce IDDM and procure islets in a nonhuman primate model. Results Twelve cynomolgus monkeys received low-dose STZ injections (60 mg/kg) simultaneously with subtotal pancreatectomy. All monkeys recovered from the procedure without complications. IDDM was induced in the animals. 57,691 ± 16,050 islets were isolated from the resected pancreas and transplanted into other monkeys. Conclusions Simultaneous subtotal pancreatectomy and low-dose STZ injection represent an effective and safe method to create an animal model of insulin dependence diabetes, while at the same time providing sufficient amounts of fresh islet cells for allotransplantation without requiring killing of additional animals. Nonhuman primate (NHP) models of insulin-dependent diabetes mellitus (IDDM) have been widely used to study islet allotransplantation and xenotransplantation [1]; [2]; [3] ;  [4]. IDDM in NHPs by injection of the β-cell toxic drug streptozotocin (STZ) or total pancreatectomy is well-documented in the literature [5]; [6] ;  [7]. Induction of IDDM in NHPs with the use of STZ is a relatively simple procedure involving intravenous injection of the drug. It does not require open abdominal surgery, and, accordingly, morbidities associated with surgery and anesthesia can be avoided. Furthermore, the peritoneal cavity remains naive, allowing for safer access during laparotomy for islet transplantation [8]. However, STZ has hepatotoxic and nephrotoxic side effects, and it is not clear what the optimal dose is of STZ for diabetes induction without invoking obvious adverse effects, because a wide range of values has been reported [9]; [10]; [11] ;  [12]. Induction of IDDM with total pancreatectomy ensures a permanent state of diabetes with the added advantage of acquiring islet cells in the process. However, this is an invasive and complicated surgical procedure that does not ensure preservation of the pancreaticoduodenal arcade. Loss of pancreatic exocrine function is another disadvantage of this procedure [7] ;  [13].  

4.1715           Lipidomic characterization and localization of phospholipids in the human lung

Zemski Berry, K.A., Murphy, R.C., Kosmider, B. and Mason, R.J.

1. Lipid Res., 58(5), 926-933 (2017)

  Lipids play a central role in lung physiology and pathology; however, a comprehensive lipidomic characterization of human pulmonary cells relevant to disease has not been performed. The cells involved in lung host defense, including alveolar macrophages (AMs), bronchial epithelial cells (BECs), and alveolar type II cells (ATIIs), were isolated from human subjects and lipidomic analysis by LC-MS and LC-MS/MS was performed. Additionally, pieces of lung tissue from the same donors were analyzed by MALDI imaging MS in order to determine lipid localization in the tissue. The unique distribution of phospholipids in ATIIs, BECs, and AMs from human subjects was accomplished by subjecting the large number of identified phospholipid molecular species to univariant statistical analysis. Specific MALDI images were generated based on the univariant statistical analysis data to reveal the location of specific cell types within the human lung slice. While the complex composition and function of the lipidome in various disease states is currently poorly understood, this method could be useful for the characterization of lipid alterations in pulmonary disease and may aid in a better understanding of disease pathogenesis.    

4.1716           Liraglutide improves liver microvascular dysfunction in cirrhosis: Evidence from translational studies

De mesquite, F.C., Guixe-Muntet, s., Fernandez-Iglesias, A., Maeso-Diaz, R., Vila, S., Hide, D., Ortega-Ribera, M., Rosa, J.L., Garcia-Pagan, J.C., Bosch, J., de Oliveira, J.R. and Gracia-Sancho, J. Scientific Reports, 7:3255 (2017)   Hepatic stellate cells (HSC) play a key role in the development of chronic liver disease (CLD). Liraglutide, well-established in type 2 diabetes, showed anti-inflammatory and anti-oxidant properties. We evaluated the effects of liraglutide on HSC phenotype and hepatic microvascular function using diverse pre-clinical models of CLD. Human and rat HSC were in vitro treated with liraglutide, or vehicle, and their phenotype, viability and proliferation were evaluated. In addition, liraglutide or vehicle was administered to rats with CLD. Liver microvascular function, fibrosis, HSC phenotype and sinusoidal endothelial phenotype were determined. Additionally, the effects of liraglutide on HSC phenotype were analysed in human precision-cut liver slices. Liraglutide markedly improved HSC phenotype and diminished cell proliferation. Cirrhotic rats receiving liraglutide exhibited significantly improved liver microvascular function, as evidenced by lower portal pressure, improved intrahepatic vascular resistance, and marked ameliorations in fibrosis, HSC phenotype and endothelial function. The anti-fibrotic effects of liraglutide were confirmed in human liver tissue and, although requiring further investigation, its underlying molecular mechanisms suggested a GLP1-R-independent and NF-κB-Sox9-dependent one. This study demonstrates for the first time that liraglutide improves the liver sinusoidal milieu in pre-clinical models of cirrhosis, encouraging its clinical evaluation in the treatment of chronic liver disease.  

4.1717           Stress Hormones Epinephrine and Corticosterone Selectively Modulate Herpes Simplex Virus 1 (HSV-1) and HSV-2 Productive Infections in Adult Sympathetic, but Not Sensory, Neurons

Ives, A.M. and Bertke, A.S.

1. Virol., 91(13), e00582-17 (2017)

  Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect and establish latency in peripheral neurons, from which they can reactivate to cause recurrent disease throughout the life of the host. Stress is associated with the exacerbation of clinical symptoms and the induction of recurrences in humans and animal models. The viruses preferentially replicate and establish latency in different subtypes of sensory neurons, as well as in neurons of the autonomic nervous system that are highly responsive to stress hormones. To determine if stress-related hormones modulate productive HSV-1 and HSV-2 infections within sensory and autonomic neurons, we analyzed viral DNA and the production of viral progeny after treatment of primary adult murine neuronal cultures with the stress hormones epinephrine and corticosterone. Both sensory trigeminal ganglion (TG) and sympathetic superior cervical ganglion (SCG) neurons expressed adrenergic receptors (activated by epinephrine) and the glucocorticoid receptor (activated by corticosterone). Productive HSV infection colocalized with these receptors in SCG but not in TG neurons. In productively infected neuronal cultures, epinephrine treatment significantly increased the levels of HSV-1 DNA replication and production of viral progeny in SCG neurons, but no significant differences were found in TG neurons. In contrast, corticosterone significantly decreased the levels of HSV-2 DNA replication and production of viral progeny in SCG neurons but not in TG neurons. Thus, the stress-related hormones epinephrine and corticosterone selectively modulate acute HSV-1 and HSV-2 infections in autonomic, but not sensory, neurons.  

4.1718           MHC II−, but not MHC II+, hepatic Stellate cells contribute to liver fibrosis of mice in infection with Schistosoma japonicum

Zhou, C-L., Kong, D-L., Liu, J-F., Lu, Z-K., Guo, H-F., Wang, W., Qiu, J-F., Liu, X-J. and Wang, Y. BBA – Molecular Basis of Disase, 1863, 1848-1857 (2017)   Hepatic stellate cells (HSCs) are considered as the main effector cells in vitamin A metabolism and liver fibrosis, as well as in hepatic immune regulation. Recently, researches have revealed that HSCs have plasticity and heterogeneity, which depend on their lobular location and whether liver is normal or injured. This research aimed to explore the biological characteristics and heterogeneity of HSCs in mice with Schistosoma japonicum (S. japonicum) infection, and determine the subpopulation of HSCs in pathogenesis of hepatic fibrosis caused by S. japonicum infection. Results revealed that HSCs significantly increased the expressions of MHC II and fibrogenic genes after S. japonicum infection, and could be classified into MHC II⁺ HSCs and MHC II⁻ HSCs subsets. Both two HSCs populations suppressed the proliferation of activated CD4⁺ T cells, whereas only MHC II⁻ HSCs displayed a myofibroblast-like phenotype. In response to IFN-γ, HSCs up-regulated the expressions of MHC II and CIITA, while down-regulated the expression of fibrogenic gene Col1. In addition, praziquantel treatment decreased the expressions of fibrogenic genes in MHC II⁻ HSCs. These results confirmed that HSCs from S. japonicum-infected mice have heterogeneity. The MHC II⁻ α-SMA⁺ HSCs were major subsets of HSCs contributing to liver fibrosis and could be considered as a potential target of praziquantel anti-fibrosis treatment.  

4.1719           The hemagglutinin-neuramidinase protein of Newcastle disease virus upregulates expression of the TRAIL gene in murine natural killer cells through the activation of Syk and NF-κB

Liang, Y., Song, D-Z., Liang, S., Zhang, Z-F., Gao, L-X. And Fan, X-H. PloS One, 12(6), e0178746 (2017)   Newcastle disease virus (NDV) is responsible for tumoricidal activity in vitro and in vivo. However, the mechanisms that lead to this activity are unclear. Natural killer cells are able to induce apoptosis of tumor cells through multiple pathways, including the tumor necrosis factor-related apoptosis-inducing ligand-death receptor pathway. We previously showed that exposure of NK and T cells to NDV resulted in enhanced tumoricidal activity that was mediated by upregulated expression of the TRAIL gene, via an interferon gamma -dependent pathway. Other pathways involved in the upregulated expression of TRAIL are yet to be identified. In the current study, we used mice in which the IFN-γ receptor one gene was inactivated functionally. We identified an IFN-γ-independent TRAIL pathway in the NDV-stimulated NK cells. Hemagglutinin-neuramidinase induced expression of the TRAIL gene in IFN-R1^-/- NK cells by binding to the NKp46 receptor. This upregulation was inhibited by pretreatment of NDV with a neutralizing monoclonal antibody against HN, or desialylation of NK cells. Phosphorylation of spleen tryosine kinases and IκBα was increased in HN-induced IFN-R1^-/- NK cells. Treatment with the HN neutralizing monoclonal antibody, pharmacological disialylation, or a Syk inhibitor decreased Syk and IκBα phosphorylation levels. We concluded that killer activation receptors pathway is involved in the IFN-γ-independent TRAIL expression of NDV-stimulated NK cells, and these are activated by Syk and NF-κB.  

4.1720           EV-3, an endogenous human erythropoietin isoform with distinct functional relevance

Bonnas, C., Wüsterfeld, L., Winkler, D., Kronstein-Wiedemann, R., Dere, E., Specht, K., Boxberg, M., Tonn, T., Ehrenreich, H., Stadler, H. and Sillaber, I. Scientific Reports, 7:3864 (2017)   Generation of multiple mRNAs by alternative splicing is well known in the group of cytokines and has recently been reported for the human erythropoietin (EPO) gene. Here, we focus on the alternatively spliced EPO transcript characterized by deletion of exon 3 (hEPOΔ3). We show co-regulation of EPO and hEPOΔ3 in human diseased tissue. The expression of hEPOΔ3 in various human samples was low under normal conditions, and distinctly increased in pathological states. Concomitant up-regulation of hEPOΔ3 and EPO in response to hypoxic conditions was also observed in HepG2 cell cultures. Using LC-ESI-MS/MS, we provide first evidence for the existence of hEPOΔ3 derived protein EV-3 in human serum from healthy donors. Contrary to EPO, recombinant EV-3 did not promote early erythroid progenitors in cultures of human CD34+ haematopoietic stem cells. Repeated intraperitoneal administration of EV-3 in mice did not affect the haematocrit. Similar to EPO, EV-3 acted anti-apoptotic in rat hippocampal neurons exposed to oxygen-glucose deprivation. Employing the touch-screen paradigm of long-term visual discrimination learning, we obtained first in vivo evidence of beneficial effects of EV-3 on cognition. This is the first report on the presence of a naturally occurring EPO protein isoform in human serum sharing non-erythropoietic functions with EPO.  

4.1721           Thymosin beta-4 regulates activation of hepatic stellate cells via hedgehog signaling

Kim, J., Hyun, J., Wang, S., Lee, C., Lee, J-W., Moon, E-Y., Cha, H., Diehl, A.M. and Jung, Y. Scientific Reports, 7:3815 (2017)   The molecular mechanisms of thymosin beta-4 (TB4) involved in regulating hepatic stellate cell (HSC) functions remain unclear. Therefore, we hypothesize that TB4 influences HSC activation through hedgehog (Hh) pathway. HSC functions declined in a TB4 siRNA-treated LX-2. TB4 suppression down-regulated both integrin linked kinase (ILK), an activator of smoothened, and phosphorylated glycogen synthase kinase 3 beta (pGSK-3B), an inactive form of GSK-3B degrading glioblastoma 2 (GLI2), followed by the decreased expression of both smoothened and GLI2. A TB4 CRISPR also blocked the activation of primary HSCs, with decreased expression of smoothened, GLI2 and ILK compared with cells transfected with nontargeting control CRISPR. Double immunostaining and an immunoprecipitation assay revealed that TB4 interacted with either smoothened at the cytoplasm or GLI2 at the nucleus in LX-2. Smoothened suppression in primary HSCs using a Hh antagonist or adenovirus transduction decreased TB4 expression with the reduced activation of HSCs. Tb4-overexpressing transgenic mice treated with CCl₄ were susceptible to the development hepatic fibrosis with higher levels of ILK, pGSK3b, and Hh activity, as compared with wild-type mice. These findings demonstrate that TB4 regulates HSC activation by influencing the activity of Smoothened and GLI2, suggesting TB4 as a novel therapeutic target in liver disease.  

4.1722           Sphingosine-1-Phosphate Prevents Egress of Hematopoietic Stem Cells From Liver to Reduce Fibrosis

King, A. ete al Gastroenterol., 153, 233-248 (2017)   Background & Aims There is growing interest in the use of bone marrow cells to treat liver fibrosis, however, little is known about their antifibrotic efficacy or the identity of their effector cell(s). Sphingosine-1-phosphate (S1P) mediates egress of immune cells from the lymphoid organs into the lymphatic vessels; we investigated its role in the response of hematopoietic stem cells (HSCs) to liver fibrosis in mice. Methods Purified (c-kit+/sca1+/lin-) HSCs were infused repeatedly into mice undergoing fibrotic liver injury. Chronic liver injury was induced in BoyJ mice by injection of carbon tetrachloride (CCl₄) or placement on a methionine-choline–deficient diet. Some mice were irradiated and given transplants of bone marrow cells from C57BL6 mice, with or without the S1P antagonist FTY720; we then studied HSC mobilization and localization. Migration of HSC lines was quantified in Transwell assays. Levels of S1P in liver, bone marrow, and lymph fluid were measured using an enzyme-linked immunosorbent assay. Liver tissues were collected and analyzed by immunohistochemical quantitative polymerase chain reaction and sphingosine kinase activity assays. We performed quantitative polymerase chain reaction analyses of the expression of sphingosine kinase 1 and 2, sphingosine-1-phosphate lyase 1, and sphingosine-1-phosphate phosphatase 1 in normal human liver and cirrhotic liver from patients with alcohol-related liver disease (n = 6). Results Infusions of HSCs into mice with liver injury reduced liver scarring based on picrosirius red staining (49.7% reduction in mice given HSCs vs control mice; P < .001), and hepatic hydroxyproline content (328 mg/g in mice given HSCs vs 428 mg/g in control mice; P < .01). HSC infusion also reduced hepatic expression of α-smooth muscle actin (0.19 ± 0.007-fold compared with controls; P < .0001) and collagen type I α 1 chain (0.29 ± 0.17-fold compared with controls; P < .0001). These antifibrotic effects were maintained with infusion of lymphoid progenitors that lack myeloid potential and were associated with increased numbers of recipient neutrophils and macrophages in liver. In studies of HSC cell lines, we found HSCs to recruit monocytes, and this process to require C-C motif chemokine receptor 2. In fibrotic liver tissue from mice and patients, hepatic S1P levels increased owing to increased hepatic sphingosine kinase-1 expression, which contributed to a reduced liver:lymph S1P gradient and limited HSC egress from the liver. Mice given the S1P antagonist (FTY720) with HSCs had increased hepatic retention of HSCs (1697 ± 247 cells in mice given FTY720 vs 982 ± 110 cells in controls; P < .05), and further reductions in fibrosis. Conclusions In studies of mice with chronic liver injury, we showed the antifibrotic effects of repeated infusions of purified HSCs. We found that HSCs promote recruitment of endogenous macrophages and neutrophils. Strategies to reduce SIP signaling and increase retention of HSCs in the liver could increase their antifibrotic activities and be developed for treatment of patients with liver fibrosis.  

4.1723           Augmented liver targeting of exosomes by surface modification with cationized pullulan

Tamura, R., Uemoto, S. and Tabata, Y. Acta Biomaterialia, 57, 274-284 (2017)   Exosomes are membrane nanoparticles containing biological substances that are employed as therapeutics in experimental inflammatory models. Surface modification of exosomes for better tissue targetability and enhancement of their therapeutic ability was recently attempted mainly using gene transfection techniques. Here, we show for the first time that the surface modification of exosomes with cationized pullulan, which has the ability to target hepatocyte asialoglycoprotein receptors, can target injured liver and enhance the therapeutic effect of exosomes. Surface modification can be achieved by a simple mixing of original exosomes and cationized pullulan and through an electrostatic interaction of both substances. The exosomes modified with cationized pullulan were internalized into HepG2 cells in vitro to a significantly greater extent than unmodified ones and this internalization was induced through the asialoglycoprotein receptor that was specifically expressed on HepG2 cells and hepatocytes. When injected intravenously into mice with concanavalin A-induced liver injury, the modified exosomes accumulated in the liver tissue, resulting in an enhanced anti-inflammatory effect in vivo. It is concluded that the surface modification with cationized pullulan promoted accumulation of the exosomes in the liver and the subsequent biological function, resulting in a greater therapeutic effect on liver injury.  

4.1724           Focal Adhesion Kinase Regulates Hepatic Stellate Cell Activation and Liver Fibrosis

Zhao, X-Ke, et al Scientific Reports, 7:4032 (2017)   Understanding the underlying molecular mechanisms of liver fibrosis is important to develop effective therapy. Herein, we show that focal-adhesion-kinse (FAK) plays a key role in promoting hepatic stellate cells (HSCs) activation in vitro and liver fibrosis progression in vivo. FAK activation is associated with increased expression of α-smooth muscle actin (α-SMA) and collagen in fibrotic live tissues. Transforming growth factor beta-1 (TGF-β1) induces FAK activation in a time and dose dependent manner. FAK activation precedes the α-SMA expression in HSCs. Inhibition of FAK activation blocks the α-SMA and collagen expression, and inhibits the formation of stress fibers in TGF-β1 treated HSCs. Furthermore, inhibition of FAK activation significantly reduces HSC migration and small GTPase activation, and induces apoptotic signaling in TGF-β1 treated HSCs. Importantly, FAK inhibitor attenuates liver fibrosis in vivo and significantly reduces collagen and α-SMA expression in an animal model of liver fibrosis. These data demonstrate that FAK plays an essential role in HSC activation and liver fibrosis progression, and FAK signaling pathway could be a potential target for liver fibrosis.  

4.1725           KCC3 loss-of-function contributes to Andermann syndrome by inducing activity-dependent neuromuscular junction defects

Bowermann, M. et al Neurobiol. Dis., 106, 35-48 (2017)   Loss-of-function mutations in the potassium-chloride cotransporter KCC3 lead to Andermann syndrome, a severe sensorimotor neuropathy characterized by areflexia, amyotrophy and locomotor abnormalities. The molecular events responsible for axonal loss remain poorly understood. Here, we establish that global or neuron-specific KCC3 loss-of-function in mice leads to early neuromuscular junction (NMJ) abnormalities and muscular atrophy that are consistent with the pre-synaptic neurotransmission defects observed in patients. KCC3 depletion does not modify chloride handling, but promotes an abnormal electrical activity among primary motoneurons and mislocalization of Na⁺/K⁺-ATPase α1 in spinal cord motoneurons. Moreover, the activity-targeting drug carbamazepine restores Na⁺/K⁺-ATPase α1 localization and reduces NMJ denervation in Slc12a6^−/− mice. We here propose that abnormal motoneuron electrical activity contributes to the peripheral neuropathy observed in Andermann syndrome.  

4.1726           Bioenergetic status modulates motor neuron vulnerability and pathogenesis in a zebrafish model of spinal muscular atrophy

Boyd, P.J. et al PloS Genetics, 13(4), e1006744 (2017)   Degeneration and loss of lower motor neurons is the major pathological hallmark of spinal muscular atrophy (SMA), resulting from low levels of ubiquitously-expressed survival motor neuron (SMN) protein. One remarkable, yet unresolved, feature of SMA is that not all motor neurons are equally affected, with some populations displaying a robust resistance to the disease. Here, we demonstrate that selective vulnerability of distinct motor neuron pools arises from fundamental modifications to their basal molecular profiles. Comparative gene expression profiling of motor neurons innervating the extensor digitorum longus (disease-resistant), gastrocnemius (intermediate vulnerability), and tibialis anterior (vulnerable) muscles in mice revealed that disease susceptibility correlates strongly with a modified bioenergetic profile. Targeting of identified bioenergetic pathways by enhancing mitochondrial biogenesis rescued motor axon defects in SMA zebrafish. Moreover, targeting of a single bioenergetic protein, phosphoglycerate kinase 1 (Pgk1), was found to modulate motor neuron vulnerability in vivo. Knockdown of pgk1 alone was sufficient to partially mimic the SMA phenotype in wild-type zebrafish. Conversely, Pgk1 overexpression, or treatment with terazosin (an FDA-approved small molecule that binds and activates Pgk1), rescued motor axon phenotypes in SMA zebrafish. We conclude that global bioenergetics pathways can be therapeutically manipulated to ameliorate SMA motor neuron phenotypes in vivo.  

4.1727           Targeting the mitochondrial pyruvate carrier attenuates fibrosis in a mouse model of nonalcoholic steatohepatitis

McCommis, K.S., Hodges, W.T., Brunt, E.M., Nalbantoglu, I., McDonald, W.G., Holley, C., Fujiwara, H., Schaffer, J.E., Colca, J.R. and Finck, B.N. Hepatology, 65(5), 1543-1556 (2017)   Diseases of the liver related to metabolic syndrome have emerged as the most common and undertreated hepatic ailments. The cause of nonalcoholic fatty liver disease is the aberrant accumulation of lipid in hepatocytes, though the mechanisms whereby this leads to hepatocyte dysfunction, death, and hepatic fibrosis are still unclear. Insulin-sensitizing thiazolidinediones have shown efficacy in treating nonalcoholic steatohepatitis (NASH), but their widespread use is constrained by dose-limiting side effects thought to be due to activation of the peroxisome proliferator–activated receptor γ. We sought to determine whether a next-generation thiazolidinedione with markedly diminished ability to activate peroxisome proliferator–activated receptor γ (MSDC-0602) would retain its efficacy for treating NASH in a rodent model. We also determined whether some or all of these beneficial effects would be mediated through an inhibitory interaction with the mitochondrial pyruvate carrier 2 (MPC2), which was recently identified as a mitochondrial binding site for thiazolidinediones, including MSDC-0602. We found that MSDC-0602 prevented and reversed liver fibrosis and suppressed expression of markers of stellate cell activation in livers of mice fed a diet rich in trans-fatty acids, fructose, and cholesterol. Moreover, mice with liver-specific deletion of MPC2 were protected from development of NASH on this diet. Finally, MSDC-0602 directly reduced hepatic stellate cell activation in vitro, and MSDC-0602 treatment or hepatocyte MPC2 deletion also limited stellate cell activation indirectly by affecting secretion of exosomes from hepatocytes. Conclusion: Collectively, these data demonstrate the effectiveness of MSDC-0602 for attenuating NASH in a rodent model and suggest that targeting hepatic MPC2 may be an effective strategy for pharmacologic development.  

4.1728           Retinoic Acid Regulates Immune Responses by Promoting IL-22 and Modulating S100 Proteins in Viral Hepatitis

Jie, Z., Liang, Y., Yi, P., Tang, H., Soong, L., Cong, Y., Zhang, K. and Sun, J.

1. Immunol., 198(9), 3448-3460 (2017)

  Although large amounts of vitamin A and its metabolite all-trans retinoic acid (RA) are stored in the liver, how RA regulates liver immune responses during viral infection remains unclear. In this study, we demonstrated that IL-22, mainly produced by hepatic γδ T cells, attenuated liver injury in adenovirus-infected mice. RA can promote γδ T cells to produce mTORC1-dependent IL-22 in the liver, but inhibits IFN-γ and IL-17. RA also affected the aptitude of T cell responses by modulating dendritic cell (DC) migration and costimulatory molecule expression. These results suggested that RA plays an immunomodulatory role in viral infection. Proteomics data revealed that RA downregulated S100 family protein expression in DCs, as well as NF-κB/ERK pathway activation in these cells. Furthermore, adoptive transfer of S100A4-repressed, virus-pulsed DCs into the hind foot of naive mice failed to prime T cell responses in draining lymph nodes. Our study has demonstrated a crucial role for RA in promoting IL-22 production and tempering DC function through downregulating S100 family proteins during viral hepatitis.  

4.1729           MicroRNA-649 promotes HSV-1 replication by directly targeting MALT1

Zhang, Y., Dai, J., Tang, J., Zhou, L. and Zhou, M.

1. Med. Virol., 89, 1069-1079 (82017)

  Herpes simplex virus type 1 (HSV-1), a member of the Herpes viridae, is associated with a wide variety of nervous system diseases including meningitis and encephalitis. The data presented here demonstrate that miR-649 promotes the replication of HSV-1 without affecting cell viability. Further mechanistic studies revealed that MALT1 (mucosa associated lymphoid tissue lymphoma translocation gene 1) is directly targeted by miR-649. We then found that MALT1 and the downstream NF-κB signaling pathway, are involved in miR-649-induced HSV-1 replication. Interestingly, miR-649 levels were downregulated after HSV-1 infection, and miR-649 expression was negatively associated with MALT1 expression in HSV-1-infected HeLa cells. Taken together, this present study indicates that miR-649 promotes HSV-1 replication through regulation of the MALT1-mediated antiviral signaling pathway and suggests a promising target for antiviral therapies.    

4.1730           IRAP+ endosomes restrict TLR9 activation and signaling

Babdor, J. et al Natuer Immunol., 18(5), 509-518 (2017)   The retention of intracellular Toll-like receptors (TLRs) in the endoplasmic reticulum prevents their activation under basal conditions. TLR9 is activated by sensing ligands in specific endosomal-lysosomal compartments. Here we identified IRAP⁺ endosomes as major cellular compartments for the early steps of TLR9 activation in dendritic cells (DCs). Both TLR9 and its ligand, the dinucleotide CpG, were present as cargo in IRAP⁺ endosomes. In the absence of the aminopeptidase IRAP, the trafficking of CpG and TLR9 to lysosomes and signaling via TLR9 were enhanced in DCs and in mice following bacterial infection. IRAP stabilized CpG-containing endosomes by interacting with the actin-nucleation factor FHOD4, which slowed the trafficking of TLR9 toward lysosomes. Thus, endosomal retention of TLR9 via the interaction of IRAP with the actin cytoskeleton is a mechanism that prevents hyper-activation of TLR9 in DCs.  

4.1731           Social network architecture of human immune cells unveiled by quantitative proteomics

Rickmann, J.C., Geiger, R., Hornburg, D., Wolf, T., Kveler, K., Jarrossay, D., Sallusto, F., Shen-Orr, S.S., Lanzavecchia, A., Mann, M. and Meissner, F. Nature Immunol., 18(5), 583-593 (2017)   The immune system is unique in its dynamic interplay between numerous cell types. However, a system-wide view of how immune cells communicate to protect against disease has not yet been established. We applied high-resolution mass-spectrometry-based proteomics to characterize 28 primary human hematopoietic cell populations in steady and activated states at a depth of >10,000 proteins in total. Protein copy numbers revealed a specialization of immune cells for ligand and receptor expression, thereby connecting distinct immune functions. By integrating total and secreted proteomes, we discovered fundamental intercellular communication structures and previously unknown connections between cell types. Our publicly accessible (http://www.immprot.org/) proteomic resource provides a framework for the orchestration of cellular interplay and a reference for altered communication associated with pathology.  

4.1732           Targeting Extracellular Cyclophilin A Reduces Neuroinflammation and Extends Survival in a Mouse Model of Amyotrophic Lateral Sclerosis

Pasetto, L. et al

1. Neurosci., 37(6), 1413-1427 (2017)

  Neuroinflammation is a major hallmark of amyotrophic lateral sclerosis (ALS), which is currently untreatable. Several anti-inflammatory compounds have been evaluated in patients and in animal models of ALS, but have been proven disappointing in part because effective targets have not yet been identified. Cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), as a foldase is beneficial intracellularly, but extracellularly has detrimental functions. We found that extracellular PPIA is a mediator of neuroinflammation in ALS. It is a major inducer of matrix metalloproteinase 9 and is selectively toxic for motor neurons. High levels of PPIA were found in the CSF of SOD1^G93A mice and rats and sporadic ALS patients, suggesting that our findings may be relevant for familial and sporadic cases. A specific inhibitor of extracellular PPIA, MM218, given at symptom onset, rescued motor neurons and extended survival in the SOD1^G93A mouse model of familial ALS by 11 d. The treatment resulted in the polarization of glia toward a prohealing phenotype associated with reduced NF-κB activation, proinflammatory markers, endoplasmic reticulum stress, and insoluble phosphorylated TDP-43. Our results indicates that extracellular PPIA is a promising druggable target for ALS and support further studies to develop a therapy to arrest or slow the progression of the disease in patients.  

4.1733           Decreased Motor Neuron Support by SMA Astrocytes due to Diminished MCP1 Secretion

Martin, J.E., Nguyen, T.T., Grunseich, C., Nofziger, J.H., Lee, P.R., Fields, D., Fishbeck, K.H. and Foran, E.

1. Neurosci., 37(21), 5309-5318 (2017)

  Spinal muscular atrophy (SMA) is an autosomal-recessive disorder characterized by severe, often fatal muscle weakness due to loss of motor neurons. SMA patients have deletions and other mutations of the survival of motor neuron 1 (SMN1) gene, resulting in decreased SMN protein. Astrocytes are the primary support cells of the CNS and are responsible for glutamate clearance, metabolic support, response to injury, and regulation of signal transmission. Astrocytes have been implicated in SMA as in in other neurodegenerative disorders. Astrocyte-specific rescue of SMN protein levels has been shown to mitigate disease manifestations in mice. However, the mechanism by which SMN deficiency in astrocytes may contribute to SMA is unclear and what aspect of astrocyte activity is lacking is unknown. Therefore, it is worthwhile to identify defects in SMN-deficient astrocytes that compromise normal function. We show here that SMA astrocyte cultures derived from mouse spinal cord of both sexes are deficient in supporting both WT and SMN-deficient motor neurons derived from male, female, and mixed-sex sources and that this deficiency may be mitigated with secreted factors. In particular, SMN-deficient astrocytes have decreased levels of monocyte chemoactive protein 1 (MCP1) secretion compared with controls and MCP1 restoration stimulates outgrowth of neurites from cultured motor neurons. Correction of MCP1 deficiency may thus be a new therapeutic approach to SMA.  

4.1734           MicroRNA Profiling Reveals Marker of Motor Neuron Disease in ALS Models

Hoye, M.L. et al

1. Neurosci., 37(22), 5574-5586 (2017)

  Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder marked by the loss of motor neurons (MNs) in the brain and spinal cord, leading to fatally debilitating weakness. Because this disease predominantly affects MNs, we aimed to characterize the distinct expression profile of that cell type to elucidate underlying disease mechanisms and to identify novel targets that inform on MN health during ALS disease time course. microRNAs (miRNAs) are short, noncoding RNAs that can shape the expression profile of a cell and thus often exhibit cell-type-enriched expression. To determine MN-enriched miRNA expression, we used Cre recombinase-dependent miRNA tagging and affinity purification in mice. By defining the in vivo miRNA expression of MNs, all neurons, astrocytes, and microglia, we then focused on MN-enriched miRNAs via a comparative analysis and found that they may functionally distinguish MNs postnatally from other spinal neurons. Characterizing the levels of the MN-enriched miRNAs in CSF harvested from ALS models of MN disease demonstrated that one miRNA (miR-218) tracked with MN loss and was responsive to an ALS therapy in rodent models. Therefore, we have used cellular expression profiling tools to define the distinct miRNA expression of MNs, which is likely to enrich future studies of MN disease. This approach enabled the development of a novel, drug-responsive marker of MN disease in ALS rodents.  

4.1735           Presence of diabetes autoantigens in extracellular vesicles derived from human islets

Hasilo, C.P., negli, S., Allaeys, I., Cloutier, N., Rutman, A.K., Gasparrini, M., Bonneil, E., Thibault, P., Boilard, E. and Paraskevas, S. Scientific Reports, 7:500 (2017)   Beta-cell (β-cell) injury is the hallmark of autoimmune diabetes. However, the mechanisms by which autoreactive responses are generated in susceptible individuals are not well understood. Extracellular vesicles (EV) are produced by mammalian cells under normal and stressed physiological states. They are an important part of cellular communication, and may serve a role in antigen processing and presentation. We hypothesized that isolated human islets in culture produce EV that contain diabetes autoantigens (DAA) from these otherwise normal, non-diabetic donors. Here we report the caspase-independent production of EV by human islets in culture, and the characterization of DAA glutamic acid decarboxylase 65 (GAD65) and zinc transporter 8 (ZnT8), as well as the β-cell resident glucose transporter 2 (Glut2), present within the EV.  

4.1736           TRAIL regulatory receptors constrain human hepatic stellate cell apoptosis

Singh, H., Otano, I., Rombouts, K., Singh, K.P., Peppa, D., Gill, U., Böttcher, K., Kennedy, P.T.F., Oben, J., Pinzani, M., Walczak, H., Fusai, G., Rosenberg, W.C. and Maini, M.K. Scientific Reports, 7:5514 (2017)   The TRAIL pathway can mediate apoptosis of hepatic stellate cells to promote the resolution of liver fibrosis. However, TRAIL has the capacity to bind to regulatory receptors in addition to death-inducing receptors; their differential roles in liver fibrosis have not been investigated. Here we have dissected the contribution of regulatory TRAIL receptors to apoptosis resistance in primary human hepatic stellate cells (hHSC). hHSC isolated from healthy margins of liver resections from different donors expressed variable levels of TRAIL-R2/3/4 (but negligible TRAIL-R1) ex vivo and after activation. The apoptotic potential of TRAIL-R2 on hHSC was confirmed by lentiviral-mediated knockdown. A functional inhibitory role for TRAIL-R3/4 was revealed by shRNA knockdown and mAb blockade, showing that these regulatory receptors limit apoptosis of hHSC in response to both oligomerised TRAIL and NK cells. A close inverse ex vivo correlation between hHSC TRAIL-R4 expression and susceptibility to apoptosis underscored its central regulatory role. Our data provide the first demonstration of non-redundant functional roles for the regulatory TRAIL receptors (TRAIL-R3/4) in a physiological setting. The potential for these inhibitory TRAIL receptors to protect hHSC from apoptosis opens new avenues for prognostic and therapeutic approaches to the management of liver fibrosis.  

4.1737           Cryptic amyloidogenic elements in mutant NEFH causing Charcot-Marie-Tooth 2 trigger aggresome formation and neuronal death

Jackuier, A. et al Acta Neuropathol.  Comm., 5:55 (2017)   Neurofilament heavy chain (NEFH) gene was recently identified to cause autosomal dominant axonal Charcot-Marie-Tooth disease (CMT2cc). However, the clinical spectrum of this condition and the physio-pathological pathway remain to be delineated. We report 12 patients from two French families with axonal dominantly inherited form of CMT caused by two new mutations in the NEFH gene. A remarkable feature was the early involvement of proximal muscles of the lower limbs associated with pyramidal signs in some patients. Nerve conduction velocity studies indicated a predominantly motor axonal neuropathy. Unique deletions of two nucleotides causing frameshifts near the end of the NEFH coding sequence were identified: in family 1, c.3008_3009del (p.Lys1003Argfs*59), and in family 2 c.3043_3044del (p.Lys1015Glyfs*47). Both frameshifts lead to 40 additional amino acids translation encoding a cryptic amyloidogenic element. Consistently, we show that these mutations cause protein aggregation which are recognised by the autophagic pathway in motoneurons and triggered caspase 3 activation leading to apoptosis in neuroblastoma cells. Using electroporation of chick embryo spinal cord, we confirm that NEFH mutants form aggregates in vivo and trigger apoptosis of spinal cord neurons. Thus, our results provide a physiological explanation for the overlap between CMT and amyotrophic lateral sclerosis (ALS) clinical features in affected patients.  

4.1738           A CXCL ortholog from Hippocampus abdominalis: Molecular features and functional delineation as a pro-inflammatory chemokine

Oh, M., bathige, S.D.N.K., Kim, Y., Lee, S., yang, H., Kim, M-J. and Lee, J. Fish & Shellfish Immunol., 67, 218-227 (2017)   Chemokines are a family of chemotactic cytokines that regulate leukocyte migration. They are classified into four groups namely, CXC, CC, C and CX₃C, based on the formation of a disulfide bridge. Among these, CXC chemokines have been identified as the largest group of chemokines in humans. In this study, we identified and functionally characterized a homolog of CXC chemokine from the big-belly seahorse, Hippocampus abdominalis, and designated it as ShCXCL. The cDNA of ShCXCL composed of a 342-bp open reading frame encoding 113 amino acids (aa). The CXC family-specific small cytokine domain (SCY) was identified from the mature peptide region, which comprised of a conserved CXC motif. As ShCXCL lacks an ELR (Glutamic acid-Leucine-Arginine) motif, it belongs to ELR⁻ subfamily. The recombinant ShCXCL protein strongly induced the nitric oxide (NO) production in macrophage cells (RAW 264.7 cell line) and showed the chemotactic effect on flounder peripheral blood leukocytes. Tissue profiling showed a ubiquitous expression pattern in all examined tissues, with a high abundance in spleen. The up-regulated mRNA expression pattern of ShCXCL was observed in blood and kidney tissues after immune stimulation by live bacteria, such as Streptococcus iniae and Edwardsiella tarda, and mitogens, such as lipopolysaccharides (LPS) and polyinosinic:polycytidylic acid (poly I:C), suggesting its important role in host immune defense against microbial infection.  

4.1739           Chemoselective functionalization of nanogels for microglia treatment

Mauri, E., Veglianese, P., Papa, S., Mariani, A., De paola, M., Rigamonti, R., Chincarini, G.M.F., Rimondo, S., Sacchetti, A. and Rossi, F. Eur. Polymer J., 94, 143-151 (2017)   The development of nanogels as nanoscale multifunctional polymer-based matrices for controlled drug and gene delivery purposes has been the subject of intense research during the last decades. Indeed, polymeric nanoparticles are capable to interact with cells to different extents, depending on their size, shape, surface properties and ligands tagged to the surface. Moreover, coating these devices using appropriate functionalization strategies can greatly improve or not their adhesion and uptake by cells. In this work, we proposed different coatings and then we studied their ability to improve or reduce microglia internalization. Nanogels (NGs) were composed by polyethylene glycol (PEG) and polyethyleneimine (PEI) conjugated with rhodamine through click chemistry reaction. Coatings were prepared using PEG monomethyl ether (mPEG), modifying its terminal hydroxyl groups with different linkers to evaluate the amount of mPEG layer chemically bond to the nanogel and its effect over microglia internalization. Nanogels were also investigated to identify which procedure is able to form networks with adequate dimensions and stability, according to the physicochemical parameters for the microglia applications in vitro. The biological experimental results showed that NGs were efficiently internalized by cells and the coating-microglia interactions allowed different cellular uptake. This outcome could be considered a promising perspective of nanogels use as carriers for drugs or genes delivery within microglia environment, improving their therapeutic effect through polymer surface modifications.  

4.1740           A new method of isolating spinal motor neurons from fetal mouse

Wang, W., Qi, B., Lv, H., Wu, F., Liu, L., Wang, W., Wang, Q., Hu, L., Hao, Y. and Wang, Y.

1. Neurosci. Methods, 288, 57-61 (2017)

  Background Isolating of primary motor neurons from animal embryos is critical for the study of neurological disease including mechanistic discovery and therapeutic development. Density gradient centrifuge taking advantage of the buoyant of motor neuron permits the enrichment of motor neurons. Despite the metrizamide, an OptiPrep medium has been introduced to separate the motor neurons by gradient centrifuge. New method We hereby used single density gradient of OptiPrep medium to isolate the spinal motor neurons from the fetal mouse. Results Single density gradient of OptiPrep medium is effective to isolate spinal motor neurons from the fetal mouse. The immunofluorescence staining analysis showed that the purity of cultured motor neurons at 72 h was between 90% and 95%. Comparison with existing method Four gradients of OptiPrep medium have been previously used to isolate the motor neurons from spinal cord of mouse. In this study, the single gradient of OptiPrep medium was demonstrated to effectively isolate spinal motor neurons from the fetal mouse. Conclusions The single gradient of OptiPrep medium is enough to produce high purity of spinal motor neurons from the fetal mouse.  

4.1741           Sigma 1 receptor activation modifies intracellular calcium exchange in the G93AhSOD1 ALS model

Tadic, V., Malci, A., Goldhammer, N., Stubendorff, B., Sengupta, S., Prell, T., Keiner, S., Liu, J., Guenther, M., Frahm, C., Witte, O.W. and Grosskreutz, J. Neuroscience, 359, 105-118 (2017)   Aberrations in intracellular calcium (Ca²⁺) have been well established within amyotrophic lateral sclerosis (ALS), a severe motor neuron disease. Intracellular Ca²⁺ concentration is controlled in part through the endoplasmic reticulum (ER) mitochondria Ca²⁺ cycle (ERMCC). The ER supplies Ca²⁺ to the mitochondria at close contacts between the two organelles, i.e. the mitochondria-associated ER membranes (MAMs). The Sigma 1 receptor (Sig1R) is enriched at MAMs, where it acts as an inter-organelle signaling modulator. However, its impact on intracellular Ca²⁺ at the cellular level remains to be thoroughly investigated. Here, we used cultured embryonic mice spinal neurons to investigate the influence of Sig1R activation on intracellular Ca²⁺ homeostasis in the presence of G93A^hSOD1 (G93A), an established ALS-causing mutation. Sig1R expression was increased in G93A motor neurons relative to non-transgenic (nontg) controls. Furthermore, we demonstrated significantly reduced bradykinin-sensitive intracellular Ca²⁺ stores in G93A spinal neurons, which were normalized by the Sig1R agonist SA4503. Moreover, SA4503 accelerated cytosolic Ca²⁺ clearance following a) AMPAR activation by kainate and b) IP₃R–mediated ER Ca²⁺ release following bradykinin stimulation in both genotypes. PRE-084 (another Sig1R agonist) did not exert any significant effects on cytosolic Ca²⁺. Both Sig1R expression and functionality were altered by the G93A mutation, indicating the centrality of Sig1R in ALS pathology. Here, we showed that intracellular Ca²⁺ shuttling can be manipulated by Sig1R activation, thus demonstrating the value of using the pharmacological manipulation of Sig1R to understand Ca²⁺ homeostasis.    

4.1742           The anti-influenza M2e antibody response is promoted by XCR1 targeting in pig skin

Deloizy, C. et al Scientific Reports, 7:7639 (2017)   XCR1 is selectively expressed on a conventional dendritic cell subset, the cDC1 subset, through phylogenetically distant species. The outcome of antigen-targeting to XCR1 may therefore be similar across species, permitting the translation of results from experimental models to human and veterinary applications. Here we evaluated in pigs the immunogenicity of bivalent protein structures made of XCL1 fused to the external portion of the influenza virus M2 proton pump, which is conserved through strains and a candidate for universal influenza vaccines. Pigs represent a relevant target of such universal vaccines as pigs can be infected by swine, human and avian strains. We found that cDC1 were the only cell type labeled by XCR1-targeted mCherry upon intradermal injection in pig skin. XCR1-targeted M2e induced higher IgG responses in seronegative and seropositive pigs as compared to non-targeted M2e. The IgG response was less significantly enhanced by CpG than by XCR1 targeting, and CpG did not further increase the response elicited by XCR1 targeting. Monophosphoryl lipid A with neutral liposomes did not have significant effect. Thus altogether M2e-targeting to XCR1 shows promises for a trans-species universal influenza vaccine strategy, possibly avoiding the use of classical adjuvants.  

4.1743           Swarm Intelligence-Enhanced Detection of Non-Small-Cell Lung Cancer Using Tumor-Educated Platelets

Best, M.G., Sol, N., In t’Veld, S.G.J.G., Vancura, A., Muller, M., Niemeijer, A-L. N., Fejes, A.V.,Tjon Kon Fat, L-A. and In t’Veld, A.E.H. Cancer Cell, 32, 238-252 (2017)   Blood-based liquid biopsies, including tumor-educated blood platelets (TEPs), have emerged as promising biomarker sources for non-invasive detection of cancer. Here we demonstrate that particle-swarm optimization (PSO)-enhanced algorithms enable efficient selection of RNA biomarker panels from platelet RNA-sequencing libraries (n = 779). This resulted in accurate TEP-based detection of early- and late-stage non-small-cell lung cancer (n = 518 late-stage validation cohort, accuracy, 88%; AUC, 0.94; 95% CI, 0.92–0.96; p < 0.001; n = 106 early-stage validation cohort, accuracy, 81%; AUC, 0.89; 95% CI, 0.83–0.95; p < 0.001), independent of age of the individuals, smoking habits, whole-blood storage time, and various inflammatory conditions. PSO enabled selection of gene panels to diagnose cancer from TEPs, suggesting that swarm intelligence may also benefit the optimization of diagnostics readout of other liquid biopsy biosources.  

4.1744           The Evaluation of Islet Purification Methods That Use Large Bottles to Create a Continuous Density Gradient

Miyagi-Shiohira, C., Kobayashi, N., Saitoh, I., Watanabe, M., Noguchi, Y., Matsushita, M. and Noguchi, H. Cell Med., 9, 45-51 (2017)   Islet purification is one of the most important steps of islet isolation for pancreatic islet transplantation. The most common method of islet purification is density gradient centrifugation using a COBE 2991 cell processor. However, this method can damage islets mechanically through its high shearing force. We recently reported that a new purification method using large plastic bottles effectively achieves a high yield of islets from the porcine pancreas. In the present study, we evaluated the methods of making a continuous density gradient. The gradient was produced with a gradient maker and two types of candy cane-shaped stainless steel pipes. One method was to use a “bent-tipped” stainless steel pipe and to load from a high-density solution to a low-density solution, uploading the stainless steel pipe. The other method was to use a regular stainless steel pipe and to load from a low-density solution to a high-density solution, leaving the stainless steel pipe in place. There were no significant differences between the two solutions in terms of the islet yield, rate of viability or purity, score, or the stimulation index after purification. Furthermore, there were no differences in the attainability or suitability of posttransplantation normoglycemia. Our study shows the equivalency of these two methods of islet purification.  

4.1745           Comparison of Purification Solutions with Different Osmolality for Porcine Islet Purification

Miyagi-Shiora, C., Kobayashi, N., Saitoh, I., Watanabe, M., Noguchi, Y., Matsushita, M. and Noguchi, H. Cell Med., 9, 53-59 (2017)   The osmolality of the purification solution is one of the most critical variables in human islet purification during islet isolation. We previously reported the effectiveness of a combined continuous density/osmolality gradient for the supplemental purification of human islets. We herein applied a combined continuous density/osmolality gradient for regular purification. The islets were purified with a continuous density gradient without osmolality preparation [continuous density/normal osmolality (CD/NO)] or continuous density/osmolality solution with osmolality preparation by 10× Hank's balanced salt solution (HBSS) [continuous density/continuous osmolality (CD/CO)]. The osmolality of the low-density solution was 400 mOsm/kg in both groups and that of the high-density solution was 410 mOsm/kg in the CD/NO group and 500 mOsm/kg in the CD/CO group. Unexpectedly, we noted no significant differences between the two solutions in terms of the islet yield, rate of viability and purity, score, stimulation index, or the attainability and suitability of posttransplantation normoglycemia. Despite reports that the endocrine and exocrine tissues of pancreata have distinct osmotic sensitivities and that high-osmolality solutions result in greater purification efficiency, the isolation and transplant outcomes did not markedly differ between the two purification solutions with different osmolalities in this study.  

4.1746           Single-cell genome sequencing at ultra-high-throughput with microfluidic droplet barcoding

Lan, F., Demaree, B., Ahmed, N. and Abate, A.R. Nature Biotech., 35(7), 640-646 (2017)   The application of single-cell genome sequencing to large cell populations has been hindered by technical challenges in isolating single cells during genome preparation. Here we present single-cell genomic sequencing (SiC-seq), which uses droplet microfluidics to isolate, fragment, and barcode the genomes of single cells, followed by Illumina sequencing of pooled DNA. We demonstrate ultra-high-throughput sequencing of >50,000 cells per run in a synthetic community of Gram-negative and Gram-positive bacteria and fungi. The sequenced genomes can be sorted in silico based on characteristic sequences. We use this approach to analyze the distributions of antibiotic-resistance genes, virulence factors, and phage sequences in microbial communities from an environmental sample. The ability to routinely sequence large populations of single cells will enable the de-convolution of genetic heterogeneity in diverse cell populations.  

4.1747           Cutting Edge: Eosinophils Undergo Caspase-1–Mediated Pyroptosis in Response to Necrotic Liver Cells

Palacios-Macapagal, D., Connor, J., Mustelin, T., Ramalingam, T.R., Wynn, T.A. and Davidson, T.S.

1. Immunol., 199(3), 847-853 (2017)

  Many chronic liver disorders are characterized by dysregulated immune responses and hepatocyte death. We used an in vivo model to study the immune response to necrotic liver injury and found that necrotic liver cells induced eosinophil recruitment. Necrotic liver induced eosinophil IL-1β and IL-18 secretion, degranulation, and cell death. Caspase-1 inhibitors blocked all of these responses. Caspase-1–mediated cell death with accompanying cytokine release is the hallmark of a novel form of cell death termed pyroptosis. To confirm this response in a disease model, we isolated eosinophils from the livers of Schistosoma mansoni–infected mice. S. mansoni eggs lodge in the hepatic sinusoids of infected mice, resulting in hepatocyte death, inflammation, and progressive liver fibrosis. This response is typified by massive eosinophilia, and we were able to confirm pyroptosis in the infiltrating eosinophils. This demonstrated that pyroptosis is a cellular pathway used by eosinophils in response to large-scale hepatic cell death.  

4.1748           A novel whole-bacterial enzyme linked-immunosorbant assay to quantify Chlamydia trachomatis specific antibodies reveals distinct differences between systemic and genital compartments

Albritton, H.L., Kozlowski, P.A:, Lillis, R.A., McGowin, C.L., Siren, J.D., Taylor, S.N., Ibana, J.A., Buckner, L.R., Shen, L and Quayle, J. PloS One, 12(8), e0183101 (2017)   Chlamydia trachomatis (CT) is the leading sexually transmitted bacterial infection. The continued global burden of CT infection strongly predicates the need for a vaccine to supplement current chlamydial control programs. The correlates of protection against CT are currently unknown, but they must be carefully defined to guide vaccine design. The localized nature of chlamydial infection in columnar epithelial cells of the genital tract necessitates investigation of immunity at the site of infection. The purpose of this study was to develop a sensitive whole bacterial enzyme-linked immunosorbent assay (ELISA) to quantify and compare CT-specific IgG and IgA in sera and genital secretions from CT-infected women. To achieve this, elementary bodies (EBs) from two of the most common genital serovars (D and E) were attached to poly-L-lysine-coated microtiter plates with glutaraldehyde. EB attachment and integrity were verified by the presence of outer membrane antigens and the absence of bacterial cytoplasmic antigens. EB-specific IgG and IgA standards were developed by pooling sera with high titers of CT-specific antibodies from infected women. Serum, endocervical and vaginal secretions, and endocervical cytobrush specimens from CT-infected women were used to quantify CT-specific IgG and IgA which were then normalized to total IgG and IgA, respectively. Analyses of paired serum and genital samples revealed significantly higher proportions of EB-specific antibodies in genital secretions compared to sera. Cervical and vaginal secretions and cytobrush specimens had similar proportions of EB-specific antibodies, suggesting any one of these genital sampling techniques could be used to quantify CT-specific antibodies when appropriate normalization methodologies are implemented. Overall, these results illustrate the need to investigate genital tract CT antibody responses, and our assay provides a useful quantitative tool to assess natural immunity in defined clinical groups and CT vaccine trials.  

4.1749           Improved yield of canine islet isolation from deceased donors

Harrington, S., Williams, S.J., Otte, V., Barchman, S., Jones, C., Ramachandran, K. and Stehno-Bittel, L. BMC Vet. Res., 13:264 (2017)   Background Canine diabetes is a strikingly prevalent and growing disease, and yet the standard treatment of a twice-daily insulin injection is both cumbersome to pet owners and only moderately effective. Islet transplantation has been performed with repeated success in canine research models, but has unfortunately not been made available to companion animals. Standard protocols for islet isolation, developed primarily for human islet transplantation, include beating-heart organ donation, vascular perfusion of preservation solutions, specialized equipment. Unfortunately, these processes are prohibitively complex and expensive for veterinary use. The aim of the study was to develop a simplified approach for isolating canine islets that is compatible with the financial and logistical restrictions inherent to veterinary medicine for the purpose of translating islet transplantation to a clinical treatment for canine diabetes. Results Here, we describe simplified strategies for isolating quality islets from deceased canine donors without vascular preservation and with up to 90 min of cold ischemia time. An average of more than 1500 islet equivalents per kg of donor bodyweight was obtained with a purity of 70% (N = 6 animals). Islets were 95% viable and responsive to glucose stimulation for a week. We found that processing only the body and tail of the pancreas increased isolation efficiency without sacrificing islet total yield. Islet yield per gram of tissue increased from 773 to 1868 islet equivalents when the head of the pancreas was discarded (N = 3/group). Conclusions In summary, this study resulted in the development of an efficient and readily accessible method for obtaining viable and functional canine islets from deceased donors. These strategies provide an ethical means for obtaining donor islets.  

4.1750           DNA strand breaks and TDP-43 mislocation are absent in the murine hSOD1G93A model of amyotrophic lateral sclerosis in vivo and in vitro

Pennendorf, D., Tadic, V., Witte, O., grosskreutz, J. and Kretz, A. PloS One, 12(8), e0183684 (2017)   Mutations in the human Cu/Zn superoxide dismutase type-1 (hSOD1) gene are common in familial amyotrophic lateral sclerosis (fALS). The pathophysiology has been linked to, e.g., organelle dysfunction, RNA metabolism and oxidative DNA damage conferred by SOD1 malfunction. However, apart from metabolically evoked DNA oxidation, it is unclear whether severe genotoxicity including DNA single-strand breaks (SSBs) and double-strand breaks (DSBs), originates from loss of function of nuclear SOD1 enzyme. Factors that endogenously interfere with DNA integrity and repair complexes in hSOD1-mediated fALS remain similarly unexplored. In this regard, uncontrolled activation of transposable elements (TEs) might contribute to DNA disintegration and neurodegeneration. The aim of this study was to elucidate the role of the fALS-causing hSOD1^G93A mutation in the generation of severe DNA damage beyond well-characterized DNA base oxidation. Therefore, DNA damage was assessed in spinal tissue of hSOD1^G93A-overexpressing mice and in corresponding motor neuron-enriched cell cultures in vitro. Overexpression of the hSOD1^G93A locus did not change the threshold for severe DNA damage per se. We found that levels of SSBs and DSBs were unaltered between hSOD1^G93A and control conditions, as demonstrated in post-mitotic motor neurons and in astrocytes susceptible to replication-dependent DNA breakage. Analogously, parameters indicative of DNA damage response processes were not activated in vivo or in vitro. Evidence for a mutation-related elevation in TE activation was not detected, in accordance with the absence of TAR DNA binding protein 43 (TDP-43) proteinopathy in terms of cytoplasmic mislocation or nuclear loss, as nuclear TDP-43 is supposed to silence TEs physiologically. Conclusively, the superoxide dismutase function of SOD1 might not be required to preserve DNA integrity in motor neurons, at least when the function of TDP-43 is unaltered. Our data establish a foundation for further investigations addressing functional TDP-43 interaction with ALS-relevant genetic mutations.  

4.1751           A versatile microfluidic device for high throughput production of microparticles and cell microencapsulation

Akbari, S., Pirbodaghi, T., Kamm, R.D. and Hammond, P.T. Lab on a Chip, 17, 2067-2075 (2017)   Biocompatible microparticles are valuable tools in biomedical research for applications such as drug delivery, cell transplantation therapy, and analytical assays. However, their translation into clinical research and the pharmaceutical industry has been slow due to the lack of techniques that can produce microparticles with controlled physicochemical properties at high throughput. We introduce a robust microfluidic platform for the production of relatively homogeneous microdroplets at a generation frequency of up to 3.1 MHz, which is about three orders of magnitude higher than the production rate of a conventional microfluidic drop maker. We demonstrated the successful implementation of our device for production of biocompatible microparticles with various crosslinking mechanisms and cell microencapsulation with high cell viability.  

4.1752           Controlled rotation and translation of spherical particles or living cells by surface acoustic waves

Bernard, I., Donikov, A.A., Marmottant, P., Rabaud, D., Poulain, C. and Thibault, P. Lab on a Chip, 17, 2470-2480 (2017)   We show experimental evidence of the acoustically-assisted micromanipulation of small objects like solid particles or blood cells, combining rotation and translation, using high frequency surface acoustic waves. This was obtained from the leakage in a microfluidic channel of two standing waves arranged perpendicularly in a LiNbO₃ piezoelectric substrate working at 36.3 MHz. By controlling the phase lag between the emitters, we could, in addition to translation, generate a swirling motion of the emitting surface which, in turn, led to the rapid rotation of spherical polystyrene Janus beads suspended in the channel and of human red and white blood cells up to several rounds per second. We show that these revolution velocities are compatible with a torque caused by the acoustic streaming that develops at the particles surface, like that first described by [F. Busse et al., J. Acoust. Soc. Am., 1981, 69(6), 1634–1638]. This device, based on standard interdigitated transducers (IDTs) adjusted to emit at equal frequencies, opens a way to a large range of applications since it allows the simultaneous control of the translation and rotation of hard objects, as well as the investigation of the response of cells to shear stress.  

4.1753           Toward a Droplet-Based Single-Cell Radiometric Assay

Gallina, M.E., Kim, T.J., Shelor, M., Vasquez, J., Mogersum, A., Kim, M., Tang, S.K.Y., Abbyad, P. and Pratx, G. Anal. Chem., 89(12), 6472-6481 (2017)   Radiotracers are widely used to track molecular processes, both in vitro and in vivo, with high sensitivity and specificity. However, most radionuclide detection methods have spatial resolution inadequate for single-cell analysis. A few existing methods can extract single-cell information from radioactive decays, but the stochastic nature of the process precludes high-throughput measurement (and sorting) of single cells. In this work, we introduce a new concept for translating radioactive decays occurring stochastically within radiolabeled single-cells into an integrated, long-lasting fluorescence signal. Single cells are encapsulated in radiofluorogenic droplets containing molecular probes sensitive to byproducts of ionizing radiation (primarily reactive oxygen species, or ROS). Different probes were examined in bulk solutions, and dihydrorhodamine 123 (DHRh 123) was selected as the lead candidate due to its sensitivity and reproducibility. Fluorescence intensity of DHRh 123 in bulk increased at a rate of 54% per Gy of X-ray radiation and 15% per MBq/ml of 2-deoxy-2-[¹⁸F]-fluoro-d-glucose ([¹⁸F]FDG). Fluorescence imaging of microfluidic droplets showed the same linear response, but droplets were less sensitive overall than the bulk ROS sensor (detection limit of 3 Gy per droplet). Finally, droplets encapsulating radiolabeled cancer cells allowed, for the first time, the detection of [¹⁸F]FDG radiotracer uptake in single cells through fluorescence activation. With further improvements, we expect this technology to enable quantitative measurement and selective sorting of single cells based on the uptake of radiolabeled small molecules.  

4.1754           Cyanidin-3-O-β-glucoside combined with its metabolite protocatechuic acid attenuated the activation of mice hepatic stellate cells

Jiang, X., Shen, T., tang, X., Yang, W., Guo, H. and Ling, w. Food Funct., 8, 2945-2957 (2017)   Previous studies indicated that cyanidin-3-O-β-glucoside (C3G) as a classical anthocyanin exerted an anti-fibrotic effect in the liver, but its bioavailability was quite low. This study was undertaken to explore the restraining effect of C3G and its metabolite protocatechuic acid (PCA) on the activation of hepatic stellate cells (HSCs). Our data demonstrated that the treatment of a carbon tetrachloride-treated mice model with C3G inhibited liver fibrosis and HSC activation. In vitro, both C3G and PCA preserved the lipid droplets and retinol in primary HSCs, and additionally inhibited the mRNA expression of α-smooth muscle actin and collagen I, but elevated the level of matrix metalloproteinase-2 and liver X receptors. Only PCA suppressed the levels of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) secreted from HSCs significantly. In addition, C3G and PCA inhibited the proliferation and migration of HSCs. In conclusion, PCA mainly explained the in vivo inhibiting effect of C3G on HSC activation and liver fibrosis.  

4.1755           Nanoemulsion-induced enzymatic crosslinking of tyramine-functionalized polymer droplets

Tom Kamperman, Sieger Henke, Bram Zoetebier, Niels Ruiterkamp, Rong Wang, Behdad Pouran, Harrie Weinans, Marcel Karperien and Jeroen Leijten

1. Mater. Chem. B, 5, 4835-4844 (2017)

  In situ gelation of water-in-oil polymer emulsions is a key method to produce hydrogel particles. Although this approach is in principle ideal for encapsulating bioactive components such as cells, the oil phase can interfere with straightforward presentation of crosslinker molecules. Several approaches have been developed to induce in-emulsion gelation by exploiting the triggered generation or release of crosslinker molecules. However, these methods typically rely on photo- or acid-based reactions that are detrimental to cell survival and functioning. In this work, we demonstrate the diffusion-based supplementation of small molecules for the in-emulsion gelation of multiple tyramine-functionalized polymers via enzymatic crosslinking using a H₂O₂/oil nanoemulsion. This strategy is compatible with various emulsification techniques, thereby readily supporting the formation of monodisperse hydrogel particles spanning multiple length scales ranging from the nano- to the millimeter. As proof of principle, we leveraged droplet microfluidics in combination with the cytocompatible nature of enzymatic crosslinking to engineer hollow cell-laden hydrogel microcapsules that support the formation of viable and functional 3D microtissues. The straightforward, universal, and cytocompatible nature of nanoemulsion-induced enzymatic crosslinking facilitates its rapid and widespread use in numerous food, pharma, and life science applications.  

4.1756           From single-molecule detection to next-generation sequencing: microfluidic droplets for high-throughput nucleic acid analysis

Ding, Y., Choo, J. and deMello, A.J. Microfluid. Nanofluid., 21:58 (2017)   Droplet-based microfluidic technologies have proved themselves to be of significant utility in the performance of high-throughput chemical and biological experiments. By encapsulating and isolating reagents within femtoliter–nanoliter droplet, millions of (bio) chemical reactions can be processed in a parallel fashion and on ultra-short timescales. Recent applications of such technologies to genetic analysis have suggested significant utility in low-cost, efficient and rapid workflows for DNA amplification, rare mutation detection, antibody screening and next-generation sequencing. To this end, we describe and highlight some of the most interesting recent developments and applications of droplet-based microfluidics in the broad area of nucleic acid analysis. In addition, we also present a cursory description of some of the most essential functional components, which allow the creation of integrated and complex workflows based on flowing streams of droplets.  

4.1757           Colonization with Helicobacter is concomitant with modified gut microbiota and drastic failure of the immune control of Mycobacterium tuberculosis

Majlessi, L., Sayes, F., Bureau, J-F., Pawlik, A., Michel, V., Jouvion, G., Huerre, M., Severgnini, M., Consolandi, C., Peano, C., Brosch, R., Touati, E. and Leclerc, C. Mucosal Immunol., 10, 1178-1189 (2017)   Epidemiological and experimental observations suggest that chronic microbial colonization can impact the immune control of other unrelated pathogens contracted in a concomitant or sequential manner. Possible interactions between Mycobacterium tuberculosis infection and persistence of other bacteria have scarcely been investigated. Here we demonstrated that natural colonization of the digestive tract with Helicobacter hepaticus in mice is concomitant with modification of the gut microbiota, subclinical inflammation, and drastic impairment of immune control of the growth of subsequently administered M. tuberculosis, which results in severe lung tissue injury. Our results provided insights upon the fact that this prior H. hepaticus colonization leads to failures in the mechanisms that could prevent the otherwise balanced cross-talk between M. tuberculosis and the immune system. Such disequilibrium ultimately leads to the inhibition of control of mycobacterial growth, outbreak of inflammation, and lung pathology. Among the dysregulated immune signatures, we noticed a correlation between the detrimental lung injury and the accumulation of activated T-lymphocytes. Our findings suggest that the impact of prior Helicobacter spp. colonization and subsequent M. tuberculosis parasitism might be greater than previously thought, which is a key point given that both species are among the most frequent invasive bacteria in human populations.  

4.1758           ADAR1 deletion induces NFκB and interferon signaling dependent liver inflammation and fibrosis

Be-Shoshan, S., Kagan, P., Sultan, M., Barabash, Z., Dor, C., Jacob-Hirsch, J., Harmelin, A., Pappo, O., Marcu-Malina, V., Ben-Ari, Z., Amariglio, N., Rechavi, G., Goldstein, I. and Safran, M. RNA Biol., 14(5), 587-602 (2017)   Adenosine deaminase acting on RNA (ADAR) 1 binds and edits double-stranded (ds) RNA secondary structures found mainly within untranslated regions of many transcripts. In the current research, our aim was to study the role of ADAR1 in liver homeostasis. As previous studies show a conserved immunoregulatory function for ADAR1 in mammalians, we focused on its role in preventing chronic hepatic inflammation and the associated activation of hepatic stellate cells to produce extracellular matrix and promote fibrosis. We show that hepatocytes specific ADAR1 knock out (KO) mice display massive liver damage with multifocal inflammation and fibrogenesis. The bioinformatics analysis of the microarray gene-expression datasets of ADAR1 KO livers reveled a type-I interferons signature and an enrichment for immune response genes compared to control littermate livers. Furthermore, we found that in vitro silencing of ADAR1 expression in HepG2 cells leads to enhanced transcription of NFκB target genes, foremost of the pro-inflammatory cytokines IL6 and IL8. We also discovered immune cell-independent paracrine signaling among ADAR1-depleted HepG2 cells and hepatic stellate cells, leading to the activation of the latter cell type to adopt a profibrogenic phenotype. This paracrine communication dependent mainly on the production and secretion of the cytokine IL6 induced by ADAR1 silencing in hepatocytes. Thus, our findings shed a new light on the vital regulatory role of ADAR1 in hepatic immune homeostasis, chiefly its inhibitory function on the crosstalk between the NFκB and type-I interferons signaling cascades, restraining the development of liver inflammation and fibrosis.  

4.1759           Thrombin-Induced Inflammation in Human Decidual Cells Is Not Affected By Heparin

Smrtka, M.P., Feng, L., Murtha, A.P. and Grotegut, C.A. Reproductive Sciences, 24(8), 1154-1163 (2017)   Objective: Thrombin (Thr) generation at the uteroplacental interface induces inflammation and weakens fetal membranes. Tissue factor (TF) is a powerful procoagulant that is increased by Thr in decidual cells (DCs). The TF expression may play an important role in modulating Thr-induced inflammation. The purpose of this study was to assess the effect of heparin, including nonanticoagulant (desulfated) heparins, on basal and Thr-induced expression of TF and inflammatory cytokines in DCs. Methods: Fetal membranes were collected from term pregnancies undergoing unlabored cesarean delivery and then DCs were isolated and cultured. Third passage DCs were conditioned in defined media for 1 week and then treated with 1 of the 4 heparins (enoxaparin, unfractionated heparin, and 2 desulfated heparins) with and without Thr (2.5 U/mL) for 24 hours. Supernatant levels of interleukin (IL) 6, IL-8, IL-10, tumor necrosis factor α, and interferon γ (IFN-γ) were determined by enzyme-linked immunosorbent assay. Western blots were performed on cell lysates to determine TF expression. A Kruskal-Wallis test was used to compare cytokine concentrations and normalized TF expression among treatments. Results: Treatment of DCs with Thr alone increased the expression of TF, IL-6, IL8, IL-10, and IFN-γ compared to basal levels (P < .05 for each). Cotreatment of DCs with Thr and any of the tested heparins did not decrease the expression of TF or inflammatory cytokines compared to treatment with Thr alone. Discussion: Heparins do not appear to affect basal or Thr-induced expression of TF or inflammatory cytokines in human term DCs. Additional work is needed to determine whether nonanticoagulant heparins can reduce inflammation and membrane weakening due to bleeding in pregnancy.  

4.1760           Double conjugated nanogels for selective intracellular drug delivery

Mauri, E., Veglianese, P., Papa, S., Mariani, A., De Paola, M., Rigamonti, R., Chincarini, G.M.F., Vismara, I., Rimondo, S., Sacchetti, A. and Rossi, F. RSC Adv., 7, 30345-30356 (2017)   One of the most important drawbacks of nanomedicine is related to the unwanted rapid diffusion of drugs loaded within nanocarriers towards the external biological environment, according to the high clearance of body fluids. Therefore, colloids can carry only a small amount of their initial content in the target district, limiting their pharmacological activity and then the therapy. To overcome this limitation, we synthesized double conjugated nanogels: the first click strategy (1,3 Huisgen cycloaddition) guarantees the traceability of nanogels while the second one (disulfide bond) links drug molecules to polymeric chains. In this study, we proposed the above-mentioned double strategy and we validated the synthesized colloids and the selective release kinetics in microglia cells, dramatically involved in several diseases of the central nervous system. Cleavable linked drugs prove to be a promising tool for the selective administration of pharmacological compounds in microglia cells and potentially in many others counteracting some relevant events.  

4.1761           EphA receptors and ephrin-A ligands are upregulated by monocytic differentiation/maturation and promote cell adhesion and protrusion formation in HL60 monocytes

Mukai, M., Suruga, N., Saeki, N. and ogawa, K. BMC Cell Biol., 18:28 (2017)   Background Eph signaling is known to induce contrasting cell behaviors such as promoting and inhibiting cell adhesion/spreading by altering F-actin organization and influencing integrin activities. We have previously demonstrated that EphA2 stimulation by ephrin-A1 promotes cell adhesion through interaction with integrins and integrin ligands in two monocyte/macrophage cell lines. Although mature mononuclear leukocytes express several members of the EphA/ephrin-A subclass, their expression has not been examined in monocytes undergoing during differentiation and maturation. Results Using RT-PCR, we have shown that EphA2, ephrin-A1, and ephrin-A2 expression was upregulated in murine bone marrow mononuclear cells during monocyte maturation. Moreover, EphA2 and EphA4 expression was induced, and ephrin-A4 expression was upregulated, in a human promyelocytic leukemia cell line, HL60, along with monocyte differentiation toward the classical CD14⁺⁺CD16⁻ monocyte subset. Using RT-PCR and flow cytometry, we have also shown that expression levels of αL, αM, αX, and β2 integrin subunits were upregulated in HL60 cells along with monocyte differentiation while those of α4, α5, α6, and β1 subunits were unchanged. Using a cell attachment stripe assay, we have shown that stimulation by EphA as well as ephrin-A, likely promoted adhesion to an integrin ligand-coated surface in HL60 monocytes. Moreover, EphA and ephrin-A stimulation likely promoted the formation of protrusions in HL60 monocytes. Conclusions Notably, this study is the first analysis of EphA/ephrin-A expression during monocytic differentiation/maturation and of ephrin-A stimulation affecting monocyte adhesion to an integrin ligand-coated surface. Thus, we propose that monocyte adhesion via integrin activation and the formation of protrusions is likely promoted by stimulation of EphA as well as of ephrin-A.  

4.1762           Non-equilibrium Inertial Separation Array for High-throughput, Large-volume Blood Fractionation

Mutlu, B.R., Smith, K.C., Edd, J.F., Nadaar, P., Dlamini, M., Kapur, R. and Toner, M. Scientific Reports, 7:9915 (2017)   Microfluidic blood processing is used in a range of applications from cancer therapeutics to infectious disease diagnostics. As these applications are being translated to clinical use, processing larger volumes of blood in shorter timescales with high-reliability and robustness is becoming a pressing need. In this work, we report a scaled, label-free cell separation mechanism called non-equilibrium inertial separation array (NISA). The NISA mechanism consists of an array of islands that exert a passive inertial lift force on proximate cells, thus enabling gentler manipulation of the cells without the need of physical contact. As the cells follow their size-based, deterministic path to their equilibrium positions, a preset fraction of the flow is siphoned to separate the smaller cells from the main flow. The NISA device was used to fractionate 400 mL of whole blood in less than 3 hours, and produce an ultrapure buffy coat (96.6% white blood cell yield, 0.0059% red blood cell carryover) by processing whole blood at 3 mL/min, or ∼300 million cells/second. This device presents a feasible alternative for fractionating blood for transfusion, cellular therapy and blood-based diagnostics, and could significantly improve the sensitivity of rare cell isolation devices by increasing the processed whole blood volume.  

4.1763           Image-based closed-loop feedback for highly mono-dispersed microdroplet production

Crawford, D.F., Smith, C.A. and Whyte, G. Scientific Reports, 7:10545 (2017)   Micron-scale droplets isolated by an immiscible liquid can provide miniaturised reaction vessels which can be manipulated in microfluidic networks, and has seen a rapid growth in development. In many experiments, the precise volume of these microdroplets is a critical parameter which can be influenced by many external factors. In this work, we demonstrate the combination of imaging-based feedback and pressure driven pumping to accurately control the size of microdroplets produced in a microfluidic device. The use of fast-response, pressure-driving pumps allows the microfluidic flow to be quickly and accurately changed, while directly measuring the droplet size allows the user to define the more meaningful parameters of droplet size and generation frequency rather than flow rates or pressures. The feedback loop enables the drift correction of pressure based pumps, and leads to a large increase in the mono-dispersity of the droplets produced over long periods. We also show how this can be extended to control multiple liquid flows, allowing the frequency of droplet formation or the average concentration of living cells per droplet to be controlled and kept constant.  

4.1764           MicroRNAs contribute to postnatal development of laminar differences and neuronal subtypes in the rat medial entorhinal cortex

Olsen, L.C., O’Reilly, K.C., Liabakk, N.B., Witter, M.P. and Sætrum, Å. Brain Struct. Funct., 222, 3107-3126 (2017)   The medial entorhinal cortex (MEC) is important in spatial navigation and memory formation and its layers have distinct neuronal subtypes, connectivity, spatial properties, and disease susceptibility. As little is known about the molecular basis for the development of these laminar differences, we analyzed microRNA (miRNA) and messenger RNA (mRNA) expression differences between rat MEC layer II and layers III–VI during postnatal development. We identified layer and age-specific regulation of gene expression by miRNAs, which included processes related to neuron specialization and locomotor behavior. Further analyses by retrograde labeling and expression profiling of layer II stellate neurons and in situ hybridization revealed that the miRNA most up-regulated in layer II, miR-143, was enriched in stellate neurons, whereas the miRNA most up-regulated in deep layers, miR-219-5p, was expressed in ependymal cells, oligodendrocytes and glia. Bioinformatics analyses of predicted mRNA targets with negatively correlated expression patterns to miR-143 found that miR-143 likely regulates the Lmo4 gene, which is known to influence hippocampal-based spatial learning.  

4.1765           Pancreatic stellate cell activation is regulated by fatty acids and ER stress

Ben-Harorosh, Y., Anosov, M., Salem, H., yatchenko, Y. and Birk, R. Exp. Cell Res., 359, 76-85 (2017)   Introduction Pancreatic pathologies are characterized by a progressive fibrosis process. Pancreatic stellate cells (PSC) play a crucial role in pancreatic fibrogenesis. Endoplasmic reticulum (ER) stress emerges as an important determinant of fibrotic remodeling. Overload of fatty acids (FA), typical to obesity, may lead to lipotoxic state and cellular stress. Aim To study the effect of different lipolytic challenges on pancreatic ER stress and PSC activation. Methods Primary PSCs were exposed to different FAs, palmitate (pal) and oleate (ole), at pathophysiological concentrations typical to obese state, and in acute caerulein-induced stress (cer). PSC activation and differentiation were analyzed by measuring fat accumulation (oil-red staining and quantitation), proliferation (cells count) and migration (wound- healing assay). PSC differentiation markers (α-sma, fibronectin, tgf-β and collagen secretion), ER stress unfolded protein response and immune indicators (Xbp1, CHOP, TNF-α, IL-6) were analyzed at the transcript and protein expression levels (quantitative RT-PCR and western blotting). Results PSC exposure to pal and ole FAs (500 µM) increased significantly fat accumulation. Proliferation and migration analysis demonstrated that ole FA retained PSC activation, while exposure to pal FA significantly halted proliferation rate and delayed migration. Cer significantly augmented PSC differentiation markers α- sma, fibronectin and collagen, and ER stress and inflammation markers including Xbp1, CHOP, TNF-α and IL-6. The ole FA treatment significantly elevated PSC differentiation markers α-sma, fibronectin and collagen secretion. PSC ER stress was demonstrated following pal treatment with significant elevation of Xbp1 splicing and CHOP levels. Conclusion Exposure to pal FA halted PSC activation and differentiation and elevated ER stress markers, while cer and ole exposure significantly induced activation, differentiation and fibrosis. Thus, dietary FA composition should be considered and optimized to regulate PSC activation and differentiation in pancreatic pathologies.  

4.1766           Protocol for High-Content Screening for the Impact of Overexpressed MicroRNAs on Primary Motor Neurons

Yardeni, T. and Hornstein, E. Neuromethods, 128, 11-19 (2017)   In this chapter we provide a protocol for the design and usage of automated, high-content microscopy screening that enables the investigation of microRNA (miRNA) impact on primary motor neuron. High-content screening (HCS) platforms facilitate superior precision in research and are scalable to study in parallel multiple genetic, molecular, or cellular conditions. miRNAs are critical for neuronal function and for brain integrity and are considered attractive candidate targets for therapy in many neuropathologies. Therefore, HCS platforms provide a novel paradigm for exploring the impact of miRNA expression, applicable for functional pathways discovery in an academic setting, or towards development of therapeutics in the pharma industry.  

4.1767           Analysis of Pathological Activities of CCN Proteins in Fibrotic Diseases: Liver Fibrosis

Chen, L. and Brigstock, D.R. Methods in Mol. Biol., 1489, 445-463 (2017)   Hepatic fibrosis is a complex pathology arising from chronic injury. Pathological features are dominated by the excessive production of extracellular matrix proteins, particularly collagens which are deposited as insoluble scar material that can compromise tissue function. Fibrosis in the liver can often be assessed by staining for collagen in tissue sections and this is an approach that is widely used for grading of fibrosis in human biopsies. However, the recognition of the molecular components that drive fibrosis, including CCN proteins, and the involvement of hepatic stellate cells (HSC) as the principal collagen-producing cells in fibrosing liver, has resulted in a wide variety of molecular and cellular approaches to study the pathogenesis of fibrosis both in vivo and in vitro.  

4.1768           Using Cloning to Amplify Neuronal Genomes for Whole-Genome Sequencing and Comprehensive Mutation Detection and Validation

Hazen, J.L., Duran, M.A., Smith, R.P., Rodriguez, A.R., Martin, G.S., Kupriyanov, S., Hall, I.M. and Baldwin, K.K. Neuromethods, 131, 163-185 (2017)   Recent studies of somatic mutation in neurons and other cell types suggest that somatic cells can acquire hundreds to thousands of new mutations over their lifetimes. Each individual mutation can have extremely low prevalence, with many mutations restricted to a single cell. Because of their rarity, somatic mutations can be challenging to detect and reliably distinguish from false-positive calls arising from amplification, sequencing, or bioinformatic methods. In these scenarios, a variety of methods are required to compensate for the limited applicability and technical artifacts inherent in any single approach. In the method we describe, somatic cell nuclear transfer (SCNT, also known as cloning) is used to reprogram single neurons to blastocysts from which we derive embryonic stem cells. Division of these cells faithfully amplifies the neuronal genome for next-generation sequencing and genome-wide mutation detection. This approach allows the detection of false positives due to amplification artifacts and is applicable to all classes of mutations. While it is both sensitive and reliable, our method is lower throughput than single-cell sequencing-based approaches and may also fail to amplify the most severely compromised neuronal genomes. In this chapter, we outline current methods for generating neuron-derived SCNT embryonic cell lines, discuss best practices for genome-wide mutation detection, and address the advantages and limitations of this approach.  

4.1769           Cellular or Exosomal microRNAs Associated with CCN Gene Expression in Liver Fibrosis

Chen, L. and Brigstock, D.R. Methods in Mol. Biol., 1489, 465-480 (2017)   Liver fibrosis occurs during chronic injury and represents, in large part, an exaggerated matrigenic output by hepatic stellate cells (HSCs) which become activated as a result of injury-induced signaling pathways in parenchymal and infl ammatory cells (hepatocytes, macrophages, etc.). The molecular components in these pathways (e.g., CCN proteins) are modulated by transcription factors as well as by factors such as microRNAs (miRs) that act posttranscriptionally. MiRs are small (~23 nt) noncoding RNAs that regulate gene expression by specifi cally interacting with the 3′ untranslated region (UTR) of target gene mRNA to repress translation or enhance mRNA cleavage. As well as acting in their cells of production, miRs (and other cellular constituents such as mRNAs and proteins) can be liberated from their cells of origin in nanovesicular membrane exosomes, which traverse the intercellular spaces, and can be delivered to neighboring cells into which they release their molecular payload, causing alterations in gene expression in the target cells. Here we summarize some of the experimental approaches for studying miR action and exosomal traff cking between hepatic cells. Insights into the mechanisms involved will yield new information about how hepatic fi brosis is regulated and, further, may identify new points of therapeutic intervention.  

4.1770           Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells

Cole, R.H., Tang, S-Y., Siltanen, C.A., Shahi, P., Zhang, J.Q., Poust, S., Gartner, Z.J. and Abate, A.R. PNAS, 114(33), 8728-8733 (2017)   Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.  

4.1771           Paired related homeobox protein 1 regulates PDGF-induced chemotaxis of hepatic stellate cells in liver fibrosis

Gong, J., Han, J., He, J., Liu, J., Han, P., Wang, Y., Li, M., Li, D., Ding, X., Du, Z., Liao, J. and Tian, D. Lab. Invest., 97(9), 1020-1032 (2017)   Activation of the platelet-derived growth factor (PDGF)/PDGF beta receptor (PDGFβR) axis has a critical role in liver fibrosis. However, the mechanisms that regulate the PDGF signaling are yet to be elucidated. The present study demonstrates that paired related homeobox protein 1 (Prrx1) is involved in PDGF-dependent hepatic stellate cell (HSCs) migration via modulation of the expression of metalloproteinases MMP2 and MMP9. PDGF elevated the level of Prrx1 through the activation of ERK/Sp1 and PI3K/Akt/Ets1 pathways. In vivo, an adenoviral-mediated Prrx1 shRNA administration attenuated liver fibrosis in thioacetamide-induced fibrotic models. These studies reveal a role of Prrx1 as a modulator of PDGF-dependent signaling in HSCs, and inhibiting its expression may offer a therapeutic approach for hepatic fibrosis.  

4.1772           Warmer night-time temperature promotes microbial heterotrophic activity and modifies stream sediment community

Freixa, A., Acuna, V., Casellas, M., Percheva, S. and Romani, A.M. Global Change Biol., 23(9), 3825-3837 (2017)   Diel temperature patterns are changing because of global warming, with higher temperatures being predicted to be more pronounced at night. Biological reactions are temperature dependent, with some occurring only during the daylight hours (e.g., light photosynthesis) and other during the entire day (e.g., respiration). Consequently, we expect the modification of daily temperature cycles to alter microbial biological reactions in stream sediments. Here, we aimed to study the effect of warming and changes of the diel temperature patterns on stream sediment biofilm functions tied to organic carbon decomposition, as well as on biofilm meiofaunal community structure. We performed an eight-week experiment with 12 artificial streams subjected to three different diel temperature patterns: warming, warmer nights and control. Significant effects of warming on biofilm function and structure were mainly detected in the long term. Our results showed that warming altered biofilm function, especially in the warmer nights’ treatment, which enhanced β-glucosidase enzyme activity. Interestingly, clear opposite diel patterns were observed for dissolved organic carbon and β-glucosidase activity, suggesting that, at night, sediment bacteria quickly consume the input of photosynthetic dissolved organic carbon labile compounds created during light-time. The biofilm structure was also altered by warming, as both warming and warmer night treatments enhanced copepod abundance and diminished abundances of turbellaria and nematodes, which, in turn, controlled bacterial, algal and ciliate communities. Overall, we conclude that warming has strong effect on sediment biofilm structure and enhanced microbial organic matter degradation which might, consequently, affect higher trophic levels and river carbon cycling.  

4.1773           Effects of supplementation of iodixanol to semen extender on quality and fertilization ability of frozen–thawed Thai native bull sperm

Chuawongboon, P., Sirisathien, S., Pongpeng, J., Sakhong, D., Nagai, T. and Vongpralub, T. Animal Sci. J., 88(9), 1311-1320 (2017)   This study investigates the effects of iodixanol supplementation in varied concentrations to Tris egg yolk (TEY) extender on the quality and fertilization ability of frozen–thawed sperm of Thai native bulls. Each ejaculate was divided into four different groups, as follows: sperm were treated with TEY extender (control group) and TEY extender supplemented with three different concentrations of iodixanol (1.25%, 2.50% and 5.00%). Semen straws were frozen in liquid nitrogen vapor. After thawing, sperm motility characteristics, viability, plasma membrane integrity and acrosome integrity were determined. Also, frozen–thawed spermatozoa from all groups were used for in vitro fertilization and artificial insemination (AI) in natural estrus Thai native cows. The results showed that the post-thaw quality of the 2.50% iodixanol group was superior to the other iodixanol groups (P < 0.05). However, iodixanol had no beneficial effect on post-thaw sperm in vitro fertilization ability and pregnancy rate after AI (P > 0.05). It can be concluded that the supplementation of 2.50% iodixanol extender significantly improves the progressive motility, viability, plasma membrane integrity and acrosome integrity of cryopreserved semen from Thai native bulls, but it has no beneficial effect on in vitro fertilization ability and pregnancy rate after AI.  

4.1774           Scaffold-Free Liver-On-A-Chip with Multiscale Organotypic Cultures

Weng, Y-S., Chang, S-F., Shih, M-C., Tseng, S-H. and Lai, C-H. Advanced Materials, 29, 1701545 (2017)   The considerable advances that have been made in the development of organotypic cultures have failed to overcome the challenges of expressing tissue-specific functions and complexities, especially for organs that require multitasking and complex biological processes, such as the liver. Primary liver cells are ideal biological building blocks for functional organotypic reconstruction, but are limited by their rapid loss of physiological integrity in vitro. Here the concept of lattice growth used in material science is applied to develop a tissue incubator, which provides physiological cues and controls the 3D assembly of primary cells. The cues include a biological growing template, spatial coculture, biomimetic radial flow, and circulation in a scaffold-free condition. The feasibility of recapitulating a multiscale physiological structural hierarchy, complex drug clearance, and zonal physiology from the cell to tissue level in long-term cultured liver-on-a-chip is demonstrated. These methods are promising for future applications in pharmacodynamics and personal medicine.  

4.1775           Complement Protein C3 Suppresses Axon Growth and Promotes Neuron Loss

Peterson, S.L., Nguyen, H.X., Mendez, O.A. and Anderson, A.J. Scientific Reports, 7:12904 (2017)   The inflammatory response to spinal cord injury (SCI) involves localization and activation of innate and adaptive immune cells and proteins, including the complement cascade. Complement C3 is important for the classical, alternative, and lectin pathways of complement activation, and its cleavage products C3a and C3b mediate several functions in the context of inflammation, but little is known about the potential functions of C3 on regeneration and survival of injured neurons after SCI. We report that 6 weeks after dorsal hemisection with peripheral conditioning lesion, C3^−/− mice demonstrated a 2-fold increase in sensory axon regeneration in the spinal cord in comparison to wildtype C3^+/+ mice. In vitro, addition of C3 tripled both myelin-mediated neurite outgrowth inhibition and neuron loss versus myelin alone, and ELISA experiments revealed that myelin serine proteases cleave C3 to generate active fragments. Addition of purified C3 cleavage products to cultured neurons suggested that C3b is responsible for the growth inhibitory and neurotoxic or anti-adhesion activities of C3. These data indicate that C3 reduces neurite outgrowth and neuronal viability in vitro and restricts axon regeneration in vivo, and demonstrate a novel, non-traditional role for this inflammatory protein in the central nervous system.  

4.1776           Antidepressants promote formation of heterocomplexes of dopamine D2 and somatostatin subtype 5 receptors in the mouse striatum

Szafran-Pilch, K., Faron-Gorecka, A., Kolasa, M., Zurawek, D., Szlachta, M., Solich, J., Kusmider, M. and Dziedzicka-Wasylewska, M. Brain Res. Bull.,135, 92-97 (2017)   The interaction between the dopaminergic and somatostatinergic systems is considered to play a potential role in mood regulation. Chronic administration of antidepressants influences release of both neurotransmitters. The molecular basis of the functional cooperation may stem from the physical interaction of somatostatin receptor subtypes and dopamine D2 receptors since they colocalize in striatal interneurons and were shown to undergo ligand-dependent heterodimerization in heterologous expression systems. In present study we adapted in situ proximity ligation assay to investigate the occurrence of D2-Sst5 receptor heterocomplexes, and their possible alterations in the striatum of mice treated acutely and repeatedly (21 days) with antidepressant drugs of different pharmacological profiles (escitalopram and desipramine). Additionally we analysed number of heterocomplexes in primary striatal neuronal cultures incubated with both antidepressant drugs for 1 h and 6 days. The studies revealed that antidepressants increase formation of D2-Sst5 receptors heterodimers. These findings provide interesting evidence that dopamine D2 and somatostatin Sst5 heterodimers may be considered as potential mediators of antidepressant effects, since the heterodimerization of these receptors occurs in native brain tissue as well as in primary striatal neuronal cultures where receptors are expressed at physiological levels.  

4.1777           Genome Editing of Plants

Songstad, D.D., Petolino, J.F., Voytas, D.F. and Reichert, N.A. Critical Reviews in Plant Sci., 36(1), 1-23 (2017)   Genome editing in organisms via random mutagenesis is a naturally occurring phenomenon. As a technology, genome editing has evolved from the use of chemical and physical mutagenic agents capable of altering DNA sequences to biological tools such as designed sequence-specific nucleases (SSN) to produce knock-out (KO) or knock-in (KI) edits and Oligonucleotide Directed Mutagenesis (ODM) where specific nucleotide changes are made in a directed manner resulting in custom single nucleotide polymorphisms (SNPs). Cibus' SU Canola™, which the US Department of Agriculture (USDA) views as non-genetically modified (non-GM), is Cibus' first commercial product arising from plant genome editing and had its test launch in 2014. Regulatory aspects of the various genome editing tools will be discussed.  

4.1778           Molecular and expression analysis of the Allograft inflammatory factor 1 (AIF-1) in the coelomocytes of the common sea urchin Paracentrotus lividus

Barca, A., Vacca, F., Viziolo, J., Grago, F., Vertrugna, c., Verri, T. and Pagliara, P. Fish & Shellfish Immunol., 71, 136-143 (2017)   Allograft inflammatory factor 1 (AIF-1) is a highly conserved gene involved in inflammation, cloned and characterized in several evolutionary distant animal species. Here, we report the molecular identification, characterization and expression of AIF-1 from the common sea urchin Paracentrotus lividus. In this species, AIF-1 encodes a predicted 151 amino acid protein with high similarity to vertebrate AIF-1 proteins. Immunocytochemical analyses on coelomocytes reveal localization of the AIF-1 protein in amoebocytes (perinuclear cytoplasmic zone) and red sphaerulocytes (inside granules), but not in vibratile cells and colorless sphaerula cells. The significant increase of AIF-1 expression (mRNA and protein) found in the coelomocytes of the sea urchin after Gram + bacterial challenge suggests the involvement of AIF-1 in the inflammatory response. Our analysis on P. lividus AIF-1 contributes to elucidate AIF-1 function along the evolutionary scale and consolidate the key evolutionary position of echinoderms throughout metazoans with respect to the common immune paths.

4.1779           Proteomic Analysis Reveals Dab2 Mediated Receptor Endocytosis Promotes Liver Sinusoidal Endothelial Cell Dedifferentiation

Lao, Y., Li, Y., Hou, Y., Chen, H., Qiu, B., Lin, W., Sun, A., Wei, H., Jiang, Y. and He, F. Scientific Reports, 7, 13456 (2017)   Sinusoidal dedifferentiation is a complicated process induced by several factors, and exists in early stage of diverse liver diseases. The mechanism of sinusoidal dedifferentiation is poorly unknown. In this study, we established a NaAsO2-induced sinusoidal dedifferentiation mice model. Liver sinusoidal endothelial cells were isolated and isobaric tag for relative and absolute quantitation (iTRAQ) based proteomic approach was adopted to globally examine the effects of arsenic on liver sinusoidal endothelial cells (LSECs) during the progression of sinusoidal dedifferentiation. In all, 4205 proteins were identified and quantified by iTRAQ combined with LC-MS/MS analysis, of which 310 proteins were significantly changed in NaAsO2 group, compared with the normal control. Validation by western blot showed increased level of clathrin-associated sorting protein Disabled 2 (Dab2) in NaAsO2 group, indicating that it may regulate receptor endocytosis, which served as a mechanism to augment intracellular VEGF signaling. Moreover, we found that knockdown of Dab2 reduced the uptake of VEGF in LSECs, furthermore blocking VEGF-mediated LSEC dedifferentiation and angiogenesis.  

4.1780           The impact of allogenic blood transfusion on the outcomes of total pancreatectomy with islet autotransplantation

Yoshimatsu, G., Shahbazov, R., Saracino, G., Lawrence, M.C., Kim, P.T., Onaca, N., Beecherl, E.E., Naziruddin, B. and Levy, M.F. Am. J. Surg., 214, 849-855 (2017)   Background Allogenic blood transfusion (ABT) may be needed for severe bleeding during total pancreatectomy with autotransplantation (TPIAT), but may induce inflammation. This study investigated the impact of ABT. Methods With a population of 83 patients who underwent TPIAT from 2006 to 2014, this study compared cytokine levels, patient characteristics, islet characteristics, metabolic outcomes, insulin requirements, and hemoglobin A1c for those who received a blood transfusion (BT) versus no blood transfusion (NBT). Results Initially, proinflammatory cytokines were moderately higher in the BT group than the NBT group. Despite longer procedures and more severe bleeding, the BT group had similar values to the NBT group for insulin requirements, serum C-peptide, hemoglobin A1c, and insulin independence rate. The probability of insulin independence was slightly higher in patients receiving ≥3 units of blood. Conclusion ABT induced elevation of proinflammatory cytokines during the perioperative period in TPIAT, but these changes did not significantly change posttransplant islet function.  

4.1781           Bio-enriched Pleurotus mushrooms for deficiency control and improved antioxidative protection of human platelets?

Poniedzialek, B., Mleczek, M., Niedzielski, P., Siwulski, M., Gasecka, m., Kozak, L. and Komosa, P. Eur. Food Res. Technol., 243, 2187-2198 (2017)   The study investigated effect of substrate supplementation with Se alone or in combination with Cu or/and Zn Se on (1) the growth of Pleurotus ostreatus and Pleurotus eryngii; (2) elements accumulation in mushrooms; (3) the antioxidant activities of bio-enriched mushroom extracts in human platelets. The accumulation of elements generally increased over concentration gradient reaching its maximum at 1.2 mM for P. ostreatus and P. eryngii: (1) over 100 and 80 mg kg⁻¹ of Se, respectively (Se supplementation); (2) over 15 and 30 mg kg⁻¹ of Cu, respectively (Se+Cu); (3) over 30 and 85 mg kg⁻¹ of Zn, respectively. Se was predominantly accumulated as an organic fraction. Contrary to P. eryngii, the P. ostreatus biomass decreased with substrate elements concentration but was satisfactory up to 0.9 mM of Se, Se+Cu and Se+Zn. The Se+Cu+Zn model yielded low biomass and elements accumulation. Extracts from mushrooms bio-enriched with Se and Se+Zn (0.6–1.2 mM) revealed significant antioxidant activities in human platelets by ameliorating reactive oxygen species (ROS) and preventing lipid peroxidation. The study demonstrated the potential application of Pleurotus mushrooms as functional food products bio-enriched with essential elements. ROS inhibition by extracts of these mushrooms may be useful in control of platelets activation cascade.  

4.1782           Bacillus anthracis lethal toxin negatively modulates ILC3 function through perturbation of IL-23-mediated MAPK signaling

Seshadri, S., Allan, D.S., Carlyle, J.R. and Zenewicz, L.A. PloS Pathogens, 13(10), e1006690 (2017)   Bacillus anthracis, the causative agent of anthrax, secretes lethal toxin that down-regulates immune functions. Translocation of B. anthracis across mucosal epithelia is key for its dissemination and pathogenesis. Group 3 innate lymphocytes (ILC3s) are important in mucosal barrier maintenance due to their expression of the cytokine IL-22, a critical regulator of tissue responses and repair during homeostasis and inflammation. We found that B. anthracis lethal toxin perturbed ILC3 function in vitro and in vivo, revealing an unknown IL-23-mediated MAPK signaling pathway. Lethal toxin had no effects on the canonical STAT3-mediated IL-23 signaling pathway. Rather lethal toxin triggered the loss of several MAP2K kinases, which correlated with reduced activation of downstream ERK1/2 and p38, respectively. Inhibition studies showed the importance of MAPK signaling in IL-23-mediated production of IL-22. Our finding that MAPK signaling is required for optimal IL-22 production in ILC3s may lead to new approaches for targeting IL-22 biology.  

4.1783           Isolation and Culture of Primary Murine Hepatic Stellate Cells

Weiskirchen, S., Tag, C.G., Sauer-Lehnen, S., Tacke, F. and Weiskirchen, R. Methods in Mol. Biol., 1627, 165-191 (2017)   Hepatic stellate cells (HSCs) are found in the perisinusoidal space of the liver (i.e., the space of Dissé). They represent 5–8% of the total number of liver cells. In normal liver, these cells have a quiescent phenotype and are characterized by numerous fat vacuoles that store vitamin A in a form of retinyl ester. In injured liver, these cells transdifferentiate into a myofibroblast phenotype, become highly proliferative and are responsible for excess collagen synthesis and deposition during fibrosis. Due to their exceptional pathophysiological relevance, several isolation and purification protocols of primary HSCs have been established that provide the basis for studying HSC biology in vitro. We here describe a method for high-purity isolation of HSCs from mice. This protocol includes the enzymatic digestion of the liver tissue by pronase and collagenase, cellular enrichment by centrifugation of the crude cell suspension through a Nycodenz density gradient, and a final (optional) flow cytometric enrichment that allows generating ultrapure HSC fractions.  

4.1784           Isolation, Purification, and Culture of Primary Murine Sensory Neurons

Katzenell, S., Cabrera, J.R., North, B.J. and Leib, D.A. Methods in Mol. Biol., 1656, 229-251 (2017)   Cultured primary neurons have been of extraordinary value for the study of neuronal anatomy, cell biology, and physiology. While use of neuronal cell lines has ease and utility, there are often caveats that arise due to their mitotic nature. This methods article presents detailed methodology for the preparation, purification, and culture of adult murine sensory neurons for the study of herpes simplex virus lytic and latent infections. While virology is the application for our laboratory, these cultures also have broad utility for neurobiologists and cell biologists. While these primary cultures have been highly informative, the methodology is challenging to many investigators. Through publication of this highly detailed protocol, it is our hope that the use of this culture system can spread in the field to allow more rapid progress in furthering our understanding of neurotropic virus infection.  

4.1785           Lectins identify distinct populations of coelomocytes in Strongylocentrotus purpuratus

Liao, W-Y., Fugmann, S.D. PloS One, 12(11), e0187987 (2017)   Coelomocytes represent the immune cells of echinoderms, but detailed knowledge about their roles during immune responses is very limited. One major challenge for studying coelomocyte biology is the lack of reagents to identify and purify distinct populations defined by objective molecular markers rather than by morphology-based classifications that are subjective at times. Glycosylation patterns are known to differ significantly between cell types in vertebrates, and furthermore they can vary depending on the developmental stage and activation states within a given lineage. Thus fluorescently labeled lectins that recognize distinct glycan structures on cell surface proteins are routinely used to identify discrete cell populations in the vertebrate immune system. Here we now employed a panel of fifteen fluorescently-labeled lectins to determine differences in the glycosylation features on the surface of Strongylocentrotus purpuratus coelomocytes by fluorescence microscopy and flow cytometry. Eight of the lectins (succinylated wheat germ agglutinin, Len culinaris lectin, Pisum sativum agglutinin, Saphora japonica agglutinin, Solanum tuberosum lectin, Lycopersicon esculentum lectin, Datura stramonium lectin, Vicia villosa lectin) showed distinct binding patterns to fixed and live cells of three major coelomocyte classes: phagocytic cells, red spherule cells, and vibratile cells. Importantly, almost all lectins bound only to a subgroup of cells within each cell type. Lastly, we established fluorescently-labeled lectin-based fluorescence activated cell sorting as a strategy to purify distinct S. purpuratus coelomocyte (sub-)populations based on molecular markers. We anticipate that this will become a routine approach in future studies focused on dissecting the roles of different coelomocytes in echinoderm immunity.  

4.1786           Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing

Tang, Q. et al

1. Exp. Med., 214(10), 2875-2887 (2017)

  Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA–protein kinase catalytic subunit (prkdc), interleukin-2 receptor γ a (il2rga), and double-homozygous–mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish.  

4.1787           A novel Cre-inducible knock-in ARL13B-tRFP fusion cilium reporter

Schmitz, F., Burtscher, I., Stauber, M., Gossler, A. and Lickert, H. Genesis, 55, e23073 (2017)   Cilia play a major role in the regulation of numerous signaling pathways and are essential for embryonic development. Mutations in genes affecting ciliary function can cause a variety of diseases in humans summarized as ciliopathies. To facilitate the detection and visualization of cilia in a temporal and spatial manner in mouse tissues, we generated a Cre-inducible cilium-specific reporter mouse line expressing an ARL13B-tRFP fusion protein driven by a CMV enhancer/chicken β actin promotor (pCAG) from the Hprt locus. We detected bright and specific ciliary signals by immunostainings of various mono- and multiciliated tissues and by time-lapse live-cell analysis of cultured embryos and organ explant cultures. Additionally, we monitored cilium assembly and disassembly in embryonic fibroblast cells using live-cell imaging. Thus, the ARL13B-tRFP reporter mouse strain is a valuable tool for the investigation of ciliary structure and function in a tissue-specific manner to understand processes, such as ciliary protein trafficking or cilium-dependent signaling in vitro and in vivo.  

4.1788           Metabolic, Reproductive, and Neurologic Abnormalities in Agpat1-Null Mice

Agarwal, A.K., Tunison, K., Dalal, J.S., Nagamma, S.S., Hamra, F.K., Sankella, S., Shao, X., Auchus, R.J., and Garg, A. Endocrinology, 158(11), 3954-3973 (2017)   Defects in the biosynthesis of phospholipids and neutral lipids are associated with cell membrane dysfunction, disrupted energy metabolism, and diseases including lipodystrophy. In these pathways, the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) enzymes transfer a fatty acid to the sn-2 carbon of sn-1-acylglycerol-3-phosphate (lysophosphatidic acid) to form sn-1, 2-acylglycerol-3-phosphate [phosphatidic acid (PA)]. PA is a precursor for key phospholipids and diacylglycerol. AGPAT1 and AGPAT2 are highly homologous isoenzymes that are both expressed in adipocytes. Genetic defects in AGPAT2 cause congenital generalized lipodystrophy, indicating that AGPAT1 cannot compensate for loss of AGPAT2 in adipocytes. To further explore the physiology of AGPAT1, we characterized a loss-of-function mouse model (Agpat1^−/−). The majority of Agpat1^−/− mice died before weaning and had low body weight and low plasma glucose levels, independent of plasma insulin and glucagon levels, with reduced percentage of body fat but not generalized lipodystrophy. These mice also had decreased hepatic messenger RNA expression of Igf-1 and Foxo1, suggesting a decrease in gluconeogenesis. In male mice, sperm development was impaired, with a late meiotic arrest near the onset of round spermatid production, and gonadotropins were elevated. Female mice showed oligoanovulation yet retained responsiveness to gonadotropins. Agpat1^−/− mice also demonstrated abnormal hippocampal neuron development and developed audiogenic seizures. In summary, Agpat1^−/− mice developed widespread disturbances of metabolism, sperm development, and neurologic function resulting from disrupted phospholipid homeostasis. AGPAT1 appears to serve important functions in the physiology of multiple organ systems. The Agpat1-deficient mouse provides an important model in which to study the contribution of phospholipid and triacylglycerol synthesis to physiology and diseases.  

4.1789           Applications of particulate oxygen-generating substances (POGS) in the bioartificial pancreas

McQuilling, J.P., Sittadjody, S., Pendergraft, S., farney, A.C. and Opara, E.C. Biomater. Sci., 5, 2437-2447 (2017)   Type-1 Diabetes (T1D) is a devastating autoimmune disorder which results in the destruction of beta cells within the pancreas. A promising treatment strategy for T1D is the replacement of the lost beta cell mass through implantation of immune-isolated microencapsulated islets referred to as the bioartificial pancreas. The goal of this approach is to restore blood glucose regulation and prevent the long-term comorbidities of T1D without the need for immunosuppressants. A major requirement in the quest to achieve this goal is to address the oxygen needs of islet cells. Islets are highly metabolically active and require a significant amount of oxygen for normal function. During the process of isolation, microencapsulation, and processing prior to transplantation, the islets’ oxygen supply is disrupted, and a large amount of islet cells are therefore lost due to extended hypoxia, thus creating a major barrier to clinical success with this treatment. In this work, we have investigated the oxygen generating compounds, sodium percarbonate (SPO) and calcium peroxide (CPO) as potential supplemental oxygen sources for islets during isolation and encapsulation before and immediately after transplantation. First, SPO particles were used as an oxygen source for islets during isolation. Secondly, silicone films containing SPO were used to provide supplemental oxygen to islets for up to 4 days in culture. Finally, CPO was used as an oxygen source for encapsulated cells by co-encapsulating CPO particles with islets in permselective alginate microspheres. These studies provide an important proof of concept for the utilization of these oxygen generating materials to prevent beta cell death caused by hypoxia.  

4.1790           Erythropoietin’s Beta Common Receptor Mediates Neuroprotection in Spinal Cord Neurons

Foley, L.S., Fullerton, D.A., mares, J., Sungelo, M., Weyant, M.J., Cleveland, J.C. and Reece, T.B. Ann. Thorac. Surg., 104, 1909-1914 (2017)   Background Paraplegia from spinal cord ischemia-reperfusion (SCIR) remains an elusive and devastating complication of complex aortic operations. Erythropoietin (EPO) attenuates this injury in models of SCIR. Upregulation of the EPO beta common receptor (βcR) is associated with reduced damage in models of neural injury. The purpose of this study was to examine whether EPO-mediated neuroprotection was dependent on βcR expression. We hypothesized that spinal cord neurons subjected to oxygen-glucose deprivation would mimic SCIR injury in aortic surgery and EPO treatment attenuates this injury in a βcR-dependent fashion. Methods Lentiviral vectors with βcR knockdown sequences were tested on neuron cell cultures. The virus with greatest βcR knockdown was selected. Spinal cord neurons from perinatal wild-type mice were harvested and cultured to maturity. They were treated with knockdown or nonsense virus and transduced cells were selected. Three groups (βcR knockdown virus, nonsense control virus, no virus control; n = 8 each) were subjected to 1 hour of oxygen-glucose deprivation. Viability was assessed. βcR expression was quantified by immunoblot. Results EPO preserved neuronal viability after oxygen-glucose deprivation (0.82 ± 0.04 versus 0.61 ± 0.01; p < 0.01). Additionally, EPO-mediated neuron preservation was similar in the nonsense virus and control mice (0.82 ± 0.04 versus 0.80 ± 0.05; p = 0.77). EPO neuron preservation was lost in βcR knockdown mice compared with nonsense control mice (0.46 ± 0.03 versus 0.80 ± 0.05; p < 0.01). Conclusions EPO attenuates neuronal loss after oxygen-glucose deprivation in a βcR-dependent fashion. This receptor holds immense clinical promise as a target for pharmacotherapies treating spinal cord ischemic injury.  

4.1791           Hyaluronic Acid/Collagen Hydrogel as an Alternative to Alginate for Long-Term Immunoprotected Islet Transplantation No Access

Harrington, S., Williams, J., Rawal, s., Ramachandran, K and Stehno-Bittel, L. Tissue Engineering: Part A, 23(19-20), 1088-1099 (2017)   Alginate has long been the material of choice for immunoprotection of islets due to its low cost and ability to easily form microspheres. Unfortunately, this seaweed-derived material is notoriously prone to fibrotic overgrowth in vivo, resulting in premature graft failure. The purpose of this study was to test an alternative, hyaluronic acid (HA-COL), for in vitro function, viability, and allogeneic islet transplant outcomes in diabetic rats. In vitro studies indicated that the HA-COL gel had diffusion characteristics that would allow small molecules such as glucose and insulin to enter and exit the gel, whereas larger molecules (70 and 500 kDa dextrans) were impeded from diffusing past the gel edge in 24 h. Islets encapsulated in HA-COL hydrogel showed significantly improved in vitro viability over unencapsulated islets and retained their morphology and glucose sensitivity for 28 days. When unencapsulated allogeneic islet transplants were administered to the omentum of outbred rats, they initially were normoglycemic, but by 11 days returned to hyperglycemia. Immunohistological examination of the grafts and surrounding tissue indicated strong graft rejection. By comparison, when using the same outbred strain of rats, allogeneic transplantation of islets within the HA-COL gel reversed long-term diabetes and prevented graft rejection in all animals. Animals were sacrificed at 40, 52, 64, and 80 weeks for evaluation, and all were non-diabetic at sacrifice. Explanted grafts revealed viable islets in the transplant site as well as intact hydrogel, with little or no evidence of fibrotic overgrowth or cellular rejection. The results of these studies demonstrate great potential for HA-COL hydrogel as an alternative to sodium alginate for long-term immunoprotected islet transplantation.  

4.1792           Differential detection photothermal spectroscopy: towards ultra-fast and sensitive label-free detection in picoliter & femtoliter droplets

Maceiczyk, R.M., Hess, D., Chiu, F.W., Stavrakis, s. and deMello, A.J. Lab on a Chip, 17, 3654-3663 (2017)   Despite the growing importance of droplet-based microfluidics in high-throughput experimentation, few current methods allow the sensitive measurement of absorbance within rapidly moving droplets. To address this significant limitation, we herein present the application of differential detection photothermal interferometry (DDPI) for single-point absorbance quantification in pL- and fL-volume droplets. To assess the efficacy of our approach, we initially measure absorbance in 100 pL droplets at frequencies in excess of 1 kHz and determine a detection limit of 1.4 μmol L⁻¹ for Erythrosin B (A = 3.8 × 10⁻⁴). Subsequently, we apply the method to the analysis of fL-volume droplets and droplets generated at frequencies in excess of 10 kHz. Finally, we demonstrate the utility of DDPI as a detection scheme for colorimetric assays. Specifically, we extract the Michaelis–Menten constant for the reaction of β-galactosidase and chlorophenol-red-β-D-galactopyranoside and monitor the metabolomic activity of a population of HL-60 cells at the single cell level. Results establish single-point absorbance detection as a powerful, sensitive and rapid alternative to fluorescence for a wide range of assays within segmented flows.  

4.1793           Potent analogues of etiprednol dicloacetate, a second generation of soft corticosteroids†

Bodor, N., Zubovics, Z., Kurucz, I., Solyom, S. and Bodor, E.

1. Pharmacy Pharmacol., 69(12), 1745-1753 82017)

  Objectives Loteprednol etabonate (LE) is the first, highly successful soft corticosteroid (SC) designed using the ‘inactive metabolite’ approach, starting with ∆¹-cortienic acid (d-CA). The next generation of SCs based on d-CA was etiprednol dicloacetate (ED). The 17α-dichloroacetyl function serves both as a unique pharmacophore and as the source of the molecule's softness. Highly potent SCs were designed based on a combination of ED and LE, introducing 6, 9 and 16 substituents in the molecule. Methods The new 6α, 9α, 16α and β 17α-dichloroacetyl 17β-esters were synthesized from the correspondingly substituted ∆¹-cortienic acids. The anti-inflammatory activity was assessed using LPS-induced TNF α-release under various conditions to determine intrinsic activity vs. systemic biological stability. In vivo anti-inflammatory activity was studied in the widely used ovalbumin-sensitized and ovalbumin-challenged Brown Norway rat model. Key findings The 6α or 9α-fluoro substitution produced highly potent corticosteroids, but the 17α-dichloroacetyl substituent provided ‘softness’ in all cases. Local application of these steroids will significantly reduce systemic activity, due to the facile hydrolytic deactivation of these molecules. Conclusions A 17α-dichloroacetyl derivative of fluticasone (FLU) is highly potent but much safer than the currently used propionate or furoate ester.  

4.1794           Up-regulation of Interleukin-21 Contributes to Liver Pathology of Schistosomiasis by Driving GC Immune Responses and Activating HSCs in Mice

Scientific Reports, 7:16682 (2017)   The pathology of schistosome egg-induced liver granuloma, fibrosis and eventually liver scarring is complicated. CD4+ helper T (Th) cells play critical roles in both host humoral immunity and cellular immunity against parasitic infection and immunopathology in schistosomiasis. Follicular helper T (Tfh) cells are another specialized subset of Th cells and involved in infectious diseases. However, the immune regulatory mechanism of Tfh cells in severe liver pathology of schistosomiasis is still poorly understood. In this study, using a S. japonicum-infected mouse model, we studied the dynamics and effects of Tfh cells in vivo and demonstrated that Tfh phenotype molecules ICOS, PD-1 and functional factor IL-21 were positively correlated with disease development by flow cytometry. Meanwhile, our results also showed that Tfh cells enriched in splenic germinal center (GC) and promoted B cells producing IgM with the progress of hepatic immunopathology by B-T co-culture experiments. More importantly, our data indicated that IL-21 contributed to the formation and development of hepatic egg granuloma and subsequent fibrosis by driving GC responses and activating HSCs by immunohistochemical detection and blocking assay in vitro. Our findings contribute to the better understanding of the immunopathogenesis of schistosomiasis and have implications for therapeutic intervention of hepatic fibrotic diseases.  

4.1795           Functional analysis of human intrafusal fiber innervation by human γ-motoneurons

Colon, A., Guo, X., Akanda, N., Cai, Y. and Hickman, J.J. Scientific Reports, 7:17202 (2017)   Investigation of neuromuscular deficits and diseases such as SMA, as well as for next generation prosthetics, utilizing in vitro phenotypic models would benefit from the development of a functional neuromuscular reflex arc. The neuromuscular reflex arc is the system that integrates the proprioceptive information for muscle length and activity (sensory afferent), to modify motoneuron output to achieve graded muscle contraction (actuation efferent). The sensory portion of the arc is composed of proprioceptive sensory neurons and the muscle spindle, which is embedded in the muscle tissue and composed of intrafusal fibers. The gamma motoneurons (γ-MNs) that innervate these fibers regulate the intrafusal fiber’s stretch so that they retain proper tension and sensitivity during muscle contraction or relaxation. This mechanism is in place to maintain the sensitivity of proprioception during dynamic muscle activity and to prevent muscular damage. In this study, a co-culture system was developed for innervation of intrafusal fibers by human γ-MNs and demonstrated by morphological and immunocytochemical analysis, then validated by functional electrophysiological evaluation. This human-based fusimotor model and its incorporation into the reflex arc allows for a more accurate recapitulation of neuromuscular function for applications in disease investigations, drug discovery, prosthetic design and neuropathic pain investigations.  

4.1796           GDF15 deficiency exacerbates chronic alcohol- and carbon tetrachloride-induced liver injury

Chung, H., Kim, J.T., Kim, H-W., Kwon, M., Kim, S.Y., Shong, M., Kim, K.S. and Yi, H-s. Scientific Reports, 7:17238 (2017)   Growth differentiation factor 15 (GDF15) has recently been shown to have an important role in the regulation of mitochondrial function and in the pathogenesis of complex human diseases. Nevertheless, the role of GDF15 in alcohol-induced or fibrotic liver diseases has yet to be determined. In this study, we demonstrate that alcohol- or carbon tetrachloride (CCl₄)-mediated hepatic GDF15 production ameliorates liver inflammation and fibrosis. Alcohol directly enhanced GDF15 expression in primary hepatocytes, which led to increased oxygen consumption. Moreover, GDF15 reduced the expression of pro-inflammatory cytokines in liver-resident macrophages, leading to an improvement in inflammation and fibrosis in the liver. GDF15 knockout (KO) mice had more TNF-α-producing T cells and more activated CD4⁺ and CD8⁺ T cells in the liver than wild-type mice. Liver-infiltrating monocytes and neutrophils were also increased in the GDF15 KO mice during liver fibrogenesis. These changes in hepatic immune cells were associated with increased tissue inflammation and fibrosis. Finally, recombinant GDF15 decreased the expression of pro-inflammatory cytokines and fibrotic mediators and prevented the activation of T cells in the livers of mice with CCl₄-induced liver fibrosis. These results suggest that GDF15 could be a potential therapeutic target for the treatment of alcohol-induced and fibrotic liver diseases.  

4.1797           Effectiveness of different molecular forms of C. histolyticum class I collagenase to recover islets

Green, M.L., Breite, A.G., Beechler, C.A., Dwuler, F.E. and McCarthy, R.C. Islets, 9(6), 177-181 (2017)   One factor that may contribute to variability between different lots of purified collagenase to recover islets is the molecular form of C. histolyticum class I (C1) collagenase used in the isolation procedure. Two different enzyme mixtures containing C1, class II (C2) collagenase and BP Protease were compared for their effectiveness to recover islets from split adult porcine pancreas. The same enzyme activities per g trimmed tissue were used for all isolations with the only difference being the mass of C1 required to achieve 25,000 collagen degradation activity U/g tissue. The results show no differences in performance of the two enzyme mixtures. The only significant difference is 19 fold more truncated C1 was required to achieve the same result as intact C1.  

4.1798           Analysis of Argonaute 2–microRNA complexes in ex vivo stored red blood cells

Vu, L., Ragupathy, V., Kulkarni, S. and Atreya, C. Transfusion, 57(12), 2995-3000 (2017)   BACKGROUND Human enucleated mature red blood cells (RBCs) contain both mature microRNAs (miRNAs) and mRNAs, and we have previously correlated RBC storage lesion processes such as eryptosis, adenosine 5′-triphosphate loss, and RBC indices with differentially expressed miRNAs. Here we have characterized Argonaute 2 (AGO2)–miRNA complexes in stored mature RBCs as a first step toward understanding their role, if any. STUDY DESIGN AND METHODS In this report AGO2-bound miRNAs in mature RBCs isolated from RBCs collected from three different healthy donors and stored for 24 hours at 4 to 6°C were identified by anti-AGO2 immunoprecipitation (IP) followed by next-generation sequencing of the RNA isolated from the IP. The data were analyzed by various bioinformatics tools. RESULTS The analysis highlighted 28 mature AGO2-bound miRNAs that are common to all three donors, representing 95.6% of the identified miRNAs. Among these, miR-16-5p (20.6%), miR-451a-5p (16.7%), miR-486-5p (12.6%), and miR-92a-3p (12.6%) are the most abundant miRNAs. Functional enrichment analysis for mRNA targets of the 28 common miRNAs identified molecules related to various diseases, biofunctions, and toxicity functions such as cardio-, hepato-, and nephrotoxicity. CONCLUSION Overall, these results demonstrate the existence of multiple intracellular AGO2-bound miRNAs in 24-hour-stored RBCs and warrant further experiments to determine whether AGO2-miRNAs are functional in RBCs.  

4.1799           Compound heterozygous CASQ2 mutations and long-term course of catecholaminergic polymorphic ventricular tachycardia

Josephs, K., Patel, K., Janson, C.M., Montagna, C. and McDonald, T.V. Mol. Genet. Genom. Med., 5(6), 788-794 (2017)   Background Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal inherited cardiac disorder characterized by episodic ventricular tachycardia during adrenergic stimulation. It is associated with significant morbidity and mortality. Knowledge of the underlying genetic cause, pathogenesis, and the natural history of the disease remains incomplete. Approximately 50% of CPVT cases are caused by dominant mutations in the cardiac ryanodine receptor (RYR2) gene, <5% of cases are accounted for by recessive mutations in cardiac calsequestrin (CASQ2) or Triadin (TRDN). Methods We report a family with two CASQ2 gene mutations. A research-based next-generation sequencing (NGS) initiative was used in a patient with a severe CPVT phenotype and her clinically unaffected son. Reverse transcription polymerase chain reaction (RT-PCR) from platelet RNA was used to assess the consequences of predicted splice variants. Results NGS revealed that the proband carried a novel c.199C>T (p.Gln67*) mutation and a previously reported splice site mutation c.532+1G>A in CASQ2. Her son is a heterozygous carrier of the c.199C>T (p.Gln67*) mutation alone and the proband was compound heterozygous at CASQ2. RNA analysis demonstrated that the splice site mutation results in the retention of intron 3 with no full-length CASQ2 mRNA. Conclusion This study describes a novel CPVT genotype and further characterizes the effect of a previously reported CASQ2 splice site mutation. The long-term follow-up of 23 years since first symptom provides additional insight into the natural history of CASQ2-associated CPVT.  

4.1800           Thirst-associated preoptic neurons encode an aversive motivational drive

Allen, W.E., Denardo, L.A., Chen, M.Z., Liu, C.D., Loh, K.M., Fenno, L.E., Ramakrisshnan, C., Deisseroth, K. and Luo, L. Science, 357(6356), 1149-1155 (2017)   Water deprivation produces a drive to seek and consume water. How neural activity creates this motivation remains poorly understood. We used activity-dependent genetic labeling to characterize neurons activated by water deprivation in the hypothalamic median preoptic nucleus (MnPO). Single-cell transcriptional profiling revealed that dehydration-activated MnPO neurons consist of a single excitatory cell type. After optogenetic activation of these neurons, mice drank water and performed an operant lever-pressing task for water reward with rates that scaled with stimulation frequency. This stimulation was aversive, and instrumentally pausing stimulation could reinforce lever-pressing. Activity of these neurons gradually decreased over the course of an operant session. Thus, the activity of dehydration-activated MnPO neurons establishes a scalable, persistent, and aversive internal state that dynamically controls thirst-motivated behavior.  

4.1801           Pro-inflammatory hepatic macrophages generate ROS through NADPH oxidase 2 via endocytosis of monomeric TLR4–MD2 complex

Kim, S.Y. et al Nature Communications, 8:2247 (2017)   Reactive oxygen species (ROS) contribute to the development of non-alcoholic fatty liver disease. ROS generation by infiltrating macrophages involves multiple mechanisms, including Toll-like receptor 4 (TLR4)-mediated NADPH oxidase (NOX) activation. Here, we show that palmitate-stimulated CD11b⁺F4/80^low hepatic infiltrating macrophages, but not CD11b⁺F4/80^high Kupffer cells, generate ROS via dynamin-mediated endocytosis of TLR4 and NOX2, independently from MyD88 and TRIF. We demonstrate that differently from LPS-mediated dimerization of the TLR4–MD2 complex, palmitate binds a monomeric TLR4–MD2 complex that triggers endocytosis, ROS generation and increases pro-interleukin-1β expression in macrophages. Palmitate-induced ROS generation in human CD68^lowCD14^high macrophages is strongly suppressed by inhibition of dynamin. Furthermore, Nox2-deficient mice are protected against high-fat diet-induced hepatic steatosis and insulin resistance. Therefore, endocytosis of TLR4 and NOX2 into macrophages might be a novel therapeutic target for non-alcoholic fatty liver disease.  

4.1802           H3.3K27M Cooperates with Trp53 Loss and PDGFRA Gain in Mouse Embryonic Neural Progenitor Cells to Induce Invasive High-Grade Gliomas

Pathania, m., De Jay, N., Maestro, N. et al Cancer Cell, 32(5), 684-700 (2017)   Gain-of-function mutations in histone 3 (H3) variants are found in a substantial proportion of pediatric high-grade gliomas (pHGG), often in association with TP53 loss and platelet-derived growth factor receptor alpha (PDGFRA) amplification. Here, we describe a somatic mouse model wherein H3.3^K27M and Trp53 loss alone are sufficient for neoplastic transformation if introduced in utero. H3.3^K27M-driven lesions are clonal, H3K27me3 depleted, Olig2 positive, highly proliferative, and diffusely spreading, thus recapitulating hallmark molecular and histopathological features of pHGG. Addition of wild-type PDGFRA decreases latency and increases tumor invasion, while ATRX knockdown is associated with more circumscribed tumors. H3.3^K27M-tumor cells serially engraft in recipient mice, and preliminary drug screening reveals mutation-specific vulnerabilities. Overall, we provide a faithful H3.3^K27M-pHGG model which enables insights into oncohistone pathogenesis and investigation of future therapies.  

4.1803           Antifibrotic effect of rapamycin containing polyethylene glycol-coated alginate microcapsule in islet xenotransplantation

Park, H-S., Kim, J-W., Lee, S-H., Yang, H.K., Ham, D-S., Sun, C-L., Hong, T.H., Khang, G., Park, C-G.   and Yoon, K-H.

1. Tissue Eng. Regen. Med., 11(4), 1274-1284 (2017)

  Islet microencapsulation is an attractive strategy for the minimization or avoidance of life-long immunosuppression after transplantation. However, the clinical implementation of this technique is currently limited by incomplete biocompatibility. Thus, the aim of the present study was to demonstrate the improved biocompatibility of rapamycin-containing polyethylene glycol (Rapa–PEG)-coating on alginate microcapsules containing xenogeneic islets. The Rapa–PEG-coating on the alginate layer was observed using scanning electron microscopy (SEM) and the molecular cut-off weight of the microcapsules was approximately 70 kDa. The viabilities of the alginate-encapsulated and Rapa–PEG-coated alginate-encapsulated islets were lower than the viability of the naked islets just after encapsulation, but these the differences diminished over time in culture dishes. Rapa–PEG-coating on the alginate capsules effectively decreased the proliferation of macrophage cells compared to the non-coating and alginate coating of xenogeneic pancreas tissues. Glucose-stimulated insulin secretion did not significantly differ among the groups prior to transplantation. The random blood glucose levels of diabetic mice significantly improved following the transplantation of alginate-encapsulated and Rapa–PEG-coated alginate-encapsulated islets, but there were no significant differences between these two groups. However, there was a significant decrease in the number of microcapsules with fibrotic cell infiltration in the Rapa–PEG-coated alginate microcapsule group compared to the alginate microcapsule group. In conclusion, Rapa–PEG-coating might be an effective technique with which to improve the biocompatibility of microcapsules containing xenogeneic islets.  

4.1804           A microfluidic-based cell encapsulation platform to achieve high long-term cell viability in photopolymerized PEGNB hydrogel microspheres

Jiang, Z., Xia, B., McBride, R.M. and Oakey, J.

1. Mater. Chem. B., 5, 173-180 (2017)

  Cell encapsulation within photopolymerized polyethylene glycol (PEG)-based hydrogel scaffolds has been demonstrated as a robust strategy for cell delivery, tissue engineering, regenerative medicine, and developing in vitro platforms to study cellular behavior and fate. Strategies to achieve spatial and temporal control over PEG hydrogel mechanical properties, chemical functionalization, and cytocompatibility have advanced considerably in recent years. Recent microfluidic technologies have enabled the miniaturization of PEG hydrogels, thus enabling the fabrication of miniaturized cell-laden vehicles. However, rapid oxygen diffusive transport times on the microscale dramatically inhibit chain growth photopolymerization of polyethylene glycol diacrylate (PEGDA), thus decreasing the viability of cells encapsulated within these microstructures. Another promising PEG-based scaffold material, PEG norbornene (PEGNB), is formed by a step-growth photopolymerization and is not inhibited by oxygen. PEGNB has also been shown to be more cytocompatible than PEGDA and allows for orthogonal addition reactions. The step-growth kinetics, however, are slow and therefore challenging to fully polymerize within droplets flowing through microfluidic devices. Here, we describe a microfluidic-based droplet fabrication platform that generates consistently monodisperse cell-laden water-in-oil emulsions. Microfluidically generated PEGNB droplets are collected and photopolymerized under UV exposure in bulk emulsions. In this work, we compare this microfluidic-based cell encapsulation platform with a vortex-based method on the basis of microgel size, uniformity, post-encapsulation cell viability and long-term cell viability. Several factors that influence post-encapsulation cell viability were identified. Finally, long-term cell viability achieved by this platform was compared to a similar cell encapsulation platform using PEGDA. We show that this PEGNB microencapsulation platform is capable of generating cell-laden hydrogel microspheres at high rates with well-controlled size distributions and high long-term cell viability.    

4.1805           Lipopolysaccharide mediates hepatic stellate cell activation by regulating autophagy and retinoic acid signaling

Chen, M., Liu, J., Yang, W. and Ling, W. Autophagy, 13(11), 1813-1827 (2017)   Bacterial translocation and lipopolysaccharide (LPS) leakage occur at a very early stage of liver fibrosis in animal models. We studied the role of LPS in hepatic stellate cell (HSC) activation and the underlying mechanisms in vitro and in vivo. Herein, we demonstrated that LPS treatment led to a dramatic increase in autophagosome formation and autophagic flux in LX-2 cells and HSCs, which was mediated through the AKT-MTOR and AMPK-ULK1 pathway. LPS significantly decreased the lipid content, including the lipid droplet (LD) number and lipid staining area in HSCs; pretreatment with macroautophagy/autophagy inhibitors or silencing ATG5 attenuated this decrease. Furthermore, lipophagy was induced by LPS through the autophagy-lysosomal pathway in LX-2 cells and HSCs. Additionally, LPS-induced autophagy further reduced retinoic acid (RA) signaling, as demonstrated by a decrease in the intracellular RA level and Rar target genes, resulting in the downregulation of Bambi and promoting the sensitization of the HSC's fibrosis response to TGFB. Compared with CCl₄ injection alone, CCl₄ plus LPS injection exaggerated liver fibrosis in mice, as demonstrated by increased Col1a1 (collagen, type I, α 1), Acta2, Tgfb and Timp1 mRNA expression, ACTA2/α-SMA and COL1A1 protein expression, and Sirius Red staining area, which could be attenuated by injection of an autophagy inhibitor. LPS also reduced lipid content in HSCs in vivo, with this change being attenuated by chloroquine (CQ) administration. In conclusion, LPS-induced autophagy resulted in LD loss, RA signaling dysfunction, and downregulation of the TGFB pseudoreceptor Bambi, thus sensitizing HSCs to TGFB signaling.  

4.1806           Elasto-Inertial Focusing of Mammalian Cells and Bacteria Using Low Molecular, Low Viscosity PEO Solutions

Holzner, G., Stavrakis, S. and Demello, A. Anal. Chem., 89(21), 11653-11663 (2017)   The ability to manipulate biological cells is critical in a diversity of biomedical and industrial applications. Microfluidic-based cell manipulations provide unique opportunities for sophisticated and high-throughput biological assays such as cell sorting, rare cell detection, and imaging flow cytometry. In this respect, cell focusing is an extremely useful functional operation preceding downstream biological analysis, since it allows the accurate lateral and axial positioning of cells moving through microfluidic channels, and thus enables sophisticated cell manipulations in a passive manner. Herein, we explore the utility of viscoelastic carrier fluids for enhanced elasto-inertial focusing of biological species within straight, rectangular cross section microfluidic channels. Since the investigated polymer solutions possess viscosities close to that of water and exhibit negligible shear thinning, focusing occurs over a wide range of elasticity numbers and a large range of Reynolds numbers. With a view to applications in the robust focusing of cells and bacteria, we assess and characterize the influence of accessible focusing parameters, including blockage ratio, volumetric flow rate, cell concentration, and polymer chain length.  

4.1807           Extremely low frequency magnetic field protects injured spinal cord from the microglia- and iron-induced tissue damage

Dey, S., Bose, S., Kumar, S., Rathmore, R., Mathur, R. and Jain, S. Electromagnetic Biol. Med., 36(4), 330-340 (2017)   Spinal cord injury (SCI) is insult to the spinal cord, which results in loss of sensory and motor function below the level of injury. SCI results in both immediate mechanical damage and secondary tissue degeneration. Following traumatic insult, activated microglia release proinflammatory cytokines and excess iron due to hemorrhage, initiating oxidative stress that contributes to secondary degeneration. Literature suggests that benefits are visible with the reduction in concentration of iron and activated microglia in SCI. Magnetic field attenuates oxidative stress and promotes axonal regeneration in vitro and in vivo. The present study demonstrates the potential of extremely low frequency magnetic field to attenuate microglia- and iron-induced secondary injury in SCI rats. Complete transection of the spinal cord (T13 level) was performed in male Wistar rats and subsequently exposed to magnetic field (50 Hz,17.96 µT) for 2 h daily for 8 weeks. At the end of the study period, spinal cords were dissected to quantify microglia, macrophage, iron content and study the architecture of lesion site. A significant improvement in locomotion was observed in rats of the SCI + MF group as compared to those in the SCI group. Histology, immunohistochemistry and flow cytometry revealed significant reduction in lesion volume, microglia, macrophage, collagen tissue and iron content, whereas, a significantly higher vascular endothelial growth factor expression around the epicenter of the lesion in SCI + MF group as compared to SCI group. These novel findings suggest that exposure to ELF-MF reduces lesion volume, inflammation and iron content in addition to facilitation of angiogenesis following SCI.  

4.1808           A proteome analysis of pig pancreatic islets and exocrine tissue by liquid chromatography with tandem mass spectrometry

Nakashima, Y., Miyagi-Shiohira, C., Kobayashi, N., Saitoh, I., Watanabe, M. and Noguchi, H. Islets, 9(6), 159-176 (2017)   Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is a proteome analysis method, and the shotgun analysis by LC-MS/MS comprehensively identifies proteins from tissues and cells with high resolving power. In this study, we analyzed the protein expression in pancreatic tissue by LC-MS/MS. Islets isolated from porcine pancreata (purity ≥95%) and exocrine tissue (purity ≥99%) were used in this study. LC-MS/MS showed that 13 proteins were expressed in pancreatic islets only (Group I), 43 proteins were expressed in both islets and exocrine tissue (Group I&E), and 102 proteins were expressed in exocrine tissue only (Group E). Proteins involved in islet differentiation and cell proliferation were identified in Group I (e.g. CLUS, CMGA, MIF). In addition, various functional proteins (e.g. SCG2, TBA1A) were identified in islet by using the new method of ‘principal component analysis (PCA)’. However, the function of such proteins on islets remains unclear. EPCAM was identified in Group E. Group E was found to include proteins involved in clinical inflammatory diseases such as pancreatitis (e.g. CBPA1, CGL, CYTB, ISK1 and PA21B). Many of these identified proteins were reported less frequently in previous studies, and HS71B, NEC2, PRAF3 and SCG1 were newly detected in Group I while CPNS1, DPEP1, GANAB, GDIB, GGT1, HSPB1, ICTL, VILI, MUTA, NDKB, PTGR1, UCHL3, VAPB and VINC were newly detected in Group E. These results show that comprehensive expression analysis of proteins by LC-MS/MS is useful as a method to investigate new factors constructing cellular component, biological process, and molecular function.   4.1808a Density separation of quiescent yeast using iodixanol Quasem, I., Luby, C.J., mace, C.R. and Fuchs, S.M. Biotechniques, 63, 169-173 (2017)   Cells undergo phenotypic changes in response to external and internal stimuli. Yeast grow exponentially when nutrients are abundant and enter stationary phase (SP) when nutrients become scarce. Under nutrient-limited conditions, a clonal population of haploid budding yeast differentiates into quiescent (Q) and nonquiescent (NQ) cells (1). Q cells are dense, unbudded daughter cells that are arrested in G₀ but are capable of re-entering the mitotic cell cycle upon nutrient replenishment. In contrast, NQ are unable to enter G_o and consist of mainly replicatively older mother cells. Q cells can survive up to 3 weeks longer in SP than NQ cells (1) and are thus used as a model for chronological aging (2). Furthermore, Q cells possess stem cell– like properties and are used to examine re-entry into the cell cycle and self-renewal (3). One important characteristic of Q cells is their resistance to a multitude of environmental stresses (4). Unlike dividing cells, Q cells are thermotolerant (5) and possess cell walls resistant to zymolyase (6). NQ cells, on the contrary, are less resistant to these treatments and are prone to genomic instability (5,7,8). Because of their short life cycle and the extensive availability of molecular tools, budding yeast are an excellent model in which to study the unique biology of quiescence (9). Rapid and inexpensive methods for separating NQ and Q yeast populations would therefore facilitate future studies. Various strategies have been employed to isolate Q cells, including cell elutriation (10) and fluorescence activated cell sorting (FACS) (6). However, these techniques require expensive equipment and specialized training. Presently, the most common method for isolating Q cells uses density-based Percoll gradients (1), which are both labor- and time-intensive. The nontoxic density gradient medium iodixanol has been used to enrich for blood monocytes (11) and motile sperm cells (12), separate peroxisomes from other organelles (13), and harvest retroviruses (14). Here, we describe an inexpensive and efficient Q-cell separation technique using a single-density, one-step gradient prepared from a solution of iodixanol   4.1808b Preservation of DCD pancreas by continuous hypothermic machine perfusion focusing on islet transplantation Akutsu, N., maruyama, M., Otsuki, K., Ishida, T., Saitoh, T., Saigo, K., Hasegawa, M., Aoyazma, H., Kenmouchi, T and Noguchi, H. Organ Biology, 24(2), 175-179 (2017)   Although clinical research of islet transplantation for type 1 diabetes has started in Japan, severe donor shortage hampers the progress. A possible solution is use of marginal donors such as donors after cardiac death (DCD). A continuous hypothermic perfusion machine, LifePort^TM (LP) has been developed and supplied by Organ Recovery Systems, Inc. and used for preservation and transportation of marginal kidney for organ transplantation. The effectiveness has been approved especially in the preservation. We applied LP to preservation of pancreas that is sensitive to ischemia reperfusion injury and found that the machine would be efficacious on islet isolation. [Method] Beagle dog pancreas was subjected to warm ischemic injury for 30min and preserved for 24hr by following three methods; continuous hypothermic perfusion by LP (LP group), simple cold storage in UW solution (UW group), two-layer method (TL group). Then, islets were isolated. [Results] Morphologically, large good quality islets were obtained in LP group. The larger amount of purified islet was recovered in LP group than UW and TL groups. In addition, when insulin secretion activities were estimated by static incubation, islets of LP group were the most functional among all. [Conclusion] In experimental DCD pancreas model, continuous hypothermic machine perfusion resulted in higher recovery and function of islets than simple cold storage and two layer method. Further improvement will achieve clinical application of the method on DCD pancreas preservation.  

4.1809           Extending antigen release from particulate vaccines results in enhanced antitumor immune response

Kapadia, C.H., Tian, S., Perry, J.L., Sailer, D., Luft, J.C. and DeSimone, J.M.

1. Controlled Release, 269, 393-404 (2018)

  Tumor-specific CD8⁺ cytotoxic T lymphocytes (CTLs) play a critical role in an anti-tumor immune response. However, vaccination intended to elicit a potent CD8⁺ T cell responses employing tumor-associated peptide antigens, are typically ineffective due to poor immunogenicity. Previously, we engineered a polyethylene glycol (PEG) hydrogel-based subunit vaccine for the delivery of an antigenic peptide and CpG (adjuvant) to elicit potent CTLs. In this study, we further examined the effect of antigen release kinetics on their induced immune responses. A CD8⁺ T cell epitope peptide from OVA (CSIINFEKL) and CpG were co-conjugated to nanoparticles utilizing either a disulfide or a thioether linkage. Subsequent studies comparing peptide release rates as a function of linker, determined that the thioether linkage provided sustained release of peptide over 72 h. Ability to control the release of peptide resulted in both higher and prolonged antigen presentation when compared to disulfide-linked peptide. Both NP vaccine formulations resulted in activation and maturation of bone marrow derived dendritic cells (BMDCs) and induced potent CD8⁺ T cell responses when compared to soluble antigen and soluble CpG. Immunization with either disulfide or thioether linked vaccine constructs effectively inhibited EG7-OVA tumor growth in mice, however only treatment with the thioether linked vaccine construct resulted in enhanced survival.  

4.1810           Increased number of tissue factor protein expressing thrombocytes in canine idiopathic immune mediated hemolytic anemia

Hennink, I., van Leeuwen, M.W., Penning, L.C. and Piek, C.J. Vet. Immunol. Immunopathol., 196, 22-29 (2018)   Dogs suffering from canine idiopathic immune mediated hemolytic anemia (cIIMHA) are at great risk of dying particularly in the first two weeks after the diagnosis is made. This high mortality risk may be associated with the development of thromboembolism (TE) and/or disseminated intravascular coagulation (DIC) resulting in organ failure. The exact mechanism of the development of TE and/or DIC in cIIMHA is still undetermined. Therefore, this study investigates the presence of tissue factor (TF) in thrombocytes of dogs suffering from cIIMHA, using OptiPrep™ for the isolation of blood cells and immunocytochemistry (ICC) to visualize TF on thrombocytes. The normalised TF quantity, acquired with ‘colour deconvolution’ (ImageJ plug in), revealed that in cIIMHA dogs the fraction TF positive thrombocytes was statistically significant higher (P < 0.001; mean 0.79; n = 7) compared to the fraction TF positive thrombocytes of the healthy dogs (mean 0.43; n = 9). We further have indications that the fraction of TF positive thrombocytes decreases with time and therapy, but that the progression rate differs individually. Since cIIMHA dogs have more thrombocytes that are TF-positive compared to healthy dogs, this may explain the increased risk to develop TE and DIC. Furthermore, it seems that the number of TF-positive thrombocytes in cIIMHA dogs remains high during the first two weeks of the disease, the time when the animals are at greatest health risk.  

4.1811           Decreased macrophage phagocytic function due to xenobiotic exposures in vitro, difference in sensitivity between various macrophage models

Berntsen, H.F., Bølling, A.K., Bjørklund, C.G., Zimmer, K., Ropstad, E., Zienolddiny, S., Becher, R., Holme, J.A., Dirve, H., Nygaard, U.C. and Bodin, J. Food Chem. Toxicol., 112, 86-90 (2018)   Both autoimmune disease prevalence and exposure to immunotoxic chemicals have increased the last decades. As a first screening of immunotoxic chemicals possibly affecting development of autoimmunity through attenuated macrophage function, we demonstrate a promising model measuring macrophage function in isolated peritoneal macrophages (PCM) from Wistar rats and C57Bl/6 mice. Immunotoxic effects of bisphenol A (BPA) and a selection of perfluoroalkyl acids (PFAAs) were analysed in vitro assessing phagocytic function of macrophages from different sources. Phagocytosis was reduced in PCM of C57Bl/6 mice and Wistar rats after BPA and perfluoroundecanoic acid (PFUnDA) exposure, but not in macrophages derived from human and rat monocyte derived macrophages (MDM). On the other hand, in vitro exposure to mixtures of persistent organic pollutants (POPs) showed similar reductions in rat PCM and rat and human MDM phagocytosis. Reduced phagocytosis was partly due to cytotoxicity. PCM isolated from non-obese diabetic (NOD) mice, interleukin 1α/β knockout (IL-1KO) mice and new-born rats were less sensitive to the xenobiotics than PCM from adult wild type rodents. Finally, in vivo studies with NOD mice verified that POP exposure also decreased the number of pancreatic macrophages in pancreatic islets, reflecting early signs of autoimmunity development, similarly as previously described for BPA.  

4.1812           Inhibition of histone deacetylase 6 (HDAC6) protects against vincristine-induced peripheral neuropathies and inhibits tumor growth

Van Helleputte, L. et al Neurobiology of Disease, 111, 59-69 (2018)   As cancer is becoming more and more a chronic disease, a large proportion of patients is confronted with devastating side effects of certain anti-cancer drugs. The most common neurological complications are painful peripheral neuropathies. Chemotherapeutics that interfere with microtubules, including plant-derived vinca-alkaloids such as vincristine, can cause these chemotherapy-induced peripheral neuropathies (CIPN). Available treatments focus on symptom alleviation and pain reduction rather than prevention of the neuropathy. The aim of this study was to investigate the potential of specific histone deacetylase 6 (HDAC6) inhibitors as a preventive therapy for CIPN using multiple rodent models for vincristine-induced peripheral neuropathies (VIPN). HDAC6 inhibition increased the levels of acetylated α-tubulin in tissues of rodents undergoing vincristine-based chemotherapy, which correlates to a reduced severity of the neurological symptoms, both at the electrophysiological and the behavioral level. Mechanistically, disturbances in axonal transport of mitochondria is considered as an important contributing factor in the pathophysiology of VIPN. As vincristine interferes with the polymerization of microtubules, we investigated whether disturbances in axonal transport could contribute to VIPN. We observed that increasing α-tubulin acetylation through HDAC6 inhibition restores vincristine-induced defects of axonal transport in cultured dorsal root ganglion neurons. Finally, we assured that HDAC6-inhibition offers neuroprotection without interfering with the anti-cancer efficacy of vincristine using a mouse model for acute lymphoblastic leukemia. Taken together, our results emphasize the therapeutic potential of HDAC6 inhibitors with beneficial effects both on vincristine-induced neurotoxicity, as well as on tumor proliferation.  

4.1813           Effect of silicon-rich water intake on the systemic and peritoneal inflammation of rats with chronic low levels of aluminum ingestion

Radvanovic, Z., Djindjic, B., Dzopalic, T., Veljkovic, A., Dunjic, M., Krstic, D., Djindjic, N., Bozic, B. and Nedeljkovic, B.

1. Trace Elements in Med. Biol., 46, 96-102 (2018)

  Background and Objectives Study evaluated effect of silicon-rich water intake on systemic inflammation and functional characteristics of peritoneal macrophages (PMs) of rats that were chronically exposed to dietary aluminum. Methods One month-old female Wistar Albino rats were administered aluminum chloride dissolved in distilled water (1.6 mg/kg body weight in 0.5 mL) by gavage for 90 days. The rats were then given standard (6 mg/L) or silicon-rich water (19 mg/L silicon) (n = 7/group). Control rats underwent sham gavage and received standard or silicon-rich water (n = 7/group). Blood was assessed for cytokine levels. Unstimulated and lipopolysaccharide (LPS)-stimulated PMs were assessed in terms of phagocytic activity and cytokine secretion in vitro. Results Chronic exposition to dietary aluminum and silicon-rich drinking water did not change serum TNF-α levels. Aluminum increased serum IL-2 and this was reversed by silicon-rich water. The aluminum-exposed rats had higher serum sICAM-1 than sham-gavaged, unrelated to type of water. LPS-stimulated PMs from aluminum-intoxicated animals exhibited low phagocytic activity and release of TNF-α, this was significantly improved by silicon-rich water intake. In the presence of silicon-rich water, LPS-stimulated and unstimulated PMs from aluminum-exposed rats produced significantly more IL-10. Conclusions Chronic ingestion of aluminum, increases systemic and peritoneal inflammation and PM dysfunction. The presence of high levels of the natural aluminum antagonist silicon in the drinking water restored IL-10 and TNF-α PM secretion, preventing prolonged inflammation. Thus, silicon intake can decrease the immunotoxicity of aluminum.  

4.1814           Targeting secreted cytokine BMP9 gates the attenuation of hepatic fibrosis

Li, P., Li, Y., Zhu, L., yang, Z., He, J., Wang, L., Shang, Q., pan, H., Wang, H., Ma, X., Li, B., Fan, X., Ge, S., Jia, R. and Zhang, H. BBA – Mol. Basis of Disease, 1864, 709-720 (2018)   Liver fibrosis is overly exuberant wound healing that leads to portal hypertension or liver cirrhosis. Recent studies have demonstrated the functions of bone morphogenetic protein 9 (BMP9) in liver fibrosis, and thus, targeting liver-specific BMP9 abnormalities will become an attractive approach for developing therapeutics to treat liver fibrosis. Here, we reveal that BMP9 serves as a valuable serum diagnostic indicator and efficient therapeutic target to attenuate liver fibrogenesis. Our analysis of biopsies from liver fibrotic patients revealed that higher BMP9 levels accompanied advanced stages of liver fibrosis. In mouse models, recombinant Bmp9 overexpression accelerated liver fibrosis, and adenovirus-mediated Bmp9 knockdown attenuated liver fibrogenesis. Intriguingly, BMP9 directly stimulated hepatic stellate cell activation via the SMAD signaling pathway to enhance hepatic fibrosis. Moreover, an inhibitory monoclonal antibody targeting Bmp9 was efficacious in treatment of mice with liver fibrosis. These observations delineate a novel model in which BMP9 directly drives SMAD/ID1 signaling in hepatic stellate cells, which modulates liver fibrogenesis development. Moreover, the findings unveil a promising surrogate biomarker for the diagnosis of hepatic fibrosis, thereby representing an efficient “BMP9 neutralization” approach in alleviating hepatic fibrosis.  

4.1815           Dairy Heifers Naturally Exposed to Fasciola hepatica Develop a Type 2 Immune Response and Concomitant Suppression of Leukocyte Proliferation

Graham-Brown, J., Hartley, C., Clough, H., Kadioglu, A., Baylis, M. and Williams, D.J.L. Infect.f Immun., 86(1), e00607-17 (2018)   Fasciola hepatica is a parasitic trematode of global importance in livestock. Control strategies reliant on anthelmintics are unsustainable due to the emergence of drug resistance. Vaccines are under development, but efficacies are variable. Evidence from experimental infection suggests that vaccine efficacy may be affected by parasite-induced immunomodulation. Little is known about the immune response to F. hepatica following natural exposure. Hence, we analyzed the immune responses over time in calves naturally exposed to F. hepatica infection. Cohorts of replacement dairy heifer calves (n = 42) with no prior exposure to F. hepatica, on three commercial dairy farms, were sampled over the course of a grazing season. Exposure was determined through an F. hepatica-specific serum antibody enzyme-linked immunosorbent assay (ELISA) and fluke egg counts. Concurrent changes in peripheral blood leukocyte subpopulations, lymphocyte proliferation, and cytokine responses were measured. Relationships between fluke infection and immune responses were analyzed by using multivariable linear mixed-effect models. All calves from one farm showed evidence of exposure, while cohorts from the remaining two farms remained negative over the grazing season. A type 2 immune response was associated with exposure, with increased interleukin-4 (IL-4) production, IL-5 transcription, and eosinophilia. Suppression of parasite-specific peripheral blood mononuclear cell (PBMC) proliferation was evident, while decreased mitogen-stimulated gamma interferon (IFN-γ) production suggested immunomodulation, which was not restricted to parasite-specific responses. Our findings show that the global immune response is modulated toward a nonproliferative type 2 state following natural challenge with F. hepatica. This has implications in terms of the timing of the administration of vaccination programs and for host susceptibility to coinfecting pathogens.  

4.1816           Recombinant human B cell repertoires enable screening for rare, specific, and natively paired antibodies

Rajan, S., Kierny, M.R., Mercer, A., Wu, J., Tovchigrechko, A., Wu, H., Dall’Acqua, W.F., Xiao, X. and Chowdhury, P.S. Communications Biol., 1:5 (2018)   The human antibody repertoire is increasingly being recognized as a valuable source of therapeutic grade antibodies. However, methods for mining primary antibody-expressing B cells are limited in their ability to rapidly isolate rare and antigen-specific binders. Here we show the encapsulation of two million primary B cells into picoliter-sized droplets, where their cognate V genes are fused in-frame to form a library of scFv cassettes. We used this approach to construct natively paired phage-display libraries from healthy donors and drove selection towards cross-reactive antibodies targeting influenza hemagglutinin. Within 4 weeks we progressed from B cell isolation to a panel of unique monoclonal antibodies, including seven that displayed broad reactivity to different clinically relevant influenza hemagglutinin subtypes. Most isolated antibody sequences were not detected by next-generation sequencing of the paired repertoire, illustrating how this method can isolate extremely rare leads not likely found by existing technologies.  

4.1817           High content image analysis reveals function of miR-124 upstream of Vimentin in regulating motor neuron mitochondria

Yardani, T. et al Scientific Reports, 8:59 (2018)   microRNAs (miRNAs) are critical for neuronal function and their dysregulation is repeatedly observed in neurodegenerative diseases. Here, we implemented high content image analysis for investigating the impact of several miRNAs in mouse primary motor neurons. This survey directed our attention to the neuron-specific miR-124, which controls axonal morphology. By performing next generation sequencing analysis and molecular studies, we characterized novel roles for miR-124 in control of mitochondria localization and function. We further demonstrated that the intermediate filament Vimentin is a key target of miR-124 in this system. Our data establishes a new pathway for control of mitochondria function in motor neurons, revealing the value of a neuron-specific miRNA gene as a mechanism for the re-shaping of otherwise ubiquitously-expressed intermediate filament network, upstream of mitochondria activity and cellular metabolism.  

4.1818           Lack of Fgf18 causes abnormal clustering of motor nerve terminals at the neuromuscular junction with reduced acetylcholine receptor clusters

Ito, K., Ohkawara, B., yagi, H., Nakashima, H., Tsushima, M., Ota, K., Konishi, H., Masuda, A., Imagama, S., Kiyama, H., Ishiguro, N. and Ohno, K. Scientific Reports, 8:434 (2018)   FGF receptor 2 is involved in the formation of the neuromuscular junction (NMJ), but its in vivo ligand remains to be determined. Laser capture microdissection of the mouse spinal motor neurons (SMNs) revealed that Fgf18 mRNA is highly expressed in SMNs in adults. Expression of Fgf18 mRNA was the highest in the spinal cord at embryonic day (E) 15.5, which gradually decreased to postnatal day 7. FGF18 protein was localized at the NMJs of the tibialis anterior muscle at E18.5 and in adults. Fgf18−/− mice at E18.5 showed decreased expressions of the NMJ-specific Chrne and Colq genes in the diaphragm. In Fgf18−/− diaphragms, the synaptophysin-positive areas at the nerve terminals and the acetylcholine receptor (AChR)-positive areas at the motor endplates were both approximately one-third of those in wild-type embryos. Fgf18−/− diaphragms ultrastructurally showed abnormal aggregation of multiple nerve terminals making a gigantic presynapse with sparse synaptic vesicles, and simplified motor endplates. In Fgf18−/− diaphragms, miniature endplate potentials were low in amplitude with markedly reduced frequency. In C2C12 myotubes, FGF18 enhanced AChR clustering, which was blocked by inhibiting FGFRs or MEK1. We propose that FGF18 plays a pivotal role in AChR clustering and NMJ formation in mouse embryogenesis.  

4.1819           The alpha7-nicotinic receptor contributes to gp120-induced neurotoxicity: implications in HIV-associated neurocognitive disorders

Capo-Velez, C.M., Morales-Vargas, B., Garcia-Gonzalez, A., Grajales-Reyes, J.G., Delgado-Velez, M., Madera, B., Baez-pagan, C.A., Quesada, O. and Lasalde-Dominicci, J.A. Scientific Reports, 8:1829 (2018)   Currently, there are no specific therapies to treat HIV-1 associated neurocognitive disorders (HAND). The HIV-1 envelope, gp120, induces neuropathological changes similar to those in HAND patients; furthermore, it triggers an upregulation of the α7-nicotinic acetylcholine receptor (α7-nAChR), facilitating intracellular calcium overload and neuronal cell death. Using a gp120_IIIB-transgenic mouse (gp120-tgm) model, we demonstrate that α7-nAChRs are upregulated on striatal neurons. Activation of α7-nAChRs leads to an increase in both intracellular calcium and percentage of apoptotic cells, which can be abrogated by antagonizing the receptor, suggesting a role for α7-nAChRs in gp120-induced neurotoxicity. Moreover, we demonstrate for the first time that gp120-tgm have learning deficiencies on a striatum-dependent behavioral task. They also show locomotor deficiencies, which improved with α7-nAChR antagonists, further supporting a role for this receptor in gp120-induced neurotoxicity. Together, these results uncover a new mechanism through which gp120-induced modulation of α7-nAChRs in the striatum can contribute to HAND development.  

4.1820           Parallel droplet microfluidics for high throughput cell encapsulation and synthetic microgel generation

Headen, D.M., Garcia, J.R. and Garcia, A.J. Microsystems & Nanoengineering, 4:17076 (2018)   Cells can be microencapsulated in synthetic hydrogel microspheres (microgels) using droplet microfluidics, but microfluidic devices with a single droplet generating geometry have limited throughput, especially as microgel diameter decreases. Here we demonstrate microencapsulation of human mesenchymal stem cells (hMSCs) in small (<100 μm diameter) microgels utilizing parallel droplet generators on a two-layer elastomer device, which has 600% increased throughput vs. single-nozzle devices. Distribution of microgel diameters were compared between products of parallel vs. single-nozzle configurations for two square nozzle widths, 35 and 100 μm. Microgels produced on parallel nozzles were equivalent to those produced on single nozzles, with substantially the same polydispersity. Microencapsulation of hMSCs was compared for parallel nozzle devices of each width. Thirty five micrometer wide nozzle devices could be operated at twice the cell concentration of 100 μm wide nozzle devices but produced more empty microgels than predicted by a Poisson distribution. Hundred micrometer wide nozzle devices produced microgels as predicted by a Poisson distribution. Polydispersity of microgels did not increase with the addition of cells for either nozzle width. hMSCs encapsulated on 35 μm wide nozzle devices had reduced viability (~70%) and a corresponding decrease in vascular endothelial growth factor (VEGF) secretion compared to hMSCs cultured on tissue culture (TC) plastic. Encapsulating hMSCs using 100 μm wide nozzle devices mitigated loss of viability and function, as measured by VEGF secretion.  

4.1821           Carnosol-mediated Sirtuin 1 activation inhibits Enhancer of Zeste Homolog 2 to attenuate liver fibrosis

Zhao, H., Wang, Z., Tang, F., Zhao, Y., Feng, D., Li, Y., Hu, Y., Wang, C., Zhou, J., Tian, X. and Yao, J. Pharmacol. Res., 128, 327-337 (2018)   Quiescent hepatic stellate cell (HSC) activation and subsequent conversion into myofibroblasts is the central event in hepatic fibrosis pathogenesis. Epithelial−mesenchymal transition (EMT), another vital participant in liver fibrosis, has the potential to initiate HSC activation, which promotes abundant myofibroblast production. Previous studies suggest that Enhancer of Zeste Homolog 2 (EZH2) plays a significant role in myofibroblast transdifferentiation; however, the underlying mechanisms remain largely unaddressed. Carnosol (CS), a compound extracted from rosemary, displays multiple pharmacological activities. This study aimed to investigate the signaling mechanisms underlying EZH2 inhibition and the anti-fibrotic effect of CS in liver fibrosis. We found that CS significantly inhibited CCl₄- and TGFβ1-induced liver fibrosis and reduced both HSC activation and EMT. EZH2 knockdown also prevented these processes induced by TGFβ1 in HSCs and AML-12 cells. Interestingly, the protective effect of CS was positively associated with Sirtuin 1 (SIRT1) activation and accompanied by EZH2 inhibition. SIRT1 knockdown attenuated the EZH2 inhibition induced by CS and increased EZH2 acetylation, which enhanced its stability. Conversely, upon TGFβ1 exposure, SIRT1 activation significantly reduced the level of EZH2 acetylation; however, EZH2 overexpression prevented the SIRT1 activation that primed myofibroblast inhibition, indicating that EZH2 is a target of SIRT1. Thus, SIRT1/EZH2 regulation could be used as a new therapeutic strategy for fibrogenesis. Together, this study provides evidence of activation of the SIRT1/EZH2 pathway by CS that inhibits myofibroblast generation, and thus, CS may represent an attractive candidate for anti-fibrotic clinical therapy.  

4.1822           Quality of cryopreserved buffalo spermatozoa improved from poor quality ejaculates

Ali, A., Ahmad, E., Ijaz, N., Ahmad, W. and Ul-hassan, F. Cryobiology, 80, 167 abstract S44 (2018)   Use of assisted reproductive technology, especially density gradient centrifugation, is being used as a technique of semen preparation in animals and humans. The technique was efficient in improving semen quality after cryopreservation. Single layer centrifugation provides a large amount of high quality sperm before conservation. Single layer centrifugation studied in stallions, boars and cow bulls, limited data available for buffalo bulls. Also bulls experience a transient reduction in semen quality, thus techniques that allow improvement in semen quality could be applied. The aim of this study was the evaluation of single layer and double layer centrifugation by the use of iodixanol, compared with conventional centrifugation and non-centrifuged semen, on the sperm characteristics during the cryopreservation process in buffalo bulls with normal and poor semen quality. Single layer centrifugation and double layer centrifugation both significantly increased the percentage of normal sperm and decreased the percentage of non-sperm cells in poor quality samples, while both were ineffective in those of normal quality. Sperm characteristics in poor quality samples increased after single and double layer centrifugation, reaching values similar to those recorded in normal samples, and this trend is maintained after equilibration and after cryopreservation. Single Layer and Double Layer Centrifugation resulted in a reduction of the sperm recovered, and this resulted in a reduction of the absolute number of sperm preserved in the normal samples, without a clear improvement in sperm characteristics in this type of sample. Data suggested that both techniques could be performed in practice, but their application should be limited to the cases in which the quality of the sperm recovered is more important than the total number of sperm.  

4.1823           Cryosurvival of Mus musculus and Peromyscus spermatozoa in the presence of Iodixanol

Agca, C., Timonin, M., Kim, S., Epperson, K. and Agca, Y. Cryobiology, 80, 185, abstract P22 (2018)   Cryopreservation of spermatozoa provides a valuable means of maintaining transgenic mouse strains used in biomedical research. Addition of Iodixanol (OptiPrep™) had beneficial effects on post-thaw motility of bull, rat and ram sperm. In the current study, we compared effects of addition of OptiPrep™ in freezing solution on Mus musculus and Peromyscus maniculatus (PM) mouse sperm post-thaw survival. Four levels of OptiPrep™ (0, 5, 15 and 25%) in combination with three levels of raffinose (18, 15.5 and 14.5%) were compared: The freezing solutions contained 18% raffinose (18R), 18% raffinose and 5% OptiPrep™ (18R5O), 15.5% raffinose and 15% OptiPrep™ (15.5R15O), and 14.5% raffinose and 25% OptiPrep™ (14.5R25O). All the freezing solutions were supplemented with 3% skim milk. The freezing solutions were tested on Mus musclus species C57BL/6, 129S1/SvImJ, and FVB/NJ and Peromyscus maniculatus. Post-thaw sperm motility, progressive motility, velocity, Live-dead sperm, acosomal integrity, mitochondrial membrane potential were determined. Mus musculus sperm post-thaw motility, progressive motility, live sperm, and acosomal integrity improved when 15.5R15O was used as the cryoprotectant. Sperm velocity and mitochondrial membrane potential were not different among sperm freezing solutions. Addition of OptiPrep™ significantly improved (P=0.05) PM live sperm concentration compared to sperm frozen in 18R. Peromyscus maniculatus post-thaw motility was significantly greater (P=0.01) for 15.5R15O compared to 18R and 18R5O. These data suggest that in addition to raffinose and skim milk, iodixanol may be an effective cryoprotectant for mouse and Peromyscus spermatozoa.  

4.1824           Liver ‘organ on a chip’

Beckwitt, C.H., Clark, A.M., Wheeler, S., taylor, D.L., Stolz, D.B., Griffith, L. and Wells, A. Exp. Cell Res., 363, 15-25 (2018)   The liver plays critical roles in both homeostasis and pathology. It is the major site of drug metabolism in the body and, as such, a common target for drug-induced toxicity and is susceptible to a wide range of diseases. In contrast to other solid organs, the liver possesses the unique ability to regenerate. The physiological importance and plasticity of this organ make it a crucial system of study to better understand human physiology, disease, and response to exogenous compounds. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems.  

4.1825           EphB2 receptor tyrosine kinase promotes hepatic fibrogenesis in mice via activation of hepatic stellate cells

Mimche, P.N., Lee, C.M., Mimche, S.M., Thapa, M., Grakoui, A., Henkemeyer, M. and Lamb, T.J. Scientific Reports, 8:2532 (2018)   Hepatic fibrosis is the result of an excessive wound-healing response subsequent to chronic liver injury. A feature of liver fibrogenesis is the secretion and deposition of extracellular matrix proteins by activated hepatic stellate cells (HSCs). Here we report that upregulation of EphB2 is a prominent feature of two mouse models of hepatic fibrosis and also observed in humans with liver cirrhosis. EphB2 is upregulated and activated in mouse HSCs following chronic carbon tetrachloride (CCl₄) exposure. Moreover, we show that EphB2 deficiency attenuates liver fibrosis and inflammation and this is correlated with an overall reduction in pro-fibrotic markers, inflammatory chemokines and cytokines. In an in vitro system of HSCs activation we observed an impaired proliferation and sub-optimal differentiation into fibrogenic myofibroblasts of HSCs isolated from EphB2−/− mice compared to HSCs isolated from wild type mice. This supports the hypothesis that EphB2 promotes liver fibrosis partly via activation of HSCs. Cellular apoptosis which is generally observed during the regression of liver fibrogenesis was increased in liver specimens of CCl₄-treated EphB2−/− mice compared to littermate controls. This data is suggestive of an active repair/regeneration system in the absence of EphB2. Altogether, our data validate this novel pro-fibrotic function of EphB2 receptor tyrosine kinase.  

4.1826           Rho-inhibiting C2IN-C3 fusion toxin inhibits chemotactic recruitment of human monocytes ex vivo and in mice in vivo

Martin, T., Möglich, A., Felix, I., Förtsch, C., Rittlinger, A., Palmer, A., Denk, S., Schneider, J., Notbohm, L.,Vogel, M., Geiger, H., Pascheke, S., Huber-lang, M. and Barth, H. Arch. Toxicol., 92(1), 323-336 (2018)   Bacterial protein toxins became valuable molecular tools for the targeted modulation of cell functions in experimental pharmacology and attractive therapeutics because of their potent and specific mode of action in human cells. C2IN-C3lim, a recombinant fusion toxin (~50 kDa) of the Rho-inhibiting C3lim from Clostridium (C.) limosum and a non-toxic portion of the C. botulinum C2 toxin (C2IN), is selectively internalized into the cytosol of monocytic cells where C3lim specifically ADP-ribosylates Rho A and -B, thereby inhibiting Rho-mediated signaling. Thus, we hypothesized that these unique features make C2IN-C3lim an attractive molecule for the targeted pharmacological down-regulation of Rho-mediated functions in monocytes. The analysis of the actin structure and the Rho ADP-ribosylation status implied that C2IN-C3lim entered the cytosol of primary human monocytes from healthy donors ex vivo within 1 h. Moreover, it inhibited the fMLP-induced chemotaxis of human monocytes in a Boyden chamber model ex vivo. Similarly, in a 3-dimensional ex vivo model of extravasation, single cell analysis revealed that C2IN-C3lim-treated cells were not able to move. In a clinically relevant mouse model of blunt chest trauma, the local application of C2IN-C3lim into the lungs after thorax trauma prevented the trauma-induced recruitment of monocytes into the lungs in vivo. Thus, C2IN-C3lim might be an attractive lead compound for novel pharmacological strategies to avoid the cellular damage response caused by monocytes in damaged tissue after trauma and during systemic inflammation. The results suggest that the pathophysiological role of clostridial C3 toxins might be a down-modulation of the innate immune system.  

4.1827           Culture of somatic cells isolated from frozen-thawed equine semen using fluorescence-assisted cell sorting

Brom-de-Luna, J., Canesin, H.S., Wright, G. and Hinrichs, K. Animal Reprod. Sci., 190, 10-17 (2017)   Nuclear transfer using somatic cells from frozen semen (FzSC) would allow cloning of animals for which no other genetic material is available. Horses are one of the few species for which cloning is commercially feasible; despite this, there is no information available on the culture of equine FzSC. After preliminary trials on equine FzSC, recovered by density-gradient centrifugation, resulted in no growth, we hypothesized that sperm in the culture system negatively affected cell proliferation. Therefore, we evaluated culture of FzSC isolated using fluorescence-assisted cell sorting. In Exp. 1, sperm were labeled using antibodies to a sperm-specific antigen, SP17, and unlabeled cells were collected. This resulted in high sperm contamination. In Exp. 2, FzSC were labeled using an anti-MHC class I antibody. This resulted in an essentially pure population of FzSC, 13–25% of which were nucleated. Culture yielded no proliferation in any of nine replicates. In Exp. 3, 5 × 10³ viable fresh, cultured horse fibroblasts were added to the frozen-thawed, washed semen, then this suspension was labeled and sorted as for Exp. 2. The enriched population had a mean of five sperm per recovered somatic cell; culture yielded formation of monolayers. In conclusion, an essentially pure population of equine FzSC could be obtained using sorting for presence of MHC class I antigens. No equine FzSC grew in culture; however, the proliferation of fibroblasts subjected to the same processing demonstrated that the labeling and sorting methods, and the presence of few sperm in culture, were compatible with cell viability.  

4.1828           PD-L1 Prevents the Development of Autoimmune Heart Disease in Graft-versus-Host Disease

Juchem, K.W., Sacirbegovic, F., Zhang, C., Sharpe, A.H., Russell, K., McNiff, J.M., Demetris, A.J., Shlomchik, M.J. and Shlomchik, W.D.

1. Immunol., 200(2), 834-846 (2018)

  Effector memory T cells (T_EM) are less capable of inducing graft-versus-host disease (GVHD) compared with naive T cells (T_N). Previously, in the TS1 TCR transgenic model of GVHD, wherein TS1 CD4 cells specific for a model minor histocompatibility Ag (miHA) induce GVHD in miHA-positive recipients, we found that cell-intrinsic properties of TS1 T_EM reduced their GVHD potency relative to TS1 T_N. Posttransplant, TS1 T_EM progeny expressed higher levels of PD-1 than did TS1 T_N progeny, leading us to test the hypothesis that T_EM induce less GVHD because of increased sensitivity to PD-ligands. In this study, we tested this hypothesis and found that indeed TS1 T_EM induced more severe skin and liver GVHD in the absence of PD-ligands. However, lack of PD-ligands did not result in early weight loss and colon GVHD comparable to that induced by TS1 T_N, indicating that additional pathways restrain alloreactive T_EM. TS1 T_N also caused more severe GVHD without PD-ligands. The absence of PD-ligands on donor bone marrow was sufficient to augment GVHD caused by either T_EM or T_N, indicating that donor PD-ligand–expressing APCs critically regulate GVHD. In the absence of PD-ligands, both TS1 T_EM and T_N induced late-onset myocarditis. Surprisingly, this was an autoimmune manifestation, because its development required non-TS1 polyclonal CD8⁺ T cells. Myocarditis development also required donor bone marrow to be PD-ligand deficient, demonstrating the importance of donor APC regulatory function. In summary, PD-ligands suppress both miHA-directed GVHD and the development of alloimmunity-induced autoimmunity after allogeneic hematopoietic transplantation.  

4.1829           Effector CD4+ T cells recognize intravascular antigen presented by patrolling monocytes

Westhorpe, C.L.V., Norman, M.U., Hall, P., Snelgrove, S.L., Finsterbusch, M., Li, A., Lo, C., Tan, Z.H., Li, S., Nilsson, S.K., Kitching, A.R. and Hiskey, M.J. Nature Communications, 9:747 (2018)   Although effector CD4⁺ T cells readily respond to antigen outside the vasculature, how they respond to intravascular antigens is unknown. Here we show the process of intravascular antigen recognition using intravital multiphoton microscopy of glomeruli. CD4⁺ T cells undergo intravascular migration within uninflamed glomeruli. Similarly, while MHCII is not expressed by intrinsic glomerular cells, intravascular MHCII-expressing immune cells patrol glomerular capillaries, interacting with CD4⁺ T cells. Following intravascular deposition of antigen in glomeruli, effector CD4⁺ T-cell responses, including NFAT1 nuclear translocation and decreased migration, are consistent with antigen recognition. Of the MHCII⁺ immune cells adherent in glomerular capillaries, only monocytes are retained for prolonged durations. These cells can also induce T-cell proliferation in vitro. Moreover, monocyte depletion reduces CD4⁺ T-cell-dependent glomerular inflammation. These findings indicate that MHCII⁺ monocytes patrolling the glomerular microvasculature can present intravascular antigen to CD4⁺ T cells within glomerular capillaries, leading to antigen-dependent inflammation.  

4.1830           Neuregulin-1 elicits a regulatory immune response following traumatic spinal cord injury

Alizadeh, A., Santhosh, K.T., Kataria, H., Gounni, A.S. and Karimi-Abdolrezaee, S.K.

1. Neuroinflamm., 15:53 (2018)

  Background Spinal cord injury (SCI) triggers a robust neuroinflammatory response that governs secondary injury mechanisms with both degenerative and pro-regenerative effects. Identifying new immunomodulatory therapies to promote the supportive aspect of immune response is critically needed for the treatment of SCI. We previously demonstrated that SCI results in acute and permanent depletion of the neuronally derived Neuregulin-1 (Nrg-1) in the spinal cord. Increasing the dysregulated level of Nrg-1 through acute intrathecal Nrg-1 treatment enhanced endogenous cell replacement and promoted white matter preservation and functional recovery in rat SCI. Moreover, we identified a neuroprotective role for Nrg-1 in moderating the activity of resident astrocytes and microglia following injury. To date, the impact of Nrg-1 on immune response in SCI has not yet been investigated. In this study, we elucidated the effect of systemic Nrg-1 therapy on the recruitment and function of macrophages, T cells, and B cells, three major leukocyte populations involved in neuroinflammatory processes following SCI. Methods We utilized a clinically relevant model of moderately severe compressive SCI in female Sprague-Dawley rats. Nrg-1 (2 μg/day) or saline was delivered subcutaneously through osmotic mini-pumps starting 30 min after SCI. We conducted flow cytometry, quantitative real-time PCR, and immunohistochemistry at acute, subacute, and chronic stages of SCI to investigate the effects of Nrg-1 treatment on systemic and spinal cord immune response as well as cytokine, chemokine, and antibody production. Results We provide novel evidence that Nrg-1 promotes a pro-regenerative immune response after SCI. Bioavailability of Nrg-1 stimulated a regulatory phenotype in T and B cells and augmented the population of M2 macrophages in the spinal cord and blood during the acute and chronic stages of SCI. Importantly, Nrg-1 fostered a more balanced microenvironment in the injured spinal cord by attenuating antibody deposition and expression of pro-inflammatory cytokines and chemokines while upregulating pro-regenerative mediators. Conclusion We provide the first evidence of a significant regulatory role for Nrg-1 in neuroinflammation after SCI. Importantly, the present study establishes the promise of systemic Nrg-1 treatment as a candidate immunotherapy for traumatic SCI and other CNS neuroinflammatory conditions.  

4.1831           Neutrophil–Hepatic Stellate Cell Interactions Promote Fibrosis in Experimental Steatohepatitis

Zhou, Z., Xu, M-J., Cai, Y., Wang, W., Jiang, J.X., Varga, Z.V., Feng, D., Pacher, P., Kunos, G., Torok, N.J. and Gao, B. Cell. Mol. Gastroenterol. Hepatol., 5, 399-413 (2018)   Background & Aims Hepatic infiltration of neutrophils is a hallmark of steatohepatitis; however, the role of neutrophils in the progression of steatohepatitis remains unknown. Methods A clinically relevant mouse model of steatohepatitis induced by high-fat diet (HFD) plus binge ethanol feeding was used. Liver fibrosis was examined. In vitro cell culture was used to analyze the interaction of hepatic stellate cells (HSCs) and neutrophils. Results HFD plus one binge ethanol (HFD+1B) feeding induced significant hepatic neutrophil infiltration, liver injury, and fibrosis. HFD plus multiple binges of ethanol (HFD+mB) caused more pronounced liver fibrosis. Microarray analyses showed that the most highly activated signaling pathway in this HFD+1B model was related to liver fibrosis and HSC activation. Blockade of chemokine (C-X-C motif) ligand 1 or intercellular adhesion molecule-1 expression reduced hepatic neutrophil infiltration and ameliorated liver injury and fibrosis. Disruption of the p47^phox gene (also called neutrophil cytosolic factor 1), a critical component of reactive oxygen species producing nicotinamide adenine dinucleotide phosphate-oxidase in neutrophils, diminished HFD+1B–induced liver injury and fibrosis. Co-culture of HSCs with neutrophils, but not with neutrophil apoptotic bodies, induced HSC activation and prolonged neutrophil survival. Mechanistic studies showed that activated HSCs produce granulocyte-macrophage colony-stimulating factor and interleukin-15 to prolong the survival of neutrophils, which may serve as a positive forward loop to promote liver damage and fibrosis. Conclusions The current data from a mouse model of HFD plus binge ethanol feeding suggest that obesity and binge drinking synergize to promote liver fibrosis, which is partially mediated via the interaction of neutrophils and HSCs. Microarray data in this article have been uploaded to NCBI’s Gene Expression Omnibus (GEO accession number: GSE98153).  

4.1832           The cytoprotective role of DJ-1 and p45 NFE2 against human primary alveolar type II cell injury and emphysema

Tan, L.H., Bahmed, K., Lin, C-R., marchatti, N., Bolla, S., Criner, G.J., kelsen, S., Madesh, M. and Kosmider, B. Scientific Reports, 8:3555 (2018)   Emphysema is characterized by irreversibly enlarged airspaces and destruction of alveolar walls. One of the factors contributing to this disease pathogenesis is an elevation in extracellular matrix (ECM) degradation in the lung. Alveolar type II (ATII) cells produce and secrete pulmonary surfactants and proliferate to restore the epithelium after damage. We isolated ATII cells from control non-smokers, smokers and patients with emphysema to determine the role of NFE2 (nuclear factor, erythroid-derived 2). NFE2 is a heterodimer composed of two subunits, a 45 kDa (p45 NFE2) and 18 kDa (p18 NFE2) polypeptides. Low expression of p45 NFE2 in patients with emphysema correlated with a high ECM degradation. Moreover, we found that NFE2 knockdown increased cell death induced by cigarette smoke extract. We also studied the cross talk between p45 NFE2 and DJ-1. DJ-1 protein is a redox-sensitive chaperone that protects cells from oxidative stress. We detected that cigarette smoke significantly increased p45 NFE2 levels in DJ-1 KO mice compared to wild-type mice. Our results indicate that p45 NFE2 expression is induced by exposure to cigarette smoke, has a cytoprotective activity against cell injury, and its downregulation in human primary ATII cells may contribute to emphysema pathogenesis.  

4.1833           Metabolic Reprogramming in Amyotrophic Lateral Sclerosis

Szelechowski, M., Amoedo, N., Obre, E., leger, C., Allard, l., Bonneu, M., Claverol, S., Lacombe, D., Oliet, S., Chevallier, S., La Masson, G. and Rossignol., R. Scientific Reports, 8:3953 (2018)   Mitochondrial dysfunction in the spinal cord is a hallmark of amyotrophic lateral sclerosis (ALS), but the neurometabolic alterations during early stages of the disease remain unknown. Here, we investigated the bioenergetic and proteomic changes in ALS mouse motor neurons and patients’ skin fibroblasts. We first observed that SODG93A mice presymptomatic motor neurons display alterations in the coupling efficiency of oxidative phosphorylation, along with fragmentation of the mitochondrial network. The proteome of presymptomatic ALS mice motor neurons also revealed a peculiar metabolic signature with upregulation of most energy-transducing enzymes, including the fatty acid oxidation (FAO) and the ketogenic components HADHA and ACAT2, respectively. Accordingly, FAO inhibition altered cell viability specifically in ALS mice motor neurons, while uncoupling protein 2 (UCP2) inhibition recovered cellular ATP levels and mitochondrial network morphology. These findings suggest a novel hypothesis of ALS bioenergetics linking FAO and UCP2. Lastly, we provide a unique set of data comparing the molecular alterations found in human ALS patients’ skin fibroblasts and SODG93A mouse motor neurons, revealing conserved changes in protein translation, folding and assembly, tRNA aminoacylation and cell adhesion processes.  

4.1834           Phytosterols Synergize With Endotoxin to Augment Inflammation in Kupffer Cells but Alone Have Limited Direct Effect on Hepatocytes

Guthrie, G., Tackett, B., Stoll, B., Martin, C., Olutoye, O. and Burrin, D.G.

1. Parenteral and Enteral Nutrition, 42(1), 37-48 (2018)

  Introduction: Phytosterols are implicated in the development of parenteral nutrition–associated liver disease. A newly proposed mechanism for phytosterol-mediated parenteral nutrition–associated liver disease is through phytosterol-facilitated hepatic proinflammatory cytokine release following exposure to intestinally derived bacteria. Whether the proinflammatory effects are liver cell specific is not known. Aim: To determine if phytosterols cause inflammation in hepatocytes or Kupffer cells independently or require costimulation by lipopolysaccharide (LPS). Methods: In an in vivo study, neonatal piglets on parenteral nutrition for 11 days received an 8-hour infusion of LPS. In the in vitro studies, neonatal piglet Kupffer cells and hepatocytes were treated with media, media + 1% soy oil, or media + 1% soy oil + 100µM phytosterols. After 24-hour incubation, cells were treated with farnesoid X receptor (FXR) agonist obeticholic acid or liver X receptor (LXR) agonist GW3965 and challenged with LPS or interleukin 1β. Results: LPS administration in piglets led to transient increases in proinflammatory cytokines and suppression of the transporters bile salt export pump and ATP-binding cassette transporter G5. In hepatocytes, phytosterols did not activate inflammation. Phytosterol treatment alone did not activate inflammation in Kupffer cells but, combined with LPS, synergistically increased interleukin 1β production. FXR and LXR agonists increased transporter expression in hepatocytes. GW3965 suppressed proinflammatory cytokine production in Kupffer cells, but obeticholic acid did not. Conclusions: LPS suppresses transporters that control bile acid and phytosterol clearance. Phytosterols alone do not cause inflammatory response. However, with costimulation by LPS, phytosterols synergistically maximize the inflammatory response in Kupffer cells.  

4.1835           HDAC6 is a therapeutic target in mutant GARS-induced Charcot-Marie-Tooth disease

Benoy, V. eet al Brain, 141, 673-687 (2018)   Peripheral nerve axons require a well-organized axonal microtubule network for efficient transport to ensure the constant crosstalk between soma and synapse. Mutations in more than 80 different genes cause Charcot-Marie-Tooth disease, which is the most common inherited disorder affecting peripheral nerves. This genetic heterogeneity has hampered the development of therapeutics for Charcot-Marie-Tooth disease. The aim of this study was to explore whether histone deacetylase 6 (HDAC6) can serve as a therapeutic target focusing on the mutant glycyl-tRNA synthetase (GlyRS/GARS)-induced peripheral neuropathy. Peripheral nerves and dorsal root ganglia from the C201R mutant Gars mouse model showed reduced acetylated α-tubulin levels. In primary dorsal root ganglion neurons, mutant GlyRS affected neurite length and disrupted normal mitochondrial transport. We demonstrated that GlyRS co-immunoprecipitated with HDAC6 and that this interaction was blocked by tubastatin A, a selective inhibitor of the deacetylating function of HDAC6. Moreover, HDAC6 inhibition restored mitochondrial axonal transport in mutant GlyRS-expressing neurons. Systemic delivery of a specific HDAC6 inhibitor increased α-tubulin acetylation in peripheral nerves and partially restored nerve conduction and motor behaviour in mutant Gars mice. Our study demonstrates that α-tubulin deacetylation and disrupted axonal transport may represent a common pathogenic mechanism underlying Charcot-Marie-Tooth disease and it broadens the therapeutic potential of selective HDAC6 inhibition to other genetic forms of axonal Charcot-Marie-Tooth disease.  

4.1836           Loss of toll-like receptor 3 aggravates hepatic inflammation but ameliorates steatosis in mice

Lee, Y-S., Kim, D-Y., Kim, T-J., Kim, S.Y., Jeong, J-M., Jeong, W-I., Jung, J-K., Choi, J.K., Yi, H-S. and Byun, J-S. Biochem. Biophys. Res. Comm., 497, 957-962 (2018)   The importance of toll-like receptor (TLR) 4 in the pathogenesis of steatohepatitis has been well documented; however, little is known about the role of TLR3. In this study, we determined whether the depletion of TLR3 modulated hepatic injury in mice and further aimed to provide mechanistic insights into the TLR3-mediated modulation of diet-induced hepatic inflammation and fat accumulation. Hepatic steatosis and inflammatory response were induced by feeding wild-type (WT) or TLR3 knockout mice a high-fat diet for 8 weeks. Primary liver resident cells, including hepatocytes, Kupffer cells, and hepatic stellate cells (HSCs), were treated with palmitic acid. TLR3 knockout mice fed a high-fat diet showed severe hepatic inflammation accompanied by nuclear factor-κB and IRF3 activation, which is mainly induced by the activation of Kupffer cells. Decreased TLR4 expression was restored in hepatic mononuclear cells and Kupffer cells in TLR3 knockout mice compared to that in the WT. Moreover, hepatic steatosis was decreased in TLR3 knockout mice. Hepatocytes from TLR3 knockout mice exhibited reduced expression of cannabinoid receptors. HSCs from TLR3 knockout mice showed decreased expression of the enzymes involved in endocannabinoid synthesis. In conclusion, this study suggests that the selective modulation of TLR3 could be a novel therapeutic target for the treatment of hepatic inflammation and steatosis.  

4.1837           Stem cell derived phenotypic human neuromuscular junction model for dose response evaluation of therapeutics

Santhanam, N., Kumanchik, L., Guo, X., Sommerhage, F., Cai, Y., Jackson, M., Martin, C., Saad, G., McAleer, C.W., Wang, Y., Lavado, A., Long, C.J. and Hickman, J.J. Biomaterials, 166, 64-78 (2018)   There are currently no functional neuromuscular junction (hNMJ) systems composed of human cells that could be used for drug evaluations or toxicity testing in vitro. These systems are needed to evaluate NMJs for diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy or other neurodegenerative diseases or injury states. There are certainly no model systems, animal or human, that allows for isolated treatment of motoneurons or muscle capable of generating dose response curves to evaluate pharmacological activity of these highly specialized functional units. A system was developed in which human myotubes and motoneurons derived from stem cells were cultured in a serum-free medium in a BioMEMS construct. The system is composed of two chambers linked by microtunnels to enable axonal outgrowth to the muscle chamber that allows separate stimulation of each component and physiological NMJ function and MN stimulated tetanus. The muscle's contractions, induced by motoneuron activation or direct electrical stimulation, were monitored by image subtraction video recording for both frequency and amplitude. Bungarotoxin, BOTOX^® and curare dose response curves were generated to demonstrate pharmacological relevance of the phenotypic screening device. This quantifiable functional hNMJ system establishes a platform for generating patient-specific NMJ models by including patient-derived iPSCs.  

4.1838           Turning off NADPH oxidase-2 by impeding p67phox activation in infected mouse macrophages reduced viral entry and inflammation

Lejal, N., Truchet, s., Bechor, E., Bouguyon, E., Khedkar, V., Bertho, N., Vidic, J., Adenot, P., Solier, S., Pick, E. and Slama-Schwok, A.

BBA – General Subject, 1862, 1263-1275 (2018)

Background

Targeting cells of the host immune system is a promising approach to fight against Influenza A virus (IAV) infection. Macrophage cells use the NADPH oxidase-2 (NOX2) enzymatic complex as a first line of defense against pathogens by generating superoxide ions O2– and releasing H2O2. Herein, we investigated whether targeting membrane -embedded NOX2 decreased IAV entry via raft domains and reduced inflammation in infected macrophages.

Methods

Confocal microscopy and western blots monitored levels of the viral nucleoprotein NP and p67phox, NOX2 activator subunit, Elisa assays quantified TNF-α levels in LPS or IAV-activated mouse or porcine alveolar macrophages pretreated with a fluorescent NOX inhibitor, called nanoshutter NS1.

Results

IAV infection in macrophages promoted p67phox translocation to the membrane, rafts clustering and activation of the NOX2 complex at early times. Disrupting rafts reduced intracellular viral NP. NS1 markedly reduced raft clustering and viral entry by binding to the C-terminal of NOX2 also characterized in vitro. NS1 decrease of TNF-α release depended on the cell type.

Conclusion

NOX2 participated in IAV entry and raft-mediated endocytosis. NOX2 inhibition by NS1 reduced viral entry. NS1 competition with p67phox for NOX2 binding shown by in silico models and cell-free assays was in agreement with NS1 inhibiting p67phox translocation to membrane-embedded NOX2 in mouse and porcine macrophages.

 

4.1839           Risk factors for early readmission after total pancreatectomy and islet auto transplantation

Shahbazov, R., Naziruddin, B., Yadav, K., Saracino, G., Yoshimatsu, G., Kansak, M.A., Beecherl, E., Kim, P.T. and Levy, M.F. HPB, 20, 166-174 (2018)   Background Little published data exist examining causes of hospital readmission following total pancreatectomy with islet autotransplantation (TPIAT). Methods A retrospective analysis was performed of a prospectively collected institutional TPIAT database. Primary outcome was unplanned readmission to the hospital within 30 days from discharge. Reasons and risk factors for readmission as well as islet function were evaluated and compared by univariate and multivariate analysis. Results 83 patients underwent TPIAT from 2006 to 2014. 21 patients (25.3%) were readmitted within 30 days. Gastrointestinal problems (52.4%) and surgical site infection (42.8%) were the most common reasons for readmission. Initial LOS and reoperation were risk factors for early readmission. Patients with delayed gastric emptying (DGE) were three times more likely to get readmitted. In multivariate analysis, patients undergoing pylorus preservation surgery were nine times more likely to be readmitted than the antrectomy group. Conclusion Early readmission after TPIAT is common (one in four patients), underscoring the complexity of this procedure. Early readmission is not detrimental to islet graft function. Patients undergoing pylorus preservation are more likely to get readmitted, perhaps due to increased incidence of delayed gastric emptying. Decision for antrectomy vs. pylorus preservation needs to be individualized.  

4.1840           Long-term donors versus non-donor men: Iron metabolism and the atherosclerotic process

Risko, P., Platenik, J., Buchal, R., Potockova, J. and Kraml, P.J. Atheresclerosis, 272, 14-20 (2018)   Background and aims The increased iron level and the labile iron pool (LIP) in circulating monocytes are connected to higher frequency of cardiovascular events. Methods The study investigates the relationship between LIP in circulating monocytes and markers of iron metabolism and atherosclerosis (inflammation, oxidative stress, endothelial dysfunction and arterial elasticity) in long-term blood donors and non-donor volunteers. Results We found that donors had significantly higher LIP values than the control group (1.89 ± 0.47 μM vs. 1.50 ± 0.41 μM, p = 0.007). Despite the observed tendency for the donor group to have higher blood pressure, cholesterol, glucose and HOMAR-IR (homeostasis model assessment of insulin resistance), the groups did not differ in inflammatory markers, markers of endothelial dysfunction and markers of impaired arterial elasticity. The donor group had significant changes in iron metabolism (higher serum Fe, ceruloplasmin, and TfR/Ft ratio (transferrin receptor/ferritin ratio) and lower hepcidin, ferritin, and CD163), indicating depletion of body iron stores and activation of iron turnover. Conclusions LIP seems to be a good marker of iron turnover activity in these individuals despite the lack of a decrease in the hemoglobin concentration. We did not find a significant correlation between LIP levels and atherosclerosis progression in the two groups. However, further studies are needed to assess long-term donorship as a protective factor against atherosclerosis.  

4.1841           Perturbing chondroitin sulfate proteoglycan signaling through LAR and PTPσ receptors promotes a beneficial inflammatory response following spinal cord injury

Dyck, S., Kataria, H., Alizadeh, A., Santhosh, K., Lang, B., Silver, J. and Karimi-Abdolrezaee, S,

1. Neuroinnflamm., 15:90 (2018)

  Background Traumatic spinal cord injury (SCI) results in upregulation of chondroitin sulfate proteoglycans (CSPGs) by reactive glia that impedes repair and regeneration in the spinal cord. Degradation of CSPGs is known to be beneficial in promoting endogenous repair mechanisms including axonal sprouting/regeneration, oligodendrocyte replacement, and remyelination, and is associated with improvements in functional outcomes after SCI. Recent evidence suggests that CSPGs may regulate secondary injury mechanisms by modulating neuroinflammation after SCI. To date, the role of CSPGs in SCI neuroinflammation remains largely unexplored. The recent discovery of CSPG-specific receptors, leukocyte common antigen-related (LAR) and protein tyrosine phosphatase-sigma (PTPσ), allows unraveling the cellular and molecular mechanisms of CSPGs in SCI. In the present study, we have employed parallel in vivo and in vitro approaches to dissect the role of CSPGs and their receptors LAR and PTPσ in modulating the inflammatory processes in the acute and subacute phases of SCI. Methods In a clinically relevant model of compressive SCI in female Sprague Dawley rats, we targeted LAR and PTPσ by two intracellular functionally blocking peptides, termed ILP and ISP, respectively. We delivered ILP and ISP treatment intrathecally to the injured spinal cord in a sustainable manner by osmotic mini-pumps for various time-points post-SCI. We employed flow cytometry, Western blotting, and immunohistochemistry in rat SCI, as well as complementary in vitro studies in primary microglia cultures to address our questions. Results We provide novel evidence that signifies a key immunomodulatory role for LAR and PTPσ receptors in SCI. We show that blocking LAR and PTPσ reduces the population of classically activated M1 microglia/macrophages, while promoting alternatively activated M2 microglia/macrophages and T regulatory cells. This shift was associated with a remarkable elevation in pro-regenerative immune mediators, interleukin-10 (IL-10), and Arginase-1. Our parallel in vitro studies in microglia identified that while CSPGs do not induce an M1 phenotype per se, they promote a pro-inflammatory phenotype. Interestingly, inhibiting LAR and PTPσ in M1 and M2 microglia positively modulates their inflammatory response in the presence of CSPGs, and harnesses their ability for phagocytosis and mobilization. Interestingly, our findings indicate that CSPGs regulate microglia, at least in part, through the activation of the Rho/ROCK pathway downstream of LAR and PTPσ. Conclusions We have unveiled a novel role for LAR and PTPσ in regulating neuroinflammation in traumatic SCI. Our findings provide new insights into the mechanisms by which manipulation of CSPG signaling can promote recovery from SCI. More importantly, this work introduces the potential of ILP/ISP as a viable strategy for modulating the immune response following SCI and other neuroinflammatory conditions of the central nervous system.  

4.1842           Cognitive impairment in metabolically-obese, normal-weight rats: identification of early biomarkers in peripheral blood mononuclear cells

Cifre, M., Palou, P. and Oliver, P. Mol. Neurodegeneration, 13:14 (2018)   Background Metabolically-obese, normal-weight (MONW) individuals are not obese in terms of weight and height but have a number of obesity-related features (e.g. greater visceral adiposity, insulin resistance, and increased risk of cardiovascular disease). The MONW phenotype is related to the intake of unbalanced diets, such as those rich in fat. Increasing evidence shows a relationship between high-fat diet consumption and mild cognitive impairment and dementia. Thus, MONW individuals could be at a greater risk of cognitive dysfunction. We aimed to evaluate whether MONW-like animals present gene expression alterations in the hippocampus associated with an increased risk of cognitive impairment, and to identify early biomarkers of cognitive dysfunction in peripheral blood mononuclear cells (PBMC). Methods Wistar rats were chronically fed with a 60% (HF60) or a 45% (HF45) high-fat diet administered isocalorically to control animals to mimic MONW features. Expression analysis of cognitive decline-related genes was performed using RT-qPCR, and working memory was assessed using a T-maze. Results High-fat diet consumption altered the pattern of gene expression in the hippocampus, clearly pointing to cognitive decline, which was accompanied by a worse performance in the T-maze in HF60 animals. Remarkably, Syn1 and Sorl1 mRNA showed the same expression pattern in both the hippocampus and the PBMC obtained at different time-points in the HF60 group, even before other pathological signs were observed. Conclusions Our results demonstrate that long-term intake of high-fat diets, even in the absence of obesity, leads to cognitive disruption that is reflected in PBMC transcriptome. Therefore, PBMC are revealed as a plausible, minimally-invasive source of early biomarkers of cognitive impairment associated with increased fat intake.  

4.1843           Paroxetine and Low-dose Risperidone Induce Serotonin 5-HT1A and Dopamine D2 Receptor Heteromerization in the Mouse Prefrontal Cortex

Kolasa, M., Solich, J., Faron-Gorecka, A., Zurawek, D., Pabian, P., Ukasiewicz, S., Kusmider, M., Szafran-Pilch, K., Szlachta, M and Dziedzicka-Wasylewska, M. Neuroscience, 377, 184-196 (2018)   Recently, it has been shown that serotonin 5-HT_1A receptor interacts with dopamine D2 receptor in vitro. However, the existence of 5-HT_1A–D2 heteromers in native tissue remains unexplored. In the present study, we investigated 5-HT_1A–D2 receptor heteromerization in mice treated acutely or chronically with paroxetine (10 mg/kg) or risperidone (0.05 mg/kg). Receptor heteromerization was visualized and quantified in the mouse brain by in situ proximity ligation assay (PLA). Additionally, we aimed to determine the cellular localization of 5-HT_1A–D2 receptor heteromers in mouse adult primary neuronal cells by immunofluorescent staining with markers for astrocytes (GFAP) and neurons (NeuN and MAP2). The results from the current study demonstrated that 5-HT_1A and D2 receptor co-localization and heteromerization occurred in the mouse prefrontal cortex. Counterstaining after PLA confirmed neuronal (pyramidal and GABAergic) as well as astrocytal localization of 5-HT_1A–D2 receptor heteromers. Chronic administration of paroxetine or risperidone increased the level of 5-HT_1A–D2 receptor heteromers in the prefrontal cortex. These changes were not accompanied by any changes in the expression of mRNAs (measured by in situ hybridization) or densities of 5-HT_1A and D2 receptors (quantified by receptor autoradiography with [3H]8-OH-DPAT and [3H]domperidone, respectively), what all indicated that paroxetine and risperidone facilitated 5-HT_1A–D2 heteromer formation independently of the receptor expression. In vitro homogenous time-resolved FRET (HTRF) study confirmed the ability of tested drugs to influence the human 5-HT_1A–D2 heteromer formation. The obtained data indicate that the increase in 5-HT_1A–D2 receptor heteromerization is a common molecular characteristic of paroxetine and low-dose risperidone treatment.  

4.1844           A licensing step links AID to transcription elongation for mutagenesis in B cells

Methlot, S.P., Litzler, L.C., Subramani, P.G., Eranki, A.K., Fifield, H., Patenaude, A-M., Gilmore, J.C., Santiago, G.E., bagci, H., Cote, J-F., Larjani, M., Verdun, R.E. and Di Noia, J.M. Nature Communications, 9:1248 (2018)   Activation-induced deaminase (AID) mutates the immunoglobulin (Ig) genes to initiate somatic hypermutation (SHM) and class switch recombination (CSR) in B cells, thus underpinning antibody responses. AID mutates a few hundred other loci, but most AID-occupied genes are spared. The mechanisms underlying productive deamination versus non-productive AID targeting are unclear. Here we show that three clustered arginine residues define a functional AID domain required for SHM, CSR, and off-target activity in B cells without affecting AID deaminase activity or Escherichia coli mutagenesis. Both wt AID and mutants with single amino acid replacements in this domain broadly associate with Spt5 and chromatin and occupy the promoter of AID target genes. However, mutant AID fails to occupy the corresponding gene bodies and loses association with transcription elongation factors. Thus AID mutagenic activity is determined not by locus occupancy but by a licensing mechanism, which couples AID to transcription elongation.  

4.1845           The draft genome of Kipferlia bialata reveals reductive genome evolution in fornicate parasites

Tanfuji, G., Takabayashi, S., Kume, K., Takagi, M., Nakayama, T., Kamikawa, R., Inagaki, Y. and Hashimoto, T. PloS One, 13(3), e0194487 (2018)   The fornicata (fornicates) is a eukaryotic group known to consist of free-living and parasitic organisms. Genome datasets of two model fornicate parasites Giardia intestinalis and Spironucleus salmonicida are well annotated, so far. The nuclear genomes of G. intestinalis assemblages and S. salmonicida are small in terms of the genome size and simple in genome structure. However, an ancestral genomic structure and gene contents, from which genomes of the fornicate parasites have evolved, remains to be clarified. In order to understand genome evolution in fornicates, here, we present the draft genome sequence of a free-living fornicate, Kipferlia bialata, the divergence of which is earlier than those of the fornicate parasites, and compare it to the genomes of G. intestinalis and S. salmonicida. Our data show that the number of protein genes and introns in K. bialata genome are the most abundant in the genomes of three fornicates, reflecting an ancestral state of fornicate genome evolution. Evasion mechanisms of host immunity found in G. intestinalis and S. salmonicida are absent in the K. bialata genome, suggesting that the two parasites acquired the complex membrane surface proteins on the line leading to the common ancestor of G. intestinalis and S. salmonicida after the divergence from K. bialata. Furthermore, the mitochondrion related organelles (MROs) of K. bialata possess more complex suites of metabolic pathways than those in Giardia and in Spironucleus. In sum, our results unveil the process of reductive evolution which shaped the current genomes in two model fornicate parasites G. intestinalis and S. salmonicida.  

4.1846           Population snapshots predict early haematopoietic and erythroid hierarchies

Tusi, B.K., Wolock, S.L., Weinreb, C., Hwang, Y., Hidalgo, D., Zilionis, R., Waisman, A., Huh, J.R., Klein, A.M. and Socolovsky, M. Nature, 555, 54-60 (2018)   The formation of red blood cells begins with the differentiation of multipotent haematopoietic progenitors. Reconstructing the steps of this differentiation represents a general challenge in stem-cell biology. Here we used single-cell transcriptomics, fate assays and a theory that allows the prediction of cell fates from population snapshots to demonstrate that mouse haematopoietic progenitors differentiate through a continuous, hierarchical structure into seven blood lineages. We uncovered coupling between the erythroid and the basophil or mast cell fates, a global haematopoietic response to erythroid stress and novel growth factor receptors that regulate erythropoiesis. We defined a flow cytometry sorting strategy to purify early stages of erythroid differentiation, completely isolating classically defined burst-forming and colony-forming progenitors. We also found that the cell cycle is progressively remodelled during erythroid development and during a sharp transcriptional switch that ends the colony-forming progenitor stage and activates terminal differentiation. Our work showcases the utility of linking transcriptomic data to predictive fate models, and provides insights into lineage development in vivo.  

4.1847           PHP14 regulates hepatic stellate cells migration in liver fibrosis via mediating TGF-β1 signaling to PI3Kγ/AKT/Rac1 pathway

Xu, A., Li, Y., Zhao, W., Hou, F., Li, X., Sun, L., Chen, W., Yang, A., Wu, S., Zhang, B., Yao, J., Wang, H. and Huang, J.

1. Mol. Med., 96(2), 119-133 (2018)

  Hepatic fibrosis is characterized by the activation of hepatic stellate cells (HSCs). Migration of the activated HSCs to the site of injury is one of the key characteristics during the wound healing process. We have previously demonstrated that 14 kDa phosphohistidine phosphatase (PHP14) is involved in migration and lamellipodia formation of HSCs. However, the role of PHP14 in liver fibrosis remains unknown. In this study, we first assessed PHP14 expression and distribution in liver fibrotic tissues using western blot, immunohistochemistry, and double immunofluorescence staining. Next, we investigated the role of PHP14 in liver fibrosis and, more specifically, the migration of HSCs by Transwell assay and 3D collagen matrices assay. Finally, we explored the possible molecular mechanisms of the effects of PHP14 on these processes. Our results show that the PHP14 expression is up-regulated in fibrotic liver and mainly in HSCs. Importantly, TGF-β1 can induce PHP14 expression in HSCs accompanied with the activation of HSCs. Consistent with the previous study, PHP14 promotes HSCs migration, especially, promotes 3D floating collagen matrices contraction but inhibits stressed-released matrices contraction. Mechanistically, the PI3Kγ/AKT/Rac1 pathway is involved in migration regulated by PHP14. Moreover, PHP14 specifically mediates the TGF-β1 signaling to PI3Kγ/AKT pathway and regulates HSC migration, and thus participates in liver fibrosis. Our study identified the role of PHP14 in liver fibrosis, particularly HSC migration, and suggested a novel mediator of transducting TGF-β1 signaling to PI3Kγ/AKT/Rac1 pathway.  

4.1848           A Macrophage Colony-Stimulating-Factor-Producing γδ T Cell Subset Prevents Malarial Parasitemic Recurrence

Mamedov, M.R., Scholzen, A., Nair, R.V. et al Immunity, 48(2), 350-363 (2018)   Despite evidence that γδ T cells play an important role during malaria, their precise role remains unclear. During murine malaria induced by Plasmodium chabaudi infection and in human P. falciparum infection, we found that γδ T cells expanded rapidly after resolution of acute parasitemia, in contrast to αβ T cells that expanded at the acute stage and then declined. Single-cell sequencing showed that TRAV15N-1 (Vδ6.3) γδ T cells were clonally expanded in mice and had convergent complementarity-determining region 3 sequences. These γδ T cells expressed specific cytokines, M-CSF, CCL5, CCL3, which are known to act on myeloid cells, indicating that this γδ T cell subset might have distinct functions. Both γδ T cells and M-CSF were necessary for preventing parasitemic recurrence. These findings point to an M-CSF-producing γδ T cell subset that fulfills a specialized protective role in the later stage of malaria infection when αβ T cells have declined.  

4.1849           Bioaccumulation of 14C-Labeled Graphene in an Aquatic Food Chain through Direct Uptake or Trophic Transfer

Dong, S., Xia, T., Yang, Y., Lin, S. and Mao, L. Environ. Sci. Technol., 52(2), 541-549 (2018)   The growing applications of graphene materials warrant a careful evaluation of their environmental fate in aquatic food webs. Escherichia coli (Bacteria), Tetrahymena thermophila (protozoa), Daphnia magna (zooplankton), and Danio rerio (vertebrate) were used to build aquatic food chains to investigate the waterborne uptake and trophic transfer of ¹⁴C-labeled graphene. Body burden factor (BBF) and trophic transfer factor (TTF) were analyzed for each organism and food chain to assess the bioaccumulation and biomagnification of graphene. The test organisms have high potential of accumulating graphene via direct uptake from culture medium with log-transformed BBF (log BBF) values of 3.66, 5.1, 3.9, and 1.62 for each organism, respectively. In the food chain from E. coli to T. thermophila, the calculated TTFs of 0.2 to 8.6 indicate the high trophic transfer potential in this aquatic food chain. However, the TTFs calculated for the food chain from T. thermophila to D. magna and from D. magna to D. rerio are much lower than 1, indicating that biomagnification was unlikely to occur in these food chains. Body burden measured for dietary uptake by T. thermophila, D. magna, and D. rerio are higher than that via waterborne exposure in a similar nominal concentration, respectively, indicating that trophic transfer is a nonnegligible route for the bioaccumulation of graphene in organisms.  

4.1850           The Chemical Potential of Plasma Membrane Cholesterol: Implications for Cell Biology

Ayuyan, A.G. and Cohen, F.S. Biophys. J., 114(4), 904-918 (2018)   Cholesterol is abundant in plasma membranes and exhibits a variety of interactions throughout the membrane. Chemical potential accounts for thermodynamic consequences of molecular interactions, and quantifies the effective concentration (i.e., activity) of any substance participating in a process. We have developed, to our knowledge, the first method to measure cholesterol chemical potential in plasma membranes. This was accomplished by complexing methyl-β-cyclodextrin with cholesterol in an aqueous solution and equilibrating it with an organic solvent containing dissolved cholesterol. The chemical potential of cholesterol was thereby equalized in the two phases. Because cholesterol is dilute in the organic phase, here activity and concentration were equivalent. This equivalence allowed the amount of cholesterol bound to methyl-β-cyclodextrin to be converted to cholesterol chemical potential. Our method was used to determine the chemical potential of cholesterol in erythrocytes and in plasma membranes of nucleated cells in culture. For erythrocytes, the chemical potential did not vary when the concentration was below a critical value. Above this value, the chemical potential progressively increased with concentration. We used standard cancer lines to characterize cholesterol chemical potential in plasma membranes of nucleated cells. This chemical potential was significantly greater for highly metastatic breast cancer cells than for nonmetastatic breast cancer cells. Chemical potential depended on density of the cancer cells. A method to alter and fix the cholesterol chemical potential to any value (i.e., a cholesterol chemical potential clamp) was also developed. Cholesterol content did not change when cells were clamped for 24–48 h. It was found that the level of activation of the transcription factor STAT3 increased with increasing cholesterol chemical potential. The cholesterol chemical potential may regulate signaling pathways.  

4.1851           Single-Cell RT-PCR in Microfluidic Droplets with Integrated Chemical Lysis

Kim, S.C., Clark, I.C., Shahi, P. and Abate, A.R. Anal. Chem., 90(2), 1273-1279 (2018)   Droplet microfluidics can identify and sort cells using digital reverse transcription polymerase chain reaction (RT-PCR) signals from individual cells. However, current methods require multiple microfabricated devices for enzymatic cell lysis and PCR reagent addition, making the process complex and prone to failure. Here, we describe a new approach that integrates all components into a single device. The method enables controlled exposure of isolated single cells to a high pH buffer, which lyses cells and inactivates reaction inhibitors but can be instantly neutralized with RT-PCR buffer. Using our chemical lysis approach, we distinguish individual cells’ gene expression with data quality equivalent to more complex two-step workflows. Our system accepts cells and produces droplets ready for amplification, making single-cell droplet RT-PCR faster and more reliable.  

4.1852           Comparison of human erythrocyte purine nucleotide metabolism and blood purine and pyrimidine degradation product concentrations before and after acute exercise in trained and sedentary subjects

Dudzinska, W., Suska, M., Lubkowska, A., Jakubowska, K., Olszewska, M., Safranow, K. and Chlubek, D.

1. Physiol. Sci., 68, 293-305 (2018)

  This study aimed at evaluating the concentration of erythrocyte purine nucleotides (ATP, ADP, AMP, IMP) in trained and sedentary subjects before and after maximal physical exercise together with measuring the activity of purine metabolism enzymes as well as the concentration of purine (hypoxanthine, xanthine, uric acid) and pyrimidine (uridine) degradation products in blood. The study included 15 male elite rowers [mean age 24.3 ± 2.56 years; maximal oxygen uptake (VO_2max) 52.8 ± 4.54 mL/kg/min; endurance and strength training 8.2 ± 0.33 h per week for 6.4 ± 2.52 years] and 15 sedentary control subjects (mean age 23.1 ± 3.41 years; VO_2max 43.2 ± 5.20 mL/kg/min). Progressive incremental exercise testing until refusal to continue exercising was conducted on a bicycle ergometer. The concentrations of ATP, ADP, AMP, IMP and the activities of adenine phosphoribosyltransferase (APRT), hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and phosphoribosyl pyrophosphate synthetase (PRPP-S) were determined in erythrocytes. The concentrations of hypoxanthine, xanthine, uric acid and uridine were determined in the whole blood before exercise, after exercise, and 30 min after exercise testing. The study demonstrated a significantly higher concentration of ATP in the erythrocytes of trained subjects which, in part, may be explained by higher metabolic activity on the purine re-synthesis pathway (significantly higher PRPP-S, APRT and HGPRT activities). The ATP concentration, just as the ATP/ADP ratio, as well as an exercise-induced increase in this ratio, correlates with the VO_2max level in these subjects which allows them to be considered as the important factors characterising physical capacity and exercise tolerance. Maximal physical exercise in the group of trained subjects results not only in a lower post-exercise increase in the concentration of hypoxanthine, xanthine and uric acid but also in that of uridine. This indicates the possibility of performing high-intensity work with a lower loss of not only purine but also pyrimidine.  

4.1853           Combined bioaugmentation with anaerobic ruminal fungi and fermentative bacteria to enhance biogas production from wheat straw and mushroom spent straw

Ferraro, A., Dottorini, G., Massini, G., Miritana, M., Signorini, A., Lembo, G. and Fabbricino, M. Bioresource Technol., 260, 364-373 (2018)   Bioaugmentation with anaerobic ruminal fungi and a pool of hydrogen-producing fermenting bacteria was tested on wheat straw (WS) and mushroom spent straw (MSS) with the aim of improving anaerobic digestion performance. Batch tests were set up to simulate a Bioaugmentation Anaerobic Digestion (BAD) treatment comparing single- (I-BAD) and two-stage (II-BAD) process configurations, at two reactor scales, 120 and 1200 ml (×10). In both cases, higher CH₄ cumulative production was obtained in the II- BAD configuration on WS (65.1 ± 8.9 Nml and 922 ± 73.8 Nml respectively). The II-BADx10 tests allowed increasing CH₄ production (≃290% and ≃330% on WS and MSS, respectively) when compared to the unaugmented condition. Final results highlighted the achievable advantages of the two stage configuration in terms of CH₄ production enhancement. Microbial community investigations confirmed the efficiency of the bioaugmentation treatment and revealed that such a result was mainly related to the Methanosarcinales increase, mostly composed by Methanosaeta.  

4.1854           Enhanced effect of human mesenchymal stem cells expressing human TNF‐αR‐Fc and HO‐1 gene on porcine islet xenotransplantation in humanized mice

Lee, H-S., Song, S., Shin, D.Y., Kim, G-S., Lee, J-H., Cho, C.W., Lee, K.W., Park, H., Ahn, C., Yang, J., Yang, H-M., park, J.B. and Kim, S-J. Xenotransplantation, 25(1), e12342 (2018)   Background Porcine islet xenotransplantation is considered an attractive alternative treatment for type 1 diabetes mellitus. However, it is largely limited because of initial rejection due to Instant Blood‐Mediated Inflammatory Reaction (IBMIR), oxidative stress, and inflammatory responses. Recently, soluble tumor necrosis factor‐ɑ receptor type I (sTNF‐αR) and heme oxygenase (HO)‐1 genes (HO‐1/sTNF‐αR) have been shown to improve the viability and functionality of porcine islets after transplantation. Methods In this study, genetically modified mesenchymal stem cells (MSCs) expressing the HO‐1/sTNF‐αR genes (HO‐1/sTNF‐αR‐MSC) were developed using an adenoviral system, and porcine islet viability and function were confirmed by in vitro tests such as GSIS, AO/PI, and the ADP/ATP ratio after coculturing with HO‐1/sTNF‐αR‐MSCs. Subsequently, isolated porcine islets were transplanted underneath the kidney capsule of diabetic humanized mice without MSCs, with MSCs or with HO‐1/sTNF‐αR‐MSCs. Results According to the results, the HO‐1/sTNF‐αR‐MSC‐treated group exhibited improved survival of porcine islets and could reverse hyperglycemia more than porcine islets not treated with MSCs or islets cotransplanted with MSCs. Moreover, the HO‐1/sTNF‐αR‐MSC group maintained its morphological characteristics and the insulin secretion pattern of transplanted porcine islets similar to endogenous islets in immunocompetent humanized mice. Conclusions Our results suggest that HO‐1/sTNF‐αR‐MSCs are efficient tools for porcine islet xenotransplantation, and this study may provide basic information for pre‐clinical animal models and future clinical trials of porcine islet xenotransplantation.  

4.1855           The effect of epitope‐based ligation of ICAM‐1 on survival and retransplantation of pig islets in nonhuman primates

Lee, J-l., Kim, J., Choi, Y-J., Park, H-J., Park, H-J., Wi, H.J., Yoon, S., Shin, J-S., Park, J.K., Jung, K.C., Lee, E.B., Kang, H.J., Hwang, E.S., Kim, S-J., Park, C-G. and Park, S.H. Xenotransplantation, 25, e12362 (2018)   Background Pig islet xenotransplantation is a promising alternative to allogeneic transplantation. However, the wide immunologic barrier between pigs and primates limits the long‐term survival of the graft. MD‐3, a novel monoclonal antibody (mAb) that recognizes a particular epitope of human ICAM‐1, can render T cells tolerant to a xenograft by arresting dendritic cell maturation. We report the long‐term survival of adult wild‐type pig islets and successful retransplantation in nonhuman primates using a protocol comprising induction with MD‐3 mAb and maintenance with anti‐CD154 mAb and sirolimus. Methods Eleven rhesus monkeys were assigned to three groups. Group 1 (n = 4) involved treatment with MD‐3 induction, short‐term (<4 months) administration of anti‐CD154 mAb, and maintenance therapy with sirolimus. Group 2 (n = 4) involved treatment with MD‐3 induction and long‐term maintenance therapy with anti‐CD154 mAb and sirolimus. Group 3 (n = 3) involved only maintenance therapy with anti‐CD154 mAb and sirolimus. Diabetes was induced in monkeys by streptozotocin, and pig islets (61 000‐112 000 IEQ/kg for each transplant; up to 280 000 IEQ/kg per recipient) were infused through the portal vein. The in vivo functional potency of the isolated islets was tested by minimal model transplant in streptozotocin‐induced diabetic NOD/SCID mice, and the mean AUC of blood glucose level divided by the number of follow‐up days was calculated. Results The islet grafts survived more than 6 months (between 225 and 727 days) in nine of 12 transplants of MD‐3‐treated groups 1 and 2, whereas in the absence of MD‐3 mAb, survival was <40 days. In three transplants of the MD‐3‐treated Group 2, functional graft survival was only for 104, 125, and 154 days. In these cases, a retrospective analysis suggested that the relatively short survival duration was associated with the relatively high AUC value in the NOD/SCID bioassay. Notably, when retransplantation was performed in Group 3, blood glucose control was extended up to 956 days, which was supported by MD‐3 mAb‐based suppression of adaptive immunity. No replication of cytomegalovirus genes was observed. Conclusions Long‐term survival of pig islet xenografts and successful retransplantation were achieved with MD‐3 mAb‐based immunosuppression regimen in this pig‐to‐monkey transplantation model. It should be emphasized that these encouraging results were achieved following the transplantation of islets from pigs that had not been genetically modified. Considering that it is possible to further substantially reduce the destruction of grafted islet using genetically modified pig islet, the islet requirement could be reduced and much longer graft survival can be achieved.  

4.1856           Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR

Zemmour, D., Zilionis, R., Kiner, E., Klein, A.M., Mathis, D. and Benoist, C. Nature Immunol., 19, 291-301 (2018)   CD4+ T regulatory cells (Treg) are central to immune homeostasis, their phenotypic heterogeneity reflecting the diverse environments and target cells that they regulate. To understand this heterogeneity, we combined single-cell RNA-seq, activation reporter and T cell receptor (TCR) analysis to profile thousands of Treg or conventional CD4+FoxP3– T cells (Tconv) from mouse lymphoid organs and human blood. Treg and Tconv pools showed areas of overlap, as resting ‘furtive’ Tregs with overall similarity to Tconvs or as a convergence of activated states. All Tregs expressed a small core of FoxP3-dependent transcripts, onto which additional programs were added less uniformly. Among suppressive functions, Il2ra and Ctla4 were quasiconstant, inhibitory cytokines being more sparsely distributed. TCR signal intensity did not affect resting/activated Treg proportions but molded activated Treg programs. The main lines of Treg heterogeneity in mice were strikingly conserved in human blood. These results reveal unexpected TCR-shaped states of activation, providing a framework to synthesize previous observations of Treg heterogeneity.  

4.1857           Oral warfarin affects some aspects of systemic immunomodulation with topical dinitrochlorobenzene (DNCB) in rats

Aleksandrov, A.P., Belij-Rammerstorfer, S., Mirkov, I., Subota, V., Kulas, J., Kataranovski, D. and Kataranovski, M. Cutaneous and Ocular Toxicol., 37(1), 29-35 (2018)   Purpose: The efficacy of topical dinitrochlorobenzene (DNCB) in the treatment of some skin dermatoses is based both on local and systemic effects. It is not known, however, whether it can be applied to patients receiving some other therapy associated with systemic immunomodulation. The aim of the present paper using a rat model was to examine whether oral warfarin (WF) intake, as shown by others and by us, had an immunomodulatory potential to interfere with effects of topical DNCB as systemic immunotherapy. Materials and methods: Rats received 3.5 mg/l of WF sodium in drinking water for 30 days and were thereafter skin-sensitized with 0.4% DNCB. Changes in the oxidative activity (myeloperoxidase/MPO, reduction of nitroblue tetrazolium/NBT and nitric oxide/NO production) as well as tumor necrosis factor (TNF) production by peripheral blood polymorphonuclear cells (PMN) were measured and compared with PMN from sensitized unexposed to WF rats. Results: WF intake enhanced some aspects of PMN activity (intracellular MPO activity and unstimulated NO production) as well as their responsiveness to exogenous stimulation (NBT reduction and TNF production from sensitized animals). However, WF also decreased PMN responsiveness of NO production to stimulation. WF affected NO and TNF production solely by PMN, as no effect on these activities of peripheral blood mononuclear cells was seen. Conclusion: Having in mind that polymorphonuclear leukocytes are the most abundant cell type in peripheral blood in humans, increase of basic aspects of PMN activity described in the present paper might be relevant for consideration of using WF as therapeutic modality in patients topically treated with DNCB.  

4.1858           In Vitro Culture, Drug Sensitivity, and Transcriptome of Plasmodium Vivax Hypnozoites

Gural, N., Mancio-Silva, L., Miller, A.B. et al Cell Host & Microbe, 23(3), 395-406 (2018)   The unique relapsing nature of Plasmodium vivax infection is a major barrier to malaria eradication. Upon infection, dormant liver-stage forms, hypnozoites, linger for weeks to months and then relapse to cause recurrent blood-stage infection. Very little is known about hypnozoite biology; definitive biomarkers are lacking and in vitro platforms that support phenotypic studies are needed. Here, we recapitulate the entire liver stage of P. vivax in vitro, using a multiwell format that incorporates micropatterned primary human hepatocyte co-cultures (MPCCs). MPCCs feature key aspects of P. vivax biology, including establishment of persistent small forms and growing schizonts, merosome release, and subsequent infection of reticulocytes. We find that the small forms exhibit previously described hallmarks of hypnozoites, and we pilot MPCCs as a tool for testing candidate anti-hypnozoite drugs. Finally, we employ a hybrid capture strategy and RNA sequencing to describe the hypnozoite transcriptome and gain insight into its biology.    

4.1859           Flow cytometry-based method for rapid and high-throughput screening of hybridoma cells secreting monoclonal antibody

Akagi, S., Nakajima, C., Tanaka, Y. and Kurihara, Y.

1. Bioscience and Bioengineering, 125(4), 464-469 (2018)

  Monoclonal antibodies (mAbs) are a valuable biomaterial for basic life sciences and industrial purposes. The production of the mAb is time and effort intensive. In this report, we established a time- and labor-saving method for the mAb production. Because membrane-type immunoglobulin on a hybridoma cell surface and its secreted form, called as antibody, share the same binding property to the antigen, the fluorescence-labeled antigen bound to membrane-type immunoglobulin can be used as a screening marker. In the method, a hybridoma labeled by a fluorescent antigen was selected and sorted singly into 96-well plate using flow cytometer. Model experiments indicated that the method is highly efficient to obtain good mAbs suitable for Western blotting and immunofluorescence. Notably, most mAbs established by this method belonged to the IgG isotype, which is preferred over the IgM counterpart. Using a high-throughput flow cytometer, the method avoids tedious repeated screening and cloning processes. Because the method uses conventional myeloma for cell fusion and all reagents required in this method are commercially available, all research laboratories can apply the method to obtain mAbs efficiently.  

4.1860           Neuronal SphK1 acetylates COX2 and contributes to pathogenesis in a model of Alzheimer’s Disease

Lee, J.Y., han, S.H., park, M.H., Baek, B., Song, I-S., Choi, M-K., Takuwa, Y., Ryu, H., Kim, S.H., He, X., Schuchman, E.H., Bae, J-S. and Jin, H.K. Nature Communications, 9:1479 (2018)   Although many reports have revealed the importance of defective microglia-mediated amyloid β phagocytosis in Alzheimer’s disease (AD), the underlying mechanism remains to be explored. Here we demonstrate that neurons in the brains of patients with AD and AD mice show reduction of sphingosine kinase1 (SphK1), leading to defective microglial phagocytosis and dysfunction of inflammation resolution due to decreased secretion of specialized proresolving mediators (SPMs). Elevation of SphK1 increased SPMs secretion, especially 15-R-Lipoxin A4, by promoting acetylation of serine residue 565 (S565) of cyclooxygenase2 (COX2) using acetyl-CoA, resulting in improvement of AD-like pathology in APP/PS1 mice. In contrast, conditional SphK1 deficiency in neurons reduced SPMs secretion and abnormal phagocytosis similar to AD. Together, these results uncover a novel mechanism of SphK1 pathogenesis in AD, in which impaired SPMs secretion leads to defective microglial phagocytosis, and suggests that SphK1 in neurons has acetyl-CoA-dependent cytoplasmic acetyltransferase activity towards COX2.  

4.1861           Scavenger receptor-mediated Ad5 entry and acLDL accumulation in monocytes/macrophages synergistically trigger innate responses against viral infection

Li, P., Feng, F., Pan, E., Fan, X., Yang, Q., Guan, M., Chen, L. and Sun, C. Virology, 519, 86-98 (2018)   Adenovirus serotype 5 (Ad5) is a common cause of respiratory tract infection, and populations worldwide have high prevalence of anti-Ad5 antibodies, implying extensively prior infection. Ad5 infection potently activates the host innate defense and inflammation, but the molecular mechanisms are not completely clarified. We report here that monocytes from Ad5-seropositive subjects upregulates the expression of scavenger receptor A (SR-A), and the increased SR-A promote the susceptibility of Ad5 entry and subsequent innate signaling activation. SR-A is also known as major receptor for lipid uptake, we therefore observed that monocytes from Ad5-seropositive subjects accumulated the acetylated low-density lipoprotein (acLDL) and had the elevated cellular stress to induce the activation of monocyte/macrophages. These findings demonstrate that SR-A-mediated Ad5 entry, innate signaling activation and acLDL accumulation synergistically trigger the robust antiviral innate and inflammatory responses, which are helpful to our understanding of the pathogenesis of adenovirus infection.  

4.1862           TGF beta inhibits expression of SP-A, SP-B, SP-C, but not SP-D in human alveolar type II cells

Correll, K.A., Edeen, K.E., Zemans, R.L., Redente, E.F., Mikels-Vigdal, A. and Mason, R.J. Biochem. Biophys. Res. Comm., 499(4), 843-846 (2018)   TGF beta is a multifunctional cytokine that regulates alveolar epithelial cells as well as immune cells and fibroblasts. TGF beta inhibits surfactant protein A, B and C expression in fetal human lung and can inhibit type II cell proliferation induced by FGF7 (KGF). However, little is known about direct effects of TGF beta on adult human type II cells. We cultured alveolar type II cells under air/liquid interface conditions to maintain their state of differentiation with or without TGF beta. TGF beta markedly decreased expression of SP-A, SP-B, SP-C, fatty acid synthase, and the phospholipid transporter ABCA3. However, TGF beta increased protein levels of SP-D with little change in mRNA levels, indicating that it is regulated independently from other components of surfactant. TGF beta is a negative regulator of both the protein and the phospholipid components of surfactant. TGF beta did not induce EMT changes in highly differentiated human type II cells. SP-D is an important host defense molecule and regulated independently from the other surfactant proteins. Taken together these data are the first report of the effect of TGF beta on highly differentiated adult human type II cells. The effects on the surfactant system are likely important in the development of fibrotic lung diseases.  

4.1863           A proteomics landscape of circadian clock in mouse liver

Ang, Y., Song, L., Lieu, M., Ge, R., Zhou, Q., Li, R., Qie, J., Zhen, B., Wang Y., He, F., Qin, J and Ding. C. Nature Communications, 9, 1553 (2018)   As a circadian organ, liver executes diverse functions in different phase of the circadian clock. This process is believed to be driven by a transcription program. Here, we present a transcription factor (TF) DNA-binding activity-centered multi-dimensional proteomics landscape of the mouse liver, which includes DNA-binding profiles of different TFs, phosphorylation, and ubiquitylation patterns, the nuclear sub-proteome, the whole proteome as well as the transcriptome, to portray the hierarchical circadian clock network of this tissue. The TF DNA-binding activity indicates diurnal oscillation in four major pathways, namely the immune response, glucose metabolism, fatty acid metabolism, and the cell cycle. We also isolate the mouse liver Kupffer cells and measure their proteomes during the circadian cycle to reveal a cell-type resolved circadian clock. These comprehensive data sets provide a rich data resource for the understanding of mouse hepatic physiology around the circadian clock.  

4.1864           IL-13 induces periostin and eotaxin expression in human primary alveolar epithelial cells: Comparison with paired airway epithelial cells

Ito, Y., Al Mubarak, R., Roberts, N., Correll, K., Janssen, W., Finigan, J., Mishra, R. and Chu, H.W. PloS One, 13(4), e0196256 (2018)   Alveolar epithelial cells are critical to the pathogenesis of pulmonary inflammation and fibrosis, which are associated with overexpression of type 2 cytokine IL-13. IL-13 is known to induce the production of profibrotic (e.g., periostin) and pro-inflammatory (e.g., eotaxin-3) mediators in human airway epithelial cells, but it remains unclear if human primary alveolar epithelial cells increase periostin and eotaxin expression following IL-13 stimulation. The goals of this study are to determine if alveolar epithelial cells increase periostin and eotaxin expression upon IL-13 stimulation, and if alveolar and airway epithelial cells from the same subjects have similar responses to IL-13. Paired alveolar and airway epithelial cells were isolated from donors without any lung disease, and cultured under submerged or air-liquid interface conditions with or without IL-13. Up-regulation of periostin protein and mRNA was observed in IL-13-stimulated alveolar epithelial cells, which was comparable to that in IL-13-stimulated paired airway epithelial cells. IL-13 also increased eotaxin-3 expression in alveolar epithelial cells, but the level of eotaxin mRNA was lower in alveolar epithelial cells than in airway epithelial cells. Our findings demonstrate that human alveolar epithelial cells are able to produce periostin and eotaxin in responses to IL-13 stimulation. This study suggests the need to further determine the contribution of alveolar epithelial cell-derived mediators to pulmonary fibrosis.  

4.1865           A Plasmodium Parasite with Complete Late Liver Stage Arrest Protects against Preerythrocytic and Erythrocytic Stage Infection in Mice

Vaughan, A.M., Sack, B.K., Dankwa, D., Minkah, N., Nguyen, T., Cardamone, H. and Kappe, S.K.I. Infect. Immun., 86(5), e00088-18 (2018)   Genetically attenuated malaria parasites (GAP) that arrest during liver stage development are powerful immunogens and afford complete and durable protection against sporozoite infection. Late liver stage-arresting GAP provide superior protection against sporozoite challenge in mice compared to early live stage-arresting attenuated parasites. However, very few late liver stage-arresting GAP have been generated to date. Therefore, identification of additional loci that are critical for late liver stage development and can be used to generate novel late liver stage-arresting GAPs is of importance. We further explored genetic attenuation in Plasmodium yoelii by combining two gene deletions, PlasMei2 and liver-specific protein 2 (LISP2), that each cause late liver stage arrest with various degrees of infrequent breakthrough to blood stage infection. The dual gene deletion resulted in a synthetic lethal phenotype that caused complete attenuation in a highly susceptible mouse strain. P. yoelii plasmei2⁻ lisp2⁻ arrested late in liver stage development and did not persist in livers beyond 3 days after infection. Immunization with this GAP elicited robust protective antibody responses in outbred and inbred mice against sporozoites, liver stages, and blood stages as well as eliciting protective liver-resident T cells. The immunization afforded protection against both sporozoite challenge and blood stage challenge. These findings provide evidence that completely attenuated late liver stage-arresting GAP are achievable via the synthetic lethal approach and might enable a path forward for the creation of a completely attenuated late liver stage-arresting P. falciparum GAP.  

4.1866           A Rift Valley fever virus Gn ectodomain-based DNA vaccine induces a partial protection not improved by APC targeting

Chrun, T., Lacote, S., Urien, c., Jouneau, L., Barc, C., Bouguyon, E., Contreras, V., Ferrier-Rembert, A., Peyrefitte, C.N., Busquets, N., Vidal, E., Pujols, J., Marianneau, P. and Schwartz-Cornil, I. Npj Vaccines, 3:14 (2018) Rift Valley fever virus, a phlebovirus endemic in Africa, causes serious diseases in ruminants and humans. Due to the high probability of new outbreaks and spread to other continents where competent vectors are present, vaccine development is an urgent priority as no licensed vaccines are available outside areas of endemicity. In this study, we evaluated in sheep the protective immunity induced by DNA vaccines encoding the extracellular portion of the Gn antigen which was either or not targeted to antigen-presenting cells. The DNA encoding untargeted antigen was the most potent at inducing IgG responses, although not neutralizing, and conferred a significant clinical and virological protection upon infectious challenge, superior to DNA vaccines encoding the targeted antigen. A statistical analysis of the challenge parameters supported that the anti-eGn IgG, rather than the T-cell response, was instrumental in protection. Altogether, this work shows that a DNA vaccine encoding the extracellular portion of the Gn antigen confers substantial—although incomplete—protective immunity in sheep, a natural host with high preclinical relevance, and provides some insights into key immune correlates useful for further vaccine improvements against the Rift Valley fever virus.  

4.1867           KLRG1+ Effector CD8+ T Cells Lose KLRG1, Differentiate into All Memory T Cell Lineages, and Convey Enhanced Protective Immunity

Herndler-Brandstetter, D., Ishigame, H., Shinnakasu, R. et al Immunity, 48(4), 716-729 (2018)   Protective immunity against pathogens depends on the efficient generation of functionally diverse effector and memory T lymphocytes. However, whether plasticity during effector-to-memory CD8+ T cell differentiation affects memory lineage specification and functional versatility remains unclear. Using genetic fate mapping analysis of highly cytotoxic KLRG1+ effector CD8+ T cells, we demonstrated that KLRG1+ cells receiving intermediate amounts of activating and inflammatory signals downregulated KLRG1 during the contraction phase in a Bach2-dependent manner and differentiated into all memory T cell linages, including CX3CR1int peripheral memory cells and tissue-resident memory cells. “ExKLRG1” memory cells retained high cytotoxic and proliferative capacity distinct from other populations, which contributed to effective anti-influenza and anti-tumor immunity. Our work demonstrates that developmental plasticity of KLRG1+ effector CD8+ T cells is important in promoting functionally versatile memory cells and long-term protective immunity.  

4.1868           Cardiomyocyte diffusible redox mediators control Trypanosoma cruzi infection: role of parasite mitochondrial iron superoxide dismutase

Estrada, D., Specker, G., Martinez, A., Dias, P.P., Hissa, B., Andrade, L.O., Radi, R. and Piacenza, L. Biochem. J., 475, 1235-1251 (2018)   Chagas disease (CD), caused by the protozoa Trypanosoma cruzi, is a chronic illness in which parasites persist in the host-infected tissues for years. T. cruzi invasion in cardiomyocytes elicits the production of pro-inflammatory mediators [TNF-α, IL-1β, IFN-γ; nitric oxide (^·NO)], leading to mitochondrial dysfunction with increased superoxide radical (O₂^·−), hydrogen peroxide (H₂O₂) and peroxynitrite generation. We hypothesize that these redox mediators may control parasite proliferation through the induction of intracellular amastigote programmed cell death (PCD). In this work, we show that T. cruzi (CL-Brener strain) infection in primary cardiomyocytes produced an early (24 h post infection) mitochondrial dysfunction with H₂O₂ generation and the establishment of an oxidative stress evidenced by FoxO3 activation and target host mitochondrial protein expression (MnSOD and peroxiredoxin 3). TNF-α/IL-1β-stimulated cardiomyocytes were able to control intracellular amastigote proliferation compared with unstimulated cardiomyocytes. In this condition leading to oxidant formation, an enhanced number of intracellular apoptotic amastigotes were detected. The ability of H₂O₂ to induce T. cruzi PCD was further confirmed in the epimastigote stage of the parasite. H₂O₂ treatment induced parasite mitochondrial dysfunction together with intra-mitochondrial O₂^·− generation. Importantly, parasites genetically engineered to overexpress mitochondrial Fe-superoxide dismutase (Fe-SODA) were more infective to TNF-α/IL-1β-stimulated cardiomyocytes with less apoptotic amastigotes; this result underscores the role of this enzyme in parasite survival. Our results indicate that cardiomyocyte-derived diffusible mediators are able to control intracellular amastigote proliferation by triggering T. cruzi PCD and that parasite Fe-SODA tilts the process toward survival as part of an antioxidant-based immune evasion mechanism.  

4.1869           Multiplexed Quantitation of Intraphagocyte Mycobacterium tuberculosis Secreted Protein Effectors

Sayes, F., Blanc, C., Ates, L.S. et al Cell Reports, 23, 1072-1084 (2018)   The pathogenic potential of Mycobacterium tuberculosis largely depends on ESX secretion systems exporting members of the multigenic Esx, Esp, and PE/PPE protein families. To study the secretion and regulation patterns of these proteins while circumventing immune cross-reactions due to their extensive sequence homologies, we developed an approach that relies on the recognition of their MHC class II epitopes by highly discriminative T cell receptors (TCRs) of a panel of T cell hybridomas. The latter were engineered so that each expresses a unique fluorescent reporter linked to specific antigen recognition. The resulting polychromatic and multiplexed imaging assay enabled us to measure the secretion of mycobacterial effectors inside infected host cells. We applied this novel technology to a large panel of mutants, clinical isolates, and host-cell types to explore the host-mycobacteria interplay and its impact on the intracellular bacterial secretome, which also revealed the unexpected capacity of phagocytes from lung granuloma to present mycobacterial antigens via MHC class II.  

4.1870           High-density lipoprotein suppresses tumor necrosis factor alpha production by mycobacteria-infected human macrophages

Inoue, M., Niki, M., Ozeki, Y., Nagi, S. et al Scientific Reports, 8:6736 (2018)   Immune responses to parasitic pathogens are affected by the host physiological condition. High-density lipoprotein (HDL) and low-density lipoprotein (LDL) are transporters of lipids between the liver and peripheral tissues, and modulate pro-inflammatory immune responses. Pathogenic mycobacteria are parasitic intracellular bacteria that can survive within macrophages for a long period. Macrophage function is thus key for host defense against mycobacteria. These basic facts suggest possible effects of HDL and LDL on mycobacterial diseases, which have not been elucidated so far. In this study, we found that HDL and not LDL enhanced mycobacterial infections in human macrophages. Nevertheless, we observed that HDL remarkably suppressed production of tumor necrosis factor alpha (TNF-α) upon mycobacterial infections. TNF-α is a critical host-protective cytokine against mycobacterial diseases. We proved that toll-like receptor (TLR)-2 is responsible for TNF-α production by human macrophages infected with mycobacteria. Subsequent analysis showed that HDL downregulates TLR2 expression and suppresses its intracellular signaling pathways. This report demonstrates for the first time the substantial action of HDL in mycobacterial infections to human macrophages.  

4.1871           Growth differentiation factor 15 ameliorates nonalcoholic steatohepatitis and related metabolic disorders in mice

Kim, K.H., Kim, S.H., Han, D.H., Jo, Y.S., Lee, Y-h. and Lee, M-S. Scientific Reports, 8:6789 (2018)   Growth differentiation factor 15 (GDF15) is an endocrine hormone belonging to the TGFβ superfamily member. GDF15 administration or GDF15 overexpression has been reported to have anti-obesity and anti-diabetic effects. Although non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) is frequently associated with obesity and insulin resistance, the functional role of endogenous GDF15 and therapeutic effect of GDF15 overexpression in NASH and related metabolic deterioration have not been evaluated. Here, we found that GDF15 expression was increased in the livers of NASH animal models and human subjects with NASH. Elevated expression of GDF15 was due to diet-induced hepatic endoplasmic reticulum (ER) stress. Gdf15-knockout mice exhibited aggravated NASH phenotypes such as increased steatosis, hepatic inflammation, fibrosis, liver injury, and metabolic deterioration. Furthermore, GDF15 directly suppressed expression of fibrosis-related genes and osteopontin (OPN), contributing factors for NASH-related fibrosis, in hepatic stellate cells in vitro and in the liver of mice in vivo. Finally, we found that GDF15-transgenic mice showed attenuation of NASH phenotypes and metabolic deterioration. Therefore, our results suggest that induction of endogenous GDF15 is a compensatory mechanism to protect against the progression of NASH and that GDF15 could be an attractive therapeutic candidate for treatment of NASH and NASH-related metabolic deterioration.  

4.1872           Polymeric nano-shielded islets with heparin-polyethylene glycol in a non-human primate model

Park, H., Haque, M.R., Park, J.B., Lee, K.W., Lee, S., Kwon, Y., Lee, H.S., Kim, G-S., Shin, D.Y., Jin, S-H., Kim, J.H., Kang, H.J., Byun, Y and Kim, S.J. Biomaterials, 171, 164-177 (2018)   Intraportal pancreatic islet transplantation incurs huge cell losses during its early stages due to instant blood-mediated inflammatory reactions (IBMIRs), which may also drive regulation of the adaptive immune system. Therefore, a method that evades IBMIR will improve clinical islet transplantation. We used a layer-by-layer approach to shield non-human primate (NHP) islets with polyethylene glycol (nano-shielded islets, NSIs) and polyethylene glycol plus heparin (heparin nano-shielded islets; HNSIs). Islets ranging from 10,000 to 20,000 IEQ/kg body weight were transplanted into 19 cynomolgus monkeys (n = 4, control; n = 5, NSI; and n = 10, HNSI). The mean C-peptide positive graft survival times were 68.5, 64 and 108 days for the control, NSI and HNSI groups, respectively (P = 0.012). HNSI also reduced the factors responsible for IBMIR in vitro. Based on these data, HNSIs in conjunction with clinically established immunosuppressive drug regimens will result in superior outcomes compared to those achieved with the current protocol for clinical islet transplantation.  

4.1873           Draft Genome Sequence of a “Candidatus Liberibacter europaeus” Strain Assembled from Broom Psyllids (Arytainilla spartiophila) from New Zealand

Frampton, R.A., Thompson, S.M., Kalamorz, f., David, C., Addison, S.M. and Smith, G.R. Genome Annoncements, 6(20), e00430-17 (2018)   Here, we report the draft genome sequence of “Candidatus Liberibacter europaeus” ASNZ1, assembled from broom psyllids (Arytainilla spartiophila) from New Zealand. The assembly comprises 15 contigs, with a total length of 1.33 Mb and a G+C content of 33.5%.  

4.1874           MicroRNA-125b Promotes Hepatic Stellate Cell Activation and Liver Fibrosis by Activating RhoA Signaling

You, K., Li, S-Y., Gong, J., Fang, J-H., Zhang, C., Zhang, M., Yuan, Y., Yang, J. and Zhuang, S-M. Molecular Therapy – Nucleic Acids, 12, 57-66 (2018)   miR-125b is frequently dysregulated in different diseases. Activation of hepatic stellate cells (HSCs) is a critical event during liver fibrogenesis. However, the function and its underlying mechanism of miR-125b in HSC activation and liver fibrosis are still unknown. Here, we showed that miR-125b was upregulated in HSCs, but not in hepatocytes, during hepatic fibrogenesis in vivo and upon culture activation in vitro. Inhibition of miR-125b suppressed the expression of profibrogenic genes in culture-activated primary HSCs and reduced the basal and transforming growth factor β (TGF-β)-induced alpha-smooth muscle actin (α-SMA) expression and cell contraction of the immortalized HSC cell line. In contrast, ectopic expression of miR-125b promoted α-SMA expression and HSC contraction. Moreover, antagonizing miR-125b in vivo significantly alleviated liver fibrosis in CCl₄-treated mice. Mechanistically, overexpression of miR-125b in HSCs enhanced RhoA activity by directly targeting StAR-related lipid transfer (START) domain containing 13 (Stard13), a RhoA-specific GTPase-activating protein, whereas knockdown of miR-125b abrogated RhoA activation. Furthermore, inhibition of RhoA or its downstream molecules, Mrtf-A and Srf, attenuated the miR-125b-induced α-SMA expression and HSC contraction. Therefore, our findings identify a miR-125b-Stard13-RhoA-α-SMA signaling cascade in HSCs and highlight its importance in hepatic fibrosis.      

4.1875           Improvement of impaired electrical activity in NPC1 mutant cortical neurons upon DHPG stimulation detected by micro-electrode array

Feng, X., Bader, B.M., Yang, F., Segura, m., Schultz, L., Schröder, O.H-U., Rolfs, A. and Luo, J. Brain Res., 1694, 87-93 (2018)   Niemann-Pick Type C1 (NPC1) disease is an autosomal recessive neurodegenerative disease characterized by an excessive accumulation of unesterified cholesterol in late endosomes/lysosomes. Patients with NPC1 disease show a series of symptoms in neuropathology, including a gradually increased loss of motor control and seizures. However, mechanism of the neurological manifestations in NPC1 disease is not fully understood yet. In this study, we utilized the micro-electrode array (MEA) to analyze the spontaneous extracellular electrical activity in cultivated cortical neurons of the NPC1 mutant (NPC1^−/−) mouse. Our results show a decrease of the spontaneous electrical activity in NPC1^−/− neuronal network when compared to wild type neurons, as indicated by the decreased spike rate, burst rate, event rate, and the increased burst period and event period. Application of 3,5-dihydroxyphenylglycine (DHPG), a specific agonist of group I metabotropic glutamate receptors, improved the electrical activity of the NPC1^−/− neuronal network, suggesting that DHPG can be used as a potential therapeutic strategy for recovery of the electrical activity in NPC1 disease.  

4.1876           Oxidative stress induces p38MAPK-dependent senescence in the feto-maternal interface cells

Jin, J., Richardson, L., Sheller-Miller. S., Zhong, N. and Menon, R. Placenta, 67, 15-23 (2018)   Objective This study tested the mechanism of the oxidative stress (OS)-induced senescence pathway at the feto-maternal interface cells. Methods Primary amnion mesenchymal cells (AMCs), chorion and decidual cells isolated from the placental membranes of women at normal term (not in labor) were exposed to OS-inducing cigarette smoke extract (CSE) for 48 h. Reactive oxygen species (ROS) was measured using 2′7′-dichlorodihydrofluorescein. Western blot analysis determined phosphorylated (P) p38MAPK and p53 expression. Senescence-associated β-Galactosidase (SA-β-Gal) and matrix metallopeptidase 9 (MMP9) histochemistry were used to measure senescence and inflammation respectively. Cotreatment of cells with the antioxidant, N-acetyl cysteine (NAC), or the p38MAPK inhibitor, SB203580 (SB), verified the activation specificity. Results CSE increased ROS production from AMCs, chorion cells, and decidual cells (P < 0.05) compared to controls. Western blot analysis determined that CSE induced p38MAPK activation (P < 0.05) and cotreatment with NAC inhibited ROS production and p38MAPK activation (P < 0.05) in all cell types. CSE did not increase p53 phosphorylation in any of the cells; however, AMCs showed constitutive P-p53 expression. CSE increased senescence in AMCs and chorion cells compared to controls (P = 0.01 and P = 0.003, respectively); however, senescence was not observed in decidual cells. Senescence was significantly reduced following cotreatment with SB and NAC (AMCs; P = 0.01 and chorion; P = 0.009). CSE increased MMP9 in all cells that was reduced by NAC. Conclusion OS induced p38MAPK activation and inflammation in all cell types that was associated with senescence in fetal cells but not in maternal cells.  

4.1877           Addition of iodixanol in bull freezing extender improves the sperm membranes integrity

Marqui, F.N., Martins Jr, A., da Cruz, T.E., Berton, T.I., U., de Paula Freitas—Dell’Aqua, C., Dell’Aqua Junior, J. and Oba, E. Animal Reprod. Sci., 194, abstract AB51, e1-e27 (2018)   This study was carried out to verify the effects of different concentrations of iodixanol added to the freezing extender on plasma and acrossomal membrane integrity of post-thawed sperm bull. Therefore, ejaculates from three Nellore bulls (n = 18) were pooled and extended in a commercial freezing medium (egg yolk-sugar-glycerol) supplemented with iodixanol at concentrations of 0% (control group), 2.5% (I-2.5), 5% (I-5), and 10% (I-10). Subsequently, semen samples were cooled for 5 h at 4 °C, filled in 0.50 ml straws and frozen in a programmable freezer machine. After that, the straws were stored in liquid nitrogen until sperm evaluation, which were performed by thawing the samples in a water bath at 37 °C/30 s. Plasma and acrossomal membrane integrity (PAMI) were simultaneously assessed using Propidium Iodide and FITC-PSA probes, respectively, while translocation of phosphatidylserine (TPS) was identified by Annexin V and plasma membrane destabilization (PMD) by YO-PRO-1. All sperm samples were analyzed by flow cytometry. ANOVA and Tukey's test were used for statistical analysis (data of six replicates), with P < 0.05 taken as significant. Higher percentage (P < 0.05) of MPAI was observed in I-10 (65 ± 1.4) than for other groups, with control group showing a lower value (56.7 ± 2.1), while I-2.5 (59.8 ± 2) did not differ from control and I-5 (62 ± 1.6) groups. Regarding to TPS, higher percentage of spermatozoa without TPS (considering intact membrane cells) was observed in I-10 (61.2 ± 1.4) with lower percentage found in control group (45.6 ± 2.4). However, I-2.5 (56.3 ± 1.9) and I-5 (57.7 ± 1.5) exhibited similar results but differed significantly from the other groups. For PMD the values of I-10 and I-5 were no different (67.8 ± 1.9 and 67 ± 1.9, respectively), but they were higher than in control (62.4 ± 2.7) and I-2.5 (61 ± 1.9) groups, both with similar results. In conclusion, the addition of iodixanol to the bovine freezing extender significantly improves the sperm membrane integrity in a dose-dependent manner. [Acknowledgements: CAPES, Tairana Artificial Insemination Station and Botupharma, Brazil.]  

4.1878           CREB controls cortical circuit plasticity and functional recovery after stroke

Caracciolo. L., Marosi, M., Mazzitelli, j., Latifi, S., Sano, y., Galvan, l., Kawaguchi, R., Holley, S., levine, M.S., Coppola, G., Portera-Calliau, C., Silva, A.J. and Carmicheal, S.T. Nature Communications, 9:2250 (2018)   Treatments that stimulate neuronal excitability enhance motor performance after stroke. cAMP-response-element binding protein (CREB) is a transcription factor that plays a key role in neuronal excitability. Increasing the levels of CREB with a viral vector in a small pool of motor neurons enhances motor recovery after stroke, while blocking CREB signaling prevents stroke recovery. Silencing CREB-transfected neurons in the peri-infarct region with the hM4Di-DREADD blocks motor recovery. Reversing this inhibition allows recovery to continue, demonstrating that by manipulating the activity of CREB-transfected neurons it is possible to turn off and on stroke recovery. CREB transfection enhances remapping of injured somatosensory and motor circuits, and induces the formation of new connections within these circuits. CREB is a central molecular node in the circuit responses after stroke that lead to recovery from motor deficits.  

4.1879           Astrocytes expressing ALS‐linked mutant FUS induce motor neuron death through release of tumor necrosis factor‐alpha

Kia, A., McAvoy, K., Krishnamurthy, K., Trotti, D. and Pasinelli, P. Glia, 66, 1016-1033 (2018)   Mutations in fused in sarcoma (FUS) are linked to amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease affecting both upper and lower motor neurons. While it is established that astrocytes contribute to the death of motor neurons in ALS, the specific contribution of mutant FUS (mutFUS) through astrocytes has not yet been studied. Here, we used primary astrocytes expressing a N‐terminally GFP tagged R521G mutant or wild‐type FUS (WTFUS) and show that mutFUS‐expressing astrocytes undergo astrogliosis, damage co‐cultured motor neurons via activation of an inflammatory response and produce conditioned medium (ACM) that is toxic to motor neurons in isolation. Time lapse imaging shows that motor neuron cultures exposed to mutFUS ACM, but not WTFUS ACM, undergo significant cell loss, which is preceded by progressive degeneration of neurites. We found that Tumor Necrosis Factor‐Alpha (TNFα) is secreted into ACM of mutFUS‐expressing astrocytes. Accordingly, mutFUS astrocyte‐mediated motor neuron toxicity is blocked by targeting soluble TNFα with neutralizing antibodies. We also found that mutant astrocytes trigger changes to motor neuron AMPA receptors (AMPAR) that render them susceptible to excitotoxicity and AMPAR‐mediated cell death. Our data provide the first evidence of astrocytic involvement in FUS‐ALS, identify TNFα as a mediator of this toxicity, and provide several potential therapeutic targets to protect motor neurons in FUS‐linked ALS.  

4.1880           An increased level of CD41-positive extracellular vesicles recovered by 100,000 ×g centrifugation from stimulated platelets

Chiang, C. and Chen, C.

5. Extracellular Vesicles, 7, Suppl. 1, abstract PF05.07 (2018)

  Background: Circulating extracellular vesicles (EVs) have been implicated in various (pro)inflammatory and metabolic conditions, includingcardiovascular diseases, pregnancy, cancers and diabetes. Platelet derived EVs have been reported to be the most abundant EVs in human blood. However, there are major inconsistencies in the numbers of circulating EVs reported in the literature, and many variables that may affect quantification of EVs. It is often presumed that circulating EVs bare the same membrane lineage markers as their originating cells, which may not be the case. In addition, the indiscrimination of large and small EVs in some studies further introduces confusion. Methods: The aim of this study was to characterize the number, size profile and compositions of circulating EVs derived from normal or calcium ionophore, A23871, stimulated platelets or red blood cells (RBCs). To obtain RBCs and platelets of high purity, an iodixanol barrier isolation method was optimized to minimize both the contamination by other cell types and platelet preactivation. EVs were collected after platelets and RBCs were incubated in Tyrode-HEPES buffer at 37°C for 30 min. EVs were enumerated using nanoparticle tracking analysis (NTA), and recovered by centrifugation at 10,000 ×g (10K pellet) or 100,000 ×g (100K pellet) to examine the presence of surface markers by Western blot. Results: We prepared EVs from above 99.92% pure platelets and RBCs and collected EVs in vitro. NTA showed that more than 70% of EVs derived from RBCs were smaller than 100 nm, while only 16% and 5% of EVs were derived from unstimulated and stimulated platelets, respectively. Each platelet secreted 64 ± 3 EVs per hour on average with the mean diameter of 146 ± 6 nm. Importantly, the expression level of CD41, a platelet-specific marker, was highly expressed in the 10K EV pellet, but not detected in the 100K EV pellet. Upon calcium ionophore stimulation, platelets secreted 3.7 times more EVs compared to normal controls. In addition, CD41 expression in the 10K pellet decreased from 1.20 to 0.94 relative to that in the platelet cell lysate. Summary/Conclusion: Among 100K blood cell-derived EV pellets, CD41 was only detected in EVs released from stimulated platelets, suggesting the expression of CD41 in the 100K pellet may be utilized as a biomarker indicating the activation of platelets. Funding: This work was funded by MOST [106-2221-E-007-003, 106-2628-E-007 -010 -MY3].  

4.1881           Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain

Raj, B., Wagner, D.E., McKenna, A., Pandey, S., Klein, A.M., Shendure, J., Gagnon, J.A. and Schier, A.F. Nature Biotech., 36(5), 442-450 (2018)   The lineage relationships among the hundreds of cell types generated during development are difficult to reconstruct. A recent method, GESTALT, used CRISPR–Cas9 barcode editing for large-scale lineage tracing, but was restricted to early development and did not identify cell types. Here we present scGESTALT, which combines the lineage recording capabilities of GESTALT with cell-type identification by single-cell RNA sequencing. The method relies on an inducible system that enables barcodes to be edited at multiple time points, capturing lineage information from later stages of development. Sequencing of ∼60,000 transcriptomes from the juvenile zebrafish brain identified >100 cell types and marker genes. Using these data, we generate lineage trees with hundreds of branches that help uncover restrictions at the level of cell types, brain regions, and gene expression cascades during differentiation. scGESTALT can be applied to other multicellular organisms to simultaneously characterize molecular identities and lineage histories of thousands of cells during development and disease.  

4.1882           The receptor tyrosine kinase TrkB signals without dimerization at the plasma membrane

Zahavi, E.E., Steinberg, N., Altman, T., Chein, M., Joshi, Y., Gradus-Pery, T. and Perlson, E. Science Signaling, 11(529), eeao4006 (2018)   Tropomyosin-related tyrosine kinase B (TrkB) is the receptor for brain-derived neurotrophic factor (BDNF) and provides critical signaling that supports the development and function of the mammalian nervous system. Like other receptor tyrosine kinases (RTKs), TrkB is thought to signal as a dimer. Using cell imaging and biochemical assays, we found that TrkB acted as a monomeric receptor at the plasma membrane regardless of its binding to BDNF and initial activation. Dimerization occurred only after the internalization and accumulation of TrkB monomers within BDNF-containing endosomes. We further showed that dynamin-mediated endocytosis of TrkB-BDNF was required for the effective activation of the kinase AKT but not of the kinase ERK1/2. Thus, we report a previously uncharacterized mode of monomeric signaling for an RTK and a specific role for the endosome in TrkB homodimerization.  

4.1883           Single blood transfusion induces the production of donor-specific alloantibodies and regulatory T cells mainly in the spleen

Ueta, H., Kitazawa, Y., Sawanobori, Ueno, T., Ueha, S., Matsushima, K. and Matsuno, K. Int. Immunol., 30(2), 53-67 (2018)   Donor-specific blood transfusion is known to induce alloresponses and lead to immunosuppression. We examined their underlying mechanisms by employing fully allogeneic rat combinations. Transfused recipients efficiently produced alloantibodies of the IgM and IgG subclasses directed against donor class I MHC. The recipients exhibited active expansion of CD4⁺ T cells and CD4⁺FOXP3⁺ regulatory T cells (T_reg cells), followed by CD45R⁺ B cells and IgM⁺ or IgG subclass⁺ antibody-forming cells mainly in the spleen. From 1.5 days, the resident MHCII⁺CD103⁺ dendritic cells (DCs) in the splenic T-cell area, periarterial lymphocyte sheath, formed clusters with recipient BrdU⁺ or 5-ethynyl-2′-deoxyuridine⁺ cells, from which the proliferative response of CD4⁺ T cells originated peaking at 3–4 days. Transfusion-induced antibodies had donor passenger cell-depleting activity in vitro and in vivo and could suppress acute GvH disease caused by donor T cells. Furthermore, T_reg cells significantly suppressed mixed leukocyte reactions in a donor-specific manner. In conclusion, single blood transfusion efficiently induced a helper T-cell-dependent anti-donor class I MHC antibody-forming cell response with immunoglobulin class switching, and a donor-specific T_reg cell response mainly in the spleen, probably by way of the indirect allorecognition via resident DCs. These antibodies and T_reg cells may be involved, at least partly, in the donor-specific transfusion-induced suppression of allograft rejection.  

4.1884           11Beta‐hydroxysteroid dehydrogenase‐1 deficiency or inhibition enhances hepatic myofibroblast activation in murine liver fibrosis

Zou, X., Ramachandran, P., Kendall, T.J., Pellicoro, A., Dora, E., Aucott, R.L., Manwani, K., Man, T.Y., Chapman, K.E., Henderson, N.C., Forbes, S.J., Webster, S.P., Iredale, J.P., Walker, B.R. and Michailidou, Z. Hepatology, 67(6), 2167-2181 (2018)   A hallmark of chronic liver injury is fibrosis, with accumulation of extracellular matrix orchestrated by activated hepatic stellate cells (HSCs). Glucocorticoids limit HSC activation in vitro, and tissue glucocorticoid levels are amplified by 11beta‐hydroxysteroid dehydrogenase‐1 (11βHSD1). Although 11βHSD1 inhibitors have been developed for type 2 diabetes mellitus and improve diet‐induced fatty liver in various mouse models, effects on the progression and/or resolution of liver injury and consequent fibrosis have not been characterized. We have used the reversible carbon tetrachloride‐induced model of hepatocyte injury and liver fibrosis to show that in two models of genetic 11βHSD1 deficiency (global, Hsd11b1^–/–, and hepatic myofibroblast‐specific, Hsd11b1^fl/fl/Pdgfrb‐cre) 11βHSD1 pharmacological inhibition in vivo exacerbates hepatic myofibroblast activation and liver fibrosis. In contrast, liver injury and fibrosis in hepatocyte‐specific Hsd11b1^fl/fl/albumin‐cre mice did not differ from that of controls, ruling out 11βHSD1 deficiency in hepatocytes as the cause of the increased fibrosis. In primary HSC culture, glucocorticoids inhibited expression of the key profibrotic genes Acta2 and Col1α1, an effect attenuated by the 11βHSD1 inhibitor [4‐(2‐chlorophenyl‐4‐fluoro‐1‐piperidinyl][5‐(1H‐pyrazol‐4‐yl)‐3‐thienyl]‐methanone. HSCs from Hsd11b1^–/– and Hsd11b1^fl/fl/Pdgfrb‐cre mice expressed higher levels of Acta2 and Col1α1 and were correspondingly more potently activated. In vivo [4‐(2‐chlorophenyl‐4‐fluoro‐1‐piperidinyl][5‐(1H‐pyrazol‐4‐yl)‐3‐thienyl]‐methanone administration prior to chemical injury recapitulated findings in Hsd11b1^–/– mice, including greater fibrosis. Conclusion: 11βHSD1 deficiency enhances myofibroblast activation and promotes initial fibrosis following chemical liver injury; hence, the effects of 11βHSD1 inhibitors on liver injury and repair are likely to be context‐dependent and deserve careful scrutiny as these compounds are developed for chronic diseases including metabolic syndrome and dementia.  

4.1885           PPARγ Contributes to Immunity Induced by Cancer Cell Vaccines That Secrete GM-CSF

Goyal, G., Wong, K., Nirschi, J., Souders, N., Neuberg, D., Anandasabatpathy, N. and Dranoff, G. Cancer Immunol. Res., 6(6), 723-732 (2018)   Peroxisome proliferator activated receptor-γ (PPARγ) is a lipid-activated nuclear receptor that promotes immune tolerance through effects on macrophages, dendritic cells (DCs), and regulatory T cells (Tregs). Granulocyte–macrophage colony stimulating factor (GM-CSF) induces PPARγ expression in multiple myeloid cell types. GM-CSF contributes to both immune tolerance and protection, but the role of PPARγ in these pathways is poorly understood. Here, we reveal an unexpected stimulatory role for PPARγ in the generation of antitumor immunity with irradiated, GM-CSF–secreting tumor-cell vaccines (GVAX). Mice harboring a deletion of pparg in lysozyme M (LysM)-expressing myeloid cells (KO) showed a decreased ratio of CD8⁺ T effectors to Tregs and impaired tumor rejection with GVAX. Diminished tumor protection was associated with altered DC responses and increased production of the Treg attracting chemokines CCL17 and CLL22. Correspondingly, the systemic administration of PPARγ agonists to vaccinated mice elevated the CD8⁺ T effector to Treg ratio through effects on myeloid cells and intensified the antitumor activity of GVAX combined with cytotoxic T lymphocyte–associated antigen-4 antibody blockade. PPARγ agonists similarly attenuated Treg induction and decreased CCL17 and CCL22 levels in cultures of human peripheral blood mononuclear cells with GM-CSF–secreting tumor cells. Together, these results highlight a key role for myeloid cell PPARγ in GM-CSF–stimulated antitumor immunity and suggest that PPARγ agonists might be useful in cancer immunotherapy.  

4.1886           Regulation of Survival Motor Neuron Protein by the Nuclear Factor-Kappa B Pathway in Mouse Spinal Cord Motoneurons

Arumugan, S., Mincheva-Tasheva, S., Periyakaruppish, A., de la Fuente, Soler, R.M. and Garcera, A. Mol. Neurobiol., 55(6), 5019-5030 (2018)   Survival motor neuron (SMN) protein deficiency causes the genetic neuromuscular disorder spinal muscular atrophy (SMA), characterized by spinal cord motoneuron degeneration. Since SMN protein level is critical to disease onset and severity, analysis of the mechanisms involved in SMN stability is one of the central goals of SMA research. Here, we describe the role of several members of the NF-κB pathway in regulating SMN in motoneurons. NF-κB is one of the main regulators of motoneuron survival and pharmacological inhibition of NF-κB pathway activity also induces mouse survival motor neuron (Smn) protein decrease. Using a lentiviral-based shRNA approach to reduce the expression of several members of NF-κB pathway, we observed that IKK and RelA knockdown caused Smn reduction in mouse-cultured motoneurons whereas IKK or RelB knockdown did not. Moreover, isolated motoneurons obtained from the severe SMA mouse model showed reduced protein levels of several NF-κB members and RelA phosphorylation. We describe the alteration of NF-κB pathway in SMA cells. In the context of recent studies suggesting regulation of altered intracellular pathways as a future pharmacological treatment of SMA, we propose the NF-κB pathway as a candidate in this new therapeutic approach.  

4.1887           BMPR2 inhibits activin and BMP signaling via wild-type ALK2

Olsen, O.E., Sankar, M., Elsaadi, S., Hella, H., Buene, G., Darvekar, S.R., Misunbd, K., Katagiri, T., Knaus, P. and Holien, T.

1. Cell Sci., 131, jcs213512 (2018)

  TGF-β/BMP superfamily ligands require heteromeric complexes of type 1 and 2 receptors for ligand-dependent downstream signaling. Activin A, a TGF-β superfamily member, inhibits growth of multiple myeloma cells, but the mechanism for this is unknown. We therefore aimed to clarify how activins affect myeloma cell survival. Activin A activates the transcription factors SMAD2/3 through the ALK4 type 1 receptor, but may also activate SMAD1/5/8 through mutated variants of the type 1 receptor ALK2 (also known as ACVR1). We demonstrate that activin A and B activate SMAD1/5/8 in myeloma cells through endogenous wild-type ALK2. Knockdown of the type 2 receptor BMPR2 strongly potentiated activin A- and activin B-induced activation of SMAD1/5/8 and subsequent cell death. Furthermore, activity of BMP6, BMP7 or BMP9, which may also signal via ALK2, was potentiated by knockdown of BMPR2. Similar results were seen in HepG2 liver carcinoma cells. We propose that BMPR2 inhibits ALK2-mediated signaling by preventing ALK2 from oligomerizing with the type 2 receptors ACVR2A and ACVR2B, which are necessary for activation of ALK2 by activins and several BMPs. In conclusion, BMPR2 could be explored as a possible target for therapy in patients with multiple myeloma.  

4.1888           In vitro inhibition of hepatic stellate cell activation by the autophagy-related lipid droplet protein ATG2A

Hong, Y., Li, S., Wang, J. and Li, Y. Scientific Reports, 8:9232 (2018)   Clinical studies have found that moderate intake of retinol or oleic acid can enlarge the lipid droplets of hepatic stellate cells and suppress their activation. However, the link between lipid droplets and cell activation is unknown. This study compared the dynamics of lipid droplet-associated protein expression between activated and reverted stellate cells. Reversion of the activated human stellate cell line LX-2 and inhibition of primary mouse stellate cell activation were induced by retinol or oleic acid, which resulted in larger lipid droplets and the downregulation of cell activation markers. Quantitative proteomics and immunoblotting were performed to compare lipid-droplet protein profiles between activated and reverted LX-2 cells. Compared to expression in activated cells, 50 lipid-droplet proteins were upregulated, whereas 28 were downregulated upon reversion. ATG2A was significantly enriched in lipid droplets of retinol/oleic acid-treated LX-2 cells and quiescent primary stellate cells. Reduced expression of α-SMA, increased expression of perilipin-3, enlarged lipid droplets, and suppression of autophagic flux were observed in ATG2A-deficient LX2 cells. Lipid-droplet protein profile changes during the reversion of activated stellate cells might provide new insights into the molecular mechanisms linking lipid droplets to liver fibrosis. ATG2A could represent a potential new drug target for hepatic fibrosis.  

4.1889           Beneficial effect of recombinant rC1rC2 collagenases on human islet function: Efficacy of low‐dose enzymes on pancreas digestion and yield

Loganathan, G., Subhashree, V., Breite, A., Tucker, W.W., Narayanan, S., Dhanasekaran, M., Mokshagundam, S., Green, M.L., Highes, M.G., Williams, S.K., Dwulet, F., McCarthy, R.C. and Balamurugan, A.N. Am. J. Transplant., 18(2), 478-485 (2018)   A high number of human islets can be isolated by using modern purified tissue dissociation enzymes; however, this requires the use of >20 Wunsch units (WU)/g of pancreas for digestion. Attempts to reduce this dose have resulted in pancreas underdigestion and poor islet recovery but improved islet function. In this study, we achieved a high number of functional islets using a low dose of recombinant collagenase enzyme mixture (RCEM‐1200 WU rC2 and 10 million collagen‐degrading activity [CDA] U of rC1 containing about 209 mg of collagenase to digest a 100‐g pancreas). The collagenase dose used in these isolations is about 42% of the natural collagenase enzyme mixture (NCEM) dose commonly used to digest a 100‐g pancreas. Low‐dose RCEM was efficient in digesting entire pancreases to obtain higher yield (5535 ± 830 and 2582 ± 925 islet equivalent/g, P < .05) and less undigested tissue (16.7 ± 5% and 37.8 ± 3%, P < .05) compared with low‐dose NCEM (12WU/g). Additionally, low‐dose RCEM islets retained better morphology (confirmed with scanning electron microscopy) and higher in vitro basal insulin release (2391 ± 1342 and 1778 ± 978 μU/mL; P < .05) compared with standard‐dose NCEM. Nude mouse bioassay demonstrated better islet function for low‐dose RCEM (area under the curve [AUC] 24 968) compared with low‐dose (AUC–38 225) or standard‐dose NCEM (AUC–38 685), P < .05. This is the first report indicating that islet function can be improved by using low‐dose rC1rC2 (RCEM).  

4.1890           Islet damage during isolation as assessed by miRNAs and the correlation of miRNA levels with posttransplantation outcome in islet autotransplantation

Saravanan, P.B., Kanak, M.A., Chang, C.A., Darden, C., Yoshimatsu, G., Lawrence, M.C. and Naziruddin, B. Am. J. Transplant., 18(4), 982-989 (2018)   High‐quality pancreatic islets are essential for better posttransplantation endocrine function in total pancreatectomy with islet autotransplantation (TPIAT), yet stress during the isolation process affects quality and yield. We analyzed islet‐enriched microRNAs (miRNAs) ‐375 and ‐200c released during isolation to assess damage and correlated the data with posttransplantation endocrine function. The absolute concentration of miR‐375, miR‐200c, and C‐peptide was measured in various islet isolation steps, including digestion, dilution, recombination, purification, and bagging, in 12 cases of TPIAT. Posttransplantation glycemic control was monitored through C‐peptide, hemoglobin A_1c, insulin requirement, and SUITO index. The amount of miR‐375 released was significantly higher during enzymatic digestion followed by the islet bagging (P < .001). Mir‐200c mirrored these changes, albeit at lower concentrations. In contrast, the C‐peptide amount was significantly higher in the purification and bagging steps (P < .001). Lower amounts of miR‐375 were associated with a lower 6‐month insulin requirement (P = .01) and lower hemoglobin A_1c (P = .04). Measurement of the absolute quantity of miRNA‐375 and ‐200c released during islet isolation is a useful tool to assess islet damage. The quantity of released miRNA is indicative of posttransplantation endocrine function in TPIAT patients.  

4.1891           Identification, Characterization, Immunolocalization, and Biological Activity of Lucilin Peptide

Tellez, G.A., Zapata, J.A., Toro, L:J., Henao, D.C., Bedoya, J.P., Rivera, D., Trujillo, J.V., Rivas-Santiago, B., Hoyos, R.O. and Castano, J.C. Acta Tropica, 185, 318-326  (2018)   Maggots from the Lucilia sp. genus are used for debridement of infected and necrotic wounds. Broad-spectrum antimicrobial activity has been described in the excretion/secretions (ES¹) of these larvae. This study identifies the genetic sequence of a cecropin-like antimicrobial peptide from Lucilia eximia. Total RNA was extracted and used for PCR-RACE amplification of a cecropin, the native peptide was immunolocalized in the tissues and secretions of the larvae, and a synthetic analog was used to explore its antimicrobial, cytotoxic, LPS neutralizing and wound-healing activities in vitro. The genetic cDNA sequence of a cecropin-like antimicrobial peptide in L. eximia called “Lucilin” was amplified, corresponding to 63 aa completed protein and 40 aa mature peptide; the structure of the mature peptide was predicted as an α-helix. The peptide was immunolocalized in the salivary glands, fat body, the ES, and hemolymph of the maggots. Lucilin synthetic peptide analog was active against E. coli DH10B with a MIC² of 7.8 μg/mL, E. coli extended spectrum b-lactamase (ESBL) (MIC: 15.6 μg/mL), and Enterobacter cloacae (MIC: 125 μg/mL), but it was not active against Pseudomonas aeruginosa and Staphylococcus epidermidis; and had no cytotoxic or hemolytic activity. It showed immunomodulatory activity against human peripheral blood mononuclear cells (PBMCs) stimulated with LPS, reducing the TNF-α production when treated at 17 μg/mL and induces cell migration of Hacat at 5 and 50 μg/mL. Lucilin is a cecropin-like peptide from L. eximia with antimicrobial activity against Gram negative bacteria and immunomodulatory activities, decreasing the TNF-α production in PBMCs and inducing cellular migration in human keratinocytes.  

4.1892           miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non–Cell-Autonomous Mechanism in ALS

Maimon, R., Ionescu, A., Bonnie, A., Sweetat, S., Wald-Altman, S., Inbar, S., gradus, T., Trotti, D., Weil, M., Behar, O. and Person, E.

1. Neurosci., 38(24), 5478-5494 (2018)

  Axon degeneration and disruption of neuromuscular junctions (NMJs) are key events in amyotrophic lateral sclerosis (ALS) pathology. Although the disease's etiology is not fully understood, it is thought to involve a non–cell-autonomous mechanism and alterations in RNA metabolism. Here, we identified reduced levels of miR126-5p in presymptomatic ALS male mice models, and an increase in its targets: axon destabilizing Type 3 Semaphorins and their coreceptor Neuropilins. Using compartmentalized in vitro cocultures, we demonstrated that myocytes expressing diverse ALS-causing mutations promote axon degeneration and NMJ dysfunction, which were inhibited by applying Neuropilin1 blocking antibody. Finally, overexpressing miR126-5p is sufficient to transiently rescue axon degeneration and NMJ disruption both in vitro and in vivo. Thus, we demonstrate a novel mechanism underlying ALS pathology, in which alterations in miR126-5p facilitate a non–cell-autonomous mechanism of motor neuron degeneration in ALS.  

4.1893           Neuronal atlas of the dorsal horn defines its architecture and links sensory input to transcriptional cell types

Häring, M., Zeisel, A., Hochgerner, H., Rinwa, P., jakobsson, J.E.T., Lönnerberg, p., La Manno, G., Sharma, N., Borgius, L., Kiehm, O., Lagerström, S. and Ernfors, P. Nature Neurosci., 21, 869-880 (2018)   The dorsal horn of the spinal cord is critical to processing distinct modalities of noxious and innocuous sensation, but little is known of the neuronal subtypes involved, hampering efforts to deduce principles governing somatic sensation. Here we used single-cell RNA sequencing to classify sensory neurons in the mouse dorsal horn. We identified 15 inhibitory and 15 excitatory molecular subtypes of neurons, equaling the complexity in cerebral cortex. Validating our classification scheme in vivo and matching cell types to anatomy of the dorsal horn by spatial transcriptomics reveals laminar enrichment for each of the cell types. Neuron types, when combined, define a multilayered organization with like neurons layered together. Employing our scheme, we find that heat and cold stimuli activate discrete sets of both excitatory and inhibitory neuron types. This work provides a systematic and comprehensive molecular classification of spinal cord sensory neurons, enabling functional interrogation of sensory processing.  

4.1894           Porcine Alveolar Macrophage-like cells are pro-inflammatory Pulmonary Intravascular Macrophages that produce large titers of Porcine Reproductive and Respiratory Syndrome Virus

Bordet, E., Maisonnasse, P., Renson, P., Bouguyon, E., Crisci, E., Tiret, M., Descamps, D., Bernelin-Cottet, C., Urien, C., Lefevre, F., Jouneau, L., Bourry, O., Leplat, J-J., Schwartz-Cornil, I. and Bertho, N. Scientific Reports, 8:10172 (2018)   Lung inflammation is frequently involved in respiratory conditions and it is strongly controlled by mononuclear phagocytes (MNP). We previously studied porcine lung MNP and described a new population of cells presenting all the features of alveolar macrophages (AM) except for their parenchymal location, that we named AM-like cells. Herein we showed that AM-like cells are macrophages phagocytosing blood-borne particles, in agreement with a pulmonary intravascular macrophages (PIM) identity. PIM have been described microscopically long time ago in species from the Laurasiatheria superorder such as bovine, swine, cats or cetaceans. We observed that PIM were more inflammatory than AM upon infection with the porcine reproductive and respiratory syndrome virus (PRRSV), a major swine pathogen. Moreover, whereas PRRSV was thought to mainly target AM, we observed that PIM were a major producer of virus. The PIM infection was more correlated with viremia in vivo than AM infection. Finally like AM, PIM-expressed genes were characteristic of an embryonic monocyte-derived macrophage population, whose turnover is independent of bone marrow-derived hematopoietic precursors. This last observation raised the interesting possibility that AM and PIM originate from the same lung precursor.  

4.1895           Cryopreservation in rhinoceros—Setting a new benchmark for sperm cryosurvival

Hermes, L., Hildebrandt, B. and Göritz, F. PloS One, 13(7), e0200154 (2018)   At times when rhinoceros are fiercely poached, when some rhinoceros species are closer than ever to extinction, and when the scientific community is in debate over the use of advanced cell technologies as a remaining resort it is time to simplify and improve existing assisted reproduction techniques to enhance breeding and genetic diversity in the living populations under our care. Semen cryopreservation has been performed in all captive rhinoceros species with limited degree of success. Here we tested three freezing extenders, containing different cryoprotectants and various freezing rates for the cryopreservation of rhinoceros sperm from 14 bulls. In experiment I, semen from 9 bulls was used to determine the most suitable diluent, cryoprotectant and freezing rate for the successful cryopreservation of rhinoceros sperm. In experiment II, semen from 5 bulls was used to assess whether the removal of seminal plasma could further improve post thaw sperm quality following cryopreservation with conditions identified in Experiment I. Semen was diluted with Berliner Cryomedia, ButoCrio® or INRA Freeze®, packaged in 0.5 mL straws and frozen 3, 4, and 5 cm over liquid nitrogen (LN) vapour or directly in a dryshipper. It was found that semen extended with ButoCrio® (containing glycerol and methylformamide) and frozen 3cm over LN vapour provided the best protection to rhinoceros spermatozoa during cryopreservation. When pooled over treatments, total and progressive post thaw motility was 75.3 ± 4.2% and 68.5 ± 5.7%, respectively marking a new benchmark for the cryopreservation of rhinoceros sperm. Post thaw total and progressive motility, viability and acrosome integrity of semen diluted in ButoCrio® was significantly higher than semen extended in Berliner Cryomedia or INRA Freeze®. The removal of seminal plasma did not improve post thaw sperm survival (p > 0.05). In conclusion, the cryosurvival of rhinoceros spermatozoa was significantly improved when using a mixture of glycerol and methylformamide in combination with a fast freezing rate at 3 cm. These results describe a new protocol for the improved cryosurvival of rhinoceros spermatozoa and will enable a more successful preservation of genetic diversity between males, especially in donors whose spermatozoa may already be compromised prior to or during collection. The successful reduction of glycerol concentration in favour of methylformamide as a cryoprotectant could be a novel suggestion for the improvement of cryopreservation techniques in other wildlife species.  

4.1896           TCR Transgenic Mice Reveal Stepwise, Multi-site Acquisition of the Distinctive Fat-Treg Phenotype

Li, C., DiSpirito, J.R., Zemmour, D. et al Cell, 174, 285-299 (2018)   Visceral adipose tissue (VAT) hosts a population of regulatory T (Treg) cells, with a unique phenotype, that controls local and systemic inflammation and metabolism. Generation of a T cell receptor transgenic mouse line, wherein VAT Tregs are highly enriched, facilitated study of their provenance, dependencies, and activities. We definitively established a role for T cell receptor specificity, uncovered an unexpected function for the primordial Treg transcription-factor, Foxp3, evidenced a cell-intrinsic role for interleukin-33 receptor, and ordered these dependencies within a coherent scenario. Genesis of the VAT-Treg phenotype entailed a priming step in the spleen, permitting them to exit the lymphoid organs and surveil nonlymphoid tissues, and a final diversification process within VAT, in response to microenvironmental cues. Understanding the principles of tissue-Treg biology is a prerequisite for precision-targeting strategies.  

4.1897           Single-Cell Microgels: Technology, Challenges, and Applications

Kamperman, T., Karperien, M., Le Gac, S. and Leijten, J. Trends in Biotechnol., 36(8), 850-865 (2018)   Single-cell-laden microgels effectively act as the engineered counterpart of the smallest living building block of life: a cell within its pericellular matrix. Recent breakthroughs have enabled the encapsulation of single cells in sub-100-μm microgels to provide physiologically relevant microniches with minimal mass transport limitations and favorable pharmacokinetic properties. Single-cell-laden microgels offer additional unprecedented advantages, including facile manipulation, culture, and analysis of individual cell within 3D microenvironments. Therefore, single-cell microgel technology is expected to be instrumental in many life science applications, including pharmacological screenings, regenerative medicine, and fundamental biological research. In this review, we discuss the latest trends, technical challenges, and breakthroughs, and present our vision of the future of single-cell microgel technology and its applications.  

4.1898           Tissue-resident NK cells differ in their expression profile of the nutrient transporters Glut1, CD98 and CD71

Salzberger, W., Martrus, G., Bachmann, K., Goebels, H., Hess, L. et al PloS One, 13(7), e0201170 (2018)   Metabolism is a critical basis for immune cell functionality. It was recently shown that NK cell subsets from peripheral blood modulate their expression of nutrient receptors following cytokine stimulation, demonstrating that NK cells can adjust to changes in metabolic requirements. As nutrient availability in blood and tissues can significantly differ, we examined NK cells isolated from paired blood-liver and blood-spleen samples and compared expression of the nutrient transporters Glut1, CD98 and CD71. CD56^bright tissue-resident (CXCR6⁺) NK cells derived from livers and spleens expressed lower levels of Glut1 but higher levels of the amino acid transporter CD98 following stimulation than CD56^bright NK cells from peripheral blood. In line with that, CD56^dim NK cells, which constitute the main NK cell population in the peripheral blood, expressed higher levels of Glut1 and lower levels of CD98 and CD71 compared to liver CD56^bright NK cells. Our results show that NK cells from peripheral blood differ from liver- and spleen-resident NK cells in the expression profile of nutrient transporters, consistent with a cell-adaptation to the different nutritional environment in these compartments.  

4.1899           Recovering Gene Interactions from Single-Cell Data Using Data Diffusion

Van Dijk, D., Sharma, R., Nainys, J. et al Cell, 174, 716-729 (2018)   Single-cell RNA sequencing technologies suffer from many sources of technical noise, including under-sampling of mRNA molecules, often termed “dropout,” which can severely obscure important gene-gene relationships. To address this, we developed MAGIC (Markov affinity-based graph imputation of cells), a method that shares information across similar cells, via data diffusion, to denoise the cell count matrix and fill in missing transcripts. We validate MAGIC on several biological systems and find it effective at recovering gene-gene relationships and additional structures. Applied to the epithilial to mesenchymal transition, MAGIC reveals a phenotypic continuum, with the majority of cells residing in intermediate states that display stem-like signatures, and infers known and previously uncharacterized regulatory interactions, demonstrating that our approach can successfully uncover regulatory relations without perturbations.  

4.1900           Chapter 6 - Liver sinusoid on a chip

Du, Y., Li, N. and Long, M. Methods in Cell. Biol., 146, 105-134 (2018)   Liver sinusoid is the main functional site in liver. Multiple types of hepatic cells are well organized in a precisely-controlled biochemical and biomechanical environment, maintaining a spectrum of hepatic functions. Here, using micro-engineering techniques, four types of primary hepatic cells are integrated into two layer channels connected by porous membrane, which recreates the sinusoidal cell composition and architecture. By incorporating shear flow into this permeable system, the blood flow in sinusoids and interstitial flow in space of Disse are recapitulated. Conventional hepatocyte-based liver-specific functions are enhanced by non-parenchymal cells co-culture and shear flow. Moreover, major immune responses in liver sinusoids, i.e., neutrophil recruitment under lipopolysaccharide (LPS) stimulation, are replicated, indicating that all types of hepatic cells contribute to this process. Thus, this liver chip provides a new in vitro model to investigate the short-duration cellular interactions under a microenvironment mimicking the physiological composition and architecture of liver organ.  

4.1901           dropEst: pipeline for accurate estimation of molecular counts in droplet-based single-cell RNA-seq experiments

Petukhov, V., Guo, J., Baryawno, N., Severe, N., scadden, D.T., Samsonova, M.G. and Kharchenko, P.V. Genome Biol., 19:78 (2018)   Recent single-cell RNA-seq protocols based on droplet microfluidics use massively multiplexed barcoding to enable simultaneous measurements of transcriptomes for thousands of individual cells. The increasing complexity of such data creates challenges for subsequent computational processing and troubleshooting of these experiments, with few software options currently available. Here, we describe a flexible pipeline for processing droplet-based transcriptome data that implements barcode corrections, classification of cell quality, and diagnostic information about the droplet libraries. We introduce advanced methods for correcting composition bias and sequencing errors affecting cellular and molecular barcodes to provide more accurate estimates of molecular counts in individual cells.  

4.1902           A Consistent Method to Identify and Isolate Mononuclear Phagocytes from Human Lung and Lymph Nodes

Gibbings, S.L. and Jakubzick, C.V. Methods in Mol. Biol., 1799, 381-395 (2018)   Mononuclear phagocytes (MP) consist of macrophages, dendritic cells (DCs), and monocytes. In all organs, including the lung, there are multiple subtypes within these categories. The existence of all these cell types suggest that there is a clear division of labor and delicate balance between the MPs under steady state and inflammatory conditions. Although great strides have been made to understand MPs in the mouse lung, and human blood, little is known about the MPs that exist in the human lung and lung-draining lymph nodes (LNs), and even less is known about their functional roles, studies of which will require a large number of sorted cells. We have comprehensively examined cell surface markers previously used in a variety of organs to identify human pulmonary MPs. In the lung, we consistently identify five extravascular pulmonary MPs and three LN MPs. These MPs were present in over 100 lungs regardless of age or gender. Notably, the human blood CD141⁺ DCs, as described in the literature, were not observed in non-diseased lungs or their draining LNs. In the lung and draining LNs, expression of CD141 was only observed on HLADR⁺ CD11c⁺ CD14⁺ extravascular monocytes (often confused in the LN as resident DCs based on the level of HLADR expression and mouse LN data). In the human lung and LNs there are at least two DC subtypes expressing HLADR, DEC205 and CD1c, along with circulating monocytes that behave as either antigen-presenting cells or macrophages. Furthermore, we demonstrate how to distinguish between alveolar macrophages and interstitial macrophage subtypes. It still remains unclear how the human pulmonary MPs identified here align with mouse MPs. Clearly, we are now past the stage of cell surface marker characterization, and future studies will need to move toward understanding what these cell types are and how they function. Our hope is that the strategy described here can help the pulmonary community take this next step.  

4.1903           Isolation of Rat and Mouse Alveolar Type II Epithelial Cells

Jansing, N.L., McClendon, J., Kage, H., Sunohara, M., Alvarez, J.R., Borok, Z. and Zemans, R.L: Methods in Mol. Biol., 1809, 69-82 (2018)   The gas exchange surface of the lungs is lined by an epithelium consisting of alveolar type (AT) I and ATII cells. ATII cells function to produce surfactant, play a role in host defense and fluid and ion transport, and serve as progenitors. ATI cells are important for gas exchange and fluid and ion transport. Our understanding of the biology of these cells depends on the investigation of isolated cells. Here, we present methods for the isolation of mouse and rat ATII cells.  

4.1904           Isolation and Characterization of Human Alveolar Type II Cells

Kosmider, B., Mason, R.J. and Bahmed, K. Methods in Mol. Biol., 1809, 83-90 (2018)   Alveolar type II (ATII) cells synthesize, store, and secrete pulmonary surfactant and restore the epithelium after damage to the alveolar epithelium. Isolation of human ATII cells provides a valuable tool to study their function under normal and pathophysiological conditions. Moreover, maintenance of their differentiated phenotype in vitro allows further study of their function. Here we describe a protocol for efficient ATII cell isolation, characterization, and culture.  

4.1905           Epithelial damage and tissue γδ T cells promote a unique tumor-protective IgE response

Crawford, G., Hayes, M.D., Seone, R.C., Ward, S., Dalessandri, T., Lai., C. et al Nature Immunol., 19, 859-870 (2018)   IgE is an ancient and conserved immunoglobulin isotype with potent immunological function. Nevertheless, the regulation of IgE responses remains an enigma, and evidence of a role for IgE in host defense is limited. Here we report that topical exposure to a common environmental DNA-damaging xenobiotic initiated stress surveillance by γδTCR+ intraepithelial lymphocytes that resulted in class switching to IgE in B cells and the accumulation of autoreactive IgE. High-throughput antibody sequencing revealed that γδ T cells shaped the IgE repertoire by supporting specific variable-diversity-joining (VDJ) rearrangements with unique characteristics of the complementarity-determining region CDRH3. This endogenous IgE response, via the IgE receptor FcεRI, provided protection against epithelial carcinogenesis, and expression of the gene encoding FcεRI in human squamous-cell carcinoma correlated with good disease prognosis. These data indicate a joint role for immunosurveillance by T cells and by B cells in epithelial tissues and suggest that IgE is part of the host defense against epithelial damage and tumor development.  

4.1906           Oscillatory inertial focusing in infinite microchannels

Mutlu, B.R., Edd, J.F. and Toner, M. PNAS, 115(30), 7682-7687 (2018)   Inertial microfluidics (i.e., migration and focusing of particles in finite Reynolds number microchannel flows) is a passive, precise, and high-throughput method for microparticle manipulation and sorting. Therefore, it has been utilized in numerous biomedical applications including phenotypic cell screening, blood fractionation, and rare-cell isolation. Nonetheless, the applications of this technology have been limited to larger bioparticles such as blood cells, circulating tumor cells, and stem cells, because smaller particles require drastically longer channels for inertial focusing, which increases the pressure requirement and the footprint of the device to the extent that the system becomes unfeasible. Inertial manipulation of smaller bioparticles such as fungi, bacteria, viruses, and other pathogens or blood components such as platelets and exosomes is of significant interest. Here, we show that using oscillatory microfluidics, inertial focusing in practically “infinite channels” can be achieved, allowing for focusing of micron-scale (i.e. hundreds of nanometers) particles. This method enables manipulation of particles at extremely low particle Reynolds number (Re_p < 0.005) flows that are otherwise unattainable by steady-flow inertial microfluidics (which has been limited to Re_p > ∼10⁻¹). Using this technique, we demonstrated that synthetic particles as small as 500 nm and a submicron bacterium, Staphylococcus aureus, can be inertially focused. Furthermore, we characterized the physics of inertial microfluidics in this newly enabled particle size and Re_p range using a Peclet-like dimensionless number (α). We experimentally observed that α >> 1 is required to overcome diffusion and be able to inertially manipulate particles.  

4.1907           Copper nanoparticles induce early fibrotic changes in the liver via TGF-β/Smad signaling and cause immunosuppressive effects in rats

Lee, I-C., Ko, J-W., Park, S-H., Shin, N-R., Shin, I-S., Moon, C., Kim, S-H., Yun, W-K., Kim, H-C. and Kim, J-C. Nanotoxicology, 12(6), 637-651 (2018)   Copper nanoparticles (Cu NPs) have various uses, including as additives in polymers/plastics, lubricants for metallic coating, and biomedical applications. We investigated the role of transforming growth factor (TGF)-β1 signaling in hepatic damage caused by Cu NPs and explored the effects of a 28-day repeated oral administration to Cu NPs on the immune response. The exposure to Cu NPs caused a dose-dependent increase in Cu levels in the liver and spleen. Cu NPs caused hepatic damage and markedly increased oxidative stress in liver tissues. Cu NPs induced activation of TGF-β1/Smad signaling by induction of vascular endothelial growth factor and matrix metalloproteinase-9. Exposure to Cu NPs also induced activation of Smad-independent pathways, phosphorylation of mitogen-activated protein kinases (MAPKs) and Akt/FoxO3. Consistent with the activation of TGF-β1/Smad-dependent and -independent pathways, Cu NPs markedly increased the deposition and induction of extracellular matrix components, α-smooth muscle actin, and collagens in liver tissues. In addition, repeated exposure to Cu NPs suppressed the proliferation of mitogenically stimulated T- or B-lymphocytes and decreased CD3⁺ (particularly, CD3⁺CD4⁺CD8⁻) and CD45⁺ population, followed by decreased levels of immunoglobulins and Th1/Th2 type cytokines. Collectively, Cu NPs caused hepatic damage and induced pro-fibrotic changes, which were closely related to the activation of oxidative stress-mediated TGF-β1/Smad-dependent and -independent pathways (MAPKs and Akt/FoxO3). We confirmed the immunosuppressive effect of Cu NPs via the inhibition of mitogen-stimulated spleen-derived lymphocyte proliferation and suppression of B- or T-lymphocyte-mediated immune responses.  

4.1908           Injectable degradable PVA microgels prepared by microfluidic technology for controlled osteogenic differentiation of mesenchymal stem cells

Hou, Y., Xie, W., Achazi, K., Cuellar-Camacho, J.L., Melzig, M.F., Chen, W. and Haag, R.

Acta Biomaterialia, 77, 28-37 (2018)

The direct injection of bone marrow mesenchymal stem cells (hMSCs) is a promising strategy for bone tissue engineering applications. Herein, we have developed injectable degradable poly(vinyl alcohol) (PVA) microgels loaded with hMSCs and growth factors and prepared by a high-throughput microfluidic technology. The PVA-based microgels with tunable mechanical and degradable properties were composed of vinyl ether acrylate-functionalized PVA (PVA-VEA) and thiolated PVA-VEA (PVA-VEA-SH) through a Michael-type crosslinking reaction under mild conditions. The hMSCs sustain high viability in PVA microgels, and cell proliferation and migration behaviors can easily be adjusted by varying crosslinking densities of PVA microgels. Additionally, bone morphogenetic protein-2 (BMP-2) co-encapsulated into the microgel environments enhanced osteogenic differentiation of hMSCs as indicated by a significant increase in alkaline phosphatase activity, calcium content, and Runx2 and OPN gene expression levels. These results demonstrate the degradable PVA microgels with tailored stem cell microenvironments and controlled release profile of the growth factor to promote and direct differentiation. These PVA-based microgels have promising potential as ideal cell vehicles for applications in regenerative medicine.

4.1909           Direct loading of CTL epitopes onto MHC class I complexes on dendritic cell surface in vivo

Wang, P., Dong, S., Zhao, P., He, X. and Chen, M. Biomaterials, 182, 92-103 (2018)   Dendritic cell (DC)-based cytotoxic T lymphocyte (CTL) epitope vaccines are effective to induce CTL responses but require complex ex vivo DC preparation and epitope-loading. To take advantage of DC-based epitope vaccines without involving the ex vivo procedures, we aimed to develop carriers to directly load CTL epitopes onto DCs in vivo. Here, we first engineered a carrier consisting of a hydrophilic polypeptide, immune-tolerant elastin-like polypeptide (iTEP) and a substrate peptide of matrix metalloproteinases-9 (sMMP). The iTEP was able to solubilize CTL epitopes. CTL epitopes were connected to the carrier, iTEP-sMMP, through sMMP so that the epitopes can be cleaved from the carrier by MMP-9. iTEP-sMMP was found to release its epitope payloads in the DC culture media, which contained MMP-9 released from DCs. iTEP-sMMP allowed for the direct loading of CTL epitopes onto the surface MHC class I complexes of DCs. Importantly, iTEP-sMMP resulted in greater epitope presentation by DCs both in vitro and in vivo than a control carrier that cannot directly load epitopes. iTEP-sMMP also induced 2-fold stronger immune responses than the control carrier. To further enhance the direct epitope-loading strategy, we furnished iTEP-sMMP with an albumin-binding domain (ABD) and found the new carrier, ABD-iTEP-sMMP, had greater lymph node (LN) accumulation than iTEP-sMMP. ABD-iTEP-sMMP also resulted in greater immune responses than iTEP-sMMP by 1.5-fold. Importantly, ABD-iTEP-sMMP-delivered CTL epitope vaccine induced stronger immune responses than free CTL epitope vaccine. Taken together, these carriers utilized two physiological features of DCs to realize direct epitope-loading in vivo: the accumulation of DCs in LNs and MMP-9 released from DCs. These carriers are a potential substitute for DC-based CTL epitope vaccines.  

4.1910           Molecular Architecture of the Mouse Nervous System

Zeisel, A., Hochgerner, H., Lönneberg, P., Ernfors, P., Marklund, U. and Linnarsson, S.

Cell, 174, 999-1014 (2018)

The mammalian nervous system executes complex behaviors controlled by specialized, precisely positioned, and interacting cell types. Here, we used RNA sequencing of half a million single cells to create a detailed census of cell types in the mouse nervous system. We mapped cell types spatially and derived a hierarchical, data-driven taxonomy. Neurons were the most diverse and were grouped by developmental anatomical units and by the expression of neurotransmitters and neuropeptides. Neuronal diversity was driven by genes encoding cell identity, synaptic connectivity, neurotransmission, and membrane conductance. We discovered seven distinct, regionally restricted astrocyte types that obeyed developmental boundaries and correlated with the spatial distribution of key glutamate and glycine neurotransmitters. In contrast, oligodendrocytes showed a loss of regional identity followed by a secondary diversification. The resource presented here lays a solid foundation for understanding the molecular architecture of the mammalian nervous system and enables genetic manipulation of specific cell types.

   

4.1911           Deconstructing Adipogenesis Induced by β3-Adrenergic Receptor Activation with Single-Cell Expression Profiling

Burl, R.B., Ramseyer, V.D., Rondini, E.A., Pique-Regi, R., Lee, Y-H. and Granneman, J.G. Cell Metab., 28(2), 300-309 (2018)   Recruitment of brown/beige adipocytes (BAs) in white adipose tissue (WAT) involves proliferation and differentiation of adipocyte stem cells (ASCs) in concert with close interactions with resident immune cells. To deconvolve stromal cell heterogeneity in a comprehensive and unbiased fashion, we performed single-cell RNA sequencing (scRNA-seq) of >33,000 stromal/vascular cells from epididymal WAT (eWAT) and inguinal WAT (iWAT) under control conditions and during β3-adrenergic receptor (ADRB3) activation. scRNA-seq identified distinct ASC subpopulations in eWAT and iWAT that appeared to be differentially poised to enter the adipogenic pathway. ADRB3 activation triggered the dramatic appearance of proliferating ASCs in eWAT, whose differentiation into BAs could be inferred from a single time point. scRNA-seq identified various immune cell types in eWAT, including a proliferating macrophage subpopulation that occupies adipogenic niches. These results demonstrate the power of scRNA-seq to deconstruct adipogenic niches and suggest novel functional interactions among resident stromal cell subpopulations.  

4.1912           Adhesion characteristics of porcine pancreatic islets and exocrine tissue to coating materials

Nakashima, Y., Miyagi-Shiohira, C., Kobayashi, N., Saitoh, I., Watanabe, M. and Noguchi, H. Islets, 10(3), e1460294 (2018)   Since the report of the Edmonton protocol in 2000, islet transplantation has been implemented worldwide, and xenotransplantation using porcine islets has also been reported. In addition, many basic experiments using pancreatic islets and exocrine tissue after isolation have been reported. Recently, exocrine cells have been found to be essential for inducing the differentiation of pancreatic islets. Therefore, the importance of the culture conditions for pancreatic tissue when conducting experiments using pancreatic tissue is also increasing. In this study, we focused on the coat material and examined the adhesive properties of porcine pancreatic islets and exocrine tissue after isolation. Porcine islet isolation was performed, and isolated islets (purity ≥95%) and exocrine tissue (purity ≥99%) were used to achieve adhesion to several extracellular matrixes, fibronectin, collagen type I, collagen type IV, laminin I, fibrinogen, and bovine serum albumin (BSA). DMEM with 0.5% FBS was used as the assay medium. For exocrine tissue, the adhesion was promoted in fibronectin, collagen type I, laminin I, and fibrinogen. The adhesive ability to fibronectin was more than twice that to BSA, while the adhesive ability to collagen type I, laminin I, and fibrinogen was less than twice that to BSA. For islets, the adhesive ability to fibronectin was weaker than that of exocrine tissue. Furthermore, the adhesion effect in fibronectin was obtained within 30 minutes and in medium containing little serum for both islets and exocrine tissues. These data suggest that fibronectin may be useful for the adhesion of pancreatic tissue.  

4.1913           A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte

Plasschaert, L.W., Zillionis, R., Choo-Wing, R., Savova, V., Knehr, J., Roma, G., Klein, A.M. and Jaffe, A.B. Nature, 560, 377-381 (2018)   The functions of epithelial tissues are dictated by the types, abundance and distribution of the differentiated cells they contain. Attempts to restore tissue function after damage require knowledge of how physiological tasks are distributed among cell types, and how cell states vary between homeostasis, injury–repair and disease. In the conducting airway, a heterogeneous basal cell population gives rise to specialized luminal cells that perform mucociliary clearance1. Here we perform single-cell profiling of human bronchial epithelial cells and mouse tracheal epithelial cells to obtain a comprehensive census of cell types in the conducting airway and their behaviour in homeostasis and regeneration. Our analysis reveals cell states that represent known and novel cell populations, delineates their heterogeneity and identifies distinct differentiation trajectories during homeostasis and tissue repair. Finally, we identified a novel, rare cell type that we call the ‘pulmonary ionocyte’, which co-expresses FOXI1, multiple subunits of the vacuolar-type H+-ATPase (V-ATPase) and CFTR, the gene that is mutated in cystic fibrosis. Using immunofluorescence, modulation of signalling pathways and electrophysiology, we show that Notch signalling is necessary and FOXI1 expression is sufficient to drive the production of the pulmonary ionocyte, and that the pulmonary ionocyte is a major source of CFTR activity in the conducting airway epithelium.  

4.1914           Endothelial Notch activation reshapes the angiocrine of sinusoidal endothelia to aggravate liver fibrosis and blunt regeneration in mice

Duan, J-L., Ruan, B., Yan, X-C., Liang, L., Song, P., Yang, Z-Y., Liu, Y., Dou, K-F., Han, h. and Wang, L. Hepatology, 68(2), 677-690 (82018)   Liver sinusoidal endothelial cells (LSECs) critically regulate liver homeostasis and diseases through angiocrine factors. Notch is critical in endothelial cells (ECs). In the current study, Notch signaling was activated by inducible EC‐specific expression of the Notch intracellular domain (NIC). We found that endothelial Notch activation damaged liver homeostasis. Notch activation resulted in decreased fenestration and increased basement membrane, and a gene expression profile with decreased LSEC‐associated genes and increased continuous EC‐associated genes, suggesting LSEC dedifferentiation. Consistently, endothelial Notch activation enhanced hepatic fibrosis (HF) induced by CCl₄. Notch activation attenuated endothelial nitric oxide synthase (eNOS)/soluble guanylate cyclase (sGC) signaling, and activation of sGC by 3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzylindazole (YC‐1) reversed the dedifferentiation phenotype. In addition, Notch activation subverted the hepatocyte‐supporting angiocrine profile of LSECs by down‐regulating critical hepatocyte mitogens, including Wnt2a, Wnt9b, and hepatocyte growth factor (HGF). This led to compromised hepatocyte proliferation under both quiescent and regenerating conditions. Whereas expression of Wnt2a and Wnt9b was dependent on eNOS‐sGC signaling, HGF expression was not rescued by the sGC activator, suggesting heterogeneous mechanisms of LSECs to maintain hepatocyte homeostasis. Conclusion: Endothelial Notch activation results in LSEC dedifferentiation and accelerated liver fibrogenesis through eNOS‐sGC signaling, and alters the angiocrine profile of LSECs to compromise hepatocyte proliferation and liver regeneration.    

4.1915           Counteracting roles of MHCI and CD8+ T cells in the peripheral and central nervous system of ALS SOD1G93A mice

Nardo, G., Trolese, M.C., Verderio, M., Mariani, A., de Paola, M., Tiva, N., Dina, G., Panini, N., Erba, E., Quattrini, A. and Bendotti, C. Molecular Neurodegeneration, 13:42 (2018)   Background The major histocompatibility complex I (MHCI) is a key molecule for the interaction of mononucleated cells with CD8⁺T lymphocytes. We previously showed that MHCI is upregulated in the spinal cord microglia and motor axons of transgenic SOD1^G93A mice. Methods To assess the role of MHCI in the disease, we examined transgenic SOD1^G93A mice crossbred with β2 microglobulin-deficient mice, which express little if any MHCI on the cell surface and are defective for CD8⁺ T cells. Results The lack of MHCI and CD8⁺ T cells in the sciatic nerve affects the motor axon stability, anticipating the muscle atrophy and the disease onset. In contrast, MHCI depletion in resident microglia and the lack of CD8⁺ T cell infiltration in the spinal cord protect the cervical motor neurons delaying the paralysis of forelimbs and prolonging the survival of SOD1^G93A mice. Conclusions We provided straightforward evidence for a dual role of MHCI in the peripheral nervous system (PNS) compared to the CNS, pointing out regional and temporal differences in the clinical responses of ALS mice. These findings offer a possible explanation for the failure of systemic immunomodulatory treatments and suggest new potential strategies to prevent the progression of ALS.  

4.1916           A droplet microfluidic platform for efficient enzymatic chromatin digestion enables robust determination of nucleosome positioning

Xu, Y., Lee, J-H., Li, Z., Wang, L., Ordog, T. and Bailey, R.C. Lab on a Chip, 18, 2583-2592 (2018)   The first step in chromatin-based epigenetic assays involves the fragmentation of chromatin to facilitate precise genomic localization of the associated DNA. Here, we report the development of a droplet microfluidic device that can rapidly and efficiently digest chromatin into single nucleosomes starting from whole-cell input material offering simplified and automated processing compared to conventional manual preparation. We demonstrate the digestion of chromatin from 2500–125 000 Jurkat cells using micrococcal nuclease for enzymatic processing. We show that the yield of mononucleosomal DNA can be optimized by controlling enzyme concentration and incubation time, with resulting mononucleosome yields exceeding 80%. Bioinformatic analysis of sequenced mononucleosomal DNA (MNase-seq) indicated a high degree of reproducibility and concordance (97–99%) compared with conventionally processed preparations. Our results demonstrate the feasibility of robust and automated nucleosome preparation using a droplet microfluidic platform for nucleosome positioning and downstream epigenomic assays.  

4.1917           Electrophysiological assessment of a peptide amphiphile nanofiber nerve graft for facial nerve repair

Greene, J.J., McClendon, M.T., Stephanopoulos, N., Alvarez, Z., Stupp, S. and Richter, C-P.

1. Tissue Eng. Regen. Med., 12(6), 1389-1401 (2018)

  Facial nerve injury can cause severe long‐term physical and psychological morbidity. There are limited repair options for an acutely transected facial nerve not amenable to primary neurorrhaphy. We hypothesize that a peptide amphiphile nanofiber neurograft may provide the nanostructure necessary to guide organized neural regeneration. Five experimental groups were compared, animals with (1) an intact nerve, (2) following resection of a nerve segment, and following resection and immediate repair with either a (3) autograft (using the resected nerve segment), (4) neurograft, or (5) empty conduit. The buccal branch of the rat facial nerve was directly stimulated with charge balanced biphasic electrical current pulses at different current amplitudes whereas nerve compound action potentials (nCAPs) and electromygraphic responses were recorded. After 8 weeks, the proximal buccal branch was surgically reexposed and electrically evoked nCAPs were recorded for groups 1–5. As expected, the intact nerves required significantly lower current amplitudes to evoke an nCAP than those repaired with the neurograft and autograft nerves. For other electrophysiologic parameters such as latency and maximum nCAP, there was no significant difference between the intact, autograft, and neurograft groups. The resected group had variable responses to electrical stimulation, and the empty tube group was electrically silent. Immunohistochemical analysis and transmission electron microscopy confirmed myelinated neural regeneration. This study demonstrates that the neuroregenerative capability of peptide amphiphile nanofiber neurografts is similar to the current clinical gold standard method of repair and holds potential as an off‐the‐shelf solution for facial reanimation and potentially peripheral nerve repair.  

4.1918           Single-Cell Map of Diverse Immune Phenotypes in the Breast Tumor Microenvironment

Azizi, E., Carr, A.J., Plitas, G., Mazutis, L., Rudensky, A. and Pe’er, D. Cell, 174, 1293-1308 (2018)   Knowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We profiled 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph nodes, using single-cell RNA-seq. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data. Despite significant similarity between normal and tumor tissue-resident immune cells, we observed continuous phenotypic expansions specific to the tumor microenvironment. Analysis of paired single-cell RNA and T cell receptor (TCR) sequencing data from 27,000 additional T cells revealed the combinatorial impact of TCR utilization on phenotypic diversity. Our results support a model of continuous activation in T cells and do not comport with the macrophage polarization model in cancer. Our results have important implications for characterizing tumor-infiltrating immune cells.  

4.1919           CX3CR1+ Macrophages and CD8+ T Cells Control Intestinal IgA Production

Kim, Y-I., Song, J-H., Ko, H-J., Kweon, M-N., Kang, Y.K., Reinecker, H-C. and Chang, S-Y.

1. Immunol., 201(4), 1287-1294 (2018)

  Secretory IgA is a key host defense mechanism that controls the intestinal microbiota. We investigated the role of CD11c⁺CX₃CR1⁺CD64⁺ macrophages in IgA production in the intestine. Intestinal CX₃CR1⁺ macrophages directly induced IgA secretion by B cells. Ag delivery to lamina propria (LP) CX₃CR1⁺ macrophages specifically induced intestinal IgA production. The induction of IgA by CX₃CR1⁺ macrophages required BAFF, a proliferation-inducing ligand, and TNF-α, but was surprisingly independent of TLR-mediated microbial recognition and retinoic acid signaling. IgA secretion by CX₃CR1⁺ macrophages was enhanced by LP CD8⁺ T cells through the secretion of IL-9 and IL-13. CX₃CR1⁺ macrophages and CD8⁺ T cells induced IgA production by B cells independently of mesenteric lymph nodes and Peyer patches. Our data reveal a previously unrecognized cellular circuitry in which LP CX₃CR1⁺ macrophages, B cells, and CD8⁺ T cells coordinate the protective Ig secretion in the small intestine upon peripheral Ag delivery.  

4.1920           IL-35 Hinders Endogenous Antitumor T-cell Immunity and Responsiveness to Immunotherapy in Pancreatic Cancer

Mirlekar, B., Michaud, D., Searcy, R., Greene, K. and Pylayeva-Gupta, Y. Cancer Immunol. Res., 6(9), 1014-1024 (2018)   Although successes in cancer immunotherapy have generated considerable excitement, this form of treatment has been largely ineffective in patients with pancreatic ductal adenocarcinoma (PDA). Mechanisms that contribute to the poor antitumor immune response in PDA are not well understood. Here, we demonstrated that cytokine IL35 is a major immunosuppressive driver in PDA and potentiates tumor growth via the suppression of endogenous antitumor T-cell responses. The growth of pancreatic tumors in mice deficient for IL35 was significantly reduced. An analysis of tumor-infiltrating immune cells revealed a role for IL35 in the expansion of regulatory T cells and the suppression of CD4⁺ effector T cells. We also detected a robust increase in both the infiltration and activation of cytotoxic CD8⁺ T cells, suggesting that targeting IL35 may be an effective strategy to convert PDA from an immunologically “cold” to “hot” tumor. Although PDA is typically resistant to anti–PD-1 immunotherapy, we demonstrated robust synergistic reduction in tumor growth when IL35 deficiency was combined with anti–PD-1 treatment. These findings provide new insight into the function of IL35 in the pathogenesis of pancreatic cancer and underscore the potential significance of IL35 as a therapeutic target for use in combination immunotherapy approaches in this deadly malignancy.    

4.1921           The Metabolic Disturbances of Motoneurons Exposed to Glutamate

Hounoum, B.M., Blasco, H., Coque, E., Vourc’h, P., Emond, E., Corcia, P., Andres, C.R., Raoul, C. and Mavel, S. Mol. Neurobiol., 55(10), 7669-7676 (2018)   Glutamate-induced excitotoxicity is considered as one of the major pathophysiological factors of motoneuron death in amyotrophic lateral sclerosis and other motoneuron diseases. In order to expand our knowledge on mechanisms of glutamate-induced excitotoxicity, the present study proposes to determine the metabolic consequences of glutamate and astrocytes in primary enriched motoneuron culture. Using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), we showed that the presence of astrocytes and glutamate profoundly modified the metabolic profile of motoneurons. Our study highlights for the first time that crosstalk between astrocytes and enriched motoneuron culture induced alterations in phenylalanine, tryptophan, purine, arginine, proline, aspartate, and glutamate metabolism in motoneurons. We observed that astrocytes modulate the sensitivity of motoneurons to glutamate, since metabolites altered by glutamate in motoneurons cultured alone were different (except 5-hydroxylysine) from those altered in co-cultured motoneurons. Our findings provide new insight into the metabolic alterations associated to astrocytes and glutamate in motoneurons and provide opportunities to identify novel therapeutic targets.  

4.1922           Role of glutamine synthetase in angiogenesis beyond glutamine synthesis

Eelen, G., Dubois, C., Cantelmo, A.R., Goveia, J., Brüning, U., DeRan, M. et al Nature, 561, 63-69 (2018)   Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.  

4.1923           Betanodavirus infection in primary neuron cultures from sole

Souto, S., Olveira, J.G., Vazques-Salgado, L., Dopazo, C.P. and Bandin, I. Vet. Res., 49:86 (2018)   Nervous necrosis virus (NNV), G. Betanodavirus, is the causative agent of viral encephalopathy and retinopathy, a disease that causes mass mortalities in a wide range of fish species. Betanodaviruses are neurotropic viruses and their replication in the susceptible fish species seems to be almost entirely restricted to nerve tissue. However, none of the cell lines used for NNV propagation has a nervous origin. In this study, first we established a protocol for the primary culture of neurons from Senegalese sole, which made it possible to further study virus-host cell interactions. Then, we compared the replication of three NNV strains with different genotypes (SJNNV, RGNNV and a RGNNV/SJNNV reassortant strain) in sole neuron primary cultures and E-11 cells. In addition, to study how two amino acid substitutions at the c-terminal of the capsid protein (positions 247 and 270) affect the binding to cell receptors, a recombinant strain was also tested. The results show that sole neural cells enabled replication of all the tested NNV strains. However, the recombinant strain shows a clearly delayed replication when compared with the wt strain. This delay was not observed in virus replicating in E-11 cells, suggesting a viral interaction with different cell receptors. The establishment of a sole primary neuronal culture protocol provides an important tool for research into betanodavirus infection in sole.  

4.1924           A novel Atg5-shRNA mouse model enables temporal control of Autophagy in vivo

Cassidy, L.D., Young, A.R.J., Perez-Mancera, P.A., Nimmervoll, B., Jaulim, A., Chen, H-C., McIntyre, D.J.O., Brais, R., Ricketts, T., Pacey, S., De La Roche, M., Gilbertson, R.J., Rubinsztein, D.C. and Narita, M. Autophagy, 14(7), 1256-1266 (2018)   Macroautophagy/autophagy is an evolutionarily conserved catabolic pathway whose modulation has been linked to diverse disease states, including age-associated disorders. Conventional and conditional whole-body knockout mouse models of key autophagy genes display perinatal death and lethal neurotoxicity, respectively, limiting their applications for in vivo studies. Here, we have developed an inducible shRNA mouse model targeting Atg5, allowing us to dynamically inhibit autophagy in vivo, termed ATG5i mice. The lack of brain-associated shRNA expression in this model circumvents the lethal phenotypes associated with complete autophagy knockouts. We show that ATG5i mice recapitulate many of the previously described phenotypes of tissue-specific knockouts. While restoration of autophagy in the liver rescues hepatomegaly and other pathologies associated with autophagy deficiency, this coincides with the development of hepatic fibrosis. These results highlight the need to consider the potential side effects of systemic anti-autophagy therapies.  

4.1925           Chronic d-serine supplementation impairs insulin secretion

Suwandhi, L., Hausmann, S., Braun, A., Gruber, T., Heinzmann, S.S. et al Mol. Metabolism, 16, 191-202 (2018)   Objective The metabolic role of d-serine, a non-proteinogenic NMDA receptor co-agonist, is poorly understood. Conversely, inhibition of pancreatic NMDA receptors as well as loss of the d-serine producing enzyme serine racemase have been shown to modulate insulin secretion. Thus, we aim to study the impact of chronic and acute d-serine supplementation on insulin secretion and other parameters of glucose homeostasis. Methods We apply MALDI FT-ICR mass spectrometry imaging, NMR based metabolomics, 16s rRNA gene sequencing of gut microbiota in combination with a detailed physiological characterization to unravel the metabolic action of d-serine in mice acutely and chronically treated with 1% d-serine in drinking water in combination with either chow or high fat diet feeding. Moreover, we identify SNPs in SRR, the enzyme converting L-to d-serine and two subunits of the NMDA receptor to associate with insulin secretion in humans, based on the analysis of 2760 non-diabetic Caucasian individuals. Results We show that chronic elevation of d-serine results in reduced high fat diet intake. In addition, d-serine leads to diet-independent hyperglycemia due to blunted insulin secretion from pancreatic beta cells. Inhibition of alpha 2-adrenergic receptors rapidly restores glycemia and glucose tolerance in d-serine supplemented mice. Moreover, we show that single nucleotide polymorphisms (SNPs) in SRR as well as in individual NMDAR subunits are associated with insulin secretion in humans. Conclusion Thus, we identify a novel role of d-serine in regulating systemic glucose metabolism through modulating insulin secretion.  

4.1926           Single cell molecular alterations reveal target cells and pathways of concussive brain injury

Arneson, D., Zhang, G., Ying, Z., Zhuang, Y., Byun, H.R., Ahn, I.S., Gomez-Pinilla, F. and Yang, X. Nature Communications, 9:3894 (2018)   The complex neuropathology of traumatic brain injury (TBI) is difficult to dissect, given the convoluted cytoarchitecture of affected brain regions such as the hippocampus. Hippocampal dysfunction during TBI results in cognitive decline that may escalate to other neurological disorders, the molecular basis of which is hidden in the genomic programs of individual cells. Using the unbiased single cell sequencing method Drop-seq, we report that concussive TBI affects previously undefined cell populations, in addition to classical hippocampal cell types. TBI also impacts cell type-specific genes and pathways and alters gene co-expression across cell types, suggesting hidden pathogenic mechanisms and therapeutic target pathways. Modulating the thyroid hormone pathway as informed by the T4 transporter transthyretin Ttr mitigates TBI-associated genomic and behavioral abnormalities. Thus, single cell genomics provides unique information about how TBI impacts diverse hippocampal cell types, adding new insights into the pathogenic pathways amenable to therapeutics in TBI and related disorders.  

4.1927           An Easy-to-Implement Protocol for Preparing Postnatal Ventral Mesencephalic Cultures

Lautenschläger, J., Mosharov, E.V., Kanter, E., Sulzer, D. and Kaminski Schierle, G.S. Frontiers in Cell. Neurosci., 12:44 (2018)   Postnatally derived cultures of ventral mesencephalic neurons offer several crucial advantages over embryonic ventral mesencephalic cultures, including a higher content of TH-positive cells and the ability to derive cells from the substantia nigra, which contains the neurons most vulnerable to Parkinson's disease. On the other hand, these cultures are more challenging to produce consistently. Here, we provide an easy-to-implement protocol for culturing postnatal ventral mesencephalic cells from the substantia nigra (SN) and the ventral tegmental area using commercially available media, dishes, and general lab equipment, avoiding extensive material and equipment purchases. The protocol can be completed in about 5 h and provides ventral midbrain neuron cultures on cortex glia feeder layers in three weeks' time. The protocol uses an optimized protease digestion, tissue storage in Hibernate A during dissection and purification of neurons on an OptiPrep density gradient.  

4.1928           Promyelocytic leukemia (PML) nuclear bodies (NBs) induce latent/quiescent HSV-1 genomes chromatinization through a PML NB/Histone H3.3/H3.3 Chaperone Axis

Cohen, C., Corpet, A., Roubille, S., Maroui, M., Poccardi N., Rousseau, A., Kleijwegt, C., Binda, O., Texier, P., Sawtell, N., Labetoulle, M. and Lomonte, P. Plos Pathogens, 14(9), e1007313 (2018)   Herpes simplex virus 1 (HSV-1) latency establishment is tightly controlled by promyelocytic leukemia (PML) nuclear bodies (NBs) (or ND10), although their exact contribution is still elusive. A hallmark of HSV-1 latency is the interaction between latent viral genomes and PML NBs, leading to the formation of viral DNA-containing PML NBs (vDCP NBs), and the complete silencing of HSV-1. Using a replication-defective HSV-1-infected human primary fibroblast model reproducing the formation of vDCP NBs, combined with an immuno-FISH approach developed to detect latent/quiescent HSV-1, we show that vDCP NBs contain both histone H3.3 and its chaperone complexes, i.e., DAXX/ATRX and HIRA complex (HIRA, UBN1, CABIN1, and ASF1a). HIRA also co-localizes with vDCP NBs present in trigeminal ganglia (TG) neurons from HSV-1-infected wild type mice. ChIP and Re-ChIP show that vDCP NBs-associated latent/quiescent viral genomes are chromatinized almost exclusively with H3.3 modified on its lysine (K) 9 by trimethylation, consistent with an interaction of the H3.3 chaperones with multiple viral loci and with the transcriptional silencing of HSV-1. Only simultaneous inactivation of both H3.3 chaperone complexes has a significant impact on the deposition of H3.3 on viral genomes, suggesting a compensation mechanism. In contrast, the sole depletion of PML significantly impacts the chromatinization of the latent/quiescent viral genomes with H3.3 without any overall replacement with H3.1. vDCP NBs-associated HSV-1 genomes are not definitively silenced since the destabilization of vDCP NBs by ICP0, which is essential for HSV-1 reactivation in vivo, allows the recovery of a transcriptional lytic program and the replication of viral genomes. Consequently, the present study demonstrates a specific chromatin regulation of vDCP NBs-associated latent/quiescent HSV-1 through an H3.3-dependent HSV-1 chromatinization involving the two H3.3 chaperones DAXX/ATRX and HIRA complexes. Additionally, the study reveals that PML NBs are major actors in latent/quiescent HSV-1 H3.3 chromatinization through a PML NB/histone H3.3/H3.3 chaperone axis.  

4.1929           Lack of Sprouty 1 and 2 enhances survival of effector CD8+ T cells and yields more protective memory cells

Shehata, H.M., Khan, S., Chen, E., Fields, P.E. and Flavell, R.A. PNAS, 115(38), E8939-E8947 (2018)   Identifying novel pathways that promote robust function and longevity of cytotoxic T cells has promising potential for immunotherapeutic strategies to combat cancer and chronic infections. We show that sprouty 1 and 2 (Spry1/2) molecules regulate the survival and function of memory CD8⁺ T cells. Spry1/2 double-knockout (DKO) ovalbumin (OVA)-specific CD8⁺ T cells (OT-I cells) mounted more vigorous autoimmune diabetes than WT OT-I cells when transferred to mice expressing OVA in their pancreatic β-islets. To determine the consequence of Spry1/2 deletion on effector and memory CD8⁺ T cell development and function, we used systemic infection with lymphocytic choriomeningitis virus (LCMV) Armstrong. Spry1/2 DKO LCMV gp33-specific P14 CD8⁺ T cells survive contraction better than WT cells and generate significantly more polyfunctional memory T cells. The larger number of Spry1/2 DKO memory T cells displayed enhanced infiltration into infected tissue, demonstrating that absence of Spry1/2 can result in increased recall capacity. Upon adoptive transfer into naive hosts, Spry1/2 DKO memory T cells controlled Listeria monocytogenes infection better than WT cells. The enhanced formation of more functional Spry1/2 DKO memory T cells was associated with significantly reduced mTORC1 activity and glucose uptake. Reduced p-AKT, p-FoxO1/3a, and T-bet expression was also consistent with enhanced survival and memory accrual. Collectively, loss of Spry1/2 enhances the survival of effector CD8⁺ T cells and results in the formation of more protective memory cells. Deleting Spry1/2 in antigen-specific CD8⁺ T cells may have therapeutic potential for enhancing the survival and functionality of effector and memory CD8⁺ T cells in vivo.  

4.1930           MicroRNA-182 Promotes Lipoprotein Lipase Expression and Atherogenesisby Targeting Histone Deacetylase 9 in Apolipoprotein E-Knockout Mice

Cheng, H-P-. Gong, D., Zhao, Z-W., He, P.P., Yu, X-H. et al Circ. J., 82, 28-38 (2018)   Background:Lipoprotein lipase (LPL) expressed in macrophages plays an important role in promoting the development of atherosclerosis or atherogenesis. MicroRNA-182 (miR-182) is involved in the regulation of lipid metabolism and inflammation. However, it remains unclear how miR-182 regulates LPL and atherogenesis. Methods and Results:Using bioinformatics analyses and a dual-luciferase reporter assay, we identified histone deacetylase 9 (HDAC9) as a target gene of miR-182. Moreover, miR-182 upregulated LPL expression by directly targetingHDAC9in THP-1 macrophages. Hematoxylin-eosin (H&E), Oil Red O and Masson’s trichrome staining showed that apolipoprotein E (ApoE)-knockout (KO) mice treated with miR-182 exhibited more severe atherosclerotic plaques. Treatment with miR-182 increased CD68 and LPL expression in atherosclerotic lesions in ApoE-KO mice, as indicated by double immunofluorescence staining in the aortic sinus. Increased miR-182-induced increases in LPL expression in ApoE-KO mice was confirmed by real-time quantitative polymerase chain reaction and western blotting analyses. Treatment with miR-182 also increased plasma concentrations of proinflammatory cytokines and lipids in ApoE-KO mice. Conclusions:The results of the present study suggest that miR-182 upregulates LPL expression, promotes lipid accumulation in atherosclerotic lesions, and increases proinflammatory cytokine secretion, likely through targetingHDAC9, leading to an acceleration of atherogenesis in ApoE-KO mice.  

4.1931           CX3CR1 differentiates F4/80low monocytes into pro-inflammatory F4/80high macrophages in the liver

Lee, Y-S., Kim, M-H., Yi, H-S., Kim, S.Y., Kim, H-H., Kim, J.H., Yeon, J.E., Byun, K.S., Byun, J-S. and Jeong, W-j. Scientifc Reports, 8:15076 (2018)   The expression of chemokine receptor CX₃CR1 is related to migration and signaling in cells of the monocyte-macrophage lineage. The precise roles of CX₃CR1 in the liver have been investigated but not clearly elucidated. Here, we investigated the roles of CX₃CR1 in hepatic macrophages and liver injury. Hepatic and splenic CX₃CR1^lowF4/80^low monocytes and CX₃CR1^lowCD16⁻ monocytes were differentiated into CX₃CR1^highF4/80^high or CX₃CR1^highCD16⁺ macrophages by co-culture with endothelial cells. Moreover, CX₃CL1 deficiency in human umbilical vein endothelial cells (HUVECs) attenuated the expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), whereas recombinant CX₃CL1 treatment reversed this expression in co-cultured monocytes. Upon treatment with clodronate liposome, hepatic F4/80^high macrophages were successfully depleted at day 2 and recovered similarly in CX₃CR1^+/GFP and CX₃CR1^GFP/GFP mice at week 4, suggesting a CX₃CR1-independent replacement. However, F4/80^high macrophages of CX₃CR1^+/GFP showed a stronger pro-inflammatory phenotype than CX₃CR1^GFP/GFP mice. In clodronate-treated chimeric CX₃CR1^+/GFP and CX₃CR1^GFP/GFP mice, CX₃CR1⁺F4/80^high macrophages showed higher expression of IL-1β and TNF-α than CX₃CR1⁻F4/80^high macrophages. In alcoholic liver injury, despite the similar frequency of hepatic F4/80^high macrophages, CX₃CR1^GFP/GFP mice showed reduced liver injury, hepatic fat accumulation, and inflammatory responses than CX₃CR1^+/GFP mice. Thus, CX₃CR1 could be a novel therapeutic target for pro-inflammatory macrophage-mediated liver injury.  

4.1932           RbAp48 Protein Is a Critical Component of GPR158/OCN Signaling and Ameliorates Age-Related Memory Loss

Kosmidis, S., Polyzos, A., Harvey, L., Youssef, M., Denny, C.A., Dranovsky, A. and Kandel, E.R. Cell Reports, 25, 959-973 (2018)   Precisely deciphering the molecular mechanisms of age-related memory loss is crucial to create appropriate therapeutic interventions. We have previously shown that the histone-binding protein RbAp48/Rbbp4 is a molecular determinant of Age-Related Memory Loss. By exploring how this protein regulates the genomic landscape of the hippocampal circuit, we find that RbAp48 controls the expression of BDNF and GPR158 proteins, both critical components of osteocalcin (OCN) signaling in the mouse hippocampus. We show that inhibition of RbAp48 in the hippocampal formation inhibits OCN’s beneficial functions in cognition and causes deficits in discrimination memory. In turn, disruption of OCN/GPR158 signaling leads to the downregulation of RbAp48 protein, mimicking the discrimination memory deficits observed in the aged hippocampus. We also show that activation of the OCN/GPR158 pathway increases the expression of RbAp48 in the aged dentate gyrus and rescues age-related memory loss.  

4.1933           Highly Angiogenic, Nonthrombogenic Bone Marrow Mononuclear Cell–Derived Spheroids in Intraportal Islet Transplantation

Oh, B.J., Jin, S-M., Hwang, Y., Choi, J.M., Lee, H-S., Kim, G., Kim, G., Park, H.J., Kim, P., Kim, S.J. and Kim, J.H. Diabetes, 67(3), 473-485 (2018)   Highly angiogenic bone marrow mononuclear cell–derived spheroids (BM-spheroids), formed by selective proliferation of the CD31⁺CD14⁺CD34⁺ monocyte subset via three-dimensional (3D) culture, have had robust angiogenetic capacity in rodent syngeneic renal subcapsular islet transplantation. We wondered whether the efficacy of BM-spheroids could be demonstrated in clinically relevant intraportal islet transplantation models without increasing the risk of portal thrombosis. The thrombogenic potential of intraportally infused BM-spheroids was compared with that of mesenchymal stem cells (MSCs) and MSC-derived spheroids (MSC-spheroids). The angiogenic efficacy and persistence in portal sinusoids of BM-spheroids were examined in rodent syngeneic and primate allogeneic intraportal islet transplantation models. In contrast to MSCs and MSC-spheroids, intraportal infusion of BM-spheroids did not evoke portal thrombosis. BM-spheroids had robust angiogenetic capacity in both the rodent and primate intraportal islet transplantation models and improved posttransplant glycemic outcomes. MRI and intravital microscopy findings revealed the persistence of intraportally infused BM-spheroids in portal sinusoids. Intraportal cotransplantation of allogeneic islets with autologous BM-spheroids in nonhuman primates further confirmed the clinical feasibility of this approach. In conclusion, cotransplantation of BM-spheroids enhances intraportal islet transplantation outcome without portal thrombosis in mice and nonhuman primates. Generating BM-spheroids by 3D culture prevented the rapid migration and disappearance of intraportally infused therapeutic cells.  

4.1934           Combination Metabolomics Approach for Identifying Endogenous Substrates of Carnitine/Organic Cation Transporter OCTN1

Masuo, Y., Ohba, Y., Yamada, K., Al-Shammanri, A.H., Seba, N., Nkamichi, N., Ogihara, T., Kunishima, M. and Kato, Y. Pharm. Res., 35(11):224 (2018)   Purpose Solute carrier SLC22A4 encodes the carnitine/organic cation transporter OCTN1 and is associated with inflammatory bowel disease, although little is known about how this gene is linked to pathogenesis. The aim of the present study was to identify endogenous substrates that are associated with gastrointestinal inflammation. Methods HEK293/OCTN1 and mock cells were incubated with colon extracts isolated from dextran sodium sulfate-induced colitis mice; the subsequent cell lysates were mixed with the amino group selective reagent 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), to selectively label OCTN1 substrates. Precursor ion scanning against the fragment ion of APDS was then used to identify candidate OCTN1 substrates. Results Over 10,000 peaks were detected by precursor ion scanning; m/z 342 had a higher signal in HEK293/OCTN1 compared to mock cells. This peak was detected as a divalent ion that contained four APDS-derived fragments and was identified as spermine. Spermine concentration in peripheral blood mononuclear cells from octn1 gene knockout mice (octn1^−/−) was significantly lower than in wild-type mice. Lipopolysaccharide-induced gene expression of inflammatory cytokines in peritoneal macrophages from octn1^−/− mice was lower than in wild-type mice. Conclusions The combination metabolomics approach can provide a novel tool to identify endogenous substrates of OCTN1.  

4.1935           MicroRNA-96 Promotes Schistosomiasis Hepatic Fibrosis in mice by suppressing Smad7

Luo, X., Zhang, D., Xie, J., Su, Q., He, X., Bai, R., Gao, G. and Pan, W. Molecular Therapy – Methods and Clin. Develop., 11, 73-82 (2018)   Infection with schistosoma causes aberrant expression of host microRNAs (miRNAs) and normalizing the levels of dysregulated miRNAs can attenuate pathology. Here, we show that the host miRNA, miR-96 is markedly up-regulated during the progression of hepatic schistosomiasis. We demonstrate that elevation of miR-96 induces hepatic fibrosis in infected mice by suppressing the expression of its target gene, Smad7. We show that infection with schistosoma induces the expression of TGF-β1, which in turn upregulates the expression of miR-96 through Smad2/3-Drosha-mediated post-transcriptional regulation. Furthermore, inhibition of miR-96 with recombinant adeno-associated virus 8 (rAAV8)-mediated delivery of Tough Decoy RNAs in mice attenuated hepatic fibrosis and prevented lethality following schistosome infection. Taken together, our data highlight the potential for rAAV8-mediated inhibition of miR-96 as a therapeutic strategy to treat hepatic schistosomiasis.  

4.1936           Impact of Escherichia coli K12 and O18:K1 on human platelets: Differential effects on platelet activation, RNAs and proteins

Fejes, A.V., Best, M.G., van der Heijden, W.A., Vancura, A., Verschueren, H., de Mast, O., Wurdinger, T. and Mannhalter, C. Scientific Reports, 8:16145 (2018)   Blood platelets can interact with bacteria, possibly leading to platelet activation, cytokine and microparticle release and immune signalling. Besides, bacteria can also affect the platelet RNA content. We investigated the impact of non-pathogenic K12 and pathogenic O18:K1 Escherichia (E.) coli strains on platelet activation, RNA expression patterns, and selected proteins. Depending on bacteria concentration, contact of platelets with E. coli K12 lead to an increase of P-selectin (24–51.3%), CD63 (15.9–24.3%), PAC-1 (3.8–14.9%) and bound fibrinogen (22.4–39%) on the surface. E. coli O18:K1 did not affect these markers. Sequencing analysis of total RNA showed that E. coli K12 caused a significant concentration change of 103 spliced mRNAs, of which 74 decreased. For the RNAs of HMBS (logFC = +5.73), ATP2C1 (logFC = −3.13) and LRCH4 (logFC = −4.07) changes were detectable by thromboSeq and Tuxedo pipelines. By Western blot we observed the conversion of HMBS protein from a 47 kDA to 40 kDa product by E. coli K12, O18:K1 and by purified lipopolysaccharide. While ATP2C1 protein was released from platelets, E. coli either reduced the secretion or broke down the released protein making it undetectable by antibodies. Our results demonstrate that different E. coli strains influence activation, RNA and protein levels differently which may affect platelet-bacteria crosstalk.  

4.1937           DNA fragmentation in epididymal freeze-dried ram spermatozoa impairs embryo development

Palazzese, L., Gosalvez, J., Anzalone, D.A., Loi, P. and Saragusty, J.

1. Reprod. Developm., 64(5), 393-400 (2018)

  Sperm freeze-drying is a revolutionary technique, which has been gaining prominence in recent years. The first related significant result was Wakayama and Yanagimachi’s demonstration in 1998 of the birth of healthy mouse offspring by Intracytoplasmic Sperm Injection (ICSI), using epididymal freeze-dried spermatozoa. Mouse, rat, and hamster models were the first small mammals born from lyophilized epididymal spermatozoa, whereas most other studies in this field used ejaculated spermatozoa. In this work, we applied this technique to ram epididymal spermatozoa, checking the correlation between DNA integrity and embryo development following ICSI. To do this, epididymal sperm from four rams was lyophilized in a trehalose, glucose, KCl, HEPES, and Trolox media. To evaluate DNA damage and fragmentation after rehydration, samples were processed for Sperm Chromatin Dispersion test (SCD), Two-Tailed Comet Assay, and were used for ICSI. Ram #2 had a higher rate of spermatozoa with intact DNA compared with rams #1, #3, and #4 (28% vs. 3.8%, 2.8%, and 5%, respectively) and the lowest rate of Single-Strand Breaks (SSBs) (70% vs. 95.9%, 92.6%, and 93% respectively). Ram #3 had a higher level of Double-Strand Breaks (DSBs) compared to Ram #1 (4.6% vs. 0.33%, respectively). Embryo development to the blastocyst stage following ICSI was only reached from rams whose sperm had higher level of intact DNA – Rams #2 and #4 (6%, 5/147 and 6.3%, 4/64, respectively). Definitively, the impact of sperm DNA damage on embryonic development depends on the balance between sperm DNA fragmentation extent, fragmentation type (SSBs or DSBs), and the oocyte’s repair capacity.  

4.1938           Droplet‐Based Microfluidic Templating of Polyglycerol‐Based Microgels for the Encapsulation of Cells: A Comparative Study

Kapourani, E., Neumann, F., Achazi, K., Dernedde, J. and Haag, R. Macromol. Bioxci., 18, 1800116 (2018)   Cell microencapsulation holds great promise as a therapeutic strategy for the controlled and sustained delivery of biologically relevant agents. The authors developed cell‐laden microgel scaffolds with excellent long‐term viabilities by combining bioorthogonal strain promoted azide–alkyne cycloaddition (SPAAC) and droplet‐based microfluidic templating. Star‐shaped polyglycerol hexaazide, α,ω‐bis azido‐linear polyglycerol or polyethylene glycol as well as dendritic polyglycerol‐(polycyclooctyne) served as bioinert hydrogel precursors. The authors demonstrate for the first time the generation of entirely polyglycerol‐based microcapsules with excellent stability and full retention of viability of the packed cells for longer than 3 weeks. As a result, our microgel particles could be used for long‐term immunoisolation of cells enabling their study during encapsulation.  

4.1939           High-throughput single-cell DNA sequencing of acute myeloid leukemia tumors with droplet microfluidics

Pellegrino, M., Sciambi, A., Treusch, S., Durruthy-Durruthy, R., Gokhale, K. et al Genome Res., 28, 1345-1352 (2018)   To enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cancer cells confined to droplets. The barcodes are then used to reassemble the genetic profiles of cells from next-generation sequencing data. By using this approach, we sequenced longitudinally collected acute myeloid leukemia (AML) tumor populations from two patients and genotyped up to 62 disease relevant loci across more than 16,000 individual cells. Targeted single-cell sequencing was able to sensitively identify cells harboring pathogenic mutations during complete remission and uncovered complex clonal evolution within AML tumors that was not observable with bulk sequencing. We anticipate that this approach will make feasible the routine analysis of AML heterogeneity, leading to improved stratification and therapy selection for the disease.  

4.1940           Polymorphisms in Receptors Involved in Opsonic and Nonopsonic Phagocytosis, and Correlation with Risk

Herrero-Sanchez, M.C., Angomas, E.B., de Ramon, C., Telleria, J.J., Corchete, L.A., Alonso, S., del Carmen Ramos, M., Penarrubia, M.J., Marquez, S., Fernandez, N., Garcia Frade, L.J. and Crespo, M.S: Infect. Immun., 86(12), e00709-18 (2018)   High-risk hematological malignancies are a privileged setting for infection by opportunistic microbes, with invasive mycosis being one of the most serious complications. Recently, genetic background has emerged as an unanticipated risk factor. For this reason, polymorphisms for genes encoding archetypal receptors involved in the opsonic and nonopsonic clearance of microbes, pentraxin-3 (PTX3) and Dectin-1, respectively, were studied and correlated with the risk of infection. Fungal, bacterial, and viral infections were registered for a group of 198 patients with high-risk hematological malignancies. Polymorphisms for the pentraxin-3 gene (PTX3) showed a significant association with the risk of fungal infection by Candida spp. and, especially, by Aspergillus spp. This link remained even for patients undergoing antifungal prophylaxis, thus demonstrating the clinical relevance of PTX3 in the defense against fungi. CLEC7A polymorphisms did not show any definite correlation with the risk of invasive mycosis, nor did they influence the expression of Dectin-1 isoforms generated by alternative splicing. The PTX3 mRNA expression level was significantly lower in samples from healthy volunteers who showed these polymorphisms, although no differences were observed in the extents of induction elicited by bacterial lipopolysaccharide and heat-killed Candida albicans, thus suggesting that the expression of PTX3 at the start of infection may influence the clinical outcome. PTX3 mRNA expression can be a good biomarker to establish proper antifungal prophylaxis in immunodepressed patients.  

4.1941           Amnion epithelial cell–derived exosomes induce inflammatory changes in uterine cells

Hadley, E.E., Sheller-Miller. S., Saade, G., Salomon, C., Mesiano, S., Taylor, R.N., Taylor, B.D. and Menon, R. Am. J. Obstet. Gynecol., 219, 478.e1-478.e21 (2018)   Background Fetal endocrine signals are generally considered to contribute to the timing of birth and the initiation of labor. Fetal tissues under oxidative stress release inflammatory mediators that lead to sterile inflammation within the maternal-fetal interface. Importantly, these inflammatory mediators are packaged into exosomes, bioactive cell-derived extra cellular vesicles that function as vectors and transport them from the fetal side to the uterine tissues where they deposit their cargo into target cells enhancing uterine inflammatory load. This exosome-mediated signaling is a novel mechanism for fetal-maternal communication. Objective This report tested the hypothesis that oxidative stress can induce fetal amnion cells to produce exosomes, which function as a paracrine intermediary between the fetus and mother and biochemically signal readiness for parturition. Study Design Primary amnion epithelial cells were grown in normal cell culture (control) or exposed to oxidative stress conditions (induced by cigarette smoke extract). Exosomes were isolated from cell supernatant by sequential ultracentrifugation. Exosomes were quantified and characterized based on size, shape, and biochemical markers. Myometrial, decidual, and placental cells (BeWo) were treated with 2 × 10⁵, 2 × 10⁷, and 2 × 10⁹ control or oxidative stress–derived amnion epithelial cell exosomes for 24 hours. Entry of amnion epithelial cell exosomes into cells was confirmed by confocal microscopy of fluorescent-labeled exosomes. The effect of amnion epithelial cell exosomes on target cell inflammatory status was determined by measuring production of interleukin-6, interleukin-8, interleukin-1β, tumor necrosis factor-α, and prostaglandin E₂ by enzyme-linked immunosorbent assay and inflammatory gene transcription factor (nuclear factor-κβ) activation status by immunoblotting for phosphorylated RelA/p65. Localization of NANOG in term human myometrium and decidua obtained from women before labor and during labor was performed using immunohistochemistry. Data were analyzed by Wilcoxon-Mann-Whitney test to compare effects of exosomes from control and oxidative stress-treated amnion epithelial cells on inflammatory status of target cells. Results Amnion epithelial cells released ∼125 nm, cup-shaped exosomes with ∼899 and 1211 exosomes released per cell from control and oxidative stress–induced cells, respectively. Amnion epithelial cell exosomes were detected in each target cell type after treatment using confocal microscopy. Treatment with amnion epithelial cell exosomes increased secretion of interleukin-6, interleukin-8, and PGE₂ and activation of NF-κβ (each P < .05) in myometrial and decidual cells. Exosome treatments had no effect on interleukin-6 and PGE₂ production in BeWo cells. NANOG staining was higher in term labor myometrium and decidua compared to tissues not in labor. Conclusion In vitro, amnion epithelial cell exosomes lead to an increased inflammatory response in maternal uterine cells whereas placental cells showed refractoriness. Fetal cell exosomes may function to signal parturition by increasing maternal gestational cell inflammation.  

4.1942           Loss of Prolyl-Hydroxylase 1 Protects against Biliary Fibrosis via Attenuated Activation of Hepatic Stellate Cells

Strowitsky, M.J., Kirschberg, J., Tuffs, C., Schiedeck, M., Ritter, A.S., Biller, M., Harness, J.M., Lasitschka, F., Schmidt, T., Radhakrishnan, P., Ulrich, A. and Schneider, M. Am. J. Pathol., 188(12), 2826-2838 (2018)   Liver fibrosis, eventually progressing to cirrhosis necessitating liver transplantation, poses a significant clinical problem. Oxygen shortage (hypoxia) and hypoxia-inducible transcription factors (HIFs) have been acknowledged as important drivers of liver fibrosis. The significance of oxygen-sensing HIF prolyl-hydroxylase (PHD) enzymes in this context has, however, remained elusive. In this study, we demonstrate that loss of PHD1 (PHD1^−/−) attenuates the development of liver fibrosis in mice subjected to chronic bile duct injury, induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine. This effect was accompanied with reduced recruitment of inflammatory leukocytes and attenuated occurrence of profibrotic myofibroblasts in PHD1^−/− livers. Further analyses focused on the significance of PHD1 in the activation of hepatic stellate cells (HSCs), which represent the driving force in liver fibrosis. Primary HSCs isolated from PHD1^−/− mice displayed significantly attenuated myofibroblast differentiation and profibrogenic properties compared with HSCs isolated from wild-type mice. Consistently, the expression of various profibrogenic and promitogenic factors was reduced in PHD1^−/− HSCs, without alterations in HIF-1α protein levels. Of importance, PHD1 protein was expressed in HSCs within human livers, and PHD1 transcript expression was significantly increased with disease severity in hepatic tissue from patients with liver fibrosis. Collectively, these findings indicate that PHD1 deficiency protects against liver fibrosis and that these effects are partly due to attenuated activation of HSCs. PHD1 may represent a therapeutic target to alleviate liver fibrosis.  

4.1943           miRNA in Rat Liver Sinusoidal Endothelial Cells and Hepatocytes and Application to Circulating Biomarkers that Discern Pathogenesis of Liver Injuries

Oda, S., Takeuchi, M., Akai, S., Shirai, Y., Tsuneyama, K. and Yokoi, T. Am. J. Pathol., 188(4), 916-928 (2018)   Sinusoidal obstruction syndrome is a serious liver injury caused by toxic injury to liver sinusoidal endothelial cells (LSECs) during clinical chemotherapy. Although circulating miRNAs, such as hepatocyte-specific miR-122-5p and miR-192-5p, have been proposed as potential noninvasive biomarkers of hepatocellular liver injury, these miRNAs may not be specific to damage to other hepatic cell types, including LSECs. We characterized miRNA expression in LSECs and hepatocytes and investigated whether cell type–specific miRNAs in plasma can discern pathogenesis of liver injuries in rats. Comprehensive miRNA expression analyses found that 66 and 12 miRNAs were highly expressed in LSECs and hepatocytes isolated from nontreated rats, respectively. An LSEC-enriched miR-511-3p was relatively liver specific according to public data. For establishing LSEC and hepatocyte injury models, rats were orally treated with monocrotaline and thioacetamide, respectively. In monocrotaline-treated rats, a sinusoidal obstruction syndrome model, LSEC damage was observed 6 hours after dosing, whereas hepatocellular damage was observed after 48 hours. Interestingly, the level of miR-511-3p in plasma was increased as early as 6 hours after monocrotaline dosing, followed by an increase of miR-122-5p after 24 hours. In the thioacetamide-induced hepatocellular injury model, the level of miR-511-3p was not altered in plasma, whereas miR-122-5p levels were increased after 6 hours. In conclusion, we identified miR-511-3p in plasma as a possible biomarker for LSEC damage.  

4.1944           Spliceosome-Associated Protein 130 Exacerbates Alcohol-Induced Liver Injury by Inducing NLRP3 Inflammasome–Mediated IL-1β in Mice

Kim, J-W., Roh, Y-S., Jeong, H., Yi, H-K., Lee, M-H., Lim, C-W. and Kim, B. Am. J. Pathol., 188(4), 967-980 (2018)   Excessive alcohol consumption leads to chronic liver diseases. Macrophage-inducible C-type lectin (Mincle) is a C-type lectin receptor that recognizes spliceosome-associated protein 130 (SAP130) known as an endogenous ligand released from dying cells. The aim was to examine the role of Mincle-SAP130 in the pathogenesis of alcoholic liver disease. Alcohol-induced liver injury was induced in wild-type (WT) and Mincle knockout (KO) mice by using a chronic-binge ethanol-feeding model. Mincle KO mice showed significant lower hepatic steatosis, inflammation with neutrophil infiltration, and fibrosis compared with WT mice after alcohol feeding. In contrast, Mincle activation exacerbated alcohol-induced liver injury. Kupffer cells (KCs) are major sources of Mincle. IL-1β expression was significantly down-regulated in Mincle KO mice compared with that in WT mice after alcohol consumption. Interestingly, expression and production of IL-1β were significantly decreased in SAP130-treated KCs isolated from leucine-rich–containing family pyrin domain containing-3–deficient mice compared with those in WT KCs. Such results were also observed in cells treated with SAP130 plus Syk inhibitor. Furthermore, infiltration of invariant natural killer T cells was decreased in livers of Mincle KO mice. Finally, inhibition of Syk signaling ameliorated alcohol-induced liver injury. Collectively, these results demonstrated that interaction between Mincle and SAP130 may promote the progression of alcoholic liver disease by IL-1β production in KCs and consequently increase inflammatory immune cell infiltration.  

4.1945           Binding of intercellular adhesion molecule 1 to β2-integrin regulates distinct cell adhesion processes on hepatic and cerebral endothelium

Tong, C-F., Zhang, Y., Lü, S-Q., Li, N., Gong, Y-X., Yang, H., Feng, S-L., Du, Y., Huang, D-D. and Long, M. Am. J. Physiol. Cell Physiol., 315, C409-C421 (2018)   Flowing polymorphonuclear neutrophils (PMNs) are forced to recruit toward inflamed tissue and adhere to vascular endothelial cells, which is primarily mediated by the binding of β₂-integrins to ICAM-1. This process is distinct among different organs such as liver and brain; however, the underlying kinetic and mechanical mechanisms regulating tissue-specific recruitment of PMNs remain unclear. Here, binding kinetics measurement showed that ICAM-1 on murine hepatic sinusoidal endothelial cells (LSECs) bound to lymphocyte function-associated antigen-1 (LFA-1) with higher on- and off-rates but lower effective affinity compared with macrophage-1 antigen (Mac-1), whereas ICAM-1 on cerebral endothelial cells (BMECs or bEnd.3 cells) bound to LFA-1 with higher on-rates, similar off-rates, and higher effective affinity compared with Mac-1. Physiologically, free crawling tests of PMN onto LSEC, BMEC, or bEnd.3 monolayers were consistent with those kinetics differences between two β₂-integrins interacting with hepatic sinusoid or cerebral endothelium. Numerical calculations and Monte Carlo simulations validated tissue-specific contributions of β₂-integrin-ICAM-1 kinetics to PMN crawling on hepatic sinusoid or cerebral endothelium. Thus, this work first quantified the biophysical regulation of PMN adhesion in hepatic sinusoids compared with cerebral endothelium.  

4.1946           Antibodies to Costimulatory Receptor 4-1BB Enhance Anti-tumor Immunity via T Regulatory Cell Depletion and Promotion of CD8 T Cell Effector Function

Buchan, S.L., Dou, L., Remer, M., Gray, J.C., Al-Shamkhani, A. and Beers, S.A. Immunity, 49, 958-970 (2018)   The costimulatory receptor 4-1BB is expressed on activated immune cells, including activated T cells. Antibodies targeting 4-1BB enhance the proliferation and survival of antigen-stimulated T cells in vitro and promote CD8 T cell-dependent anti-tumor immunity in pre-clinical cancer models. We found that T regulatory (Treg) cells infiltrating human or murine tumors expressed high amounts of 4-1BB. Intra-tumoral Treg cells were preferentially depleted by anti-4-1BB mAbs in vivo. Anti-4-1BB mAbs also promoted effector T cell agonism to promote tumor rejection. These distinct mechanisms were competitive and dependent on antibody isotype and FcγR availability. Administration of anti-4-1BB IgG2a, which preferentially depletes Treg cells, followed by either agonistic anti-4-1BB IgG1 or anti-PD-1 mAb augmented anti-tumor responses in multiple solid tumor models. An antibody engineered to optimize both FcγR-dependent Treg cell depleting capacity and FcγR-independent agonism delivered enhanced anti-tumor therapy. These insights into the effector mechanisms of anti-4-1BB mAbs lay the groundwork for translation into the clinic.  

4.1947           Large-scale reconstruction of cell lineages using single-cell readout of transcriptomes and CRISPR–Cas9 barcodes by scGESTALT

Raj, B., Gagnon, J.A., and Schier, A.F. Nature Protocols, 13, 2685-2713 (2018)   Lineage relationships among the large number of heterogeneous cell types generated during development are difficult to reconstruct in a high-throughput manner. We recently established a method, scGESTALT, that combines cumulative editing of a lineage barcode array by CRISPR–Cas9 with large-scale transcriptional profiling using droplet-based single-cell RNA sequencing (scRNA-seq). The technique generates edits in the barcode array over multiple timepoints using Cas9 and pools of single-guide RNAs (sgRNAs) introduced during early and late zebrafish embryonic development, which distinguishes it from similar Cas9 lineage-tracing methods. The recorded lineages are captured, along with thousands of cellular transcriptomes, to build lineage trees with hundreds of branches representing relationships among profiled cell types. Here, we provide details for (i) generating transgenic zebrafish; (ii) performing multi-timepoint barcode editing; (iii) building scRNA-seq libraries from brain tissue; and (iv) concurrently amplifying lineage barcodes from captured single cells. Generating transgenic lines takes 6 months, and performing barcode editing and generating single-cell libraries involve 7 d of hands-on time. scGESTALT provides a scalable platform to map lineage relationships between cell types in any system that permits genome editing during development, regeneration, or disease.  

4.1948           Correction of Hyperglycemia in Diabetic Rats With the Use of Microencapsulated Young Market Pig Islets

Jiang, X-F., Qian, T-L., Chen, D., Lu, H-W., Xue, P., Yang, X-W., Zhang, L-H., Hu, Y-Z. and Zhang, D-W. Transplant. Proceedings, 50(10), 3895-3899 (2018)   Objective The aim of this work was to investigate the transplantation efficacy of microencapsulated young market pig islets in a diabetic rat model. Methods Islets were isolated and purified from young market pigs obtained from a local slaughterhouse. The islets were encapsulated in barium alginate and subjected to a glucose-induced insulin release functional assay in culture. Microencapsulated islets were transplanted into diabetic Sprague-Dawley rats and removed after 30 days for histologic examination. Results The mean islet equivalent (IEQ) yield per gram of digested tissue was 3,125 ± 617 IEQ/g after isolation and 2,618 ± 917 IEQ/g after purification, respectively. Host rats' blood glucose concentrations normalized (from 22.3 ± 2.7 mmol/L to 5.1 ± 0.67 mmol/L) following encapsulated islet transplantation. After graft removal, hyperglycemia recurred in the rats, indicating that the grafts were responsible for maintaining euglycemia. Histology revealed viable islets in the capsules 30 days after graft removal. Immunolabeling of insulin verified that β-cells within the capsules remained well granulated. No fibrosis or immune cells were found in histopathology. Conclusions Barium alginate encapsulation of young market pig islets can normalize glucose regulation in diabetic rats without fibrosis or an immunologic response.  

4.1949           Quantitative assessment of cell population diversity in single-cell landscapes

Liu, Q., Herring, C.A., Sheng, Q., Ping, J., Simmons, A.J., Chen, B., Banerjee, A., Li, W., Gu, G., Coffey, R.J., Shyr, Y. and Lau, K.S. PloS Biology, 16(10), e2006687 (2018)   Single-cell RNA sequencing (scRNA-seq) has become a powerful tool for the systematic investigation of cellular diversity. As a number of computational tools have been developed to identify and visualize cell populations within a single scRNA-seq dataset, there is a need for methods to quantitatively and statistically define proportional shifts in cell population structures across datasets, such as expansion or shrinkage or emergence or disappearance of cell populations. Here we present sc-UniFrac, a framework to statistically quantify compositional diversity in cell populations between single-cell transcriptome landscapes. sc-UniFrac enables sensitive and robust quantification in simulated and experimental datasets in terms of both population identity and quantity. We have demonstrated the utility of sc-UniFrac in multiple applications, including assessment of biological and technical replicates, classification of tissue phenotypes and regional specification, identification and definition of altered cell infiltrates in tumorigenesis, and benchmarking batch-correction tools. sc-UniFrac provides a framework for quantifying diversity or alterations in cell populations across conditions and has broad utility for gaining insight into tissue-level perturbations at the single-cell resolution.  

4.1950           Directed evolution of excited state lifetime and brightness in FusionRed using a microfluidic sorter

Mmanna, P., Hung, S-T., Mukherjee, S., Friis, P., Simpson, D.M., Lo, M.N., Palmer, A.E. and Jimenez, R. Integr.Biol., 10, 516-526 (2018)   Green fluorescent proteins (GFP) and their blue, cyan and red counterparts offer unprecedented advantages as biological markers owing to their genetic encodability and straightforward expression in different organisms. Although significant advancements have been made towards engineering the key photo-physical properties of red fluorescent proteins (RFPs), they continue to perform sub-optimally relative to GFP variants. Advanced engineering strategies are needed for further evolution of RFPs in the pursuit of improving their photo-physics. In this report, a microfluidic sorter that discriminates members of a cell-based library based on their excited state lifetime and fluorescence intensity is used for the directed evolution of the photo-physical properties of FusionRed. In-flow measurements of the fluorescence lifetime are performed in a frequency-domain approach with sub-millisecond sampling times. Promising clones are sorted by optical force trapping with an infrared laser. Using this microfluidic sorter, mutants are generated with longer lifetimes than their precursor, FusionRed. This improvement in the excited state lifetime of the mutants leads to an increase in their fluorescence quantum yield up to 1.8-fold. In the course of evolution, we also identified one key mutation (L177M), which generated a mutant (FusionRed-M) that displayed ∼2-fold higher brightness than its precursor upon expression in mammalian (HeLa) cells. Photo-physical and mutational analyses of clones isolated at the different stages of mutagenesis reveal the photo-physical evolution towards higher in vivo brightness.  

4.1951           Targeting Endothelial Erk1/2-Akt Axis as a Regeneration Strategy to Bypass Fibrosis during Chronic Liver Injury in Mice

Lao, Y., Li, Y., Zhang, P., Shao, Q., Lin, W., Qiu, B., Lv, Y. et al Molecular Therapy, 26(12), 2779-2797 (2018)   Liver sinusoidal endothelial cells (LSECs) have great capacity for liver regeneration, and this capacity can easily switch to profibrotic phenotype, which is still poorly understood. In this study, we elucidated a potential target in LSECs for regenerative treatment that can bypass fibrosis during chronic liver injury. Proregenerative LSECs can be transformed to profibrotic phenotype after 4 weeks of carbon tetrachloride administration or 10 days of bile duct ligation. This phenotypic alternation of LSECs was mediated by extracellular regulated protein kinases 1 and 2 (Erk1/2)-Akt axis switch in LSECs during chronic liver injury; Erk1/2 was normally associated with maintenance of the LSEC proregenerative phenotype, inhibiting hepatic stellate cell (HSC) activation and promoting tissue repair by enhancing nitric oxide (NO)/reactive oxygen species (ROS) ratio and increasing expression of hepatic growth factor (HGF) and Wingless-type MMTV integration site family member 2 (Wnt2). Alternatively, Akt induced LSEC profibrotic phenotype, which mainly stimulated HSC activation and concomitant senescence by reducing NO/ROS ratio and decreasing HGF/Wnt2 expression. LSEC-targeted adenovirus or drug particle to promote Erk1/2 activity can alleviate liver fibrosis, accelerate fibrosis resolution, and enhance liver regeneration. This study demonstrated that the Erk1/2-Akt axis acted as a switch to regulate the proregenerative and profibrotic phenotypes of LSECs, and targeted therapy promoted liver regeneration while bypassing fibrosis, providing clues for a more effective treatment of liver diseases.  

4.1952           Engineering PD-1-Presenting Platelets for Cancer Immunotherapy

Zhang, X., Wang, J., Chen, Z., Hu, Q., Wang, C., Yan, J., Dotti, G., Huang, p. and Gu, Z. Nano Lett., 18, 5716-5725 82018)   Radical surgery still represents the treatment choice for several malignancies. However, local and distant tumor relapses remain the major causes of treatment failure, indicating that a postsurgery consolidation treatment is necessary. Immunotherapy with checkpoint inhibitors has elicited impressive clinical responses in several types of human malignancies and may represent the ideal consolidation treatment after surgery. Here, we genetically engineered platelets from megakaryocyte (MK) progenitor cells to express the programmed cell death protein 1 (PD-1). The PD-1 platelet and its derived microparticle could accumulate within the tumor surgical wound and revert exhausted CD8⁺ T cells, leading to the eradication of residual tumor cells. Furthermore, when a low dose of cyclophosphamide (CP) was loaded into PD-1-expressing platelets to deplete regulatory T cells (Tregs), an increased frequency of reinvigorated CD8⁺ lymphocyte cells were observed within the postsurgery tumor microenvironment, directly preventing tumor relapse.  

4.1953           Effects of Transplanted Islets Nano-Encapsulated with Hyperbranched Polyethylene Glycol and Heparin on Microenvironment Reconstruction and Glucose Control

Haque, M.R., Jeong, J-H., Lee, K.W., Shin, D.Y., Kim, G-S., Kim, S.J. and Byun, Y. Bioconjugate Chem., 29, 2945-2953 (2018)   The microenvironment of pancreatic islets gets disrupted during enzyme digestion and causes islets to remain in a vulnerable state, leading to poor outcome in the initial days of transplantation. To avoid immune invasion while allowing the reconstruction of the microenvironment of the transplanted site, we propose immunoisolation polymers, which can nanoencapsulate islets quickly without cytotoxicity. Here, nonhuman primate (NHP) islets were nanoencapsulated with hyperbranched polyethylene glycol (hb-PEG) and heparin by layer-by-layer technology and transplanted into the kidney subcapsular space of diabetic C57BL/6 mice. An immunosuppressive drug protocol was applied to increase the survival time until the animals were sacrificed. The recipients of NHP islets exhibited high nonfasting blood glucose level (BGL) for 2–3 weeks, which was normalized afterward. Immunohistochemical (IHC) analysis revealed an immature vascular basement membrane and cell surface integrins directly associated with poor initial insulin production. The transplanted grafts regained their own microenvironment within a month without any outside stimuli. No lymphocyte infiltration was observed in the grafts at any time. Humoral and cell-mediated immune responses were prominently diminished by the hb-PEG/Heparin nanoencapsulated islets. Immunoisolation accompanied by an immunosuppressive drug protocol protects islets by helping them avoid immunogenesis while at the same time allowing them to reconstruct their microenvironment.  

4.1954           KDM5 Histone Demethylase Activity Links Cellular Transcriptomic Heterogeneity to Therapeutic Resistance

Hinohara, K., Wu, H-J., Vigneua, S., Gimelbrant, A.A., Michor, F. and Polyak, K. Cancer Cell, 34, 939-953 (2018)   Members of the KDM5 histone H3 lysine 4 demethylase family are associated with therapeutic resistance, including endocrine resistance in breast cancer, but the underlying mechanism is poorly defined. Here we show that genetic deletion of KDM5A/B or inhibition of KDM5 activity increases sensitivity to anti-estrogens by modulating estrogen receptor (ER) signaling and by decreasing cellular transcriptomic heterogeneity. Higher KDM5B expression levels are associated with higher transcriptomic heterogeneity and poor prognosis in ER ⁺ breast tumors. Single-cell RNA sequencing, cellular barcoding, and mathematical modeling demonstrate that endocrine resistance is due to selection for pre-existing genetically distinct cells, while KDM5 inhibitor resistance is acquired. Our findings highlight the importance of cellular phenotypic heterogeneity in therapeutic resistance and identify KDM5A/B as key regulators of this process.  

4.1955           In vitro characterization of neonatal, juvenile, and adult porcine islet oxygen demand, β‐cell function, and transcriptomes

Smith, K.E., Purvis, W.G., Davis, M.A., Min, C.G., Cooksey, A.M. et al Xenotransplantation, 25(6), e12432 (2018)   Background There is currently a shortage of human donor pancreata which limits the broad application of islet transplantation as a treatment for type 1 diabetes. Porcine islets have demonstrated potential as an alternative source, but a study evaluating islets from different donor ages under unified protocols has yet to be conducted. Methods Neonatal porcine islets (NPI; 1‐3 days), juvenile porcine islets (JPI; 18‐21 days), and adult porcine islets (API; 2+ years) were compared in vitro, including assessments of oxygen consumption rate, membrane integrity determined by FDA/PI staining, β‐cell proliferation, dynamic glucose‐stimulated insulin secretion, and RNA sequencing. Results Oxygen consumption rate normalized to DNA was not significantly different between ages. Membrane integrity was age dependent, and API had the highest percentage of intact cells. API also had the highest glucose‐stimulated insulin secretion response during a dynamic insulin secretion assay and had 50‐fold higher total insulin content compared to NPI and JPI. NPI and JPI had similar glucose responsiveness, β‐cell percentage, and β‐cell proliferation rate. Transcriptome analysis was consistent with physiological assessments. API transcriptomes were enriched for cellular metabolic and insulin secretory pathways, while NPI exhibited higher expression of genes associated with proliferation. Conclusions The oxygen demand, membrane integrity, β‐cell function and proliferation, and transcriptomes of islets from API, JPI, and NPI provide a comprehensive physiological comparison for future studies. These assessments will inform the optimal application of each age of porcine islet to expand the availability of islet transplantation.  

4.1956           Smart Hydrogel Microfluidics for Single‐Cell Multiplexed Secretomic Analysis with High Sensitivity

Hsu, M.N., Wei, S-C., Guo, S., Phan, D-T., Zhang, Y. and Chen, C-H. Small, 14(49), 1802918 (2018)   Secreted proteins determine a range of cellular functionalities correlated with human health and disease progression. Because of cell heterogeneity, it is essential to measure low abundant protein secretions from individual cells to determine single‐cell activities. In this study, an integrated platform consisting of smart hydrogel immunosensors for the sensitive detection of single‐cell secretions is developed. A single cell and smart hydrogel microparticles are encapsulated within a droplet. After incubation, target secreted proteins from the cell are captured in the smart hydrogel particle for immunoassay. The temperature‐induced volume phase transition of the hydrogel biosensor allows the concentration of analytes within the gel matrix to increase, enabling high‐sensitivity measurements. Distinct heterogeneity for live cell secretions is determined from 6000 cells within 1 h. This method is tested for low abundant essential secretions, such as interleukin‐6, interleukin‐8, and monocyte chemoattractant protein‐1 secretions of both suspended cells (HL60) and adherent cells (MCF7 and MDA‐MB‐231). This platform is highly flexible and can be used to simultaneously measure a wide range of clinically relevant cellular secretions; it thus represents a novel tool for precise biological assays.  

4.1957           Re‐evaluation of mouse tissue factor pathway inhibitor and comparison of mouse and human tissue factor pathway inhibitor physiology

Girard, T.J., Grunz, K., Lasky, N.M., Malone, J.P. and Broze Jr., G.J.

1. Thromb. Hemostasis, 16, 2246-2257 (2018)

  Background Mouse models can provide insight into the pathophysiology of human thrombosis and hemostasis. Tissue factor pathway inhibitor (TFPI) regulates coagulation through protein S (PS)‐enhanced factor (F) Xa inhibition and FXa‐dependent inhibition of FVIIa/tissue factor (TF) activity. TFPI is expressed as isoforms α and β in man, and α, β and γ in the mouse. Objective Assess the reliability of extending TFPI‐related studies in mice to humans. Method Compare mouse and human TFPI physiology using a variety of methods. Results Mouse TFPI and human TFPI are similar in regard to: (i) the mechanisms for FVIIa/TF and FXa inhibition; (ii) TFPIα is a soluble form and TFPIβ is glycosyl phosphatidyl inositol (GPI) membrane anchored; (iii) the predominant circulating form of TFPI in plasma is lipoprotein‐associated; (iv) low levels of TFPIα circulate in plasma and increase following heparin treatment; and (v) TFPIα is the isoform in platelets. They differ in that: (i) mouse TFPI circulates at a ~20‐fold higher concentration; (ii) mouse lines with isolated isoform deletions show this circulating mouse TFPI is derived from TFPIγ; (iii) sequences homologous to the mouse TFPIγ exon are present in many species, including man, but in primates are unfavorable for splicing; and (iv) tandem mass spectrometry (MS/MS) detects sequences for TFPI isoforms α and β in human plasma and α and γ in mouse plasma. Conclusion To dissect the pathophysiological roles of human TFPIα and TFPIβ, studies in TFPIγ null mice, expressing only α and β, only α or only β should better reflect the human situation.  

4.1958           Comprehensive analysis of serum microRNAs in hepatic sinusoidal obstruction syndrome (SOS) in rats: implication as early phase biomarkers for SOS

Takeuchi, M., Oda, S., Tsuneyama, K. and Yokoi, T. Arch. Toxicol., 92(9), 2947-2962 (2018)   Sinusoidal obstruction syndrome (SOS) is a liver injury caused by clinical chemotherapy, of which pathogenesis is associated with the damage in liver sinusoidal endothelial cells (LSEC). The unavailability of appropriate specific biomarkers for the early diagnosis of SOS may potentially overlook SOS patients. In this study, we sought to find serum microRNAs (miRNAs) as non-invasive biomarkers for investigating SOS in rats. Male Sprague–Dawley rats were orally administered monocrotaline, and then, their livers and sera were collected after 0.25, 0.5, 1, 2, 4, and 7 days. The rats showed a typical SOS phenotype including LSEC damage as early as day 0.25, followed by severe hepatocyte damage on day 2, and developed hepatic fibrosis from days 4 to 7. The miRNA microarray showed that 65 serum miRNAs were increased in their levels on day 0.25, when LSEC damage was observed, while hepatocyte damage was absent. Among the increased serum miRNAs on days 0.25–1, miR-511-3p was enriched in normal LSECs and miR-21-5p was in both LSECs and hepatocytes, suggesting that they were released into blood from the damaged LSECs. The miR-122-5p, miR-192-5p, and miR-101b-3p, which were enriched in hepatocytes, reached the highest levels in serum on day 2, suggesting their utility as indicators for hepatocyte damage. No miRNA showing an increasing trend from days 4 to 7 was found as a biomarker for fibrosis. In conclusion, we found that LSEC-derived miR-21-5p and especially miR-511-3p in serum would serve as early phase biomarkers for SOS in response to LSEC damage.  

4.1959           In-droplet microparticle washing and enrichment using surface acoustic wave-driven acoustic radiation force

Sung, S.H.J. ete al Lab Chip, 18, 2936-2945 (2018)   Washing and enrichment of particles and cells are crucial sample preparation procedures in biomedical and biochemical assays. On-chip in-droplet microparticle washing and enrichment have been pursued but remained problematic due to technical difficulties, especially simultaneous and precise control over the droplet interface and in-droplet samples. Here, we have achieved a breakthrough in label-free, continuous, on-demand, in-droplet microparticle washing and enrichment using surface acoustic waves. When exposed to the acoustic field, the droplet and suspended particles experience acoustic radiation force arising from inhomogeneous wave scattering at the liquid/liquid and liquid/solid interfaces. Based on these acoustophoretic phenomena, we have demonstrated in-droplet microparticle washing and enrichment in an acoustofluidic device. We expect that the proposed acoustic method will offer new perspectives to sample washing and enrichment by performing the operation in microscale droplets.  

4.1960           Microfluidic bead encapsulation above 20 kHz with triggered drop formation

Clark, C. and Abate, A.R: Lab. Chip, 18, 3598-3605 (2018)   Microsphere beads are functionalized with oligonucleotides, antibodies, and other moieties to enable specific detection of analytes. Droplet microfluidics leverages this for single-molecule or -cell analysis by pairing beads and targets in water-in-oil droplets. Pairing is achieved with devices operating in the dripping regime, limiting throughput. Here, we describe a pairing method that uses beads to trigger the breakup of a jet into monodispersed droplets. We use the method to pair 10⁵ Human T cells with polyacrylamide beads ten times faster than methods operating in the dripping regime. Our method improves the throughput of bead-based droplet workflows, enabling analysis of large populations and the detection of rare events.  

4.1961           An optofluidic system with integrated microlens arrays for parallel imaging flow cytometry

Holzner, G., Du, Y., Cao, X., Choo, J., deMello, A.J. and Stavrakis, S. Lab. Chip, 18, 3631-3637 (2018)   In recent years, high-speed imaging has become increasingly effective for the rapid analysis of single cells in flowing environments. Single cell imaging methods typically incorporate a minimum magnification of 10× when extracting sizing and morphological information. Although information content may be significantly enhanced by increasing magnification, this is accompanied by a corresponding reduction in field of view, and thus a decrease in the number of cells assayed per unit time. Accordingly, the acquisition of high resolution data from wide field views remains an unsolved challenge. To address this issue, we present an optofluidic flow cytometer integrating a refractive, microlens array (MLA) for imaging cells at high linear velocities, whilst maximizing the number of cells per field of view. To achieve this, we adopt an elasto-inertial approach for cell focusing within an array of parallel microfluidic channels, each equipped with a microlens. We characterize the optical performance of the microlenses in terms of image formation, magnification and resolution using both ray-tracing simulations and experimental measurements. Results demonstrate that the optofluidic platform can efficiently count and magnify micron-sized objects up to 4 times. Finally, we demonstrate the capabilities of the platform as an imaging flow cyclometer, demonstrating the efficient discrimination of hB and Jurkat cells at throughputs up to 50 000 cells per second.  

4.1962           Functional TCR T cell screening using single-cell droplet microfluidics

Segaliny, A.I., Li, G., Kong, l., Ren, C., Chen, X., Wang, J.K., Baltimore, D., Wu, G. and Zhao, W. Lab Chip, 18, 3733-3749 (2018)   Adoptive T cell transfer, in particular TCR T cell therapy, holds great promise for cancer immunotherapy with encouraging clinical results. However, finding the right TCR T cell clone is a tedious, time-consuming, and costly process. Thus, there is a critical need for single cell technologies to conduct fast and multiplexed functional analyses followed by recovery of the clone of interest. Here, we use droplet microfluidics for functional screening and real-time monitoring of single TCR T cell activation upon recognition of target tumor cells. Notably, our platform includes a tracking system for each clone as well as a sorting procedure with 100% specificity validated by downstream single cell reverse-transcription PCR and sequencing of TCR chains. Our TCR screening prototype will facilitate immunotherapeutic screening and development of T cell therapies.  

4.1963           Acoustic impedance-based size-independent isolation of circulating tumour cells from blood using acoustophoresis

Karthick, S., Pradeep, P.N., Kanchana, P. and Sen, A.K: Lab Chip, 18, 3802-3813 82018)   Label-free isolation of CTCs from blood is critical for the development of diagnostic and prognostic tools for cancer. Here, we report a label-free method based on acoustic impedance contrast for the isolation of CTCs from peripheral blood mononuclear cells (PBMCs) in a microchannel using acoustophoresis. We describe a method in which the acoustophoretic migration of PBMCs is arrested by matching their acoustic impedance with that of the sample medium, and CTCs that have different acoustic impedance compared to PBMCs migrate toward the pressure node or antinode and thus become isolated. We show that acoustic streaming which can adversely affect the CTC isolation is suppressed owing to the inhomogeneous liquid flow configuration. We establish a method for isolation of CTCs that have higher or lower acoustic impedance compared to PBMCs by controlling the acoustic impedance contrast of the liquids across the channel. Applying this method, we demonstrate label-free isolation of HeLa and MDA-MB-231 cells from PBMCs (collected from 2.0 mL of blood) within one hour yielding a recovery of >86% and >50-fold enrichment. Combined impedance and size-based sorting is proposed as a promising tool for the effective isolation of CTCs from blood.  

4.1964           Electrospun nanofibers facilitate better alignment, differentiation, and long-term culture in an in vitro model of the neuromuscular junction (NMJ)

Luo, B., Tian, L., Chen, N., Ramakrisna, S., Thakor, N. and Yang, I.H. Biomater Sci., 6, 3262-3272 (2018)   The neuromuscular junction (NMJ) is a specialized synapse between motor neurons and the muscle fibers they innervate. Due to the complexity of various signalling molecules and pathways, in vivo NMJs are difficult to study. Therefore, in vitro motor neuron–muscle co-culture plays a pivotal role in studying the mechanisms of NMJ formation associated with neurodegenerative diseases. There is a growing need to develop novel methodologies that can be used to develop long-term cultures of NMJs. To date, there have been few studies on NMJ development and long-term maintenance of the system, which is also the main challenge for the current in vitro models of NMJs. In this study, we demonstrate a long-term co-culture system of primary embryonic motor neurons from Sprague-Dawley rats and C2C12 cells on both random and aligned electrospun polylactic acid (PLA) nanofibrous scaffolds. This is the first study to explore the role of electrospun nanofibers in the long-term maintenance of NMJs. PLA nanofibrous scaffolds provide better contact guidance for C2C12 cells aligning along the fibers, thus guiding myotube formation. We can only maintain the co-culture system on a conventional glass substrate for 2 weeks, whilst 55% and 70% of the cells still survived on random and aligned PLA substrates after 7 weeks. Our nanofiber-based long-term co-culture system is used as an important tool for the fundamental research of NMJs.  

4.1965           Droplet microfluidics in thermoplastics: device fabrication, droplet generation, and content manipulation using integrated electric and magnetic fields

Sahone, V., Doonan, S.R. and Bailey, R.C: Anal. Methods, 10, 4264-4274 (2018)   We have developed droplet microfluidic devices in thermoplastics and demonstrated the integration of key functional components that not only facilitate droplet generation, but also include electric field-assisted reagent injection, droplet splitting, and magnetic field-assisted bead extraction. We manufactured devices in poly(methyl methacrylate) and cyclic olefin polymer using a hot-embossing procedure employing silicon masters fabricated via photolithography and deep reactive ion etching techniques. Device characterization showed robust fabrication with uniform feature transfer and good embossing yield. Channel modification with heptadecafluoro-1,1,2,2-tetrahydrodecyltrichlorosilane increased device hydrophobicity, allowing stable generation of 330 pL aqueous droplets using T-junction configuration. Picoinjector and K-channel motifs were also both successfully integrated into the thermoplastic devices, allowing for robust control over electric field-assisted reagent injection, as well as droplet splitting with the K-channel. A magnetic field was also introduced to the K-channel geometry to allow for selective concentration of magnetic beads while decanting waste volume through droplet splitting. To show the ability to link multiple, modular features in a single thermoplastic device, we integrated droplet generation, reagent injection, and magnetic field-assisted droplet splitting on a single device, realizing a magnetic bead washing scheme to selectively exchange the fluid composition around the magnetic particles, analogous to the washing steps in many common biochemical assays. Finally, integrated devices were used to perform a proof-of-concept in-droplet β-galactosidase enzymatic assay combining enzyme-magnetic bead containing droplet generation, resorufin-β-D-galactopyranoside substrate injection, enzyme–substrate reaction, and enzyme-magnetic bead washing. By integrating multiple droplet operations and actuation forces we have demonstrated the potential of thermoplastic droplet microfluidic devices for complex (bio)chemical analysis, and we envision a path toward mass fabrication of droplet microfluidic devices for a range of (bio)chemical applications.  

4.1966           Isolation of Conventional Murine Lung Dendritic Cell Subsets

Bosteels, C., Lambrecht, B.N. and Hammad, H. Current Protocols in Immunology, 120(1), 3.7B.1-3.7B.16 (2018)   The lungs are continuously exposed to environmental threats, requiring an adequate and stringent immune response of a heterogeneous set of effector cells. Dendritic cells (DCs) form a dense network in the respiratory mucosa and act as the central regulators of the different components of this response, both sensing the nature of the threats and precisely coordinating the effector mechanisms best suited for overcoming it. The DCs are classically subdivided in two main groups, plasmacytoid DCs (pDCs) and conventional DCs (cDCs), the latter being further subdivided into cDC1s and cDC2s based on ontogeny and their distinct non‐redundant functions. This protocol provides different enrichment methods and represents an up‐to‐date, universal framework that uses a minimal set of highly specific lineage markers to discriminate and sort pure cDC subsets from the murine lung but also across tissues and species which is an added value in intra‐ and interspecies comparative research.  

4.1967           Comparison of Tissue Loading Before and After the Creation of a Continuous Density Gradient in Porcine Islet Purification

Miyagi-Shiohira, C., Nakashima, Y., Ebi, N., Hamada, E., Tamaki, Y., Kuwae, K., Kobayashi, N., Saitoh, I., Watanabe, M., Konjo, T. and Noguchi, H. Cell Med., 10, xxxx-xxxx (2018)   The purification step is one of the most important and difficult procedures in islet isolation for pancreatic islet transplantation. We previously reported that a purification method using large plastic bottles effectively achieved a high yield of islets from the porcine pancreas. In this study, we evaluated the impact of the timing of tissue loading on porcine islet purification using large plastic bottles. One method involved loading digested tissue after creating a continuous density gradient (tissue after gradient [TAG]). The other method involved loading digested tissue before creating a continuous density gradient (tissue before gradient [TBG]). There were no significant differences between TAG and TBG in terms of the islet yield, rates of viability and purity, score, and in the stimulation index after purification. Furthermore, there were no marked differences in the attainability or suitability of post-transplantation normoglycemia. Our study shows the equivalency of these two methods of islet purification.  

4.1968           A Comparison of Pancreatic Islet Purification using Iodixanol with University of Wisconsin Solution and with Na-Lactobionate and Histidine Solution

Hakashima, Y., Miyagi-Shiohira, C., Ebi, N., Harmada, E., Tamaki, Y., Kuwae, K., Kobayashi, N., Saitoh, I., Watanabe, M., Kinjo, T. and Noguchi, H. Cell Med., 10, xxx-xxx (2018)   Purification of pancreatic islets is an important step in islet isolation for islet transplantation. In this study, to investigate how a solution composed mainly of Na-lactobionate and histidine (HL) influences the purification of islets, iodixanol was added to a purified solution for porcine islet isolation. A solution (IU) made by adding iodixanol to University of Wisconsin solution and a solution (IHL) made by adding iodixanol to HL solution were used to evaluate the islet isolation performance. We noted no significant differences between the two purification methods with regard to the islet yield, survival rate or purity, score, or stimulation index. These results show that IHL solution is as useful as IU solution for islet purification.  

4.1969           Evaluation of Islet Purification Methods for Making a Continuous Density Gradient and Loading Tissue

Ebi, N., Miyagi-Shiohira, C., Harmada, E., Tamaki, Y., Masamoto, M., Makishi, E., Nakashima, Y., Kobayasha, N., Saitoh, I., Watanabe, M., Noguchi, Y., Kinjo, T and Noguchi, H. Cell Med., 10, xxx-xxx (2018)   Islet purification is one of the most important steps of islet isolation for pancreatic islet transplantation. We previously reported that a purification method using large plastic bottles effectively achieved a high yield of islets from porcine pancreas. In this study, we evaluated the methods for making a continuous density gradient and loading tissue. One method involved loading digested tissue on top of a continuous density gradient (top loading). The other method involved mixing digested tissue with low-density solution and then making a continuous gradient (mixed loading). There were no significant differences between the 2 purification methods in terms of the islet yield, rate of viability or purity, score, or in the stimulation index after purification. Furthermore, there were no marked differences in the attainability or suitability of posttransplantation normoglycemia. Our study shows the equivalency of these 2 methods of islet purification.    

4.1970           Preservation of Gametes and Embryos

Arav, A. and Saragusty, J. Animal Biotechnology 1. Springer, Cham, 235-267 (2018)   Cryopreservation is the practical implementation of the scientific field of cryobiology. It was developed particularly over the last two centuries, having major milestones in the field of animal reproduction. Technologies such as directional freezing of sperm and sperm desiccation, as well as oocyte and embryo freezing and vitrification, are discussed and described in this chapter. Hereinafter, we describe the major breakthroughs of the past two centuries and our foresight for the near future.  

4.1971           Human Somatic Stem Cell Neural Differentiation Potential

Eve, K D.J., Sanberg, P.R., Buzanska, L., Sarnowska, A. and Domanska-Janik, K. Human Neural Stem Cells. Results and Problems in Cell Differentiation, Springer, Cham, 66, 21-87 (2018)   Human somatic stem cells can be identified and isolated from different types of tissues and are grouped here based on their developmental maturation and ability to undergo neural differentiation. The first group will represent afterbirth somatic tissues, which are perinatal stem cells including placental blood and tissue, amniotic fluid and tissue, and umbilical cord blood- and umbilical cord tissue-derived cells. The second group of cells discussed in this chapter is the adult stem cells, generally those in a transient period of development, thus placing them in the special position of transitioning from the perinatal to young somatic tissue, and they include the menstrual blood-, the peripheral blood-, and the bone marrow-derived stem cells.  

4.1972           Preparation of Induced Pluripotent Stem Cells Using Human Peripheral Blood Monocytes

Isogai, S., Yamamoto, N., Hiramatsu, N., Goto, Y., Hayashi, M., Kondo, M. and Imaizumi, K. Cellular Programming, 20(6), 347-355 (2018)   Since induced pluripotent stem (iPS) cells have been established, in recent years, clinical transplantation of cells differentiated from iPS cells derived from human skin fibroblasts is been in progress. On the contrary, monocytes have complete genome information without damage and gene recombination, they are contained in the peripheral blood by ∼3%–8% and differentiate into dendritic cells that are the type of control tower for immune cells. However, generation of monocyte-derived iPS cells has only been successful when special persistent Sendai virus vectors have been used. Therefore, in this study, as a preculture method for monocytes, a culture method for maintaining activity without using any cytokine was established, and using a commercially available vector without genetic toxicity without damaging the chromosome of the cell, iPS cells derived from monocytes were successfully produced. This cell has the ability to differentiate into three germ layers, and when compared with commercially available iPS cells, there was no significant difference between self-renewal and gene expression in the three germ layers. In future, we will compare the differentiation induction of monocyte-derived iPS cells with dendritic cells and investigate the production of dendritic cells that can cope with various antigens.  

4.1973           Immune Response to Extracellular Vesicles from Human Islets of Langerhans in Patients with Type 1 Diabetes

Rutman, A.K., Negi, S., Gasparrini, M., Hasilo, C.P., Tchervenkov, J. and Paraskevas, S. Endocrinology, 159(11), 3834-3847 (2018)   The autoimmune response that characterizes type 1 diabetes (T1D) has no clear cause. Extracellular vesicles (EVs) play an important role in triggering the immune response in other contexts. Here, we propose a model by which EVs isolated from human islets stimulate proinflammatory immune responses and lead to peripheral blood mononuclear cell (PBMC) activation. We show that human islet EVs are internalized by monocytes and B cells and lead to an increase in T-helper 1, 2, and 17 cytokine expression, as well as T and B cell proliferation. Importantly, we demonstrate memory T and B cell activation by EVs selectively in PBMCs of patients with T1D. Additionally, human islet EVs induce an increase in antibodies against glutamic acid decarboxylase 65 (GAD65) in T1D PBMCs. Furthermore, pretreatment of T1D PBMCs with ibrutinib, an inhibitor of Bruton tyrosine kinase, dampens EV-induced memory B cell activation and GAD65 antibody production. Collectively, our findings indicate a role for human islet EVs in mediating activation of B and T cells and GAD65 autoantibody production.  

4.1974           TDP-43 levels are higher in platelets from patients with sporadic amyotrophic lateral sclerosis than in healthy controls

Hishizawa, M., Yamashita, H., Akizuki, M., Urushitani, M. and Takahashi, R. Neurochem. Int., 224, 41.45 (2019)   TAR DNA-binding protein 43 (TDP-43) is a major pathological protein of ubiquitinated inclusions in motor neurons of sporadic amyotrophic lateral sclerosis (ALS). TDP-43 is ubiquitously expressed and the majority of TDP-43 is normally localized to the nucleus. In motor neurons of patients with ALS, TDP-43 is not localized in the nucleus, relocates to the cytoplasm, and accumulates as cytoplasmic inclusions. Based on recent reports that TDP-43 is increased in the cytoplasmic fraction of peripheral blood mononuclear cells in sporadic ALS, and several studies on platelet dysfunction in ALS patients, we investigated the TDP-43 levels in platelets from patients with sporadic ALS. We measured TDP-43 levels with a sandwich enzyme-linked immunosorbent assay in platelets separated from whole blood, and compared the TDP-43 level in platelets from sporadic ALS (n = 19) patients with platelets from non-ALS controls (n = 21). The TDP-43 concentration in platelets was significantly higher in patients with ALS compared to age-matched controls. According to sub-analysis, the TDP-43 concentration in platelets tended to increase in ALS patients with longer disease duration, as well as with lower score on the ALS Functional Rating Scale Revised (ALSFRS-R), though the differences were not statistically significant. These results suggest that ALS also affects platelets in addition to motor neurons.  

4.1975           Investigation of mitochondrial calcium uniporter role in embryonic and adult motor neurons from G93AhSOD1 mice

Tadic, V., Adam, A., Goldhammer, N., Lautenschlaeger, J., Oberstadt, M., Malci, A., Le, T.T.L., Sengupta, S., Stubendorff, B., Keiner, S., Witte, O.W: and Grosskreutz, J. Neurobiology of Aging, 75, 209-222 (2019)   Amyotrophic lateral sclerosis is characterized by progressive death of motor neurons (MNs) with glutamate excitotoxicity and mitochondrial Ca²⁺ overload as critical mechanisms in disease pathophysiology. We used MNs from G93A^hSOD1 and nontransgenic embryonic cultures and adult mice to analyze the expression of the main mitochondrial calcium uniporter (MCU). MCU was overexpressed in cultured embryonic G93A^hSOD1 MNs compared to nontransgenic MNs but downregulated in MNs from adult G93A^hSOD1 mice. Furthermore, cultured embryonic G93A^hSOD1 were rescued from kainate-induced excitotoxicity by the Ca²⁺/calmodulin-dependent protein kinase type II inhibitor; KN-62, which reduced MCU expression in G93A^hSOD1 MNs. MCU activation via kaempferol neither altered MCU expression nor influenced MN survival. However, its acute application served as a fine tool to study spontaneous Ca²⁺ activity in cultured neurons which was significantly altered by the mutated hSOD1. Pharmacological manipulation of MCU expression might open new possibilities to fight excitotoxic damage in amyotrophic lateral sclerosis.  

4.1976           Different aspects of platelet evaluation in dengue: Measurement of circulating mediators, ability to interact with the virus, the degree of activation and quantification of intraplatelet protein content

Da Costa Barros, T., de Oliveira Batista, D., de Carvalho, A.T., da Costa Faria, N.R. et al Virus Res., 260, 163-172 (2019)   Platelets play a role in hemostasis, coagulation, angiogenesis, inflammation and immune response is one of the most affected cells in dengue. Here we describe some aspects of platelets by observing their specific circulating mediators, the ability to interact with the virus and morphological consequences of this interaction, activation markers and intraplatelet protein contents in dengue. We conducted this study using dengue-patients as well as healthy donors. Immunoenzymatic assay, flow cytometry, transmission electron microscopy and intraplatelet proteins expression assays were carried out. Briefly, we found an increase in sCD62L, NO or TBX2 ratio in platelet count, mostly in patients with the worse clinical outcome. After in vitro DENV infection or during natural infection, platelets underwent morphological alteration with increased expression of platelet activation markers, particularly in natural infections. Analysis of intraplatelet protein contents revealed different angiogenic and inflammatory profiles, maintaining or not extracellular matrix integrity between DF and DFWS patients. Thus, platelets are frequently affected by dengue, either by altering their own functionality, as "carrier" of the virus, or as an antiviral and mediator-secreting effector cell. Thus, strategies aimed at recovering platelet amounts in dengue seem to be essential for a better clinical outcome of the patients.  

4.1977           Chapter 56 - Islet Cell Transplantation

Emamaullee, J.A., Pepper, A. and Shapiro, A.M.J. Principle of Regenerative Med., third Ed., 987-1007 (2019)   The Edmonton Protocol for clinical islet transplantation offers hope for patients with diabetes that a life without insulin injections is possible. More than 1000 patients with type 1 diabetes have been transplanted successfully worldwide since 1999. However, islet transplantation is reserved only for those with complications of recalcitrant glycemic lability or hypoglycemic unawareness. Although up to 80% of recipients may achieve insulin independence for the first year after transplant, many have to return to small amounts of insulin over time. Many facets of islet transplantation are being investigated, including strategies to expand the donor pool, optimize the implantation site, and develop new sources of β cells, including stem cell–derived therapies, and strategic immunomodulation. This chapter describes the current state of clinical and experimental islet transplantation and explores areas of ongoing research to expand the availability of this procedure.  

4.1978           Chapter 10 - Understanding GPCR dimerization

Faron-Gorecka, A., Sziachta, M., Kolasa, M., Solich, J., Gorecki, A., Kusmider, M., Zurawek, D. and Dziedzicka-Wasylewska, M. Methods in Cell Biol., 149, 155-178 (2019)   Initially G protein-coupled receptors, GPCRs, were thought to act as monomers, but recently strong evidence has been gathered indicating that they are capable of forming homo- and heterodimers or higher order oligomeric complexes, and that the dimerization phenomenon can modulate the pharmacological response and function of these receptors. In this chapter we point to the great potential of alternative therapeutic approach targeted at GPCR dimers, which is especially important in the field of neuropsychopharmacology. We also included a brief description of methods used for studying the phenomenon of GPCR oligomerization, with particular attention paid to the proximity ligation assay, PLA, the procedure which allows the study of interactions between receptors not only in vitro but also in vivo, with good anatomical resolution, what is especially important in the studies of various GPCRs involved in central neurotransmission.  

4.1979           Effect of antioxidant supplements on lipid peroxidation levels in primary cortical neuron cultures

Foret, M.K., Do Carmo, S., Lincoln, R., Greene, L.E., Zhang, W., Cuello, A.C. and Cosa, G. Free Radical Biol. Med., 130, 471-477 (2019)   Oxidative stress, specifically lipid peroxidation, is a major driving force in neurodegenerative processes. However, the exact role of lipid peroxidation remains elusive as reliable real-time detection and quantification of lipid peroxyl radicals proves to be challenging in vitro and in vivo. Motivated by this methodological limitation, we have optimized conditions for real-time imaging and quantification of lipid peroxyl radical generation in primary neuron cultures using the lipophilic fluorogenic antioxidant H₄BPMHC (8-((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-methyl)-1,5-di(3-chloropropyl)-pyrromethene fluoroborate), an α-tocopherol analog probe. By subjecting neurons to different antioxidant conditions in the presence and absence of lipid peroxidation inducing stressors (Haber-Weiss reagents), we maximized H₄BPMHC sensitivity and confirmed its potential to temporally resolve subtle and marked differences in lipid peroxidation levels in real-time. Herein we report imaging and quantification of homeostatic and induced lipid peroxidation in primary neuron cultures, supporting the use of this probe for investigating healthy and diseased states. Overall these results provide the necessary foundation and impetus towards using H₄BPMHC for elucidating and mapping lipid peroxyl radical contributions to ROS-associated pathological processes in neurons.  

4.1980           The phagocytic state of brain myeloid cells after ischemia revealed by superresolution structured illumination microscopy

Fumagalli, S., Fiordaliso, F., Perego, C., Corbelli, A., Mariani, A., De Paola, M. and De Simoni, M-G.

1. Neuroinflammation, 16:9 (2019)

  Background Phagocytosis is a key function of myeloid cells and is highly involved in brain ischemic injury. It has been scarcely studied in vivo, thus preventing a deep knowledge of the processes occurring in the ischemic environment. Structured illumination microscopy (SIM) is a superresolution technique which helps study phagocytosis, a process involving the recruitment of vesicles sized below the resolution limits of standard confocal microscopy. Methods Mice underwent permanent occlusion of the middle cerebral artery and were sacrificed at 48 h or 7 days after insult. Immunofluorescence for CD11b, myeloid cell membrane marker, and CD68, lysosomal marker was done in the ischemic area. Images were acquired using a SIM system and verified with SIM check. Lysosomal distribution was measured in the ischemic area by the gray level co-occurrence matrix (GLCM). SIM dataset was compared with transmission electron microscopy images of macrophages in the ischemic tissue at the same time points. Cultured microglia were stimulated with LPS to uptake 100 nm fluorescent beads and imaged by time-lapse SIM. GLCM was used to analyze bead distribution over the cytoplasm. Results SIM images reached a resolution of 130 nm and passed the quality control diagnose, ruling out possible artifacts. After ischemia, GLCM applied to the CD68 images showed that myeloid cells at 48 h had higher angular second moment (ASM), inverse difference moment (IDM), and lower entropy than myeloid cells at 7 days indicating higher lysosomal clustering at 48 h. At this time point, lysosomal clustering was proximal (< 700 nm) to the cell membrane indicating active target internalization, while at 7 days, it was perinuclear, consistent with final stages of phagocytosis or autophagy. Electron microscopy images indicated a similar pattern of lysosomal distribution thus validating the SIM dataset. GLCM on time-lapse SIM from phagocytic microglia cultures revealed a temporal decrease in ASM and IDM and increase in entropy, as beads were uptaken, indicating that GLCM informs on the progression of phagocytosis. Conclusions GLCM analysis on SIM dataset quantitatively described different phases of macrophage phagocytic behavior revealing the dynamics of lysosomal movements in the ischemic brain indicating initial active internalization vs. final digestion/autophagy.  

4.1981           Tonsil conventional dendritic cells are not infected by porcine reproductive and respiratory syndrome virus

Puebla-Clark, L., Parra-Sanchez, H., Resendiz, M., Valenzuela, O. and Hernandez, J. Virology, 529, 65-72 (2019)   Porcine reproductive and respiratory syndrome virus (PRRSV) infects monocyte-derived DCs, and previous reports have shown that PRRSV does not infect conventional DCs (cDCs) in vitro, but the effects on cDCs from lymphoid tissues are unknown. This study analyzed the response and susceptibility of tonsil DEC205⁺cDCs from infected pigs. We confirmed the phenotype and lineage of bona fide tonsil cDCs with the mRNA expression of FLT3⁺ and the phenotype MHCII⁺CADM1^highDEC205⁺ (DEC205⁺cDCs). These cells were not infected by PRRSV, whereas CD163⁺ tonsil cells were infected. The numbers of tonsil cDCs and CD163⁺ cells were not affected by PRRSV, in contrast to the reduction in alveolar macrophage numbers. DEC205⁺cDCs exhibited an increase in the expression of IL-12 at 5 days postinfection, suggesting a proinflammatory response by these cells to the virus. In summary, this study confirms that, in vitro and in vivo, cDCs are not susceptible to PRRSV but can respond against it.  

4.1982           Cellular heterogeneity identified by single-cell alkaline phosphatase (ALP) via a SERRS-microfluidic droplet platform

Sun, D., Cao, F., Cong, L., Xu, W., Chen, Q., Shi, W. and Xu, S. Lab Chip, 19(2), 335-342 (2019)   Alkaline phosphatase (ALP) is a useful indicator for disease state diagnosis and clinical outcome. Investigation of ALP expression among cells is still challenging since ALP expression in a single cell is too low to be detectable. In our work, an ultrasensitive, high-throughput analytical method was applied for ALP determination in a single cell by using a surface-enhanced resonance Raman scattering (SERRS)-based microfluidic droplet technique. An ALP catalyzed substrate (5-bromo-4-chloro-3-indolyl phosphate, BCIP) was used to evaluate ALP activity in the cell within one droplet. When BCIP was incubated with cells, ALP can catalyze a hydrolysis reaction of colorless BCIP and oxidize the intermediate compound to form blue 5,5′-dibromo-4,4′-dichloro-1H,1H-[2,2′]biindolylidene-3,3′-dione (BCI), which is a resonant Raman-active species. The encapsulation of BCI in droplets is favorable for detecting extremely low levels of molecules due to an accumulation effect along with reaction time. The ALP concentration as low as 1.0 × 10⁻¹⁵ M can be successfully detected in a uniform droplet. In addition, cellular heterogeneity profiled by ALP expression on single-cell resolution was monitored with this SERRS-based microfluidic droplet technique. Ultrasensitive determination of ALP secreted from individual cells can help us to understand cell-to-cell heterogeneity.  

4.1983           Ultrasensitive and Simultaneous Detection of Two Cytokines Secreted by Single Cell in Microfluidic Droplets via Magnetic-Field Amplified SERS

Sun, D., Cao, F., Xu, W., Chen, Q., Shi, W. and Xu, S. Anal. Chem., 91(3), 2251-2258 (2019)   A surface-enhanced Raman scattering (SERS)-microfluidic droplet platform for the rapid, ultrasensitive and simultaneous detection of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) secreted by a single cell is presented. The high-throughput water-in-oil droplets containing an individual cell along with four kinds of immune-particles (antibody-conjugated silver nanoparticles or magnetic beads, AgNPs@Ab₁ and MNs@Ab₂) in each were achieved by a cross-typed microfluidic chip, and then they were captured by a collection channel array for SERS measurements. In the appearance of cytokines secreted by one cell, AgNPs@Ab₁ can be linked onto the surface of MNs@Ab₂ through the immune-recognition to form an immune-sandwich, which makes the “turn on” SERS signal of the Raman reporters previously laid on the surface of MNs due to the adjacent AgNPs. Furthermore, the second SERS signal amplification is from the magnetic field-induced spontaneous collection effect, which brings 75 times enhancement for SERS signal. Additionally, the encapsulation of cytokines in an isolated droplet permits an accumulation effect of targets with time. Owing to the dual signal enhancement and the accumulation effect, such few cytokines secreted by a single cell become detectable and a limit of detection is achieved as 1.0 fg/mL in one droplet. By using this ultrasensitive SERS-microdroplet method, the VEGF and IL-8 secretions from several cells in one droplet were explored and the data show that the cell–cell interaction may promote angiogenesis of cancer cells through the up-regulation of VEGF and IL-8.  

4.1984           Ginkgolide A Prevents the Amyloid-β-Induced Depolarization of Cortical Neurons

Kuo, L-C., Song, Y-Q., Yao, C-A., Cheng, I.H., Chien, C-T., Lee, G-C., Yang, W-C. and Lin, Y.

1. Agric. Food Chem., 67(1), 81-89 (2019)

  Utilizing the N-methyl-d-aspartate (NMDA) receptor antagonist as a strategy, memantine is the only agent available for clinically treating mild to severe Alzheimer’s disease (AD). Our aim was to develop novel similar herb-based drugs. Using a screening platform, ginkgolide A (GA), a pure compound extracted from Ginkgo biloba, was found to attenuate amyloid β (Aβ)-induced abnormal depolarization in mouse primary cortical neurons. Using receptor agonists, it was determined that GA inhibits both NMDA receptors and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. Furthermore, the Aβ-induced increase in c-Jun N-terminal kinase phosphorylation in neurons was prevented by GA. Body weight, glutamate oxaloacetate transaminase, glutamic–pyruvic transaminase, liver histology, and kidney histology were similar when the wild-type/AD animal model mice with and without GA treatment were compared. This pure compound improves the memory of wild-type mice. Our findings indicate that GA has great potential clinically for the treatment of AD because it might target NMDA receptors just like memantine.  

4.1985           Methods for collection, handling, and analysis of sea urchin coelomocytes

Smith, L.C., Hawley, T.S., Henson, J.H., Majeske, A.J., Oren, M. and Rosental, B. Methods in Cell Biol., 150, 357-388 (2019)  

Sea urchin coelomocytes can be collected in large numbers from adult sea urchins of the species, Strongylocentrotus purpuratus, which typically has 12–40 mL of coelomic fluid. Coelomocytes are used for analysis of immune reactions and immune gene expression in addition to basic functions of cells, in particular for understanding structure and modifications of the cytoskeleton in phagocytes. The methods described here include coelomocyte isolation, blocking the clotting reaction, establishing and maintaining primary cultures, separation of different types of coelomocytes into fractions, processing live coelomocytes for light microscopy, fixation and staining for light and electron microscopy, analysis of coelomocyte populations by flow cytometry, and sorting single cells for more detailed follow-up analyses including transcriptomics or genomic characteristics. These methods are provided to make working with coelomocytes accessible to researchers who are unfamiliar with these cells and perhaps to aid others who have worked extensively with invertebrate cells.

 

4.1986           Intrahepatic Delivery of Pegylated Catalase Is Protective in a Rat Ischemia/Reperfusion Injury Model

Akateh, C., Beal, E.W., Kim, J-L., Reader, B.F., Maynard, K., Zweier, J.L., Whitson, B.A. and Black, S.M.

1. Surg. Res., 238, 152-163 (2019)

  Background Ischemia/reperfusion injury (IRI) can occur during liver surgery. Endogenous catalase is important to cellular antioxidant defenses and is critical to IRI prevention. Pegylation of catalase (PEG-CAT) improves its therapeutic potential by extending plasma half-life, but systemic administration of exogenous PEG-CAT has been only mildly therapeutic for hepatic IRI. Here, we investigated the protective effects of direct intrahepatic delivery of PEG-CAT during IRI using a rat hilar clamp model. Materials and methods PEG-CAT was tested in vitro and in vivo. In vitro, enriched rat liver cell populations were subjected to oxidative stress injury (H₂O₂), and measures of cell health and viability were assessed. In vivo, rats underwent segmental (70%) hepatic warm ischemia for 1 h, followed by 6 h of reperfusion, and plasma alanine aminotransferase and aspartate aminotransferase, tissue malondialdehyde, adenosine triphosphate, and GSH, and histology were assessed. Results In vitro, PEG-CAT pretreatment of liver cells showed substantial uptake and protection against oxidative stress injury. In vivo, direct intrahepatic, but not systemic, delivery of PEG-CAT during IRI significantly reduced alanine aminotransferase and aspartate aminotransferase in a time-dependent manner (P < 0.01, P < 0.0001, respectively, for all time points) compared to control. Similarly, tissue malondialdehyde (P = 0.0048), adenosine triphosphate (P = 0.019), and GSH (P = 0.0015), and the degree of centrilobular necrosis, were improved by intrahepatic compared to systemic PEG-CAT delivery. Conclusions Direct intrahepatic administration of PEG-CAT achieved significant protection against IRI by reducing the volume distribution and taking advantage of the substantial hepatic first-pass uptake of this molecule. The mode of delivery was an important factor for protection against hepatic IRI by PEG-CAT.  

4.1987           Retrograde transport of Akt by a neuronal Rab5-APPL1 endosome

Goto-Silvia, L., McShane, M.P.P., Salinas, S., Kalaidzidis, Y., Schiavo, G. and Zerial, M. Scientific Reports, 9:2433 (2019)   Long-distance axonal trafficking plays a critical role in neuronal function and transport defects have been linked to neurodegenerative disorders. Various lines of evidence suggest that the small GTPase Rab5 plays a role in neuronal signaling via early endosomal transport. Here, we characterized the motility of Rab5 endosomes in primary cultures of mouse hippocampal pyramidal cells by live-cell imaging and showed that they exhibit bi-directional long-range motility in axons, with a strong bias toward retrograde transport. Characterization of key Rab5 effectors revealed that endogenous Rabankyrin-5, Rabenosyn-5 and APPL1 are all present in axons. Further analysis of APPL1-positive endosomes showed that, similar to Rab5-endosomes, they display more frequent long-range retrograde than anterograde movement, with the endosomal levels of APPL1 correlated with faster retrograde movement. Interestingly, APPL1-endosomes transport the neurotrophin receptor TrkB and mediate retrograde axonal transport of the kinase Akt1. FRET analysis revealed that APPL1 and Akt1 interact in an endocytosis-dependent manner. We conclude that Rab5-APPL1 endosomes exhibit the hallmarks of axonal signaling endosomes to transport Akt1 in hippocampal pyramidal cells.  

4.1988           Excessive Cell Growth Causes Cytoplasm Dilution and Contributes to Senescence

Neurohr, G.E., Terry, R.L., Lengefeld, J., van Werven, F.J., Holt, L.J. and Amon, A. Cell, 176, 1083-1097 (2019)   Cell size varies greatly between cell types, yet within a specific cell type and growth condition, cell size is narrowly distributed. Why maintenance of a cell-type specific cell size is important remains poorly understood. Here we show that growing budding yeast and primary mammalian cells beyond a certain size impairs gene induction, cell-cycle progression, and cell signaling. These defects are due to the inability of large cells to scale nucleic acid and protein biosynthesis in accordance with cell volume increase, which effectively leads to cytoplasm dilution. We further show that loss of scaling beyond a certain critical size is due to DNA becoming limiting. Based on the observation that senescent cells are large and exhibit many of the phenotypes of large cells, we propose that the range of DNA:cytoplasm ratio that supports optimal cell function is limited and that ratios outside these bounds contribute to aging.    

4.1989           Interferon‐γ Receptor 1 and GluR1 upregulated in motor neurons of symptomatic hSOD1G93A mice

Sengupta, S., Le, T.T., Adam, A., Tadic, V., Stubendorff, B., Keiner, S., Kloss, L., Prell, T., Witte, O.W. and Grosskreutz, J. Eur. J. Neurosci., 49, 62-78 (2019)   Motor neurons are markedly vulnerable to excitotoxicity mostly by alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic receptor (AMPAR) stimulation and are principal targets in the neurodegenerative disease Amyotrophic Lateral Sclerosis. Interferon‐gamma (IFN‐γ), a pro‐inflammatory cytokine, can independently cause neuronal dysfunction by triggering calcium influx through a calcium‐permeable complex of IFN‐γ receptor 1(IFNGR1) subunit and AMPAR subunit GluR1. This receptor complex is formed via a non‐canonical neuron‐specific IFN‐γ pathway that involves Jak1/Stat1 and Protein Kinase A. In this study, we explore the expression of the pathway's participants for the first time in the hSOD1G93A Amyotrophic Lateral Sclerosis mouse model. Elevated IFNGR1 and GluR1 are detected in motor neurons of hSOD1G93A symptomatic mice ex vivo, unlike the downstream targets ‐ Jak1, Stat1, and Protein Kinase A. We, also, determine effects of IFN‐γ alone or in the presence of an excitotoxic agent, kainate, on motor neuron survival in vitro. IFN‐γ induces neuronal damage, but does not influence kainate‐mediated excitotoxicity. Increased IFNGR1 can most likely sensitize motor neurons to excitotoxic insults involving GluR1 and/or pathways mediated by IFN‐γ, thus, serving as a potential direct link between neurodegeneration and inflammation in Amyotrophic Lateral Sclerosis.  

4.1990           4 in 1: Antibody‐free protocol for isolating the main hepatic cells from healthy and cirrhotic single rat livers

Fernandez-Iglesias, A., Ortega-Ribera, M., Guixe-Muntet, S. and Gracia-Sancho, J.

1. Cell Mol. Med., 23(2), 877-886 (2019)

  Liver cells isolated from pre‐clinical models are essential tools for studying liver (patho)physiology, and also for screening new therapeutic options. We aimed at developing a new antibody‐free isolation method able to obtain the four main hepatic cell types (hepatocytes, liver sinusoidal endothelial cells [LSEC], hepatic macrophages [HMΦ] and hepatic stellate cells [HSC]) from a single rat liver. Control and cirrhotic (CCl₄ and TAA) rat livers (n = 6) were perfused, digested with collagenase and mechanically disaggregated obtaining a multicellular suspension. Hepatocytes were purified by low revolution centrifugations while non‐parenchymal cells were subjected to differential centrifugation. Two different fractions were obtained: HSC and mixed LSEC + HMΦ. Further LSEC and HMΦ enrichment was achieved by selective adherence time to collagen‐coated substrates. Isolated cells showed high viability (80%‐95%) and purity (>95%) and were characterized as functional: hepatocytes synthetized albumin and urea, LSEC maintained endocytic capacity and in vivo fenestrae distribution, HMΦ increased expression of inflammatory markers in response to LPS and HSC were activated upon in vitro culture. The 4 in 1 protocol allows the simultaneous isolation of highly pure and functional hepatic cell sub‐populations from control or cirrhotic single livers without antibody selection.  

4.1991           Liver‐Selective MMP‐9 Inhibition in the Rat Eliminates Ischemia‐Reperfusion Injury and Accelerates Liver Regeneration

Wang, X., Maretti-Mira, A.C., Wang, L. and DeLeve, L. Hepatology, 69(1), 314-328 (2019)   Recruitment of liver sinusoidal endothelial cell progenitor cells (sprocs) from the bone marrow by vascular endothelial growth factor‐stromal cell‐derived factor‐1 (VEGF‐sdf‐1) signaling promotes recovery from injury and drives liver regeneration. Matrix metalloproteinases (MMPs) can proteolytically cleave VEGF, which might inhibit progenitor cell recruitment, but systemic matrix metalloproteinase inhibition might prevent efflux of progenitors from the bone marrow. The hypothesis for this study was that liver‐selective MMP‐9 inhibition would protect the hepatic VEGF‐sdf‐1 signaling pathway, enhance bone marrow sproc recruitment, and thereby ameliorate liver injury and accelerate liver regeneration, whereas systemic MMP inhibition would impair bone marrow sproc mobilization and therefore have less benefit or be detrimental. We found that liver‐selective MMP‐9 inhibition accelerated liver regeneration after partial hepatectomy by 40%, whereas systemic MMP inhibition impaired liver regeneration. Liver‐selective MMP‐9 inhibition largely abolished warm ischemia‐reperfusion injury. In the extended hepatectomy model, liver‐selective MMP‐9 inhibition restored liver sinusoidal endothelial cell integrity, enhanced liver regeneration, and reduced ascites. Liver‐selective MMP‐9 inhibition markedly increased recruitment and engraftment of bone marrow sprocs, whereas systemic MMP inhibition impaired mobilization of bone marrow sprocs and their hepatic engraftment. Hepatic MMP‐9 proteolytically cleaved VEGF after partial hepatectomy. Liver‐selective MMP‐9 inhibition prevented VEGF cleavage and doubled protein expression of VEGF and its downstream signaling partner sdf‐1. In contrast, systemic MMP inhibition enhanced recruitment and engraftment of infused allogeneic progenitors. Conclusion: Liver‐selective MMP inhibition prevents proteolytic cleavage of hepatic VEGF, which enhances recruitment and engraftment of bone marrow sprocs after liver injury. This ameliorates injury and accelerates liver regeneration. Liver‐selective MMP‐9 inhibition may be a therapeutic tool for liver injury that damages the vasculature, whereas systemic MMP inhibition can enhance the benefit of stem cell therapy with endothelial progenitor cells.  

4.1992           Targeting the Sigma-1 Receptor via Pridopidine Ameliorates Central Features of ALS Pathology in a SOD1G93A Model

Inoescu, A., Gradus, T., Altman, T., Maimon, R., Avraham, N.S., Geva, M., Hayden, M. and Perlson, E. Cell Death & Disease, 10:210 (2019)   Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease affecting both the upper and lower motor neurons (MNs), with no effective treatment currently available. Early pathological events in ALS include perturbations in axonal transport (AT), formation of toxic protein aggregates and Neuromuscular Junction (NMJ) disruption, which all lead to axonal degeneration and motor neuron death. Pridopidine is a small molecule that has been clinically developed for Huntington disease. Here we tested the efficacy of pridopidine for ALS using in vitro and in vivo models. Pridopidine beneficially modulates AT deficits and diminishes NMJ disruption, as well as motor neuron death in SOD1G93A MNs and in neuromuscular co-cultures. Furthermore, we demonstrate that pridopidine activates the ERK pathway and mediates its beneficial effects through the sigma-1 receptor (S1R). Strikingly, in vivo evaluation of pridopidine in SOD1G93A mice reveals a profound reduction in mutant SOD1 aggregation in the spinal cord, and attenuation of NMJ disruption, as well as subsequent muscle wasting. Taken together, we demonstrate for the first time that pridopidine improves several cellular and histological hallmark pathologies of ALS through the S1R.  

4.1993           Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase

Finelli, M.J., Paramo, T., Pires, E., Ryan, B.J., Wade-martins, R., Biggin, P.C., McCullagh, J. and Oliver, P.L. Mol. Neuurobiol., 56, 1558-1577 (2019)   Glucose metabolism is essential for the brain: it not only provides the required energy for cellular function and communication but also participates in balancing the levels of oxidative stress in neurons. Defects in glucose metabolism have been described in neurodegenerative disease; however, it remains unclear how this fundamental process contributes to neuronal cell death in these disorders. Here, we investigated the molecular mechanisms driving the selective neurodegeneration in an ataxic mouse model lacking oxidation resistance 1 (Oxr1) and discovered an unexpected function for this protein as a regulator of the glycolytic enzyme, glucose-6-phosphate isomerase (GPI/Gpi1). Initially, we present a dysregulation of metabolites of glucose metabolism at the pre-symptomatic stage in the Oxr1 knockout cerebellum. We then demonstrate that Oxr1 and Gpi1 physically and functionally interact and that the level of Gpi1 oligomerisation is disrupted when Oxr1 is deleted in vivo. Furthermore, we show that Oxr1 modulates the additional and less well-understood roles of Gpi1 as a cytokine and neuroprotective factor. Overall, our data identify a new molecular function for Oxr1, establishing this protein as important player in neuronal survival, regulating both oxidative stress and glucose metabolism in the brain.  

4.1994           Key role of streambed moisture and flash storms for microbial resistance and resilience to long‐term drought

Gionchetta, G., Oliva, f., Menendez, M., Laseras, P.L. and Romani, A.M. Freshwater Biology, 64(2), 306-322 (2019)  

1. Spatial and temporal widening of drought periods together with occurrence of flash storm events are consequences of global change affecting temporary stream ecosystems. Streambed heterotrophic microbes and the key biogeochemical processes they carry on could be endangered by the strengthening of drought episodes.
2. Here, we performed a 165‐day experiment through 12 streambed sediment columns to study heterotrophic microbial functional and structural responses to long‐term drought. Two sediment depths (surface and hyporheic) and leaf litter were monitored under three treatments: control (maintained in wet, pool‐like conditions), dry (5 months of drought), and dry–storm (5 months of drought including two flash storms). All treatments were then followed by rewetting.
3. Surface sediment followed by leaf litter was the most affected by the long‐term drought as shown by the reduction of polysaccharidic enzyme activities and litter decomposition rate, although lignin decomposition was not affected. This resulted in a greater use of recalcitrant compounds which might be due to reduced labile organic matter sources and the greater resistance of fungi than bacteria to drought. Moreover, in surface sediment, bacterial viability and algal biomass were reduced while the production of extracellular polymeric substances was intensified with dryness, suggesting its potential role as survival strategy.
4. Hyporheic sediments appeared more resistant to long‐term drought, and this might be linked to its slightly higher water content (2.5%) than the surface (0.5%) during drying together with a greater content of fine material that allowed fungi and bacteria to survive and extracellular enzyme activities to be maintained.
5. Microbial resilience to long‐term drought in sediment and leaf litter was promoted by the flash storms as shown by the fast recovery of enzyme activities, bacterial viability and leaf litter decomposition rate in the dry–storm treatment, once rewetted. Storms might liberate previously occluded carbon sources that fuel the fast microbial reactivation as well as providing sediment moisture for microbial survival.
6. Our results highlight that long‐term drought may compromise stream biogeochemical processes linked to organic matter decomposition and that microbes are highly sensitive to minimal water content changes. Thus, long‐term drought consequences could be mitigated by occasional precipitations or by natural streambed moisture.

 

4.1995           Dendritic cell-based immunization induces Coccidioides Ag2/PRA-specific immune response

Awasthi, S., Vilekar, P., Conkleton, A. and Rahman, N. Vaccine, 37, 1685-1691 (2019)   Valley Fever, or coccidioidomycosis, is caused by a soil-borne, highly virulent fungal pathogen, Coccidioides spp. Infection with Coccidioides can be life-threatening. Since an effective treatment is not available and the T cell-mediated immune response is protective, vaccine development is of interest. In this study, a primary dendritic cell (DC)-vaccine was evaluated for its ability to stimulate Coccidioides antigen-specific immune response in an extremely susceptible BALB/c mouse model. The DC-vaccine (Ag2-DC) was prepared by non-virally transfecting the primary bone marrow-derived DCs with a plasmid DNA encoding Ag2/PRA (protective epitope of Coccidioides). Mice were intranasally immunized with Ag2-DC on days 2 and 10. Immunized mice were necropsied on days 8, 32, and 44. Major organs and blood samples were harvested. The most common indicators of injury (protein, lactate, and albumin), Ag/PRA-specific cytokine-secreting cells, and IgG and its isotypes were determined by biochemical and immunologic assays, respectively. No signs of sickness were noted. Similarly, no significant changes were observed in the levels of total lung protein, lactate, and albumin, in immunized mice compared with healthy control mice. Interferon (IFN-γ), and interleukin (IL)-4 and IL-17 cytokine-secreting cells were observed in lung and lymph nodes upon Ag2-DC immunization. Our results showed that the levels of serum IgG and its isotypes were increased in Ag2-DC-immunized mice. This report provides evidence of DC immunization-stimulated Ag2/PRA-specific immune responses.  

4.1996           Effects of remote ischaemic preconditioning on intraportal islet transplantation in a rat model

Delaune, V., Lacotte, S., Gex, Q., Slits, F., Kahler-Quesada, A., lavallard, V., Peloso, A., Orci, L.A., Berney, T. and Toso, C. Transplant. Int., 32(3), 323-333 (2019)   Remote ischaemic preconditioning (RIPC), which is the intermittent interruption of blood flow to a site distant from the target organ, is known to improve solid organ resistance to ischaemia‐reperfusion injury. This procedure could be of interest in islet transplantation to mitigate hypoxia‐related loss of islet mass after isolation and transplantation. Islets isolated from control or RIPC donors were analyzed for yield, metabolic activity, gene expression and high mobility group box‐1 (HMGB1) content. Syngeneic marginal mass transplantation was performed in four streptozotocin‐induced diabetic groups: control, RIPC in donor only, RIPC in recipient only, and RIPC in donor and recipient. Islets isolated from RIPC donors had an increased yield of 20% after 24 h of culture compared to control donors (P = 0.007), linked to less cell death (P = 0.08), decreased expression of hypoxia‐related genes (Hif1a P = 0.04; IRP94 P = 0.008), and increased intra‐cellular (P = 0.04) and nuclear HMGB1. The use of RIPC in recipients only did not allow for reversal of diabetes, with increased serum HMGB1 at day 1; the three other groups demonstrated significantly better outcomes. Performing RIPC in the donors increases islet yield and resistance to hypoxia. Validation is needed, but this strategy could help to decrease the number of donors per islet recipient.  

4.1997           Artificial MicroRNA-Mediated Tgfbr2 and Pdgfrb Co-Silencing Ameliorates Carbon Tetrachloride–Induced Hepatic Fibrosis in Mice

Jiang, Y., Zhao, Y., He, F. and Wang, H. Human Gene Therapy, 30(2), 179-196 (2019)   Hepatic stellate cells (HSCs) are the primary cell type responsible for liver fibrogenesis. Transforming growth factor beta 1 (TGF-β1) and platelet-derived growth factor (PDGF) are key profibrotic cytokines that regulate HSC activation and proliferation with functional convergence. Dual RNA interference against their receptors may achieve therapeutic effects. A novel RNAi strategy based on HSC-specific GFAP promoter-driven and lentiviral-expressed artificial microRNAs (amiRNAs) was devised that consists of an microRNA-30a backbone and effective shRNAs against mouse Pdgfrβ and Tgfbr2. Then, its antifibrotic efficacy was tested in primary and cultured HSCs and in mice affected with carbon tetrachloride–induced hepatic fibrosis. The study shows that amiRNA-mediated Pdgfrβ and Tgfbr2 co-silencing inhibits HSC activation and proliferation. After recombinant lentiviral particles were delivered into the liver via tail-vein injection, therapeutic amiRNAs were preferentially expressed in HSCs and efficiently co-knocked down in situTgfbr2 and Pdgfrβ expression, which correlates with downregulated expression of target or effector genes of their signaling, which include Pai-1, P70S6K, and D-cyclins. amiRNA-based HSC-specific co-silencing of Tgfbr2 and Pdgfrβ significantly suppressed hepatic expression of fibrotic markers α-Sma and Col1a1, extracellular matrix regulators Mmps and Timp1, and phenotypically ameliorated liver fibrosis, as indicated by reductions in serum alanine aminotransferase activity, collagen deposition, and α-Sma-positive staining. The findings provide proof of concept for the use of amiRNA-mediated co-silencing of two profibrogenic pathways in liver fibrosis treatment and highlight the therapeutic potential of concatenated amiRNAs for gene therapy.  

4.1998           H19/miR‐148a/USP4 axis facilitates liver fibrosis by enhancing TGF‐β signaling in both hepatic stellate cells and hepatocytes

Zhu, J., Luo, Z., Pan, Y., Zhewng, W., Li, W., Zhang, Z., Xiong, P., Xu, D., Du, M., Wang, B., Yu, J., Zhang, J. and Liu, J.

1. Cell. Physiol., 234(6), 9698-9710 (2019)

  Liver fibrosis is a wound‐healing response represented by excessive extracellular matrix deposition. Activation of hepatic stellate cell (HSC) is the critical cellular basis for hepatic fibrogenesis, whereas hepatocyte undergoes epithelial‐mesenchymal transition (EMT) which is also involved in chronic liver injury. Long noncoding RNA H19 has been found to be associated with cholestatic liver fibrosis lately. However, the role of H19 in liver fibrosis remains largely to be elucidated. In this study, we found that the expression of H19 was significantly upregulated in the liver tissue of CCl₄‐induced mice, a toxicant‐induced liver fibrogenesis model. Overexpression of H19 significantly aggravated activation of HSC and EMT of hepatocyte both by stimulating transforming growth factor‐β (TGF‐β) pathway. In terms of mechanism, H19 functioned as a competing endogenous RNA to sponge miR‐148a and subsequently sustained the level of ubiquitin‐specific protease 4 (USP4), which was an identified target of miR‐148a and was able to stabilize TGF‐β receptor I. In conclusion, our findings revealed a novel H19/miR‐148a/USP4 axis which promoted liver fibrosis via TGF‐β pathway in both HSC and hepatocyte, indicating that H19 could become a promising target for the treatment of liver fibrosis.  

4.1999           Roles of Progesterone Receptor Membrane Component 1 in Oxidative Stress–Induced Aging in Chorion Cells

Feng, L., Allen, T.K., Marinello, W.P. and Murtha, A.P. Reproduction Sci., 26(3), 394-403 (2019)   Introduction: Oxidative stress–mediated fetal membrane cell aging is activated prematurely in preterm premature rupture of membranes (PPROMs). The mechanism of this phenomenon is largely understudied. Progesterone receptor membrane component 1 (PGRMC1) has been recognized as a potential protective component for maintaining fetal membrane integrity and healthy pregnancies. We aimed to investigate the effects of oxidative stress (represented by hydrogen peroxide [H₂O2]) on fetal membrane and chorion cell senescence, p38 mitogen-activated protein kinase (MAPK) phosphorylation, and sirtuin 3 (SIRT3) and to examine the roles of PGRMC1 in these effects. Methods: Following serum starvation for 24 hours, full-thickness fetal membrane explants and primary chorion cells were treated with H₂O2 at 100, 300, and 500 µM for 24 hours. Cells were fixed for cell senescence-associated β-galactosidase assay. Cell lysates were harvested for quantitive reverse transcription polymerase chain reaction to quantify SIRT3 messenger RNA. Cell lysates were harvested for Western blot to semi-quantify SIRT3 protein and p38 MAPK phosphorylation levels, respectively. To examine the role of PGRMC1, primary chorion cells underwent the same treatment mentioned above following PGRMC1 knockdown using validated PGRMC1-specific small-interfering RNA. Results: Hydrogen peroxide significantly induced cell senescence and p38 MAPK phosphorylation, and it significantly decreased SIRT3 expression in full-thickness fetal membrane explants and chorion cells. These effects were enhanced by PGRMC1 knockdown. Discussion: This study further demonstrated that oxidative stress–induced cell aging is one of the mechanisms of PPROM and PGRMC1 acts as a protective element for maintaining fetal membrane integrity by inhibiting oxidative stress–induced chorion cell aging.  

4.2000           Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade

Miller, B., Sen, D., Al Abosy, R., Bi, K., Virkud, Y.V. et al Nature Immunology, 20, 326-336 (2019)   T cell dysfunction is a hallmark of many cancers, but the basis for T cell dysfunction and the mechanisms by which antibody blockade of the inhibitory receptor PD-1 (anti-PD-1) reinvigorates T cells are not fully understood. Here we show that such therapy acts on a specific subpopulation of exhausted CD8+ tumor-infiltrating lymphocytes (TILs). Dysfunctional CD8+ TILs possess canonical epigenetic and transcriptional features of exhaustion that mirror those seen in chronic viral infection. Exhausted CD8+ TILs include a subpopulation of ‘progenitor exhausted’ cells that retain polyfunctionality, persist long term and differentiate into ‘terminally exhausted’ TILs. Consequently, progenitor exhausted CD8+ TILs are better able to control tumor growth than are terminally exhausted T cells. Progenitor exhausted TILs can respond to anti-PD-1 therapy, but terminally exhausted TILs cannot. Patients with melanoma who have a higher percentage of progenitor exhausted cells experience a longer duration of response to checkpoint-blockade therapy. Thus, approaches to expand the population of progenitor exhausted CD8+ T cells might be an important component of improving the response to checkpoint blockade.

4.2001           Gene expression analysis reveals early dysregulation of disease pathways and links Chmp7 to pathogenesis of spinal and bulbar muscular atrophy

Malik, B., Devine, H., Patani, R., La Spada, A.R., Hanna, M.G. and Greensmith, L. Scientific Reports, 9:3539 (2019)   Spinal and bulbar muscular atrophy (SBMA) results from a CAG repeat expansion within the androgen receptor gene (AR). It is unclear why motor neurons selectively degenerate and there are currently no treatments for this debilitating disease. To uncover the causative genes and pathways involved in motor neuron dysfunction, we undertook transcriptomic profiling of primary embryonic motor neurons from SBMA mice. We show that transcriptional dysregulation occurs early during development in SBMA motor neurons. One gene found to be dysregulated, Chmp7, was also altered in vivo in spinal cord before symptom onset in SBMA mice, and crucially in motor neuron precursor cells derived from SBMA patient stem cells, suggesting that Chmp7 may play a causal role in disease pathogenesis by disrupting the endosome-lysosome system. Furthermore, genes were enriched in SBMA motor neurons in several key pathways including p53, DNA repair, WNT and mitochondrial function. SBMA embryonic motor neurons also displayed dysfunctional mitochondria along with DNA damage, possibly resulting from DNA repair gene dysregulation and/or mitochondrial dysfunction. This indicates that a coordinated dysregulation of multiple pathways leads to development of SBMA. Importantly, our findings suggest that the identified pathways and genes, in particular Chmp7, may serve as potential therapeutic targets in SBMA.  

4.2002           Mitochondrial Modulation by Dichloroacetate Reduces Toxicity of Aberrant Glial Cells and Gliosis in the SOD1G93A Rat Model of Amyotrophic Lateral Sclerosis

Martinez-Palma, L., Miquel, E., Lagos-Rodriguez, V., Barbeiro, L., Cassina, A. and Cassina, P. Neurotherapeutics, 16(1), 203-215 (2019)   Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron (MN) degeneration and gliosis. Neonatal astrocytes obtained from the SOD1G93A rat model of ALS exhibit mitochondrial dysfunction and neurotoxicity that can be reduced by dichloroacetate (DCA), a metabolic modulator that has been used in humans, and shows beneficial effects on disease outcome in SOD1G93A mice. Aberrant glial cells (AbGC) isolated from the spinal cords of adult paralytic SOD1G93A rats exhibit highly proliferative and neurotoxic properties and may contribute to disease progression. Here we analyze the mitochondrial activity of AbGC and whether metabolic modulation would modify their phenotypic profile. Our studies revealed fragmented mitochondria and lower respiratory control ratio in AbGC compared to neonatal SOD1G93A and nontransgenic rat astrocytes. DCA (5 mM) exposure improved AbGC mitochondrial function, reduced their proliferative rate, and importantly, decreased their toxicity to MNs. Furthermore, oral DCA administration (100 mg/kg, 10 days) to symptomatic SOD1G93A rats reduced MN degeneration, gliosis, and the number of GFAP/S100β double-labeled hypertrophic glial cells in the spinal cord. DCA treatment of AbGC reduced extracellular lactate levels indicating that the main recognized DCA action, targeting the pyruvate dehydrogenase kinase/pyruvate dehydrogenase complex, may underlie our findings. Our results show that AbGC metabolic phenotype is related to their toxicity to MNs and indicate that its modulation can reduce glial mediated pathology in the spinal cord. Together with previous findings, these results further support glial metabolic modulation as a valid therapeutic strategy in ALS.  

4.2003           Migration velocity of red blood cells in microchannels

Losserand, S., Coupier, G. and Podgorsky, T. Microvasc. Res., 124, 30-36 (2019)   The lateral migration of red blood cells (RBCs) in confined channel flows is an important ingredient of microcirculatory hydrodynamics and is involved in the development of a cell free layer near vessel walls and influences the distribution of RBCs in networks. It is also relevant to a number of lab-on-chip applications. This migration is a consequence of their deformability and is due to the combined effects of hydrodynamic wall repulsion and the curvature of the fluid velocity profile. We performed microfluidic experiments with dilute suspensions of RBCs in which the trajectories and migration away from the channel wall are analyzed to extract the mean behavior, from which we propose a generic scaling law for the transverse migration velocity valid in a whole range of parameters relevant to microcirculatory and practical situations. Experiments with RBCs of different mechanical properties (separated by density gradient sedimentation or fixed with glutaraldehyde) show the influence of this parameter which can induce significant dispersion of the trajectories.  

4.2004           A Macrophage-Pericyte Axis Directs Tissue Restoration via Amphiregulin-Induced Transforming Growth Factor Beta Activation

Minutti, C.M., Modak, R.V., Macdonald, F., Li, F., Smyth, D.J. et al Immunity, 50(3), 645-654.e6 (2019)   The epidermal growth factor receptor ligand Amphiregulin has a well-documented role in the restoration of tissue homeostasis after injury; however, the mechanism by which Amphiregulin contributes to wound repair remains unknown. Here we show that Amphiregulin functioned by releasing bioactive transforming growth factor beta (TGF-β) from latent complexes via integrin-α_V activation. Using acute injury models in two different tissues, we found that by inducing TGF-β activation on mesenchymal stromal cells (pericytes), Amphiregulin induced their differentiation into myofibroblasts, thereby selectively contributing to the restoration of vascular barrier function within injured tissue. Furthermore, we identified macrophages as a critical source of Amphiregulin, revealing a direct effector mechanism by which these cells contribute to tissue restoration after acute injury. Combined, these observations expose a so far under-appreciated mechanism of how cells of the immune system selectively control the differentiation of tissue progenitor cells during tissue repair and inflammation.  

4.2005           STING-mediated intestinal barrier dysfunction contributes to lethal sepsis

Hu, Q., Ren, H., Li, G., Wang, D., Zhou, Q., Wu, J., Zheng, J., Huang, J., Slade, D.A., Wu, X. and Ren, J. EBioMedicine, 41, 497-508 (2019)   Background Gut integrity is compromised in abdominal sepsis with increased cellular apoptosis and altered barrier permeability. Intestinal epithelial cells (IEC) form a physiochemical barrier that separates the intestinal lumen from the host's internal milieu and is strongly involved in the mucosal inflammatory response and immune response. Recent research indicates the involvement of the stimulator of interferons genes (STING) pathway in uncontrolled inflammation and gut mucosal immune response. Methods We investigated the role of STING signaling in sepsis and intestinal barrier function using intestinal biopsies from human patients with abdominal sepsis and with an established model of abdominal sepsis in mice. Findings In human abdominal sepsis, STING expression was elevated in peripheral blood mononuclear cells and intestinal biopsies compared with healthy controls, and the degree of STING expression in the human intestinal lamina propria correlated with the intestinal inflammation in septic patients. Moreover, elevated STING expression was associated with high levels of serum intestinal fatty acid binding protein that served as a marker of enterocyte damage. In mice, the intestinal STING signaling pathway was markedly activated following the induction of sepsis induced by cecal ligation perforation (CLP). STING knockout mice showed an alleviated inflammatory response, attenuated gut permeability, and decreased bacterial translocation. Whereas mice treated with a STING agonist (DMXAA) following CLP developed greater intestinal apoptosis and a more severe systemic inflammatory response. We demonstrated that mitochondrial DNA (mtDNA) was released during sepsis, inducing the intestinal inflammatory response through activating the STING pathway. We finally investigated DNase I administration at 5 hours post CLP surgery, showing that it reduced systemic mtDNA and inflammatory cytokines levels, organ damage, and bacterial translocation, suggesting that inhibition of mtDNA-STING signaling pathway protects against CLP-induced intestinal barrier dysfunction. Interpretation Our results indicate that the STING signaling pathway can contribute to lethal sepsis by promoting IEC apoptosis and through disrupting the intestinal barrier. Our findings suggest that regulation of the mtDNA-STING pathway may be a promising therapeutic strategy to promote mucosal healing and protect the intestinal barrier in septic patients.  

4.2006           Age- and AD-related redox state of NADH in subcellular compartments by fluorescence lifetime imaging microscopy

Dong, Y., Digman, M.A. and Brewer, G.J. GeroScience, 41(1), 51-67 (2019)   Nicotinamide adenine dinucleotide (reduced form: NADH) serves as a vital redox-energy currency for reduction-oxidation homeostasis and fulfilling energetic demands. While NADH exists as free and bound forms, only free NADH is utilized for complex I to power oxidative phosphorylation, especially important in neurons. Here, we studied how much free NADH remains available for energy production in mitochondria of old living neurons. We hypothesize that free NADH in neurons from old mice is lower than the levels in young mice and even lower in neurons from the 3xTg-AD Alzheimer’s disease (AD) mouse model. To assess free NADH, we used lifetime imaging of NADH autofluorescence with 2-photon excitation to be able to resolve the pool of NADH in mitochondria, cytoplasm, and nuclei. Primary neurons from old mice were characterized by a lower free/bound NADH ratio than young neurons from both non-transgenic (NTg) and more so in 3xTg-AD mice. Mitochondrial compartments maintained 26 to 41% more reducing NADH redox state than cytoplasm for each age, genotype, and sex. Aging diminished the mitochondrial free NADH concentration in NTg neurons by 43% and in 3xTg-AD by 50%. The lower free NADH with age suggests a decline in capacity to regenerate free NADH for energetic supply to power oxidative phosphorylation which further worsens in AD. Applying this non-invasive approach, we showed the most explicit measures yet of bioenergetic deficits in free NADH with aging at the subcellular level in live neurons from in-bred mice and an AD model.  

4.2007           Structural Principles in Robo Activation and Auto-inhibition

Barak, R., Yom-Tov, G., Guez-Haddad, J., Henis-Korenblit, S., Isupov, M.N. and Opataowsky, Y. Cell, 177(2), 272-285 (2019)   Proper brain function requires high-precision neuronal expansion and wiring, processes controlled by the transmembrane Roundabout (Robo) receptor family and their Slit ligands. Despite their great importance, the molecular mechanism by which Robos’ switch from “off” to “on” states remains unclear. Here, we report a 3.6 Å crystal structure of the intact human Robo2 ectodomain (domains D1–8). We demonstrate that Robo cis dimerization via D4 is conserved through hRobo1, 2, and 3 and the C. elegans homolog SAX-3 and is essential for SAX-3 function in vivo. The structure reveals two levels of auto-inhibition that prevent premature activation: (1) cis blocking of the D4 dimerization interface and (2) trans interactions between opposing Robo receptors that fasten the D4-blocked conformation. Complementary experiments in mouse primary neurons and C. elegans support the auto-inhibition model. These results suggest that Slit stimulation primarily drives the release of Robo auto-inhibition required for dimerization and activation.  

4.2008           Micro-Particle Operations Using Asymmetric Traps

Lee, J., Mena, S.E: and Burns, M.A.

Scientific Reports, 9:1278 (2019)

Micro-particle operations in many lab-on-a-chip devices require active-type techniques that are accompanied by complex fabrication and operation. The present study describes an alternative method using a passive microfluidic scheme that allows for simpler operation and, therefore, potentially less expensive devices. We present three practical micro-particle operations using our previously developed passive mechanical trap, the asymmetric trap, in a non-acoustic oscillatory flow field. First, we demonstrate size-based segregation of both binary and ternary micro-particle mixtures using size-dependent trap-particle interactions to induce different transport speeds for each particle type. The degree of segregation, yield, and purity of the binary segregations are 0.97 ± 0.02, 0.96 ± 0.06, and 0.95 ± 0.05, respectively. Next, we perform a solution exchange by displacing particles from one solution into another in a trap array. Lastly, we focus and split groups of micro-particles by exploiting the transport polarity of asymmetric traps. These operations can be implemented in any closed fluidic circuit containing asymmetric traps using non-acoustic oscillatory flow, and they open new opportunities to flexibly control micro-particles in integrated lab-on-a-chip platforms with minimal external equipment.

 

4.2009           Deletion of C-C Motif Chemokine Ligand 5 Worsens Invariant Natural Killer T–Cell Mediated Hepatitis via Compensatory Up-regulation of CXCR2–Related Chemokine Activity

Chen, L., Gu, J., Qian, Y., Li, M., Qian, Y., Xu, M., Li, J., Wen, Y., Xia, L., Li, J., Xia, Q., Kong, X. and Wu, H. Cell. Mol. Gastroenterol. Hepatol., 7(3), 623-639 (2019)   Background & Aims Chemokine-mediated immune cell recruitment plays pivotal roles in liver inflammation. C-C motif chemokine ligand 5 (CCL5) has been shown to be responsible for the recruitment of monocytes/macrophages and has been implicated in various liver diseases, including nonalcoholic fatty liver disease, fibrosis, and hepatocellular carcinoma. Previous studies have also shown that inhibition of CCL5 appears to be a promising therapeutic approach for several chronic liver diseases. However, whether blocking CCL5 could benefit immune cell–mediated hepatitis remains largely elusive. Methods By adopting a specific agonist, alpha-galactosylceramide (α-Galcer), of invariant natural killer T cells (iNKTs), we investigated the function and mechanism of CCL5 in the iNKT induced murine hepatitis model. Results We found significantly increased CCL5 expression in α-Galcer–induced hepatitis murine model. Such an increase in CCL5 is mainly enriched in non-parenchymal cells such as macrophages and iNKTs but not in hepatocytes. Surprisingly, CCL5 blockage by genetic deletion of Ccl5 does not affect the α-Galcer–induced iNKT activation but greatly worsens α-Galcer–induced liver injury accompanied by an increased hepatic neutrophil infiltration. Mechanistically, we demonstrated that greater neutrophil accumulation in the liver is responsible for the enhanced liver injury in Ccl5^-/- mice. Such an increased hepatic neutrophil infiltration is mainly caused by an enhanced CXCL1-CXCR2 signal in Ccl5^-/- mice. Therapeutically, either antibody-mediated neutrophil depletion or a CXCR2 antagonist, SB225002, mediated CXCR2 signaling blockage significantly ameliorated α-Galcer–induced liver injury in Ccl5^-/- mice. Conclusions Our present study demonstrates that (1) α-Galcer–induced murine hepatitis could greatly induce CCL5 production in macrophages and iNKT cells; (2) loss of CCL5 could enhance CXCL1 expression in hepatocytes and activate CXCL1-CXCR2 axis in neutrophils to augment their hepatic infiltration; and (3) neutrophil depletion or blockage of CXCL1-CXCR2 axis greatly improves α-Galcer–induced liver injury in Ccl5^-/- mice. This study suggests that clinical utilization of CCL5 blockage may compensatorily induce the activation of other chemokine pathways to enhance neutrophil recruitment and liver injury in hepatitis.  

4.2010           Th17 cell frequency is associated with low bone mass in primary sclerosing cholangitis

Schmidt, T., Schwinge, D., Rolvien, T., Schramm, C., Schinke, T. and Amling, M.

1. Hepatol., 70, 941-953 (2019)

  Background & Aims Osteoporotic fractures are a major cause of morbidity and reduced quality of life in patients with primary sclerosing cholangitis (PSC), a progressive bile duct disease of unknown origin. Although it is generally assumed that this pathology is a consequence of impaired calcium homeostasis and malabsorption, the cellular and molecular causes of PSC-associated osteoporosis are unknown. Methods We determined bone mineral density by dual-X-ray absorptiometry and assessed bone microstructure by high-resolution peripheral quantitative computed tomography in patients with PSC. Laboratory markers of liver and bone metabolism were measured, and liver stiffness was assessed by FibroScan. We determined the frequency of Th17 cells by the ex vivo stimulation of peripheral blood mononuclear cells in a subgroup of 40 patients with PSC. To investigate the potential involvement of IL-17 in PSC-associated bone loss, we analyzed the skeletal phenotype of mice lacking Abcb4 and/or Il-17. Results Unlike in patients with primary biliary cholangitis, bone loss in patients with PSC was not associated with disease duration or liver fibrosis. However, we observed a significant negative correlation between the bone resorption biomarker deoxypyridinoline and bone mineral density in the PSC cohort, indicating increased bone resorption. Importantly, the frequency of Th17 cells in peripheral blood was positively correlated with the urinary deoxypyridinoline level and negatively correlated with bone mass. We observed that Abcb4-deficient mice displayed a low-bone-mass phenotype, which was corrected by an additional Il-17 deficiency or anti-IL-17 treatment, whereas the liver pathology was unaffected. Conclusions Our findings demonstrate that an increased frequency of Th17 cells is associated with bone resorption in PSC. Whether antibody-based IL-17 blockade is beneficial against bone loss in patients with PSC should be addressed in future studies.  

4.2011           Incomplete Differentiation of Engrafted Bone Marrow Endothelial Progenitor Cells Initiates Hepatic Fibrosis in the Rat

Maretti-Mira, A.C., Wang, X., Wang, l. and Deleve, L.D. Hepatology, 69(3), 1259-1272 (2019)   Normal liver sinusoidal endothelial cells (LSECs) promote quiescence of hepatic stellate cells (HSCs). Prior to fibrosis, LSECs undergo capillarization, which is permissive for HSC activation, the proximate event in hepatic fibrosis. The aims of this study were to elucidate the nature of and mechanisms leading to capillarization and to determine how LSECs promote HSC quiescence and why “capillarized LSECs” lose control of HSC activation. The contribution of bone marrow (BM) endothelial progenitor cells to capillarization was identified using rats transplanted with transgenic enhanced green fluorescent protein–positive BM. Shotgun proteomics and informatics were used to identify the LSEC mediator that maintains HSC quiescence. The study shows that capillarization is due to repair of injured LSECs by BM endothelial progenitors that engraft but fail to fully mature. Lack of maturation of BM‐derived LSECs is due to cell autonomous pathways that inhibit the nitric oxide pathway. We identify heparin binding epidermal growth factor–like growth factor (HB‐EGF) as the signal that maintains HSC quiescence and show that immature LSECs are unable to shed HB‐EGF from the cytosolic membrane. Conclusion: Chronic liver injury can recruit BM progenitors of LSECs that engraft and fail to fully differentiate, which creates an environment that is permissive for hepatic fibrosis; elucidation of these early events in the fibrotic process will provide targets for treatment of hepatic fibrosis.  

4.2012           Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri‐islet extracellular matrix

Loganathan, G., Subhashree, V., narayanan, S., Tweed, B., Goedde, M.A., Gunaratnam, B., Tucker, W.W., Goli, P., Mokshagundam, S., McCarthy, R.C., Williams, S.K., Hughes, M.G. and Balamurugan, A.N. Am. J. Transplant., 19(3), 831-843 (2019)   Human islet isolation from young donor pancreases (YDP) utilizing the current purified standard dose of collagenase‐protease enzyme mixtures often results in the release of a high percentage of mantled islets. Mantled islets are those surrounded by exocrine tissue and are difficult to purify by density gradient centrifugation, leading to poor islet recovery. Based on difference in extracellular matrix, and total collagen content between YDP and old donor pancreas (ODP, > 35 Y) led us to compare results from islet isolation using increased collagenase combination (ICC) or increased protease combination (IPC), to the standard enzyme combination (SEC) in a “trisected” pancreas model to overcome the donor‐to‐donor variability. These results showed a reduced percentage of mantled islets (17% ± 7.5%) and higher postpurification islet recovery (83.8% ± 5.6%) with IPC. Furthermore, these results were confirmed in 13 consecutive whole pancreas islet isolations utilizing IPC from VitaCyte, Roche, or SERVA collagenase‐protease enzyme mixtures. Results obtained from in vitro and in vivo islet functional assessment indicated that islets isolated using IPC retained normal islet morphology, insulin secretion, and the ability to reverse diabetes after transplantation in diabetic nude mice. This is the first report utilizing trisected pancreas to assess the effectiveness of different enzyme combinations to improve islet recovery from young donor pancreases.  

4.2013           An integrin-based nanoparticle that targets activated hepatic stellate cells and alleviates liver fibrosis

Li, Y., Pu, S., Liu, Q., Li, R., Zhang, J., Wu, T., Chen, L., Li, H., Yang, X., Zou, M., Xiao, J., Xie, W. and He, J.

1. Controlled Release, 303, 77-90 (2019)

  Activation of hepatic stellate cells (HSCs) contributes to the development of liver fibrosis. Because of a relatively small population of HSCs in the liver and the lack of specific membrane targeting proteins, HSC-targeted therapy remains a major clinical challenge. Here we first showed that a hallmark of activated HSC (aHSC) is their increased expression of integrin αvβ3. Thus we established sterically stable liposomes that contain the cyclic peptides (cRGDyK) with a high affinity to αvβ3 to achieve aHSC-specific delivery. Our results showed that the cRGDyK-guided liposomes were preferentially internalized by activated HSCs in vitro and in vivo, and the internalization was abolished by excess free cRGDyK or knockdown of αvβ3. In contrast, quiescent HSCs, hepatocytes, Kupffer cells, sinusoidal endothelial cells, or biliary cells showed minimal uptake of the cRGDyK-guided liposomes. When loaded with the hedgehog inhibitor vismodegib, the cRGDyK-guided liposomes inhibited hedgehog pathway signaling specifically in activated HSCs. Moreover, treatment of mice with vismodegib-loaded cRGDyK-liposomes markedly inhibited the fibrogenic phenotype in bile duct ligation- or thioacetamide-treated mice. We conclude that the cRGDyK-guided liposomes can specifically target the activated HSCs, but not quiescent HSCs. This nanoparticle system showed great promise to deliver therapeutic agents to aHSC to treat liver fibrosis.  

4.2014           Nicotinamide mononucleotide (NMN) supplementation rescues cerebromicrovascular endothelial function and neurovascular coupling responses and improves cognitive function in aged mice

Tarantini, S., Valcarcel-Ares, M., Toth, P., Yabluchanskiy, A., Tucsek, Z., Kiss, T., Hertelendy, P., Kinter, M., Ballabh, p., Süle, Z., Farkas, E., Baur, J.A., Sinclair, D.A., Csiszar, A. and Ungvari, Z. Redox Biology, 24, 101192 (2019)   Adjustment of cerebral blood flow (CBF) to neuronal activity via neurovascular coupling (NVC) has an essential role in maintenance of healthy cognitive function. In aging increased oxidative stress and cerebromicrovascular endothelial dysfunction impair NVC, contributing to cognitive decline. There is increasing evidence showing that a decrease in NAD⁺ availability with age plays a critical role in a range of age-related cellular impairments but its role in impaired NVC responses remains unexplored. The present study was designed to test the hypothesis that restoring NAD⁺ concentration may exert beneficial effects on NVC responses in aging. To test this hypothesis 24-month-old C57BL/6 mice were treated with nicotinamide mononucleotide (NMN), a key NAD⁺ intermediate, for 2 weeks. NVC was assessed by measuring CBF responses (laser Doppler flowmetry) evoked by contralateral whisker stimulation. We found that NVC responses were significantly impaired in aged mice. NMN supplementation rescued NVC responses by increasing endothelial NO-mediated vasodilation, which was associated with significantly improved spatial working memory and gait coordination. These findings are paralleled by the sirtuin-dependent protective effects of NMN on mitochondrial production of reactive oxygen species and mitochondrial bioenergetics in cultured cerebromicrovascular endothelial cells derived from aged animals. Thus, a decrease in NAD⁺ availability contributes to age-related cerebromicrovascular dysfunction, exacerbating cognitive decline. The cerebromicrovascular protective effects of NMN highlight the preventive and therapeutic potential of NAD⁺ intermediates as effective interventions in patients at risk for vascular cognitive impairment (VCI).  

4.2015           Qualitative and quantitative proteomic analyses of Schistosoma japonicum eggs and egg-derived secretory-excretory proteins

De Marco Verissimo, C., Potriquet, J., You, H., McManus, D.P., Mulvenna, J. and Jones, M.K. Parasites & Vectors, 12:173 (2019)   Background Schistosome parasites lay up to a thousand eggs per day inside the veins of their mammalian hosts. The immature eggs deposited by females against endothelia of venules will embryonate within days. Approximately 30% of the eggs will migrate to the lumen of the intestine to continue the parasite life-cycle. Many eggs, however, are trapped in the liver and intestine causing the main pathology associated with schistosomiasis mansoni and japonica, the liver granulomatous response. Excretory-secretory egg proteins drive much of egg-induced pathogenesis of schistosomiasis mansoni, and Schistosoma japonicum induce a markedly distinct granulomatous response to that of S. mansoni. Methods To explore the basis of variations in this responsiveness, we investigated the proteome of eggs of S. japonicum. Using mass spectrometry qualitative and quantitative (SWATH) analyses, we describe the protein composition of S. japonicum eggs secretory proteins (ESP), and the differential expression of proteins by fully mature and immature eggs, isolated from faeces and ex vivo adults. Results Of 957 egg-related proteins identified, 95 were exclusively found in S. japonicum ESP which imply that they are accessible to host immune system effector elements. An in-silico analysis implies that ESP are able of stimulating the innate and adaptive immune system through several different pathways. While quantitative SWATH analysis revealed 124 proteins that are differentially expressed by mature and immature S. japonicum eggs, illuminating some important aspects of eggs biology and infection, we also show that mature eggs are more likely than immature eggs to stimulate host immune responses. Conclusions Here we present a list of potential targets that can be used to develop better strategies to avoid severe morbidity during S. japonicum infection, as well as improving diagnosis, treatment and control of schistosomiasis japonica.  

4.2016           Csf1r or Mer inhibition delays liver regeneration via suppression of Kupffer cells

Santamaria-Barria, J.A., Zeng, S., Greer, J.B., Beckman, M.J., Seifert, A.M., Cohen, N.A., Zhang, J.Q., Crawley, M.H., Green, B.L., Loo, J.K., Maltbaek, J.H. and DeMatteo, R.P. PloS One, 14(5), e0216275 (2019)   Introduction Murine Kupffer cells (KCs) comprise CD11b^hi and F4/80^hi subsets. Tissue-resident macrophages are known to express the tyrosine kinase receptors colony-stimulating factor 1 receptor (Csf1r) and Mer. However, the expression of Csf1r and Mer on KC subsets and the importance of these tyrosine kinases during liver regeneration (LR) are unknown. Methods KCs from wild-type and Csf1r-GFP mice were characterized by flow cytometry. Partial hepatectomy (PH) was performed in mice treated with clodronate liposomes, a Csf1r small molecule inhibitor or depleting antibody, or a small molecule Mer inhibitor. Sera and livers were analyzed. The function of sorted KC subsets was tested in vitro. Results Mer was specifically expressed on tissue-resident F4/80^hi KCs, 55% of which also expressed Csf1r. Mer⁺Csf1r⁺ and Mer⁺Csf1r^- KCs had distinct expression of macrophage markers. Csf1r inhibition in mice reduced F4/80^hi KCs by approximately 50%, but did not affect CD11b^hi KCs. Clodronate liposomes depleted F4/80^hi KCs, but also altered levels of other intrahepatic leukocytes. Csf1r inhibition delayed LR, as demonstrated by a 20% reduction in liver-to-body weight ratios 7 days after PH. At 36h after PH, Csf1r inhibition increased serum ALT and histological liver injury, and decreased liver cell proliferation. A small molecule inhibitor of Mer did not alter the percentage of KCs or their proliferation and just modestly delayed LR. In vitro, Csf1r or Mer inhibition did not decrease KC viability, but did attenuate their cytokine response to stimulation. Conclusions F4/80^hi KCs are Mer⁺ and can be subdivided based on Csf1r expression. Csf1r or Mer inhibition each reduces KC cytokine production and delays LR.  

4.2017           Distinct Cortical-Thalamic-Striatal Circuits through the Parafascicular Nucleus

Mandelbaum, G., Taranda, J., Haynes, T.M., Robertson, K., Osten, P. and Sabatini, B.L. Neuron, 102, 636-652 (2019)   The thalamic parafascicular nucleus (PF), an excitatory input to the basal ganglia, is targeted with deep-brain stimulation to alleviate a range of neuropsychiatric symptoms. Furthermore, PF lesions disrupt the execution of correct motor actions in uncertain environments. Nevertheless, the circuitry of the PF and its contribution to action selection are poorly understood. We find that, in mice, PF has the highest density of striatum-projecting neurons among all sub-cortical structures. This projection arises from transcriptionally and physiologically distinct classes of PF neurons that are also reciprocally connected with functionally distinct cortical regions, differentially innervate striatal neurons, and are not synaptically connected in PF. Thus, mouse PF contains heterogeneous neurons that are organized into parallel and independent associative, limbic, and somatosensory circuits. Furthermore, these subcircuits share motifs of cortical-PF-cortical and cortical-PF-striatum organization that allow each PF subregion, via its precise connectivity with cortex, to coordinate diverse inputs to striatum.  

4.2018           Charting cellular identity during human in vitro β-cell differentiation

Veres, A., Faust, A.L., Bushnell, H.L., Engquist, E.N., Hyoje-Ruy Kenty, J., harb, G., Poh, Y-C., Sintov, E., Gürtler, M., Pagliuca, F.W., Peterson, Q.P. and Melton, D.A. Nature, 569, 368-373 (2019)   In vitro differentiation of human stem cells can produce pancreatic β-cells; the loss of this insulin-secreting cell type underlies type 1 diabetes. Here, as a step towards understanding this differentiation process, we report the transcriptional profiling of more than 100,000 human cells undergoing in vitro β-cell differentiation, and describe the cells that emerged. We resolve populations that correspond to β-cells, α-like poly-hormonal cells, non-endocrine cells that resemble pancreatic exocrine cells and a previously unreported population that resembles enterochromaffin cells. We show that endocrine cells maintain their identity in culture in the absence of exogenous growth factors, and that changes in gene expression associated with in vivo β-cell maturation are recapitulated in vitro. We implement a scalable re-aggregation technique to deplete non-endocrine cells and identify CD49a (also known as ITGA1) as a surface marker of the β-cell population, which allows magnetic sorting to a purity of 80%. Finally, we use a high-resolution sequencing time course to characterize gene-expression dynamics during the induction of human pancreatic endocrine cells, from which we develop a lineage model of in vitro β-cell differentiation. This study provides a perspective on human stem-cell differentiation, and will guide future endeavours that focus on the differentiation of pancreatic islet cells, and their applications in regenerative medicine.  

4.2019           An Atlas of Vagal Sensory Neurons and Their Molecular Specialization

Kupari, J., Häring, M., Agirre, E., Castelo-Branco, G. and Ernfors, P. Cell Reports, 27, 2508-2523 (2019)   Sensory functions of the vagus nerve are critical for conscious perceptions and for monitoring visceral functions in the cardio-pulmonary and gastrointestinal systems. Here, we present a comprehensive identification, classification, and validation of the neuron types in the neural crest (jugular) and placode (nodose) derived vagal ganglia by single-cell RNA sequencing (scRNA-seq) transcriptomic analysis. Our results reveal major differences between neurons derived from different embryonic origins. Jugular neurons exhibit fundamental similarities to the somatosensory spinal neurons, including major types, such as C-low threshold mechanoreceptors (C-LTMRs), A-LTMRs, Aδ-nociceptors, and cold-, and mechano-heat C-nociceptors. In contrast, the nodose ganglion contains 18 distinct types dedicated to surveying the physiological state of the internal body. Our results reveal a vast diversity of vagal neuron types, including many previously unanticipated types, as well as proposed types that are consistent with chemoreceptors, nutrient detectors, baroreceptors, and stretch and volume mechanoreceptors of the respiratory, gastrointestinal, and cardiovascular systems.  

4.2020           Aged‐senescent cells contribute to impaired heart regeneration

Lewis-McDoughall, F.C., Ruchaya, P.J., Domenjo-Vila, E., Teoh, T.S., Prata, l., Cottle, B.J., Clark, J.E., Punjabi, P.P., Awad, W., Torella, D., Tchkonia, T., Kirkland, J.L. and Ellison-Hughes, G.M. Aging Cell, 18, e12931 (2019)   Aging leads to increased cellular senescence and is associated with decreased potency of tissue‐specific stem/progenitor cells. Here, we have done an extensive analysis of cardiac progenitor cells (CPCs) isolated from human subjects with cardiovascular disease, aged 32–86 years. In aged subjects (>70 years old), over half of CPCs are senescent (p16^INK4A, SA‐β‐gal, DNA damage γH2AX, telomere length, senescence‐associated secretory phenotype [SASP]), unable to replicate, differentiate, regenerate or restore cardiac function following transplantation into the infarcted heart. SASP factors secreted by senescent CPCs renders otherwise healthy CPCs to senescence. Elimination of senescent CPCs using senolytics abrogates the SASP and its debilitative effect in vitro. Global elimination of senescent cells in aged mice (INK‐ATTAC or wild‐type mice treated with D + Q senolytics) in vivo activates resident CPCs and increased the number of small Ki67‐, EdU‐positive cardiomyocytes. Therapeutic approaches that eliminate senescent cells may alleviate cardiac deterioration with aging and restore the regenerative capacity of the heart.  

4.2021           Single-Cell Transcriptomics of Human and Mouse Lung Cancers Reveals Conserved Myeloid Populations across Individuals and Species

Zilionis, R., Engblom, C., Pfirschke, C., Savova, V., Levantini, E., Pittet, M.J. and Klein, A.M. Immunity, 50(5), 1317-1334 (2019)   Tumor-infiltrating myeloid cells (TIMs) comprise monocytes, macrophages, dendritic cells, and neutrophils, and have emerged as key regulators of cancer growth. These cells can diversify into a spectrum of states, which might promote or limit tumor outgrowth but remain poorly understood. Here, we used single-cell RNA sequencing (scRNA-seq) to map TIMs in non-small-cell lung cancer patients. We uncovered 25 TIM states, most of which were reproducibly found across patients. To facilitate translational research of these populations, we also profiled TIMs in mice. In comparing TIMs across species, we identified a near-complete congruence of population structures among dendritic cells and monocytes; conserved neutrophil subsets; and species differences among macrophages. By contrast, myeloid cell population structures in patients’ blood showed limited overlap with those of TIMs. This study determines the lung TIM landscape and sets the stage for future investigations into the potential of TIMs as immunotherapy targets.  

4.2022           Synthetic poly(ethylene glycol)‐based microfluidic islet encapsulation reduces graft volume for delivery to highly vascularized and retrievable transplant site

Weaver, J.D., Headen, D.M., Coronel, M.M., Hunckler, M.D., Shirwan, H. and Garcia, A.J. Am. J. Transplant.,19(5), 1315-1327 (2019)   Transplant of hydrogel‐encapsulated allogeneic islets has been explored to reduce or eliminate the need for chronic systemic immunosuppression by creating a physical barrier that prevents direct antigen presentation. Although successful in rodents, translation of alginate microencapsulation to large animals and humans has been hindered by large capsule sizes (≥500 μm diameter) that result in suboptimal nutrient diffusion in the intraperitoneal space. We developed a microfluidic encapsulation system that generates synthetic poly(ethylene glycol)‐based microgels with smaller diameters (310 ± 14 μm) that improve encapsulated islet insulin responsiveness over alginate capsules and allow transplant within vascularized tissue spaces, thereby reducing islet mass requirements and graft volumes. By delivering poly(ethylene glycol)‐encapsulated islets to an isolated, retrievable, and highly vascularized site via a vasculogenic delivery vehicle, we demonstrate that a single pancreatic donor syngeneic islet mass exhibits improved long‐term function over conventional alginate capsules and close integration with transplant site vasculature. In vivo tracking of bioluminescent allogeneic encapsulated islets in an autoimmune type 1 diabetes murine model showed enhanced cell survival over unencapsulated islets in the absence of chronic systemic immunosuppression. This method demonstrates a translatable alternative to intraperitoneal encapsulated islet transplant.  

4.2023           Stevioside inhibits experimental fibrosis by down‐regulating profibrotic Smad pathways and blocking hepatic stellate cell activation

Casas-Grajales, S., Alvarez-Suarez, D., Ramos-Tovar, E., Dayana, L., Buendia-Montano, B., Reyes-Gordillo, K., Camacho, K., Camacho, j., Tsutsumi, V., Laksshman, M.R. and Muriel, P. Basic. Clin. Pharmacol. Toxicol., 24(6), 670-680 (2019)   Liver cirrhosis is associated with increased morbidity and mortality with important health and social consequences; however, an effective treatment has not been found yet. Previous reports have shown some beneficial effects of stevioside (SVT) in different diseases, but the ability of SVT to inhibit liver cirrhosis has not been reported. Therefore, we studied the potential of this diterpenoid to inhibit liver cirrhosis induced by thioacetamide, a model that shares many similarities with the human disease, and investigated the possible underlying molecular mechanism using in vivo and in vitro approaches. Cirrhosis was induced in male Wistar rats by chronic thioacetamide administration (200 mg/kg) intraperitoneally three times per week. Rats received saline or SVT (20 mg/kg) two times daily intraperitoneally. In addition, co‐cultures were incubated with either lipopolysaccharide or ethanol. Liver fibrosis, hepatic stellate cells activation, metalloproteinases activity, canonical and non‐canonical Smads pathway and expression of several profibrogenic genes were evaluated. Thioacetamide activated hepatic stellate cells and distorted the liver parenchyma with the presence of abundant thick bands of collagen. In addition, thioacetamide up‐regulated the protein expression of α‐smooth muscle actin, transforming growth factor‐β1, metalloproteinases‐9,‐2 and ‐13 and overstimulate the canonical and non‐canonical Smad pathways. SVT administration inhibited all of these changes. In vitro, SVT inhibited the up‐regulation of several genes implicated in cirrhosis when cells were exposed to lipopolysaccharides or ethanol. We conclude that SVT inhibited liver damage by blocking hepatic stellate cells activation, down‐regulating canonical and non‐canonical profibrotic Smad pathways.  

4.2024           Cell‐specific elevation of Runx2 promotes hepatic infiltration of macrophages by upregulating MCP‐1 in high‐fat diet‐induced mice NAFLD

Zhong, L., Huang, L., Xue, Q., Liu, C., Xu, K., Shen, W. and Deng, L.

1. Cell. Biochem., 120(7), 11761-11774 (2019)

  Objective We have demonstrated runt‐related transcription factor 2 (Runx2) plays important role in atherosclerosis. It has been indicated that atherosclerosis shares the similar histopathology with nonalcoholic steatohepatitis (NASH), a progressive stage of nonalcoholic fatty liver disease (NAFLD), on macrophages infiltration. However, the function of Runx2 in NAFLD is completely unknown. Here, we investigated the underlying mechanism of Runx2 triggering macrophages infiltration in the development of NAFLD. Methods Mice were fed with high‐fat diet (HFD) for a long time. Histopathologic features, macrophages infiltration, expression of monocyte chemotactic protein 1 (MCP‐1), and Runx2 were, respectively, analyzed in vivo. Lentivirus or short interfering RNA were transfected in murine hepatic stellate cells (HSCs) and the transwell assay was performed to verify the contribution of Runx2 for macrophages migration in vitro. Results Long‐term treatment with HFD induced the progression of NAFLD, and NASH was initiated from 8 months on diet. HFD increased the expression of F4/80 upon HFD feeding, indicated HFD promotes hepatic infiltration of macrophages in NAFLD. In addition, HFD upregulated the expression of MCP‐1 and Runx2 during NAFLD development. Unexpectedly, Runx2 upregulation is cell‐type depended in NAFLD, and only abundantly elevated in activated HSCs. Furthermore, we found that Runx2 could increase or decrease the expression of MCP‐1 in HSCs, and regulate macrophages migration by influencing MCP‐1 production in vitro. Conclusions Our results give evidence that the upregulation of Runx2 specific in activated HSCs promotes hepatic infiltration of macrophages by increasing MCP‐1 expression in NAFLD, which reveals a novel mechanism and provides a cell‐specific therapeutic target for NAFLD.  

4.2025           Multitranscriptome analyses reveal prioritized genes specifically associated with liver fibrosis progression independent of etiology

Chen, W., Wu, X., Yan, X., Xu, A., Yang, A. and You, H. Am. J. Physiol. Gastroentest. Liver Physiol., 316, G744-G754 (2019)   Elimination or suppression of causative factors can raise the possibility of liver fibrosis regression. However, different injurious stimuli will give fibrosis from somewhat different etiologies, which, in turn, may hamper the discovery of liver fibrosis-specific therapeutic drugs. Therefore, the analogical cellular and molecular events shared by various etiology-evoked liver fibrosis should be clarified. Our present study systematically integrated five publicly available transcriptomic data sets regarding liver fibrosis with different etiologies from the Gene Expression Omnibus database and performed a series of bioinformatics analyses and experimental verifications. A total of 111 significantly upregulated and 16 downregulated genes were identified specific to liver fibrosis independent of any etiology. These genes were predominately enriched in some Kyoto Encyclopedia of Genes and Genomes pathways, including the “PI3K-AKT signaling pathway,” “Focal adhesion,” and “ECM-receptor interaction.” Subsequently, five prioritized liver fibrosis-specific genes, including COL4A2, THBS2, ITGAV, LAMB1, and PDGFRA, were screened. These genes were positively associated with each other and liver fibrosis progression. In addition, they could robustly separate all stages of samples in both training and validation data sets with diverse etiologies when they were regarded as observed variables applied to principal component analysis plots. Expressions of all five genes were confirmed in activated primary mouse hepatic stellate cells (HSCs) and transforming growth factor β1-treated LX-2 cells. Moreover, THBS2 protein was enhanced in liver fibrosis rodent models, which could promote HSC activation and proliferation and facilitate NOTCH1/JAG1 expression in HSCs. Overall, our current study may provide potential targets for liver fibrosis therapy and aid to a deeper understanding of the molecular underpinnings of liver fibrosis.NEW & NOTEWORTHY Prioritized liver fibrosis-specific genes THBS2, COL4A2, ITGAV, LAMB1, and PDGFRA were identified and significantly associated with liver fibrosis progression and could be combined to discriminate liver fibrosis stages regardless of any etiology. Among the identified prioritized liver fibrosis-specific targets, THBS2 protein was confirmed to be enhanced in liver fibrosis rodent models, which could promote hepatic stellate cell (HSC) activation and proliferation and facilitate NOTCH1/JAG1 expression in HSCs.Prioritized liver fibrosis-specific genes THBS2, COL4A2, ITGAV, LAMB1, and PDGFRA were identified and significantly associated with liver fibrosis progression and could be combined to discriminate liver fibrosis stages regardless of any etiology. Among the identified prioritized liver fibrosis-specific targets, THBS2 protein was confirmed to be enhanced in liver fibrosis rodent models, which could promote hepatic stellate cell (HSC) activation and proliferation and facilitate NOTCH1/JAG1 expression in HSCs.  

4.2026           Enhancing immunogenicity and protective efficacy of inactivated avian influenza H9N2vaccine with recombinant chicken IFN-α in chicken

Gan, L., Tian, Y., Zhao, Y., Shan, X-q., Zhou, W., Xia, B-B., Chen, J., Wang, M-L. and Zhao, J. Vet. Microbiol., 234, 77-82 (2019)   Control of currently circulating re-assorted low-pathogenicity avian influenza (LPAI) H9N2 is a major concern for both animal and human health. Thus, an improved LPAI H9N2 vaccination strategy is needed to induce complete immunity in chickens against LPAI H9N2 virus strains. Cytokines play a crucial role in mounting both the type and extent of an immune response generated following infection with a pathogen or after vaccination. To improve the efficacy of inactivated LPAI H9N2 vaccine, prokaryotic expression recombination chicken interferon-α (rchIFN-α) was used as vaccine adjuvant.In this study chIFN-α was used as adjuvant in inactivated AI H9N2 vaccine, modulated the immune response of chickens against the vaccine antigen through enhanced humoral and Th1-biased cell-mediated immunity, compared to chickens that received single AI H9N2 vaccine. To further test the protective efficacy of this improved vaccination regimen, immunized chickens were challenged with a high dose of LPAI H9N2 virus. Combined administration rchIFN-α showed markedly enhanced protection compared to single administration of the vaccine, as determined by mortality, clinical severity, and feed and water intake. This enhancement of protective immunity was further confirmed by reduced rectal shedding and replication of AIV H9N2 in challenged chickens. Our results indicate the value of combined administration of rchIFN-α to generate an effective immunization strategy in chickens against LPAI H9N2.  

4.2027           Chromulinavorax destructans, a pathogen of microzooplankton that provides a window into the enigmatic candidate phylum Dependentiae

Deeg, C.M., Zimmer, M.M., George, E.E., Husnik, F., Keeling, P.J. and Suttle, C.A. PloS Pathogens, 15(5), e1007801 (2019)   Members of the major candidate phylum Dependentiae (a.k.a. TM6) are widespread across diverse environments from showerheads to peat bogs; yet, with the exception of two isolates infecting amoebae, they are only known from metagenomic data. The limited knowledge of their biology indicates that they have a long evolutionary history of parasitism. Here, we present Chromulinavorax destructans (Strain SeV1) the first isolate of this phylum to infect a representative from a widespread and ecologically significant group of heterotrophic flagellates, the microzooplankter Spumella elongata (Strain CCAP 955/1). Chromulinavorax destructans has a reduced 1.2 Mb genome that is so specialized for infection that it shows no evidence of complete metabolic pathways, but encodes an extensive transporter system for importing nutrients and energy in the form of ATP from the host. Its replication causes extensive reorganization and expansion of the mitochondrion, effectively surrounding the pathogen, consistent with its dependency on the host for energy. Nearly half (44%) of the inferred proteins contain signal sequences for secretion, including many without recognizable similarity to proteins of known function, as well as 98 copies of proteins with an ankyrin-repeat domain; ankyrin-repeats are known effectors of host modulation, suggesting the presence of an extensive host-manipulation apparatus. These observations help to cement members of this phylum as widespread and diverse parasites infecting a broad range of eukaryotic microbes.  

4.2028           Compromised DNA Repair and Signalling in Human Granulocytes

Ponath, V., Heylmann, D., Haak, T., Woods, K., Becker, H. and Kaina, B.

1. Innate Immun., 11, 74-85 (2019)

  In previous studies, we showed impaired DNA repair in human monocytes. Here, we addressed the question of whether human neutrophilic granulocytes that arise from the same precursor as monocytes exhibit a similar phenotype and are impaired in repairing their DNA. We show that neutrophilic granulocytes isolated from peripheral blood display a lack of the same repair proteins that are missing in monocytes and do not show repair of their DNA when damaged by ionising radiation (IR) or chemical ROS. Contrary to T cells, we observed no decline in the number of single-strand breaks following γ-radiation. Also, granulocytes did not show γH2AX foci formation while T cells and peripheral blood lymphocytes (PBL) responded. In comparison to PBL, XRCC1, PARP-1 and ligase III were not expressed and there was also no discernible signal for key damage response proteins ATM, ATR and DNA-PK_CS as well as γH2AX in neutrophils. Time course and dose-response experiments confirmed the absence of H2AX phosphorylation after radiation treatment although an accumulation of double-strand breaks was detected in the neutral Comet assay. Overall, the data indicate that terminally differentiated neutrophilic granulocytes in the peripheral blood display strong downregulation of DNA repair and DNA damage response factors, which should be taken into account if studies with whole peripheral blood containing granulocytes are performed, causing a significant intra-experimental variation in the cellular repair capacity.  

4.2029           A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway

Healing, E., Charlier, C.F., Meira, L.B. and Elliott, R.M. Nucleic Acid Res., 47(11), e61 (2019)   DNA repair is essential for the maintenance of genomic integrity, and evidence suggest that inter-individual variation in DNA repair efficiency may contribute to disease risk. However, robust assays suitable for quantitative determination of DNA repair capacity in large cohort and clinical trials are needed to evaluate these apparent associations fully. We describe here a set of microplate-based oligonucleotide assays for high-throughput, non-radioactive and quantitative determination of repair enzyme activity at individual steps and over multiple steps of the DNA base excision repair pathway. The assays are highly sensitive: using HepG2 nuclear extract, enzyme activities were quantifiable at concentrations of 0.0002 to 0.181 μg per reaction, depending on the enzyme being measured. Assay coefficients of variation are comparable with other microplate-based assays. The assay format requires no specialist equipment and has the potential to be extended for analysis of a wide range of DNA repair enzyme activities. As such, these assays hold considerable promise for gaining new mechanistic insights into how DNA repair is related to individual genetics, disease status or progression and other environmental factors and investigating whether DNA repair activities can be used a biomarker of disease risk.  

4.2030           A Guide for Ex Vivo Handling and Storage of Stool Samples Intended for Fecal Microbiota Transplantation

Burz, S.D., Abraham, A-L., Fonseca, F., David, O., Chapron, A. et al Scientific Reports, 9:8897 (2019)   Owing to the growing recognition of the gut microbiota as a main partner of human health, we are expecting that the number of indications for fecal microbiota transplantation (FMT) will increase. Thus, there is an urgent need for standardization of the entire process of fecal transplant production. This study provides a complete standardized procedure to prepare and store live and ready-to-use transplants that meet the standard requirements of good practices to applied use in pharmaceutical industry. We show that, if time before transformation to transplants would exceed 24 hours, fresh samples should not be exposed to temperatures above 20 °C, and refrigeration at 4 °C can be a safe solution. Oxygen-free atmosphere was not necessary and simply removing air above collected samples was sufficient to preserve viability. Transplants prepared in maltodextrin-trehalose solutions, stored in a -80 °C standard freezer and then rapidly thawed at 37 °C, retained the best revivification potential as  proven by 16S rRNA profiles, metabolomic fingerprints, and flow cytometry assays over a 3-month observation period. Maltodextrin-trehalose containing cryoprotectants were also efficient in preserving viability of lyophilized transplants, either in their crude or purified form, an option that can be attractive for fecal transplant biobanking and oral formulation.  

4.2031           Microencapsulated Immunoassays for Detection of Cytokines in Human Blood

Rahimiam, A., Siltanen, c., Feyzizarnagh, h., Escalante, P. and Revzin, A. ASC Sens., 4, 578-585 (2019)   Cytokines are produced by leukocytes in blood and may be used as indicators of malignancies or infections. The objective of this study was to develop a strategy for immunosensing cytokines in whole, unprocessed human blood. Microfluidic droplet generation was employed to fabricate ∼400 μm diameter microcapsules with a hydrogel shell and an aqueous core containing sensing microbeads. The hydrogel shell was composed of poly(ethylene glycol) forming a thin (∼10 μm) immunoisolation layer protecting antibody-modified microbeads inside the capsule from immune cells on the outside. The microbeads were functionalized with antibodies against cytokines of interest: interferon (IFN)-γ and tumor necrosis factor (TNF)-α. While nonfouling, a hydrogel shell was permeable to cytokine molecules; these molecules were captured on microbeads and were detected with fluorescently labeled secondary antibodies. Calibration of encapsulated immunoassays with known concentrations of cytokines revealed a limit of detection of 14.8 and 14.4 pM for IFN-γ and TNF-α, respectively. We also demonstrated the concept of multi-cytokine detection by fabricating distinct populations of capsules carrying either anti-IFN-γ or anti-TNF-α microbeads and dispensing these capsules into a solution containing both cytokine types. Importantly, when placed into whole blood for 16 h, microcapsules were free of leukocytes, effectively protecting sensing beads from the blood components. To further demonstrate utility of this strategy, encapsulated microbeads were used for detection of IFN-γ in blood of patients with latent tuberculosis infection (LTBI) and unexposed healthy controls. When compared to gold standard technology (interferon gamma release assay or IGRA), our encapsulated immunoassay accurately predicted LTBI diagnosis in 11 out of 14 patients. Overall, encapsulation of immunoassays represents a promising strategy for keeping sensing elements operational in a highly fouling complex environment such as blood.  

4.2032           Dean Flow Assisted Single Cell and Bead Encapsulation for High Performance Single Cell Expression Profiling

Li, L., Wu, P., Luo, Z., Wang, L., Ding, W., Wu, T., Chen, J., He, J., He, Y., Wang, H., Chen, Y., Li, G., Li, Z. and He, L. ACS Sens., 4, 1299-1305 (2019)   Droplet microfluidics-based platform (Drop-seq) has been shown to be a powerful tool for single cell expression profiling. Nevertheless, this platform required the simultaneous encapsulation of single cell and single barcoded bead, the incidence of which was very low, limiting its efficiency. Spiral channels were reported to focus the barcoded beads and thus increased the efficiency, but focusing of cells was not demonstrated, which could potentially further enhance the performance. Here, we designed spiral and serpentine channels to focus both bead and cell solutions and implemented this microfluidic design on Drop-seq. We characterized the effect of cell/bead concentration on encapsulation results and tested the performance by coencapsulating barcoded beads and human–mouse cell mixtures followed by sequencing. The results showed ∼300% and ∼40% increase in cell utilization rate compared to the traditional Drop-seq device and the device focusing beads alone, respectively. This chip design showed great potential for high efficiency single cell expression profiling.  

4.2033           Galectin-3 Inhibits Cancer Metastasis by Negatively Regulating Integrin β3 Expression

Hayashi, Y., Jia, W., Kidoya, H., Muramatsu, F., Tsukada, Y. and Takakura, N. Am. J. Pathol., 189(4), 900-910 (2019)   Galectin-3 (Gal-3; gene LGALS3) is a member of the β-galactose–binding lectin family. Previous studies showed that Gal-3 is expressed in several tissues across species and functions as a regulator of cell proliferation, apoptosis, adhesion, and migration, thus affecting many aspects of events, such as angiogenesis and tumorigenesis. Although several reports have suggested that the level of Gal-3 expression correlates positively with tumor progression, herein we show that highly metastatic mouse melanoma B16/BL6 cells express less Gal-3 than B16 cells with a lower metastatic potential. It was found that overexpression of Gal-3 in melanoma cells in fact suppresses metastasis. In contrast, knocking out Gal-3 expression in cancer cells promoted cell aggregation mediated through interactions with platelets and fibrinogen in vitro and increased the number of metastatic foci in vivo. Thus, reduced Gal-3 expression results in the up-regulation of β3 integrin expression, and this contributes to metastatic potential. These findings indicate that changes of Gal-3 expression in cancer cells during tumor progression influence the characteristics of metastatic cells.    

4.2034           Identification of Novel Protein Targets of Dimethyl Fumarate Modification in Neurons and Astrocytes Reveals Actions Independent of Nrf2 Stabilization

Piroli, G.G., Manual, A.M., Patel, T., Walla, M.D., Shi, L., Lanci, S.A., Wang, J., Galloway, A., Ortinski, P.I., Smith, D.S. and Frizzell, N. Mol. Cell. Proteomics, 18, 504-519 (2019)   The fumarate ester dimethyl fumarate (DMF) has been introduced recently as a treatment for relapsing remitting multiple sclerosis (RRMS), a chronic inflammatory condition that results in neuronal demyelination and axonal loss. DMF is known to act by depleting intracellular glutathione and modifying thiols on Keap1 protein, resulting in the stabilization of the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. We have previously shown that DMF reacts with a wide range of protein thiols, suggesting that the complete mechanisms of action of DMF are unknown. Here, we investigated other intracellular thiol residues that may also be irreversibly modified by DMF in neurons and astrocytes. Using mass spectrometry, we identified 24 novel proteins that were modified by DMF in neurons and astrocytes, including cofilin-1, tubulin and collapsin response mediator protein 2 (CRMP2). Using an in vitro functional assay, we demonstrated that DMF-modified cofilin-1 loses its activity and generates less monomeric actin, potentially inhibiting its cytoskeletal remodeling activity, which could be beneficial in the modulation of myelination during RRMS. DMF modification of tubulin did not significantly impact axonal lysosomal trafficking. We found that the oxygen consumption rate of N1E-115 neurons and the levels of proteins related to mitochondrial energy production were only slightly affected by the highest doses of DMF, confirming that DMF treatment does not impair cellular respiratory function. In summary, our work provides new insights into the mechanisms supporting the neuroprotective and remyelination benefits associated with DMF treatment in addition to the antioxidant response by Nrf2.  

4.2035           Sensory lesioning induces microglial synapse elimination via ADAM10 and fractalkine signaling

Gunner, G., Cheadle, l., Johnson, K.M., Ayata, P., Badimon, A., Mondo, E. et al Nature Neurosci., 22, 1075-1088 (2019)   Microglia rapidly respond to changes in neural activity and inflammation to regulate synaptic connectivity. The extracellular signals, particularly neuron-derived molecules, that drive these microglial functions at synapses remain a key open question. Here we show that whisker lesioning, known to dampen cortical activity, induces microglia-mediated synapse elimination. This synapse elimination is dependent on signaling by CX3CR1, the receptor for microglial fractalkine (also known as CXCL1), but not complement receptor 3. Furthermore, mice deficient in CX3CL1 have profound defects in synapse elimination. Single-cell RNA sequencing revealed that Cx3cl1 is derived from cortical neurons, and ADAM10, a metalloprotease that cleaves CX3CL1 into a secreted form, is upregulated specifically in layer IV neurons and in microglia following whisker lesioning. Finally, inhibition of ADAM10 phenocopies Cx3cr1^−/− and Cx3cl1^−/− synapse elimination defects. Together, these results identify neuron-to-microglia signaling necessary for cortical synaptic remodeling and reveal that context-dependent immune mechanisms are utilized to remodel synapses in the mammalian brain.  

4.2036           Targeting SYK signaling in myeloid cells protects against liver fibrosis and hepatocarcinogenesis

Torres-Hernandez, A., Wang, W., Nikiforov, Y., Tejeda, K., Torres, l., Kalabin, A. et al Oncogene, 38, 4512-4526 (2019)   Liver fibrosis and fibrosis-associated hepatocarcinogenesis are driven by chronic inflammation and are leading causes of morbidity and death worldwide. SYK signaling regulates critical processes in innate and adaptive immunity, as well as parenchymal cells. We discovered high SYK expression in the parenchymal hepatocyte, hepatic stellate cell (HSC), and the inflammatory compartments in the fibrotic liver. We postulated that targeting SYK would mitigate hepatic fibrosis and oncogenic progression. We found that inhibition of SYK with the selective small molecule inhibitors Piceatannol and PRT062607 markedly protected against toxin-induced hepatic fibrosis, associated hepatocellular injury and intra-hepatic inflammation, and hepatocarcinogenesis. SYK inhibition resulted in increased intra-tumoral expression of the p16 and p53 but decreased expression of Bcl-xL and SMAD4. Further, hepatic expression of genes regulating angiogenesis, apoptosis, cell cycle regulation, and cellular senescence were affected by targeting SYK. We found that SYK inhibition mitigated both HSC trans-differentiation and acquisition of an inflammatory phenotype in T cells, B cells, and myeloid cells. However, in vivo experiments employing selective targeted deletion of SYK indicated that only SYK deletion in the myeloid compartment was sufficient to confer protection against fibrogenic progression. Targeting SYK promoted myeloid cell differentiation into hepato-protective TNFα^low CD206^hi phenotype downregulating mTOR, IL-8 signaling and oxidative phosphorylation. Collectively, these data suggest that SYK is an attractive target for experimental therapeutics in treating hepatic fibrosis and oncogenesis.  

4.2037           Engineering the cellular mechanical microenvironment – from bulk mechanics to the nanoscale

Matellan, C. and del Rio Hernandez, A.E.

1. Cell. Sci., 132, jcs229013 (2019)

  The field of mechanobiology studies how mechanical properties of the extracellular matrix (ECM), such as stiffness, and other mechanical stimuli regulate cell behaviour. Recent advancements in the field and the development of novel biomaterials and nanofabrication techniques have enabled researchers to recapitulate the mechanical properties of the microenvironment with an increasing degree of complexity on more biologically relevant dimensions and time scales. In this Review, we discuss different strategies to engineer substrates that mimic the mechanical properties of the ECM and outline how these substrates have been applied to gain further insight into the biomechanical interaction between the cell and its microenvironment  

4.2038           The RNA-binding protein FUS/TLS undergoes calcium-mediated nuclear egress during excitotoxic stress and is required for GRIA2 mRNA processing

Tischbein, M., baron, D.M., Lin, Y-C., Gall, K.V., landers, J.E., Fallini, C. and Bosco, D.A.

1. Biol. Chem., 294(26), 10194-10210 (2019)

  Excitotoxic levels of glutamate represent a physiological stress that is strongly linked to amyotrophic lateral sclerosis (ALS) and other neurological disorders. Emerging evidence indicates a role for neurodegenerative disease–linked RNA-binding proteins (RBPs) in the cellular stress response. However, the relationships between excitotoxicity, RBP function, and disease have not been explored. Here, using primary cortical and motor neurons, we found that excitotoxicity induced the translocation of select ALS-linked RBPs from the nucleus to the cytoplasm within neurons. RBPs affected by excitotoxicity included TAR DNA–binding protein 43 (TDP-43) and, most robustly, fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS). We noted that FUS is translocated through a calcium-dependent mechanism and that its translocation coincides with striking alterations in nucleocytoplasmic transport. Furthermore, glutamate-induced up-regulation of glutamate ionotropic receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)–type subunit 2 (GRIA2) in neurons depended on FUS expression, consistent with a functional role for FUS in excitotoxic stress. These findings reveal molecular links among prominent factors in neurodegenerative diseases, namely excitotoxicity, disease-associated RBPs, and nucleocytoplasmic transport.  

4.2039           Establishment and Characterization of Four Novel Thyroid Cancer Cell Lines and PDX Models Expressing the RET/PTC1 Rearrangement, BRAFV600E, or RASQ61R as Drivers

Schweppe, R.E., Pozdeyev, N., Pike, L.A., Korch, C., Zhou, Q., Sams, S.B. et al Mol. Cancer Res., 17(5), 1036-1048 (2019)   Cancer cell lines are critical models to study tumor progression and response to therapy. In 2008, we showed that approximately 50% of thyroid cancer cell lines were redundant or not of thyroid cancer origin. We therefore generated new authenticated thyroid cancer cell lines and patient-derived xenograft (PDX) models using in vitro and feeder cell approaches and characterized these models in vitro and in vivo. We developed four thyroid cancer cell lines, two derived from 2 different patients with papillary thyroid cancer (PTC) pleural effusions, CUTC5, and CUTC48; one derived from a patient with anaplastic thyroid cancer (ATC), CUTC60; and one derived from a patient with follicular thyroid cancer (FTC), CUTC61. One PDX model (CUTC60-PDX) was also developed. Short tandem repeat (STR) genotyping showed that each cell line and PDX is unique and match the original patient tissue. The CUTC5 and CUTC60 cells harbor the BRAF (V600E) mutation, the CUTC48 cell line expresses the RET/PTC1 rearrangement, and the CUTC61 cells have the HRAS (Q61R) mutation. Moderate to high levels of PAX8 and variable levels of NKX2-1 were detected in each cell line and PDX. The CUTC5 and CUTC60 cell lines form tumors in orthotopic and flank xenograft mouse models.  

4.2040           Bioinspired neuron-like electronics

Yang, X., Zhou, T., Zwang, T.J., Hong, G., Zhao, Y., Viveros, R.D., Fu, T-M., Gao, T. and Lieber, C.M. Nature Materials, 18, 510-517 (2019)   As an important application of functional biomaterials, neural probes have contributed substantially to studying the brain. Bioinspired and biomimetic strategies have begun to be applied to the development of neural probes, although these and previous generations of probes have had structural and mechanical dissimilarities from their neuron targets that lead to neuronal loss, neuroinflammatory responses and measurement instabilities. Here, we present a bioinspired design for neural probes—neuron-like electronics (NeuE)—where the key building blocks mimic the subcellular structural features and mechanical properties of neurons. Full three-dimensional mapping of implanted NeuE–brain interfaces highlights the structural indistinguishability and intimate interpenetration of NeuE and neurons. Time-dependent histology and electrophysiology studies further reveal a structurally and functionally stable interface with the neuronal and glial networks shortly following implantation, thus opening opportunities for next-generation brain–machine interfaces. Finally, the NeuE subcellular structural features are shown to facilitate migration of endogenous neural progenitor cells, thus holding promise as an electrically active platform for transplantation-free regenerative medicine.  

4.2041           Neuroinflammation in the pathogenesis of axonal Charcot-Marie-Tooth disease caused by lack of GDAP1

Fernandez-Lizarbe, S., Civera-Tregon, A., Cantarero, l., Herrer, I., Juarez, P., Hoenicka, J and Palau, F. Exp. Neurol., 320, 113004 (2019)   Mutations in the GDAP1 mitochondrial outer membrane gene cause Charcot-Marie-Tooth (CMT) neuropathy. Reduction or absence of GDAP1 has been associated with abnormal changes in the mitochondrial morphology and dynamics, oxidative stress and changes in calcium homeostasis. Neuroinflammation has been described in rodent models of genetic demyelinating CMT neuropathies but not in CMT primarily associated with axonopathy. Inflammatory processes have also been related to mitochondrial changes and oxidative stress in central neurodegenerative disorders. Here we investigated the presence of neuroinflammation in the axonal neuropathy of the Gdap1^−/− mice. We showed by transcriptome profile of spinal cord and the in vivo detection of activated phagocytes that the absence of GDAP1 is associated with upregulation of inflammatory pathways. We observed reactive gliosis in spinal cord with increase of the astroglia markers GFAP and S100B, and the microglia marker IBA1. Additionally, we found significant increase of inflammatory mediators such as TNF-α and pERK, and C1qa and C1qb proteins of the complement system. Importantly, we observed an increased expression of CD206 and CD86 as M2 and M1 microglia and macrophage response markers, respectively, in Gdap1^−/− mice. These inflammatory changes were also associated with abnormal molecular changes in synapses. In summary, we demonstrate that inflammation in spinal cord and sciatic nerve, but not in brain and cerebellum, is part of the pathophysiology of axonal GDAP1-related CMT.  

4.2042           An optimised tissue disaggregation and data processing pipeline for characterising fibroblast phenotypes using single-cell RNA sequencing

Waise, S., parker, R., Rose-Zerilli, M.J.J., Layfiled, D.M., Wood, O., West, J., Ottensmeier, C.H., Thomas, G.J. and Hanley, C.J. Scientific Reports, 9:9580 (2019)   Single-cell RNA sequencing (scRNA-Seq) provides a valuable platform for characterising multicellular ecosystems. Fibroblasts are a heterogeneous cell type involved in many physiological and pathological processes, but remain poorly-characterised. Analysis of fibroblasts is challenging: these cells are difficult to isolate from tissues, and are therefore commonly under-represented in scRNA-seq datasets. Here, we describe an optimised approach for fibroblast isolation from human lung tissues. We demonstrate the potential for this procedure in characterising stromal cell phenotypes using scRNA-Seq, analyse the effect of tissue disaggregation on gene expression, and optimise data processing to improve clustering quality. We also assess the impact of in vitro culture conditions on stromal cell gene expression and proliferation, showing that altering these conditions can skew phenotypes.  

4.2043           Single-Cell Analysis of the Liver Epithelium Reveals Dynamic Heterogeneity and an Essential Role for YAP in Homeostasis and Regeneration

Pepe-Mooney, B.J., Dill, M.T., Alemany, A., Shalek, A.K., van Oudenaarden, A. and Camargo, F.D. Cell Stem Cell, 25, 23-38 (2019)   The liver can substantially regenerate after injury, with both main epithelial cell types, hepatocytes and biliary epithelial cells (BECs), playing important roles in parenchymal regeneration. Beyond metabolic functions, BECs exhibit substantial plasticity and in some contexts can drive hepatic repopulation. Here, we performed single-cell RNA sequencing to examine BEC and hepatocyte heterogeneity during homeostasis and after injury. Instead of evidence for a transcriptionally defined progenitor-like BEC cell, we found significant homeostatic BEC heterogeneity that reflects fluctuating activation of a YAP-dependent program. This transcriptional signature defines a dynamic cellular state during homeostasis and is highly responsive to injury. YAP signaling is induced by physiological bile acids (BAs), required for BEC survival in response to BA exposure, and is necessary for hepatocyte reprogramming into biliary progenitors upon injury. Together, these findings uncover molecular heterogeneity within the ductal epithelium and reveal YAP as a protective rheostat and regenerative regulator in the mammalian liver.  

4.2044           The Cytoplasmic DNA Sensor cGAS Promotes Mitotic Cell Death

Zierhut, c., Yamaguchi, N., Paredes, M., Luo, J-D., Carroll, T. and Funabiki, H. Cell, 178, 302-315 (2019)   Pathogenic and other cytoplasmic DNAs activate the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway to induce inflammation via transcriptional activation by IRF3 and nuclear factor κB (NF-κB), but the functional consequences of exposing cGAS to chromosomes upon mitotic nuclear envelope breakdown are unknown. Here, we show that nucleosomes competitively inhibit DNA-dependent cGAS activation and that the cGAS-STING pathway is not effectively activated during normal mitosis. However, during mitotic arrest, low level cGAS-dependent IRF3 phosphorylation slowly accumulates without triggering inflammation. Phosphorylated IRF3, independently of its DNA-binding domain, stimulates apoptosis through alleviating Bcl-xL-dependent suppression of mitochondrial outer membrane permeabilization. We propose that slow accumulation of phosphorylated IRF3, normally not sufficient for inducing inflammation, can trigger transcription-independent induction of apoptosis upon mitotic aberrations. Accordingly, expression of cGAS and IRF3 in cancer cells makes mouse xenograft tumors responsive to the anti-mitotic agent Taxol. The Cancer Genome Atlas (TCGA) datasets for non-small cell lung cancer patients also suggest an effect of cGAS expression on taxane response.  

4.2045           Inhibition of allogeneic islet graft rejection by VISTA-conjugated liposome

Guo, M., Han, S., Liu, Y., Guo, W., Zhao, Y., Liu, F., Shi, X., Ding, G. and Wang, Q. Biochem. Biophys. Res. Comm., 516, 914-920 (2019)   The Ig superfamily member V-domain Ig-containing suppressor of T-cell activation (VISTA) is a negative regulator with broad-spectrum activities and has reported that blockade of VISTA or combination with other negative checkpoint receptors sufficiently break tumor tolerance. However, it remains unclear whether VISTA could induce allogeneic T-cell hyporesponsiveness and inhibit allograft rejection. Here we found VISTA treatment significantly inhibited lymphocyte proliferation and activation in allogeneic MLR assay through impairing SYK-VAV pathway. Interestingly, though neither VISTA protein nor VISTA-Fc fusion protein administration exerted satisfactory immunosuppressive effect on allograft survival due to their short half-life in circulation, this problem was solved by conjugating VISTA protein on liposome by biotin-streptavidin system, which markedly prolonged its circulating half-life to 60 h. With islet transplant model, administration of VISTA-conjugated liposome could markedly prolong allograft survival by inhibition of SYK-VAV pathway, thus maintained the normal blood glucose level of recipients during treatment period. The results indicate VISTA is a promising therapeutic target to treat allograft rejection of islet transplantation.  

4.2046           A Multi-center Study on the Reproducibility of Drug-Response Assays in Mammalian Cell Lines

Niepel, M., hafner, m., Mills, C.E., Birtwistle, M.R., heiser, L.M. and Sorger, P.K. Cell Systems, 9, 35-48 (2019)   Evidence that some high-impact biomedical results cannot be repeated has stimulated interest in practices that generate findable, accessible, interoperable, and reusable (FAIR) data. Multiple papers have identified specific examples of irreproducibility, but practical ways to make data more reproducible have not been widely studied. Here, five research centers in the NIH LINCS Program Consortium investigate the reproducibility of a prototypical perturbational assay: quantifying the responsiveness of cultured cells to anti-cancer drugs. Such assays are important for drug development, studying cellular networks, and patient stratification. While many experimental and computational factors impact intra- and inter-center reproducibility, the factors most difficult to identify and control are those with a strong dependency on biological context. These factors often vary in magnitude with the drug being analyzed and with growth conditions. We provide ways to identify such context-sensitive factors, thereby improving both the theory and practice of reproducible cell-based assays.  

4.2047           Synaptogenesis Stimulates a Proteasome-Mediated Ribosome Reduction in Axons

Costa, R.O., Martins, H., Martins, L.F., Cancedda, L., Jaffrey, S.R. and Almeida, R.D. Cell Reports, 28, 864-876 (2019)   Ribosomes and a subset of cellular mRNAs are trafficked into axons of developing neurons. The axonal localization of translational machinery allows new proteins to be rapidly and locally synthesized during axonal growth and pathfinding. However, in mature neurons, axonal ribosomes are significantly reduced or even absent. The mechanism that elicits this removal is currently unknown. Here, we demonstrate that synapse formation is the trigger for ribosome reduction in mature axons. In vivo analysis shows that axonal ribosome levels decrease in rat brain at a developmental stage coincident with synapse formation. Next, we observe in vitro that different synaptogenic inducers trigger an overall decrease of ribosomal proteins and rRNA in the axons of spinal motor neurons. We further observe that this process is dependent on the ubiquitin-proteasome system but not on autophagy. Together, these data identify synaptogenesis as the long missing biological trigger that leads to ribosome disappearance during axonal maturation.  

4.2048           Class IIa HDACs regulate learning and memory through dynamic experience-dependent repression of transcription

Zhu, Y., Huang, M., Bushong, E., Phan, S., Uytiepo, M., beutter, E., Boemer, D., Tsui, K., Ellisman, M. and Maximov, A. Nature Communications, 10:3469 (2019)   The formation of new memories requires transcription. However, the mechanisms that limit signaling of relevant gene programs in space and time for precision of information coding remain poorly understood. We found that, during learning, the cellular patterns of expression of early response genes (ERGs) are regulated by class IIa HDACs 4 and 5, transcriptional repressors that transiently enter neuronal nuclei from cytoplasm after sensory input. Mice lacking these repressors in the forebrain have abnormally broad experience-dependent expression of ERGs, altered synaptic architecture and function, elevated anxiety, and severely impaired memory. By acutely manipulating the nuclear activity of class IIa HDACs in behaving animals using a chemical-genetic technique, we further demonstrate that rapid induction of transcriptional programs is critical for memory acquisition but these programs may become dispensable when a stable memory is formed. These results provide new insights into the molecular basis of memory storage.  

4.2049           Reversibility of Age-related Oxidized Free NADH Redox States in Alzheimer’s Disease Neurons by Imposed External Cys/CySS Redox Shifts

Dong, Y., Sameni, S., Digman, M.A. and Brewer, G.J. Scientific Reports, 9:11274 (2019)   Redox systems including extracellular cysteine/cystine (Cys/CySS), intracellular glutathione/oxidized glutathione (GSH/GSSG) and nicotinamide adenine dinucleotide reduced/oxidized forms (NADH/NAD⁺) are critical for maintaining redox homeostasis. Aging as a major risk factor for Alzheimer’s disease (AD) is associated with oxidative shifts, decreases in anti-oxidant protection and dysfunction of mitochondria. Here, we examined the flexibility of mitochondrial-specific free NADH in live neurons from non-transgenic (NTg) or triple transgenic AD-like mice (3xTg-AD) of different ages under an imposed extracellular Cys/CySS oxidative or reductive condition. We used phasor fluorescence lifetime imaging microscopy (FLIM) to distinguish free and bound NADH in mitochondria, nuclei and cytoplasm. Under an external oxidative stress, a lower capacity for maintaining mitochondrial free NADH levels was found in old compared to young neurons and a further decline with genetic load. Remarkably, an imposed Cys/CySS reductive state rejuvenated the mitochondrial free NADH levels of old NTg neurons by 71% and old 3xTg-AD neurons by 89% to levels corresponding to the young neurons. Using FLIM as a non-invasive approach, we were able to measure the reversibility of aging subcellular free NADH levels in live neurons. Our results suggest a potential reductive treatment to reverse the loss of free NADH in old and Alzheimer’s neurons.      

4.2050           Glial pathology and retinal neurotoxicity in the anterior visual pathway in experimental autoimmune encephalomyelitis

Jin, J., Smith, M.D., Kersbergen, C.J., Kam, T-i., Viswanathan, M., Martin, K., Dawson, T.M., Dawson, V., Zack, D.J., Whartenby, K. and Calebresi, P.A. Acta Neuuropathol. Comm., 7:125 (2019)   The animal model experimental autoimmune encephalomyelitis (EAE) has been used extensively in the past to test mechanisms that target peripheral immune cells for treatment of multiple sclerosis (MS). While there have been some notable successes in relapsing MS, the development of therapies for progressive multiple sclerosis (MS) has been hampered by lack of an appropriate animal model. Further, the mechanisms underlying CNS inflammation and neuronal injury remain incompletely elucidated. It is known that the MOG _35–55 EAE mouse model does not have insidious behavioral progression as occurs in people with MS, but there is significant neuronal and axonal injury in EAE, as a result of the inflammation. In the present study, we describe the time course of glial activation and retinal neurodegeneration in the EAE model, and highlight the utility of studying the anterior visual pathway for modeling mechanisms of neuronal injury that may recapitulate critical aspects of the pathology described in people with MS following optic neuritis and subclinical optic neuropathy. We show that A1 neurotoxic astrocytes are prevalent in optic nerve tissue and retina, and are associated with subsequent RGC loss in the most commonly used form of the EAE model induced by MOG _35–55 peptide in C57/B6 mice. We developed a semi-automatic method to quantify retinal ganglion cells (RGC) and show that RGCs remain intact at peak EAE (PID 16) but are significantly reduced in late EAE (PID 42). Postsynaptic proteins and neurites were also compromised in the retina of late EAE mice. The retinal pathology manifests weeks after the microglial and astrocyte activation, which were prominent in optic nerve tissues at PID 16. Microglia expressed iNOS and had increased gene expression of C1q, TNF-α, and IL-1α. Astrocytes expressed high levels of complement component 3 and other genes associated with A1 neurotoxic astrocytes. Our data suggest that EAE can be used to study the pathobiology of optic neuropathy and to examine the preclinical neuroprotective effects of drugs that target activation of neurotoxic A1 astrocytes.  

4.2051           Antibody repertoire analysis of mouse immunization protocols using microfluidics and molecular genomics

Asensio, M.A., Lim, Y.W., Wayham, N., Stadtmiller, K., Edgar, R.C., Leong, J., Leong, R., Mizrahi, R.A., Adams, M.S., Simons, J.F., Spindler, M.J., Johnson, D.S. and Adler, A.S. mAbs, 11(5), 870-883 (2019)   Immunization of mice followed by hybridoma or B-cell screening is one of the most common antibody discovery methods used to generate therapeutic monoclonal antibody (mAb) candidates. There are a multitude of different immunization protocols that can generate an immune response in animals. However, an extensive analysis of the antibody repertoires that these alternative immunization protocols can generate has not been performed. In this study, we immunized mice that transgenically express human antibodies with either programmed cell death 1 protein or cytotoxic T-lymphocyte associated protein 4 using four different immunization protocols, and then utilized a single cell microfluidic platform to generate tissue-specific, natively paired immunoglobulin (Ig) repertoires from each method and enriched for target-specific binders using yeast single-chain variable fragment (scFv) display. We deep sequenced the scFv repertoires from both the pre-sort and post-sort libraries. All methods and both targets yielded similar oligoclonality, variable (V) and joining (J) gene usage, and divergence from germline of enriched libraries. However, there were differences between targets and/or immunization protocols for overall clonal counts, complementarity-determining region 3 (CDR3) length, and antibody/CDR3 sequence diversity. Our data suggest that, although different immunization protocols may generate a response to an antigen, performing multiple immunization protocols in parallel can yield greater Ig diversity. We conclude that modern microfluidic methods, followed by an extensive molecular genomic analysis of antibody repertoires, can be used to quickly analyze new immunization protocols or mouse platforms.  

4.2052           A predicted Francisella tularensis DXD-motif glycosyltransferase blocks immune activation

Nau, G.J., Horzemba, J., O’Dee, D., Brown, M.J., Russo, B.C., Hernandez, A., Dillon, S.T., Cheng, J., Kane, L.P., Sanker, S. and Hukriede, N.A. Virulence, 10(1), 643-656 (2019)   Pathogens enhance their survival during infections by manipulating host defenses. Francisella tularensis evades innate immune responses, which we have found to be dependent on an understudied gene ybeX (FTL_0883/FTT_0615c). To understand the function of YbeX, we sought protein interactors in F. tularensis subsp. holarctica live vaccine strain (LVS). An unstudied Francisella protein co-immunoprecipitated with recombinant YbeX, which is a predicted glycosyltransferase with a DXD-motif. There are up to four genomic copies of this gene with identical sequence in strains of F. tularensis pathogenic to humans, despite ongoing genome decay. Disruption mutations were generated by intron insertion into all three copies of this glycosyltransferase domain containing gene in LVS, gdcA_1-3. The resulting strains stimulated more cytokines from macrophages in vitro than wild-type LVS and were attenuated in two in vivo infection models. GdcA was released from LVS during culture and was sufficient to block NF-κB activation when expressed in eukaryotic cells. When co-expressed in zebrafish, GdcA and YbeX were synergistically lethal to embryo development. Glycosyltransferases with DXD-motifs are found in a variety of pathogens including NleB, an Escherichia coli type-III secretion system effector that inhibits NF-κB by antagonizing death receptor signaling. To our knowledge, GdcA is the first DXD-motif glycosyltransferase that inhibits NF-κB in immune cells. Together, these findings suggest DXD-motif glycosyltransferases may be a conserved virulence mechanism used by pathogenic bacteria to remodel host defenses.  

4.2053           IL‐7‐dependent compositional changes within the γδ T cell pool in lymph nodes during ageing lead to an unbalanced anti‐tumour response

Chen, H-C., Eling, N., Martinez-Jimenez, C.P., McNeill O’Brian, L., Carbonara, V., Marioni, J.C., Odom, D.T. and de la Roche, M. EMBO Reports, 20, e47379 (2019)   How the age‐associated decline of immune function leads to increased cancer incidence is poorly understood. Here, we have characterised the cellular composition of the γδ T‐cell pool in peripheral lymph nodes (pLNs) upon ageing. We find that ageing has minimal cell‐intrinsic effects on function and global gene expression of γδ T cells, and γδTCR diversity remains stable. However, ageing alters TCRδ chain usage and clonal structure of γδ T‐cell subsets. Importantly, IL‐17‐producing γδ17 T cells dominate the γδ T‐cell pool of aged mice—mainly due to the selective expansion of Vγ6⁺ γδ17 T cells and augmented γδ17 polarisation of Vγ4⁺ T cells. Expansion of the γδ17 T‐cell compartment is mediated by increased IL‐7 expression in the T‐cell zone of old mice. In a Lewis lung cancer model, pro‐tumourigenic Vγ6⁺ γδ17 T cells are exclusively activated in the tumour‐draining LN and their infiltration into the tumour correlates with increased tumour size in aged mice. Thus, upon ageing, substantial compositional changes in γδ T‐cell pool in the pLN lead to an unbalanced γδ T‐cell response in the tumour that is associated with accelerated tumour growth.  

4.2054           Blocking Triggering Receptor Expressed on Myeloid Cells‐1‐Positive Tumor‐Associated Macrophages Induced by Hypoxia Reverses Immunosuppression and Anti‐Programmed Cell Death Ligand 1 Resistance in Liver Cancer

Wu, Q., Zhou, W., Yin, S., Zhou, Y., Chen, T., Qian, J., Su, R., Hong, L., Lu, H., Zhang, F., Xie, H., Zhou, L. and Zheng, S. Hepatology, 70(1), 198-214 (2019)   Tumor‐associated macrophages (TAMs) are recognized as antitumor suppressors, but how TAMs behave in the hypoxic environment of hepatocellular carcinoma (HCC) remains unclear. Here, we demonstrated that hypoxia inducible factor 1α induced increased expression of triggering receptor expressed on myeloid cells‐1 (TREM‐1) in TAMs, resulting in immunosuppression. Specifically, TREM‐1‐positive (TREM‐1⁺) TAMs abundant at advanced stages of HCC progression indirectly impaired the cytotoxic functions of CD8⁺ T cells and induced CD8⁺ T‐cells apoptosis. Biological and functional assays showed that TREM‐1⁺ TAMs had higher expression of programmed cell death ligand 1 (PD‐L1) under hypoxic environment. However, TREM‐1⁺ TAMs could abrogate spontaneous and PD‐L1‐blockade‐mediated antitumor effects in vivo, suggesting that TREM‐1⁺ TAM‐induced immunosuppression was dependent on a pathway separate from PD‐L1/programmed cell death 1 axis. Moreover, TREM‐1⁺ TAM‐associated regulatory T cells (Tregs) were crucial for HCC resistance to anti‐PD‐L1 therapy. Mechanistically, TREM‐1⁺ TAMs elevated chemokine (C‐C motif) ligand 20 expression through the extracellular signal‐regulated kinase/NF‐κβ pathway in response to hypoxia and tumor metabolites leading to CCR6⁺Foxp3⁺ Treg accumulation. Blocking the TREM‐1 pathway could significantly inhibit tumor progression, reduce CCR6⁺Foxp3⁺ Treg recruitment, and improve the therapeutic efficacy of PD‐L1 blockade. Thus, these data demonstrated that CCR6⁺Foxp3⁺ Treg recruitment was crucial for TREM‐1⁺ TAM‐mediated anti‐PD‐L1 resistance and immunosuppression in hypoxic tumor environment. Conclusion: This study highlighted that the hypoxic environment initiated the onset of tumor immunosuppression through TREM‐1⁺ TAMs attracting CCR6⁺Foxp3⁺ Tregs, and TREM‐1⁺ TAMs endowed HCC with anti‐PD‐L1 therapy resistance.  

4.2055           The affinity, intrinsic activity and selectivity of a structurally novel EP2 receptor agonist at human prostanoid receptors

Coleman, R.A., Woodrooffe, A.J., Clark, K.L., Toris, C.B., Fan, S., Wang, J.W. and Woodward, D.F. Br. J. Pharmacol., 176, 687-698 (2019)   Background and Purpose Prostanoid EP₂ receptor agonists exhibit several activities including ocular hypotension, tocolysis and anti‐inflammatory activity. This report describes the affinity and selectivity of a structurally novel, non‐prostanoid EP₂ receptor agonist, PGN‐9856, and its therapeutic potential. Experimental Approach The pharmacology of a series of non‐prostanoid EP₂ receptor agonists was determined according to functional and radioligand binding studies, mostly using human recombinant prostanoid receptor transfectants. The selectivity of PGN‐9856, as the preferred compound, was subsequently determined by using a diverse variety of non‐prostanoid target proteins. The therapeutic potential of PGN‐9856 was addressed by determining its activity in relevant primate cell, tissue and disease models. Key Results PGN‐9856 was a selective and high affinity (pKi ≥ 8.3) ligand at human recombinant EP₂ receptors. In addition to high affinity binding, it was a potent and full EP₂ receptor agonist with a high level of selectivity at EP₁, EP₃, EP₄, DP, FP, IP and TP receptors. In cells overexpressing human recombinant EP₂ receptors, PGN‐9856 displayed a potency (pEC₅₀≥ 8.5) and a maximal response (increase in cAMP) comparable to that of the endogenous agonist PGE₂. PGN‐9856 exhibited no appreciable affinity (up 10 μM) for a range of 53 other receptors, ion channels and enzymes. Finally, PGN‐9856 exhibited tocolytic, anti‐inflammatory and long‐acting ocular hypotensive properties consistent with its potent EP₂ receptor agonist properties. Conclusions and Implications PGN‐9856 is a potent, selective and efficacious prostanoid EP₂ receptor agonist with diverse potential therapeutic applications: tocolytic, anti‐inflammatory and notably anti‐glaucoma.  

4.2056           Characterization of the Subventricular-Thalamo-Cortical Circuit in the NP-C Mouse Brain, and New Insights Regarding Treatment

Park, M.H., Choi, B.J., Jeong, M.S., Lee, J.Y., Jung, I.K., Park, K.H., Lee, H.W., Yamaguchi, T., Marti, H.H., Lee, B.H., Schuchman, E.H., Jin, H.K. and Bae, J-s. Molecular Therapy, 37(8), 1507-1526 (2019)   Gliosis in Niemann-Pick type C (NP-C) disease is characterized by marked changes in microglia and astrocytes. However, the gliosis onset and progression in NP-C has not been systematically studied, nor has the mechanism underlying this finding. Here, we found early gliosis in the subventricular zone (SVZ) of NP-C mice. Neural progenitor damage by Npc1 mutation suppressed vascular endothelial growth factor (VEGF) expression and further induced microglia activation followed by astrogliosis. Interestingly, excessive astrogliosis in the SVZ induced neural progenitor retention and/or migration into thalamus via astrocyte-derived VEGF, resulting in acceleration of thalamic and cortical gliosis through thalamo-cortical pathways. Transplantation of VEGF-overexpressing neural stem cells into the SVZ improved whole-brain pathology of NP-C mice. Overall, our data provide a new pathological perspective on NP-C neural pathology, revealing abnormalities in the subventricular-thalamo-cortical circuit of NP-C mouse brain and highlighting the importance of the SVZ microenvironment as a therapeutic target for NP-C disease.  

4.2057           An upstream enhancer regulates Gpihbp1 expression in a tissue-specific manner

Allan, C.M., Heizer, P.J., Tu, Y., Sandoval, N.P., Jung, R.S., Morales, J.E., Sajti, E., Troutman, T.D., Saunders, T.L., Cusanovich, D.A., Beigneux, A.P., Romanski, C.E., Fong, L.G. and Young, S.G.

1. Lipid Res., 60, 869-879 (2019)

  Glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 (GPIHBP1), the protein that shuttles LPL to the capillary lumen, is essential for plasma triglyceride metabolism. When GPIHBP1 is absent, LPL remains stranded within the interstitial spaces and plasma triglyceride hydrolysis is impaired, resulting in severe hypertriglyceridemia. While the functions of GPIHBP1 in intravascular lipolysis are reasonably well understood, no one has yet identified DNA sequences regulating GPIHBP1 expression. In the current studies, we identified an enhancer element located ∼3.6 kb upstream from exon 1 of mouse Gpihbp1. To examine the importance of the enhancer, we used CRISPR/Cas9 genome editing to create mice lacking the enhancer (Gpihbp1^Enh/Enh). Removing the enhancer reduced Gpihbp1 expression by >90% in the liver and by ∼50% in heart and brown adipose tissue. The reduced expression of GPIHBP1 was insufficient to prevent LPL from reaching the capillary lumen, and it did not lead to hypertriglyceridemia—even when mice were fed a high-fat diet. Compound heterozygotes (Gpihbp1^Enh/− mice) displayed further reductions in Gpihbp1 expression and exhibited partial mislocalization of LPL (increased amounts of LPL within the interstitial spaces of the heart), but the plasma triglyceride levels were not perturbed. The enhancer element that we identified represents the first insight into DNA sequences controlling Gpihbp1 expression.  

4.2058           Transitional human alveolar type II epithelial cells suppress extracellular matrix and growth factor gene expression in lung fibroblasts

Correll, K.A., Edeen, K.E., Zemans, R.L., Redente, E.F., Serban, K.A., Curran-Everett, D., Edelman, B.L., Mikels-Vigdal, A. and Mason, R.J. Am. J. Physiol. Lung Cell Mol. Physiol., 317, L283-L294 (2019)   Epithelial-fibroblast interactions are thought to be very important in the adult lung in response to injury, but the specifics of these interactions are not well defined. We developed coculture systems to define the interactions of adult human alveolar epithelial cells with lung fibroblasts. Alveolar type II cells cultured on floating collagen gels reduced the expression of type 1 collagen (COL1A1) and α-smooth muscle actin (ACTA2) in fibroblasts. They also reduced fibroblast expression of hepatocyte growth factor (HGF), fibroblast growth factor 7 (FGF7, KGF), and FGF10. When type II cells were cultured at an air-liquid interface to maintain high levels of surfactant protein expression, this inhibitory activity was lost. When type II cells were cultured on collagen-coated tissue culture wells to reduce surfactant protein expression further and increase the expression of some type I cell markers, the epithelial cells suppressed transforming growth factor-β (TGF-β)-stimulated ACTA2 and connective tissue growth factor (CTGF) expression in lung fibroblasts. Our results suggest that transitional alveolar type II cells and likely type I cells but not fully differentiated type II cells inhibit matrix and growth factor expression in fibroblasts. These cells express markers of both type II cells and type I cells. This is probably a normal homeostatic mechanism to inhibit the fibrotic response in the resolution phase of wound healing. Defining how transitional type II cells convert activated fibroblasts into a quiescent state and inhibit the effects of TGF-β may provide another approach to limiting the development of fibrosis after alveolar injury.  

4.2059           Native State Single-Cell Printing System and Analysis for Matrix Effects

Li, Q., Tang, F., Huo, X., Huang, X., Zhang, Y., Wang, X. and Zhang, X. Anal. Chem., 91(13), 8115-8122 (2019)   Mass spectrometry is subject to matrix effects, which causes severe limitations on the analysis of live single cells in their native state. Here, we propose a three-phase droplet-based single-cell printing analysis system (TP-SCP), which can package, extract, separate, print, and analyze live single cells in saline matrixes (such as phosphate buffered saline) with matrix-assisted laser desorption/ionization mass spectrometry. This method can eliminate matrix effects to obtain information on a single cell in their native state. We report that a cell packaging percentage of 44% and single-cell packaging percentage of 88% can be achieved by TP-SCP. The system was capable of processing three to four single cells per second, which was 30 to 40 times higher than the traditional droplet-based microextraction (about 10 s/cell). Additionally, the MCF-7, A2780, 293, and 4T1 cells were screened in our system. The effect of cell viability and heterogeneity analysis was investigated, suggesting that the concentration of monounsaturated phosphatidylinositol and phosphatidylethanolamine both increase in cancer cells. Compared with conventional mass spectrometry, TP-SCP can ensure the accuracy of heterogeneity analysis of live single cells in their native state. Both a principal component analysis and a linear discriminant analysis were used to perform classification and identification of cells with an accuracy of 100%. This method provides an innovative framework for research on cell quality control, cell biology, cancer diagnosis, and prevention.  

4.2060           Flow cytometric analysis of the leukocyte landscape during bleomycin-induced lung injury and fibrosis in the rat

Kloth, C., Gruben, N., Ochs, M., Knudsen, L. and Lopez-Rodriguez, E. Am. J. Physiol. Lung Cell. Mol. Physiol., 317, L109-L126 (2019)   Bleomycin-induced lung injury and fibrosis is a well-described model to investigate lung inflammatory and remodeling mechanisms. Rat models are clinically relevant and are also widely used, but rat bronchoalveolar lavage (BAL) cells are not fully characterized with flow cytometry due to the limited availability of antibodies for this species. We optimized a comprehensive time-dependent flow cytometric analysis of cells after bleomycin challenge, confirming previous studies in other species and correlating them to histological staining, cytokine profiling, and collagen accumulation analysis in rat lungs. For this purpose, we describe a novel panel of rat surface markers and a strategy to identify and follow BAL cells over time. By combining surface markers in rat alveolar cells (CD45⁺), granulocytes and other myeloid cells, monocytes and macrophages can be identified by the expression of CD11b/c. Moreover, different activation states of macrophages (CD163⁺) can be observed: steady state (CD86⁻MHC-II^low), activation during inflammation (CD86⁺,MHC-II^high), activation during remodeling (CD86⁺MHC-II^low), and a population of newly recruited monocytes (CD163⁻α-granulocyte⁻). Hydroxyproline measured as marker of collagen content in lung tissue showed positive correlation with the reparative phase (CD163⁻ cells and tissue inhibitor of metalloproteinases (TIMP) and IL-10 increase). In conclusion, after a very early granulocytic recruitment, inflammation in rat lungs is observed by activated macrophages, and high release of IL-6 and fibrotic remodeling is characterized by recovery of the macrophage population together with TIMP, IL-10, and IL-18 production. Recruited monocytes and a second peak of granulocytes appear in the transitioning phase, correlating with immunostaining of arginase-1 in the tissue, revealing the importance of events leading the changes from injury to aberrant repair.  

4.2061           Distinguishing cancer cell lines at a single living cell level via detection of sialic acid by dual-channel plasmonic imaging and by using a SERS-microfluidic droplet platform

Cong, L., Liang, L., Cao, F., Sun, D., Yue, J., Xu, W., Liang, C.and Xu, S. Microchimica Acta, 186(6), 367 (2019)   A high-throughput, dual-channel single cell analytical method is described for the detection of sialic acid (SA) on single cell based on the use of microfluidic droplets integrated with plasmonic imaging and surface-enhanced Raman spectroscopy (SERS) with the assistance of a multifunctional metal nanoparticle-based probe. The multifunctional plasmonic nanoprobe was prepared by modifying silver nanoparticles (AgNPs) with 4-mercaptophenylboronic acid (MPBA) that both warrants SA recognition and acts as a Raman reporter. This nanoprobe is a high-contrast indicator under bright field imaging due to the strong energy loss feature of AgNPs, and also owns possesses a strong SERS enhancement capability toward MPBA. Cells incubated with the plasmonic nanoprobes were isolated in water-in-oil droplets and then were re-dispersed in a chamber array chip. High-precision profiles of SA on a single cell in one droplet were obtained by the bright field imaging and image processing. The SA expression levels on different cell lines (MCF-7, HepG2, SGC and BNL.CL2) traced by SERS spectroscopy were compared. The statistical data among different cell lines confirm that the SA expression levels on cancer cells are much higher than that on normal cells. Single cell analysis further revealed that the cell-to-cell variations are more obvious in cancer cell lines. This study provides a valuable tool for understanding glycan-related biochemical processes.  

4.2062           IL‐25 protects against high‐fat diet‐induced hepatic steatosis in mice by inducing IL‐25 and M2a macrophage production

Zheng, X-L., Wu, J-P., Gong, Y., Hong, J-B., Xiao, H-Y., Zhong, J-W., Xie, B., Li, B-M., Guo, G.H., Zhu, X. and Wang, A-J. Immunol. Cell Biol., 97(2), 165-177 (2019)   Interleukin (IL)‐25 is a cytokine that has previously been shown to have a protective role against nonalcoholic fatty liver disease (NAFLD), which is associated with the induction of M2 macrophage differentiation. However, the direct relationships between IL‐25 expression regulation, M2 induction and NAFLD remain unknown. In this study, we demonstrate that IL‐25 promotes hepatic macrophage differentiation into M2a macrophages both in vivo and in vitro via the IL‐13/STAT6 pathway. M2 macrophages that were differentiated in vitro were able to ameliorate high‐fat diet HFD‐induced hepatic steatosis. Furthermore, we found that IL‐25 treatment, both in vitro and in vivo, promotes direct binding of STAT6 to the IL‐25 gene promoter region. This binding of STAT6 in response to IL‐25 treatment also resulted in the increase of IL‐25 expression in hepatocytes. Together, these findings identify IL‐25 as a protective factor against HFD‐induced hepatic steatosis by inducing an increase of IL‐25 expression in hepatocytes and through promotion of M2a macrophage production.  

4.2063           A Stromal Niche Defined by Expression of the Transcription Factor WT1 Mediates Programming and Homeostasis of Cavity-Resident Macrophages

Buechler, M.B., Kim, K-W., Onufer, E.J., Junttila, M.R., Randolph, G.J. and Turley, S.J. Immunity, 51(1), 119-130 (2019)   Tissue-resident macrophages require specific milieus for the maintenance of defining gene-expression programs. Expression of the transcription factor GATA6 is required for the homeostasis, function and localization of peritoneal cavity-resident macrophages. Gata6 expression is maintained in a non-cell autonomous manner and is elicited by the vitamin A metabolite, retinoic acid. Here, we found that the GATA6 transcriptional program is a common feature of macrophages residing in all visceral body cavities. Retinoic acid-dependent and -independent hallmark genes of GATA6 ⁺ macrophages were induced by mesothelial and fibroblastic stromal cells that express the transcription factor Wilms’ Tumor 1 (WT1), which drives the expression of two rate-limiting enzymes in retinol metabolism. Depletion of Wt1 ⁺ stromal cells reduced the frequency of GATA6 ⁺ macrophages in the peritoneal, pleural and pericardial cavities. Thus, Wt1 ⁺ mesothelial and fibroblastic stromal cells constitute essential niche components supporting the tissue-specifying transcriptional landscape and homeostasis of cavity-resident macrophages.  

4.2064           IRF4-dependent dendritic cells regulate CD8+ T-cell differentiation and memory responses in influenza infection

Ainsua-Enrich, E., Hatipouglu, I., Kaaclei, S., Turner, S., Paul, J., Singh, S., Bagavant, H. and Kovats, S. Mucosal Immunol., 12, 1025-1037 (2019)   Acute respiratory disease caused by influenza viruses is imperfectly mitigated by annual vaccination to select strains. Development of vaccines that elicit lung-resident memory CD8⁺ T cells (T_RM) would offer more universal protection to seasonal and emerging pandemic viruses. Understanding how lung-resident dendritic cells (DCs) regulate T_RM differentiation would be an important step in this process. Here, we used CD11c-cre-Irf4^f/f (KO) mice, which lack lung-resident IRF4-dependent CD11b⁺CD24^hi DCs and show IRF4 deficiency in other lung cDC subsets, to determine if IRF4-expressing DCs regulate CD8⁺ memory precursor cells and T_RM during influenza A virus (IAV) infection. KO mice showed defective CD8⁺ T-cell memory, stemming from a deficit of T regulatory cells and memory precursor cells with decreased Foxo1 expression. Transfer of wild-type CD11b⁺CD24^hi DCs into KO mice restored CD8⁺ memory precursor cell numbers to wild-type levels. KO mice recovered from a primary infection harbored reduced numbers of CD8⁺ T_RM and showed deficient expansion of IFNγ⁺CD8⁺ T cells and increased lung pathology upon challenge with heterosubtypic IAV. Thus, vaccination strategies that harness the function of IRF4-dependent DCs could promote the differentiation of CD8⁺ T_RM during IAV infection.  

4.2065           A temporal thymic selection switch and ligand binding kinetics constrain neonatal Foxp3+ Treg cell development

Stadinski, B.D., Blevins, S.J., Spidale, N.A., Duke, B.R., Huseby, P.G., Stern, L.J. and Huseby, E.S. Nature Immunol., 20, 1046-1058 (2019)   The neonatal thymus generates Foxp3⁺ regulatory T (tT_reg) cells that are critical in controlling immune homeostasis and preventing multiorgan autoimmunity. The role of antigen specificity on neonatal tT_reg cell selection is unresolved. Here we identify 17 self-peptides recognized by neonatal tT_reg cells, and reveal ligand specificity patterns that include self-antigens presented in an age- and inflammation-dependent manner. Fate-mapping studies of neonatal peptidyl arginine deiminase type IV (Padi4)-specific thymocytes reveal disparate fate choices. Neonatal thymocytes expressing T cell receptors that engage IA^b-Padi4 with moderate dwell times within a conventional docking orientation are exported as tT_reg cells. In contrast, Padi4-specific T cell receptors with short dwell times are expressed on CD4⁺ T cells, while long dwell times induce negative selection. Temporally, Padi4-specific thymocytes are subject to a developmental stage-specific change in negative selection, which precludes tT_reg cell development. Thus, a temporal switch in negative selection and ligand binding kinetics constrains the neonatal tT_reg selection window.  

4.2066           Early life alcohol exposure primes hypothalamic microglia to later-life hypersensitivity to immune stress: possible epigenetic mechanism

Chastain, L.G., Franklin, T., gangisetty, O., Cabrera, M.A., Mukherjee, S., Shrivastava, P., Jabbar, S. and Sarkar, D.K. Neuropsychopharmacol., 44, 1579-1588 (2019)   Growing evidence has shown that developmental alcohol exposure induces central nervous system inflammation and microglia activation, which may contribute to long-term health conditions, such as fetal alcohol spectrum disorders. These studies sought to investigate whether neonatal alcohol exposure during postnatal days (PND) 2–6 in rats (third trimester human equivalent) leads to long-term disruption of the neuroimmune response by microglia. Exposure to neonatal alcohol resulted in acute increases in activation and inflammatory gene expression in hypothalamic microglia including tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). Adults with neonatal alcohol pre-exposure (alcohol fed; AF) animals showed an exaggerated peripheral stress hormonal response to an immune challenge (lipopolysaccharides; LPS). In addition, there were significantly more microglia present in the hypothalamus of adult AF animals, and their hypothalamic microglia showed more cluster of differentiation molecule 11b (Cd11b) activation, TNF-α expression, and IL-6 expression in response to LPS. Interestingly, blocking microglia activation with minocycline treatment during PND 2–6 alcohol exposure ameliorated the hormonal and microglial hypersensitivity to LPS in AF adult animals. Investigation of possible epigenetic programming mechanisms by alcohol revealed neonatal alcohol decreased several repressive regulators of transcription in hypothalamic microglia, while concomitantly increasing histone H3 acetyl lysine 9 (H3K9ac) enrichment at TNF-α and IL-6 promoter regions. Importantly, adult hypothalamic microglia from AF animals showed enduring increases in H3K9ac enrichment of TNF-α and IL-6 promoters both at baseline and after LPS exposure, suggesting a possible epigenetic mechanism for the long-term immune disruption due to hypothalamic microglial priming.  

4.2067           Targeting p16-induced senescence prevents cigarette smoke-induced emphysema by promoting IGF1/Akt1 signaling in mice

Cottage, C., Peterson, N., Kearley, J., Berlin, A., Xiong, X., Huntley, A., Zhao, W., Brown, C., Migneault, A., Zerrouki, K., Criner, G., Kolbeck, R., Connor, J. and Lemaire, R. Communications Biol., 2:307 (2019)   Senescence is a mechanism associated with aging that alters tissue regeneration by depleting the stem cell pool. Chronic obstructive pulmonary disease (COPD) displays hallmarks of senescence, including a diminished stem cell population. DNA damage from cigarette smoke (CS) induces senescence via the p16 pathway. This study evaluated the contribution of p16 to CS-associated lung pathologies. p16 expression was prominent in human COPD lungs compared with normal subjects. CS induces impaired pulmonary function, emphysema, and increased alveolar epithelial cell (AECII) senescence in wild-type mice, whereas CS-exposed p16^−/− mice exhibit normal pulmonary function, reduced emphysema, diminished AECII senescence, and increased pro-growth IGF1 signaling, suggesting that improved lung function in p16^−/− mice was due to increased alveolar progenitor cell proliferation. In conclusion, our study suggests that targeting senescence may facilitate alveolar regeneration in COPD emphysema by promoting IGF1 proliferative signaling.  

4.2068           Sorting by interfacial tension (SIFT): label-free selection of live cells based on single-cell metabolism

Pan, C.W., Horvath, D.G., Braza, S., Moore, T., Lynch, A., Feit, C. and Abbyad, P. Lab on a Chip, 19(8), 1317-1351 (2019)   Selection of live cells from a population is critical in many biological studies and biotechnologies. We present here a novel droplet microfluidic approach that allows for label-free and passive selection of live cells using the glycolytic activity of individual cells. It was observed that with the use of a specific surfactant utilized to stabilize droplet formation, the interfacial tension of droplets was very sensitive to pH. After incubation, cellular lactate release results in droplets containing a live cell to attain a lower pH than other droplets. This enables the sorting of droplets containing live cells when confined droplets flow over a microfabricated trench oriented diagonally with respect to the direction of flow. The technique is demonstrated with human U87 glioblastoma cells for the selection of only droplets containing a live cell while excluding either empty droplets or droplets containing a dead cell. This label-free sorting method, dubbed sorting by interfacial tension (SIFT) presents a new strategy to sort diverse cell types based on metabolic activity.  

4.2069           Microfluidic blood vasculature replicas using backside lithography

Fenech, M., Girod, V., Claveria, V., Meance, S., Abkarian, M. and Charlot, B. Lab on a Chip, 19, 2096-2106 (2019)   Blood vessels in living tissues are an organized and hierarchical network of arteries, arterioles, capillaries, veinules and veins. Their sizes, lengths, shapes and connectivity are set up for an optimum perfusion of the tissues in which they deploy. In order to study the hemodynamics and hemophysics of blood flows and also to investigate artificial vasculature for organs on a chip, it is essential to reproduce most of these geometric features. Common microfluidic techniques produce channels with a uniform height and a rectangular cross section that do not capture the size hierarchy observed in vivo. This paper presents a new single-mask photolithography process using an optical diffuser to produce a backside exposure leading to microchannels with both a rounded cross section and a direct proportionality between local height and local width, allowing a one-step design of intrinsically hierarchical networks.  

4.2070           Traveling surface acoustic wave (TSAW) microfluidic fluorescence activated cell sorter (μFACS)

Mutafolos, K., Spink, P., Logstrom, C.D., Lu, P.J., Lu, H., Sharpe, J.C., Franke, T. and Weitz, D.A. Lab on a Chip, 19, 2435-2443 (2019)   We report a microfluidic fluorescence activated cell-sorting (μFACS) device that employs traveling surface acoustic waves (TSAW) to sort cells at rates comparable to conventional jet-in-air FACS machines, with high purity and viability. The device combines inertial flow focusing and sheath flow to align and evenly space cells, improving the sorting accuracy and screening rate. We sort with an interdigital transducer (IDT) whose tapered geometry allows precise positioning of the TSAW for optimal cell sorting. We sort three different cell lines at several kHz, at cell velocities exceeding one meter per second, while maintaining both sorting purity and cell viability at around 90% simultaneously.  

4.2071           High-throughput stem cell-based phenotypic screening through microniches

Kolb, L., Allazetta, S., karlsson, M., Girgin, M., Weber, W. and Lutolf, M.P. Biomater. Sci., 7, 3471-3479 (2019)   As the field of tissue engineering develops, methods for screening combinations of signals for their effects on stem cell behavior are needed. We introduce a microgel-based screening platform for testing combinations of in situ-generated proteins on stem cell fate in ultrahigh-throughput. Compartmentalizing individual sets of growth factors was addressed by encapsulating aggregates of stable recombinant cell lines secreting individual glycoproteins into microgels through an on-chip polymerization. When these ‘microniches’ are cultured with a cell type of interest, fluorescence reporters indicate positive niches that perform the desired function, and the underlying producer cell lines of these selected microniches are analyzed by barcoded RNA sequencing. The microniche-based screening work-flow was validated via a model system based on engineered mammalian cells expressing yellow fluorescent protein (YFP) upon anti-inflammatory cytokine interleukin 4 (IL4)-based activation.  

4.2072           Strategies for robust and accurate experimental approaches to quantify nanomaterial bioaccumulation across a broad range of organisms

Petersen, E.J., Mortimer, M., Burgess, R.M., Handy, R., Hanna, S. et al Environ. Sci. Nano, 6, 1619-1656 (2019)   One of the key components for environmental risk assessment of engineered nanomaterials (ENMs) is data on bioaccumulation potential. Accurately measuring bioaccumulation can be critical for regulatory decision-making regarding material hazard and risk, and for understanding the mechanism of toxicity. This perspective provides expert guidance for performing ENM bioaccumulation measurements across a broad range of test organisms and species. To accomplish this aim, we critically evaluated ENM bioaccumulation within three categories of organisms: single-celled species, multicellular species excluding plants, and multicellular plants. For aqueous exposures of suspended single-celled and small multicellular species, it is critical to perform a robust procedure to separate suspended ENMs and small organisms to avoid overestimating bioaccumulation. For many multicellular organisms, it is essential to differentiate between the ENMs adsorbed to external surfaces or in the digestive tract and the amount absorbed across epithelial tissues. For multicellular plants, key considerations include how exposure route and the role of the rhizosphere may affect the quantitative measurement of uptake, and that the efficiency of washing procedures to remove loosely attached ENMs to the roots is not well understood. Within each organism category, case studies are provided to illustrate key methodological considerations for conducting robust bioaccumulation experiments for different species within each major group. The full scope of ENM bioaccumulation measurements and interpretations are discussed including conducting the organism exposure, separating organisms from the ENMs in the test media after exposure, analytical methods to quantify ENMs in the tissues or cells, and modeling the ENM bioaccumulation results. One key finding to improve bioaccumulation measurements was the critical need for further analytical method development to identify and quantify ENMs in complex matrices. Overall, the discussion, suggestions, and case studies described herein will help improve the robustness of ENM bioaccumulation studies.  

4.2073           Droplet CAR-Wash: continuous picoliter-scale immunocapture and washing

Doonan, S.R., Lin, M. and Bailey, R.C. Lab on a Chip, 19, 1589-1598 (2019)   To address current limitations in adapting solid phase sample capture and washing techniques to continuously flowing droplet microfluidics, we have developed the “Coalesce-Attract-Resegment Wash” (CAR-Wash) approach. This module provides efficient, high-throughput magnetic washing by electrocoalescing magnetic bead-laden input droplets with a washing buffer flow and magnetophoretically transporting beads through the buffer into a secondary droplet formation streamline. In this work, we first characterized the technology in terms of throughput, sample retention, and flow-based exclusion of waste volume, demonstrating >500 Hz droplet processing with >98% bead retention and >100-fold dilution in final droplets. Next, we showed that the technique can be adapted to alternative commercially available magnetic beads with lower magnetite content per particle. Then, we demonstrated the CAR-Wash module's effectiveness in washing away a small molecule competitive inhibitor to restore the activity of magnetic bead-immobilized β-galactosidase. Finally, we applied the system to immunomagnetically enrich a green fluorescent protein–histone H2B fusion protein from cell lysate while washing away mCherry and other lysate components. We believe this approach will bridge the gap between powerful biochemical and bioanalytical techniques and current droplet microfluidic capabilities, and we envision future application in droplet-based immunoassays, solid phase extraction, and other complex, multi-step operations.  

4.2074           Landscape of Intercellular Crosstalk in Healthy and NASH Liver Revealed by Single-Cell Secretome Gene Analysis

Xiong, X., Kuang, H., Ansari, S., Yu, Y., Li, J.Z. and Lin, J.D. Molecular Cell, 75(3), 644-660 (2019)   Cell-cell communication via ligand-receptor signaling is a fundamental feature of complex organs. Despite this, the global landscape of intercellular signaling in mammalian liver has not been elucidated. Here we perform single-cell RNA sequencing on non-parenchymal cells isolated from healthy and NASH mouse livers. Secretome gene analysis revealed a highly connected network of intrahepatic signaling and disruption of vascular signaling in NASH. We uncovered the emergence of NASH-associated macrophages (NAMs), which are marked by high expression of triggering receptors expressed on myeloid cells 2 (Trem2), as a feature of mouse and human NASH that is linked to disease severity and highly responsive to pharmacological and dietary interventions. Finally, hepatic stellate cells (HSCs) serve as a hub of intrahepatic signaling via HSC-derived stellakines and their responsiveness to vasoactive hormones. These results provide unprecedented insights into the landscape of intercellular crosstalk and reprogramming of liver cells in health and disease.  

4.2075           Mitochondrial dysfunction in human primary alveolar type II cells in emphysema

Kosmider, B., Lin, C-R., Karim, L., Tomar, D., Vlasenko, L., Marchetti, N., Bolla, S., Madesh, M., Criner, G.J. and Bahmed, K. EBioMed., 46, 305-316 (2019)   Background Cigarette smoke is the main risk factor of pulmonary emphysema development, which is characterized by alveolar wall destruction. Mitochondria are important for alveolar type II (ATII) cell metabolism due to ATP generation. Methods We isolated ATII cells from control non-smoker and smoker organ donors, and after lung transplant of patients with emphysema to determine mitochondrial function, dynamics and mitochondrial (mt) DNA damage. Findings We found high mitochondrial superoxide generation and mtDNA damage in ATII cells in emphysema. This correlated with decreased mtDNA amount. We also detected high TOP1-cc and low TDP1 levels in mitochondria in ATII cells in emphysema. This contributed to the decreased resolution of TOP1-cc leading to accumulation of mtDNA damage and mitochondrial dysfunction. Moreover, we used lung tissue obtained from areas with mild and severe emphysema from the same patients. We found a correlation between the impaired fusion and fission as indicated by low MFN1, OPA1, FIS1, and p-DRP1 levels and this disease severity. We detected lower TDP1 expression in severe compared to mild emphysema. Interpretation We found high DNA damage and impairment of DNA damage repair in mitochondria in ATII cells isolated from emphysema patients, which contribute to abnormal mitochondrial dynamics. Our findings provide molecular mechanisms of mitochondrial dysfunction in this disease.  

4.2076           Single-cell RT-LAMP mRNA detection by integrated droplet sorting and merging

Chung, M.T., Kurabayashi, K. and Cai, D. Lab. Chip., 19, 2425-2434 (2019)   Recent advances in transcriptomic analysis at single-cell resolution reveal cell-to-cell heterogeneity in a biological sample with unprecedented resolution. Partitioning single cells in individual micro-droplets and harvesting each cell's mRNA molecules for next-generation sequencing has proven to be an effective method for profiling transcriptomes from a large number of cells at high throughput. However, the assays to recover the full transcriptomes are time-consuming in sample preparation and require expensive reagents and sequencing cost. Many biomedical applications, such as pathogen detection, prefer highly sensitive, reliable and low-cost detection of selected genes. Here, we present a droplet-based microfluidic platform that permits seamless on-chip droplet sorting and merging, which enables completing multi-step reaction assays within a short time. By sequentially adding lysis buffers and reactant mixtures to micro-droplet reactors, we developed a novel workflow of single-cell reverse transcription loop-mediated-isothermal amplification (scRT-LAMP) to quantify specific mRNA expression levels in different cell types within one hour. Including single cell encapsulation, sorting, lysing, reactant addition, and quantitative mRNA detection, the fully on-chip workflow provides a rapid, robust, and high-throughput experimental approach for a wide variety of biomedical studies.  

4.2077           Sp1-regulated expression of p11 contributes to motor neuron degeneration by membrane insertion of TASK1

Garcia-Morales, V., Rodriguez-Bey, G., Gomez-Perez, L., Dominguez-Vias, G., Gonzalez-Forero, D., Portillo, F., Carmpos-Caro, A., Gento-Caro, A., Issaoui, N., Soler, R.M., Garcera, A. and Moreno-Lopez, B. Nature Communications, 10:3784 (2019)   Disruption in membrane excitability contributes to malfunction and differential vulnerability of specific neuronal subpopulations in a number of neurological diseases. The adaptor protein p11, and background potassium channel TASK1, have overlapping distributions in the CNS. Here, we report that the transcription factor Sp1 controls p11 expression, which impacts on excitability by hampering functional expression of TASK1. In the SOD1-G93A mouse model of ALS, Sp1-p11-TASK1 dysregulation contributes to increased excitability and vulnerability of motor neurons. Interference with either Sp1 or p11 is neuroprotective, delaying neuron loss and prolonging lifespan in this model. Nitrosative stress, a potential factor in human neurodegeneration, stimulated Sp1 expression and human p11 promoter activity, at least in part, through a Sp1-binding site. Disruption of Sp1 or p11 also has neuroprotective effects in a traumatic model of motor neuron degeneration. Together our work suggests the Sp1-p11-TASK1 pathway is a potential target for treatment of degeneration of motor neurons.  

4.2078           Modulation of actin polymerization affects nucleocytoplasmic transport in multiple forms of amyotrophic lateral sclerosis

Giampetruzzi, A., Danielson, E.W., Gumina, V., Jeon, M., Boopathy, S., Brown, R.H., Ratti, A., Landers, J.E. and Fallini, C. Nature Communications, 10:3827 (2019)   Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown etiology. Although defects in nucleocytoplasmic transport (NCT) may be central to the pathogenesis of ALS and other neurodegenerative diseases, the molecular mechanisms modulating the nuclear pore function are still largely unknown. Here we show that genetic and pharmacological modulation of actin polymerization disrupts nuclear pore integrity, nuclear import, and downstream pathways such as mRNA post-transcriptional regulation. Importantly, we demonstrate that modulation of actin homeostasis can rescue nuclear pore instability and dysfunction caused by mutant PFN1 as well as by C9ORF72 repeat expansion, the most common mutation in ALS patients. Collectively, our data link NCT defects to ALS-associated cellular pathology and propose the regulation of actin homeostasis as a novel therapeutic strategy for ALS and other neurodegenerative diseases.  

4.2079           Thromboxane A2 receptor signaling in endothelial cells attenuates monocrotaline-induced liver injury

Otaka, F., Ito, Y., Inoue, T., Ohkubo, H., Nishizawa, N., Kojo, K., Betto, T., Yamane, S., Narumiya, S., Koizumi, W and Majima, M. Toxicol. Appl. Pharmacol., 381, 114733 (2019)   Sinusoidal obstruction syndrome (SOS) is a major complication of chemotherapy and hematopoietic stem cell transplantation. The early stage of SOS is characterized by liver sinusoidal endothelial cell (LSEC) injury accompanied by platelet aggregation. Thromboxane A₂ (TxA₂) induces platelet aggregation through the thromboxane prostanoid (TP) receptor. In this study, we explored the role of TP signaling in a monocrotaline (MCT)-induced mouse model of SOS. Relative to wild-type (WT) mice, TP-deficient (TP^−/−) mice exhibited more severe MCT-liver injury, as indicated by elevated levels of alanine aminotransferase (ALT) and coagulative necrosis. Extensive accumulation of platelets in the liver was observed in both WT and TP^−/− mice. TP expression co-localized with CD31-positive LSECs. MCT treatment caused LSEC destruction, concomitant with elevated expression of matrix metalloproteinases (MMPs) and adhesion molecules in WT mice, and LSEC damage was further exacerbated in TP^−/− mice. Viability of isolated LSECs was lower in cells from TP^−/− mice, whereas mRNA levels of MMPs and adhesion molecules were higher; U46619, a TxA₂ agonist, reduced these levels in WT mice. These data suggest that TP signaling has no effect on platelet accumulation during MCT-induced liver injury, but instead prevents injury by suppressing LSEC damage.  

4.2080           Hesperetin derivative attenuates CCl4-induced hepatic fibrosis and inflammation by Gli-1-dependent mechanisms

Chen, X., Li, X-F., Chen, Y., Zhu, S., Li, H-D., Chen, S-Y., Wang, J-N., Pan, X-Y., Bu, F-T., Huang, C. and Li, J. Int. Immunopharmacol., 76, 105838 (2019)   Hepatic fibrosis, a common pathological feature and leading cause of various chronic liver diseases, still lacks effective therapy. Hesperetin derivative (HD) is a derivative of Traditional Chinese Medicine monomer isolated from the fruit peel of Citrusaurantium L. (Rutaceae). In the present study, we revealed the anti-fibrotic effects of HD in CCl₄-induced mouse hepatic fibrosis model and in TGF-β1-activated LX-2 cells, in vivo and in vitro. Results showed that HD prevented CCl₄-induced liver injury and histological damage. Consistently, HD inhibited the up-regulation of liver fibrogenesis markers α-SMA, Col1α1, Col3α1 and TIMP-1 in primary hepatic stellate cells (HSCs) and suppressed inflammatory responses in primary liver macrophages from hepatic fibrosis mice. Furthermore, HD promoted the apoptosis of activated HSCs, a key step in the onset of fibrosis regression. Mechanistically, the Hedgehog pathway was involved in HD-treated hepatic fibrosis, and HD specifically contributed to attenuate the aberrant expression of Glioma associated oncogene-1 (Gli-1). Interestingly, blockade of Gli-1 removed the inhibitory effect of HD on activated HSCs, indicating that Gli-1 may play a pivotal role in mediating the anti-fibrotic effect of HD in hepatic fibrosis. Collectively, our results suggest that HD may be a potential anti-fibrotic Traditional Chinese Medicine monomer for the treatment of hepatic fibrosis.  

4.2081           The effect of cysteine oxidation on DJ-1 cytoprotective function in human alveolar type II cells

Bahmed, K., Boukhenouna, S., Karim, L., Andrews, T., Lin, J., Powers, R., Wilson, M. A., Lin, C-R., Messier, E., Reisdorph, N., Powell, R.L., Tang, H-Y., Mason, R.J., Criner, G.J. and Kosmider, B. Cell Death & Disease, 10:638 (2019)   DJ-1 is a multifunctional protein with cytoprotective functions. It is localized in the cytoplasm, nucleus, and mitochondria. The conserved cysteine residue at position 106 (Cys106) within DJ-1 serves as a sensor of redox state and can be oxidized to both the sulfinate (-SO₂⁻) and sulfonate (-SO₃⁻) forms. DJ-1 with Cys106-SO₂⁻ has cytoprotective activity but high levels of reactive oxygen species can induce its overoxidation to Cys106-SO₃⁻. We found increased oxidative stress in alveolar type II (ATII) cells isolated from emphysema patients as determined by 4-HNE expression. DJ-1 with Cys106-SO₃⁻ was detected in these cells by mass spectrometry analysis. Moreover, ubiquitination of Cys106-SO₃⁻ DJ-1 was identified, which suggests that this oxidized isoform is targeted for proteasomal destruction. Furthermore, we performed controlled oxidation using H₂O₂ in A549 cells with DJ-1 knockout generated using CRISPR-Cas9 strategy. Lack of DJ-1 sensitized cells to apoptosis induced by H₂O₂ as detected using Annexin V and propidium iodide by flow cytometry analysis. This treatment also decreased both mitochondrial DNA amount and mitochondrial ND1 (NADH dehydrogenase 1, subunit 1) gene expression, as well as increased mitochondrial DNA damage. Consistent with the decreased cytoprotective function of overoxidized DJ-1, recombinant Cys106-SO₃⁻ DJ-1 exhibited a loss of its thermal unfolding transition, mild diminution of secondary structure in CD spectroscopy, and an increase in picosecond–nanosecond timescale dynamics as determined using NMR. Altogether, our data indicate that very high oxidative stress in ATII cells in emphysema patients induces DJ-1 overoxidation to the Cys106-SO₃⁻ form, leading to increased protein flexibility and loss of its cytoprotective function, which may contribute to this disease pathogenesis.  

4.2082           Cell type-specific transcriptional programs in mouse prefrontal cortex during adolescence and addiction

Bhattacherjee, A., Djekidel, M.N., Chen, R., Chen, W., Tuesta, L.M. and Zhang, Y. Nature Communications, 10:4169 (2019)   Coordinated activity-induced transcriptional changes across multiple neuron subtypes of the prefrontal cortex (PFC) play a pivotal role in encoding and regulating major cognitive behaviors. Yet, the specific transcriptional programs in each neuron subtype remain unknown. Using single-cell RNA sequencing (scRNA-seq), here we comprehensively classify all unique cell subtypes in the PFC. We analyze transcriptional dynamics of each cell subtype under a naturally adaptive and an induced condition. Adaptive changes during adolescence (between P21 and P60), a highly dynamic phase of postnatal neuroplasticity, profoundly impacted transcription in each neuron subtype, including cell type-specific regulation of genes implicated in major neuropsychiatric disorders. On the other hand, an induced plasticity evoked by chronic cocaine addiction resulted in progressive transcriptional changes in multiple neuron subtypes and became most pronounced upon prolonged drug withdrawal. Our findings lay a foundation for understanding cell type-specific postnatal transcriptional dynamics under normal PFC function and in neuropsychiatric disease states.  

4.2083           TrkB agonistic antibodies superior to BDNF: Utility in treating motoneuron degeneration

Guo, W., Pang, K., Chen, Y., Wang, S., Li, H., Xu, Y. et al Neurobiology of Disease, 132, 104590 (2019)   While Brain-derived Neurotrophic Factor (BDNF) has long been implicated in treating neurological diseases, recombinant BDNF protein has failed in multiple clinical trials. In addition to its unstable and adhesive nature, BDNF can activate p75^NTR, a receptor mediating cellular functions opposite to those of TrkB. We have now identified TrkB agonistic antibodies (TrkB-agoAbs) with several properties superior to BDNF: They exhibit blood half-life of days instead of hours, diffuse centimeters in neural tissues instead millimeters, and bind and activate TrkB, but not p75^NTR. In addition, TrkB-agoAbs elicit much longer TrkB activation, reduced TrkB internalization and less intracellular degradation, compared with BDNF. More importantly, some of these TrkB-agoAbs bind TrkB epitopes distinct from that by BDNF, and work cooperatively with endogenous BDNF. Unlike BDNF, the TrkB-agoAbs exhibit a half-life of days/weeks and diffused readily in nerve tissues. We tested one of TrkB-agoAbs further and showed that it enhanced motoneuron survival in the spinal-root avulsion model for motoneuron degeneration in vivo. Thus, TrkB-agoAbs are promising drug candidates for the treatment of neural injury.  

4.2084           Platelet P-selectin triggers rapid surface exposure of tissue factor in monocytes

Ivanov, I.I., Apta, B.H.R., Bonna, A.M. and Harper, M.T. Scientific Reports, 9:13397 (2019)   Tissue factor (TF) plays a central role in haemostasis and thrombosis. Following vascular damage, vessel wall TF initiates the extrinsic coagulation cascade. TF can also be exposed by monocytes. Inflammatory or infectious stimuli trigger synthesis of new TF protein by monocytes over the course of hours. It has also been suggested that monocytes can expose TF within minutes when stimulated by activated platelets. Here, we have confirmed that monocytes rapidly expose TF in whole blood and further demonstrate that platelet P-selectin exposure is necessary and sufficient. Monocyte TF exposure increased within five minutes in response to platelet activation by PAR1-AP, PAR4-AP or CRP-XL. PAR1-AP did not trigger TF exposure on isolated monocytes unless platelets were also present. In whole blood, PAR1-AP-triggered TF exposure required P-selectin and PGSL-1. In isolated monocytes, although soluble recombinant P-selectin had no effect, P-selectin coupled to 2 µm beads triggered TF exposure. Cycloheximide did not affect rapid TF exposure, indicating that de novo protein synthesis was not required. These data show that P-selectin on activated platelets rapidly triggers TF exposure on monocytes. This may represent a mechanism by which platelets and monocytes rapidly contribute to intravascular coagulation.  

4.2085           Activation of the intrinsic fibroinflammatory program in adult pancreatic acinar cells triggered by Hippo signaling disruption

Liu, J., Gao, M., Nipper, M., Deng, J., Sharkey, F.E., Johnson, R.L., Crawford, H.C., Chen, Y. and Wang, P. PloS Biology, 17(9), e3000418 (2019)   Damaged acinar cells play a passive role in activating pancreatic stellate cells (PSCs) via recruitment of immune cells that subsequently activate PSCs. However, whether acinar cells directly contribute to PSC activation is unknown. Here, we report that the Hippo pathway, a well-known regulator of proliferation, is essential for suppression of expression of inflammation and fibrosis-associated genes in adult pancreatic acinar cells. Hippo inactivation in acinar cells induced yes-associated protein 1 (YAP1)/transcriptional coactivator with PDZ binding motif (TAZ)-dependent, irreversible fibrosis and inflammation, which was initiated by Hippo-mediated acinar-stromal communications and ameliorated by blocking YAP1/TAZ target connective tissue growth factor (CTGF). Hippo disruption promotes acinar cells to secrete fibroinflammatory factors and induce stromal activation, which precedes acinar proliferation and metaplasia. We found that Hippo disruption did not induce cell-autonomous proliferation but primed acinar cells to exogenous pro-proliferative stimuli, implying a well-orchestrated scenario in which Hippo signaling acts as an intrinsic link to coordinate fibroinflammatory response and proliferation for maintenance of the tissue integrity. Our findings suggest that the fibroinflammatory program in pancreatic acinar cells is suppressed under normal physiological conditions. While transient activation of inflammatory gene expression during tissue injury may contribute to the control of damage and tissue repair, its persistent activation may result in tissue fibrosis and failure of regeneration.  

4.2086           UVB-Induced Tumor Heterogeneity Diminishes Immune Response in Melanoma

Wolf, Y., Bartok, O., Patkar, S., Swanton, C., Ruppin, E. and Samuels, Y. Cell, 179, 219-235 (2019)   Although clonal neo-antigen burden is associated with improved response to immune therapy, the functional basis for this remains unclear. Here we study this question in a novel controlled mouse melanoma model that enables us to explore the effects of intra-tumor heterogeneity (ITH) on tumor aggressiveness and immunity independent of tumor mutational burden. Induction of UVB-derived mutations yields highly aggressive tumors with decreased anti-tumor activity. However, single-cell-derived tumors with reduced ITH are swiftly rejected. Their rejection is accompanied by increased T cell reactivity and a less suppressive microenvironment. Using phylogenetic analyses and mixing experiments of single-cell clones, we dissect two characteristics of ITH: the number of clones forming the tumor and their clonal diversity. Our analysis of melanoma patient tumor data recapitulates our results in terms of overall survival and response to immune checkpoint therapy. These findings highlight the importance of clonal mutations in robust immune surveillance and the need to quantify patient ITH to determine the response to checkpoint blockade.  

4.2087           Recombinant Virus-like Particles Presenting IL-33 Successfully Modify the Tumor Microenvironment and Facilitate Antitumor Immunity in a Model of Breast Cancer

Winnbust, J., Bas, E., Ferreira, T.A., Spruston, N., Svoboda, K. and Chandrashekar, J. Cell, 179, 268-281 (2019)   Recently, interleukin (IL)-33 has been closely associated with a variety of clinical cancers. IL-33 presents both protumorigenic, and less frequently, antitumorigenic functions depending on disease conditions. IL-33 signaling appears to be a possible target for the treatment of applicable tumor diseases. This study aimed to develop an effective approach to intervene in IL-33 functioning in tumors and reveal the immunotherapeutic potential of anti-IL-33 active immunization. Recombinant truncated hepatitis B virus core antigen (HBcAg), presenting mature IL-33 molecules on the surface of virus-like particles (VLPs), was prepared and used to immunize BALB/c mice in a model of murine 4T1 breast cancer. The immunization was performed through either a preventive or therapeutic strategy in two separate studies. Anti-IL-33 immunization with VLPs elicited a persistent and highly titrated specific antibody response and significantly suppressed orthotopic tumor growth in the preventive study and lung metastasis in both studies. The underlying mechanisms might include promoting tumor-specific Th1 and CTL-mediated cellular responses and the expression of the effector molecule interferon-γ (IFN-γ), suppressing T-helper type 2 (Th2) responses, and significantly reducing the infiltration of immunosuppressive Treg (regulatory T) cells and myeloid-derived suppressor cells (MDSCs) into tumor tissues in the immunized mice. In conclusion, anti-IL-33 active immunization employing recombinant VLPs as an antigen delivery platform effectively modified the tumor microenvironment and promoted antitumor immunity, indicating the potential of this approach as a new and promising immunotherapeutic strategy for the treatment of cancers where IL-33 plays a definite protumorigenic role.  

4.2088           A rapid and sensitive method to detect Toxoplasma gondii oocysts in soil samples

Escotte-Binet, S., Da Silva, A.M., Cances, B., Aubert, D., Dubey, J., La Carbona, S., Villena, I. and Poulle, M-L. Vet. Parasitol., 274, 108904 (2019)   Documenting the extent of soil contamination by Toxoplasma gondii oocysts is a key issue to prevent the worldwide infection caused by this protozoan. Our aim was to improve the practicability and sensitivity of a low-cost method to detect T. gondii DNA in soil samples developed a few years ago. Various parameters of the reference protocol were modified to determine their effect on the detection of T. gondii DNA in soil samples (“natural soil” and “sand”) spiked with oocysts. We tested i) filtration using stomacher bags, ii) Tween 80, Tween 20, SDS and Triton X100 as dispersion solutions, iii) sucrose solution, zinc chloride solution, Optiprep and Percoll as density gradients, iv) freeze/thaw versus mechanical grinding as lysis methods, and v) Qiagen versus Fastprep as extraction kits The optimized protocol is quicker and easier to use than the previous one, and includes the following items: 0.1% Tween80/PBS for dispersion, sucrose solution for flotation, mechanical grinding, and FastDNA spin kit for extraction. It accurately detects T. gondii DNA in both fresh and frozen soil samples and displays a detection limit below 1 oocyst/g of fresh soil.  

4.2089           Interferon-γ-dependent immune responses contribute to the pathogenesis of sclerosing cholangitis in mice

Ravichandran, G., Neumann, K., Berkhout, L.K., Schramm, C., Altfeld, m. and Tiegs, G.

1. Hepatol., 71, 773-782 (2019)

  Background and Aims Primary sclerosing cholangitis (PSC) is an idiopathic, chronic cholestatic liver disorder characterized by biliary inflammation and fibrosis. Increased numbers of intrahepatic interferon-γ- (IFNγ) producing lymphocytes have been documented in patients with PSC, yet their functional role remains to be determined. Methods Liver tissue samples were collected from patients with PSC. The contribution of lymphocytes to liver pathology was assessed in Mdr2^−/− x Rag1^−/− mice, which lack T and B cells, and following depletion of CD90.2⁺ or natural killer (NK)p46⁺ cells in Mdr2^−/− mice. Liver pathology was also determined in Mdr2^−/− x Ifng^−/− mice and following anti-IFNγ antibody treatment of Mdr2^−/− mice. Immune cell composition was analysed by multi-colour flow cytometry. Liver injury and fibrosis were determined by standard assays. Results Patients with PSC showed increased IFNγ serum levels and elevated numbers of hepatic CD56^bright NK cells. In Mdr2^−/− mice, hepatic CD8⁺ T cells and NK cells were the primary source of IFNγ. Depletion of CD90.2⁺ cells reduced hepatic Ifng expression, NK cell cytotoxicity and liver injury similar to Mdr2^−/− x Rag1^−/− mice. Depletion of NK cells resulted in reduced CD8⁺ T cell cytotoxicity and liver fibrosis. The complete absence of IFNγ in Mdr2^−/−x Ifng^−/− mice reduced NK cell and CD8⁺ T cell frequencies expressing the cytotoxic effector molecules granzyme B and TRAIL and prevented liver fibrosis. The antifibrotic effect of IFNγ was also observed upon antibody-dependent neutralisation in Mdr2^−/− mice. Conclusion IFNγ changed the phenotype of hepatic CD8⁺ T cells and NK cells towards increased cytotoxicity and its absence attenuated liver fibrosis in chronic sclerosing cholangitis. Therefore, unravelling the immunopathogenesis of PSC with a particular focus on IFNγ might help to develop novel treatment options.  

4.2090           DNA Damage and Repair in Patients with Coronary Artery Disease: Correlation with Plaque Morphology Using Optical Coherence Tomography (DECODE Study)

Shah, N., Meira, L.B., Elliott, R.M., Hoole, S.P., West, N.E., Brown, A.J., Bennett, M.R., Garcia-Garcia, H.M., Kuku, K.O., Dan, K., Kohn, P., Mariathas, M., Curzen, N. and Mahmoudi, M. Cardiovasc. Revascularization Med., 20, 812-818 (2019)   Objective The aim of this study was to examine DNA ligase activity and expression of DNA damage response pathway (DDR) genes in patients with stable angina (SA) and non-ST elevation myocardial infarction (NSTEMI) and determine whether they correlate with plaque morphology. Background Patients with coronary artery disease (CAD) have evidence of deoxyribonucleic acid (DNA) damage in peripheral blood mononuclear cells (PBMCs). It is unclear whether this represents excess damage or defective DNA repair activity. Methods DNA ligase activity and the expression of 22 DDR genes were measured in PBMCs of patients (both SA (n = 47) and NSTEMI (n = 42)) and in age and gender-matched controls (n = 35). Target lesion anatomical assessment was undertaken with frequency domain optical coherent tomography. Results DNA ligase activity was different across the three groups of patients (control = 119 ± 53, NSTEMI = 115.6 ± 85.1, SA = 81 ± 55.7 units/g of nuclear protein; ANOVA p = 0.023). Pair wise comparison demonstrated that this significance is due to differences between the control and SA patients (p = 0.046). Genes involved in double strand break repair and nucleotide excision repair pathways were differentially expressed in patients with SA and NSTEMI. In SA patients, fibrocalcific plaques were strongly associated with GTSE1, DDB1, MLH3 and ERCC1 expression. By contrast, in NSTEMI patients the strongest association was observed between fibrous plaques and ATM and XPA expression. Conclusion PBMCs from patients with CAD exhibit differences in DNA ligase activity and expression of DDR genes. Expression levels of certain DDR genes are strongly associated with plaque morphology and may play a role in plaque development and progression.  

4.2091           Liver-Derived Signals Sequentially Reprogram Myeloid Enhancers to Initiate and Maintain Kupffer Cell Identity

Sakai, M., Troutman, T.D., Seidman, J.S., McDonald, J.G., Geissmann, F. and Glass, C.K. Immunity, 51, 655-670 (2019)   Tissue environment plays a powerful role in establishing and maintaining the distinct phenotypes of resident macrophages, but the underlying molecular mechanisms remain poorly understood. Here, we characterized transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. We obtained evidence that combinatorial interactions of the Notch ligand DLL4 and transforming growth factor-b (TGF-β) family ligands produced by sinusoidal endothelial cells and endogenous LXR ligands were required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of the Notch transcriptional effector RBPJ activated poised enhancers to rapidly induce LXRα and other Kupffer cell lineage-determining factors. These factors in turn reprogrammed the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes.  

4.2092           New methods of removing debris and high-throughput counting of cyst nematode eggs extracted from field soil

Kalwa, U., Legner, C., Wiezien, E., Tylka, G. and Pandey, S. PloS One, 14(10), e0223386 (2019)   The soybean cyst nematode (SCN), Heterodera glycines, is the most damaging pathogen of soybeans in the United States. To assess the severity of nematode infestations in the field, SCN egg population densities are determined. Cysts (dead females) of the nematode must be extracted from soil samples and then ground to extract the eggs within. Sucrose centrifugation commonly is used to separate debris from suspensions of extracted nematode eggs. We present a method using OptiPrep as a density gradient medium with improved separation and recovery of extracted eggs compared to the sucrose centrifugation technique. Also, computerized methods were developed to automate the identification and counting of nematode eggs from the processed samples. In one approach, a high-resolution scanner was used to take static images of extracted eggs and debris on filter papers, and a deep learning network was trained to identify and count the eggs among the debris. In the second approach, a lensless imaging setup was developed using off-the-shelf components, and the processed egg samples were passed through a microfluidic flow chip made from double-sided adhesive tape. Holographic videos were recorded of the passing eggs and debris, and the videos were reconstructed and processed by custom software program to obtain egg counts. The performance of the software programs for egg counting was characterized with SCN-infested soil collected from two farms, and the results using these methods were compared with those obtained through manual counting.  

4.2093           High-Throughput Mapping of Long-Range Neuronal Projection Using in Situ Sequencing

Chen, X., Sun, Y-C., Zhan, H., Huang, Z.J., Gillis, J. and Zador, A.M. Cell, 179, 772-786 (2019)   Understanding neural circuits requires deciphering interactions among myriad cell types defined by spatial organization, connectivity, gene expression, and other properties. Resolving these cell types requires both single-neuron resolution and high throughput, a challenging combination with conventional methods. Here, we introduce barcoded anatomy resolved by sequencing (BARseq), a multiplexed method based on RNA barcoding for mapping projections of thousands of spatially resolved neurons in a single brain and relating those projections to other properties such as gene or Cre expression. Mapping the projections to 11 areas of 3,579 neurons in mouse auditory cortex using BARseq confirmed the laminar organization of the three top classes (intratelencephalic [IT], pyramidal tract-like [PT-like], and corticothalamic [CT]) of projection neurons. In depth analysis uncovered a projection type restricted almost exclusively to transcriptionally defined subtypes of IT neurons. By bridging anatomical and transcriptomic approaches at cellular resolution with high throughput, BARseq can potentially uncover the organizing principles underlying the structure and formation of neural circuits.  

4.2094           Soluble egg antigen of Schistosoma japonicum induces pyroptosis in hepatic stellate cells by modulating ROS production

Kong, D-L., Kong, F-Y., Liu, X-Y., Yan, C., Cui, J., Tang, R-X. and Zheng, K-Y. Parasites & Vectors, 12:475 (2019)   Background Inflammation-induced dysfunction of hepatic stellate cells (HSCs) is involved in schistosomiasis-associated liver fibrosis, and soluble egg antigen (SEA) is a crucial pathogen-associated molecular pattern associated with liver injury in schistosomiasis. In addition, numerous studies have shown that caspase-1-mediated pyroptosis participates in the development of multiple inflammation-related diseases. However, whether pyroptotic cell death of HSCs is involved in SEA-mediated liver damage is not well understood. Methods Primary cultured HSCs and Schistosoma japonicum-infected mouse liver tissue were analysed for histological changes and caspase-1 activation, and the role of pyroptosis in the mechanisms underlying SEA-induced HSC death was investigated. Accumulation of reactive oxygen species (ROS) in infected livers and SEA-stimulated HSCs was measured by flow cytometry and immunofluorescence. Results Caspase-1 activity was elevated in both liver tissues and HSCs of S. japonicum-infected mice. Furthermore, SEA stimulation increased the proportion of pyroptotic HSCs, as shown by lactate dehydrogenase (LDH) release assays and by flow cytometric analysis of propidium iodide (PI) and caspase-1 double staining in cells. In addition, ROS generation was elevated in infected liver tissues and SEA-stimulated HSCs, and ROS inhibition downregulated SEA-induced caspase-1 activation and pyroptosis in HSCs. Conclusions Our present study demonstrates that pyroptotic cell death in HSCs induced by SEA via ROS-mediated caspase-1 activation may serve as a significant mechanism to initiate the inflammatory response and thereby exacerbate liver injury during S. japonicum infection.  

4.2095           Inflammation in acute coronary syndrome: Expression of TLR2 mRNA is increased in platelets of patients with ACS

Heger, L.A., Hortmann, M., Albrecht, M., Colberg, C., Peter, K., Witsch, T., Stallmann, D., Zirlik, A., Bode, C., Duerschmied, D. and Ahrens, I PloS One, 14(10), e0224181 (2019)   Background Platelets are key components in atherogenesis and determine the course of its clinical sequelae acute coronary syndrome (ACS). Components of the innate immune system—the superfamily of TLR receptors–are present in platelets and represent a link between atherothrombosis and inflammation. We hypothesize that alteration in platelet TLR mRNA expression is a result of inflammation driving coronary atherosclerosis and may represent an alternative platelet activation pathway in ACS. TLR2-, TLR4- and TLR9- mRNA-expression was determined in ACS patients and compared to patients with invasive exclusion of atherosclerotic lesions of coronary arteries. Methods A total of fifty-four patients were enrolled in this clinical retrospective cohort single centre study. Total RNA from sepharose-filtered highly purified platelets was isolated using acid guanidinium thiocyanate-phenol-chloroform extraction and transcribed to cDNA using a first strand cDNA synthesis kit. To determine absolute copy numbers of TLR2, TLR4 and TLR9 we used plasmid based quantitative PCR with normalisation to an internal control. Results We found that mRNA expression levels of TLR2 but not TLR 4 and 9 are up-regulated in platelets of patients with ACS when compared to patients without coronary atherosclerosis. Conclusion Our results suggest elevated TLR2 mRNA expression in platelets as a biomarker reflecting the underlying inflammation in ACS and possibly severity of coronary atherosclerosis. Platelet TLR2 may represent a link between inflammation and atherothrombosis in ACS.  

4.2096           The role of DJ-1 in human primary alveolar type II cell injury induced by e-cigarette aerosol

Bahmed, K., Lin, C-R., Simborio, H., Karim, L., Aksoy, M., Kelsen, S., Tomar, D., Madesh, M., Elrod, J., Messier, E., Mason, R., Unterwald, E.M., Eisenstein, T.K., Criner, G.J. and Kosmider, B. Am. J. Physiol. Lung Cell Mol. Physiol., 317, L475-L485 (2019)   alveolus participates in gas exchange, which can be impaired by environmental factors and toxins. There is an increase in using electronic cigarettes (e-cigarettes); however, their effect on human primary alveolar epithelial cells is unknown. Human lungs were obtained from nonsmoker organ donors to isolate alveolar type II (ATII) cells. ATII cells produce and secrete pulmonary surfactant and restore the epithelium after damage, and mitochondrial function is important for their metabolism. Our data indicate that human ATII cell exposure to e-cigarette aerosol increased IL-8 levels and induced DNA damage and apoptosis. We also studied the cytoprotective effect of DJ-1 against ATII cell injury. DJ-1 knockdown in human primary ATII cells sensitized cells to mitochondrial dysfunction as detected by high mitochondrial superoxide production, decreased mitochondrial membrane potential, and calcium elevation. DJ-1 knockout (KO) mice were more susceptible to ATII cell apoptosis and lung injury induced by e-cigarette aerosol compared with wild-type mice. Regulation of the oxidative phosphorylation (OXPHOS) is important for mitochondrial function and protection against oxidative stress. Major subunits of the OXPHOS system are encoded by both nuclear and mitochondrial DNA. We found dysregulation of OXPHOS complexes in DJ-1 KO mice after exposure to e-cigarette aerosol, which could disrupt the nuclear/mitochondrial stoichiometry, resulting in mitochondrial dysfunction. Together, our results indicate that DJ-1 deficiency sensitizes ATII cells to damage induced by e-cigarette aerosol leading to lung injury.  

4.2097           Activated CD8+ T Cells Cause Long-Term Neurological Impairment after Traumatic Brain Injury in Mice

Daglas, M., Draxler, D.F., Ho, H., Alderuccio, F., Sashindranath, m. and Medcalf, R.L. Cell Reports, 29, 1178-1191 (2019)   Traumatic brain injury (TBI) leaves many survivors with long-term disabilities. A prolonged immune response in the brain may cause neurodegeneration, resulting in chronic neurological disturbances. In this study, using a TBI mouse model, we correlate changes in the local immune response with neurodegeneration/neurological dysfunction over an 8-month period. Flow cytometric analysis reveals a protracted increase in effector/memory CD8⁺ T cells (expressing granzyme B) in the injured brain. This precedes interleukin-17⁺CD4⁺ T cell infiltration and is associated with progressive neurological/motor impairment, increased circulating brain-specific autoantibodies, and myelin-related pathology. Genetic deficiency or pharmacological depletion of CD8⁺ T cells, but not depletion of CD4⁺ T cells, improves neurological outcomes and produces a neuroprotective Th2/Th17 immunological shift, indicating a persistent detrimental role for cytotoxic T cells post-TBI. B cell deficiency results in severe neurological dysfunction and a heightened immune reaction. Targeting these adaptive immune cells offers a promising approach to improve recovery following TBI.  

4.2098           Fenestral diaphragms and PLVAP associations in liver sinusoidal endothelial cells are developmentally regulated

Auvinen, K., Lokka, E., Mokkala, E., Jäppinen, N., Tyystjärvi, S., Saine, H., Peurla, M., Shetty, S., Elima, K., Rantakari, P. and Salmi, M. Scientific Reports, 9:15698 (2019)   Endothelial cells contain several nanoscale domains such as caveolae, fenestrations and transendothelial channels, which regulate signaling and transendothelial permeability. These structures can be covered by filter-like diaphragms. A transmembrane PLVAP (plasmalemma vesicle associated protein) protein has been shown to be necessary for the formation of diaphragms. The expression, subcellular localization and fenestra-forming role of PLVAP in liver sinusoidal endothelial cells (LSEC) have remained controversial. Here we show that fenestrations in LSEC contain PLVAP-diaphragms during the fetal angiogenesis, but they lose the diaphragms at birth. Although it is thought that PLVAP only localizes to diaphragms, we found luminal localization of PLVAP in adult LSEC using several imaging techniques. Plvap-deficient mice revealed that the absence of PLVAP and diaphragms did not affect the morphology, the number of fenestrations or the overall vascular architecture in the liver sinusoids. Nevertheless, PLVAP in fetal LSEC (fenestrations with diaphragms) associated with LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1), neuropilin-1 and VEGFR2 (vascular endothelial growth factor receptor 2), whereas in the adult LSEC (fenestrations without diaphragms) these complexes disappeared. Collectively, our data show that PLVAP can be expressed on endothelial cells without diaphragms, contradict the prevailing concept that biogenesis of fenestrae would be PLVAP-dependent, and reveal previously unknown PLVAP-dependent molecular complexes in LSEC during angiogenesis.  

4.2099           Resilient Phenotype in Chronic Mild Stress Paradigm Is Associated with Altered Expression Levels of miR-18a-5p and Serotonin 5-HT1a Receptor in Dorsal Part of the Hippocampus

Zurawek, D., Gruca, P., Antkiewicz-Michaluk, L. and Dzidzicka-Wasylewska, M. Mol. Neurobiol., 56, 7680-7683 (2019)   Disturbed serotonergic signaling in the hippocampus observed in many individuals vulnerable to stress has been suggested as one of the primary factors contributing to the development of depression. However, little is known about the physiology of the brain in the resilient phenotype. Resilient subjects maintain a positive mood and psychological balance despite being under the stress influence. In our study, we generated stress-vulnerable and resilient rats by using a chronic mild stress (CMS) paradigm. Using different molecular approaches, we revealed that resilient animals exhibited a significantly decreased expression level of miR-18a-5p and, in the same time, an elevated level of 5-HT1AR in dorsal, but not ventral, part of the hippocampus. Described biochemical changes were not observed in animals behaviorally vulnerable to stress. Further, in vitro analysis showed that miR-18a-5p may be a negative epigenetic regulator of 5-HT1AR since the treatment of adult hippocampal neurons with miR-18a-5p mimic significantly lowered the expression level of mRNA encoding 5-HT1AR. Moreover, bioinformatic analysis of potential target genes expressed in the hippocampus and being regulated by miR-18a-5p showed that this microRNA may regulate biological processes, such as axonogenesis, which are important in the functioning of the hippocampus in both rats and humans. All these molecular features may contribute to serotonergic homeostatic balance at the level of serotonin turnover observed in hippocampi of resilient but not stress-vulnerable rats. Delineation of further molecular and biochemical markers underlying resilience to stress may contribute to the development of new antidepressant strategies which will restore resilient phenotype in depressed patients.  

4.2100           Sensitivity of Kupffer cells and liver sinusoidal endothelial cells to ricin toxin and ricin toxin–Ab complexes

Mooney, B., Torres-Velez, F.J., Doering, J., Ehrbar, D.J. and Mantis, N.J.

1. Leukoc. Biol., 106(5), 1161-1176 (2019)

  Ricin toxin is a plant‐derived, ribosome‐inactivating protein that is rapidly cleared from circulation by Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs)—with fatal consequences. Rather than being inactivated, ricin evades normal degradative pathways and kills both KCs and LSECs with remarkable efficiency. Uptake of ricin by these 2 specialized cell types in the liver occurs by 2 parallel routes: a “lactose‐sensitive” pathway mediated by ricin's galactose/N‐acetylgalactosamine‐specific lectin subunit (RTB), and a “mannose‐sensitive” pathway mediated by the mannose receptor (MR; CD206) or other C‐type lectins capable of recognizing the mannose‐side chains displayed on ricin's A (RTA) and B subunits. In this report, we investigated the capacity of a collection of ricin‐specific mouse MAb and camelid single‐domain (V_HH) antibodies to protect KCs and LSECs from ricin‐induced killing. In the case of KCs, individual MAbs against RTA or RTB afforded near complete protection against ricin in ex vivo and in vivo challenge studies. In contrast, individual MAbs or V_HHs afforded little (<40%) or even no protection to LSECs against ricin‐induced death. Complete protection of LSECs was only achieved with MAb or V_HH cocktails, with the most effective mixtures targeting RTA and RTB simultaneously. Although the exact mechanisms of protection of LSECs remain unknown, evidence indicates that the Ab cocktails exert their effects on the mannose‐sensitive uptake pathway without the need for Fcγ receptor involvement. In addition to advancing our understanding of how toxins and small immune complexes are processed by KCs and LSECs, our study has important implications for the development of Ab‐based therapies designed to prevent or treat ricin exposure should the toxin be weaponized.  

4.2101           PTPN2 regulates the generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity

LaFleur, M.W., Nguyen, T.H., Coxe, M.A., Miller, B.C., Yates, K.B., Gillis, J.E., Sen, D.R., Gaudiano, E.F., Al Abosy, R., Freeman, G.J., Haining, W.N. and Sharpe, A.H. Nature Immunol., 20, 1335-1347 (2019)   CD8⁺ T cell exhaustion is a state of dysfunction acquired in chronic viral infection and cancer, characterized by the formation of Slamf6⁺ progenitor exhausted and Tim-3⁺ terminally exhausted subpopulations through unknown mechanisms. Here we establish the phosphatase PTPN2 as a new regulator of the differentiation of the terminally exhausted subpopulation that functions by attenuating type 1 interferon signaling. Deletion of Ptpn2 in CD8⁺ T cells increased the generation, proliferative capacity and cytotoxicity of Tim-3⁺ cells without altering Slamf6⁺ numbers during lymphocytic choriomeningitis virus clone 13 infection. Likewise, Ptpn2 deletion in CD8⁺ T cells enhanced Tim-3⁺ anti-tumor responses and improved tumor control. Deletion of Ptpn2 throughout the immune system resulted in MC38 tumor clearance and improved programmed cell death-1 checkpoint blockade responses to B16 tumors. Our results indicate that increasing the number of cytotoxic Tim-3⁺CD8⁺ T cells can promote effective anti-tumor immunity and implicate PTPN2 in immune cells as an attractive cancer immunotherapy target.  

4.2102           Glutamate Signaling in Hepatic Stellate Cells Drives Alcoholic Steatosis

Choi, W-M., Kim, H-H., Kim, M-H., Kim, S.K., Kunos, G. and Jeong, W-l., Cell Metabolism, 30, 877-889 (2019)   Activation of hepatocyte cannabinoid receptor-1 (CB₁R) by hepatic stellate cell (HSC)-derived 2-arachidonoylglycerol (2-AG) drives de novo lipogenesis in alcoholic liver disease (ALD). How alcohol stimulates 2-AG production in HSCs is unknown. Here, we report that chronic alcohol consumption induced hepatic cysteine deficiency and subsequent glutathione depletion by impaired transsulfuration pathway. A compensatory increase in hepatic cystine-glutamate anti-porter xCT boosted extracellular glutamate levels coupled to cystine uptake both in mice and in patients with ALD. Alcohol also induced the selective expression of metabotropic glutamate receptor-5 (mGluR5) in HSCs where mGluR5 activation stimulated 2-AG production. Consistently, genetic or pharmacologic inhibition of mGluR5 or xCT attenuated alcoholic steatosis in mice via the suppression of 2-AG production and subsequent CB₁R-mediated de novo lipogenesis. We conclude that a bidirectional signaling operates at a metabolic synapse between hepatocytes and HSCs through xCT-mediated glutamate-mGluR5 signaling to produce 2-AG, which induces CB₁R-mediated alcoholic steatosis.  

4.2103           Hepatocyte-derived exosomal MiR-194 activates PMVECs and promotes angiogenesis in hepatopulmonary syndrome

Chen, L., Han, Y., Li, Y., Chen, B., Bai, X., Belguise, K., Wang, X., Chen, Y., Yi, B. and Lu, K. Cell Death & Disease, 10:853 (2019)   Hepatopulmonary syndrome (HPS) is a serious vascular complication in the setting of liver disease. Factors produced by the liver are essential to regulate pulmonary angiogenesis in the pathogenesis of HPS; however, the pathogenic mechanisms of pulmonary angiogenesis are not fully understood. We investigated the role of HPS rat serum exosomes (HEs) and sham-operated rat serum exosomes (SEs) in the regulation of angiogenesis. We found that HEs significantly enhance PMVEC proliferation, migration, and tube formation. We further identified miR-194 was the most notably increased miRNA in HEs compared to SEs. Once released, hepatocyte-derived exosomal miR-194 was internalized by PMVECs, leading to the promotion of PMVEC proliferation, migration, and tube formation through direct targeting of THBS1, STAT1, and LIF. Importantly, the pathogenic role of exosomal miR-194 in initiating angiogenesis was reversed by P53 inhibition, exosome secretion inhibition or miR-194 inhibition. Additionally, high levels of miR-194 were found in serum exosomes and were positively correlated with P(A-a)O₂ in HPS patients and rats. Thus, our results highlight that the exosome/miR-194 axis plays a critical pathologic role in pulmonary angiogenesis, representing a new therapeutic target for HPS.  

4.2104           The Role of Kupffer Cells as Mediators of Adipose Tissue Lipolysis

Ma, W., Zhao, D., He, F. and Tang, L.

1. Immunol., 203, 2689-2700 (2019)

  Kupffer cells (KCs) are the resident macrophages of the liver, and they respond to and counteract metabolic stresses, such as those imposed by high-fat diet feeding in mouse models. However, little is known regarding the role of these cells in maintaining metabolic homeostasis under metabolically normal conditions. In this study, we found that depletion of KCs in vivo led to enhanced lipolysis in adipose tissue by increasing the expression of FGF21, a metabolic regulator, in hepatocytes. IL-1β secreted from KCs contributed to the suppression of FGF21 expression in hepatocytes. FGF21 overexpression led to a lean phenotype and enhanced lipolysis in mice. KC depletion resulted in a lack of IL-1β signaling in the liver, leading to elevated expression of FGF21 in hepatocytes. FGF21 promoted lipolysis in adipose tissue and led to hyperlipidemia and decreased body weight. The secretion of IL-1β in KCs was mediated by bacterial products. Antibiotic treatment also led to enhanced lipolysis. Therefore, the current study identified a physiological role of KCs in the regulation of adipose lipolysis.  

4.2105           Dietary zerumbone, a sesquiterpene, ameliorates hepatotoxin‐mediated acute and chronic liver injury in mice

Kim, J-W., Yang, D., Jeong, h., park, I.S., Lee, M-H., Lim, C.W. and Kim, B. Phytotherapy Res., 33(5), 1538-1550 (2019)   Acute liver injury (ALI) is a life‐threatening clinical syndrome. Long‐lasting liver injury can lead to chronic hepatic inflammation and fibrogenic responses. Zerumbone (ZER), the main constituent of rhizomes of Zingiber zerumbet Smith, has a variety of functions including anticancer activity. We investigated the role of ZER on the progression of hepatotoxin‐induced liver injury. Single or repeated injection of CCl₄ was used to induce acute or chronic liver injury, respectively. Mice were orally administered with ZER (10, 50 mg/kg) during the experimental period. Histopathologic analysis and serum biochemical levels revealed that ZER had hepatoprotective activities against ALI. Similar effects of ZER on injured livers were confirmed by analyses of inflammation and apoptosis‐related genes. Western blot analysis showed that protein levels of apoptotic molecules were decreased, whereas antiapoptotic protein levels were conversely increased in injured livers treated with ZER. Furthermore, chronic liver injury and its associated fibrogenesis in mice were reduced by ZER treatment. These findings from our in vivo experiments further indicate that ZER could alleviate hepatocellular toxicity and inhibit activation of primary hepatic stellate cells. Our results suggest that ZER might have potential as a safe and prophylactic alternative to prevent acute and chronic liver injury.  

4.2106           Hepatitis B virus-induced modulation of liver macrophage function promotes hepatocyte infection

Faure-Dupuy, S., Delphin, M., Aillot, L., Heikenwälder, M., Durantel. D. and Licifora, J.

1. Hepatol., 71, 1086-1098 (2019)

  Background & Aims Liver macrophages can be involved in both pathogen clearance and/or pathogenesis. To get further insight on their role during chronic hepatitis B virus (HBV) infections, our aim was to phenotypically and functionally characterize in vivo and ex vivo the interplay between HBV, primary human liver macrophages (PLMs) and primary blood monocytes differentiated into pro-inflammatory or anti-inflammatory macrophages (M1-MDMs or M2-MDMs, respectively). Methods PLMs or primary blood monocytes, either ex vivo differentiated into M1-MDMs or M2-MDMs, were exposed to HBV and their activation followed by ELISA or quantitative reverse transcription PCR (RT-qPCR). Liver biopsies from HBV-infected patients were analysed by RT-qPCR or immunohistochemistry. Viral parameters in HBV-infected primary human hepatocytes and differentiated HepaRG cells were followed by ELISA, qPCR and RT-qPCR analyses. Results HBc protein was present within the macrophages of liver biopsies taken from HBV-infected patients. Macrophages from HBV-infected patients also expressed higher levels of anti-inflammatory macrophage markers than those from non-infected patients. Ex vivo exposure of naive PLMs to HBV led to reduced secretion of pro-inflammatory cytokines. Upon exposure to HBV or HBV-producing cells during differentiation and activation, M1-MDMs secreted less IL-6 and IL-1β, whereas M2-MDMs secreted more IL-10 when exposed to HBV during activation. Finally, cytokines produced by M1-MDMs, but not those produced by HBV-exposed M1-MDMs, decreased HBV infection of hepatocytes. Conclusions Altogether, our data strongly suggest that HBV modulates liver macrophage functions to favour the establishment of infection.  

4.2107           Muscleblind acts as a modifier of FUS toxicity by modulating stress granule dynamics and SMN localization

Casci, I., Krishnamurthy, Kour, S., Tripathy, V., Ramesh, N., Anderson, E.N. et al Nature Communications, 10:5583 (2019)   Mutations in fused in sarcoma (FUS) lead to amyotrophic lateral sclerosis (ALS) with varying ages of onset, progression and severity. This suggests that unknown genetic factors contribute to disease pathogenesis. Here we show the identification of muscleblind as a novel modifier of FUS-mediated neurodegeneration in vivo. Muscleblind regulates cytoplasmic mislocalization of mutant FUS and subsequent accumulation in stress granules, dendritic morphology and toxicity in mammalian neuronal and human iPSC-derived neurons. Interestingly, genetic modulation of endogenous muscleblind was sufficient to restore survival motor neuron (SMN) protein localization in neurons expressing pathogenic mutations in FUS, suggesting a potential mode of suppression of FUS toxicity. Upregulation of SMN suppressed FUS toxicity in Drosophila and primary cortical neurons, indicating a link between FUS and SMN. Our data provide in vivo evidence that muscleblind is a dominant modifier of FUS-mediated neurodegeneration by regulating FUS-mediated ALS pathogenesis.  

4.2108           High-resolution label-free 3D mapping of extracellular pH of single living cells

Zhang, Y., Takahashi, Y., Hong, S.P., Liu, F., Bednarska, J., Goff, P.S., Novak, P. et al Nature Communications, 10:5610 (2019)   Dynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H⁺ in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity > 0.01 units, 2 ms response time, and 50 nm spatial resolution. The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependance via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells.  

4.2109           Construction of Thymus Organoids from Decellularized Thymus Scaffolds

Tajima, A., Pradhan, I., Geng, X., Trucca, M. and Fan, Y. Methods in Mol. Biol., 1576, 33-42 (2019)   One of the hallmarks of modern medicine is the development of therapeutics that can modulate immune responses, especially the adaptive arm of immunity, for disease intervention and prevention. While tremendous progress has been made in the past decades, manipulating the thymus, the primary lymphoid organ responsible for the development and education of T lymphocytes, remains a challenge. One of the major obstacles is the difficulty to reproduce its unique extracellular matrix (ECM) microenvironment that is essential for maintaining the function and survival of thymic epithelial cells (TECs), the predominant population of cells in the thymic stroma. Here, we describe the construction of functional thymus organoids from decellularized thymus scaffolds repopulated with isolated TECs. Thymus decellularization was achieved by freeze–thaw cycles to induce intracellular ice crystal formation, followed by detergent-induced cell lysis. Cellular debris was removed with extensive wash. The decellularized thymus scaffolds can largely retain the 3D extracellular matrix (ECM) microenvironment that can support the recolonization of TECs. When transplanted into athymic nude mice, the reconstructed thymus organoids can effectively promote the homing of bone marrow-derived lymphocyte progenitors and support the development of a diverse and functional T cell repertoire. Bioengineering of thymus organoids can be a promising approach to rejuvenate/modulate the function of T-cell mediated adaptive immunity in regenerative medicine.    

4.2110           An in vitro compartmental system underlines the contribution of mitochondrial immobility to the ATP supply in the NMJ

Altman, T., Geller, D., Kleeblatt, E., Gradus-Perry, T. and Perlson, E.

1. Cell Sci., 132, jcs234492 (2019)

  The neuromuscular junction (NMJ) is the largest, most-complex synapse in the human body. Motor neuron (MN) diseases, such as amyotrophic lateral sclerosis (ALS), specifically target MNs and the NMJs. However, little is known about the reasons for MN-selective neuronal and synaptic vulnerability in MN diseases. Here, utilizing a compartmental microfluidic in vitro co-culture system, we provide a possible explanation for why the NMJ, other than its unusual dimensions, differs from other synapses. By using live-imaging techniques, we discovered that cultured MNs display higher axonal and synaptic mitochondrial immobility compared with sympathetic neurons (SNs), leading to a profound enrichment of mitochondria only in the MN NMJ. Furthermore, by employing a synaptic ATP sensor, we show that mitochondrial respiration is the key contributor to ATP production in MN NMJs but not in SN synapses. Taken together, our data suggest that mitochondrial localization underlies the unique and specific qualities of MN NMJs. Our findings shed light on the role of mitochondria in MN and NMJ maintenance, and possibly indicate how mitochondria may serve as a source for selective MN vulnerability in neurodegenerative diseases  

4.2111           A Layer-by-Layer Single-Cell Coating Technique To Produce Injectable Beating Mini Heart Tissues via Microfluidics

Guerzoni, L.P., Tsukamoto, Y., Gehlen, D.B., Rommel, D., haraszti, T., Akashi, M. and De Laporte, L. Biomacromolecules,l 20(10), 3746-3754 (2019)   Human induced pluripotent stem cells (hiPSCs) are used as an alternative for human embryonic stem cells. Cardiomyocytes derived from hiPSCs are employed in cardiac tissue regeneration constructs due to the heart’s low regeneration capacity after infarction. A coculture of hiPSC-CM and primary dermal fibroblasts is encapsulated in injectable poly(ethylene glycol)-based microgels via microfluidics to enhance the efficiency of regenerative cell transplantations. The microgels are prepared via Michael-type addition of multi-arm PEG-based molecules with an enzymatically degradable peptide as a cross-linker and modified with a cell-adhesive peptide. Cell–cell interactions and, consequently, cell viability are improved by a thin extracellular matrix (ECM) coating formed on the cell surfaces via layer-by-layer (LbL) deposition. The beating strength of encapsulated cardiomyocytes (∼60 BPM) increases by 2-fold compared to noncoated cells. The combination of microfluidics with the LbL technique offers a new technology to fabricate functional cardiac mini tissues for cell transplantation therapies.  

4.2112           Single-Cell Phenotypic Profiling of CTCs in Whole Blood Using an Integrated Microfluidic Device

Pei, H., Li, L., Wang, Y., Sheng, R., Wang, Y., Xie, S., Shui, L., Si, H. and Tang, B. Anal. Chem., 91(17), 11078-11084 (2019)   Single-cell phenotypic profiling of circulating tumor cells (CTCs) in the blood of cancer patients can reveal vital tumor biology information. Even though various approaches have been provided to enrich and detect CTCs, it remains challenging for consecutive CTC sorting, enumeration, and single-cell characterizations. Here, we report an integrated microfluidic device (IMD) for single-cell phenotypic profiling of CTCs that enables automated CTCs sorting from whole blood following continuous single-cell phenotypic analysis while satisfying the requirements of both high purity (92 ± 3%) of cell sorting and high-throughput processing capacity (5 mL whole blood/3 h). Using this new technique we test the phenotypes of individual CTCs collected from xenograft tumor-bearing mice and colorectal (CRC) patients at different tumor stages. We obtained a correlation between CTC characterization and clinical tumor stage and treatment response. The developed IMD offers a high-throughput, convenient, and rapid strategy to study individual CTCs toward minimally invasive cancer therapy prediction and disease monitoring and has the potential to be translated to clinic for liquid biopsy.  

4.2113           “Basicles”: Microbial Growth and Production Monitoring in Giant Lipid Vesicles

Juskova, P., Schmid, Y.R.F., Stucki, A., Schmitt, S., Held, M. and Dittrich, P.S. ACS Appl. Mater Interfaces, 11, 34698-34706 (2019)   We present an optimized protocol to encapsulate bacteria inside giant unilamellar lipid vesicles combined with a microfluidic platform for real-time monitoring of microbial growth and production. The microfluidic device allows us to immobilize the lipid vesicles and record bacterial growth and production using automated microscopy. Moreover, the lipid vesicles retain hydrophilic molecules and therefore can be used to accumulate products of microbial biosynthesis, which we demonstrate here for a riboflavin-producing bacterial strain. We show that stimulation as well as inhibition of bacterial production can be performed through the liposomal membrane simply by passive diffusion of inducing or antibiotic compounds, respectively. The possibility to introduce as well as accumulate compounds in liposomal cultivation compartments represents great advantage over the current state of the art systems, emulsion droplets, and gel beads. Additionally, the encapsulation of bacteria and monitoring of individual lipid vesicles have been accomplished on a single microfluidic device. The presented system paves the way toward highly parallel microbial cultivation and monitoring as required in biotechnology, basic research, or drug discovery.  

4.2114           T cell fate following Salmonella infection is determined by a STING-IRF1 signaling axis in mice

Park, S-M., Omatsu, T., Zhao, Y., Yoshida, N., Shah, p., Zagani, R., and Reinecker, H-C. Comminications Biol., 2:464 (2019)   The innate immune response following infection with entero-invasive bacterial species is triggered upon release of cyclic di-guanylate monophosphate (c-di-GMP) into the host cell cytosol. Bacterial c-di-GMP activates the intracellular Sensor Stimulator of Interferon Genes (STING), encoded by Tmem173 in mice. Here we identify Interferon Regulatory Factor (IRF) 1 as a critical effector of STING-mediated microbial DNA sensing that is responsible for T_H17 cell generation in the mucosal immune system. We find that STING activation induces IRF1-dependent transcriptional programs in dendritic cells (DCs) that define T cell fate determination, including induction of Gasdermin D, IL-1 family member cytokines, and enzymes for eicosanoid synthesis. Our results show that IRF1-dependent transcriptional programs in DCs are a prerequisite for antigen-specific T_H17 subspecification in response to microbial c-di-GMP and Salmonella typhimurium infection. Our identification of a STING-IRF1 signaling axis for adaptive host defense control will aid further understanding of infectious disease mechanisms.  

4.2115           Self-Organization of Mouse Stem Cells into an Extended Potential Blastoid

Sozen, B., Cox, A.L., De Jonghe, J., Bao, M., Hollfelder, F., Glover, D.M. and Zermicka-Goetz, M. Developmental Cell, 51, 698-712 (2019)   Mammalian blastocysts comprise three distinct cell lineages essential for development beyond implantation: the pluripotent epiblast, which generates the future embryo, and surrounding it the extra-embryonic primitive endoderm and the trophectoderm tissues. Embryonic stem cells can reintegrate into embryogenesis but contribute primarily to epiblast lineages. Here, we show that mouse embryonic stem cells cultured under extended pluripotent conditions (EPSCs) can be partnered with trophoblast stem cells to self-organize into blastocyst-like structures with all three embryonic and extra-embryonic lineages. Morphogenetic and transcriptome profiling analyses reveal that these blastocyst-like structures show distinct embryonic-abembryonic axes and primitive endoderm differentiation and can initiate the transition from the pre- to post-implantation egg cylinder morphology in vitro.  

4.2116           Upregulation of cancer-associated gene expression in activated fibroblasts in a mouse model of non-alcoholic steatohepatitis

Asakawa, M., Itoh, M., Suganami, T., Sakia, T., Kanai, S., Shirakawa, I. Scientific Reports, 9:19601 (2019)   Non-alcoholic steatohepatitis (NASH), characterized by chronic inflammation and fibrosis, is predicted to be the leading cause of cirrhosis and hepatocellular carcinoma (HCC) in the next decade. Although recent evidence suggests the importance of fibrosis as the strongest determinant of HCC development, the molecular mechanisms underlying NASH-induced carcinogenesis still remain unclear. Here we performed RNA sequencing analysis to compare gene expression profiles of activated fibroblasts prepared from two distinct liver fibrosis models: carbon tetrachloride–induced fibrosis as a model without obesity and HCC and genetically obese melanocortin 4 receptor–deficient (MC4R-KO) mice fed Western diet, which develop steatosis, NASH, and eventually HCC. Our data showed that activated fibroblasts exhibited distinct gene expression patterns in each etiology, and that the ‘pathways in cancer’ were selectively upregulated in the activated fibroblasts from MC4R-KO mice. The most upregulated gene in these pathways was fibroblast growth factor 9 (FGF9), which was induced by metabolic stress such as palmitate. FGF9 exerted anti-apoptotic and pro-migratory effects in fibroblasts and hepatoma cells in vitro and accelerated tumor growth in a subcutaneous xenograft model. This study reveals upregulation of cancer-associated gene expression in activated fibroblasts in NASH, which would contribute to the progression from NASH to HCC.  

4.2117           Restoration of liver sinusoidal cell phenotypes by statins improves portal hypertension and histology in rats with NASH

Bravo, M., Raurell, I., Hide, D., Fernandez-Iglesias, A., Gil, M., Barbera, A., Salcedo, M.T., Augustin, S., Genesca, J. and Martell, M. Scientific Reports, 9:20183 (2019)   Non-alcoholic steatohepatitis (NASH) is a common chronic liver disorder in developed countries, with the associated clinical complications driven by portal hypertension (PH). PH may precede fibrosis development, probably due to endothelial dysfunction at early stages of the disease. Our aim was to characterize liver sinusoidal endothelial cell (LSEC) dedifferentiation/capillarization and its contribution to PH in NASH, together with assessing statins capability to revert endothelial function improving early NASH stages. Sprague-Dawley rats were fed with high fat glucose-fructose diet (HFGFD), or control diet (CD) for 8 weeks and then treated with simvastatin (sim) (10 mg·kg⁻¹·day⁻¹), atorvastatin (ato) (10 mg·kg⁻¹·day⁻¹) or vehicle during 2 weeks. Biochemical, histological and hemodynamic determinations were carried out. Sinusoidal endothelial dysfunction was assessed in individualized sorted LSEC and hepatic stellate cells (HSC) from animal groups and in whole liver samples. HFGFD rats showed full NASH features without fibrosis but with significantly increased portal pressure compared with CD rats (10.47 ± 0.37 mmHg vs 8.30 ± 0.22 mmHg; p < 0.001). Moreover, HFGFD rats showed a higher percentage of capillarized (CD32b⁻/CD11b⁻) LSEC (8% vs 1%, p = 0.005) showing a contractile phenotype associated to HSC activation. Statin treatments caused a significant portal pressure reduction (sim: 9.29 ± 0.25 mmHg, p < 0.01; ato: 8.85 ± 0.30 mmHg, p < 0.001), NASH histology reversion, along with significant recovery of LSEC differentiation and a regression of HSC activation to a more quiescent phenotype. In an early NASH model without fibrosis with PH, LSEC transition to capillarization and HSC activation are reverted by statin treatment inducing portal pressure decrease and NASH features improvement.  

4.2118           Isolation of senescent cells by iodixanol (OptiPrep) density gradient‐based separation

Kovacovicova, K. and Vinciguerra, M. Cell Proliferation, 52, e12674 (2019)   Objectives Chemotherapeutic drugs induce senescence in cancer cells but, unlike replicative senescence or oncogene‐induced senescence, do so rather inefficiently and depending on DNA damage. A thorough understanding of the biology of chemotherapy‐induced senescent cells requires their isolation from a mixed population of adjacent senescent and non‐senescent cancer cells. Materials and methods We have developed and optimized a rapid iodixanol (OptiPrep)‐based gradient centrifugation system to identify, isolate and characterize doxorubicin (DXR)‐induced senescent hepatocellular carcinoma (HCC) cells (HepG2 and Huh‐7) in vitro. Results After cellular exposure to DXR, we used iodixanol gradient‐based centrifugation to isolate and re‐plate cells on collagen‐coated flasks, despite their low or null proliferative capacity. The isolated cell populations were enriched for DXR‐induced senescent HCC cells, as confirmed by proliferation arrest assay, and β‐galactosidase and DNA damage‐dependent γH2A.X staining. Conclusions Analysing pure cultures of chemotherapy‐induced senescent versus non‐responsive cancer cells will increase our knowledge on chemotherapeutic mechanisms of action, and help refine current therapeutic strategies.  

4.2119           Nicotinamide mononucleotide (NMN) treatment attenuates oxidative stress and rescues angiogenic capacity in aged cerebromicrovascular endothelial cells: a potential mechanism for the prevention of vascular cognitive impairment

Kiss, T., Balasubrammanian, P., Valcarcel-Ares, M.N., Tarantini, S., Yabluchanskiy, A., Csipo, T., Lipecz, A., Reglodi, D., Zhang, X.A., bari, F., Farkas, E., Csiszar, A. and Ungvari, Z. GeroScience, 41(5), 619-630 (2019)   Age-related impairment of angiogenesis likely has a critical role in cerebromicrovascular rarefaction and development of vascular cognitive impairment and dementia (VCID) in the elderly. Recently, we demonstrated that aging is associated with NAD⁺ depletion in the vasculature and that administration of NAD⁺ precursors exerts potent anti-aging vascular effects, rescuing endothelium-mediated vasodilation in the cerebral circulation and improving cerebral blood supply. The present study was designed to elucidate how treatment with nicotinamide mononucleotide (NMN), a key NAD⁺ intermediate, impacts age-related impairment of endothelial angiogenic processes. Using cerebromicrovascular endothelial cells (CMVECs) isolated from young and aged F344xBN rats, we demonstrated that compared with young cells, aged CMVECs exhibit impaired proliferation, cellular migration (measured by a wound-healing assay using electric cell-substrate impedance sensing [ECIS] technology), impaired ability to form capillary-like structures, and increased oxidative stress. NMN treatment in aged CMVECs significantly improved angiogenic processes and attenuated H₂O₂ production. We also found that pre-treatment with EX-527, a pharmacological inhibitor of SIRT1, prevented NMN-mediated restoration of angiogenic processes in aged CMVECs. Collectively, we find that normal cellular NAD⁺ levels are essential for normal endothelial angiogenic processes, suggesting that age-related cellular NAD⁺ depletion and consequential SIRT1 dysregulation may be a potentially reversible mechanism underlying impaired angiogenesis and cerebromicrovascular rarefaction in aging. We recommend that pro-angiogenic effects of NAD⁺ boosters should be considered in both preclinical and clinical studies.  

4.2120           Injection and Self‐Assembly of Bioinspired Stem Cell‐Laden Gelatin/Hyaluronic Acid Hybrid Microgels Promote Cartilage Repair In Vivo

Feng, Q., Li, Q., Wen, H., Chen, J., Liang, M., Huang, H., Lan, D., Dong, H. and Cao, X. Adv. Funct.Mater., 29, 1906690 (2019)   In this paper, a novel bioinspired stem cell‐laden microgel and related in vivo cartilage repair strategy are proposed. In particular, herein the preparation of new stem cell‐laden microgels, which can be injected into the chondral defect site in a minimally invasive way, and more importantly, capable of in situ self‐assembly into 3D macroporous scaffold without external stimuli, is presented. Specifically, thiolated gelatin (Gel‐SH) and vinyl sulfonated hyaluronic acid (HA‐VS) are first synthesized, and then stem cell‐laden gelatin/hyaluronic acid hybrid microgels (Gel‐HA) are generated by mixing Gel‐SH, HA‐VS, and bone mesenchymal stem cells (BMSCs) together via droplet‐based microfluidic approach, followed by gelation through fast and efficient thiol‐Michael addition reaction. The encapsulated BMSCs show high viability, proliferation, and chondrogenic differentiation potential in the microgels. Moreover, the in vitro test proves that BMSC‐laden Gel‐HA microgels are injectable without sacrificing BMSC viability, and more importantly, can self‐assemble into cartilage‐like scaffolds via cell–cell interconnectivity. In vivo experiments further confirm that the self‐assembled microgels can inhibit vascularization and hypertrophy. The Gel‐HA microgels and relevant cartilage repair strategy, i.e., injecting BMSC‐laden microgels separately and reconstructing chondral defect structure by microgel self‐assembly, provides a simple and effective method for cartilage tissue engineering and regenerative medicine.  

4.2121           Stretch growth of motor axons in custom mechanobioreactors to generate long‐projecting axonal constructs

Katiyar, K., Struzyna, L.A., Das, S. and Cullen, D.K.

1. Tissue Eng. Regen. Med., 13(11), 2040-2054 (2019)

  The central feature of peripheral motor axons is their remarkable lengths as they project from a motor neuron residing in the spinal cord to distant target muscle. However, current in vitro models have not replicated this feature owing to challenges in generating motor axon tracts beyond a few millimeters in length. To address this, we have developed a novel combination of microtissue engineering and mechanically assisted growth techniques to create long‐projecting centimeter‐scale motor axon tracts. Here, primary motor neurons were isolated from rat spinal cords and induced to form engineered microspheres via forced aggregation in custom microwells. This technique yielded healthy motor neurons projecting dense, fasciculated axonal tracts. Within our custom‐built mechanobioreactors, motor neuron culture conditions, neuronal/axonal architecture, and mechanical growth conditions were optimized to generate parameters for robust and efficient stretch growth of motor axons. We found that axons projecting from motor neuron aggregates were able to tolerate displacement rates at least 10 times greater than those by axons projecting from dissociated motor neurons. The growth and structural characteristics of these stretch‐grown motor axons were compared with that of benchmark stretch‐grown sensory axons, revealing increased motor axon fasciculation. Finally, motor axons were integrated with myocytes and stretch grown to create novel long‐projecting axonal‐myocyte constructs that recreate characteristic dimensions of native nerve‐muscle anatomy. This is the first demonstration of mechanical elongation of spinal motor axons and may have applications as anatomically inspired in vitro testbeds or as tissue‐engineered living scaffolds for targeted axon tract reconstruction following nervous system injury or disease.  

4.2122           IDOL regulates systemic energy balance through control of neuronal VLDLR expression

Lee, S.D., Priest, C., Bjursell, M., Gao, J., Arneson, D.V., Ahn, I.S. et al Nature Metabolism, 1, 1089-1100 (2019)   Liver X receptors limit cellular lipid uptake by stimulating the transcription of inducible degrader of the low-density lipoprotein receptor (IDOL), an E3 ubiquitin ligase that targets lipoprotein receptors for degradation. The function of IDOL in systemic metabolism is incompletely understood. Here we show that loss of IDOL in mice protects against the development of diet-induced obesity and metabolic dysfunction by altering food intake and thermogenesis. Unexpectedly, analysis of tissue-specific knockout mice revealed that IDOL affects energy balance, not through its actions in peripheral metabolic tissues (liver, adipose tissue, endothelium, intestine, and skeletal muscle) but by controlling lipoprotein receptor abundance in neurons. Single-cell RNA sequencing of the hypothalamus demonstrated that IDOL deletion altered gene expression linked to the control of metabolism. Finally, we identified very low-density lipoprotein receptor (VLDLR) rather than low-density lipoprotein receptor (LDLR) as the primary mediator of the effects of IDOL on energy balance. These data identify a role for the neuronal IDOL–VLDLR pathway in metabolic homoeostasis and diet-induced obesity.  

4.2123           Interleukin‐9 blockage reduces early hepatic granuloma formation and fibrosis during Schistosoma japonicum infection in mice

Zhan, T., Ma, H., Jiang, S., Zhong, Z., Wang, X., Li, C., Yu, D., Liu, L. Xu, J. and Xia, J. Immunology, 158, 296-303 (2019)   Hepatic fibrosis induced by schistosomes is regulated by a complex network of cytokines. T helper type 9 (Th9) cells are a new type of effector T helper cells, which mainly secrete the specific cytokine interleukin‐9 (IL‐9). Interleukin‐9 has been shown to contribute to liver fibrosis in patients with chronic hepatitis B and in a mouse model due to carbon tetrachloride. However, the role of IL‐9 in schistosomiasis fibrosis remains unknown. In this study, we investigated the roles of IL‐9 in schistosomiasis through in vivo and in vitro studies. The in vivo studies found that neutralization of IL‐9 reduced liver granulomatous inflammation and collagen deposition around parasite eggs. The in vitro studies found that the treatment of primary hepatic stellate cells with IL‐9 induced a significant increase of collagen and α‐smooth‐muscle actin. Moreover, we also described the dynamics and relevance of IL‐9 and IL‐4 in mice infected with Schistosoma japonicum. We found that IL‐9 might appear more quickly and at higher levels than IL‐4. Hence, our findings indicated that IL‐9 might play a role in regulating hepatic fibrosis in early‐stage schistosomiasis and become a promising approach for regulating hepatic fibrosis caused by S. japonicum.  

4.2124           The relationship between DJ-1 and S100A8 in human primary alveolar type II cells in emphysema

Lin, C-R., Bahmed, K., Tomar, D., Marchetti, N., Criner, G.J., Bolla, S., Wilson, M.A., Madesh, M. and Kosmider, B. Am. J. Physiol. Lung Cell Med. Physiol., 317, L791-L804 (2019)   Pulmonary emphysema is characterized by alveolar type II (ATII) cell death, destruction of alveolar wall septa, and irreversible airflow limitation. Cigarette smoke induces oxidative stress and is the main risk factor for this disease development. ATII cells isolated from nonsmokers, smokers, and patients with emphysema were used for this study. ATII cell apoptosis in individuals with this disease was detected. DJ-1 and S100A8 have cytoprotective functions against oxidative stress-induced cell injury. Reduced DJ-1 and S100A8 interaction was found in ATII cells in patients with emphysema. The molecular function of S100A8 was determined by an analysis of the oxidation status of its cysteine residues using chemoselective probes. Decreased S100A8 sulfination was observed in emphysema patients. In addition, its lower levels correlated with higher cell apoptosis induced by cigarette smoke extract in vitro. Cysteine at position 106 within DJ-1 is a central redox-sensitive residue. DJ-1 C106A mutant construct abolished the cytoprotective activity of DJ-1 against cell injury induced by cigarette smoke extract. Furthermore, a molecular and complementary relationship between DJ-1 and S100A8 was detected using gain- and loss-of-function studies. DJ-1 knockdown sensitized cells to apoptosis induced by cigarette smoke extract, and S100A8 overexpression provided cytoprotection in the absence of DJ-1. DJ-1 knockout mice were more susceptible to ATII cell apoptosis induced by cigarette smoke compared with wild-type mice. Our results indicate that the impairment of DJ-1 and S100A8 function may contribute to cigarette smoke-induced ATII cell injury and emphysema pathogenesis.  

4.2125           Strategies to Minimize Various Stress-Related Freeze–Thaw Damages During Conventional Cryopreservation of Mammalian Spermatozoa

Kumar, A., Prasad, J.K., Srivastava, N. and Ghosh, S.K: Biopreservation and Biobanking, 17(6), 603-612 (2019)   The aim of the article is to report a review on different sperm cryopreservation techniques, various stress-related freeze–thaw damages altering sperm structure and function during conventional cryopreservation, and strategies to minimize these stresses. Sperm cryopreservation has allowed indefinite storage and successful transportation of valuable germplasm from proven sites at distant locations, for genetic upgradation through implementation of reproductive techniques, such as artificial insemination. Different techniques for sperm cryopreservation have been proposed such as conventional freezing techniques, directional freezing, and sperm vitrification. Drawbacks related to conventional freezing methods, such as heterogeneous ice nucleation and repeated freeze–thaw cycles at the ice front that disrupts and kill sperm cells, led to the emergence of the directional freezing technique. Sperm vitrification is advantageous as there is no ice crystal-induced physical damages to sperm. However, sperm vitrification has less applicability as encouraging results are only reported in human, dog, and cat. In spite of several drawbacks, conventional freezing techniques are still most widely used for sperm cryopreservation. Spermatozoa experience stresses in the form of cold shock, osmotic stress, and mainly oxidative stress during conventional cryopreservation ultimately reduces the sperm viability and fertility. Several attempts have been made in the past to minimize all these stresses individually or in combination. Membrane fluidity was increased to prevent the cold shock and cryocapacitation-like changes by the addition of cholesterol to the membrane. Antifreeze proteins were added in semen extender to minimize freeze–thaw damages due to heterogeneous ice nucleation and ice recrystallization. Oxidative stress was reduced either by neutralizing reactive oxygen species (ROS) through enzymatic, nonenzymatic, plant-based antioxidants or reductants; or by minimizing the level of sources like the semen radiation exposure, leucocytes, and dead and defective spermatozoa, which lead to ROS production during the semen cryopreservation process. A novel approach of minimizing oxidative stress was to reduce the oxygen tension in sperm microenvironment that is, extender by partial deoxygenation process, as a number of literatures pointed out direct link of O₂ with ROS production. When compared with other strategies, partial deoxygenation of semen extender with N₂ gassing is found as a cost-effective, comparatively easy and a potential approach to large-scale frozen semen production.  

4.2126           Excellent Islet Yields after 18-h Porcine Pancreas Preservation by Ductal Injection, Pancreas Preservation with MK Solution, Bottle Purification, and Islet Purification Using Iodixanol with UW Solution and Iodixanol with MK Solution

Kuwae, K., Miyagi-Shiohira, C., Hamada, E., Tamaki, Y., Nishime, K., Sakai, M., Yonaha, T., Makishi, E., Saitoh, I., Watanabe, M.a nd Noguchi, H.

1. Clin. Med., 8(10), E1561 (2019)

  Successful islet isolation is the key to successful islet transplantation. Our group recently modified the islet isolation protocol to include pancreatic ductal injection of the preservation solution, pancreas storage in modified extracellular-type trehalose-containing Kyoto (MK) solution, and use of an iodixanol-based purification solution and bottle purification. In this study, we applied these methods to porcine islet isolation after 18-h pancreas preservation and compared two solutions with different compositions in bottle purification. Islet yield before purification was 651,661 ± 157,719 islet equivalents (IE) and 5576 ± 1538 IE/g pancreas weight. An IU solution was made by adding iodixanol to University of Wisconsin solution and an IK solution was made by adding iodixanol to MK solution. The efficacy of the two solutions for islet isolation was compared. There were no significant differences between the two purification methods with regard to islet yield, survival rate, purity, score, or stimulation index. These results indicate that our isolation protocol produces efficient islet yields from prolonged cold-stored pancreas and that IU and IK solutions are equally useful for islet purification.  

4.2127           Decreased erythrocyte binding of Siglec-9 increases neutrophil activation in sickle cell disease

Kiser, Z.M., Lizcano, A., Nguyen, J., Becker, G.L., Belcher, J.D., Varki, A.P. and Vercellotti, G.M. Blood Cells, Molecules and Diseases, 81, 102399 (2020)   Oxidative stress and inflammation promote vaso-occlusion in sickle cell disease (SCD). CD33-related Sialic acid-binding immunoglobulin-type lectins (CD33rSiglecs) are cell surface proteins that recognize sialic acids inhibit innate immune cell functions. We have shown that Siglec-9 on human neutrophils interact with erythrocyte sialic acids (prominently glycophorin-A (GYPA) to suppress neutrophil reactive oxygen species (ROS). We hypothesized that altered sickle erythrocyte membrane sialic acid leads to decreased Siglec-9 binding capability, and thus a decreased neutrophil oxidative burst. SS erythrocytes express significantly more sialic acid than AA erythrocytes (p = 0.02). SS erythrocytes displayed significantly less Siglec-9-Fc binding 39% ± 11 (mean ± SEM) compared to AA erythrocytes 78% ± 5 (p = 0.009). Treatment of AA erythrocytes with sialidase to remove sialic acid decreased binding to 3% ± 7.9 (p ≤ 0.001). When freshly isolated neutrophils were incubated with AA erythrocytes, neutrophils achieved 16% ± 6 of the oxidative burst exhibited by a stimulated neutrophil without erythrocytes. In contrast, neutrophils incubated with SS erythrocytes achieved 47% ± 6 of the oxidative burst (AA versus SS, p = 0.03). Stimulated neutrophils incubated with AA erythrocytes showed minimal NET formation while with SS erythrocytes NETs increased. SS erythrocytes are deficient in binding to neutrophil Siglec-9 which may contribute to the increased oxidative stress in SCD.  

4.2128           A rapid and accurate method to quantify neurite outgrowth from cell and tissue cultures: Two image analytic approaches using adaptive thresholds or machine learning

Ossinger, A., Bajic, A., Pan, S., Andersson, B., Ranefall, P., Hailer, N.P.and Schizas, N.

1. Neurosci. Methods, 331, 108522 (2020)

  Background Assessments of axonal outgrowth and dendritic development are essential readouts in many in vitro models in the field of neuroscience. Available analysis software is based on the assessment of fixed immunolabelled tissue samples, making it impossible to follow the dynamic development of neurite outgrowth. Thus, automated algorithms that efficiently analyse brightfield images, such as those obtained during time-lapse microscopy, are needed. New method We developed and validated algorithms to quantitatively assess neurite outgrowth from living and unstained spinal cord slice cultures (SCSCs) and dorsal root ganglion cultures (DRGCs) based on an adaptive thresholding approach called NeuriteSegmantation. We used a machine learning approach to evaluate dendritic development from dissociate neuron cultures. Results NeuriteSegmentation successfully recognized axons in brightfield images of SCSCs and DRGCs. The temporal pattern of axonal growth was successfully assessed. In dissociate neuron cultures the total number of cells and their outgrowth of dendrites were successfully assessed using machine learning. Comparison with existing methods The methods were positively correlated and were more time-saving than manual counts, having performing times varying from 0.5−2 min. In addition, NeuriteSegmentation was compared to NeuriteJ®, that uses global thresholding, being more reliable in recognizing axons in areas of intense background. Conclusion The developed image analysis methods were more time-saving and user-independent than established approaches. Moreover, by using adaptive thresholding, we could assess images with large variations in background intensity. These tools may prove valuable in the quantitative analysis of axonal and dendritic outgrowth from numerous in vitro models used in neuroscience.  

4.2129           The impact of surgical complications on the outcome of total pancreatectomy with islet autotransplantation

Shahbazov, R., Naziruddin, B., Salam, O., Saracino, G., lwvy, M.F., Beecheri, E. and Onaca, N. Am. J. Surg., 219, 99-105 (2020)   Total pancreatectomy with islet autotransplantation is a promising treatment for refractory chronic pancreatitis. We analyzed postoperative complications in 83 TPIAT patients and their impact on islet graft function. We examined patient demographics, preoperative risk factors, intraoperative variables, and 30- and 90-day postoperative morbidity and mortality. Daily insulin requirement, HbA1c, C-peptide levels, and narcotic requirements were analyzed before and after surgery. Adverse events were recorded, with postoperative complications graded according to the Clavien-Dindo classification. There was no mortality in this patient group. Postoperative complications occurred in 38 patients (45.7%). Patients with postoperative complications were readmitted significantly more often within 30 days (p = 0.01) and 90 days posttransplant (p < 0.0003) and had a significantly longer hospital stay (p = 0.004) and intensive care unit stay (p = 0.001). Insulin dependence and graft function assessed by HbA1c, C-Peptide and insulin requirements did not differ significantly by these complications. Postoperative complications after TPIAT are associated with longer hospital and intensive care unit stay and with readmission; however, the surgical complications do not affect islet graft function.  

4.2130           Effects of primary amine-based coatings on microglia internalization of nanogels

Mauri, E., Veglianese, P., Papa, S., Rossetti, A., De Paola, M., Mariani, A., Posel, Z., Psocco, P., Sachetti, A. and Rossi, F. Colloids and Surfaces B: Bioinerfaces, 185, 110574 (2020)   Nanogels represent a pivotal class of biomaterials in the therapeutic intracellular treatment of many diseases, especially those involving the central nervous system (CNS). Their biocompatibility and synergy with the biological environment encourage their cellular uptake, releasing the curative cargo in the desired area. As a main drawback, microglia are generally able to phagocytize any foreign element overcoming the blood brain barrier (BBB), including these materials, drastically limiting their bioavailability for the target cells. In this work, we investigated the opportunity to tune and therefore reduce nanogel internalization in microglia cultures, exploiting the orthogonal chemical functionalization with primary amine groups, as a surface coating strategy. Nanogels are designed by following two methods: the direct grafting of aliphatic primary amines and the linkage of -NH₂ modified PEG on the nanogel surface. The latter synthesis was proposed to evaluate the combination of PEGylation with the basic nitrogen atom. The achieved results indicate the possibility of effectively modulating the uptake of nanogels, in particular limiting their internalization using the PEG-NH₂ coating. This outcome could be considered a promising strategy for the development of carriers for drugs or gene delivery that could overcome microglia scavenging.  

4.2131           Pancreatic cancer-associated inflammation drives dynamic regulation of p35 and Ebi3

Michaud, D., Mirlekar, B., Bischoff, S., Cowley, D.O., Vignali, D.A.A. and Pylayeva-Gupta, Y. Cytokine, 125, 154817 (2020)   B cells are important modulators of immune responses both in autoimmunity and cancer. We have previously shown that B regulatory (Breg) cells promote pancreatic cancer via production of IL35, a heterodimeric cytokine comprised of the subunits p35 (Il12a) and Ebi3. However, it is not known how production of IL35 is regulated in vivo in the context of cancer-associated inflammation. To begin addressing this question, we have generated a knock-in mouse model, Il12a^GFP, where an IRES-emGFP gene was inserted within the 3′ UTR of the Il12a locus. EmGFP signal in B cells from the Il12a^GFP mice correlated with expression of p35 mRNA and protein. Using this model, we observed that in addition to Bregs, expression of GFP (p35) is upregulated in several other B cell subtypes in response to cancer. We assessed the expression of the other IL35 subunit, Ebi3, using a published tdTomato reporter model. We determined that Ebi3 expression was more tightly regulated in vivo and in vitro, suggesting that stimuli affecting Ebi3 upregulation are more likely to result in production of full IL35 heterodimer. We were also able to detect GFP and Tomato signal in myeloid & T cell lineages suggesting that these reporter models could also be used for tracking IL12-, IL27- and IL35-producing cells. Furthermore, using primary B cells isolated from reporter mice, we identified BCR, CD40 and TLR pathways as potential drivers of IL35 expression. These findings highlight the importance of pancreatic cancer-associated inflammatory processes as drivers of cytokine expression and provide a tool to dissect both disease-associated regulation of IL12- and IL35-competent lineage cells as well as establish assays for pharmacological targeting of individual subunits of heterodimeric IL12 family cytokines.  

4.2132           Chapter 7 - Assisted Reproductive Technologies and Genetic Modifications in Rats

Agca, Y. The Laboratory Rat (Third Edition) American College of Laboratory Animal Medicine, 181-213 (2020)   Over the past two centuries rats have been recognized as an excellent experimental animal model in numerous biomedical research applications and translational medicine for better understanding of mammalian reproductive systems and germ-line gene modification to study human diseases and disorders. In almost every aspect of reproductive sciences, rats have commonly been used in studies involving sex steroid hormone action, gonadotropin-releasing hormone (GnRH) action mechanism, age-related changes in GnRH neurosecretory function, and menopause. Furthermore, rats have been extensively used to study cellular, molecular, morphological, physiological, and functional changes associated with male and female gamete and embryo development, adverse effects of environmentally relevant hazardous substances, reproductive cycle, epigenetic transgenerational inheritance, and phenotype. Moreover, significant progress has been made in follicular isolation, in vitro oocyte maturation, spermatogonial stem cell culture, establishment of embryonic stem cells, preimplantation embryo development, microinjection and insemination, and effective germplasm cryopreservation. These state-of-the-art germplasm biotechnologies in combination with novel genome editing technologies have accelerated the creation of new transgenic and knockout rat models without strain limitation. This chapter will review assisted reproductive technologies, germplasm cryobanking, and genetic modification in rats.  

4.2133           Chapter 4 - Islet isolation for autotransplantation, following total or near total pancreatectomy

Linetsky, E. and Ricordi, C. Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, 2, 67-87 (2020)   Human islet isolation for autotransplantation (IAT) is performed to prevent surgically induced diabetes following near total or total pancreatectomy. The latter is performed to alleviate intractable pain and suffering and prolong life in patients suffering from chronic pancreatitis. Since 1997, when it was first performed at the University of Minnesota, islet isolation for IAT following pancreatic resection has been demonstrated as the best option to improve pain, alleviate the risk of “brittle diabetes,” and offer freedom from exogenous insulin. The most critical factor for a favorable metabolic outcome as a result of IAT is the transplanted islet cell mass, making islet isolation yield of outmost importance. This chapter discusses current islet cell isolation techniques utilized in processing of autologous pancreata in our center and others, with the goal of optimizing the isolation outcome.  

4.2134           Genomic programming of IRF4-expressing human Langerhans cells

Sirvent, S., Vallejo, A.F., Davies, J., Clayton, K., Wu, Z., Woo, J. et al Nature Communications, 11:313 (2020)   Langerhans cells (LC) can prime tolerogenic as well as immunogenic responses in skin, but the genomic states and transcription factors (TF) regulating these context-specific responses are unclear. Bulk and single-cell transcriptional profiling demonstrates that human migratory LCs are robustly programmed for MHC-I and MHC-II antigen presentation. Chromatin analysis reveals enrichment of ETS-IRF and AP1-IRF composite regulatory elements in antigen-presentation genes, coinciding with expression of the TFs, PU.1, IRF4 and BATF3 but not IRF8. Migration of LCs from the epidermis is accompanied by upregulation of IRF4, antigen processing components and co-stimulatory molecules. TNF stimulation augments LC cross-presentation while attenuating IRF4 expression. CRISPR-mediated editing reveals IRF4 to positively regulate the LC activation programme but repress NF2EL2 and NF-kB pathway genes that promote responsiveness to oxidative stress and inflammatory cytokines. Thus, IRF4-dependent genomic programming of human migratory LCs appears to enable LC maturation while attenuating excessive inflammatory and immunogenic responses in the epidermis.  

4.2135           Single‐Cell Analysis Using Droplet Microfluidics

Matula, K., Rivello, F. and Huck, W.T.S. Adv. Biosys., 4(1), 1900188 (2020)   Droplet microfluidics has revolutionized the study of single cells. In article number 1900188, Wilhelm T. S. Huck, Kinga Matuła, and Francesca Rivello provide an overview of droplet microfluidics approaches in genomics, epigenetics, transcriptomics, proteomics (secreted antibodies and cytokines), and metabolomics. The review is addressed to both novice researchers in the field, but also experienced scientists who wish to explore new combinations of single‐cell studies.  

4.2136           Quercetin Attenuates Decrease of Thioredoxin Expression Following Focal Cerebral Ischemia and Glutamate-induced Neuronal Cell Damage

Park, D-J., Kang, J-B., Shah, F-A., Jin, Y-B. and Koh, P-O. Neuroscience, 428, 38-49 (2020)   Quercetin is a bioactive flavonoid which abundantly exists in vegetables and fruits. Quercetin exerts a neuroprotective effect against cerebral ischemia. Thioredoxin acts as antioxidant by regulating redox signaling. This study investigated whether quercetin regulates thioredoxin expression in focal cerebral ischemia and glutamate-induced neuronal cell death. Male Sprague Dawley rats (210–230 g) were intraperitoneally injected with vehicle or quercetin (10 mg/kg) 1 h prior to middle cerebral artery occlusion (MCAO). Cerebral cortex was collected 24 h after MCAO. MCAO led to neurological movement deficits, brain edema, and serious histopathological damages in cerebral cortex, and quercetin alleviated these damages following MCAO. We observed the change of thioredoxin expression in MCAO animals with quercetin using proteomic approach, reverse-transcription PCR, and Western blot analyses. Thioredoxin expression decreased in vehicle-treated MCAO animals, while quercetin attenuated this decrease. Moreover, quercetin treatment alleviated the decrease in the number of thioredoxin-positive cells in cerebral cortex of MCAO animals. Furthermore, immunoprecipitation analysis demonstrated that interaction of apoptosis signal-regulating kinase 1 (ASK1) and thioredoxin was decreased in MCAO animals with vehicle, while quercetin prevented MCAO-induced decrease in these binding. In addition, quercetin also alleviated the reduction of cell viability and the decrease in thioredoxin expression in glutamate-treated hippocampal cell line and primary cultures of cortical neurons. However, in thioredoxin-silenced cortical neuron, anti-apoptotic effect of quercetin was decreased. Thus, changes of thioredoxin expression by quercetin may contribute to the neuroprotective effect of quercetin in focal cerebral ischemia. Our findings suggest that quercetin mediates its neuroprotective function by regulation of thioredoxin expression and maintenance of interaction between ASK1 and thioredoxin.  

4.2137           Human Liver Memory CD8+ T Cells Use Autophagy for Tissue Residence

Swadling, L., Pallett, L.J., Diniz, M.O., Schurich, A., Simon, A.K. and Maini, M.K. Cell Reports, 30, 687-698 (2020)   Tissue-resident memory T cells have critical roles in long-term pathogen and tumor immune surveillance in the liver. We investigate the role of autophagy in equipping human memory T cells to acquire tissue residence and maintain functionality in the immunosuppressive liver environment. By performing ex vivo staining of freshly isolated cells from human liver tissue, we find that an increased rate of basal autophagy is a hallmark of intrahepatic lymphocytes, particularly liver-resident CD8⁺ T cells. CD8⁺ T cells with increased autophagy are those best able to proliferate and mediate cytotoxicity and cytokine production. Conversely, blocking autophagy induction results in the accumulation of depolarized mitochondria, a feature of exhausted T cells. Primary hepatic stellate cells or the prototypic hepatic cytokine interleukin (IL)-15 induce autophagy in parallel with tissue-homing/retention markers. Inhibition of T cell autophagy abrogates tissue-residence programming. Thus, upregulation of autophagy adapts CD8⁺ T cells to combat mitochondrial depolarization, optimize functionality, and acquire tissue residence.  

4.2138           Torin2 Exploits Replication and Checkpoint Vulnerabilities to Cause Death of PI3K-Activated Triple-Negative Breast Cancer Cells

Chopra, S.S., Jenney, A., Palmer, A., Asara, J.M., Gray, N.S. and Sorger, P.K. Cell Systems, 10, 66-81 (2020)   Frequent mutation of PI3K/AKT/mTOR signaling pathway genes in human cancers has stimulated large investments in targeted drugs but clinical successes are rare. As a result, many cancers with high PI3K pathway activity, such as triple-negative breast cancer (TNBC), are treated primarily with chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors, we find that one drug, Torin2, is unusually effective because it inhibits both mTOR and other PI3K-like kinases (PIKKs). In contrast to mTOR-selective inhibitors, Torin2 exploits dependencies on several kinases for S-phase progression and cell-cycle checkpoints, thereby causing accumulation of single-stranded DNA and death by replication catastrophe or mitotic failure. Thus, Torin2 and its chemical analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors.  

4.2139           Neutralization of Oxidized Phospholipids Ameliorates Non-alcoholic Steatohepatitis

Sun, X., Seidman, J.S., Zhao, P.…, Tsimikas, S., Glass, C.K. and Witztum, J.L. Cell Metabolism. 31(1), 189-206 (2020)   Oxidized phospholipids (OxPLs), which arise due to oxidative stress, are proinflammatory and proatherogenic, but their roles in non-alcoholic steatohepatitis (NASH) are unknown. Here, we show that OxPLs accumulate in human and mouse NASH. Using a transgenic mouse that expresses a functional single-chain variable fragment of E06, a natural antibody that neutralizes OxPLs, we demonstrate the causal role of OxPLs in NASH. Targeting OxPLs in hyperlipidemic Ldlr ^−/− mice improved multiple aspects of NASH, including steatosis, inflammation, fibrosis, hepatocyte death, and progression to hepatocellular carcinoma. Mechanistically, we found that OxPLs promote ROS accumulation to induce mitochondrial dysfunction in hepatocytes. Neutralizing OxPLs in AMLN-diet-fed Ldlr ^−/− mice reduced oxidative stress, improved hepatic and adipose-tissue mitochondrial function, and fatty-acid oxidation. These results suggest targeting OxPLs may be an effective therapeutic strategy for NASH.  

4.2140           Age and sex determine CD4+ T cell stimulatory and polarizing capacity of rat splenic dendritic cells

Stojic-Vukanic, Z., Pilipovic, I., Bufan, B., Stojanovic, M., And Leposavic, G. Biogerontology, 21, 83-107 (2020)   The study investigated influence of sex and age on splenic myeloid dendritic cells (DCs) from Dark Agouti rats. Freshly isolated DCs from young males exhibited less mature phenotype and greater endocytic capacity compared with those from age-matched females. Upon LPS stimulation in vitro they were less potent in stimulating allogeneic CD4+ cells in mixed leukocyte reaction (MLR), due to lower expression of MHC II, and greater NO and IL-10 production. In accordance with higher TGF-β production, young male rat DCs were less potent in stimulating IL-17 production in MLR than those from young females. Irrespective of sex, endocytic capacity and responsiveness of DCs to LPS stimulation in culture, judging by their allostimulatory capacity in MLR decreased with age, reflecting decline in MHC II surface density followed by their greater NO production; the effects more prominent in females. Additionally, compared with LPS-stimulated DCs from young rats, those from sex-matched aged rats were more potent in stimulating IL-10 production in MLR, whereas capacity of DCs from aged female and male rats to stimulate IL-17 production remained unaltered and decreased, respectively. This reflected age-related shift in IL-6/TGF-β production level ratio in LPS-stimulated DC cultures towards TGF-β, and sex-specific age-related remodeling CD4+ cell cytokine pathways. Additionally, compared with LPS-stimulated DCs from young rats, those cells from sex-matched aged rats were less potent in stimulating IFN-γ production in MLR, the effect particularly prominent in MLRs encompassing male rat DCs. The study showed that stimulatory and polarizing capacity of DCs depends on rat sex and age.  

4.2141           Hypoxia induces the activation of hepatic stellate cells through the PVT1-miR-152-ATG14 signaling pathway

Yu, F., Dong, B., Dong, P., He, Y., Zheng, J. and Xu, P. Mol. Cell. Biochem., 465, 115-123 (2020)   Increasing studies have indicated that hypoxia serves as a pivotal microenvironmental factor that facilitates activation of hepatic stellate cells (HSCs). However, the mechanism by which hypoxia activates HSCs is not clear. Here, we demonstrated that plasmacytoma variant translocation 1 (PVT1) and autophagy were overexpressed in liver fibrotic specimens. In primary mouse HSCs, both PVT1 and autophagy were induced by hypoxia. Further study showed that hypoxia-induced autophagy depended on expression of PVT1 and miR-152 in HSCs. Luciferase reporter assay indicated that autophagy-related gene 14 (ATG14) was a direct target of miR-152. In addition, inhibition of autophagy by 3‐methyladenine and Beclin-1 siRNA impeded activation of HSCs cultured in 1% O₂. Taken together, autophagy induction via the PVT1-miR-152-ATG14 signaling pathway contributes to activation of HSCs under hypoxia condition.  

4.2142           Epilepsy in a melanocyte-lineage mTOR hyperactivation mouse model: A novel epilepsy model

Yang, F., Yang, L., Wataya-Kaneda, M., Teng, L. and Katayama, I. PloS One, 15(1), e0228204 (2020)   Objective To clarify the complex mechanism underlying epileptogeneis, a novel animal model was generated. Methods In our previous research, we have generated a melanocyte-lineage mTOR hyperactivation mouse model (Mitf-M-Cre Tsc2 KO mice; cKO mice) to investigate mTOR pathway in melanogenesis regulation, markedly reduced skin pigmentation was observed. Very unexpectedly, spontaneous recurrent epilepsy was also developed in this mouse model. Results Compared with control littermates, no change was found in either brain size or brain mass in cKO mice. Hematoxylin staining revealed no obvious aberrant histologic features in the whole brains of cKO mice. Histoimmunofluorescence staining and electron microscopy examination revealed markedly increased mTOR signaling and hyperproliferation of mitochondria in cKO mice, especially in the hippocampus. Furthermore, rapamycin treatment reversed these abnormalities. Conclusions This study suggests that our melanocyte-lineage mTOR hyperactivation mouse is a novel animal model of epilepsy, which may promote the progress of both epilepsy and neurophysiology research.  

4.2143           Spinal Motoneuron TMEM16F Acts at C-boutons to Modulate Motor Resistance and Contributes to ALS Pathogenesis

Soulard, C., Salsac, C., Mouzat, K., Lumbroso, S., Raoul, C. and Scamps, F. Cell Reports, 30, 2581-2593 (2020)   Neuronal Ca²⁺ entry elicited by electrical activity contributes to information coding via activation of K⁺ and Cl⁻ channels. While Ca²⁺-dependent K⁺ channels have been extensively studied, the molecular identity and role of Ca²⁺-activated Cl⁻ channels (CaCCs) remain unclear. Here, we demonstrate that TMEM16F governs a Ca²⁺-activated Cl⁻ conductance in spinal motoneurons. We show that TMEM16F is expressed in synaptic clusters facing pre-synaptic cholinergic C-boutons in α-motoneurons of the spinal cord. Mice with targeted exon deletion in Tmem16f display decreased motor performance under high-demanding tasks attributable to an increase in the recruitment threshold of fast α-motoneurons. Remarkably, loss of TMEM16F function in a mouse model of amyotrophic lateral sclerosis (ALS) significantly reduces expression of an activity-dependent early stress marker and muscle denervation, delays disease onset, and preserves muscular strength only in male ALS mice. Thus, TMEM16F controls motoneuron excitability and impacts motor resistance as well as motor deterioration in ALS.  

4.2144           Toll-like Receptor-6 Signaling Prevents Inflammation and Impacts Composition of the Microbiota During Inflammation-Induced Colorectal Cancer

Kim, J-H., Kordahl, M.C., Chac, D. and DePaolo, R.W. Cancer Prev. Res., 26, 25-40 (2020)   Tightly regulated immune responses must occur in the intestine to avoid unwanted inflammation, which may cause chronic sequela leading to diseases such as colorectal cancer. Toll-like receptors play an important role in preventing aberrant immune responses in the intestine by sensing endogenous commensal microbiota and delivering important regulatory signals to the tissue. However, the role that specific innate receptors may play in the development of chronic inflammation and their impact on the composition of the colonic microbiota is not well understood. Using a model of inflammation-induced colorectal cancer, we found that Lactobacillus species are lost more quickly in wild-type (WT) mice than TLR6-deficient mice resulting in overall differences in bacterial composition. Despite the longer retention of Lactobacillus, the TLR6-deficient mice presented with more tumors and a worse overall outcome. Restoration of the lost Lactobacillus species suppressed inflammation, reduced tumor number, and prevented change in the abundance of Proteobacteria only when given to WT mice, indicating the effect of these Lactobacillus are TLR6 dependent. We found that the TLR6-dependent effects of Lactobacillus could be dissociated from one another via the involvement of IL10, which was necessary to dampen the inflammatory microenvironment, but had no effect on bacterial composition. Altogether, these data suggest that innate immune signals can shape the composition of the microbiota under chronic inflammatory conditions, bias the cytokine milieu of the tissue microenvironment, and influence the response to microbiota-associated therapies.  

4.2145           Macrophage galactose lectin is critical for Kupffer cells to clear aged platelets

Deppermann, C., Kratofil, R.M., Peiseler, M., David, B.A., Zindel, J., Castanheira, K F.V.E.S., van der Wal, F., Carestia, A., Jenne, C.N., Marth, J.D. and Kubes, P.

1. Exp. Med., 217(4), e20190723 (2020)

  Every day, megakaryocytes produce billions of platelets that circulate for several days and eventually are cleared by the liver. The exact removal mechanism, however, remains unclear. Loss of sialic acid residues is thought to feature in the aging and clearance of platelets. Using state-of-the-art spinning disk intravital microscopy to delineate the different compartments and cells of the mouse liver, we observed rapid accumulation of desialylated platelets predominantly on Kupffer cells, with only a few on endothelial cells and none on hepatocytes. Kupffer cell depletion prevented the removal of aged platelets from circulation. Ashwell-Morell receptor (AMR) deficiency alone had little effect on platelet uptake. Macrophage galactose lectin (MGL) together with AMR mediated clearance of desialylated or cold-stored platelets by Kupffer cells. Effective clearance is critical, as mice with an aged platelet population displayed a bleeding phenotype. Our data provide evidence that the MGL of Kupffer cells plays a significant role in the removal of desialylated platelets through a collaboration with the AMR, thereby maintaining a healthy and functional platelet compartment.  

4.2146           T cell receptor and cytokine signal integration in CD8+ T cells is mediated by the protein Themis

Brzostek, J., Gautam, N., Zhao, X., Chen, E.W., Mehta, M., Tung, D.W.H., Chua, Y.L., Yap, J., Cho, S.H., Sankaran, S., Rybakin, V., Fu, G. and Gascoigne, N.R.J. Nature Immunol., 21, 186-198 (2020)   T cell homeostasis and functional responsiveness require signals from self-peptide–major histocompatibility complex (self-pMHC) and cytokines, but the mechanisms controlling this signal integration are unknown. Using a conditional deletion of the T cell lineage-specific protein Themis, we show that Themis is required for the maintenance of peripheral CD8⁺ T cells and for proliferative CD8⁺ T cell responses to low-affinity pMHC aided by cytokines. Themis-deficient peripheral T cells show a phenotype indicative of reduced tonic signaling from self-pMHC, strongly suggesting that Themis is a positive regulator of T cell receptor signal strength in response to low-affinity self-pMHC in peripheral T cells. Signals from low-affinity pMHC and cytokines synergistically induce phosphorylation of the kinase Akt, metabolic changes and c-Myc transcription factor induction in CD8⁺ T cells only in the presence of Themis. This function of Themis is mediated through Shp1 phosphatase, as peripheral Themis and Shp1 double deletion rescues the peripheral CD8⁺ T cell maintenance.  

4.2147           Regenerative lineages and immune-mediated pruning in lung cancer metastasis

Laughney, A.M., Hu, J., Campbell, N.R., Bakhoum, S., Setty, M. et al Nature Med., 26, 259-269 (2020)   Developmental processes underlying normal tissue regeneration have been implicated in cancer, but the degree of their enactment during tumor progression and under the selective pressures of immune surveillance, remain unknown. Here we show that human primary lung adenocarcinomas are characterized by the emergence of regenerative cell types, typically seen in response to lung injury, and by striking infidelity among transcription factors specifying most alveolar and bronchial epithelial lineages. In contrast, metastases are enriched for key endoderm and lung-specifying transcription factors, SOX2 and SOX9, and recapitulate more primitive transcriptional programs spanning stem-like to regenerative pulmonary epithelial progenitor states. This developmental continuum mirrors the progressive stages of spontaneous outbreak from metastatic dormancy in a mouse model and exhibits SOX9-dependent resistance to natural killer cells. Loss of developmental stage-specific constraint in macrometastases triggered by natural killer cell depletion suggests a dynamic interplay between developmental plasticity and immune-mediated pruning during metastasis.  

4.2148           Determining histone H4 acetylation patterns in human peripheral blood mononuclear cells using mass spectrometry

Bux, E.M., Solis-Mezarino, V., Kuhm, C., Northoff, B.H., Karin, I., Klopstock, T., Holdt, L.M., Völker-Albert, M., Imhof, A. and Peleg, S. Clin. Mass Spectrometry, 15, 54-60 (2020)   Misregulated chromatin remodeling via erroneous histone acetylation has been linked with multiple diseases including many age-associated maladies. Previous studies on histone acetylation have utilized specific histone acetylation antibodies as a primary quantification tool. However, antibodies that target specific histone modifications frequently lack sufficient sensitivity and specificity, making signal quantification and data interpretation difficult. Here, we used mass spectrometry to quantify the acetylation patterns of histone H4 in peripheral blood mononuclear cells (PBMCs) from human patients of varying age. We observed that mono-acetylation of H4K16ac is lower in midlife human PBMCs, and that various changes occur in the H4 acetylation signature using PBMCs prepared from old patients. Our data corroborates previous work suggesting that ageing might be characterized by specific changes in the acetylation states of histone H4 and shows that targeted mass spectrometry together with appropriate quantification methods can be used to sensitively measure such changes.  

4.2149           Recombinant collagenase from Grimontia hollisae as a tissue dissociation enzyme for isolating primary cells

Tanaka, K., Okitsu, T., Teramura, N., Iijima, K., Hayashida, O., Teramae, H. and Hattori, S. Scientific Reports, 10:3927 (2020)   Collagenase products are crucial to isolate primary cells in basic research and clinical therapies, where their stability in collagenolytic activity is required. However, currently standard collagenase products from Clostridium histolyticum lack such stability. Previously, we produced a recombinant 74-kDa collagenase from Grimontia hollisae, which spontaneously became truncated to ~60 kDa and possessed no stability. In this study, to generate G. hollisae collagenase useful as a collagenase product, we designed recombinant 62-kDa collagenase consisting only of the catalytic domain, which exhibits high production efficiency. We demonstrated that this recombinant collagenase is stable and active under physiological conditions. Moreover, it possesses higher specific activity against collagen and cleaves a wider variety of collagens than a standard collagenase product from C. histolyticum. Furthermore, it dissociated murine pancreata by digesting the collagens within the pancreata in a dose-dependent manner, and this dissociation facilitated isolation of pancreatic islets with masses and numbers comparable to those isolated using the standard collagenase from C. histolyticum. Implantation of these isolated islets into five diabetic mice led to normalisation of the blood glucose concentrations of all the recipients. These findings suggest that recombinant 62-kDa collagenase from G. hollisae can be used as a collagenase product to isolate primary cells.  

4.2150           Two-step magnetic bead-based (2MBB) techniques for immunocapture of extracellular vesicles and quantification of microRNAs for cardiovascular diseases: A pilot study

Chen, S., Shiesh, S-C., Lee, G-B. and Chen, C. PloS One, 15(2), e0229610 (2020)   Targeted gene therapy using recombinant adeno-associated virus (rAAV) vectors is a potential therapeutic strategy for treating cancer, and tissue-specific promoters may help with tissue targeting. Medullary thyroid carcinoma (MTC) is a disease of the calcitonin secreting thyroid C cells, and calcitonin is highly expressed in MTC tumors compared to other cells. To target MTC cells, we evaluated an rAAV serotype 2 vector (rAAV2-pM+104GFP) containing a modified calcitonin/calcitonin gene related peptide promoter (pM+104) and a green fluorescent protein (GFP) reporter gene. In vitro transduction experiments comparing the MTC TT cell line with non-MTC cell lines demonstrated that rAAV2-pM+104-GFP infection yielded significantly (p<0.05) higher GFP expression in TT cells than in non-MTC cell lines (HEK293 and HeLa), and significantly higher expression than in TT cells infected with the positive control rAAV2-pCBA-GFP vector. The rAAV2-pCBA-GFP control vector included a well-characterized, ubiquitously expresses control promoter, the chicken beta actin promoter with a cytomegalovirus enhancer (pCBA). In vivo experiments using a TT cell xenograft tumor mouse model showed that tumors directly injected with 2 x 1010 vg of rAAV2-pM+104-GFP vector resulted in GFP expression detected in 21.7% of cells, 48 hours after the injection. Furthermore, GFP expression was significantly higher for rAAV-pM +104-GFP treatments with a longer vector treatment duration and higher vector dose, with up to 52.6% (q<0.05) GFP cells detected 72 hours after injecting 1x 1011 vg/tumor. These data show that we have developed an rAAV vector with improved selectivity for MTC.  

4.2151           Mitochondrial activity is impaired in lymphocytes of MS patients in correlation with disease severity

Armon-Omar, A., Neuman, H., Sharabi-Nov, A. and Shahien, R. Multiple Sclerosis and Related Disorders, 41, 102025 (2020)   Background Multiple sclerosis (MS) is a multifactorial disease of the central nervous system in young adults. Mitochondrial respiration provides fuel necessary for cellular function and is especially important in cells with large energy demand including neurons. Various studies suggest that the pathogenesis of MS may be associated with mitochondrial dysfunction. Methods We examined 145 volunteers including 62 MS patients and healthy controls. MS patients were divided into two groups according to their disease severity: those with mild disability (EDSS=0–3.0) and those with moderate-severe MS (EDSS=3.5–8). After signing an informed consent, blood was taken and was separated to platelets and lymphocytes. Mitochondria activity was monitored as mitochondrial transmembrane potential following staining with JC1 dye in platelets and lymphocytes utilizing flow cytometry. Results We examined mitochondria activity as JC1 values from all separated lymphocyte samples and found significantly higher levels of mitochondrial activity in lymphocytes separated from healthy controls vs. MS patients (mean of 87.9% vs. 75.6%, p = 0.001). Significant differences in mitochondrial activity were also found when comparing means of groups divided according to MS disease severity. Interestingly, there were no significant differences in mitochondrial activity between patients treated with diverse medications or untreated patients. Mitochondrial activity was also examined in platelets, but no significant differences were found between groups. Conclusions Results obtained here show that mitochondrial activity was significantly lower in MS patients in comparison to healthy controls. In addition, there was a significant difference in mitochondrial activity depending on MS degree of disability. These initial findings in a peripheral examination hold potential for new diagnostic biomarkers to be considered in the future.  

4.2152           Neurokinin-1 Receptor Antagonism Ameliorates Dry Eye Disease by Inhibiting Antigen-Presenting Cell Maturation and T Helper 17 Cell Activation

Yu, M., Lee, S-M., Lee, H., Amouzegar, A., Nakao, T., Chen, Y. and Dana, R. Am. J. Pathol., 190(1), 125-133 (2020)   Neuroinflammation plays an important role in the pathogenesis of ocular surface disease, including dry eye disease (DED), but little is known about the contribution of substance P (SP) to DED. In this study, we investigated the expression of SP at the ocular surface and evaluated its effect on maturation of antigen-presenting cells (APCs), the key cell component involved in the induction of type 17 helper T-cell (Th17) response in DED. The effect of topical blockade of SP signaling was further investigated using neurokinin-1 receptor (NK1R) inhibitors on APC maturation, Th17 cell activation, and disease severity in a mouse model of DED. The results demonstrate that SP is constitutively expressed at the ocular surface, and trigeminal ganglion neurons are the major source of SP in DED. SP derived from trigeminal ganglion enhanced the expression of major histocompatibility complex class II maturation marker by bone marrow–derived dendritic cells, an effect that is abrogated by blockade of SP signaling using NK1R antagonist spantide. Finally, using a well-established murine model of DED, topical treatment of DED mice with NK1R antagonists CP-99,994 and L-733,060 suppressed APC acquisition of major histocompatibility complex class II, reduced Th17 cell activity, and ameliorated DED severity. These findings are of translational value, as they suggest that antagonizing NK1R-mediated SP signaling may be an effective strategy in suppressing Th17-mediated ocular surface disease.  

4.2153           miR-22 inhibition reduces hepatic steatosis via FGF21 and FGFR1 induction

Hu, Y., Liu, H-X., Jena, P.K., Sheng, L., Ali, M.R. and Wan, Y-J.Y. JHEP Reports, 2(2), 100093 (2020)   Background & Aims Metabolism supports cell proliferation and growth. Surprisingly, the tumor suppressor miR-22 is induced by metabolic stimulators like bile acids. Thus, this study examines whether miR-22 could be a metabolic silencer. Methods The relationship between miR-22 and the expression of fibroblast growth factor 21 (FGF21) and its receptor FGFR1 was studied in cells and fatty livers obtained from patients and mouse models. We evaluated the effect of an miR-22 inhibitor alone and in combination with obeticholic acid (OCA) for the treatment of steatosis. Results The levels of miR-22 were inversely correlated with those of FGF21, FGFR1, and PGC1α in human and mouse fatty livers, suggesting that hepatic miR-22 acts as a metabolic silencer. Indeed, miR-22 reduced FGFR1 by direct targeting and decreased FGF21 by reducing the recruitment of PPARα and PGC1α to their binding motifs. In contrast, an miR-22 inhibitor increases hepatic FGF21 and FGFR1, leading to AMPK and ERK1/2 activation, which was effective in treating alcoholic steatosis in mouse models. The farnesoid x receptor-agonist OCA induced FGF21 and FGFR1, as well as their inhibitor miR-22. An miR-22 inhibitor and OCA were effective in treating diet-induced steatosis, both alone and in combination. The combined treatment was the most effective at improving insulin sensitivity, releasing glucagon-like peptide 1, and reducing hepatic triglyceride in obese mice. Conclusion The simultaneous induction of miR-22, FGF21 and FGFR1 by metabolic stimulators may maintain FGF21 homeostasis and restrict ERK1/2 activation. Reducing miR-22 enhances hepatic FGF21 and activates AMPK, which could be a novel approach to treat steatosis and insulin resistance.  

4.2154           Sirtuin 1 activation alleviates primary biliary cholangitis via the blocking of the NF-κB signaling pathway

Li, Y., Xi, Y., Tao, G., Xu, G., Yang, Z., Fu, X., Liang, Y., Qian, J., Cui, Y. and Jiang, T. Int. Immunopharmacol., 83, 106386 (2020)   This report sought to establish the mechanistic role of sirtuin-1 (Sirt1), a NAD⁺-dependent deacetylase in the modulation of primary biliary cholangitis (PBC) pathogenesis. 64 PBC patients (diagnosed based on practice guidelines for American Association for the Study of Liver Diseases) and 60 healthy controls were included in this study. Clinically, the mRNA expression level of Sirt1 in macrophages differentiated from peripheral blood mononuclear cells (PBMCs) of PBC subjects substantially decreased when compared with the healthy controls but not in other Sirt family genes (Sirt2-7). Consistent with clinical results, a PBC murine model showed that levels of Sirt1 significantly decreased in the liver and Kupffer cells of mice treated with polyinosinic/polycytidylic acid (poly I:C) for 16 weeks. A TAK1 inhibitor (NG25) prevented the poly I:C-induced Sirt1 protein level decreasing in Kupffer cells but not MAPK inhibitor. Sirt1 activators resveratrol (RSV) and SRT1720 (SRT) ameliorated poly I:C-induced hepatic injury observed via histopathologic analysis and decreased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the PBC murine model. Furthermore, Sirt1 activators significantly reduced pro-inflammatory cytokines levels such as interleukin-1 beta (IL-1β), IL-6, interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in serum in poly I:C-induced mice. In addition, Sirt1 activators significantly inhibited the phosphorylated and acetylated levels of the RelA/p65 subunit of the nuclear transcription factor (NF-κB) but not the interferon regulatory factor (IRF) 3 in poly I:C-injured mice livers. Significantly, RSV improved the interaction between Sirt1 and p65, which may contribute to the decreased activity of NF-κB. In summary, the Sirt1 signaling pathway plays an essential role in the development of PBC and this may represent a novel approach and target for the treatment of PBC.  

4.2155           Ectopic expression of the Stabilin2 gene triggered by an intracisternal A particle (IAP) element in DBA/2J strain of mice

Maeda-Smithies, N., Hiller, S., Dong, S., Kim, H-S., Bennett, B.J. and Kayashima, Y. Mammalian Genome, 31, 2-16 (2020)   Stabilin2 (Stab2) encodes a large transmembrane protein which is predominantly expressed in the liver sinusoidal endothelial cells (LSECs) and functions as a scavenger receptor for various macromolecules including hyaluronans (HA). In DBA/2J mice, plasma HA concentration is ten times higher than in 129S6 or C57BL/6J mice, and this phenotype is genetically linked to the Stab2 locus. Stab2 mRNA in the LSECs was significantly lower in DBA/2J than in 129S6, leading to reduced STAB2 proteins in the DBA/2J LSECs. We found a retrovirus-derived transposable element, intracisternal A particle (IAP), in the promoter region of Stab2^DBA which likely interferes with normal expression in the LSECs. In contrast, in other tissues of DBA/2J mice, the IAP drives high ectopic Stab2^DBA transcription starting within the 5′ long terminal repeat of IAP in a reverse orientation and continuing through the downstream Stab2^DBA. Ectopic transcription requires the Stab2-IAP element but is dominantly suppressed by the presence of loci on 59.7–73.0 Mb of chromosome (Chr) 13 from C57BL/6J, while the same region in 129S6 requires additional loci for complete suppression. Chr13:59.9–73 Mb contains a large number of genes encoding Krüppel-associated box-domain zinc-finger proteins that target transposable elements-derived sequences and repress their expression. Despite the high amount of ectopic Stab2^DBA transcript in tissues other than liver, STAB2 protein was undetectable and unlikely to contribute to the plasma HA levels of DBA/2J mice. Nevertheless, the IAP insertion and its effects on the transcription of the downstream Stab2^DBA exemplify that stochastic evolutional events could significantly influence susceptibility to complex but common diseases.  

4.2156           Extended pharmacodynamic responses observed upon PROTAC-mediated degradation of RIPK2

Mares, A., Miah, A.H., Smith, I.E.D., Rackham, M., Thawani, A.R., Cryan, J. et al Communications Biology, 3:140 (2020)   Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound. This disconnect between PK and PD, when coupled with low nanomolar potency, offers the potential for low human doses and infrequent dosing regimens with PROTAC medicines.  

4.2157           The Transcription Factor Promyelocytic Leukemia Zinc Finger Protein Is Associated with Expression of Liver‐Homing Receptors on Human Blood CD56bright Natural Killer Cells

Hess, L.U., Matrus, G., Ziegler, A.F., Langeneckert, A.E., Salzberger, W., Goebels, H. et al Hepatol. Comm., 4(3), 409-424 (2020)   The transcription factor promyelocytic leukemia zinc finger protein (PLZF) is involved in the development of natural killer (NK) cells and innate lymphoid cells, including liver‐resident NK cells in mice. In human NK cells, the role of PLZF in liver residency is still unknown. Expression of PLZF in matched human peripheral blood‐ and liver‐derived NK cells and the association of PLZF expression with surface molecules and transcription factors relevant for tissue residency were investigated using multiparameter flow cytometry and assessing single‐cell messenger RNA (mRNA) levels. Intrahepatic cluster of differentiation (CD)56^bright NK cells expressed significantly higher levels of PLZF than peripheral blood CD56^bright NK cells, which were predominantly PLZF^lo. Expression of PLZF was highest within C‐X‐C motif chemokine receptor 6 (CXCR6)⁺CD69⁺ liver‐resident NK cells among intrahepatic CD56^bright NK cell populations. Association of PLZF with liver‐residency markers was also reflected at mRNA levels. A small PLZF^hiCD56^bright NK cell population was identified in peripheral blood that also expressed the liver‐residency markers CXCR6 and CD69 and shared functional characteristics with liver‐resident NK cells. Conclusion: PLZF is implicated as part of a transcriptional network that promotes liver residency of human NK cells. Expression of liver‐homing markers on peripheral blood PLZF^hiCD56^bright NK cells identifies an intermediate population potentially contributing to the maintenance of liver‐resident NK cells.  

4.2158           Dual TBK1/IKKɛ inhibitor amlexanox attenuates the severity of hepatotoxin‐induced liver fibrosis and biliary fibrosis in mice

Zhou, Z., Qi, J., Zhao, J., Lim, C.W., Kim, J-W. and Kim, B.

1. Cell Med. Med., 24, 1383-1398 (2020)

  Although numerous studies have suggested that canonical IκB kinases (IKK) play a key role in the progression of liver fibrosis, the role of non‐canonical IKKε and TANK‐binding kinase 1 (TBK1) on the development and progression of liver fibrosis remains unclear. To demonstrate such issue, repeated injection of CCl₄ was used to induce hepatotoxin‐mediated chronic liver injury and biliary fibrosis was induced by 0.1% diethoxycarbonyl‐1, 4‐dihydrocollidine diet feeding for 4 weeks. Mice were orally administered with amlexanox (25, 50, and 100 mg/kg) during experimental period. Significantly increased levels of TBK1 and IKKε were observed in fibrotic livers or hepatic stellate cells (HSCs) isolated from fibrotic livers. Interestingly, amlexanox treatment significantly inhibited the phosphorylation of TBK1 and IKKε accompanied by reduced liver injury as confirmed by histopathologic analysis, decreased serum biochemical levels and fibro‐inflammatory responses. Additionally, treatment of amlexanox promoted the fibrosis resolution. In accordance with these findings, amlexanox treatment suppressed HSC activation and its related fibrogenic responses by partially inhibiting signal transducer and activator of transcription 3. Furthermore, amlexanox decreased the activation and inflammatory responses in Kupffer cells. Collectively, we found that inhibition of the TBK1 and IKKε by amlexanox is a promising therapeutic strategy to cure liver fibrosis.  

4.2159           Phase II study of α-galactosylceramide-pulsed antigen-presenting cells in patients with advanced or recurrent non-small cell lung cancer

Toyoda, T., Karmata, T., Tanaka, K., Ihara, F., Takami, M., Suzuki, H., Nakajima, T., Ikeuchi, T., Kawasaki, Y., Hanaoka, H., Nakayama, T., Yoshino, I. and Motohashi, S.

1. Immunother. Cancer, 8, e000316 (2020)

  Background Invariant natural killer T (iNKT) cells produce copious amounts of cytokines in response to specific glycolipid antigens such as α-galactosylceramide (αGalCer) presented by CD1d-expressing antigen-presenting cells (APCs), thus orchestrating other immune cells to fight tumors. Because of their ability to induce strong antitumor responses activated by αGalCer, iNKT cells have been studied for their application in cancer immunotherapy. In our previous phase I/II trial in non-small cell lung cancer (NSCLC) patients who had completed the standard treatment, we showed a relatively long median survival time without severe treatment-related adverse events. Based on these results, we performed a phase II trial to evaluate clinical responses, safety profiles and immune responses as a second-line treatment for advanced NSCLC. Methods Patients with advanced or recurrent NSCLC refractory to first-line chemotherapy were eligible. αGalCer-pulsed APCs were intravenously administered four times. Overall survival time was evaluated as the primary endpoint. The safety profile and immune responses after APC injection were also monitored. This study was an open label, single-arm, phase II clinical trial performed at Chiba University Hospital, Japan. Results Thirty-five patients were enrolled in this study, of which 32 (91.4%) completed the trial. No severe adverse events related to the treatment were observed. The estimated median survival time of the 35 cases was 21.9 months (95% CI, 14.8 to 26.0). One case (2.9%) showed a partial response, 14 cases (40.0%) remained as stable disease, and 19 cases (54.3%) were evaluated as progressive disease. The geometric mean number of iNKT cells in all cases was significantly decreased and the mean numbers of natural killer (NK) cells, interferon-γ-producing cells in response to αGalCer, and effector CD8+ T cells were significantly increased after the administration of αGalCer-pulsed APCs. Conclusions The intravenous administration of αGalCer-pulsed APCs was well-tolerated and was accompanied by prolonged overall survival. These results are encouraging and warrant further evaluation in a randomized phase III trial to demonstrate the survival benefit of this immunotherapy.  

4.2160           Deep learning guided image-based droplet sorting for on-demand selection and analysis of single cells and 3D cell cultures

Anagnostidis, V., Sherlock, B., Metz, J., Mair, P., Hollfelder, F. and Gielen, F. Lab Chip, 20, 889-900 (2020)   Uncovering the heterogeneity of cellular populations and multicellular constructs is a long-standing goal in fields ranging from antimicrobial resistance to cancer research. Emerging technology platforms such as droplet microfluidics hold the promise to decipher such heterogeneities at ultra-high-throughput. However, there is a lack of methods able to rapidly identify and isolate single cells or 3D cell cultures. Here we demonstrate that deep neural networks can accurately classify single droplet images in real-time based on the presence and number of micro-objects including single mammalian cells and multicellular spheroids. This approach also enables the identification of specific objects within mixtures of objects of different types and sizes. The training sets for the neural networks consisted of a few hundred images manually picked and augmented to up to thousands of images per training class. Training required less than 10 minutes using a single GPU, and yielded accuracies of over 90% for single mammalian cell identification. Crucially, the same model could be used to classify different types of objects such as polystyrene spheres, polyacrylamide beads and MCF-7 cells. We applied the developed method for the selection of 3D cell cultures generated with Hek293FT cells encapsulated in agarose gel beads, highlighting the potential of the technology for the selection of objects with a high diversity of visual appearances. The real-time sorting of single droplets was in-line with droplet generation and occurred at rates up to 40 per second independently of image size up to 480 × 480 pixels. The presented microfluidic device also enabled storage of sorted droplets to allow for downstream analyses.  

4.2161           Improving Single-Cell Encapsulation Efficiency and Reliability through Neutral Buoyancy of Suspension

Liu, H., Li, M., Wang, Y., Piper, J. and Jiang, L. Micromachines, 11(1), 94 (2020)   Single-cell analysis is of critical importance in revealing cell-to-cell heterogeneity by characterizing individual cells and identifying minority sub-populations of interest. Droplet-based microfluidics has been widely used in the past decade to achieve high-throughput single-cell analysis. However, to maximize the proportion of single-cell emulsification is challenging due to cell sedimentation and aggregation. The purpose of this study was to investigate the influence of single-cell encapsulation and incubation through the use of neutral buoyancy. As a proof of concept, OptiPrep™ was used to create neutrally buoyant cell suspensions of THP-1, a human monocytic leukemia cell line, for single-cell encapsulation and incubation. We found that using a neutrally buoyant suspension greatly increased the efficiency of single-cell encapsulation in microdroplets and eliminated unnecessary cell loss. Moreover, the presence of OptiPrep™ was shown to not affect cellular viability. This method significantly improved the effectiveness of single-cell study in a non-toxic environment and is expected to broadly facilitate single-cell analysis.  

4.2162           Proteolytic Processing of Neuregulin 2

Czarnek, M. and Bereta, J. Mol. Neurobiol., 57, 1799-1813 (2020)   Neuregulin 2 (NRG2) belongs to the EGF family of growth factors. Most of this family members require proteolytic cleavage to liberate their ectodomains capable of binding and activating their cognate ErbB receptors. To date, most of the studies investigating proteolytic processing of neuregulins focused on NRG1, which was shown to undergo ectodomain shedding by several ADAM proteases and BACE1 and the remaining fragment was further cleaved by γ-secretase. Recently, NRG2 attracted more attention due to its role in the neurogenesis and modulation of behaviors associated with psychiatric disorders. In this study, we used genetic engineering methods to identify proteases involved in proteolytic processing of murine NRG2. Using non-neuronal cell lines as well as cultures of primary hippocampal neurons, we demonstrated that the major proteases responsible for releasing NRG2 ectodomain are ADAM10 and BACE2. Co-expression of NRG2 and BACE2 in neurons of certain brain structures including medulla oblongata and cerebellar deep nuclei was confirmed via immunohistochemical staining. The cleavage of NRG2 by ADAM10 or BACE2 generates a C-terminal fragment that serves as a substrate for γ-secretase. We also showed that murine NRG2 is subject to post-translational modifications, substantial glycosylation of its extracellular part, and phosphorylation of the cytoplasmic tail.  

4.2163           Expression of ALS-linked SOD1 Mutation in Motoneurons or Myotubes Induces Differential Effects on Neuromuscular Function In vitro

Benlefki, S., Sanchez-Vicente, A., Milla, V., Lucas, O., Soulard, C., Younes, R., Gergely, C., Bowerman, M., Raoul, C., Scamps, F. and Hilaire, C. Neuroscience, 435, 33-43 (2020)   Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that selectively affects upper and lower motoneurons. Dismantlement of the neuromuscular junction (NMJ) is an early pathological hallmark of the disease whose cellular origin remains still debated. We developed an in vitro NMJ model to investigate the differential contribution of motoneurons and muscle cells expressing ALS-causing mutation in the superoxide dismutase 1 (SOD1) to neuromuscular dysfunction. The primary co-culture system allows the formation of functional NMJs and fosters the expression of the ALS-sensitive fast fatigable type II-b myosin heavy chain (MHC) isoform. Expression of SOD1^G93A in myotubes does not prevent the formation of a functional NMJ but leads to decreased contraction frequency and lowers the slow type I MHC isoform transcript levels. Expression of SOD1^G93A in both motoneurons and myotubes or in motoneurons alone however alters the formation of a functional NMJ. Our results strongly suggest that motoneurons are a major factor involved in the process of NMJ dismantlement in an experimental model of ALS.  

4.2164           Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells

Belabed, M., Mauvais, F-X., Maschalidi, S., Kurowaka, M., Goudin, N., Huang, J-D., Fischer, A., de Saint Basile, G., van Endert, P., Sepulveda, F.E. and Menasche, G. Nature Communicataions, 11:1817 (2020)   Dendritic cells (DCs) constitute a specialized population of immune cells that present exogenous antigen (Ag) on major histocompatibility complex (MHC) class I molecules to initiate CD8 + T cell responses against pathogens and tumours. Although cross-presentation depends critically on the trafficking of Ag-containing intracellular vesicular compartments, the molecular machinery that regulates vesicular transport is incompletely understood. Here, we demonstrate that mice lacking Kif5b (the heavy chain of kinesin-1) in their DCs exhibit a major impairment in cross-presentation and thus a poor in vivo anti-tumour response. We find that kinesin-1 critically regulates antigen cross-presentation in DCs, by controlling Ag degradation, the endosomal pH, and MHC-I recycling. Mechanistically, kinesin-1 appears to regulate early endosome maturation by allowing the scission of endosomal tubulations. Our results highlight kinesin-1’s role as a molecular checkpoint that modulates the balance between antigen degradation and cross-presentation.  

4.2165           The platelet receptor CLEC-2 blocks neutrophil mediated hepatic recovery in acetaminophen induced acute liver failure

Chauhan, A., Sheriff, L., Hussain, M.T., Webb, G.J., Patten, A., Shephard, E.L., Shaw, R., Weston, C.J., Haldar, D., Bourke, S., Bhandari, R., Watson, S., Adams, D.H., Watson, S.P. and Lalor, P.F. Nature Communications, 11:1939 (2020)   Acetaminophen (APAP) is the main cause of acute liver failure in the West. Specific efficacious therapies for acute liver failure (ALF) are limited and time-dependent. The mechanisms that drive irreversible acute liver failure remain poorly characterized. Here we report that the recently discovered platelet receptor CLEC-2 (C-type lectin-like receptor) perpetuates and worsens liver damage after toxic liver injury. Our data demonstrate that blocking platelet CLEC-2 signalling enhances liver recovery from acute toxic liver injuries (APAP and carbon tetrachloride) by increasing tumour necrosis factor-α (TNF-α) production which then enhances reparative hepatic neutrophil recruitment. We provide data from humans and mice demonstrating that platelet CLEC-2 influences the hepatic sterile inflammatory response and that this can be manipulated for therapeutic benefit in acute liver injury. Since CLEC-2 mediated platelet activation is independent of major haemostatic pathways, blocking this pathway represents a coagulopathy-sparing, specific and novel therapy in acute liver failure.  

4.2166           Rab27a plays a dual role in metastatic propensity of pancreatic cancer

Kren, N., Michaud, D., Bagchi, S., Greene, K. and Pylayeva-Gupta, Y. Scientific Reports, 10:7390 (2020)   Pancreatic cancer is an aggressive malignancy, often diagnosed at metastatic stages. Several studies have implicated systemic factors, such as extracellular vesicle release and myeloid cell expansion, in the establishment of pre-metastatic niches in cancer. The Rab27a GTPase is overexpressed in advanced cancers, can regulate vesicle trafficking, and has been previously linked to non-cell autonomous control of tumor growth and metastasis, however, the role of Rab27a itself in the metastatic propensity of pancreatic cancer is not well understood. Here, we have established a model to study how Rab27a directs formation of the pre-metastatic niche. Loss of Rab27a in pancreatic cancer cells did not decrease tumor growth in vivo, but resulted in altered systemic myeloid cell expansion, both in the primary tumors and at the distant organ sites. In metastasis assays, loss of Rab27a expression in tumor cells injected into circulation compromised efficient outgrowth of metastatic lesions. However, Rab27a knockdown cells had an unexpected advantage at initial steps of metastatic seeding, suggesting that Rab27a may alter cell-autonomous invasive properties of the tumor cells. Gene expression analysis of gene expression revealed that downregulation of Rab27a increased expression of genes involved in epithelial-to-mesenchymal transition pathways, consistent with our findings that primary tumors arising from Rab27a knockdown cells were more invasive. Overall, these data reveal that Rab27a can play divergent roles in regulating pro-metastatic propensity of pancreatic cancer cells: by generating pro-metastatic environment at the distant organ sites, and by suppressing invasive properties of the cancer cells.  

4.2167           Targeting DUSP7 signaling alleviates hepatic steatosis, inflammation and oxidative stress in high fat diet (HFD)-fed mice via suppression of TAK1

Wu, L., Liu, Y., Zhao, Y., Li, M. and Guo, L. Free Rad. Biol. Med., 153, 140-158 (2020)   The non-alcoholic fatty liver disease (NAFLD), as a critical liver disease, is still lack of effective treatments because the molecular mechanism revealing the NAFLD pathogenesis remains unclear. Dual specific phosphatase 6 (DUSP7) shows effects on inflammatory response and is a negative feedback mechanism of the mitogen-activated protein kinase (MAPK) superfamily, which are critical factors in regulating NAFLD progression. However, the effects of DUSP7 on hepatic steatosis are still not fully understood. Here, we found that DUSP7 functioned as a negative regulator of NAFLD and in various metabolic disorders. DUSP7 expression was markedly reduced in liver samples from patients with simple hepatic steatosis or non-alcoholic steatohepatitis (NASH), as well as in liver tissues from high fat diet (HFD)-challenged mice or genetically obese (ob/ob) mice. DUSP7 knockout markedly accelerated insulin resistance, glucose intolerance, liver dysfunction, fibrosis and hepatic steatosis in HFD-fed mice. In addition, inflammatory response was significantly exacerbated in HFD-challenged mice with DUSP7 deletion, which was associated with the elevated activation of nuclear factor-κB (NF-κB) and MAPKs signaling pathways. Moreover, oxidative stress was detected in liver of HFD-induced mice, and this phenomenon was aggravated in mice with DUSP7 knockout. Importantly, we demonstrated that DUSP7 physically interacted with transforming growth factor β (TGF-β)-activated kinase (TAK1). DUSP7 deletion considerably promoted the activation of TAK1 in mice after HFD feeding, contributing to the lipid deposition, inflammatory response and reactive oxygen species (ROS) production. Taken together, DUSP7 might function as a protective factor against NAFLD development and metabolic disorder through alleviating dyslipidemia, inflammation and oxidative stress by directly interacting with TAK1 in hepatocytes, which was involved in the suppression of fibrosis. Thus, we may provide an effective strategy for the treatment of hepatic steatosis via targeting DUSP7.    

4.2168           Lysosomal acid lipase is the major acid retinyl ester hydrolase in cultured human hepatic stellate cells but not essential for retinyl ester degradation

Wagner, C., Hois, V., Pajed, L., Pusch, L-M., Wolinski, H., Trauner, m., Zimmermann, R., Taschler, U. and Lass, A. BBA-Mol. Cell Biol. Lipids, 1865, 158730 (2020)   Vitamin A is stored as retinyl esters (REs) in lipid droplets of hepatic stellate cells (HSCs). To date, two different pathways are known to facilitate the breakdown of REs: (i) Hydrolysis of REs by neutral lipases, and (ii) whole lipid droplet degradation in autolysosomes by acid hydrolysis. In this study, we evaluated the contribution of neutral and acid RE hydrolases to the breakdown of REs in human HSCs. (R)-Bromoenol lactone (R-BEL), inhibitor of adipose triglyceride lipase (ATGL) and patatin-like phospholipase domain-containing 3 (PNPLA3), the hormone-sensitive lipase (HSL) inhibitor 76-0079, as well as the serine-hydrolase inhibitor Orlistat reduced neutral RE hydrolase activity of LX-2 cell-lysates between 20 and 50%. Interestingly, in pulse-chase experiments, R-BEL, 76-0079, as well as Orlistat exerted little to no effect on cellular RE breakdown of LX-2 cells as well as primary human HSCs. In contrast, Lalistat2, a specific lysosomal acid lipase (LAL) inhibitor, virtually blunted acid in vitro RE hydrolase activity of LX-2 cells. Accordingly, HSCs isolated from LAL-deficient mice showed RE accumulation and were virtually devoid of acidic RE hydrolase activity. In pulse-chase experiments however, LAL-deficient HSCs, similar to LX-2 cells and primary human HSCs, were not defective in degrading REs. In summary, results demonstrate that ATGL, PNPLA3, and HSL contribute to neutral RE hydrolysis of human HSCs. LAL is the major acid RE hydrolase in HSCs. Yet, LAL is not limiting for RE degradation under serum-starvation. Together, results suggest that RE breakdown of HSCs is facilitated by (a) so far unknown, non-Orlistat inhibitable RE-hydrolase(s).  

4.2169           N-AS-triggered SPMs are direct regulators of microglia in a model of Alzheimer’s disease

Lee, J.Y., Han, S.H., Park, M.H., Song, I-S., Choi, M-K., Yu, E., Park, C-M., Kim, H-J., Kim, S.H., Schuchman, E.H., Jin, H.K. and Bae, J-s. Nature Communications, 11:2358 (2020)   Sphingosine kinase1 (SphK1) is an acetyl-CoA dependent acetyltransferase which acts on cyclooxygenase2 (COX2) in neurons in a model of Alzheimer’s disease (AD). However, the mechanism underlying this activity was unexplored. Here we show that N-acetyl sphingosine (N-AS) is first generated by acetyl-CoA and sphingosine through SphK1. N-AS then acetylates serine 565 (S565) of COX2, and the N-AS-acetylated COX2 induces the production of specialized pro-resolving mediators (SPMs). In a mouse model of AD, microglia show a reduction in N-AS generation, leading to decreased acetyl-S565 COX2 and SPM production. Treatment with N-AS increases acetylated COX2 and N-AS-triggered SPMs in microglia of AD mice, leading to resolution of neuroinflammation, an increase in microglial phagocytosis, and improved memory. Taken together, these results identify a role of N-AS in the dysfunction of microglia in AD.  

4.2170           A phase I study of loco-regional immunotherapy by transbronchial injection of α-galactosylceramide-pulsed antigen presenting cells in patients with lung cancer

Ishibashi, F., Sakairi, Y., Iwata, T., Moriya, Y., Mizobauchi, T., Hoshino, H., Yoshida, S., Hanaoka, H., Yoshino, I. and Motohashi, S. Clin. Immunol., 215, 108457 (2020)   We conducted a phase I study of the trans-bronchial injection of α-galactosylceramide (αGalCer)-pulsed antigen presenting cells (APCs) to evaluate their safety, immune responses, and anti-tumor activities. Patients with advanced or recurrent non-small cell lung cancer (NSCLC) refractory to standard treatments were eligible. αGalCer-pulsed APCs were administered intratumorally or intranodally by bronchoscopy. Twenty-one patients were enrolled in this study. No severe adverse events related to the cell therapy were observed during this study in any patient. After αGalCer-pulsed APCs were administrated, increased iNKT cell numbers were observed in PBMCs from eight cases, and IFN-γ producing cells were increased in the peripheral blood of 10 cases. Regarding clinical responses, one case exhibited a partial response and eight were classified as stable disease. In the tumor microenvironment, IFN-γ expression was upregulated after treatment in partial response or stable disease cases and TGF-β was upregulated in progressive disease cases.  

4.2171           B cell–Derived IL35 Drives STAT3-Dependent CD8+ T-cell Exclusion in Pancreatic Cancer

Mirlekar, B., Michaud, D., Lee, S.L., Kren, N.P., Harris, C., Greene, K., Goldman, E.C. et al Cancer Immunol. Res., 8(3), 292-308 (2020)   Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy characterized by a paucity of tumor-proximal CD8⁺ T cells and resistance to immunotherapeutic interventions. Cancer-associated mechanisms that elicit CD8⁺ T-cell exclusion and resistance to immunotherapy are not well-known. Here, using a Kras- and p53-driven model of PDA, we describe a mechanism of action for the protumorigenic cytokine IL35 through STAT3 activation in CD8⁺ T cells. Distinct from its action on CD4⁺ T cells, IL35 signaling in gp130⁺CD8⁺ T cells activated the transcription factor STAT3, which antagonized intratumoral infiltration and effector function of CD8⁺ T cells via suppression of CXCR3, CCR5, and IFNγ expression. Inhibition of STAT3 signaling in tumor-educated CD8⁺ T cells improved PDA growth control upon adoptive transfer to tumor-bearing mice. We showed that activation of STAT3 in CD8⁺ T cells was driven by B cell– but not regulatory T cell–specific production of IL35. We also demonstrated that B cell–specific deletion of IL35 facilitated CD8⁺ T-cell activation independently of effector or regulatory CD4⁺ T cells and was sufficient to phenocopy therapeutic anti-IL35 blockade in overcoming resistance to anti–PD-1 immunotherapy. Finally, we identified a circulating IL35⁺ B-cell subset in patients with PDA and demonstrated that the presence of IL35⁺ cells predicted increased occurrence of phosphorylated (p)Stat3⁺CXCR3⁻CD8⁺ T cells in tumors and inversely correlated with a cytotoxic T-cell signature in patients. Together, these data identified B cell–mediated IL35/gp130/STAT3 signaling as an important direct link to CD8⁺ T-cell exclusion and immunotherapy resistance in PDA.  

4.2172           Single-cell RNA-seq analysis of the brainstem of mutant SOD1 mice reveals perturbed cell types and pathways of amyotrophic lateral sclerosis

Liu, W., Venugopal, S., Majid, S., Ahn, I.S., Diamante, G., Hong, J., Yang, X. and Chandler, S.H. Neurobiol. of Disease, 141, 104877 (2020)   Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons throughout the brain and spinal cord progressively degenerate resulting in muscle atrophy, paralysis and death. Recent studies using animal models of ALS implicate multiple cell-types (e.g., astrocytes and microglia) in ALS pathogenesis in the spinal motor systems. To ascertain cellular vulnerability and cell-type specific mechanisms of ALS in the brainstem that orchestrates oral-motor functions, we conducted parallel single cell RNA sequencing (scRNA-seq) analysis using the high-throughput Drop-seq method. We isolated 1894 and 3199 cells from the brainstem of wildtype and mutant SOD1 symptomatic mice respectively, at postnatal day 100. We recovered major known cell types and neuronal subpopulations, such as interneurons and motor neurons, and trigeminal ganglion (TG) peripheral sensory neurons, as well as, previously uncharacterized interneuron subtypes. We found that the majority of the cell types displayed transcriptomic alterations in ALS mice. Differentially expressed genes (DEGs) of individual cell populations revealed cell-type specific alterations in numerous pathways, including previously known ALS pathways such as inflammation (in microglia), stress response (ependymal and an uncharacterized cell population), neurogenesis (astrocytes, oligodendrocytes, neurons), synapse organization and transmission (microglia, oligodendrocyte precursor cells, and neuronal subtypes), and mitochondrial function (uncharacterized cell populations). Other cell-type specific processes altered in SOD1 mutant brainstem include those from motor neurons (axon regeneration, voltage-gated sodium and potassium channels underlying excitability, potassium ion transport), trigeminal sensory neurons (detection of temperature stimulus involved in sensory perception), and cellular response to toxic substances (uncharacterized cell populations). DEGs consistently altered across cell types (e.g., Malat1), as well as cell-type specific DEGs, were identified. Importantly, DEGs from various cell types overlapped with known ALS genes from the literature and with top hits from an existing human ALS genome-wide association study (GWAS), implicating the potential cell types in which the ALS genes function with ALS pathogenesis. Our molecular investigation at single cell resolution provides comprehensive insights into the cell types, genes and pathways altered in the brainstem in a widely used ALS mouse model.  

4.2173           Amyloid-β and p-Tau Anti-Threat Response to Herpes Simplex Virus 1 Infection in Primary Adult Murine Hippocampal Neurons

Powell-Doherty, R., Abbott, A.R.N., Nelson, L.A. and Bertke, A.S.

1. Virol., 94(9), e01874-19 (2020)

  Alzheimer’s Disease (AD) is the sixth leading cause of death in the United States. Recent studies have established a potential link between herpes simplex virus 1 (HSV-1) infection and the development of AD. HSV-1 DNA has been detected in AD amyloid plaques in human brains, and treatment with the antiviral acyclovir (ACV) was reported to block the accumulation of the AD-associated proteins beta-amyloid (Aβ) and hyper-phosphorylated tau (p-tau) in Vero and glioblastoma cells. Our goal was to determine whether the accumulation of AD-related proteins is attributable to acute and/or latent HSV-1 infection in mature hippocampal neurons, a region of the brain severely impacted by AD. Primary adult murine hippocampal neuronal cultures infected with HSV-1, with or without antivirals, were assessed for Aβ and p-tau expression over 7 days postinfection. P-tau expression was transiently elevated in HSV-1-infected neurons, as well as in the presence of antivirals alone. Infected neurons, as well as uninfected neurons treated with antivirals, had a greater accumulation of Aβ₄₂ than uninfected untreated neurons. Furthermore, Aβ₄₂ colocalized with HSV-1 latency-associated transcript (LAT) expression. These studies suggest that p-tau potentially acts as an acute response to any perceived danger-associated molecular pattern (DAMP) in primary adult hippocampal neurons, while Aβ aggregation is a long-term response to persistent threats, including HSV-1 infection.  

4.2174           Image-Based Single Cell Sorting Automation in Droplet Microfluidics

Sesen, M. and Whyte, G. Scientific Reports, 10:8736 (2020)   The recent boom in single-cell omics has brought researchers one step closer to understanding the biological mechanisms associated with cell heterogeneity. Rare cells that have historically been obscured by bulk measurement techniques are being studied by single cell analysis and providing valuable insight into cell function. To support this progress, novel upstream capabilities are required for single cell preparation for analysis. Presented here is a droplet microfluidic, image-based single-cell sorting technique that is flexible and programmable. The automated system performs real-time dual-camera imaging (brightfield & fluorescent), processing, decision making and sorting verification. To demonstrate capabilities, the system was used to overcome the Poisson loading problem by sorting for droplets containing a single red blood cell with 85% purity. Furthermore, fluorescent imaging and machine learning was used to load single K562 cells amongst clusters based on their instantaneous size and circularity. The presented system aspires to replace manual cell handling techniques by translating expert knowledge into cell sorting automation via machine learning algorithms. This powerful technique finds application in the enrichment of single cells based on their micrographs for further downstream processing and analysis.  

4.2175           Bacillus subtilis spores as adjuvants against avian influenza H9N2 induce antigen-specific antibody and T cell responses in White Leghorn chickens

Lee, J.E., Kye, Y-C., Park, S-M., Shim, B-S., Yoo, S., Hwang, E., Kim, H., Kim, S-J., Han, S.H., Park, T.S., Oark, B-C. and Yun, C-H. Vet. Res., 51:68 (2020)   Low-pathogenicity avian influenza H9N2 remains an endemic disease worldwide despite continuous vaccination, indicating the need for an improved vaccine strategy. Bacillus subtilis (B. subtilis), a gram-positive and endospore-forming bacterium, is a non-pathogenic species that has been used in probiotic formulations for both animals and humans. The objective of the present study was to elucidate the effect of B. subtilis spores as adjuvants in chickens administered inactivated avian influenza virus H9N2. Herein, the adjuvanticity of B. subtilis spores in chickens was demonstrated by enhancement of H9N2 virus-specific IgG responses. B. subtilis spores enhanced the proportion of B cells and the innate cell population in splenocytes from chickens administered both inactivated H9N2 and B. subtilis spores (Spore + H9N2). Furthermore, the H9N2 and spore administration induced significantly increased expression of the pro-inflammatory cytokines IL-1β and IL-6 compared to that in the H9N2 only group. Additionally, total splenocytes from chickens immunized with inactivated H9N2 in the presence or absence of B. subtilis spores were re-stimulated with inactivated H9N2. The subsequent results showed that the extent of antigen-specific CD4⁺ and CD8⁺ T cell proliferation was higher in the Spore + H9N2 group than in the group administered only H9N2. Taken together, these data demonstrate that B. subtilis spores, as adjuvants, enhance not only H9N2 virus-specific IgG but also CD4⁺ and CD8⁺ T cell responses, with an increase in pro-inflammatory cytokine production. This approach to vaccination with inactivated H9N2 together with a B. subtilis spore adjuvant in chickens produces a significant effect on antigen-specific antibody and T cell responses against avian influenza virus.  

4.2176           Microbiota-Induced Type I Interferons Instruct a Poised Basal State of Dendritic Cells

Schaupp, L., Muth, S., Rogell, L., Schild, H., Diefenbach, A. and Probst, H.C. Cell, 181, 1080-1096 (2020)   Environmental signals shape host physiology and fitness. Microbiota-derived cues are required to program conventional dendritic cells (cDCs) during the steady state so that they can promptly respond and initiate adaptive immune responses when encountering pathogens. However, the molecular underpinnings of microbiota-guided instructive programs are not well understood. Here, we report that the indigenous microbiota controls constitutive production of type I interferons (IFN-I) by plasmacytoid DCs. Using genome-wide analysis of transcriptional and epigenetic regulomes of cDCs from germ-free and IFN-I receptor (IFNAR)-deficient mice, we found that tonic IFNAR signaling instructs a specific epigenomic and metabolic basal state that poises cDCs for future pathogen combat. However, such beneficial biological function comes with a trade-off. Instructed cDCs can prime T cell responses against harmless peripheral antigens when removing roadblocks of peripheral tolerance. Our data provide fresh insights into the evolutionary trade-offs that come with successful adaptation of vertebrates to their microbial environment.  

4.2177           Downregulation of the Arg/N-degron Pathway Sensitizes Cancer Cells to Chemotherapy In Vivo

Leboeuf, D., Abakumova, T., Prikazchikova, T., Rhym, L., Anderson, D.G., Zatsepin, T. and Piatkov, K.I. Molecular Therapy, 28(4), 1092-1104 (2020)   The N-degron pathway is an emerging target for anti-tumor therapies, because of its capacity to positively regulate many hallmarks of cancer, including angiogenesis, cell proliferation, motility, and survival. Thus, inhibition of the N-degron pathway offers the potential to be a highly effective anti-cancer treatment. With the use of a small interfering RNA (siRNA)-mediated approach for selective downregulation of the four Arg/N-degron-dependent ubiquitin ligases, UBR1, UBR2, UBR4, and UBR5, we demonstrated decreased cell migration and proliferation and increased spontaneous apoptosis in cancer cells. Chronic treatment with lipid nanoparticles (LNPs) loaded with siRNA in mice efficiently downregulates the expression of UBR-ubiquitin ligases in the liver without any significant toxic effects but engages the immune system and causes inflammation. However, when used in a lower dose, in combination with a chemotherapeutic drug, downregulation of the Arg/N-degron pathway E3 ligases successfully reduced tumor load by decreasing proliferation and increasing apoptosis in a mouse model of hepatocellular carcinoma, while avoiding the inflammatory response. Our study demonstrates that UBR-ubiquitin ligases of the Arg/N-degron pathway are promising targets for the development of improved therapies for many cancer types.  

4.2178           Millimeter-thick xenoislet-laden fibers as retrievable transplants mitigate foreign body reactions for long-term glycemic control in diabetic mice

Watanabe, T., Okitsu, T., Ozawa, F., Nagata, S., Matsunari, H., Nagashima, H., Nagaya, M., Teramae, H. and Takeuchi, S. Biomaterials, 255, 120162 (2020)   Transplantation technologies of pancreatic islets as well as stem cell-derived pancreatic beta cells encapsulated in hydrogel for the induction of immunoprotection could advance to treat type 1 diabetes mellitus, if the hydrogel transplants acquire retrievability through mitigating foreign body reactions after transplantation. Here, we demonstrate that the diameter of the fiber-shaped hydrogel transplants determines both in vivo cellular deposition onto themselves and their retrievability. Specifically, we found that the in vivo cellular deposition is significantly mitigated when the diameter is 1.0 mm and larger, and that 1.0 mm-thick xenoislet-laden fiber-shaped hydrogel transplants can be retrieved after being placed in the intraperitoneal cavities of immunocompetent diabetic mice for more than 100 days, during which period the hydrogel transplants can normalize the blood glucose concentrations of the mice. These findings Vagal sensory neurons relay viscero- and somatosensory information from within the body and play a key role in maintaining physiological homeostasis. We recently characterized the diversity of vagal sensory neurons in the mouse using a single-cell transcriptomics approach. Here, we provide an in-depth protocol for the extraction of mouse vagal ganglia and the production of high-quality single-cell suspensions from this tissue. This effective protocol can also be applied for use with other peripheral and central neuron populations with few modifications.cell-derived functional cells through improving their in vivo efficacy and safety.  

4.2179           Protocol to Prepare Single-Cell Suspensions from Mouse Vagal Sensory Ganglia for Transcriptomic Studies

Häring, M., Fatt, M. and Kupari, J. STAR Protocols, 1, 100030 (2020)   Vagal sensory neurons relay viscero- and somatosensory information from within the body and play a key role in maintaining physiological homeostasis. We recently characterized the diversity of vagal sensory neurons in the mouse using a single-cell transcriptomics approach. Here, we provide an in-depth protocol for the extraction of mouse vagal ganglia and the production of high-quality single-cell suspensions from this tissue. This effective protocol can also be applied for use with other peripheral and central neuron populations with few modifications.  

4.2180           A novel enrichment approach for anaerobic digestion of lignocellulosic biomass: Process performance enhancement through an inoculum habitat selection

Ferraro, A., Massini, G., Miritana, V.M., Rosa, S., Signorini, A., Fabbricino, M. Bioresource Technol., 313, 123703 (2020)   Inocula enrichment was performed using an innovative habitat-based selection approach to improve wheat straw (WS) anaerobic digestion (AD) efficiency. The procedure was carried out by sequentially re-inoculating the primary microbial community seven times in subsequent anaerobic reactors containing untreated WS. Re-inocula were performed at different re-inoculum times (24, 48, and 96 h) by moving a porous support mimicking a rumen structure from one batch to the next (S-tests) or re-inoculating only the culture medium (C-tests). Highest H₂ production yields were observed after four and five re-inocula (0.08 ± 0.02 NmL h⁻¹ gVS⁻¹ and 0.09 ± 0.02 NmL h⁻¹ gVS⁻¹) for S-24 and S-48, respectively. For S-96, higher CH₄ yields were observed after the start-up test and sixth re-inoculum (0.05 ± 0.003 NmL h⁻¹ gVS⁻¹ and 0.04 ± 0.005 NmL h⁻¹ gVS⁻¹, respectively). Accordingly, S-96 showed the highest active Archaea component (7%). C-test microbial communities were dominated by fermenting, hydrogen-producing bacteria and showed lower microbial community diversity than S-tests.  

4.2181           Pre-innervated tissue-engineered muscle promotes a pro-regenerative microenvironment following volumetric muscle loss

Das, S., Browne, K.D., Laimo, F.A., Maggiore, J.C., Hilman, M.C., Kaisaier, H., Aguilar, C.A., Ali, Z.S., Mourkioti, F. and Cullen, D.K. Communications Biol., 3:330 (2020)   Volumetric muscle loss (VML) is the traumatic or surgical loss of skeletal muscle beyond the inherent regenerative capacity of the body, generally leading to severe functional deficit. Formation of appropriate somato-motor innervations remains one of the biggest challenges for both autologous grafts as well as tissue-engineered muscle constructs. We aim to address this challenge by developing pre-innervated tissue-engineered muscle comprised of long aligned networks of spinal motor neurons and skeletal myocytes on aligned nanofibrous scaffolds. Motor neurons led to enhanced differentiation and maturation of skeletal myocytes in vitro. These pre-innervated tissue-engineered muscle constructs when implanted in a rat VML model significantly increased satellite cell density, neuromuscular junction maintenance, graft revascularization, and muscle volume over three weeks as compared to myocyte-only constructs and nanofiber scaffolds alone. These pro-regenerative effects may enhance functional neuromuscular regeneration following VML, thereby improving the levels of functional recovery following these devastating injuries.  

4.2182           Cushioned centrifugation during sperm selection increases the fertilization and cleavage rates of cattle embryos produced in vitro

Pavin, C.U.M., Leivas, F.G., Santos, F.W., Missio, D., Mesquita, F.S., dos Santos Brum, D. Animal Reprod. Sci., 219, 106508 (2020)   This study was conducted to evaluate the effect of utilization of an iodixanol-based solution as a cushioning method during the sperm selection utilizing discontinuous Percoll gradient centrifugation in in vitro production (IVP) of cattle embryos. In Experiment I, all aliquots of thawed semen were subjected to sperm selection using the same discontinuous Percoll® gradients, except for the following four conditions: presence of cushioning solution (Cushion Fluid, Minitube) during the first centrifugation process (C1), presence of cushioning solution during the second centrifugation process (C2), inclusion of cushioning solution in both centrifugation steps (C1-2), and no addiction of cushioning solution (C; control group). Recovery rates, sperm kinetics, and reactive oxygen species (ROS) production were evaluated. In Experiment II, sperm cells were processed using sperm selection conditions C and C1, and fertilization rates and embryonic development kinetics were compared between experimental groups. With use of condition C1, there was improvement in fertilization and cleavage rates when compared to use of condition C (56.4% compared with 45.5% and 80.0% compared 64.7%, respectively). In conclusion, results indicate the use of a cushioning solution during sperm selection positively affects the developmental potential of embryos.  

4.2183           LncRNA FTX represses the progression of non-alcoholic fatty liver disease to hepatocellular carcinoma via regulating the M1/M2 polarization of Kupffer cells

Wu, H., Zhong, Z., Wang, A., Yuan, C., Ning, K., Hu, H., Wang, C. and Yin, X. Cancer Cell Int., 20:266 (2020)   Background The effect of lncRNA FTX on non-alcoholic fatty liver disease (NAFLD) conversion to hepatocellular carcinoma (HCC) is unclear. Methods In our study, C57BL/6 mice was fed with high fat diet for obtaining NAFLD mouse model, and diethylnitrosamine induced the formation of HCC tumor. The expression of iNOS and CD206 in tissues were examined using immunohistochemistry. In addition, qRT-PCR was implemented to detect the expression of FTX and mRNAs. The percentage of M1 and M2 Kupffer cells (KCs) were determined using flow cytometry. The pathological change in liver tissues was displayed by H&E staining. Besides, immunofluorescence assay was performed to ensure the primary KCs through labeling F4/80. Results Here, we found that the expression of FTX and the ratio of M1/M2 KCs in liver tissues from NAFLD-transformed HCC (NAFLD-HCC) patients lower than in liver tissues from NAFLD patients. Subsequently, we revealed that the expression of FTX and M1/M2 KCs ratio were downregulated during NAFLD conversion to HCC. Importantly, increasing of FTX inhibited HCC tumor growth, improved liver damage and promoted M1 polarization of KCs during NAFLD conversion to HCC, while these effects of FTX were reversed by inactivating of KCs. Finally, in vitro experiments, our data indicated that FTX facilitated the M1 polarization of KCs. Conclusion In conclusion, our results demonstrated that upregulation of FTX suppressed NAFLD conversion to HCC though promoting M1 polarization of KCs. Our findings presented a new regulatory mechanism for NAFLD conversion to HCC and provided a new biomarker for inhibiting this conversion.  

4.2184           Microwell-based pancreas-on-chip model enhances genes expression and functionality of rat islets of Langerhans

Essaouiba, A., Okitsu, T., Jellali, R., Shinohara, M., Danoy, M., Tauran, Y., Legallais, C., Sakai, Y. and Leclerc, E. Mol. Cell. Endocrinol., 514, 110892 (2020)   Organ-on-chip technology is a promising tool for investigating physiological in vitro responses in drug screening development, and in advanced disease models. Within this framework, we investigated the behavior of rat islets of Langerhans in an organ-on-chip model. The islets were trapped by sedimentation in a biochip with a microstructure based on microwells, and perfused for 5 days of culture. The live/dead assay confirmed the high viability of the islets in the biochip cultures. The microfluidic culture leads to upregulation of mRNA levels of important pancreatic islet genes: Ins1, App, Insr, Gcgr, Reg3a and Neurod. Furthermore, insulin and glucagon secretion were higher in the biochips compared to the Petri conditions after 5 days of culture. We also confirmed glucose-induced insulin secretion in biochips via high and low glucose stimulations leading to high/low insulin secretion. The high responsiveness of the pancreatic islets to glucagon-like peptide 1 (GLP-1) stimulation in the biochips was reflected by the upregulation of mRNA levels of Gcgr, Reg3a, Neurog3, Ins1, Ins2, Stt and Glp-1r and by increased insulin secretion. The results obtained highlighted the functionality of the islets in the biochips and illustrated the potential of our pancreas-on-chip model for future pancreatic disease modeling and anti-diabetic drugs screening.  

4.2185           Lineage dynamics of the endosymbiotic cell type in the soft coral Xenia

Hu, M., Zheng, X., Fan, C-M. and Zheng, Y. Nature, 582, 534-538 (2020)   Many corals harbour symbiotic dinoflagellate algae. The algae live inside coral cells in a specialized membrane compartment known as the symbiosome, which shares the photosynthetically fixed carbon with coral host cells while host cells provide inorganic carbon to the algae for photosynthesis1. This endosymbiosis—which is critical for the maintenance of coral reef ecosystems—is increasingly threatened by environmental stressors that lead to coral bleaching (that is, the disruption of endosymbiosis), which in turn leads to coral death and the degradation of marine ecosystems2. The molecular pathways that orchestrate the recognition, uptake and maintenance of algae in coral cells remain poorly understood. Here we report the chromosome-level genome assembly of a Xenia species of fast-growing soft coral3, and use this species as a model to investigate coral–alga endosymbiosis. Single-cell RNA sequencing identified 16 cell clusters, including gastrodermal cells and cnidocytes, in Xenia sp. We identified the endosymbiotic cell type, which expresses a distinct set of genes that are implicated in the recognition, phagocytosis and/or endocytosis, and maintenance of algae, as well as in the immune modulation of host coral cells. By coupling Xenia sp. regeneration and single-cell RNA sequencing, we observed a dynamic lineage progression of the endosymbiotic cells. The conserved genes associated with endosymbiosis that are reported here may help to reveal common principles by which different corals take up or lose their endosymbionts.  

4.2186           Fetal Membrane Organ-On-Chip: An Innovative Approach to Study Cellular Interactions

Richardson, L., Gnecco, J., Ding, T., Osteen, K., Rogers, L.M., Aronoff, D.M. and Menon, R. Reproductive Sciences, 27, 1562-1569 (2020)   Objective: Fetal membranes, a vital component that helps maintain pregnancy and contribute to parturition signaling, are often studied in segments due to its structural complexity. Transwells are traditionally used to study cell interactions; however, their usefulness is limited. To overcome these difficulties, a fetal membrane-organ-on-chip (FM-OO-C) was created to study interactive properties of amnion epithelial cells (AECs) and decidual cells compared to transwell systems. Methods: Primary AECs and decidual cells from term, nonlaboring fetal membranes were cultured in a 2-chamber (AEC/decidual cell) FM-OO-C device and sandwiched between a semipermeable membrane. Cells were treated with cigarette smoke extract (CSE) or dioxin, and membrane permeability and cellular senescence were measured after 48 hours. The same experiments were conducted in transwells for comparisons. Results: Compared to transwell cultures, FM-OO-C model produced better membrane permeability readings regardless of the side of treatment or time point. Membrane permeabilization was higher in AECs directly treated with CSE (1.6 fold) compared to similar treatment on the decidual side (1.2 fold). In FM-OO-C, treatments forced changes between cellular layers. This was evident when CSE and dioxin-induced senescence on one side of the chamber produced similar changes on the opposite side. This effect was minimal in the transwell system. Conclusion: The controlled environment of an FM-OO-C allows for improved signal propagation between cells by minimizing noise and highlighting the small changes between treatments that cannot be seen in conventional transwell devices. Fetal membrane-organ-on-chip provides a better interaction between cell types that can be used to study fetal–maternal signaling during pregnancy in future studies.  

4.2187           Synaptic dysfunction induced by glycine‐alanine dipeptides in C9orf72‐ALS /FTD is rescued by SV 2 replenishment

Jensen, B.K., Schuldi, M.H., McAvoy, K., Russell, K.A., Boehringer, A., Curran, B.M., Krishnamurthy, K., Wen, X., Westergard, T., Ma, L., Haeusler, A.R., Edbauer, D., Pasinelli, P. and Trotti, D.

EMBO Mol. Med., 12:e10722 (2020)

 

The most common cause of amyotrophic lateral sclerosis (ALS ) and frontotemporal dementia (FTD ) is an intronic hexanucleotide repeat expansion in the C9orf72 gene. In disease, RNA transcripts containing this expanded region undergo repeat‐associated non‐AUG translation to produce dipeptide repeat proteins (DPR s), which are detected in brain and spinal cord of patients and are neurotoxic both in vitro and in vivo paradigms. We reveal here a novel pathogenic mechanism for the most abundantly detected DPR in ALS /FTD autopsy tissues, poly‐glycine‐alanine (GA ). Previously, we showed motor dysfunction in a GA mouse model without loss of motor neurons. Here, we demonstrate that mobile GA aggregates are present within neurites, evoke a reduction in synaptic vesicle‐associated protein 2 (SV 2), and alter Ca²⁺ influx and synaptic vesicle release. These phenotypes could be corrected by restoring SV 2 levels. In GA mice, loss of SV 2 was observed without reduction of motor neuron number. Notably, reduction in SV 2 was seen in cortical and motor neurons derived from patient induced pluripotent stem cell lines, suggesting synaptic alterations also occur in patients.

 

4.2188           Globally Abundant “Candidatus Udaeobacter” Benefits from Release of Antibiotics in Soil and Potentially Performs Trace Gas Scavenging

Willms, I.M., Rudolph, A.Y., Göschel, I., Bolz, S.H., Schneider, D., Penone, C., Poehlein, A., Schönong, I. and Nacke, H. mSphere, 5(4), e00186-20 (2020)   Verrucomicrobia affiliated with “Candidatus Udaeobacter” belong to the most abundant soil bacteria worldwide. Although the synthesis of antibiotics presumably evolved in soil, and environmental pollution with antimicrobials increases, the impact of these complex molecules on “Ca. Udaeobacter” remains to be elucidated. In this study, we demonstrate that “Ca. Udaeobacter” representatives residing in grassland as well as forest soil ecosystems show multidrug resistance and even take advantage of antibiotics release. Soils treated with up to six different antibiotics exhibited a higher “Ca. Udaeobacter” abundance than corresponding controls after 3, 8, and 20 days of incubation. In this context, we provide evidence that “Ca. Udaeobacter” representatives may utilize nutrients which are released due to antibiotic-driven lysis of other soil microbes and thereby reduce energetically expensive synthesis of required biomolecules. Moreover, genomic analysis revealed the presence of genes conferring resistance to multiple classes of antibiotics and indicated that “Ca. Udaeobacter” representatives most likely oxidize the trace gas H₂ to generate energy. This energy might be required for long-term persistence in terrestrial habitats, as already suggested for other dominant soil bacteria. Our study illustrates, for the first time, that globally abundant “Ca. Udaeobacter” benefits from release of antibiotics, which confers advantages over other soil bacteria and represents a so-far overlooked fundamental lifestyle feature of this poorly characterized verrucomicrobial genus. Furthermore, our study suggests that “Ca. Udaeobacter” representatives can utilize H₂ as an alternative electron donor.  

4.2189           PLK1 regulates hepatic stellate cell activation and liver fibrosis through Wnt/β‐catenin signalling pathway

Chen, Y., Chen, X., Ji, Y-R., Zhu, S., Bu, F-T., Du, X-S., Meng, X-M., Huang, C. and Li, J.

1. Cell Mol. Med., 24, 7405-7416 (2020)

  As an outcome of chronic liver disease, liver fibrosis involves the activation of hepatic stellate cells (HSCs) caused by a variety of chronic liver injuries. It is important to explore approaches to inhibit the activation and proliferation of HSCs for the treatment of liver fibrosis. PLK1 is overexpressed in many human tumour cells and has become a popular drug target in tumour therapy. Therefore, further study of the function of PLK1 in the cell cycle is valid. In the present study, we found that PLK1 expression was elevated in primary HSCs isolated from CCl₄‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. Knockdown of PLK1 inhibited α‐SMA and Col1α1 expression and reduced the activation of HSCs in CCl₄‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. We further showed that inhibiting the expression of PLK1 reduced the proliferation of HSCs and promoted HSCs apoptosis in vivo and in vitro. Furthermore, we found that the Wnt/β‐catenin signalling pathway may be essential for PLK1‐mediated HSCs activation. Together, blocking PLK1 effectively suppressed liver fibrosis by inhibiting HSC activation, which may provide a new treatment strategy for liver fibrosis.  

4.2190           Bone Scaffolds Based on Degradable Vaterite/PEG‐Composite Microgels

Stengelin, E., Kuzmina, A., Beltramo, G.L., Koziol, M.F., Besch, L., Schröder, R., Unger, R.E., Tremel, W. and Seiffert, S. Adv. Healthcare Mater., 9, 1901820 (2020)   Vaterite, a metastable modification of calcium carbonate, embedded in a flexible microgel packaging with adjustable mechanical properties, functionality, and biocompatibility, provides a powerful scaffolding for bone tissue regeneration, as it is easily convertible to bone‐like hydroxyapatite (HA). In this study, the synthesis and physical analysis of a packaging material to encapsulate vaterite particles and osteoblast cells into monodisperse, sub‐millimeter‐sized microgels, is described whereby a systematic approach is used to tailor the microgel properties. The size and shape of the microgels is controlled via droplet‐based microfluidics. Key requirements for the polymer system, such as absence of cytotoxicity as well as biocompatibility and biodegradability, are accomplished with functionalized poly(ethylene glycol) (PEG), which reacts in a cytocompatible thiol–ene Michael addition. On a mesoscopic level, the microgel stiffness and gelation times are adjusted to obtain high cellular viabilities. The co‐encapsulation of living cells provides i) an in vitro platform for the study of cellular metabolic processes which can be applied to bone formation and ii) an in vitro foundation for novel tissue‐regenerative therapies. Finally, the degradability of the microgels at physiological conditions caused by hydrolysis‐sensitive ester groups in the polymer network is examined.  

4.2191           Cell‐type‐resolved proteomic analysis of the human liver

Ölander, M., Wisniewski, J.R. and Artursson, P. Liver Int., 40, 1770-1780 (2020)   Background & Aims The human liver functions through a complex interplay between parenchymal and non‐parenchymal cells. Mass spectrometry‐based proteomic analysis of intact tissue has provided an in‐depth view of the human liver proteome. However, the predominance of parenchymal cells (hepatocytes) means that the total tissue proteome mainly reflects hepatocyte expression. Here we therefore set out to analyse the proteomes of the major parenchymal and non‐parenchymal cell types in the human liver. Methods We applied quantitative label‐free proteomic analysis on the major cell types of the human liver: hepatocytes, liver endothelial cells, Kupffer cells and hepatic stellate cells. Results We identified 9791 proteins, revealing distinct protein expression profiles across cell types, whose in vivo relevance was shown by the presence of cell‐type‐specific proteins. Analysis of proteins related to the immune system indicated that mechanisms of immune‐mediated liver injury include the involvement of several cell types. Furthermore, in‐depth investigation of proteins related to the absorption, distribution, metabolism, excretion and toxicity (ADMET) of xenobiotics showed that ADMET‐related tasks are not exclusively confined to hepatocytes, and that non‐parenchymal cells may contribute to drug transport and metabolism. Conclusions Overall, the data we provide constitute a unique resource for exploring the proteomes of the major types of human liver cells, which will facilitate an improved understanding of the human liver in health and disease.  

4.2192           Epigenetic homogeneity in histone methylation underlies sperm programming for embryonic transcription

Oikawa, M., Simeone, A., Hormanseder, E., Teperek, M., Gaggioli, V., O’Doherty, A. et al Nature Communications, 11:3491 (2020)   Sperm contributes genetic and epigenetic information to the embryo to efficiently support development. However, the mechanism underlying such developmental competence remains elusive. Here, we investigated whether all sperm cells have a common epigenetic configuration that primes transcriptional program for embryonic development. Using calibrated ChIP-seq, we show that remodelling of histones during spermiogenesis results in the retention of methylated histone H3 at the same genomic location in most sperm cell. This homogeneously methylated fraction of histone H3 in the sperm genome is maintained during early embryonic replication. Such methylated histone fraction resisting post-fertilisation reprogramming marks developmental genes whose expression is perturbed upon experimental reduction of histone methylation. A similar homogeneously methylated histone H3 fraction is detected in human sperm. Altogether, we uncover a conserved mechanism of paternal epigenetic information transmission to the embryo through the homogeneous retention of methylated histone in a sperm cells population.  

4.2193           Mitomycin C treatment improves pancreatic islet graft longevity in intraportal islet transplantation by suppressing proinflammatory response

Yamane, K., Anazawa, T., Tada, S., Fujimoto, N., Inoguchi, K., Emoto, N., Nagai, K., Masui, T., Okajima, H., Takaori, K., Sumi, S. and Uemoto, S. Scientific Reports, 10:12086 (2020)   The in vitro culture period prior to cell transplantation (i.e. pancreatic islet transplantation) enables cell modification and is thus advantageous. However, the islet preconditioning method has not been fully explored. Here we present a simple approach for islet preconditioning that uses the antibiotic mitomycin C (MMC), which has antitumor activity, to reduce islet immunogenicity and prevent proinflammatory events in an intraportal islet transplantation model. Freshly isolated mice islets were treated for 30 min with 10 μg/mL MMC or not, cultured for 20 h and transplanted into the livers of syngeneic or allogeneic diabetic mouse recipients. In the allogeneic model, MMC preconditioning significantly prolonged graft survival without requiring immunosuppressants. In vitro, MMC treatment suppressed the expression of proinflammatory cytokines in islet allografts, while immunohistochemical studies revealed the suppression of inflammatory cell infiltration into MMC-treated allografts relative to untreated allografts. Furthermore, MMC preconditioning significantly suppressed the mRNA expression of proinflammatory cytokines into the transplant site and induced the differentiation of regulatory T cells with the ability to suppress CD4⁺ T cell-mediated immune responses. In conclusion, islet preconditioning with MMC prolonged graft survival in an intraportal islet transplantation model by suppressing proinflammatory events and inducing potentially regulatory lymphocytes.  

4.2194           Tβ4 suppresses lincRNA-p21-mediated hepatic apoptosis and fibrosis by inhibiting PI3K-AKT-NF-κB pathway

Yang, L., Fu, W-l., Zhu, Y. and Wang, X-g. Gene, 758, 144946 (2020)   Hepatic injury is one of the most challenging diseases in clinical medicine. Hepatic injury is accompanied by hepatocyte apoptosis and leads to hepatic fibrosis and cirrhosis, which may cause liver cancer and increased mortality. Therefore, it is essential to investigate the regulation mechanism and therapeutic strategies for hepatic injury. In the study, the effects of Thymosin β4 (Tβ4) on Long intergenic noncoding RNA-p21 (lincRNA-p21)-mediated liver injury were investigated. Results showed that lincRNA-p21 overexpression promoted hepatocytes apoptosis, which was blocked by Tβ4. Besides, Tβ4 reversed the levels of cleaved caspase-3 and caspase-9 induced by lincRNA-p21. LincRNA-p21 overexpression also caused the pathological injury and fibrosis in hepatic tissues and increased the levels of fibrosis-related proteins (Collagen I, α-SMA and TIMP-1), and induced hydroxyproline and ALT production. However, Tβ4 reversed the effects of overexpression of lincRNA-p21 on hepatic injury and fibrosis. In vitro experiments, after lincRNA-p21 was overexpressed in hepatic stellate cells (HSCs), the proliferation ability and the levels of HSCs markers α-SMA and Desmin were increased. However, Tβ4 reversed the effects of lincRNA-p21 on HSCs. Furthermore, the PI3K-AKT-NF-κB pathway was activated by lincRNA-p21, which was then reversed by the Tβ4 administration. After the mice treated by insulin-like growth factor-1 (IGF-1) (the activator of PI3K-AKT), the inhibitory effect of Tβ4 on activated the PI3K-AKT-NF-κB pathway was abrogated. Besides, IGF-1 abolished the protective effects of Tβ4 on hepatic apoptosis and fibrosis induced by lincRNA-p21. Therefore, Tβ4 reversed. lincRNA-p21-mediated liver injury through inhibiting PI3K-AKT-NF-κB pathway. Tβ4 may be a promising drug for fibrosis therapy.  

4.2195           Molecular expression analysis and characterization of rockfish (Sebastes schlegelii) B cell activating factor

Madhuranga, W.S.P., Tharuka, M.D.N., yang, h., Lim, C., Wan, Q., Bathige, S.D.N.K. and Lee, J. Com. Biochem. Physiol., Part B, 250, 110480 (2020)   B cell activating factor (BAFF) is recognized as a member of the TNF superfamily proteins that mediate the immune responses. In this study, BAFF from rockfish (Sebastes schlegelii) (SsBAFF) was characterized based on its functional aspects. The open reading frame of SsBAFF is 804 bp in length and encodes a 267 long amino acid residue protein with predicted molecular weight of 29.48 kDa. The deduced protein sequence comprises with transmembrane domain, furin cleavage site and TNF domain carrying Flap binding site that unique to TNF family. Recombinant SsBAFF (rSsBAFF) significantly enhanced rockfish lymphocytes proliferation and viability in a concentration dependent-manner according to the results from water soluble tetrazolium salt (WST-1) assay and flow cytometric assay. rSsBAFF also modulated the expression of genes involved in anti-inflammatory (IL-10 and NFκB-2) and anti-apoptotic (Bcl-2 and Bax) signal pathways. SsBAFF mRNA expression was detected ubiquitously in all analyzed rockfish tissues, with the highest levels in the spleen and head kidney. Further, the expression of SsBAFF in spleen were significantly induced following LPS, poly (I:C) and Streptococcus iniae challenges. These findings strongly suggest that SsBAFF might play an important role in rockfish immune system through regulating the inflammatory response and proliferation of immune cells.  

4.2196           Engineering the cellular mechanical microenvironment to regulate stem cell chondrogenesis: Insights from a microgel model

Feng, Q., Gao, H., Wen, H., Huang, H., Li, Q., Liang, M., Liu, Y., Dong, H. and Cao, X. Acta Biomaterialia, 113, 393-406 (2020)   Biophysical cues (especially mechanical cues) embedded in cellular microenvironments show a critical impact on stem cell fate. Despite the capability of traditional hydrogels to mimic the feature of extracellular matrix (ECM) and tune their physicochemical properties via diverse approaches, their relatively large size not only induces biased results, but also hinders high-throughput screening and analysis. In this paper, a microgel model is proposed to recapitulate the role of 3D mechanical microenvironment on stem cell behaviors especially chondrogenesis in vitro. The small diameter of microgels brings the high surface area to volume ratio and then the enlarged diffusion area and shortened diffusion distance of soluble molecules, leading to uniform distribution of nutrients and negligible biochemical gradient inside microgels. To construct ECM-like microenvironment with tunable mechanical strength, three gelatin/hyaluronic acid hybrid microgels with low, medium and high crosslinking densities, i.e., Gel-HA(L), Gel-HA(M) and Gel-HA(H), are fabricated in microfluidic devices by Michael addition reaction between thiolated gelatin (Gel-SH) and ethylsulfated hyaluronic acid (HA-VS) with different substitution degrees of vinyl sulfone groups. Our results show that mouse bone marrow mesenchymal stem cell (BMSC) proliferation, distribution and chondrogenesis are all closely dependent on mechanical microenvironments in microgels. Noteworthily, BMSCs show a clear trend of differentiating into hyaline cartilage in Gel-HA(L) and fibrocartilage in Gel-HA(M) and Gel-HA(H). Whole transcriptome RNA sequencing reveals that mechanical microenvironment of microgels affects BMSC differentiation via TGF-β/Smad signaling pathway, Hippo signaling pathway and Integrin/YAP/TAZ signaling pathway. We believe this microgel model provides a new way to further explore the interaction between cells and 3D microenvironment.  

4.2197           Characterizing liver sinusoidal endothelial cell fenestrae on soft substrates upon AFM imaging and deep learning

Li, P., Zhou, J., Li, W., Wu, H., Hu, J., Ding, Q., Lü, S., Pan, J., Zhang, C., Li, N. and Long, M. BBA-General Subjects, 1864, 129702 (2020)   Background Liver sinusoidal endothelial cells (LSECs) display unique fenestrated morphology. Alterations in the size and number of fenestrae play a crucial role in the progression of various liver diseases. While their features have been visualized using atomic force microscopy (AFM), the in situ imaging methods and off-line analyses are further required for fenestra quantification. Methods Primary mouse LSECs were cultured on a collagen-I-coated culture dish, or a polydimethylsiloxane (PDMS) or polyacrylamide (PA) hydrogel substrate. An AFM contact mode was applied to visualize fenestrae on individual fixed LSECs. Collected images were analyzed using an in-house developed image recognition program based on fully convolutional networks (FCN). Results Key scanning parameters were first optimized for visualizing the fenestrae on LSECs on culture dish, which was also applicable for the LSECs cultured on various hydrogels. The intermediate-magnification morphology images of LSECs were used for developing the FCN-based, fenestra recognition program. This program enabled us to recognize the vast majority of fenestrae from AFM images after twice trainings at a typical accuracy of 81.6% on soft substrate and also quantify the statistics of porosity, number of fenestrae and distribution of fenestra diameter. Conclusions Combining AFM imaging with FCN training is able to quantify the morphological distributions of LSEC fenestrae on various substrates.  

4.2198           Self-Reporting Transposons Enable Simultaneous Readout of Gene Expression and Transcription Factor Binding in Single Cells

Moudgil, A., Wilkinson, M.N., Chen, X., Morris, S.A., Dougherty, J.D. and Mitra, R.D. Cell, 182, 992-1008 (2020)   Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells. The genomic locations of SRTs are recovered from mRNA, and SRTs deposited by exogenous, TF-transposase fusions can be used to map TFBS. We then present scCC, which map SRTs from scRNA-seq libraries, simultaneously identifying cell types and TFBS in those same cells. We benchmark multiple TFs with this technique. Next, we use scCC to discover BRD4-mediated cell-state transitions in K562 cells. Finally, we map BRD4 binding sites in the mouse cortex at single-cell resolution, establishing a new method for studying TF biology in situ.    

4.2199           Motoneuron expression profiling identifies an association between an axonal splice variant of HDGF-related protein 3 and peripheral myelination

Kerman, B.E., Genoud, S., Vatandaslar, B.K., Denli, A.M., Ghosh, S.G., Xu, X., Yeo, G.W., Aimone, J.B. and Gage, F.H.

1. Biol. Chem., 295(34), 12233-12246 (2020)

  Disorders that disrupt myelin formation during development or in adulthood, such as multiple sclerosis and peripheral neuropathies, lead to severe pathologies, illustrating myelin's crucial role in normal neural functioning. However, although our understanding of glial biology is increasing, the signals that emanate from axons and regulate myelination remain largely unknown. To identify the core components of the myelination process, here we adopted a microarray analysis approach combined with laser-capture microdissection of spinal motoneurons during the myelinogenic phase of development. We identified neuronal genes whose expression was enriched during myelination and further investigated hepatoma-derived growth factor-related protein 3 (HRP3 or HDGFRP3). HRP3 was strongly expressed in the white matter fiber tracts of the peripheral (PNS) and central (CNS) nervous systems during myelination and remyelination in a cuprizone-induced demyelination model. The dynamic localization of HPR3 between axons and nuclei during myelination was consistent with its axonal localization during neuritogenesis. To study this phenomenon, we identified two splice variants encoded by the HRP3 gene: the canonical isoform HRP3-I and a newly recognized isoform, HRP3-II. HRP3-I remained solely in the nucleus, whereas HRP3-II displayed distinct axonal localization both before and during myelination. Interestingly, HRP3-II remained in the nuclei of unmyelinated neurons and glial cells, suggesting the existence of a molecular machinery that transfers it to and retains it in the axons of neurons fated for myelination. Overexpression of HRP3-II, but not of HRP3-I, increased Schwann cell numbers and myelination in PNS neuron–glia co-cultures. However, HRP3-II overexpression in CNS co-cultures did not alter myelination.  

4.2200           The Monocyte-Derived Exosomal CLMAT3 Activates the CtBP2-p300-NF-κB Transcriptional Complex to Induce Proinflammatory Cytokines in ALI

Chen, Z., Dong, W-H., Qin, Z-M. and Li, Q-G. Molecular Therapy-Nucleic Acids, 21, 1100-1110 (2020)   Monocytes and macrophages are the two major cell types involved in innate immunity. Exosomes act as signaling molecules to regulate cell-to-cell communication by releasing proteins, mRNAs, microRNAs (miRNAs), and long noncoding RNAs (lncRNAs). However, it is still unclear whether monocyte-derived exosomes are involved in the communication between monocytes and macrophages. In this study, we analyzed the differentially expressed lncRNA profiles in monocytes isolated from blood samples of healthy controls and acute lung injury (ALI) patients. We focused our study on investigating the signaling downstream of CLMAT3 (colorectal liver metastasis-associated transcript 3), a lncRNA that regulated proinflammatory cytokine genes. We revealed that CLMAT3 specifically targeted CtBP2 (C-terminal binding protein 2) and repressed its expression. Elevated CtBP2 acted as a coactivator to assemble a transcriptional complex with histone acetyltransferase p300 and NF-κB (nuclear factor κB) subunits. In vitro coculture and in vivo injection of ALI monocyte-derived exosomes increased the production of proinflammatory cytokines. Importantly, the administration of two CtBP2 inhibitors, NSC95397 and MTOB, could significantly reverse CtBP2-mediated transactivation. Collectively, our results support a model in which monocyte-derived exosomal CLMAT3 activates the CtBP2-p300-NF-κB complex to induce proinflammatory cytokines, thus contributing to the pathogenesis of ALI.  

4.2201           Plasmacytoid dendritic cells regulate colitis-associated tumorigenesis by controlling myeloid-derived suppressor cell infiltration

Ko, H-J., Hong, E-H., Cho, J., Ahn, J-h., Kwon, B-E., Kweon, M-N., Seo, S-U., Yoon, B-l. and Chang, S-Y. Cancer Lett., 493, 102-112 (2020)   Toll-like receptor (TLR)3 and TLR7 are important for stimulating plasmacytoid dendritic cells (pDCs), which secrete type I interferon. Mice deficient for TLR3 and TLR7 (TLR3^−/−TLR7^−/−) reportedly exhibit deteriorated colitis because of impaired pDCs. However, the role of pDCs in tumorigenesis-associated inflammation progression has not been studied. We treated wild-type or TLR3^−/−TLR7^−/− mice with dextran sulfate sodium (DSS) and/or azoxymethane (AOM) and examined colon mucosa, measured body weight and colon length of mice, and examined pDC and myeloid-derived suppressor cell (MDSC) accumulation. Further, we depleted pDCs in AOM/DSS-treated wild-type mice by treating them with anti-PDCA-1 antibodies. We found that MDSCs significantly increased, while pDCs decreased in TLR3^−/−TLR7^−/− mice. Moreover, TLR3^−/−TLR7^−/− mice developed colitis-associated colon cancer following AOM/DSS treatment. Additionally, we showed that a defect in TLR7 of pDCs is responsible for the aggravation of colitis-associated colon cancer. Further, we showed that TLR7 ligand mitigates colitis-associated colon cancer. Collectively, our results demonstrate that gut pDCs play a crucial role in reducing colorectal cancer development via the regulation of infiltrating MDSCs.  

4.2202           The cellular and molecular landscape of hypothalamic patterning and differentiation from embryonic to late postnatal development

Kim, D.W., Washington, P.W., Wang, Z.Q., Lin, S.H., Sun, C., Ismaial, B.T., Wang, H., Jiang, L. and Blackshaw, S. Nature Communications, 11:4360 (2020)   The hypothalamus is a central regulator of many innate behaviors essential for survival, but the molecular mechanisms controlling hypothalamic patterning and cell fate specification are poorly understood. To identify genes that control hypothalamic development, we have used single-cell RNA sequencing (scRNA-Seq) to profile mouse hypothalamic gene expression across 12 developmental time points between embryonic day 10 and postnatal day 45. This identified genes that delineated clear developmental trajectories for all major hypothalamic cell types, and readily distinguished major regional subdivisions of the developing hypothalamus. By using our developmental dataset, we were able to rapidly annotate previously unidentified clusters from existing scRNA-Seq datasets collected during development and to identify the developmental origins of major neuronal populations of the ventromedial hypothalamus. We further show that our approach can rapidly and comprehensively characterize mutants that have altered hypothalamic patterning, identifying Nkx2.1 as a negative regulator of prethalamic identity. These data serve as a resource for further studies of hypothalamic development, physiology, and dysfunction.  

4.2203           BMP-9 Modulates the Hepatic Responses to LPS

Gaitantzi, H., Karch, J., Germann, L., Cai, C., Rausch, V., Birgin, E., Rahbari, N. et al Cells, 9, 617 (2020)   It was previously shown that Bone Morphogenetic Protein (BMP)-9 is constitutively produced and secreted by hepatic stellate cells (HSC). Upon acute liver damage, BMP-9 expression is transiently down-regulated and blocking BMP-9 under conditions of chronic damage ameliorated liver fibrogenesis in C57BL/6 mice. Thereby, BMP-9 acted as a pro-fibrogenic cytokine in the liver but without directly activating isolated HSC in vitro. Lipopolysaccharide (LPS), an endotoxin derived from the membrane of Gram-negative bacteria in the gut, is known to be essential in the pathogenesis of diverse kinds of liver diseases. The aim of the present project was therefore to investigate how high levels of BMP-9 in the context of LPS signalling might result in enhanced liver damage. For this purpose, we stimulated human liver sinusoidal endothelial cells (LSEC) with LPS and incubated primary human liver myofibroblasts (MF) with the conditioned medium of these cells. We found that LPS led to the secretion of factors from LSEC that upregulate BMP-9 expression in MF. At least one of these BMP-9 enhancing factors was defined to be IL-6. High BMP-9 in turn, especially in combination with LPS stimulation, induced the expression of certain capillarization markers in LSEC and enhanced the LPS-mediated induction of pro-inflammatory cytokines in primary human macrophages. In LSEC, pre-treatment with BMP-9 reduced the LPS-mediated activation of the NfkB pathway, whereas in macrophages, LPS partially inhibited the BMP-9/Smad-1 signaling cascade. In vivo, in mice, BMP-9 led to the enhanced presence of F4/80-positive cells in the liver and it modulated the LPS-mediated regulation of inflammatory mediators. In summary, our data point to BMP-9 being a complex and highly dynamic modulator of hepatic responses to LPS: Initial effects of LPS on LSEC led to the upregulation of BMP-9 in MF but sustained high levels of BMP-9 in turn promote pro-inflammatory reactions of macrophages. Thereby, the spatial and timely fine-tuned presence (or absence) of BMP-9 is needed for efficient wound-healing responses in the liver.  

4.2204           Exogenous Liposomal Ceramide-C6 Ameliorates Lipidomic Profile, Energy Homeostasis, and Anti-Oxidant Systems in NASH

Zanieri, F., Levi, A., Montefusco, D., Longato, L., De Chiara, F., Frenguelli, L., Omenetti, S. et al Cells, 9, 1237 (2020)   In non-alcoholic steatohepatitis (NASH), many lines of investigation have reported a dysregulation in lipid homeostasis, leading to intrahepatic lipid accumulation. Recently, the role of dysfunctional sphingolipid metabolism has also been proposed. Human and animal models of NASH have been associated with elevated levels of long chain ceramides and pro-apoptotic sphingolipid metabolites, implicated in regulating fatty acid oxidation and inflammation. Importantly, inhibition of de novo ceramide biosynthesis or knock-down of ceramide synthases reverse some of the pathology of NASH. In contrast, cell permeable, short chain ceramides have shown anti-inflammatory actions in multiple models of inflammatory disease. Here, we investigated non-apoptotic doses of a liposome containing short chain C6-Ceramide (Lip-C6) administered to human hepatic stellate cells (hHSC), a key effector of hepatic fibrogenesis, and an animal model characterized by inflammation and elevated liver fat content. On the basis of the results from unbiased liver transcriptomic studies from non-alcoholic fatty liver disease patients, we chose to focus on adenosine monophosphate activated kinase (AMPK) and nuclear factor-erythroid 2-related factor (Nrf2) signaling pathways, which showed an abnormal profile. Lip-C6 administration inhibited hHSC proliferation while improving anti-oxidant protection and energy homeostasis, as indicated by upregulation of Nrf2, activation of AMPK and an increase in ATP. To confirm these in vitro data, we investigated the effect of a single tail-vein injection of Lip-C6 in the methionine-choline deficient (MCD) diet mouse model. Lip-C6, but not control liposomes, upregulated phospho-AMPK, without inducing liver toxicity, apoptosis, or exacerbating inflammatory signaling pathways. Alluding to mechanism, mass spectrometry lipidomics showed that Lip-C6-treatment reversed the imbalance in hepatic phosphatidylcholines and diacylglycerides species induced by the MCD-fed diet. These results reveal that short-term Lip-C6 administration reverses energy/metabolic depletion and increases protective anti-oxidant signaling pathways, possibly by restoring homeostatic lipid function in a model of liver inflammation with fat accumulation.  

4.2205           Platelet Transforming Growth Factor-β1 Induces Liver Sinusoidal Endothelial Cells to Secrete Interleukin-6

Balaphas, A., Meyer, J., Perozzo, R., Zeisser-Labouebe, M., Berndt, S., Turzi, A., Fontana, P., Scapozza, l., Gonelle-Gispert, C. and Bühleer, L.H. Cells, 9, 1311 (82020)   The roles and interactions of platelets and liver sinusoidal endothelial cells in liver regeneration are unclear, and the trigger that initiates hepatocyte proliferation is unknown. We aimed to identify the key factors released by activated platelets that induce liver sinusoidal endothelial cells to produce interleukin-6 (IL-6), a cytokine implicated in the early phase of liver regeneration. We characterized the releasate of activated platelets inducing the in vitro production of IL-6 by mouse liver sinusoidal endothelial cells and observed that the stimulating factor was a thermolabile protein. Following gel filtration, a single fraction of activated platelet releasate induced a maximal IL-6 secretion by liver sinusoidal endothelial cells (90.2 ± 13.9 versus control with buffer, 9.0 ± 0.8 pg/mL, p < 0.05). Mass spectroscopy analysis of this fraction, followed by in silico processing, resulted in a reduced list of 18 candidates. Several proteins from the list were tested, and only recombinant transforming growth factor β1 (TGF-β1) resulted in an increased IL-6 production up to 242.7 ± 30.5 pg/mL, which was comparable to non-fractionated platelet releasate effect. Using neutralizing anti-TGF-β1 antibody or a TGF-β1 receptor inhibitor, IL-6 production by liver sinusoidal endothelial cells was dramatically reduced. These results support a role of platelet TGF-β1 β1 in the priming phase of liver regeneration.  

4.2206           Evaluation of Multi-Layered Pancreatic Islets and Adipose-Derived Stem Cell Sheets Transplanted on Various Sites for Diabetes Treatment

Lee, Y., Yi, H-J., Kim, Y.H., Lee, S., Oh, J., Okano, T., Shim, I.K. and Kim, S.C. Cells, 9, 1999 (2020)   Islet cell transplantation is considered an ideal treatment for insulin-deficient diabetes, but implantation sites are limited and show low graft survival. Cell sheet technology and adipose-derived stem cells (ADSCs) can be useful tools for improving islet cell transplantation outcomes since both can increase implantation efficacy and graft survival. Herein, the optimal transplantation site in diabetic mice was investigated using islets and stem cell sheets. We constructed multi-layered cell sheets using rat/human islets and human ADSCs. Cell sheets were fabricated using temperature-responsive culture dishes. Islet/ADSC sheet (AI sheet) group showed higher viability and glucose-stimulated insulin secretion than islet-only group. Compared to islet transplantation alone, subcutaneous AI sheet transplantation showed better blood glucose control and CD31+ vascular traits. Because of the adhesive properties of cell sheets, AI sheets were easily applied on liver and peritoneal surfaces. Liver or peritoneal surface grafts showed better glucose control, weight gain, and intraperitoneal glucose tolerance test (IPGTT) profiles than subcutaneous site grafts using both rat and human islets. Stem cell sheets increased the therapeutic efficacy of islets in vivo because mesenchymal stem cells enhance islet function and induce neovascularization around transplanted islets. The liver and peritoneal surface can be used more effectively than the subcutaneous site in future clinical applications.  

4.2207           Biomarkers of Browning in Cold Exposed Siberian Adults

Efremova, A., Colleluori, G., Thomsky, M., Perugini, J., Protasoni, M., Reguzzoni, M., Faragalli, A., Carle, F., Giiordano, A. and Cinti, S. Nutrients, 12, 2161 (2020)   Cold-exposure promotes energy expenditure by inducing brown adipose tissue (BAT) thermogenesis, which over time, is also sustained by browning, the appearance, or increase, of brown-like cells into white fat depots. Identification of circulating markers reflecting BAT activity and browning is crucial to study this phenomenon and its triggers, also holding possible implications for the therapy of obesity and metabolic diseases. Using RT-qPCR, we evaluated the peripheral blood mononuclear cells (PBMC) expression profile of regulators of BAT activity (CIDEA, PRDM16), white adipocytes browning (HOXC9 and SLC27A1), and fatty acid β-oxidation (CPT1A) in 150 Siberian healthy miners living at extremely cold temperatures compared to 29 healthy subjects living in thermoneutral conditions. Anthropometric parameters, glucose, and lipid profiles were also assessed. The cold-exposed group showed significantly lower weight, BMI, hip circumference, and PBMC expression of CIDEA, but higher expression of HOXC9 and higher circulating glucose compared to controls. Within the cold-exposed group, BMI, total cholesterol, and the atherogenic coefficient were lower in individuals exposed to low temperatures for a longer time. In conclusion, human PBMC expresses the brown adipocytes marker CIDEA and the browning marker HOXC9, which, varying according to cold-exposure, possibly reflect changes in BAT activation and white fat browning.  

4.2208           Dysmetabolic Circulating Tumor Cells Are Prognostic in Metastatic Breast Cancer

Brisotto, G., Biscontin, E., Rossi, E., Bulfoni, M., Piruska, A., Spazzpan, S., Poggiana, C., Vidotto, R., Steffan, A., Colombatti, A., Huck, W.T.S., Ceselli, D., Zamarchi, R., Turetta, M. and Del Ben, F. Cancers, 12, 1005 (2020)   Circulating tumor cells (CTCs) belong to a heterogeneous pool of rare cells, and a unequivocal phenotypic definition of CTC is lacking. Here, we present a definition of metabolically-altered CTC (MBA-CTCs) as CD45-negative cells with an increased extracellular acidification rate, detected with a single-cell droplet microfluidic technique. We tested the prognostic value of MBA-CTCs in 31 metastatic breast cancer patients before starting a new systemic therapy (T0) and 3–4 weeks after (T1), comparing results with a parallel FDA-approved CellSearch (CS) approach. An increased level of MBA-CTCs was associated with: i) a shorter median PFS pre-therapy (123 days vs. 306; p < 0.0001) and during therapy (139 vs. 266 days; p = 0.0009); ii) a worse OS pre-therapy (p = 0.0003, 82% survival vs. 20%) and during therapy (p = 0.0301, 67% survival vs. 38%); iii) good agreement with therapy response (kappa = 0.685). The trend of MBA-CTCs over time (combining data at T0 and T1) added information with respect to separate evaluation of T0 and T1. The combined results of the two assays (MBA and CS) increased stratification accuracy, while correlation between MBA and CS was not significant, suggesting that the two assays are detecting different CTC subsets. In conclusion, this study suggests that MBA allows detection of both EpCAM-negative and EpCAM-positive, viable and label-free CTCs, which provide clinical information apparently equivalent and complementary to CS. A further validation of proposed method and cut-offs is needed in a larger, separate study.  

4.2209           Control of Persistent Salmonella Infection Relies on Constant Thymic Output Despite Increased Peripheral Antigen-Specific T Cell Immunity

Goggins, J.A., Kurtz, J.R. and Mclachlan, J.B. Pathogens, 9, 605 (2020)   Recent thymic emigrants are the youngest subset of peripheral T cells and their involvement in combating persistent bacterial infections has not been explored. Here, we hypothesized that CD4+ recent thymic emigrants are essential immune mediators during persistent Salmonella infection. To test this, we thymectomized adult mice either prior to, or during, persistent Salmonella infection. We found that thymic output is crucial in the formation of protective immune responses during the early formation of a Salmonella infection but is dispensable once persistent Salmonella infection is established. Further, we show that thymectomized mice demonstrate increased infection-associated mortality and bacterial burdens. Unexpectedly, numbers of Salmonella-specific CD4+ T cells were significantly increased in thymectomized mice compared to sham control mice. Lastly, we found that T cells from thymectomized mice may be impaired in producing the effector cytokine IL-17 at early time points of infection, compared to thymically intact mice. Together, these results imply a unique role for thymic output in the formation of immune responses against a persistent, enteric pathogen.  

4.2210           Intraglomerular Monocyte/Macrophage Infiltration and Macrophage–Myofibroblast Transition during Diabetic Nephropathy Is Regulated by the A2B Adenosine Receptor

Torres, A., Munoz, K., Nahuelpan, Y., Saez, A-P.R., Mendoza, P., Jara, C., Capelli, C., Suarez, R., Oyarzun, C., Quezada, C. and Martin, R.S. Cells, 9, 1051 (2020)   Diabetic nephropathy (DN) is considered the main cause of kidney disease in which myofibroblasts lead to renal fibrosis. Macrophages were recently identified as the major source of myofibroblasts in a process known as macrophage–myofibroblast transition (MMT). Adenosine levels increase during DN and in vivo administration of MRS1754, an antagonist of the A_2B adenosine receptor (A_2BAR), attenuated glomerular fibrosis (glomerulosclerosis). We aimed to investigate the association between A_2BAR and MMT in glomerulosclerosis during DN. Kidneys/glomeruli of non-diabetic, diabetic, and MRS1754-treated diabetic (DM+MRS1754) rats were processed for histopathologic, transcriptomic, flow cytometry, and cellular in vitro analyses. Macrophages were used for in vitro cell migration/transmigration assays and MMT studies. In vivo MRS1754 treatment attenuated the clinical and histopathological signs of glomerulosclerosis in DN rats. Transcriptomic analysis demonstrated a decrease in chemokine-chemoattractants/cell-adhesion genes of monocytes/macrophages in DM+MRS1754 glomeruli. The number of intraglomerular infiltrated macrophages and MMT cells increased in diabetic rats. This was reverted by MRS1754 treatment. In vitro cell migration/transmigration decreased in macrophages treated with MRS1754. Human macrophages cultured with adenosine and/or TGF-β induced MMT, a process which was reduced by MRS1754. We concluded that pharmacologic blockade of A_2BAR attenuated some clinical signs of renal dysfunction and glomerulosclerosis, and decreased intraglomerular macrophage infiltration and MMT in DN rats.  

4.2211           Generation, localization and functions of macrophages during the development of testis

Lokka, E., Lintukorpi, L., Cisneros-Montalvo, S., Mäkelä, J-A., Tuustjärvi, S., Ojasalo, V., Gerke, H., Toppari, J., Rantakari, P., and Salmi, M. Nature Communications, 11:4375 (2020)   development, and later replaced by bone marrow-derived macrophages. By contrast, the peritubular macrophages have been reported to emerge first in the postnatal testis and solely represent descendants of bone marrow-derived monocytes. Here, we define new monocyte and macrophage types in the fetal and postnatal testis using high-dimensional single-cell analyses. Our results show that interstitial macrophages have a dominant contribution from fetal liver-derived precursors, while peritubular macrophages are generated already at birth from embryonic precursors. We find that bone marrow-derived monocytes do not substantially contribute to the replenishment of the testicular macrophage pool even after systemic macrophage depletion. The presence of macrophages prenatally, but not postnatally, is necessary for normal spermatogenesis. Our multifaceted data thus challenge the current paradigms in testicular macrophage biology by delineating their differentiation, homeostasis and functions.  

4.2212           CSS: cluster similarity spectrum integration of single-cell genomics data

He, Z., Brazovskaja, A., Ebert, S., Camp, J.G. and Treutlein, B. Genome Biology, 21:224 (2020)   It is a major challenge to integrate single-cell sequencing data across experiments, conditions, batches, time points, and other technical considerations. New computational methods are required that can integrate samples while simultaneously preserving biological information. Here, we propose an unsupervised reference-free data representation, cluster similarity spectrum (CSS), where each cell is represented by its similarities to clusters independently identified across samples. We show that CSS can be used to assess cellular heterogeneity and enable reconstruction of differentiation trajectories from cerebral organoid and other single-cell transcriptomic data, and to integrate data across experimental conditions and human individuals.  

4.2213           Tbet promotes CXCR6 expression in immature natural killer cells and natural killer cell egress from the bone marrow

Cuff, A.O., Perchet, T., Dertschnig, S., Golub, R. and Male, V. Immunology, 161, 28-38 (82020)   Tbet‐deficient mice have reduced natural killer (NK) cells in blood and spleen, but increased NK cells in bone marrow and lymph nodes, a phenotype that is thought to be the result of defective migration. Here, we revisit the role of Tbet in NK cell bone marrow egress. We definitively show that the accumulation of NK cells in the bone marrow of Tbet‐deficient Tbx21^−/− animals occurs because of a migration defect and identify a module of genes, co‐ordinated by Tbet, which affects the localization of NK cells in the bone marrow. Cxcr6 is approximately 125‐fold underexpressed in Tbx21^−/−, compared with wild‐type, immature NK cells. Immature NK cells accumulate in the bone marrow of CXCR6‐deficient mice, and CXCR6‐deficient progenitors are less able to reconstitute the peripheral NK cell compartment than their wild‐type counterparts. However, the CXCR6 phenotype is largely confined to immature NK cells, whereas the Tbet phenotype is present in both immature and mature NK cells, suggesting that genes identified as being more differentially expressed in mature NK cells, such as S1pr5, Cx3cr1, Sell and Cd69, may be the major drivers of the phenotype.  

4.2214           Rate zonal centrifugation can partially separate platelets from platelet‐derived vesicles

Rikkert, L., Engelaer, M., Hau, C.M., terstappen, L.W.M.M., Nieuwland, R. and Coumans, F.A.W. Res. Pract. Thromb. Haemost., 4, 1053-1059 (2020)   Background Centrifugation is commonly used as a first step to enrich biomarkers from blood. Biomarkers are separated on the basis of density and/or diameter. However, the centrifugation protocol affects the yield and purity of biomarkers, for example, isolation of platelets results in co‐isolation with extracellular vesicles (EVs). Objective To assess the ability of rate zonal centrifugation (RZC) to separate platelets from co‐isolated EVs. Methods Using a linear Optiprep gradient, RZC was able to separate a mixture of beads with different diameters but similar density. Next, RZC was applied to samples containing both platelets and platelet‐derived EVs (n = 3). After RZC, all fractions were collected and stained with anti‐CD61‐Alexa 488 to measure the concentrations of platelets and platelet‐derived EVs by flow cytometry. Results We confirm that RZC separates polystyrene beads with diameters of 140 nm, 380 nm and 1,000 nm. Next, we show that the majority of platelets occur in fractions 8‐19, whereas the majority of platelet‐derived EVs are detectable in fractions 1‐7. Furthermore, each fraction contains a different diameter range of platelets, which suggests that separation is indeed diameter based. Conclusion RZC can partially separate platelets from EVs.  

4.2215           Relationship between normal weight obesity and mild cognitive impairment is reflected in cognitive‐related genes in human peripheral blood mononuclear cells

Zhang, S., Zhao, M., Wang, F., Liu, J., Zhheng, H. and Lei, P. Psychogeriatrics, 20, 35-43 (2020)   Aim Obesity contributes to the development of mild cognitive impairment, but the potential role of normal weight obesity in this disease has not been explored in humans. The aim of the study was to reveal the relationship between normal weight obesity and mild cognitive impairment in elderly individuals. Methods This study consisted of 360 patients with amnestic mild cognitive impairment and 360 cognitively normal controls. Normal weight obesity was defined as having metabolic syndrome but a normal weight. Metabolic health meant having no metabolic syndrome. Reverse transcription quantitative real‐time polymerase chain reaction was adopted to measure the messenger RNA expression of four cognitive‐related genes (amyloid precursor protein, cyclic adenosine monophosphate‐responsive element‐binding protein 1, sortilin‐related receptor 1, and synapsin I) in peripheral blood mononuclear cells. Results Normal weight obesity was related to a higher risk of amnestic mild cognitive impairment (odds ratio = 3.14, 95% confidence interval: 2.13–4.60). In the patients, the expression of each gene in the peripheral blood mononuclear cells was linearly related to Mini‐Mental State Examination and Montreal Cognitive Assessment scores (P < 0.05). The expression of these genes in the patients with metabolic health deviated from the normal levels found in the controls (P < 0.05), and the deviations were more significant in the patients with normal weight obesity (P < 0.05). Conclusion Normal weight obesity may be a potential risk factor for amnestic mild cognitive impairment in elderly. This relationship was reflected in the abnormal expression of several cognitive‐related genes in peripheral blood mononuclear cells.  

4.2216           Prospecting potential links between PRRSV infection susceptibility of alveolar macrophages and other respiratory infectious agents present in conventionally reared pigs

Museau, L., Hervet, C., Saade, G., Menard, D., Belloc, C., Meurens, F. and Bertho, N. Vet. Immunol. Immunopathol., 229, 110114 (2020)   Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is one of the main component of the porcine respiratory disease complex (PRDC), which strongly impact the pig production. Although PRRSV is often considered as a primary infection that eases subsequent respiratory coinfections, the possibility that other PRDC components may facilitate PRRSV infection has been largely overlooked. The main cellular targets of PRRSV are respiratory macrophages among them alveolar macrophages (AM) and pulmonary intravascular macrophages (PIM). AM, contrarily to PIM, are directly exposed to the external respiratory environment, among them co-infectious agents. In order to explore the possibility of a co-infections impact on the capacity of respiratory macrophages to replicate PRRSV, we proceed to in vitro infection of AM and PIM sampled from animals presenting different sanitary status, and tested the presence in the respiratory tract of these animals of the most common porcine respiratory pathogens (PCV2, Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma floculare, Pasteurella multocida, Bordetella bronchiseptica, Streptoccocus suis). In this exploratory study with a limited number of animals, no statistic differences were observed between AM and PIM susceptibility to in vitro PRRSV infection, nor between AM coming from animals presenting very contrasting respiratory coinfection loads.  

4.2217           ABCA1 plays an anti-inflammatory role by affecting TLR4 at the feto–maternal interface

Ding, N., Liu, N., Yang, L., Han, X., Lin, L. and Long, Y. Life Sciences, 259, 118390 (2020)   Aims This study aimed to evaluate the function and pathway of ATP-binding cassette transporter member A1 (ABCA1)-induced anti-inflammatory response in cells at the feto–maternal interface. Main methods The primary amniotic mesenchymal cells (AMCs), chorion cells and decidual cells were isolated from placental membranes of women with uncomplicated pregnancies at full-term (not in labor) using enzymatic digestion. Flow cytometry was used to measure the purity of isolated cells. Immunofluorescence assay was performed to detect the location of ABCA1 and toll-like receptor 4 (TLR4). Reverse transcription PCR and western blotting analyses were used to examine ABCA1, TLR4 and inflammatory factor expression in primary cells. ELISA was used to detect cytokine secretions from the primary cells. Key findings ABCA1 and TLR4 were mainly located in the cell nucleus and cytoplasm of feto-maternal interface cells. ABCA1 expression remained the highest in chorion cells, medium in decidual cells, and weakest in AMCs. Upregulated expression of ABCA1 decreased expression of TLR4 and the levels of pro-inflammatory factors, but increased cytoprotective factors in all cell types. In contrast, downregulated expression of ABCA1 increased the expression of TLR4 and pro-inflammatory factors, but decreased the levels of cytoprotective factors. Downregulated ABCA1 expression followed by decreased TLR4 expression using a small interference RNA (siRNA) induced reduction of interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) in all cell types. Significance ABCA1 at feto-maternal interface acts as an anti-inflammatory role by reducing the expression of TLR4 in uncomplicated pregnancies. ABCA1 might be a potential therapeutic target for preventing gestational diseases.  

4.2218           Development of a pancreas-liver organ-on-chip coculture model for organ-to-organ interaction studies

Essaouiba, A., Okitsu, T., Kinoshita, R., Jellali, R., Shinohara, M., Danoy, M., Leggggallais, C., Sakai, Y. and Leclerc, E. Biochem. Eng. J., 164, 107783 (2020)   Type 2 diabetes mellitus (T2DM) is a widespread chronic disease with a high prevalence of comorbidity and mortality. The exponential increase of TD2M represents an important public health challenge and leads a strong demand for the development of relevant in vitro models to improve mechanistic understanding of diabetes and identify new anti-diabetic drugs and therapies. These models involve considering the multi-organ characteristic of T2DM. The organ-on-chip technology has made it possible to connect several organs thanks to dedicated microbioreactors interconnected by microfluidic network. Here, we developed pancreas-liver coculture model in a microfluidic biochip, using rat islets of Langerhans and hepatocytes. The behavior and functionality of the model were compared to islets and hepatocytes (with/without insulin) monocultures. Compared to monoculture, the islets coculture presented high C-peptide and insulin secretions, and downregulation of Pdx1, Glut2, App, Ins1, Neurod, Neurog3 and Gcgr genes. In the hepatic compartment, the monocultures without insulin were negative to CK18 staining and displayed a weaker albumin production, compared to monoculture with insulin. The hepatocytes cocultures were highly positive to INSR, GLUT2, CK18 and CYP3A2 immunostaining and allowed to recover mRNA levels similar to monocultures with insulin. The result showed that islets could produce insulin to supplement the culture medium and recover hepatic functionality. This model illustrated the potential of organ-on-chip technology for reproducing crosstalk between liver and pancreas.  

4.2219           Influence of the Core Formulation on Features and Drug Delivery Ability of Carbamate-Based Nanogels

Pinelli, F., Pizetti, F., Ortola, O.F., Marchetti, A., Rossetti, A., Sacchetti, A. and Rossi, F. Int. J. Mol. Sci., 21, 6621 (2020)   In the last years, nanogels have emerged as one of the most promising classes of novel drug delivery vehicles since they can be employed in multiple fields, such as various therapeutics or diagnostics, and with different classes of compounds and active molecules. Their features, such as a high volume to surface ratio, excellent drug loading and release ability, as well as biocompatibility and tunable behavior, are unique, and, nowadays, great efforts are made to develop new formulations that can be employed in a wider range of applications. Polyethylene glycol (PEG)-polyethylenimine (PEI) nanogels probably represent the baseline of this class of biomaterials and they are still largely employed and studied. In any way, the possibility to exploit new core formulations for nanogels is certainly very interesting in order to understand the influence of different polymer chains on the final properties of the system. In this research, we explore and make a comparison between PEG-PEI nanogels and two other different formulations: pluronic F127-PEI nanogels and PEG-Jeffamine nanogels. We propose nanogels synthesis methods, their chemical and physical characterization, as well as their stability analysis, and we focus on the different drug delivery ability that these structures exhibit working with different typologies of drug mimetics.  

4.2220           Blocking exposed PD-L1 elicited by nanosecond pulsed electric field reverses dysfunction of CD8+ T cells in liver cancer

Qian, J., Chen, T., Wu, Q., Zhou, l., Zhou, W., Wu, L., Wang, S., Lu, J., Wang, W., Li, D., Xie, H., Su, R., Guo, D., Liu, Z., He, N., Yin, S. and Zheng, S. Cancer Lett., 495, 1-11 (2020)   As a promising method for local tumor treatment, nanosecond pulsed electric field (nsPEF) ablation elicits a potent anti-tumor immune response. However, the mechanism of the nsPEF-mediated anti-tumor immune response and its effects on the tumor microenvironment remains unclear. Here, we demonstrated that nsPEF treatment increased the level of membrane PD-L1 in liver cancer cells. Furthermore, nsPEF induced the release of PD-L1-associated extra-cellular vesicles, leading to the dysfunction of CD8⁺ T cells, which could potentially be reversed by PD-L1 blockade. Biological and functional assays also demonstrated that nsPEF treatment resulted in the increased PD-L1 level and dysfunction of infiltrated CD8⁺ T cells in tumor tissues in vivo, indicating the long term antitumor efficacy of nsPEF treatment. A combination of nsPEF treatment and PD-L1 blockade effectively inhibited tumor growth and improved the survival of the tumor-bearing mouse. In conclusion, nsPEF treatment induced the translocation and release of PD-L1 and contributed to the dysfunction of infiltrated CD8⁺ T cells, resulting in tumor progression at later stages. The combination of nsPEF treatment and PD-L1 blockade is a promising therapeutic strategy for liver cancer.    

4.2221           G4C2 Repeat RNA Initiates a POM121-Mediated Reduction in Specific Nucleoporins in C9orf72 ALS/FTD

Coyne, A.N., Zaepfel, B.L., Hayes, l., Svendsen, C.N., Sareen, D. and Rothstein, J.D. Neuron, 107, 1124-1140 (2020)   Through mechanisms that remain poorly defined, defects in nucleocytoplasmic transport and accumulations of specific nuclear-pore-complex-associated proteins have been reported in multiple neurodegenerative diseases, including C9orf72 Amyotrophic Lateral Sclerosis and Frontotemporal Dementia (ALS/FTD). Using super-resolution structured illumination microscopy, we have explored the mechanism by which nucleoporins are altered in nuclei isolated from C9orf72 induced pluripotent stem-cell-derived neurons (iPSNs). Of the 23 nucleoporins evaluated, we observed a reduction in a subset of 8, including key components of the nuclear pore complex scaffold and the transmembrane nucleoporin POM121. Reduction in POM121 appears to initiate a decrease in the expression of seven additional nucleoporins, ultimately affecting the localization of Ran GTPase and subsequent cellular toxicity in C9orf72 iPSNs. Collectively, our data suggest that the expression of expanded C9orf72 ALS/FTD repeat RNA alone affects nuclear POM121 expression in the initiation of a pathological cascade affecting nucleoporin levels within neuronal nuclei and ultimately downstream neuronal survival.  

4.2222           Functions of Small Organic Compounds that Mimic the HNK-1 Glycan

Wang, M., Theis, Kabat, M., Loers, G., Agre, L.A. and Schachner, M. Int. J. Mol. Sci., 21, 7018 (2020)   Because of the importance of the HNK-1 carbohydrate for preferential motor reinnervation after injury of the femoral nerve in mammals, we screened NIH Clinical Collection 1 and 2 Libraries and a Natural Product library comprising small organic compounds for identification of pharmacologically useful reagents. The reason for this attempt was to obviate the difficult chemical synthesis of the HNK-1 carbohydrate and its isolation from natural sources, with the hope to render such compounds clinically useful. We identified six compounds that enhanced neurite outgrowth from cultured spinal motor neurons at nM concentrations and increased their neurite diameter, but not their neurite branch points. Axons of dorsal root ganglion neurons did not respond to these compounds, a feature that is in agreement with their biological role after injury. We refer to the positive functions of some of these compounds in animal models of injury and delineate the intracellular signaling responses elicited by application of compounds to cultured murine central nervous system neurons. Altogether, these results point to the potential of the HNK-1 carbohydrate mimetics in clinically-oriented settings.  

4.2223           Targeting Mitochondria-Located circRNA SCAR Alleviates NASH via Reducing mROS Output

Zhao, Q., Liu, J., Deng, H., Xu, X., Gao, Z. and Su, S. Cell, 183, 76-93 (2020)   Mitochondria, which play central roles in immunometabolic diseases, have their own genome. However, the functions of mitochondria-located noncoding RNAs are largely unknown due to the absence of a specific delivery system. By circular RNA (circRNA) expression profile analysis of liver fibroblasts from patients with nonalcoholic steatohepatitis (NASH), we observe that mitochondrial circRNAs account for a considerable fraction of downregulated circRNAs in NASH fibroblasts. By constructing mitochondria-targeting nanoparticles, we observe that Steatohepatitis-associated circRNA ATP5B Regulator (SCAR), which is located in mitochondria, inhibits mitochondrial ROS (mROS) output and fibroblast activation. circRNA SCAR, mediated by PGC-1α, binds to ATP5B and shuts down mPTP by blocking CypD-mPTP interaction. Lipid overload inhibits PGC-1α by endoplasmic reticulum (ER) stress-induced CHOP. In vivo, targeting circRNA SCAR alleviates high fat diet-induced cirrhosis and insulin resistance. Clinically, circRNA SCAR is associated with steatosis-to-NASH progression. Collectively, we identify a mitochondrial circRNA that drives metaflammation and serves as a therapeutic target for NASH.  

4.2224           The MEK Inhibitor Trametinib Suppresses Major Histocompatibility Antigen-mismatched Rejection Following Pancreatic Islet Transplantation

Tada, S., Anazawa, T., Shindo, T., Yamane, K., Inoguchi, K., Fujimoto, N., Nagai, K., Masui, T., Okajima, H., Takaori, K., Sumi, S. and Uemoto, S. Trandsplantation Direct, 6, e591 (2020)   Background.  Potential adverse effects, such as functional impairment of islets, render conventional immunosuppressive drugs unsuitable for use in islet transplantation. In addition, as a single therapy, they cannot prolong islet allograft survival. Here, we investigated the utility of the mitogen-activated protein kinase inhibitor trametinib and asked whether it ameliorates acute rejection of transplanted islets without the need for conventional immunosuppressants. Methods.  Islets from fully major histocompatibility complex-mismatched BALB/c mice were transplanted into streptozotocin-induced diabetic C57BL/6 mice via the portal vein. These mice received trametinib or vehicle (orally) for 28 days. Isolated islets from BALB/c mice were incubated in vitro with different concentrations of trametinib to determine viability and function. Results.  Trametinib (0.1 and 0.3 mg/kg) prolonged graft survival significantly (P = 0.0007 and P = 0.005, respectively) when compared with vehicle. Histologic analyses revealed that cellular infiltration of the graft by lymphocytes was inhibited significantly on day 7 (P < 0.05). In addition, trametinib suppressed functional differentiation of naive CD4⁺ T cells in recipients. Expression of mRNA encoding inflammatory cytokines interleukin (IL)-2, tumor necrosis factor α, and interferon γ in recipients treated with trametinib was also inhibited (P < 0.001, P < 0.05, and P < 0.01, respectively). Trametinib also increased production of IL-4 and IL-10 (P < 0.05 and P = 0.20, respectively). In vitro, islets incubated with different concentrations of trametinib exhibited no harmful effects with respect to viability and function. Conclusions.  Trametinib delayed islet graft rejection by inhibiting functional differentiation of naive CD4⁺ T cells and regulating inflammatory cytokines. Trametinib might be a promising candidate for maintenance immunosuppressive therapy after allogeneic islet transplantation.  

4.2225           Therapeutic IDOL Reduction Ameliorates Amyloidosis and Improves Cognitive Function in APP/PS1 Mice

Gao, J., Littman, R., Diamante, G., Xiao, X., Ahn, I.S., Yang, X., Cole, T.A. and Tontonoz, P. Mol. Cell. Biol., 40(8), e00518-19 (2020)   Brain lipoprotein receptors have been shown to regulate the metabolism of ApoE and β-amyloid (Aβ) and are potential therapeutic targets for Alzheimer’s disease (AD). Previously, we identified E3 ubiquitin ligase IDOL as a negative regulator of brain lipoprotein receptors. Genetic ablation of Idol increases low-density lipoprotein receptor protein levels, which facilitates Aβ uptake and clearance by microglia. In this study, we utilized an antisense oligonucleotide (ASO) to reduce IDOL expression therapeutically in the brains of APP/PS1 male mice. ASO treatment led to decreased Aβ pathology and improved spatial learning and memory. Single-cell transcriptomic analysis of hippocampus revealed that IDOL inhibition upregulated lysosomal/phagocytic genes in microglia. Furthermore, clustering of microglia revealed that IDOL-ASO treatment shifted the composition of the microglia population by increasing the prevalence of disease-associated microglia. Our results suggest that reducing IDOL expression in the adult brain promotes the phagocytic clearance of Aβ and ameliorates Aβ-dependent pathology. Pharmacological inhibition of IDOL activity in the brain may represent a therapeutic strategy for the treatment of AD.  

4.2226           From the Inside Out: an Epibiotic Bdellovibrio Predator with an Expanded Genomic Complement

Deeg, C.M., Le, T.T., Zimmer, M.M. and Suttle, C.A.

1. Bacteriol., 202(8), e00565-19 (2020)

  Bdellovibrio and like organisms are abundant environmental parasitoids of prokaryotes that show diverse predation strategies. The vast majority of studied Bdellovibrio bacteria and like organisms deploy intraperiplasmic replication inside the prey cell, while few isolates with smaller genomes consume their prey from the outside in an epibiotic manner. The novel parasitoid “Candidatus Bdellovibrio qaytius” was isolated from a eutrophic freshwater pond in British Columbia, where it was a continual part of the microbial community. “Ca. Bdellovibrio qaytius” was found to preferentially prey on the betaproteobacterium Paraburkholderia fungorum without entering the periplasm. Despite its epibiotic replication strategy, “Ca. Bdellovibrio” encodes a large genomic complement more similar to that of complex periplasmic predators. Functional genomic annotation further revealed several biosynthesis pathways not previously found in epibiotic predators, indicating that “Ca. Bdellovibrio” represents an intermediate phenotype and at the same time narrowing down the genomic complement specific to epibiotic predators. In phylogenetic analysis, “Ca. Bdellovibrio qaytius” occupies a widely distributed, but poorly characterized, basal cluster within the genus Bdellovibrio. This suggests that epibiotic predation might be a common predation type in nature and that epibiotic predation could be the ancestral predation type in the genus.  

4.2227           Activation of ASC Inflammasome Driven by Toll-Like Receptor 4 Contributes to Host Immunity against Rickettsial Infection

Rumfield, C., Hyseni, I., McBride, J.W., Walker, D.H. and Fang, R. Infect. Immune., 88(4), e00886-19 (2020)   Rickettsiae are cytosolically replicating, obligately intracellular bacteria causing human infections worldwide with potentially fatal outcomes. We previously showed that Rickettsia australis activates ASC inflammasome in macrophages. In the present study, host susceptibility of ASC inflammasome-deficient mice to R. australis was significantly greater than that of C57BL/6 (B6) controls and was accompanied by increased rickettsial loads in various organs. Impaired host control of R. australis in vivo in ASC^−/− mice was associated with dramatically reduced levels of interleukin 1β (IL-1β), IL-18, and gamma interferon (IFN-γ) in sera. The intracellular concentrations of R. australis in bone marrow-derived macrophages (BMMs) of TLR4^−/− and ASC^−/− mice were significantly greater than those in BMMs of B6 controls, highlighting the important role of inflammasome and these molecules in controlling rickettsiae in macrophages. Compared to B6 BMMs, TLR4^−/− BMMs failed to secrete a significant level of IL-1β and had reduced expression levels of pro-IL-1β in response to infection with R. australis, suggesting that rickettsiae activate ASC inflammasome via a Toll-like receptor 4 (TLR4)-dependent mechanism. Further mechanistic studies suggest that the lipopolysaccharide (LPS) purified from R. australis together with ATP stimulation led to cleavage of pro-caspase-1 and pro-IL-1β, resulting in TLR4-dependent secretion of IL-1β. Taken together, these observations indicate that activation of ASC inflammasome, most likely driven by interaction of TLR4 with rickettsial LPS, contributes to host protective immunity against R. australis. These findings provide key insights into defining the interactions of rickettsiae with the host innate immune system.    

4.2228           High-Resolution Mapping of Multiway Enhancer-Promoter Interactions Regulating Pathogen Detection

Vangala, P., Murphy, R., Quinodoz, S.A., Gellatly, K., McDonel, P., Guttman, M. and Garber, M. Molecular Cell, 80, 359-373 (2020)   Eukaryotic gene expression regulation involves thousands of distal regulatory elements. Understanding the quantitative contribution of individual enhancers to gene expression is critical for assessing the role of disease-associated genetic risk variants. Yet, we lack the ability to accurately link genes with their distal regulatory elements. To address this, we used 3D enhancer-promoter (E-P) associations identified using split-pool recognition of interactions by tag extension (SPRITE) to build a predictive model of gene expression. Our model dramatically outperforms models using genomic proximity and can be used to determine the quantitative impact of enhancer loss on gene expression in different genetic backgrounds. We show that genes that form stable E-P hubs have less cell-to-cell variability in gene expression. Finally, we identified transcription factors that regulate stimulation-dependent E-P interactions. Together, our results provide a framework for understanding quantitative contributions of E-P interactions and associated genetic variants to gene expression.  

4.2229           Ischemic Preconditioning Improves Microvascular Endothelial Function in Remote Vasculature by Enhanced Prostacyclin Production

Rytter, N., Carter, H., Piil, P., Sørensen, H., Ehlers, T., Holmegaard, F., Tuxen, C., Jones, H., Thijssen, D., Gliemann, L. and Hellsten, Y.

1. Am. Heart. Assoc., 9, e016017 (2020)

  BACKGROUND The mechanisms underlying the effect of preconditioning on remote microvasculature remains undisclosed. The primary objective was to document the remote effect of ischemic preconditioning on microvascular function in humans. The secondary objective was to test if exercise also induces remote microvascular effects. METHODS AND RESULTS A total of 12 healthy young men and women participated in 2 experimental days in a random counterbalanced order. On one day the participants underwent 4×5 minutes of forearm ischemic preconditioning, and on the other day they completed 4×5 minutes of hand‐grip exercise. On both days, catheters were placed in the brachial and femoral artery and vein for infusion of acetylcholine, sodium nitroprusside, and epoprostenol. Vascular conductance was calculated from blood flow measurements with ultrasound Doppler and arterial and venous blood pressures. Ischemic preconditioning enhanced (P<0.05) the remote vasodilator response to intra‐arterial acetylcholine in the leg at 5 and 90 minutes after application. The enhanced response was associated with a 6‐fold increase (P<0.05) in femoral venous plasma prostacyclin levels and with a transient increase (P<0.05) in arterial plasma levels of brain‐derived neurotrophic factor and vascular endothelial growth factor. In contrast, hand‐grip exercise did not influence remote microvascular function. CONCLUSIONS These findings demonstrate that ischemic preconditioning of the forearm improves remote microvascular endothelial function and suggest that one of the underlying mechanisms is a humoral‐mediated potentiation of prostacyclin formation.  

4.2230           Method for Passive Droplet Sorting after Photo-Tagging

Dobson, C., Zielke, C., Pan, C.W., Feit, C. and Abbyad, P. Micromachines, 11:964 (2020)   We present a method to photo-tag individual microfluidic droplets for latter selection by passive sorting. The use of a specific surfactant leads to the interfacial tension to be very sensitive to droplet pH. The photoexcitation of droplets containing a photoacid, pyranine, leads to a decrease in droplet pH. The concurrent increase in droplet interfacial tension enables the passive selection of irradiated droplets. The technique is used to select individual droplets within a droplet array as illuminated droplets remain in the wells while other droplets are eluted by the flow of the external oil. This method was used to select droplets in an array containing cells at a specific stage of apoptosis. The technique is also adaptable to continuous-flow sorting. By passing confined droplets over a microfabricated trench positioned diagonally in relation to the direction of flow, photo-tagged droplets were directed toward a different chip exit based on their lateral movement. The technique can be performed on a conventional fluorescence microscope and uncouples the observation and selection of droplets, thus enabling the selection on a large variety of signals, or based on qualitative user-defined features.  

4.2231           Improved 3D cellular morphometry of Caenorhabditis elegans embryos using a refractive index matching medium

Xiong, R. and Sugloka, K. PloS One, 15(9), e0238955 (2020)   Cell shape change is one of the driving forces of animal morphogenesis, and the model organism Caenorhabditis elegans has played a significant role in analyzing the underlying mechanisms involved. The analysis of cell shape change requires quantification of cellular shape descriptors, a method known as cellular morphometry. However, standard C. elegans live imaging methods limit the capability of cellular morphometry in 3D, as spherical aberrations generated by samples and the surrounding medium misalign optical paths. Here, we report a 3D live imaging method for C. elegans embryos that minimized spherical aberrations caused by refractive index (RI) mismatch. We determined the composition of a refractive index matching medium (RIMM) for C. elegans live imaging. The 3D live imaging with the RIMM resulted in a higher signal intensity in the deeper cell layers. We also found that the obtained images improved the 3D cell segmentation quality. Furthermore, our 3D cellular morphometry and 2D cell shape simulation indicated that the germ cell precursor P₄ had exceptionally high cortical tension. Our results demonstrate that the RIMM is a cost-effective solution to improve the 3D cellular morphometry of C. elegans. The application of this method should facilitate understanding of C. elegans morphogenesis from the perspective of cell shape changes.  

4.2232           Acoustic separation of living and dead cells using high density medium

Olofsson, K., Hammarström, B. and Wiklund, M. Lab on a Chip, 20, 1981-1990 (2020)   The acoustic radiation force, originating from ultrasonic standing waves and utilized in numerous cell oriented acoustofluidic applications, is dependent on the acoustic contrast factor which describes the relationship between the acousto-mechanical properties of a particle and its surrounding medium. The acousto-mechanical properties of a cell population are known to be heterogeneously distributed but are often assumed to be constant over time. In this paper, we use microchannel acoustophoresis to show that the cell state within a cell population, in our case living and dead cells, influences the mechanical phenotype. By investigating the trapping location of viable and dead K562, MCF-7 and A498 cells as a function of the suspension medium density, we observed that beyond a specific medium density the viable cells were driven to the pressure anti-node while the dead cells were retained in the pressure node. Using this information, we were able to calculate the effective acoustic impedance of viable K562 and MCF-7 cells. The spatial separation between viable and dead cells along the channel width demonstrates a novel acoustophoresis approach for binary separation of viable and dead cells in a cell-size independent and robust manner.  

4.2233           Gelatin-Modified Calcium/Strontium Hydrogen Phosphates Stimulate Bone Regeneration in Osteoblast/Osteoclast Co-Culture and in Osteoporotic Rat Femur Defects—In Vitro to In Vivo Translation

Kruppke, B., Ray, S., Alt, V., Rohnke, M., Kern, C., Kampschulte, M., Heinemann, C., Budak, M., Adam, J., Döhner, N., Franz-Forsthoffer, L., El Khassawna, T., Heiss, C., Hanke, T. and Thormann, U. Molecules, 25, 5103 (2020)   The development and characterization of biomaterials for bone replacement in case of large defects in preconditioned bone (e.g., osteoporosis) require close cooperation of various disciplines. Of particular interest are effects observed in vitro at the cellular level and their in vivo representation in animal experiments. In the present case, the material-based alteration of the ratio of osteoblasts to osteoclasts in vitro in the context of their co-cultivation was examined and showed equivalence to the material-based stimulation of bone regeneration in a bone defect of osteoporotic rats. Gelatin-modified calcium/strontium phosphates with a Ca:Sr ratio in their precipitation solutions of 5:5 and 3:7 caused a pro-osteogenic reaction on both levels in vitro and in vivo. Stimulation of osteoblasts and inhibition of osteoclast activity were proven during culture on materials with higher strontium content. The same material caused a decrease in osteoclast activity in vitro. In vivo, a positive effect of the material with increased strontium content was observed by immunohistochemistry, e.g., by significantly increased bone volume to tissue volume ratio, increased bone morphogenetic protein-2 (BMP2) expression, and significantly reduced receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio. In addition, material degradation and bone regeneration were examined after 6 weeks using stage scans with ToF-SIMS and µ-CT imaging. The remaining material in the defects and strontium signals, which originate from areas exceeding the defect area, indicate the incorporation of strontium ions into the surrounding mineralized tissue. Thus, the material inherent properties (release of biologically active ions, solubility and degradability, mechanical strength) directly influenced the cellular reaction in vitro and also bone regeneration in vivo. Based on this, in the future, materials might be synthesized and specifically adapted to patient-specific needs and their bone status.  

4.2234           Platelets prevent the development of monocrotaline-induced liver injury in mice

Otaka, F., Ito, Y., Goto, T., Eshima, K., Amano, H., Koizumi, W. and Majima, M. Toxicol. Lett., 335, 71-81 (2020)   Destruction of liver sinusoidal endothelial cells (LSECs) is an initial event in sinusoidal obstruction syndrome (SOS) that leads to accumulation of platelets in the liver. Herein, we explored the role of platelets during progression of experimental SOS induced by monocrotaline (MCT) in mice. Depletion of platelets using an anti-CD41 antibody or anti-thrombocyte serum exacerbated MCT-induced liver injury in C57BL/6 mice, as indicated by an increase in the alanine transaminase (ALT) level, which was associated with hemorrhagic necrosis. Thrombocytosis induced by thrombopoietin (TPO) or the TPO receptor agonist romiplostim (ROM) attenuated MCT-induced liver injury, as evidenced by lower levels of ALT and mRNA encoding matrix metalloproteinase (MMP) 9, and higher levels of mRNA encoding vascular endothelial growth factor receptor (VEGFR) 2 and VEGFR3. The level of activated hepatic platelets was higher in TPO- and ROM-treated mice than in saline-treated mice. Co-culture with a high number of platelets increased the viability of LSECs and their mRNA levels of CD31, VEGFR2, and VEGFR3, and decreased their mRNA level of MMP9. The level of VEGF-A was increased in the culture medium of LSECs co-cultured with platelets. These results indicate that platelets attenuate MCT-induced liver injury by minimizing damage to LSECs.  

4.2235           Inhibition of aquaporin-3 in macrophages by a monoclonal antibody as potential therapy for liver injury

Hara-Chikuma, M., Tanaka, M., Verkman, A.S. and Yasui, M. Nature Communications, 11:5666 (2020)   Aquaporin 3 (AQP3) is a transporter of water, glycerol and hydrogen peroxide (H₂O₂) that is expressed in various epithelial cells and in macrophages. Here, we developed an anti-AQP3 monoclonal antibody (mAb) that inhibited AQP3-facilitated H₂O₂ and glycerol transport, and prevented liver injury in experimental animal models. Using AQP3 knockout mice in a model of liver injury and fibrosis produced by CCl₄, we obtained evidence for involvement of AQP3 expression in nuclear factor-κB (NF-κB) cell signaling, hepatic oxidative stress and inflammation in macrophages during liver injury. The activated macrophages caused stellate cell activation, leading to liver injury, by a mechanism involving AQP3-mediated H₂O₂ transport. Administration of an anti-AQP3 mAb, which targeted an extracellular epitope on AQP3, prevented liver injury by inhibition of AQP3-mediated H₂O₂ transport and macrophage activation. These findings implicate the involvement of macrophage AQP3 in liver injury, and provide evidence for mAb inhibition of AQP3-mediated H₂O₂ transport as therapy for macrophage-dependent liver injury.  

4.2236           Loss of Class III Phosphoinositide 3-Kinase Vps34 Results in Cone Degeneration

Rajala, A., He, F., Anderson, R.E., Wensel, T.G. and Rajala, R.V. Biology, 9:384 (2020)   The major pathway for the production of the low-abundance membrane lipid phosphatidylinositol 3-phosphate (PI(3)P) synthesis is catalyzed by class III phosphoinositide 3-kinase (PI3K) Vps34. The absence of Vps34 was previously found to disrupt autophagy and other membrane-trafficking pathways in some sensory neurons, but the roles of phosphatidylinositol 3-phosphate and Vps34 in cone photoreceptor cells have not previously been explored. We found that the deletion of Vps34 in neighboring rods in mouse retina did not disrupt cone function up to 8 weeks after birth, despite diminished rod function. Immunoblotting and lipid analysis of cones isolated from the cone-dominant retinas of the neural retina leucine zipper gene knockout mice revealed that both PI(3)P and Vps34 protein are present in mouse cones. To determine whether Vps34 and PI(3)P are important for cone function, we conditionally deleted Vps34 in cone photoreceptor cells of the mouse retina. Overall retinal morphology and rod function appeared to be unaffected. However, the loss of Vps34 in cones resulted in the loss of structure and function. There was a substantial reduction throughout the retina in the number of cones staining for M-opsin, S-opsin, cone arrestin, and peanut agglutinin, revealing degeneration of cones. These studies indicate that class III PI3K, and presumably PI(3)P, play essential roles in cone photoreceptor cell function and survival.  

4.2237           CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

Trolese, M., Mariani, A., Terao, M., de Paola, M., Fabbrizio, P., Sironi, F., Kurosi, M., Bonanno, S., Marcuzzo, S., Bernasconi, P., Trojsi, F., Aronica, E., Bendotti, C. and Nardo, G. EBioMed., 62, 103097 (2020)   Background CXCL13 is a B and T lymphocyte chemokine that mediates neuroinflammation through its receptor CXCR5. This chemokine is highly expressed by motoneurons (MNs) in Amyotrophic Lateral Sclerosis (ALS) SOD1G93A (mSOD1) mice during the disease, particularly in fast-progressing mice. Accordingly, in this study, we investigated the role of this chemokine in ALS. Methods We used in vitro and in vivo experimental paradigms derived from ALS mice and patients to investigate the expression level and distribution of CXCL13/CXCR5 axis and its role in MN death and disease progression. Moreover, we compared the levels of CXCL13 in the CSF and serum of ALS patients and controls. Findings CXCL13 and CXCR5 are overexpressed in the spinal MNs and peripheral axons in mSOD1 mice. CXCL13 inhibition in the CNS of ALS mice resulted in the exacerbation of motor impairment (n = 4/group;Mean_Diff.=27.81) and decrease survival (n = 14_Treated:19.2 ± 1.05wks, n = 17_Controls:20.2 ± 0.6wks; 95% CI: 0.4687–1.929). This was corroborated by evidence from primary spinal cultures where the inhibition or activation of CXCL13 exacerbated or prevented the MN loss. Besides, we found that CXCL13/CXCR5 axis is overexpressed in the spinal cord MNs of ALS patients, and CXCL13 levels in the CSF discriminate ALS (n = 30) from Multiple Sclerosis (n = 16) patients with a sensitivity of 97.56%. Interpretation We hypothesise that MNs activate CXCL13 signalling to attenuate CNS inflammation and prevent the neuromuscular denervation. The low levels of CXCL13 in the CSF of ALS patients might reflect the MN dysfunction, suggesting this chemokine as a potential clinical adjunct to discriminate ALS from other neurological diseases.  

4.2238           RNA sequencing reveals changes in the microRNAome of transdifferentiating hepatic stellate cells that are conserved between human and rat

Sabater, L., Locatelli, L., Oakley, F., hardy, T., French, J., Robinson, S.M., Sen, G., Mann, D.A. and Mann, J. Scientific Reports, 10:21708 (2020)   MicroRNAs are small (~ 22nt long) noncoding RNAs (ncRNAs) that regulate gene expression at the post-transcriptional level. Over 2000 microRNAs have been described in humans and many are implicated in human pathologies including tissue fibrosis. Hepatic stellate cells (HSC) are the major cellular contributors to excess extracellular matrix deposition in the diseased liver and as such are important in the progression of liver fibrosis. We employed next generation sequencing to map alterations in the expression of microRNAs occurring across a detailed time course of culture-induced transdifferentiation of primary human HSC, this a key event in fibrogenesis. Furthermore, we compared profiling of human HSC microRNAs with that of rat HSC so as to identify those molecules that are conserved with respect to modulation of expression. Our analysis reveals that a total of 229 human microRNAs display altered expression as a consequence of HSC transdifferentiation and of these 104 were modulated early during the initiation phase. Typically modulated microRNAs were targeting kinases, transcription factors, chromatin factors, cell cycle regulators and growth factors. 162 microRNAs changed in expression during transdifferentiation of rat HSC, however only 17 underwent changes that were conserved in human HSC. Our study therefore identifies widespread changes in the expression of HSC microRNAs in fibrogenesis, but suggests a need for caution when translating data obtained from rodent HSC to events occurring in human cells.  

4.2239           Spindle Scaling Is Governed by Cell Boundary Regulation of Microtubule Nucleation

Rieckhoff, E.M., Berndt, F., Elsner, M., Golfier, S., Decker, F., Ishihara, K and Brugues, J. Current Biol., 30, 4973-4983 (2020)   Cellular organelles such as the mitotic spindle adjust their size to the dimensions of the cell. It is widely understood that spindle scaling is governed by regulation of microtubule polymerization. Here, we use quantitative microscopy in living zebrafish embryos and Xenopus egg extracts in combination with theory to show that microtubule polymerization dynamics are insufficient to scale spindles and only contribute below a critical cell size. In contrast, microtubule nucleation governs spindle scaling for all cell sizes. We show that this hierarchical regulation arises from the partitioning of a nucleation inhibitor to the cell membrane. Our results reveal that cells differentially regulate microtubule number and length using distinct geometric cues to maintain a functional spindle architecture over a large range of cell sizes.  

4.2240           Exercise alters LPS-induced glial activation in the mouse brain

Mota, B.C. and Kelly, A.M. Neuronal Signalling, 4, NS2020003 (2020)   Experimental and epidemiological evidence suggest that modifiable lifestyle factors, including physical exercise, can build structural and cognitive reserve in the brain, increasing resilience to injury and insult. Accordingly, exercise can reduce the increased expression of proinflammatory cytokines in the brain associated with ageing or experimentally induced neuroinflammation. However, the cellular mechanisms by which exercise exerts this effect are unknown, including the effects of exercise on classic or alternative activation of astrocytes and microglia. In the present study, we assess the effects of nine consecutive days of treadmill running on the glial cell response to a single systemic injection of lipopolysaccharide (LPS) and, in parallel, the effects on spatial learning and memory. We show that prior exercise protects against LPS-induced impairment of performance in the object displacement task concomitant with attenuation of IL-1β, TNFα and IL-10 mRNA expression in the hippocampus. Assessment of isolated astrocytes and microglia revealed that LPS induced a proinflammatory response in these cells that was not observed in cells prepared from the brains of mice who had undergone prior exercise. The results suggest that exercise modulates neuroinflammation by reducing the proinflammatory microglial response, suggesting a mechanism by which exercise may be neuroprotective.  

4.2241           Probing single-cell metabolism reveals prognostic value of highly metabolically active circulating stromal cells in prostate cancer

Rivello, F.R., Matula, K., Piruska, A., SMits, M., Mehra, N. and Huck, W.T.S. Sci. Adv., 6:eaaz3849 (2020)   Despite their important role in metastatic disease, no general method to detect circulating stromal cells (CStCs) exists. Here, we present the Metabolic Assay-Chip (MA-Chip) as a label-free, droplet-based microfluidic approach allowing single-cell extracellular pH measurement for the detection and isolation of highly metabolically active cells (hm-cells) from the tumor microenvironment. Single-cell mRNA-sequencing analysis of the hm-cells from metastatic prostate cancer patients revealed that approximately 10% were canonical EpCAM⁺ hm-CTCs, 3% were EpCAM⁻ hm-CTCs with up-regulation of prostate-related genes, and 87% were hm-CStCs with profiles characteristic for cancer-associated fibroblasts, mesenchymal stem cells, and endothelial cells. Kaplan-Meier analysis shows that metastatic prostate cancer patients with more than five hm-cells have a significantly poorer survival probability than those with zero to five hm-cells. Thus, prevalence of hm-cells is a prognosticator of poor outcome in prostate cancer, and a potentially predictive and therapy response biomarker for agents cotargeting stromal components and preventing epithelial-to-mesenchymal transition.  

4.2242           Tumor Cell–Derived TGFβ1 Attenuates Antitumor Immune Activity of T Cells via Regulation of PD-1 mRNA

Wu, P., Geng, B., Chen, Q., Zhao, E., Liu, J., Sun, C., Zha, C., Shao, Y., You, B., Zhang, W., Li, L., Meng, X., Cai, J. and Li, X. Cancer Immunol. Res., 8, 1470-1484 (2020)   Dysfunction in T-cell antitumor activity contributes to the tumorigenesis, progression, and poor outcome of clear cell renal cell carcinoma (ccRCC), with this dysfunction resulting from high expression of programmed cell death-1 (PD-1) in T cells. However, the molecular mechanisms maintaining high PD-1 expression in T cells have not been fully investigated in ccRCC. Here, we describe a mechanism underlying the regulation of PD-1 at the mRNA level and demonstrated its impact on T-cell dysfunction. Transcriptomic analysis identified a correlation between TGFβ1 and PD-1 mRNA levels in ccRCC samples. The mechanism underlying the regulation of PD-1 mRNA was then investigated in vitro and in vivo using syngeneic tumor models. We also observed that TGFβ1 had prognostic significance in patients with ccRCC, and its expression was associated with PD-1 mRNA expression. CcRCC-derived TGFβ1 activated P38 and induced the phosphorylation of Ser¹⁰ on H3, which recruited p65 to increase SRSF3 and SRSF5 expression in T cells. As a result, the half-life of PD-1 mRNA in T cells was prolonged. SRSF3 coordinated with NXF1 to induce PD-1 mRNA extranuclear transport in T cells. We then demonstrated that TGFβ1 could induce SRSF3 expression to restrict the antitumor activity of T cells, which influenced immunotherapy outcomes in ccRCC mouse models. Our findings highlight that tumor-derived TGFβ1 mediates immune evasion and has potential as a prognostic biomarker and therapeutic target in ccRCC.  

4.2243           Salmonella Persistence and Host Immunity Are Dictated by the Anatomical Microenvironment

Kurtz, J.R., Nieves, W., Bauer, D.L., Israel, K.E., Adcox, H.E., Gunn, J.S., Morici, L.A. and McLachlan, J.B. Infect. Immun., 88(8), e00026-20 (2020)   The intracellular bacterial pathogen Salmonella is able to evade the immune system and persist within the host. In some cases, these persistent infections are asymptomatic for long periods and represent a significant public health hazard because the hosts are potential chronic carriers, yet the mechanisms that control persistence are incompletely understood. Using a mouse model of chronic typhoid fever combined with major histocompatibility complex (MHC) class II tetramers to interrogate endogenous, Salmonella-specific CD4⁺ helper T cells, we show that certain host microenvironments may favorably contribute to a pathogen’s ability to persist in vivo. We demonstrate that the environment in the hepatobiliary system may contribute to the persistence of Salmonella enterica subsp. enterica serovar Typhimurium through liver-resident immunoregulatory CD4⁺ helper T cells, alternatively activated macrophages, and impaired bactericidal activity. This contrasts with lymphoid organs, such as the spleen and mesenteric lymph nodes, where these same cells appear to have a greater capacity for bacterial killing, which may contribute to control of bacteria in these organs. We also found that, following an extended period of infection of more than 2 years, the liver appeared to be the only site that harbored Salmonella bacteria. This work establishes a potential role for nonlymphoid organ immunity in regulating chronic bacterial infections and provides further evidence for the hepatobiliary system as the site of chronic Salmonella infection.  

4.2244           Histone H3.3G34-Mutant Interneuron Progenitors Co-opt PDGFRA for Gliomagenesis

Chen, C.C.L., Deshmukh, S., Jessa, S., Salomoni, P., Kleinman, C. and Jabado, N. Cell, 183, 1617-1633 (2020)   Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here we show that 50% of G34R/V tumors (n = 95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. Although considered gliomas, G34R/V tumors actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V mutations impair neuronal differentiation. The lineage of origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V tumors harbor dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell fate specification. G34R/V may become dispensable for tumor maintenance, whereas mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumors. G34R/V gliomas are neuronal malignancies where interneuron progenitors are stalled in differentiation by G34R/V mutations and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signaling.  

4.2245           Birefringence Changes of Dendrites in Mouse Hippocampal Slices Revealed with Polarizing Microscopy

Koike-Tani, M., Tominaga, T., Oldenbourg, R. and Tani, T. Biophys. J., 118, 2366-2384 (2020)   Intrinsic optical signal (IOS) imaging has been widely used to map the patterns of brain activity in vivo in a label-free manner. Traditional IOS refers to changes in light transmission, absorption, reflectance, and scattering of the brain tissue. Here, we use polarized light for IOS imaging to monitor structural changes of cellular and subcellular architectures due to their neuronal activity in isolated brain slices. To reveal fast spatiotemporal changes of subcellular structures associated with neuronal activity, we developed the instantaneous polarized light microscope (PolScope), which allows us to observe birefringence changes in neuronal cells and tissues while stimulating neuronal activity. The instantaneous PolScope records changes in transmission, birefringence, and slow axis orientation in tissue at a high spatial and temporal resolution using a single camera exposure. These capabilities enabled us to correlate polarization-sensitive IOS with traditional IOS on the same preparations. We detected reproducible spatiotemporal changes in both IOSs at the stratum radiatum in mouse hippocampal slices evoked by electrical stimulation at Schaffer collaterals. Upon stimulation, changes in traditional IOS signals were broadly uniform across the area, whereas birefringence imaging revealed local variations not seen in traditional IOS. Locations with high resting birefringence produced larger stimulation-evoked birefringence changes than those produced at low resting birefringence. Local application of glutamate to the synaptic region in CA1 induced an increase in both transmittance and birefringence signals. Blocking synaptic transmission with inhibitors CNQX (for AMPA-type glutamate receptor) and D-APV (for NMDA-type glutamate receptor) reduced the peak amplitude of the optical signals. Changes in both IOSs were enhanced by an inhibitor of the membranous glutamate transporter, DL-TBOA. Our results indicate that the detection of activity-induced structural changes of the subcellular architecture in dendrites is possible in a label-free manner.  

4.2246           Reconstructed Single-Cell Fate Trajectories Define Lineage Plasticity Windows during Differentiation of Human PSC-Derived Distal Lung

Hurley, K., Ding, J., Villacorta-Martin, C., Camargo, F., Bar-Joseph, Z. and Kotton, D.N. Cell Stem Cell, 26, 593-608 (2020)   Alveolar epithelial type 2 cells (AEC2s) are the facultative progenitors responsible for maintaining lung alveoli throughout life but are difficult to isolate from patients. Here, we engineer AEC2s from human pluripotent stem cells (PSCs) in vitro and use time-series single-cell RNA sequencing with lentiviral barcoding to profile the kinetics of their differentiation in comparison to primary fetal and adult AEC2 benchmarks. We observe bifurcating cell-fate trajectories as primordial lung progenitors differentiate in vitro, with some progeny reaching their AEC2 fate target, while others diverge to alternative non-lung endodermal fates. We develop a Continuous State Hidden Markov model to identify the timing and type of signals, such as overexuberant Wnt responses, that induce some early multipotent NKX2-1⁺ progenitors to lose lung fate. Finally, we find that this initial developmental plasticity is regulatable and subsides over time, ultimately resulting in PSC-derived AEC2s that exhibit a stable phenotype and nearly limitless self-renewal capacity.  

4.2247           Early pathogenesis of cystic fibrosis gallbladder disease in a porcine model

Zarei, K., Stroik, M.R., Gansemer, N.D., Thurman, A.L., Ostegaard, L.S., Ernst, S.E., Thornell, I.M., Powers, L.S., Pezzulo, A.A., Meyerholz, D.K. and Stolz, D. Lab. Invest, 100, 1388-1399 (2020)   Hepatobiliary disease causes significant morbidity in people with cystic fibrosis (CF), yet this problem remains understudied. We previously found that newborn CF pigs have microgallbladders with significant luminal obstruction in the absence of infection and consistent inflammation. In this study, we sought to better understand the early pathogenesis of CF pig gallbladder disease. We hypothesized that loss of CFTR would impair gallbladder epithelium anion/liquid secretion and increase mucin production. CFTR was expressed apically in non-CF pig gallbladder epithelium but was absent in CF. CF pig gallbladders lacked cAMP-stimulated anion transport. Using a novel gallbladder epithelial organoid model, we found that Cl⁻ or HCO₃⁻ was sufficient for non-CF organoid swelling. This response was absent for non-CF organoids in Cl⁻/HCO₃⁻-free conditions and in CF. Single-cell RNA-sequencing revealed a single epithelial cell type in non-CF gallbladders that coexpressed CFTR, MUC5AC, and MUC5B. Despite CF gallbladders having increased luminal MUC5AC and MUC5B accumulation, there was no significant difference in the epithelial expression of gel-forming mucins between non-CF and CF pig gallbladders. In conclusion, these data suggest that loss of CFTR-mediated anion transport and fluid secretion contribute to microgallbladder development and luminal mucus accumulation in CF.  

4.2248           Targeted pharmacological therapy restores β-cell function for diabetes remission

Sachs, S., Bastidas-Ponce, A., Tritschler, S., Bakhti, M., Böttcher, A., Sanchez-Garrido, M.A., Tarquis-Medina, M. et al Nature Metabolism, 2, 192-209 (2020)   Dedifferentiation of insulin-secreting β cells in the islets of Langerhans has been proposed to be a major mechanism of β-cell dysfunction. Whether dedifferentiated β cells can be targeted by pharmacological intervention for diabetes remission, and ways in which this could be accomplished, are unknown as yet. Here we report the use of streptozotocin-induced diabetes to study β-cell dedifferentiation in mice. Single-cell RNA sequencing (scRNA-seq) of islets identified markers and pathways associated with β-cell dedifferentiation and dysfunction. Single and combinatorial pharmacology further show that insulin treatment triggers insulin receptor pathway activation in β cells and restores maturation and function for diabetes remission. Additional β-cell selective delivery of oestrogen by Glucagon-like peptide-1 (GLP-1–oestrogen conjugate) decreases daily insulin requirements by 60%, triggers oestrogen-specific activation of the endoplasmic-reticulum-associated protein degradation system, and further increases β-cell survival and regeneration. GLP-1–oestrogen also protects human β cells against cytokine-induced dysfunction. This study not only describes mechanisms of β-cell dedifferentiation and regeneration, but also reveals pharmacological entry points to target dedifferentiated β cells for diabetes remission.  

4.2249           Bone morphogenetic protein 8B promotes the progression of non-alcoholic steatohepatitis

Vacca, M., Leslie, J., Virtue, S., lam, B.Y.H., Govaere, O., Tiniakos, D. et al Nature Metabolism, 2, 514-531 (2020)   Non-alcoholic steatohepatitis (NASH) is characterized by lipotoxicity, inflammation and fibrosis, ultimately leading to end-stage liver disease. The molecular mechanisms promoting NASH are poorly understood, and treatment options are limited. Here, we demonstrate that hepatic expression of bone morphogenetic protein 8B (BMP8B), a member of the transforming growth factor beta (TGFβ)–BMP superfamily, increases proportionally to disease stage in people and animal models with NASH. BMP8B signals via both SMAD2/3 and SMAD1/5/9 branches of the TGFβ–BMP pathway in hepatic stellate cells (HSCs), promoting their proinflammatory phenotype. In vivo, the absence of BMP8B prevents HSC activation, reduces inflammation and affects the wound-healing responses, thereby limiting NASH progression. Evidence is featured in primary human 3D microtissues modelling NASH, when challenged with recombinant BMP8. Our data show that BMP8B is a major contributor to NASH progression. Owing to the near absence of BMP8B in healthy livers, inhibition of BMP8B may represent a promising new therapeutic avenue for NASH treatment.  

4.2250           Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap

Bounab, Y., Eyer, K., Dixneut, S., Rybczynska, M., Chauvel, C., Mistretta, M. et al Nature Protocols, 15, 2920-2955 (2020)   Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8–10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.  

4.2251           Massively parallel interrogation and mining of natively paired human TCRαβ repertoires

Spindler, M.J., Nelson, A.L., Wagner, E.K., Oppermans, N., Bridgeman, J.S., Heather, J.M., Adler, A.S., Asensio, M.A., Edgar, R.C., Lim, Y.W., Meyer, E.H., Hawkins, R.E., Cobbold, M. and Johnson, D.S. Nature Biotechnol., 38, 609-619 (2020)   T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach used massively parallel microfluidics to generate libraries of natively paired, full-length TCRαβ clones, from millions of primary T cells, which were then expressed in Jurkat cells. The TCRαβ–Jurkat libraries enabled repeated screening and panning for antigen-reactive TCRs using peptide major histocompatibility complex binding and cellular activation. We captured more than 2.9 million natively paired TCRαβ clonotypes from six healthy human donors and identified rare (<0.001% frequency) viral-antigen-reactive TCRs. We also mined a tumor-infiltrating lymphocyte sample from a patient with melanoma and identified several tumor-specific TCRs, which, after expression in primary T cells, led to tumor cell killing.  

4.2252           High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics

Gerard, A., Woolfe, A., Mottet, G., Reichen, M., Castrillon, C., Menrath, V. et al Nature Biotechnol., 38, 715-721 (2020)   Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100–1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450–900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.    

4.2253           Illumination guidelines for ultrafast pump–probe experiments by serial femtosecond crystallography

Grünbein, M.L., Stricker, M., Kovacs, G.N., Kloos, M., Doak, R.B., Shoeman, R.L., Reinstein, J., Lecler, S., Haacke, S. and Schlichting, I. Nature Methods, 17, 681-684 (2020)   Time-resolved crystallography with X-ray free-electron lasers enables structural characterization of light-induced reactions on ultrafast timescales. To be biologically and chemically relevant, such studies must be carried out in an appropriate photoexcitation regime to avoid multiphoton artifacts, a common issue in recent studies. We describe numerical and experimental approaches to determine how many photons are needed for single-photon excitation in microcrystals, taking into account losses by scattering.  

4.2254           IL-1R Regulates Disease Tolerance and Cachexia in Toxoplasma gondii Infection

Melchor, S.J., Saunders, C.M., Sanders, I., hatter, J.A., Byrnes, K.A., Coutermarsh-Ott, S. and ewald, S.E.

1. Immunol., 204, 3329-3338 (2020)

  Toxoplasma gondii is an obligate intracellular parasite that establishes life-long infection in a wide range of hosts, including humans and rodents. To establish a chronic infection, pathogens often exploit the trade-off between resistance mechanisms, which promote inflammation and kill microbes, and tolerance mechanisms, which mitigate inflammatory stress. Signaling through the type I IL-1R has recently been shown to control disease tolerance pathways in endotoxemia and Salmonella infection. However, the role of the IL-1 axis in T. gondii infection is unclear. In this study we show that IL-1R^−/− mice can control T. gondii burden throughout infection. Compared with wild-type mice, IL-1R^−/− mice have more severe liver and adipose tissue pathology during acute infection, consistent with a role in acute disease tolerance. Surprisingly, IL-1R^−/− mice had better long-term survival than wild-type mice during chronic infection. This was due to the ability of IL-1R^−/− mice to recover from cachexia, an immune-metabolic disease of muscle wasting that impairs fitness of wild-type mice. Together, our data indicate a role for IL-1R as a regulator of host homeostasis and point to cachexia as a cost of long-term reliance on IL-1–mediated tolerance mechanisms.  

4.2255           Microfluidic Platform for the Isolation of Cancer-Cell Subpopulations Based on Single-Cell Glycolysis

Zielke, C., Pan, C.W., Ramirez, A.J.G., Feit, C., Dobson, C., Davidson, C., Sandel, B. and Abbyad, P. Anal. Chem., 92, 6949-6957 (2020)   High rates of glycolysis in tumors have been associated with cancer metastasis, tumor recurrence, and poor outcomes. In this light, single cells that exhibit high glycolysis are specific targets for therapy. However, the study of these cells requires efficient tools for their isolation. We use a droplet microfluidic technique developed in our lab, Sorting by Interfacial Tension (SIFT), to isolate cancer cell subpopulations based on glycolysis without the use of labels or active sorting components. By controlling the flow conditions on chip, the threshold of selection can be modified, enabling the isolation of cells with different levels of glycolysis. Hypoxia in tumors, that can be simulated with treatment with CoCl₂, leads to an increase in glycolysis, and more dangerous tumors. The device was used to enrich CoCl₂ treated MDA-MB 231 breast cancer cells from an untreated population. It is also used to sort K562 human chronic myelogenous leukemia cells that have either been treated or untreated with 2-deoxy-d-glucose (2DG), a pharmaceutical that targets cell metabolism. The technique provides a facile and robust way of separating cells based on elevated glycolytic activity; a biomarker associated with cancer cell malignancy.  

4.2256            Three-Dimensional Microtumors for Probing Heterogeneity of Invasive Bladder Cancer

Torab, P., Yan, Y., Yamashita, H., Warrick, J.I., Raman, J.D., DeGraff, D.J. and Wong, P.K. Anal. Chem., 92, 8768-8775 (2020)   Bladder cancer is an increasingly common malignancy, and muscle invasive bladder cancer is associated with particularly high rates of morbidity and mortality. The morphologic and molecular diversity of bladder cancer poses significant challenges in elucidating the invasion mechanisms responsible for disease progression. Furthermore, conventional invasion assays do not provide a physiological context for studying bladder cancer invasion within 3D microenvironments and have limited ability to capture the contribution of cellular phenotypic heterogeneity to disease progression. Here, we describe the development of a 3D microtumor invasion model suitable for the analysis of cellular phenotypic heterogeneity in cell lines and primary tumor cells from bladder cancer patients. This model incorporates a self-assembly approach for recapitulating features of bladder cancer invasion in 3D microenvironments and probing the invasive cell subpopulations. The gene expression profiles of invading microtumors were analyzed by incorporating a gold nanorod-locked nucleic acid biosensor. The incorporation of the single cell biosensor and transient gene knockdown into the system revealed the formation of invasive leader cells with upregulated Delta-like ligand 4 (DLL4) expression as well as the role of NOTCH1-DLL4 signaling in collective bladder cancer invasion. The involvement of DLL4 expressing cells in bladder cancer invasion was also observed in patient samples obtained from transurethral resection. Collectively, our study demonstrates a 3D microtumor invasion model for investigating intracellular heterogeneity of bladder cancer invasion and analyzing patient derived samples toward personalized medicine applications.  

4.2257           Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS

Brower, K.K., Khariton, M., Suzuki, P.H., Still, C., Kim, G., Calhoun, S.C.K., Qi, L.S., Wang, B. and Fordyce, P.M. Anal. Chem., 92, 13262-13270 (2020)   In the past five years, droplet microfluidic techniques have unlocked new opportunities for the high-throughput genome-wide analysis of single cells, transforming our understanding of cellular diversity and function. However, the field lacks an accessible method to screen and sort droplets based on cellular phenotype upstream of genetic analysis, particularly for large and complex cells. To meet this need, we developed Dropception, a robust, easy-to-use workflow for precise single-cell encapsulation into picoliter-scale double emulsion droplets compatible with high-throughput screening via fluorescence-activated cell sorting (FACS). We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying sizes and morphologies as well as a heterogeneous cell mixture of a whole dissociated flatworm (5–25 μm in diameter) within highly monodisperse double emulsions (35 μm in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events (<2% of cell-containing droplets), thereby allowing direct linkage of cellular phenotype to genotype. Across all cell lines, cell loading efficiency approaches the theoretical limit with no observable bias by cell size. FACS measurements reveal the ability to discriminate empty droplets from those containing cells with good agreement to single-cell occupancies quantified via microscopy, establishing robust droplet screening at single-cell resolution. High-throughput FACS screening of cellular picoreactors has the potential to shift the landscape of single-cell droplet microfluidics by expanding the repertoire of current nucleic acid droplet assays to include functional phenotyping.  

4.2258           Targeted Single-Cell RNA and DNA Sequencing With Fluorescence-Activated Droplet Merger

Clark, I.C., Delley, C.L., Sun, C., Thakur, R., Stott, S.L., Thaploo, S., Li, Z., Quintana, F.J. and Abate, A.R. Anal. Chem., 92, 14616-14623 (2020)   Analyzing every cell in a diverse sample provides insight into population-level heterogeneity, but abundant cell types dominate the analysis and rarer populations are scarcely represented in the data. To focus on specific cell types, the current paradigm is to physically isolate subsets of interest prior to analysis; however, it remains difficult to isolate and then single-cell sequence such populations because of compounding losses. Here, we describe an alternative approach that selectively merges cells with reagents to achieve enzymatic reactions without having to physically isolate cells. We apply this technique to perform single-cell transcriptome and genome sequencing of specific cell subsets. Our method for analyzing heterogeneous populations obviates the need for pre- or post-enrichment and simplifies single-cell workflows, making it useful for other applications in single-cell biology, combinatorial chemical synthesis, and drug screening.  

4.2259           Selective Modulation of A1 Astrocytes by Drug-Loaded Nano-Structured Gel in Spinal Cord Injury

Vismara, I., Papa, S., Veneruso, V., Mauri, E., Mariani, A., De Paola, M., Affatato, R., Rosetti, A., Sponchioni, M., Moscatelli, D., Sacchetti, A., Rossi, F., Forloni, G. and Veglianese, P. ACS Nano, 14, 360-371 (2020)   Astrogliosis has a very dynamic response during the progression of spinal cord injury, with beneficial or detrimental effects on recovery. It is therefore important to develop strategies to target activated astrocytes and their harmful molecular mechanisms so as to promote a protective environment to counteract the progression of the secondary injury. The challenge is to formulate an effective therapy with maximum protective effects, but reduced side effects. In this study, a functionalized nanogel-based nanovector was selectively internalized in activated mouse or human astrocytes. Rolipram, an anti-inflammatory drug, when administered by these nanovectors limited the inflammatory response in A1 astrocytes, reducing iNOS and Lcn2, which in turn reverses the toxic effect of proinflammatory astrocytes on motor neurons in vitro, showing advantages over conventionally administered anti-inflammatory therapy. When tested acutely in a spinal cord injury mouse model, it improved motor performance, but only in the early stage after injury, reducing the astrocytosis and preserving neuronal cells.  

4.2260           Engineering E. coli for Magnetic Control and the Spatial Localization of Functions

Aubry, M., Wang, W-A., Guyodo, Y., Delacou, E., Guigner, J-M., Espeli, O., Lebreton, A., Guyot, F. and Gueroui, Z. ACS Synth. Biol., 9, 3030-3041 (2020)   The fast-developing field of synthetic biology enables broad applications of programmed microorganisms including the development of whole-cell biosensors, delivery vehicles for therapeutics, or diagnostic agents. However, the lack of spatial control required for localizing microbial functions could limit their use and induce their dilution leading to ineffective action or dissemination. To overcome this limitation, the integration of magnetic properties into living systems enables a contact-less and orthogonal method for spatiotemporal control. Here, we generated a magnetic-sensing Escherichia coli by driving the formation of iron-rich bodies into bacteria. We found that these bacteria could be spatially controlled by magnetic forces and sustained cell growth and division, by transmitting asymmetrically their magnetic properties to one daughter cell. We combined the spatial control of bacteria with genetically encoded-adhesion properties to achieve the magnetic capture of specific target bacteria as well as the spatial modulation of human cell invasions.  

4.2261           Nanoplasmon-enhanced drop-screen for high throughput single-cell nucleocytoplasmic miRNA profiling

Jia Liu, Guoyun Sun, Shih-Chung Wei, Song Guo, Weikang Nicholas Lin and Chia-Hung Che Lab. Chip, 20, 1939-1946 (2020)   Cell nucleocytoplasmic profiles of microRNAs (miRNAs) are critical to determining a single cell's essential functionalities, such as cellular transcription, nucleus export and degradation, which gives a comprehensive view of cellular processes. Despite the importance of addressing nucleocytoplasmic heterogeneity, the challenge of high-throughput screening remains. Although a droplet-based approach was developed for single-cell miRNA assays, the challenge of quantifying miRNA with high sensitivity to indicate nucleocytoplasmic heterogeneity remains. In this study, a nanoplasmon-enhanced droplet screening platform was developed to quantify single-cell nucleocytoplasmic heterogeneity with the high sensitivity of 0.1 nM. Droplet screening and multiplexed plasmonic assays are synergistic: droplet screening is used to isolate single cells for high-throughput screening, while enhanced nanoplasmonic assays are conducted to precisely determine different types of miRNAs, addressing the cell nucleocytoplasmic profile. Here, two nucleic acid-functionalized plasmonic nanosensors, silver nanoparticles functionalized with designed sequences to target miRNAs, are synthesized. After the targets are bound, competitive formation of sensor-target hybrids interferes with plasmonic coupling between the nanoparticles, decreasing a fluorescence signal and thus enabling high-sensitivity single-cell miRNA quantification. Using the fluorescence signal change as a readout allows continuous-flow measurement to provide a single-cell nucleocytoplasmic profile in a high-throughput manner (∼100 cells per minute) for effective quantitative cell biology.  

4.2262           Modeling ascending infection with a feto-maternal interface organ-on-chip

Richardson, L.S., Kim, S., Han, A. and Menon, R. Lab. Chip, 20, 4486-4501 (2020)   Maternal infection (i.e., ascending infection) and the resulting host inflammatory response are risk factors associated with spontaneous preterm birth (PTB), a major pregnancy complication. However, the path of infection and its propagation from the maternal side to the fetal side have been difficult to study due to the lack of appropriate in vitro models and limitations of animal models. A better understanding of the propagation kinetics of infectious agents and development of the host inflammatory response at the feto-maternal (amniochorion-decidua, respectively) interface (FMi) is critical in curtailing host inflammatory responses that can lead to PTB. To model ascending infection and determine inflammatory responses at the FMi, we developed a microfluidic organ-on-chip (OOC) device containing primary cells from the FMi (decidua, chorion, and amnion [mesenchyme and epithelium]) and collagen matrix harvested from primary tissue. The FMi-OOC is composed of four concentric circular cell/collagen chambers designed to mimic the thickness and cell density of the FMi in vivo. Each layer is connected by arrays of microchannels filled with type IV collagen to recreate the basement membrane of the amniochorion. Cellular characteristics (viability, morphology, production of nascent collagen, cellular transitions, and migration) in the OOC were similar to those seen in utero, validating the physiological relevance and utility of the developed FMi-OOC. The ascending infection model of the FMi-OOC, triggered by exposing the maternal (decidua) side of the OOC to lipopolysaccharide (LPS, 100 ng mL⁻¹), shows that LPS propagated through the chorion, amnion mesenchyme, and reached the fetal amnion within 72 h. LPS induced time-dependent and cell-type-specific pro-inflammatory cytokine production (24 h decidua: IL-6, 48 h chorion: GM-CSF and IL-6, and 72 h amnion mesenchyme and epithelium: GM-CSF and IL-6). Collectively, this OOC model and study successfully modeled ascending infection, its propagation, and distinct inflammatory response at the FMi indicative of pathologic pathways of PTB. This OOC model provides a novel platform to study physiological and pathological cell status at the FMi, and is expected to have broad utility in the field of obstetrics.  

4.2263           Microfluidics for label-free sorting of rare circulating tumor cells

Zhu, S., Jiang, F., Han, Y., Xiang, N. and Ni, Z. Analyst, 145, 7103-7124 (2020)   Circulating tumor cells (CTCs) have been widely considered as promising novel biomarkers for molecular research and clinical diagnosis of cancer. However, the sorting of CTCs is very challenging due to the rarity of CTCs in blood and the morphological similarity to blood cells. Although affinity-based CTC sorting methods could capture CTCs using specific biochemical markers, there are limitations such as the loss of cell viability after labeling and a requirement for expensive biochemical marker reagents. Emerging label-free CTC sorting methods rely on the physical properties of cells and can potentially overcome the aforementioned limitations. In this review, we highlight recent advances in label-free CTC sorting methods, with emphasis on device structures and performances. Specifically, we present a detailed discussion on label-free CTC sorting methods, including passive ones that depend on the channel structure or specific fluidic effects and active ones that use external force fields, as well as provide an overview of the principles, advantages, limitations, and applications of state-of-art label-free CTC sorting devices. Finally, we provide a future perspective of microfluidics for label-free CTC sorting and hope to inspire readers to develop new devices for applications in clinical cancer diagnoses and research.  

4.2264           Treatment of Full-Thickness Skin Wounds with Blood-Derived CD34+ Precursor Cells Enhances Healing with Hair Follicle Regeneration

Li, S., Hu, M. and Lorenz, H.P. Adv. Wound Care, 9(5), 264-276 (2020)   Objective: Epidermal CD34⁺ stem cells located in the hair follicle (HF) bulge area are capable of inducing HF neogenesis and enhancing wound healing after transplantation. In this study, we observed CD34⁺ cells derived from blood directly participate in dermal regeneration during full-thickness excisional wound healing. Approach: We isolated and in vitro expanded a subset of hematopoietic stem cell (HSC)-like precursor cells from the peripheral blood of adult mice with the surface markers: CD34⁺, leucine rich repeat containing G protein-coupled receptor 5 (LGR5)⁺, CD44⁺, c-kit⁺, lineage negative (lin⁻), and E-cadherin⁻. These blood-derived precursor cells (BDPCs), can be further differentiated into epithelial-like cells (eBDPCs) and secret fibroblast growth factor 9 (Fgf9) protein. Result: When transplanted into full-thickness skin wounds, eBDPC treatment produced accelerated healing and enhanced skin structure regeneration with less dermal scar formation. Also, HF neogenesis (HFN) was observed with incorporation of labeled BDPCs in the wound area.  

4.2265            An interspecies translation model implicates integrin signaling in infliximab-resistant inflammatory bowel disease

Brubaker, D.K., Kumar, M.P., Chiswick, E.L., Gregg, C., Starchenko, A., Vega, P.N., Southard-Smith, A.N., Simmons, A.J., Scoville, E.A., Coburn, L.A., Wilson, K.T., Lau, K.S. and lauffenberger, D.A. Sci.Signal., 13, eaay3258 (2020)   Anti–tumor necrosis factor (anti-TNF) therapy resistance is a major clinical challenge in inflammatory bowel disease (IBD), due, in part, to insufficient understanding of disease-site, protein-level mechanisms. Although proteomics data from IBD mouse models exist, data and phenotype discrepancies contribute to confounding translation from preclinical animal models of disease to clinical cohorts. We developed an approach called translatable components regression (TransComp-R) to overcome interspecies and trans-omic discrepancies between mouse models and human subjects. TransComp-R combines mouse proteomic data with patient pretreatment transcriptomic data to identify molecular features discernable in the mouse data that are predictive of patient response to therapy. Interrogating the TransComp-R models revealed activated integrin pathway signaling in patients with anti–TNF-resistant colonic Crohn’s disease (cCD) and ulcerative colitis (UC). As a step toward validation, we performed single-cell RNA sequencing (scRNA-seq) on biopsies from a patient with cCD and analyzed publicly available immune cell proteomics data to characterize the immune and intestinal cell types contributing to anti-TNF resistance. We found that ITGA1 was expressed in T cells and that interactions between these cells and intestinal cell types were associated with resistance to anti-TNF therapy. We experimentally showed that the α₁ integrin subunit mediated the effectiveness of anti-TNF therapy in human immune cells. Thus, TransComp-R identified an integrin signaling mechanism with potential therapeutic implications for overcoming anti-TNF therapy resistance. We suggest that TransComp-R is a generalizable framework for addressing species, molecular, and phenotypic discrepancies between model systems and patients to translationally deliver relevant biological insights.  

4.2266           Cross-sectional focusing of red blood cells in a constricted microfluidic channel

Abay, A., Recktenwald, S.M., John, T., Kaestner, L. and Wagner, C. Soft Matter, 16, 534-543 (2020)   Constrictions in blood vessels and microfluidic devices can dramatically change the spatial distribution of passing cells or particles and are commonly used in biomedical cell sorting applications. However, the three-dimensional nature of cell focusing in the channel cross-section remains poorly investigated. Here, we explore the cross-sectional distribution of living and rigid red blood cells passing a constricted microfluidic channel by tracking individual cells in multiple layers across the channel depth and across the channel width. While cells are homogeneously distributed in the channel cross-section pre-contraction, we observe a strong geometry-induced focusing towards the four channel faces post-contraction. The magnitude of this cross-sectional focusing effect increases with increasing Reynolds number for both living and rigid red blood cells. We discuss how this non-uniform cell distribution downstream of the contraction results in an apparent double-peaked velocity profile in particle image velocimetry analysis and show that trapping of red blood cells in the recirculation zones of the abrupt construction depends on cell deformability.  

4.2267           Arachidonate 12S-lipoxygenase of platelet-type in hepatic stellate cells of methionine and choline-deficient diet-fed mice

Mori, Y., Kawakami, Y., Kanzaki, K., Otsuki, A., Kimura, Y., Kanji, H., Tanaka, R., Tsukayama, I., Hojo, N., Suzuki-Yamamoto, T., Kawakami, T. and Takahashi, Y.

1. Biochem., 168(5), 455-463 (2020)

  A role of 12-lipoxygenase in the progression of non-alcoholic steatohepatitis (NASH) is suggested, although the underlying mechanism is not entirely understood. The catalytic activity of 12S-lipoxygenase that was hardly observed in liver cytosol of normal chow-fed mice was clearly detectable in that of NASH model mice prepared by feeding a methionine and choline-deficient (MCD) diet. The product profile, substrate specificity and immunogenicity indicated that the enzyme was the platelet-type isoform. The expression levels of mRNA and protein of platelet-type 12S-lipoxygenase in the liver of MCD diet-fed mice were significantly increased compared with those of normal chow-fed mice. Immunohistochemical analysis showed that platelet-type 12S-lipoxygenase colocalized with α-smooth muscle actin as well as vitamin A in the cells distributing along liver sinusoids. These results indicate that the expression level of platelet-type 12S-lipoxygenase in hepatic stellate cells was increased during the cell activation in MCD diet-fed mice, suggesting a possible role of the enzyme in pathophysiology of liver fibrosis.  

4.2268           In vivo stress granule misprocessing evidenced in a FUS knock-in ALS mouse model

Zhang, X., Wang, F., Hu, Y., Chen, R., Meng, D., Guo, L., Lv, H., Guan, J. and Jia, Y. Brain, 143, 1350-1367 (2020)   Many RNA-binding proteins, including TDP-43, FUS, and TIA1, are stress granule components, dysfunction of which causes amyotrophic lateral sclerosis (ALS). However, whether a mutant RNA-binding protein disrupts stress granule processing in vivo in pathogenesis is unknown. Here we establish a FUS ALS mutation, p.R521C, knock-in mouse model that carries impaired motor ability and late-onset motor neuron loss. In disease-susceptible neurons, stress induces mislocalization of mutant FUS into stress granules and upregulation of ubiquitin, two hallmarks of disease pathology. Additionally, stress aggravates motor performance decline in the mutant mouse. By using two-photon imaging in TIA1-EGFP transduced animals, we document more intensely TIA1-EGFP-positive granules formed hours but cleared weeks after stress challenge in neurons in the mutant cortex. Moreover, neurons with severe granule misprocessing die days after stress challenge. Therefore, we argue that stress granule misprocessing is pathogenic in ALS, and the model we provide here is sound for further disease mechanistic study.  

4.2269           Single-cell RNA sequencing identifies senescent cerebromicrovascular endothelial cells in the aged mouse brain

Kiss, T., Nyul-Toth, A., Balassubramanian, P., Tarantini, S., Ahire, C., DelFavero, J., Yabluchanskiy, A., Csipo, T., Farkas, E., Wiley, G., Garman, l., Csisar, A. and Ungvari, Z. GeroScience, 42, 429-444 (2020)   Age-related phenotypic changes of cerebromicrovascular endothelial cells lead to dysregulation of cerebral blood flow and blood-brain barrier disruption, promoting the pathogenesis of vascular cognitive impairment (VCI). In recent years, endothelial cell senescence has emerged as a potential mechanism contributing to microvascular pathologies opening the avenue to the therapeutic exploitation of senolytic drugs in preclinical studies. However, difficulties with the detection of senescent endothelial cells in wild type mouse models of aging hinder the assessment of the efficiency of senolytic treatments. To detect senescent endothelial cells in the aging mouse brain, we analyzed 4233 cells in fractions enriched for cerebromicrovascular endothelial cells and other cells associated with the neurovascular unit obtained from young (3-month-old) and aged (28-month-old) C57BL/6 mice. We define 13 transcriptomic cell types by deep, single-cell RNA sequencing. We match transcriptomic signatures of cellular senescence to endothelial cells identified on the basis of their gene expression profile. Our study demonstrates that with advanced aging, there is an increased ratio of senescent endothelial cells (~ 10%) in the mouse cerebral microcirculation. We propose that our single-cell RNA sequencing–based method can be adapted to study the effect of aging on senescence in various brain cell types as well as to evaluate the efficiency of various senolytic regimens in multiple tissues.  

4.2270           Nicotinamide mononucleotide (NMN) supplementation promotes neurovascular rejuvenation in aged mice: transcriptional footprint of SIRT1 activation, mitochondrial protection, anti-inflammatory, and anti-apoptotic effectsss, T., Nyul-Toth, A., Balasubramanian, P., Tarantini, S., Ahire, C., Yabluchanskiy, A., Csipo, T., Farkas, E., Wren, J.D., Garman, L., Csiszar, A. and Ungvari, Z.

GeroScience, 42, 527-546 (2020)   Aging-induced structural and functional alterations of the neurovascular unit lead to impairment of neurovascular coupling responses, dysregulation of cerebral blood flow, and increased neuroinflammation, all of which contribute importantly to the pathogenesis of age-related vascular cognitive impairment (VCI). There is increasing evidence showing that a decrease in NAD⁺ availability with age plays a critical role in age-related neurovascular and cerebromicrovascular dysfunction. Our recent studies demonstrate that restoring cellular NAD⁺ levels in aged mice rescues neurovascular function, increases cerebral blood flow, and improves performance on cognitive tasks. To determine the effects of restoring cellular NAD⁺ levels on neurovascular gene expression profiles, 24-month-old C57BL/6 mice were treated with nicotinamide mononucleotide (NMN), a key NAD⁺ intermediate, for 2 weeks. Transcriptome analysis of preparations enriched for cells of the neurovascular unit was performed by RNA-seq. Neurovascular gene expression signatures in NMN-treated aged mice were compared with those in untreated young and aged control mice. We identified 590 genes differentially expressed in the aged neurovascular unit, 204 of which are restored toward youthful expression levels by NMN treatment. The transcriptional footprint of NMN treatment indicates that increased NAD⁺ levels promote SIRT1 activation in the neurovascular unit, as demonstrated by analysis of upstream regulators of differentially expressed genes as well as analysis of the expression of known SIRT1-dependent genes. Pathway analysis predicts that neurovascular protective effects of NMN are mediated by the induction of genes involved in mitochondrial rejuvenation, anti-inflammatory, and anti-apoptotic pathways. In conclusion, the recently demonstrated protective effects of NMN treatment on neurovascular function can be attributed to multifaceted sirtuin-mediated anti-aging changes in the neurovascular transcriptome. Our present findings taken together with the results of recent studies using mitochondria-targeted interventions suggest that mitochondrial rejuvenation is a critical mechanism to restore neurovascular health and improve cerebral blood flow in aging.  

4.2271           Comprehensive characterization of hepatocyte-derived extracellular vesicles identifies direct miRNA-based regulation of hepatic stellate cells and DAMP-based hepatic macrophage IL-1β and IL-17 upregulation in alcoholic hepatitis mice

Eguchi, A., Yan, R., Pan, S.Q., Wu, R., Kim, J., Chen, Y., Ansong, C., Smith, R.D., Tempaku, M., Ohno-Marchado, L., takei, Y., Feldstein, A.E. and Tsukamoto, H.

1. Mol. Med., 98, 1021-1034 (2020)

  Extracellular vesicles (EVs) have been growingly recognized as biomarkers and mediators of alcoholic liver disease (ALD) in human and mice. Here we characterized hepatocyte-derived EVs (HC-EVs) and their cargo for their biological functions in a novel murine model that closely resembles liver pathology observed in patients with alcoholic hepatitis (AH), the most severe spectrum of ALD. The numbers of circulating EVs and HC-EVs were significantly increased by 10-fold in AH mice compared with control mice. The miRNA (miR)–seq analysis detected 20 upregulated and 4 downregulated miRNAs (P < 0.001–0.05) in AH-HC-EVs. Treatment of murine primary hepatic stellate cells (HSCs) with AH-HC-EVs induced α-SMA (P < 0.05) and Col1a1 (P < 0.001). Smad7 and Nr1d2 genes, which were downregulated in HSCs from the AH mice, were predicted targets of 20 miRs upregulated in AH-HC-EVs. Among them were miR-27a and miR-181 which upon transfection in HSCs, indeed repressed Nr1d2, the quiescent HSC marker. AH-HC-EVs were also enriched with organelle proteins and mitochondrial DNA (10-fold, P < 0.05) and upregulated IL-1β and IL-17 production by hepatic macrophages (HMs) from AH mice in a TLR9-dependent manner. These results demonstrate HC-EV release is intensified in AH and suggest that AH-HC-EVs orchestrate liver fibrogenesis by directly targeting the quiescent HSC transcripts via a unique set of miRNAs and by amplifying HSC activation via DAMP-based induction of profibrogenic IL-1β and IL-17 by HMs.  

4.2272           An analysis of monocytes and dendritic cells differentiated from human peripheral blood monocyte-derived induced pluripotent stem cells

Hiramatsu, N., Yamamoto, N., Isogai, S., Onouchi, T., Hirayama, M., Maeda, S., Ina, T., Kondo, M. and Imaizumi, K. Med. Mol. Morph., 53, 63-72 (2020)   Dendritic cell-based immunotherapy, which uses a patient's own immune cells, can be used for cancer treatment and allergy control, such as autoimmune disease and rejection associated with transplantation. However, these treatments create a burden on patients due to repeated blood collection. We used cell biological analysis of monocytes with few mutations obtained from minimal blood collection for genome recombination. Next, we established human peripheral blood monocyte-derived induced pluripotent stem cells (iPSCs) using a commercial vector and standard culture method. We found that when established iPSCs were induced to differentiate, monocytes showed phagocytic properties and expressed CD14 and CX3CR1. Further, the generated dendritic cells (DCs) expressed CCL17 and highly expressed HLA-DR following the addition of the mite antigen. Taken together, these data show that monocyte-derived iPS cells can be used to differentiate into monocytes and DCs. In addition, the use of these cells can be applied to the pathological analysis of dendritic cell therapy and monocyte diseases.  

4.2273           Trisected pancreas model for testing tissue dissociation enzyme combinations: a novel methodology for improving human islet yield for clinical islet transplantation

Loganathan, G., Venugopal, S. and Balamurugan, A.N.

1. Diabetes & Metabolic Disorders, 19, 381-389 (2020)

  Purpose Human islet isolation requires a defined collagenase-protease enzyme combination for obtaining a successful islet yield. While different islet laboratories use different enzyme combinations, a systematic methodology to identify optimal enzyme combinations and their concentrations within a single donor pancreas has not been tested. In this study, we designed a trisected pancreas model to test efficacy of three clinical grade enzyme blends (VitaCyte, Roche, SERVA) within a single pancreas. Methods Islet isolations were performed using brain-dead donor pancreases (n = 15) applying the enzyme-related design of experiments (DOEs) and the trisected model approach. After trimming, split each pancreas into three individual lobes (head, body, tail). As per the DOEs, the lobes were altered between different experiments, to minimize anatomical bias. Islets isolated from each lobe (27 lobes totally) were subjected to functional assessments. Insulin staining and islet area fraction were determined for tissue sections obtained from each lobe. Results Utilizing the trisected model, we identified that the collagenase dose from three different vendors did not affect the pancreas digestion and islet yield, but islet morphology after isolation with the neutral protease and BP-protease was better than thermolysin. In addition, the head lobe yielded a lower islet mass and higher tissue volume compared to other two lobes, irrespective of enzyme combination used. Conclusions This study demonstrates that the trisected model is a promising methodology in assessing donor and isolation associated parameters. Based on this study, we conclude that the donor characteristics and an optimal enzyme dose play a critical role in achieving higher islet yields.  

4.2274           Pancreas vs. Islet Transplantation: the False Dilemma

Wijkstrom, M. Current Transplant. Reports, 7, 230-236 (2020)   Purpose of Review Pancreas and islet cell transplantation are the only treatment modalities that can restore euglycemia and allow freedom from hypoglycemic events. Transplantation for diabetes has decreased in the USA over the last 6 years due to several factors. Reasons for this and possible future paths will be explored. Recent Findings Clinical application of islet transplantation is mostly limited by regulatory hurdles in the USA. The limitations of pancreas transplantation are multifactorial and more nuanced. Both types of transplants are limited by the number of suitable organ donors; however, pancreas and islet transplantation do not compete for the same organs. Only 10.7% of available pancreata are currently being utilized for clinical application. Summary Current technology if utilized more efficiently could significantly increase the available donor pancreata for both whole organ and islets. Considering that severely diseased pancreata are routinely processed for islets and subsequent transplanted in chronic pancreatitis patients, and have significant metabolic impact in these patients, there is an opportunity to widen the organ use beyond what is currently done. Optimizing access to β-cell replacement therapy has significant potential to improve outcomes for carefully selected patients.  

4.2275           The peripheral CB1 receptor antagonist JD5037 attenuates liver fibrosis via a CB1 receptor/β-arrestin1/Akt pathway

Tan, S., Liu, H., Ke, B., Jiang, J. and Wu, B. Br. J. Pharmacol., 177(12), 2830-2847 (2020)   Background and Purpose Liver fibrosis is a serious cause of morbidity and mortality worldwide and has no adequate treatment. Accumulating evidence suggests that cannabinoid CB₁ receptors regulate a variety of physiological and pathological processes in the liver, and blockage of CB₁ receptor signalling shows promise as a new therapy for several liver diseases. The aim of this study was to investigate the potential therapeutic effects of CB₁ receptors and a peripheral CB₁ receptor antagonist JD5037 in liver fibrogenesis. Experimental Approach Liver samples from both humans and mouse models were investigated. The peripheral CB₁ receptor antagonist JD5037, β-arr1 wild type (β-arr1-WT) and β-arr1 knockout (β-arr1-KO) littermate models, and primary hepatic stellate cells (HSCs) were also used. The mechanisms underlying CB₁ receptor-regulated HSCs activation in fibrosis and the therapeutic potential of JD5037 were further analysed. Key Results CB₁ receptors were induced in samples from patients with liver fibrosis and from mouse models. These receptors promoted activation of HSCs in liver fibrosis via recruiting β-arrestin1 and Akt signalling, while blockage of CB₁ receptors with JD5037 attenuated CB₁ receptor-regulated HSCs activation and liver fibrosis by suppressing β-arrestin1/Akt signalling. Conclusions and Implications CB₁ receptors promote the activation of HSCs and liver fibrosis via the β-arrestin1/Akt signalling pathway. The peripheral CB₁ receptor antagonist JD5037 blocked this pathway, the activation of HSCs and liver fibrosis. This compound and the associated pathway may be a novel approach to the treatment of liver fibrosis.    

4.2276           Susceptibility of Rat Steatotic Liver to Ischemia–Reperfusion Is Treatable With Liver-Selective Matrix Metalloproteinase Inhibition

Wang, X., Walkey, C., Maretti-Mira, A.C., Wang, L., Johnson, D.L. and DeLeve, L.D. Hepatology, 72(5), 1771-1785 (2020)   Background and Aims This study examined whether enhanced susceptibility of steatotic liver to ischemia–reperfusion (I/R) injury is due to impaired recruitment of bone marrow (BM) progenitors of liver sinusoidal endothelial cells (LSECs, also called sinusoidal endothelial cell progenitor cells [sprocs]) with diminished repair of injured LSECs and whether restoring signaling to recruit BM sprocs reduces I/R injury. Approach and Results Hepatic vessels were clamped for 1 hour in rats fed a high-fat, high-fructose (HFHF) diet for 5, 10, or 15 weeks. Matrix metalloproteinase 9 (MMP-9) antisense oligonucleotides (ASO) or an MMP inhibitor were used to induce liver-selective MMP-9 inhibition. HFHF rats had mild, moderate, and severe steatosis, respectively, at 5, 10, and 15 weeks. I/R injury was enhanced in HFHF rats; this was accompanied by complete absence of hepatic vascular endothelial growth factor (VEGF)–stromal cell–derived factor 1 (sdf1) signaling, leading to lack of BM sproc recruitment. Liver-selective MMP-9 inhibition to protect against proteolytic cleavage of hepatic VEGF using either MMP-9 ASO or intraportal MMP inhibitor in 5-week and 10-week HFHF rats enhanced hepatic VEGF–sdf1 signaling, increased BM sproc recruitment, and reduced alanine aminotransferase (ALT) by 92% and 77% at 5 weeks and by 80% and 64% at 10 weeks of the HFHF diet, respectively. After I/R injury in 15-week HFHF rats, the MMP inhibitor reduced active MMP-9 expression by 97%, ameliorated histologic evidence of injury, and reduced ALT by 58%, which is comparable to control rats sustaining I/R injury. Rescue therapy with intraportal MMP inhibitor, given after ischemia, in the 5-week HFHF rat reduced ALT by 71% and reduced necrosis. Conclusions Lack of signaling to recruit BM sprocs that repair injured LSECs renders steatotic liver more susceptible to I/R injury. Liver-selective MMP-9 inhibition enhances VEGF–sdf1 signaling and recruitment of BM sprocs, which markedly protects against I/R injury, even in severely steatotic rats.  

4.2277           River biofilms adapted to anthropogenic disturbances are more resistant to WWTP inputs

Freixa, A., Perujo, N., Langenheder, S. and Romani, A.M. FEMS Microbiol. Ecol., 96, fiaa152 (2020)   The sensitivity and spatial recovery of river sediment biofilms along 1 km after the input of two wastewater treatment plants (WWTPs) located in two river reaches with different degrees of anthropogenic influence were investigated. First, at the upper reach, we observed an inhibition of some microbial functions (microbial respiration and extracellular enzyme activities) and strong shifts in bacterial community composition (16S rRNA gene), whereas an increase in microbial biomass and activity and less pronounced effect on microbial diversity and community composition were seen at the lower reach. Second, at the lower reach we observed a quick spatial recovery (around 200 m downstream of the effluent) as most of the functions and community composition were similar to those from reference sites. On the other hand, bacterial community composition and water quality at the upper reach was still altered 1 km from the WWTP effluent. Our results indicate that biofilms in the upstream sites were more sensitive to the effect of WWTPs due to a lower degree of tolerance after a disturbance than communities located in more anthropogenically impacted sites.  

4.2278           Myelin-associated glycoprotein inhibits neurite outgrowth through inactivation of the small GTPase Rap1

Nikulina, E., Gkioka, V., Siddiq, M.M., Mellado, W., Hilaire, M., Cain, C.R., Hannila, S.S. and Filbin, M.T. FEBS Lett., 594(9), 1389-1402 (2020)   Rap1 is a small GTPase that has been implicated in dendritic development and plasticity. In this study, we investigated the role of Rap1 in axonal growth and its activation in response to neurotrophins and myelin-associated inhibitors. We report that Rap1 is activated by brain-derived neurotrophic factor and that this activation can be blocked by myelin-associated glycoprotein (MAG) or central nervous system myelin, which also induced increases in Rap1GAP1 levels. In addition, we demonstrate that adenoviral overexpression of Rap1 enhances neurite outgrowth in the presence of MAG and myelin, while inhibition of Rap1 activity through overexpression of Rap1GAP1 blocks neurite outgrowth. These findings suggest that Rap1GAP1 negatively regulates neurite outgrowth, making it a potential therapeutic target to promote axonal regeneration.  

4.2279           Evaluation of synergic effects of iodixanol and trehalose on cryosurvival of electroejaculated ram semen

Özmen, M., Cirit, U., Arici, R., Demir, K., Kurt, D., Pabuccuoglu, S. and Ak, K. Andrologia, 52(9), e13656 (2020)   The primary aim of the study was to investigate whether iodixanol and trehalose would have a synergic effect on the cryosurvival of electroejaculated ram semen. Tris-based diluter was used to prepare 9 different extenders by the addition of iodixanol or trehalose alone or varying combinations of these substances. Diluters were prepared as follows: Tris (control), Io5 (5% iodixanol), Tr25 (25 mmol/L trehalose), Tr50 (50 mmol/L trehalose), Tr50 + Io1.25 (50 mmol/L trehalose and 1.25% iodixanol), Tr50 + Io2.5 (50 mmol/L trehalose and 2.5% iodixanol), Tr50 + Io5 (50 mmol/L trehalose and 5% iodixanol), Tr25 + Io5 (25 mmol/L trehalose and 5% iodixanol) and Tr12.5 + Io5 (12.5 mmol/L trehalose and 5% iodixanol). Supplementation of the freezing extender with trehalose or iodixanol alone supported the protection of both morphological and functional integrity of ram spermatozoa and total motility at 1 and 4 hr post-thawing respectively. However, beyond these positive effects, the combination of trehalose (25 mmol/L) and iodixanol (5%) significantly increased post-thaw sperm longevity and motion properties at the end of 4-hr incubation. The results of the study clearly showed that there was positive synergic effect of iodixanol and trehalose on cryosurvival of ram semen.  

4.2280           Single-Cell RNA Sequencing Reveals Stromal Evolution into LRRC15+ Myofibroblasts as a Determinant of Patient Response to Cancer Immunotherapy

Dominguez, C.X., Müller, S., Keerthivasan, S., Koeppen, H., Hung, J., Gierke, S., Breart, B. et al Cancer Discov., 10, 232-253 (2020)   With only a fraction of patients responding to cancer immunotherapy, a better understanding of the entire tumor microenvironment is needed. Using single-cell transcriptomics, we chart the fibroblastic landscape during pancreatic ductal adenocarcinoma (PDAC) progression in animal models. We identify a population of carcinoma-associated fibroblasts (CAF) that are programmed by TGFβ and express the leucine-rich repeat containing 15 (LRRC15) protein. These LRRC15⁺ CAFs surround tumor islets and are absent from normal pancreatic tissue. The presence of LRRC15⁺ CAFs in human patients was confirmed in >80,000 single cells from 22 patients with PDAC as well as by using IHC on samples from 70 patients. Furthermore, immunotherapy clinical trials comprising more than 600 patients across six cancer types revealed elevated levels of the LRRC15⁺ CAF signature correlated with poor response to anti–PD-L1 therapy. This work has important implications for targeting nonimmune elements of the tumor microenvironment to boost responses of patients with cancer to immune checkpoint blockade therapy.  

4.2281           On-Demand Droplet Collection for Capturing Single Cells

Nan, L., Lai, M.Y.A., Tang, Y.H., Chan, Y.K., Poon, L.L.M. and Shum, H.C. Small, 16, 1902889 (2020)   Droplet-based microfluidic techniques are extensively used in efficient manipulation and genome-wide analysis of individual cells, probing the heterogeneity among populations of individuals. However, the extraction and isolation of single cells from individual droplets remains difficult due to the inevitable sample loss during processing. Herein, an automated system for accurate collection of defined numbers of droplets containing single cells is presented. Based on alternate sorting and dispensing in three branch channels, the droplet number can be precisely controlled down to single-droplet resolution. While encapsulating single cells and reserving one branch as a waste channel, sorting can be seamlessly integrated to enable on-demand collection of single cells. Combined with a lossless recovery strategy, this technique achieves capture and culture of individual cells with a harvest rate of over 95%. The on-demand droplet collection technique has great potential to realize quantitative processing and analysis of single cells for elucidating the role of cell-to-cell variations.  

4.2282           Irradiated and CCl4-treated bone marrow-derived liver macrophages exhibit different gene expression patterns and phenotypes

Fan, X., Shan, S., Wu, P., Lin, W., Jia, J. and He, F. Scand. J. Immunol., 92, e12916 (2020)   Myeloid cells infiltrate into the liver and differentiate into macrophages in different liver injury mouse models. However, the heterogeneity of bone marrow (BM)-derived LMs populations remains to be understood. To investigate this and understand the impact of the macrophage niche on the properties of recruited BM-derived macrophages, we used a non-myeloablation BM transplantation model to label and trace BM-derived LMs. Subsequently, we quantified the number of embryonic-derived liver-resident macrophages, BM-derived LMs and total LMs in CCl₄ and irradiated acute liver injury mouse models, respectively. Finally, we compared the cell fate, gene expression patterns, chemokine signals, and surface markers of irradiated and CCl₄-treated BM-derived LMs. We observed that, as compared to CCl_4, radiation generated a macrophage niche by depleting embryonic-derived liver-resident macrophages and induced the recruitment of BM-derived LMs that further settled in the liver. Irradiated and CCl₄-treated BM-derived LMs are different with respect to their cell fates, gene expression patterns, and chemokine expression and recruitment. They also have different surface markers shortly after differentiating from their progenitors. Our findings suggest that irradiated and CCl₄-treated LM populations derived from the bone marrow display different patterns of gene expression and phenotypes; these differences may be due to the availability of macrophage niche.  

4.2283           Rapid remote conditioning mediates modulation of blood cell paracrine activity and leads to the production of a secretome with neuroprotective features

Bonova, P., Jachova, J., Nemethova, M., Macakova, L., Bona, M. and Gottlieb, M.

1. Neurochem., 154, 99-111 (2020)

  The indirect use of the protective potential of stem cells in the form of cell secretomes has become an attractive strategy in regenerative medicine. In the present work, we studied the paracrine activity of blood cells that could be modulated towards a neuroprotective nature using in vivo remote conditioning (i.e. tolerant blood cells). The increased neuronal survival mediated by the tolerant secretome was clearly confirmed in vitro in a model of glutamate toxicity in a primary culture of rat cortical neurons and in vivo in a pre- and post-treatment of rats that were subjected to transient occlusion of the middle cerebral artery. Bioinformatic-based analysis of the protein profile revealed higher amounts of proteins released by the tolerant blood cells; 29 proteins were recognised as secreted. More than half of these secreted proteins were involved in the biological processes of the response to the stimulus (GO:0050896) and the response to chemicals (GO:0042221). The protective phenotype was most likely mediated by the synergistic effect of multiple identified proteins, including unique to the tolerant secretome (ceruloplasmin, D-3-phosphoglycerate dehydrogenase) and was promoted by the co-participation of several reaction pathways. The most probably of these pathways were post-translation protein modification, MAP2K and MAPK activation and platelet activation. Taken together, our results demonstrate that properly stimulated blood cells could serve as a source for cell-free-based therapies of regenerative medicine.  

4.2284           Impact of microbial contamination of the islet product during total pancreatectomy with islet autotransplantation

Kumano, K., Takita, M., Vasu, S., Darden, C., lawrence, M., Beecherl, E., Gupta, A., Onaca, N. and Naziruddin, B.

1. Hepatobiliary Pancreat. Sci., 27, 211-218 (2020)

  Background The combined use of interleukin-1β and tumor necrosis factor–α blockers in the peritransplant period has improved outcomes of total pancreatectomy with islet autotransplantation (TPIAT). However, these drugs may suppress the immune system, resulting in severe infection. Methods We retrospectively investigated the impact of microbial-contaminated islet product on posttransplant complications and metabolic outcomes of TPIAT patients receiving the IL-1β and TNF-blockade treatment at our center. Results Among 108 TPIAT patients, 37 patients (34%) received contaminated products. Preoperative stent treatment and fibrosis score were independent risk factors for the contamination. There were no significant differences between the contaminated and noncontaminated product groups in posttransplant infectious complication rate, length of hospitalization, or readmission rate. However, islet equivalents (P < .0001) and insulin independence rate (P = .036) at 6 months were significantly lower for patients receiving contaminated product. Conclusions These results suggest that combined anti-inflammatory drug use is safe and well tolerated in TPIAT patients who receive contaminated islet product and does not increase the rate of infectious complications; however, contaminated islet product is associated with poor metabolic outcomes.  

4.2285           Current situation of clinical islet transplantation from allogeneic toward xenogeneic

Matsumoto, S. and Shimoda, M.

1. Diabetes, 12(10), 733-741 (2020)

  Currently, type 1 diabetes requires lifelong insulin injection and careful blood glucose control to prevent secondary complications, but islet transplantation could make a type 1 diabetic patient insulin independent. On the other hand, islet transplantation needs human donors and donor shortage is the most serious issue. To alleviate the donor shortage, non-heart-beating and living donors were used; in addition, the efficacy of islet isolation and transplantation has been improved. However, the donor shortage issue will not be solved as long as human donors are the only source.   To solve the donor shortage issue, islet xenotransplantation using porcine islets was initiated in 1994. Islet xenotransplantation has a potential to cure many type 1 diabetic patients, although there is the risk of developing serious or novel infection. Therefore, the World Health Organization has been interested in xenotransplantation, and the International Xenotransplantation Association (IXA) has published consensus statements to initiate xenogeneic islet transplantation. Clinical islet xenotransplantation was conducted under the official regulation, and safety and efficacy data have been accumulated. Currently an efficient method to overcome xenorejection is an important research target. In addition to traditional immunosuppressive drugs and immune isolation methods, the gene modification with CRISPR and blastocyst complementation have been investigated with promising outcomes. Once the xenorejection issue is overcome, islet xenotransplantation should become a curative treatment for type 1 diabetic patients.  

4.2286           Dendritic cell-derived TGF-β mediates the induction of mucosal regulatory T-cell response to Helicobacter infection essential for maintenance of immune tolerance in mice

Owyang, S.Y., Zhang, M., El-Zaatari, M., Eaton, K.A., Bishu, S., Hou, G., Grasberger, H. and Kao, J.Y. Helicobacter, 25, e12763 (2020)   Background Helicobacter pylori infection leads to regulatory T-cell (Treg) induction in infected mice, which contributes to H. pylori immune escape. However, the mechanisms responsible for H. pylori induction of Treg and immune tolerance remain unclear. We hypothesized DC-produced TGF-β may be responsible for Treg induction and immune tolerance. Materials and Methods To test this hypothesis, we generated TGF-β^∆DC mice (CD11c⁺ DC-specific TGF-β deletion) and assessed the impact of DC-specific TGF-β deletion on DC function during Helicobacter infection in vitro and in vivo. To examine the T cell–independent DC function, we crossed TGF-β^∆DC mice onto Rag1KO background to generate TGF-β^∆DCxRag1KO mice. Results When stimulated with H. pylori, TGF-β^∆DC BMDC/splenocyte cocultures showed increased levels of proinflammatory cytokines and decreased levels of anti-inflammatory cytokines compared to control, indicating a proinflammatory DC phenotype. Following 6 months of H. felis infection, TGF-β^∆DC mice developed more severe gastritis and a trend toward more metaplasia compared to TGF-β^fl/fl with increased levels of inflammatory Th1 cytokine mRNA and lower gastric H. felis colonization compared to infected TGF-β^fl/fl mice. In a T cell–deficient background using TGF-β^∆DCxRag1KO mice, H. felis colonization was significantly lower when DC-derived TGF-β was absent, revealing a direct, innate function of DC in controlling H. felis infection independent of Treg induction. Conclusions Our findings indicate that DC-derived TGF-β mediates Helicobacter-induced Treg response and attenuates the inflammatory Th1 response. We also demonstrated a previously unrecognized innate role of DC controlling Helicobacter colonization via a Treg-independent mechanism. DC TGF-β signaling may represent an important target in the management of H. pylori.  

4.2287           Fasting- and ghrelin-induced food intake is regulated by NAMPT in the hypothalamus

De Guia, R.M., Hassing, A.S., Skov, L.J., Ratner, C., Plucinska, K., Madsen, S., Diep, T.A., Dela Cruz, G.V., Trammell, S.A.J., Sustarsic, E.G., Emanuelli, B., Gillum, M.P., Gerhart-Hines, Z., Holst, B. and Treebak, J.T: Acta Physiologica, 228, e13437 (2020)   Aim Neurons in the arcuate nucleus of the hypothalamus are involved in regulation of food intake and energy expenditure, and dysregulation of signalling in these neurons promotes development of obesity. The role of the rate-limiting enzyme in the NAD⁺ salvage pathway, nicotinamide phosphoribosyltransferase (NAMPT), for regulation energy homeostasis by the hypothalamus has not been extensively studied. Methods We determined whether Nampt mRNA or protein levels in the hypothalamus of mice were affected by diet-induced obesity, by fasting and re-feeding, and by leptin and ghrelin treatment. Primary hypothalamic neurons were treated with FK866, a selective inhibitor of NAMPT, or rAAV carrying shRNA directed against Nampt, and levels of reactive oxygen species (ROS) and mitochondrial respiration were assessed. Fasting and ghrelin-induced food intake was measured in mice in metabolic cages after intracerebroventricular (ICV)-mediated FK866 administration. Results NAMPT levels in the hypothalamus were elevated by administration of ghrelin and leptin. In diet-induced obese mice, both protein and mRNA levels of NAMPT decreased in the hypothalamus. NAMPT inhibition in primary hypothalamic neurons significantly reduced levels of NAD⁺, increased levels of ROS, and affected the expression of Agrp, Pomc and genes related to mitochondrial function. Finally, ICV-induced NAMPT inhibition by FK866 did not cause malaise or anhedonia, but completely ablated fasting- and ghrelin-induced increases in food intake. Conclusion Our findings indicate that regulation of NAMPT levels in hypothalamic neurons is important for the control of fasting- and ghrelin-induced food intake.  

4.2288           Antibody-induced NKG2D blockade in a rat model of intraportal islet transplantation leads to a deleterious reaction

Delaune, V., Toso, C., Kahler-Quesada, A., Slits, F., Gex, Q., Kaya, G., lavallard, V., Orci, L.A., Peloso, A. and Lacotte, S. Transplant. Int., 33(6), 675-688 (2020)   Intraportal islet transplantation is plagued by an acute destruction of transplanted islets. Amongst the first responders, NK cells and macrophages harbour an activating receptor, NKG2D, recognizing ligands expressed by stressed cells. We aimed to determine whether islet NKG2D ligand expression increases with culture time, and to analyse the impact of antibody-induced NKG2D blockade in islet transplantation. NKG2D-ligand expression was analysed in rat and human islets. Syngeneic marginal mass intraportal islet transplantations were performed in rats: control group, recipients transplanted with NKG2D-recombinant-treated islets (recombinant group), and recipients treated with a mouse anti-rat anti-NKG2D antibody and transplanted with recombinant-treated islets (antibody-recombinant group). Islets demonstrated increased gene expression of NKG2D ligands with culture time. Blockade of NKG2D on NK cells decreased in vitro cytotoxicity against islets. Recipients from the control and recombinant groups showed similar metabolic results; conversely, treatment with the antibody resulted in lower diabetes reversal. The antibody depleted circulating and liver NK cells in recipients, who displayed increased macrophage infiltration of recipient origin around the transplanted islets. In vitro blockade of NKG2D ligands had no impact on early graft function. Systemic treatment of recipients with an anti-NKG2D antibody was deleterious to the islet graft, possibly through an antibody-dependent cell-mediated cytotoxicity reaction.  

4.2289           Production of the Cytokine VEGF-A by CD4+ T and Myeloid Cells Disrupts the Corneal Nerve Landscape and Promotes Herpes Stromal Keratitis

Yun, H., Yee, M.B., Lathrop, K.L., Kinchington, P.R., Hendricks, R.L. and Leger, A.J. Immunity, 53, 1050-1062 (2020)   Herpes simplex virus type 1 (HSV-1)-infected corneas can develop a blinding immunoinflammatory condition called herpes stromal keratitis (HSK), which involves the loss of corneal sensitivity due to retraction of sensory nerves and subsequent hyperinnervation with sympathetic nerves. Increased concentrations of the cytokine VEGF-A in the cornea are associated with HSK severity. Here, we examined the impact of VEGF-A on neurologic changes that underly HSK using a mouse model of HSV-1 corneal infection. Both CD4⁺ T cells and myeloid cells produced pathogenic levels of VEGF-A within HSV-1-infected corneas, and CD4⁺ cell depletion promoted reinnervation of HSK corneas with sensory nerves. In vitro, VEGF-A from infected corneas repressed sensory nerve growth and promoted sympathetic nerve growth. Neutralizing VEGF-A in vivo using bevacizumab inhibited sympathetic innervation, promoted sensory nerve regeneration, and alleviated disease. Thus, VEGF-A can shape the sensory and sympathetic nerve landscape within the cornea, with implications for the treatment of blinding corneal disease.  

4.2290           Vitamin D Receptor Activation in Liver Macrophages Ameliorates Hepatic Inflammation, Steatosis, and Insulin Resistance in Mice

Dong, B., Zhou, Y., Wang, W., Scott, J., Kim, K, Sun, Z., Guo, Q., Lu, Y., Gonzales, N.M., Wu, H., Hartig, A.M., York, R.B., Yang, F. and Moore, D.D. Hepatology, 71(5), 1559-1574 (2020)   Background and Aims Obesity-induced chronic inflammation is a key component in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and insulin resistance. Increased secretion of proinflammatory cytokines by macrophages in metabolic tissues promotes disease progression. In the diet-induced obesity (DIO) mouse model, activation of liver resident macrophages, or Kupffer cells (KCs), drives inflammatory responses, which recruits circulating macrophages and promotes fatty liver development, and ultimately contributes to impaired hepatic insulin sensitivity. Hepatic macrophages express the highest level of vitamin D receptors (VDRs) among nonparenchymal cells, whereas VDR expression is very low in hepatocytes. VDR activation exerts anti-inflammatory effects in immune cells. Approach and Results Here we found that VDR activation exhibits strong anti-inflammatory effects in mouse hepatic macrophages, including those isolated from DIO livers, and mice with genetic loss of Vdr developed spontaneous hepatic inflammation at 6 months of age. Under the chronic inflammation conditions of the DIO model, VDR activation by the vitamin D analog calcipotriol reduced liver inflammation and hepatic steatosis, significantly improving insulin sensitivity. The hyperinsulinemic euglycemic clamp revealed that VDR activation greatly increased the glucose infusion rate, while hepatic glucose production was remarkably decreased. Glucose uptake in muscle and adipose did not show similar effects, suggesting that improved hepatic insulin sensitivity is the primary contributor to the beneficial effects of VDR activation. Finally, specifically ablating liver macrophages by treatment with clodronate liposomes largely abolished the beneficial metabolic effects of calcipotriol, confirming that VDR activation in liver macrophages is required for the antidiabetic effect. Conclusions Activation of liver macrophage VDRs by vitamin D ligands ameliorates liver inflammation, steatosis and insulin resistance. Our results suggest therapeutic paradigms for treatment of NAFLD and type 2 diabetes mellitus.  

4.2291           Discordance between platelet-supported and vesicle-supported factor VIII activity in the presence of anti-C2 domain inhibitory antibodies

Chatterjee, M., Meeks, S., Novakovic, V.A. and Gilbert, G.E.

1. Thromb. Haemost., 18(12), 3184-3193 (2020)

  Background We recently reported that factor VIII (FVIII) binds to a macromolecular complex including fibrin on thrombin-stimulated platelets and that two antibodies against FVIII diminish platelet-supported FVIII activity more than vesicle-supported activity. The C2 domain of FVIII is known to bind to phospholipid membrane and also binds fibrin. Objectives We asked whether the degree of inhibition by anti-C2 antibodies would show differences between platelet-supported and the standard activated partial thromboplastin time (aPTT) assay. Methods We evaluated the inhibition by a well-defined panel of monoclonal anti-C2 domain antibodies encompassing the major epitopes of the C2 domain. Activity was measured in an activated platelet time (aPT) assay containing fresh, density gradient-purified human platelets. Results The aPT exhibited a log-linear relationship between FVIII and time to fibrin formation over a 4-log range, encompassing 0.01% to 100% plasma FVIII. Nine of 10 mAbs inhibited 89% to 96% of FVIII activity, whereas mAb F85 did not. There was no correlation between the degree of inhibition in the aPTT-based assay and the platelet assay. In particular, four mAbs did not inhibit the aPTT assay, yet inhibited 90% of platelet-based activity. Residual FVIII activity in purified-protein assays, relying on platelets, correlated with the aPT assay. Conclusions The degree of FVIII impairment by some inhibitor antibodies is substantially different on platelet membranes vs synthetic vesicles. Thus, current inhibitor assays may underestimate the frequency of significant inhibitors, and a platelet-based assay may more accurately assess bleeding risk.  

4.2292           Mitochondria-specific delivery system for targeted regulation of mitochondrial gene expression

Su, S., Cao, S., Liu, J. and Xu, X. STAR Protocols, 2, 100275 (2021)   Targeted regulation of mitochondrial gene expression is challenging due to the lack of a mitochondria-specific delivery system. We have previously developed various stimuli-responsive nanoparticle (NP)-based delivery systems to transport nucleic acids for regulation of target gene expression. This protocol describes the design and preparation of an NP platform for mitochondria-specific gene delivery (mito-NP). We use mito-NP in primary liver fibroblasts that are transplanted into mice. Mito-NP can be used to deliver various nucleic acid therapeutics and to treat mitochondria-regulated diseases.  

4.2293           Purification of mouse hepatic non-parenchymal cells or nuclei for use in ChIP-seq and other next-generation sequencing approaches

Troutman, T.D., Bennett, H., Sakai, M., Seidman, J., Heinz, S. and Glass, C.K. STAR Protocols, 2, 100363 (2021)   Significant advancements in understanding disease mechanisms can occur through combined analysis of next-generation sequencing datasets generated using purified cell populations. Here, we detail our optimized protocol for purification of mouse hepatic macrophages (or other liver non-parenchymal populations) suitable for use in various next-generation sequencing protocols. An alternative framework is described for sorting pre-fixed hepatic nuclei populations. This strategy has the advantage of rapidly preserving the nuclei and can facilitate success with ChIP-seq for more challenging molecules.  

4.2294           CloneSeq - Single-cell clonal 3D culture and analysis protocol

Sun, X., Bavli, D., Kozulin, C., Motzik, A., Buxboim, A. and Ram, O. STAR Protocols, 2, 100794 (2021)   This CloneSeq protocol combines clonal expansion inside 3D hydrogel spheres and droplet-based RNA sequencing to resolve the limited sensitivity of single-cell approaches. CloneSeq can reveal rare subpopulations and support cellular stemness. CloneSeq can be adapted to different biological systems to discover rare subpopulations by leveraging clonal enhanced sensitivity. Important considerations include the hydrogel composition, adaptation of 3D cultured clones to the inDrops system, and inherent adhesive properties of the cells. CloneSeq is only validated for cell lines so far.  

4.2295           Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8+ T cells

Ghilas, S., Ambrosini, M., Cancel, J-C., Lelouard, H., Dalod, M. and Crozat, K. iScience, 24, 103059 (2021)   Successful immune responses rely on a regulated delivery of the right signals to the right cells at the right time. Here we show that natural killer (NK) and dendritic epidermal γδ T cells (DETCs) use similar mechanisms to spatiotemporally orchestrate conventional type 1 dendritic cell (cDC1) functions in the spleen, skin, and its draining lymph nodes (dLNs). Upon MCMV infection in the spleen, cDC1 clusterize with activated NK cells in marginal zones. This XCR1-dependent repositioning of cDC1 toward NK cells allows contact delivery of IL-12 and IL-15/IL-15Rα by cDC1, which is critical for NK cell responses. NK cells deliver granulocyte-macrophage colony-stimulating factor (GM-CSF) to cDC1, guiding their CCR7-dependent relocalization into the T cell zone. In MCMV-infected skin, XCL1-secreting DETCs promote cDC1 migration from the skin to the dLNs. This XCR1-dependent licensing of cDC1 both in the spleen and skin accelerates antiviral CD8⁺ T cell responses, revealing an additional mechanism through which cDC1 bridge innate and adaptive immunity.  

4.2296           Single-cell intracellular epitope and transcript detection reveals signal transduction dynamics

Rivello, F., van Buljtenen, E., Matula, K., van Eenennaam, H., Mulder, K.W. and Huck, W.T.S. Cell Reports Methods, 1, 100070 (2021)   To further our understanding of how biochemical information flows through cells upon external stimulation, we require single-cell multi-omics methods that concurrently map changes in (phospho)protein levels across signaling networks and the associated gene expression profiles. Here, we present quantification of RNA and intracellular epitopes by sequencing (QuRIE-seq), a droplet-based platform for single-cell RNA and intra- and extracellular (phospho)protein quantification through sequencing. We applied QuRIE-seq to quantify cell-state changes at both the signaling and the transcriptome level after 2-, 4-, 6-, 60-, and 180-min stimulation of the B cell receptor pathway in Burkitt lymphoma cells. Using the multi-omics factor analysis (MOFA+) framework, we delineated changes in single-cell (phospho)protein and gene expression patterns over multiple timescales and revealed the effect of an inhibitory drug (ibrutinib) on signaling and gene expression landscapes.  

4.2297           Tim4 recognizes carbon nanotubes and mediates phagocytosis leading to granuloma formation

Omori, S., Tsugita, M., Hoshikawa, Y., Nagata, S., Kinoshita, K. and Nakayama, M. Cell Reports, 34(6), 108734 (2021)   Macrophage recognition and phagocytosis of crystals is critical for the associated fibrosis and cancer. Of note, multi-walled carbon nanotubes (MWCNTs), the highly representative products of nanotechnology, induce macrophage NLRP3 inflammasome activation and cause asbestosis-like pathogenesis. However, it remains largely unknown how macrophages efficiently recognize MWCNTs on their cell surfaces. Here, we identify by a targeted screening of phagocyte receptors the phosphatidylserine receptors T cell immunoglobulin mucin 4 (Tim4) and Tim1 as the pattern-recognition receptors for carbon crystals. Docking simulation studies reveal spatiotemporally stable interfaces between aromatic residues in the extracellular IgV domain of Tim4 and one-dimensional carbon crystals. Further, CRISPR-Cas9-mediated deletion of Tim4 and Tim1 reveals that Tim4, but not Tim1, critically contributes to the recognition of MWCNTs by peritoneal macrophages and to granuloma development in a mouse model of direct mesothelium exposure to MWCNTs. These results suggest that Tim4 recognizes MWCNTs through aromatic interactions and mediates phagocytosis leading to granulomas.  

4.2298           High-throughput multiparametric imaging flow cytometry: toward diffraction-limited sub-cellular detection and monitoring of sub-cellular processes

Holzner, G., mateescu, B., van Leeuwen, D., Cereghetti, G., Dechant, R., Stavrakis, S. and DeMello, A. Cell Reports, 34, 108824 (2021)   We present a sheathless, microfluidic imaging flow cytometer that incorporates stroboscopic illumination for blur-free fluorescence detection at ultra-high analytical throughput. The imaging platform is capable of multiparametric fluorescence quantification and sub-cellular localization of these structures down to 500 nm with microscopy image quality. We demonstrate the efficacy of the approach through the analysis and localization of P-bodies and stress granules in yeast and human cells using fluorescence and bright-field detection at analytical throughputs in excess of 60,000 and 400,000 cells/s, respectively. Results highlight the utility of our imaging flow cytometer in directly investigating phase-separated compartments within cellular environments and screening rare events at the sub-cellular level for a range of diagnostic applications.  

4.2299           Functional Genomics Identifies Metabolic Vulnerabilities in Pancreatic Cancer

Zhu, X.G., Chudnovskiy, A., Baudrier, L., Victora, G.D., Gooddarzi, H. and Birsoy, K. Cell Metabolism, 33(1), 211-221 (2021)   Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer characterized by complex metabolic adaptations that promote survival in a severely hypoxic and nutrient-limited tumor microenvironment (TME). Modeling microenvironmental influences in cell culture has been challenging, and technical limitations have hampered the comprehensive study of tumor-specific metabolism in vivo. To systematically interrogate metabolic vulnerabilities in PDA, we employed parallel CRISPR-Cas9 screens using in vivo and in vitro systems. This work revealed striking overlap of in vivo metabolic dependencies with those in vitro. Moreover, we identified that intercellular nutrient sharing can mask dependencies in pooled screens, highlighting a limitation of this approach to study tumor metabolism. Furthermore, metabolic dependencies were similar between 2D and 3D culture, although 3D culture may better model vulnerabilities that influence certain oncogenic signaling pathways. Lastly, our work demonstrates the power of genetic screening approaches to define in vivo metabolic dependencies and pathways that may have therapeutic utility.  

4.2300           CD81 marks immature and dedifferentiated pancreatic β-cells

Salinno, C., Büttner, M., Cota, P., Tritschler, S., tarquis-Medina, M., Bastidas-Ponce, A., Schelbner, K., Burtscher, I., Böttcher, A., Theis, F.J., Bakhti, M. and Lickert, H. Mol. Metabolism, 49, 101188 (2021)   Objective Islets of Langerhans contain heterogeneous populations of insulin-producing β-cells. Surface markers and respective antibodies for isolation, tracking, and analysis are urgently needed to study β-cell heterogeneity and explore the mechanisms to harness the regenerative potential of immature β-cells. Methods We performed single-cell mRNA profiling of early postnatal mouse islets and re-analyzed several single-cell mRNA sequencing datasets from mouse and human pancreas and islets. We used mouse primary islets, iPSC-derived endocrine cells, Min6 insulinoma, and human EndoC-βH1 β-cell lines and performed FAC sorting, Western blotting, and imaging to support and complement the findings from the data analyses. Results We found that all endocrine cell types expressed the cluster of differentiation 81 (CD81) during pancreas development, but the expression levels of this protein were gradually reduced in β-cells during postnatal maturation. Single-cell gene expression profiling and high-resolution imaging revealed an immature signature of β-cells expressing high levels of CD81 (CD81^high) compared to a more mature population expressing no or low levels of this protein (CD81^low/-). Analysis of β-cells from different diabetic mouse models and in vitro β-cell stress assays indicated an upregulation of CD81 expression levels in stressed and dedifferentiated β-cells. Similarly, CD81 was upregulated and marked stressed human β-cells in vitro. Conclusions We identified CD81 as a novel surface marker that labels immature, stressed, and dedifferentiated β-cells in the adult mouse and human islets. This novel surface marker will allow us to better study β-cell heterogeneity in healthy subjects and diabetes progression.  

4.2301           Vertical sleeve gastrectomy triggers fast β-cell recovery upon overt diabetes

Oppenländer, L., Palit, S., Stemmer, K., Greisle, T., Starr, M., Salinno, C., Bastidas-Ponce, A., Feuchtinger, A., Böttcher, A., Ansarullah, Theis, F.J. and Lickert, H. Mol. Metabolism, 54, 101330 (2021)   Objective The effectiveness of bariatric surgery in restoring β-cell function has been described in type-2 diabetes (T2D) patients and animal models for years, whereas the mechanistic underpinnings are largely unknown. The possibility of vertical sleeve gastrectomy (VSG) to rescue far-progressed, clinically-relevant T2D and to promote β-cell recovery has not been investigated on a single-cell level. Nevertheless, characterization of the heterogeneity and functional states of β-cells after VSG is a fundamental step to understand mechanisms of glycaemic recovery and to ultimately develop alternative, less-invasive therapies. Methods We performed VSG in late-stage diabetic db/db mice and analyzed the islet transcriptome using single-cell RNA sequencing (scRNA-seq). Immunohistochemical analyses and quantification of β-cell area and proliferation complement our findings from scRNA-seq. Results We report that VSG was superior to calorie restriction in late-stage T2D and rapidly restored normoglycaemia in morbidly obese and overt diabetic db/db mice. Single-cell profiling of islets of Langerhans showed that VSG induced distinct, intrinsic changes in the β-cell transcriptome, but not in that of α-, δ-, and PP-cells. VSG triggered fast β-cell redifferentiation and functional improvement within only two weeks of intervention, which is not seen upon calorie restriction. Furthermore, VSG expanded β-cell area by means of redifferentiation and by creating a proliferation competent β-cell state. Conclusion Collectively, our study reveals the superiority of VSG in the remission of far-progressed T2D and presents paths of β-cell regeneration and molecular pathways underlying the glycaemic benefits of VSG.  

4.2302           A point mutation in the Pdia6 gene results in loss of pancreatic β-cell identity causing overt diabetes

Chhlabra, N., Amend, A-L., Bastidas-Ponce, A., Sabrautzki, S., Tarquis-Medina, M., Sachs, S., Rubey, M., Lorenz-Deplereux, B., Feudhtinger, A., Bakhti, M., Lickert, H., Przemeck, G.K.H. and de Angelis, M.H. Mol. Metabolism, 54, 101334 (2021)   Objective Protein disulfide isomerases (PDIs) are oxidoreductases that are involved in catalyzing the formation and rearrangement of disulfide bonds during protein folding. One of the PDI members is the PDI-associated 6 (PDIA6) protein, which has been shown to play a vital role in β-cell dysfunction and diabetes. However, very little is known about the function of this protein in β-cells in vivo. This study aimed to describe the consequences of a point mutation in Pdia6 on β-cell development and function. Methods We generated an ENU mouse model carrying a missense mutation (Phe175Ser) in the second thioredoxin domain of the Pdia6 gene. Using biochemical and molecular tools, we determined the effects of the mutation on the β-cell development at embryonic day (E)18.5 and β-cell identity as well as function at postnatal stages. Results Mice homozygous for the Phe175Ser (F175S) mutation were mildly hyperglycemic at weaning and subsequently became hypoinsulinemic and overtly diabetic at the adult stage. Although no developmental phenotype was detected during embryogenesis, mutant mice displayed reduced insulin-expressing β-cells at P14 and P21 without any changes in the rate of cell death and proliferation. Further analysis revealed an increase in BiP and the PDI family member PDIA4, but without any concomitant apoptosis and cell death. Instead, the expression of prominent markers of β-cell maturation and function, such as Ins2, Mafa, and Slc2a2, along with increased expression of α-cell markers, Mafb, and glucagon was observed in adult mice, suggesting loss of β-cell identity. Conclusions The results demonstrate that a global Pdia6 mutation renders mice hypoinsulinemic and hyperglycemic. This occurs due to the loss of pancreatic β-cell function and identity, suggesting a critical role of PDIA6 specifically for β-cells.  

4.2303           p38α in macrophages aggravates arterial endothelium injury by releasing IL-6 through phosphorylating megakaryocytic leukemia 1

Zhang, M., Gao, J., Zhao, X., Zhao, M., Ma, D., Zhang, X.,Tian, D., Pan, B., Yan, X., Wu, J., Meng, X., Yin, H. and Zheng, L. Redox Biol., 38, 101775 (2021)   Background Macrophages regulate the inflammatory response and affect re-endothelialization. Inflammation and macrophages play important roles in promoting tissue repair, but p38α mitogen-activated protein kinase's role in re-endothelialization is unknown. Methods and results Wire injuries of carotid arteries and Evans blue staining were performed in macrophage-specific p38α-knockout (p38α^fl/flLysMCre^+/-) mice and control mice (p38α^fl/fl). Re-endothelialization of the carotid arteries at 3, 5 and 7 days was significantly promoted in p38α^fl/flLysMCre^+/- mice. In vitro experiments indicated that both the proliferation and migration of endothelial cells were enhanced in conditioned medium from peritoneal macrophages of p38α^fl/flLysMCre^+/- mice. Interleukin-6 (IL-6) level was decreased significantly in macrophages of p38α^fl/flLysMCre^+/- mice and an IL-6-neutralizing antibody promoted endothelial cell migration in vitro and re-endothelialization in p38α^fl/fl mice in vivo. Phosphoproteomics revealed that the phosphorylation level of S544/T545/S549 sites in megakaryocytic leukemia 1 (MKL1) was decreased in p38α^fl/flLysMCre^+/- mice. The mutation of either S544/S549 or T545/S549 sites could reduce the expression of IL-6 and the inhibition of MKL1 reduced the expression of IL-6 in vitro and promoted re-endothelialization in vivo. Conclusion p38α in macrophages aggravates injury of arteries by phosphorylating MKL1, and increasing IL-6 expression after vascular injury.    

4.2304           The protective effects of fibroblast growth factor 10 against hepatic ischemia-reperfusion injury in mice

Li, S., Zhu, Z., Xue, M., Pan, X., Tong, G., Yi, X. et al Redox Biol., 40, 101859 (2021)   Hepatic ischemia-reperfusion injury (IRI) is a major complication of liver surgery and transplantation. IRI leads to hepatic parenchymal cell death, resulting in liver failure, and lacks effective therapeutic approaches. Fibroblast growth factor 10 (FGF10) is a paracrine factor which is well-characterized with respect to its pro-proliferative effects during embryonic liver development and liver regeneration, but its role in hepatic IRI remains unknown. In this study, we investigated the role of FGF10 in liver IRI and identified signaling pathways regulated by FGF10. In a mouse model of warm liver IRI, FGF10 was highly expressed during the reperfusion phase. In vitro experiments demonstrated that FGF10 was primarily secreted by hepatic stellate cells and acted on hepatocytes. The role of FGF10 in liver IRI was further examined using adeno-associated virus-mediated gene silencing and overexpression. Overexpression of FGF10 alleviated liver dysfunction, reduced necrosis and inflammation, and protected hepatocytes from apoptosis in the early acute injury phase of IRI. Furthermore, in the late phase of IRI, FGF10 overexpression also promoted hepatocyte proliferation. Meanwhile, gene silencing of FGF10 had the opposite effect. Further studies revealed that overexpression of FGF10 activated nuclear factor-erythroid 2-related factor 2 (NRF2) and decreased oxidative stress, mainly through activation of the phosphatidylinositol-3-kinase/AKT pathway, and the protective effects of FGF10 overexpression were largely abrogated in NRF2 knockout mice. These results demonstrate the protective effects of FGF10 in liver IRI, and reveal the important role of NRF2 in FGF10-mediated hepatic protection during IRI.  

4.2305           Enhancing antibody-dependent cellular phagocytosis by Re-education of tumor-associated macrophages with resiquimod-encapsulated liposomes

Li, H., Somiya, M and Kuroda, S. Biomaterials, 268, 120601 (2021)   Tumor-associated macrophages (TAMs) exist in nearly all tumors, and form a major part of the tumor microenvironment. TAMs are divided into two groups: tumor-suppressing M1 type and tumor-promoting M2 type. Most TAMs are educated by the tumor cells to become M2 type, which support tumor growth and make immunotherapy ineffective. Antibody-dependent cellular phagocytosis (ADCP) is an important mechanism for antibody cancer therapy, and this mechanism is dependent on TAMs. In this study, we found that the M1 type macrophages elicit a more efficient ADCP response than the M2 type, which was confirmed by three tumor cell lines, Raji, A431, and SKBR3, along with their corresponding therapeutic antibody Rituximab, anti-EGFR mouse monoclonal antibody (clone 528), and Trastuzumab, respectively. Resiquimod (R848), an immune system activating agent, has been shown to stimulate the M1 type macrophages, and re-educate the TAMs from M2 type to M1 type. By treating TAMs with R848, the ADCP response increased significantly in vitro and in in vivo mouse xenograft models. R848 encapsulated liposomes (R848-LPs) not only accumulated efficiently in the tumor tissues, but also distributed in the TAMs. Synergizing the R848-LPs with the anti-EGFR mouse monoclonal antibody (clone 528) significantly inhibited WiDr-tumor growth in vivo. Our study also revealed that the TAM-targeted delivery of R848 is able to re-educate the TAMs to M1 type, enhance the ADCP effect of the antibodies, and hence, enhance the anti-tumor effect of the therapeutic antibodies.  

4.2306           Bile acid-receptor TGR5 deficiency worsens liver injury in alcohol-fed mice by inducing intestinal microbiota dysbiosis

Spatz, M., Ciocan, D., Merlen, G., Rainteau, D., Humbert, L., Gomes-Rochette, N. et al JHEP Reports, 3(2), 100230 (2021)   Background & Aims Bile-acid metabolism and the intestinal microbiota are impaired in alcohol-related liver disease. Activation of the bile-acid receptor TGR5 (or GPBAR1) controls both biliary homeostasis and inflammatory processes. We examined the role of TGR5 in alcohol-induced liver injury in mice. Methods We used TGR5-deficient (TGR5-KO) and wild-type (WT) female mice, fed alcohol or not, to study the involvement of liver macrophages, the intestinal microbiota (16S sequencing), and bile-acid profiles (high-performance liquid chromatography coupled to tandem mass spectrometry). Hepatic triglyceride accumulation and inflammatory processes were assessed in parallel. Results TGR5 deficiency worsened liver injury, as shown by greater steatosis and inflammation than in WT mice. Isolation of liver macrophages from WT and TGR5-KO alcohol-fed mice showed that TGR5 deficiency did not increase the pro-inflammatory phenotype of liver macrophages but increased their recruitment to the liver. TGR5 deficiency induced dysbiosis, independently of alcohol intake, and transplantation of the TGR5-KO intestinal microbiota to WT mice was sufficient to worsen alcohol-induced liver inflammation. Secondary bile-acid levels were markedly lower in alcohol-fed TGR5-KO than normally fed WT and TGR5-KO mice. Consistent with these results, predictive analysis showed the abundance of bacterial genes involved in bile-acid transformation to be lower in alcohol-fed TGR5-KO than WT mice. This altered bile-acid profile may explain, in particular, why bile-acid synthesis was not repressed and inflammatory processes were exacerbated. Conclusions A lack of TGR5 was associated with worsening of alcohol-induced liver injury, a phenotype mainly related to intestinal microbiota dysbiosis and an altered bile-acid profile, following the consumption of alcohol.  

4.2307           GIP-GIPR promotes neurite outgrowth of cortical neurons in Akt dependent manner

Teng, L., Guan, T., Guo, B., Ma, C., Wu, R., Xu, M., Liu, M. and Liu, Y. Biochem. Biophys.Res. Comm, 534, 121-127 (2021)   The intrinsic capacity of axonal growth is varied among the neurons form different tissues or different developmental stages. In this study, we established an in vitro model to compare the axonal growth of neurons from embryonic 18 days, post-natal 1 day and post-natal 3 days rat. The E18 neurons showed powerful ability of neuritogenensis and axon outgrowth and the ability decreased rapidly along with development. The transcriptome profile of these neurons revealed a set of genes positively correlated with the capacity of neurite outgrowth. Glucose-dependent insulinotropic polypeptide receptor (GIPR) is identified as a gene to promote neurite outgrowth, which was approved by siRNA knock down assay in E18 neuron. Glucose-dependent insulinotropic polypeptide (GIP), a ligand of GIPR secreted from enteroendocrine K cells, is well-known for its role in nutrient sensing and intake. To verify the effect of GIP-GIPR signal on neurite outgrowth, we administrated GIP to stimulate the E18 neurons, the results showed that GIP significantly improved extension of axon. We further revealed that GIP increased Rac1/Cdc42 phosphorylation in Akt dependent manner. In summary, our study established an in vitro model to screen the genes involved in neurite outgrowth, and we provided mechanical insight on the GIP-GIPR axis to promote axonal outgrowth.  

4.2308           Functionalized nanogel for treating activated astrocytes in spinal cord injury

Papa, S., Veneruso, V., Mauri, E., Cremonesi, G., Mingaj, X., Mariani, A., De Paola, M., Rossetti, A., Sacchetti, A., Rossi, F., Forloni, G. and Veglianse, P.

1. Controlled release, 330, 218-228 (2021)

  Astrogliosis has a unique reaction during spinal cord damage, with helpful or adverse impacts on recovery. There is consequently a pressing need for treatment to target activated astrocytes and their unsafe response after injury to ensure some preservative effect during the progressive damage. We specifically developed and characterized a functionalized nanogel-based nanovector in vitro and in vivo, demonstrating its selectivity towards astrocytes, and limited uptake by macrophages when functionalized with both NH₂ and Cy5 groups. In vitro experiments showed that the internalization was mediated by a clathrin-dependent endocytic pathway. After internalization into the cytoplasm of astrocytes, nanogels undergo lysosomal degradation and release compounds with potential therapeutic efficacy.  

4.2309           Use of antioxidants to augment semen efficiency during liquid storage and cryopreservation in livestock animals: A review

Al-Mutary, M.G.

1. King Saud Univ.-Science, 33, 101226 (2021)

  Despite the widespread use of frozen or refrigerated mammalian spermatozoa, their quality remains low as they are highly sensitivity to injuries during preservation and thawing. Moreover, in vitro conditions during spermatozoa storage may affect sperm quality and thereby oocyte fertilization, cleavage, and blastocyst development. Recently, antioxidants have been employed during semen extenders for livestock in different storage protocols to compensate for the depletion of endogenous antioxidant concentration in seminal plasma due to dilution as well as to counteract in vitro oxidative stress and minimize free radical generation. The present article reviews the most effective enzymatic or non-enzymatic antioxidants used during livestock spermatozoa preservation.  

4.2310           Aryl Hydrocarbon Receptor Activity in Hepatocytes Sensitizes to Hyperacute Acetaminophen-Induced Hepatotoxicity in Mice

Schuran, F.A., Lommetz, C., Steudter, A., Ghallab, A., Wieschendorf, B., Schwinge, D., Zuehlke, S., Reinders, J., Heeren, J., Lohse, A.W., Schramm, C., Herkel, J. and Carambia, A. Cell. Mol. Gastroenterol. Hepatol., 11(2), 371-388 (2021)   Background & Aims Acetaminophen (APAP)-induced liver injury is one of the most common causes of acute liver failure, however, a clear definition of sensitizing risk factors is lacking. Here, we investigated the role of the ligand-activated transcription factor aryl hydrocarbon receptor (Ahr) in APAP-induced liver injury. We hypothesized that Ahr, which integrates environmental, dietary, microbial and metabolic signals into complex cellular transcriptional programs, might act as a rheostat for APAP-toxicity. Methods Wildtype or conditional Ahr knockout mice lacking Ahr in hepatocytes (Alb^Δ/ΔAhr) or myeloid cells (LysM^Δ/ΔAhr) were treated with the specific Ahr ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) together with APAP. Results Ahr activation by ITE, which by itself was non-toxic, exacerbated APAP-induced hepatotoxicity compared to vehicle-treated controls, causing 80% vs. 0% mortality after administration of a normally sublethal APAP overdose. Of note, Ahr activation induced hepatocyte death even at APAP doses within the therapeutic range. Aggravated liver injury was associated with significant neutrophil infiltration; however, lack of Ahr in myeloid cells did not protect LysM^Δ/ΔAhr mice from exacerbated APAP hepatotoxicity. In contrast, Alb^Δ/ΔAhr mice were largely protected from ITE-induced aggravated liver damage, indicating that Ahr activation in hepatocytes, but not in myeloid cells, was instrumental for disease exacerbation. Mechanistically, Ahr activation fueled hepatic accumulation of toxic APAP metabolites by up-regulating expression of the APAP-metabolizing enzyme Cyp1a2, a direct Ahr downstream target. Conclusions Ahr activation in hepatocytes potentiates APAP-induced hepatotoxicity. Thus, individual exposition to environmental Ahr ligands might explain individual sensitivity to hyperacute liver failure.  

4.2311           Protoplast isolation, transient transformation, and flow-cytometric analysis of reporter-gene activation in Cannabis sativa L.

Beard, K.M., Boling, A.W.H., Bargmann, B.O.R. Industrial Crops & Products, 164, 113360 (2021)   Cannabis sativa L. is a valuable, up-and-coming industrial crop with a substantially growing market. However, due to an extended period of legal restriction, research with cannabis has been limited, particularly in laboratory settings. Expanding the application of biotechnological techniques to cannabis can facilitate addressing species-specific impediments to improving crop traits and further fundamental understanding of its intricacies. Here, we describe application of protoplast transformation for the study of transient gene expression in a low-THC cannabis cultivar. To produce explant tissue as a source of protoplasts, a method for hormone-free in vitro micropropagation is established. Protoplasts are isolated from young leaves of the micropropagated stocks and transiently transformed with plasmid DNA carrying a fluorescent marker gene. This is the first report of protoplast transformation in this species. A protoplast isolation yield is achieved of up to 2 × 10⁶ cells per gram of leaf material, vitality staining shows that up to 82 % of isolated protoplasts are viable, and quantification of the cells expressing a fluorescent protein indicates that up to 31 % of the cells can be successfully transformed. Additionally, protoplasts are transformed with an auxin-responsive reporter gene and the reaction to treatment with indole-3-acetic acid is quantified using flow cytometry. This work demonstrates that relatively minor modification of standard techniques can be used to study this important emerging crop.  

4.2312           Mitochondria and calcium defects correlate with axonal dysfunction in GDAP1-related Charcot-Marie-Tooth mouse model

Civera-tregon, A., Dominguez, L., martinez-valero, P., Serrano, C., Vallmitjana, A., Benitez, R., Hoenicka, J. Satrustegui, J. and Palau, F. Nerobiology of Disease, 152, 105300 (2021)   Ganglioside-induced differentiation associated protein 1 (GDAP1) gene encodes a protein of the mitochondrial outer membrane and of the mitochondrial membrane contacts with the endoplasmic reticulum (MAMs) and lysosomes. Since mutations in GDAP1 cause Charcot–Marie–Tooth, an inherited motor and sensory neuropathy, its function is essential for peripheral nerve physiology. Our previous studies showed structural and functional defects in mitochondria and their contacts when GDAP1 is depleted. Nevertheless, the underlying axonal pathophysiological events remain unclear. Here, we have used embryonic motor neurons (eMNs) cultures from Gdap1 knockout (Gdap1^−/−) mice to investigate in vivo mitochondria and calcium homeostasis in the axons. We imaged mitochondrial axonal transport and we found a defective pattern in the Gdap1^−/− eMNs. We also detected pathological and functional mitochondria membrane abnormalities with a drop in ATP production and a deteriorated bioenergetic status. Another consequence of the loss of GDAP1 in the soma and axons of eMNs was the in vivo increase calcium levels in both basal conditions and during recovery after neuronal stimulation with glutamate. Further, we found that glutamate-stimulation of respiration was lower in Gdap1^−/− eMNs showing that the basal bioenergetics failure jeopardizes a full respiratory response and prevents a rapid return of calcium to basal levels. Together, our results demonstrate that the loss of GDAP1 critically compromises the morphology and function of mitochondria and its relationship with calcium homeostasis in the soma and axons, offering important insight into the cellular mechanisms associated with axonal degeneration of GDAP1-related CMT neuropathies and the relevance that axon length may have.  

4.2313           Efficient establishment of reactivatable latency by an acyclovir-resistant herpes simplex virus 1 thymidine kinase substitution mutant with reduced neuronal replication

Wang, S., Hou, F., Yao, Y-F. and Pan, D. Virology, 556, 140-148 (2021)   Herpes simplex virus 1 causes recurrent diseases by reactivating from latency, which requires the viral thymidine kinase (TK) gene. An acyclovir-resistant mutation in TK, V204G, was previously repeatedly identified in a patient with recurrent herpetic keratitis. We found that compared with its parental strain KOS, a laboratory-derived V204G mutant virus was impaired in replication in cultured neurons despite little defect in non-neuronal cells. After corneal inoculation of mice, V204G exhibited defects in ocular replication that were modest over the first three days but severe afterward. Acute replication of V204G in trigeminal ganglia was significantly impaired. However, V204G established latency with viral loads as high as KOS and reactivated with high frequency albeit reduced kinetics. Acyclovir treatment that drastically decreased ocular and ganglionic replication of KOS had little effect on V204G. Thus, despite reduced neuronal replication due to impaired TK activity, this clinically relevant drug-resistant mutant can efficiently establish reactivatable latency.  

4.2314           Platelets immune response against Mycobacterium tuberculosis infection

Torres-Juarez, F., trejo-Martinez, L.A., Layseca-Espinosa, E., Leon-Contreras, J.C., Enciso-Moreno, J.A., Hernandez.Pando, R. and Rivs-Santiago, B. Microbial Pathogenesis, 153, 104768 (2021)   Tuberculosis (TB) is the first cause of death by a single infectious agent. Previous reports have highlighted the presence of platelets within Tb granulomas, albeit the immune-associated platelet response to Mycobacterium tuberculosis (Mtb) has not been deeply studied. Our results showed that platelets are recruited into the granuloma in the late stages of tuberculosis. Furthermore, electron-microscopy studies showed that platelets can internalize Mtb and produce host defense peptides (HDPs), such as RNase 7, HBD2 and hPF-4 that bind to the internalized Mtb. Mtb-infected platelets exhibited higher transcription and secretion of IL-1β and TNF-α, whereas IL-10 and IL-6 protein levels decreased. These results suggest that platelets participate in the immune response against Mtb through HDPs and cytokines production.  

4.2315           Cyanidin attenuates IL-17A cytokine signaling mediated monocyte migration and differentiation into mature osteoclasts in rheumatoid arthritis

Samarpita, S. and Rasool, M. Cytokine, 142, 155502 (2021)   Interleukin (IL)-17A signaling pathway plays a critical role in the initiation and progression of rheumatoid arthritis (RA) and represents a viable target for RA therapy. Cyanidin, a flavonoid compound, is a novel inhibitor of IL-17A/IL-17RA (receptor subunit A) interaction in several inflammatory diseases. However, the therapeutic efficacy of cyanidin on IL-17A cytokine signaling induced monocyte migration and fibroblast-like synoviocytes (FLS) released RANKL mediated osteoclastogenesis in RA has not yet been deciphered. In the present study, cyanidin impeded IL-17A induced migration of monocytes isolated from adjuvant-induced arthritic (AA) rats. At the molecular level, cyanidin blocked the activation of p38MAPK signaling in response to IL-17A. Importantly, cyanidin downregulated IL-17A induced expression of HSP27, CXCR4, and CCR7 in AA monocytes via modulating IL-17/p38 MAPK signaling axis. Alternatively, cyanidin significantly suppressed the formation of matured osteoclasts and bone resorption in a coculture system consisting of IL‐17 treated AA‐FLS and rat bone marrow-derived monocytes/macrophages. Further, cyanidin significantly inhibited the expression of RANKL and increased the expression of OPG in AA-FLS via blunted activation of IL-17A/STAT-3 signaling cascade. Interestingly, cyanidin impaired IL-17A induced overexpression of IL-17RA. Taken together, our study proposes a novel therapeutic function of cyanidin towards targeted inhibition of IL-17A/IL-17RA signaling mediated disease severity and bone erosion in RA.  

4.2316           sEVs from tonsil-derived mesenchymal stromal cells alleviate activation of hepatic stellate cells and liver fibrosis through miR-486-5p

Kim, J., Lee, C., Shin, Y., Wang, S., Han, J., Kim, M., Kim, J.M., Shin, S-C., Lee, B-J., Kim, T-J. and Jung, Y. Molecular Therapy, 29(4), 1471-1486 (2021)   Mesenchymal stromal cells (MSCs) are considered as a promising therapeutic tool for liver fibrosis, a main feature of chronic liver disease. Because small extracellular vesicles (sEVs) harboring a variety of proteins and RNAs are known to have similar functions with their derived cells, MSC-derived sEVs carry out the regenerative capacities of MSCs. Human tonsil-derived MSCs (T-MSCs) are reported as a novel source of MSCs, but their effects on liver fibrosis remain unclear. In the present study, we investigated the effects of T-MSC-derived sEVs on liver fibrosis. The expression of profibrotic genes decreased in human primary hepatic stellate cells (pHSCs) co-cultured with T-MSCs. Treatment of T-MSC-sEVs inactivated human and mouse pHSCs. Administration of T-MSC-sEVs ameliorated hepatic injuries and fibrosis in chronically damaged liver induced by carbon tetrachloride (CCl₄). miR-486-5p highly enriched in T-MSC-sEVs targeting the hedgehog receptor, smoothened (Smo), was upregulated, whereas Smo and Gli2, the hedgehog target gene, were downregulated in pHSCs and liver tissues treated with T-MSC-sEVs or miR-486-5p mimic, indicating that sEV-miR-486 inactivates HSCs by suppressing hedgehog signaling. Our results showed that T-MSCs attenuate HSC activation and liver fibrosis by delivering sEVs, and miR-486 in the sEVs inactivates hedgehog signaling, suggesting that T-MSCs and their sEVs are novel anti-fibrotic therapeutics for treating chronic liver disease.  

4.2317           Improvement strategy of a microfluidic sorter using a pneumatic bilayer valve

Jin, S.H., Lee, B., Kim, J.S. and Lee, C-S. Chem. Engineering Science, 245, 116384 (2021)   Here, we describe the study of a pneumatic bilayer valve-based microfluidic sorter to improve sorting performance and stability. Unlike conventional microfluidic fluorescence-activated droplet sorters, the mechanical flow shifting characteristics of the pneumatic bilayer valve are less affected by the physical properties of samples, making it widely applicable to various sample types. The optimization of the valve channel structure improves sorting accuracy and efficiency at higher throughput by reducing the actuation time by three times, and alignment of fluid flows reduces false-positive errors by 5.5 times which can directly affect sorting accuracy and efficiency. Under these optimized conditions, we achieve both sorting accuracy and efficiency more than 98% at 50 Hz throughput. This microfluidic sorter allows the sorting of various sample types, such as cell suspensions, cell-laden hydrogels, and cell-encapsulated droplets.  

4.2318           Characterization of Hevin (SPARCL1) Immunoreactivity in Postmortem Human Brain Homogenates

Nunez-delMoral, A., Brocos-Mosquera, I., Viakou, V., Callado, L.F. and Erdozain, A.M. Neuroscience, 467, 91-109 (2021)   Hevin is a matricellular glycoprotein that plays important roles in neural developmental processes such as neuronal migration, synaptogenesis and synaptic plasticity. In contrast to other matricellular proteins whose expression decreases when development is complete, hevin remains highly expressed, suggesting its involvement in adult brain function. In vitro studies have shown that hevin can have different post-translational modifications. However, the glycosylation pattern of hevin in the human brain remains unknown, as well as its relative distribution and localization. The present study provides the first thorough characterization of hevin protein expression by Western blot in postmortem adult human brain. Our results demonstrated two major specific immunoreactive bands for hevin: an intense band migrating around 130 kDa, and a band migrating around 100 kDa. Biochemical assays revealed that both hevin bands have a different glycosylation pattern. Subcellular fractionation showed greater expression in membrane-enriched fraction than in cytosolic preparation, and a higher expression in prefrontal cortex (PFC) compared to hippocampus (HIP), caudate nucleus (CAU) and cerebellum (CB). We confirmed that a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) and matrixmetalloproteinase 3 (MMP-3) proteases digestion led to an intense double band with similar molecular weight to that described as SPARC-like fragment (SLF). Finally, hevin immunoreactivity was also detected in human astrocytoma, meningioma, cerebrospinal fluid and serum samples, but was absent from any blood cell type.  

4.2319           Male reproductive systems of Macaca mulatta: Gonadal development, spermatogenesis and applications in spermatogonia stem cell transplantation

Wei, Y.L., She, Z-Y., Huang, T., Zhang, H-T. and Wang, X-R. Res. Vet. Science, 137, 127-137 (2021)   Rhesus macaque (Macaca mulatta) is widely applied in animal model construction of infertility, spermatogonia stem cell transplantation and male reproductive diseases. In this review, we describe the seasonal changes of the reproductive system in rhesus macaques, the regular pattern of spermatogenesis and spermatozoa maturation, and the differentiation of spermatogonia and spermatocytes. The duration of the M. mulatta spermatogenesis is approximately 10 days and seminiferous epithelium cycles mainly consist of 12 stages, which provide a suitable model for reproductive studies in non-human primates. Here, we summarize the features of gonadal development and sperm maturation in the rhesus monkeys, which provide important information in the studies of reproductive biology. Rhesus macaque is an excellent animal model in spermatogonia stem cell transplantation. We discuss the applications and progresses of assisted reproductive technologies in sperm liquefaction, semen cryopreservation and spermatogonia stem cell transplantation of rhesus macaques. Besides, we sort out recent proteomic analyses of male reproductive systems and semen samples in rhesus macaques. This review mainly focuses on male reproductive biology and application studies using M. mulatta, which would promote the development of new therapeutic interventions on assisted reproduction and reproductive disease studies in the future.  

4.2320           Novel antimicrobial cecropins derived from O. curvicornis and D. satanas dung beetles

Arias, D.C.H., Toro, L.J., Ramirez, G.A.T., Osorio-Mendez, J.F., Rodriguez-Carlos, A., Valle, J., Martin-Luevano, S.P., Rivas-Santiago, B., Andreu, D. and Isorio, J.C.C. Peptides, 145, 170626 (2021)   Antibiotic resistance is an increasing global problem and therapeutic alternatives to traditional antibiotics are needed. Antimicrobial and host defense peptides represent an attractive source for new therapeutic strategies, given their wide range of activities including antimicrobial, antitumoral and immunomodulatory. Insects produce several families of these peptides, including cecropins. Herein, we characterized the sequence, structure, and biological activity of three cecropins called satanin 1, 2, and curvicin, found in the transcriptome of two dung beetle species Dichotomius satanas and Onthophagus curvicornis. Sequence and circular dichroism analyses show that they have typical features of the cecropin family: short length (38–39 amino acids), positive charge, and amphipathic α-helical structure. They are active mainly against Gram-negative bacteria (3.12–12.5 μg/mL), with low toxicity on eukaryotic cells resulting in high therapeutic indexes (TI > 30). Peptides also showed effects on TNFα production in LPS-stimulated PBMCs. The biological activity of Satanin 1, 2 and Curvicin makes them interesting leads for antimicrobial strategies.  

4.2321           Echinococcus multilocularis infection induces UBE2N suppression via exosomal emu-miR-4989

Cai, M., Ding, J., Li, Y., He, G., Yang, J., Liu, T., Guo, X., Yang, X., Wang, X., Cho, W.C., Harandi, M.F. and Zheng, Y. Acta Tropica, 223, 106087 (2021)   Echinococcus multilocularis metacestodes mainly reside in liver in humans and animals, and cause serious damages. UBE2N was herein shown to be downregulated in response to the infection. UBE2N was further shown to be predominantly expressed in the hepatocytes, which was also significantly downregulated during the infection. UBE2N was a target of emu-miR-4989, which was loaded into the exosomes secreted by parasites. These emu-miR-4989-encapsulating exosomes were internalized by hepatocytes, and induced a significant decrease of relative luciferase activity in the cells transfected with the construct containing a wild type of UBE2N 3’-UTR compared to the control (p < 0.05). These results demonstrate that emu-miR-4989 is involved in the UBE2N inhibition in the hepatocytes during E. multilocularis through exosomes.  

4.2322           Sirt6 Alleviated Liver Fibrosis by Deacetylating Conserved Lysine 54 on Smad2 in Hepatic Stellate Cells

Zhang, J., Li, Y., Liu, Q., Huang, Y., Li, R., Wu, T. et al Hepatology, 73(3), 1140-1157 (2021)   Backgrounds and Aims Activation of hepatic stellate cells (HSCs) is a central driver of fibrosis. This study aimed to elucidate the role of the deacetylase sirtuin 6 (Sirt6) in HSC activation and liver fibrosis. Approach and Results Gain-of-function and loss-of-function models were used to study the function of Sirt6 in HSC activation. Mass spectrometry was used to determine the specific acetylation site. The lecithin retinol acyltransferase–driven cyclization recombination recombinase construct (CreERT2) mouse line was created to generate HSC-specific conditional Sirt6-knockout mice (Sirt6^△HSC). We found that Sirt6 is most abundantly expressed in HSCs as compared with other liver cell types. The expression of Sirt6 was decreased in activated HSCs and fibrotic livers of mice and humans. Sirt6 knockdown and Sirt6 overexpression increased and decreased fibrogenic gene expression, respectively, in HSCs. Mechanistically, Sirt6 inhibited the phosphorylation and nuclear localization of mothers against decapentaplegic homolog (Smad) 2. Further study demonstrated that Sirt6 could directly interact with Smad2, deacetylate Smad2, and decrease the transcription of transforming growth factor β/Smad2 signaling. Mass spectrometry revealed that Sirt6 deacetylated conserved lysine 54 on Smad2. Mutation of lysine 54 to Arginine in Smad2 abolished the regulatory effect of Sirt6. In vivo, specific ablation of Sirt6 in HSCs exacerbated hepatocyte injury and cholestasis-induced liver fibrosis in mice. With targeted delivery of the Sirt6 agonist MDL-800, its concentration was 9.28-fold higher in HSCs as compared with other liver cells and alleviated hepatic fibrosis. Conclusions Sirt6 plays a key role in HSC activation and liver fibrosis by deacetylating the profibrogenic transcription factor Smad2. Sirt6 may be a potential therapeutic target for liver fibrosis.  

4.2323           Toward whole tissue imaging of axolotl regeneration

Masselink, W. and Tanaka, E.M. Developmental Dynamics, 250, 800-806 (2021)   The axolotl is a highly regenerative organism and has been studied in laboratories for over 150 years. Despite a long-standing fascination with regeneration in general and axolotl specifically, we are still scratching the surface trying to visualize and understand the complex cellular behavior that underlies axolotl regeneration. In this review, we will discuss the progress that has been made in visualizing these processes focusing on four major aspects: cell labeling approaches, the removal of pigmentation, reductionist approaches to perform live cell imaging, and finally recent developments applying tissue clearing strategies to visualize the processes that underly regeneration. We also provide several suggestions that the community could consider exploring, notably the generation of novel alleles that further reduce pigmentation as well as improvements in tissue clearing strategies.  

4.2324           Shapiro's Laws Revisited: Conventional and Unconventional Cytometry at CYTO2020

Galbraith, D.W. Cytometry Part A, 99A, 129-132 (2021)   Extracting relevant information from a very large excess of irrelevant debris. Judicious gating of PI-stained Arabidopsis leaf homogenates defines the position of a very minor proportion of nuclei within a two-dimensional frequency distribution, whose properties can then be finely dissected. From: Galbraith (2009). Cytometry Part A.  

4.2325           An autophagy enhancer ameliorates diabetes of human IAPP-transgenic mice through clearance of amyloidogenic oligomer

Kim, J., Park, K., Kim, M.J., Lim, H., Kim, K.H., Kim, S-W., Lee, E-S., Kim, H., Kim, S.J., Hur, K.Y., Kim, J.H., Ahn, J., Yoon, J-W. and Lee, M-S. Nature Comm., 12:183 (2021)   We have reported that autophagy is crucial for clearance of amyloidogenic human IAPP (hIAPP) oligomer, suggesting that an autophagy enhancer could be a therapeutic modality against human diabetes with amyloid accumulation. Here, we show that a recently identified autophagy enhancer (MSL-7) reduces hIAPP oligomer accumulation in human induced pluripotent stem cell-derived β-cells (hiPSC-β-cells) and diminishes oligomer-mediated apoptosis of β-cells. Protective effects of MSL-7 against hIAPP oligomer accumulation and hIAPP oligomer-mediated β-cell death are significantly reduced in cells with knockout of MiTF/TFE family members such as Tfeb or Tfe3. MSL-7 improves glucose tolerance and β-cell function of hIAPP⁺ mice on high-fat diet, accompanied by reduced hIAPP oligomer/amyloid accumulation and β-cell apoptosis. Protective effects of MSL-7 against hIAPP oligomer-mediated β-cell death and the development of diabetes are also significantly reduced by β-cell-specific knockout of Tfeb. These results suggest that an autophagy enhancer could have therapeutic potential against human diabetes characterized by islet amyloid accumulation.  

4.2326           Machine learning identifies candidates for drug repurposing in Alzheimer’s disease

Rodriguez. S., Hug, C., Todorov, P., Moret, N., Boswell, S.A., Evans, K., Zhou, G., Johnson, N.T., Hyman, B.T., Sorger, P.K., Albers, M.W. and Sokolov, A. Nature Comm., 12:1033 (2021)   Clinical trials of novel therapeutics for Alzheimer’s Disease (AD) have consumed a large amount of time and resources with largely negative results. Repurposing drugs already approved by the Food and Drug Administration (FDA) for another indication is a more rapid and less expensive option. We present DRIAD (Drug Repurposing In AD), a machine learning framework that quantifies potential associations between the pathology of AD severity (the Braak stage) and molecular mechanisms as encoded in lists of gene names. DRIAD is applied to lists of genes arising from perturbations in differentiated human neural cell cultures by 80 FDA-approved and clinically tested drugs, producing a ranked list of possible repurposing candidates. Top-scoring drugs are inspected for common trends among their targets. We propose that the DRIAD method can be used to nominate drugs that, after additional validation and identification of relevant pharmacodynamic biomarker(s), could be readily evaluated in a clinical trial.  

4.2327           Transcription organizes euchromatin via microphase separation

Hilbert, L., Sato, Y., Kuznetsova, K., Bianucci, T., Kimura, H., Jülicher, F., Honigmann, A., Zaburdaev, V. and Vastenhouw, N.L. Nature Comm., 12:1360 (2021)   In eukaryotes, DNA is packed inside the cell nucleus in the form of chromatin, which consists of DNA, proteins such as histones, and RNA. Euchromatin, which is permissive for transcription, is spatially organized into transcriptionally inactive domains interspersed with pockets of transcriptional activity. While transcription and RNA have been implicated in euchromatin organization, it remains unclear how their interplay forms and maintains transcription pockets. Here we combine theory and experiment to analyze the dynamics of euchromatin organization as pluripotent zebrafish cells exit mitosis and begin transcription. We show that accumulation of RNA induces formation of transcription pockets which displace transcriptionally inactive chromatin. We propose that the accumulating RNA recruits RNA-binding proteins that together tend to separate from transcriptionally inactive euchromatin. Full phase separation is prevented because RNA remains tethered to transcribed euchromatin through RNA polymerases. Instead, smaller scale microphases emerge that do not grow further and form the typical pattern of euchromatin organization.  

4.2328           Single cell transcriptomics of primate sensory neurons identifies cell types associated with chronic pain

Kupari, J., Usoskin, D., Parisien, M., Lou, D., Hu, Y., Fatt, M. et al Nature Comm., 12:1510 (2021)   Distinct types of dorsal root ganglion sensory neurons may have unique contributions to chronic pain. Identification of primate sensory neuron types is critical for understanding the cellular origin and heritability of chronic pain. However, molecular insights into the primate sensory neurons are missing. Here we classify non-human primate dorsal root ganglion sensory neurons based on their transcriptome and map human pain heritability to neuronal types. First, we identified cell correlates between two major datasets for mouse sensory neuron types. Machine learning exposes an overall cross-species conservation of somatosensory neurons between primate and mouse, although with differences at individual gene level, highlighting the importance of primate data for clinical translation. We map genomic loci associated with chronic pain in human onto primate sensory neuron types to identify the cellular origin of chronic pain. Genome-wide associations for chronic pain converge on two different neuronal types distributed between pain disorders that display different genetic susceptibilities, suggesting both unique and shared mechanisms between different pain conditions.  

4.2329           Orphan GPR116 mediates the insulin sensitizing effects of the hepatokine FNDC4 in adipose tissue

Georgiadi, A., Lopez-Salazar, V., El-Merahbi, R., Karikari, R.A., Ma, X. et al Nature Comm., 12:2999 (2021)   The proper functional interaction between different tissues represents a key component in systemic metabolic control. Indeed, disruption of endocrine inter-tissue communication is a hallmark of severe metabolic dysfunction in obesity and diabetes. Here, we show that the FNDC4-GPR116, liver-white adipose tissue endocrine axis controls glucose homeostasis. We found that the liver primarily controlled the circulating levels of soluble FNDC4 (sFNDC4) and lowering of the hepatokine FNDC4 led to prediabetes in mice. Further, we identified the orphan adhesion GPCR GPR116 as a receptor of sFNDC4 in the white adipose tissue. Upon direct and high affinity binding of sFNDC4 to GPR116, sFNDC4 promoted insulin signaling and insulin-mediated glucose uptake in white adipocytes. Indeed, supplementation with FcsFNDC4 in prediabetic mice improved glucose tolerance and inflammatory markers in a white-adipocyte selective and GPR116-dependent manner. Of note, the sFNDC4-GPR116, liver-adipose tissue axis was dampened in (pre) diabetic human patients. Thus our findings will now allow for harnessing this endocrine circuit for alternative therapeutic strategies in obesity-related pre-diabetes.  

4.2330           Loss of polycomb repressive complex 1 activity and chromosomal instability drive uveal melanoma progression

Bakhoum, M.F., Francis, J.H., Agustinus, A., Earlie, E.M., Di Bona, M., Abrahamson, D.H., Duran, M., Masilionis, I., Molina, E., Shoushtari, A.N., Goldbaum, M.H., Mischel, P.S., Bakhoum, S.F. and Laugney, A.M. Nature Comm., 12:5402 (2021)   Chromosomal instability (CIN) and epigenetic alterations have been implicated in tumor progression and metastasis; yet how these two hallmarks of cancer are related remains poorly understood. By integrating genetic, epigenetic, and functional analyses at the single cell level, we show that progression of uveal melanoma (UM), the most common intraocular primary cancer in adults, is driven by loss of Polycomb Repressive Complex 1 (PRC1) in a subpopulation of tumor cells. This leads to transcriptional de-repression of PRC1-target genes and mitotic chromosome segregation errors. Ensuing CIN leads to the formation of rupture-prone micronuclei, exposing genomic double-stranded DNA (dsDNA) to the cytosol. This provokes tumor cell-intrinsic inflammatory signaling, mediated by aberrant activation of the cGAS-STING pathway. PRC1 inhibition promotes nuclear enlargement, induces a transcriptional response that is associated with significantly worse patient survival and clinical outcomes, and enhances migration that is rescued upon pharmacologic inhibition of CIN or STING. Thus, deregulation of PRC1 can promote tumor progression by inducing CIN and represents an opportunity for early therapeutic intervention.  

4.2331           Comparison of DNA repair and radiosensitivity of different blood cell populations

Heylmann, D., Ponath, V., Kindler, T. and Kaina, B. Scientific Reports, 11:2478 (2021)   Despite the frequent use of ionising radiation (IR) in therapy and diagnostics and the unavoidable exposure to external radiation sources, our knowledge regarding the radiosensitivity of human blood cell populations is limited and published data, obtained under different experimental conditions, are heterogeneous. To compare the radiosensitivity of different hematopoietic cell populations, we set out to determine the responses of cells obtained from peripheral blood of healthy volunteers under identical conditions (resting, non-stimulated cells). First, we measured the radiation response of T cells (Treg, Th, CTL), B cells, NK cells, CD34+ progenitor cells and monocytes obtained from peripheral blood and monocyte-derived macrophages (Mph) and immature dendritic cells (iDC) ex vivo and show that T and B cells are highly sensitive, starting to undergo apoptosis following IR with a dose as low as 0.125 Gy. Importantly, there was no clear threshold dose and cell death/apoptosis increased up to a saturation level with a dose of 2 Gy. The sensitivity decreased in the order of T cells > NK and B cells > monocytes > macrophages and iDC. The data confirm a previous report that Mph and iDC are radiation-resistant compared to their progenitor monocytes. Although non-stimulated T and B cells were highly radiation-sensitive compared to monocytes and macrophages, they were competent in the repair of DNA double-strand breaks, as shown by a decline in γH2AX foci in the post-exposure period. CD34+ cells obtained from peripheral blood also showed γH2AX decline post-exposure, indicating they are repair competent. Granulocytes (CD15+) did not display any γH2AX staining following IR. Although peripheral blood lymphocytes, the main fraction are T cells, were significantly more radiation-sensitive than monocytes, they displayed the expression of the repair proteins XRCC1, ligase III and PARP-1, which were nearly non-expressed in monocytes. To assess whether monocytes are depleted in vivo following IR, we measured the amount of T cells and monocytes in cancer patients who received total-body radiation (TBR, 6 × 2 Gy). We observed that the number of T cells in the peripheral blood significantly declined already after the first day of TBR and remained at a low level, which was accompanied by an increase in the number of γH2AX foci in the surviving CD3+ T cell fraction. In contrast, the number of monocytes did not decline extensively, reflecting their radiation resistance compared to T cells. Monocytes also showed an accumulation of γH2AX foci in vivo, but the levels were significantly lower than in T cells. CD56+ NK cells displayed a response similar to T cells. The data support the notion that unstimulated T cell subfractions are nearly equally radiation sensitive. There are, however, remarkable differences in the radiation sensitivity between the lymphoid and the myeloid lineage, with lymphoid cells being significantly more sensitive than cells of the myeloid lineage. In the myeloid lineage, macrophages and iDCs were the most radio-resistant cell types.  

4.2332           Disulfide HMGB1 acts via TLR2/4 receptors to reduce the numbers of oligodendrocyte progenitor cells after traumatic injury in vitro

Ved, R., Sharouf, F., Harari, B., Muzaffar, M., Manivannan, S., Ormonde, C., Gray, W.P. and Zaben, M. Scientific Reports, 11:6181 (2021)   Traumatic brain injury (TBI) is associated with poor clinical outcomes; autopsy studies of TBI victims demonstrate significant oligodendrocyte progenitor cell (OPC) death post TBI; an observation, which may explain the lack of meaningful repair of injured axons. Whilst high-mobility group box-1 (HMGB1) and its key receptors TLR2/4 are identified as key initiators of neuroinflammation post-TBI, they have been identified as attractive targets for development of novel therapeutic approaches to improve post-TBI clinical outcomes. In this report we establish unequivocal evidence that HMGB1 released in vitro impairs OPC response to mechanical injury; an effect that is pharmacologically reversible. We show that needle scratch injury hyper-acutely induced microglial HMGB1 nucleus-to-cytoplasm translocation and subsequent release into culture medium. Application of injury-conditioned media resulted in significant decreases in OPC number through anti-proliferative effects. This effect was reversed by co-treatment with the TLR2/4 receptor antagonist BoxA. Furthermore, whilst injury conditioned medium drove OPCs towards an activated reactive morphology, this was also abolished after BoxA co-treatment. We conclude that HMGB1, through TLR2/4 dependant mechanisms, may be detrimental to OPC proliferation following injury in vitro, negatively affecting the potential for restoring a mature oligodendrocyte population, and subsequent axonal remyelination. Further study is required to assess how HMGB1-TLR signalling influences OPC maturation and myelination capacity.  

4.2333           Core–shell hydrogel microcapsules enable formation of human pluripotent stem cell spheroids and their cultivation in a stirred bioreactor

Fattahi, P., Rahimian, A., Slama, M.O., Gwon, K., Gonzelez-Suarez, A.M., Wolf, J., Baskaran, H., Duffy, C.D., Stybayeva, G., Peterson, Q.P. and Revzin, A. Scientific Reports, 11:7177 (2021)   Cellular therapies based on human pluripotent stem cells (hPSCs) offer considerable promise for treating numerous diseases including diabetes and end stage liver failure. Stem cell spheroids may be cultured in stirred bioreactors to scale up cell production to cell numbers relevant for use in humans. Despite significant progress in bioreactor culture of stem cells, areas for improvement remain. In this study, we demonstrate that microfluidic encapsulation of hPSCs and formation of spheroids. A co-axial droplet microfluidic device was used to fabricate 400 μm diameter capsules with a poly(ethylene glycol) hydrogel shell and an aqueous core. Spheroid formation was demonstrated for three hPSC lines to highlight broad utility of this encapsulation technology. In-capsule differentiation of stem cell spheroids into pancreatic β-cells in suspension culture was also demonstrated.  

4.2334           Profilin2 regulates actin rod assembly in neuronal cells

Walter, L.M., Rademacher, S., Pich, A. and Claus, P. Scientific Reports, 11:10287 (2021)   Nuclear and cytoplasmic actin-cofilin rods are formed transiently under stress conditions to reduce actin filament turnover and ATP hydrolysis. The persistence of these structures has been implicated in disease pathology of several neurological disorders. Recently, the presence of actin rods has been discovered in Spinal Muscular Atrophy (SMA), a neurodegenerative disease affecting predominantly motoneurons leading to muscle weakness and atrophy. This finding underlined the importance of dysregulated actin dynamics in motoneuron loss in SMA. In this study, we characterized actin rods formed in a SMA cell culture model analyzing their composition by LC–MS-based proteomics. Besides actin and cofilin, we identified proteins involved in processes such as ubiquitination, translation or protein folding to be bound to actin rods. This suggests their sequestration to actin rods, thus impairing important cellular functions. Moreover, we showed the involvement of the cytoskeletal protein profilin2 and its upstream effectors RhoA/ROCK in actin rod assembly in SMA. These findings implicate that the formation of actin rods exerts detrimental effects on motoneuron homeostasis by affecting actin dynamics and disturbing essential cellular pathways.  

4.2335           Modular barcode beads for microfluidic single cell genomics

Delley, C.L. and Abate, A.R. Scientific Reports, 11:10857 (2021)   Barcode beads allow efficient nucleic acid tagging in single cell genomics. Current barcode designs, however, are fabricated with a particular application in mind. Repurposing to novel targets, or altering to add additional targets as information is obtained is possible but the result is suboptimal. Here, we describe a modular framework that simplifies generation of multifunctional beads and allows their easy extension to new targets.  

4.2336           Identification of a targetable KRAS-mutant epithelial population in non-small cell lung cancer

Maroni, G., Bassal, M.A., Krishnan, I., Fhu, C.W., Savova, V. et al Communications Biol., 4:370 (2021)   Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencing in aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer. We previously reported a key role for the oncogene BMI-1 in adenocarcinomas. We therefore investigated the effects of in vivo PTC596 treatment, which affects BMI-1 activity, in our murine model. Post-treatment, MRI analysis showed decreased tumor size, while single cell transcriptomics concomitantly detected near complete ablation of the mutant-KRAS-associated subpopulation, signifying the presence of a pharmacologically targetable, tumor-associated subpopulation. Our findings therefore hold promise for the development of a targeted therapy for KRAS-mutant adenocarcinomas.  

4.2337           Auranofin prevents liver fibrosis by system Xc-mediated inhibition of NLRP3 inflammasome

Kim, H.Y., Choi, Y.J., Kim, S.K.H., Jun, D. W., Yoon, K., Kim, N., Hwang, J., Kim, Y-M., Lim, S.C. and Kang, K.W. Communications Biol., 4:824 (2021)   Demand for a cure of liver fibrosis is rising with its increasing morbidity and mortality. Therefore, it is an urgent issue to investigate its therapeutic candidates. Liver fibrosis progresses following ‘multi-hit’ processes involving hepatic stellate cells, macrophages, and hepatocytes. The NOD-like receptor protein 3 (NLRP3) inflammasome is emerging as a therapeutic target in liver fibrosis. Previous studies showed that the anti-rheumatic agent auranofin inhibits the NLRP3 inflammasome; thus, this study evaluates the antifibrotic effect of auranofin in vivo and explores the underlying molecular mechanism. The antifibrotic effect of auranofin is assessed in thioacetamide- and carbon tetrachloride-induced liver fibrosis models. Moreover, hepatic stellate cell (HSC), bone marrow-derived macrophage (BMDM), kupffer cell, and hepatocyte are used to examine the underlying mechanism of auranofin. Auranofin potently inhibits activation of the NLRP3 inflammasome in BMDM and kupffer cell. It also reduces the migration of HSC. The underlying molecular mechanism was inhibition of cystine-glutamate antiporter, system Xc. Auranofin inhibits system Xc activity and instantly induced oxidative burst, which mediated inhibition of the NLRP3 inflammasome in macrophages and HSCs. Therefore, to the best of our knowledge, we propose the use of auranofin as an anti-liver fibrotic agent.  

4.2338           Essential roles of plexin-B3+ oligodendrocyte precursor cells in the pathogenesis of Alzheimer’s disease

Nihonmatsu-Kikuchi, N., Yu, X-J., Matsuda, Y., Ozawa, N., Ito, T., Satou, K. et al Commincations Biol., 4:870 (2021)   The role of oligodendrocyte lineage cells, the largest glial population in the adult central nervous system (CNS), in the pathogenesis of Alzheimer’s disease (AD) remains elusive. Here, we developed a culture method for adult oligodendrocyte progenitor cells (aOPCs). Fibroblast growth factor 2 (FGF2) promotes survival and proliferation of NG2⁺ aOPCs in a serum-free defined medium; a subpopulation (~5%) of plexin-B3⁺ aOPCs was also found. FGF2 withdrawal decreased NG2⁺, but increased plexin-B3⁺ aOPCs and Aβ1-42 secretion. Plexin-B3⁺ aOPCs were distributed throughout the adult rat brain, although less densely than NG2⁺ aOPCs. Spreading depolarization induced delayed cortical plexin-B3⁺ aOPC gliosis in the ipsilateral remote cortex. Furthermore, extracellular Aβ1-42 accumulation was occasionally found around plexin-B3⁺ aOPCs near the lesions. In AD brains, virtually all cortical SPs were immunostained for plexin-B3, and plexin-B3 levels increased significantly in the Sarkosyl-soluble fractions. These findings suggest that plexin-B3⁺ aOPCs may play essential roles in AD pathogenesis, as natural Aβ-secreting cells.  

4.2339           Fas/FasL mediates NF-κBp65/PUMA-modulated hepatocytes apoptosis via autophagy to drive liver fibrosis

Tan, S., Liu, X., Chen, L., Wu, X., Tao, L., Pan, X., Tan, S., Liu, H., Jiang, J. and Wu, B. Cell Death & Disease, 12:474 (2021)   Fas/Fas ligand (FasL)-mediated cell apoptosis involves a variety of physiological and pathological processes including chronic hepatic diseases, and hepatocytes apoptosis contributes to the development of liver fibrosis following various causes. However, the mechanism of the Fas/FasL signaling and hepatocytes apoptosis in liver fibrogenesis remains unclear. The Fas/FasL signaling and hepatocytes apoptosis in liver samples from both human sections and mouse models were investigated. NF-κBp65 wild-type mice (p65^f/f), hepatocytes specific NF-κBp65 deletion mice (p65Δhepa), p53-upregulated modulator of apoptosis (PUMA) wild-type (PUMA-WT) and PUMA knockout (PUMA-KO) littermate models, and primary hepatic stellate cells (HSCs) were also used. The mechanism underlying Fas/FasL-regulated hepatocytes apoptosis to drive HSCs activation in fibrosis was further analyzed. We found Fas/FasL promoted PUMA-mediated hepatocytes apoptosis via regulating autophagy signaling and NF-κBp65 phosphorylation, while inhibition of autophagy or PUMA deficiency attenuated Fas/FasL-modulated hepatocytes apoptosis and liver fibrosis. Furthermore, NF-κBp65 in hepatocytes repressed PUMA-mediated hepatocytes apoptosis via regulating the Bcl-2 family, while NF-κBp65 deficiency in hepatocytes promoted PUMA-mediated hepatocytes apoptosis and enhanced apoptosis-linked inflammatory response, which contributed to the activation of HSCs and liver fibrogenesis. These results suggest that Fas/FasL contributes to NF-κBp65/PUMA-modulated hepatocytes apoptosis via autophagy to enhance liver fibrogenesis, and this network could be a potential therapeutic target for liver fibrosis.  

4.2340           Haploinsufficiency of the schizophrenia and autism risk gene Cyfip1 causes abnormal postnatal hippocampal neurogenesis through microglial and Arp2/3 mediated actin dependent mechanisms

Haan, N., Westacott, L.J., Carter, J., Owen, M.J., Gray, W:P., Hall, J. and Wilkinson, L.S: Translational Psychiatry, 11:313 (2021)   Genetic risk factors can significantly increase chances of developing psychiatric disorders, but the underlying biological processes through which this risk is effected remain largely unknown. Here we show that haploinsufficiency of Cyfip1, a candidate risk gene present in the pathogenic 15q11.2(BP1–BP2) deletion may impact on psychopathology via abnormalities in cell survival and migration of newborn neurons during postnatal hippocampal neurogenesis. We demonstrate that haploinsufficiency of Cyfip1 leads to increased numbers of adult-born hippocampal neurons due to reduced apoptosis, without altering proliferation. We show this is due to a cell autonomous failure of microglia to induce apoptosis through the secretion of the appropriate factors, a previously undescribed mechanism. Furthermore, we show an abnormal migration of adult-born neurons due to altered Arp2/3 mediated actin dynamics. Together, our findings throw new light on how the genetic risk candidate Cyfip1 may influence the hippocampus, a brain region with strong evidence for involvement in psychopathology.  

4.2341           The establishment of variant surface glycoprotein monoallelic expression revealed by single-cell RNA-seq of Trypanosoma brucei in the tsetse fly salivary glands

Hutchinson, S., Foulon, S., Crouzols, A., Menafra, R., Rotureau, B., Griffiths, A.D. and Bastin, P. PloS Pathogens, 17(9), e1009904 (2021)   The long and complex Trypanosoma brucei development in the tsetse fly vector culminates when parasites gain mammalian infectivity in the salivary glands. A key step in this process is the establishment of monoallelic variant surface glycoprotein (VSG) expression and the formation of the VSG coat. The establishment of VSG monoallelic expression is complex and poorly understood, due to the multiple parasite stages present in the salivary glands. Therefore, we sought to further our understanding of this phenomenon by performing single-cell RNA-sequencing (scRNA-seq) on these trypanosome populations. We were able to capture the developmental program of trypanosomes in the salivary glands, identifying populations of epimastigote, gamete, pre-metacyclic and metacyclic cells. Our results show that parasite metabolism is dramatically remodeled during development in the salivary glands, with a shift in transcript abundance from tricarboxylic acid metabolism to glycolytic metabolism. Analysis of VSG gene expression in pre-metacyclic and metacyclic cells revealed a dynamic VSG gene activation program. Strikingly, we found that pre-metacyclic cells contain transcripts from multiple VSG genes, which resolves to singular VSG gene expression in mature metacyclic cells. Single molecule RNA fluorescence in situ hybridisation (smRNA-FISH) of VSG gene expression following in vitro metacyclogenesis confirmed this finding. Our data demonstrate that multiple VSG genes are transcribed before a single gene is chosen. We propose a transcriptional race model governs the initiation of monoallelic expression.  

4.2342           DNA methylation patterns expose variations in enhancer-chromatin modifications during embryonic stem cell differentiation

Alajem, A., Roth, H., Ratgauzer, S., Bavli, D., Motzik, A., Lahav, S., Peled, I. and Ram, O. PloS Genetics, 17(4), e1009498 (2021)   In mammals, cellular identity is defined through strict regulation of chromatin modifications and DNA methylation that control gene expression. Methylation of cytosines at CpG sites in the genome is mainly associated with suppression; however, the reason for enhancer-specific methylation is not fully understood. We used sequential ChIP-bisulfite-sequencing for H3K4me1 and H3K27ac histone marks. By collecting data from the same genomic region, we identified enhancers differentially methylated between these two marks. We observed a global gain of CpG methylation primarily in H3K4me1-marked nucleosomes during mouse embryonic stem cell differentiation. This gain occurred largely in enhancer regions that regulate genes critical for differentiation. The higher levels of DNA methylation in H3K4me1- versus H3K27ac-marked enhancers, despite it being the same genomic region, indicates cellular heterogeneity of enhancer states. Analysis of single-cell RNA-seq profiles demonstrated that this heterogeneity correlates with gene expression during differentiation. Furthermore, heterogeneity of enhancer methylation correlates with transcription start site methylation. Our results provide insights into enhancer-based functional variation in complex biological systems.  

4.2343           Microfluidic-based imaging of complete Caenorhabditis elegans larval development

Berger, S., Spiri, S., DeMello, A. and Hajnal, A. Development, 148, dev199674 (2021)   Several microfluidic-based methods for Caenorhabditis elegans imaging have recently been introduced. Existing methods either permit imaging across multiple larval stages without maintaining a stable worm orientation, or allow for very good immobilization but are only suitable for shorter experiments. Here, we present a novel microfluidic imaging method that allows parallel live-imaging across multiple larval stages, while maintaining worm orientation and identity over time. This is achieved through an array of microfluidic trap channels carefully tuned to maintain worms in a stable orientation, while allowing growth and molting to occur. Immobilization is supported by an active hydraulic valve, which presses worms onto the cover glass during image acquisition only. In this way, excellent quality images can be acquired with minimal impact on worm viability or developmental timing. The capabilities of the devices are demonstrated by observing the hypodermal seam and P-cell divisions and, for the first time, the entire process of vulval development from induction to the end of morphogenesis. Moreover, we demonstrate feasibility of on-chip RNAi by perturbing basement membrane breaching during anchor cell invasion.  

4.2344           Neonatal low-density granulocytes internalize and kill bacteria but suppress monocyte function using extracellular DNA

Seman, B.G., Vance, J.K., Akers, S.M. and Robinson, C.M.

1. Cell Sci., 134, jcs252528 (2021)

  Low-density granulocytes (LDGs) are found abundantly in neonatal blood; however, there is limited mechanistic understanding of LDG interactions with bacteria and innate immune cells during acute infection. We aimed to determine how human neonatal LDGs may influence control of the bacterial burden at sites of infection, both individually and in the presence of mononuclear phagocytes. LDGs from human umbilical cord blood do phagocytose Escherichia coli O1:K1:H7 and traffic bacteria into acidic compartments. However, LDGs were significantly less efficient at bacterial uptake and killing compared to monocytes, and this activity was associated with a reduced inflammatory cytokine response. The presence of bacteria triggered the release of DNA (eDNA) from LDGs into the extracellular space that resembled neutrophil extracellular traps, but had limited anti-bacterial activity. Instead, eDNA significantly impaired monocyte control of bacteria during co-culture. These results suggest that LDG recruitment to sites of bacterial infection may compromise host protection in the neonate. Furthermore, our findings reveal novel insights into LDG activity during infection, clarify their inflammatory contributions relative to monocytes, and identify a novel LDG mechanism of immunosuppression.  

4.2345           Synergic effect of atorvastatin and ambrisentan on sinusoidal and hemodynamic alterations in a rat model of NASH

Bravo, M., Raurell, I., Barbera, A., Hide, D., Gil, M., Estrella, F., Salcedo, M.T., Augustin, S., Genesca, J. and Martell, M. Dis. Model. Mech., 14(5), dmm048884 (2021)   In non-alcoholic steatohepatitis (NASH), decreased nitric oxide and increased endothelin-1 (ET-1, also known as EDN1) released by sinusoidal endothelial cells (LSEC) induce hepatic stellate cell (HSC) contraction and contribute to portal hypertension (PH). Statins improve LSEC function, and ambrisentan is a selective endothelin-receptor-A antagonist. We aimed to analyse the combined effects of atorvastatin and ambrisentan on liver histopathology and hemodynamics, together with assessing the underlying mechanism in a rat NASH model. Diet-induced NASH rats were treated with atorvastatin (10 mg/kg/day), ambrisentan (30 mg/kg/day or 2 mg/kg/day) or a combination of both for 2 weeks. Hemodynamic parameters were registered and liver histology and serum biochemical determinations analysed. Expression of proteins were studied by immunoblotting. Conditioned media experiments were performed with LSEC. HSCs were characterized by RT-PCR, and a collagen lattice contraction assay was performed. Atorvastatin and ambrisentan act synergistically in combination to completely normalize liver hemodynamics and reverse histological NASH by 75%. Atorvastatin reversed the sinusoidal contractile phenotype, thus improving endothelial function, whereas ambrisentan prevented the contractile response in HSCs by blocking ET-1 response. Additionally, ambrisentan also increased eNOS (also known as Nos3) phosphorylation levels in LSEC, via facilitating the stimulation of endothelin-receptor-B in these cells. Furthermore, the serum alanine aminotransferase of the combined treatment group decreased to normal levels, and this group exhibited a restoration of the HSC quiescent phenotype. The combination of atorvastatin and ambrisentan remarkably improves liver histology and PH in a diet-induced NASH model. By recovering LSEC function, together with inhibiting the activation and contraction of HSC, this combined treatment may be an effective treatment for NASH patients.  

4.2346           CD8+CD103+ tissue-resident memory T cells convey reduced protective immunity in cutaneous squamous cell carcinoma

Lai, C., Coltart, G., Shapanis, A., Healy, C., Alabdukareem, A., Selvendran, S., Theaker, J., Sommerlad, M., Rose-Zerilli, M., Al-Shamkhani, A. and Healy, E.

1. Immunother. Cancer, 9:e001807 (2021)

  Background Tumor infiltrating lymphocytes play a key role in antitumor responses; however, while several memory T-cell subtypes have been reported in inflammatory and neoplastic conditions, the proportional representation of the different subsets of memory T cells and their functional significance in cancer is unclear. Keratinocyte skin cancer is one of the most common cancers globally, with cutaneous squamous cell cancer (cSCC) among the most frequent malignancies capable of metastasis. Methods Memory T-cell subsets were delineated in human cSCCs and, for comparison, in non-lesional skin and blood using flow cytometry. Immunohistochemistry was conducted to quantify CD103+ cells in primary human cSCCs which had metastasized (P-M) and primary cSCCs which had not metastasized (P-NM). TIMER2.0 (timer.cistrome.org) was used to analyze TCGA cancer survival data based on ITGAE expression. Immunofluorescence microscopy was performed to determine frequencies of CD8+CD103+ cells in P-M and P-NM cSCCs. Results Despite intertumoral heterogeneity, most cSCC T cells were CCR7−/CD45RA− effector/resident memory (TRM) lymphocytes, with naive, CD45RA+/CCR7− effector memory re-expressing CD45RA, CCR7+/L-selectin+ central memory and CCR7+/L-selectin− migratory memory lymphocytes accounting for smaller T-cell subsets. The cSCC CD8+ T-cell population contained a higher proportion of CD69+/CD103+ TRMs than that in non-lesional skin and blood. These cSCC CD69+/CD103+ TRMs exhibited increased IL-10 production, and higher CD39, CTLA-4 and PD-1 expression compared with CD103− TRMs in the tumor. CD103+ cells were more frequent in P-M than P-NM cSCCs. Analysis of TCGA data demonstrated that high expression of ITGAE (encoding CD103) was associated with reduced survival in primary cutaneous melanoma, breast carcinoma, renal cell carcinoma, kidney chromophobe cancer, adrenocortical carcinoma and lower grade glioma. Immunofluorescence microscopy showed that the majority of CD103 was present on CD8+ T cells and that CD8+CD103+ cells were significantly more frequent in P-M than P-NM cSCCs. Conclusion These results highlight CD8+CD103+ TRMs as an important functional T-cell subset associated with poorer clinical outcome in this cancer.  

4.2347           Mesoporous silica nanoparticles inflame tumors to overcome anti-PD-1 resistance through TLR4-NFκB axis

Sun, M., Gu, P., Yang, Y., Yu, L., Jiang, Z., Li, J., Le, Y., Chen, Y., Ba, Q. and Wang, H.

1. Immunother. Cancer, 9:e002508 (2021)

  Background The clinical benefits of antiprogrammed cell death protein 1 (PD-1) therapy are compromised by resistance in immunologically cold tumors. Convergence of immunotherapy and bioengineering is potential to overcome the resistance. Mesoporous silica nanoparticles (MSNs) are considered the most promising inorganic biological nanomaterials for clinical transformation, however, the fundamental influence of MSNs on immunotherapy is unclear. In this study, we aimed to investigate the role of MSNs in tumor resensitization and explore the feasibility of MSNs combined with anti-PD-1 in cancer therapy. Methods Intrinsic and acquired resistant tumors, as well as spontaneous and secondary tumor recurrence models, were used to evaluate the influence of MSNs and the synergistical effect with anti-PD-1 therapy. The roles of CD8⁺ cytotoxic T-lymphocytes (CTLs) and macrophages were assessed in Rag-1^-/- mice, ovalbumin/OT-1 TCR transgenic T-cell system, and other blocking mice models. Mechanistic studies were processed by transcriptomics analysis and conducted in primary cells, in vitro coculture systems, and Toll-like receptor 4 (TLR4) knockout mice. Results Both granular and rod-shaped MSNs efficiently overcame tumor resistance with dependence on diameter and aspect ratio. Only once injection of MSNs in prior to anti-PD-1 markedly improved the treatment efficacy, protective immunity, and prognosis. MSNs per se boosted infiltration of CTLs as the early event (days 2–3); and synergistically with anti-PD-1 therapy, MSNs rapidly established a T cell-inflamed microenvironment with abundant high-activated (interferon-γ/tumor necrosis factor-α/Perforin/GranzymeB) and low-exhausted (PD-1/lymphocyte-activation gene 3 (LAG-3)/T-cell immunoglobulin and mucin-domain containing-3 (TIM-3)) CTLs. Chemokines Ccl5/Cxcl9/Cxcl10, which were produced predominantly by macrophages, promoted MSNs-induced CTLs infiltration. MSNs led to high Ccl5/Cxcl9/Cxcl10 production in vitro and in mice through regulating TLR4-NFκB axis. Blocking TLR4-NFκB axis in macrophages or CTLs infiltration abrogated MSNs-induced resensitization to anti-PD-1 therapy. Conclusions MSNs efficiently and rapidly inflame immunologically cold tumors and resensitize them to anti-PD-1 therapy through TLR4-NFκB-Ccl5/Cxcl9/Cxcl10 axis. MSNs-based theranostic agents can serve as sensitizers for patients with resistant tumors to improve immunotherapy.  

4.2348           A High Activity Level Is Required for Augmented Muscle Capillarization in Older Women

Gliemann, L., Rytter, N., Yujia, L., Tamariz-Ellemann, A., Carter, H. and Hellstein, Y Med. & Science in Sports & Exercise, 53(5), 894-903 (2021)   Purpose This study aimed to evaluate the influence of lifelong regular physical activity on skeletal muscle capillarization in women. Methods Postmenopausal women, 61±4 yr old, were divided according to self-reported physical activity level over the past 20 yrs: sedentary (SED; n = 14), moderately active (MOD; n = 12), and very active (VERY; n = 15). Leg blood flow (LBF) was determined by ultrasound Doppler, and blood samples were drawn from the femoral artery and vein for calculation of leg oxygen uptake (LVO2) at rest and during one-legged knee extensor exercise. A skeletal muscle biopsy was obtained from the vastus lateralis and analyzed for capillarization and vascular endothelial growth factor (VEGF) and mitochondrial OXPHOS proteins. Platelets were isolated from venous blood and analyzed for VEGF content and effect on endothelial cell proliferation. Results The exercise-induced rise in LBF and LVO2 was faster (P = 0.008) in VERY compared with SED and MOD. Steady-state LBF and LVO2 were lower (P < 0.04) in MOD and VERY compared with SED. Capillary–fiber ratio and capillary density were greater (P < 0.03) in VERY (1.65 ± 0.48 and 409.3 ± 57.5) compared with MOD (1.30 ± 0.19 and 365.0 ± 40.2) and SED (1.30 ± 0.30 and 356.2 ± 66.3). Skeletal muscle VEGF and OXPHOS complexes I, II, and V were ~1.6-fold and ~1.25-fold (P < 0.01) higher, respectively, in VERY compared with SED. Platelets from all groups induced an approximately nine-fold (P < 0.001) increase in endothelial cell proliferation. Conclusion A very active lifestyle is associated with superior skeletal muscle exercise hemodynamics and greater potential for oxygen extraction concurrent with a higher skeletal muscle capillarization and mitochondrial capacity.  

4.2349           Longitudinal Peripheral Blood Transcriptional Analysis Reveals Molecular Signatures of Disease Progression in COVID-19 Patients

Yan, Q., Li, P., Ye, X., Huang, X., Feng, B. et al

1. Immunol., 206, 2146-2159 (2021)

  Coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with some patients developing severe illness or even death. Disease severity has been associated with increased levels of proinflammatory cytokines and lymphopenia. To elucidate the atlas of peripheral immune response and pathways that might lead to immunopathology during COVID-19 disease course, we performed a peripheral blood RNA sequencing analysis of the same patient’s samples collected from symptom onset to full recovery. We found that PBMCs at different disease stages exhibited unique transcriptome characteristics. We observed that SARS-CoV-2 infection caused excessive release of inflammatory cytokines and lipid mediators as well as an aberrant increase of low-density neutrophils. Further analysis revealed an increased expression of RNA sensors and robust IFN-stimulated genes expression but a repressed type I IFN production. SARS-CoV-2 infection activated T and B cell responses during the early onset but resulted in transient adaptive immunosuppression during severe disease state. Activation of apoptotic pathways and functional exhaustion may contribute to the reduction of lymphocytes and dysfunction of adaptive immunity, whereas increase in IL2, IL7, and IL15 may facilitate the recovery of the number and function of lymphocytes. Our study provides comprehensive transcriptional signatures of peripheral blood response in patients with moderate COVID-19.  

4.2350           A Versatile Microencapsulation Platform for Hyaluronic Acid and Polyethylene Glycol

Harrington, S., Ott, L., Karanu, F., Ramachandran, K. and Stehno-Bittel, L: Tissue Engineering: Part A, 27(3-4), 153-164 (2021)   Cell microencapsulation is a rapidly expanding field with broad potential for stem cell therapies and tissue engineering research. Traditional alginate microspheres suffer from poor biocompatibility, and microencapsulation of more advanced hydrogels is challenging due to their slower gelation rates. We have developed a novel, noncytotoxic, nonemulsion-based method to produce hydrogel microspheres compatible with a wide variety of materials, called core-shell spherification (CSS). Fabrication of microspheres by CSS derived from two slow-hardening hydrogels, hyaluronic acid (HA) and polyethylene glycol diacrylate (PEGDA), was characterized. HA microspheres were manufactured with two different crosslinking methods: thiolation and methacrylation. Microspheres of methacrylated HA (MeHA) had the greatest swelling ratio, the largest average diameter, and the lowest diffusion barrier. In contrast, PEGDA microspheres had the smallest diameters, the lowest swelling ratio, and the highest diffusion barrier, while microspheres of thiolated HA had characteristics that were in between the other two groups. To test the ability of the hydrogels to protect cells, while promoting function, diabetic NOD mice received intraperitoneal injections of PEGDA or MeHA microencapsulated canine islets. PEGDA microspheres reversed diabetes for the length of the study (up to 16 weeks). In contrast, islets encapsulated in MeHA microspheres at the same dose restored normoglycemia, but only transiently (3–4 weeks). Nonencapsulated canine islet transplanted at the same dose did not restore normoglycemia for any length of time. In conclusion, CSS provides a nontoxic microencapsulation procedure compatible with various hydrogel types.  

4.2351           Suppression of Th1 and Th17 Proinflammatory Cytokines and Upregulation of FOXP3 Expression by a Humanized Anti-DNAM-1 Monoclonal Antibody

Yamashita-Kaanemaru, Y., Oh-oka, K., Abe, F., Shibuya, K. and Shibuya, A. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 40(2). 52-59 (2021)   DNAM-1 is an activating immunoreceptor expressed on hematopoietic cells, including both CD4⁺ and CD8⁺ T cells, natural killer cells, and platelets. Since DNAM-1 is involved in the pathogenesis of various inflammatory diseases and cancers in humans as well as mouse models, it is a potential target for immunotherapy for these diseases. In this study, we generated a humanized neutralizing antihuman DNAM-1 monoclonal antibody (mAb), named TNAX101A, which contains an engineered Fc portion of human IgG1 to reduce Fc-mediated effector functions. We show that TNAX101A efficiently interfered the binding of DNAM-1 to its ligand CD155 and showed unique functions; it decreased production of the inflammatory cytokines such as interferon-gamma, tumor necrosis factor alpha, interleukin (IL)-6, IL-17A, and IL-17F by anti-CD3 antibody-stimulated or alloantigen-stimulated T cells and increased FOXP3 expression in anti-CD3-stimulated regulatory T (Treg) cells. These dual functions of TNAX101A may be advantageous for the treatment of T cell-mediated inflammatory diseases through both downregulation of effector T cell function and upregulation of Treg cell function.  

4.2352           CSF1R defines the mononuclear phagocyte system lineage in human blood in health and COVID-19

Combes, T.W., Orsenigo, F., Stewart, A., Mendis, A.S.J.R., Dunn-Walters, D., Gordon, S. and Martinez, F.O. Immunotherapy Advances, 1(1), ltab003 (2021)   Mononuclear phagocytes defend tissues, present antigens, and mediate recovery and healing. To date, we lack a marker to unify mononuclear phagocytes in humans or that informs us about their origin. Here, we reassess mononuclear phagocyte ontogeny in human blood through the lineage receptor CSF1R, in the steady state and in COVID-19. We define CSF1R as the first sensitive and reproducible pan-phagocyte lineage marker, to identify and enumerate all conventional monocytes, and the myeloid dendritic cells. In the steady state, CSF1R is sufficient for sorting and immuno-magnetic isolation. In pathology, changes in CSF1R are more sensitive than CD14 and CD16. In COVID-19, a significant drop in membrane CSF1R is useful for stratifying patients, beyond the power of cell categories published thus far, which fail to capture COVID-19 specific events. Importantly, CSF1R defines cells which are neither conventional monocytes nor DCs, which are missed in published analysis. CSF1R decrease can be linked ex vivo to high CSF1 levels. Blood assessment of CSF1R+ cells opens a developmental window to the Mononuclear Phagocyte System in transit from bone marrow to tissues, supports isolation and phenotypic characterisation, identifies novel cell types, and singles out CSF1R inhibition as therapeutic target in COVID-19 and other diseases.  

4.2353           Bound2Learn: a machine learning approach for classification of DNA-bound proteins from single-molecule tracking experiments

Kapadia, N., El-Hajj, Z.W. and Reyes-Lamothe, R. Nucleic Acids Res., 49(14), e79 (2021)   DNA-bound proteins are essential elements for the maintenance, regulation, and use of the genome. The time they spend bound to DNA provides useful information on their stability within protein complexes and insight into the understanding of biological processes. Single-particle tracking allows for direct visualization of protein–DNA kinetics, however, identifying whether a molecule is bound to DNA can be non-trivial. Further complications arise when tracking molecules for extended durations in processes with slow kinetics. We developed a machine learning approach, termed Bound2Learn, using output from a widely used tracking software, to robustly classify tracks in order to accurately estimate residence times. We validated our approach in silico, and in live-cell data from Escherichia coli and Saccharomyces cerevisiae. Our method has the potential for broad utility and is applicable to other organisms.  

4.2354           Induction of cytotoxic effector cells towards cholangiocellular, pancreatic, and colorectal tumor cells by activation of the immune checkpoint CD40/CD40L on dendritic cells

Sadeghlar, F., Vogt, A., Mohr, R.U., Mahn, R., van Beekum, K., Kornek, M., Weismüller, T.J., Braanchi, V., Matthaei, H., Toma, M., Schmidt-Wolf, I.G.H., Kalff, J., Strassburg, C.P. and Gonzalez-Carmona, M.A. Cancer Immunol. Immunother., 70, 1451-2464 (2021)   Introduction Gastrointestinal (GI) malignancies, such as cholangiocarcinoma, pancreatic carcinoma, and metastatic colorectal carcinoma, have a poor prognosis and effective therapeutic approaches are still challenging. Checkpoint inhibition with PD-1 or PDL-1 antibodies revealed promising results in different tumor entities; however, only few patients with GI tumors can potentially benefit from PD1/PDL1 inhibiting immunotherapy. Further immunotherapeutic strategies for GI malignancies are urgently needed. The aim of this study was to demonstrate that in vitro activation of the immune checkpoint CD40/CD40L can improve DC action towards bile duct, pancreas, and colorectal carcinoma. Methods Human DC were isolated from buffy coats from healthy donors, pulsed with tumor lysates and then transduced with adenoviruses encoding human CD40L (Ad-hCD40L). Using transwell assays, the effects of (m)CD40L on DC immunoactivation compared to (s)CD40L were analyzed. Surface marker and cytokine/chemokine expression were measured by flow cytometry, ELISA and cytokine arrays. Capacity of Ad-hCD40L-transduced DC to induce tumor-specific effector cells was tested using MTT proliferation assay and cytotoxicity assays. Apoptosis induction on tumor cells after culturing with supernatants of Ad-hCD40L-transduced DC was analyzed by flow cytometry. Results Ad-hCD40L transduction induced a high expression of (s)CD40L and (m)CD40L on DC and seemed to induce a strong cellular CD40/CD40L interaction among DC, leading to the formation of cell aggregates. Due to the CD40/CD40L interaction, a significant upregulation of DC maturation markers and a Th1-shift on cytokines/chemokines in the supernatant of DC were achieved. Interestingly, a pure Th1-shift was only achieved, when a cellular CD40/CD40L interaction among DC took place. (s)CD40L induced almost no upregulation of maturation markers and rather resulted in a Th2-cytokine expression, such as IL-10. Correspondingly, (m)CD40L-expressing DC led to significant proliferation and stimulation of tumor-specific effector cells with increased cytotoxicity towards pancreatic, bile duct and colorectal tumor cells. Supernatants of Ad-hCD40L-transduced DC could also induce apoptosis in the different tumor cells in vitro. Conclusion Stimulation of the immune checkpoint CD40L/CD40 by endogenous expression of (m)CD40L provokes a cellular interaction, which increases the immunomodulatory capacity of DC. A Th1 cytokine/chemokine expression is induced, leading to a significant proliferation and enabling cytotoxicity of effector cells towards human bile duct, pancreatic and colorectal tumor cells. The present data point to the promising approach for DC-based immunotherapy of gastrointestinal malignances by activating the CD40/CD40L immune checkpoint.  

4.2355           A Practical Guide to Rodent Islet Isolation and Assessment Revisited

Corbin, K.L., West, H.L., Brodsky, S., Whitticar, N.B., Koch, W.J. and Nunemaker, C.S. Biol. Procedures Online, 23:7 (2021)   Insufficient insulin secretion is a key component of both type 1 and type 2 diabetes. Since insulin is released by the islets of Langerhans, obtaining viable and functional islets is critical for research and transplantation. The effective and efficient isolation of these small islands of endocrine cells from the sea of exocrine tissue that is the rest of the pancreas is not necessarily simple or quick. Choosing and administering the digestive enzyme, separation of the islets from acinar tissue, and culture of islets are all things that must be considered. The purpose of this review is to provide a history of the development of islet isolation procedures and to serve as a practical guide to rodent islet research for newcomers to islet biology. We discuss key elements of mouse islet isolation including choosing collagenase, the digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews techniques for assessing islet viability and function such as visual assessment, glucose-stimulated insulin secretion and intracellular calcium measurements. A detailed protocol is provided that describes a common method our laboratory uses to obtain viable and functional mouse islets for in vitro study. This review thus provides a strong foundation for successful procurement and purification of high-quality mouse islets for research purposes.  

4.2356           Assessment of the impact of mitochondrial genotype upon drug-induced mitochondrial dysfunction in platelets derived from healthy volunteers

Ball, A.L., Bloch, K.M., Rainbow, L., Liu, X., Kenny, J., Lyon, J.J., Gregory, R., Alfirevic, A. and Chadwick, A.E. Arch. Toxicol., 95, 1335-1347 (2021)   Mitochondrial DNA (mtDNA) is highly polymorphic and encodes 13 proteins which are critical to the production of ATP via oxidative phosphorylation. As mtDNA is maternally inherited and undergoes negligible recombination, acquired mutations have subdivided the human population into several discrete haplogroups. Mitochondrial haplogroup has been found to significantly alter mitochondrial function and impact susceptibility to adverse drug reactions. Despite these findings, there are currently limited models to assess the effect of mtDNA variation upon susceptibility to adverse drug reactions. Platelets offer a potential personalised model of this variation, as their anucleate nature offers a source of mtDNA without interference from the nuclear genome. This study, therefore, aimed to determine the effect of mtDNA variation upon mitochondrial function and drug-induced mitochondrial dysfunction in a platelet model. The mtDNA haplogroup of 383 healthy volunteers was determined using next-generation mtDNA sequencing (Illumina MiSeq). Subsequently, 30 of these volunteers from mitochondrial haplogroups H, J, T and U were recalled to donate fresh, whole blood from which platelets were isolated. Platelet mitochondrial function was tested at basal state and upon treatment with compounds associated with both mitochondrial dysfunction and adverse drug reactions, flutamide, 2-hydroxyflutamide and tolcapone (10–250 μM) using extracellular flux analysis. This study has demonstrated that freshly-isolated platelets are a practical, primary cell model, which is amenable to the study of drug-induced mitochondrial dysfunction. Specifically, platelets from donors of haplogroup J have been found to have increased susceptibility to the inhibition of complex I-driven respiration by 2-hydroxyflutamide. At a time when individual susceptibility to adverse drug reactions is not fully understood, this study provides evidence that inter-individual variation in mitochondrial genotype could be a factor in determining sensitivity to mitochondrial toxicants associated with costly adverse drug reactions.  

4.2357           Isolation of bacteria from artificial bronchoalveolar lavage fluid using density gradient centrifugation and their accessibility by Raman spectroscopy

Wichmann, C., Rösch, P. and Popp, J. Anal. Bioanal. Chem., 413, 5193-5200 (2021)   Raman spectroscopy is an analytical method to identify medical samples of bacteria. Because Raman spectroscopy detects the biochemical properties of a cell, there are many factors that can influence and modify the Raman spectra of bacteria. One possible influence is a proper method for isolation of the bacteria. Medical samples in particular never occur in purified form, so a Raman-compatible isolation method is needed which does not affect the bacteria and thus the resulting spectra. In this study, we present a Raman-compatible method for isolation of bacteria from bronchoalveolar lavage (BAL) fluid using density gradient centrifugation. In addition to measuring the bacteria from a patient sample, the yield and the spectral influence of the isolation on the bacteria were investigated. Bacteria isolated from BAL fluid show additional peaks in comparison to pure culture bacteria, which can be attributed to components in the BAL sample. The isolation gradient itself has no effect on the spectra, and with a yield of 63% and 78%, the method is suitable for isolation of low concentrations of bacteria from a complex matrix.  

4.2358           Selective involution of thymic medulla by cyclosporine A with a decrease of mature thymic epithelia, XCR1+ dendritic cells, and epithelium-free areas containing Foxp3+ thymic regulatory T cells

Sawanobori, Y., Kitazawa, Y., Ueta, H., matsuno, K. and Tokuda, N: Histochem. Cell Biol., 156, 133-146 (2021)   Immunosuppressive drugs such as cyclosporine A (CSA) can disrupt thymic structure and functions, ultimately inducing syngeneic/autologous graft-versus-host disease together with involuted medullas. To elucidate the effects of CSA on the thymus more precisely, we analyzed the effects of CSA on the thymus and T cell system using rats. In addition to confirming the phenomena already reported, we newly found that the proportion of recent thymic emigrants also greatly decreased, suggesting impaired supply. Immunohistologically, the medullary thymic epithelial cells (mTECs) presented with a relative decrease in the subset with a competent phenotype and downregulation of class II major histocompatibility complex molecules. In control rats, thymic dendritic cells (DCs) comprised two subsets, XCR1⁺SIRP1α⁻CD4⁻ and XCR1⁻SIRP1α⁺CD4⁺. The former had a tendency to selectively localize in the previously-reported epithelium-containing areas of the rat medullas, and the number was significantly reduced by CSA treatment. The epithelium-free areas, another unique domains in the rat medullas, contained significantly more Foxp3⁺ thymic Tregs. With CSA treatment, the epithelium-free areas presented strong involution, and the number and distribution of Tregs in the medulla were greatly reduced. These results suggest that CSA inhibits the production of single-positive thymocytes, including Tregs, and disturbs the microenvironment of the thymic medulla, with a decrease of the competent mTECs and disorganization of epithelium-free areas and DC subsets, leading to a generation of autoreactive T cells with selective medullary involution.  

4.2359           Hepatic progenitor cells promote the repair of schistosomiasis liver injury by inhibiting IL-33 secretion in mice

Zhang, B., Wu, X., Li, J., Ning, A., Zhang, B., Liu, J., Song, L., Yan, C., Sun, X., Zheng, K. and Wu, Z. Stem Cell Res. Therapy, 12:546 (2021)   Background Hepatic schistosomiasis, a chronic liver injury induced by long-term Schistosoma japonicum (S. japonicum) infection, is characterized by egg granulomas and fibrotic pathology. Hepatic progenitor cells (HPCs), which are nearly absent or quiescent in normal liver, play vital roles in chronic and severe liver injury. But their role in the progression of liver injury during infection remains unknown. Methods In this study, the hepatic egg granulomas, fibrosis and proliferation of HPCs were analyzed in the mice model of S. japonicum infection at different infectious stages. For validating the role of HPCs in hepatic injury, tumor necrosis factor-like-weak inducer of apoptosis (TWEAK) and TWEAK blocking antibody were used to manipulate the proliferation of HPCs in wild-type and IL-33^−/− mice infected with S. japonicum. Results We found that the proliferation of HPCs was accompanied by inflammatory granulomas and fibrosis formation. HPCs expansion promoted liver regeneration and inhibited inflammatory egg granulomas, as well as the deposition of fibrotic collagen. Interestingly, the expression of IL-33 was negatively associated with HPCs’ expansion. There were no obvious differences of liver injury caused by infection between wild-type and IL-33^−/− mice with HPCs’ expansion. However, liver injury was more attenuated in IL-33^−/− mice than wild-type mice when the proliferation of HPCs was inhibited by anti-TWEAK. Conclusions Our data uncovered a protective role of HPCs in hepatic schistosomiasis in an IL-33-dependent manner, which might provide a promising progenitor cell therapy for hepatic schistosomiasis.  

4.2360           Metataxonomic analysis of tissue-associated microbiota in grooved carpet-shell (Ruditapes decussatus) and Manila (Ruditapes philippinarum) clams

Gerpe, D., Lasa, A., Lema, A. and Romalde, J.L. Int. Microbiol., 23, 607-618 (2021)   Culture-dependent techniques only permit the study of a low percentage of the microbiota diversity in the environment. The introduction of next generation sequencing (NGS) technologies shed light into this hidden microbial world, providing a better knowledge on the general microbiota and, specifically, on the microbial populations of clams. Tissue-associated microbiota of Ruditapes decussatus and Ruditapes philippinarum (mantle, gills, gonad and hepatopancreas) was analysed in two different locations of Galicia (northwest of Spain) during Spring (April) and Autumn (October), employing a metataxonomic approach. High bacterial diversity and richness were found in all samples where a total of 22,044 OTUs were obtained. In most samples, phylum Proteobacteria was most frequently retrieved, although other phyla as Actinobacteria, Bacteroidetes, Tenericutes, Firmicutes or Chlamydiae also appeared at high relative abundances in the samples. At genus level, great variation was found across tissues and sampling periods. A Nonmetric Multidimensional Scaling (NMDS) and a hierarchical clustering analysis allowed to further analyse the factors responsible for the differences among groups of samples in the different sites. Results showed sample ordination based on tissue origin and sampling periods, pointing out that the microbiota was influenced by these factors. Indeed, predominance of certain genera was observed, such as Endozoicomonas or Methylobacterium in gills and gonads, respectively, suggesting that selection of specific bacterial taxa is likely to occur. So far, this study provided a general picture of the tissue associated microbial population structure in R. decussatus and R. philippinarum clams, which, ultimately, allowed the identification of specific tissue-related taxa.  

4.2361           Pathogenic roles and therapeutic potential of the CCL8–CCR8 axis in a murine model of IgG4-related sialadenitis

Honda, F., Tsuboi, H., Ono, Y., Abe, S., Takahashi, H., Ito, K., et al Arthritis Res. Ther., 23:214 (2021)   Background Our previous studies reveal that CCL18-CCR8 chemokine axis is upregulated in patients of immunoglobulin G4-related disease (IgG4-RD), suggesting that the CCL18–CCR8 axis is implicated in the etiology of IgG4-RD, although whether this axis has a potential as a therapeutic target remains unclear. Our purpose was to clarify the pathogenic roles and therapeutic potential of the murine CCL8 (analog of human CCL18)–CCR8 axis by using an animal model of IgG4-RD (LAT Y136F knockin mice; LAT mice). Methods We compared the infiltration of inflammatory cells and the fibrosis of the salivary glands of 6-week-old LAT mice and littermate mice. The expressions of Ccl8 and Ccr8 were also compared. Next, we investigated the therapeutic effects of intravenous administration of anti-CCL8 neutralizing antibody in LAT mice against inflammation and fibrosis of the salivary glands. We also investigated the effects of stimulation with recombinant mouse CCL8 on the collagen production in a mouse fibroblast cell line (NIH/3 T3) in vitro. Results When compared with the littermates, the LAT mice showed apparent infiltration of inflammatory cells and fibrosis in the salivary glands. The focus and fibrosis score in the salivary glands were significantly higher in the LAT mice than in the littermates. The expression levels of Ccl8 in the spleen and of Ccr8 in the salivary glands were significantly higher in the LAT mice than in the littermates. Anti-CCL8 antibody significantly improved the focus and fibrosis score in the salivary glands of the LAT mice. In vitro, stimulation with recombinant mouse CCL8 significantly increased the expression of collagen and ERK1/2 phosphorylation in NIH/3 T3. Conclusion We clarified the overexpression and therapeutic potential of the mouse CCL8–CCR8 axis in LAT mice, which could play a crucial role in fibrosis via ERK1/2 phosphorylation, as well as the chemotaxis of inflammatory cells. The human CCL18–CCR8 axis might be a novel therapeutic target for IgG4-RD.  

4.2362           Increasing toll-like receptor 2 on astrocytes induced by Schwann cell-derived exosomes promotes recovery by inhibiting CSPGs deposition after spinal cord injury

Pan, D., Li, Y., Yang, F., Lv, Z., Zhu, S., Shao, Y., Huang, Y., Ning, G. and Feng, S.

1. Neuroinflamm., 18:172 (2021)

  Background Traumatic spinal cord injury (SCI) is a severely disabling disease that leads to loss of sensation, motor, and autonomic function. As exosomes have great potential in diagnosis, prognosis, and treatment of SCI because of their ability to easily cross the blood–brain barrier, the function of Schwann cell-derived exosomes (SCDEs) is still largely unknown. Methods A T10 spinal cord contusion was established in adult female mice. SCDEs were injected into the tail veins of mice three times a week for 4 weeks after the induction of SCI, and the control group was injected with PBS. High-resolution transmission electron microscope and western blot were used to characterize the SCDEs. Toll-like receptor 2 (TLR2) expression on astrocytes, chondroitin sulfate proteoglycans (CSPGs) deposition and neurological function recovery were measured in the spinal cord tissues of each group by immunofluorescence staining of TLR2, GFAP, CS56, 5-HT, and β-III-tublin, respectively. TLR2^f/f mice were crossed to the GFAP-Cre strain to generate astrocyte specific TLR2 knockout mice (TLR2^−/−). Finally, western blot analysis was used to determine the expression of signaling proteins and IKKβ inhibitor SC-514 was used to validate the involved signaling pathway. Results Here, we found that TLR2 increased significantly on astrocytes post-SCI. SCDEs treatment can promote functional recovery and induce the expression of TLR2 on astrocytes accompanied with decreased CSPGs deposition. The specific knockout of TLR2 on astrocytes abolished the decreasing CSPGs deposition and neurological functional recovery post-SCI. In addition, the signaling pathway of NF-κB/PI3K involved in the TLR2 activation was validated by western blot. Furthermore, IKKβ inhibitor SC-514 was also used to validate this signaling pathway. Conclusion Thus, our results uncovered that SCDEs can promote functional recovery of mice post-SCI by decreasing the CSPGs deposition via increasing the TLR2 expression on astrocytes through NF-κB/PI3K signaling pathway.  

4.2363           Efficient isolation and purification of tissue-specific protoplasts from tea plants (Camellia sinensis (L.) O. Kuntze)

Xu, X-f., Zhu, H-y., Ren, Y-f., Feng, C., Ye, Z-h., Cai, H-m., Wan, X-c. and Peng, C-y. Plant Methods, 17:84 (2021)   Background Plant protoplasts constitute unique single-cell systems that can be subjected to genomic, proteomic, and metabolomic analysis. An effective and sustainable method for preparing protoplasts from tea plants has yet to be established. The protoplasts were osmotically isolated, and the isolation and purification procedures were optimized. Various potential factors affecting protoplast preparation, including enzymatic composition and type, enzymatic hydrolysis duration, mannitol concentration in the enzyme solution, and iodixanol concentration, were evaluated. Results The optimal conditions were 1.5% (w/v) cellulase and 0.4–0.6% (w/v) macerozyme in a solution containing 0.4 M mannitol, enzymatic hydrolysis over 10 h, and an iodixanol concentration of 65%. The highest protoplast yield was 3.27 × 10⁶ protoplasts g⁻¹ fresh weight. As determined through fluorescein diacetate staining, maximal cell viability was 92.94%. The isolated protoplasts were round and regularly shaped without agglomeration, and they were less than 20 μm in diameter. Differences in preparation, with regard to yield and viability in the tissues (roots, branches, and leaves), cultivars, and cultivation method, were also observed. Conclusions In summary, we reported on a simple, efficient method for preparing protoplasts of whole-organ tissue from tea plant. The findings are expected to contribute to the rapid development of tea plant biology.  

4.2364           Pancreatic Islet Purification from Large Mammals and Humans Using a COBE 2991 Cell Processor versus Large Plastic Bottles

Noguchi, H. et al

1. Clin. Med., 10:10 (2021)

  The islet purification step in clinical islet isolation is important for minimizing the risks associated with intraportal infusion. Continuous density gradient with a COBE 2991 cell processor is commonly used for clinical islet purification. However, the high shear force involved in the purification method using the COBE 2991 cell processor causes mechanical damage to the islets. We and other groups have shown human/porcine islet purification using large cylindrical plastic bottles. Shear stress can be minimized or eliminated using large cylindrical plastic bottles because the bottles do not have a narrow segment and no centrifugation is required during tissue loading and the collection processes of islet purification. This review describes current advances in islet purification from large mammals and humans using a COBE 2991 cell processor versus large cylindrical plastic bottles.  

4.2365           Regional Delivery of Anti-PD-1 Agent for Colorectal Liver Metastases Improves Therapeutic Index and Anti-Tumor Activity

Chai, L.F., hardaway, J.C., Heatherton, K.R., O’Connell, K.P., Lopes, M.C., Rabinowitz, B.A., Ghosh, C.C., Guha, P., Jaroch, D.J., Cox, B.F. and Katz, S.C. Vaccines, 9:807 (2021)   Metastatic liver tumors have presented challenges with the use of checkpoint inhibitors (CPIs), with only limited success. We hypothesize that regional delivery (RD) of CPIs can improve activity in the liver and minimize systemic exposure, thereby reducing immune-related adverse events (irAE). Using a murine model of colorectal cancer liver metastases (LM), we confirmed high levels of PD-L1 expression on the tumor cells and liver myeloid-derived suppressor cells (L-MDSC). In vivo, we detected improved LM response at 3 mg/kg on PTD7 via portal vein (PV) regional delivery as compared to 3 mg/kg via tail vein (TV) systemic delivery (p = 0.04). The minimal effective dose at PTD7 was 5 mg/kg (p = 0.01) via TV and 0.3 mg/kg (p = 0.02) via PV. We detected 6.7-fold lower circulating CPI antibody levels in the serum using the 0.3 mg/kg PV treatment compared to the 5 mg/kg TV cohort (p < 0.001) without increased liver toxicity. Additionally, 3 mg/kg PV treatment resulted in increased tumor cell apoptotic signaling compared to 5 mg/kg TV (p < 0.05). Therefore, RD of an anti-PD-1 CPI therapy for CRCLM may improve the therapeutic index by reducing the total dose required and limiting the systemic exposure. These advantages could expand CPI indications for liver tumors.  

4.2366           Interleukin-17A Triggers the Release of Platelet-Derived Factors Driving Vascular Endothelial Cells toward a Pro-Angiogenic State

Gatsiou, A., Sopova, K., Tselepis, A. and Stellos, K. Cells, 10:1855 (2021)   Platelets comprise a highly interactive immune cell subset of the circulatory system traditionally known for their unique haemostatic properties. Although platelets are considered as a vault of growth factors, cytokines and chemokines with pivotal role in vascular regeneration and angiogenesis, the exact mechanisms by which they influence vascular endothelial cells (ECs) function remain underappreciated. In the present study, we examined the role of human IL-17A/IL-17RA axis in platelet-mediated pro-angiogenic responses. We reveal that IL-17A receptor (IL-17RA) mRNA is present in platelets transcriptome and a profound increase is documented on the surface of activated platelets. By quantifying the protein levels of several factors, involved in angiogenesis, we identified that IL-17A/IL17RA axis selectively induces the release of vascular endothelial growth factor, interleukin -2 and -4, as well as monocyte chemoattractant protein -1 from treated platelets. However, IL-17A exerted no effect on the release of IL-10, an anti-inflammatory factor with potentially anti-angiogenic properties, from platelets. Treatment of human endothelial cell two-dimensional tubule networks or three-dimensional spheroid and mouse aortic ring structures with IL-17A-induced platelet releasate evoked pro-angiogenic responses of ECs. Our findings suggest that IL-17A may critically affect platelet release of pro-angiogenic factors driving ECs towards a pro-angiogenic state  

4.2367           Poplar-Assisted Bioremediation for Recovering a PCB and Heavy-Metal-Contaminated Area

Ancona, V., Rascio, I., Aimola, G., Campanale, C., Grenni, P., Di Lenola, M., Garbini, G.L., Uricchio, V.F. and Caracciolo, A.B. Agriculture, 11:689 (2021)   A Monviso clone has been applied to promote PCB degradation in a soil historically contaminated by polychlorinated biphenyls (PCBs) and heavy metals (HMs). The multi-contaminated area is located in Southern Italy. PCBs, HMs, and the soil microbial community (abundance, viability, and structure) were analysed in selected plots of the poplar-treated area. At 900 days after poplar planting, chemical analyses showed that PCBs and most of HMs diminished under the Italian legal limits. The overall results suggest that the poplar clone was effective in promoting PCB rhizodegradation and HM phytostabilization. Organic carbon content increased strongly in the rhizosphere of the planted plots. Microbiological results highlighted an overall increase in microbial abundance, cell viability, and the presence of bacterial groups involved in PCB degradation. The poplar-based bioremediation technology is a nature-based solution able to promote the recovery of soil quality in terms of contaminant removal, increase in organic carbon, and stimulation of autochthonous bacterial groups able to transform PCBs.  

4.2368           Thermophilic Anaerobic Digestion of Second Cheese Whey: Microbial Community Response to H2 Addition in a Partially Immobilized Anaerobic Hybrid Reactor

Lembo, G., Rosa, S., Miritana, V.m., Marone, A., Massini, G., Fenice, M. and Signorini, A. Process, 9:43 (2021)   In this study, we investigated thermophilic (55 °C) anaerobic digestion (AD) performance and microbial community structure, before and after hydrogen addition, in a novel hybrid gas-stirred tank reactor (GSTR) implemented with a partial immobilization of the microbial community and fed with second cheese whey (SCW). The results showed that H₂ addition led to a 25% increase in the methane production rate and to a decrease of 13% in the CH₄ concentration as compared with the control. The recovery of methane content (56%) was reached by decreasing the H₂ flow rate. The microbial community investigations were performed on effluent (EF) and on interstitial matrix (IM) inside the immobilized area. Before H₂ addition, the Anaerobaculaceae (42%) and Lachnospiraceae (27%) families dominated among bacteria in the effluent, and the Thermodesulfobiaceae (32%) and Lachnospiraceae (30%) families dominated in the interstitial matrix. After H₂ addition, microbial abundance showed an increase in the bacteria and archaea communities in the interstitial matrix. The Thermodesulfobiaceae family (29%)remained dominant in the interstitial matrix, suggesting its crucial role in the immobilized community and the SHA-31 family was enriched in both the effluent (36%) and the interstitial matrix (15%). The predominance of archaea Methanothermobacter thermoautrophicus indicated that CH₄ was produced almost exclusively by the hydrogenotrophic pathway  

4.2369           Generation of a Novel Nkx6-1 Venus Fusion Reporter Mouse Line

Burtscher, I., Tarquis-Medina, M., Salinno, C., Schirge, S., Beckenbauer, J., Bakhti, M. and Lickert, H. Int. J. Mol. Sci., 22:3434 (2021)   Nkx6-1 is a member of the Nkx family of homeodomain transcription factors (TFs) that regulates motor neuron development, neuron specification and pancreatic endocrine and β-cell differentiation. To facilitate the isolation and tracking of Nkx6-1-expressing cells, we have generated a novel Nkx6-1 Venus fusion (Nkx6-1-VF) reporter allele. The Nkx6-1-VF knock-in reporter is regulated by endogenous cis-regulatory elements of Nkx6-1 and the fluorescent protein fusion does not interfere with the TF function, as homozygous mice are viable and fertile. The nuclear localization of Nkx6-1-VF protein reflects the endogenous Nkx6-1 protein distribution. During embryonic pancreas development, the reporter protein marks the pancreatic ductal progenitors and the endocrine lineage, but is absent in the exocrine compartment. As expected, the levels of Nkx6-1-VF reporter are upregulated upon β-cell differentiation during the major wave of endocrinogenesis. In the adult islets of Langerhans, the reporter protein is exclusively found in insulin-secreting β-cells. Importantly, the Venus reporter activities allow successful tracking of β-cells in live-cell imaging and their specific isolation by flow sorting. In summary, the generation of the Nkx6-1-VF reporter line reflects the expression pattern and dynamics of the endogenous protein and thus provides a unique tool to study the spatio-temporal expression pattern of this TF during organ development and enables isolation and tracking of Nkx6-1-expressing cells such as pancreatic β-cells, but also neurons and motor neurons in health and disease.  

4.2370           Sperm Oxidative Stress during In Vitro Manipulation and Its Effects on Sperm Function and Embryo Development

Gualtieri, R., Kalthur, G., Barbato, V., Longobardi, S., Di Rella, F., Adiga, S.K. and Talevi, R. Antioxidants, 10:1025 (2021)   Reactive oxygen species (ROS) generated at low levels during mitochondrial respiration have key roles in several signaling pathways. Oxidative stress (OS) arises when the generation of ROS exceeds the cell’s antioxidant scavenging ability and leads to cell damage. Physiological ROS production in spermatozoa regulates essential functional characteristics such as motility, capacitation, acrosome reaction, hyperactivation, and sperm-oocyte fusion. OS can have detrimental effects on sperm function through lipid peroxidation, protein damage, and DNA strand breakage, which can eventually affect the fertility of an individual. Substantial evidence in the literature indicates that spermatozoa experiencing OS during in vitro manipulation procedures in human- and animal-assisted reproduction are increasingly associated with iatrogenic ROS production and eventual impairment of sperm function. Although a direct association between sperm OS and human assisted reproductive techniques (ART) outcomes after in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI) is still a matter of debate, studies in animal models provide enough evidence on the adverse effects of sperm OS in vitro and defective fertilization and embryo development. This review summarized the literature on sperm OS in vitro, its effects on functional ability and embryo development, and the approaches that have been proposed to reduce iatrogenic sperm damage and altered embryonic development.  

4.2371           Polymerized Albumin Receptor of Hepatitis B Virus for Evading the Reticuloendothelial System

Takagi, K., Somiya, M., Jung, J., Iljima, M. and Kuroda, S. Pharmaceuticals, 14:408 (2021)   Various strategies, such as optimization of surface chemistry, size, shape, and charge, have been undertaken to develop nanoparticles (NPs) as DDS (drug delivery system) nanocarriers for evading the reticuloendothelial system (RES) in vivo. We previously developed a hollow NP composed of hepatitis B virus (HBV) surface antigen L proteins and lipid bilayers, hereinafter referred to as bio-nanocapsule (BNC), as a nonviral DDS nanocarrier. Such a BNC harbors the HBV-derived human hepatic cell-specific infection mechanism, and intravenously injected BNCs by themselves were shown to avoid clearance by RES-rich organs and accumulate in target tissues. In this study, since the surface modification with albumins is known to prolong the circulation time of nanomedicines, we examined whether the polymerized albumin receptor (PAR) of BNCs contributes to RES evasion in mouse liver. Our results show that NPs conjugated with peptides possessing sufficient PAR activity were captured by Kupffer cells less efficiently in vitro and were able to circulate for a longer period of time in vivo. Comparing with polyethylene glycol, PAR peptides were shown to reduce the recognition by RES to equal content. Taken together, our results strongly suggest that the PAR domain of BNCs, as well as HBV, harbors an innate RES evasion mechanism. Therefore, the surface modification with PAR peptides could be an alternative strategy for improving the pharmacodynamics and pharmacokinetics of forthcoming nanomedicines.  

4.2372           A Modified Flotation Density Gradient Centrifugation Technique Improves the Semen Quality of Stallions with a High DNA Fragmentation Index

Umair, M., Henning, H., Stout, T.A.E. and Claes, A. Animals, 11:1973 (2021)   Sperm DNA fragmentation compromises fertilization and early embryo development. Since spermatozoa lack the machinery to repair DNA damage, to improve the likelihood of establishing a healthy pregnancy, it is preferable to process ejaculates of stallions with a high sperm DNA fragmentation index (DFI) before artificial insemination or intracytoplasmic sperm injection. The aim of this study was to examine a modified flotation density gradient centrifugation (DGC) technique in which semen was diluted with a colloid solution (Opti-prep^TM) to increase its density prior to layering between colloid layers of lower and higher density. The optimal Opti-prep^TM solution (20–60%) for use as the bottom/cushion layer was first determined, followed by a comparison between a modified sedimentation DGC and the modified flotation DGC technique, using different Opti-prep^TM solutions (20%, 25% and 30%) as the top layer. Finally, the most efficient DGC technique was selected to process ejaculates from Friesian stallions (n = 3) with high sperm DFI (>20%). The optimal Opti-prep^TM solution for the cushion layer was 40%. The modified sedimentation technique resulted in two different sperm populations, whereas the modified flotation technique yielded three populations. Among the variants tested, the modified flotation DGC using 20% Opti-prep^TM as the top layer yielded the best results; the average sperm recovery was 57%; the DFI decreased significantly (from 12% to 4%) and the other sperm quality parameters, including progressive and total motility, percentages of spermatozoa with normal morphology and viable spermatozoa with an intact acrosome, all increased (p < 0.05). In Friesian stallions with high sperm DFI, the modified flotation DGC markedly decreased the DFI (from 31% to 5%) and significantly improved the other semen quality parameters, although sperm recovery was low (approximately 20%). In conclusion, stallion sperm DFI and other sperm quality parameters can be markedly improved using a modified flotation DGC technique employing a 40% Opti-prep^TM cushion and a 20% top layer.  

4.2373           Rickettsia parkeri with a Genetically Disrupted Phage Integrase Gene Exhibits Attenuated Virulence and Induces Protective Immunity against Fatal Rickettsioses in Mice

Arroyave, E., Hyseni, I., Burkhardt, N., Kuo, Y-F., Wang, T., Munderloh, U and Fang, R. Pathogens, 10:819 (2021)   Although rickettsiae can cause life-threatening infections in humans worldwide, no licensed vaccine is currently available. To evaluate the suitability of live-attenuated vaccine candidates against rickettsioses, we generated a Rickettsia parkeri mutant RPATATE_0245::pLoxHimar (named 3A2) by insertion of a modified pLoxHimar transposon into the gene encoding a phage integrase protein. For visualization and selection, R. parkeri 3A2 expressed mCherry fluorescence and resistance to spectinomycin. Compared to the parent wild type (WT) R. parkeri, the virulence of R. parkeri 3A2 was significantly attenuated as demonstrated by significantly smaller size of plaque, failure to grow in human macrophage-like cells, rapid elimination of Rickettsia and ameliorated histopathological changes in tissues in intravenously infected mice. A single dose intradermal (i.d.) immunization of R. parkeri 3A2 conferred complete protection against both fatal R. parkeri and R. conorii rickettsioses in mice, in association with a robust and durable rickettsiae-specific IgG antibody response. In summary, the disruption of RPATATE_0245 in R. parkeri resulted in a mutant with a significantly attenuated phenotype, potent immunogenicity and protective efficacy against two spotted fever group rickettsioses. Overall, this proof-of-concept study highlights the potential of R. parkeri mutants as a live-attenuated and multivalent vaccine platform in response to emergence of life-threatening spotted fever rickettsioses.  

4.2374           Pro-Inflammatory Cytokines and Antibodies Induce hnRNP A1 Dysfunction in Mouse Primary Cortical Neurons

Li, M., Haamilton, R., Salapa, H.E. and Levin, M.C. Brain Sciences, 11:1282 (2021)   Multiple sclerosis (MS) is an inflammatory disease of the central nervous system with a significant neurodegenerative component. Dysfunctional RNA-binding proteins (RBPs) are causally linked to neuronal damage and are a feature of MS, including the mislocalization of the RBP heterogeneous nuclear ribonucleoprotein A1 (A1). Here, we show that primary neurons exposed to pro-inflammatory cytokines and anti-A1 antibodies, both characteristic of an MS autoimmune response, displayed increased A1 mislocalization, stress granule formation, and decreased neurite length, a marker of neurodegeneration. These findings illustrate a significant relationship between secreted immune factors, A1 dysfunction, and neuronal damage in a disease-relevant model system.  

4.2375           m6A mRNA methylation-directed myeloid cell activation controls progression of NAFLD and obesity

Qin, Y., Li, B., Arumugam, S., Flavell, R.A., Li, H-B. and Ouyang, X. Cell Reports, 37, 109968 (2021)   N⁶-methyladenosine (m⁶A) RNA modification is a fundamental determinant of mRNA metabolism, but its role in innate immunity-driven non-alcoholic fatty liver disease (NAFLD) and obesity is not known. Here, we show that myeloid lineage-restricted deletion of the m⁶A “writer” protein Methyltransferase Like 3 (METTL3) prevents age-related and diet-induced development of NAFLD and obesity in mice with improved inflammatory and metabolic phenotypes. Mechanistically, loss of METTL3 results in the differential expression of multiple mRNA transcripts marked with m⁶A, with a notable increase of DNA Damage Inducible Transcript 4 (DDIT4) mRNA level. In METTL3-deficient macrophages, there is a significant downregulation of mammalian target of rapamycin (mTOR) and nuclear factor κB (NF-κB) pathway activity in response to cellular stress and cytokine stimulation, which can be restored by knockdown of DDIT4. Taken together, our findings identify the contribution of METTL3-mediated m⁶A modification of Ddit4 mRNA to macrophage metabolic reprogramming in NAFLD and obesity.  

4.2376           Mapping the evolution of T cell states during response and resistance to adoptive cellular therapy

Achireddy, P., Azizi, E., Burdziak, C., Alyea, E:P., Pe’er, D. and Wu, C.J. Cell Reports, 37, 109992 (2021)   To elucidate mechanisms by which T cells eliminate leukemia, we study donor lymphocyte infusion (DLI), an established immunotherapy for relapsed leukemia. We model T cell dynamics by integrating longitudinal, multimodal data from 94,517 bone marrow-derived single T cell transcriptomes in addition to chromatin accessibility and single T cell receptor sequencing from patients undergoing DLI. We find that responsive tumors are defined by enrichment of late-differentiated T cells before DLI and rapid, durable expansion of early differentiated T cells after treatment, highly similar to “terminal” and “precursor” exhausted subsets, respectively. Resistance, in contrast, is defined by heterogeneous T cell dysfunction. Surprisingly, early differentiated T cells in responders mainly originate from pre-existing and novel clonotypes recruited to the leukemic microenvironment, rather than the infusion. Our work provides a paradigm for analyzing longitudinal single-cell profiling of scenarios beyond adoptive cell therapy and introduces Symphony, a Bayesian approach to infer regulatory circuitry underlying T cell subsets, with broad relevance to exhaustion antagonists across cancers.  

4.2377           Survivin expression is essential for early activation of hepatic stellate cells and fibrosis progression in chronic liver injury

Sharma, S., Ghufran, S.M., Das, B., Roy, B., Ghose, S. and Biswas, S. Life Sciences, 287, 120119 (2021)   Aim Hepatic fibrosis in injured liver is characterized by the activation of hepatic stellate cells (HSCs) from their quiescent state. Survivin (BIRC5) is one of the key genes that are upregulated during activation of HSCs but their role in HSC activation and fibrosis progression is unknown. Here, we have investigated the role of survivin protein in early fibrogenic activation of HSCs and fibrosis progression in chronic liver injury. Materials & methods Primary quiescent HSCs were isolated from healthy mice liver through perfusion and cultured for fibrogenic activation. Survivin expression was suppressed by its pharmacological suppressant, YM155. We developed chronic liver injury induced fibrotic mice model through administrating repeated dose of CCl₄ for 2 weeks and 4 weeks. Mice were pre-treated with YM155 a week before CCl₄ administration till 2nd week of dosing and then discontinued. Hepatic parameters were characterized and underlying mechanisms were investigated. Key findings Survivin expression gradually increased along with the expression of αSMA, collagen I activation maker in HSCs during their activation from quiescent state. Survivin suppression through YM155 downregulated αSMA, collagen I. Pre-treatment of YM155 in mice ceased the early activation of HSCs and onset of fibrosis in injured liver. However, discontinuation of YM155 initiated the activation of HSCs and fibrosis progression that shows survivin expression in HSCs is essential for their early activation and onset of liver fibrosis. Significance Survivin expression induces with activation of HSCs and drives onset of liver fibrosis in injured liver. Targeting survivin protein in activated HSCs could be a potential anti-fibrotic therapeutic approach in chronic liver injury.  

4.2378           Comprehensive analysis of differentially expressed mRNA and circRNA in Ankylosing spondylitis patients’ platelets

Wang, T., Meng, S., Chen, P., Wei, L., Liu, C., Tang, D., Liu, D., Jiang, Z. and Hong, X. Exp. Cell Res., 409, 112895 (2021)   Ankylosing spondylitis (AS) is a chronic inflammatory disease significantly decreasing the quality of life. Platelets play an important and active role in the development of AS. Accumulating evidence demonstrated platelets contain diverse RNA repository inherited from megakaryocytes or microvesicles. Platelet RNAs are dynamically affected by pathological conditions and could be used as diagnostic or prognostic biomarkers. However, the role of the platelet RNAs in AS is elusive. In this study, we compared mRNA and circRNA profiles in platelets between AS patients and healthy controls using RNA sequencing and bioinformatic analysis, and found 4996 mRNAs and 2942 circRNAs were differently expressed. The significantly over-expressed mRNAs in AS patients are involved in platelet activity, gap junction, focal adhesion, rap1 and toll and Imd signaling pathway. The previous identified platelet-derived immune mediators such as P2Y1, P2Y12, PF4, GPIbα, CD40L, ICAM2, CCL5 (RANTES), TGF-β (TGF-β1 and TGF-β2) and PDGF (PDGFB and PDGFA) are also included in these over expressed mRNAs, implying these factors may trigger inflammatory cascades and promote the development of AS. Additionally, we found two down-regulated circRNA (circPTPN22 and circFCHSD2) from the intersection analyses of platelets and spinal ligament tissues of AS patients. The circRNA-miRNA-mRNA regulatory network of these two circRNAs was constructed, and the target mRNAs were enriched in Th17 cell differentiation, inflammatory bowel disease, cell adhesion molecules, cytokine-cytokine receptor interaction, Jak-STAT and Wnt signaling pathway, all these pathways participate in the bone remodeling and pro/anti-inflammatory immune regulation in AS. Then, qRT-PCR was performed to validate the expression of selected key mRNAs and circRNAs and the results demonstrated that the expression levels of P2Y12, GPIbα, circPTPN22 and circFCHSD2 were consistent with the sequencing analysis. In addition, the high expression of five predicted miRNAs interacting with circPTPN22 and circFCHSD2 were also detected in AS by qRT-PCR. Taken together, our study presents a comprehensive overview of mRNAs and circRNAs in platelets in AS patients and offers new insight into the mechanisms of platelet involving in the pathogenesis of AS. The mRNAs and circRNAs identified in this study may serve as candidates for diagnosis and targeted treatment of AS.  

4.2379           A weakly supervised deep learning approach for label-free imaging flow-cytometry-based blood diagnostics

Otesteanu, C.F., Ugrinic, M., Holzner, G., Stavrakis, S., DeMello, A. and Claassen, M. Cell Reports Methods, 1, 100094 (2021)   The application of machine learning approaches to imaging flow cytometry (IFC) data has the potential to transform the diagnosis of hematological diseases. However, the need for manually labeled single-cell images for machine learning model training has severely limited its clinical application. To address this, we present iCellCnn, a weakly supervised deep learning approach for label-free IFC-based blood diagnostics. We demonstrate the capability of iCellCnn to achieve diagnosis of Sézary syndrome (SS) from patient samples on the basis of bright-field IFC images of T cells obtained after fluorescence-activated cell sorting of human peripheral blood mononuclear cell specimens. With a sample size of four healthy donors and five SS patients, iCellCnn achieved a 100% classification accuracy. As iCellCnn is not restricted to the diagnosis of SS, we expect such weakly supervised approaches to tap the diagnostic potential of IFC by providing automatic data-driven diagnosis of diseases with so-far unknown morphological manifestations.  

4.2380           Axonal TDP-43 condensates drive neuromuscular junction disruption through inhibition of local synthesis of nuclear encoded mitochondrial proteins

Altman, T., Ionescu, A., Ibraheem, A., Priesmann, D., Gradus-Pery, T. et al Nature Comm., 12:6914 (2021)   Mislocalization of the predominantly nuclear RNA/DNA binding protein, TDP-43, occurs in motor neurons of ~95% of amyotrophic lateral sclerosis (ALS) patients, but the contribution of axonal TDP-43 to this neurodegenerative disease is unclear. Here, we show TDP-43 accumulation in intra-muscular nerves from ALS patients and in axons of human iPSC-derived motor neurons of ALS patient, as well as in motor neurons and neuromuscular junctions (NMJs) of a TDP-43 mislocalization mouse model. In axons, TDP-43 is hyper-phosphorylated and promotes G3BP1-positive ribonucleoprotein (RNP) condensate assembly, consequently inhibiting local protein synthesis in distal axons and NMJs. Specifically, the axonal and synaptic levels of nuclear-encoded mitochondrial proteins are reduced. Clearance of axonal TDP-43 or dissociation of G3BP1 condensates restored local translation and resolved TDP-43-derived toxicity in both axons and NMJs. These findings support an axonal gain of function of TDP-43 in ALS, which can be targeted for therapeutic development.  

4.2381           Formation of a protein corona on the surface of extracellular vesicles in blood plasma

Toth, E.A., Turiak, L., Visnovitz, T., Cserep, C., Mazlo, A. et al

1. Extracell. Vesicles, 10, e12140 (2021)

  In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein ‘contamination’ of EV preparations and may add a new perspective to EV research.  

4.2382           A CRMP4-dependent retrograde axon-to-soma death signal in amyotrophic lateral sclerosis

Maimon, R., Ankol, L., Pery, T.G., Altman, T., Ionescu, A. et al EMBO J., 40, e107586 (2021)   Amyotrophic lateral sclerosis (ALS) is a fatal non-cell-autonomous neurodegenerative disease characterized by the loss of motor neurons (MNs). Mutations in CRMP4 are associated with ALS in patients, and elevated levels of CRMP4 are suggested to affect MN health in the SOD1^G93A-ALS mouse model. However, the mechanism by which CRMP4 mediates toxicity in ALS MNs is poorly understood. Here, by using tissue from human patients with sporadic ALS, MNs derived from C9orf72-mutant patients, and the SOD1^G93A-ALS mouse model, we demonstrate that subcellular changes in CRMP4 levels promote MN loss in ALS. First, we show that while expression of CRMP4 protein is increased in cell bodies of ALS-affected MN, CRMP4 levels are decreased in the distal axons. Cellular mislocalization of CRMP4 is caused by increased interaction with the retrograde motor protein, dynein, which mediates CRMP4 transport from distal axons to the soma and thereby promotes MN loss. Blocking the CRMP4-dynein interaction reduces MN loss in human-derived MNs (C9orf72) and in ALS model mice. Thus, we demonstrate a novel CRMP4-dependent retrograde death signal that underlies MN loss in ALS.  

4.2383           Liver Sinusoidal Endothelial Cells Suppress Bone Morphogenetic Protein 2 Production in Response to TGFβ Pathway Activation

Colucci, S.M Altamura, S., Marques, O., Dropmann, A., Horvat, N.K., Müdder, K., Hammad, S., Dooley, S. and Muckenthaler, M.U. Hepatology, 74(4), 2186-2200 (2021)   Background and Aims TGFβ/bone morphogenetic protein (BMP) signaling in the liver plays a critical role in liver disease. Growth factors, such as BMP2, BMP6, and TGFβ1, are released from LSECs and signal in a paracrine manner to hepatocytes and hepatic stellate cells to control systemic iron homeostasis and fibrotic processes, respectively. The misregulation of the TGFβ/BMP pathway affects expression of the iron-regulated hormone hepcidin, causing frequent iron overload and deficiency diseases. However, whether LSEC-secreted factors can act in an autocrine manner to maintain liver homeostasis has not been addressed so far. Approach and Results We analyzed publicly available RNA-sequencing data of mouse LSECs for ligand-receptor interactions and identified members of the TGFβ family (BMP2, BMP6, and TGFβ1) as ligands with the highest expression levels in LSECs that may signal in an autocrine manner. We next tested the soluble factors identified through in silico analysis in optimized murine LSEC primary cultures and mice. Exposure of murine LSEC primary cultures to these ligands shows that autocrine responses to BMP2 and BMP6 are blocked despite high expression levels of the required receptor complexes partially involving the inhibitor FK-506–binding protein 12. By contrast, LSECs respond efficiently to TGFβ1 treatment, which causes reduced expression of BMP2 through activation of activin receptor-like kinase 5. Conclusions These findings reveal that TGFβ1 signaling is functionally interlinked with BMP signaling in LSECs, suggesting druggable targets for the treatment of iron overload diseases associated with deficiency of the BMP2-regulated hormone hepcidin, such as hereditary hemochromatosis, β-thalassemia, and chronic liver diseases.  

4.2384           In Vivo Efficacy of Neutrophil-Mediated Bone Regeneration Using a Rabbit Calvarial Defect Model

Herath, T.D., Saigo, L., Schaller, B., larbi, A., Teoh, S.H., Kirkpatrick, C.J. and Goh, B.T. Inr. J. Mol. Sci., 22:13016 (2021)   Reconstruction of bone due to surgical removal or disease-related bony defects is a clinical challenge. It is known that the immune system exerts positive immunomodulatory effects on tissue repair and regeneration. In this study, we evaluated the in vivo efficacy of autologous neutrophils on bone regeneration using a rabbit calvarial defect model. Methods: Twelve rabbits, each with two surgically created calvarial bone defects (10 mm diameter), were randomly divided into two groups; (i) single application of neutrophils (SA-NP) vs. SA-NP control, and (ii) repetitive application of neutrophils (RA-NP) vs. RA-NP control. The animals were euthanized at 4 and 8 weeks post-operatively and the treatment outcomes were evaluated by micro-computed tomography, histology, and histomorphometric analyses. Results: The micro-CT analysis showed a significantly higher bone volume fraction (bone volume/total volume) in the neutrophil-treated groups, i.e., median interquartile range (IQR) SA-NP (18) and RA-NP (24), compared with the untreated controls, i.e., SA-NP (7) and RA-NP (14) at 4 weeks (p < 0.05). Similarly, new bone area fraction (bone area/total area) was significantly higher in neutrophil-treated groups at 4 weeks (p < 0.05). Both SA-NP and RA-NP had a considerably higher bone volume and bone area at 8 weeks, although the difference was not statistically significant. In addition, immunohistochemical analysis at 8 weeks revealed a higher expression of osteocalcin in both SA-NP and RA-NP groups. Conclusions: The present study provides first hand evidence that autologous neutrophils may have a positive effect on promoting new bone formation. Future studies should be performed with a larger sample size in non-human primate models. If proven feasible, this new promising strategy could bring clinical benefits for bone defects to the field of oral and maxillofacial surgery.  

4.2385           Synaptotagmin-13 Is a Neuroendocrine Marker in Brain, Intestine and Pancreas

Tarquis-Medina, M., Scheibner, K., Gonzelez-Garcia, I., Bastidas-Ponce, A., Sterr, M., Jaki, J., Schirge, S., Garcia-Caceres, C. and Lickert, H. Int. J. Mol. Sci., 22:12526 (2021)   Synaptotagmin-13 (Syt13) is an atypical member of the vesicle trafficking synaptotagmin protein family. The expression pattern and the biological function of this Ca²⁺-independent protein are not well resolved. Here, we have generated a novel Syt13-Venus fusion (Syt13-VF) fluorescence reporter allele to track and isolate tissues and cells expressing Syt13 protein. The reporter allele is regulated by endogenous cis-regulatory elements of Syt13 and the fusion protein follows an identical expression pattern of the endogenous Syt13 protein. The homozygous reporter mice are viable and fertile. We identify the expression of the Syt13-VF reporter in different regions of the brain with high expression in tyrosine hydroxylase (TH)-expressing and oxytocin-producing neuroendocrine cells. Moreover, Syt13-VF is highly restricted to all enteroendocrine cells in the adult intestine that can be traced in live imaging. Finally, Syt13-VF protein is expressed in the pancreatic endocrine lineage, allowing their specific isolation by flow sorting. These findings demonstrate high expression levels of Syt13 in the endocrine lineages in three major organs harboring these secretory cells. Collectively, the Syt13-VF reporter mouse line provides a unique and reliable tool to dissect the spatio-temporal expression pattern of Syt13 and enables isolation of Syt13-expressing cells that will aid in deciphering the molecular functions of this protein in the neuroendocrine system.  

4.2386           Generalizing hydrogel microparticles into a new class of bioinks for extrusion bioprinting

Xin, S., Deo, K.A., Dai, J., Pandian, N.K.R., Chimene, D., Moebius, R.M., Jain, A., Han, A., Gaharwar, A.K. and Alge, D.L. Sci. Adv., 7, eabk3087 (2021)   Hydrogel microparticles (HMPs) are an emerging bioink that can allow three-dimensional (3D) printing of most soft biomaterials by improving physical support and maintaining biological functions. However, the mechanisms of HMP jamming within printing nozzles and yielding to flow remain underexplored. Here, we present an in-depth investigation via both experimental and computational methods on the HMP dissipation process during printing as a result of (i) external resistance from the printing apparatus and (ii) internal physicochemical properties of HMPs. In general, a small syringe opening, large or polydisperse size of HMPs, and less deformable HMPs induce high resistance and closer HMP packing, which improves printing fidelity and stability due to increased interparticle adhesion. However, smooth extrusion and preserving viability of encapsulated cells require low resistance during printing, which is associated with less shear stress. These findings can be used to improve printability of HMPs and facilitate their broader use in 3D bioprinting.  

4.2387           Repair of peripheral nerve defects by nerve grafts incorporated with extracellular vesicles from skin-derived precursor Schwann cells

Yu, M., Gu, G., Cong, M., Du, M., Wang, W., Shen, M., Zhang, Q., Shi, H., Gu, X. and Ding, F. Acta Biomaterialia, 134, 190-203 (2021)   Our previous studies have shown that extracellular vesicles from skin-derived precursor Schwann cells (SKP-SC-EVs) promote neurite outgrowth of sensory and motor neurons in vitro. This study was aimed at generating an artificial nerve graft incorporated with SKP-SC-EVs to examine in vivo effects of SKP-SC-EVs on peripheral nerve regeneration. Here SKP-SC-EVs were isolated and then identified by morphological observation and phenotypic marker expression. Following co-culture with SCs or motoneurons, SKP-SC-EVs were internalized, showing the capability to enhance SC viability or motoneuron neurite outgrowth. In vitro, SKP-SC-EVs released from Matrigel could maintain cellular uptake property and neural activity. Nerve grafts were developed by incorporating Matrigel-encapsulated SKP-SC-EVs into silicone conduits. Functional evaluation, histological investigation, and morphometric analysis were performed to compare the nerve regenerative outcome after bridging the 10-mm long sciatic nerve defect in rats with our developed nerve grafts, silicone conduits (filled with vehicle), and autografts respectively. Our developed nerve grafts significantly accelerated the recovery of motor, sensory, and electrophysiological functions of rats, facilitated outgrowth and myelination of regenerated axons, and alleviated denervation-induced atrophy of target muscles. Collectively, our findings suggested that incorporation of SKP-SC-EVs into nerve grafts might represent a promising paradigm for peripheral nerve injury repair.  

4.2388           Early upregulation of cytosolic phospholipase A2α in motor neurons is induced by misfolded SOD1 in a mouse model of amyotrophic lateral sclerosis

Edelstein, Y.F.M., Solomonov, Y., Hadad, N., Alfahei, L., Israelson, A. and Levy, R.

1. Neuroimmflammation, 18:274 (2021)

  Background Amyotrophic lateral sclerosis (ALS) is a fatal multifactorial neurodegenerative disease characterized by the selective death of motor neurons. Cytosolic phospholipase A₂ alpha (cPLA₂α) upregulation and activation in the spinal cord of ALS patients has been reported. We have previously shown that cPLA₂α upregulation in the spinal cord of mutant SOD1 transgenic mice (SOD1^G93A) was detected long before the development of the disease, and inhibition of cPLA₂α upregulation delayed the disease’s onset. The aim of the present study was to determine the mechanism for cPLA₂α upregulation. Methods Immunofluorescence analysis and western blot analysis of misfolded SOD1, cPLA₂α and inflammatory markers were performed in the spinal cord sections of SOD1^G93A transgenic mice and in primary motor neurons. Over expression of mutant SOD1 was performed by induction or transfection in primary motor neurons and in differentiated NSC34 motor neuron like cells. Results Misfolded SOD1 was detected in the spinal cord of 3 weeks old mutant SOD1^G93A mice before cPLA₂α upregulation. Elevated expression of both misfolded SOD1 and cPLA₂α was specifically detected in the motor neurons at 6 weeks with a high correlation between them. Elevated TNFα levels were detected in the spinal cord lysates of 6 weeks old mutant SOD1^G93A mice. Elevated TNFα was specifically detected in the motor neurons and its expression was highly correlated with cPLA₂α expression at 6 weeks. Induction of mutant SOD1 in primary motor neurons induced cPLA₂α and TNFα upregulation. Over expression of mutant SOD1 in NSC34 cells caused cPLA₂α upregulation which was prevented by antibodies against TNFα. The addition of TNFα to NSC34 cells caused cPLA₂α upregulation in a dose dependent manner. Conclusions Motor neurons expressing elevated cPLA₂α and TNFα are in an inflammatory state as early as at 6 weeks old mutant SOD1^G93A mice long before the development of the disease. Accumulated misfolded SOD1 in the motor neurons induced cPLA₂α upregulation via induction of TNFα.  

4.2389           Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

Cao, L.-L., Guan, P-P., Zhang, S-Q., Yang, Y., Huang, X-S. and Wang, P.

1. Neuroinnflammation, 18:281 (2021)

  Background Neuroinflammation is thought to be a cause of Alzheimer’s disease (AD), which is partly caused by inadequate mitophagy. As a receptor of mitophagy, we aimed to reveal the regulatory roles of optineurin (OPTN) on neuroinflammation in the pathogenesis of AD. Methods BV2 cells and APP/PS1 transgenic (Tg) mice were used as in vitro and in vivo experimental models to determine the regulatory roles of OPTN in neuroinflammation of AD. Sophisticated molecular technologies including quantitative (q) RT-PCR, western blot, enzyme linked immunosorbent assay (ELISA), co-immunoprecipitation (Co-IP) and immunofluorescence (IF) were employed to reveal the inherent mechanisms. Results As a consequence, key roles of OPTN in regulating neuroinflammation were identified by depressing the activity of absent in melanoma 2 (AIM2) inflammasomes and receptor interacting serine/threonine kinase 1 (RIPK1)-mediated NF-κB inflammatory mechanisms. In detail, we found that expression of OPTN was downregulated, which resulted in activation of AIM2 inflammasomes due to a deficiency in mitophagy in APP/PS1 Tg mice. By ectopic expression, OPTN blocks the effects of Aβ oligomer (Aβo) on activating AIM2 inflammasomes by inhibiting mRNA expression of AIM2 and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), leading to a reduction in the active form of caspase-1 and interleukin (IL)-1β in microglial cells. Moreover, RIPK1 was also found to be negatively regulated by OPTN via ubiquitin protease hydrolysis, resulting in the synthesis of IL-1β by activating the transcriptional activity of NF-κB in BV2 cells. As an E3 ligase, the UBAN domain of OPTN binds to the death domain (DD) of RIPK1 to facilitate its ubiquitination. Based on these observations, ectopically expressed OPTN in APP/PS1 Tg mice deactivated microglial cells and astrocytes via the AIM2 inflammasome and RIPK-dependent NF-κB pathways, leading to reduce neuroinflammation. Conclusions These results suggest that OPTN can alleviate neuroinflammation through AIM2 and RIPK1 pathways, suggesting that OPTN deficiency may be a potential factor leading to the occurrence of AD.  

4.2390           Myeloid-associated differentiation marker is a novel SP-A-associated transmembrane protein whose expression on airway epithelial cells correlates with asthma severity

Dy, A.B.C., langlais, P.R., Barker, N.K., Addison, K.J., Tanyaratsrisakul, S., Boitono, S., Christenson, S.A., Kraft, M., Meyers, D., Bleecker, E.R., Li, X. and Ledford, J.G. Scientific Reports, 11:23392 82021)   Surfactant protein A (SP-A) is well-known for its protective role in pulmonary immunity. Previous studies from our group have shown that SP-A mediates eosinophil activities, including degranulation and apoptosis. In order to identify potential binding partners on eosinophils for SP-A, eosinophil lysates were subjected to SP-A pull-down and tandem mass spectrometry (MS/MS) analysis. We identified one membrane-bound protein, myeloid-associated differentiation marker (MYADM), as a candidate SP-A binding partner. Blocking MYADM on mouse and human eosinophils ex vivo prevented SP-A from inducing apoptosis; blocking MYADM in vivo led to increased persistence of eosinophilia and airway hyper-responsiveness in an ovalbumin (OVA) allergy model and increased airways resistance and mucus production in a house dust mite (HDM) asthma model. Examination of a subset of participants in the Severe Asthma Research Program (SARP) cohort revealed a significant association between epithelial expression of MYADM in asthma patients and parameters of airway inflammation, including: peripheral blood eosinophilia, exhaled nitric oxide (FeNO) and the number of exacerbations in the past 12 months. Taken together, our studies provide the first evidence of MYADM as a novel SP-A-associated protein that is necessary for SP-A to induce eosinophil apoptosis and we bring to light the potential importance of this previously unrecognized transmembrane protein in patients with asthma.  

4.2391           Chemical characterization of red cells from the black sea urchin Arbacia lixula by X-ray photoelectron spectroscopy

Pagliara, P., Chrizzi, D. and Guascito, M.R. RSC Adv., 11, 27074-27083 (2021)   Red spherula cells (RSC) from sea urchin coelomic fluid have attracted great interest for their specific and intriguing properties, such as for example antimicrobial activities and immune response, that probably tie in with their red characteristic pigments. Although to date different studies have been reported aimed to chemically characterize their pigments extracted from the cells, few data are available about the chemical characterization of the cell surface. In this work, a systematic chemical characterization of the RSC surface by X-ray photoelectron spectroscopy (XPS) analysis is described. The results were compared with data on colorless cells from the same coelomic fluid sample. Our observations evidenced that the two cell types were characterized by the presence of different chemical functional groups. In particular, the colorless cells are dominated by the presence of alkyl, alcohol, amide, and carboxyl groups in accordance with other similar cell types, enriched in Na⁺ and Cl⁻ ions. Traces of elements like S (sulphonates) and P (phosphates) are also present. On the other hand, the RSC in addition to the alkyl groups show a reduction in the content of amide groups, accompanied by the anomalous presence of keto-enolic groups that probably can be associated with the presence of quinones/hydro-quinones from red pigments. A chemical enrichment in elements such as Cl⁻ and Mg²⁺ and sulphate groups (–R–O–SO₃⁻), as well as the presence of sulphides and phosphates traces, is evident. The absence of carbonate groups is also observed in both cell populations, confirming the absence of sodium and magnesium carbonate salts. No traces of toxic elements (i.e., heavy metals) have been revealed.  

4.2392           Hepatic nonparenchymal cells isolated from rodent livers and characterized for the development

of primary hepatic cocultures

Krimmling, T., Ullrich, A. and Runge, D. Toxicol. Lett., 350S, P15-11, S1-S276 (2021)   Liver cells are established as in vitro models for toxicology studies in 2D and 3D cultures. The goal is to approach the in vivo situation as good as possible. To expand the specificity of the liver models, the total cell population of the liver has to be taken into account. Cultivation and verification of these nonparenchymal cells, like Liver Sinusoidal Endothelial Cells (LSEC), Kupffer cells (KC) and Stellate cells (SC) remain an issue. Percoll gradients for cell separation of nonparenchymal cells have been established, but the yield of NPC per gram liver tissue can be low. At PRIMACYT, internal experiments comparing Percoll- and Iodixanol gradients in rat liver showed a slightly higher yield (20%) of the whole NPC population when cells were isolated by an Iodixanol gradient compared to Percoll. In mice, this effect was more obvious by providing about 54% higher NPC yield with Iodixanol. We examined assays to prove purity and viability of the KC, LSEC and SC fractions of the rats and mice after purification by Iodixanol. In a first step, Kupffer cells were tested over culture time for their stimulation by LPS, followed by a phagocytosis test. LSEC fractions were tested equally to proof the absence of phagocytic cells. Stellate cells were tested for the presence of lipid droplets by Oil Red O staining. Further analysis was done by immunofluorescence by CD68 (KC), CD31 (LSEC) and Vimentin (SC). Finally, the fluorescence was used to count the cell nuclei against positively stained cells by a cell counter system to indicate the proportions of cells present in the purified cell fractions. Together, the tests could give rise to a clear characterization of the liver cells. Finally, the approach to utilize tissue-like cocultures should reduce the number of laboratory animals used for toxicity studies and drug development.  

4.2393           Extracellular histones, a new class of inhibitory molecules of CNS axonal regeneration

Siddiq, M., Hannila, S.S., Zorina, Y., Nikulina, E., Rabinovich, V. et al Brain Comm., 3(4), fcab271 (2021)   Axonal regeneration in the mature CNS is limited by extracellular inhibitory factors. Triple knockout mice lacking the major myelin-associated inhibitors do not display spontaneous regeneration after injury, indicating the presence of other inhibitors. Searching for such inhibitors, we have detected elevated levels of histone H3 in human CSF 24 h after spinal cord injury. Following dorsal column lesions in mice and optic nerve crushes in rats, elevated levels of extracellular histone H3 were detected at the injury site. Similar to myelin-associated inhibitors, these extracellular histones induced growth cone collapse and inhibited neurite outgrowth. Histones mediate inhibition through the transcription factor Y-box-binding protein 1 and Toll-like receptor 2, and these effects are independent of the Nogo receptor. Histone-mediated inhibition can be reversed by the addition of activated protein C in vitro, and activated protein C treatment promotes axonal regeneration in the crushed optic nerve in vivo. These findings identify extracellular histones as a new class of nerve regeneration-inhibiting molecules within the injured CNS.  

4.2394           Methylome inheritance and enhancer dememorization reset an epigenetic gate safeguarding embryonic programs

Wu, X., Zhang, H., Zhang, B., Zhang, Y., Wang, Q., Shen, W. et al Sci.Adv., 7, eabl3858 (2021)   Marked epigenetic reprogramming is essential to convert terminally differentiated gametes to totipotent embryos. It remains puzzling why postfertilization global DNA reprogramming occurs in mammals but not in nonmammalian vertebrates. In zebrafish, global methylome inheritance is however accompanied by extensive enhancer “dememorization” as they become fully methylated. By depleting maternal dnmt1 using oocyte microinjection, we eliminated DNA methylation in early embryos, which died around gastrulation with severe differentiation defects. Notably, methylation deficiency leads to derepression of adult tissue–specific genes and CG-rich enhancers, which acquire ectopic transcription factor binding and, unexpectedly, histone H3 lysine 4 trimethylation (H3K4me3). By contrast, embryonic enhancers are generally CG-poor and evade DNA methylation repression. Hence, global DNA hypermethylation inheritance coupled with enhancer dememorization installs an epigenetic gate that safeguards embryonic programs and ensures temporally ordered gene expression. We propose that “enhancer dememorization” underlies and unifies distinct epigenetic reprogramming modes in early development between mammals and nonmammals.  

4.2395           Bruton’s tyrosine kinase drives neuroinflammation and anxiogenic behavior in mouse models of stress

Ghosh, S., Mohammed, Z. and Singh, I.

1. Neuroinflammation, 18:289 (2021)

  Background Current therapies targeting several neurotransmitter systems are only able to partially mitigate the symptoms of stress- and trauma-related disorder. Stress and trauma-related disorders lead to a prominent inflammatory response in humans, and in pre-clinical models. However, mechanisms underlying the induction of neuroinflammatory response in PTSD and anxiety disorders are not clearly understood. The present study investigated the mechanism underlying the activation of proinflammatory NLRP3 inflammasome and IL1β in mouse models of stress. Methods We used two mouse models of stress, i.e., mice subjected to physical restraint stress with brief underwater submersion, and predator odor stress. Mice were injected with MCC950, a small molecule specific inhibitor of NLRP3 activation. To pharmacologically inhibit BTK, a specific inhibitor ibrutinib was used. To validate the observation from ibrutinib studies, a separate group of mice was injected with another BTK-specific inhibitor LFM-A13. Seven days after the induction of stress, mice were examined for anxious behavior using open field test (OFT), light–dark test (LDT), and elevated plus maze test (EPM). Following the behavior tests, hippocampus and amygdale were extracted and analyzed for various components of NLRP3–caspase 1–IL1β pathway. Plasma and peripheral blood mononuclear cells were also used to assess the induction of NLRP3–Caspase 1–IL-1β pathway in stressed mice. Results Using two different pre-clinical models of stress, we demonstrate heightened anxious behavior in female mice as compared to their male counterparts. Stressed animals exhibited upregulation of proinflammatory IL1β, IL-6, Caspase 1 activity and NLRP3 inflammasome activation in brain, which were significantly higher in female mice. Pharmacological inhibition of NLRP3 inflammasome activation led to anxiolysis as well as attenuated neuroinflammatory response. Further, we observed induction of activated Bruton’s tyrosine kinase (BTK), an upstream positive-regulator of NLRP3 inflammasome activation, in hippocampus and amygdala of stressed mice. Next, we conducted proof-of-concept pharmacological BTK inhibitor studies with ibrutinib and LFM-A13. In both sets of experiments, we found BTK inhibition led to anxiolysis and attenuated neuroinflammation, as indicated by significant reduction of NLRP3 inflammasome and proinflammatory IL-1β in hippocampus and amygdala. Analysis of plasma and peripheral blood mononuclear cells indicated peripheral induction of NLRP3–caspase 1–IL1β pathway in stressed mice. Conclusion Our study identified BTK as a key upstream regulator of neuroinflammation, which drives anxiogenic behavior in mouse model of stress. Further, we demonstrated the sexually divergent activation of BTK, providing a clue to heightened neuroinflammation and anxiogenic response to stress in females as compared to their male counterparts. Our data from the pharmacological inhibition studies suggest BTK as a novel target for the development of potential clinical treatment of PTSD and anxiety disorders. Induction of pBTK and NLRP3 in peripheral blood mononuclear cells of stressed mice suggest the potential effect of stress on systemic inflammation.  

4.2396           Necroptosis contributes to chronic inflammation and fibrosis in aging liver

Mohammed, S., Thadathil, N., Selvarani, R., Nicklas, E.H., Wang, d., Miller, B.F., Richardson, A. and Deepa, S.S. Aging Cell, 20, e13512 (2021)   Inflammaging, characterized by an increase in low-grade chronic inflammation with age, is a hallmark of aging and is strongly associated with various age-related diseases, including chronic liver disease (CLD) and hepatocellular carcinoma (HCC). Because necroptosis is a cell death pathway that induces inflammation through the release of DAMPs, we tested the hypothesis that age-associated increase in necroptosis contributes to chronic inflammation in aging liver. Phosphorylation of MLKL and MLKL oligomers, markers of necroptosis, as well as phosphorylation of RIPK3 and RIPK1 were significantly upregulated in the livers of old mice relative to young mice and this increase occurred in the later half of life (i.e., after 18 months of age). Markers of M1 macrophages, expression of pro-inflammatory cytokines (TNFα, IL6 and IL1β), and markers of fibrosis were all significantly upregulated in the liver with age and the change in necroptosis paralleled the changes in inflammation and fibrosis. Hepatocytes and liver macrophages isolated from old mice showed elevated levels of necroptosis markers as well as increased expression of pro-inflammatory cytokines relative to young mice. Short-term treatment with the necroptosis inhibitor, necrostatin-1s (Nec-1s), reduced necroptosis, markers of M1 macrophages, fibrosis, and cell senescence as well as reducing the expression of pro-inflammatory cytokines in the livers of old mice. Thus, our data show for the first time that liver aging is associated with increased necroptosis and necroptosis contributes to chronic inflammation in the liver, which in turn appears to contribute to liver fibrosis and possibly CLD.  

4.2397           Inceptor counteracts insulin signalling in β-cells to control glycaemia

Ansarullah, Jain, C., Far, F.F., Holmberg, S., Wissmiller, K., von Hahn, F.G: et al Nature, 590, 326-331 (2021)   Resistance to insulin and insulin-like growth factor 1 (IGF1) in pancreatic β-cells causes overt diabetes in mice; thus, therapies that sensitize β-cells to insulin may protect patients with diabetes against β-cell failure1^,2^,3. Here we identify an inhibitor of insulin receptor (INSR) and IGF1 receptor (IGF1R) signalling in mouse β-cells, which we name the insulin inhibitory receptor (inceptor; encoded by the gene Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to INSR and IGF1R4, and a mannose 6-phosphate receptor domain that is also found in the IGF2 receptor (IGF2R)5. Knockout mice that lack inceptor (Iir^−/−) exhibit signs of hyperinsulinaemia and hypoglycaemia, and die within a few hours of birth. Molecular and cellular analyses of embryonic and postnatal pancreases from Iir^−/− mice showed an increase in the activation of INSR–IGF1R in Iir^−/− pancreatic tissue, resulting in an increase in the proliferation and mass of β-cells. Similarly, inducible β-cell-specific Iir^−/− knockout in adult mice and in ex vivo islets led to an increase in the activation of INSR–IGF1R and increased proliferation of β-cells, resulting in improved glucose tolerance in vivo. Mechanistically, inceptor interacts with INSR–IGF1R to facilitate clathrin-mediated endocytosis for receptor desensitization. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of inceptor resulted in the retention of inceptor and INSR at the plasma membrane to sustain the activation of INSR–IGF1R in β-cells. Together, our findings show that inceptor shields insulin-producing β-cells from constitutive pathway activation and identify inceptor as a potential molecular target for INSR–IGF1R sensitization and diabetes therapy.  

4.2398           Skin-resident innate lymphoid cells converge on a pathogenic effector state

Biolecki, P., Risenfeld, S.J., Hütter, J-C., Triglia, E.T., Kowalczyk, M.S. et al Nature, 592, 128-132 (2021)   Tissue-resident innate lymphoid cells (ILCs) help sustain barrier function and respond to local signals. ILCs are traditionally classified as ILC1, ILC2 or ILC3 on the basis of their expression of specific transcription factors and cytokines1. In the skin, disease-specific production of ILC3-associated cytokines interleukin (IL)-17 and IL-22 in response to IL-23 signalling contributes to dermal inflammation in psoriasis. However, it is not known whether this response is initiated by pre-committed ILCs or by cell-state transitions. Here we show that the induction of psoriasis in mice by IL-23 or imiquimod reconfigures a spectrum of skin ILCs, which converge on a pathogenic ILC3-like state. Tissue-resident ILCs were necessary and sufficient, in the absence of circulatory ILCs, to drive pathology. Single-cell RNA-sequencing (scRNA-seq) profiles of skin ILCs along a time course of psoriatic inflammation formed a dense transcriptional continuum—even at steady state—reflecting fluid ILC states, including a naive or quiescent-like state and an ILC2 effector state. Upon disease induction, the continuum shifted rapidly to span a mixed, ILC3-like subset also expressing cytokines characteristic of ILC2s, which we inferred as arising through multiple trajectories. We confirmed the transition potential of quiescent-like and ILC2 states using in vitro experiments, single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) and in vivo fate mapping. Our results highlight the range and flexibility of skin ILC responses, suggesting that immune activities primed in healthy tissues dynamically adapt to provocations and left unchecked, drive pathological remodelling.  

4.2399           Cross-tissue organization of the fibroblast lineage

Buechler, M.B., Pradhan, R.N., Krishnamurty, A.T., Cox, C. et al Nature, 593, 575-579 (2021)   Fibroblasts are non-haematopoietic structural cells that define the architecture of organs, support the homeostasis of tissue-resident cells and have key roles in fibrosis, cancer, autoimmunity and wound healing1. Recent studies have described fibroblast heterogeneity within individual tissues1. However, the field lacks a characterization of fibroblasts at single-cell resolution across tissues in healthy and diseased organs. Here we constructed fibroblast atlases by integrating single-cell transcriptomic data from about 230,000 fibroblasts across 17 tissues, 50 datasets, 11 disease states and 2 species. Mouse fibroblast atlases and a Dpt^IRESCreERT2 knock-in mouse identified two universal fibroblast transcriptional subtypes across tissues. Our analysis suggests that these cells can serve as a reservoir that can yield specialized fibroblasts across a broad range of steady-state tissues and activated fibroblasts in disease. Comparison to an atlas of human fibroblasts from perturbed states showed that fibroblast transcriptional states are conserved between mice and humans, including universal fibroblasts and activated phenotypes associated with pathogenicity in human cancer, fibrosis, arthritis and inflammation. In summary, a cross-species and pan-tissue approach to transcriptomics at single-cell resolution has identified key organizing principles of the fibroblast lineage in health and disease.  

4.2400           Molecular architecture of the developing mouse brain

La Manno, G., Siletti, K., Furlan, A., Gyllborg, D., VInsland, E., Albiach, A.M. et al Nature, 596, 92-96 (2021)   The mammalian brain develops through a complex interplay of spatial cues generated by diffusible morphogens, cell–cell interactions and intrinsic genetic programs that result in probably more than a thousand distinct cell types. A complete understanding of this process requires a systematic characterization of cell states over the entire spatiotemporal range of brain development. The ability of single-cell RNA sequencing and spatial transcriptomics to reveal the molecular heterogeneity of complex tissues has therefore been particularly powerful in the nervous system. Previous studies have explored development in specific brain regions1^,2^,3^,4^,5^,6^,7^,8, the whole adult brain9 and even entire embryos10. Here we report a comprehensive single-cell transcriptomic atlas of the embryonic mouse brain between gastrulation and birth. We identified almost eight hundred cellular states that describe a developmental program for the functional elements of the brain and its enclosing membranes, including the early neuroepithelium, region-specific secondary organizers, and both neurogenic and gliogenic progenitors. We also used in situ mRNA sequencing to map the spatial expression patterns of key developmental genes. Integrating the in situ data with our single-cell clusters revealed the precise spatial organization of neural progenitors during the patterning of the nervous system.  

4.2401           Regulation of host and virus genes by neuronal miR-138 favours herpes simplex virus 1 latency

Sun, B., Yang, X., Hou, F., Yu, X., Wang, Q., Oh, H.S. et al Nature Microbiol., 6, 682-696 (2021)   MicroRNA miR-138, which is highly expressed in neurons, represses herpes simplex virus 1 (HSV-1) lytic cycle genes by targeting viral ICP0 messenger RNA, thereby promoting viral latency in mice. We found that overexpressed miR-138 also represses lytic processes independently of ICP0 in murine and human neuronal cells; therefore, we investigated whether miR-138 has targets besides ICP0. Using genome-wide RNA sequencing/photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation followed by short interfering RNA knockdown of candidate targets, we identified the host Oct-1 and Foxc1 messenger mRNAs as miR-138’s targets, whose gene products are transcription factors important for HSV-1 replication in neuronal cells. OCT-1 has a known role in the initiation of HSV transcription. Overexpression of FOXC1, which was not known to affect HSV-1, promoted HSV-1 replication in murine neurons and ganglia. CRISPR–Cas9 knockout of FOXC1 reduced viral replication, lytic gene expression and miR-138 repression in murine neuronal cells. FOXC1 also collaborated with ICP0 to decrease heterochromatin on viral genes and compensated for the defect of an ICP0-null virus. In summary, miR-138 targets ICP0, Oct-1 and Foxc1 to repress HSV-1 lytic cycle genes and promote epigenetic gene silencing, which together enable favourable conditions for latent infection.  

4.2402           Activity-dependent regulome of human GABAergic neurons reveals new patterns of gene regulation and neurological disease heritability

Boulting, G.L., Durresi, E., Ataman, B., Sherman, M.A., Mei, K. harmin, D.A. et al Nature Neurosci., 24, 437-448 (2021)   Neuronal activity-dependent gene expression is essential for brain development. Although transcriptional and epigenetic effects of neuronal activity have been explored in mice, such an investigation is lacking in humans. Because alterations in GABAergic neuronal circuits are implicated in neurological disorders, we conducted a comprehensive activity-dependent transcriptional and epigenetic profiling of human induced pluripotent stem cell-derived GABAergic neurons similar to those of the early developing striatum. We identified genes whose expression is inducible after membrane depolarization, some of which have specifically evolved in primates and/or are associated with neurological diseases, including schizophrenia and autism spectrum disorder (ASD). We define the genome-wide profile of human neuronal activity-dependent enhancers, promoters and the transcription factors CREB and CRTC1. We found significant heritability enrichment for ASD in the inducible promoters. Our results suggest that sequence variation within activity-inducible promoters of developing human forebrain GABAergic neurons contributes to ASD risk.  

4.2403           Integrating barcoded neuroanatomy with spatial transcriptional profiling enables identification of gene correlates of projections

Sun, Y-C., Chen, X., Fisher, S., Lu, S., Zhan, H., Gillis, J. and Zador, A.M. Nature Neurosci., 24, 873-885 (2021)   Functional circuits consist of neurons with diverse axonal projections and gene expression. Understanding the molecular signature of projections requires high-throughput interrogation of both gene expression and projections to multiple targets in the same cells at cellular resolution, which is difficult to achieve using current technology. Here, we introduce BARseq2, a technique that simultaneously maps projections and detects multiplexed gene expression by in situ sequencing. We determined the expression of cadherins and cell-type markers in 29,933 cells and the projections of 3,164 cells in both the mouse motor cortex and auditory cortex. Associating gene expression and projections in 1,349 neurons revealed shared cadherin signatures of homologous projections across the two cortical areas. These cadherins were enriched across multiple branches of the transcriptomic taxonomy. By correlating multigene expression and projections to many targets in single neurons with high throughput, BARseq2 provides a potential path to uncovering the molecular logic underlying neuronal circuits.  

4.2404           Decoding molecular and cellular heterogeneity of mouse nucleus accumbens

Chen, R., Blosser, T.R., Djekidel, M.N., Hao, J., Bhattacherjee, A., Chen, W., Tuesta, L.M., Zhuang, X. and Zhang, Y. Nature Neurosci., 24, 1757-1771 (2021)   The nucleus accumbens (NAc) plays an important role in regulating multiple behaviors, and its dysfunction has been linked to many neural disorders. However, the molecular, cellular and anatomic heterogeneity underlying its functional diversity remains incompletely understood. In this study, we generated a cell census of the mouse NAc using single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization, revealing a high level of cell heterogeneity in this brain region. Here we show that the transcriptional and spatial diversity of neuron subtypes underlie the NAc’s anatomic and functional heterogeneity. These findings explain how the seemingly simple neuronal composition of the NAc achieves its highly heterogenous structure and diverse functions. Collectively, our study generates a spatially resolved cell taxonomy for understanding the structure and function of the NAc, which demonstrates the importance of combining molecular and spatial information in revealing the fundamental features of the nervous system.  

4.2405           A RIPK1-regulated inflammatory microglial state in amyotrophic lateral sclerosis

Mifflin, L., Hu, Z., Dufort, C., Hession, C.C., Walker, A.J., Hiu, K., Zhu, H., Liu, N., Liu, J.S., levin, J.Z., Stevens, B., Yuan, J. and Zou, C. PNAS, 18(13), e2025102118 (2021)   Microglial-derived inflammation has been linked to a broad range of neurodegenerative and neuropsychiatric conditions, including amyotrophic lateral sclerosis (ALS). Using single-cell RNA sequencing, a class of Disease-Associated Microglia (DAMs) have been characterized in neurodegeneration. However, the DAM phenotype alone is insufficient to explain the functional complexity of microglia, particularly with regard to regulating inflammation that is a hallmark of many neurodegenerative diseases. Here, we identify a subclass of microglia in mouse models of ALS which we term RIPK1-Regulated Inflammatory Microglia (RRIMs). RRIMs show significant up-regulation of classical proinflammatory pathways, including increased levels of Tnf and Il1b RNA and protein. We find that RRIMs are highly regulated by TNFα signaling and that the prevalence of these microglia can be suppressed by inhibiting receptor-interacting protein kinase 1 (RIPK1) activity downstream of the TNF receptor 1. These findings help to elucidate a mechanism by which RIPK1 kinase inhibition has been shown to provide therapeutic benefit in mouse models of ALS and may provide an additional biomarker for analysis in ongoing phase 2 clinical trials of RIPK1 inhibitors in ALS.  

4.2406           ADAM9 enhances Th17 cell differentiation and autoimmunity by activating TGF-β1

Umeda, M., Yoshida, N., Hisada, R., Burbano, C., Orite, S.Y.K., Kono, M., Kyttaris, V.C., Krisfield, S., Owen, C.A. and Tsokos, G.C. PNAS, 118(18), e2023230118 (2021)   The a disintegrin and metalloproteinase (ADAM) family of proteinases alter the extracellular environment and are involved in the development of T cells and autoimmunity. The role of ADAM family members in Th17 cell differentiation is unknown. We identified ADAM9 to be specifically expressed and to promote Th17 differentiation. Mechanistically, we found that ADAM9 cleaved the latency-associated peptide to produce bioactive transforming growth factor β1, which promoted SMAD2/3 phosphorylation and activation. A transcription factor inducible cAMP early repressor was found to bind directly to the ADAM9 promoter and to promote its transcription. Adam9-deficient mice displayed mitigated experimental autoimmune encephalomyelitis, and transfer of Adam9-deficient myelin oligodendrocyte globulin-specific T cells into Rag1^−/− mice failed to induce disease. At the translational level, an increased abundance of ADAM9 levels was observed in CD4⁺ T cells from patients with systemic lupus erythematosus, and ADAM9 gene deletion in lupus primary CD4⁺ T cells clearly attenuated their ability to differentiate into Th17 cells. These findings revealed that ADAM9 as a proteinase provides Th17 cells with an ability to activate transforming growth factor β1 and accelerates its differentiation, resulting in aberrant autoimmunity.  

4.2407           Sorting for secreted molecule production using a biosensor-in-microdroplet approach

Bowman, E.K., Wagner, J.M., Yuan, S-F., Deaner, M., Palmer, C.M., D’Oeelsnitz, S., Cordova, l., Li, X., Craig, F.F. and Alper, H.S. PNAS, 118(36), e2106818118 (2021)   Sorting large libraries of cells for improved small molecule secretion is throughput limited. Here, we combine producer/secretor cell libraries with whole-cell biosensors using a microfluidic-based screening workflow. This approach enables a mix-and-match capability using off-the-shelf biosensors through either coencapsulation or pico-injection. We demonstrate the cell type and library agnostic nature of this workflow by utilizing single-guide RNA, transposon, and ethyl-methyl sulfonate mutagenesis libraries across three distinct microbes (Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica), biosensors from two organisms (E. coli and S. cerevisiae), and three products (triacetic acid lactone, naringenin, and L-DOPA) to identify targets improving production/secretion.  

4.2408           One-to-one innervation of vocal muscles allows precise control of birdsong

Adam, I., Mexwell, A., Rössler, H., Hansen, E.B., Vellema, M., Brewer, J. and Elemans, C.P.H. Current Biology, 31, 3115-3124 (2021)   The motor control resolution of any animal behavior is limited to the minimal force step available when activating muscles, which is set by the number and size distribution of motor units (MUs) and muscle-specific force. Birdsong is an excellent model system for understanding acquisition and maintenance of complex fine motor skills, but we know surprisingly little about how the motor pool controlling the syrinx is organized and how MU recruitment drives changes in vocal output. Here we developed an experimental paradigm to measure MU size distribution using spatiotemporal imaging of intracellular calcium concentration in cross-sections of living intact syrinx muscles. We combined these measurements with muscle stress and an in vitro syrinx preparation to determine the control resolution of fundamental frequency (f_o), a key vocal parameter, in zebra finches. We show that syringeal muscles have extremely small MUs, with 40%–50% innervating ≤3 and 13%–17% innervating a single muscle fiber. Combined with the lowest specific stress (5 mN/mm²) known to skeletal vertebrate muscle, small force steps by the major f_o controlling muscle provide control of 50-mHz to 7.3-Hz steps per MU. We show that the song system has the highest motor control resolution possible in the vertebrate nervous system and suggest this evolved due to strong selection on fine gradation of vocal output. Furthermore, we propose that high-resolution motor control was a key feature contributing to the radiation of songbirds that allowed diversification of song and speciation by vocal space expansion.  

4.2409           Diversity of developing peripheral glia revealed by single-cell RNA sequencing

Tasdemir-Yilmaz, O.E., Druckenbrod, N.R., Olukoya, O.O., Kharchenko, P.V., Goodrich, L.V. and Segal, R. Developmental Cell, 56, 2516-2536 (2021)   The peripheral nervous system responds to a wide variety of sensory stimuli, a process that requires great neuronal diversity. These diverse neurons are closely associated with glial cells originating from the neural crest. However, the molecular nature and diversity among peripheral glia are not understood. Here, we used single-cell RNA sequencing to profile developing and mature glia from somatosensory dorsal root ganglia and auditory spiral ganglia. We found that glial precursors (GPs) in these two systems differ in their transcriptional profiles. Despite their unique features, somatosensory and auditory GPs undergo convergent differentiation to generate molecularly uniform myelinating and non-myelinating Schwann cells. By contrast, somatosensory and auditory satellite glial cells retain system-specific features. Lastly, we identified a glial signature gene set, providing new insights into commonalities among glia across the nervous system. This survey of gene expression in peripheral glia constitutes a resource for understanding functions of glia across different sensory modalities.  

4.2410           CloneSeq: A highly sensitive analysis platform for the characterization of 3D-cultured single-cell-derived clones

Bavli, D., Sun, X., Kozulin, C., Meshorer, E., Buxboim, A. and Ram, O. Developmental Cell, 56, 1804-1817 (2021)   Single-cell assays have revealed the importance of heterogeneity in many biological systems. However, limited sensitivity is a major hurdle for uncovering cellular variation. To overcome it, we developed CloneSeq, combining clonal expansion inside 3D hydrogel spheres and droplet-based RNA sequencing (RNA-seq). We show that clonal cells maintain similar transcriptional profiles and cell states. CloneSeq of lung cancer cells revealed cancer-specific subpopulations, including cancer stem-like cells, that were not revealed by scRNA-seq. Clonal expansion within 3D soft microenvironments supported cellular stemness of embryonic stem cells (ESCs) even without pluripotent media, and it improved epigenetic reprogramming efficiency of mouse embryonic fibroblasts. CloneSeq of ESCs revealed that the differentiation decision is made early during Oct4 downregulation and is maintained during early clonal expansion. Together, we show CloneSeq can be adapted to different biological systems to discover rare subpopulations by leveraging the enhanced sensitivity within clones.  

4.2411           In vitro assay for single-cell characterization of impaired deformability in red blood cells under recurrent episodes of hypoxia

Qiang, Y., Liu, J., Dao, M. and Du, E. Lab Chip, 21, 3458-3470 (2021)   Red blood cells (RBCs) are subjected to recurrent changes in shear stress and oxygen tension during blood circulation. The cyclic shear stress has been identified as an important factor that alone can weaken cell mechanical deformability. The effects of cyclic hypoxia on cellular biomechanics have yet to be fully investigated. As the oxygen affinity of hemoglobin plays a key role in the biological function and mechanical performance of RBCs, the repeated transitions of hemoglobin between its R (high oxygen tension) and T (low oxygen tension) states may impact their mechanical behavior. The present study focuses on developing a novel microfluidic-based assay for characterization of the effects of cyclic hypoxia on cell biomechanics. The capability of this assay is demonstrated by a longitudinal study of individual RBCs in health and sickle cell disease subjected to cyclic hypoxia conditions of various durations and levels of low oxygen tension. The viscoelastic properties of cell membranes are extracted from tensile stretching and relaxation processes of RBCs induced by the electrodeformation technique. Results demonstrate that cyclic hypoxia alone can significantly reduce cell deformability, similar to the fatigue damage accumulated through cyclic mechanical loading. RBCs affected by sickle cell disease are less deformable (significantly higher membrane shear modulus and viscosity) than normal RBCs. The fatigue resistance of sickle RBCs to the cyclic hypoxia challenge is significantly inferior to that of normal RBCs, and this trend is more significant in mature erythrocytes of sickle cells. When the oxygen affinity of sickle hemoglobin is enhanced by anti-sickling drug treatment of 5-hydroxymethyl-2-furfural (5-HMF), sickle RBCs show ameliorated resistance to fatigue damage induced by cyclic hypoxia. These results indicate an important biophysical mechanism underlying RBC senescence in which the cyclic hypoxia challenge alone can lead to mechanical degradation of the RBC membrane. We envision that the application of this assay can be further extended to RBCs in other blood diseases and other cell types.  

4.2412           Synthetic cellular communication-based screening for strains with improved 3-hydroxypropionic acid secretion

Kim, S., Jin, S.H., Lim, H.G., Lee, B., Kim, J., Yang, J., Seo, S.W., Lee, C-S. and Jung, G.Y. Lab Chip, 21, 4455-4463 (2021)   Although cellular secretion is important in industrial biotechnology, its assessment is difficult due to the lack of efficient analytical methods. This study describes a synthetic cellular communication-based microfluidic platform for screening strains with the improved secretion of 3-hydroxypropionic acid (3-HP), an industry-relevant platform chemical. 3-HP-secreting cells were compartmentalized in droplets, with receiving cells equipped with a genetic circuit that converts the 3-HP secretion level into an easily detectable signal. This platform was applied to identify Escherichia coli genes that enhance the secretion of 3-HP. As a result, two genes (setA, encoding a sugar exporter, and yjcO, encoding a Sel1 repeat-containing protein) found by this platform enhance the secretion of 3-HP and its production. Given the increasing design capability for chemical-detecting cells, this platform has considerable potential in identifying efflux pumps for not only 3-HP but also many important chemicals.  

4.2413           A droplet-based microfluidic flow cytometry enabling absolute quantification of single-cell proteins leveraging constriction channel

Yang, H., Wei, Y., Fan, B., Liu, L., Zhang, T., Chen, D., Wang, J. and Chen, J. Microfluidics and Nanofluidics, 25:30 (2021)   Measurements of single-cell proteins provide key insights in studies related to cellular heterogeneities, while the absolute quantification of single-cell non-surface proteins remains elusive. This paper presents a droplet-based microfluidic flow cytometry enabling high-throughput quantification of both surface and intracellular proteins of single cell. Gradient solutions of fluorescently labeled proteins were first flushed into constriction channels with fluorescence signals detected to obtain calibration curves. Then, droplets encapsulating single cells stained with fluorescently labeled proteins were aspirated into constriction channels with excited fluorescence signals detected and translated into numbers of proteins based on the calibration curves. Based on this approach, both suspended and adherent cells with both surface and intracellular staining were processed. More specifically, the numbers of ConA binding sites of K562 cells, anti-β-actin antibody binding sites of A549 and HeLa cells were quantified as 3.43 ± 4.07 × 10⁶/cell (N_c = 530); 9.20 ± 5.21 × 10⁵/cell (N_c = 1073); and 1.44 ± 0.75 × 10⁶/cell (N_c = 1138), respectively. As a high-throughput microfluidic platform, this technique can add a quantitative approach to measure single-cell proteins with arbitrary distributions within cells.  

4.2414           Characterisation of circulating tumour cell phenotypes identifies a partial-EMT sub-population for clinical stratification of pancreatic cancer

Seman, A., Bernard, V., Kim, D.U., Lee, J.J., Huang, J. et al Br. J. Cancer, 124, 1970-1977 (2021)   Background Limited accessibility of the tumour precludes longitudinal characterisation for therapy guidance in pancreatic ductal adenocarcinoma (PDAC). Methods We utilised dielectrophoresis-field flow fractionation (DEP-FFF) to isolate circulating tumour cells (CTCs) in 272 blood draws from 74 PDAC patients (41 localised, 33 metastatic) to non-invasively monitor disease progression. Results Analysis using multiplex imaging flow cytometry revealed four distinct sub-populations of CTCs: epithelial (E-CTC), mesenchymal (M-CTC), partial epithelial-mesenchymal transition (pEMT-CTC) and stem cell-like (SC-CTC). Overall, CTC detection rate was 76.8% (209/272 draws) and total CTC counts did not correlate with any clinicopathological variables. However, the proportion of pEMT-CTCs (prop-pEMT) was correlated with advanced disease, worse progression-free and overall survival in all patients, and earlier recurrence after resection. Conclusion Our results underscore the importance of immunophenotyping and quantifying specific CTC sub-populations in PDAC.  

4.2415           Long-term control of diabetes in a nonhuman primate by two separate transplantations of porcine adult islets under immunosuppression

Kim, J-M., Hong, S-H., Shin, J-S., Min, B-H., Kim, H.J., Chung, H., Kim, J., Bang, Y.J., Seo, S., Hwang, E.S., Kang, H-J., Ha, J. and Park, C-G. Am. J. Transplant., 21, 3561-3572 (2021)   Porcine islet transplantation is an alternative to allo-islet transplantation. Retransplantation of islets is a routine clinical practice in islet allotransplantation in immunosuppressed recipients and will most likely be required in islet xenotransplantation in immunosuppressed recipients. We examined whether a second infusion of porcine islets could restore normoglycemia and further evaluated the efficacy of a clinically available immunosuppression regimen including anti-thymocyte globulin for induction; belimumab, sirolimus, and tofacitinib for maintenance and adalimumab, anakinra, IVIg, and tocilizumab for inflammation control in a pig to nonhuman primate transplantation setting. Of note, all nonhuman primates were normoglycemic after the retransplantation of porcine islets without induction therapy. Graft survival was >100 days for all 3 recipients, and 1 of the 3 monkeys showed insulin independence for >237 days. Serious lymphodepletion was not observed, and rhesus cytomegalovirus reactivation was controlled without any serious adverse effects throughout the observation period in all recipients. These results support the clinical applicability of additional infusions of porcine islets. The maintenance immunosuppression regimen we used could protect the reinfused islets from acute rejection.  

4.2416           Selective depletion of hepatic stellate cells specific LOXL1 alleviates liver fibrosis

Yang, A., Yan, X., Xu, H., Fan, X., Zhang, M., Huang, T., Li, W., Chen, W., Jia, J. and You, H. FASEB J., 35, e21918 (2021)   The role of LOXL1 in fibrosis via mediating ECM crosslinking and stabilization is well established; however, the role of hepatic stellate cells (HSCs)-specific LOXL1 in the development of fibrosis remains unknown. We generated HSCs-specific Loxl1-depleted mice (Loxl1^Gfap-cre mice) to investigate the HSCs-specific contribution of LOXL1 in the pathogenesis of fibrosis. Loxl1^fl/fl mice were used as the control. Furthermore, we used RNA sequencing to explore the underlying changes in the transcriptome. Results of the sirius red staining, type I collagen immunolabeling, and hydroxyproline content analysis, coupled with the reduced expression of profibrogenic genes revealed that Loxl1^Gfap-cre mice with CCl₄-induced fibrosis exhibited decreased hepatic fibrosis. In addition, Loxl1^Gfap-cre mice exhibited reduced macrophage tissue infiltration by CD68-positive cells and decreased expression of inflammatory genes compared with the controls. RNA sequencing identified integrin α8 (ITGA8) as a key modulator of LOXL1-mediated liver fibrosis. Functional analyses showed that siRNA silencing of Itga8 in cultured fibroblasts led to a decline in the LOXL1 expression and inhibition of fibroblast activation. Mechanistic analyses indicated that LOXL1 activated the FAK/PI3K/AKT/HIF1a signaling pathway, and the addition of inhibitors of FAK or PI3K reversed these results via downregulation of LOXL1. Furthermore, HIF1a directly interacted with LOXL1 and upregulated its expression, indicating that LOXL1 can positively self-regulate by forming a positive feedback loop with the FAK/PI3K/AKT/HIF1a pathway. We demonstrated that HSCs-specific Loxl1 deficiency prevented fibrosis, inflammation and that ITGA8/FAK/PI3K/AKT/HIF1a was essential for the function and expression of LOXL1. Knowledge of this approach can provide novel mechanisms and targets to treat fibrosis in the future.  

4.2417           Rapid, Simple, and Inexpensive Spatial Patterning of Wettability in Microfluidic Devices for Double Emulsion Generation

Liu, H., Piper, J.A. and Li, M. Anal. Chem., 93, 10955-10965 (2021)   Water-in-oil-in-water (w/o/w) double emulsion (DE) encapsulation has been widely used as a promising platform technology for various applications in the fields of food, cosmetics, pharmacy, chemical engineering, materials science, and synthetic biology. Unfortunately, DEs formed by conventional emulsion generation approaches in most cases are highly polydisperse, making them less desirable for quantitative assays, controlled biomaterial synthesis, and entrapped ingredient release. Microfluidic devices can generate monodisperse DEs with controllable size, morphology, and production rate, but these generally require multistep fabrication processes and use of different solvents or bulky external instrumentation to pattern channel wettability. To overcome these limitations, we propose a rapid, simple, and inexpensive method to spatially pattern wettability in microfluidic devices for the continuous generation of monodisperse DEs. This is achieved by applying corona–plasma treatment to a select zone of the microchannel surface aided by a custom-designed corona resistance microchannel to strictly confine the plasma-treatment zone in a single polydimethylsiloxane (PDMS) microfluidic device. The properties of PDMS channel surfaces and key microchannel regions for DE generation are characterized under different levels of treatment. The size, shell thickness, and number of inner cores of generated DEs are shown to be highly controllable by tuning the phase flow rate ratios. Using DEs as templates, we successfully achieve a one-step generation and collection of gelatin microgels. Additionally, we demonstrate the biological capability of generated DEs by flow cytometric screening of the encapsulation and growth of yeast cells within DEs. We expect that the proposed approach will be widely used to create microfluidic devices with more complex wettability patterns.  

4.2418           Highly Efficient Transfection of Human Primary T Lymphocytes Using Droplet-Enabled Mechanoporation

Joo, B., Hur, J., Kim, G-B., Yun, S.G. and Chung, A.J. ACS Nano, 15, 12888-12898 (2021)   Whole-cell-based therapy has been extensively used as an effective disease treatment approach, and it has rapidly changed the therapeutic paradigm. To fully accommodate this shift, advances in genome modification and cell reprogramming methodologies are critical. Traditionally, molecular tools such as viral and polymer nanocarriers and electroporation have been the norm for internalizing external biomolecules into cells for cellular engineering. However, these approaches are not fully satisfactory considering their cytotoxicity, high cost, low scalability, and/or inconsistent and ineffective delivery and transfection. To address these challenges, we present an approach that leverages droplet microfluidics with cell mechanoporation, bringing intracellular delivery to the next level. In our approach, cells and external cargos such as mRNAs and plasmid DNAs are coencapsulated into droplets, and as they pass through a series of narrow constrictions, the cell membrane is mechanically permeabilized where the cargos in the vicinity are internalized via convective solution exchange enhanced by recirculation flows developed in the droplets. Using this principle, we demonstrated a high level of functional macromolecule delivery into various immune cells, including human primary T cells. By utilizing droplets, the cargo consumption was drastically reduced, and near-zero clogging was realized. Furthermore, high scalability without sacrificing cell viability and superior delivery over state-of-the-art methods and benchtop techniques were demonstrated. Notably, the droplet-based intracellular delivery strategy presented here can be further applied to other mechanoporation microfluidic techniques, highlighting its potential for cellular engineering and cell-based therapies.  

4.2419           Recent advances in droplet microfluidics for enzyme and cell factory engineering

Yang, J., Tu, R., Yuan, H., Wang, Q. and Zhu, L. Critical Reviews in BIotechnol., 41(7), 1023-1045 (2021)   Enzymes and cell factories play essential roles in industrial biotechnology for the production of chemicals and fuels. The properties of natural enzymes and cells often cannot meet the requirements of different industrial processes in terms of cost-effectiveness and high durability. To rapidly improve their properties and performances, laboratory evolution equipped with high-throughput screening methods and facilities is commonly used to tailor the desired properties of enzymes and cell factories, addressing the challenges of achieving high titer and the yield of the target products at high/low temperatures or extreme pH, in unnatural environments or in the presence of unconventional media. Droplet microfluidic screening (DMFS) systems have demonstrated great potential for exploring vast genetic diversity in a high-throughput manner (>10⁶/h) for laboratory evolution and have been increasingly used in recent years, contributing to the identification of extraordinary mutants. This review highlights the recent advances in concepts and methods of DMFS for library screening, including the key factors in droplet generation and manipulation, signal sources for sensitive detection and sorting, and a comprehensive summary of success stories of DMFS implementation for engineering enzymes and cell factories during the past decade.  

4.2420           Gut Microbiome Directs Hepatocytes to Recruit MDSCs and Promote Cholangiocarcinoma

Qianfei Zhang, Chi Ma, Yi Duan, Bernd Heinrich, Umberto Rosato, Laurence P. Diggs, Lichun Ma et al Cancer Discovery, 11, 1248-1467 (2021)   Gut dysbiosis is commonly observed in patients with cirrhosis and chronic gastrointestinal disorders; however, its effect on antitumor immunity in the liver is largely unknown. Here we studied how the gut microbiome affects antitumor immunity in cholangiocarcinoma. Primary sclerosing cholangitis (PSC) or colitis, two known risk factors for cholangiocarcinoma which promote tumor development in mice, caused an accumulation of CXCR2⁺ polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC). A decrease in gut barrier function observed in mice with PSC and colitis allowed gut-derived bacteria and lipopolysaccharide to appear in the liver and induced CXCL1 expression in hepatocytes through a TLR4-dependent mechanism and an accumulation of CXCR2⁺ PMN-MDSCs. In contrast, neomycin treatment blocked CXCL1 expression and PMN-MDSC accumulation and inhibited tumor growth even in the absence of liver disease or colitis. Our study demonstrates that the gut microbiome controls hepatocytes to form an immunosuppressive environment by increasing PMN-MDSCs to promote liver cancer.  

4.2421           AICAR and compound C negatively modulate HCC-induced primary human hepatic stellate cell activation in vitro

Böttcher, K., Longato, L., marrone, G., Mazza, G., Ghemtio, L., Hall, A. et al Am. J. Physiol. Gastrointest. Physiol., 320, G543-G556 (2021)   Tumor stroma and microenvironment have been shown to affect hepatocellular carcinoma (HCC) growth, with activated hepatic stellate cells (HSC) as a major contributor in this process. Recent evidence suggests that the energy sensor adenosine monophosphate-activated kinase (AMPK) may mediate a series of essential processes during carcinogenesis and HCC progression. Here, we investigated the effect of different HCC cell lines with known TP53 or CTNBB1 mutations on primary human HSC activation, proliferation, and AMPK activation. We show that conditioned media obtained from multiple HCC cell lines differently modulate human hepatic stellate cell (hHSC) proliferation and hHSC AMPK activity in a paracrine manner. Pharmacological treatment of hHSC with AICAR and Compound C inhibited the HCC-induced proliferation/activation of hHSC through AMPK-dependent and AMPK-independent mechanisms, which was further confirmed using mouse embryonic fibroblasts (MEFs) deficient of both catalytic AMPKα isoforms (AMPK^{α1/α2−/−}) and wild type (wt) MEF. Both compounds induced S-phase cell-cycle arrest and, in addition, AICAR inhibited the mTORC1 pathway by inhibiting phosphorylation of 4E-BP1 and S6 in hHSC and wt MEF. Data mining of the Cancer Genome Atlas (TCGA) and the Liver Cancer (LICA-FR) showed that AMPKα1 (PRKAA1) and AMPKα2 (PRKAA2) expression differed depending on the mutation (TP53 or CTNNB1), tumor grading, and G1-G6 classification, reflecting the heterogeneity in human HCC. Overall, we provide evidence that AMPK modulating pharmacological agents negatively modulate HCC-induced hHSC activation and may therefore provide a novel approach to target the mutual, tumor-promoting interactions between hHSC and HCC.  

4.2422           Microfluidic Single-cell Trapping and Cultivation for the Analysis of Host-viral Interactions

Ganguly, R., Lee, B., Kang, S., Kim, Y.S., Jeong, S-G., Kim, J.S., Park, S.Y., Yohei, Y. and Lee, C-S. Biotechnology and Bioprocess Engineering, 26, 179-187 (2021)   The isolation of single cells and their further cultivation in confined chambers are essential to the collection of statistically reliable temporal information in cell-based biological experiments. In this work, we present a hydrodynamic single-cell trapping and culturing platform that facilitates biological analysis and experimentation of virus infection into host cells. To find the optimum design of the cell trap at the microscale, we evaluated hook traps with different widths and trap intervals to obtain a high trapping efficiency of a single cell. The proposed design leverages the stochastic position of the cells as they flow into the structured microfluidic channels, where hundreds of single cells are then arrayed in nanoliter chambers for simultaneous cell-specific data collection. Optimum design is used to devise and implement a hydrodynamic cell-trapping mechanism that is minimally detrimental to the cell viability and retains a high trapping efficiency (90%), with the capability of reaching high fill factors (90%) in short loading times (10 min) in a 450-trap device. Finally, we perform an analysis of host-viral interactions under the treatment of a drug concentration gradient as a proof of concept.  

4.2423           scRNA-seq of human vitiligo reveals complex networks of subclinical immune activation and a role for CCR5 in Treg function

Gellatly, K.J., Strassner, J.P., Essien, K., Refat, M.A., Murphy, R.L. et al Sci. Transl. Med., 13, eabd8995 (2021)   Vitiligo is an autoimmune skin disease characterized by the targeted destruction of melanocytes by T cells. Cytokine signaling between keratinocytes and T cells results in CD8⁺ T cell infiltration of vitiligo lesions, but the full scope of signals required to coordinate autoimmune responses is not completely understood. We performed single-cell RNA sequencing on affected and unaffected skin from patients with vitiligo, as well as healthy controls, to define the role of each cell type in coordinating autoimmunity during disease progression. We confirmed that type 1 cytokine signaling occupied a central role in disease, but we also found that this pathway was used by regulatory T cells (T_regs) to restrain disease progression in nonlesional skin. We determined that CCL5-CCR5 signaling served as a chemokine circuit between effector CD8⁺ T cells and T_regs, and mechanistic studies in a mouse model of vitiligo revealed that CCR5 expression on T_regs was required to suppress disease in vivo but not in vitro. CCR5 was not required for T_reg recruitment to skin but appeared to facilitate T_reg function by properly positioning these cells within the skin. Our data provide critical insights into the pathogenesis of vitiligo and uncover potential opportunities for therapeutic interventions.  

4.2424           Regional Delivery of CAR-T Effectively Controls Tumor Growth in Colorectal Liver Metastasis Model

Chai, L.F., Hardaway, J.C., Heatherton, K.R., O’Connell, K.P., LaPorte, J.P., Guha, P., Lopes, M.C., Rabinowitz, B.A., Jaroch, D., Cox, B.F., Knight, R. and Katz, S.C.

1. Surg. Res., 272, 37-50 (2022)

  Background Effective treatment of solid tumors requires multi-modality approaches. In many patients with stage IV liver disease, current treatments are not curative. Chimeric antigen receptor T cells (CAR-T) are an intriguing option following success in hematological malignancies, but this has not been translated to solid tumors. Limitations include sub-optimal delivery and elevated interstitial fluid pressures. We developed a murine model to test the impact of high-pressure regional delivery (HPRD) on trafficking to liver metastases (LM) and tumor response. Materials and Methods CAR-T were generated from CD45.1 mice and adoptively transferred into LM-bearing CD45.2 mice via regional or systemic delivery (RD, SD). Trafficking, tumor growth, and toxicity were evaluated with flow cytometry, tumor bioluminescence (TB, photons/sec log₂-foldover baseline), and liver function tests (LFTs). Results RD of CAR-T was more effective at controlling tumor growth versus SD from post-treatment days (PTD) 2-7 (P = 0.002). HPRD resulted in increased CAR-T penetration versus low-pressure RD (LPRD, P = 0.004), suppression of tumor proliferation (P = 0.03), and trended toward improved long-term control at PTD17 (TB=3.7 versus 6.1, P = 0.47). No LFT increase was noted utilizing HPRD versus LPRD (AST/ALT P = 0.65/0.84) while improved LFTs in RD versus SD groups suggested better tumor control (HPRD AST/ALT P = 0.04/0.04, LPRD AST/ALT P = 0.02/0.02). Conclusions Cellular immunotherapy is an emerging option for solid tumors. Our model suggests RD and HPRD improved CAR-T penetration into solid tumors with improved short-term tumor control. Barriers associated with SD can be overcome using RD techniques to maximize therapeutic delivery and HPRD may further augment efficacy without increased toxicity.  

4.2425           Intracellular IL-32 regulates mitochondrial metabolism, proliferation, and differentiation of malignant plasma cells

Aass, K.R., Mjelle, R., Kastnes, M.H., Slørdal, T.S., Waage, A. and Standal, T. iScience, 25, 103605 (2022)   Interleukin-32 (IL-32) is a nonclassical cytokine expressed in cancers, inflammatory diseases, and infections. Its expression is regulated by two different oxygen sensing systems; HIF1α and cysteamine dioxygenase (ADO), indicating that IL-32 may be involved in the response to hypoxia. We here demonstrate that endogenously expressed, intracellular IL-32 interacts with components of the mitochondrial respiratory chain and promotes oxidative phosphorylation. Knocking out IL-32 in three myeloma cell lines reduced cell survival and proliferation in vitro and in vivo. High-throughput transcriptomic and MS-metabolomic profiling of IL-32 KO cells revealed that cells depleted of IL-32 had perturbations in metabolic pathways, with accumulation of lipids, pyruvate precursors, and citrate. IL-32 was expressed in a subgroup of myeloma patients with inferior survival, and primary myeloma cells expressing IL-32 had a gene signature associated with immaturity, proliferation, and oxidative phosphorylation. In conclusion, we demonstrate a previously unrecognized role of IL-32 in the regulation of plasma cell metabolism.  

4.2426           Whole and fractionated human platelet lysate biomaterials-based biotherapy induces strong neuroprotection in experimental models of amyotrophic lateral sclerosis

Goul, F., Timmermann, K., Gosset, p., Raoul, C., Dutheil, M., Jonneaux, A., Garcon, G., Moreau, C., Danel-Brunaud, V., Duce, J., Burouf, T., Devedjian, J-C. and Devos, D. Biomaterials, 280, 121311 (2022)   Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease of motor neurons leading to death within 3 years and without a curative treatment. Neurotrophic growth factors (NTFs) are pivotal for cell survival. A reason for the lack of patient efficacy with single recombinant NTF brain infusion is likely to be due to the synergistic neuroprotective action of multiple NTFs on a diverse set of signaling pathways. Fractionated (protein size <50, <30, <10, <3 kDa) heat-treated human platelet lysate (HHPL) preparations were adapted for use in brain tissue with the aim of demonstrating therapeutic value in ALS models and further elucidation of the mechanisms of action. In neuronal culture all fractions induced Akt-dependent neuroprotection as well as a strong anti-apoptotic and anti-ferroptotic action. In the <3 kDa fraction anti-ferroptotic properties were shown to be GPX4 dependent highlighting a role for other platelet elements associated with NTFs. In the SOD1^G86R mouse model, lifespan was strongly increased by intracerebroventricular delivery of HHPL or by intranasal administration of <3 kDa fraction. Our results suggest that the platelet lysate biomaterials are neuroprotective in ALS. Further studies would now validate theragnostic biomarker on its antiferroptotic action, for further clinical development.  

4.2427           Amyloid beta increases ABCA1 and HMGCR protein expression, and cholesterol synthesis and accumulation in mice neurons and astrocytes

Azizidoost, S., Babaahmadi-Rezaei, H., Nazeri, Z., Cheraghzadeh, M. and Kheirrollah, A. BBA-Mol. Cell Biol. Lipids, 1867, 159069 (2022)   Introduction Imbalanced cholesterol metabolism in the brain is one of the main pathophysiological mechanisms involved in Alzheimer's disease. We investigated the effect of amyloid-beta (Aβ) on the main proteins involved in regulation of cholesterol metabolism along with cholesterol content in astrocytes and neurons. Methods Astrocytes and neurons were cultured and treated with Aβ. Apolipoprotein E (apoE) level in the cells and conditioned media, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), ATP-binding cassette transporter A1 (ABCA1), and cytochrome P450 46A1 (CYP46A1) in cell lysates were determined using immunoblotting. Astrocyte media was added to the Aβ-pretreated neurons then, HMGCR was assessed. Cholesterol was measured in both cells and media. Results Aβ caused a significant increase in HMGCR and ABCA1 protein levels and cholesterol content in both cells without increasing cholesterol efflux. A similar increase was seen for cellular apoE level in astrocytes with no changes in media with a significant reduction of cholesterol efflux. HMGCR level was restored to near control level when Aβ-pretreated neurons were exposed to media from culture astrocytes. Conclusion Almost all events related to cholesterol homeostasis in neurons and astrocytes, are somehow affected by Aβ. However, because ABCA1 has the most important role(s) in brain cholesterol homeostasis, all subsequent events associated with astrocytes-cholesterol synthesis and its shuttling to neurons are influenced by the effects of Aβ on ABCA1 which could likely be responsible for altered brain cholesterol metabolism in Alzheimer's disease.  

4.2428           Arabic gum plus colistin coated moxifloxacin-loaded nanoparticles for the treatment of bone infection caused by Escherichia coli

Aguilera-Correa, J.J., Gisbert-Garzaran, M., Mediero, A., Carias-Calix, R.A., Jimenez-Jimenez, C., Esteban, J. and Vallet-Regi, M. Acta Biomaterialia, 137, 218-237 (2022)   Osteomyelitis is an inflammatory process of bone and bone marrow that may even lead to patient death. Even though this disease is mainly caused by Gram-positive organisms, the proportion of bone infections caused by Gram-negative bacteria, such as Escherichia coli, has significantly increased in recent years. In this work, mesoporous silica nanoparticles have been employed as platform to engineer a nanomedicine able to eradicate E. coli- related bone infections. For that purpose, the nanoparticles have been loaded with moxifloxacin and further functionalized with Arabic gum and colistin (AG+CO-coated MX-loaded MSNs). The nanosystem demonstrated high affinity toward E. coli biofilm matrix, thanks to AG coating, and marked antibacterial effect because of the bactericidal effect of moxifloxacin and the disaggregating effect of colistin. AG+CO-coated MX-loaded MSNs were able to eradicate the infection developed on a trabecular bone in vitro and showed pronounced antibacterial efficacy in vivo against an osteomyelitis provoked by E. coli. Furthermore, AG+CO-coated MX-loaded MSNs were shown to be essentially non-cytotoxic with only slight effect on cell proliferation and mild hepatotoxicity, which might be attributed to the nature of both antibiotics. In view of these results, these nanoparticles may be considered as a promising treatment for bone infections caused by enterobacteria, such as E. coli, and introduce a general strategy against bone infections based on the implementation of antibiotics with different but complementary activity into a single nanocarrier.  

4.2429           A role for BET proteins in regulating basal, dopamine-induced and cAMP/PKA-dependent transcription in rat striatal neurons

Jones-Tabah, J., Martin, R.D., Chen, J.J., Tanny, J.C., Clarke, P.B.S. and Hebert, T.E. Cellular Signalling, 91, 110226 (2022)   The activity of striatal medium-spiny projection neurons is regulated by D1 and D2 dopamine receptors. The D1 receptor (D1R) is a Gα_s/olf-coupled GPCR which activates a cAMP/PKA/DARPP-32 signalling cascade that increases excitability and facilitates plasticity, partly through the regulation of transcription. Upon activation via D1R, PKA can translocate to the nucleus to regulate transcription through the phosphorylation of various targets. One candidate effector of PKA-dependent transcriptional regulation is the BET protein Brd4. It is known that when Brd4 is activated by phosphorylation, it binds more readily to acetylated histones at promoters and enhancers; moreover, in non-neuronal cells, PKA signalling has been shown to increase recruitment of Brd4 to chromatin. However, it is unknown whether BET proteins, or Brd4 specifically, are involved in transcriptional activation by cAMP/PKA in neurons. Here, we demonstrate that in adult rats, inhibition of BET proteins with the bromodomain inhibitor JQ1 suppressed the expression of ~25% of D1R-upregulated genes, while also increasing the expression of a subset of immediate-early genes. We further found that cAMP/PKA signalling promotes Brd4 recruitment to dopamine-induced genes in striatal neurons, and that knockdown of Brd4 attenuates D1R-induced gene expression. Finally, we report that JQ1 treatment downregulated expression of many GPCRs and also impaired ERK1/2 signalling in striatal neurons. Our findings identify the BET protein family, and Brd4 in particular, as novel regulators of basal and D1R-dependent transcription in rat striatal neurons, and delineate complex bi-directional effects of bromodomain inhibitors on neuronal transcription.  

4.2430           Protocol for the assessment of human T cell activation by real-time metabolic flux analysis

Kong, B.S., Lee, C., and Cho, Y.M. STAR Protocols, 3, 101084 (2022)   The elevation of glycolysis in autoreactive T cells is a key target for the prevention and treatment of T cell-related autoimmune diseases, such as type 1 diabetes (T1D). Here, we describe a simple and efficient protocol for isolating human peripheral blood mononuclear cells (PBMCs) and T cells, and the subsequent assessment of T cell glycolysis using Seahorse analyzer. This protocol is useful to analyze different subsets of T cells and applicable to different autoimmune disease models (i.e., T1D, multiple sclerosis). For complete details    

4.2431           Identification and function analysis of an immune deficiency homolog in swimming crab, Portunus trituberculatus

Zhou, S-M., Zhao, J-J., Wang, Y., Jin, S., Zhou, Q-C. and Yin, F. Fish and Shellfish Immunol., 121, 245-253 (2022)   The immune deficiency (IMD) pathway is involved in both antiviral and antibacterial immune responses in Drosophila. IMD protein is the key adaptor to link the extracellular signal and the intracellular reaction to initiate the signal transduction in IMD pathway. In present study, the cDNA of the IMD (Pt-IMD) was identified from a marine crab, Portunus trituberculatus. The Pt-IMD is predicted to encode 170 amino acids with a death domain. Real-Time quantitative PCR analysis showed that Pt-IMD was constitutively expressed in hemocytes, intestine, gill, heart, muscle and hepatopancreas in normal crab. Moreover, the transcript of Pt-IMD in large-granule hemocytes is approximately 6-fold higher than semi-granular cells and agranular cells. Intracellular localization showed Pt-IMD was distributed mainly in the cytoplasm when it was over-expressed in Drosophila Schneider 2 (S2) cell. Functionally, over-expression of Pt-IMD could activate the promoters of Drosophila antimicrobial peptide genes (AMPs) in S2 cell. Furthermore, Pt-IMD expression was also knock-down by RNAi to determine the function of Pt-IMD on regulation of the expression of different antimicrobial peptides (AMPs) in crab. In the primary cultured hemocytes challenged with or without Vibrio alginolyticus, after Pt-IMD was knocked-down by specific long double strand RNA, the expression of anti-lipopolysaccharide factor1 (ALF1), ALF3, crustin1, crustin3, arasin2, hyastatin1and hyastatin3 have been significantly inhibited in normal cell or bacterial infected cell, while the expression of lysozyme was normal in non-infected cells and was significantly induced in bacterial infected cells, which compared to the non-specific dsRNA treated cells.  

4.2432           Macrophages activated by hepatitis B virus have distinct metabolic profiles and suppress the virus via IL-1β to downregulate PPARα and FOXO3

Li, Y., Zhu, Y., Feng, S., Wang, S., Ann, D.K. and Ou, J-h. J. Cell Reports, 38, 110284 (2022)   Macrophages display phenotypic plasticity and can be induced by hepatitis B virus (HBV) to undergo either M1-like pro-inflammatory or M2-like anti-inflammatory polarization. Here, we report that M1-like macrophages stimulated by HBV exhibit a strong HBV-suppressive effect, which is diminished in M2-like macrophages. Transcriptomic analysis reveals that HBV induces the expression of interleukin-1β (IL-1β) in M1-like macrophages, which display a high oxidative phosphorylation (OXPHOS) activity distinct from that of conventional M1-like macrophages. Further analysis indicates that OXPHOS attenuates the expression of IL-1β, which suppresses the expression of peroxisome proliferator-activated receptor α (PPARα) and forkhead box O3 (FOXO3) in hepatocytes to suppress HBV gene expression and replication. Moreover, multiple HBV proteins can induce the expression of IL-1β in macrophages. Our results thus indicate that macrophages can respond to HBV by producing IL-1β to suppress HBV replication. However, HBV can also metabolically reprogram macrophages to enhance OXPHOS to minimize this host antiviral response.  

4.2433           A flow cytometry-based approach to identify distinct coelomocyte subsets of the purple sea urchin, Strongylocentrotus purpuratus

Hudgell, M.A.B., Grayfer, L. and Smith, L.C. Developmental and Cmnparative Immunol., 130, 104352 (2022)   The sea urchin, Strongylocentrotus purpuratus, possesses at least seven distinguishable cell populations in the coelomic fluid, which vary in morphology, size, and function. Of these, the large phagocytes, small phagocytes, and red spherule cells are thought to be key to the echinoid immune response. Because there are currently no effective and rapid means of evaluating sea urchin coelomocytes, we developed a flow cytometry based approach to identify these subsets from unseparated, unstained, live cells. In particular our gating strategy distinguishes between the large phagocytes, small phagocytes, red spherule cells, and a mixed population of vibratile cells and colorless spherule cells. This flow cytometry based analysis increases the speed and improves the reliability of coelomocyte analysis compared to differential cell counts by microscopy.  

4.2434           A cell atlas of microbe-responsive processes in the zebrafish intestine

Willms, R.J., Jones, L.O., Hocking, J.C. and Foley, E. Cell Reports, 38, 1100311 (2022)   Gut microbial products direct growth, differentiation, and development in animal hosts. However, we lack system-wide understanding of cell-specific responses to the microbiome. We profiled cell transcriptomes from the intestine, and associated tissue, of zebrafish larvae raised in the presence or absence of a microbiome. We uncovered extensive cellular heterogeneity in the conventional zebrafish intestinal epithelium, including previously undescribed cell types with known mammalian homologs. By comparing conventional to germ-free profiles, we mapped microbial impacts on transcriptional activity in each cell population. We revealed intricate degrees of cellular specificity in host responses to the microbiome that included regulatory effects on patterning and on metabolic and immune activity. For example, we showed that the absence of microbes hindered pro-angiogenic signals in the developing vasculature, causing impaired intestinal vascularization. Our work provides a high-resolution atlas of intestinal cellular composition in the developing fish gut and details the effects of the microbiome on each cell type.  

4.2435           Sphingosine 1-Phosphate Receptor 4 Promotes Nonalcoholic Steatohepatitis by Activating NLRP3 Inflammasome

Hong, C.H., Ko, M.S., Kim, J.H., Cho, H., Lee, C-H. eet al Cell. Mol. Gastroenterol. Hepatol., 33(2), 925-947 (2022)   Background & Aims Sphingosine 1-phosphate receptors (S1PRs) are a group of G-protein–coupled receptors that confer a broad range of functional effects in chronic inflammatory and metabolic diseases. S1PRs also may mediate the development of nonalcoholic steatohepatitis (NASH), but the specific subtypes involved and the mechanism of action are unclear. Methods We investigated which type of S1PR isoforms is activated in various murine models of NASH. The mechanism of action of S1PR4 was examined in hepatic macrophages isolated from high-fat, high-cholesterol diet (HFHCD)-fed mice. We developed a selective S1PR4 functional antagonist by screening the fingolimod (2-amino-2-[2-(4- n -octylphenyl)ethyl]-1,3- propanediol hydrochloride)-like sphingolipid-focused library. Results The livers of various mouse models of NASH as well as hepatic macrophages showed high expression of S1pr4. Moreover, in a cohort of NASH patients, expression of S1PR4 was 6-fold higher than those of healthy controls. S1pr4^+/- mice were protected from HFHCD-induced NASH and hepatic fibrosis without changes in steatosis. S1pr4 depletion in hepatic macrophages inhibited lipopolysaccharide-mediated Ca⁺⁺ release and deactivated the Nod-like receptor pyrin domain-containning protein 3 (NLRP3) inflammasome. S1P increased the expression of S1pr4 in hepatic macrophages and activated NLRP3 inflammasome through inositol trisphosphate/inositol trisphosphate–receptor–dependent [Ca⁺⁺] signaling. To further clarify the biological function of S1PR4, we developed SLB736, a novel selective functional antagonist of SIPR4. Similar to S1pr4^+/- mice, administration of SLB736 to HFHCD-fed mice prevented the development of NASH and hepatic fibrosis, but not steatosis, by deactivating the NLRP3 inflammasome. Conclusions S1PR4 may be a new therapeutic target for NASH that mediates the activation of NLRP3 inflammasome in hepatic macrophages.  

4.2436           Rab31 promotes activation of hepatic stellate cells by accelerating TGF-β receptor II complex endocytosis

Qin, C., Liu, Y., Huang, S., Ning, B., He, S. and Zhong, L. Int. J. Biochem. Cell Biol., 144, 106170 (2022)   Background & Aims Hepatic stellate cells activation is the key process of liver fibrosis, revealing the molecular mechanism of which is helpful to provide an effective target for inhibiting liver fibrosis. Rab31, a small GTPase, regulates the specificity of intracellular vesicular transport system, and is crucial for signal transduction. However, whether Rab31 is involved in hepatic stellate cells activation is unknown. Methods & Results Analysis of the differences in gene expression between human healthy and fibrotic liver tissues by sequencing revealed that Rab31 was significantly upregulated in fibrotic tissues. Immunohistochemistry and immunofluorescence analysis confirmed that Rab31 positively correlated with hepatic fibrosis. Next, mouse primary hepatic stellate cells were prepared, and their continuous activation was accompanied by Rab31 expression increased. Interestingly, knockdown of Rab31 by lentivirus can significantly restrict those cell activation. Subsequently, the vary of signal transduction after Rab31 knockdown was detected, its presented that the TGF-β/Smads signaling was obviously affected. Following experiments identified that Rab31 knockdown significantly inhibited the TGF-β activation and led to the failure of hepatic stellate cells activation. Importantly, we revealed that Rab31 knockdown could inhibit TGF-β receptor II complex endocytosis, a prerequisite for the activation of TGF-β signaling. Finally, in a mouse CCl₄ fibrosis model, we proved that Rab31 knockdown markedly inhibited hepatic fibrosis. Conclusions Our study demonstrated that Rab31 could promote hepatic stellate cells activation by accelerating TGF-β Receptor II complex endocytosis, suggesting that interfering with Rab31 could be an effectively strategy to inhibit hepatic fibrosis progression.  

4.2437           Identification of functional pathways for regenerative bioactivity of selected renal cells

Sha, W., Bertram, T., Jain, D., Brouwer, C. and Basu, J. Stem Cell Res. & Ther., 13:72 (2022)   Background Selected renal cells (SRC) are in Phase II clinical trials as a kidney-sourced, autologous, tubular epithelial cell-enriched cell-based therapy for chronic kidney disease (CKD). In preclinical studies with rodent models of CKD, SRC have been shown to positively modulate key renal biomarkers associated with development of the chronic disease condition. Methods A comparative bioinformatic analysis of transcripts specifically enriched or depleted in SRC component sub-populations relative to the initial, biopsy-derived cell source was conducted. Results Outcomes associated with therapeutically relevant bioactivity from a systematic, genome-wide transcriptomic profiling of rodent SRC are reported. Key transcriptomic networks and concomitant signaling pathways that may underlie SRC mechanism of action as manifested by reparative, restorative, and regenerative bioactivity in rodent models of chronic kidney disease are identified. These include genes and gene networks associated with cell cycle control, transcriptional control, inflammation, ECM–receptor interaction, immune response, actin polymerization, regeneration, cell adhesion, and morphogenesis. Conclusions These data indicate that gene networks associated with development of the kidney are also leveraged for SRC regenerative bioactivity, providing evidence of potential mechanisms of action.  

4.2438           KIAA1363 affects retinyl ester turnover in cultured murine and human hepatic stellate cells

Wagner, C., Hois, V., Eggeling, A., Pusch, L-M., Pajed, l., Starlinger, P., Claudel, T., Trauner, M., Zimmermann, R., Taschler, U. and Lass, A.

1. Lipid Res., 63(3), 100173 (2022)

  Large quantities of vitamin A are stored as retinyl esters (REs) in specialized liver cells, the hepatic stellate cells (HSCs). To date, the enzymes controlling RE degradation in HSCs are poorly understood. In this study, we identified KIAA1363 (also annotated as arylacetamide deacetylase 1 or neutral cholesterol ester hydrolase 1) as a novel RE hydrolase. We show that KIAA1363 is expressed in the liver, mainly in HSCs, and exhibits RE hydrolase activity at neutral pH. Accordingly, addition of the KIAA1363-specific inhibitor JW480 largely reduced RE hydrolase activity in lysates of cultured murine and human HSCs. Furthermore, cell fractionation experiments and confocal microscopy studies showed that KIAA1363 localizes to the endoplasmic reticulum. We demonstrate that overexpression of KIAA1363 in cells led to lower cellular RE content after a retinol loading period. Conversely, pharmacological inhibition or shRNA-mediated silencing of KIAA1363 expression in cultured murine and human HSCs attenuated RE degradation. Together, our data suggest that KIAA1363 affects vitamin A metabolism of HSCs by hydrolyzing REs at the endoplasmic reticulum, thereby counteracting retinol esterification and RE storage in lipid droplets.  

4.2439           An ancient, Antarctic-specific species complex: large divergences between multiple Antarctic lineages of the tardigrade genus Mesobiotus

Short, K.A., Sands, C.J., McInnes, S.J., Pisani, D., Stevens, M.I. and Convey, P. Mol. Phylogenetics Evolution, 170, 107429 (2022)   Antarctica has been isolated and progressively glaciated for over 30 million years, with only approximately 0.3 % of its area currently ice-free and capable of supporting terrestrial ecosystems. As a result, invertebrate populations have become isolated and fragmented, in some cases leading to speciation. Terrestrial invertebrate species currently found in Antarctica often show multi-million year, and even Gondwanan, heritage, with little evidence of recent colonisation. Mesobiotus is a globally distributed tardigrade genus. It has commonly been divided into two “groups”, referred to as harmsworthi and furciger, with both groups currently considered cosmopolitan, with global reports including from both the Arctic and the Antarctic. However, some authors considered that Meb. furciger, as originally described, may represent an Antarctic-specific lineage. Using collections of tardigrades from across the Antarctic continent and publicly available sequences obtained from online databases, we use mitochondrial and nuclear ribosomal sequence data to clarify the relationships of Antarctic Mesobiotus species. Our analyses show that all Antarctic members belong to a single lineage, evolving separately from non-Antarctic representatives. Within this Antarctic lineage there are further deep divisions among geographic regions of the continent, consistent with the presence of a species complex. Based on our data confirming the deep divisions between this Antarctic lineage, which includes representatives of both groups, we recommend that the use of furciger and harmsworthi group terminology is now abandoned, as it leads to systematic and biogeographical confusion.  

4.2440           Inhibition of endothelial Nox2 activation by LMH001 protects mice from angiotensin II-induced vascular oxidative stress, hypertension and aortic aneurysm

Fan, L.M., Liu, F., Du, J., Geng, L. and Li, J-M. Redox Biology, 51, 102269 (2022)   Endothelial oxidative stress and inflammation attributable to the activation of a Nox2-NADPH oxidase are key features of many cardiovascular diseases. Here, we report a novel small chemical compound (LMH001, MW = 290.079), by blocking phosphorylated p47^phox interaction with p22^phox, inhibited effectively angiotensin II (AngII)-induced endothelial Nox2 activation and superoxide production at a small dose (IC50 = 0.25 μM) without effect on peripheral leucocyte oxidative response to pathogens. The therapeutic potential of LMH001 was tested using a mouse model (C57BL/6J, 7-month-old) of AngII infusion (0.8 mg/kg/d, 14 days)-induced vascular oxidative stress, hypertension and aortic aneurysm. Age-matched littermates of p47^phox knockout mice were used as controls of Nox2 inhibition. LMH001 (2.5 mg/kg/d, ip. once) showed no effect on control mice, but inhibited completely AngII infusion-induced excess ROS production in vital organs, hypertension, aortic walls inflammation and reduced incidences of aortic aneurysm. LMH001 effects on reducing vascular oxidative stress was due to its inhibition of Nox2 activation and was abrogated by knockout of p47^phox. LMH001 has the potential to be developed as a novel drug candidate to treat oxidative stress-related cardiovascular diseases.  

4.2441           Notch-mediated lactate metabolism regulates MDSC development through the Hes1/MCT2/c-Jun axis

Zhao, J-L., Ye, Y-C., Gao, C-C-. Zhang, W., Han, H. and Qin, H-Y. Cell Reports, 38, 110451 (2022)   Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) play critical roles in tumorigenesis. However, the mechanisms underlying MDSC and TAM development and function remain unclear. In this study, we find that myeloid-specific activation of Notch/RBP-J signaling downregulates lactate transporter MCT2 transcription via its downstream molecule Hes1, leading to reduced intracellular lactate levels, blunted granulocytic MDSC (G-MDSC) differentiation, and enhanced TAM maturation. We identify c-Jun as a novel intracellular sensor of lactate in myeloid cells using liquid-chromatography-mass spectrometry (LC-MS) followed by CRISPR-Cas9-mediated gene disruption. Meanwhile, lactate interacts with c-Jun to protect from FBW7 ubiquitin-ligase-mediated degradation. Activation of Notch signaling and blockade of lactate import repress tumor progression by remodeling myeloid development. Consistently, the relationship between the Notch-MCT2/lactate-c-Jun axis in myeloid cells and tumorigenesis is also confirmed in clinical lung cancer biopsies. Taken together, our current study shows that lactate metabolism regulated by activated Notch signaling might participate in MDSC differentiation and TAM maturation.  

4.2442           Histone Variant macroH2A1.1 Enhances Nonhomologous End Joining-dependent DNA Double-strand-break Repair and Reprogramming Efficiency of Human iPSCs

Giallongo, S., Rehakova, D., Biagini, T., Lo Re, O., Raina, P. et al Stem Cells, 40, 35-48 (2022)   DNA damage repair (DDR) is a safeguard for genome integrity maintenance. Increasing DDR efficiency could increase the yield of induced pluripotent stem cells (iPSC) upon reprogramming from somatic cells. The epigenetic mechanisms governing DDR during iPSC reprogramming are not completely understood. Our goal was to evaluate the splicing isoforms of histone variant macroH2A1, macroH2A1.1, and macroH2A1.2, as potential regulators of DDR during iPSC reprogramming. GFP-Trap one-step isolation of mtagGFP-macroH2A1.1 or mtagGFP-macroH2A1.2 fusion proteins from overexpressing human cell lines, followed by liquid chromatography-tandem mass spectrometry analysis, uncovered macroH2A1.1 exclusive interaction with Poly-ADP Ribose Polymerase 1 (PARP1) and X-ray cross-complementing protein 1 (XRCC1). MacroH2A1.1 overexpression in U2OS-GFP reporter cells enhanced specifically nonhomologous end joining (NHEJ) repair pathway, while macroH2A1.1 knock-out (KO) mice showed an impaired DDR capacity. The exclusive interaction of macroH2A1.1, but not macroH2A1.2, with PARP1/XRCC1, was confirmed in human umbilical vein endothelial cells (HUVEC) undergoing reprogramming into iPSC through episomal vectors. In HUVEC, macroH2A1.1 overexpression activated transcriptional programs that enhanced DDR and reprogramming. Consistently, macroH2A1.1 but not macroH2A1.2 overexpression improved iPSC reprogramming. We propose the macroH2A1 splicing isoform macroH2A1.1 as a promising epigenetic target to improve iPSC genome stability and therapeutic potential.  

4.2443           Cell type–specific mechanism of Setd1a heterozygosity in schizophrenia pathogenesis

Chen, R., Liu, Y., Djekidel, M.N., Chen, W., Bhattacherjee, A., Chen, Z., Scolnik, E. and Zhang, Y. Sci. Adv., 8, eabm1077 (2022)   Schizophrenia (SCZ) is a chronic, serious mental disorder. Although more than 200 SCZ-associated genes have been identified, the underlying molecular and cellular mechanisms remain largely unknown. Here, we generated a Setd1a (SET domain containing 1A) haploinsufficiency mouse model to understand how this SCZ-associated epigenetic factor affects gene expression in brain regions highly relevant to SCZ. Single-cell RNA sequencing revealed that Setd1a heterozygosity causes highly variable transcriptional adaptations across different cell types in prefrontal cortex (PFC) and striatum. The Foxp2⁺ neurons exhibit the most prominent gene expression changes among the different neuron subtypes in PFC, which correlate with changes in histone H3 lysine 4 trimethylation. Many of the genes dysregulated in Setd1a^+/− mice are involved in neuron morphogenesis and synaptic function. Consistently, Setd1a^+/− mice exhibit certain behavioral features of patients with SCZ. Collectively, our study establishes Setd1a^+/− mice as a model for understanding SCZ and uncovers a complex brain region– and cell type–specific dysregulation that potentially underlies SCZ pathogenesis.  

4.2444           Active single cell encapsulation using SAW overcoming the limitations of Poisson distribution

Link, A., McGrath, J.S., Zaimagaoglu, M. and Franke, T. Lab Chip, 22, 193-200 (2022)   We demonstrate the use of an acoustic device to actively encapsulate single red blood cells into individual droplets in a T-junction. We compare the active encapsulation with the passive encapsulation depending on the number of loaded cells as well as the created droplet volumes. This method overcomes the Poisson limitation statistical loading of cells for the passive encapsulation. In our experiments we reach a single cell encapsulation efficiency of 97.9 ± 2.1% at droplet formation rates exceeding 15 Hz.  

4.2445           The miR-23b/27b/24-1 Cluster Inhibits Hepatic Fibrosis by Inactivating Hepatic Stellate Cells

Wan, L-Y., Peng, H., Ni, Y-R., Jiang, X-P., Wang, J-J., Zhang, Y-Q. et al Cell. Mol. Gastroenterol. Hepatol., 13, 1393-1412 (2022)   Background & Aims Hepatic fibrosis is characterized by hepatic stellate cell (HSC) activation and transdifferentiation-mediated extracellular matrix (ECM) deposition, which both contribute to cirrhosis. However, no antifibrotic regimen is available in the clinic. microRNA-23b/27b/24-1 cluster inhibition of transforming growth factor-β (TGF-β) signaling during hepatic development prompted us to explore whether this cluster inhibits HSC activation and hepatic fibrosis. Methods Experimental fibrosis was studied in carbon tetrachloride (CCl₄)-treated C57BL/6 mice. After administration of miR-23b/27b/24-1 lentivirus or vehicle, animals were euthanized for liver histology. In primary rat HSC and HSC-T6, the anti-fibrotic effect of miR-23b/27b/24-1 cluster was furtherly investigated by RNA-sequencing, luciferase reporter assay, western blotting and bioinformatic means. Results In this study, we showed that increasing the miR-23b/27b/24-1 level through intravenous delivery of miR-23b/27b/24-1 lentivirus ameliorated mouse hepatic fibrosis. Mechanistically, the miR-23b/27b/24-1 cluster directly targeted messenger RNAs, which reduced the protein expression of 5 secretory profibrotic genes (TGF-β2, Gremlin1, LOX, Itgα2, and Itgα5) in HSCs. Suppression of the TGF-β signaling pathway by down-regulation of TGF-β2, Itgα2, and Itgα5, and activation of the bone morphogenetic protein signaling pathway by inhibition of Gremlin1, decreased extracellular matrix secretion of HSCs. Furthermore, down-regulation of LOX expression softened the ECM. Moreover, a reduction in tissue inhibitors of metalloproteinase 1 expression owing to weakened TGF-β signaling increased ECM degradation. Conclusions Hepatic overexpression of the miR-23b/27b/24-1 cluster blocked hepatic fibrosis and may be a novel therapeutic regimen for patients with hepatic fibrosis.  

4.2446           Engineered neuronal microtissue provides exogenous axons for delayed nerve fusion and rapid neuromuscular recovery in rats

Burrell, J.C., Das, S., Laimo, F.A., Katiyar, K.S., Browne, K.D., Shultz, R.B., Tien, V.J., Vu, P.T., Petrov, D., Ali, Z.A., Rosen, J.M. and Cullen, D.K. Bioactive Materials, 15, 339-353 (2022)   Nerve injury requiring surgical repair often results in poor functional recovery due to the inability of host axons to re-grow long distances and reform meaningful connections with the target muscle. While surgeons can re-route local axon fascicles to the target muscle, there are no technologies to provide an exogenous source of axons without sacrificing healthy nerves. Accordingly, we have developed tissue engineered neuromuscular interfaces (TE-NMIs) as the first injectable microtissue containing motor and sensory neurons in an anatomically-inspired architecture. TE-NMIs provide axon tracts that are intended to integrate with denervated distal structures and preserve regenerative capacity during prolonged periods without host innervation. Following implant, we found that TE-NMI axons promoted Schwann cell maintenance, integrated with distal muscle, and preserved an evoked muscle response out to 20-weeks post nerve transection in absence of innervation from host axons. By repopulating the distal sheath with exogenous axons, TE-NMIs also enabled putative delayed fusion with proximal host axons, a phenomenon previously not achievable in delayed repair scenarios due to distal axon degeneration. Here, we found immediate electrophysiological recovery after fusion with proximal host axons and improved axon maturation and muscle reinnervation at 24-weeks post-transection (4-weeks following delayed nerve fusion). These findings show that TE-NMIs provide the potential to improve functional recovery following delayed nerve repair.  

4.2447           Bioactive hydrogel microcapsules for guiding stem cell fate decisions by release and reloading of growth factors

Gwon, K., Hong, H.J., Gonzalez-Suarez, A.M., Slama, M.Q., Choi, D., Hong, J., Baskaran, H., Stybayeva, G., Peterson, Q.P. and Revzin, A. Bioactive Materials,15, 1-14 (2022)   Human pluripotent stem cells (hPSC) hold considerable promise as a source of adult cells for treatment of diseases ranging from diabetes to liver failure. Some of the challenges that limit the clinical/translational impact of hPSCs are high cost and difficulty in scaling-up of existing differentiation protocols. In this paper, we sought to address these challenges through the development of bioactive microcapsules. A co-axial flow focusing microfluidic device was used to encapsulate hPSCs in microcapsules comprised of an aqueous core and a hydrogel shell. Importantly, the shell contained heparin moieties for growth factor (GF) binding and release. The aqueous core enabled rapid aggregation of hPSCs into 3D spheroids while the bioactive hydrogel shell was used to load inductive cues driving pluripotency maintenance and endodermal differentiation. Specifically, we demonstrated that one-time, 1 h long loading of pluripotency signals, fibroblast growth factor (FGF)-2 and transforming growth factor (TGF)-β1, into bioactive microcapsules was sufficient to induce and maintain pluripotency of hPSCs over the course of 5 days at levels similar to or better than a standard protocol with soluble GFs. Furthermore, stem cell-carrying microcapsules that previously contained pluripotency signals could be reloaded with an endodermal cue, Nodal, resulting in higher levels of endodermal markers compared to stem cells differentiated in a standard protocol. Overall, bioactive heparin-containing core-shell microcapsules decreased GF usage five-fold while improving stem cell phenotype and are well suited for 3D cultivation of hPSCs.    

4.2448           A mouse model with widespread expression of the C9orf72-linked glycine–arginine dipeptide displays non-lethal ALS/FTD-like phenotypes

Verdone, B.M., Cicardi, M.E., Wen, X., Sriramoji, S., Russell, K., Markandaiah, S.S., Jensen, B.K., Krishnamurthy, K., Haeusler, A.R., Pasinelli, P. and Trotti, D. Scientific Reports, 12:5644 (2022)   Translation of the hexanucleotide G4C2 expansion associated with C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) produces five different dipeptide repeat protein (DPR) species that can confer toxicity. There is yet much to learn about the contribution of a single DPR to disease pathogenesis. We show here that a short repeat length is sufficient for the DPR poly-GR to confer neurotoxicity in vitro, a phenomenon previously unobserved. This toxicity is also reported in vivo in our novel knock-in mouse model characterized by widespread central nervous system (CNS) expression of the short-length poly-GR. We observe sex-specific chronic ALS/FTD-like phenotypes in these mice, including mild motor neuron loss, but no TDP-43 mis-localization, as well as motor and cognitive impairments. We suggest that this model can serve as the foundation for phenotypic exacerbation through second-hit forms of stress.  

4.2449           Transplantation of human iPSC-derived muscle stem cells in the diaphragm of Duchenne muscular dystrophy model mice

Miura, Y., Sato, M., Kuwahara, T., Ebata, T., Tabata, Y. and Sakurai, H. PloS One, 17(4), e0266391 (2022)   Duchenne muscular dystrophy (DMD) is an intractable genetic muscular disorder characterized by the loss of DYSTROPHIN. The restoration of DYSTROPHIN is expected to be a curative therapy for DMD. Because muscle stem cells (MuSCs) can regenerate damaged myofibers with full-length DYSTROPHIN in vivo, their transplantation is being explored as such a therapy. As for the transplanted cells, primary satellite cells have been considered, but donor shortage limits their clinical application. We previously developed a protocol that differentiates induced pluripotent stem cells (iPSCs) to MuSCs (iMuSCs). To ameliorate the respiratory function of DMD patients, cell transplantation to the diaphragm is necessary but difficult, because the diaphragm is thin and rapidly moves. In the present study, we explored the transplantation of iMuSCs into the diaphragm. First, we show direct cell injection into the diaphragm of mouse was feasible. Then, to enhance the engraftment of the transplanted cells in a rapidly moving diaphragm, we mixed polymer solutions of hyaluronic acid, alginate and gelatin to the cell suspension, finding a solution of 20% dissolved hyaluronic acid and 80% dissolved gelatin improved the engraftment. Thus, we established a method for cell transplantation into mouse diaphragm and show that an injectable hyaluronic acid-gelatin solution enables the engraftment of iMuSCs in the diaphragm.  

4.2450           The Annexin A2-Notch regulatory loop in hepatocytes promotes liver fibrosis in NAFLD by increasing osteopontin expression

Wang, G., Duan, J., Pu, G., Ye, C., Li, Y., Xiu, W., Xu, J., Liu, B., Zhu, Y. and Wang, C. BBA-Mol. Basis of Disease, 1868, 166413 (2022)   Background The mechanisms underlying the progression of liver disease from simple hepatic steatosis to advanced nonalcoholic steatohepatitis (NASH) and liver fibrosis warrant further investigation. Increased mRNA levels of Annexin A2 protein (Anxa2) have been observed in patients with NASH. However, the role of Anxa2 in NASH remains unclear. Methods The protein levels of Anxa2 were analyzed in the livers of mice and patients with NASH. Anxa2-knockout and -knockdown mice were generated, and NASH was induced through a high fructose, palmitate, and cholesterol (FPC) diet or methionine- and choline-deficient (MCD) diet. Findings We found elevated expression of Anxa2 in the livers of patients and mice with NASH. Anxa2 knockdown but not knockout ameliorated liver fibrosis in both FPC and MCD diet–fed mice. Liver-specific Anxa2 overexpression increased collagen deposition in mice fed a normal diet. Mechanistically, Anxa2 overexpression in hepatocytes promoted hepatic stellate cell activation in a paracrine manner by increasing osteopontin expression. Notch inhibition suppressed the exogenous overexpression of Anxa2-induced osteopontin and endogenous Anxa2 expression. Additionally, Anxa2 overexpression accelerated the progression of nonalcoholic fatty liver disease (NAFLD) in mice fed a high-fat diet. Moreover, Anxa2 levels were higher in NAFLD patients with advanced liver fibrosis than in those with mild liver fibrosis, as determined using the Gene Expression Omnibus database. Interpretation In conclusion, we found increased Anxa2 expression in hepatocytes promoted liver fibrosis in NASH mice by increasing osteopontin expression. The Anxa2-Notch positive regulatory loop contributes to this process and represents a novel target for the treatment of NASH-related liver fibrosis.  

4.2451           Biocatalytic living materials built by compartmentalized microorganisms in annealable granular hydrogels

Li, Y., Di, Z., Yan, X., Wen, H., Cheng, W., Zhang, j. and Yu, Z Chem. Engineering J., 445, 136822 (2022)   The field of living materials aims to use microorganisms as cell factories for drawing energy from their environment and to modulate the performance of the materials in some manner. Although the emergence of bioprinting techniques has given rise to the creating of living materials with rationally designed properties, a challenge is the controlling of the printability of the cell-laden bioinks while maintaining the high viability of cells. Here, we present an annealable granular hydrogel system that can encapsulate and compartment microorganisms for the 3D printing of biocatalytic living materials. Yeast-laden hydrogel microparticles (HMPs) are generated by a droplet-based microfluidic preparation process and then are jammed into granular hydrogels with shear-thinning and self-recovery behaviors. Upon extrusion-based 3D printing, the jammed HMPs are able to deposit into a designated structure and can be further annealed by interparticle cross-linking. Using HMPs as microorganism carriers, yeast cells are protected by encapsulation and survive shear forces during 3D printing. Further, printed constructs display enhanced catalytic activity, which show increased ethanol production due to improved mass transfer. The combination of annealable granular hydrogels and 3D printing should enable novel routes to produce microorganisms-based living materials with further applications in bioremediation, biosensing and biomedicine.  

4.2452           Analysis of the interplay between hepatitis B virus-positive hepatocytes and Kupffer cells ex vivo using mice as a model

Jiyoung, Y.L., Doumet, L., Helou, G., Akbari, O., Ou, J.J. STAR Protocols, 2, 101364 (2022)   Kupffer cells play critical roles in both hepatitis B virus (HBV) persistence and clearance. Here, we provide a protocol for studying the interplay between Kupffer cells and HBV-positive hepatocytes ex vivo using mice as a model. This protocol includes hydrodynamic injection of HBV DNA into mouse hepatocytes, liver perfusion for isolating hepatocytes and Kupffer cells, and Seahorse metabolic analysis of Kupffer cells. This protocol allows the detailed analysis of how HBV-positive hepatocytes and Kupffer cells impact each other ex vivo.  

4.2453           Notch-Regulated c-Kit–Positive Liver Sinusoidal Endothelial Cells Contribute to Liver Zonation and Regeneration

Dun, J-L., Zhou, Z-Y., Ruan, B., Fang, Z-Q., Ding, J., Liu, J-J., Song, P., Xu, H., Xu, C., Yue, Z-S., Han, H., Dou, G-R. and Wang, L. Cell. Mol., Gastroenterol. Hepatol., 13, 1741-1756 (2022)   Background & Aims Liver sinusoidal endothelial cells (SECs) promote the proliferation of hepatocytes during liver regeneration. However, the specific subset of SECs and its mechanisms during the process remain unclear. In this study, we investigated the potential role of c-kit⁺ SECs, a newly identified subset of SECs in liver regeneration. Methods Partial hepatectomy mice models were established to induce liver regeneration. Hepatic c-kit expression was detected by quantitative reverse-transcription polymerase chain reaction, immunofluorescent staining, and fluorescence-activated cell sorting. VE-cadherin-cyclization recombinase-estrogen receptor (Cdh5-Cre-ERT) Notch intracellular domain and Cdh5-Cre recombination signal binding protein Jκ^floxp mice were introduced to mutate Notch signaling. c-Kit⁺ SECs were isolated by magnetic beads. Single-cell RNA sequencing was performed on isolated SECs. Liver injuries were induced by CCl₄ or quantitative polymerase chain reaction injection. Results Hepatic c-kit is expressed predominantly in SECs. Liver resident SECs contribute to the increase of c-kit during partial hepatectomy–induced liver regeneration. Isolated c-kit⁺ SECs promote hepatocyte proliferation in vivo and in vitro by facilitating angiocrine. The distribution of c-kit shows distinct spatial differences that are highly coincident with the liver zonation marker wingless-type MMTV integration site family, member2 (Wnt2). Notch mutation reshapes the c-kit distribution and liver zonation, resulting in altered hepatocyte proliferation. c-Kit⁺ SECs were shown to regulate hepatocyte regeneration through angiocrine in a Wnt2-dependent manner. Activation of the Notch signaling pathway weakens liver regeneration by inhibiting positive regulatory effects of c-kit⁺ SECs on hepatocytes. Furthermore, c-kit⁺ SEC infusion attenuates toxin-induced liver injuries in mice. Conclusions Our results suggest that c-kit⁺ SECs contributes to liver zonation and regeneration through Wnt2 and is regulated by Notch signaling, providing opportunities for novel therapeutic approaches to liver injury in the future. Transcript profiling: GEO (accession number: GSE134037).  

4.2454           Chapter 5 - Allogeneic islet isolation: Methods to improve islet cell transplantation with new technologies in organ transplant retrieval and isolation techniques

Balamurugan,. A.N., Samaga, K.K., Narayanan, S., Kodipol, A.A., Moksagundan, S.P. and nathan, J.D. Pancreas and Beta Cell Replacement, 1, 81-96 (2022)   Islet cell transplantation (IT) is a proven therapy in preventing severe hypoglycemia and restoring insulin independence for the subgroups of patients with type 1 diabetes and severe chronic pancreatitis. Several studies have reported promising clinical outcomes for patients undergoing this procedure. IT involves a sequential series of steps, including donor pancreas procurement and islet isolation, purification, culture, and infusion. Several factors impact the success of human islet isolation, including the quality of the pancreas, the skills of the islet isolation team, and the dose and composition of the pancreatic tissue-dissociation enzyme blends. This chapter presents an overview of some new developments that have contributed to improvements in particular for allogeneic IT success, particularly focusing on donor selection, pancreas procurement, and islet isolation techniques, with a discussion of future areas of progress to improve overall outcomes.  

4.2455           Human pancreatic microenvironment promotes β-cell differentiation via non-canonical WNT5A/JNK and BMP signaling

Chmielowiec, J., Szlachcic, W.J., Yang, D., Scavuzzo, M.A., Wamble, k., Sarrion-Perdigones, A., Sabek, O.M., Venken, K.J.T. and Borowiak, M. Nature Comm., 13:1952 (2022)   In vitro derivation of pancreatic β-cells from human pluripotent stem cells holds promise as diabetes treatment. Despite recent progress, efforts to generate physiologically competent β-cells are still hindered by incomplete understanding of the microenvironment’s role in β-cell development and maturation. Here, we analyze the human mesenchymal and endothelial primary cells from weeks 9-20 fetal pancreas and identify a time point-specific microenvironment that permits β-cell differentiation. Further, we uncover unique factors that guide in vitro development of endocrine progenitors, with WNT5A markedly improving human β-cell differentiation. WNT5A initially acts through the non-canonical (JNK/c-JUN) WNT signaling and cooperates with Gremlin1 to inhibit the BMP pathway during β-cell maturation. Interestingly, we also identify the endothelial-derived Endocan as a SST+ cell promoting factor. Overall, our study shows that the pancreatic microenvironment-derived factors can mimic in vivo conditions in an in vitro system to generate bona fide β-cells for translational applications.  

4.2456           Rapid switching and durable on-chip spark-cavitation-bubble cell sorter

Jiao, Z., Han, Y., Zhao, J., Chao, Z., Tarnok, A. and You, Z. Microsystems & Nanoengineering, 8:52 (82022)   Precise and high-speed sorting of individual target cells from heterogeneous populations plays an imperative role in cell research. Although the conventional fluorescence-activated cell sorter (FACS) is capable of rapid and accurate cell sorting, it occupies a large volume of the instrument and inherently brings in aerosol generation as well as cross-contamination among samples. The sorting completed in a fully enclosed and disposable microfluidic chip has the potential to eliminate the above concerns. However, current microfluidic cell sorters are hindered by the high complexities of the fabrication procedure and the off-chip setup. In this paper, a spark-cavitation-bubble-based fluorescence-activated cell sorter is developed to perform fast and accurate sorting in a microfluidic chip. It features a simple structure and an easy operation. This microfluidic sorter comprises a positive electrode of platinum and a negative electrode of tungsten, which are placed on the side of the main channel. By applying a high-voltage discharge on the pair of electrodes, a single spark cavitation bubble is created to deflect the target particle into the downstream collection channel. The sorter has a short switching time of 150 μs and a long lifespan of more than 100 million workable actions. In addition, a novel control strategy is proposed to dynamically adjust the discharge time to stabilize the size of the cavitation bubble for continuous sorting. The dynamic control of continuously triggering the sorter, the optimal delay time between fluorescence detection and cell sorting, and a theoretical model to predict the ideal sorting recovery and purity are studied to improve and evaluate the sorter performance. The experiments demonstrate that the sorting rate of target particles achieves 1200 eps, the total analysis throughput is up to 10,000 eps, the particles sorted at 4000 eps exhibit a purity greater than 80% and a recovery rate greater than 90%, and the sorting effect on the viability of HeLa cells is negligible.  

4.2457           MyD88 in hepatic stellate cells enhances liver fibrosis via promoting macrophage M1 polarization

Zhang, J., Liu, Y., Chen, H., Yuan, Q., Wang, J., Niu, M., Hou, L., Gu, J. and Zhang, J. Cell Death and Disease, 13:411 (2022)   During liver fibrosis, quiescent HSCs (qHSCs) are activated to become activated HSCs (aHSCs)/myofibroblasts. The signal adapter MyD88, an essential component of TLR signaling, plays an important role in liver fibrosis. However, far less is known about the specific effects of MyD88 signaling in both qHSCs and aHSCs in the progress of liver fibrosis. Here, we used a CCl₄-induced mouse fibrosis model in which MyD88 was selectively depleted in qHSCs (GFAP^MyD88−/− mice) or aHSCs (α-SMA^MyD88−/− mice). MyD88 deficiency in qHSCs or aHSCs attenuated liver fibrosis in mice and inhibited α-SMA-positive cell activation. Inhibition of MyD88 in HSCs decreased α-SMA and collagen I levels, inflammatory cell infiltration, and pro-inflammatory gene expression. Furthermore, MyD88 signaling in HSCs increased the secretion of CXCL10, which promoted macrophage M1 polarization through CXCR3, leading to activation of the JAK/STAT1 pathway. Inhibition of CXCL10 attenuated macrophage M1 polarization and reduced liver fibrosis. Thus, MyD88 signaling in HSCs crucially contributes to liver fibrosis and provides a promising therapeutic target for the prevention and treatment of liver fibrosis.  

4.2458           Dissolvable microgel-templated macroporous hydrogels for controlled cell assembly

Jiang, Z., Lin, F-Y., Jiang, K., Nguyen, H., Chang, C-Y and Lin, C-C. Biomaterials Adv., 134, 112712 (2022)   Mesenchymal stem cells (MSCs)-based therapies have been widely used to promote tissue regeneration and to modulate immune/inflammatory response. The therapeutic potential of MSCs can be further improved by forming multi-cellular spheroids. Meanwhile, hydrogels with macroporous structures are advantageous for improving mass transport properties for the cell-laden matrices. Herein, we report the fabrication of MSC-laden macroporous hydrogel scaffolds through incorporating rapidly dissolvable spherical cell-laden microgels. Dissolvable microgels were fabricated by tandem droplet-microfluidics and thiol-norbornene photopolymerization using a novel fast-degrading macromer poly(ethylene glycol)-norbornene-dopamine (PEGNB-Dopa). The cell-laden PEGNB-Dopa microgels were subsequently encapsulated within another bulk hydrogel matrix, whose porous structure was generated efficiently by the rapid degradation of the PEGNB-Dopa microgels. The cytocompatibility of this in situ pore-forming approach was demonstrated with multiple cell types. Furthermore, adjusting the stiffness and cell adhesiveness of the bulk hydrogels afforded the formation of solid cell spheroids or hollow spheres. The assembly of solid or hollow MSC spheroids led to differential activation of AKT pathway. Finally, MSCs solid spheroids formed in situ within the macroporous hydrogels exhibited robust secretion of HGF, VEGF-A, IL-6, IL-8, and TIMP-2. In summary, this platform provides an innovative method for forming cell-laden macroporous hydrogels for a variety of future biomedical applications.  

4.2459           Stress Hormones Epinephrine and Corticosterone Selectively Reactivate HSV-1 and HSV-2 in Sympathetic and Sensory Neurons

Goswani, P., Ives, A.M., Abbott, A.R.N. and Bertke, A.S. Viruses, 14:1115 (2022)   Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) establish latency in sensory and autonomic neurons, from which they can reactivate to cause recurrent disease throughout the life of the host. Stress is strongly associated with HSV recurrences in humans and animal models. However, the mechanisms through which stress hormones act on the latent virus to cause reactivation are unknown. We show that the stress hormones epinephrine (EPI) and corticosterone (CORT) induce HSV-1 reactivation selectively in sympathetic neurons, but not sensory or parasympathetic neurons. Activation of multiple adrenergic receptors is necessary for EPI-induced HSV-1 reactivation, while CORT requires the glucocorticoid receptor. In contrast, CORT, but not EPI, induces HSV-2 reactivation in both sensory and sympathetic neurons through either glucocorticoid or mineralocorticoid receptors. Reactivation is dependent on different transcription factors for EPI and CORT, and coincides with rapid changes in viral gene expression, although genes differ for HSV-1 and HSV-2, and temporal kinetics differ for EPI and CORT. Thus, stress-induced reactivation mechanisms are neuron-specific, stimulus-specific and virus-specific. These findings have implications for differences in HSV-1 and HSV-2 recurrent disease patterns and frequencies, as well as development of targeted, more effective antivirals that may act on different responses in different types of neurons  

4.2460           Antigen Targeting of Porcine Skin DEC205+ Dendritic Cells

Melgoza-Gonzalez, E.A., Resendiz-Sandoval, M., Hinojosa-Trujillo, D., Hernansez-Valenzuela, S., Garica-Vega, M., Mata-Haro, V., Tepale-Segura, A., Bonifaz, L.C., Perez-Torres, A. and Hernandez, J. Vaccines, 10:684 (2022)   Dendritic cell (DC) targeting by DEC205⁺ cells effectively promotes the internalization of antigens that may trigger a specific immune response. In this study, we evaluated the ability of a recombinant antibody, anti-DEC205 (rAb ZH9F7), to trigger cellular endocytosis in subpopulations of DCs and targeted cells after intradermal injection and subsequent migration toward lymph nodes. Furthermore, the cellular immune response was evaluated in pigs after intradermal application of the antigenized rAb ZH9F7 combined with porcine circovirus type 2 cap antigen (rAb ZH9F7-Cap). We demonstrated that rAb ZH9F7 recognized conventional type 1 and 2 DCs from the blood and skin and monocytes. It promoted receptor-mediated endocytosis and migration of cDCs and moDCs toward regional lymph nodes. Intradermal application of rAb ZH9F7-Cap induced a higher frequency of IFN-γ-secreting CD4⁺CD8⁺ T lymphocytes and antibodies against Cap protein than that in the control group. In conclusion, the rAb ZH9F7-Cap system promoted the target of skin cDC1 and cDC2, provoking migration to the regional lymph nodes and inducing a Th1 response, as evidenced by the proliferation of double-positive CD4⁺CD8⁺ T cells, which correlates with an enhanced ability to target the cDC1 subset both in vitro and in vivo  

4.2461           Modern Metaproteomics: A Unique Tool to Characterize the Active Microbiome in Health and Diseases, and Pave the Road towards New Biomarkers—Example of Crohn’s Disease and Ulcerative Colitis Flare-Ups

Henry, C., Bassignani, A., Berland, M., Langlella, O., Sokol, H. and Juste, C. Cells, 11:1340 (2022)   Thanks to the latest developments in mass spectrometry, software and standards, metaproteomics is emerging as the vital complement of metagenomics, to make headway in understanding the actual functioning of living and active microbial communities. Modern metaproteomics offers new possibilities in the area of clinical diagnosis. This is illustrated here, for the still highly challenging diagnosis of intestinal bowel diseases (IBDs). Using bottom-up proteomics, we analyzed the gut metaproteomes of the same twenty faecal specimens processed either fresh or after a two-month freezing period. We focused on metaproteomes of microbial cell envelopes since it is an outstanding way of capturing host and host–microbe interaction signals. The protein profiles of pairs of fresh and frozen-thawed samples were closely related, making feasible deferred analysis in a distant diagnosis centre. The taxonomic and functional landscape of microbes in diverse IBD phenotypes—active ulcerative colitis, or active Crohn’s disease either with ileo-colonic or exclusive colonic localization—differed from each other and from the controls. Based on their specific peptides, we could identify proteins that were either strictly overrepresented or underrepresented in all samples of one clinical group compared to all samples of another group, paving the road for promising additional diagnostic tool for IBDs

5           Viruses

 

5.1           Isolation of a retrovirus from multiple sclerosis patients in self-generated iodixanol gradients.

Møller-Larsen, A. and Christensen, T.

1. Virol. Methods, 73(2), 151-161 (1998)

  The use of iodixanol, a relatively new iodinated gradient medium, is described for isolation of a retrovirus, which was harvested from the supernatant of lymphoid cell lines originating from patients with multiple sclerosis (MS). The virus is produced in low amounts and has been shown to be fragile, as manifested in a loss of surface glycoproteins when purified in other gradient media. The gradient fractions were analysed after centrifugation in iodixanol by incorporation of 3H-UTP, reverse transcriptase (RT) assays and electron microscopy (EM) and it was found that iodixanol does not cause the degree of damage to the particles observed previously. These most favourable conditions are probably due to low viscosity and almost iso-osmotic conditions even in high concentrations. Furthermore, these advantages go together with higher reproducibility in self-forming gradients, easier handling and shorter centrifugation time. Iodixanol can also be used for preparation of HTLV-1.  

5.2           Sustained secretion of human alpha-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors

Song, S. et al Proc. Natl. Acad. Sci. USA, 95, 14384-14388 (1998)   Recombinant adeno-associated virus (AAV) vectors have been used to transduce murine skeletal muscle as a platform for secretion of therapeutic proteins.  The utility of this approach for treating alpha-l-antitrypsin (AAT) deficiency was tested in murine myocytes in vitro and in vivo.  AAV vectors expressing the human AAT gene from either the cytomegalovirus (CMV) promoter (AAV-C-AT) or the human elongation factor 1-a promoter (AAV-E-AT) were examined. In vitro in C2Cl2 murine myoblasts, the expression levels in transient transfections were similar between the two vectors.  One month after transduction, however, the human elongation factor 1 promoter mediated 10-fold higher stable human AAT expression than the CMV promoter. In vivo transduction was performed by injecting doses of up to 1.4 x 10¹³ particles into skeletal muscles of several mouse strains (C57BL/6, BALB/c, and SCID). In vivo, the CMV vector mediated higher levels of expression, with sustained serum levels over 800 mg/ml in SCID and over 400 mg/ml in C57BL/6 mice.  These serum concentrations are 100,000-fold higher than those previously observed with AAV vectors in muscle and are at levels which would be therapeutic if achieved in humans.  High level expression was delayed for several weeks but was sustained for over 15 wk.  Immune responses were dependent upon the mouse strain and the vector dosage.  These data suggest that recombinant AAV vector transduction of skeletal muscle could provide a means for replacing AAT or other essential serum proteins but that immune responses may be elicited under certain conditions.  

5.3           Assembly of a tailed bacterial virus and its genome release studied in three dimensions

Tao, Y. et al Cell, 95, 431-437 (1998)   We present the first three-dimensional reconstruction of a prolate, tailed phage, and its empty prohead precursor by cryo-electron microscopy. The head-tail connector, the central component of the DNA packaging machine, is visualized for the first time in situ within the Bacillus subtilis dsDNA phage f29. The connector, with 12- or 13-fold symmetry, appears to fit loosely into a pentameric vertex of the head, a symmetry mismatch that may be required to rotate the connector to package DNA. The prolate head of f29 has 10 hexameric units in its cylindrical equatorial region, and 11 pentameric and 20 hexameric units comprise icosahedral end-caps with T-3 quasi-symmetry. Reconstruction of an emptied phage particle shows that the connector and neck/tail assembly undergo significant conformational changes upon ejection of DNA.    

5.4           Highly purified human immunodeficiency virus type 1 reveals a virtual absence of Vif virions

Dettenhofer, M. and Yu, X.F.

1. Virology, 73(2), 1460-1467 (1999)

  The Vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary blood-derived lymphocytes, macrophages, and certain human T-cell lines. It has been shown that Vif is associated with HIV-1 virions purified by sucrose density-equilibrium gradient analysis. However, the specificity of Vif incorporation into virions has not been determined. Moreover, recent studies have demonstrated that standard HIV-1 particle preparations created with sucrose density-equilibrium gradients are contaminated with cell-derived microvesicles. Here we demonstrate, as previously reported, that Vif cosediments with HIV-1 particles in sucrose density-equilibrium gradient analysis. However, we also found that, when Vif was expressed in the absence of all other HIV-1 encoded gene products and then isolated by sucrose density-equilibrium gradient centrifugation from extracellular supernatants, its sedimentation pattern was largely unaltered, suggesting that Vif can be secreted from cells. Using a newly developed OptiPrep velocity gradient method, we were able to physically separate most of the extracellular Vif from the HIV-1 virions without disrupting the infectivity of the virus. By titrating serial dilutions of purified Vif and Gag against the viral peak fraction in the OptiPrep gradient, we demonstrate that <1.0 Vif molecule per virion was present. This study shows that Vif is not significantly present in HIV-1 virions, a finding which is consistent with the idea that Vif functions predominantly in the virus-producing cells during virus assembly. The OptiPrep velocity gradient technique described here could be an easy and rapid way to purify HIV and other enveloped viruses from microvesicles and/or cell debris.  

5.5           Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield

Zolotukhin, S. et al Gene Therapy. 6, 973-985 (1999)   Conventional methods for rAAV purification that are based on cesium chloride ultracentrifugation have often produced vector preparations of variable quality and resulted in significant loss of particle infectivity. We report here several novel purification strategies that involve the use of non-ionic iodixanol gradients followed by ion exchange or heparin affinity chromatography by either conventional or HPLC columns. These methods result in more than 50% recovery of rAAV from a crude lysate and routinely produce vector that is more than 99% pure. More importantly, the new purification procedures consistently produce rAAV stocks with particle-to-infectivity ratios of less than 100, which is significantly better than conventional methods. The new protocol increases the overall yield of infectious rAAV by at least 10-fold and allows for the complete purification of rAAV in 1 working day. Several of these methods should also be useful for large-scale production.  

5.6           Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system

Hermens, W.T. et al. Hum. Gen. Ther., 10(11), 1885-1891 (1999)   Recombinant adeno-associated virus (rAAV) vectors have become attractive tools for in vivo gene transfer. The production and purification of high-titer rAAV vector stocks for experimental and therapeutic gene transfer continue to undergo improvement. Standard rAAV vector purification protocols include the purification of the vector by cesium chloride (CsCl)-density gradient centrifugation followed by extensive desalination via dialysis against a physiological buffer for in vivo use. These procedures are extremely time consuming and frequently result in a substantial loss of the infectious vector titer. As an alternative to CsCl we have investigated the use of Iodixanol, an X-ray contrast solution, as density gradient medium. Purification of rAAV vectors by Iodixanol shortened the centrifugation period to 3 hr and resulted in reproducible concentration and purification of rAAV-vector stocks. We show that injection of rAAV derived from an Iodixanol gradient can be used for in vivo gene transfer applications in the brain and spinal cord without detectable cytopathic effects and directing stable transgene expression for at least 2 months.  

5.7           Purification and protein composition of PM2, the first lipid-containing bacterial virus to be isolated

Kivel@, H.M., M@nnist`, R.H., Kalkkinen, N. and Bamford, D.H. Virology, 262(2), 364-374 (1999)   The marine, icosahedral bacteriophage PM2 was isolated in the late 1960s. It was the first phage for which lipids were firmly demonstrated to be part of the virion structure and it has been classified as the type organism of the Corticoviridae family. The host, Pseudoalteromonas espejiana BAL-31, belongs to a common group of marine bacteria. We developed a purification method producing virions with specific infectivity approximately as high as that of the lipid-containing phages PRD1 and f6. The sensitivity of the virus to normally used purification media such as those containing sucrose is demonstrated. We also present an alternative host, a pseudoalteromonad, that allows enhanced purification of the virus under reduced salt conditions. We show, using N-terminal amino acid sequencing and comparison with the genomic sequence, that there are at least eight structural proteins in the infectious virus.  

5.8           Influenza viruses select ordered lipid domains during budding from the plasma membrane

Scheiffele, P., Rietveld, A., Wilk, T. and Simons, K.

1. Biol. Chem., 274(4), 2038-2044 (1999)

  During the budding of enveloped viruses from the plasma membrane, the lipids are not randomly incorporated into the envelope, but virions seem to have a lipid composition different from the host membrane.  Here, we have analyzed lipid assemblies in three different viruses: fowl plague virus (FPV) from the influenza virus family, vesicular stomatitis virus (VSV), and Semliki Forest virus (SFV).  Analysis of detergent extractability of proteins, cholesterol, phosphoglycerolipids, and sphingomyelin in virions showed that FPV contains high amounts of detergent-insoluble complexes, whereas such complexes are largely absent from VSV or SFV.  Cholesterol depletion from the viral envelope by methyl-b-cyclodextrin results in increased solubility of sphingomyelin and of the glycoproteins in the FPV envelope.  This biochemical behavior suggests that so-called raft-lipid domains are selectively incorporated into the influenza virus envelope.  The "fluidity" of the FPV envelope, as measured by the fluorescence polarization of diphenylhexatriene, was significantly lower than compared with VSV or SFV.  Furthermore, influenza virus hemagglutinin incorporated into the envelope of recombinant VSV was largely detergent-soluble, indicating the depletion of raft-lipid assemblies from this membrane.  The results provide a model for lipid selectivity during virus budding and support the view of lipid rafts as cholesterol-dependent, ordered domains in biological membranes.  

5.9           Opposing effects of human immunodeficiency virus type 1 matrix mutations support a myristyl switch model of Gag membrane targeting

Paillart, J-C. and G`ttlinger, H.G.

1. Virol., 73(4), 2604-2612 (1999)

  Targeting of the human immunodeficiency virus type I (HIV-1) Gag precursor Pr55^gag to the plasma membrane, the site of virus assembly, is primarily mediated by the N-terminal matrix (MA) domain.  N-myristylation of MA is essential for the stable association of Pr55^gag with membranes and for virus assembly. We now show that single amino acid substitutions near the N terminus of MA can dramatically impair assembly without compromising myristylation.  Subcellular fractionation demonstrated that Gag membrane binding was compromised to a similar extent as in the absence of the myristyl acceptor site, indicating that the myristyl group was not available for membrane insertion.  Remarkably, the effects of the N-terminal modifications could be completely suppressed by second-site mutations in the globular core of MA.  The compensatory mutations enhanced Gag membrane binding and increased viral particle yields above wild-type levels, consistent with an increase in the exposure of the myristyl group.  Our results support a model in which the compact globular core of MA sequesters the myristyl group to prevent aberrant binding to intracellular membranes, while the N terminus is critical to allow the controlled exposure of the myristyl group for insertion into the plasma membrane.  

5.10           The Gag domains required for avian retroviral RNA encapsidation determined by using two independent assays

Lee, E-G., Yeo, A., Kraemer, B., Wickens, M. and Linial, M.L.

1. Virol, 73(8), 6282-6292 (1999)

  The Rous sarcoma virus (RSV) Gag precursor polyprotein is the only viral protein which is necessary for specific packaging of genomic RNA.  To map domains within Gag which are important for packaging, we constructed a series of Gag mutations in conjunction with a protease (PR) active-site point mutation in a full-length viral construct.  We found that deletion of either the matrix (MA), the capsid (CA), or the protease (PR) domain did not abrogate packaging, although the MA domain is likely to be required for proper assembly.  A previously characterized deletion of both Cys-His motifs in RSV nucleocapsid protein (NC) reduced both the efficiency of particle release and specific RNA packaging by 6- to 10-fold, consistent with previous observations that the NC Cys-His motifs played a role in assembly and RNA packaging.  Most strikingly, when amino acid changes at Arg 549 and 551 immediately downstream of the distal NC Cys-His box were made, RNA packaging was reduced by more than 25-fold with no defect in particle release, demonstrating the importance of this basic amino acid region in packaging.  We also used the yeast three-hybrid system to study avian retroviral RNA-Gag interactions.  Using this assay, we found that the interactions of the minimal packaging region My with Gag are of high affinity and specificity.  Using a number of My and Gag mutants, we have found a clear correlation between a reporter gene activation in a yeast three-hybrid binding system and an in vivo packaging assay.  Our results showed that the binding assay provides a rapid genetic assay of both RNA and protein components for specific encapsulation.  

5.11           Proteolytic activity, the carboxy terminus of Gag, and the primer binding site are not required for Pol incorporation into foamy virus particles

Baldwin, D.N. and Linial, M.L.

1. Virol., 73(8), 6387-6393 (1999)

  Human foamy virus (HFV) is the prototype member of the spumaviruses.  While similar in genomic organization to other complex retroviruses, foamy viruses share several features with their more distant relatives, the hepadnaviruses such as human hepatitis B virus (HBV).  Both HFV and HBV express their Pol proteins independently from the structural proteins.  However unlike HBV, Pol is not required for assembly of HFV core particles or for packaging of viral RNA.  These results suggest that the assembly of Pol into HFV particles must occur by a mechanism different from those used by retroviruses and hepadnaviruses.  We have examined possible mechanisms for HFV Pol incorporation, including the role of proteolysis in assembly of Pol and the role of initiation of reverse transcription.  We have found that proteolytic activity is not required for Pol incorporation. p4 Gag and the residues immediately upstream of the cleavage site in Gag are also not important.  Deletion of the primer binding site had no effect on assembly, ruling out early steps of reverse transcription in the process of Pol incorporation.    

5.12           Incorporation of wild-type and C-terminally truncated human epidermal growth factor receptor into human immunodeficiency virus-like particles: insight into the processes governing glycoproteins incorporation into retroviral particles

Henriksson, P., Pfeiffer, T., Zentgraf, H., Alke, A. And Bosch, V.

1. Virol, 73(11), 9294-9302 (1999)

  Previous results have indicated that incorporation of surface glycoprotein into retroviral particles is not a specific process and that many heterologous viral and cellular glycoproteins can be incorporated as long as they do not have long cytoplasmic C-terminal regions which were presumed to be sterically inhibitory.  In this study, this concept has been directly examined by analyzing the incorporation of the wild-type human epidermal growth factor receptor (Wt-EGFR) and of a C-terminally truncated mutant of Wt-EGFR (Tr-EGFR) into human immunodeficiency virus (HIV)-like particles.  Incorporation was directly analyzed at the protein level and by immunogold labelling of enriched HIV-like particles.  In agreement with the above concept, Tr-EGFR, with only 7 C-terminal amino acids (aa), was efficiently incorporated into HIV-like particles.  Incorporation of the Wt-EGFR species, with 542 C-terminal cytoplasmic aa, was reduced by a factor of about 5 in comparison to that of the Tr-EGFR species.  However, the Wt-EGFR species was still very significantly present in the HIV-like particles.  A series of control experiments verified that this represents genuine incorporation of Wt-EGFR into the membrane of HIV-like particles.  These observations allow further speculation as to the processes governing glycoprotein incorporation into retroviral particles and indicate that the internal virus structure of HIV (in particular the matrix layer [MA]) can accommodate much larger heterologous cytoplasmic domains in incorporated glycoproteins than previously assumed.  

5.13           Viral receptors and vector purification: New approaches for generating clinical-grade reagents

Summerford, C. and Samulski, R.J. Nature Medicine 5, 587-588 (1999)   Extract of text Two independent laboratories have developed protocols in which heparin, an analog of the natural receptor for AAV, is used as the affinity matrix for AAV vector purification. In each protocol, a commercially available heparin affinity col­umn is used (POROS HE/M heparin, Boehringer Mannheim, Germany). Given that heparan sulfate proteoglycan is known to serve as viral receptor for AAV, the success of heparin columns in rAAV purification and their superiority over cellulose sulfate or ion-exchange columns is not surpris­ing. A potential problem with the use of this affinity approach is that many cellular proteins are also known to associate physically with heparin. Thus, use of a heparin column requires the incorporation of a specific strategy to remove contaminating heparin-binding proteins. Each laboratory has taken a different successful approach to address this problem. In one study, Zolotukhin et al. semi-purified virus from a ‘freeze/thaw’ cell lysate by centrifu­gation in a density step-gradient of non-ionic media (iodix­anol). The semi-pure preparation was then applied to an HPLC heparin column (POROS HE/M heparin) for further purification. The procedure takes less than 1 day and is supe­rior to traditional CsCl centrifugation in that it results in a higher yield of virus (greater than tenfold) that is more infectious (lower total particle-to-infectious particle ratio). The particle-to-infectivity ratios are consistently less than 100:1, which is considerably better than the 1,000:1 ratio often resulting from traditional purification strategies. The reduced particle-to-infectious particle ratio is presumably due to the use of more benign conditions, as well as the use of a mole­cule for purification that is essentially analogous to the nat­ural receptor of the virus. The iodixanol/heparin procedure for rAAV purification is fast (completion in I working day), convenient (uses a commercially available column), repro­ducible and results in 50-70% recovery of virus (yield) that is greater than 99% pure.  

5.14           Comparison of antibody titers determined by hemagglutination inhibition and enzyme immunoassay for JC virus and BK virus

Hamilton, R.S., Gravell, M. and Major, E.O.

1. Clin. Microbiol., 38(1), 105-109 (1999)

  A comparison of antibody titers to JC virus (JCV) or BK virus (BKV) was made by hemagglutination inhibition (HI) and enzyme immunoassay (EIA) with 114 human plasma samples.  Antibody titers to JCV or BKV determined by HI were lower than those determined by EIA.  Nevertheless, as HI titers increased so did EIA titers.  When antibody data were compared by the Spearman rank correlation test, highly significant correlations were found between HI and EIA titers.  Results obtained by plotting EIA antibody titers for JCV against those for BKV generally showed a reciprocal relationship, i.e., samples with high antibody titers to JCV had lower antibody titers to BKV and vice versa.  Some samples, however, had antibody titers to both viruses.  Of the samples tested, 25.4% (25 of 114) had HI and EIA antibody titers to JCV and BKV which were identical or closely related.  This is not the scenario one would expect for cross-reactive epitopes shared by the two viruses, but one suggesting that these samples were from individuals who had experienced infections by both viruses. Adsorption with concentrated JCV or BKV antigen of sera with high antibody titers to both JCV and BKV and testing by JCV and BKV EIA gave results which support this conclusion.  Although 52.6% (51 of 97) of the samples from the Japanese population tested had very high antibody titers (³ 40,960) to either JCV or BKV, none of the samples were found by a dot blot immunoassay to have antibodies which cross-reacted with simian virus 40.  The results from this study, in agreement with those of others, suggest that humans infected by JCV or BKV produce antibodies to species-specific epitopes on their VP1 capsid protein, which is associated with hemagglutination and cellular binding.  

5.15           Entry of the two infectious forms of vaccinia virus at the plasma membrane is signaling-dependent for the IMV but not the EEV

Krijnse Locker, J. et al Mol. Biol. Cell, 11, 2497-2511 (2000)   The simpler of the two infectious forms of vaccinia virus, the intracellular mature virus (IMV) is known to infect cells less efficiently than the extracellular enveloped virus (EEV), which is surrounded by an additional, TGN-derived membrane.  We show here that when the IMV binds HeLa cells, it activates a signaling cascade that is regulated by the GTPase rac1 and rhoA, ezrin, and both tyrosine and protein kinase C phosphorylation.  These cascades are linked to the formation of actin and ezrin containing protrusions at the plasma membrane that seem to be essential for the entry of IMV cores.  The identical cores of the EEV also appear to enter at the cell surface, but surprisingly, without the need for signaling and actin/membrane rearrangements.  Thus, in addition to its known role in wrapping the IMV and the formation of intracellular actin comets, the membrane of the EEV seems to have evolved the capacity to enter cells silently, without a need for signaling.  

5.16           The double-stranded RNA-binding protein Staufen is incorporated in human Immunodeficiency virus type 1: evidence for a role in genomic encapsidation

Mouland, A.J., Mercier, J., Luo, M., Bernier, L., DesGroseillers, L and Cohen E.

1. Virol., 74(12), 5441-5451 (2000)

  Human Staufen (hStau), a double-stranded RNA (dsRNA)-binding protein that is involved in mRNA transport, is incorporated in human immunodeficiency virus type 1 (HIV-1) and in other retroviruses, including HIV-2 and Moloney murine leukemia virus. Sucrose and OptiPrep gradient analyses reveal co-sedimentation of hStau with purified HIV-1, while subtilisin assays demonstrate that it is internalized. hStau incorporation in HIV-1 is selective, is dependent on an intact functional dsRNA-binding domain, and quantitatively correlates with levels of encapsidated HIV-1 genomic RNA. By co-immunoprecipitation and reverse transcription-PCR analyses, we demonstrate that hStau is associated with HIV-1 genomic RNA in HIV-1 expressing cells and purified virus. Over-expression of hStau enhances virion incorporation levels, and a corresponding, threefold increase in HIV-1 genomic RNA encapsidation levels. This coordinated increase in hStau and genomic RNA packaging had a significant negative effect on viral infectivity. This study is the first to describe hStau within HIV-1 particles and provides evidence that hStau binds HIV-1 genomic RNA, indicating that it may be implicated in retroviral genome selection and packaging into assembling virions.  

5.17           Minimal exclusion of plasma membrane proteins during retrovirus envelope formation

Hammarstedt, M, Wallengren, K., Pedersen, K.W., Roos, N. And Garoff, H. Proc. Natl. Acad. Sci. USA, 97(13), 7527-7532 (2000)   The retrovirus forms its envelope by budding at the plasma membrane (PM). This process is primarily driven by its cytoplasmic core-precursor protein, Gag, as shown by the efficient formation of virus-like Gag particles in the absence of its envelope protein, Env. More interestingly, several studies have demonstrated incorporation of various PM proteins into retrovirus, but the underlying mechanism of this phenomenon has remained elusive. We have purified Moloney murine leukemia virus Gag particles by sedimentation in an iodixanol gradient and donor PMs by flotation in a sucrose gradient and compared their protein compositions at equal lipid basis. We found that most PM proteins are presented at similar density in both membranes. The inclusion of PM proteins was unaffected by incorporation of Env protein into the envelope of the Gag particles and whether these were produced at high or low level in the cells. These findings indicate that most PM proteins become incorporated into retrovirus envelope without significant sorting. This feature of retrovirus assembly should be considered when studying retrovirus functions and developing retrovirus vectors.  

5.18           Kinetic analysis of human immunodeficiency virus type 1 assembly reveals the presence of sequential intermediates

Tritel, M. and Resh, M.D.

1. Virol., 74(13), 5845-5855 (2000)

  The assembly and budding of lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), are mediated by the Gag protein precursor, but the molecular details of these processes remain poorly defined. In this study, we combined pulse-chase techniques with density gradient centrifugation to identify, isolate, and characterize sequential kinetic intermediates in the lentivirus assembly process. We show that newly synthesized HIV-1 Gag rapidly form cytoplasmic protein complexes that are resistant to detergent treatment, sensitive to protease digestion, and degraded intracellularly. A subpopulation of newly synthesized Gag binds membranes within 5 to 10 min and over several hours assembles into membrane-bound complexes of increasing size and/or density that can be resolved on OptiPrep density gradients. These complexes likely represent assembly intermediates because they are not observed with assembly-defective Gag mutants and can be chased into extracellular virus-like particles. At steady state, nearly all of the Gag is present as membrane-bound complexes in various stages of assembly. The identification of sequential assembly intermediates provides the first demonstration that HIV-1 particle assembly proceeds via a ordered process. Assembly intermediates should serve as attractive targets for the design of antiviral agents that interfere with the process of particle production.  

5.19           Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and  construction of AAV2 vectors with altered tropism

Wu, P. et al

1. Virol., 74(18), 8635-8647 (2000)

  Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy.  Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors.  To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis.  Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants.  Analysis of these mutants revealed eight separate phenotypes.  Infectious titers of the mutants revealed four classes.  Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious.  Further analysis revealed some of the defects in the class 2, 3, and 4 mutants.  Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be b-barrel structures in the capsid protein VP3.  Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability.  Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles.  One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA.  Additionally, live mutants were defective in heparan binding, a step that is believed to be essential for viral entry.  These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3.  The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591.  In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid.  These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664.  Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3.  The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or co-receptor binding.  Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.  

5.20           Human immunodeficiency virus type 1 Vpr protein is incorporated into the virion in significantly smaller amounts than Gag and is phosphorylated in infected cells

Mhller, B., Tessmer, U., Schubert, U. and Kr@usslich, H.G.

1. Virol., 74(20), 9727-9731 (2000)

  Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) is a small accessory protein involved in the nuclear import of viral DNA and the growth arrest of host cells. Several studies have demonstrated that a significant amount of Vpr is incorporated into the virus particle via interaction with the p6 domain of Gag, and it is generally assumed that Vpr is packaged in equimolar ratio to Gag. We have quantitated the relative amount of Vpr in purified virions following [³⁵S] cysteine labeling of infected MT-4 cells, as well as by quantitative immunoblotting and found that Vpr is present in a molar ration of approx. 1:7 compared to capsid. Analysis of isolated core particles showed that Vpr is associated with the mature viral core, despite quantitative loss of p6 from core preparations. Metabolic labeling of infected cells with ortho[³²P]phosphate revealed that a small fraction of Vpr is phosphorylated in virions and infected cells.  

5.21           Genetically modified CD34⁺ cells as cellular vehicles for gene delivery into areas of angiogenesis in a rhesus model

Gomez-Navarro, J. et al Gene Therapy, 7, 43-52 (2000).   To develop a cellular vehicle able to reach systemically dis­seminated areas of angiogenesis, we sought to exploit the natural tropism of circulating endothelial progenitor cells (EPCs). Primate CD34⁺ EPCs were genetically modified with high efficiency and minimal toxicity using a non-replicative herpes virus vector. These EPCs localized in a skin autograft model of angiogenesis in rhesus monkeys, and sustained the expression of a reporter gene for several weeks while circulating in the blood. In animals infused with autologous CD34⁺ EPCs transduced with a thymidine kinase-encoding herpes virus, skin autografts and subcutaneous Matrigel pellets impregnated with vascular growth factors underwent necrosis or accelerated regression after administration of ganciclovir. Importantly, the whole intervention was perfectly well tolerated. The accessibility, easy manipulation, lack of immunogenicity of the autologous CD34⁺ cell vehicles, and tropism for areas of angiogenesis render autologous CD34⁺ circulating endothelial progenitors as ideal candidates for exploration of their use as cellular vehicles when systemic gene delivery to those areas is required.  

5.22           Use of the NADH-quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae as a possible cure for complex I defects in human cells

Seo, B.B., Wang, J.M., Flotte, T.R., Yagi, T. and Matsuno-Yagi, A.

1. Biol. Chem.,275(48), 37774-37778 (2000)

  The NDi1 enzyme of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane.  We have shown previously that the NDI1 gene can be functionally expressed in Chinese hamster cells [Seo et al. (1998) Proc. Natl. Acad. Sci. USA, 95, 9167-9171] and human embryonal kidney 293 (HEK 293) cells [Seo et al. (1999) Biochim. Biophys. Acta, 1412, 56-65] and that the NDI1 protein is capable of compensating respiratory deficiencies caused by defects in the host NADH-quinone oxidoreductase (complex I).  To extend the potential of use of this enzyme to repair complex I deficiencies in vivo, we constructed a recombinant adeno-associated virus vector carrying the NDI1 gene (rAAV-NDI1).  With rAAV-NDI1 as the gene delivery method, we were able to achieve high transduction efficiencies (nearly 100%) even in 143B cells that are difficult to transfect by lipofection or calcium phosphate precipitation methods.  The NDI1 gene was successfully introduced into non-proliferating human cells using rAAV-NDI1. The expressed Ndi1 protein was shown to be functionally active just as seen for proliferating cells.  Furthermore, when cells were cultured under the conditions where energy has to be provided by respiration, the NDI1 -transduced cells were able to grow even in the presence of added complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion. In contrast, control cells that did not receive the NDI1 gene failed to survive as anticipated.  The NDI1 protein has a great potential as a molecular remedy for complex I defects and it is highly likely that the same strategy can be extended to correction of other mitochondrial disorders.  

5.23           Efficient gene transfer into human cord blood CD34⁺ cells and the CD34⁺ CD38^- subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV

Nathwani, A.C. et al Gene Therapy, 7, 183-195 (2000)   Recombinant adeno-associated viral (rAAV) vectors have been evaluated for their ability to transduce primitive hematopoietic cells. Early studies documented rAAV-mediated gene expression during progenitor derived colony formation in vitro, but studies examining genome integration and long-term gene expression in hematopoietic cells have yielded conflicting results. Such studies were performed with crude vector preparations. Using improved methodology, we have generated high titer, biologically active preparations of rAAV free of wild-type AAV (less than 1/10⁷ particles) and adenovirus. Transduction of CD34⁺ cells from umbilical cord blood was evaluated with a bicistronic rAAV vector encoding the green fluorescent protein (GFP) and a trimetrexate resistant variant of dihydrofolate reductase (DHFR). Freshly isolated quiescent CD34⁺ cells were resistant to transduction (less than 4%), but transduction increased to 23 ± 2% after 2 days of cytokine stimulation and was further augmented by addition of tumor necrosis factor a (51 ± 4%) at a multiplicity of infection of 10⁶ . rAAV-mediated gene expression was transient in that progenitor derived colony formation was inhibited by trimetrexate. Primitive CD34⁺ and CD34⁺ , CD38^-  subsets were sequentially transduced with a rAAV vector encoding the murine ecotropic receptor followed by transduction with an ecotropic retroviral vector encoding GFP and DHFR. Under optimal conditions 41 ± 7% of CD34⁺  progenitors and 21 ± 6% of CD34⁺ , CD38^-  progenitors  became trimetrexate resistant. These results document that highly purified rAAV transduce primitive human hematopoietic cells efficiently but gene expression appears to be transient.  

5.24           Identification of a novel consensus sequence at the cleavage site of the Lassa virus  glycoprotein

Lenz, O., Ter Meulen, J., Feldmann, H., Klenk, H-D. and Garten, W.

1. Virol., 74(23), 11418-11421 (2000)

  The Lassa virus glycoprotein consists of an amino-terminal and a carboxy-terminal cleavage fragment designated GP-1 and GP-2, respectively, that are derived by proteolysis from the precursor GP-C. The membrane-anchored GP-2 obtained from purified virions of the Josiah strain revealed the N-terminal tripeptide GTF₂₆₂ when analyzed by Edman degradation. Upstream of this site, GP-C contains the tetrapeptide sequence RRLI₂₅₉ which is conserved in all Lassa virus isolates published to date. Systematic mutational analysis of vector-expressed GP-C revealed that the motif R-X (L/I/V)-L₂₅₉ (where X stands for L, I, or V) is essential for cleavage of the peptide bond between leucine₂₅₉ and glycine₂₆₀. This cleavage motif is homologous to the consensus sequence recognized by a novel class of cellular endoproteases which have so far not been implicated in the processing of viral glycoproteins.  

5.25           Protection of Macaca nemestrina from disease following pathogenic simian immunodeficiency virus (SIV) challenge: utilization of SIV nucleocapsid DNA vaccines with and without an SIV protein boost

Gorelick, R.J. et al

1. Virol., 74(24), 11935-11949 (2000)

  Molecular clones were constructed that express nucleocapsid (NC) deletion mutant simian immunodeficiency viruses (SIVs) that are replication defective but capable of completing virtually all of the steps of a single viral infection cycle. These steps include production of particles that are viral RNA deficient yet contain a full complement of processed viral proteins. The mutant particles are ultrastructurally indistinguishable from wild-type virus. Similar to a live attenuated vaccine, this approach should allow immunological presentation of a full range of viral epitopes, without the safety risks of replication virus. A total of 11 Macaca nemestrina macaques were inoculated with NC mutant SIV expressing DNA, intramuscularly (i.m.) in one study and i.m. and subcutaneously in another study. Six control animals received vector DNA lacking SIV sequences. Only modest and inconsistent humoral responses and no cellular immune responses were observed prior to challenge. Following intravenous challenge with 20 animal infectious doses of the pathogenic SIV(Mne) in a long-term study, all control animals became infected and three of four animals developed progressive SIV disease leading to death. All 11 NC mutant SIV DNA immunized animals became infected following challenge but typically showed decreased initial peak plasma SIV RNA levels compared to those of control animals (P = 0.0007). In the long-term study, most of the immunized animals had low or undetectable postacute levels of plasma SIV RNA, and no CD4⁺ T-cell depletion or clinical evidence of progressive disease, over more than 2 years of observation. Although a subset of immunized and control animals were boosted with SIV(Mne) proteins, no apparent protective benefit was observed. Immunization of macaques with DNA that codes for replication-defective but structurally complete virions appears to protect from or at least delay the onset of AIDS after infection with pathogenic immunodeficiency virus. With further optimization, this may be a promising approach for vaccine development.  

5.26           Genetically modified CD34⁺ cells exert a cytotoxic bystander effect on human endothelial and cancer cells

Arafat, W.O. et al Clin. Can. Res., 6, 4442-4448 (2000)   We and others have proposed mammalian cells as gene delivery vehicles with the potential for overcoming physiological barriers to viral vectors. To that end, we previously have shown the potential of CD34⁺ endothelial progenitors for systemic gene delivery in a primate angiogenesis model. Here we seek to explore the utility of CD34⁺ cells of human origin as vehicles for toxin genes and, in particular, to measure their capacity to effect a cytotoxic bystander effect in human endothelium and tumor cells. To this end, CD34⁺ cells were transduced with TOZ.1, a nonreplicative herpes simplex vector encoding thymidine kinase. To test the capacity of CD34⁺ cells to induce a cytotoxic bystander effect in target cells, we performed mixing experiments, whereby TOZ.1-transduced CD34⁺ cells were mixed with either human vascular endothelial cells or human ovarian tumor cells (SKOV3.ip1). Cell viability was measured by the MTS assay. Lastly, mixtures of TOZ.1-transduced CD34⁺ and SKOV3.ip1 tumor cells were injected s.c. to evaluate the bystander effect in vivo. After transduction of CD34⁺ cells with TOZ.1, treatment with ganciclovir induced the killing of 99% of cells. In cell-mixing experiments, a linear correlation was observed between the percentages of TOZ.1-transduced CD34⁺ cells and total cell killing. For example, when 50% of CD34⁺ transduced cells were mixed with nontransduced SKOV3.ip1, >70% of the cells died. Similarly, when the same percentage was mixed with human vascular endothelial cells, >80 of the total number of cells died. In vivo studies showed an abrogation of tumor formation when TOZ.1-transduced CD34⁺ cells and ganciclovir were administered. Our observations establish the feasibility of a method for cell-based toxin gene delivery into disseminated areas of tumor angiogenesis.  

5.27           Adeno-associated virus vectors: activity and applications in the CNS

Peel, A.L. and Klein, R.L.

1. Neurosci. Meth., 98, 95-104 (2000)

  Transgenic strategies are useful for functional studies and they may also lead to the novel therapies. Controlling transgene expression in defined cell populations over time is increasingly important for both functional and gene therapy experiments. The Adeno-associated virus (AAV) vector may provide sufficient spatio-temporal control of gene expression for these purposes. This paper reviews in vivo somatic gene transfer methodology using AAV. Advantageous features of this system include neuronal gene expression that is: (1) efficient; (2) long-lived; and (3) non-toxic. Thus, AAV-mediated gene transfer is a good method for functional genomic research. From characterizing vector activity in the brain using different combinations of promoters and transgenes in the mid to late 1990’s, researchers continue to discover novel uses of AAV for both basic and clinical neuroscience.  

5.28           Long-term differential modulation of genes encoding orexigenic and anorexigenic peptides by leptin delivered by rAAV vector in ob/ob mice

Dhillon, H. et al Regulatory Peptides, 92, 97-105 (2000)   We investigated the long-term effects of physiological levels of leptin produced by gene therapy on body weight (BW) and expression of genes that encode orexigenic peptides in the hypothalamus. Recombinant adeno-associated viral vector (rAAV), a non-pathogenic and non-immunogenic vector, encoding leptin (bOb) was generated and administered iv to ob/ob mice lacking endogenous leptin. Whereas the lowest dose of rAAV- bOb (6 x 10⁹ particles) was ineffective, the middle dose (6 x 10¹⁰ particles) curbed BW gain without affecting food consumption for 75 days of observation. A ten-fold higher dose (6 x 10¹¹ particles) resulted in increased blood leptin levels and suppressed both BW gain and food consumption throughout the duration of the experiment. rAAV-bOb doses that either curbed BW without affecting food consumption or evoked BW loss and reduced food intake, decreased the expression of genes encoding the orexigenic peptides, neuropeptide Y and agouti-related peptide in the ARC, and the two doses were equally effective. Concomitantly, the expression of genes encoding the anorexigenic peptide, a-melanocyte stimulating hormone and cocaine-and-amphetamine regulatory transcript, was augmented with the latter gene displaying a dose0dependent response. These results document the efficacy of delivering biologically active leptin for extended periods by an iv injection of rAAV-bOb and show that physiological leptin concentration simultaneously exert a tonic inhibitory effect on orexigenic and a stimulatory effect on anorexigenic signaling in the hypothalamus. This intricate dynamic interplay induced by leptin regulates BW with or without an effect on food intake in leptin-deficient ob/ob mice. Further, these results suggest that gene therapy is an effective mode of delivery to the hypothalamus of those therapeutic proteins that cross the blood-brain barrier to ameliorate neuroendocrine disorders.  

5.29           AAV vectors: is clinical success on the horizon?

Monahan, P.E. and Samulski, R.J. Gene Therapy, 7, 24-30 (2000)   Potential applications and impact of the adeno-associated virus (AAV) as a gene transfer vector have expanded rapidly in the last decade. Recent advances in the production of high-titer purified rAAV vector stocks have made the transition to human clinical trials a reality in the last moments of the millenium. Production improvements will be complemented in the coming years with understanding of and innovations in the targeting and packaging of rAAV, the design of transgene cassettes, and the host immune response to the vectors. These expected areas of progress are discussed, with special attention to clinical applications for which rAAV vectors may help close the gap towards successful gene therapy.  

5.30           Inhibition of recombinant adeno-associated virus (rAAV) transduction by bronchial secretions from cystic fibrosis patients

Virella-Lowell, I., Poirier, A., Chesnut, K.A., Brantly, M. and Flotte, T.R. Gene Therapy, 7, 1783-1789 (2000)   The conducting airways are the primary target for gene transfer in cystic fibrosis (CF), yet the inflammation associated with CF lung disease could potentially pose a significant barrier to gene transfer vectors, such as recombinant adeno-associated virus (rAAV). In order to investigate this possibility, aliquots of bronchoalveolar lavage (BAL) fluid from eight individuals with CF were tested for their in vitro inhibitory effects on rAAV transduction, along with BAL from non-CF individuals. While the non-CF BAL fluid was not inhibitory, seven of eight CF BAL samples had significant inhibitory activity, resulting in a five- to 20-fold reduction in transduction events. Inhibition of rAAV transduction by CF BAL could be reversed by alpha-1-antitrypsin (AAT), but not by DNase. When neutrophil elastase and neutrophil alpha defensins (human neutrophil peptides, HNP) were measured in these samples, they were elevated by 500- and 10000-fold, respectively. The levels of HNP correlated inversely with the amount of rAAV transduction. Furthermore, rAAV transduction could be blocked by purified HNP in an AAT-reversible manner at HNP concentrations within the range measured in these fluids. We conclude that products of inflammation in CF BAL fluid are inhibitory to rAAV transduction, and that these effects may be reversible by AAT.  

5.31           The adeno-associated virus vector for orthopaedic gene therapy

Schwarz, E. M. Clin. Orthopaed. Related. Res., #379S, S31-S39 (2000).   During the last decade researchers working with recombinant adeno-associated virus have shown the use of this vector for efficient and long-term gene transfer in various tissues Including lung, muscle, brain, spinal cord, retina, and liver. In 1999 the first results documenting the use of this vector in transducing joint cells were published. Additional advantages of recombinant adeno-associated virus for in vivo gene therapy are: (1) its ability to transduce nondividing cells; (2) site-specific integration into the host genome; (3) high viral titer (>10¹³/mL); and (4) the vector is not cytotoxic and does not provoke a significant immune response. Most important, several groups have documented the ability to deliver sustained trans gene expression in an immunocompetent host for more than 1 year, and that curative levels of gene product (factor IX), from one injection is sustained long-term in a large animal (hemophilia B dog). Comparable results have not been achieved with any other vector to date. As a re suit of this work the first Phase I clinical trials using recombinant adeno-associated virus are under way for cystic fibrosis. The history of the recombinant adeno-associated virus vector and its future promise for orthopaedic gene therapies are described. The goal of the current review is to provide the reader with an understanding of the advantages and disadvantages of this vector for treatment of musculoskeletal diseases. Additional information concerning re- combinant adeno-associated virus can be obtained in more general reviews.  

5.32           Molecular characterization of HERV-H variants associated with multiple sclerosis

Christensen, T. et al Acta Neurol. Scand., 101, 229-238 (2000)   Our objective was to characterize retroviral sequences by RT-PCR with gag and env primers on RNA from RT-positive retroviral particles produced by multiple sclerosis (MS) derived B-lymphoblastoid cell lines. Sequence variants with high homology to the potentially functional subgroup RGH of the human endogenous retrovirus RTVL-H/HERV-H family were found. The same sequences were also specifically found in the particulate fraction of a series of MS patient plasma samples and were absent in controls. South-Western blots demonstrate the presence of a nucleic acid binding protein, corresponding in size and function to the nucleocapsid protein, Gag NC, of the retrovirus. We also present indications for transmission of the retrovirus to PHA-stimulated lymphocytes from healthy individuals.  

5.33           Effect of DNA-dependent protein kinase on the molecular fate of the rAAV2 genome in skeletal muscle

Song, S., Laipis, P.J., Berns, K.I. and Flotte, T.R. Proc. Natl. Acad. Sci., 98, 4084-4088 (2001)   We report that the DNA-dependent protein kinase (DNA-PK) affects the molecular fate of the recombinant adeno-associated virus (rAAV) genome in skeletal muscle. RAAV-human a1-antitrypsin (rAAV-hAAT) vectors were delivered by intramuscular injection to either C57BL/6 (DNA-PKcs⁺) or C57BL/6-SCID [severe combined immunodeficient (SCID), DNA-PKcs^-] mice. In both strains, high levels of transgene expression were sustained for up to 1 year after a single injection. Southern blot analysis showed that rAAV genomes persisted as linear episomes for more than 1 year in SCID mice, whereas only circular episomal forms were observed in the C57BL/6 strain. These results indicate that DNA-PK is involved in the formation of circular rAAV episomes.  

5.34           Incorporation of lysyl-tRNA synthetase into human immunodeficiency virus type 1

Chen, S. et al

1. Virol., 75(11), 5043-5048 (2001)

  During human immunodeficiency virus type 1 (HIV-1) assembly, tRNA^Lys isoacceptors are selectively incorporated into virions and tRNA₃^Lys is used as the primer for reverse transcription. We show herein that the tRNA^Lys-binding protein, lysyl-tRNA synthetase (LysRS), is also selectively packaged into HIV-1. The viral precursor protein Pr55^gag alone will package LysRS into Pr55^gagparticles, independently of tRNA^Lys. With the additional presence of the viral precursor protein Pr160^gag-pol, tRNA^Lys and LysRS are both packaged into the particle. While the predominant cytoplasmic LysRS has an apparent M_r of 70,000, viral LysRS associated with tRNA^Lys packaging is shorter, with an apparent M_r of 63,000. The truncation occurs independently of viral protease and might be required to facilitate interactions involved in the selective packaging and genomic placement of primer tRNA₃^Lys.  

5.35           The late stage of human immunodeficiency virus type 1 assembly is an energy-dependent process

Tritel, M. and Resh, M.D.

1. Virol., 75(12), 5473-5481 (2001)

  Several recent studies have indicated the involvement of host cell factors in human immunodeficiency virus type 1 (HIV-1) assembly. To ascertain whether ATP-dependent factors play a role in this process, we quantified virus-like particle (VLP) production by ATP-depleted cells. Pharmacological ATP depletion abrogated VLP production without affecting cell viability or inducing degradation of HIV-1 Gag protein. This effect occurred even when the ATP-depleting agents were added 1 h into the assembly process, and it was reversed by removal of these agents. ATP depletion did not affect Gag membrane binding or multimerization. Density gradient analysis indicated that HIV-1 assembly intermediates were stalled late in the assembly process. This conclusion was further supported by electron microscopy analysis, which revealed a preponderance of plasma membrane-associated stalk-like structures in the ATP-depleted cells. Since no HIV-1 proteins bind or hydrolyze ATP, these findings indicate that an ATP-requiring cellular factor is an obligatory participant in the HIV-1 assembly process.  

5.36           Vif is largely absent from human immunodeficiency virus type 1 mature virions and associates with viral particles containing unprocessed Gag

Sova, P., Volsky, D.J., Wang, L. and Chao, W.

1. Virol., 75(12), 5504-5517 (2001)

  Vif is a human immunodeficiency virus type 1 (HIV-1) protein that is essential for the production of infectious virus. Most of Vif synthesized during HIV infection localizes within cells, and the extent of Vif packaging into virions and its function there remain controversial. Here we show that a small but detectable amount of Vif remains associated with purified virions even after their treatment with the protease subtilisin. However, treatment of these virions with 1% Triton X-100 revealed that most of the virion-associated Vif segregated with detergent-resistant virus particles consisting of unprocessed Gag, indicating that detergent soluble, mature virions contain very little Vif. To investigate the control of Vif packaging in immature virus particles, we tested its association with Gag-containing virus-like particles (VLPs) in a Vif and Gag co-expression system in human cells. Only a small proportion of Vif molecules synthesized in this system became packaged into VLPs, and the VLP-associated Vif was protected from exogenous protease and detergent treatment, indicating that it is stably incorporated into immature virion-like cores. About 10-fold more Vpr than Vif was packaged into VLPs but most of the VLP-associated Vpr was removed by treatment with detergent. Mutagenesis of the C-terminal sequences in Gag previously shown to be responsible for interaction with Vif did not reduce the extent of Vif packaging into Gag VLPs. Surprisingly, short deletions in the capsid domain (CA) of Gag (amino acid residues 284 to 304 and 350 to 362) increased Vif packaging over 10-fold. The 350 to 363 deletion introduced into CA in HIV provirus also increased Vif incorporation into purified virions. Our results show that Vif can be packaged at low levels into aberrant virus particles or immature virions and that Vif is not present significantly in mature virions. Overall these results indicate that the Vif content in virions is tightly regulated and also argue against a function of virion-associated Vif.  

5.37           A particle-associated glycoprotein signal peptide essential for virus maturation and infectivity

Lindemann, D. et al

1. Virol., 75(13), 5762-5771 (2001)

  Signal peptides (SP) are key determinants for targeting glycoproteins to the secretory pathway. Here we describe the involvement in particle maturation as an additional function of a viral glycoprotein SP. The SP of foamy virus (FV) envelope glycoprotein is predicted to be unusually long. Using an SP-specific antiserum, we demonstrate that its proteolytic removal occurs post-translationally by a cellular protease and that the major N-terminal cleavage product, gp18, is found in purified viral particles. Analysis of mutants in proposed signal peptidase cleavage positions and N-glycosylation sites revealed an SP about 148 amino acids (aa) in length. FV particle release from infected cells requires the presence of cognate envelope protein and cleavage of its SP sequence. An N-terminal 15-aa SP domain with two conserved tryptophan residues was found to be essential for the egress of FV particles. While the SP N terminus was found to mediate the specificity of FV Env to interact with FV capsids, it was dispensable for Env targeting to the secretory pathway and FV envelope-mediated infectivity of murine leukemia virus pseudotypes.  

5.38           Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins

Latham, T. and Galarza, J.M.

1. Virol., 75(13), 6154-6165 (2001)

  We are studying the structural proteins and molecular interactions required for formation and release of influenza virus-like particles (VLPs) from the cell surface. To investigate these events, we generated a quadruple baculovirus recombinant that simultaneously expresses in Sf9 cells the hemagglutinin (HA), neuraminidase (NA), matrix (M1), and M2 proteins of influenza virus A/Udorn/72 (H3N2). Using this quadruple recombinant, we have been able to demonstrate by double-labeling immunofluorescence that matrix protein (M1) localizes in nuclei as well as at discrete areas of the plasma membrane where HA and NA colocalize at the cell surface. Western blot analysis of cell supernatant showed that M1, HA, and NA were secreted into the culture medium. Furthermore, these proteins comigrated in similar fractions when concentrated supernatant was subjected to differential centrifugation. Electron microscopic examination (EM) of these fractions revealed influenza VLPs bearing surface projections that closely resemble those of wild-type influenza virus. Immunogold labeling and EM demonstrated that the HA and NA were present on the surface of the VLPs. We further investigated the minimal number of structural proteins necessary for VLP assembly and release using single-gene baculovirus recombinants. Expression of M1 protein alone led to the release of vesicular particles, which in gradient centrifugation analysis migrated in a similar pattern to that of VLPs. Immuno-precipitation of M1 proteins from purified M1 vesicles, VLPs, or influenza virus showed that the relative amount of M1 protein associated with M1 vesicles or VLPs was higher than that associated with virions, suggesting that particle formation and budding is a very frequent event. Finally, the HA gene within the quadruple recombinant was replaced either by a gene encoding the G protein of vesicular stomatitis virus or by a hybrid gene containing the cytoplasmic tail and transmembrane domain of the HA and the ectodomain of the G protein. Each of these constructs was able to drive the assembly and release of VLPs, although enhanced recruitment of the G glycoprotein onto the surface of the particle was observed with the recombinant carrying a G/HA chimeric gene. The described approach to assembly of wild-type and chimeric influenza VLPs may provide a valuable tool for further investigation of viral morphogenesis and genome packaging as well as for the development of novel vaccines.  

5.39           Efficient ex vivo transduction of pancreatic islet cells with recombinant adeno-associated virus vectors

Flotte, T. et al Diabetes, 50, 515-520 (2001)   The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islet cells may allow enhanced posttransplantation survival of islet allografts and inhibition of recurrent autoimmune destruction of these cells in type 1 diabetes. However, transient transgene expression and the tendency to induce host inflammatory responses have limited previous gene delivery studies using viral transfer vectors. We demonstrate here that recombinant adeno-associated virus (rAAV) serotype 2, a vector that can overcome these limitations, effectively transduces both human and murine pancreatic islet cells with reporter genes as well as potentially important immunoregulatory cytokine genes (interleukin-4, interleukin-10), although a very high multiplicity of infection (10,000 infectious units/islet equivalent) was required. This requirement was alleviated by switching to rAAV serotype 5, which efficiently transduced islets at a multiplicity of infection of 100. Although adenovirus (Ad) coinfection was required for efficient ex vivo expression at early time points, islets transduced without Ad expressed efficiently when they were transplanted under the renal capsule and allowed to survive in vivo. The rAAV-delivered transgenes did not interfere with islet cell insulin production and were expressed in both b- and non-b-cells. We believe rAAV will provide a useful tool to deliver therapeutic genes for modulating immune responses against islet cells and markedly enhance long-term graft survival.  

5.40           Central leptin gene therapy suppresses body weight gain, adiposity and serum insulin without affecting food consumption in normal rats

Dhillon, H. et al Regulatory Peptides, 99, 69-77 (2001)   The weight-reducing effects of leptin are predominantly mediated through the hypothalamus in the brain. Gene therapy strategies designed for weight control have so far tested the short-term effect of peripherally delivered viral vectors encoding the leptin gene. In order to circumvent the multiple peripheral effects of hyperleptinemia and to overcome the age-related development of leptin resistance due to multiple factors, including defective leptin transport across the blood brain barrier, we determined whether delivery of viral vectors directly into the brain is a viable therapeutic strategy for long-term weight control in normal wild-type rats. A recombinant adeno-associated virus (rAAV) vector encoding rat leptin (Ob) cDNA was generated (rAVV-bOb). When administered once intracerebroventricularly (i.c.v.), rAAV-bOb suppressed the normal time-related weight gain for extended periods of time in adult Sprague-Dawley rats. The vector expression was confirmed by immunocytochemical localization of GFP and RT-PCR analysis of leptin in the hypothalamus. This sustained restraint on weight gain was not due to shifts in caloric consumption because food-intake was similar in rAAV-bOb treated and rAAV-GFP treated control rats throughout the experiment. Weight gain suppression, first apparent after 2 weeks, was a result of reduced white fat depots and was accompanied by drastically reduced serum leptin and insulin concentrations in conjunction with normoglycemia. Additionally, there was a marked increase in uncoupling protein-1 (UCP1) mRNA expression in brown adipose tissue, thereby indicating increased energy expenditure through thermogenesis. Seemingly, a selective enhancement in energy expenditure following central delivery of the leptin gene is a viable therapeutic strategy to control the age-related weight gain and provide protection from the accompanying multiple peripheral effects of hyperleptinemia and hyperinsulinemia.  

5.41           Specific interaction of a novel foamy virus env leader protein with the N-terminal Gag domain

Wilk, T. et al

1. Virol., 75(17), 7995-8007 (2001)

  Cryoelectron micrographs of purified human foamy virus (HFV) and feline foamy virus (FFV) particles revealed distinct radial arrangements of Gag proteins. The capsids were surrounded by an internal Gag layer that in turn was surrounded by, and separated from, the viral membrane. The width of this layer was about 8 nm for HFV and 3.8 nm for FFV. This difference in width is assumed to reflect the different sizes of the HFV and FFV MA domains: the HFV MA domain is about 130 residues longer than that of FFV. The distances between the MA layer and the edge of the capsid were identical in different particle classes. In contrast, only particles with a distended envelope displayed an invariant, close spacing between the MA layer and the Env membrane which was absent in the majority of particles. This indicates a specific interaction between MA and Env at an unknown step of morphogenesis. This observation was supported by surface plasmon resonance studies. The purified N-terminal domain of FFV Gag specially interacted with synthetic peptides and a defined protein domain derived from the N-terminal Env leader protein. The specificity of this interaction was demonstrated by using peptides varying in the conserved Trp residues that are known to be required for HFV budding. The interaction with Gag required residues within the novel virion-associated FFV Env leader protein of about 16.5 kDa.  

5.42           Gene therapy: Promises and problems

Pfeifer, A. and Verma, I.M. Annu.Rev.Genomics Hum. Genet., 2, 177-211 (2001)   Gene therapy can be broadly defined as the transfer of genetic material to cure a disease or at least to improve the clinical status of a patient. One of the basic concepts of gene therapy is to transform viruses into genetic shuttles, which will deliver the gene of interest into the target cells. Based on the nature of the viral genome, these gene therapy vectors can be divided into RNA and DNA viral vectors. The majority of RNA virus-based vectors have been derived from simple retroviruses like murine leukemia virus. A major shortcoming of these vectors is that they are not able to transduce nondividing cells. This problem may be overcome by the use of novel retroviral vectors derived from lentivirus, such as human immunodeficiency virus (HIV). The most commonly used DNA virus vectors are based on adenoviruses and adeno-associated viruses. Although the available vector systems are able to deliver genes in vivo into cells, the ideal delivery vehicle has not been found. Thus, the present viral vectors should be used only with great caution in human beings and further progress in vector development is necessary.  

5.43           A model for gene therapy of human hereditary lymphedema

Karkkkainen, M.J et al Proc. Natl. Acad. Sci., 98, 12677-12682 (2001)   Primary human lymphedema (Milroy’s disease), characterized by a chronic and disfiguring swelling of the extremities, is associated with heterozygous inactivating missense mutations of the gene encoding vascular endothelial growth factor C/D receptor (VEGFR-3). Here, we describe a mouse model and a possible treatment for primary lymphedema. Like the human patients, the lymphedema (Chy) mice have an inactivating Vegfr3 mutation in their germ line, and swelling of the limbs because of hypoplastic cutaneous, but not visceral, lymphatic vessels. Neuropilin (NRP)-2 bound VEGF-C and was expressed in the visceral, but not in the cutaneous, lymphatic endothelia, suggesting that it may participate in the pathogenesis of lymphedema. By using virus-mediated VEGF-C gene therapy, we were able to generate functional lymphatic vessels in the lymphedema mice. Our results suggest that growth factor gene therapy is applicable to human lymphedema and provide a paradigm for other diseases associated with mutant receptors.  

5.44           Identification of Aleutian mink disease parvovirus capsid sequences mediating antibody-dependent enhancement of infection, virus neutralization, and immune complex formation

Bloom, M.E. et al

1. Virol., 75(22), 11116-11127 (2001)

  Aleutian mink disease parvovirus (ADV) causes a persistent infection associated with circulating immune complexes, immune complex disease, hypergammaglobulinemia, and high levels of antiviral antibody. Although antibody can neutralize ADV infectivity in Crandell feline kidney cells in vitro, virus is not cleared in vivo, and capsid-based vaccines have proven uniformly ineffective. Antiviral antibody also enables ADV to infect macrophages, the target cells for persistent infection, by Fc-receptor-mediated antibody-dependent enhancement (ADE). The antibodies involved in these unique aspects of ADV pathogenesis may have specific targets on the ADV capsid. Prominent differences exist between the structure of ADV and other, more typical parvoviruses, which can be accounted for by short peptide sequences in the flexible loop regions of the capsid proteins. In order to determine whether these short sequences are target for antibodies involved in ADV pathogenesis, we studied heterologous antibodies against several peptides present in the major capsid protein, VP2. Of these antibodies, a polyclonal rabbit antibody to peptide VP2:428-446 was the most interesting. The anti-VP2: 428-446 antibody aggregated virus particles into immune complexes, mediated ADE, and neutralized virus infectivity in vitro. Thus, antibody against this short peptide can be implicated in key facts of ADV pathogenesis. Structural modeling suggested that surface-exposed residues of VP2: 428-446 is readily accessible for antibody binding. The observation that antibodies against a single target peptide in the ADV capsid can mediate both neutralization and ADE may explain the failure of capsid-based vaccines.  

5.45           N-terminal cleavage fragment of glycosylated Gag is incorporated into murine oncornavirus particles

Fujisawa, R., McAtee, F.J., Favara, C., Hayes, S.F. and Portis, J.L.

1. Virol., 75(22), 11239-11243 (2001)

  Glycosylated Gag (Glycogag) is a transmembrane protein encoded by murine and feline oncornaviruses.  While the protein is dispensible for virus replication, Glycogag-null mutants of a neurovirulent murine oncornavirus are slow to spread in vivo and exhibit a loss of pathogenicity. The function of this protein in the virus life cycle, however, is not understood. Glycogag is expressed at the plasma membrane of infected cells but has not been detected in virions. In the present study we have reexamined this issue and have found an N-terminal cleavage fragment of Glycogag, which was pelleted by high-speed centrifugation and sedimented in sucrose density gradients at the same buoyant density as virus particles. Its association with virions was confirmed by velocity sedimentation through iodixanol, which effectively separated membrane microvesicles from virus particles. Furthermore, the apparent molecular weight of the virion-associated protein was different from that of the protein extracted from the plasma membrane, suggesting some level of specificity or selectivity of incorporation.  

5.46           Recombinant adeno-associated virus-mediated gene delivery of apolipoprotein B mRNA site-specific ribozyme

Sun, S., Ford, T., Davis, A and Teng, B-B. American Society of Gene Therapy, 4^th Annual Meeting, abstract 430, (2001)   Apolipoprotein B (apoB) plays an obligatory role in the production of triglyceride-rich lipoprotein particles and it is necessary for the transport of lipids and nutrients in the circulation. However, overproduction of apoB is strongly associated with premature coronary artery diseases. Patients with familial hypercholesterolemia have markedly elevated plasma levels of cholesterol and apoB and develop atherosclerosis. To modulate apoB production, we designed a hammerhead ribozyme targeted at GUA6679q of apoB mRNA (designated RB15) to cleave apoB mRNA in vivo. From our previous study, we used E1-deleted adenovirus vector to deliver RB15 to a dyslipidemia mouse model. The study showed that RB15 cleaved apoB mRNA efficiently. There was a marked reduction of apoB gene expression and decrease plasma levels of cholesterol, triglyceride, and human apoB100. Therefore, apoB mRNA-specific hammerhead ribozyme can be used as a potential therapeutic agent to modulate apoB gene expression and to treat hyperlipidemia. To have a long-term gene expression, no immune response, and no toxicity in gene therapy, in this study, we sought to construct a liver-specific adeno-associated virus (AAV) vector to deliver RB15 to HepG2 cells and animals. RB15 is driven by transthyretin liver-specific promoter (TTR) and a 2773-bp human genomic fragment of hypoxanthine guanine phosphoribosyltransferase (HPRT) was inserted downstream of 5’ ITR of AAV vector (pAAV-TTR-RB15). We produced rAAV-TTR-RB15 by co-transfection of pAAV-TTR-RB15 with helper plasmid pDG in 293 cells, followed by purification using non-ionic iodixanol gradient and by ion exchange with heparin affinity chromatography. The virus titer was 1x10¹² particles/ml, determined by both real-time PCR and dot-blot hybridization. We characterized the rAAV viral capsid proteins (VP1, VP2 and VP3) by western blotting.   The rAAV-TRR-RB15 (1x10⁸ particles) was used to infect HepG2 cells. Total RNA was extracted at days 3 and 7 after injection. Using RNase protection assay the levels of apoB mRNA on day 7 was barely detectable (7.6% compared to that of non-treated samples). The rAAV-TTR-RB15 (8x10¹⁰ particles) was used to transduce mouse overexpressing human apoB gene. Using both PCR and real-time PCR RB15 DNA was detectable in the mouse liver on day 45 after treatment. The RB15 RNA was also detected in mouse liver on day 45 after treatment by RT/PCR. Southern blot analysis show that rAAV-TTR-RB15 was stably transduced into the liver. Using Western blot analysis, the levels of human apoB decreased on days 7,14, and 28 to 13%, 40%, and 63%, respectively, compared to that of day 0 before treatment. In conclusion, the expressed ribozyme RB15 RNA was active, which decreased apoB production.  

5.47           Homologous gene targeting using an autonomous parvovirus vector

Hendrie, P.C. and Russell, D.W. American Society of Gene Therapy, 4^th Annual Meeting, abstract 515, (2001)   Our group has previously demonstrated efficient gene targeting with vectors based on adeno-associated virus (AAV) (Russell and Hirata Nat. Genet. 18: 325-330). In order to evaluate the phenomenon in other parvovirus vectors, we constructed a gene-targeting vector using terminal hairpins of the autonomous parvovirus, minute virus of mice, prototype strain (MVMp) which differs from AAV in that only one strand is packaged in virions. We used a dual-functioning vector containing an MSCV-LTR-driven green fluorescent protein (GFP) transgene cassette to monitor gene addition and a 2.5kb sequence homologous to alkaline phosphatase target loci introduced by retroviral vectors to assay for gene targeting. Vector stocks were prepared by transient transfection with all viral proteins provided in trans from a packaging plasmid devoid of MVM termini. Cell lysates were purified by centrifugation through iodixanol gradients, filtered and concentrated. 10¹⁰ full-length vector genomes were obtained from 10⁷ transfected cells. Stocks were used to infect a human HT 1080 cell line that contained an integrated placental alkaline phosphatase gene with a 4 base pair deletion in the coding sequence. After infection, no significant cytopathic effect or decrease in plating efficiency was observed in vector-treated cells versus controls. GFP transgene expression was detected by flow cytometry in up to 3% of cells in a dose dependent fashion. Alkaline phosphatase expression due to gene targeting was seen in up to 0.3% of foci, also in a dose dependent fashion. MVMp’s property of packaging predominantly a single “minus strand” DNA molecule was used to compare the relative targeting rates of coding and non-coding DNA strands, alone and in combination. These experiments demonstrate efficient transduction using MVMp based vectors and prove that the introduction of single-stranded genomes of either orientation is sufficient to carry out gene targeting.  

5.48           Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra

Neurons

Furler, S., Paterna, J-C., Weibel, M. and Bheler, H. Gene Therapy, 8, 864-873 (2001)   Recombinant adeno-associated viruses (rAAVs) are promising vectors for gene therapy since they efficiently and stably transduce a variety of tissues of immunocompetent animals. The major disadvantages of rAAVs are their limited capacity to package foreign DNA (£5 kb). Often, co-expression of two or more genes from a single viral vector is desirable to achieve maximal therapeutic efficacy or to track transduced cells in vivo by suitable reporter genes. The internal ribosome entry site (IRES) sequence of encephalomyocarditis virus has been widely used to construct bicistronic viral vectors. However, the IRES is rather long and IRES-mediated translation can be relatively inefficient when compared with cap-dependent translation. As an alternative to the IRES for in vivo gene expression, we studied the 16 amino-acid long 2A peptide of foot and mouth disease virus (FMDV). The 2A peptide mediates the primary cis-“cleavage” of the FMDV polyprotein in a cascade of processing events that ultimately generate the mature FMDV proteins. We have generated several different rAAV genomes in which two coding regions are fused in-frame via the FMDV 2A sequence. We show that FMDV 2A efficiently mediates the generation of the expected cleavage products from the artificial fusion proteins in cells. Furthermore, we find that both EGFP and a-synuclein are expressed at substantially higher levels from 2A vectors than from the corresponding IRES-based vectors, while DOD-1 is expressed at comparable or slightly higher levels. Finally, we demonstrate for the first time, that the 2A sequence results in effective bicistronic gene expression in vivo after injection of 2A-dependent rAAV into rat substantia nigra. We conclude that 2A-containing rAAVs may represent an attractive alternative to IRES-dependent vectors for ex vivo and in vivo gene expression and gene therapy.  

5.49           Prevention of systemic clinical disease in MPS VII mice following AAV-mediated neonatal gene transfer

Daly, T.M., Ohlemiller, K.K., Roberts, M.S., Vogler, C.A. and Sands, M.S. Gene Therapy, 8, 1291-1298 (2001)   For many inborn errors of metabolism, early treatment is critical to prevent long-term developmental sequelae. We have previously shown that systemic treatment of neonatal mucopolysaccharidosis type VII (MPS VII) mice with recombinant adeno-associated virus (AAV) vectors results in relatively long-term expression of b-glucuronidase (GUSB) in multiple tissues, and a reduction in lysosomal storage. Here, we demonstrate that therapeutic levels of enzyme persist for at least 1 year following a single intravenous injection of virus in neonatal MPS VII mice. The level and distribution of GUSB expression achieved is sufficient to prevent the development of many aspects of clinical disease over the life of the animal. Following treatment, bone lengths, weights and retinal function were maintained at nearly normal levels throughout the life of the animal. In addition, significant improvements in survival and auditory function were seen in AAV-treated MPS VII mice when compared with untreated mutant siblings. These data suggest that AAV-mediated gene transfer in the neonatal period can lead to prevention of many of the clinical symptoms associated with MPS VII in the murine model of this disease.  

5.50           Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors

Song, S. et al Gene Therapy, 8, 1299-1306 (2001)   Previous work from our group showed that recombinant adeno-associated virus (rAAV) vectors mediated long-term secretion of therapeutic serum levels of human alpha-1 antitrypsin (hAAT) after a single injection in murine muscle. We hypothesized that hepatocyte transduction could be even more efficient, since these cells represent the natural site of AAT production and secretion. To test this hypothesis, rAAV vectors containing the hAAT cDNA driven by either the human elongation factor 1 alpha promoter, the human cytomegalovirus immediate-early promoter (CMV), or the CMV-chicken beta actin hybrid (CB) promoter were injected into the portal or tail veins of adult C57BI/6 mice. Potentially therapeutic serum levels of hAAT (600 ug/ml) were achieved after portal vein injection of doses of 4x10⁹ infectious units (IU), a 10-fold lower dose than required for similar levels of expression via the i.m. route. Serum levels greater than 1 mg/ml were achieved at doses of 3x10¹⁰ IU. Southern blotting of liver DNA revealed the presence of circular episomal vector genomes. Immunostaining showed that transgene expression was scattered throughout the liver parenchyma. Similar results were obtained with a rAAV-CB-green fluorescent protein (GFP) vector. There was no evidence of hepatic toxicity. These data indicate that liver-directed rAAV-based gene therapy is effective in the murine model, and hence might be feasible for treatment of human AAT deficiency.  

5.51           Gene therapy restores vision in a canine model of childhood blindness

Acland, G.M. et al Nature Gen., 28, 92-95 (2001)   The relationship between the neurosensory photoreceptors and the adjacent retinal pigment epithelium (RPE) controls not only normal retinal function, but also the pathogenesis of hereditary retinal degenerations. The molecular bases for both primary photoreceptor and RPE diseases that cause blindness have been identified. Gene therapy has been used successfully to slow degeneration in rodent models of primary photoreceptor diseases, but efficacy of gene therapy directed at photoreceptors and RPE in a large-animal model of human disease has not been reported. Here we study one of the most clinically severe retinal degenerations, Leber congenital amaurosis (LCA). LCA causes near total blindness in infancy and can result from mutations in RPE65 (LCA, type II; MIM 180069 and 204100). A naturally occurring animal model, the RPE65^-/- dog, suffers from early and severe visual impairment similar to that seen in human LCA. We used a recombinant adeno-associated virus (AAV) carrying wild-type RPE65 (AAV-RPE65) to test the efficacy of gene therapy in this model. Our results indicate that visual function was restored in this large animal model of childhood blindness.  

5.52           Dose-dependent effects of central leptin gene therapy on genes that regulate body weight and appetite in the hypothalamus

Dhillon, H., Kalra, S.P. and Kalra, P.S. Mol. Ther., 4(2), 139-145 (2001)   We have examined the dose-dependent effects and central action of intraventricular administration of a recombinant adeno-associated virus encoding rat leptin (rAAV-leptin) in suppressing body weight (BW) gain in adult female rats. A low dose of rAAV-leptin (5x10(10) particles) suppressed weight gain (15%) without changing daily food intake (FI), but a twofold higher dose decreased BW by 30% along with a reduction in daily FI. Reduced BW was due to a loss in body adiposity because serum leptin was reduced. Serum insulin levels were decreased (96%) by only the high dose along with a slight reduction in glucose. Uncoupling protein-1 (UCP-1) mRNA expression in brown adipose tissue (BAT), reflecting energy expenditure through thermogenesis, was upregulated to the same magnitude by the two rAAV-leptin doses. We analyzed by in situ hybridization the expression in the hypothalamus of genes encoding the appetite-regulating neuropeptides. Only the high dose decreased expression of neuropeptide Y (NPY), the orexigenic peptide, and increased proopiomelanocortin (POMC), precursor of the an orexigenic peptide, alpha-MSH. Our studies show for the first time that increased availability of leptin within the hypothalamus through central leptin gene therapy dose-dependently decreases weight gain, adiposity, and serum insulin by increasing energy expenditure and decreasing FI. The decrease in FI occurs only when NPY is reduced and alpha-MSH is increased in the hypothalamus by the high dose of rAAV-leptin. Delivery of the leptin gene centrally through rAAV vectors is a viable therapeutic modality for long-term control of weight and metabolic hormones.  

5.53           CMV-b-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1a promoter and results in therapeutic levels of human factor X in mice

Xu, L. et al Human Gen. Ther., 12, 563-573 (2001)   Although AAV vectors show promise for hepatic gene therapy, the optimal transcriptional regulatory elements have not yet been identified. In this study, we show that an AAV vector with the CMV enhancer/chicken beta-actin promoter results in 9.5-fold higher expression after portal vein injection than an AAV vector with the EF1 alpha promoter, and 137-fold higher expression than an AAV vector with the CMV promoter/enhancer. Although induction of the acute-phase response with the administration of lipopolysaccharide (LPS) activated the CMV promoter/enhancer from the context of an adenoviral vector in a previous study, LPS resulted in only a modest induction of this promoter from an AAV vector in vivo. An AAV vector with the CMV-beta-actin promoter upstream of the coagulation protein human factor X (hFX) was injected intravenously into neonatal mice. This resulted in expression of hFX at 548 ng/ml (6.8% of normal) for up to 1.2 years, and 0.6 copies of AAV vector per diploid genome in the liver at the time of sacrifice. Neonatal intramuscular injection resulted in expression of hFX at 248 ng/ml (3.1% of normal), which derived from both liver and muscle. We conclude that neonatal gene therapy with an AAV vector with the CMV-beta-actin promoter might correct hemophilia due to hFX deficiency.  

5.54           Cochlear function and transgene expression in the guinea pig cochlea, using adenovirus- and adeno-associated virus-directed gene transfer

Luebke, A.E., Foster, P.K., Muller, C.D. and Peel, A.L. Human Gen. Ther., 12, 773-781 (2001)   Development of a viral vector that can infect hair cells of the cochlea without producing viral-associated ototoxic effects is crucial for utilizing gene replacement therapy as a treatment for certain forms of hereditary deafness. In the present study, cochlear function was monitored using distortion-product otoacoustic emissions (DPOAEs) in guinea pigs that received infusions of either (E1(-), E3(-)) adenovirus, or adeno-associated virus (AAV), directly into the scala tympani. Replication-deficient (E1(-), E3(-)) adenovirus-directed gene transfer, using the cytomegalovirus (CMV) promoter, drove transgene expression to inner hair cells and pillar cells of the cochlea. AAV transduction was tested with several promoters, such as platelet-derived growth factor (PDGF), neuron-specific enolase (NSE), and elongation factor 1a (EF-1a) promoters; which drove transgene expression to cochlear blood vessels, nerve fibers, and certain spiral limbus cells, respectively. AAV transgene expression was visualized by green fluorescent protein immunostaining. Immunocytochemistry to heparan sulfate confirmed the absence of proteoglycans in guinea pig hair cells, indicating that the receptor for AAV was not present on these cells. However, the heparan sulfate proteoglycan expression pattern mimicked the AAV transduction pattern. An overall finding was that cochlear function was not altered throughout the infection period using AAV titers as high as 5 x 10⁸ IP/infused cochlea. In contrast, cochlear function was severely compromised by 8 days postinfection with adenoviral titers of 5 x 10⁸ PFU/infused cochlea, and outer hair cells were eliminated. Thus, cochlear hair cells are amenable to in vivo gene transfer using a replication-deficient (E1^-, E3^-) adenovirus. However, replication-defective or gutted adenovirus vectors must be employed to overcome the ototoxic effects of (E1^-, E3^-) adenovirus vectors.  

5.55           Insertional mutagenesis of the adeno-associated virus type 2 (AAV2) capsid gene and generation of AAV2 vectors targeted to alternative cell-surface receptors

Shi, W., Arnold, G.S. and Bartlett, J.S. Human Gen. Ther., 12, 1697-1711 (2001)   Recombinant adeno-associated virus (AAV) vectors are of interest in the context of gene therapy because of their ability to mediate efficient transfer and stable expression of therapeutic genes in a wide variety of tissues. However, AAV-mediated gene delivery to specific cell populations is often precluded by the widespread distribution of heparan sulfate proteoglycan (HSPG), the primary cellular receptor for the virus. Conversely, an increasing number of cell types are being identified that do not express HSPG and are therefore poor targets for AAV-mediated gene transfer. To address these issues, we have developed strategies to physically modify AAV vectors and allow efficient, HSPG-independent, receptor-targeted infection. We began by generating a series of 38 virus capsid mutants containing peptide insertions at 25 unique sites within the AAV capsid protein. The mutant viruses were characterized on the basis of their phenotypes and grouped into three classes: class I mutants (4 of 38) did not assemble particles; class II mutants (14 of 38) assembled noninfectious particles; and class III mutants (20 of 38) assembled fully infectious particles. We examined the HSPG-binding characteristics of the class II mutants and showed that a defect in receptor binding was a common reason for their lack of infectivity. The display of foreign peptide epitopes on the surface of the mutant AAV particles was found to be highly dependent on the inclusion of appropriate scaffolding sequences. Optimal scaffolding sequences and five preferred sites for the insertion of targeting peptide epitopes were identified. These sites are located within each of the three AAV capsid proteins, and thus display inserted epitopes 3, 6, or 60 times per vector particle. Modified AAV vectors displaying a 15-amino acid peptide, which binds to the human luteinizing hormone receptor (LH-R), were generated and assessed for their ability to target gene delivery to receptor-bearing cell lines. Titers of these mutant vectors were essentially the same as wild-type vector. The LH-R-targeted vector was able to transduce ovarian cancer cells (OVCAR-3) in an HSPG-independent manner. Furthermore, transduction was shown to proceed via the LH-R and therefore treatment of OVCAR-3 cells with progesterone, to increase LH-R expression, accordingly increased LH mutant-mediated gene transfer. This technology may have a significant impact on the use of AAV vectors for human gene therapy.  

5.56           Herpes simplex virus mediated nerve growth factor expression in bladder and affererent neurons: potential treatment for diabetic bladder dysfunction

Goins, W. F. et al

1. Urol., 165, 1748-1754 (2001)

  Purpose: Diabetic cystopathy resulting from sensory neuropathy may potentially be treated by direct gene therapy. It has been suggested that nerve growth factor (NGF) has an ameliorative effect in preventing the death in diabetes of afferent dorsal root ganglion neurons, which control bladder function. We investigated NGF gene transfer to the bladder and bladder afferent pathways for treating diabetic cystopathy. We used replication competent and replication defec­tive herpes simplex virus type 1 (HSV-1) vectors that express a functionally active form of the b-subunit of mouse NGF (b-NGF) to examine the level and duration of therapeutic gene expres­sion after administration of the vectors. Materials and Methods: NGF expression during acute (3 days) and latent (21 days) infections was assessed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemical testing after the injection of 1x10⁶ to 1x10⁸ pfu HSV-NGF expression vectors into the bladder wall of adult rats. Results: HSV vectors with the strong human cytomegalovirus immediate early promoter used to drive b-NGF gene expression exhibited increased NGF 3 days after infection in the bladder and L6 to S1 dorsal root ganglia, where bladder afferent neurons are located. ELISA analysis revealed that NGF in the bladder tissue and dorsal root ganglia was increased 7 to 9 and 2 to 4-fold, respectively, over the control vector. Increased NGF expression in L6 to S1 dorsal root ganglia neurons was also detected by immunohistochemical staining with antiNGF antibodies. Extended NGF expression was detected by ELISA 21 days after injection. Replication defective vectors containing HSV-1 latency promoter (LAP-2) driving NGF expressed NGF in the bladder and dorsal root ganglia 21 days after bladder injection. ELISA analysis confirmed an approxi­mate 2 to 3-fold increase of NGF expression in the bladder and L6 to S1 dorsal root ganglia. Conclusions: The NGF gene may be transferred and expressed in the bladder and bladder afferent pathways using HSV vectors. To our knowledge our study represents the first demon­stration of the effectiveness of gene therapy for altering neurotrophic expression in visceral sensory neurons. This technique of gene transfer may be useful for treating certain types of neurogenic bladder dysfunction, such as diabetic cystopathy, in which decreased NGF transport may be a causative factor.  

5.57           Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity

Rabinowitz, J.E. et al

1. Virol., 76, 791-801 (2002)

  The serotypes of adeno-associated virus (AAV) have the potential to become important resources for clinical gene therapy. In an effort to compare the role of serotype-specific virion shells on vector transduction, we cloned each of the serotype capsid coding domains into a common vector backbone containing AAV type 2 replication genes. This strategy allowed the packaging of AAV2 inverted terminal repeat vectors into each serotype-specific virions. Each of these helper plasmids (pXR1 through pXR5) efficiently replicated the transgene DNA and expressed helper proteins at nearly equivalent levels. In this study, we observed a correlation between the amount of transgene replication and packaging efficiency. The physical titer of these hybrid vectors ranged between 1.3 x 10¹¹ and 9.8 x 10¹²/ml (types 1 and 2, respectively). Of thefive serotype vectors, only types 2 and 3 were efficiently purifiedby heparin-Sepharose column chromatography, illustrating thehigh degree of similarity between these virions. We analyzedvector transduction in reference and mutant Chinese hamsterovary cells deficient in heparan sulfate proteoglycan and sawa correlation between transduction and heparan sulfate bindingdata. In this analysis, types 1 and 5 were most consistent intransduction efficiency across all cell lines tested. In vivoeach serotype was ranked after comparison of transgene levelsby using different routes of injection and strains of rodents.Overall, in this analysis, type 1 was superior for efficienttransduction of liver and muscle, followed in order by types5, 3, 2, and 4. Surprisingly, this order changed when vectorwas introduced into rat retina. Types 5 and 4 were most efficient,followed by type 1. These data established a hierarchy for efficientserotype-specific vector transduction depending on the targettissue. These data also strongly support the need for extendingthese analyses to additional animal models and human tissue.The development of these helper plasmids should facilitate directcomparisons of serotypes, as well as begin the standardizationof production for further clinical development.  

5.58           The late-domain-containing protein p6 is the predominant phosphoprotein of human immunodeficiency virus type 1 particles

Mhller, B., Patschinsky, T. and Kr@usslich, H-G.

1. Virol., 76(3), 1015-1024 (2002)

  The Gag-derived protein p6 of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the release of virions from the membranes of infected cells. It is presumed that p6 and functionally related proteins from other viruses act as adapters, recruiting cellular factors to the budding site. This interaction is mediated by so-called late domains within the viral proteins. Previous studies had suggested that virus release from the plasma membrane shares elements with the cellular endocytosis machinery. Since protein phosphorylation is known to be a regulatory mechanism in these processes, we have investigated the phosphorylation of HIV-1 structural proteins. Here we show that p6 is the major phosphoprotein of HIV-1 particles. After metabolic labelling of infected cells with [ortho-³²P]phosphate, we found that phosphorylated p6 from infected cells and from virus particles consisted of several forms, suggesting differential phosphorylation at multiple sites. Apparently, phosphorylation occurred shortly before or after the release of p6 from Gag and involved only a minor fraction of the total virion-associated p6 molecules. Phosphoamino acid analysis indicated phosphorylation at Ser and Thr, as well as a trace of Tyr phosphorylation, supporting the conclusion that multiple phosphorylation events do occur. In vitro experiments using purified virus revealed that endogenous or exogenously added p6 was efficiently phosphorylated by virion-associated cellular kinase(s). Inhibition experiments suggested that a cyclin-dependent kinase or a related kinase, most likely ERK2, was involved in p6 phosphorylation by virion-associated enzymes.  

5.59           Cells in human postmortem brain tissue slices remain alive for several weeks in culture

Verwer, R.W.H. et al FASEB J., 16, 54-60 (2002)   Animal models for human neurological and psychiatric diseases only partially mimic the underlying pathogenic processes. Therefore, we investigated the potential use of cultured postmortem brain tissue from adult neurological patients and controls. The present study shows that human brain tissue slices obtained by autopsy within 8 h after death can be maintained in vitro for extended periods (up to 78 days) and can be manipulated experimentally. We report for the first time that 1) neurons and glia in such cultures could be induced to express the reporter gene LacZ after transduction with adeno-associated viral vectors and 2) cytochrome oxidase activity could be enhanced by the addition of pyruvate to the medium. These slice cultures offer new opportunities to study the cellular and molecular mechanisms of neurological and psychiatric diseases and new therapeutic strategies.  

5.60           Inhibition of atherosclerosis in apolipoprotein-E-deficient mice following muscle transduction with adeno-associated virus vectors encoding human apolipoprotein-E

Harris, J.D. et al Gene Therapy, 9, 21-29 (2002)   Apolipoprotein E (apoE) is a multifunctional plasma glycoprotein involved in lipoprotein metabolism and a range of cell signaling phenomena. ApoE-deficient (apoE^-/-) mice exhibit severe hypercholesterolaemia and are an excellent model of human atherosclerosis. ApoE somatic gene transfer and bone marrow transplantation in apoE^-/- mice results in reversal of hypercholesterolaemia, inhibition of atherogenesis and regression of atherosclerotic plaque density. Replication defective adeno-associated virus vectors (rAAVs) are an attractive system currently in clinical trials for muscle-based heterologous gene therapy to express secreted recombinant plasma proteins. Here we have applied rAAV transduction of skeletal muscle to express wild-type (Î3) and a defective receptor-binding mutant (Î2) human apoE transgene in apoE^-/- mice. In treated animals, apoE mRNA was present in transduced muscles and, although plasma levels of recombinant apoE fell below the detection levels of our ELISA (i.e. < 10 ng/ml), circulating antibodies to human apoE and rAAV were induced. Up to 3 months after a single administration of rAAV/apoE3, a significant reduction in atherosclerotic plaque density in aortas of treated animals was observed (approximately 30%), indicating that low-level rAAV-mediated apoE3 expression from skeletal muscle can retard atherosclerotic progression in this well-defined genetic model.  

5.61           Structural requirements for the assembly of Norwalk virus-like particles

Bertolotti-Ciarlet, A., White, L.J., Chen, R., Prasad, B.V.V. and Estes, M.K.

1. Virol., 76(8), 4044-4055 (2002)

  Norwalk virus (NV) is the prototype strain of a group of human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant NV virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. The X-ray structure of the rNV VLPs has revealed that the capsid protein folds into two principal domains: a shell (S) domain and a protruding (P) domain (B. V. V. Prasad, M. E. Hardy, T. Dokland, J. Bella, M. G. Rossmann, and M. K. Estes, Science 286:287-290, 1999). To investigate the structural requirements for the assembly of rNV VLPs, we performed mutational analyses of the capsid protein. We examined the ability of 10 deletion mutants of the capsid protein to assemble into VLPs in insect cell cultures. Deletion of the N-terminal 20 residues, suggested by the X-ray structure to be involved in a switching mechanism during assembly, did not affect the ability of the mutant capsid protein to self-assemble into 38-nm VLPs with a T=3 icosahedral symmetry. Further deletions in the N-terminal region affected particle assembly. Deletions in the C-terminal regions of the P domain, involved in the interactions between the P and S domains, did not block the assembly process, but they affected the size and stability of the particles. Mutants carrying three internal deletion mutations in the P domain, involved in maintaining dimeric interactions, produced significantly larger 45-nm particles, albeit in low yields. The complete removal of the protruding domain resulted in the formation of smooth particles with a diameter that is slightly smaller than the 30-nm diameter expected from the rNV structure. These studies indicate that the shell domain of the NV capsid protein contains everything required to initiate the assembly of the capsid, whereas the entire protruding domain contributes to the increased stability of the capsid by adding intermolecular contacts between the dimeric subunits and may control the size of the capsid.  

5.62           Adeno-associated virus capsids displaying immunoglobulin-binding domains permit antibody-mediated vector retargeting to specific cell surface receptors

Ried, M.U., Girod, A., Leike., K., Bhning, H. And Hallek, M.

1. Virol., 76(9), 4559-4566 (2002)

  Recombinant adeno-associated virus type 2 (rAAV2) is a promising vector for human somatic gene therapy. However, its broad host range is a disadvantage for some applications, because it reduces the specificity of the gene transfer. To overcome this limitation, we sought to create a versatile rAAV vector targeting system which would allow us to redirect rAAV binding to specific cell surface receptors by simple coupling of different ligands to its capsid. For this purpose, an immunoglobulin G (IgG) binding domain of protein A, Z34C, was inserted into the AAV2 capsid at amino acid position 587. The resulting AAV2-Z34C mutants could be packaged and purified to high titers and bound to IgG molecules. rAAV2-Z34C vectors coupled to antibodies against CD29 (ß₁-integrin), CD117 (c-kit receptor), and CXCR4 specifically transduced distinct human hematopoietic cell lines. In marked contrast, no transduction was seen in the absence of antibodies or in the presence of specific blocking reagents. These results demonstrate for the first time that an immunoglobulin binding domain can be inserted into the AAV2 capsid and coupled to various antibodies, which mediate the retargeting of rAAV vectors to specific cell surface receptors.  

5.63           Specific incorporation of heat shock protein 70 family members into primate Lentiviral virions

Gurer, C., Cimarelli, A. and Luban, J.

1. Virol., 76, 4666-4670 (2002)

  To determine if any heat shock proteins are incorporated into human immunodeficiency virus type 1 (HIV-1) virions in a manner similar to that of the peptidyl-prolyl isomerase cyclophilin A, we probed purified virions with antibodies against heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70, Hsc70, and Hsp90. Of these proteins, Hsp60, Hsp70, and Hsc70 associated with virions purified based on either particle density or size and were shown to be incorporated within the virion membrane, where they were protected from digestion by exogenous protease. Virion incorporation of Hsp70 was also observed with HIV-2 and with simian immunodeficiency viruses SIV_MAC and SIV_AGM, but it appears to be specific for primate lentiviruses, since Hsp70 was not detected in association with Moloney murine leukemia virus virions. Of the HIV-1 genes, gag was found to be sufficient for Hsp70 incorporation, though Hsp70 was roughly equimolar with pol-encoded proteins in virions.  

5.64           Adeno-associated virus vector gene transfer and sarcolemmal expression of a 144 kDa micro-dystrophin effectively restores the dystrophin-associated protein complex and inhibits myofibre degeneration in nude/mdx mice

Fabb, S.A., Wells, D.J., Serpente, P. and Dickson, G. Hum. Mol. Genet., 11(7), 733-741 (2002)   Duchenne muscular dystrophy is a severe life-threatening X-linked recessive disorder, caused by mutations in the dystrophin gene, for which currently there is no effective treatment. Because of the large size of the dystrophin cDNA (14 kb) this precluded it from being used in early adenovirus- or retrovirus-based gene therapy vectors. However, some therapeutic success has been achieved in mdx mice using adenovirus- and retrovirus-mediated transfer of a 6.3 kb recombinant mini-dystrophin cDNA. Despite this, problems with immunogenicity and inefficient transduction of mature myofibres make these vectors less than ideal for gene transfer to skeletal muscle. Adeno-associated viral (AAV) vectors overcome many of the problems associated with other vector systems. However, AAV vectors can only accommodate <5 kb of foreign DNA. For this reason we have produced a micro-dystrophin cDNA gene construct that is <3.8 kb. This construct, driven by a CMV promoter, was introduced into the skeletal muscle of 12-day-old nude/mdx mice using an AAV vector, resulting in specific sarcolemmal expression of micro-dystrophin in >50% of myofibres up to 20 weeks of age, and effective restoration of the dystrophin-associated protein (DAP) complex components. Additionally, evaluation of central nucleation indicated a significant inhibition of degenerative dystrophic muscle pathology. We have therefore shown that the current micro-dystrophin gene delivered in vivo using an AAV vector is not only capable of restoring sarcolemmal DAP complexes, but can also ameliorate dystrophic pathology at the cellular level.  

5.65           Inhibition of retinal neovascularisation by gene transfer of soluble VEGF receptor sFlt-1

Bainbridge, J.W.B. et al Gene Therapy, 9(5), 320-326 (2002)   Retinal angiogenesis is a central feature of the leading causes of blindness. Current treatments for these conditions are of limited efficacy and cause significant adverse effects. In this study, we evaluated the angiostatic effect of gene transfer of the soluble VEGF receptor sFlt-1 in a mouse model of ischaemia-induced retinal neovascularisation using adenovirus and adeno-associated virus (AAV) vectors. We induced proliferative retinopathy in mice by exposure to 75% oxygen from postnatal day 7 (p7) to p12 and injected intravitreally recombinant viral vectors expressing the reporter green fluorescent protein (GFP) or vectors expressing the VEGF inhibitor sFlt-1. Efficient adenovirus-mediated GFP expression was evident in cells of the corneal endothelium and iris pigment epithelium. AAV-mediated GFP expression was evident in ganglion cells and cells of the inner nuclear layer of the retina. Vector-mediated sFlt-1 expression was confirmed by ELISA of pooled homogenised whole eyes. Injection of either vector expressing sFlt-1 resulted in a reduction in the number of neovascular endothelial cells by 56% and 52% for adenovirus and AAV vectors, respectively (P < 0.05). Local gene transfer of sFlt-1 consistently inhibits experimental retinal neovascularisation by approximately 50% and offers a powerful novel approach to the clinical management of retinal neovascular disorders.  

5.66           Central leptin gene delivery evokes persistent leptin signal transduction in young and aged-obese rats but physiological responses become attenuated over time in aged-obese rats

Scarpace, P.J. et al Neuropharmacol., 42, 548-561 (2002)   The purpose of this study was to determine if long-term leptin treatment desensitizes leptin signal transduction and the subsequent downstream anorexic and thermogenic responses in normal and leptin-resistant age-related obese rats. To this end, we administered, i.c.v., recombinant adeno-associated virus encoding rat leptin cDNA (rAAV-leptin) or control virus into young and aged0obese rats and after 9 or 46 days, examined food intake, oxygen consumption, body weight, serum leptin, STAT3 phosphorylation, hypothalamic NPY and POMC mRNAs, and UCP1 expression and protein level in brown adipose tissue (BAT). In young rats, rAAV-leptin depleted body fat and both anorexic and thermogenic mechanisms contributed to this effect. Moreover, leptin signal transduction was not desensitized, and there were persistent physiological responses. Similarly, in the aged-obese rats, there was unabated leptin signal transduction, however, both the anorexic and thermogenic responses completely attenuated sometime after day 9. This attenuation, downstream of the leptin receptor, may be contributing to the leptin-resistance and age-related weight gain in these aged-obese rats. Finally, in young rats, although the initial responses to rAAV-leptin were dominated by anorexic responses, by 46 days, the predominant response was thermogenic rather than anorexic, suggesting that energy expenditure may be an important component of long-term weight maintenance.  

5.67           Virosome-mediated delivery of protein antigens to dendritic cells

Bungener, L. et al Vaccine, 20, 2287-2295 (2002)   Virosomes are reconstituted viral membranes in which protein can be encapsulated. Fusion-active virosomes, fusion-inactive virosomes and liposomes were used to study the conditions needed for delivery of encapsulated protein antigen ovalbumin (OVA) to dendritic cells (DCs) for MHC class I and II presentation. Fusion-active virosomes, but not fusion-inactive virosomes, were able to deliver OVA to DCs for MHC class I presentation at picomolar OVA concentrations. Fusion activity of virosomes was not required for MHC class II presentation of antigen. Therefore, virosomes are an efficient system for delivery of protein antigens for stimulation of both helper and CTL responses.  

5.68           Central leptin gene therapy blocks high-fat diet-induced weight gain, hyperleptinemia, and hyperinsulinemia

Dube, M.G. et al Diabetes, 51, 1729-1736 (2002)   Recombinant adeno-associated virus (rAAV), encoding either rat leptin (rAAV-lep) or green fluorescent protein (rAAV-GFP, control), was injected intracerebroventricularly in rats consuming a high-fat diet (HFD; 45 kcal%). Caloric consumption and body weight were monitored weekly until the rats were killed at 9 weeks. Untreated control rats consuming regular rat diet (RCD; 11 kcal%) were monitored in parallel. Body weight gain was accelerated in rAAV-GFP + HFD control rats relative to those consuming RCD, despite equivalent kcal consumption. At 9 weeks, serum leptin, free fatty acids, triglycerides, and insulin were elevated in HFD control rats. In contrast, rAAV-lep treatment reduced intake and blocked the HFD-induced increase in weight, adiposity, and metabolic variables. Blood glucose was slightly reduced but within the normal range, and serum ghrelin levels were significantly elevated in rAAV-lep + HFD rats. Uncoupling protein-1 (UCP1) mRNA in brown adipose tissue (BAT), an index of energy expenditure through nonshivering thermogenesis, was decreased in rats consuming HFD. Treatment with rAAV-lep significantly augmented BAT UCP1 mRNA expression, indicating increased thermogenic energy expenditure. These findings demonstrate that central leptin gene therapy efficiently prevents weight gain, increased adiposity, and hyperinsulinemia in rats consuming an HFD by decreasing energy intake and increasing thermogenic energy expenditure.  

5.69           TrkB gene transfer protects retinal ganglion cells from axotomy-induced death in vivo

Cheng, L., Sapieha, P., Kittlerova, P., Hauswirth, W.W. and Di Polo, A.

1. Neurosci., 22(10), 3977-3986 (2002)

  Injury-induced downregulation of neurotrophin receptors may limit the response of neurons to trophic factors, compromising their ability to survive. We tested this hypothesis in a model of CNS injury: retinal ganglion cell (RGC) death after transfection of the adult rat optic nerve. TrkB mRNA rapisly decreased in axotomized RGCs to ~50% of the level in intact retinas. TrkB gene transfer into RGCs combined with exogenous BDNF administration markedly increased neuronal survival: 76% of RGCs remained alive at 2 weeks after axotomy, a time when >90% of these neurons are lost without treatment. Activation of mitogen-activated protein kinase, but no phosphatidylinositol-3 kinase, was required for TrkB-induced survival. These data provide proof-of-principle that enhancing the capacity of injured neurons to respond to trophic factors can be an effective neuroprotective strategy in the adult CNS.  

5.70           Novel monoclonal antibody directed at the receptor binding site on the avian sarcoma and leukosis virus env complex

Ochsenbauer-Jambor, C., Delos, S.E., Accavitti, M.A., White, J.M. and Hunter, E.

1. Virol., 76(15), 7518-7527 (2002)

  We report here on the generation of a mouse monoclonal antibodydirected against Rous sarcoma virus (RSV) subgroup A Env thatwill be useful in functional and structural analysis of RSVEnv, as well as in approaches employing the RCAS/Tva systemfor gene targeting. BALB/c mice were primed and given boosterstwice with EnvA-expressing NIH 3T3 cells. Resulting hybridomaswere tested by enzyme-linked immunosorbent assay against RCANBPvirions and SU-A-immunoglobulin G immunoadhesin. One highlyreactive hybridoma clone, mc8C5, was subcloned and tested inimmunofluorescence, immunoprecipitation (IP), and Western blottingassays. In all three assays, mc8C5-4 subgroup-specifically recognizesSR-A Env, through the SU domain, expressed from different vectorsin both avian and mammalian cells. This multifunctionality isnotable for a mouse monoclonal. We furthermore observed a preferencefor binding to terminally glycosylated Env over core-glycosylatedEnv precursor in IPs, suggesting that the epitope is at leastpartially conformational and dependent on glycosylation. Mostimportantly, we found mc8C5-4 inhib-ited Env function: in vitro,the monoclonal not only interferes with binding of the EnvAreceptor, Tva, but it also blocks the Tva-induced conformationalchange required for activation of the fusion peptide, withoutinducing that change itself. Infection of Tva-expressing avianor mammalian cells by avian sarcoma and leukosis virus (ASLV)or EnvA-pseudotyped murine leukemia virus, respectively, isefficiently inhibited by mc8C5-4. The apparent interferenceof the monoclonal with the EnvA-Tva complex formation suggeststhat the epitope seen by mc8C5 overlaps with the receptor bindingsite. This is supported by the observa-tion that mutations ofbasic residues in hr2 or of the downstream glycosylation site,which both impair Tva-binding to EnvA, have similar effectson the binding of mc8C5. Thus, anti-ASLV-SU-A mc8C5-4 provesto be a unique new immunoreagent that targets receptor-bindingsite on a prototypical retroviral envelope.

5.71           Bacteriophage PM2 has a protein capsid surrounding a spherical proteinaceous lipid core

Kivelä, H.M., Kalkkinen, N. and Bamford, D.H.

1. Virol., 76(16), 8169-8178 (2002)

  The marine double-stranded DNA (dsDNA) bacteriophage PM2, studied since 1968, is the type organism of the family Corticoviridae, infecting two gram-negative Pseudoalteromonas species. The virion contains a membrane underneath an icosahedral protein capsid composed of two structural proteins. The purified major capsid protein, P2, appears as a trimer, and the receptor binding protein, P1, appears as a monomer. The C-terminal part of P1 is distal and is responsible for receptor binding activity. The rest of the structural proteins are associated with the internal phospholipid membrane enclosing the viral genome. This internal particle is designated the lipid core. The overall structural organization of phage PM2 resembles that of dsDNA bacteriophage PRD1, the type organism of the family Tectiviridae.  

5.72           The block in assembly of modified vaccinia virus Ankara in HeLa cells reveals new insights into vaccinia virus morphogenesis

Sancho, M.C., Schleich, S., Griffiths, G. and Krijinse-Locker, J

1. Virol., 76, 8318-8334 (2002)

  It has previously been shown that upon infection of HeLa cellswith modified vaccinia virus Ankara (MVA), assembly is blockedat a late stage of infection and immature virions (IVs) accumulate(G. Sutter and B. Moss, Proc. Natl. Acad. Sci. USA 89:10847-10851,1992). In the present study the morphogenesis of MVA in HeLacells was studied in more detail and compared to that undertwo conditions that permit the production of infectious particles:infection of HeLa cells with the WR strain of vaccinia virus(VV) and infection of BHK cells with MVA. Using several quantitativeand qualitative assays, we show that early in infection, MVAin HeLa cells behaves in a manner identical to that under thepermissive conditions. By immunofluorescence microscopy (IF)at late times of infection, the labelings for an abundant membraneprotein of the intracellular mature virus, p16/A14L, and theviral DNA colocalize under permissive conditions, whereas inHeLa cells infected with MVA these two structures do not colocalizeto the same extent. In both permissive and nonpermissive infection,p16-labeled IVs first appear at 5 h postinfection. In HeLa cellsinfected with MVA, IVs accumulated predominantly outside theDNA regions, whereas under permissive conditions they were associatedwith the viral DNA. At 4 h 30 min, the earliest time at whichp16 is detected, the p16 labeling was found predominantly ina small number of distinct puncta by IF, which were distinctfrom the sites of DNA in both permissive and nonpermissive infection.By electron microscopy, no crescents or IVs were found at thistime, and the p16-labeled structures were found to consist ofmembrane-rich vesicles that were in continuity with the cellularendoplasmic reticulum. Over the next 30 min of infection, alarge number of p16-labeled crescents and IVs appeared abruptlyunder both permissive and nonpermissive conditions. Under permissiveconditions, these IVs were in close association with the sitesof DNA, and a significant amount of these IVs engulfed the viralDNA. In contrast, under nonpermissive conditions, the IVs andDNA were mostly in separate locations and relatively few IVsacquired DNA. Our data show that in HeLa cells MVA forms normalDNA replication sites and normal viral precursor membranes butthe transport between these two structures is inhibited.  

5.73           Targeted transgene insertion into human chromosomes by adeno-associated virus vectors

Hirata, R., Chamberlain, J., Dong, R. and Russell, D.W. Nature Biotech., 20, 735-738 (2002)   Efficient methods are needed for the precise genetic manipulation of diploid human cells, in which cellular senescence and low conventional gene targeting rates limit experimental and therapeutic options. We have shown previously that linear, single-stranded DNA vectors based on adeno-associated virus (AAV) could accurately introduce small (<20 bp) genetic modifications into homologous human chromosomal sequences^1-4. Here we have used AAV vectors to introduce large (>1 kb) functional transgene cassettes into the hypoxanthine phosphoribosyl transferase (HPRT) and Type I collagen (COL1A1) loci in normal human fibroblasts. The transgene cassettes are inserted at high frequencies (1% of the total cell population under optimal conditions) and without secondary mutations. Selection for the inserted transgene cassette can be used to enrich for targeting events, such that >70% of surviving cells have undergone gene targeting with an appropriately designed vector. This approach should prove useful both for functional genomic analysis in diploid human cells and for therapeutic gene targeting.  

5.74           Efficient gene transfer of CD40 ligand into primary B-CLL cells using recombinant adeno-associated virus (rAAV) vectors

Wendtner, C-W. et al Blood, 100(5), 1655-1661 (2002)   B cells of chronic lymphocytic leukemia (B-CLL) are resistant to transduction with most currently available vector systems. Using an optimized adenovirus-free packaging system, recombinant adeno-associated virus (rAAV) vectors coding for the enhanced green fluorescent protein (AAV/EGFP) and CD40 ligand (AAV/CD40L) were packaged and highly purified resulting in genomic titers up to 3 × 10¹¹/mL. Cells obtained from 24 patients with B-CLL were infected with AAV/EGFP or AAV/CD40L at a multiplicity of infection (MOI) of 100 resulting in transgene expression in up to 97% of cells as detected by flow cytometry 48 hours after infection. Viral transduction could be specifically blocked by heparin. Transduction with AAV/CD40L resulted in up-regulation of the costimulatory molecule CD80 not only on infected CLL cells but also on noninfected bystander leukemia B cells, whereas this effect induced specific proliferation of HLA-matched allogeneic T cells. Vaccination strategies for patients with B-CLL using leukemia cells infected ex vivo by rAAV vectors now seems possible in the near future.  

5.75           Leptin-induced leptin resistance reveals separate roles for the anorexic and thermogenic responses in weight maintenance

Scarpace, P.J. et al Endocrinology, 143(8), 3026-3035 (2002)   The purpose of this study was to determine whether leptin inducesleptin resistance by examining the temporal attenuation of theanorexic and energy expenditure responses to leptin. We administeredrecombinant adeno-associated virus encoding rat leptin cDNAor control viral vector into mildly obese rats for 138 d andcompared these results with those from pair-fed rats. We measuredfood consumption, body weight, oxygen consumption, leptin signaltransduction, and brown adipose tissue uncoupling protein 1.The anorexic response attenuated by d 25, whereas the increasein energy expenditure persisted for 83 d before attenuating.Despite attenuation of physiological responses, phosphorylatedsignal transducer and activator of transcription-3 remainedelevated for the duration of the study. The temporal differentialattenuation of the anorexic and thermogenic responses allowedus to determine the relative contributions of each responseto weight maintenance. The anorexic response predominantly mediatedthe initial loss of body weight, but only the energy expenditureresponse was necessary to maintain the reduced weight. Thisstudy provides evidence that leptin induces leptin resistance.The leptin resistance was associated with persistent elevationin hypothalamic phosphorylated signal transducer and activatorof transcription-3 and was characterized by a rapid attenuationof the anorexic response and slower onset for the attenuationof the energy expenditure response. We propose that both elevatedleptin and obesity may be necessary for the development of leptinresistance.  

5.76           Transduction of human and mouse pancreatic islet cells using a bicistronic recombinant adeno-associated viral vector

Kapturczak, M. et al Mol. Ther., 5(2), 154-160 (2002)   Recent reports indicate successful transduction of pancreatic islets using recombinant adeno-associated viral (rAAV) vectors. This advance offers new possibilities in rendering islets resistant to rejection and recurrence of autoimmune destruction in the setting of islet transplantation as treatment of type 1 diabetes. Most gene delivery approaches using islets have thus far involved transduction with a single gene. However, the concomitant delivery of more than one gene encoding cytoprotective and/or immunoregulatory molecules may offer superior clinical utility. Here, we have generated a bicistronic rAAV (serotype 2) vector incorporating a viral internal ribosome entry site (IRES), derived from polio virus type 1, to allow for translation of two coupled cDNAs from a single mRNA transcript. Our study demonstrates the ability of this vector to produce significant expression of two reporter proteins in human and mouse islets in vitro. This expression did not interfere with beta-cell function. Transduction was maintained in vivo following transplantation of mouse islets. These data are the first report of efficient islet cell transduction with two genes using a single bicistronic rAAV vector and have direct implications for strategies aimed at enhancing islet transplant survival.  

5.77           The assembly of Ebola virus nucleocapsid requires virion-associated proteins 35 and 24 and posttranslational modification of nucleoprotein

Huang, Y. et al Mol. Cell, 10, 307-316 (2002)   Ebola virus encodes seven viral structural and regulatory proteins that support its high rates of replication, but little is known about nucleocapsid assembly of this virus in infected cells. We report here that three viral proteins are necessary and sufficient for formation of Ebola virus particles and that intracellular posttranslational modification regulates this process. Expression of the nucleoprotein (NP) and virion-associated proteins VP35 and VP24 led to spontaneous assembly of nucleocapsids in transfected 293T cells by transmission electron microscopy. A specific biochemical interaction of these three proteins was demonstrated, and, interestingly, O-glycosylation and sialation of NP were demonstrated and necessary for their association. This distinct mechanism of regulation for filovirus assembly suggests new approaches for viral therapies and vaccines for Ebola and related viruses.  

5.78           Expression of lecithin cholesterol acyltransferase and/or apoA-I mediated by recombinant adeno-associated virus in myogenic cells

Wang, L.F., Fan, L.M., Chen, B.R., Wang, R.N. and Wei, E.H. Acta Biochim et Biophys Sinica, 34(1), 33-38 (2002)   Lecithin cholesterol acyltransferase (LCAT) is the major enzyme producing most plasma cholesterol esters(CE) and a key participant in the process of reverse cholesterol transfer (RCT). The aim of this research is to co-express LCAT and it's natural activator apoA-I, with the recombinant adeno-associated virus vectors in the skeletal muscle cells, in order to pave a new way for gene therapy of the primary or secondary LCAT deficiency. 293T cells was cotransfected with pDG and rAAVAIL/rAAVL plasmids to produce infectious rAAV, and non-ionic iodixanol gradient centrifugation, followed by heparin affinity chromatography, were performed for separation, purification and concentration of rAAV. The particle numbers of rAAV, assayed by dot blot, were 7 x 10¹⁴/L (rAAVAIL) and 1 x 10¹⁴/L (rAAVL). These vectors were then transduced into C2C12 myoblasts. The results of ELISA and Western blot for human apoA-I, and [3H]-cholesterol-labeled radiochemical methods for LCAT activity, showed that the expression of human apoA-I cDNA and/or human LCAT cDNA in transduced C2C12 cells lasted for 30 days, even after myoblasts were differentiated into myotubes. PCR products for the transgene indicated the long-term persistence of transduced vector sequences. The results indicate that the methods used for production and purification of rAAV is efficient, and rAAV vector mediated the expression and secretion of LCAT and apoA-I gene in C2C12 myoblasts successfully. It suggests that the use of rAAV vectors mediating the high efficiency, long-term expression of human LCAT cDNA and/or apoA-I cDNA in skeletal muscle in vivo can be a safe and fesible strategy for the gene therapy of LCAT deficiency.  

5.79           Viral vector-mediated gene expression in olfactory ensheathing glia implants in the lesioned rat spinal cord

Ruitenberg, M.J. et al Gen. Ther., 9, 135-146 (2002)   Implantation of olfactory ensheathing glia (OEG) is a promising strategy to augment long-distance regeneration in the injured spinal cord. In this study, implantation of OEG following unilateral hemisection of the dorsal cervical spinal cord was combined with ex vivo gene transfer techniques. We report, to our knowledge for the first time, that purified cultures of primary OEG are capable of expressing a foreign gene following adenoviral (AdV) and lentiviral (LV) vector-mediated gene transfer. OEG implants subjected to AdV vector-mediated gene transfer expressed high levels of transgenic protein in both intact and lesioned spinal cord at 7 days after implantation. However, the levels of transgene expression gradually declined between 7 and 30 days after implantation in lesioned spinal cord. Infection with LV vectors resulted in stable transduction of primary OEG cultures and transgene expression persisted for at least 4 months after implantation. Genetic engineering of OEG opens the possibility of expressing additional neurotrophic genes and create optimal 'bridging' substrates to support spinal axon regeneration. Furthermore, stable transduction of OEG allows us to reliably study the behaviour of implanted cells and to obtain better understanding of their regeneration supporting properties.  

5.80           Adeno-associated virus-mediated gene transfer of endostatin inhibits angiogenesis and tumor growth in vivo

Shi, W., Teschendorf, C., Muzyczka, N. and Siemann, D. Can. Gen. Ther., 9, 513-521 (2002)   A variety of approaches has demonstrated that interfering with tumor-induced angiogenesis may be an effective strategy in cancer therapy. However, it is likely that to be most effective such strategies will require extended suppression of the angiogenic process. Gene therapy offers a possible approach to achieve sustained release of a therapeutically potent transferred gene product. In the present study the angiogenesis inhibitor endostatin was expressed through a recombinant adeno-associated viral (rAAV) vector and shown to be biologically active in vitro and in vivo. Intramuscular injection of rAAV – HuEndo (1x10⁹ i.u.) led to a sustained serum both in the initiation and subsequent growth of a human colorectal cancer model.  

5.81           Sustained hepatic and renal glucose-6-phosphatase expression corrects glycogen storage disease type Ia in mice

Sun, M-S. et al Human Mol. Gen., 11(18), 2155-2164 (2002)   Deficiency of glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis, causes glycogen storage disease type Ia (GSD-Ia), an autosomal recessive disorder characterized by growth retardation, hypoglycemia, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. G6Pase is an endoplasmic reticulum-associated transmembrane protein expressed primarily in the liver and the kidney. Therefore, enzyme replacement therapy is not feasible using current strategies, but somatic gene therapy, targeting G6Pase to the liver and the kidney, is an attractive possibility. Previously, we reported the development of a mouse model of G6Pase deficiency that closely mimics human GSD-Ia. Using neonatal GSD-Ia mice, we now demonstrate that a combined adeno virus and adeno-associated virus vector-mediated gene transfer leads to sustained G6Pase expression in both the liver and the kidney and corrects the murine GSD-Ia disease for at least 12 months. Our results suggest that human GSD-Ia would be treatable by gene therapy.  

5.82           Amelioration of chronic neuropathic pain after partial nerve injury by adeno-associated viral (AVV) vector-mediated over-expression of BDNF in the rat spinal cord

Eaton, M.J., Blits, B., Ruitenberg, M.J., Verhaagen, J. and Oudega, M. Gene Therapy, 9, 1387-1395 (2002)   Changing the levels of neurotrophins in the spinal cord micro-environment after nervous system injury has been proposed to recover normal function, such that behavioral response to peripheral stimuli does not lead to chronic pain. We have investigated the effects of recombinant adeno-associated viral (rAAV)-mediated over-expression of brain-derived neurotrophic factor (BDNF) in the spinal cord on chronic neuropathic pain after unilateral chronic constriction injury (CCI) of the sciatic nerve. The rAAV-BDNF vector was injected into the dorsal horn at the thirteenth thoracic spinal cord vertebra (L₁ level) 1 week after CCI. Allodynia and hyperalgesia induced by CCI in the hindpaws were permanently reversed, beginning 1 week after vector injection, compared with a similar injection of a control rAAV-GFP vector (green fluorescent protein) or saline. In situ hybridization for BDNF demonstrated that both dorsal and ventral lumbar spinal neurons contained an intense signal for BDNF mRNA, at 1 to 8 weeks after vector injection. There was no similar BDNF mRNA over-expression associated with either injections of saline or rAAV-GFP. These data suggest that chronic neuropathic pain is sensitive to early spinal BDNF levels after partial nerve injury and that rAAV-mediated gene transfer could potentially be used to reverse chronic pain after nervous system injuries in humans.  

5.83           Intravitreal injection of adeno-associated viral vectors results in the transduction of different types of retinal neurons in neonatal and adult rats: A comparison with Lentivirus vectors

Harvey, A.R. et al Mol. Cell. Neurosci., 21, 141-157 (2002)   Replication-deficient viral vectors encoding the marker gene green fluorescent protein (GFP) were injected into the vitreous of newborn, juvenile (P14), and adult rats. We tested two different types of modified virus: adeno-associated viral-2-GFP (AAV-GFP) and lentiviral-GFP vectors (LV-GFP). The extent of retinal cell transduction in different-aged animals was compared 7, 21, and 70 days after eye injections. At all postinjection times, LV-GFP transduction was mostly limited to pigment epithelium and cells in sclera and choroid. In contrast, transduction of large numbers of neural retinal cells was seen 21 and 70 days after AAV-GFP injections. AAV-GFP predominantly transduced neurons, although GFP-positive Müller cells were seen. All neuronal classes were labeled, but the extent of transduction for a given class varied depending on injection age. After P0 injections about 50% of transduced cells were photoreceptors and 30-40% were amacrine or bipolar cells. After adult injections 60-70% of transduced cells were retinal ganglion cells. In adults many GFP-positive retinal axons were traced through the optic nerve/tract and terminal arbors were visualized in central targets.  

5.84           Fast and reliable titration of recombinant adeno-associated virus type-2 using quantitative real-time PCR

Rohr, U-P. et al

1. Virol. Meth., 106, 81-88 (2002)

  In this study, a quantitative real-time PCR (qPCR) was developed to determine genomic rAAV-2 titers using the Light-Cycler technology. Since the CMV promoter is the most commonly used promoter in gene therapeutic approaches, primers were designed which hybridize with the human CMV promoter sequence. PCR products were detected by the addition of SYBR green. qPCR of a 5 log spanning serial dilution of the vector plasmid containing one CMV promoter per plasmid molecule yielded a high amplification efficiency of 1.99 per cycle. To quantify the copy number of viral genomes, the qPCR curves of adeno-associated virus type 2 (AAV-2) samples were related to a standard curve assessed by the 5 log spanning serial vector plasmid dilution (0.01-100 pg DNA). For validation of the method, rAAV-2 preparations were analyzed by a standard method and qPCR in parallel. As standard method, flow cytometry was used for titration of infectious viral particles on HeLa cells using the Enhanced Green Fluorescent Protein as a marker. A significant correlation was found between the results obtained by flow cytometry and the results from the qPCR over a 5 log range (r=0.85, P<0.0001). The mean ratio between infectious rAAV-2 particles titrated via flow cytometry and genomic copies of rAAV-2 measured by qPCR of the same sample was 1:253. The higher titers found by qPCR might be due to multiple transduction of a single cell or to non-infectious particles generated during rAAV-2 preparation. In conclusion, qPCR is a fast and reliable method for determination of rAAV-2 titers and might be a powerful tool for standardization of rAAV-2 preparations particularly in the context of clinical studies.  

5.85           Use of HSV vectors to modify the nervous system

Glorioso, J.C and Fink, D.J. Curr. Opinion in Drug Discovery & Development, 5(2), (2002)   Herpes simplex virus (HSV) is a natural human pathogen that efficiently infects sensory neurons to establish a life-long latent state. Recombinant replication-defective vectors, created by disruption of critical viral gene functions, nonetheless target neurons and can be used to express transgenes to alter the structure and/or function of the nervous system. Specific applications of these vectors to models of neurodegeneration (Parkinson’s disease), trauma, (spinal root avulsion), peripheral neuropathy and neuronal function (pain) have been published within the last year. With these applications and the clinical experience in human tumor trials with HSV vectors, the stage is set for the use of HSV-based vectors to treat neurologic disease in humans in the near future.  

5.86           Adenovirus-facilitated nuclear translocation of adeno-associated virus type 2

Xiao, W., Warrington, K.H., Hearing, P-. Hughes, J. and Muzyczka, N.

1. Virol., 76, 11505-11517 (2002)

  We examined cytoplasmic trafficking and nuclear translocation of adeno-associated virus type 2 (AAV) by using Alexa Fluor 488-conjugated wild-type AAV, A20 monoclonal antibody immunocytochemistry, and subcellular fractionation techniques followed by DNA hybridization. Our results indicated that in the absence of adenovirus (Ad), AAV enters the cell rapidly and escapes from early endosomes with a t_1/2 of about 10 min postinfection. Cytoplasmically distributed AAV accumulated around the nucleus and persisted perinuclearly for 16 to 24 h. Viral uncoating occurred before or during nuclear entry beginning about 12 h postinfection, when viral protein and DNA were readily detected in the nucleus. Few, if any, intact AAV capsids were found in the nucleus. In the presence of Ad, however, cytoplasmic AAV quickly translocated into the nucleus as intact particles as early as 40 min after coinfection, and this facilitated nuclear translocation of AAV was not blocked by the nuclear pore complex inhibitor thapsigargan. The rapid nuclear translocation of intact AAV capsids in the presence of Ad suggested that one or more Ad capsid proteins might be altering trafficking. Indeed, coinfection with empty Ad capsids also resulted in the appearance of AAV DNA in nuclei within 40 min. Escape from early endosomes did not seem to be affected by Ad coinfection.  

5.87           Cell-type-specific characteristics modulate the transduction efficiency of adeno-associated virus type 2 and restrain infection of endothelial cells

Pajusola, K. et al

1. Virol., 76, 11530-11540 (2002)

  Adeno-associated viruses (AAVs) are promising vectors for variousgene therapy applications due to their long-lasting transgeneexpression and wide spectrum of target cells. Recently, however,it has become apparent that there are considerable differencesin the efficiencies of transduction of different cell typesby AAVs. Here, we analyzed the efficiencies of transductionand the transport mechanisms of AAV type 2 (AAV-2) in differentcell types, emphasizing endothelial cells. Expression analysesin both cultured cells and the rabbit carotid artery assay showeda remarkably low level of endothelial cell transduction in comparisonto the highly permissive cell types. The study of the endosomalpathways of AAV-2 with fluorescently labeled virus showed cleartargeting of the Golgi area in permissive cell lines, but thisphenomenon was absent in the endothelial cell line EAhy-926.On the other hand, the response to the block of endosomal acidificationby bafilomycin A1 also showed differences among the permissivecell types. We also analyzed the effect of proteasome inhibitorson endothelial cells, but their impact on the primary cellsand in vivo was not significant. On the contrary, analysis ofthe expression pattern of heparan sulfate proteoglycans (HSPGs),the primary receptors of AAV-2, revealed massive deposits ofHSPG in the extracellular matrix of endothelial cells. The matrix-associatedreceptors may therefore compete for virus binding and reducetransduction in endothelial cells. Accordingly, in endothelialcells detached from their matrix, AAV-2 transduction was significantlyincreased. Altogether, these results point to a more complexcell-type-specific mode of transduction of AAV-2 than previouslyappreciated.  

5.88           Evidence for the existence of distinct central appetite, energy expenditure, and ghrelin stimulation pathways as revealed by hypothalamic site-specific leptin gene therapy

Bagnasco, M, Dube, M.G., Kalra, P.S. and Kalra, S.P. Endocrinology, 143, 4409-4421 (2002)   To identify the specific hypothalamic sites in which leptin acts to decrease energy intake and/or increase energy expenditure, recombinant adeno-associated virus vector-encoding leptin was microinjected bilaterally into one of four hypothalamic sites in female rats. Leptin transgene expression in the ventromedial  nucleus and paraventricular nucleus induced comparable decreases in daily food intake (FI; 18–20%) and body weight (BW; 26–29%), accompanied by drastic reductions in serum leptin (81–97%), insulin (92–93%), free fatty acids (35–36%), and normoglycemia. Leptin transgene expression in the arcuate nucleus (ARC) decreased BW gain (21%) and FI (11%) to a lesser range, but the metabolic hormones were suppressed to the same extent. Leptin transgene expression in the medial preoptic area (MPOA) decreased BW and metabolic hormones without decreasing FI. Finally, leptin transgene expression in all four sites augmented serum ghrelin and thermogenic energy expenditure, as shown by uncoupling protein-1 mRNA expression in brown adipose tissue. Proopiomelanocortin gene expression in the ARC was up-regulated by leptin expression in all four sites, but neuropeptide Y gene expression in the ARC was suppressed by leptin transgene expression in the ARC but not in the MPOA. Thus, whereas leptin expression in the paraventricular nucleus, ventromedial nucleus, or ARC suppresses adiposity and insulin by decreasing energy intake and increasing energy expenditure, in the MPOA it suppresses these variables by increasing energy expenditure alone.  

5.89           Adaptation of laboratory grade recombinant AAV production to manufacture of vector for human administration

De, B., Mendez, B., Hackett, N.R., Kaminsky, S.M. and Crystal, R.G. Abstract, 5th Annual Meeting of the American Society of Gene Therapy (Academic Press) no. 147 (2002)   Gene transfer using AAV2 vectors is applicable to the treatment of human genetic disease due to the persistent expression of the transgene in many organs. However, laboratory grade AAV2 production uses a number of protocols that are not practical to scale to large batch size for vector in quantities relevant to human applications, and the process is difficult to adapt to Good Manufacturing Practice. The objective of this work is the transition of laboratory grade AAV2 vector production to GMP production including the identification and control of possible sources of contaminants, identification of critical control parameters in the production process and demonstrating that the overall process is robust. The simplest AAV2 production method was deemed to be the two plasmid cotransfection method in which the recombinant AAV2 plasmid consisting of the expression cassette for the therapeutic gene between the AAV2 inverted terminal repeats is contransfected with a helper plasmid providing the AAV2 rep and cap proteins driven by the mouse mammary tumor virus (MMTV) promoter and the Ad E2, E4 and VA genes driven by their own promoter. Two different GMP certified 293 derived cell lines were assessed and shown to produce similar yields of recombinant AAV2 vector (by anti-capsid ELISA) after cotransfection by either the CaPO₄ or Polyfect (QIAGEN) method. The rAAV yield was limited by the amount and purity of the plasmids used for cotransfection and yield increased in linear manner with plasmid quantity from 0.4 to 16 mg per 15 cm plate of cells. The optimal purification method consisted of making a crude viral lysate by freeze thaw followed by a discontinuous iodixanol gradient. Isolated rAAV is affinity purified using a salt gradient on a heparin agarose column and desalted by spin gel filtration. The discontinuous iodixanol gradient was optimized for separation of infectious rAAV (assessed by gene transduction) relative to empty AAV2 capsids. Excluding deoxycholate from the purification process, to avoid testing for residual deoxycholate in the product, was assessed, but high purity product was not obtained. On that basis, several batches of rAAV expressing a variety of different reporter genes and therapeutic genes have been produced. Purity assessed by SDS-PAGE was >95% and average yield (n=34 preparations) was 3.9x10³ particle units by ELISA per transfected cell. Preparations were also assessed for genome content by TaqMan realtime quantitative PCR using primers and probes specific to the promoter and therefore applicable to a number of different vectors. Electron microscopy provides an independent measure of the relative abundance of empty capsid particles and a general measure of the quality of the lot. These data show the existence of a robust process for rAAV production in which critical production parameters have been identified, raw materials identified and a batch record written based on an extensive series of laboratory grade preparations.  

5.90           Dopaminergic cell loss induced by human A30P a-synuclein gene transfer to the rat substantia nigra

Klein, R.L., King, M.A:, Hamby, M.E. and Meyer, E.M. Hum. Gen Ther., 13, 605-612 (2002)   Somatic cell gene transfer was used to express a mutant form of α-synuclein (α-syn) that is associated with Parkinson's disease (PD) in the rat substantia nigra (SN), a brain region that, in humans, degenerates during PD. DNA encoding the A30P mutant of human α-syn linked to familial PD was incorporated into an adeno-associated virus vector, which was injected into the adult rat midbrain. The cytomegalovirus/chicken β-actin promoter was used to drive transgene expression. Over a 1-year time course, this treatment produced three significant features relevant to PD: (1) accumulation of α-syn in SN neuron perikarya, (2) Lewy-like dystrophic neurites in the SN and the striatum, and (3) a 53% loss of SN dopamine neurons. However, motor dysfunction was not found in either rotational or rotating rod testing. The lack of behavioral deficits, despite the significant cell loss, may reflect pathogenesis similar to that of PD, where greater than 50% losses occur before motor behavior is affected.  

5.91           Visualization of the intracellular behavior of HIV in living cells

McDonald, D. et al

1. Cell Biol., 159, 441-452 (2002)

  To track the behavior of human immunodeficiency virus (HIV)-1 in the cytoplasm of infected cells, we have tagged virions by incorporation of HIV Vpr fused to the GFP. Observation of the GFP-labeled particles in living cells revealed that they moved in curvilinear paths in the cytoplasm and accumulated in the perinuclear region, often near the microtubule-organizing center. Further studies show that HIV uses cytoplasmic dynein and the microtubule network to migrate toward the nucleus. By combining GFP fused to the NH₂ terminus of HIV-1 Vpr tagging with other labeling techniques, it was possible to determine the state of progression of individual particles through the viral life cycle. Correlation of immunofluorescent and electron micrographs allowed high resolution imaging of microtubule-associated structures that are proposed to be reverse transcription complexes. Based on these observations, we propose that HIV uses dynein and the microtubule network to facilitate the delivery of the viral genome to the nucleus of the cell during early postentry steps of the HIV life cycle.  

5.92           Production and purification of serotype 1,2, and 5 recombinant adeno-associated viral vectors

Zolotukhin, S. et al Methods, 28, 158-167 (2002)   Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1×10¹² to 1×10¹³ vector genomes/ml.  

5.93           Adeno-associated viral vectors as agents for gene delivery: application in disorders and trauma of the central nervous system

Ruitenberg, M.J., Eggers, R., Boer, G.J. and Verhaagen, J. Methods, 28, 182-194 (2002)   The use of viral vectors as agents for gene delivery provides a direct approach to manipulate gene expression in the mammalian central nervous system (CNS). The present article describes in detail the methodology for the injection of viral vectors, in particular adeno-associated virus (AAV) vectors, into the adult rat brain and spinal cord to obtain reproducible and successful transduction of neural tissue. Surgical and injection procedures are based on the extensive experience of our laboratory to deliver viral vectors to the adult rat CNS and have been optimized over the years. First, a brief overview is presented on the use and potential of viral vectors to treat neurological disorders or trauma of the CNS. Next, methods to deliver AAV vectors to the rat brain and spinal cord are described in great detail with the intent of providing a practical guide to potential users. Finally, some data on the experimental outcomes following AAV vector-mediated gene transfer to the adult rat CNS are presented as is a brief discussion on both the advantages and limitations of AAV vectors as tools for somatic gene transfer.  

5.94           Recombinant adeno-associated virus vector design and gene expression in the mammalian brain

Paterna, J-C. and Büeler, H. Methods, 28, 208-218 (2002)   Efficiency and stability of recombinant adeno-associated virus (rAAV)-mediated gene expression within the mammalian brain are determined by several factors. These include the dose of infectious particles, the purity of the vector stock, the serotype of rAAV, the route of administration, and the intrinsic properties, most notably the rAAV receptor density, of the targeted area. Furthermore, the choice of appropriate regulatory elements in rAAV vector design is of fundamental importance to achieve high-level sustained in vivo transcription and translation. This review summarizes the characteristics of various transcriptional and posttranscriptional regulatory elements, and highlights their influence on the expression performance of rAAV vectors in the mammalian brain.  

5.95           Autonomous parvovirus vectors

Maxwell, I.H., Terrell, K.L. and Maxwell, F. Methods, 28, 168-181 (2002)   Parvoviruses are small, icosahedral viruses (»25 nm) containing a single-strand DNA genome (»5 kb) with hairpin termini. Autonomous parvoviruses (APVs) are found in many species; they do not require a helper virus for replication but they do require proliferating cells (S-phase functions) and, in some cases, tissue-specific factors. APVs can protect animals from spontaneous or experimental tumors, leading to consideration of these viruses, and vectors derived from them, as anticancer agents. Vector development has focused on three rodent APVs that can infect human cells, namely, LuIII, MVM, and H1. LuIII-based vectors with complete replacement of the viral coding sequences can direct transient or persistent expression of transgenes in cell culture. MVM-based and H1-based vectors with substitution of transgenes for the viral capsid sequences retain viral nonstructural (NS) coding sequences and express the NS1 protein. The latter serves to amplify the vector genome in target cells, potentially contributing to antitumor activity. APV vectors have packaging capacity for foreign DNA of »4.8 kb, a limit that probably cannot be exceeded by more than a few percent. LuIII vectors can be pseudotyped with capsid proteins from related APVs, a promising strategy for controlling tissue tropism and circumventing immune responses to repeated administration. Initial success has been achieved in targeting such a pseudotyped vector by genetic modification of the capsid. Subject to advances in production and purification methods, APV vectors have potential as gene transfer agents for experimental and therapeutic use, particularly for cancer therapy.  

5.96           Production of recombinant H1 parvovirus stocks devoid of replication-competent viruses

Brown, C.S. et al Human Gen. Ther., 13, 2135-2145 (2002)   Vector and helper plasmids for the production of recombinant H1 (rH1) parvovirus, an oncolytic virus and candidate vector for cancer gene therapy, were constructed with the aim of reducing the contamination of these preparations with replication-competent viruses (RCV). Split-helper plasmids were constructed by manipulating the splicing signals for the capsid proteins such that VP1 and VP2 were expressed from separate plasmids. H1 vectors with similarly mutated splice sites were packaged, using the split-helper plasmids, and the resulting recombinant H1 viruses were completely free of RCV because the generation of recombinants expressing both capsid proteins was prevented. Vector yields of rH1 produced with split-helper plasmids in combination with splice site-modified vectors were similar (in the range of 107 replication units/ml) to yields of rH1 produced with the standard vector/helper pair, in which case significant levels of RCV were generated (104-105 plaque-forming units/ml). To assess the functionality of this approach in vivo, rH1 was produced that contained the human interleukin 2 (IL-2) transgene and that was devoid of RCV. This IL-2-carrying rH1 vector expressed IL-2 efficiently in human tumor cells (HeLa) in vitro and generated antitumor responses in nude mice xenografted with HeLa cells that had been infected ex vivo with this virus. These results should allow the large-scale production of recombinant oncotropic parvoviruses and their assessment for the gene therapy of cancer in a clinical setting.  

5.97           Adeno-associated virus vectors for gene transfer to the brain

Okada, T. et al Methods, 28, 237-247 (2002) Gene therapy is a novel method under investigation for the treatment of neurological disorders. Considerable interest has focused on the possibility of using viral vectors to deliver genes to the central nervous system. Adeno-associated virus (AAV) is a potentially useful gene transfer vehicle for neurologic gene therapies. The advantages of AAV vector include the lack of any associated disease with a wild-type virus, the ability to transduce nondividing cells, the possible integration of the gene into the host genome, and the long-term expression of transgenes. The development of novel therapeutic strategies for  neuro-logical disorder by using AAV vector has an increasing impact on gene therapy research. This article describes methods that can be used to generate rodent and nonhuman primate models for testing treatment strategies linked to pathophysiological events in the ischemic brain and neurodegenerative disorders such as Parkinson’s disease.  

5.98           Recent advances in recombinant adeno-associated virus vector production

1. R. Clark

Kidney Int., 61, Symposium 1, S9-S15 (2002)   Adeno-associated virus (AAV) is a replication-defective parvovirus that is being developed as a vector for human gene transfer. Recombinant AAV (rAAV) vectors are being proposed as a gene transfer vehicle for an array of human diseases. The recent interest in rAAV has been driven by the unexpected finding that these simple vectors can efficiently transduce a variety of postmitotic cells, resulting in long-lived, robust gene expression. However, a major obstacle to common­place usage of rAAV vectors was the production in sufficient quantities for preclinical and human trials. Fortunately, several recent technological advances in vector production, purifica­tion, and titration have resulted in significant increases (>10-fold) in production capacity. Thus, there are several methods for the production of rAAV in excess of 10⁴ particles/cell, levels that should permit widespread use of this technology for clinical applications.    

5.99           Neonatal porcine pancreatic cell clusters as a potential source for transplantation in humans: characterization of proliferation, apoptosis, xenoantigen expression and gene delivery with recombinant AAV

Vizzardelli, C. et al Xenotransplant. 9, 14-24 (2002)   Neonatal porcine islets are characterized by reproducible isolation success and high yields, sizable advantages over adult islets. In this work we have analyzed selected phenotypic and functional characteristics of porcine neonatal islets relevant to their possible use for transplant in humans. We show that porcine islet cells proliferate in culture, and synthesize and store islet-specific hormones. Proliferating beta cells can be easily identified. Implant of cultured neonatal islets in immunodeficient rodents results in the reversal of diabetes, albeit with delay. We also show that measurable apoptosis occurs in cultured neonatal porcine islets. Further, antigens recognized by human natural antibodies are expressed in a dynamic fashion over the culture period analyzed and are not limited to the alpha-Gal epitope. Lastly, we demonstrate that a recombinant Adeno-Associated virus can be used to efficiently deliver a reporter gene in porcine islets. This characterization might be helpful in the definition of the potential use of neonatal porcine islets for human transplantation.  

5.100           Tissue cultures from adult human postmortem subcortical brain areas

Verwer, R.W.H., Dubelaar, E.J.G., Hermens, W.T.J.M.C. and Swaab, D.F.

1. Cell. Mol. Med., 6, 429-432 (2002)

  Animal models used to study human aging and neurodegeneration do not display all symptoms of these processes as they are found in humans. Recently, we have shown that many cells in neocortical slices from adult human postmortem brain may survive for extensive periods in vitro. Such cultures may enable us to study age and disease related processes directly in human brain tissue. Here, we present observations on subcortical brain tissue.  

5.101           Virion-associated cholesterol is critical for the maintenance of HIV-1 structure and infectivity

Campbell, S. M., Crowe, S. M. and Mak, J. AIDS, 16, 2253-2261 (2002)   Objective:  HIV-l particles are enriched with cholesterol; however, the significance of this cholesterol enrichment is unknown. This study examines the structural and functional roles of cholesterol in HIV-1 replication. Methods:   Using methyl-b-cyclodextrin (CD) to remove cholesterol from the HIV-1 envelope, buoyant density and infectivity of the cholesterol-deficient HIV-1 particles were compared with the untreated control. The specificity and requirement of cholesterol as an HIV-1 -associated lipid were investigated by replenishing cholesterol-deficient HIV-1 with cholesterol, cholestenone (a cholesterol structural analogue) or sphingomyelin (a structurally unrelated yet virion-associated lipid). Results:     CD-mediated removal of virion cholesterol increased the buoyant density of virion particles and reduced HIV-1 infectivity. Trans-supplementation of exogenous cholesterol rescued the defects associated with CD-induced cholesterol depletion in HIV-1. However, the restoration of viral infectivity could not be achieved by trans­supplementation of either cholestenone or sphingomyelin. Conclusion: This study provides the first direct evidence that HIV-1 -associated choles­terol is important for the maintenance of virion structure and infectivity. While the buoyant density of cholesterol-defective HIV-1 can be restored by a cholesterol structural analogue, cholestenone, the requirement for cholesterol is essential for HIV­-1 infectivity.  

5.102           AAV2 vectors mediate efficient and sustained transduction of rat embryonic ventral mesencephalon

Lehtonen, E. et al Neuroreport, 13, 1503-1507 (2002)   The success of transplantation of human embryonic mesencephalic tissue to treat Parkinsonian patients is limited by the poor survival of the transplant. We show that an AAV2 vector mediates efficient expression of the egfp reporter gene in organotypic cultures of freshly explanted solid fragments of rat embryonic ventral mesencephalon (VM).We observed early and sustained transgene expression (4 days to ³6 weeks). Furthermore, rAAV-infected rat embryonic VM transplanted in the adult striatum continued toexpress EGFP for ³3 months. More than 95% of the transduced cells were neurons. Dopaminergic neurons were transduced at low frequency at earlier time points. This method of gene delivery could prove useful to achieve local, continuous secretion of neurotrophic factors at physiologically relevant doses to treat Parkinson’s disease.  

5.103           Phenotypic rescue after adeno-associated virus-mediated delivery of 4-sulfatase to the retinal pigment epithelium of feline mucopolysaccharidosis VI

Ho, T.T., Maguire, A.M., Aguirre, G.D., surface, E.M., Anand, V., Zeng, Y., Salvetti, A., Hopwood, J.J., Haskins, M.E. and Bennett, J.

1. Gene Med., 4(6), 613-621 (2002)

  Background Mucopolysaccharidosis VI (MPS VI), due to recessively inherited 4-sulfatase (4S) deficiency, results in lysosomal storage of dermatan sulfate in numerous tissues. Retinal involvement is limited to the retinal pigment epithelium (RPE). This study aimed to determine whether recombinant adeno-associated virus (AAV)-mediated delivery of 4S would reverse the RPE pathology seen in MPS VI cats. Methods AAV.f4S, containing the feline 4S cDNA, was delivered unilaterally to eyes of affected cats by subretinal or intravitreal injection. Contralateral eyes received AAV with the green fluorescent protein (GFP) reporter gene as control. At 2–11 months post-injection, the cats were sacrificed and the treatment effects were evaluated histologically. Results By ophthalmoscopy and histological analyses, GFP was evident as early as 4 weeks and persisted through the latest time point (11 months). Untreated and AAV.GFP-treated diseased retinas contained massively hypertrophied RPE cells secondary to accumulation of dilated lysosomal inclusions containing dermatan sulfate. MPS VI eyes treated subretinally with AAV.f4S had minimal RPE cell inclusions and, consequently, were not hypertrophied. Conclusions AAV-mediated subretinal delivery of f4S provided correction of the disease phenotype in RPE cells of feline MPS VI, supporting the utility of AAV as a vector for the treatment of RPE-specific as well as lysosomal storage diseases.  

5.104           Overexpression of Parkinson's disease-associated -Synuclein^A53T by recombinant adeno-associated virus in mice does not increase the vulnerability of dopaminergic neurons to MPTP

Dong, Z., Ferger, B., Feldon, J. and Büeler, H.

1. Neurobiol., 53(1), 1-10 (2002)

  Mutations in the -synuclein gene are linked to a rare dominant form of familial Parkinson's disease, and -synuclein is aggregated in Lewy bodies of both sporadic and dominant Parkinson's disease. It has been proposed that mutated -synuclein causes dopaminergic neuron loss by enhancing the vulnerability of these neurons to a variety of insults, including oxidative stress, apoptotic stimuli, and selective dopaminergic neurotoxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To test this hypothesis in vivo, we overexpressed human -synucleinA53T in the substantia nigra of normal and MPTP-treated mice by rAAV-mediated gene transfer. Determination of dopaminergic neuron survival, striatal tyrosine hydroxylase fiber density, and striatal content of dopamine and its metabolites in rAAV-injected and uninjected hemispheres demonstrated that -synucleinA53T does not increase the susceptibility of dopaminergic neurons to MPTP. Our findings argue against a direct detrimental role for (mutant) -synuclein in oxidative stress and/or apoptotic pathways triggered by MPTP, but do not rule out the possibility that -synuclein aggregation in neurons exposed to oxidative stress for long periods of time may be neurotoxic.  

5.105           High-Titer Stocks of Adeno-Associated Virus from Replicating Amplicons and Herpes Vectors

Mistry, A.R., De Alwis, M., Feudner, E., Ali, R.R. and Thrasher, A.J. Methods in Mol. Med., 69, 445-460 (2002)   The adeno-associated virus (AAV) is a nonpathogenic member of the Parvoviridae family (for review, see ref. 1) Recently this virus has gained considerable interest and has been developed as a gene delivery vector (2). Six primate AAV serotypes (designated AAV types 1–6) have so far been identified and characterized in the literature (3,4). The most extensively studied of these isolates is AAV type 2. The vast majority of the transduction studies have been carried out using recombinant vectors (rAAV) based on serotype 2. These studies have shown that rAAV2 has the ability to transduce a wide range of both dividing and nondividing cells, achieving efficient long-term gene expression in vivo in a variety of tissues including retina (5), muscle (6), central nervous system (7), and liver (8). The range of tissues transduced by recombinant AAV vectors based on other serotypes is currently being investigated by several laboratories (9). It is hoped that rAAV vectors produced from these serotypes may prove to be useful for the transduction of tissues that are poorly infected by AAV2. A major problem associated with the use of rAAV has been the difficulty in producing large quantities of high-titer stock (10,11). This has become an important issue as vectors based on rAAV2 have now reached the stage at which they are starting to be used in human clinical trials (12). This chapter describes the use of the herpes simplex virus type 1 (HSV-1) amplicons that have been developed in our laboratory to attain high-titer stocks of rAAV2 (13). A brief description of the background and basis of the system is given in the first section.  

5.106           Development of Replication-Defective Herpes Simplex Virus Vectors

Goins, W.F., Krisky, D.M., Wolfe, D.P., Fink, D.J. and Glorioso, J.C. Methods in Mol. Med., 69, 481-507 (2002)   A greater understanding of the molecular, biochemical, and genetic factors involved in the progression of a specific disease state has led to the development of genetic therapies using direct gene transfer to ameliorate the disease condition or correct a genetic defect in situ. Effective gene therapy approaches require delivery strategies and vehicles that 1) efficiently deliver the therapeutic gene(s) to a sufficient number of dividing or nondividing cells to achieve the desired therapeutic effect; 2) persist long term within the cell without disturbing host cell functions; and 3) can regulate the level and duration of therapeutic gene expression for diseases that may either require high-level transient transgene expression or continuous low-level synthesis of the therapeutic product. Numerous viral and nonviral vectors have been employed to treat a variety of genetic and acquired diseases. Each vector system has its own particular advantages and disadvantages that will suit it to a specific therapeutic application. Herpes simplex virus type 1 (HSV-1) possesses a number of practical advantages for in vivo gene therapy to the nervous system and other tissues. HSV-1 can infect a wide variety of both dividing and postmitotic cell types and canbe propagated to high titers on complementing cell lines. It also has a large genome size (152 kb), which allows the virus to accommodate large (1) or numerous therapeutic gene sequences (>35 kb) (2). In addition, the natural biology of HSV-1 infection involves long-term persistence of the viral genome in a latent, nonintegrated state in neuronal cell nuclei (3–5) and other postmitotic cell types in the absence of viral protein synthesis, genome integration, or interference with host cell biology. Since the virus does not disrupt normal host cell biology or express viral antigens during latency, cells harboring latent virus will not be attacked by the host’s immune system and should allow persistence of the viral genome for the lifetime of the host, obviating the need for repeat dosing of the virus vector. During latency the viral genome is transcriptionally silent except for expression of a set of viral latency-associated transcripts (LATs) (6–11). Since the LATs are not required for establishment or maintenance of this latent state (12–18), it should be possible to delete these genes and replace them with the desired therapeutic gene to drive expression of this gene product from an otherwise quiescent genome using the latency and neuronal cell-specific promoter complex resident within the vector genome.    

5.107           Evidence for packaging of rep-cap sequences into adeno-associated virus (AAV) type 2 capsids in the absence of inverted terminal repeats: a model for generation of rep-positive AAV particles

Nony, P., Chadeuf, G., Tessier, J., Moullier, P. and Salvetti, A.

1. Virol., 77, 776-781 (2003)

  We previously reported that a 350-bp region of the adeno-associated virus (AAV) type 2 rep gene contains a cis-acting element responsible for the Rep-dependent replication of a transiently transfected rep-cap plasmid. In this study, we further report that replicated rep-cap sequences can be packaged into AAV capsids in the absence of the inverted terminal repeats.  

5.108           Foamy virus envelope glycoprotein is sufficient for particle budding and release

Shaw, K.L., Lindemann, D., Mulligan, M.J. and Goepfert, P.A.

1. Virol., 77(4), 2338-2348 (2003)

  Foamy viruses (FVs) are classified in the family Retroviridae, but recent data have shown that they are not conventional retroviruses. Notably, several characteristics of their particle replication strategies are more similar to those of hepatitis B virus (HBV) than those of typical retroviruses. Compared to conventional retroviruses, which require only Gag proteins for budding and release of virus-like particles (VLPs), both FV and HBV require Env proteins. In the case of HBV, Env (S protein) alone is sufficient to form subviral particles (SVPs). Because FVs also depend on Env for budding, we tested whether FV Env alone could produce SVPs. The Env proteins of FV and murine leukemia virus (MuLV) were both released into cell culture supernatants and migrated into isopycnic gradients; however, unlike MuLV Env, FV Env displayed characteristics of SVPs. FV Env particles were of greater density than those of MuLV (1.11 versus 1.07 g/ml, respectively), which strongly suggested that the released proteins of FV Env were particulate. When we examined FV SVPs by immunoelectron microscopy, we found particles that were consistent in morphology, size, and staining with gold beads, similar to FV VLPs and unlike the particle-like structures of MuLV Env, which were more consistent with vesicles produced from nonspecific membrane "blebbing." Taken together, our results demonstrated that FV Env alone is sufficient for particle budding. This finding is unique among retroviruses and further demonstrated the similarities between FV and HBV.  

5.109           Tetracycline-inducible transgene expression mediated by a single AAV vector

Chtarto, A., Bender, H.U., Hanemann, C.O., Kemp, T., Lehtonen, E., Livivier, M., Brotchi, J., Velu, T. and Tenenbaum, L. Gene Therapy, 10, 84-94 (2003)   Regulated gene delivery systems are usually made of two elements: an inducible promoter and a transactivator. In order to optimize gene delivery and regulation, a single viral vector ensuring adequate stoichiometry of the two elements is required. However, efficient regulation is hampered by interferences between the inducible promoter and (i) the promoter used to express the transactivator and/or (ii) promoter/enhancer elements present in the viral vector backbone. We describe a single AAV vector in which transcription of both the reverse tetracycline transactivator (rtTA) and the transgene is initiated from a bidirectional tetracycline-responsive promoter and terminated at bidirectional SV40 polyadenylation sites flanking both ITRs. Up to 50-fold induction of gene expression in human tumor cell lines and 100-fold in primary cultures of rat Schwann cells was demonstrated. In addition an 80-fold induction in vivo in the rat brain has been obtained. In vitro, the autoregulatory vector exhibits an induced expression level superior to that obtained using the constitutive CMV promoter. Although extinction of the transgene after removal of tetracycline was rapid (less than 3 days), inducibility after addition of tetracycline was slow (about 14 days). This kinetics is suitable for therapeutic gene expression in slowly progressive diseases while allowing rapid switch-off in case of undesirable effects. As compared to previously described autoregulatory tet-repressible (tetOFF) AAV vectors, the tet-inducible (tetON) vector prevents chronic antibiotic administration in the uninduced state.  

5.110           Gene therapy delivery of endostatin enhances the treatment efficacy of radiation

Shi, W., Teschendorf, C., Muzyczka, N. and Siemann, D.W. Radiother. Oncol., 66, 1-9 (2003)   Background and purpose: To evaluate whether sustained expression of mouse endostatin by adeno-associated virus (AAV)-mediated gene transfer can enhance the treatment efficacy of ionizing radiation. Materials and methods: Mouse endostatin was cloned into recombinant AAV (rAAV) under the control of CMV b-actin promoter. Recombinant mouse endostatin expressed via AAV gene transfer was tested for biological activity in endothelial cells. The impact of elevated serum levels of endostatin on tumor-induced angiogenesis was evaluated using an in vivo angiogenesis assay. The anti-tumor efficacy of combining rAAV-mediated endostatin delivery with radiation was evaluated in a human colorectal tumor model (HT29). Results: Recombinant mouse endostatin expressed through an AAV vector (rAAV-mEndo) inhibited endothelial cell proliferation (by 40–45%) and migration (by 22–33%). Intramuscular injection of rAAV-mEndo (1×10⁹ i.u.) led to a sustained serum endostatin level of ~500 ng/ml. Compared to control animals this endostatin level was sufficient to inhibit tumor cell-induced vessel formation (37 vs. 28.5, P<0.05) and delay the growth of HT29 xenografts (time from 200 to 1000 mm³, 21 vs. 34.5 days, P<0.05). When combined with ionizing radiation, elevated serum endostatin levels significantly enhanced the time for tumors to grow from 200 to 1000 mm³ (radiation, 34 days; endostatin plus radiation, 50 days, P<0.05). Conclusion: The delivery of endostatin via rAAV vectors may provide an effective means of enhancing the anti-tumor efficacy of radiation therapy.  

5.111           Functional role of HIV-1 virion-associated uracil DNA glycosylase 2 in the correction of G:U mispairs to G:C pairs

Priet, S., Navarro, J-M., Gros, N., Querat, G. and Sire, J.

1. Biol. Chem., 278, 4566-4571 (2003)

  Human monocytes/macrophages are target cells for HIV-1 infection. As other non-dividing cells, they are characterized by low and imbalanced intracellular dNTP pool levels and an excess of dUTP. The replication of HIV-1 in this cellular context favors misincorporation of uracil residues into viral DNA because of the use of dUTP in place of dCTP. We have previously reported that the host uracil DNA glycosylase enzyme UNG2 is packaged into HIV-1 viral particles via a specific association with the integrase domain of the Gag-Pol precursor. In this study, we investigated whether virion-associated UNG2 plays a role similar to that of its cellular counterpart. We show that the L172A mutation of integrase impaired the packaging of UNG2 into viral particles. Using a primer-template DNA substrate containing G:U mispairs, we demonstrate that wild-type viral lysate has the ability to repair G:U mismatched pairs to G:C matched pairs, in contrast to UNG2-deficient viral lysate. Moreover, no correction of G:T mispairs by wildtype HIV-1 viral lysate was observed, which argues for the specificity of the repair process. We also show that UNG2 physically associates with the viral reverse transcriptase enzyme. Altogether our data indicate for the first time that a uracil repair pathway is specifically associated with HIV-1 viral particles. However, the molecular mechanism of this process remains to be characterized further.  

5.112           Efficient large scale production and concentration of HIV-1-based lentiviral vectors for use in vivo

Coleman J.E. et al Physiol. Genomics, 12, 221-228 (2003)   The aim of this study was to develop an efficient method for packaging and concentrating lentiviral vectors that consistently yields high-titer virus on a scale suitable for in vivo applications. Transient cotransfection of 293T packaging cells with DNA plasmids encoding lentiviral vector components was optimized using SuperFect, an activated dendrimer-based transfection reagent. The use of SuperFect allowed reproducible and efficient production of high-titer lentiviral vector at concentrations greater than 1 x 10⁷ transducing units per ml (TU/ml) and required less than one-third of the total amount of DNA used in traditional calcium phosphate transfection methods. Viral titers were further increased using a novel concentration protocol that yielded an average final titer of 1.4 x 10¹⁰ TU/ml. Lentiviruses produced using these methods exhibited efficient transduction of central nervous system and peripheral tissues in vivo. The method is reproducible and can be scaled up to facilitate the use of these vectors in animal studies.  

5.113           Interactions between the transmembrane segments of the alphavirus E1 and E2 proteins play a role in virus budding and fusion

Sjøberg, M. and Garoff, H.

1. Virol., 77(6), 3441-3450 (2003)

  The alphavirus envelope is built by heterodimers of the membrane proteins E1 and E2. The complex is formed as a p62E1 precursor in the endoplasmic reticulum. During transit to the plasma membrane (PM), it is cleaved into mature E1-E2 heterodimers, which are oligomerized into trimeric complexes, so-called spikes that bind both to each other and, at the PM, also to nucleocapsid (NC) structures under the membrane. These interactions drive the budding of new virus particles from the cell surface. The virus enters new cells by a low-pH-induced membrane fusion event where both inter- and intraheterodimer interactions are reorganized to establish a fusion-active membrane protein complex. There are no intact heterodimers left after fusion activation; instead, an E1 homotrimer remains in the cellular (or viral) membrane. We analyzed whether these transitions depend on interactions in the transmembrane (TM) region of the heterodimer. We observed a pattern of conserved glycines in the TM region of E1 and made two mutants where either the glycines only (SFV/E1^4L) or the whole segment around the glycines (SFV/E1^11L) was replaced by leucines. We found that both mutations decreased the stability of the heterodimer and increased the formation of the E1 homotrimer at a suboptimal fusion pH, while the fusion activity was decreased. This suggested that TM interactions play a role in virus assembly and entry and that anomalous or uncoordinated protein reorganizations take place in the mutants. In addition, the SFV/E1^11L mutant was completely deficient in budding, which may reflect an inability to form multivalent NC interactions at the PM.    

5.114           Chimeric and pseudotyped parvoviruses minimize the contamination of recombinant stocks with replication-competent viruses and identify a DNA sequence that restricts parvovirus H-1 in mouse cells

Wrzesinski, C. et al

1. Virol., 77(6), 3851-3858 (2003)

  Recent studies demonstrated the ability of the recombinant autonomous parvoviruses MVMp (fibrotropic variant of the minute virus of mice) and H-1 to transduce therapeutic genes in tumor cells. However, recombinant vector stocks are contaminated by replication-competent viruses (RCVs) generated during the production procedure. To reduce the levels of RCVs, chimeric recombinant vector genomes were designed by replacing the right-hand region of H-1 virus DNA with that of the closely related MVMp virus DNA and conversely. Recombinant H-1 and MVMp virus pseudotypes were also produced with this aim. In both cases, the levels of RCVs contaminating the virus stocks were considerably reduced (virus was not detected in pseudotyped virus stocks, even after two amplification steps), while the yields of vector viruses produced were not affected. H-1 virus could be distinguished from MVMp virus by its restriction in mouse cells at an early stage of infection prior to detectable viral DNA replication and gene expression. The analysis of the composite viruses showed that this restriction could be assigned to a specific genomic determinant(s). Unlike MVMp virus, H-1 virus capsids were found to be a major determinant of the greater permissiveness of various human cell lines for this virus.  

5.115           Signal peptide of Lassa virus glycoprotein GP-C exhibits an unusual length

Eichler, R., Lenz, O., Strecker, T. And Garten, W. FEBS Lett., 538, 203-206 (2003)   Lassa virus glycoprotein is synthesized as precursor GP-C into the lumen of the endoplasmic reticulum and cleaved posttranslationally into the N-terminal subunit GP-1 and the C-terminal subunit GP-2 by subtilase SKI-1/S1P. The N-terminal portion of the primary translation product preGP-C contains a signal peptide of unknown length. In order to demonstrate the signal peptide cleavage site, purified viral GP-1 isolated from Lassa virus particles was N-terminally sequenced as TSLYKGV, identical to amino acids 59–65 of GP-C. Mutational analysis of the amino acid residues flanking the putative cleavage site led to non-cleavable preGP-C indicating that no other signal peptide cleavage site exists. Interestingly, GP-C mutants with a non-cleavable signal peptide were not further processed by SKI-1/S1P. This observation suggests that the signal peptide cleavage is necessary for GP-C maturation and hence for Lassa virus replication.

5.116           Antibodies against a human endogenous retrovirus and the preponderance of env splice variants in multiple sclerosis patients

Christensen, T., Sørensen, P.D., Hansen, H.J. and Møller-Larsen, A. Multiple Sclerosis, 9, 6-15 (2003)   The human endogenous retrovirus HERV-H is associated with multiple sclerosis (MS). Previously performed reverse transcriptase-polymerase chain reactions (RT-PCR) on virion-RNA demonstrated sequence variants of the HERV-H family located in the particulate fraction of MS patient plasma samples and not in controls. In this study a significantly elevated level of antibodies towards peptides derived from HERV-H/RGH-2 DNA sequences in serum and cerebrospinal fluid (CSF) from MS patients is demonstrated. Further, Wistar rats immunized with purified virions develop a specific serologic response, indicating that some virion proteins are encoded by HERV-H-related sequences. Also shown is that in RNA from blood cells, a HERV-H protease-env splice variant can be found together with an env splice variant in about 40% of MS patients but only in 10% of controls. The results substantiate the association between activated HERV-H and MS, but a causal relationship is yet to be demonstrated. HERV-H could represent a causal factor either by eliciting an autoimmune response or through the pathogenic potential of the retrovirus itself.    

5.117           Optic neuropathy induced by reductions in mitochondrial superoxide dismutase

Qi, X., Lewin, A., Hauswirth, W.W. and Guy, J. Invest. Ophthal. Vis. Sci., 44, 1088-1096 (2003)   PURPOSE. Reactive oxygen species (ROS) are suspected to playa pivotal role in the pathogenesis of Leber hereditary opticneuropathy (LHON), caused by mutated complex I subunit genes.It seems surprising that optic neuropathy has not been describedin animals with a knockout of genes encoding critical anti-ROSdefenses. If ROS have a role in the optic nerve injury of LHON,then increasing mitochondrial levels of ROS should induce opticneuropathy. METHODS. To develop an animal model system for study of oxidativeinjury to the optic nerve, mitochondrial defenses were decreasedagainst ROS by designing hammerhead ribozymes to degrade SOD2mRNA. Several potential ribozymes were analyzed in vitro. Theone with the best kinetic characteristics was cloned into arecombinant adeno-associated virus (rAAV) vector for deliveryand testing in cells and animals. The effects of the AAV-expressingribozyme on murine cell growth, SOD2 mRNA and protein, cellularROS levels, and apoptosis were evaluated by RNase protectionassay, immunoblot analysis, and ROS- and apoptosis-activatedfluorescent probes. The rAAV-ribozyme was then injected intothe eyes of DBA/1J mice, and the effect on the optic nerve wasevaluated by ocular histopathologic examination. RESULTS. The AAV-expressing ribozyme decreased SOD2 mRNA andprotein levels by as much as 85%, increased cellular superoxide,reduced mitochondrial membrane potential, and culminated inthe death of infected cell lines by apoptosis without significantlyaltering complex I and III activity, somewhat spared in themost common LHON mutation (G11778A), although adenosine triphosphate(ATP) synthesis is markedly reduced. When inoculated into theeyes of mice, the AAV-expressing ribozyme led to loss of axonsand myelin in the optic nerve and ganglion cells in the retina,the hallmarks of optic nerves examined at autopsy of patientswith LHON. CONCLUSIONS. The striking similarity of the optic neuropathyto the histopathology of LHON is powerful evidence supportingROS as a key factor in the pathogenesis of LHON.  

5.118           Gene delivery in renal tubular epithelial cells using recombinant adeno-associated viral vectors

Chen, S. et al

1. Am. Soc. Nephrol., 14, 947-958 (2003)

  Gene therapy has the potential to provide a therapeutic strategy for numerous renal diseases such as diabetic nephropathy, chronic rejection, Alport syndrome, polycystic kidney disease, and inherited tubular disorders. In previous studies using cationic liposomes or adenoviral or retroviral vectors to deliver genes into the kidney, transgene expression has been transient and often associated with adverse host immune responses, particularly with the use of adenoviral vectors. The unique properties of recombinant adeno-associated viral (rAAV) vectors permit long-term stable transgene expression with a relatively low host immune response. The purpose of the present study was to evaluate gene expression in the rat kidney after intrarenal arterial infusion of a rAAV (serotype 2) vector encoding green fluorescence protein (GFP) induced by a cytomegalovirus-chicken beta-actin hybrid promoter. The left kidney of experimental animals was treated with either saline or transduced with rAAV2-GFP (0.125 ml/100 g body wt, 1 x 10¹⁰/ml infectious units) through the renal artery. A time-dependent expression of GFP was observed in all kidneys injected with rAAV2-GFP, with maximal expression observed at 6 wk posttransduction. The expression of GFP was restricted to cells in the S₃ segment of the proximal tubule and intercalated cells in the collecting duct, the latter identified by co-localization with H⁺-ATPase. No transduction was observed in the glomeruli or the intrarenal vasculature. These studies demonstrate successful transgene expression in tubular epithelial cells, specifically in the S₃ segment of the proximal tubule and intercalated cells, after intrarenal administration of a rAAV vector and provide the impetus for further studies to exploit its use as a tool for gene therapy in the kidney.  

5.119           Packaging of human chromosome 19-specific adeno-associated virus (AAV) integration sites in AAV virions during AAV wild-type and recombinant AAV vector production

Hüser, D., Weger, S. and Heilbronn, R.   Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus on human chromosome 19, called AAVS1. During the development of a sensitive real-time PCR assay for site-specific integration, AAV-AAVS1 junctions were reproducibly detected in highly purified AAV wild-type and recombinant AAV vector stocks. A series of controls documented that the junctions were packaged in AAV capsids and were newly generated during a single round of AAV production. Cloned junctions displayed variable AAV sequences fused to AAVS1. These data suggest that packaged junctions represent footprints of AAV integration during productive infection. Apparently, AAV latency established by site-specific integration and the helper virus-dependent, productive AAV cycle are more closely related than previously thought.  

5.120           Cytotoxic immune response after retroviral-mediated hepatic gene transfer in rat does not preclude expression from adeno-associated virus 1 transducted muscles

Aubert, D., Pichard, V., Durand, S., Moullier, P. and Ferry, N. Hum. Gen. Ther., 14, 473-481 (2003)   Intravenous delivery of nls-lacZ retroviral vectors to the regenerating liver triggers a cytotoxic immune response directed against transduced hepatocytes. We sought to determine whether prior immunization with retroviral vectors impacted on adeno-associated virus (AAV)-mediated muscular expression of the same transgene. The first group of rats first received nls-lacZ retroviral vectors intravenously after a partial hepatectomy. Thirty days later they received AAV vectors intramuscularly in both legs. In the second group, animals received the same vectors in the opposite sequence (i.e., AAV first and retroviruses 20 days later). In the first group, immune response occurred after retrovirus delivery with appearance of anti-β-galactosidase antibodies and elimination of transduced hepatocytes. However, the immune response did not prevent sustained (9-month) β-galactosidase expression in AAV-injected muscles. In the second group, AAV injections did not induce immune response and resulted in β-galactosidase expression in myofibers. In this group, subsequent delivery of retroviral vectors triggered appearance of immune response and elimination of transduced hepatocytes. However, the immune response did not modify β-galactosidase expression in AAV-transduced myofibers for up to 9 months. These results demonstrate a differential susceptibility between retrovirally transduced liver and AAV-transduced muscles to immune response against the transgene product.  

5.121           Packaging of an AAV vector encoding human acid a-glucosidase for gene therapy in glycogen storage disease type II with a modified hybrid adenovirus-AAV vector

Sun, B. et al Mol. Ther., 7(4), 467-477 (2003)   We have developed an improved method for packaging adeno-associated virus (AAV) vectors with a replication-defective adenovirus-AAV (Ad-AAV) hybrid virus. The AAV vector encoding human acid a-glucosidase (hGAA) was cloned into an E1, polymerase/preterminal protein-deleted adenovirus, such that it is packaged as an Ad vector. Importantly, the Ad-AAV hybrid cannot replicate during AAV vector packaging in 293 cells, because of deletion of polymerase/preterminal protein. The residual Ad-AAV in the AAV vector stock was reduced to <1 infectious particle per 10¹⁰ AAV vector particles. These modifications resulted in ~30-fold increased packaging of the AAV vector for the hybrid Ad-AAV vector method as compared with standard transfection-only methods. Similarly improved packaging was demonstrated for pseudotyping the AAV vector as AAV6, and for AAV vector packaging with a second Ad-AAV vector encoding canine glucose-6-phosphatase. Liver-targeted delivery of either the Ad-AAV hybrid or AAV vector particles in acid a-glucosidase-knockout (GAA-KO) mice revealed secretion of hGAA with the Ad-AAV vector, and sustained secretion of hGAA with an AAV vector in hGAA-tolerant GAA-KO mice. Further development of hybrid Ad-AAV vectors could offer distinct advantages for gene therapy in glycogen storage diseases.  

5.122           RGD inclusion in VP3 provides adeno-associated virus type 2 (AAV2)-based vectors with a heparan sulfate-independent cell entry mechanism

Shi, W. and Bartlett, J.S. Mol. Ther., 7(4), 515-525 (2003)   Recombinant adeno-associated virus (AAV) has become an attractive vector system for a number of gene therapy paradigms. However, the utility of AAV vectors is often limited by the absence of heparan sulfate proteoglycan (HSPG), the virus's primary attachment receptor, on the desired target cell population. In order to achieve HSPG-independent gene delivery, several groups have shown that the endogenous tropism of AAV can be expand by genetically altering the viral capsid. However, the parameters of this developing technology have yet to be defined and it has not yet been determined if these modified vectors actually infect cells via these engineered interactions. Previously we constructed a series of insertion mutants spanning the AAV capsid protein gene and identified specific sites that can tolerate the insertion of small exogenous peptides. Here we describe a number of sites within the AAV capsid gene that can be used for the insertion of integrin-targeting peptide epitopes. Incorporation of an Arg-Gly-Asp (RGD)-containing peptide at these sites enables AAV to infect integrin-expressing cells independent of HSPG. Mutant AAV vectors displaying these peptide ligands can be produced to wild-type titer and have been shown to specifically interact with the targeted integrin receptors and mediate infection via this interaction. We report significant increases in gene transfer to Raji, K562, and SKOV-3 cell lines that express integrin, but little HSPG, suggesting that rAAV vectors displaying RGD peptides may be of great utility for treatment of neoplasms characterized by the deficiency of HSPG expression. We have also demonstrated that due to their expanded tropism, these novel vectors are capable of efficient transduction of AAV2-resistant tumors in vivo suggesting that they may offer significant therapeutic advantages.  

5.123           Adeno-asociated virus-mediated aspartoacylase gene transfer to the brain of knockout mouse for canavan disease

Matalon, R. et al Mol. Ther., 7(5), 580-587 (2003)   Canavan disease (CD) is an autosomal recessive leukodystrophy caused by deficiency of aspartoacylase (ASPA). Deficiency of ASPA leads to elevation of N-acetyl-L-aspartic acid (NAA) in the brain and urine. To explore the feasibility of gene transfer to replace ASPA in CD, we generated a knockout mouse and constructed an AAV vector that encodes human ASPA cDNA (hASPA) followed by green fluorescent protein (GFP) after an intraribosomal entry site. We injected CD mice with rAAV-hASPA-GFP in the striatum and thalamus or injected rAAV-GFP identically into control animals. Three to five months after the injection, we determined the presence of ASPA in the CD mouse brain by ASPA activity assay, GFP expression, and Western blot analysis. While rAAV-GFP-injected animals displayed undetectable levels of ASPA, all detection methods revealed significant ASPA levels in rAAV-hASPA-GFP-injected CD mice. We evaluated the functional effects of rAAV-hASPA-GFP-mediated ASPA expression by standard histological methods, magnetic resonance spectroscopy (MRS) for in vivo NAA levels, and magnetic resonance imaging of CD mice. rAAV-hASPA-injected animals displayed a remarkable lack of spongiform degeneration in the thalamus. However, pathology in sites unrelated to the injected areas showed no improvement in histopathology. The improvement in thalamic neuropathology was also detectable via in vivo MRI. MRS revealed that in vivo NAA levels were also reduced. These data indicate that rAAV-mediated ASPA delivery may be an interesting avenue for the treatment of CD.  

5.124           Human gene targeting by adeno-associated virus vectors is enhanced by DVA double-strand breaks

Miller, D.G., Petek, L.M. and Russell, D.W. Mol. Biol. Cell, 23(10),. 3550-3557 (2003)   The use of adeno-associated virus (AAV) to package gene-targeting vectors as single-stranded linear molecules has led to significant improvements in mammalian gene-targeting frequencies. However, the molecular basis for the high targeting frequencies obtained is poorly understood, and there could be important mechanistic differences between AAV-mediated gene targeting and conventional gene targeting with transfected double-stranded DNA constructs. Conventional gene targeting is thought to occur by the double-strand break (DSB) model of homologous recombination, as this can explain the higher targeting frequencies observed when DSBs are present in the targeting construct or target locus. Here we compare AAV-mediated gene-targeting frequencies in the presence and absence of induced target site DSBs. Retroviral vectors were used to introduce a mutant lacZ gene containing an I-SceI cleavage site and to efficiently deliver the I-SceI endonuclease, allowing us to carry out these studies with normal and transformed human cells. Creation of DSBs by I-SceI increased AAV-mediated gene-targeting frequencies 60- to 100-fold and resulted in a precise correction of the mutant lacZ reporter gene. These experiments demonstrate that AAV-mediated gene targeting can result in repair of a DNA DSB and that this form of gene targeting exhibits fundamental similarities to conventional gene targeting. In addition, our findings suggest that the selective creation of DSBs by using viral delivery systems can increase gene-targeting frequencies in scientific and therapeutic applications.  

5.125           Efficient gene targeting mediated by adeno-associated virus and DNA double-stranded breaks

Porteus, M.H., Cathomen, T., Weitsman, M.D. and Baltimore, D. Mol. Cell. Biol., 23(10), 3558-3565 (2003)   Gene targeting is the in situ manipulation of the sequence ofan endogenous gene by the introduction of homologous exogenousDNA. Presently, the rate of gene targeting is too low for itto be broadly used in mammalian somatic cell genetics or tocure genetic diseases. Recently, it has been demonstrated thatinfection with recombinant adeno-associated virus (rAAV) vectorscan mediate gene targeting in somatic cells, but the mechanismis unclear. This paper explores the balance between random integrationand gene targeting with rAAV. Both random integration and spontaneousgene targeting are dependent on the multiplicity of infection(MOI) of rAAV. It has previously been shown that the introductionof a DNA double-stranded break (DSB) in a target gene can stimulategene targeting by several-thousand-fold in somatic cells. Creationof a DSB stimulates the frequency of rAAV-mediated gene targetingby over 100-fold, suggesting that the mechanism of rAAV-mediatedgene targeting involves, at least in part, the repair of DSBsby homologous recombination. Absolute gene targeting frequenciesreach 0.8% with a dual vector system in which one rAAV vectorprovides a gene targeting substrate and a second vector expressesthe nuclease that creates a DSB in the target gene. The frequenciesof gene targeting that we achieved with relatively low MOIssuggest that combining rAAV vectors with DSBs is a promisingstrategy to broaden the application of gene targeting.  

5.126           Structural and functional protection of photoreceptors from MNU-induced retinal degeneration by the X-linked inhibitor of apoptosis

Petrin, D. et al. Invest. Ophthalmol., 44, 2757-2763 (2003)   PURPOSE. To evaluate the neuroprotective effects of adenoasso­ciated virus delivery of XIAP in N-methyl-N-nitrosourea (MNU)­induced retinal degeneration in Sprague-Dawley rats.   METHODS. Spraguc-Dawley rats were injected subretinally with recombinant adenoassociated virus (rAAV) encoding either XIAP or green fluorescent protein (GFP; injection control). Six weeks after injection, the animals received an intraperitoneal injection of MNU, a DNA methylating agent, at a dose of 6() mg/kg. Electroretinograms (ERGs) were recorded at 0, 24, 48 and 72 hours and 1 week after MNU. The rats were killed after the ERG was performed and were perfused with 4% parafor­maldehyde. Eyes were then enucleated and embedded for cryosectioning. Eye sections were analyzed by TUNEL and histologic techniques. Real-time PCR and Western analysis were performed to confirm the overexpression of XIAP in injected eyes.   RESULTS. Real-time PCR and Western analysis confirmed the overexpression of XIAP in virus-injected eyes in comparison to uninjected control eyes. At 24 hours after MNU injection, fewer cells had undergone apoptosis in the XIAP-treated eyes in comparison with GFP-injected or uninjected eyes. Hematox­ylin and eosin staining revealed that the uninjected and GFP­injected photoreceptors were destroyed by 72 hours after injection of MNU, whereas the AAV-XIAP-injected eyes showed structural protection of the photoreceptors at all time points throughout the 1-week sampling period. ERGs showed func­tional protection up to 1 week after MNU injection in the AAV-XIAP-injected eye, whereas no response was observed in the control eye.   CONCLUSIONS. The results suggest that XIAP is protective against this potent chemotoxic agent and holds promise as a therapeu­tic agent in gene therapy approaches to treating retinitis.  

5.127           Features of the Env leader protein and the N-terminal Gag domain of feline foamy virus important for virus morphogenesis

Geiselhart, V., Schwantes, A., Bastone, P., Frech, M. and Lochelt, M. Virol., 310, 235-244 (2003)   Previous studies have shown that foamy virus (FV) particle budding, especially the involvement of the viral Env glycoprotein, is different from that of other (ortho) retroviruses: the N-terminal Env leader protein Elp is a constituent of released FV particles. A defined sequence in Elp required for particle budding binds to the MA domain of Gag. To extend these findings, we show that feline FV Elp is a membrane-anchored protein with the N-terminus located inside the particle. Thus, the intemal/cytoplasmic domain of Elp has the correct topology for interacting with Gag during budding. In addition to Elp, an Elp-related protein of about 9 kDa was shown to be virion associated and is probably generated by cellular signal peptidases. Besides the function of Elp binding, the N-terminal domain of Gag was shown to be required for proper localization of feline FV Gag to the cytoplasm and the perinuclear/nuclear region.    

5.128           Reversion of the lethal phenotype of an HIV-1 integrase mutant virus by overexpression of the same integrase mutant protein

Priet,S., Navarro, J-M., Querat, G. and Sire, J.

1. Biol. Chem., 278, 20724-20730 (2003)

  The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is essential for integration of viral DNA into host cell chromatin. We have reported previously (Priet, S., Navarro, J. M., Gros, N., Querat, G., and Sire, J. (2003) J. Biol. Chem. 278, 4566-4571) that IN also plays a role in the packaging of the host uracil DNA glycosylase UNG2 into viral particles and that the region of IN en­compassing residues 170-180 was responsible for the interaction with UNG2 and for its packaging into viri­ons. In this work, we aimed to investigate the replica­tion of HIV-1 viruses rendered deficient in virion~asso­ciated UNG2 by single or double point mutations in the region 170-180 of IN. We show that the L172A/K173A IN mutant virus was deficient for UNG2 packaging and was defective for replication because of a blockage at the stage of proviral DNA integration in host cell DNA. In vitro assays using long term repeat mimics, however, demonstrate that the L172A/K173A IN mutant was cata­lytically active. Moreover, trans-complementation ex­periments show that the viral propagation of L172A/K173A viruses could be rescued by the overexpression of VprL172A/K173A IN fusion protein in a dose-dependent manner and that this rescue is independent of UNG2 packaging. Altogether, our data indicate that L172A/K173A mutations of IN induce a subtle defect in the function of IN, which nevertheless dramatically impairs viral replication. Unexpectedly, this blockage of replica­tion could be overcome by forcing the packaging of higher amounts of this same mutated integrase. This is the first study reporting that blockage of the integration process of HIV-1 provirus carrying a mutation of IN could be alleviated by increasing amounts of IN even carrying the same mutations.  

5.129           Helper virus-free, optically controllable and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6

Grimm, D., Kay, M.A. and Kleinschmidt, J.A. Mol. Therapy, 7, 839-850 (2003)   We present a simple and safe strategy for producing high-titer adeno-associated virus (AAV) vectors derived from six different AAV serotypes (AAV-1 to AAV-6). The method, referred to as “HOT,” is helper virus free, optically controllable, and based on transfection of only two plasmids, i.e., an AAV vector construct and one of six novel AAV helper plasmids. The latter were engineered to carry AAV serotype rep and cap genes together with adenoviral helper functions, as well as unique fluorescent protein expression cassettes, allowing confirmation of successful transfection and identification of the transfected plasmid. Cross-packaging of vector DNA derived from AAV-2, -3, or -6 was up to 10-fold more efficient using our novel plasmids, compared to a conservative adenovirus-dependent method. We also identified a variety of useful antibodies, allowing detec­tion of Rep or VP proteins, or assembled capsids, of all six AAV serotypes. Finally, we describe unique cell tropisms and kinetics of transgene expression for AAV serotype vectors in primary or transformed cells from four different species. In sum, the HOT strategy and the antibodies presented here, together with the reported findings, should facilitate and support the further development of AAV serotype vectors as powerful new tools for human gene therapy.  

5.130           Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding

Opie, S.R., Warrington, K.H., Agbandje-McKenna, M., Zolotukhin, S. and Muzyczka, N.

1. Virol., 77, 6995-7006 (2003)

  The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette. Intact capsids were assayed for their ability to bind to heparin-agarose in vitro, and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. We found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose. We observed a general correlation between heparin-agarose binding and infectivity as measured by GFP transduction; however, a subset of mutants that partially bound heparin-agarose (R484A and K532A) were completely noninfectious, suggesting that they had additional blocks to infectivity that were unrelated to heparin binding. Conservative mutation of positions R585 and R588 to lysine slightly reduced heparin-agarose binding and had comparable effects on infectivity. Substitution of AAV2 residues 585 through 590 into a location predicted to be structurally equivalent in AAV5 generated a hybrid virus that bound to heparin-agarose efficiently and was able to package DNA but was noninfectious. Taken together, our results suggest that residues R585 and R588 are primarily responsible for heparin sulfate binding and that mutation of these residues has little effect on other aspects of the viral life cycle. Interactive computer graphics examination of the AAV2 VP3 atomic coordinates revealed that residues which contribute to heparin binding formed a cluster of five basic amino acids that presented toward the icosahedral threefold axis from the surrounding spike protrusion. Three other kinds of mutants were identified. Mutants R459A, H509A, and H526A/K527A bound heparin at levels comparable to that of wild-type virus but were defective for transduc­tion. Another mutant, H358A, was defective for capsid assembly. Finally, an R459A mutant produced signif­icantly lower levels of full capsids, suggesting a packaging defect.  

5.131           Immune responses to adeno-associated virus and its recombinant vectors

Sun, J.Y., Anand-Jawa, V., Chatterjee, S. and Wong, K.K. Gene Therapy, 10, 964-976 (2003)   Recombinant adeno-associated virus (rAAV) vectors have emerged as highly promising for use in gene transfer for a variety of reasons, including lack of pathogenicity and wide host range. In addition, all virus-encoded genes have been removed from standard rAAV vectors, resulting in their comparatively low intrinsic immunogenicity. For gene replacement strategies, transgenes encoded by rAAV vectors may induce less robust host immune responses than other vectors in vivo However, under appropriate conditions, host immune responses can be generated against rAAV-encoded transgenes, raising the potential for their use in vaccine development. In this review, we summarize current under­ standing of the generation of both undesirable and beneficial host immune responses directed against rAAV and encoded transgenes, and how they might be exploited for optimal use of this promising vector system.  

5.132           In vitro selection of viral vectors with modified tropism: the adeno-associated virus display

Perabo, L. et al. Mol. Therapy, 8, 151-157 (2003)   Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy. In an effort to generate viral mutants with controlled tropism we produced a library of adeno­associated virus (AAV) clones with randomly modified capsids and used it for the selection of receptor-targeting mutants. After several rounds of selection on different cell lines that were resistant to infection by wild-type (wt) AAV, infectious mutants were harvested at high titers. These mutants transduced target cells with an up to 100-fold increased efficiency, in a receptor­specific manner and without interacting with the primary receptor for wt AAV. The results demonstrate for the first time that a combinatorial approach based on a eukaryotic virus library allows one to generate efficient, receptor-specific targeting vectors with desired tropism.  

5.133           Receptor targeting of adeno-associated virus vectors

Buning, H. et al. Gene Therapy 10, 1142-1151 (2003)   Adeno-associated virus (AAV) is a promising vector for human somatic gene therapy. However, its broad host range is a disadvantage for in vivo gene therapy, because it does not allow the selective tissue- or organ-restricted trans-duction required to enhance the safety and efficiency of the gene transfer. Therefore, increasing efforts are being made to target AAV-2-based vectors to specific receptors. The studies summarized in this review show that it is possible to target MV-2 to a specific cell. So far, the most promising approach is the genetic modification of the viral capsid. However, the currently available AAV-2 targeting vectors need to be improved with regard to the elimination of the wild-type AAV-2 tropism and the improvement of infectious titers. The creation of highly efficient AAV-2 targeting vectors will also require a better understanding of the trans-membrane and intracellular processing of this virus.  

5.134           Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts

Mangeat, B. et al. Nature, 424, 99-103 (2003)   Viral replication usually requires that innate intracellular lines of defence be overcome, a task usually accomplished by specialized viral gene products. The virion infectivity factor (Vif) protein of human immunodeficiency virus (HIV) is required during the late stages of viral production to counter the antiviral activity of APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; also known as CEM15), a protein expressed notably in human T Iymphocytes. When produced in the presence of APOBEC3G, vif-defective virus is non-infectious. APOBEC3G is closely related to APOBEC1, the central com­ponent of an RNA-editing complex that deaminates a cytosine residue in apoB messenger RNA. APOBEC family members also have potent DNA mutator activity through dC deamination however, whether the editing potential of APOBEC3G has any relevance to HIV inhibition is unknown. Here, we demonstrate that it does, as APOBEC3G exerts its antiviral effect during reverse transcription to trigger G-to-A hypermutation in the nascent retroviral DNA. We also find that APOBEC3G can act on a broad range of retroviruses in addition to HIV suggesting that hypermutation by editing is a general innate defence mechanism against this important group of pathogens.  

5.135           Anterograde delivery of brain-derived neurotrophic factor to striatum via nigral transduction of recombinant adeno-associated virus increases neuronal death but promotes neurogenic response following stroke

Gustafsson, E. et al. Eur. J. Neurosci., 17, 2667-2678 (2003)   To explore the role of brain-derived neurotrophic factor for survival and generation of striatal neurons after stroke, recombinant adeno­-associated viral vectors carrying brain-derived neurotrophic factor or green fluorescent protein genes were injected into right rat substantia nigra 4-5 weeks prior to 30 min ipsilateral of middle cerebral artery occlusion. The brain-derived neurotrophic factor- recombinant adeno-associated viral transduction markedly increased the production of brain-derived neurotrophic factor protein by nigral cells. Brain-derived neurotrophic factor was transported anterogradely to the striatum and released in biologically active form, as revealed by the hypertrophic response of striatal neuropeptide Y-positive interneurons. Animals transduced with brain-derived neurotrophic factor-recombinant adeno-associated virus also exhibited abnormalities in body posture and movements, including tilted body to the right, choreiform movements of left forelimb and head, and spontaneous, so-called ‘barrel’ rotation along their long axis. The continuous delivery of brain-derived neurotrophic factor had no effect on the survival of striatal projection neurons after stroke, but exaggerated the loss of cholinergic, and parvalbumin- and neuropeptide Y-positive, g-aminobutyric acid-ergic interneurons. The high brain-derived neurotrophic factor levels in the animals subjected to stroke also gave rise to an increased number of striatal cells expressing doublecortin, a marker for migrating neuroblasts, and cells double-labelled with the mitotic marker, 5-bromo-2’-deoxyuridine-5’monophosphate, and early neuronal (Hu) or striatal neuronal (Meis2) markers. Our findings indicate that long-term anterograde delivery of high levels of brain-derived neurotrophic factor increases the vulnerability of striatal interneurons to stroke-induced damage. Concomitantly, brain-derived neurotrophic factor potentiates the stroke-induced neurogenic response, at least at early stages.  

5.136           Shuttle PCR-based cloning of the infectious adeno-associated virus type 5 genome

Lee, K., Kim, Y-G., Jo, E-C.

1. Virol. Methods, 111, 75-84 (2003)

  Adeno-associated virus type 5 (AAV5), which is distinct from the other serotypes of AAV, has attracted considerable interest as a premier gene delivery vector. As do the other serotypes, AAV5 contains its 4.7 kb-sized, single-stranded genome flanked with inverted terminal repeats (ITRs) in a hairpin conformation, which serves frequently as pause and arrest sites for DNA polymerases during PCR. To amplify the full-length of the AAV5 genome in single step, we established a shuttled, long and accurate PCR (LA­PCR) procedure in the present study. Furthermore, helper oligonucleotides, which hybridize with the palindromic sequence elements in ITR, were designed and employed in PCR to prevent the formation of hairpin structures by highly GC-rich ITRs. Consequently, a 4.7 kb-sized PCR product was amplified successfully, and cloned into a pBluescript® II KS(+) plasmid. Six plasmids, harboring the full-length AAV5 genome, rescued wild type AAV5 viruses on transfection to HeLa and HEK 293 cells, which were co-infected with helper adenoviruses. Western and Southern blot analyses supported further the fact that the pAAV5 plasmids harbored the full-length AAV5 genome. The PCR method described in this study is applicable for the cloning of genomes containing variable palindromic structures, in addition to AAV genomes of other serotypes.  

5.137           Recombinant AAV serotype 1 transduction efficiency and tropism in the murine brain

Wang, C., Wang, C-M., Clark, K.R. and Sferra, T.J. Gen. Ther., 10, 1528-1534 (2003)   Recombinant adeno-associated virus serotype 2 (rAAV2) vectors have shown promise as therapeutic agents for neurologic disorders. However, intracerebral administration of this vector leads to preferential transduction of neurons and a restricted region of transgene expression. The recently developed rAAV vectors based upon nonserotype 2 viruses have the potential to overcome these limitations. Therefore, we directly compared a rAAV type 1 to a type 2 vector in the murine brain. The vectors were engineered to carry identical genomes (AAV2 terminal repeat elements flanking an enhanced green fluorescent protein expression cassette) and were administered by stereotaxic-guided intracerebral injection. We found that the rAAV1 vector (rAAV1-GFP) had a 13- to 35-fold greater transduction efficiency than that of the rAAV2 vector (rAAV2-GFP). Also, rAAV1-transduced cells were observed at a greater distance from the injection site than rAAV2-transduced cells. Neurons were the predominant cell type transduced by both vector types. However, in contrast to rAAV2-GFP, rAAV1-GFP was capable of transducing glial and ependymal cells. Thus, rAAV1-based vectors have biologic properties within the brain distinct from that of rAAV2. These differences might be capitalized upon to develop novel gene transfer strategies for neurologic disorders.  

5.138           Targeting recombinant adeno-associated virus vectors to enhance gene transfer to pancreatic islets and liver

Loiler, S.A. et al Gen. Ther., 10, 1551-1558 (2003)   Human pancreatic islet cells and hepatocytes represent the two most likely target cells for genetic therapy of type I diabetes. However, limits to the efficiency of rAAV serotype 2 (rAAV2)-mediated gene transfer have been reported for both of these cell targets. Here we report that nonserotype 2 AAV capsids can mediate more efficient transduction of islet cells, with AAV1 being the most efficient serotype in murine islets, suggesting that receptor abundance could be limiting. In order to test this, we generated rAAV particles that display a ligand (ApoE) that targets the low-density lipoprotein receptor, which is present on both of these cell types. The rAAVApoE viruses greatly enhanced the efficiency of transduction of both islet cells ex vivo and murine hepatocytes in vivo when compared to native rAAV2 serotype (220- and four-fold, respectively). The use of receptor-targeted rAAV particles may circumvent the lower abundance of receptors on certain nonpermissive cell types.  

5.139           Attenuation of seizures and neuronal death by adeno-associated virus vector galanin expression and secretion

Haberman, R.P., Samulski, R.J. and McCown, T.J. Nature Med., 9(8), 1076-1080 (2003)   Seizure disorders present an attractive gene therapy target, particularly because viral vectors such as adeno-associated virus (AAV) and lentivirus can stably transduce neurons^1-3. When we targeted the N-methyl-D-aspartic acid (NMDA) excitatory amino acid receptor with an AAV-delivered antisense oligonucleotide, however, the promoter determined whether focal seizure sensitivity was significantly attenuated or facilitated⁴. One potential means to circumvent this liability would be to express an inhibitory neuroactive peptide and constitutively secrete the peptide from the transduced cell. The neuropeptide galanin can modulate seizure activity in vivo^{5, 6}, and the laminar protein fibronectin is usually secreted through a constitutive pathway^{7, 8}. Initially, inclusion of the fibronectin secretory signal sequence (FIB)⁹ in an AAV vector caused significant gene product secretion in vitro. More importantly, the combination of this secretory signal with the coding sequence for the active galanin peptide significantly attenuated in vivo focal seizure sensitivity, even with different promoters, and prevented kainic acid–induced hilar cell death. Thus, neuroactive peptide expression and local secretion provides a new gene therapy platform for the treatment of neurological disorders.  

5.140           The Mason-Pfizer monkey virus PPPY and PSAP motifs both contribute to virus release

Gottwein, E. et al

1. Virol., 77(17), 9474-9485 (2003)

  Late (L) domains are required for the efficient release of several groups of enveloped viruses. Three amino acid motifs have been shown to provide L-domain function, namely, PPXY, PT/SAP, or YPDL. The retrovirus Mason-Pfizer monkey virus (MPMV) carries closely spaced PPPY and PSAP motifs. Mutation of the PPPY motif results in a complete loss of virus release. Here, we show that the PSAP motif acts as an additional L domain and promotes the efficient release of MPMV but requires an intact PPPY motif to perform its function. Examination of HeLaP4 cells expressing PSAP mutant virus by electron microscopy revealed mostly late budding structures and chains of viruses accumulating at the cell surface with little free virus. In the case of the PPPY mutant virus, budding appeared to be mostly arrested at an earlier stage before induction of membrane curvature. The cellular protein TSG101, which interacts with the human immunodeficiency virus type 1 (HIV-1) PTAP L domain, was packaged into MPMV in a PSAP-dependent manner. Since TSG101 is crucial for HIV-1 release, this result suggests that the Gag-TSG101 interaction is responsible for the virus release function of the MPMV PSAP motif. Nedd4, which has been shown to interact with viral PPPY motifs, was also detected in MPMV particles, albeit at much lower levels. Consistent with a role of VPS4A in the budding of both PPPY and PTAP motif-containing viruses, the overexpression of ATPase-defective GFP-VPS4A fusion proteins blocked both wild-type and PSAP mutant virus release.  

5.141           Anti-apoptotic effects of CNTF gene transfer on photoreceptor degeneration in experimental antibody-induced retinopathy

Adamus, G., Sugden, B., Shiraga, S., Timmers, A.M. and Hauswirth, W.W.

1. Autoimmun., 21, 121-129 (2003)

  Autoantibodies against recoverin are found in the sera of patients with cancer-associated retinopathy syndrome, a paraneoplastic disease associated with retinal degeneration. We have previously shown that anti-recoverin autoantibodies induced photoreceptor apoptotic cell death after injection into the vitreous of Lewis rats. Ciliary neurotrophic factor (CNTF) has been shown to promote the survival of a number of neuronal cell types, including photoreceptors. In this study, we examined whether an adeno-associated virus (AAV)-mediated delivery of gene encoding the human CNTF protected photoreceptor cells from anti-recoverin antibody-induced death. One month after subretinal injection of the AAV–CNTF gene into one eye and a control vector into the other eye, an anti-recoverin antibody was injected to induce retinal cell death in Lewis rats. Subretinal administration of the virus led to an efficient transduction of photoreceptors, as indicated by immunostaining of retinas with anti-CNTF. Histological examination of the corresponding retinas showed that photoreceptor cells were significantly protected from apoptotic death in the CNTF-treated eyes. CNTF treatment of the retinas resulted in a time-dependent activation of STAT 3. The present study shows that an AAV-mediated delivery of CNTF may protect photoreceptors from antibody-induced cell death through the activation of STAT3 and the suppression of caspase 3 activity, a key caspase leading to apoptosis. Thus, CNTF may be a useful treatment for human antibody- mediated retinal degeneration.  

5.142           Practical considerations of recombinant adeno-associated virus-mediated gene transfer for treatment of retinal degenerations

Shen, W-Y. et al

1. Gene Med.,5, 576-587 (2003)

  BACKGROUND: Photoreceptor (PR) and retinal pigment epithelium (RPE) are the principal cell targets in retinal gene therapy. Recombinant adeno-associated virus (rAAV) has emerged as a very promising vector for gene therapy in hereditary retinal diseases. Gene transfer at different stages of the disease is a practical consideration for future clinical application. METHODS: A rAAV carrying the enhanced green fluorescent protein gene driven by a cytomegalovirus promoter was produced by either co-infecting the 293 cell line with E1-defective adenovirus and purified by CsCl(2) density gradient (CsCl(2)-rAAV), or by transfecting with an adenoviral helper plasmid and purified by iodixanol density gradient followed by heparin column chromatography (heparin-rAAV). The impact of different virus preparations on the patterns of transgene expression was investigated after subretinal injection. Furthermore, rAAV-mediated gene transfer was evaluated at both early and advanced stages of retinal degeneration in four disease models including the RCS rat, rd, RPE(65) (-)/(-) and cathepsin D mutant mice that are associated with PR- or RPE-related gene defects. RESULTS: CsCl(2)-rAAV predominantly transduced RPE and with less efficiency in PR. In contrast, heparin-rAAV predominantly transduced PR but with much less efficiency in RPE. Subretinal injection of either rAAV preparation induced no changes to retinal morphology and retinal-choroidal vasculature. The product of transgene, however, could be observed in multiple tracts in the brain. In the four disease models, target cells were efficiently transduced not only at the early stage, but also at the late stage of disease as long as the target cells were present. CONCLUSIONS: Different preparations of rAAV have an impact on the patterns of transgene expression after subretinal injection. Patients at advanced stages of retinal degeneration may still benefit from rAAV-mediated gene therapy. The possible side effects of transgenic products on the central nervous system should be carefully monitored once therapeutic genes are employed.  

5.143           Construction and characterization of a full-length infectious molecular clone from a fast replicating, X4-tropic HIV-1 CRF02.AG primary isolate

Tebit, D.M., Zekeng, L., Kaptue, J., Fräusslich, H-G- and Herchenröder, O. Virology, 313, 645-652 (2003)   Based on our previous analysis of HIV-1 isolates from Cameroon, we constructed a full-length infectious molecular clone from a primary isolate belonging to the CRF02.AG group of recombinant viruses which dominate the HIV-epidemic in West and Central Africa. The virus derived by transfection of the proviral clone pBD6-15 replicated with similar efficiency compared to its parental isolate and used CXCR4 as coreceptor as well. Furthermore, HIV-1 BD6-15 exhibited similar replication properties and virus yield as the reference B-type HIV-1 strain NL4-3. Sequence analysis revealed open reading frames for all structural and accessory genes apart from vpr. Phylogenetic and bootscanning analyses confirmed that BD6-15 clusters with CRF02.AG recombinant strains from West and Central Africa with similar cross-over points as described for the CRF02.AG prototype strain lbNG. Thus, pBD6-15 represents the first non-subtype B infectious molecular clone of a fast replicating, high producer, X4-tropic primary HIV-1 isolate, which had only been briefly passaged in primary cells.  

5.144           Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors

Müller, O.J. et al Nature Biotechnology, 21(9), 1040-1046 (2003)   Characterizing the molecular diversity of the cell surface is critical for targeting gene therapy. Cell type–specific binding ligands can be used to target gene therapy vectors. However, targeting systems in which optimum eukaryotic vectors can be selected on the cells of interest are not available. Here, we introduce and validate a random adeno-associated virus (AAV) peptide library in which each virus particle displays a random peptide at the capsid surface. This library was generated in a three-step system that ensures encoding of displayed peptides by the packaged DNA. As proof-of-concept, we screened AAV-libraries on human coronary artery endothelial cells. We observed selection of particular peptide motifs. The selected peptides enhanced transduction in coronary endothelial cells but not in control nonendothelial cells. This vector targeting strategy has advantages over other combinatorial approaches such as phage display because selection occurs within the context of the capsid and may have a broad range of applications in biotechnology and medicine.  

5.145           Novel tools for production and purification of recombinant adeno-associated viral vectors

Harris, J.D., Beattie, S.G. and Dickson, J.G. Methods Mol. Med., 76(7), 255-267 (2003)   No abstract available  

5.146           Gene therapy with brian-derived neurotrophic factor as a protection: retinal ganglion cells in a rat glaucoma model

Martin, K.R.G. et al Invest. Ophtalmol. Vis. Sci., 44 4357-4365 (2003)   PURPOSE. To develop a modified adenoassociated viral (AAV) vectorcapable of efficient transfection of retinal ganglion cells(RGCs) and to test the hypothesis that use of this vector toexpress brain-derived neurotrophic factor (BDNF) could be protectivein experimental glaucoma. METHODS. Ninety-three rats received one unilateral, intravitreal injection of either normal saline (n = 30), AAV-BDNF-woodchuck hepatitis posttranscriptional regulatory element (WPRE; n = 30), or AAV-green fluorescent protein (GFP)-WPRE (n = 33). Twoweeks later, experimental glaucoma was induced in the injectedeye by laser application to the trabecular meshwork. Survivalof RGCs was estimated by counting axons in optic nerve crosssections after 4 weeks of glaucoma. Transgene expression wasassessed by immunohistochemistry, Western blot analysis, anddirect visualization of GFP. RESULTS. The density of GFP-positive cells in retinal wholemounts was 1,828 ± 299 cells/mm² (72,273 ± 11,814 cells/retina). Exposure to elevated intraocular pressure was similar in all groups. Four weeks after initial laser treatment, axon loss was 52.3% ± 27.1% in the saline-treated group (n = 25) and 52.3% ± 24.2% in the AAV-GFP-WPRE group (n = 30), but only 32.3% ± 23.0% in the AAV-BDNF-WPRE group (n = 27). Survival in AAV-BDNF-WPRE animals increased markedly and the difference was significant compared with those receiving either AAV-GFP-WPRE (P = 0.002, t-test) or saline (P = 0.006, t-test). CONCLUSIONS. Overexpression of the BDNF gene protects RGC asestimated by axon counts in a rat glaucoma model, further supportingthe potential feasibility of neurotrophic therapy as a complementto the lowering of IOP in the treatment of glaucoma.  

5.147           Identification of a heparin-binding motif on adeno-associated virus type 2 capsids

Kern, A. et al

1. Virol., 77(20), 11072-11081 (2003)

  Infection of cells with adeno-associated virus (AAV) type 2 (AAV-2) is mediated by binding to heparan sulfate proteoglycan and can be competed by heparin. Mutational analysis of AAV-2 capsid proteins showed that a group of basic amino acids (arginines 484, 487, 585, and 588 and lysine 532) contribute to heparin and HeLa cell binding. These amino acids are positioned in three clusters at the threefold spike region of the AAV-2 capsid. According to the recently resolved atomic structure for AAV-2, arginines 484 and 487 and lysine 532 on one site and arginines 585 and 588 on the other site belong to different capsid protein subunits. These data suggest that the formation of the heparin-binding motifs depends on the correct assembly of VP trimers or even of capsids. In contrast, arginine 475, which also strongly reduces heparin binding as well as viral infectivity upon mutation to alanine, is located inside the capsid structure at the border of adjacent VP subunits and most likely influences heparin binding indirectly by disturbing correct subunit assembly. Computer simulation of heparin docking to the AAV-2 capsid suggests that heparin associates with the three basic clusters along a channel-like cavity flanked by the basic amino acids. With few exceptions, mutant infectivities correlated with their heparin- and cell-binding properties. The tissue distribution in mice of recombinant AAV-2 mutated in R484 and R585 indicated markedly reduced infection of the liver, compared to infection with wild-type recombinant AAV, but continued infection of the heart. These results suggest that although heparin binding influences the infectivity of AAV-2, it seems not to be necessary.  

5.148           HIV-1 Vif blocks the antiviral activity of APOBEC3G by impairing both its translation and intracellular stability

Stopak, K., de Noronha, C., Yonemoto, W. And Greene, W.C. Mol. Cell., 12, 591-601 (2003)   The human immunodeficiency virus type 1 (HIV-1) relies on Vif (viral infectivity factor) to overcome the potent antiviral function of APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, also known as CEM15). Using an APOBEC3G-specific antiserum, we now show that Vif prevents virion incorporation of endogenous APOBEC3G by effectively depleting the intracellular levels of this enzyme in HIV-1-infected T cells. Vif achieves this depletion by both impairing the translation of APOBEC3G mRNA and accelerating the posttranslational degradation of the APOBEC3G protein by the 26S proteasome. Vif physically interacts with APOBEC3G, and expression of Vif alone in the absence of other HIV-1 proteins is sufficient to cause depletion of APOBEC3G. These findings highlight how the bimodal translational and posttranslational inhibitory effects of Vif on APOBEC3G combine to markedly suppress the expression of this potent antiviral enzyme in virally infected cells, thereby effectively curtailing the incorporation of APOBEC3G into newly formed HIV-1 virions.  

5.149           The protein network of HIV budding

Von Schwedler, U.K. et al Cell, 114, 701-713 (2003)   HIV release requires TSG101, a cellular factor that sorts proteins into vesicles that bud into multivesicular bodies (MVB). To test whether other proteins involved in MVB biogenesis (the class E proteins) also participate in HIV release, we identified 22 candidate human class E proteins. These proteins were connected into a coherent network by 43 different protein-protein interactions, with AIP1 playing a key role in linking complexes that act early (TSG101/ESCRT-I) and late (CHMP4/ESCRT-III) in the pathway. AIP1 also binds the HIV-1 p6^Gag and EIAV p9^Gag proteins, indicating that it can function directly in virus budding. Human class E proteins were found in HIV-1 particles, and dominant-negative mutants of late-acting human class E proteins arrested HIV-1 budding through plasmal and endosomal membranes. These studies define a protein network required for human MVB biogenesis and indicate that the entire network participates in the release of HIV and probably many other viruses.  

5.150           Central leptin gene therapy fails to oversome leptin resistance associated with diet-induced obesity

Wilsey, J., Zolotukhin, S., Prima, V. and Scarpace, P.J. Am. J. Physiol. Interg. Comp. Physiol.285, R1101-R1020 (2003)   The objective of this study was to determine if central overexpression of leptin could overcome the leptin resistance caused by 100 days of high-fat feeding. Three-month old-F344XBN male rats were fed either control low fat chow (Chow), which provides 15% of energy as fat, or a high-fat/high-sucrose diet (HF), which provides 59% of energy as fat. Over several weeks, the HF-fed animals spontaneously split into two groups of animals: those that became obese on the HF diet (DIO) and those that did not gain extra weight on the HF diet [diet resistant (DR)]. After 100 days of HF feeding, animals were given a single intracerebroventricular injection containing 5.75E10 particles of rAAV encoding leptin (rAAV-leptin) or control virus (rAAV-con). Chow animals responded robustly to rAAV-leptin, including significant anorexia, weight loss, and lipopenia. In contrast, DIO were completely unresponsive to rAAV-leptin. DR rats responded to rAAV-leptin, but in a more variable fashion than Chow. Unlike what was observed in Chow, the anorectic response to rAAV-leptin rapidly attenuated and was no longer significant by day 14 postvector delivery. Both DIO and DR animals were found to have reduced long-form leptin receptor expression and enhanced basal P-STAT-3 in the hypothalamus with respect to Chow. rAAV-leptin caused an increase in STAT3 phosphorylation and proopiomelanocortin expression in the hypothalamus and an increase in uncoupling protein-1 in brown adipose tissue in both Chow and DR animals, but failed to do so in DIO. This suggests that central overexpression of leptin is not a viable strategy to reverse diet-induced obesity.  

5.151           Lassa virus Z protein is a matrix protein sufficient for the release of virus-like particles

Strecker, T. et al

1. Virol., 77(19), 10700-10705 (2003)

  Lassa virus is an enveloped virus with glycoprotein spikes on its surface. It contains an RNA ambisense genome that encodes the glycoprotein precursor GP-C, the nucleoprotein NP, the polymerase L, and the Z protein. Here we demonstrate that the Lassa virus Z protein (i) is abundant in viral particles, (ii) is strongly membrane associated, (iii) is sufficient in the absence of all other viral proteins to release enveloped particles, and (iv) contains two late domains, PTAP and PPXY, necessary for the release of virus-like particles. Our data provide evidence that Z is the Lassa virus matrix protein that is the driving force for virus particle release.  

5.152           Dissociation of rabies virus matrix protein functions in regulation of viral RNA synthesis and virus assembly

Finke, S. and Conzelmann, K-K.

1. Virol., 77(22), 12704-12082 (2003)

  Recently, we have shown that the rabies virus (RV) matrix (M) protein regulates the balance of virus RNA synthesis by shifting synthesis activity from transcription to replication (S. Finke, R. Mueller-Waldeck, and K. K. Conzelmann, J. Gen. Virol. 84:1613-1621, 2003). Here we describe the identification of an M residue critical for regulation of RV RNA synthesis. By analyzing the phenotype of heterotypic RV M proteins with respect to RNA synthesis of RV SAD L16, we identified the M proteins of the RV ERA and PV strains as deficient. Comparison of M sequences suggested that a single residue, arginine 58, was critical. A recombinant virus having this amino acid exchanged with a glycine, SAD M(R58G), has lost the abilities to downregulate RV transcription and to stimulate replication. This resulted in an increase in the transcription rate of more than 15-fold, as previously observed for M deletion mutants. Most importantly, the efficiencies of virus assembly and budding were equal for wild-type M and M(R58G), as determined in assays studying the transient complementation of an M- and G-deficient RV construct, NPgrL. In addition, virus particle density, protein composition, and specific infectivity of SAD L16 and SAD M(R58G) viruses were identical. Thus, we have identified mutations that affect the function of M only in regulation of RNA synthesis, but not in assembly and budding, providing evidence that these functions are genetically separable.  

5.153           Recombinant adeno.associated virus: formulation challenges and strategies for a gene therapy vector

Fraser Wright, J., Chunlin Tang, G.Q. and Sommer, J.M. Curr. Opin. Drug Discov. Devel., 6(2), 174-178 (2003)   Recombinant adeno-associated virus (AAV)-based vectors capable of expressing therapeutic gene products in vivo have shown significant promise for human gene therapy. One challenge facing the field is the development of vector formulations to achieve optimal vector safety, stability and efficacy. Formulation challenges for AAV vectors can be divided into those relating to maintaining vector activity during purification and storage, and those relating to efficient target tissue transduction in vivo. AAV vectors are potentially susceptible to loss of activity through aggregation, proteolysis and oxidation, as well as through non-specific binding to product contact materials used for vector purification and storage. These deleterious changes need to be thoroughly characterized, and the conditions and excipients to prevent them need to be identified. For in vivo administration, major vector formulation challenges include optimization of efficiency and specificity of target tissue transduction, and the ability to overcome host immune responses.  

5.154           Brain-derived neurotrophic factor in the ventral midbrain-nucleus accumbens pathway: a role in depression

Eisch, A.J. et al Biol. Psychiatry., 54, 994-1005 (2003)   Previous work has shown that brain-derived neurotrophic factor (BDNF) and its receptor, tyrosine kinase receptor B (TrkB), are involved in appetitive behavior. Here we show that BDNF in the ventral tegmental area–nucleus accumbens (VTA–NAc) pathway is also involved in the development of a depression-like phenotype. Brain-derived neurotrophic factor signaling in the VTA–NAc pathway was altered in two complementary ways. One group of rats received intra-VTA infusion of vehicle or BDNF for 1 week. A second group of rats received intra-NAc injections of vehicle or adeno-associated viral vectors encoding full-length (TrkB.FL) or truncated (TrkB.T1) TrkB; the latter is kinase deficient and serves as a dominant-negative receptor. Rats were examined in the forced swim test and other behavioral tests. Intra-VTA infusions of BDNF resulted in 57% shorter latency to immobility relative to control animals, a depression-like effect. Intra-NAc injections of TrkB.T1 resulted in and almost fivefold longer latency to immobility relative to TrkB.FL and control animals, an antidepressant-like effect. No effect on anxiety-like behaviors or locomotion was seen. These data suggest that BDNF action in the VTA–NAc pathway might be related to development of a depression-like phenotype. This interpretation is intriguing in that it suggests a role for BDNF in the VTA–NAc that is opposite of the proposed role for BDNF in the hippocampus.  

5.155           Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo

McCarty, D.M. et al Gen. Ther., 10, 2112-2118 (2003)   An important limitation of recombinant adeno-associated virus (rAAV) vector efficiency is the requirement of hostcell-mediated synthesis of double-stranded DNA from the single-stranded genome. We have bypassed this step in a specialized self-complementary rAAV (scAAV) vector, by utilizing the tendency of AAV to package DNA dimers when the replicating genome is half the length of the wild type (wt). To produce these vectors efficiently, we have deleted the terminal resolution site (trs) from one rAAV TR, preventing the initiation of replication at the mutated end. These constructs generate single-stranded, inverted repeat genomes, with a wt TR at each end, and a mutated TR in the middle. After uncoating, the viral DNA folds through intramolecular base pairing within the mutant TR, which then proceeds through the genome to form a double-stranded molecule. We have used the scAAV to investigate barriers to rAAV transduction in the mouse liver, muscle and brain. In each tissue, scAAV was characterized by faster onset of gene expression and higher transduction efficiency. This study confirms earlier predictions that complementary-strand DNA synthesis is the primary barrier to rAAV-2 transduction. The scAAV is unaffected by this barrier, and provides an extremely efficient vector for gene transfer into many types of cells in vivo.    

5.156           Genetic modifications of the adeno-associated virus type 2 capsid reduce the affinity and the neutralizing effects of human serum antibodies

Huttner, N.A. et al Gen. Ther., 10, 2139-2147 820039   The high prevalence of human serum antibodies against adeno-associated virus type 2 (AAV) vectors represents a potential limitation for in vivo applications. Consequently, the development of AAV vectors able to escape antibody binding and neutralization is of importance. To identify capsid domains which contain major immunogenic epitopes, six AAV capsid mutants carrying peptide insertions in surface exposed loop regions (I-261, I-381, I-447, I-534, I-573, I-587) were analyzed. Two of these mutants, I-534 and I-573, showed an up to 70% reduced affinity for AAV antibodies as compared to wild-type AAV in the majority of serum samples. In addition, AAV mutant I-587 but not wild-type AAV efficiently transduced cells despite the presence of neutralizing antisera. Taken together, the results show that major neutralizing effects of human AAV antisera might be overcome by the use of AAV capsid mutants.  

5.157           Chromsomal integration and homologous gene targeting by replication-incompetent vectors based on the autonomous parvovirus minute virus mice

Hendric, P.C., Hirata, R.K. and Russell, D.W.

1. Virol., 77(24), 13136-13145 (2003)

  The molecular mechanisms responsible for random integration and gene targeting by recombinant adeno-associated virus (AAV) vectors are largely unknown, and whether vectors derived from autonomous parvoviruses transduce cells by similar pathways has not been investigated. In this report, we constructed vectors based on the autonomous parvovirus minute virus of mice (MVM) that were designed to introduce a neomycin resistance expression cassette (neo) into the X-linked human hypoxanthine phosphoribosyl transferase (HPRT) locus. High-titer, replication-incompetent MVM vector stocks were generated with a two-plasmid transfection system that preserved the wild-type characteristic of packaging only one DNA strand. Vectors with inserts in the forward or reverse orientations packaged noncoding or coding strands, respectively. In human HT-1080 cells, MVM vector random integration frequencies (neo⁺ colonies) were comparable to those obtained with AAV vectors, and no difference was observed for noncoding and coding strands. HPRT gene-targeting frequencies (HPRT mutant colonies) were lower with MVM vectors, and the noncoding strand frequency was threefold greater than that of the coding strand. Random integration and gene-targeting events were confirmed by Southern blot analysis of G418- and 6-thioguanine (6TG)-resistant clones. In separate experiments, correction of an alkaline phosphatase (AP) gene by gene targeting was nine times more effective with a coding strand vector. The data suggest that single-stranded parvoviral vector genomes are substrates for gene targeting and possibly for random integration as well.  

5.158           Impurity of recombinant adeno-associated virus type 2 affects the transduction characteristics following subretinal injection in the rat

Shen, W-Y., Lai, Y.K.Y., Lai, C-M. and Rakoczy, P.E. Vision Res., 44, 339-348 (2003)   We recently reported that different purification methods of recombinant adeno-associated virus type 2 (rAAV2) affect the transduction characteristics following subretinal injection. In this study, we examined the roles of contaminant proteins from the HEK-293 cells and helper adenovirus, inactivation of helper adenovirus and cell stress induced by DNA-damaging agents in rAAV-mediated retinal transduction. Our results showed that contaminating factors/proteins resulting from the helper E1 deleted adenovirus are possibly responsible for efficient RPE transduction. Future studies of these factors will undoubtedly lead to development of new therapeutic approaches to PR- and RPE-specific retinal diseases.  

5.159           Local gene knowdown in the brain using viral-mediated RNA interference

Hommel, J.D., Sears, R.M., Georgescu, D., Simmoms, D.L. and DiLeone, R.J. Nature Med., 9(12), 1539-1544 (2003)   Conditional mutant techniques that allow spatial and temporal control over gene expression can be used to create mice with restricted genetic modifications. These mice serve as powerful disease models in which gene function in adult tissues can be specifically dissected. Current strategies for conditional genetic manipulation are inefficient, however, and often lack sufficient spatial control. Here we use viral-mediated RNA interference (RNAi) to generate a specific knockdown of Th, the gene encoding the dopamine synthesis enzyme tyrosine hydroxylase, within midbrain neurons of adult mice. This localized gene knockdown resulted in behavioral changes, including a motor performance deficit and reduced response to a psychostimulant. These results underscore the potential of using viral-mediated RNAi for the rapid production and testing of new genetic disease models. Similar strategies may be used in other model species, and may ultimately find applications in human gene therapy.  

5.160           Non-small lung cancer cells are prime targets for p53 gene transfer mediated by a recombinant adeno-associated virus type-2 vector

Rohr, U-P. et al Cenc. Gen. Ther., 10, 898-906 (2003)   In this study, we elucidated the potential of recombinant adeno-associated virus type-2 (rAAV-2) vectors for lung cancer gene therapy. Cell lines of the three major histological subtypes of non-small cell lung cancer (NSCLC) were highly susceptible for rAAV-2 showing transduction rates between 63.4 and 98.9%. In contrast, cell lines of small cell carcinomas were resistant to rAAV-2 infection. For restoration of p53 function in p53 deficient NSCLC, a rAAV-2 vector was constructed containing wt p53 cDNA. Following transduction with rAAV-p53, cell growth of all NSCLC cell lines was significantly reduced in a dose-dependent manner between 44 and 71.7% in comparison with rAAV-GFP transduced cells. The reduction of tumor cell growth was associated with increased apoptosis. Adding cisplatin to rAAV-p53-infected cells led to a significant growth inhibition between 81 and 91% indicating a synergistic effect between cisplatin and rAAV-p53. Interestingly, the tumor cells surviving cisplatin and rAAV-p53 treatment were inhibited in their ability to form colonies as reflected by a reduction of colony growth between 57 and 90.4%. In conclusion, rAAV-2 vectors exhibit a strong tropism for NSCLC. Successful inhibition of tumor cell growth following transduction with a rAAV-p53 vector underlines the potential role of rAAV-2 in cancer gene therapy.  

5.161           Enhancement of gene transfer with recombinant adeno-associated virus (rAAV) vectors into primary B-cell chronic lymphocytic leukemia cells by CpG-oligodeoxynucleotides

Theiss, H.D. et al Exp. Hematol., 31, 1223-1229 (2003)  

Objective

Transduction of primary B-cell chronic lymphocytic leukemia (B-CLL) cells with recombinant adeno-associated virus (rAAV) vectors is dependent on preactivation of leukemic cells by CD40L. CpG-oligodeoxynucleotides (CpG-ODNs) are able to activate cytokine production and proliferation of B-CLL cells. Therefore CpG-ODNs were tested for their potential to enhance transgene expression in CLL cells.

Materials and methods

Using an optimized adenovirus-free packaging system, rAAV vectors coding for the enhanced green fluorescent protein (AAV/EGFP) were packaged and highly purified resulting in infectious titers up to 5×10⁹/mL. Cells obtained from patients with B-CLL were infected with AAV/EGFP at a multiplicity of infection of 100 while being stimulated with CpG-ODNs and/or CD40L-expressing HeLa/SF cells. Transgene expression was assessed after 48 hours by flow cytometry.

Results

Stimulation of B-CLL cells by CpG-ODNs resulted in up-regulation of costimulatory molecules and G₁/S-phase transition at similar levels compared to activation by HeLa/SF cells, but use of CpG-ODNs alone did not result in any efficient AAV/EGFP transduction. Combined stimulation of B-CLL cells with HeLa/SF cells and CpG-ODNs during AAV/EGFP transduction significantly enhanced transgene expression compared to feeder stimulation alone (p = 0.004). In addition, the copy number per single cell was significantly increased by addition of CpG-ODNs as detected by quantitative real-time PCR (p = 0.04). Use of self-complementary AAV vectors that are not dependent on target cell DNA synthesis did not result in increased transgene expression compared to single-stranded AAV vectors (p = 0.30).

Conclusion

Stimulation by CD40L is crucial for efficient gene transfer into B-CLL cells by rAAV vectors, whereas transduction efficiency can be significantly enhanced by CpG-ODNs.  

5.162           Adeno-associated virus-mediated gene transfer of a secreted decoy human macrophage scavenger receptor reduces atherosclerotic lesion formation in LDL receptor knockout mice

Jalkanen, J. Et al Mol. Ther., 8(6), 903-910 (2003)   Macrophage scavenger receptors (MSR) promote atherosclerotic lesion formation, and modulation of MSR activity has been shown to influence atherosclerosis. Soluble receptors are effective in inhibiting receptor-mediated functions in various diseases. We have generated a secreted macrophage scavenger receptor (sMSR) that consists of the bovine growth hormone signal sequence and the human MSR A I extracellular domains. sMSR reduces degradation of atherogenic modified low-density lipoproteins and monocyte/macrophage adhesion on endothelial cells in vitro. To test long-term effects of sMSR, atherosclerosis-susceptible LDLR knockout mice were transduced via the tail vein with an adeno-associated virus (AAV) expressing sMSR or control enhanced green fluorescent protein (EGFP), and a Western-type diet was started. Gene transfer caused a temporary elevation in alkaline phosphatase and aspartate amino transferase values without a change in C-reactive protein. sMSR protein was detected in the plasma of the transduced mice by a specific ELISA 6 months after the gene transfer. AAV-mediated sMSR gene transfer reduced atherosclerotic lesion area in the aorta by 21% (P < 0.05) compared to EGFP-transduced control mice. Even though eradication of established disease was not possible, atherosclerotic lesion formation could be modified using AAV-mediated gene transfer of the decoy sMSR.  

5.163           An endogenous retrovirus derived from human melanoma cells

Muster, T. et al Cancer Res., 63, 8735-8741 (2003)   We show that human melanoma cells produce retrovirus-like particles that exhibit reverse transcriptase activity, package sequences homologous to human endogenous retrovirus K (HERV-K), and contain mature forms of the Gag and Env proteins. We also demonstrate expression of the pol gene and of Gag, Env, and Rec proteins in human melanomas and metastases but not in melanocytes or normal lymph nodes. The data suggest that expression of retroviral genes and production of retroviral particles is activated during development of melanoma.  

5.164           The Vif protein of human immunodeficiency virus type 1 (HIV-1): enigmas and solutions

Baraz, L. and Kotler, M. Current Medicinal Chem. 11, 221-231 (2003)   HIV-1 and other complex retroviruses express six auxiliary genes in addition to the canonical retroviral genes, gag, pol and env. Vif (virion infectivity factor) protein is absolutely essential for productive HIV-1 infection of peripheral blood lymphocytes and macrophages, the two major HIV-1 target cells in vivo. However, Vif is not required for production of infectious particles in several human cell lines. In spite of the prominent phenotype of Vif mutations, the mechanism of its action remains unknown. During the last decade several models were suggested to explain the mechanism of Vif activity. One view holds  that Vif  is  active  in  virions  after  budding or  after  entry into target cells during the early stages of HIV-1 replications. The second view places the action of Vif at the late stage of HIV-1 replication in virus producing cells, which affects the production of infectious virus. According to this view, Vif either compensates the cell factor required for production of infectious virus, or alternatively, it neutralizes a cell factor, which prevents the production of infectious particles in these cells. This review is addressed to summarize the models envisioned to explain Vif activities. The findings described here, that Vif interacts with viral and cellular components, elaborates the importance of Vif as a novel target for developing anti HIV-1 drugs.  

5.165           Sustained tetracycline-regulated transgene expression in vivo in rat retinal ganglion cells using a single type 2 adeno-associated viral vector

Folliot, S., Briot, D., Conrath, H., Provost, N., Cherel, Y., Moullier, P. and Rolling, F.

1. Gene Med., 5(6), 493-501 (2003)

  Background Viral vector delivery of neurotrophic-expressing transgenes in the retina may retard or prevent the onset of blindness associated with photoreceptor degeneration. A key safety issue is to achieve regulated expression of these genes in the retina. The purpose of our study was to evaluate whether a single recombinant AAV-2 (rAAV) encoding for a tetracycline (Tet)-regulated destabilized reporter gene could provide quantitative profiles of gene regulation targeted to the rat neuroretina. Methods A rAAV vector carrying a destabilized green fluorescent protein (dgfp) under a tet-regulatable promoter and the tetracycline-repressed transactivator (tTA) was generated (rAAVtetoff.dgfp) and administered intravitreally in nine Wistar rats. Retinas were monitored for 6 months using noninvasive fluorescence imaging and the animals were subjected to two cycles of doxycycline (Dox), a tetracycline analog. Eyes were ultimately examined by histology. Results Intravitreal injection of rAAVtetoff.dgfp resulted in effective transduction of ganglion cells. Following full expression of the transgene in the absence of Dox, 95% of the GFP signal was shut down 48 h post Dox administration and the signal was undetectable 7 days later. Initial levels of GFP expression were restored 21 days after Dox administration ceased. This pattern of expression was repeated twice over a period of 6 months. Conclusions This report demonstrates that rAAVtetoff.dgfp intravitreally injected rats displayed tight and sustained long-term regulation of the reporter gene in ganglion cells. These findings may have important implications regarding rAAV-mediated gene therapy using neuroprotective approaches for retinitis pigmentosa and glaucoma.  

5.166           Suppression of complex I gene expression induces optic neuropathy

Qi, X., Lewin, A.S.., Hauswirth, W.W. and Guy, J. Ann. Neurol., 53, 198-205 (2003)   Optic nerve degeneration is a feature common to diseases with mutations in genes that encode complex I of the respiratory chain. Vulnerability of this central nervous system tract is a mystery, because of the paucity of animal models used to investigate effects of the mutated DNA in tissues rather than isolated in cultured cells. Using a ribozyme designed to degrade the mRNA encoding a critical nuclear-encoded subunit gene of complex I (NDUFA1), we tested whether oxidative phosphorylation deficiency can recapitulate the optic neuropathy of mitochondrial disease. Injection of adenoassociated virus expressing this ribozyme led to axonal destruction and demyelination, the hallmarks of Leber hereditary optic neuropathy.  

5.167           Use of the Herpes Simplex Viral Genome to Construct Gene Therapy Vectors

Burton, E.A., Huang, S., Goins, W.F. and Glorioso, J.C. Methods in Mol. Med., 76, 1-31 (2003)   Abstract not available.  

5.168           Targeted Integration by Adeno-Associated Virus

Weitzman, M., Young Jr., S.M., Cathhomen, T. and Samulski, T.J. Methods in Mol. Med., 76, 201-219 (2003)   The integration of foreign DNA into the genomes of host cells is of fundamental importance for viral oncology, evolution, transgenic organisms, and gene therapy applications. Expression of foreign genes in eukaryotic cells is highly dependent upon the effi ciency of integration events and the site of insertion. Integration can sometimes have detrimental effects on the host cell, such as insertional mutagenesis or activation of proto-oncogenes. For gene therapy, it would therefore be attractive to target insertion to innocuous chromosomal sites. Understanding the mechanisms for integration of foreign DNA and target site selection is crucial for approaches where long-term expression from delivered transgenes is required. One of the most promising viral vector systems is based on the adenoassociated virus (AAV). AAV vectors consist of a transgene cassette fl anked by viral inverted terminal repeats (ITRs). AAV is unique in that the wild-type virus can preferentially integrate its genome in a site-specifi c manner into an integration locus (AAVS1) on human chromosome 19. Understanding the requirements and mechanism for site-specifi c integration will provide insights into how targeting can be incorporated into gene therapy vectors. Targeted vectors will avoid the potential hazards of insertional mutagenesis and will remove the positional effects on gene expression from the integrated provirus. This chapter describes what is known about targeted integration by AAV and assays that have been developed to study this process, in order to harness it for transgene integration.  

5.169           Rapid neurofibrillary tangle formation after localized gene transfer of mutated Tau

Klein, R.L. et al Am. J. Pathol., 164(1), 347-353 (2004)   Neurofibrillary pathology was produced in the brains of adult rats after localized gene transfer of human tau carrying the P301L mutation, which is associated with frontotemporal dementia with parkinsonism. Within 1 month of in situ transfection ofthe basal forebrain region of normal rats, tau-immunoreactiveand argyrophilic neuronal lesions formed. The fibrillar lesionshad features of neurofibrillary tangles and tau immunoreactivityat light and electron microscopic levels. In addition to neurofibrillarytangles, other tau pathology, including pretangles and neuropilthreads, was abundant and widespread. Tau gene transfer to thehippocampal region of amyloid-depositing transgenic mice producedpretangles and threads, as well as intensely tau-immunoreactiveneurites in amyloid plaques. The ability to produce neurofibrillarypathology in adult rodents makes this a useful method to studytau-related neurodegeneration.  

5.170           Spatial and temporal organization of adeno-associated virus DNA replication in live cells

Fraefel, C. Et al

1. Virol., 78(1), 389-398 (2004)

  Upon cell entry, the genomes of herpes simplex virus type 1 (HSV-1) and adenovirus (Ad) associate with distinct nuclear structures termed ND10 or promyelocytic leukemia (PML) nuclear bodies (NBs). PML NB morphology is altered or disrupted by specific viral proteins as replication proceeds. We examined whether adeno-associated virus (AAV) replication compartments also associate with PML NBs, and whether modification or disruption of these by HSV-1 or Ad, both of which are helper viruses for AAV, is necessary at all. Furthermore, to add a fourth dimension to our present view of AAV replication, we established an assay that allows visualization of AAV replication in live cells. A recombinant AAV containing 40 lac repressor binding sites between the AAV inverted terminal repeats was constructed. AAV Rep protein and helper virus-mediated replication of this recombinant AAV genome was visualized by binding of enhanced yellow fluorescent protein-lac repressor fusion protein to double-stranded AAVreplication intermediates. We demonstrate in live cells thatAAV DNA replication occurs in compartments which colocalizewith AAV Rep. Early after infection, the replication compartmentswere small and varied in numbers from 2 to more than 40 percell nucleus. Within 4 to 8 h, individual small replicationcompartments expanded and fused to larger structures which filledout much of the cell nucleus. We also show that AAV replicationcompartments can associate with modified PML NBs in Ad-infectedcells. In wild-type HSV-1-infected cells, AAV replication compartmentsand PML NBs did not coexist, presumably because PML was completelydisrupted by the HSV-1 ICP0 protein. However, alteration ordisruption of PML appears not to be a prerequisite for AAV replication,as the formation of replication compartments was normal whenthe ICP0 mutants HSV-1 dl1403 and HSV-1 FXE, which do not affectPML NBs, were used as the helper viruses; under these conditions,AAV replication compartments did not associate with PML NBs.  

5.171           The Rep protein of adeno-associated virus type 2 interacts with single-stranded DNA-binding proteins that enhance viral replication

Stracker, T.H. et al

1. Virol., 78(1), 441-453 (2004)

  Adeno-associated virus (AAV) type 2 is a human parvovirus whose replication is dependent upon cellular proteins as well as functions supplied by helper viruses. The minimal herpes simplex virus type 1 (HSV-1) proteins that support AAV replication in cell culture are the helicase-primase complex of UL5, UL8, and UL52, together with the UL29 gene product ICP8. We show that AAV and HSV-1 replication proteins colocalize at discrete intranuclear sites. Transfections with mutant genes demonstrate that enzymatic functions of the helicase-primase are not essential. The ICP8 protein alone enhances AAV replication in an in vitro assay. We also show localization of the cellular replication protein A (RPA) at AAV centers under a variety of conditions that support replication. In vitro assays demonstrate that the AAV Rep68 and Rep78 proteins interact with the single-stranded DNA-binding proteins (ssDBPs) of Ad (Ad-DBP), HSV-1 (ICP8), and the cell (RPA) and that these proteins enhance binding and nicking of Rep proteins at the origin. These results highlight the importance of intranuclear localization and suggest that Rep interaction with multiple ssDBPs allows AAV to replicate under a diverse set of conditions.  

5.172           Efficient intracellular assembly of papillomaviral vectors

Buck, C.B., Pastrana, D.V., Lowy, D.R. and Schiller, J.T.

1. Virol., 78(2) 751-757 (2004)

  Although the papillomavirus structural proteins, L1 and L2, can spontaneously coassemble to form virus-like particles, currently available methods for production of L1/L2 particles capable of transducing reporter plasmids into mammalian cells are technically demanding and relatively low-yield. In this report, we describe a simple 293 cell transfection method for efficient intracellular production of papillomaviral-based gene transfer vectors carrying reporter plasmids. Using bovine papillomavirus type 1 (BPV1) and human papillomavirus type 16 as model papillomaviruses, we have developed a system for producing papillomaviral vector stocks with titers of several billion transducing units per milliliter. Production of these vectors requires both L1 and L2, and transduction can be prevented by papillomavirus-neutralizing antibodies. The stocks can be purified by an iodixanol (OptiPrep) gradient centrifugation procedure that is substantially more effective than standard cesium chloride gradient purification. Although earlier data had suggested a potential role for the viral early protein E2, we found that E2 protein expression did not enhance the intracellular production of BPV1 vectors. It was also possible to encapsidate reporter plasmids devoid of BPV1 DNA sequences. BPV1 vector production efficiency was significantly influenced by the size of the target plasmid being packaged. Use of 6-kb target plasmids resulted in BPV1 vector yields that were higher than those with target plasmids closer to the native 7.9-kb size of papillomavirus genomes. The results suggest that the intracellular assembly of papillomavirus structural proteins around heterologous reporter plasmids is surprisingly promiscuous and may be driven primarily by a size discrimination mechanism.  

5.173           Immunity to adeno-associated virus serotype 2 delivered transgenes impared by genetic predisposition to autoimmunity

Zhang, Y.C. et al Gene Ther., 11, 233-240 (2004) Adeno-associated virus (AAV) is widely considered a promising vector for therapeutic gene delivery. This promise is based on previous studies assessing AAVs safety and toxicity, ability to infect nondividing cells, elicit a limited immune response and provide long-term gene expression. However, we now find that earlier studies underappreciated the degree of AAV immunogenicity as well as the extent to which genetic background, through regulation of immune responsiveness, influences the duration of gene expression and thereby the effectiveness of AAV-mediated gene therapy. We evaluated antibody responses in 12 mouse strains to AAV serotype 2 (AAV2) and AAV2-expressed transgene products including green fluorescent protein (GFP), human 1-antitrypsin and murine interleukin-10. As expected, all immunocompetent mice administered AAV2 developed serologic evidence of immune responsiveness to the virus. However, a previously unidentified serologic prozone effect was observed suggesting that the concentrations of anti-AAV2 antibodies may have historically been subject to marked underestimation. Furthermore, strains with genetic predisposition to autoimmunity (eg, NOD, NZW, MRL-lpr) specifically imparted a functionally deleterious immune response to AAV-delivered transgene products. These findings suggest that more thorough studies of anti-AAV immunity should be performed, and that genetic predisposition to autoimmunity should be considered when assessing AAV efficacy and safety in humans.

5.174           Model of unidirectional transluminal gene transfer

Arap, M.A. et al Mol. Ther., 9(2), 305-310 (2004)   transfer assays in vitro are poor indicators of transduction efficacy observed in vivo. We designed and optimized an intermediate model for assessing and quantifying unidirectional transduction ex vivo. The model enables simultaneous transmucosal evaluation of up to 96 different variables under the same tissue conditions. We show that the model is versatile and suitable for use with different vectors (adenovirus and AAV), different reporter genes ( -galactosidase and green fluorescent protein), and viscera with various tissue features such as peritoneum and urothelium. Ex vivo transduction assays may correlate better with in vivo gene transfer results. Because the experimental model described here can be performed in small samples, it may enable translational applications in tissues of human origin.  

5.175           Development of efficient viral vectors selective for vascular smooth muscle cells

Work, L.M. et al Mol. Ther., 9(2) 198-208 (2004)   The vascular smooth muscle cell (SMC) is integral to the pathogenesis of neointimal formation associated with late vein graft failure, in-stent restenosis, and transplant arteriopathy. Viral vectors transduce SMC with low efficiency and hence, there is a need for improvement. We aimed to enhance the efficiency and selectivity of gene delivery to human SMC. Targeting ligands were identified using phage display on primary human saphenous vein SMC with linear and cyclic libraries. Two linear peptides, EYHHYNK (EYH) and GETRAPL (GET), were incorporated into the HI loop of adenovirus (Ad) fibers and the capsid protein of adeno-associated virus-2 (AAV-2). Exposure of human venous SMC to EYH-modified (but not the GET-modified) Ad vector resulted in a significant increase in transgene expression levels at short, clinically relevant exposure times. Similarly, the EYH-modified AAV vector resulted in enhanced gene transfer to human venous SMC but not endothelial cells in a time- and dose-dependent manner. The EYH-modified AAV vector also enhanced (up to 70-fold) gene delivery to primary human arterial SMC. Hence, incorporation of EYH into Ad and AAV capsids resulted in a significant and selective enhancement in transduction of SMC and has implications for improving local gene delivery to the vasculature.  

5.176           CD46 is a cellular receptor for bovine viral diarrhea virus

Maurer, K., Krey, T., Moennig, V., Thiel, H-J. and Rümenapf, T.

1. Virol., 78(4), 1792-1799 (2004)

  Various monoclonal antibodies (MAbs) that recognize cell surface proteins on bovine cells were previously shown to efficiently block infection with bovine viral diarrhea virus (BVDV) (C. Schelp, I. Greiser-Wilke, G. Wolf, M. Beer, V. Moennig, and B. Liess, Arch. Virol. 140:1997-2009, 1995). With one of these MAbs, a 50- to 58-kDa protein was purified from calf thymus by immunoaffinity chromatography. Microchemical analysis of two internal peptides revealed significant sequence homology to porcine and human CD46. The cDNA of bovine CD46 (CD46_bov) was cloned and further characterized. Heterologously expressed CD46_bov was detected by the MAb used for purification. A putative function of CD46_bov as a BVDV receptor was studied with respect to virus binding and susceptibility of nonpermissive cells. While the expression of CD46_bov correlated well with the binding of [³H]uridine-labeled BVDV, the susceptibility of cells nonpermissive for BVDV was not observed. However, the expression of CD46_bov resulted in a significant increase in the susceptibility of porcine cells to BVDV. These results provide strong evidence that CD46_bov serves as a cellular receptor for BVDV.  

5.177           Role of viral vectors and virion shells in cellular gene expression

Stilwell, J.L. and Samulski, R.J. Mol. Ther., 9(3), 337-346 (2004)   The role of the virion shell in viral pathogenesis is relatively unknown yet the use of viral vectors in human gene transfer experiments requires an understanding of these interactions. In this study, we used DNA microarrays to identify genes modulated during pathogenic adenovirus or nonpathogenic adeno-associated virus infections. Responses to wt viruses, recombinant vectors, or empty virion particles were compared. Adeno-associated virus shells induced nearly the full complement of changes elicited by the intact virus. The cellular genes elicited a nonpathogenic response, with antiproliferative genes being induced as a cluster. In contrast, adenovirus and adenovirus empty capsid infection yielded a broader response and subset, respectively, including induction of immune and stress-response genes associated with pathogenic effects. Our studies show that the impact of the viral capsid on cellular gene expression, and potential host toxicity, must be considered independent of the vector genome for safe gene transfer in the clinic.

5.178           Optimal design of a single recombinant adeno-associated virus derived from serotypes 1 and 2 to achieve more tightly regulated transgene expression from nonhuman primate muscle

Chenuaud, P. et al Mol. Ther., 9(3), 410-418 (2004)   Recombinant adeno-associated virus (rAAV) vector supports long-term transgene expression from skeletal muscle in most mammals, including human. In some instances, the requirement for tight control of the transgene expression is expected. The original tetracycline-dependent system using the rtTA (Dox-on) transactivator displayed a baseline activity in the off state but improved versions are now available and need to be evaluated in a single-rAAV-vector strategy. In the present study we cloned, in three different orientations, the two expression cassettes responsible for doxycycline-mediated transgene regulation and further evaluated the basal and inducible activity of the recently described rtTA2^S-S2, rtTA2^S-M2, and rtTA2^S-M2nls transactivators. Evaluations were conducted in vivo in mice and nonhuman primates using the respective homologous erythropoietin cDNA as a reporter gene because of its sensitive detection by ELISA. The woodchuck hepatitis virus posttranscriptional regulatory element sequence was also introduced to enhance further the stringency with respect to basal activity in the absence of inducer.    

5.179           Adeno-associated virus 2-mediated antiangiogenic cancer gene therapy: long-term efficacy of a vector encoding angiostatin and endostatin over vectors encoding a single factor

Ponnazhagan, S. et al Can. Res., 64, 1781-1787 (2004)   Angiogenesis is characteristic of solid tumor growth and a surrogate marker for metastasis in many human cancers. Inhibition of tumor angiogenesis using antiangiogenic drugs and gene transfer approaches has suggested the potential of this form of therapy in controlling tumor growth. However, for long-term tumor-free survival by antiangiogenic therapy, the factors controlling tumor neovasculature need to be systemically maintained at stable therapeutic levels. Here we show sustained expression of the antiangiogenic factors angiostatin and endostatin as secretory proteins by recombinant adeno-associated virus 2 (rAAV)-mediated gene transfer. Both vectors provided significant protective efficacy in a mouse tumor xenograft model. Stable transgene persistence and systemic levels of both angiostatin and endostatin were confirmed by in situ hybridization of the vector-injected tissues and by serum ELISA measurements, respectively. Whereas treatment with rAAV containing either endostatin or angiostatin alone resulted in moderate to significant protection, the combination of endostatin and angiostatin gene transfer from a single vector resulted in a complete protection. These data suggest that AAV-mediated long-term expression of both endostatin and angiostatin may have clinical utility against recurrence of cancers after primary therapies and may represent rational adjuvant therapies in combination with radiation or chemotherapy.  

5.180           T cells from a high proportion of apparently naive cattle can be activated by modified vaccinia virus Ankara (MVA)

Sandbulte, M.R., Platt, R. and Roth, J.A. Viral immunol., 17(1), 39-49 (2004)   Modified vaccinia virus Ankara (MVA) was used as a vector to express genes from bovine respiratory syncytial virus (BRSV). Using these recombinant viruses as recall antigens for cells from BRSv-immuned cattle proved to be problematic because non-recombinant MVA itself frequently stimulated high levels of T lymphocyte activation. This phenomenon was observed in a high percentage of cattle from multiple herds. Gamma delta TCR⁺ T cells were more sensitive to activation by MVA than other classes of T cells. A serological assay for MVA neutralization detected low, fluctuating titers of serum virus neutralizing (SVN) activity toward MVA in some cattle, but these were lower titers than those observed in cattle that underwent MVA vaccination. T clee reactivity in non-vaccinated cattle did not correlate significantly (p > 0.05) with SVN activity, undermining the notion that any adaptive immune response was responsible for the observed T cell sensitivity. More probable explanations are that MVA has mitogenic or superantigenic properties, or that the virus induces gd TCR⁺ T cell activation through interactions with innate pattern recognition receptors.  

5.181           Differential modulation of energy balance by leptin, ciliary neurothophic factor, and leukemia inhibitory factor gene delivery: microarray deoxyribonucleic acid-chip analysis of gene expression

Prima, V. et al Endocrinol., 145(4), 2035-2045 (2004)   Most obese animal models, whether associated with genetic, diet-induced, or age-related obesity, display pronounced leptin resistance, rendering leptin supplement therapy ineffective in treating obesity. Ciliary neurotrophic factor (CNTF) has been recently used to invoke leptin-like signaling pathways, thereby circumventing leptin resistance. In the current study, we characterize immediate and long-term molecular events in the hypothalamus of rats exposed to the sustained ectopic expression of leptin, CNTF, or leukemia inhibitory factor, another neurocytokine of IL-6 family, all delivered centrally via a viral vector. The respective transgene-encoded ligands induced similar but not identical metabolic responses as assessed by the reduction in body weight gain and changes in food intake. To define molecular mechanisms of weight-reducing and anorexigenic action of cytokines, we have analyzed the gene expression profiles of 1300 brain-specific genes in the hypothalami of normal rats subjected to the prolonged cytokine action for 10 wk. We present evidence that constitutive expression of cytokines in the brain induces changes in gene expression characteristic of chronic inflammation leading to either temporal weight reduction (CNTF) or severe cachexia (leukemia inhibitory factor). Our results convey a cautionary note regarding potential use of the tested cytokines in therapeutic applications.    

5.182           Selective modification of variable loops alters tropism and enhances immunogenicity of human immunodeficiency virus type 1 envelope

Yang, Z-Y. Et al

1. Virol., 78(8), 4029-4036 (2004)

  Although the B clade of human immunodeficiency virus type 1 (HIV-1) envelopes (Env) includes five highly variable regions, each of these domains contains a subset of sequences that remain conserved. The V3 loop has been much studied for its ability to elicit neutralizing antibodies, which are often restricted to a limited number of closely related strains, likely because a large number of antigenic structures are generated from the diverse amino acid sequences in this region. Despite these strain-specific determinants, subregions of V3 are highly conserved, and the effects of different portions of the V3 loop on Env tropism and immunogenicity have not been well delineated. For this report, selective deletions in V3 were introduced by shortening of the stem of the V3 loop. These mutations were explored in combination with deletions of selected V regions. Progressive shortening of the stem of V3 abolished the immunogenicity as well as the functional activity of HIV Env; however, two small deletions on both arms of the V3 stem altered the tropism of the dualtropic 89.6P viral strain so that it infected only CXCR4⁺ cells. When this smaller deletion was combined with removal of the V1 and V2 loops and used as an immunogen in guinea pigs, the antisera were able to neutralize multiple independent clade B isolates with a higher potency. These findings suggest that highly conserved subregions within V3 may be relevant targets for eliciting neutralizing antibody responses, affecting HIV tropism, and increasing the immunogenicity of AIDS vaccines.  

5.183           Reactivity of human sera in a sensitive, high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18

Pastrana, D.V. et al Virology, 321, 205-216 (2004)   Sensitive high-throughput neutralization assays, based upon pseudoviruses carrying a secreted alkaline phosphatase (SEAP) reporter gene, were developed and validated for human papillomavirus (HPV)16, HPV18, and bovine papillomavirus 1 (BPV1). SEAP pseudoviruses were produced by transient transfection of codon-modified papillomavirus structural genes into an SV40 T antigen expressing line derived from 293 cells, yielding sufficient pseudovirus from one flask for thousands of titrations. In a 96-well plate format, in this initial characterization, the assay was reproducible and appears to be as sensitive as, but more specific than, a standard papillomavirus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA). The neutralization assay detected type-specific HPV16 or HPV18 neutralizing antibodies (titers of 160–10240) in sera of the majority of a group of women infected with the corresponding HPV type, but not in virgin women. Sera from HPV16 VLP vaccinees had high anti-HPV16 neutralizing titers (mean: 45000; range: 5120–163840), but no anti-HPV18 neutralizing activity. The SEAP pseudovirus-based neutralization assay should be a practical method for quantifying potentially protective antibody responses in HPV natural history and prophylactic vaccine studies.  

5.184           Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors

Ahmed, B.Y. et al BMC Neuroscience, 5(4), 1-11 (2004)   Background Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV) and lentiviral (LV) vectors into discrete regions of the forebrain. Results Recombinant AAV-Cre, AAV-GFP (green fluorescent protein) and LV-Cre-EGFP (enhanced GFP) were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. Conclusion AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.  

5.185           The potential of gene transfer into primary B-CLL cells using recombinant virus vectors

Wendtner, C.M., Kofler, D.M., Mayr, C., Bund, D. And Hallek, M. Leukemia & Lymphona, 45(5), 897-904 (2004)   Despite recent advances, chronic lymphocytic leukemia (CLL) as the most common leukemia remains a largely incurable disease. Modern treatment options include novel drugs like purine analogues, monoclonal antibodies and transplantation strategies. Moreover, gene transfer of immunostimulatory molecules is another, but still experimental approach that can be used to potentiate immune responses against leukemic cells. CD40 ligand (CD40L) was shown to be a promising molecule for immunotherapy of B-CLL playing a critical role in immune activation. However, CLL B cells are resistant to transduction with most currently available vector systems. Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy in this area. Using replication defective adenovirus encoding CD40L (Ad-CD40L), immunologic and clinical responses were seen in CLL patients after infusion of autologous Ad-CD40L-CLL cells in a recent phase I trial. Due to the immunogenic nature of adenovirus vectors, alternative vector systems are currently explored. Recombinant adeno-associated virus (rAAV) was shown to enable efficient transduction of primary B-CLL cells. By use of a library of AAV clones with randomly modified capsids, receptor-targeting mutants with a tropism for CLL cells can be selected. Furthermore, helper-virus free Epstein-Barr virus (EBV)-based gene transfer vectors hold promise for development of CLL-targeted vaccines after remaining safety issues will be resolved. Herpes simplex virus (HSV)-based vectors, especially HSV amplicons, have favorable features for B-CLL gene transfer including high transduction efficiency, ability to infect postmitotic cells and a large packaging capacity. The challenge for the future will be to transfer these alternative vector systems into clinic and allow the detection of a CLL-specific immune response by use of defined tumor antigens. This will make it possible to establish the potential clinical role of gene therapy for CLL patients.  

5.186           Recombinant adeno-associated virus as delivery vector for gene therapy – a review

Lu, Y. Stem Cells and Development, 13, 133-145 (2004)   Recombinant adeno-associated virus (rAAV) is one of the most promising delivery vectors for gene therapy, due to its nonpathogenic property, nonimmunogenecity to host, and broad cell and tissue tropisms. This article summarizes the biological characteristics of AAV; the procedures to prepare, purify, and characterize the rAAV for gene therapy applications; and some of the clinical trials utilizing rAAV as delivery vehicles. Also discussed are the current efforts to modify rAAV to change its tropism, the application of different promoters to accommodate specific transgene expression, and the strategy to expand its capacity.  

5.187           Efficient PRNP gene targeting in bovine fibroblasts by adeno-associated virus vectors

Hirata, R.K. et al Cloning and Stem cells, 6(1), 31-36 (2004)   Abstract: Gene-targeted livestock can be created by combining ex vivo manipulation of cultured nuclear donor cells with cloning by nuclear transfer. However, this process can be limited by the low gene targeting frequencies obtained by transfection methods, and the limited ex vivo life span of the normal nuclear donor cells. We have developed an alternative gene targeting method based on the delivery of linear, single-stranded DNA molecules by adeno-associated virus (AAV) vectors, which can be used to introduce a variety of different mutations at single copy loci in normal human cells. Here we show that AAV vectors can efficiently target the PRNP gene encoding the prion protein PrP in bovine fetal fibroblasts, which can be used as nuclear donors to clone cattle. Cattle with both PRNP genes disrupted should be resistant to bovine spongiform encephalopathyAbstract: Gene-targeted livestock can be created by combining ex vivo manipulation of cultured nuclear donor cells with cloning by nuclear transfer. However, this process can be limited by the low gene targeting frequencies obtained by transfection methods, and the limited ex vivo life span of the normal nuclear donor cells. We have developed an alternative gene targeting method based on the delivery of linear, single-stranded DNA molecules by adeno-associated virus (AAV) vectors, which can be used to introduce a variety of different mutations at single copy loci in normal human cells. Here we show that AAV vectors can efficiently target the PRNP gene encoding the prion protein PrP in bovine fetal fibroblasts, which can be used as nuclear donors to clone cattle. Cattle with both PRNP genes disrupted should be resistant to bovine spongiform encephalopathy.  

5.188           pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-sign

Yang, Z-Y. Et al

1. Virol., 78(11), 5642-5680 (2004)

  The severe acute respiratory syndrome coronavirus (SARS-CoV) synthesizes several putative viral envelope proteins, including the spike (S), membrane (M), and small envelope (E) glycoproteins. Although these proteins likely are essential for viral replication, their specific roles in SARS-CoV entry have not been defined. In this report, we show that the SARS-CoV S glycoprotein mediates viral entry through pH-dependent endocytosis. Further, we define its cellular tropism and demonstrate that virus transmission occurs through cell-mediated transfer by dendritic cells. The S glycoprotein was used successfully to pseudotype replication-defective retroviral and lentiviral vectors that readily infected Vero cells as well as primary pulmonary and renal epithelial cells from human, nonhuman primate, and, to a lesser extent, feline species. The tropism of this reporter virus was similar to that of wild-type, replication-competent SARS-CoV, and binding of purified S to susceptible target cells was demonstrated by flow cytometry. Although myeloid dendritic cells were able to interact with S and to bind virus, these cells could not be infected by SARS-CoV. However, these cells were able to transfer the virus to susceptible target cells through a synapse-like structure. Both cell-mediated infection and direct infection were inhibited by anti-S antisera, indicating that strategies directed toward this gene product are likely to confer a therapeutic benefit for antiviral drugs or the development of a SARS vaccine.  

5.189           Recombinant adeno-associated virus type 2-mediated gene delivery into the Rpe65^-/- knockout mouse eye results in limited rescue

Lai, C-M. et al Genetic Vaccines and Therapy, 2(3), xx-xx( 2004)   Background Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function. Methods rAAV.RPE65 was injected into the subretinal space of Rpe65^-/- knockout mice and control mice. Histological and immunohistological analyses were performed to evaluate any rescue of photoreceptors and to determine longevity and pattern of transgene expression. Electron microscopy was used to examine ultrastructural changes, and electroretinography was used to measure changes in visual function following rAAV.RPE65 injection. Results rAAV-mediated RPE65 expression was detected for up to 18 months post injection. The delivery of rAAV.RPE65 to Rpe65^-/- mouse retinas resulted in a transient improvement in the maximum b-wave amplitude under both scotopic and photopic conditions (76% and 59% increase above uninjected controls, respectively) but no changes were observed in a-wave amplitude. However, this increase in b-wave amplitude was not accompanied by any slow down in photoreceptor degeneration or apoptotic cell death. Delivery of rAAV.RPE65 also resulted in a decrease in retinyl ester lipid droplets and an increase in short wavelength cone opsin-positive cells, suggesting that the recovery of RPE65 expression has long-term benefits for retinal health. Conclusion This work demonstrated the potential benefits of using the Rpe65^-/- mice to study the effects and mechanism of rAAV.RPE65-mediated gene delivery into the retina. Although the functional recovery in this model was not as robust as in the dog model, these experiments provided important clues about the long-term physiological benefits of restoration of RPE65 expression in the retina.  

5.190           Nef stimulates human immunodeficiency virus type 1 replication in primary T cells by enhancing virion-associated gp120 levels: coreceptor-dependent requirement for Nef in viral replication

Lundquist, C.A., Zhou, J. and Aiken, C.

1. Virol., 78(12), 6287-6296 (2004)

  The Nef protein enhances human immunodeficiency virus type 1 (HIV-1) replication through an unknown mechanism. We and others have previously reported that efficient HIV-1 replication in activated primary CD4⁺ T cells depends on the ability of Nef to downregulate CD4 from the cell surface. Here we demonstrate that Nef greatly enhances the infectivity of HIV-1 particles produced in primary T cells. Nef-defective HIV-1 particles contained significantly reduced quantities of gp120 on their surface; however, Nef did not affect the levels of virion-associated gp41, indicating that Nef indirectly stabilizes the association of gp120 with gp41. Surprisingly, Nef was not required for efficient replication of viruses that use CCR5 for entry, nor did Nef influence the infectivity or gp120 content of these virions. Nef also inhibited the incorporation of CD4 into HIV-1 particles released from primary T cells. We propose that Nef, by downregulating cell surface CD4, enhances HIV-1 replication by inhibiting CD4-induced dissociation of gp120 from gp41. The preferential requirement for Nef in the replication of X4-tropic HIV-1 suggests that the ability of Nef to downregulate CD4 may be most important at later stages of disease when X4-tropic viruses emerge.  

5.191           Circulating anti-wild type adeno-associated virus type 2 (AAV2) antibodies inhibit recombinant AAV2 (rAAV2)-mediated, but not rAAV5-mediated, gene transfer in the brain

Peden, C.S., Burger, C., Muzyczka, N. and Mandel, R.J.

1. Virol., 78(12), 6344-6359 (2004)

  Epidemiological studies report that 80% of the population maintainsantibodies (Ab) to wild-type (wt) adeno-associated virus type2 (AAV2), with 30% expressing neutralizing Ab (NAb). The blood-brainbarrier (BBB) provides limited immune privilege to brain parenchyma,and the immune response to recombinant AAV (rAAV) administrationin the brain of a naive animal is minimal. However, centralnervous system transduction in preimmunized animals remainsunstudied. Vector administration may disrupt the BBB sufficientlyto promote an immune response in a previously immunized animal.We tested the hypothesis that intracerebral rAAV administrationand readministration would not be affected by the presence ofcirculating Ab to wt AAV2. Rats peripherally immunized withlive wt AAV2 and naive controls were tested with single intrastriatalinjections of rAAV2 encoding human glial cell line-derived neurotrophicfactor (GDNF) or green fluorescent protein (GFP). Striatal readministrationof rAAV2-GDNF was also tested in preimmunized and naive rats.Finally, serotype specificity of the immunization against wtAAV2 was examined by single injections of rAAV5-GFP. Preimmunizationresulted in high levels of circulating NAb and prevented transductionby rAAV2 as assessed by striatal GDNF levels. rAAV2-GFP striataltransduction was also prevented by immunization, while rAAV5-GFP-mediatedtransduction, as assessed by stereological cell counting, wasunaffected. Additionally, inflammatory markers were presentin those animals that received repeated administrations of rAAV2,including markers of a cell-mediated immune response and cytotoxicdamage. A live virus immunization protocol generated the circulatinganti-wt-AAV Ab seen in this experiment, while human titers arecommonly acquired via natural infection. Regardless, the datashow that the presence of high levels of NAb against wt AAVcan reduce rAAV-mediated transduction in the brain and shouldbe accounted for in future experiments utilizing this vector.  

5.192           Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus

Warrington, K.H. et al

1. Virol., 78(12), 6595-6609 (2004)

  Direct insertion of amino acid sequences into the adeno-associated virus type 2 (AAV) capsid open reading frame (cap ORF) is one strategy currently being developed for retargeting this prototypical gene therapy vector. While this approach has successfully resulted in the formation of AAV particles that have expanded or retargeted viral tropism, the inserted sequences have been relatively short, linear receptor binding ligands. Since many receptor-ligand interactions involve nonlinear, conformation-dependent binding domains, we investigated the insertion of full-length peptides into the AAV cap ORF. To minimize disruption of critical VP3 structural domains, we confined the insertions to residue 138 within the VP1-VP2 overlap, which has been shown to be on the surface of the particle following insertion of smaller epitopes. The insertion of coding sequences for the 8-kDa chemokine binding domain of rat fractalkine (CX3CL1), the 18-kDa human hormone leptin, and the 30-kDa green fluorescent protein (GFP) after residue 138 failed to lead to formation of particles due to the loss of VP3 expression. To test the ability to complement these insertions with the missing capsid proteins in trans, we designed a system for producing AAV vectors in which expression of one capsid protein is isolated and combined with the remaining two capsid proteins expressed separately. Such an approach allows for genetic modification of a specific capsid protein across its entire coding sequence leaving the remaining capsid proteins unaffected. An examination of particle formation from the individual components of the system revealed that genome-containing particles formed as long as the VP3 capsid protein was present and demonstrated that the VP2 capsid protein is nonessential for viral infectivity. Viable particles composed of all three capsid proteins were obtained from the capsid complementation groups regardless of which capsid proteins were supplied separately in trans. Significant overexpression of VP2 resulted in the formation of particles with altered capsid protein stoichiometry. The key finding was that by using this system we successfully obtained nearly wild-type levels of recombinant AAV-like particles with large ligands inserted after residue 138 in VP1 and VP2 or in VP2 exclusively. While insertions at residue 138 in VP1 significantly decreased infectivity, insertions at residue 138 that were exclusively in VP2 had a minimal effect on viral assembly or infectivity. Finally, insertion of GFP into VP1 and VP2 resulted in a particle whose trafficking could be temporally monitored by using confocal microscopy. Thus, we have demonstrated a method that can be used to insert large (up to 30-kDa) peptide ligands into the AAV particle. This system allows greater flexibility than current approaches in genetically manipulating the composition of the AAV particle and, in particular, may allow vector retargeting to alternative receptors requiring interaction with full-length conformation-dependent peptide ligands.  

5.193           Characterization of the genome and structural proteins of hepatitis C virus resolved from infected human liver

Nielsen, S.U., Bassendine, F., Burt, A.D., Bevitt, D.J. and Toms, G.L.

1. Gen. Virol., 85, 1497-1507 (2004)

  In the absence of satisfactory cell culture systems for hepatitis C virus (HCV), virtually all that is known about the proteins of the virus has been learned by the study of recombinant proteins. Characterization of virus proteins from patients with HCV has been retarded by the low virus titre in blood and limited availability of infected tissue. Here, the authors have identified a primary infection in a liver transplanted into an immunodeficient patient with chronic HCV. The patient required re-transplant and the infected liver, removed 6 weeks after the initial transplant, had a very high titre of HCV, 5x10⁹ International Units (IU) per gram of liver. The density distribution of HCV in iodixanol gradients showed a peak at 1·04 g ml^–1 with 73 % of virus below 1·08 g ml^–1. Full-length HCV RNA was detected by Northern blotting and the ratio between positive- and negative-strand HCV RNA was determined as 60. HCV was partially purified by precipitation with heparin/Mn²⁺ and a single species of each of the three structural proteins, core, E1 and E2, was detected by Western blotting. The molecular mass of core was 20 kDa, which corresponds to the mature form from recombinant sources. The molecular mass of glycoprotein E1 was 31 kDa before and 21 kDa after deglycosylation with PNGase F or endoglycosidase H. Glycoprotein E2 was 62 kDa before and 36 kDa after deglycosylation, but E2-P7 was not detected. This was in contrast to recombinant sources of E2 which contain E2-P7.  

5.194           Transduction profiles of recombinant adeno-associated virus vectors derived from serotypes 2 and 5 in the nigrostriatal system of rats

Paterna, J-C., Feldon, J. and Büeler, H.

1. Virol., 78(13), 6808-6817 (2004)

  We compared the transduction efficiencies and tropisms of titer-matched recombinant adeno-associated viruses (rAAV) derived from serotypes 2 and 5 (rAAV-2 and rAAV-5, respectively) within the rat nigrostriatal system. The two serotypes (expressing enhanced green fluorescent protein [EGFP]) were delivered by stereotaxic surgery into the same animals but different hemispheres of the striatum (STR), the substantia nigra (SN), or the medial forebrain bundle (MFB). While both serotypes transduced neurons effectively within the STR, rAAV-5 resulted in a much larger EGFP-expressing area than did rAAV-2. However, neurons transduced with rAAV-2 vectors expressed higher levels of EGFP. Consistent with this result, EGFP-positive projections emanating from transduced striatal neurons covered a larger area of the SN pars reticulata (SNr) after striatal delivery of rAAV-5, but EGFP levels in fibers of the SNr were higher after striatal injection of rAAV-2. We also compared the potentials of the two vectors for retrograde transduction and found that striatal delivery of rAAV-5 resulted in significantly more transduced dopaminergic cell bodies within the SN pars compacta and ventral tegmental area. Similarly, EGFP-transduced striatal neurons were detected only after nigral delivery of rAAV-5. Furthermore, we demonstrate that after striatal AAV-5 vector delivery, the transduction profiles were stable for as long as 9 months. Finally, although we did not target the hippocampus directly, efficient and widespread transduction of hippocampal neurons was observed after delivery of rAAV-5, but not rAAV-2, into the MFB.  

5.195           Correction of metabolic, craniofacial, and neurologic abnormalities in MPS I mice treated at birth with adeno-associated virus vector transducing the human a-L-iduronidase gene

Hartung, S.D. et al Mol. Ther., 9(6), 866-875 (2004)   Murine models of lysosomal storage diseases provide an opportunity to evaluate the potential for gene therapy to prevent systemic manifestations of the disease. To determine the potential for treatment of mucopolysaccharidosis type I using a gene delivery approach, a recombinant adeno-associated virus (AAV) vector, vTRCA1, transducing the human iduronidase (IDUA) gene was constructed and 1 × 10¹⁰ particles were injected intravenously into 1-day-old Idua^−/− mice. High levels of IDUA activity were present in the plasma of vTRCA1-treated animals that persisted for the 5-month duration of the study, with heart and lung of this group demonstrating the highest tissue levels of gene transfer and enzyme activity overall. vTRCA1-treated Idua^−/− animals with measurable plasma IDUA activity exhibited histopathological evidence of reduced lysosomal storage in a number of tissues and were normalized with respect to urinary GAG excretion, craniofacial bony parameters, and body weight. In an open field test, vTRCA1-treated Idua^−/− animals exhibited a significant reduction in total squares covered and a trend toward normalization in rearing events and grooming time compared to control-treated Idua^−/− animals. We conclude that AAV-mediated transduction of the IDUA gene in newborn Idua^−/− mice was sufficient to have a major curative impact on several of the most important parameters of the disease.  

5.196           A conserved leucine that constricts the pore through the capsid fivefold cylinder plays a central role in parvoviral infection

Farr, G.A. and Tattersall, P. Virology, 323, 243-256 (2004)   The atomic structure of the DNA-containing T = 1 particle of the parvovirus minute virus of mice (MVM) reveals cylindrical projections at each fivefold symmetry axis, each containing an 8 Å pore through which runs 10 amino acids of a single VP2 N-terminus. The tightest constriction of this pore is formed at its inner end by the juxtaposition of leucine side chains from position 172 of five independent VP2 molecules. To test whether L172 modulates the extrusion of VP N-termini, we constructed and analyzed a complete set of amino acid substitution mutants at this highly conserved residue. All but one mutant produced DNA-containing virions, but only two, L172V and L172I, were infectious, the others being blocked for viral entry. Several mutants were significantly defective for assembly at 39 °C, but not at 32 °C. L172W significantly impaired genome encapsidation, indicating that the fivefold cylinder may also be the DNA packaging portal. Although tryptic cleavage of the VP2 N-terminus was not affected for the mutants, VP1 was degraded during proteolysis of mutant, but not wild-type, virions.  

5.197           Adeno-associated virus 2-mediated gene therapy decreases autofluorescent storage material and increases brain mass in a murine model of infantile neuronal ceroid lipofuscinosis

Griffey, M. et al Neurobiol.of  Disease, 16, 360-369 (2004)   Infantile neuronal ceroid lipofuscinosis (INCL) is the earliest onset form of a class of inherited neurodegenerative disease called Batten disease. INCL is caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). Autofluorescent storage material accumulates in virtually all tissues in INCL patients, including the brain, and leads to widespread neuronal loss and cortical atrophy. To determine the efficacy of viral-mediated gene therapy, we injected a recombinant adeno-associated virus 2 vector encoding human PPT1 (rAAV–PPT1) intracranially (I.C.) into a murine model of INCL. INCL mice given four I.C. injections of rAAV–PPT1 as newborns exhibited PPT1 activity near the injection sites and decreased secondary elevations of another lysosomal enzyme. In addition, storage material was decreased in cortical, hippocampal, and cerebellar neurons, and brain weights and cortical thicknesses were increased. These data demonstrate that an adeno-associated virus 2 (AAV2)-mediated gene therapy approach may provide some therapeutic benefit for INCL.  

5.198           CD154 signaling regulates the Th1 response to herpes simplex virus-1 and inflammation in infected corneas

Xu, M., Lepisto, A.J. and Hendricks, R.L.

1. Immunol., 173, 1232-1239 (2004)

  Approximately 7 days after HSV-1 corneal infection, BALB/c mice develop tissue-destructive inflammation in the cornea termed herpes stromal keratitis (HSK), as well as periocular skin lesions that are characterized by vesicles, edema, and fur loss. CD4⁺ T cells and Th1 cytokines contribute to both the immunopathology in the cornea and the eradication of viral replication in the skin. We demonstrate that disruption of CD40/CD154 signaling does not impact the initial expansion of CD4⁺ T cells in the draining lymph nodes, but dramatically reduces the persistence and Th1 polarization of these cells. Despite the reduced Th1 response, CD154^–/– mice developed HSK and periocular skin disease with similar kinetics and severity (as assessed by clinical examination) as wild-type (WT) mice. However, when the composition of the inflammatory infiltrate was examined by flow cytometric analysis, CD154^–/– mice exhibited significantly fewer CD4⁺ and CD8⁺ T cells and neutrophils than WT mice at the peak of HSK. Moreover, CD4⁺ T cells from infected corneas of CD154^–/– mice produced significantly less IFN- than those of WT mice when stimulated with viral Ags in vitro. The IFN- production of cells from infected corneas of WT mice was not affected by addition of anti-CD154 mAb to the stimulation cultures. This suggests that CD154 signaling is required at the inductive phase, but not at the effector phase, of the Th1 response within the infected cornea. We conclude that local disruption of CD40/CD154 signaling is not likely to be a useful therapy for HSK.  

5.199           Development and characterization of neutralizing monoclonal antibody to the SARs-coronavirus

Berry, J.D. et al

1. Virol. Methods., 120, 87-96 (2004)

  There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.  

5.200           AAV-mediated intravitreal gene therapy reduces lysosomal storage in the retinal pigmented epithelium and improves retinal function in adult MPS VII mice

Hennig, A.K. et al Mol. Ther., 10(1), 106-116 (2004)   The -glucuronidase-deficient mucopolysaccharidosis type VII (MPS VII) mouse accumulates partially degraded glycosaminoglycans in many cell types, including retinal pigmented epithelial (RPE) cells in the eye. This lysosomal storage in RPE cells leads to progressive retinal degeneration and reduced function as measured by flash electroretinography (ERG). The impact of AAV-mediated intraocular gene therapy on pathology and retinal function was examined in normal and MPS VII mice treated at 4 weeks of age, when lysosomal storage is evident but functional impairment is minimal in affected animals. At 16 weeks, an age at which untreated MPS VII mice have advanced histologic lesions and significantly reduced ERG amplitudes, treated eyes had nearly normal levels of -glucuronidase activity, preservation of cells in the outer nuclear layer of the retina, and decreased lysosomal storage within the RPE. The AAV-treated MPS VII mice also had significantly increased dark-adapted ERG amplitudes compared to untreated MPS VII mice. Although retinal function was improved, the efficacy of the treatment depended heavily on parameters related to the injection procedure, such as the injection volume, injection site, and vector dose. These data suggest that intraocular AAV-mediated therapy may be efficacious for treating the retinal disease associated with certain lysosomal storage diseases.  

5.201           Simulteneous presence of endogenous retrovirus and herpes virus antigens has profound effect on cell-mediated immune responses: implications for multiple sclerosis

Brudek, T., Christensen, T., Hansen, H.J., Bobecka, J. And Møller-Larsen, A. AIDS Res. and Human Retroviruses, 20(4), 415-423 (2004)   Retroviruses have been suggested as possible pathogenic factors in multiple sclerosis (MS), supported by the observation that endogenous retroviruses are activated in MS patients. Different members of the herpes family of which several are neurotropic have also been suggested as factors in MS pathogenesis. Further, interactions between retroviruses and herpes viruses have been implied in the development of MS. The objective of the study was investigation of cell-mediated immune responses of MS patients to retrovirus and herpes virus antigens, particularly antigen combinations, with analyses of the influence of retrovirus antigens on cellular immunological reactivity toward other viral antigens. Cellular immunity as measured by blast transformation assays was analyzed using freshly isolated peripheral blood mononuclear cells from 47 MS patients and 36 healthy volunteers. Combinations of the endogenous retrovirus HERV-H and herpes virus antigens resulted in highly increased cellular immune responses among both the MS patients and healthy subjects. The increase was synergistic in character in most samples. Very pronounced effects were obtained using HHV-6A and HSV-1 antigens. Blast transformation assays combining antigens from two different herpes viruses or combinations of measles and herpes antigens showed no synergy. The obtained data indicate a pronounced synergistic effect on the cellular immune response when retrovirus and herpes antigens are present together. The cause of the synergy is unknown so far. The effect on the immune response may influence the disease progression.  

5.202           The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead rbozyme: a self-complementary AAV2 vector improves the gene expression

Zhong, S., Sun, S. and Teng, B-B. Genetic Vaccines and Therapy, 2(5), 1-11 (2004)   In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA⁶⁶⁷⁹ (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene expression in the mice livers, which did not produce the therapeutic effects on alteration the lipid levels or the inhibition of atherosclerosis development. In contrast, the ribozyme RB15 RNA mediated by scAAV2-TTR-RB15 vector was expressed immediately at day-1 after transduction in HepG2 cells. The apoB mRNA levels were decreased 47% (p = 0.001), compared to the control vector scAAV2-TTR-RB15-mutant. This study provided evidence that the rAAV2 single-strand vector mediated a prolonged but not efficient transduction in mouse liver. However, the scAAV2 double-strand vector mediated a rapid and efficient gene expression in liver cells. This strategy using scAAV2 vectors represents a better approach to express small molecules such as ribozyme.  

5.203           Specific targeted binding of herpes simplex virus type 1 ro hepatocytes via the human hepatitis B virus preS1 peptide

Argnani, R., Boccafogli, L., Marconi, P.C. and Manservigi, R. Gene Therapy, 11, 1087-1098 (2004)   To improve the utility of herpes simplex virus type 1 (HSV-1) vectors for gene therapy, the viral envelope needs to be manipulated to achieve cell-specific gene delivery. In this report, we have engineered an HSV-1 mutant virus, KgBpK^- gC^-, deleted for the glycoprotein C (gC) and the heparan sulfate-binding domain (pK) of gB, in order to express gC:preS1 and gC:preS1 active peptide (preS1ap) fusion molecules. PreS1, and a 27 amino acid active peptide inside preS1 (preS1ap), are supposed to be the molecules that the human hepatitis B virus (HBV) needs to bind specifically to hepatocytes. Biochemical analysis demonstrated that the gC:preS1ap fusion molecule was expressed and incorporated into the envelope of the recombinant HSV-1 virus KgBpK^-gC:preS1ap. Moreover, KgBpK^-gC:preS1ap recombinant virus gained a specific binding activity to an hepatoblastoma cell line (HepG2) with a consequent productive infection. In addition, anti-preS1-specific antibodies were shown to neutralize recombinant virus infectivity, and a synthetic preS1ap peptide was able to elute KgBpK^-gC:preS1ap virus bound on HpeG2 cells. These data provide further evidence that HSV-1 can productively infect cells through a specific binding to a non-HSV-1 receptor. Furthermore, these data strongly support the hypothesis that the HBV preS1ap molecule is an HBV ligand to hepatocytes.  

5.204           Adeno-associated virus vectors integrate at chromosome breakage sites

Miller, D.G., Petek, L.M. and Russell, D.W. Nature Gen., 36(7), 767-773 (2004)   Adeno-associated virus (AAV) vectors transduce cells by multiple pathways, including integration at nonhomologous chromosomal locations by an unknown mechanism^{1, 2, 3, 4, 5}. We reasoned that spontaneous chromosome breaks may facilitate vector integration and investigated this in cells containing a specific chromosomal double-strand break created by the endonuclease I-SceI or multiple breaks created by treatment with etoposide or -irradiation. Vector proviruses were found at I-SceI cleavage sites, and sequencing of vector-chromosome junctions detected microhomologies, deletions and insertions that were similar when integration occurred spontaneously at random locations or at induced double-strand breaks. Infection with AAV vectors did not increase mutation rates in normal human cells. Our results establish a mechanism for integration and suggest that AAV vectors can integrate at existing chromosome breaks rather than causing breaks themselves, which has implications for their clinical use.  

5.205           Adeno-associated virus site-specific integration and AAVS1 disruption

Hamilton, H., Gomos, J., Berns, K.I.and Falck-Pedersen, E.

1. Virol., 78(15), 7874-7882 (2004)

  Adeno-associated virus (AAV) is a single-stranded DNA virus with a unique biphasic lifestyle consisting of both a productive and a latent phase. Typically, the productive phase requires coinfection with a helper virus, for instance adenovirus, while the latent phase dominates in healthy cells. In the latent state, AAV is found integrated site specifically into the host genome at chromosome 19q13.4 qtr (AAVS1), the only animal virus known to integrate in a defined location. In this study we investigated the latent phase of serotype 2 AAV, focusing on three areas: AAV infection, rescue, and integration efficiency as a function of viral multiplicity of infection (MOI); efficiency of site-specific integration; and disruption of the AAVS1 locus. As expected, increasing the AAV MOI resulted in an increase in the percentage of cells infected, with 80% of cells infected at an MOI of 10. Additional MOI only marginally effected a further increase in percentage of infected cells. In contrast to infection, we found very low levels of integration at MOIs of less than 10. At an MOI of 10, at which 80% of cells are infected, less than 5% of clonal cell lines contained integrated AAV DNA. At an MOI of 100 or greater, however, 35 to 40% of clonal cell lines contained integrated AAV DNA. Integration and the ability to rescue viral genomes were highly correlated. Analysis of integrated AAV indicated that essentially all integrants were AAVS1 site specific. Although maximal integration efficiency approached 40% of clonal cell lines (essentially 50% of infected cells), over 80% of cell lines contained a genomic disruption at the AAVS1 integration locus on chromosome 19 ( 100% of infected cells). Rep expression by itself and in the presence of a plasmid integration substrate was able to mediate this disruption of the AAVS1 site. We further characterized the disruption event and demonstrated that it resulted in amplification of the AAVS1 locus. The data are consistent with a revised model of AAV integration that includes preliminary expansion of a defined region in AAVS1.  

5.206           The host cell MAP konase ERK-2 regulates viral assembly and release by phosphorylating the p6^gag protein of HIV-1

Hemonnot, B. et al

1. Biol. Chem., 279(31), 32426-32434 (2004)

  The host cell MAP kinase ERK-2 incorporated within human immunodeficiency virus type 1 particles plays a critical role in virus infectivity by phosphorylating viral proteins. Recently, a fraction of the virus incorporated late (L) domain-containing p6^gag protein, which has an essential function in the release of viral particles from the cell surface, was reported to be phosphorylated by an unknown virus-associated cellular protein kinase (Muller, B., Patschinsky, T., and Krausslich, H. G. (2002) J. Virol. 76, 1015–1024). The present study demonstrates the contribution of the MAP kinase ERK-2 in p6^gag phosphorylation. According to mutational analysis, a single ERK-2-phosphorylated threonine residue, belonging to a highly conserved phosphorylation MAP kinase consensus site, was identified at position 23 within p6^gag. Substitution by an alanine of the Thr²³ phosphorylable residue within the pNL4.3 molecular clone was found to decrease viral release from various cell types. As observed from electron microscopy experiments, most virions produced from this molecular clone remained incompletely separated from the host cell membrane with an immature morphology and displayed a reduced infectivity in single round infection experiments. Analysis of protein processing by Western blotting experiments revealed an incomplete Pr55^gag maturation and a reduction in the virion-associated reverse transcriptase proteins was observed that was not related to differences in intracellular viral protein expression. Altogether, these data suggest that phosphorylation of p6^gag protein by virus-associated ERK-2 is involved in the budding stage of HIV-1 life cycle.  

5.207           Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2 and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system.

Burger, C. et al Mol. Ther., 10(2), 302-317 (2004)   Recombinant adeno-associated virus 2 (rAAV2) has been shown to deliver genes to neurons effectively in the brain, retina, and spinal cord. The characterization of new AAV serotypes has revealed that they have different patterns of transduction in diverse tissues. We have investigated the tropism and transduction frequency in the central nervous system (CNS) of three different rAAV vector serotypes. The vectors contained AAV2 terminal repeats flanking a green fluorescent protein expression cassette under the control of the synthetic CBA promoter, in AAV1, AAV2, or AAV5 capsids, producing the pseudotypes rAAV2/1, rAAV2/2, and rAAV2/5. Rats were injected with rAAV2/1, rAAV2/2, or rAAV2/5 into selected regions of the CNS, including the hippocampus (HPC), substantia nigra (SN), striatum, globus pallidus, and spinal cord. In all regions injected, the three vectors transduced neurons almost exclusively. All three vectors transduced the SN pars compacta with high efficiency, but rAAV2/1 and rAAV2/5 also transduced the pars reticulata. Moreover, rAAV2/1 showed widespread distribution throughout the entire midbrain. In the HPC, rAAV2/1 and rAAV2/5 targeted the pyramidal cell layers in the CA1–CA3 regions, whereas AAV2/2 primarily transduced the hilar region of the dentate gyrus. In general, rAAV2/1 and rAAV2/5 exhibited higher transduction frequencies than rAAV2/2 in all regions injected, although the differences were marginal in some regions. Retrograde transport of rAAV1 and rAAV5 was also observed in particular CNS areas. These results suggest that vectors based on distinct AAV serotypes can be chosen for specific applications in the nervous system.  

5.208           The human endosomal sorting complex required for transport (ESCRT-I) and its role in HIV-1 budding

Stuchell, M.D. et al

1. Biol. Chem., 279(34), 36059-36071 (2004)

  Efficient human immunodeficiency virus type 1 (HIV-1) budding requires an interaction between the PTAP late domain in the viral p6^Gag protein and the cellular protein TSG101. In yeast, Vps23p/TSG101 binds both Vps28p and Vps37p to form the soluble ESCRT-I complex, which functions in sorting ubiquitylated protein cargoes into multivesicular bodies. Human cells also contain ESCRT-I, but the VPS37 component(s) have not been identified. Bioinformatics and yeast two-hybrid screening methods were therefore used to identify four novel human proteins (VPS37A–D) that share weak but significant sequence similarity with yeast Vps37p and to demonstrate that VPS37A and VPS37B bind TSG101. Detailed studies produced four lines of evidence that human VPS37B is a Vps37p ortholog. 1) TSG101 bound to several different sites on VPS37B, including a putative coiled-coil region and a PTAP motif. 2) TSG101 and VPS28 co-immunoprecipitated with VPS37B-FLAG, and the three proteins comigrated together in soluble complexes of the correct size for human ESCRT-I ( 350 kDa). 3) Like TGS101, VPS37B became trapped on aberrant endosomal compartments in the presence of VPS4A proteins lacking ATPase activity. 4) Finally, VPS37B could recruit TSG101/ESCRT-I activity and thereby rescue the budding of both mutant Gag particles and HIV-1 viruses lacking native late domains. Further studies of ESCRT-I revealed that TSG101 mutations that inhibited PTAP or VPS28 binding blocked HIV-1 budding. Taken together, these experiments define new components of the human ESCRT-I complex and characterize several TSG101 protein/protein interactions required for HIV-1 budding and infectivity.  

5.209           Differential myocardial gene delivery by recombinant serotype-specific adeno-assocaited viral vectors

Du, L. et al Mol. Ther., 10(3), 604-608 (2004)   Recombinant cross-packaging of adeno-associated virus (AAV) genome of one serotype into other AAV serotypes has the potential to optimize tissue-specific gene transduction and expression in the heart. To evaluate the role of AAV1 to 5 virion shells on AAV2 transgene transduction, we constructed hybrid vectors in which each serotype capsid coding domain was cloned into a common vector backbone containing AAV2 replication genes. Constructs were tested for expression in: (1) adult murine heart in vivo using direct injection of virus, (2) neonatal and adult murine ventricular cardiomyocytes in vitro, and (3) adult human ventricular cardiomyocytes in vitro, using green fluorescent protein (GFP) as the measurable transgene. Serotype 1 virus demonstrated the highest transduction efficiency in adult murine cardiomyocytes both in vitro and in vivo, while serotype 2 virus had the greater transduction efficiency in neonatal cardiomyocytes in vitro. Prolonged in vivo myocardial GFP expression was observed for up to 12 months using serotype 1 and 2 vectors only. In human cardiomyocytes, serotype 1 vector was superior in transduction efficiency, followed by types 2, 5, 4, and 3. These data establish a hierarchy for efficient serotype-specific vector transduction in myocardial tissue. AAV1 serotype packaging results in more efficient transduction of genes in the murine and human adult heart, compared to other AAV serotypes. Our results suggest that adult human cardiac gene therapy may be enhanced by the use of serotype 1-specific AAV vectors.  

5.210           Engagement of the B-cell antigen receptor (BCR) allows efficient transduction of ZAP-70-positive primary B-CLL cells by recombinant adeno-associated virus (rAAV) vectors

Kofler, D.M. et al Gen. Ther., 11, 1415-1424 (2004)   Engagement of the B-cell antigen receptor (BCR) by crosslinking of the surface immunoglobulin (sIg) homodimer was studied for recombinant adeno-associated virus (rAAV)-mediated gene transfer into B-cell chronic lymphocytic leukaemia (B-CLL) cells. Leukemic cells obtained from 20 patients were stimulated with anti-sIg-directed antibodies and transduced with rAAV vectors coding for enhanced green fluorescent protein (EGFP) (AAV/EGFP) or CD40L (AAV/CD40L). Transduction of B-CLL cells was enhanced after BCR engagement compared to unstimulated controls (P=0.0356). BCR crosslinking induced a significant, dose- and time-dependent upregulation of heparan sulfate proteoglycan (HSPG), the primary receptor for AAV, on B-CLL cells (mean: 38.2 versus 1.7%; P=0.0006). A correlation of HSPG expression after BCR crosslinking with transduction efficiency by AAV/EGFP (P=0.0153) and AAV/CD40L (P=0.0347) was observed. High expression of zeta-associated protein 70 (ZAP-70) in B-CLL cells correlated with a better transduction efficiency by AAV/EGFP (P<0.0001) and AAV/CD40L (P=0.002), respectively: 48 h after transduction of ZAP-70-positive samples, transgene expression was seen in a mean of 33.8% (s.e.m. 3.7%) and 28.9% (s.e.m. 6.7%) of cells, respectively, and could be specifically blocked by heparin, a soluble competitor of HSPG (P<0.0001). In summary, engagement of the BCR on ZAP-70 positive B-CLL cells allows efficient rAAV-mediated gene delivery.  

5.211           The raft-promoting property of virion-associated cholesterol, but not the presence of virion-associated Brij 98 rafts, is a determinant of human immunodeficiency virus type 1 infectivity

Campbell, S. et al

1. Virol., 78(19), 10556-10565 (2004)

  Lipid rafts are enriched in cholesterol and sphingomyelin and are isolated on the basis of insolubility in detergents, such as Brij 98 and Triton X-100. Recent work by Holm et al. has shown that rafts insoluble in Brig 98 can be found in human immunodeficiency virus type 1 (HIV-1) virus-like particles, although it is not known whether raft-like structures are present in authentic HIV-1 and it is unclear whether a virion-associated raft-like structure is required for HIV replication. Independently, it was previously reported that virion-associated cholesterol is critical for HIV-1 infectivity, although the specific requirement of virion cholesterol in HIV-1 was not examined. In the present study, we have demonstrated that infectious wild-type HIV-1 contains Brij 98 rafts but only minimal amounts of Triton X-100 rafts. To directly assess the functional requirement of virion-associated rafts and various features of cholesterol on HIV-1 replication, we replaced virion cholesterol with exogenous cholesterol analogues that have demonstrated either raft-promoting or -inhibiting capacity in model membranes. We observed that variable concentrations of exogenous analogues are required to replace a defined amount of virion-associated cholesterol, showing that structurally diverse cholesterol analogues have various affinities toward HIV-1. We found that replacement of 50% of virion cholesterol with these exogenous cholesterol analogues did not eliminate the presence of Brij 98 rafts in HIV-1. However, the infectivity levels of the lipid-modified HIV-1s directly correlate with the raft-promoting capacities of these cholesterol analogues. Our data provide the first direct assessment of virion-associated Brij 98 rafts in retroviral replication and illustrate the importance of the raft-promoting property of virion-associated cholesterol in HIV-1 replication.  

5.212           HIV-1 virion fusion assay: uncoating not required and no effect of Nef on fusion

Cavrois, M., Neidleman, J., Yonemoto, W., Fenard, D. and Greene, W.C. Virology, 328, 36-44 (2004)   We recently described a sensitive and specific assay that detects the fusion of HIV-1 virions to a broad range of target cells, including primary CD4 cells. This assay involves the use of virions containing β-lactamase-Vpr (BlaM-Vpr) and the loading of target cells with CCF2, a fluorogenic substrate of β-lactamase. Since Vpr strongly associates with the viral core, uncoating of the viral particle might be required for effective cleavage of CCF2 by BlaM-Vpr. Here, we show that BlaM-Vpr within mature viral cores effectively cleaves CCF2, indicating that this assay measures virion fusion independently of uncoating. We also show that wildtype and Nef-deficient HIV-1 virions fuse with equivalent efficiency to HeLa-CD4 cells, SupT1 T cells, and primary CD4 T cells. Since Nef enhances cytoplasmic delivery of viral cores and increases viral infectivity, these findings indicate that Nef enhances an early post-fusion event in the multistep process of viral entry. Possible sites of Nef action include enlargement of the fusion pore, enhanced uncoating of viral particles, and more efficient passage of viral cores through the dense cortical actin network located immediately beneath the plasma membrane.  

5.213           Cloning and characterization of a secreted form of angiotensin-converting enzyne 2

Huentelmann, M.J., Zubcevic, J., Katovich, M.J. and Raizada, M.K. Regulatory Peptides, 122, 61-67 (2004)   Angiotensin-converting enzyme 2 (ACE2) is a newly discovered, membrane-bound aminopeptidase responsible for the production of vasodilatory peptides such as angiotensin 1–7 (Ang 1–7). Thus, ACE2 is important in counteracting the adverse, vasoconstrictor effects of angiotensin II (Ang II). The objective of the present study was to clone and characterize a constitutively secreted form of ACE2 as a prelude to an investigation into its therapeutic potential in hypertension. A truncated form of ACE2 was cloned into a lentiviral vector behind the human elongation factor 1 alpha promoter (lenti-shACE2). Transfection experiments demonstrated that secreted human ACE2 (shACE2) was secreted constitutively into the medium. The kinetic properties of shACE2 were comparable to the human recombinant enzyme (rACE2). Transduction of human coronary artery endothelial cells and rat cardiomyocytes with lenti-shACE2 showed a significant secretion of the enzyme into the medium compared to its native, membrane-bound homolog (human ACE2 [hACE2]). In addition, systemic administration of lenti-shACE2 into neonatal rats resulted in a eightfold increase in ACE2 activity in the serum above control values. These observations establish that lenti-shACE2 can be used to transduce cardiovascularly relevant cells for the secretion of functional ACE2 enzyme both in vitro and in vivo. Collectively, these results set the stage for the use of these vectors to investigate the consequences of ACE2 over-expression in the pathogenesis of hypertension

5.214           Gene therapy using replication-defective herpes simplex virus vectors expressing nerve growth factor in a rat model of diabetic cystopathy

Sasaki, K. et al Diabetes, 53, 2723-2730 (2004)   Diabetic cystopathy is one of the common complications of diabetes and current therapy is limited. In the present study, the effects of gene therapy, using replication-defective herpes simplex virus type 1 (HSV-1) vectors to deliver and express the nerve growth factor (NGF) gene (HSV-NGF) on tissue NGF levels and bladder function, were evaluated in streptozotocin (STZ)-induced diabetic rats. Diabetic rats exhibited a significant decrease in NGF levels in the bladder and lumbosacral dorsal root ganglia (DRG) detected by enzyme-linked immunosorbent assay and displayed marked bladder dysfunction 12 weeks after STZ injection. In contrast, rats with bladder wall injection of the NGF expression vector 8 weeks after STZ treatment exhibited a significant increase of NGF levels in the bladder and L6 DRG 4 weeks after HSV-NGF injection. Along with the restoration of tissue NGF expression, in metabolic cage studies and cystometry, HSV-NGF–injected rats also showed significantly reduced bladder capacity and postvoid residual volume than diabetic rats injected with the control vector (HSV-lacZ), indicating that voiding function was improved after HSV vector–mediated NGF gene delivery. Thus, HSV vector–mediated NGF gene therapy may prove useful to restore decreased NGF expression in the bladder and bladder afferent pathways, thereby improving hypoactive bladder function in diabetes.  

5.215           Establishment of papillomavirus infection is enhanced by promyelocytic leukemia protein (PML) expression

Day, P.M., Baker, C:C:, Lowy, D.R. and Schiller, J.T. PNAS, 101(39), 14252-14257 (2004)   Previous studies have suggested that most papillomaviruses enter the host cell via clathrin-dependent receptor-mediated endocytosis but have not addressed later steps in viral entry. To examine these events, we followed the localization of L2 and packaged DNA after entry of infectious virions or L1/L2 pseudovirions. Confocal microscopic analyses of HeLa cells showed a time-dependent uncoating of capsids in cytoplasmic vesicles and the accumulation of both L2 and viral DNA at distinct nuclear domains identified as nuclear domain 10 (ND10). Both L2 and the pseudogenome had a punctate distribution and localized to ND10 in promyelocytic leukemia protein (PML)-expressing cells, whereas L2 had a diffuse nuclear distribution in PML–/– cells. The number of pseudovirus-infected cells was an order of magnitude higher in the PML+ cells compared with the PML–/– cells, and viral genome transcription after infection with authentic bovine papillomavirus virions was similarly elevated in PML+ cells. The results identify a role for PML in the enhancement of viral infectivity in the early part of the life cycle. We propose a model in which L2 chaperones the viral genome to ND10 to efficiently initiate viral transcription.  

5.216           Recombinant adeno-associated virus 2-mediated transfer of the human superoxide-dismutase gene does not confer radioresistance on HeLa cervical carcinoma cells

Veldwijk, M.R. et al Radiother. & Oncol., 72, 341-350 (2004)   Background and purpose The success rate of any therapeutic approach depends on the therapeutic window, which can be increased by either raising the resistance of the normal tissue without protecting the tumor cells or by sensitizing the tumor cells but not the normal cells. Two promising candidate genes for normal tissue protection against radiation-induced damage may be the copper–zinc (CuZnSOD) and manganese superoxide-dismutase genes (MnSOD). The recombinant adeno-associated virus 2 (rAAV-2) offers attractive advantages over other vector systems: low immunogenicity, ability to infect dividing and non-dividing tissues and a low chance of insertional mutagenesis, due to extra-chromosomal localization. We report the production of novel rAAV-2-SOD vectors and the investigation of their modulating effects on HeLa-RC cells after irradiation.

Material and methods

rAAV-2 vectors were cloned containing the human CuZnSOD or MnSOD as transgene and vector stocks were produced. In the initial experiments human cervix carcinoma (HeLa-RC) cells were chosen for their susceptibility to rAAV-2. On day 0, cells were seeded and transduced with the rAAV-2-SOD vectors. On day 3, cells were harvested, irradiated (0.5–8 Gy) and reseeded in different assays (FACS, SOD, MTT and colony assays).   Results Although >70% of all cells expressed SOD and significant amounts of functional SOD protein were detected, no radioprotective effect of SOD was observed after transduction of HeLa-RC cells. Conclusions Novel rAAV-2-SOD vectors that could be produced at high titer, were able to efficiently infect cells and express the SOD genes. The absence of a radioprotective effect in HeLa-RC cancer cells indicates an additional safety feature and suggests that rAAV-mediated MnSOD overexpression might contribute to increasing the therapeutic index when applied for normal tissue protection.  

5.217           Analysis of the interaction between adeno-associated virus and heparan sulfate using atomic force microscopy

Negishi, A. et al Glycobiology, 14(11), 969-977 (2004)   Adeno-associated virus (AAV) has been widely used as a viral vector to deliver genes to animal and human tissues in gene therapy studies. Both AAV-2 and AAV-3 use cell surface heparan sulfate (HS), a highly sulfated polysaccharide, as a receptor to establish infections. In this study, we used atomic force microscopy (AFM) to investigate the interaction of HS and AAV. A silicon chip functionalized with HS was used as a substrate for binding AAV for AFM analysis. To validate our approach, we found that the binding of AAV-2 to the HS surface was effectively competed by soluble HS, suggesting that the binding of AAV-2 to the functionalized surface was specific. In addition, we examined the binding of various AAV serotypes, including AAV-1, AAV-2, AAV-3, and AAV-5, to the HS surface. As expected, only AAV-2 and AAV-3 bound, whereas AAV-1 and AAV-5 did not. This observation was consistent with the previous conclusion that AAV-1 and AAV-5 do not use HS as a receptor for infection. In conclusion, we developed a novel approach to investigate the interaction of AAV virus with its polysaccharide-based receptor at the level of a single viral particle. Given that HSs serve as receptor for numerous viruses, this approach has the potential to become a generalized method for studying interactions between the viral particle and HS, as well as other virus–cell interactions, and potentially serve as a platform for screening antiviral therapies.  

5.218           Recombinant adeno-associated viral (rAAv) vectors as therapeutic tools for duchenne muscular dystrophy (DMD)

Athanasopoulos, T., Graham, I.R., Foster, H. and Dickson, G. Gen. Ther., 11, 109-121 (2004)   Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder caused by recessive mutations in the dystrophin gene. The size of the gene (2.4 Mb) and mRNA (14 kb) in addition to immunogenicity problems and inefficient transduction of mature myofibres by currently available vector systems are formidable obstacles to the development of efficient gene therapy approaches. Adeno-associated viral (AAV) vectors overcome many of the problems associated with other vector systems (nonpathogenicity and minimal immunogenicity, extensive cell and tissue tropism) but accommodate limited transgene capacity (<5 kb). As a result of these observations, a number of laboratories worldwide have engineered a series of microdystrophin cDNAs based on genotype-phenotype relationship in Duchenne (DMD) and Becker (BMD) dystrophic patients, and transgenic studies in mdx mice. Recent progress in characterization of AAV serotypes from various species has demonstrated that alternative AAV serotypes are far more efficient in transducing muscle than the traditionally used AAV2. This article summarizes the current progress in the field of recombinant adeno-associated viral (rAAV) delivery for DMD, including optimization of recombinant AAV-microdystrophin vector systems/cassettes targeting the skeletal and cardiac musculature.  

5.219           Susceptibility of mesothelioma cell lines to adeno-associated virus 2 vector-based suicide gene therapy

Berlinghoff, S. et al Lung Cancer, 46, 179-186 (2004)   Although great efforts have been made to improve conventional therapy for diffuse malignant pleural mesothelioma, the median survival time of the patients after appearance of clinical symptoms remains poor. Due to confinement of the primary tumor to the pleural space, locoregional approaches are attractive strategies to improve the clinical outcome. In this context locoregional gene therapy using the recombinant adeno-associated virus 2 (rAAV-2) may be a new approach. Vectors were constructed containing a fusion gene, consisting of the Herpes simplex virus thymidine kinase (HSV-TK) and the green fluorescent protein (GFP) genes; the former serving as suicide gene by converting the prodrug ganciclovir (GCV) into a toxic agent, thereby killing infected cells. Among a number of different tumor cell lines, rAAV-2 achieved high GFP expression levels in three mesothelioma cell lines (H-Meso-1, MSTO-211H, NCI-H28). A variety of rAAV-2-constructs containing different promoters were tested. The vector with the elongation factor-1 (EF-1 ) promoter showed the highest expression rates. Expression could be further increased by addition of the tyrosine kinase inhibitor genistein. Using the rAAV-2-based suicide system, a nearly complete eradication of transduced and GCV-treated mesothelioma cells was observed. rAAV-2-based suicide gene therapy may be a new approach for locoregional treatment of mesothelioma.  

5.220           Infection of specific dendritic cells by CCR5-tropic human immunodeficiency virus type 1 promotes cell-mediated transmission of virus resistant to broadly neutralizing antibodies

Ganesh, L. et al

1. Virol., 78(21), 11980-11987 (2004)

  The tropism of human immunodeficiency virus type 1 for chemokine receptors plays an important role in the transmission of AIDS. Although CXCR4-tropic virus is more cytopathic for T cells, CCR5-tropic strains are transmitted more frequently in humans for reasons that are not understood. Phenotypically immature myeloid dendritic cells (mDCs) are preferentially infected by CCR5-tropic virus, in contrast to mature mDCs, which are not susceptible to infection but instead internalize virus into a protected intracellular compartment and enhance the infection of T cells. Here, we define a mechanism to explain preferential transmission of CCR5-tropic viruses based on their interaction with mDCs and sensitivity to neutralizing antibodies. Infected immature mDCs differentiated normally and were found to enhance CCR5-tropic but not CXCR4-tropic virus infection of T cells even in the continuous presence of neutralizing antibodies. Infectious synapses also formed normally in the presence of such antibodies. Infection of immature mDCs by CCR5-tropic virus can therefore establish a pool of infected cells that can efficiently transfer virus at the same time that they protect virus from antibody neutralization. This property of DCs may enhance infection, contribute to immune evasion, and could provide a selective advantage for CCR5-tropic virus transmission.  

5.221           Recombinant HIV-1 Pr55^gag virus-like particles: potent stimulators of innate and acquired immune responses

Deml. L., Speth, C., Dierich, M.P., Wolf, H. and Wagner, R. Mol. Immunol., 42, 259-277 (2005)   Several previous reports have clearly demonstrated the strong effectiveness of human immunodeficiency virus (HIV) Gag polyprotein-based virus-like particles (VLP) to stimulate humoral and cellular immune responses in complete absence of additional adjuvants. Yet, the mechanisms underlying the strong immunogenicity of these particulate antigens are still not very clear. However, current reports strongly indicate that these VLP act as “danger signals” to trigger the innate immune system and possess potent adjuvant activity to enhance the immunogenicity of per se only weakly immunogenic peptides and proteins. Here, we review the current understanding of how various particle-associated substances and other impurities may contribute to the observed immune-activating properties of these complex immunogens.  

5.222           Molecular characterization of a panel of murine monoclonal antibodies specific for the SARS-coronavirus

Gubbins, M.J. et al Mol. Immunol., 42, 125-136 (2005)   availability of monoclonal antibodies (mAbs) specific for the SARS-coronavirus (SARS-CoV) is important for the development of both diagnostic tools and treatment of infection. A molecular characterization of nine monoclonal antibodies raised in immune mice, using highly purified, inactivated SARS-CoV as the inoculating antigen, is presented in this report. These antibodies are specific for numerous viral protein targets, and six of them are able to effectively neutralize SARS-CoV in vitro, including one with a neutralizing titre of 0.075 nM. A phylogenetic analysis of the heavy and light chain sequences reveals that the mAbs share considerable homology. The majority of the heavy chains belong to a single Ig germline V-gene family, while considerably more sequence variation is evident in the light chain sequences. These analyses demonstrate that neutralization ability can be correlated with specific murine V_H-gene alleles. For instance, one evident trend is high sequence conservation in the V_H chains of the neutralizing mAbs, particularly in CDR-1 and CDR-2. The results suggest that optimization of murine mAbs for neutralization of SARS-CoV infection will likely be possible, and will aid in the development of diagnostic tools and passive treatments for SARS-CoV infection.  

5.223           Effect of virus-specific antibodies on attachment, internalization and infection of porcine reproductive and respiratory syndrome virus in primary macrophages

Delputte, P.L., Meerts, P., Costers, S. and Nauwynck, H.J. Vet. Immunol. Immunopathol., 102, 179-188 (2004) Porcine reproductive and respiratory syndrome virus (PRRSV) induces respiratory distress in young pigs  and reproductive failure in sows. In PRRSV infected pigs, virus persists for several weeks to several months. Although IPMA antibodies are detected from 7 days post inoculation (pi), virus neutralizing (VN) antibodies are commonly detected starting from 3 weeks pi with an SN test on Marc-145 cells. Since infection of Marc-145 cells is quite different compared to infection of macrophages, the in vivo target cell, the role of these VN antibodies in in vivo protection is questionable. In our study, we demonstrated that antibodies from pigs early in infection with PRRSV Lelystad virus (14 days pi) showed no neutralization in the SN test on Marc-145 cells, but partially reduced Lelystad virus infection of porcine alveolar macrophages. At 72 days pi, VN antibodies were detected by the SN test on Marc-145 cells, and these protected macrophages completely against Lelystad virus infection. In contrast, these VN antibodies only partially reduced porcine alveolar macrophage infection of a Belgian PRRSV isolate (homologous virus), and had no effect on infection of porcine alveolar macrophages with the American type VR-2332 strain (heterologous virus). Confocal analysis of Lelystad virus attachment and internalization in macrophages showed that antibodies blocked infection through both a reduction in virus attachment, and a reduction of PRRSV internalization. Western immunoblotting analysis revealed that sera from 14 days pi, which showed no neutralization in the SN test on Marc-145 cells but partially reduced Lelystad virus infection of macrophages, predominantly recognized the Lelystad virus N protein, and reacted faintly with the M envelope protein. Sera from 72 days pi, with VN antibodies that blocked infection of Marc-145 cells and PAM, reacted with the N protein and the two major envelope proteins M and GP₅. Using the Belgian PRRSV isolate 94V360 an identical but less intense reactivity profile was obtained. VN sera also recognized the VR-2332 N and M protein, but not the GP₅ protein.

5.224           Tracking fluorescence-labeled rabies virus: enhanced green fluorescent protein-tagged phosphoprotein P supports virus gene expression and formation of infectious particles

Finke, S., Brzozka, K. And Conzelmann, K-K.

1. Virol., 78(22), 12333-12343 (2004)

  Rhabdoviruses such as rabies virus (RV) encode only five multifunctional proteins accomplishing viral gene expression and virus formation. The viral phosphoprotein, P, is a structural component of the viral ribonucleoprotein (RNP) complex and an essential cofactor for the viral RNA-dependent RNA polymerase. We show here that RV P fused to enhanced green fluorescent protein (eGFP) can substitute for P throughout the viral life cycle, allowing fluorescence labeling and tracking of RV RNPs under live cell conditions. To first assess the functions of P fusion constructs, a recombinant RV lacking the P gene, SAD P, was complemented in cell lines constitutively expressing eGFP-P or P-eGFP fusion proteins. P-eGFP supported the rapid accumulation of viral mRNAs but led to low infectious-virus titers, suggesting impairment of virus formation. In contrast, complementation with eGFP-P resulted in slower accumulation of mRNAs but similar infectious titers, suggesting interference with polymerase activity rather than with virus formation. Fluorescence microscopy allowed the detection of eGFP-P-labeled extracellular virus particles and tracking of cell binding and temperature-dependent internalization into intracellular vesicles. Recombinant RVs expressing eGFP-P or an eGFP-P mutant lacking the binding site for dynein light chain 1 (DLC1) instead of P were used to track interaction with cellular proteins. In cells expressing a DsRed-labeled DLC1, colocalization of DLC1 with eGFP-P but not with the mutant P was observed. Fluorescent labeling of RV RNPs will allow further dissection of virus entry, replication, and egress under live-cell conditions as well as cell interactions.  

5.225           Generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production

Huang, Y., Yang, Z-Y., Kong, W-P. and Nabel, G.J.

1. Virol., 78(22), 12557-12565 (2004)

  The recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV) contains four structural genes, two replicase-transcriptase open reading frames, and more than five potential genes of unknown function. Despite this relative simplicity, the molecular regulation of SARS-CoV replication and assembly is not understood. Here, we report that two viral genes, encoding the SARS-CoV membrane (M) and nucleocapsid (N) proteins, are necessary and sufficient for formation of virus-like particles. Expression vectors encoding these two proteins were synthesized by using preferred human codons. When M and N expression plasmids were cotransfected into human 293 renal epithelial cells, pseudoparticles formed readily. The addition of a third gene, encoding the spike (S) glycoprotein, facilitated budding of particles that contained a corona-like halo resembling SARS-CoV when examined by transmission electron microscopy, with a buoyant density characteristic of coronaviruses. Specific biochemical interactions of these proteins were also shown in vitro. The S, M, and N proteins of the SARS-CoV are, therefore, necessary and sufficient for pseudovirus assembly. These findings advance the understanding of the morphogenesis of SARS-CoV and enable the generation of safe, conformational mimetics of the SARS virus that may facilitate the development of vaccines and antiviral drugs.  

5.226           Recombinant adeno-associated virus serotype 2 effectively transduces primary rat brain astrocytes and microglia

Gong, Y. et al Brain Res. Protocols, 14, 18-24 (2004)   Recombinant adeno-associated virus-2 (rAAV2) under control of the chicken beta actin promoter/truncated CMV enhancer (CBA) was investigated for its ability to transduce primary cultures of rat brain neurons, microglia and astrocytes. This vector was highly effective in all three cell types in heparin-sensitive manners (astrocytes, microglia and neurons transduced by >98%, 75%, and 95%, respectively). However, astrocytes co-cultured with neurons were not transduced. rAAV2/CBA is an important new method for genetic manipulation of brain cells, though this may be modulated by interactions among cell types.  

5.227           Functional characterization of a recombinant adeno-associated virus 5-pseudotyped cystic fibrosis transmembrane conductance regulator vector

Sirninger, J. et al Human Gen. Ther., 15, 832-841 (2004)   Despite extensive experience with recombinant adeno-associated virus (rAAV) 2 vectors in the lung, gene expression has been low in the context of cystic fibrosis (CF) gene therapy, where the large size of the cystic fibrosis transmembrane conductance regulator (CFTR) coding sequence has prompted the use of compact endogenous promoter elements. We evaluated the possibility that gene expression from recombinant adeno-associated virus (rAAV) could be improved by using alternate AAV capsid serotypes that target different cell-surface receptors (i.e., rAAV5) and/or using stronger promoters. The relative activities of the cytomegalovirus (CMV) Rous sarcoma virus (RSV) promoter, the CMV enhancer/β-actin (CB) promoter combination, and the CMV enhancer/RSV promoter hybrid were assessed in vitro in a CF bronchial cell line. The CB promoter was the most efficient. AAV capsid serotypes, rAAV2 and rAAV5, were also compared, and rAAV5 was found to be significantly more efficient. Based on these studies a rAAV5-CB-promoter-driven CFTR minigene vector was then used to correct the CF chloride transport defect in vitro, as well as the hyperinflammatory lung phenotype in Pseudomonas-agarose bead challenged CF mouse lungs in vivo. These studies provide functional characterization of a new version of rAAV-CFTR vectors.  

5.228           Augmentation of antitumor activity of a recombinant adeno-associated virus carcinoembryonic antigen vaccine with plasmid adjuvant

Ponnazhagan, S. et al Human Gen. Ther., 15, 856-864 (2004)   Recombinant adeno-associated virus 2 (rAAV) vectors have been successfully used for sustained expression of therapeutic genes. The potential of using rAAV as a cancer vaccine vector and the impact of a bacterial plasmid adjuvant on this activity were investigated. C57BL/6 mice received a single intramuscular injection of rAAV expressing the human tumor-associated antigen, carcinoembryonic antigen (CEA). Three weeks later, when CEA expression was optimal, a bacterial plasmid containing methylated DNA motifs was injected into the same muscle. Mice were challenged 1 week later with syngeneic MC38 tumor cells stably expressing CEA. Immunization with rAAV-CEA alone resulted in sustained transgene expression and the elicitation of a humoral immune response to CEA. Cellular immune response, however, was weak, and tumor protection was not significant. In contrast, immunization with rAAV-CEA and the plasmid adjuvant resulted in stronger cellular immune response to CEA and tumor protection. The addition of plasmid adjuvant increased both myeloid dendritic cell recruitment in situ and CEA-specific T-helper-1-associated immune response. These data indicate that robust rAAV transgene expression of a tumor antigen followed by transient plasmid delivery to recruit and activate dendritic cells is an effective method of eliciting antitumor cellular immune responses.  

5.229           Functional expression of the single subunit NADH dehydrogenase in mitochondria in vivo: a potential therapy for complex I deficiencies

Seo, B.B. et al Human Gen. Ther., 15, 887-895 (2004)   It has been reported that defects of mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) are involved in many human diseases (such as encephalomyopathies and sporadic Parkinson's disease). However, no effective remedies have been established for complex I deficiencies. We have adopted a gene therapy approach utilizing the NDI1 gene that codes for the single subunit NADH dehydrogenase of Saccharomyces cerevisiae (Ndi1). Our earlier experiments show that the Ndi1 protein can replace or supplement the functionality of complex I in various cultured cells. For this approach to be useful, it is important to demonstrate in vivo that the mature protein is correctly placed in mitochondria. In this study, we have attempted in vivo expression of the NDI1 gene in skeletal muscles and brains (substantia nigra and striatum) of rodents. In all tissues tested, the Ndi1 protein was identified in the injected area by immunohistochemical staining at 1-2 weeks after the injection. Sustained expression was observed for at least 7 months. Double-staining of the sections using antibodies against Ndi1 and F₁-ATPase revealed that the expressed Ndi1 protein was predominantly localized to mitochondria. In addition, the tissue cells expressing the Ndi1 protein stimulated the NADH dehydrogenase activity, suggesting that the expressed Ndi1 is functionally active. It was also confirmed that the Ndi1 expression induced no inflammatory response in the tissues examined. The data indicate that the NDI1 gene will be a promising therapeutic tool in the treatment of encephalomyopathies and neurodegenerative diseases caused by complex I impairments.  

5.230           Long-term correction of murine lipoprotein lipase deficiency with AAV1-mediated gene transfer of the naturally occurring LPL^S447X beneficial mutation

Ross, C.J.D. et al Human Gen. Ther., 15, 906-919 (2004)   Human lipoprotein lipase (LPL) deficiency causes profound hypertriglyceridemia and life-threatening pancreatitis. We recently developed an adult murine model for LPL deficiency: LPL -/- mice display grossly elevated plasma triglyceride (TG) levels (>200-fold) and very low high-density lipoprotein cholesterol (HDL-C < 10% of normal). We used this animal model to test the efficacy of adeno-associated virus-mediated expression of hLPL^S447X (AAV1-LPL^S447X) in muscle for the treatment of LPL deficiency. Intramuscular administration of AAV1-LPL^S447X resulted in dose-dependent expression of hLPL protein and LPL activity (up to 33% of normal murine levels) in postheparin plasma. Remarkably, visible hyperlipidemia was resolved within 1 week; plasma TG was reduced to near-normal levels (from 99.0 to 1.8 mmol/L), and plasma HDL-C was increased 6-fold (from 0.2 to 1.1 mmol/L). At 8 months after administration of AAV1-LPL^S447X, an intravenous lipid challenge showed efficient, near-normal clearance of plasma TG. Histologic analyses of injected muscle further indicated that abnormal muscle morphology observed in LPL -/- mice was reversed after treatment. Expression of therapeutic levels of LPL^S447X, and the subsequent beneficial effect on plasma lipid levels, has lasted for more than 1 year. We therefore conclude that AAV1-mediated transfer of LPL^S447X into murine skeletal muscle results in long-term near-correction of dyslipidemia associated with LPL deficiency.  

5.231           Induction of brain region-specific forms of obesity by Agouti

Kas, M.J.H. et al

1. Neurosci., 24(45), 10176-10181 (2004)

  Disruption of melanocortin (MC) signaling, such as by ectopic Agouti overexpression, leads to an obesity syndrome with hyperphagia, obesity, and accelerated body weight gain during high-fat diet. To investigate where in the brain disruption of MC signaling results in obesity, long-term Agouti expression was induced after local injections of recombinant adeno-associated viral particles in selected brain nuclei of adult rats. Agouti expression in the paraventricular nucleus, a hypothalamic region with a high density of MC receptors, induced acute onset hyperphagia and rapid weight gain that persisted for at least 6 weeks. In contrast, obesity and hyperphagia developed with a 3 week delay when Agouti was expressed in the dorsal medial hypothalamus. Agouti expression in the lateral hypothalamus (LH) did not affect food intake and body weight during regular diet, despite the presence of MC receptors in this region. However, during exposure to a high-fat diet, animals with Agouti expression in the LH exhibited a marked increase in body weight. Here we show that the LH is important for the protection against diet-induced obesity by controlling caloric intake during consumption of a high-fat diet. Together, this study provides evidence that different aspects of the Agouti-induced obesity syndrome, such as hyperphagia and diet responsiveness, are mediated by distinct brain regions and opens challenging opportunities for further understanding of pathophysiological processes in the development of the obesity syndrome.  

5.232           Improved behavior and neuropathology in the mouse model of Sanfilippo type IIIb disease after adeno-associated virus-mediated gene transfer in the striatum

Cressant, A. et al

1. Neurosci., 24(45), 10229-10239 (2004)

  Sanfilippo syndrome is a mucopolysaccharidosis (MPS) caused by a lysosomal enzyme defect interrupting the degradation pathway of heparan sulfates. Affected children develop hyperactivity, aggressiveness, delayed development, and severe neuropathology. We observed relevant behaviors in the mouse model of Sanfilippo syndrome type B (MPSIIIB), in which the gene coding for -N-acetylglucosaminidase (NaGlu) is invalidated. We addressed the feasibility of gene therapy in these animals. Vectors derived from adeno-associated virus serotype 2 (AAV2) or 5 (AAV5) coding for NaGlu were injected at a single site in the putamen of 45 6-week-old MPSIIIB mice. Normal behavior was observed in treated mice. High NaGlu activity, far above physiological levels, was measured in the brain and persisted at 38 weeks of age. NaGlu immunoreactivity was detected in neuron intracellular organelles, including lysosomes. Enzyme activity spread beyond vector diffusion areas. Delivery to the entire brain was reproducibly obtained with both vector types. NaGlu activity was higher and distribution was broader with AAV5-NaGlu than with AAV2-NaGlu vectors. The compensatory increase in the activity of various lysosomal enzymes was improved. The accumulation of gangliosides GM2 and GM3 present before treatment and possibly participating in neuropathology was reversed. Characteristic vacuolations in microglia, perivascular cells, and neurons, which were prominent before the age of treatment, disappeared in areas in which NaGlu was present. However, improvement was only partial in some animals, in contrast to high NaGlu activity. These results indicate that NaGlu delivery from intracerebral sources has the capacity to alleviate most disease manifestations in the MPSIIIB mouse model.  

5.233           Improved efficiency of a Salmonella-based vaccine against human papillomavirus type 16 virus-like particles achieved by using a codon-optimized version of L1

Baud, D., Ponci, F., Bobst, M., De Gandhi, P. And Nardelli-Haefliger, D.

1. Virol., 78(23), 12901-12909 (2004)

  Cervical cancer results from cervical infection by human papillomaviruses (HPVs), especially HPV16. An effective vaccine against these HPVs is expected to have a dramatic impact on the incidence of this cancer and its precursor lesions. The leading candidate, a subunit prophylactic HPV virus-like particle (VLP) vaccine, can protect women from HPV infection. An alternative improved vaccine that avoids parenteral injection, that is efficient with a single dose, and that induces mucosal immunity might greatly facilitate vaccine implementation in different settings. In this study, we have constructed a new generation of recombinant Salmonella organisms that assemble HPV16 VLPs and induce high titers of neutralizing antibodies in mice after a single nasal or oral immunization with live bacteria. This was achieved through the expression of a HPV16 L1 capsid gene whose codon usage was optimized to fit with the most frequently used codons in Salmonella. Interestingly, the high immunogenicity of the new recombinant bacteria did not correlate with an increased expression of L1 VLPs but with a greater stability of the L1-expressing plasmid in vitro and in vivo in absence of antibiotic selection. Anti-HPV16 humoral and neutralizing responses were also observed with different Salmonella enterica serovar Typhimurium strains whose attenuating deletions have already been shown to be safe after oral vaccination of humans. Thus, our findings are a promising improvement toward a vaccine strain that could be tested in human volunteers.  

5.234           Therapeutic levels for a1-antitrypsin following intrapleural administration of a non-human primate serotype rh10 AAV vector expressing a1-antitrypsin

De, B.P et al Mol. Ther., 9, Suppl. 1, 338, S128 (2004) Alpha 1-antitrypsin ( 1AT), a serine protease inhibitor synthesized and secreted by the liver, protects the lung from degradation by neutrophil proteases. 1AT deficiency is a common autosomal recessive disorder associated with the accelerated development of emphysema if serum 1AT levels are <11 M (570 g/ml). In this study, we test the hypothesis that persistent expression of 1AT at therapeutic levels can be achieved in mice by intrapleural administration of AAV vectors expressing the cDNA for 1AT at vector doses that can be safely scaled up to humans. In initial studies, mice were injected with 10¹¹ genome copies (gc) of an adeno-associated virus seroytpe 2 (AAV2) based vector expressing human cDNA for 1AT from a cytomegalovirus-chicken -actin hybrid promoter. Serum levels of 80 ± 19 g/ml (mean ± SD) were achieved 8 wk following intrapleural administration, a route chosen to minimize safety issues while providing proximity to the lung. Since this fell short of the target expression level, an alternate approach was developed using the same vector construct pseudotyped by the capsid of AAVrh.10. AAVrh.10 is derived from rhesus macaque and utilizes cell surface receptor(s) distinct from that of AAV2 and is not recognized by preexisting anti-AAV2 neutralizing antibodies. The AAVrh.10 1AT vector was prepared by triple transfection with a yield of 15,000 ± 5,000 gc/cell and purified by iodixanol gradient and ion exchange chromatography. The AAVrh.10 1AT vector was administered in C57Bl/6 mice (n=4/group) at a dose of 5 × 10¹⁰ gc, via intrapleural, intramuscular, and intratracheal route and serum 1AT levels measured by ELISA. At 2 wk post-injection, the level of 1AT in intrapleural injected animals was 200 ± 10 g/ml, compared to 1.2 ± 0.6 g/ml for intratracheal injected animals and 60 ± 15 g/ml for intramuscular injected animals (p < 0.01 all pairwise comparisons). Intrapleural administration of AAVrh.1 1AT vector at a higher dose (10¹¹ gc) showed serum 1AT levels of 2,200 ± 300 g/ml after 8 wk, higher than the target for therapy. To determine the relative distribution of the vector in lung following intrapleural administration, an AAVrh.10 vector expressing luciferase was injected intrapleurally in C57Bl/6 mice (n = 4/group) and after 8 wk, luciferase activity was measured in tissue homogenates. The luciferase activity in the diaphragm was 7 × 10⁶ RLU/mg, in the left lung 7.5 × 10⁶ RLU/mg, in the right lung 1.7 × 10⁶ RLU/mg whereas in liver and kidney luciferase activities were 0.8 × 10⁶ RLU/mg and 0.02 × 10⁶ RLU/mg, respectively, indicating that diaphragm and lung are the major sites of gene transfer. These data indicate that intrapleural administration of an AAVrh.10 vector may be a more efficient and readily scalable strategy for delivery of 1AT to the lung than by other routes including direct intratracheal administration.

5.235           Incorporation of the green fluorescent protein into the adeno-associated virus type 2 capsid

Lux, K. et al Mol. Ther., 9, Suppl. 1, (2004)   Adeno-Associated Virus type 2 (AAV) is a small, nonenveloped, icosahedral virus of approximately 25 nm in diameter that packages a single-stranded DNA. Until now, no human disease caused by AAV has been detected. This and other features as e.g. its ability to transduce both dividing and non-dividing cells, its low immunogenicity and its broad tropism make of AAV a promising system for the development as gene therapy vector. However, many aspects of its infectious biology still remain to be elucidated. Green fluorescent protein (GFP) has been extensively used to study intracellular trafficking of proteins. Therefore, we incorporated GFP into the AAV capsid in order to allow a direct visualization of the infectious process. Based on earlier results, obtained by Yang et al. (1998), we generated an N-terminal fusion of the GFP protein with the second largest capsid protein, named VP2. We could show by transient transfection assays that this fusion protein is tranported into the nucleus like wild type capsid protein. Next, we generated viral particles containing the GFP-VP2 fusion protein. Viral progeny was obtained with titers comparable to wild type AAV and could be purified by iodixanol gradient centrifugation or heparin affinity chromatography. The GFP-VP2 fusion protein was detected together with the other wild type capsid proteins in Western Blot analysis of purified viral preparations. The particle to capsid ratio showed that the fusion protein does not interfere with viral genome packaging. Furthermore, HeLa cells infected with GFP-VP2 containing virions resulted in eGFP positive cells measurable by FACS analysis. Such infections could be inhibited by the addition of heparin. Finally, fluorescent viral particles could be visualized by live cell imaging and fluorescent microscopy. First results will be presented.  

5.236           Universal purification of AAV serotypes 1-5 modified to contain a heparin binding epitope

Faust, S.M. et al Mol. Ther., 9 Suppl. 1, (2004)   To directly evaluate the utility of distinct serotypes of adeno-associated virus (AAV) for gene therapy applications, it will be necessary to purify each in a similar manner. The methodology to obtain highly purified rAAV2 for use in clinical trials has been established using heparin column affinity binding in conjunction with iodixanol gradients. With the exception of AAV3, the other serotypes of AAV do not bind heparin. Recently the amino acids responsible for the ability of AAV2 to bind to heparin were identified (Kern et al 2003, J Virol 77:11072–81) and tested in the context of rAAV5 (Opie et al 2003, J Virol 77:6995–7006). To assess whether all serotypes could be modified to bind heparin, the AAV2 amino acids R585 R588 and A590 were substituted into the homologous positions in serotypes 1, 3, 4, and 5 to generate the pxr1RRA, pxr3RRA, pxr4RRA, and pxr5RRA plasmids. These helper vectors were then used to produce recombinant eGFP virus which were initially purified by either cesium chloride or iodixanol gradients followed by dialysis. 1 × 1010 viral genome-containing particles from serotypes 1–5 and the analogous RRA-containing serotypes were applied to three types of commercially available heparin agarose (Sigma: Heparin type I (cat # H6508), II-S (cat # H3025), and III-S (cat # H1277)). As expected, rAAV2 and rAAV3 bound all three types of heparin with rAAV2 binding type III-S and rAAV3 binding type I heparin most efficiently. As expected AAV1, 4, and 5 did not bind to most types of heparin; however, there were some exceptions. Most notably, approximately 70% of rAAV4 bound type III-S heparin while approximately 40% of AAV1, 4, and 5 bound to and eluted from type II-S heparin. These results suggest that other types of heparin should be considered in the optimal purification of AAV serotypes. AAV serotypes modified to contain the RRA epitope bound and eluted from all types of heparin agarose tested in a profile similar to rAAV2. The binding affinity of rAAV5 RRA was the least efficient with approximately 60% of total virus eluting from the columns. rAAV RRA 1, 3, and 4 bound with greater efficiency (between 80–85%). The ability to purify these AAV serotypes in similar manners will allow more accurate comparisons to be made regarding tissue tropisms. In addition, since every purification method utilized for clinical trials must undergo its own certification, a universal purification scheme for all AAV serotypes would eliminate this need.  

5.237           Intrapleural administration of a serotype 5 adeno-associated virus coding for a1-antitrypsin mediates persistent, high lung and serum levels of a1-antitrypsin

De, B. et al Mol. Ther., 10(6), 1003-1010 (2004)   α1-Antitrypsin (α1AT) is a serine proteinase inhibitor that protects the lung from degradation by neutrophil proteases. In α1AT deficiency, an autosomal recessive disorder resulting from mutations in the α1AT (approved symbol SERPINA1) gene, serum α1AT levels of < 570 μg/ml are associated with development of emphysema. Adeno-associated virus (AAV) serotype 2 (AAV2) vectors expressing α1AT administered intramuscularly or intravenously mediate sustained serum levels of α1AT in experimental animals. Since the lung is only 2% of the body weight, AAV vector delivery to the muscle or liver is inefficient, as most of the α1AT does not reach the lung. The present study evaluates AAV2- and AAV5-mediated delivery of human α1AT (hα1AT) to C57BL/6 mice using the intrapleural space as a platform for local production of α1AT. Intrapleural administration of either an AAV5-hα1AT or an AAV2-hα1AT vector achieves higher lung and serum levels of α1AT than intramuscular delivery. AAV5-mediated serum and lung α1AT levels were 10-fold higher than those achieved by AAV2 delivery via either route. The diaphragm, lung, and heart are the major sites of transgene expression following intrapleural administration of an AAV5 reporter vector. At 40 weeks postadministration, intrapleural administration of the AAV5-hα1AT vector mediated serum α1AT levels of 900 ± 50 μg/ml, 1.6-fold higher than the accepted therapeutic level of 570 μg/ml. In the context that the pleura is a safe site for administration, intrapleural administration using AAV5 vectors may represent an attractive gene therapy strategy for α1AT deficiency in humans.  

5.238           Furin-mediated cleavage of the feline foamy virus Env leader protein

Geiselhart, V., Bastone, P., Kempf, T., Schnölzer, M. and Löchelt, M.

1. Virol., 78(24), 13573-13581 (2004)

  The molecular biology of spuma or foamy retroviruses is different from that of the other members of the Retroviridae. Among thedistinguishing features, the N-terminal domain of the foamyvirus Env glycoprotein, the 16-kDa Env leader protein Elp, isa component of released, infectious virions and is requiredfor particle budding. The transmembrane protein Elp specificallyinteracts with N-terminal Gag sequences during morphogenesis.In this study, we investigate the mechanism of Elp release fromthe Env precursor protein. By a combination of genetic, biochemical,and biophysical methods, we show that the feline foamy virus(FFV) Elp is released by a cellular furin-like protease, mostlikely furin itself, generating an Elp protein consisting of127 amino acid residues. The cleavage site fully conforms tothe rules for an optimal furin site. Proteolytic processingat the furin cleavage site is required for full infectivityof FFV. However, utilization of other furin proteases and/orcleavage at a suboptimal signal peptidase cleavage site canpartially rescue virus viability. In addition, we show thatFFV Elp carries an N-linked oligosaccharide that is not conservedamong the known foamy viruses.  

5.239           Progress in the use of adeno-associated viral vectors for gene therapy

Büning, H., Braun-Falco, M. and Hallek, M. Cells Tissues Organs, 177, 139-150 (2004)   The development of safe and efficient gene transfer vectors is crucial for the success of gene therapy trials. A viral vector system promising to meet these requirements is based on the apathogenic adeno-associated virus (AAV-2), a member of the parvovirus family. The advantages of this vector system is the stability of the viral capsid, the low immunogenicity, the ability to transduce both dividing and non-dividing cells, the potential to integrate site specifically and to achieve long-term gene expression even in vivo, and its broad tropism allowing the efficient transduction of diverse organs including the skin. All this makes AAV-2 attractive and efficient for in vitro gene transfer and local injection in vivo. This review covers the progress made in AAV vector technology including the development of AAV vectors based on other serotypes, summarizes the results obtained by AAV targeting vectors and outlines potential applications in the field of cutaneous gene therapy.  

5.240           Osteogenic differentiation of recombinant adeno-associated virus 2-transduced murine mesenchymal stem cells and development of an immunocomponent mouse model for ex vivo osteoporosis gene therapy

Kumar, S., Mahendra, G., Nagy, T.R. and ponnazhagan, S. Hum. Gen. Ther., 15, 1197-1206 (2004) Gene therapy for osteopenic conditions including osteoporosis is a potential alternative to pharmacotherapy for cost effectiveness, long-term viability, and the ability to enhance bone mass by anabolic approaches. Increased understanding of mesenchymal stem cell (MSC) lineage differentiation during osteogenesis, and of the molecular pathways involved in bone cell production, provides an opportunity for the advancement of gene therapy approaches for osteopenic conditions. The potential of MSCs in osteoblast differentiation and the relative ease of MSC isolation and culturing offer a promising resource for the development of ex vivo gene therapy for bone defects. In an effort to develop ex vivo gene therapy for osteoporosis, we used genemodified MSCs in a preclinical mouse model to determine the efficiency of transduction of murine MSCs by recombinant adeno-associated virus 2 (AAV) vectors carrying reporter genes and determined their osteogenic potential after recombinant AAV-mediated expression of bone morphogenic protein 2, known to induce osteoblast differentiation. Although surgical ovariectomy is believed to induce progressive bone loss in mouse models, similar to an osteoporosis-like phenotype in humans, several factors, including hormonal alteration and dietary habits, significantly affect both the onset and progression of the disease. Thus, in the present study, we determined the influence of these factors and developed an immunocompetent mouse model of osteoporosis with degenerative bone loss as in the human pathology.

5.241           Gene transfer into rabbit arteries with adeno-associated virus and adenovirus vectors

Gruchala, M., bhardwaj, S., Pajusola, K., Roy, H., Rissanen, T.T., Kokina, I., Kholova, I., Markkanen, J.E., Rutanen, J., Heikura, T., Alitalo, K., Büeler, H. and Ylä-Herttula, S.

1. Gene Med., 6(5), 545-554 (2004)

  Background Gene transfer offers considerable potential for altering vessel wall physiology and intervention in vascular disease. Therefore, there is great interest in developing optimal strategies and vectors for efficient, targeted gene delivery into a vessel wall. Methods We studied adeno-associated viruses (AAV; 9 × 108 to 4 × 109 TU/ml) for their usefulness to transduce rabbit arteries in vivo in comparison with adenoviruses (Adv; 1 × 109 to 1 × 1010 pfu/ml). 100 µl of viruses or placebo solution were injected intraluminally into transiently isolated carotid segments. Results In normal arteries AAV transduced mainly medial smooth muscle cells (SMC) while Adv transduced exclusively endothelial cells (EC). Mechanical injury to EC layer and internal elastic lamina enabled Adv to penetrate and transduce medial SMC. Transgene expression in EC after the AAV-mediated gene transfer was very low. The use of the EC-specific Tie-1 promoter did not lead to specific transgene expression in EC. Transgene expression in SMC persisted for at least 100 days after the AAV treatment whereas the Adv-mediated effect diminished in 14 days. AAV caused only a modest increase in EC VCAM-1 expression and proliferation rate of vascular cells as compared with the mock-treated arteries while Adv caused an extensive inflammatory cell infiltration, VCAM-1 expression, vascular cell proliferation and morphological damages. Conclusions Significant differences were observed between the AAV and the Adv vectors in their patterns of arterial transduction and consequent inflammatory responses. These distinct properties may be utilized for different applications in vascular biology research and gene therapy for cardiovascular diseases.  

5.242           Identification of a replication-defective herpes simplex virus for recombinant adeno-associated virus type 2 (rAAV2) particle assembly using stable producer cell lines

Toublanc, E., Benraiss, A., Bonnin, D., Blouin, V., Brument, N., Cartier, N., Epstein, A.L., Moullier, P. and Salvetti, A.

1. Gene Med., 6(5), 555-564 (2004)

  Background The development of stable producer cell lines for recombinant adeno-associated virus (rAAV) assembly is a strategy followed by many groups to develop scalable production methods suitable for good manufacturing practice (GMP) requirements. The major drawback of this method lies in the requirement for replicating adenovirus (Ad) for rAAV assembly. In the present study, we analyzed the ability of several replication-defective herpes simplex type 1 (HSV-1) helper viruses to induce rAAV2 particle production from stable producer cell lines. Methods Several stable rAAV producer cell clones were infected with wild-type and replication-defective HSV strains and analyzed for rep-cap gene amplification, viral protein synthesis and rAAV titers achieved. In vivo analysis following rAAV injection in the murine brain was also conducted to evaluate the toxicity and biopotency of the rAAV stocks. Results We demonstrated that an HSV strain mutated in the UL30 polymerase gene could efficiently be used in this context, resulting in rAAV titers similar to those measured with wild-type HSV or Ad. Importantly, with respect to clinical developments, the use of this mutant resulted in rAAV stocks which were consistently devoid of contaminating HSV particles and fully active in vivo in the murine central nervous system with no detectable toxicity. Conclusions This study, together with our previous report describing a rAAV chromatography-based purification process, contributes to the definition of an entirely scalable process for the generation of rAAV particles.  

5.243           Autonomous parvovirus vectors: preventing the generation of wild-type or replication-competent virus

Brandenburger, A. and Velu, T.

1. Gene Med., 6, S203-S211 (2004)

  The preferential expression of autonomous parvoviruses in tumour cells and their oncolytic activity has attracted attention to the potential use of these viruses as vectors for cancer gene therapy. Moreover, they are non-pathogenic in adult animals and they seem to be associated with low or no immunogenicity. Other interesting features are their episomal replication and high stability. Vectors derived from the autonomous parvoviruses MVM(p) or H1 express proteins that can directly or indirectly interfere with tumour development. They retain cis- and trans-acting sequences required for viral DNA amplification; the transgene replaces part of the capsid coding genes. Their development has been hampered by low titres and contamination with replication-competent virus (RCV) that is generated through homologous recombination with helper plasmids. Several approaches have been used to avoid recombination between vectors and helpers. In most instances, reducing the homology up- or downstream of the transgene in either the vector or the helper did not significantly affect RCV production. However, completely eliminating homology downstream of the transgene, splitting VP genes on different helpers or pseudotyping vectors resulted in the production of RCV-free stocks. Although VP-containing particles could sometimes be identified in these stocks by in situ hybridisation, they did not amplify and are therefore not true RCV. The integration of capsid-coding sequences into packaging cells also reduced contamination by RCV and allowed for the amplification of vectors through serial infections. Great progress has been made recently towards the generation of truly RCV-free stocks of vectors derived from autonomous parvoviruses H1 and MVMp. Combining these new vectors with a new packaging cell line should greatly facilitate their development.  

5.244           Prevention of neuropathology in the mouse model of hurler syndrome

Desmaris, N., Verot, L., Puech, J.P., Caillaud, C., Vanier, M.T. and Heard, J.M. Ann. Neurol., 56(1), 68-76 (2004)   A defect of the lysosomal enzyme -L-iduronidase (IDUA) interrupts heparan and dermatan sulfate degradation and causes neuropathology in children with severe forms of mucopolysaccharidosis type I (MPSI, Hurler syndrome). Enzyme substitution therapy is beneficial but ineffective on the central nervous system. We could deliver the missing enzyme to virtually the entire brain of MPSI mice through a single injection of gene transfer vectors derived from adenoassociated virus serotype 2 (AAV2) or 5 (AAV5) coding for human IDUA. This result was reproducibly achieved with both vector types in 46 mice and persisted for at least 26 weeks. Success was more frequent, enzyme activity was higher, and corrected areas were broader with AAV5 than with AAV2 vectors. Treatment presumably reversed and certainly prevented the accumulation of GM2 and GM3 gangliosides, which presumably participates to neuropathology. Lysosomal distension, which already was present at the time of treatment, had disappeared from both brain hemispheres and was minimal in the cerebellum in mice analyzed 26 weeks after injection. This study shows that pathology associated with MPSI can be prevented in the entire mouse brain by a single AAV vector injection, providing a preliminary evaluation of the feasibility of gene therapy to stop neuropathology in Hurler syndrome.  

5.245           SOD2 gene transfer protects against optic neuropathy induced by deficiency of complex I

Qi, X., Lewin, A.S., Sun, L., Hauswirth, W.W. and Guy, J. Ann. Neurol., 56(2), 182-191 (2004)   Mutations in genes encoding the NADH ubiquinone oxidoreductase, complex I of the respiratory chain, cause a diverse group of diseases. They include Leber hereditary optic neuropathy, Leigh syndrome, and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes. There is no effective treatment for these or any other mitochondrial disorder. Using a unique animal model of severe complex I deficiency induced by ribozymes targeted against a critical complex I subunit gene (NDUFA1), we attempted rescue of the optic nerve degeneration associated with Leber hereditary optic neuropathy. We used adenoassociated virus to deliver the human gene for SOD2 to the visual system of disease-induced mice. Relative to mock infection, SOD2 reduced apoptosis of retinal ganglion cells and degeneration of optic nerve fibers, the hallmarks of this disease. Rescue of this animal model supports a critical role for oxidative injury in disorders with complex I deficiency and shows that a respiratory deficit may be effectively treated in mammals, thus offering hope to patients.  

5.246           Delivery of herpes simplex virus-based vectors to the nervous system

Goss, J.R., Natsume, A., Wolfe, D., mata, M., Glorioso, J.C. and Fink, D. Methods Mol. Biol., 246, 309-322 (2004)   Gene transfer to the nervous system is an attractive option to treat a wide variety of neurological insults. The expression of trophic factor and/or antiapoptotic genes may be beneficial in halting the slow neurodegeneration in such conditions as Parkinson's disease (4,5), the rapid neuronal cell death following trauma to the brain or spinal cord (6,7), or in treating peripheral neuropathies associated with diabetes or use of chemotherapeutic agents (8,9). Introduction of dominant-negative mutant genes or antisense RNA to treat diseases such as Huntington's disease, or transfer of genes to replace lost or mutated endogenous proteins to treat disorders such as lysosomal storage diseases, may prove useful. In addition, gene transfer to overexpress endogenous antinociceptive proteins has great potential in pain management. The problem faced by all of these applications is finding a suitable methodology that will facilitate the transfer of exogenous genes to the appropriate nerve cells; virusbased vectors have proven quite efficient in transferring genes to many different cell types.  

5.247           Efficacy of an adeno-associated virus 8-pseudotyped vector in glycogen storage disease type II

Sun, B. et al Mol. Ther., 11(1), 57-65 (2005)   Glycogen storage disease type II (GSD-II; Pompe disease) causes death in infancy from cardiorespiratory failure. The underlying deficiency of acid α-glucosidase (GAA; acid maltase) can be corrected by liver-targeted gene therapy in GSD-II, if secretion of GAA is accompanied by receptor-mediated uptake in cardiac and skeletal muscle. An adeno-associated virus (AAV) vector encoding human (h) GAA was pseudotyped as AAV8 (AAV2/8) and injected intravenously into immunodeficient GSD-II mice. High levels of hGAA were maintained in plasma for 24 weeks following AAV2/8 vector administration. A marked increase in vector copy number in the liver was demonstrated for the AAV2/8 vector compared to the analogous AAV2/2 vector. GAA deficiency in the heart and skeletal muscle was corrected with the AAV2/8 vector in male GSD-II mice, consistent with receptor-mediated uptake of hGAA. Male GSD-II mice demonstrated complete correction of glycogen storage in heart and diaphragm with the AAV2/8 vector, while female GSD-II mice had correction only in the heart. A biomarker for GSD-II was reduced in both sexes following AAV2/8 vector administration. Therefore, GAA production with an AAV2/8 vector in a depot organ, the liver, generated evidence for efficacious gene therapy in a mouse model for GSD-II.  

5.248           Antiangiogenic cancer gene therapy by adeno-associated virus 2-mediated stable expression of the soluble FMS-like tyrosine kinase-1 receptor

Mahendra, G. et al Cancer Gene Therapy, 12, 26-34 (2005)   Antiangiogenic gene transfer has the potential to be more efficacious than protein-based therapies or pharmacotherapies for the control of solid tumor growth, invasion and metastasis. For a sustained antiangiogenic effect, a vector capable of long-term expression without vector-associated immunity or toxicity is advantageous. The present study evaluated the potential of a recombinant adeno-associated virus-2 (rAAV) encoding the human soluble FMS-like tyrosine kinase receptor 1 (sFlt-1), which functions by both sequestering vascular endothelial growth factor (VEGF) and forming inactive heterodimers with other membrane-spanning VEGF receptors, in vitro and in vivo. Results indicated significant growth inhibitory activity of the transgenic factor in a human umbilical vein endothelial cell proliferation assay in vitro and protection against the growth of an angiogenesis-dependent human ovarian cancer cell line, SKOV3.ip1, xenograft in vivo with increased disease-free survival. Stable expression of the secretory factor and transgene persistence were confirmed by immunohistochemistry and in situ hybridization analyses, respectively. Increased therapeutic effects on both the growth index of the implanted tumor cells and tumor-free survival also correlated with an increasing dose of the vector used. These studies indicate that rAAV-mediated sFlt-1 gene therapy may be a feasible approach for inhibiting tumor angiogenesis, particularly as an adjuvant/therapy.  

5.249           Adeno-associated virus-mediated delivery of a mutant endodtatin suppresses ovarian carcinoma growth in mice

Subramanian, I.V., Ghebre, R. and Ramakrishnan, S. Gene Ther., 12, 30-38 (2005)   Earlier studies have shown that a point mutation in human endostatin at position 125 (human endostatin wherein proline 125 was substituted with alanine, P125A-endostatin) improves endothelial cell binding and antiangiogenic activity. In the present study, we investigated the effect of recombinant adeno-associated virus (rAAV)-mediated gene delivery of P125A-endostatin (rAAV-P125Aendo) in a mouse model of ovarian carcinoma. Intramuscular (i.m.) injection of rAAV-P125Aendo resulted in a dose-dependent increase in serum endostatin levels. Consequently, vascular endothelial growth factor- and basic fibroblast growth factor-mediated angiogenesis was significantly inhibited in mice injected with rAAV-P125Aendo as compared to control mice injected with rAAV-LacZ. Furthermore, gene therapy using rAAV-P125Aendo construct showed sustained secretion of P125A-endostatin for up to 9 weeks after a single i.m. administration. Recombinant AAV-P125Aendo injection significantly inhibited the growth of human ovarian cancer cells in athymic nude mice. Immunofluorescence studies of residual tumors surgically removed from the rAAV-P125Aendo-treated animals showed decreased number of vessel ends and vessel length, indicating inhibition of angiogenesis. These studies suggest that recombinant AAV-mediated antiangiogenic gene therapy methods can be used to inhibit ovarian cancer growth.    

5.250           HSV vector-mediated transduction and GDNF secretion from adipose cells

Fradette, J. et al Gene Ther., 12, 48-58 (2005)   The accessibility of adipose tissue and its ability to secrete various bioactive molecules suggest that adipose cells may be attractive targets for gene therapy applications. Here, we report the use of highly defective herpes simplex virus (HSV) vectors as suitable gene transfer agents for adipose cells in culture and fat tissue in animals. Using an in vitro model of human adipose differentiation, we first demonstrated that mature adipocytes and their precursor cells express the two principal HSV viral entry receptors HveA and HveC (nectin-1) and are efficiently transduced at a low multiplicity of infection by HSV-lacZ reporter gene and glial cell line-derived neurotrophic factor (GDNF) gene vectors. Extended expression of -galactosidase and secretion of GDNF occurred in transduced fat tissue explants from rabbits. In vivo gene transfer to rabbit subcutaneous adipose tissue resulted in local GDNF expression for at least 2 months. These experiments establish the efficient transduction of adipose cells by HSV vectors and suggest that fat tissue may represent a useful site for HSV-mediated gene delivery with potential for therapeutic applications.  

5.251           The infectivity and lytic activity of minute virus of mice wild-type and derived vector particles are strikingly different

Lang, S. et al

1. Virol., 79(1), 289-298 (2005)

  Gene therapy vectors have been developed from autonomous rodent parvoviruses that carry a therapeutic gene or a marker gene in place of the genes encoding the capsid proteins. These vectors are currently evaluated in preclinical experiments. The infectivity of the vector particles deriving from the fibroblastic strain of minute virus of mice (MVMp) (produced by transfection in human cells) was found to be far less (approximately 50-fold-less) infectious than that of wild-type virus particles routinely produced by infection of A9 mouse fibroblasts. Similarly, wild-type MVMp produced by transfection also had a low infectivity in mouse cells, indicating that the method and producer cells influence the infectivity of the virus produced. Interestingly, producer cells made as many full vector particles as wild-type particles, arguing against deficient packaging being responsible for the low infectivity of viruses recovered from transfected cells. The hurdle to infection with full particles produced through transfection was found to take place at an early step following entry and limiting viral DNA replication and gene expression. Infections with transfection or infection-derived virus stocks normalized for their replication ability yielded similar monomer and dimer DNA amplification and gene expression levels. Surprisingly, at equivalent replication units, the capacity of parvovirus vectors to kill tumor cells was lower than that of the parental wild-type virus produced under the same transfection conditions, suggesting that beside the viral nonstructural proteins, the capsid proteins, assembled capsids, or the corresponding coding region contribute to the lytic activity of these viruses.  

5.252           Conditional cytomegalovirus replication in vitro and in vivo

Rupp, B., Ruzsics, Z., Sacher, T. and Koszinowski, U.H.

1. Virol., 79(1), 486-494 (2005)

  We have established a conditional gene expression system for cytomegalovirus which allows regulation of genes independently from the viral replication program. Due to the combination of all elements required for regulated expression in the same viral genome, conditional viruses can be studied in different cell lines in vitro and in the natural host in vivo. The combination of a self-sufficient tetracycline-regulated expression cassette and Flp recombinase-mediated insertion into the viral genome allowed fast construction of recombinant murine cytomegaloviruses carrying different conditional genes. The regulation of two reporter genes, the essential viral M50 gene and a dominant-negative mutant gene (m48.2) encoding the small capsid protein, was analyzed in more detail. In vitro, viral growth was regulated by the conditional expression of M50 by 3 orders of magnitude and up to a millionfold when the dominant-negative small capsid protein mutant was used. In vivo, viral growth of the dominant-negative mutant was reduced to detection limits in response to the presence of doxycycline in the organs of mice. We believe that this conditional expression system is applicable to genetic studies of large DNA viruses in general.        

5.253           Comparative immunogenicity of human immunodeficiency virus particles and corresponding polypeptides in a DNA vaccine

Akahata, W., Yang, Z-y. and Nabel, G.J.

1. Virol., 79(1), 626-631 (2005)

  The immunogenicity of a plasmid DNA expression vector encoding both Gag and envelope (Env), which produced human immunodeficiency virus (HIV) type 1 virus-like particles (VLP), was compared to vectors expressing Gag and Env individually, which presented the same gene products as polypeptides. Vaccination with plasmids that generated VLP showed cellular immunity comparable to that of Gag and cell-mediated or humoral responses similar to those of Env as immunization with separate vectors. These data suggest that DNA vaccines encoding separated HIV polypeptides generate immune responses similar to those generated by viral particles.  

5.254           Virosome-mediated delivery of protein antigens in vivo: efficient induction of class I MHC-restricted cytotoxic T lymphocyte activity

Bungener, L. et al Vaccine, 23, 1232-1241 (2005)   Induction of CTL responses against protein antigens is an important aim in vaccine development. In this paper we present fusion-active virosomes as a vaccine delivery system capable of efficient induction of CTL responses in vivo. Virosomes are reconstituted viral membranes, which do not contain the genetic material of the virus they are derived from. Foreign macromolecules, including protein antigens, can be encapsulated in virosomes during the reconstitution process. Functionally reconstituted virosomes retain the cell binding and fusion characteristics of the native virus. Thus, upon uptake by cells through receptor-mediated endocytosis, virosomes will deliver their content to the cell cytosol. In a previous study, we demonstrated that protein antigens delivered in this manner to dendritic cells are efficiently processed for both MHC class I and class II presentation. Here, we studied in vivo induction of cellular immune responses against virosome-encapsulated ovalbumin (OVA) in mice. As little as 0.75 μg OVA delivered by fusion-active virosomes was sufficient to induce a powerful class I MHC-restricted CTL response. All immunization routes that were used (i.m., i.p. and s.c.) resulted in efficient induction of CTL activity. The CTLs induced were cytotoxic in a standard ⁵¹Cr-release assay and produced IFNγ in response to OVA peptide. Thus, virosomes represent an ideal antigen delivery system for induction of cellular immunity against encapsulated protein antigens.  

5.255           AAV mediated expression of anti-sense neuropeptide Y cRNA in the arcuate of rats results in decreased weight gain and food intake

Gardiner, J.V. et al Biochem. Biophys. Res. Comm., 327, 1088-1093 (2005)   Neuropeptide Y (NPY) is the most potent stimulant of feeding when administered by intracerebroventricular injection. Despite this, there is conflicting evidence as to its importance in the regulation of daily food intake and energy balance. It has been suggested that whilst it is important in the response to starvation it has little role in the regulation of daily food intake. To investigate the role of NPY in the regulation of food intake, anti-sense cRNA to NPY was expressed in the arcuate nucleus of adult male rats. The anti-sense NPY (AS-NPY) construct was initially tested in vitro and there was a decrease of approximately 50% in NPY release from anti-sense treated cells compared to controls (16.3 ± 2.0 fmol/L [AS-NPY] vs 37.3 ± 7.7 fmol/L [control], mean ± SEM p < 0.05). NPY release from hypothalamic explants from anti-sense injected animals was decreased by over 50% compared to those from controls at both 15 and 20 days after AAV injection (15 days 42% ± 6.5% [AS-NPY] vs 100% ± 36% [control], 20 days 41% ± 6% [AS-NPY] vs 100% ± 27% [control] mean ± SEM, p < 0.05). In a study lasting for 50 days, weight gain was significantly lower in anti-sense injected animals from day 16 (day 16: 6.25 ± 1.10 g [AS-NPY] vs 9.42 ± 0.65 g [control] mean ± SEM, p < 0.05) and remained so until the end of the study when they had gained approximately 40% less weight than controls (day 50: 52.0 ± 9.6 g [AS-NPY] vs 82.0 ± 6.3 g [control] mean ± SEM, p < 0.01). Cumulative food intake was significantly lower in the anti-sense injected animals from day 23 (day 23: 225.8 ± 1.9 g [AS-NPY] vs 250.6 ± 8.7 g [control], mean ± SEM, p < 0.05) and remained so until the end of the study (day 50: 834.5 ± 14.8 g [AS-NPY] vs 926.0 ± 31.7 g [control], mean ± SEM, p < 0.05). Similarly mean daily food intake was also reduced in the anti-sense injected animals (days 7–14: 24.9 ± 0.4 g/day [AS-NPY] vs 27.2 ± 0.4 g/day [control], mean ± SEM, p < 0.01). These data are supportive of a role for NPY in the regulation of daily food intake as well as in response to starvation.

5.256           Expression profiling of human hepatoma cells reveals global repression of genes involved in cell proliferation, growth, and apotosis upon infection with parvovirus H-1

Li, J. et al

1. Virol., 79(4), 2274-2286 (2005)

  Autonomous parvoviruses are characterized by their stringent dependency on host cell S phase and their cytopathic effects on neoplastic cells. To better understand the interactions between the virus and its host cell, we used oligonucleotide arrays that carry more than 19,000 unique human gene sequences to profile the gene expression of the human hepatocellular carcinoma cell line QGY-7703 at two time points after parvovirus H-1 infection. At the 6-h time point, a single gene was differentially expressed with a >2.5-fold change. At 12 h, 105 distinct genes were differentially expressed in virus-infected cells compared to mock-treated cells, with 93% of these genes being down-regulated. These repressed genes clustered mainly into classes involved in transcriptional regulation, signal transduction, immune and stress response, and apoptosis, as exemplified by genes encoding the transcription factors Myc, Jun, Fos, Ids, and CEBPs. Quantitative real-time reverse transcription-PCR analysis on selected genes validated the array data and allowed the changes in cellular gene expression to be correlated with the accumulation of viral transcripts and NS1 protein. Western blot analysis of several cellular proteins supported the array results and substantiated the evidence given by these and other data to suggest that the H-1 virus kills QGY-7703 cells by a nonapoptotic process. The promoter regions of most of the differentially expressed genes analyzed fail to harbor any motif for sequence-specific binding of NS1, suggesting that direct binding of NS1 to cellular promoters may not participate in the modulation of cellular gene expression in H-1 virus-infected cells.  

5.257           Mutational analysis of narrow pores at the fivefold symmetry axes of adeno-associated virus type 2 capsids reveals a dual role in genome packaging and activation of phospholipase A2 activity

Bleker, S., Sonntag, F. And Kleinschmidt, J.A.

1. Virol., 79(4), 2528-2540 (2005)

  Adeno-associated virus type 2 (AAV2) capsids show 12 pores at the fivefold axes of symmetry. We mutated amino acids which constitute these pores to investigate possible functions of these structures within the AAV2 life cycle. Mutants with alterations in conserved residues were impaired mainly in genome packaging or infectivity, whereas few mutants were affected in capsid assembly. The packaging phenotype was characterized by increased capsid-per-genome ratios. Analysis of capsid-associated DNA versus encapsidated DNA revealed that this observation was due to reduced and not partial DNA encapsidation. Most mutants with impaired infectivity showed a decreased capability to expose their VP1 N termini. As a consequence, the activation of phospholipase A2 (PLA2) activity, which is essential for efficient infection, was affected on intact capsids. In a few mutants, the exposure of VP1 N termini and the development of PLA2 activity were associated with enhanced capsid instability, which is obviously also deleterious for virus infection. Therefore, PLA2 activity seems to be required on intact capsids for efficient infection. In conclusion, these results suggest that the pores at the fivefold axes function not only as portals for AAV2 single-stranded DNA packaging but also as channels for presentation of the PLA2 domain on AAV2 virions during infection.  

5.258           Maturation of papillomavirus capsids

Buck, C.B., Thompson, C.D., Pang, Y-Y-s., Lowy, D.R. and Schiller, J.T.

1. Virol., 79(5), 2839-2846 (2005)

  The papillomavirus capsid is a nonenveloped icosahedral shell formed by the viral major structural protein, L1. It is known that disulfide bonds between neighboring L1 molecules help to stabilize the capsid. However, the kinetics of inter-L1 disulfide bond formation during particle morphogenesis have not previously been examined. We have recently described a system for producing high-titer papillomavirus-based gene transfer vectors (also known as pseudoviruses) in mammalian cells. Here we show that papillomavirus capsids produced using this system undergo a maturation process in which the formation of inter-L1 disulfide bonds drives condensation and stabilization of the capsid. Fully mature capsids exhibit improved regularity and resistance to proteolytic digestion. Although capsid maturation for other virus types has been reported to occur in seconds or minutes, papillomavirus capsid maturation requires overnight incubation. Maturation of the capsids of human papillomavirus types 16 and 18 proceeds through an ordered accumulation of dimeric and trimeric L1 species, whereas the capsid of bovine papillomavirus type 1 matures into more extensively cross-linked forms. The presence of encapsidated DNA or the minor capsid protein, L2, did not have major effects on the kinetics or extent of capsid maturation. Immature capsids and capsids formed from L1 mutants with impaired disulfide bond formation are infectious but physically fragile. Consequently, capsid maturation is essential for efficient purification of papillomavirus-based gene transfer vectors. Despite their obvious morphological differences, mature and immature capsids are similarly neutralizable by various L1- and L2-specific antibodies.  

5.259           Recombinant adeno-associated virus 2-mediated antiangiogenic prevention in a mouse model of intraperitoneal ovarian cancer

Isayeva, T., Ren, C. and Ponnazhagan, S. Clin. Cancer Res., 11, 1342-1347 (2005)   Purpose: In the present study, we sought to determine the potentialof sustained transgene expression by a single i.m. administrationof recombinant adeno-associated virus 2 (rAAV) encoding angiostatinand endostatin in inhibiting i.p. ovarian cancer growth anddissemination in a preclinical mouse model. Experimental Design: Cohorts of female athymic nude mice received either no virus or 1.2 x 10¹¹ particles of rAAV encoding green fluorescence protein or endostatin plus angiostatin, i.m. Three weeks later, the mice were i.p. injected with 10⁶ human epithelialovarian cancer cell line SKOV3.ip1. As a measure of effectivenessof the therapy, tumor weight, abdominal distension, ascitesvolume and vascular endothelial growth factor level, and tumorweight were determined. Immunohistochemistry was done to determinetumor cell apoptosis and endothelial cell proliferation followingthe therapy. Tumor-free survival was recorded as the end point. Results: Results indicated a significant tumor-free survival (P < 0.003) following therapy with rAAV encoding endostatinand angiostatin compared with untreated or rAAV-green fluorescenceprotein–treated mice. Ascites volume in rAAV endostatinand angiostatin–treated mice was significantly lower thannaive mice and contained less hemorrhage and tumor conglomerates.The level of vascular endothelial growth factor in the ascitesof antiangiogenic vector treated mice was also significantlyless compared with the untreated mice. Immunohistochemical analysesindicated increased tumor cell apoptosis and decreased bloodvasculature following rAAV endostatin and angiostatin treatment. Conclusion: The results indicate that antiangiogenic geneticprevention from stable systemic levels of angiostatin and endostatinby i.m. administration of rAAV can be used for the treatmentof i.p. ovarian cancer growth and dissemination.  

5.260           Long-term in vivo inhibition of CNS neurodegradation by Bcl-X_L gene transfer

Malik, J.M.I., Shevtsova, Z., Bähr, M. and Kügler, S. Mol. Ther., 11(3), 373-381 (2005)   The inherently low regenerative capacity of the CNS demands effective strategies to inhibit neurodegeneration in acute lesions but also in slowly progressive neurological disorders. Therefore, therapeutic targets that can interact with the degeneration cascade to block, not just postpone, neuronal degeneration need to be defined. Bcl-X_L, a protein protecting the integrity of the mitochondrial membrane potential, was investigated for its neuroprotective properties in a long-term in vivo model of neuronal cell death. An AAV-2-based vector was used to express both Bcl-X_L and EGFP in retinal ganglion cells (RGCs) of the adult rat retina. Transection of the optic nerve results in degeneration of RGCs in control retinae, while Bcl-X_L-overexpressing ganglion cells were protected from degeneration. At 2 weeks after axotomy, 94% of the transduced RGCs survived the lesion (15% in controls). For the first time, we investigated RGC survival up to 8 weeks after axotomy and detected that 46% of the Bcl-X_L-overexpressing RGCs still survived, representing significantly increased neuroprotection compared to neurotrophin-based approaches. We could also show that the axons of AAV-Bcl-X_L-transduced RGCs remained morphologically intact after the lesion, thus providing the basis for regeneration-inducing attempts.  

5.261           Valproic acid enhances gene expression from viral gene transfer vectors

Fan, S. et al

1. Virol. Methods, 125(1), 23-33 (2005)

  Viral vectors represent an efficient delivery method for in vitro and in vivo gene transfer, and their utility may be further enhanced through the use of pharmacologic agents that increase gene expression. Here, we demonstrate that valproic acid (VPA), a drug which is widely used for the treatment of epilepsy and mood disorders, enhances and prolongs expression of exogenous genes in cells transduced with various gene transfer agents, including adenovirus, adeno-associated virus and herpesvirus vectors. This effect occurs in a wide range of cell types, including both primary cells and cell lines, and appears to be associated with VPA's ability to function as a histone deacetylase inhibitor (HDACi). VPA treatment also enhanced adenovirally-vectored expression of a luciferase reporter gene in mice, as demonstrated by in vivo imaging. VPA was also less cytotoxic than a commonly used HDAC inhibitor, TSA, suggesting its use as a safer alternative. Taken together, these results suggest that VPA treatment may represent a useful approach to various gene transfer approaches in which enhanced transgene expression is desirable.  

5.262           Structural organization of an encephalitic human isolate of Banna virus (genus Seadornavirus, family Reoviridae)

Jaafar, F.M., Attoui, H., Mertens, P.P.C., de Micco, P. and de Lamballerie, X.

1. Gen. Virol., 86, 1147-1157 (2005)

  Banna virus (BAV) is the type species of the genus Seadornavirus within the family Reoviridae. The Chinese BAV isolate (BAV-Ch), which causes encephalitis in humans, was shown to have a structural organization and particle morphology reminiscent of that of rotaviruses, with fibre proteins projecting from the surface of the particle. Intact BAV-Ch virus particles contain seven structural proteins, two of which (VP4 and VP9) form the outer coat. The inner (core) particles contain five additional proteins (VP1, VP2, VP3, VP8 and VP10) and are ‘non-turreted’, with a relatively smooth surface appearance. VP2 is the ‘T=2’ protein that forms the innermost ‘subcore’ layer, whilst VP8 is the ‘T=13’ protein forming the core-surface layer. Sequence comparisons indicate that BAV VP9 and VP10 are equivalent to the VP8* and VP5* domains, respectively, of rotavirus outer-coat protein VP4 (GenBank accession no. P12976). VP9 has also been shown to be responsible for virus attachment to the host-cell surface and may be involved in internalization. These similarities reveal a previously unreported genetic link between the genera Rotavirus and Seadornavirus, although the expressionof BAV VP9 and VP10 from two separate genome segments, ratherthan by the proteolytic cleavage of a single gene product (asseen in rotavirus VP4), suggests a significant evolutionaryjump between the members of these two genera.  

5.263           Effects of defective herpes simplex vectors expressing neurotrophic factors on the proliferation and differentiation of nervous cells in vivo

Marconi, P. et al Gen. Ther., 12, 559-569 (2005)   Neurotrophic factors (NTFs) are known to govern the processes involved in central nervous system cell proliferation and differentiation. Thus, they represent very attractive candidates for use in the study and therapy of neurological disorders. We constructed recombinant herpesvirus-based-vectors capable of expressing fibroblast growth factor-2 (FGF-2) and ciliary neurotrophic factor (CNTF) alone or in combinations. In vitro, vectors expressing FGF-2 and CNTF together, but not those expressing either NTF alone, caused proliferation of O-2A progenitors. Furthermore, based on double-labeling experiments performed using markers for neurons (MAP-2), oligodendrocytes (CNPase) and astrocytes (GFAP), most of the new cells were identified as astrocytes, but many expressed neuronal or oligodendrocytic markers. In vivo, vectors have been injected in the rat hippocampus. At 1 month after inoculation, a highly significant increase in BrdU-positive cells was observed in the dentate gyrus of animals injected with the vector expressing FGF-2 and CNTF together, but not in those injected with vectors expressing the single NTFs. Furthermore, double-labeling experiments confirmed in vitro data, that is, most of the new cells identified as astrocytes, some as neurons or oligodendrocytes. These data show the feasibility of the vector approach to induce proliferation and differentiation of neurons and/or oligodendrocytes in vivo.  

5.264           HIV type 1 can act as an APC upon acquisition from the host cell of peptid-loaded HLA-DR and CD86 molecules

Roy, J. et al

1. Immunol., 174, 4779-4788 (2005)

  It is well documented that a wide range of host-derived cell surface constituents is inserted within HIV type 1 (HIV-1) and located on the exterior of the virion. Although no virus-associated protein of host origin has been shown to be absolutely required for virus replication, studies have revealed that many of these proteins are functional and can affect several steps of the virus life cycle. In this study, we found that HIV-1 acquires peptide-loaded class II MHC (MHC-II) and the costimulatory CD86 molecules from the host cell. Moreover, we present evidence that virions bearing such peptide-loaded MHC-II and CD86 proteins can lead to activation of the transcription factors NF- B and NF-AT in an Ag-specific human T cell line. A linear correlation was found between activation of NF- B and the amount of peptide-loaded MHC-II molecules inserted within HIV-1. Finally, transcription of unintegrated and integrated HIV-1 DNA was promoted upon exposure of peptide-specific human T cells to viruses bearing both peptide-loaded MHC-II and CD86 proteins. These data suggest that HIV-1 can operate as an APC depending on the nature of virus-anchored host cell membrane components. It can be proposed that HIV-1 can manipulate one of its primary targets through the process of incorporation of host-derived proteins.  

5.265           A conformational  change in the adeno-associated virus type 2 capsid leads to the exposure of hidden VP1 N termini

Kronenberger, S., Böttcher, B., von der Lieth, C.W., Bleker, S. and Kleinschmidt, J.A.

1. Virol., 79(9), 5296-5303 (2005)

  The complex infection process of parvoviruses is not well understood so far. An important role has been attributed to a phospholipase A₂ domain which is located within the unique N terminus of the capsid protein VP1. Based on the structural difference between adeno-associated virus type 2 wild-type capsids and capsids lacking VP1 or VP2, we show via electron cryomicroscopy that the N termini of VP1 and VP2 are involved in forming globules inside the capsids of empty and full particles. Upon limited heat shock, VP1 and possibly VP2 become exposed on the outsides of full but not empty capsids, which is correlated with the disappearance of the globules in the inner surfaces of the capsids. Using molecular modeling, we discuss the constraints on the release of the globularly organized VP1-unique N termini through the channels at the fivefold symmetry axes outside of the capsid.  

5.266           Combination gene therapy for glioblastoma involving herpes simplex virus vector-mediated codelivery of mutant IkBa and HSV thymidine kinase

Moriuchi, S. et al Can. Gen. Ther., 12, 487-496 (2005)   To improve the effectiveness of herpes simplex virus (HSV) thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy, the replication-defective HSV vector TOI B expressing both HSV-TK and a mutant form of the NF- B inhibitor I B (I B M) was developed. TOI B was constructed by recombining the I B M gene into the U_L41 locus of a replication-defective lacZ expression vector, TOZ.1. Expression of I B M was confirmed by Western blotting, and the ability of the mutant protein to inhibit NF- B nuclear translocation was examined by electrophoretic mobility shift assay. In human glioblastoma U-87MG cells, the p50/p50 dimer of NF- B was already translocated to the nucleus without receptor-dependent signaling by TNF- . Following infection with TOI B, nuclear translocation of NF- B in U-87MG cells was significantly inhibited and caspase-3 activity increased compared with TOZ.1-infected cells. The cytotoxicity of TOI B for U-87MG cells was investigated by colorimetric MTT assay. At an MOI of 3, TOI B infection killed 85% of the cells compared to 20% killed by TOZ.1 infection. In the presence of GCV, these numbers increased to 95-100% for TOI B and 80-85% for TOZ.1. TOI B neurotoxicity measured on cultured murine neurons was relatively low and similar to that of TOZ.1. The survival of nude mice implanted into the brain with U-87MG tumor cells was markedly prolonged by intratumoral TOI B injection and GCV administration. Survival of TOI B+GCV group was significantly longer (P<.02, Wilcoxon test) than for the control groups (TOZ.1 or TOI B only, PBS or PBS+GCV). These results suggest that I B M expression may be a safe enhancement of replication-defective HSV-based suicide gene therapy in vitro and in vivo.  

5.267           Association of human endogenous retroviruses with multiple sclerosis and possible interactions with herpes virus

Christensen, T. Rev. Med. Virol., 15, 179-211 (2005)   The hypothesis that human endogenous retrovirus (HERVs) play a role in autoimmune diseases is subject to increasing attention. HERVs represent both putative susceptibility genes and putative pathogenic viruses in  the immune-mediated neurological disease multiple sclerosis (MS). Gammaretroviral HERV sequences are found in reverse transcriptase-positive virions produced by cultured mononuclear cells from MS patients, and they have been isolated from MS samples of plasma, serum and CSF, and characterised to some extent at the nucleotide, protein/enzyme, virion and immunogenic level. Two types of sequences, HERV-H and HERV-W, have been reported. No known HERV-H or HERV-W copy contains  complete ORFs in all prerequisite genes, although  several copies have coding potential, and several such  sequences are specifically activated in MS, apparently resulting in the production of complete, competent  virions. Increased antibody reactivity to specific Gammaretroviral HERV epitopes is found in MS serum and CSF, and cell-mediated immune responses have also been  reported. Further, HERV-encoded proteins can have neuropathogenic effects. The activating factor(s) in the process resulting in  protein or virion production may be members of the Herpesviridae. Several herpes viruses, such as HSV-1, VZV, EBV and HHV-6, have been associated with MS pathogenesis, and retroviruses and herpes viruses have complex interactions. The current understanding of HERVs, and specifically the investigations of HERV activation and expression in MS are the major subjects of this review, which also proposes to synergise the herpes and HERV findings, and presents several possible pathogenic mechanisms for HERVs in MS.  

5.268           Encapsidation of minute virus of mice DNA: Aspects of the translocation mechanism revelaed by the structure of partially packaged genomes

Cotmore, S.F. and Tattersall, P. Virology, 336, 100-112 (2005)   Minute virus of mice (MVM) packages a single, negative-sense copy of its linear single-stranded DNA genome, but a chimeric virus, MML, in which >95% MVM sequence was fused to the right-hand terminus of LuIII, packages >40% positive-sense DNA. While encapsidation of both MML strands begins efficiently, genome translocation frequently stalls at specific sites in positive-sense DNA. Internalized sequences, derived from the 3′ end of the strand, ranged from 1 to 5 kb in length, with species of around 2 kb predominating. When nuclease activity during isolation was minimized, these truncated species were found to be part of pre-excised 5 kb single-strands. Similarly, some partially encapsidated negative-sense DNAs were observed, forming a continuum of protected 3′ sequences between 1 and 3 kb in length, but these were less abundant and more uniformly distributed than their positive-sense counterparts, indicating that the negative strand has evolved for efficient internalization. The paucity of protected DNAs shorter than 1–2 kb suggests that translocation is biphasic, proceeding efficiently through the first (3′) third of the genome, but prone to stall thereafter. Sequences with conspicuous secondary structure, including stem–loop and guanidine rich regions, were found to interrupt packaging, especially when positioned near the 5′ end of the strand. Since VP2 amino-terminal peptides were exposed at the particle surface in all packaging intermediates, extrusion of this peptide precedes translocation of the full-length strand.  

5.269           Quantitative real-time PCR for titration of infectious recombinant AAV-2 particles

Rohr; U-P. et al

1. Virol. Meth., 127, 40-45 (2005)

  In this report, we present a fast, reliable and easy to perform method to quantify infectious titers of recombinant AAV-2 (rAAV-2) particles using the LightCycler technology, which is independent from the therapeutic transgene and without the presence of a marker gene. The method is based on the life cycle of AAV-2: after infection of the host cell, the single stranded (ss) AAV-2 genome is converted into a double stranded (ds) form. Following infection with rAAV-2, HeLa cells were lysed and ssDNA of transcriptionally inactive particles were efficiently removed by ssDNA-specific S1 nuclease digestion. The remaining viral dsDNA can be quantified by quantitative real-time PCR (qPCR). For validation of the new method, rAAV-2 preparations were analyzed by two other standard methods for titration of infectious particles in parallel, i.e. the infectious center assay (ICA) as well as flow cytometry using GFP as a marker. Comparing the infectious titers of 40 different AAV-2 fractions assessed by qPCR with the titers determined by FACS analysis a significant correlation (r = 0.87, p < 0.001) with a mean ratio of the titers assessed by qPCR and FACS of 1.92 (S.D. ± 1.59) was found. Further, the titers of seven rAAV-2 fractions using qPCR and ICA covering 5 log ranges were compared and a significant correlation was found between the results (r = 0.80, p < 0.001) with a mean ratio of 3.38 (S.D. ± 1.79), respectively.  

5.270           Interleukin 10 attenuates neointimal proliferation and inflammation in aortic allografts by a heme oxygenase-dependent pathway

Chen, S. et al PNAS, 102(20), 7251-7256 (2005)   Interleukin 10 (IL-10) is a pleiotropic cytokine with well known antiinflammatory, immunosuppressive, and immunostimulatory properties. Chronic allograft rejection, characterized by vascular neointimal proliferation, is a major cause of organ transplant loss, particularly in heart and kidney transplant recipients. In a Dark Agouti to Lewis rat model of aortic transplantation, we evaluated the effects of a single intramuscular injection of a recombinant adeno-associated viral vector (serotype 1) encoding IL-10 (rAAV1-IL-10) on neointimal proliferation and inflammation. rAAV1-IL-10 treatment resulted in a significant reduction of neointimal proliferation and graft infiltration with macrophages and T and B lymphocytes. The mechanism underlying the protective effects of IL-10 in aortic allografts involved heme oxygenase 1 (HO-1) because inhibition of HO activity reversed not only neointimal proliferation but also inflammatory cell infiltration. Our results indicate that IL-10 attenuates neointimal proliferation and inflammatory infiltration and strongly imply that HO-1 is an important intermediary through which IL-10 regulates the inflammatory responses associated with chronic vascular rejection.  

5.271           Effects of adeno-associated virus DNA hairpin structure on recombination

Choi, V.W., Samulski, R.J. and McCarty, D.

1. Virol., 79(11), 6801-6807 (2005)

  Hairpin DNA ends are evolutionarily conserved intermediates in DNA recombination. The hairpin structures present on the ends of the adeno-associated virus (AAV) genome are substrates for recombination that give rise to persistent circular and concatemeric DNA episomes through intramolecular and intermolecular recombination, respectively. We have developed circularization-dependent and orientation-specific self-complementary AAV (scAAV) vectors as a reporter system to examine recombination events involving distinct hairpin structures, i.e., closed versus open hairpins. The results suggest that intramolecular recombination (circularization) is far more efficient than intermolecular recombination (concatemerization). Among all possible combinations of terminal repeats (TRs) involved in intermolecular recombination, the closed-closed TR structures are twice as efficient as the open-open TR substrates for recombination. In addition, both intramolecular recombination and intermolecular recombination exhibit the common dependency on specific DNA polymerases and topoisomerases. The circularization-dependent and orientation-specific scAAV vectors can serve as an efficient and controlled system for the delivery of DNA structures that mimic mammalian recombination intermediates and should be useful in assaying recombination in different experimental settings as well as elucidating the molecular mechanism of recombinant AAV genome persistence.  

5.272           Adeno-associated virus-mediated gene transfer to hair cells and support cells of the murine cochlea

Stone, I.M., Lurie, D.I., Kelley, M.W. and Poulsen, D.J. Mol. Ther., 11(6), 843-848 (2005)   More than 28 million Americans suffer from various forms of hearing loss. The lack of effective treatments for many forms of hearing disorders has prompted interest in the potential application of gene delivery techniques to treat both inherited and pathological hearing disorders. However, to develop a gene therapy strategy that will successfully treat hearing disorders, appropriate vectors that are capable of transducing cochlear hair cells and support cells must be identified. In the present study, we examined the efficiency with which AAV vectors (serotypes 1, 2, and 5) transduce hair cells and support cells in cochlear explants from P0 and E13 mice. We further examined the ability of the CBA and GFAP promoters to drive expression of a GFP marker gene in hair cells and support cells. Robust GFP expression was observed in hair cells and support cells following transduction of primary murine cochlear explants with AAV serotypes 1 and 2, but not serotype 5. The CBA promoter predominantly drove GFP expression in hair cells. In contrast, strong expression from the GFAP promoter was observed primarily in support cells. Thus, using AAV vectors and specific promoters, cell-type-specific expression of transgenes can be established within the cochlea.  

5.273           Altering AAV tropism with mosaic viral capsids

Gigout, L. et al Mol. Ther., 11(6), 856-865 (2005)   Over the past decade, AAV-based vectors have emerged as promising candidates for gene therapeutic applications. Despite the broad tropism of the first eight serotypes identified, certain cell types are refractory to transduction with AAV-based vectors. Furthermore, for certain applications the targeting of specific cell types is desirable. To improve on present methods to alter AAV2 tropism, we take advantage of AAV2 mosaics. Here, we show that AAV2 mosaics have improved infectivity compared with all-mutant virions. Using an AAV2 mutant that contains the immunoglobulin-binding Z34C fragment of protein A, we demonstrate the utility of AAV2 mosaics to alter AAV2 tropism. This system allows us to transduce selectively and efficiently MO7e and Jurkat cells. The use of AAV2 mosaics with a protein A fragment inserted into their capsid, together with targeting antibodies, is a versatile method that allows the specific transduction of a wide array of cell types.  

5.274           Virus-like particle (VLP) vaccine conferred complete protection against a lethal influenza virus challenge

Galarza, J.M., Latham, T. and Cupo, A. Viral Immunol., 18(1), 244-251 (2005)   We have previously demonstrated the formation and release of influenza virus-like particles (VLPs) from the surface of Sf9 cells infected with either a quadruple baculovirus recombinant that simultaneously expresses the influenza structural proteins hemagglutinin (HA), neuraminidase (NA), matrix 1 (M1) and M2, or a combination of single recombinants that include the M1 protein. In this work, we present data on the immunogenicity and protective efficacy afforded by VLPs (formed by M1 and HA) following immunization of mice. VLP vaccine ( 1 µg HA) were formulated with or without IL-12 as adjuvant and administered twice, at two weeks intervals, by either intranasal instillation or intramuscular injection. All VLP-vaccinated and influenza-immunized control mice demonstrated high antibody titers to the HA protein; however, intranasal instillation of VLPs elicited antibody titers that were higher than those induced by either intramuscular inoculation of VLPs or intranasal inoculation with two sub-lethal doses of the challenge influenza virus (control group). Antibody responses were enhanced when VLP vaccine was formulated with IL12 as adjuvant. All mice were challenged with 5 LD50 of a mouse-adapted influenza A/Hong Kong/68 (H3N2) virus. Intramuscular administration of VLP vaccine formulated with or without IL-12 afforded 100% protection against a lethal influenza virus challenge. Similarly, intranasal instillation of VLP vaccine alone protected 100% of the mice, whereas VLP formulated with IL-12 protected 90% of the vaccinated mice. Not only do these results suggest a novel approach to the development of VLP vaccines for diverse influenza virus strains, but also the creation of multivalent vaccines by decoration of the surface of the VLPs with antigens from other pathogens.  

5.275           Impact of humoral immune response on distribution and efficacy of recombinant adeno-associated virus-derived a-glucosidese in a model of glycogen storage disease type II

Creshawn, K.O. et al Hum. Gen. Ther., 16, 68-80 (2005)   Glycogen storage disease type II (GSDII) is a lysosomal storage disease caused by a deficiency in acid -glucosidase (GAA), and leads to cardiorespiratory failure by the age of 2 years. In this study, we investigate the impact of anti-GAA antibody formation on cross-correction of the heart, diaphragm, and hind-limb muscles from liver-directed delivery of recombinant adeno-associated virus (rAAV)5- and rAAV8-GAA vectors. GAA^-/- mice receiving 1 10¹² vector genomes of rAAV5- or rAAV8-DHBV-hGAA were analyzed for anti-GAA antibody response, GAA levels, glycogen reduction, and contractile function. We demonstrate that restoration of GAA to the affected muscles is dependent on the presence or absence of the antibody response. Immune-tolerant mice had significantly increased enzyme levels in the heart and skeletal muscles, whereas immune-responsive mice had background levels of GAA in all tissues except the diaphragm. The increased levels of activity in immune-tolerant mice correlated with reduced glycogen in the heart and diaphragm and, overall, contractile function of the soleus muscle was significantly improved. These findings highlight the importance of the immune response to rAAV-encoded GAA in correcting GSDII and provide additional understanding of the approach to treatment of GSDII.  

5.276           Efficient transduction of vascular endothelial cells with recombinant adeno-associated virus serotype 1 and 5 vectors

Chen, S. et al Hum. Gen. Ther., 16, 235-247 (2005)   Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC). rAAV vectors with AAV2 inverted terminal repeats containing the human ₁-antitrypsin (hAAT) gene were transcapsidated using helper plasmids to provide viral capsids for the AAV1 through 5 serotypes. True type rAAV2 and 5 vectors encoding -galactosidase or green fluorescence protein were also studied. Infection with rAAV1 resulted in the most efficient transduction in both HAEC and RAEC compared to other serotypes (p < 0.001) at 7 days posttransduction. Interestingly, expression was increased in cells transduced with rAAV5 to levels surpassing rAAV1 by day 14 and 21. Transduction with rAAV1 was completely inhibited by removal of sialic acid with sialidase, while heparin had no effect. These studies are the first demonstration that sialic acid residues are required for rAAV1 transduction in endothelial cells. Transduction of rat aortic segments ex vivo and in vivo demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors, in comparison to rAAV2. These results suggest the unique potential of rAAV1 and rAAV5-based vectors for vascular-targeted gene-based therapeutic strategies.  

5.277           Production of recombinant adeno-associated virus vectors

Zolotukhin, S. Hum. Gen. Ther., 16, 551-557 (2005)   Recombinant adeno-associated virus (rAAV) is a prototypical gene therapy vector characterized by excellent safety profiles, wide host range, and the ability to transduce differentiated cells. Numerous rAAV-based vectors providing efficient and sustained expression of transgenes in target tissues have been developed for preclinical studies. Interest in rAAV has been driven by advances in production methods originally developed for rAAV serotype 2 vectors and expanded to include alternative serotypes. The transition to clinical trials is dependent on the development of scalable production methods of Good Manufacturing Practice-grade vectors described in this review.  

5.278           Cross-neutralization of cutaneous and mucosal Papillomavirus types with anti-sea to the amino terminus of L2

Pastrana, D.V. et al Virology, 337, 365-372 (2005)   Vaccination with papillomavirus L2 has been shown to induce neutralizing antibodies that protect against homologous type infection and cross-neutralize a limited number of genital HPVs. Surprisingly, we found that antibodies to bovine papillomavirus (BPV1) L2 amino acids 1–88 induced similar titers of neutralizing antibodies against Human papillomavirus (HPV)16 and 18 and BPV1 pseudoviruses and also neutralized HPV11 native virions. These antibodies also neutralized each of the other pseudovirus types tested, HPV31, HPV6 and Cottontail rabbit papillomavirus (CRPV) pseudoviruses, albeit with lower titers. HPV16, HPV18, HPV31, HPV6 and CRPV L2 anti-sera also displayed some cross-neutralization, but the titers were lower and did not encompass all pseudoviruses tested. This study demonstrates the presence of broadly cross-neutralizing epitopes at the N-terminus of L2 that are shared by cutaneous and mucosal types and by types that infect divergent species. BPV1 L2 was exceptionally effective at inducing cross-neutralizing antibodies to these shared epitopes.  

5.279           Perigestational suppression of weight gain with central leptin gene therapy results in lower weight F1 generation

Lecklin, A., Dube, M.G., Torto, R.N., Kalra, P.S. and Kalra, S.P Peptides, 26, 1176-1187 (2005)   The efficacy of central leptin therapy on weight homeostasis through various phases of reproduction, pregnancy outcome and postnatal, prepubertal and pubertal growth of offspring was assessed. Enhanced leptin transgene expression after a single intracerebroventricular injection of recombinant adeno-associated virus vector encoding the leptin gene (rAAV-lep) decreased calorie intake and weight in adult nulliparous female rats. rAAV-lep treated rats conceived normally, displayed unremarkable pregnancy rate, parturition and delivered normal sized litters. Significantly lower weight was maintained through gestation, lactation, and post-lactation periods. The maintenance of a modest weight reduction was accompanied by voluntarily reduced calorie intake, increased thermogenic energy expenditure, decreased adiposity as reflected by drastically reduced leptin levels, and suppressed insulin and insulin-like growth factor 1 levels through lactation and post-lactation in rAAV-lep treated dams. The offspring at birth weighed significantly less than those of controls and this lower weight range was sustained during postnatal, prepubertal, pubertal and adult (3 months old) periods, contemporaneous with metabolic circulating hormones in the normal range. For the first time we show the persistent efficacy of central leptin gene therapy to suppress weight gain through all phases of reproduction, lactation and post-lactation in dams and reveal the potential imprinting link to producing lower weight in the F1 generation.          

5.280           Production of infectious human papillomavirus independently of viral replication and epithelial cell differentiation

Pyeon, D., Lambert, P.F. and Ahlquist, P. PNAS, 102(26), 9311-9316 (2005)   Papillomaviruses are small DNA viruses that are associated with benign and malignant epithelial lesions, including >95% of cervical cancers and 20% of head and neck cancers. Because papillomavirus replication and virion production are tied to epithelial cell differentiation, infectious papillomavirus virion production has been limited to cumbersome organotypic cultures and mouse xenografts. Consequent difficulties in obtaining useful amounts of wild-type or mutant human papillomavirus (HPV) virions have greatly limited studies on many aspects of papillomavirus biology. To overcome these limitations, we developed a system to encapsidate the full-length papillomaviral genome into infectious virions, independently of viral DNA replication and epithelial differentiation. This transient-transfection-based system produces >1,000 times more infectious virus per cell culture dish than the much more labor-intensive organotypic culture. Furthermore, we show that this method allows the facile generation of infectious particles containing wild-type, mutant, or chimeric papillomaviral genomes, overcoming barriers to studying many facets of replication, host interactions, and vaccine and drug development, which has been limited by the insufficient availability of infectious virions.  

5.281           The impact of envelope glycoprotein cleavage on the antigenicity, infectivity, and neutralization sensitivity of Env-pseudotyped human immunodeficiency virus type 1 particles

Herrera, C. et al Virology, 338, 154-172 (2005)   Endoproteolytic processing of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoproteins is an obligate part of the biosynthetic pathway that generates functional, fusion-competent Env complexes, which are then incorporated into infectious virions. We have examined the influence of cleavage on Env-specific antibody reactivity, Env incorporation into pseudovirions, and the infectivity and neutralization sensitivity of Env-pseudotyped viruses. To do so, we have used both incompletely processed wild-type (Wt) Env and engineered, cleavage-defective Env mutants. We find that there is no simple association between antibody reactivity to cell surface-expressed Env, and the ability of the same antibody to neutralize virus pseudotyped with the same Env proteins. One explanation for the absence of such an association is the diverse array of Env species present on the surface of transiently transfected cells. We also confirm that cleavage-defective mutants are antigenically different from Wt Env. These findings have implications for the use of Env binding assays as predictors of neutralizing activity, and for the development of cleavage-defective Env trimers for use as subunit immunogens.  

5.282           Targeted measles virus vector displaying echistatin infects endothelial cells via avb3 and leads to tumor regression

Hallak, L.K., Merchan, J.R., Storgard, C.M., Loftus, J.C. and Russell, S.J. Cancer Res., 65(12), 5292-5300 (2005)   Targeting tumor-associated vascular endothelium by replication-competent viral vectors is a promising strategy for cancer gene therapy. Here we describe the development of a viral vector based on the Edmonston vaccine strain of measles virus targeted to integrin vß3, which is expressed abundantly on activated but not quiescent vascular endothelium. We displayed a disintegrin, M28L echistatin that binds with a high affinity to integrin vß3 on the COOH terminus of the viral attachment (H) protein and rescued the replication-competent recombinant virus by reverse genetics. The new targeted virus was named measles virus echistatin vector (MV-ERV). Its native binding to CD46 was purposefully retained to allow virus infection of tumor cells expressing this receptor. MV-ERV correctly displayed echistatin on the outer surface of its envelope and produced interesting ring formation phenomena due to cell detachment upon infection of susceptible Vero cells in vitro. MV-ERV grew to 10⁶ plaque-formingunits/mL, slightly lower than the parental Edmonston strainof measles virus (MV-Edm), but it selectively infected Chinesehamster ovary cells expressing integrin vß3. It alsoselectively infected both bovine and human endothelial cellson matrigels and unlike MV-Edm, MV-ERV infected newly formedblood vessels in chorioallantoic membrane assays. In animalmodels, MV-ERV but not the control MV-Edm caused the regressionof s.c. xenografts of resistant multiple myeloma tumors (MM1)in severe combined immunodeficient mice. The tumors were eithercompletely eradicated or their growth was significantly retarded.The specificity, potency, and feasibility of MV-ERV infectionclearly show the potential use of MV-ERV in gene therapy fortargeting tumor-associated vasculature for the treatment ofsolid tumors.  

5.283           Packaging capacity of adeno-associated virus serotypes: impact of larger genomes on infectivity and postentry steps

Grieger, J.C. and Samulski, R.J.

1. Virol., 79(15), 9933-9944 (2005)

  The limited packaging capacity of adeno-associated virus (AAV)precludes the design of vectors for the treatment of diseasesassociated with larger genes. Autonomous parvoviruses, suchas minute virus of mice and B19, while identical in size (25nm), are known to package larger genomes of 5.1 and 5.6 kb,respectively, compared to AAV genomes of 4.7 kb. One primarydifference is the fact that wild-type (wt) AAV utilizes threecapsid subunits instead of two to form the virion shell. Inthis study, we have characterized the packaging capacity ofAAV serotypes 1 through 5 with and without the Vp2 subunit.Using reporter transgene cassettes that range in size from 4.4to 6.0 kb, we determined that serotypes 1 through 5 with andwithout Vp2 could successfully package, replicate in, and transducecells. Dot blot analysis established that packaging efficiencywas similar for all vector cassettes and that the integrityof encapsidated genomes was intact regardless of size. Althoughphysical characterization determined that virion structureswere indistinguishable from wt, transduction experiments determinedthat all serotype vectors carrying larger genomes (5.3 kb andhigher) transduced cells less efficiently (within a log) thanAAV encapsidating wt size genomes. This result was not uniqueto reporter genes and was observed for CFTR vector cassettesranging in size from 5.1 to 5.9 kb. No apparent advantage inpackaging efficiency was observed when Vp2 was present or absentfrom the virion. Further analysis determined that a postentrystep was responsible for the block in infection and specifictreatment of cells upon infection with proteasome inhibitorsincreased transduction of AAV encapsidating larger DNA templatesto wt levels, suggesting a preferential degradation of virionsencapsidating larger-than-wt genomes. This study illustratesthat AAV is capable of packaging and protecting recombinantgenomes as large as 6.0 kb but the larger genome-containingvirions are preferentially degraded by the proteasome and thatthis block can be overcome by the addition of proteasome inhibitors.  

5.284           AAV serotype-dependent apolipoprotein A-I_Milano gene expression

Sharifi, B.G. et al Atherosclerosis, 181, 261-269 (2005)   Recent evidence from a double-blind, randomized study showed that treatment with apolipoprotein A-I_Milano (ApoA-I_Milano) in a complex with phospholipids produced significant regression of the coronary atheroma burden in patients with acute coronary syndromes. We previously showed similar regression of atherosclerosis in an animal model. Here, we examined a viral vector-based gene delivery system as a basis for ApoA-I_Milano gene therapy. Comparing levels of expression using combinations of the cytomegalovirus (CMV) promoter in a recombinant serotype 2 adeno-associated virus (rAAV2) linked to ApoA-I_Milano or the enhanced green fluorescent protein (EGFP) genes, we found that a promoter construct of two CMV core promoters sharing a CMV enhancer was more active than other combinations or a single CMV promoter. In vivo assessment of this optimal CMV construct using rAAV2 virus particles for intravenous (IV) or intramuscular (IM) routes of delivery produced high circulating levels of ApoA-I_Milano protein for extended periods (up to 220 ng/ml at 22 weeks p.i.) by IV delivery while the IM route resulted in a relatively short period of very low-level ApoA-I_Milano expression. Since there was no difference in the immune response between the two routes of delivery, we reasoned that tissue tropism might be responsible for this differential gene expression. To explore this possibility, we investigated the effect of different AAV serotypes on ApoA-I_Milano gene expression in vivo. It found that rAAV1-mediated expression of ApoA-I_Milano was approximately 15- and 9-fold higher than rAAV2 and rAAV5, respectively when IM injection routes were compared while all three AAV serotypes produced substantial levels of ApoA-I_Milano expression from IV injection. These studies demonstrate that by modifying the promoter and serotype, increases in the efficiency of AAV-directed transgene expression could be achieved and support the potential of AAV-mediated gene therapy.  

5.285           The long terminal repeat-containing retrotransposon Tf1 possesses amino acids in gag that regulate nucleat localization and particle formation

Kim, M-K., Claiborn, K.C. and Levin, H.L.

1. Virol., 79(15), 9540-9555 (2005)

  Tf1 is a long terminal repeat-containing retrotransposon of Schizosaccharomyces pombe that is studied to further our understanding of retrovirus propagation. One important application is to examine Tf1 as a model for how human immunodeficiency virus type 1 proteins enter the nucleus. The accumulation of Tf1 Gag in the nucleus requires an N-terminal nuclear localization signal (NLS) and the nuclear pore factor Nup124p. Here, we report that NLS activity is regulated by adjacent residues. Five mutant transposons were made, each with sequential tracts of four amino acids in Gag replaced by alanines. All five versions of Tf1 transposed with frequencies that were significantly lower than that of the wild type. Although all five made normal amounts of Gag, two of the mutations did not make cDNA, indicating that Gag contributed to reverse transcription. The localization of the Gag in the nucleus was significantly reduced by mutations A1, A2, and A3. These results identified residues in Gag that contribute to the function of the NLS. The Gags of A4 and A5 localized within the nucleus but exhibited severe defects in the formation of virus-like particles. Of particular interest was that the mutations in Gag-A4 and Gag-A5 caused their nuclear localization to become independent of Nup124p. These results suggested that Nup124p was only required for import of Tf1 Gag because of its extensive multimerization.  

5.286           Gamma interferon can block herpes simplex virus type 1 reactivation from latency, even in the presence of late gene expression

Decman, V., Kinchington, P.R., Harvey, S.A. and Hendricks, R.L.

1. Virol., 79(16), 10339-10347 (2005)

  Herpes simplex virus type 1 (HSV-1)-specific CD8⁺ T cells and the cytokine gamma interferon (IFN- ) are persistently present in trigeminal ganglia (TG) harboring latent HSV-1. We define "latency" as the retention of functional viral genomes in sensory neurons without the production of infectious virions and "reactivation" as a multistep process leading from latency to virion assembly. CD8⁺ T cells can block HSV-1 reactivation in ex vivo mouse TG cultures and appear to be the sole source of IFN- in these cultures. Here we demonstrate that IFN- alone can block HSV-1 reactivation in some latently infected neurons, and we identify points of intervention in the life cycle of the reactivating virus. Cell suspensions of TG that were latently infected with recombinant RE HSV-1 expressing enhanced green fluorescent protein from the promoter for infected cell protein 0 (ICP0) or glycoprotein C (gC) were depleted of endogenous CD8⁺ or CD45⁺ cells and cultured in the presence or absence of IFN- . Our results demonstrate that IFN- acts on latently infected neurons to inhibit (i) HSV-1 reactivation, (ii) ICP0 promoter activity, (iii) gC promoter activity, and (iv) reactivation in neurons in which the ICP0 or gC promoter is active. Interestingly, we detected transcripts for ICP0, ICP4, and gH in neurons that expressed the ICP0 promoter but were prevented by IFN- from reactivation and virion formation. Thus, the IFN- blockade of HSV-1 reactivation from latency in neurons is associated with an inhibition of the expression of the ICP0 gene (required for reactivation) and a blockade of a step that occurs after the expression of at least some viral structural genes.  

5.287           Complete replication of hepatitis C virus in cell culture

Lindenbach, B.D. et al Science, 309, 623-626 (2005)   Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 10⁵ infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon- and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals.  

5.288           Enhanced expression of glutamate decarboxylase 65 improves symptoms of rat parkinsonian models

Lee, B. et al Gene Ther.,12, 1215-1222 (2005)   In this study, we report the amelioration of parkinsonian symptoms in rat Parkinson's disease (PD) models, as a result of the expression of glutamate decarboxylase (GAD) 65 with a modified cytomegalovirus (CMV) promoter. The transfer of the gene for gamma-amino butryic acid (GAD), the rate-limiting enzyme in gama-amino butrylic acid (GABA) production, has been investigated as a means to increase inhibitory synaptic activity. Electrophysiological evidence suggests that the transfer of the GAD65 gene to the subthalamic nucleus (STN) can change the excitatory output of this nucleus to inhibitory output. Our in vitro results also demonstrated higher GAD65 expression in cells transfected with the JDK promoter, as compared to cells transfected with the CMV promoter. Also, a rat PD model in which recombinant adeno-associated virus-2 (rAAV2)-JDK-GAD65 was delivered into the STN exhibited significant behavioral improvements, as compared to the saline-injected group. Interestingly, we observed that these behavioral improvements were more obvious in rat PD models in which rAAV2-JDK-GAD65 was injected into the STN than in rat PD models in which rAAV2-CMV-GAD65 was injected into the STN. Moreover, according to electrophysiological data, the rAAV2-JDK-GAD65-injected group exhibited more constant improvements in firing rates than did the rAAV2-CMV-GAD65-injected group. These data indicate that the JDK promoter, when coupled with GAD65 expression, is more effective with regard to parkinsonian symptoms than is the CMV promoter.  

5.289           Virus like particle vaccine conferred complete protection against a lethal influenza virus challenge

Galarza, J.M., Latham, T. And Cupo, A. Viral Immunol., 18(2), 365-372 (2005)   We have previously demonstrated the formation and release of influenza virus-like particles (VLPs) from the surface of Sf9 cells infected with either a quadruple baculovirus recombinant that simultaneously expresses the influenza structural proteins hemagglutinin (HA), neuraminidase (NA), matrix 1 (M1), and matrix 2 (M2), or a combination of single recombinants that include the M1 protein. In this work, we present data on the immunogenicity and protective efficacy afforded by VLPs (formed by M1 and HA) after immunization of mice. VLP vaccine ( 1 µg HA) were formulated with or without IL-12 as adjuvant and administered twice, at 2-week intervals, by either intranasal instillation or intramuscular injection. All VLP-vaccinated and influenza-immunized control mice demonstrated high antibody titers to the HA protein; however, intranasal instillation of VLPs elicited antibody titers that were higher than those induced by either intramuscular inoculation of VLPs or intranasal inoculation with two sub-lethal doses of the challenge influenza virus (control group). Antibody responses were enhanced when VLP vaccine was formulated with IL12 as adjuvant. All mice were challenged with 5 LD50 of a mouse-adapted influenza A/Hong Kong/68 (H3N2) virus. Intramuscular administration of VLP vaccine formulated with or without IL-12 afforded 100% protection against a lethal influenza virus challenge. Similarly, intranasal instillation of VLP vaccine alone protected 100% of the mice, whereas VLP formulated with IL-12 protected 90% of the vaccinated mice. Not only do these results suggest a novel approach to the development of VLP vaccines for diverse influenza virus strains, but also the creation of multivalent vaccines by decoration of the surface of the VLPs with antigens from other pathogens.  

5.290           AAV2-mediated ocular gene therapy for infantile neuronal ceroid lipofuscinosis

Griffey, M., Shannon, L., Macauley, J.M.O. ands Sands, M.S. Mol. Ther., 12(3), 413-421 (2005)   Infantile neuronal ceroid lipofuscinosis (INCL) is a neurodegenerative disorder caused by mutations in the gene encoding the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). The earliest clinical sign in INCL is blindness, followed by seizures, cognitive deficits, and early death. Little is known about the progression of the visual deficits in INCL. Here we characterize the progressive retinal dysfunction and examine the efficacy of AAV2-mediated ocular gene therapy in the murine model of INCL. Significant decreases in both mixed rod/cone and pure cone electroretinographic amplitudes were observed at as early as 2 months of age. Intravitreal injection of AAV2-PPT1 increased enzyme levels in the eye to greater than normal levels. The increased PPT1 activity correlated with improvements in the histological abnormalities as well as both mixed rod/cone and pure cone functions. We also demonstrated that palmitoyl protein thioesterase-1 activity was detected in the brain following intravitreal injection. The brain activity is likely due to anterograde axonal transport along the optic tracts. Interestingly, the degree of neurodegeneration throughout the visual pathways of the brain was greatly reduced in AAV-treated INCL mice. Therefore, intravitreal AAV-mediated gene therapy has direct benefits to the eye and to distal sites in the brain along the visual pathways.  

5.291           Localized gene expression following administration of adeno-associated viral vectors via pancreatic ducts

Loiler, S.A. et al Mol. Ther., 12(3), 519-527 (2005)   Gene transfer into pancreatic cells in vivo could be of immense therapeutic benefit in cases of type 1 diabetes (T1D) through the production of molecules capable of interrupting the progression of autoimmunity or promoting regeneration of insulin-secreting β cells. We adapted a clinically relevant surgical technique (endoscopic retrograde cholangiopancreatography) to deliver rAAV encoding human α1-antitrypsin (approved gene symbol SERPINA1) to the pancreas of 3-week-old Fisher 344 rats and C57BL/6 mice. We compared natural as well as bioengineered serotypes of rAAV (rAAV1, rAAV2/Apo (B. R. Burkhardt et al., 2003, Ann. N. Y. Acad. Sci. 1005, 237–241) [1], rAAV8) as well as different promoters (chicken β-actin, human insulin) for their expression in vivo. Rats injected with rAAV1 showed the highest hAAT expression (week 2, rAAV1/CB-AT, 579 ± 457 ng/ml). In mice, rAAV8 vector delivered the highest serum concentration of hAAT (week 2, rAAV8/CB-AT, 19 ± 6 μg/ml). The chicken β-actin promoter provided the highest expression in both rodent experiments. Immunohistochemical staining indicated transduction primarily of pancreatic acinar cells with either the rAAV1/CB-AT vector in the rat or the rAAV8/CB-AT vector in the mouse. This study demonstrates that rAAV vectors can be designed to deliver therapeutic genes efficiently to the pancreas and achieve high levels of gene expression and may be useful in treating pancreatic disorders, including T1D.  

5.292           Determination of the relative amounts of Gag and Pol proteins in foamy virus particles

Cartelliery, M., Rudolph, W., Herchenröder, O., Lindemann, D. and Rethwilm, A. Retrovirol., 2(44), 1-7 (2005)   We determined the relative ratios of Gag and Pol molecules in highly purified virions of spumaretroviruses or foamy viruses (FVs) using monoclonal antibodies and bacterially expressed reference proteins. We found that the cleaved p68^Gag moiety dominates in infectious FVs. Furthermore, approximate mean ratios in FV are 16:1 (pr71^Gag plus p68^Gag:p85^RT),12:1 (p68^Gag:p85^RT), and 10:1 (pr71^Gag plus p68^Gag:p40^IN). Thus, the results indicate that FVs have found a way to incorporate approximately as much Pol protein into their capsids as orthoretroviruses, despite a completely different Pol expression strategy.  

5.293           Key Golgi factors for structural and functional maturation of  bunyamwera virus

Novoa, R.R., Calderita, G., Cabezas, P., Elliott, R.M. and Risco, C.

1. Virol., 79(17), 10852-10863 (2005)

  Several complex enveloped viruses assemble in the membranes of the secretory pathway, such as the Golgi apparatus. Among them, bunyaviruses form immature viral particles that change their structure in a trans-Golgi-dependent manner. To identify key Golgi factors for viral structural maturation, we have purified and characterized the three viral forms assembled in infected cells, two intracellular intermediates and the extracellular mature virion. The first viral form is a pleomorphic structure with fully endo-ß-N-acetylglucosaminidase H (Endo-H)-sensitive, nonsialylated glycoproteins. The second viral intermediate is a structure with hexagonal and pentagonal contours and partially Endo-H-resistant glycoproteins. Sialic acid is incorporated into the small glycoprotein of this second viral form. Growing the virus in glycosylation-deficient cells confirmed that acquisition of Endo-H resistance but not sialylation is critical for the trans-Golgi-dependent structural maturation and release of mature viruses. Conformational changes in viral glycoproteins triggered by changes in sugar composition would then induce the assembly of a compact viral particle of angular contours. These structures would be competent for the second maturation step, taking place during exit from cells, that originates fully infectious virions.  

5.294           Large-scale analysis of adeno-associated virus vector integration sites in normal human cells

Miller, D.G. et al

1. Virol., 79(17), 1143411442 (2005)

  The integration sites of viral vectors used in human gene therapy can have important consequences for safety and efficacy. However, an extensive evaluation of adeno-associated virus (AAV) vector integration sites has not been completed, despite the ongoing use of AAV vectors in clinical trials. Here we have used a shuttle vector system to isolate and analyze 977 unique AAV vector-chromosome integration junctions from normal human fibroblasts and describe their genomic distribution. We found a significant preference for integrating within CpG islands and the first 1 kb of genes, but only a slight overall preference for transcribed sequences. Integration sites were clustered throughout the genome, including a major preference for integration in ribosomal DNA repeats, and 13 other hotspots that contained three or more proviruses within a 500-kb window. Both junctions were localized from 323 proviruses, allowing us to characterize the chromosomal deletions, insertions, and translocations associated with vector integration. These studies establish a profile of insertional mutagenesis for AAV vectors and provide unique insight into the chromosomal distribution of DNA strand breaks that may facilitate integration.  

5.295           Host range mutants of minute virus of mice with a single VP2 amino acid change require additional silent mutations that regulate NS2 accumulation

D’Abramo Jr., A.M., Ali, A.A., Wang, F., Cotmore, S.F. and Tattersall, P. Virology, 340, 143-154 (2005)   Two host range switch mutants of the immunosuppressive strain of parvovirus Minute Virus of Mice (MVMi) were isolated from plaques on A9 fibroblasts. Both carried a single coding mutation at residue D399 in VP2, to alanine and glycine in hr105 and hr107, respectively, and a second, non-coding, guanine-to-adenine change at nucleotide 1970 in hr105 and 1967 in hr107. These mutations were recreated in a wild type MVMi infectious plasmid clone, both alone and as pairs, in either the original or switched combinations. All single mutants failed to replicate productively in fibroblasts, but the two pairs of changes were functionally equivalent. Single D399 mutations allowed the viruses to initiate infection in fibroblasts, but NS2 expression was severely restricted and correlated with poor accumulation and release of progeny virus. Mutations at 1967 or 1970 enhanced NS2 accumulation, and allowed efficient progeny production and release. Conversely, the D399 mutations destroyed the viruses' ability to infect EL4 lymphocytes. In all productive EL4 infections, NS2 was expressed at high ratios even in the absence of upstream mutations, and progeny accumulation was efficient. However, EL4 cells lack a mechanism for early progeny release, potentially explaining why virus amplification in these cells is slow.    

5.296           Adeno-associated virus vectors are able to restore fatty aldehyde dehydrogenase-deficiency, implications for gene therapy in Sjögre-Larsson ayndrome

Haug, S. and Braun-Falco, M. Arch. Dermatol. Res., 296, 568-572 (2005)   Sjögren-Larsson Syndrome (SLS) is caused by an autosomal recessive defect in the gene coding for fatty aldehyde dehydrogenase (FALDH), an enzyme necessary for the oxidation of long-chain aliphatic aldehydes to fatty acid as one enzyme of the fatty alcohol:nicotinamide-adenine dinucleotide (NAD+)-oxidoreductase complex (FAO). The impaired activity of FALDH leads to the clinical symptom triad of generalized ichthyosis, mental retardation, and spastic diplegia or tetraplegia. Treatment options are primarily symptomatic. Gene therapy by means of genetic reintroduction of the functional FALDH gene into defective cells has so far not been considered as a therapeutic modality. In order to pursue such an approach for SLS, we constructed a recombinant adeno-associated virus-2 vector containing the human cDNA of functional FALDH and evaluated its capability to restore the enzyme-deficiency in a FALDH-deficient cell line resembling the gene defect of SLS. rAAV-2 transduction of FALDH-deficient cells, usually exhibiting less than 10% of normal FALDH activity, resulted in an increase of FALDH activity within the range of unaffected cells. Moreover, FALDH-transduced cells regained resistance over exposure to long chain aldehydes, which are otherwise toxic to FALDH-deficient cells. These results indicated that rAAV-2 vectors are able to restore FALDH-deficiency in a cell system resembling SLS. The findings give the first support to the concept that gene therapy might be a future option for the treatment of SLS.  

5.297           Recombinant adeno-associated virus type 2-mediated gene transfer into human keratinocytes is influenced by both the ubiquitin/proteasome pathway and epidermal growth factor receptor tyrosine kinase

Braun-Falco, M., Eisenried, A., Büning, H. and Ring, J. Arch. Dermatol. Res., 296, 528-535 (2005)   Efficient gene delivery into keratinocytes is a prerequisite for successful skin gene therapy. Vectors based on recombinant adeno-associated virus type 2 (rAAV-2) offer several promising features that make them attractive for cutaneous applications. However, highly efficient gene delivery may be hampered by different cellular factors, including lack of viral receptors, impairment of cytoplasmic trafficking or limitations in viral second-strand synthesis. This study was undertaken to find factors that influence rAAV-2-mediated in vitro gene transfer into human keratinocytes and, consequently, ways to optimize gene delivery. Transduction experiments using rAAV-2 vectors expressing green fluorescent protein (GFP) demonstrated that impaired cellular trafficking of vector particles and high levels of autophosphorylation at epidermal growth factor receptor tyrosine kinase (EGF-R TK) have a negative influence on gene transfer into keratinocytes. Treatment of keratinocytes with proteasome inhibitor MG132 resulted in a transient augmentation of GFP expression in up to 37% of cells. Treatment with EGF-R TK inhibitors (quinazoline type) enhanced transgene expression in 10–14.5% of the cells. Gene expression was stable for more than 10 weeks and persisted until proliferative senescence occurred. This stable gene expression allows speculation that keratinocyte stem cells have initially been transduced. These findings might have relevance for the use of rAAV-2 vectors in skin gene therapy: transient enhancement of rAAV-2 transduction with proteasome inhibitors might be useful for genetic promotion of wound healing or skin-directed vaccination. Treatment with quinazolines may increase rAAV-2 transduction of keratinocyte stem cells, which is important for gene therapy approaches to inherited diseases.  

5.298           Tau gene transfer, but not alpha-synuclein, induces both progressive dopamine neuron degeneration and rotational behavior in the rat

Klein, R.L., Dayton, R.D., Lin, W-L. and Dickson, D.W. Neurobiol. Dis., 20(1), 64-73 (2005)   Using a viral vector for mutant (P301L) tau, we studied the effects of gene transfer to the rat substantia nigra in terms of structural and functional properties of dopaminergic neurons. The mutant tau vector caused progressive loss of pars compacta dopaminergic neurons over time, reduced striatal dopamine content, and amphetamine-stimulated rotational behavior consistent with a specific lesion effect. In addition, structural studies demonstrated neurofibrillary tangles and neuritic pathology. Wild-type tau had similar effects on neuronal loss and rotational behavior. In contrast, mutant α-synuclein vectors did not induce rotational behavior, although α-synuclein filaments formed in nigrostriatal axons. Dopamine neuron function is affected by tau gene transfer and appears to be more susceptible to tau- rather than α-synuclein-related damage in this model. Both tau and α-synuclein are important for substantia nigra neurodegeneration models in rats, further indicating their potential as therapeutic targets for human diseases involving loss of dopamine neurons.  

5.299           Genomic stability of self-complementary adeno-associated virus 2 during early stages of transduction in mouse muscle in vivo

Ren, C., Kumar, S., Shaw, D.R. and ponnazhagan, S. Hum. Gen. Ther., 16, 1-11 (2005)   Studies have demonstrated that packaging of recombinant adeno-associated virus 2 (rAAV) as self-complementary duplex strand (sc) results in early transgene expression, possibly eliminating rate-limiting secondstrand synthesis. In the present study, we evaluated the molecular organization, stability of the sc AAV genome, and transgene expression in the quadriceps muscle of C57BL/6J mice in vivo as compared with single-stranded (ss) AAV. Studies were carried out with rAAV encoding green fluorescent protein (GFP) or human carcinoembryonic antigen (CEA) either as single-stranded or self-complementary duplex strand structures, encapsidated in AAV-2 capsids. Mice were injected with 10¹¹ particles of the respective viruses and the vector-injected muscles were harvested 1 week, 2 weeks, 3 weeks, or 2 months later. Tissues were processed for total DNA isolation for the analyses of vector genomic configuration and copy number, and for immunostaining of transgene expression. ELISA was done on serum samples to quantitate CEA-specific humoral immune response as a correlate of transgene expression. Results of Southern blot and PCR analyses indicated more disintegration of the monomeric ss AAV DNA in vivo compared with linear sc AAV DNA. The results also indicated efficient conversion of the self-complementary duplex-stranded vector genome to dimer during early time points. As expected, transgene expression was detected at early time points with self-complementary duplex-stranded vector and persisted stably. However, the advantage of higher transgene expression from sc AAV was balanced over time by the single-stranded vector. These data demonstrate that sc AAV provides better stability for transgene structure during the initial stages of transduction and may have better utility in AAV gene therapy in situations, which mandate early transgene expression.  

5.300           Green fluorescent protein-tagged adeno-associated virus particles allow the study of cytosolic and nuclear trafficking

Lux, K. et al

1. Virol., 79(18), 11776-11787 (2005)

  To allow the direct visualization of viral trafficking, we genetically incorporated enhanced green fluorescent protein (GFP) into the adeno-associated virus (AAV) capsid by replacement of wild-type VP2 by GFP-VP2 fusion proteins. High-titer virus progeny was obtained and used to elucidate the process of nuclear entry. In the absence of adenovirus 5 (Ad5), nuclear translocation of AAV capsids was a slow and inefficient process: at 2 h and 4 h postinfection (p.i.), GFP-VP2-AAV particles were found in the perinuclear area and in nuclear invaginations but not within the nucleus. In Ad5-coinfected cells, isolated GFP-VP2-AAV particles were already detectable in the nucleus at 2 h p.i., suggesting that Ad5 enhanced the nuclear translocation of AAV capsids. The number of cells displaying viral capsids within the nucleus increased slightly over time, independently of helper virus levels, but the majority of the AAV capsids remained in the perinuclear area under all conditions analyzed. In contrast, independently of helper virus and with 10 times less virions per cell already observed at 2 h p.i., viral genomes were visible within the nucleus. Under these conditions and even with prolonged incubation times (up to 11 h p.i.), no intact viral capsids were detectable within the nucleus. In summary, the results show that GFP-tagged AAV particles can be used to study the cellular trafficking and nuclear entry of AAV. Moreover, our findings argue against an efficient nuclear entry mechanism of intact AAV capsids and favor the occurrence of viral uncoating before or during nuclear entry.  

5.301           Intrastriatal rAAV-mediated delivery of anti-huntingtin shRNAs induces partial reversal of disease progression in R6/1 Huntington’s disease transgenic mice

Rodriguez-Lebron, E., Denovan-Wright, E.M., Nash, K., Lewin, A.S. and Mandel, R.J. Mol. Ther., 12(4), 618-633 (2005)   Huntington's disease (HD) is a fatal neurodegenerative disorder caused by the presence of an abnormally expanded polyglutamine domain in the N-terminus of huntingtin. We developed a recombinant adeno-associated viral serotype 5 (rAAV5) gene transfer strategy to posttranscriptionally suppress the levels of striatal mutant huntingtin (mHtt) in the R6/1 HD transgenic mouse via RNA interference. Transient cotransfection of HEK293 cells with plasmids expressing a portion of human mHtt derived from R6/1 transgenic HD mice and a short-hairpin RNA directed against the 5′ UTR of the mHtt mRNA (siHUNT-1) resulted in reduction in the levels of mHtt mRNA (−75%) and protein (−60%). Long-term in vivo rAAV5-mediated expression of siHUNT-1 in the striatum of R6/1 mice reduced the levels of mHtt mRNA (−78%) and protein (−28%) as determined by quantitative RT-PCR and Western blot analysis, respectively. The reduction in mHtt was concomitant with a reduction in the size and number of neuronal intranuclear inclusions and a small but significant normalization of the steady-state levels of preproenkephalin and dopamine- and cAMP-responsive phosphoprotein 32 kDa mRNA. Finally, bilateral expression of rAAV5-siHUNT-1 resulted in delayed onset of the rear paw clasping phenotype exhibited by the R6/1 mice. These results suggest that a reduction in the levels of striatal mHtt can ameliorate the HD phenotype of R6/1 mice.  

5.302           Prolonged recovery of retinal structure/function after gene therapy in an Rs1h-deficient mouse model of X-linked juvenile retinoschisis

Min, S.H. et al Mol. Ther., 12(4), 644-651 (2005)   X-linked juvenile retinoschisis (RS) is a common cause of juvenile macular degeneration in males. RS is characterized by cystic spoke-wheel-like maculopathy, peripheral schisis, and a negative (b-wave more reduced than a-wave) electroretinogram (ERG). These symptoms are due to mutations in the RS1 gene in Xp22.2 leading to loss of functional protein. No medical treatment is currently available. We show here that in an Rs1h-deficient mouse model of human RS, delivery of the human RS1 cDNA with an AAV vector restored expression of retinoschisin to both photoreceptors and the inner retina essentially identical to that seen in wild-type mice. More importantly, unlike an earlier study with a different AAV vector and promoter, this work shows for the first time that therapeutic gene delivery using a highly specific AAV5–opsin promoter vector leads to progressive and significant improvement in both retinal function (ERG) and morphology, with preservation of photoreceptor cells that, without treatment, progressively degenerate.  

5.303           A combination of mutations enhances the neurotropism of AAV-2

Xu, J., Ma, C., Bass, C. and Terwilliger, E.F. Virology, 341(2), 203-214 (2005)   There is strong interest in developing practical strategies for gene delivery to the central nervous system (CNS). Direct delivery into the brain or spinal cord is highly invasive as well as inefficient or hazardous using most current vector systems. Our objective was to generate innocuous gene vehicles that would be effectively taken up by axons and then home to the neuron cell bodies. Vectors derived from Adeno-Associated Virus (AAV), a harmless human parvovirus, offer strong starting candidates for deriving such vehicles. Enhancing the axonal uptake of AAV, and conferring more efficient retrograde transport capabilities upon the virus, should produce near ideal gene transfer vehicles for the CNS. To enhance retrograde transport of the virus, peptides mimicking binding domains for cytoplasmic dynein were inserted in the capsid by directed mutagenesis. In separate clones, peptides derived from an NMDA receptor antagonist were also introduced to provide a specific affinity for this receptor. When combined, these two functionally distinct classes of mutation enabled efficient gene transfer into neurons under conditions not permissive for standard AAV-2 vectors prepared under the same conditions. These results hold strong promise for the development of safe, convenient vehicles to target genes and other sequences to neurons, enabling new and novel approaches for the treatment of multiple neurological disorders.  

5.304           Human herpesvirus 8 enhances human immunodeficiency virus replication in acutely infected cells and induces reactivation in latently infected cells

Caselli, E. et al Blood, 106(8), 2790-2797 (2005)   Human herpesvirus 8 (HHV-8) is etiologically associated with Kaposi sarcoma (KS), the most common AIDS-associated malignancy. Previous results indicate that the HHV-8 viral transactivator ORF50 interacts synergistically with Tat protein in the transactivation of human immunodeficiency virus (HIV) long terminal repeat (LTR), leading to increased cell susceptibility to HIV infection. Here, we analyze the effect of HHV-8 infection on HIV replication in monocyte-macrophage and endothelial cells, as potential targets of coinfection. Primary or transformed monocytic and endothelial cells were infected with a cell-free HHV-8 inoculum and subsequently infected with lymphotropic or monocytotropic strains of HIV. The results show that HHV-8 coinfection markedly increases HIV replication in both cell types. HHV-8 infection induces also HIV reactivation in chronically infected cell lines and in peripheral blood mononuclear cells (PBMCs) from patients with asymptomatic HIV, suggesting the possibility that similar interactions might take place also in vivo. Furthermore, coinfection is not an essential condition, since contiguity of differently infected cells is sufficient for HIV reactivation. The results suggest that HHV-8 might be a cofactor for HIV progression and that HHV-8-infected endothelial cells might play a relevant role in transendothelial HIV spread.  

5.305           Nonrandom packaging of host RNAs in moloney murine leukemia virus

Onafuwa-Nuga, A.A., King, S.R. and Telesnitsky, A.

1. Virol., 79(21), 13528-13537 (2005)

  Moloney murine leukemia virus (MLV) particles contain both viral genomic RNA and an assortment of host cell RNAs. Packaging of virus-encoded RNA is selective, with virions virtually devoid of spliced env mRNA and highly enriched for unspliced genome. Except for primer tRNA, it is unclear whether packaged host RNAs are randomly sampled from the cell or specifically encapsidated. To address possible biases in host RNA sampling, the relative abundances of several host RNAs in MLV particles and in producer cells were compared. Using 7SL RNA as a standard, some cellular RNAs, such as those of the Ro RNP, were found to be enriched in MLV particles in that their ratios relative to 7SL differed little, if at all, from their ratios in cells. Some RNAs were underrepresented, with ratios relative to 7SL several orders of magnitude lower in virions than in cells, while others displayed intermediate values. At least some enriched RNAs were encapsidated by genome-defective nucleocapsid mutants. Virion RNAs were not a random sample of the cytosol as a whole, since some cytoplasmic RNAs like tRNA^Met were vastly underrepresented, while U6 spliceosomal RNA, which functions in the nucleus, was enriched. Real-time PCR demonstrated that env mRNA, although several orders of magnitude less abundant than unspliced viral RNA, was slightly enriched relative to actin mRNA in virions. These data demonstrate that certain host RNAs are nearly as enriched in virions as genomic RNA and suggest that ^– mRNAs and some other host RNAs may be specifically excluded from assembly sites.  

5.306           Chromatograpahic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications

Burova, E. and Ioffe, E. Gen. Ther., 12, S5-S17 (2005)   In recent years, recombinant adenoviral and adeno-associated viral (AAV) vectors have been exploited in a number of gene delivery approaches. The use of these vectors in clinical gene transfer has increased the demand for their characterization, production and purification. Although the classical method of adenovirus or AAV purification by density gradient centrifugation is effective on a small scale, chromatographic separation is the most versatile and powerful method for large-scale production of recombinant adenovirus or AAV. This review describes different chromatographic modes for adenovirus or AAV purification and process development, as well as the utility of different purification steps for virus production. Advances in the development of viral vectors for gene therapy, such as the discovery of new AAV serotypes, adenoviral and AAV retargeting and improved production of helper-dependent adenoviral vectors, require further development of efficient purification methods.  

5.307           Purification of adenovirus and adeno-associated virus: comparison of novel membrane-based technology to conventional techniques

Duffy, A.M., O’Doherty, A.M., O’Brien, T. and Strappe, P.M. Gen. Ther., 12, S62-S72 (2005)   Adenovirus (Ad) and Adeno-associated virus (AAV) are efficient gene delivery systems; manipulation of the wild-type genome allows their use as vectors for the overexpression of desirable transgenes. Generation and purification of such viral vectors can be labour intensive, costly and require specialized equipment, but a new generation of membrane-mediated ion exchange kits for purification of recombinant virus may facilitate this process. Here, we examine the yields, transgene expression and purity of preparations of Ad and AAV purified using commercially available kits in comparison to other established techniques for purification of recombinant viral vectors. We demonstrate comparable results for Ad and AAV respectively in all parameters investigated, with a substantial reduction in purification time for the kit-based technology. Such approaches are attractive methods for small-scale purification of recombinant Ad and AAV viral vectors.

5.308           Downstream processing of viral vectors and vaccines

Morenweiser, R. Gen. Ther., 12, S103-S110 (2005)   Viral vectors and viral vaccines more and more play an important role in current medical approaches. Gene vectors like adenoviruses, adeno-associated viruses or retroviruses are the vehicles being developed for delivering genetic material to the target cell in gene therapy. Viral vaccines, like attenuated or inactivated rabies virus, influenza virus or hepatitis virus vaccines, are powerful tools to limit the number of serious viral infections and pandemics. Higher safety demands, that is, reduction of side effects, by regulatory authorities like Food and Drug Administration (FDA) and European Agency for the Evaluation of Medicinal Products (EMEA), nowadays force developers as well as manufacturers to improve their production and purification processes for viral vectors and vaccines. Like for influenza viral vaccines, manufacturers begin to switch from egg cultivation to mammalian cell culture systems. Also within the purification procedure, a clear trend from classical purification methods like sucrose gradient centrifugation towards more sophisticated techniques like tangential flow filtration and liquid chromatography can be observed.  

5.309           Current issues in adeno-assocaited viral vector production

Merten, O-W., Geny-Fiamma, C. and Douar, A.M. Gen. Ther., 12, S51-S61 (2005)   Adeno-associated virus (AAV) is currently one of the most promising systems for human gene therapy. Numerous preclinical studies have documented the excellent safety profile of these vectors along with their impressive performances in their favored target, consisting of highly differentiated postmitotic tissues such as muscle, central nervous system and liver. Clinical trials have been conducted confirming these data, but also emphasizing the requirement of further high-tech developments of the production and purification procedures that would allow both scaling-up and improvement of vector batch quality, necessary to human application. The scope of this review will be the state of the art in the various production methods of recombinant AAV (rAAV), delimiting their respective perimeter of application and also their main advantages and drawbacks, and thereby shedding light on the main challenges to take in the near future to bring AAV vectors more widely into the clinics.  

5.310           Efficient hepatic delivery and expression from a recombinant adeno-associated virus 8 pseudotyped a1-antitrypsin vector

Conlon, T.J: et al Mol. Ther., 12(5), 867-875 (2005)   α1-Antitrypsin (AAT) deficiency is a single-gene disorder in which a mutation in the AAT (approved symbol SERPINA1) gene (PI*Z) leads to misfolding of the protein, loss of the protective antiprotease effect of AAT for the lungs, and a toxic effect on hepatocytes. Optimal therapy for AAT deficiency will require a high percentage of hepatocyte transduction to be effective for liver and lung disease. Recently, rAAV genomes pseudotyped with capsids from serotypes 7 and 8 showed efficient hepatic transduction. We hypothesized that upon portal vein injection to target hepatocytes, serotype 8 would better transduce target cells and therefore express hAAT in both a greater percentage of cells and greater amounts. AAV2 and pseudotyped vectors for serotypes 1, 5, and 8 carrying the human AAT transgene were injected at 1 × 10¹⁰ particle doses into C57Bl/6 mice. Circulating hAAT from AAV2/8-injected animals showed a 2-log advantage over AAV2 and 3-log increase over AAV2/1 and 5 for the 24-week study. Most significantly, up to 40% of total liver cells stained positive for the transgene in AAV2/8 subjects while remaining primarily episomal. Therefore, pseudotyped AAV8 provides a vehicle to infect a high percentage of hepatocytes stably and thereby express therapeutic molecules to modify AAT PiZ transcripts.  

5.311           Caveolin-1 is not essential for biosynthetic apical membrane transport

Manninen, A. et al Mol. Cell. Biol., 25(22), 10087-10096 (2005)   Caveolin-1 has been implicated in apical transport of glycosylphosphatidylinositol (GPI)-anchored proteins and influenza virus hemagglutinin (HA). Here we have studied the role of caveolin-1 in apical membrane transport by generating caveolin-1-deficient Madin-Darby canine kidney (MDCK) cells using retrovirus-mediated RNA interference. The caveolin-1 knockdown (cav1-KD) MDCK cells were devoid of caveolae. In addition, caveolin-2 was retained in the Golgi apparatus in cav1-KD MDCK cells. However, we found no significant alterations in the apical transport kinetics of GPI-anchored proteins or HA upon depletion of caveolin-1. Similar results were obtained using embryonic fibroblasts from caveolin-1-knockout mice. Thus, we conclude that caveolin-1 does not play a major role in lipid raft-mediated biosynthetic membrane trafficking.  

5.312           AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in long-term TPP-I expression compatible with therapy for LINCL

Sondhi, D. et al Gen. Ther., 12, 1618-1632 (2005)   Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal, autosomal recessive disease resulting from mutations in the CLN2 gene with consequent deficiency in its product tripeptidyl peptidase I (TPP-I). In the central nervous system (CNS), the deficiency of TPP-I results in the accumulation of proteins in lysosomes leading to a loss of neurons causing progressive neurological decline, and death by ages 10–12 years. To establish the feasibility of treating the CNS manifestations of LINCL by gene transfer, an adeno-associated virus 2 (AAV2) vector encoding the human CLN2 cDNA (AAV2_CUhCLN2) was assessed for its ability to establish therapeutic levels of TPP-I in the brain. In vitro studies demonstrated that AAV2_CUhCLN2 expressed CLN2 and produced biologically active TPP-I protein of which a fraction was secreted as the pro-TPP-I precursor and was taken up by nontransduced cells (ie, cross-correction). Following AAV2-mediated CLN2 delivery to the rat striatum, enzymatically active TPP-I protein was detected. By immunohistochemistry TPP-I protein was detected in striatal neurons (encompassing nearly half of the target structure) for up to 18 months. At the longer time points following striatal administration, TPP-I-positive cell bodies were also observed in the substantia nigra, frontal cerebral cortex and thalamus of the injected hemisphere, and the frontal cerebral cortex of the noninjected hemisphere. These areas of the brain contain neurons that extend axons into the striatum, suggesting that CNS circuitry may aid the distribution of the gene product. To assess the feasibility of human CNS delivery, a total of 3.6 10¹¹ particle units of AAV2_CUhCLN2 was administered to the CNS of African green monkeys in 12 distributed doses. Assessment at 5 and 13 weeks demonstrated widespread detection of TPP-I in neurons, but not glial cells, at all regions of injection. The distribution of TPP-I-positive cells was similar between the two time points at all injection sites. Together, these data support the development of direct CNS gene transfer using an AAV2 vector expressing the CLN2 cDNA for the CNS manifestations of LINCL.  

5.313           Molecular characterization of adeno-associated viruses infecting children

Chen, C-L. et al

1. Virol., 79(23), 14781-14792 (2005)

  Although adeno-associated virus (AAV) infection is common in humans, the biology of natural infection is poorly understood. Since it is likely that many primary AAV infections occur during childhood, we set out to characterize the frequency and complexity of circulating AAV isolates in fresh and archived frozen human pediatric tissues. Total cellular DNA was isolated from 175 tissue samples including freshly collected tonsils (n = 101) and archived frozen samples representing spleen (n = 21), lung (n = 16), muscle (n = 15), liver (n = 19), and heart (n = 3). Samples were screened for the presence of AAV and adenovirus sequences by PCR using degenerate primers. AAV DNA was detected in 7 of 101 (7%) tonsil samples and two of 74 other tissues (one spleen and one lung). Adenovirus sequences were identified in 19 of 101 tonsils (19%), but not in any other tissues. Complete capsid gene sequences were recovered from all nine AAV-positive tissues. Sequence analyses showed that eight of the capsid sequences were AAV2-like ( 98% amino acid identity), while the single spleen isolate was intermediate between serotypes 2 and 3. Comparison to the available AAV2 crystal structure revealed that the majority of the amino acid substitutions mapped to surface-exposed hypervariable domains. To further characterize the AAV capsid structure in these samples, we used a novel linear rolling-circle amplification method to amplify episomal AAV DNA and isolate infectious molecular clones from several human tissues. Serotype 2-like viruses were generated from these DNA clones and interestingly, failed to bind to a heparin sulfate column. Inspection of the capsid sequence from these two clones (and the other six AAV2-like isolates) revealed that they lacked arginine residues at positions 585 and 588 of the capsid protein, which are thought to be essential for interaction with the heparin sulfate proteoglycan coreceptor. These data provide a framework with which to explore wild-type AAV persistence in vivo and provide additional tools to further define the biodistribution and form of AAV in human tissues.  

5.314           Enhanced gene transfer to arthritic joints using adeno-associated virus type 5: implications for intra-articular gene therapy

Adriaansen, J. et al Ann. Rheum. Dis., 64, 1677-1684 (2005)   Background: Gene therapy of the joint has great potential asa new therapeutic approach for the treatment of rheumatoid arthritis(RA). The vector chosen is of crucial importance for clinicalsuccess. Objective: To investigate the tropism and transduction efficiencyin arthritic joints in vivo, and in synovial cells in vitro,using five different serotypes of recombinant adeno-associatedvirus (rAAV) encoding ß-galactosidase or green fluorescentprotein genes. Methods: rAAV was injected into the ankle joints of rats withadjuvant arthritis after the onset of disease. Synovial tissuewas examined at different time points for ß-galactosidaseprotein and gene expression by in situ staining and polymerasechain reaction (PCR) analysis, respectively. In addition, theability of rAAV to transduce primary human fibroblast-like synoviocytesfrom patients with RA was investigated in vitro. Results: Intra-articular injection of the rAAV5 serotype resultedin the highest synovial transduction, followed by much lowerexpression using rAAV2. Expression of the transgene was alreadydetectable 7 days after injection and lasted for at least 4weeks. Only background staining was seen for serotypes 1, 3,and 4. Importantly, there was a minimal humoral immune responseto rAAV5 compared with rAAV2. Additionally, it was found thatboth rAAV2 and rAAV5 can efficiently transduce human fibroblast-likesynoviocytes obtained from patients with RA. Conclusion: Intra-articular rAAV mediated gene therapy in RAmight be improved by using rAAV5 rather than other serotypes.  

5.315           AAV2/5-mediated NGF gene delivery protects septal cholinergic neurons following axotomy

Wu, K. et al Brain Res., 1061(2), 107-113 (2005)   Nerve growth factor (NGF) therapy has been proposed to treat cognitive impairments in aged patients including those with Alzheimer's disease. Various viral vectors, including adeno-associated virus serotype 2 (AAV2), have been investigated for their ability to deliver NGF in brain. In this study, hybrid vectors (AAV2/5) consisting of the genome of recombinant AAV2 and the capsid of AAV serotype 5 were evaluated for their ability to deliver NGF and green fluorescent protein (GFP) genes into brain. Compared to AAV2, AAV2/5 consistently led to more septal neurons being transduced with GFP over a wider range of distribution. However, both types of vector provided similar levels of long-term (17 weeks) protection of septal cholinergic neurons from axotomy and led to similar levels of NGF accumulation in this region. These results demonstrate that rAAV-mediated NGF gene delivery is neuroprotective for an extended period of time, but that factors other than transduction efficiency appear to determine transgenic NGF expression in septum.  

5.316           Expansion of family reoviridae to include nine-segmented dsRNA viruses: isolation and characterization of a new virus designated aedes pseudoscutellaris reovirus assigned to a proposed genes (dinovernavirus)

Attoui, H. et al Virology, 343, 212-223 (2005)   Family Reoviridae is known, by definition, to contain dsRNA viruses with 10–12 genome segments. We report here the characterization of the first member of this family with a nine-segmented genome. This virus was isolated from Aedes pseudoscutellaris mosquito cells and designated aedes pseudoscutellaris reovirus (APRV). Virions are single-shelled with turrets but are non-occluded by contrast to cypoviruses. APRV replicates in various mosquito cell lines, but not in mice or mammalian cells. Complete sequence analysis showed that APRV is phylogenetically related to cypoviruses, fijiviruses and oryzaviruses. The maximum amino acid identities with cypoviruses, oryzaviruses or fijiviruses in the polymerase, are compatible with values observed between these genera and lower than values within a given genus. This suggests that APRV should be classified within a new genus that we designated Dinovernavirus (sigla from D: Double-stranded, i: insect, nove: nine from the latin “novem”, rna: RNA, virus) in family Reoviridae.  

5.317           Long-term restoration of rod and cone vision by single dose rAAV-mediated gene transfer to the retina in a canine model of childhood blindness

Acland, G.M. et al Mol. Ther., 12(6), 1072-1082 (2005)   The short- and long-term effects of gene therapy using AAV-mediated RPE65 transfer to canine retinal pigment epithelium were investigated in dogs affected with disease caused by RPE65 deficiency. Results with AAV 2/2, 2/1, and 2/5 vector pseudotypes, human or canine RPE65 cDNA, and constitutive or tissue-specific promoters were similar. Subretinally administered vectors restored retinal function in 23 of 26 eyes, but intravitreal injections consistently did not. Photoreceptoral and postreceptoral function in both rod and cone systems improved with therapy. In dogs followed electroretinographically for 3 years, responses remained stable. Biochemical analysis of retinal retinoids indicates that mutant dogs have no detectable 11-cis-retinal, but markedly elevated retinyl esters. Subretinal AAV-RPE65 treatment resulted in detectable 11-cis-retinal expression, limited to treated areas. RPE65 protein expression was limited to retinal pigment epithelium of treated areas. Subretinal AAV-RPE65 vector is well tolerated and does not elicit high antibody levels to the vector or the protein in ocular fluids or serum. In long-term studies, wild-type cDNA is expressed only in target cells. Successful, stable restoration of rod and cone photoreceptor function in these dogs has important implications for treatment of human patients affected with Leber congenital amaurosis caused by RPE65 mutations.  

5.318           Successful production of pseudotyped rAAV vectors using a modified Baculovirus expression system

Kohlbrenner, E. et al Mol. Ther., 12(6), 1217-1225 (2005)   Scalable production of rAAV vectors remains a major obstacle to the clinical application of this prototypical gene therapy vector. A recently developed baculovirus-based production protocol (M. Urabe et al., 2002, Hum. Gene Ther. 13, 1935–1943) found limited applications due to the system's design. Here we report a detailed analysis of the stability of the original baculovirus system components BacRep, BacVP, and transgene cassette-containing BacGFP. All of the baculovirus helpers analyzed were prone to passage-dependent loss-of-function deletions resulting in considerable decreases in rAAV titers. To alleviate the instability and to extend the baculovirus platform to other rAAV serotypes, we have modified both Rep- and Cap-encoding components of the original system. The modifications include a parvoviral phospholipase A2 domain swap allowing production of infectious rAAV8 vectors in vivo. Alternatively, an infectious rAAV8 (or rAAV5) vector incorporating the AAV2 VP1 capsid protein in a mosaic vector particle with AAV8 capsid proteins was produced using a novel baculovirus vector. In this vector, the level of AAV2 VP1 expression is controlled with a “riboswitch,” a self-cleaving ribozyme controlled by toyocamycin in the “ON” mode. The redesigned baculovirus system improves our capacity for rAAV manufacturing by making this production platform more applicable to other existing serotypes.  

5.319           Sensory neurons regulate the effector functions of CD8⁺ T cells in controlling HSV-1 latency ex vivo

Prabhakaran, K. et al Immunity, 23, 515-525 (2005)   We provide evidence that sensory neurons regulate the effector functions and phenotype of CD8⁺ T cells during active immunosurveillance of HSV-1 latency. Low-level viral gene expression in latently infected sensory ganglia gives rise to a unique, functionally active CD8⁺ T cell population. Surprisingly, distinct neuronal subsets require different CD8 effector mechanisms to maintain viral latency, with some requiring IFN-γ and others requiring lytic granules (LG). This nonredundant efficacy of CD8⁺ T cell effector mechanisms in maintaining viral latency is explained as follows: (1) a subset of neurons that expresses IFN-γ receptors (IFN-γR⁺) and Qa1 responds to IFN-γ, but Qa1 engagement of CD94/NKG2a blocks LG exocytosis by CD8⁺ T cells; (2) another neuronal subset is responsive to LG because it lacks Qa1 and is refractory to IFN-γ because it also lacks IFN-γR. In the latter subset, LG appear to provide a nonlethal block of viral reactivation.  

5.320           Parvoviral virions deploy a capsid-tetrered lipolytic wnzyme to breach the endosomal membrane during cell entry

Farr, G.A., Zhang, L-G. and Tattersall, P. PNAS, 102(47), 17148-17153 (2005)   Enveloped viruses deliver their virions into the host cell by fusion with the cellular plasma or endosomal membrane, thus creating topological continuity between the cytosol and the inside of the viral envelope. Nonenveloped viruses are, by their very nature, denied this strategy and must employ alternative methods to breach their host cell's delimiting membrane. We show here that the compact icosahedral parvoviral virion gains entry by deploying a lipolytic enzyme, phospholipase A₂ (PLA₂), that is expressed at the N terminus of VP1, the minor coat protein. This region of VP1 is normally sequestered within the viral shell but is extruded during the entry process as a capsid-tethered domain. A single amino acid substitution in the active site of the VP1 PLA₂ inactivates enzymatic activity and abrogates infectivity. We have used transencapsidation of a vector expressing green fluorescent protein to show that infection by this PLA₂-defective mutant can be complemented by coinfection with wild-type or mutant full virions, provided they can express a functional PLA₂. Even though wild-type empty capsids contain an active form of the enzyme, it is not externalized under physiological conditions, and such capsids are not able to complement the PLA₂ mutant. Significantly, highly efficient rescue can be achieved by polyethyleneimine-induced endosome rupture or by coinfection with adenovirus as long as uptake of the two viruses is simultaneous and the adenovirus is capable of deploying pVI, a capsid protein with endosomolytic activity. Together, these results demonstrate a previously unrecognized enzymatic mechanism for nonenveloped virus penetration.  

5.321           Determination of osteoprogenitor-specific promoter activity in mouse mesenchymal stem cells by recombinant adeno-associated virus transduction

Kumar, S., Mahendra, G. and Ponnazhagan, S. Biochim. Biophys. Acta, 1731(2), 95-103 (2005)   Towards utilizing gene-targeted, repopulating mesenchymal stem cells (MSC) to increase osteogenesis, we evaluated the expression of bone-specific promoters during MSC differentiation. Multi-lineage potential of cultured MSC was confirmed by osteogenic, adipogenic and chondrogenic differentiation under controlled conditions. Recombinant adeno-associated virus (rAAV) encoding luciferase under the human cytomegalovirus (CMV), mouse alkaline phosphatase (ALP), Runx-2/cbfa1 (RUNX), osteopontin (OPN), collagen type 1a (COL), and osteocalcin (OCN) promoters was used to transduce mouse MSC. Replicate cultures were maintained undifferentiated or differentiated to osteoblast lineage. Luciferase expression was determined on days 1, 2, 3, 7, 14, or 21 as a measure of promoter activity. Expression of osteogenic markers and mineralization was determined as correlates of osteopoiesis. Results indicated expression from CMV promoter in undifferentiated and differentiated cultures at early stage. However, expression from COL and RUNX promoters was abundant only in differentiating cultures as early as 24 h but declined gradually. Expression from OPN and ALP promoters was evident 24 h following osteogenic differentiation and peaked gradually until 2 weeks before declining. Expression from OC promoter was evident only after 7 days of differentiation but remained until final analysis on day 21. That rAAV transduction of MSC does not induce differentiation was also confirmed by quantitative reverse-transcription polymerase chain reaction (QRT-PCR). The observed stage-specific expression of analyzed promoters was not significant when the MSC were differentiated to adipocytes. Thus, the use of RUNX2 or COL promoter to stably express osteoinductive factors in MSC may allow both self-renewal of modified MSC and enrichment of osteoblast commitment.  

5.322           IL-10 suppresses chemokines, inflammation, and fibrosis in a model of chronic disease

Mu, W. et al

1. Am. Soc. Nephrol., 16, 3651-3660 (2005)

  IL-10 is a pluripotent cytokine that plays a pivotal role in the regulation of immune and inflammatory responses. Whereas short-term administration of IL-10 has shown benefit in acute glomerulonephritis, no studies have addressed the potential benefits of IL-10 in chronic renal disease. Chronically elevated blood levels of IL-10 in rats were achieved by administration of a recombinant adeno-associated virus serotype 1 IL-10 (rAAV1–IL-10) vector. Control rats were given a similar dose of rAAV1-GFP. Four weeks after injection, IL-10 levels in serum were measured by ELISA, and chronic renal disease was induced by a 5/6 nephrectomy (n = 6 in each group). Eight weeks later, rats were killed and renal tissue was obtained for RNA, protein, and immunohistochemical analysis. Serum levels of IL-10 were 12-fold greater in the rAAV1–IL-10 group by 4 wk after rAAV1–IL-10 administration (345 ± 169 versus 28 ± 15 pg/ml; P = 0.001), and levels were maintained throughout the experiment. rAAV1–IL-10 treatment resulted in less proteinuria (P < 0.05), lower serum creatinine (P < 0.05), and higher creatinine clearances (P < 0.01) compared with rAAV1-GFP–treated rats. Renal interstitial infiltration was significantly attenuated by rAAV1–IL-10 administration as assessed by numbers of CD4⁺, CD8⁺, monocyte-macrophages (ED-1⁺) and dendritic (OX-62⁺) cells (P < 0.05), and this correlated with reductions in the renal expression of monocyte (renal monocyte chemoattractant protein-1 mRNA and protein) and T cell (RANTES mRNA) chemokines. rAAV1–IL-10 administration decreased mRNA levels of IFN- and IL-2 in the kidney. The reduction in inflammatory cells was associated with a significant reduction in glomerulosclerosis and interstitial fibrosis. It is concluded that IL-10 blocks inflammation and improves renal function in this model of chronic renal disease. The feasibility of long-term overexpression of a gene using the AAV serotype 1 vector system in a model of renal disease is also demonstrated.  

5.323           Modulation of muscle regeneration, myogenesis, and adipogenesis by the Rho family nucleotide exchange factor GEFT

Bryan, B. et al Mol. Cell. Biol., 25(24), 11089-11101 (2005)   Rho family guanine nucleotide exchange factors (GEFs) regulate diverse cellular processes including cytoskeletal reorganization, cell adhesion, and differentiation via activation of the Rho GTPases. However, no studies have yet implicated Rho-GEFs as molecular regulators of the mesenchymal cell fate decisions which occur during development and repair of tissue damage. In this study, we demonstrate that the steady-state protein level of the Rho-specific GEF GEFT is modulated during skeletal muscle regeneration and that gene transfer of GEFT into cardiotoxin-injured mouse tibialis anterior muscle exerts a powerful promotion of skeletal muscle regeneration in vivo. In order to molecularly characterize this regenerative effect, we extrapolate the mechanism of action by examining the consequence of GEFT expression in multipotent cell lines capable of differentiating into a number of cell types, including muscle and adipocyte lineages. Our data demonstrate that endogenous GEFT is transcriptionally upregulated during myogenic differentiation and downregulated during adipogenic differentiation. Exogenous expression of GEFT promotes myogenesis of C2C12 cells via activation of RhoA, Rac1, and Cdc42 and their downstream effector proteins, while a dominant-negative mutant of GEFT inhibits this process. Moreover, we show that GEFT inhibits insulin-induced adipogenesis in 3T3L1 preadipocytes. In summary, we provide the first evidence that the Rho family signaling pathways act as potential regulators of skeletal muscle regeneration and provide the first reported molecular mechanism illustrating how a mammalian Rho family GEF controls this process by modulating mesenchymal cell fate decisions.  

5.324           Generation of HPV pseudovirions using transfection and their use in neutralization assays

Pastrana, B.C.B., Lowy, D.R. and Schiller, J.T. Methods Mol. Med., 119, 445-462 (2005)   It has recently become possible to generate high-titer papillomavirus-based gene-transfer vectors. The vectors, also known as papillomavirus pseudoviruses (PsV), have been useful for studying papillomavirus assembly, entry, and neutralization, and may have future utility as laboratory gene-transfer tools or vaccine vehicles. This chapter outlines a simple method for production of PsV and their use in a high-throughput papillomavirus neutralization assay. The production method is based on transfection of a 293 cell line, 293TT, engineered to express high levels of SV40 large T antigen. The cells are co-transfected with codon-modified papillomavirus capsid genes, L1 and L2, together with a pseudogenome plasmid containing the SV40 origin of replication. Pseudogenome encapsidation within L1/L2 capsids is largely sequence independent, and plasmids entirely lacking PV sequences can be packaged efficiently, provided they are less than 8 kilobases in size. Non-infectious virus-like particles (VLPs) can also be produced after transfection of 293TT cells with L1 alone. Efficient purification of the PsV or VLPs is achieved by Optiprep (iodixanol) density gradient ultracentrifugation. Using these methods, it is possible to produce highly purified PsV with yields of at least 10(9) transducing units from a single 75-cm2 flask of cells. PsV encapsidating a secreted alkaline phosphatase (SEAP) reporter plasmid were used to develop a high-throughput in vitro neutralization assay in a 96-well plate format. Infection of 293TT cells is monitored by SEAP activity in the culture supernatant, using a highly sensitive chemiluminescent reporter system. Antibody-mediated PsV neutralization is detected by a reduction in SEAP activity. The neutralization assay has similar analytic sensitivity to, and higher specificity than, a standard VLP-based enzyme-linked immunosorbent assay (ELISA).    

5.325           Safety of direct administration of AAV2_CUhCLN2, a candidate treatment for the central nervous system manifestations of late infantile neuronal ceroid lipofuscinosis, to brain of rats and nonhuman primates

Hackett, N.R. et al Hum. Gen. Ther., 16, 1484-1503 (2005)   Late infantile neuronal ceroid lipofuscinosis (LINCL), a pediatric autosomal recessive neurodegenerative lysosomal storage disorder, results from mutations in the CLN2 gene and consequent deficiency in tripeptidyl-peptidase I (TPP-I) and progressive destruction of neurons. We have previously demonstrated that CNS gene transfer of AAV2_CUhCLN2 (an AAV2-based vector expressing the human CLN2 cDNA) in rats and nonhuman primates mediates long-term TPP-I expression in the CNS neurons [Sondhi, D., Peterson, D.A., Giannaris, E.L., Sanders, C.T., Mendez, B.S., De, B., Rostkowski, A., Blancard, B., Bjugstad, K., Sladek, J.R., Redmond, D.E., Leopold, P.L., Kaminsky, S.M., Hackett, N.R., and Crystal, R.G. (2005). Gene Ther. 12, 1618–1632]. The present study tests the hypothesis that direct CNS administration of a clinical-grade AAV2_CUhCLN2 vector to the CNS of rats and nonhuman primates at doses scalable to humans has a long-term safety profile acceptable for initiating clinical trials. Fischer 344 rats were injected bilaterally via the striatum with 2 × 10¹⁰ particle units (PU) of AAV2_CUhCLN2, using saline as a control. At 13, 26, and 52 weeks, vector and phosphate-buffered salineinjected rats were killed (n = 6 per time point), and blood, brain, and distant organs were assessed. There were no biologically significant differences between control and vector groups for complete blood count, serum chemistry, and neutralizing anti-AAV2 antibody levels. CNS administration of AAV2 CUhCLN2 did not result in any pathological changes in the brain that were attributable to the vector, although microscopic changes were observed along the track consistent with needle trauma. A total dose of 3.6 × 10¹⁰ or 3.6 × 10¹¹ PU of AAV2_CUhCLN2 was administered to the CNS of African Green monkeys at 12 locations, targeting the caudate nucleus, hippocampus, and overlying cortices. Monkeys (n = 3 at each dose) were killed 1, 13, 26, or 52 weeks after injection. Controls included sham-injected, saline-injected, and AAV2_CUNull-injected (3.6 × 10¹¹ PU) monkeys. There were no biologically significant differences among vector-injected and control groups in any parameter of the general assessment, complete blood count, or serum chemistry assessed at multiple time points after vector administration. Importantly, no abnormal behavior was observed in any group in videotaped neurological assessment, where behaviors were quantified before administration and at multiple time points afterward. Histopathological examination of the CNS demonstrated that 1 week after administration, AAV2_CUhCLN2 produced transient minor white matter edema with reactive glial cells in the corona radiata of the cerebrum along the injection track and in the surrounding white matter. This abnormality was not observed at 13, 26, or 52 weeks. Together with the long-term gene expression after gene transfer, these findings supported the initiation of clinical trials to assess the safety of AAV2_CUhCLN2 administration to individuals with LINCL.  

5.326           Enhancing rAAV vector expression in the lung

Virella-Lowell, I., Zusman, B., Foust, K., Loiler, S., Conlon, T., Song, S., Chesnut, K.A., Ferkol, T. and Flotte, T.R:

1. Gene Med., 7(7), 842-850 (2005)

  Despite favorable DNA transfer efficiency, gene expression from recombinant adeno-associated virus (rAAV2) vectors in the lung has been variable in the context of cystic fibrosis (CF) gene therapy. This is due, in part, to the large size of the CF transmembrane regulator (CFTR)-coding sequence which necessitates the use of compact endogenous promoter elements versus stronger exogenous promoters. We evaluated the possibility that gene expression from rAAV could be improved by using AAV capsid serotypes with greater tropism for the apical surface of airway cells (i.e. rAAV5 or rAAV1) and/or using strong promoters such as the cytomegalovirus (CMV) enhancer/chicken beta-actin hybrid (Cbeta) promoter. The relative activity of the CMV immediate-early (CMVie) promoter, the Cbeta promoter, and the Cbeta promoter with a downstream woodchuck hepatitis virus post-transcriptional regulatory element (wpre) were assessed in vitro and in vivo in C57\Bl6 mice using human alpha-1 antitrypsin (hAAT) as a secreted reporter. In vivo, the Cbeta-AAT-wpre group achieved maximum serum levels of 1.5 mg/ml of hAAT. AAV capsid serotypes were then compared in vivo utilizing the transcriptionally optimized CB-wpre cassette in rAAV serotype 1, 2 or 5 capsids (rAAV1, rAAV2, and rAAV5), utilizing luciferase as a reporter to compare expression over a wide dynamic range. The pulmonary luciferase levels at 8 weeks were similar in rAAV5 and rAAV1 groups (2.9 x 10(6) relative light units (RLU)/g tissue and 2.7 x 10(6) RLU/g tissue, respectively), both of which were much higher than rAAV2. Although the advantage of rAAV5 over rAAV2 in the lung has already been described, the availability of another serotype (rAAV1) capable of efficient gene transfer in the lung could be useful.  

5.327           Adeno-associated virus serotypes 1 to 5 mediated tumor cell directed gene transfer and improvement of transduction efficiency

Hacker, U.T., Wingenfeld, L., Kofler, D. M., Schuhmann, N.K., Lutz, S., Herold, T., King, S.B.S., garner, F.M., Perabo, L., Rabinowitz, J., McCarty, D.M., Samulski, R.J., Hallek, M. and Büning, H.

1. Gene Med., 7(11), 1429-1438 (2005)

  Background Gene therapy is an attractive new approach for the treatment of cancer. Therefore, the development of efficient vector systems is of crucial importance in this field. Different adeno-associated virus (AAV) serotypes have been characterized so far, which show considerable differences in tissue tropism. Consequently, we aimed to characterize the most efficient serotype for this application. Methods To exclude all influences other than those provided by the capsid, all serotypes contained the same transgene cassette flanked by the AAV2 inverted terminal repeats. We systematically compared these vectors for efficiency in human cancer cell directed gene transfer. In order to identify limiting steps, the influence of second-strand synthesis and proteasomal degradation of AAV in a poorly transducible cell line were examined. Results AAV2 was the most efficient serotype in all solid tumor cells and primary melanoma cells with transduction rates up to 98 ± 0.3%. Transduction above 70% could be reached with serotypes 1 (in cervical and prostate carcinoma) and 3 (in cervical, breast, prostate and colon carcinoma) using 1000 genomic particles per cell. In the colon carcinoma cell line HT-29 proteasomal degradation limited AAV1–AAV4-mediated gene transfer. Moreover, inefficient second-strand synthesis prevents AAV2-mediated transgene expression in this cell line. Conclusions Recent advances in AAV-vector technology suggest that AAV-based vectors can be used for cancer gene therapy. Our comparative analysis revealed that, although AAV2 is the most promising candidate for such an application, serotypes 1 and 3 are valid alternatives. Furthermore, the use of self-complementary AAV vectors and proteasome inhibitors significantly improves cancer cell transduction.  

5.328           Recombinant adeno-associated virus (rAAV) expressing TFPI-2 inhibits invasion, angiogenesis and tumor growth in a human glioblastoma cell line

Yanamandra, N., Kondraganti, S., Gondi, C.S., Gujrati, M., Olivero, W.C., Dinh, D.H. and Rao, J.S. Int. J. Cancer, 115(6), 998-1005 (2005)   Recombinant adeno-associated viruses (rAAV) have become the vector of choice for many gene therapy protocols. rAAVs have a number of attractive features including long-term transgene expression and the ability to transduce both dividing and non-dividing cells. We have shown previously the anti-cancer role of tissue factor pathway inhibitor-2 (TFPI-2), a matrix-associated serine protease inhibitor, in human glioblastomas. As a result of our present study, in which 0.8-kb fragment of human TFPI-2 was cloned into the adeno-associated viral vectors (rAAA-TFPI-2), rAAV-TFPI-2 infection of SNB19 cells significantly increased TFPI-2 as determined by Western blotting. As assessed by spheroid and Matrigel assays, infection of SNB19 cells with rAAV-TFPI-2 significantly reduced migration and invasion in a dose-dependent manner. Tumor spheroids infected with rAAV-TFPI-2 and co-cultured with fetal rat brain aggregates did not invade rat brain aggregates, whereas 90–95% of the mock and AAV-CMV infected cells invaded rat brain aggregates. In vitro angiogenesis studies (tumor cells co-cultured with endothelial cells or endothelial cells seeded on matrigel) showed reduction of capillary-like structure formation in rAAV-TFPI-2-treated cells as compared to parental and mock-transfected cells. In in vivo angiogenesis results demonstrated the formation of microvessels in SNB19 parental cells and this formation was inhibited when the SNB19 cells were infected with rAAV-TFPI-2. Further, we observed a large reduction of tumor growth in SNB19 cells treated with rAAV-TFPI-2 virus injected intracerebrally when compared to controls. Our study demonstrates that rAAV-TFPI-2-mediated gene therapy offers a novel tool for the treatment of brain tumors.  

5.329           Production, purification, crystallization and preliminary X-ray analysis of adeno-associated virus serotype 8

Lane, M.D., Nam, H-J., Padron, E., Gurda-Whitaker, B., Kohlbrenner, E., Aslanidi, G., Byrne, B., McKenna, R., Muzycka, N., Zolotukhin, S. and Agbandje-McKenna, M. Acta Cryst., F61, 558-561 (2005)   Adeno-associated viruses (AAVs) are actively being developed for clinical gene-therapy applications and the efficiencies of the vectors could be significantly improved by a detailed understanding of their viral capsid structures and the structural determinants of their tissue-transduction interactions. AAV8 is ~80% identical to the more widely studied AAV2, but its liver-transduction efficiency is significantly greater than that of AAV2 and other serotypes. The production, purification, crystallization and preliminary X-ray crystallographic analysis of AAV8 viral capsids are reported. The crystals diffract X-rays to 3.0 Å resolution using synchrotron radiation and belong to the hexagonal space group P6322, with unit-cell parameters a = 257.5, c = 443.5 Å. The unit cell contains two viral particles, with ten capsid viral protein monomers per crystallographic asymmetric unit.  

5.330           VP2 cleavage and the leucine ring at the base of the fivefold cylinder control pH-dependent externalization of both the VP1 N terminus and the genome of minute virus of mice

Farr, G.A., Cotmore, S.F. and Tattersall, P.

1. Virol., 80(1), 161-171 (2006)

  Cylindrical projections surrounding the fivefold-symmetry axes in minute virus of mice (MVM) harbor central pores that penetrate through the virion shell. In newly released DNA-containing particles, these pores contain residues 28 to 38 belonging to a single copy of VP2, disposed so that its extreme N-terminal domain projects outside the particle. Virions are metastable, initially sequestering internally the N termini of all copies of the minor capsid protein, VP1, that is essential for entry. This VP1 domain can be externalized in vitro in response to limited heating, and we show here that the efficiency of this transition is greatly enhanced by proteolysis of VP2 N termini to yield VP3. This step also renders the VP1 rearrangement pH dependent, indicating that VP2 cleavage is a maturation step required to prime subsequent emergence of the VP1 "entry" domain. The tightest constriction within the cylinder is created by VP2 leucine 172, the five symmetry-related copies of which form a portal that resembles an iris diaphragm across the base of the pore. In MVMp, threonine substitution at this position, L172T, yields infectious particles following transfection at 37°C, but these can initiate infection only at 32°C, and this process can be blocked by exposing virions to a cellular factor(s) at 37°C during the first 8 h after entry. At 32°C, the mutant particle is highly infectious, and it remains stable prior to VP2 cleavage or following cleavage at pH 5.5 or below. However, upon exposure to neutral pH following VP2 cleavage, its VP1-specific sequences and genome are extruded even at room temperature, underscoring the significance of the VP2 cleavage step for MVM particle dynamics.  

5.331           Liao ning, a new Chinese seadornavirus that replicates in transformed and embryonic mammalian cells

Attoui, H. et al

1. Gen. Virol., 87, 199-208 (2006)

  Seadornaviruses are emerging arboviral pathogens from the south-east of Asia. The genus Seadornavirus contains two distinct species, Banna virus (BAV) isolated from humans with encephalitis and Kadipiro virus. BAV replicates within insect cells and mice but not in cultured mammalian cells. Here, the discovery of Liao ning virus (LNV), a new seadornavirus from the Aedes dorsalis mosquito, which was completely sequenced and was found to be related to BAV and Kadipiro virus, is reported. Two serotypes of LNV could be distinguished by a serum neutralization assay. According to amino acid identity with other seadornaviruses, and to criteria set by the ICTV for species delineation, LNV was identified as a member of a new species of virus. Its morphology was characterized by electron microscopy and found to be similar to that of BAV. LNV is the first reported seadornavirus that replicates in mammalian cells, leading to massive cytopathic effect in all transformed or embryonic cell lines tested. LNV- and BAV-infected mice producing a viraemia lasting for 5 days was followed by viral clearance. Mice infection generated virus quasi-species for LNV (the first reported observation for quasi-species in the family Reoviridae) but not for BAV. Challenge with BAVin mice immunized against BAV did not lead to productive infection.However, challenge with LNV in mice immunized against LNV waslethal with a new phase of viraemia and massive haemorrhage. A table showing the sequences used in RdRps phylogenetic analysis of seadornaviruses is available as supplementary material in JGV Online.  

5.332           High levels of persistent expression of a1antitrypsin mediated by the nonhuman primate serotype rh.10 adeno-associated virus despite preexisting immunity to common human adeno-associated viruses

De, B.P. et al Mol. Ther., 13(1), 67-76 (2006)   α1-Antitrypsin (α1AT) deficiency is a genetic disorder causing emphysema if serum α1AT levels are <570 μg/ml. We have shown that intrapleural administration of an AAV5α1AT vector yielded persistent therapeutic α1AT serum levels. Since anti-AAV2 and -AAV5 antibodies prevalent in humans may limit the use of these common serotypes in gene therapy, we screened 25 AAV vectors derived from humans and nonhuman primates for α1AT expression following intrapleural administration to mice. The rhesus AAVrh.10 serotype yielded the highest levels and was chosen for further study. Following intrapleural administration, 77% of total body transgene expression was in the chest wall, diaphragm, lung, and heart. Intrapleural administration of AAVrh.10α1AT provided long-term, therapeutic α1AT expression in mice, although higher doses were required to achieve therapeutic levels in female mice than in male mice. Intrapleural administration of AAVrh.10α1AT produced the same levels in AAV2/AAV5-preimmune and naive mice. In mice administered with AAV5α1AT and subsequently “boosted” with the AAVrh.10α1AT vector, serum levels were increased by 300%. These data indicate that AAVrh.10 is the most effective known AAV vector for intrapleural gene delivery and has the advantage of circumventing human immunity to AAV.  

5.333           Systemic correction of a fatty acid oxidation defect by intramuscular injection of a recombinant adeno-associated virus vector

Conlon, T.I. et al Hum. Gen. Ther., 17, 71-80 (2006)   Mitochondrial β-oxidation of fatty acids is required to meet physiologic energy requirements during illness and periods of fasting or physiologic stress, and is most active in liver and striated muscle. Acyl-CoA dehydrogenases of varying chain-length specificities represent the first step in the mitochondria for each round of β-oxidation, each of which removes two-carbon units as acetyl-CoA for entry into the tricarboxylic acid cycle. We have used recombinant adeno-associated virus (rAAV) vectors expressing short-chain acyl-CoA dehydrogenase (SCAD) to correct the accumulation of fatty acyl-CoA intermediates in deficient cell lines. The rAAV-SCAD vector was then packaged into either rAAV serotype 1 or 2 capsids and injected intramuscularly into SCAD-deficient mice. A systemic effect was observed as judged by restoration of circulating butyryl- carnitine levels to normal. Total lipid content at the injection site was also decreased as demonstrated by noninvasive magnetic resonance spectroscopy (MRS). SCAD enzyme activity in the injected muscle was found at necropsy to be above the normal control mouse level. This study is the first to demonstrate the systemic correction of a fatty acid oxidation disorder with rAAV and the utility of MRS as a noninvasive method to monitor SCAD correction after in vivo gene therapy.  

5.334           Intracranial delivery of CLN2 reduces brain pathology in a mouse model of classical late infantile neuronal ceroid lipofuscinosis

Passini, M.A. et al

1. Neurosci., 26(5), 1334-1342 (2006)

  Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a lysosomal storage disorder caused by mutations in CLN2, which encodes lysosomal tripeptidyl peptidase I (TPP1). Lack of TPP1 results in accumulation of autofluorescent storage material and curvilinear bodies in cells throughout the CNS, leading to progressive neurodegeneration and death typically in childhood. In this study, we injected adeno-associated virus (AAV) vectors containing the human CLN2 cDNA into the brains of CLN2^–/– mice to determine therapeutic efficacy. AAV2_CUhCLN2 or AAV5_CUhCLN2 were stereotaxically injected into the motor cortex, thalamus, and cerebellum of both hemispheres at 6 weeks of age, and mice were then killed at 13 weeks after injection. Mice treated with AAV2_CUhCLN2 and AAV5_CUhCLN2 contained TPP1 activity at each injection tract that was equivalent to 0.5- and 2-fold that of CLN2^+/+ control mice, respectively. Lysosome-associated membrane protein 1 immunostaining and confocal microscopy showed intracellular targeting of TPP1 to the lysosomal compartment. Compared with control animals, there was a marked reduction of autofluorescent storage in the AAV2_CUhCLN2 and AAV5_CUhCLN2 injected brain regions, as well as adjacent regions, including the striatum and hippocampus. Analysis by electron microscopy confirmed a significant decrease in pathological curvilinear bodies in cells. This study demonstrates that AAV-mediated TPP1 enzyme replacement corrects the hallmark cellular pathologies of cLINCL in the mouse model and raises the possibility of using AAV gene therapy to treat cLINCL patients.  

5.335           Long-term adeno-associated viral vector-mediated expression of truncated TrkB in the adult rat facial nucleus results in motor neuron degeneration

De Wit, J., Eggers, R., Evers, R., Castren, E. and Verhaagen, J.

1. Neurosci., 26(5), 1516-1530 (2006)

  Adult facial motor neurons continue to express full-length TrkB tyrosine kinase receptor (TrkB.FL), the high-affinity receptor for the neurotrophins BDNF and neurotrophic factor-4/5 (NT-4/5), suggesting that they remain dependent on target-derived and locally produced neurotrophins in adulthood. Studies on the role of TrkB signaling in the adult CNS have been hampered by the early lethality of bdnf, nt-4/5, and trkB knock-out mice.We disrupted TrkB.FL signaling in adult facial motor neuronsusing adeno-associated viral vector-mediated overexpressionof a naturally occurring dominant-negative TrkB receptor, TrkB.T1.Expression of TrkB.T1 resulted in neuronal atrophy and downregulationof NeuN (neuronal-specific nuclear protein) and ChAT expressionin facial motor neurons. A subset of transduced neurons displayedsigns of motor neuron degeneration that included dendritic beadingand rounding of the soma at 2 months of TrkB.T1 expression.Cell counts revealed a significant reduction in motor neuronnumber in the facial nucleus at 4 months after onset of expressionof TrkB.T1, suggesting that a proportion of TrkB.T1-expressingmotor neurons became undetectable as a result of severe atrophyor was lost because of cell death. In contrast, overexpressionof TrkB.FL did not result in a decrease in facial motor neuronnumber. Our results indicate that a subset of facial motor neuronsremains dependent on TrkB ligands for the maintenance of structuraland molecular characteristics in adulthood.  

5.336           Hypothalamic rAAV-mediated GDNF gene delivery ameliorates age-related obesity

Tümer, N. et al Neurobiol. Of Aging, 27(3), 459-470 (2006)   Intraventricular delivery of glial cell line-derived neurotrophic factor (GDNF) results in weight loss. We hypothesized that this effect of GDNF was likely mediated via its effects on dopaminergic neurons in the hypothalamus. Continuous rAAV-mediated GDNF expression in the hypothalamus of young and senescent rats resulted in weight loss compared to controls. However, GDNF-induced weight loss was unrelated to alterations in hypothalamic dopamine levels. The weight loss was associated with decreased food intake and increased energy expenditure, but these effects were not mediated by changes in hypothalamic NPY or POMC expression. Moreover, uncoupling protein 1 levels were unchanged in brown adipose tissue (BAT). The reduction in weight and adiposity were as great or greater in the aged rats even though aged rats are generally resistant to weight loss therapies. In summary, central GDNF gene delivery reduces weight and adiposity in young and aged rats through decreased food intake and increased energy expenditure. Our observations in aged rats suggest that GDNF may be especially effective in reducing obesity in aged obese rats.  

5.337           Association between hepatitis C virus and very-low-density lipoprotein (VLDL)/LDL analyzed in iodixanol density gradients

Nielsen, S.U. et al

1. Virol., 80(5), 2418-2428 (2006)

  Hepatitis C virus (HCV) RNA circulates in the blood of persistently infected patients in lipoviroparticles (LVPs), which are heterogeneous in density and associated with host lipoproteins and antibodies. The variability and lability of these virus-host complexes on fractionation has hindered our understanding of the structure of LVP and determination of the physicochemical properties of the HCV virion. In this study, HCV from an antibody-negative immunodeficient patient was analyzed using three fractionation techniques, NaBr gradients, isotonic iodixanol, and sucrose gradient centrifugation. Iodixanol gradients were shown to best preserve host lipoprotein-virus complexes, and all HCV RNA was found at densities below 1.13 g/ml, with the majority at low density, 1.08 g/ml. Immunoprecipitation with polyclonal antibodies against human ApoB and ApoE precipitated 91.8% and 95.0% of HCV with low density, respectively, suggesting that host lipoprotein is closely associated with HCV in a particle resembling VLDL. Immunoprecipitation with antibodies against glycoprotein E2 precipitated 25% of HCV with low density, providing evidence for the presence of E2 in LVPs. Treatment of serum with 0.5% deoxycholic acid in the absence of salt produced HCV with a density of 1.12 g/ml and a sedimentation coefficient of 215S. The diameters of these particles were calculated as 54 nm. Treatment of serum with 0.18% NP-40 produced HCV with a density of 1.18 g/ml, a sedimentation coefficient of 180S, and a diameter of 42 nm. Immunoprecipitation analysis showed that ApoB remained associated with HCV after treatment of serum with deoxycholic acid or NP-40, whereas ApoE was removed from HCV with these detergents.  

5.338           Molecular disruption of hypothalamic nutrient sensing induces obesity

He, W., Lam, T.K.T., Obici, S. and Rossetti, L. Nature Neurosci., 9(2), 227-233 (2006)   The sensing of circulating nutrients within the mediobasal hypothalamus may be critical for energy homeostasis. To induce a sustained impairment in hypothalamic nutrient sensing, adeno-associated viruses (AAV) expressing malonyl–coenzyme A decarboxylase (MCD; an enzyme involved in the degradation of malonyl coenzyme A) were injected bilaterally into the mediobasal hypothalamus of rats. MCD overexpression led to decreased abundance of long-chain fatty acyl–coenzyme A in the mediobasal hypothalamus and blunted the hypothalamic responses to increased lipid availability. The enhanced expression of MCD within this hypothalamic region induced a rapid increase in food intake and progressive weight gain. Obesity was sustained for at least 4 months and occurred despite increased plasma concentrations of leptin and insulin. These findings indicate that nutritional modulation of the hypothalamic abundance of malonyl–coenzyme A is required to restrain food intake and that a primary impairment in this central nutrient-sensing pathway is sufficient to disrupt energy homeostasis and induce obesity.  

5.339           Directed evolution of adeno-associated virus yields enhanced gene delivery vectors

Maheshri, N., Koerber, J.T., Kaspar, B.K. and Schaffer, D.V. Nature Biotechnol., 24(2), 198-204 (2006)   Adeno-associated viral vectors are highly safe and efficient gene delivery vehicles. However, numerous challenges in vector design remain, including neutralizing antibody responses, tissue transport and infection of resistant cell types. Changes must be made to the viral capsid to overcome these problems; however, very often insufficient information is available for rational design of improvements. We therefore applied a directed evolution approach involving the generation of large mutant capsid libraries and selection of adeno-associated virus (AAV) 2 variants with enhanced properties. High-throughput selection processes were designed to isolate mutants within the library with altered affinities for heparin or the ability to evade antibody neutralization and deliver genes more efficiently than wild-type capsid in the presence of anti-AAV serum. This approach, which can be extended to additional gene delivery challenges and serotypes, directs viral evolution to generate 'designer' gene delivery vectors with specified, enhanced properties.  

5.340           Long-term correction of murine glycogen storage disease type Ia by recombinant adeno-associated virus-1-mediated gene transfer

Ghosh, A. et al Gen. Ther., 13, 321-329 (2006)   Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in glucose-6-phosphatase- (G6Pase- ), a nine-transmembrane domain, endoplasmic reticulum-associated protein expressed primarily in the liver and kidney. Previously, we showed that infusion of an adeno-associated virus (AAV) serotype 2 vector carrying murine G6Pase- (AAV2-G6Pase- ) into neonatal GSD-Ia mice failed to sustain their life beyond weaning. We now show that neonatal infusion of GSD-Ia mice with an AAV serotype 1-G6Pase- (AAV1-G6Pase- ) or AAV serotype 8-G6Pase- (AAV8-G6Pase- ) results in hepatic expression of the G6Pase- transgene and markedly improves the survival of the mice. However, only AAV1-G6Pase- can achieve significant renal transgene expression. A more effective strategy, in which a neonatal AAV1-G6Pase- infusion is followed by a second infusion at age one week, provides sustained expression of a complete, functional, G6Pase- system in both the liver and kidney and corrects the metabolic abnormalities in GSD-Ia mice for the 57 week length of the study. This effective use of gene therapy to correct metabolic imbalances and disease progression in GSD-Ia mice holds promise for the future of gene therapy in humans.  

5.341           XIAP-mediated neuroprotection in retinal ischemia

Renwick, J. et al Gen. Ther., 13, 339-347 (2006)   Retinal ischemia results in the loss of vision in a number of ocular diseases including acute glaucoma, diabetic retinopathy, hypertensive retinopathy and retinal vascular occlusion. Recent studies have shown that most of the neuronal death that leads to loss of vision results from apoptosis. XIAP-mediated gene therapy has been shown to protect a number of neuronal types from apoptosis but has never been assessed in retinal neurons following ischemic-induced cell death. We injected an adeno-associated viral vector expressing XIAP or GFP into rat eyes and 6 weeks later, rendered them ischemic by raising intraocular pressure. Functional analysis revealed that XIAP-treated eyes retained larger b-wave amplitudes than GFP-treated eyes up to 4 weeks post-ischemia. The number of cells in the inner nuclear layer (INL) and the thickness of the inner retina were significantly preserved in XIAP-treated eyes compared to GFP-treated eyes. Similarly, there was no significant reduction in optic nerve axon numbers in XIAP-treated eyes. There were also significantly fewer TUNEL (TdT-dUTP terminal nick end labeling) positive cells in the INL of XIAP-treated retinas at 24 h post-ischemia. Thus, XIAP-mediated gene therapy imparts both functional and structural protection to the retina after a transient ischemic episode.  

5.342           Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells

Yi, M., Villanueva, R.A., Thomas, D.L., Wakita, T. and Lemon, S.M. PNAS, 103(7), 2310-2315 (2006)   Infections with hepatitis C virus (HCV) are marked by frequent viral persistence, chronic liver disease, and extraordinary viral genetic diversity. Although much has been learned about HCV since its discovery, progress has been slowed by a lack of permissive cell culture systems supporting its replication. Productive infections have been achieved recently with genotype 2a virus, but cirrhosis and liver cancer are typically associated with genotype 1 HCV, which is more prevalent and relatively resistant to IFN therapy. We describe production of infectious genotype 1a HCV in cells transfected with synthetic RNA derived from a prototype virus (H77-S). Viral proteins accumulated more slowly in H77-S transfected cells than in cells transfected with genotype 2a (JFH-1) RNA, but substantially more H77-S RNA was secreted into supernatant fluids. Most secreted RNA was noninfectious, banding in isopycnic gradients at a density of 1.04–1.07 gm/cm³, but infectivity was associated with H77-S particles possessing a density of 1.13–1.14 gm/cm³. The specific infectivity of H77-S particles (5.4 x 10⁴ RNA copies per focus-forming unit) was significantly lower than JFH-1 virus (1.4 x 10² RNA copies per focus-forming unit). Infection with either virus was blocked by CD81 antibody. Sera from genotype 1a-infected individuals neutralized H77-S virus, but had little activity against genotype 2a virus, suggesting that these genotypes represent different serotypes. The ability of this genotype 1a virus to infect cultured cells will substantially benefit antiviral and vaccine discovery programs.    

5.343           Protective efficacy of an oral vaccine to reduce carriage of Borrelia burgdorferi (strain N40) on mouse and tick reservoirs

Scheckelhoff, M.R., Telford, S.R. and Hu, L.T. Vaccine, 24(11), 1949-1957 (2006)   Lyme disease is caused by the spirochete Borrelia burgdorferi, which is transmitted through the bite of infected Ixodes ticks. Vaccination of mice with outer surface protein A (OspA) of B. burgdorferi has been shown to both protect mice against B. burgdorferi infection and reduce carriage of the organism in feeding ticks. Here we report the development of a murine-targeted OspA vaccine utilizing Vaccinia virus to interrupt transmission of disease in the reservoir hosts, thus reducing incidence of human disease. Oral vaccination of mice with a single dose of Vaccinia expressing OspA resulted in high antibody titers to OspA, 100% protection of vaccinated mice from infection with B. burgdorferi, and significant clearance of B. burgdorferi from infected ticks fed on vaccinated animals. The results indicate the vaccine is effective and may provide a manner to reduce incidence of Lyme disease.  

5.344           The HIV lipodome: a raft with an unusual composition

Brügger, B. et al PNAS, 108(8), 2641-2646 (2006)   The lipids of enveloped viruses play critical roles in viral morphogenesis and infectivity. They are derived from the host membranes from which virus budding occurs, but the precise lipid composition has not been determined for any virus. Employing mass spectrometry, this study provides a quantitative analysis of the lipid constituents of HIV and a comprehensive comparison with its host membranes. Both a substantial enrichment of the unusual sphingolipid dihydrosphingomyelin and a loss of viral infectivity upon inhibition of sphingolipid biosynthesis in host cells are reported, establishing a critical role for this lipid class in the HIV replication cycle. Intriguingly, the overall lipid composition of native HIV membranes resembles detergent-resistant membrane microdomains and is strikingly different from that of host cell membranes. With this composition, the HIV lipidome provides strong evidence for the existence of lipid rafts in living cells.  

5.345           Efficient neuronal gene transfer with AAV8 leads to neurotoxic levels of tau or green fluorescent proteins

Klein, R.L. et al Mol. Ther., 13(3), 517-527 (2006)   Adeno-associated virus (AAV) serotype 8 appears to be the strongest of the natural serotypes reported to date for gene transfer in liver and muscle. In this study, we evaluated AAV8 in the brain by several methods, including biophotonic imaging of green fluorescent protein (GFP). In the adult rat hippocampus, levels of GFP expressed were clearly greater with AAV8 than with AAV2 or AAV5 by Western blot and biophotonic imaging and slightly but significantly greater than AAV1 by Western blot. In the substantia nigra, the GFP expression conferred by AAV8 was toxic to dopamine neurons, although toxicity could be avoided with dose titration. At the low dose at which there was no GFP toxicity from the GFP vector, another AAV8 vector for a disease-related (P301L) form of the microtubule-associated protein tau caused a 78% loss of dopamine neurons and significant amphetamine-stimulated rotational behavior. The AAV8 tau vector-induced cell loss was greater than that from AAV2 or AAV5 tau vectors, demonstrating that the increased gene transfer was functional. While the toxicity observed with GFP expression warrants great caution, the efficient AAV8 is promising for animal models of neurodegenerative diseases and potentially as well for gene therapy of brain diseases.  

5.346           Adeno-associated virus vectors serotyped with AAV8 capsid are more efficient than AAV-1 or –2 serotypes for widespread gene delivery to the neonatal mouse brain

Broekman, M.L.D., Comer, L.A., Hyman, B.T. and Sena-Esteves, M. Neuroscience, 138, 501-510 (2006)   Adeno-associated virus (AAV) vectors have gained a preeminent position in the field of gene delivery to the normal brain through their ability to achieve extensive transduction of neurons and to mediate long-term gene expression with no apparent toxicity. In adult animals direct infusion of AAV vectors into the brain parenchyma results in highly efficient transduction of target structures. However AAV-mediated global delivery to the adult brain has been an elusive goal. In contrast, widespread global gene delivery has been obtained by i.c.v. injection of AAV1 or AAV2 in neonates. Among the novel AAV serotypes cloned and engineered for production of recombinant vectors, AAV8 has shown a tremendous potential for in vivo gene delivery with nearly complete transduction of many tissues in rodents after intravascular infusion. Here we compare the efficiency of an AAV8 serotyped vector with that of AAV1 and AAV2 serotyped vectors for the extent of gene delivery to the brain after neonatal injection into the lateral ventricles. The vectors all encoded green fluorescent protein (GFP) under control of a hybrid CMV enhancer/chicken beta-actin promoter with AAV2 inverted terminal repeats, but differed from each other with respect to the capsid type. A total of 6.8×10¹⁰ genome copies were injected into the lateral ventricles of postnatal day 0 mice. Mice were killed at postnatal day 30 and brains analyzed for distribution of GFP-positive cells. AAV8 proved to be more efficient than AAV1 or AAV2 vectors for gene delivery to all of the structures analyzed, including the cerebral cortex, hippocampus, olfactory bulb, and cerebellum. Moreover the intensity of gene expression, assessed using a microarray reader, was considerably higher for AAV8 in all structures analyzed. In conclusion, the enhanced transduction achieved by AAV8 compared with AAV1 and AAV2 indicates that AAV8 is the superior serotype for gene delivery to the CNS.  

5.347           Human a-defensins block papillomavirus infection

Buck, C.B. et al PNAS, 103(5), 1516-1521 (2006)   Sexually transmitted human papillomaviruses (HPVs) are the primary cause of cervical cancer. Recent advances in techniques for production of papillomaviral vectors [known as pseudoviruses (PsVs)] have made it possible to perform high-throughput screens for compounds that might block the initial stages of papillomavirus infection. We have used PsVs to screen a variety of compounds that might function as inhibitors of HPV infection, with emphasis on human peptides previously implicated in innate antimicrobial immunity. Little is known about the possible activity of these peptides against nonenveloped viruses, such as HPVs. Our screen revealed that human -defensins 1-3 [known as human neutrophil peptides (HNPs) 1-3] and human -defensin 5 (HD-5) are potent antagonists of infection by both cutaneous and mucosal papillomavirus types. In contrast, human -defensins 1 and 2 displayed little or no anti-HPV activity. HD-5 was particularly active against sexually transmitted HPV types, with 50% inhibitory doses in the high ng/ml range. Microscopic studies of PsV inhibition by the -defensins revealed that they block virion escape from endocytic vesicles but not virion binding or internalization. Consistent with this finding, PsVs remained susceptible to inhibition by -defensins for many hours after initial binding to cells. HNPs 1-3 and HD-5 have been reported to be present in the female genital tract at levels that overlap those that inhibit HPVs in vitro, suggesting that they could present a natural barrier to the sexual transmission of HPV and could serve as the basis of a broad-spectrum topical microbicide.  

5.348           Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro

Lindenbach, B.D. et al PNAS, 103(10), 3805-3809 (2006)   Hepatitis C virus (HCV) is a major cause of chronic liver disease, frequently progressing to cirrhosis and increased risk of hepatocellular carcinoma. Current therapies are inadequate and progress in the field has been hampered by the lack of efficient HCV culture systems. By using a recently described HCV genotype 2a infectious clone that replicates and produces infectious virus in cell culture (HCVcc), we report here that HCVcc strain FL-J6/JFH can establish long-term infections in chimpanzees and in mice containing human liver grafts. Importantly, virus recovered from these animals was highly infectious in cell culture, demonstrating efficient ex vivo culture of HCV. The improved infectivity of animal-derived HCV correlated with virions of a lower average buoyant density than HCVcc, suggesting that physical association with low-density factors influences viral infectivity. These results greatly extend the utility of the HCVcc genetic system to allow the complete in vitro and in vivo dissection of the HCV life cycle.  

5.349           Insertional mutagenesis at position 520 and 584 of adeno-associated virus type 2 (AAV2) capsid gene and generation of AAV2 vectors with eliminated heparin-binding ability and introduced novel tropism

Shi, X., Fang, G. And Shi, W. Human. Gen. Ther., 17, 353-361 (2006)   Recombinant adeno-associated virus (AAV) vectors are promising in the context of gene therapy because of their ability to mediate efficient gene transfer and stable gene expression. AAV2 uses heparin sulfate as its primary receptor, which is widely expressed on the various tissues and organs. This limits the application of AAV2 in targeting specific tissues. To make an AAV2 vector with modified tropism, we constructed various AAV2 capsid mutants by inserting RGD-4C peptide at position 520 and/or at position 584. Eight mutants were generated, identified, and characterized. Heparin-binding ability was completely abrogated in five mutants, and partially reduced in three mutants. Solid-phase ELISA and gene transduction assays confirmed that the novel tropism is determined by the introduced RGD epitope, which binds to cellular integrin receptor. Our observations suggest that simultaneous modification at both sites, tentatively involved in heparin binding, results in altered tropism and improved transduction efficiency in vitro.  

5.350           Improved cardiac gene transfer by transcriptional and transductional targeting of adeno-associated viral vectors

Müller, O.J. et al Cardiovasc. Res., 70, 70-78 (2006)   Objective Vectors based on recombinant adeno-associated virus 2 (AAV-2) are a promising tool for cardiac gene transfer. However, potential therapeutic applications need to consider the predominant transduction of the liver once AAV-2 vectors enter the systemic circulation. We therefore aimed to increase efficiency and specificity of cardiac vector delivery by combining transcriptional and cell surface targeting. Methods For analysis of transcriptional targeting, recombinant AAV vectors were generated harboring a luciferase reporter gene under control of the cytomegalovirus (CMV) promoter or the 1.5-kb cardiac myosin light chain promoter fused to the CMV immediate-early enhancer (CMV_enh/MLC1.5). Luciferase activities were determined in representative organs three weeks after intravenous injection of the vector into adult mice. Transductional targeting was studied using luciferase-reporter constructs crosspackaged into capsids of AAV serotypes 1 to 6 and modified AAV-2 capsids devoid of binding their primary receptor heparan sulfate proteoglycan. Results Intravenous injections of AAV-2 vectors harboring the CMV_enh/MLC1.5 promoter enabled a specific and 50-fold higher reporter gene expression in left ventricular myocardium of adult mice compared to vectors containing the CMV promoter. Comparison of AAV-2 vector genomes crosspackaged into capsids of AAV-1 to -6 showed that AAV-1, -4, -5, and -6 capsids increased cardiac transduction efficiency by about 10-fold. However, transduction of other organs such as the liver was also increased after systemic administration. In contrast, AAV-2-based vectors with ablated binding to their primary receptor heparan sulfate proteoglycan enabled a significantly increased efficiency of cardiac gene transfer and reduced transduction of the liver. Conclusions Combining transcriptional targeting by the CMV_enh/MLC1.5 promoter and AAV vectors devoid of binding the AAV-2 primary receptor results in an efficient cardiac gene transfer with a significantly reduced hepatic transduction.  

5.351           7SL RNA, but not the 54-kd signal recognition particle protein, is an abundant component of both infectious HIV-1 and minimal virus-like particles

Onafuwa-Nuga, A.A., Telesnitsky, A. and King, S.R. RNA, 12, 542-546 (2006)   The virion incorporation of 7SL, the RNA component of the hostsignal recognition particle (SRP), has been shown for severalsimple retroviruses. Data here demonstrate that 7SL is alsopackaged by HIV-1, in sevenfold molar excess of genomic RNA.Viral determinants of HIV-1 genome and primer tRNA packagingwere not required for 7SL incorporation, as virus-like particleswith only minimal assembly components efficiently packaged 7SL.The majority of 7SL within cells resides in ribonucleoproteincomplexes bound by SRP proteins, and most SRP protein existsin signal recognition particles. However, Western blot comparisonof virion and cell samples revealed that there is at least 25-foldless SRP p54 protein per 7SL RNA in HIV-1 particles than incells. Comparing 7SL:actin mRNA ratios in virions and cellsrevealed that 7SL RNA appears selectively enriched in virions.            

5.352           Vascular bed-targeted in vivo gene delivery using tropism-modified adeno-associated viruses

Work, L.M. et al Mol. Ther., 13(4), 683-693 (2006)   Virus-mediated gene delivery is restricted by the infectivity profile of the chosen vector. Targeting the vascular endothelium via systemic delivery has been attempted using peptides isolated in vitro (using either phage or vector display) and implicit reliance on target receptor expression in vivo. This has limited application since endothelial cells in vitro and in vivo differ vastly in receptor profiles and because of the existence of complex endothelial “zip codes” in vivo. We therefore tested whether in vivo phage display combined with adeno-associated virus (AAV) capsid modifications would allow in vivo homing to the endothelium residing in defined organs. Extensive in vivo biopanning in rats identified four consensus peptides homing to the lung or brain. Each was incorporated into the VP3 region of the AAV-2 capsid to display the peptide at the virion surface. Peptides that conferred heparan independence were shown to retarget virus to the expected vascular bed in vivo in a preferential manner, determined 28 days post-systemic injection by both virion DNA and transgene expression profiling. Our findings significantly impact the design of viral vectors for targeting individual vascular beds in vivo.  

5.353           Downstream processing of oncoretroviral and lentiviral gene therapy vectors

De la Mercedes Segura, M., Kamen, A. and Garnier, A. Biotech. Advances, 24, 321-337 (2006)   Retroviral vectors from both oncoretroviral and lentiviral origins have a great potential as gene delivery vehicles. A number of research groups have devoted considerable effort to the development of large-scale production strategies for retroviral vectors. However, the manufacturing of clinical-grade vectors for gene therapy, especially for in vivo applications, additionally requires scaleable purification strategies to remove the contaminants present in the harvested supernatants while preserving the functionality of the vectors. In this article, we review recent advances made in the field of downstream processing of retroviral vectors. The methods currently described in the literature for clarification, concentration and purification of retroviral vectors will be presented, with special emphasis on novel chromatography methods that open up the possibility to selectively and efficiently purify retroviruses on a large-scale. Problems associated with stability and quantification of retroviral particles will be outlined and future challenges will be discussed.  

5.354           Activators of viral gene expression in polarized epithelial monolayers identified by rapid-throughput drug screening

Sorscher, E.J. et al Gene Ther., 13, 781-788 (2006)   Epithelial polarity and tight junction formation limit the ability of adenovirus, retrovirus and adeno-associated virus (AAV) to deliver and express virally encoded genes. Using an extended half-life luciferase assay and high-throughput luminometry, we screened 23 000 compounds and natural product extracts as potentiators to overcome this barrier. Seven strong activators were discovered (up to several hundred fold above control) and two of these exhibited spectrum of activity in multiple cell types (HeLa (human cervical carcinoma), cystic fibrosis bronchial epithelial (human bronchial), HT29 (human colonic carcinoma), Calu3 (airway serous glandular)). Enhanced transduction by unrelated gene transfer vectors (adenovirus, lentivirus, AAV, liposomal) was also observed. These results establish a strategy for identifying compounds that improve viral gene transfer to resistant cell types, and provide new tools for examining epithelial defense against viral infection. The compounds should have broad usefulness in experimental therapies for cancer and genetic diseases.  

5.355           Cholesterol-induced caveolin targeting to lipid droplets in adipocytes: a role for caveolar endocytosis

Le Lay, S. et al Traffic, 7, 549-561 (2006)   We have investigated the targeting of caveolin to lipid bodies in adipocytes that express high levels of caveolins and contain well-developed lipid droplets. We observed that the lipid droplets isolated from adipocytes of caveolin-1 knock out mice contained dramatically reduced levels of cholesterol, indicating that caveolin is required for maintaining the cholesterol content of this organelle. Analysis of caveolin distribution by cell fractionation and fluorescent light microscopy in 3T3-L1 adipocytes indicated that addition of cholesterol rapidly stimulated translocation of caveolin to lipid droplets. The cholesterol-induced trafficking of caveolins to lipid droplets was shown to be dynamin- and protein kinase C (PKC)-dependent and modulated by src tyrosine kinase activation, suggesting a role for caveolar endocytosis in this novel trafficking pathway. Consistent with this, caveolae budding was stimulated by cholesterol addition. The present data identify lipid droplets as potential target organelles for caveolar endocytosis and demonstrate a role for caveolin-1 in the maintenance of free cholesterol levels in adipocyte lipid droplets.  

5.356           Purification and characterization of retrovirus vector particles by rate zonal ultracentrifugation

De la Mercedes Segura, M., Garnier, A. and Kamen, A.

1. Virol. Methods., 133, 82-91 (2006)

  Sucrose equilibrium density ultracentrifugation remains the most widely used technique for retrovirus purification. However, purified virus preparations obtained by this routine method usually contain considerable amounts of contaminating cell membrane vesicles. In addition, sucrose solutions are highly viscous and hyperosmotic which jeopardizes the integrity and functionality of the retrovirus particle. In order to overcome these limitations, an alternative purification technique using rate zonal ultracentrifugation and iodixanol as gradient medium was developed. Recombinant retrovirus particles were produced by 293-GPG packaging cells grown in suspension in the presence of 10% FBS. Concentrated supernatants were purified by rate zonal sedimentation on a 10–30% continuous iodixanol gradient. Virus particles were recovered intact and active from the central fractions of the gradient. By using this strategy, high levels of purification were achieved, with no evident contamination with cell membrane vesicles as indicated by subtilisin treatment studies. The level of purity of the retrovirus preparation is over 95% as shown by SDS-PAGE analysis and size-exclusion chromatography. Purified particles appear homogenous in size and morphology according to negative stain electron microscopy. In addition, large amounts of defective retrovirus particles produced by 293-GPG packaging cells can be separated from functional retrovirus particles using this purification strategy.  

5.357           Gene transfer into rat mesenchymal stem cells: a comparative study of viral and nonviral vectors

McMahon, J.M. et al Stem Cells and Develop., 15, 87-96 (2006)   Mesenchymal stem cells (MSCs) have been proposed for use in combinatorial gene and cell therapy protocols for the treatment of disease and promotion of repair. The efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression with minimal cell death. The study was carried out on bone-marrow derived rat MSCs, and a range of vectors was tested on the same stem cell preparation. Adenovirus, adeno-associated virus (AAV; serotypes 1, 2, 4, 5, and 6), lentivirus, and nonviral vectors were compared. Lentivirus proved to be most effective with transduction efficiencies of up to 95%, concurrent with low levels of cell toxicity. Adenovirus also proved effective, but a significant increase in cell death was seen with increasing viral titer. Rat MSCs remained refractory to transduction by all AAV serotypes, in contrast to rabbit MSCs tested at the same time. Lipofection of plasmid DNA gave moderate transfection levels but was also accompanied by cell death. Electroporative gene transfer proved ineffective at the parameters tested and resulted in high cell death. High and moderate levels of cell transduction using lentivirus vectors did not affect the ability of the cells to differentiate down the adipogenic pathway.  

5.358           Direct gene therapy for repair of the spinal cord

Blits, B. and Bartlett Bunge, M.

1. Neurotrauma, 23(3/4), 508-520 (2006)

  For regrowth of injured nerve fibers following spinal cord injury (SCI), the environment must be favorable for axonal growth. The delivery of a therapeutic gene, beneficial for axonal growth, into the central nervous system for repair can be accomplished in many ways. Perhaps the most simple and elegant strategy is the so-called direct gene therapy approach that uses a single injection for delivery of a gene therapy vehicle. Among the vectors that have been used to transduce neural tissue in vivo are non-viral, herpes simplex viral, adeno-associated viral, adenoviral, and lentiviral vectors, each with their own merits and limitations. Many studies have been undertaken using direct gene therapy, ranging from strategies for neuroprotection to axonal growth promotion at the injury site, dorsal root injury repair, and initiation of a growth-supporting genetic program. The limitations and successes of direct gene transfer for spinal cord repair are discussed in this review.      

5.359           Identification of human papillomavirus type 16 L1 surface loops required for neutralization by human sera

Carter, J.J. et al

1. Virol., 80(10), 4664-4672 (2006)

  The variable surface loops on human papillomavirus (HPV) virions required for type-specific neutralization by human sera remain poorly defined. To determine which loops are required for neutralization, a series of hybrid virus-like particles (VLPs) were used to adsorb neutralizing activity from HPV type 16 (HPV16)-reactive human sera before being tested in an HPV16 pseudovirion neutralization assay. The hybrid VLPs used were composed of L1 sequences of either HPV16 or HPV31, on which one or two regions were replaced with homologous sequences from the other type. The regions chosen for substitution were the five known loops that form surface epitopes recognized by monoclonal antibodies and two additional variable regions between residues 400 and 450. Pretreatment of human sera, previously found to react to HPV16 VLPs in enzyme-linked immunosorbent assays, with wild-type HPV16 VLPs and hybrid VLPs that retained the neutralizing epitopes reduced or eliminated the ability of sera to inhibit pseudovirus infection in vitro. Surprisingly, substitution of a single loop often ablated the ability of VLPs to adsorb neutralizing antibodies from human sera. However, for all sera tested, multiple surface loops were found to be important for neutralizing activity. Three regions, defined by loops DE, FG, and HI, were most frequently identified as being essential for binding by neutralizing antibodies. These observations are consistent with the existence of multiple neutralizing epitopes on the HPV virion surface.  

5.360           Parkin is protective for substantia nigra dopamine neurons in a tau gene transfer neurodegeneration model

Klein, R.L., Dayton, R.D., Henderson, K.M. and Petrucelli, L. Neurosci. Lett., 401(1-2), 130-135 (2006)   Parkin is a ubiquitin ligase involved in the ubiquitin-proteasome system. Elevating parkin expression in cells reduces markers of oxidative stress while blocking parkin expression increases oxidative stress. In parkin gene knock down mouse and fly models, mitochondria function is deficient. Parkin is neuroprotective against a variety of toxic insults, while it remains unclear which of the above properties of parkin may mediate the protective actions. One of the models for which parkin is protective is overexpression of alpha-synuclein, a protein that self-aggregates in Parkinson disease. The microtubule-associated protein tau is another protein that self-aggregates in specific neurodegenerative diseases that also involve loss of dopamine neurons such as frontotemporal dementia with parkinsonism linked to chromosome 17, progressive supranuclear palsy and corticobasal degeneration. We recently developed a tau-induced dopaminergic degeneration model in rats using adeno-associated virus vectors. In this study, we successfully targeted either a mixed tau/parkin vector or mixed tau/control vector to the rat substantia nigra. While there was significant loss of dopamine neurons in the tau/control group relative to uninjected substantia nigra, there was no cell loss in the tau/parkin group. We found no difference in total tau levels between tau/control and tau/parkin groups. Parkin therefore protects dopamine neurons against tau as it does against alpha-synuclein, which further supports parkin as a therapeutic target for diseases involving loss of dopamine neurons.  

5.361           Separate Basic Region Motifs within the Adeno-Associated Virus Capsid Proteins Are Essential for Infectivity and Assembly

Grieger, J.C., Snowdy, S. and Samulski, R.J.

1. Virol., 80(11), 5190-5210 (2006)

  Adeno-associated virus (AAV) is gaining momentum as a gene therapy vector for human applications. However, there remain impediments to the development of this virus as a vector. One of these is the incomplete understanding of the biology of the virus, including nuclear targeting of the incoming virion during initial infection, as well as assembly of progeny virions from structural components in the nucleus. Toward this end, we have identified four basic regions (BR) on the AAV2 capsid that represent possible nuclear localization sequence (NLS) motifs. Mutagenesis of BR1 (¹²⁰QAKKRVL¹²⁶) and BR2 (¹⁴⁰PGKKRPV¹⁴⁶) had minor effects on viral infectivity ( 4- and 10-fold, respectively), whereas BR3 (¹⁶⁶PARKRLN¹⁷²) and BR4 (³⁰⁷RPKRLN³¹²) were found to be essential for infectivity and virion assembly, respectively. Mutagenesis of BR3, which is located in Vp1 and Vp2 capsid proteins, does not interfere with viral production or trafficking of intact AAV capsids to the nuclear periphery but does inhibit transfer of encapsidated DNA into the nucleus. Substitution of the canine parvovirus NLS rescued the BR3 mutant to wild-type (wt) levels, supporting the role of an AAV NLS motif. In addition, rAAV2 containing a mutant form of BR3 in Vp1 and a wt BR3 in Vp2 was found to be infectious, suggesting that the function of BR3 is redundant between Vp1 and Vp2 and that Vp2 may play a role in infectivity. Mutagenesis of BR4 was found to inhibit virion assembly in the nucleus of transfected cells. This affect was not completely due to the inefficient nuclear import of capsid subunits based on Western blot analysis. In fact, aberrant capsid foci were observed in the cytoplasm of transfected cells, compared to the wild type, suggesting a defect in early viral assembly or trafficking. Using three-dimensional structural analysis, the lysine- and arginine-to-asparagine change disrupts hydrogen bonding between these basic residues and adjacent beta strand glutamine residues that may prevent assembly of intact virions. Taken together, these data support that the BR4 domain is essential for virion assembly. Each BR was also found to be conserved in serotypes 1 to 11, suggesting that these regions are significant and function similarly in each serotype. This study establishes the importance of two BR motifs on the AAV2 capsid that are essential for infectivity and virion assembly.  

5.362           In vivo complementation of complex I by the yeast Ndi enzyme

Boo Seo, B., Nakamura-Ogiso, E., Flotte, T.R., Matsuno-Yagi, A. and Yagi, T.

1. Biol. Chem.,281(20), 14250-14255 (2006)

  Recent studies suggest that dysfunction of the NADH-quinone oxidoreductase (complex I) is associated with a number of human diseases, including neurodegenerative disorders such as Parkinson disease. We have shown previously that the single subunit rotenone-insensitive NADH-quinone oxidoreductase (Ndi1) of Saccharomyces cerevisiae mitochondria can restore NADH oxidation in complex I-deficient mammalian cells. The Ndi1 enzyme is insensitive to complex I inhibitors such as rotenone and 1-methyl-4-phenylpyridinium ion, known as a metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To test the possible use of the NDI1 gene as a therapeutic agent in vivo, we chose a mouse model of Parkinson disease. The NDI1-recombinant adeno-associated virus particles (rAAV-NDI1)were injected unilaterally into the substantia nigra of mice.The animals were then subjected to treatment with MPTP. Thedegree of neurodegeneration in the nigrostriatal system wasassessed immunohistochemically through the analysis of tyrosinehydroxylase and glial fibrillary acidic protein. It was evidentthat the substantia nigra neurons on the side used for injectionof rAAV-NDI1 retained a high level of tyrosine hydroxylase-positivecells, and the ipsilateral striatum exhibited significantlyless denervation than the contralateral striatum. Furthermore,striatal concentrations of dopamine and its metabolites in thehemisphere that received rAAV-NDI1 were substantially higherthan those of the untreated hemisphere, reaching more than 50%of the normal levels. These results indicate that the expressedNdi1 protein elicits resistance to MPTP-induced neuronal injury.The present study is the first successful demonstration of complementationof complex I by the Ndi1 enzyme in animals.  

5.363           Correction of Feline lipoprotein lipase deficiency with adeno-associated virus serotype 1-mediated gene transfer of the lipoprotein lipase S447X beneficial mutation

Ross, C.J.D. et al Human Gen. Ther., 17, 487-499 (2006)   Human lipoprotein lipase (hLPL) deficiency, for which there currently exists no adequate treatment, leads to excessive plasma triglycerides (TGs), recurrent abdominal pain, and life-threatening pancreatitis. We have shown that a single intramuscular administration of adeno-associated virus (AAV) serotype 1 vector, encoding the human LPL^S447X variant, results in complete, long-term normalization of dyslipidemia in LPL ^/ mice. As a prelude to gene therapy for human LPL deficiency, we tested the efficacy of AAV1-LPL^S447X in LPL ^/ cats, which demonstrate hypertriglyceridemia (plasma TGs, >10,000 mg/dl) and clinical symptoms similar to LPL deficiency in humans, including pancreatitis. Male LPL ^/ cats were injected intramuscularly with saline or AAV1-LPL^S447X (1 × 10¹¹–1.7 × 10¹² genome copies [GC]/kg), combined with oral doses of cyclophosphamide (0–200 mg/m² per week) to inhibit an immune response against hLPL. Within 3–7 days after administration of ≥5 × 1011 GC of AAV1-LPL^S447X per kilogram, the visible plasma lipemia was completely resolved and plasma TG levels were reduced by >99% to normal levels (10–20 mg/dl); intermediate efficacy (95% reduction) was achieved with 1 × 10¹¹ GC/kg. Injection in two sites, greatly limiting the amount of transduced muscle, was sufficient to completely correct the dyslipidemia. By varying the dose per site, linear LPL expression was demonstrated over a wide range of local doses (4 × 10¹⁰–1 × 10¹² GC/site). However, efficacy was transient, because of an anti-hLPL immune response blunting LPL expression. The level and duration of efficacy were significantly improved with cyclophosphamide immunosuppression. We conclude that AAV1-mediated delivery of LPL^S447X in muscle is an effective means to correct the hypertriglyceridemia associated with feline LPL deficiency.  

5.364           Mosaic vectors comprised of modified AAV1 capsid proteins for efficient vector purification and targeting to vascular endothelial cells

Stachler, M.D. and Bartlett, J.S. Gen. Ther., 13, 926-931 (2006)   Vascular-targeted gene therapies have the potential to treat many of the leading causes of mortality in the western world. Unfortunately, these therapies have been ineffective due to poor vascular gene transfer. The use of alternative virus serotypes and the incorporation of vascular targeting ligands into vectors has resulted in only modest increases in vascular gene transfer. Adeno-associated virus (AAV) 1 has shown the most promise among the AAV vectors for the transduction of vascular endothelial cells. However, no straightforward small-scale purification strategy exists for AAV1 as it does for AAV2 making it difficult to quickly produce AAV1 vector for analysis. Here we have combined two AAV1 capsid protein modifications to enhance vascular gene transfer and allow easy purification of vector particles. Mosaic vector particles have been produced comprised of capsid proteins containing the well-characterized RGD4C modification to target integrins present on the vasculature, and capsid proteins containing a modification that permits metabolic biotinylation and efficient purification of mosaic particles by avidin affinity chromatography. We show that the RGD modification results in a 50–100-fold enhancement in endothelial cell gene transfer that is maintained in biotinylated mosaic AAV1 particles. These results suggest that mosaic virions hold significant promise for targeted gene delivery to the vasculature.  

5.365           Phosphorylation of the HTLV-1 matrix L-domain-containing protein by virus-associated ERK-2 kinase

Hemonnot, B. et al Virology, 349(2), 430-439 (2006)   L-domain-containing proteins from animal retroviruses play a critical role in the recruitment of the host cell endocytic machinery that is required for retroviruses budding. We recently demonstrated that phosphorylation of the p6^gag protein containing the L-domain of the human immunodeficiency virus type 1 regulates viral assembly and budding. Here, we investigated whether or not the L-domain-containing protein from another human retrovirus, namely the matrix protein of the human T-cell leukemia virus type 1, that contains the canonical PTAP and PPPY L-domain motifs, shares similar functional properties. We found that MA is phosphorylated at several sites. We identified one phosphorylated amino acid in the HTLV-1 MA protein as being S105, located in the close vicinity to the L-domain sequence. S105 phosphorylation was found to be mediated by the cellular kinase ERK-2 that is incorporated within HTLV-1 virus particles in an active form. Mutation of the ERK-2 target S105 residue into an alanine was found to decrease viral release and budding efficiency of the HTLV-1_ACH molecular clone from transfected cells. Our data thus support the postulate that phosphorylation of retroviral L-domain proteins is a common feature to retroviruses that participates in the regulation of viral budding.  

5.366           Safety of recombinant adeno-associated virus type-2 RPE65 vector delivered by ocular subretinal injection

Jacobson, S.G. et al Mol. Ther., 13(6), 1074-1084 (2006)   AAV2 delivery of the RPE65 gene to the retina of blind RPE65-deficient animals restores vision. This strategy is being considered for human trials in RPE65-associated Leber congenital amaurosis (LCA), but toxicity and dose efficacy have not been defined. We studied ocular delivery of AAV-2/2.RPE65 in RPE65-mutant dogs. There was no systemic toxicity. Ocular examinations showed mild or moderate inflammation that resolved over 3 months. Retinal histopathology indicated that traumatic lesions from the injection were common, but thinning within the injection region occurred only at the two highest vector doses. Biodistribution studies at 3 months postinjection showed no vector in optic nerve or visual centers in the brain and only isolated non-dose-related detection in other organs. We also performed biodistribution studies in normal rats at about 2 weeks and 2 months postinjection and vector was not widespread outside the injected eye. Dose–response results in RPE65-mutant dogs indicated that the highest 1.5-log unit range of vector doses proved efficacious. The efficacy and toxicity limits defined in this study lead to suggestions for the design of a subretinal AAV-2/2.RPE65 human trial of RPE65-associated LCA.  

5.367           Adeno-associated virus vector serotypes mediate sustained correction of bilirubin UDP glucuronosyltransferase deficiency in rats

Seppen, J. et al Mol. Ther., 13(6), 1085-1092 (2006)   Crigler–Najjar (CN) patients have no bilirubin UDP glucuronosyltransferase (UGT1A1) activity and suffer brain damage because of bilirubin toxicity. Vectors based on adeno-associated virus (AAV) serotype 2 transduce liver cells with relatively low efficiency. Recently, AAV serotypes 1, 6, and 8 have been shown to be more efficient for liver cell transduction. We compared AAV serotypes 1, 2, 6, and 8 for correction of UGT1A1 deficiency in the Gunn rat model of CN disease. Adult Gunn rats were injected with CMV-UGT1A1 AAV vectors. Serum bilirubin was decreased over the first year by 64% for AAV1, 16% for AAV2, 25% for AAV6, and 35% for AAV8. Antibodies to UGT1A1 were detected after injection of all AAV serotypes. An AAV1 UGT1A1 vector with the liver-specific albumin promoter corrected serum bilirubin levels but did not induce UGT1A1 antibodies. Two years after injection of AAV vectors all animals had large lipid deposits in the liver. These lipid deposits were not seen in age-matched control animals. AAV1 vectors are promising candidates for CN gene therapy because they can mediate a reduction in serum bilirubin levels in Gunn rats that would be therapeutic in humans.  

5.368           A rapid and efficient method for purification of recombinant adenovirus with arginine–glycine–aspartic acid-modified fibers

Peng, H.H. et al Anal. Biochem., 354(1), 140-147 (2006)   Recombinant adenoviral vectors (adenovectors) have been subject to various genetic modifications to improve their transduction efficiency and targeting capacity. Production and purification of adenovectors with modified capsid proteins can be problematic using conventional two-cycle CsCl gradient ultracentrifugation. We have developed a new method for purifying recombinant adenovectors in two steps: iodixanol discontinuous density gradient ultracentrifugation and size exclusion column chromatography. The purity and infectious activity of adenovectors isolated by the two methods were comparable. The new method yielded three to four times more adenovectors with arginine–glycine–aspartic acid (RGD)-modified fiber proteins than did the conventional CsCl method. For other fiber-modified and wild-type adenovectors, the yields of the two methods were comparable. Thus, the iodixanol-based method can be used not only to improve the production of RGD-modified adenovectors but also to purify adenovectors with or without fiber modifications. Moreover, the whole procedure can be completed in 3 h. Therefore, this method is rapid and efficient for production of recombination adenovectors, especially those with RGD-modified fibers.  

5.369           Common protective and diverse smooth muscle cell effects of AAV-mediated angiopoietin-1 and -2 expression in rat cardiac allograft vasculopathy

Nykänen, A.I. et al Circ.Res., 98, 1373-1380 (2006)   Angiopoietin-1 (Ang1) and Ang2 regulate the maintenance of normal vasculature by direct endothelial and indirect smooth muscle cell (SMC) effects. Dysfunction of vascular wall cells is considered central in cardiac allograft vasculopathy (CAV), where inflammation and arterial injury initiate subsequent intimal SMC proliferation. In this study, we investigated the effect of exogenous Ang1 and Ang2 in chronically rejecting rat cardiac allografts by intracoronary adeno-associated virus (AAV)-mediated gene transfer. Bioluminescent imaging of AAV-transfected syngeneic grafts revealed gradual and stable transgene expression in graft cardiomyocytes. In cardiac allografts, both AAV-Ang1 and AAV-Ang2 decreased inflammation and increased antiapoptotic Bcl-2 mRNA and Bcl-2/Bax ratio at 8 weeks. Only AAV-Ang2 decreased the development of CAV, whereas AAV-Ang1 activated arterial SMC and increased PDGF-A mRNA in the allograft. Collectively, our results show that exogenous Ang1 and Ang2 have similar antiinflammatory and antiapoptotic effects in cardiac allografts. Prolonged AAV-mediated Ang1 transgene expression also induced SMC activation, whereas AAV-Ang2 lacked the SMC activating effects and decreased CAV. Our results thus highlight the common protective and diverse SMC effects of Ang1 and Ang2 in cardiac allograft microenvironment and the importance of timing of angiopoietins to achieve therapeutic effects.      

5.370           Identification of a Dynein Interacting Domain in the Papillomavirus Minor Capsid Protein L2

Florin, L. et al

1. Virol., 80(13), 6691-6696 (2006)

  Papillomaviruses enter cells via endocytosis (H. C. Selinka et al., Virology 299:279-287, 2002). After egress from endosomes, the minor capsid protein L2 accompanies the viral DNA to the nucleus and subsequently to the subnuclear promyelocytic leukemia protein bodies (P. M. Day et al., Proc. Natl. Acad. Sci. USA 101:14252-14257, 2004), suggesting that this protein may be involved in the intracytoplasmic transport of the viral genome. We now demonstrate that the L2 protein is able to interact with the microtubule network via the motor protein dynein. L2 protein was found attached to microtubules after uncoating of incoming human papillomavirus pseudovirions. Based on immunofluorescence and coimmunoprecipitation analyses, the L2 region interacting with dynein is mapped to the C-terminal 40 amino acids. Mutations within this region abrogating the L2/dynein interaction strongly reduce the infectivity of pseudoviruses, indicating that this interaction mediates the minus-end-directed transport of the viral genome along microtubules towards the nucleus.  

5.371           Adeno-associated Virus-Mediated Expression and Constitutive Secretion of Galanin Suppresses Limbic Seizure Activity in Vivo

McCown, T.J. Mol. Ther., 14(1), 63-68 (2006)   Intractable temporal lobe epilepsy presents an ideal target for gene therapy, but therapeutic success depends upon the ability to suppress limbic seizure activity. Adeno-associated virus vectors (AAV) were constructed in which the fibronectin secretory signal sequence (FIB) preceded the coding sequence for galanin (AAV-FIB-GAL) or green fluorescent protein (AAV-FIB-GFP), constructs that express and constitutively secrete the gene product. Bilateral AAV-FIB-GAL infusion into the rat piriform cortex (2 μl/side) significantly attenuated kainic acid-induced seizures (10 mg/kg, ip) such that 11/12 rats exhibited no limbic seizures, while the remaining rat exhibited only a brief, single class III seizure. This AAV-FIB-GAL infusion also prevented electrographic seizure activity. In contrast, bilateral AAV-FIB-GFP infusion did not alter either behavioral or electrographic seizure activity. Since prior seizure exposure could influence vector efficacy, another group of rats received daily electrical stimulation of the piriform cortex until three consecutive class V seizures were elicited. Subsequently, AAV-FIB-GAL or AAV-FIB-GFP (3 μl/30 min) was infused into the area of the electrode. One week later the AAV-FIB-GAL rats exhibited a significant increase in the stimulation current necessary to evoke limbic seizure activity, while AAV-FIB-GFP did not alter the seizure threshold. Thus, AAV-mediated galanin expression and secretion significantly suppress limbic seizure activity in vivo.  

5.372           Metabolic Biotinylation Provides a Unique Platform for the Purification and Targeting of Multiple AAV Vector Serotypes

Arnold, G.S., Sasser, A.K., Stachler, M.D. and Bartlett, J.S. Mol. Ther., 14(1), 97-106 (2006)   The development of rationally designed targeted gene delivery vectors is an important focus for gene therapy. While genetic modification of AAV can produce vectors with modified tropism, incorporation of targeting peptides into the structural context of the AAV virion often results in loss of function or loss of virion integrity. To address this issue, we have developed a targeting system using metabolically biotinylated AAV. We generated serotype 1, 2, 3, 4, and 5 AAV capsids with small peptide insertions that are metabolically biotinylated in packaging cells during vector production by coexpression of the Escherichia coli BirA, biotin ligase, gene. Biotin moieties are exposed on the surface of assembled AAV particles and can interact with avidin. Metabolically biotinylated AAV vectors produced in this manner maintained endogenous titer and tissue tropism, could be purified on monomeric avidin resin, and could be retargeted to cells engineered to express an artificial avidin–biotin receptor. This technology provides not only a single platform for the purification of multiple AAV vector serotypes, but also a means for the development of multiple targeted AAV vectors utilizing a single capsid modification via straightforward avidin–biotin ligand coupling.          

5.373           α 1-Antitrypsin Gene Therapy Modulates Cellular Immunity and Efficiently Prevents Type 1 Diabetes in Nonobese Diabetic Mice

Lu, Y. et al Human Gen. Ther., 17, 625-634 (2006)   An imbalance of the immune-regulatory pathways plays an important role in the development of type 1 diabetes. Therefore, immunoregulatory and antiinflammatory strategies hold great potential for the prevention of this autoimmune disease. Studies have demonstrated that two serine proteinase inhibitors, α ₁-antitrypsin (AAT) and elafin, act as potent antiinflammatory agents. In the present study, we sought to develop an efficient gene therapy approach to prevent type 1 diabetes. Cohorts of 4-week-old female nonobese diabetic (NOD) mice were injected intramuscularly with rAAV1-CB-hAAT, rAAV1-CB-hElafin, or saline. AAV1 vector mediated sustained high levels of transgene expression, sufficient to overcome a humoral immune response against hAAT. AAT gene therapy, contrary to elafin and saline, was remarkably effective in preventing type 1 diabetes. T cell receptor spectratyping indicated that AAT gene therapy altered T cell repertoire diversity in splenocytes from NOD mice. Adoptive transfer experiments demonstrated that AAT gene therapy attenuated cellular immunity associated with beta cell destruction. This study demonstrates that AAT gene therapy attenuates cell-mediated autoimmunity, alters the T cell receptor repertoire, and efficiently prevents type 1 diabetes in the NOD mouse model. These results strongly suggest that rAAV1-mediated AAT gene therapy may be useful as a novel approach to prevent type 1 diabetes.  

5.374           Retrovirus infection strongly enhances scrapie infectivity release in cell culture

Leblanc, P. et al EMBO J., 25, 2674-2685 (2006)   Prion diseases are neurodegenerative disorders associated in most cases with the accumulation in the central nervous system of PrP^Sc (conformationally altered isoform of cellular prion protein (PrP^C); Sc for scrapie), a partially protease-resistant isoform of the PrP^C. PrP^Sc is thought to be the causative agent of transmissible spongiform encephalopathies. The mechanisms involved in the intercellular transfer of PrP^Sc are still enigmatic. Recently, small cellular vesicles of endosomal origin called exosomes have been proposed to contribute to the spread of prions in cell culture models. Retroviruses such as murine leukemia virus (MuLV) or human immunodeficiency virus type 1 (HIV-1) have been shown to assemble and bud into detergent-resistant microdomains and into intracellular compartments such as late endosomes/multivesicular bodies. Here we report that moloney murine leukemia virus (MoMuLV) infection strongly enhances the release of scrapie infectivity in the supernatant of coinfected cells. Under these conditions, we found that PrP^C, PrP^Sc and scrapie infectivity are recruited by both MuLV virions and exosomes. We propose that retroviruses can be important cofactors involved in the spread of the pathological prion agent.  

5.375           Restoration of fatty aldehyde dehydrogenase deficiency in Sjögren–Larsson syndrome

Haug, S. and Braun-Falco, M. Gen. Ther., 13, 1021-1026 (2006)   Sjögren–Larsson syndrome (SLS) is an autosomal recessive neurocutaneous disorder caused by mutation in the ALDH3A2 gene that codes for human fatty aldehyde dehydrogenase (FALDH). Sjögren–Larsson syndrome patients lack FALDH, which catalyzes the oxidation of long-chain aliphatic aldehydes to fatty acids. The impaired FALDH activity leads to congenital ichthyosis, mental retardation and spasticity. The current lack of treatment is an impetus to develop gene therapy strategies by introducing functional FALDH into defective cells. We delivered human FALDH into keratinocytes of SLS patients using recombinant adeno-associated virus-2 vectors. Transduction of SLS keratinocytes resulted in an augmentation of FALDH activity comparable to phenotypically normal heterozygous carriers. Toxicity of long-chain aldehydes for FALDH-deficient cells decreased almost to the level of unaffected keratinocytes. Three-dimensional culture of corrected SLS keratinocytes revealed an ameliorated FALDH expression. These studies demonstrate the restoration of FALDH in human SLS cells supporting the concept of gene therapy as a potential future treatment option for SLS.          

5.376           Serial Passage through Human Glioma Xenografts Selects for a 134.5 Herpes Simplex Virus Type 1 Mutant That Exhibits Decreased Neurotoxicity and Prolongs Survival of Mice with Experimental Brain Tumors

Shah, A.C. et al

1. Virol., 80(15), 7308-7315 (2006)

  Previous studies have described in vitro serial passage of a ₁34.5 herpes simplex virus type 1 (HSV-1) strain in SK-N-SH neuroblastoma cells and selection of mutants that have acquired the ability to infect and replicate in this previously nonpermissive cell line. Here we describe the selection of a mutant HSV-1 strain by in vivo serial passage, which prolongs survival in two separate experimental murine brain tumor models. Two conditionally replication-competent ₁34.5 viruses, M002, which expresses murine interleukin-12, and its parent virus, R3659, were serially passaged within human malignant glioma D54-MG cell lines in vitro or flank tumor xenografts in vivo. The major findings are (i) viruses passaged in vivo demonstrate decreased neurovirulence, whereas those passaged in vitro demonstrate a partial recovery of the neurovirulence associated with HSV-1; and (ii) vvD54-M002, the virus selected after in vivo serial passage of M002 in D54-MG tumors, improves survival in two independent murine brain tumor models compared to the parent (unpassaged) M002. Additionally, in vitro-passaged, but not in vivo-passaged, M002 displayed changes in the protein synthesis profile in previously nonpermissive cell lines, as well as early U_S11 transcription. Thus, a mutant HSV-1 strain expressing a foreign gene can be selected for enhanced antitumor efficacy via in vivo serial passage within flank D54-MG tumor xenografts. The enhanced antitumor efficacy of vvD54-M002 is not due to restoration of protein synthesis or early U_S11 expression. This finding emphasizes the contribution of the in vivo tumor environment for selecting novel oncolytic HSV specifically adapted for tumor cell destruction in vivo.  

5.377           Homologous recombination is required for AAV-mediated gene targeting

Vasileva, A., Linden, R.M. and Jessberger, R. Nucleic Acid Res., 34(11), 3345-3360 (2006)   High frequencies of gene targeting can be achieved by infection of mammalian cells with recombinant adeno-associated virus (rAAV) vectors [D. W. Russell and R. K. Hirata (1998) Nature Genet., 18, 325–330; D. W. Russell and R. K. Hirata (2000) J. Virol., 74, 4612–4620; R. Hirata et al. (2002) Nat. Biotechnol., 20, 735–738], but the mechanism of targeting is unclearand random integration often occurs in parallel. We assessedthe role of specific DNA repair and recombination pathways inrAAV gene targeting by measuring correction of a mutated enhancedgreen fluorescent protein (EGFP) gene in cells where homologousrecombination (HR) or non-homologous end-joining (NHEJ) hadbeen suppressed by RNAi. EGFP-negative cells were transducedwith rAAV vectors carrying a different inactivating deletionin the EGFP, and in parallel with rAAV vectors carrying redfluorescent protein (RFP). Expression of RFP accounted for viraltransduction efficiency and long-term random integration. Approximately0.02% of the infected GFP-negative cells were stably convertedto GFP positive cells. Silencing of the essential NHEJ componentDNA-PK had no significant effect on the frequency of targetingat any time point examined. Silencing of the SNF2/SWI2 familymembers RAD54L or RAD54B, which are important for HR, reducedthe rate of stable rAAV gene targeting 5-fold. Further, partialsilencing of the Rad51 paralogue XRCC3 completely abolishedstable long-term EGFP expression. These results show that rAAVgene targeting requires the Rad51/Rad54 pathway of HR.  

5.378           Dominant-negative effect of hetero-oligomerization on the function of the human immunodeficiency virus type 1 envelope glycoprotein complex

Herrera, C. Et al Virology, 351, 121-132 (2006)   The human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein forms trimers that mediate interactions with the CD4 receptor and a co-receptor on the target cell surface, thereby triggering viral fusion with the cell membrane. Cleavage of Env into its surface, gp120, and transmembrane, gp41, moieties is necessary for activation of its fusogenicity. Here, we produced pseudoviruses with phenotypically mixed wild-type (Wt) and mutant, cleavage-incompetent Env in order to quantify the effects of incorporating uncleaved Env on virion infectivity, antigenicity and neutralization sensitivity. We modeled the relative infectivity of three such phenotypically mixed viral strains, JR-FL, HXBc2 and a derivative of the latter, 3.2P, as a function of the relative amount of Wt Env. The data were fit very closely (R² > 0.99) by models which assumed that only Wt homotrimers were functional, with different approximate thresholds of critical numbers of functional trimers per virion for the three strains. We also produced 3.2P pseudoviruses containing both a cleavage-competent Env that is defective for binding the neutralizing monoclonal antibody (NAb) 2G12, and a cleavage-incompetent Env that binds 2G12. The 2G12 NAb was not able to reduce the infectivity of these pseudoviruses detectably. Their neutralization by the CD4-binding site-directed agents CD4-IgG2 and NAb b12 was also unaffected by 2G12 binding to uncleaved Env. These results further strengthen the conclusion that only homotrimers consisting of cleaved Env are functional. They also imply that the function of a trimer is unaffected sterically by the binding of an antibody to an adjacent trimer.  

5.379           Enhanced humoral and memory B cellular immunity using HPV16/18 L1 VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only

Giannini, S.L. et al Vaccine, 24, 5937-5949 (2006)   An effective virus-like particle (VLP) based prophylactic vaccine designed to protect against persistent infection with human papillomavirus (HPV) types 16 and 18 and subsequent lesion development will need to induce a strong humoral and cellular immune response capable of providing long-term protection. Our objective was to evaluate the ability of an HPV16/18 L1 VLP vaccine formulated with the AS04 adjuvant system (3-O-desacyl-4′-monophosphoryl lipid A (MPL) and aluminium salt) to induce an immune response of higher magnitude and persistence compared to a vaccine formulated with aluminium salt only. We demonstrated that MPL adsorbed onto aluminium salt retains its capacity to activate an innate immune response as assessed by the production of TNFα by human monocytes (U937). In addition, vaccination of mice, monkeys or human subjects with AS04 formulations induced higher total anti-L1 VLP16 and L1 VLP18 antibody responses (1.6–8.5-fold) than the aluminium salt only formulations. The enhanced antibody response induced by the AS04 vaccine formulation (1.6–4.1-fold) in monkeys and humans was shown to be targeted to functional neutralising L1 VLP16 and L1 VLP18 epitopes as assessed by V5/J4 specific ELISAs or HPV16 and HPV18 pseudo-neutralization assays. The enhanced immune profile observed with the AS04 formulation in terms of both total, V5/J4 specific and neutralizing antibodies was shown to persist for at least 3.5-year post-vaccination in human subjects. Finally, using the newly developed B cell ELISPOT assay we also demonstrated that the AS04 formulation elicited an increased frequency (2.2–5.2-fold) of HPV L1 VLP specific memory B cells when compared with the aluminium salt only formulations. These data strongly support the role of the AS04 adjuvant, which includes the immunostimulant MPL, in triggering a persistent vaccine-induced immune response of high quality.  

5.380           CD8 T Cells Mediate Transient Herpes Stromal Keratitis in CD4-Deficient Mice

Lepisto, A.J., Frank, G.M., Xu, M., Stuart, P.M. and Hendricks, R.L. Invest. Ophthalmol. Vis. Sci., 47, 3400-3409 (2006)   PURPOSE. To evaluate the role of CD4⁺ T cells in the developmentof murine herpes stromal keratitis (HSK). METHODS. The corneas of wild-type (WT) BALB/c mice and three types of CD4-deficient BALB/c mice (CD4^–/–, CD4-depleted,CD4 and CD8 double-depleted) were infected with different dosesof HSV-1 RE, and HSK incidence and severity were monitored.Corneal infiltrates were quantitatively and functionally assayedby flow cytometric analysis of individually digested diseasedcorneas and documented histologically. RESULTS. At a relatively high infectious dose (1 x 10⁵ pfu/cornea): (1) CD4-deficient and WT BALB/c mice had severe HSK with a similar incidence (80%–100%), whereas HSK did not develop in mice deficient in both CD4⁺ and CD8⁺ T cells; (2) neutrophils were the predominate leukocyte in the corneas of CD4-deficient and WT mice; (3) the corneas of WT mice had activated, HSV-1-specific CD4⁺ T cells, but few if any CD8⁺ T cells; (4) the corneas of CD4-deficient mice had activated, HSV-1-specific CD8⁺ T cells; and (5) HSK in CD4-deficient mice was transient, showing loss of CD8⁺ T cells at 2 to 3 weeks after infection (pi) followed by a loss of neutrophils. At a relatively low infectious dose of HSV-1 (10³ pfu/cornea) severe HSK developed in 80% to 90%of WT mice, but in only 30% to 40% of CD4-deficient mice. CONCLUSIONS. CD4⁺ T cells preferentially mediate HSK, but, in their absence, a high infectious dose of HSV-1 can induce histologically similar but transient HSK that is mediated by CD8⁺ T cells.            

5.381           Oncolytic murine autonomous parvovirus, a candidate vector for glioma gene therapy, is innocuous to normal and immunocompetent mouse glial cells

Abschuetz, A. et al Cell Tissue Res., 325, 423-436 (2006)   The sensitivity of brain tumour cells to wild-type or recombinant parvoviruses H1-PV and MVMp makes these agents promising candidates for gene therapy of astrocytoma. This application raises the question of whether parvoviruses exert deleterious or bystander effects on normal glial cells surrounding tumours. We addressed this question in the mouse model by using cell cultures derived from BALB/c, C57BL/6 and VM/Dk strains. Astrocytes and a large proportion of microglia cultures were competent for MVMp uptake. Infection was, however, abortive as replication-associated viral proteins synthesis took place in less than 10% of astrocytes and no progeny virions were produced. This restriction was even more pronounced for microglia in which no viral protein expression could be detected, save for a minute fraction of VM/Dk-derived cells. Infection with MVMp had no significant effect on glial cell survival and did not interfere with their immune potential. Indeed, neither the lipopolysaccharide (LPS)/interferon (IFN-γ)-induced cytotoxicity of VM/Dk-derived microglia towards the mouse glioma (MT539MG) cell line, nor the glial cells capacity for tumour necrosis factor α production upon LPS stimulation or LPS/IFN-γ stimulation were affected by infection with MVMp. Moreover, stimulation with LPS and/or IFN-γ resulted in a decreased expression of the viral replicative and cytotoxic protein NS1. Together, our data indicate that, in the natural host, a majority of normal glial cells are not competent for MVMp replication and that the abortive infection taking place in a minor fraction of these cells fails to impede their survival and immunocompetence, giving credit to the consideration of autonomous parvoviruses for glioma therapy.  

5.382           Regulated Synthesis and Functions of Laminin 5 in Polarized Madin-Darby Canine Kidney Epithelial Cells

Mak, G.Z. et al Mol. Biol. Cell, 17(8), 3664-3677 (2006)   Renal tubular epithelial cells synthesize laminin (LN)5 during regeneration of the epithelium after ischemic injury. LN5 is a truncated laminin isoform of particular importance in the epidermis, but it is also constitutively expressed in a number of other epithelia. To investigate the role of LN5 in morphogenesis of a simple renal epithelium, we examined the synthesis and function of LN5 in the spreading, proliferation, wound-edge migration, and apical–basal polarization of Madin-Darby canine kidney (MDCK) cells. MDCK cells synthesize LN5 only when subconfluent, and they degrade the existing LN5 matrix when confluent. Through the use of small-interfering RNA to knockdown the LN5 3 subunit, we were able to demonstrate that LN5 is necessary for cell proliferation and efficient wound-edge migration, but not apical–basal polarization. Surprisingly, suppression of LN5 production caused cells to spread much more extensively than normal on uncoated surfaces, and exogenous keratinocyte LN5 was unable to rescue this phenotype. MDCK cells also synthesized laminin 5, a component of LN10, that independent studies suggest may form an assembled basal lamina important for polarization. Overall, our findings indicate that LN5 is likely to play an important role in regulating cell spreading, migration, and proliferation during reconstitution of a continuous epithelium.  

5.383           Gene-Eluting Stents: Comparison of Adenoviral and Adeno- Associated Viral Gene Delivery to the Blood Vessel Wall In Vivo

Sharif, F. et al Human Gene Ther., 17, 741-750 (2006)   Gene-eluting stents are being evaluated in animals as an alternative approach to inhibiting in-stent restenosis. Adeno-associated virus type 2 (AAV2) and adenovirus are commonly used for gene transfer applications. We tested the hypothesis that these vectors can achieve prolonged and localized gene delivery to the vessel wall, using stents as delivery platforms. AdβGal (5 × 10⁹ plaque-forming units) and AAV2βGal (5.3 × 10⁹ DNase-resistant particles) were used to coat BiodivYsio stents with matrix HI coating (Abbott Vascular Devices, Galway, Ireland). After balloon injury, external iliac arteries of New Zealand White rabbits were stented. The reverse transcription-polymerase chain reaction was used to assess viral spread. Expression of LacZ was demonstrated with both vectors at five time points (3, 7, 14, 21, and 28 days). In the adenovirus group the median percentage of cells expressing the transgene on day 3 was 2.73%, which increased to a median expression of 7.31% at 28 days (p > 0.05). Expression was localized to medial cells on day 3, but was observed predominantly in neointimal cells on day 28. In the AAV group, day 3 expression was 5.78%, which decreased to 2.12% on day 28 (p = 0.05). No systemic dissemination of virus was seen in any group. Adenovirus- and AAV2-coated stents can be used to deliver genes to the blood vessel wall for up to 28 days.  

5.384           Memory-related deficits following selective hippocampal expression of Swedish mutation amyloid precursor protein in the rat

Gong, Y. et al Exp. Neurol., 200, 371-377 (2006)   The gene encoding for the Swedish double mutation (K595N/M596L) of amyloid precursor protein (APP695Swe) was expressed bilaterally in adult rat hippocampus to determine its long-term effects on memory-related behavior as well as amyloid deposition. Recombinant adeno-associated viral serotype 2 (rAAV2) vectors were injected that contained either non-expressing DNA or cDNA encoding for APP695Swe under control of a chicken beta actin/cytomegalovirus promoter/enhancer. Immunolabeling human APP with the antibody 6E10 was observed throughout the cytoplasm of aspiny and, to a lesser extent, spine-bearing hippocampal neurons 6 and 12 months post-injection of the APP695Swe but not control vector. Aβ1–42 immunolabeling was identified in unusual immunoreactive objects within the hilus of the dentate gyrus and in the granule cell layer, proximal to the injection site. At 12 months post-transduction, rats that received the APP695Swe gene also demonstrated significant deficits in the acquisition and probe components of the spatial-memory-related Morris water task compared to control animals. These behavioral deficits occurred in the absence of any amyloid plaques, gliosis, or FluoroJade labeling of dying neurons. In conclusion, prolonged and localized APP695Swe expression in hippocampal neurons is sufficient to produce memory deficits without plaque formation or neuronal loss.  

5.385           Gene targeting in vivo by adeno-associated virus vectors

Miller, D.G. et al Nature Biotecnol., 24(8), 1022-1026   Therapeutic gene delivery typically involves the addition of a transgene expression cassette to mutant cells. This approach is complicated by transgene silencing, aberrant transcriptional regulation and insertional mutagenesis. An alternative strategy is to correct mutations through homologous recombination, allowing for normal regulation of gene expression from the endogenous locus. Adeno-associated virus (AAV) vectors containing single-stranded DNA efficiently transduce cells in vivo and have been shown to target homologous chromosomal sequences in cultured cells¹. To determine whether AAV-mediated gene targeting can occur in vivo, we developed a mouse model that contains a mutant, nuclear-localized lacZ gene inserted at the ubiquitously expressed ROSA26 locus. Foci of -galactosidase-positive hepatocytes were observed in these mice after injection with an AAV vector containing a lacZ gene fragment, and precise correction of the 4-bp deletion was demonstrated by gene sequencing. We also used AAV gene-targeting vectors to correct the naturally occurring GusB gene mutation responsible for murine mucopolysaccharidosis type VII².  

5.386           Adeno-associated Virus Serotypes: Vector Toolkit for Human Gene Therapy

Wu, Z., Asokan, A. and Samulski, R.J. Mol. Ther., 14(3), 316-327 (2006)   Recombinant adeno-associated viral (AAV) vectors have rapidly advanced to the forefront of gene therapy in the past decade. The exponential progress of AAV-based vectors has been made possible by the isolation of several naturally occurring AAV serotypes and over 100 AAV variants from different animal species. These isolates are ideally suited to development into human gene therapy vectors due to their diverse tissue tropisms and potential to evade preexisting neutralizing antibodies against the common human AAV serotype 2. Despite their prolific application in several animal models of disease, the mechanisms underlying selective tropisms of AAV serotypes remain largely unknown. Efforts to understand cell surface receptor usage and intracellular trafficking pathways exploited by AAV continue to provide significant insight into the biology of AAV vectors. Such unique traits are thought to arise from differences in surface topology of the capsids of AAV serotypes and variants. In addition to the aforementioned naturally evolved AAV isolates, several strategies to engineer hybrid AAV serotype vectors have been formulated in recent years. The generation of mosaic or chimeric vectors through the transcapsidation or marker-rescue/domain-swapping approach, respectively, is notable in this regard. More recently, combinatorial strategies for engineering AAV vectors using error-prone PCR, DNA shuffling, and other molecular cloning techniques have been established. The latter library-based approaches can serve as powerful tools in the generation of low-immunogenic and cell/tissue type-specific AAV vectors for gene delivery. This review is focused on recent developments in the isolation of novel AAV serotypes and isolates, their production and purification, diverse tissue tropisms, mechanisms of cellular entry/trafficking, and capsid structure. Strategies for engineering hybrid AAV vectors derived from AAV serotypes and potential implications of the rapidly expanding AAV vector toolkit are discussed.  

5.387           Anti-Aβ single-chain antibody delivery via adeno-associated virus for treatment of Alzheimer's disease

Fukuchi, K-i. et al Neurobiol. Disease, 23, 502-511 (2006)   Immunization of mouse models of Alzheimer disease (AD) with amyloid-peptide (Aβ) reduces Aβ deposits and attenuates their memory and learning deficits. Recent clinical trials were halted due to meningoencephalitis, presumably induced by T cell mediated and/or Fc-mediated immune responses. Because injection of anti-Aβ F(ab')₂ antibodies also induces clearance of amyloid plaques in AD mouse models, we have tested a novel gene therapy modality where an adeno-associated virus (AAV) encoding anti-Aβ single-chain antibody (scFv) is injected into the corticohippocampal regions of AD mouse models. One year after injection, expression of scFv was readily detectable in the neurons of the hippocampus without discernible neurotoxicity. AD mouse models subjected to AAV injection had much less amyloid deposits at the injection sites than the mouse models subjected to PBS injection. Because the scFv lacks the Fc portion of the immunoglobulin molecule, this modality may be a feasible solution for AD without eliciting inflammation.  

5.388           How does hepatitis C virus enter cells?

Diedrich, G. FEBS J., 273, 3871-3885 (2006)   Hepatitis C virus (HCV) exists in different forms in the circulation of infected people: lipoprotein bound and lipoprotein free, enveloped and nonenveloped. Viral particles with the highest infectivity are associated with lipoproteins, whereas lipoprotein-free virions are poorly infectious. The detection of HCV's envelope proteins E1 and E2 in lipoprotein-associated virions has been challenging. Because lipoproteins are readily endocytosed, some forms of HCV might utilize their association with lipoproteins rather than E1 and E2 for cell attachment and internalization. However, vaccination of chimpanzees with recombinant envelope proteins protected the animals from hepatitis C infection, suggesting an important role for E1 and E2 in cell entry. It seems possible that different forms of HCV use different receptors to attach to and enter cells. The putative receptors and the assays used for their validation are discussed in this review.  

5.389           Keratinocyte-Secreted Laminin 5 Can Function as a Transient Receptor for Human Papillomaviruses by Binding Virions and Transferring Them to Adjacent Cells

Culp, T.D., Budgeon, L.R., Marinkovich, M.P., Meneguzzi, G. and Christensen, N.D.

1. Virol., 80(18), 8940-8950 (2006)

  Human papillomaviruses (HPVs) replicate only in the terminally differentiating epithelium of the skin and mucosa. While infection of basal keratinocytes is considered a requirement for permissive infection, it remains unclear whether virions can specifically target basal cells for adsorption and uptake following epithelial wounding. We present evidence that HPV binds specifically to laminin 5 (LN5), a component of the extracellular matrix (ECM) secreted by migrating and basal keratinocytes. HPV type 11 capsids colocalized with LN5 in the ECM secreted by vaginal keratinocytes. Binding of both virions and virus-like particles to purified LN5 and to the LN5-rich ECM secreted by cultured keratinocytes was effectively blocked by pretreatment with anti-LN5 antibodies. HPV capsid binding to human cervical mucosa sections included the basement membrane which contains LN5. Cultured keratinocytes expressing 6 integrin, a transmembrane protein known to bind LN5, were readily infected by virions preadsorbed to LN5-containing substrates, whereas mutant keratinocytes lacking 6 integrin were relatively resistant to infection via this route. These findings suggest a model of natural HPV infection in which proliferating keratinocytes expressing 6 integrin at the site of epithelial wounding might be targeted by virions adsorbed transiently to LN5 secreted by migrating keratinocytes.          

5.390           Amelioration of Arthritis by Intraarticular Dominant Negative IKKβ Gene Therapy Using Adeno-Associated Virus Type 5

Tas, S.W. et al Human Gene Ther., 17, 821-832 (2006)   Nuclear factor (NF)-κB is highly activated in the synovium of rheumatoid arthritis (RA) patients, and can induce transcription of many proinflammatory molecules. Phosphorylation of inhibitor of κB (IκB) proteins is an important step in NF-κB activation and under inflammatory conditions is regulated predominantly by IκB kinase (IKK)β. Consequently, specific targeting of IKKβ in the joint, using gene therapy, presents a sophisticated treatment option for arthritis. In the present study we investigated the effect of inhibiting IKKβ in adjuvant arthritis (AA) in rats, using recombinant adeno-associated virus (rAAV)-mediated intraarticular gene therapy. For this purpose rAAV5 carrying the dominant negative IKKβ gene (AAV5.IKKβdn) or control AAV5.eGFP was injected into the right ankle joint. Rats treated with AAV5.IKKβdn in early arthritis exhibited significantly reduced paw swelling (p < 0.05). Immunohistochemical analysis of synovial tissue revealed reduced levels of interleukin (IL)-6 (p = 0.005) and tumor necrosis factor-α (TNF-α) (p = 0.03), whereas IL-10 levels were not affected. No significant effect was found on cartilage and bone destruction, or on matrix metalloproteinase-3 and tissue inhibitor of matrix metalloproteinase-1 expression. Injection of AAV5.IKKβdn in the preclinical phase showed only a marginal effect on arthritis. Importantly, in this study we also demonstrate for the first time that our vector is capable of transducing human RA whole synovial tissue biopsies ex vivo, resulting in reduced IL-6 production after TNF-α stimulation (p = 0.03). In conclusion, we are the first to demonstrate that rAAV5 can be used to successfully deliver a therapeutic gene (IKKβdn) to the synovium, resulting in reduced severity of inflammation in AA in vivo and proinflammatory cytokine production in human RA synovial tissue ex vivo. This translational research represents a crucial next step toward the development of gene therapy for application in humans.  

5.391           Studying cellular architecture in three dimensions with improved resolution: Ta replicas revisited

Cabezas, P. and Risco, C. Cell Biol. Int., 30(9), 747-754 (2006)   Metal replicas have been used for surface analysis of biological structures with a variety of spatial resolutions. Platinum (Pt) has been the metal of choice because it provides very stable replicas and images of high contrast. Some other metals, such as tantalum (Ta) have been reported to provide better resolution on isolated macromolecular complexes and cellular structures. Our goal is to study the gain in detail with Ta and to evaluate if it provides enough detail and resolution to assist in the study of complex volumes of intact cellular structures obtained by methods that reach molecular resolution. To this purpose Pt and Ta replicas of cellular structures and viruses have been studied by transmission electron microscopy (TEM). Replicas of Ta show new details on the surface of two types of isolated viral particles such as 100 nm bunyaviruses and large, >300 nm, vaccinia virus (VV). Inside cells, the structural pieces that build VV immature particles are visualized only in Ta replicas. Looking for smaller intracellular complexes, new details are also seen in nuclear pores from Ta replicas. Additional masses, most likely representing the cargo during transport, are distinguished in some of the pores. Visualization of proteins in plasma membranes strongly suggests that detail and resolution of Ta replicas are similar to those estimated for 3D maps currently obtained by electron tomography of viruses and cells.  

5.392           Leptin Receptor Signaling in Midbrain Dopamine Neurons Regulates Feeding

Hommel, J.D. et al Neuron, 51, 801-810 (2006)   The leptin hormone is critical for normal food intake and metabolism. While leptin receptor (Lepr) function has been well studied in the hypothalamus, the functional relevance of Lepr expression in the ventral tegmental area (VTA) has not been investigated. The VTA contains dopamine neurons that are important in modulating motivated behavior, addiction, and reward. Here, we show that VTA dopamine neurons express Lepr mRNA and respond to leptin with activation of an intracellular JAK-STAT pathway and a reduction in firing rate. Direct administration of leptin to the VTA caused decreased food intake while long-term RNAi-mediated knockdown of Lepr in the VTA led to increased food intake, locomotor activity, and sensitivity to highly palatable food. These data support a critical role for VTA Lepr in regulating feeding behavior and provide functional evidence for direct action of a peripheral metabolic signal on VTA dopamine neurons.  

5.393           a-1 Antitrypsin Inhibits Caspase-3 Activity, Preventing Lung Endothelial Cell Apoptosis

Petrache, I. et al Am. J. Pathol., 169(4), 1155-1166 (2006)   a-1 Antitrypsin (A1AT) is an abundant circulating serpin with a postulated function in the lung of potently inhibiting neutrophil-derived proteases. Emphysema attributable to A1AT deficiency led to the concept that a protease/anti-protease imbalance mediates cigarette smoke-induced emphysema. We hypothesized that A1AT has other pathobiological relevant functions in addition to elastase inhibition. We demonstrate a direct prosurvival effect of A1AT through inhibition of lung alveolar endothelial cell apoptosis. Primary pulmonary endothelial cells internalized human A1AT, which co-localized with and inhibited staurosporine-induced caspase-3 activation. In cell-free studies, native A1AT, but not conformers lacking an intact reactive center loop, inhibited the interaction of recombinant active caspase-3 with its specific substrate. Furthermore, overexpression of human A1AT via replication-deficient adeno-associated virus markedly attenuated alveolar wall destruction and oxidative stress caused by caspase-3 instillation in a mouse model of apoptosis-dependent emphysema. Our findings suggest that direct inhibition of active caspase-3 by A1AT may represent a novel anti-apoptotic mechanism relevant to disease processes characterized by excessive structural cell apoptosis, oxidative stress, and inflammation, such as pulmonary emphysema.  

5.394           AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells

Leaver, S.G. et al Gene Therapy, 13, 1328-1341 (2006)   We compared the effects of intravitreal injection of bi-cistronic adeno-associated viral (AAV-2) vectors encoding enhanced green fluorescent protein (GFP) and either ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43) on adult retinal ganglion cell (RGC) survival and regeneration following (i) optic nerve (ON) crush or (ii) after ON cut and attachment of a peripheral nerve (PN). At 7 weeks after ON crush, quantification of III-tubulin immunostaining revealed that, compared to AAV-GFP controls, RGC survival was not enhanced by AAV-GAP43-GFP but was increased in AAV-CNTF-GFP (mean RGCs/retina: 17 450 358 s.e.m.) and AAV-BDNF-GFP injected eyes (10 200 4064 RGCs/retina). Consistent with increased RGC viability in AAV-CNTF-GFP and AAV-BDNF-GFP injected eyes, these animals possessed many III-tubulin- and GFP-positive fibres proximal to the ON crush. However, only in the AAV-CNTF-GFP group were regenerating RGC axons seen in distal ON (1135 367 axons/nerve, 0.5 mm post-crush), some reaching the optic chiasm. RGCs were immunoreactive for CNTF and quantitative RT-PCR revealed a substantial increase in CNTF mRNA expression in retinas transduced with AAV-CNTF-GFP. The combination of AAV-CNTF-GFP transduction of RGCs with autologous PN-ON transplantation resulted in even greater RGC survival and regeneration. At 7 weeks after PN transplantation there were 27 954 ( 2833) surviving RGCs/retina, about 25% of the adult RGC population. Of these, 13 352 ( 1868) RGCs/retina were retrogradely labelled after fluorogold injections into PN grafts. In summary, AAV-mediated expression of CNTF promotes long-term survival and regeneration of injured adult RGCs, effects that are substantially enhanced by combining gene and cell-based therapies/interventions.  

5.395           Virus-like particles: Designing an effective AIDS vaccine

Young, K.R., McBurney, S.P., Karkhanis, L.U. and Ross, T.M. Methods, 40, 98-117 (2006)   Viruses that infect eukaryotic organisms have the unique characteristic of self-assembling into particles. The mammalian immune system is highly attuned to recognizing and attacking these viral particles following infection. The use of particle-based immunogens, often delivered as live-attenuated viruses, has been an effective vaccination strategy for a variety of viruses. The development of an effective vaccine against the human immunodeficiency virus (HIV) has proven to be a challenge, since HIV infects cells of the immune system causing severe immunodeficiency resulting in the syndrome known as AIDS. In addition, the ability of the virus to adapt to immune pressure and reside in an integrated form in host cells presents hurdles for vaccinologists to overcome. A particle-based vaccine strategy has promise for eliciting high titer, long-lived, immune responses to a diverse number of viral epitopes against different HIV antigens. Live-attenuated viruses are effective at generating both cellular and humoral immune responses. However, while these vaccines stimulate immunity, challenged animals rarely clear the viral infection and the degree of attenuation directly correlates with protection from disease. Further, a live-attenuated vaccine has the potential to revert to a pathogenic form. Alternatively, virus-like particles (VLPs) mimic the viral particle without causing an immunodeficiency disease. VLPs are self-assembling, non-replicating, non-pathogenic particles that are similar in size and conformation to intact virions. A variety of VLPs for lentiviruses are currently in preclinical and clinical trials. This review focuses on our current status of VLP-based AIDS vaccines, regarding issues of purification and immune design for animal and clinical trials.  

5.396           Identification of host cell proteins in purified infectious humanherpesvirus 6A (HHV-6A) viral particles

Ahlquist, J., Hammarstadt, M., Jacobson, S., Garoff, H. and Fogdell-Hahn, A.

1. Immunoimmunol., 178, Suppl. 1, page 114, abstr. 0S6B-01 (2006)

  An association between the autoimmune disease multiple sclerosis and HHV-6A has been suggested. In vivo, HHV-6A has been detected in myelin producing cells, i.e. the main cells that are affected in MS. HHV-6A might incorporate host cell proteins into the viral particles during replication. When the virus is detected by the immune system, the incorporated protein will be presented for the immune system. This could be one mechanism for induction of autoimmunity. Incorporation of host cell proteins have been shown for other viruses, for example the complement proteins CD55 and CD59 that are incorporated into HCMV, HTLV-1 and HIV-1. However, the purity of the virus preparations has been debated since cellular vesicles might contaminate the viral preparations during purification in sucrose gradients. In this study, we used iso-osmotic iodixanol gradients to purify T-cell cultured HHV-6A. In our gradient peak fraction we detect high levels of viral DNA by real-time PCR, expression of gp60/110 byWestern blot and intact viral particles by electron microscopy. The purified virus particles were also able to re-infect T cells with good efficiency. We have low cellular contamination in the viral particle containing fractions as seen by analyzing viral and mock preparations by SDS-PAGE. Immunoblotting with anti-CD46 and other anti-host cell protein antibodies gave clear bands in the viral containing fractions compared to the mock containing fraction. In conclusion, this method may be used to study the protein content of viral particles and that may give important clues for how autoimmunity is induced.  

5.397           Unique Biologic Properties of Recombinant AAV1 Transduction in Polarized Human Airway Epithelia

Yan, Z. et al

1. Biol. Chem., 281(40), 29684-29692 (2006)

  The choice of adeno-associated virus serotypes for clinical applications is influenced by the animal model and model system used to evaluate various serotypes. In the present study, we sought to compare the biologic properties of rAAV2/1, rAAV2/2, and rAAV2/5 transduction in polarized human airway epithelia using viruses purified by a newly developed common column chromatography method. Results demonstrated that apical transduction of human airway epithelia with rAAV2/1 was 100-fold more efficient than rAAV2/2 and rAAV2/5. This transduction profile in human airway epithelia (rAAV2/1 >> rAAV2/2 = rAAV2/5) was significantly different from that seen following nasal administration of these vectors to mouse lung (rAAV2/5 > rAAV2/1 >> rAAV2/2), emphasizing differences in transduction of these serotypes between these two species. In stark contrast to rAAV2/2 and rAAV2/5, rAAV2/1 transduced both the apical and basolateral membrane of human airway epithelia with similar efficiency. However, the overall level of transduction across serotypes did not correlate with vector internalization. We hypothesized that differences in post-entry processing of these serotypes might influence the efficiency of apical transduction. To this end, we tested the effectiveness of proteasome inhibitors to augment nuclear translocation and gene expression from the three serotypes. Augmentation of rAAV2/1 apical transduction of human polarized airway epithelia was 10-fold lower than that for rAAV2/2 and rAAV2/5. Cellular fractionation studies demonstrated that proteasome inhibitors more significantly enhanced rAAV2/2 and rAAV2/5 translocation to the nucleus than rAAV2/1. These results demonstrate that AAV1 transduction biology in human airway epithelia differs from that of AAV2 and AAV5 by virtue of altered ubiquitin/proteasome sensitivities that influence nuclear translocation.              

5.398           Macrophage Transcriptional Responses following In Vitro Infection with a Highly Virulent African Swine

Zhang, F. et al

1. Virol., 80(21), 10514-10521 (2006)

  We used a porcine microarray containing 2,880 cDNAs to investigate the response of macrophages to infection by a virulent African swine fever virus (ASFV) isolate, Malawi LIL20/1. One hundred twenty-five targets were found to be significantly altered at either or both 4 h and 16 h postinfection compared with targets after mock infection. These targets were assigned into three groups according to their temporal expression profiles. Eighty-six targets showed increased expression levels at 4 h postinfection but returned to expression levels similar to those in mock-infected cells at 16 h postinfection. These encoded several proinflammatory cytokines and chemokines, surface proteins, and proteins involved in cell signaling and trafficking pathways. Thirty-four targets showed increased expression levels at 16 h postinfection compared to levels at 4 h postinfection and in mock-infected cells. One host gene showed increased expression levels at both 4 and 16 h postinfection compared to levels in mock-infected cells. The microarray results were validated for 12 selected genes by quantitative real-time PCR. Levels of protein expression and secretion were measured for two proinflammatory cytokines, interleukin 1beta and tumor necrosis factor alpha, during a time course of infection with either the virulent Malawi LIL20/1 isolate or the OUR T88/3 nonpathogenic isolate. The results revealed differences between these two ASFV isolates in the amounts of these cytokines secreted from infected cells.  

5.399           Host cell DNA repair pathways in adeno-associated viral genome processing

Choi, V.W., McCarty, D.M. and Samulski, R.J.

1. Virol., 80(21), 10346-10356 (2006)

  Recent studies have shown that wild-type and recombinant adeno-associated virus (AAV and rAAV) genomes persist in human tissue predominantly as double-stranded (ds) circular episomes derived from input linear single-stranded virion DNA. Using self-complementary recombinant AAV (scAAV) vectors, we generated intermediates that directly transition to ds circular episomes. The scAAV genome ends are palindromic hairpin-structured terminal repeats, resembling a double-stranded break repair intermediate. Utilizing this substrate, we found cellular DNA recombination and repair factors to be essential for generating circular episomal products. To identify the specific cellular proteins involved, the scAAV circularization-dependent vector was used as a reporter in 19 mammalian DNA repair-deficient cell lines. The results show that RecQ helicase family members (BLM and WRN), Mre11 and NBS1 of the Mre11-Rad50-Nbs1 (MRN) complex, and ATM are required for efficient scAAV genome circularization. We further demonstrated that the scAAV genome requires ATM and DNA-PK(CS), but not NBS1, to efficiently convert to a circular form in nondividing cells in vivo using transgenic mice. These studies identify specific pathways involved for further elucidating viral and cellular mechanisms of DNA maintenance important to the viral life cycle and vector utilizations.  

5.400           Characterization of neutralizing epitopes within the major capsid protein of human papillomavirus type 33

Roth, S.D., Sapp, M., Streeck, R.E. and Selinka, H-C. Virol. J., 3(83), 1-11 (2006)   Background Infections with papillomaviruses induce type-specific immune responses, mainly directed against the major capsid protein, L1. Based on the propensity of the L1 protein to self-assemble into virus-like particles (VLPs), type-specific vaccines have already been developed. In order to generate vaccines that target a broader spectrum of HPV types, extended knowledge of neutralizing epitopes is required. Despite the association of human papillomavirus type 33 (HPV33) with cervical carcinomas, fine mapping of neutralizing conformational epitopes on HPV33 has not been reported yet. By loop swapping between HPV33 and HPV16 capsid proteins, we have identified amino acid sequences critical for the binding of conformation-dependent type-specific neutralizing antibodies to surface-exposed hyper variable loops of HPV33 capsid protein L1. Results Reactivities of monoclonal antibodies (mAbs) H33.B6, H33.E12, H33.J3 and H16.56E with HPV16:33 and HPV33:16 hybrid L1 VLPs revealed the complex structures of their conformational epitopes as well as the major residues contributing to their binding sites. Whereas the epitope of mAb H33.J3 was determined by amino acids (aa) 51–58 in the BC loop of HPV33 L1, sequences of at least two hyper variable loops, DE (aa 132–140) and FGb (aa 282–291), were found to be essential for binding of H33.B6. The epitope of H33.E12 was even more complex, requiring sequences of the FGa loop (aa 260–270), in addition to loops DE and FGb. Conclusion These data demonstrate that neutralizing epitopes in HPV33 L1 are mainly located on the tip of the capsomere and that several hyper variable loops contribute to form these conformational epitopes. Knowledge of the antigenic structure of HPV is crucial for designing hybrid particles as a basis for intertypic HPV vaccines.  

5.401           Adeno-Associated Virus Type 2 Capsids with Externalized VP1/VP2 Trafficking Domains Are Generated prior to Passage through the Cytoplasm and Are Maintained until Uncoating Occurs in the Nucleus

Sonntag, F., Bleker, S., Leuchs, B., Fischer, R. And Kleinschmidt, J.A.

1. Virol., 80(22), 11040-11054 (2006)

  Common features of parvovirus capsids are open pores at the fivefold symmetry axes that traverse the virion shell. Upon limited heat treatment in vitro, the pores can function as portals to externalize VP1/VP2 protein N-terminal sequences which harbor infection-relevant functional domains, such as a phospholipase A₂ catalytic domain. Here we show that adeno-associated virus type 2 (AAV2) also exposes its VP1/VP2 N termini in vivo during infection, presumably in the endosomal compartment. This conformational change is influenced by treatment with lysosomotropic reagents. While incubation of cells with bafilomycin A1 reduced exposure of VP1/VP2 N termini, incubation with chloroquine stimulated externalization transiently. N-terminally located basic amino acid clusters with nuclear localization activity also become exposed in this process and are accessible on the virus capsid when it enters the cytoplasm. This is an obligatory step in AAV2 infection. However, a direct role of these sequences in nuclear translocation of viral capsids could not be determined by microinjection of wild-type or mutant viruses. This suggests that further modifications of the capsid have to take place in a precytoplasmic entry step that prepares the virus for nuclear entry. Microinjection of several capsid-specific antibodies into the cell nucleus blocked AAV2 infection completely, supporting the conclusion that AAV2 capsids bring the infectious genome into the nucleus.  

5.402           Differential Biophysical Properties of Infectious Intracellular and Secreted Hepatitis C Virus Particles

Gastaminza, P., Kapadia, S.B. and Chisari, F.

1. Virol., 80(22), 11074-11081 (2006)

  The recent development of a cell culture infection model for hepatitis C virus (HCV) permits the production of infectious particles in vitro. In this report, we demonstrate that infectious particles are present both within the infected cells and in the supernatant. Kinetic analysis indicates that intracellular particles constitute precursors of the secreted infectious virus. Ultracentrifugation analyses indicate that intracellular infectious viral particles are similar in size ( 65 to 70 nm) but different in buoyant density ( 1.15 to 1.20 g/ml) from extracellular particles ( 1.03 to 1.16 g/ml). These results indicate that infectious HCV particles are assembled intracellularly and that their biochemical composition is altered during viral egress.  

5.403           Papillomavirus Particles Assembled in 293TT Cells Are Infectious In Vivo

Culp, T.D. et al

1. Virol., 80(22), 11381-11384 (2006)

  Papillomaviruses (PVs) demonstrate both tissue and species tropisms. Because PVs replicate only in terminally differentiating epithelium, the recent production of infectious PV particles in 293 cells marks an important breakthrough. In this article, we demonstrate that infectious PV particles produced in 293TT cells can cause papillomatous growths in the natural host animal. Moreover, we show that species-matched PV genomes can be successfully delivered in vivo by a heterologous, species-mismatched PV capsid. Additionally, our results indicate that the addition of the simian virus 40 origin of replication to the papillomavirus genome increases the production of infectious papillomavirus particles by increasing genome amplification in the transfected 293TT cells.  

5.404           HIV Induces Maturation of Monocyte-Derived Dendritic Cells and Langerhans Cells

Harman, A.N. et al

1. Immunol., 177, 7103-7113 (2006)

  In HIV infection, dendritic cells (DCs) may play multiple roles, probably including initial HIV uptake in the anogenital mucosa, transport to lymph nodes, and subsequent transfer to T cells. The effects of HIV-1 on DC maturation are controversial, with several recent conflicting reports in the literature. In this study, microarray studies, confirmed by real-time PCR, demonstrated that the genes encoding DC surface maturation markers were among the most differentially expressed in monocyte-derived dendritic cells (MDDCs), derived from human blood, treated with live or aldrithriol-2-inactivated HIV-1_BaL. These effects translated to enhanced cell surface expression of these proteins but differential expression of maturation markers was only partial compared with the effects of a conventional potent maturation stimulus. Such partially mature MDDCs can be converted to fully mature cells by this same potent stimulus. Furthermore, live HIV-1 stimulated greater changes in maturation marker surface expression than aldrithriol-2-inactivated HIV-1 and this enhanced stimulation by live HIV-1 was mediated via CCR5, thus suggesting both viral replication-dependent and -independent mechanisms. These partially mature MDDCs demonstrated enhanced CCR7-mediated migration and are also able to stimulate interacting T cells in a MLR, suggesting DCs harboring HIV-1 might prepare CD4 lymphocytes for transfer of HIV-1. Increased maturation marker surface expression was also demonstrated in native DCs, ex vivo Langerhans cells derived from human skin. Thus, HIV initiates maturation of DCs which could facilitate subsequent enhanced transfer to T cells.  

5.405           Controlling bacteriophage phi29 DNA-packaging motor by addition or discharge of a peptide at N-terminus of connector protein that interacts with pRNA

Sun, J., Cai, Y., Moll, W-D. and Guo, P. Nucleic Acids Res., 34(19), 5482-5490 (2006)   Bacteriophage phi29 utilizes a motor to translocate genomic DNA into a preformed procapsid. The motor contains six pRNAs, an enzyme and one 12-subunit connector with a central channel for DNA transportation. A 20-residue peptide containing a His-tag was fused to the N-terminus of the connector protein gp10. This fusion neither interfered with procapsid assembly nor affected the morphology of the prolate-shaped procapsid. However, the pRNA binding and virion assembly activity were greatly reduced. Such decreased functions can be switched back on by the removal of the tag via protease cleavage, supporting the previous finding that the N-terminus of gp10 is essential for the pRNA binding. The DNA-packaging efficiency with dimeric pRNA was more seriously affected by the extension than with monomeric pRNA. It is speculated that the fusion of the tag generated physical hindrance to pRNA binding, with greater influence for the dimers than the monomers due to their size. These results reveal a potential to turn off and turn on the motor by attaching or removing, respectively, a component to outer part of the motor, and offers an approach for the inhibition of viral replication by using a drug or a small peptide targeted to motor components.  

5.406           Potentiation of in vivo neuroprotection by BclXL and GDNF co-expression depends on post-lesion time in deafferentiated CNS neurons

Shevtsova, Z. Et al Gene Therapy, 13, 1569-1578 (2006)   To elucidate effective and long-lasting neuroprotective strategies, we analysed a combination of mitochondrial protection and neurotrophic support in two well-defined animal models of neurodegeneration, traumatic lesion of optic nerve and complete 6-hydroxydopamine (6-OHDA) lesion of nigrostriatal pathway. Neuroprotection by BclX_L, Glial cell line-derived neurotrophic factor (GDNF) or BclX_L plus GDNF co-expression were studied at 2 weeks and at 6–8 weeks after lesions. In both lesion paradigms, the efficacy of this combination approach significantly differed depending on post-lesion time. We show that BclX_L expression is more important for neuronal survival in the early phase after lesions, whereas GDNF-mediated neuroprotection becomes more prominent in the advanced state of neurodegeneration. BclX_L expression was not sufficient to finally inhibit degeneration of deafferentiated central nervous system neurons. Long-lasting GDNF-mediated neuroprotection depended on BclX_L co-expression in the traumatic lesion paradigm, but was independent of BclX_L in the 6-OHDA lesion model. The results demonstrate that neuroprotection studies in animal models of neurodegenerative diseases should generally be performed over extended periods of time in order to reveal the actual potency of a therapeutic approach.

5.407           Intracranial Adeno-Associated Virus-Mediated Delivery of Anti-Pan Amyloid , Amyloid 40, and Amyloid 42 Single-Chain Variable Fragments Attenuates Plaque Pathology in Amyloid Precursor Protein Mice

Levites, Y. et al

1. Neurosci., 26(46), 11923-11928 (2006)

  Accumulation of amyloid protein (A ) aggregates is hypothesized to trigger a pathological cascade that causes Alzheimer's disease (AD). Active or passive immunizations targeting A are therefore of great interest as potential therapeutic strategies. We have evaluated the use of recombinant anti-A single-chain variable fragments (scFvs) as a potentially safer form of anti-A immunotherapy. We have generated and characterized three anti-A scFvs that recognize A 1–16, A x-40, or A x-42. To achieve widespread brain delivery, constructs expressing these anti-A scFvs were packaged into adeno-associated virus (AAV) vectors and injected into the ventricles of postnatal day 0 (P0) amyloid precursor protein CRND8-transgenic mice. Intracranial delivery of AAV to neonatal mice resulted in widespread neuronal delivery. In situ expression of each of the anti-A scFvs after intracerebroventricular AAV serotype 1 delivery to P0 pups decreased A deposition by 25–50%. These data suggest that intracranial anti-A scFv expression is an effective strategy to attenuate amyloid deposition. As opposed to transgenic approaches, these studies also establish a "somatic brain transgenic" paradigm to rapidly and cost-effectively evaluate potential modifiers of AD-like pathology in AD mouse models.  

5.408           Proteolytic Mapping of the Adeno-associated Virus Capsid

Van Vliet, K., Blouin, V., Agbandje-McKenna, M. and Snyder, R.O. Mol. Ther., 14(6), 809-821 (2006)   The three-dimensional structures of the viral capsid of three AAV serotypes have previously been determined by X-ray crystallography or cryoelectron microscopy. These studies of AAV and similar studies of autonomous parvoviruses have yielded important structural information about the virions in a low-energy conformation. However, there is little information on the structural properties of AAV virions in solution under physiological conditions. We demonstrate that proteolytic digestion of AAV2 virions with trypsin results in cleavage at a specific site on the capsid surface while the capsid remains intact. The products of digestion were mapped using unique antibodies, protein sequencing, mass spectroscopy, and 3D structure modeling to a region on a surface loop that is common to all three AAV2 structural proteins. Empty AAV2 capsids could be distinguished from full (DNA-containing) capsids, having an increased susceptibility of VP2 to trypsin and being digested more rapidly by chymotrypsin. Proteolytic analysis utilizing trypsin or chymotrypsin was also capable of distinguishing AAV2 from AAV1 and AAV5, as seen by differential susceptibility and unique fragment patterns. These data demonstrate a novel approach for studying the structure of AAV capsids in solution and should be valuable in the testing and engineering of AAV vectors for gene transfer.  

5.409           Selective and Quickly Reversible Inactivation of Mammalian Neurons In Vivo Using the Drosophila Allatostatin Receptor

Tan, E.M: et al Neuron, 51, 157-170 (2006)   Genetic strategies for perturbing activity of selected neurons hold great promise for understanding circuitry and behavior. Several such strategies exist, but there has been no direct demonstration of reversible inactivation of mammalian neurons in vivo. We previously reported quickly reversible inactivation of neurons in vitro using expression of the Drosophila allatostatin receptor (AlstR). Here, adeno-associated viral vectors are used to express AlstR in vivo in cortical and thalamic neurons of rats, ferrets, and monkeys. Application of the receptor's ligand, allatostatin (AL), leads to a dramatic reduction in neural activity, including responses of visual neurons to optimized visual stimuli. Additionally, AL eliminates activity in spinal cords of transgenic mice conditionally expressing AlstR. This reduction occurs selectively in AlstR-expressing neurons. Inactivation can be reversed within minutes upon washout of the ligand and is repeatable, demonstrating that the AlstR/AL system is effective for selective, quick, and reversible silencing of mammalian neurons in vivo.        

5.410           Expression of human immunodeficiency virus type 1 tat from a replication-deficient herpes simplex type 1 vector induces antigen-specific T cell responses

Bozac, A. et al Vaccine, 24(49-50), 7148-7158 (2006)   Herpes simplex type-1 virus (HSV-1) based vectors have been widely used in different gene therapy approaches and also as experimental vaccines against HSV-1 infection. Recent advances in the HSV-1 technology do support the use of replication defective HSV-1 as vaccine vectors for delivery of foreign antigens. We have examined the ability of a recombinant replication-defective HSV-1 vector expressing the HIV-1 Tat protein to induce long-term Tat-specific immune responses in the Balb/c murine model. The results showed that vector administration by the subcutaneous route elicits anti-Tat specific T-cell mediated immune responses in mice characterized by the presence of the Tat-specific cytotoxic activity and production of high levels of IFN-γ.  

5.411           Preclinical Model To Test Human Papillomavirus Virus (HPV) Capsid Vaccines In Vivo Using Infectious HPV/Cottontail Rabbit Papillomavirus Chimeric Papillomavirus Particles

Mejia, A.F. et al

1. Virol., 80(24), 12393-12397 (2006)

  A human papillomavirus (HPV) vaccine consisting of virus-like particles (VLPs) was recently approved for human use. It is generally assumed that VLP vaccines protect by inducing type-specific neutralizing antibodies. Preclinical animal models cannot be used to test for protection against HPV infections due to species restriction. We developed a model using chimeric HPV capsid/cottontail rabbit papillomavirus (CRPV) genome particles to permit the direct testing of HPV VLP vaccines in rabbits. Animals vaccinated with CRPV, HPV type 16 (HPV-16), or HPV-11 VLPs were challenged with both homologous (CRPV capsid) and chimeric (HPV-16 capsid) particles. Strong type-specific protection was observed, demonstrating the potential application of this approach.  

5.412           Capsid modifications overcome low heterogeneous expression of heparan sulfate proteoglycan that limits AAV2-mediated gene transfer and therapeutic efficacy in human ovarian carcinoma

Shi, W., Hemminki, A. and Bartlett, J.S. Gynecol. Oncol., 103, 1054-1062 (2006)   Objectives. Capsid-modified AAV vectors can mediate enhanced gene transfer to neoplasms characterized by low AAV receptor expression. Here we sought to determine the therapeutic potential of a capsid-modified AAV vector for gene therapy of ovarian carcinoma (OvCa). Methods. We tested a panel of OvCa cell lines for AAV2-mediated gene transduction and for sensitivity to ganciclovir (GCV) following AAVHSVtk administration. Levels of AAV internalization and attachment receptor were assessed by flow cytometry and immunohistochemistry. The role of receptors in AAV-mediated gene transfer was assessed by competition assays. Finally, we examined the ability of a modified vector with an integrin-binding RGD motif inserted into the AAV capsid to improve gene delivery to OvCa and enhance AAVHSVtk/GCV-mediated killing by cytotoxicity assay. Results. All OvCa cell lines were poorly transduced with AAV2 vectors and showed variably sensitive to AAVHSVtk/GCV. While OvCa cell lines expressed AAV2 internalization receptors (α_v integrins), expression of the AAV2 attachment receptor, HSPG, was variable and not detected on many lines. Analysis of archived clinical specimens showed no detectable HSPG expression on approximately 45% of primary human tumors. Gene transfer to OvCa was increased several fold using the RGD-modified vector. Gene transfer was independent of HSPG and specific to the targeted receptor. Importantly, the RGD-modified capsid markedly increased the ability of the AAVHSVtk to kill OvCa cells in the presence of GCV. Conclusions. The development of AAV vectors targeted to cell surface receptors other than HSPG will be critical to the advancement of AAV-mediated gene therapy for treating OvCa.  

5.413           Latent Virus Influences the Generation and Maintenance of CD8+ T Cell Memory

Sheridan, B.S., Khanna, K.M., Frank, G.M. and Hendricks, R.L.

1. Immunol., 177, 8356-8364 (2006)

  The influence of latent virus on CD8⁺ T cell memory is poorly understood. HSV type 1 specifically establishes latency in trigeminal ganglia (TG) after corneal infection of mice. In latently infected TG, IL-15 deprivation reduced the following: 1) accumulation of HSV-specific CD8⁺ effector T cells (HSV-CD8_eff), 2) accumulation of CD127⁺ putative HSV-CD8 memory precursors, and 3) the size and functionality of the memory (HSV-CD8_mem) population. Although compromised in IL-15^–/– mice, the HSV-CD8_mem pool persisted in latently infected tissue, but not in noninfected tissue of the same mice. Anti-IL-2 treatment also dramatically reduced the size of the HSV-CD8_eff population in the TG, but did not influence the concomitant generation of the CD127⁺ putative HSV-CD8_mem precursor population or the size or functionality of the HSV-CD8_mem pool. Thus, the size of the memory pool appears to be determined by the size of the CD127⁺ CD8_mem precursor population and not by the size of the overall CD8_eff pool. HSV-CD8_mem showed a higher basal rate of proliferation in latently infected than noninfected tissue, which was associated with a reduced population of CD4⁺FoxP3⁺ regulatory T cells. Thus, the generation, maintenance, and function of memory CD8⁺ T cells is markedly influenced by latent virus.  

5.414           Generation and characterization of a stable cell population releasing fluorescent HIV-1-based Virus Like Particles in an inducible way

Muratori, C. et al BMC Biotechnol., 6(52), xxx (2006)   Background The availability of cell lines releasing fluorescent viral particles can significantly support a variety of investigations, including the study of virus-cell interaction and the screening of antiviral compounds. Regarding HIV-1, the recovery of such biologic reagents represents a very hard challenge due to the intrinsic cytotoxicity of many HIV-1 products. We sought to overcome such a limitation by using a cell line releasing HIV-1 particles in an inducible way, and by exploiting the ability of a HIV-1 Nef mutant to be incorporated in virions at quite high levels. Results Here, we report the isolation and characterization of a HIV-1 packaging cell line, termed 18-4s, able to release valuable amounts of fluorescent HIV-1 based Virus-Like Particles (VLPs) in an inducible way. 18-4s cells were recovered by constitutively expressing the HIV-1 NefG3C mutant fused with the enhanced-green fluorescent protein (NefG3C-GFP) in a previously isolated inducible HIV-1 packaging cell line. The G3C mutation creates a palmitoylation site which results in NefG3C-GFP incorporation into virions greatly exceeding that of the wild type counterpart. Upon induction of 18-4s cells with ponasterone A and sodium butyrate, up to 4 microg/ml of VLPs, which had incorporated about 150 molecules of NefG3C-GFP per viral particle, were released into the culture supernatant. Due to their intrinsic strong fluorescence, the 18-4s VLPs were easily detectable by a novel cytofluorometric-based assay developed here. The treatment of target cells with fluorescent 18-4 VLPs pseudotyped with different glycoprotein receptors resulted in these becoming fluorescent as early as two hours post-challenge. Conclusions We created a stable cell line releasing fluorescent HIV-1 based VLPs upon induction useful for several applications including the study of virus-cell interactions and the screening of antiviral compounds.  

5.415           Construction of diverse adeno-associated viral libraries for directed evolution of enhanced gene delivery vehicles

Koerber, J.T., Maheshri, N., Kaspar, B.K. and Schaffer, D.V. Nature Protocols, 1(2), 701-706 (2006)   Rational design of improved gene delivery vehicles is a challenging and potentially time-consuming process. As an alternative approach, directed evolution can provide a rapid and efficient means for identifying novel proteins with improved function. Here we describe a methodology for generating very large, random adeno-associated viral (AAV) libraries that can be selected for a desired function. First, the AAV2 cap gene is amplified in an error-prone PCR reaction and further diversified through a staggered extension process. The resulting PCR product is then cloned into pSub2 to generate a diverse (>10⁶) AAV2 plasmid library. Finally, the AAV2 plasmid library is used to package a diverse pool of mutant AAV2 virions, such that particles are composed of a mutant AAV genome surrounded by the capsid proteins encoded in that genome, which can be used for functional screening and evolution. This procedure can be performed in approximately 2 weeks.  

5.416           Production and characterization of adeno-associated viral vectors

Grieger, J.C., Choi, V.W. and Samulski, R.J. Nature Protocols, 1(3), 1412-1428 (2006)   The adeno-associated virus (AAV) is one of the most promising viral vectors for human gene therapy. As with any potential therapeutic system, a thorough understanding of it at the in vitro and in vivo levels is required. Over the years, numerous methods have been developed to better characterize AAV vectors. These methods have paved the way to a better understanding of the vector and, ultimately, its use in clinical applications. This review provides an up-to-date, detailed description of essential methods such as production, purification and titering and their application to characterize current AAV vectors for preclinical and clinical use.  

5.417           Adeno-associated virus-mediated gene transfer of a secreted form of TRAIL inhibits tumor growth and occurrence in an experimental tumor model

Yoo, J., Choi, S., Hwang, K-S., Cho, W-K., Jung, C-R., Kwon, S-T. and Im, D-S.

1. Gene Med., 8(2), 163-174 (2006)

  Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cell death in various tumor cells, but relatively spares normal cells. Recombinant adeno-associated virus (rAAV) vectors have a number of advantages including in vivo long-term gene expression. Here, we assessed the biological activity of a novel, secreted form of TRAIL (sTRAIL) for cancer gene therapy using a rAAV2 vector. Methods A plasmid and rAAV2 vectors were constructed encoding sTRAIL composed of a leader sequence, the isoleucine zipper, and the active domain of TRAIL (aa 95–281). The functionality of sTRAIL was validated by cell viability, FACS analysis, caspase-3 activity, and TUNEL staining. rAAV-sTRAIL was injected intratumorally to nude mice bearing human A549 lung tumor cells. Nude mice received A549 tumor cells after intravenous delivery of rAAV-sTRAIL. The antitumor effect was then evaluated by measuring tumor regression and occurrence in the experimental animal. Results sTRAIL was released from cells transfected with the sTRAIL expression construct or transduced with rAAV-sTRAIL, and induced apoptosis in cancer cells, but spared normal fibroblast cells. Secreted sTRAIL formed oligomers including trimers with intersubunit disulfide. Purified sTRAIL exerted much lower cytotoxicity on primary human hepatocytes compared to recombinant TRAIL. Intratumoral delivery of rAAV-sTRAIL significantly inhibited growth of A549 tumors established in nude mice. A number of apoptotic tumor cells were detected by TUNEL staining in mice treated with rAAV-sTRAIL. Systemic pretreatment with rAAV-sTRAIL significantly inhibited tumor formation in nude mice. Conclusion The results suggest that rAAV-sTRAIL may be useful for local or systemic cancer gene therapy for treating TRAIL-sensitive tumors.  

5.418           Mechanisms of AAV transduction in glaucoma-associated human trabecular meshwork cells

Borras, T., Xue, W., Choi, V.W., Bartlett, J.S., Li, G., Samulski, R.J. and Chisolm, S.S.

1. Gene Med., 8(5), 589-602 (2006), 589-602 (2006)

  Background Glaucoma is a chronic eye disease which leads to irreversible blindness. The trabecular meshwork tissue controls intraocular pressure (IOP), which is the major risk factor for glaucoma. Gene therapy treatment of chronic diseases requires the use of long-term expression, low toxicity and lack of immune response vectors. Adeno-associated viruses (AAV) possess these characteristics but have been unable to transduce the trabecular meshwork. Because of the importance of regulating elevated IOP by long-term gene therapy, we investigated mechanisms of AAV transduction to the human trabecular meshwork (TM). Methods Primary human trabecular meshwork cells (HTM) and perfused organ cultures were infected with rAAV2-GFP, RGD-pseudotyped rAAV2-GFP alone, or combined with recombinant ΔE1/E3 adenoviruses. Intracellular rAAV2 DNA and RNA were measured by relative quantitative and real-time TaqMan polymerase chain reaction (PCR). Host transcriptome was analyzed using high-density oligonucleotide microarrays. One transduction mechanism was tested using self-complementary AAV (scAAV). Results The dramatic transduction enhancement obtained upon co-infection of rAAV2 with ΔE1/E3 adenoviruses provides insights into transduction mechanisms in the HTM. Even if not transduced, rAAV2 enters TM cells. GeneChip analysis showed significant changes in host genes involved in cell cycle and DNA replication. Consequently, scAAV-GFP transduction was highly efficient. Other transduction-enhancement genes included coxsackie adenovirus receptor (CAR) and genes relevant to trabecular meshwork function. Conclusions The rate-limiting step of AAV transduction was not viral entry failure but, at least in part, host downregulation of DNA replication. Additional specific host genes might be involved. Our study revealed genes and mechanisms which led for the first time to efficient AAV transduction of the HTM.  

5.419           Humoral immune responses against minute virus of mice vectors

Lang, S.I., Giese, N.A., Rommelaere, J., Dinsart, C. and Cornelis, J.J.

1. Gene Med., 8(9), 1141-1150 (2006)

  Background Owing to their oncolytic properties, autonomous rodent parvoviruses and derived vectors constitute potential anti-tumor agents. Methods Humoral immune responses to minute virus of mice (MVMp) were characterized. In particular, the generation of neutralizing antibodies on subsequent therapeutic virus applications was evaluated in a mouse melanoma model. Mice bearing subcutaneous melanomas were injected intratumorally with virus and re-injected 10 days later in a second tumor on the other flank. Four days after the first or second injection, the tumors and lymph nodes were analyzed by RT-PCR for gene expression. Results Injection of MVMp in tumor-bearing B6 mice resulted in viral gene expression in tumors and draining lymph nodes. A repeated virus administration did not lead to detectable viral transcription if it was preceded by a virus infection 10 days earlier. This protection correlated with the induction of virus-neutralizing antibodies following the first virus application. The restrictions on viral gene expression after a consecutive MVMp injection could be alleviated in subsequent applications by the use of viruses consisting of MVMp genomes packaged into capsids of a related parvovirus. Neutralizing antibody induction was irrespective of the route of administration and of the presence of a tumor and persisted at significant levels at least up to 26 weeks after the viral infection. MVMp infection of B6 mice stimulated the generation of IgM and IgG anti-viral antibodies, the latter mainly of the T-helper (Th) 1-dependent IgG2, and the T-cell-independent IgG3 subclasses. Conclusions Neutralizing antibodies impede the effectiveness of a subsequent virus administration, but can be overcome by pseudotyping.  

5.420           Isolation of targeted AAV2 vectors from novel virus display libraries

Waterkamp, D.A., Müller, O.J., Ying, Y., Trepel, M. and Kleinschmidt, J.A.

1. Gene Med., 8(11), 1307-1319 (2006)

  Random peptide ligands displayed on viral capsids are emerging tools for selection of targeted gene transfer vectors even without prior knowledge of the potential target cell receptor. We have previously introduced adeno-associated viral (AAV)-displayed peptide libraries that ensure encoding of displayed peptides by the packaged AAV genome. A major limitation of these libraries is their contamination with wild-type (wt) AAV. Here we describe a novel and improved library production system that reliably avoids generation of wt AAV by use of a synthetic cap gene. Selection of targeted AAV vectors from wt-containing and the novel wt-free libraries on cell types with different permissivity for wt AAV2 replication suggested the superiority of the wt-free library. However, from both libraries highly specific peptide sequence motifs were selected which improved transduction of cells with moderate or low permissivity for AAV2 replication. Strong reduction of HeLa cell transduction compared to wt AAV2 and only low level transduction of non-target cells by some selected clones showed that not only the efficiency but also the specificity of gene transfer was improved. In conclusion, our study validates and improves the unique potential of virus display libraries for the development of targeted gene transfer vectors.  

5.421           Combined prophylactic and therapeutic cancer vaccine: Enhancing CTL responses to HPV16 E2 using a chimeric VLP in HLA-A2 mice

Qian, J., Dong, Y., Pang, Y-Y.s., Ibrahim, R., Berzofsky, J.A., Schiller, J.T. and Kheif, S.N. Int. J. Cancer, 118(12), 3022-3029 (2006)   We identified the strategies to induce a CTL response to human papillomavirus (HPV) 16 E2 in HLA-A2 transgenic mice (AAD). A chimeric HPV16 virus-like particle (VLP) that includes full length HPV16 E7 and E2 (VLP-E7E2) was generated. The combination of E2 and E7 has the advantage that E2 is expressed in early dysplasia and neoplasia lesions, where E7 is expressed in more advance lesions. Since T cell response to E2 is less defined, we first evaluated the strategies to enhancing CD8+ T cell responses to HPV E7, using different combinations of immune-modulators with VLP-E7E2. Data showed that the CTL response to E7 could be significantly enhanced by coinjection of GM-CSF and antiCD40 antibodies with chimeric VLP-E7E2 without adjuvant. However, using the same combination, a low level of CD8+ T cell response to E2 was detected. To enhance the CD8+ T cell response to E2, we analyzed T cell epitopes from E2 sequence. A heterogonous prime-boost with chimeric VLP-E7E2 and E2 peptides was performed. The data showed that the priming with chimeric VLP-E7E2, followed by boosting with E2 peptides, gave a better CTL response than 2 immunizations with E2 peptides. The enhanced immunity is due to the increase of CD11c+ and CD11c+ CD40+ double positive dendritic cells in mice that received immune-modulators, GM-CSF and antiCD40. Furthermore, the level of anti-L1 antibodies remains similar in mice immunized with chimeric VLP with/without immune-modulators. Thus, the data suggested that the chimeric VLP-E7E2 has a therapeutic potential for the treatment of HPV-associated CINs and cancer without diminishing VLPs potential as a prophylactic vaccine by inducing anti-L1 antibodies against free virus.      

5.422           Suppression of ovarian cancer by muscle-mediated expression of soluble VEGFR-1/Flt-1 using adeno-associated virus serotype 1-derived vector

Takei, Y., Mizukami, H., Saga, Y., Yoshimura, I., Hasumi, Y., Takayama, T., Kohno, T., Matsushita, T., Okada, T., Kume, A., Suzuki, M. and Ozawa, K. Int. J. Cancer, 120, 278-284 (2006)   Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis in a variety of tumors. A soluble form of Flt-1 (sFlt-1), a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidences suggest the applicability of sFlt-1 in tumor suppression by means of anti-angiogenesis. We previously demonstrated the efficacy of sflt-1 gene expression in situ to suppress tumor growth and ascites in ovarian cancer. Here, we demonstrate the therapeutic applicability of muscle-mediated expression of sFlt-1 in tumor-bearing mice. Initially, tumor suppressive action was confirmed by inoculating sFlt-1-expressing ovarian cancer (SHIN-3) cells into mice, both subcutaneously and intraperitoneally. To validate the therapeutic efficacy in a more clinically relevant model, adeno-associated virus vectors encoding sflt-1 were introduced into mouse skeletal muscles and were subsequently inoculated with tumor cells. As a result, high serum sFlt-1 levels were constantly observed, and the growth of both subcutaneously- and intraperitoneally-inoculated tumors was significantly suppressed. No delay in wound healing or adverse events of neuromuscular damage were noted, body weight did not change, and laboratory data, such as those representing liver and renal functions, were not affected. These results indicate that sFlt-1 suppresses growth and peritoneal dissemination of ovarian cancer by the inhibition of angiogenesis, and thus suggest the usefulness of gene therapy for ovarian cancer.  

5.423           Long-Lasting Regeneration After Ischemia in the Cerebral Cortex

Leker, R.R. et al Stroke, 38, 153-161 (2007)   Background and Purpose— Because fibroblast growth factor2 is a mitogen for central nervous system stem cells, we exploredwhether long-term fibroblast growth factor 2 delivery to thebrain can improve functional outcome and induce cortical neurogenesisafter ischemia. Methods— Rats underwent permanent distal middle cerebralartery occlusion resulting in an ischemic injury limited tothe cortex. We used an adeno-associated virus transfection systemto induce long-term fibroblast growth factor 2 expression andmonitored behavioral and histological changes. Results— Treatment increased the number of proliferatingcells and improved motor behavior. Neurogenesis continued throughout90 days after the ischemia, and the occurrence of newly generatedcells with characteristics of neural precursors and immatureneurons was most evident 90 days after treatment. Conclusions— Focal cortical ischemia elicits an ongoing neurogenic response that can be enhanced with fibroblast growth factor 2 leading to improved functional outcome.  

5.424           Compensatory Mutations in E1, p7, NS2, and NS3 Enhance Yields of Cell Culture-Infectious Intergenotypic Chimeric Hepatitis C Virus

Yi, M., Ma, Y., Yates, J. and Lemon, S.M.

1. Virol., 81(2), 629-638 (2007)

  There is little understanding of mechanisms underlying the assembly and release of infectious hepatitis C virus (HCV) from cultured cells. Cells transfected with synthetic genomic RNA from a unique genotype 2a virus (JFH1) produce high titers of virus, while virus yields are much lower with a prototype genotype 1a RNA containing multiple cell culture-adaptive mutations (H77S). To characterize the basis for this difference in infectious particle production, we constructed chimeric genomes encoding the structural proteins of H77S within the background of JFH1. RNAs encoding polyproteins fused at the NS2/NS3 junction ("H-NS2/NS3-J") and at a site of natural, intergenotypic recombination within NS2 ["H-(NS2)-J"] produced infectious virus. In contrast, no virus was produced by a chimera fused at the p7-NS2 junction. Chimera H-NS2/NS3-J virus (vH-NS2/NS3-J) recovered from transfected cultures contained compensatory mutations in E1 and NS3 that were essential for the production of infectious virus, while yields of infectious vH-(NS2)-J were enhanced by mutations within p7 and NS2. These compensatory mutations were chimera specific and did not enhance viral RNA replication or polyprotein processing; thus, they likely compensate for incompatibilities between proteins of different genotypes at sites of interactions essential for virus assembly and/or release. Mutations in p7 and NS2 acted additively and increased the specific infectivity of vH-(NS2)-J particles, while having less impact on the numbers of particles released. We conclude that interactions between NS2 and E1 and p7 as well as between NS2 and NS3 are essential for virus assembly and/or release and that each of these viral proteins plays an important role in this process.  

5.425           Characterization of herpes simplex virus type 1 recombinants that express and incorporate high levels of HCV E2-gC chimeric proteins

Kouvatsis, V. et al Virus Res., 123(1), 40-39 (2007)   We report the construction of two HSV-1 recombinants encoding chimeric forms of the E2 glycoprotein of HCV-1a composed of the ectodomain of E2 (aa384–611 or 384–711) fused to different parts of the transmembrane and cytoplasmic domain of the HSV-1 gC glycoprotein (gC). The parental HSV-1, known as KgBpK⁻gC⁻, is deleted for gC and the main heparan sulphate (HS) binding domain of gB, and it exhibits impaired binding (ca. 80%) to HS compared to the wild type virus KOS [Laquerre, S., Argnani, R., Anderson, D.B., Zucchini, S., Manservigi, R., Glorioso, J.C., 1998. Heparan sulphate proteoglycan binding by herpes simplex virus type 1 glycoproteins B and C, which differ in their contributions to virus attachment, penetration, and cell-to-cell spread. J. Virol. 72, 6119–6130]. We show that gC:E2 proteins are efficiently expressed and transported to the cell surface. We also demonstrate that HSV-1 can incorporate both gC:E2 chimeric proteins into particles and show that incorporation of both chimeric molecules in the viral envelope partially restored binding (ca. 20%) of the HSV-1 recombinants to heparan sulphate. Finally, we showed that the gC:E2ScaI chimeric glycoprotein was able to bind a recombinant form of hCD81 and virion-expressed gC:E2ScaI permitted the binding of the HSV-1 recombinant virus to the hCD81 molecule.  

5.426           Purification and immunogenicity study of human papillomavirus type 16 L1 protein in Saccharomyces cerevisiae

Kim, S.N., Jeong, H.S., Park, S.N. and Kim, H-J.

1. Virol. Methods, 139(1), 24-30 (2007)

  Human papillomavirus 16 virus-like particle (HPV16 VLP) vaccines expressed in Saccharomyces cerevisiae are under Phase III trial and are expected to be on the market in the near future. We have established a convenient and economical system for the prophylactic study of vaccines derived from HPV16 VLPs, and neutralization tests to standardize HPV serological methodology as a measure of validation. To purify HPV16 VLPs, yeast cells expressing HPV16 L1 protein were cultured and purified on a small scale by ultracentrifugation and size-exclusion and cation-exchange chromatography using open columns. The highly purified HPV16 L1 protein was identified by SDS-PAGE and Western blotting, and electron microscopic analysis confirmed that they self-assembled into VLPs. To test the efficacy of the purified VLPs as a vaccine and their ability to induce humoral immunity, we performed ELISA assays and observed a significant increase in the titer of anti-HPV16 VLPs antibodies in the sera of immunized mice. High anti-HPV16 neutralizing titers were found in the sera of vaccinated mice, as measured by a SEAP-based pseudovirus neutralization assay. These results would be useful in the evaluation of the immunogenicity of HPV vaccine candidates, and provide an international reference standard for HPV serological methods.  

5.427           Neuronal specificity of α-synuclein toxicity and effect of Parkin co-expression in primates

Yasuda, T. et al Neurosci., 144(2), 743-753 (2007)   Recombinant adeno-associated viral (rAAV) vector-mediated overexpression of α-synuclein (αSyn) protein has been shown to cause neurodegeneration of the nigrostriatal dopaminergic pathway in rodents and primates. Using serotype-2 rAAV vectors, we recently reported the protective effect of Parkin on αSyn-induced nigral dopaminergic neurodegeneration in a rat model. Here we investigated the neuronal specificity of αSyn toxicity and the effect of Parkin co-expression in a primate model. We used another serotype (type-1) of AAV vector that was confirmed to deliver genes of interest anterogradely and retrogradely to neurons in rats. The serotype-1 rAAV (rAAV1) carrying αSyn cDNA (rAAV1-αSyn), and a cocktail of rAAV1-αSyn and rAAV1 carrying parkin cDNA (rAAV1-parkin) were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral GABAergic and nigrostriatal dopaminergic neurons. Injection of rAAV1-αSyn alone decreased tyrosine hydroxylase immunoreactivity in the striatum compared with the contralateral side injected with a cocktail of rAAV1-αSyn and rAAV1-parkin. Immunostaining of striatonigral GABAergic neurons was similar on both sides. Overexpression of Parkin in GABAergic neurons was associated with less accumulation of αSyn protein and/or phosphorylation at Ser129 residue. Our results suggest that the toxicity of accumulated αSyn is not induced in non-dopaminergic neurons and that the αSyn-ablating effect of Parkin is exerted in virtually all neurons in primates.

5.428           Baculovirus-based Vaccination Vectors Allow for Efficient Induction of Immune Responses Against Plasmodium falciparum Circumsporozoite Protein

Strauss, R. et al Mol. Ther., 15(1), 193-202 (2007)   Baculovirus vectors are able to transduce a large variety of mammalian cell types and express transgenes placed under the control of heterologous promoters. In this study, we evaluated the potential of baculovirus vectors for malaria vaccination. To induce efficient CD4(+) and CD8(+) T-cell responses, we produced a series of vectors that display the Plasmodium falciparum circumsporozoite (CS) protein in the virion envelope and/or allow for CS expression upon transduction of mammalian cells. We found that baculovirus vectors can transduce professional antigen-presenting cells and trigger their maturation, which is a prerequisite for efficient antigen presentation. Upon intramuscular injection into mice, the vector that both displayed and expressed CS induced higher anti-CS antibody titers (of the immunoglobulin (IgG)1 and IgG2a type) and a higher frequency of interferon- -producing T cells specific to CS, than the vectors which either only displayed or only expressed CS. The baculovirus CS display/expression vector was also superior in inducing CS-specific CD4(+) and CD8(+) T-cell responses in vitro using human peripheral blood mononuclear cells from naive donors. This, together with the absence of pre-existing immunity to baculoviruses in humans, the absence of viral gene expression in mammalian cells, and the relative low immunogenicity of baculovirus virions, makes these vectors promising tools for vaccination. Furthermore, the ability to produce large amounts in serum-free medium at a low cost adds a further advantage to this vector system.  

5.429           AAV1 Mediated Co-expression of Formylglycine-Generating Enzyme and Arylsulfatase A Efficiently Corrects Sulfatide Storage in a Mouse Model of Metachromatic Leukodystr

Kurai, T. et al Mol. Ther., 15(1), 38-43 (2007)   Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA) and is characterized by deposition of sulfatide in all organs, particularly the nervous system. Recently, formylglycine-generating enzyme (FGE) was found to be essential for activation of sulfatases. This study examined the utility of FGE co-expression in AAV type 1 vector (AAV1)-mediated gene therapy of ASA knockout (MLD) mice. AAV1-ASA alone or AAV1-ASA and AAV1-FGE were co-injected into a single site of the hippocampus. Enzyme assay and immunohistochemical analysis showed that ASA was detected not only in the injected hemisphere but also in the non-injected hemisphere by 7 months after injection. Level of ASA activity and extent of ASA distribution were significantly enhanced by co-introduction of AAV1-FGE. Marked reductions in sulfatide levels were observed throughout the entire brain. The unexpectedly widespread distribution of ASA may be due to a combination of diffusion in extracellular spaces, transport through axons, and circulation in cerebrospinal fluid. The rotarod test revealed improvement of neurological functions. These results demonstrate that direct injection of AAV1 vectors expressing ASA and FGE represents a highly promising approach with significant implications for the development of clinical protocols for MLD gene therapy.  

5.430           Sequence variability of retroviral particles derived from human melanoma cells: Melanoma-associated retrovirus

Hirschl, S. et al Virus Res., 123(2), 211-215 (2007)   We have shown that melanoma cells produce viral particles that contain sequences which are homologous to human endogenous retroviruses. In this study particles derived from different melanoma cell lines and from melanoma cells of a lymph node metastasis were characterized. We determined the density and the reverse transcriptase (RT) activity of viral particles. Furthermore, we analyzed the sequence variability of multiple clones of each particle preparation. The particles were found to package sequences, which vary for each of the analyzed cell lines. Moreover, even particles derived from the same cell line contain heterologous sequences.  

5.431           Lysis of Human Immunodeficiency Virus Type 1 by a Specific Secreted Human Phospholipase A2

Kim, J-O. Et al

1. Virol., 81(3), 1441-1450 (2007)

  Phospholipase A₂ (PLA₂) proteins affect cellular activation, signal transduction, and possibly innate immunity. A specific secretory PLA₂, sPLA₂-X, is shown here to neutralize human immunodeficiency virus type 1 (HIV-1) through degradation of the viral membrane. Catalytic function was required for antiviral activity, and the target cells of infection were unaffected. sPLA₂-X potently reduced gene transfer of HIV-1 Env-pseudotyped lentivirus vectors and inhibited the replication of both CCR5- and CXCR4-tropic HIV-1 in human CD4⁺ T cells. Virions resistant to damage by antibody and complement were sensitive to lysis by sPLA₂-X, suggesting a novel mechanism of antiviral surveillance independent of the acquired immune system.  

5.432           Hybrid Adeno-Associated Virus Bearing Nonhomologous Inverted Terminal Repeats Enhances Dual-Vector Reconstruction of Minigenes In Vivo

Yan, Z, Lei-Butter, D.C.M., Zhang, Y., Zak, R. and Engelhardt, J.F. Hum. Gen. Ther., 18, 81-87 (2007)   We have previously demonstrated that hybrid adeno-associated viral (AAV) vectors bearing nonhomologous inverted terminal repeats (ITRs) enhance directional intermolecular recombination and the efficiency of dual-AAV vector trans-splicing in cultured cells. Using hybrid-ITR vectors carrying two exons of a lacZ minigene, we demonstrate that this dual-vector approach also mediates higher levels (3- to 6-fold) of gene reconstitution in mouse skeletal muscle, liver, and heart. Inhibition of the proteasome by systemic administration of Doxil (Food and Drug Administration-approved lipid-formulated doxorubicin) further enhanced dual-vector trans-splicing 6- to 12-fold in two mouse strains. Hence, using hybrid-ITR AAV vectors in combination with proteasome modulation enhanced dual-vector delivery of a transgene 36-fold over the current dual-vector trans-splicing approaches. These data provide in vivo evidence that ITR sequence-dependent homologous recombination, rather than nonhomologous end joining, is the predominant mechanism for AAV genome heterodimerization. Hence, enhanced directional recombination provided by hybrid-ITR vectors may be a useful in vivo strategy for improving dual-vector delivery of transgenes larger than the AAV packaging limit.  

5.433           Intraperitoneal gene therapy by rAAV provides long-term survival against epithelial ovarian cancer independently of survivin pathway

Isayeva, T. and Ponnazhagan, S. et al Gen. Ther., 14, 138-146 (2007)   Epithelial ovarian carcinoma is the leading cause of death from gynecological malignancies. Owing to the lack of an effective screening method, insidious onset, and non-specific symptoms, a majority of women present with advanced stage disease. Despite improvements from cytoreductive surgery and chemotherapy, recurrent disease remains a formidable challenge. In the present study, we demonstrate for the first time that stable intra-abdominal genetic transfer of endostatin and angiostatin (E+A) by recombinant adeno-associated virus (rAAV) provides sustained antitumor effects on the growth and dissemination of epithelial ovarian cancer in a mouse model. Further, when combined with paclitaxel (taxol), the effect of this therapy was dramatically increased and resulted in long-term tumor-free survival overcoming prior limitations of chemotherapy and gene therapy. The combined effects of angiosuppressive therapy and chemotherapy were found to be independently of survivin pathway. Evidence for the superior effects of the combination therapy was indicated by significantly lower ascites volume with less hemorrhage and tumor conglomerates, lower ascites vascular endothelial growth factor, higher tumor cell apoptosis and decreased blood vasculature, and long-term disease-free survival. Histopathology of visceral organs and liver enzyme assays indicated no toxicity or pathology.  

5.434           Neutralization of HPV16, 18, 31, and 58 pseudovirions with antisera induced by immunizing rabbits with synthetic peptides representing segments of the HPV16 minor capsid protein L2 surface region

Kondo, K. et al Virology, 358(2), 266-272 (2007)   Neutralizing antibody against human papillomavirus (HPV) minor capsid protein L2 can cross-neutralize different HPV genotypes in vitro. To identify the segments containing the cross-neutralization epitopes of HPV16 L2, we characterized antisera obtained by immunizing two rabbits with each of the ten synthetic peptides of 14 to 20 amino acids (aa) long, which represents a part of the HPV16 L2 sequence from aa 14 to 144. The antisera against the peptides within the region from aa 18 to 144 efficiently bound to HPV16 L1/L2-capsids and neutralized HPV16 pseudovirions, indicating that the region is displayed on the surface of the capsids and contains several neutralization epitopes. Antiserum against the peptide from aa 18 to 38 (anti-P18/38) cross-neutralized HPV18. Anti-P56/75 cross-neutralized HPV18, 31, and 58. Anti-P61/75 and anti-P64/81 cross-neutralized HPV18 and 58. Anti-P96/115 and the antiserum induced by a mutant P96/115 (S and T at aa 101 and 112 were replaced with L and S, respectively) cross-neutralized HPV31 and 58. The mixture of equal volumes of three antisera, anti-P18/38, anti-P56/75, and anti-mutant P96/115, neutralized HPV16, 18, 31, and 58 more efficiently than anti-P56/75 alone, suggesting that there is a synergistic effect of antibodies on the cross-neutralization. The cross-neutralization appears to be correlated with conserved aa sequences among HPV types. The data in this study provide a basis for designing vaccine antigens effective against a broader spectrum of the high-risk HPVs.  

5.435           The use of recombinant adeno-associated virus for skeletal gene therapy

Dai, J. and Rabie, A.B.M. Orthod. Craniofacial Res., 10, 1-14 (2007)   Objectives – To provide a comprehensive literature review describing recent developments of the recombinant adeno-associated virus (rAAV) vector and exploring the therapeutic application of rAAV for bone defects, cartilage lesions and rheumatoid arthritis. Design – Narrative review. Result – The review outlines the serotypes and genome of AAV, integration and life cycle of the rAAV vectors, the immune response and regulating system for AAV gene therapy. Furthermore, the advancements of rAAV gene therapy for bone growth together with cartilage repair are summarized. Conclusion – Recombinant adeno-associated virus vector is perceived to be one of the most promising vector systems for bone and cartilage gene therapy approaches and further investigations need to be carried out for craniofacial research.  

5.436           The Membrane Anchor R7BP Controls the Proteolytic Stability of the Striatal Specific RGS Protein, RGS9-2

Anderson., G.R., Semenov, A., Song, J.H. and Martemyanov, K.A.

1. Biol. Chem., 282(7), 4772-4791 (2007)

  A member of the RGS (regulators of G protein signaling) family, RGS9-2 is a critical regulator of G protein signaling pathways that control locomotion and reward signaling in the brain. RGS9-2 is specifically expressed in striatal neurons where it forms complexes with its newly discovered partner, R7BP (R7 family binding protein). Interaction with R7BP is important for the subcellular targeting of RGS9-2, which in native neurons is found in plasma membrane and its specializations, postsynaptic densities. Here we report that R7BP plays an additional important role in determining proteolytic stability of RGS9-2. We have found that co-expression with R7BP dramatically elevates the levels of RGS9-2 and its constitutive subunit, G 5. Measurement of the RGS9-2 degradation kinetics in cells indicates that R7BP markedly reduces the rate of RGS9-2·G 5 proteolysis. Lentivirus-mediated RNA interference knockdown of the R7BP expression in native striatal neurons results in the corresponding decrease in RGS9-2 protein levels. Analysis of the molecular determinants that mediate R7BP/RGS9-2 binding to result in proteolytic protection have identified that the binding site for R7BP in RGS proteins is formed by pairing of the DEP (Disheveled, EGL-10, Pleckstrin) domain with the R7H (R7 homology), a domain of previously unknown function that interacts with four putative -helices of the R7BP core. These findings provide a mechanism for the regulation of the RGS9 protein stability in the striatal neurons.  

5.437           α7 Nicotinic receptor gene delivery into mouse hippocampal neurons leads to functional receptor expression, improved spatial memory-related performance, and tau hyperphosphorylation

Ren, K. et al Neurosci., 145, 314.322 (2007)   Brain α7 nicotinic receptors have become therapeutic targets for Alzheimer’s disease (AD) based on their memory-enhancing and neuroprotective actions. This study investigated the feasibility of increasing neuronal α7 receptor functions using a gene delivery approach based on neuron-selective recombinant adeno-associated virus (rAAV)–derived vectors. In order to determine whether α7 receptor-mediated cytotoxicity was dependent on receptor density, rat α7 nicotinic receptors were expressed at high concentrations in GH4C1 cells as measured with nicotine-displaceable [³H]methyllycaconitine (MLA) binding. The potency of GTS-21 (an α7 receptor agonist) to induce cell loss was similar in these cells to that seen in pheochromocytoma (PC12) cells expressing nine-times-lower receptor levels, suggesting that cytotoxicity was more dependent on agonist concentration than receptor density. Hippocampal transduction with rat α7 nicotinic receptors increased [³H]MLA binding in this region in wild type and α7 receptor-knockout (KO) mice without apparent cytotoxicity. No difference was observed in Kd values for MLA binding between endogenous and transgenic receptors. Single cell recordings demonstrated that dentate granule cells that normally have no α7 receptor response did so following α7 receptor gene delivery in wild type mice. Recovery of α7 function was also observed in stratum oriens and stratum radiatum neurons of KO mice following gene delivery. Wild type mice exhibited improved acquisition performance in the Morris water task 1 month after bilateral hippocampal transductions with the rat α7 receptor gene compared with green fluorescent protein–transduced controls. However, both groups reached similar training levels and there was no difference in subsequent probe performance. Finally, this gene delivery approach was used to test whether α7 receptors affect tau-phosphorylation. Chronic (i.e. 2 month but not 2 week) expression of high levels of α7 receptors in hippocampus increased AT8 staining characteristic of hyperphosphorylated tau in that region, indicating that endogenous agonist-mediated receptor activation may be able to modulate this process.  

5.438           Leukocyte-specific protein 1 interacts with DC-SIGN and mediates transport of HIV to the proteasome in dendritic cells

Smith, A.L. et al

1. Exp. Med., 204(2), 421-430 (2007)

  Dendritic cells (DCs) capture and internalize human immunodeficiency virus (HIV)-1 through C-type lectins, including DC-SIGN. These cells mediate efficient infection of T cells by concentrating the delivery of virus through the infectious synapse, a process dependent on the cytoplasmic domain of DC-SIGN. Here, we identify a cellular protein that binds specifically to the cytoplasmic region of DC-SIGN and directs internalized virus to the proteasome. This cellular protein, leukocyte-specific protein 1 (LSP1), was defined biochemically by immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. LSP1 is an F-actin binding protein involved in leukocyte motility and found on the cytoplasmic surface of the plasma membrane. LSP1 interacted specifically with DC-SIGN and other C-type lectins, but not the inactive mutant DC-SIGN 35, which lacks a cytoplasmic domain and shows altered virus transport in DCs. LSP1 diverts HIV-1 to the proteasome. Down-regulation of LSP1 with specific small interfering RNAs in human DCs enhanced HIV-1 transfer to T cells, and bone marrow DCs from lsp1^–/– mice also showed an increase in transfer of HIV-1_BaL to a human T cell line. Proteasome inhibitors increased retention of viral proteins in lsp1^+/+ DCs, and substantial colocalization of virus to the proteasome was observed in wild-type compared with LSP1-deficient cells. Collectively, these data suggest that LSP1 protein facilitates virus transport into the proteasome after its interaction with DC-SIGN through its interaction with cytoskeletal proteins.  

5.439           Protein kinase A activates protein phosphatase 2A by phosphorylation of the B56 subunit

Ahn, J-H. et al PNAS, 104(8), 2979-2984 (2007)   Our previous studies of DARPP-32 in striatal slices have shown that activation of D1 receptors leads to cAMP-dependent dephosphorylation of Thr-75, the Cdk5 site in DARPP-32. In the current study, we have elucidated a mechanism whereby protein phosphatase 2A (PP2A) is activated by a cAMP/PKA-dependent pathway, leading to dephosphorylation of Thr-75. PP2A consists of a catalytic C subunit that associates with the scaffolding A subunit and a variety of B subunits. We have found that the A/C subunits of PP2A, in association with the B56 (or PPP2R5D) regulatory subunit, is an active DARPP-32 phosphatase. The B56 subunit expressed in HEK293 cells forms a heterotrimeric assembly that catalyzes PKA-mediated dephosphorylation at Thr-75 in DARPP-32 (also cotransfected into HEK293 cells). The B56 subunit is phosphorylated by PKA, and this increases the overall activity of PP2A in vitro and in vivo. Among four PKA-phosphorylation sites identified in B56 in vitro, Ser-566 was found to be critical for the regulation of PP2A activity. Moreover, Ser-566 was phosphorylated by PKA in response to activation of D1 receptors in striatal slices. Based on these studies, we propose that the B56 /A/C PP2A complex regulates the dephosphorylation of DARPP-32 at Thr-75, thereby helping coordinate the efficacy of dopaminergic neurotransmission in striatal neurons. Moreover, stimulation of protein phosphatase activity by this mechanism may represent an important signaling pathway regulated by cAMP in neurons and other types of cell.            

5.440           Controlled delivery of glial cell line-derived neurotrophic factor by a single tetracycline-inducible AAV vector

Chtarto, A. et al Exp. Neurol., 204, 387-399 (2007)   An autoregulated tetracycline-inducible recombinant adeno-associated viral vector (rAAV-pTet_bidiON) utilizing the rtTAM2 reverse tetracycline transactivator (rAAV-rtTAM2) was used to conditionally express the human GDNF cDNA. Doxycycline, a tetracycline analog, induced a time- and dose-dependent release of GDNF in vitro in human glioma cells infected with rAAV-rtTAM2 serotype 2 virus. Introducing the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) downstream to the rtTAM2 coding sequence, resulted in a more rapid induction and a higher basal expression level. In vivo, 8 weeks after a single injection of the rAAV-rtTAM2-GDNF vector encapsidated into AAV serotype 1 capsids in the rat striatum, the GDNF protein level was 60 pg/mg tissue in doxycycline-treated animals whereas in untreated animals, it was undistinguishable from the endogenous level (  4 pg/mg tissue). However, a residual GDNF expression in the uninduced animals was evidenced by a sensitive immunohistochemical staining. As compared to rAAV1-rtTAM2-GDNF, the rAAV1-rtTAM2-WPRE-GDNF vector expressed a similar concentration of GDNF in the induced state (with doxycycline) but a basal level (without doxycycline)  2.5-fold higher than the endogenous striatal level. As a proof for biological activity, for both vectors, downregulation of tyrosine hydroxylase was evidenced in dopaminergic terminals of doxycycline-treated but not untreated animals. In conclusion, the rAAV1-rtTAM2 vector which expressed biologically relevant doses of GDNF in the striatum in response to doxycycline with a basal level undistinguishable from the endogenous striatal level, as measured by quantitative ELISA assay, constitutes an interesting tool for local conditional transgenesis.  

5.441           Expression and function of the OX40/OX40L costimulatory pair during herpes stromal keratitis

Lepisto, A.J., Xu, M., Yagita, H., Weinberg, A.D. and Hendricks, R.L.

1. Leukoc. Biol., 81(3), 766-774 (2007)

  Herpes stromal keratitis (HSK) is an immunopathological disease regulated by Th1 CD4 T cells, which require APC and costimulation within the infected cornea to mediate disease. Recent studies suggest the OX40:OX40 ligand (OX40L) interaction enhances effector cell cytokine secretion at inflammatory sites. OX40⁺ cells were detected in HSV-1-infected mouse corneas as early as 3 days postinfection (dpi), prior to the onset of HSK, and their frequency increased through 15 dpi, when all mice exhibited severe HSK. OX40L⁺ cells were first detected at 7 dpi, coincident with the initiation of HSK. It is interesting that the OX40L⁺ cells did not coexpress MHC Class II or the dendritic cell (DC) marker CD11c. Our findings demonstrate rapid infiltration of activated (OX40⁺) CD4⁺ T cells into HSV-1-infected corneas and expression of OX40L on MHC Class II-negative cells but surprisingly, not on MHC Class II⁺ CD11c⁺ DC, which are present in the infected corneas and required for HSK. Moreover, neither local nor systemic treatment of mice with a blocking antibody to OX40L or with a blocking fusion protein altered the course of HSK significantly, possibly as a result of a lack of OX40L expression on functional APC.  

5.442           The proteasome inhibitor bortezomib acts differently in combination with p53 gene transfer or cytotoxic chemotherapy on NSCLC cells

Neukirchen, J. et al Cancer Gene Therapy, 14, 431-439 (2007)   In this report, the effects of a combined treatment with the proteasome inhibitor bortezomib and either a recombinant adeno-associated virus type 2 (rAAV-2)-mediated p53 gene transfer or chemotherapeutic agents, docetaxel and pemetrexed, were tested on p53 positive and p53negative non-small cell lung cancer (NSCLC) cell lines. The combination of bortezomib and rAAV-p53 led to a significant synergistic inhibition of cell growth between 62–82% depending on the p53 status of the cell line and drug concentration. Surviving cells of the combined treatment showed a significant reduced ability to form colonies. Enhanced cell toxicity was associated with a 5.3–14.4-fold increase of the apoptotic rate and intracellular p53 level up to 50.4% following vector-mediated p53 restoration and bortezomib treatment. In contrast, an antagonistic effect on tumor cell growth and colony formation was observed for the combination of bortezomib and docetaxel or pemetrexed as a reduction of cell growth between 31 and 48% was found in comparison to 50% using the single agents. Lower cytotoxic effects were associated with significantly reduced apoptosis and an increase of clonogenic growth. The observed antagonistic effects between bortezomib and docetaxel or pemetrexed might influence clinical trials using these compounds. Conversely, p53 restoration and bortezomib treatment led to enhanced, synergistic tumor cell toxicity.

5.443           Long-term consequences of human alpha-synuclein overexpression in the primate ventral midbrain

Eslamboli, A. et al Brain, 130, 799-815 (2007)   Overexpression of human -synuclein ( -syn) using recombinant adeno-associated viral (rAAV) vectors provides a novel tool to study neurodegenerative processes seen in Parkinson's disease and other synucleinopathies. We used a pseudotyped rAAV2/5 vector to express human wild-type (wt) -syn, A53T mutated -syn, or the green fluorescent protein (GFP) in the primate ventral midbrain. Twenty-four adult common marmosets (Callithrix jacchus) were followed with regular behavioural tests for 1 year after transduction. -Syn overexpression affected motor behaviour such that all animals remained asymptomatic for at least 9 weeks, then motor bias comprising head position bias and full body rotations were seen in wt- -syn expressing animals between 15 and 27 weeks; in the later phase, the animals overexpressing the A53T -syn, in particular, showed a gradual worsening of motor performance, with increased motor coordination errors. Histological analysis from animals overexpressing either the wt or A53T -syn showed prominent degeneration of dopaminergic fibres in the striatum. In the ventral midbrain, however, the dopaminergic neurodegeneration was more prominent in the A53T group than in the WT group suggesting differential toxicity of these two proteins in the primate brain. The surviving cell bodies and their processes in the substantia nigra were stained by antibodies to the pathological form of -syn that is phosphorylated at Ser position 129. Moreover, we found, for the first time, ubiquitin containing aggregates after overexpression of -syn in the primate midbrain. There was also a variable loss of oligodendroglial cells in the cerebral peduncle. These histological and behavioural data suggest that this model provides unique opportunities to study progressive neurodegeneration in the dopaminergic system and deposition of -syn and ubiquitin similar to that seen in Parkinson's disease, and to test novel therapeutic targets for neuroprotective strategies.  

5.444           Human herpesvirus 8 acute infection of endothelial cells induces monocyte chemoattractant protein 1–dependent capillary-like structure formation: role of the IKK/NF- B pathway

Caselli, E. et al Blood, 109(7), 2718-2726 (2007)   Human herpesvirus 8 (HHV-8) is considered the causative agent of Kaposi sarcoma, a highly vascularized neoplasm characterized by spindle-shaped cells of endothelial origin and inflammatory cell infiltration. The cell transforming ability of HHV-8 has been associated with the activation of NF- B, a nuclear factor playing a pivotal role in promoting inflammation and cell proliferation; however, little is known about NF- B activation during acute HHV-8 infection. In the present study, we used a recently established in vitro model of HHV-8 acute productive infection in endothelial cells to investigate the effect of HHV-8 on NF- B activity and function. HHV-8 rapidly and potently induced NF- B activity in endothelial cells via stimulation of the I B kinase (IKK). Following IKK activation, HHV-8 selectively triggered the production of high levels of monocyte chemoattractant protein 1 (MCP-1), whereas it did not affect the expression of other NF- B–dependent proinflammatory proteins, including TNF- , IL-8, and RANTES. Deletion of NF- B–binding sites in the MCP-1 enhancer resulted in significant inhibition of HHV-8–induced transcription. Furthermore, MCP-1 production was accompanied by virus-induced capillary-like structure formation at early stages of infection. The results suggest that HHV-8–induced MCP-1 may play an important role in promoting inflammation and pathogenic angiogenesis typical of HHV-8–associated lesions.  

5.445           Expression of a synapsin IIb site 1 phosphorylation mutant in 3T3-L1 adipocytes inhibits basal intracellular retention of Glut4

Muretta, J.M., Romenskaia, I., Cassiday, P.A. and Mastick, C.C.

1. Cell Sci., 120, 1168-1177 (2007)

  Glut4 exocytosis in adipocytes uses protein machinery that is shared with other regulated secretory processes. Synapsins are phosphoproteins that regulate a `reserve pool' of vesicles clustered behind the active zone in neurons. We found that adipocytes (primary cells and the 3T3-L1 cell line) express synapsin IIb mRNA and protein. Synapsin IIb co-localizes with Glut4 in perinuclear vesicle clusters. To test whether synapsin plays a role in Glut4 traffic, a site 1 phosphorylation mutant (S10A synapsin) was expressed in 3T3-L1 adipocytes. Interestingly, expression of S10A synapsin increased basal cell surface Glut4 almost fourfold (50% maximal insulin effect). Insulin caused a further twofold translocation of Glut4 in these cells. Expression of the N-terminus of S10A synapsin (amino acids 1-118) was sufficient to inhibit basal Glut4 retention. Neither wild-type nor S10D synapsin redistributed Glut4. S10A synapsin did not elevate surface levels of the transferrin receptor in adipocytes or Glut4 in fibroblasts. Therefore, S10A synapsin is inhibiting the specialized process of basal intracellular retention of Glut4 in adipocytes, without affecting general endocytic cycling. While mutant forms of many proteins inhibit Glut4 exocytosis in response to insulin, S10A synapsin is one of only a few that specifically inhibits Glut4 retention in basal adipocytes. These data indicate that the synapsins are important regulators of membrane traffic in many cell types.  

5.446           HPV16 L1 capsid protein expressed from viable adenovirus recombinants elicits neutralizing antibody in mice

Berg, M. et al Vaccine, 25, 3501-3510 (2007)   Immunization against human papillomavirus (HPV) infection promises to reduce the worldwide burden of cervical cancer. To evaluate the potential of live recombinant adenoviruses for induction of HPV infection-blocking immunity, we prepared viable adenovirus recombinants that express the HPV16 L1 gene from the adenovirus major late transcriptional unit. Adenovirus-produced HPV16 L1 assembles into virus-like particles (VLPs) in infected cells in culture. Purified HPV16 VLPs are recognized by HPV16 neutralizing antibodies and induce high neutralizing titers when injected intraperitoneally into mice. Canine oral papillomavirus VLPs derived from previously described recombinants also induce strong antibody responses in mice. These data support our suggestion that viable adenovirus recombinants will be able to induce protective immunity to papillomavirus infection during replication in human vaccinees.  

5.447           Production of Infectious Hepatitis C Virus of Various Genotypes in Cell Cultures

Kato, T. et al

1. Virol., 81(9), 4405-4411 (2007)

  A unique hepatitis C virus (HCV) strain JFH-1 has been shown to replicate efficiently in cell culture with production of infectious HCV. We previously developed a DNA expression system containing HCV cDNA flanked by two self-cleaving ribozymes to generate HCV particles in cell culture. In this study, we produced HCV particles of various genotypes, including 1a (H77), 1b (CG1b), and 2a (J6 and JFH-1), in the HCV-ribozyme system. The constructs also contain the secreted alkaline phosphatase gene to control for transfection efficiency and the effects of culture conditions. After transfection into the Huh7-derived cell line Huh7.5.1, continuous HCV replication and secretion were confirmed by the detection of HCV RNA and core antigen in the culture medium. HCV replication levels of strains H77, CG1b, and J6 were comparable, whereas the JFH-1 strain replicates at a substantially higher level than the other strains. To evaluate the infectivity in vitro, the culture medium of JFH-1-transfected cells was inoculated into naive Huh7.5.1 cells. HCV proteins were detected by immunofluorescence 3 days after inoculation. To evaluate the infectivity in vivo, the culture medium from HCV genotype 1b-transfected cells was inoculated into a chimpanzee and caused a typical course of HCV infection. The HCV 1b propagated in vitro and in vivo had sequences identical to those of the HCV genomic cDNA used for cell culture transfection. The development of culture systems for production of various HCV genotypes provides a valuable tool not only to study the replication and pathogenesis of HCV but also to screen for antivirals.  

5.448           Live Covisualization of Competing Adeno-Associated Virus and Herpes Simplex Virus Type 1 DNA Replication: Molecular Mechanisms of Interaction

Glauser, D.L. et al

1. Virol., 81(9), 4732-4743 (2007)

  We performed live cell visualization assays to directly assess the interaction between competing adeno-associated virus (AAV) and herpes simplex virus type 1 (HSV-1) DNA replication. Our studies reveal the formation of separate AAV and HSV-1 replication compartments and the inhibition of HSV-1 replication compartment formation in the presence of AAV. AAV Rep is recruited into AAV replication compartments but not into those of HSV-1, while the single-stranded DNA-binding protein HSV-1 ICP8 is recruited into both AAV and HSV-1 replication compartments, although with differential staining patterns. Slot blot analysis of coinfected cells revealed a dose-dependent inhibition of HSV-1 DNA replication by wild-type AAV but not by rep-negative recombinant AAV. Consistent with this, Western blot analysis indicated that wild-type AAV affects the levels of the HSV-1 immediate-early protein ICP4 and the early protein ICP8 only modestly but strongly inhibits the accumulation of the late proteins VP16 and gC. Furthermore, we demonstrate that the presence of Rep in the absence of AAV DNA replication is sufficient for the inhibition of HSV-1. In particular, Rep68/78 proteins severely inhibit the formation of mature HSV-1 replication compartments and lead to the accumulation of ICP8 at sites of cellular DNA synthesis, a phenomenon previously observed in the presence of viral polymerase inhibitors. Taken together, our results suggest that AAV and HSV-1 replicate in separate compartments and that AAV Rep inhibits HSV-1 at the level of DNA replication.  

5.449           From the Cover: Mania-like behavior induced by disruption of CLOCK

Roybal, K. et al PNAS, 104(15), 6406-6411 (2007)   Circadian rhythms and the genes that make up the molecular clock have long been implicated in bipolar disorder. Genetic evidence in bipolar patients suggests that the central transcriptional activator of molecular rhythms, CLOCK, may be particularly important. However, the exact role of this gene in the development of this disorder remains unclear. Here we show that mice carrying a mutation in the Clock gene display an overall behavioral profile that is strikingly similar to human mania, including hyperactivity, decreased sleep, lowered depression-like behavior, lower anxiety, and an increase in the reward value for cocaine, sucrose, and medial forebrain bundle stimulation. Chronic administration of the mood stabilizer lithium returns many of these behavioral responses to wild-type levels. In addition, the Clock mutant mice have an increase in dopaminergic activity in the ventral tegmental area, and their behavioral abnormalities are rescued by expressing a functional CLOCK protein via viral-mediated gene transfer specifically in the ventral tegmental area. These findings establish the Clock mutant mice as a previously unrecognized model of human mania and reveal an important role for CLOCK in the dopaminergic system in regulating behavior and mood.  

5.450           Exploring the contribution of distal P4 promoter elements to the oncoselectivity of Minute Virus of Mice

Paglino, J., Burnett, E. and Tattersall, P. Virology, 361(1), 174-184 (2007)   Minute Virus of Mice (MVM) shares inherent oncotropic properties with other members of the genus Parvovirus. Two elements responsible, at least in part, for this oncoselectivity have been mapped to an Ets1 binding site adjacent to the P4 TATA box of the initiating promoter, P4, and to a more distal cyclic AMP responsive element (CRE), located within the telomeric hairpin stem. Here the CRE overlaps one half-site for the binding of parvoviral initiation factor (PIF), which is essential for viral DNA replication. We used a degenerate oligonucleotide selection approach to show that CRE binding protein (CREB) selects the sequence ACGTCAC within this context, rather than its more generally accepted palindromic TGACGTCA recognition site. We have developed strategies for manipulating these sequences directly within the left-end palindrome of the MVM infectious clone and used them to clone mutants whose CRE either matches the symmetric consensus sequence or is scrambled, or in which the PIF binding site is incrementally weakened with respect to the CRE. The panel of mutants were tested for fitness relative to wildtype in normal murine fibroblasts A9 or transformed human fibroblasts 324 K, through multiple rounds of growth in co-infected cultures, using a differential real-time quantitative PCR assay. We confirmed that inactivating the CRE substantially abrogates oncoselectivity, but found that improving its fit to the palindromic consensus is somewhat debilitating in either cell type. We also confirmed that reducing the PIF half-site spacing by one basepair enhances oncoselectivity, but found that a further basepair deletion significantly reduces this effect.  

5.451           Engineering Adeno-Associated Virus for One-Step Purification via Immobilized Metal Affinity Chromatography

Koerber, J.T., Jang, J-H., Yu, J.H., Kane, R.S. and Schaffer, D.V. Human Gene Ther., 18, 367-378 (2007)   Adeno-associated virus (AAV) is a promising vehicle for gene therapy, which will rely on the generation of high-titer, high-purity recombinant vectors. However, numerous purification protocols can involve challenging optimization or scalability issues, and most AAV serotypes do not bind heparin or sialic acid, used for AAV2/3 or AAV4/5 purification, requiring the development of new chromatography strategies. Immobilized metal affinity chromatography (IMAC) allows for robust protein purification via affinity tags such as the hexahistidine (His₆) sequence. Through the combination of a diverse AAV2 library and rational peptide insertions, we have located an optimal His₆ tag insertion site within the viral capsid. This mutant and a related AAV8 variant can be purified from clarified cell lysate in a single gravity column step at infectious particle yields exceeding 90%. Furthermore, injection of IMAC-purified vector into the brain demonstrates that it mediates high-efficiency gene delivery in vivo, equivalent to that of wild-type capsid, with minimal immune cell activation. This affinity chromatography method may offer advantages in ease of purification, final vector purity, and process scalability. Moreover, a combined rational design and high-throughput library selection approach can aid in the design of enhanced viral gene delivery vectors.  

5.452           Binding and neutralization efficiencies of monoclonal antibodies, Fab fragments, and scFv specific for L1 epitopes on the capsid of infectious HPV particles

Culp, T.D., Spatz, C.M., Reed, C.A. and Christensen, N.D. Virology, 361, 435-446 (2007)   We compared the neutralization abilities of individual monoclonal antibodies (MAb) of two large panels reactive with L1 epitopes of HPV-11 or HPV-16. Binding titers were compared using both L1-only VLPs and L1/L2 pseudovirions. While the VLPs were antigenically similar to the pseudovirions, clear differences in the surface exposure of some epitopes were evident with the HPV-16 particles. To determine whether all antibody binding events are equivalent in their neutralizing effect on infectious HPV virions or pseudovirions, the binding and neutralization titers for individual MAbs were used to calculate the relative neutralization efficiency for each antibody. HPV neutralization was achieved by all MAbs capable of strong binding to either linear or conformation-sensitive epitopes on pseudovirus particles. Our data suggest, however, that some L1 epitopes may be more neutralization-sensitive than other surface epitopes, in that successful infection can be blocked by varying degrees of epitope saturation. Additionally, the effective neutralization of virions by several monovalent Fab fragments and single-chain variable fragments (scFv) demonstrates that viral neutralization does not require HPV particle aggregation or L1 crosslinking. Identification of capsid protein structures rich in neutralization-sensitive epitopes may aid in the development of improved recombinant vaccines capable of eliciting effective and long-term antibody-mediated protection against multiple HPV types.  

5.453           SIK1 is a class II HDAC kinase that promotes survival of skeletal myocytes

Berdeaux, R. et al Nature Med., 13(5), 597-603 (2007)   During physical exercise, increases in motor neuron activity stimulate the expression of muscle-specific genes through the myocyte enhancer factor 2 (MEF2) family of transcription factors. Elevations in intracellular calcium increase MEF2 activity via the phosphorylation-dependent inactivation of class II histone deacetylases (HDACs). In studies to determine the role of the cAMP responsive element binding protein (CREB) in skeletal muscle, we found that mice expressing a dominant-negative CREB transgene (M-ACREB mice) exhibited a dystrophic phenotype along with reduced MEF2 activity. Class II HDAC phosphorylation was decreased in M-ACREB myofibers due to a reduction in amounts of Snf1lk (encoding salt inducible kinase, SIK1), a CREB target gene that functions as a class II HDAC kinase. Inhibiting class II HDAC activity either by viral expression of Snf1lk or by the administration of a small molecule antagonist improved the dystrophic phenotype in M-ACREB mice, pointing to an important role for the SIK1-HDAC pathway in regulating muscle function.  

5.454           Targeted high-efficiency, homogeneous myocardial gene transfer

Sasano, T., Kikuchi, K., McDonald, A.D., Lai, S. and Donahue, J.K.

1. Mol. Cell. Cardiol., 42(5), 954-961 (2007)

  Myocardial gene therapy continues to show promise as a tool for investigation and treatment of cardiac disease. Progress toward clinical approval has been slowed by limited in vivo delivery methods. We investigated the problem in a porcine model, with an objective of developing a method for high efficiency, homogeneous myocardial gene transfer that could be used in large mammals, and ultimately in humans. Eighty-one piglets underwent coronary catheterization for delivery of viral vectors into the left anterior descending artery and/or the great cardiac vein. The animals were followed for 5 or 28 days, and then transgene efficiency was quantified from histological samples. The baseline protocol included treatment with VEGF, nitroglycerin, and adenosine followed by adenovirus infusion into the LAD. Gene transfer efficiency varied with choice of viral vector, with use of VEGF, adenosine, or nitroglycerin, and with calcium concentration. The best results were obtained by manipulation of physical parameters. Simultaneous infusion of adenovirus through both left anterior descending artery and great cardiac vein resulted in gene transfer to 78 ± 6% of myocytes in a larger target area. This method was well tolerated by the animals. We demonstrate targeted, homogeneous, high efficiency gene transfer using a method that should be transferable for eventual human usage.  

5.455           Immunization with hepatitis C virus-like particles results in control of hepatitis C virus infection in chimpanzees

Elmowalid, G.A. et al PNAS, 104(20), 8427-8432 (2007)   Recombinant hepatitis C virus (HCV)-like particles (HCV-LPs) containing HCV structural proteins (core, E1, and E2) produced in insect cells resemble the putative HCV virions and are capable of inducing strong and broad humoral and cellular immune responses in mice and baboons. Here, we present evidence on the immunogenicity and induction of protective immunity by HCV-LPs in chimpanzees. Chimpanzees (two in each group), were immunized with HCV-LPs or HCV-LPs plus AS01B adjuvant. After immunizations, all animals developed an HCV-specific immune response including IFN- ⁺, IL-2⁺, CD4⁺, and CD8⁺ T cell and proliferative lymphocyte responses against core, E1, and E2. Upon challenge with an infectious HCV inoculum, one chimpanzee developed transient viremia with low HCV RNA titers (10³ to 10⁴ copies per ml) in the third and fourth weeks after the challenge. The three other chimpanzees became infected with higher levels of viremia (10⁴ to 10⁵ copies per ml), but their viral levels became unquantifiable (<10³ copies per ml) 10 weeks after the challenge. After the HCV challenge, all four chimpanzees demonstrated a significant increase in peripheral and intrahepatic T cell and proliferative responses against the HCV structural proteins. These T cell responses coincided with the fall in HCV RNA levels. Four naïve chimpanzees were infected with the same HCV inoculum, and three developed persistent infection with higher viremia in the range of 10⁵ to 10⁶ copies per ml. Our study suggests that HCV-LP immunization induces HCV-specific cellular immune responses that can control HCV challenge in the chimpanzee model.  

5.456           Functional Requirements of the Yellow Fever Virus Capsid Protein

Patkar, C.G., Jones, C.T., Chang, Y-h., Warrier, R. and Kuhn, R.J.

1. Virol., 81(12), 6471-6481 (2007)

  Although it is known that the flavivirus capsid protein is essential for genome packaging and formation of infectious particles, the minimal requirements of the dimeric capsid protein for virus assembly/disassembly have not been characterized. By use of a trans-packaging system that involved packaging a yellow fever virus (YFV) replicon into pseudo-infectious particles by supplying the YFV structural proteins using a Sindbis virus helper construct, the functional elements within the YFV capsid protein (YFC) were characterized. Various N- and C-terminal truncations, internal deletions, and point mutations of YFC were analyzed for their ability to package the YFV replicon. Consistent with previous reports on the tick-borne encephalitis virus capsid protein, YFC demonstrates remarkable functional flexibility. Nearly 40 residues of YFC could be removed from the N terminus while the ability to package replicon RNA was retained. Additionally, YFC containing a deletion of approximately 27 residues of the C terminus, including a complete deletion of C-terminal helix 4, was functional. Internal deletions encompassing the internal hydrophobic sequence in YFC were, in general, tolerated to a lesser extent. Site-directed mutagenesis of helix 4 residues predicted to be involved in intermonomeric interactions were also analyzed, and although single mutations did not affect packaging, a YFC with the double mutation of leucine 81 and valine 88 was nonfunctional. The effects of mutations in YFC on the viability of YFV infection were also analyzed, and these results were similar to those obtained using the replicon packaging system, thus underscoring the flexibility of YFC with respect to the requirements for its functioning.  

5.457           Brain area, age and viral vector-specific glial cell-line-derived neurotrophic factor expression and transport in rat

Kanter-Schlifke, I., Georgievska, B., Kirik, D. And Kokaia, M. Neuroreport, 18(9), 845-850 (2007)   We investigated the feasibility of viral vector-mediated expression and axonal transport of the glial cell-line-derived neurotrophic factor, a potential antiepileptic agent, to the hippocampus and the piriform cortex, areas involved in the induction and spread of seizure activity. Glial cell-line-derived neurotrophic factor overexpression was induced by injections of recombinant vectors derived from serotype 2 adeno-associated virus or lentivirus. We found that recombinant adeno-associated viral vector was able to effectively transduce mitral cells of the olfactory bulb and pyramidal cells of CA1, resulting in transport of glial cell-line-derived neurotrophic factor to the piriform cortex and to the contralateral CA1 area, respectively. These data suggest that the recombinant adeno-associated viral vector vector system is an optimal alternative for therapeutic glial cell-line-derived neurotrophic factor gene transduction and transport of the protein to the epileptogenic brain areas.

5.458           The B''/PR72 subunit mediates Ca2+-dependent dephosphorylation of DARPP-32 by protein phosphatase 2A

Ahn, J-H. et al PNAS, 104(23), 9876-9881 (2007)   In dopaminoceptive neurons, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) plays a central role in integrating the effects of dopamine and other neurotransmitters. Phosphorylation of DARPP-32 at Thr-34 by protein kinase A results in inhibition of protein phosphatase 1 (PP1), and phosphorylation at Thr-75 by Cdk5 (cyclin-dependent kinase 5) results in inhibition of protein kinase A. Dephosphorylation at Thr-34 involves primarily the Ca²⁺-dependent protein phosphatase, PP2B (calcineurin), whereas dephosphorylation of Thr-75 involves primarily PP2A, the latter being subject to control by both cAMP- and Ca²⁺-dependent regulatory mechanisms. In the present study, we have investigated the mechanism of Ca²⁺-dependent regulation of Thr-75 by PP2A. We show that the PR72 (or B'' or PPP2R3A) regulatory subunit of PP2A is highly expressed in striatum. Through the use of overexpression and down-regulation by using RNAi, we show that PP2A, in a heterotrimeric complex with the PR72 subunit, mediates Ca²⁺-dependent dephosphorylation at Thr-75 of DARPP-32. The PR72 subunit contains two Ca²⁺ binding sites formed by E and F helices (EF-hands 1 and 2), and we show that the former is necessary for the ability of PP2A activity to be regulated by Ca²⁺, both in vitro and in vivo. Our studies also indicate that the PR72-containing form of PP2A is necessary for the ability of glutamate acting at -amino-3-hydroxy-5-methylisoxazole-4-propionic acid and NMDA receptors to regulate Thr-75 dephosphorylation. These studies further our understanding of the complex signal transduction pathways that regulate DARPP-32. In addition, our studies reveal an alternative intracellular mechanism whereby Ca²⁺ can activate serine/threonine phosphatase activity.  

5.459           Seizure Suppression by GDNF Gene Therapy in Animal Models of Epilepsy

Kanter-Schlifke, I., Georgievska, B., Kirik, D. and Kokaia, M. Mol. Ther., 15(6), 1106-1113 (2007)   Temporal lobe epilepsy patients remain refractory to available anti-epileptic drugs in 30% of cases, indicating a need for novel therapeutic strategies. In this context, glial cell line–derived neurotrophic factor (GDNF) emerges as a possible new agent for epilepsy treatment. However, a limited number of studies, use of different epilepsy models, and different methods of GDNF delivery preclude understanding of the mechanisms for the seizure-suppressant action of GDNF. Here we show that recombinant adeno-associated viral (rAAV) vector–based GDNF overexpression in the rat hippocampus suppresses seizures in two models of temporal lobe epilepsy. First, when rAAV-GDNF was injected before hippocampal kindling, the number of generalized seizures decreased, and the prolongation of behavioral convulsions in fully kindled animals was prevented. Second, injection of rAAV-GDNF after kindling increased the seizure induction threshold. Third, rAAV-GDNF decreased the frequency of generalized seizures during the self-sustained phase of status epilepticus. Our data demonstrate the complexity of mechanisms and the beneficial action of GDNF in epilepsy. Furthermore, we show that ectopic rAAV-mediated GDNF gene expression in the seizure focus is a feasible way to mitigate seizures and provides proof of principle that the neurotrophic factor–based gene therapy approach has the potential to be developed as alternative strategy for epilepsy treatment.  

5.460           Immunogenicity of recombinant human immunodeficiency virus type 1-like particles expressing gp41 derivatives in a pre-fusion state

Kum, M., Qiao, Z., Yu, J., Montefiori, D. and Reinherz, E. L. Vaccine, 25, 5102-5114 (2007)   The conserved membrane proximal external region (MPER) of the ectodomain of human immunodeficiency virus type 1 (HIV-1) gp41 is the target of two broadly neutralizing antibodies, 2F5 and 4E10. However, no neutralizing antibodies have been elicited against immunogens bearing these epitopes. Given that structural and biochemical studies suggest that the lipid membrane of the virion is involved in their proper configuration, HIV-1 gp41 derivatives in a pre-fusion state were expressed on the surface of immature virus like particles (VLP) derived from Sf9 cells. Guinea pigs were immunized with three doses of VLPs or Sf9 cells presenting gp41 derivatives with or without E. coli heat-labile enterotoxin (LT) as an adjuvant. While immune sera contained high titer anti-VLP antibodies, the specific anti-gp41 antibody responses were low with no neutralizing antibodies detected. An explanation for this absence may be the low level of gp41 expression relative to the many other proteins derived from host cells which are incorporated onto the VLP surface. In addition, the anti-gp41 immune response was preferentially directed to the C-helical domain, away from the MPER. Future vaccine design needs to contend with the complexity of epitope display as well as immunodominance.  

5.461           Effects of Sustained Antiangiogenic Therapy in Multistage Prostate Cancer in TRAMP Model

Isayeva, T., Chanda, D., Kallmann, L., Eltoum, I-E.A. and Ponnazhagan, S. Cancer Res., 67(12), 5789-5797 (2007)   Antiangiogenic therapy is a promising alternative for prostate cancer growth and metastasis and holds great promise as an adjuvant therapy. The present study evaluated the potential of stable expression of angiostatin and endostatin before the onset of neoplasia and during the early and late stages of prostate cancer progression in transgenic adenocarcinoma of mouse prostate (TRAMP) mice. Groups of 5-, 10-, and 18-week-old male TRAMP mice received recombinant adeno-associated virus-6 encoding mouse endostatin plus angiostatin (E+A) by i.m. injection. The effects of therapy were determined by sacrificing groups of treated mice at defined stages of tumor progression and following cohorts of similarly treated mice for long-term survival. Results indicated remarkable survival after recombinant adeno-associated virus–(E+A) therapy only when the treatment was given at an earlier time, before the onset of high-grade neoplasia, compared with treatment given for invasive cancer. Interestingly, early-stage antiangiogenic therapy arrested the progression of moderately differentiated carcinoma to poorly differentiated state and distant metastasis. Immunohistochemical analysis of the prostate from treated mice indicated significantly lower endothelial cell proliferation and increased tumor cell apoptosis. Vascular endothelial growth factor receptor (VEGFR)-2 expression was significantly down-regulated in tumor endothelium after treatment but not VEGFR-1. Analysis of the neuroendocrine marker synaptophysin expression indicated that antiangiogenic therapy given at an early-stage disease reduced neuroendocrine transition of the epithelial tumors. These studies indicate that stable endostatin and angiostatin gene therapy may be more effective for minimally invasive tumors rather than advanced-stage disease.  

5.462           Restoration of Tissue Factor Pathway Inhibitor-2 in a Human Glioblastoma Cell Line Triggers Caspase-Mediated Pathway and Apoptosis

George, J., Gondi, C.S., Dinh, D.H., Gujrati, M. and Rao, J.S. Clin. Cancer Res., 13(12), 3507-3517 (2007)   Purpose: The induction of apoptotic pathways in cancer cellsoffers a novel and potentially useful approach to improve patientresponses to conventional chemotherapy. Tissue factor pathwayinhibitor-2 (TFPI-2) is a protease inhibitor that is abundantin the extracellular matrix and highly expressed in noninvasivecells but absent or undetectable in highly invasive human glioblastomacells. Experimental Design: Using a recombinant adeno-associated viralvector carrying human TFPI-2 cDNA, we stably expressed TFPI-2in U-251 cells, a highly invasive human glioblastoma cell line.Our previous studies showed that restoration of TFPI-2 in glioblastomaseffectively prevents cell proliferation, angiogenesis, and tumorinvasion. In this study, we determined whether TFPI-2 restorationcould induce apoptosis through the caspase-mediated signalingpathway. Results: The results from nuclear chromatin staining, terminaldeoxynucleotidyl transferase–mediated dUTP nick end labelingassay, and fluorescence-activated cell sorting analysis showedincreased apoptosis in U-251 cells after restoration of TFPI-2.Caspase-9 and caspase-3 activity assays showed increased activity,indicating enhanced apoptosis. Immunofluorescence for cleavedcaspase-9 and caspase-3 depicted increased expression and colocalizationof both molecules. Western blot analysis showed increased transcriptionalactivities of Fas ligand, tumor necrosis factor-, Bax, Fas-associateddeath domain, and tumor necrosis factor receptor 1–associateddeath domain as well as elevated levels of cleaved caspasesand poly(ADP-ribose) polymerase. Semiquantitative reverse transcription-PCRdepicted increased expression of tumor necrosis factor- andFas ligand and the related death domains tumor necrosis factorreceptor 1–associated death domain and Fas-associateddeath domain. Conclusions: Taken together, these results show that restorationof TFPI-2 activates both intrinsic and extrinsic caspase-mediated,proapoptotic signaling pathways and induces apoptosis in U-251cells. Furthermore, our study suggests that recombinant adeno-associatedviral vector–mediated gene expression offers a novel toolfor cancer gene therapy.  

5.463           Bovine Papillomavirus Type 1 Infection Is Mediated by SNARE Syntaxin 18

Laniosz, V., Nguyen, K.C. and Meneses, P.I.

1. Virol., 81(14), 7435-7448 (2007)

  Events that lead to viral infections include the binding of the virus to the target cells, internalization of the virus into the cells, and the ability of the viral genome to be expressed. These steps are mediated by cellular and viral proteins and are temporally regulated. The papillomavirus capsid consists of two virally encoded capsid proteins, L1 and L2. Much is known about the role of the major capsid protein L1 compared to what is known of the role of the L2 protein. We identified the interaction of the L2 protein with SNARE protein syntaxin 18, which mediates the trafficking of vesicles and their cargo between the endoplasmic reticulum, the cis-Golgi compartment, and possibly the plasma membrane. Mutations of L2 residues 41 to 44 prevented the interaction of L2 protein with syntaxin 18 in cotransfection experiments and resulted in noninfectious pseudovirions. In this paper, we describe that syntaxin 18 colocalizes with infectious bovine papillomavirus type 1 (BPV1) pseudovirions during infection but does not colocalize with the noninfectious BPV1 pseudovirions made with an L2 mutant at residues 41 to 44. We show that an antibody against BPV1 L2 residues 36 to 49 ( L2 36-49) binds to in vitro-generated BPV1 pseudoviral capsids and does not coimmunoprecipitate syntaxin 18- and BPV1 L2-transfected proteins. L2 36-49 was able to partially or completely neutralize infection of BPV1 pseudovirions and genuine virions. These results support the dependence of syntaxin 18 during BPV1 infection and the ability to interfere with infection by targeting the L2-syntaxin 18 interaction and further define the infectious route of BPV1 mediated by the L2 protein.  

5.464           Human Immunodeficiency Virus Type 1 Replication in Dendritic Cell-T-Cell Cocultures Is Increased upon Incorporation of Host LFA-1 due to Higher Levels of Virus Production in Immature Dendritic Cells

Gilbert, C., Cantin, R., Barat, C. and Tremblay, M.J.

1. Virol., 81(14), 7672-7682 (2007)

  Dendritic cells (DCs) act as a portal for invasion by human immunodeficiency virus type-1 (HIV-1). Here, we investigated whether virion-incorporated host cell membrane proteins can affect virus replication in DC-T-cell cocultures. Using isogenic viruses either devoid of or bearing host-derived leukocyte function-associated antigen 1 (LFA-1), we showed that HIV-1 production is augmented when LFA-1-bearing virions are used compared to that for viral entities lacking this adhesion molecule. This phenomenon was observed in immature monocyte-derived DCs (IM-MDDCs) only and not in DCs displaying a mature phenotype. The increase is not due to higher virus production in responder CD4⁺ T cells but rather is linked with a more important productive infection of IM-MDDCs. We provided evidence that virus-associated host LFA-1 molecules do not affect a late event in the HIV-1 life cycle but rather exert an effect on an early step in virus replication. We demonstrated that the enhancement of productive infection of IM-MDDCs that is conferred by virus-anchored host LFA-1 involves the protein kinase A (PKA) and PKC signal transduction pathways. The biological significance of this phenomenon was established by performing experiments with virus stocks produced in primary human cells and anti-LFA-1 antibodies. Together, our results indicate that the association between some virus-bound host proteins and their natural cognate ligands can modulate de novo HIV-1 production by IM-MDDCs. Therefore, the additional interactions between virus-bound host cell membrane constituents and counter receptors on the surfaces of DCs can influence HIV-1 replication in IM-MDDC-T-cell cocultures.  

5.465           Ultracentrifugation of Serum Samples Allows Detection of Hepatitis C Virus RNA in Patients with Occult Hepatitis C

Bartolome, J. et al

1. Virol., 81(14), 7710-7715 (2007)

  Occult hepatitis C virus (HCV) infection of patients with abnormal liver function tests of unknown origin who are anti-HCV and serum HCV RNA negative but who have HCV RNA in the liver has been described. As HCV replicates in the liver cells of these patients, it could be that the amount of circulating viral particles is under the detection limit of the most sensitive techniques. To prove this hypothesis, serum samples from 106 patients with occult HCV infection were analyzed. Two milliliters of serum was ultracentrifuged over a 10% sucrose cushion for 17 h at 100,000 x g_av, where av means average, and HCV RNA detection was performed by strand-specific real-time PCR. Out of the 106 patients, 62 (58.5%) had detectable serum HCV RNA levels after ultracentrifugation, with a median load of 70.5 copies/ml (range, 18 to 192). Iodixanol density gradient studies revealed that HCV RNA was positive at densities of 1.03 to 1.04 and from 1.08 to 1.19 g/ml, which were very similar to those found in the sera of patients with classical chronic HCV infection. Antigenomic HCV RNA was found in the livers of 56 of 62 (90.3%) patients with detectable serum HCV RNA levels after ultracentrifugation, compared to 27 of 44 (61.4%) negative patients (P < 0.001). No differences in the median loads of antigenomic HCV RNA between patients with an those without serum HCV RNA (4.5 x 10⁴ [range, 7.9 x 10² to 1.0 x 10⁶] versus 2.3 x 10⁴ [range, 4.0 x 10² to 2.2 x 10⁵]) were found. Alanine aminotransferase and gamma-glutamyl transpeptidase levels, liver necroinflammatory activity, and fibrosis did not differ between both groups. In conclusion, HCV RNA can be detected in the sera of patients with occult HCV infection after circulating viral particles are concentrated by ultracentrifugation.  

5.466           Surface-Exposed Adeno-Associated Virus Vp1-NLS Capsid Fusion Protein Rescues Infectivity of Noninfectious Wild-Type Vp2/Vp3 and Vp3-Only Capsids but Not That of Fivefold Pore Mutant Virions

Grieger, J.C.,Gurda-Whittaker, B., Agbandje-McKenna, M. and Samulski, R.J.

1. Virol., 81(15), 7833-7843 (2007)

  Over the past 2 decades, significant effort has been dedicated to the development of adeno-associated virus (AAV) as a vector for human gene therapy. However, understanding of the virus with respect to the functional domains of the capsid remains incomplete. In this study, the goal was to further examine the role of the unique Vp1 N terminus, the N terminus plus the recently identified nuclear localization signal (NLS) (J. C. Grieger, S. Snowdy, and R. J. Samulski, J. Virol 80:5199-5210, 2006), and the virion pore at the fivefold axis in infection. We generated two Vp1 fusion proteins (Vp1 and Vp1NLS) linked to the 8-kDa chemokine domain of rat fractalkine (FKN) for the purpose of surface exposure upon assembly of the virion, as previously described (K. H. Warrington, Jr., O. S. Gorbatyuk, J. K. Harrison, S. R. Opie, S. Zolotukhin, and N. Muzyczka, J. Virol 78:6595-6609, 2004). The unique Vp1 N termini were found to be exposed on the surfaces of these capsids and maintained their phospholipase A2 (PLA2) activity, as determined by native dot blot Western and PLA2 assays, respectively. Incorporation of the fusions into AAV type 2 capsids lacking a wild-type Vp1, i.e., Vp2/Vp3 and Vp3 capsid only, increased infectivity by 3- to 5-fold (Vp1FKN) and 10- to 100-fold (Vp1NLSFKN), respectively. However, the surface-exposed fusions did not restore infectivity to AAV virions containing mutations at a conserved leucine (Leu336Ala, Leu336Cys, or Leu336Trp) located at the base of the fivefold pore. EM analyses suggest that Leu336 may play a role in global structural changes to the virion directly impacting downstream conformational changes essential for infectivity and not only have local effects within the pore, as previously suggested.  

5.467           Early Innate Immune Responses to Sin Nombre Hantavirus Occur Independently of IFN Regulatory Factor 3, Characterized Pattern Recognition Receptors, and Viral Entry

Prescott, J.B., Hall, P.R., Bondu-Havkins, V.S., Ye, C. and  Hjelle, B.

1. Immunol., 179, 1796-1802 (2007)

  Sin Nombre virus (SNV) is a highly pathogenic New World virus and etiologic agent of hantavirus cardiopulmonary syndrome. We have previously shown that replication-defective virus particles are able to induce a strong IFN-stimulated gene (ISG) response in human primary cells. RNA viruses often stimulate the innate immune response by interactions between viral nucleic acids, acting as a pathogen-associated molecular pattern, and cellular pattern-recognition receptors (PRRs). Ligand binding to PRRs activates transcription factors which regulate the expression of antiviral genes, and in all systems examined thus far, IFN regulatory factor 3 (IRF3) has been described as an essential intermediate for induction of ISG expression. However, we now describe a model in which IRF3 is dispensable for the induction of ISG transcription in response to viral particles. IRF3-independent ISG transcription in human hepatoma cell lines is initiated early after exposure to SNV virus particles in an entry- and replication-independent fashion. Furthermore, using gene knockdown, we discovered that this activation is independent of the best-characterized RNA- and protein-sensing PRRs including the cytoplasmic caspase recruitment domain-containing RNA helicases and the TLRs. SNV particles engage a heretofore unrecognized PRR, likely located at the cell surface, and engage a novel IRF3-independent pathway that activates the innate immune response.  

5.468           The Nucleoside Triphosphate Diphosphohydrolase-1/CD39 Is Incorporated into Human Immunodeficiency Type 1 Particles, Where It Remains Biologically Active

Barat, C., Martin, G., Beaudoin, A.R., Sevigny, J. and Tremblay, M.J.

1. Mol. Biol., 371, 269-282 (2007)

  Human immunodeficiency virus type 1 (HIV-1) carries a variety of host proteins in addition to virus-encoded structural proteins, both in its envelope and inside the viral particle. Previous studies have reported that the HIV-1 life-cycle is affected by such virus-associated host cell surface proteins. The nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), also known as CD39, is a plasma membrane-bound ectoenzyme that hydrolyzes extracellular ATP and ADP to AMP. It has been shown that CD39 inhibits platelet function, and is thus a critical thromboregulatory molecule. We demonstrate here that host-derived CD39 is acquired by both laboratory-adapted and clinical variants of HIV-1 produced in cellular reservoirs of the virus. Moreover, purified CD39-bearing virions, but not isogenic viruses lacking CD39, display strong ATPase and ADPase activities. It is of particular interest that virions bearing this cellular enzyme can inhibit ADP-induced platelet aggregation, an effect blocked by an NTPDase inhibitor. On the basis of published and the present data on the functionality of human cellular proteins embedded within HIV-1, it can be proposed that these proteins might contribute to some of the immunologic deficiencies seen in infected individuals.  

5.469           Novel rat Alzheimer's disease models based on AAV-mediated gene transfer to selectively increase hippocampal Aβ levels

Lawlor, P.A. et al Molecular Neurodegeneration, 2(11), 1-13 (2007)   Background Alzheimer's disease (AD) is characterized by a decline in cognitive function and accumulation of amyloid-β peptide (Aβ) in extracellular plaques. Mutations in amyloid precursor protein (APP) and presenilins alter APP metabolism resulting in accumulation of Aβ42, a peptide essential for the formation of amyloid deposits and proposed to initiate the cascade leading to AD. However, the role of Aβ40, the more prevalent Aβ peptide secreted by cells and a major component of cerebral Aβ deposits, is less clear. In this study, virally-mediated gene transfer was used to selectively increase hippocampal levels of human Aβ42 and Aβ40 in adult Wistar rats, allowing examination of the contribution of each to the cognitive deficits and pathology seen in AD. Results Adeno-associated viral (AAV) vectors encoding BRI-Aβ cDNAs were generated resulting in high-level hippocampal expression and secretion of the specific encoded Aβ peptide. As a comparison the effect of AAV-mediated overexpression of APPsw was also examined. Animals were tested for development of learning and memory deficits (open field, Morris water maze, passive avoidance, novel object recognition) three months after infusion of AAV. A range of impairments was found, with the most pronounced deficits observed in animals co-injected with both AAV-BRI-Aβ40 and AAV-BRI-Aβ42. Brain tissue was analyzed by ELISA and immunohistochemistry to quantify levels of detergent soluble and insoluble Aβ peptides. BRI-Aβ42 and the combination of BRI-Aβ40+42 overexpression resulted in elevated levels of detergent-insoluble Aβ. No significant increase in detergent-insoluble Aβ was seen in the rats expressing APPsw or BRI-Aβ40. No pathological features were noted in any rats, except the AAV-BRI-Aβ42 rats which showed focal, amorphous, Thioflavin-negative Aβ42 deposits. Conclusion The results show that AAV-mediated gene transfer is a valuable tool to model aspects of AD pathology in vivo, and demonstrate that whilst expression of Aβ42 alone is sufficient to initiate Aβ deposition, both Aβ40 and Aβ42 may contribute to cognitive deficits.  

5.470           Antitumor effects of non-replicative herpes simplex vectors expressing antiangiogenic proteins and thymidine kinase on Lewis lung carcinoma establishment and growth

Berto, E. et al Cancer Gene Therapy, 14, 791-801 (2007)   There is growing evidence that combinations of antiangiogenic proteins with other antineoplastic treatments such as chemo- or radiotherapy and suicide genes-mediated tumor cytotoxicity lead to synergistic effects. In the present work, we tested the activity of two non-replicative herpes simplex virus (HSV)-1-based vectors, encoding human endostatin angiostatin or endostatin kringle5 fusion proteins in combination with HSV-1 thymidine kinase (TK) molecule, on endothelial cells (ECs) and Lewis lung carcinoma (LLC) cells. We observed a significant reduction of the in vitro growth, migration and tube formation by primary ECs upon direct infection with the two recombinant vectors or cultivation with conditioned media obtained from the vector-infected LLC cells. Moreover, direct cytotoxic effect of HSV-1 TK on both LLC and ECs was demonstrated. We then tested the vectors in vivo in two experimental settings, that is, LLC tumor growth or establishment, in C57BL/6 mice. The treatment of pre-established subcutaneous tumors with the recombinant vectors with ganciclovir (GCV) induced a significant reduction of tumor growth rate, while the in vitro infection of LLC cells with the antiangiogenic vectors before their implantation in mice flanks, either in presence or absence of GCV, completely abolished the tumor establishment.

5.471           AAV-Mediated Expression Targeting of Rod and Cone Photoreceptors with a Human Rhodopsin Kinase Promoter

Khani, S.C. et al Invest. Ophthalmol. Vis. Sci., 48(9), 3954-3961 (2007)   PURPOSE. Gene therapy for retinal degeneration requires well-definedpromoters that drive expression in rod and cone photoreceptors.This study was undertaken to develop short, active derivativesof the human rhodopsin kinase (RK) gene promoter for targetingtransgene expression in rods and cones. RK, also known as Gprotein–coupled receptor kinase 1 (GRK1), is a componentof the light adaptation pathway expressed in rods and cones. METHODS. Human RK (hRK) promoter and its concatemers or derivatives extending into the conserved 5' untranslated region (5'-UTR) were assayed for promoter activity in WERI retinoblastoma or Crx/Sp1-supplemented HEK-293 cells. The derivative displaying the highest activity was linked to a GFP reporter and packaged in a pseudotyped adenoassociated viral vector (AAV2/5). The AAV vector was tested in vivo by subretinal injections in wild-type mice, in the all-cone Nrl^–/– mice, and in the cone-rich diurnal Nile grass rat (Arvicanthis niloticus). Control eyesreceived a similar AAV2/5 vector carrying a mouse rod opsin(mOps) promoter-controlled GFP reporter. RESULTS. The hRK promoter with the full 5' untranslated sequence(–112 to +180) was the most active in cell culture. Deliveredby the AAV2/5 vector, RK promoter drove GFP expression specificallyin photoreceptors. In rods, hRK promoter-mediated expressionwas as efficient as, but appeared more uniform than, mOps promoter-mediatedexpression. In cones, the hRK promoter drove expression, whereasthe mOps promoter did not. CONCLUSIONS. The hRK promoter is active and specific for rodand cone photoreceptors. Because of its small size and provenactivity in cones, it is a promoter of choice for somatic genetransfer and gene therapy targeting rods and cones.  

5.472           Bidirectional changes in water-maze learning following recombinant adenovirus-associated viral vector (rAAV)-mediated brain-derived neurotrophic factor expression in the rat hippocampus

Pietropaolo, S., Paterna, J-C., Büeler, H., Feldon, J. and Yee, B.K. Behavioural Pharmacol., 18, 533-547 (2007)   Alterations in hippocampal brain-derived neurotrophic factor (BDNF) expression have been implicated in the pathogenesis of emotional and cognitive dysfunction. Here, we induced BDNF overexpression in the rat hippocampus using recombinant adenovirus-associated viral (rAAV) vectors, and studied its long-term (2 months postinduction) effects on anxiety-related behaviour, exploration in the open field, and spatial learning in the water maze. Although the treatment successfully led to substantial elevation of hippocampal BDNF levels, its effect on spatial learning was bidirectional: a subset of rAAV-induced BDNF-overexpressing rats performed well above control level, whereas the rest were clearly impaired. This behavioural distinction corresponded to two markedly different levels of BDNF overexpression. The increase in dorsal hippocampal BDNF content achieved in the 'water-maze-impaired' subgroup was twice that attained in the 'water-maze-improved' rats. Although neither subgroup of rAAV-induced BDNF-overexpressing rats differed from controls in the open field, the 'water-maze-impaired' subgroup also showed a significant anxiolytic effect. Our results suggest that hippocampal BDNF elevation significantly affects cognitive and emotional behaviours, but the direction and magnitude of the effects critically depend on the precise levels of overexpression. This factor must be taken into account in future studies examining the functional consequences of hippocampal BDNF overexpression.  

5.473           Ectopic expression of Wnt10b decreases adiposity and improves glucose homeostasis in obese rats

Aslanidi, G. et al Am.J. Physiol. Endocrinol. Metab., 293, E726-E736 (2007)   The Wnt family of secreted glycoproteins had previously been shown to regulate diverse processes during early development. Wnt signaling also plays a key role in the homeostasis of adult tissues maintaining stem cell pluripotency and determining differentiating cell fate. The age-related decrease in Wnt signaling may contribute to increased muscle adiposity and diminished bone strength. In the current study, we investigated the long-term metabolic consequences of the upregulated Wnt/ -catenin signaling in skeletal muscles of adult diet-induced obese (DIO) rats. To this end, we generated a recombinant adeno-associated virus (rAAV) vector encoding murine Wnt10b cDNA. The long-term expression of rAAV1-Wnt10b was tested after intramuscular injection in the female DIO rat. Animals fed high-fat diet and treated with rAAV1-Wnt10b showed a sustained reduction in body weight compared with controls, and expression of Wnt10b was accompanied by a reduction in hyperinsulinemia and triglyceride plasma levels as well as improved glucose homeostasis. Nuclear magnetic resonance methods revealed that ectopic expression of Wnt10b resulted in a decrease in both global and muscular fat deposits in DIO rats. The long-range effect of locally expressed Wnt10b was also manifested through the increased bone mineral density. The detailed analysis of molecular markers revealed fibroblast growth factor-4 and vascular endothelial growth factor as possible mediators of the systemic effect of Wnt10b transgene expression. Our data demonstrate that altering Wnt/ -catenin signaling in the skeletal muscle of an adult animal invokes moderate responses with favorable metabolic profile, bringing the notion of alternative therapeutic modality in the treatment of obesity, diabetes, and osteoporosis.  

5.474           Differential internalization and nuclear uncoating of self-complementary adeno-associated virus pseudotype vectors as determinants of cardiac cell transduction

Sipo, I. et al Gene Therapy, 14, 1319-1329 (2007)   Recently it was shown that several new pseudotyped adeno-associated virus (AAV) vectors support cardioselective expression of transgenes. The molecular mechanisms underlying this propensity for cardiac cell transduction are not well understood. We comparatively analyzed AAV vector attachment, internalization, intracellular trafficking, and nuclear uncoating of recombinant self-complementary (sc) AAV2.2 versus pseudotyped scAAV2.6 vectors expressing green fluorescence protein (GFP) in cells of cardiac origin. In cardiac-derived HL-1 cells and primary neonatal rat cardiomyocytes (PNCMs), expression of GFP increased rapidly after incubation with scAAV2.6-GFP, but remained low after scAAV2.2-GFP. Internalization of scAAV2.6-GFP was more efficient than that of scAAV2.2-GFP. Nuclear translocation was similarly efficient for both, but differential nuclear uncoating rates emerged as a key additional determinant of transduction: 30% of all scAAV2.6-GFP genomes translocated to the nucleus became uncoated within 48 h, but only 16% of scAAV2.2-GFP genomes. In contrast to this situation in cells of cardiac origin, scAAV2.2-GFP displayed more efficient internalization and similar (tumor cell line HeLa) or higher (human microvascular endothelial cell (HMEC)) uncoating rates than scAAV.2.6-GFP in non-cardiac cell types. In summary, both internalization and nuclear uncoating are key determinants of cardiac transduction by scAAV2.6 vectors. Any in vitro screening for the AAV pseudotype most suitable for cardiac gene therapy – which is desirable since it may allow significant reductions in vector load in upcoming clinical trials – needs to quantitate both key steps in transduction.  

5.475           An Immunogenic and Protective Alphavirus Replicon Particle-Based Dengue Vaccine Overcomes Maternal Antibody Interference in Weanling Mice

White, L.J. et al

1. Virol., 81(19), 10329-10339 (2007)

  A candidate pediatric dengue virus (DENV) vaccine based on nonpropagating Venezuelan equine encephalitis virus replicon particles (VRP) was tested for immunogenicity and protective efficacy in weanling mice in the presence and absence of potentially interfering maternal antibodies. A gene cassette encoding envelope proteins prM and E from mouse-adapted DENV type 2 (DENV2) strain NGC was cloned into a VEE replicon vector and packaged into VRP, which programmed proper in vitro expression and processing of DENV2 envelope proteins upon infection of Vero cells. Primary immunization of 3-week-old weanling BALB/c mice in the footpad with DENV2 VRP resulted in high levels of DENV-specific serum immunoglobulin G antibodies and significant titers of neutralizing antibodies in all vaccinates. A booster immunization 12 weeks after the prime immunization resulted in increased neutralizing antibodies that were sustained for at least 30 weeks. Immunization at a range of doses of DENV2 VRP protected mice from an otherwise-lethal intracranial DENV2 challenge. To model vaccination in the presence of maternal antibodies, weanling pups born to DENV2-immune or DENV2-naïve dams were immunized with either DENV2 VRP or live DENV2 given peripherally. The DENV2 VRP vaccine induced neutralizing-antibody responses in young mice regardless of the maternal immune status. In contrast, live-DENV2 vaccination performed poorly in the presence of preexisting anti-DENV2 antibodies. This study demonstrates the feasibility of a VRP vaccine approach as an early-life DENV vaccine in populations with high levels of circulating DENV antibodies and suggests the utility of VRP-based vaccines in other instances where maternal antibodies make early vaccination problematic.        

5.476           Filamentous Influenza A Virus Infection Predisposes Mice to Fatal Septicemia following Superinfection with Streptococcus pneumoniae Serotype 3

Speshock, J.L., Doyon-Reale, N., Rabah, R., Neely, M.N. and Roberts, P.C. Infect. Immun., 75(6), 3102-3111 (2007)   Previous studies have demonstrated that animals exposed to Streptococcus pneumoniae while recovering from influenza A virus infection exhibit exacerbated disease symptoms. However, many of the current animal models exploring dual viral and bacterial synergistic exacerbations of respiratory disease have utilized mouse-adapted influenza virus and strains of Streptococcus pneumoniae that in themselves are highly lethal to mice. Here we describe a mouse model of bacterial superinfection in which a mild, self-limiting influenza virus infection is followed by mild, self-limiting superinfection with S. pneumoniae serotype 3. S. pneumoniae superinfection results in rapid dissemination of the bacterium from the respiratory tract and systemic spread to all major organs of the mice, resulting in fatal septicemia. This phenomenon in mice was observed in superinfected animals undergoing an active viral infection as well as in mice that had completely cleared the virus 7 to 8 days prior to superinfection. Neutrophils were the predominant cellular inflammatory infiltrate in the lungs of superinfected mice compared to singly infected animals. Among other cytokines and chemokines, the neutrophil activator granulocyte colony-stimulating factor (G-CSF) was found to be significantly overexpressed in the spleens, lungs, and brains of superinfected animals. High G-CSF protein levels were observed in sera and lung lavage fluid from superinfected animals, suggesting that G-CSF is a major contributor to synergistic exacerbation of disease leading to fatal septicemia.  

5.477           Surface-exposed Amino Acid Residues of HPV16 L1 Protein Mediating Interaction with Cell Surface Heparan Sulfate

Knappe, M. et al

1. Biol. Chem., 282(38), 27913-27922 (2007)

  Efficient infection of cells by human papillomaviruses (HPVs) and pseudovirions requires primary interaction with cell surface proteoglycans with apparent preference for species carrying heparan sulfate (HS) side chains. To identify residues contributing to virus/cell interaction, we performed point mutational analysis of the HPV16 major capsid protein, L1, targeting surface-exposed amino acid residues. Replacement of lysine residues 278, 356, or 361 for alanine reduced cell binding and infectivity of pseudovirions. Various combinations of these amino acid exchanges further decreased cell attachment and infectivity with residual infectivity of less than 5% for the triple mutant, suggesting that these lysine residues cooperate in HS binding. Single, double, or triple exchanges for arginine did not impair infectivity, demonstrating that interaction is dependent on charge distribution rather than sequence-specific. The lysine residues are located within a pocket on the capsomere surface, which was previously proposed as the putative receptor binding site. Fab fragments of binding-neutralizing antibody H16.56E that recognize an epitope directly adjacent to lysine residues strongly reduced HS-mediated cell binding, further corroborating our findings. In contrast, mutation of basic surface residues located in the cleft between capsomeres outside this pocket did not significantly reduce interaction with HS or resulted in assembly-deficient proteins. Computer-simulated heparin docking suggested that all three lysine residues can form hydrogen bonds with 2-O-, 6-O-, and N-sulfate groups of a single HS molecule with a minimal saccharide domain length of eight monomer units. This prediction was experimentally confirmed in binding experiments using capsid protein, heparin molecules of defined length, and sulfate group modifications.  

5.478           Cryo-electron microscopy and three-dimensional reconstructions of hepatitis C virus particles

Yu, X. et al Virology, 367, 126-134 (2007)   The structural details of hepatitis C virus (HCV) have been elusive because of the lack of a robust tissue culture system for producing an adequate amount of virions from infectious sources for in-depth three-dimensional (3D) structural analysis. Using both negative-stain and cryo-electron microscopy (cryoEM), we show that HCV virions isolated from cell culture have a rather uniform size of 500 Å in diameter and that recombinantly expressed HCV-like particles (HCV-LPs) have similar morphologic, biophysical and antigenic features in spite of the varying sizes of the particles. 3D reconstructions were obtained from HCV-LPs with the same size as the HCV virions in the presence and absence of monoclonal antibodies bound to the E1 glycoprotein. The 3D reconstruction of HCV-LP reveals a multilayered architecture, with smooth outer-layer densities arranged in a ‘fishbone’ configuration. Reconstruction of the particles in complex with anti-E1 antibodies shows that sites of the E1 epitope are exposed and surround the 5-, 3- and 2-fold axes. The binding pattern of the anti-E1 antibody and the fitting of the structure of the dengue virus E glycoprotein into our 3D reconstructions further suggest that the HCV-LP E1 and E2 proteins form a tetramer (or dimer of heterodimers) that corresponds morphologically and functionally to the flavivirus E homodimer. This first 3D structural analysis of HCV particles offers important insights into the elusive mechanisms of HCV assembly and maturation.  

5.479           rAAV-mediated nigral human parkin over-expression partially ameliorates motor deficits via enhanced dopamine neurotransmission in a rat model of Parkinson's disease

Manfredson, F.P. et al Exp. Neurol., 207, 289-301 (2007)   We hypothesized that over-expressing the E3 ligase, parkin, whose functional loss leads to Parkinson's disease, in the nigrostriatal tract might be protective in the unilateral 6-hydroxydopamine (6-OHDA) rat lesion model. Recombinant adeno-associated virus (rAAV) encoding human parkin or green fluorescent protein (GFP) was injected into the rat substantia nigra 6 weeks prior to a four-site striatal 6-OHDA lesion. Vector-mediated parkin over-expression significantly ameliorated motor deficits as measured by amphetamine-induced rotational behavior and spontaneous behavior in the cylinder test but forelimb akinesia as assessed by the stepping test was unaffected. rAAV-mediated human parkin was expressed in the nigrostriatal tract, the substantia pars reticulata, and the subthalamic nucleus. However, in lesioned animals, there was no difference between nigral parkin and GFP-transduction on lesion-induced striatal tyrosine hydroxylase (TH) innervation or nigral TH positive surviving neurons. A second lesion experiment was performed to determine if striatal dopamine (DA) neurotransmission was enhanced as measured biochemically. In this second group of parkin and GFP treated rats, behavioral improvement was again observed. In addition, striatal TH and DA levels were slightly increased in the parkin-transduced group. In a third experiment, we evaluated parkin and GFP transduced rats 6 weeks after vector injection without DA depletion. When challenged with amphetamine, parkin treated rats tended to display asymmetries biased away from the treated hemisphere. Nigral parkin over-expression induced increases in both striatal TH and DA levels. Therefore, while parkin over-expression exerted no protective effect on the nigrostriatal DA system, parkin appeared to enhance the efficiency of nigrostriatal DA transmission in intact nigral DA neurons likely due to the observed increases in TH.  

5.480           Inhibition of Transfer to Secondary Receptors by Heparan Sulfate-Binding Drug or Antibody Induces Noninfectious Uptake of Human Papillomavirus

Selinka, H-C. Et al

1. Virol., 81(20), 10970-10980 (2007)

  Infection with various human papillomaviruses (HPVs) induces cervical cancers. Cell surface heparan sulfates (HS) have been shown to serve as primary attachment receptors, and molecules with structural similarity to cell surface HS, like heparin, function as competitive inhibitors of HPV infection. Here we demonstrate that the N,N'-bisheteryl derivative of dispirotripiperazine, DSTP27, efficiently blocks papillomavirus infection by binding to HS moieties, with 50% inhibitory doses of up to 0.4 µg/ml. In contrast to short-term inhibitory effects of heparin, pretreatment of cells with DSTP27 significantly reduced HPV infection for more than 30 h. Using DSTP27 and heparinase, we furthermore demonstrate that HS moieties, rather than laminin 5, present in the extracellular matrix (ECM) secreted by keratinocytes are essential for infectious transfer of ECM-bound virions to cells. Prior binding to ECM components, especially HS, partially alleviated the requirement for cell surface HS. DSTP27 blocks infection by cell-bound virions by feeding into a noninfectious entry pathway. Under these conditions, virus colocalized with HS moieties in endocytic vesicles. Similarly, postattachment treatment of cells with heparinase, cytochalasin D, or neutralizing antibodies resulted in uptake of virions without infection, indicating that deviation into a noninfectious entry pathway is a major mode of postattachment neutralization. In untreated cells, initial colocalization of virions with HS on the cell surface and in endocytic vesicles was lost with time. Our data suggest that initial attachment of HPV to HS proteoglycans (HSPGs) must be followed by secondary interaction with additional HS side chains and transfer to a non-HSPG receptor for successful infection.            

5.481           Salmonella enterica Serovar Typhi Ty21a Expressing Human Papillomavirus Type 16 L1 as a Potential Live Vaccine against Cervical Cancer and Typhoid Fever

Fraillery, D. et al Clin. Vaccine Immunol., 14(10), 1285-1295 (2007)   Human papillomavirus (HPV) vaccines based on L1 virus-like particles (VLPs) can prevent HPV-induced genital neoplasias, the precursors of cervical cancer. However, most cervical cancers occur in developing countries, where the implementation of expensive vaccines requiring multiple injections will be difficult. A live Salmonella-based vaccine could be a lower-cost alternative. We previously demonstrated that high HPV type 16 (HPV16)-neutralizing titers are induced after a single oral immunization of mice with attenuated Salmonella enterica serovar Typhimurium strains expressing a codon-optimized version of HPV16 L1 (L1S). To allow the testing of this type of vaccine in women, we constructed a new L1-expressing plasmid, kanL1S, and tested kanL1S recombinants of three Salmonella enterica serovar Typhi vaccine strains shown to be safe in humans, i.e., Ty21a, the actual licensed typhoid vaccine, and two highly immunogenic typhoid vaccine candidates, Ty800 and CVD908-htrA. In an intranasal mouse model of Salmonella serovar Typhi infection, Ty21a kanL1S was unique in inducing HPV16-neutralizing antibodies in serum and genital secretions, while anti-Salmonella responses were similar to those against the parental Ty21a vaccine. Electron microscopy examination of Ty21a kanL1S lysates showed that L1 assembled in capsomers and capsomer aggregates but not well-ordered VLPs. Comparison to the neutralizing antibody response induced by purified HPV16 L1 VLP immunizations in mice suggests that Ty21a kanL1S may be an effective prophylactic HPV vaccine. Ty21a has been widely used against typhoid fever in humans with a remarkable safety record. These finds encourage clinical testing of Ty21a kanL1S as a combined typhoid fever/cervical cancer vaccine with the potential for worldwide application.  

5.482           Timing of Therapeutic Intervention Determines Functional and Survival Outcomes in a Mouse Model of Late Infantile Batten Disease

Cabrera-Salazar, M.A. et al Mol. Ther., 15(10), 1782-1788 (2007)   Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a monogenic disorder caused by the loss of tripeptidyl peptidase 1 (TPP1) activity as a result of mutations in CLN2. Absence of TPP1 results in lysosomal storage with an accompanying axonal degeneration throughout the central nervous system (CNS), which leads to progressive neurodegeneration and early death. In this study, we compared the efficacies of pre- and post-symptomatic injections of recombinant adeno-associated virus (AAV) for treating the cellular and functional abnormalities of CLN2 mutant mice. Intracranial injection of AAV1-hCLN2 resulted in widespread human TPP1 (hTPP1) activity in the brain that was 10–100-fold above wild-type levels. Injections before disease onset prevented storage and spared neurons from axonal degeneration, reflected by the preservation of motor function. Furthermore, the majority of CLN2 mutant mice treated pre-symptomatically lived for at least 330 days, compared with a median survival of 151 days in untreated CLN2 mutant controls. In contrast, although injection after disease onset ameliorated lysosomal storage, there was evidence of axonal degeneration, motor function showed limited recovery, and the animals had a median lifespan of 216 days. These data illustrate the importance of early intervention for enhanced therapeutic benefit, which may provide guidance in designing novel treatment strategies for cLINCL patients.  

5.483           Systemic Cancer Gene Therapy Using Adeno-associated Virus Type 1 Vector Expressing MDA-7/IL24

Tahara, I. et al Mol. Ther., 15(10), 1805-1811 (2007)   Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL24), selectively induces apoptosis in cancer cells without harming normal cells. It also exerts immunomodulatory and antiangiogenic effects, as well as potent antitumor bystander effects, making it an ideal candidate for a new anticancer gene therapy. Here, we examined the feasibility of adeno-associated virus type 1 (AAV1) vector-mediated systemic gene therapy using mda-7/IL24. In vitro studies showed that medium conditioned by AAV1-mda7-transducedC2C12 cells induces tumor cell-specific apoptosis and inhibits angiogenesis in a human umbilical vein endothelial cell tube formation assay. To assess the in vivo effects of AAV1-mediated systemic delivery of MDA-7/IL24, we generated a subcutaneous tumor model by injecting Ehrlich ascites tumor cells into the dorsum of DDY mice. A single intravenous injection of AAV1-mda7 (2.0 10¹¹ viral genomes) significantly inhibited tumor growth. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and immunohistochemical analyses showed significant induction of tumor-cell-specific apoptosis and reduction of microvessel formation within the tumors, and there was a significant increase in survival among the AAV1-mda7-treated mice. These results clearly demonstrate that continuous systemic delivery of MDA-7/IL24 can serve as an effective treatment for cancer. Thus, AAV1 vector-mediated systemic delivery of MDA-7/IL24 represents a potentially important new approach to anticancer therapy.  

5.484           Genus Molluscipoxvirus

Bugert, J.J. Poxviruses, Birkhäuser Basel, 89-112 (2007)   Molluscum contagiosum (MC) is a common wart-like skin infection mainly seen in children and caused by Molluscum contagiosum virus (MCV). The typical poxvirus particle morphology and genome organization of MCV led to its classification as a member of the family Poxviridae where it is the sole member of the genus Molluscipoxvirus. The genome of MCV type 1 (MCV 1/80) has been completely sequenced (GenBank accession U60315). Of 182 hypothetical MCV open reading frames (> 45 amino acids) only 35 have a significant homology to coding sequences of other poxviruses. Unique MCV genes include mc159, an apoptosis inhibitor (vFLIP), mc054, a viral IL-18 binding protein, mc148, a soluble IL-8 antagonist, and mc162, a Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) binding protein. MCV does not encode an epidermal growth factor (EGF) homolog. MCV shares a number of genes only with para- and avipoxviruses and stands out as phylogenetically distinct from all other poxviruses. This is reflected in a number of unique biological characteristics that set MCV apart from other poxviruses: MCV replication in vivo is limited to differentiating keratinocytes of the spineous layer of the human epidermis. MCV induces an enhanced rate of mitosis in keratinocytes, possibly by way of EGF receptor up-regulation, and interferes with the normal epidermal cell differentiation program. The lack of local inflammation gives typical MCV lesions a pearly bland appearance. MC infection can persist in human skin for years. An inflammatory reaction, spontaneous or induced by trauma, frequently leads to the sudden and complete disappearance of MCV lesions. The local, subacute and proliferative nature of the MC infection puts MCV close to a group of animal poxviruses causing slow growing skin tumors. MCV replicates inefficiently in skin xenotranplants to immunodeficient mice. There is currently no cell- or tissue culture system that supports replication of MCV in vitro.  

5.485           Liver-Directed Gene Transfer of Atheroprotective Human ApoA-Imilano

Osman, E., Graham, I., Athanasopoulos, T., Nathwani, A.C. and Owen, J.S. Human Gene Therapy, 18(10), 955-993 (2007)   Background: Apolipoprotein (Apo) A-I (ApoA-I) is the major constituent of plasma HDL, which protects against coronary heart disease (CHD). A natural variant, ApoA-IMilano (ApoAIM), is highly anti-atherogenic and has an Arg-173Cys substitution, which allows formation of disulfide-linked dimers. This confers HDL with unique structural and functional properties, although the mechanism(s) for enhanced atheroprotection remains ill-defined. ApoA-IM is thus an excellent gene therapy candidate to treat CHD. Aim: To inhibit development of atherosclerotic lesions in hyperlipidemic apoE_/_ mice by hepatic expression of human ApoA-IM. Methods and Results: Our choice of vector for high, sustained liver-specific expression was self-complementary, adeno-associated virus serotype-8 (scAAV8) driven by the strong, but short, promoter LP1 containing a hepatic control region and the alpha-1-anti-trypsin gene promoter/enhancer. First, we cloned ApoA-I and ApoA-IM into a scAAV-based plasmid and verified construct viability by transfecting cultured cells in vitro; this confirmed secretion of ApoA-I protein and, for ApoA-IM, formation of dimeric ApoA-I (_56 kDa). Viability was also demonstrated in vivo by hydrodynamic tail-vein injections into mice. Transgene expression was confirmed by the appearance of human ApoA-I protein in plasma, as detected by western blotting 10 days post-injection, and by mRNA transcripts in the livers. Packaging into scAAV8.LP1.ApoA-I and scAAV8.LP1.ApoA-IM viral vectors was accomplished by triple plasmid transfection of 293T cells, purifying the particles via iodixanol ultracentrifugation. The vectors (1011 viral genomes/mouse) were then injected into apoE_/_ mice for a 12-week study to evaluate their ability to inhibit development of atherogenesis. Analysis of blood at 0, 3, 6, and 12 weeks and histological measurements of the atherosclerotic plaque are in progress. Conclusions: We have successfully packaged human ApoAI and ApoA-IM into the potent gene transfer vector, scAAV8.LP1, and demonstrated functionality in vitro and in vivo. A preclinical trial to assess its therapeutic potential is near completion and will be presented.  

5.486           A Novel Intranasal Virus-Like Particle (VLP) Vaccine Designed to Protect against the Pandemic 1918 Influenza A Virus (H1N1)

Matassov, D., Cupo, A. and Galarza, J.M. Viral Immunol., 20(3), 441-452 (2007)   We have prepared a virus-like particle (VLP) vaccine bearing the surface glycoproteins HA and NA of the 1918 influenza A virus by infecting Sf9 cells with a quadruple recombinant baculovirus that expresses the four influenza proteins (HA, NA, M1, and M2) required for the assembly and budding of the VLPs. The presence of HA and M1 in the purified VLPs was confirmed by Western blot, and that of NA by a neuraminidase enzymatic assay. For in vivo studies, the 1918 VLP vaccine was formulated with or without an oligonucleotide containing two CpG motifs and administered in two doses 2 wk apart via the intranasal route. The antibody titers in mice immunized with VLP vaccines were higher than in mice vaccinated with an inactivated swine virus (H1N1) control, when CHO cells expressing 1918 HA were used as antigen. The opposite result was obtained when disrupted swine virus was the antigen for the ELISA test. Vaccine efficacy was evaluated by challenging immunized mice with the 1918 antigenically related influenza virus A/swine/Iowa/15/30 (H1N1) and measuring viral titers in the upper and lower respiratory tract. Mice immunized with VLP vaccine plus CpG demonstrated significantly lower viral titers in the nose and lungs than did the control on days 2 and 4 postchallenge and completely cleared the virus by day 6. Furthermore, they did not show symptoms of disease although there was a minor decrease in body weight. Mice vaccinated with VLP alone also demonstrated significantly lower viral titers in the nose and lungs than did the placebo group as well as the inactivated virus group on days 4 and 6 postchallenge. These results suggest that it is feasible to make a safe and immunogenic vaccine to protect against the extremely virulent 1918 virus, using a novel and safe cell-based technology.  

5.487           Detection of L1, infectious virions and anti-L1 antibody in domestic rabbits infected with cottontail rabbit papillomavirus

Hu, J. et al

1. Gen. Virol., 88, 3286-3298 (2007)

  Shope papillomavirus or cottontail rabbit papillomavirus (CRPV) is one of the first small DNA tumour viruses to be characterized. Although the natural host for CRPV is the cottontail rabbit (Sylvilagus floridanus), CRPV can infect domestic laboratory rabbits (Oryctolagus cuniculus) and induce tumour outgrowth and cancer development. In previous studies, investigators attempted to passage CRPV in domestic rabbits, but achieved very limited success, leading to the suggestion that CRPV infection in domestic rabbits was abortive. The persistence of specific anti-L1 antibody in sera from rabbits infected with either virus or viral DNA led us to revisit the questions as to whether L1 and infectious CRPV can be produced in domestic rabbit tissues. We detected various levels of L1 protein in most papillomas from CRPV-infected rabbits using recently developed monoclonal antibodies. Sensitive in vitro infectivity assays additionally confirmed that extracts from these papillomas were infectious. These studies demonstrated that the CRPV/New Zealand White rabbit model could be used as an in vivo model to study natural virus infection and viral life cycle of CRPV and not be limited to studies on abortive infections.  

5.488           A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy

Rodino-Klapac, L.R. et al

1. Translational Med., 5(45), 1-11 (2007)

  Background Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation. Methods The capacity of AAV1, AAV6 or AAV8 to cross the vascular endothelial barrier carrying a micro-dystrophin cDNA was compared under identical conditions with delivery through a catheter placed in the femoral artery of the mdx mouse. Transduction efficiency was assessed by immuno-staining using an antibody (Manex1a) that recognizes the N-terminus of micro-dystrophin. The degree of physiologic correction was assessed by measuring tetanic force and protection from eccentric contraction in the extensor digitorum longus muscle (EDL). The vascular delivery paradigm found successful in the mouse was carried to the non-human primate to test its potential translation to boys with DMD. Results Regional vascular delivery resulted in transduction by rAAV8.micro-dystrophin reaching 94.5 ± 0.9 (1 month), 91.3 ± 3.1 (2 months), and 89.6 ± 1.6% (3 months). rAAV6.micro-dystrophin treated animals demonstrated 87.7 ± 6.8 (1 month), 78.9 ± 7.4 (2 months), and 81.2 ± 6.2% (3 months) transduction. In striking contrast, rAAV1 demonstrated very low transduction efficiency [0.9 ± 0.3 (1 month), 2.1 ± 0.8 (2 months), and 2.1 ± 0.7% (3 months)] by vascular delivery. Micro-dystrophin delivered by rAAV8 and rAAV6 through the femoral artery significantly improved tetanic force and protected against eccentric contraction. Mouse studies translated to the hindlimb of cynamologous macaques using a similar vascular delivery paradigm. rAAV8 carrying eGFP in doses proportional to the mouse (5 × 10¹² vg/kg in mouse vs 2 × 10¹² vg/kg in monkey) demonstrated widespread gene expression [medial gastrocnemius – 63.8 ± 4.9%, lateral gastrocnemius – 66.0 ± 4.5%, EDL – 80.2 ± 3.1%, soleus – 86.4 ± 1.9%, TA – 72.2 ± 4.0%. Conclusion These studies demonstrate regional vascular gene delivery with AAV serotype(s) in mouse and non-human primate at doses, pressures and volumes applicable for clinical trials in children with DMD.  

5.489           Cdk5 Modulates Cocaine Reward, Motivation, and Striatal Neuron Excitability

Benavides, D.R. et al

1. Neurosci., 27(47), 12967-12976 (2007)

  Cyclin-dependent kinase 5 (Cdk5) regulates dopamine neurotransmission and has been suggested to serve as a homeostatic target of chronic psychostimulant exposure. To study the role of Cdk5 in the modulation of the cellular and behavioral effects of psychoactive drugs of abuse, we developed Cre/loxP conditional knock-out systems that allow temporal and spatial control of Cdk5 expression in the adult brain. Here, we report the generation of Cdk5 conditional knock-out (cKO) mice using the CaMKII promoter-driven Cre transgenic line (CaMKII-Cre). In this model system, loss of Cdk5 in the adult forebrain increased the psychomotor-activating effects of cocaine. Additionally, these CaMKII-Cre Cdk5 cKO mice show enhanced incentive motivation for food as assessed by instrumental responding on a progressive ratio schedule of reinforcement. Behavioral changes were accompanied by increased excitability of medium spiny neurons in the nucleus accumbens (NAc) in Cdk5 cKO mice. To study NAc-specific effects of Cdk5, another model system was used in which recombinant adeno-associated viruses expressing Cre recombinase caused restricted loss of Cdk5 in NAc neurons. Targeted knock-out of Cdk5 in the NAc facilitated cocaine-induced locomotor sensitization and conditioned place preference for cocaine. These results suggest that Cdk5 acts as a negative regulator of neuronal excitability in the NAc and that Cdk5 may govern the behavioral effects of cocaine and motivation for reinforcement.  

5.490           Vectors selected from adeno-associated viral display peptide libraries for leukemia cell–targeted cytotoxic gene therapy

Michelfelder, S. et al Exp. Hematol., 35, 1766-1776 (2007)   Objective For acute myeloid leukemia (AML), gene therapy may be used to treat patients refractory to conventional chemotherapy. However, availability of vectors sufficiently and specifically transducing this cell type is very limited. Method Here we report the selection of capsid-modified adeno-associated viral (AAV) vectors targeting Kasumi-1 AML cells by screening random AAV displayed peptide libraries. Results The peptide inserts of the enriched capsid mutants share a common sequence motif. The same motif was selected in an independent library screening on HL-60 AML cells. Recombinant targeted vectors displaying the selected peptides transduced the target leukemia cells they have been selected on up to 500-fold more efficiently compared to AAV vectors with control peptide inserts. One of the selected clones (NQVGSWS) also efficiently transduced all members of a panel of four other AML cell lines. Binding and blocking experiments showed that NQVGSWS binding to leukemia cells is independent of the wild-type AAV-2 receptor heparin sulfate proteoglycan. Transduction assays on a panel of hematopoietic and nonhematopoietic cell lines showed that the NQVGSWS capsid was able to overcome resistance to AAV transduction, especially in hematopoietic cancer cells, whereas normal peripheral blood mononuclear cells and CD34⁺ hematopoietic progenitor cells were not transduced. Conclusions Consequently, recombinant targeted NQVGSWS AAV vectors harboring a suicide gene conferred selective killing to Kasumi-1 cells, but not to control cells. This suggests that the AAV mutant selected here may be used as a tool to target therapeutic genes to AML cells.  

5.491           Thiol-reactive reagents inhibits intracellular trafficking of human papillomavirus type 16 pseudovirions by binding to cysteine residues of major capsid protein L1

Ishii, Y. et al Virol. J., 4(1), 110-120 (2007)   Background A human papillomavirus (HPV) virion is composed of capsid proteins L1 and L2. Several cysteine residues are located on L1 of various HPVs at markedly similar relative positions, suggesting their important functions. Although the authentic virions cannot be studied with cultured cells, surrogate pseudovirions consisting of capsid and reporter plasmid are available for studies dealing with infectivity. Results HPV type16-pseudovirions (16PVs) were found to lose their infectivity after incubation with thiol-reactive reagents [biotin polyethyleneoxide iodoacetamide (BPEOIA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM), 4-(N-maleimido)benzyl-trimethylammonium iodide (MBTA), and [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET)]. A labelled streptavidin was detected to bind to the complex of BPEOIA and L1 of the 16PVs incubated with BPEOIA. The analysis of molecular mass of trypsin-fragments derived from the complex of the BPEOIA and L1 indicated that BPEOIA bound to at least C146, C225, and C229. No appreciable change of the 16PVs carrying DTNB or NEM was detected by sedimentation analysis or electron microscopy. The 16PVs carrying DTNB or NEM were able to bind to and enter HeLa cells but degraded before they reached the perinuclear region. Conclusion HPV16 L1 C146, C225, and C229 have free thiol, which are accessible to BPEOIA, DTNB, NEM, MBTA, and MTSET. Binding of DTNB or NEM to the thiols may cause conformational changes that result in the inhibition of the entry and trafficking of the 16PVs.  

5.492           Purification of infectious human herpesvirus 6A virions and association of host cell proteins

Hammarstadt, M., Ahlqvist, J., Jacobson, S., Garoff, H. and Fogdell-Hahn, A. Virology J., 4, 101-111 (2007)   Viruses that are incorporating host cell proteins might trigger autoimmune diseases. It is therefore of interest to identify possible host proteins associated with viruses, especially for enveloped viruses that have been suggested to play a role in autoimmune diseases, like human herpesvirus 6A (HHV-6A) in multiple sclerosis (MS). RESULTS: We have established a method for rapid and morphology preserving purification of HHV-6A virions, which in combination with parallel analyses with background control material released from mock-infected cells facilitates qualitative and quantitative investigations of the protein content of HHV-6A virions. In our iodixanol gradient purified preparation, we detected high levels of viral DNA by real-time PCR and viral proteins by metabolic labelling, silver staining and western blots. In contrast, the background level of cellular contamination was low in the purified samples as demonstrated by the silver staining and metabolic labelling analyses. Western blot analyses showed that the cellular complement protein CD46, the receptor for HHV-6A, is associated with the purified and infectious virions. Also, the cellular proteins clathrin, ezrin and Tsg101 are associated with intact HHV-6A virions. CONCLUSION: Cellular proteins are associated with HHV-6A virions. The relevance of the association in disease and especially in autoimmunity will be further investigated.  

5.493           Isolated HIV-1 core is active for reverse transcription

Warrilow, D., Stenzel, D. and Harrich, D. Retrovirology, 4, 77-81 (2007)   Whether purified HIV-1 virion cores are capable of reverse transcription or require uncoating to be activated is currently controversial. To address this question we purified cores from a virus culture and tested for the ability to generate authentic reverse transcription products. A dense fraction (approximately 1.28 g/ml) prepared without detergent, possibly derived from disrupted virions, was found to naturally occur as a minor sub-fraction in our preparations. Core-like particles were identified in this active fraction by electron microscopy. We are the first to report the detection of authentic strong-stop, first-strand transfer and full-length minus strand products in this core fraction without requirement for an uncoating activity.  

5.494           Adeno-Associated Viral Vectors Engineered For Macrolide-Adjustable Transgene Expression In Mammalian Cells and Mice

Fluri, D.A., Daoud-el baba, M. And Fussenegger, M. BMC Biotech., 7, 75-  (2007)   Background Adjustable gene expression is crucial in a number of applications such as de- or transdifferentiation of cell phenotypes, tissue engineering, various production processes as well as gene-therapy initiatives. Viral vectors, based on the Adeno-Associated Virus (AAV) type 2, have emerged as one of the most promising types of vectors for therapeutic applications due to excellent transduction efficiencies of a broad variety of dividing and mitotically inert cell types and due to their unique safety features. Results We designed recombinant adeno-associated virus (rAAV) vectors for the regulated expression of transgenes in different configurations. We integrated the macrolide-responsive E.REX systems (EON and EOFF) into rAAV backbones and investigated the delivery and expression of intracellular as well as secreted transgenes for binary set-ups and for self- and auto-regulated one-vector configurations. Extensive quantitative analysis of an array of vectors revealed a high level of adjustability as well as tight transgene regulation with low levels of leaky expression, both crucial for therapeutical applications. We tested the performance of the different vectors in selected biotechnologically and therapeutically relevant cell types (CHO-K1, HT-1080, NHDF, MCF-7). Moreover, we investigated key characteristics of the systems, such as reversibility and adjustability to the regulating agent, to determine promising candidates for in vivo studies. To validate the functionality of delivery and regulation we performed in vivo studies by injecting particles, coding for compact self-regulated expression units, into mice and adjusting transgene expression. Conclusion Capitalizing on established safety features and a track record of high transduction efficiencies of mammalian cells, adeno- associated virus type 2 were successfully engineered to provide new powerful tools for macrolide-adjustable transgene expression in mammalian cells as well as in mice.  

5.495           WHO workshop and practical course on Human Papillomavirus (HPV) genetyping and HPV16/18 serology

Lausanne, Switzerland, June 2007   The first meeting of the global HPV laboratory network (LabNet) was organized as a joint workshop and practical course. The aims of the workshop were to discuss laboratory aspects linked to the introduction, follow-up and surveillance of the HPV prophylactic vaccines and the role of the global HPV LabNet in promoting internationally recognized quality of the laboratory functions needed. The practical course allowed the participants to provide input on both theoretical and practical issues involved in international standardization and quality control of HPV laboratory methodologies. The course provided an opportunity for participants to conduct HPV genotyping on patient samples and a candidate HPV LabNet DNA proficiency panel using consensus primer PCR and reverse hybridization methodology. Participants also performed HPV serology on patient samples and a candidate International Standard (IS) for HPV16 antibody using both a virus-like particle (VLP)-based enzyme-linked immunosorbent assay (ELISA) and a pseudovirion (PsV)-based neutralization assay. The workshop reviewed the tasks of the appointed HPV LabNet members and made a number of practical decision regarding organization and implementation of the specific tasks of the HPV LabNet, such as the organization of the writing of the WHO Laboratory Manual for HPV diagnosis and the design of the HPV LabNet communication strategy including a bi-annual Newsletter. The workshop/practical course was positively evaluated by the participants and can be used as a template for forthcoming workshops and practical courses for newly appointed HPV LabNet members or trainees involved in HPV surveillance.  

5.496           XIAP Protection of Photoreceptors in Animal Models of Retinitis Pigmentosa

Leonard, K.C. et al PloS One, 3, e314 (2007)   Background Retinitis pigmentosa (RP) is a blinding genetic disorder that is caused by the death of photoreceptors in the outer nuclear layer of the retina. To date, 39 different genetic loci have been associated with the disease, and 28 mutated genes have been identified. Despite the complexity of the underlying genetic basis for RP, the final common pathway is photoreceptor cell death via apoptosis. Methodology/Principal Findings In this study, P23H and S334ter rhodopsin transgenic rat models of RP were used to test the neuroprotective effects of anti-apoptotic gene therapy. Adeno-associated viruses (AAV) carrying the X-linked inhibitor of apoptosis (XIAP) or green fluorescent protein (GFP) were delivered subretinally into the eye of transgenic rat pups. Histological and functional measures were used to assess neuroprotection. XIAP is known to block apoptosis by inhibiting the action of caspases-3, -7 and -9. The results show that XIAP gene therapy provides long-term neuroprotection of photoreceptors at both structural and functional levels. Conclusions/Significance Our gene therapy strategy targets the apoptotic cascade, which is the final common pathway in all forms of retinitis pigmentosa. This strategy holds great promise for the treatment of RP, as it allows for the broad protection of photoreceptors, regardless of the initial disease causing mutation.  

5.497           Conformational Reorganization of the SARS Coronavirus Spike Following Receptor Binding: Implications for Membrane Fusion

Beniac, D.R., deVarennes, S.L., Andonov, A., He, R. and Booth, T.F. PloS One, 10, e1082 (2007)   The SARS coronavirus (SARS-CoV) spike is the largest known viral spike molecule, and shares a similar function with all class 1 viral fusion proteins. Previous structural studies of membrane fusion proteins have largely used crystallography of static molecular fragments, in isolation of their transmembrane domains. In this study we have produced purified, irradiated SARS-CoV virions that retain their morphology, and are fusogenic in cell culture. We used cryo-electron microscopy and image processing to investigate conformational changes that occur in the entire spike of intact virions when they bind to the viral receptor, angiotensin-converting enzyme 2 (ACE2). We have shown that ACE2 binding results in structural changes that appear to be the initial step in viral membrane fusion, and precisely localized the receptor-binding and fusion core domains within the entire spike. Furthermore, our results show that receptor binding and subsequent membrane fusion are distinct steps, and that each spike can bind up to three ACE2 molecules. The SARS-CoV spike provides an ideal model system to study receptor binding and membrane fusion in the native state, employing cryo-electron microscopy and single-particle image analysis.  

5.498           Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

Soros, V.B., Yonemoto, W. and Greene, W.C. PloS Pathogens, 3(2), e15 (2007)   APOBEC3G (A3G) is a potent antiretroviral deoxycytidine deaminase that, when incorporated into HIV virions, hypermutates nascent viral DNA formed during reverse transcription. HIV Vif counters the effect of A3G by depleting intracellular stores of the enzyme, thereby blocking its virion incorporation. Through pulse-chase analyses, we demonstrate that virion A3G is mainly recruited from the cellular pool of newly synthesized enzyme compared to older “mature” A3G already residing in high-molecular-mass RNA–protein complexes. Virion-incorporated A3G forms a large complex with viral genomic RNA that is clearly distinct from cellular HMM A3G complexes, as revealed by both gel filtration and biochemical fractionation. Unexpectedly, the enzymatic activity of virion-incorporated A3G is lost upon its stable association with HIV RNA. The activity of the latent A3G enzyme is ultimately restored during reverse transcription by the action of HIV RNase H. Degradation of the viral genomic RNA by RNase H not only generates the minus-strand DNA substrate targeted by A3G for hypermutation but also removes the inhibitory RNA bound to A3G, thereby enabling its function as a deoxycytidine deaminase. These findings highlight an unexpected interplay between host and virus where initiation of antiviral enzymatic activity is dependent on the action of an essential viral enzyme.  

5.499           Carrageenan Is a Potent Inhibitor of Papillomavirus Infection

Buck, C.B. et al PloS Pathogens, 2(7), e69 (2007)   Certain sexually transmitted human papillomavirus (HPV) types are causally associated with the development of cervical cancer. Our recent development of high-titer HPV pseudoviruses has made it possible to perform high-throughput in vitro screens to identify HPV infection inhibitors. Comparison of a variety of compounds revealed that carrageenan, a type of sulfated polysaccharide extracted from red algae, is an extremely potent infection inhibitor for a broad range of sexually transmitted HPVs. Although carrageenan can inhibit herpes simplex viruses and some strains of HIV in vitro, genital HPVs are about a thousand-fold more susceptible, with 50% inhibitory doses in the low ng/ml range. Carrageenan acts primarily by preventing the binding of HPV virions to cells. This finding is consistent with the fact that carrageenan resembles heparan sulfate, an HPV cell-attachment factor. However, carrageenan is three orders of magnitude more potent than heparin, a form of cell-free heparan sulfate that has been regarded as a highly effective model HPV inhibitor. Carrageenan can also block HPV infection through a second, postattachment heparan sulfate–independent effect. Carrageenan is in widespread commercial use as a thickener in a variety of cosmetic and food products, ranging from sexual lubricants to infant feeding formulas. Some of these products block HPV infectivity in vitro, even when diluted a million-fold. Clinical trials are needed to determine whether carrageenan-based products are effective as topical microbicides against genital HPVs.  

5.500           MCP-3 (CCL7) delivered by parvovirus MVMp reduces tumorigenicity of mouse melanoma cells through activation of T lymphocytes and NK cells

Wetzel, K., Struyf, S., Van Damme, J., Kayser, T., Vecchi, A., Sozzani, S., Rommelaere, J., Cornelius, J.J. and Dinsart, C. Int. J. Cancer, 120(6), 1364-1371 (2007)   Monocyte chemotactic protein 3 (MCP-3/CCL7), a CC chemokine able to attract and activate a large panel of leukocytes including natural killer cells and T lymphocytes, could be beneficial in antitumor therapy. Vectors were constructed based on the autonomous parvovirus minute virus of mice (MVMp), carrying the human (MCP-3) cDNA. These vectors were subsequently evaluated in the poorly immunogenic mouse melanoma model B78/H1. The infection of the tumor cells with MCP3-transducing vector at low virus input multiplicities, but not with wild-type virus, strongly inhibited tumor growth after implantation in euthymic mice. In a therapeutic B78/H1 model, repeated intratumoral injections of MCP3-tranducing virus prevented further tumor expansion as long as the treatment was pursued. The antitumor effects of the MCP-3-transducing vector were not restricted to this tumor model since they could also be observed in the K1735 melanoma. The depletion of CD4, CD8, NK cells and of interferon (IFN ) in mice implanted with MVMp/MCP3-infected B78/H1 cells abolished the antitumor activity of the vector. The latter data, together with tumor growth in nude mice and reverse-transcriptase (RT)-PCR analyses of MVMp/MCP3-treated tumors, clearly showed that activated CD4, CD8 and NK cells were indispensable for the antineoplastic effect in the B78/H1 tumor. Altogether, our results show that MCP3-transducing parvovirus vectors may be quite potent against poorly or nonimmunogenic tumors, even in conditions where only a fraction of the tumor cell population is efficiently infected with recombinant parvoviruses.  

5.501           In vivo expression of human ATP:cob(I)alamin adenosyltransferase (ATR) using recombinant adeno-associated virus (rAAV) serotypes 2 and 8

Erger, K.E., Conlon, T.J., Leal, N.A., Zori, R., Bobik, T.A. and Flotte, T.R.

1. Gene Med., 9(6), 462-469 (2007)

  Background Methylmalonic aciduria (MMA) is an autosomal recessive disease with symptoms that include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000-fold greater than normal. CblB MMA, a subset of the mutations leading to MMA, is caused by a deficiency in the enzyme cob(I)alamin adenosyltransferase (ATR). No animal model currently exists for this disease. ATR functions within the mitochondria matrix in the final conversion of cobalamin into coenzyme B12, adenosylcobalamin (AdoCbl). AdoCbl is a required coenzyme for the mitochondrial enzyme methylmalonyl-CoA mutase (MCM). Methods The human ATR cDNA was cloned into a recombinant adeno-associated virus (rAAV) vector and packaged into AAV 2 or 8 capsids and delivered by portal vein injection to C57/Bl6 mice at a dose of 1 × 1010 and 1 × 1011 particles. Eight weeks post-injection RNA, genomic DNA and protein were then extracted and analyzed. Results Using primer pairs specific to the cytomegalovirus (CMV) enhancer/chicken β-actin (CBAT) promoter within the rAAV vectors, genome copy numbers were found to be 0.03, 2.03 and 0.10 per cell in liver for the rAAV8 low dose, rAAV8 high dose and rAAV2 high dose, respectively. Western blotting performed on mitochondrial protein extracts demonstrated protein levels were comparable to control levels in the rAAV8 low dose and rAAV2 high dose animals and 3- to 5-fold higher than control levels were observed in high dose animals. Immunostaining demonstrated enhanced transduction efficiency of hepatocytes to over 40% in the rAAV8 high dose animals, compared to 9% and 5% transduction in rAAV2 high dose and rAAV8 low dose animals, respectively. Conclusions These data demonstrate the feasibility of efficient ATR gene transfer to the liver as a prelude to future gene therapy experiments.  

5.502           Enhanced preparation of adeno-associated viral vectors by using high hydrostatic pressure to selectively inactivate helper adenovirus

Leonard, J.N., Ferstl, P., Delgado, A. and Schaffer, D.V. Biotechnololgy and Bioengineering, 97(5), 1170-1179 (2007)   Gene delivery vectors based on adeno-associated virus (AAV) have significant therapeutic potential, but much room for improvement remains in the areas of vector engineering and production. AAV production requires complementation with either helper virus, such as adenovirus, or plasmids containing helper genes, and helper virus-based approaches have distinct advantages in the use of bioreactors to produce large quantities of AAV vectors for clinical applications. However, helper viruses must eventually be inactivated and removed from AAV preparations to ensure safety. The current practice of thermally inactivating adenovirus is problematic as it can also inactivate AAV. Here, we report a novel method using high hydrostatic pressure (HHP) to selectively and completely inactivate helper adenovirus without any detectable loss of functional AAV vectors. The pressure inactivation kinetics of human adenovirus serotype 5 and the high-pressure stabilities of AAV serotypes 2 and 5 (AAV2, AAV5), which were previously unknown, were characterized. Adenovirus was inactivated beyond detection at 260 MPa or higher, whereas AAV2 was stable up to ∼450 MPa, and surprisingly, AAV5 was stable up to at least 700 MPa. The viral genomic DNA of pressure-inactivated AAV2 was made sensitive to DNAse I digestion, suggesting that gross changes in particle structure had occurred, and this hypothesis was further supported by transmission electron microscopy. This approach should be useful in the laboratory- and clinical-scale production of AAV gene delivery vectors. Moreover, HHP provides a tool for probing the biophysical properties of AAV, which may facilitate understanding and improving the functions of this important virus.  

5.503           InXy and SeXy, compact heterologous reporter proteins for mammalian cells

Fluri, D.A., Kelm, J.M., Lesage, G., Daoud-El Baba, M. and Fussenegger, M. Biotechnology and Bioingineering, 98(3), 655-667 (2007)   Mammalian reporter proteins are essential for gene-function analysis, drugscreening initiatives and as model product proteins for biopharmaceutical manufacturing. Bacillus subtilis can maintain its metabolism by secreting Xylanase A (XynA), which converts xylan into shorter xylose oligosaccharides. XynA is a family 11 xylanase monospecific for D-xylose containing substrates. Mammalian cells transgenic for constitutive expression of wild-type xynA showed substantial secretion of this prokaryotic enzyme. Deletion analysis confirmed that a prokaryotic signal sequence encoded within the first 81 nucleotides was compatible with the secretory pathway of mammalian cells. Codon optimization combined with elimination of the prokaryotic signal sequence resulted in an exclusively intracellular mammalian Xylanase A variant (InXy) while replacement by an immunoglobulin-derived secretion signal created an optimal secreted Xylanase A derivative (SeXy). A variety of chromogenic and fluorescence-based assays adapted for use with mammalian cells detected InXy and SeXy with high sensitivity and showed that both reporter proteins resisted repeated freeze/thaw cycles, remained active over wide temperature and pH ranges, were extremely stable in human serum stored at room temperature and could independently be quantified in samples also containing other prominent reporter proteins such as the human placental alkaline phosphatase (SEAP) and the Bacillus stearothermophilus-derived secreted -amylase (SAMY). Glycoprofiling revealed that SeXy produced in mammalian cells was N- glycosylated at four different sites, mutation of which resulted in impaired secretion. SeXy was successfully expressed in a variety of mammalian cell lines and primary cells following transient transfection and transduction with adeno-associated virus particles (AAV) engineered for constitutive SeXy expression. Intramuscular injection of transgenic AAVs into mice showed significant SeXy levels in the bloodstream. InXy and SeXy are highly sensitive, compact and robust reporter proteins, fully compatible with pre-existing marker genes and can be assayed in high-throughput formats using very small sample volumes.    

5.504           Double-Labeled Rabies Virus: Live Tracking of Enveloped Virus Transport

Klingen, Y., Conzelmann, K-K. and Finke, S.

1. Virol., 82(1), 237-245 (2008)

  Here we describe a strategy to fluorescently label the envelope of rabies virus (RV), of the Rhabdoviridae family, in order to track the transport of single enveloped viruses in living cells. Red fluorescent proteins (tm-RFP) were engineered to comprise the N-terminal signal sequence and C-terminal transmembrane spanning and cytoplasmic domain sequences of the RV glycoprotein (G). Two variants of tm-RFP were transported to and anchored in the cell surface membrane, independent of glycosylation. As shown by confocal microscopy, tm-RFP colocalized at the cell surface with the RV matrix and G protein and was incorporated into G gene-deficient virus particles. Recombinant RV expressing the membrane-anchored tm-RFP in addition to G yielded infectious viruses with mosaic envelopes containing both tm-RFP and G. Viable double-labeled virus particles comprising a red fluorescent envelope and a green fluorescent ribonucleoprotein were generated by expressing in addition an enhanced green fluorescent protein-phosphoprotein fusion construct (S. Finke, K. Brzozka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004). Individual enveloped virus particles were observed under live cell conditions as extracellular particles and inside endosomal vesicles. Importantly, double-labeled RVs were transported in the retrograde direction over long distances in neurites of in vitro-differentiated NS20Y neuroblastoma cells. This indicates that the typical retrograde axonal transport of RV to the central nervous system involves neuronal transport vesicles in which complete enveloped RV particles are carried as a cargo.  

5.505           Cardio-specific long-term gene expression in a porcine model after selective pressure-regulated retroinfusion of adeno-associated viral (AAV) vectors

Raake, P.W. et al Gene Therapy, 15, 12-17 (2008)   Cornerstone for an efficient cardiac gene therapy is the need for a vector system, which enables selective and long-term expression of the gene of interest. In rodent animal models adeno-associated viral (AAV) vectors like AAV-6 have been shown to efficiently transduce cardiomyocytes. However, since significant species-dependent differences in transduction characteristics exist, large animal models are of imminent need for preclinical evaluations. We compared gene transfer efficiencies of AAV-6 and heparin binding site-deleted AAV-2 vectors in a porcine model. Application of the AAVs was performed by pressure-regulated retroinfusion of the anterior interventricular cardiac vein, which has been previously shown to efficiently deliver genes to the myocardium (3.5 10¹⁰ viral genomes per animal; n=5 animals per group). All vectors harbored a luciferase reporter gene under control of a cytomegalovirus (CMV)-enhanced 1.5 kb rat myosin light chain promoter (CMV-MLC2v). Expression levels were evaluated 4 weeks after gene transfer by determining luciferase activities. To rule out a systemic spillover peripheral tissue was analyzed by PCR for the presence of vector genomes. Selective retroinfusion of AAV serotype 6 vectors into the anterior cardiac vein substantially increased reporter gene expression in the targeted distal left anterior descending (LAD) territory (65 943 31 122 vs control territory 294 69, P<0.05). Retroinfusion of AAV-2 vectors showed lower transgene expression, which could be increased with coadministration of recombinant human vascular endothelial growth factor (1365 707 no vascular endothelial growth factor (VEGF) vs 38 760 2448 with VEGF, P<0.05). Significant transgene expression was not detected in other organs than the heart, although vector genomes were detected also in the lung and liver. Thus, selective retroinfusion of AAV-6 into the coronary vein led to efficient long-term myocardial reporter gene expression in the targeted LAD area of the porcine heart. Coapplication of VEGF significantly increased transduction efficiency of AAV-2.  

5.506           AAV8, 9, Rh10, Rh43 Vector Gene Transfer in the Rat Brain: Effects of Serotype, Promoter and Purification Method

Klein, R.-L., Dayton, R.D., Tatom, J.B., Henderson, K.M. and Henning, P.P. Molecular Therapy, 16(1), 89-96 (2008)   We compared adeno-associated virus (AAV) serotypes for expression levels of green fluorescent protein (GFP) in the adult rat hippocampus by biophotonic imaging. Preparations of AAV serotypes 8, 9, Rh10, and Rh43 incorporating cytomegalovirus (CMV) promoter–driven GFP were purified by a CsCl method. Neither AAV Rh10 nor AAV Rh43 produced greater levels of GFP than AAV8, which was used as a reference. For AAV9, there was an increase relative to AAV8. The CsCl-purified AAV8 displayed an astroglial transduction pattern in contrast to the expected neuronal expression of other AAVs. After preparing the same CMV-GFP plasmid in AAV8 with an iodixanol purification method, the expected neuronal pattern resulted. The astroglial expression with the CsCl AAV8 was probably due to relatively high levels of protein impurities. We compared the CMV promoter with the CMV/chicken -actin (CBA) promoter in the context of AAV8, both prepared by iodixanol, and found the CBA promoter to produce stronger GFP expression. At two doses of vectors optimized for serotype, promoter and purification, we did not observe serotype differences among AAV8, AAV9, or AAV Rh10. The purification method can therefore impact the transduction pattern as well as the results when comparing serotype strengths.  

5.507           Rapid/Sustained Anti-anthrax Passive Immunity Mediated by Co-administration of Ad/AAV

De, B.P., Hackett, N.R., Crystal, R.G. and Boyer, J.L. Molecular Therapy, 16(1), 203-209 (2008)   Achieving both immediate and sustained protection against diseases caused by bacterial toxins and extracellular pathogens is a challenge in developing biodefense therapeutics. We hypothesized that a single co-administration of an adenovirus (Ad) vector and an adeno-associated virus (AAV) vector, both expressing a pathogen-specific monoclonal antibody, would provide rapid, persistent passive immunotherapy against the pathogen. In order to test this strategy, we used the lethal toxin of Bacillus anthracis as a target of a monoclonal antibody directed against the protective antigen (PA) component of the toxin, using co-administration of an Ad vector encoding an anti-PA monoclonal antibody (Ad PA) and an AAV vector encoding an anti-PA monoclonal antibody (AAVrh.10 PA). As early as 1 day after co-administration of Ad PA and AAVrh.10 PA to mice, serum anti-PA antibody levels were detectable, and were sustained through 6 months. Importantly, animals that received both vectors were protected against toxin challenge as early as 1 day after administration and throughout the 6 month duration of the experiment. These data provide a new paradigm of genetic passive immunotherapy by co-administration of Ad and AAV vectors, each encoding a pathogen-specific monoclonal antibody, as an effective approach for both rapid and sustained protection against a bio-terror attack.  

5.508           Purification of human respiratory syncytial virus by ultracentrifugation in iodixanol density gradient

Gias, E., Nielsen, S.U., Morgan, L.A.F. and Toms, G.L.

1. Virol. Methods, 147, 328-332 (2008)

  Ultracentrifugation in sucrose density gradient remains the most commonly used technique for hRSV purification. However, the high viscosity and hyper-osmotic property of sucrose can cause damage to the extremely labile virus leading to loss of infectivity. To overcome these limitations, an alternative purification technique was developed using iodixanol as gradient medium, incorporating MgSO₄ as a stabilizing agent and EDTA to disaggregate the virus prior to infectivity assay. Virus particles were banded at the 20–36% interface after purification of polyethylene glycol-concentrated viruses by rate zonal ultracentrifugation on a 20–52% discontinuous iodixanol gradient. The presence of the virus was confirmed by viral fusion glycoprotein content using ELISA. After further purification by buoyant density ultracentrifugation on a 20–52% continuous gradient, the virus was recovered in the region of density 1.15–1.19 g/ml and this was confirmed by the coincidence of the infectivity titre, viral genome and fusion glycoprotein peaks. Analysis of recovery rates showed that the use of iodixanol increased the virus yield up to 69%. Iodixanol was also found to be non-toxic to HeLa cells used in infectivity assay, eliminating the need of its downstream removal by dialysis.  

5.509           Generation and characterization of a preventive and therapeutic HPV DNA vaccine

Kim, D. et al Vaccine, 26(3), 351-360 (2008)  

Cervical cancer is one of the most common cancers in women worldwide. Persistent infection with human papillomavirus (HPV) is considered to be the etiological factor for cervical cancer. Therefore, an effective vaccine against HPV infections may lead to the control of cervical cancer. An ideal HPV vaccine should aim to generate both humoral immune response to prevent new infections as well as cell-mediated immunity to eliminate established infection or HPV-related disease. In the current study, we have generated a potential preventive and therapeutic HPV DNA vaccine using human calreticulin (CRT) linked to HPV16 early proteins, E6 and E7 and the late protein L2 (hCRTE6E7L2). We found that vaccination with hCRTE6E7L2 DNA vaccine induced a potent E6/E7-specific CD8+ T cell immune response, resulting in a significant therapeutic effect against E6/E7 expressing tumor cells. In addition, vaccination with hCRTE6E7L2 DNA generated significant L2-specific neutralizing antibody responses, protecting against pseudovirion infection. Thus, the hCRTE6E7L2 DNA vaccines are capable of generating potent preventive and therapeutic effects in vaccinated mice. Our data has significant clinical implications.

5.510           In vivo gene delivery for development of mammalian models for Parkinson's disease

Ulusoy, A., Bjorklund, T., Hermening, S. and Kirik, D. Exp. Neurol., 209, 89-100 (2008)   During the last decade, identification of the genes involved in familial forms of Parkinson's disease (PD) has advanced our understanding of the mechanisms underlying the development of different aspects of PD. However the available animal models still remain as the main limiting factor for the development of neuroprotective therapies that can halt the progression of the disease, through which we wish to provide a better quality of life for the PD patients. Here, we review the recently developed animal models based on overexpression of PD-associated genes using recombinant viral vectors. Recombinant adeno-associated viral vectors, in particular, have been very useful in targeting the nigral dopamine neurons both in the rodent and the primate brain. In order to provide insights into the establishment of these models in the laboratory, we will not only give an overview of the results from these studies but also cover practical issues related to the production and handling of the viral vectors, which are critical for the successful application of this approach.      

5.511           Identification of Host Proteins Associated with Retroviral Vector Particles by Proteomic Analysis of Highly Purified Vector Preparations

Segura, M.M. et al

1. Virol., 82(3), 1107-1117 (2008)

  The Moloney murine leukemia virus (MMLV) belongs to the Retroviridaefamily of enveloped viruses, which is known to acquire minuteamounts of host cellular proteins both on the surface and insidethe virion. Despite the extensive use of retroviral vectorsin experimental and clinical applications, the repertoire ofhost proteins incorporated into MMLV vector particles remainsunexplored. We report here the identification of host proteinsfrom highly purified retroviral vector preparations obtainedby rate-zonal ultracentrifugation. Viral proteins were fractionatedby one-dimensional sodium dodecyl sulfate-polyacrylamide gelelectrophoresis, in-gel tryptic digested, and subjected to liquidchromatography/tandem mass spectrometry analysis. Immunogoldelectron microscopy studies confirmed the presence of severalhost membrane proteins exposed at the vector surface. Thesestudies led to the identification of 27 host proteins on MMLVvector particles derived from 293 HEK cells, including 5 proteinspreviously described as part of wild-type MMLV. Nineteen hostproteins identified corresponded to intracellular proteins.A total of eight host membrane proteins were identified, includingcell adhesion proteins integrin β1 (fibronectin receptorsubunit beta) and HMFG-E8, tetraspanins CD81 and CD9, and lateendosomal markers CD63 and Lamp-2. Identification of membraneproteins on the retroviral surface is particularly attractive,since they can serve as anchoring sites for the insertion oftags for targeting or purification purposes. The implicationsof our findings for retrovirus-mediated gene therapy are discussed.  

5.512           Cell Factors Stimulate Human Immunodeficiency Virus Type 1 Reverse Transcription In Vitro

Warrilow, D. et al

1. Virol., 82(3), 1425-1437 (2008)

  After fusion of the human immunodeficiency virus type 1 (HIV-1) envelope with the host cell membrane, the HIV-1 core enters the cell cytoplasm. Core components are then restructured to form the reverse transcription complex (RTC); the biochemical details of this process are currently unclear. To investigate early RTC formation, we characterized the endogenous reverse transcription activity of virions, which was less efficient than reverse transcription during cell infection and suggested a requirement for a cell factor. The addition of detergent to virions released reverse transcriptase and capsid, and reverse transcription products became susceptible to the action of exogenous nucleases, indicating virion disruption. Disruption was coincident with the loss of the endogenous reverse transcription activity of virions, particularly late reverse transcription products. Consistent with this observation, the use of a modified "spin thru" method, which uses brief detergent exposure, also disrupted virions. The addition of lysates made from mammalian cell lines (Jurkat, HEK293T, and NIH 3T3 cells) to virions delipidated by detergent stimulated late reverse transcription efficiency. A complex with reverse transcription activity that was slower sedimenting than virions on a velocity gradient was greatly stimulated to generate full-length reverse transcription products and was associated with only relatively small amounts of capsid. These experiments suggest that cell factors are required for efficient reverse transcription of HIV-1.  

5.513           Integrin _Vβ₃ Binds to the RGD Motif of Glycoprotein B of Kaposi's Sarcoma-Associated Herpesvirus and Functions as an RGD-Dependent Entry Receptor

Garrigues, H.J., Rubinchikova, Y.E., DiPersio, C. and Rose, T.M.

1. Virol., 82(3), 1570-1580 (2008)

  Kaposi's sarcoma-associated herpesvirus (KSHV) envelope-associated glycoprotein B (gB) is involved in the initial steps of binding to host cells during KSHV infection. gB contains an RGD motif reported to bind the integrin ₃β₁ during virus entry. Although the ligand specificity of ₃β₁ has been controversial, current literature indicates that ₃β₁ ligand recognition is independent of RGD. We compared ₃β₁ to the RGD-binding integrin, _Vβ₃, for binding to envelope-associated gB and a gB(RGD) peptide. Adhesion assays demonstrated that β₃-CHO cells overexpressing _Vβ₃ specifically bound gB(RGD), whereas ₃-CHO cells overexpressing ₃β₁ did not. Function-blocking antibodies to _Vβ₃ inhibited the adhesion of HT1080 fibrosarcoma cells to gB(RGD), while antibodies to ₃β₁ did not. Using affinity-purified integrins and confocal microscopy, _Vβ₃ bound to gB(RGD) and KSHV virions, demonstrating direct receptor-ligand interactions. Specific _Vβ₃ antagonists, including cyclic and dicyclic RGD peptides and _Vβ₃ function-blocking antibodies, inhibited KSHV infection by 70 to 80%. Keratinocytes from ₃-null mice lacking ₃β₁ were fully competent for infection by KSHV, and reconstitution of ₃β₁ function by transfection with ₃ cDNA reduced KSHV infectivity from 74% to 55%. Additional inhibitory effects of ₃β₁ on the cell surface expression of _Vβ₃ and on _Vβ₃-mediated adhesion of ₃-CHO cells overexpressing ₃β₁ were detected, consistent with previous reports of transdominant inhibition of _Vβ₃ function by ₃β₁. These observations may explain previous reports of an inhibition of KSHV infection by soluble ₃β₁. Our studies demonstrate that _Vβ₃ is a cellular receptor mediating both the cell adhesion and entry of KSHV into target cells through binding the virion-associated gB(RGD).  

5.514           Neonatal Intraperitoneal or Intravenous Injections of Recombinant Adeno-Associated Virus Type 8 Transduce Dorsal Root Ganglia and Lower Motor Neurons

Foust, K.D., Poirier, A., Pacak, C.A., Mandel, R.J. and Flotte, T.R. Human Gene Therapy, 19(1), 61-69 (2008)   Targeting lower motor neurons (LMNs) for gene delivery could be useful for disorders such as spinal muscular atrophy and amyotrophic lateral sclerosis. LMNs reside in the ventral gray matter of the spinal cord and send axonal projections to innervate skeletal muscle. Studies have used intramuscular injections of adeno-associated virus type 2 (AAV2) to deliver viral vectors to LMNs via retrograde transport. However, treating large areas of the spinal cord in a human would require numerous intramuscular injections, thereby increasing viral titer and risk of immune response. New AAV serotypes, such as AAV8, have a dispersed transduction pattern after intravenous or intraperitoneal injection in neonatal mice, and may transduce LMNs by retrograde transport or through entry into the nervous system. To test LMN transduction after systemic injection, we administered recombinant AAV8 (rAAV8) carrying the green fluorescent protein (GFP) gene by intravenous or intraperitoneal injection to neonatal mice on postnatal day 1. Tissues were harvested 5 and 14 days postinjection and analyzed by real-time polymerase chain reaction and GFP immunohistochemistry to assess the presence of AAV genomes and GFP expression, respectively. Spinal cords were positive for AAV genomes at both time points. GFP immunohistochemistry revealed infrequent labeling of LMNs across all time points and injection routes. Somewhat surprisingly, there was extensive labeling of fibers in the dorsal horns and columns, indicating dorsal root ganglion transduction across all time points and injection routes. Our data suggest that systemic injection of rAAV8 is not an effective delivery route to target lower motor neurons, but could be useful for targeting sensory pathways in chronic pain.  

5.515           Recombinant Adeno-Asociated Virus-Mediated Global Anterograde Delivery of Glial Cell Line-Derived Neurotrophic Factor to the Spinal Cord: Comparison of Rubrospinal and Corticospinal Tracts in the Rat

Foust, K.D., Flotte, T.R., Reier, P.J. and Mandel, R.J. Human Gene Therapy, 19(1), 71-81 (2008)   Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of spinal lower motoneurons. Gene delivery is a promising strategy to deliver therapeutic molecules to these vulnerable cells. However, definition of an optimal route of delivery capable of accessing neurons over a considerable extent of the neuraxis represents a significant logistical problem. Intramuscular vector injections are not ideal as this approach would involve hundreds of injections to completely treat an ALS patient and also would be dependent on retrograde transport of the viral platform of choice. Alternatively, upper motoneurons could deliver trophic factors over considerable distances by anterograde transport after a relatively localized intracerebral injection. To test this approach, the present study was designed to compare the corticospinal (CST) and rubrospinal (RST) tracts for their ability to transport recombinant adeno-associated virus serotype 5 (rAAV5)-derived green fluorescent protein (GFP) or glial cell line-derived neurotrophic factor (GDNF) to the spinal cord. Unilateral injections of rAAV5-GFP into the red nucleus (RN) or motor cortex of normal rats produced GFP-positive fibers in the appropriate descending tracts extending to the lumbar spinal cord. For both tracts, GFP-positive axonal projections into the spinal gray matter were consistently observed. GDNF immunohistochemistry demonstrated that confirmed RN injections resulted in GDNF-positive fibers projecting into spinal gray matter as seen in the GFP group. In contrast, confirmed cortical rAAV5-GDNF injections resulted in less evident staining in spinal cord. Spinal cord GDNF levels were elevated at distances up to 72 mm from the injection sites, and confirmed that RST-related GDNF transport to spinal cord surpassed CST-associated delivery.  

5.516           An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation

Ryu, B.Y. et al Blood, 111(4), 1866-1875 (2008)   Pathogenic activation of the LMO2 proto-oncogene by an oncoretroviral vector insertion in a clinical trial for X-linked severe combined immunodeficiency (X-SCID) has prompted safety concerns. We used an adeno-associated virus vector to achieve targeted insertion of a -retroviral long terminal repeat (LTR) driving a GFP expression cassette with flanking loxP sites in a human T-cell line at the precise location of vector integration in one of the patients with X-SCID. The LTR-GFP cassette was inserted into the first intron of the LMO2 gene, resulting in strong activation of LMO2. Cre-mediated cassette exchange was used to replace the original LTR-GFP cassette with one flanked by insulator elements leading to a several fold reduction in LMO2 expression. The LTR-GFP cassette was also replaced with a globin gene regulatory cassette that failed to activate the LMO2 gene in lymphoid cells. A -retroviral vector with 2 intact LTRs resulted in activation of the LMO2 gene when inserted into the first intron, but a self-inactivating lentiviral vector with an internal cellular promoter and flanking insulator elements did not activate the LMO2 gene. Thus, this system is useful for comparing the safety profiles of vector cassettes with various regulatory elements for their potential for proto-oncogene activation.  

5.517           Cone-specific expression using a human red opsin promoter in recombinant AAV

Li, Q., Timmers, A.M., Guy, J., Pang, J. and Hauswirth, W.W. Vision Res., 48(3), 332-338 (2008)   Purpose To determine the feasibility of targeting gene expression specifically to cone photoreceptors using recombinant adeno-associated virus (rAAV) as the vector. Methods An rAAV vector was constructed that contains a 2.1 kb upstream sequence of the human red opsin gene to direct green fluorescent protein (GFP) expression. A control construct containing a 472 bp mouse rod opsin promoter, previously shown to drive photoreceptor-specific expression, was also used. Each recombinant virus was injected into the subretinal space of rat, ferret or guinea pig eyes. GFP expression was analyzed 4–6 weeks after injection microscopically. Result The human 2.1 kb cone opsin gene upstream sequence targeted GFP expression only to a subset of photoreceptors. Cone-specific expression was shown by co-localization of GFP fluorescence and cone-specific opsin antibody staining. Additionally, in rats, expression was specific for L/M-cones whereas no S-cones exhibited GFP fluorescence. The efficiency of rAAV mediated cone transduction surrounding the injection site was high since every L/M-cone antibody-staining cone was also positive for GFP expression. Conclusion The human red/green opsin gene promoter used in this study is sufficient to direct efficient cone-specific gene expression in several mammalian species, suggesting that key cell-type specific regulatory elements must be broadly conserved in mammals. These observations have significance in devising gene therapy strategies for retinal dystrophies that primarily affect cones and point toward a way to functionally dissect the cone opsin promoter in vivo.  

5.518           Human RNA "Rumor" Viruses: the Search for Novel Human Retroviruses in Chronic Disease

Voisset, C., Weiss, R.A. and Griffiths, D.J. Microbiol. Mol. Biol. Rev., 72(1), 157-196 (2008)   Summary: Retroviruses are an important group of pathogens that cause a variety of diseases in humans and animals. Four human retroviruses are currently known, including human immunodeficiency virus type 1, which causes AIDS, and human T-lymphotropic virus type 1, which causes cancer and inflammatory disease. For many years, there have been sporadic reports of additional human retroviral infections, particularly in cancer and other chronic diseases. Unfortunately, many of these putative viruses remain unproven and controversial, and some retrovirologists have dismissed them as merely "human rumor viruses." Work in this field was last reviewed in depth in 1984, and since then, the molecular techniques available for identifying and characterizing retroviruses have improved enormously in sensitivity. The advent of PCR in particular has dramatically enhanced our ability to detect novel viral sequences in human tissues. However, DNA amplification techniques have also increased the potential for false-positive detection due to contamination. In addition, the presence of many families of human endogenous retroviruses (HERVs) within our DNA can obstruct attempts to identify and validate novel human retroviruses. Here, we aim to bring together the data on "novel" retroviral infections in humans by critically examining the evidence for those putative viruses that have been linked with disease and the likelihood that they represent genuine human infections. We provide a background to the field and a discussion of potential confounding factors along with some technical guidelines. In addition, some of the difficulties associated with obtaining formal proof of causation for common or ubiquitous agents such as HERVs are discussed.  

5.519           Engineering adeno-associated virus 2 vectors for targeted gene delivery to atherosclerotic lesions

White, K. et al Gene Therapy, 15, 443-451 (2008)   Targeted delivery of biological agents to atherosclerotic plaques may provide a novel treatment and/or useful tool for imaging of atherosclerosis in vivo. However, there are no known viral vectors that possess the desired tropism. Two plaque-targeting peptides, CAPGPSKSC (CAP) and CNHRYMQMC (CNH) were inserted into the capsid of adeno-associated virus 2 (AAV2) to assess vector retargeting. AAV2-CNH produced significantly higher levels of transduction than unmodified AAV2 in human, murine and rat endothelial cells, whereas transduction of nontarget HeLa cells was unaltered. Transduction studies and surface plasmon resonance suggest that AAV2-CNH uses membrane type 1 matrix metalloproteinase as a surface receptor. AAV2-CAP only produced higher levels of transduction in rat endothelial cells, possibly because the virus was found to be affected by proteasomal degradation. In vivo substantially higher levels of both peptide-modified AAV2 vectors was detected in the brachiocephalic artery (site of advanced atherosclerotic plaques) and aorta, whereas reduced levels were detected in all other organs examined. These results suggest that in the AAV2 platform the peptides are exposed on the capsid surface in a way that enables efficient receptor binding and so creates effective atherosclerotic plaque targeted vectors.  

5.520           Scavenger Receptor Class B Is Required for Hepatitis C Virus Uptake and Cross-Presentation by Human Dendritic Cells

Barth, H. et al

1. Virol., 82(7), 3466-3479 (2008)

  Class B scavenger receptors (SR-Bs) bind lipoproteins and play an important role in lipid metabolism. Most recently, SR-B type I (SR-BI) and its splicing variant SR-BII have been found to mediate bacterial adhesion and cytosolic bacterial invasion in mammalian cells. In this study, we demonstrate that SR-BI is a key host factor required for hepatitis C virus (HCV) uptake and cross-presentation by human dendritic cells (DCs). Whereas monocytes and T and B cells were characterized by very low or undetectable SR-BI expression levels, human DCs demonstrated a high level of cell surface expression of SR-BI similar to that of primary human hepatocytes. Antibodies targeting the extracellular loop of SR-BI efficiently inhibited HCV-like particle binding, uptake, and cross-presentation by human DCs. Moreover, human high-density lipoprotein specifically modulated HCV-like particle binding to DCs, indicating an interplay of HCV with the lipid transfer function of SR-BI in DCs. Finally, we demonstrate that anti-SR-BI antibodies inhibit the uptake of cell culture-derived HCV (HCVcc) in DCs. In conclusion, these findings identify a novel function of SR-BI for viral antigen uptake and recognition and may have an important impact on the design of HCV vaccines and immunotherapeutic approaches aiming at the induction of efficient antiviral immune responses.  

5.521           Detection of viral bioagents using a shear horizontal surface acoustic wave biosensor

Bisoffi, M. et al Biosensors and Bioelectronics, 23, 1397-1403 (2008)   Viruses are of high medical and biodefense concern and their detection at concentrations well below the threshold necessary to cause health hazards continues to be a challenge with respect to sensitivity, specificity, and selectivity. Ideally, assays for accurate and real time detection of viral agents would not necessitate any pre-processing of the analyte, which would make them applicable for example to bodily fluids (blood, sputum) and man-made as well as naturally occurring bodies of water (pools, rivers). We describe herein a robust biosensor that combines the sensitivity of surface acoustic waves (SAW) generated at a frequency of 325 MHz with the specificity provided by antibodies for the detection of viral agents. A lithium tantalate-based SAW transducer with silicon dioxide waveguide sensor platform featuring three test and one reference delay lines was used to adsorb antibodies directed against either Coxsackie virus B4 or the category A bioagent Sin Nombre virus (SNV), a member of the genus Hantavirus, family Bunyaviridae, negative-stranded RNA viruses. Rapid detection (within seconds) of increasing concentrations of viral particles was linear over a range of order of magnitude for both viruses, although the sensor was approximately 5 × 10⁵-fold more sensitive for the detection of SNV. For both pathogens, the sensor's selectivity for its target was not compromised by the presence of confounding Herpes Simplex virus type 1. The biosensor was able to detect SNV at doses lower than the load of virus typically found in a human patient suffering from hantavirus cardiopulmonary syndrome (HCPS). Further, in a proof-of-principle real world application, the SAW biosensor was capable to selectively detect SNV agents in complex solutions, such as naturally occurring bodies of water (river, sewage effluent) without analyte pre-processing. This is the first study that reports on the detection of viral agents using an antibody-based SAW biosensor that has the potential to be used as a hand-held and self-contained device for rapid viral detection in the field.  

5.522           Augmented transgene expression in transformed cells using a parvoviral hybrid vector

Krüger, L. et al Cancer Gene Therapy, 15, 252-267 (2008)   Autonomous parvoviruses possess an intrinsic oncotropism based on viral genetic elements controlling gene expression and genome replication. We constructed a hybrid vector consisting of the H1 parvovirus-derived expression cassette comprising the p4 promoter, the ns1 gene and the p38 promoter flanked by the adeno-associated viruses 2 (AAV2) inverted terminal repeats and packaged into AAV2 capsids. Gene transduction using this vector could be stimulated by coinfection with adenovirus, by irradiation or treatment with genotoxic agents, similar to standard AAV2 vectors. However, the latter were in most cases less efficient in gene transduction than the hybrid vector. With the new vector, tumor cell-selective increase in transgene expression was observed in pairs of transformed and non-transformed cells, leading to selective killing of the transformed cells after expression of a prodrug-converting enzyme. Preferential gene expression in tumor versus normal liver tissue was also observed in vivo in a syngeneic rat model. Comparative transduction of a panel of different tumor cell lines with the H1 and the H1/AAV hybrid vector showed a preference of each vector for distinct cell types, probably reflecting the dependence of the viral tropism on capsid determinants.  

5.523           A combined therapeutic approach for pyruvate dehydrogenase deficiency using self-complementary adeno-associated virus serotype-specific vectors and dichloroacetate

Han, Z. et al Mol. Gen. Metabol., 93, 381-387 (2008)   We determined the ability of self-complementary adeno-associated virus (scAAV) vectors to deliver and express the pyruvate dehydrogenase E1α subunit gene (PDHA1) in primary cultures of skin fibroblasts from 3 patients with defined mutations in PHDA1 and 3 healthy subjects. Cells were transduced with scAAV vectors containing the cytomegalovirus promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a vector:cell ratio of 200. Transgene expression was measured 72 h later. The transduction efficiency of scAAV2 and scAAV6 vectors was 3- to 5-fold higher than that of the other serotypes, which were subsequently used to transduce fibroblasts with wild-type PDHA1 cDNA under the control of the chicken beta-action (CBA) promoter at a vector:cell ratio of 1000. Total PDH-specific activity and E1α protein expression were determined 10 days post-transduction. Both vectors increased E1α expression 40–60% in both control and patient cells, and increased PDH activity in two patient cell lines. We also used dichloroacetate (DCA) to maximally activate PDH through dephosphorylation of E1α. Exposure for 24 h to 5 mM DCA increased PDH activity in non-transduced control (mean 37% increase) and PDH deficient (mean 44% increase) cells. Exposure of transduced patient fibroblasts to DCA increased PDH activity up to 90% of the activity measured in untreated control cells. DCA also increased expression of E1α protein and, to variable extents, that of other components of the PDH complex in both non-transduced and transduced cells. These data suggest that a combined gene delivery and pharmacological approach may hold promise for the treatment of PDH deficiency.  

5.524           Stable Integration of Recombinant Adeno-Associated Virus Vector Genomes After Transduction of Murine Hematopoietic Stem Cells

Han, Z. et al Human Gene Therapy, 19, 267-278 (2008)   We previously reported that among single-stranded adeno-associated virus (ssAAV) vectors, serotypes 1 through 5, ssAAV1 is the most efficient in transducing murine hematopoietic stem cells (HSCs), but viral second-strand DNA synthesis remains a rate-limiting step. Subsequently, using double-stranded, self-complementary AAV (scAAV) vectors, serotypes 7 through 10, we observed that scAAV7 vectors also transduce murine HSCs efficiently. In the present study, we used scAAV1 and scAAV7 shuttle vectors to transduce HSCs in a murine bone marrow serial transplant model in vivo, which allowed examination of the AAV proviral integration pattern in the mouse genome, as well as recovery and nucleotide sequence analyses of AAV–HSC DNA junction fragments. The proviral genomes were stably integrated, and integration sites were localized to different mouse chromosomes. None of the integration sites was found to be in a transcribed gene, or near a cellular oncogene. None of the animals, monitored for up to 1 year, exhibited pathological abnormalities. Thus, AAV proviral integration-induced risk of oncogenesis was not found in our study, which provides functional confirmation of stable transduction of self-renewing multipotential HSCs by scAAV vectors as well as promise for the use of these vectors in the potential treatment of disorders of the hematopoietic system.  

5.525           Sulfated K5 Escherichia coli Polysaccharide Derivatives as Wide-Range Inhibitors of Genital Types of Human Papillomavirus

Lembo, D. et al Antimicrob. Agents Chemother., 52, 1374-1381 (2008)   Genital human papillomaviruses (HPV) represent the most common sexually transmitted agents and are classified into low or high risk by their propensity to cause genital warts or cervical cancer, respectively. Topical microbicides against HPV may be a useful adjunct to the newly licensed HPV vaccine. A main objective in the development of novel microbicides is to block HPV entry into epithelial cells through cell surface heparan sulfate proteoglycans. In this study, selective chemical modification of the Escherichia coli K5 capsular polysaccharide was integrated with innovative biochemical and biological assays to prepare a collection of sulfated K5 derivatives with a backbone structure resembling the heparin/heparan biosynthetic precursor and to test them for their anti-HPV activity. Surface plasmon resonance assays revealed that O-sulfated K5 with a high degree of sulfation [K5-OS(H)] and N,O-sulfated K5 with a high [K5-N,OS(H)] or low [K5-N,OS(L)] sulfation degree, but not unmodified K5, N-sulfated K5, and O-sulfated K5 with low levels of sulfation, prevented the interaction between HPV-16 pseudovirions and immobilized heparin. In cell-based assays, K5-OS(H), K5-N,OS(H), and K5-N,OS(L) inhibited HPV-16, HPV-18, and HPV-6 pseudovirion infection. Their 50% inhibitory concentration was between 0.1 and 0.9 µg/ml, without evidence of cytotoxicity. These findings provide insights into the design of novel, safe, and broad-spectrum microbicides against genital HPV infections.  

5.526           Modulation of spontaneous hippocampal synaptic events with 5-hydroxyindole, 4OH-GTS-21, and rAAV-mediated α7 nicotinic receptor gene transfer

Thinschmidt, J. et al Brain Res., 1203, 51-60 (2008)   One approach to treatment of negative cognitive effects associated with Alzheimer's disease and schizophrenia may involve activation of neuronal α7 nicotinic acetylcholine receptors (nAChRs). We used the α7-selective partial agonist 3-(4-hydroxy, 2-methoxy-benzylidene)anabaseine (4OH-GTS-21), the α7 modulator 5-hydroxyindole (5-HI), and recombinant adeno-associated virus (rAAV)-mediated α7 gene transfer in order to test the hypothesis whether combining these strategies would significantly increase indirect measures of α7 nAChR function, including measures of spontaneous synaptic events in CA1 pyramidal cells. 5-HI (1 mM), and 5-HI (1 mM) + 4OH-GTS-21 (5 μM) increased the frequency of APV- and NBQX-sensitive currents, while 5-HI + 4OH-GTS-21 increased the frequency and amplitude of bicuculline-sensitive currents. Effects on EPSCs were blocked with tetrodotoxin (TTX) (1 μM), but not by methyllycaconitine (MLA) (50 nM). Neither TTX nor MLA reduced the potentiation of IPSC frequencies. However, TTX blocked, and in some cases MLA reduced, the potentiation of IPSC amplitudes. These data suggest that effects of 5-HI + 4OH-GTS-21 on EPSC frequency were associated with action potential-dependent transmitter release produced by 5HI, and that potentiation of IPSC amplitudes resulted at least in part, from activation of α7 nAChRs. Finally, rAAV-mediated α7 gene transfer did not alter the magnitude of effects produced by 5-HI or 5-HI + 4OH-GTS-21. Thus, although we previously showed that direct measures of α7 nAChR function were enhanced by α7 gene transfer, indirect measures of α7 nAChRs function were not significantly enhanced by combining α7 gene transfer with either agonist activation or positive allosteric modulation of α7 nAChRs.    

5.527           The Role of the Adeno-Associated Virus Capsid in Gene Transfer

Van Vliet, K.M., Blouin, V., Brument, N., Agbandje-McKenna, M. and Snyder, R.O. Methods Mol Biol., 437, 51-91 (2008)   Adeno-associated virus (AAV) is one of the most promising viral gene transfer vectors that has been shown to effect long-term gene expression and disease correction with low toxicity in animal models, and is well tolerated in human clinical trials. The surface of the AAV capsid is an essential component that is involved in cell binding, internalization, and trafficking within the targeted cell. Prior to developing a gene therapy strategy that utilizes AAV, the serotype should be carefully considered since each capsid exhibits a unique tissue tropism and transduction efficiency. Several approaches have been undertaken in an effort to target AAV vectors to specific cell types, including utilizing natural serotypes that target a desired cellular receptor, producing pseudotyped vectors, and engineering chimeric and mosaic AAV capsids. These capsid modifications are being incorporated into vector production and purification methods that provide for the ability to scale-up the manufacturing process to support human clinical trials. Protocols for small-scale and large-scale production of AAV, as well as assays to characterize the final vector product, are presented here. The structures of AAV2, AAV4, and AAV5 have been solved by X-ray crystallography or cryo-electron microscopy (cryo-EM), and provide a basis for rational vector design in developing customized capsids for specific targeting of AAV vectors. The capsid of AAV has been shown to be remarkably stable, which is a desirable characteristic for a gene therapy vector; however, recently it has been shown that the AAV serotypes exhibit differential susceptibility to proteases. The capsid fragmentation pattern when exposed to various proteases, as well as the susceptibility of the serotypes to a series of proteases, provides a unique fingerprint for each serotype that can be used for capsid identity validation. In addition to serotype identification, protease susceptibility can also be utilized to study dynamic structural changes that must occur for the AAV capsid to perform its various functions during the virus life cycle. The use of proteases for structural studies in solution complements the crystal structural studies of the virus. A generic protocol based on proteolysis for AAV serotype identification is provided here.  

5.528           Tau expression levels from various adeno-associated virus vector serotypes produce graded neurodegenerative disease states

Klein, R.L., Dayton, R.D., Tatom, J.B., Diacczynsky, C.G. and Salvatore, M.F. Eur. J. Neurosci., 27, 1615-1625 (2008)   Neurodegenerative diseases involving neurofibrillary tangle pathology are pernicious. By expressing the microtubule-associated protein tau, a major component of tangles, with a viral vector, we induce neuropathological sequelae in rats that are similar to those seen in human tauopathies. We tested several variants of the adeno-associated virus (AAV) vector for tau expression in the nigrostriatal system in order to develop models with graded onset and completeness. Whereas previous studies with AAV2 tau vectors produced partial lesions of the nigrostriatal system, AAV9 or AAV10 tau vectors were more robust. These vectors had formidable efficacy relative to 6-hydroxydopamine for dopamine loss in the striatum. Time-courses for tau transgene expression, dopamine loss and rotational behavior tracked the disease progression with the AAV9 tau vector. There was a nearly complete lesion over a delayed time-course relative to 6-hydroxydopamine, with a sequence of tau expression by 1 week, dopamine loss by 2 weeks and then behavior effect by 3–4 weeks. Relative to AAV2 or AAV8, tau expression from AAV9 or AAV10 peaked earlier and caused more dopamine loss. Varying vector efficiencies produced graded states of disease up to nearly complete. The disease models stemming from the AAV variants AAV9 or AAV10 may be useful for rapid drug screening, particularly for tau diseases that affect the nigrostriatal system, such as progressive supranuclear palsy.  

5.529           Activated Inflammatory Infiltrate in HSV-1-Infected Corneas without Herpes Stromal Keratitis

Divito, S.J. and Hendricks, R.L. Invest. Ophthalmol. Vis. Sci., 49(4), 1488-1495 (2008)   PURPOSE. To investigate herpes stromal keratitis (HSK) immunopathologyby studying HSV-1-infected corneas that fail to develop HSK. METHODS. Plaque assay quantified HSV-1 in the tear film of infectedmice. FACS analysis enumerated corneal leukocytic infiltrateand characterized infiltrate phenotypically after staining foractivation and regulatory T cell (Treg) markers and for markersof antigen-presenting cell (APC) maturation. Treg cells weredepleted in vivo using anti-CD25 mAb. Luminex analysis quantifiedthe amount of cytokines and chemokines expressed in cornealtissue homogenate. RESULTS. Infected corneas without HSK exhibited a pronounced leukocytic infiltrate containing a significantly higher proportion and nearly identical absolute number of activated CD4⁺ T cells 15 days after infection when compared with those with HSK. Moreover, the frequency and absolute number of regulatory CD4⁺ T cells (Tregs) was lower in nondiseased corneas, and Treg depletion did not influence HSK incidence. The frequency of mature, immunogenic DCs and the ratio of mature DCs to CD4⁺ T cells were nearlyidentical in corneas with and without HSK. The authors observeda reduced population of neutrophils and reduced expression ofneutrophil chemoattractants MIP-1β and keratinocyte chemoattractantand the neutrophil-attracting cytokine IL-6 in corneas withoutHSK. CONCLUSIONS. These findings demonstrate that HSV-1-infected corneas can retain clarity in the presence of a substantial secondary leukocytic infiltrate, that activated CD4⁺ T cells,while necessary, are not sufficient for HSK development, thatsusceptibility to HSK is not determined by Tregs, and that clinicaldisease correlates with the accumulation of a critical massof neutrophils through chemoattraction.  

5.530           R5 and X4 HIV Viruses Differentially Modulate Host Gene Expression in Resting CD4+ T Cells

Sirois, M. et al AIDS Research and Human Retroviruses, 24(3), 485-493 (2008)   During HIV-1 infection, distinct biological phenotypes are observed between R5 and X4 HIV-1 strains with respect to pathogenicity and tropism. In this study, temporal changes of the expression levels of the complete human transcriptome, representing 47,000 well-characterized human transcripts, were monitored in the first 24 h during HIV-1 R5 and X4 exposition in resting primary CD4⁺ T cells. We provide evidence that R5 viruses modulate, to a greater extent than X4 viruses, the level of mRNA of the resting CD4⁺ T cells. Indeed, modulation of the TCR signaling and the actin organization involving the WAVE/ABI complex and the ARP2/3 complex appeared to be associated with R5 exposition. The data suggest that the ability of R5 viruses to modulate TCR-mediated actin polymerization and signaling creates a favorable environment for CD4⁺ T cell activation after TCR stimulation and may partly explain why R5 is the primary strain observed early in the natural infection process.  

5.531           Adenovirus and adeno-associated virus-mediated delivery of human myophosphorylase cDNA and LacZ cDNA to muscle in the ovine model of McArdle’s disease: Expression and re-expression of glycogen phosphorylase

McC Howell, J. et al Neuromuscular Disorders, 18, 248-258 (2008)   At present there is no satisfactory treatment for McArdle’s disease, deficiency of myophosphorylase. Injection of modified adenovirus 5 (AdV5) and adeno-associated virus 2 (AAV2) vectors containing myophosphorylase expression cassettes, into semitendinosus muscle of sheep with McArdle’s disease, produced expression of functional myophosphorylase and some re-expression of the non-muscle glycogen phosphorylase isoforms (both liver and brain) in regenerating fibres. Expression of both non-muscle isoforms was also seen after control injections of AdV5LacZ vectors. There was up to an order of magnitude greater expression of phosphorylase after myophosphorylase vector injection than after LacZ controls (62% of sections with over 1000 positive muscle fibres, versus 7%). The results presented here suggest that the use of viral vector-mediated phosphorylase gene transfer may be applicable to the treatment of McArdle’s disease and that sustained re-expression of the brain and liver isoforms should also be investigated as a possible treatment.  

5.532           Virus-Like Display of a Neo-Self Antigen Reverses B Cell Anergy in a B Cell Receptor Transgenic Mouse Model

Chackerian, B., Durfee, M.R. and Schiller, J.T.

1. Immunol., 180, 5816-5825 (2008)

  The ability to distinguish between self and foreign Ags is a central feature of immune recognition. For B cells, however, immune tolerance is not absolute, and factors that include Ag valency, the availability of T help, and polyclonal B cell stimuli can influence the induction of autoantibody responses. Here, we evaluated whether multivalent virus-like particle (VLP)-based immunogens could induce autoantibody responses in well-characterized transgenic (Tg) mice that express a soluble form of hen egg lysozyme (HEL) and in which B cell tolerance to HEL is maintained by anergy. Immunization with multivalent VLP-arrayed HEL, but not a trivalent form of HEL, induced high-titer Ab responses against HEL in both soluble HEL Tg mice and double Tg mice that also express a monoclonal HEL-specific BCR. Induction of autoantibodies against HEL was not dependent on coadministration of strong adjuvants, such as CFA. In contrast to previous data showing the T-independent induction of Abs to foreign epitopes on VLPs, the ability of HEL-conjugated VLPs to induce anti-HEL Abs in tolerant mice was dependent on the presence of CD4⁺ Th cells, and could be enhanced by the presence of pre-existing cognate T cells. In in vitro studies, VLP-conjugated HEL was more potent than trivalent HEL in up-regulating surface activation markers on purified anergic B cells. Moreover, immunization with VLP-HEL reversed B cell anergy in vivo in an adoptive transfer model. Thus, Ag multivalency and T help cooperate to reverse B cell anergy, a major mechanism of B cell tolerance.  

5.533           Adeno-associated Virus of a Single-polarity DNA Genome Is Capable of Transduction In Vivo

Zhou, X. et al Molecular Therapy, 16(3), 494-499 (2008)   The adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. AAV vectors with wild-type inverted terminal repeats package either the plus- or the minus-strand DNA genomes with equal frequency. By creating a series of deletions within the, we have developed a genetic approach that can generate an AAV vector that packages its single-stranded DNA genome predominantly in a single polarity (99.4%). This novel reagent efficiently transduced muscle, brain and liver in whole animals. The transduction efficiencies were similar to those of the control mixed-polarity vectors. Our results showed that reannealing of plus- and minus-strand DNA was not required for AAV-mediated transduction in vivo, supporting the hypothesis that second-strand DNA synthesis is a primary pathway in converting the single-stranded AAV genome into double-stranded forms. The availability of the single-polarity AAV vector would aid further studies on the mechanism of AAV transduction as well as the application of AAV vector for gene replacement therapy.  

5.534           Noninvasive In Vivo Delivery of Transgene via Adeno-Associated Virus into Supporting Cells of the Neonatal Mouse Cochlea

Izuka, T. et al Human Gene Therapy, 19, 384-390 (2008)   There are a number of genetic diseases that affect the cochlea early in life, which require normal gene transfer in the early developmental stage to prevent deafness. The delivery of adenovirus (AdV) and adeno-associated virus (AAV) was investigated to elucidate the efficiency and cellular specificity of transgene expression in the neonatal mouse cochlea. The extent of AdV transfection is comparable to that obtained with adult mice. AAV-directed gene transfer after injection into the scala media through a cochleostomy showed transgene expression in the supporting cells, inner hair cells (IHCs), and lateral wall with resulting hearing loss. On the other hand, gene expression was observed in Deiters cells, IHCs, and lateral wall without hearing loss after the application of AAV into the scala tympani through the round window. These findings indicate that injection of AAV into the scala tympani of the neonatal mouse cochlea therefore has the potential to efficiently and noninvasively introduce transgenes to the cochlear supporting cells, and this modality is thus considered to be a promising strategy to prevent hereditary prelingual deafness.  

5.535           Surface Loop Dynamics in Adeno-Associated Virus Capsid Assembly

DiPrimio, N., Asokan, A., Govindasamy, L., Agbandje-McKenna, M. And samulski, R.J.

1. Virol., 82(11), 5178-5189 (2008)

  The HI loop is a prominent domain on the adeno-associated virus (AAV) capsid surface that extends from each viral protein (VP) subunit overlapping the neighboring fivefold VP. Despite the highly conserved nature of the residues at the fivefold pore, the HI loops surrounding this critical region vary significantly in amino acid sequence between the AAV serotypes. In order to understand the role of this unique capsid domain, we ablated side chain interactions between the HI loop and the underlying EF loop in the neighboring VP subunit by generating a collection of deletion, insertion, and substitution mutants. A mutant lacking the HI loop was unable to assemble particles, while a substitution mutant (10 glycine residues) assembled particles but was unable to package viral genomes. Substitution mutants carrying corresponding regions from AAV1, AAV4, AAV5, and AAV8 yielded (i) particles with titers and infectivity identical to those of AAV2 (AAV2 HI1 and HI8), (ii) particles with a decreased virus titer (1 log) but normal infectivity (HI4), and (iii) particles that synthesized VPs but were unable to assemble into intact capsids (HI5). AAV5 HI is shorter than all other HI loops by one amino acid. Replacing the missing residue (threonine) in AAV2 HI5 resulted in a moderate particle assembly rescue. In addition, we replaced the HI loop with peptides varying in length and amino acid sequence. This region tolerated seven-amino-acid peptide substitutions unless they spanned a conserved phenylalanine at amino acid position 661. Mutation of this highly conserved phenylalanine to a glycine resulted in a modest decrease in virus titer but a substantial decrease (1 log order) in infectivity. Subsequently, confocal studies revealed that AAV2 F661G is incapable of efficiently completing a key step in the infectious pathway nuclear entry, hinting at a possible perturbation of VP1 phospholipase activity. Molecular modeling studies with the F661G mutant suggest that disruption of interactions between F661 and an underlying P373 residue in the EF loop of the neighboring subunit might adversely affect incorporation of the VP1 subunit at the fivefold axis. Western blot analysis confirmed inefficient incorporation of VP1, as well as a proteolytically processed VP1 subunit that could account for the markedly reduced infectivity. In summary, our studies show that the HI loop, while flexible in amino acid sequence, is critical for AAV capsid assembly, proper VP1 subunit incorporation, and viral genome packaging, all of which implies a potential role for this unique surface domain in viral infectivity.  

5.536           Arrangement of L2 within the Papillomavirus Capsid

Buck, C.B. et al

1. Virol., 82(11), 5190-5197 (2008)

  Papillomaviruses are a family of nonenveloped DNA tumor viruses.Some sexually transmitted human papillomavirus (HPV) types,including HPV type 16 (HPV16), cause cancer of the uterine cervix.Papillomaviruses encode two capsid proteins, L1 and L2. Themajor capsid protein, L1, can assemble spontaneously into a72-pentamer icosahedral structure that closely resembles nativevirions. Although the minor capsid protein, L2, is not requiredfor capsid formation, it is thought to participate in encapsidationof the viral genome and plays a number of essential roles inthe viral infectious entry pathway. The abundance of L2 andits arrangement within the virion remain unclear. To addressthese questions, we developed methods for serial propagationof infectious HPV16 capsids (pseudoviruses) in cultured humancell lines. Biochemical analysis of capsid preparations producedusing various methods showed that up to 72 molecules of L2 canbe incorporated per capsid. Cryoelectron microscopy and imagereconstruction analysis of purified capsids revealed an icosahedrallyordered L2-specific density beneath the axial lumen of eachL1 capsomer. The relatively close proximity of these L2 densitybuttons to one another raised the possibility of homotypic L2interactions within assembled virions. The concept that theN and C termini of neighboring L2 molecules can be closely apposedwithin the capsid was supported using bimolecular fluorescencecomplementation or "split GFP" technology. This structural informationshould facilitate investigation of L2 function during the assemblyand entry phases of the papillomavirus life cycle.  

5.537           An Alteration of Human Immunodeficiency Virus gp41 Leads to Reduced CCR5 Dependence and CD4 Independence

Taylor, B.M. et al

1. Virol., 82(11), 5460-5471 (2008)

  Human immunodeficiency virus (HIV) type 1 infection requires functional interactions of the viral surface (gp120) glycoprotein with cell surface CD4 and a chemokine coreceptor (usually CCR5 or CXCR4) and of the viral transmembrane (gp41) glycoprotein with the target cell membrane. Extensive genetic variability, generally in gp120 and the gp41 ectodomain, can result in altered coreceptor use, fusion kinetics, and neutralization sensitivity. Here we describe an R5 HIV variant that, in contrast to its parental virus, infects T-cell lines expressing low levels of cell surface CCR5. This correlated with an ability to infect cells in the absence of CD4, increased sensitivity to a neutralizing antibody recognizing the coreceptor binding site of gp120, and increased resistance to the fusion inhibitor T-20. Surprisingly, these properties were determined by alterations in gp41, including the cytoplasmic tail, a region not previously shown to influence coreceptor use. These data indicate that HIV infection of cells with limiting levels of cell surface CCR5 can be facilitated by gp41 sequences that are not exposed on the envelope ectodomain yet induce allosteric changes in gp120 that facilitate exposure of the CCR5 binding site.  

5.538           HIV-1 Assembly: Viral Glycoproteins Segregate Quantally to Lipid Rafts that Associate Individually with HIV-1 Capsids and Virions

Leung, K. et al Cell Host & Microbe, 3, 285-292 (2008)   HIV-1 assembly depends on its structural protein, Gag, which after synthesis on ribosomes, traffics to the late endosome/plasma membrane, associates with HIV Env glycoprotein, and forms infectious virions. While Env and Gag migrate to lipid microdomains, their stoichiometry and specificity of interaction are unknown. Pseudotyped viral particles can be made with one viral core surrounded by heterologous envelope proteins. Taking advantage of this property, we analyzed the association of HIV Env and Ebola glycoprotein (GP), with HIV-1 Gag coexpressed in the same cell. Though both viral glycoproteins were expressed, each associated independently with Gag, giving rise to distinct virion populations, each with a single glycoprotein type. Confocal imaging demonstrated that Env and GP localized to distinct lipid raft microdomains within the same cell where they associated with different virions. Thus, a single Gag particle associates “quantally” with one lipid raft, containing homogeneous trimeric viral envelope proteins, to assemble functional virions.  

5.539           Multivalent Presentation of Antihantavirus Peptides on Nanoparticles Enhances Infection Blockade

Hall, P.R. et al Antimicrob. Agents Chemother., 52(6), 2079-2088 (2008)   Viral entry into susceptible host cells typically results from multivalent interactions between viral surface proteins and host entry receptors. In the case of Sin Nombre virus (SNV), a New World hantavirus that causes hantavirus cardiopulmonary syndrome, infection involves the interaction between viral membrane surface glycoproteins and the human integrin _vβ₃. Currently, there are no therapeutic agents available which specifically target SNV. To address this problem, we used phage display selection of cyclic nonapeptides to identify peptides that bound SNV and specifically prevented SNV infection in vitro. We synthesized cyclic nonapeptides based on peptide sequences of phage demonstrating the strongest inhibition of infection, and in all cases, the isolated peptides were less effective at blocking infection (9.0% to 27.6% inhibition) than were the same peptides presented by phage (74.0% to 82.6% inhibition). Since peptides presented by the phage were pentavalent, we determined whether the identified peptides would show greater inhibition if presented in a multivalent format. We used carboxyl linkages to conjugate selected cyclic peptides to multivalent nanoparticles and tested infection inhibition. Two of the peptides, CLVRNLAWC and CQATTARNC, showed inhibition that was improved over that of the free format when presented on nanoparticles at a 4:1 nanoparticle-to-virus ratio (9.0% to 32.5% and 27.6% to 37.6%, respectively), with CQATTARNC inhibition surpassing 50% when nanoparticles were used at a 20:1 ratio versus virus. These data illustrate that multivalent inhibitors may disrupt polyvalent protein-protein interactions, such as those utilized for viral infection of host cells, and may represent a useful therapeutic approach.  

5.540           HSP70 and Constitutively Active HSF1 Mediate Protection Against CDCrel-1-mediated Toxicity

Jung, A.E., Fitzsimons, H.L., Bland, R. J., During, M.J: and Young, D. Mol. Therapy, 16(6), 1048-1055 (2008)   Defects in cellular quality control mechanisms are thought to contribute to the neuropathology of Parkinson's disease (PD). Overexpressing heat shock proteins (HSPs) may constitute a powerful therapeutic strategy for PD, because they boost the ability of the cell to eliminate unwanted proteins. We investigated the neuroprotective potential of HSP70, HSP40, and H-BH, a constitutively active form of heat shock factor 1, in a rat model of PD based on adeno-associated virus (AAV) vector–mediated overexpression of CDCrel-1, a parkin substrate known to be toxic to dopaminergic neurons. AAV vector–mediated overexpression of H-BH and of HSP70 afforded similar levels of protection against CDCrel-1 toxicity, with 20% improvement in survival of dopaminergic neurons as compared to the controls. The assessment of protection conferred was made using tyrosine hydroxylase (TH) and HuC/D immunohistochemistry and Fluoro-Gold retrograde tracing, and by observing the extent of preservation of spontaneous function and also the extent of drug-induced motor function. In contrast to H-BH and HSP70, HSP40 overexpression exacerbated CDCrel-1-mediated cell death. Real-time reverse transcriptase (RT)-PCR analysis showed that H-BH had the effect of upregulating endogenous HSP70 and HSP40 mRNA levels 10-fold and 4-fold over basal levels, respectively, whereas AAV vector–mediated HSP70 and HSP40 mRNA levels were over 100-fold higher. Our results suggest that a comparatively modest upregulation of multiple HSPs may be an effective approach for achieving significant neuroprotection in PD.  

5.541           Construction and Production of Recombinant Herpes Simplex Virus Vectors

Goins, W.F., Krisky, D.M., Wechuck, J.B., Huang, S. and Glorioso, J.C. Methods in Mol. Biol., 434, 97-113 (2008)   Virus vectors have been employed as gene transfer vehicles for various pre-clinical and clinical gene therapy applications. Replication-competent herpes simplex virus (HSV) vectors that replicate specifically in actively dividing glial tumor cells have been used in Phase I–II human trials in patients with glioblastoma multiforme (GBM), a fatal form of brain cancer. Research during the last decade on the development of HSV vectors has resulted in the engineering of recombinant vectors that are totally replication defective, non-toxic, and capable of long-term transgene expression. This chapter describes methods for the construction of recombinant genomic HSV vectors based on the HSV-1 replication-defective vector backbones, steps in their purification, and their small-scale production for use in cell culture experiments as well as studies in animals.  

5.542           BRI2 (ITM2b) Inhibits Aβ Deposition In Vivo

Kim, J. et al

1. Neurosci., 28(23), 6030-6036 (2008)

  Analyses of the biologic effects of mutations in the BRI2 (ITM2b) and the amyloid β precursor protein (APP) genes support the hypothesis that cerebral accumulation of amyloidogenic peptides in familial British and familial Danish dementias and Alzheimer's disease (AD) is associated with neurodegeneration. We have used somatic brain transgenic technology to express the BRI2 and BRI2-Aβ1–40 transgenes in APP mouse models. Expression of BRI2-Aβ1–40 mimics the suppressive effect previously observed using conventional transgenic methods, further validating the somatic brain transgenic methodology. Unexpectedly, we also find that expression of wild-type human BRI2 reduces cerebral Aβ deposition in an AD mouse model. Additional data indicate that the 23 aa peptide, Bri23, released from BRI2 by normal processing, is present in human CSF, inhibits Aβ aggregation in vitro and mediates its anti-amyloidogenic effect in vivo. These studies demonstrate that BRI2 is a novel mediator of Aβ deposition in vivo.  

5.543           Cellular Proteins in Influenza Virus Particles

Shaw, M.L.., Stone, K.L., Colangelo, C.M., Gulcicek, E.E. and Palese, P. PLOSpathogens, 4(6), e1000085 (2008)   Virions are thought to contain all the essential proteins that govern virus egress from the host cell and initiation of replication in the target cell. It has been known for some time that influenza virions contain nine viral proteins; however, analyses of other enveloped viruses have revealed that proteins from the host cell can also be detected in virions. To address whether the same is true for influenza virus, we used two complementary mass spectrometry approaches to perform a comprehensive proteomic analysis of purified influenza virus particles. In addition to the aforementioned nine virus-encoded proteins, we detected the presence of 36 host-encoded proteins. These include both cytoplasmic and membrane-bound proteins that can be grouped into several functional categories, such as cytoskeletal proteins, annexins, glycolytic enzymes, and tetraspanins. Interestingly, a significant number of these have also been reported to be present in virions of other virus families. Protease treatment of virions combined with immunoblot analysis was used to verify the presence of the cellular protein and also to determine whether it is located in the core of the influenza virus particle. Immunogold labeling confirmed the presence of membrane-bound host proteins on the influenza virus envelope. The identification of cellular constituents of influenza virions has important implications for understanding the interactions of influenza virus with its host and brings us a step closer to defining the cellular requirements for influenza virus replication. While not all of the host proteins are necessarily incorporated specifically, those that are and are found to have an essential role represent novel targets for antiviral drugs and for attenuation of viruses for vaccine purposes.  

5.544           Mutations in the Amino Terminus of Foamy Virus Gag Disrupt Morphology and Infectivity but Do Not Target Assembly

Life, R.B., Lee, E-G., Eastman, S.W. and Linial, M.L.

1. Virol., 82(13), 6109-6119 (2008)

  Foamy viruses (FVs) assemble using pathways distinct from those of orthoretroviruses. FV capsid assembly takes place near the host microtubule-organizing center (MTOC). Assembled capsids then migrate by an unknown mechanism to the trans-Golgi network to colocalize with the FV glycoprotein, Env. Interaction with Env is required for FV capsid egress from cells; the amino terminus of FV Gag contains a cytoplasmic targeting/retention signal that is responsible for targeting assembly to the MTOC. A mutant Gag was constructed by addition of a myristylation (M) signal in an attempt to target assembly to the plasma membrane and potentially overcome the dependence upon Env for budding (S. W. Eastman and M. L. Linial, J. Virol. 75:6857-6864, 2001). Using this and additional mutants, we now show that assembly is not redirected to the plasma membrane. Addition of an M signal leads to gross morphological defects. The aberrant particles still assemble near the MTOC but do not produce infectious virus. Although extracellular Gag can be detected in a pelletable form in the absence of Env, the mutant particles contain very little genomic RNA and are less dense. Our analyses indicate that the amino terminus of Gag contains an Env interaction domain that is critical for bona fide egress of assembled capsids.  

5.545           Monitoring Early Fusion Dynamics of Human Immunodeficiency Virus Type 1 at Single-Molecule Resolution

Dobrowsky, T.M., Zhou, Y., Sun, S.X., Siliciano, R.F. and Wirtz, D.

1. Virol., 82(14), 7022-7033 (2008)

  The fusion of human immunodeficiency virus type 1 (HIV-1) to host cells is a dynamic process governed by the interaction between glycoproteins on the viral envelope and the major receptor, CD4, and coreceptor on the surface of the cell. How these receptors organize at the virion-cell interface to promote a fusion-competent site is not well understood. Using single-molecule force spectroscopy, we map the tensile strengths, lifetimes, and energy barriers of individual intermolecular bonds between CCR5-tropic HIV-1 gp120 and its receptors CD4 and CCR5 or CXCR4 as a function of the interaction time with the cell. According to the Bell model, at short times of contact between cell and virion, the gp120-CD4 bond is able to withstand forces up to 35 pN and has an initial lifetime of 0.27 s and an intermolecular length of interaction of 0.34 nm. The initial bond also has an energy barrier of 6.7 k_BT (where k_B is Boltzmann's constant and T is absolute temperature). However, within 0.3 s, individual gp120-CD4 bonds undergo rapid destabilization accompanied by a shortened lifetime and a lowered tensile strength. This destabilization is significantly enhanced by the coreceptor CCR5, not by CXCR4 or fusion inhibitors, which suggests that it is directly related to a conformational change in the gp120-CD4 bond. These measurements highlight the instability and low tensile strength of gp120-receptor bonds, uncover a synergistic role for CCR5 in the progression of the gp120-CD4 bond, and suggest that the cell-virus adhesion complex is functionally arranged about a long-lived gp120-coreceptor bond.  

5.546           Efficient trans-Encapsidation of Hepatitis C Virus RNAs into Infectious Virus-Like Particles

Steinmann, E., Brohm, C., Kallis, S., Bartenschlager, R. And Pietschmann, T.

1. Virol., 82(14), 7034-7046 (2008)

  Recently, complete replication of hepatitis C virus (HCV) in tissue culture was established using the JFH1 isolate. To analyze determinants of HCV genome packaging and virion assembly, we developed a system that supports particle production based on trans-packaging of subgenomic viral RNAs. Using JFH1 helper viruses, we show that subgenomic JFH1 replicons lacking the entire core to NS2 coding region are efficiently encapsidated into infectious virus-like particles. Similarly, chimeric helper viruses with heterologous structural proteins trans-package subgenomic JFH1 replicons. Like authentic cell culture-produced HCV (HCVcc) particles, these trans-complemented HCV particles (HCV_TCP) penetrate target cells in a CD81 receptor-dependent fashion. Since HCV_TCP production was limited by competition between the helper and subgenomic RNA and to avoid contamination of HCV_TCP stocks with helper viruses, we created HCV packaging cells. These cells encapsidate various HCV replicons with high efficiency, reaching infectivity titers up to 10⁶ tissue culture infectious doses 50 per milliliter. The produced particles display a buoyant density comparable to HCVcc particles and can be propagated in the packaging cell line but support only a single-round infection in naïve cells. Together, this work demonstrates that subgenomic HCV replicons are assembly competent, thus excluding cis-acting RNA elements in the core-to-NS2 genomic region essential for RNA packaging. The experimental system described here should be helpful to decipher the mechanisms of HCV assembly and to identify RNA elements and viral proteins involved in particle formation. Similar to other vector systems of plus-strand RNA viruses, HCV_TCP may prove valuable for gene delivery or vaccination approaches.  

5.547           Annexin II Incorporated into Influenza Virus Particles Supports Virus Replication by Converting Plasminogen into Plasmin

LeBouder, F. et al

1. Virol., 82(14), 6820-6828 (2008)

  For influenza viruses to become infectious, the proteolytic cleavage of hemagglutinin (HA) is essential. This usually is mediated by trypsin-like proteases in the respiratory tract. The binding of plasminogen to influenza virus A/WSN/33 leads to the cleavage of HA, a feature determining its pathogenicity and neurotropism in mice. Here, we demonstrate that plasminogen also promotes the replication of other influenza virus strains. The inhibition of the conversion of plasminogen into plasmin blocked influenza virus replication. Evidence is provided that the activation of plasminogen is mediated by the host cellular protein annexin II, which is incorporated into the virus particles. Indeed, the inhibition of plasminogen binding to annexin II by using a competitive inhibitor inhibits plasminogen activation into plasmin. Collectively, these results indicate that the annexin II-mediated activation of plasminogen supports the replication of influenza viruses, which may contribute to their pathogenicity.  

5.548           Human Apolipoprotein E Expression from Mouse Skeletal Muscle by Electrotransfer of Nonviral DNA (Plasmid) and Pseudotyped Recombinant Adeno-Associated Virus (AAV2/7)

Evans, V. et al Human Gene Therapy, 19, 569-578 (2008)   Plasma apolipoprotein E (apoE) has multiple atheroprotective actions. However, although liver-directed adenoviral gene transfer of apoE reverses hypercholesterolemia and inhibits atherogenesis in apoE-deficient (apoE ^/ ) mice, safety considerations have revived interest in nonviral DNA (plasmid) and nonpathogenic adeno-associated viral (AAV) vectors. Here, we assess the effectiveness of these two delivery vehicles by minimally invasive intramuscular injection. First, we constructed AAV2-based expression plasmids harboring human apoE3 cDNA, driven by two muscle-specific promoters (CK6 and C5-12) and one ubiquitous promoter (CAG); each efficiently expressed apoE3 in transfected cultured C2C12 mouse myoblasts, although muscle-specific promoters were active only in differentiated multinucleate myotubes. Second, a pilot study verified that electrotransfer of the CAG-driven plasmid (p.CAG.apoE3) into tibialis anterior muscles, pretreated with hyaluronidase, of apoE ^/ mice significantly enhanced (p < 0.001) local intramuscular expression of apoE3. However, in a 7-day experiment, the CK6- and C5-12-driven plasmids produced less apoE3 in muscle than did p.CAG.apoE3 (0.61 ± 0.38 and 0.45 ± 0.38 vs. 13.38 ± 7.46 μg of apoE3 per muscle, respectively), but plasma apoE3 levels were below our detection limit (<15 ng/ml) in all mice and did not reverse the hyperlipidemia. Finally, we showed that intramuscular injection of a cross-packaged AAV serotype 7 viral vector, expressing human apoE3 from the CAG promoter, resulted in increasing levels of apoE3 in plasma over 4 weeks, although the concentration reached (1.40 ± 0.35 μg/ml) was just below the threshold level needed to reduce the hypercholesterolemia. We conclude that skeletal muscle can serve as an effective secretory platform to express the apoE3 transgene, but that improved gene transfer vectors are needed to achieve full therapeutic levels of plasma apoE3 protein.  

5.549           Biochemical Correction of Short-Chain Acyl-Coenzyme A Dehydrogenase Deficiency after Portal Vein Injection of rAAV8-SCAD

Beattie, S.G. et al Human Gene Therapy, 19, 579-588 (2008)   Recombinant adeno-associated viral vectors pseudotyped with serotype 5 and 8 capsids (AAV5 and AAV8) have been shown to be efficient gene transfer reagents for the liver. We have produced AAV5 and AAV8 vectors that express mouse short-chain acyl-CoA dehydrogenase (mSCAD) cDNA under the transcriptional control of the cytomegalovirus–chicken β-actin hybrid promoter. We hypothesized that these vectors would produce sufficient hepatocyte transduction (after administration via the portal vein) and thus sufficient SCAD enzyme to correct the phenotype observed in the SCAD-deficient (BALB/cByJ) mouse, which includes elevated blood butyrylcarnitine and hepatic steatosis. Ten weeks after portal vein injection into 8-week-old mice, AAV8-treated livers contained acyl-CoA dehydrogenase activity (14.3 mU/mg) toward butyryl-CoA, compared with 7.6 mU/mg in mice that received phosphate-buffered saline. Immunohistochemistry showed expression of mSCAD within rAAV8-mSCAD-transduced hepatocytes, as seen by light microscopy. A significant reduction of circulating butyrylcarnitine was seen in AAV5-mSCAD- and AAV8-mSCAD-injected mice. Magnetic resonance spectroscopy of fasted mice demonstrated a significant reduction in relative lipid content within the livers of AAV8-mSCAD-treated mice. These results demonstrate biochemical correction of SCAD deficiency after AAV8-mediated SCAD gene delivery.  

5.550           Optimized Lentiviral Transduction of Mouse Bone Marrow-Derived Mesenchymal Stem Cells

Ricks, D.M., Kutner, R., Zhang, X-Y., Welsh, D.A. and Reiser, J. Stem Cells and Development, 17, 441-450 (2008)   Mesenchymal stem cells (MSCs) have attracted much attention as potential platforms for transgene delivery and cell-based therapy for human disease. MSCs have the capability to self-renew and retain multipotency after extensive expansion in vitro, making them attractive targets for ex vivo modification and autologous transplantation. Viral vectors, including lentiviral vectors, provide an efficient means for transgene delivery into human MSCs. In contrast, mouse MSCs have proven more difficult to transduce with lentiviral vectors than their human counterparts, and because many studies use mouse models of human disease, an improved method of transduction would facilitate studies using ex vivo-modified mouse MSCs. We have worked toward improving the production of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors and optimizing transduction conditions for mouse MSCs using lentivirus vectors pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G), the ecotropic murine leukemia virus envelope glycoprotein (MLV-E), and the glycoproteins derived from the Armstrong and WE strains of lymphocytic choriomeningitis virus (LCMV-Arm, LCMV-WE). Mouse MSCs were readily transduced following overnight incubation using a multiplicity of infection of at least 40. Alternatively, mouse MSCs in suspension were readily transduced after a 1-h exposure to lentiviral pseudotypes immediately following trypsin treatment or retrieval from storage in liquid nitrogen. LCMV-WE pseudotypes resulted in efficient transduction of mouse MSCs with less toxicity than VSV-G pseudotypes. In conclusion, our improved production and transduction conditions for lentiviral vectors resulted in efficient transduction of mouse MSCs, and these improvements should facilitate the application of such cells in the context of mouse models of human disease.        

5.551           NS3 Helicase Domains Involved in Infectious Intracellular Hepatitis C Virus Particle Assembly

Ma, Y., Yates, J., Liang, Y., Lemon, S.M. and Yi, M.

1. Virol., 82(15), 7624-7639 (2008)

  A mutation within subdomain 1 of the hepatitis C virus (HCV) NS3 helicase (NS3-Q221L) (M. Yi, Y. Ma, J. Yates, and S. M. Lemon, J. Virol. 81:629-638, 2007) rescues a defect in production of infectious virus by an intergenotypic chimeric RNA (HJ3). Although NS3-Gln-221 is highly conserved across HCV genotypes, the Leu-221 substitution had no effect on RNA replication or NS3-associated enzymatic activities. However, while transfection of unmodified HJ3 RNA failed to produce either extracellular or intracellular infectious virus, transfection of HJ3 RNA containing the Q221L substitution (HJ3/QL) resulted in rapid accumulation of intracellular infectious particles with release into extracellular fluids. In the absence of the Q221L mutation, both NS5A and NS3 were recruited to core protein on the surface of lipid droplets, but there was no assembly of core into high-density, rapidly sedimenting particles. Further analysis demonstrated that a Q221N mutation minimally rescued virus production and led to a second-site I399V mutation in subdomain 2 of the helicase. Similarly, I399V alone allowed only low-level virus production and led to selection of an I286V mutation in subdomain 1 of the helicase which fully restored virus production, confirming the involvement of both major helicase subdomains in the assembly process. Thus, multiple mutations in the helicase rescue a defect in an early-intermediate step in virus assembly that follows the recruitment of NS5A to lipid droplets and precedes the formation of dense intracellular viral particles. These data reveal a previously unsuspected role for the NS3 helicase in early virion morphogenesis and provide a new perspective on HCV assembly.  

5.552           Generation of efficient human blood progenitor–targeted recombinant adeno-associated viral vectors (AAV) by applying an AAV random peptide library on primary human hematopoietic progenitor cells

Sellner, L. et al Exp. Hematol., 36, 957-964 (2008)   Objective Currently standard recombinant adeno-associated virus serotype 2(rAAV2)–based vectors lack the efficiency for gene transfer into primary human CD34⁺ peripheral blood progenitor cells (PBPC). Materials and Methods An advancement in vector development now allows the generation of rAAV capsid mutants that offer higher target cell efficiency and specificity. To increase the gene transfer into hematopoietic progenitor cells, we applied this method for the first time on primary human CD34⁺ PBPC cells. Results On a panel of leukemia cell lines (CML/AML), significantly higher gene transfer efficiency of the rAAV capsid mutants (up to 100% gene transfer) was observed compared to standard rAAV2 vectors. A higher transduction efficiency in the imatinib-resistant cell line LAMA84-R than in their sensitive counterpart LAMA84-S and a pronounced difference in susceptibility for the capsid mutants vs rAAV2 in LAMA84-S were particularly striking. On solid tumor cell lines, on the other hand, rAAV2 was more efficient than the capsid mutants, suggesting an increased specificity of our capsid mutants for hematopoietic progenitor cells. On primary human CD34⁺ PBPC significantly higher (up to eightfold; 16% green fluorescent protein–positive) gene transfer could be obtained with the newly generated vectors compared to standard rAAV2 vectors. Conclusion These novel vectors may enable efficient gene transfer using rAAV-based vectors into primary human blood progenitor cells for a future clinical application.  

5.553           HPV16 and BPV1 Infection Can Be Blocked by the Dynamin Inhibitor Dynasore

Abban, C.Y., Bradbury, N.A. and Meneses, P.I. Am. J. Therapeut., 15, 304-311 (2008)   The initial entry of papillomaviruses into their target cells has been shown to occur by clathrin-mediated endocytosis and caveolae-mediated endocytosis. These mechanisms entail the formation of nascent-coated vesicles at the plasma membrane. Such coated vesicles, clathrin or caveolin, form and pinch-off in a controlled mechanism that involves several proteins including dynamin. Dynamin is a GTPase that forms a dynamin ring at the stem connecting the nascent vesicle to the plasma membrane. In a still not fully characterized mechanism, dynamin's contraction and twisting results in the scission of the vesicle. In an effort to better characterize the role and molecular mechanisms of dynamin's function, researchers have identified dynasore, a dynamin GTPase inhibitor that prevents the scission of dynamin-dependent endocytic vesicles. Here, we have tested if infection by pseudovirus corresponding to the oncogenic human papillomavirus type 16 and bovine papillomavirus type 1 can be blocked by dynasore. We present data demonstrating that dynasore can block infection of human papillomavirus type 16 and bovine papillomavirus type 1 pseudovirions in a dose- and time-dependent manner with equal efficiency. Presently, there is no available therapy that can block infection by a wide range of papillomavirus regardless of species or genotypes. Targeting dynamin may lead to the rational design of drug able to prevent infection by papillomaviruses, and by other infectious agents dependent on this protein for initial internalization into target cells. Whether such an approach will prove successful needs further investigation.  

5.554           Lack of toxicity of alpha-sarcoglycan overexpression supports clinical gene transfer trial in LGMD2D

Rodino-Klapac, L.R., Lee, J-S., Mulligan, R.C., Clark, K.R. and Mendell, J.R. Neurology, 71, 240-247 (2008)   Background: Alpha-sarcoglycan (-SG) deficiency (limb-girdlemuscular dystrophy [LGMD] type 2D) is the most common form ofsarcoglycan-LGMD. No treatment is currently available. Priorstudies suggest that overexpression of -SG via adeno-associatedvirus (AAV)-mediated gene transfer results in poorly sustainedgene expression related to transgene toxicity. These findingspotentially preclude gene therapy as a treatment approach forLGMD2D. Methods: The human -SG gene (h -SG) was directly transferred to the tibialis anterior muscle of 4- to 5-week-old -SG KO mice using AAV, type 1. The gene was placed under control of either the ubiquitously expressed cytomegalovirus (CMV) promoter or muscle specific promoters that included desmin, muscle creatine kinase (MCK), and its further modification, truncated MCK (tMCK). Low (3 x 10⁹ vg) and high (3 x 10¹⁰ vg) doses of AAV1.h-SG wereadministered. Results: Sustained gene expression was observed irrespectiveof promoters at 6 and 12 weeks post gene transfer. Quantitationof -SG gene expression by fiber counts yielded similar levelsof myofiber transduction for both MCK promoters (60 to 70%),while 34% of fibers were transduced with the DES promoter. Therewas a trend toward lower expression at the 12-week time pointwith the CMV promoter. Western blot analysis revealed -SG overexpressionusing CMV and both the MCK promoters. Conclusion: Our data demonstrate robust and sustained adeno-associatedvirus type 1 alpha-sarcoglycan gene expression under controlof muscle creatine kinase promoters, without evidence of cytotoxicity.These findings support the use of gene therapy as a potentialtreatment approach for limb-girdle muscular dystrophy type 2D.  

5.555           Direct Infection and Replication of Naturally Occurring Hepatitis C Virus Genotypes 1, 2, 3 and 4 in Normal Human Hepatocyte Cultures

Buck, M. PlosOne, 3(7), e2660 (2008)   Background Hepatitis C virus (HCV) infection afflicts about 170 million individuals worldwide. However, the HCV life cycle is only partially understood because it has not been possible to infect normal human hepatocytes in culture. The current Huh-7 systems use cloned, synthetic HCV RNA expressed in hepatocellular carcinoma cells to produce virions, but these cells cannot be infected with naturally occurring HCV obtained from infected patients. Methodology/Principal Findings Here, we describe a human hepatocyte culture permissible to the direct infection with naturally occurring HCV genotypes 1, 2, 3 and 4 in the blood of HCV-infected patients. The culture system mimics the biology and kinetics of HCV infection in humans, and produces infectious virions that can infect naïve human hepatocytes. Conclusions/Significance This culture system should complement the existing systems, and may facilitate the understanding of the HCV life cycle, its effects in the natural host cell, the hepatocyte, as well as the development of novel therapeutics and vaccines.        

5.556           Systemic Insulin-like Growth Factor-1 Reverses Hypoalgesia and Improves Mobility in a Mouse Model of Diabetic Peripheral Neuropathy

Chu, Q. et al Mol. Ther., 16(8), 1400-1408 (2008)   Peripheral neuropathy is a particularly debilitating complication of both type 1 and type 2 diabetes characterized by sensory and motor neuron damage and decreased circulating levels of insulin-like growth factor 1 (IGF-1). Quite often, an early hyperalgesia is followed by hypoalgesia and muscle weakness. Hypoalgesia can lead to significant morbidity for which there is no current treatment. Hyperglycemic, streptozotocin (STZ)-induced rodent models reproduce these symptoms. We investigated whether increasing systemic IGF-1 could improve neuronal function in hyper- and hypoalgesic STZ-treated mice. Increased circulating levels of IGF-1 were achieved by delivering a plasmid or adeno-associated viral (AAV) vector bearing mouse IGF-1 to the liver. Treating mice in the hyperalgesia stage prevented later hypoalgesia. Treating mice in the hypoalgesia stage reversed existing hypoalgesia. This latter effect could be seen by merely restoring IGF-1 serum levels to normalcy, which was possible to achieve by IGF-1 gene therapy or insulin treatment. Sensory nerve functional correction was seen to be correlated with attenuated Schwann cell vacuolization and demyelination in peripheral sensory nerve fibers. A further increase in serum IGF-1 levels with gene therapy also improved motor function, consistent with the observed prevention of both muscle atrophy and peripheral motor nerve fiber demyelination. These results suggest that the restoration of systemic levels of IGF-1 may prove to be a highly effective therapeutic modality for treating diabetic peripheral neuropathy.  

5.557           Site-specific Modification of AAV Vector Particles with Biophysical Probes and Targeting Ligands Using Biotin Ligase

Stachler, M., Chen. I., Ting, A.Y. and Bartlett, J.S. Mol. Ther., 16(8), 1467-1473 (2008)   We have developed a highly specific and robust new method for labeling adeno-associated virus (AAV) vector particles with either biophysical probes or targeting ligands. Our approach uses the Escherichia coli enzyme biotin ligase (BirA), which ligates biotin to a 15-amino-acid biotin acceptor peptide (BAP) in a sequence-specific manner. In this study we demonstrate that by using a ketone isotere of biotin as a cofactor we can ligate this probe to BAP-modified AAV capsids. Because ketones are absent from AAV, BAP-modified AAV particles can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage modification methodology in the context of a mammalian cell lysate for the labeling of AAV vector particles with various fluorophores, and for the attachment of a synthetic cyclic arginine–glycine–aspartate (RGD) peptide (c(RGDfC)) to target integrin receptors that are present on neovasculature. Fluorophore labeling allowed the straightforward determination of intracellular particle distribution. Ligand conjugation mediated a significant increase in the transduction of endothelial cells in vitro, and permitted the intravascular targeting of AAV vectors to tumor-associated vasculature in vivo. These results suggest that this approach holds significant promise for future studies aimed at understanding and modifying AAV vector–cellular interactions.  

5.558           RNAi-mediated knockdown of dystrophin expression in adult mice does not lead to overt muscular dystrophy pathology

Seno, M.M.G.  et al Human Mol. Genet., 17(17), 2622-2632 (2008)   Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. DMD has a complex and as yet incompletely defined molecular pathophysiology. The peak of the pathology attributed to dystrophin deficiency happens between 3 and 8 weeks of age in mdx mice, the animal model of DMD. Accordingly, we hypothesized that the pathology observed with dystrophin deficiency may be developmentally regulated. Initially, we demonstrated that profound small interfering RNA-mediated dystrophin knockdown could be achieved in mouse primary muscle cultures. The use of adeno-associated virus vectors to express short-hairpin RNAs targeting dystrophin in skeletal muscle in vivo yielded a potent and specific dystrophin knockdown, but only after 5 months, indicating the very long half-life of dystrophin. Interestingly, and in contrast to what is observed in congenital dystrophin deficiency, long-term ( 1 year) dystrophin knockdown in adult mice did not result, per se, in overt dystrophic pathology or upregulation of utrophin. This supports our hypothesis and suggests new pathophysiology of the disease. Furthermore, taking into account the rather long half-life of dystrophin, and the notion that the development of pathology is age-dependent, it indicates that a single gene therapy approach before the onset of pathology might convey a long-term cure for DMD.  

5.559           Protein Kinase A-Dependent Step(s) in Hepatitis C Virus Entry and Infectivity

Farquhar, M.J.

1. Virol., 82(17), 8797-8811 (2008)

  Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.  

5.560           SV40 vectors carrying minimal sequence of viral origin with exchangeable capsids

Nakanishi, A. et al Virology, 379, 110-117 (2008)   Polyomaviral vectors are generated by transfecting 293T cells with three sets of DNAs: DNA for the expression of simian virus 40 (SV40) T antigen; DNA for the expression of SV40 capsid proteins, and vector DNA harboring a reporter gene expression cassette carrying a SV40 origin. The vector DNA harbors a minimal sequence originating from SV40, and thus can carry a longer transgene. Moreover, the viable recombinants are not detectable in the vector preparation, and the vectors can transduce the DNA with efficiency similar to that of virions. Vector particles bearing capsid proteins of BK virus, JC virus, and B-lymphotropic papovavirus instead of SV40 were prepared, and they exhibited differential efficiency of gene transduction to the target cells. This method can be used to develop a surrogate system to study the functions of capsid proteins of polyomaviruses and to generate a set of polyomaviral vectors targeted at specific cell types.  

5.561           Role of NMDA Receptors in Dopamine Neurons for Plasticity and Addictive Behaviors

Zweifel, L.S., Argilli, E., Bonci, E. and Palmiter, R.D. Neuron, 59, 486-496 (2008)   A single exposure to drugs of abuse produces an NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) of AMPA receptor (AMPAR) currents in DA neurons; however, the importance of LTP for various aspects of drug addiction is unclear. To test the role of NMDAR-dependent plasticity in addictive behavior, we genetically inactivated functional NMDAR signaling exclusively in DA neurons (KO mice). Inactivation of NMDARs results in increased AMPAR-mediated transmission that is indistinguishable from the increases associated with a single cocaine exposure, yet locomotor responses to multiple drugs of abuse were unaltered in the KO mice. The initial phase of locomotor sensitization to cocaine is intact; however, the delayed sensitization that occurs with prolonged cocaine withdrawal did not occur. Conditioned behavioral responses for cocaine-testing environment were also absent in the KO mice. These findings provide evidence for a role of NMDAR signaling in DA neurons for specific behavioral modifications associated with drug seeking behaviors.  

5.562           Selective overexpression of excitatory amino acid transporter 2 (EAAT2) in astrocytes enhances neuroprotection from moderate but not severe hypoxia–ischemia

Weller, M.L. et al Neuroscience, 155, 1204-1211 (2008)   Attempts have been made to elevate excitatory amino acid transporter 2 (EAAT2) expression in an effort to compensate for loss of function and expression associated with disease or pathology. Increased EAAT2 expression has been noted following treatment with β-lactam antibiotics, and during ischemic preconditioning (IPC). However, both of these conditions induce multiple changes in addition to alterations in EAAT2 expression that could potentially contribute to neuroprotection. Therefore, the aim of this study was to selectively overexpress EAAT2 in astrocytes and characterize the cell type specific contribution of this transporter to neuroprotection. To accomplish this we used a recombinant adeno-associated virus vector, AAV1–glial fibrillary acidic protein (GFAP)–EAAT2, designed to selectively drive the overexpression of EAAT2 within astrocytes. Both viral-mediated gene delivery and β-lactam antibiotic (penicillin-G) treatment of rat hippocampal slice cultures resulted in a significant increase in both the expression of EAAT2, and dihydrokainate (DHK) sensitive glutamate uptake. Penicillin-G provided significant neuroprotection in rat hippocampal slice cultures under conditions of both moderate and severe oxygen glucose deprivation (OGD). In contrast, viral-mediated overexpression of EAAT2 in astrocytes provided enhanced neuroprotection only following a moderate OGD insult. These results indicate that functional EAAT2 can be selectively overexpressed in astrocytes, leading to enhanced neuroprotection. However, this cell type specific increase in EAAT2 expression offers only limited protection compared to treatment with penicillin-G.  

5.563           Cardiac-targeted RNA interference mediated by an AAV9 vector improves cardiac function in coxsackievirus B3 cardiomyopathy

Fechner, H. et al

1. Mol. Med., 86, 987-997 (2008)

  RNA interference (RNAi) has potential to be a novel therapeutic strategy in diverse areas of medicine. In this paper, we report on targeted RNAi for the treatment of a viral cardiomyopathy, which is a major cause of sudden cardiac death or terminal heart failure in children and young adults. RNAi therapy employs small regulatory RNAs to achieve its effect, but in vivo use of synthetic small interfering RNAs is limited by instability in plasma and low transfer into target cells. We instead evaluated an RNAi strategy using short hairpin RNA (shRdRp) directed at the RNA polymerase (RdRP) of coxsackievirus B3 (CoxB3) in HeLa cells, primary rat cardiomyocytes (PNCMs) and CoxB3-infected mice in vivo. A conventional AAV2 vector expressing shRdRp protected HeLa against virus-induced death, but this vector type was unable to transduce PNCMs. In contrast, an analogous pseudotyped AAV2.6 vector was protective also in PNCMs and reduced virus replication by >3 log10 steps. Finally, we evaluated the intravenous treatment of mice with an AAV2.9-shRdRp vector because AAV9 carries the most cardiotropic AAV capsid currently known for in vivo use. Mice with CoxB3 cardiomyopathy had disturbed left ventricular (LV) function with impaired parameters of contractility (dP/dt max=3,006±287 vs. 7,482±487 mmHg/s, p<0.01) and diastolic relaxation (dP/dt min=−2,224±195 vs. −6,456±356 mmHg/s, p<0.01 and Tau=16.2 ± 1.1 vs. 10.7±0.6 ms, p<0.01) compared to control mice. AAV2.9-shRdRp treatment significantly attenuated the cardiac dysfunction compared to control vector-treated mice on day 10 after CoxB3 infection: dP/dt max=3,865±354 vs. 3,006±287 mmHg/s (p<0.05), dP/dt min=−3,245±231 vs. −2,224±195 mmHg/s (p<0.05) and Tau=11.9±0.5 vs. 16.2±1.1 ms (p<0.01). The data show, for the first time, that intravenously injected AAV9 has the potential to target RNAi to the heart and suggest AAV9-shRNA vectors as a novel therapeutic approach for cardiac disorders.  

5.564           Discrimination between exosomes and HIV-1: Purification of both vesicles from cell-free supernatants

Cantin, R., Diou, J., Belanger, D., Tremblay, A.M. and Gilbert, C.

J. Immunol. Methods, 338, 21-30 (2008)

Although enveloped retroviruses bud from the cell surface of T lymphocytes, they use the endocytic pathway and the internal membrane of multivesicular bodies for their assembly and release from macrophages and dendritic cells (DCs). Exosomes, physiological nanoparticles produced by hematopoietic cells, egress from this same pathway and are similar to retroviruses in terms of size, density, the molecules they incorporate and their ability to activate immune cells. Retroviruses are therefore likely to contaminate in vitro preparations of exosomes and vice versa and sucrose gradients are inefficient at separating them. However, we have found that their sedimentation velocities in an iodixanol (Optiprep™) velocity gradient are sufficiently different to allow separation and purification of both vesicles. Using acetylcholinesterase as an exosome marker, we demonstrate that Optiprep™ velocity gradients are very efficient in separating exosomes from HIV-1 particles produced on 293T cells, primary CD4+ T cells, macrophages or DCs, with exosomes collecting at 8.4–12% iodixanol and HIV-1 at 15.6%. We also show that immunodepletion with an anti-acetylcholinesterase antibody rapidly produces highly purified preparations of HIV-1 or exosomes. These findings have applications in fundamental research on exosomes and/or AIDS, as well as in clinical applications where exosomes are involved, more specifically in tumour therapy or in gene therapy using exosomes generated from DCs genetically modified by transfection with virus.

 

5.565           Measurement of the Humoral Immune Response following an Incident Human Papillomavirus Type 16 or 18 Infection in Young Women by a Pseudovirion-Based Neutralizing Antibody Assay

Steele, J. et al Clin. Vaccine Immunol., 15(9), 1387-1390 (2008)   We have evaluated a neutralizing antibody assay which uses human papillomavirus (HPV) type 16 (HPV-16) and HPV-18 pseudovirions carrying a secretory alkaline phosphatase reporter gene and which can potentially measure functionally relevant HPV type-specific neutralizing antibodies. The reproducibility of the assay was excellent; for HPV-16, the intra- and interassay kappa values were 0.95 and 0.90, respectively; and for HPV-18, the corresponding values were 0.90 and 0.90. This assay was used to describe the kinetics of the neutralizing antibody response in a cohort of 42 young women who were recruited soon after first intercourse and who first tested positive for HPV-16 DNA or HPV-18 DNA, or both, during follow-up. Most women seroconverted following the first detection of type-specific HPV DNA and remained seropositive until the end of follow-up. Our findings are broadly consistent with those of two other cohort studies which have measured the serological response following an incident infection by using the technically simpler virus-like-particle-based enzyme-linked immunosorbent assay.  

5.566           Therapeutic Potential of Mesenchymal Stem Cells Producing Interferon- in a Mouse Melanoma Lung Metastasis Model

Ren, C. et al Stem Cells, 26, 2332-2338 (2008)   Adult stem cells represent a potential source for cell-based therapy of cancer. The present study evaluated the potential of bone marrow-derived mesenchymal stem cells (MSC), genetically modified to express interferon (IFN)- , for the treatment of lung metastasis in an immunocompetent mouse model of metastatic melanoma. A recombinant adeno-associated virus (rAAV) 6 vector encoding IFN- was used to transduce mouse bone marrow-derived MSC ex vivo. Expression and bioactivity of the transgenic protein from rAAV-transduced MSC were confirmed prior to in vivo studies. A lung metastasis model of melanoma was developed by i.v. injection of B16F10 cells into 8-week-old C57BL/6 mice. Ten days later, MSC transduced with rAAV-IFN- or green fluorescent protein were intravenously injected. One cohort of mice was sacrificed to determine the effects of the therapy at an earlier time point, and another cohort was observed for long-term survival. Results indicated that systemic administration of MSC producing IFN- reduced the growth of B16F10 melanoma cells and significantly prolonged survival. Immunohistochemistry analysis of the tumors from MSC-IFN- -treated animals indicated an increase in apoptosis and a decrease in proliferation and blood vasculature. These data demonstrate the potential of adult MSC constitutively producing IFN- to reduce the growth of lung metastasis in melanoma.  

5.567           Human Endogenous Retrovirus K (HML-2) Elements in the Plasma of People with Lymphoma and Breast Cancer

Contreras-Galindo, r. et al

1. Virol., 82(19), 9329-9336 (2008)

  Actively replicating endogenous retroviruses entered the human genome millions of years ago and became a stable part of the inherited genetic material. They subsequently acquired multiple mutations, leading to the assumption that these viruses no longer replicate. However, certain human tumor cell lines have been shown to release endogenous retroviral particles. Here we show that RNA from human endogenous retrovirus K (HERV-K) (HML-2), a relatively recent entrant into the human genome, can be found in very high titers in the plasma of patients with lymphomas and breast cancer as measured by either reverse transcriptase PCR or nucleic acid sequence-based amplification. Further, these titers drop dramatically with cancer treatment. We also demonstrate the presence of reverse transcriptase and viral RNA in plasma fractions that contain both immature and correctly processed HERV-K (HML-2) Gag and envelope proteins. Finally, using immunoelectron microscopy, we show the presence of HERV-K (HML-2) virus-like particles in the plasma of lymphoma patients. Taken together, these findings demonstrate that elements of the endogenous retrovirus HERV-K (HML-2) can be found in the blood of modern-day humans with certain cancers.  

5.568           Characterization of hepatitis C RNA-containing particles from human liver by density and size

Nielsen, S.U. et al

1. Gen. Virol., 89, 2507-2517 (2008)

  Hepatitis C virus (HCV) particles found in vivo are heterogeneous in density and size, but their detailed characterization has been restricted by the low titre of HCV in human serum. Previously, our group has found that HCV circulates in blood in association with very-low-density lipoprotein (VLDL). Our aim in this study was to characterize HCV RNA-containing membranes and particles in human liver by both density and size and to identify the subcellular compartment(s) where the association with VLDL occurs. HCV was purified by density using iodixanol gradients and by size using gel filtration. Both positive-strand HCV RNA (present in virus particles) and negative-strand HCV RNA (an intermediate in virus replication) were found with densities below 1.08 g ml^–1. Viral structural and non-structural proteins, host proteins ApoB, ApoE and caveolin-2, as well as cholesterol, triglyceride and phospholipids were also detected in these low density fractions. After fractionation by size with Superose gel filtration, HCV RNA and viral proteins co-fractionated with endoplasmic reticulum proteins and VLDL. Fractionation on Toyopearl, which separates particles with diameters up to 200 nm, showed that 78 % of HCV RNA from liver was >100 nm in size, with a positive-/negative-strand ratio of 6 : 1. Also, 8 % of HCV RNA was found in particles with diameters between 40 nm and 70 nm and a positive-/negative-strand ratio of 45 : 1. This HCV was associated with ApoB, ApoE and viral glycoprotein E2, similar to viral particles circulating in serum. Our results indicate that the association between HCV and VLDL occurs in the liver.  

5.569           Coactivator PGC-1α regulates the fasting inducible xenobiotic-metabolizing enzyme CYP2A5 in mouse primary hepatocytes

Arpiainen, S. et al Toxicol Appl. Pharmacol., 232, 135-141 (2008)   The nutritional state of organisms and energy balance related diseases such as diabetes regulate the metabolism of xenobiotics such as drugs, toxins and carcinogens. However, the mechanisms behind this regulation are mostly unknown. The xenobiotic-metabolizing cytochrome P450 (CYP) 2A5 enzyme has been shown to be induced by fasting and by glucagon and cyclic AMP (cAMP), which mediate numerous fasting responses. Peroxisome proliferator-activated receptor γ coactivator (PGC)-1α triggers many of the important hepatic fasting effects in response to elevated cAMP levels. In the present study, we were able to show that cAMP causes a coordinated induction of PGC-1α and CYP2A5 mRNAs in murine primary hepatocytes. Furthermore, the elevation of the PGC-1α expression level by adenovirus mediated gene transfer increased CYP2A5 transcription. Co-transfection of Cyp2a5 5′ promoter constructs with the PGC-1α expression vector demonstrated that PGC-1α is able to activate Cyp2a5 transcription through the hepatocyte nuclear factor (HNF)-4α response element in the proximal promoter of the Cyp2a5 gene. Chromatin immunoprecipitation assays showed that PGC-1α binds, together with HNF-4α, to the same region at the Cyp2a5 proximal promoter. In conclusion, PGC-1α mediates the expression of Cyp2a5 induced by cAMP in mouse hepatocytes through coactivation of transcription factor HNF-4α. This strongly suggests that PGC-1α is the major factor mediating the fasting response of CYP2A5.  

5.570           Dissection and identification of regions required to form pseudoparticles by the interaction between the nucleocapsid (N) and membrane (M) proteins of SARS coronavirus

Hatakeyama, S. et al Virology, 380(1), 99-108 (2008)   When expressed in mammalian cells, the nucleocapsid (N) and membrane (M) proteins of the severe acute respiratory syndrome coronavirus (SARS-CoV) are sufficient to form pseudoparticles. To identify region(s) of the N molecule required for pseudoparticle formation, we performed biochemical analysis of the interaction of N mutants and M in HEK293 cells. Using a peptide library derived from N, we found that amino acids 101–115 constituted a novel binding site for M. We examined the ability of N mutants to interact with M and form pseudoparticles, and our observations indicated that M bound to NΔ(101–115), N1–150, N151–300, and N301–422, but not to N1–150Δ(101–115). However, pseudoparticles were formed when NΔ(101–115) or N301–422, but not N1–150 or N151–300, were expressed with M in HEK293 cells. These results indicated that the minimum portion of N required for the interaction with M and pseudoparticle formation consists of amino acids 301–422.  

5.571           The effect of point mutations within the N-terminal domain of Mason-Pfizer monkey virus capsid protein on virus core assembly and infectivity

Wildova, M. et al Virology, 380(1), 157-163 (2008)   Retroviral capsid protein (CA) mediates protein interactions driving the assembly of both immature viral particles and the core of the mature virions. Structurally conserved N-terminal domains of several retroviruses refold after proteolytic cleavage into a β-hairpin, stabilized by a salt bridge between conserved N-terminal Pro and Asp residues. Based on comparison with other retroviral CA, we identified Asp50 and Asp57 as putative interacting partners for Pro1 in Mason-Pfizer monkey virus (M-PMV) CA. To investigate the importance of CA Pro1 and its interacting Asp in M-PMV core assembly and infectivity, P1A, P1Y, D50A, T54A and D57A mutations were introduced into M-PMV. The P1A and D57A mutations partially blocked Gag processing and the released viral particles exhibited aberrant cores and were non-infectious. These data indicate that the region spanning residues Asp50–Asp57 plays an important role in stabilization of the β-hairpin and that Asp57 likely forms a salt-bridge with P1 in M-PMVCA.  

5.572           DNA Shuffling of Adeno-associated Virus Yields Functionally Diverse Viral Progeny

Koerber, J.T., Jang, J-H. And Schaffer, D.V. Molecular Therapy, 16(10), 1703-1709 (2008)   Adeno-associated virus (AAV) vectors are extremely effective gene-delivery vehicles for a broad range of applications. However, the therapeutic efficacy of these and other vectors is currently limited by barriers to safe, efficient gene delivery, including pre-existing antiviral immunity, and infection of off-target cells. Recently, we have implemented directed evolution of AAV, involving the generation of randomly mutagenized viral libraries based on serotype 2 and high-throughput selection, to engineer enhanced viral vectors. Here, we significantly extend this capability by performing high-efficiency in vitro recombination to create a large (10⁷), diverse library of random chimeras of numerous parent AAV serotypes (AAV1, 2, 4–6, 8, and 9). In order to analyze the extent to which such highly chimeric viruses can be viable, we selected the library for efficient viral packaging and infection, and successfully recovered numerous novel chimeras. These new viruses exhibited a broad range of cell tropism both in vitro and in vivo and enhanced resistance to human intravenous immunoglobulin (IVIG), highlighting numerous functional differences between these chimeras and their parent serotypes. Thus, directed evolution can potentially yield unlimited numbers of new AAV variants with novel gene-delivery properties, and subsequent analysis of these variants can further extend basic knowledge of AAV biology.  

5.573           Application of a haematopoetic progenitor cell-targeted adeno-associated viral (AAV) vector established by selection of an AAV random peptide library on a leukaemia cell line

Stiefelhagen, M. et al Genetic Vaccines and Therapy, 6, 12-21 (2008)   Background For many promising target cells (e.g.: haematopoeitic progenitors), the susceptibility to standard adeno-associated viral (AAV) vectors is low. Advancements in vector development now allows the generation of target cell-selected AAV capsid mutants. Methods To determine its suitability, the method was applied on a chronic myelogenous leukaemia (CML) cell line (K562) to obtain a CML-targeted vector and the resulting vectors tested on leukaemia, non-leukaemia, primary human CML and CD34⁺ peripheral blood progenitor cells (PBPC); standard AAV2 and a random capsid mutant vector served as controls. Results Transduction of CML (BV173, EM3, K562 and Lama84) and AML (HL60 and KG1a) cell lines with the capsid mutants resulted in an up to 36-fold increase in CML transduction efficiency (K562: 2-fold, 60% ± 2% green fluorescent protein (GFP)⁺ cells; BV173: 9-fold, 37% ± 2% GFP⁺ cells; Lama84: 36-fold, 29% ± 2% GFP⁺ cells) compared to controls. For AML (KG1a, HL60) and one CML cell line (EM3), no significant transduction (<1% GFP⁺ cells) was observed for any vector. Although the capsid mutant clone was established on a cell line, proof-of-principle experiments using primary human cells were performed. For CML (3.2-fold, mutant: 1.75% ± 0.45% GFP⁺ cells, p = 0.03) and PBPC (3.5-fold, mutant: 4.21% ± 3.40% GFP⁺ cells) a moderate increase in gene transfer of the capsid mutant compared to control vectors was observed. Conclusion Using an AAV random peptide library on a CML cell line, we were able to generate a capsid mutant, which transduced CML cell lines and primary human haematopoietic progenitor cells with higher efficiency than standard recombinant AAV vectors.  

5.574           Clathrin- and Caveolin-Independent Entry of Human Papillomavirus Type 16—Involvement of Tetraspanin-Enriched Microdomains (TEMs)

Spoden, G. et al PloS One, 3(10), e3313 (2008)   Background Infectious entry of human papillomaviruses into their host cells is an important step in the viral life cycle. For cell binding these viruses use proteoglycans as initial attachment sites. Subsequent transfer to a secondary receptor molecule seems to be involved in virus uptake. Depending on the papillomavirus subtype, it has been reported that entry occurs by clathrin- or caveolin-mediated mechanisms. Regarding human papillomavirus type 16 (HPV16), the primary etiologic agent for development of cervical cancer, clathrin-mediated endocytosis was described as infectious entry pathway. Methodology/Principal Findings Using immunofluorescence and infection studies we show in contrast to published data that infectious entry of HPV16 occurs in a clathrin- and caveolin-independent manner. Inhibition of clathrin- and caveolin/raft-dependent endocytic pathways by dominant-negative mutants and siRNA-mediated knockdown, as well as inhibition of dynamin function, did not impair infection. Rather, we provide evidence for involvement of tetraspanin-enriched microdomains (TEMs) in HPV16 endocytosis. Following cell attachment, HPV16 particles colocalized with the tetraspanins CD63 and CD151 on the cell surface. Notably, tetraspanin-specific antibodies and siRNA inhibited HPV16 cell entry and infection, confirming the importance of TEMs for infectious endocytosis of HPV16. Conclusions/Significance Tetraspanins fulfill various roles in the life cycle of a number of important viral pathogens, including human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, their involvement in endocytosis of viral particles has not been proven. Our data indicate TEMs as a novel clathrin- and caveolin-independent invasion route for viral pathogens and especially HPV16.  

5.575           Targeting Dyrk1A with AAVshRNA Attenuates Motor Alterations in TgDyrk1A, a Mouse Model of Down Syndrome

Ortiz-Abalia, J. et al Am. J. Human Genetics, 83(4), 479-488 (2008)   Genetic-dissection studies carried out with Down syndrome (DS) murine models point to the critical contribution of Dyrk1A overexpression to the motor abnormalities and cognitive deficits displayed in DS individuals. In the present study we have used a murine model overexpressing Dyrk1A (TgDyrk1A mice) to evaluate whether functional CNS defects could be corrected with an inhibitory RNA against Dyrk1A, delivered by bilateral intrastriatal injections of adeno-associated virus type 2 (AAVshDyrk1A). We report that AAVshDyrk1A efficiently transduced HEK293 cells and primary neuronal cultures, triggering the specific inhibition of Dyrk1A expression. Injecting the vector into the striata of TgDyrk1A mice resulted in a restricted, long-term transduction of the striatum. This gene therapy was found to be devoid of toxicity and succeeded in normalizing Dyrk1A protein levels in TgDyrk1A mice. Importantly, the behavioral studies of the adult TgDyrk1A mice treated showed a reversal of corticostriatal-dependent phenotypes, as revealed by the attenuation of their hyperactive behavior, the restoration of motor-coordination defects, and an improvement in sensorimotor gating. Taken together, the data demonstrate that normalizing Dyrk1A gene expression in the striatum of adult TgDyrk1A mice, by means of AAVshRNA, clearly reverses motor impairment. Furthermore, these results identify Dyrk1A as a potential target for therapy in DS.  

5.576           Three-Dimensional Organization of Rift Valley Fever Virus Revealed by Cryoelectron Tomography

Freiberg, A.N., Sherman, M.B., Morais, M.C., Holbrook, M.R. and Watowich, S.J.

1. Virol., 82(21), 10341-10348 (2008)

  Rift Valley fever virus (RVFV) is a member of the Bunyaviridae virus family (genus Phlebovirus) and is considered to be one of the most important pathogens in Africa, causing viral zoonoses in livestock and humans. Here, we report the characterization of the three-dimensional structural organization of RVFV vaccine strain MP-12 by cryoelectron tomography. Vitrified-hydrated virions were found to be spherical, with an average diameter of 100 nm. The virus glycoproteins formed cylindrical hollow spikes that clustered into distinct capsomeres. In contrast to previous assertions that RVFV is pleomorphic, the structure of RVFV MP-12 was found to be highly ordered. The three-dimensional map was resolved to a resolution of 6.1 nm, and capsomeres were observed to be arranged on the virus surface in an icosahedral lattice with clear T=12 quasisymmetry. All icosahedral symmetry axes were visible in self-rotation functions calculated using the Fourier transform of the RVFV MP-12 tomogram. To the best of our knowledge, a triangulation number of 12 had previously been reported only for Uukuniemi virus, a bunyavirus also within the Phlebovirus genus. The results presented in this study demonstrate that RVFV MP-12 possesses T=12 icosahedral symmetry and suggest that other members of the Phlebovirus genus, as well as of the Bunyaviridae family, may adopt icosahedral symmetry. Knowledge of the virus architecture may provide a structural template to develop vaccines and diagnostics, since no effective anti-RVFV treatments are available for human use.  

5.577           Detecting Small Changes and Additional Peptides in the Canine Parvovirus Capsid Structure

Nelson, C.D.S. et al

1. Virol., 82(21), 10397-10407 (2008)

  Parvovirus capsids are assembled from multiple forms of a single protein and are quite stable structurally. However, in order to infect cells, conformational plasticity of the capsid is required and this likely involves the exposure of structures that are buried within the structural models. The presence of functional asymmetry in the otherwise icosahedral capsid has also been proposed. Here we examined the protein composition of canine parvovirus capsids and evaluated their structural variation and permeability by protease sensitivity, spectrofluorometry, and negative staining electron microscopy. Additional protein forms identified included an apparent smaller variant of the virus protein 1 (VP1) and a small proportion of a cleaved form of VP2. Only a small percentage of the proteins in intact capsids were cleaved by any of the proteases tested. The capsid susceptibility to proteolysis varied with temperature but new cleavages were not revealed. No global change in the capsid structure was observed by analysis of Trp fluorescence when capsids were heated between 40°C and 60°C. However, increased polarity of empty capsids was indicated by bis-ANS binding, something not seen for DNA-containing capsids. Removal of calcium with EGTA or exposure to pHs as low as 5.0 had little effect on the structure, but at pH 4.0 changes were revealed by proteinase K digestion. Exposure of viral DNA to the external environment started above 50°C. Some negative stains showed increased permeability of empty capsids at higher temperatures, but no effects were seen after EGTA treatment.  

5.578           Cutaneous and mucosal human papillomaviruses differ in net surface charge, potential impact on tropism

Mistry, N., Wibom, C. and evander, M. Virol. J., 5, 118-123 (2008)   Papillomaviruses can roughly be divided into two tropism groups, those infecting the skin, including the genus beta PVs, and those infecting the mucosa, predominantly genus alpha PVs. The L1 capsid protein determines the phylogenetic separation between beta types and alpha types and the L1 protein is most probably responsible for the first interaction with the cell surface. Virus entry is a known determinant for tissue tropism and to study if interactions of the viral capsid with the cell surface could affect HPV tropism, the net surface charge of the HPV L1 capsid proteins was analyzed and HPV-16 (alpha) and HPV-5 (beta) with a mucosal and cutaneous tropism respectively were used to study heparin inhibition of uptake. The negatively charged L1 proteins were all found among HPVs with cutaneous tropism from the beta- and gamma-PV genus, while all alpha HPVs were positively charged at pH 7.4. The linear sequence of the HPV-5 L1 capsid protein had a predicted isoelectric point (pI) of 6.59 and a charge of -2.74 at pH 7.4, while HPV-16 had a pI of 7.95 with a charge of +2.98, suggesting no interaction between HPV-5 and the highly negative charged heparin. Furthermore, 3D-modelling indicated that HPV-5 L1 exposed more negatively charged amino acids than HPV-16. Uptake of HPV-5 (beta) and HPV-16 (alpha) was studied in vitro by using a pseudovirus (PsV) assay. Uptake of HPV-5 PsV was not inhibited by heparin in C33A cells and only minor inhibition was detected in HaCaT cells. HPV-16 PsV uptake was significantly more inhibited by heparin in both cells and completely blocked in C33A cells.  

5.579           Cancer gene therapy using mesenchymal stem cells expressing interferon- in a mouse prostate cancer lung metastasis model

Ren, C. et al Gene Therapy, 15, 1446-1453 (2008)   Cell-based therapy for cancer is a promising new field. Among cell types that can be used for this purpose, mesenchymal stem cells (MSCs) appear to hold great advantage for reasons including easier propagation in culture, possible genetic modification to express therapeutic proteins and preferential homing to sites of cancer growth upon in vivo transfer. The present study evaluated the potential of genetically modified MSC, constitutively expressing interferon (IFN)- , in an immunocompetent mouse model of prostate cancer lung metastasis. A recombinant adeno-associated virus (rAAV) encoding mouse IFN- was constructed and initially tested in vitro for high-level expression and bioactivity of the transgenic protein. MSCs were transduced by the rAAV-IFN- or green fluorescent protein ex vivo and used as cellular vehicles to target lung metastasis of TRAMP-C2 prostate cancer cells in a therapy model. Cohorts of mice were killed on days 30 and 75 to determine the effect of therapy by measurement of tumor volume, histology, immunohistochemistry, enzyme-linked immunosorbent assay and flow cytometry. Results indicated a significant reduction in tumor volume in lungs following IFN- -expressing MSC therapy. Immunohistochemistry of the lung demonstrated increased tumor cell apoptosis and decreased tumor cell proliferation and blood vessel counts. A significant increase in the natural kill cell activity was observed following IFN- therapy correlating the antitumor effect. Systemic level of IFN- was not significantly elevated from this targeted cell therapy. These data demonstrate the potential of MSC-based IFN- therapy for prostate cancer lung metastasis.  

5.580           Codon and mRNA Sequence Optimization of Microdystrophin Transgenes Improves Expression and Physiological Outcome in Dystrophic mdx Mice Following AAV2/8 Gene Transfer

Foster, H. et al Molecular Therapy, 16, 1825-1832 (2008)   Duchenne muscular dystrophy is a fatal muscle-wasting disorder. Lack of dystrophin compromises the integrity of the sarcolemma and results in myofibers that are highly prone to contraction-induced injury. Recombinant adeno-associated virus (rAAV)-mediated dystrophin gene transfer strategies to muscle for the treatment of Duchenne muscular dystrophy (DMD) have been limited by the small cloning capacity of rAAV vectors and high titers necessary to achieve efficient systemic gene transfer. In this study, we assess the impact of codon optimization on microdystrophin ( AB/R3-R18/ CT) expression and function in the mdx mouse and compare the function of two different configurations of codon-optimized microdystrophin genes ( AB/R3-R18/ CT and R4-R23/ CT) under the control of a muscle-restrictive promoter (Spc5-12). Codon optimization of microdystrophin significantly increases levels of microdystrophin mRNA and protein after intramuscular and systemic administration of plasmid DNA or rAAV2/8. Physiological assessment demonstrates that codon optimization of AB/R3-R18/ CT results in significant improvement in specific force, but does not improve resistance to eccentric contractions compared with noncodon-optimized AB/R3-R18/ CT. However, codon-optimized microdystrophin R4-R23/ CT completely restored specific force generation and provided substantial protection from contraction-induced injury. These results demonstrate that codon optimization of microdystrophin under the control of a muscle-specific promoter can significantly improve expression levels such that reduced titers of rAAV vectors will be required for efficient systemic administration.  

5.581           BAG1 promotes axonal outgrowth and regeneration in vivo via Raf-1 and reduction of ROCK activity

Planchamp, V. et al Brain, 131, 2606-2619 (2008)   Improved survival of injured neurons and the inhibition of repulsive environmental signalling are prerequisites for functional regeneration. BAG1 (Bcl-2-associated athanogene-1) is an Hsp70/Hsc70-binding protein, which has been shown to suppress apoptosis and enhance neuronal differentiation. We investigated BAG1 as a therapeutic molecule in the lesioned visual system in vivo. Using an adeno-associated viral vector, BAG1 (AAV.BAG1) was expressed in retinal ganglion cells (RGC) and then tested in models of optic nerve axotomy and optic nerve crush. BAG1 significantly increased RGC survival as compared to adeno-associated viral vector enhanced green fluorescent protein (AAV.EGFP) treated controls and this was independently confirmed in transgenic mice over-expressing BAG1 in neurons. The numbers and lengths of regenerating axons after optic nerve crush were also significantly increased in the AAV.BAG1 group. In pRGC cultures, BAG1-over-expression resulted in a 3-fold increase in neurite length and growth cone surface. Interestingly, BAG1 induced an intracellular translocation of Raf-1 and ROCK2 and ROCK activity was decreased in a Raf-1-dependent manner by BAG1-over-expression. In summary, we show that BAG1 acts in a dual role by inhibition of lesion-induced apoptosis and interaction with the inhibitory ROCK signalling cascade. BAG1 is therefore a promising molecule to be further examined as a putative therapeutic tool in neurorestorative strategies.  

5.582           Role of the Hepatitis C Virus Core+1 Open Reading Frame and Core cis-Acting RNA Elements in Viral RNA Translation and Replication

Vassilaki, N. et al

1. Virol., 82(23), 11503-11515 (2008)

  Four conserved RNA stem-loop structures designated SL47, SL87, SL248, and SL443 have been predicted in the hepatitis C virus (HCV) core encoding region. Moreover, alternative translation products have been detected from a reading frame overlapping the core gene (core+1/ARFP/F). To study the importance of the core+1 frame and core-RNA structures for HCV replication in cell culture and in vivo, a panel of core gene silent mutations predicted to abolish core+1 translation and affecting core-RNA stem-loops were introduced into infectious-HCV genomes of the isolate JFH1. A mutation disrupting translation of all known forms of core+1 and affecting SL248 did not alter virus production in Huh7 cells and in mice xenografted with human liver tissue. However, a combination of mutations affecting core+1 at multiple codons and at the same time, SL47, SL87, and SL248, delayed RNA replication kinetics and substantially reduced virus titers. The in vivo infectivity of this mutant was impaired, and in virus genomes recovered from inoculated mice, SL87 was restored by reversion and pseudoreversion. Mutations disrupting the integrity of this stem-loop, as well as that of SL47, were detrimental for virus viability, whereas mutations disrupting SL248 and SL443 had no effect. This phenotype was not due to impaired RNA stability but to reduced RNA translation. Thus, SL47 and SL87 are important RNA elements contributing to HCV genome translation and robust replication in cell culture and in vivo.  

5.583           Functional importance of dengue virus maturation: infectious properties of immature virions

Zybert, I.A., van der Ende-Metselaar, H., Wilschut, J. And Smit, J.M.

1. Gen. Virol., 89, 3047-3051 (2008)

  Prior to the release of flavivirus particles from infected cells, the viral surface protein prM is cleaved to M by the cellular enzyme furin. For dengue virus (DENV), this maturation process appears to be very inefficient since a high proportion of progeny virions contain uncleaved prM. Furthermore, it has been reported that prM-containing DENV particles are infectious. These observations contradict the general assumption that prM processing is required to render virus particles infectious. Therefore, in this study, we reinvestigated the infectious properties of immature DENV virions. DENV particles were produced in furin-deficient LoVo cells. We observed that DENV-infected LoVo cells secrete high numbers of prM-containing particles. Subsequent analysis of the infectious titre revealed that immature particles lack the ability to infect cells, the infectious unit to particle ratio being 10 000-fold reduced compared with that of wild-type virus. Our results indicate that cleavage of prM to M is required for DENV infectivity.  

5.584           Augmentation of AAV-mediated cardiac gene transfer after systemic administration in adult rats

Müller, O.J., Schinkel, S., Kleinschmidt, J.A., Katus, H.A. and Bekeredjian, R. Gene Therapy, 15, 1558-1565 (2008)   Adeno-associated virus (AAV)-6 or -9-pseudotyped vectors are suitable for efficient cardiac gene transfer after intravenous injection in mice. However, a systemic application in larger animals or humans would require very high doses of viral particles. Therefore, the aim of our study was to test if ultrasound-targeted microbubble destruction could augment cardiac transduction of AAV vectors after intravenous administration in rats. To analyze efficiency and specificity of gene transfer, microbubbles loaded with AAV-6 or -9 harboring a luciferase or enhanced green fluorescent protein (EGFP) reporter gene were infused into the jugular vein of adult Sprague–Dawley rats. During the infusion, high mechanical index ultrasound was administered to the heart. Control rats received the same amount of virus without microbubbles, but with ultrasound. After 4 weeks, organs were harvested and analyzed for reporter gene expression. In contrast to low cardiac expression after systemic transfer of the vector solution without microbubbles, ultrasound-targeted destruction of microbubbles significantly increased cardiac reporter activities between 6- and 20-fold. Analysis of spatial distribution of transgene expression using an AAV-9 vector encoding for EGFP revealed transmural expression predominantly in the left ventricular anterior wall. In conclusion, ultrasound targeted microbubble destruction augments cardiac transduction of AAV vectors in rats. This approach may be suitable for efficient, specific and noninvasive AAV-mediated gene transfer in larger animals or humans.  

5.585           Identification of a Residue in Hepatitis C Virus E2 Glycoprotein That Determines Scavenger Receptor BI and CD81 Receptor Dependency and Sensitivity to Neutralizing Antibodies

Grove, J. et al

1. Virol., 82(24), 12020-12029 (2008)

  Hepatitis C virus (HCV) infection is dependent on at least three coreceptors: CD81, scavenger receptor BI (SR-BI), and claudin-1. The mechanism of how these molecules coordinate HCV entry is unknown. In this study we demonstrate that a cell culture-adapted JFH-1 mutant, with an amino acid change in E2 at position 451 (G451R), has a reduced dependency on SR-BI. This altered receptor dependency is accompanied by an increased sensitivity to neutralization by soluble CD81 and enhanced binding of recombinant E2 to cell surface-expressed and soluble CD81. Fractionation of HCV by density gradient centrifugation allows the analysis of particle-lipoprotein associations. The cell culture-adapted mutation alters the relationship between particle density and infectivity, with the peak infectivity occurring at higher density than the parental virus. No association was observed between particle density and SR-BI or CD81 coreceptor dependence. JFH-1 G451R is highly sensitive to neutralization by gp-specific antibodies, suggesting increased epitope exposure at the virion surface. Finally, an association was observed between JFH-1 particle density and sensitivity to neutralizing antibodies (NAbs), suggesting that lipoprotein association reduces the sensitivity of particles to NAbs. In summary, mutation of E2 at position 451 alters the relationship between particle density and infectivity, disrupts coreceptor dependence, and increases virion sensitivity to receptor mimics and NAbs. Our data suggest that a balanced interplay between HCV particles, lipoprotein components, and viral receptors allows the evasion of host immune responses.  

5.586           Heparan Sulfate-Independent Cell Binding and Infection with Furin-Precleaved Papillomavirus Capsids

Day, P.M., Lowy, D.R. and Schiller, J.T.

1. Virol., 82(24), 12565-12568 (2008)

  Papillomavirus infection normally involves virion binding to cell surface heparan sulfate proteoglycans (HSPGs). However, we found that human papillomavirus type 16 pseudovirions efficiently bound and infected cells lacking HSPGs if their L2 capsid protein was precleaved by furin, a cellular protease required for infection. The inability of pseudovirions to efficiently bind and infect cultured primary keratinocytes was also overcome by furin precleavage, suggesting that the defect involves altered HSPG modification. We conclude that the primary function of HSPG binding is to enable cell surface furin cleavage of L2 and that binding to a distinct cell surface receptor(s) is a subsequent step of papillomavirus infection.  

5.587           Genetic Modification of Adeno-Associated Viral Vector Type 2 Capsid Enhances Gene Transfer Efficiency in Polarized Human Airway Epithelial Cells

White, A.F., Mazur, M., Sorcher, E.J., Zinn, K.R. and Ponnazhagan, S. Human Gene Therapy, 19, 1407-1414 (2008)   Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.  

5.588           The C Terminus of Foamy Retrovirus Gag Contains Determinants for Encapsidation of Pol Protein into Virions

Lee, E-G. and Lineal, M.L.

1. Virol., 82(21), 10803-10810 (2008)

  Foamy viruses (FV) differ from orthoretroviruses in many aspects of their replication cycle. A major difference is in the mode of Pol expression, regulation, and encapsidation into virions. Orthoretroviruses synthesize Pol as a Gag-Pol fusion protein so that Pol is encapsidated into virus particles through Gag assembly domains. However, as FV express Pol independently of Gag from a spliced mRNA, packaging occurs through a distinct mechanism. FV genomic RNA contains cis-acting sequences that are required for Pol packaging, suggesting that Pol binds to RNA for its encapsidation. However, it is not known whether Gag is directly involved in Pol packaging. Previously our laboratory showed that sequences flanking the three glycine-arginine-rich (GR) boxes at the C terminus of FV Gag contain domains important for RNA packaging and Pol expression, cleavage, and packaging. We have now shown that both deletion and substitution mutations in the first GR box (GR1) prevented neither the assembly of particles with wild-type density nor packaging of RNA genomes but led to a defect in Pol packaging. Site-directed mutagenesis of GR1 indicated that the clustered positively charged amino acids in GR1 play important roles in Pol packaging. Our results suggest that GR1 contains a Pol interaction domain and that a Gag-Pol complex is formed and binds to RNA for incorporation into virions.  

5.589           Three-Dimensional Analysis of Budding Sites and Released Virus Suggests a Revised Model for HIV-1 Morphogenesis

Carlson, L-A., Briggs, J.A.G., Glass, B., Riches, J.D., Simon, M.N., Johnson, M.C., Müller, B., Grünewald, K. and Kräisslich, H-G. Cell Host and Microbe, 4, 592-599 (2008)   Current models of HIV-1 morphogenesis hold that newly synthesized viral Gag polyproteins traffic to and assemble at the cell membrane into spherical protein shells. The resulting late-budding structure is thought to be released by the cellular ESCRT machinery severing the membrane tether connecting it to the producer cell. Using electron tomography and scanning transmission electron microscopy, we find that virions have a morphology and composition distinct from late-budding sites. Gag is arranged as a continuous but incomplete sphere in the released virion. In contrast, late-budding sites lacking functional ESCRT exhibited a nearly closed Gag sphere. The results lead us to propose that budding is initiated by Gag assembly, but is completed in an ESCRT-dependent manner before the Gag sphere is complete. This suggests that ESCRT functions early in HIV-1 release akin to its role in vesicle formation and is not restricted to severing the thin membrane tether.  

5.590           AAV-mediated knockdown of phospholamban leads to improved contractility and calcium handling in cardiomyocytes

Andino, L.M., Takeda, M., Kashara, H., Jakymiw, A., Byrne, B.J. and Lewin, A.S.

1. Gene Med., 10(2), 132-142 (2008)

  Background Reduced contractility due to dysregulation of intracellular calcium (Ca2+) is a common pathologic feature of chronic heart failure. Calcium stores in the sarcoplasmic reticulum play a major role in regulating cardiac contractility. Several animal models of heart failure have been treated by altering the regulation of the sarcoplamic reticulum ATPase through ablation or down-regulation of its inhibitor peptide, phospholamban (PLN). Methods We have designed two small hairpin RNAs (shRNAs) to block the synthesis of PLN via RNA interference. These were tested in cell culture using a co-transfection assay and using adeno-associated virus (AAV)-mediated delivery to cardiomyocytes. Reverse-transcription polymerase chain reaction (RT-PCR) and Western blots were used to measure reduction in PLN mRNA and protein levels. Reduction of PLN was also documented by indirect immunofluorescence. Free cytosolic calcium and contractile properties of transduced cardiomyocytes was examined on fura-2-loaded cells. Direct cardiac injection was used to deliver AAV1-shRNAs to mice, and reduction of PLN was measured by indirect immunofluorescence. Results Both siRNAs led to significant reduction of PLN RNA and protein levels in cultured cells. Down-regulation of PLN led to enhanced cell shortening and relaxation and to a decrease in the time constant of calcium decay, signs of improved contractility and calcium handling. In the hearts of AAV-infected mice, shRNA-transduced cells showed significant reduction in the level of PLN. Conclusions Our results suggest that AAV-delivered shRNAs mediated physiologically significant suppression of phospholamban that may be useful in combating the effects of chronic heart failure.  

5.591           Gene delivery to the vasculature mediated by low-titre adeno-associated virus serotypes 1 and 5

Sen, S., Contoy, S., Hynes, S.O., McMahon, J., O’Doherty, A., Bartlett, J.S., Akhtar, Y., Adegbola, T., Conolly, C.E., Sultan, S., Barry, F., Katusic, Z.S. and O’Brien, T.

1. Gene Med., 10(2), 143-151 (2008)

  Background Vascular gene therapy requires safe and efficient gene transfer in vivo. Recombinant adeno-associated virus (AAV) is a promising viral vector but its use in the vasculature has produced conflicting results and serotypes other than AAV2 have not been intensively studied. We investigated the efficiency of alternative AAV serotypes for vascular gene delivery in vitro and in vivo. Methods Vascular cell lines were transduced in vitro with AAV vectors. Rabbit carotid arteries were transduced with AAV1, 2 and 5 encoding enhanced green fluorescent protein (eGFP) (∼1.4 × 10⁹ DNAse-resistant particles (drp)). Gene transfer in vivo was assessed at 14 and 28 days. High-titre doses of AAV2 encoding β-galactosidase in vivo were also studied. Results In vitro, transgene expression was not observed in endothelial cells using AAV2 whereas the use of serotypes 1 and 5 resulted in detectable levels of transgene expression. Coronary artery smooth muscle cells (CASMCs) transduced with AAV2 demonstrated higher levels of GFP expression than AAV1 or 5. Transgene expression in vivo was noted using low-titre AAV1 and AAV5 (∼1.4 × 10⁹ drp) in the media and adventitia. Only delivery of AAV1eGFP resulted in neointimal formation (3/7 vessels examined), with transgene expression noted in the neointima. Transgene expression with AAV2 was not detected in any layer of the blood vessel wall using low titre (∼10⁹ drp). However, high-titre (∼10¹¹ drp) AAV2 resulted in transduction of cells in the media and adventitia but not the endothelium. Conclusions AAV1 and AAV5 have advantages over AAV2 for vascular gene delivery at low titres.  

5.592           Recent developments in adeno-associated virus vector technology

Büning, H., Perabo, L., Coutelle, O., Quadt-Humme, S. and Hallek, M.

1. Gene Med., 10(6), 717-733 (2008)

  Adeno-associated virus (AAV), a single-stranded DNA parvovirus, is emerging as one of the leading gene therapy vectors owing to its nonpathogenicity and low immunogenicity, stability and the potential to integrate site-specifically without known side-effects. A portfolio of recombinant AAV vector types has been developed with the aim of optimizing efficiency, specificity and thereby also the safety of in vitro and in vivo gene transfer. More and more information is now becoming available about the mechanism of AAV/host cell interaction improving the efficacy of recombinant AAV vector (rAAV) mediated gene delivery. This review summarizes the current knowledge of the infectious biology of AAV, provides an overview of the latest developments in the field of AAV vector technology and discusses remaining challenges.  

5.593           Recombinant adeno-associated virus-mediated gene delivery of long chain acyl coenzyme A dehydrogenase (LCAD) into LCAD-deficient mice

Beattie, S.G., Goetzman, E., Tang, Q., Conlon, T., Campbell-Thompson, M., Matern, D., Vockley, J. and Flotte, T.R.

1. Gene Med., 10(10), 1113-1123 (2008)

  Background Very long chain acyl coenzyme A (CoA) dehydrogenase (VLCAD) deficiency is a relatively common mitochondrial β-oxidation disorder. The most severe form of VLCAD deficiency presents with neonatal cardiomyopathy and hepatic failure and is generally fatal within the first year of life. Mice deficient for long chain acyl CoA dehydrogenase (LCAD) closely resemble the clinical syndrome observed in VLCAD-deficient humans. Recombinant adeno-associated viral (rAAV) vectors with pseudotype capsids were investigated for their potential towards correcting the phenotype observed in mice heterozygous (+/−) for LCAD (i.e. liver and muscle steatosis). Methods rAAV containing the mouse LCAD cDNA (mLCAD) under the transcriptional control of the CMV/chicken β-actin hybrid promoter were injected intramuscularly into the tibialis anterior (TA) muscle of LCAD+/− mice or injected into the portal vein to transduce hepatocytes. Results Ten weeks post-injection of rAAV1-mLCAD into the TA muscle, significantly increased levels of mLCAD within mitochondria were demonstrated by immunostaining of TA sections, immunoblotting of mitochondrial isolates and by the electron transfer flavoprotein (ETF) fluorescence reduction enzyme activity assay. Magnetic resonance spectroscopy of vector-injected TA muscle demonstrated a reduction in the lipid content compared to phosphate-buffered saline-injected mice, whereas a systemic effect was observed as a reduction in liver macrosteatosis. Eight weeks after portal vein injection of rAAV8-mLCAD into LCAD+/− mice, increased levels of mLCAD within hepatocyte mitochondria were demonstrated by immunostaining and also by the ETF assay. Scoring of the hepatosteatosis observed in partially deficient LCAD mice indicated a reduction in the lipid content within livers of vector-treated mice. Conclusions These studies show that rAAV-mediated delivery of mLCAD was efficient and led to an amelioration of local and systemic pathologies observed in partially deficient LCAD mice.  

5.594           Therapeutic Potential of Mesenchymal Stem Cells Producing Interferon- in a Mouse Melanoma Lung Metastasis Model

Ren, C., Kumar, S., Chanda, D., Chen, J., Mountz, J.D. and Ponnazhagan, S. Stem Cells, 26(9), 2332-2338 (2008)   Adult stem cells represent a potential source for cell-based therapy of cancer. The present study evaluated the potential of bone marrow-derived mesenchymal stem cells (MSC), genetically modified to express interferon (IFN)- , for the treatment of lung metastasis in an immunocompetent mouse model of metastatic melanoma. A recombinant adeno-associated virus (rAAV) 6 vector encoding IFN- was used to transduce mouse bone marrow-derived MSC ex vivo. Expression and bioactivity of the transgenic protein from rAAV-transduced MSC were confirmed prior to in vivo studies. A lung metastasis model of melanoma was developed by i.v. injection of B16F10 cells into 8-week-old C57BL/6 mice. Ten days later, MSC transduced with rAAV-IFN- or green fluorescent protein were intravenously injected. One cohort of mice was sacrificed to determine the effects of the therapy at an earlier time point, and another cohort was observed for long-term survival. Results indicated that systemic administration of MSC producing IFN- reduced the growth of B16F10 melanoma cells and significantly prolonged survival. Immunohistochemistry analysis of the tumors from MSC-IFN- -treated animals indicated an increase in apoptosis and a decrease in proliferation and blood vasculature. These data demonstrate the potential of adult MSC constitutively producing IFN- to reduce the growth of lung metastasis in melanoma.  

5.595           Estrogen plays a critical role in AAV2-mediated gene transfer in ovarian cancer

Shi, W-F. and Bartlett, J.S. Acta Pharmacol. Sin., 29(12), 1440-1450 (2008)   AIM: The aim of our study was to develop an effective gene delivery system for ovarian cancer gene therapy. METHODS: The expression of heparin sulfate proteoglycan (HSPG) and integrins alpha(upsilon)beta(3) and alpha(upsilon)beta(5) were analyzed with flow cytometry on 2 human ovarian cancer cell lines (OVCAR-3 and SKOV-3ip). The gene transduction efficiencies were evaluated with recombinant adeno-associated viral vector (rAAV)2-green fluorescent protein or rAAV2-lactase Z followed by flow cytometry or cytohistochemistry staining. The effect of 17beta-estradiol on ovarian cancer cell proliferation, HSPG, the expressions of integrins alpha(upsilon)beta(3) and alpha(upsilon)beta(5), and adeno-associated viral vector (AAV)2-mediated gene transduction were determined. RESULTS: In the present study, we found: (1) a variation in HSPG and the expressions of integrins alpha(upsilon)beta(3) and alpha(upsilon)beta(5) between OVCAR-3 and SKOV-3ip; (2) that 17beta-estradiol was shown to significantly stimulate cell proliferation and integrin beta(5) expression in certain ovarian cancer cell lines; and (3) integrintargeted A520/N584RGD-rAAV2, which has alternative interactivity with integrins and abrogates the binding capacity HSPG, showed much higher gene transduction efficiency in ovarian cancer cells than rAAV2 in the presence/absence of 17beta-estradiol. Moreover, this RGD-modified rAAV2 exerted more efficient transduction in ovarian cancer cells in response to 17beta-estradiol. CONCLUSION: Our findings implied that A520/N584RGD-rAAV2 may offer great potential for ovarian cancer treatment in vivo.  

5.596           Functional Cystic Fibrosis Transmembrane Conductance Regulator Expression in Cystic Fibrosis Airway Epithelial Cells by AAV6.2-Mediated Segmental Trans-Splicing

Song, Y., Lou, H.H., Boyer, J.L., Limberis, M.P., Vandenberghe, L.H.m Hackett, N.R., Leopold, P.L., Wilson, J.M. and Crystal, R.G. Human Gene Therapy, 20, 267-281 (2009)   Cystic fibrosis is characterized by deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl− transporter. The packaging constraints of adeno-associated viral (AAV) vectors preclude delivery of both an active promoter and CFTR cDNA to target cells. We hypothesized that segmental trans-splicing, in which two AAV vectors deliver the 5′ and 3′ halves of the CFTR cDNA, could mediate splicing of two pre-mRNAs into a full-length, functional CFTR mRNA. Using a segmental trans-splicing 5′ donor–3′ acceptor pair that split the CFTR cDNA between exons 14a and 14b, cotransfection of donor and acceptor plasmids into CFTR− cells resulted in full-length CFTR message and protein. Microinjection of plasmids into CFTR− cells produced cAMP-activated Cl− conductance. Vectors created with an engineered human serotype, AAV6.2, were used to deliver CFTR donor and acceptor constructs, resulting in full-length CFTR mRNA and protein as well as cAMP-activated Cl− conductance in CFTR− cells, including human CF airway epithelial IB3-1 cells. Thus, segmental trans-splicing can be used with AAV vectors to mediate expression of CFTR, a strategy potentially applicable to individuals with CF.  

5.597           Artificial MicroRNAs as siRNA Shuttles: Improved Safety as Compared to shRNAs In vitro and In vivo

Boudreau, R.L., Martins, I. and Davidson, B.L. Molecular Therapy. 17(1), 169-175 (2009)   RNA interference (RNAi) provides a promising therapeutic approach to human diseases. However, data from recent reports demonstrate that short-hairpin RNAs (shRNAs) may cause cellular toxicity, and this warrants further investigation of the safety of using RNAi vectors. Earlier, in comparing hairpin-based RNAi vectors, we noted that shRNAs are highly expressed and yield an abundance of unprocessed precursors, whereas artificial microRNAs (miRNAs) are expressed at lower levels and are processed efficiently. We hypothesized that unprocessed shRNAs arise from the saturation of endogenous RNAi machinery, which poses likely a burden to cells. In this study, we tested that hypothesis by assessing the relative effects of shRNAs and artificial miRNAs on the processing and function of miRNAs. In competition assays, shRNAs disrupted miRNA biogenesis and function, whereas artificial miRNAs avoided this interference even when dosed to silence as effectively as shRNAs. We next compared the safety of these vectors in mouse cerebella, and found that shRNAs cause Purkinje cell neurotoxicity. By contrast, artificial miRNA expression was well tolerated, resulting in effective target gene silencing in Purkinje cells. These findings, together with data from earlier work in mouse striata, suggest that miRNA-based platforms are better suited for therapeutic silencing in the mammalian brain.  

5.598           Novel anti-VEGF chimeric molecules delivered by AAV vectors for inhibition of retinal neovascularization

Pechan, P., Rubin, H., Lukason, M., Ardinger, J., DuFresne, E., Hauswirth, W.W., Wadsworth, S.C. and Scaria, A. Gene Therapy, 16, 10-16 (2009)   Vascular endothelial growth factor (VEGF) is important in pathological neovascularization, which is a key component of diseases such as the wet form of age-related macular degeneration, proliferative diabetic retinopathy and cancer. One of the most potent naturally occurring VEGF binders is VEGF receptor Flt-1. We have generated two novel chimeric VEGF-binding molecules, sFLT01 and sFLT02, which consist of the second immunoglobulin (IgG)-like domain of Flt-1 fused either to a human IgG1 Fc or solely to the CH3 domain of IgG1 Fc through a polyglycine linker 9Gly. In vitro analysis showed that these novel molecules are high-affinity VEGF binders. We have demonstrated that adeno-associated virus serotype 2 (AAV2)-mediated intravitreal gene delivery of sFLT01 efficiently inhibits angiogenesis in the mouse oxygen-induced retinopathy model. There were no histological observations of toxicity upon persistent ocular expression of sFLT01 for up to 12 months following intravitreal AAV2-based delivery in the rodent eye. Our data suggest that AAV2-mediated intravitreal gene delivery of our novel molecules may be a safe and effective treatment for retinal neovascularization.  

5.599           Enhancing transduction of the liver by adeno-associated viral vectors

Nathwani, A.C., Cochrane, M., McIntosh, J., Ng, C.Y.C., Zhou, J., Gray, J.T. and Davidoff, A.M. Gene Therapy, 16, 60-69 (2009)   A number of distinct factors acting at different stages of the adeno-associated virus vector (AAV)-mediated gene transfer process were found to influence murine hepatocyte transduction. Foremost among these was the viral capsid protein. Self-complementary (sc) AAV pseudotyped with capsid from serotype 8 or rh.10 mediated fourfold greater hepatocyte transduction for a given vector dose when compared with vector packaged with AAV7 capsid. An almost linear relationship between vector dose and transgene expression was noted for all serotypes with vector doses as low as 1 10⁷ vg per mouse (4 10⁸ vg kg^-1) mediating therapeutic levels of human FIX (hFIX) expression. Gender significantly influenced scAAV-mediated transgene expression, with twofold higher levels of expression observed in male compared with female mice. Pretreatment of mice with the proteasome inhibitor bortezomib increased scAAV-mediated hFIX expression from 4 0.6 to 9 2  g ml^-1 in female mice, although the effect of this agent was less profound in males. Exposure of mice to adenovirus 10–20 weeks after gene transfer with AAV vectors augmented AAV transgene expression twofold by increasing the level of proviral mRNA. Hence, optimization of individual steps in the AAV gene transfer process can further enhance the potency of AAV-mediated transgene expression, thus increasing the probability of successful gene therapy.  

5.600           A Novel Method to Incorporate Bioactive Cytokines as Adjuvants on the Surface of Virus Particles

Yang, Y., Leggat, D., Herbert, A., Roberts, P.C. and Sundick, R.S.

1. Interferon & Cytokine Res., 29(1), 9-22 (2009)

  Cytokines have been used extensively as adjuvants in vaccines. However, practical considerations limit their use; diffusion from antigen, short half-lives and additional production costs. To address these problems we have developed a technology that efficiently produces inactivated, whole-virus influenza vaccine bearing membrane-bound cytokines. To provide “proof of principle,” we chose chicken interleukin-2 (IL-2) and chicken granulocyte–macrophage colony-stimulating factor. Fusion constructs were generated in which their coding regions were linked to the influenza virus transmembrane encoding domains of the neuraminidase and hemagglutinin genes, respectively. These fusion constructs were used to establish stable Madin–Darby Canine Kidney cell lines, constitutively expressing membrane-bound cytokine. Cell surface expression was verified by immunofluorescence and cytokine-specific bioassays. Influenza virus harvested from infected cytokine-bearing cells was purified, inactivated, and confirmed to include membrane-bound cytokine by immunofluorescence, Western blotting and bioassay. Cytokine bioactivity was preserved using several standard virus inactivation protocols. Both cytokine-bearing influenza vaccines are now being tested for immunogenicity in vivo. Initial experiments indicate that chickens injected with IL-2-bearing influenza have elevated antiviral antibody levels, compared to chickens given conventional vaccine. In conclusion, this technology offers a novel method to utilize cytokines and other immunostimulatory molecules as adjuvants for viral vaccines.  

5.601           Angiostatin overexpression is associated with an improvement in chronic kidney injury by an anti-inflammatory mechanism

Mu, W., Long, D.A., Ouyang, X., Agarwal, A., Cruz, P.E., Roncal, C.A., Nakagawa, T., Yu, X., Hauswirth, W.W. and Johnson, R.J. Am. J. Physiol. Renal Physiol., 296, F145-F152 (2009)   Angiostatin, a proteolytic fragment of plasminogen, is a potent anti-angiogenic factor recently shown also to have an inhibitory effect on leukocyte recruitment and macrophage migration. Because both angiogenesis and inflammation play key roles in the progression of chronic kidney disease, we evaluated the effect of angiostatin treatment in the rat remnant kidney model. Rats were pretreated for 4 wk with recombinant adeno-associated viruses expressing either angiostatin or green fluorescence protein. Chronic renal disease was then induced by a subtotal nephrectomy, and rats were killed 8 wk later for analysis. Angiostatin treatment was associated with significantly less proteinuria but no alterations in serum creatinine, creatinine clearance, and blood urea nitrogen levels. Treatment with angiostatin reduced renal peritubular capillary number and decreased urinary nitric oxide levels. Despite reducing capillary density, angiostatin diminished interstitial fibrosis in association with reduced macrophage and T-cell infiltration and renal monocyte chemoattractant protein-1 mRNA levels. In conclusion, angiostatin overexpression was associated with attenuated renal disease progression in a model of chronic kidney injury, likely because of its anti-inflammatory actions. However, its anti-angiogenic actions suggest countering effects that could partially offset its benefit in chronic kidney diseases.  

5.602           Identification of Cellular Proteins That Interact with the Adeno-Associated Virus Rep Protein

Nash, K., Chen, W., Salganik, M. and Muzyczka, N.

1. Virol., 83(1), 454-469 (2009)

  Adeno-associated virus (AAV) codes for four related nonstructural Rep proteins. AAV both replicates and assembles in the nucleus and requires coinfection with a helper virus, either adenovirus (Ad) or herpesvirus, for a productive infection. Like other more complex DNA viruses, it is believed that AAV interacts or modifies host cell proteins to carry out its infection cycle. To date, relatively little is known about the host proteins that interact with the viral Rep proteins, which are known to be directly involved in DNA replication, control of viral and cellular transcription, splicing, and protein translation. In this study, we used affinity-tagged Rep protein to purify cellular protein complexes that were associated with Rep in cells that had been infected with Ad and AAV. In all, we identified 188 cellular proteins from 16 functional categories, including 14 transcription factors, 6 translation factors, 15 potential splicing proteins, 5 proteins involved in protein degradation, and 13 proteins involved in DNA replication or repair. This dramatically increases the number of potential interactions over the current number of approximately 26. Twelve of the novel proteins found were further tested by coimmunoprecipitation or colocalization using confocal immunomicroscopy. Of these, 10 were confirmed as proteins that formed complexes with Rep, including proteins of the MCM complex (DNA replication), RCN1 (membrane transport), SMC2 (chromatin dynamics), EDD1 (ubiquitin ligase), IRS4 (signal transduction), and FUS (splicing). Computer analysis suggested that 45 and 28 of the 188 proteins could be placed in a pathway of interacting proteins involved in DNA replication and protein synthesis, respectively. Of the proteins involved in DNA replication, all of the previously identified proteins involved in AAV DNA replication were found, except Ad DBP. The only Ad protein found to interact with Rep was the E1b55K protein. In addition, we confirmed that Rep interacts with Ku70/80 helicase. In vitro DNA synthesis assays demonstrated that although Ku helicase activity could substitute for MCM to promote strand displacement synthesis, its presence was not essential. Our study suggests that the interaction of AAV with cellular proteins is much more complex than previously suspected and provides a resource for further studies of the AAV life cycle.  

5.603           Incorporation of CD40 Ligand into the Envelope of Pseudotyped Single-Cycle Simian Immunodeficiency Viruses Enhances Immunogenicity

Lin, F-C., Pengh, Y., Jones, L.A., Verardi, P.H. and Yilma, T.D.

1. Virol., 83(3), 1216-1227 (2009)

  A vaccine for the prevention of human immunodeficiency virus (HIV) infection is desperately needed to control the AIDS pandemic. To address this problem, we developed vesicular stomatitis virus glycoprotein-pseudotyped replication-defective simian immunodeficiency viruses (dSIVs) as an AIDS vaccine strategy. The dSIVs retain characteristics of a live attenuated virus without the drawbacks of potential virulence caused by replicating virus. To improve vaccine immunogenicity, we incorporated CD40 ligand (CD40L) into the dSIV envelope. CD40L is one of the most potent stimuli for dendritic cell (DC) maturation and activation. Binding of CD40L to its receptor upregulates expression of major histocompatibility complex class I, class II, and costimulatory molecules on DCs and increases production of proinflammatory cytokines and chemokines, especially interleukin 12 (IL-12). This cytokine polarizes CD4⁺ T cells to Th1-type immune responses. DC activation and mixed lymphocyte reaction (MLR) studies were performed to evaluate the immunogenicity of CD40L-dSIV in vitro. Expression levels of CD80, CD86, HLA-DR, and CD54 on DCs transduced with the dSIV incorporating CD40L (CD40L-dSIV) were significantly higher than on those transduced with dSIV. Moreover, CD40L-dSIV-transduced DCs expressed up to 10-fold more IL-12 than dSIV-transduced DCs. CD40L-dSIV-transduced DCs enhanced proliferation and gamma interferon secretion by naive T cells in an MLR. In addition, CD40L-dSIV-immunized mice exhibited stronger humoral and cell-mediated immune responses than dSIV-vaccinated animals. The results show that incorporating CD40L into the dSIV envelope significantly enhances immunogenicity. As a result, CD40L-dSIVs can be strong candidates for development of a safe and highly immunogenic AIDS vaccine.  

5.604           Infectious Molecular Clones of Adeno-Associated Virus Isolated Directly from Human Tissues

Schnepp, B.C., Jensen, R.L., Clark, K.R. and Johnson, P.R.

1. Virol., 83(3), 1456-1464 (2009)

  Adeno-associated virus (AAV) replication and biology have been extensively studied using cell culture systems, but there is precious little known about AAV biology in natural hosts. As part of our ongoing interest in the in vivo biology of AAV, we previously described the existence of extrachromosomal proviral AAV genomes in human tissues. In the current work, we describe the molecular structure of infectious DNA clones derived directly from these tissues. Sequence-specific linear rolling-circle amplification was utilized to isolate clones of native circular AAV DNA. Several molecular clones containing unit-length viral genomes directed the production of infectious wild-type AAV upon DNA transfection in the presence of adenovirus help. DNA sequence analysis of the molecular clones revealed the ubiquitous presence of a double-D inverted terminal repeat (ITR) structure, which implied a mechanism by which the virus is able to maintain ITR sequence continuity and persist in the absence of host chromosome integration. These data suggest that the natural life cycle of AAV, unlike that of retroviruses, might not have genome integration as an obligatory component.  

5.605           Respiratory Syncytial Virus Activates Innate Immunity through Toll-Like Receptor 2

Murawski, M., Bowen, G.N., Cerny, A.M., Anderson, L.J., Haynes, L.M., Tripp, R.A., Kurt-Jones, E.A. and Finberg, R.W.

1. Virol., 83(3), 1492-1500 (2009)

  Respiratory syncytial virus (RSV) is a common cause of infection that is associated with a range of respiratory illnesses, from common cold-like symptoms to serious lower respiratory tract illnesses such as pneumonia and bronchiolitis. RSV is the single most important cause of serious lower respiratory tract illness in children <1 year of age. Host innate and acquired immune responses activated following RSV infection have been suspected to contribute to RSV disease. Toll-like receptors (TLRs) activate innate and acquired immunity and are candidates for playing key roles in the host immune response to RSV. Leukocytes express TLRs, including TLR2, TLR6, TLR3, TLR4, and TLR7, that can interact with RSV and promote immune responses following infection. Using knockout mice, we have demonstrated that TLR2 and TLR6 signaling in leukocytes can activate innate immunity against RSV by promoting tumor necrosis factor alpha, interleukin-6, CCL2 (monocyte chemoattractant protein 1), and CCL5 (RANTES). As previously noted, TLR4 also contributes to cytokine activation (L. M. Haynes, D. D. Moore, E. A. Kurt-Jones, R. W. Finberg, L. J. Anderson, and R. A. Tripp, J. Virol. 75:10730-10737, 2001, and E. A. Kurt-Jones, L. Popova, L. Kwinn, L. M. Haynes, L. P. Jones, R. A. Tripp, E. E. Walsh, M. W. Freeman, D. T. Golenbock, L. J. Anderson, and R. W. Finberg, Nat. Immunol. 1:398-401, 2000). Furthermore, we demonstrated that signals generated following TLR2 and TLR6 activation were important for controlling viral replication in vivo. Additionally, TLR2 interactions with RSV promoted neutrophil migration and dendritic cell activation within the lung. Collectively, these studies indicate that TLR2 is involved in RSV recognition and subsequent innate immune activation.  

5.606           Vaccination with HPV16 L2E6E7 fusion protein in GPI-0100 adjuvant elicits protective humoral and cell-mediated immunity

Karanam, B., Gambhira, R., Peng, S., Jagu, S., Kim, D-J., Ketner, G.W., Stern, P.L., Adams, R.J. and Roden, R.B.S. Vaccine, 27, 1040-1049 (2009)   A vaccine comprising human papillomavirus type 16 (HPV16) L2, E6 and E7 in a single tandem fusion protein (termed TA-CIN) has the potential advantages of both broad cross-protection against HPV transmission through induction of L2 antibodies able to cross neutralize different HPV types and of therapy by stimulating T cell responses targeting HPV16 early proteins. However, patients vaccinated with TA-CIN alone develop weak HPV neutralizing antibody and E6/E7-specific T cell responses. Here we test TA-CIN formulated along with the adjuvant GPI-0100, a semi-synthetic quillaja saponin analog that was developed to promote both humoral and cellular immune responses. Subcutaneous administration to mice of TA-CIN (20 μg) with 50 μg GPI-0100, three times at biweekly intervals, elicited high titer HPV16 neutralizing serum antibody, robust neutralizing titers for other HPV16-related types, including HPV31 and HPV58, and neutralized to a lesser extent other genital mucosatropic papillomaviruses like HPV18, HPV45, HPV6 and HPV11. Notably, vaccination with TA-CIN in GPI-0100 protected mice from cutaneous HPV16 challenge as effectively as HPV16 L1 VLP without adjuvant. Formulation of TA-CIN with GPI-0100 enhanced the production of E7-specific, interferon γ producing CD8⁺ T cell precursors by 20-fold. Vaccination with TA-CIN in GPI-0100 also completely prevented tumor growth after challenge with 5 × 10⁴ HPV16-transformed TC-1 tumor cells, whereas vaccination with TA-CIN alone delayed tumor growth. Furthermore, three monthly vaccinations with 125 μg of TA-CIN and 1000 μg GPI-0100 were well tolerated by pigtail macaques and induced both HPV16 E6/E7-specific T cell responses and serum antibodies that neutralized all HPV types tested.  

5.607           Heparin binding induces conformational changes in Adeno-associated virus serotype 2

LEVY, h.c., Bowman, V.D., Govindasamy, L., McKenna, R., Nash, K., Warrington, K., Chen, W., Muzyczka, N., Yan, X., Baker, T.S. and Agbandje-McKenna, M.

1. Struct. Biol., 165, 145-156 (2009)

  Adeno-associated virus serotype 2 (AAV2) uses heparan sulfate proteoglycan as a cell surface-attachment receptor. In this study the structures of AAV2 alone and complexed with heparin were determined to 18 Å resolution using cryo-electron microscopy and three-dimensional image reconstruction. A difference map showed positive density, modeled as heparin, close to the icosahedral twofold axes and between the protrusions that surround the threefold axes of the capsid. Regions of the model near the threefold place the receptor in close proximity to basic residues previously identified as part of the heparin binding site. The region of the model near the twofold axes identifies a second contact site, not previously characterized but which is also possibly configured by heparin binding. The difference map also revealed two significant conformational changes: (I) at the tops of the threefold protrusions, which have become flattened in the complex, and (II) at the fivefold axes where the top of the channel is widened possibly in response to movement of the HI loops in the capsid proteins. Ordered density in the interior of the capsid in the AAV2–heparin complex was interpreted as nucleic acid, consistent with the presence of non-viral DNA in the expressed capsids.  

5.608           A Novel Method for Targeted Gene Therapy in Ischemic Tissues through Viral Transfection of an Expression Cassette Containing Multiple Repetitions of Hypoxia Response Element

Cross, K.J., Bomsztyk, E.D., Weinstein, A.L., Teo, E.H., Spector, J.A. and Lyden, D.C. Plastic & Reconstrt. Surg., 123, Suppl, 76S-82S (2009)   Background: Increased levels of the transcription factor hypoxia inducible factor (HIF)-1 occur only in hypoxic tissue. The authors propose a therapeutic strategy that relies on HIF-1, the enhancer hypoxia response element (HRE), and the delivery vector adeno-associated virus-2 (AAV2) to direct ischemia specific gene therapy to skin. Methods: An expression cassette containing the CMV promoter driving the reporter gene green fluorescent protein (GFP) was used to assess cutaneous tropism of AAV2. Transfection of dermal fibroblasts and immortalized keratinocytes (HaCat) was assessed with flow cytometry. Human embryonic kidney 293 (HEK) cells were used to produce vector stocks and test the authors' therapeutic strategy in quadruplicate. An expression cassette with nine repeats of HRE linked to β-galactosidase (LacZ) within the AAV2 vector was constructed. HEK cells were transfected and exposed to normoxic (21% oxygen) and hypoxic (1% oxygen) conditions. LacZ activity was measured by conversion of galactoside red-β-D-galactopyranoside. Results: Approximately 50 percent of dermal fibroblasts and HaCat cells were transfected when treated with 1 × 10⁴ genome copies/cell of AAV2-CMV-GFP. Using the same titration of AAV2-9HRE-LacZ, transfected HEK cells demonstrated LacZ activity of 0.496 ± 0.068 U/μg in normoxia and 2.9 ± 0.58 U/μg in hypoxia. Transfected cells exposed to 24 hours of hypoxia show greater than an 11-fold increase in LacZ activity (p < 0.05) compared with baseline normoxic controls. Conclusions: The authors' results confirm that AAV2 has in vitro tropism for skin-derived cell lines. Furthermore, HRE will drive gene expression in ischemia but not normoxia. This is the first step toward the authors' goal of HIF-1-regulated gene therapy to prevent ischemia related skin injury.  

5.609           Secretion of Hepatitis C Virus Envelope Glycoproteins Depends on Assembly of Apolipoprotein B Positive Lipoproteins

Icard, V., Diaz, O., Scholtes, C., Perrin-Cocon, L., Ramiere, C., Bartenschlager, R., Penin, F., Lotteau, V. and Andre, P. PloSOne, 4(1), e4233 (2009)   The density of circulating hepatitis C virus (HCV) particles in the blood of chronically infected patients is very heterogeneous. The very low density of some particles has been attributed to an association of the virus with apolipoprotein B (apoB) positive and triglyceride rich lipoproteins (TRL) likely resulting in hybrid lipoproteins known as lipo-viro-particles (LVP) containing the viral envelope glycoproteins E1 and E2, capsid and viral RNA. The specific infectivity of these particles has been shown to be higher than the infectivity of particles of higher density. The nature of the association of HCV particles with lipoproteins remains elusive and the role of apolipoproteins in the synthesis and assembly of the viral particles is unknown. The human intestinal Caco-2 cell line differentiates in vitro into polarized and apoB secreting cells during asymmetric culture on porous filters. By using this cell culture system, cells stably expressing E1 and E2 secreted the glycoproteins into the basal culture medium after one week of differentiation concomitantly with TRL secretion. Secreted glycoproteins were only detected in apoB containing density fractions. The E1–E2 and apoB containing particles were unique complexes bearing the envelope glycoproteins at their surface since apoB could be co-immunoprecipitated with E2-specific antibodies. Envelope protein secretion was reduced by inhibiting the lipidation of apoB with an inhibitor of the microsomal triglyceride transfer protein. HCV glycoproteins were similarly secreted in association with TRL from the human liver cell line HepG2 but not by Huh-7 and Huh-7.5 hepatoma cells that proved deficient for lipoprotein assembly. These data indicate that HCV envelope glycoproteins have the intrinsic capacity to utilize apoB synthesis and lipoprotein assembly machinery even in the absence of the other HCV proteins. A model for LVP assembly is proposed.  

5.610           Recombinant Adeno-Associated Virus-Based Gene Transfer of Cathelicidin Induces Therapeutic Neovascularization Preferentially via Potent Collateral Growth

Pinkenburg, O., Pfosser, A., Hinkel, R., Böttcher, M., Dingees, C., Lebherz, C., Sultana, S., Enssle, J., El-Aouni, C., Büning, H., Boekstegers, P., Bals, R. and Kupatt, C. Human Gene Therapy, 20, 159-167 (2009)   Therapeutic neovascularization is a concept well validated in animal models, however, without clear-cut success in clinical studies. To achieve prolonged transgene expression, recombinant adeno-associated virus (rAAV) was used in a chronic ischemic hind-limb model and the human antimicrobial peptide cathelicidin (LL-37/hCAP-18) was used as proangiogenic factor. Seven days after femoral artery excision, 0.5 × 10¹¹ rAAV particles encoding for green fluorescent protein (rAAV.GFP), cathelicidin (rAAV.cath), or vascular endothelial growth factor A (rAAV.VEGF-A) were retroinfused into the anterior tibial vein of rabbits (n = 5 per group). In addition, one rAAV.cath-treated group obtained a constant infusion with the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin into the ischemic tissue starting on day 7. On day 7 and day 35 angiography of both hind limbs was performed for collateral quantification and frame count score (cinedensitometry). Capillary-to-muscle fiber ratios were obtained on day 35. Compared with controls, application of rAAV.cath induced a gain of perfusion (153 ± 12 vs. 107 + 9% of day 7 controls) via increased collateral growth (length index, 161 ± 14 vs. 97 ± 9%, controls), but no significant capillary growth (1.16 ± 0.09 vs. 0.99 ± 0.08, controls). Wortmannin application completely abolished the effects of rAAV.cath, indicating the involvement of the PI3K signal pathway. In conclusion, rAAV-mediated cathelicidin expression is capable of inducing functionally relevant neovascularization, preferentially by collateral growth. The rAAV-based vectors as long-expressing vector expression systems and cathelicidin as proangiogenic factor provide a promising new combination in the treatment of peripheral artery disease.  

5.611           Role of Heparan Sulfate in Attachment to and Infection of the Murine Female Genital Tract by Human Papillomavirus

Johnson, K.M., Kines, R.C., Roberts, J.N., Lowy, D.R., Schiller, J.T. and Day, P.M.

1. Virol., 83(5), 2067-2074 (2009)

  The host factors required for in vivo infection have not been investigated for any papillomavirus. Using a recently developed murine cervicovaginal challenge model, we evaluated the importance of heparan sulfate proteoglycans (HSPGs) in human papillomavirus (HPV) infection of the murine female genital tract. We examined HPV type 16 (HPV16) as well as HPV31 and HPV5, for which some evidence suggests that they may differ from HPV16 in their utilization of HSPGs as their primary attachment factor in vitro. Luciferase-expressing pseudovirus of all three types infected the mouse genital tract, although HPV5, which normally infects nongenital epidermis, was less efficient. Heparinase III treatment of the genital tract significantly inhibited infection of all three types by greater than 90% and clearly inhibited virion attachment to the basement membrane and cell surfaces, establishing that HSPGs are the primary attachment factors for these three viruses in vivo. However, the pseudoviruses differed in their responses to treatment with various forms of heparin, a soluble analog of heparan sulfate. HPV16 and HPV31 infections were effectively inhibited by a highly sulfated form of heparin, but HPV5 was not, although it bound the compound. In contrast, a N-desulfated and N-acylated variant preferentially inhibited HPV5. Inhibition of infection paralleled the relative ability of the variants to inhibit basement membrane and cell surface binding. We speculate that cutaneous HPVs, such as HPV5, and genital mucosal HPVs, such as HPV16 and -31, may have evolved to recognize different forms of HSPGs to enable them to preferentially infect keratinocytes at different anatomical sites.  

5.612           Reevaluating the CD8 T-Cell Response to Herpes Simplex Virus Type 1: Involvement of CD8 T Cells Reactive to Subdominant Epitopes

 Sheridan, B.S., Cherpes, T.L., Urban, J., Kalinski, P. and Hendricks, P.L.

1. Virol., 83(5), 2237-2245 (2009)

  In C57BL/6 (B6) mice, most herpes simplex virus (HSV)-specific CD8 T cells recognize a strongly immunodominant epitope on glycoprotein B (gB₄₉₈) and can inhibit HSV type 1 (HSV-1) reactivation from latency in trigeminal ganglia (TG). However, half of the CD8 T cells retained in latently infected TG of B6 mice are not gB₄₉₈ specific and have been largely ignored. The following observations from our current study indicate that these gB₄₉₈-nonspecific CD8 T cells are HSV specific and may contribute to the control of HSV-1 latency. First, following corneal infection, OVA₂₅₇-specific OT-1 CD8 T cells do not infiltrate the infected TG unless mice are simultaneously immunized with OVA₂₅₇ peptide, and then they are not retained. Second, 30% of CD8 T cells in acutely infected TG that produce gamma interferon in response to HSV-1 stimulation directly ex vivo are gB₄₉₈ nonspecific, and these cells maintain an activation phenotype during viral latency. Finally, gB₄₉₈-nonspecific CD8 T cells are expanded in ex vivo cultures of latently infected TG and inhibit HSV-1 reactivation from latency in the absence of gB₄₉₈-specific CD8 T cells. We conclude that many of the CD8 T cells that infiltrate and are retained in infected TG are HSV specific and potentially contribute to maintenance of HSV-1 latency. Identification of the viral proteins recognized by these cells will contribute to a better understanding of the dynamics of HSV-1 latency.  

5.613           Enhancement of Adeno-Associated Virus Infection by Mobilizing Capsids into and Out of the Nucleolus

Johnson, J.S. and Samulski, R.J.

1. Virol., 83(6), 2632-2644 (2009)

  Adeno-associated virus (AAV) serotypes are being tailored for numerous therapeutic applications, but the parameters governing the subcellular fate of even the most highly characterized serotype, AAV2, remain unclear. To understand how cellular conditions control capsid trafficking, we have tracked the subcellular fate of recombinant AAV2 (rAAV2) vectors using confocal immunofluorescence, three-dimensional infection analysis, and subcellular fractionation. Here we report that a population of rAAV2 virions enters the nucleus and accumulates in the nucleolus after infection, whereas empty capsids are excluded from nuclear entry. Remarkably, after subcellular fractionation, virions accumulating in nucleoli were found to retain infectivity in secondary infections. Proteasome inhibitors known to enhance transduction were found to potentiate nucleolar accumulation. In contrast, hydroxyurea, which also increases transduction, mobilized virions into the nucleoplasm, suggesting that two separate pathways influence vector delivery in the nucleus. Using a small interfering RNA (siRNA) approach, we then evaluated whether nucleolar proteins B23/nucleophosmin and nucleolin, previously shown to interact with AAV2 capsids, affect trafficking and transduction efficiency. Similar to effects observed with proteasome inhibition, siRNA-mediated knockdown of nucleophosmin potentiated nucleolar accumulation and increased transduction 5- to 15-fold. Parallel to effects from hydroxyurea, knockdown of nucleolin mobilized capsids to the nucleoplasm and increased transduction 10- to 30-fold. Moreover, affecting both pathways simultaneously using drug and siRNA combinations was synergistic and increased transduction over 50-fold. Taken together, these results support the hypothesis that rAAV2 virions enter the nucleus intact and can be sequestered in the nucleolus in stable form. Mobilization from the nucleolus to nucleoplasmic sites likely permits uncoating and subsequent gene expression or genome degradation. In summary, with these studies we have refined our understanding of AAV2 trafficking dynamics and have identified cellular parameters that mobilize virions in the nucleus and significantly influence AAV infection.  

5.614           Gag p27-Specific B- and T-Cell Responses in Simian Immunodeficiency Virus SIVagm-Infected African Green Monkeys

Lozana Reina, J-M., Favre, D., Kasakow, Z., Mayau, V., Nugeyre, M-T., Ka, T., Faye, A., Miller, C.J., Scott-Algara, D., McCune, J.M., Barre-Sinoussi, F., Diop, O.M. and Müller-Trutwin, M.C.

1. Virol., 83(6), 2770-2777 (2009)

  Nonpathogenic simian immunodeficiency virus SIVagm infection of African green monkeys (AGMs) is characterized by the absence of a robust antibody response against Gag p27. To determine if this is accompanied by a selective loss of T-cell responses to Gag p27, we studied CD4⁺ and CD8⁺ T-cell responses against Gag p27 and other SIVagm antigens in the peripheral blood and lymph nodes of acutely and chronically infected AGMs. Our data show that AGMs can mount a T-cell response against Gag p27, indicating that the absence of anti-p27 antibodies is not due to the absence of Gag p27-specific T cells.  

5.615           Establishment of Human Papillomavirus Infection Requires Cell Cycle Progression

Pyeon, D., Pearce, S.M., Lank, S.M., Ahlquist, P. and Lambert, P.F. PloSPathogens, 5(2), e100318 (2009)   Human papillomaviruses (HPVs) are DNA viruses associated with major human cancers. As such there is a strong interest in developing new means, such as vaccines and microbicides, to prevent HPV infections. Developing the latter requires a better understanding of the infectious life cycle of HPVs. The HPV infectious life cycle is closely linked to the differentiation state of the stratified epithelium it infects, with progeny virus only made in the terminally differentiating suprabasal compartment. It has long been recognized that HPV must first establish its infection within the basal layer of stratified epithelium, but why this is the case has not been understood. In part this restriction might reflect specificity of expression of entry receptors. However, this hypothesis could not fully explain the differentiation restriction of HPV infection, since many cell types can be infected with HPVs in monolayer cell culture. Here, we used chemical biology approaches to reveal that cell cycle progression through mitosis is critical for HPV infection. Using infectious HPV16 particles containing the intact viral genome, G1-synchronized human keratinocytes as hosts, and early viral gene expression as a readout for infection, we learned that the recipient cell must enter M phase (mitosis) for HPV infection to take place. Late M phase inhibitors had no effect on infection, whereas G1, S, G2, and early M phase cell cycle inhibitors efficiently prevented infection. We conclude that host cells need to pass through early prophase for successful onset of transcription of the HPV encapsidated genes. These findings provide one reason why HPVs initially establish infections in the basal compartment of stratified epithelia. Only this compartment of the epithelium contains cells progressing through the cell cycle, and therefore it is only in these cells that HPVs can establish their infection. By defining a major condition for cell susceptibility to HPV infection, these results also have potentially important implications for HPV control.        

5.616           High-efficiency Transduction of the Mouse Retina by Tyrosine-mutant AAV Serotype Vectors

Petrs-Silva, H., Dinulescu, A., Li, Q., Min, S-H., Chiodo, V., Pang, J-J., Zhong, L., Zolotukhin, S., Srivastava, A., Lewin, A.S. and Hauswirth, W.W. Molecular Therapy, 17(3), 463-471(2009)   Vectors derived from adeno-associated viruses (AAVs) have become important gene delivery tools for the treatment of many inherited ocular diseases in well-characterized animal models. Previous studies have determined that the viral capsid plays an essential role in the cellular tropism and efficiency of transgene expression. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV2 capsid targets the viral particles for ubiquitination and proteasome- mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo. Because the tyrosine residues are highly conserved in other AAV serotypes, in this study we evaluated the intraocular transduction characteristics of vectors containing point mutations in surface- exposed capsid tyrosine residues in AAV serotypes 2, 8, and 9. Several of these novel AAV mutants were found to display a strong and widespread transgene expression in many retinal cells after subretinal or intravitreal delivery compared with their wild-type counterparts. For the first time, we show efficient transduction of the ganglion cell layer by AAV serotype 8 or 9 mutant vectors, thus providing additional tools besides AAV2 for targeting these cells. These enhanced AAV vectors have a great potential for future therapeutic applications for retinal degenerations and ocular neovascular diseases.  

5.617           Directed evolution of adeno-associated virus to an infectious respiratory virus

Excoffon, K.J.D.A., Koerber, J.T., Dickey, D.D., Murtha, M., Keshavjee, S., Kaspar, B.K., Zabner, J. and Schaffer, D.V. PNAS, 106(10), 3865-3870 (2009)   Respiratory viruses evolve to maintain infectivity levels that permit spread yet prevent host and virus extinction, resulting in surprisingly low infection rates. Respiratory viruses harnessed as gene therapy vectors have illustrated this limitation. We used directed evolution in an organotypic human airway model to generate a highly infectious adeno-associated virus. This virus mediated gene transfer more than 100-fold better than parental strains and corrected the cystic fibrosis epithelial Cl⁻ transport defect. Thus, under appropriate selective pressures, viruses can evolve to be more infectious than observed in nature, a finding that holds significant implications for designing vectors for gene therapy and for understanding emerging pathogens.  

5.618           PET imaging in rats to discern temporal onset differences between 6-hydroxydopamine and tau gene vector neurodegeneration models

Klein, R.L., Dayton, R.D., Terry, T.L., Vascoe, C., Sunderland, J.J. and Tainter, K.H. Brain Res., 1259, 113-122 (2009)   We attempted to monitor the nigrostriatal dopaminergic system in rats with positron emission tomography (PET) during the progression of two experimental disease states. One model was 6-hydroxydopamine (6-OHDA) lesioning and the other was direct gene transfer of the microtubule-associated protein tau to the substantia nigra using an adeno-associated virus vector (AAV9). The PET ligand was 6-[18F]fluoro-l-m-tyrosine (FMT), imaged prior to, and at two intervals after initiating dopaminergic neurodegeneration. The striatum was delineated with the aid of repeated PET imaging (FMT and sodium fluoride for bone), realignment to subsequent computed axial tomography scans, and registration to an atlas, which proved essential to tracking disease progression. The striata on the two sides of the brain were compared over time after unilateral lesioning treatments. 6-OHDA reduced uptake on the ipsilateral side relative to the untreated contralateral side at both 1 and 4 weeks after lesioning, while the AAV9 tau led to reduced uptake of the tracer in the striatum at 4 weeks, but not 1 week after treatment. The amplitude of the loss of FMT uptake in striatum at 4 weeks with either model was subtle relative to the postmortem histological analysis of the tissue, but the multi-modal imaging analysis yielded statistical effects that matched well with the histology in terms of the timing of the loss of dopaminergic markers. Live longitudinal imaging successfully tracked two distinct types of disease progression in individual rats, although the FMT is not a sensitive ligand to monitor the extent of the lesion.  

5.619           Neuroprotective Effects of Inositol 1,4,5-Trisphosphate Receptor C-Terminal Fragment in a Huntington's Disease Mouse Model

Tang, T-S., Guo, C., Chen, X. and Bezprovanny, I.

1. Neurosci., 29(5), 1257-1266 (2009)

  Huntington's disease (HD) is a dominantly inherited, progressive neurodegenerative disease caused by an expanded polyglutamine tract in huntingtin protein (Htt). Medium spiny striatal neurons (MSNs) are primarily affected in HD. Mutant huntingtin protein (Htt^exp) specifically binds to and activates type 1 inositol 1,4,5-trisphosphate receptor (InsP₃R1), an intracellular Ca²⁺ release channel. Htt^exp–InsP₃R1 association is mediated by a cytosolic C-terminal tail of InsP₃R1 (a 122-aa-long IC10 fragment). To evaluate an importance of Htt^exp association with InsP₃R1 for HD pathology, we generated lentiviral and adeno-associated viruses expressing GFP-IC10 fusion protein and performed a series of experiments with YAC128 HD transgenic mouse. Infection with Lenti-GFP-IC10 virus stabilized Ca²⁺ signaling in cultured YAC128 MSNs and protected YAC128 MSNs from glutamate-induced apoptosis. Intrastriatal injections of AAV1-GFP-IC10 significantly alleviated motor deficits and reduced MSN loss and shrinkage in YAC128 mice. Our results demonstrate an importance of InsP₃R1–Htt^exp association for HD pathogenesis and suggested that InsP₃R1 is a potential therapeutic target for HD. Our data also support potential use of IC10 peptide as a novel HD therapeutic agent.  

5.620           Positron Emission Tomography Imaging Demonstrates Correlation between Behavioral Recovery and Correction of Dopamine Neurotransmission after Gene Therapy

Leriche, L., Björklund, T., Breysse, N., Besret, L., Gregoire, M-C., Carlsson, T., Dolle, F., Mandel, R.J., Deglon, N., Hantraye, P. and Kirik, D.

1. Neurosci., 29(5), 1544-1553 (2009)

  In vivo gene transfer using viral vectors is an emerging therapy for neurodegenerative diseases with a clinical impact recently demonstrated in Parkinson's disease patients. Recombinant adeno-associated viral (rAAV) vectors, in particular, provide an excellent tool for long-term expression of therapeutic genes in the brain. Here we used the [¹¹C]raclopride [(S)-(–)-3,5-dichloro-N-((1-ethyl-2-pyrrolidinyl)methyl)-2-hydroxy-6-methoxybenzamide] micro-positron emission tomography (PET) technique to demonstrate that delivery of the tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1) enzymes using an rAAV5 vector normalizes the increased [¹¹C]raclopride binding in hemiparkinsonian rats. Importantly, we show in vivo by microPET imaging and postmortem by classical binding assays performed in the very same animals that the changes in [¹¹C]raclopride after viral vector-based enzyme replacement therapy is attributable to a decrease in the affinity of the tracer binding to the D₂ receptors, providing evidence for reconstitution of a functional pool of endogenous dopamine in the striatum. Moreover, the extent of the normalization in this non-invasive imaging measure was highly correlated with the functional recovery in motor behavior. The PET imaging protocol used in this study is fully adaptable to humans and thus can serve as an in vivo imaging technique to follow TH + GCH1 gene therapy in PD patients and provide an additional objective measure to a potential clinical trial using rAAV vectors to deliver L-3,4-dihydroxyphenylanaline in the brain.  

5.621           Functional Complementation of Glra1spd-ot, a Glycine Receptor Subunit Mutant, by Independently Expressed C-Terminal Domains

Villman, C., Oertel, J., Ma-Högemeier, Z-L., Hollmann, M., Sprengel, R., Becker, K., Breitinger, H-G. and Becker, C-M.

1. Neurosci., 29(8), 2440-2452 (2009)

  The oscillator mouse (Glra1^spd-ot) carries a 9 bp microdeletion plus a 2 bp microinsertion in the glycine receptor 1 subunit gene, resulting in the absence of functional 1 polypeptides from the CNS and lethality 3 weeks after birth. Depending on differential use of two splice acceptor sites in exon 9 of the Glra1 gene, the mutant allele encodes either a truncated 1 subunit (spd^ot-trc) or a polypeptide with a C-terminal missense sequence (spd^ot-elg). During recombinant expression, both splice variants fail to form ion channels. In complementation studies, a tail construct, encoding the deleted C-terminal sequence, was coexpressed with both mutants. Coexpression with spd^ot-trc produced glycine-gated ion channels. Rescue efficiency was increased by inclusion of the wild-type motif RRKRRH. In cultured spinal cord neurons from oscillator homozygotes, viral infection with recombinant C-terminal tail constructs resulted in appearance of endogenous 1 antigen. The functional rescue of 1 mutants by the C-terminal tail polypeptides argues for a modular subunit architecture of members of the Cys-loop receptor family.  

5.622           Long-Term Cardiac-Targeted RNA Interference for the Treatment of Heart Failure Restores Cardiac Function and Reduces Pathological Hypertrophy

Suckau, L. et al Circulation, 119, 1241-1252 (2009)   Background— RNA interference (RNAi) has the potential to be a novel therapeutic strategy in diverse areas of medicine. Here, we report on targeted RNAi for the treatment of heart failure, an important disorder in humans that results from multiple causes. Successful treatment of heart failure is demonstrated in a rat model of transaortic banding by RNAi targeting of phospholamban, a key regulator of cardiac Ca²⁺ homeostasis. Whereas gene therapyrests on recombinant protein expression as its basic principle,RNAi therapy uses regulatory RNAs to achieve its effect. Methods and Results— We describe structural requirements to obtain high RNAi activity from adenoviral and adeno-associated virus (AAV9) vectors and show that an adenoviral short hairpin RNA vector (AdV-shRNA) silenced phospholamban in cardiomyocytes (primary neonatal rat cardiomyocytes) and improved hemodynamics in heart-failure rats 1 month after aortic root injection. For simplified long-term therapy, we developed a dimeric cardiotropic adeno-associated virus vector (rAAV9-shPLB) to deliver RNAi activity to the heart via intravenous injection. Cardiac phospholamban protein was reduced to 25%, and suppression of sacroplasmic reticulum Ca²⁺ ATPase in the HF groups was rescued. In contrast to traditional vectors, rAAV9 showed high affinity for myocardium but low affinity for liver and other organs. rAAV9-shPLB therapy restored diastolic (left ventricular end-diastolic pressure, dp/dt_min, and ) and systolic (fractional shortening) functionalparameters to normal ranges. The massive cardiac dilation wasnormalized, and cardiac hypertrophy, cardiomyocyte diameter,and cardiac fibrosis were reduced significantly. Importantly,no evidence was found of microRNA deregulation or hepatotoxicityduring these RNAi therapies.  

5.623           Improvement of Gemcitabine-Based Therapy of Pancreatic Carcinoma by Means of Oncolytic Parvovirus H-1PV

Angelova, A.L., Aprahamian, M., Grekova, S.P., Hajri, A., Leuchs, B., Giese, N.A., Dinsart, C., Hermann, A., Balboni, G., Rommelaere, J. and Raykov, Z. Clin. Cancer Res., 15(2), 511-519 (2009)   Pancreatic carcinoma is a gastrointestinal malignancy with poorprognosis. Treatment with gemcitabine, the most potent chemotherapeuticagainst this cancer up to date, is not curative, and resistancemay appear. Complementary treatment with an oncolytic virus,such as the rat parvovirus H-1PV, which is infectious but nonpathogenicin humans, emerges as an innovative option. Purpose: To prove that combining gemcitabine and H-1PV in amodel of pancreatic carcinoma may reduce the dosage of the toxicdrug and/or improve the overall anticancer effect. Experimental Design: Pancreatic tumors were implanted orthotopically in Lewis rats or subcutaneously in nude mice and treated with gemcitabine, H-1PV, or both according to different regimens. Tumor size was monitored by micro-computed tomography, whereas bone marrow, liver, and kidney functions were monitored by measuring clinically relevant markers. Human pancreatic cell lines and gemcitabine-resistant derivatives were tested in vitro for sensitivityto H-1PV infection with or without gemcitabine. Results: In vitro studies proved that combining gemcitabinewith H-1PV resulted in synergistic cytotoxic effects and achievedan up to 15-fold reduction in the 50% effective concentrationof the drug, with drug-resistant cells remaining sensitive tovirus killing. Toxicologic screening showed that H-1PV had anexcellent safety profile when applied alone or in combinationwith gemcitabine. The benefits of applying H-1PV as a second-linetreatment after gemcitabine included reduction of tumor growth,prolonged survival of the animals, and absence of metastaseson CT-scans. Conclusion: In addition to their potential use as monotherapyfor pancreatic cancer, parvoviruses can be best combined withgemcitabine in a two-step protocol.  

5.624           Striatal Readministration of rAAV Vectors Reveals an Immune Response Against AAV2 Capsids That Can Be Circumvented

Peden, C.S., Manfredsson, F.P., Reimsnider, S.K., Poirier, A.E., Burger, C., Muzyczka, N. and Mandel, R.J. Mol. Therapy, 17(3), 524-537 (2009)   Recombinant adeno-associated virus (rAAV) expresses no viral genes after transduction. In addition, because the brain is relatively immunoprivileged, intracranial rAAV transduction may be immunologically benign due to a lack of antigen presentation. However, preexposure to AAV allows neutralizing antibodies (nAbs) to block brain transduction and rAAV readministration in the brain leads to an inflammatory response in the second-injection site. In this study, we replicate our striatal rAAV2/2-GDNF readministration results and extend this effect to a second transgene, green fluorescent protein (GFP). Unlike rAAV2/2-GDNF readministration, striatal rAAV2/2-GFP readministration leads to a loss of transgene in the second site in the absence of detectable circulating nAbs. In order to determine whether the transgene or the AAV2 capsid is the antigenic stimulus in brain for the immune response in the second site, we readministered rAAV2/2-GFP using two different rAAV serotypes (rAAV2/2 followed by rAAV2/5). In this case, there was no striatal inflammation or transgene loss detected in the second-injection site. In addition, striatal readministration of rAAV2/5-GFP also resulted in no detectable immune response. Furthermore, delaying rAAV2/2 striatal readministration to a 11-week interval abrogated the immune response in the second-injection site. Finally, while striatal readministration of rAAV2/2 leads to significant loss of transgene in the second-injection site, this effect is not due to loss of vector genomes as determined by quantitative real-time PCR. We conclude that intracellular processing of AAV capsids after transduction is the immunogenic antigen and capsid serotypes that are processed more quickly than rAAV2/2 are less immunogenic.  

5.625           An efficient rHSV-based complementation system for the production of multiple rAAV vector serotypes

Kang, W., Wang, L., Harrrell, H., Liu, J., Thonmas, D.L., Mayfield, T.L., Scotti,, M.M., Ye, G.J., veres, G. and Knop, D.R. Gene Therapy, 16, 229-239 (2009)   Recombinant herpes simplex virus type 1 (rHSV)-assisted recombinant adeno-associated virus (rAAV) vector production provides a highly efficient and scalable method for manufacture of clinical grade rAAV vectors. Here, we present an rHSV co-infection system for rAAV production, which uses two ICP27-deficient rHSV constructs, one bearing the rep2 and cap (1, 2 or 9) genes of rAAV, and the second bearing an AAV2 ITR-gene of interest (GOI) cassette. The optimum rAAV production parameters were defined by producing rAAV2/GFP in HEK293 cells, yielding greater than 9000 infectious particles per cell with a 14:1 DNase resistance particle to infectious particle (DRP/ip) ratio. The optimized co-infection parameters were then used to generate large-scale stocks of rAAV1/AAT, which encode the human -1-antitrypsin (hAAT) protein, and purified by column chromatography. The purified vector was extensively characterized by rAAV- and rHSV-specific assays and compared to transfection-made vector for in vivo efficacy in mice through intramuscular injection. The co-infection method was also used to produce rAAV9/AAT for comparison to rAAV1/AAT in vivo. Intramuscular administration of 1 10¹¹ DRP per animal of rHSV-produced rAAV1/AAT and rAAV9/AAT resulted in hAAT protein expression of 5.4 10⁴ and 9.4 10⁵ ng ml^-1 serum respectively, the latter being clinically relevant.  

5.626           TNF- and the IFN-_γ-inducible protein 10 (IP-10/CXCL-10) delivered by parvoviral vectors act in synergy to induce antitumor effects in mouse glioblastoma

Enderlin, M., Kleinmann, E.V., Struyf, S., Buracchi, C., Vecchi, A., Kinscherf, R., Kiessling, F., Paschek, S., Sozzani, S., Rommelaere, J., Cornelis, J.J., Van Damme, J. and Dinsart, C. Cancer Gene Therapy, 16, 149-160 (2009)   Interferon- -inducible protein 10 is a potent chemoattractant for natural killer cells and activated T lymphocytes. It also displays angiostatic properties and some antitumor activity. Tumor necrosis factor- (TNF- ) is a powerful immunomodulating cytokine with demonstrated tumoricidal activity in various tumor models and the ability to induce strong immune responses. This prompted us to evaluate the antitumor effects of recombinant parvoviruses designed to deliver IP-10 or TNF- into a glioblastoma. When Gl261 murine glioma cells were infected in vitro with an IP-10- or TNF- -transducing parvoviral vector and were subcutaneously implanted in mice, tumor growth was significantly delayed. Complete tumor regression was observed when the glioma cells were coinfected with both the vectors, demonstrating synergistic antitumor activity. In an established in vivo glioma model, however, repeated simultaneous peritumoral injection of the IP-10- and TNF- -delivering parvoviruses failed to improve the therapeutic effect as compared with the use of a single cytokine-delivering vector. In this tumor model, cytokine-mediated immunostimulation, rather than inhibition of vascularization, is likely responsible for the therapeutic efficacy.      

5.627           Effects of UCH-L1 on [alpha]-synuclein over-expression mouse model of Parkinson's disease

Yasuda, T., Nihira, T., Ren, Y-R., Cao, X-Q., Wada, K., Setsuie, R., Kabuta, T., Wada, K., Hattori, N., Mizuno, Y. and Mochizuki, H.

1. Neurochem., 108, 932-944 (2009)

  The rare inherited form of Parkinson's disease (PD), PARK5, is caused by a missense mutation in ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) gene, resulting in Ile93Met substitution in its gene product (UCH-L1^Ile93Met). PARK5 is inherited in an autosomal-dominant mode, but whether the Ile93Met mutation gives rise to a gain-of-toxic-function or loss-of-function of UCH-L1 protein remains controversial. Here, we investigated the selective vulnerabilities of dopaminergic (DA) neurons in UCH-L1-transgenic (Tg) and spontaneous UCH-L1-null gracile axonal dystrophy mice to an important PD-causing insult, abnormal accumulation of α-synuclein (αSyn). Immunohistochemistry of midbrain sections of a patient with sporadic PD showed αSyn- and UCH-L1-double-positive Lewy bodies in nigral DA neurons, suggesting physical and/or functional interaction between the two proteins in human PD brain. Recombinant adeno-associated viral vector-mediated over-expression of αSyn for 4 weeks significantly enhanced the loss of nigral DA cell bodies in UCH-L1^Ile93Met-Tg mice, but had weak effects in age-matched UCH-L1^wild-type-Tg mice and non-Tg littermates. In contrast, the extent of αSyn-induced DA cell loss in gracile axonal dystrophy mice was not significantly different from wild-type littermates at 13-weeks post-injection. Our results support the hypothesis that PARK5 is caused by a gain-of-toxic-function of UCH-L1^Ile93Met mutant, and suggest that regulation of UCH-L1 in nigral DA cells could be a future target for treatment of PD.  

5.628           Overexpression of Galgt2 in skeletal muscle prevents injury resulting from eccentric contractions in both mdx and wild-type mice

Martin, P.T., Xu, R., Rodino-klapac, L.R., Oglesbay, E., Camboni, M., Montgomery, C.L., Shontz, K., Chicoine, L.G., Reed Clark, K., Sahenk, Z., Mendell, J.R. and Janssen, P.M.L. Am. J. Physiol. Cell Physiol., 296, C476-C488 (2009)   The cytotoxic T cell (CT) GalNAc transferase, or Galgt2, is a UDP-GalNAc:β1,4-N-acetylgalactosaminyltransferase that is localized to the neuromuscular synapse in adult skeletal muscle, where it creates the synaptic CT carbohydrate antigen {GalNAcβ1,4[NeuAc(orGc) 2, 3]Galβ1,4GlcNAcβ-}. Overexpression of Galgt2 in the skeletal muscles of transgenic mice inhibits the development of muscular dystrophy in mdx mice, a model for Duchenne muscular dystrophy. Here, we provide physiological evidence as to how Galgt2 may inhibit the development of muscle pathology in mdx animals. Both Galgt2 transgenic wild-type and mdx skeletal muscles showed a marked improvement in normalized isometric force during repetitive eccentric contractions relative to nontransgenic littermates, even using a paradigm where nontransgenic muscles had force reductions of 95% or more. Muscles from Galgt2 transgenic mice, however, showed a significant decrement in normalized specific force and in hindlimb and forelimb grip strength at some ages. Overexpression of Galgt2 in muscles of young adult mdx mice, where Galgt2 has no effect on muscle size, also caused a significant decrease in force drop during eccentric contractions and increased normalized specific force. A comparison of Galgt2 and microdystrophin overexpression using a therapeutically relevant intravascular gene delivery protocol showed Galgt2 was as effective as microdystrophin at preventing loss of force during eccentric contractions. These experiments provide a mechanism to explain why Galgt2 overexpression inhibits muscular dystrophy in mdx muscles. That overexpression also prevents loss of force in nondystrophic muscles suggests that Galgt2 is a therapeutic target with broad potential applications.  

5.629           Expression of hepatitis C virus (HCV) structural proteins in trans facilitates encapsidation and transmission of HCV subgenomic RNA

Adair, R., Patel, A.H., Corless, L., Griffin, S., Rowlands, D.J. and McCormick, J.

1. Gen. Virol., 90, 833-842 (2009)

  A characteristic of many positive-strand RNA viruses is that, whilst replication of the viral genome is dependent on the expression of the majority of non-structural proteins in cis, virus particle formation can occur when most or all of the structural proteins are co-expressed in trans. Making use of a recently identified hepatitis C virus (HCV) isolate (JFH1) that can be propagated in tissue culture, this study sought to establish whether this is also the case for hepaciviruses. Stable cell lines containing one of two bicistronic replicons derived from the JFH1 isolate were generated that expressed non-structural proteins NS3–5B or NS2–5B. Release and transmission of these replicons to naïve Huh7 cells could then be demonstrated when baculovirus transduction was used to express the HCV proteins absent from the subgenomic replicons. Transmission could be blocked by a neutralizing antibody targeted at the E2 envelope protein, consistent with this phenomenon occurring via trans-encapsidation of replicon RNA into virus-like particles. Transmission was also dependent on expression of NS2, which was most effective at promoting virus particle formation when expressed in cis on the replicon RNA compared with in trans via baculovirus delivery. Density gradient analysis of the particles revealed the presence of a broad infectious peak between 1.06 and 1.11 g ml^–1, comparable to that seen when propagating full-length virus in tissue culture. In summary, the trans-encapsidation system described offers a complementary and safer approach to study HCV particle formation and transmission in tissue culture.  

5.630           Rectal and vaginal immunization of mice with human papillomavirus L1 virus-like particles

Fraillery, D., Zosso, N. and Nardelli-Haefliger, D. Vaccine, 27, 2326-2334 (2009)   Human papillomavirus (HPV) vaccines based on L1 virus-like particle (VLP) can prevent genital HPV infection and associated lesions after three intramuscular injections. Needle-free administration might facilitate vaccine implementation, especially in developing countries. Here we have investigated rectal and vaginal administration of HPV16 L1 VLPs in mice and their ability to induce anti-VLP and HPV16-neutralizing antibodies in serum and in genital, rectal and oral secretions. Rectal and vaginal immunizations were not effective in the absence of adjuvant. Cholera toxin was able to enhance systemic and mucosal anti-VLPs responses after rectal immunization, but not after vaginal immunization. Rectal immunization with Resiquimod and to a lesser extent Imiquimod, but not monophosphoryl lipid A, induced anti-HPV16 VLP antibodies in serum and secretions. Vaginal immunization was immunogenic only if administered in mice treated with nonoxynol-9, a disrupter of the cervico-vaginal epithelium. Our findings show that rectal and vaginal administration of VLPs can induce significant HPV16-neutralizing antibody levels in secretions, despite the fact that low titers are induced in serum. Imidazoquinolines, largely used to treat genital and anal warts, and nonoxonol-9, used as genital microbicide/spermicide were identified as adjuvants that could be safely used by the rectal or vaginal route, respectively.  

5.631           Indexing TNF-α gene expression using a gene-targeted reporter cell line

Yan, Z., Lei-Butters, D., Engelhardt, J.F. and Leno, G.H. BMC Biol., 7, 8-17 (2009)   Background Current cell-based drug screening technologies utilize randomly integrated reporter genes to index transcriptional activity of an endogenous gene of interest. In this context, reporter expression is controlled by known genetic elements that may only partially capture gene regulation and by unknown features of chromatin specific to the integration site. As an alternative technology, we applied highly efficient gene-targeting with recombinant adeno-associated virus to precisely integrate a luciferase reporter gene into exon 1 of the HeLa cell tumor necrosis factor-alpha (TNF-α) gene. Drugs known to induce TNF-α expression were then used to compare the authenticity of gene-targeted and randomly integrated transcriptional reporters. Results TNF-α-targeted reporter activity reflected endogenous TNF-α mRNA expression, whereas randomly integrated TNF-α reporter lines gave variable expression in response to transcriptional and epigenetic regulators. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), currently used in cancer clinical trials to induce TNF-α gene transcription, was only effective at inducing reporter expression from TNF-α gene-targeted cells. Conclusion We conclude that gene-targeted reporter cell lines provide predictive indexing of gene transcription for drug discovery.  

5.632           Successful Expansion but Not Complete Restriction of Tropism of Adeno-Associated Virus by In Vivo Biopanning of Random Virus Display Peptide Libraries

Michelfelder, S., Kohlschütter, J., Skorupa, A., Phennings, S., Müller,O., Kleinschmidt, J.A. and Trepel, M. PloSOne, 4(4), e5122 (2009)   Targeting viral vectors to certain tissues in vivo has been a major challenge in gene therapy. Cell type-directed vector capsids can be selected from random peptide libraries displayed on viral capsids in vitro but so far this system could not easily be translated to in vivo applications. Using a novel, PCR-based amplification protocol for peptide libraries displayed on adeno-associated virus (AAV), we selected vectors for optimized transduction of primary tumor cells in vitro. However, these vectors were not suitable for transduction of the same target cells under in vivo conditions. We therefore performed selections of AAV peptide libraries in vivo in living animals after intravenous administration using tumor and lung tissue as prototype targets. Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues. The selected clones indeed conferred gene expression in the target tissue while gene expression was undetectable in animals injected with control vectors. However, all of the vectors selected for tumor transduction also transduced heart tissue and the vectors selected for lung transduction also transduced a number of other tissues, particularly and invariably the heart. This suggests that modification of the heparin binding motif by target-binding peptide insertion is necessary but not sufficient to achieve tissue-specific transgene expression. While the approach presented here does not yield vectors whose expression is confined to one target tissue, it is a useful tool for in vivo tissue transduction when expression in tissues other than the primary target is uncritical.  

5.633           Preliminary evaluation of a self-complementary AAV2/8 vector for hepatic gene transfer of human apoE3 to inhibit atherosclerotic lesion development in apoE-deficient mice

Osman, E., Evans, V., Graham, I.R., Athanasopoulos, T., McIntosh, Nathwani, A.C., Simons, J.P., Dickson, G. and Owen, J.S. Atherosclerosis, 204, 121-126 (2009)   Hepatic gene transfer of atheroprotective human apoE by recombinant viral vectors can reverse hypercholesterolaemia and inhibit atherogenesis in apoE-deficient (apoE^−/−) mice. Here, in preliminary studies we assess the effectiveness of a recently developed self-complementary adeno-associated virus (scAAV) serotype 8 vector, driven by a hepatocyte-specific promoter (LP1), for liver-directed gene delivery of human apoE3. Vector viability was validated by transducing cultured HepG2 cells and measuring secretion of apoE3 protein. Male and female apoE^−/− mice, 6-month old and fed on normal chow, were intravenously injected with 1 × 10¹¹ vg (vector genomes) of scAAV2/8.LP1.apoE3; age-matched untreated mice served as controls. In male mice, plasma apoE3 levels were sufficiently high (up to 17 μg/ml) to normalize plasma total cholesterol and ameliorate their proatherogenic lipoprotein profile, by reducing VLDL/LDL and increasing HDL 5-fold. At termination (12 weeks) development of aortic atherosclerosis was significantly retarded by 58% (aortic lesion area 8.2 ± 1.4% vs. 19.3 ± 2.4% in control males; P < 0.001). Qualitatively similar anti-atherogenic effects were noted when female mice were treated, but the benefits were less marked and aortic lesions, for example, were reduced by only 33% (15.7 ± 3.7% vs. 23.6 ± 6.9%). Although group numbers were small (n = 4/5), this gender-specific difference reflected two to three times less apoE3 in plasma of female mice at weeks 3 and 6, implying that gene transfer to female liver using scAAV vectors may require additional optimization, despite their established superior potency to conventional single-stranded (ssAAV) vectors.  

5.634           Calsyntenins Mediate TGN Exit of APP in a Kinesin-1-Dependent Manner

Ludwig, A., Blume, J., Diep, T-M., Yuan, J., Mateos, J.M., Leuthäuser, K., Steuble, M., Streit, P. and Sonderegger, P. Traffic, 10, 572-589 (2009)   Kinesin motors are required for the export of membranous cargo from the trans-Golgi network (TGN), yet information about how kinesins are recruited to forming transport intermediates is sparse. Here we show that the Kinesin-1 docking protein calsyntenin-1 localizes to the TGN in vivo and directly and specifically recruits Kinesin-1 to Golgi/TGN membranes as well as to dynamic post-Golgi carriers. Overexpression of various calsyntenin chimeras and kinesin light chain 1 (KLC1) at high levels caused the formation of aberrant membrane stacks at the endoplasmic reticulum (ER) or the Golgi, disrupted overall Golgi structure and blocked exit of calsyntenin from the TGN. Intriguingly, this blockade of calsyntenin exit strongly and selectively impeded TGN exit of amyloid precursor protein (APP). Using live cell microscopy we found that calsyntenins exit the TGN in Kinesin-1-decorated tubular structures which may serve as carriers for calsyntenin-1-mediated post-TGN transport of APP. Abrogation of this pathway via virus-mediated knockdown of calsyntenin-1 expression in primary cultured neurons caused a marked elevation of APP C-terminal fragments. Together, these results indicate a role for calsyntenin-1 in Kinesin-1-dependent TGN exit and post-Golgi transport of APP-containing organelles and further suggest that distinct intracellular routes may exhibit different capacities for proteolytic processing of APP.  

5.635           Comparison of transduction efficiency of recombinant AAV serotypes 1, 2, 5, and 8 in the rat nigrostriatal system

McFarland, N., Lee, J-S., Hyman, B.T. and McLean, P.J.

1. Neurochem., 109, 838-845 (2009)

  Enhanced delivery and expression of genes in specific neuronal systems is critical for the development of genetic models of neurodegenerative disease and potential gene therapy. Recent discovery of new recombinant adeno-associated viral (rAAV) capsid serotypes has resulted in improved transduction efficiency, but expression levels, spread of transgene, and potential toxicity can differ depending on brain region and among species. We compared the transduction efficiency of titer-matched rAAV 2/1, 2/5, and 2/8 to the commonly used rAAV2/2 in the rat nigrostriatal system via expression of the reporter transgene, enhanced green fluorescent protein. Newer rAAV serotypes 2/1, 2/5, and 2/8 demonstrated marked increase in transduction and spread of enhanced green fluorescent protein expression in dopaminergic nigrostriatal neurons and projections to the striatum and globus pallidus compared to rAAV2/2 at 2 weeks post-injection. The number of nigral cells transduced was greatest for rAAV2/1, but for serotypes 2/5 and 2/8 was still two- to threefold higher than that for 2/2. Enhanced transduction did not cause an increase in glial cell response or toxicity. New rAAV serotypes thus promise improved gene delivery to nigrostriatal system with the potential for better models and therapeutics for Parkinson disease and other neurodegenerative disorders.  

5.636           Mimicking Aspects of Frontotemporal Lobar Degeneration and Lou Gehrig's Disease in Rats via TDP-43 Overexpression

Tatom, J.B., Wang, D.B., Dayton, R.D., Skalli, O., Hutton, M.L., Dickson, D.W. and Klein, R.L. Molecular Therapy, 17(4), 607-613 (2009)   Since the discovery of neuropathological lesions made of TDP-43 and ubiquitin proteins in cases of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), there is a burst of effort on finding related familial mutations and developing animal models. We used an adeno-associated virus (AAV) vector for human TDP-43 expression targeted to the substantia nigra (SN) of rats. Though TDP-43 was expressed mainly in neuronal nuclei as expected, it was also expressed in the cytoplasm, and dotted along the plasma membrane of neurons. Cytoplasmic staining was both diffuse and granular, indicative of preinclusion lesions, over 4 weeks. Ubiquitin deposited in the cytoplasm, specifically in the TDP-43 group, and staining for microglia was increased dose-dependently by 1–2 logs in the TDP-43 group, while neurons were selectively obliterated. Neuronal death induced by TDP-43 was pyknotic and apoptotic. TDP-43 gene transfer caused loss of dopaminergic neurons in the SN and their axons in the striatum. Behavioral motor dysfunction resulted after TDP-43 gene transfer that was vector dose-dependent and progressive over time. The cytoplasmic expression, ubiquitination, and neurodegeneration mimicked features of the TDP-43 diseases, and the gliosis, apoptosis, and motor impairment may also be relevant to TDP-43 disease forms involving nigrostriatal degeneration.  

5.637           Effect of CNTF on Retinal Ganglion Cell Survival in Experimental Glaucoma

Pease, M.E., Zack, D.J., Berlinicke, C., Bloom, K., Cone, F., Wang, Y., Klein, R.L., Hauswirth, W.W. and Quigley, H.A. Invest. Ophthalmol. Vis. Sci., 50(5), 2194-2200 (2009)   PURPOSE. To assess the neuroprotective effect of virally mediatedoverexpression of ciliary-derived neurotrophic factor (CNTF)and brain-derived neurotrophic factor (BDNF) in experimentalrat glaucoma. METHODS. Laser-induced glaucoma was produced in one eye of 224Wistar rats after injection of adenoassociated viral vectors(type 2) containing either CNTF, BDNF, or both, with saline-injectedeyes and noninjected glaucomatous eyes serving as the control.IOP was measured with a hand-held tonometer, and semiautomatedoptic nerve axon counts were performed by masked observers.IOP exposure over time was adjusted in multivariate regressionanalysis to calculate the effect of CNTF and BDNF. RESULTS. By multivariate regression, CNTF had a significant protective effect, with 15% less RGC axon death (P < 0.01).Both combined CNTF-BDNF and BDNF overexpression alone had nostatistically significant improvement in RGC axon survival.By Western blot, there was a quantitative increase in CNTF andBDNF expression in retinas exposed to single viral vectors carryingeach gene, but no increase with sequential injection of bothvectors. CONCLUSIONS. These data confirm that CNTF can exert a protectiveeffect in experimental glaucoma. The reason for the lack ofobserved effect in the BDNF overexpression groups is unclear,but it may be a function of the level of neurotrophin expressionachieved.  

5.638           Localized delivery of fibroblast growth factor–2 and brain-derived neurotrophic factor reduces spontaneous seizures in an epilepsy model

Paradiso, B. et al PNAS, 106(17), 7191-7196 (2009)   A loss of neurons is observed in the hippocampus of many patients with epilepsies of temporal lobe origin. It has been hypothesized that damage limitation or repair, for example using neurotrophic factors (NTFs), may prevent the transformation of a normal tissue into epileptic (epileptogenesis). Here, we used viral vectors to locally supplement two NTFs, fibroblast growth factor–2 (FGF-2) and brain-derived neurotrophic factor (BDNF), when epileptogenic damage was already in place. These vectors were first characterized in vitro, where they increased proliferation of neural progenitors and favored their differentiation into neurons, and they were then tested in a model of status epilepticus-induced neurodegeneration and epileptogenesis. When injected in a lesioned hippocampus, FGF-2/BDNF expressing vectors increased neuronogenesis, embanked neuronal damage, and reduced epileptogenesis. It is concluded that reduction of damage reduces epileptogenesis and that supplementing specific NTFs in lesion areas represents a new approach to the therapy of neuronal damage and of its consequences.  

5.639           Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injection

Hellström, M., Ruitenberg, M.J., Pollett, M.A., Ehlert, E.M.E., Twisk, J., Verhaagen, J. and Harvey, A.R. Gene Therapy, 16, 521-532 (2009)   Recombinant adeno-associated virus (rAAV) vectors are increasingly being used as tools for gene therapy, and clinical trials have begun in patients with genetically linked retinal disorders. Intravitreal injection is optimal for the transduction of retinal ganglion cells (RGCs), although complete selectivity has not been achieved. There may also be advantages in using intravitreal approaches for the transduction of photoreceptors. Here we compared the cellular tropism and transduction efficiency of rAAV2/1, -2/2, -2/3, -2/4, -2/5, -2/6 and -2/8 in adult rat retina after intravitreal injection. Each vector encoded green fluorescent protein (GFP), and the number, laminar distribution and morphology of transduced GFP⁺ cells were determined using fluorescent microscopy. Assessment of transduced cell phenotype was based on cell morphology and immunohistochemistry. rAAV2/2 and rAAV2/6 transduced the greatest number of cells, whereas rAAV2/5 and rAAV2/8 were least efficient. Most vectors primarily transduced RGCs; however, rAAV2/6 had a more diverse tropism profile, with 46% identified as amacrine or bipolar cells, 23% as RGCs and 22% as Müller cells. Müller cells were also frequently transduced by rAAV2/4. The highest photoreceptor transduction was seen after intravitreal rAAV2/3 injection. These data facilitate the design and selection of rAAV vectors to target specific retinal cells, potentially leading to an improved gene therapy for various human retinal pathologies.  

5.640           Incorporation of membrane-bound, mammalian-derived immunomodulatory proteins into influenza whole virus vaccines boosts immunogenicity and protection against lethal challenge

Herbert, A., Heffron, L., Sundick, R. and Roberts, P.C. Virology J., 6, 42-58 (2009)   Background: Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1), demonstrates the need for a much improved, more sophisticated influenza vaccine. We have developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC). Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Results: We have evaluated the immunogenicity of inactivated cytokine-bearing influenza vaccines using a mouse model of lethal influenza virus challenge. CYT-IVACs were produced by stably transfecting MDCK cell lines with mouse-derived cytokines (GM-CSF, IL-2 and IL-4) fused to the membrane-anchoring domain of the viral hemagglutinin. Influenza virus replication in these cell lines resulted in the uptake of the bioactive membrane-bound cytokines during virus budding and release. In vivo efficacy studies revealed that a single low dose of IL-2 or IL-4-bearing CYT-IVAC is superior at providing protection against lethal influenza challenge in a mouse model and provides a more balanced Th1/Th2 humoral immune response, similar to live virus infections. Conclusion: We have validated the protective efficacy of CYT-IVACs in a mammalian model of influenza virus infection. This technology has broad applications in current influenza virus vaccine development and may prove particularly useful in boosting immune responses in the elderly, where current vaccines are minimally effective.    

5.641           DDX3 DEAD-Box RNA Helicase Inhibits Hepatitis B Virus Reverse Transcription by Incorporation into Nucleocapsids

Wang, H., Kim, S. and Ryu, W-S.

1. Virol., 83(11), 5815-5824 (2009)

  Viruses utilize host factors in many steps of their life cycles. Yet, little is known about host factors that contribute to the life cycle of hepatitis B virus (HBV), which replicates its genome by reverse transcription. To identify host factors that contribute to viral reverse transcription, we sought to identify cellular proteins that interact with HBV polymerase (Pol) by using affinity purification coupled with mass spectrometry. One of the HBV Pol-interacting host factors identified was DDX3 DEAD-box RNA helicase, which unwinds RNA in an ATPase-dependent manner. Recently, it was shown that DDX3 is essential for both human immunodeficiency virus and hepatitis C virus infection. In contrast, we found that the ectopic expression of DDX3 led to significantly reduced viral DNA synthesis. The DDX3-mediated inhibition of viral DNA synthesis did not affect RNA encapsidation, a step prior to reverse transcription, and indicated that DDX3 inhibits HBV reverse transcription. Mutational analysis revealed that mutant DDX3 with an inactive ATPase motif, but not that with an inactive RNA helicase motif, failed to inhibit viral DNA synthesis. Our interpretation is that DDX3 inhibits viral DNA synthesis at a step following ATP hydrolysis but prior to RNA unwinding. Finally, OptiPrep density gradient analysis revealed that DDX3 was incorporated into nucleocapsids, suggesting that DDX3 inhibits viral reverse transcription following nucleocapsid assembly. Thus, DDX3 represents a novel host restriction factor that limits HBV infection.  

5.642           Site-specific integration of adeno-associated virus involves partial duplication of the target locus

Henckaerts, E., Dutheil, N., Zeltner, N., Kattman, S., Kohlbrenner, E., Ward, P., Clement, N., Rebollo, P., Kennedy, M., Keller, G.M. and Linden, R.M. PNAS, 106(18), 7571-7576 (2009)   A variety of viruses establish latency by integrating their genome into the host genome. The integration event generally occurs in a nonspecific manner, precluding the prediction of functional consequences from resulting disruptions of affected host genes. The nonpathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene. To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved despite the resulting rearrangements. A latently infected, Mbs85-targeted mouse ES cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multilineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a viral strategy for targeted genome addition with the apparent absence of functional consequences.  

5.643           A novel sorting strategy of trichosanthin for hijacking human immunodeficiency virus type 1

Zhao, W-L., Zhang, F., Feng, D., Shan, J.W., Chen, S. and Sui, S-F. Biochem. Biophys. Res. Comm.,384, 347-351 (2009)   Trichosanthin (TCS) is a type I ribosome-inactivating protein that plays dual role of plant toxin and anti-viral peptide. The sorting mechanism of such an exogenous protein is in long pursuit. Here, we examined TCS trafficking in cells expressing the HIV-1 scaffold protein Gag, and we found that TCS preferentially targets the Gag budding sites at plasma membrane or late endosomes depending on cell types. Lipid raft membrane but not the Gag protein mediates the association of TCS with viral components. After Gag budding, TCS is then released in association with the virus-like particles to generate TCS-enriched virions. The resulting TCS-enriched HIV-1 exhibits severely impaired infectivity. Overall, the observations indicate the existence of a unique and elaborate sorting strategy for hijacking HIV-1.  

5.644           Efficient gene transfer to periodontal ligament cells and human gingival fibroblasts by adeno-associated virus vectors

Kunze, M., Huber, A., Krajewski, A., Lowden, E., Schhuhmann, N., Buening, H., Hallek, M., Noack, M. and Perabo, L.

1. Dentistry, 37, 502-508 (2009)

  Objectives We explored for the first time the possibility to deliver a reporter gene (Green Fluorescence Protein) to human primary periodontal ligament (PDL) cells and human gingival fibroblasts (HGF) using shuttle vectors derived from adeno-associated virus (AAV). Since AAV transduction rates on other human primary fibroblasts have been previously shown to depend on the particular cell lineage and on the employed viral serotype, we determined the most effective AAV variant for periodontal cells comparing different vector types. Methods AAV serotypes 1–5 encoding GFP in single stranded (ss) and self-complementary (sc) vector genome conformations were used to infect primary HGF and PDL cells. Two days post-infection, the percentage of GFP expressing cells was determined by flow cytometry. Results Highest transduction rates for both cell types were achieved with self-complementary vectors derived from AAV-2, resulting in GFP expression in up to 86% of PDL cells and 50% of HGF. Transgene expression could be observed by optical microscopy for 2 months after infection. Lower but detectable rates were obtained with serotypes 1, 3 and 5. Conclusions The efficacy demonstrated here and the safety and versatility of AAV technology indicated in previous studies clearly suggest the potential of AAV vectors as tools for gene transfer to periodontal tissues.  

5.645           Preferential labeling of inhibitory and excitatory cortical neurons by endogenous tropism of adeno-associated virus and lentivirus vectors

Nathanson, J.L., Yanagawa, Y., Obata, K. and Callaway, E.M. Neurosci., 161, 441-450 (2009)   Despite increasingly widespread use of recombinant adeno-associated virus (AAV) and lentiviral (LV) vectors for transduction of neurons in a wide range of brain structures and species, the diversity of cell types within a given brain structure is rarely considered. For example, the ability of a vector to transduce neurons within a brain structure is often assumed to indicate that all neuron types within the structure are transduced. We have characterized the transduction of mouse somatosensory cortical neuron types by recombinant AAV pseudotyped with serotype 1 capsid (rAAV2/1) and by recombinant lentivirus pseudotyped with the vesicular stomatitis virus (VSV) glycoprotein. Both vectors used human synapsin (hSyn) promoter driving DsRed-Express. We demonstrate that high titer rAAV2/1-hSyn efficiently transduces both cortical excitatory and inhibitory neuronal populations, but use of lower titers exposes a strong preference for transduction of cortical inhibitory neurons and layer 5 pyramidal neurons. In contrast, we find that VSV-G-LV-hSyn principally labels excitatory cortical neurons at the highest viral titer generated. These findings demonstrate that endogenous tropism of rAAV2/1 and VSV-G-LV can be used to obtain preferential gene expression in mouse somatosensory cortical inhibitory and excitatory neuron populations, respectively.  

5.646           CHARACTERIZATION OF HEPATITIS C VIRUS PARTICLES IN HUMAN PLASMA: ASSOCIATION WITH IMMUNOGLOBULINS G1, G3 & M AND APOLIPOPROTEINS A-I, A-II, B, C-I AND E

Nielsen, s., Sheridan, D., Bridge, S., Felmlee, D., Neely, D., Toms, G. and Bassendine, M.

1. Hepatol., 50, Suppl. 1, S317-S317 (2009)

  Background and Aims: HCV circulating in blood is heterogeneous in density and size with virus of low density, associated with host apolipoproteins, being implicated as the infectious form. Our aim was to explore the influence of the host immune response to HCV on the density, size and biochemical composition of virus particles from patients with chronic infection. Patients and Methods: We compared circulating HCV genotype 1 in blood from three immunocompetent and one immunodeficient patient. Such characterization of HCV has hitherto been hindered by the low titre of HCV in plasma, but we used 500 ml venesected blood from immunocompetent patients with coexistent genetic haemochromatosis, yielding 108 IU of HCV. Our methods are density analysis by iodixanol gradient centrifugation, size analysis by Superose gel filtration and biochemical analysis by immunoprecipitation and SDS PAGE. Results: 82% of HCV in plasma from the immunodeficient patient had density <1.06 g/ml, compared to 1.0% in immunocompetent patients, in whom 74% of viral RNA had density between 1.08 and 1.12 g/ml. Gel filtration showed that all HCV with density <1.12 g/ml co-purified with very low density lipoprotein (VLDL), with a cholesterol, triglyceride and phospholipid composition similar to large VLDL. This VLDL fraction contained large amounts of IgG1, IgG3 and IgM, which were not found on VLDL purified from controls without HCV. In the immunodeficient patient viral RNA immunoprecipitated with antibodies to apolipoprotein (apo) B, C-I and E, normally found on VLDL, as well as with anti-apoAI and antiapoAII, normally found on high density lipoproteins. In immunocompetent patients with chronic HCV, virus particles were associated with antibodies and immunoprecipitated with protein G (which binds all subclasses of IgG). In contrast immunoprecipitation with anti-apoB was poor, suggesting epitopes on HCV/VLDL complexes were masked by antibodies. Conclusion: This analysis show that HCV circulates in association with host VLDL and that HCV/VLDL complexes in chronic infection are associated with large amounts of IgG1, IgG3 and IgM of unknown specificity. These antibodies which may react with virus or host proteins, increase the density of HCV/VLDL complexes and their specificity is of relevance in vaccine development.  

5.647           EVALUATION OF AAV-MEDIATED IFNa-GENE THERAPY EFFICACY IN HBV TRANSGENIC MICE: CONSTITUTIVE VERSUS INDUCIBLE EXPRESSION

Vanrell, L., Olague, C., Vales, A., Paneda, A., Tenembaum, L. and Gonzales-Aseguinolaza, G.

1. Hepatol., 50, Suppl. 1,S214-S215 (2009)

  Background: Hepatitis B virus (HBV) and hepatitis C virus (HCV) constitute the main etiologic factors for the chronic viral hepatitis affecting more than 550 million people worldwide. Interferon alpha (IFNa) has demonstrated therapeutic efficacy in both chronic hepatitis B and C. However, the monotherapy response rate (25−35%) can be increased, and side effects limited, by improving the pharmacokinetic of IFNalpha therapy with stabilising ligands. Gene therapy allows a continuous in vivo expression of the transgene at the desired site. AAV vectors lack pathogenicity in humans and have demonstrated prolonged expression of numerous transgenes, in several tissues and immunocompetent animal models. Aims: Therapeutic efficacy evaluation of a recombinant AAV expressing murine IFN alpha (AAVIFN) under the control of a constitutive or an inducible promoter in HBV transgenic mice. Methods: We have produced AAV vectors expressing luciferase or murine IFN-alpha1 into two different expression cassettes (AAVIFN). The first one contains the promoter for the human Elongation Factor-1 alpha (hEF1alpfa), the transgene, and the SV40 polyadenilation signal sequence. The second one contains a CMV-TetON promoter, the transgene, and the SV40 polyadenilation signal. AAV serotype 8 virus production was performed by cotransfection into 293 T cells of the plasmid containing the expression cassette flanked by the AAV ITRs, and the plasmids containing the necessary AAV and adenoviral proteins, using linear polyethylenimine (25 kDa). Viruses were purified by iodixanol centrifugation gradient and viral titers were determined by real time PCR. IFN-alpha concentration was determined by the cytopathic effect (CPE) reduction assay. Serum viral load was determined by qPCR after viral DNA purification from serum samples. Results: AAV constitutively expressing murine IFN-alpha was able to inhibit HBV viral replication to undetectable levels in HBV transgenic mice, while the administration of AAV expressing luciferase had no effect over viral replication. However, prolonged IFN-alpha expression resulted in severe side effects as shown by a profound leukopenia that led to animals death. Experiments are now being performed to determine the antiviral efficacy and safety of the AAV vector expressing IFN-alpha in an inducible manner. Conclusion: This report highlights the promises and the limitations of IFNa-gene therapy in viral hepatitis.  

5.648           Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors

Craig, A.T. et al. Virology J., 6, 61-70 (2009)   Background Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. Results We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgfβ1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. Conclusion AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation.    

5.649           trans-Complementation of an NS2 Defect in a Late Step in Hepatitis C Virus (HCV) Particle Assembly and Maturation

Yi, M., Ma, Y., Yates, J. and Lemon, S.M. PLOSPathogens, 5(5), e1000403 (2009)   Recent studies using cell culture infection systems that recapitulate the entire life cycle of hepatitis C virus (HCV) indicate that several nonstructural viral proteins, including NS2, NS3, and NS5A, are involved in the process of viral assembly and release. Other recent work suggests that Ser-168 of NS2 is a target of CK2 kinase–mediated phosphorylation, and that this controls the stability of the genotype 1a NS2 protein. Here, we show that Ser-168 is a critical determinant in the production of infectious virus particles. Substitution of Ser-168 with Ala (or Gly) ablated production of infectious virus by cells transfected with a chimeric viral RNA (HJ3-5) containing core-NS2 sequences from the genotype 1a H77 virus within the background of genotype 2a JFH1 virus. An S168A substitution also impaired production of virus by cells transfected with JFH1 RNA. This mutation did not alter polyprotein processing or genome replication. This defect in virus production could be rescued by expression of wt NS2 in trans from an alphavirus replicon. The trans-complementing activities of NS2 from genotypes 1a and 2a demonstrated strong preferences for rescue of the homologous genotype. Importantly, the S168A mutation did not alter the association of core or NS5A proteins with host cell lipid droplets, nor prevent the assembly of core into particles with sedimentation and buoyant density properties similar to infectious virus, indicating that NS2 acts subsequent to the involvement of core, NS5A, and NS3 in particle assembly. Second-site mutations in NS2 as well as in NS5A can rescue the defect in virus production imposed by the S168G mutation. In aggregate, these results indicate that NS2 functions in trans, in a late-post assembly maturation step, perhaps in concert with NS5A, to confer infectivity to the HCV particle.  

5.650           A Therapeutic Antibody against West Nile Virus Neutralizes Infection by Blocking Fusion within Endosomes

Thompson, B.S., Moesker, B., Smit, J.M., Wilschut, J., Diamond, M.S. and Fremont, D.H. PLOSPathogens, 5(5), e1000453 (2009)   Defining the precise cellular mechanisms of neutralization by potently inhibitory antibodies is important for understanding how the immune system successfully limits viral infections. We recently described a potently inhibitory monoclonal antibody (MAb E16) against the envelope (E) protein of West Nile virus (WNV) that neutralizes infection even after virus has spread to the central nervous system. Herein, we define its mechanism of inhibition. E16 blocks infection primarily at a post-attachment step as antibody-opsonized WNV enters permissive cells but cannot escape from endocytic compartments. These cellular experiments suggest that E16 blocks the acid-catalyzed fusion step that is required for nucleocapsid entry into the cytoplasm. Indeed, E16 directly inhibits fusion of WNV with liposomes. Additionally, low-pH exposure of E16–WNV complexes in the absence of target membranes did not fully inactivate infectious virus, further suggesting that E16 prevents a structural transition required for fusion. Thus, a strongly neutralizing anti–WNV MAb with therapeutic potential is potently inhibitory because it blocks viral fusion and thereby promotes clearance by delivering virus to the lysosome for destruction.  

5.651           Adaptive Mutations in the JC Virus Protein Capsid Are Associated with Progressive Multifocal Leukoencephalopathy (PML)

Sunyaev, S., Lugovskoy, A., Simon, K. and Gorelik, L. PLOSGenetics, 5(2), e1000368 (2009)   PML is a progressive and mostly fatal demyelinating disease caused by JC virus infection and destruction of infected oligodendrocytes in multiple brain foci of susceptible individuals. While JC virus is highly prevalent in the human population, PML is a rare disease that exclusively afflicts only a small percentage of immunocompromised individuals including those affected by HIV (AIDS) or immunosuppressive drugs. Viral- and/or host-specific factors, and not simply immune status, must be at play to account for the very large discrepancy between viral prevalence and low disease incidence. Here, we show that several amino acids on the surface of the JC virus capsid protein VP1 display accelerated evolution in viral sequences isolated from PML patients but not in sequences isolated from healthy subjects. We provide strong evidence that at least some of these mutations are involved in binding of sialic acid, a known receptor for the JC virus. Using statistical methods of molecular evolution, we performed a comprehensive analysis of JC virus VP1 sequences isolated from 55 PML patients and 253 sequences isolated from the urine of healthy individuals and found that a subset of amino acids found exclusively among PML VP1 sequences is acquired via adaptive evolution. By modeling of the 3-D structure of the JC virus capsid, we showed that these residues are located within the sialic acid binding site, a JC virus receptor for cell infection. Finally, we go on to demonstrate the involvement of some of these sites in receptor binding by demonstrating a profound reduction in hemagglutination properties of viral-like particles made of the VP1 protein carrying these mutations. Collectively, these results suggest that a more virulent PML causing phenotype of JC virus is acquired via adaptive evolution that changes viral specificity for its cellular receptor(s).  

5.652           Human Monoclonal Antibodies against West Nile Virus Induced by Natural Infection Neutralize at a Postattachment Step

Vogt, M.R., Moesker, B., Goudsmit, J., Jongeneelen, M., Austin, S.K., Oliphant, T., Nelson, S., Pierson, T.C., Wilschut, J., Throsby, M. and Diamond, M.S.

1. Virol., 83(13), 6494-6507 (2009)

  West Nile virus (WNV) is a neurotropic flavivirus that is now a primary cause of epidemic encephalitis in North America. Studies of mice have demonstrated that the humoral immune response against WNV limits primary infection and protects against a secondary challenge. The most-potent neutralizing mouse monoclonal antibodies (MAbs) recognize an epitope on the lateral ridge of domain III (DIII-lr) of the envelope (E) protein. However, studies with serum from human patients show that antibodies against the DIII-lr epitope comprise, at best, a minor component of the human anti-WNV antibody response. Herein, we characterize in detail two WNV-specific human MAbs, CR4348 and CR4354, that were isolated from B-cell populations of convalescent patients. These MAbs strongly neutralize WNV infection of cultured cells, protect mice against lethal infection in vivo, and yet poorly recognize recombinant forms of the E protein. Instead, CR4348 and CR4354 bind determinants on intact WNV virions and subviral particles in a pH-sensitive manner, and neutralization is altered by mutations at the dimer interface in domain II and the hinge between domains I and II, respectively. CR4348 and CR4354 human MAbs neutralize infection at a postattachment step in the viral life cycle, likely by inhibiting acid-induced fusion within the endosome.  

5.653           Tumultuous Relationship between the Human Immunodeficiency Virus Type 1 Viral Infectivity Factor (Vif) and the Human APOBEC-3G and APOBEC-3F Restriction Factors

Henriet, S., Mercenne, G., Bernacchi, S., Paillart, J-C. and Marquet, R. Microbiol. Mol. Biol. Reviews, 73(2), 211-232 (2009)   The viral infectivity factor (Vif) is dispensable for human immunodeficiency virus type 1 (HIV-1) replication in so-called permissive cells but is required for replication in nonpermissive cell lines and for pathogenesis. Virions produced in the absence of Vif have an aberrant morphology and an unstable core and are unable to complete reverse transcription. Recent studies demonstrated that human APOBEC-3G (hA3G) and APOBEC-3F (hA3F), which are selectively expressed in nonpermissive cells, possess strong anti-HIV-1 activity and are sufficient to confer a nonpermissive phenotype. Vif induces the degradation of hA3G and hA3F, suggesting that its main function is to counteract these cellular factors. Most studies focused on the hypermutation induced by the cytidine deaminase activity of hA3G and hA3F and on their Vif-induced degradation by the proteasome. However, recent studies suggested that several mechanisms are involved both in the antiviral activity of hA3G and hA3F and in the way Vif counteracts these antiviral factors. Attempts to reconcile the studies involving Vif in virus assembly and stability with these recent findings suggest that hA3G and hA3F partially exert their antiviral activity independently of their catalytic activity by destabilizing the viral core and the reverse transcription complex, possibly by interfering with the assembly and/or maturation of the viral particles. Vif could then counteract hA3G and hA3F by excluding them from the viral assembly intermediates through competition for the viral genomic RNA, by regulating the proteolytic processing of Pr55^Gag, by enhancing the efficiency of the reverse transcription process, and by inhibiting the enzymatic activities of hA3G and hA3F.  

5.654           [alpha]-Synuclein S129 Phosphorylation Mutants Do Not Alter Nigrostriatal Toxicity in a Rat Model of Parkinson Disease

McFarland, N.R., Fan, Z., Xu, K., Schwarzschild, M.A., Feany, M.B., Hyman, B.T. and McLean, P.J.

1. Neuropathol. Exp. Neurol., 68(5), 515-524 (2009)

  Lewy bodies are found in Parkinson disease and related disorders and are extensively phosphorylated at Ser-129 (S129), but whether S129 phosphorylation mediates [alpha]-synuclein aggregation and neurotoxicity has been controversial. We used recombinant adeno-associated virus to overexpress [alpha]-synuclein in the rat nigrostriatal system. Rats were injected with recombinant adeno-associated virus 2/8 expressing either human wild-type (wt) or mutant [alpha]-synuclein with S129 replaced by alanine (S129A) or aspartate (S129D). Contralateral substantia nigra injections containing empty vector served as controls. Both wt and S129 mutants resulted in significant dopaminergic cell loss in the recipients by 6 weeks, but there were only small decreases in nigrostriatal terminal density and tyrosine hydroxylase expression. There were no significant differences in dopaminergic cell loss, nigrostriatal terminal density, or tyrosine hydroxylase expression among the wt and S129 mutants. Furthermore, we did not observe any differences in [alpha]-synuclein aggregate formation or distribution among wt and either S129 mutant. These findings contrast with those from previous studies and suggest that injections of both S129 phosphorylation mutants result in dopaminergic neurotoxicity similar to wt injections. Further study is needed to clarify the effects of these S129 mutants and [alpha]-synuclein phosphorylation in mammalian systems.  

5.655           Replication and Assembly of Human Papillomaviruses

Conway, M.J. and Meyers, C.

1. Dental Res., 88(4), 307-317 (2009)

  Human papillomaviruses (HPVs) are small dsDNA tumor viruses, which are the etiologic agents of most cervical cancers and are associated with a growing percentage of oropharyngeal cancers. The HPV capsid is non-enveloped, having a T=7 icosahedral symmetry formed via the interaction among 72 pentamers of the major capsid protein, L1. The minor capsid protein L2 associates with L1 pentamers, although it is not known if each L1 pentamer contains a single L2 protein. The HPV life cycle strictly adheres to the host cell differentiation program, and as such, native HPV virions are only produced in vivo or in organotypic "raft" culture. Research producing synthetic papillomavirus particles—such as virus-like particles (VLPs), papillomavirus-based gene transfer vectors, known as pseudovirions (PsV), and papillomavirus genome-containing quasivirions (QV)—has bypassed the need for stratifyingand differentiating host tissue in viral assembly and has allowedfor the rapid analysis of HPV infectivity pathways, transmission,immunogenicity, and viral structure.  

5.656           Glycoengineered Acid -Glucosidase With Improved Efficacy at Correcting the Metabolic Aberrations and Motor Function Deficits in a Mouse Model of Pompe Disease

Zhu, Y., Jiang, J-L.,Gumlaw, N.K., Zhang, J., Bercury, S.D., Ziegler, R.J., Lee, K., Kudo, M., Canfield, W.M., Edmubds, T., Jiang, C., Mattaliano, R.J. and Cheng, S.H. Molecular Therapy, 17(6), 954-963 (2009)   Improving the delivery of therapeutics to disease-affected tissues can increase their efficacy and safety. Here, we show that chemical conjugation of a synthetic oligosaccharide harboring mannose 6-phosphate (M6P) residues onto recombinant human acid -glucosidase (rhGAA) via oxime chemistry significantly improved its affinity for the cation-independent mannose 6-phosphate receptor (CI-MPR) and subsequent uptake by muscle cells. Administration of the carbohydrate-remodeled enzyme (oxime-neo-rhGAA) into Pompe mice resulted in an approximately fivefold higher clearance of lysosomal glycogen in muscles when compared to the unmodified counterpart. Importantly, treatment of immunotolerized Pompe mice with oxime-neo-rhGAA translated to greater improvements in muscle function and strength. Treating older, symptomatic Pompe mice also reduced tissue glycogen levels but provided only modest improvements in motor function. Examination of the muscle pathology suggested that the poor response in the older animals might have been due to a reduced regenerative capacity of the skeletal muscles. These findings lend support to early therapeutic intervention with a targeted enzyme as important considerations in the management of Pompe disease.  

5.657           Nigrostriatal rAAV-mediated GDNF Overexpression Induces Robust Weight Loss in a Rat Model of Age-related Obesity

Manfredsson, F.P., Turner, N., erdos, B., Landa, T., Broxson, C.S., Sullivan, L.F., Rising, A.C., Foust, K.D., Zhang, Y., Muzycka, N., gorbatyuk, O.S., Scarpace, P.J. and Mandel, R.J. Molecular Therapy, 17(6), 980-991 (2009)   Intraventricular administration of glial cell line–derived neurotrophic factor (GDNF) in primate and humans to study Parkinson's disease (PD) has revealed the potential for GDNF to induce weight loss. Our previous data indicate that bilateral continuous hypothalamic GDNF overexpression via recombinant adeno-associated virus (rAAV) results in significant failure to gain weight in young rats and weight loss in aged rats. Based on these previous results, we hypothesized that because the nigrostriatal tract passes through the lateral hypothalamus, motor hyperactivity mediated by nigrostriatal dopamine (DA) may have been responsible for the previously observed effect on body weight. In this study, we compared bilateral injections of rAAV2/5-GDNF in hypothalamus versus substantia nigra (SN) in aged Brown-Norway X Fisher 344 rats. Nigrostriatal GDNF overexpression resulted in significantly greater weight loss than rats treated in hypothalamus. The nigral or hypothalamic GDNF-induced weight loss was unrelated to motor activity levels of the rats, though some of the weight loss could be attributed to a transient reduction in food intake. Forebrain DA levels did not account for the observed effects on body weight, although GDNF-induced increases in nucleus accumbens DA may have partially contributed to this effect in the hypothalamic GDNF-treated group. However, only nigrostriatal GDNF overexpression induced activation of phosphorylated extracellular signal-regulated kinase (p-ERK) in a small population of corticotrophin-releasing factor [corticotrophin-releasing hormone (CRH)] neurons located specifically in the medial parvocellullar division (MPD) of the paraventricular nucleus of the hypothalamus. Activation of these hypothalamic CRH neurons likely accounted for the observed metabolic effects leading to weight loss in obese rats.  

5.658           Low pH-dependent Hepatitis C Virus Membrane Fusion Depends on E2 Integrity, Target Lipid Composition, and Density of Virus Particles

Haid, S., Pietschmann, T. and Pecheur, E-I.

1. Biol. Chem., 284(26), 17657-17667 (2009)

  Hepatitis C virus (HCV) is an enveloped, positive strand RNA virus of about 9.6 kb. Like all enveloped viruses, the HCV membrane fuses with the host cell membrane during the entry process and thereby releases the genome into the cytoplasm, initiating the viral replication cycle. To investigate the features of HCV membrane fusion, we developed an in vitro fusion assay using cell culture-produced HCV and fluorescently labeled liposomes. With this model we could show that HCV-mediated fusion can be triggered in a receptor-independent but pH-dependent manner and that fusion of the HCV particles with liposomes is dependent on the viral dose and on the lipid composition of the target membranes. In addition CBH-5, an HCV E2-specific antibody, inhibited fusion in a dose-dependent manner. Interestingly, point mutations in E2, known to abrogate HCV glycoprotein-mediated fusion in a cell-based assay, altered or even abolished fusion in the liposome-based assay. When assaying the fusion properties of HCV particles with different buoyant density, we noted higher fusogenicity of particles with lower density. This could be attributable to inherently different properties of low density particles, to association of these particles with factors stimulating fusion, or to co-floatation of factors enhancing fusion activity in trans. Taken together, these data show the important role of lipids of both the viral and target membranes in HCV-mediated fusion, point to a crucial role played by the E2 glycoprotein in the process of HCV fusion, and reveal an important behavior of HCV of different densities with regard to fusion.  

5.659           Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations

Pietschmann, T., Zayas, M., Meuleman, P., Long, G., Appel, N., Koutsoudakis, G., Kallis, S., Leroux-Roels, G., Lohmann, V. and Bartenschlager, R. PloSPathogens, 5(6), e1000475 (2009)   With the advent of subgenomic hepatitis C virus (HCV) replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs), previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A), but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I) as well as NS5A (S2204R), whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes.  

5.660           Features Distinguishing Epstein-Barr Virus Infections of Epithelial Cells and B Cells: Viral Genome Expression, Genome Maintenance, and Genome Amplification

Shannon-Lowe, C., Adland, E., Bell, A.I., Delecluse, H-J., Rickinson, A.B. and Rowe, M.

1. Virol., 83(15), 7749-7760 (2009)

  Epstein-Barr virus (EBV) is associated with malignant diseases of lymphoid and epithelial cell origin. The tropism of EBV is due to B-cell-restricted expression of CD21, the major receptor molecule for the virus. However, efficient infection of CD21– epithelial cells can be achieved via transfer from EBV-coated B cells. We compare and contrast here the early events following in vitro infection of primary B cells and epithelial cells. Using sensitive, quantitative reverse transcription-PCR assays for several latent and lytic transcripts and two-color immunofluorescence staining to analyze expression at the single cell level, we confirmed and extended previous reports indicating that the two cell types support different patterns of transcription. Furthermore, whereas infection of B cells with one or two copies of EBV resulted in rapid amplification of the viral genome to >20 copies per cell, such amplification was not normally observed after infection of primary epithelial cells or undifferentiated epithelial lines. In epithelial cells, EBNA1 expression was detected in only ca. 40% of EBER+ cells, and the EBV genome was subsequently lost during prolonged culture. One exception was that infection of AGS, a gastric carcinoma line, resulted in maintenance of EBNA1 expression and amplification of the EBV episome. In contrast to B cells, where amplification of the EBV episome occurred even with a replication-defective BZLF1-knockout virus, amplification in AGS cells was dependent upon early lytic cycle gene expression. These data highlight the influence of the host cell on the outcome of EBV infection with regard to genome expression, amplification, and maintenance.    

5.661           A novel sorting strategy of trichosanthin for hijacking human immunodeficiency virus type 1

Zhao, W-L., Zhang, F., Feng, D., Shan, J.W., Chen, S. and Sui, S-F. Biochem. Biophys. Res. Comm.,384, 347-351 (2009)   Trichosanthin (TCS) is a type I ribosome-inactivating protein that plays dual role of plant toxin and anti-viral peptide. The sorting mechanism of such an exogenous protein is in long pursuit. Here, we examined TCS trafficking in cells expressing the HIV-1 scaffold protein Gag, and we found that TCS preferentially targets the Gag budding sites at plasma membrane or late endosomes depending on cell types. Lipid raft membrane but not the Gag protein mediates the association of TCS with viral components. After Gag budding, TCS is then released in association with the virus-like particles to generate TCS-enriched virions. The resulting TCS-enriched HIV-1 exhibits severely impaired infectivity. Overall, the observations indicate the existence of a unique and elaborate sorting strategy for hijacking HIV-1.  

5.662           Expression of a Modified Form of CD4 Results in the Release of an Anti-HIV Factor Derived from the Env Sequence

Zaldivar, I., Munoz-Fernandez, M.A., Alarcon, B. and San Jose, E.

1. Immunol., 183, 1188-1196 (2009)

  We have studied the inhibitory effect of a CD4 chimera (CD4 15) on HIV replication. This chimera is retained in the endoplasmic reticulum and traps the HIV envelope precursor gp160, preventing its maturation. Retroviral expression of the chimera strongly inhibited HIV replication even when it is expressed by only a minority of the T cell population. This protective effect on bystander nontransduced cells is mediated by a soluble factor that we identified as a fragment of HIV gp120 envelope protein and accordingly, we named this factor Env-derived antiviral factor (EDAF). Biochemical and immunoreactivity data show that EDAF is comprised of the gp120 C3-C5 regions and indeed, a recombinant protein bearing this sequence reproduces the anti-HIV properties of EDAF. Surprisingly, three tryptic peptides derived from EDAF are homologous but not identical with the corresponding sequences of the HIV isolate used to generate EDAF. We propose that EDAF results from an alternative intracellular processing of the Env protein provoked by its association to CD4 15 and the selection of the best fitted Env protein sequences contained within the HIV isolate. The presence of EDAF improves the therapeutic potential of the CD4 15 gene and it opens new possibilities for antiviral treatment and vaccine development.  

5.663           Oncolytic Rat Parvovirus H-1PV, a Candidate for the Treatment of Human Lymphoma: In Vitro and In Vivo Studies

Angelova, A.L., Aprahamian, M., Balboni, G., Deleques, H.-j., Feederle, R., Kiprianova, I., Grekova, S.P., Galabov, A.S., Witzens-Harig, M., Ho, A.D., Rommelaere, J. and Raykov, Z. Molecular Therapy, 17(7), 1164-1172 (2009)   The incidence of lymphomas developing in both immunocompetent and immunosuppressed patients continues to steadily increase worldwide. Current chemotherapy and immunotherapy approaches have several limitations, such as severe side toxicity and selection of resistant cell variants. Autonomous parvoviruses (PVs), in particular the rat parvovirus H-1PV, have emerged as promising anticancer agents. Although it is apathogenic in humans, H-1PV has been shown to infect and suppress various rat and human tumors in animal models. In this study, we demonstrate the capacity of H-1PV for efficiently killing, through necrosis, cell cultures originating from Burkitt's lymphoma (BL), while sparing normal B lymphocytes. The cytotoxic effect was generally accompanied by a productive H-1PV infection. Remarkably, parvovirus-based monotherapy efficiently suppressed established BL at an advanced stage in a severe combined immunodeficient (SCID) mouse model of the disease. The data show for the first time that an oncolytic parvovirus deserves further consideration as a potential tool for the treatment of some non-Hodgkin B-cell lymphomas, including those resistant to apoptosis induction by rituximab.  

5.664           Faithful Expression of Multiple Proteins via 2A-Peptide Self-Processing: A Versatile and Reliable Method for Manipulating Brain Circuits

Tang, W., Ehrlich, I., Wolff, S.B.E., Michalski, A-M., Wölfl, S., Hasan, M.T., Lüthi, A. and Sprengel, R.

1. Neurosci., 29(27), 8629-8621 (2009)

  We have studied the inhibitory effect of a CD4 chimera (CD4_15) on HIV replication. This chimera is retained in the endoplasmic reticulum and traps the HIV envelope precursor gp160, preventing its maturation. Retroviral expression of the chimera strongly inhibited HIV replication even when it is expressed by only a minority of the T cell population. This protective effect on bystander nontransduced cells is mediated by a soluble factor that we identified as a fragment of HIV gp120 envelope protein and accordingly, we named this factor Env-derived antiviral factor (EDAF). Biochemical and immunoreactivity data show that EDAF is comprised of the gp120 C3-C5 regions and indeed, a recombinant protein bearing this sequence reproduces the anti-HIV properties of EDAF. Surprisingly, three tryptic peptides derived from EDAF are homologous but not identical with the corresponding sequences of the HIV isolate used to generate EDAF. We propose that EDAF results from an alternative intracellular processing of the Env protein provoked by its association to CD4_15 and the selection of the best fitted Env protein sequences contained within the HIV isolate. The presence of EDAF improves the therapeutic potential of the CD4_15 gene and it opens new possibilities for antiviral treatment and vaccine development.  

5.665           The Lipidomes of Vesicular Stomatitis Virus, Semliki Forest Virus, and the Host Plasma Membrane Analyzed by Quantitative Shotgun Mass Spectrometry

Kalvodova, L., Sampaio, J.L., Cordo, S., Ejsing, C.S., Shevchenko, A. and Simons, K.

1. Virol., 83(16), 7996-8003 (2009)

  Although enveloped virus assembly in the host cell is a crucial step in the virus life cycle, it remains poorly understood. One issue is how viruses include lipids in their membranes during budding from infected host cells. To analyze this issue, we took advantage of the fact that baby hamster kidney cells can be infected by two different viruses, namely, vesicular stomatitis virus and Semliki Forest virus, from the Rhabdoviridae and Togaviridae families, respectively. We purified the host plasma membrane and the two different viruses after exit from the host cells and analyzed the lipid compositions of the membranes by quantitative shotgun mass spectrometry. We observed that the lipid compositions of these otherwise structurally different viruses are virtually indistinguishable, and only slight differences were detected between the viral lipid composition and that of the plasma membrane. Taken together, the facts that the lipid compositions of the two viruses are so similar and that they strongly resemble the composition of the plasma membrane suggest that these viruses exert little selection in including lipids in their envelopes.  

5.666           Four Conserved Cysteine Residues of the Hepatitis B Virus Polymerase Are Critical for RNA Pregenome Encapsidation

Kim, S., Lee, J. and Ryu, W-S.

1. Virol., 83(16), 8032-8040 (2009)

  Hepadnaviruses replicate via reverse transcription of an RNA template, the pregenomic RNA (pgRNA). Although hepadnaviral polymerase (Pol) and retroviral reverse transcriptase are distantly related, some of their features are distinct. In particular, Pol contains two additional N-terminal subdomains, the terminal protein and spacer subdomains. Since much of the spacer subdomain can be deleted without detrimental effects to hepatitis B virus (HBV) replication, this subdomain was previously thought to serve only as a spacer that links the terminal protein and reverse transcriptase subdomains. Unexpectedly, we found that the C terminus of the spacer subdomain is indispensable for the encapsidation of pgRNA. Alanine-scanning mutagenesis revealed that four conserved cysteine residues, three at the C terminus of the spacer subdomain and one at the N terminus of the reverse transcriptase subdomain, are critical for encapsidation. The inability of the mutant Pol proteins to incorporate into nucleocapsid particles, together with other evidence, argued that the four conserved cysteine residues are critical for RNA binding. One implication is that these four cysteine residues might form a putative zinc finger motif. Based on these findings, we speculate that the RNA binding activity of HBV Pol may be mediated by this newly identified putative zinc finger motif.  

5.667           TOM

5.668           Rapid, reproducible transduction of select forebrain regions by targeted recombinant virus injection into the neonatal mouse brain

Pipel, N., Landeck, N., Klugmann, M., Seeburg, P.H. and Schwarz, M.K.

1. Neurosci. Methods, 182, 55-63 (2009)

  Viral vectors can mediate long-term gene expression in different regions of the brain. Recombinant adeno-associated virus (rAAV) and Lenti virus (LV) have both gained prominence due to their ability to achieve specific transduction of various neuronal populations. Whilst widespread gene delivery has been obtained by targeted injection of rAAV in various brain structures, LV has also been utilized for infection of stem cell populations for cell lineage tracing. Both viral vector systems are most commonly used for gene delivery in mature brains, but the great potential of somatic gene delivery into the neonate brain has not been systematically exploited. Here we provide a systematic guideline for efficient stereotaxic virus delivery into different neuronal populations of the neonate brain. We demonstrate region specific recombination of a ‘stop-floxed’ Rosa26 reporter allele upon targeted injection of rAAV vectors expressing Cre-recombinase at postnatal day zero (P0). In addition, utilizing LV, we show efficient transduction of P0 subventricular zone stem cells with subsequent labeling of 20% of migrating neuroblasts along the rostral migratory stream (RMS) into the olfactory bulb. In summary, we report on an optimized protocol for facile, reproducible, high-throughput virus-based gene transfer into neonatal brains of wild-type and genetically altered mice, which allows targeted transduction of different brain regions and distinct neuronal populations.  

5.669           Probing HIV-1 Membrane Liquid Order by Laurdan Staining Reveals Producer Cell-dependent Differences

Lorizate, M., Brügger, B., Akiyama, H., Glass, B., Müller, B., Anderluh, G., Wieland, F.T. and Kräusslich, H-G.

1. Biol. Chem., 284(33), 22238-22247 (2009)

  Viruses acquire their envelope by budding from a host cell membrane,but viral lipid composition may differ from that of the buddingmembrane. We have previously reported that the HIV-1 membraneis highly enriched in cholesterol, sphingolipids, and otherraft lipids, suggesting that the virus may bud from pre-existingor virus-induced lipid rafts. Here, we employed the environmentallysensitive fluorescent dye Laurdan to study the membrane lateralstructure of HIV-1 derived from different cell lines. Differencesin viral membrane order detected by Laurdan staining were shownby mass spectrometry to be due to differences in lipid composition.Isogenic viruses from two different cell lines were both stronglyenriched in raft lipids and displayed a liquid-ordered membrane,but these effects were significantly more pronounced for HIV-1from the T-cell line MT-4 compared with virus from 293T cells.Host-dependent differences in the lipidomes predominantly affectedthe ratio of sphingomyelins (including dihydrosphingomyelin)to phosphatidylcholine, whereas cholesterol contents were similar.Accordingly, treatment of infectious HIV-1 with the sphingomyelin-bindingtoxins Equinatoxin-II or lysenin showed differential inhibitionof infectivity. Liposomes consisting of lipids that had beenextracted from viral particles exhibited slightly less liquidorder than the respective viral membranes, which is likely tobe due to absence of membrane proteins and to loss of lipidasymmetry. Synthetic liposomes consisting of a quaternary lipidmixture emulating the viral lipids showed a liquid order similarto liposomes derived from virion lipids. Thus, Laurdan stainingrepresents a rapid and quantitative method to probe viral membraneliquid order and may prove useful in the search for lipid activedrugs.  

5.670           Target Cell Cyclophilins Facilitate Human Papillomavirus Type 16 Infection

Bienkowska-Haba, M., Patel, H.D. and Sapp, M. PloSPathogens, 5(7), e1000524 (2009)   Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV–induced diseases.  

5.671           Comparison of Viral and Nonviral Vectors for Gene Transfer to Human Endothelial Progenitor Cells

Kealy, B., Liew, A., McMahon, J.M., Ritter, T., O’Doherty, A., Hoare, M., Greiser, U., Vaughan, E.E., Maenz, M., O’Shea, C., Barry, F. and O’Brien, T. Tissue Eng. Part C, 15(2), 223-231 (2009)   Background/Aims: The ability of endothelial progenitor cells (EPCs) to home to sites of neoangiogenesis makes them attractive candidates for use in the field of gene therapy. The efficacy of this approach depends on the efficiency of the vector used for transgene delivery. Methods/Results: In this study, we have compared the efficiency of adenovirus, five serotypes of AAV2, VSVG-pseudotyped lentivirus, and nonviral plasmid/liposome DNA vectors to deliver the green fluorescence protein reporter gene to human early EPCs to determine efficacy and vector-related cell toxicity. Adenovirus proved most effective with efficiencies of up to 80% with low levels of cell death. Lower levels of expression were seen with other vectors. Electroporation proved unsuitable at the parameters tested. We have also identified at least two distinct subpopulations that exist in the heterogeneous parent EPC culture, one of which is amenable to transduction with adenovirus and one that is not. In addition, adenoviral transduction did not disrupt the ability of the cells to incorporate into endothelial structures in vitro. Conclusion: We have found adenovirus to be the most efficient of the vector systems tested for gene delivery to EPCs, an effect that is mediated almost entirely by one of two identified subpopulations.  

5.672           The role of NH₄Cl and cysteine proteases in Human Papillomavirus type 16 infection

Dabydeen, S.A. and Meneses, P.I. Virol. J., 6, 109-120 (2009)   Background The infectious pathway of the non-enveloped Human Papillomavirus Type 16 (HPV16) includes binding to the cell surface, clathrin-mediated endocytosis, and penetration into an endosome. HPV16 infection was shown to decrease in the presence of the lysosomotrophic neutralizing agent ammonium chloride (NH₄Cl). NH₄Cl neutralizes acidic endo-lysosome compartments, thus suggesting that pH was responsible for PV capsid conformational changes leading endosome escape. Results However, our data suggested that NH₄Cl blocked infection by preventing the movement of PV viral particles from the early endosome to the caveosome as was shown for JC virus [1,2]. We have confirmed that HPV 16 infection requires the trafficking of reporter-virions to the caveosome as is the case for BPV1 [3,4]. In this manuscript we propose that the observed decrease in infection of PV in the presence of NH₄Cl was due to a loss of movement of reporter-virions to caveosomes. We also demonstrate that cysteine proteases are involved in the infectious process, and that cathepsin B treatment of viral particles was shown to overcome the block of infection observed in the presence of furin inhibition. We confirmed the need for cathepsin B in HPV16 infection using cathepsin B null mouse embryonic fibroblasts. Conclusion We present data that suggest HPV16 infection is in part mediated by cysteine proteases, and that NH₄Cl blocks the intracellular trafficking of infectious viral particles. To our knowledge this is the first demonstration that cysteine proteases influence the infection of a non-enveloped virus.  

5.673           Hepatitis C Virus entry: the early steps in the viral replication cycle

Sabahi, A. Virol. J., 6, 117-127 (2009)   Approximately 170 million are infected with the hepatitis C virus (HCV) world wide and an estimated 2.7 million are HCV RNA positive in the United States alone. The acute phase of the HCV infection, in majority of individuals, is asymptomatic. A large percentage of those infected with HCV are unable to clear the virus and become chronically infected. The study of the HCV replication cycle was hampered due to difficulties in growing and propagating the virus in an in vitro setting. The advent of the HCV pseudo particle (HCVpp) and HCV cell culture (HCVcc) systems have made possible the study of the HCV replication cycle, in vitro. Studies utilizing the HCVpp and HCVcc systems have increased our insight into the early steps of the viral replication cycle of HCV, such as the identification of cellular co-receptors for binding and entry. The aim of this article is to provide a review of the outstanding literature on HCV entry, specifically looking at cellular co-receptors involved and putting the data in the context of the systems used (purified viral envelope proteins, HCVpp system, HCVcc system and/or patient sera) and to also give a brief description of the cellular co-receptors themselves.    

5.674           Dengue virus neutralization by human immune sera: Role of envelope protein domain III-reactive antibody

Wahala, W.M.P.B., Kraus, A.A., Haymore, L.B., Accavitti-Loper, M.A. and de Silva, A.M. Virology, 392, 103-113 (2009)   Dengue viruses (DENV) are the etiological agents of dengue fever (DF) and dengue hemorrhagic fever (DHF). The DENV complex consists of four closely related viruses designated DENV serotypes 1 through 4. Although infection with one serotype induces cross reactive antibody to all 4 serotypes, the long-term protective antibody response is restricted to the serotype responsible for infection. Cross reactive antibodies appear to enhance infection during a second infection with a different serotype. The goal of the present study was to characterize the binding specificity and functional properties of human DENV immune sera. The study focused on domain III of the viral envelope protein (EDIII), as this region has a well characterized epitope that is recognized by strongly neutralizing serotype-specific mouse monoclonal antibodies (Mabs). Our results demonstrate that EDIII-reactive antibodies are present in primary and secondary DENV immune human sera. Human antibodies bound to a serotype specific epitope on EDIII after primary infection and a serotype cross reactive epitope on EDIII after secondary infection. However, EDIII binding antibodies constituted only a small fraction of the total antibody in immune sera binding to DENV. Studies with complete and EDIII antibody depleted human immune sera demonstrated that EDIII binding antibodies play a minor role in DENV neutralization. We propose that human antibodies directed to other epitopes on the virus are primarily responsible for DENV neutralization. Our results have implications for understanding protective immunity following natural DENV infection and for evaluating DENV vaccines.  

5.675           Expression of Neprilysin in Skeletal Muscle Reduces Amyloid Burden in a Transgenic Mouse Model of Alzheimer Disease

Liu, Y., Studzinski, C., Beckett, T., Guan, H., Hersh, M.A., Murphy, M.P., Klein, R. and Hersh, L.B. Molecular Therapy, 17(8), 1381-1386 (2009)   Neprilysin (NEP) is a zinc metallopeptidase that efficiently degrades the amyloid (A ) peptides believed to be involved in the etiology of Alzheimer disease (AD). The focus of this study was to develop a new and tractable therapeutic approach for treating AD using NEP gene therapy. We have introduced adeno-associated virus (AAV) expressing the mouse NEP gene into the hindlimb muscle of 6-month-old human amyloid precursor protein (hAPP) (3X-Tg-AD) mice, an age which correlates with early stage AD. Overexpression of NEP in muscle decreased brain soluble A peptide levels by ~60% and decreased amyloid deposits by ~50%, with no apparent adverse effects. Expression of NEP on muscle did not affect the levels of a number of other physiological peptides known to be in vitro substrates. These findings demonstrate that peripheral expression of NEP and likely other peptidases represents an alternative to direct administration into brain and illustrates the potential for using NEP expression in muscle for the prevention and treatment of AD.  

5.676           Allele-specific RNAi Mitigates Phenotypic Progression in a Transgenic Model of Alzheimer's Disease

Rodriguez-Lebron, E., Gouvion, C.M., Moore, S.A., Davidson, B.L. and Paulson, H.L. Molecular Therapy, 17(9), 1563-1573 (2009)   Despite recent advances suggesting new therapeutic targets, Alzheimer's disease (AD) remains incurable. Aberrant production and accumulation of the A peptide resulting from altered processing of the amyloid precursor protein (APP) is central to the pathogenesis of disease, particularly in dominantly inherited forms of AD. Thus, modulating the production of APP is a potential route to effective AD therapy. Here, we describe the successful use of an allele-specific RNA interference (RNAi) approach targeting the Swedish variant of APP (APPsw) in a transgenic mouse model of AD. Using recombinant adeno-associated virus (rAAV), we delivered an anti-APPsw short-hairpin RNA (shRNA) to the hippocampus of AD transgenic mice (APP/PS1). In short- and long-term transduction experiments, reduced levels of APPsw transprotein were observed throughout targeted regions of the hippocampus while levels of wild-type murine APP remained unaltered. Moreover, intracellular production of transfer RNA (tRNA)-valine promoter–driven shRNAs did not lead to detectable neuronal toxicity. Finally, long-term bilateral hippocampal expression of anti-APPsw shRNA mitigated abnormal behaviors in this mouse model of AD. The difference in phenotype progression was associated with reduced levels of soluble A but not with a reduced number of amyloid plaques. Our results support the development of allele-specific RNAi strategies to treat familial AD and other dominantly inherited neurodegenerative diseases.  

5.677           Dose Optimization for Long-term rAAV-mediated RNA Interference in the Nigrostriatal Projection Neurons

Ulusoy, A., Sahin, G., Björklund, T., Aebischer, P. and Kirik, D. Molecular Therapy, 17(9), 1574-1584 (2009)   Short-hairpin RNA (shRNA)–mediated gene knockdown is a powerful tool for targeted gene silencing and an emerging novel therapeutic strategy. Recent publications, however, reported unexpected toxicity after utilizing viral-mediated shRNA knockdown in vivo. Thus, it is currently unclear whether shRNA-mediated knockdown strategy can be used as a safe and efficient tool for gene silencing. In this study, we have generated rAAV vectors expressing shRNAs targeting the rat tyrosine hydroxylase (TH) mRNA (shTH) for testing the efficacy of in vivo TH knockdown in the nigral dopaminergic neurons. At high titers, not only the shTH vectors but also the scrambled and green fluorescence protein (GFP)–only controls caused cell death. In a dose–response study, we identified a dose window leading to >60% decrease in TH⁺ neurons without any change in vesicular monoamine transporter-2 (VMAT2) expression. Moreover, using the safe and efficient dose, we showed that dopamine (DA) synthesis rate was significantly reduced and this lead to emergence of motor deficits in the shTH-expressing rats. Interestingly, these animals showed very robust and long-lasting recovery after a single systemic L-3,4-dihydroxyphenylalanine (L-DOPA) administration beyond what can be achieved in 6-hydroxydopamine (6-OHDA)–lesioned rats. Our results have implications for both mechanistic and therapeutic studies utilizing long-term shRNA-mediated gene silencing in the nigrostriatal projection system.  

5.678           Genome sequences of two novel phages infecting marine roseobacters

Zhao, Y., Wang, K., Jiao, N.and Chen, F. Environment. Microbiol., 11(8), 2055-2064 (2009)   Two bacteriophages, DSS3[PHI]2 and EE36[PHI]1, which infect marine roseobacters Silicibacter pomeroyi DSS-3 and Sulfitobacter sp. EE-36, respectively, were isolated from Baltimore Inner Harbor water. These two roseophages resemble bacteriophage N4, a large, short-tailed phage infecting Escherichia coli K12, in terms of their morphology and genomic structure. The full genome sequences of DSS3[PHI]2 and EE36[PHI]1 reveal that their genome sizes are 74.6 and 73.3 kb, respectively, and they both contain a highly conserved N4-like DNA replication and transcription system. Both roseophages contain a large virion-encapsidated RNA polymerase gene (> 10 kb), which was first discovered in N4. DSS3[PHI]2 and EE36[PHI]1 also possess several genes (i.e. ribonucleotide reductase and thioredoxin) that are most similar to the genes in roseobacters. Overall, the two roseophages are highly closely related, and share 80-94% nucleotide sequence identity over 85% of their ORFs. This is the first report of N4-like phages infecting marine bacteria and the second report of N4-like phage since the discovery of phage N4 40 years ago. The finding of these two N4-like roseophages will allow us to further explore the specific phage-host interaction and evolution for this unique group of bacteriophages.  

5.679           Chimeric L1-L2 Virus-Like Particles as Potential Broad-Spectrum Human Papillomavirus Vaccines

Schellenbacher, C., Rsoden, R. and Kirnbauer, R.

1. Virol., 83(19), 10085-10095 (2009)

  The amino (N) terminus of the human papillomavirus (HPV) minor capsid protein L2 can induce low-titer, cross-neutralizing antibodies. The aim of this study was to improve immunogenicity of L2 peptides by surface display on highly ordered, self-assembled virus-like particles (VLP) of major capsid protein L1, and to more completely characterize neutralization epitopes of L2. Overlapping peptides comprising amino acids (aa) 2 to 22 (hereafter, chimera or peptide 2-22), 13 to 107, 18 to 31, 17 to 36, 35 to 75, 75 to 112, 115 to 154, 149 to 175, and 172 to 200 of HPV type 16 (HPV16) L2 were genetically engineered into the DE surface loop of bovine papillomavirus type 1 L1 VLP. Except for chimeras 35-75 and 13-107, recombinant fusion proteins assembled into VLP. Vaccination of rabbits with Freund's adjuvanted native VLP induced higher L2-specific antibody titers than vaccination with corresponding sodium dodecyl sulfate-denatured proteins. Immune sera to epitopes within residues 13 to 154 neutralized HPV16 in pseudovirion neutralization assays, whereas chimera 17-36 induced additional cross-neutralization to divergent high-risk HPV18, -31, -45, -52, and -58; low-risk HPV11; and beta-type HPV5 (titers of 50 to 10,000). Aluminum hydroxide-monophosphoryl lipid A (Alum-MPL)-adjuvanted VLP induced similar patterns of neutralization in both rabbits and mice, albeit with 100-fold-lower titers than Freund's adjuvant. Importantly, Alum-MPL-adjuvanted immunization with chimeric HPV16L1-HPV16L2 (peptide 17-36) VLP induced neutralization or cross-neutralization of HPV16, -18, -31, -45, -52, and -58; HPV6 and -11; and HPV5 (titers of 50 to 100,000). Immunization with HPV16 L1-HPV16 L2 (chimera 17-36) VLP in adjuvant applicable for human use induces broad-spectrum neutralizing antibodies against HPV types evolutionarily divergent to HPV16 and thus may protect against infection with mucosal high-risk, low-risk, and beta HPV types and associated disease.  

5.680           Functional Analysis of N-Terminal Residues of Ty1 Integrase

Moore, S.P., and Garfinkel, D.J.

1. Virol., 83(18), 9502-9511 (2009)

  The Ty1 retrotransposon of Saccharomyces cerevisiae is comprised of structural and enzymatic proteins that are functionally similar to those of retroviruses. Despite overall sequence divergence, certain motifs are highly conserved. We have examined the Ty1 integrase (IN) zinc binding domain by mutating the definitive histidine and cysteine residues and thirteen residues in the intervening (X32) sequence between IN-H22 and IN-C55. Mutation of the zinc-coordinating histidine or cysteine residues reduced transposition by more than 4,000-fold and led to IN and reverse transcriptase (RT) instability as well as inefficient proteolytic processing. Alanine substitution of the hydrophobic residues I28, L32, I37 and V45 in the X32 region reduced transposition 85- to 688-fold. Three of these residues, L32, I37, and V45, are highly conserved among retroviruses, although their effects on integration or viral infectivity have not been characterized. In contrast to the HHCC mutants, all the X32 mutants exhibited stable IN and RT, and protein processing and cDNA production were unaffected. However, glutathione S-transferase pulldowns and intragenic complementation analysis of selected transposition-defective X32 mutants revealed decreased IN-IN interactions. Furthermore, virus-like particles with in-L32A and in-V45A mutations did not exhibit substantial levels of concerted integration products in vitro. Our results suggest that the histidine/cysteine residues are important for steps in transposition prior to integration, while the hydrophobic residues function in IN multimerization.  

5.681           Gene targeting in human pluripotent stem cells with adeno-associated virus vectors

Mitsui, K., Ssuzuki, K., Aizawa, E., Kawase, E., Suemori, H., Nakatsuji, N. and Mitani, K. Biochem. Biophys. Res. Comm., 388, 711-717 (2009)   Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the ability to differentiate into various cell types, and will become a potential source of cellular materials for regenerative medicine. To make full use of hESCs or hiPSCs for both basic and clinical research, genetic modification, especially gene targeting via homologous recombination (HR), would be an essential technique. This report describes the successful gene targeting of the hypoxanthine phosphoribosyl transferase 1 (HPRT1) and the NANOG loci in human pluripotent stem cells with adeno-associated virus (AAV) vectors. At the HPRT1 locus, up to 1% of stable transformants were targeted via HR with an AAV-HPRT1 targeting vector, without loss of pluripotency. On the other hand, 20–87% of stable transformants were targeted using an AAV-NANOG-targeting vector designed for the promoter-trap strategy. In the KhES-3 cell line, which shows particularly high fragility to experimental manipulation, gene targeting was successful only by using an AAV vector but not by electroporation. In addition to hESC, gene targeting was achieved in hiPSC lines at similar frequencies. These data indicate that AAV vectors may therefore be a useful tool to introduce genetic modifications in hESCs and hiPSCs.  

5.682           Use of GFP to Analyze Morphology, Connectivity, and Function of Cells in the Central Nervous System

Harvey, A.R., Ehlert, E., de Wit, J., Drummond, E.S., Pollett, M.A., Ruitenberg, M., Plant, G.W., Verhaagen, J. and Levelt, C.N. Methods in Mol. Biol., 515, 63-95 (2009)   We here describe various approaches using GFP that are being used in the morphological and functional analysis of specific cell types in the normal and injured central nervous system. Incorporation of GFP into viral vectors allows phenotypic characterization of transduced cells and can be used to label their axons and terminal projections. Characterization of transduced cell morphology can be enhanced by intracellular injection of living GFP-labeled cells with appropriate fluorescent dyes. Ex vivo labeling of precursor or glial cells using viral vectors that encode GFP permits long-term identification of these cells after transplantation into the brain or spinal cord. In utero electroporation methods result in expression of gene products in developing animals, allowing both functional and morphological studies to be carried out. GFPCre has been developed as a marker gene for viral vector-mediated expression of the bacterial recombinase Cre in the brain of adult mice with “floxed” transgenes. GFPCre-mediated induction of transgene expression can be monitored by GFP expression in defined populations of neurons in the adult brain. Finally, GFP can be used to tag proteins, permitting dynamic visualization of the protein of interest in living cells.  

5.683           Fragile X mental retardation protein replacement restores hippocampal synaptic function in a mouse model of fragile X syndrome

Zeier, Z., Kumar, A., Bodhinathan, K., Feller, J.A., Foster, T.C. and Bloom, D.C. Gene Therapy, 16(9), 1122-1129 (2009)   Fragile X syndrome (FXS) is caused by a mutation that silences the fragile X mental retardation gene (FMR1), which encodes the fragile X mental retardation protein (FMRP). To determine whether FMRP replacement can rescue phenotypic deficits in a fmr1-knockout (KO) mouse model of FXS, we constructed an adeno-associated virus-based viral vector that expresses the major central nervous system (CNS) isoform of FMRP. Using this vector, we tested whether FMRP replacement could rescue the fmr1-KO phenotype of enhanced long-term depression (LTD), a form of synaptic plasticity that may be linked to cognitive impairments associated with FXS. Extracellular excitatory postsynaptic field potentials were recorded from CA3–CA1 synaptic contacts in hippocampal slices from wild-type (WT) and fmr1-KO mice in the presence of AP-5 and anisomycin. Paired-pulse low-frequency stimulation (PP-LFS)-induced LTD is enhanced in slices obtained from fmr1 KO compared with WT mice. Analyses of hippocampal synaptic function in fmr1-KO mice that received hippocampal injections of vector showed that the PP-LFS-induced LTD was restored to WT levels. These results indicate that expression of the major CNS isoform of FMRP alone is sufficient to rescue this phenotype and suggest that post-developmental protein replacement may have the potential to improve cognitive function in FXS.  

5.684           Large-Scale Adeno-Associated Viral Vector Production Using a Herpesvirus-Based System Enables Manufacturing for Clinical Studies

Clement, N., Knop, D.R. and Byrne, B.J. Human Gene Therapy, 20(8), 796-806 (2009)   The ability of recombinant adeno-associated viral (rAAV) vectors to exhibit minimal immunogenicity and little to no toxicity or inflammation while eliciting robust, multiyear gene expression in vivo are only a few of the salient features that make them ideally suited for many gene therapy applications. A major hurdle for the use of rAAV in sizeable research and clinical applications is the lack of efficient and versatile large-scale production systems. Continued progression toward flexible, scalable production techniques is a prerequisite to support human clinical evaluation of these novel biotherapeutics. This review examines the current state of large-scale production methods that employ the herpes simplex virus type 1 (HSV) platform to produce rAAV vectors for gene delivery. Improvements have substantially advanced the HSV/AAV hybrid method for large-scale rAAV manufacture, facilitating the generation of highly potent, clinical-grade purity rAAV vector stocks. At least one human clinical trial employing rAAV generated via rHSV helper-assisted replication is poised to commence, highlighting the advances and relevance of this production method.  

5.685           Effect of Adeno-Associated Virus Serotype and Genomic Structure on Liver Transduction and Biodistribution in Mice of Both Genders

Paneda, A., Vanrell, L., Mauleon, I., Crettaz, J.S., Berraondo, P., Timmermanns, E.J., Beattie, S.G., Twisk, J., van Deventer, S., Prieto, J., Fontanellas, A., Rodriguez-Pena, M.S. and Gonzales-Aseguinolaza, G. Human Gene Therapy, 20(8), 908-917 (2009)   Recombinant adeno-associated viral (AAV) vectors have unique properties, which make them suitable vectors for gene transfer. Here we assess the liver transduction efficiency and biodistribution of AAV-pseudotyped capsids (serotypes) 1, 5, 6, and 8, combined with single-stranded and double-stranded genomic AAV2 structures carrying the luciferase reporter gene after systemic administration. The analysis was performed in vivo and ex vivo, in male and female mice. Gender-related differences in AAV-mediated transduction and biodistribution were shown for the four serotypes. Our data confirm the superiority of AAV8 over the rest of the serotypes, as well as a significant advantage of double-stranded genomes in terms of liver transduction efficiency, particularly in females. Regarding biodistribution, AAV5 displayed a narrower tropism than the other serotypes tested, transducing, almost exclusively, the liver. Interestingly, AAV1 and AAV8, in particular those having single-stranded genomes, showed high transduction efficiency of female gonads. However, no inadvertent germ line transmission of AAV genomes was observed after breeding single-stranded AAV8-injected female mice with untreated males. In conclusion, double-stranded AAV8 vectors led to the highest levels of liver transduction in mice, as demonstrated by luciferase expression. Nevertheless, the transduction of other organs with AAV8 vectors could favor the use of less efficient serotypes, such as AAV5, which display a narrow tropism.  

5.686           Adenovirus-based virotherapy enabled by cellular YB-1 expression in vitro and in vivo

Rognoni, E., Widmaier, M., Haczek, C., Mantwill, K., Holzmüller, R., Gansbacher, B., Kolk, A., Sachuster, T., Schmid, R.M., Saur, D., Kaszubiak, A., Lage, H. and Holm, P.S. Cancer Gene Therapy, 16, 753-763 (2009)   We have earlier described the oncolytic adenovirus vector dl520 that was rendered cancer-specific by deletion of the transactivation domain CR3 of the adenoviral E1A13S protein; this deletion causes antitumor activity in drug-resistant cells displaying nuclear YB-1 expression. We hypothesized that the anticancer activity of dl520 could be further improved by introducing the RGD motif in the fiber knob and by deletion of the adenoviral E1B19K protein (Ad-Delo3-RGD). In this study, the in vitro and in vivo antitumor activity of Ad-Delo3-RGD was investigated focussing on two pancreatic cancer cell lines MiaPaCa-2 and BxPC3 alone and in combination with cytotoxic drugs. Furthermore, luciferin-based bioluminescence imaging was established to study the therapeutic response in vivo. In addition, to confirm the specificity of Ad-Delo3-RGD for YB-1 a tetracycline-inducible anti-YB-1 shRNA-expressing cell variant EPG85-257RDB/tetR/YB-1 was used. This TetON regulatable expression system allows us to measure adenoviral replication by real-time PCR in the absence of YB-1 expression. The results confirmed the YB-1 dependency of Ad-Delo3-RGD and showed that Ad-Delo3-RGD has potent activity against human pancreatic cancer cells in vitro and in vivo, which was augmented by the addition of paclitaxel. However, although high replication capacity was measured in vitro and in vivo, complete tumor regression was not achieved, indicating the need for further improvements to treat pancreatic cancer effectively.  

5.687           Tissue-Spanning Redox Gradient-Dependent Assembly of Native Human Papillomavirus Type 16 Virions

Conway, M.J., Alam, S., Ryndock, E.J., Cruz, L., Christensen, N.D., Roden, R.B. and Meyers, C.

1. Virol., 83(20), 10515-10526 (2009)

  Papillomavirus capsids are composed of 72 pentamers reinforced through inter- and intrapentameric disulfide bonds. Recent research suggests that virus-like particles and pseudovirions (PsV) can undergo a redox-dependent conformational change involving disulfide interactions. We present here evidence that native virions exploit a tissue-spanning redox gradient that facilitates assembly events in the context of the complete papillomavirus life cycle. DNA encapsidation and infectivity titers are redox dependent in that they can be temporally modulated via treatment of organotypic cultures with oxidized glutathione. These data provide evidence that papillomavirus assembly and maturation is redox-dependent, utilizing multiple steps within both suprabasal and cornified layers.  

5.688           Quantitation of Human Seroresponsiveness to Merkel Cell Polyomavirus

Pastrana, D.V., Tolstov, Y.L., Becker, J.C., Moore, P.S., Chang, Y. and Buck, C.B. PloSPathogens, 5(9), e1000578 (2009)   Merkel cell carcinoma (MCC) is a relatively uncommon but highly lethal form of skin cancer. A majority of MCC tumors carry DNA sequences derived from a newly identified virus called Merkel cell polyomavirus (MCV or MCPyV), a candidate etiologic agent underlying the development of MCC. To further investigate the role of MCV infection in the development of MCC, we developed a reporter vector-based neutralization assay to quantitate MCV-specific serum antibody responses in human subjects. Our results showed that 21 MCC patients whose tumors harbored MCV DNA all displayed vigorous MCV-specific antibody responses. Although 88% (42/48) of adult subjects without MCC were MCV seropositive, the geometric mean titer of the control group was 59-fold lower than the MCC patient group (p<0.0001). Only 4% (2/48) of control subjects displayed neutralizing titers greater than the mean titer of the MCV-positive MCC patient population. MCC tumors were found not to express detectable amounts of MCV VP1 capsid protein, suggesting that the strong humoral responses observed in MCC patients were primed by an unusually immunogenic MCV infection, and not by viral antigen expressed by the MCC tumor itself. The occurrence of highly immunogenic MCV infection in MCC patients is unlikely to reflect a failure to control polyomavirus infections in general, as seroreactivity to BK polyomavirus was similar among MCC patients and control subjects. The results support the concept that MCV infection is a causative factor in the development of most cases of MCC. Although MCC tumorigenesis can evidently proceed in the face of effective MCV-specific antibody responses, a small pilot animal immunization study revealed that a candidate vaccine based on MCV virus-like particles (VLPs) elicits antibody responses that robustly neutralize MCV reporter vectors in vitro. This suggests that a VLP-based vaccine could be effective for preventing the initial establishment of MCV infection.  

5.689           N-Linked glycans on dengue viruses grown in mammalian and insect cells

Hacker, K., White, L. and de Silva, A.M.

1. Gen. Virol., 90, 2097-2106 (2009)

  This study compared the ability of mosquito and mammalian cell-derived dengue virus (DENV) to infect human dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN)-expressing cells and characterized the structure of envelope (E) protein N-linked glycans on DENV derived from the two cell types. DENVs derived from both cell types were equally effective at infecting DC-SIGN-expressing human monocytes and dendritic cells. The N-linked glycans on mosquito cell-derived virus were a mix of high-mannose and paucimannose glycans. In virus derived from mammalian cells, the N-linked glycans were a mix of high-mannose and complex glycans. These results indicate that N-linked glycans are incompletely processed during DENV egress from cells, resulting in high-mannose glycans on viruses derived from both cell types. Studies with full-length and truncated E protein demonstrated that incomplete processing was most likely a result of the poor accessibility of glycans on the membrane-anchored protein.  

5.690           AAV-Tau Mediates Pyramidal Neurodegeneration by Cell-Cycle Re-Entry without Neurofibrillary Tangle Formation in Wild-Type Mice

Jaworski, T., Dewachter, I., Lechat, B., Croes, S., Termont, A., Demedts, D., Borghgraef, P., Devijver, H., Filipkowski, R.K., Kaczmarek, L., Kügler, S. and Van Leuven, F. PlosOne, 4(10), e7280 (2009)   In Alzheimer's disease tauopathy is considered secondary to amyloid, and the duality obscures their relation and the definition of their respective contributions. Transgenic mouse models do not resolve this problem conclusively, i.e. the relative hierarchy of amyloid and tau pathology depends on the actual model and the genes expressed or inactivated. Here, we approached the problem in non-transgenic models by intracerebral injection of adeno-associated viral vectors to express protein tau or amyloid precursor protein in the hippocampus in vivo. AAV-APP mutant caused neuronal accumulation of amyloid peptides, and eventually amyloid plaques at 6 months post-injection, but with only marginal hippocampal cell-death. In contrast, AAV-Tau, either wild-type or mutant P301L, provoked dramatic degeneration of pyramidal neurons in CA1/2 and cortex within weeks. Tau-mediated neurodegeneration proceeded without formation of large fibrillar tau-aggregates or tangles, but with increased expression of cell-cycle markers. We present novel AAV-based models, which demonstrate that protein tau mediates pyramidal neurodegeneration in vivo. The data firmly support the unifying hypothesis that post-mitotic neurons are forced to re-enter the cell-cycle in primary and secondary tauopathies, including Alzheimer's disease.  

5.691           Adeno-associated virus serotype 2 induces cell-mediated immune responses directed against multiple epitopes of the capsid protein VP1

Madsen, D., Cantwell, E.R., O’Brien, T., Johnson, P.A. and Mahon, B.P.

1. Gen. Virol., 90, 2622-2633 (2009)

  Adeno-associated virus serotype 2 (AAV-2) has been developed as a gene therapy vector. Antibody and cell-mediated immune responses to AAV-2 or AAV-2-transfected cells may confound the therapeutic use of such vectors in clinical practice. In one of the most detailed examinations of AAV-2 immunity in humans to date, cell-mediated and humoral immune responses to AAV-2 were characterized from a panel of healthy blood donors. The extent of AAV-2-specific antibody in humans was determined by examination of circulating AAV-2-specific total IgG levels in plasma from 45 normal donors. Forty-one donors were seropositive and responses were dominated by IgG1 and IgG2 subclasses. Conversely, AAV-2-specific IgG3 levels were consistently low in all donors. Cell-mediated immune recall responses were detectable in nearly half the population studied. In vitro restimulation with AAV-2 of peripheral blood mononuclear cell cultures from 16 donors elicited gamma interferon (IFN- ) (ten donors), interleukin-10 (IL-10) (eight donors) and interleukin-13 (IL-13) (four donors) responses. Using a series of overlapping peptides derived from the sequence of the VP1 viral capsid protein, a total of 59 candidate T-cell epitopes were identified. Human leukocyte antigen characterization of donors revealed that the population studied included diverse haplotypes, but that at least 17 epitopes were recognized by multiple donors and could be regarded as immunodominant. These data indicate that robust immunological memory to AAV-2 is established. The diversity of sequences recognized suggests that attempts to modify the AAV-2 capsid, as a strategy to avoid confounding immunity, will not be feasible.  

5.692           Distinct immune responses to transgene products from rAAV1 and rAAV8 vectors

Lu, Y. and Song, S. PNAS, 106(40), 17158-17162 (2009)   Recently developed serotypes of recombinant adeno-associated virus (rAAV) vectors have significantly enhanced the use of rAAV vectors for gene therapy. However, host immune responses to the transgene products from different serotypes remain uncharacterized. In the present study, we evaluated the differential immune responses to the transgene products from rAAV1 and rAAV8 vectors. In non-obese diabetic (NOD) mice, which have a hypersensitive immunity, rAAV serotype 1 vector (rAAV1-hAAT) induced high levels of both humoral and cellular responses, while rAAV8-hAAT did not. In vitro studies showed that rAAV1, but not rAAV8 vector transduced dendritic cells (DCs) efficiently. In vivo studies indicated that vector transduction of DCs was essential for the immune responses; while the presence of a transgene product (or foreign gene product produced by host cells) was not immunogenic. Intriguingly, preimmunization with rAAV8-hAAT vector or with serum of hAAT transgenic NOD mouse induced immune tolerance to rAAV1-hAAT injection. These results demonstrate the immunogenic differences of rAAV1 and rAAV8 and imply tremendous potential for these vectors in different applications, where an immune response to transgene is to be either elicited or avoided.  

5.693           A Reporter of Local Dendritic Translocation Shows Plaque- Related Loss of Neural System Function in APP-Transgenic Mice

Meyer-Luehmann, M., Mielke, M., Spires-Jones, T.L., Stoothoff, W., Jones, P., Bacskai, B.J. and Hyman, B.T.

1. Neurosci., 29(40), 12636-12640 (2009)

  Although neuronal communication is thought to be summated within local dendritic segments, no technique is currently available to monitor activity in vivo at this level of resolution. To overcome this challenge, we developed an optical reporter of neuronal activity using the coding sequence of Venus, flanked by short stretches of the 5'- and 3'-untranslated regions from calcium/calmodulin-dependent kinase II (CAMKII ). This reporter takes advantage of the fact that CAMKII mRNA is transported to the dendrite and locally translated in an activity-dependent manner. Using adeno-associated virus, we used this reporter to study neuronal activity in adult mice. Exposure of the mice to an enriched environment led to enhancement of Venus expression in dendritic segments of somatosensory cortex, demonstrating in vivo that dendritic mRNA translocation and local translation occur in response to physiologically relevant stimuli. We then used this system to examine the impact of Alzheimer-related local amyloid-β deposits on neural system function to test the hypothesis that plaques are toxic. In APPswe/PS1dE9 (APP/PS1) mice, neurons close to plaques, and dendritic segments close to plaques, both showed diminished fluorescent intensity and therefore neuronal activity. In contrast to wild-type mice, fluorescent intensity in neurons near plaques in transgenic mice did not increase after environmental enrichment. These data indicate that neuronal activity in dendritic segments and neurons in the vicinity of a plaque is decreased compared with wild-type mice, supporting the idea that plaques are a focal lesion leading to impaired neural system function.  

5.694           A quantitative PCR assay for SV40 neutralization adaptable for high-throughput applications

Murata, H., Teferedegne, B., Lewis Jr, A.M. and Peden, K.

1. Virol. Methods, 162, 236-244 (2009)

  A neutralization assay incorporating a quantitative SYBR Green PCR endpoint has been developed for SV40. The present study demonstrates that crude virus samples can serve as suitable amplification templates for quantitative PCR without the need for nucleic acid extraction. The denaturation temperature of thermocycling appears to be sufficient to release the encapsidated viral genome and allow its availability as a PCR template. Issues arising from inhibitors of PCR present in crude virus samples can be circumvented easily by a 100-fold dilution step. Using a streamlined procedure that eliminates sample nucleic acid extraction (a hitherto rate-limiting step that diminishes throughput substantially), quantitative PCR was applied in order to assess: (1) the replication kinetics of SV40 and (2) the inhibition of SV40 productive infection by neutralizing antibodies. A similar high-throughput approach might be feasible for related polyomaviruses (e.g., BKV and JCV) as well as for other families of viruses.  

5.695           Overlapping and independent structural roles for human papillomavirus type 16 L2 conserved cysteines

Conway, M.J., Alam, S., Christensen, N.D. and Meyers, C. Virology, 393, 295-303 (2009)   Cryoelectron microscopy images of HPV16 pseudovirions (PsV) depict that each pentamer of L1 can be occluded with a monomer of L2. Further research suggests that an N-terminal external loop of L2 exists, which is the target of neutralizing and cross-neutralizing antibodies. Here we show that N-terminal L2 cysteine residues, Cys22 and Cys28, have overlapping and independent structural roles, which affect both early- and late-stage assembly events. Substitution of either cysteine residue enhances infectivity markedly in comparison to wild-type HPV16. However, only Cys22Ser 20-day virions become nearly as stable as wild type. In addition, Cys22Ser, and Cys22,28Ser 20-day virions have lost their susceptibility to neutralization by anti-L2 antibodies, whereas Cys28Ser 20-day virions remain partially susceptible. These results suggest that Cys28 is necessary for late-stage stabilization of capsids, while Cys22 is necessary for proper display of L2 neutralizing epitopes.  

5.696           Apolipoprotein E on hepatitis C virion facilitates infection through interaction with low-density lipoprotein receptor

Owen, D.M., Huang, H., Ye, J. and Gale Jr., M. Virology, 394, 99-108 (2009)   Hepatitis C virus (HCV) infection is a major cause of liver disease. HCV associates with host apolipoproteins and enters hepatocytes through complex processes involving some combination of CD81, claudin-1, occludin, and scavenger receptor BI. Here we show that infectious HCV resembles very low density lipoprotein (VLDL) and that entry involves co-receptor function of the low-density lipoprotein receptor (LDL-R). Blocking experiments demonstrate that β-VLDL itself or anti-apolipoprotein E (apoE) antibody can block HCV entry. Knockdown of the LDL-R by treatment with 25-hydroxycholesterol or siRNA ablated ligand uptake and reduced HCV infection of cells, whereas infection was rescued upon cell ectopic LDL-R expression. Analyses of gradient-fractionated HCV demonstrate that apoE is associated with HCV virions exhibiting peak infectivity and dependence upon the LDL-R for cell entry. Our results define the LDL-R as a cooperative HCV co-receptor that supports viral entry and infectivity through interaction with apoE ligand present in an infectious HCV/lipoprotein complex comprising the virion. Disruption of HCV/LDL-R interactions by altering lipoprotein metabolism may therefore represent a focus for future therapy.  

5.697           AAV Recombineering with Single Strand Oligonucleotides

Hirsch, M.L., Storici, F., Li, C., Choi, V.W. and Samulski, R.J. PloSOne, 4(11), e7705 (2009)   Adeno-associated virus (AAV) transduction initiates a signaling cascade that culminates in a transient DNA damage response. During this time, host DNA repair proteins convert the linear single-strand AAV genomes to double-strand circular monomers and concatemers in processes stimulated by the AAV inverted terminal repeats (ITRs). As the orientation of AAV genome concatemerization appears unbiased, the likelihood of concatemerization in a desired orientation is low (less than 1 in 6). Using a novel recombineering method, Oligo-Assisted AAV Genome Recombination (OAGR), this work demonstrates the ability to direct concatemerization specifically to a desired orientation in human cells. This was achieved by a single-strand DNA oligonucleotide (oligo) displaying homology to distinct AAV genomes capable of forming an intermolecular bridge for recombination. This DNA repair process results in concatemers with genomic junctions corresponding to the sequence of oligo homology. Furthermore, OAGR was restricted to single-strand, not duplexed, AAV genomes suggestive of replication-dependent recombination. Consistent with this process, OAGR demonstrated oligo polarity biases in all tested configurations except when a portion of the oligo targeted the ITR. This approach, in addition to being useful for the elucidation of intermolecular homologous recombination, may find eventual relevance for AAV mediated large gene therapy.  

5.698           A Novel Adeno-Associated Viral Variant for Efficient and Selective Intravitreal Transduction of Rat Müller Cells

Klimsak, R.R., Koerberr, J.T., Dalkara, D., Flannery, J.G. and Schaffer, D.V. PloSOne, 4(10), e7467 (2009)   Background The pathologies of numerous retinal degenerative diseases can be attributed to a multitude of genetic factors, and individualized treatment options for afflicted patients are limited and cost-inefficient. In light of the shared neurodegenerative phenotype among these disorders, a safe and broad-based neuroprotective approach would be desirable to overcome these obstacles. As a result, gene delivery of secretable-neuroprotective factors to Müller cells, a type of retinal glia that contacts all classes of retinal neurons, represents an ideal approach to mediate protection of the entire retina through a simple and innocuous intraocular, or intravitreal, injection of an efficient vehicle such as an adeno-associated viral vector (AAV). Although several naturally occurring AAV variants have been isolated with a variety of tropisms, or cellular specificities, these vectors inefficiently infect Müller cells via intravitreal injection. Methodology/Principal Findings We have previously applied directed evolution to create several novel AAV variants capable of efficient infection of both rat and human astrocytes through iterative selection of a panel of highly diverse AAV libraries. Here, in vivo and in vitro characterization of these isolated variants identifies a previously unreported AAV variant ShH10, closely related to AAV serotype 6 (AAV6), capable of efficient, selective Müller cell infection through intravitreal injection. Importantly, this new variant shows significantly improved transduction relative to AAV2 (>60%) and AAV6. Conclusions/Significance Our findings demonstrate that AAV is a highly versatile vector capable of powerful shifts in tropism from minor sequence changes. This isolated variant represents a new therapeutic vector to treat retinal degenerative diseases through secretion of neuroprotective factors from Müller cells as well as provides new opportunities to study their biological functions in the retina.  

5.699           Role of L2 cysteines in papillomavirus infection and neutralization

Gambhira, R., Jagu, S., Karanam, B., Day, P.M. and Roden, R. Virol. J., 6, 176-181 (2009)   Vaccination of mice with minor capsid protein L2 or passive transfer with the L2-specific neutralizing monoclonal antibody RG-1 protects against human papillomavirus type 16 (HPV16) challenge. Here we explored the nature of the RG-1 epitope and its contribution to viral infectivity. RG-1 bound equivalently HPV16 L2 residues 17-36 with or without an intact C22-C28 disulphide bridge. HPV16 L2 mutations K20A, C22A, C22S, C28A, C28S, or P29A prevented RG-1 binding, whereas Y19A, K23A or Q24A had no impact. Mutation of either C22 or C28 to alanine or serine compromises HPV16 pseudoviral infectivity both in vitro and in the murine vaginal tract, but does not impact pseudovirion assembly. Despite their lack of infectivity, HPV16 pseudovirions containing C22S or C28S mutant L2 bind to cell surfaces, are taken up, and expose the 17-36 region on the virion surface as for wild type HPV16 pseudovirions suggesting normal furin cleavage of L2. Mutation of the second cysteine residue in Bovine papillomavirus type 1 (BPV1) L2 to serine (C25S) dramatically reduced the infectivity of BPV1 pseudovirions. Surprisingly, in contrast to the double mutation in HPV16 L2, the BPV1 L2 C19S, C25S double mutation reduced BPV1 pseudovirion infectivity of 293TT cells by only half.  

5.700           Novel and scalable approach to research grade AAV vector manufacturing and separation of distinct AAV serotypes

Toelen, J., Lock, M., Vandenberghe, L., Carlon, M., Wilson, J. And Debyser, Z. Human Gene Therapy, ESGCT, DGGT, GSZ, and ISCT 2009 Poster Presentations 1417-1545, poster 52 (2009)   Background: AAV vector manufacturing protocols of research grade vector for which several combinations of serotype and genome may be required, still involve laborintensive processes. Several desired outcomes of the downstream process, e.g. purification of multiple serotypes or separation of empty from full particles, are difficult to obtain and must be individually tailored for each serotype. Our observations that AAV is found in the culture supernatant during production of many serotypes, suggested that the supernatant represents a relatively pure source of vector in comparison with cell-derived material. Methods: Here we describe a serum-free AAV production system based upon PEI-mediated transfection of HEK 293 cells in 10-layer Hyperflasks. The supernatant is collected after 5 days, followed by a 50 fold concentration using tangential flow filtration (TFF) and loaded to an optimized iodixanol step-gradient. After ultracentrifugation the gradient fractions containing vector are identified, pooled and subjected to further concentration and buffer exchange. Results: Process conditions have been optimized such that final yields of several vector serotypes approach 70% with >90% capsid protein purity from a single gradient as assessed with EM and PAGE analysis. AAV serotypes 1, 2, 5, 6, 7, 8 and 9 have been produced using this protocol and used for in vivo application in the murine brain and lung. Conclusions: We present a fast and broadly applicable AAV production protocol based on the isolation of AAV from the supernatant and a purification using a single iodixanol gradient. This process had the capacity for upscaling since TFF enables a >100 fold concentration. AAV vectors produced with this method are successfully used in small animal models.  

5.701           Versatile Somatic Gene Transfer for Modeling Neurodegenerative Diseases

Klein, R.L., Wang, D.B. and King, M.A. Neurotox. Res., 16, 329-342 (2009)   A growing variety of technical approaches allow control over the expression of selected genes in living organisms. The ability to deliver functional exogenous genes involved in neurodegenerative diseases has opened pathological processes to experimental analysis and targeted therapeutic development in rodent and primate preclinical models. Biological adaptability, economic animal use, and reduced model development costs complement improved control over spatial and temporal gene expression compared with conventional transgenic models. A review of viral vector studies, typically adeno-associated virus or lentivirus, for expression of three proteins that are central to major neurodegenerative diseases, will illustrate how this approach has powered new advances and opportunities in CNS disease research.  

5.702           Intrabody Gene Therapy Ameliorates Motor, Cognitive, and Neuropathological Symptoms in Multiple Mouse Models of Huntington's Disease

Southwell, A.L., Ko, J. and Patterson, P.H.

1. Neurosci., 29(43), 13589-13602 (2009)

  Huntington's disease (HD) is an autosomal dominant neurodegenerative disease resulting from the expansion of a glutamine repeat in the huntingtin (Htt) protein. Current therapies are directed at managing symptoms such as chorea and psychiatric disturbances. In an effort to develop a therapy directed at disease prevention we investigated the utility of highly specific, anti-Htt intracellular antibodies (intrabodies). We previously showed that V_L12.3, an intrabody recognizing the N terminus of Htt, and Happ1, an intrabody recognizing the proline-rich domain of Htt, both reduce mHtt-induced toxicity and aggregation in cell culture and brain slice models of HD. Due to the different mechanisms of action of these two intrabodies, we then tested both in the brains of five mouse models of HD using a chimeric adeno-associated virus 2/1 (AAV2/1) vector with a modified CMV enhancer/chicken β-actin promoter. V_L12.3 treatment, while beneficial in a lentiviral model of HD, has no effect on the YAC128 HD model and actually increases severity of phenotype and mortality in the R6/2 HD model. In contrast, Happ1 treatment confers significant beneficial effects in a variety of assays of motor and cognitive deficits. Happ1 also strongly ameliorates the neuropathology found in the lentiviral, R6/2, N171-82Q, YAC128, and BACHD models of HD. Moreover, Happ1 significantly prolongs the life span of N171-82Q mice. These results indicate that increasing the turnover of mHtt using AAV-Happ1 gene therapy represents a highly specific and effective treatment in diverse mouse models of HD.  

5.703           Follistatin Gene Delivery Enhances Muscle Growth and Strength in Nonhuman Primates

Kota, J. et al Scienve Translational Medicine, 1(6), 6ra15 (2009)   Antagonists of myostatin, a blood-borne negative regulator of muscle growth produced in muscle cells, have shown considerable promise for enhancing muscle mass and strength in rodent studies and could serve as potential therapeutic agents for human muscle diseases. One of the most potent of these agents, follistatin, is both safe and effective in mice, but similar tests have not been performed in nonhuman primates. To assess this important criterion for clinical translation, we tested an alternatively spliced form of human follistatin that affects skeletal muscle but that has only minimal effects on nonmuscle cells. When injected into the quadriceps of cynomolgus macaque monkeys, a follistatin isoform expressed from an adeno-associated virus serotype 1 vector, AAV1-FS344, induced pronounced and durable increases in muscle size and strength. Long-term expression of the transgene did not produce any abnormal changes in the morphology or function of key organs, indicating the safety of gene delivery by intramuscular injection of an AAV1 vector. Our results, together with the findings in mice, suggest that therapy with AAV1-FS344 may improve muscle mass and function in patients with certain degenerative muscle disorders.  

5.704           Tetherin Inhibits HIV-1 Release by Directly Tethering Virions to Cells

Perez-Caballero, D., Zang, T., Ebrahimi, A., McNatt, M.W., Gregory, D.A., Johnson, M.C. and Bieniasz, P.D. Cell, 139, 499-511 (2009)   Tetherin is an interferon-induced protein whose expression blocks the release of HIV-1 and other enveloped viral particles. The underlying mechanism by which tetherin functions and whether it directly or indirectly causes virion retention are unknown. Here, we elucidate the mechanism by which tetherin exerts its antiviral activity. We demonstrate, through mutational analyses and domain replacement experiments, that tetherin configuration rather than primary sequence is critical for antiviral activity. These findings allowed the design of a completely artificial protein, lacking sequence homology with native tetherin, that nevertheless mimicked its antiviral activity. We further show that tetherin is incorporated into HIV-1 particles as a parallel homodimer using either of its two membrane anchors. These results indicate that tetherin functions autonomously and directly and that infiltration of virion envelopes by one or both of tetherin's membrane anchors is necessary, and likely sufficient, to tether enveloped virus particles that bud through the plasma membrane.  

5.705           Efficient Gene Delivery and Selective Transduction of Glial Cells in the Mammalian Brain by AAV Serotypes Isolated From Nonhuman Primates

Lawlor, P.A., Bland, R.J., Mouravlev, A., Young, D. and During, M.J. Molecular Therapy, 17(10), 1692-1702 (2009)   Adeno-associated viral (AAV) vectors have become the primary delivery agent for somatic gene transfer into the central nervous system (CNS). To date, AAV-mediated gene delivery to the CNS is based on serotypes 1–9, with efficient gene transfer to neurons only—selective and widespread transduction of glial cells have not been observed. Recently, additional endogenous AAVs have been isolated from nonhuman primate tissues. In this study, transduction obtained with AAV serotypes bb2, cy5, rh20, rh39, and rh43 was compared to that obtained with AAV8, another nonhuman primate isolate previously shown to perform well in mammalian brain. Titer-matched vectors encoding the enhanced green fluorescent protein (EGFP) reporter, driven by the constitutive CAG promoter, were injected into the hippocampus, striatum, or substantia nigra (SN) of adult rats. More widespread neuronal transduction was observed following infusion of cy5, rh20, and rh39 than observed with AAV8. Of interest, preferential transduction of astrocytes was observed with rh43. To optimize glial transduction, vector stocks driven by cell-specific promoters were generated—widespread and targeted transduction of astrocytes and oligodendrocytes was observed using rh43 and AAV8, driven by the glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) promoters, expanding the utility of AAV for modeling and treating diseases involving glial cell pathology.  

5.706           Molecular characterization of glycoprotein genes and phylogenetic analysis of two swine paramyxoviruses isolated from United States

Qiao, D., Janke, B.H. and Elankumaran, S. Virus Genes, 39, 53-65 (2009)   Two swine paramyxoviruses (SPMV)—(81-19252 (Texas-81) and 92-7783 (ISU-92)—were isolated from encephalitic pigs in the United States in 1981 and 1992. Antigenic, morphologic, and biological characteristics of these two viruses were essentially similar to members of the family Paramyxoviridae. Antigenic analysis by indirect fluorescent antibody, immunoblot, and one-way cross-neutralization tests placed these viruses along with bovine parainfluenza 3 (BPIV3) viruses. Purified virions were 50–300 nm in size and morphologically indistinguishable from other paramyxoviruses. These two viruses hemagglutinated red blood cells and had neuraminidase activity. The gene junctions of fusion (F) and hemagglutinin (HN) glycoprotein genes of these viruses contained highly conserved transcription start and stop signal sequences and trinucleotide intergenic regions similar to other Paramyxoviridae. The F gene of ISU-92 was longer than Texas-81 due to insertion of a 24-nucleotide “U”-rich 3′ untranslated region. Structure-based sequence alignment of glycoproteins of these two SPMVs indicated that they are essentially similar in structure and function to parainfluenzaviruses. The Texas-81 strain was closely related to BPIV3 Shipping Fever (SF) strain at nucleotide and amino acid level, while the ISU-92 strain was more closely related to BPIV3 910N strain. The envelope glycoproteins of ISU-92 had only ~92 and ~96% identity at nucleotide and amino acid levels with BPIV3-SF strain, respectively. The high sequence identities to BPIV3 indicated cross-species infection in pigs. Phylogenetic analyses based on both F protein and HN protein suggested the classification of these viruses into the subfamily Paramyxovirinae, genus Respirovirus, and genotype A of BPIV3.  

5.707           Nuclear location of minor capsid protein L2 is required for expression of a reporter plasmid packaged in HPV51 pseudovirions

Kondo, K., Ishii, Y., Mori, S., Shimabukuro, S., Yoshikawa, H. and Kanda, T. Virology, 394, 259-265 (2009)   The deduced amino acid (aa) sequence of L2 of the newly sequenced HPV51 strain, isolated by Matsukura and Sugase (Ma-strain), was markedly different from that of the prototype HPV51 isolated by Nuovo et al. (Nu-strain) (GenBank M62877) in two regions: aa 95–99 (region I) and aa 179–186 (region II). The two regions of Ma-strain were homologous to those of the other mucosal HPVs. The aa sequences of the N-terminal and C-terminal regions of Ma-L2 and Nu-L2 were identical and contained the nuclear localizing signal (NLS). When expressed in HEK293 cells, Ma-strain L2 (Ma-L2) was located in the nucleus but Nu-strain L2 (Nu-L2), in the cytoplasm. The chimeric L2s having both Nu-L2 regions I and II were located in the cytoplasm, and those having one of them were located both in the nucleus and cytoplasm, suggesting that Nu-L2 regions I and II inhibit the NLS function. For a better understanding of a role of L2 in infection, pseudovirion (PV) preparations were produced with a reporter, Ma-strain L1, and various L2s (Ma-L2, Nu-L2, or the chimeric L2s). These PV preparations contained structurally similar particles composed of L1 and L2 and the packaged reporter plasmid at a similar level. The reporter expression was not induced in HEK293 cells after inoculation with PVs containing the L2s that are incapable of localizing in the nucleus when expressed alone. Among PVs containing L2s capable of localizing in the nucleus, the reporter expression was induced only by PVs containing Ma-L2 region I. Thus, the results indicate that the expression of the reporter in the HPV51 PV requires the nuclear localizing ability of L2 and another unknown function associated with region I.  

5.708           B cells and monocytes from patients with active multiple sclerosis exhibit increased surface expression of both HERV-H Env and HERV-W Env, accompanied by increased seroreactivity

Brudek, T., Christensen, T., Aagaard, L., Petersen, T., Hansen, H.J. and Møller-Larsen, A. Retrovirology, 6, 104-116 (2009)   Background The etiology of the neurogenerative disease multiple sclerosis (MS) is unknown. The leading hypotheses suggest that MS is the result of exposure of genetically susceptible individuals to certain environmental factor(s). Herpesviruses and human endogenous retroviruses (HERVs) represent potentially important factors in MS development. Herpesviruses can activate HERVs, and HERVs are activated in MS patients. Results Using flow cytometry, we have analyzed HERV-H Env and HERV-W Env epitope expression on the surface of PBMCs from MS patients with active and stable disease, and from control individuals. We have also analyzed serum antibody levels to the expressed HERV-H and HERV-W Env epitopes. We found a significantly higher expression of HERV-H and HERV-W Env epitopes on B cells and monocytes from patients with active MS compared with patients with stable MS or control individuals. Furthermore, patients with active disease had relatively higher numbers of B cells in the PBMC population, and higher antibody reactivities towards HERV-H Env and HERV-W Env epitopes. The higher antibody reactivities in sera from patients with active MS correlate with the higher levels of HERV-H Env and HERV-W Env expression on B cells and monocytes. We did not find such correlations for stable MS patients or for controls. Conclusion These findings indicate that both HERV-H Env and HERV-W Env are expressed in higher quantities on the surface of B cells and monocytes in patients with active MS, and that the expression of these proteins may be associated with exacerbation of the disease.  

5.709           Suppression of hippocampal TRPM7 protein prevents delayed neuronal death in brain ischemia

Sun, H-S., Jackson, M.F., Martin, L.J., Jansen, K., Teves, L., Cui, H., Kiyonaka, S., Mori, Y., Jones, M., Forder, J.P., Golde, T.E., Orser, B.A., MacDonald, J.F. and Tymianski, M. Nature Neurosci., 12(10), 1300-1307 (2009)   Cardiac arrest victims may experience transient brain hypoperfusion leading to delayed death of hippocampal CA1 neurons and cognitive impairment. We prevented this in adult rats by inhibiting the expression of transient receptor potential melastatin 7 (TRPM7), a transient receptor potential channel that is essential for embryonic development, is necessary for cell survival and trace ion homeostasis in vitro, and whose global deletion in mice is lethal. TRPM7 was suppressed in CA1 neurons by intrahippocampal injections of viral vectors bearing shRNA specific for TRPM7. This had no ill effect on animal survival, neuronal and dendritic morphology, neuronal excitability, or synaptic plasticity, as exemplified by robust long-term potentiation (LTP). However, TRPM7 suppression made neurons resistant to ischemic death after brain ischemia and preserved neuronal morphology and function. Also, it prevented ischemia-induced deficits in LTP and preserved performance in fear-associated and spatial-navigational memory tasks. Thus, regional suppression of TRPM7 is feasible, well tolerated and inhibits delayed neuronal death in vivo.  

5.710           Blockade of Protein Phosphatase 2B Activity in the Amygdala Increases Anxiety- and Depression-Like Behaviors in Mice

Bahi, A., Mineur, YS. And Picciotto, M.R. Biol. Psychiatry, 66, 1139-1146 (2009)   Background Organ transplant patients receive chronic administration of the calcineurin inhibitor cyclosporin-A (CsA) and demonstrate increased incidence of mood disorders. Significant calcineurin expression can be observed with immunohistochemistry in the amygdala, a brain area important for behaviors related to mood disorders and anxiety. It is therefore important to determine whether chronic blockade of calcineurin might contribute to symptoms of anxiety and depression in these patients. Methods Pharmacological CsA and viral-mediated gene transfer (adeno-associated viral expression of short hairpin RNA [AAV-shRNA]) approaches were used to inhibit calcineurin activity globally and selectively in the amygdala of the mouse brain to determine the role of calcineurin in behaviors related to depression and anxiety. Results Systemic inhibition of calcineurin activity with CsA or local downregulation of calcineurin levels in the amygdala with AAV-delivered shRNAs targeting calcineurin A increased behavioral measures of anxiety in both the elevated plus maze and light/dark tests with no changes in locomotor activity. In the forced swim and tail suspension models of depression-like behavior, calcineurin blockade in the amygdala increased immobility similarly to manipulations that lead to a depression-like phenotype. Conclusions Taken together, these data demonstrate that decreasing calcineurin activity in the amygdala increases anxiety- and depression-like behaviors. These studies suggest that chronic administration of CsA to organ transplant patients could have significant effects on anxiety and mood and that this should be recognized as a clinical consequence of treatment to prevent transplant rejection.  

5.711           The ORF59 DNA polymerase processivity factor homologs of Old World primate RV2 rhadinoviruses are highly conserved nuclear antigens expressed in differentiated epithelium in infected macaques

Bruce, A.G., Bakke, A.M., Gravett, C.A., DeMAster, L.K., Bielefeldt-Ohmann, H., Burnside, K:L. And Rose, T.M. Virol. J., 6, 205-224 (2009)   Background ORF59 DNA polymerase processivity factor of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is required for efficient copying of the genome during virus replication. KSHV ORF59 is antigenic in the infected host and is used as a marker for virus activation and replication. Results We cloned, sequenced and expressed the genes encoding related ORF59 proteins from the RV1 rhadinovirus homologs of KSHV from chimpanzee (PtrRV1) and three species of macaques (RFHVMm, RFHVMn and RFHVMf), and have compared them with ORF59 proteins obtained from members of the more distantly-related RV2 rhadinovirus lineage infecting the same non-human primate species (PtrRV2, RRV, MneRV2, and MfaRV2, respectively). We found that ORF59 homologs of the RV1 and RV2 Old World primate rhadinoviruses are highly conserved with distinct phylogenetic clustering of the two rhadinovirus lineages. RV1 and RV2 ORF59 C-terminal domains exhibit a strong lineage-specific conservation. Rabbit antiserum was developed against a C-terminal polypeptide that is highly conserved between the macaque RV2 ORF59 sequences. This anti-serum showed strong reactivity towards ORF59 encoded by the macaque RV2 rhadinoviruses, RRV (rhesus) and MneRV2 (pig-tail), with no cross reaction to human or macaque RV1 ORF59 proteins. Using this antiserum and RT-qPCR, we determined that RRV ORF59 is expressed early after permissive infection of both rhesus primary fetal fibroblasts and African green monkey kidney epithelial cells (Vero) in vitro. RRV- and MneRV2-infected foci showed strong nuclear expression of ORF59 that correlated with production of infectious progeny virus. Immunohistochemical studies of an MneRV2-infected macaque revealed strong nuclear expression of ORF59 in infected cells within the differentiating layer of epidermis corroborating previous observations that differentiated epithelial cells are permissive for replication of KSHV-like rhadinoviruses. Conclusion The ORF59 DNA polymerase processivity factor homologs of the Old World primate RV1 and RV2 rhadinovirus lineages are phylogenetically distinct yet demonstrate similar expression and localization characteristics that correlate with their use as lineage-specific markers for permissive infection and virus replication. These studies will aid in the characterization of virus activation from latency to the replicative state, an important step for understanding the biology and transmission of rhadinoviruses, such as KSHV.  

5.712           Molecular Evolution of Adeno-associated Virus for Enhanced Glial Gene Delivery

Koerber, J.T., Klimczak, R., Jang, J-H., Dalkara, D., Flannery, J.G. and Schaffer, D.V. Molecular Therapy, 17(12), 2088-2095 (2009)   The natural tropism of most viral vectors, including adeno-associated viral (AAV) vectors, leads to predominant transduction of neurons and epithelia within the central nervous system (CNS) and retina. Despite the clinical relevance of glia for homeostasis in neural tissue, and as causal contributors in genetic disorders such as Alzheimer's and amyotrophic lateral sclerosis, efforts to develop more efficient gene delivery vectors for glia have met with limited success. Recently, viral vector engineering involving high-throughput random diversification and selection has enabled the rapid creation of AAV vectors with valuable new gene delivery properties. We have engineered novel AAV variants capable of efficient glia transduction by employing directed evolution with a panel of four distinct AAV libraries, including a new semi-random peptide replacement strategy. These variants transduced both human and rat astrocytes in vitro up to 15-fold higher than their parent serotypes, and injection into the rat striatum yielded astrocyte transduction levels up to 16% of the total transduced cell population, despite the human astrocyte selection platform. Furthermore, one variant exhibited a substantial shift in tropism toward Müller glia within the retina, further highlighting the general utility of these variants for efficient glia transduction in multiple species within the CNS and retina.  

5.713           Inner Limiting Membrane Barriers to AAV-mediated Retinal Transduction From the Vitreous

Dalkara, D., Kolstad, K.D., caporale, N., Visel, M., Klimczak, R.R., Schaffer, D.V. and Flannery, J.G. Molecular Therapy, 17(12), 2096-2102 (2009)   Adeno-associated viral gene therapy has shown great promise in treating retinal disorders, with three promising clinical trials in progress. Numerous adeno-associated virus (AAV) serotypes can infect various cells of the retina when administered subretinally, but the retinal detachment accompanying this injection induces changes that negatively impact the microenvironment and survival of retinal neurons. Intravitreal administration could circumvent this problem, but only AAV2 can infect retinal cells from the vitreous, and transduction is limited to the inner retina. We therefore sought to investigate and reduce barriers to transduction from the vitreous. We fluorescently labeled several AAV serotype capsids and followed their retinal distribution after intravitreal injection. AAV2, 8, and 9 accumulate at the vitreoretinal junction. AAV1 and 5 show no accumulation, indicating a lack of appropriate receptors at the inner limiting membrane (ILM). Importantly, mild digestion of the ILM with a nonspecific protease enabled substantially enhanced transduction of multiple retinal cell types from the vitreous, with AAV5 mediating particularly remarkable expression in all retinal layers. This protease treatment has no effect on retinal function as shown by electroretinogram (ERG) and visual cortex cell population responses. These findings may help avoid limitations, risks, and damage associated with subretinal injections currently necessary for clinical gene therapy.  

5.714           Global diffuse distribution in the brain and efficient gene delivery to the dorsal root ganglia by intrathecal injection of adeno-associated viral vector serotype 1

Iwamoto, N., Watanabe, A., Yamamoto, M., Miyake, N., Kurai, T., teramoto, A. and Shimada, T.

1. Gene Med., 11(6), 498-505 (2009)

  Background The success of gene therapy for inherited neurodegenerative diseases such as metachromatic leukodystrophy (MLD) depends on the development of efficient gene delivery throughout the brain guarded by the blood–brain barrier and achieves distribution of the deficient enzyme throughout the brain. Direct injection of viral vector into the brain parenchyma is too invasive and may not be sufficient to treat the entire brain. As an alternative approach, we examined the feasibility of intrathecal (IT) injection of adeno-associated viral vector serotype 1 (AAV1). Methods AAV1 vector expressing arylsulfatase A (ASA) and green fluorescence protein (GFP) was intrathecally injected into ASA knockout MLD model mice. Expression of GFP was assessed by fluorescence microscopy and immunohistochemical methods, whereas the concentration of ASA was determined by a quantitative enzyme-linked immunosorbent assay. Results Broad distribution of GFP expression was seen throughout the brain after IT injection of AAV1 vector. In addition, a large number of nerve fibers in the dorsal spinal cord and many neural cell bodies in the dorsal root ganglia were efficiently transduced. Widespread distribution of ASA activity and a significant reduction of sulfatide content were confirmed in treated MLD model mice. Conclusions IT injection of AAV1 vector is a useful and non-invasive method for widespread gene delivery to the brain and dorsal root ganglia.  

5.715           Intra-articular gene delivery and expression of interleukin-1Ra mediated by self-complementary adeno-associated virus

Kay, J.D., Gouze, E., Oligino, T.J., Gouz, J-N., Watson, R.S., Levings, P.P., Bush, M.L., Dacanay, A., Nickerson, D.M., Robbins, P.D. and Ghivizanni, S.C.

1. Gene Med., 11(7), 605-614 (2009)

  Background The adeno-associated virus (AAV) has many safety features that favor its use in the treatment of arthritic conditions; however, the conventional, single-stranded vector is inefficient for gene delivery to fibroblastic cells that primarily populate articular tissues. This has been attributed to the inability of these cells to convert the vector to a double-stranded form. To overcome this, we evaluated double-stranded self-complementary (sc) AAV as a vehicle for intra-articular gene delivery. Methods Conventional and scAAV vectors were used to infect lapine articular fibroblasts in culture to determine transduction efficiency, transgene expression levels, and nuclear trafficking. scAAV containing the cDNA for interleukin (IL)-1 receptor antagonist (Ra) was delivered to the joints of naïve rabbits and those with IL-1β-induced arthritis. From lavage of the joint space, levels of transgenic expression and persistence were measured by enzyme-linked immunosorbent assay. Infiltrating leukocytes were quantified using a hemocytometer. Results Transgene expression from scAAV had an earlier onset and was approximately 25-fold greater than conventional AAV despite the presence of similar numbers of viral genomes in the nuclei of infected cells. Fibroblasts transduced with scAAV produced amounts of IL1-Ra comparable to those transduced with adenoviral and lentiviral vectors. IL1-Ra was present in lavage fluid of most animals for 2 weeks in sufficient quantities to inhibit inflammation of the IL-1β-driven model. Once lost, neither subsequent inflammatory events, nor re-administration of the virus could re-establish transgene expression. Conclusions scAAV-mediated intra-articular gene transfer is robust and similarly efficient in both normal and inflamed joints; the resulting transgenic expression is sufficient to achieve biological relevance in joints of human proportion.  

5.716           AAV gene therapy as a means to increase apolipoprotein (Apo) A-I and high-density lipoprotein-cholesterol levels: correction of murine ApoA-I deficiency

Vaessen, S.F., Veldman, R.J., Comijn, E.M., Snapper, J., Sierts, J.A., van den Oever, K., Beattie, S.G., Twisk, J. and Kuivenhoven, J.A.

1. Gene Med., 11(8), 697-707 (2009)

  Background Inherited apolipoprotein (Apo) A-I deficiency is an orphan disorder characterized by high-density lipoprotein (HDL)-cholesterol deficiency and premature atherosclerosis. Constitutive over-expression of ApoA-I might provide a means to treat this disease. The present study provides a comprehensive evaluation of adeno-associated virus (AAV)-mediated ApoA-I gene delivery to express human (h)ApoA-I and correct the low HDL-cholesterol phenotype associated with ApoA-I deficiency. Methods In an effort to maximize AAV-mediated gene expression, we performed head-to-head comparisons of recombinant AAVs with pseudotype capsids 1, 2, 6 and 8 administered by different routes with the use of five different liver-specific promoters in addition to cytomegalovirus as single-stranded or as self-complementary (sc) AAV vectors. Results Intravenous administration of 1 × 1013 gc/kg scAAV8, in combination with the liver-specific promoter LP1, in female ApoA-I−/− mice resulted in hApoA-I expression levels of 634 ± 69 mg/l, which persisted for the duration of the study (15 weeks). This treatment resulted in full recovery of HDL-cholesterol levels with correction of HDL particle size and apolipoprotein composition. In addition, we observed increased adrenal cholesterol content and a significant increase in bodyweight in treated mice. Conclusions The present study demonstrates that systemic delivery of a scAAV8 vector provides a means for efficient liver expression of hApoA-I, thereby correcting the lipid abnormalities associated with murine ApoA-I deficiency. Importantly, the study demonstrates that AAV-based gene therapy can be used to express therapeutic proteins at a high level for a prolonged period of time and, as such, provides a basis for further development of this strategy to treat hApoA-I deficiency.  

5.717           Correction of mutant Fanconi anemia gene by homologous recombination in human hematopoietic cells using adeno-associated virus vector

Paiboonsukwong, K., Ohbayashi, F., Shiiba, H., Aizawa, E., Yamashita, T. and Mitani, K.

1. Gene Med., 11(11), 1012-1019 (2009)

  Background Adeno-associated virus (AAV) vectors have been shown to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which can overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR. Methods We infected a human B-lymphoblastoid cell line (BCL) derived from a normal subject with an AAV, which disrupts the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus, to measure the frequency of AAV-mediated HR in BCL cells. We subsequently constructed an AAV vector encoding the normal sequences from the Fanconi anemia group A (FANCA) locus to correct a mutation in the gene in BCL derived from a FANCA patient. Results Under optimal conditions, approximately 50% of BCL cells were transduced with an AAV serotype 2 (AAV-2) vector. In FANCA BCL cells, up to 0.016% of infected cells were gene-corrected by HR. AAV-mediated restoration of normal genotypic and phenotypic characteristics in FANCA-mutant cells was confirmed at the DNA, protein and functional levels. Conclusions The results obtained in the present study indicate that AAV vectors may be applicable for gene correction therapy of inherited hematopoietic disorders.  

5.718           Engineering adeno-associated virus serotype 2-based targeting vectors using a new insertion site-position 453-and single point mutations

Boucas, J., Lux, K., Huber, A., Schievenbusch, S., von Freyend, M.J., Perabo, L., Quadt-Humme, S., Odenthal, M., Hallek, M. and Büning, H.

1. Gene Med., 11(12), 1103-1113 (2009)

  Background Genetic modification of capsid proteins by peptide insertion has created the possibility of using adeno-associated viral (AAV) vectors for receptor specific gene transfer (AAV targeting). The most common site used for insertion in AAV serotype 2 capsids are amino acid positions 587 and 588 located at the second highest capsid protrusion. Reasoning that peptide insertions at the most exposed position augments target receptor interaction, we explored position 453 as a new insertion site. Methods Position 453 was identified in silico. Capsid mutants carrying the model ligand RGD-4C in position 453 with and without R585A/R588A substitutions were compared with respective mutants carrying the ligand in position 587. The accessibility of the inserted ligand was determined by an enzyme-linked immunosorbent assay, whereas the transduction efficiency and specificity of receptor binding were assayed by gene transfer and competition experiments, respectively. Vector biodistribution was determined in mice by quantitative polymerase chain reaction analysis. Results Initially, RGD-4C, inserted at position 453, failed to efficiently bind its target receptor. R585 and R588, located at the neighboring peak and known to mediate primary receptor binding, were identified as interfering residues. R585A and R588A substitutions rendered position 453 mutants superior to those with the ligand in position 587 in target receptor binding and cell transduction efficiency. The in vivo biodistribution was independent of the insertion site, but directed by the inserted ligand when primary receptor binding was avoided. Conclusions Position 453 emerged as a prominent site for the development of targeting mutants. Furthermore, we show for the first time that linearly distant residues can be critical for the efficiency of inserted peptide ligands.  

5.719           Limb-girdle muscular dystrophy type 2D gene therapy restores -sarcoglycan and associated proteins

Mendell, J.R., Rodino-Klapac, L.R., Rosales-Quintero, X., Kota, J., Coley, B.D., Galloway, G., Craenen, J.M., Lewis, S., Malik, V., Shilling, C., Byrne, B.J., Conlon, T., Campbell, K.J., Bremer, W.G., Viollet, L., Walker, C.M., Sahenk, Z. and Clark, K.R. Ann. Neurol., 66(3), 290-297 (2009)   Objective -Sarcoglycan deficiency results in a severe form of muscular dystrophy (limb-girdle muscular dystrophy type 2D [LGMD2D]) without treatment. Gene replacement represents a strategy for correcting the underlying defect. Questions related to this approach were addressed in this clinical trial, particularly the need for immunotherapy and persistence of gene expression. Methods A double-blind, randomized controlled trial using rAAV1.tMCK.hSGCA injected into the extensor digitorum brevis muscle was conducted. Control sides received saline. A 3-day course of methylprednisolone accompanied gene transfer without further immune suppression. Results No adverse events were encountered. SGCA gene expression increased 4–5-fold over control sides when examined at 6 weeks (2 subjects) and 3 months (1 subject). The full sarcoglycan complex was restored in all subjects, and muscle fiber size was increased in the 3-month subject. Adeno-associated virus serotype 1 (AAV1)-neutralizing antibodies were seen as early as 2 weeks. Neither CD4+ nor CD8+ cells were increased over contralateral sides. Scattered foci of inflammation could be found, but showed features of programmed cell death. Enzyme-linked immunospot (ELISpot) showed no interferon- response to -SG or AAV1 capsid peptide pools, with the exception of a minimal capsid response in 1 subject. Restimulation to detect low-frequency capsid-specific T cells by ELISpot assays was negative. Results of the first 3 subjects successfully achieved study aims, precluding the need for additional enrollment. Interpretation The finding of this gene replacement study in LGMD2D has important implications for muscular dystrophy. Sustained gene expression was seen, but studies over longer time periods without immunotherapy will be required for design of vascular delivery gene therapy trials.  

5.720           Human Merkel cell polyomavirus infection II. MCV is a common human infection that can be detected by conformational capsid epitope immunoassays

Tolstov, Y., Pastrana, D.V., Feng, H., Becker, J.C., Jenkins, F.J., Moschos, S., Chang, Y., Bick, C.B. and Moore, P.S. Int. J. Cancer, 125(6), 1250-1256 (2009)   Merkel cell polyomavirus (MCV) is a newly-discovered human tumor virus found in ∼80% of Merkel cell carcinoma (MCC). The rate of MCV infection among persons without MCC is unknown. We developed a MCV virus-like particle (VLP) enzyme-linked immunoassay (EIA) that does not cross-react with human BK or murine polyomaviruses. Peptide mapping of the MCV VP1 gene and immunoblotting with denatured MCV VLP are less sensitive than the MCV EIA in detecting MCV antibodies suggesting antibody reactivity in this assay primarily targets conformational but not linear epitopes. Among MCC patients, all 21 (100%) patients tested with MCV-positive tumors had high serum MCV IgG but not high MCV IgM levels. Only 3 of 6 (50%) MCC patients with MCV-negative tumors were positive for MCV antibodies. Sera from most adults, including 107 of 166 (64%) blood donors, 63 of 100 (63%) commercial donors and 37 of 50 (74%) systemic lupus erythematosus patients, show evidence for prior MCV exposure. Age-specific MCV prevalence was determined by examining a cross-sectional distribution of 150 Langerhans cell histiocytosis (an unrelated neoplasm) patient sera. MCV prevalence increases from 50% among children age 15 years or younger to 80% among persons older than 50 years. We did not find evidence for vertical transmission among infants. Although past exposure to MCV is common among all adult groups, MCC patients have a markedly elevated MCV IgG response compared with control patients. Our study demonstrates that MCV is a widespread but previously unrecognized human infection.  

5.721           Viral vectors: from virology to transgene expression

Bouard, D., Alazard-Dany, N. and Cosset, F-L. Br. J: Pharmacol., 157(2), 153-165 (2009)   In the late 1970s, it was predicted that gene therapy would be applied to humans within a decade. However, despite some success, gene therapy has still not become a routine practise in medicine. In this review, we will examine the problems, both experimental and clinical, associated with the use of viral material for transgenic insertion. We shall also discuss the development of viral vectors involving the most important vector types derived from retroviruses, adenoviruses, herpes simplex viruses and adeno‐associated viruses.  

5.722           Activation of an Antiviral Response in Normal but Not Transformed Mouse Cells: a New Determinant of Minute Virus of Mice Oncotropism

Grekova, S., Zawatzky, R., Hörlein, R., Cziepluch, C., Mincberg, M., Davis, C., Rommelaere, J. and Daeffler, L.

1. Virol., 84(1), 516-531 (2010)

  Parvovirus minute virus of mice (MVMp) is endowed with oncotropic properties so far ascribed only to the dependency of the virus life cycle on cellular factors expressed during S phase and/or modulated by malignant transformation. For other viruses oncotropism relies on their inability to circumvent type I interferon (IFN)-induced innate antiviral mechanisms, the first line of defense triggered by normal cells against viral infections. These agents propagate, therefore, preferentially in transformed/tumor cells, which often lack functional antiviral mechanisms. The present study aimed at investigating whether antiviral processes also contribute to MVMp oncotropism. Our results demonstrate that in contrast to MVMp-permissive transformed mouse A9 fibroblasts, freshly isolated normal counterparts (mouse embryonic fibroblasts [MEFs]) mount, through production and release of type I IFNs upon their infection, an antiviral response against MVMp lytic multiplication. Pretreatment of MEFs with a type I IFN-β-neutralizing antibody, prior to MVMp infection, inhibits the virus-triggered antiviral response and improves the fulfillment of the MVMp life cycle. Our results also show that part of the A9 permissiveness to MVMp relies on the inability to produce type I IFNs upon parvovirus infection, a feature related either to an A9 intrinsic deficiency of this process or to an MVMp-triggered inhibitory mechanism, since stimulation of these cells by exogenous IFN-β strongly inhibits the parvovirus life cycle. Taken together, our results demonstrate for the first time that parvovirus infection triggers an innate antiviral response in normal cells and suggest that the MVMp oncotropism depends at least in part on the failure of infected transformed cells to mount such a response.  

5.723           Mouse-Specific Residues of Claudin-1 Limit Hepatitis C Virus Genotype 2a Infection in a Human Hepatocyte Cell Line

Haid, S., Windisch, M.P., Bartenschlager, R. and Pietschmann, T.

1. Virol., 84(2), 964-975 (2010)

  Recently, claudin-1 (CLDN1) was identified as a host protein essential for hepatitis C virus (HCV) infection. To evaluate CLDN1 function during virus entry, we searched for hepatocyte cell lines permissive for HCV RNA replication but with limiting endogenous CLDN1 expression, thus permitting receptor complementation assays. These criteria were met by the human hepatoblastoma cell line HuH6, which (i) displays low endogenous CLDN1 levels, (ii) efficiently replicates HCV RNA, and (iii) produces HCV particles with properties similar to those of particles generated in Huh-7.5 cells. Importantly, naïve cells are resistant to HCV genotype 2a infection unless CLDN1 is expressed. Interestingly, complementation of HCV entry by human, rat, or hamster CLDN1 was highly efficient, while mouse CLDN1 (mCLDN1) supported HCV genotype 2a infection with only moderate efficiency. These differences were observed irrespective of whether cells were infected with HCV pseudoparticles (HCVpp) or cell culture-derived HCV (HCVcc). Comparatively low entry function of mCLDN1 was observed in HuH6 but not 293T cells, suggesting that species-specific usage of CLDN1 is cell type dependent. Moreover, it was linked to three mouse-specific residues in the second extracellular loop (L152, I155) and the fourth transmembrane helix (V180) of the protein. These determinants could modulate the exposure or affinity of a putative viral binding site on CLDN1 or prevent optimal interaction of CLDN1 with other human cofactors, thus precluding highly efficient infection. HuH6 cells represent a valuable model for analysis of the complete HCV replication cycle in vitro and in particular for analysis of CLDN1 function in HCV cell entry.  

5.724           Early CD4+ T Cell Help Prevents Partial CD8+ T Cell Exhaustion and Promotes Maintenance of Herpes Simplex Virus 1 Latency

Frank, G.M., Lepisto, A.J., Freeman, M.L., Sheridan, B.S., Cherpes, T.L. and Hendricks, R.L.

1. Immunol., 184, 277-286 (2010)

  HSV-specific CD8⁺ T cells provide constant immunosurveillance of HSV-1 latently infected neurons in sensory ganglia, and their functional properties are influenced by the presence of latent virus. In this study, we show that ganglionic HSV-specific CD8⁺ T cells exhibit a higher functional avidity (ability to respond to low epitope density) than their counterparts in noninfected lungs, satisfying a need for memory effector cells that can respond to low densities of viral epitopes on latently infected neurons. We further show that lack of CD4⁺ T cell help during priming leads to a transient inability to control latent virus, which was associated with a PD-1/PD-L1 mediated reduced functional avidity of ganglionic HSV-specific CD8⁺ T cells. CD4⁺ T cells are not needed to maintain CD8⁺ T cell memory through 34 d after infection, nor do they have a direct involvement in the maintenance of HSV-1 latency.  

5.725           Self-complementary AAV Virus (scAAV) Safe and Long-term Gene Transfer in the Trabecular Meshwork of Living Rats and Monkeys

Buie, L.K., Rasmussen, C.A., Porterfield, E.C., Ramgolam, V.S., Choi, V.W., Markovic-Plese, S., Samulski, R.J., Kaufman, P.L. and Borras, T. Invest. Ophthalmol. Vis. Asci., 51(1), 236-248 (2010)   Purpose. AAV vectors produce stable transgene expression and elicit lowimmune response in many tissues. AAVs have been the vectorsof choice for gene therapy for the eye, in particular the retina.scAAVs are modified AAVs that bypass the required second-strandDNA synthesis to achieve transcription of the transgene. Thegoal was to investigate the ability of AAV vectors to inducelong-term, safe delivery of transgenes to the trabecular meshworkof living animals. Methods. Single doses of AAV2.GFP and AAV2.RGD.GFP/Ad5.LacZ were injected intracamerally (IC) into rats (n = 28 eyes). A single dose of scAAV.GFP was IC-injected into rats (n = 72 eyes) and cynomolgus monkeys (n = 3). GFP expression was evaluated by fluorescence,immunohistochemistry, and noninvasive gonioscopy. Intraocularpressure (IOP) was measured with calibrated tonometer (rats)and Goldmann tonometer (monkeys). Differential expression ofscAAV-infected human trabecular meshwork cells (HTM) was determinedby microarrays. Humoral and cell-mediated immune responses wereevaluated by ELISA and peripheral blood proliferation assays. Results. No GFP transduction was observed on the anterior segment tissuesof AAV-injected rats up to 27 days after injection. In contrast,scAAV2 transduced the trabecular meshwork very efficiently,with a fast onset (4 days). Eyes remained clear and no adverseeffects were observed. Transgene expression lasted >3.5 monthsin rats and >2.35 years in monkeys. Conclusions. The scAAV viral vector provides prolonged and safe transductionin the trabecular meshwork of rats and monkeys. The stable expressionand safe properties of this vector could facilitate the developmentof trabecular meshwork drugs for gene therapy for glaucoma.  

5.726           Adeno-associated viral vector (AAV)-mediated gene transfer in the red nucleus of the adult rat brain: Comparative analysis of the transduction properties of seven AAV serotypes and lentiviral vectors

Blits, B., Derks, S., Twisk, J., Ehlert, E., Prins, J. and Verhaagen, J.

1. Neurosci. Methods, 185, 257-263 (2010)

  Recombinant adeno-associated viral vectors (AAVs) are very promising gene transfer tools for the nervous system. We have compared the efficiency of gene expression of seven AAV serotypes in young adult rats following a single injection in a major nucleus of the mid brain, the red nucleus, which is the origin of the rubrospinal tract. AAV serotypes 1–6 and 8 and a lentiviral vector (LV) were used, all encoding green fluorescent protein (GFP) under control of the cytomegalovirus (CMV) promoter. AAV vectors were titer matched at 5 × 10¹¹ genomic copies (GC)/ml and 1 μl was injected into the red nucleus. The proportion of transduced neurons in the red nucleus was determined at 1 and 4 weeks post-injection. AAV1 would be the vector of choice if the aim would be to overexpress a transgene at high level for a longer period of time. AAV5 and AAV8 would be the preferred serotype if onset of expression is should be somewhat delayed. The use of lentiviral vectors should be considered when transduction of both glial cells and neurons is required. Serotypes 3 and 4 did not transduce red nucleus neurons. AAV1, AAV6 and LV would be the vectors of choice if the aim of the experiment would be to rapidly express a transgene. The current data are important for the design of experiments that aim to study the effects of transgene products on the regenerative capacity of injured red nucleus neurons.  

5.727           Searching for a "Hidden" Prophage in a Marine Bacterium

Zhao, Y., Wang, K., Ackermann, H-W., Halden, R.U., Jiao, N. and Chen, F. Appl. Envir. Microbiol., 76(2), 589-595 (2010   Prophages are common in many bacterial genomes. Distinguishing putatively viable prophages from nonviable sequences can be a challenge, since some prophages are remnants of once-functional prophages that have been rendered inactive by mutational changes. In some cases, a putative prophage may be missed due to the lack of recognizable prophage loci. The genome of a marine roseobacter, Roseovarius nubinhibens ISM (hereinafter referred to as ISM), was recently sequenced and was reported to contain no intact prophage based on customary bioinformatic analysis. However, prophage induction experiments performed with this organism led to a different conclusion. In the laboratory, virus-like particles in the ISM culture increased more than 3 orders of magnitude following induction with mitomycin C. After careful examination of the ISM genome sequence, a putative prophage (ISM-pro1) was identified. Although this prophage contains only minimal phage-like genes, we demonstrated that this "hidden" prophage is inducible. Genomic analysis and reannotation showed that most of the ISM-pro1 open reading frames (ORFs) display the highest sequence similarity with Rhodobacterales bacterial genes and some ORFs are only distantly related to genes of other known phages or prophages. Comparative genomic analyses indicated that ISM-pro1-like prophages or prophage remnants are also present in other Rhodobacterales genomes. In addition, the lysis of ISM by this previously unrecognized prophage appeared to increase the production of gene transfer agents (GTAs). Our study suggests that a combination of in silico genomic analyses and experimental laboratory work is needed to fully understand the lysogenic features of a given bacterium.  

5.728           Immature Dengue Virus: A Veiled Pathogen?

Rodenhuis-Zybert, I.A., van der Schaar, H.M., da Silva Voorham, J.M., van der Ende-Metselaar, H., Lei, H-Y., Wilschut, J. and Smit, J.M. PloSPathogens, 6(1), e1000718 (2010)   Cells infected with dengue virus release a high proportion of immature prM-containing virions. In accordance, substantial levels of prM antibodies are found in sera of infected humans. Furthermore, it has been recently described that the rates of prM antibody responses are significantly higher in patients with secondary infection compared to those with primary infection. This suggests that immature dengue virus may play a role in disease pathogenesis. Interestingly, however, numerous functional studies have revealed that immature particles lack the ability to infect cells. In this report, we show that fully immature dengue particles become highly infectious upon interaction with prM antibodies. We demonstrate that prM antibodies facilitate efficient binding and cell entry of immature particles into Fc-receptor-expressing cells. In addition, enzymatic activity of furin is critical to render the internalized immature virus infectious. Together, these data suggest that during a secondary infection or primary infection of infants born to dengue-immune mothers, immature particles have the potential to be highly infectious and hence may contribute to the development of severe disease.  

5.729           Gene therapy with a promoter targeting both rods and cones rescues retinal degeneration caused by AIPL1 mutations

Sun, X., Pawlyk, B., Xu, X., Liu, X., Bulgalov, O.V., Adamian, M., Sandberg, M.A., khani, S.C., Tan, M-H., Smith, A.J., Ali, R.R. and Li, T. Gene Therapy, 17(1), 117-131 (2010)   Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is required for the biosynthesis of photoreceptor phosphodiesterase (PDE). Gene defects in AIPL1 cause a heterogeneous set of conditions ranging from Leber's congenital amaurosis (LCA), the severest form of early-onset retinal degeneration, to milder forms such as retinitis pigmentosa (RP) and cone-rod dystrophy. In mice, null and hypomorphic alleles cause retinal degeneration similar to human LCA and RP, respectively. Thus these mouse models represent two ends of the disease spectrum associated with AIPL1 gene defects in humans. We evaluated whether adeno-associated virus (AAV)-mediated gene replacement therapy in these models could restore PDE biosynthesis in rods and cones and thereby improve photoreceptor survival. We validated the efficacy of human AIPL1 (isoform 1) replacement gene controlled by a promoter derived from the human rhodopsin kinase (RK) gene, which is active in both rods and cones. We found substantial and long-term rescue of the disease phenotype as a result of transgene expression. This is the first gene therapy study in which both rods and cones were targeted successfully with a single photoreceptor-specific promoter. We propose that the vector and construct design used in this study could serve as a prototype for a human clinical trial.  

5.730           Efficient transduction of non-human primate motor neurons after intramuscular delivery of recombinant AAV serotype 6

Towne, C., Schneider, B.L., Kieran, D., Redmond Jr., D.E. and Aebischer, P. Gene Therapy, 17(1), 141-146 (2010)   Retrograde transport of viral vectors in the rodent spinal cord provides a powerful means to administer a therapeutic transgene from the innervated musculature. With the aim of scaling up this approach to non-human primates, we have injected recombinant adeno-associated vectors (rAAV) serotype 6 expressing enhanced green fluorescent protein (eGFP) into the gastrocnemius muscle of African green monkeys to determine whether this results in efficient transgene delivery to lumbar motor neurons. Cells expressing eGFP were observed across more than 1 cm of the spinal cord 4 weeks after intramuscular injection, reaching more than half of motor neurons in some cross-sections. Furthermore, quantitative PCR on the spinal cord tissue confirmed that eGFP expression within motor neurons was due to bona fide retrograde transport of the vector genome from the muscle. Although infiltrations of macrophages and lymphocytes were observed in the rAAV2/6-injected muscle, there was no detectable immune response within the transduced region of the spinal cord. These findings imply that retrograde delivery of rAAV serotype 6 in a primate species constitutes a non-invasive and robust approach to transduce motor neurons, a crucial target cell population in neurodegenerative disorders, such as amyotrophic lateral sclerosis and spinal muscular atrophy.  

5.731           Endogenous Leptin Signaling in the Caudal Nucleus Tractus Solitarius and Area Postrema Is Required for Energy Balance Regulation

Hayes, M.R., Skibicka, K.P., Leichner, T.M., Guarnieri, D.J., DiLeone, R.J., Bence, K.K. and Grill. H.J. Cell Metabolism, 11, 77-83 (2010)   Medial nucleus tractus solitarius (mNTS) neurons express leptin receptors (LepRs), and intra-mNTS delivery of leptin reduces food intake and body weight. Here, the contribution of endogenous LepR signaling in mNTS neurons to energy balance control was examined. Knockdown of LepR in mNTS and area postrema (AP) neurons of rats (LepRKD) via adeno-associated virus short hairpin RNA-interference (AAV-shRNAi) resulted in significant hyperphagia for chow, high-fat, and sucrose diets, yielding increased body weight and adiposity. The chronic hyperphagia of mNTS/AP LepRKD rats is likely mediated by a reduction in leptin potentiation of gastrointestinal satiation signaling, as LepRKD rats showed decreased sensitivity to the intake-reducing effects of cholecystokinin. LepRKD rats showed increased basal AMP-kinase activity in mNTS/AP micropunches, and pharmacological data suggest that this increase provides a likely mechanism for their chronic hyperphagia. Overall these findings demonstrate that LepRs in mNTS and AP neurons are required for normal energy balance control.  

5.732           AAV9-mediated erythropoietin gene delivery into the brain protects nigral dopaminergic neurons in a rat model of Parkinson's disease

Xue, Y-Q., Ma, B-F., Zhao, L-R., Tatom, J.B., Li, B., Jiang, L-X., Klein, R.L. and Duan, W-M. Gene Therapy, 17(1), 83-94 (2010)   We have recently shown that intrastriatal injection of recombinant human erythropoietin (EPO) protects dopaminergic (DA) neurons in the substantia nigra (SN) from 6-hydroxydopamine (6-OHDA) toxicity in a rat model of Parkinson's disease. However, systemic administration of EPO did not protect nigral DA neurons, suggesting that the blood–brain barrier limits the passage of EPO protein into the brain. In the present study, we used an adeno-associated viral (AAV) serotype 9 (AAV9) vector to deliver the human EPO gene into the brain of 6-OHDA-lesioned rats. We observed that expression of the human EPO gene was robust and stable in the striatum and the SN for up to 10 weeks. EPO-immunoreactive (IR) cells were widespread throughout the injected striatum, and EPO-IR neurons and fibers were also found in the ipsilateral SN. Enzyme-linked immunosorbent assay and western blot analyses exhibited dramatic levels of EPO protein in the injected striatum. As a result, nigral DA neurons were protected against 6-OHDA-induced toxicity. Amphetamine-induced rotational asymmetry and spontaneous forelimb use asymmetry were both attenuated. Interestingly, we also observed that intrastriatal injection of AAV9-EPO vectors led to increased numbers of red blood cells in peripheral blood. This highlights the importance of using an inducible gene delivery system for EPO gene delivery.  

5.733           Optimization of stealth adeno-associated virus vectors by randomization of immunogenic epitopes

Maersch, S., Huber, A., Büning, H., Hallek, M. and Perabo, L. Virology, 397, 167-175 (2010)   Therapeutic gene transfer by adeno-associated virus of serotype 2 (AAV-2) vectors is hampered in patients with pre-existing immunity. Molecular engineering was recently used to identify key immunogenic amino acid residues of the viral capsid and generate mutants with decreased antibody recognition. Here we explored the importance of finely tuning amino acid identity at immunogenic sites to optimize vector phenotype. A capsid library was generated by codon randomization at five positions where substitutions were shown to yield antibody evading phenotypes. Screening this library to isolate immune-escaping mutants allowed an exhaustive scan of combinations of the 20 natural amino acids at each position and yielded variants that remained infectious when incubated with serum or IVIG concentrations that completely neutralize AAV-2. Clones obtained replacing different residues at the same positions displayed strikingly different phenotypes, demonstrating that a precise choice of amino acid substitutions is fundamental to optimize immune-escaping, packaging ability, infectivity and tropism.  

5.734           Therapeutic potential of genetically modified adult stem cells for osteopenia

Kumar, S., Nagy, T.R. and Ponnazhagan, S. Gene Therapy, 17, 105-116 (2010)   Adult stem cells have therapeutic potential because of their intrinsic capacity for self-renewal, especially for bone regeneration. The present study shows the utility of ex vivo modified mesenchymal stem cells (MSC) to enhance bone density in an immunocompetent mouse model of osteopenia. MSC were transduced ex vivo with a recombinant adeno-associated virus 2 (rAAV2) expressing bone morphogenetic protein 2 (BMP2) under the transcriptional control of collagen type-1α promoter. To enrich bone homing in vivo, we further modified the cells to transiently express the mouse α4 integrin. The modified MSC were systemically administered to ovariectomized, female C57BL/6 mice. Effects of the therapy were determined by dual-energy X-ray absorptiometry, 3D micro-CT, histology and immunohistochemistry for up to 6 months. Results indicated that mice transplanted with MSC expressing BMP2 showed significant increase in bone mineral density and bone mineral content (P<0.001) with relatively better proliferative capabilities of bone marrow stromal cells and higher osteocompetent pool of cells compared to control animals. Micro-CT analysis of femora and other bone histomorphometric analyses indicated more trabecular bone following MSC-BMP2 therapy. Results obtained by transplanting genetically modified MSC from green fluorescent protein transgenic mouse suggested that production of BMP2 from transplanted MSC also influenced the mobilization of endogenous progenitors for new bone formation.  

5.735           Depletion of Virion-Associated Divalent Cations Induces Parvovirus Minute Virus of Mice To Eject Its Genome in a 3'-to-5' Direction from an Otherwise Intact Viral Particle

Cotmore, S.F., Hafenstein, S. and Tattersall, P.

1. Virol., 84(4), 1945-1956 (2010)

  We describe a structural rearrangement that can occur in parvovirus minute virus of mice (MVMp) virions following prolonged exposure to buffers containing 0.5 mM EDTA. Such particles remain stable at 4°C but undergo a conformational shift upon heating to 37°C at pH 7.2 that leads to the ejection of much of the viral genome in a 3'-to-5' direction, leaving the DNA tightly associated with the otherwise intact capsid. This rearrangement can be prevented by the addition of 1 mM CaCl2 or MgCl2 prior to incubation at 37°C, suggesting that readily accessible divalent cation binding sites in the particle are critical for genome retention. Uncoating was not seen following the incubation of virions at pH 5.5 and 37°C or at pH 7.2 and 37°C in particles with subgenomic DNA, suggesting that pressure exerted by the full-length genome may influence this process. Uncoated genomes support complementary-strand synthesis by T7 DNA polymerase, but synthesis aborts upstream of the right-hand end, which remains capsid associated. We conclude that viral genomes are positioned so that their 3' termini and coding sequences can be released from intact particles at physiological temperatures by a limited conformational rearrangement. In the presence of divalent cations, incremental heating between 45°C and 65°C induces structural transitions that first lead to the extrusion of VP1 N termini, followed by genome exposure. However, in cation-depleted virions, the sequence of these shifts is blurred. Moreover, cation-depleted particles that have been induced to eject their genomes at 37°C continue to sequester their VP1 N termini within the intact capsid, suggesting that these two extrusion events represent separable processes.  

5.736           B Cell Transduction of Capsid Mutant (Y730F) Adeno-Associated Virus-2 Vectors with Mage-A3 Gene for Immunotherapy of Colorectal Cancer

Batchu, R.B., Qazi, A.M., Seward, S., Haider, M., Khalil, K., Semaan, A., Steffes, C., Madhu, P. and Weaver, D.W.

1. Surg. Res., 158(2), 197

  Introduction: Colorectal cancer (CRC) is the second most common cause of mortality in cancers. Systemic chemotherapy with drug combinations increases overall survival, however it is associated with unsatisfactory toxicity profiles. Immunotherapy exploits naturally occurring defense system, embodies an ideal nontoxic treatment capable of evoking tumor-specific cytotoxic T lymphocyte (CTL) responses to kill cancer cells. Although dendritic cells (DCs) have primary cellular responsibility as antigen presenting cells (APC) for T-cell priming in vivo, activated B cells also have ability to function as APCs. Further activated B cells have advantages over DCs for their ability to expand in vitro and also have been safely piloted as part of vaccines. MAGE-A3 antigen is specifically expressed in tumor cells of a majority of colon cancer patients, making it as ideal target for immunotherapy of CRC. Although self-complimentary adeno-associated viral vectors-2 (scAAV-2) vectors have been successfully used in clinical trails, it require large vector doses since majority of the vector particles are subjected degradation by ubiquitin pathway after tyrosine phosphorylation of its VP3 capsid protein at 730 amino acid. Here we show an efficient transduction of activated B cells with scAAV-2 vector carrying MAGE-A3 with tyrosine to phenylalanine capsid mutation at amino acid 730 (Y730F) to circumvent ubiquitin mediated degradation resulting in high trans-gene expression. Methods: 293-CD40 cell line used for the generation of activated B cells was cultured in IMDM medium supplemented with 10% FCS. MAGE-A3 gene was cloned in scAAV-2 vector and recombinant viral particles were generated in 293 cell line and purified by iodixanol density gradient centrifugation. Transduction of activated B cells was conducted both with wild type and Y730F mutant scAAV-2 recombinant viral vectors carrying MAGE-A3 gene. Intracellular staining and FACS analysis were conducted to see the expression of antigen. Results: Expression of MAGE-A3 tumor specific antigen was confirmed in several colon cancer cell lines. Various AAV serotypes were tested for B cell transduction and confirmed efficient transduction with AAV-2 serotype with self-complimentary vector. Further we observed an efficient transduction of activated B cells with Y530F mutant scAAV-2 compared with wild type AAV-2 vectors. IFN γ secretion was confirmed in co-cultures of T cell and transduced B cell indicating the generation CTLs. Generation of MAGE-A3-specific cytotoxic T cells using scAAV-2 as gene delivery vehicle with CD40-activated B cells as antigen presenting cells holds the promise of specifically killing CRC cells. Conclusions: For the first time we showed an efficient B cell transduction of new generation scAAV-2 MAGE-A3 (Y730F) vectors. Use of B cells as APCs allowed us to expand them in vitro unlike DCs and we further we significantly enhanced the MAGE-A3 trans-gene expression by using Y730F capsid mutant scAAV-2. Gene therapy with novel AAV vector transduced B cells to enhance anti-tumor immunity is a promising new approach to treat CRC.  

5.737           A nontoxic derivative of lipopolysaccharide increases immune responses to Gardasil^® HPV vaccine in mice

Han, J.E., Kim, H.K., Park, S.A., Lee, S.J., Kim, H.J., Son, G.H., Kim, Y.T., Cho, Y.J., Kim, H-J. and Lee, N.G. Int. Immunopharmacol., 10(2), 169-176 (2010)   Human papillomavirus (HPV) is the causative agent of cervical cancer, the second most common cause of cancer death in women worldwide. The licensed HPV vaccine Gardasil^® from Merck & Co. is a quadrivalent vaccine containing virus-like particles (VLPs) of the L1 proteins from HPV types 6, 11, 16, and 18 adsorbed on aluminum salts (alum). CIA07 is an immunostimulatory agent comprised of bacterial DNA fragments (CIA02) and a nontoxic derivative of lipopolysaccharide (CIA05) that has been shown to have antitumor activity and adjuvant activity for viral and bacterial vaccine antigens. We investigated whether these CIAs are capable of promoting the immune response to Gardasil^®. Balb/c mice were immunized intramuscularly twice three weeks apart with 1/20 human dose of Gardasil^® alone or in combination with CIA02, CIA05 or both, and immune responses were assessed. The serum anti-HPV16 L1 VLP IgG antibody titer was significantly higher in mice administered CIA05 or CIA05 plus CIA02, but not in those given CIA02, compared with mice given Gardasil^® alone. A secreted alkaline phosphatase (SEAP)-based pseudovirus neutralization assay showed increased neutralizing antibody titers in both CIA05 and CIA05 plus CIA02 groups. Coadministration of CIA05 with Gardasil^® led to a marked increase in serum IgG2a antibody titer and the percentage of interferon (IFN)-γ⁺ cells in the spleen, indicating that CIA05 effectively promotes Th1-type immune responses. These data indicate that CIA05, in synergy with alum, enhances the immune response to HPV L1 VLPs and suggest its potential as an adjuvant for the development of a potent prophylactic HPV vaccine.  

5.738           Microglia Acquire Distinct Activation Profiles Depending on the Degree of α-Synuclein Neuropathology in a rAAV Based Model of Parkinson's Disease

PloSOne, 5(1), e8784 (2010)   Post-mortem analysis of brains from Parkinson's disease (PD) patients strongly supports microglia activation and adaptive immunity as factors contributing to disease progression. Such responses may be triggered by α-synuclein (α-syn), which is known to be the main constituent of the aggregated proteins found in Lewy bodies in the brains of PD patients. To investigate this we used a recombinant viral vector to express human α-syn in rat midbrain at levels that induced neuronal pathology either in the absence or the presence of dopaminergic cell death, thereby mimicking early or late stages of the disease. Microglia activation was assessed by stereological quantification of Mac1+ cells, as well as the expression patterns of CD68 and MCH II. In our study, when α-syn induced neuronal pathology but not cell death, a fast transient increase in microglia cell numbers resulted in the long-term induction of MHC II+ microglia, denoting antigen-presenting ability. On the other hand, when α-syn induced both neuronal pathology and cell death, there was a delayed increase in microglia cell numbers, which correlated with long-lasting CD68 expression and a morphology reminiscent of peripheral macrophages. In addition T-lymphocyte infiltration, as judged by the presence of CD4+ and CD8+ cells, showed distinct kinetics depending on the degree of neurodegeneration, and was significantly higher when cell death occurred. We have thus for the first time shown that the microglial response differs depending on whether α-syn expression results on cell death or not, suggesting that microglia may play different roles during disease progression. Furthermore, our data suggest that the microglial response is modulated by early events related to α-syn expression in substantia nigra and persists at the long term.  

5.739           Directed evolution of adeno-associated virus for glioma cell transduction

Maguire, C.A., Gianni, D., Meijer, D.H., Shaket, L.A., Wakimoto, H., Rabkin, S.D., Gao, G. and Sena-Esteves, M.

1. Neurooncol., 96, 337-347 (2010)

  Glioblastoma multiforme (GBM) is a serious form of brain cancer for which there is currently no effective treatment. Alternative strategies such as adeno-associated virus (AAV) vector mediated-genetic modification of brain tumor cells with genes encoding anti-tumor proteins have shown promising results in preclinical models of GBM, although the transduction efficiency of these tumors is often low. As higher transduction efficiency of tumor cells should lead to enhanced therapeutic efficacy, a means to rapidly engineer AAV vectors with improved transduction efficiency for individual tumors is an attractive strategy. Here we tested the possibility of identifying high-efficiency AAV vectors for human U87 glioma cells by selection in culture of a newly constructed chimeric AAV capsid library generated by DNA shuffling of six different AAV cap genes (AAV1, AAV2, AAV5, AAVrh.8, AAV9, AAVrh.10). After seven rounds of selection, we obtained a chimeric AAV capsid that transduces U87 cells at high efficiency (97% at a dose of 104 genome copies/cell), and at low doses it was 1.45–1.6-fold better than AAV2, which proved to be the most efficient parental capsid. Interestingly, the new AAV capsid displayed robust gene delivery properties to all glioma cells tested (including primary glioma cells) with relative fluorescence indices ranging from 1- to 14-fold higher than AAV2. The selected vector should be useful for in vitro glioma research when efficient transduction of several cell lines is required, and provides proof-of-concept that an AAV library can be used to generate AAV vectors with enhanced transduction efficiency of glioma cells.  

5.740           Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain

Kornum, B.R., Stott, S.R.W., Mattson, B., Wisman, L., Ettrup, A., Hermening, S., Knudsen, G.M. and Kirik, D. Exp. Neurol., 222, 70-85 (2010)   Viral vector-mediated gene transfer has emerged as a powerful means to target transgene expression in the central nervous system. Here we characterized the efficacy of serotypes 1 and 5 recombinant adeno-associated virus (rAAV) vectors encoding green fluorescent protein (GFP) after stereotaxic delivery to the neonatal rat and minipig striatum. The efficiency of GFP expression and the phenotype of GFP-positive cells were assessed within the forebrain at different time points up to 12 months after surgery. Both rAAV1-GFP and rAAV5-GFP delivery resulted in transduction of the striatum as well as striatal input and output areas, including large parts of the cortex. In both species, rAAV5 resulted in a more widespread transgene expression compared to rAAV1. In neonatal rats, rAAV5 also transduced several other areas such as the olfactory bulbs, hippocampus, and septum. Phenotypic analysis of the GFP-positive cells, performed using immunohistochemistry and confocal microscopy, showed that most of the GFP-positive cells by either serotype were NeuN-positive neuronal profiles. The rAAV5 vector further displayed the ability to transduce non-neuronal cell types in both rats and pigs, albeit at a low frequency. Our results show that striatal delivery of rAAV5 vectors in the neonatal brain represents a useful tool to express genes of interest both in the basal ganglia and the neocortex. Furthermore, we apply, for the first time, viral vector-mediated gene transfer to the pig brain providing the opportunity to study effects of genetic manipulation in this non-primate large animal species. Finally, we generated an atlas of the Göttingen minipig brain for guiding future studies in this large animal species.  

5.741           Immune responses to JC virus in patients with multiple sclerosis treated with natalizumab: a cross-sectional and longitudinal study

Jilek, S., Jaquiery, E., Hirsch, H.H., Lysandropoulos, A., Canales, M., Guignard, L., Schluep, M., Pantaleo, G. and Du Pasquier, R.A. Lancet Neurol., 9, 264-272 (2010)   Background Natalizumab is used to prevent relapses and progression of disability in patients with multiple sclerosis but has been associated with progressive multifocal leukoencephalopathy (PML). We aimed to better understand the associations between JC virus, which causes PML, and natalizumab treatment. Methods We prospectively assessed patients with multiple sclerosis who started treatment with natalizumab. Blood and urine samples were tested for the presence of JC virus DNA with quantitative real-time PCR before treatment and at regular intervals after treatment onset for up to 18 months. At the same timepoints, by use of proliferation and enzyme-linked immunospot assays, the cellular immune responses against JC virus, Epstein-Barr virus, cytomegalovirus, myelin oligodendrocyte glycoprotein, and myelin oligodendrocyte basic protein (MOBP) were assessed. Humoral immune response specific to JC virus was assessed with an enzyme immunoassay. The same experiments were done on blood samples from patients with multiple sclerosis before and 10 months after the start of interferon beta treatment. Findings We assessed 24 patients with multiple sclerosis who received natalizumab and 16 who received interferon beta. In patients treated with natalizumab, JC virus DNA was not detected in the blood at any timepoint. However, JC virus DNA was present in the urine of six patients and in most of these patients the concentrations of JC virus DNA were stable over time. Compared with pretreatment values, the cellular immune response was increased to cytomegalovirus at 6 months, to JC virus at 1, 9, and 12 months, and to Epstein-Barr virus and MOBP at 12 months. Humoral responses remained stable. There were no increases in cellular immune responses specific to the viruses or myelin proteins in the 16 patients treated with interferon beta. Interpretation Natalizumab increases cellular immune responses specific to viruses and myelin proteins in the peripheral blood after 1 year, without evidence of viral reactivation.  

5.742           A broad-spectrum antiviral targeting entry of enveloped viruses

Wolf, M.C. et al PNAS, 107(7), 3157-3162 (2010)   We describe an antiviral small molecule, LJ001, effective against numerous enveloped viruses including Influenza A, filoviruses, poxviruses, arenaviruses, bunyaviruses, paramyxoviruses, flaviviruses, and HIV-1. In sharp contrast, the compound had no effect on the infection of nonenveloped viruses. In vitro and in vivo assays showed no overt toxicity. LJ001 specifically intercalated into viral membranes, irreversibly inactivated virions while leaving functionally intact envelope proteins, and inhibited viral entry at a step after virus binding but before virus–cell fusion. LJ001 pretreatment also prevented virus-induced mortality from Ebola and Rift Valley fever viruses. Structure–activity relationship analyses of LJ001, a rhodanine derivative, implicated both the polar and nonpolar ends of LJ001 in its antiviral activity. LJ001 specifically inhibited virus–cell but not cell–cell fusion, and further studies with lipid biosynthesis inhibitors indicated that LJ001 exploits the therapeutic window that exists between static viral membranes and biogenic cellular membranes with reparative capacity. In sum, our data reveal a class of broad-spectrum antivirals effective against enveloped viruses that target the viral lipid membrane and compromises its ability to mediate virus–cell fusion.  

5.743           Optimized adeno-associated viral vector-mediated striatal DOPA delivery restores sensorimotor function and prevents dyskinesias in a model of advanced Parkinson’s disease

Björklund, T.,Carlsson, T., Cederfjäll, E.A., Carta, M. and Kirik, D. Brain, 133(2), 496-511 (2010)   Viral vector-mediated gene transfer utilizing adeno-associatedviral vectors has recently entered clinical testing as a noveltool for delivery of therapeutic agents to the brain. Clinicaltrials in Parkinson’s disease using adeno-associated viralvector-based gene therapy have shown the safety of the approach.Further efforts in this area will show if gene-based approachescan rival the therapeutic efficacy achieved with the best pharmacologicaltherapy or other, already established, surgical interventions.One of the strategies under development for clinical applicationis continuous 3,4-dihydroxyphenylalanine delivery. This approachhas been shown to be efficient in restoring motor function andreducing established dyskinesias in rats with a partial lesionof the nigrostriatal dopamine projection. Here we utilized highpurity recombinant adeno-associated viral vectors serotype 5coding for tyrosine hydroxylase and its co-factor synthesizingenzyme guanosine-5'-triphosphate cyclohydrolase-1, deliveredat an optimal ratio of 5 : 1, to show that the enhanced 3,4-dihydroxyphenylalanineproduction obtained with this optimized delivery system resultsin robust recovery of function in spontaneous motor tests aftercomplete dopamine denervation. We found that the therapeuticefficacy was substantial and could be maintained for at least6 months. The tyrosine hydroxylase plus guanosine-5'-triphosphatecyclohydrolase-1 treated animals were resistant to developingdyskinesias upon peripheral L-3,4-dihydroxyphenylalanine drugchallenge, which is consistent with the interpretation thatcontinuous dopamine stimulation resulted in a normalizationof the post-synaptic response. Interestingly, recovery of forelimbuse in the stepping test observed here was maintained even aftera second lesion depleting the serotonin input to the forebrain,suggesting that the therapeutic efficacy was not solely dependenton dopamine synthesis and release from striatal serotonergicterminals. Taken together these results show that vector-mediatedcontinuous 3,4-dihydroxyphenylalanine delivery has the potentialto provide significant symptomatic relief even in advanced stagesof Parkinson’s disease.  

5.744           R7BP Complexes with RGS9-2 and RGS7 in the Striatum Differentially Control Motor Learning and Locomotor Responses to Cocaine

Anderson, G.R., Cao, Y., Davidson, S., Truong, H.V., Pravetoni, M., Thomas, M.J., Wickman, K., Giesler Jr, G.J. and Martemyanov, K.A. Neuropsychopharmacol., 35, 1040-1050 (2010)   In the striatum, signaling through G protein-coupled dopamine receptors mediates motor and reward behavior, and underlies the effects of addictive drugs. The extent of receptor responses is determined by RGS9-2/Gβ5 complexes, a striatally enriched regulator that limits the lifetime of activated G proteins. Recent studies suggest that the function of RGS9-2/Gβ5 is controlled by the association with an additional subunit, R7BP, making elucidation of its contribution to striatal signaling essential for understanding molecular mechanisms of behaviors mediated by the striatum. In this study, we report that elimination of R7BP in mice results in motor coordination deficits and greater locomotor response to morphine administration, consistent with the essential role of R7BP in maintaining RGS9-2 expression in the striatum. However, in contrast to previously reported observations with RGS9-2 knockouts, mice lacking R7BP do not show higher sensitivity to locomotor-stimulating effects of cocaine. Using a striatum-specific knockdown approach, we show that the sensitivity of motor stimulation to cocaine is instead dependent on RGS7, whose complex formation with R7BP is dictated by RGS9-2 expression. These results indicate that dopamine signaling in the striatum is controlled by concerted interplay between two RGS proteins, RGS7 and RGS9-2, which are balanced by a common subunit, R7BP.  

5.745           Lipidomic study of intracellular Singapore grouper iridovirus

Wu, J., Chan, R., Wenk, M.R. and Hew, C-L. Virology, 399, 248-256 (2010)   Singapore grouper iridoviruses (SGIV) infected grouper cells release few enveloped extracellular viruses by budding and many unenveloped intracellular viruses following cell lysis. The lipid composition and function of such unenveloped intracellular viruses remain unknown. Detergent treatment of the intracellular viruses triggered the loss of viral lipids, capsid proteins and infectivity. Enzymatic digestion of the viral lipids with phospholipases and sphingomyelinase retained the viral capsid proteins but reduced infectivity. Over 220 lipid species were identified and quantified from the viruses and its producer cells by electrospray ionization mass spectrometry. Ten caspid proteins that dissociated from the viruses following the detergent treatments were identified by MALDI-TOF/TOF-MS/MS. Five of them were demonstrated to be lipid-binding proteins. This is the first research detailing the lipidome and lipid–protein interactions of an unenveloped virus. The identified lipid species and lipid-binding proteins will facilitate further studies of the viral assembly, egress and entry.  

5.746           In-Solution Virus Capture Assay Helps Deconstruct Heterogeneous Antibody Recognition of Human Immunodeficiency Virus Type 1

Leaman, D.P., Kinkead, H. and Zwick, M.B.

1. Virol., 84(7), 3382-3395 (2010)

  Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) on whole virions is heterogeneous, so molecular analysis of Env with monoclonal antibodies (MAbs) is challenging. Virus capture assays (VCAs) involving immobilized MAbs are typically used, but these assays suffer from immobilization artifacts and do not provide binding constants. Furthermore, we show here that certain HIV-1 neutralizing MAbs, including 2G12, 4E10, 2F5, Z13e1, and D5, will capture virion particles completely devoid of Env. We modified the VCA such that MAbs and virions are incubated in solution, and unbound MAbs are removed prior to the capture step. This modification nearly eliminated evidence of Env-independent binding by MAbs to virions and allowed determination of apparent affinity constants in solution. Three important qualitative observations were further revealed. First, neutralizing MAbs 2F5, 4E10, and Z13e1 against the membrane-proximal external region (MPER) of HIV-1 gp41 were found to capture virions efficiently only if a significant amount of uncleaved gp160 or synthetic MPER peptide was present. Second, we show how non-native forms of Env vary by Env genotype and that Env from HIV-1JR-FL is more homogeneously trimeric than that from HIV-1JR-CSF. Third, we determined that Env containing all or parts of gp41, including uncleaved gp160, binds spontaneously to free virions. This exogenous Env is an indiscriminate molecular "bridge" between Env-specific Ab and virions and can affect VCA analyses, particularly using pseudotyped virions. Heterogeneity in Env from endogenous and exogenous sources might also subvert humoral immunity to HIV-1, so in-solution VCAs may help to dissect this heterogeneity for vaccine design purposes.  

5.747           Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system

Ehlert, E.M., Eggers, R., Niclou, S.P. and Verhaagen, J. BMC Neuroscience, 11, 20-30 (2010)   Background After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs) and several axon guidance molecules, including all members of the secreted (class 3) Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi) is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV) mediated expression of short hairpin RNAs (shRNAs) to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1) and Neuropilin 2 (Npn-2). Results We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG) of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents. Conclusions RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation.  

5.748           An efficient process for the purification of helper-dependent adenoviral vector and removal of helper virus by iodixanol ultracentrifugation

Dormond, E., Chahal, P., Bernier, A., Tran, R., Perrier, M. and Kamen, A.

1. Virol. Methods, 165, 83-89 (2010)

  The preparation of large amount of purified helper-dependent adenoviral vector material is hampered by the lack of development of downstream processes with proven records on separation and recovery efficiencies. In order to facilitate the use of clinical-grade helper-dependent virus material for large-scale in vivo studies, a three-step purification scheme consisting of (1) an anion-exchange chromatography for initial capturing of virus, (2) a shallow iodixanol density gradient ultracentrifugation for the removal of helper virus from helper-dependent virus, and (3) a size-exclusion chromatography for the removal of iodixanol and residual protein contaminants as a polishing step was developed. The use of a fast iodixanol density ultracentrifugation step was highly effective in separating infectious helper-dependent virus from contaminating helper virus. The overall downstream processing scheme gave 80% infectious particle yield. The contamination ratio of helper virus in the helper-dependent virus preparation are reduced from 2.57 to 0.03% corresponding to a reduction of helper virus by factors of 85 by two iodixanol purification steps. It was also demonstrated that size-exclusion chromatography is an excellent step for the removal of iodixanol and polishing of the final helper-dependent virus preparation.  

5.749           Differential Transduction Following Basal Ganglia Administration of Distinct Pseudotyped AAV Capsid Serotypes in Nonhuman PrimatesSerotype Transduction in Nonhuman Primates

Dodiya, H.B., Bjorklund, T., Stansell, J., Mandel, R.J., Kirik, D. and Kordower, J.H. Molecular Therapy, 18(3), 579-587 (2010)   We examined the transduction efficiency of different adeno-associated virus (AAV) capsid serotypes encoding for green fluorescent protein (GFP) flanked by AAV2 inverted terminal repeats in the nonhuman primate basal ganglia as a prelude to translational studies, as well as clinical trials in patients with Parkinson's disease (PD). Six intact young adult cynomolgus monkeys received a single 10 µl injection of AAV2/1-GFP, AAV2/5-GFP, or AAV2/8-GFP pseudotyped vectors into the caudate nucleus and putamen bilaterally in a pattern that resulted in each capsid serotype being injected into at least four striatal sites. GFP immunohistochemistry revealed excellent transduction rates for each AAV pseudotype. Stereological estimates of GFP⁺ cells within the striatum revealed that AAV2/5-GFP transduces significantly higher number of cells than AAV2/8-GFP (P < 0.05) and there was no significant difference between AAV2/5-GFP and AAV2/1-GFP (P = 0.348). Consistent with this result, Cavalieri estimates revealed that AAV2/5-GFP resulted in a significantly larger transduction volume than AAV2/8-GFP (P < 0.05). Each pseudotype transduced striatal neurons effectively [>95% GFP⁺ cells colocalized neuron-specific nuclear protein (NeuN)]. The current data suggest that AAV2/5 and AAV2/1 are superior to AAV2/8 for gene delivery to the nonhuman primate striatum and therefore better candidates for therapeutic applications targeting this structure.  

5.750           Dual Reporter Comparative Indexing of rAAV Pseudotyped Vectors in Chimpanzee AirwayComparative Indexing of rAAV in Chimpanzee Airway

Flotte, T.R., Fischer, A.C., Boetzmann, J., Mueller, C., Cebotaru, L., Yan, Z., Wang, L., Wilson, J.M., Guggino, W.B. and Engelhardt, J.F. Molecular Therapy, 18(3), 594-600 (2010)   Selecting the most efficient recombinant adeno-associated virus (rAAV) serotype for airway gene therapy has been difficult due to cross-specific differences in tropism and immune response between humans and animal models. Chimpanzees—the closest surviving genetic relative of humans—provide a valuable opportunity to select the most effective serotypes for clinical trials in humans. However, designing informative experiments using this protected species is challenging due to limited availability and experimental regulations. We have developed a method using Renilla luciferase (RL) and firefly luciferase (FL) reporters to directly index the relative transduction and immune response of two promising rAAV serotypes following lung coinfection. Analysis of differential luciferase activity in chimpanzee airway brushings demonstrated a 20-fold higher efficiency for rAAV1 over rAAV5 at 90 days, a finding that was similar in polarized human airway epithelia. T-cell responses to AAV5 capsid were stronger than AAV1 capsid. This dual vector indexing approach may be useful in selecting lead vector serotypes for clinical gene therapy and suggests rAAV1 is preferred for cystic fibrosis.  

5.751           Natural Strain Variation and Antibody Neutralization of Dengue Serotype 3 Viruses

Wahala, W.M.P.B., Donaldson, E.F., de Alwis, R., Accavitti-Loper, M.A., Baric, R.S. and de Silva, A.M. PloSPathogens, 6(3), e1000821 (2010)   Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge “type specific” epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.  

5.752           Use of the piggyBac transposon to create HIV-1 gag transgenic insect cell lines for continuous VLP production

Lynch, A.G., Tanzer, F., Fraser, M.J., Shephard, E.G., Williamson, A-L. and  Rybicki, E.P. BMC Biotechnol., 10, 30-42 (2010)   Background Insect baculovirus-produced Human immunodeficiency virus type 1 (HIV-1) Gag virus-like-particles (VLPs) stimulate good humoral and cell-mediated immune responses in animals and are thought to be suitable as a vaccine candidate. Drawbacks to this production system include contamination of VLP preparations with baculovirus and the necessity for routine maintenance of infectious baculovirus stock. We used piggyBac transposition as a novel method to create transgenic insect cell lines for continuous VLP production as an alternative to the baculovirus system. Results Transgenic cell lines maintained stable gag transgene integration and expression up to 100 cell passages, and although the level of VLPs produced was low compared to baculovirus-produced VLPs, they appeared similar in size and morphology to baculovirus-expressed VLPs. In a murine immunogenicity study, whereas baculovirus-produced VLPs elicited good CD4 immune responses in mice when used to boost a prime with a DNA vaccine, no boost response was elicited by transgenically produced VLPs. Conclusion Transgenic insect cells are stable and can produce HIV Pr55 Gag VLPs for over 100 passages: this novel result may simplify strategies aimed at making protein subunit vaccines for HIV. Immunogenicity of the Gag VLPs in mice was less than that of baculovirus-produced VLPs, which may be due to lack of baculovirus glycoprotein incorporation in the transgenic cell VLPs. Improved yield and immunogenicity of transgenic cell-produced VLPs may be achieved with the addition of further genetic elements into the piggyBac integron.  

5.753           CULTURE OF HEPATITIS C VIRUS (HCV) IN PRIMARY HUMAN ADULT HEPATOCYTES: A PHYSIOLOGICAL MODEL FOR THE PRODUCTION OF AUTHENTIC INFECTIOUS PARTICLES

Podevin, P., Carpentier, A., Pene, v., Aoudjehane, L., Hernandez, C., Calle, V., Demignot, S., Scatton, O., Meritet, J-F., Bartenschlager, R., Wakita, T., Conti, F., Calmus, Y. and Rosenberg, A.R.

1. Hepatol., 52, Suppl.1, S259 (2010)

  Background and Aims: In the blood of HCV-infected patients, infectivity is mainly supported by particles of exceptionally low buoyant density corresponding to virus associated with host lipoproteins containing apolipoprotein B (ApoB). These complexes are believed to assemble within the hepatocyte, the cell type specialized in the secretion of very-low-density lipoproteins (VLDL). HCV can be grown in vitro in the hepatocarcinoma-derived cell line Huh-7, but the buoyant density of viral RNA-containing particles is higher for the virus produced in this cell-culture-based system (HCVcc) than for HCV in vivo. We have recently shown that HCV can also be grown in differentiated human hepatocytes maintained in primary culture upon inoculation with HCVcc. Here we have characterized the virus produced in primary culture (HCVpc) in comparison with HCVcc. Methods: The virus buoyant densities were determined in iodixanol gradients. Specific infectivity was calculated as the ratio of infectivity titer (focus-formation assay) to the viral RNA amount (viral load assay). Cells were compared for the secretion of ApoBcontaining lipoproteins by sequential ultracentrifugation of the culture supernatants to separate the VLDL, LDL, and high density lipoprotein (HDL) fractions. Results: Regardless of both the source of primary hepatocytes and the HCV genotype, the progeny virus HCVpc had higher specific infectivity than the input virus HCVcc, correlating with lower buoyant density of the viral RNA-containing particles. Both of these properties were lost after re-culture of HCVpc in Huh-7 cells. Irrespective of whether the cells were infected or not, ApoB was found mainly in the VLDL fraction for primary hepatocytes, whereas it was found only in the LDL and HDL fractions for Huh-7 cells. Upon infection, the majority of HCVRNA produced from primary hepatocytes and Huh-7 cells was found in the VLDL and HDL fraction, respectively. Conclusions: HCVpc has properties that distinguish it from HCVcc and more closely mimic those of HCV in vivo, consistent with the fact that differentiated human hepatocytes, contrary to Huh-7 transformed cells, have the ability to secrete authentic VLDL. Thus, our system of productive infection in primary human hepatocytes provides a most relevant in vitro model for studying the morphogenesis of authentic HCV.  

5.754           INSULIN RESISTANCE CORRELATES WITH LOW DENSITY HEPATITIS C VIRUS PARTICLES IN GENOTYPE 1 INFECTION

Bridge, S.H., Sheridan, D.A., Felmlee, D.J. and Nielsen, S.U. J: Hepatol., 52, Suppl. 1, S417 (2010)   Background: Chronic infection with hepatitis C virus (HCV) can induce insulin resistance (IR) in a genotype dependent manner. IR contributes to resistance to therapy and fibrosis progression and is more frequent in genotype 1 (G1) infections. HCV isolated from the blood of patients with chronic hepatitis C (CHC) is very heterogeneous in density (d). The population of virus particles at d < 1.07g/mL are associated with apolipoprotein B and are referred to as lipo-viro-particles (LVP). HCV-LVP have a higher specific infectivity than high density HCV particles in vivo for chimpanzees and in vitro in the HCVcc system. Aims: To evaluate the relationship between metabolic markers and  HCV-LVP in a well characterised cohort of G1 CHC patients. Methods: Fasting plasma samples were collected from 51 G1 CHC patients. A range of metabolic parameters were measured including insulin resistance as assessed by HOMA IR, total cholesterol, HDL and non HDL cholesterol, triglycerides, non-esterified fatty acids, apolipoproteins B and A1 (apoB and apoA1). HCV-LVP was measured on the same fasting sample by density ultracentrifugation using isotonic iodixanol gradients. The gradient was fractionated by a density cutoff d < 1.07g/mL and HCVRNA was quantitated in this fraction (HCV-LVP) and plasma (total viral load). Results: The mean fasting total viral load was 5.98±0.57 HCVRNA (log10 IU/mL) of which 24.1% was HCV-LVP (range 2.9–74.0%). HCV-LVP (log10 IU/mL) significantly correlated with HOMA IR (r = 0.383, p = 0.006). 18/51 G1 CHC patients had a HOMA IR >2. The correlation with HOMA IR remained significant after exclusion of 12 G1 CHC patients with additional metabolic syndrome as defined by International Diabetes Federation criteria (p = 0.015). HCV-LVP also significantly correlated with serum triglycerides (r = 0.265, p = 0.006), but this did not remain significant after exclusion of patients with metabolic syndrome (p = 0.136). There was no significant correlation with apoB, total cholesterol, HDL and non HDL cholesterol or with apoA1. Conclusion: In genotype 1 CHC patients insulin resistance positively correlates with HCV-LVP, a putative surrogate marker of infectious virus. This may explain why IR is associated with resistance to therapy and fibrosis progression.  

5.755           Comparison of AAV Serotypes for Gene Delivery to Dorsal Root Ganglion Neurons

Mason, M.R.J., Ehlert, E.M.E., Eggers, R., Pool, C.W., Hermening, S., Huseinovic, A., Timmermans, E., Blits, B. and Verhaagen, J. Molecular Therapy, 18(4), 715-724 (2010)   For many experiments in the study of the peripheral nervous system, it would be useful to genetically manipulate primary sensory neurons. We have compared vectors based on adeno-associated virus (AAV) serotypes 1, 2, 3, 4, 5, 6, and 8, and lentivirus (LV), all expressing green fluorescent protein (GFP), for efficiency of transduction of sensory neurons, expression level, cellular tropism, and persistence of transgene expression following direct injection into the dorsal root ganglia (DRG), using histological quantification and qPCR. Two weeks after injection, AAV1, AAV5, and AAV6 had transduced the most neurons. The time course of GFP expression from these three vectors was studied from 1 to 12 weeks after injection. AAV5 was the most effective serotype overall, followed by AAV1. Both these serotypes showed increasing neuronal transduction rates at later time points, with some injections of AAV5 yielding over 90% of DRG neurons GFP⁺ at 12 weeks. AAV6 performed well initially, but transduction rates declined dramatically between 4 and 12 weeks. AAV1 and AAV5 both transduced large-diameter neurons, IB4⁺ neurons, and CGRP⁺ neurons. In conclusion, AAV5 is a highly effective gene therapy vector for primary sensory neurons following direct injection into the DRG.  

5.756           Self-assembly of Severe Acute Respiratory Syndrome Coronavirus Membrane Protein

Tseng, Y-T., Wang, S-M., Huang, K-J., Lee, A. I-R., Chiang, C-C- and Wang, C-T.

1. Biol. Chem., 285(17), 12862-12872 (2010)

  Coronavirus membrane (M) protein can form virus-like particles (VLPs) when coexpressed with nucleocapsid (N) or envelope (E) proteins, suggesting a pivotal role for M in virion assembly. Here we demonstrate the self-assembly and release of severe acute respiratory syndrome coronavirus (SARS-CoV) M protein in medium in the form of membrane-enveloped vesicles with densities lower than those of VLPs formed by M plus N. Although efficient N-N interactions require the presence of RNA, we found that M-M interactions were RNA-independent. SARS-CoV M was observed in both the Golgi area and plasma membranes of a variety of cells. Blocking M glycosylation does not appear to significantly affect M plasma membrane labeling intensity, M-containing vesicle release, or VLP formation. Results from a genetic analysis indicate involvement of the third transmembrane domain of M in plasma membrane-targeting signal. Fusion proteins containing M amino-terminal 50 residues encompassing the first transmembrane domain were found to be sufficient for membrane binding, multimerization, and Golgi retention. Surprisingly, we found that fusion proteins lacking all three transmembrane domains were still capable of membrane binding, Golgi retention, and interacting with M. The data suggest that multiple SARS-CoV M regions are involved in M self-assembly and subcellular localization.  

5.757           Electron Cryotomography of Tula Hantavirus Suggests a Unique Assembly Paradigm for Enveloped Viruses

Huiskonen, J.T., Hepojoki, J., Laurinmäki, P., Vaheri, A., Lankinen, H., Butcher, S.J. and Grünewald, K.

1. Virol., 84(10), 4889-4897 (2010)

  Hantaviruses (family Bunyaviridae) are rodent-borne emerging viruses that cause a serious, worldwide threat to human health. Hantavirus diseases include hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Virions are enveloped and contain a tripartite single-stranded negative-sense RNA genome. Two types of glycoproteins, GN and GC, are embedded in the viral membrane and.  protrusions, or "spikes." The membrane encloses a ribonucleoprotein core, which consists of the RNA segments, the nucleocapsid protein, and the RNA-dependent RNA polymerase. Detailed information on hantavirus virion structure and glycoprotein spike composition is scarce. Here, we have studied the structures of Tula hantavirus virions using electron cryomicroscopy and tomography. Three-dimensional density maps show how the hantavirus surface glycoproteins, membrane, and ribonucleoprotein are organized. The structure of the GN-GC spike complex was solved to 3.6-nm resolution by averaging tomographic subvolumes. Each spike complex is a square-shaped assembly with 4-fold symmetry. Spike complexes formed ordered patches on the viral membrane by means of specific lateral interactions. These interactions may be sufficient for creating membrane curvature during virus budding. In conclusion, the structure and assembly principles of Tula hantavirus exemplify a unique assembly paradigm for enveloped viruses.  

5.758           Scalable production of influenza virus in HEK-293 cells for efficient vaccine manufacturing

Le Ru, A., Jacob, D., Transfiguracion, J. and Ansorge, S. Vaccine, 28, 3661-3671 (2010)   Cell culture processes offer an attractive alternative to conventional chicken egg-based influenza vaccine production methods. However, most protocols still rely on the use of adherent cells, which makes process scale-up a challenging issue. In this study, it is demonstrated that the HEK-293 human cell line is able to efficiently replicate influenza virus. Production in serum-free suspension of HEK-293 cultures resulted in high titers of infectious influenza viruses for different subtypes and variants including A/H1, A/H3 and B strains. After virus adaptation and optimization of infection conditions, production in 3-L bioreactor resulted in titers of up to 10⁹ IVP/mL demonstrating the scale-up potential of the process.  

5.759           A Differential Role for Macropinocytosis in Mediating Entry of the Two Forms of Vaccinia Virus into Dendritic Cells

Sandgren, K.J., Wilkinson, J., Miranda-Saksena, M., McInerney, G.M., Byth-Wilson, K., Robinson, P.J. and Cunningham, A.L. PloSPathogens, 6(4), e1000866 (2010)   Vaccinia virus (VACV) is being developed as a recombinant viral vaccine vector for several key pathogens. Dendritic cells (DCs) are specialised antigen presenting cells that are crucial for the initiation of primary immune responses; however, the mechanisms of uptake of VACV by these cells are unclear. Therefore we examined the binding and entry of both the intracellular mature virus (MV) and extracellular enveloped virus (EV) forms of VACV into vesicular compartments of monocyte-derived DCs. Using a panel of inhibitors, flow cytometry and confocal microscopy we have shown that neither MV nor EV binds to the highly expressed C-type lectin receptors on DCs that are responsible for capturing many other viruses. We also found that both forms of VACV enter DCs via a clathrin-, caveolin-, flotillin- and dynamin-independent pathway that is dependent on actin, intracellular calcium and host-cell cholesterol. Both MV and EV entry were inhibited by the macropinocytosis inhibitors rottlerin and dimethyl amiloride and depended on phosphotidylinositol-3-kinase (PI(3)K), and both colocalised with dextran but not transferrin. VACV was not delivered to the classical endolysosomal pathway, failing to colocalise with EEA1 or Lamp2. Finally, expression of early viral genes was not affected by bafilomycin A, indicating that the virus does not depend on low pH to deliver cores to the cytoplasm. From these collective results we conclude that VACV enters DCs via macropinocytosis. However, MV was consistently less sensitive to inhibition and is likely to utilise at least one other entry pathway. Definition and future manipulation of these pathways may assist in enhancing the activity of recombinant vaccinia vectors through effects on antigen presentation.  

5.760           Hepatitis C Virus Hypervariable Region 1 Modulates Receptor Interactions, Conceals the CD81 Binding Site, and Protects Conserved Neutralizing Epitopes

Bankwitz, D., Steinmann, E., Bitzegeio, J., Ciesek, S., Friesland, M., Herrmann, E., Zeisel, M.B., Baumert, T.F., Keck, Z-y., Foung, S.K.H., Pecheur, E-I- and Pietschmann, T.

1. Virol., 84(11), 5751-5763 (2010)

  The variability of the hepatitis C virus (HCV), which likely contributes to immune escape, is most pronounced in hypervariable region 1 (HVR1) of viral envelope protein 2. This domain is the target for neutralizing antibodies, and its deletion attenuates replication in vivo. Here we characterized the relevance of HVR1 for virus replication in vitro using cell culture-derived HCV. We show that HVR1 is dispensable for RNA replication. However, viruses lacking HVR1 ( HVR1) are less infectious, and separation by density gradients revealed that the population of HVR1 virions comprises fewer particles with low density. Strikingly, HVR1 particles with intermediate density (1.12 g/ml) are as infectious as wild-type virions, while those with low density (1.02 to 1.08 g/ml) are poorly infectious, despite quantities of RNA and core similar to those in wild-type particles. Moreover, HVR1 particles exhibited impaired fusion, a defect that was partially restored by an E1 mutation (I347L), which also rescues infectivity and which was selected during long-term culture. Finally, HVR1 particles were no longer neutralized by SR-B1-specific immunoglobulins but were more prone to neutralization and precipitation by soluble CD81, E2-specific monoclonal antibodies, and patient sera. These results suggest that HVR1 influences the biophysical properties of released viruses and that this domain is particularly important for infectivity of low-density particles. Moreover, they indicate that HVR1 obstructs the viral CD81 binding site and conserved neutralizing epitopes. These functions likely optimize virus replication, facilitate immune escape, and thus foster establishment and maintenance of a chronic infection.  

5.761           Frequent Endonuclease Cleavage at Off-target Locations In Vivo

Petek, L.M., Russell, D.W. and Miller, D.G. Molecular Therapy, 18(5), 983-986 (2010)   Target-site DNA breaks increase recombination frequencies, however, the specificity of the enzymes used to create them remains poorly defined. The location and frequency of off-target cleavage events are especially important when rare-cutting endonucleases are used in clinical settings. Here, we identify noncanonical cleavage sites of I-SceI that are frequently cut in the human genome by localizing adeno-associated virus (AAV) vector-chromosome junctions, demonstrating the importance of in vivo characterization of enzyme cleavage specificity.  

5.762           Mesenchymal Stem Cells Expressing Osteogenic and Angiogenic Factors Synergistically Enhance Bone Formation in a Mouse Model of Segmental Bone Defect

Kumar, S., Wan, C., Ramaswamy, G., Clemens, T.L. and Ponnazhagan, S. Molecular Therapy, 18(5), 1026-1034 (2010)   The potential of mesenchymal stem cells (MSC) in tissue regeneration is increasingly gaining attention. There is now accumulating evidence that MSC make an important contribution to postnatal vasculogenesis. During bone development and fracture healing, vascularization is observed before bone formation. The present study determined the potential of MSC, transduced ex vivo with a recombinant adeno-associated virus 6 (rAAV6) encoding bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) in a mouse model of segmental bone defect created in the tibiae of athymic nude mice. Mouse MSC that were mock-transduced or transduced with rAAV6-BMP2:VEGF were systemically transplanted following radiographic confirmation of the osteotomy. Effects of the therapy were determined by enzyme-linked immunosorbent assay measurements for BMP2 and VEGF, dual-energy X-ray absorptiometry (DXA) for bone density, three-dimensional microcomputed tomography (µCT) for bone and capillary architecture, and histomorphometry for bone remodeling. Results of these analyses indicated enhanced bone formation in the group that received BMP2+VEGF-expressing MSC compared to other groups. The therapeutic effects were accompanied by increased vascularity and osteoblastogenesis, indicating its potential for effective use while treating difficult nonunion bone defects in humans.  

5.763           High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency

Ayuso, E., Mingozzi, F., Montane, J., Leon, X., Anguela, X.M., Haurigot, V., Edmonson, S.A., Africa, L., Zhou, S., High, K.A., Bosch, F. and Wright, J.F. Gene Therapy, 17, 503-510 (2010)   The purity of adeno-associated virus (AAV) vector preparations has important implications for both safety and efficacy of clinical gene transfer. Early-stage screening of candidates for AAV-based therapeutics ideally requires a purification method that is flexible and also provides vectors comparable in purity and potency to the prospective investigational product manufactured for clinical studies. The use of cesium chloride (CsCl) gradient-based protocols provides the flexibility for purification of different serotypes; however, a commonly used first-generation CsCl-based protocol was found to result in AAV vectors containing large amounts of protein and DNA impurities and low transduction efficiency in vitro and in vivo. Here, we describe and characterize an optimized, second-generation CsCl protocol that incorporates differential precipitation of AAV particles by polyethylene glycol, resulting in higher yield and markedly higher vector purity that correlated with better transduction efficiency observed with several AAV serotypes in multiple tissues and species. Vectors purified by the optimized CsCl protocol were found to be comparable in purity and functional activity to those prepared by more scalable, but less flexible serotype-specific purification processes developed for manufacture of clinical vectors, and are therefore ideally suited for pre-clinical studies supporting translational research.  

5.764           Changes in Adeno-Associated Virus-Mediated Gene Delivery in Retinal Degeneration

Kolstad, K.D., Dalkara, D., Guerin, K., Visel, M., Hoffmann, N., Schaffer, D.V. and Flannery, J.G. Human Gene Therapy, 21, 571-578 (2010)   Gene therapies for retinal degeneration have relied on subretinal delivery of viral vectors carrying therapeutic DNA. The subretinal injection is clearly not ideal as it limits the viral transduction profile to a focal region at the injection site and negatively affects the neural retina by detaching it from the supportive retinal pigment epithelium (RPE). We assessed changes in adeno-associated virus (AAV) dispersion and transduction in the degenerating rat retina after intravitreal delivery. We observed a significant increase in AAV-mediated gene transfer in the diseased compared with normal retina, the extent of which depends on the AAV serotype injected. We also identified key structural changes that correspond to increased viral infectivity. Particle diffusion and transgene accumulation in normal and diseased retina were monitored via fluorescent labeling of viral capsids and quantitative PCR. Viral particles were observed to accumulate at the vitreoretinal junction in normal retina, whereas particles spread into the outer retina and RPE in degenerated tissue. Immunohistochemistry illustrates remarkable changes in the architecture of the inner limiting membrane, which are likely to underlie the increased viral transduction in diseased retina. These data highlight the importance of characterizing gene delivery vectors in diseased tissue as structural and biochemical changes can alter viral vector transduction patterns. Furthermore, these results indicate that gene delivery to the outer nuclear layer may be achieved by noninvasive intravitreal AAV administration in the diseased state.  

5.765           IFN- Promotes Complement Expression and Attenuates Amyloid Plaque Deposition in Amyloid β Precursor Protein Transgenic Mice

Chakrabarty, P., Ceballos-Diaz, C., Beccard, a., Janus, C., Dickson, D., Golde, T.E. and Das, P.

1. Immunol., 184, 5333-5343 (2010)

  Reactive gliosis surrounding amyloid β (Aβ) plaques is an early feature of Alzheimer’s disease pathogenesis and has been postulated to represent activation of the innate immune system in an apparently ineffective attempt to clear or neutralize Aβ aggregates. To evaluate the role of IFN- –mediated neuroinflammation on the evolution of Aβ pathology in transgenic (Tg) mice, we have expressed murine IFN- (mIFN- ) in the brains of Aβ precursor protein (APP) Tg mice using recombinant adeno-associated virus serotype 1. Expression of mIFN- in brains of APP TgCRND8 mice results in robust noncell autonomous activation of microglia and astrocytes, and a concomitant significant suppression of Aβ deposition. In these mice, mIFN- expression upregulated multiple glial activation markers, early components of the complement cascade as well as led to infiltration of Ly-6c positive peripheral monocytes but no significant effects on APP levels, APP processing or steady-state Aβ levels were noticed in vivo. Taken together, these results suggest that mIFN- expression in the brain suppresses Aβ accumulation through synergistic effects of activated glia and components of the innate immune system that enhance Aβ aggregate phagocytosis.  

5.766           Adeno-Associated Vector (Type 8)-Mediated Expression of Soluble Flt-1 Efficiently Inhibits Neovascularization in a Murine Choroidal Neovascularization Model

Igarashi, T., Miyake, K., Masuda, I., Takahashi, H. and Shimada, T. Human Gene Therapy, 21, 631-637 (2010)   To assess the feasibility of a gene therapeutic approach to treating choroidal neovascularization (CNV), we generated a recombinant adeno-associated viral (AAV) vector (type 8) encoding soluble Flt-1 (AAV-sflt-1), and determined its ability to inhibit angiogenesis. When we treated human umbilical vein endothelial cells (HUVECs) with the supernatant of cells transduced with AAV-sflt-1 or AAV-EGFP (control), we found that tube formation was significantly inhibited by the former but not the latter (area: 25,121 ± 557 vs. 68,628 ± 1357 pixels [p < 0.01]; length: 4811 ± 246 vs. 10,894 ± 297 pixels [p < 0.01]). CNV was induced in C57BL/6 mice by making four separate choroidal burns around the optic nerve in each eye, using a diode laser. Thereafter, 2 μl (5 × 10¹¹ vector genomes/ml) of AAV-sflt-1 (n = 11) or control AAV-LacZ (n = 12) was injected into the subretinal space, and 2 weeks later the eyes were removed for flatmount analysis of CNV surface area. Notably, subretinal delivery of AAV-sflt-1 significantly diminished CNV at the laser lesions, as compared with AAV-LacZ (555 ± 304 vs. 1470 ± 1000 μm²; p = 0.007). These results suggest that there was diffusion of the secreted sFlt-1 across the retina and that long-term suppression of CNV is possible through the use of stable rAAV-mediated sflt-1 expression. In vivo gene therapy thus appears to be a feasible approach to the clinical management of CNV in conditions such as age-related macular degeneration.  

5.767           CD317/Tetherin Is Enriched in the HIV-1 Envelope and Downregulated from the Plasma Membrane upon Virus Infection

Habermann, A., Krijnse-Locker, J., Oberwinkler, H., Eckhardt, M., Homann, S., Andrew, A., Strebel, K. and Kräusslich, H-G.

1. Virol., 84(9), 4616-4658 (2010)

  CD317/Bst-2/tetherin is a host factor that restricts the release of human immunodeficiency virus type 1 (HIV-1) by trapping virions at the plasma membrane of certain producer cells. It is antagonized by the HIV-1 accessory protein Vpu. Previous light microscopy studies localized CD317 to the plasma membrane and the endosomal compartment and showed Vpu induced downregulation. In the present study, we performed quantitative immunoelectron microscopy of CD317 in cells producing wild-type or Vpu-defective HIV-1 and in control cells. Double-labeling experiments revealed that CD317 localizes to the plasma membrane, to early and recycling endosomes, and to the trans-Golgi network. CD317 largely relocated to endosomes upon HIV-1 infection, and this effect was partly counteracted by Vpu. Unexpectedly, CD317 was enriched in the membrane of viral buds and cell-associated and cell-free viruses compared to the respective plasma membrane, and this enrichment was independent of Vpu. These results suggest that the tethering activity of CD317 critically depends on its density at the cell surface and appears to be less affected by its density in the virion membrane.  

5.768           Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis via Accumulation of Reactive Oxygen Species

Hristov, G., Kr¨mer, M., Li, J., El-Andoloussi, N., Mora, R., Daeffler, L., Zentgraf, H., Rommelaere, J. and Marchini, A.

1. Virol., 84(12), 5909-5922 (2010)

  The rat parvovirus H-1 (H-1PV) attracts high attention as an anticancer agent, because it is not pathogenic for humans and has oncotropic and oncosuppressive properties. The viral nonstructural NS1 protein is thought to mediate H-1PV cytotoxicity, but its exact contribution to this process remains undefined. In this study, we analyzed the effects of the H-1PV NS1 protein on human cell proliferation and cell viability. We show that NS1 expression is sufficient to induce the accumulation of cells in G2 phase, apoptosis via caspase 9 and 3 activation, and cell lysis. Similarly, cells infected with wild-type H-1PV arrest in G2 phase and undergo apoptosis. Furthermore, we also show that both expression of NS1 and H-1PV infection lead to higher levels of intracellular reactive oxygen species (ROS), associated with DNA double-strand breaks. Antioxidant treatment reduces ROS levels and strongly decreases NS1- and virus-induced DNA damage, cell cycle arrest, and apoptosis, indicating that NS1-induced ROS are important mediators of H-1PV cytotoxicity.  

5.769           Recognition of decay accelerating factor and α_vβ₃ by inactivated hantaviruses: Toward the development of high-throughput screening flow cytometry assays

Buranda, T., Wu, Y., Perez, D., Jett, S.D., BonduHawkins, V., Ye, C., Edwards, B., Hall, P., Larson, R.S., Lopez, G.P., Sklar, L.A. and Hjelle, B. Anal. Biochem., 402, 151-160 (2010)   Hantaviruses cause two severe diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The lack of vaccines or specific drugs to prevent or treat HFRS and HCPS and the requirement for conducting experiments in a biosafety level 3 laboratory (BSL-3) limit the ability to probe the mechanism of infection and disease pathogenesis. In this study, we developed a generalizable spectroscopic assay to quantify saturable fluorophore sites solubilized in envelope membranes of Sin Nombre virus (SNV) particles. We then used flow cytometry and live cell confocal fluorescence microscopy imaging to show that ultraviolet (UV)-killed SNV particles bind to the cognate receptors of live virions, namely, decay accelerating factor (DAF/CD55) expressed on Tanoue B cells and α_vβ₃ integrins expressed on Vero E6 cells. SNV binding to DAF is multivalent and of high affinity (K_d   26 pM). Self-exchange competition binding assays between fluorescently labeled SNV and unlabeled SNV are used to evaluate an infectious unit-to-particle ratio of approximately 1:14,000. We configured the assay for measuring the binding of fluorescently labeled SNV to Tanoue B suspension cells using a high-throughput flow cytometer. In this way, we established a proof-of-principle high-throughput screening (HTS) assay for binding inhibition. This is a first step toward developing HTS format assays for small molecule inhibitors of viral–cell interactions as well as dissecting the mechanism of infection in a BSL-2 environment.  

5.770           Usage of heparan sulfate, integrins, and FAK in HPV16 infection

Abban, C.Y., and Meneses, P.I. Virology, 403, 1-16 (2010)   Human papillomavirus type 16 (HPV16) is the major causative agent of cervical cancer. Studies regarding the early binding and signaling molecules that play a significant role in infection are still lacking. The current study analyzes the role of heparan sulfate, integrins, and the signaling molecule FAK in HPV16 infection of human adult keratinocytes cell line (HaCaTs). Our data demonstrate that infection requires the binding of viral particles to heparan sulfate followed by activation of focal adhesion kinase through an integrin. Infections were reduced in the presence of the FAK inhibitor, TAE226. TAE226 was observed to inhibit viral entry to the early endosome a known infectious route. These findings suggest that FAK can serve as a novel target for antiviral therapy.  

5.771           A High-throughput Pharmacoviral Approach Identifies Novel Oncolytic Virus Sensitizers

Diallo, J-S., Le Boeuf, F., Lai, F., Cox, J., Vaha-Koskela, M., Abdelbary, H., MacTavish, H., Waite, K., Falls, T., Wang, J., Brown, R., Blanchard, J.E., Brown, E.D., Kirn, D.H., Hiscott, J., Atkins, H., Lichty, B.D. and Bell, J.C. Molecular Therapy, 18(6), 1123-1129 (2010)   Oncolytic viruses (OVs) are promising anticancer agents but like other cancer monotherapies, the genetic heterogeneity of human malignancies can lead to treatment resistance. We used a virus/cell-based assay to screen diverse chemical libraries to identify small molecules that could act in synergy with OVs to destroy tumor cells that resist viral infection. Several molecules were identified that aid in viral oncolysis, enhancing virus replication and spread as much as 1,000-fold in tumor cells. One of these molecules we named virus-sensitizers 1 (VSe1), was found to target tumor innate immune response and could enhance OV efficacy in animal tumor models and within primary human tumor explants while remaining benign to normal tissues. We believe this is the first example of a virus/cell-based “pharmacoviral” screen aimed to identify small molecules that modulate cellular response to virus infection and enhance oncolytic virotherapy.  

5.772           The Central and Basolateral Amygdala Are Critical Sites of Neuropeptide Y/Y2 Receptor-Mediated Regulation of Anxiety and Depression

Tasan, R.O., Nguyen, N.K., Weger, S., Sartori, S.B., Singewald, N., Heilbronn, R., Herzog, H. and Sperk, G.

1. Neurosci., 30(18), 6282-6290 (2010)

  Anxiety is integrated in the amygdaloid nuclei and involves the interplay of the amygdala and various other areas of the brain. Neuropeptides play a critical role in regulating this process. Neuropeptide Y (NPY), a 36 aa peptide, is highly expressed in the amygdala. It exerts potent anxiolytic effects through cognate postsynaptic Y1 receptors, but augments anxiety through presynaptic Y2 receptors. To identify the precise anatomical site(s) of Y2-mediated anxiogenic action, we investigated the effect of site-specific deletion of the Y2 gene in amygdaloid nuclei on anxiety and depression-related behaviors in mice. Ablating the Y2 gene in the basolateral and central amygdala resulted in an anxiolytic phenotype, whereas deletion in the medial amygdala or in the bed nucleus of the stria terminalis had no obvious effect on emotion-related behavior. Deleting the Y2 receptor gene in the central amygdala, but not in any other amygdaloid nucleus, resulted in an added antidepressant-like effect. It was associated with a reduction of presumably presynaptic Y2 receptors in the stria terminalis/bed nucleus of the stria terminalis, the nucleus accumbens, and the locus ceruleus. Our results are evidence of the highly site-specific nature of the Y2-mediated function of NPY in the modulation of anxiety- and depression-related behavior. The activity of NPY is likely mediated by the presynaptic inhibition of GABA and/or NPY release from interneurons and/or efferent projection neurons of the basolateral and central amygdala.  

5.773           Rotaviruses Associate with Cellular Lipid Droplet Components To Replicate in Viroplasms, and Compounds Disrupting or Blocking Lipid Droplets Inhibit Viroplasm Formation and Viral Replication

Cheung, W., Gill, M., Esposito, A., Kaminski, C.F., Courousse, N., Chwetzoff, S., Trugnan, G., Keshavan, N., Lever, A. and Desselberger, U.

1. Virol., 84(13), 6782-6798 (2010)

  Rotaviruses are a major cause of acute gastroenteritis in children worldwide. Early stages of rotavirus assembly in infected cells occur in viroplasms. Confocal microscopy demonstrated that viroplasms associate with lipids and proteins (perilipin A, ADRP) characteristic of lipid droplets (LDs). LD-associated proteins were also found to colocalize with viroplasms containing a rotaviral NSP5-enhanced green fluorescent protein (EGFP) fusion protein and with viroplasm-like structures in uninfected cells coexpressing viral NSP2 and NSP5. Close spatial proximity of NSP5-EGFP and cellular perilipin A was confirmed by fluorescence resonance energy transfer. Viroplasms appear to recruit LD components during the time course of rotavirus infection. NSP5-specific siRNA blocked association of perilipin A with NSP5 in viroplasms. Viral double-stranded RNA (dsRNA), NSP5, and perilipin A cosedimented in low-density gradient fractions of rotavirus-infected cell extracts. Chemical compounds interfering with LD formation (isoproterenol plus isobutylmethylxanthine; triacsin C) decreased the number of viroplasms and inhibited dsRNA replication and the production of infectious progeny virus; this effect correlated with significant protection of cells from virus-associated cytopathicity. Rotaviruses represent a genus of another virus family utilizing LD components for replication, pointing at novel therapeutic targets for these pathogens.  

5.774           Transcriptome analysis of a tau overexpression model in rats implicates an early pro-inflammatory response

Wang, D.B., Dayton, R.D., Zweig, R.M. and Klein, R.L. Exp. Neurol., 224, 197-206 (2010)   Neurofibrillary tangles comprised of the microtubule-associated protein tau are pathological features of Alzheimer's disease and several other neurodegenerative diseases, such as progressive supranuclear palsy. We previously overexpressed tau in the substantia nigra of rats and mimicked some of the neurodegenerative sequelae that occur in humans such as tangle formation, loss of dopamine neurons, and microgliosis. To study molecular changes involved in the tau-induced disease state, we used DNA microarrays at an early stage of the disease process. A range of adeno-associated virus (AAV9) vector doses for tau were injected in groups of rats with a survival interval of 2 weeks. Specific decreases in messages for dopamine-related genes validated the technique with respect to the dopaminergic cell loss observed. Of the mRNAs upregulated, there was a dose-dependent effect on multiple genes involved in immune response such as chemokines, interferon-inducible genes and leukocyte markers, only in the tau vector groups and not in dose-matched controls of either transgene-less empty vector or control green fluorescent protein vector. Histological staining for dopamine neurons and microglia matched the loss of dopaminergic markers and upregulation of immune response mRNAs in the microarray data, respectively. RT-PCR for selected markers confirmed the microarray results, with similar changes found by either technique. The mRNA data correlate well with previous findings, and underscore microgliosis and immune response in the degenerative process following tau overexpression.  

5.775           Optimization of Adeno-Associated Viral Vector-Mediated Gene Delivery to the Hypothalamus

De Backer, M.W.A., Brans, M.A.D., Luijendijk, M.C., garner, K.M. and Adan, R.A. Human Gene Therapy, 21, 673-682 (2010)   To efficiently deliver genes and short hairpin RNAs to the hypothalamus we aimed to optimize the transduction efficiency of adeno-associated virus (AAV) in the rat hypothalamus. We compared the transduction efficiencies of AAV2 vectors pseudotyped with AAV1, AAV8, and mosaic AAV1/2 and AAV2/8 coats with that of an AAV2 coated vector after injection into the lateral hypothalamus of rats. In addition, we determined the transduction areas and the percentage of neurons infected after injection of various titers and volumes of two AAV1-pseudotyped vectors in the paraventricular hypothalamus (PVN). Successful gene delivery to the hypothalamus was achieved with AAV1-pseudotyped AAV vectors. The optimal approach to transduce an area, with the size of the PVN, was to inject 1 × 10⁹ genomic copies of an AAV1-pseudotyped vector in a volume of 1 μl. At a radius of 0.05 mm from the injection site almost all neurons were transduced. In addition, overexpression of AgRP with the optimal approach resulted in an increase in food intake and body weight when compared with AAV-GFP.  

5.776           Validation of multiplexed human papillomavirus serology using pseudovirions bound to heparin-coated beads

Faust, H., Knekt, P., Forslund, O. and Dillner, J.

1. Gen. Virol., 91, 1840-1848 (2010)

  This study developed and validated a high-throughput human papillomavirus (HPV) serology method based on Luminex technology, using pseudovirions (PsVs) of eight mucosal HPV types (HPV-6, -11, -16, -18, -31, -45, -52 and -58) and two cutaneous HPV types (HPV-5 and -38) bound to heparin-coated beads. Analysis with neutralizing type-specific monoclonal antibodies against the included HPV types indicated the type specificity of the assay. Analysis of negative-control serum samples from 63 children and 71 middle-aged women with up to one lifetime sexual partner indicated high specificity. Positive-control serum samples from subjects with known HPV DNA status or clinical diagnosis found expected sensitivities for most of the HPV types in 219 European serum samples, but lower than expected in 124 samples from Africa. HPV-45 and -52 did not react as expected with the human serum samples. The PsV-Luminex method was used to determine the HPV-seropositivity-associated relative risk for future cervical cancer using 208 serum samples from a prospective study of 18 814 women followed for 23 years, analysed previously with standard HPV-16 ELISA. The PsV-Luminex method gave similar results to ELISA ( =0.77). As expected, HPV seropositivities assayed using the PsV-Luminex method found an increased risk of cervical cancer for HPV-16 [odds ratio (OR)=7.7, 95 % confidence interval (CI)=2.6–23] and HPV-31 (OR=4.1, 95 % CI=1.6–10.8), non-significant tendencies for increased risk for other mucosal HPV types and no risk for the cutaneous HPV types. In summary, multiplexed HPV serology using mammalian-derived PsVs selected for native conformation by binding to heparin-coated beads was validated as a high-throughput HPV serological method for most of the analysed HPV types.  

5.777           Merkel Cell Polyomavirus and Two Previously Unknown Polyomaviruses Are Chronically Shed from Human Skin

Schowalter, R.M., Pastrana, D.V., Pumphrey, K.A., Moyer, A.L. and Buck, C.B. Cell Host & Microbe, 7, 509-515 (2010)   Mounting evidence indicates that Merkel cell polyomavirus (MCV), a circular double-stranded DNA virus, is a causal factor underlying a highly lethal form of skin cancer known as Merkel cell carcinoma. To explore the possibility that MCV and other polyomaviruses commonly inhabit healthy human skin, we developed an improved rolling circle amplification (RCA) technique to isolate circular DNA viral genomes from human skin swabs. Complete MCV genomes were recovered from 40% of healthy adult volunteers tested, providing full-length, apparently wild-type cloned MCV genomes. RCA analysis also identified two previously unknown polyomavirus species that we name human polyomavirus-6 (HPyV6) and HPyV7. Biochemical experiments show that polyomavirus DNA is shed from the skin in the form of assembled virions. A pilot serological study indicates that infection or coinfection with these three skin-tropic polyomaviruses is very common. Thus, at least three polyomavirus species are constituents of the human skin microbiome.  

5.778           The ISG15 Conjugation System Broadly Targets Newly Synthesized Proteins: Implications for the Antiviral Function of ISG15

Durfee, L.A., Lyon, N., Seo, K. and Huibregste, J.M. Mol. Cell, 38, 722-732 (2010)   ISG15 is an interferon-induced and antiviral ubiquitin-like protein (Ubl). Herc5, the major E3 enzyme for ISG15, mediates the ISGylation of more than 300 proteins in interferon-stimulated cells. In addressing this broad substrate selectivity of Herc5, we found that: (1) the range of substrates extends even further and includes many exogenously expressed foreign proteins, (2) ISG15 conjugation is restricted to newly synthesized pools of proteins, and (3) Herc5 is physically associated with polyribosomes. These results lead to a model for ISGylation in which Herc5 broadly modifies newly synthesized proteins in a cotranslational manner. This further suggests that, in the context of an interferon-stimulated cell, newly translated viral proteins may be primary targets of ISG15. Consistent with this, we demonstrate that ISGylation of human papillomavirus (HPV) L1 capsid protein has a dominant-inhibitory effect on the infectivity of HPV16 pseudoviruses.    

5.779           A Novel p40-Independent Function of IL-12p35 Is Required for Progression and Maintenance of Herpes Stromal Keratitis

Frank, G.M., Divito, S.J., Maker, D.M., Xu, M. and Hendricks, R.L. Invest. Ophthalmol. Vis. Sci., 51(7), 3591-3598 (2010)   Purpose. Interleukin (IL)-12p40 can couple with IL-12p35 or p19 chains to form the molecules IL-12p70 and IL-23, respectively, which promote T_H1 cytokine responses. IL-12p35 can bind to EBI3 to form the anti-inflammatory molecule IL-35, but a proinflammatory function of IL-12p35 independent of IL-12p40 has not been described. Here such a function in a mouse model of herpes stromal keratitis (HSK), a CD4⁺ T_H1 cell–dependent corneal inflammation,is demonstrated. Methods. Corneas of wild-type (WT), IL-12p40^–/–, IL-12p35^–/–, and IL-12p35^–/–p40^–/– (double knockout)mice were infected with the RE strain of HSV-1, and HSK wasmonitored based on corneal opacity, neovascularization, leukocyticinfiltrate, and cytokine/chemokine levels. Results. All mouse strains developed moderate HSK by 11 days after infection (dpi). However, from 11 to 21 dpi, HSK progressed in WT and IL-12p40^–/– mice but regressed in IL-12p35^–/– and IL-12p35^–/–p40^–/– mice. HSK regression was characterized by reductions in neutrophils and CD4⁺ T cellsand attenuation of blood vessels, which was associated withreduced levels of the chemokines KC (CXCL3), Mip-2 (CXCL2),and MCP-1 (CCL2) and the angiogenic factor vascular endothelialgrowth factor. Conclusions. HSK development does not require IL-12p40 and is thus independentof IL-12p70 and IL-23. However, late HSK progression does requirea previously unrecognized IL-12p40–independent, proinflammatoryfunction of IL-12p35.  

5.780           Adaptation of Hepatitis C Virus to Mouse CD81 Permits Infection of Mouse Cells in the Absence of Human Entry Factors

Bitzegeio, J., Bankwitz, D., Hueging, K., Haid, S., Brohm, C., Zeisel, M.B., Herrmann, E., Iken, M., Ott, M., Baumert, T.F. and Pietschmann, T. PloSPathogens, 6(7), e1000978 (2010)   Hepatitis C virus (HCV) naturally infects only humans and chimpanzees. The determinants responsible for this narrow species tropism are not well defined. Virus cell entry involves human scavenger receptor class B type I (SR-BI), CD81, claudin-1 and occludin. Among these, at least CD81 and occludin are utilized in a highly species-specific fashion, thus contributing to the narrow host range of HCV. We adapted HCV to mouse CD81 and identified three envelope glycoprotein mutations which together enhance infection of cells with mouse or other rodent receptors approximately 100-fold. These mutations enhanced interaction with human CD81 and increased exposure of the binding site for CD81 on the surface of virus particles. These changes were accompanied by augmented susceptibility of adapted HCV to neutralization by E2-specific antibodies indicative of major conformational changes of virus-resident E1/E2-complexes. Neutralization with CD81, SR-BI- and claudin-1-specific antibodies and knock down of occludin expression by siRNAs indicate that the adapted virus remains dependent on these host factors but apparently utilizes CD81, SR-BI and occludin with increased efficiency. Importantly, adapted E1/E2 complexes mediate HCV cell entry into mouse cells in the absence of human entry factors. These results further our knowledge of HCV receptor interactions and indicate that three glycoprotein mutations are sufficient to overcome the species-specific restriction of HCV cell entry into mouse cells. Moreover, these findings should contribute to the development of an immunocompetent small animal model fully permissive to HCV.  

5.781           Characterization of monoclonal antibodies specific for the Merkel cell polyomavirus capsid

Pastrana, D.V., Pumphrey, K.A., Cuburu, N., Schowalter, R.M. and Buck, C.B. Virology, 405, 20-25 (2010)   Merkel cell polyomavirus (MCV) has been implicated as a causative agent in Merkel cell carcinoma. Robust polyclonal antibody responses against MCV have been documented in human subjects, but monoclonal antibodies (mAbs) specific for the VP1 capsid protein have not yet been characterized. We generated 12 mAbs capable of binding recombinant MCV virus-like particles. The use of a short immunogenic priming schedule was important for production of the mAbs. Ten of the 12 mAbs were highly effective for immunofluorescent staining of cells expressing capsid proteins. An overlapping set of 10 mAbs were able to neutralize the infectivity of MCV-based reporter vectors, with 50% effective doses in the low picomolar range. Three mAbs interfered with the binding of MCV virus-like particles to cells. This panel of anti-capsid antibodies should provide a useful set of tools for the study of MCV.  

5.782           In Vivo RNAi-Mediated α-Synuclein Silencing Induces Nigrostriatal Degeneration

Gorbatyuk, O.S., Li, S., Nash, K., Gorbatyuk, M., Lewin, A.S., Sullivan, L.F., Mandel, R.J., Chen, W., Meyers, C., Manfredsson, F.P. and Muzyczka, N. Molecular Therapy, 18(8), 1450-1457 (2010)   Two small-interfering RNAs (siRNAs) targeting α-synuclein (α-syn) and three control siRNAs were cloned in an adeno-associated virus (AAV) vector and unilaterally injected into rat substantia nigra pars compacta (SNc). Reduction of α-syn resulted in a rapid (4 week) reduction in the number of tyrosine hydroxylase (TH) positive cells and striatal dopamine (DA) on the injected side. The level of neurodegeneration induced by the different siRNAs correlated with their ability to downregulate α-syn protein and mRNA in tissue culture and in vivo. Examination of various SNc neuronal markers indicated that neurodegeneration was due to cell loss and not just downregulation of DA synthesis. Reduction of α-syn also resulted in a pronounced amphetamine induced behavioral asymmetry consistent with the level of neurodegeneration. In contrast, none of the three control siRNAs, which targeted genes not normally expressed in SNc, showed evidence of neurodegeneration or behavioral asymmetry, even at longer survival times. Moreover, co-expression of both rat α-syn and α-syn siRNA partially reversed the neurodegenerative and behavioral effects of α-syn siRNA alone. Our data show that α-syn plays an important role in the rat SNc and suggest that both up- and downregulation of wild-type α-syn expression increase the risk of nigrostriatal pathology.  

5.783           An adeno-associated viral vector transduces the rat hypothalamus and amygdala more efficient than a lentiviral vector

De Backer, M.W.A., Fitzsimons, C., Brans, M.A.D., Luijendijk, C.M., Garner, K.M., Vreugdenhil, E. and Adan, R.A.H. BMC Neurosci., 11, 81-88 (2010)   Background This study compared the transduction efficiencies of an adeno-associated viral (AAV) vector, which was pseudotyped with an AAV1 capsid and encoded the green fluorescent protein (GFP), with a lentiviral (LV) vector, which was pseudotyped with a VSV-G envelop and encoded the discosoma red fluorescent protein (dsRed), to investigate which viral vector transduced the lateral hypothalamus or the amygdala more efficiently. The LV-dsRed and AAV1-GFP vector were mixed and injected into the lateral hypothalamus or into the amygdala of adult rats. The titers that were injected were 1 × 10⁸ or 1 × 10⁹ genomic copies of AAV1-GFP and 1 × 10⁵ transducing units of LV-dsRed. Results Immunostaining for GFP and dsRed showed that AAV1-GFP transduced significantly more cells than LV-dsRed in both the lateral hypothalamus and the amygdala. In addition, the number of LV particles that were injected can not easily be increased, while the number of AAV1 particles can be increased easily with a factor 100 to 1000. Both viral vectors appear to predominantly transduce neurons. Conclusions This study showed that AAV1 vectors are better tools to overexpress or knockdown genes in the lateral hypothalamus and amygdala of adult rats, since more cells can be transduced with AAV1 than with LV vectors and the titer of AAV1 vectors can easily be increased to transduce the area of interest.  

5.784           Differential Effects of DNA Double-Strand Break Repair Pathways on Single-Strand and Self-Complementary Adeno-Associated Virus Vector Genomes

Cataldi, M and McCarty, D.M.

1. Virol., 84(17), 8673-8682 (2010)

  The linear DNA genomes of recombinant adeno-associated virus (rAAV) gene delivery vectors are acted upon by multiple DNA repair and recombination pathways upon release into the host nucleus, resulting in circularization, concatemer formation, or chromosomal integration. We have compared the fates of single-strand rAAV (ssAAV) and self-complementary AAV (scAAV) genomes in cell lines deficient in each of three signaling factors, ATM, ATR, and DNA-PKCS, orchestrating major DNA double-strand break (DSB) repair pathways. In cells deficient in ATM, transduction as scored by green fluorescent protein (GFP) expression is increased relative to that in wild-type (wt) cells by 2.6-fold for ssAAV and 6.6-fold for scAAV vectors, arguing against a mechanism related to second-strand synthesis. The augmented transduction is not reflected in Southern blots of nuclear vector DNA, suggesting that interactions with ATM lead to silencing in normal cells. The additional functional genomes in ATM–/– cells remain linear, and the number of circularized genomes is not affected by the mutation, consistent with compartmentalization of genomes into different DNA repair pathways. A similar effect is observed in ATR-deficient cells but is specific for ssAAV vector. Conversely, a large decrease in transduction is observed in cells deficient in DNA-PKCS, which is involved in DSB repair by nonhomologous end joining rather than homologous recombination. The mutations also have differential effects on chromosomal integration of ssAAV versus scAAV vector genomes. Integration of ssAAV was specifically reduced in ATM–/– cells, while scAAV integration was more profoundly inhibited in DNA-PKCS–/– cells. Taken together, the results suggest that productive rAAV genome circularization is mediated primarily by nonhomologous end joining.  

5.785           Distinct Intracellular Trafficking of Hepatitis C Virus in Myeloid and Plasmacytoid Dendritic Cells

Lambotin, M., Baumert, T.F. and Barth, H.

1. Virol., 84(17), 8964-8969 (2010)

  Dendritic cells (DCs) are of pivotal importance for the initiation of immune responses to control and eliminate viral infections. The molecular mechanisms of hepatitis C virus (HCV) antigen uptake and processing by blood DCs are poorly defined. Here we show that human blood DC subsets acquire HCV independent of the classical HCV entry factors. Following HCV uptake, human plasmacytoid and myeloid DC subsets deliver HCV antigen into distinct endocytotic compartments, which are dedicated to presentation to CD4+ or CD8+ T cells. Our findings support a model of HCV antigen processing and presentation in which DC subsets fulfill distinct functions.  

5.786           Gene transfer into human cord blood−derived CD34⁺ cells by adeno-associated viral vectors

Schuhmann, N., Pozzoli, O., Sallach, J., Huber, A., Avitabile, D., Perabo, L., Rappl, G., Capogrossi, M.C., Hallek, M., Pesce, M. and Büning, H. Exp. Hematol., 38, 707-717 (2010)   Objective Bone marrow−derived CD34⁺ cells are currently used in clinical trials in patients with ischemic heart disease. An option to enhance activity of injected progenitors may be offered by genetic engineering of progenitor cells with angiogenic growth factors. Recombinant adeno-associated viral vectors (rAAV) have emerged as a leading gene transfer systems. In contrast to other vector systems in use for genetic engineering of CD34⁺ cells, rAAV-mediated gene expression does not depend on vector integration. This is relevant for application in regenerative medicine of ischemic tissues, where transient transgene expression is likely sufficient to achieve therapeutic benefits. Materials and Methods We compared three different human AAV serotypes, packaged as pseudotypes by a helper virus-free production method, for their transduction efficiency in human cord blood−derived CD34⁺ cells. We further assessed the impact of vector genome conformation, of α_vβ₅ and α₅β₁ integrin availability and of the transcription-modulating drugs retinoic acid and Trichostatin A on rAAV-mediated human CD34⁺ cell transduction. Results We provide, for the first time, evidence that hCD34⁺ cells can be reproducibly transduced with high efficiency by self-complementary rAAV2 without inducing cytotoxicity or interfering with their differentiation potential. We further show the involvement of α₅β₁ integrin as a crucial AAV2 internalization receptor and a function for transcription-modulating drugs in enhancing rAAV-mediated transgene expression. Conclusion This study represents a first step toward translation of a combined cellular/rAAV-based therapy of ischemic disease.  

5.787           Circulating neprilysin clears brain amyloid

Liu, Y., Stuzinski, C., Beckett, T., Murphy, M.P., Klein, R.L. and Hersh, L.B. Mol. Cell. Neurosci., 45, 101-107 (2010)   The use of the peptidase neprilysin (NEP) as a therapeutic for lowering brain amyloid burden is receiving increasing attention. We have previously demonstrated that peripheral expression of NEP on the surface of hindlimb muscle lowers brain amyloid burden in a transgenic mouse model of Alzheimer's disease. In this study we now show that using adeno-associated virus expressing a soluble secreted form of NEP (secNEP-AAV8), NEP secreted into plasma is effective in clearing brain Aβ. Soluble NEP expression in plasma was sustained over the 3-month time period it was measured. Secreted NEP decreased plasma Aβ by 30%, soluble brain Aβ by  28%, insoluble brain Aβ by  55%, by 12%. This secNEP did not change plasma levels of substance oligomers and Aβ  P or bradykinin, nor did it alter blood pressure. No NEP was detected in CSF, nor did the AAV virus produce brain expression of NEP. Thus the lowering of brain Aβ was due to plasma NEP which altered blood-brain Aβ transport dynamics. Expressing NEP in plasma provides a convenient way to monitor enzyme activity during the course of its therapeutic testing.  

5.788           Release of Genotype 1 Hepatitis E Virus from Cultured Hepatoma and Polarized Intestinal Cells Depends on Open Reading Frame 3 Protein and Requires an Intact PXXP Motif

Emerson, S.U., Nguyen, H.T., Torian, U., Burke, D., Engle, R. and Purcell, R.H.

1. Virol., 84(18), 9059-9069 (2010)

  Hepatitis E virus genotype 1 strain Sar55 replicated in subcloned Caco-2 intestinal cells and Huh7 hepatoma cells that had been transfected with in vitro transcribed viral genomes, and hepatitis E virions were released into the culture medium of both cell lines. Virus egress from cells depended on open reading frame 3 (ORF3) protein, and a proline-rich sequence in ORF3 was important for egress from cultured cells and for infection of macaques. Both intracellular ORF3 protein accumulation and virus release occurred at the apical membrane of polarized Caco-2 cells. ORF3 protein and lipids were intimately associated with virus particles produced in either cell line; ORF2 epitopes were masked in these particles and could not be immunoprecipitated with anti-ORF2.  

5.789           Presynaptic dopaminergic compartment determines the susceptibility to L-DOPA–induced dyskinesia in rats

Ulusoy, A., Sahin, G. and Kirik, D. PNAS, 107(29), 13159-13164 (2010)   Drug-induced dyskinesias in dopamine-denervated animals are known to depend on both pre- and postsynaptic changes of the nigrostriatal circuitry. In lesion models used thus far, changes occur in both of these compartments and, therefore, it has not been possible to dissect the individual contribution of each compartment in the pathophysiology of dyskinesias. Here we silenced the nigrostriatal dopamine neurotransmission without affecting the anatomical integrity of the presynaptic terminals using a short-hairpin RNA-mediated knockdown of tyrosine hydroxylase enzyme (shTH). This treatment resulted in significant reduction (by about 70%) in extracellular dopamine concentration in the striatum as measured by on-line microdialysis. Under these conditions, the animals remained nondyskinetic after chronic L-DOPA treatment, whereas partial intrastriatal 6-hydoxydopamine lesioned rats with comparable reduction in extracellular dopamine levels developed dyskinesias. On the other hand, apomorphine caused moderate to severe dyskinesias in both groups. Importantly, single-dose L-DOPA challenge in apomorphine-primed shTH animals failed to activate the already established abnormal postsynaptic responses. Taken together, these data provide direct evidence that the status of the presynaptic, DA releasing compartment is a critical determinant of both the induction and maintenance of L-DOPA–induced dyskinesias.  

5.790           Induction of Rapid and Highly Efficient Expression of the Human ND4 Complex I Subunit in the Mouse Visual System by Self-complementary Adeno-Associated Virus

Koilkonda, R.D., Chou, T-H., Porciatti, V., Hauswirth, W.W. and Guy, J. Arch. Ophthalmol., 128(7), 876-883 (2010)   Objective  To demonstrate the high efficiency and rapidityof allotopic expression of a normal human ND4 subunit of complexI in the vertebrate retina using a self-complementary adeno-associatedvirus (scAAV) vector for ocular gene delivery to treat acutevisual loss in Leber hereditary optic neuropathy (LHON). Methods  The nuclear-encoded human ND4 subunit fused tothe P1 isoform of subunit C of adenosine triphosphate synthase(ATPc) mitochondrial targeting sequence and FLAG epitope waspackaged in scAAV2 capsids or single-stranded (ss) AAV2 capsids.These constructs were injected into the vitreous cavities ofmice. The contralateral eyes were injected with scAAV–greenfluorescent protein (GFP). One week later, pattern electroretinogramsand gene expression of the human ND4 subunit and GFP were evaluated.Quantitative analysis of ND4FLAG-injected eyes was assessedrelative to Thy1.2-labeled retinal ganglion cells (RGCs). Results  Pattern electroretinogram amplitudes remainednormal in eyes inoculated with scAAV-ND4FLAG, ssAAV-ND4FLAG,and GFP. Confocal microscopy revealed the typical perinuclearmitochondrial expression of scAAV-ND4FLAG in almost the entireretinal flat mount. In contrast, scAAV-GFP expression was cytoplasmicand nuclear. Relative to Thy1.2-positive RGCs, quantificationof scAAV-ND4FLAG–positive RGCs was 91% and that of ssAAV-ND4FLAG–positiveRGCs was 51%. Conclusion  Treatment of acute visual loss due to LHON may be possible with a normal human ND4 subunit gene of complexI, mutated in most cases of LHON, when delivered by an scAAVvector. Clinical Relevance  Unlike most retinal degenerations thatresult in slowly progressive loss of vision over many years,LHON due to mutated mitochondrial DNA results in apoplectic,bilateral severe and usually irreversible visual loss. For rescueof acute visual loss in LHON, a highly efficient and rapid geneexpression system is required.  

5.791           Brain Microglial Cytokines in Neurogenic Hypertension

Shi, P., Diez-Freire, C., Jun, J.Y., Qi, Y., Katovich, M.J., Li, Q., Sriramula, S., Francis, J., Sumners, C. and Raizada, M.K. Hypertension, 56, 297-303 (2010)   Accumulating evidence indicates a key role of inflammation in hypertension and cardiovascular disorders. However, the role of inflammatory processes in neurogenic hypertension remains to be determined. Thus, our objective in the present study was to test the hypothesis that activation of microglial cells and the generation of proinflammatory cytokines in the paraventricular nucleus (PVN) contribute to neurogenic hypertension. Intracerebroventricular infusion of minocycline, an anti-inflammatory antibiotic, caused a significant attenuation of mean arterial pressure, cardiac hypertrophy, and plasma norepinephrine induced by chronic angiotensin II infusion. This was associated with decreases in the numbers of activated microglia and mRNAs for interleukin (IL) 1β, IL-6, and tumor necrosis factor- , and an increase in the mRNA for IL-10 in the PVN. Overexpression of IL-10 induced by recombinant adenoassociated virus-mediated gene transfer in the PVN mimicked the antihypertensive effects of minocycline. Furthermore, acute application of a proinflammatory cytokine, IL-1β, into the left ventricle or the PVN in normal rats resulted in a significant increase in mean arterial pressure. Collectively, this indicates that angiotensin II induced hypertension involves activation of microglia and increases in proinflammatory cytokines in the PVN. These data have significant implications on the development of innovative therapeutic strategies for the control of neurogenic hypertension.  

5.792           Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral display peptide library

Ying, Y., Müller, O.J., Goehringer, C., Leuchs, B., Trepel, M., Katus, H.A. and Kleinschmidt, J.A. Gene Therapy, 17, 980-990 (2010)   Selection of targeted vectors from virus display peptide libraries is a versatile and efficient approach to improve vector specificity and efficiency. This strategy has been used to target various cell types in vitro. Here, we report the screening of an adeno-associated virus type 2 (AAV2) display peptide library in vivo to select vectors specifically homing to heart tissue after systemic application in mice. Selected library clones indicated superior specificity of gene transfer compared with wild-type AAV2, AAV9 and a heparin binding-deficient AAV2 mutant. Such targeted vectors were able to reconstitute expression of δ-sarcoglycan in the heart of adult δ-sarcoglycan knockout mice after systemic gene transfer in vivo, attesting to the therapeutic potential of this approach.  

5.793           Near-perfect infectivity of wild-type AAV as benchmark for infectivity of recombinant AAV vectors

Zeltner, N., Kohlbrenner, E., Clement, N., weber, T. and Linden, R.M. Gene Therapy, 17, 872-879 (2010)   Viral vectors derived from adeno-associated viruses (AAVs) are widely used for gene transfer both in vitro and in vivo. The increasing use of AAV as a gene transfer vector, as well as recently shown immunological complications in clinical trials, highlight the necessity to define the specific activity of vector preparations beyond current standards. In this report, we determined the infectious, physical and genome-containing particle titers of several wild-type AAV type 2 (wtAAV2) and recombinant AAV type 2 (rAAV2) preparations that were produced and purified by standard methods. We found that the infectivity of wtAAV2 approaches a physical-to-infectious particle ratio of one. This near-perfect physical-to-infectious particle ratio defines a ‘ceiling’ for the theoretically achievable quality of recombinant AAV vectors. In comparison, for rAAV2, only approximately 50 out of 100 viral particles contained a genome and, more strikingly, only approximately 1 of the 100 viral particles was infectious. Our findings suggest that current strategies for rAAV vector design, production and/or purification should be amenable to improvements. Ultimately, this could result in the generation of near-perfect vector particles, a prospect with significant implications for gene therapy.  

5.794           Co-expression of C-terminal truncated alpha-synuclein enhances full-length alpha-synuclein-induced pathology

Ulusoy, A., Febbraro, F., Jensen, P.H., Kirik, D. and Romero-Ramos, M. Eur. J. Neurosci., 32(3), 409-422 (2010)   Lewy bodies, which are a pathological hallmark of Parkinson’s disease, contain insoluble polymers of alpha-synuclein ( syn). Among the different modifications that can promote the formation of toxic syn species, C-terminal truncation is among the most abundant alterations in patients with Parkinson’s disease. In vitro, C-terminal truncated syn aggregates faster and sub-stoichiometric amounts of C-terminal truncated syn promote aggregation of the full-length syn ( synFL) and induce neuronal toxicity. To address in vivo the putative stimulation of syn-induced pathology by the presence of truncated syn, we used recombinant adeno-associated virus to express either synFL or a C-terminal truncated syn (1-110) in rats. We adjusted the recombinant adeno-associated virus vector concentrations so that either protein alone led to only mild to moderate axonal pathology in the terminals of nigrostriatal dopamine neurons without frank cell loss. When these two forms of syn were co-expressed at these pre-determined levels, it resulted in a more aggressive pathology in fiber terminals as well as dopaminergic cell loss in the substantia nigra. Using an antibody that did not detect the C-terminal truncated syn (1-110) but only synFL, we demonstrated that the co-expressed truncated protein promoted the progressive accumulation of synFL and formation of larger pathological accumulations. Moreover, in the co-expression group, three of the eight animals showed apomorphine-induced turning, suggesting prominent post-synaptic alterations due to impairments in the dopamine release, whereas the mild pathology induced by either form alone did not cause motor abnormalities. Taken together these data suggest that C-terminal truncated syn can interact with and exacerbate the formation of pathological accumulations containing synFL in vivo.  

5.795           Morphological Characterization and Fusion Properties of Triglyceride-rich Lipoproteins Obtained from Cells Transduced with Hepatitis C Virus Glycoproteins

Pechuer, E-I., Diaz, O., Molle, J., Icard, V., Bonnafous, P., Lambert, O. and Andre, P.

1. Biol. Chem., 285(33), 25802-25811 (2010)

  The density of hepatitis C virus (HCV) particles circulating in the blood of chronically infected patients and of cell-culture produced HCV is heterogeneous. Specific infectivity and fusion of low density particles are higher than those of high density particles. We recently characterized hybrid particles produced by Caco-2 colon or Huh-7.5 liver cells transduced with HCV E1 and E2 envelope glycoproteins. Caco-2-derived particles, called empty lipo-viral particles (eLVP), are composed of triglyceride-rich lipoproteins positive for apolipoproteins B (i.e. apoB100 and apoB48) and contain HCV E1 and E2. Here we aimed at characterizing the morphology and in vitro fusion properties of eLVP using electron microscopy and fluorescence spectroscopy. They displayed the aspect of β-lipoproteins, and immunogold labeling confirmed the presence of apoB and HCV E1 and E2 at their surface. These particles are able to fuse with lipid bilayers (liposomes) in a fusion process leading to the coalescence of internal contents of triglyceride-rich lipoproteins particles and liposomes. Fusion was pH-dependent and could be inhibited by either Z-fFG, a peptide known to inhibit viral fusion, or by monoclonal antibodies directed against HCV E2 or the apolipoprotein moiety of the hybrid particle. Interestingly, particles derived from Huh-7.5 cells failed to display equivalent efficient fusion. Optimal fusion activity is, thus, observed when HCV envelope proteins are associated to apoB-positive hybrid particles. Our results, therefore, point to a crucial role of the E1 and E2 proteins in HCV fusion with a subtle interplay with the apolipoprotein part of eLVP.  

5.796           The effect of purification method on the completeness of the immature HIV-1 Gag shell

Kol, N., Tsvitov, M., hevroni, L., Wolf, S.G., Pang, H-B., Kay, M.S. and Rousso, I.

1. Virol. Methods, 169, 244-247 (2010)

  Elucidating the structure of the immature HIV-1 Gag core is an important aspect of understanding the biology of this virus. In doing so, preservation of the fragile Gag lattice is essential. In this study, the effects of purification methods on the structural and mechanical integrity of immature HIV-1 are examined. The results show that the morphological and mechanical properties of the virion are preserved to a significantly higher degree by Iodixanol (OptiPrep) purification compared to the standard sucrose method. In conclusion, these results indicate that OptiPrep instead of sucrose purification should be employed when conducting structural studies on the HIV-1 virion.  

5.797           AAVrh.10-mediated genetic delivery of bevacizumab to the pleura to provide local anti-VEGF to suppress growth of metastatic lung tumors

Watanabe, M., Boyer, J.L: and Crystal, R.G. Gene Therapy, 17, 1042-1051 (2010)   Vascular endothelial growth factor (VEGF) produced by tumor cells has a central role in stimulating angiogenesis required for tumor growth. Humanized monoclonal anti-VEGF antibody (bevacizumab, Avastin), approved as a treatment for non-squamous, non-small cell lung cancer, requires administration every 3 weeks. We hypothesized that an intrapleural administration of an adeno-associated virus (AAV) vector expressing an anti-VEGF-A antibody equivalent of bevacizumab would result in sustained anti-VEGF-A localized expression within the lung and suppress metastatic tumor growth. The AAV vector AAVrh.10αVEGF encodes the light chain and heavy chain complementary DNAs of monoclonal antibody A.4.6.1, a murine antibody that specifically recognizes human VEGF-A with the same antigen-binding site as bevacizumab. A metastatic lung tumor model was established in severe combined immunodeficient mice by intravenous administration of human DU145 prostate carcinoma cells. Intrapleural administration of AAVrh.10αVEGF directed long-term expression of the anti-human VEGF-A antibody in lung, as shown by sustained, high-level anti-human VEGF titers in lung epithelial lining fluid for 40 weeks, which was the duration of the study. In the AAVrh.10αVEGF-treated animals, tumor growth was significantly suppressed (P<0.05), the numbers of blood vessels and mitotic nuclei in the tumor was decreased (P<0.05) and there was increased survival (P<0.05). Thus, intrapleural administration of an AAVrh.10 vector, encoding the murine monoclonal antibody equivalent of bevacizumab, effectively suppresses the growth of metastatic lung tumors, suggesting AAV-mediated gene transfer to the pleura to deliver bevacizumab locally to the lung as a novel alternative platform to conventional monoclonal antibody therapy.  

5.798           Combined Paracrine and Endocrine AAV9 mediated Expression of Hepatocyte Growth Factor for the Treatment of Renal Fibrosis

Schievenbusch, S., Strack, I., Scheffler, M., Nischt, R., Coutelle, O., Hösel, M., Hallek, M., Fries, J.W.U., Dienes, H-P., Odenthal, M and Büning, H. Molecular Therapy, 18(7), 1302-1309 (2010)   In chronic renal disease, tubulointerstitial fibrosis is a leading cause of renal failure. Here, we made use of one of the most promising gene therapy vector platforms, the adeno-associated viral (AAV) vector system, and the COL4A3-deficient mice, a genetic mouse model of renal tubulointerstitial fibrosis, to develop a novel bidirectional treatment strategy to prevent renal fibrosis. By comparing different AAV serotypes in reporter studies, we identified AAV9 as the most suitable delivery vector to simultaneously target liver parenchyma for endocrine and renal tubular epithelium for paracrine therapeutic expression of the antifibrogenic cytokine human hepatocyte growth factor (hHGF). We used transcriptional targeting to drive hHGF expression from the newly developed CMV-enhancer-Ksp-cadherin-promoter (CMV-Ksp) in renal and hepatic tissue following tail vein injection of rAAV9-CMV-Ksp-hHGF into COL4A3-deficient mice. The therapeutic efficiency of our approach was demonstrated by a remarkable attenuation of tubulointerstitial fibrosis and repression of fibrotic markers such as collagen1α1 (Col1A1), platelet-derived growth factor receptor-β (PDGFR-β), and α-smooth muscle actin (SMA). Taken together, our results show the great potential of rAAV9 as an intravenously applicable vector for the combined paracrine and endocrine expression of antifibrogenic factors in the treatment of renal failure caused by tubulointerstitial fibrosis.  

5.799           Mucosal delivery of human papillomavirus pseudovirus-encapsidated plasmids improves the potency of DNA vaccination

Graham, B.S., Kines, R.C., Corbett, K.S., Nicewonger, J., Johnson, T.R., Chen, M., La Vigne, D., Roberts, J.N., Cuburu, N., Schiller, J.T. and Buck, C.B. Mucosal Immunol., 3(5), 475-486 (2010)   Mucosal immunization may be important for protection against pathogens whose transmission and pathogenesis target the mucosal tissue. The capsid proteins of human papillomavirus (HPV) confer tropism for the basal epithelium and can encapsidate DNA during self-assembly to form pseudovirions (PsVs). Therefore, we produced mucosal vaccine vectors by HPV PsV encapsidation of DNA plasmids expressing an experimental antigen derived from the M and M2 proteins of respiratory syncytial virus (RSV). Intravaginal (IVag) delivery elicited local and systemic M–M2-specific CD8+ T-cell and antibody responses in mice that were comparable to an ~10,000-fold higher dose of naked DNA. A single HPV PsV IVag immunization primed for M–M2-specific-IgA in nasal and vaginal secretions. Based on light emission and immunofluorescent microscopy, immunization with HPV PsV-encapsidated luciferase- and red fluorescent protein (RFP)-expressing plasmids resulted in transient antigen expression (<5 days), which was restricted to the vaginal epithelium. HPV PsV encapsidation of plasmid DNA is a novel strategy for mucosal immunization that could provide new vaccine options for selected mucosal pathogens.  

5.800           Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles

Pejawar-Gaddy, S., Rajawat, Y., Hilioti, Z., Xue, J., Gaddy, D.F., Finn, R.P., Viscidi, R.P. and Bissis, I. Cancer Immunol. Immunother., 59, 1685-1696 (2010)   Virus-like particles (VLPs) are promising vaccine technology due to their safety and ability to elicit strong immune responses. Chimeric VLPs can extend this technology to low immunogenicity foreign antigens. However, insertion of foreign epitopes into the sequence of self-assembling proteins can have unpredictable effects on the assembly process. We aimed to generate chimeric bovine papillomavirus (BPV) VLPs displaying a repetitive array of polyanionic docking sites on their surface. These VLPs can serve as platform for covalent coupling of polycationic fusion proteins. We generated baculoviruses expressing chimeric BPV L1 protein with insertion of a polyglutamic-cysteine residue in the BC, DE, HI loops and the H4 helix. Expression in insect cells yielded assembled VLPs only from insertion in HI loop. Insertion in DE loop and H4 helix resulted in partially formed VLPs and capsomeres, respectively. The polyanionic sites on the surface of VLPs and capsomeres were decorated with a polycationic MUC1 peptide containing a polyarginine-cysteine residue fused to 20 amino acids of the MUC1 tandem repeat through electrostatic interactions and redox-induced disulfide bond formation. MUC1-conjugated fully assembled VLPs induced robust activation of bone marrow-derived dendritic cells, which could then present MUC1 antigen to MUC1-specific T cell hybridomas and primary naïve MUC1-specific T cells obtained from a MUC1-specific TCR transgenic mice. Immunization of human MUC1 transgenic mice, where MUC1 is a self-antigen, with the VLP vaccine induced MUC1-specific CTL, delayed the growth of MUC1 transplanted tumors and elicited complete tumor rejection in some animals.  

5.801           Adeno-associated virus gene transfer in Morquio A disease – effect of promoters and sulfatase-modifying factor 1

Almeciga-Diaz, C., Montano, A.M., Tomatsu, S. and Barrera, L.A. FEBS J., 277(17), 3608-3619 (2010)   Mucopolysaccharidosis (MPS) IVA is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme N-acetylgalatosamine-6-sulfate sulfatase (GALNS), which leads to the accumulation of keratan sulfate and chondroitin 6-sulfate, mainly in bone. To explore the possibility of gene therapy for Morquio A disease, we transduced the GALNS gene into HEK293 cells, human MPS IVA fibroblasts and murine MPS IVA chondrocytes by using adeno-associated virus (AAV)-based vectors, which carry human GALNS cDNA. The effects of the promoter and the cotransduction with the sulfatase-modifying factor 1 gene (SUMF1) on GALNS activity levels was evaluated. Downregulation of the cytomegalovirus (CMV) immediate early enhancer/promoter was not observed for 10 days post-transduction. The eukaryotic promoters induced equal or higher levels of GALNS activity than those induced by the CMV promoter in HEK293 cells. Transduction of human MPS IVA fibroblasts induced GALNS activity levels that were 15–54% of those of normal human fibroblasts, whereas in transduced murine MPS IVA chondrocytes, the enzyme activities increased up to 70% of normal levels. Cotransduction with SUMF1 vector yielded an additional four-fold increase in enzyme activity, although the level of elevation depended on the transduced cell type. These findings suggest the potential application of AAV vectors for the treatment of Morquio A disease, depending on the combined choice of transduced cell type, selection of promoter, and cotransduction of SUMF1.  

5.802           Efficacy of Recombinant Adeno-Associated Viral Vectors Serotypes 1, 2, and 5 for the Transduction of Pancreatic and Colon Carcinoma Cells

Teschendorf, C., Emons, b., Muzycka, N., Graeven, U. and Schmiegel, W. Anticancer Res., 30, 1931-1936 (2010)   Background: The development of efficient and specific vector systems remains a central issue in gene therapy. Several different adeno-associated virus (AAV) serotypes have so far been characterized so far which show different tissue tropisms. Materials and Methods: The vectors used here contained AAV2 transgene cassette containing green fluorescent protein (GFP) in AAV1, AAV2, or AAV5 capsids, producing the recombinant pseudotypes rAAV2/1, rAAV2/2, and rAAV2/5. The transduction efficiency of the different pseudotyped AAV vectors was tested in vitro in pancreatic and colon cancer cells lines (HT-29, BXPC3, and Hs766T). Results: For all three serotypes, the percentage of GFP-positive cells was below 10% at multiplicities of infection (MOI) 100 rAAV vectors when used alone for infection. However, transduction efficiency for rAAV vectors increased dramatically when the cells were co-infected with wild-type adenovirus (wtAd). The percentage of GFP-positive cells ranged from 19.8-65.3% for AAV2/1 and 16.9-70.2% for AAV2/5, respectively. It was highest for rAAV2/2, at 40.9-88.4%. Variation between the cell lines was observed, with BXPC3 scoring the highest transduction rates and HT-29 the lowest. Conclusion: This study indicates that vectors based on distinct AAV serotypes 1, 2, and 5 all transduce pancreatic and colon cell lines poorly when used alone. Co-infection with wtAd increase transduction rates dramatically indicating that slow second-strand synthesis is a reason for the poor transduction efficiency. Due to the poor transduction rates, none of the rAAV serotypes tested here seem to be feasible for the treatment of malignant tumors.  

5.803           Replacement Gene Therapy with a Human RPGRIP1 Sequence Slows Photoreceptor Degeneration in a Murine Model of Leber Congenital Amaurosis

Pawlyk, B.S., Bulgakov, O.V., Liu, X., Xu, X., Adasmian, M., Sun, X., Khani, S.C., Berson, E.L., Sandberg, M.A. and Li, T. Human Gene Therapy, 21, 993-1004 (2010)   RPGR-interacting protein-1 (RPGRIP1) is localized in the photoreceptor-connecting cilium, where it anchors the RPGR (retinitis pigmentosa GTPase regulator) protein, and its function is essential for photoreceptor maintenance. Genetic defect in RPGRIP1 is a known cause of Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. We evaluated the efficacy of replacement gene therapy in a murine model of LCA carrying a targeted disruption of RPGRIP1. The replacement construct, packaged in an adeno-associated virus serotype 8 (AAV8) vector, used a rhodopsin kinase gene promoter to drive RPGRIP1 expression. Both promoter and transgene were of human origin. After subretinal delivery of the replacement gene in the mutant mice, human RPGRIP1 was expressed specifically in photoreceptors, localized correctly in the connecting cilia, and restored the normal localization of RPGR. Electroretinogram and histological examinations showed better preservation of rod and cone photoreceptor function and improved photoreceptor survival in the treated eyes. This study demonstrates the efficacy of human gene replacement therapy and validates a gene therapy design for future clinical trials in patients afflicted with this condition. Our results also have therapeutic implications for other forms of retinal degenerations attributable to a ciliary defect.  

5.804           Inhibition of nuclear entry of HPV16 pseudovirus-packaged DNA by an anti-HPV16 L2 neutralizing antibody

Ishii, Y., Tanaka, K., Kondo, K., Takeuchi, T., Mori, S. and Kanda, T. Virology, 406, 181-188 (2010)   Rabbit anti-HPV16 L2 serum (anti-P56/75) neutralizes multiple oncogenic human papillomaviruses (HPVs). We inoculated HeLa cells with HPV16 pseudovirus (16PV) and with anti-P56/75-bound 16PV (16PV-Ab). Both 16PV and 16PV-Ab attached equally well to the cell surface. However, the cell-attached L1 protein of 16PV became trypsin-resistant after incubation at 37 °C, whereas approximately 20% of the cell-attached 16PV-Ab L1 remained trypsin-sensitive. Confocal microscopy of HeLa cells inoculated with 16PV revealed packaged DNA in the nucleus at 22 h after inoculation; however, nuclear DNA was not detected in cells inoculated with 16PV-Ab. Electron microscopy of HeLa cells inoculated with 16PV showed particles located in multivesicular bodies, lamellar bodies, and the cytosol after 4 h; no cytosolic particles were detected after inoculation with 16PV-Ab. These data suggest that anti-P56/75 inhibits HPV infection partly by blocking viral entry and primarily by blocking the transport of the viral genome to the nucleus.  

5.805           Structure of a Venezuelan equine encephalitis virus assembly intermediate isolated from infected cells

Lamb, K., Lokesh, G.L., Sherman, M. and Watowich, S. Virology, 406, 261-269 (2010)   Venezuelan equine encephalitis virus (VEEV) is a prototypical enveloped ssRNA virus of the family Togaviridae. To better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. These particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. Purified pre-viral nucleocapsids were spherical with a unimodal diameter distribution. The structure of one class of pre-viral nucleocapsids was determined with single particle reconstruction of cryo-electron microscopy images. These studies showed that pre-viral nucleocapsids assembled into an icosahedral structure with a capsid stoichiometry similar to the mature nucleocapsid. However, the individual capsomers were organized significantly differently within the pre-viral and mature nucleocapsids. The pre-viral nucleocapsid structure implies that nucleocapsids are highly plastic and undergo glycoprotein and/or lipid-driven rearrangements during virus self-assembly. This mechanism of self-assembly may be general for other enveloped viruses.  

5.806           Identification of Two APOBEC3F Splice Variants Displaying HIV-1 Antiviral Activity and Contrasting Sensitivity to Vif

Lassen, K.G., Wissing, S., Lobritz, M.A., Santiago, M. and Greene, W.C.

1. Biol. Chem., 285(38), 29326-29335 (2010)

  Approximately half of all human genes undergo alternative mRNA splicing. This process often yields homologous gene products exhibiting diverse functions. Alternative splicing of APOBEC3G (A3G) and APOBEC3F (A3F), the major host resistance factors targeted by the HIV-1 protein Vif, has not been explored. We investigated the effects of alternative splicing on A3G/A3F gene expression and antiviral activity. Three alternatively spliced A3G mRNAs and two alternatively spliced A3F mRNAs were detected in peripheral blood mononuclear cells in each of 10 uninfected, healthy donors. Expression of these splice variants was altered in different cell subsets and in response to cellular stimulation. Alternatively spliced A3G variants were insensitive to degradation by Vif but displayed no antiviral activity against HIV-1. Conversely, alternative splicing of A3F produced a 37-kDa variant lacking exon 2 (A3FΔ2) that was prominently expressed in macrophages and monocytes and was resistant to Vif-mediated degradation. Alternative splicing also produced a 24-kDa variant of A3F lacking exons 2–4 (A3FΔ2–4) that was highly sensitive to Vif. Both A3FΔ2 and A3FΔ2–4 displayed reduced cytidine deaminase activity and moderate antiviral activity. These alternatively spliced A3F gene products, particularly A3FΔ2, were incorporated into HIV virions, albeit at levels less than wild-type A3F. Thus, alternative splicing of A3F mRNA generates truncated antiviral proteins that differ sharply in their sensitivity to Vif.  

5.807           Evaluation of Systemic Follistatin as an Adjuvant to Stimulate Muscle Repair and Improve Motor Function in Pompe Mice

Foley, J.W., Bercury, S.D., Finn, P., Cheng, S.H., Scheule, R.K. and Ziegler, R.J. Molecular Therapy, 18(9), 1584-1591 (2010)   Due to the lack of acid α-glucosidase (GAA) activity, Pompe mice develop glycogen storage pathology and progressive skeletal muscle dysfunction with age. Applying either gene or enzyme therapy to reconstitute GAA levels in older, symptomatic Pompe mice effectively reduces glycogen storage in skeletal muscle but provides only modest improvements in motor function. As strategies to stimulate muscle hypertrophy, such as by myostatin inhibition, have been shown to improve muscle pathology and strength in mouse models of muscular dystrophy, we sought to determine whether these benefits might be similarly realized in Pompe mice. Administration of a recombinant adeno-associated virus serotype 8 vector encoding follistatin, an inhibitor of myostatin, increased muscle mass and strength but only in Pompe mice that were treated before 10 months of age. Younger Pompe mice showed significant muscle fiber hypertrophy in response to treatment with follistatin, but maximal gains in muscle strength were achieved only when concomitant GAA administration reduced glycogen storage in the affected muscles. Despite increased grip strength, follistatin treatment failed to improve rotarod performance. These findings highlight the importance of treating Pompe skeletal muscle before pathology becomes irreversible, and suggest that adjunctive therapies may not be effective without first clearing skeletal muscle glycogen storage with GAA.  

5.808           Efficient KRT14 Targeting and Functional Characterization of Transplanted Human Keratinocytes for the Treatment of Epidermolysis Bullosa Simplex

Petek, L.M., Fleckman, P. and Miller, D.G. Molecular Therapy, 18(9), 1624-1632 (2010)   Inherited skin blistering conditions collectively named epidermolysis bullosa (EB) cause significant morbidity and mortality due to the compromise of the skin's barrier function, the pain of blisters, inflammation, and in some cases scaring and cancer. The simplex form of EB is usually caused by dominantly inherited mutations in KRT5 or KRT14. These mutations result in the production of proteins with dominant-negative activity that disrupt polymerization of intermediate filaments in the basal keratinocyte layer and result in a weak epidermal–dermal junction. The genome of adeno-associated virus (AAV) vectors can recombine with chromosomal sequence so that mutations can be corrected, or production of proteins with dominant-negative activity can be disrupted. We demonstrate a clinically feasible strategy for efficient targeting of the KRT14 gene in normal and EB-affected human keratinocytes. Using a gene-targeting vector with promoter trap design, targeted alteration of one allele of KRT14 occurred in 100% of transduced cells and transduction frequencies ranged from 0.1 to 0.6% of total cells. EBS patient keratinocytes with precise modifications of the mutant allele are preferentially recovered from targeted cell populations. Single epidermal stem cell clones produced histologically normal skin grafts after transplantation to athymic mice and could generate a sufficient number of cells to transplant the entire skin surface of an individual.  

5.809           Rapid Construction of Adeno-Associated Virus Vectors Expressing Multiple Short Hairpin RNAs with High Antiviral Activity Against Echovirus 30

Rothe, D., Wajant, G., Grunert, H-P., Zeichhhardt, H., Fechner, H. and Kurreck, J. Oligonucleotides, 20(4), 191-198 (2010)   RNA interference has proven to be a powerful tool to inhibit viruses. For the prevention of viral escape, multiple short hairpin RNAs (shRNAs) will have to be employed. This article describes a rapid procedure for the generation of shRNA expression cassettes by parallel cloning as well as a simple strategy for the combination of selected units. After delivery of the shRNA expression cassettes with adeno-associated virus vectors, inhibition of echovirus 30 as well as silencing of an important cellular cofactor of virus replication were achieved. The procedure has the potential to be generally applicable for silencing of multiple endogenous targets or viruses.  

5.810           Parvovirus H1 selectively induces cytotoxic effects on human neuroblastoma cells

Lacroix, J., Leuchs, B., Li, J., Hristov, G., Deubzer, H.E., Kulozik, A.E., Rommelaere, J., Schleofer, J.R. and Witt, O. Int. J.Cancer, 127, 1230-1239 (2010)   Despite multimodal therapeutic concepts, advanced localized and high-risk neuroblastoma remains a therapeutic challenge with a long-term survival rate below 50%. Consequently, new modalities for the treatment of neuroblastoma, e.g., oncolytic virotherapy are urgently required. H-1PV is a rodent parvovirus devoid of relevant pathogenic effects in infected adult animals. In contrast, the virus has oncolytic properties and is particularly cytotoxic for transformed or tumor-derived cells of various species including cells of human origin. Here, a preclinical in vitro assessment of the application of oncolytic H-1PV for the treatment of neuroblastoma cells was performed. Infection efficiency, viral replication and lytic activity of H-1PV were analyzed in 11 neuroblastoma cell lines with different MYCN status. Oncoselectivity of the virus was confirmed by the infection of short term cultures of nonmalignant infant cells of different origin. In these nontransformed cells, no effect of H-1PV on viability or morphology of the cells was observed. In contrast, a lytic infection was induced in all neuroblastoma cell lines examined at MOIs between 0.001 and 10 pfu/cell. H-1PV actively replicated with virus titres increasing up to 5,000-fold within 48–96 hr after infection. The lytic effect of H-1PV was observed independent of MYCN oncogene amplification or differentiation status. Moreover, a significant G2-arrest and induction of apoptosis could be demonstrated. Infection efficiency, rapid virus replication and exhaustive lytic effects on neuroblastoma cells together with the low toxicity of H-1PV for nontransformed cells, render this parvovirus a promising candidate for oncolytic virotherapy of neuroblastoma.  

5.811           Combined vascular endothelial growth factor-A and fibroblast growth factor 4 gene transfer improves wound healing in diabetic mice

Jazwa, A., Kucharzewska, P., Leja, J., Zagorska, A., Sierpniowska, A., Stepniewski, J., Kozakowska, M., Taha, H., Ochiya, T., Derlacz, R., Vahakangas, E., Yla-Herttuala, S., Jozkowicz, A. and Dulak, J. GeneticVaccines and Therapy, 8, 6-21 (2010)   Background Impaired wound healing in diabetes is related to decreased production of growth factors. Hence, gene therapy is considered as promising treatment modality. So far, efforts concentrated on single gene therapy with particular emphasis on vascular endothelial growth factor-A (VEGF-A). However, as multiple proteins are involved in this process it is rational to test new approaches. Therefore, the aim of this study was to investigate whether single AAV vector-mediated simultaneous transfer of VEGF-A and fibroblast growth factor 4 (FGF4) coding sequences will improve the wound healing over the effect of VEGF-A in diabetic (db/db) mice. Methods Leptin receptor-deficient db/db mice were randomized to receive intradermal injections of PBS or AAVs carrying β-galactosidase gene (AAV-LacZ), VEGF-A (AAV-VEGF-A), FGF-4 (AAV-FGF4-IRES-GFP) or both therapeutic genes (AAV-FGF4-IRES-VEGF-A). Wound healing kinetics was analyzed until day 21 when all animals were sacrificed for biochemical and histological examination. Results Complete wound closure in animals treated with AAV-VEGF-A was achieved earlier (day 19) than in control mice or animals injected with AAV harboring FGF4 (both on day 21). However, the fastest healing was observed in mice injected with bicistronic AAV-FGF4-IRES-VEGF-A vector (day 17). This was paralleled by significantly increased granulation tissue formation, vascularity and dermal matrix deposition. Mechanistically, as shown in vitro, FGF4 stimulated matrix metalloproteinase-9 (MMP-9) and VEGF receptor-1 expression in mouse dermal fibroblasts and when delivered in combination with VEGF-A, enhanced their migration. Conclusion Combined gene transfer of VEGF-A and FGF4 can improve reparative processes in the wounded skin of diabetic mice better than single agent treatment.  

5.812           In Vivo Mechanisms of Vaccine-Induced Protection against HPV Infection

Day, P.M., Kines, R.C., Thompson, C.D., Jagu, S., Roden, R.B., Lowy, D.R. and Schiller, J.T. Cell Host & Microbe, 8, 260-270 (2010)   Using a human papillomavirus (HPV) cervicovaginal murine challenge model, we microscopically examined the in vivo mechanisms of L1 virus-like particle (VLP) and L2 vaccine-induced inhibition of infection. In vivo HPV infection requires an initial association with the acellular basement membrane (BM) to induce conformational changes in the virion that permit its association with the keratinocyte cell surface. By passive transfer of immune serum, we determined that anti-L1 antibodies can interfere with infection at two stages. Similarly to active VLP immunization, transfer of high L1 antibody concentrations prevented BM binding. However, in the presence of low concentrations of anti-L1, virions associated with the BM, but to the epithelial cell surface was not detected. Regardless of the concentration, L2 vaccine-induced antibodies allow BM association but prevent association with the cell surface. Thus, we have revealed distinct mechanisms of vaccine-induced inhibition of virus infection in vivo.  

5.813           Regulation of the activity of an adeno-associated virus vector cancer vaccine administered with synthetic Toll-like receptor agonists

Triozzi, P.L., Aldrich, W. and Ponnazhagan, S. Vaccine, 28(50), 7837-7843 (2010)   Recombinant adeno-associated virus (rAAV) is being tested as a vaccine vector, but the cellular immune responses elicited in animal tumor models have not been completely protective. The adjuvant effects of the TLR7 agonist, imiquimod, and the TLR9 agonist, ODN1826, were tested with rAAV expressing the melanoma antigen, Trp2. Mice immunized with rAAV-TRP2 and either TLR agonist alone generated T-helper-1 antitumor immune responses. Antitumor activity in all experiments was still incomplete. Furthermore, antitumor activity was not achieved when the combination of ODN1826 and imiquimod was used as adjuvant. In vitro, the combination increased IL-10 production by dendritic cells. In vivo, the combination reduced T-helper-1 response and dendritic cell activation and increased myeloid suppressor cells; regulatory T cells were not significantly modulated. Depletion of myeloid derived suppressor cells enhanced the antitumor activity of immunization with rAAV-TRP2 and the imiquimod-ODN1826 combination; depletion of regulatory T cells did not. TLR7 and TLR9 agonists can be used to enhance the immune response to rAAV immunogens, but antagonism can be observed when combined. Suppressor mechanisms, including those mediated by myeloid cells, may negatively regulate the antitumor immune response.    

5.814           Biodistribution and safety assessment of AAV2-GAD following intrasubthalamic injection in the rat

Fitzsimons, H.L., Riban, V., Bland, R.J., Wendelken, J.L., Sapan, C.V. and During, M.J:

1. Gene Med., 12, 385-398 (2010)

  Background The steps necessary to translate promising new biological therapies to the clinic are poorly documented. For gene therapy, there are unique aspects that need to be addressed in biodistribution studies. Notably, the spread of the vector beyond the intended target cells or tissue may result in persistent unwanted biological activity or unpredictable biological events; thus, it is critical to evaluate the risks associated with viral vector-mediated gene transfer prior to embarking on human clinical trials. Methods In the present study, we conducted a comprehensive assessment of vector biodistribution throughout the brain, blood and major organs of rats that had been injected via the subthalamic nucleus with recombinant adeno-associated virus (AAV) expressing glutamic acid decarboxylase (GAD). In addition, behavioral and histological analyses were also performed. Results AAV genomes were not detected in blood or cerebrospinal fluid, and did not disseminate to organs outside of the brain in the majority of animals. In the brain, an average of 97.3% of AAV2-GAD genomes were restricted to the area of the ipsilateral subthalamic nucleus (STN). There were no discernable effects of AAV2-GAD on general health, and a behavioral assessment of the animals did not reveal any alteration in general behavior, exploration, locomotion or motor symmetry. Conclusions The present study met Food and Drug Administration requirements, in addition to efficacy and toxicity studies in rodents and nonhuman primates, to support and supplement a Phase II clinical trial invloving the gene transfer of AAV2-GAD to the human STN for the potential therapy of Parkinson's disease.  

5.815           Modulation of coxsackie and adenovirus receptor expression for gene transfer to normal and dystrophic skeletal muscle

Larochelle, N., Teng, Q., Gilbert, R., Deol, J.R., Karpati, G., Holland, P.C. and Nalbantoglu, J.

1. Gene Med., 12, 266-275 (2010)

  Background Efficient adenovirus (AdV)-mediated gene transfer is possible only in immature muscle or regenerating muscle, suggesting that a developmentally regulated event plays a major role in limiting AdV uptake in mature skeletal muscle. Previously, we showed that the expression of the primary coxsackie and adenovirus receptor (CAR) is severely down-regulated during muscle maturation and that, in muscle-specific CAR transgenic mice, there is significant enhancement of AdV-mediated gene transfer to mature skeletal muscle. Methods To evaluate whether increasing CAR expression can also augment gene transfer to dystrophic muscle that has many regenerating fibers, we crossed CAR transgenics with dystrophin-deficient mice (mdx/CAR). We also tested a two-step protocol in which CAR levels were increased in the target muscle, prior to administration of AdV, through the use of recombinant adeno-associated virus (AAV2) expressing CAR. Lastly, we assessed the effect of histone deacetylase inhibitors on CAR and AdV transduction efficiency in myoblasts and mdx muscle. Results Although somewhat higher rates of transduction can be achieved in adult mdx mice than in normal mice as a result of ongoing muscle regeneration in these animals, CAR expression in the mdx background (mdx/CAR transgenics) still markedly improved the susceptibility of mature muscle to AdV-mediated gene transfer of dystrophin. Prior administration of AAV2-CAR to normal muscle led to significantly increased transduction by subsequent injection of AdV. The histone deacetylase inhibitor valproate increased CAR transcript and protein levels in myoblasts and mdx muscle, and also increased AdV-mediated gene transfer. Conclusions We have developed a method of increasing CAR levels in both normal and regenerating muscle.  

5.816           Recombinant mammalian cell derived hepatitis C virus-like particles induce neutralizing antibody responses to hepatitis C virus

Johnson, D.F., Chin, R., Earnest-Siveira, L., Zentgraf, H., Bock, T., Chua, B., Jackson, D.C.and Torresi, J. Clin. Microbiol. Infect., 16, S319 (2010)   Objective: Clearance of Hepatitis C virus (HCV) requires a strong and broadly cross-reactive CD4+, CD8+ T cell and neutralising antibody (Ab) responses. Virus like particles (VLPs) resemble mature parent virus inducing protective humoral and cellular immune responses against HCV and provide a viable prophylactic vaccine candidate. Methods: Recombinant adenoviruses expressing HCVcore-E1-E2 were used to infect Huh7 (hepatoma) cells and produce HCV VLPs. These were isolated from cell lysates and purified by Iodixanol density gradient ultracentrifugation. E1 and E2 glycoproteins of the correct size in HCV VLPs were confirmed by Western immunoblot. HCV VLPs were analysed by testing for maturation of dendritic cells (DC) and mice were immunised with HCV VLPs alone and with alum and Freunds adjuvants. The mice were assessed for (1) humoral responses against both VLPs and a recombinant E2 protein of HCV, (2) production of mouse antibody secreting cells (memory B cells) in splenocytes using B cell Elispot assays and (3) neutralizing Ab in a Huh 7 cell entry assay. A second group of mice were immunised with VLPs and 2 novel adjuvants. Results: We have produced, purified and confirmed the presence of HCV VLPs of genotype 1a by western immunoblot, electron microscopy (EM) and immunogold EM. HCV VLPs efficiently stimulate the maturation of dendritic cells to level that are comparable to lipopolysaccharide  (LPS). Mice immunised induced strong humoral responses to E2 and VLPs and mouse anti-HCV VLP serum neutralized VLP entry into Huh7 cells. B cell elispot assays, using mouse splenocytes, demonstrated production of mouse antibody secreting cells / memory B cells. Mice immunised with VLPs and 2 novel adjuvants demonstrated a greater humoral response and increased levels of mouse antibody secreting cells compared with mice immunised with HCV VLPs alone or in Alum. Conclusion: Mammalian cell derived HCV VLPs exhibit similar morphological, biophysical and immunological properties as putative HCV virions and are a viable vaccine strategy for HCV. HCV VLPs of genotypes 1b, 3a and 4 are currently being produced. Studies of CD8 responses and methods to improve VLP immunogenicity and alternative adjuvanting are ongoing.  

5.817           Identification of a Dendrimeric Heparan Sulfate-Binding Peptide That Inhibits Infectivity of Genital Types of Human Papillomaviruses

Donalisio, M., Rusnati, M., Civra, A., Bugatti, A., Allemand, D., Pirri, G., Giuliani, A., Landolfo, S. and limbo, D. Antimicrob. Agents Chemother., 54(10), 4290-4299 (2010)   Peptide dendrimers consist of a peptidyl branching core and/or covalently attached surface functional units. They show a variety of biological properties, including antiviral activity. In this study, a minilibrary of linear, dimeric, and dendrimeric peptides containing clusters of basic amino acids was evaluated for in vitro activity against human papillomaviruses (HPVs). The peptide dendrimer SB105-A10 was found to be a potent inhibitor of genital HPV types (i.e., types 16, 18, and 6) in pseudovirus-based neutralization assays. The 50% inhibitory concentration was between 2.8 and 4.2 µg/ml (0.59 and 0.88 µM), and no evidence of cytotoxicity was observed. SB105-A10 interacts with immobilized heparin and with heparan sulfates exposed on the cell surface, most likely preventing virus attachment. The findings from this study indicate SB105-A10 to be a leading candidate compound for further development as an active ingredient of a topical microbicide against HPV and other sexually transmitted viral infections.  

5.818           Papillomavirus Infection Requires Secretase

Karanam, B., Peng, S., Li, T., Buck, C., day, P.M. and Roden, R.B.S.

1. Virol., 84(20), 10661-10670 (2010)

  The mechanism by which papillomaviruses breach cellular membranes to deliver their genomic cargo to the nucleus is poorly understood. Here, we show that infection by a broad range of papillomavirus types requires the intramembrane protease secretase. The -secretase inhibitor (S,S)-2-[2-(3,5-difluorophenyl)-acetylamino]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-propionamide (compound XXI) inhibits infection in vitro by all types of papillomavirus pseudovirions tested, with a 50% inhibitory concentration (IC50) of 130 to 1,000 pM, regardless of reporter construct and without impacting cellular viability. Conversely, XXI does not inhibit in vitro infection by adenovirus or pseudovirions derived from the BK or Merkel cell polyomaviruses. Vaginal application of XXI prevents infection of the mouse genital tract by human papillomavirus type 16 (HPV16) pseudovirions. Nicastrin and presenilin-1 are essential components of the -secretase complex, and mouse embryo fibroblasts deficient in any one of these components were not infected by HPV16, whereas wild-type and β-secretase (BACE1)-deficient cells were susceptible. Neither the uptake of HPV16 into Lamp-1-positive perinuclear vesicles nor the disassembly of capsid to reveal both internal L1 and L2 epitopes and bromodeoxyuridine (BrdU)-labeled encapsidated DNA is dependent upon -secretase activity. However, blockade of -secretase activity by XXI prevents the BrdU-labeled DNA encapsidated by HPV16 from reaching the ND10 subnuclear domains. Since prior studies indicate that L2 is critical for endosomal escape and targeting of the viral DNA to ND10 and that secretase is located in endosomal membranes, our findings suggest that either L2 or an intracellular receptor are cleaved by secretase as papillomavirus escapes the endosome.  

5.819           SMAD4: a predictive marker of PDAC cell permissiveness for oncolytic infection with parvovirus H-1PV

Dempe, S., Stroh-Dege, A.Y., Schwarz, E., Rommelaere, J. and Dinsart, C. Int. J. Cancer, 126, 2914-2927 (2010)   Pancreatic ductal adenocarcinoma (PDAC) represents the eighth frequent solid tumor and fourth leading cause of cancer death. Because current treatments against PDAC are still unsatisfactory, new anticancer strategies are required, including oncolytic viruses. Among these, autonomous parvoviruses (PV), like MVMp (minute virus of mice) and H-1PV are being explored as candidates for cancer gene therapy. Human PDAC cell lines were identified to display various susceptibilities to an infection with H-1PV. The correlation between the integrity of the transcription factor SMAD4, mutated in 50% of all PDAC, and H-1PV permissiveness was particularly striking. Indeed, mutation or deletion of SMAD4 dramatically reduced the activity of the P4 promoter and, consequently, the accumulation of the pivotal NS1 protein. By means of DNA affinity immunoblotting, novel binding sites for SMAD4 and c-JUN transcription factors could be identified in the P4 promoter of H-1PV. The overexpression of wild-type SMAD4 in deficient cell lines (AsPC-1, Capan-1) stimulated the activity of the P4 promoter, whereas interference of endogenous SMAD4 function with a dominant-negative mutant decreased the viral promoter activity in wild-type SMAD4-expressing cells (Panc-1, MiaPaCa-2) reducing progeny virus production. In conclusion, the importance of members of the SMAD family for H-1PV early promoter P4 activity should guide us to select SMAD4-positive PDACs, which may be possible targets for an H-1PV-based cancer therapy.  

5.820           Combination of alpha-1 antitrypsin and doxycycline suppresses collagen-induced arthritis

Grimstein, C., Choi, Y-K., Satoh, M., Lu, Y., Wang, X., Campbell-Thompson, M. and Song, S.

1. Gene Med., 12, 35-44 (2010)

  Background Rheumatoid arthritis (RA) is a complex disease characterized by autoimmune inflammation and joint destruction. Despite recent advances in RA treatment, current therapies require further improvement to overcome adverse events and ineffectiveness in some cases. By targeting different pathways/molecules using drug combinations, a better treatment can be obtained, whereas adverse events are reduced. In order to develop a new treatment option, the present study employs a gene therapy-based combination therapy using doxycycline and human alpha-1 antitrypsin (hAAT). Methods DBA/1 mice were immunized with type II collagen to induce arthritis. Four weeks before immunization, they received a doxycycline containing diet and a single injection of adeno-associated virus vector expressing hAAT under the control of a tetracycline-dependent promoter. Control groups received doxycycline alone or saline. Macroscopic arthritis development as well as histopathological changes in the joint were evaluated. In addition, the effects of hAAT and doxycycline on lipopolysaccharide (LPS)- or tumor necrosis factor- -induced interleukin (IL)-6 production from mouse fibroblast cells were also determined. Results Combination therapy significantly reduced arthritis development and progression compared to the control group in respect to macroscopic as well as histopathological changes. Doxycycline and hAAT in combination also inhibited IL-6 expression from LPS-stimulated NIH/3T3 mouse fibroblast cells, indicating a contributing mechanism of arthritis inhibition. Conclusions The results obtained in the present study indicate that a combination therapy using AAT and doxycycline holds promising potential as a new therapy for RA.  

5.821           Apolipoprotein E interacts with hepatitis C virus nonstructural protein 5A and determines assembly of infectious particles

Benga, W.J.A., Krieger, S.E., Dimitrova, M., Zeisel, M.B., Parnot, M., Lupberger, J., Hildt, E., Luo, G., McLauchlan, J., Baumert, T.F. and Schuster, C. Hepatology, 51, 43-53 (2010)   Chronic hepatitis C virus (HCV) infection is a major cause of liver disease worldwide. Restriction of HCV infection to human hepatocytes suggests that liver-specific host factors play a role in the viral life cycle. Using a yeast-two-hybrid system, we identified apolipoprotein E (apoE) as a liver-derived host factor specifically interacting with HCV nonstructural protein 5A (NS5A) but not with other viral proteins. The relevance of apoE–NS5A interaction for viral infection was confirmed by co-immunoprecipitation and co-localization studies of apoE and NS5A in an infectious HCV cell culture model system. Silencing apoE expression resulted in marked inhibition of infectious particle production without affecting viral entry and replication. Analysis of particle production in liver-derived cells with silenced apoE expression showed impairment of infectious particle assembly and release. The functional relevance of the apoE–NS5A interaction for production of viral particles was supported by loss or decrease of apoE–NS5A binding in assembly-defective viral mutants. Conclusion: These results suggest that recruitment of apoE by NS5A is important for viral assembly and release of infectious viral particles. These findings have important implications for understanding the HCV life cycle and the development of novel antiviral strategies targeting HCV–lipoprotein interaction.  

5.822           Projections of preBötzinger Complex neurons in adult rats

Tan, W., Pagliardini, S., Yang, P., Janczewski, W.A. and Feldman, J.L.

1. Comp. Neurol., 518, 1862-1878 (2010)

  The preBötzinger Complex (preBötC) contains neural microcircuitry essential for normal respiratory rhythm generation in rodents. A subpopulation of preBötC neurons expresses somatostatin, a neuropeptide with a modulatory action on breathing. Acute silencing of a subpopulation of preBötC neurons transfected by a virus driving protein expression under the somatostatin promoter results in persistent apnea in awake adult rats. Given the profound effect of silencing these neurons, their projections are of interest. We used an adeno-associated virus to overexpress enhanced green fluorescent protein driven by the somatostatin promoter in preBötC neurons to label their axons and terminal fields. These neurons send brainstem projections to: 1) contralateral preBötC; 2) ipsi- and contralateral Bötzinger Complex; 3) ventral respiratory column caudal to preBötC; 4) parafacial respiratory group / retrotrapezoid nucleus; 5) parahypoglossal nucleus/nucleus of the solitary tract; 6) parabrachial/Kölliker-Fuse nuclei; and 7) periaqueductal gray. We did not find major projections to either cerebellum or spinal cord. We conclude that there are widespread projections from preBötC somatostatin-expressing neurons specifically targeted to brainstem regions implicated in control of breathing, and provide a network basis for the profound effects and the essential role of the preBötC in breathing.  

5.823           The rate of hepatitis C virus infection initiation in vitro is directly related to particle density

Sabahi, A., Marsh, K.A., Dahari, H., Corcoran, P., Lamora, J.M., Yu, X., Garry, R.F. and Uprichard, S.L. Virology, 407, 110-119 (2010)   To gain a more complete understanding of hepatitis C virus (HCV) entry, we initially assessed the rate at which HCV initiates productive attachment/infection in vitro and discovered it to be slower than most viruses. Since HCV, including cell culture-derived HCV (HCVcc), exhibits a broad-density profile (1.01–1.16 g/ml), we hypothesized that the varying densities of the HCVcc particles present in the inoculum may be responsible for this prolonged entry phenotype. To test this hypothesis, we show that during infection, particles of high density disappeared from the viral inoculum sooner and initiated productive infection faster than virions of low density. Moreover, we could alter the rate of attachment/infection initiation by increasing or decreasing the density of the cell culture medium. Together, these findings demonstrate that the relationship between the density of HCVcc and the density of the extracellular milieu can significantly impact the rate at which HCVcc productively interacts with target cells in vitro.  

5.824           Lipoprotein lipase and hepatic triglyceride lipase reduce the infectivity of hepatitis C virus (HCV) through their catalytic activities on HCV-associated lipoproteins

Shimizu, Y., Hishiki, T., Sugiyama, K., Ogawa, K., Funami, K., Kato, A., Ohsaki, Y., Fujimoto, T., Takaku, H. and Shimotohno, K. Virology, 407, 152-159 (2010)   The effect of lipolysis by lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) on hepatitis C virus (HCV) infection was evaluated. First, medium from HuH7.5 cells bearing HCV genome replication was treated with LPL. LPL treatment led to reduced HCV infectivity, shifted HCV to higher densities, and lowered the amount of apolipoprotein E-associated HCV. The effect of endogenous HTGL secreted from HuH7.5 on HCV infectivity was next examined. Neutralization of HTGL by an anti-HTGL antibody resulted in suppression of LPL-induced reduction in infectivity of HCV-bearing medium, while knockdown of HTGL by siRNA led to increased HCV infectivity irrespective of LPL. HCV in medium from HTGL knockdown cells was found in fractions with a lower density. These results indicate that changes in the nature of HCV-associated lipoproteins by LPL and/or HTGL affect HCV infectivity, suggesting that association of HCV with specific lipoproteins is important for HCV infectivity.  

5.825           GFAP-Driven GFP Expression in Activated Mouse Müller Glial Cells Aligning Retinal Blood Vessels Following Intravitreal Injection of AAV2/6 VectorsMüller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Müller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy.We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Müller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Müller glial cells, several other inner retinal cell types were transduced. To obtain Müller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1^−/− retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels.Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.

Aartsen, W.M., van Cleef, K.W.R., Pellissier, L.P., Hoek, R.M., Vos, R.M., Blits, B., Rhlert, E.M.E., Balaggan, K.S., Ali, R.R., Verhaagen, J. and Wijnholds, J. PloSOne, 5(8), e12387 (2010)   Background Müller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Müller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy. Methodology/Principal Findings We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Müller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Müller glial cells, several other inner retinal cell types were transduced. To obtain Müller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1−/− retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels. Conclusions/Significance Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.  

5.826           Polyunsaturated liposomes are antiviral against hepatitis B and C viruses and HIV by decreasing cholesterol levels in infected cells

Pollock, S., Nichita, N.B., Böhmer, A., Radulescu, C., Dwek, R.A. and Zitzmann, N. PNAS, 107(40), 17176-17181 (2010)   The pressing need for broad-spectrum antivirals could be met by targeting host rather than viral processes. Cholesterol biosynthesis within the infected cell is one promising target for a large number of viral systems, including hepatitis C virus (HCV), hepatitis B virus (HBV) and HIV. Liposomes developed for intracellular, endoplasmic reticulum (ER)-targeted in vivo drug delivery have been modified to include polyunsaturated fatty acids that exert an independent antiviral activity through the reduction of cellular cholesterol. These polyunsaturated ER liposomes (PERLs) have greater activity than lovastatin (Mevacor, Altoprev), which is clinically approved for lowering cholesterol and preventing cardiovascular disease. Treatment of HCV, HBV, and HIV infections with PERLs significantly decreased viral secretion and infectivity, and pretreatment of naïve cells reduced the ability of both HCV and HIV to establish infections because of the decreased levels of plasma membrane cholesterol. Direct competition for cellular receptors was an added effect of PERLs against HCV infections. The greatest antiviral activity in all three systems was the inhibition of viral infectivity through the reduction of virus-associated cholesterol. Our study demonstrates that PERLs are a broadly effective antiviral therapy and should be developed further in combination with encapsulated drug mixtures for enhanced in vivo efficacy.  

5.827           Inhibition of gamma secretase blocks HPV infection

Huang, H-S., Buck, C.B, and Lambert, P.F. Virology, 407, 391-396 (2010)   Human papillomaviruses (HPV) are common sexually transmitted pathogens that predispose women to cervical and other anogenital cancers. HPV vaccines can prevent infection by some but not other sexually transmitted HPVs but are too costly for use in much of the world at greatest risk to HPV-associated cancers. Microbicides provide an inexpensive alternative to vaccines. In a high throughput screen, drugs that inhibit the cellular protein complex known as gamma secretase were identified as potential HPV microbicides. gamma Secretase inhibitors (GSIs) inhibited the infectivity of HPV pseudoviruses both in human keratinocytes and in mouse cells, with IC₅₀ values in the picomolar to the nanomolar range. Using a mouse model, we observed that a GSI could inhibit HPV infection to the same degree as its effectiveness in inhibiting gamma secretase activity in vivo. We conclude that gamma secretase activity is required for HPV infection and that GSIs are effective microbicides against anogenital HPVs.  

5.828           Structure of Penaeus stylirostris Densovirus, a Shrimp Pathogen

KaufmaNN, b., Bowman, V.D., Li, Y., Szelei, J., Waddell, P.J., Tijssen, P. and Rossmann, M.G.

1. Virol., 84(21), 11289-11296 (2010)

  Penaeus stylirostris densovirus (PstDNV), a pathogen of penaeid shrimp, causes significant damage to farmed and wild shrimp populations. In contrast to other parvoviruses, PstDNV probably has only one type of capsid protein that lacks the phospholipase A2 activity that has been implicated as a requirement during parvoviral host cell infection. The structure of recombinant virus-like particles, composed of 60 copies of the 37.5-kDa coat protein, the smallest parvoviral capsid protein reported thus far, was determined to 2.5-Å resolution by X-ray crystallography. The structure represents the first near-atomic resolution structure within the genus Brevidensovirus. The capsid protein has a β-barrel "jelly roll" motif similar to that found in many icosahedral viruses, including other parvoviruses. The N-terminal portion of the PstDNV coat protein adopts a "domain-swapped" conformation relative to its twofold-related neighbor similar to the insect parvovirus Galleria mellonella densovirus (GmDNV) but in stark contrast to vertebrate parvoviruses. However, most of the surface loops have little structural resemblance to any of the known parvoviral capsid proteins.  

5.829           Galectin-1 and HIV-1 Infection

St.-Pierre, C., Ouellet, M., Tremblay, M.J. and Sato, S. Methods in Enzymol., 480, 267-294 (2010)   Initial binding of human immunodeficiency virus-1 (HIV-1) to its susceptible CD4⁺ cells is the limiting step for the establishment of infection as the avidity of viral envelope gp120 for CD4 is not high and the number of viral envelope spikes on the surface is found to be low compared to highly infectious viruses. Several host factors, such as C-type lectins, are listed as being able to enforce or facilitate the crucial interaction of HIV-1 to the susceptible cell. Recent works suggest that a host soluble β-galactoside-binding lectin, galectin-1, also facilitates both virion binding and the infection of target cells in a manner dependent on lactose but not mannose, suggesting that this soluble galectin can be considered as a host factor that influences HIV-1 pathogenesis. In this chapter, we describe methods used to investigate the potential role of the galectin family in HIV-1-mediated disease progression.  

5.830           Production of Infectious Hepatitis C Virus in Primary Cultures of Human Adult Hepatocytes

Podevin, P., Carpentier, A., Pene, V., Aoudjehane, L., Carriere, M., Zaidi, S., Hernandez, C., Calle, V., Meritet, J-F., Scatton, O., Dreux, M., Cosset, F-L., Wakita, T., Bartenschlager, R., Dermignot, S., Conti, F., Rosenberg, A.R. and Calmus, Y. Gastroenterol., 139(9), 1355-1364 (2010)   Background & Aims Although hepatitis C virus (HCV) can be grown in the hepatocarcinoma-derived cell line Huh-7, a cell-culture model is needed that supports its complete, productive infection cycle in normal, quiescent, highly differentiated human hepatocytes. We sought to develop such a system. Methods Primary cultures of human adult hepatocytes were inoculated with HCV derived from Huh-7 cell culture (HCVcc) and monitored for expression of hepatocyte differentiation markers and replication of HCV. Culture supernatants were assayed for HCV RNA, core antigen, and infectivity titer. The buoyant densities of input and progeny virus were compared in iodixanol gradients. Results While retaining expression of differentiation markers, primary hepatocytes supported the complete infectious cycle of HCV, including production of significant titers of new infectious progeny virus, which was called primary-culture–derived virus (HCVpc). Compared with HCVcc, HCVpc had lower average buoyant density and higher specific infectivity; this was similar to the characteristics of virus particles associated with the very-low-density lipoproteins that are produced during in vivo infection. These properties were lost after re-culture of HCVpc in poorly differentiated Huh-7 cells, suggesting that authentic virions can be produced only by normal hepatocytes that secrete authentic very-low-density lipoproteins. Conclusions We have established a cell-culture–based system that allows production of infectious HCV in physiologically relevant human hepatocytes. This provides a useful tool for the study of HCV interactions with its natural host cell and for the development of antiviral therapies.  

5.831           Dengue Virus Ensures Its Fusion in Late Endosomes Using Compartment-Specific Lipids

Zaitseva, E., Yang, S-T., Melikov, K., Pourmal, S. and Chernomordik, L.V. PloSPathogens, 6(10), e1001131 (2010)   Many enveloped viruses invade cells via endocytosis and use different environmental factors as triggers for virus-endosome fusion that delivers viral genome into cytosol. Intriguingly, dengue virus (DEN), the most prevalent mosquito-borne virus that infects up to 100 million people each year, fuses only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors that time DEN fusion to virion entry into late endosomes? Here we show that DEN utilizes bis(monoacylglycero)phosphate, a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery. Effective virus fusion to plasma- and intracellular- membranes, as well as to protein-free liposomes, requires the target membrane to contain anionic lipids such as bis(monoacylglycero)phosphate and phosphatidylserine. Anionic lipids act downstream of low-pH-dependent fusion stages and promote the advance from the earliest hemifusion intermediates to the fusion pore opening. To reach anionic lipid-enriched late endosomes, DEN travels through acidified early endosomes, but we found that low pH-dependent loss of fusogenic properties of DEN is relatively slow in the presence of anionic lipid-free target membranes. We propose that anionic lipid-dependence of DEN fusion machinery protects it against premature irreversible restructuring and inactivation and ensures viral fusion in late endosomes, where the virus encounters anionic lipids for the first time during entry. Currently there are neither vaccines nor effective therapies for DEN, and the essential role of the newly identified DEN-bis(monoacylglycero)phosphate interactions in viral genome escape from the endosome suggests a novel target for drug design.  

5.832           Strand Transfer and Elongation of HIV-1 Reverse Transcription Is Facilitated by Cell Factors In Vitro

Warrilow, D., Warren, K. and Harrich, D. PloSOne, 5(10), e13229 (2010)   Recent work suggests a role for multiple host factors in facilitating HIV-1 reverse transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 reverse transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor reverse transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s) did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of reverse transcription. Instead, the cell factor(s) enhanced first strand transfer and synthesis of late reverse transcription suggesting it stabilized the reverse transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous reverse transcription (ERT) system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded reverse transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s) suggesting a lack of involvement of the capsid in the in vitro assay. In addition, reverse transcription products were found to be resistant to exogenous DNase I activity when the active fraction was present in the ERT assay. These results indicate that the cell factor(s) may improve reverse transcription by facilitating DNA strand transfer and DNA synthesis. It also had a protective function for the reverse transcription products, but it is unclear if this is related to improved DNA synthesis.  

5.833           The Extracellular Matrix Glycoprotein Tenascin-C Is Beneficial for Spinal Cord Regeneration

Chen, J., Lee, H.J., Jakovcevski, I., Shah, R., Bhagat, N., Loers, G., Liu, H-Y., Meiners, S., Taschenberger, G., Kügler, S., Irintchev, A. and ASchachner, M. Molecular Therapy, 18(10), 1769-1777 (2010)   Tenascin-C (TNC), a major component of the extracellular matrix, is strongly upregulated after injuries of the central nervous system (CNS) but its role in tissue repair is not understood. Both regeneration promoting and inhibiting roles of TNC have been proposed considering its abilities to both support and restrict neurite outgrowth in vitro. Here, we show that spontaneous recovery of locomotor functions after spinal cord injury is impaired in adult TNC-deficient (TNC^–/–) mice in comparison to wild-type (TNC^+/+) mice. The impaired recovery was associated with attenuated excitability of the plantar Hoffmann reflex (H-reflex), reduced glutamatergic input, reduced sprouting of monaminergic axons in the lumbar spinal cord and enhanced post-traumatic degeneration of corticospinal axons. The degeneration of corticospinal axons in TNC^–/– mice was normalized to TNC^+/+ levels by application of the alternatively spliced TNC fibronectin type III homologous domain D (fnD). Finally, overexpression of TNC-fnD via adeno-associated virus in wild-type mice improved locomotor recovery, increased monaminergic axons sprouting, and reduced lesion scar volume after spinal cord injury. The functional efficacy of the viral-mediated TNC indicates a potentially useful approach for treatment of spinal cord injury.  

5.834           Infectivity of Hepatitis C Virus Is Influenced by Association with Apolipoprotein E Isoforms

Hishiki, T., Shimizu, Y., Tobita, R., Sugiyama, K., Ogawa, K., Funami, K., Ohsaki, Y., Fijimoto, T., Takaku, H., Wakita, T., Baumert, T.F., Miyanari, Y. and Shimotohno, K.

1. Virol., 84(22), 12048-12057 (2010)

  Hepatitis C virus (HCV) is a causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV in circulating blood associates with lipoproteins such as very low density lipoprotein (VLDL) and low-density lipoprotein (LDL). Although these associations suggest that lipoproteins are important for HCV infectivity, the roles of lipoproteins in HCV production and infectivity are not fully understood. To clarify the roles of lipoprotein in the HCV life cycle, we analyzed the effect of apolipoprotein E (ApoE), a component of lipoprotein, on virus production and infectivity. The production of infectious HCV was significantly reduced by the knockdown of ApoE. When an ApoE mutant that fails to be secreted into the culture medium was used, the amount of infectious HCV in the culture medium was dramatically reduced; the infectious HCV accumulated inside these cells, suggesting that infectious HCV must associate with ApoE prior to virus release. We performed rescue experiments in which ApoE isoforms were ectopically expressed in cells depleted of endogenous ApoE. The ectopic expression of the ApoE2 isoform, which has low affinity for the LDL receptor (LDLR), resulted in poor recovery of infectious HCV, whereas the expression of other isoforms, ApoE3 and ApoE4, rescued the production of infectious virus, raising it to an almost normal level. Furthermore, we found that the infectivity of HCV required both the LDLR and scavenger receptor class B, member I (SR-BI), ligands for ApoE. These findings indicate that ApoE is an essential apolipoprotein for HCV infectivity.  

5.835           Blue Native PAGE and Biomolecular Complementation Reveal a Tetrameric or Higher-Order Oligomer Organization of the Physiological Measles Virus Attachment Protein H

Brindley, M.A. and Plemper, R.K.

1. Virol., 84(23), 12174-12184 (2010)

  Members of the Paramyxovirinae subfamily rely on the concerted action of two envelope glycoprotein complexes, attachment protein H and the fusion (F) protein oligomer, to achieve membrane fusion for viral entry. Despite advances in X-ray information, the organization of the physiological attachment (H) oligomer in functional fusion complexes and the molecular mechanism linking H receptor binding with F triggering remain unknown. Here, we have applied an integrated approach based on biochemical and functional assays to the problem. Blue native PAGE analysis indicates that native H complexes extract predominantly in the form of loosely assembled tetramers from purified measles virus (MeV) particles and cells transiently expressing the viral envelope glycoproteins. To gain functional insight, we have established a bimolecular complementation (BiC) assay for MeV H, on the basis of the hypothesis that physical interaction of H with F complexes, F triggering, and receptor binding constitute distinct events. Having experimentally confirmed three distinct H complementation groups, implementation of H BiC (H-BiC) reveals that a high-affinity receptor-to-paramyxovirus H monomer stoichiometry below parity is sufficient for fusion initiation, that F binding and fusion initiation are separable in H oligomers, and that a higher relative amount of F binding-competent than F fusion initiation- or receptor binding-competent H monomers per oligomer is required for optimal fusion. By capitalizing on these findings, H-BiC activity profiles confirm the organization of H into tetramers or higher-order multimers in functional fusion complexes. Results are interpreted in light of a model in which receptor binding may affect the oligomeric organization of the attachment protein complex.  

5.836           Low density Hepatitis C virus particles (lipoviral particles) associate with insulin resistance in genotype 1 infection

Bridge, S.H., Sheridan, D.A., Felmlee, D.J., Toms, G.L., Neely, R.D.G. and Bassendine, M.F. Atherosclerosis, 213(1), e4 (2010)   Background and aims: In hepatitis C virus (HCV) infection the buoyant density of virus particles in serum is heterogeneous due to physical association of virions with lipoproteins. Evidence from animal models and cell culture suggest that lower density apolipoprotein B-associated HCV (lipo-viro-particles (LVP)) have higher specific infectivity than high density HCV. We sought to quantitate LVP in patients with CHC genotype 1 (CHC-G1) and to examine metabolic determinants of LVP load. Methods: Serum lipid and apolipoprotein levels were determined in 51 fasting patients with CHC-G1. Insulin resistance index (HOMA-IR: homeostasis model of assessment) was calculated. LVP load was quantitated by real time RT-PCR of the fraction of plasma at density d < 1.07 g/mL following iodixanol density gradient centrifugation. Results: The contribution of LVP to serum HCV viral load (% LVP) in fasting patients is highly variable (2.9–74%). % LVP correlated with HOMA-IR (P = .004), serum triglyceride concentration (P = .022) and TG:HDL-C ratio (P = .004), which is a characteristic feature of IR. Patients with metabolic syndrome (Met-S) (n = 12) exhibited higher % LVP than non Met-S subjects (P = .025). Lower % LVP was associated with early virological response (EVR) to treatment (EVR 20.1%, nonresponders 33.8%, P = .031). Conclusions: This study offers further insight into the life cycle of HCV in vivo. It suggests that HCV preferentially hijacks the VDLD₁ pathway, which is modulated by IR. This may explain why IR is associated with poorer treatment outcomes, and suggests that these outcomes could be amenable to therapeutic modulation of LVP levels.  

5.837           Transduction of the inner mouse retina using AAVrh8 and AAVrh10 via intravitreal injection

Giove, T.J., Sena-Esteves, M. and Eldred, W.D. Exp. Eye Res., 91, 652-659 (2010)   Adeno-associated virus (AAV) is a proven, safe and effective vector for gene delivery in the retina. There are over 100 serotypes of AAV, and AAV2 through AAV9 have been evaluated in the retina. Each AAV serotype has different cell tropism and transduction efficiency. Intravitreal injections of AAV into the eye tend to transduce cells in the ganglion cell layer (GCL), while subretinal injections tend to transduce retinal pigment epithelium and photoreceptors. Efficient transduction of the inner retina beyond the GCL is not well established with the current methodologies and serotypes used to date. In this study, we compared the cellular tropism of AAVrh8 and AAVrh10 vectors encoding enhanced green fluorescent protein (EGFP) using intravitreal injections. We found that AAVrh8 largely transduced cells in the GCL and also amacrine cells in the inner nuclear layer (INL), as well as Müller and horizontal cells. Inner retinal transduction with AAVrh10 was similar to AAVrh8, but AAVrh10 appeared to also transduce bipolar cells. The transduction efficiency as measured by the intensity of EGFP signal was 3.5 fold higher in horizontal cells transduced with AAVrh10 than AAVrh8. Glial fibrillary accessory protein (GFAP) levels were increased in Müller cells in transduced areas for both serotypes. The results of this study suggest that AAVrh8 and AAVrh10 may be excellent vector candidates to deliver genetic material to the INL, particularly for amacrine and horizontal cells, however they may also cause cellular stress as shown by increased glial GFAP expression.  

5.838           Pronounced microgliosis and neurodegeneration in aged rats after tau gene transfer

Klein, R.L., Dayton, R.D., Diaczynsky, C.G. and Wang, D.B. Neurobiology of Aging, 31, 2091-2102 (2010)   Microtubule-associated protein tau gene transfer to the substantia nigra of rats using the adeno-associated virus (AAV) vector previously led to neuropathology and neurodegeneration in young rats. In this study, we compared equal tau gene transfer in either 3 or 20-month-old rats, in order to test the hypothesis that late middle-aged rats are more susceptible to neurodegeneration. Two intervals and two vector doses of the tau vector probed for age-related differences in the initial sensitivity to low-level tau expression. Gene transfer efficiency was similar for both ages, but the tau vector caused more dopaminergic cell loss and a greater behavioral deficit in aged rats at specific doses and time points. Tau gene transfer caused microgliosis relative to the control vector, and to a greater extent in aged rats. The maximal microglial response occurred at 2 weeks preceding the peak dopaminergic cell loss by 8 weeks. The cellular and behavioral outcomes were more severe in the aged rats, validating the model for studies of age-related diseases.  

5.839           HIV-1 is budded from CD4+ T lymphocytes independently of exosomes

Park, I-W. and He, J.J. Virology J., 7, 234-238 (2010)   The convergence of HIV-1 budding and exosome biogenesis at late endosomal compartments called multivesicular bodies has fueled the debate on whether HIV-1 is budded from its target cells and transmitted in the form of exosomes. The point of contention appears to primarily derive from the types of target cells in question and lack of a well-defined protocol to separate exosomes from HIV-1. In this study, we adapted and established a simplified protocol to define the relationship between HIV-1 production and exosome biogenesis. Importantly, we took advantage of the newly established protocol to unequivocally show that HIV-1 was produced from CD4+ T lymphocytes Jurkat cells independently of exosomes. Thus, this study not only presents a simplified way to obtain highly purified HIV-1 virions for identification of host proteins packaged into virions, but also provides a technical platform that can be employed to define the relationship between exosome biogenesis and budding of HIV-1 or other viruses and its contributions to viral pathogenesis.  

5.840           Expression of human A53T alpha-synuclein in the rat substantia nigra using a novel AAV1/2 vector produces a rapidly evolving pathology with protein aggregation, dystrophic neurite architecture and nigrostriatal degeneration with potential to model the pathology of Parkinson's disease

Koprich, J.B., Johnston, T.H., Reyes, M.G., Sun, X. and Brotchie, J.M. Mol. Neurodegeneration, 5, 43-54 (2010)   Background The pathological hallmarks of Parkinson's disease (PD) include the presence of alpha-synuclein (α-syn) rich Lewy bodies and neurites and the loss of dopaminergic (DA) neurons of the substantia nigra (SN). Animal models of PD based on viral vector-mediated over-expression of α-syn have been developed and show evidence of DA toxicity to varying degrees depending on the type of virus used, its concentration, and the serotype of vector employed. To date these models have been variable, difficult to reproduce, and slow in their evolution to achieve a desired phenotype, hindering their use as a model for testing novel therapeutics. To address these issues we have taken a novel vector in this context, that can be prepared in high titer and which possesses an ability to produce neuronally-directed expression, with expression dynamics optimised to provide a rapid rise in gene product expression. Thus, in the current study, we have used a high titer chimeric AAV1/2 vector, to express human A53T α-syn, an empty vector control (EV), or green fluorescent protein (GFP), the latter to control for the possibility that high levels of protein in themselves might contribute to damage. Results We show that following a single 2 μl injection into the rat SN there is near complete coverage of the structure and expression of A53T α-syn or GFP appears throughout the striatum. Within 3 weeks of SN delivery of their respective vectors, aggregations of insoluble α-syn were observed in SN DA neurons. The numbers of DA neurons in the SN were significantly reduced by expression of A53T α-syn (52%), and to a lesser extent by GFP (24%), compared to EV controls (both P < 0.01). At the level of the striatum, AAV1/2-A53T α-syn injection produced dystrophic neurites and a significant reduction in tyrosine hydroxylase levels (by 53%, P < 0.01), this was not seen in the AAV1/2-GFP condition. Conclusions In the current implementation of the model, we recapitulate the primary pathological hallmarks of PD, although a proportion of the SN damage may relate to general protein overload and may not be specific for A53T α-syn. Future studies will thus be required to optimise the dose of AAV1/2 employed before fully characterizing this model. The dynamics of the evolution of the pathology however, provide advantages over current models with respect to providing an initial screen to assess efficacy of novel treatments that might prevent/reverse α-syn aggregation.  

5.841           Increasing cholesterol synthesis in 7-dehydrosterol reductase (DHCR7) deficient mouse models through gene transfer

Matabosch, X., Ying, L., Serra, M., Wassif, C.A., Porter, F.D., Shackleton, C. and Watson, G.

1. Steroid Biochem. Mol. Biol., 122 303-309 (2010)

  Smith–Lemli–Opitz syndrome (SLOS) is caused by deficiency in the terminal step of cholesterol biosynthesis: the conversion of 7-dehydrocholesterol (7DHC) to cholesterol (C), catalyzed by 7-dehydrocholesterol reductase (DHCR7). This disorder exhibits several phenotypic traits including dysmorphia and mental retardation with a broad range of severity. There are few proven treatment options. That most commonly used is a high cholesterol diet that seems to enhance the quality of life and improve behavioral characteristics of patients, although these positive effects are controversial. The goal of our study was to investigate the possibility of restoring DHCR7 activity by gene transfer. We constructed an adeno-associated virus (AAV) vector containing the DHCR7 gene. After we infused this vector into affected mice, the introduced DHCR7 gene could be identified in liver, mRNA was expressed and a functional enzyme was produced. Evidence of functionality came from the ability to partially normalize the serum ratio of 7DHC/C in treated animals, apparently by increasing cholesterol production with concomitant decrease in 7DHC precursor. By 5 weeks after treatment the mean ratio (for 7 animals) had fallen to 0.05 while the ratio for untreated littermate controls had risen to 0.14. This provides proof of principle that gene transfer can ameliorate the genetic defect causing SLOS and provides a new experimental tool for studying the pathogenesis of this disease. If effective in humans, it might also offer a possible alternative to exogenous cholesterol therapy. However, it would not offer a complete cure for the disorder as many of the negative implications of defective synthesis are already established during prenatal development.  

5.842           Measurements of low density apolipoprotein B associated hepatitis C virus lipoviral particles in genetype 1 infection is more clinically relevant than total viral load

Sheridan D., Bridge, S., Sheridan, D.A., Felmlee, D., Thomas, H., Taylor-Robinson, S., Dermot, R., Neely, G., Toms, G.L. and Bassendine, M.F. Gut, 59, A6 (2010)   Introduction The density of hepatitis C virus (HCV) in plasma is heterogeneous but the factors that influence this are poorly understood. Evidence from animal models and cell culture suggest that low-density apolipoprotein B (apoB)-associated HCV lipoviral particles (LVP) are more infectious than high density HCV. Aim We measured HCV LVP in patients with chronic hepatitis C genotype 1 (CHC-G1) and examined metabolic determinants of LVP load and clinical correlates. Method Fasting lipid profiles and HOMA-IR (homeostasis model assessment of insulin resistance) were determined in 51 CHC-G1 patients. LVP and non-LVP viral load were quantitated by real-time RT-PCR of plasma at density d<1.07 g/ml and d>1.07 g/ml, respectively, following iodixanol density gradient ultracentrifugation. The LVP ratio was calculated using: LVP/(LVP+non- LVP)¼LVP ratio. Results The mean LVP ratio was 0.241 but varied 25-fold (0.029 to 0.74). When divided above and below the median value of 0.177, those with high LVP ratio had metabolic syndrome characteristics, higher liver stiffness and poorer early virological response rates (EVR) (see Abstract OP14 table 1). Univariate analysis showed LVP ratio correlated with HOMA-IR (p¼.004) and triglyceride (TG)/HDL-C ratio (p¼0.004), but not with apoB. In multivariate analysis HOMA-IR was the main determinant of LVP load (log10 IU/ml) (p¼0.037; R2¼16.6%) but TG/HDL-C ratio was the strongest predictor of LVP ratio (p¼0.019; R2¼24.4%). Higher LVP ratios were associated with non-response to antiviral therapy (p¼0.037) and with greater liver stiffness (p¼0.001). There was no association between total viral load and host clinical and metabolic parameters. Conclusion Measurement of HCV LVP is of more direct clinical relevance than total HCV viral load. Insulin resistance and associated dyslipidaemia are the major determinants of low-density apoBassociated LVP in fasting plasma. This provides a novel mechanism to explain why insulin resistance is associated with more rapidly progressive liver disease and poorer treatment outcomes.  

5.843           The carboxy-terminal fragment of inhibitor-2 of protein phosphatase-2A induces Alzheimer disease pathology and cognitive impair

Wang, X., Blanchard, J., Kohlbrenner, E., Clement, N., Linden, R.M., Radu, A., Grundke-Iqbal, I. and Iqbal, K. FASEB J., 24, 4420-4432 (2010)   Development of rational therapeutic treatments of Alzheimer disease (AD) requires the elucidation of the etiopathogenic mechanisms of neurofibrillary degeneration and β-amyloidosis, the two hallmarks of this disease. Here we show, employing an adeno-associated virus serotype 1 (AAV1)-induced expression of the C-terminal fragment (I_2CTF) of I₂^PP2A, also called SET, in rat brain, decrease in protein phosphatase 2A (PP2A) activity, abnormal hyperphosphorylation of tau, and neurodegeneration; littermates treated identically but with vector only, i.e., AAV1-enhanced green fluorescent protein (GFP), served as a control. Furthermore, there was an increase in the level of activated glycogen synthase kinase-3β and enhanced expression of intraneuronal Aβ in AAV1-I_2CTF animals. Morris water maze behavioral test revealed that infection with AAV1-I_2CTF induced spatial reference memory and memory consolidation deficits and a decrease in the brain level of pSer133-CREB. These findings suggest a novel etiopathogenic mechanism of AD, which is initiated by the cleavage of I₂^PP2A, producing I_2CTF, and describe a novel disease-relevant nontransgenic animal model of AD.—Wang, X., Blanchard, J., Kohlbrenner, E., Clement, N., Linden, R. M., Radu, A., Grundke-Iqbal, I., Iqbal, K. The carboxy-terminal fragment of inhibitor-2 of protein phosphatase-2A induces Alzheimer disease pathology and cognitive impairment.  

5.844           Sustained alpha-sarcoglycan gene expression after gene transfer in limb-girdle muscular dystrophy, type 2D

Mendell, J.R., Rodino-Klapac, L.R., Rosales, X.Q., Coley, B.D., Galloway, G., Lewis, S., Malik, V., Shilling, C., Byrne, B.J., Conlon, T., Campbell, K.J., Bremer, W.G., Taylor, L.E., Flanigan, K.M., Gastier-Foster, J.M., Astbury, C., Kota, J., Sahenk, Z., Walker, C.M. and Clark, K.R. Ann. Neurol., 68(5), 629-638 (2010)   Objective: The aim of this study was to attain long-lasting alpha-sarcoglycan gene expression in limb-girdle muscular dystrophy, type 2D (LGMD2D) subjects mediated by adeno-associated virus (AAV) gene transfer under control of a muscle specific promoter (tMCK). Methods: rAAV1.tMCK.hSGCA (3.25 × 10¹¹ vector genomes) was delivered to the extensor digitorum brevis muscle of 3 subjects with documented SGCA mutations via a double-blind, randomized, placebo controlled trial. Control sides received saline. The blind was not broken until the study was completed at 6 months and all results were reported to the oversight committee. Results: Persistent alpha-sarcoglycan gene expression was achieved for 6 months in 2 of 3 LGMD2D subjects. Markers for muscle fiber transduction other than alpha-sarcoglycan included expression of major histocompatibility complex I, increase in muscle fiber size, and restoration of the full sarcoglycan complex. Mononuclear inflammatory cells recruited to the site of gene transfer appeared to undergo programmed cell death, demonstrated by terminal deoxynucleotide transferase–mediated deoxyuridine triphosphate nick-end labeling and caspase-3 staining. A patient failing gene transfer demonstrated an early rise in neutralizing antibody titers and T-cell immunity to AAV, validated by enzyme-linked immunospot on the second day after gene injection. This was in clear distinction to other participants with satisfactory gene expression. Interpretation: The findings of this gene replacement study in LGMD2D subjects have important implications not previously demonstrated in muscular dystrophy. Long-term, sustainable gene expression of alpha-sarcoglycan was observed following gene transfer mediated by AAV. The merit of a muscle-specific tMCK promoter, not previously used in a clinical trial, was evident, and the potential for reversal of disease was displayed    

5.845           Intravascular Transfer Contributes to Postprandial Increase in Numbers of Very-Low-Density Hepatitis C Virus Particles

Felmlee, D.J., Sheridan, D.A., Bridge, S.H., Nielsen, S.U., Milne, R.W., Packard, C.J., Caslake, M.J., McLauchlan, J., Toms, G.L., Dermot, R., Neely, G. and Bassendine, M.F. Gastroenterology, 139, 1774-1783 (2010)   Background & Aims The physical association of hepatitis C virus (HCV) particles with lipoproteins in plasma results in distribution of HCV in a broad range of buoyant densities. This association is thought to increase virion infectivity by mediating cell entry via lipoprotein receptors. We sought to determine if factors that affect triglyceride-rich lipoprotein (TRL) metabolism alter the density and dynamics of HCV particles in the plasma of patients with chronic HCV infection. Methods Fasting patients (n = 10) consumed a high-fat milkshake; plasma was collected and fractionated by density gradients. HCV- RNA was measured in the very-low-density fraction (VLDF, d < 1.025 g/mL) before and at 7 serial time points postprandially. Results The amount of HCV RNA in the VLDF (HCVVLDF) increased a mean of 26-fold, peaking 180 minutes after the meal (P < .01). Quantification of HCV RNA throughout the density gradient fractions revealed that HCVVLDF rapidly disappeared, rather than migrating into the adjacent density fraction. Immuno-affinity separation of the VLDF, using antibodies that recognize apolipoprotein B–100 and not apolipoprotein B–48, showed that HCVVLDF is composed of chylomicron- and VLDL-associated HCV particles; peaking 120 and 180 minutes after the meal, respectively. Plasma from fasting HCV-infected patients mixed with uninfected plasma increased the quantity of HCVVLDF, compared with that mixed with phosphate-buffered saline, showing extracellular assembly of HCVVLDF. Conclusions Dietary triglyceride alters the density and dynamics of HCV in plasma. The rapid clearance rate of HCVVLDF indicates that association with TRL is important for HCV infectivity. HCV particles, such as exchangeable apolipoproteins, appear to reassociate with TRLs in the vascular compartment.  

5.846           Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale

Lock, M., Alvira, M., Vandenberghe, L.H., Samanta, A., Toelen, J., Debyser, Z. and Wilson, J.M. Human Gene Therapy, 21, 1259-1271 (2010)   Adeno-associated viral (AAV) manufacturing at scale continues to hinder the application of AAV technology to gene therapy studies. Although scalable systems based on AAV–adenovirus, AAV–herpesvirus, and AAV–baculovirus hybrids hold promise for clinical applications, they require time-consuming generation of reagents and are not highly suited to intermediate-scale preclinical studies in large animals, in which several combinations of serotype and genome may need to be tested. We observed that during production of many AAV serotypes, large amounts of vector are found in the culture supernatant, a relatively pure source of vector in comparison with cell-derived material. Here we describe a high-yielding, recombinant AAV production process based on polyethylenimine (PEI)-mediated transfection of HEK293 cells and iodixanol gradient centrifugation of concentrated culture supernatant. The entire process can be completed in 1 week and the steps involved are universal for a number of different AAV serotypes. Process conditions have been optimized such that final purified yields are routinely greater than 1 × 10¹⁴ genome copies per run, with capsid protein purity exceeding 90%. Initial experiments with vectors produced by the new process demonstrate equivalent or better transduction both in vitro and in vivo when compared with small-scale, CsCl gradient-purified vectors. In addition, the iodixanol gradient purification process described effectively separates infectious particles from empty capsids, a desirable property for reducing toxicity and unwanted immune responses during preclinical studies.  

5.847           Preexisting Immunity and Low Expression in Primates Highlight Translational Challenges for Liver-directed AAV8-mediated Gene Therapy

Hurlbut, G.D., Ziegler, R.J., Nietupski, J.B., Foley, J.W., Woodworth, L.A., Meyers, E., Bercury, S.D., Pande, N.N., Souza, D.W., Bree, M.P., Lukason, M.J., Marshall, J., Cheng, S.H. and Scheule, R.K. Mol. Therapy, 18(11), 1983-1994 (2010)   Liver-directed gene therapy with adeno-associated virus (AAV) vectors effectively treats mouse models of lysosomal storage diseases (LSDs). We asked whether these results were likely to translate to patients. To understand to what extent preexisting anti-AAV8 antibodies could impede AAV8-mediated liver transduction in primates, commonly preexposed to AAV, we quantified the effects of preexisting antibodies on liver transduction and subsequent transgene expression in mouse and nonhuman primate (NHP) models. Using the highest viral dose previously reported in a clinical trial, passive transfer of NHP sera containing relatively low anti-AAV8 titers into mice blocked liver transduction, which could be partially overcome by increasing vector dose tenfold. Based on this and a survey of anti-AAV8 titers in 112 humans, we predict that high-dose systemic gene therapy would successfully transduce liver in >50% of human patients. However, although high-dose AAV8 administration to mice and monkeys with equivalent anti-AAV8 titers led to comparable liver vector copy numbers, the resulting transgene expression in primates was ~1.5-logs lower than mice. This suggests vector fate differs in these species and that strategies focused solely on overcoming preexisting vector-specific antibodies may be insufficient to achieve clinically meaningful expression levels of LSD genes using a liver-directed gene therapy approach in patients.  

5.848           Induction of Immune Tolerance to a Therapeutic Protein by Intrathymic Gene Delivery

Chu, Q., Moreland, R.J., Gao, L., Taylor, K.M., Meyers, E., Cheng, S.H. and Scheule, R.K. Mol. Therapy, 18(12), 2146-2154 (2010)   The efficacy of recombinant enzyme therapy for genetic diseases is limited in some patients by the generation of a humoral immune response to the therapeutic protein. Inducing immune tolerance to the protein prior to treatment has the potential to increase therapeutic efficacy. Using an AAV8 vector encoding human acid α-glucosidase (hGAA), we have evaluated direct intrathymic injection for inducing tolerance. We have also compared the final tolerogenic states achieved by intrathymic and intravenous injection. Intrathymic vector delivery induced tolerance equivalent to that generated by intravenous delivery, but at a 25-fold lower dose, the thymic hGAA expression level was 10,000-fold lower than the liver expression necessary for systemic tolerance induction. Splenic regulatory T cells (Tregs) were apparent after delivery by both routes, but with different phenotypes. Intrathymic delivery resulted in Tregs with higher FoxP3, TGFβ, and IL-10 mRNA levels. These differences may account for the differences noted in splenic T cells, where only intravenous delivery appeared to inhibit their activation. Our results imply that different mechanisms may be operating to generate immune tolerance by intrathymic and intravenous delivery of an AAV vector, and suggest that the intrathymic route may hold promise for decreasing the humoral immune response to therapeutic proteins in genetic disease indications.  

5.849           Restoration of Cone Vision in the CNGA3^−/− Mouse Model of Congenital Complete Lack of Cone Photoreceptor Function

Michalakis, S., Mühlfriedel, R., Tanimoto, N., Krisnamoorthy, V., Koch, S., Fischer, M.D., Becirovic, E., Bai, L., Huber, G., Beck, S.C., Fahl, E., Büning, H., Paquet-Durand, F., Zong, X., Gollisch, T., Biel, M. and Seeliger, M.W. Molecular Therapy, 18(12), 2057-2063 (2010)   Congenital absence of cone photoreceptor function is associated with strongly impaired daylight vision and loss of color discrimination in human achromatopsia. Here, we introduce viral gene replacement therapy as a potential treatment for this disease in the CNGA3^−/− mouse model. We show that such therapy can restore cone-specific visual processing in the central nervous system even if cone photoreceptors had been nonfunctional from birth. The restoration of cone vision was assessed at different stages along the visual pathway. Treated CNGA3^−/− mice were able to generate cone photoreceptor responses and to transfer these signals to bipolar cells. In support, we found morphologically that treated cones expressed regular cyclic nucleotide-gated (CNG) channel complexes and opsins in outer segments, which previously they did not. Moreover, expression of CNGA3 normalized cyclic guanosine monophosphate (cGMP) levels in cones, delayed cone cell death and reduced the inflammatory response of Müller glia cells that is typical of retinal degenerations. Furthermore, ganglion cells from treated, but not from untreated, CNGA3^−/− mice displayed cone-driven, light-evoked, spiking activity, indicating that signals generated in the outer retina are transmitted to the brain. Finally, we demonstrate that this newly acquired sensory information was translated into cone-mediated, vision-guided behavior.  

5.850           Efficient Gene Transfer Into the Mouse Lung by Fetal Intratracheal Injection of rAAV2/6.2

Carlon, M., Toelen, J., Van der Perren, A., Vandenberghe, L.H., Reumers, V., Sbragia, L., Gijsbers, R., Baekelandt, V., Himmelreich, U., Wilson, J.M., Deprest, J. and Debyser, Z. Molecular Therapy, 18(12), 2130-2138 (2010)   Fetal gene therapy is one of the possible new therapeutic strategies for congenital or perinatal diseases with high mortality or morbidity. We developed a novel delivery strategy to inject directly into the fetal mouse trachea. Intratracheal (i.t.) injection at embryonic day 18 (E18) was more efficient in targeting the fetal lung than conventional intra-amniotic (i.a.) delivery. Viral vectors derived from adeno-associated virus serotype 6.2, with tropism for the airway epithelium and not earlier tested in the fetal mouse lung, were injected into the fetal trachea. Bioluminescence (BL) imaging (BLI) was combined with magnetic resonance (MR) imaging (MRI) for noninvasive and accurate localization of transgene expression in vivo. Histological analysis for β-galactosidase (β-gal) revealed 17.5% of epithelial cells transduced in the conducting airways and 1.5% in the alveolar cells. Stable gene expression was observed up to 1 month after injection. This study demonstrates that direct injection of rAAV2/6.2 in the fetal mouse trachea is superior to i.a. delivery for transducing the lung. Second, as stable gene transfer was detected up to 1 postnatal month, this approach may be useful to evaluate fetal gene therapy for pulmonary diseases such as cystic fibrosis, requiring both substantial numbers of transduced cells as well as prolonged gene expression to obtain a stable phenotypic effect.  

5.851           Adenovirus Targeting to Prostate-Specific Membrane Antigen through Virus-Displayed, Semirandom Peptide Library Screening

Wu, P., Kudrolli, T.A., Chowdhury, W.H., Liu, M.M., Rodriguez, R. and Lupold, S.E. Cancer Res., 70(23), 9549-9553 (2010)   The convergence of phage-displayed peptide libraries and recombinant viral vectors launched a promising new direction in targeted viral gene therapeutics, but the translation of targeting peptides to functional cancer therapeutic agents has been challenging. Here, we report progress in developing a successful strategy to optimize targeted viral infection through adenovirus-displayed, semirandom peptide libraries. A phage-derived peptide targeting the prostate-specific membrane antigen (PSMA) was genetically incorporated into the adenoviral capsid Fiber protein and flanked by random peptide cassettes. The resulting adenovirus library was biopanned against PSMA-expressing cells and tumors to identify a PSMA-retargeted adenovirus. While the initial peptide alone could not target viral infection, the selected virus preferentially infects PSMA-expressing cells through the targeting peptide and infects LNCaP tumors after intravenous injection. Our results indicate that virus-displayed, semirandom peptide libraries can be used to optimize targeting infection. This approach represents a novel principle for developing targeted agents in a variety of disease models.  

5.852           Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy

Yukl, S.A., Shergill, A.K., McQuaid, K., Gianella, S., Lampiris, H., Hare, C.B., Ppandori, M., Sinclair, E., Günthard, H.F., Fischer, M., Wong, J.K. and Havlir, D.V. AIDS, 24(16), 2451-2460 (2010)   Objective: To determine whether raltegravir-containing antiretroviral therapy (ART) intensification reduces HIV levels in the gut. Design: Open-label study in HIV-positive adults on ART with plasma HIV RNA below 40 copies/ml. Methods: Seven HIV-positive adults received 12 weeks of ART intensification with raltegravir alone or in combination with efavirenz or darunavir. Gut cells were obtained by upper and lower endoscopy with biopsies from duodenum, ileum, colon, and rectum at baseline and 12 weeks. Study outcomes included plasma HIV RNA, HIV DNA and RNA from peripheral blood mononuclear cells (PBMC) and four gut sites, T-cell subsets, and activation markers. Results: Intensification produced no consistent decrease in HIV RNA in the plasma, PBMC, duodenum, colon, or rectum. However, five of seven participants had a decrease in unspliced HIV RNA per 10⁶ CD4⁺ T cells in the ileum. There was a trend towards decreased T-cell activation in all sites, which was greatest for CD8⁺ T cells in the ileum and PBMC, and a trend towards increased CD4⁺ T cells in the ileum. Conclusion: Most HIV RNA and DNA in the blood and gut is not the result of ongoing replication that can be impacted by short-term intensification with raltegravir. However, the ileum may support ongoing productive infection in some patients on ART, even if the contribution to plasma RNA is not discernible.  

5.853           Differences in HIV Burden and Immune Activation within the Gut of HIV-Positive Patients Receiving Suppressive Antiretroviral Therapy

Yukl, S.A., Gianella, S., Sinclair, E., Epling, L., Li, Q., Duan, L., Choi, A.L.M., Girling, V., Ho, T., Li, P., Fujimoto, K., Lampris, H., Hare, C.B., Pandori, M., Haase, A.T., Günthard, H.F., Fischer, M., Shergill, A.K., McQuaid, K., Havlir, D. and Wong, J.K.

1. Infectious Diseases,10, 1553-1561 (2010)

  Background. The gut is a major reservoir for human immunodeficiency virus (HIV) in patients receiving antiretroviral therapy (ART). We hypothesized that distinct immune environments within the gut may support varying levels of HIV. Methods. In 8 HIV-1-positive adults who were receiving ART and had CD4⁺ T cell counts of >200 cells/µL and plasma viral loads of <40 copies/mL, levels of HIV and T cell activation were measured in blood samples and endoscopic biopsy specimens from the duodenum, ileum, ascending colon, and rectum. Results. HIV DNA and RNA levels per CD4⁺ T cell were higher in all 4 gut sites compared with those in the blood. HIV DNA levels increased from the duodenum to the rectum, whereas the median HIV RNA level peaked in the ileum. HIV DNA levels correlated positively with T cell activation markers in peripheral blood mononuclear cells (PBMCs) but negatively with T cell activation markers in the gut. Multiply spliced RNA was infrequently detected in gut, and ratios of unspliced RNA to DNA were lower in the colon and rectum than in PBMCs, which reflects paradoxically low HIV transcription, given the higher level of T cell activation in the gut. Conclusions. HIV DNA and RNA are both concentrated in the gut, but the inverse relationship between HIV DNA levels and T cell activation in the gut and the paradoxically low levels of HIV expression in the large bowel suggest that different processes drive HIV persistence in the blood and gut.  

5.854           Optimized Transduction of Human Monocyte-Derived Dendritic Cells by Recombinant Adeno-Associated Virus Serotype 6

Ussher, J.E. and Taylor, J.A. Human Gene Therapy, 21, 1675-1686 (2010)   Dendritic cells are the key antigen-presenting cells involved in the initiation of the adaptive immune response. Recombinant adeno-associated viruses (rAAVs) can transduce dendritic cells and have gained attention as potential vaccines capable of stimulating T cell immunity. Here we show that rAAV2 pseudotyped with type 6 capsid (rAAV2/6) exhibits significantly higher tropism for human monocyte-derived dendritic cells (MoDCs) than other serotypes and variants. Transduction was abolished by a single lysine-to-alanine mutation within the AAV6 capsid previously shown to inhibit binding to heparin. However, unlike rAAV2, soluble heparin did not inhibit rAAV2/6 transduction of MoDCs. Further enhancement of MoDC transduction was observed after mutation of Tyr-731 in the capsid of AAV6 consistent with a report that tyrosine residues are phosphorylated, leading to ubiquitination of capsids during uptake. Pseudotyped rAAV2/6 vectors containing a Y731F mutation minimally altered the immunophenotype of MoDCs, which retained their immunostimulatory ability and were able to stimulate an antigen-specific CD8⁺ T cell clone. These findings should assist in the development of rAAV2/6 as a vaccine vector.      

5.855           Structural Analysis of HIV-1 Maturation Using Cryo-Electron Tomography

De Marco, A., Müller, B., Glass, B., Riches, J.D., Kräusslich, H-G. and Briggs, J.A.G. PloSPathogens, 6(11), e1001215 (2010)   HIV-1 buds form infected cells in an immature, non-infectious form. Maturation into an infectious virion requires proteolytic cleavage of the Gag polyprotein at five positions, leading to a dramatic change in virus morphology. Immature virions contain an incomplete spherical shell where Gag is arranged with the N-terminal MA domain adjacent to the membrane, the CA domain adopting a hexameric lattice below the membrane, and beneath this, the NC domain and viral RNA forming a disordered layer. After maturation, NC and RNA are condensed within the particle surrounded by a conical CA core. Little is known about the sequence of structural changes that take place during maturation, however. Here we have used cryo-electron tomography and subtomogram averaging to resolve the structure of the Gag lattice in a panel of viruses containing point mutations abolishing cleavage at individual or multiple Gag cleavage sites. These studies describe the structural intermediates correlating with the ordered processing events that occur during the HIV-1 maturation process. After the first cleavage between SP1 and NC, the condensed NC-RNA may retain a link to the remaining Gag lattice. Initiation of disassembly of the immature Gag lattice requires cleavage to occur on both sides of CA-SP1, while assembly of the mature core also requires cleavage of SP1 from CA.  

5.856           Suppressor of cytokine signaling 3 knockdown in the mediobasal hypothalamus: counterintuitive effects on energy balance

De Backer, M.W.A., Brans, M.A.D., van Rosen, A.J., van der Zwaal, E.M., Luijendijk, M.C.M., Garner, K.G., de Krom, M., van Beekum, O., la Fleur, S.E. and Adan, R.A.H.

1. Mol. Endocrinol., 45, 341-353 (2010)

  An increase in brain suppressor of cytokine signaling 3 (SOCS3) has been implicated in the development of both leptin and insulin resistance. Socs3 mRNA is localized throughout the brain, and it remains unclear which brain areas are involved in the effect of SOCS3 levels on energy balance. We investigated the role of SOCS3 expressed in the mediobasal hypothalamus (MBH) in the development of diet-induced obesity in adult rats. Socs3 mRNA was down-regulated by local injection of adeno-associated viral vectors expressing a short hairpin directed against Socs3, after which we determined the response to high-fat high-sucrose choice diet. In contrast to neuronal Socs3 knockout mice, rats with SOCS3 knockdown limited to the MBH showed increased body weight gain, larger amounts of white adipose tissue, and higher leptin concentrations at the end of the experiment. These effects were partly due to the decrease in locomotor activity, as 24 h food intake was comparable with controls. In addition, rats with Socs3 knockdown in the MBH showed alterations in their meal patterns: average meal size in the light period was increased and was accompanied by a compensatory decrease in meal frequency in the dark phase. In addition, neuropeptide Y (Npy) mRNA levels were significantly increased in the arcuate nucleus of Socs3 knockdown rats. Since leptin is known to stimulate Npy transcription in the absence of Socs3, these data suggest that knockdown of Socs3 mRNA limited to the MBH increases Npy mRNA levels, which subsequently decreases locomotor activity and alters feeding patterns.  

5.857           Efficient delivery of DNA vaccines using human papillomavirus pseudovirions

Peng, S., Monie, A., Kang, T.H., Hung, C-F., Roden, r. and Wu, T-C. Gene Therapy, 17, 1453-1464 (2010)   We have examined non-replicative human papillomavirus (HPV) pseudovirions as an approach in the delivery of naked DNA vaccines without safety concerns associated with live viral vectors. In this study, we have generated HPV-16 pseudovirions encapsidating a DNA vaccine encoding the model antigen, ovalbumin (OVA) (HPV16-OVA pseudovirions). Vaccination with HPV16-OVA pseudovirions subcutaneously elicited significantly stronger OVA-specific CD8+ T-cell immune responses compared with OVA DNA vaccination via gene gun in a dose-dependent manner. We showed that a single amino acid mutation in the L2 minor capsid protein that eliminates the infectivity of HPV16-OVA pseudovirion significantly decreased the antigen-specific CD8+ T-cell responses in vaccinated mice. Furthermore, a subset of CD11c+ cells and B220+ cells in draining lymph nodes became labeled on vaccination with fluorescein isothiocyanate-labeled HPV16-OVA pseudovirions in injected mice. HPV pseudovirions were found to infect bone marrow-derived dendritic cells (BMDCs) in vitro. We also showed that pretreatment of HPV16-GFP pseudovirions with furin leads to enhanced HPV16-OVA pseudovirion infection of BMDCs and OVA antigen presentation. Our data suggest that DNA vaccines delivered using HPV pseudovirions represent an efficient delivery system that can potentially affect the field of DNA vaccine delivery.   Purification of viruses by centrifugation Lawrence, J.E. and Steward, G.F. Manual of Aquatic Viral Ecology, Chapter 17, 166-181 (2010)   Ultracentrifugation provides a means to concentrate, analyze, and purify viruses in solution, and therefore represents an invaluable tool for aquatic virologists. This chapter reviews the theory of ultracentrifugation and presents the technical knowledge necessary for an investigator to adapt or develop methods to meet his or her needs. Detailed protocols for the purification of viruses from culture lysates and vial assemblages from natural water samples are provided.  

5.858           Intrinsic phospholipase A2 activity of adeno-associated virus is involved in endosomal escape of incoming particles

Stahnke, S., Lux, K., Uhrig, S., Kreppel, F., Hösel, M., Coutelle, O., Ogris, M., Hallek, M. and Büning, H. Virology, 409, 77-83 (2011)   The unique region of the VP1 capsid protein of adeno-associated viruses (AAV) in common with autonomously replicating parvoviruses comprises a secreted phospholipase A2 (sPLA2) homology domain. While the sPLA2 domain of Minute Virus of Mice has recently been shown to mediate endosomal escape by lipolytic pore formation, experimental evidence for a similar function in AAV infection is still lacking. Here, we explored the function of the sPLA2 domain of AAV by making use of the serotype 2 mutant ⁷⁶HD/AN. The sPLA2 defect in ⁷⁶HD/AN, which severely impairs AAV's infectivity, could be complemented in trans by co-infection with wild-type AAV2. Furthermore, co-infection with endosomolytically active, but not with inactive adenoviral variants partially rescued ⁷⁶HD/AN, providing the first evidence for a function of this domain in endosomal escape of incoming AAV particles.  

5.859           Focal expression of mutated tau in entorhinal cortex neurons of rats impairs spatial working memory

Ramirez, J.J., Poulton, W.E., Knelson, E., Barton, C., King, M.A. and Klein, R.L Behavioural Brain Res., 216, 332-340 (2011)   Entorhinal cortex neuropathology begins very early in Alzheimer's disease (AD), a disorder characterized by severe memory disruption. Indeed, loss of entorhinal volume is predictive of AD and two of the hallmark neuroanatomical markers of AD, amyloid plaques and neurofibrillary tangles (NFTs), are particularly prevalent in the entorhinal area of AD-afflicted brains. Gene transfer techniques were used to create a model neurofibrillary tauopathy by injecting a recombinant adeno-associated viral vector with a mutated human tau gene (P301L) into the entorhinal cortex of adult rats. The objective of the present investigation was to determine whether adult onset, spatially restricted tauopathy could be sufficient to reproduce progressive deficits in mnemonic function. Spatial memory on a Y-maze was tested for approximately 3 months post-surgery. Upon completion of behavioral testing the brains were assessed for expression of human tau and evidence of tauopathy. Rats injected with the tau vector became persistently impaired on the task after about 6 weeks of postoperative testing, whereas the control rats injected with a green fluorescent protein vector performed at criterion levels during that period. Histological analysis confirmed the presence of hyperphosphorylated tau and NFTs in the entorhinal cortex and neighboring retrohippocampal areas as well as limited synaptic degeneration of the perforant path. Thus, highly restricted vector-induced tauopathy in retrohippocampal areas is sufficient for producing progressive impairment in mnemonic ability in rats, successfully mimicking a key aspect of tauopathies such as AD.  

5.860           Hepatitis C virus expressing flag-tagged envelope protein 2 has unaltered infectivity and density, is specifically neutralized by flag antibodies and can be purified by affinity chromatography

Prentoe, J. and Bukh, J. Virology, 409, 148-155 (2011)   Hepatitis C virus (HCV) purification by ultracentrifugation is difficult because of the low and heterogeneous density of native and cultured viruses. It was recently shown that inserting flag tag into envelope protein 2 (E2) of HCV permitted virus purification by affinity chromatography. However, flag-tagged viruses had drastically altered properties, and purification yield was low. In this study, we found that insertion of flag tag at the N-terminus of E2 in HCV recombinant J6/JFH1 did not affect viability in Huh7.5 cells, and that flag-tagged virus had physiochemical properties similar to the original virus. Flag-tagged virus was susceptible to flag-specific antibody neutralization, and infected cells could be immuno-stained by anti-flag antibodies. Using affinity chromatography with anti-flag resin we repeatedly obtained ~ 30% recovery of infectious particles. The full viability and unaltered physiochemical properties of flag-tagged HCV is an important improvement for utilizing these viruses for imaging, virion composition analysis and possibly vaccine development.  

5.861           Enhancement of NK cell antitumor responses using an oncolytic parvovirus

Bhat, R., Dempe, S., Dinsart, C. and Rommelaere, J. Int. J. Cancer, 128, 908-919 (2011)   Natural killer (NK) cells play a vital role in the rejection of tumors. Pancreatic ductal adenocarcinoma (PDAC), however, remains a poor prognosis malignancy, due to its resistance to radio- and chemotherapy, and low immunogenicity. We demonstrate here that IL-2-activated human NK cells are able to kill PDAC cells. Currently, novel strategies are being pursued to combat PDAC. In this regard, oncolytic viruses, in addition to killing tumor cells, may also have the potential to augment antitumor immune responses. We found that, besides having an intrinsic oncolytic activity, parvovirus H-1PV is able to enhance NK cell-mediated killing of PDAC cells. Our results show that H-1PV infection of Panc-1 cells increases NK cell capacity to release IFN- , TNF- and MIP-1 /β. Multiple activating receptors are involved in the NK cell-mediated killing of Panc-1 cells. Indeed, blocking of the natural cytotoxicity receptors—NKp30, 44 and 46 in combination, and NKG2D and DNAM1 alone inhibit the killing of Panc-1 cells. Interestingly, H-1PV infection of Panc-1 cells overcomes the part of inhibitory effects suggesting that parvovirus may induce additional NK cell ligands on Panc-1 cells. The enhanced sensitivity of H-1PV-infected PDAC cells to NK cell-dependent killing could be traced back to the upregulation of the DNAM-1 ligand, CD155 and to the downregulation of MHC class I expression. Our data suggests that NK cells display antitumor potential against PDAC and that H-1PV-based oncolytic immunotherapy could further boost NK cell-mediated immune responses and help to develop a combinatorial therapeutic approach against PDAC.  

5.862           Identification of the dynein light chains required for human papillomavirus infection

Schneider, M.A., Spoden, G.A., Florin, L. and Lambert, C. Cell. Microbiol., 13(1), 32-46 (2011)   Human papillomaviruses (HPVs) are a family of small non-enveloped DNA viruses. Some genital HPV types, including HPV type 16 (HPV16), are the causative agent for the development of cancer at the site of infection. HPVs encode two capsid proteins, L1 and L2. After endocytic cell entry and egress from endosomes, L2 accompanies the viral DNA to the nucleus where replication is initiated. For cytoplasmic transport, L2 interacts with the microtubule network via the motor protein complex dynein. We have performed yeast two-hybrid screening and identified the dynein light chain DYNLT1 (previously called Tctex1) as interaction partner of HPV16 L2. Using co-immunoprecipitation and immunofluorescence colocalization studies we confirmed the L2–DYNLT1 interaction in mammalian cells. Further studies revealed that DYNLT3, the second member of the Tctex-light chain family, also interacts with L2 in vitro and in vivo, whereas other constituents of the dynein complex were not found to associate with L2. Depletion of DYNLT1 and DYNLT3 by specific siRNAs or cytosolic delivery of light chain-specific antibodies inhibited infection of HPV16. Therefore, this work identified two host cell proteins involved in HPV16 infection that are most likely required for transport purposes towards the nucleus.  

5.863           Expression of E1E2 on Hepatitis C RNA-Containing Particles Released from Primary Cultured Human Hepatocytes Derived from Infected Cirrhotic Livers

Ndongo, N., Selliah, S., Bertillon, P., Raymond, V-A., Trepe, C., Bilodeau, M. and Petit, M-A. Intervirology, 54, 1-9 (2011)   Objective: To determine whether liver-derived hepatitis C RNA-containing particles express the E1E2 discontinuous antigenic determinant defined by unique monoclonal antibody (mAb) D32.10 which recognizes three highly conserved segments in E1 (aa297–306) and E2 (aa480–494 and aa613–621) envelope glycoproteins. Methods: Human hepatocytes were isolated from HCV-infected cirrhotic explanted livers. The liver-derived hepatitis C virus (HCV) particles released from three distinct cultures (genotypes 1b and 2b) were characterized. HCV RNA+ was quantified by real-time RT-PCR. The E1E2 antigenic activity was assessed by indirect ELISA and immunoblotting using D32.10. The density distributions of HCV RNA and E1E2 antigen were determined by isopycnic sucrose density gradients. HCV E1E2, E2 and core antigens were detected in the cells by immunochemical staining. Results: Liver-derived HCV particles contained HCV RNA (10⁶–10⁷ copies/mg of protein) and core proteins and expressed the E1E2/D32.10 epitope. HCV RNA and E1E2 cosedimented between 1.15 and 1.25 g/ml in sucrose gradients. Moreover, the mAb D32.10 detected E1E2 by immunostaining in HCV-infected hepatocytes in parallel with E2 and core antigens. Conclusion: Our results provide evidence that the mAb D32.10 recognizes E1E2 envelope complexes expressed in the cell cytoplasm and on the surface of HCV RNA-containing particles released from short-term cultures of in vivo infected hepatocytes.    

5.864           Biochemical and Morphological Properties of Hepatitis C Virus Particles and Determination of Their Lipidome

Merz, A., Long, G., Hiet, M-S-. Brügger, B., Chlanda, P., Andre, P., Wieland, F., Krijnse-Locker, J. and Bartenschlager, R.

1. Biol. Chem., 286(4), 3018-3032 (2011)

  A hallmark of hepatitis C virus (HCV) particles is their association with host cell lipids, most notably lipoprotein components. It is thought that this property accounts for the low density of virus particles and their large heterogeneity. However, the composition of infectious virions and their biochemical and morphological properties are largely unknown. We developed a system in which the envelope glycoprotein E2 was N-terminally tagged with a FLAG epitope. This virus, designated Jc1E2^FLAG, produced infectivity titers to wild type levels and allowed affinity purification of virus particles that were analyzed for their protein and lipid composition. By using mass spectrometry, we found the lipid composition of Jc1E2^FLAG particles to resemble the one very low- and low density-lipoprotein with cholesteryl esters accounting for almost half of the total HCV lipids. Thus, HCV particles possess a unique lipid composition that is very distinct from all other viruses analyzed so far and from the human liver cells in which HCV was produced. By electron microscopy (EM), we found purified Jc1E2^FLAG particles to be heterogeneous, mostly spherical structures, with an average diameter of about 73 nm. Importantly, the majority of E2-containing particles also contained apoE on their surface as assessed by immuno-EM. Taken together, we describe a rapid and efficient system for the production of large quantities of affinity-purified HCV allowing a comprehensive analysis of the infectious virion, including the determination of its lipid composition.  

5.865           Intravenous scAAV9 delivery of a codon-optimized SMN1 sequence rescues SMA mice

Dominguez, E., Marais, T., Chatauret, N., Benkhelifa-Ziyyat, S., Duque, S., Ravassard, P., Carcenac, R., Astord, S., Pereira de Moura, A., Voit, T. and Barkats, M. Hum. Mol. Genet., 20(4), 681-693 (2011)   Spinal muscular atrophy (SMA) is the most common genetic disease leading to infant mortality. This neuromuscular disorder is caused by the loss or mutation of the telomeric copy of the ‘survival of motor neuron’ (Smn) gene, termed SMN1. Loss of SMN1 leads to reduced SMN protein levels, inducing degeneration of motor neurons (MN) and progressive muscle weakness and atrophy. To date, SMA remains incurable due to the lack of a method to deliver therapeutically active molecules to the spinal cord. Gene therapy, consisting of reintroducing SMN1 in MNs, is an attractive approach for SMA. Here we used postnatal day 1 systemic injection of self-complementary adeno-associated virus (scAAV9) vectors carrying a codon-optimized SMN1 sequence and a chimeric intron placed downstream of the strong phosphoglycerate kinase (PGK) promoter (SMNopti) to overexpress the human SMN protein in a mouse model of severe SMA. Survival analysis showed that this treatment rescued 100% of the mice, increasing life expectancy from 27 to over 340 days (median survival of 199 days) in mice that normally survive about 13 days. The systemic scAAV9 therapy mediated complete correction of motor function, prevented MN death and rescued the weight loss phenotype close to normal. This study reports the most efficient rescue of SMA mice to date after a single intravenous injection of an optimized SMN-encoding scAAV9, highlighting the considerable potential of this method for the treatment of human SMA.  

5.866           Robust cardiomyocyte-specific gene expression following systemic injection of AAV: in vivo gene delivery follows a Poisson distribution

Presad, K-MR., Xu, Y., Yang, Z., Acton, S.T. and French, B.A. Gene Therapy, 18, 43-52 (2011)   Newly isolated serotypes of AAV readily cross the endothelial barrier to provide efficient transgene delivery throughout the body. However, tissue-specific expression is preferred in most experimental studies and gene therapy protocols. Previous efforts to restrict gene expression to the myocardium often relied on direct injection into heart muscle or intracoronary perfusion. Here, we report an AAV vector system employing the cardiac troponin T (cTnT) promoter. Using luciferase and enhanced green fluorescence protein (eGFP), the efficiency and specificity of cardiac reporter gene expression using AAV serotype capsids: AAV-1, 2, 6, 8 or 9 were tested after systemic administration to 1-week-old mice. Luciferase assays showed that the cTnT promoter worked in combination with each of the AAV serotype capsids to provide cardiomyocyte-specific gene expression, but AAV-9 followed closely by AAV-8 was the most efficient. AAV9-mediated gene expression from the cTnT promoter was 640-fold greater in the heart compared with the next highest tissue (liver). eGFP fluorescence indicated a transduction efficiency of 96% using AAV-9 at a dose of only 3.15 × 10¹⁰ viral particles per mouse. Moreover, the intensity of cardiomyocyte eGFP fluorescence measured on a cell-by-cell basis revealed that AAV-mediated gene expression in the heart can be modeled as a Poisson distribution, requiring an average of nearly two vector genomes per cell to attain an 85% transduction efficiency.  

5.867           In Vivo Application of an RNAi Strategy for the Selective Suppression of a Mutant Allele

Kubodera, T., Yamada, H., Anzai, M., Ohira, S., Yokoto, S., Hirai, Y., Mochizuki, H., Shimada, T., Mitani, T., Mizusawa, H. and Yokota, T. Human Gene Therapy, 22, 27-34 (2011)   Gene therapy for dominantly inherited diseases with small interfering RNA (siRNA) requires mutant allele-specific suppression when genes in which mutation causes disease normally have an important role. We previously proposed a strategy for selective suppression of mutant alleles; both mutant and wild-type alleles are inhibited by most effective siRNA, and wild-type protein is restored using mRNA mutated to be resistant to the siRNA. Here, to prove the principle of this strategy in vivo, we applied it to our previously reported anti–copper/zinc superoxide dismutase (SOD1) short hairpin RNA (shRNA) transgenic (Tg) mice, in which the expression of the endogenous wild-type SOD1 gene was inhibited by more than 80%. These shRNA Tg mice showed hepatic lipid accumulation with mild liver dysfunction due to downregulation of endogenous wild-type SOD1. To rescue this side effect, we generated siRNA-resistant SOD1 Tg mice and crossed them with anti-SOD1 shRNA Tg mice, resulting in the disappearance of lipid accumulation in the liver. Furthermore, we also succeeded in mutant SOD1-specific gene suppression in the liver of SOD1^G93A Tg mice, a model for amyotrophic lateral sclerosis, using intravenously administered viral vectors. Our method may prove useful for siRNA-based gene therapy for dominantly inherited diseases.  

5.868           Recruitment of DNA replication and damage response proteins to viral replication centers during infection with NS2 mutants of Minute Virus of Mice (MVM)

Riuz, Z., Mihaylov, I.S., Cotmore, S.F. and Tattersall, P. Virology, 410, 375-384 (2011)   MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA₃₂, which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins.  

5.869           Novel Properties of Tyrosine-mutant AAV2 Vectors in the Mouse Retina

Petrs-Silva, H., Dinculescu, A., Li, Q., Deng, W-T., Pang, J-J., Min, S-H., Chiodo, V., Beeley, A.W., Govindasamy, L., Bennett, A., Agbandje-McKenna, M., Zhong, L., Li, B., Jayandharan, G.R., Srivastava, A., Lewin, A.S. and Hauswirth, W.W. Molecular Therapy, 19(2), 293-301 (2011)   Vectors based on adeno-associated virus serotype 2 (AAV2) have been used extensively in many gene-delivery applications, including several successful clinical trials for one type of Leber congenital amaurosis in the retina. Many studies have focused on improving AAV2 transduction efficiency and cellular specificity by genetically engineering its capsid. We have previously shown that vectors-containing single-point mutations of capsid surface tyrosines in serotypes AAV2, AAV8, and AAV9 displayed significantly increased transduction efficiency in the retina compared with their wild-type counterparts. In the present study, we evaluated the transduction characteristics of AAV2 vectors containing combinations of multiple tyrosine to phenylalanine mutations in seven highly conserved surface-exposed capsid tyrosine residues following subretinal or intravitreal delivery in adult mice. The multiply mutated vectors exhibited different in vivo transduction properties, with some having a unique ability of transgene expression in all retinal layers. Such novel vectors may be useful in developing valuable new therapeutic strategies for the treatment of many genetic diseases.  

5.870           Sustained Enzymatic Correction by rAAV-Mediated Liver Gene Therapy Protects Against Induced Motor Neuropathy in Acute Porphyria Mice

Unzu, C., Sampedro, A., Mauleon, I., Alegre, M., Beattie, S.G., de Salamanca, R.E., Snapper, J., Twisk, J., Petry, H., Gonzalez-Aseguinolaza, G., Artieda, J., Rodriguez-Pena, M., Prieto, J. and Fontanellas, A. Molecular Therapy, 19(2), 243-250 (2011)   Acute intermittent porphyria (AIP) is characterized by a hereditary deficiency of hepatic porphobilinogen deaminase (PBGD) activity. Clinical features are acute neurovisceral attacks accompanied by overproduction of porphyrin precursors in the liver. Recurrent life-threatening attacks can be cured only by liver transplantation. We developed recombinant adeno-associated virus (rAAV) vectors expressing human PBGD protein driven by a liver-specific promoter to provide sustained protection against induced attacks in a predictive model for AIP. Phenobarbital injections in AIP mice induced porphyrin precursor accumulation, functional block of nerve conduction, and progressive loss of large-caliber axons in the sciatic nerve. Hepatocyte transduction showed no gender variation after rAAV2/8 injection, while rAAV2/5 showed lower transduction efficiency in females than males. Full protection against induced phenobarbital-attacks was achieved in animals showing over 10% of hepatocytes expressing high amounts of PBGD. More importantly, sustained hepatic expression of hPBGD protected against loss of large-caliber axons in the sciatic nerve and disturbances in nerve conduction velocity as induced by recurrent phenobarbital administrations. These data show for the first time that porphyrin precursors generated in the liver interfere with motor function. rAAV2/5-hPBGD vector can be produced in sufficient quantity for an intended gene therapy trial in patients with recurrent life-threatening porphyria attacks.  

5.871           Safe, Efficient, and Reproducible Gene Therapy of the Brain in the Dog Models of Sanfilippo and Hurler Syndromes

Ellinwood, N.M., Ausseil, J., Desmaris, N., Bigou, S., Liu, S., Jens, J.K., Snella, E.M., Mohammed, E.E.A., Thomson, C.B., Raoul, S., Joussemet, B., Roux, F., Cherel, Y., Lajat, Y., Piraud, M., Benchaouir, R., Hermening, S., Petry, H., Froissart, R., Tardieu, M., Ciron, C., Moullier, P., Parkes, J., Kline, K.L., Maire, I., Vanier, M-T., Heard, J-M. and Colle, M-A. Molecular Therapy, 19(2), 251-259 (2011)   Recent trials in patients with neurodegenerative diseases documented the safety of gene therapy based on adeno-associated virus (AAV) vectors deposited into the brain. Inborn errors of the metabolism are the most frequent causes of neurodegeneration in pre-adulthood. In Sanfilippo syndrome, a lysosomal storage disease in which heparan sulfate oligosaccharides accumulate, the onset of clinical manifestation is before 5 years. Studies in the mouse model showed that gene therapy providing the missing enzyme α-N-acetyl-glucosaminidase to brain cells prevents neurodegeneration and improves behavior. We now document safety and efficacy in affected dogs. Animals received eight deposits of a serotype 5 AAV vector, including vector prepared in insect Sf9 cells. As shown previously in dogs with the closely related Hurler syndrome, immunosuppression was necessary to prevent neuroinflammation and elimination of transduced cells. In immunosuppressed dogs, vector was efficiently delivered throughout the brain, induced α-N-acetyl-glucosaminidase production, cleared stored compounds and storage lesions. The suitability of the procedure for clinical application was further assessed in Hurler dogs, providing information on reproducibility, tolerance, appropriate vector type and dosage, and optimal age for treatment in a total number of 25 treated dogs. Results strongly support projects of human trials aimed at assessing this treatment in Sanfilippo syndrome.  

5.872           Hypervariable Region 1 Differentially Impacts Viability of Hepatitis C Virus Strains of Genotypes 1 to 6 and Impairs Virus Neutralization

Prentoe, J., Jensen, T.B., Meuleman, P., Serre, S.B.N., Scheel, T.K.H., Lereoux-Roels, G., Gottwein, J.M. and Bukh, J.

1. Virol., 85(5), 2224-2234 (2011)

  Hypervariable region 1 (HVR1) of hepatitis C virus (HCV) E2 envelope glycoprotein has been implicated in virus neutralization and persistence. We deleted HVR1 from JFH1-based HCV recombinants expressing Core/E1/E2/p7/NS2 of genotypes 1 to 6, previously found to grow efficiently in human hepatoma Huh7.5 cells. The 2a HVR1, 5a HVR1, and 6a HVR1 Core-NS2 recombinants retained viability in Huh7.5 cells, whereas 1a HVR1, 1b HVR1, 2b HVR1, 3a HVR1, and 4a HVR1 recombinants were severely attenuated. However, except for recombinant 4a HVR1, viruses eventually spread, and reverse genetics studies revealed adaptive envelope mutations that rescued the infectivity of 1a HVR1, 1b HVR1, 2b HVR1, and 3a HVR1 recombinants. Thus, HVR1 might have distinct functional roles for different HCV isolates. Ultracentrifugation studies showed that deletion of HVR1 did not alter HCV RNA density distribution, whereas infectious particle density changed from a range of 1.0 to 1.1 g/ml to a single peak at 1.1 g/ml, suggesting that HVR1 was critical for low-density HCV particle infectivity. Using chronic-phase HCV patient sera, we found three distinct neutralization profiles for the original viruses with these genotypes. In contrast, all HVR1-deleted viruses were highly sensitive with similar neutralization profiles. In vivo relevance for the role of HVR1 in protecting HCV from neutralization was demonstrated by ex vivo neutralization of 2a and 2a HVR1 produced in human liver chimeric mice. Due to the high density and neutralization susceptibility of HVR1-deleted viruses, we investigated whether a correlation existed between density and neutralization susceptibility for the original viruses with genotypes 1 to 6. Only the 2a virus displayed such a correlation. Our findings indicate that HVR1 of HCV shields important conserved neutralization epitopes with implications for viral persistence, immunotherapy, and vaccine development.  

5.873           Vaccine protection against lethal homologous and heterologous challenge using recombinant AAV vectors expressing codon-optimized genes from pandemic swine origin influenza virus (SOIV)

Sipo, I., Knauf, M., Fechner, H., Poller, W., Planz, O., Kurth, R. and Norley, S. Vaccine, 29, 1690-1699 (2011)   The recent H1N1 influenza pandemic and the inevitable delay between identification of the virus and production of the specific vaccine have highlighted the urgent need for new generation influenza vaccines that can preemptively induce broad immunity to different strains of the virus. In this study we have produced AAV-based vectors expressing the A/Mexico/4603/2009 (H1N1) hemagglutinin (HA), nucleocapsid (NP) and the matrix protein M1 and have evaluated their ability to induce specific immune response and protect mice against homologous and heterologous challenge. Each of the vaccine vectors elicited potent cellular and humoral immune responses in mice. Although immunization with AAV-M1 did not improve survival after challenge with the homologous strain, immunization with the AAV-H1 and AAV-NP vectors resulted in survival of all mice, as did inoculation with a combination of all three vectors. Furthermore, trivalent vaccination also conferred partial protection against challenge with the highly heterologous and virulent A/PR/8/34 strain of H1N1 influenza.  

5.874           Transactivation of human parvovirus B19 gene expression in endothelial cells by adenoviral helper functions

Pozzuto, T., von Kietzell, K., Bock, T., Schmidt-Lucke, C., Poller, W., Zobel, T., Lassner, D., Zeichhardt, H., wager, S. and Fechner, H. Virology, 411, 50-64 (2011)   Human parvovirus B19 (B19V) DNA is highly prevalent in endothelial cells lining up intramyocardial arterioles and postcapillary venules of patients with chronic myocarditis and cardiomyopathies. We addressed the question of a possible stimulation of B19V gene expression in endothelial cells by infection with adenoviruses. Adenovirus infection led to a strong augmentation of B19V structural and nonstructural proteins in individual endothelial cells infected with B19V or transfected with an infectious B19V genome. Transactivation was mostly mediated at the level of transcription and not due to adenovirus-mediated induction of second-strand synthesis from the single-stranded parvoviral genome. The main adenoviral functions required were E1A and E4orf6, which displayed synergistic effects. Furthermore, a limited B19V genome replication could be demonstrated in endothelial cells and adenovirus infection induced the appearance of putative dimeric replication intermediates. Thus the almost complete block in B19V gene expression seen in endothelial cells can be abrogated by infection with other viruses.  

5.875           The Red clover necrotic mosaic virus Capsid as a Multifunctional Cell Targeting Plant Viral Nanoparticle

Lockney, D.M., Guenther, R.N., Loo, L., Overton, W., Antonelli, R., Clark, J., Hu, M., Luft, C., Lommel, S.A. and Franzen, S. Bioconjugate Chem., 22(1), 66.73 (2011)   Multifunctional nanoparticles hold promise as the next generation of therapeutic delivery and imaging agents. Nanoparticles comprising many types of materials are being tested for this purpose, including plant viral capsids. It has been found that Red clover necrotic mosaic virus (RCNMV) can be loaded with significant amounts of therapeutic molecules with molecular weights of 600 or even greater. Formulation of RCNMV into a plant viral nanoparticle (PVN) involves the loading of cargo and attachment of peptides. In this study, we show that targeting peptides (less than 16 amino acids) can be conjugated to the capsid using the heterobifunctional chemical linker sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The uptake of both native RCNMV capsids and peptide-conjugated RCNMV was tested in the HeLa cell line for peptides with and without fluorescent labels. Uptake of RCNMV conjugate with a CD46 targeting peptide was monitored by flow cytometry. When formulated PVNs loaded with doxorubicin and armed with a targeting peptide were delivered to HeLa cells, a cytotoxic effect was observed. The ability to modify RCNMV for specific cell targeting and cargo delivery offers a method for the intracellular delivery of reagents for research assays as well as diagnostic and therapeutic applications.  

5.876           Intraperitoneal AAV9-shRNA inhibits target expression in neonatal skeletal and cardiac muscles

Mayra, A., Tomimitsu, H., Kubodera, T., Kobaayashi, M., Piao, W., Sunaga, F., Hirai, Y., Shimada, T., Mizusawa, H. and Yokota, T. Biochem. Biophys. Res. Comm., 405, 204-209 (2011)   Systemic injections of AAV vectors generally transduce to the liver more effectively than to cardiac and skeletal muscles. The short hairpin RNA (shRNA)-expressing AAV9 (shRNA-AAV9) can also reduce target gene expression in the liver, but not enough in cardiac or skeletal muscles. Higher doses of shRNA-AAV9 required for inhibiting target genes in cardiac and skeletal muscles often results in shRNA-related toxicity including microRNA oversaturation that can induce fetal liver failure. In this study, we injected high-dose shRNA-AAV9 to neonates and efficiently silenced genes in cardiac and skeletal muscles without inducing liver toxicity. This is because AAV is most likely diluted or degraded in the liver than in cardiac or skeletal muscle during cell division after birth. We report that this systemically injected shRNA-AAV method does not induce any major side effects, such as liver dysfunction, and the dose of shRNA-AAV is sufficient for gene silencing in skeletal and cardiac muscle tissues. This novel method may be useful for generating gene knockdown in skeletal and cardiac mouse tissues, thus providing mouse models useful for analyzing diseases caused by loss-of-function of target genes.  

5.877           Cross-Neutralization Potential of Native Human Papillomavirus N-Terminal L2 Epitopes

Conway, M.J., Cruz, L., Alam, S., Christensen, N.D. and Meyers, C. PloSOne, 6(2), e16405 (2011)   Background Human papillomavirus (HPV) capsids are composed of 72 pentamers of the major capsid protein L1, and an unknown number of L2 minor capsid proteins. An N-terminal “external loop” of L2 contains cross-neutralizing epitopes, and native HPV16 virions extracted from 20-day-old organotypic tissues are neutralized by anti-HPV16 L2 antibodies but virus from 10-day-old cultures are not, suggesting that L2 epitopes are more exposed in mature, 20-day virions. This current study was undertaken to determine whether cross-neutralization of other HPV types is similarly dependent on time of harvest and to screen for the most effective cross-neutralizing epitope in native virions. Methodology and Principal Findings Neutralization assays support that although HPV16 L2 epitopes were only exposed in 20-day virions, HPV31 or HPV18 epitopes behaved differently. Instead, HPV31 and HPV18 L2 epitopes were exposed in 10-day virions and remained so in 20-day virions. In contrast, presumably due to sequence divergence, HPV45 was not cross-neutralized by any of the anti-HPV16 L2 antibodies. We found that the most effective cross-neutralizing antibody was a polyclonal antibody named anti-P56/75 #1, which was raised against a peptide consisting of highly conserved HPV16 L2 amino acids 56 to 75. Conclusions and Significance This is the first study to determine the susceptibility of multiple, native high-risk HPV types to neutralization by L2 antibodies. Multiple anti-L2 antibodies were able to cross-neutralize HPV16, HPV31, and HPV18. Only neutralization of HPV16 depended on the time of tissue harvest. These data should inform attempts to produce a second-generation, L2-based vaccine.  

5.878           rAAV2-mediated restoration of LEKTI in LEKTI-deficient cells from Netherton patients

Roedl, D., Oji, V., Buters, J.T.M., Behrendt, H. and Braun-Falco, M:

1. Dermatol. Sci., 61(3), 194-198 (2011)

  Background Netherton syndrome (NS, MIM 256500) is a potential live threatening autosomal-recessive skin disorder clinically characterized by the trias of congenital erythroderma, hair shaft anomalies and atopic diathesis. It is caused by mutations in the gene SPINK5 resulting in a deficiency of its processed protein named lympho-epithelial Kazal-type related inhibitor (LEKTI). LEKTI controls the activity of several serine proteases in the skin that are involved in terminal differentiation. Loss of LEKTI results in protease hyperactivity, increased degradation of intercellular junctions, reduced stratum corneum adhesion and impaired skin barrier function. Today NS can only be treated symptomatically. Objective Does gene transfer offer a therapeutic option for NS in the future? Methods A recombinant adeno-associated virus type 2 vector was constructed containing the full length cDNA (rAAV2/C-SPINK5) of functional human LEKTI. Infectious virus particles were used for transfection of LEKTI-deficient-keratinocytes of NS patients in vitro. Results Gene transfer of SPINK5 in NS-keratinocytes led to a five-fold increase in mRNA expression of SPINK5 reaching almost 75% of normal value. The functionality of the expressed LEKTI was proven in a hydrolytic activity assay demonstrating that the activity of LEKTI after gene transfer increased closely to the level seen in keratinocytes of healthy individuals. Conclusion The results provide first evidence that gene transfer of SPINK5 results in increased LEKTI activity in NS-keratinocytes, thus offering a rational to further pursue such a gene therapy approach for NS.  

5.879           Evaluation and Optimization of the Administration of Recombinant Adeno-Associated Viral Vectors (Serotypes 2/1, 2/2, 2/rh8, 2/9, and 2/rh10) by Convection-Enhanced Delivery to the Striatum

White, E., Bienemann, A., Sena-Esteves, M., Taylor, H., Bunnen, C., Castrique, E and Gill, S. Human Gene Therapy, 22, 237-251 (2011)   Convection-enhanced delivery (CED) of recombinant adeno-associated virus (rAAV) vectors is a promising approach for delivery of therapeutic transgenes to the brain. In this study we have systematically examined vector dosing in vivo. Infusions of rAAV serotypes 2/1, 2/2, 2/rh8, 2/9, and 2/rh10 expressing an enhanced green fluorescent protein reporter gene were undertaken into the striatum of rats and pigs using CED. Vector distribution, as defined by the volume of distribution and number of transduced cells following each infusion, was determined using stereological methods. Immunohistochemistry was used to determine the transductional tropism of serotypes and to evaluate for the presence of immune cell infiltration into the brain. Vector distribution was highly variable between serotypes. Infusion rate had no significant effect on vector distribution or the occurrence of tissue damage. For serotypes 2/1, 2/2 and 2/rh10, as the vector concentration was increased beyond 1012 vg/ml, no increase in vector distribution was observed. In contrast, for serotypes 2/rh8 and 2/9, retrograde axonal transport was observed above this threshold concentration. Cell transduction was principally neuronal for all serotypes and was associated with a low-level immune response. In planning clinical trials it is critical that these observations are considered in order to achieve optimal vector dosing.  

5.880           Long-term persistence of human papillomavirus in environments

Ding, D-C., Chang, Y-C., Liu, H-W. and Chu, T-Y. Gynecologic Oncology, 121, 148-151 (2011)   Objective The possibility of its indirect transmission of human papillomavirus (HPV) via formites has been widely raised but with no biological proof. This study explored the durability of HPV16 pseudoviruses and native viruses in different environmental contamination scenarios. Methods Pseudoviruses were mixed with PBS, cervico-vaginal secretion (CVS), or serum to simulate contamination by genital warts, vaginal discharge or menstruation, respectively, and subjected to in-vitro cell infection assay. The integrity of native HPV16 from CVS of infected women was detected by conformation-specific antibody. Results In viruses exposed to PBS, a persistent infectivity of 30% was noted for at least 7 days. A similar persistence but lower (18%) infectivity was noted in those exposed to CVS. In serum-containing medium, the infection ratio rose initially, remained stable for three more days then rapidly decreased thereafter. Upon desiccation, infectivity was persistently low (10%). Finally, intact native HPV was detectable after 5 days of environmental exposure. Conclusion This study demonstrated the high environmental survivability of HPV. However, survivability was lower in viruses exposed to CVS or desiccation.  

5.881           Novel Mutations in a Tissue Culture-Adapted Hepatitis C Virus Strain Improve Infectious-Virus Stability and Markedly Enhance Infection Kinetics

Pokrovskii, M.V., Bush, C.O:, Beran, R.K.F., Robinson, M.F., Cheng, G., Tirunagari, N., Fenaoux, M:, Greenstein, A.E., Zhong, W., Delaney, W.E. and Paulson, M.S.

1. Virol., 85(8), 3978-3985 (2011)

  Hepatitis C virus (HCV) establishes persistent infections and leads to chronic liver disease. It only recently became possible to study the entire HCV life cycle due to the ability of a unique cloned patient isolate (JFH-1) to produce infectious particles in tissue culture. However, despite efficient RNA replication, yields of infectious virus particles remain modest. This presents a challenge for large-scale tissue culture efforts, such as inhibitor screening. Starting with a J6/JFH-1 chimeric virus, we used serial passaging to generate a virus with substantially enhanced infectivity and faster infection kinetics compared to the parental stock. The selected virus clone possessed seven novel amino acid mutations. We analyzed the contribution of individual mutations and identified three specific mutations, core K78E, NS2 W879R, and NS4B V1761L, which were necessary and sufficient for the adapted phenotype. These three mutations conferred a 100-fold increase in specific infectivity compared to the parental J6/JFH-1 virus, and media collected from cells infected with the adapted virus yielded infectious titers as high as 1 x 10⁸ 50% tissue culture infective doses (TCID₅₀)/ml. Further analyses indicated that the adapted virus has longer infectious stability at 37°C than the wild type. Given that the adapted phenotype resulted from a combination of mutations in structural and nonstructural proteins, these data suggest that the improved viral titers are likely due to differences in virus particle assembly that result in significantly improved infectious particle stability. This adapted virus will facilitate further studies of the HCV life cycle, virus structure, and high-throughput drug screening.  

5.882           AAV-mediated gene targeting methods for human cells

Khan, I.F., Hirata, R.K. and Russell, D.W. Nature Protocols, 6(4), 482-500 (2011)   Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10⁻⁵ to 10⁻² per infected cell. These targeting frequencies are 1–4 logs higher than those obtained by conventional transfection or electroporation approaches. A wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. Optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by Southern blots. This protocol (from vector design through a single round of targeting and screening) can be completed in ~10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks.  

5.883           Quantitative Proteomic Analysis of Tumor Reversion in Multiple Myeloma Cells

Ge, F., Zhang, L., Tao, S-C., Kitazato, K., Zhang, Z-P., Zhang, X-E. and Bi, L-J.

1. Proteome Res., 10(2), 845-855 (2011)

  Tumor reversion is defined as the process by which cancer cells lose their malignant phenotype. However, relatively little is known about the cellular proteome changes that occur during the reversion process. A biological model of multiple myeloma (MM) reversion was established by using the H-1 parvovirus as a tool to select for revertant cells from MM cells. Isolated revertant cells displayed a strongly suppressed malignant phenotype both in vitro and in vivo. To explore possible mechanisms of MM reversion, the protein profiles of the revertant and parental MM cells were compared using a quantitative proteomic strategy termed SILAC-MS. Our results revealed that 379 proteins were either activated or inhibited during the reversion process, with a much greater proportion of the proteins, including STAT3, TCTP, CDC2, BAG2, and PCNA, being inhibited. Of these, STAT3, which is significantly down regulated, was selected for further functional studies. Inhibition of STAT3 expression by RNA interference resulted in suppression of the malignant phenotype and concomitant down regulation of TCTP expression, suggesting that myeloma reversion operates, at least in part, through inhibition of STAT3. Our results provide novel insights into the mechanisms of tumor reversion and suggest new alternative approaches for MM treatment.  

5.884           Adenosine kinase as a target for therapeutic antisense strategies in epilepsy

Theofilas, P., Brar, S., Stewart, K-A., Shen, H-Y., Sandau, U.S., Poulsen, D. and Boison, D. Epilepsoa, 52(3), 589-601 (2011)   Purpose: Given the high incidence of refractory epilepsy, novel therapeutic approaches and concepts are urgently needed. To date, viral-mediated delivery and endogenous expression of antisense sequences as a strategy to prevent seizures have received little attention in epilepsy therapy development efforts. Here we validate adenosine kinase (ADK), the astrocyte-based key negative regulator of the brain’s endogenous anticonvulsant adenosine, as a potential therapeutic target for antisense-mediated seizure suppression. Methods: We developed adenoassociated virus 8 (AAV8)-based gene therapy vectors to selectively modulate ADK expression in astrocytes. Cell type selectivity was achieved by expressing an Adk-cDNA in sense or antisense orientation under the control of an astrocyte-specific gfaABC1D promoter. Viral vectors where injected into the CA3 of wild-type mice or spontaneously epileptic Adk-tg transgenic mice that overexpress ADK in brain. After virus injection, ADK expression was assessed histologically and biochemically. In addition, intracranial electroencephalography (EEG) recordings were obtained. Key Findings: We demonstrate in wild-type mice that viral overexpression of ADK within astrocytes is sufficient to trigger spontaneous recurrent seizures in the absence of any other epileptogenic event, whereas ADK downregulation via AAV8-mediated RNA interference almost completely abolished spontaneous recurrent seizures in Adk-tg mice. Significance: Our data demonstrate that modulation of astrocytic ADK expression can trigger or prevent seizures, respectively. This is the first study to use an antisense approach to validate ADK as a rational therapeutic target for the treatment of epilepsy and suggests that gene therapies based on the knock down of ADK might be a feasible approach to control seizures in refractory epilepsy.  

5.885           Long-Term Cardiac pro-B-Type Natriuretic Peptide Gene Delivery Prevents the Development of Hypertensive Heart Disease in Spontaneously Hypertensive Rats

Cataliotti, A., Tonne, J.M., Bellavia, D., Martin, F.L., Oehleer, E.A., Harders, G.E., Campbell, J.M., Peng, K-W., Russell, S.J., Malatino, L.S., Burnett, J.C. and Ikeda, Y. Circulation, 123, 1297-1305 (2011)   Background— Diastolic dysfunction associated with high blood pressure (BP)leads to cardiac remodeling and fibrosis and progression tocongestive heart failure. B-type natriuretic peptide (BNP) hasBP-lowering, antifibrotic, and antihypertrophic properties,which makes BNP an attractive agent for attenuating the adversecardiac remodeling associated with hypertension. In the currentstudy, we tested the effects of sustained cardiac proBNP genedelivery on BP, cardiac function, and remodeling in spontaneouslyhypertensive rats (SHR). Methods and Results— We used the myocardium-tropic adeno-associated virus serotype9 (AAV9) vector to achieve continuously enhanced cardiac ratproBNP expression. In SHR, a single systemic administrationof AAV9 vector allowed long-term cardiac BNP overexpression,resulting in reductions in systolic and diastolic BP for 9 monthsafter injection. Left ventricular (LV) thickness, LV end-systolicdimensions, and LV mass were reduced, whereas ejection fractionwas significantly increased, in BNP-treated compared with untreatedSHR. Circumferential systolic strain and strain rate of theearly phase of diastole were improved in BNP-treated comparedwith untreated SHR. Noncardiac overexpression of BNP via AAV2vector was not associated with changes in BP and plasma BNPin SHR. Furthermore, normal Wistar rats injected with AAV9 proBNPvector showed significantly reduced heart weights 4 weeks afterinjection without BP reduction. Conclusions— AAV9 vector facilitates sustained cardiac proBNP overexpressionand improves LV function in hypertensive heart disease. Long-termproBNP delivery improved both systolic and diastolic function.The effects on cardiac structure and function occurred independentlyof BP-lowering effects in normal Wistar rats.  

5.886           Defining the Herpes Simplex Virus-Specific CD8+ T Cell Repertoire in C57BL/6 Mice

St. Leger, A.J., Peters, B., Sidney, J., Sette, A. and Hendricks, R.L.

1. Immunol., 186, 3927-3933 (2011)

  HSV type 1 (HSV-1) expresses its genes sequentially as immediate early (α), early (β), leaky late (γ1), and true late (γ2), where viral DNA synthesis is an absolute prerequisite only for γ2 gene expression. The γ1 protein glycoprotein B (gB) contains a strongly immunodominant CD8⁺ T cell epitope (gB_498–505) that is recognized by 50% of both the CD8⁺ effector T cells in acutely infected trigeminal ganglia (TG) and the CD8⁺ memory T cells in latently infected TG. Of 376 predicted HSV-1 CD8⁺ T cell epitopes in C57BL/6 mice, 19 (gB_498–505 and 18 subdominant epitopes) stimulated CD8⁺ T cells in the spleens and TG of HSV-1 acutely infected mice. These 19 epitopes identified virtually all CD8⁺ T cells in the infected TG that represent all or the vast majority of the HSV-specific CD8⁺ TCR repertoire. Only 11 of ∼84 HSV-1 proteins are recognized by CD8⁺ T cells, and most (∼80%) are expressed before viral DNA synthesis. Neither the immunodominance of gB_498–505 nor the dominance hierarchy of the subdominant epitopes is due solely to MHC or TCR affinity. We conclude that the vast majority of CD8⁺ T cells in HSV-1 acutely infected TG are HSV specific, that HSV-1 β and γ1 proteins that are expressed before viral DNA synthesis are favored targets of CD8⁺ T cells, and that dominance within the TCR repertoire is likely due to the frequency or expansion and survival characteristics of CD8⁺ T cell precursors.  

5.887           Structure of a Packaging-Defective Mutant of Minute Virus of Mice Indicates that the Genome Is Packaged via a Pore at a 5-Fold Axis

Plevka, P., Hafenstein, S., Li, L., D’Abramo Jr., A., Cotmore, S.F., Rossmann, M.G. and Tattersall, P.

1. Virol., 85(10), 4822-4827 (2011)

  The parvovirus minute virus of mice (MVM) packages a single copy of its linear single-stranded DNA genome into preformed capsids, in a process that is probably driven by a virus-encoded helicase. Parvoviruses have a roughly cylindrically shaped pore that surrounds each of the 12 5-fold vertices. The pore, which penetrates the virion shell, is created by the juxtaposition of 10 antiparallel β-strands, two from each of the 5-fold-related capsid proteins. There is a bottleneck in the channel formed by the symmetry-related side chains of the leucines at position 172. We report here the X-ray crystal structure of the particles produced by a leucine-to-tryptophan mutation at position 172 and the analysis of its biochemical properties. The mutant capsid had its 5-fold channel blocked, and the particles were unable to package DNA, strongly suggesting that the 5-fold pore is the packaging portal for genome entry.  

5.888           Adeno-associated virus serotypes 7 and 8 outperform serotype 9 in expressing atheroprotective human apoE3 from mouse skeletal muscle

Evans, V.C., Graham, I.R., Athanasopoulos, T., Galley, D.J., Jackson, C.L., Simons, J.P., Dickson, G. and Owen, J.S. Metabolism, 60(4), 491-498 (2011)   Intramuscular injection of adeno-associated viral (AAV) vectors is potentially a safe, minimally invasive procedure for the long-term gene expression of circulating antiatherogenic proteins. Here, we compare secretion and atheroprotective effects of human apoE3 after injection of 3 pseudotyped AAV vectors (AAV2/7, AAV2/8, or AAV2/9), driven by the CMV enhancer/chicken β-actin (CAG) promoter, into skeletal muscle of hyperlipidemic apolipoprotein E–deficient (apoE^−/−) mice. Vector viabilities were verified by transducing cultured C2C12 mouse myotubes and assessing secretion of human apoE3 protein. Both hind limb tibialis anterior muscles of female C57BL/6 apoE^−/− mice, 2 months old and fed a high-fat diet, were each injected with 1 × 10¹⁰ vector genomes of AAV vector. Identical noninjected mice served as controls; and blood was collected at weeks 0, 1, 2, 4, and 13. At termination (13 weeks), the brachiocephalic artery was excised; and after staining sections, plaque morphometry and fractional lipid content were quantified by computerized image analysis. Intramuscular injection of AAV2/7 and AAV2/8 vectors produced up to 2 μg human apoE3 per milliliter plasma, just below the threshold to reverse dyslipoproteinemia. AAV2/9 was notably less effective, mice having a 3-fold lower level of plasma apoE3 at 13 weeks and a 50% greater burden of atherosclerotic plaque lipid in their brachiocephalic arteries. We conclude that although vector refinement is needed to exploit fully apoE3 atheroprotective functions, AAV2/7 and AAV2/8 are promising gene transfer vectors for muscle-based expression of antiatherogenic circulating proteins.  

5.889           Plaque purification as a method to mitigate the risk of adventitious-agent contamination in influenza vaccine virus seeds

Murata, H., Macauley, J., Lewis Jr., A.M. and Peden, K. Vaccine, 29, 3155-3161 (2011)   At present, the seed viruses for the manufacture of licensed seasonal inactivated influenza vaccines in the United States are derived from primary egg isolates as a result of concerns associated with adventitious agents. According to the prevailing view, the passage of influenza viruses through eggs serves as a filtering step to remove potential contaminating viruses. We have investigated the feasibility of addressing adventitious-agent risk by subjecting influenza virus to a plaque-purification procedure using MDCK cells. SV40 and canine adenovirus-1 (representing viruses for which MDCK cells are non-permissive and permissive, respectively) were used as challenge viruses to model agents of concern that might be co-isolated along with the influenza virus. By mixing influenza virus strain A/PR/8/34 with varying amounts of each challenge virus and then performing a plaque assay for influenza virus using MDCK cells, we have attempted to determine the efficiency by which the challenge virus is removed. Our data suggest that substantial removal can be achieved even after a single round of plaque purification. If cell-derived isolates were deemed to be acceptable following a plaque-purification procedure, the manufacture of seasonal influenza vaccine would be facilitated by: (1) the expansion of the repertoire of viruses from which seed virus candidates could be generated for licensed egg-derived vaccines as well as for vaccines manufactured in mammalian cells; and (2) the mitigation of adventitious-agent risk associated with the seed virus, and hence the elimination of the need to passage seed viruses in eggs for vaccines manufactured in mammalian cells.  

5.890           Global gene transfer into the CNS across the BBB after neonatal systemic delivery of single-stranded AAV vectors

Miyake, N., Miyake, K., Yamamoto, M., Hirai, Y. and Shimada, T. Brain Res., 1389, 19-26 (2011)   Central nervous system (CNS) disorders are important targets for gene therapy; however, delivery of therapeutic proteins and/or genes to the brain remains a major challenge due to the difficulty of efficiently delivering viral vectors across the blood–brain barrier (BBB). In the present work, we tested the ability of several single-stranded adeno-associated viral (ssAAV) serotypes to deliver transgenes to the brain and spinal cord in neonatal mice. We injected ssAAV vectors encoding GFP (serotype-1, -8, -9 and -10: 1.5 × 10¹¹ vector genomes each) into the jugular vein of neonatal mice and assessed GFP expression immunohistochemically. Strong GFP signals were detected in both the brain and spinal cord after injection of any of these serotypes. ssAAV serotype-9 mediated gene transfer was the most efficient. GFP expression was detected throughout the brain, including the cortex, cerebellum, olfactory bulb and brainstem and was sustained for at least 18 months. Immunohistochemical staining showed that the GFP signals were detected in GFAP positive astrocytes, NeuN positive neurons, and Calbindin positive purkinje cells. Our data suggest that systemic neonatal injection of ssAAV is an effective strategy for delivering transgenes to target neuronal systems that are not accessible to viral vectors in adult animals. These vectors should prove highly useful for efficient and long-term overexpression or downregulation of genes in CNS and spinal cord and could be a useful means of treating genetic neurological diseases.  

5.891           Cellular fusion for gene delivery to SCA1 affected Purkinje neurons

Chen, K.A., Cruz, P.E., Lanuto, D.J., Flotte, T.R., Borchelt, D.R., Srivastava, A., Zhang, J., Steindler, D.A. and Zheng, T. Mol. Cell. Neurosci., 47, 61-70 (2011)   Cerebellar Purkinje neurons (PNs) possess a well characterized propensity to fuse with bone marrow-derived cells (BMDCs), producing heterokaryons with Purkinje cell identities. This offers the potential to rescue/repair at risk or degenerating PNs in the inherited ataxias, including Spinocerebellar Ataxia 1 (SCA1), by introducing therapeutic factors through BMDCs to potentially halt or reverse disease progression. In this study, we combined gene therapy and a stem cell-based treatment to attempt repair of at-risk PNs through cell–cell fusion in a Sca1^154Q/2Q knock-in mouse model. BMDCs enriched for the hematopoietic stem cell (HSC) population were genetically modified using adeno-associated viral vector 7 (AAV7) to carry SCA1 modifier genes and transplanted into irradiated Sca1^154Q/2Q mice. Binucleated Purkinje heterokaryons with sex-mismatched donor Y chromosomes were detected and successfully expressed the modifier genes in vivo. Potential effects of the new genome within Purkinje heterokaryons were evaluated using nuclear inclusions (NIs) as a biological marker to reflect possible modifications of the SCA1 disease process. An overall decrease in number of NIs and an increase in the number of surviving PNs were observed in treated Sca1^154Q/2Q. Furthermore, Bergmann glia were found to have fusogenic potential with the donor population and reveal another potential route of therapeutic entry into at-risk cells of the SCA1 cerebellum. This study presents a first step towards a proof-of-principle that combines somatic cellular fusion events with a neuroprotective gene therapy approach for providing potential neuronal protection/repair in a variety of neurodegenerative disorders.  

5.892           Efficacious and Safe Tissue-Selective Controlled Gene Therapy Approaches for the Cornea

Mohan, R.R., Sinha, S., Tandon, A., Gupta, R., Tovey, J.C.K. and Sharma, A. PloSOne, 6(4), e18771 (2011)   Untargeted and uncontrolled gene delivery is a major cause of gene therapy failure. This study aimed to define efficient and safe tissue-selective targeted gene therapy approaches for delivering genes into keratocytes of the cornea in vivo using a normal or diseased rabbit model. New Zealand White rabbits, adeno-associated virus serotype 5 (AAV5), and a minimally invasive hair-dryer based vector-delivery technique were used. Fifty microliters of AAV5 titer (6.5×10¹² vg/ml) expressing green fluorescent protein gene (GFP) was topically applied onto normal or diseased (fibrotic or neovascularized) rabbit corneas for 2-minutes with a custom vector-delivery technique. Corneal fibrosis and neovascularization in rabbit eyes were induced with photorefractive keratectomy using excimer laser and VEGF (630 ng) using micropocket assay, respectively. Slit-lamp biomicroscopy and immunocytochemistry were used to confirm fibrosis and neovascularization in rabbit corneas. The levels, location and duration of delivered-GFP gene expression in the rabbit stroma were measured with immunocytochemistry and/or western blotting. Slot-blot measured delivered-GFP gene copy number. Confocal microscopy performed in whole-mounts of cornea and thick corneal sections determined geometric and spatial localization of delivered-GFP in three-dimensional arrangement. AAV5 toxicity and safety were evaluated with clinical eye exam, stereomicroscopy, slit-lamp biomicroscopy, and H&E staining. A single 2-minute AAV5 topical application via custom delivery-technique efficiently and selectively transduced keratocytes in the anterior stroma of normal and diseased rabbit corneas as evident from immunocytochemistry and confocal microscopy. Transgene expression was first detected at day 3, peaked at day 7, and was maintained up to 16 weeks (longest tested time point). Clinical and slit-lamp eye examination in live rabbits and H&E staining did not reveal any significant changes between AAV5-treated and untreated control corneas. These findings suggest that defined gene therapy approaches are safe for delivering genes into keratocytes in vivo and has potential for treating corneal disorders in human patients.  

5.893           Epstein-Barr Virus Infection of Polarized Epithelial Cells via the Basolateral Surface by Memory B Cell-Mediated Transfer InfectionEpstein-Barr virus (EBV) is an important human pathogen that is carried as a latent infection of B cells by most adults worldwide. Infection of epithelial cells is also believed to be important in the normal life-cycle of EBV, and is certainly associated with the pathogenesis of some epithelial tumours. Whilst EBV binds to and infects B cells that express CD21, a receptor for the gp350 viral glycoprotein, binding of EBV to CD21-negative epithelial cells is inefficient. However, we have identified an efficient process of ‘transfer infection’. This process involves EBV first binding to B cells, resulting in CD21-mediated capping of virus and activation of adhesion molecules, which facilitates conjugate formation between B cells and epithelial cells and the subsequent entry of EBV into epithelial cells. We have characterised the molecular processes involved in transfer infection, both in unpolarized cells modelling pre-malignant epithelial cells, and in normal polarized epithelial cells. The details of the molecular interactions in these infection models led us to identify a subset of B cells, memory B cells, as being the primary vehicles for transfer infection. The results reinforce the likely physiological significance of transfer infection of epithelial cells in healthy persistence and in EBV pathogenesis.

Shannon-Lowe, C. and Rowe, M. PloSPathogens, 7(5), e1001338 (2011)   Epstein Barr virus (EBV) exhibits a distinct tropism for both B cells and epithelial cells. The virus persists as a latent infection of memory B cells in healthy individuals, but a role for infection of normal epithelial is also likely. Infection of B cells is initiated by the interaction of the major EBV glycoprotein gp350 with CD21 on the B cell surface. Fusion is triggered by the interaction of the EBV glycoprotein, gp42 with HLA class II, and is thereafter mediated by the core fusion complex, gH/gL/gp42. In contrast, direct infection of CD21-negative epithelial cells is inefficient, but efficient infection can be achieved by a process called transfer infection. In this study, we characterise the molecular interactions involved in the three stages of transfer infection of epithelial cells: (i) CD21-mediated co-capping of EBV and integrins on B cells, and activation of the adhesion molecules, (ii) conjugate formation between EBV-loaded B cells and epithelial cells via the capped adhesion molecules, and (iii) interaction of EBV glycoproteins with epithelial cells, with subsequent fusion and uptake of virions. Infection of epithelial cells required the EBV gH and gL glycoproteins, but not gp42. Using an in vitro model of normal polarized epithelia, we demonstrated that polarization of the EBV receptor(s) and adhesion molecules restricted transfer infection to the basolateral surface. Furthermore, the adhesions between EBV-loaded B cells and the basolateral surface of epithelial cells included CD11b on the B cell interacting with heparan sulphate moieties of CD44v3 and LEEP-CAM on epithelial cells. Consequently, transfer infection was efficiently mediated via CD11b-positive memory B cells but not by CD11b–negative naïve B cells. Together, these findings have important implications for understanding the mechanisms of EBV infection of normal and pre-malignant epithelial cells in vivo.  

5.894           An Evolved Adeno-associated Viral Variant Enhances Gene Delivery and Gene Targeting in Neural Stem Cells

Jang, J-H., Koerber, J.T., Kim, J-S., Asuri, P., Vazin, T., Bartel, M., Keung, A., Kwon, I., Park, K.I. and Schaffer, D.V. Molecular Therapy, 19(4), 667-675 (2011)   Gene delivery to, and gene targeting in, stem cells would be a highly enabling technology for basic science and biomedical application. Adeno-associated viral (AAV) vectors have demonstrated the capacity for efficient delivery to numerous cells, but their application to stem cells has been limited by low transduction efficiency. Due to their considerable advantages, however, engineering AAV delivery systems to enhance gene delivery to stem cells may have an impact in stem cell biology and therapy. Therefore, using several diverse AAV capsid libraries—including randomly mutagenized, DNA shuffled, and random peptide insertion variants—we applied directed evolution to create a “designer” AAV vector with enhanced delivery efficiency for neural stem cells (NSCs). A novel AAV variant, carrying an insertion of a selected peptide sequence on the surface of the threefold spike within the heparin-binding site, emerged from this evolution. Importantly, this evolved AAV variant mediated efficient gene delivery to rat, mouse, and human NSCs, as well as efficient gene targeting within adult NSCs, and it is thus promising for applications ranging from basic stem cell biology to clinical translation.  

5.895           Apolipoprotein B Knockdown by AAV-delivered shRNA Lowers Plasma Cholesterol in Mice

Koorneef, A., Maczuga, P., van Logtenstein, R., Borel, F., Blits, B., Ritsema, T., van Deventer, S., petry, H. and Konstantinova, P. Molecular Therapy, 19(4), 731-740 (2011)   Serum low-density lipoprotein cholesterol (LDL-C) levels are proportionate to the risk of atherosclerotic cardiovascular disease. In order to reduce serum total cholesterol and LDL-C levels in mice, RNA interference (RNAi) was used to inhibit expression of the structural protein of LDL-C, apolipoprotein B100 (ApoB). We developed and screened 19 short hairpin RNAs (shRNAs) targeting conserved sequences in human, mouse, and macaque ApoB mRNAs (shApoB) and subsequently narrowed our focus to one candidate for in vivo testing. Self-complementary adeno-associated virus serotype 8 (scAAV8) was used for long-term transduction of murine liver with shApoB. A strong dose-dependent knockdown of ApoB mRNA and protein was observed, which correlated with a reduction in total cholesterol levels, without obvious signs of toxicity. Furthermore, shApoB was found to specifically reduce LDL-C in diet-induced dyslipidemic mice, whereas high-density lipoprotein cholesterol (HDL-C) remained unaffected. Finally, elevated lipid accumulation was shown in murine liver transduced with shApoB, a known phenotypic side effect of lowering ApoB levels. These results demonstrate a robust dose-dependent knockdown of ApoB by AAV-delivered shRNA in murine liver, thus providing an excellent candidate for development of RNAi-based gene therapy for the treatment of hypercholesterolemia.  

5.896           Orexin Gene Transfer into Zona Incerta Neurons Suppresses Muscle Paralysis in Narcoleptic Mice

Liu, M., Blanco-Centurion, C., Konadhode, R., Begum, S., Pelluru, D., Geashchenko, D., Sakurai, T., Yanagisawa, M., van den Pol, A.N. and Shiromani, P.J.

1. Neurosci., 31(16), 6028-6040 (2011)

  Cataplexy, a sudden unexpected muscle paralysis, is a debilitating symptom of the neurodegenerative sleep disorder, narcolepsy. During these attacks, the person is paralyzed, but fully conscious and aware of their surroundings. To identify potential neurons that might serve as surrogate orexin neurons to suppress such attacks, the gene for orexin (hypocretin), a peptide lost in most human narcoleptics, was delivered into the brains of the orexin-ataxin-3 transgenic mouse model of human narcolepsy. Three weeks after the recombinant adenoassociated virus (rAAV)-mediated orexin gene transfer, sleep–wake behavior was assessed. rAAV-orexin gene delivery into neurons of the zona incerta (ZI), or the lateral hypothalamus (LH) blocked cataplexy. Orexin gene transfer into the striatum or in the melanin-concentrating hormone neurons in the ZI or LH had no such effect, indicating site specificity. In transgenic mice lacking orexin neurons but given rAAV-orexin, detectable levels of orexin-A were evident in the CSF, indicating release of the peptide from the surrogate neurons. Retrograde tracer studies showed that the amygdala innervates the ZI consistent with evidence that strong emotions trigger cataplexy. In turn, the ZI projects to the locus ceruleus, indicating that the ZI is part of a circuit that stabilizes motor tone. Our results indicate that these neurons might also be recruited to block the muscle paralysis in narcolepsy.  

5.897           Whirlin Replacement Restores the Formation of the USH2 Protein Complex in Whirlin Knockout Photoreceptors

Zou, J., Luo, L., Shen, Z., Chiodo, V.A., Ambati, B.K., hauswirth, W.W. and Yang, J. Invest. Ophthalmol. Vis. Sci., 52(5), 2343-2351 (2011)   Purpose. Whirlin is the causative gene for Usher syndrome type IID (USH2D), a condition manifested as both retinitis pigmentosa and congenital deafness. Mutations in this gene cause disruption of the USH2 protein complex composed of USH2A and VLGR1 at the periciliary membrane complex (PMC) in photoreceptors. In this study, the adeno-associated virus (AAV)-mediated whirlin replacement was evaluated as a treatment option. Methods. Murine whirlin cDNA driven by the human rhodopsin kinase promoter (hRK) was packaged as an AAV2/5 vector and delivered into the whirlin knockout retina through subretinal injection. The efficiency, efficacy, and safety of this treatment were examined using immunofluorescent staining, confocal imaging, immunoelectron microscopy, Western blot analysis, histologic analysis, and electroretinogram. Results. The AAV-mediated whirlin expression started at two weeks, reached its maximum level at 10 weeks, and lasted up to six months post injection. The transgenic whirlin product had a molecular size and an expression level comparable to the wild-type. It was distributed at the PMC in both rod and cone photoreceptors from the central to peripheral retina. Importantly, the transgenic whirlin restored the cellular localization and expression level of both USH2A and VLGR1 and did not cause defects in the retinal histology and function in the whirlin knockout mouse. Conclusions. Whirlin transgene recruits USH2A and VLGR1 to the PMC and is sufficient for the formation of the USH2 protein complex in photoreceptors. The combined hRK and AAV gene delivery system could be an effective gene therapy approach to treat retinal degeneration in USH2D patients.  

5.898           Intravitreal Injection of AAV2 Transduces Macaque Inner Retina

Yin, L. et al Invest. Ophthalmol Vis. Sci., 52(5), 2775-2783 (2011)   Purpose. Adeno-associated virus serotype 2 (AAV2) has been shown to be effective in transducing inner retinal neurons after intravitreal injection in several species. However, results in nonprimates may not be predictive of transduction in the human inner retina, because of differences in eye size and the specialized morphology of the high-acuity human fovea. This was a study of inner retina transduction in the macaque, a primate with ocular characteristics most similar to that of humans. Methods. In vivo imaging and histology were used to examine GFP expression in the macaque inner retina after intravitreal injection of AAV vectors containing five distinct promoters. Results. AAV2 produced pronounced GFP expression in inner retinal cells of the fovea, no expression in the central retina beyond the fovea, and variable expression in the peripheral retina. AAV2 vector incorporating the neuronal promoter human connexin 36 (hCx36) transduced ganglion cells within a dense annulus around the fovea center, whereas AAV2 containing the ubiquitous promoter hybrid cytomegalovirus (CMV) enhancer/chicken-β-actin (CBA) transduced both Müller and ganglion cells in a dense circular disc centered on the fovea. With three shorter promoters—human synapsin (hSYN) and the shortened CBA and hCx36 promoters (smCBA and hCx36sh)—AAV2 produced visible transduction, as seen in fundus images, only when the retina was altered by ganglion cell loss or enzymatic vitreolysis. Conclusions. The results in the macaque suggest that intravitreal injection of AAV2 would produce high levels of gene expression at the human fovea, important in retinal gene therapy, but not in the central retina beyond the fovea.  

5.899           Insulin resistance and low-density apolipoprotein B-associated lipoviral particles in hepatitis C virus genotype 1 infection

Bridge, S.H., Sheridan, D.A., Felmlee, D.J., Nielsen, S.U., Thomas, H.C., Taylor-Robinson, S.D., Neely, R.D.G., Toms, G.L. and Bassendine, M.F. Gut, 60, 680-687 (2011)   Background The density of hepatitis C virus (HCV) in plasma is heterogeneous but the factors which influence this are poorly understood. Evidence from animal models and cell culture suggest that low-density apolipoprotein B (apoB)-associated HCV lipoviral particles (LVP) are more infectious than high-density HCV. Objective To measure LVP in patients with chronic hepatitis C genotype 1 (CHC-G1) and examine metabolic determinants of LVP load. Patients 51 patients with CHC-G1 infection. Methods Fasting lipid profiles and homeostasis model assessment of insulin resistance (HOMA-IR) were determined in 51 patients with CHC-G1. LVP and non-LVP viral load were measured by real-time PCR of plasma at density <1.07 g/ml and >1.07 g/ml, respectively, following iodixanol density gradient ultracentrifugation. The LVP ratio was calculated using the formula: LVP/(LVP + non-LVP). Results The mean LVP ratio was 0.241 but varied 25-fold (from 0.029 to 0.74). Univariate analysis showed that the LVP ratio correlated with HOMA-IR (p=0.004) and the triglyceride/high-density lipoprotein cholesterol (TG/HDL-C) ratio (p=0.004), but not with apoB. In multivariate analysis, HOMA-IR was the main determinant of LVP load (log₁₀IU/ml) (R²=16.6%; p=0.037) but the TG/HDL-C ratio was the strongest predictor of the LVP ratio (R²=24.4%; p=0.019). Higher LVP ratios were associated with non-response to antiviral therapy (p=0.037) and with greater liver stiffness (p=0.001). Conclusion IR and associated dyslipidaemia are the major determinants of low-density apoB-associated LVP in fasting plasma. This provides a possible mechanism to explain why IR is associated with more rapidly progressive liver disease and poorer treatment outcomes.  

5.900           Rescue of Infectious Particles from Preassembled Alphavirus Nucleocapsid Cores

Snyder, J.E., Azisgolshani, O., Wu, B., He, Y., Lee, A.C., Jose, J., Suter, D.M., Knobler, C.M., Gelbart, W.M. and Kuhn, R.J. J: Virol., 85(12), 5773-5781 (2011)   Alphaviruses are small, spherical, enveloped, positive-sense, single-stranded, RNA viruses responsible for considerable human and animal disease. Using microinjection of preassembled cores as a tool, a system has been established to study the assembly and budding process of Sindbis virus, the type member of the alphaviruses. We demonstrate the release of infectious virus-like particles from cells expressing Sindbis virus envelope glycoproteins following microinjection of Sindbis virus nucleocapsids purified from the cytoplasm of infected cells. Furthermore, it is shown that nucleocapsids assembled in vitro mimic those isolated in the cytoplasm of infected cells with respect to their ability to be incorporated into enveloped virions following microinjection. This system allows for the study of the alphavirus budding process independent of an authentic infection and provides a platform to study viral and host requirements for budding.  

5.901           Direct gene transfer to the CNS prevents emergence of neurologic disease in a murine model of mucopolysaccharidosis type I

Wolf, D., Lenander, A.W., Nan, Z., Belur, L.R., Whitley, C.B., Gupta, P., Low, W.C. and McIvor, R.S. Neurobiol. Disease, 43, 123-133 (2011)   The mucopolysaccharidoses (MPSs) are a group of 11 storage diseases caused by disruptions in glycosaminoglycan (GAG) catabolism, leading to their accumulation in lysosomes. Resultant multisystemic disease is manifested by growth delay, hepatosplenomegaly, skeletal dysplasias, cardiopulmonary obstruction, and, in severe MPS I, II, III, and VII, progressive neurocognitive decline. Some MPSs are treated by allogeneic hematopoietic stem cell transplantation (HSCT) and/or recombinant enzyme replacement therapy (ERT), but effectiveness is limited by central nervous system (CNS) access across the blood-brain barrier. To provide a high level of gene product to the CNS, we tested neonatal intracerebroventricular (ICV) infusion of an adeno-associated virus (AAV) serotype 8 vector transducing the human α-l-iduronidase gene in MPS I mice. Supranormal levels of iduronidase activity in the brain (including 40× normal levels in the hippocampus) were associated with transduction of neurons in motor and limbic areas identifiable by immunofluorescence staining. The treatment prevented accumulation of GAG and GM3 ganglioside storage materials and emergence of neurocognitive dysfunction in a modified Morris water maze test. The results suggest the potential of improved outcome for MPSs and other neurological diseases when a high level of gene expression can be achieved by direct, early administration of vector to the CNS.  

5.902           A Large and Intact Viral Particle Penetrates the Endoplasmic Reticulum Membrane to Reach the Cytosol

Inoue, T. and Tsai, B. PloS Pathogens, 7(5), e1002037 (2011)   Biological membranes represent a major barrier during viral infection. While the mechanism by which an enveloped virus breaches the limiting membrane of a host cell is well-characterized, this membrane penetration process is poorly understood for non-enveloped viruses. Indeed, most available insights on membrane transport of non-enveloped viruses are built upon in vitro studies. Here we established a cell-based assay to elucidate the molecular mechanism by which the non-enveloped SV40 penetrates the endoplasmic reticulum (ER) membrane to access the cytosol, a critical step in infection. Strikingly, we uncovered SV40 breaches the ER membrane as a large and intact viral particle, despite the conformational changes it experiences in the ER lumen. This result suggests that the ER membrane can accommodate translocation of a large protein complex, possibly through either a sizeable protein channel or the ER membrane bilayer. In addition to this finding, we also pinpoint viral and host components that control the ER-to-cytosol membrane transport event. Together, our data illuminate the cellular mechanism by which a non-enveloped virus penetrates the limiting membrane of a target cell during infection.  

5.903           An alpha-synuclein AAV gene silencing vector ameliorates a behavioral deficit in a rat model of Parkinson's disease, but displays toxicity in dopamine neurons

Khodr, C.E., Sapru, M.K., Pedapati, J., Han, Y., West, N.C., Kells, A.P., Bankiewicz, K.S. and Bohn, M.C. Brain Res., 1395, 94-107 (2011)   Effects of silencing ectopically expressed hSNCA in rat substantia nigra (SN) were examined as a novel therapeutic approach to Parkinson's disease (PD). AAV-hSNCA with or without an AAV harboring a short-hairpin (sh)RNA targeting hSNCA or luciferase was injected into one SN. At 9 weeks, hSNCA-expressing rats had reduced SN dopamine (DA) neurons and exhibited a forelimb deficit. AAV-shRNA-SNCA silenced hSNCA and protected against the forelimb deficit. However, AAV-shRNA-SNCA also led to DA neuron loss suggesting undesirable effects of chronic shRNA expression. Effects on nigrostriatal-projecting neurons were examined using a retrograde tract tracer. Loss of striatal-projecting DA neurons was evident in the vector injection site, whereas DA neurons outside this site were lost in hSNCA-expressing rats, but not in hSNCA-silenced rats. These observations suggest that high levels of shRNA-SNCA were toxic to DA neurons, while neighboring neurons exposed to lower levels were protected by hSNCA gene silencing. Also, data collected on DA levels suggest that neurons other than or in addition to nigrostriatal DA neurons contributed to protection of forelimb use. Our observations suggest that while hSNCA gene silencing in DA neurons holds promise as a novel PD therapy, further development of silencing technology is required.  

5.904           Comparison of IL-10 and MCP-1-7ND gene transfer with AAV9 vectors for protection from murine autoimmune myocarditis

Kaya, Z., Leib, C., Werfel, S., Göser, S., Öttl, R., Leuchs, B., Pfitzer, G., Katus, H.A. and Müller, O.J. Cardivasc. Res., 91, 116-123 (2011)   Aims Overexpression of therapeutic genes with potential disease-limiting effects, specifically at the site of inflammation, remains a major clinical challenge. In this study, we investigate the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of the anti-inflammatory mediators interleukin (IL)-10 and a dominant-negative inhibitor of monocyte chemoattractant protein-1 (MCP1-7ND) on prevention of autoimmune myocarditis. Methods and results Autoimmune myocarditis was induced by immunizing A/J mice with subcutaneous injection of 120 µg cardiac Troponin I (cTnI) on Days 0, 7, and 14. Two weeks prior to initial immunization, each mouse received a single systemic dose of 10¹² AAV9 vectors carrying the coding sequence of IL-10 or MCP1-7ND transcriptionally targeted to the heart. Mice were sacrificed 28 days after initial immunization for further analysis. Only expression of IL-10 resulted in a highly significant decrease in myocardial inflammation and fibrosis, as well as an increased ejection fraction compared with controls. Further analyses of cytokine profiles of cTnI-stimulated splenocytes from IL-10 and MCP1-7ND-treated mice revealed significant alterations compared with controls. In addition, transcript levels of chemokine receptor CCR4 and T-cell activation gene were significantly reduced in hearts of IL-10-treated mice as determined by quantitative real-time PCR. Conclusion Our study suggests that cardiac expression of IL-10 with AAV9 vectors is a promising therapeutic approach for autoimmune myocarditis.  

5.905           Large-scale recombinant adeno-associated virus production

Kotin, R Hum. Mol. Genet., 20(Rev. Issue 1), R2-R6 (2011)   Since recombinant adeno-associated virus (rAAV) was first described as a potential mammalian cell transducing system, frequent reports purportedly solving the problems of scalable production have appeared. Yet few of these processes have enabled the development of robust and economical rAAV production. Two production platforms have emerged that have gained broad support for producing both research and clinical grade vectors. These processes differ fundamentally in several aspects. One approach is based on adherent mammalian cells and uses optimized chemical transient transfection for introducing the essential genetic components into the cells. The other approach utilizes suspension cultures of invertebrate cells. Baculovirus expression vectors are used for introducing the AAV genes into the cells. In addition, the baculovirus provides the helper functions necessary for efficient AAV DNA replication. The use of suspension cell culture provides an intrinsically more scalable platform system than using adherent cells. The upstream processes for suspension cultures are amenable for automation and are easily monitored and regulated to maintain optimum conditions that produce consistent yields of rAAV. Issues relating to developing new and improving existing rAAV production methods are discussed.  

5.906           The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9–dependent innate immune responses in the liver

Martino, A.T., Suzuki, M., Markusic, D.M., Zolotukhin, I., Ryals, R.C., Moghimi, B., Ertl, H.C.J., Muruve, D.A., Lee, B. and Herzog, R.W. Blood, 117, 6459-6468 (2011)   Although adeno-associated viral (AAV) vectors have been successfully used in hepatic gene transfer for treatment of hemophilia and other diseases in animals, adaptive immune responses blocked long-term transgene expression in patients on administration of single-stranded AAV serotype-2 vector. More efficient vectors have been developed using alternate capsids and self-complimentary (sc) genomes. This study investigated their effects on the innate immune profile on hepatic gene transfer to mice. A mild and transient up-regulation of myeloid differentiation primary response gene (88), TLR9, TNF-α, monocyte chemotactic protein-1, IFN-γ inducible protein-10, and IFN-α/β expression in the liver was found after single-stranded AAV vector administration, regardless of the capsid sequence. In contrast, scAAV vectors induced higher increases of these transcripts, upregulated additional proinflammatory genes, and increased circulating IL-6. Neutrophil, macrophage, and natural killer cell liver infiltrates were substantially higher on injection of scAAV. Some but not all of these responses were Kupffer cell dependent. Independent of the capsid or expression cassette, scAAV vectors induced dose-dependent innate responses by signaling through TLR9. Increased innate responses to scAAV correlated with stronger adaptive immune responses against capsid (but not against the transgene product). However, these could be blunted by transient inhibition of TLR9.  

5.907           Cytotoxic CD4+ T Cell Responses to EBV Contrast with CD8 Responses in Breadth of Lytic Cycle Antigen Choice and in Lytic Cycle Recognition

Long, H.M., Leese, A.M., Chagoury, O.L., Connerty, S.R., Quarcoopome, J., Quinn, L., Shannon-Lowe, C. and Rickinson, A.B.

1. Immunol., 187, 92-101 (2011)

  EBV, a B lymphotropic herpesvirus, encodes two immediate early (IE)-, >30 early (E)-, and >30 late (L)-phase proteins during its replication (lytic) cycle. Despite this, lytic Ag-induced CD8 responses are strongly skewed toward IE and a few E proteins only, all expressed before HLA I presentation is blocked in lytically infected cells. For comparison, we examined CD4⁺ T cell responses to eight IE, E, or L proteins, screening 14 virus-immune donors to overlapping peptide pools in IFN-γ ELISPOT assays, and established CD4⁺ T cell clones against 12 defined epitopes for target-recognition assays. We found that the lytic Ag-specific CD4⁺ T cell response differs radically from its CD8 counterpart in that it is widely distributed across IE, E, and L Ag targets, often with multiple reactivities detectable per donor and with IE, E, or L epitope responses being numerically dominant, and that all CD4⁺ T cell clones, whether IE, E, or L epitope-specific, show strong recognition of EBV-transformed B cell lines, despite the lines containing only a small fraction of lytically infected cells. Efficient recognition occurs because lytic Ags are released into the culture and are acquired and processed by neighboring latently infected cells. These findings suggested that lytic Ag-specific CD4 responses are driven by a different route of Ag display than drives CD8 responses and that such CD4 effectors could be therapeutically useful against EBV-driven lymphoproliferative disease lesions, which contain similarly small fractions of EBV-transformed cells entering the lytic cycle.  

5.908           Efficient and stable transduction of dopaminergic neurons in rat substantia nigra by rAAV 2/1, 2/2, 2/5, 2/6.2, 2/7, 2/8 and 2/9

Deer Perren, A., Toelen, J., Carlon, M., Van den Haute, C., Coun, F., Heeman, B., Reumers, V., Vandenberghe, L.H., Wilson, J.M., Debyser, Z. and Baeklandt, V. Gene Therapy, 18, 517-527 (2011)   Dysfunction of the nigrostriatal system is the major cause of Parkinson's disease (PD). This brain region is therefore an important target for gene delivery aiming at disease modeling and gene therapy. Recombinant adeno-associated viral (rAAV) vectors have been developed as efficient vehicles for gene transfer into the central nervous system. Recently, several serotypes have been described, with varying tropism for brain transduction. In light of the further development of a viral vector-mediated rat model for PD, we performed a comprehensive comparison of the transduction and tropism for dopaminergic neurons (DNs) in the adult Wistar rat substantia nigra (SN) of seven rAAV vector serotypes (rAAV 2/1, 2/2, 2/5, 2/6.2, 2/7, 2/8 and 2/9). All vectors were normalized by titer and volume, and stereotactically injected into the SN. Gene expression was assessed non-invasively and quantitatively in vivo by bioluminescence imaging at 2 and 5 weeks after injection, and was found to be stable over time. Immunohistochemistry at 6 weeks following injection revealed the most widespread enhanced green fluorescence protein expression and the highest number of positive nigral cells using rAAV 2/7, 2/9 and 2/1. The area transduced by rAAV 2/8 was smaller, but nevertheless almost equal numbers of nigral cells were targeted. Detailed confocal analysis revealed that serotype 2/7, 2/9, 2/1 and 2/8 transduced at least 70% of the DNs. In conclusion, these results show that various rAAV serotypes efficiently transduce nigral DNs, but significant differences in transgene expression pattern and level were observed.  

5.909           Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear

Kilpatrick, L.A., Li, Q., Yang, J., Goddard, J.C., Fekete, D.M. and Lang, H. Gene Therapy, 18, 569-578 (2011)   Murine models are ideal for studying cochlear gene transfer, as many hearing loss-related mutations have been discovered and mapped within the mouse genome. However, because of the small size and delicate nature, the membranous labyrinth of the mouse is a challenging target for the delivery of viral vectors. To minimize injection trauma, we developed a procedure for the controlled release of adeno-associated viruses (AAVs) into the scala media of adult mice. This procedure poses minimal risk of injury to structures of the cochlea and middle ear, and allows for near-complete preservation of low and middle frequency hearing. In this study, transduction efficiency and cellular specificity of AAV vectors (serotypes 1, 2, 5, 6 and 8) were investigated in normal and drug-deafened ears. Using the cytomegalovirus promoter to drive gene expression, a variety of cell types were transduced successfully, including sensory hair cells and supporting cells, as well as cells in the auditory nerve and spiral ligament. Among all five serotypes, inner hair cells were the most effectively transduced cochlear cell type. All five serotypes of AAV vectors transduced cells of the auditory nerve, though serotype 8 was the most efficient vector for transduction. Our findings indicate that efficient AAV inoculation (via the scala media) can be performed in adult mouse ears, with hearing preservation a realistic goal. The procedure we describe may also have applications for intra-endolymphatic drug delivery in many mouse models of human deafness.  

5.910           Phenotypic Correction of a Mouse Model for Primary Hyperoxaluria With Adeno-associated Virus Gene Transfer

Salido, E., Rodriguez-Pena, M., Santana, A., Beattie, S.G., Petry, H. and Torres, A. Molecular Therapy, 19(5), 870-875 (2011)   Primary hyperoxaluria type I (PH1) is an inborn error of metabolism caused by deficiency of the hepatic enzyme alanine-glyoxylate aminotransferase (AGXT or AGT) which leads to overproduction of oxalate by the liver and subsequent urolithiasis and renal failure. The current therapy largely depends on liver transplantation, which is associated with significant morbidity and mortality. To explore an alternative treatment, we used somatic gene transfer in a mouse genetic model for PH1 (Agxt1KO). Recombinant adeno-associated virus (AAV) vectors containing the human AGXT complementary DNA (cDNA) were pseudotyped with capsids from either serotype 8 or 5, and delivered to the livers of Agxt1KO mice via the tail vein. Both AAV8-AGXT and AAV5-AGXT vectors were able to reduce oxaluria to normal levels. In addition, treated mice showed blunted increase of oxaluria after challenge with ethylene glycol (EG), a glyoxylate precursor. In mice, AGT enzyme activity in whole liver extracts were restored to normal without hepatic toxicity nor immunogenicity for the 50 day follow-up. In summary, this study demonstrates the correction of primary hyperoxaluria in mice treated with either AAV5 or AAV8 vectors.  

5.911           Modification of the Abbott RealTime assay for detection of HIV-1 plasma RNA viral loads less than one copy per milliliter

Yukl, S.A., Li, P., Fujimoto, K., Lampiris, H., Lu, C.M., Hare, C.B., Deeks, S.G., Liegler, T., Pandori, M., Havlir, D.V. and Wong, J.K.

1. Virol. Methods, 175, 261-265 (2011)

  Although commercial tests are approved for detection of HIV-1 plasma viral loads ≥20 copies per milliliter (ml), only one specialized research assay has been reported to detect plasma viral loads as low as 1 copy/ml. This manuscript describes a method of concentrating HIV-1 virions from up to 30 ml of plasma, which can be combined with a commercial viral load test to create a widely available, reproducible assay for quantifying plasma HIV RNA levels less than 1 copy/ml. Using this pre-analytically modified assay, samples with a known level of 0.5 copy/ml were detected in 8 of 12 replicates (mean 0.47 copy/ml; 95% confidence interval (CI) 0.14–0.81 copy/ml) and samples with a known level of 1.0 copy/ml were detected in 13 of 13 replicates (mean 1.96 copy/ml; 95% CI 1.42–2.50 copy/ml). By concentrating virus from 30 ml of plasma, HIV RNA could be measured in 16 of 19 samples (84%) from 12 of 12 subjects (mean 2.77 copy/ml; 95% CI 0.86–4.68 copy/ml). The measured viral load correlated inversely (r = −0.78; p = 0.028) with the total duration of viral suppression (viral load < 40 copies/ml).  

5.912           Increased Expression of Wild-Type or a Centronuclear Myopathy Mutant of Dynamin 2 in Skeletal Muscle of Adult Mice Leads to Structural Defects and Muscle Weakness

Crowling, B.S., Toussaaint, A., Amoasii, L., Koebel, P., Ferry, A., Davignon, L., Nishino, I., Mandel, J-L. and Laporte, J. Am. J. Pathol., 178(5), 2224-2235 (2011)   Dynamin 2 (DNM2) is a large GTPase implicated in many cellular functions, including cytoskeleton regulation and endocytosis. Although ubiquitously expressed, DNM2 was found mutated in two genetic disorders affecting different tissues: autosomal dominant centronuclear myopathy (ADCNM; skeletal muscle) and peripheral Charcot-Marie-Tooth neuropathy (peripheral nerve). To gain insight into the function of DNM2 in skeletal muscle and the pathological mechanisms leading to ADCNM, we introduced wild-type DNM2 (WT-DNM2) or R465W DNM2 (RW-DNM2), the most common ADCNM mutation, into adult wild-type mouse skeletal muscle by intramuscular adeno-associated virus injections. We detected altered localization of RW-DNM2 in mouse muscle. Several ADCNM features were present in RW-DNM2 mice: fiber atrophy, nuclear mislocalization, and altered mitochondrial staining, with a corresponding reduction in specific maximal muscle force. The sarcomere and triad structures were also altered. We report similar findings in muscle biopsy specimens from an ADCNM patient with the R465W mutation. In addition, expression of wild-type DNM2 induced some muscle defects, albeit to a lesser extent than RW-DNM2, suggesting that the R465W mutation has enhanced activity in vivo. In conclusion, we show the RW-DNM2 mutation acts in a dominant manner to cause ADCNM in adult muscle, and the disease arises from a primary defect in skeletal muscle rather than secondary to peripheral nerve involvement. Therefore, DNM2 plays important roles in the maintenance of adult muscle fibers.  

5.913           Long-term in vivo resistin overexpression induces myocardial dysfunction and remodeling in rats

Chemaly, E.R., Hadri, L., Zhang, AS., Kim, M., Kohlbrenner, E., Sheng, J., Liang, L., Chen, J., K-Raman, P., Hajjar, R.J. and Lebeche, D.

1. Mol. Cell. Cardiol., 51, 144-155 (2011)

  We have previously reported that resistin induces hypertrophy and impairs contractility in isolated rat    cardiomyocytes. To examine the long-term cardiovascular effects of resistin, we induced in vivo  overexpression of resistin using adeno-associated virus serotype 9 injected by tail vein in rats and compared to control animals. Ten weeks after viral injection, overexpression of resistin was associated with increased ratio of left ventricular (LV) weight/body weight, increased end-systolic LV volume and significant decrease in LV contractility, measured by the end-systolic pressure volume relationship slope in LV pressure volume loops, compared to controls. At the molecular level, mRNA expression of ANF and β-MHC, and protein levels of phospholamban were increased in the resistin group without a change in the level of SERCA2a protein expression. Increased fibrosis by histology, associated with increased mRNA levels of collagen, fibronectin and connective tissue growth factor were observed in the resistin-overexpressing hearts. Resistin overexpression was also associated with increased apoptosis in vivo, along with an apoptotic molecular phenotype in vivo and in vitro. Resistin-overexpressing LV tissue had higher levels of TNF-α receptor 1 and iNOS, and reduced levels of eNOS. Cardiomyocytes overexpressing resistin in vitro produced larger amounts of TNFα in the medium, had increased phosphorylation of IκBα and displayed increased intracellular reactive oxygen species (ROS) content with increased expression and activity of ROS-producing NADPH oxidases compared to controls. Long-term resistin overexpression is associated with a complex phenotype of oxidative stress, inflammation, fibrosis, apoptosis and myocardial remodeling and dysfunction in rats. This phenotype recapitulates key features of diabetic cardiomyopathy.  

5.914           Lipoprotein component associated with hepatitis C virus is essential for virus infectivity

Shimizu, Y., Hishiki, T., Ujino, S., Sugiuama, K., Funami, K. and Shimotohno, K. Current Opinion in Virology, 1, 19-26 (2011)   Many chronic hepatitis patients with hepatitis C virus (HCV) are observed to have a degree of steatosis which is a factor in the progression of liver diseases. Transgenic mice expressing HCV core protein develop liver steatosis before the onset of hepatocellular carcinoma, suggesting active involvement of HCV in the de-regulation of lipid metabolism in host cells. However, the role of lipid metabolism in HCV life cycle has not been fully understood until the establishment of in vitro HCV infection and replication system. In this review we focus on HCV production with regard to modification of lipid metabolism observed in an in vitro HCV infection and replication system. The importance of lipid droplet to HCV production has been recognized, possibly at the stage of virus assembly, although the precise mechanism of lipid droplet for virus production remains elusive. Association of lipoprotein with HCV in circulating blood in chronic hepatitis C patients is observed. In fact, HCV released from culture medium is also associated with lipoprotein. The fact that treatment of HCV fraction with lipoprotein lipase (LPL) abolished infectivity indicates the essential role of lipoprotein's association with virus particle in the virus life cycle. In particular, apolipoprotein E (ApoE), a component of lipoprotein associated with HCV plays a pivotal role in HCV infectivity by functioning as a virus ligand to lipoprotein receptor that also functions as HCV receptor. These results strongly suggest the direct involvement of lipid metabolism in the regulation of the HCV life cycle.      

5.915           Myocardial gene delivery using molecular cardiac surgery with recombinant adeno-associated virus vectors in vivo

White, J.D. et al Gene Therapy, 18, 546-552 (2011)   We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy.  

5.916           Schwann cell targeting via intrasciatic injection of AAV8 as gene therapy strategy for peripheral nerve regeneration

Homs, J., Ariza, L., Pages, G., Udina, E., Navarro, X., Chillon, M. and Bosch, A.

Gene Therapy, 18, 622-630 (2011)   Efficient transduction of the peripheral nervous system (PNS) is required for gene therapy of acquired and inherited neuropathies, neuromuscular diseases and for pain treatment. We have characterized the tropism and transduction efficiency of different adeno-associated vectors (AAV) pseudotypes after sciatic nerve injection in the mouse. Among the pseudotypes tested, AAV2/1 transduced both Schwann cells and neurons, AAV2/2 infected only sensory neurons and AAV2/8 preferentially transduced Schwann cells. AAV2/8 expression in the sciatic nerve was detected up to 10 weeks after administration, the latest time point analyzed. The injected mice developed neutralizing antibodies against all AAVs tested; the titers were higher against AAV2/1 than AAV2/2 and were the lowest for AAV2/8, correlating with a higher transgene expression overtime. AAV2/8 coding for ciliary neurotrophic factor (CNTF) led to an upregulation of P0 and PMP22 myelin proteins, four weeks after transduction of injured sciatic nerves. Importantly, CNTF-transduced mice showed a significant increase in both GAP43 expression in sensory neurons, a marker of axonal regeneration, and the compound muscle action potential. These results prove the utility of AAV8 as a gene therapy vector for Schwann cells to treat myelin disorders or to improve nerve regeneration.  

5.917           Reprogramming Virus Nanoparticles to Bind Metal Ions upon Activation with Heat

Musick, M.A., McConnell, K.I., Lue, J.K., Wei, F., Chen, C. and Suh, J. BioMacromolecules, 12, 2153-2158 (2011)   We have reprogrammed the stimulus-responsive conformational change property of a virus nanoparticle (VNP) to enable the surface exposure of metal binding motifs upon activation with heat. The VNP is based on the widely investigated adeno-associated virus (AAV). An intrinsic bioactive functionality of AAV was genetically replaced with a hexahistidine (His) tag. The peptide domain with the inserted His tag is normally inaccessible. Upon external stimulation with heat, the VNP undergoes a conformational change, resulting in externalization of His tag-containing domains and the conferred ability to bind metal. We show that beyond this newfound functionality of the capsid, the VNPs maintain many of the wild-type capsid properties. Our work lays the groundwork for developing stimulus-responsive VNPs that can be used as “smart” building blocks for the creation of higher order structures.    

5.918           Manufacturing of Adenovirus Vectors: Production and Purification of Helper Dependent Adenovirus

Dormond, E. and Kamen, A.A. Methods in Mol. Biol., 737, 139-156 (2011)   Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride secretion is critical to maintaining airway surface hydration and efficient mucociliary clearance in the upper airways. Mutations in CFTR in cystic fibrosis lead to reduced expression of functional CFTR channels at the apical plasma membrane of the airway epithelium, leading to dehydration of the airway surface liquid and diminished mucociliary clearance. Cell surface CFTR is modulated by changes in CFTR endocytosis and recycling, effectively altering the cell surface abundance of the channel. This chapter examines current methods employed to measure the cell surface expression of CFTR, as well as methods to monitor CFTR movement through the endocytic pathway.  

5.919           Herpes Simplex Virus Type 1-Derived Recombinant and Amplicon Vectors

Fraefel, C., Marconi, P. and Epstein, A.L. Methods in Mol. Biol., 737, 303-343 (2011)   Herpes simplex virus type 1 (HSV-1) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153 kbp double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes (1) the two approaches most commonly used to prepare recombinant vectors through homologous recombination, either in eukaryotic cells or in bacteria, and (2) the two methodologies currently used to generate helper-free amplicon vectors, either using a bacterial artificial chromosome (BAC)-based approach or a Cre/loxP site-specific recombination strategy.  

5.920           A simple and rapid method for isolation of caliciviruses from liver of infected rabbits

Teixeira, L., Marques, R.M., Aguas, A.P., and Ferreira, P.G. Res.Vet. Sci.,91, 164-166 (2011)   Rabbit Haemorrhagic Disease Virus (RHDV), a member of the Caliciviridae family, is the etiologic agent of Rabbit Haemorrhagic Disease (RHD); this viral disease is highly contagious and kills more than 90% of infected adult rabbits. Research on experimental calicivirus infection uses inocula obtained from livers of rabbits dying from calicivirus infection. This implies that caliciviruses have to be purified from liver homogenates. Current methods to isolate caliciviruses from rabbit livers are time consuming. We propose here a new procedure for fast purification of rabbit caliciviruses from liver homogenates that uses centrifugation through an iodixanol gradient. This method offers in approximately 2 h a sample with a high degree of calicivirus purity, as shown by its biochemical and immunocytochemistry analysis, which is also able to kill adult rabbits from RHD within 48 h of inoculation.  

5.921           Cardiac AAV9-S100A1 Gene Therapy Rescues Post-Ischemic Heart Failure in a Preclinical Large Animal Model

Pleger, S.T. et al Science Translational Medicine, 3(92), 92ra64 (2011)   As a prerequisite for clinical application, we determined the long-term therapeutic effectiveness and safety of adeno-associated virus (AAV)–S100A1 gene therapy in a preclinical large animal model of heart failure. S100A1, a positive inotropic regulator of myocardial contractility, becomes depleted in failing cardiomyocytes in humans and animals, and myocardial-targeted S100A1 gene transfer rescues cardiac contractile function by restoring sarcoplasmic reticulum calcium (Ca²⁺) handling in acutely and chronically failing hearts in small animal models. We induced heart failure in domestic pigs by balloon occlusion of the left circumflex coronary artery, resulting in myocardial infarction. After 2 weeks, when the pigs displayed significant left ventricular contractile dysfunction, we administered, by retrograde coronary venous delivery, AAV serotype 9 (AAV9)–S100A1 to the left ventricular, non-infarcted myocardium. AAV9-luciferase and saline treatment served as control. At 14 weeks, both control groups showed significantly decreased myocardial S100A1 protein expression along with progressive deterioration of cardiac performance and left ventricular remodeling. AAV9-S100A1 treatment prevented and reversed these functional and structural changes by restoring cardiac S100A1 protein levels. S100A1 treatment normalized cardiomyocyte Ca²⁺ cycling, sarcoplasmic reticulum calcium handling, and energy homeostasis. Transgene expression was restricted to cardiac tissue, and extracardiac organ function was uncompromised. This translational study shows the preclinical feasibility of long-term therapeutic effectiveness of and a favorable safety profile for cardiac AAV9-S100A1 gene therapy in a preclinical model of heart failure. Our results present a strong rationale for a clinical trial of S100A1 gene therapy for human heart failure that could potentially complement current strategies to treat end-stage heart failure.  

5.922           A Prime-Boost Strategy Using Virus-Like Particles Pseudotyped for HCV Proteins Triggers Broadly Neutralizing Antibodies in Macaques

Garrone, P. et al Science Translational Medicine, 3(94), 94ra71 (2011)   Chronic hepatitis C virus (HCV) infection, with its cohort of life-threatening complications, affects more than 200 million persons worldwide and has a prevalence of more than 10% in certain countries. Preventive and therapeutic vaccines against HCV are thus much needed. Neutralizing antibodies (NAbs) are the foundation for successful disease prevention for most established vaccines. However, for viruses that cause chronic infection such as HIV or HCV, induction of broad NAbs from recombinant vaccines has remained elusive. We developed a vaccine platform specifically aimed at inducing NAbs based on pseudotyped virus-like particles (VLPs) made with retroviral Gag. We report that VLPs pseudotyped with E2 and/or E1 HCV envelope glycoproteins induced high-titer anti-E2 and/or anti-E1 antibodies, as well as NAbs, in both mouse and macaque. The NAbs, which were raised against HCV 1a, cross-neutralized the five other genotypes tested (1b, 2a, 2b, 4, and 5). Thus, the described VLP platform, which can be pseudotyped with a vast array of virus envelope glycoproteins, represents a new approach to viral vaccine development.  

5.923           Glutathione depletion and overproduction both initiate degeneration of nigral dopaminergic neurons

Garrido, M., Tereshchenko, Y., Zhevtsova, Z., Taschenberger, G., Bähr, M. and Kügler, S. Acta Neuropathol., 121, 475-485 (2011)   Parkinson’s disease is a neurodegenerative disorder characterized by severe motor deficits mainly due to degeneration of dopaminergic neurons in the substantia nigra. Decreased levels of the cell’s most important antioxidant, glutathione, have been detected in nigral neurons of Parkinson patients, but it is unknown if they are the cause or merely the consequence of the disease. To elucidate if glutathione depletion causes selective degeneration of nigral dopaminergic neurons, we down-regulated glutathione synthesis in different brain areas of adult rats by a viral vector-based RNAi approach. Decreased glutathione synthesis resulted in progressive degeneration of nigral dopaminergic neurons, while extra-nigral and striatal neurons were significantly less vulnerable. Degeneration of dopaminergic neurons was accompanied by progressive protein aggregate formation and functional motor deficits and was partially rescued by a-synuclein. That the survival of nigral dopaminergic neurons depends on the precise control of glutathione levels was further demonstrated by significant degeneration induced through moderate overproduction of glutathione. Over-expression of either of the two subunits of glutamate–cysteine ligase induced aberrant glutathiolation of cellular proteins and significant degeneration of dopaminergic neurons. Thus, while glutathione depletion was demonstrated to be a selective trigger for dopaminergic neuron degeneration, a glutathione replacement approach as a potential treatment option for Parkinson’s patients must be considered with great care. In conclusion, our data demonstrate that survival of nigral dopaminergic neurons crucially depends on a tight regulation of their glutathione levels and that the depleted glutathione content detected in the brains of Parkinson’s disease patients can be a causative insult for neuronal degeneration.  

5.924           Differentiation-Dependent Interpentameric Disulfide Bond Stabilizes Native Human Papillomavirus Type 16

Conway, M.J., Cruz, L., Alam, S., Christensen, N.D. and Meyers, C. PloS One, 6(7), e22427 (2011)   Genetic and biochemical analyses of human papillomavirus type 16 (HPV16) capsids have shown that certain conserved L1 cysteine residues are critical for capsid assembly, integrity, and maturation. Since previous studies utilized HPV capsids produced in monolayer culture-based protein expression systems, the ascribed roles for these cysteine residues were not placed in the temporal context of the natural host environment for HPV, stratifying and differentiating human tissue. Here we extend upon previous observation, that HPV16 capsids mature and become stabilized over time (10-day to 20-day) in a naturally occurring tissue-spanning redox gradient, by identifying temporal roles for individual L1 cysteine residues. Specifically, the C175S substitution severely undermined wild-type titers of the virus within both 10 and 20-day tissue, while C428S, C185S, and C175,185S substitutions severely undermined wild-type titers only within 20-day tissue. All mutations led to 20-day virions that were less stable than wild-type and failed to form L1 multimers via nonreducing SDS-PAGE. Furthermore, Optiprep-fractionated 20-day C428S, C175S, and C175,185S capsids appeared permeable to endonucleases in comparison to wild-type and C185S capsids. Exposure to an oxidizing environment failed to enhance infectious titers of any of the cysteine mutants over time as with wild-type. Introduction of these cys mutants results in failure of the virus to mature.  

5.925           Cellular and Viral Factors Regulating Merkel Cell Polyomavirus Replication

Feng, H., Kwun, H.J., Liu, X., Gjoerup, O., Stolz, D.B., Chang, Y. and Moore, P.S. PloS One, 6(7), e22468 (2011)   Merkel cell polyomavirus (MCV), a previously unrecognized component of the human viral skin flora, was discovered as a mutated and clonally-integrated virus inserted into Merkel cell carcinoma (MCC) genomes. We reconstructed a replicating MCV clone (MCV-HF), and then mutated viral sites required for replication or interaction with cellular proteins to examine replication efficiency and viral gene expression. Three days after MCV-HF transfection into 293 cells, although replication is not robust, encapsidated viral DNA and protein can be readily isolated by density gradient centrifugation and typical ~40 nm diameter polyomavirus virions are identified by electron microscopy. The virus has an orderly gene expression cascade during replication in which large T (LT) and 57kT proteins are first expressed by day 2, followed by expression of small T (sT) and VP1 proteins. VP1 and sT proteins are not detected, and spliced 57kT is markedly diminished, in the replication-defective virus suggesting that early gene splicing and late gene transcription may be dependent on viral DNA replication. MCV replication and encapsidation is increased by overexpression of MCV sT, consistent with sT being a limiting factor during virus replication. Mutation of the MCV LT vacuolar sorting protein hVam6p (Vps39) binding site also enhances MCV replication while exogenous hVam6p overexpression reduces MCV virion production by >90%. Although MCV-HF generates encapsidated wild-type MCV virions, we did not find conditions for persistent transmission to recipient cell lines suggesting that MCV has a highly restricted tropism. These studies identify and highlight the role of polyomavirus DNA replication in viral gene expression and show that viral sT and cellular hVam6p are important factors regulating MCV replication. MCV-HF is a molecular clone that can be readily manipulated to investigate factors affecting MCV replication.  

5.926           Clathrin Facilitates the Morphogenesis of Retrovirus Particles

Zhang, F., Zang, T., Wilson, S.J., Johnson, M.C. and Bieniasz, P.D. PloS Pathogens, 7(6), e1002119 (2011)   The morphogenesis of retroviral particles is driven by Gag and GagPol proteins that provide the major structural component and enzymatic activities required for particle assembly and maturation. In addition, a number of cellular proteins are found in retrovirus particles; some of these are important for viral replication, but many lack a known functional role. One such protein is clathrin, which is assumed to be passively incorporated into virions due to its abundance at the plasma membrane. We found that clathrin is not only exceptionally abundant in highly purified HIV-1 particles but is recruited with high specificity. In particular, the HIV-1 Pol protein was absolutely required for clathrin incorporation and point mutations in reverse transcriptase or integrase domains of Pol could abolish incorporation. Clathrin was also specifically incorporated into other retrovirus particles, including members of the lentivirus (simian immunodeficiency virus, SIVmac), gammaretrovirus (murine leukemia virus, MLV) and betaretrovirus (Mason-Pfizer monkey virus, M-PMV) genera. However, unlike HIV-1, these other retroviruses recruited clathrin primarily using peptide motifs in their respective Gag proteins that mimicked motifs found in cellular clathrin adaptors. Perturbation of clathrin incorporation into these retroviruses, via mutagenesis of viral proteins, siRNA based clathrin depletion or adaptor protein (AP180) induced clathrin sequestration, had a range of effects on the accuracy of particle morphogenesis. These effects varied according to which retrovirus was examined, and included Gag and/or Pol protein destabilization, inhibition of particle assembly and reduction in virion infectivity. For each retrovirus examined, clathrin incorporation appeared to be important for optimal replication. These data indicate that a number of retroviruses employ clathrin to facilitate the accurate morphogenesis of infectious particles. We propose a model in which clathrin contributes to the spatial organization of Gag and Pol proteins, and thereby regulates proteolytic processing of virion components during particle assembly.  

5.927           Glycosaminoglycans and Sialylated Glycans Sequentially Facilitate Merkel Cell Polyomavirus Infectious Entry

Schowalter, R.M., pastrana, D.V. and Buck, C.B. PloS Pathogens, 7(7), e1002161 (2011)   Merkel cell polyomavirus (MCV or MCPyV) appears to be a causal factor in the development of Merkel cell carcinoma, a rare but highly lethal form of skin cancer. Although recent reports indicate that MCV virions are commonly shed from apparently healthy human skin, the precise cellular tropism of the virus in healthy subjects remains unclear. To begin to explore this question, we set out to identify the cellular receptors or co-receptors required for the infectious entry of MCV. Although several previously studied polyomavirus species have been shown to bind to cell surface sialic acid residues associated with glycolipids or glycoproteins, we found that sialylated glycans are not required for initial attachment of MCV virions to cultured human cell lines. Instead, glycosaminoglycans (GAGs), such as heparan sulfate (HS) and chondroitin sulfate (CS), serve as initial attachment receptors during the MCV infectious entry process. Using cell lines deficient in GAG biosynthesis, we found that N-sulfated and/or 6-O-sulfated forms of HS mediate infectious entry of MCV reporter vectors, while CS appears to be dispensable. Intriguingly, although cell lines deficient in sialylated glycans readily bind MCV capsids, the cells are highly resistant to MCV reporter vector-mediated gene transduction. This suggests that sialylated glycans play a post-attachment role in the infectious entry process. Results observed using MCV reporter vectors were confirmed using a novel system for infectious propagation of native MCV virions. Taken together, the findings suggest a model in which MCV infectious entry occurs via initial cell binding mediated primarily by HS, followed by secondary interactions with a sialylated entry co-factor. The study should facilitate the development of inhibitors of MCV infection and help shed light on the infectious entry pathways and cellular tropism of the virus.  

5.928           Enhanced Sialic Acid-Dependent Endocytosis Explains the Increased Efficiency of Infection of Airway Epithelia by a Novel Adeno-Associated Virus

Dickey, D.D., Excoffon, J.D.A., Koerber, J.T., Bergen, J., Steines, B., Klesney-Tait, J., Schaffer, D.V. and Zabner, J.

1. Virol., 85(17), 9023-9030 (2011)

  We previously used directed evolution in human airway epithelia to create adeno-associated virus 2.5T (AAV2.5T), a highly infectious chimera of AAV2 and AAV5 with one point mutation (A581T). We hypothesized that the mechanism for its increased infection may be a higher binding affinity to the surface of airway epithelia than its parent AAV5. Here, we show that, like AAV5, AAV2.5T, uses 2,3N-linked sialic acid as its primary receptor; however, AAV2.5T binds to the apical surface of human airway epithelia at higher levels and has more receptors than AAV5. Furthermore, its binding affinity is similar to that of AAV5. An alternative hypothesis is that AAV2.5T interaction with 2,3N-linked sialic acid may instead be required for cellular internalization. Consistent with this, AAV2.5T binds but fails to be internalized by CHO cells that lack surface expression of sialic acid. Moreover, whereas AAV2.5T binds similarly to human (rich in 2,3N-linked sialic acid) and pig airway epithelia (2,6N-linked sialic acid), significantly more virus was internalized by human airway. Subsequent transduction correlated with the level of internalized rather than surface-bound virus. We also found that human airway epithelia internalized significantly more AAV2.5T than AAV5. These data suggest that AAV2.5T has evolved to utilize specific 2,3N-linked sialic acid residues on the surface of airway epithelia that mediate rapid internalization and subsequent infection. Thus, sialic acid serves as not just an attachment factor but is also required for AAV2.5T internalization, possibly representing an important rate-limiting step for other viruses that use sialic acids.  

5.929           Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo

Mittmann, W., Wallace, D.J., Czubayko, U., Herb, J.T., Schaefer, A.T., Looger, L.L., Denk, W. and Kerr, J.N.D. Nature Neurosci., 14(8), 1089-1093 (2011)   Multiphoton imaging (MPI) is widely used for recording activity simultaneously from many neurons in superficial cortical layers in vivo. We combined regenerative amplification multiphoton microscopy (RAMM) with genetically encoded calcium indicators to extend MPI of neuronal population activity into layer 5 (L5) of adult mouse somatosensory cortex. We found that this approach could be used to record and quantify spontaneous and sensory-evoked activity in populations of L5 neuronal somata located as much as 800 μm below the pia. In addition, we found that RAMM could be used to simultaneously image activity from large (~80) populations of apical dendrites and follow these dendrites down to their somata of origin.  

5.930           Virally delivered Channelrhodopsin-2 Safely and Effectively Restores Visual Function in Multiple Mouse Models of Blindness

Doroudchi, M.M. et al Molecular Therapy, 19(7), 1220-1229 (2011)   Previous work established retinal expression of channelrhodopsin-2 (ChR2), an algal cation channel gated by light, restored physiological and behavioral visual responses in otherwise blind rd1 mice. However, a viable ChR2-based human therapy must meet several key criteria: (i) ChR2 expression must be targeted, robust, and long-term, (ii) ChR2 must provide long-term and continuous therapeutic efficacy, and (iii) both viral vector delivery and ChR2 expression must be safe. Here, we demonstrate the development of a clinically relevant therapy for late stage retinal degeneration using ChR2. We achieved specific and stable expression of ChR2 in ON bipolar cells using a recombinant adeno-associated viral vector (rAAV) packaged in a tyrosine-mutated capsid. Targeted expression led to ChR2-driven electrophysiological ON responses in postsynaptic retinal ganglion cells and significant improvement in visually guided behavior for multiple models of blindness up to 10 months postinjection. Light levels to elicit visually guided behavioral responses were within the physiological range of cone photoreceptors. Finally, chronic ChR2 expression was nontoxic, with transgene biodistribution limited to the eye. No measurable immune or inflammatory response was observed following intraocular vector administration. Together, these data indicate that virally delivered ChR2 can provide a viable and efficacious clinical therapy for photoreceptor disease-related blindness.  

5.931           Overview of Current Scalable Methods for Purification of Viral Vectors

Segura, M.M., Kamen, A.A. and Garnier, A. Methods in Mol. Biol., 737, 89-116 (2011)   As a result of the growing interest in the use of viruses for gene therapy and vaccines, many virus-based products are being developed. The manufacturing of viruses poses new challenges for process developers and regulating authorities that need to be addressed to ensure quality, efficacy, and safety of the final product. The design of suitable purification strategies will depend on a multitude of variables including the vector production system and the nature of the virus. In this chapter, we provide an overview of the most commonly used purification methods for viral gene therapy vectors. Current chromatography options available for large-scale purification of g-retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes simplex virus, baculovirus, and poxvirus vectors are presented.  

5.932           Arousal Effect of Caffeine Depends on Adenosine A2A Receptors in the Shell of the Nucleus Accumbens

Lazarus, M. et al

1. Neurosci., 31(27), 10067-10075 (2011)

  Caffeine, the most widely used psychoactive compound, is an adenosine receptor antagonist. It promotes wakefulness by blocking adenosine A_2A receptors (A_2ARs) in the brain, but the specific neurons on which caffeine acts to produce arousal have not been identified. Using selective gene deletion strategies based on the Cre/loxP technology in mice and focal RNA interference to silence the expression of A_2ARs in rats by local infection with adeno-associated virus carrying short-hairpin RNA, we report that the A_2ARs in the shell region of the nucleus accumbens (NAc) are responsible for the effect of caffeine on wakefulness. Caffeine-induced arousal was not affected in rats when A_2ARs were focally removed from the NAc core or other A_2AR-positive areas of the basal ganglia. Our observations suggest that caffeine promotes arousal by activating pathways that traditionally have been associated with motivational and motor responses in the brain.  

5.933           Parkin-Mediated Protection of Dopaminergic Neurons in a Chronic MPTP-Minipump Mouse Model of Parkinson Disease

Yasuda, T. et al

1. Neuropathol. Exp. Neurol., 70(8), 686-697 (2011)

  Loss-of-function mutations in the ubiquitin ligase parkin are the major cause of recessively inherited early-onset Parkinson disease (PD). Impairment of parkin activity caused by nitrosative or dopamine-related modifications may also be responsible for the loss of dopaminergic (DA) neurons in sporadic PD. Previous studies have shown that viral vector-mediated delivery of parkin prevented DA neurodegeneration in several animal models, but little is known about theneuroprotective actions of parkin in vivo. Here, we investigated mechanisms of neuroprotection of overexpressed parkin in a modifiedlong-term mouse model of PD using osmotic minipump administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Recombinant adeno-associated viral vector-mediated intranigral delivery of parkin prevented motor deficits and DA cell loss in the mice. Ser129-phosphorylated α-synuclein-immunoreactive cells were increased in the substantia nigra of parkin-treated mice. Moreover, delivery of parkin alleviated the MPTP-induced decrease of the active phosphorylated form of Akt. On the other hand, upregulation of p53 and mitochondrial alterations induced by chronic MPTP administration were barely suppressed by parkin. These results suggest that the neuroprotective actions of parkin may be impaired in severe PD.    

5.934           Effect of Pap Smear Collection and Carrageenan on Cervicovaginal Human Papillomavirus-16 Infection in a Rhesus Macaque Model

Roberts, J.N., Kines, R.C., Katki, H.A., Lowy, D.R. and Schiller, J.T.

1. Natl. Cancer Inst., 103(9), 737-743 (2011)

  Background Human papillomavirus (HPV) infection of the genital mucosa is thought to require trauma to the cervicovaginal epithelium. Therefore, we determined whether a cytology specimen collection procedure (Pap smear), which disrupts the epithelium by design, renders the cervix more susceptible to HPV infection in a primate model. Methods In a series of female rhesus macaques, a speculum examination was performed with (n = 8) or without (n = 4) a cytology specimen collection procedure as it is commonly practiced in a gynecology clinic. An internal digital examination was performed after specimen collection using Surgilube (n = 4) or 1% iota-carrageenan, a previously indentified HPV inhibitor (n = 4) as the lubricant. The cervix was then inoculated with HPV16 pseudovirions expressing red fluorescent protein. After 3 days, the reproductive tracts were excised and the cervix was cryosectioned. Sections were analyzed by fluorescent confocal microscopy for the number of red fluorescent protein–positive keratinocytes. Results Substantial infection of the ectocervix, the transformation zone, and the endocervix was detected, but only in conjunction with the cytology specimen collection procedure (cytology using Surgilube vs without cytology using Surgilube, mean = 84 infectious events per section vs mean = 0.05 infectious events per section, difference = 84 infectious events per section, 95% confidence interval = 19 to 384 infectious events per section). When the carrageenan gel was substituted for Surgilube for an internal digital examination, the mean number of infectious events decreased (carrageenan gel vs Surgilube, mean = 3.5 events per section vs mean = 84 infectious events per section difference = 81 events per section, 95% confidence interval = 33 to 213 events per section). Conclusions These findings indicate that cytology screening in women might lead to a transient enhancement of susceptibility to HPV infection and that use of a carrageenan-based gel during the examination might mitigate this enhancement.  

5.935           A single direct injection into the left ventricular wall of an adeno-associated virus 9 (AAV9) vector expressing extracellular superoxide dismutase from the cardiac troponin-T promoter protects mice against myocardial infarction

Prasad, K-M.R., Smith, R.S., Xu, Y. and French, B.A.

1. Gene Med., 13(6), 333-341 (2011)

  Background Localized administration of a highly efficient gene delivery system in combination with a cardiac-selective promoter may provide a favorable biosafety profile in clinical applications such as coronary artery bypass graft surgery, where regions of myocardium can be readily injected to protect them against the potential threat of future ischemic events. Methods Adeno-associated virus (AAV) vectors expressing luciferase or enhanced green fluorescent protein (eGFP) packaged into AAV serotypes 1, 2, 6, 8 and 9 were injected into the left ventricular (LV) wall of adult mice to determine the time course, magnitude and distribution of gene expression. An AAV9 vector expressing extracellular superoxide dismutase (EcSOD) from the cardiac troponin T (cTnT) promoter was then directly injected into the LV wall of adult mice. Myocardial infarction was induced 4 weeks after injection and infarct size was determined by triphenyltetrazolium chloride and phthalo blue staining. Results Serotypes AAV 9, 8, 1 and 6 provided early onset of gene expression in the heart with minimal extra-cardiac gene expression. AAV9 provided the highest magnitude of gene expression. Immunostaining for eGFP showed expression spanning the anterior to posterior walls from the mid ventricle to the apex. A single direct injection of the AAV9 vector bearing EcSOD (n = 5) decreased the mean infarct size by 50% compared to the eGFP control group (n = 8) (44 ± 7% versus 22 ± 5%; p = 0.04). Conclusions AAV serotype 9 is highly efficient for cardiac gene delivery, as evidenced by early onset and high-level gene expression. AAV9-mediated, cardiac selective overexpression of EcSOD from the cTnT promoter significantly reduced infarct size in mice.  

5.936           Sumoylation inhibits α-synuclein aggregation and toxicity

Krumova, P. et al

1. Cell Biol., 194(1), 49-60 (2011)

  Posttranslational modification of proteins by attachment of small ubiquitin-related modifier (SUMO) contributes to numerous cellular phenomena. Sumoylation sometimes creates and abolishes binding interfaces, but increasing evidence points to another role for sumoylation in promoting the solubility of aggregation-prone proteins. Using purified α-synuclein, an aggregation-prone protein implicated in Parkinson’s disease that was previously reported to be sumoylated upon overexpression, we compared the aggregation kinetics of unmodified and modified α-synuclein. Whereas unmodified α-synuclein formed fibrils, modified α-synuclein remained soluble. The presence of as little as 10% sumoylated α-synuclein was sufficient to delay aggregation significantly in vitro. We mapped SUMO acceptor sites in α-synuclein and showed that simultaneous mutation of lysines 96 and 102 to arginine significantly impaired α-synuclein sumoylation in vitro and in cells. Importantly, this double mutant showed increased propensity for aggregation and cytotoxicity in a cell-based assay and increased cytotoxicity in dopaminergic neurons of the substantia nigra in vivo. These findings strongly support the model that sumoylation promotes protein solubility and suggest that defects in sumoylation may contribute to aggregation-induced diseases.  

5.937           A synthetic prestin reveals protein domains and molecular operation of outer hair cell piezoelectricity

Schaechinger, T., Gorbunov, D., Halaszovich, C.R., Moser, T., Kügler, S., Fakler, B. and Oliver, D. EMBO J., 30(14), 2793-2804 (2011)   Prestin, a transporter-like protein of the SLC26A family, acts as a piezoelectric transducer that mediates the fast electromotility of outer hair cells required for cochlear amplification and auditory acuity in mammals. Non-mammalian prestin orthologues are anion transporters without piezoelectric activity. Here, we generated synthetic prestin (SynPres), a chimera of mammalian and non-mammalian prestin exhibiting both, piezoelectric properties and anion transport. SynPres delineates two distinct domains in the protein's transmembrane core that are necessary and sufficient for generating electromotility and associated non-linear charge movement (NLC). Functional analysis of SynPres showed that the amplitude of NLC and hence electromotility are determined by the transport of monovalent anions. Thus, prestin-mediated electromotility is a dual-step process: transport of anions by an alternate access cycle, followed by an anion-dependent transition generating electromotility. The findings define structural and functional determinants of prestin's piezoelectric activity and indicate that the electromechanical process evolved from the ancestral transport mechanism.    

5.938           Long-term propagation of serum hepatitis C virus (HCV) with production of enveloped HCV particles in human HepaRG hepatocytes

Ndongo-Thiam, N., Berthillon, P., Errazuriz, E., Bordes, I., De Sequeira, S., Trepo, C. and Petit, M-A. Hepatology, 54(2), 406-417 (2011)   HepaRG human liver progenitor cells exhibit morphology and functionality of adult hepatocytes. We investigated the susceptibility of HepaRG hepatocytes to in vitro infection with serum-derived hepatitis C virus (HCV) particles (HCVsp) and the potential neutralizing activity of the E1E2-specific monoclonal antibody (mAb) D32.10. The infection was performed using HCVsp when the cells actively divided at day 3 postplating. HCV RNA, E1E2, and core antigens were quantified in HCV particles recovered from culture supernatants of differentiated cells for up to 66 days. The density distributions of particles were analyzed on iodixanol or sucrose gradients. Electron microscopy (EM) and immune-EM studies were performed for ultrastructural analysis of cells and localization of HCV E1E2 proteins in thin sections. HCV infection of HepaRG cells was documented by increasing production of E1E2-core-RNA(+) HCV particles from day 21 to day 63. Infectious particles sedimented between 1.06 and 1.12 g/mL in iodixanol gradients. E1E2 and core antigens were expressed in 50% of HCV-infected cells at day 31. The D32.10 mAb strongly inhibited HCV RNA production in HepaRG culture supernatants. Infected HepaRG cells frozen at day 56 were reseeded at low density. After only 1-3 subcultures and induction of a cell differentiation process the HepaRG cells produced high titer HCV RNA and thus showed to be sustainably infected. Apolipoprotein B-associated empty E1E2 and complete HCV particles were secreted. Characteristic virus-induced intracellular membrane changes and E1E2 protein-association to vesicles were observed. Conclusion: HepaRG progenitor cells permit HCVsp infection. Differentiated HepaRG cells support long-term production of infectious lipoprotein-associated enveloped HCV particles. The E1E2-specific D32.10 mAb neutralizes the infection and this cellular model could be used as a surrogate infection system for the screening of entry inhibitors.  

5.939           Significant Inhibition of Corneal Scarring In Vivo with Tissue-Selective, Targeted AAV5 Decorin Gene Therapy

Mohan, R.R., Tandon, A., Sharma, A., Cowden, J.W. and Tovey, J.C.K. Invest. Ophthalmol. Vis. Sci., 52(7), 4833-4841 (2011)   Purpose. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the stroma with adeno-associated virus serotype 5 (AAV5) inhibits corneal fibrosis in vivo without significant side effects. Methods. An in vivo rabbit model of corneal fibrosis was used. Targeted decorin gene therapy was delivered to the rabbit cornea by a single topical application of AAV5 (100 μL; 6.5 × 10¹² μg/mL) onto the bare stroma for 2 minutes. The levels of corneal fibrosis were determined with stereomicroscopy, slit lamp biomicroscopy, α-smooth muscle actin (αSMA), fibronectin, and F-actin immunocytochemistry, and/or immunoblotting. CD11b, F4/80 immunocytochemistry, and TUNEL assay were used to examine immunogenicity and cytotoxicity of AAV5 to the cornea. Transmission electron microscopy (TEM) was used to investigate ultrastructural features. Slot-blot–quantified the copy number of AAV5-delivered decorin genes. Results. Selective decorin delivery into the stroma showed a significant (P < 0.01) decrease in corneal haze (1.3 ± 0.3) compared with the no-decorin-delivered control rabbit corneas (3 ± 0.4) quantified using slit lamp biomicroscopy. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA, F-actin, and fibronectin proteins (59%–73%; P < 0.001 or <0.01) in decorin-delivered rabbit corneas compared with the no-decorin-delivered controls. The visual clinical eye examination, slit lamp clinical studies, TUNEL, CD11b, and F4/80 assays revealed that AAV5-mediated decorin gene therapy is nonimmunogenic and nontoxic for the cornea. TEM studies suggested that decorin gene delivery does not jeopardize collagen fibrillogenesis as no significant differences in collagen fibril diameter and arrangement were observed in decorin-delivered and no-decorin-delivered control corneas. Conclusions. Tissue-targeted AAV5-mediated decorin gene therapy is effective and safe for treating corneal fibrosis in vivo.  

5.940           Cell selective targeting of a simian virus 40 virus-like particle conjugated to epidermal growth factor

Kitai, Y., Fukuda, H., Enomoto, T., Asakawa, Y., Suzuki, T., Inouye, S. and Handa, H.

1. Biotechnol., 155, 251-256 (2011)

  Simian virus 40 (SV40) virus-like particles (VLPs) are efficient nanocarriers for gene delivery. VLPs conjugated to human epidermal growth factor (hEGF) were prepared and the cell selectivity of the VLP was examined using human epithelial carcinoma A431 cells, which overexpress the EGF receptor. The endocytic efficiency was determined by the level of Gaussia luciferase activity from the encapsulated protein in hEGF-conjugated VLPs. EGF receptor-mediated endocytosis of hEGF-conjugated VLPs was significantly increased and was confirmed by fluorescence imaging using mCherry encapsulated in hEGF-conjugated VLPs. These results suggest that VLPs of SV40 conjugated to a specific ligand could be used for cell selective gene delivery.  

5.941           DNA vaccines delivered by human papillomavirus pseudovirions as a promising approach for generating antigen-specific CD8+ T cell immunity

Peng, S., Ma, B., Chen, S-H., Hung, C-F. and Wu, T.C. Cell & Bioscience, 1, 26-36 (2011)   Background Human papillomavirus (HPV) pseudovirions have recently been shown to deliver DNA efficiently in vivo, resulting in the priming of antigen-specific CD8+ T cells in vaccinated mice. In the current study, we compare the different preparation methods for the generation of HPV pseudovirions for their ability to efficiently infect cells. We also compare the antigen-specific CD8+ T cell immune responses generated by different DNA delivery methods and several commonly used forms of vaccination with that of HPV pseudovirions. Results We found that the preparation method of pseudovirions is important for the efficient delivery of encapsidated DNA. We have shown that vaccination with DNA encoding model antigen ovalbumin (OVA) delivered by HPV-16 pseudovirions was capable of generating therapeutic antitumor effects against OVA-expressing tumor. In addition, vaccination with DNA encoding OVA delivered by HPV-16 pseudovirions generated the highest number of OVA-specific CD8+ T cells in mice in our system compared to DNA delivered by other delivery methods. We also found that vaccination with OVA DNA delivered by HPV-16 pseudovirions generated the highest number of OVA-specific CD8+ T cells in mice compared to other forms of antigen-specific vaccines. Furthermore, HPV-16 pseudovirions were capable of carrying DNA vaccine encoding clinically relevant antigen, telomerase reverse transcriptase, to generate antigen-specific CD8+ T cell immune responses. Conclusions Our data suggest that DNA vaccines delivered by HPV-16 pseudovirions may be advantageous compared to other delivery methods and other forms of antigen-specific vaccines for application to antigen-specific immunotherapy.  

5.942           Peptide Ligands Incorporated into the Threefold Spike Capsid Domain to Re-Direct Gene Transduction of AAV8 and AAV9 In Vivo

Michelfelder, S., Varadi, K., Raupp, C., Hunger, A., Köbelin, J., Pahrmann, C., Schrepfer, S., Müller, O.J., Kleinschmidt, J.A. and Trepel, M. PloS One, 6(8), e23101 (2011)   Efficiency and specificity of viral vectors are vital issues in gene therapy. Insertion of peptide ligands into the adeno-associated viral (AAV) capsid at receptor binding sites can re-target AAV2-derived vectors to alternative cell types. Also, the use of serotypes AAV8 and -9 is more efficient than AAV2 for gene transfer to certain tissues in vivo. Consequently, re-targeting of these serotypes by ligand insertion could be a promising approach but has not been explored so far. Here, we generated AAV8 and -9 vectors displaying peptides in the threefold spike capsid domain. These peptides had been selected from peptide libraries displayed on capsids of AAV serotype 2 to optimize systemic gene delivery to murine lung tissue and to breast cancer tissue in PymT transgenic mice (PymT). Such peptide insertions at position 590 of the AAV8 capsid and position 589 of the AAV9 capsid changed the transduction properties of both serotypes. However, both peptides inserted in AAV8 did not result in the same changes of tissue tropism as they did in AAV2. While the AAV2 peptides selected on murine lung tissue did not alter tropism of serotypes 8 and -9, insertion of the AAV2-derived peptide selected on breast cancer tissue augmented tumor gene delivery in both serotypes. Further, this peptide mediated a strong but unspecific in vivo gene transfer for AAV8 and abrogated transduction of various control tissues for AAV9. Our findings indicate that peptide insertion into defined sites of AAV8 and -9 capsids can change and improve their efficiency and specificity compared to their wild type variants and to AAV2, making these insertion sites attractive for the generation of novel targeted vectors in these serotypes.  

5.943           Regression of Glioma in Rat Models by Intranasal Application of Parvovirus H-1

Kiprianova, I., Thomas, N. and Ayache, A. et al Clin. Cancer Res., 17(16), 5333-5342 (2011)   Purpose: In previous studies, we have shown that the apathogenic rat parvovirus H-1 (H-1PV) is capable to induce regression of advanced symptomatic rat and human gliomas in a rat model, when the virus was injected in the tumor (intracranially) or intravenously. Infection with H-1PV did not provoke any pathology in nontumor tissue. This study addresses the question whether also intranasal application of this oncolytic virus is suitable and sufficient for treating gliomas in this animal model. Experimental Design: Rat (RG-2) or human (U87) glioma cells were grafted stereotactically in the brain of rats (Wistar or RNU, respectively), and after development of tumors visible by MRI, H-1PV was instilled intranasally. Tumor regression was monitored by MRI, and survival was analyzed by Kaplan–Meier analysis. Brains from sacrificed animals were analyzed for histologic alterations, presence of viral DNA and proteins and infectious virions. In addition, distribution of virus to other organs was determined. Results: A single intranasal instillation of H-1PV was sufficient to induce efficient regression of rat glioma, leading to significant prolongation of survival without any toxicity for other tissues. It is shown that the virus reaches brain and other tissues, and that the viral replication-associated (and oncolysis-associated) regulatory proteins are exclusively expressed in the tumor tissue. In rats with xenografts of human glioma, oncolytic activity of H-1PV was less pronounced, however, leading to significant prolongation of survival. Conclusion: In view of an ongoing clinical trial on the use of H-1PV for oncolytic virotherapy of glioma, the option of applying the virus intranasally may be a valuable alternative to invasive routes of infection.  

5.944           Mouse Hepatic Cells Support Assembly of Infectious Hepatitis C Virus Particles

Long, G., Hiet, M-S., Windisch, M.P., Lee, J-Y., Lohmann, V. and Bartenschlager, R. Gatroenterology, 141(3), 1057-1066 (2011)   Background & Aims Hepatitis C virus (HCV) has a high propensity to establish persistence; better understanding of this process requires the development of a fully permissive and immunocompetent small animal model. Mouse cells can be engineered to express the human orthologs of the entry molecules CD81 and occludin to allow entry of HCV. However, RNA replication is poor in mouse cells, and it is not clear whether they support assembly and release of infectious HCV particles. We used a trans-complementation-based system to demonstrate HCV assembly competence of mouse liver cell lines. Methods A panel of 3 mouse hepatoma cell lines that contain a stable subgenomic HCV replicon was used for ectopic expression of the HCV structural proteins, p7, nonstructural protein 2, and/or apolipoprotein E (apoE). Assembly and release of infectious HCV particles was determined by measuring viral RNA, proteins, and infectivity of virus released into the culture supernatant. Results Mouse replicon cells released low amounts of HCV particles, but ectopic expression of apoE increased release of infectious HCV to levels observed in the human hepatoma cell line Huh7.5. Thus, apoE is the limiting factor for assembly of HCV in mouse hepatoma cells but probably not in primary mouse hepatocytes. Products of all 3 human alleles of apoE and mouse apoE support HCV assembly with comparable efficiency. Mouse and human cell-derived HCV particles have similar biophysical properties, dependency on entry factors, and levels of association with apoE. Conclusions Mouse hepatic cells permit HCV assembly and might be developed to create an immunocompetent and fully permissive mouse model of HCV infection.  

5.945           Development of a Liver-specific Tet-On Inducible System for AAV Vectors and Its Application in the Treatment of Liver Cancer

Vanrell, L. et al Molecular Therapy, 19(7), 1245-1253 (2011)   Recombinant adeno-associated virus (rAAV) are effective gene delivery vehicles that can mediate long-lasting transgene expression. However, tight regulation and tissue-specific transgene expression is required for certain therapeutic applications. For regulatable expression from the liver we designed a hepatospecific bidirectional and autoregulatory tetracycline (Tet)-On system (Tet_bidirAlb) flanked by AAV inverted terminal repeats (ITRs). We characterized the inducible hepatospecific system in comparison with an inducible ubiquitous expression system (Tet_bidirCMV) using luciferase (luc). Although the ubiquitous system led to luc expression throughout the mouse, luc expression derived from the hepatospecific system was restricted to the liver. Interestingly, the induction rate of the Tet_bidirAlb was significantly higher than that of Tet_bidirCMV, whereas leakage of Tet_bidirAlb was significantly lower. To evaluate the therapeutic potential of this vector, an AAV-Tet_bidir-Alb-expressing interleukin-12 (IL-12) was tested in a murine model for hepatic colorectal metastasis. The vector induced dose-dependent levels of IL-12 and interferon-γ (IFN-γ), showing no significant toxicity. AAV-Tet_bidir-Alb-IL-12 was highly efficient in preventing establishment of metastasis in the liver and induced an efficient T-cell memory response to tumor cells. Thus, we have demonstrated persistent, and inducible in vivo expression of a gene from a liver-specific Tet-On inducible construct delivered via an AAV vector and proved to be an efficient tool for treating liver cancer.  

5.946           Transduction of Human Adipose-Derived Mesenchymal Stem Cells by Recombinant Adeno-Associated Virus Vectors

Locke, M., Ussher, J.E., Mistry, R., Taylor, J.A. and Dunbar, P.R. Tissue Engineering; Part C, 17(9), 949-959 (2011)   Human adipose-derived stem cells (ASCs) are attractive targets for genetic manipulation and cellular therapies. However, current methods of gene transfer are limited by lack of efficiency, toxicity, or safety concerns. Recombinant adeno-associated virus (rAAV) has been extensively assessed as a gene therapy vector and has an excellent safety profile. This study reports the efficient transduction of well-characterized, homogeneous cultures of human ASCs by rAAV serotypes 2, 5, and 6. Transduction with rAAV2 at high multiplicity of infection was associated with reduced cell viability; however, no adverse effect was seen with serotypes 5 and 6. A further increase in transduction efficiency was observed using a rAAV6 Y731F tyrosine capsid mutant. rAAV-transduced ASCs retained their adipogenic potential. Therefore, rAAV serotypes 2, 5, and 6 should be considered the vectors of choice for genetic manipulation of ASCs.  

5.947           GDNF fails to exert neuroprotection in a rat α-synuclein model of Parkinson’s disease

Decressac, M., Ulusoy, A., Mattsson, B., Georievska, B., Romero-Ramos, M., Kirik, D. and Bjørklund, A. Brain, 134, 2302-2311 (2011)   The neuroprotective effect of the glial cell line-derived neurotrophic factor has been extensively studied in various toxic models of Parkinson’s disease. However, it remains unclear whether this neurotrophic factor can protect against the toxicity induced by the aggregation-prone protein α-synuclein. Targeted overexpression of human wild-type α-synuclein in the nigrostriatal system, using adeno-associated viral vectors, causes a progressive degeneration of the nigral dopamine neurons and the development of axonal pathology in the striatum. In the present study, we investigated, using different paradigms of delivery, whether glial cell line-derived neurotrophic factor can protect against the neurodegenerative changes and the cellular stress induced by α-synuclein. We found that viral vector-mediated delivery of glial cell line-derived neurotrophic factor into substantia nigra and/or striatum, administered 2–3 weeks before α-synuclein, was inefficient in preventing the wild-type α-synuclein-induced loss of dopamine neurons and terminals. In addition, glial cell line-derived neurotrophic factor overexpression did not ameliorate the behavioural deficit in this rat model of Parkinson’s disease. Quantification of striatal α-synuclein-positive aggregates revealed that glial cell line-derived neurotrophic factor had no effect on α-synuclein aggregation. These data provide the evidence for the lack of neuroprotective effect of glial cell line-derived neurotrophic factor against the toxicity of human wild-type α-synuclein in an in vivo model of Parkinson’s disease. The difference in neuroprotective efficacy of glial cell line-derived neurotrophic factor seen in our model and the commonly used neurotoxin models of Parkinson’s disease, raises important issues pertinent to the interpretation of the results obtained in preclinical models of Parkinson’s disease, and their relevance for the therapeutic use glial cell line-derived neurotrophic factor in patients with Parkinson’s disease.  

5.948           P44 Use of Intralipid infusion to analyse apolipoprotein B (apoB) and HCV RNA kinetics in chronic infection

Felmlee, D., Sheridan, D., Bridge, S., Packard, C., Caslake, M., Toms, G., Neely, D. and Bassendine, M. Gut, 60, A21 (2011)   Introduction Production of infectious HCV is dependent on hepatocytes VLDL assembly, maturation, and secretory machinery. ApoB-100 is the structural apolipoprotein of large VLDL (VLDL1), small VLDL2, and LDL. Although HCV production is dependent on VLDL, chronic HCV infection clinically manifests in lower VLDL and LDL levels, particularly in genotype 3. Aim To determine the production and clearance rates of apoB and triglyceride (TG) in VLDL1 in chronic HCV infected patients compared to uninfected volunteers. In addition, to observe if altering the VLDL1 kinetics would affect low-density HCV RNA quantities. Method VLDL1 kinetics were analysed using a protocol involving an IV infusion of a chylomicron-like lipid emulsion (Intralipid) for 120 min to prevent the clearance of VLDL1 by lipoprotein lipase.¹ Multiple blood samples were taken during and for 4 h after the infusion. Lipoprotein kinetics were examined by cumulative flotation ultracentrifugation and the clearance of HCV RNA from different density fractions was studied by iodixanol gradient ultracentrifugation.² Results VLDL1 TG production rate was lower for HCV patients [n=6] compared to healthy subjects1 [n=10], but the production rate of VLDL1 apoB was similar. Chronic HCV patients cleared Intralipid at a slower rate than uninfected controls. Plasma HCV RNA accumulated linearly in the serum during the 6 h experiment [14% increase per hour], indicating that Intralipid infusion either stimulated virion production, diminished virion clearance or both. Immediately after the Intralipid infusion ceased, triglyceride cleared exponentially, but at a slower rate than in uninfected individuals. HCV RNA in a very-low density fraction (VLDF, d<1.025 g/ml) also immediately cleared, but linearly, parallelling VLDL1 clearance (t1/2=77 min). However, HCV RNA in the high density fraction continued to accumulate [19.8% increase per hr] during the post-infusion period. Conclusion VLDL1 TG production and clearance rates are impaired in patients with chronic HCV infection. HCV associates with large TG-rich lipoproteins in vivo, and clears from the plasma via this route. However, competitive inhibition of lipoprotein clearance results in accumulation of HCV particles in the vascular compartment, particularly those that lack association with TG-rich lipoproteins. Intralipid effectively uncouples the interaction of higher density de novo produced particles, with VLDL. We hypothesise that HCV particles need to transfer onto very low density ‘acceptor’ particles to facilitate clearance via the remnant pathway.  

5.949           P50 Apolipoprotein E and low-density, apolipoprotein B associated lipoviral particles in chronic hepatitis C infection: evidence for genotype-specific modulation of lipid pathways

Bridge, S., Sheridan, D., Felmlee, D., Crossey, M., Thomas, H., Taylor-Robinson, S., Toms, G., Neely, D. and Bessendine,M. Gut, 60, A24 (2011)   Introduction Hepatitis C virus (HCV) co-opts the VLDL assembly, maturation, degradation, and secretory machinery of hepatocytes. Infectious low density particles have been termed lipoviral particles (LVP). LVPs in vivo are triglyceride (TG) rich and contain at least viral RNA, HCV core protein and the VLDL components apolipoprotein B (apoB) and apoE. ApoE is a constituent of infectious HCV particles produced in cell culture, and production of infectious particles is dramatically impaired from cells in which apoE expression has been genetically silenced. Aim To examine the relationship between LVP and apoE in vivo. Method Fasting plasma samples were obtained from 39 chronic HCV genotype (G) three patients and 51 HCV G1 patients. LVP were measured using iodixanol density gradient ultracentrifugation as recently described.¹ ApoE levels were determined by an automated immunonephlometric method. Demographic data were recorded and liver biochemical tests, lipid profiles and HOMA-IR were measured in all patients. Results The mean LVP in HCV G3 was 5.2 log10 IU/ml with a mean LVP ratio of 0.286, but showed wide variation (0.03–0.96). This was not significantly different to LVP variation in HCV G1 we have previously reported.¹ In HCV G1 we found a strong positive correlation of LVP HCV RNA with apoE levels (r=0.488, p=0.001) and also with LVP ratio (r=0.428, p=0.001). In contrast in HCV G3 we found a significant negative correlation of LVP HCV RNA with apoE levels (r=−0.428, p=0.013), suggesting different utilisation of lipoprotein pathways. We also found a negative correlation of LVP in HCV G3 with HDL cholesterol (r= −0.468, p=0.003) and its structural lipoprotein apoA1 (r= −0.479, p=0.002) whereas we have reported no correlation of LVP in cHCVG1 with HDL or apoA1.¹ Furthermore in HCV G3 we found low TG levels compared to G1 (1.00±0.71 vs 1.35±0.76) and no correlation of LVP with TG or HOMA-IR, again contrasting to G1 infection¹. Conclusion This study suggests that while serum apoE quantity is a positive determinant for LVP quantity in HCV G1 infection, it is a negative determinant in HCV G3 infection. Furthermore, LVP quantity in HCV G3 is largely based on the paucity of HDL quantity and their components, rather than the parameters associated with TRL levels as in HCV G1. These differences highlight that interaction with host lipoprotein metabolism is important for HCV infection in different genotypes, but in genotype specific ways.  

5.950           Methods for assessing feline immunodeficiency virus infection, infectivity and purification

Ammersbach, M. and Bienzle, D. Vet. Immunol. Immunopathol., 143, 202-214 82011)   Infection of cats with the feline immunodeficiency virus (FIV) recapitulates many aspects of infection of humans with HIV, including highly activated but ineffectual immune responses. Infected hosts remain seropositive for life, and detection of antibodies is the mainstay of diagnosis. However, to quantify virus for research or prognosis, viral proteins, nucleic acids or enzymes, are typically measured by ELISA, PCR or activity, respectively. While such assays are in wide use, they do not distinguish whole, infectious viral particles from defective or disrupted viruses. Titers of infectious viral particles may be estimated from tissue culture infectious doses or by enumerating cell-associated viral proteins, viral transcriptional activity or formation of syncytia. To analyze the viral proteome and the incorporation of host components into viral envelopes, pure lentiviral preparations are required. Methods for purifying lentiviruses include ultracentrifugation to separate particles by size, mass and/or density; chromatography to separate particles by charge, affinity or size; and additional removal of extraviral proteins and exosomes through subtilisin digestion or immunoaffinity. This article reviews advantages and disadvantages of different approaches to purification of lentiviruses with special reference to suitability for FIV, and highlights effects of purification on immune responses and immune assays.  

5.951           Adeno-associated viral vector-mediated gene transduction in mesencephalic slice culture

Nihira, T., Yasuda, T., Hirai, Y., Shimada, T., Mizuno, Y. and Mochizuki, H.

1. Neurosci. Methods, 201, 55-60 (2011)

  Adeno-associated viral (AAV) vector is a non-pathogenic vehicle that is suitable for the delivery of foreign genes into non-dividing neuronal cells. This vector has been utilized for in vivo neurological research and in clinical trials of gene therapy for neurodegenerative disorders. Viral vector-mediated gene delivery has the limitation that progressive changes in cellular phenotype cannot be monitored in living animals. To visualize living neurons transduced with foreign genes in vitro, we used cultured mesencephalic tissue harboring living dopaminergic (DA) neurons and examined cellular tropism of serotype-1 and serotype-2 AAV vectors in a culture system. The viability of DA neurons was evaluated using transgenic mice carrying enhanced green fluorescent protein under the control of the rat tyrosine hydroxylase (TH) promoter, which enables the visualization of living DA cells in the substantia nigra. Apoptosis of a subset of neuronal cells was noted within one day of culture. After 7 days, the serotype-1 AAV vector had successfully delivered the foreign gene into neurons and astrocytes, and serotype-2 AAV vector was able to transduce TH-positive DA neurons efficiently. Our method should be useful for in vitro investigations of pathological changes in DA neurons following transduction with foreign genes.  

5.952           Adeno-associated virus serotype 9-mediated overexpression of extracellular superoxide dismutase improves recovery from surgical hind-limb ischemia in BALB/c mice

Saqib, A., Prasad, K-M.M., Katwal, A.B., Sanders, J.M., Lye, J., French, B.A. and Annex, B.H.

1. Vasc. Surg., 54, 810-818 (2011)

  Objective Neovascularization is a physiologic repair process that partly depends on nitric oxide. Extracellular superoxide dismutase (EcSOD) is the major scavenger of superoxide. It is an important regulator of nitric oxide bioavailability and thus protects against vascular dysfunction. We hypothesized that overexpression of EcSOD in skeletal muscle would improve recovery from hind-limb ischemia. Methods Adeno-associated virus serotype 9 (AAV9) vectors expressing EcSOD or luciferase (control) from the cytomegalovirus promoter were cross-packaged into AAV9 capsids and injected intramuscularly into the hind-limb muscles (1 × 10¹¹ viral genomes/limb) of 12-week-old mice. Ischemia was induced after intramuscular injections. Laser Doppler was used to measure limb perfusion on days 0, 7, and 14 after injection. Values were expressed as a ratio relative to the nonischemic limb. EcSOD expression was measured by Western blotting. Capillary density was documented by immunohistochemical staining for platelet endothelial cell adhesion molecule. Apoptosis was assessed by terminal deoxynucleotide transferase-mediated biotin-deoxy uridine triphosphate nick-end labeling and necrosis was visually evaluated daily. Results EcSOD expression was twofold upregulated in EcSOD treated vs control ischemic muscles at day 14. Capillary density (capillaries/fiber) was 1.9-fold higher in treated (1.65 ± 0.02) vs control muscle (0.78 ± 0.17, P < .05). Recovery of perfusion ratio at day 14 after ischemia was 1.5-fold greater in EcSOD vs control mice (P < .05). The percentage of apoptotic nuclei was 1.3% ± 0.4% in EcSOD-treated mice compared with 4.2% ± 0.2% in controls (P < .001). Limb necrosis was also significantly lower in EcSOD vs control mice. Conclusion AAV9-mediated overexpression of EcSOD in skeletal muscle significantly improves recovery from hind-limb ischemia in mice, consistent with improved capillary density and perfusion ratios in treated mice. Clinical Relevance Atherosclerosis remains a major cause of morbidity and mortality in the Western world. Extensive research has been conducted to attempt to harness the ability to facilitate the growth of blood vessels to limit the complications that follow an arterial occlusion. In no area of medicine is this needed greater than in peripheral arterial disease (PAD), where patients continue to experience high rates of poor wound healing and amputation. Gene therapy has been attempted in humans, with mixed results to date. Our study used a preclinical model of surgically induced hind-limb ischemia in a strain of mice that have a low rate of endogenous perfusion recovery from ischemia and a high risk of necrosis. We used an adeno-associated virus serotype 9 (AAV9) as the vector to deliver the gene expressing extracellular superoxide dismutase (EcSOD). Intramuscular EcSOD demonstrated a beneficial effect in this preclinical model. The AAV9 was able to induce high levels of gene expression without inducing detectable inflammation. This study may serve as a foundation for the future investigation of EcSOD, AAV9, or both, in PAD.  

5.953           AAV Mediated GDNF Secretion From Retinal Glia Slows Down Retinal Degeneration in a Rat Model of Retinitis Pigmentosa

Dalkara, D., Kolstad, K.D., Guerin, K.I., Hoffmann, N.V., Visel, M., Klimczak, R.R., Schaffer, D.V. and Flannery, J.G. Molecular Therapy, 19(9), 1602-1608 (2011)   Mutations in over 80 identified genes can induce apoptosis in photoreceptors, resulting in blindness with a prevalence of 1 in 3,000 individuals. This broad genetic heterogeneity of disease impacting a wide range of photoreceptor functions renders the design of gene-specific therapies for photoreceptor degeneration impractical and necessitates the development of mutation-independent treatments to slow photoreceptor cell death. One promising strategy for photoreceptor neuroprotection is neurotrophin secretion from Müller cells, the primary retinal glia. Müller glia are excellent targets for secreting neurotrophins as they span the entire tissue, ensheath all neuronal populations, are numerous, and persist through retinal degeneration. We previously engineered an adeno-associated virus (AAV) variant (ShH10) capable of efficient and selective glial cell transduction through intravitreal injection. ShH10-mediated glial-derived neurotrophic factor (GDNF) secretion from glia, generates high GDNF levels in treated retinas, leading to sustained functional rescue for over 5 months. This GDNF secretion from glia following intravitreal vector administration is a safe and effective means to slow the progression of retinal degeneration in a rat model of retinitis pigmentosa (RP) and shows significant promise as a gene therapy to treat human retinal degenerations. These findings also demonstrate for the first time that glia-mediated secretion of neurotrophins is a promising treatment that may be applicable to other neurodegenerative conditions.  

5.954           High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Ling, C., Lu, Y., Cheng, B., McGoogan, K.E., Gee, S.W.Y., Ma, W., Li, B., Aslanidi, G.V. and Srivastava, A.

1. Vis. Exp., 49, (2011), http://www.jove.com/details.php?id=2538

  Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models¹. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo. However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells^2,3. In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.  

5.955           Lentiviral gene transfer into the dorsal root ganglion of adult rats

Yu, H., Fischer, G., Jia, G., Reiser, J., Park, F. and Hogan, Q.H. Molecular Pain, 7, 63 (2011)   Background Lentivector-mediated gene delivery into the dorsal root ganglion (DRG) is a promising method for exploring pain pathophysiology and for genetic treatment of chronic neuropathic pain. In this study, a series of modified lentivector particles with different cellular promoters, envelope glycoproteins, and viral accessory proteins were generated to evaluate the requirements for efficient transduction into neuronal cells in vitro and adult rat DRG in vivo. Results In vitro, lentivectors expressing enhanced green fluorescent protein (EGFP) under control of the human elongation factor 1α (EF1α) promoter and pseudotyped with the conventional vesicular stomatitis virus G protein (VSV-G) envelope exhibited the best performance in the transfer of EGFP into an immortalized DRG sensory neuron cell line at low multiplicities of infection (MOIs), and into primary cultured DRG neurons at higher MOIs. In vivo, injection of either first or second-generation EF1α-EGFP lentivectors directly into adult rat DRGs led to transduction rates of 19 ± 9% and 20 ± 8% EGFP-positive DRG neurons, respectively, detected at 4 weeks post injection. Transduced cells included a full range of neuronal phenotypes, including myelinated neurons as well as both non-peptidergic and peptidergic nociceptive unmyelinated neurons. Conclusion VSV-G pseudotyped lentivectors containing the human elongation factor 1α (EF1α)-EGFP expression cassette demonstrated relatively efficient transduction to sensory neurons following direct injection into the DRG. These results clearly show the potential of lentivectors as a viable system for delivering target genes into DRGs to explore basic mechanisms of neuropathic pain, with the potential for future clinical use in treating chronic pain.  

5.956           Construction of Capsid-Modified Adenoviruses by Recombination in Yeast and Purification by Iodixanol-Gradient

Gimenez-Alejandre, M., Gros, A. and Alemany, R. Methods in Mol. Biol., 797, 21-34 (2011)   Adenovirus represents a valuable tool for the treatment of cancer, but tumor targeting remains a pending issue. Most common procedures to modify adenovirus genome are time-consuming due to the requirement of multiple cloning steps, and the low efficacy of the recombination process. Here, we present a new method for homologous recombination in yeast to fast construct recombinant adenoviruses. Also, an alternative procedure to purify viral stocks, based on iodixanol gradient is described. Compared to classical methods, iodixanol is nontoxic to cells, which avoids desalting to use in vitro and in vivo. Moreover, viral stocks are more viable and it can be used for large-scale purifications. Finally, a protocol for analyzing blood persistence of modified vector in in vivo biodistribution is presented.  

5.957           Propagation, Purification, and In Vivo Testing of Oncolytic Vesicular Stomatitis Virus Strains

Diallo, J-S., Vähä-Koskela, M., Le Boeuf, F. and Bell, J. Methods in Mol. Biol., 797, 127-140 (2011)   Oncolytic viruses are self-amplifying therapeutics that specifically replicate in and kill cancer cells. We have previously shown that vesicular stomatitis virus (VSV) can be used as an oncolytic virus. A strain of VSV harboring a mutation in the M protein (VSV􀀤51) was found to exhibit enhanced tumor selectivity over its wild-type counterpart due to its inability to overcome antiviral programs in normal cells and due to the frequent defects in antiviral signaling pathways observed in the majority of tumors. VSV􀀤51 can harbor transgenes, is easily propagated and purified to high titers, and shows potent oncolytic activity in several mouse models, including syngeneic CT26-lacZ subcutaneous colon carcinoma models. However, VSVneutralizing antibodies targeting mainly the VSV-G surface glycoprotein arise within 3–5 days following the initial dose. This should be considered for strategies aiming at increasing the effectiveness of VSV through delivery of additional doses of virus or aiming to prolong VSV replication in vivo.    

5.958           Recombinant Adeno-Associated Viral Vectors

De Backer, M.W.A., garner, K.M., Luijendijk, M.C.M. and Adan, R.A.H. Methods in Mol. Biol., 789, 357-376 (2011)   Recombinant adeno-associated viral (rAAV) vectors can be used to locally or systemically enhance or silence gene expression. They are relatively nonimmunogenic and can transduce dividing and nondividing cells, and different rAAV serotypes may transduce diverse cell types. Therefore, rAAV vectors are excellent tools to study the function of neuropeptides in local brain areas. In this chapter, we describe a protocol to produce high-titer, in vivo grade, rAAV vector stocks. The protocol includes an Iodixanol gradient, an anion exchange column and a desalting/concentration step and can be used for every serotype. In addition, a short protocol for rAAV injections into the brain and directions on how to detect and localize transduced cells are given.  

5.959           Expression of codon optimized major capsid protein (L1) of human papillomavirus type 16 and 18 in Pichia pastoris; purification and characterization of the virus-like particles

Rao, N.H., Babu, P.B., Rajendra, L., Sriraman, R., Pang, Y-Y.S., Schiller, J.T. and Srinivasan, V.A: Vaccine, 29, 7326-7334 (2011)   The major capsid protein (L1) of human papillomaviruses (HPV) expressed in heterologous systems assembles into virus-like particles (VLPs). We report cloning and expression of codon optimized HPV L1 genes of the two high-risk HPV types 16 and 18 in methylotropic yeast, Pichia pastoris. The VLPs produced in P. pastoris were subjected to three step purification method involving density gradient centrifugations and size exclusion chromatography. The enriched VLPs were characterized using conformation-specific monoclonal antibodies in ELISA and by transmission electron microscopy. Mice immunized with a bivalent HPV16 and HPV18 VLPs developed high serum antibody titers to both HPV types that persisted for 190 days post vaccination. Serum of mice immunized with the HPV-VLP preparations could neutralize homologous pseudoviruses in an in vitro assays. Our results demonstrate that the L1 proteins expressed in P. pastoris fold properly as evidenced by assembly into VLPs and induction of type-specific neutralizing antibody response in mice. This work constitutes a step towards developing an alternate production platform for generating an affordable HPV vaccine to meet the needs of developing countries.  

5.960           In vitro reconstitution of SV40 particles that are composed of VP1/2/3 capsid proteins and nucleosomal DNA and direct efficient gene transfer

Enomoto, T., Kukimoto, I., Kawano, M-a., Yamaguchi, Y., Berk, A.J. and Handa, H. Virology, 420, 1-9 (2011)   SV40 is comprised of the viral minichromosome and the capsid proteins VP1, VP2, and VP3. Complete reconstitution of SV40 virions in vitro remains a challenge. Here we describe in vitro reconstitution of SV40 particles that contain ~ 5-kb circular nucleosomal DNA with hyperacetylated histones and are encapsidated in a coat composed of VP1, VP2, and VP3, closely mimicking the characteristics of authentic SV40 virions. When inoculated into mammalian cells, VP1/2/3 particles containing nucleosomal DNA carrying a reporter gene yielded a significantly higher level of gene expression than VP1-only particles containing the corresponding naked DNA. The elevated gene expression resulted mainly from enhanced association of the particles with the cell surface and from facilitation of subsequent uptake into cells. Thus, the in vitro reconstitution system reported here should be useful for the elucidation of Polyomaviridae assembly mechanisms and for the development of novel carriers for gene delivery.  

5.961           A multimeric L2 vaccine for prevention of animal papillomavirus infections

Jagu, S., Malandro, N., Kwak, K., Yuan, H., Schlegel, R., palmer, K.E., Huh, W.K., Campo, M.S. and Roden, R.B.S. Virology, 420, 43-50 (2011)   It is unclear what level of neutralizing antibody is sufficient to protect cattle from experimental bovine papillomavirus type 4 (BPV4) challenge. Markedly lower, and often undetected, serum neutralizing antibody titers were associated with protection in cattle vaccinated with BPV4 L2 as compared to L1 VLP. We hypothesized that vaccination with concatemers of the N-terminal protective epitopes of L2 derived from multiple animal papillomavirus types would enhance the breadth and strength of immunity. Therefore we generated a multimeric L2 antigen derived from three bovine and three canine papillomavirus types with divergent phenotypes and purified it from bacteria. Mice vaccinated three times with this six type L2 vaccine formulated in alum or RIBI adjuvant generated robust serum neutralizing antibody titers against BPV1, BPV4 and canine oral papillomavirus (COPV). Furthermore, vaccination with this six type L2 vaccine formulated in adjuvant, like BPV1 L1 VLP, protected the mice from experimental challenge with BPV1 pseudovirus.  

5.962           Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

Nam, H-J., Gurda, B.L., McKenna, R., Potter, M., Byrne, B., Salganik, M., Muzyczka, N. and Agbandje-McKenna, M.

1. Virol., 85(22), 11791-11799 (2011)

  The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.  

5.963           Orthoretroviral-like prototype foamy virus gag-pol expression is compatible with viral replication

Swiersly, A., Wiek, C., Rweh, J., Zentgraf, H. and Lindemann, D. Retrovirology, 8, 66-79 (2011)   Background Foamy viruses (FVs) unlike orthoretroviruses express Pol as a separate precursor protein and not as a Gag-Pol fusion protein. A unique packaging strategy, involving recognition of briding viral RNA by both Pol precursor and Gag as well as potential Gag-Pol protein interactions, ensures Pol particle encapsidation. Results Several Prototype FV (PFV) Gag-Pol fusion protein constructs were generated to examine whether PFV replication is compatible with an orthoretroviral-like Pol expression. During their analysis, non-particle-associated secreted Pol precursor protein was discovered in extracellular wild type PFV particle preparations of different origin, copurifying in simple virion enrichment protocols. Different analysis methods suggest that extracellular wild type PFV particles contain predominantly mature p85^PR-RT and p40^IN Pol subunits. Characterization of various PFV Gag-Pol fusion constructs revealed that PFV Pol expression in an orthoretroviral manner is compatible with PFV replication as long as a proteolytic processing between Gag and Pol proteins is possible. PFV Gag-Pol translation by a HIV-1 like ribosomal frameshift signal resulted in production of replication-competent virions, although cell- and particle-associated Pol levels were reduced in comparison to wild type. In-frame fusion of PFV Gag and Pol ORFs led to increased cellular Pol levels, but particle incorporation was only marginally elevated. Unlike that reported for similar orthoretroviral constructs, a full-length in-frame PFV Gag-Pol fusion construct showed wildtype-like particle release and infectivity characteristics. In contrast, in-frame PFV Gag-Pol fusion with C-terminal Gag ORF truncations or non-removable Gag peptide addition to Pol displayed wildtype particle release, but reduced particle infectivity. PFV Gag-Pol precursor fusion proteins with inactivated protease were highly deficient in regular particle release, although coexpression of p71^Gag resulted in a significant copackaging of these proteins. Conclusions Non-particle associated PFV Pol appears to be naturally released from infected cells by a yet unknown mechanism. The absence of particle-associated Pol precursor suggests its rapid processing upon particle incorporation. Analysis of different PFV Gag-Pol fusion constructs demonstrates that orthoretroviral-like Pol expression is compatible with FV replication in principal as long as fusion protein processing is possible. Furthermore, unlike orthoretroviruses, PFV particle release and infectivity tolerate larger differences in relative cellular Gag/Pol levels.  

5.964           SUMO1-dependent modulation of SERCA2a in heart failure

Kho, C., Lee, A., Jeong, D., Oh, J.G., Chaanine, A.H., Kizana, E., Park, W.J. and Haijar, R.J. Nature, 477, 601-606 (2011)   The calcium-transporting ATPase ATP2A2, also known as SERCA2a, is a critical ATPase responsible for Ca²⁺ re-uptake during excitation–contraction coupling. Impaired Ca²⁺ uptake resulting from decreased expression and reduced activity of SERCA2a is a hallmark of heart failure¹. Accordingly, restoration of SERCA2a expression by gene transfer has proved to be effective in improving cardiac function in heart-failure patients², as well as in animal models³. The small ubiquitin-related modifier (SUMO) can be conjugated to lysine residues of target proteins⁴, and is involved in many cellular processes⁵. Here we show that SERCA2a is SUMOylated at lysines 480 and 585 and that this SUMOylation is essential for preserving SERCA2a ATPase activity and stability in mouse and human cells. The levels of SUMO1 and the SUMOylation of SERCA2a itself were greatly reduced in failing hearts. SUMO1 restitution by adeno-associated-virus-mediated gene delivery maintained the protein abundance of SERCA2a and markedly improved cardiac function in mice with heart failure. This effect was comparable to SERCA2A gene delivery. Moreover, SUMO1 overexpression in isolated cardiomyocytes augmented contractility and accelerated Ca²⁺ decay. Transgene-mediated SUMO1 overexpression rescued cardiac dysfunction induced by pressure overload concomitantly with increased SERCA2a function. By contrast, downregulation of SUMO1 using small hairpin RNA (shRNA) accelerated pressure-overload-induced deterioration of cardiac function and was accompanied by decreased SERCA2a function. However, knockdown of SERCA2a resulted in severe contractile dysfunction both in vitro and in vivo, which was not rescued by overexpression of SUMO1. Taken together, our data show that SUMOylation is a critical post-translational modification that regulates SERCA2a function, and provide a platform for the design of novel therapeutic strategies for heart failure.  

5.965           Age-Specific Seroprevalence of Merkel Cell Polyomavirus, BK Virus, and JC Virus

Viscidi, R.P., Rollison, D.E., Sondak, V.K., Silver, B., Messina, J.L., Giuliano, A.R., Fulp, W., Ajidahun, A. and Rivanera, D. Clin. Vacc. Immunol., 18(10), 1737-1743 (2011)   We produced capsids of Merkel cell polyomavirus (MCPyV) in a baculovirus expression system and developed a virus-like particle (VLP) enzyme-linked immunosorbent assay (ELISA). To determine age-specific seroprevalence, serum samples were collected from 947 individuals attending hospital outpatient clinics and ranging in age from 1 to 93 years. To evaluate the association between exposure to MCPyV and Merkel cell cancer (MCC), plasma samples were obtained from 33 MCC patients and 37 controls. MCPyV seroprevalence was 45% in children under 10 years of age, increased to 60% in the next decade of life, and peaked at 81% among those 60 to 69 years of age. Levels of MCPyV capsid antibodies were positively correlated with age (P = 0.007). Virus specificity of MCPyV seroreactivity was supported by competitive inhibition of reactivity by MCPyV VLPs and not by BK polyomavirus (BKPyV) VLPs. MCPyV seroprevalence was greater among MCC patients (91%) than controls (68%; age-adjusted P value, 0.32); the mean level of MCPyV antibodies was also greater (P = 0.04). The age-specific seroprevalence of MCPyV shares with previously known polyomaviruses, BKPyV and JC polyomavirus (JCPyV), evidence of widespread exposure in human populations beginning early in life. MCPyV age-specific seroprevalence also has unique features. Seroprevalence among children is higher than that of JCPyV but lower than that of BKPyV. Among older adults, MCPyV seroprevalence remains high, while that of BKPyV declines and that of JCPyV continues to rise. In agreement with results from other studies, we found an association between MCPyV seropositivity and MCC, and higher levels of serum MCPyV capsid antibodies in MCC patients than in controls.  

5.966           A Genetic Interaction between the Core and NS3 Proteins of Hepatitis C Virus Is Essential for Production of Infectious Virus

Jones, D.M., Atoom, A.M., Zhang, X., Kottilil, S. and Russell, R.S.

1. Virol., 85(23), 12351-12361 (2011)

  By analogy to other members of the Flaviviridae family, the hepatitis C virus (HCV) core protein is presumed to oligomerize to form the viral nucleocapsid, which encloses the single-stranded RNA genome. Core protein is directed to lipid droplets (LDs) by domain 2 (D2) of the protein, and this process is critical for virus production. Domain 1 (D1) of core is also important for infectious particle morphogenesis, although its precise contribution to this process is poorly understood. In this study, we mutated amino acids 64 to 75 within D1 of core and examined the ability of these mutants to produce infectious virus. We found that residues 64 to 66 are critical for generation of infectious progeny, whereas 67 to 75 were dispensable for this process. Further investigation of the defective 64 to 66 mutant (termed JFH1_T-64–66) revealed it to be incapable of producing infectious intracellular virions, suggesting a fault during HCV assembly. Furthermore, isopycnic gradient analyses revealed that JFH1_T-64–66 assembled dense intracellular species of core, presumably representing nucleocapsids. Thus, amino acids 64 to 66 are seemingly not involved in core oligomerization/nucleocapsid assembly. Passaging of JFH1_T-64–66 led to the emergence of a single compensatory mutation (K1302R) within the helicase domain of NS3 that completely rescued its ability to produce infectious virus. Importantly, the same NS3 mutation abrogated virus production in the context of wild-type core protein. Together, our results suggest that residues 64 to 66 of core D1 form a highly specific interaction with the NS3 helicase that is essential for the generation of infectious HCV particles at a stage downstream of nucleocapsid assembly.  

5.967           Epstein-Barr Virus BamHI W Repeat Number Limits EBNA2/EBNA-LP Coexpression in Newly Infected B Cells and the Efficiency of B-Cell Transformation: a Rationale for the Multiple W Repeats in Wild-Type Virus Strains

Tierney, R.J., Kao, K-Y., nagra, J.K. and Rickinson, A.B.

1. Virol., 85(23), 12362-12375 (2011)

  The genome of Epstein-Barr virus (EBV), a gammaherpesvirus with potent B-cell growth-transforming ability, contains multiple copies of a 3-kb BamHI W repeat sequence; each repeat carries (i) a promoter (Wp) that initiates transformation by driving EBNA-LP and EBNA2 expression and (ii) the W1W2 exons encoding the functionally active repeat domain of EBNA-LP. The W repeat copy number of a virus therefore influences two potential determinants of its transforming ability: the number of available Wp copies and the maximum size of the encoded EBNA-LP. Here, using recombinant EBVs, we show that optimal B-cell transformation requires a minimum of 5 W repeats (5W); the levels of transforming ability fall progressively with viruses carrying 4, 3, and 2 W repeats, as do the levels of Wp-initiated transcripts expressed early postinfection (p.i.), while viruses with 1 copy of the wild-type W repeat (1W) and 0W are completely nontransforming. We therefore suggest that genetic analyses of EBV transforming function should ensure that wild-type and mutant strains have equal numbers (ideally at least 5) of W copies if the analysis is not to be compromised. Attempts to enhance the transforming function of low-W-copy-number viruses, via the activity of helper EBV strains or by gene repair, suggested that the critical defect is not related to EBNA-LP size but to the failure to achieve sufficiently strong coexpression of EBNA-LP and EBNA2 early postinfection. We further show by the results of ex vivo assays that EBV strains in the blood of infected individuals typically have a mean of 5 to 8 W copies, consistent with the view that evolution has selected for viruses with an optimal transforming function.  

5.968           Neutralization of non-vaccine human papillomavirus pseudoviruses from the A7 and A9 species groups by bivalent HPV vaccine sera

Draper, E., Bissett, S.L., Howell-Jones, R., Edwards, D., Munslow, G., Soldan, K. and Beddows, S. Vaccine, 29(47), 8585-8590 (2011)   The majority of cervical cancers are associated with infection by one or more Human Papillomavirus (HPV) types from just two distinct Alpha-Papillomavirus species groups, A7 and A9. The extent to which the current HPV16/18 vaccines will protect against other genetically related HPV types is of interest to inform vaccine implementation, cervical disease surveillance and the development of second generation HPV vaccines. The aim of this study was to determine the frequency and titer of neutralizing antibodies against a range of A7 (18, 39, 45, 59, 68) and A9 (16, 31, 33, 35, 52, 58) HPV types using sera from individuals immunized with the bivalent HPV vaccine within the school-based, UK national HPV immunization programme. Serum samples were collected from 69 girls aged 13–14 years, a median 5.9 months (inter-quartile range, IQR, 5.7–6.0) after their third vaccine dose. Cross-neutralizing antibodies against HPV31, HPV33, HPV35 and HPV45 were common and strongly associated with the titer for the related vaccine-type, but were considerably lower (<1%) than their related vaccine type-specific response. The low prevalence of these HPV types in the population and the ages within the study cohort suggest these responses are due to vaccination. It is unclear whether such low levels of neutralizing antibodies would be sufficient to protect at the site of infection in the absence of other immune effectors but the coincidence with HPV types reported from efficacy studies is intriguing. The utility of neutralizing antibodies as surrogate markers of protection remains to be determined.  

5.969           Monitoring virus entry into living cells using DiD-labeled dengue virus particles

Ayala-Nunez, N.V., Wilschut, J. and Smit, J.M. Methods, 55(2), 137-143 (2011)   A variety of approaches can be applied to investigate the multiple steps and interactions that occur during virus entry into the host cell. Single-virus tracking is a powerful real-time imaging technique that offers the possibility to monitor virus-cell binding, internalization, intracellular trafficking behavior, and the moment of membrane fusion of single virus particles in living cells. Here we describe the development and applications of a single-virus tracking assay based on the use of DiD-labeled dengue virus (DENV) in BS-C-1 cells. In addition – and using the same experimental setup – we present a binding and fusion assay that can be used to obtain a rapid insight into the relative extent of virus binding to the cell surface and membrane fusion. Details of virus labeling and characterization, microscopy setup, protocols, data analysis, and hints for troubleshooting are described throughout the paper.  

5.970           Protein Kinase C-Dependent Dephosphorylation of Tyrosine Hydroxylase Requires the B56δ Heterotrimeric Form of Protein Phosphatase 2A

Ahn, J-H., Kim, Y., Kim, H-S., Greengard, P and Nairn, A.C. PloS One, 6(10), e26292 (2011)   Tyrosine hydroxylase, which plays a critical role in regulation of dopamine synthesis, is known to be controlled by phosphorylation at several critical sites. One of these sites, Ser40, is phosphorylated by a number of protein kinases, including protein kinase A. The major protein phosphatase that dephosphorylates Ser40 is protein phosphatase-2A (PP2A). A recent study has also linked protein kinase C to the dephosphorylation of Ser40 [1], but the mechanism is unclear. PP2A isoforms are comprised of catalytic, scaffold, and regulatory subunits, the regulatory B subunits being able to influence cellular localization and substrate selection. In the current study, we find that protein kinase C is able to phosphorylate a key regulatory site in the B56δ subunit leading to activation of PP2A. In turn, activation of the B56δ-containing heterotrimeric form of PP2A is responsible for enhanced dephosphorylation of Ser40 of tyrosine hydroylase in response to stimulation of PKC. In support of this mechanism, down-regulation of B56δ expression in N27 cells using RNAi was found to increase dopamine synthesis. Together these studies reveal molecular details of how protein kinase C is linked to reduced tyrosine hydroxylase activity via control of PP2A, and also add to the complexity of protein kinase/protein phosphatase interactions.  

5.971           Targeted Decorin Gene Therapy Delivered with Adeno-Associated Virus Effectively Retards Corneal Neovascularization In Vivo

Mohan, R.R., Tovey, J.C.K., Sharma, A., Schultz, G.S., Cowden, J.W. and Tandon, A. PloS One, 6(10), e26432 (2011)   Decorin, small leucine-rich proteoglycan, has been shown to modulate angiogenesis in nonocular tissues. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the rabbit stroma with adeno-associated virus serotype 5 (AAV5) impedes corneal neovascularization (CNV) in vivo without significant side effects. An established rabbit CNV model was used. Targeted decorin gene therapy in the rabbit stroma was delivered with a single topical AAV5 titer (100 µl; 5×10¹² vg/ml) application onto the stroma for two minutes after removing corneal epithelium. The levels of CNV were examined with stereomicroscopy, H&E staining, lectin, collagen type IV, CD31 immunocytochemistry and CD31 immunoblotting. Real-time PCR quantified mRNA expression of pro- and anti-angiogenic genes. Corneal health in live animals was monitored with clinical, slit-lamp and optical coherence tomography biomicroscopic examinations. Selective decorin delivery into stroma showed significant 52% (p<0.05), 66% (p<0.001), and 63% (p<0.01) reduction at early (day 5), mid (day 10), and late (day 14) stages of CNV in decorin-delivered rabbit corneas compared to control (no decorin delivered) corneas in morphometric analysis. The H&E staining, lectin, collagen type IV, CD31 immunostaining (57–65, p<0.5), and CD31 immunoblotting (62–67%, p<0.05) supported morphometric findings. Quantitative PCR studies demonstrated decorin gene therapy down-regulated expression of VEGF, MCP1 and angiopoietin (pro-angiogenic) and up-regulated PEDF (anti-angiogenic) genes. The clinical, biomicroscopy and transmission electron microscopy studies revealed that AAV5–mediated decorin gene therapy is safe for the cornea. Tissue-targeted AAV5-mediated decorin gene therapy decreases CNV with no major side effects, and could potentially be used for treating patients.  

5.972           Lipoprotein Lipase Inhibits Hepatitis C Virus (HCV) Infection by Blocking Virus Cell Entry

Maillard, P., Walic, M., Meuleman, P., Roohvand, F., Huby, T., Le Goff, W., Leroux-Roels, G., Pecheur, E-I. and Budkowska, A. PloS One, 6(10), e26637 (2011)   A distinctive feature of HCV is that its life cycle depends on lipoprotein metabolism. Viral morphogenesis and secretion follow the very low-density lipoprotein (VLDL) biogenesis pathway and, consequently, infectious HCV in the serum is associated with triglyceride-rich lipoproteins (TRL). Lipoprotein lipase (LPL) hydrolyzes TRL within chylomicrons and VLDL but, independently of its catalytic activity, it has a bridging activity, mediating the hepatic uptake of chylomicrons and VLDL remnants. We previously showed that exogenously added LPL increases HCV binding to hepatoma cells by acting as a bridge between virus-associated lipoproteins and cell surface heparan sulfate, while simultaneously decreasing infection levels. We show here that LPL efficiently inhibits cell infection with two HCV strains produced in hepatoma cells or in primary human hepatocytes transplanted into uPA-SCID mice with fully functional human ApoB-lipoprotein profiles. Viruses produced in vitro or in vivo were separated on iodixanol gradients into low and higher density populations, and the infection of Huh 7.5 cells by both virus populations was inhibited by LPL. The effect of LPL depended on its enzymatic activity. However, the lipase inhibitor tetrahydrolipstatin restored only a minor part of HCV infectivity, suggesting an important role of the LPL bridging function in the inhibition of infection. We followed HCV cell entry by immunoelectron microscopy with anti-envelope and anti-core antibodies. These analyses demonstrated the internalization of virus particles into hepatoma cells and their presence in intracellular vesicles and associated with lipid droplets. In the presence of LPL, HCV was retained at the cell surface. We conclude that LPL efficiently inhibits HCV infection by acting on TRL associated with HCV particles through mechanisms involving its lipolytic function, but mostly its bridging function. These mechanisms lead to immobilization of the virus at the cell surface. HCV-associated lipoproteins may therefore be a promising target for the development of new therapeutic approaches.  

5.973           Capsomer Vaccines Protect Mice from Vaginal Challenge with Human Papillomavirus

Wu, W-H., Gersch, E., Kwak, K., jaguar, S., karanam, B., Huh, W.K., Garcea, R.L. and Roden, R.B.S. PloS One, 6(10), e27141 (2011)   Capsomers were produced in bacteria as glutathione-S-transferase (GST) fusion proteins with human papillomavirus type 16 L1 lacking the first nine and final 29 residues (GST-HPV16L1Δ) alone or linked with residues 13–47 of HPV18, HPV31 and HPV45 L2 in tandem (GST-HPV16L1Δ-L2x3). Subcutaneous immunization of mice with GST-HPV16L1Δ or GST-HPV16L1Δ-L2x3 in alum and monophosphoryl lipid A induced similarly high titers of HPV16 neutralizing antibodies. GST-HPV16L1Δ-L2x3 also elicited moderate L2-specific antibody titers. Intravaginal challenge studies showed that immunization of mice with GST-HPV16 L1Δ or GST-HPV16L1Δ-L2x3 capsomers, like Cervarix®, provided complete protection against HPV16. Conversely, vaccination with GST-HPV16 L1Δ capsomers failed to protect against HPV18 challenge, whereas mice immunized with either GST-HPV16L1Δ-L2x3 capsomers or Cervarix® were each completely protected. Thus, while the L2-specific response was moderate, it did not interfere with immunity to L1 in the context of GST-HPV16L1Δ-L2x3 and is sufficient to mediate L2-dependent protection against an experimental vaginal challenge with HPV18.  

5.974           Trafficking of Hepatitis C Virus Core Protein during Virus Particle Assembly

Counihan, N.A., Rawlinson, S.M. and Lindenbach, B.D. PloS One, 7(10), e1002302 (2011)   Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production. However, the process by which core is recruited from LD into nascent virus particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus particle assembly.  

5.975           Electron cryotomography of measles virus reveals how matrix protein coats the ribonucleocapsid within intact virions

Liljeroos, L., Huiskonen, J.T., Ora, A., Susi, P. and Butcher, S.J. PNAS, 108(44), 18085-18090 (2011)   Measles virus is a highly infectious, enveloped, pleomorphic virus. We combined electron cryotomography with subvolume averaging and immunosorbent electron microscopy to characterize the 3D ultrastructure of the virion. We show that the matrix protein forms helices coating the helical ribonucleocapsid rather than coating the inner leaflet of the membrane, as previously thought. The ribonucleocapsid is folded into tight bundles through matrix–matrix interactions. The implications for virus assembly are that the matrix already tightly interacts with the ribonucleocapsid in the cytoplasm, providing a structural basis for the previously observed regulation of RNA transcription by the matrix protein. Next, the matrix-covered ribonucleocapsids are transported to the plasma membrane, where the matrix interacts with the envelope glycoproteins during budding. These results are relevant to the nucleocapsid organization and budding of other paramyxoviruses, where isolated matrix has been observed to form helices.  

5.976           Microvesicles and Viral Infection

Meckes Jr, D.G. and Raab-Traub, N.

1. Virol., 85(24), 12844-12854 (2011)

  Cells secrete various membrane-enclosed microvesicles from their cell surface (shedding microvesicles) and from internal, endosome-derived membranes (exosomes). Intriguingly, these vesicles have many characteristics in common with enveloped viruses, including biophysical properties, biogenesis, and uptake by cells. Recent discoveries describing the microvesicle-mediated intercellular transfer of functional cellular proteins, RNAs, and mRNAs have revealed additional similarities between viruses and cellular microvesicles. Apparent differences include the complexity of viral entry, temporally regulated viral expression, and self-replication proceeding to infection of new cells. Interestingly, many virally infected cells secrete microvesicles that differ in content from their virion counterparts but may contain various viral proteins and RNAs. For the most part, these particles have not been analyzed for their content or functions during viral infection. However, early studies of microvesicles (L-particles) secreted from herpes simplex virus-infected cells provided the first evidence of microvesicle-mediated intercellular communication. In the case of Epstein-Barr virus, recent evidence suggests that this tumorigenic herpesvirus also utilizes exosomes as a mechanism of cell-to-cell communication through the transfer of signaling competent proteins and functional microRNAs to uninfected cells. This review focuses on aspects of the biology of microvesicles with an emphasis on their potential contributions to viral infection and pathogenesis.  

5.977           Site-Specific Proteolytic Cleavage of the Amino Terminus of Herpes Simplex Virus Glycoprotein K on Virion Particles Inhibits Virus Entry

Jambunathan, N., Chowdhury, S., Subramanian, R., Chouljenko, V.N., Walker, J.D. and Kousoulas, K.G.

1. Virol., 85(24), 12910-12918 (2011)

  Herpes simplex virus 1 (HSV-1) glycoprotein K (gK) is expressed on virions and functions in entry, inasmuch as HSV-1(KOS) virions devoid of gK enter cells substantially slower than is the case for the parental KOS virus (T. P. Foster, G. V. Rybachuk, and K. G. Kousoulas, J. Virol. 75:12431–12438, 2001). Deletion of the amino-terminal 68-amino-acid (aa) portion of gK caused a reduction in efficiency and kinetics of virus entry similar to that of the gK-null virus in comparison to the HSV-1(F) parental virus. The UL20 membrane protein and gK were readily detected on double-gradient-purified virion preparations. Immuno-electron microscopy confirmed the presence of gK and UL20 on purified virions. Coimmunoprecipitation experiments using purified virions revealed that gK interacted with UL20, as has been shown in virus-infected cells (T. P. Foster, V. N. Chouljenko, and K. G. Kousoulas, J. Virol. 82:6310–6323, 2008). Scanning of the HSV-1(F) viral genome revealed the presence of a single putative tobacco etch virus (TEV) protease site within gD, while additional TEV predicted sites were found within the UL5 (helicase-primase helicase subunit), UL23 (thymidine kinase), UL25 (DNA packaging tegument protein), and UL52 (helicase-primase primase subunit) proteins. The recombinant virus gDΔTEV was engineered to eliminate the single predicted gD TEV protease site without appreciably affecting its replication characteristics. The mutant virus gK-V5-TEV was subsequently constructed by insertion of a gene sequence encoding a V5 epitope tag in frame with the TEV protease site immediately after gK amino acid 68. The gK-V5-TEV, R-gK-V5-TEV (revertant virus), and gDΔTEV viruses exhibited similar plaque morphologies and replication characteristics. Treatment of the gK-V5-TEV virions with TEV protease caused approximately 32 to 34% reduction of virus entry, while treatment of gDΔTEV virions caused slightly increased virus entry. These results provide direct evidence that the gK and UL20 proteins, which are genetically and functionally linked to gB-mediated virus-induced cell fusion, are structural components of virions and function in virus entry. Site-specific cleavage of viral glycoproteins on mature and fully infectious virions utilizing unique protease sites may serve as a generalizable method of uncoupling the roles of viral glycoproteins in virus entry and virion assembly.  

5.978           A Virus-Like Particle-Based Epstein-Barr Virus Vaccine

Ruiss, R., Jochum, S., Wanner, G., Reisbach, G., hammerschmidt, W. and Zeidler, R.

1. Virol., 85(24), 13105-13113 (2011)

  Epstein-Barr Virus (EBV) is an ubiquitous human herpesvirus which can lead to infectious mononucleosis and different cancers. In immunocompromised individuals, this virus is a major cause for morbidity and mortality. Transplant patients who did not encounter EBV prior to immunosuppression frequently develop EBV-associated malignancies, but a prophylactic EBV vaccination might reduce this risk considerably. Virus-like particles (VLPs) mimic the structure of the parental virus but lack the viral genome. Therefore, VLPs are considered safe and efficient vaccine candidates. We engineered a dedicated producer cell line for EBV-derived VLPs. This cell line contains a genetically modified EBV genome which is devoid of all potential viral oncogenes but provides viral proteins essential for the assembly and release of VLPs via the endosomal sorting complex required for transport (ESCRT). Human B cells readily take up EBV-based VLPs and present viral epitopes in association with HLA molecules to T cells. Consequently, EBV-based VLPs are highly immunogenic and elicit humoral and strong CD8⁺ and CD4⁺ T cell responses in vitro and in a preclinical murine model in vivo. Our findings suggest that VLP formulations might be attractive candidates to develop a safe and effective polyvalent vaccine against EBV.  

5.979           A Murine Genital-Challenge Model Is a Sensitive Measure of Protective Antibodies against Human Papillomavirus Infection

Longet, S., Schiller, J.T., Bobst, M., Jichlinski, P. and Nardelli-Haefliger, D.

1. Virol., 85(24), 13253-13259 (2011)

  The available virus-like particle (VLP)-based prophylactic vaccines against specific human papillomavirus (HPV) types afford close to 100% protection against the type-associated lesions and disease. Based on papillomavirus animal models, it is likely that protection against genital lesions in humans is mediated by HPV type-restricted neutralizing antibodies that transudate or exudate at the sites of genital infection. However, a correlate of protection was not established in the clinical trials because few disease cases occurred, and true incident infection could not be reliably distinguished from the emergence or reactivation of prevalent infection. In addition, the current assays for measuring vaccine-induced antibodies, even the gold standard HPV pseudovirion (PsV) in vitro neutralization assay, may not be sensitive enough to measure the minimum level of antibodies needed for protection. Here, we characterize the recently developed model of genital challenge with HPV PsV and determine the minimal amounts of VLP-induced neutralizing antibodies that can afford protection from genital infection in vivo after transfer into recipient mice. Our data show that serum antibody levels >100-fold lower than those detectable by in vitro PsV neutralization assays are sufficient to confer protection against an HPV PsV genital infection in this model. The results clearly demonstrate that, remarkably, the in vivo assay is substantially more sensitive than in vitro PsV neutralization and thus may be better suited for studies to establish correlates of protection.  

5.980           The Cellular Protein Lyric Interacts with HIV-1 Gag

Engeland, C.E., Oberwinkler, H., Schümann, M., Krawuse, E., Müller, G.A. and Kräusslich, H-G.

1. Virol., 85(24), 13322-13332 (2011)

  Human immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein driving assembly and release of virions from infected cells. Gag alone is capable of self-assembly in vitro, but host factors have been shown to play a role in efficient viral replication and particle morphogenesis within the living cell. In a series of affinity purification experiments, we identified the cellular protein Lyric to be an HIV-1 Gag-interacting protein. Lyric was previously described to be an HIV-inducible gene and is involved in various signaling pathways. Gag interacts with endogenous Lyric via its matrix (MA) and nucleocapsid (NC) domains. This interactio, n requires Gag multimerization and Lyric amino acids 101 to 289. Endogenous Lyric is incorporated into HIV-1 virions and is cleaved by the viral protease. Gag-Lyric interaction was also observed for murine leukemia virus and equine infectious anemia virus, suggesting that it represents a conserved feature among retroviruses. Expression of the Gag binding domain of Lyric increased Gag expression levels and viral infectivity, whereas expression of a Lyric mutant lacking the Gag binding site resulted in lower Gag expression and decreased viral infectivity. The results of the current study identify Lyric to be a cellular interaction partner of HIV-1 Gag and hint at a potential role in regulating infectivity. Further experiments are needed to elucidate the precise role of this interaction.  

5.981           Viruses as Nanomaterials for Drug Delivery

Lockney, D., Franzen, S. and Lommel, S. Methods in Mol. Biol., 726, 207-221 82011)   Virus delivery vectors are one among the many nanomaterials that are being developed as drug delivery materials. This chapter focuses on methods utilizing plant virus nanoparticles (PVNs) synthesized from the Red clover necrotic mosaic virus (RCNMV). A successful vector must be able to effectively carry and subsequently deliver a drug cargo to a specific target. In the case of the PVNs, we describe two types of ways cargo can be loaded within these structures: encapsidation and infusion. Several targeting approaches have been used for PVNs based on bioconjugate chemistry. Herein, examples of such approaches will be given that have been used for RCNMV as well as for other PVNs in the literature. Further, we describe characterization of PVNs, in vitro cell studies that can be used to test the efficacy of a targeting vector, and potential routes for animal administration.  

5.982           Adeno-Associated Virus Biology

Weitzman, M.D. and Linden, R.M. Methods in Mol. Biol., 807, 1-23 (2011)   Adeno-associated virus (AAV) was fi rst discovered as a contaminant of adenovirus stocks in the 1960s. The development of recombinant AAV vectors (rAAV) was facilitated by early studies that generated infectious molecular clones, determined the sequence of the genome, and defi ned the genetic elements of the virus. The refi nement of methods and protocols for the production and application of rAAV vectors has come from years of studies that explored the basic biology of this virus and its interaction with host cells. Interest in improving vector performance has in turn driven studies that have provided tremendous insights into the basic biology of the AAV lifecycle. In this chapter, we review the background on AAV biology and its exploitation for vectors and gene delivery.  

5.983           AAV Capsid Structure and Cell Interactions

Agbandje-McKenna, M. and Kleinschmidt, J. Methods in Mol. Biol., 807, 47-92 (2011)   The Adeno-associated viruses (AAVs) are not associated with any diseases, and their ability to package non-genomic DNA and to transduce different cell/tissue populations has generated signifi cant interest in understanding their basic biology in efforts to improve their utilization for corrective gene delivery. This includes their capsid structure, cellular tropism and interactions for entry, uncoating, replication, DNA packaging, capsid assembly, and antibody neutralization. The human and nonhuman primate AAVs are clustered into serologically distinct genetic clade and serotype groups, which have distinct cellular/tissue tropisms and transduction effi ciencies. These properties are highly dependent upon the AAV capsid amino acid sequence, their capsid structure, and their interactions with host cell factors, including cell surface receptors, co-receptors, signaling molecules, proteins involved in host DNA replication, and host-derived antibodies. This chapter reviews the current structural information on AAV capsids and the capsid viral protein regions playing a role in the cellular interactions conferring an infective phenotype, which are then used to annotate the functional regions of the capsid. Based on the current data, the indication is that the AAVs, like other members of the Parvoviridae and other ssDNA viruses that form a T = 1 capsid, have evolved a multifunctional capsid with conserved core regions as is required for effi cient capsid traffi cking, capsid assembly, and genome packaging. Disparate surface loop structures confer differential receptor recognition and are involved in antibody recognition. The role of structural regions in capsid uncoating remains to be elucidated.  

5.984           Adeno-Associated Virus Vector Delivery to the Heart

Bish, L.T., Sweeney, H.L., Müller, O.J. and Bekeredjian, R. Methods in Mol. Biol., 807, 219-237 (2011)   Cardiac gene transfer may serve as a novel therapeutic approach in the treatment of heart disease. For it to reach its full potential, methods for highly effi cient cardiac gene transfer must be available to investigators so that informative preclinical data can be collected and evaluated. We have recently optimized AAVmediated cardiac gene transfer protocols in both the mouse and rat. In the mouse, we have developed a procedure for intrapericardial delivery of vector in the neonate and successfully applied intravenous injections in adult animals. In the rat, we have developed a procedure for direct injection of vector into the myocardium in adults and established a protocol for vector delivery into the left ventricular anterior wall by ultrasound-targeted destruction of microbubbles loaded with AAV. Each protocol can be used to achieve safe and effi cient cardiac gene transfer in the model of choice.  

5.985           Modification and Labeling of AAV Vector Particles

Büning, H., Bolyard, C.M., Hallek, M. and Bartlett, J.S. Methos in Mol. Biol., 807, 273-300 (2011)   Adeno-associated virus (AAV) has become a versatile vector platform. In recent years, powerful techniques for the generation of tropism-modifi ed vectors (rAAV-targeting vectors) and for investigation of virus–cell interaction were developed. The following chapter describes strategies for insertion of peptide ligands into the viral capsid and the subsequent characterization of capsid mutants, for producing mosaic capsids and for labeling the viral capsid chemically or genetically.  

5.986           Production and Purification of Recombinant Adeno-Associated Vectors

Wang, L., Blouin, V., Brument, N., Bello-Roufai, M. and Francois, A. Methos in Mol. Biol., 807, 361-404 (2011)   The use of recombinant adeno-associated virus (rAAV) vectors in gene therapy for preclinical studies in animal models and human clinical trials is increasing, as these vectors have been shown to be safe and to mediate persistent transgene expression in vivo. Constant improvement in rAAV manufacturing processes (upstream production and downstream purifi cation) has paralleled this evolution to meet the needs for larger vector batches, higher vector titer, and improved vector quality and safety. This chapter provides an overview of existing production and purifi cation systems used for adeno-associated virus (AAV) vectors, and the advantages and disadvantages of each system are outlined. Regulatory guidelines that apply to the use of these systems for clinical trials are also presented. The methods described are examples of protocols that have been utilized for establishing rAAV packaging cell lines, production of rAAV vectors using recombinant HSV infection, and for chromatographic purifi cation of various AAV vector serotypes. A protocol for the production of clinical-grade rAAV type 2 vectors using transient transfection and centrifugationbased purifi cation is also described.  

5.987           Authentically Phosphorylated α-Synuclein at Ser129 Accelerates Neurodegeneration in a Rat Model of Familial Parkinson's Disease

Sato, H., Araawaka, S., Hara, S., Fukushima, S., Koga, K., Koyama, S. and Kato, T.

1. Neuroscience, 31(46), 16884-16894 (2011)

  Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra (SN) and the appearance of fibrillar aggregates of insoluble α-synuclein (α-syn) called Lewy bodies (LBs). Approximately 90% of α-syn deposited in LBs is phosphorylated at serine 129 (Ser129). In contrast, only 4% of total α-syn is phosphorylated in normal brain, suggesting that accumulation of Ser129-phosphorylated α-syn is involved in the pathogenesis of PD. However, the role of Ser129 phosphorylation in α-syn neurotoxicity remains unclear. In this study, we coexpressed familial PD-linked A53T α-syn and G-protein-coupled receptor kinase 6 (GRK6) in the rat SN pars compacta using recombinant adeno-associated virus 2. Coexpression of these proteins yielded abundant Ser129-phosphorylated α-syn and significantly exacerbated degeneration of dopaminergic neurons when compared with coexpression of A53T α-syn and GFP. Immunohistochemical analysis revealed that Ser129-phosphorylated α-syn was preferentially distributed to swollen neurites. However, biochemical analysis showed that the increased expression of Ser129-phosphorylated α-syn did not promote accumulation of detergent-insoluble α-syn. Coexpression of catalytically inactive K215R mutant GRK6 failed to accelerate A53T α-syn-induced degeneration. Furthermore, introducing a phosphorylation-incompetent mutation, S129A, into A53T α-syn did not alter the pace of degeneration, even when GRK6 was coexpressed. Our study demonstrates that authentically Ser129-phosphorylated α-syn accelerates A53T α-syn neurotoxicity without the formation of detergent-insoluble α-syn, and suggests that the degenerative process could be constrained by inhibiting the kinase that phosphorylates α-syn at Ser129.  

5.988           The combined effects of oncolytic reovirus plus Newcastle disease virus and reovirus plus parvovirus on U87 and U373 cells in vitro and in vivo

Alkassar, M., Gärtner, B., Roemer, K., Graesser, F., Rommerlaere, J., Kaestner, L., Haeckel, I. and Graf, N.

1. Neurooncol., 104, 715-727 (2011)

  Previous results had documented oncolytic capacity of reovirus, parvovirus and Newcastle disease virus (NDV) on several tumor cell types. To test whether combinations of these viruses may increase this capacity, human U87- and U373-glioblastoma cells, in vitro or xenografted into immuno-compromised mice, were subjected to simultaneous double infections and analyzed. Our results show that reovirus (serotype-3) plus NDV (Hitcher-B1) and reovirus plus parvovirus-H1 lead to a significant increase in tumor cell killing in vitro in both cell lines (Kruskal–Wallis test, P\0.01) and in vivo. Immunofluorescence and flow cytometry analyses demonstrated the simultaneous replication of the viruses in nearly all cells ([95%) after combined infection. These data thus indicate that a synergistic anti-tumor effect can be achieved by the combined infection with oncolytic viruses.  

5.989           Delivery of AAV2/9-Microdystrophin Genes Incorporating Helix 1 of the Coiled-Coil Motif in the C-Terminal Domain of Dystrophin Improves Muscle Pathology and Restores the Level of α1-Syntrophin and α-Dystrobrevin in Skeletal Muscles of mdx Mice

Koo, T., malerba, A., Athanasopoulos, T., Trollet, C., Boldrin, L., Ferry, A., Popplewell, L., Foster, H., Foster, K. and Dickson, G. Human Gene Therapy, 22, 1379-1388 (2011)   Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence–optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signaling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain–extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction–induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.  

5.990           Molecular Links between the E2 Envelope Glycoprotein and Nucleocapsid Core in Sindbis Virus

Tang, J., Jose, J., Chipman, P., Zhang, W., Kuhn, R.J. and Baker, T.S.

1. Mol. Biol., 414, 442-459 (2011)

  A three-dimensional reconstruction of Sindbis virus at 7.0 Å resolution presented here provides a detailed view of the virion structure and includes structural evidence for key interactions that occur between the capsid protein (CP) and transmembrane (TM) glycoproteins E1 and E2. Based on crystal structures of component proteins and homology modeling, we constructed a nearly complete, pseudo-atomic model of the virus. Notably, this includes identification of the 33-residue cytoplasmic domain of E2 (cdE2), which follows a path from the E2 TM helix to the CP where it enters and exits the CP hydrophobic pocket and then folds back to contact the viral membrane. Modeling analysis identified three major contact regions between cdE2 and CP, and the roles of specific residues were probed by molecular genetics. This identified R393 and E395 of cdE2 and Y162 and K252 of CP as critical for virus assembly. The N-termini of the CPs form a contiguous network that interconnects 12 pentameric and 30 hexameric CP capsomers. A single glycoprotein spike cross-links three neighboring CP capsomers as might occur during initiation of virus budding.  

5.991           Activation of the human immune system by chemotherapeutic or targeted agents combined with the oncolytic parvovirus H-1

Moehler, M., Sieben, M., Roth, S., Springsguth, F., Leuchs, B., Zeidler, M., Dinsaart, C., Rommelaere, J. and Galle, P.R. BMC Cancer, 11, 464-477 (2011)   Background Parvovirus H-1 (H-1PV) infects and lyses human tumor cells including melanoma, hepatoma, gastric, colorectal, cervix and pancreatic cancers. We assessed whether the beneficial effects of chemotherapeutic agents or targeted agents could be combined with the oncolytic and immunostimmulatory properties of H-1PV. Methods Using human ex vivo models we evaluated the biological and immunological effects of H-1PV-induced tumor cell lysis alone or in combination with chemotherapeutic or targeted agents in human melanoma cells +/- characterized human cytotoxic T-cells (CTL) and HLA-A2-restricted dendritic cells (DC). Results H-1PV-infected MZ7-Mel cells showed a clear reduction in cell viability of >50%, which appeared to occur primarily through apoptosis. This correlated with viral NS1 expression levels and was enhanced by combination with chemotherapeutic agents or sunitinib. Tumor cell preparations were phagocytosed by DC whose maturation was measured according to the treatment administered. Immature DC incubated with H-1PV-induced MZ7-Mel lysates significantly increased DC maturation compared with non-infected or necrotic MZ7-Mel cells. Tumor necrosis factor-α and interleukin-6 release was clearly increased by DC incubated with H-1PV-induced SK29-Mel tumor cell lysates (TCL) and was also high with DC-CTL co-cultures incubated with H-1PV-induced TCL. Similarly, DC co-cultures with TCL incubated with H-1PV combined with cytotoxic agents or sunitinib enhanced DC maturation to a greater extent than cytotoxic agents or sunitinib alone. Again, these combinations increased pro-inflammatory responses in DC-CTL co-cultures compared with chemotherapy or sunitinib alone. Conclusions In our human models, chemotherapeutic or targeted agents did not only interfere with the pronounced immunomodulatory properties of H-1PV, but also reinforced drug-induced tumor cell killing. H-1PV combined with cisplatin, vincristine or sunitinib induced effective immunostimulation via a pronounced DC maturation, better cytokine release and cytotoxic T-cell activation compared with agents alone. Thus, the clinical assessment of H-1PV oncolytic tumor therapy not only alone but also in combination strategies is warranted.  

5.992           TRAF6 and IRF7 Control HIV Replication in Macrophages

Sirois, M., Robitaille, L., Allary, R., Shah, M., Woelk, C.H., Estaquier, J. and Corbell, J. PloS Pne, 6(11), e28125 (2011)   The innate immune system recognizes virus infection and evokes antiviral responses which include producing type I interferons (IFNs). The induction of IFN provides a crucial mechanism of antiviral defense by upregulating interferon-stimulated genes (ISGs) that restrict viral replication. ISGs inhibit the replication of many viruses by acting at different steps of their viral cycle. Specifically, IFN treatment prior to in vitro human immunodeficiency virus (HIV) infection stops or significantly delays HIV-1 production indicating that potent inhibitory factors are generated. We report that HIV-1 infection of primary human macrophages decreases tumor necrosis factor receptor-associated factor 6 (TRAF6) and virus-induced signaling adaptor (VISA) expression, which are both components of the IFN signaling pathway controlling viral replication. Knocking down the expression of TRAF6 in macrophages increased HIV-1 replication and augmented the expression of IRF7 but not IRF3. Suppressing VISA had no impact on viral replication. Overexpression of IRF7 resulted in enhanced viral replication while knocking down IRF7 expression in macrophages significantly reduced viral output. These findings are the first demonstration that TRAF6 can regulate HIV-1 production and furthermore that expression of IRF7 promotes HIV-1 replication.  

5.993           Adeno-Associated Virus-Mediated Rescue of the Cognitive Defects in a Mouse Model for Angelman Syndrome

Daily, J.L., Nash, K., Jinwal, U., Golde, T., Rogers, J., Peters, M.M., Burdine, R.D., Dickey, C., Banko, J.L. and Weeber, E.J. PloS One, 6(12), e27221 (2011)   Angelman syndrome (AS), a genetic disorder occurring in approximately one in every 15,000 births, is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes for E6-AP, an ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. However, we have determined that abnormal calcium/calmodulin-dependent protein kinase II (CaMKII) regulation is seen in the maternal UBE3A deletion AS mouse model and is responsible for the major phenotypes. Specifically, there is an increased αCaMKII phosphorylation at the autophosphorylation sites Thr²⁸⁶ and Thr^305/306, resulting in an overall decrease in CaMKII activity. CaMKII is not produced until after birth, indicating that the deficits associated with AS are not the result of developmental abnormalities. The present studies are focused on exploring the potential to rescue the learning and memory deficits in the adult AS mouse model through the use of an adeno-associated virus (AAV) vector to increase neuronal UBE3A expression. These studies show that increasing the levels of E6-AP in the brain using an exogenous vector can improve the cognitive deficits associated with AS. Specifically, the associative learning deficit was ameliorated in the treated AS mice compared to the control AS mice, indicating that therapeutic intervention may be possible in older AS patients.  

5.994           Preclinical Safety of RNAi-Mediated HTT Suppression in the Rhesus Macaque as a Potential Therapy for Huntington's Disease

McBride, J.L., Pitzer, M.R., Boudreau, R.L:, Dufour, B., Hobbs, T., Ojeda, S.R. and Davidson, B.L. Molecular Therapy, 19(12), 2152-2162 (2011)   To date, a therapy for Huntington's disease (HD), a genetic, neurodegenerative disorder, remains elusive. HD is characterized by cell loss in the basal ganglia, with particular damage to the putamen, an area of the brain responsible for initiating and refining motor movements. Consequently, patients exhibit a hyperkinetic movement disorder. RNA interference (RNAi) offers therapeutic potential for this disorder by reducing the expression of HTT, the disease-causing gene. We have previously demonstrated that partial suppression of both wild-type and mutant HTT in the striatum prevents behavioral and neuropathological abnormalities in rodent models of HD. However, given the role of HTT in various cellular processes, it remains unknown whether a partial suppression of both alleles will be safe in mammals whose neurophysiology, basal ganglia anatomy, and behavioral repertoire more closely resembles that of a human. Here, we investigate whether a partial reduction of HTT in the normal non-human primate putamen is safe. We demonstrate that a 45% reduction of rhesus HTT expression in the mid- and caudal putamen does not induce motor deficits, neuronal degeneration, astrogliosis, or an immune response. Together, these data suggest that partial suppression of wild-type HTT expression is well tolerated in the primate putamen and further supports RNAi as a therapy for HD.  

5.995           Rational Design of Therapeutic siRNAs: Minimizing Off-targeting Potential to Improve the Safety of RNAi Therapy for Huntington's Disease

Boudreau, R., Spengler, R.M. and Davidson, B.L. Molecular Therapy, 19(12), 2169-2177 (2011)   RNA interference (RNAi) provides an approach for the treatment of many human diseases. However, the safety of RNAi-based therapies can be hampered by the ability of small inhibitory RNAs (siRNAs) to bind to unintended mRNAs and reduce their expression, an effect known as off-target gene silencing. Off-targeting primarily occurs when the seed region (nucleotides 2–8 of the small RNA) pairs with sequences in 3′-UTRs of unintended mRNAs and directs translational repression and destabilization of those transcripts. To date, most therapeutic RNAi sequences are selected primarily for gene silencing efficacy, and later evaluated for safety. Here, in designing siRNAs to treat Huntington's disease (HD), a dominant neurodegenerative disorder, we prioritized selection of sequences with minimal off-targeting potentials (i.e., those with a scarcity of seed complements within all known human 3′-UTRs). We identified new promising therapeutic candidate sequences which show potent silencing in cell culture and mouse brain. Furthermore, we present microarray data demonstrating that off-targeting is significantly minimized by using siRNAs that contain “safe” seeds, an important strategy to consider during preclinical development of RNAi-based therapeutics.  

5.996           Searching for Presynaptic NMDA Receptors in the Nucleus Accumbens

Huang, Y.H., Ishikawa, M., Lee, B.R., Nakanishi, N., Schlüter, O.M. and Dong, Y.

1. Neurosci., 31(50), 18453-18463 (2011)

  The nucleus accumbens shell (NAc) is a key brain region mediating emotional and motivational learning. In rodent models, dynamic alterations have been observed in synaptic NMDA receptors (NMDARs) within the NAc following incentive stimuli, and some of these alterations are critical for acquiring new emotional/motivational states. NMDARs are prominent molecular devices for controlling neural plasticity and memory formation. Although synaptic NMDARs are predominately located postsynaptically, recent evidence suggests that they may also exist at presynaptic terminals and reshape excitatory synaptic transmission by regulating presynaptic glutamate release. However, it remains unknown whether presynaptic NMDARs exist in the NAc and contribute to emotional and motivational learning. In an attempt to identify presynaptically located NMDARs in the NAc, the present study uses slice electrophysiology combined with pharmacological and genetic tools to examine the physiological role of the putative presynaptic NMDARs in rats. Our results show that application of glycine, the glycine-site agonist of NMDARs, potentiated presynaptic release of glutamate at excitatory synapses on NAc neurons, whereas application of 5,7-dichlorokynurenic acid or 7-chlorokynurenic acid, the glycine-site antagonists of NMDARs, produced the opposite effect. However, these seemingly presynaptic NMDAR-mediated effects could not be prevented by application of d-APV, the glutamate-site NMDAR antagonist, and were still present in the mice in which NMDAR NR1 or NR3 subunits were genetically deleted. Thus, rather than suggesting the existence of presynaptic NMDARs, our results support the idea that an unidentified type of glycine-activated substrate may account for the presynaptic effects appearing to be mediated by NMDARs.  

5.997           The green tea polyphenol, epigallocatechin-3-gallate, inhibits hepatitis C virus entry

Ciesek, S., von Hahn, T., Colpitts, C.C., Schang, L.M., Friesland, M., Steinmann, J., Manns, M.P., Ott, M., Wedemeyer, H., Meuleman, P., Pietschmann, T. and Steinmann, E. Hepatology, 54(6), 1947-1955 (2011)   Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Current antiviral therapy fails to clear infection in a substantial proportion of cases. Drug development is focused on nonstructural proteins required for RNA replication. Individuals undergoing orthotopic liver transplantation face rapid, universal reinfection of the graft. Therefore, antiviral strategies targeting the early stages of infection are urgently needed for the prevention of HCV infection. In this study, we identified the polyphenol, epigallocatechin-3-gallate (EGCG), as an inhibitor of HCV entry. Green tea catechins, such as EGCG and its derivatives, epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC), have been previously found to exert antiviral and antioncogenic properties. EGCG had no effect on HCV RNA replication, assembly, or release of progeny virions. However, it potently inhibited Cell-culture–derived HCV (HCVcc) entry into hepatoma cell lines as well as primary human hepatocytes. The effect was independent of the HCV genotype, and both infection of cells by extracellular virions and cell-to-cell spread were blocked. Pretreatment of cells with EGCG before HCV inoculation did not reduce HCV infection, whereas the application of EGCG during inoculation strongly inhibited HCV infectivity. Moreover, treatment with EGCG directly during inoculation strongly inhibited HCV infectivity. Expression levels of all known HCV (co-)receptors were unaltered by EGCG. Finally, we showed that EGCG inhibits viral attachment to the cell, thus disrupting the initial step of HCV cell entry. Conclusion: The green tea molecule, EGCG, potently inhibits HCV entry and could be part of an antiviral strategy aimed at the prevention of HCV reinfection after liver transplantation  

5.998           Long-term VEGF-A expression promotes aberrant angiogenesis and fibrosis in skeletal muscle

Karvinen, H., Pasanen, E., Rissanen, T.T., Korpisalo, P., Vähäkangas, E., Jazwa, A., Giacca, M. and Ylä-Herttuala, S. Gene Therapy, 18(12), 1166-1172 (2011)   Vascular endothelial growth factor A (VEGF-A) induces strong angiogenesis and it has been widely used in proangiogenic gene therapy studies. However, little is known about long-term effects of VEGF-A expression in skeletal muscle. Here the long- term effects of adeno-associated virus (AAV) encoding human VEGF-A₁₆₅ (AAV-VEGF-A) gene transfer in normal and ischemic rabbit hindlimb skeletal muscles were studied. AAV-LacZ was used as a control. In one-year follow-up, a remarkable increase in skeletal muscle perfusion compared with AAV-LacZ was observed measured with Doppler and contrast pulse sequence ultrasound. Angiogenesis was also seen in histology as enlarged and sprouting capillaries. In addition to favorable angiogenic effects, aberrant vascular structures with CD31 positive cell layers were seen inside muscle fibers after AAV-VEGF-A gene transfer. Importantly, we found increased amounts of extracellular matrix with a high number of macrophages and fibrosis in AAV-VEGF-A transduced muscles. No changes in skeletal muscle morphology were detected in AAV-LacZ transduced muscles. Our results indicate that local AAV-VEGF-A gene transfer efficiently promotes long-term angiogenesis in large animal model. However, non-regulated expression of VEGF-A causes unfavorable changes in muscle morphology, which suggests the need for regulation of the transgene expression in long-term AAV-mediated VEGF-A gene transfer applications.  

5.999           Alix regulates egress of hepatitis B virus naked capsid particles in an ESCRT-independent manner

Bardens, A., Döring, T., Stieler, J and Prange, R.

Cell. Microbiol., 13(4), 602-619 (2011)

Hepatitis B virus (HBV) is an enveloped DNA virus that exploits the endosomal sorting complexes required for transport (ESCRT) pathway for budding. In addition to infectious particles, HBV-replicating cells release non-enveloped (nucleo)capsids, but their functional implication and pathways of release are unclear. Here, we focused on the molecular mechanisms and found that the sole expression of the HBV core protein is sufficient for capsid release. Unexpectedly, released capsids are devoid of a detectable membrane bilayer, implicating a non-vesicular exocytosis process. Unlike virions, naked capsid budding does not require the ESCRT machinery. Rather, we identified Alix, a multifunctional protein with key roles in membrane biology, as a regulator of capsid budding. Ectopic overexpression of Alix enhanced capsid egress, while its depletion inhibited capsid release. Notably, the loss of Alix did not impair HBV production, furthermore indicating that virions and capsids use diverse export routes. By mapping of Alix domains responsible for its capsid release-mediating activity, its Bro1 domain was found to be required and sufficient. Alix binds to core via its Bro1 domain and retained its activity even if its ESCRT-III binding site is disrupted. Together, the boomerang-shaped Bro1 domain of Alix appears to escort capsids without ESCRT.

 

5.1000           Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

Neumann, F., Borchert, S., Schmidt, C., Reimer, r., Hohenberg, H., Fischer, N. and Grundhoff, A. PloS One, 6(12), e29112 (2011)   Merkel Cell Polyomavirus (MCPyV) genomes are clonally integrated in tumor tissues of approximately 85% of all Merkel cell carcinoma (MCC) cases, a highly aggressive tumor of the skin which predominantly afflicts elderly and immunosuppressed patients. All integrated viral genomes recovered from MCC tissue or MCC cell lines harbor signature mutations in the early gene transcript encoding for the large T-Antigen (LT-Ag). These mutations selectively abrogate the ability of LT-Ag to support viral replication while still maintaining its Rb-binding activity, suggesting a continuous requirement for LT-Ag mediated cell cycle deregulation during MCC pathogenesis. To gain a better understanding of MCPyV biology, in vitro MCPyV replication systems are required. We have generated a synthetic MCPyV genomic clone (MCVSyn) based on the consensus sequence of MCC-derived sequences deposited in the NCBI database. Here, we demonstrate that transfection of recircularized MCVSyn DNA into some human cell lines recapitulates efficient replication of the viral genome, early and late gene expression together with virus particle formation. However, serial transmission of infectious virus was not observed. This in vitro culturing system allows the study of viral replication and will facilitate the molecular dissection of important aspects of the MCPyV lifecycle.  

5.1001           Persistent Suppression of Ocular Neovascularization with Intravitreal Administration of AAVrh.10 Coding for Bevacizumab

Mao, Y., Kiss, S., Boyer, J.L., Hackett, N.R., Qiu, J., Carbone, A., Mezey, J.G., Kaminsky, S.M., D’Amico, D.J. and Crystal, R.G. Human Gene Therapy, 22, 1525-1535 (2011)   Vascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of neovascular age-related macular degeneration and diabetic retinopathy. Bevacizumab, an anti-VEGF monoclonal antibody, is efficacious for these disorders, but requires monthly intravitreal administration, with associated discomfort, cost, and adverse event risk. We hypothesized that a single intravitreal administration of adeno-associated virus (AAV) vector expressing bevacizumab would result in persistent eye expression of bevacizumab and suppress VEGF-induced retinal neovascularization. We constructed an AAV rhesus serotype rh.10 vector to deliver bevacizumab (AAVrh.10BevMab) and assessed its ability to suppress neovascularization in transgenic mice overexpressing human VEGF165 in photoreceptors. Intravitreal AAVrh.10BevMab directed long-term bevacizumab expression in the retinal pigmented epithelium. Treated homozygous mice had reduced levels of neovascularization, with 90±4% reduction 168 days following treatment. Thus, a single administration of AAVrh.10BevMab provides long-term suppression of neovascularization without the costs and risks associated with the multiple administrations required for the current conventional bevacizumab monoclonal drug delivery.  

5.1002           Mutations at the Base of the Icosahedral Five-Fold Cylinders of Minute Virus of Mice Induce 3′-to-5′ Genome Uncoating and Critically Impair Entry Functions

Cotmore, S.F. and Tattersall, P.

1. Virol., 86(1), 69-80 (2012)

  The linear single-stranded DNA genome of minute virus of mice can be ejected, in a 3′-to-5′ direction, via a cation-linked uncoating reaction that leaves the 5′ end of the DNA firmly complexed with its otherwise intact protein capsid. Here we compare the phenotypes of four mutants, L172T, V40A, N149A, and N170A, which perturb the base of cylinders surrounding the icosahedral 5-fold axes of the virus, and show that these structures are strongly implicated in 3′-to-5′ release. Although noninfectious at 37°C, all mutants were viable at 32°C, showed a temperature-sensitive cell entry defect, and, after proteolysis of externalized VP2 N termini, were unable to protect the VP1 domain, which is essential for bilayer penetration. Mutant virus yields from multiple-round infections were low and were characterized by the accumulation of virions containing subgenomic DNAs of specific sizes. In V40A, these derived exclusively from the 5′ end of the genome, indicative of 3′-to-5′ uncoating, while L172T, the most impaired mutant, had long subgenomic DNAs originating from both termini, suggesting additional packaging portal defects. Compared to the wild type, genome release in vitro following cation depletion was enhanced for all mutants, while only L172T released DNA, in both directions, without cation depletion following proteolysis at 37°C. Analysis of progeny from single-round infections showed that uncoating did not occur during virion assembly, release, or extraction. However, unlike the wild type, the V40A mutant extensively uncoated during cell entry, indicating that the V40-L172 interaction restrains an uncoating trigger mechanism within the endosomal compartment.  

5.1003           A Novel Self-Replicating Chimeric Lentivirus-Like Particle

Jurgens, C.K., Young, K.R., Madden, V.J., Johnson, P.R. and Johnston, R.E.

1. Virol., 86(1), 246-261 (2012)

  Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4⁺ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.    

5.1004           Innate Sensing of Foamy Viruses by Human Hematopoietic Cells

Rua, R., Lepelley, A., Gessain, A. and Schwartz, O.

1. Virol., 86(2), 909-918 (2012)

  Foamy viruses (FV) are nonpathogenic retroviruses that have cospeciated with primates for millions of years. FV can be transmitted through severe bites from monkeys to humans. Viral loads remain generally low in infected humans, and no secondary transmission has been reported. Very little is known about the ability of FV to trigger an innate immune response in human cells. A few previous reports suggested that FV do not induce type I interferon (IFN) in nonhematopoietic cells. Here, we examined how human hematopoietic cells sense FV particles and FV-infected cells. We show that peripheral blood mononuclear cells (PBMCs), plasmacytoid dendritic cells (pDCs), and the pDC-like cell line Gen2.2 detect FV, produce high levels of type I IFN, and express the IFN-stimulated gene MxA. Fewer than 20 FV-infected cells are sufficient to trigger an IFN response. Both prototypic and primary viruses stimulated IFN release. Donor cells expressing a replication-defective virus, carrying a mutated reverse transcriptase, induced IFN production by target cells as potently as wild-type virus. In contrast, an FV strain with env deleted, which does not produce viral particles, was inactive. IFN production was blocked by an inhibitor of endosomal acidification (bafilomycin A1) and by an endosomal Toll-like receptor (TLR) antagonist (A151). Silencing experiments in Gen2.2 further demonstrated that TLR7 is involved in FV recognition. Therefore, FV are potent inducers of type I IFN by pDCs and by PBMCs. This previously underestimated activation of the innate immune response may be involved in the control of viral replication in humans.  

5.1005           An adaptive mutation in NS2 is essential for efficient production of infectious 1b/2a

chimeric hepatitis C virus in cell culture

Chan, K., Cheng, G., Beran, R.K.F., Yang, H., Appleby, T.C., Pokrovskii, M.V., Mo, H., Zhonmg, W. and Delaney IV, W.E. Virology, 422, 224-234 (2012)   The development of JFH1 based intergenotypic recombinants which exploit the unique replication characteristics of JFH1 has made it possible to study infectious HCV encoding the structural genes of additional HCV genotypes including genotype 1b. Although, intergenotypic 1b/2a chimeric genomes replicate efficiently in transfected cells they produce very low viral titers, limiting the utility of this system. Here, intergenotypic 1b/2a variants were generated by serially passaging the virus in a novel highly permissive Huh-7 cell clone. The adapted virus was 1000-fold more infectious than the parental unadapted virus and six adapted mutations were identified throughout the genome. Of the mutations identified, L839S in the NS2 gene was the most critical for the adapted phenotype by enhancing the infectivity of assembled viral particles. Overall, the efficient production of infectious 1b/2a virus particles will facilitate the discovery and characterization of inhibitors targeting steps that involve the structural genes of genotype 1b HCV.  

5.1006           The high risk HPV16 L2 minor capsid protein has multiple transport signals that mediate its nucleocytoplasmic traffic

Mamoor, S., Onder, Z., Karanam, B., Kwak, K., Bordeaux, J., Crosby, L., Roden, R.B.S. and Moroianu, J. Virology, 422(2), 413-424 (2012)   In this study we examined the transport signals contributing to HPV16 L2 nucleocytoplasmic traffic using confocal microscopy analysis of enhanced green fluorescent protein—L2 (EGFP-L2) fusions expressed in HeLa cells. We confirmed that both nuclear localization signals (NLSs), the nNLS (1MRHKRSAKRTKR12) and cNLS (456RKRRKR461), previously characterized in vitro (Darshan et al., 2004), function independently in vivo. We discovered that a middle region rich in arginine residues (296SRRTGIRYSRIGNKQTLRTRS316) functions as a nuclear retention sequence (NRS), as mutagenesis of critical arginine residues within this NRS reduced the fraction of L2 in the nucleus despite the presence of both NLSs. Significantly, the infectivity of HPV16 pseudoviruses containing either RR297AA or RR297EE within the L2 NRS was strongly reduced both in HaCaT cells and in a murine challenge model. Experiments using Ratjadone A nuclear export inhibitor and mutation-localization analysis lead to the discovery of a leucine-rich nuclear export signal (₄₆₂LPYFFSDVSL) mediating 16L2 nuclear export. These data indicate that HPV16 L2 nucleocytoplasmic traffic is dependent on multiple functional transport signals.      

5.1007           Spinal Delivery of AAV Vector Restores Enzyme Activity and Increases Ventilation in Pompe Mice

Qiu, K., Falk, D.J., Reier, P.J., Byrne, B.J. ansd Fuller, D.D. Molecular Therapy, 20(1), 21-27 (2012)   Pompe disease is a form of muscular dystrophy due to lysosomal storage of glycogen caused by deficiency of acid α-glucosidase (GAA). Respiratory failure in Pompe disease has been attributed to respiratory muscle dysfunction. However, evaluation of spinal tissue from Pompe patients and animal models indicates glycogen accumulation and lower motoneuron pathology. We hypothesized that restoring GAA enzyme activity in the region of the phrenic motor nucleus could lead to improved breathing in a murine Pompe model (the Gaa^−/− mouse). Adeno-associated virus serotype 5 (AAV5), encoding either GAA or green fluorescent protein (GFP), was delivered at the C₃–C₄ spinal level of adult Gaa^−/− mice and the spinal cords were harvested 4 weeks later. AAV5-GAA injection restored spinal GAA enzyme activity and GAA immunostaining was evident throughout the cervical ventral horn. The periodic acid Schiff (PAS) method was used to examine neuronal glycogen accumulation, and spinal PAS staining was attenuated after AAV5-GAA injection. Lastly, plethysmography revealed that minute ventilation was greater in unanesthetized AAV5-GAA versus AAV5-GFP treated Gaa^−/− mice at 1–4 months postinjection. These results support the hypothesis that spinal cord pathology substantially contributes to ventilatory dysfunction in Gaa^−/− mice and therefore requires further detailed evaluation in patients with Pompe disease.  

5.1008           Neutralizing Antibodies Against AAV Serotypes 1, 2, 6, and 9 in Sera of Commonly Used Animal Models

Rapti, K., Loius-Jeune, V., Kohlbrenner, E., Ishikawa, K., Ladage, D., Zolotukhin, S., Hajjar, R.J. and Weber, T. Molecular Therapy, 20(1), 73-83 (2012)   Adeno-associated virus (AAV)-based vectors are promising gene delivery vehicles for human gene transfer. One significant obstacle to AAV-based gene therapy is the high prevalence of neutralizing antibodies in humans. Until now, it was thought that, except for nonhuman primates, pre-existing neutralizing antibodies are not a problem in small or large animal models for gene therapy. Here, we demonstrate that sera of several animal models of cardiovascular diseases harbor pre-existing antibodies against the cardiotropic AAV serotypes AAV1, AAV6, and AAV9 and against AAV2. The neutralizing antibody titers vary widely both between species and between serotypes. Of all species tested, rats displayed the lowest levels of neutralizing antibodies. Surprisingly, naive mice obtained directly from commercial vendors harbored neutralizing antibodies. Of the large animal models tested, the neutralization of AAV6 transduction by dog sera was especially pronounced. Sera of sheep and rabbits showed modest neutralization of AAV transduction whereas porcine sera strongly inhibited transduction by all AAV serotypes and displayed the largest variation between individual animals. Importantly, neutralizing antibody titers as low as 1/4 completely prevented in vivo transduction by AAV9 in rats. Our results suggest that prescreening of animals for neutralizing antibodies will be important for future gene transfer experiments in these animal models.  

5.1009           CREB-activity and nmnat2 transcription are down-regulated prior to neurodegeneration, while NMNAT2 over-expression is neuroprotective, in a mouse model of human tauopathy

Ljungberg, M.C., Ali, Y.O., Zhu, J., WU, C-S., Oka, K., Zhai, R.G. and Lu, H-C. Human. Mol. Genet., 21(2), 251-267 (2012)   Tauopathies, characterized by neurofibrillary tangles (NFTs) of phosphorylated tau proteins, are a group of neurodegenerative diseases, including frontotemporal dementia and both sporadic and familial Alzheimer's disease. Forebrain-specific over-expression of human tau_P301L, a mutation associated with frontotemporal dementia with parkinsonism linked to chromosome 17, in rTg4510 mice results in the formation of NFTs, learning and memory impairment and massive neuronal death. Here, we show that the mRNA and protein levels of NMNAT2 (nicotinamide mononucleotide adenylyltransferase 2), a recently identified survival factor for maintaining neuronal health in peripheral nerves, are reduced in rTg4510 mice prior to the onset of neurodegeneration or cognitive deficits. Two functional cAMP-response elements (CREs) were identified in the nmnat2 promoter region. Both the total amount of phospho-CRE binding protein (CREB) and the pCREB bound to nmnat2 CRE sites in the cortex and the hippocampus of rTg4510 mice are significantly reduced, suggesting that NMNAT2 is a direct target of CREB under physiological conditions and that tau_P301L overexpression down-regulates CREB-mediated transcription. We found that over-expressing NMNAT2 or its homolog NMNAT1, but not NMNAT3, in rTg4510 hippocampi from 6 weeks of age using recombinant adeno-associated viral vectors significantly reduced neurodegeneration caused by tau_P301L over-expression at 5 months of age. In summary, our studies strongly support a protective role of NMNAT2 in the mammalian central nervous system. Decreased endogenous NMNAT2 function caused by reduced CREB signaling during pathological insults may be one of underlying mechanisms for neuronal death in tauopathies.  

5.1010           AAV8 vector expressing IL24 efficiently suppresses tumor growth mediated by specific mechanisms in MLL/AF4-positive ALL model mice

Tamia, H., Miyake, K., Yamaguchi, H., Takatori, M., Dan, K., Inokuchi, K. and Shimada, T. Blood, 119(1), 64-71 (2012)   Mixed-lineage leukemia (MLL)/AF4-positive acute lymphoblastic leukemia (ALL) is a common type of leukemia in infants, which is associated with a high relapse rate and poor prognosis. IL24 selectively induces apoptosis in cancer cells and exerts immunomodulatory and antiangiogenic effects. We examined the effects of adeno-associated virus type 8 (AAV8) vector-mediated muscle-directed systemic gene therapy in MLL/AF4-positive ALL using IL24. In a series of in vitro studies, we examined the effects of AAV8-IL24–transduced C2C12 cell-conditioned medium. We also examined the effects of AAV8-IL24 in MLL/AF4 transgenic mice. The results revealed the effects of AAV8-IL24 in MLL/AF4-positive ALL both in vitro and in vivo. With regard to the mechanism of therapy using AAV8-IL24 in MLL/AF4-positive ALL, we demonstrated the antiangiogenicity and effects on the ER stress pathway and unreported pathways through inhibition of S100A6 and HOXA9, which is specific to MLL/AF4-positive ALL. Inhibition of S100A6 by IL24 was dependent on TNF-α and induced acetylation of p53 followed by activation of the caspase 8–caspase 3 apoptotic pathway. Inhibition of HOXA9 by IL24, which was independent of TNF-α, induced MEIS1 activation followed by activation of the caspase 8–caspase 3 apoptotic pathway. Thus, gene therapy using AAV8-IL24 is a promising treatment for MLL/AF4-positive ALL.  

5.1011           Polyethylenimine Is a Strong Inhibitor of Human Papillomavirus and Cytomegalovirus Infection

Spoden, G.A., Besold, K., Krauter, S., Plachter, B., Hanik, N., Kilbinger, A.F.M., Lambert, C. and Florin, L. Antimicrob. Agents Chemother., 56(1), 75-82 (2012)   Polyethylenimines are cationic polymers with potential as delivery vectors in gene therapy and with proven antimicrobial activity. However, the antiviral activity of polyethylenimines has not been addressed in detail thus far. We have studied the inhibitory effects of a linear 25-kDa polyethylenimine on infections with human papillomaviruses and human cytomegaloviruses. Preincubation of cells with polyethylenimine blocked primary attachment of both viruses to cells, resulting in a significant reduction of infection. In addition, the dissemination of human cytomegalovirus in culture cells was efficiently reduced by recurrent administration of polyethylenimine. Polyethylenimine concentrations required for inhibition of human papillomavirus and cytomegalovirus did not cause any cytotoxic effects. Polyethylenimines and their derivatives may thus be attractive molecules for the development of antiviral microbicides.  

5.1012           The importance of being short: The role of rabies virus phosphoprotein isoforms assessed by differential IRES translation initiation

Marschalek, A., Drechsel, L. and Conzelmann, K-K. Eur. J. Immunol., 91(1), 17-23 (2012)   The rabies virus (RV) phosphoprotein P is a multifunctional protein involved in viral RNA synthesis and in counteracting host innate immune responses. We have previously shown that RV P gene expression levels can be regulated by using picornavirus internal ribosome entry site (IRES) elements. Here we exploited a particular feature of the foot-and-mouth disease virus (FMDV) IRES, namely, preferential initiation at a downstream initiation codon, to address the role of N-terminally truncated RV phosphoproteins usually generated in RV-infected cells through ribosomal leaky scanning. Recombinant RVs in which P synthesis was directed by the poliovirus or FMDV IRES produced full-length P (P1) or a truncated form (P2), as the dominant product, respectively. While the P2 overexpressing virus showed attenuated growth in interferon-incompetent cells, it was superior to the P1 overexpressing virus in preventing expression of host interferon-stimulated genes. This indicates that in RV infected cells the availability of the truncated P2 protein is critical for viral resistance to interferon.    

5.1013           Rescue of synaptic plasticity and spatial learning deficits in the hippocampus of Homer1 knockout mice by recombinant Adeno-associated viral gene delivery of Homer1c

Gerstein, H., O’Riordan, K., Osting, S., Schwarz, M. and Burger, C. Neurobiology of Learning and Memory, 97(1), 17-29 (2012)   Homer1 belongs to a family of scaffolding proteins that interact with various post-synaptic density proteins including group I metabotropic glutamate receptors (mGluR1/5). Previous research in our laboratory implicates the Homer1c isoform in spatial learning. Homer1 knockout mice (H1-KO) display cognitive impairments, but their synaptic plasticity properties have not been described. Here, we investigated the role of Homer1 in long-term potentiation (LTP) in the hippocampal CA1 region of H1-KO mice in vitro. We found that late-phase LTP elicited by high frequency stimulation (HFS) was impaired, and that the induction and maintenance of theta burst stimulation (TBS) LTP were reduced in H1-KO. To test the hypothesis that Homer1c was sufficient to rescue these LTP deficits, we delivered Homer1c to the hippocampus of H1-KO using recombinant adeno-associated virus (rAAV). We found that rAAV-Homer1c rescued HFS and TBS-LTP in H1-KO animals. Next, we tested whether the LTP rescue by Homer1c was occurring via mGluR1/5. A selective mGluR5 antagonist, but not an mGluR1 antagonist, blocked the Homer1c-induced recovery of late-LTP, suggesting that Homer1c mediates functional effects on plasticity via mGluR5. To investigate the role of Homer1c in spatial learning, we injected rAAV-Homer1c to the hippocampus of H1-KO. We found that rAAV-Homer1c significantly improved H1-KO performance in the Radial Arm Water Maze. These results point to a significant role for Homer1c in synaptic plasticity and learning.  

5.1014           Viral Vectors for RNA Interference Applications in Cancer Research and Therapy

Fechner, H. and Kurreck, J.  Drug Delivery in Oncology: From Basic Research to Cancer Therapy, 1415-1442 (2012)   No abstract available  

5.1015           Gene Therapy Restores Missing Cone-Mediated Vision in the CNGA3−/− Mouse Model of Achromatopsia

Michalakis, S. et al Advances in Experimental Medicine and Biology, 723(3), 183-189 (2012)   The absence of cyclic nucleotide-gated (CNG) channels in cone photoreceptor outer segments leads to achromatopsia, a severely disabling disease associated with the complete lack of cone photoreceptor function. In a common form, loss of the CNGA3 subunit disrupts visual transduction in cones and causes progressive degeneration. Here, we show that adeno-associated viral vector-mediated gene replacement therapy added the lacking sensual quality, cone-mediated vision, in the CNGA3^−/− mouse model of the human disease. The functional rescue of cone vision was assessed at different sites along the visual pathway. In particular, we show electrophysiologically that treated CNGA3^−/− mice became able to generate cone-mediated responses and to transfer these signals to bipolar and finally ganglion cells. In support, we found morphologically that expression of CNGA3 delayed cone cell death. Finally, we show in a behavioral test that treated mice acquired photopic vision suggesting that achromatopsia patients may as well benefit from gene replacement therapy.  

5.1016           Positive correlation between Merkel cell polyomavirus viral load and capsid-specific antibody titer

Pastrana, D.V., Wieland, U., Silling, S., Buck, C.B. and Pfister, H. Med. Microbiol. Immunol., 201, 17-23 (2012)   Merkel cell polyomavirus (MCPyV or MCV) is the first polyomavirus to be clearly implicated as a causal agent underlying a human cancer, Merkel cell carcinoma (MCC). Infection with MCPyV is common in the general population, and a majority of adults shed MCPyV from the surface of their skin. In this study, we quantitated MCPyV DNA in skin swab specimens from healthy volunteers sampled at different anatomical sites over time periods ranging from 3 months to 4 years. The volunteers were also tested using a serological assay that detects antibodies specific for the MCPyV virion. There was a positive correlation between MCPyV virion-specific antibody titers and viral load at all anatomical sites tested (dorsal portion of the hands, forehead, and buttocks) (Spearman’s r 0.644, P < 0.0001). The study results are consistent with previous findings suggesting that the skin is primary site of chronic MCPyV infection in healthy adults and suggest that the magnitude of an individual’s seroresponsiveness against the MCPyV virion generally reflects the overall MCPyV DNA load across wide areas of the skin. In light of previous reports indicating a correlation between MCC and strong MCPyV-specific seroresponsiveness, this model suggests that poorly controlled chronic MCPyV infection might be a risk factor in the development of MCC.  

5.1017           Corticospinal tract transduction: a comparison of seven adeno-associated viral vector serotypes and a non-integrating lentiviral vector

Hutson, T.H., Verhaagen, J., Yanez-Munoz, R.J. and Moon, L.D.F. Gene Therapy, 19(1), 49-60 (2012)   The corticospinal tract (CST) is extensively used as a model system for assessing potential therapies to enhance neuronal regeneration and functional recovery following spinal cord injury (SCI). However, efficient transduction of the CST is challenging and remains to be optimised. Recombinant adeno-associated viral (AAV) vectors and integration-deficient lentiviral vectors are promising therapeutic delivery systems for gene therapy to the central nervous system (CNS). In the present study the cellular tropism and transduction efficiency of seven AAV vector serotypes (AAV1, 2, 3, 4, 5, 6, 8) and an integration-deficient lentiviral vector were assessed for their ability to transduce corticospinal neurons (CSNs) following intracortical injection. AAV1 was identified as the optimal serotype for transducing cortical and CSNs with green fluorescent protein (GFP) expression detectable in fibres projecting through the dorsal CST (dCST) of the cervical spinal cord. In contrast, AAV3 and AAV4 demonstrated a low efficacy for transducing CNS cells and AAV8 presented a potential tropism for oligodendrocytes. Furthermore, it was shown that neither AAV nor lentiviral vectors generate a significant microglial response. The identification of AAV1 as the optimal serotype for transducing CSNs should facilitate the design of future gene therapy strategies targeting the CST for the treatment of SCI.  

5.1018           Preclinical evaluation of a clinical candidate AAV8 vector for ornithine transcarbamylase (OTC) deficiency reveals functional enzyme from each persisting vector genome

Wang, L., Morizono, H., Lin, J., Bell, P., Jones, D., MCMenamin, D., Yu, H., Batshaw, M.L. and Wilson, J.M. Molecular Genetics and Metabolism, 105, 203-211 (2012)   Ornithine transcarbamylase deficiency (OTCD), the most common and severe urea cycle disorder, is an excellent model for developing liver-directed gene therapy. No curative therapy exists except for liver transplantation which is limited by available donors and carries significant risk of mortality and morbidity. Adeno-associated virus 8 (AAV8) has been shown to be the most efficient vector for liver-directed gene transfer and is currently being evaluated in a clinical trial for treating hemophilia B. In this study, we generated a clinical candidate vector for a proposed OTC gene therapy trial in humans based on a self-complementary AAV8 vector expressing codon-optimized human OTC (hOTCco) under the control of a liver-specific promoter. Codon-optimization dramatically improved the efficacy of OTC gene therapy. Supraphysiological expression levels and activity of hOTC were achieved in adult spfash mice following a single intravenous injection of hOTCco vector. Vector doses as low as 1 × 10¹⁰ genome copies (GC) achieved robust and sustained correction of the OTCD biomarker orotic aciduria and clinical protection against an ammonia challenge. Functional expression of hOTC in 40% of liver areas was found in mice treated with a low vector dose of 1 × 10⁹ GC. We suggest that the clinical candidate vector we have developed has the potential to achieve therapeutic effects in OTCD patients.  

5.1019           Frontotemporal lobar degeneration-related proteins induce only subtle memory-related deficits when bilaterally overexpressed in the dorsal hippocampus

Dayton, R.D., Wang, D.B., Cain, C.D., Schrott, L.M., Ramirez, J.J., King, M.A. and Klein, R.L. Exp. Neurol., 233, 807-814 (2012)   Frontotemporal lobar degeneration (FTLD) is a neurodegenerative disease that involves cognitive decline and dementia. To model the hippocampal neurodegeneration and memory-related behavioral impairment that occurs in FTLD and other tau and TDP-43 proteinopathy diseases, we used an adeno-associated virus serotype 9 (AAV9) vector to induce bilateral expression of either microtubule-associated protein tau or transactive response DNA binding protein 43 kDa (TDP-43) in adult rat dorsal hippocampus. Human wild-type forms of tau or TDP-43 were expressed. The vectors/doses were designed for moderate expression levels within neurons. Rats were evaluated for acquisition and retention in the Morris water task over 12 weeks after gene transfer. Neither vector altered acquisition performance compared to controls. In measurements of retention, there was impairment in the TDP-43 group. Histological examination revealed specific loss of dentate gyrus granule cells and concomitant gliosis proximal to the injection site in the TDP-43 group, with shrinkage of the dorsal hippocampus. Despite specific tau pathology, the tau gene transfer surprisingly did not cause obvious neuronal loss or behavioral impairment. The data demonstrate that TDP-43 produced mild behavioral impairment and hippocampal neurodegeneration in rats, whereas tau did not. The models could be of value for studying mechanisms of FTLD and other diseases with tau and TDP-43 pathology in the hippocampus including Alzheimer's disease, with relevance to early stage mild impairment.  

5.1020           Novel antivirals inhibit early steps in HPV infection

Huang, H-S., Pyeon, D., Pearce, S.M., lank, S.M., Griffin, L.M., Ahlquist, P. and Lambert, P.F. Antiviral Res., 93, 280-287 (2012)   The future incidence of cervical cancer is forecast to decline because of the remarkably effective prophylactic vaccines against human papillomaviruses. However, lack of access to these expensive vaccines in the developing countries where cervical cancer is most frequent, and the restricted genotypes these vaccines protect against, will limit their impact. Clearly, there is still a need for identifying other modalities for preventing HPV infections. Ready access to effective, inexpensive antivirals represents one potentially valuable approach to the prevention of genital HPV infections. We developed a well-validated high throughput screening (HTS) assay for identifying compounds that inhibit HPV infection and applied this assay to identify lead compounds that act by inhibiting an early step in infection. We screened over 40,000 small molecules that were available at the University of Wisconsin Small Molecule Screening Facility (UW-SMSF). The top 22 compounds were chosen for further analyses based upon the pharmacological property, scaffold diversity, strength of the inhibitory activity and lack of nonspecific cytotoxicity. Of these compounds, #13 and #14 had the most acceptable properties of low to submicromolar IC₅₀’s and low cytotoxicity. Optimal antiviral activities were elicited by exposure of cells to the #13 and #14 during the initial 12 h following infection. Twenty-nine #13-like and 15 #14-like analogs were identified in silico and tested for their antiviral activities corresponded to the altered structures comparing to #13 and #14, informing on the pharmacophore structure of each compound. Studies indicate that both compounds inhibit infection post-entry.  

5.1021           Derivation of non-infectious envelope proteins from virions isolated from plasma negative for HIV antibodies

Vyas, G.N., Stoddart, C.A., Killian, M.S., Brennan, T.V., Goldberg, T., Ziman, A. and Bryson, Y. Biologicals, 40(1), 15-20 (2012)   Natural membrane-bound HIV-1 envelope proteins (mHIVenv) could be used to produce an effective subunit vaccine against HIV infection, akin to effective vaccination against HBV infection using the hepatitis B surface antigen. The quaternary structure of mHIVenv is postulated to elicit broadly neutralizing antibodies protective against HIV-1 transmission. The founder virus transmitted to infected individuals during acute HIV-1 infection is genetically homogeneous and restricted to CCR5-tropic phenotype. Therefore, isolates of plasma-derived HIV-1 (PHIV) from infected blood donors while negative for antibodies to HIV proteins were selected for expansion in primary lymphocytes as an optimized cell substrate (OCS). Virions in the culture supernatants were purified by removing contaminating microvesicles using immunomagnetic beads coated with anti-CD45. Membrane cholesterol was extracted from purified virions with beta-cyclodextrin to permeabilize them and expel p24, RT and viral RNA, and permit protease-free Benzonase to hydrolyze the residual viral/host DNA/RNA without loss of gp120. The resultant mHIVenv, containing gp120 bound to native gp41 in immunoreactive form, was free from infectivity in vitro in co-cultures with OCS and in vivo after inoculating SCID-hu Thy/Liv mice. These data should help development of mHIVenv as a virally safe immunogen and enable preparation of polyclonal hyper-immune globulins for immunoprophylaxis against HIV-1 infection.  

5.1022           Progressive neurodegenerative and behavioural changes induced by AAV-mediated overexpression of α-synuclein in midbrain dopamine neurons

Decressac, M., Mattsson, B., Lundblad, m., Weikop, P. and Björklund, A. Neurobiology of Disease, 45, 939-953 (2012)   Parkinson's disease (PD) is characterised by the progressive loss of nigral dopamine neurons and the presence of synucleinopathy. Overexpression of α-synuclein in vivo using viral vectors has opened interesting possibilities to model PD-like pathology in rodents. However, the attempts made so far have failed to show a consistent behavioural phenotype and pronounced dopamine neurodegeneration. Using a more efficient adeno-associated viral (AAV) vector construct, which includes a WPRE enhancer element and uses the neuron-specific synapsin-1 promoter to drive the expression of human wild-type α-synuclein, we have now been able to achieve increased levels of α-synuclein in the transduced midbrain dopamine neurons sufficient to induce profound deficits in motor function, accompanied by reduced expression of proteins involved in dopamine neurotransmission and a time-dependent loss of nigral dopamine neurons, that develop progressively over 2–4 months after vector injection. As in human PD, nigral cell loss was preceded by degenerative changes in striatal axons and terminals, and the appearance of α-synuclein positive inclusions in dystrophic axons and dendrites, supporting the idea that α-synuclein-induced pathology hits the axons and terminals first and later progresses to involve also the cell bodies. The time-course of changes seen in the AAV-α-synuclein treated animals defines distinct stages of disease progression that matches the pre-symptomatic, early symptomatic, and advanced stages seen in PD patients. This model provides new interesting possibilities for studies of stage-specific pathologic mechanisms and identification of targets for disease-modifying therapeutic interventions linked to early or late stages of the disease.  

5.1023           Matrigel-embedded 3D culture of Huh-7 cells as a hepatocyte-like polarized system to study hepatitis C virus cycle

Molina-Jimenez, F., Benedicto, I., Thi, V.L.D., Gondar, V., Lavillette, D., Marin, J.J., Briz, O., Moreno-Otero, R., Aldabe, r., Baumert, T.F., Cosset, F-L., Lopez-Cabrera, M. and Majano, P.L. Virology, 425, 31-39 (2012)   Hepatocytes are highly polarized cells where intercellular junctions, including tight junctions (TJs), determine the polarity. Recently, the TJ-associated proteins claudin-1 and occludin have been implicated in hepatitis C virus (HCV) entry and spread. Nevertheless, cell line-based experimental systems that exhibit hepatocyte-like polarity and permit robust infection and virion production are not currently available. Thus, we sought to determine whether cell line-based, Matrigel-embedded cultures could be used to study hepatitis C virus (HCV) infection and virion production in a context of hepatocyte-like polarized cells. In contrast to standard bidimensional cultures, Matrigel-cultured Huh-7 cells adopted hepatocyte polarization features forming a continuous network of functional proto-bile canaliculi structures. These 3D cultures supported HCV infection by JFH-1 virus and produced infective viral particles which shifted towards lower densities with higher associated specific infectivity. In conclusion, our findings describe a novel use of Matrigel to study the entire HCV cycle in a more relevant context.  

5.1024           Characterization of Human Endogenous Retroviral Elements in the Blood of HIV-1-Infected Individuals

Contrera-Galindo, R., Kaplan, M.H., Contreras-Galindo, A.C., Gonzales-Hernandez, M.J., Ferlenghi, I., Giusti, F., Lorenzo, E., Gitlin, S.D., Dosik, M.H., Yamamura, Y. and Markovitz, D.M.

1. Virol., 86(1), 262-276 (2012)

  We previously reported finding the RNA of a type K human endogenous retrovirus, HERV-K (HML-2), at high titers in the plasma of HIV-1-infected and cancer patients (R. Contreras-Galindo et al., J. Virol. 82:9329–9236, 2008.). The extent to which the HERV-K (HML-2) proviruses become activated and the nature of their activated viral RNAs remain important questions. Therefore, we amplified and sequenced the full-length RNA of the env gene of the type 1 and 2 HERV-K (HML-2) viruses collected from the plasma of seven HIV-1-infected patients over a period of 1 to 3 years and from five breast cancer patients in order to reconstruct the genetic evolution of these viruses. HERV-K (HML-2) RNA was found in plasma fractions of HIV-1 patients at a density of ∼1.16 g/ml that contained both immature and correctly processed HERV-K (HML-2) proteins and virus-like particles that were recognized by anti-HERV-K (HML-2) antibodies. RNA sequences from novel HERV-K (HML-2) proviruses were discovered, including K111, which is specifically active during HIV-1 infection. Viral RNA arose from complete proviruses and proviruses devoid of a 5′ long terminal repeat, suggesting that the expression of HERV-K (HML-2) RNA in these patients may involve sense and antisense transcription. In HIV-1-infected individuals, the HERV-K (HML-2) viral RNA showed evidence of frequent recombination, accumulation of synonymous rather than nonsynonymous mutations, and conserved N-glycosylation sites, suggesting that some of the HERV-K (HML-2) viral RNAs have undergone reverse transcription and are under purifying selection. In contrast, HERV-K (HML-2) RNA sequences found in the blood of breast cancer patients showed no evidence of recombination and exhibited only sporadic viral mutations. This study suggests that HERV-K (HML-2) is active in HIV-1-infected patients, and the resulting RNA message reveals previously undiscovered HERV-K (HML-2) genomic sequences.    

5.1025           Conserved Glycine 33 Residue in Flexible Domain I of Hepatitis C Virus Core Protein Is Critical for Virus Infectivity

Angus, A.G.N., Loquet, A., Stack, S.J., Dalrympie, D., Gatherer, D., Penin, F. and Patel, A.H.

1. Virol., 86(2), 679-690 (2012)

  Hepatitis C virus core protein forms the viral nucleocapsid and plays a critical role in the formation of infectious particles. In this study, we demonstrate that the highly conserved residue G33, located within domain 1 of the core protein, is important for the production of cell culture-infectious virus (HCVcc). Alanine substitution at this position in the JFH1 genome did not alter viral RNA replication but reduced infectivity by ∼2 logs. Virus production by this core mutant could be rescued by compensatory mutations located immediately upstream and downstream of the original G33A mutation. The examination of the helix-loop-helix motif observed in the core protein structure (residues 15 to 41; Protein Data Bank entry 1CWX) indicated that the residues G33 and F24 are in close contact with each other, and that the G33A mutation induces a steric clash with F24. Molecular simulations revealed that the compensatory mutations increase the helix-loop-helix flexibility, allowing rescue of the core active conformation required for efficient virus production. Taken together, these data highlight the plasticity of core domain 1 conformation and illustrate the relationship between its structural tolerance to mutations and virus infectivity.  

5.1026           Espirito Santo Virus: a New Birnavirus That Replicates in Insect Cells

Vancini, R., Paredes, A., Ribeiro, M., Blackburn, K., Ferreira, D., Kononchik Jr., J.P., Hernandez, R. and Brown, D.

1. Virol., 86(5), 2390-2399 (2012)

  Espirito Santo virus (ESV) is a newly discovered virus recovered as contamination in a sample of a virulent strain of dengue-2 virus (strain 44/2), which was recovered from a patient in the state of Espirito Santo, Brazil, and amplified in insect cells. ESV was found to be dependent upon coinfection with a virulent strain of dengue-2 virus and to replicate in C6/36 insect cells but not in mammalian Vero cells. A sequence of the genome has been produced by de novo assembly and was not found to match to any known viral sequence. An incomplete match to the nucleotide sequence of the RNA-dependent RNA polymerase from Drosophila X virus (DXV), another birnavirus, could be detected. Mass spectrometry analysis of ESV proteins found no matches in the protein data banks. However, peptides recovered by mass spectrometry corresponded to the de novo-assembled sequence by BLAST analysis. The composition and three-dimensional structure of ESV are presented, and its sequence is compared to those of other members of the birnavirus family. Although the virus was found to belong to the family Birnaviridae, biochemical and sequence information for ESV differed from that of DXV, the representative species of the genus Entomobirnavirus. Thus, significant differences underscore the uniqueness of this infectious agent, and its relationship to the coinfecting virus is discussed.  

5.1027           Interactions of the Cytoplasmic Domain of Sindbis Virus E2 with Nucleocapsid Cores Promote Alphavirus Budding

Jose, J., Przybyla, L., Edwards, T.J., Perera, R., Burgner II, J.W. and Kuhn, R.J.

1. Virol., 86(5), 2585-2599 (2012)

  Alphavirus budding from the plasma membrane occurs through the specific interaction of the nucleocapsid core with the cytoplasmic domain of the E2 glycoprotein (cdE2). Structural studies of the Sindbis virus capsid protein (CP) have suggested that these critical interactions are mediated by the binding of cdE2 into a hydrophobic pocket in the CP. Several molecular genetic studies have implicated amino acids Y400 and L402 in cdE2 as important for the budding of alphaviruses. In this study, we characterized the role of cdE2 residues in structural polyprotein processing, glycoprotein transport, and capsid interactions. Along with hydrophobic residues, charged residues in the N terminus of cdE2 were critical for the effective interaction of cores with cdE2, a process required for virus budding. Mutations in the C-terminal signal sequence region of cdE2 affected E2 protein transport to the plasma membrane, while nonbudding mutants that were defective in cdE2-CP interaction accumulated E2 on the plasma membrane. The interaction of cdE2 with cytoplasmic cores purified from infected cells and in vitro-assembled core-like particles suggests that cdE2 interacts with assembled cores to mediate budding. We hypothesize that these cdE2 interactions induce a change in the organization of the nucleocapsid core upon binding leading to particle budding and priming of the nucleocapsid cores for disassembly that is required for virus infection.    

5.1028           Adeno-associated virus-mediated brain delivery of 5-lipoxygenase modulates the AD-like phenotype of APP mice

Chu, J., Giannopoulos, P.F., Ceballos-Diaz, C., Golde, T.E. and Pratico, D. Mol. Neurogeneration, 7, 1-10 (2012)   Background The 5-lipoxygenase (5LO) enzymatic pathway is widely distributed within the central nervous system. Previous works showed that this protein is up-regulated in Alzheimer's disease (AD), and that its genetic absence results in a reduction of Amyloid beta (Aβ) levels in the Tg2576 mice. Here by employing an adeno-associated viral (AAV) vector system to over-express 5LO in the same mouse model, we examined its contribution to their cognitive impairments and brain AD-like amyloid pathology. Results Our results showed that compared with controls, 5LO-targeted gene brain over-expression in Tg2576 mice results in significant memory deficits. On the other hand, brain tissues had a significant elevation in the levels of Aβ peptides and deposition, no change in the steady state levels of amyloid-β precursor protein (APP), BACE-1 or ADAM-10, but a significant increase in PS1, nicastrin, and Pen-2, three major components of the γ-secretase complex. Additional data indicate that the transcription factor CREB was elevated and so were the mRNA levels for PS1, nicastrin and Pen-2. Conclusions These data demonstrate that neuronal 5LO plays a functional role in the pathogenesis of AD-like amyloidotic phenotype by modulating the γ-secretase pathway. They support the hypothesis that this enzyme is a novel therapeutic target for the treatment and prevention of AD.  

5.1029           Structural Organization of DNA in Chlorella Viruses

Wulfmeyer, T., Polzer, C., Hiepler, g., Hamacher, K., Shoeman, R., Dunigan, D.D., Van Etten, J.L., Lolicato, M., Moroni, A., Thiel, G. and Meckel, T. PloS One, 7(2), e30133 (2012)   Chlorella viruses have icosahedral capsids with an internal membrane enclosing their large dsDNA genomes and associated proteins. Their genomes are packaged in the particles with a predicted DNA density of ca. 0.2 bp nm⁻³. Occasionally infection of an algal cell by an individual particle fails and the viral DNA is dynamically ejected from the capsid. This shows that the release of the DNA generates a force, which can aid in the transfer of the genome into the host in a successful infection. Imaging of ejected viral DNA indicates that it is intimately associated with proteins in a periodic fashion. The bulk of the protein particles detected by atomic force microscopy have a size of ~60 kDa and two proteins (A278L and A282L) of about this size are among 6 basic putative DNA binding proteins found in a proteomic analysis of DNA binding proteins packaged in the virion. A combination of fluorescence images of ejected DNA and a bioinformatics analysis of the DNA reveal periodic patterns in the viral DNA. The periodic distribution of GC rich regions in the genome provides potential binding sites for basic proteins. This DNA/protein aggregation could be responsible for the periodic concentration of fluorescently labeled DNA observed in ejected viral DNA. Collectively the data indicate that the large chlorella viruses have a DNA packaging strategy that differs from bacteriophages; it involves proteins and share similarities to that of chromatin structure in eukaryotes.  

5.1030           Activation of a Helper and Not Regulatory Human CD4+ T Cell Response by Oncolytic H-1 Parvovirus

Morales, O., Richard, A., Martin, N., Mrizak, D., Senechal, M., Miroux, C., Pancre, V., Rommelaere, J., Caillet-Fauquet, P., de Launoit, Y. and Delhem, N. PloS One, 7(2), e32197 (2012)   Background H-1 parvovirus (H-1 PV), a rodent autonomous oncolytic parvovirus, has emerged as a novel class of promising anticancer agents, because of its ability to selectively find and destroy malignant cells. However, to probe H-1 PV multimodal antitumor potential one of the major prerequisites is to decipher H-1 PV direct interplay with human immune system, and so prevent any risk of impairment. Methodology/Principal findings Non activated peripheral blood mononuclear cells (PBMCs) are not sensitive to H-1 PV cytotoxic effect. However, the virus impairs both activated PBMC proliferation ability and viability. This effect is related to H-1 PV infection as evidenced by Western blotting detection of H-1 PV main protein NS1. However, TCID50 experiments did not allow newly generated virions to be detected. Moreover, flow cytometry has shown that H-1 PV preferentially targets B lymphocytes. Despite seeming harmful at first sight, H-1 PV seems to affect very few NK cells and CD8+ T lymphocytes and, above all, clearly does not affect human neutrophils and one of the major CD4+ T lymphocyte subpopulation. Very interestingly, flow cytometry analysis and ELISA assays proved that it even activates human CD4+ T cells by increasing activation marker expression (CD69 and CD30) and both effective Th1 and Th2 cytokine secretion (IL-2, IFN-γ and IL-4). In addition, H-1 PV action does not come with any sign of immunosuppressive side effect. Finally, we have shown the efficiency of H-1 PV on xenotransplanted human nasopharyngeal carcinoma, in a SCID mouse model reconstituted with human PBMC. Conclusions/Significance Our results show for the first time that a wild-type oncolytic virus impairs some immune cell subpopulations while directly activating a Helper CD4+ T cell response. Thus, our data open numerous gripping perspectives of investigation and strongly argue for the use of H-1 PV as an anticancer treatment.  

5.1031           Early Responding Dendritic Cells Direct the Local NK Response To Control Herpes Simplex Virus 1 Infection within the Cornea

Frank, G.M., Buela, K-A.g., Maker, D.M., Harvey, S.A.k. and Hendricks, R.L.

1. Immunol., 188(3), 1350-1359 (2012)

  Dendritic cells (DCs) regulate both innate and adaptive immune responses. In this article, we exploit the unique avascularity of the cornea to examine a role for local or very early infiltrating DCs in regulating the migration of blood-derived innate immune cells toward HSV-1 lesions. A single systemic diphtheria toxin treatment 2 d before HSV-1 corneal infection transiently depleted CD11c⁺ DCs from both the cornea and lymphoid organs of CD11c-DTR bone marrow chimeric mice for up to 24 h postinfection. Transient DC depletion significantly delayed HSV-1 clearance from the cornea through 6 d postinfection. No further compromise of viral clearance was observed when DCs were continuously depleted throughout the first week of infection. DC depletion did not influence extravasation of NK cells, inflammatory monocytes, or neutrophils into the peripheral cornea, but it did significantly reduce migration of NK cells and inflammatory monocytes, but not neutrophils, toward the HSV-1 lesion in the central cornea. Depletion of NK cells resulted in similar loss of viral control to transient DC ablation. Our findings demonstrate that resident corneal DCs and/or those that infiltrate the cornea during the first 24 h after HSV-1 infection contribute to the migration of NK cells and inflammatory monocytes into the central cornea, and are consistent with a role for NK cells and possibly inflammatory monocytes, but not polymorphonuclear neutrophils, in clearing HSV-1 from the infected cornea.  

5.1032           Identification of adeno-associated viral vectors suitable for intestinal gene delivery and modulation of experimental colitis

Polyak, S., Mach, A., Porvasnik, S., Dixon, L., Conlon, T., Erger, K.E., Acosta, A., Wright, A.J., Campbell-Thompson, M., Zolotukhin, I., Wasserfall, C. and Mah, C. Am. J. Physiol. Gastrointest. Liver Physiol., 302(3), G296-G308 (2012)   Effective gene transfer with sustained gene expression is an important adjunct to the study of intestinal inflammation and future therapy in inflammatory bowel disease. Recombinant adeno-associated virus (AAV) vectors are ideal for gene transfer and long-term transgene expression. The purpose of our study was to identify optimal AAV pseudotypes for transduction of the epithelium in the small intestine and colon, which could be used for studies in experimental colitis. The tropism and transduction efficiencies of AAV pseudotypes 1–10 were examined in murine small intestine and colon 8 wk after administration by real-time PCR and immunohistochemistry. The clinical and histopathological effects of IL-10-mediated intestinal transduction delivered by AAVrh10 were examined in the murine IL-10^−/− enterocolitis model. Serum IL-10 levels and IL-10 expression were followed by ELISA and real-time PCR, respectively. AAV pseudotypes 4, 7, 8, 9, and 10 demonstrated optimal intestinal transduction. Transgene expression was sustained 8 wk after administration and was frequently observed in enteroendocrine cells. Long-term IL-10 gene expression and serum IL-10 levels were observed following AAV transduction in an IL-10^−/− model of enterocolitis. Animals treated with AAVrh10-IL-10 had lower disease activity index scores, higher colon weight-to-length ratios, and lower microscopic inflammation scores. This study identifies novel AAV pseudotypes with small intestine and colon tropism and sustained transgene expression capable of modulating mucosal inflammation in a murine model of enterocolitis.      

5.1033           The recombinant lectin-like domain of thrombomodulin inhibits angiogenesis through interaction with Lewis Y antigen

Kuo, C-H., Chen, P-K., Chang, B-I., Sung, M-C., Shi, C-S., Lee, J-S., Chang, C-F., Shi, G.-Y. and Wu, H-L. Blood, 119(5), 1302-1313 82012)   Lewis Y Ag (LeY) is a cell-surface tetrasaccharide that participates in angiogenesis. Recently, we demonstrated that LeY is a specific ligand of the recombinant lectin-like domain of thrombomodulin (TM). However, the biologic function of interaction between LeY and TM in endothelial cells has never been investigated. Therefore, the role of LeY in tube formation and the role of the recombinant lectin-like domain of TM—TM domain 1 (rTMD1)—in antiangiogenesis were investigated. The recombinant TM ectodomain exhibited lower angiogenic activity than did the recombinant TM domains 2 and 3. rTMD1 interacted with soluble LeY and membrane-bound LeY and inhibited soluble LeY-mediated chemotaxis of endothelial cells. LeY was highly expressed on membrane ruffles and protrusions during tube formation on Matrigel. Blockade of LeY with rTMD1 or Ab against LeY inhibited endothelial tube formation in vitro. Epidermal growth factor (EGF) receptor in HUVECs was LeY modified. rTMD1 inhibited EGF receptor signaling, chemotaxis, and tube formation in vitro, and EGF-mediated angiogenesis and tumor angiogenesis in vivo. We concluded that LeY is involved in vascular endothelial tube formation and rTMD1 inhibits angiogenesis via interaction with LeY. Administration of rTMD1 or recombinant adeno-associated virus vector carrying TMD1 could be a promising antiangiogenesis strategy.  

5.1034           TPV1, the first virus isolated from the hyperthermophilic genus Thermococcus

Gorlas, A., Koonin, E.V., Bienvenu, N., Priur, D. and Geslin, C. Environmental Microbiol., 14(2), 503-516 (2012)   We describe a novel virus, TPV1 (Thermococcus prieurii virus 1), which was discovered in a hyperthermophilic euryarchaeote isolated from a deep-sea hydrothermal chimney sample collected at a depth of 2700 m at the East Pacific Rise. TPV1 is the first virus isolated and characterized from the hyperthermophilic euryarchaeal genus Thermococcus. TPV1 particles have a lemon-shaped morphology (140 nm × 80 nm) similar to the structures previously reported for Fuselloviruses and for the unclassified virus-like particle PAV1 (Pyrococcus abyssi virus 1). The infection with TPV1 does not cause host lysis and viral replication can be induced by UV irradiation. TPV1 contains a double-stranded circular DNA of 21.5 kb, which is also present in high copy number in a free form in the host cell. The TPV1 genome encompasses 28 predicted genes; the protein sequences encoded in 16 of these genes show no significant similarity to proteins in public databases. Proteins predicted to be involved in genome replication were identified as well as transcriptional regulators. TPV1 encodes also a predicted integrase of the tyrosine recombinase family. The only two genes that are homologous between TPV1 and PAV1 are TPV1-22 and TPV1-23, which encode proteins containing a concanavalin A-like lectin/glucanase domain that might be involved in virus–host recognition.  

5.1035           Enhanced gene delivery to the neonatal retina through systemic administration of tyrosine-mutated AAV9

Dalkara, D., Byrne, L.C., Lee, T., Hoffmann, N.V., Schaffer, D.V. and Flannery, J.G. Gene Therapy, 19(2), 176-181 (2012)   Delivery of therapeutic genes to a large region of the retina with minimal damage from intraocular surgery is a central goal of treatment for retinal degenerations. Recent studies have shown that AAV9 can reach the central nervous system (CNS) and retina when administered systemically to neonates, which is a promising strategy for some retinal diseases. We investigated whether the retinal transduction efficiency of systemically delivered AAV9 could be improved by mutating capsid surface tyrosines, previously shown to increase the infectivity of several AAV vectors. Specifically, we evaluated retinal transduction following neonatal intravascular administration of AAV9 vectors containing tyrosine to phenylalanine mutations at two highly conserved sites. Our results show that a novel, double tyrosine mutant of AAV9 significantly enhanced gene delivery to the CNS and retina, and that gene expression can be restricted to rod photoreceptor cells by incorporating a rhodopsin promoter. This approach provides a new methodology for the development of retinal gene therapies or creation of animal models of neurodegenerative disease.  

5.1036           Identification of the Niemann-Pick C1–like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor

Sainz Jr., B., Barretto, N., Martin, D.N., Hiraga, N., Imamura, M., Hussain, S., Marsh, K.A., Yu, X., Chayama, K., Alrefai, W.A. and Uprichard, S.L. Nature Medicine, 18(2), 281-286 (2012)   Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. With ~170 million individuals infected and current interferon-based treatment having toxic side effects and marginal efficacy, more effective antivirals are crucially needed¹. Although HCV protease inhibitors were just approved by the US Food and Drug Administration (FDA), optimal HCV therapy, analogous to HIV therapy, will probably require a combination of antivirals targeting multiple aspects of the viral lifecycle. Viral entry represents a potential multifaceted target for antiviral intervention; however, to date, FDA-approved inhibitors of HCV cell entry are unavailable. Here we show that the cellular Niemann-Pick C1–like 1 (NPC1L1) cholesterol uptake receptor is an HCV entry factor amendable to therapeutic intervention. Specifically, NPC1L1 expression is necessary for HCV infection, as silencing or antibody-mediated blocking of NPC1L1 impairs cell culture–derived HCV (HCVcc) infection initiation. In addition, the clinically available FDA-approved NPC1L1 antagonist ezetimibe^{2, 3} potently blocks HCV uptake in vitro via a virion cholesterol–dependent step before virion-cell membrane fusion. Moreover, ezetimibe inhibits infection by all major HCV genotypes in vitro and in vivo delays the establishment of HCV genotype 1b infection in mice with human liver grafts. Thus, we have not only identified NPC1L1 as an HCV cell entry factor but also discovered a new antiviral target and potential therapeutic agent.      

5.1037           Non-human primate model of amyotrophic lateral sclerosis with cytoplasmic mislocalization of TDP-43

Uchida, A. et al Brain, 135, 833-846 (2012)   Amyotrophic lateral sclerosis is a fatal neurodegenerative disease characterized by progressive motoneuron loss. Redistribution of transactive response deoxyribonucleic acid-binding protein 43 from the nucleus to the cytoplasm and the presence of cystatin C-positive Bunina bodies are considered pathological hallmarks of amyotrophic lateral sclerosis, but their significance has not been fully elucidated. Since all reported rodent transgenic models using wild-type transactive response deoxyribonucleic acid-binding protein 43 failed to recapitulate these features, we expected a species difference and aimed to make a non-human primate model of amyotrophic lateral sclerosis. We overexpressed wild-type human transactive response deoxyribonucleic acid-binding protein 43 in spinal cords of cynomolgus monkeys and rats by injecting adeno-associated virus vector into the cervical cord, and examined the phenotype using behavioural, electrophysiological, neuropathological and biochemical analyses. These monkeys developed progressive motor weakness and muscle atrophy with fasciculation in distal hand muscles first. They also showed regional cytoplasmic transactive response deoxyribonucleic acid-binding protein 43 mislocalization with loss of nuclear transactive response deoxyribonucleic acid-binding protein 43 staining in the lateral nuclear group of spinal cord innervating distal hand muscles and cystatin C-positive cytoplasmic aggregates, reminiscent of the spinal cord pathology of patients with amyotrophic lateral sclerosis. Transactive response deoxyribonucleic acid-binding protein 43 mislocalization was an early or presymptomatic event and was later associated with neuron loss. These findings suggest that the transactive response deoxyribonucleic acid-binding protein 43 mislocalization leads to α-motoneuron degeneration. Furthermore, truncation of transactive response deoxyribonucleic acid-binding protein 43 was not a prerequisite for motoneuronal degeneration, and phosphorylation of transactive response deoxyribonucleic acid-binding protein 43 occurred after degeneration had begun. In contrast, similarly prepared rat models expressed transactive response deoxyribonucleic acid-binding protein 43 only in the nucleus of motoneurons. There is thus a species difference in transactive response deoxyribonucleic acid-binding protein 43 pathology, and our monkey model recapitulates amyotrophic lateral sclerosis pathology to a greater extent than rodent models, providing a valuable tool for studying the pathogenesis of sporadic amyotrophic lateral sclerosis.  

5.1038           Efficient Gene Therapy for Parkinson's Disease Using Astrocytes as Hosts for Localized Neurotrophic Factor Delivery

Drinkut, A., Tereshchenko, Y., Schulz, J.B., Bähr, M. and Kügler, S. Molecular Therapy, 20(3), 534-543 (2012)   Current gene therapy approaches for Parkinson's disease (PD) deliver neurotrophic factors like glial cell line-derived neurotrophic factor (GDNF) or neurturin via neuronal transgene expression. Since these potent signaling-inducing neurotrophic factors can be distributed through long-distance neuronal projections to unaffected brain sites, this mode of delivery may eventually cause side effects. To explore a localized and thus potentially safer alternative for gene therapy of PD, we expressed GDNF exclusively in astrocytes and evaluated the efficacy of this approach in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rat 6-hydroxy-dopamine (6-OHDA) models of PD. In terms of protection of dopaminergic cell bodies and projections, dopamine (DA) synthesis and behaviour, astrocyte-derived GDNF demonstrated the same efficacy as neuron-derived GDNF. In terms of safety, unilateral striatal GDNF expression in astrocytes did not result in delivery of bio-active GDNF to the contralateral hemispheres (potential off-target sites) as happened when GDNF was expressed in neurons. Thus, astrocytic GDNF expression represents a localized but efficient alternative to current gene therapeutic strategies for the treatment of PD, especially if viral vectors with enhanced tissue penetration are considered. Astrocytic neurotrophic factor expression may open new venues for neurotrophic factor-based gene therapy targeting severe diseases of the brain.  

5.1039           Striatal Pleiotrophin Overexpression Provides Functional and Morphological Neuroprotection in the 6-Hydroxydopamine Model

Gombash, S.E., Lipton, J.W., Collier, T.J., Madhavan, L., Steece-Collier, K., Cole-Strauss, A., Terpstra, B.T., Speiles-Engemann, A.L., Daley, B.F., Wohlgenant, S.L., Thompson, V.B., Manfredsson, F., Mandel, R.J. and Sortwell, C.E. Molecular Therapy, 20(3), 544-554 (2012)   Current gene therapy approaches for Parkinson's disease (PD) deliver neurotrophic factors like glial cell line-derived neurotrophic factor (GDNF) or neurturin via neuronal transgene expression. Since these potent signaling-inducing neurotrophic factors can be distributed through long-distance neuronal projections to unaffected brain sites, this mode of delivery may eventually cause side effects. To explore a localized and thus potentially safer alternative for gene therapy of PD, we expressed GDNF exclusively in astrocytes and evaluated the efficacy of this approach in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rat 6-hydroxy-dopamine (6-OHDA) models of PD. In terms of protection of dopaminergic cell bodies and projections, dopamine (DA) synthesis and behaviour, astrocyte-derived GDNF demonstrated the same efficacy as neuron-derived GDNF. In terms of safety, unilateral striatal GDNF expression in astrocytes did not result in delivery of bio-active GDNF to the contralateral hemispheres (potential off-target sites) as happened when GDNF was expressed in neurons. Thus, astrocytic GDNF expression represents a localized but efficient alternative to current gene therapeutic strategies for the treatment of PD, especially if viral vectors with enhanced tissue penetration are considered. Astrocytic neurotrophic factor expression may open new venues for neurotrophic factor-based gene therapy targeting severe diseases of the brain.  

5.1040           A phase I trial of adeno-associated virus serotype 1-γ-sarcoglycan gene therapy for limb girdle muscular dystrophy type 2C

Herson, S. et al Brain, 135(2), 483-492 (2012 )   γ-Sarcoglycanopathy or limb girdle muscular dystrophy type 2C is an untreatable disease caused by autosomal recessively inherited mutations of the γ-sarcoglycan gene. Nine non-ambulatory patients (two males, seven females, mean age 27 years; range 16–38 years) with del525T homozygous mutation of the γ-sarcoglycan gene and no γ-sarcoglycan immunostaining on muscle biopsy were divided into three equal groups to receive three escalating doses of an adeno-associated virus serotype 1 vector expressing the human γ-sarcoglycan gene under the control of the desmin promoter, by local injection into the extensor carpi radialis muscle. The first group received a single injection of 3 × 10⁹ viral genomes in 100 µl, the second group received a single injection of 1.5 × 10¹⁰ viral genomes in 100 µl, and the third group received three simultaneous 100-µl injections at the same site, delivering a total dose of 4.5 × 10¹⁰ viral genomes. No serious adverse effects occurred during 6 months of follow-up. All nine patients became adeno-associated virus serotype 1 seropositive and one developed a cytotoxic response to the adeno-associated virus serotype 1 capsid. Thirty days later, immunohistochemical analysis of injected-muscle biopsy specimens showed γ-sarcoglycan expression in all three patients who received the highest dose (4.7–10.5% positively stained fibres), while real-time polymerase chain reaction detected γ-sarcoglycan messenger RNA. In one patient, γ-sarcoglycan protein was detected by western blot. For two other patients who received the low and intermediate doses, discrete levels of γ-sarcoglycan expression (<1% positively stained fibres) were also detectable. Expression of γ-sarcoglycan protein can be induced in patients with limb girdle muscular dystrophy type 2C by adeno-associated virus serotype 1 gene transfer, with no serious adverse effects.  

5.1041           Retargeting of Rat Parvovirus H-1PV to Cancer Cells through Genetic Engineering of the Viral Capsid

Alllaume, X., El-Andaloussi, N., Leuchs, B., Bonifati, S., Kulkarni, A., Marttila, T., Kaufmann, J.K., Nettelbeck, D.M., Kleinschmidt, J., Rommelaere, J. and Marchini, A.

1. Virol., 86(7), 3452-3465 (2012)

  The rat parvovirus H-1PV is a promising anticancer agent given its oncosuppressive properties and the absence of known side effects in humans. H-1PV replicates preferentially in transformed cells, but the virus can enter both normal and cancer cells. Uptake by normal cells sequesters a significant portion of the administered viral dose away from the tumor target. Hence, targeting H-1PV entry specifically to tumor cells is important to increase the efficacy of parvovirus-based treatments. In this study, we first found that sialic acid plays a key role in H-1PV entry. We then genetically engineered the H-1PV capsid to improve its affinity for human tumor cells. By analogy with the resolved crystal structure of the closely related parvovirus minute virus of mice, we developed an in silico three-dimensional (3D) model of the H-1PV wild-type capsid. Based on this model, we identified putative amino acids involved in cell membrane recognition and virus entry at the level of the 2-fold axis of symmetry of the capsid, within the so-called dimple region. In situ mutagenesis of these residues significantly reduced the binding and entry of H-1PV into permissive cells. We then engineered an entry-deficient viral capsid and inserted a cyclic RGD-4C peptide at the level of its 3-fold axis spike. This peptide binds α_vβ₃ and α_vβ₅ integrins, which are overexpressed in cancer cells and growing blood vessels. The insertion of the peptide rescued viral infectivity toward cells overexpressing α_vβ₅ integrins, resulting in the efficient killing of these cells by the reengineered virus. This work demonstrates that H-1PV can be genetically retargeted through the modification of its capsid, showing great promise for a more efficient use of this virus in cancer therapy.  

5.1042           Distinct Neuronal Coding Schemes in Memory Revealed by Selective Erasure of Fast Synchronous Synaptic Transmission

Xu, W., Morishita, W., Buckmaster, P.S., Pang, Z.P., Malenka, R.C. and Südof, T.C. Neuron, 73(5), 990-1001 (2012)   Neurons encode information by firing spikes in isolation or bursts and propagate information by spike-triggered neurotransmitter release that initiates synaptic transmission. Isolated spikes trigger neurotransmitter release unreliably but with high temporal precision. In contrast, bursts of spikes trigger neurotransmission reliably (i.e., boost transmission fidelity), but the resulting synaptic responses are temporally imprecise. However, the relative physiological importance of different spike-firing modes remains unclear. Here, we show that knockdown of synaptotagmin-1, the major Ca²⁺ sensor for neurotransmitter release, abrogated neurotransmission evoked by isolated spikes but only delayed, without abolishing, neurotransmission evoked by bursts of spikes. Nevertheless, knockdown of synaptotagmin-1 in the hippocampal CA1 region did not impede acquisition of recent contextual fear memories, although it did impair the precision of such memories. In contrast, knockdown of synaptotagmin-1 in the prefrontal cortex impaired all remote fear memories. These results indicate that different brain circuits and types of memory employ distinct spike-coding schemes to encode and transmit information.  

5.1043           (−)-Epigallocatechin-3-gallate is a new inhibitor of hepatitis C virus entry

Calland, N., Albecka, A., Belouzard, S., Wychowski, C., Duverlie, G., Descamps, V., Hober, D., Dubuisson, J., Rouille, Y. and Seron, K. Hepatology, 55(3), 720-729 (2012)   Here, we identify (−)-epigallocatechin-3-gallate (EGCG) as a new inhibitor of hepatitis C virus (HCV) entry. EGCG is a flavonoid present in green tea extract belonging to the subclass of catechins, which has many properties. Particularly, EGCG possesses antiviral activity and impairs cellular lipid metabolism. Because of close links between HCV life cycle and lipid metabolism, we postulated that EGCG may interfere with HCV infection. We demonstrate that a concentration of 50 μM of EGCG inhibits HCV infectivity by more than 90% at an early step of the viral life cycle, most likely the entry step. This inhibition was not observed with other members of the Flaviviridae family tested. The antiviral activity of EGCG on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition, using binding assays at 4°C, we demonstrate that EGCG prevents attachment of the virus to the cell surface, probably by acting directly on the particle. We also show that EGCG has no effect on viral replication and virion secretion. By inhibiting cell-free virus transmission using agarose or neutralizing antibodies, we show that EGCG inhibits HCV cell-to-cell spread. Finally, by successive inoculation of naïve cells with supernatant of HCV-infected cells in the presence of EGCG, we observed that EGCG leads to undetectable levels of infection after four passages. Conclusion: EGCG is a new, interesting anti-HCV molecule that could be used in combination with other direct-acting antivirals. Furthermore, it is a novel tool to further dissect the mechanisms of HCV entry into the hepatocyte.  

5.1044           Human Papillomavirus Antibody Reference Reagents for Use in Postvaccination Surveillance Serology

Bissett, S.L., Wilkinson, D., Tettmar, K.I., Jones, N., Stanford, E., Panicker, G., Faust, H., Borrow, R., Soldan, K., Unger, E.R., Dillner, J., Minor, P. and Beddows, S. Clin. Vaccine Immunol., 19(3), 449-451 (2012)   Suitably controlled serosurveillance surveys are essential for evaluating human papillomavirus (HPV) immunization programs. A panel of plasma samples from 18-year-old females was assembled, the majority of the samples being from recipients of the bivalent HPV vaccine. Antibody specificities were evaluated by three independent laboratories, and 3 pools that displayed no antibodies to any HPV type tested or intermediate or high levels of antibody to HPV16, HPV18, HPV31, and HPV45 were created. These pools will be useful as control reagents for HPV serology.    

5.1045           Using Recombinant Adeno-Associated Viral Vectors for Gene Expression in the Brain

Van der Perren, A., Toelen, J., Taymans, J-M. and Baekelandt, V. Neuromethods, 65, 47-68 (2012)   Recombinant AAV vectors currently enjoy an excellent track record in brain applications such as generating preclinical models of neurodegeneration and gene therapy for brain disorders. Indeed, rAAV vectors have been useful in modeling diseases such as Parkinson’s disease (discussed below) and have also been tested in various phases of clinical development for Parkinson’s disease (Christine et al., Neurology 73:1662–1669, 2009; Kaplitt et al., Lancet 369:2097–2105, 2007) and Alzheimer’s disease (Mandel 2010 , Curr Opin Mol Ther 12:240–247, 2010). In this review, we will discuss the vectorology of rAAV, rAAV production, and purifi cation of the different rAAV serotypes. We will also describe locoregional transduction of the brain using rAAV vectors and illustrate these techniques with specifi c examples of applications such as non-invasive imaging of reporter genes and disease modeling in Parkinson’s disease.  

5.1046           Profiling of HIV Proteins in Cerebrospinal Fluid

Wojtkiewicz, M. and Ciborowski, P. Neuromethods, 64, 225-244 (2012)   HIV-1 proteins are rarely identifi ed during mass spectrometry-based proteomic profi ling studies of body fl uids from HIV-1-infected people even when elaborated fractionation schema and highly sensitive instruments are used. Genotyping of HIV-1 isolated from body fl uids does not provide exact information about characteristics of circulating proteins and is a limiting factor in expanding an important segment of our knowledge about the course of infection. Therefore, we propose that in vitro amplifi cation of freshly isolated virus followed by sucrose cushion purifi cation will yield suffi cient amounts of viral proteins for mass spectrometric characterization. This chapter provides protocols for virus propagation using CD4+ T cell line or human macrophages, virus purifi cation, and preparation of samples for two-dimensional electrophoresis and mass spectrometry analyses.  

5.1047           Chimeric calicivirus-like particles elicit specific immune responses in pigs

Crisci, E., Fraile, L., Moreno, N., Blanco, N., Cabezon, R., Costa, C., Mussa, T., Baratelli, M., Martinez-Orellana, P., Ganges, L., Martinez, J., Barcena, J. and Montoya, M. Vaccine, 30, 2427-2439 (2012)   Virus-like particles (VLPs) have received considerable attention due to their potential application in veterinary vaccines and, in particular, VLPs from rabbit haemorrhagic disease virus (RHDV) have successfully shown to be good platforms for inducing immune responses against an inserted foreign epitope in mice. The aim of this study was to assess the immunogenicity of chimeric RHDV-VLPs as vaccine vectors in pigs. For this purpose, we have generated chimeric VLPs containing a well-known T epitope of 3A protein of foot-and-mouth disease virus (FMDV). Firstly, RHDV-VLPs were able to activate immature porcine bone marrow-derived dendritic cells (poBMDCs) in vitro. Secondly, pigs were inoculated twice in a two-week interval with chimeric RHDV-VLPs at different doses intranasally or intramuscularly. One intramuscularly treated group was also inoculated with adjuvant Montanide™ ISA 206 at the same time. Specific IgG and IgA antibodies against RHDV-VLPs were induced and such levels were higher in the adjuvanted group compared with other groups. Interestingly, anti-RHDV-VLP IgA responses were higher in groups inoculated intramuscularly than those that received the VLPs intranasally. Two weeks after the last immunisation, specific IFN-γ-secreting cells against 3A epitope and against RHDV-VLPs were detected in PBMCs by ELISPOT. The adjuvanted group exhibited the highest IFN-γ-secreting cell numbers and lymphoproliferative specific T cell responses against 3A epitope and RHDV-VLP. This is the first immunological report on the potential use of chimeric RHDV-VLPs as antigen carriers in pigs.  

5.1048           Activation of Protein Kinase C (PKC)α or PKCε as an Approach to Increase Morphine Tolerance in Respiratory Depression and Lethal Overdose

Lin, H-Y., Law, P-Y. and Loh, H.H.

1. Pharmacol. Exp. Ther., 341(1), 115-125 (2012)

  Long-term use of opioids is hindered by respiratory depression and the possibility for fatal overdose in drug abusers. This is attributed to higher levels of tolerance that develops against antinociception than to respiratory depression. Identifying important mechanisms that would increase morphine respiratory depression and overdose tolerance could lead to the safer use of opioids. Because protein kinase C (PKC) activity mediates the development and maintenance of morphine antinociceptive tolerance, we hypothesized that activating PKCα or PKCε at the pre-Bötzinger complex (preBötC) can increase morphine tolerance in respiration and overdose. Laser microdissection and quantitative reverse transcriptase-polymerase chain reaction were used to compare the relative mRNA abundances of PKCα, γ, and ε between ventrolateral periaqueductal gray (vlPAG) and preBötC. To test whether PKCα or ε could enhance morphine tolerance in respiratory depression and overdose, lentivirus carrying the wild type, constitutively activated mutants, and small interference RNA against PKCα or ε was stereotaxically injected into the preBötC. Expression of constitutively active PKC (CAPKC) α or ε, but not wild-type PKC (WTPKC) α or ε, at the preBötC allowed rats to develop tolerance to morphine respiratory depression. In terms of lethality, expression of WTPKCε, CAPKCα, or CAPKCε at preBötC increased morphine tolerance to lethal overdose. CAPKCε-expressing rats developed the highest level of respiratory depression tolerance. Furthermore, when CAPKCε lentivirus was injected into the vlPAG, rats were able to develop significant antinociceptive tolerance at low doses of morphine that normally do not cause tolerance. The approach of increasing morphine respiratory depression and lethality tolerance by increasing PKCα or ε activity at preBötC could be used to make opioids safer for long-term use.  

5.1049           Universal Real-Time PCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2-Derived Inverted Terminal Repeat Sequences

Aurnhammer, C., Haase, M., Muether, N., Hausl, M., Raussschhuber, C.,  Huber, I., Nitschko, H., Busch, U., Sing, A., Ehrhardt, A. and Baiker, A. Human Gene Therapy MethodsPart  B, 23(1), 18-28 (2012)   Viral vectors based on various naturally occurring adeno-associated virus (AAV) serotypes are among the most promising tools in human gene therapy. For the production of recombinant AAV (rAAV) vectors, researchers are focusing predominantly on cross-packaging an artificial AAV genome based on serotype 2 (AAV2) into capsids derived from other serotypes. Within the packaged genome the inverted terminal repeats (ITRs) are the only cis-acting viral elements required for rAAV vector generation and depict the lowest common denominator of all AAV2-derived vector genomes. Up to now, no quantitative PCR (qPCR) for the detection and quantification of AAV2 ITRs could be established because of their extensive secondary hairpin structure formation. Current qPCR-based methods are therefore targeting vector-encoded transgenes or regulatory elements. Herein we establish a molecular biological method that allows accurate and reproducible quantification of AAV2 genomes on the basis of an AAV2 ITR sequence-specific qPCR. Primers and labeled probe are located within the ITR sequence and have been designed to detect both wild-type AAV2 and AAV2-based vectors. This method is suitable for detecting single-stranded DNA derived from AAV2 vector particles and double-stranded DNA derived from vector plasmids. The limit of detection has been determined as 50 ITR sequence copies per reaction, by comparison with a plasmid standard. In conclusion, this method describes the first qPCR system facilitating the detection and quantification of AAV2 ITR sequences. Because this method can be used universally for all AAV2 genome-based vectors, it will significantly simplify rAAV2 vector titrations in the future.  

5.1050           Analysis of Particle Content of Recombinant Adeno-Associated Virus Serotype 8 Vectors by Ion-Exchange Chromatography

Lock, M., Alvira, M.R. and Wilson, J.M. Human Gene Therapy Methods Part B., 23(1), 56-64 (2012)   Advances in adeno-associated virus (AAV)-mediated gene therapy have brought the possibility of commercial manufacturing of AAV vectors one step closer. To realize this prospect, a parallel effort with the goal of ever-increasing sophistication for AAV vector production technology and supporting assays will be required. Among the important release assays for a clinical gene therapy product, those monitoring potentially hazardous contaminants are most critical for patient safety. A prominent contaminant in many AAV vector preparations is vector particles lacking a genome, which can substantially increase the dose of AAV capsid proteins and lead to possible unwanted immunological consequences. Current methods to determine empty particle content suffer from inconsistency, are adversely affected by contaminants, or are not applicable to all serotypes. Here we describe the development of an ion-exchange chromatography-based assay that permits the rapid separation and relative quantification of AAV8 empty and full vector particles through the application of shallow gradients and a strong anion-exchange monolith chromatography medium.    

5.1051           Systemic and Mucosal Immune Responses to Sublingual or Intramuscular Human Papilloma Virus Antigens in Healthy Female Volunteers

Huo, Z., Bissett, S.L., Giemza, R., Beddows, S., Oeser, C. and Lewis, D.J.M. PloS One, 7(3), e33736 (2012)   The sublingual route has been proposed as a needle-free option to induce systemic and mucosal immune protection against viral infections. In a translational study of systemic and mucosal humoral immune responses to sublingual or systemically administered viral antigens, eighteen healthy female volunteers aged 19–31 years received three immunizations with a quadravalent Human Papilloma Virus vaccine at 0, 4 and 16 weeks as sublingual drops (SL, n = 12) or intramuscular injection (IM, n = 6). IM antigen delivery induced or boosted HPV-specific serum IgG and pseudovirus-neutralizing antibodies, HPV-specific cervical and vaginal IgG, and elicited circulating IgG and IgA antibody secreting cells. SL antigens induced ~38-fold lower serum and ~2-fold lower cervical/vaginal IgG than IM delivery, and induced or boosted serum virus neutralizing antibody in only 3/12 subjects. Neither route reproducibly induced HPV-specific mucosal IgA. Alternative delivery systems and adjuvants will be required to enhance and evaluate immune responses following sublingual immunization in humans.  

5.1052           Long-term polarization of microglia upon α-synuclein overexpression in nonhuman primates

Barkholt, P., Sanchez-Guajardo, V., Kirik, D. and Romero-Ramos, M. Neurosci., 208, 85-96 (2012)   We have previously shown that persistent α-synuclein overexpression in ventral midbrain of marmoset leads to a distinctive neurodegenerative process and motor defects. The neurodegeneration was confined to caudate putamen dopaminergic fibers in animals overexpressing wild-type (wt) α-synuclein. However, A53T α-synuclein overexpression induced neurodegeneration that resulted in nigral dopaminergic cell death. Here, we analyze the microglia population in the midbrain of these animals by stereological quantification of Iba1+ cells. Our data here show that monkeys overexpressing A53T α-synuclein showed a long-term increase in microglia presenting macrophagic morphology. However, wt α-synuclein overexpression, despite the absence of dopaminergic cell death, resulted in a permanent robust increase of the microglia population characterized by a range of distinct morphological types that persisted after 1 year. These results confirm that the microglial response differs depending on the type of α-synuclein (wt/A53T) and/or whether α-synuclein expression results in cell death or not, suggesting that microglia may play different roles during disease progression. Furthermore, the microglial response is modulated by events related to α-synuclein expression in substantia nigra and persists in the long term. The data presented here is in agreement with that previously observed in a recombinant adeno-associated virus (rAAV) α-synuclein rat model, thereby validating both the findings and the model, and highlighting the translational potential of the rodent model to higher species closer to humans.  

5.1053           Induced Pluripotent Stem Cell Clones Reprogrammed via Recombinant Adeno-Associated Virus-Mediated Transduction Contain Integrated Vector Sequences

Weltner, J., Anisimov, A., Alitalo, K., Otonkoski, T. and Trokovic, RS.

1. Virol., 86(8), 4463-4467 (2012)

  Fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSC) by ectopic expression of key transcription factors. Current methods for the generation of integration-free iPSC are limited by the low efficiency of iPSC generation and by challenges in reprogramming methodology. Recombinant adeno-associated virus (rAAV) is a potent gene delivery vehicle capable of efficient transduction of transgenic DNA into cells. rAAV stays mainly as an episome in nondividing cells, and the extent of integration is still poorly defined for various replicating cells. In this study, we aimed to induce iPSC from mouse and human fibroblasts by using rAAV vector-mediated transient delivery of reprogramming factors. We succeeded in deriving induced pluripotent stem cells from mouse but not human fibroblasts. Unexpectedly, the rAAV vector-mediated reprogramming led to frequent genomic integration of vector sequences during the reprogramming process, independent of the amount of virus used, and to persistent expression of reprogramming factors in generated iPSC clones. It thus appears that rAAV vectors are not compatible with the derivation of integration-free iPSC.  

5.1054           Role of low-density lipoprotein receptor in the hepatitis C virus life cycle

Albecka, A., Belouzard, S., de Beeck, A.O., Descamps, V., Goueslain, L., Bertrand-Michel, J., Terce, F., Duverlie, G., Rouille, Y. and Dubuisson, J. Hepatology, 55(4), 998-1007 (2012)   Hepatitis C virus (HCV) particles are known to be in complex with lipoproteins. As a result of this interaction, the low-density lipoprotein (LDL) receptor (LDLR) has been proposed as a potential entry factor for HCV; however, its implication in virus entry remains unclear. Here, we reinvestigated the role of the LDLR in the HCV life cycle by comparing virus entry to the mechanism of lipoprotein uptake. A small interfering RNA targeting the LDLR in Huh-7 cells reduced HCV infectivity, confirming that this receptor plays a role in the life cycle of HCV generated in cell culture. However, kinetics of internalization were much faster for lipoproteins than for infectious HCV particles. Furthermore, a decrease in HCV RNA replication was observed by blocking the LDLR with a specific antibody, and this was associated with an increase in the ratio of phosphatidylethanolamine to phosphatidylcholine in host cells. Nevertheless, a soluble form of the LDLR inhibited both HCV entry into the hepatocytes and its binding to the LDLR expressed on Chinese hamster ovary cells, suggesting a direct interaction between the HCV particle and the LDLR. Finally, we showed that modification of HCV particles by lipoprotein lipase (LPL) reduces HCV infectivity and increases HCV binding to LDLR. Importantly, LPL treatment also induced an increase in RNA internalization, suggesting that LDLR, at least in some conditions, leads to nonproductive internalization of HCV. Conclusion: The LDLR is not essential for infectious HCV particle entry, whereas the physiological function of this receptor is important for optimal replication of the HCV genome.

5.1055           Tetradecanoylphorbol-13-acetate (TPA) significantly increases AAV2/5 transduction of human neuronal cells in vitro

You, Q., Brown, L.A., McClements, M., Hankins, M.W. and MacLaren, R.E. Exp. Eye Res., 97, 148-153 (2012)   Recombinant adeno-associated virus type 2 (AAV2) vectors have shown great promise in current ophthalmology clinical trials targeting gene delivery to the retinal pigment epithelium (RPE). To treat the majority of retinal diseases, however, gene delivery would need to be targeted to photoreceptor neurons of the outer retina. AAV2 pseudotyped with the AAV5 capsid (AAV2/5) has shown far greater transduction efficiency in photoreceptors compared to standard AAV2 vectors. For clinical trial applications using gene therapy, it is helpful to generate pre-clinical data in human cells wherever possible. There is however very little data, indeed some controversy, as to whether AAV2/5 can be used effectively in differentiated neurons in culture. In this study we show that transduction of the human neuroblastoma cell line SH-SY5Y with recombinant AAV2/5 expressing GFP is well tolerated. Furthermore, we explore the mechanism whereby exposure to retinoic acid (RA) and the phorbol ester 12-O-Tetradecanoylphorbol-13- acetate (TPA) can induce this cell line to differentiate into a stable population of human neurons, with significantly increased levels of AAV2/5 transduction. These observations may be helpful for assessing AAV2/5 vectors in vitro, particularly where it is necessary to generate pre-clinical data for clinical trials of gene therapy to the human central nervous system.    

5.1056           AAV8gfp preferentially targets large diameter dorsal root ganglion neurones after both intra-dorsal root ganglion and intrathecal injection

Jacques, S.J., Ahmed, Z., Forbes, A., Douglas, M.R., Vigenswara, V., Berry, M. and Logan, A. Mol. Cell. Neurosci., 49, 464-474 (2012)   Adeno-associated viral vectors (AAV) are increasingly used to deliver therapeutic genes to the central nervous system (CNS) where they promote transgene expression in post mitotic neurones for long periods with little or no toxicity. In adult rat dorsal root ganglia (DRG), we investigated the cellular tropism of AAV8 containing the green fluorescent protein gene (gfp) after either intra-lumbar DRG or intrathecal injection and showed that transduced DRG neurones (DRGN) expressed GFP irrespective of the delivery route, while non-neuronal cells were GFP⁻. After intra-DRG delivery of AAV8gfp, the mean DRGN transduction rate was 11%, while intrathecal delivery transduced a mean of 1.5% DRGN. After intra-DRG injection, 2% of small DRGN (< 30 μm in diameter) were GFP⁺ compared with 32% of large DRGN (> 60 μm in diameter). Axons of transduced DRGN were also GFP⁺; no intra-spinal neurones were transduced. A small number of contralateral DRGN were transduced after intra-DRG injection, suggesting that AAV8 may diffuse from injected DRG into the spinal canal. Microglia and astrocytes were highly ramified with increased GFAP⁺ immunoreactivity (i.e. activated) in the neuropil around GFP⁺ DRG axon projections within the cord after intra-DRG injection. This study showed that after both intra-DRG and intrathecal delivery, strong preferential AAV8 tropism exists for large DRGN unassociated with cell death, but GFP⁺ axons projecting in the spinal cord induced local glial activation. These results open up opportunities for targeted delivery of therapeutics such as neurotrophic factors to the injured spinal cord.  

5.1057           Conjugation of paclitaxel on adeno-associated virus (AAV) nanoparticles for co-delivery of genes and drugs

Wei, F., McConnell, K.I., Yu, T-K. and Suh, J. Eur. J. Pharmaceut. Sci., 46, 167-172 (2012)   We have investigated the use of adeno-associated virus (AAV) nanoparticles as platforms for the co-delivery of genes and drugs to cancer cells. With its regular geometry, nanoscale dimensions, lack of pathogenicity, and high infection efficiency in a wide range of human cells and tissues, AAV is a promising vector for such applications. We tested the covalent conjugation of paclitaxel onto surface-exposed lysine residues present on the virus capsid. Immunoblotting results suggest successful attachment of drug molecules to the virus nanoparticles. Favorably, the reaction conditions did not reduce the gene delivery efficiency of the AAV vectors. Unfortunately, decrease in cancer cell viability was not observed with our AAV–taxol conjugates. For future attempts at conjugating drugs to the AAV nanoparticle, we have identified several improvements than can be considered to achieve the desired cytotoxicity in target cells.    

5.1058           Neutralization Serotyping of BK Polyomavirus Infection in Kidney Transplant Recipients

Pastrana, D.V., Brennan, D.C., Cuburu, N., Storch, G.A., Viscidi, R.P., Randhawa, P.S. and Buck, C.B. PloS Pathogens, 8(4), e1002650 (2012)   BK polyomavirus (BKV or BKPyV) associated nephropathy affects up to 10% of kidney transplant recipients (KTRs). BKV isolates are categorized into four genotypes. It is currently unclear whether the four genotypes are also serotypes. To address this issue, we developed high-throughput serological assays based on antibody-mediated neutralization of BKV genotype I and IV reporter vectors (pseudoviruses). Neutralization-based testing of sera from mice immunized with BKV-I or BKV-IV virus-like particles (VLPs) or sera from naturally infected human subjects revealed that BKV-I specific serum antibodies are poorly neutralizing against BKV-IV and vice versa. The fact that BKV-I and BKV-IV are distinct serotypes was less evident in traditional VLP-based ELISAs. BKV-I and BKV-IV neutralization assays were used to examine BKV type-specific neutralizing antibody responses in KTRs at various time points after transplantation. At study entry, sera from 5% and 49% of KTRs showed no detectable neutralizing activity for BKV-I or BKV-IV neutralization, respectively. By one year after transplantation, all KTRs were neutralization seropositive for BKV-I, and 43% of the initially BKV-IV seronegative subjects showed evidence of acute seroconversion for BKV-IV neutralization. The results suggest a model in which BKV-IV-specific seroconversion reflects a de novo BKV-IV infection in KTRs who initially lack protective antibody responses capable of neutralizing genotype IV BKVs. If this model is correct, it suggests that pre-vaccinating prospective KTRs with a multivalent VLP-based vaccine against all BKV serotypes, or administration of BKV-neutralizing antibodies, might offer protection against graft loss or dysfunction due to BKV associated nephropathy.  

5.1059           Single-cell resolution fluorescence imaging of circadian rhythms detected with a Nipkow spinning disk confocal system

Enoki, R., Ono, D., Hasan, M.T., Honma, S. and Honma, K-i.

1. Neuroscience Methods, 207, 72-79 (2012)

  Single-point laser scanning confocal imaging produces signals with high spatial resolution in living organisms. However, photo-induced toxicity, bleaching, and focus drift remain challenges, especially when recording over several days for monitoring circadian rhythms. Bioluminescence imaging is a tool widely used for this purpose, and does not cause photo-induced difficulties. However, bioluminescence signals are dimmer than fluorescence signals, and are potentially affected by levels of cofactors, including ATP, O₂, and the substrate, luciferin. Here we describe a novel time-lapse confocal imaging technique to monitor circadian rhythms in living tissues. The imaging system comprises a multipoint scanning Nipkow spinning disk confocal unit and a high-sensitivity EM-CCD camera mounted on an inverted microscope with auto-focusing function. Brain slices of the suprachiasmatic nucleus (SCN), the central circadian clock, were prepared from transgenic mice expressing a clock gene, Period 1 (Per1), and fluorescence reporter protein (Per1::d2EGFP). The SCN slices were cut out together with membrane, flipped over, and transferred to the collagen-coated glass dishes to obtain signals with a high signal-to-noise ratio and to minimize focus drift. The imaging technique and improved culture method enabled us to monitor the circadian rhythm of Per1::d2EGFP from optically confirmed single SCN neurons without noticeable photo-induced effects or focus drift. Using recombinant adeno-associated virus carrying a genetically encoded calcium indicator, we also monitored calcium circadian rhythms at a single-cell level in a large population of SCN neurons. Thus, the Nipkow spinning disk confocal imaging system developed here facilitates long-term visualization of circadian rhythms in living cells.  

5.1060           Oral administration of HPV-16 L2 displayed on Lactobacillus casei induces systematic and mucosal cross-neutralizing effects in Balb/c mice

Yoon, S-W., Lee, T-Y., Kim, S-J., Lee, Il-H., Sung, M-H., park, J-S. and Poo, H. Vaccine, 30, 3286-3294 (2012)   The human papillomavirus (HPV) minor capsid protein, L2, is a good candidate for prophylactic vaccine development because L2-specific antibodies have cross-neutralizing activity against diverse HPV types. Here, we developed a HPV mucosal vaccine candidate using the poly-γ-glutamic acid synthetase A (pgsA) protein to display a partial HPV-16 L2 protein (N-terminal 1–224 amino acid) on the surface of Lactobacillus casei (L. casei). The oral immunization with L. casei-L2 induced productions of L2-specific serum IgG and vaginal IgG and IgA in Balb/c mice. To examine cross-neutralizing activity, we used a sensitive high-throughput neutralization assay based on HPV-16, -18, -45, -58, and bovine papillomavirus 1 (BPV1) pseudovirions. Our results revealed that mice vaccinated with L. casei-L2 not only generated neutralizing antibodies against HPV-16, but they also produced antibodies capable of cross-neutralizing the HPV-18, -45, and -58 pseudovirions. Consistent with previous reports, vaccination with HPV-16 L1 virus-like particles (VLPs) failed to show cross-neutralizing activity. Finally, we found that oral administration of L. casei-L2 induced significant neutralizing activities against genital infection by HPV-16, -18, -45, and -58 pseudovirions encoding a fluorescence reporter gene. These results collectively indicate that oral administration of L2 displayed on L. casei induces systemic and mucosal cross-neutralizing effects in mice.  

5.1061           HIV-2 Genome Dimerization Is Required for the Correct Processing of Gag: a Second-Site Reversion in Matrix Can Restore Both Processes in Dimerization-Impaired Mutant Viruses

L’Hernault, A., Weiss, E.U., Greatorex, J.S. and Lever, A.M.

1. Virol., 86(10), 5867-5876 (2012)

  A unique feature of retroviruses is the packaging of two copies of their genome, noncovalently linked at their 5′ ends. In vitro, dimerization of human immunodeficiency virus type 2 (HIV-2) RNA occurs by interaction of a self-complementary sequence exposed in the loop of stem-loop 1 (SL-1), also termed the dimer initiation site (DIS). However, in virions, HIV-2 genome dimerization does not depend on the DIS. Instead, a palindrome located within the packaging signal (Psi) is the essential motif for genome dimerization. We reported previously that a mutation within Psi decreasing genome dimerization and packaging also resulted in a reduced proportion of mature particles (A. L'Hernault, J. S. Greatorex, R. A. Crowther, and A. M. Lever, Retrovirology 4:90, 2007). In this study, we investigated further the relationship between HIV-2 genome dimerization, particle maturation, and infectivity by using a series of targeted mutations in SL-1. Our results show that disruption of a purine-rich (₃₉₂-GGAG-₃₉₅) motif within Psi causes a severe reduction in genome dimerization and a replication defect. Maintaining the extended SL-1 structure in combination with the ₃₉₂-GGAG-₃₉₅ motif enhanced packaging. Unlike that of HIV-1, which can replicate despite mutation of the DIS, HIV-2 replication depends critically on genome dimerization rather than just packaging efficiency. Gag processing was altered in the HIV-2 dimerization mutants, resulting in the accumulation of the MA-CA-p2 processing intermediate and suggesting a link between genome dimerization and particle assembly. Analysis of revertant SL-1 mutant viruses revealed that a compensatory mutation in matrix (70TI) could rescue viral replication and partially restore genome dimerization and Gag processing. Our results are consistent with interdependence between HIV-2 RNA dimerization and the correct proteolytic cleavage of the Gag polyprotein.  

5.1062           Comparison of the behavioural and histological characteristics of the 6-OHDA and α-synuclein rat models of Parkinson's disease

Decressac, M., Mattsson, B. and Björklund, A. Exp. Neurol., 235, 306-315 (2012)   Development of relevant models of Parkinson's disease (PD) is essential for a better understanding of the pathological processes underlying the human disease and for the evaluation of promising targets for therapeutic intervention. To date, most pre-clinical studies have been performed in the well-established rodent and non-human primate models using injection of 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydroxypyridine (MPTP). Overexpression of the disease-causing protein α-synuclein (α-syn), using adeno-associated viral (AAV) vectors, has provided a novel model that recapitulates many features of the human disease. In the present study we compared the AAV-α-syn rat model with models where the nigro-striatal pathway is lesioned by injection of 6-OHDA in the striatum (partial lesion) or the medial forebrain bundle (full lesion). Examination of the behavioural changes over time revealed a different progression and magnitude of the motor impairment. Interestingly, dopamine (DA) neuron loss is prominent in both the toxin and the AAV-α-syn models. However, α-syn overexpressing animals were seen to exhibit less cell and terminal loss for an equivalent level of motor abnormalities. Prominent and persistent axonal pathology is only observed in the α-syn rat model. We suggest that, while neuronal and terminal loss mainly accounts for the behavioural impairment in the toxin-based model, similar motor deficits result from the combination of cell death and dysfunction of the remaining nigro-striatal neurons in the AAV-α-syn model. While the two models have been developed to mimic DA neuron deficiency, they differ in their temporal and neuropathological characteristics, and replicate different aspects of the pathophysiology of the human disease. This study suggests that the AAV-α-syn model replicates the human pathology more closely than either of the other two 6-OHDA lesion models.      

5.1063           The Choice of Resin-Bound Ligand Affects the Structure and Immunogenicity of Column-Purified Human Papillomavirus Type 16 Virus-Like Particles

Kim, H.J., Lim, S.J., Kwag, H-L. and Kim, H-J. PloS One, 7(4), e35893 (2012)   Cell growth conditions and purification methods are important in determining biopharmaceutical activity. However, in studies aimed at manufacturing virus-like particles (VLPs) for the purpose of creating a prophylactic vaccine and antigen for human papillomavirus (HPV), the effects of the presence of a resin-bound ligand during purification have never been investigated. In this study, we compared the structural integrity and immunogenicity of two kinds of VLPs derived from HPV type 16 (HPV16 VLPs): one VLP was purified by heparin chromatography (hHPV16 VLP) and the other by cation-exchange chromatography (cHPV16 VLP). The reactivity of anti-HPV16 neutralizing monoclonal antibodies (H16.V5 and H16.E70) towards hHPV16 VLP were significantly higher than the observed cHPV16 VLP reactivities, implying that hHPV16 VLP possesses a greater number of neutralizing epitopes and has a greater potential to elicit anti-HPV16 neutralizing antibodies. After the application of heparin chromatography, HPV16 VLP has a higher affinity for H16.V5 and H16.E70. This result indicates that heparin chromatography is valuable in selecting functional HPV16 VLPs. In regard to VLP immunogenicity, the anti-HPV16 L1 IgG and neutralizing antibody levels elicited by immunizations of mice with hHPV16 VLPs were higher than those elicited by cHPV16 VLP with and without adjuvant. Therefore, the ability of hHPV16 VLP to elicit humoral immune responses was superior to that of cHPV16 VLP. We conclude that the specific chromatographic technique employed for the purification of HPV16 VLPs is an important factor in determining the structural characteristics and immunogenicity of column-purified VLPs.  

5.1064           Corneal Transduction by Intra-Stromal Injection of AAV Vectors In Vivo in the Mouse and Ex Vivo in Human Explants

Hippert, C., Ibanes, S., Serratrice, N., Court, F., Malecaze, F., Kremer, E.J. and Kkalatzis, V. PloS One, 7(4), e35318 (2012)   The cornea is a transparent, avascular tissue that acts as the major refractive surface of the eye. Corneal transparency, assured by the inner stroma, is vital for this role. Disruption in stromal transparency can occur in some inherited or acquired diseases. As a consequence, light entering the eye is blocked or distorted, leading to decreased visual acuity. Possible treatment for restoring transparency could be via viral-based gene therapy. The stroma is particularly amenable to this strategy due to its immunoprivileged nature and low turnover rate. We assayed the potential of AAV vectors to transduce keratocytes following intra-stromal injection in vivo in the mouse cornea and ex vivo in human explants. In murine and human corneas, we transduced the entire stroma using a single injection, preferentially targeted keratocytes and achieved long-term gene transfer (up to 17 months in vivo in mice). Of the serotypes tested, AAV2/8 was the most promising for gene transfer in both mouse and man. Furthermore, transgene expression could be transiently increased following aggression to the cornea.  

5.1065           Rescue of severely affected dystrophin/utrophin-deficient mice through scAAV-U7snRNA-mediated exon skipping

Goyenvalle, A., Babbs, A., Wright, J., Wilkins, V., Powell, D., Garcia, L. and Davies, K.E. Hum. Mol. Genet., 21(11), 2559-2571 (2012)   Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD and recent clinical trials have demonstrated encouraging results. However, antisense oligonucleotide-mediated exon skipping for DMD still faces major hurdles such as extremely low efficacy in the cardiac muscle, poor cellular uptake and relatively rapid clearance from circulation, which means that repeated administrations are required to achieve some therapeutic efficacy. To overcome these limitations, we previously proposed the use of small nuclear RNAs (snRNAs), especially U7snRNA to shuttle the antisense sequences after vectorization into adeno-associated virus (AAV) vectors. In this study, we report for the first time the efficiency of the AAV-mediated exon skipping approach in the utrophin/dystrophin double-knockout (dKO) mouse which is a very severe and progressive mouse model of DMD. Following a single intravenous injection of scAAV9-U7ex23 in dKO mice, near-normal levels of dystrophin expression were restored in all muscles examined, including the heart. This resulted in a considerable improvement of their muscle function and dystrophic pathology as well as a remarkable extension of the dKO mice lifespan. These findings suggest great potential for AAV-U7 in systemic treatment of the DMD phenotype.  

5.1066           High-efficiency transduction of human monocyte-derived dendritic cells by capsid-modified recombinant AAV2 vectors

Aslanidi, G.V., Rivers, A.E., Ortiz, L., Govindasamy, L., Ling, C., Jayandharan, G.R., Zolotukhin, S., Agbandje-McKenna, M and Srivastava, A. Vaccine, 30, 3908-3917 (2012)   Phosphorylation of surface-exposed tyrosine residues negatively impacts the transduction efficiency of recombinant AAV2 vectors. Pre-treatment of cells with specific cellular serine/threonine kinase inhibitors also significantly increased the transduction efficiency of AAV2 vectors. We reasoned that site-directed mutagenesis of surface-exposed serine residues might allow the vectors to evade phosphorylation and thus lead to higher transduction efficiency. Each of the 15 surface-exposed serine (S) residues was substituted with valine (V) residues, and the transduction efficiency of three of these mutants, S458V, S492V and S662V, was increased by up to ∼20-fold in different cell types. The S662V mutant was efficient in transducing human monocyte-derived dendritic cells (moDCs), a cell type not readily amenable to transduction by the conventional AAV vectors, and did not induce any phenotypic changes in these cells. Recombinant S662V-AAV2 vectors encoding a truncated human telomerase (hTERT) gene were generated and used to stimulate cytotoxic T cells (CTLs) against target cells. S662V-AAV2-hTERT vector-transduced DCs resulted in rapid, specific T-cell clone proliferation and generation of robust CTLs, which led to specific cell lysis of K562 cells. These studies suggest that high-efficiency transduction of moDCs by serine-modified AAV2 vectors is feasible, which supports the potential utility of these vectors for future human DCs vaccine studies.  

5.1067           Sialyllactose in Viral Membrane Gangliosides Is a Novel Molecular Recognition Pattern for Mature Dendritic Cell Capture of HIV-1

Izquierdo-Useros, N., Lorizate, M., Contreras, F-X., Rodriguez-Plata, M.T., Glass, B., Erkizia, I., Prado, J.G., Casas, J., Fabrias, G., Kräusslich, H-G., Martinez-Picado, J. PloS Biology, 10(4), e1001315 (2012)   HIV-1 is internalized into mature dendritic cells (mDCs) via an as yet undefined mechanism with subsequent transfer of stored, infectious virus to CD4⁺ T lymphocytes. Thus, HIV-1 subverts a DC antigen capture mechanism to promote viral spread. Here, we show that gangliosides in the HIV-1 membrane are the key molecules for mDC uptake. HIV-1 virus-like particles and liposomes mimicking the HIV-1 lipid composition were shown to use a common internalization pathway and the same trafficking route within mDCs. Hence, these results demonstrate that gangliosides can act as viral attachment factors, in addition to their well known function as cellular receptors for certain viruses. Furthermore, the sialyllactose molecule present in specific gangliosides was identified as the determinant moiety for mDC HIV-1 uptake. Thus, sialyllactose represents a novel molecular recognition pattern for mDC capture, and may be crucial both for antigen presentation leading to immunity against pathogens and for succumbing to subversion by HIV-1.  

5.1068           Dysregulation of dopamine receptor D2 as a sensitive measure for Huntington disease pathology in model mice

Crook, Z.R. and Housman, D.E. PNAS, 109(9), 7487-7492 (2012)   The ability to quantitatively evaluate the impact of a potential therapeutic intervention for Huntington disease (HD) in animal models for the disease is a critical step in the pathway to development of an effective therapy for this devastating neurodegenerative disorder. We report here an approach that combines a cell-based assay’s quantitative accuracy and direct relationship to molecular processes with the ability to directly monitor effects in HD model mouse neurons. To accomplish this goal, we have developed an accurate quantitative reporter assay for a transcript known to be down-regulated as an early consequence of mutant huntingtin expression. This system uses mouse strains carrying a GFP reporter for the expression of the dopamine receptor D2, expressed in the medium spiny neurons of the basal ganglion. This receptor consistently demonstrates reduced expression in patients and murine models, and the FACS-based assay gives a highly accurate and quantitative readout of this pathology in mouse neurons expressing mutant huntingtin. For four genetic models and one viral model, a highly reproducible time course of loss of reporter expression is observed. This quantitative measure of HD pathology can be used to measure the effects of HD therapeutics in small cohorts with high confidence. We further demonstrate that the introduction of an shRNA against the huntingtin transgene by virus can improve this pathological status in medium spiny neurons transduced with the construct. We believe this system can be of great utility in the validation of effective therapeutic interventions for HD.  

5.1069           Comparative analysis of recombinant Human Papillomavirus 8 L1 production in plants by a variety of expression systems and purification methods

Matic, S., Masenga, V., Poli, A., Rinaldi, R., Milne, R.G., Vecchiati, M. and Noris, E. Plant Biotech. J., 10(4), 410-421 (2012)   Human papillomavirus 8 (HPV-8), one of the high-risk cutaneous papillomaviruses (cHPVs), is associated with epidermodysplasia verruciformis and nonmelanoma skin cancer in immuno-compromised individuals. Currently, no vaccines against cHPVs have been reported; however, recent studies on cross-neutralizing properties of their capsid proteins (CP) have fostered an interest in vaccine production against these viruses. We examined the potential of producing HPV-8 major CP L1 in Nicotiana benthamiana by agroinfiltration of different transient expression vectors: (i) the binary vector pBIN19 with or without silencing suppressor constructs, (ii) the nonreplicating Cowpea mosaic virus-derived expression vector pEAQ-HT and (iii) a replicating Tobacco mosaic virus (TMV)-based vector alone or with signal peptides. Although HPV-8 L1 was successfully expressed using pEAQ-HT and TMV, a 15-fold increase was obtained with pEAQ-HT. In contrast, no L1 protein could be immune detected using pBIN19 irrespective of whether silencing suppressors were coexpressed, although such constructs were required for identifying L1-specific transcripts. A fourfold yield increase in L1 expression was obtained when 22 C-terminal amino acids were deleted (L1ΔC22), possibly eliminating a nuclear localization signal. Electron microscopy showed that plant-made HPV-8 L1 proteins assembled in appropriate virus-like particles (VLPs) of T = 1 or T = 7 symmetry. Ultrathin sections of L1ΔC22-expressing cells revealed their accumulation in the cytoplasm in the form of VLPs or paracrystalline arrays. These results show for the first time the production and localization of HPV-8 L1 protein in planta and its assembly into VLPs representing promising candidate for potential vaccine production.  

5.1070           Foamy Virus Pol Protein Expressed as a Gag-Pol Fusion Retains Enzymatic Activities, Allowing for Infectious Virus Production

Lee, E-G., Sinicrope, A., Jackson, D.L., Yu, S.F. and Linial, M.L.

1. Virol., 86(11), 5992-6001 (2012)

  Foamy viruses (FV) synthesize Pol from a spliced pol mRNA independently of Gag, unlike orthoretroviruses, which synthesize Pol as a Gag-Pol protein that coassembles with Gag. We found that prototype FV (PFV) mutants expressing Gag and Pol only as a Gag-Pol protein without the spliced Pol contain protease activity equivalent to that of wild-type (WT) Pol. Regardless of the presence or absence of the spliced Pol, the PFV Gag-Pol proteins can assemble into virus-like particles (VLPs), in contrast to the orthoretroviral Gag-Pol proteins, which cannot form VLPs. However, the PFV Gag-Pol VLPs have aberrant morphologies and are not infectious. In the absence of the spliced Pol, coexpression of a PFV Gag-Pol protein with Gag can produce infectious virions. Our results suggest that enzymes encoded by PFV pol (protease, reverse transcriptase, and integrase) are enzymatically active if they are synthesized as part of a Gag-Pol protein.  

5.1071           Carbohydrate response element-binding protein (ChREBP) plays a pivotal role in beta cell glucotoxicity

Poungvarin, N., Lee, J.K., Yechoor, V.K., Li, M.V., Assavapoke, T., Suksaranjit, P., Thepsongwajja, J.J., Saha, P.K., Oka, K. and Chan, L. Diabetologia, 55(6), 1783-1796 (2012)   Aims/hypothesis This study was aimed at the elucidation of the pathogenesis of glucotoxicity, i.e. the mechanism whereby hyperglycaemia damages pancreatic beta cells. The identification of pathways in the process may help identify targets for beta cell-protective therapy. Carbohydrate response element-binding protein (ChREBP), a transcription factor that regulates the expression of multiple hyperglycaemia-induced genes, is produced in abundance in pancreatic beta cells. We hypothesise that ChREBP plays a pivotal role in mediating beta cell glucotoxicity. Methods We assessed the role of ChREBP in glucotoxicity in 832/13 beta cells, isolated mouse islets and human pancreas tissue sections using multiple complementary approaches under control and high-glucose-challenge conditions as well as in adeno-associated virus-induced beta cell-specific overexpression of Chrebp (also known as Mlxipl) in mice. Results Under both in vitro and in vivo conditions, ChREBP activates downstream target genes, including fatty acid synthase and thioredoxin-interacting protein, leading to lipid accumulation, increased oxidative stress, reduced insulin gene transcription/secretion and enhanced caspase activity and apoptosis, processes that collectively define glucotoxicity. Immunoreactive ChREBP is enriched in the nucleuses of beta cells in pancreatic tissue sections from diabetic individuals compared with non-diabetic individuals. Finally, we demonstrate that induced beta cell-specific Chrebp overexpression is sufficient to phenocopy the glucotoxicity manifestations of hyperglycaemia in mice in vivo. Conclusions/interpretation These data indicate that ChREBP is a key transcription factor that mediates many of the hyperglycaemia-induced activations in a gene expression programme that underlies beta cell glucotoxicity at the molecular, cellular and whole animal levels.  

5.1072           Inclusion of a portion of the native SNCA 3′UTR reduces toxicity of human S129A SNCA on striatal-projecting dopamine neurons in rat substantia Nigra

Khodr, C.E., Pedapati, J., Han, Y. and Bohn, M.C. Develop. Neurobiol., 72(6), 906-917 (2012)   Experimental models of Parkinson's disease (PD) created by aberrant expression of the alpha-synuclein (SNCA) coding region have been reported. However, noncoding regions function in normal physiology and recent in vitro studies have shown that microRNAs-7 and -153 regulate SNCA expression by binding the 3′UTR. Here, effects of different hSNCA forms were examined in vivo. Adult, male rats were injected into one substantia nigra (SN) with AAV-wtSNCA, AAV-S129A hSNCA, or AAV-S129D hSNCA either with or without a portion of the native 3′UTR. DA neurons in SN that maintained striatal (ST) projections at the end of treatment were retrogradely labeled by bilateral ST fluorogold (FG) injections and FG-positive DA neurons in SN were counted. At 5 weeks, hSNCA coding vectors reduced numbers of FG-positive neurons in injected SN compared with uninjected SN (wtSNCA, p = 0.05; S129A/D hSNCA, p = 0.01). At 7 and 9 weeks, wtSNCA- and S129D hSNCA-treated rats exhibited recovery, but S129A hSNCA-injected rats did not (p = 0.01). In contrast, numbers of FG-positive neurons were unaffected by hSNCA expression when the 3′UTR was included. When FG-positive neurons were expressed as the ratio of numbers in injected to uninjected sides, the S129A hSNCA coding vector resulted in the highest decrease at 9 weeks versus wtSNCA (p = 0.05) or S129D hSNCA (p = 0.01). Inclusion of the 3′UTR resulted in no significant differences in FG-positive neuron ratios. These data suggest that inclusion of the 3′UTR protects against S129A hSNCA-induced loss of nigrostriatal-projecting DA neurons in vivo and that mis-regulation of hSNCA expression and function at noncoding regions contribute to PD pathogenesis.  

5.1073           Gene delivery to mitochondria by targeting modified adenoassociated virus suppresses Leber’s hereditary optic neuropathy in a mouse model

Yu, H., Koilkonda, R., Chou, T-H., Porciatti, V., Ozdemir, S.S., Chiiodo, V., Boye, S.L., Boye, S.E., Hauswirth, W.W., Lewin, A.S. and Guy, J. PNAS Plus, 109, E1238-E1247 (2012)   To introduce DNA into mitochondria efficiently, we fused adenoassociated virus capsid VP2 with a mitochondrial targeting sequence to carry the mitochondrial gene encoding the human NADH ubiquinone oxidoreductase subunit 4 (ND4). Expression of WT ND4 in cells with the G11778A mutation in ND4 led to restoration of defective ATP synthesis. Furthermore, with injection into the rodent eye, human ND4 DNA levels in mitochondria reached 80% of its mouse homolog. The construct expressed in most inner retinal neurons, and it also suppressed visual loss and optic atrophy induced by a mutant ND4 homolog. The adenoassociated virus cassette accommodates genes of up to ∼5 kb in length, thus providing a platform for introduction of almost any mitochondrial gene and perhaps even allowing insertion of DNA encompassing large deletions of mtDNA, some associated with aging, into the organelle of adults.  

5.1074           Adult-onset focal expression of mutated human tau in the hippocampus impairs spatial working memory of rats

Mustroph, M:L., King, M.A., Klein, R.L. and Ramirez, J.J. Behavioural Brain Res., 233(1), 141-148 (2012)   Tauopathy in the hippocampus is one of the earliest cardinal features of Alzheimer's disease (AD), a condition characterized by progressive memory impairments. In fact, density of tau neurofibrillary tangles (NFTs) in the hippocampus strongly correlates with severity of cognitive impairments in AD. In the present study, we employed a somatic cell gene transfer technique to create a rodent model of tauopathy by injecting a recombinant adeno-associated viral vector with a mutated human tau gene (P301L) into the hippocampus of adult rats. The P301L mutation is causal for frontotemporal dementia with parkinsonism-17 (FTDP-17), but it has been used for studying memory effects characteristic of AD in transgenic mice. To ascertain if P301L-induced mnemonic deficits are persistent, animals were tested for 6 months. It was hypothesized that adult-onset, spatially restricted tau expression in the hippocampus would produce progressive spatial working memory deficits on a learned alternation task. Rats injected with the tau vector exhibited persistent impairments on the hippocampal-dependent task beginning at about 6 weeks post-transduction compared to rats injected with a green fluorescent protein vector. Histological analysis of brains for expression of human tau revealed hyperphosphorylated human tau and NFTs in the hippocampus in experimental animals only. Thus, adult-onset, vector-induced tauopathy spatially restricted to the hippocampus progressively impaired spatial working memory in rats. We conclude that the model faithfully reproduces histological and behavioral findings characteristic of dementing tauopathies. The rapid onset of sustained memory impairment establishes a preclinical model particularly suited to the development of potential tauopathy therapeutics.  

5.1075           AAV2 mediated retrograde transduction of corticospinal motor neurons reveals initial and selective apical dendrite degeneration in ALS

Jara, J.H., Villa, S.R., Khan, N.A., Bohn, M.C. and Özdinler, P.H. Neurobiololgy of Disease, 47, 174-183 (2012)   Corticospinal motor neurons (CSMN) are the cortical component of motor neuron circuitry, which controls voluntary movement and degenerates in diseases such as amyotrophic lateral sclerosis, primary lateral sclerosis and hereditary spastic paraplegia. By using dual labeling combined with molecular marker analysis, we identified AAV2–2 mediated retrograde transduction as an effective approach to selectively target CSMN without affecting other neuron populations both in wild-type and hSOD1^G93A transgenic ALS mice. This approach reveals very precise details of cytoarchitectural defects within vulnerable neurons in vivo. We report that CSMN vulnerability is marked by selective degeneration of apical dendrites especially in layer II/III of the hSOD1^G93A mouse motor cortex, where cortical input to CSMN function is vastly modulated. While our findings confirm the presence of astrogliosis and microglia activation, they do not lend support to their direct role for the initiation of CSMN vulnerability. This study enables development of targeted gene replacement strategies to CSMN in the cerebral cortex, and reveals CSMN cortical modulation defects as a potential cause of neuronal vulnerability in ALS.  

5.1076           Microvesicle-associated AAV Vector as a Novel Gene Delivery System

Maguire, C.A., Balaj, L., Sivaraman, S., Crommentuijn, M.H.W., Ericsson, M., Mincheva-Nilsson, L., Baranov, V., Gianni, D., Tannous, B.A., Sena-Esteves, M., Breakefiled, X.O. and Skog, J.

Molecular Therapy, 20(5), 960-971 (2012)

Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery.

 

5.1077           Functional mechanisms of the cellular prion protein (PrPC) associated anti-HIV-1 properties

Alais, S., Soto-Rifo, R., Balter, V., Gruffat, H., Manet, E., Schaeffer, L., Darlix, J.L., Cimarelli, A., Raposo, G., Ohlmann, T. and Leblanc, P.

Cell. Mol. Life Sci., 69(8), 1331-1352 (2012)

The cellular prion protein PrPC/CD230 is a GPI-anchor protein highly expressed in cells from the nervous and immune systems and well conserved among vertebrates. In the last decade, several studies suggested that PrPC displays antiviral properties by restricting the replication of different viruses, and in particular retroviruses such as murine leukemia virus (MuLV) and the human immunodeficiency virus type 1 (HIV-1). In this context, we previously showed that PrPC displays important similarities with the HIV-1 nucleocapsid protein and found that PrPC expression in a human cell line strongly reduced HIV-1 expression and virus production. Using different PrPC mutants, we report here that the anti-HIV-1 properties are mostly associated with the amino-terminal 24-KRPKP-28 basic domain. In agreement with its reported RNA chaperone activity, we found that PrPC binds to the viral genomic RNA of HIV-1 and negatively affects its translation. Using a combination of biochemical and cell imaging strategies, we found that PrPC colocalizes with the virus assembly machinery at the plasma membrane and at the virological synapse in infected T cells. Depletion of PrPC in infected T cells and microglial cells favors HIV-1 replication, confirming its negative impact on the HIV-1 life cycle.

5.1078           One influenza virus particle packages eight unique viral RNAs as shown by FISH analysis

Chou, Y-y., Vafabakhsh, R., Dogänay, S., Gao, Q., Ha, T. and Palese, P. PNAS, 109(23), 9101-9106 (2012)   Influenza A virus possesses a segmented genome of eight negative-sense, single-stranded RNAs. The eight segments have been shown to be represented in approximately equal molar ratios in a virus population; however, the exact copy number of each viral RNA segment per individual virus particles has not been determined. We have established an experimental approach based on multicolor single-molecule fluorescent in situ hybridization (FISH) to study the composition of viral RNAs at single-virus particle resolution. Colocalization analysis showed that a high percentage of virus particles package all eight different segments of viral RNAs. To determine the copy number of each RNA segment within individual virus particles, we measured the photobleaching steps of individual virus particles hybridized with fluorescent probes targeting a specific viral RNA. By comparing the photobleaching profiles of probes against the HA RNA segment for the wild-type influenza A/Puerto Rico/8/34 (PR8) and a recombinant PR8 virus carrying two copies of the HA segment, we concluded that only one copy of HA segment is packaged into a wild type virus particle. Our results showed similar photobleaching behaviors for other RNA segments, suggesting that for the majority of the virus particles, only one copy of each RNA segment is packaged into one virus particle. Together, our results support that the packaging of influenza viral genome is a selective process.  

5.1079           Human Galectin 3 Binding Protein Interacts with Recombinant Adeno-Associated Virus Type 6

Denard, J., Beley, C., Kotin, R., Lai-Kuen, R., Blot, S., Leh, H., Asokan, A., Samulski, J., Moullier, P., Voit, T., Garcia, L.a dnSvinartchouk, F.

1. Virol., 86(12), 6620-6631 (2012)

  Recombinant adeno-associated viruses (rAAVs) hold enormous potential for human gene therapy. Despite the well-established safety and efficacy of rAAVs for in vivo gene transfer, there is still little information concerning the fate of vectors in blood following systemic delivery. We screened for serum proteins interacting with different AAV serotypes in humans, macaques, dogs, and mice. We report that serotypes rAAV-1, -5, and -6 but not serotypes rAAV-2, -7, -8, -9, and -10 interact in human sera with galectin 3 binding protein (hu-G3BP), a soluble scavenger receptor. Among the three serotypes, rAAV-6 has the most important capacities for binding to G3BP. rAAV-6 also bound G3BP in dog sera but not in macaque and mouse sera. In mice, rAAV-6 interacted with another protein of the innate immune system, C-reactive protein (CRP). Furthermore, interaction of hu-G3BP with rAAV-6 led to the formation of aggregates and hampered transduction when the two were codelivered into the mouse. Based on these data, we propose that species-specific interactions of AAVs with blood proteins may differentially impact vector distribution and efficacy in different animal models.  

5.1080           LuIII Parvovirus Selectively and Efficiently Targets, Replicates in, and Kills Human Glioma Cells

Paglino, J.C., Ozduman, K. and van den Pol, A.N.

1. Virol., 86(13), 7280-7291 (2012)

  Because productive infection by parvoviruses requires cell division and is enhanced by oncogenic transformation, some parvoviruses may have potential utility in killing cancer cells. To identify the parvovirus(es) with the optimal oncolytic effect against human glioblastomas, we screened 12 parvoviruses at a high multiplicity of infection (MOI). MVMi, MVMc, MVM-G17, tumor virus X (TVX), canine parvovirus (CPV), porcine parvovirus (PPV), rat parvovirus 1A (RPV1A), and H-3 were relatively ineffective. The four viruses with the greatest oncolytic activity, LuIII, H-1, MVMp, and MVM-G52, were tested for the ability, at a low MOI, to progressively infect the culture over time, causing cell death at a rate higher than that of cell proliferation. LuIII alone was effective in all five human glioblastomas tested. H-1 progressively infected only two of five; MVMp and MVM-G52 were ineffective in all five. To investigate the underlying mechanism of LuIII's phenotype, we used recombinant parvoviruses with the LuIII capsid replacing the MVMp capsid or with molecular alteration of the P4 promoter. The LuIII capsid enhanced efficient replication and oncolysis in MO59J gliomas cells; other gliomas tested required the entire LuIII genome to exhibit enhanced infection. LuIII selectively infected glioma cells over normal glial cells in vitro. In mouse models, human glioblastoma xenografts were selectively infected by LuIII when administered intratumorally; LuIII reduced tumor growth by 75%. LuIII also had the capacity to selectively infect subcutaneous or intracranial gliomas after intravenous inoculation. Intravenous or intracranial LuIII caused no adverse effects. Intracranial LuIII caused no infection of mature mouse neurons or glia in vivo but showed a modest infection of developing neurons.  

5.1081           Gene Transfer Targeting Mouse Vestibule Using Adenovirus and Adeno-Associated Virus Vectors

Okada, H., Iizuka, T., Mochizuki, H., Nihira, T., Kamiya, K., Inoshita, A., Kasagi, H., Kasai, M. and Ikeda, K. Otology & Neurology, 33(4), 655-659 (2012)   Hypothesis: The present study assessed how to inject a gene into the mouse vestibule and which is the optimum gene to the mouse vestibule adenovirus (AdV) vector or adeno-associated virus (AAV) vector. Background: Loss of vestibular hair cell is seen in various balance disorder diseases. There have been some reports concerning gene delivery to the mouse vestibule in recent years. To effectively induce transgene expression at the vestibule, we assessed the efficiency of inoculating the mouse inner ear using various methods. Methods: We employed an AdV- and AAV-carrying green fluorescent protein using a semicircular canal approach (via a canalostomy) and round window approach. Results: AAV injection via canalostomy induced gene expression at the hair cells, supporting cells, and fibrocytes at the vestibular organs without auditory or balance dysfunction, suggesting it was the most suitable transfection method. This method is thus considered to be a promising strategy to prevent balance dysfunction. Conclusion: AAV injection via canalostomy to the vestibule is the noninvasive and highly efficient transfection method, and this study may have the potential to repair balance disorders in human in the future.  

5.1082           CMV Late Phase-Induced mTOR Activation Is Essential for Efficient Virus Replication in Polarized Human Macrophages

Poglitsch, M., Weichhart, T., Hecking, M., Werzowa, J., Katholnig, K., Antlanger, M., Krmpotic, A., Jonjic, S., Hörl, W.H., Zlabinger, G.J., Puchhammer, E. and Säemann, M.D. Am. J. Transplant., 12(6), 1458-1468 (2012)   Human cytomegalovirus (CMV) remains one of the most important pathogens following solid-organ transplantation. Mounting evidence indicates that mammalian target of rapamycin (mTOR) inhibitors may decrease the incidence of CMV infection in solid-organ recipients. Here we aimed at elucidating the molecular mechanisms of this effect by employing a human CMV (HCMV) infection model in human macrophages, since myeloid cells are the principal in vivo targets of HCMV. We demonstrate a highly divergent host cell permissiveness for HCMV with optimal infection susceptibility in M2 but not M1 polarized macrophages. Employing an ultrahigh purified HCMV stock we observed rapamycin-independent viral entry and induction of IFN-β transcripts, but no proinflammatory cytokines or mitogen-activated protein kinases and mTOR activation early after infection. However, in the late infection phase, sustained mTOR activation was observed in HCMV-infected cells and was required for efficient viral protein synthesis including the viral late phase proteins pUL-44 and pp65. Accordingly, rapamycin strongly suppressed CMV replication 3 and 5 days postinfection in macrophages. In conclusion, these data indicate that mTOR is essential for virus replication during late phases of the viral cycle in myeloid cells and might explain the potent anti-CMV effects of mTOR inhibitors after organ transplantation.  

5.1083           Molecular and behavioral changes associated with adult hippocampus-specific SynGAP1 knockout

Muhia, M., Willadt, S. and Yee, B.K. Learn. Mem., 19(7), 268-281 (2012)   The synaptic Ras/Rap-GTPase-activating protein (SynGAP1) plays a unique role in regulating specific downstream intracellular events in response to N-methyl-D-aspartate receptor (NMDAR) activation. Constitutive heterozygous loss of SynGAP1 disrupts NMDAR-mediated physiological and behavioral processes, but the disruptions might be of developmental origin. Therefore, the precise role of SynGAP1 in the adult brain, including its relative functional significance within specific brain regions, remains unexplored. The present study constitutes the first attempt in achieving adult hippocampal-specific SynGAP1 knockout using the Cre/loxP approach. Here, we report that this manipulation led to a significant numerical increase in both small and large GluA1 and NR1 immunoreactive clusters, many of which were non-opposed to presynaptic terminals. In parallel, the observed marked decline in the amplitude of spontaneous excitatory currents (sEPSCs) and inter-event intervals supported the impression that SynGAP1 loss might facilitate the accumulation of extrasynaptic glutamatergic receptors. In addition, SynGAP1-mediated signaling appears to be critical for the proper integration and survival of newborn neurons. The manipulation impaired reversal learning in the probe test of the water maze and induced a delay-dependent impairment in spatial recognition memory. It did not significantly affect anxiety or reference memory acquisition but induced a substantial elevation in spontaneous locomotor activity in the open field test. Thus, the present study demonstrates the functional significance of SynGAP1 signaling in the adult brain by capturing several changes that are dependent on NMDAR and hippocampal integrity.  

5.1084           Virus-mediated gene delivery for human gene therapy

Giacca, M. and  Zacchigna, S.

1. Controlled Release, 161, 377-388 (2012)

  After over 20 years from the first application of gene transfer in humans, gene therapy is now a mature discipline, which has progressively overcome several of the hurdles that prevented clinical success in the early stages of application. So far, the vast majority of gene therapy clinical trials have exploited viral vectors as very efficient nucleic acid delivery vehicles both in vivo and ex vivo. Here we summarize the current status of viral gene transfer for clinical applications, with special emphasis on the molecular properties of the major classes of viral vectors and the information so far obtained from gene therapy clinical trials.  

5.1085           Apolipoprotein-E and hepatitis C lipoviral particles in genotype 1 infection: Evidence for an association with interferon sensitivity

Sheridan, D.A., Bridge, S.H., Felmlee, D.J., Crossey, M.M.E., Thomas, H.C., Taylor-Robinson, S.D., Toms, G.L., Neely, T.R,D,G. and Bassendine, M.F:

1. Hepatol., 57, 32-38 (2012)

  Background & Aims Hepatitis C virus (HCV) interacts with apolipoproteins B (apoB) and E (apoE) to form infectious lipoviral particles (LVP). Response to peginterferon is influenced by interferon-stimulated genes (ISGs) and IL28B genotype. LDL cholesterol (LDL-C) also predicts interferon response, therefore we hypothesised that LVP may also be associated with interferon sensitivity. Methods LVP (HCV RNA density ⩽1.07 g/ml) and ‘non-LVP’ (d >1.07 g/ml) were measured in 72 fasted HCV-G1 patients by iodixanol density gradient ultracentrifugation and the LVP ratio (LVP/LVP + non-LVP) was calculated. Fasting lipid profiles and apolipoproteins B and E were measured. Interferon-gamma-inducible protein 10 kDa (IP10), a marker of ISGs, was measured by ELISA. Results Complete early virological response (EVR) was associated with lower apoE (23.9 ± 7.7 vs. 36.1 ± 15.3 mg/L, p = 0.013), higher LDL-C (p = 0.039) and lower LVP ratios (p = 0.022) compared to null responders. In multivariate linear regression analysis, apoE was independently associated with LVP (R² 19.5%, p = 0.003) and LVP ratio (p = 0.042), and negatively with LDL-C (p <0.001). IP10 was significantly associated with ApoB (p = 0.001) and liver stiffness (p = 0.032). IL28B rs12979860 CC was associated with complete EVR (p = 0.044), low apoE (CC 28 ± 11 vs. CT/TT 35 ± 13 mg/L, p = 0.048) and higher non-LVP (p = 0.008). Logistic regression analysis indicated that patients with high LVP ratios were less likely to have EVR (odds ratio 0.01, p = 0.018). Conclusions In HCV-G1, interferon sensitivity is characterised by low LVP ratios and low apoE levels in addition to higher LDL-C and IL28B rs12979860 CC. Null-response is associated with increased LVP ratio. The association of apoE and LVP with peginterferon treatment response suggests that lipid modulation is a potential target to modify interferon sensitivity.  

5.1086           PICOT increases cardiac contractility by inhibiting PKCζ activity

Oh, J.G., Jeong, D., Cha, H., Kim, J.M., Lifirsu, E., Kim, J., Yang, D.K., Park, C.S., Kho, C., Park, S., Yoo, Y.J., Kim, D.H., Kim, J., Hajjar, R.J. and Park, W.J.

1. Mol. Cell. Cardiol., 53, 53-63 (2012)

  Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT) has distinct anti-hypertrophic and inotropic functions. We have previously shown that PICOT exerts its anti-hypertrophic effect by inhibiting calcineurin-NFAT signaling through its C-terminal glutaredoxin domain. However, the mechanism underlying the inotropic effect of PICOT is unknown. The results of protein pull-down experiments showed that PICOT directly binds to the catalytic domain of PKCζ through its N-terminal thioredoxin-like domain. Purified PICOT protein inhibited the kinase activity of PKCζ in vitro, which indicated that PICOT is an endogenous inhibitor of PKCζ. The inhibition of PKCζ activity with a PKCζ-specific pseudosubstrate peptide inhibitor was sufficient to increase the cardiac contractility in vitro and ex vivo. Overexpression of PICOT or inhibition of PKCζ activity down-regulated PKCα activity, which led to the elevation of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) 2a activity, concomitant with the increased phosphorylation of phospholamban (PLB). Overexpression of PICOT or inhibition of PKCζ activity also down-regulated protein phosphatase (PP) 2A activity, which subsequently resulted in the increased phosphorylation of troponin (Tn) I and T, key myofilament proteins associated with the regulation of contractility. PICOT appeared to inhibit PP2A activity through the disruption of the functional PKCζ/PP2A complex. In contrast to the overexpression of PICOT or inhibition of PKCζ, reduced PICOT expression resulted in up-regulation of PKCα and PP2A activities, followed by decreased phosphorylation of PLB, and TnI and T, respectively, supporting the physiological relevance of these events. Transgene- or adeno-associated virus (AAV)-mediated overexpression of PICOT restored the impaired contractility and prevented further morphological and functional deterioration of the failing hearts. Taken together, the results of the present study suggest that PICOT exerts its inotropic effect by negatively regulating PKCα and PP2A activities through the inhibition of PKCζ activity. This finding provides a novel insight into the regulation of cardiac contractility.  

5.1087           Homologous Recombination Mediates Functional Recovery of Dysferlin Deficiency following AAV5 Gene Transfer

Grose, W.E., Clark, K.R., Griffin, D., Malik, V., Shontz, K.M., Montgomery, C.L., Lewis, S., Brown Jr., R.H., Janssen, P.M., Mendell, J.R. and Rodino-Klapac, L.R. PloS One, 7(6), e39233 (2012)   The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV) gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb) negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9). Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes.  

5.1088           Dysregulated dopamine storage increases the vulnerability to α-synuclein in nigral neurons

Ulusoy, A., Björklund, T., Buck, K. and Kirik, D. Neurobiology of Disease, 47, 367-377 (2012)   Impairments in the capacity of dopaminergic neurons to handle cytoplasmic dopamine may be a critical factor underlying the selective vulnerability of midbrain dopamine neurons in Parkinson's disease. Furthermore, toxicity of α-synuclein in dopaminergic neurons has been suggested to be mediated by direct interaction between dopamine and α-synuclein through formation of abnormal α-synuclein species, although direct in vivo evidence to support this hypothesis is lacking. Here, we investigated the role of dopamine availability on α-synuclein mediated neurodegeneration in vivo. We found that overexpression of α-synuclein in nigral dopamine neurons in mice with deficient vesicular storage of dopamine led to a significant increase in dopaminergic neurodegeneration. Importantly, silencing the tyrosine hydroxylase enzyme – thereby reducing dopamine content in the nigral neurons – reversed the increased vulnerability back to the baseline level observed in wild-type littermates, but failed to eliminate it completely. Importantly, TH knockdown was not effective in altering the toxicity in the wild-type animals. Taken together, our data suggest that under normal circumstances, in healthy dopamine neurons, cytoplasmic dopamine is tightly controlled such that it does not contribute significantly to α-synuclein mediated toxicity. Dysregulation of the dopamine machinery in the substantia nigra, on the other hand, could act as a trigger for induction of increased toxicity in these neurons and could explain how these neurons become more vulnerable and die in the disease process.  

5.1089           Alpha-Synuclein Cell-to-Cell Transfer and Seeding in Grafted Dopaminergic Neurons In Vivo

Angot, e., Steiner, J.A., Tome, C.M.L., Ekström, P., Mattson, B., Björklund, A. and Brundlin, P. PloS One, 7(6), e39465 (2012)   Several people with Parkinson’s disease have been treated with intrastriatal grafts of fetal dopaminergic neurons. Following autopsy, 10–22 years after surgery, some of the grafted neurons contained Lewy bodies similar to those observed in the host brain. Numerous studies have attempted to explain these findings in cell and animal models. In cell culture, α-synuclein has been found to transfer from one cell to another, via mechanisms that include exosomal transport and endocytosis, and in certain cases seed aggregation in the recipient cell. In animal models, transfer of α-synuclein from host brain cells to grafted neurons has been shown, but the reported frequency of the event has been relatively low and little is known about the underlying mechanisms as well as the fate of the transferred α-synuclein. We now demonstrate frequent transfer of α-synuclein from a rat brain engineered to overexpress human α-synuclein to grafted dopaminergic neurons. Further, we show that this model can be used to explore mechanisms underlying cell-to-cell transfer of α-synuclein. Thus, we present evidence both for the involvement of endocytosis in α-synuclein uptake in vivo, and for seeding of aggregation of endogenous α-synuclein in the recipient neuron by the transferred α-synuclein. Finally, we show that, at least in a subset of the studied cells, the transmitted α-synuclein is sensitive to proteinase K. Our new model system could be used to test compounds that inhibit cell-to-cell transfer of α-synuclein and therefore might retard progression of Parkinson neuropathology.  

5.1090           AAVrh.10-Mediated Expression of an Anti-Cocaine Antibody Mediates Persistent Passive Immunization That Suppresses Cocaine-Induced Behavior

Rosenberg, J., Hicks, M.J., De, B.P., Pagovich, O., Frenk, E., Janda, K.D., Wee, S., Koob, G.F., Hackett, N.R., Kaminsky, S.M., Worgali, S., Tignor, N., Mezey, J.G. and Crystal, R.G. Human Gene Therapy, 23, 451-459 (2012)   Cocaine addiction is a major problem affecting all societal and economic classes for which there is no effective therapy. We hypothesized an effective anti-cocaine vaccine could be developed by using an adeno-associated virus (AAV) gene transfer vector as the delivery vehicle to persistently express an anti-cocaine monoclonal antibody in vivo, which would sequester cocaine in the blood, preventing access to cognate receptors in the brain. To accomplish this, we constructed AAVrh.10antiCoc.Mab, an AAVrh.10 gene transfer vector expressing the heavy and light chains of the high affinity anti-cocaine monoclonal antibody GNC92H2. Intravenous administration of AAVrh.10antiCoc.Mab to mice mediated high, persistent serum levels of high-affinity, cocaine-specific antibodies that sequestered intravenously administered cocaine in the blood. With repeated intravenous cocaine challenge, naive mice exhibited hyperactivity, while the AAVrh.10antiCoc.Mab-vaccinated mice were completely resistant to the cocaine. These observations demonstrate a novel strategy for cocaine addiction by requiring only a single administration of an AAV vector mediating persistent, systemic anti-cocaine passive immunity.

5.1091           Development and Validation of Novel AAV2 Random Libraries Displaying Peptides of Diverse Lengths and at Diverse Capsid Positions

Naumer, M., Ying, Y., Michelfelder, S., Reuter, A., Trepel, M., Müller, O.J. and Kleinschmidt, J.A. Human Gene Therapy, 23, 492-507 (2012)   Libraries based on the insertion of random peptide ligands into the capsid of adeno-associated virus type 2 (AAV2) have been widely used to improve the efficiency and selectivity of the AAV vector system. However, so far only libraries of 7-mer peptide ligands have been inserted at one well-characterized capsid position. Here, we expanded the combinatorial AAV2 display system to a panel of novel AAV libraries, displaying peptides of 5, 7, 12, 19, or 26 amino acids in length at capsid position 588 or displaying 7-mer peptides at position 453, the most prominently exposed region of the viral capsid. Library selections on two unrelated cell types—human coronary artery endothelial cells and rat cardiomyoblasts—revealed the isolation of cell type–characteristic peptides of different lengths mediating strongly improved target-cell transduction, except for the 26-mer peptide ligands. Characterization of vector selectivity by transduction of nontarget cells and comparative gene-transduction analysis using a panel of 44 human tumor cell lines revealed that insertion of different-length peptides allows targeting of distinct cellular receptors for cell entry with similar efficiency, but with different selectivity. The application of such novel AAV2 libraries broadens the spectrum of targetable receptors by capsid-modified AAV vectors and provides the opportunity to choose the best suited targeting ligand for a certain application from a number of different candidates.  

5.1092           The ephrin receptor tyrosine kinase A2 is a cellular receptor for Kaposi's sarcoma–associated herpesvirus

Hahn, A.S., Kaufmann, J.K., Wies, E., Naschberger, E., Panteleev-Ivlev, J., Schmidt, K., Holzer, A., Schmidt, M., Chen, J., König, S., Ensser, A., Myoung, J., Brockmeyer, N.H., Stürzl, M., Fleckenstein, B. and Neipel, F. Nature Med., 18(6), 961-966 (2012)   Kaposi's sarcoma–associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma¹, a highly vascularized tumor originating from lymphatic endothelial cells, and of at least two different B cell malignancies^{2, 3}. A dimeric complex formed by the envelope glycoproteins H and L (gH-gL) is required for entry of herpesviruses into host cells⁴. We show that the ephrin receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV gH-gL. EphA2 co-precipitated with both gH-gL and KSHV virions. Infection of human epithelial cells with a GFP-expressing recombinant KSHV strain, as measured by FACS analysis, was increased upon overexpression of EphA2. Antibodies against EphA2 and siRNAs directed against EphA2 inhibited infection of endothelial cells. Pretreatment of KSHV with soluble EphA2 resulted in inhibition of KSHV infection by up to 90%. This marked reduction of KSHV infection was seen with all the different epithelial and endothelial cells used in this study. Similarly, pretreating epithelial or endothelial cells with the soluble EphA2 ligand ephrinA4 impaired KSHV infection. Deletion of the gene encoding EphA2 essentially abolished KSHV infection of mouse endothelial cells. Binding of gH-gL to EphA2 triggered EphA2 phosphorylation and endocytosis, a major pathway of KSHV entry^{5, 6}. Quantitative RT-PCR and in situ histochemistry revealed a close correlation between KSHV infection and EphA2 expression both in cultured cells derived from human Kaposi's sarcoma lesions or unaffected human lymphatic endothelium, and in situ in Kaposi's sarcoma specimens, respectively. Taken together, our results identify EphA2, a tyrosine kinase with known functions in neovascularization and oncogenesis, as an entry receptor for KSHV.  

5.1093           Mucin Biopolymers As Broad-Spectrum Antiviral Agents

Lieleg, O., Lieleg, C., Bloom, J., Buck, C.B. and Ribbeck, K. Biomacromolecules, 13, 1724-1732 (2012)   Mucus is a porous biopolymer matrix that coats all wet epithelia in the human body and serves as the first line of defense against many pathogenic bacteria and viruses. However, under certain conditions viruses are able to penetrate this infection barrier, which compromises the protective function of native mucus. Here, we find that isolated porcine gastric mucin polymers, key structural components of native mucus, can protect an underlying cell layer from infection by small viruses such as human papillomavirus (HPV), Merkel cell polyomavirus (MCV), or a strain of influenza A virus. Single particle analysis of virus mobility inside the mucin barrier reveals that this shielding effect is in part based on a retardation of virus diffusion inside the biopolymer matrix. Our findings suggest that purified mucins may be used as a broad-range antiviral supplement to personal hygiene products, baby formula or lubricants to support our immune system.  

5.1094           An In-Frame Deletion in the NS Protein-Coding Sequence of Parvovirus H-1PV Efficiently Stimulates Export and Infectivity of Progeny Virions

Weiss, N., Stroh-Dege, A., Rommelaere, J., Dinsart, C. and Salome, N.

1. Virol., 86(14), 7554-7564 (2012)

  An in-frame, 114-nucleotide-long deletion that affects the NS-coding sequence was created in the infectious molecular clone of the standard parvovirus H-1PV, thereby generating Del H-1PV. The plasmid was transfected and further propagated in permissive human cell lines in order to analyze the effects of the deletion on virus fitness. Our results show key benefits of this deletion, as Del H-1PV proved to exhibit (i) higher infectivity (lower particle-to-infectivity ratio) in vitro and (ii) enhanced tumor growth suppression in vivo compared to wild-type H-1PV. This increased infectivity correlated with an accelerated egress of Del H-1PV progeny virions in producer cells and with an overall stimulation of the viral life cycle in subsequently infected cells. Indeed, virus adsorption and internalization were significantly improved with Del H-1PV, which may account for the earlier appearance of viral DNA replicative forms that was observed with Del H-1PV than wild-type H-1PV. We hypothesize that the internal deletion within the NS2 and/or NS1 protein expressed by Del H-1PV results in the stimulation of some step(s) of the viral life cycle, in particular, a maturation step(s), leading to more efficient nuclear export of infectious viral particles and increased fitness of the virus produced.  

5.1095           Engineering Multiple U7snRNA Constructs to Induce Single and Multiexon-skipping for Duchenne Muscular Dystrophy

Goyenvalle, A., Wright, J., Babbs, A., Wilkins, V., Garcia, L. and Davies, K.E. Molecular Therapy, 20(6), 1212-1221 (2012)   Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD but still faces personalized medicine challenges as different mutations found in DMD patients require skipping of different exons. However, 70% of DMD patients harbor dystrophin gene deletions in a mutation-rich area or “hot-spot” in the central genomic region. In this study, we have developed 11 different U7 small-nuclear RNA, to shuttle antisense sequences designed to mask key elements involved in the splicing of exons 45 to 55. We demonstrate that these constructs induce efficient exon skipping both in vitro in DMD patients' myoblasts and in vivo in human DMD (hDMD) mice and that they can be combined into a single vector to achieve a multi skipping of at least 3 exons. These very encouraging results provide proof of principle that efficient multiexon-skipping can be achieved using adeno-associated viral (AAV) vectors encoding multiple U7 small-nuclear RNAs (U7snRNAs), offering therefore very promising tools for clinical treatment of DMD.  

5.1096           Activation of a glioma-specific immune response by oncolytic parvovirus Minute Virus of Mice infection

Grekova, S.P., Raykov, Z., Zawatzky, R., Rommelaere, J. and Koch, U. Cancer Gene Therapy, 19(7), 468-475 (2012)   Rodent autonomous parvoviruses (PVs) are endowed with oncotropic properties and represent virotherapeutics with inherent oncolytic features. This work aimed to evaluate the capacity of Minute Virus of Mice (MVMp) to act as an adjuvant stimulating a mouse glioblastoma-specific immune response. MVMp was shown to induce cell death through apoptosis in glioma GL261 cells. Antigen-presenting cells (APCs) provide the initial cue for innate and adaptive immune responses, and thus MVMp-infected GL261 cells were tested for their ability to activate dendritic cells (DCs) and microglia (MG), two distinct cell types that are able to act as APCs. MG and discrete DC subsets were activated after co-culture with MVMp-infected glioma GL261 cells, as evidenced by upregulation of specific activation markers (CD80, CD86) and release of proinflammatory cytokines (tumor necrosis factor-α and interleukin-6). The in vivo analysis of immunodeficient and immunocompetent mice revealed a clear difference in their susceptibility to MVMp-mediated tumor suppression. Immunocompetent mice were fully protected from tumor outgrowth of GL261 cells infected ex vivo with MVMp. In contrast, immunodeficient animals were less competent for MVMp-dependent tumor inhibition, with only 20% of the recipients being protected, arguing for an additional immune component to allow full tumor suppression. In keeping with this conclusion, immunocompetent mice engrafted with MVMp-infected glioma cells developed a level of anti-tumor immunity with isolated splenocytes producing elevated levels of interferon-γ. In rechallenge experiments using uninfected GL261 cells, we could show complete protection against the tumor, arguing for the induction of a T-cell-mediated, tumor-specific, long-term memory response. These findings indicate that the anticancer effect of PVs can be traced back not only for their direct oncolytic effect, but also to their ability to break tumor tolerance.  

5.1097           Neuronal Nogo-A upregulation does not contribute to ER stress-associated apoptosis but participates in the regenerative response in the axotomized adult retina

Pernet, V., Joly, S., Dalkara, D., Schwarz, O., Christ, F., Schaffer, D., Flannery, J.G. and Schwab, M.E. Cell Death and Differentiation, 19(7), 1096-1108 (2012)   Nogo-A, an axonal growth inhibitory protein known to be mostly present in CNS myelin, was upregulated in retinal ganglion cells (RGCs) after optic nerve injury in adult mice. Nogo-A increased concomitantly with the endoplasmic reticulum stress (ER stress) marker C/EBP homologous protein (CHOP), but CHOP immunostaining and the apoptosis marker annexin V did not co-localize with Nogo-A in individual RGC cell bodies, suggesting that injury-induced Nogo-A upregulation is not involved in axotomy-induced cell death. Silencing Nogo-A with an adeno-associated virus serotype 2 containing a short hairpin RNA (AAV2.shRNA-Nogo-A) or Nogo-A gene ablation in knock-out (KO) animals had little effect on the lesion-induced cell stress or death. On the other hand, Nogo-A overexpression mediated by AAV2.Nogo-A exacerbated RGC cell death after injury. Strikingly, however, injury-induced sprouting of the cut axons and the expression of growth-associated molecules were markedly reduced by AAV2.shRNA-Nogo-A. The axonal growth in the optic nerve activated by the intraocular injection of the inflammatory molecule Pam3Cys tended to be lower in Nogo-A KO mice than in WT mice. Nogo-A overexpression in RGCs in vivo or in the neuronal cell line F11 in vitro promoted regeneration, demonstrating a positive, cell-autonomous role for neuronal Nogo-A in the modulation of axonal regeneration.  

5.1098           Critical Determinants of Human α-Defensin 5 Activity against Non-enveloped Viruses

Gounder, A.P., Wiens, M.E., Wilson, S.S., Lu, W. and Smith, J.G.

1. Biol. Chem., 287(29), 24554-24562 (2012)

  Human α-defensins, such as human α-defensin 5 (HD5), block infection of non-enveloped viruses, including human adenoviruses (AdV), papillomaviruses (HPV), and polyomaviruses. Through mutational analysis of HD5, we have identified arginine residues that contribute to antiviral activity against AdV and HPV. Of two arginine residues paired on one face of HD5, Arg-28 is critical for both viruses, while Arg-9 is only important for AdV. Two arginine residues on the opposite face of the molecule (Arg-13 and Arg-32) and unpaired Arg-25 are less important for both. In addition, hydrophobicity at residue 29 is a major determinant of anti-adenoviral activity, and a chemical modification that prevents HD5 self-association was strongly attenuating. Although HD5 binds to the capsid of AdV, the molecular basis for this interaction is undefined. Capsid binding by HD5 is not purely charge-dependent, as substitution of lysine for Arg-9 and Arg-28 was deleterious. Analysis of HD5 analogs that retained varying levels of potency demonstrated that anti-adenoviral activity is directly correlated with HD5 binding to the virus, confirming that the viral capsid rather than the cell is the relevant target. Also, AdV aggregation induced by HD5 binding is not sufficient for neutralization. Rather, these studies confirm that the major mechanism of HD5-mediated neutralization of AdV depends upon specific binding to the viral capsid through interactions mediated in part by critical arginine residues, hydrophobicity at residue 29, and multimerization of HD5, which increases initial binding of virus to the cell but prevents subsequent viral uncoating and genome delivery to the nucleus.  

5.1099           Factors influencing helper-independent adeno-associated virus replication

Nicolas, A., Jolinon, N., Alazard-Dany, N., Barateau, V., Epstein, A., Greco, A., Büning, H. and Salvetti, A. Virology, 432, 1-9 (2012)   The inability of Adeno-Associated Virus (AAV) to replicate on its own is a strong argument in favor of the use of recombinant AAV vectors for in vivo gene transfer. However, some previous studies suggested that AAV may become replication competent in cells exposed to a genotoxic stress even in the absence of co-infection with a helper virus. To comprehensively explore this phenomenon, we examined AAV genome replication in several human cell lines exposed to different genotoxic conditions. We found that all treatments induced only negligible levels of AAV replication never exceeding ten fold above background. Further investigation indicated that induction of helper-independent AAV replication relied on the synergistic contribution of several extrinsic factors linked to the origin of the cell line and the quality of the AAV preparation. These results further support the notion that helper independent AAV replication cannot occur at significant levels in vivo.  

5.1100           Cholecystokinin knock-down in the basolateral amygdala has anxiolytic and antidepressant-like effects in mice

Del Boca, C., Lutz, P.E., Le Merrer, Koebel, P. and Kieffer, B.L. Neuroscience, 218, 185-195 (2012)   Cholecystokinin (CCK) is a neuropeptide widely distributed in the mammalian brain. This peptide regulates many physiological functions and behaviors, such as cardio-respiratory control, thermoregulation, nociception, feeding, memory processes and motivational responses, and plays a prominent role in emotional responses including anxiety and depression. CCK-expressing brain regions involved in these functions remain unclear and their identification represents an important step towards understanding CCK function in the brain. The basolateral amygdala (BLA) is strongly involved in emotional processing and expresses high levels of CCK. In this study we examined the contribution of CCK expressed in this brain region to emotional responses in mice. To knockdown CCK specifically in the BLA, we used stereotaxic delivery of recombinant adeno-associated viral vectors expressing a CCK-targeted shRNA. This procedure efficiently reduced CCK levels locally. shCCK-treated animals showed reduced levels of anxiety in the elevated plus-maze, and lower despair-like behavior in the forced swim test. Our data demonstrate that CCK expressed in the BLA represents a key brain substrate for anxiogenic and depressant effects of the peptide. The study also suggests that elevated amygdalar CCK could contribute to panic and major depressive disorders that have been associated with CCK dysfunction in humans.  

5.1101           Regulatory T cells are decreased in acute RHDV lethal infection of adult rabbits

Teixeira, L., Marques, R.M., Aguas, A.P. and Ferreira, P.G. Vet. Immunol. Immunopathol., 148, 343-347 (2012)   Rabbit hemorrhagic disease virus (RHDV) is the etiologic agent of rabbit hemorrhagic disease (RHD), an acute lethal infection that kills 90% of adult rabbits due to severe acute liver inflammation. Interestingly, young rabbits are naturally resistant to RHDV infection. Here, we have compared naturally occurring CD4⁺Foxp3⁺ regulatory T cells (Tregs) between young and adult rabbits after infection by RHDV. The number and frequency of Tregs was decreased in the spleen of adult rabbits 24 h after the RHDV infection; this was in contrast with the unchanged number and frequency of splenic Tregs found in young rabbits after the same infection. Also, serum levels of IL-10 and TGF-β were enhanced in the infected adult rabbits whereas no alteration was observed in infected young rabbits. However, this increase is accompanied by a burst of pro-inflammatory cytokines, but seems not able to prevent the death of the animals with severe acute liver inflammation in few days after infection. Since Tregs downregulate inflammation, we conclude that their decrease may contribute to the natural susceptibility of adult rabbits to RHDV infection.  

5.1102           CXCR4 gene transfer prevents pressure overload induced heart failure

LaRocca, T.J., Jeong, D., Kohlbrenner, E., Lee, A., Chen, J., Hajjar, R.J. and Tarzami, S.T.

1. Mol. Cell. Cardiol., 53, 223-232 (2012)

  Stem cell and gene therapies are being pursued as strategies for repairing damaged cardiac tissue following myocardial infarction in an attempt to prevent heart failure. The chemokine receptor-4 (CXCR4) and its ligand, CXCL12, play a critical role in stem cell recruitment post-acute myocardial infarction. Whereas progenitor cell migration via the CXCL12/CXCR4 axis is well characterized, little is known about the molecular mechanisms of CXCR4 mediated modulation of cardiac hypertrophy and failure. We used gene therapy to test the effects of CXCR4 gene delivery on adverse ventricular remodeling due to pressure overload. We assessed the effect of cardiac overexpression of CXCR4 during trans-aortic constriction (TAC) using a cardiotropic adeno-associated viral vector (AAV9) carrying the CXCR4 gene. Cardiac overexpression of CXCR4 in mice with pressure overload prevented ventricular remodeling, preserved capillary density and maintained function as determined by echocardiography and in vivo hemodynamics. In isolated adult rat cardiac myocytes, CXCL12 treatment prevented isoproterenol induced hypertrophy and interrupted the calcineurin/NFAT pathway. Finally, a complex involving the L-type calcium channel, β2-adrenoceptor, and CXCR4 (Cav1.2/β2AR/CXCR4) was identified in healthy cardiac myocytes and was shown to dissociate as a consequence of heart failure. CXCR4 administered to the heart via gene transfer prevents pressure overload induced heart failure. The identification of CXCR4 participation in a Cav1.2-β2AR regulatory complex provides further insight into the mechanism by which CXCR4 modulates calcium homeostasis and chronic pressure overload responses in the cardiac myocyte. Together these results suggest that AAV9.CXCR4 gene therapy is a potential therapeutic approach for congestive heart failure.  

5.1103           AAV-Directed Persistent Expression of a Gene Encoding Anti-Nicotine Antibody for Smoking Cessation

Hicks, M.J., Rosenberg, J.B., De, B.P., Pagovich, O.E., Young, C.N., Qiu, J-p., Kaminsky, S.M., Hackett, N.R., Worgall, S., Janda, K.D., Davisson, R.L. and Crystal, R.G. Science Translation Medicine, 4(140), 140ra87 (2012)   Current strategies to help tobacco smokers quit have limited success as a result of the addictive properties of the nicotine in cigarette smoke. We hypothesized that a single administration of an adeno-associated virus (AAV) gene transfer vector expressing high levels of an anti-nicotine antibody would persistently prevent nicotine from reaching its receptors in the brain. To test this hypothesis, we constructed an AAVrh.10 vector that expressed a full-length, high-affinity, anti-nicotine antibody derived from the Fab fragment of the anti-nicotine monoclonal antibody NIC9D9 (AAVantiNic). In mice treated with this vector, blood concentrations of the anti-nicotine antibody were dose-dependent, and the antibody showed high specificity and affinity for nicotine. The antibody shielded the brain from systemically administered nicotine, reducing brain nicotine concentrations to 15% of those in naïve mice. The amount of nicotine sequestered in the serum of vector-treated mice was more than seven times greater than that in untreated mice, with 83% of serum nicotine bound to immunoglobulin G. Treatment with the AAVantiNic vector blocked nicotine-mediated alterations in arterial blood pressure, heart rate, and locomotor activity. In summary, a single administration of a gene transfer vector expressing a high-affinity anti-nicotine monoclonal antibody elicited persistent (18 weeks), high titers of an anti-nicotine antibody that obviated the physiologic effects of nicotine. If this degree of efficacy translates to humans, AAVantiNic could be an effective preventative therapy for nicotine addiction.  

5.1104           Mapping a Neutralizing Epitope onto the Capsid of Adeno-Associated Virus Serotype 8

Gurda, B.L., Raupp, C., Popa-Wagner, R., Naumer, M., Olson, N.H., Ng, R., McKenna, R., Baker, T.S., Kleinschmidt, J.A. and Agbandje-McKenna, M.

1. Virol., 86(15), 7739-7751 (2012)

  Adeno-associated viruses (AAVs) are small single-stranded DNA viruses that can package and deliver nongenomic DNA for therapeutic gene delivery. AAV8, a liver-tropic vector, has shown great promise for the treatment of hemophilia A and B. However, as with other AAV vectors, host anti-capsid immune responses are a deterrent to therapeutic success. To characterize the antigenic structure of this vector, cryo-electron microscopy and image reconstruction (cryo-reconstruction) combined with molecular genetics, biochemistry, and in vivo approaches were used to define an antigenic epitope on the AAV8 capsid surface for a neutralizing monoclonal antibody, ADK8. Docking of the crystal structures of AAV8 and a generic Fab into the cryo-reconstruction for the AAV8-ADK8 complex identified a footprint on the prominent protrusions that flank the 3-fold axes of the icosahedrally symmetric capsid. Mutagenesis and cell-binding studies, along with in vitro and in vivo transduction assays, showed that the major ADK8 epitope is formed by an AAV variable region, VRVIII (amino acids 586 to 591 [AAV8 VP1 numbering]), which lies on the surface of the protrusions facing the 3-fold axis. This region plays a role in AAV2 and AAV8 cellular transduction. Coincidently, cell binding and trafficking assays indicate that ADK8 affects a postentry step required for successful virus trafficking to the nucleus, suggesting a probable mechanism of neutralization. This structure-directed strategy for characterizing the antigenic regions of AAVs can thus generate useful information to help re-engineer vectors that escape host neutralization and are hence more efficacious.  

5.1105           Structures of Merkel Cell Polyomavirus VP1 Complexes Define a Sialic Acid Binding Site Required for Infection

Neu, U., Hengel, H., Blaum, B.S., Schowalter, R.M., Macejak, D., Gilbert, M., Wakarchuk, W.W., Imamura, A., Ando, H., Kiso, M., Arnberg, N., Garcea, R.L., peters, T., Buck, C.B. and Stehle, T. PloS Pathogens, 8(7), e1002738 (2012)   The recently discovered human Merkel cell polyomavirus (MCPyV or MCV) causes the aggressive Merkel cell carcinoma (MCC) in the skin of immunocompromised individuals. Conflicting reports suggest that cellular glycans containing sialic acid (Neu5Ac) may play a role in MCPyV infectious entry. To address this question, we solved X-ray structures of the MCPyV major capsid protein VP1 both alone and in complex with several sialylated oligosaccharides. A shallow binding site on the apical surface of the VP1 capsomer recognizes the disaccharide Neu5Ac-α2,3-Gal through a complex network of interactions. MCPyV engages Neu5Ac in an orientation and with contacts that differ markedly from those observed in other polyomavirus complexes with sialylated receptors. Mutations in the Neu5Ac binding site abolish MCPyV infection, highlighting the relevance of the Neu5Ac interaction for MCPyV entry. Our study thus provides a powerful platform for the development of MCPyV-specific vaccines and antivirals. Interestingly, engagement of sialic acid does not interfere with initial attachment of MCPyV to cells, consistent with a previous proposal that attachment is mediated by a class of non-sialylated carbohydrates called glycosaminoglycans. Our results therefore suggest a model in which sialylated glycans serve as secondary, post-attachment co-receptors during MCPyV infectious entry. Since cell-surface glycans typically serve as primary attachment receptors for many viruses, we identify here a new role for glycans in mediating, and perhaps even modulating, post-attachment entry processes.  

5.1106           MAP-Kinase Regulated Cytosolic Phospholipase A2 Activity Is Essential for Production of Infectious Hepatitis C Virus Particles

Mmenzel, N., Fishl, W., Hueging, K., Bankwitz, D., Frentzen, A., Haid, S., Gentzsch, J., Kaderali, L., Bartenschlager, r. and Pietschmann, T. PloS Pathogens, 8(7), e1002829 (2012)   Hepatitis C virus (HCV) has infected around 160 million individuals. Current therapies have limited efficacy and are fraught with side effects. To identify cellular HCV dependency factors, possible therapeutic targets, we manipulated signaling cascades with pathway-specific inhibitors. Using this approach we identified the MAPK/ERK regulated, cytosolic, calcium-dependent, group IVA phospholipase A2 (PLA2G4A) as a novel HCV dependency factor. Inhibition of PLA2G4A activity reduced core protein abundance at lipid droplets, core envelopment and secretion of particles. Moreover, released particles displayed aberrant protein composition and were 100-fold less infectious. Exogenous addition of arachidonic acid, the cleavage product of PLA2G4A-catalyzed lipolysis, but not other related poly-unsaturated fatty acids restored infectivity. Strikingly, production of infectious Dengue virus, a relative of HCV, was also dependent on PLA2G4A. These results highlight previously unrecognized parallels in the assembly pathways of these human pathogens, and define PLA2G4A-dependent lipolysis as crucial prerequisite for production of highly infectious viral progeny.  

5.1107           Development of AAVLP(HPV16/31L2) Particles as Broadly Protective HPV Vaccine Candidate

Nieto, K., Weghofer, M., Sehr, P., Ritter, M., Sedlmeier, S., Karanam, B., Seitz, H., Müller, M., Kellner, M., Hörer, M., Michaelis, U., Roden, R.B.S., Gissmann, L and Kleinschmidt, J.A. PloS One, 7(6), e39741 (2012)   The human papillomavirus (HPV) minor capsid protein L2 is a promising candidate for a broadly protective HPV vaccine yet the titers obtained in most experimental systems are rather low. Here we examine the potential of empty AAV2 particles (AAVLPs), assembled from VP3 alone, for display of L2 epitopes to enhance their immunogenicity. Insertion of a neutralizing epitope (amino acids 17–36) from L2 of HPV16 and HPV31 into VP3 at positions 587 and 453, respectively, permitted assembly into empty AAV particles (AAVLP(HPV16/31L2)). Intramuscularly vaccination of mice and rabbits with AAVLP(HPV16/31L2)s in montanide adjuvant, induced high titers of HPV16 L2 antibodies as measured by ELISA. Sera obtained from animals vaccinated with the AAVLP(HPV16/31L2)s neutralized infections with several HPV types in a pseudovirion infection assay. Lyophilized AAVLP(HPV16/31L2) particles retained their immunogenicity upon reconstitution. Interestingly, vaccination of animals that were pre-immunized with AAV2 - simulating the high prevalence of AAV2 antibodies in the population - even increased cross neutralization against HPV31, 45 and 58 types. Finally, passive transfer of rabbit antisera directed against AAVLP(HPV16/31L2)s protected naïve mice from vaginal challenge with HPV16 pseudovirions. In conclusion, AAVLP(HPV16/31L2) particles have the potential as a broadly protective vaccine candidate regardless of prior exposure to AAV.  

5.1108           A Beta-Herpesvirus with Fluorescent Capsids to Study Transport in Living Cells

Bosse, J.B., Bauerfeind, R., Popilka, L., Marcinowski, L., Taeglich, M., Jung, C., Striebinger, H., von Einem, J., Gaul, U., Walther, P., Koszinowski, U.H. and Ruzsics, Z. PloS One, 7(7), e40585 (2012)   Fluorescent tagging of viral particles by genetic means enables the study of virus dynamics in living cells. However, the study of beta-herpesvirus entry and morphogenesis by this method is currently limited. This is due to the lack of replication competent, capsid-tagged fluorescent viruses. Here, we report on viable recombinant MCMVs carrying ectopic insertions of the small capsid protein (SCP) fused to fluorescent proteins (FPs). The FPs were inserted into an internal position which allowed the production of viable, fluorescently labeled cytomegaloviruses, which replicated with wild type kinetics in cell culture. Fluorescent particles were readily detectable by several methods. Moreover, in a spread assay, labeled capsids accumulated around the nucleus of the newly infected cells without any detectable viral gene expression suggesting normal entry and particle trafficking. These recombinants were used to record particle dynamics by live-cell microscopy during MCMV egress with high spatial as well as temporal resolution. From the resulting tracks we obtained not only mean track velocities but also their mean square displacements and diffusion coefficients. With this key information, we were able to describe particle behavior at high detail and discriminate between particle tracks exhibiting directed movement and tracks in which particles exhibited free or anomalous diffusion.  

5.1109           Ovarian Cancer Gene Therapy Using HPV-16 Pseudovirion Carrying the HSV-tk Gene

Hung, C-F., Chiang, A.J., Tsai, H-H., Pomper, M.G., Kang, T.H., Roden, R.R. and Wu, T-C. PloS One, 7(7), e40983 (2012)   Ovarian cancer is the leading cause of death from all gynecological cancers and conventional therapies such as surgery, chemotherapy, and radiotherapy usually fail to control advanced stages of the disease. Thus, there is an urgent need for alternative and innovative therapeutic options. We reason that cancer gene therapy using a vector capable of specifically delivering an enzyme-encoding gene to ovarian cancer cells will allow the cancer cell to metabolize a harmless prodrug into a potent cytotoxin, which will lead to therapeutic effects. In the current study, we explore the use of a human papillomavirus (HPV) pseudovirion to deliver a herpes simplex virus thymidine kinase (HSV-tk) gene to ovarian tumor cells. We found that the HPV-16 pseudovirion was able to preferentially infect murine and human ovarian tumor cells when administered intraperitoneally. Furthermore, intraperitoneal injection of HPV-16 pseudovirions carrying the HSV-tk gene followed by treatment with ganciclovir led to significant therapeutic anti-tumor effects in murine ovarian cancer-bearing mice. Our data suggest that HPV pseudovirion may serve as a potential delivery vehicle for ovarian cancer gene therapy.  

5.1110           (Pro)renin Receptor Triggers Distinct Angiotensin II-Independent Extracellular Matrix Remodeling and Deterioration of Cardiac Function

Moilanen, A-M., Rysä, J., Serpi, R., Mustonen, E., Szabo, Z., Aro, J., Näpänkangas, J., Tenhunen, O., Sutinen, M., Salo, T. and Ruskoaho, H. PloS One, 7(7), e41404 (2012)   Background Activation of the renin-angiotensin-system (RAS) plays a key pathophysiological role in heart failure in patients with hypertension and myocardial infarction. However, the function of (pro)renin receptor ((P)RR) is not yet solved. We determined here the direct functional and structural effects of (P)RR in the heart. Methodology/Principal Findings (P)RR was overexpressed by using adenovirus-mediated gene delivery in normal adult rat hearts up to 2 weeks. (P)RR gene delivery into the anterior wall of the left ventricle decreased ejection fraction (P<0.01), fractional shortening (P<0.01), and intraventricular septum diastolic and systolic thickness, associated with approximately 2–fold increase in left ventricular (P)RR protein levels at 2 weeks. To test whether the worsening of cardiac function and structure by (P)RR gene overexpression was mediated by angiotensin II (Ang II), we infused an AT₁ receptor blocker losartan via osmotic minipumps. Remarkably, cardiac function deteriorated in losartan-treated (P)RR overexpressing animals as well. Intramyocardial (P)RR gene delivery also resulted in Ang II-independent activation of extracellular-signal-regulated kinase1/2 phosphorylation and myocardial fibrosis, and the expression of transforming growth factor-β1 and connective tissue growth factor genes. In contrast, activation of heat shock protein 27 phosphorylation and apoptotic cell death by (P)RR gene delivery was Ang II-dependent. Finally, (P)RR overexpression significantly increased direct protein–protein interaction between (P)RR and promyelocytic zinc-finger protein. Conclusions/Significance These results indicate for the first time that (P)RR triggers distinct Ang II-independent myocardial fibrosis and deterioration of cardiac function in normal adult heart and identify (P)RR as a novel therapeutic target to optimize RAS blockade in failing hearts.  

5.1111           Entry Tropism of BK and Merkel Cell Polyomaviruses in Cell Culture

Schowalter, R.M., Reinhold, W.C. and Buck, C.B. PloS One, 7(7), e42181 (2012)   Merkel Cell Polyomavirus (MCV or MCPyV) was recently discovered in an aggressive form of skin cancer known as Merkel cell carcinoma (MCC). Integration of MCV DNA into the host genome likely contributes to the development of MCC in humans. MCV infection is common and many healthy people shed MCV virions from the surface of their skin. MCV DNA has also been detected in samples from a variety of other tissues. Although MCC tumors serve as a record that MCV can infect the Merkel cell lineage, the true tissue tropism and natural reservoirs of MCV infection in the host are not known. In an effort to gain insight into the tissue tropism of MCV, and to possibly identify cellular factors responsible for mediating infectious entry of the virus, the infection potential of human cells derived from a variety of tissues was evaluated. MCV gene transfer vectors (pseudoviruses) carrying reporter plasmid DNA encoding GFP or luciferase genes were used to transduce keratinocytes and melanocytes, as well as lines derived from MCC tumors and the NCI-60 panel of human tumor cell lines. MCV transduction was compared to transduction with pseudoviruses based on the better-studied human BK polyomavirus (BKV). The efficiency of MCV and BKV transduction of various cell types occasionally overlapped, but often differed greatly, and no clear tissue type preference emerged. Application of native MCV virions to a subset of highly transducible cell types suggested that the lines do not support robust replication of MCV, consistent with recent proposals that the MCV late phase may be governed by cellular differentiation in vivo. The availability of carefully curated gene expression data for the NCI-60 panel should make the MCV and BKV transduction data for these lines a useful reference for future studies aimed at elucidation of the infectious entry pathways of these viruses.  

5.1112           Silencing Relaxin-3 in Nucleus Incertus of Adult Rodents: A Viral Vector-based Approach to Investigate Neuropeptide Function

Callander, G.E., Ma, S., Ganella, D.E., Wimmer, V.C., Gundlach, A.L., Thomas, W.G. and Bathgate, R.A.D. PloS One, 7(8), e42300 (2012)   Relaxin-3, the most recently identified member of the relaxin peptide family, is produced by GABAergic projection neurons in the nucleus incertus (NI), in the pontine periventricular gray. Previous studies suggest relaxin-3 is a modulator of stress responses, metabolism, arousal and behavioural activation. Knockout mice and peptide infusions in vivo have significantly contributed to understanding the function of this conserved neuropeptide. Yet, a definitive role remains elusive due to discrepancies between models and a propensity to investigate pharmacological effects over endogenous function. To investigate the endogenous function of relaxin-3, we generated a recombinant adeno-associated viral (rAAV) vector expressing microRNA against relaxin-3 and validated its use to knock down relaxin-3 in adult rats. Bilateral stereotaxic infusion of rAAV1/2 EmGFP miR499 into the NI resulted in significant reductions in relaxin-3 expression as demonstrated by ablation of relaxin-3-like immunoreactivity at 3, 6 and 9 weeks and by qRT-PCR at 12 weeks. Neuronal health was unaffected as transduced neurons in all groups retained expression of NeuN and stained for Nissl bodies. Importantly, qRT-PCR confirmed that relaxin-3 receptor expression levels were not altered to compensate for reduced relaxin-3. Behavioural experiments confirmed no detrimental effects on general health or well-being and therefore several behavioural modalities previously associated with relaxin-3 function were investigated. The validation of this viral vector-based model provides a valuable alternative to existing in vivo approaches and promotes a shift towards more physiologically relevant investigations of endogenous neuropeptide function.  

5.1113           Different Pattern of Immunoglobulin Gene Usage by HIV-1 Compared to Non-HIV-1 Antibodies Derived from the Same Infected Subject

Li, L., Wang, X-H., Banerjee, S., Volsky, B., Williams, C., Virland, D., Nadas, A., Seaman, M.S., Chen, X., Spearman, P. and Zolla-Pazner, S. PloS One, 7(6)., e39534 (2012)   A biased usage of immunoglobulin (Ig) genes is observed in human anti-HIV-1 monoclonal antibodies (mAbs) resulting probably from compensation to reduced usage of the VH3 family genes, while the other alternative suggests that this bias usage is due to antigen requirements. If the antigen structure is responsible for the preferential usage of particular Ig genes, it may have certain implications for HIV vaccine development by the targeting of particular Ig gene-encoded B cell receptors to induce neutralizing anti-HIV-1 antibodies. To address this issue, we have produced HIV-1 specific and non-HIV-1 mAbs from an infected individual and analyzed the Ig gene usage. Green-fluorescence labeled virus-like particles (VLP) expressing HIV-1 envelope (Env) proteins of JRFL and BaL and control VLPs (without Env) were used to select single B cells for the production of 68 recombinant mAbs. Ten of these mAbs were HIV-1 Env specific with neutralizing activity against V3 and the CD4 binding site, as well as non-neutralizing mAbs to gp41. The remaining 58 mAbs were non-HIV-1 Env mAbs with undefined specificities. Analysis revealed that biased usage of Ig genes was restricted only to anti-HIV-1 but not to non-HIV-1 mAbs. The VH1 family genes were dominantly used, followed by VH3, VH4, and VH5 among anti-HIV-1 mAbs, while non-HIV-1 specific mAbs preferentially used VH3 family genes, followed by VH4, VH1 and VH5 families in a pattern identical to Abs derived from healthy individuals. This observation suggests that the biased usage of Ig genes by anti-HIV-1 mAbs is driven by structural requirements of the virus antigens rather than by compensation to any depletion of VH3 B cells due to autoreactive mechanisms, according to the gp120 superantigen hypothesis.  

5.1114           Long-Term Retinal PEDF Overexpression Prevents Neovascularization in a Murine Adult Model of Retinopathy

Haurigot, V., Villacampa, P., Ribera, A., Bosch, A., Ramos, D., Ruberte, J. and Bosch, F. PloS One, 7(7), e41511 (2012)   Neovascularization associated with diabetic retinopathy (DR) and other ocular disorders is a leading cause of visual impairment and adult-onset blindness. Currently available treatments are merely palliative and offer temporary solutions. Here, we tested the efficacy of antiangiogenic gene transfer in an animal model that mimics the chronic progression of human DR. Adeno-associated viral (AAV) vectors of serotype 2 coding for antiangiogenic Pigment Epithelium Derived Factor (PEDF) were injected in the vitreous of a 1.5 month-old transgenic model of retinopathy that develops progressive neovascularization. A single intravitreal injection led to long-term production of PEDF and to a striking inhibition of intravitreal neovascularization, normalization of retinal capillary density, and prevention of retinal detachment. This was parallel to a reduction in the intraocular levels of Vascular Endothelial Growth Factor (VEGF). Normalization of VEGF was consistent with a downregulation of downstream effectors of angiogenesis, such as the activity of Matrix Metalloproteinases (MMP) 2 and 9 and the content of Connective Tissue Growth Factor (CTGF). These results demonstrate long-term efficacy of AAV-mediated PEDF overexpression in counteracting retinal neovascularization in a relevant animal model, and provides evidence towards the use of this strategy to treat angiogenesis in DR and other chronic proliferative retinal disorders.  

5.1115           Paramecium bursaria Chlorella Virus 1 Proteome Reveals Novel Architectural and Regulatory Features of a Giant Virus

Dunigan, D.D:, Cerny, R.L., Bauman, A.T:, Roach, J.C., Lane, L.C., Agarkova, I.V., Wulser, K., Yanai-Balser, G.M., Gurnon, J.R., Vitek, J.C., Kronschnabel, B.J., Jeanniard, A., Blanc, G., Upton, C., Duncan, G.A., McClung, D.O., Ma, F. and Van Etten, J.L.

1. Virol., 86(16), 8821-8834 (2012)

  The 331-kbp chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) genome was resequenced and annotated to correct errors in the original 15-year-old sequence; 40 codons was considered the minimum protein size of an open reading frame. PBCV-1 has 416 predicted protein-encoding sequences and 11 tRNAs. A proteome analysis was also conducted on highly purified PBCV-1 virions using two mass spectrometry-based protocols. The mass spectrometry-derived data were compared to PBCV-1 and its host Chlorella variabilis NC64A predicted proteomes. Combined, these analyses revealed 148 unique virus-encoded proteins associated with the virion (about 35% of the coding capacity of the virus) and 1 host protein. Some of these proteins appear to be structural/architectural, whereas others have enzymatic, chromatin modification, and signal transduction functions. Most (106) of the proteins have no known function or homologs in the existing gene databases except as orthologs with proteins of other chloroviruses, phycodnaviruses, and nuclear-cytoplasmic large DNA viruses. The genes encoding these proteins are dispersed throughout the virus genome, and most are transcribed late or early-late in the infection cycle, which is consistent with virion morphogenesis.  

5.1116           A Specific Domain of the Chikungunya Virus E2 Protein Regulates Particle Formation in Human Cells: Implications for Alphavirus Vaccine Design

Akahata, W. and Nabel, G.J.

1. Virol., 86(16), 8879-8883 (2012)

  Virus-like particles (VLPs) can be generated from Chikungunya virus (CHIKV), but different strains yield variable quantities of particles. Here, we define the genetic basis for these differences and show that amino acid 234 in E2 substantially affects VLP production. This site is located within the acid-sensitive region (ASR) known to initiate a major conformational change in E1/E2. Selected other mutations in the ASR, or changes in pH, also increased VLP yield. These results demonstrate that the ASR of E2 plays an important role in regulating particle generation.  

5.1117           Impact of VP1-Specific Protein Sequence Motifs on Adeno-Associated Virus Type 2 Intracellular Trafficking and Nuclear Entry

Popa-Wagner, R., Porwal, M., Kann, M., Reuss, M., Weimer, M., Florin, L. and Kleinschmidt, J.A.

1. Virol., 86(17), 9163-9174 (2012)

  Adeno-associated virus type 2 (AAV2) has gained much interest as a gene delivery vector. A hallmark of AAV2-mediated gene transfer is an intracellular conformational change of the virus capsid, leading to the exposure of infection-relevant protein domains. These protein domains, which are located on the N-terminal portion of the structural proteins VP1 and VP2, include a catalytic phospholipase A₂ domain and three clusters of basic amino acids. We have identified additional protein sequence motifs located on the VP1/2 N terminus that also proved to be obligatory for virus infectivity. These motifs include signals that are known to be involved in protein interaction, endosomal sorting and signal transduction in eukaryotic cells. Among different AAV serotypes they are highly conserved and mutation of critical amino acids of the respective motifs led to a severe infection-deficient phenotype. In particular, mutation of a YXXQ-sequence motif significantly reduced accumulation of virus capsids around the nucleus in comparison to wild-type AAV2. Interestingly, intracellular trafficking of AAV2 was shown to be independent of PLA₂ activity. Moreover, mutation of three PDZ-binding motifs, which are located consecutively at the very tip of the VP1 N terminus, revealed a nuclear transport-defective phenotype, suggesting a role in nuclear uptake of the virus through an as-yet-unknown mechanism.  

5.1118           The Threefold Protrusions of Adeno-Associated Virus Type 8 Are Involved in Cell Surface Targeting as Well as Postattachment Processing

Raupp, C., Naumer, M., Müller, O.J., Gurda, L., Agbandje-Mckenna, M. and Kleinschmidt, J.A.

1. Virol., 86(17), 9396-9408 (2012)

  Adeno-associated virus (AAV) has attracted considerable interest as a vector for gene therapy owing its lack of pathogenicity and the wealth of available serotypes with distinct tissue tropisms. One of the most promising isolates for vector development, based on its superior gene transfer efficiency to the liver in small animals compared to AAV type 2 (AAV2), is AAV8. Comparison of the in vivo gene transduction of rAAV2 and rAAV8 in mice showed that single amino acid exchanges in the 3-fold protrusions of AAV8 in the surface loops comprised of residues 581 to 584 and 589 to 592 to the corresponding amino acids of AAV2 and vice versa had a strong influence on transduction efficiency and tissue tropism. Surprisingly, not only did conversion of AAV8 to AAV2 cap sequences increase the transduction efficiency and change tissue tropism but so did the reciprocal conversion of AAV2 to AAV8. Insertion of new peptide motifs at position 590 in AAV8 also enabled retargeting of AAV8 capsids to specific tissues, suggesting that these sequences can interact with receptors on the cell surface. However, a neutralizing monoclonal antibody that binds to amino acids ₅₈₈QQNTA₅₉₂ of AAV8 does not prevent cell binding and virus uptake, indicating that this region is not necessary for receptor binding but rather that the antibody interferes with an essential step of postattachment processing in which the 3-fold protrusion is also involved. This study supports a multifunctional role of the 3-fold region of AAV capsids in the infection process.  

5.1119           Endogenous leptin receptor signaling in the medial nucleus tractus solitarius affects meal size and potentiates intestinal satiation signals

Kanoski, S.E., Zhao, S., Guarnieri, D.J., DiLeone, R.J., Yan, J., De Jonghe, B.C., Bence, K.K., Hayes, M.R. and Grill, H.J. Am. J. Physiol, Endocrinol. Metab., 303, E496-E503 (2012)   Leptin receptor (LepRb) signaling in the hindbrain is required for energy balance control. Yet the specific hindbrain neurons and the behavioral processes mediating energy balance control by hindbrain leptin signaling are unknown. Studies here employ genetic [adeno-associated virally mediated RNA interference (AAV-RNAi)] and pharmacological methodologies to specify the neurons and the mechanisms through which hindbrain LepRb signaling contributes to the control of food intake. Results show that AAV-RNAi-mediated LepRb knockdown targeting a region encompassing the mNTS and area postrema (AP) (mNTS/AP LepRbKD) increases overall cumulative food intake by increasing the size of spontaneous meals. Other results show that pharmacological hindbrain leptin delivery and RNAi-mediated mNTS/AP LepRb knockdown increased and decreased the intake-suppressive effects of intraduodenal nutrient infusion, respectively. These meal size and intestinally derived signal amplification effects are likely mediated by LepRb signaling in the mNTS and not the AP, since 4th icv and mNTS parenchymal leptin (0.5 μg) administration reduced food intake, whereas this dose did not influence food intake when injected into the AP. Overall, these findings deepen the understanding of the distributed neuronal systems and behavioral mechanisms that mediate the effects of leptin receptor signaling on the control of food intake.  

5.1120           Design of a Single AAV Vector for Coexpression of TH and GCH1 to Establish Continuous DOPA Synthesis in a Rat Model of Parkinson's Disease

Cederfjäll, E., Sahin, G., Kirik, D. and Björklund, T. Molecular Therapy, 20(7), 1315-1326 (2012)   Preclinical efficacy of continuous delivery of 3,4-dihydroxyphenylalanine (DOPA) with adeno-associated viral (AAV) vectors has recently been documented in animal models of Parkinson's disease (PD). So far, all studies have utilized a mix of two monocistronic vectors expressing either of the two genes, tyrosine hydroxylase (TH) and GTP cyclohydrolase-1 (GCH1), needed for DOPA production. Here, we present a novel vector design that enables efficient DOPA production from a single AAV vector in rats with complete unilateral dopamine (DA) lesions. Functional efficacy was assessed with drug-induced and spontaneous motor behavioral tests where vector-treated animals showed near complete and stable recovery within 1 month. Recovery of motor function was associated with restoration of extracellular DA levels as assessed by online microdialysis. Histological analysis showed robust transgene expression not only in the striatum but also in overlying cortical areas. In globus pallidus, we noted loss of NeuN staining, which might be due to different sensitivity in neuronal populations to transgene expression. Taken together, we present a single AAV vector design that result in efficient DOPA production and wide-spread transduction. This is a favorable starting point for continued translation toward a therapeutic application, although future studies need to carefully review target region, vector spread and dilution with this approach.  

5.1121           Sialic Acid Deposition Impairs the Utility of AAV9, but Not Peptide-modified AAVs for Brain Gene Therapy in a Mouse Model of Lysosomal Storage Disease

Chen, Y.H., Claflin, K., Geoghegan, J.C. and Davidson, B.L. Molecular Therapy, 20(7), 1393-1399 (2012)   Recombinant vector systems have been recently identified that when delivered systemically can transduce neurons, glia, and endothelia in the central nervous system (CNS), providing an opportunity to develop therapies for diseases affecting the brain without performing direct intracranial injections. Vector systems based on adeno-associated virus (AAV) include AAV serotype 9 (AAV9) and AAVs that have been re-engineered at the capsid level for CNS tropism. Here, we performed a head-to-head comparison of AAV9 and a capsid modified AAV for their abilities to rescue CNS and peripheral disease in an animal model of lysosomal storage disease (LSD), the mucopolysacharidoses (MPS) VII mouse. While the peptide-modified AAV reversed cognitive deficits, improved storage burden in the brain, and substantially prolonged survival, we were surprised to find that AAV9 provided no CNS benefit. Additional experiments demonstrated that sialic acid, a known inhibitor of AAV9, is elevated in the CNS of MPS VII mice. These studies highlight how disease manifestations can dramatically impact the known tropism of recombinant vectors, and raise awareness to assuming similar transduction profiles between normal and disease models.  

5.1122           Anhedonia requires MC4R-mediated synaptic adaptations in nucleus accumbens

Lim, B.K., Huang, K.W., Grueter, B.A., Rothwell, P.E. and Malenka, R.C. Nature, 487, 183-198 (2012)   Chronic stress is a strong diathesis for depression in humans and is used to generate animal models of depression. It commonly leads to several major symptoms of depression, including dysregulated feeding behaviour, anhedonia and behavioural despair. Although hypotheses defining the neural pathophysiology of depression have been proposed, the critical synaptic adaptations in key brain circuits that mediate stress-induced depressive symptoms remain poorly understood. Here we show that chronic stress in mice decreases the strength of excitatory synapses on D1 dopamine receptor-expressing nucleus accumbens medium spiny neurons owing to activation of the melanocortin 4 receptor. Stress-elicited increases in behavioural measurements of anhedonia, but not increases in measurements of behavioural despair, are prevented by blocking these melanocortin 4 receptor-mediated synaptic changes in vivo. These results establish that stress-elicited anhedonia requires a neuropeptide-triggered, cell-type-specific synaptic adaptation in the nucleus accumbens and that distinct circuit adaptations mediate other major symptoms of stress-elicited depression.  

5.1123           Altered profile of basket cell afferent synapses in hyper-excitable dentate gyrus revealed by optogenetic and two-pathway stimulations

Ledri, M., Nikitidou, L., Erdelyi, F., Szabo, G., Kirik, D., Deisseroth, K. and Kokaia, M. Eur. J. Neurosci., 36(1), 1971-1983 (2012)   Cholecystokinin (CCK-) positive basket cells form a distinct class of inhibitory GABAergic interneurons, proposed to act as fine-tuning devices of hippocampal gamma-frequency (30–90 Hz) oscillations, which can convert into higher frequency seizure activity. Therefore, CCK-basket cells may play an important role in regulation of hyper-excitability and seizures in the hippocampus. In normal conditions, the endogenous excitability regulator neuropeptide Y (NPY) has been shown to modulate afferent inputs onto dentate gyrus CCK-basket cells, providing a possible novel mechanism for excitability control in the hippocampus. Using GAD65-GFP mice for CCK-basket cell identification, and whole-cell patch-clamp recordings, we explored whether the effect of NPY on afferent synapses to CCK-basket cells is modified in the hyper-excitable dentate gyrus. To induce a hyper-excitable state, recurrent seizures were evoked by electrical stimulation of the hippocampus using the well-characterized rapid kindling protocol. The frequency of spontaneous and miniature excitatory and inhibitory post-synaptic currents recorded in CCK-basket cells was decreased by NPY. The excitatory post-synaptic currents evoked in CCK-basket cells by optogenetic activation of principal neurons were also decreased in amplitude. Interestingly, we observed an increased proportion of spontaneous inhibitory post-synaptic currents with slower rise times, indicating that NPY may inhibit gamma aminobutyric acid release preferentially in peri-somatic synapses. These findings indicate that increased levels and release of NPY observed after seizures can modulate afferent inputs to CCK-basket cells, and therefore alter their impact on the oscillatory network activity and excitability in the hippocampus.  

5.1124           Hoechst increases adeno-associated virus-mediated transgene expression in airway epithelia by inducing the cytomegalovirus promoter

Dickey, D.D., Excoffon, K.J.D.A., Young, K.R., Parekh, K.R. and Zabner, J.

1. Gene Med., 14(6), 366-373 (2012)

  Background In airway epithelia, the kinetics of recombinant adeno-associated virus (AAV) transgene expression is slow. This has negative practical implications for research, as well as for translation into therapy. The DNA minor groove-binding agent Hoechst-33342 has been shown to enhance AAV transgene expression. In the present study, we investigated the mechanism of Hoechst-related augmentation of AAV-mediated transgene expression. Methods We investigated the effect of Hoechst-33342 on HT1080, COS-7, mouse and human airway epithelia transduced with different AAV serotypes encoding enhanced green fluorescent protein (eGFP). We exposed cells to increasing concentrations of Hoechst-33342 at different time points. We evaluated the effect on second-strand DNA synthesis using AAV with a self-complementary genome. We also investigated the effect on expression from transfected plasmids with and without AAV2 inverted terminal repeats (ITRs). Results We found that Hoechst-33342 significantly accelerated AAV transgene expression for all serotypes tested. Hoechst-33342 only had an effect when the treatment was given during or after transduction, even 120 days post-transduction, suggesting an effect on transgene expression regulation. Hoechst-33342 increased transgene expression when cells were transduced with a self-complementary AAV with the cytomegalovirus promoter, although there was no effect on cells transduced with conventional single-stranded AAV encoding the Rous sarcoma virus promoter. Finally, Hoechst-33342 increases gene expression from transfected plasmids regardless of the presence of AAV2 ITRs. Conclusions Hoechst dramatically augments and accelerates AAV-mediated transgene expression in airway epithelia without altering AAV-mediated gene transfer. Hoechst activation of the cytomegalovirus promoter is seen in plasmids, although it is drastically enhanced in the context of AAV.

5.1125           Phylogenetically diverse TT virus viremia among pregnant women

Bzhalava, D., Ekström, J., Lysholm, F., Hultin, E., Faust, H., Persson, B., Lehtinen, M., de Villiers, E-M. and Dillner, J. Virology, 432, 427-434 (2012)   Infections during pregnancy have been suggested to be involved in childhood leukemias. We used high-throughput sequencing to describe the viruses most readily detectable in serum samples of pregnant women. Serum DNA of 112 mothers to leukemic children was amplified using whole genome amplification. Sequencing identified one TT virus (TTV) isolate belonging to a known type and two putatively new TTVs. For 22 mothers, we also performed TTV amplification by general primer PCR before sequencing. This detected 39 TTVs, two of which were identical to the TTVs found after whole genome amplification. Altogether, we found 40 TTV isolates, 29 of which were putatively new types (similarities ranging from 89% to 69%). In conclusion, high throughput sequencing is useful to describe the known or unknown viruses that are present in serum samples of pregnant women.  

5.1126           An acidic oligopeptide displayed on AAV2 improves axial muscle tropism after systemic delivery

Lee, N-C., Falk, D.J., Byrne, B.J., Conlon, T.J., Clement, N., Porvasnik, S., Jorgensen, M.L., Potter, M., Erger, K., Watson, R., Ghivizzani, S.C., Chiu, H-C., Chien, Y-H., and Hwu, W-L. Genetic Vaccines Therapy, 10, 3-8 (2012)   Background The appropriate tropism of adeno-associated virus (AAV) vectors that are systemically injected is crucial for successful gene therapy when local injection is not practical. Acidic oligopeptides have been shown to enhance drug delivery to bones. Methods In this study six-L aspartic acids (D6) were inserted into the AAV2 capsid protein sequence between amino acid residues 587 and 588. 129SVE mice were injected with double-stranded wild-type- (WT-) or D6-AAV2 mCherry expression vectors (3.24 x 10¹⁰ vg per animal) via the superficial temporal vein within 24 hours of birth. Results Fluorescence microscopy and quantitative polymerase chain reaction confirmed higher levels of mCherry expression in the paraspinal and gluteus muscles in the D6-AAV2 injected mice. The results revealed that although D6-AAV2 was less efficient in the transduction of immortalized cells stronger mCherry signals were detected over the spine and pelvis by live imaging in the D6-AAV2-injected mice than were detected in the WT-AAV2-injected mice. In addition, D6-AAV2 lost the liver tropism observed for WT-AAV2. Conclusions An acidic oligopeptide displayed on AAV2 improves axial muscle tropism and decreases liver tropism after systemic delivery. This modification should be useful in creating AAV vectors that are suitable for gene therapy for diseases involving the proximal muscles.  

5.1127           AAV-Mediated Knock-Down of HRC Exacerbates Transverse Aorta Constriction-Induced Heart Failure

Park, C.S., Cha, H., Kwown, E.J., Jeong, D., Hajjar, R.J., Kranias, E.G., Cho, C., park, W.J. and Kim, D.H. PloS One, 7(8), e43282 (2012)   Background Histidine-rich calcium binding protein (HRC) is located in the lumen of sarcoplasmic reticulum (SR) that binds to both triadin (TRN) and SERCA affecting Ca²⁺ cycling in the SR. Chronic overexpression of HRC that may disrupt intracellular Ca²⁺ homeostasis is implicated in pathogenesis of cardiac hypertrophy. Ablation of HRC showed relatively normal phenotypes under basal condition, but exhibited a significantly increased susceptibility to isoproterenol-induced cardiac hypertrophy. In the present study, we characterized the functions of HRC related to Ca²⁺ cycling and pathogenesis of cardiac hypertrophy using the in vitro siRNA- and the in vivo adeno-associated virus (AAV)-mediated HRC knock-down (KD) systems, respectively. Methodology/Principal Findings AAV-mediated HRC-KD system was used with or without C57BL/6 mouse model of transverse aortic constriction-induced failing heart (TAC-FH) to examine whether HRC-KD could enhance cardiac function in failing heart (FH). Initially we expected that HRC-KD could elicit cardiac functional recovery in failing heart (FH), since predesigned siRNA-mediated HRC-KD enhanced Ca²⁺ cycling and increased activities of RyR2 and SERCA2 without change in SR Ca²⁺ load in neonatal rat ventricular cells (NRVCs) and HL-1 cells. However, AAV9-mediated HRC-KD in TAC-FH was associated with decreased fractional shortening and increased cardiac fibrosis compared with control. We found that phospho-RyR2, phospho-CaMKII, phospho-p38 MAPK, and phospho-PLB were significantly upregulated by HRC-KD in TAC-FH. A significantly increased level of cleaved caspase-3, a cardiac cell death marker was also found, consistent with the result of TUNEL assay. Conclusions/Significance Increased Ca²⁺ leak and cytosolic Ca²⁺ concentration due to a partial KD of HRC could enhance activity of CaMKII and phosphorylation of p38 MAPK, causing the mitochondrial death pathway observed in TAC-FH. Our results present evidence that down-regulation of HRC could deteriorate cardiac function in TAC-FH through perturbed SR-mediated Ca²⁺ cycling.  

5.1128           Human Anti-CCR4 Minibody Gene Transfer for the Treatment of Cutaneous T-Cell Lymphoma

Han, T., Abdel-Motal, U.M., Chang, D-K., Sui, J., Muvaffak, A., Campbell, J., Zhu, Q., Kupper, T.S. and Marasco, W.A. PloS One, 7(9), e44455 (2012)   Background Although several therapeutic options have become available for patients with Cutaneous T-cell Lymphoma (CTCL), no therapy has been curative. Recent studies have demonstrated that CTCL cells overexpress the CC chemokine receptor 4 (CCR4). Methodology/Principal Findings In this study, a xenograft model of CTCL was established and a recombinant adeno-associated viral serotype 8 (AAV8) vector expressing a humanized single-chain variable fragment (scFv)-Fc fusion (scFvFc or “minibody”) of anti-CCR4 monoclonal antibody (mAb) h1567 was evaluated for curative treatment. Human CCR4⁺ tumor-bearing mice treated once with intravenous infusion of AAV8 virions encoding the h1567 (AAV8-h1567) minibody showed anti-tumor activity in vivo and increased survival. The AAV8-h1567 minibody notably increased the number of tumor-infiltrating Ly-6G⁺ FcγRIIIa(CD16A)⁺ murine neutrophils in the tumor xenografts over that of AAV8-control minibody treated mice. Furthermore, in CCR4⁺ tumor-bearing mice co-treated with AAV8-h1567 minibody and infused with human peripheral blood mononuclear cells (PBMCs), marked tumor infiltration of human CD16A⁺ CD56⁺ NK cells was observed. The h1567 minibody also induced in vitro ADCC activity through both mouse neutrophils and human NK cells. Conclusions/Significance Overall, our data demonstrate that the in vivo anti-tumor activity of h1567 minibody is mediated, at least in part, through CD16A⁺ immune effector cell ADCC mechanisms. These data further demonstrate the utility of the AAV-minibody gene transfer system in the rapid evaluation of candidate anti-tumor mAbs and the potency of h1567 as a potential novel therapy for CTCL.  

5.1129           Characterization of Hepatitis C Virus Particle Subpopulations Reveals Multiple Usage of the Scavenger Receptor BI for Entry Steps

Thi, V.L.D., Granier, C., Zeisel, M.B., Guerin, M., Mancip, J., Granio, O., Penin, F., Lavilette, D., Bartenschlager, R., Baumert, T.F., Cosset, F.L. and Dreux, M.

1. Biol. Chem., 287(37), 31242-31257 (2012)

  Hepatitis C virus (HCV) particles assemble along the very low density lipoprotein pathway and are released from hepatocytes as entities varying in their degree of lipid and apolipoprotein (apo) association as well as buoyant densities. Little is known about the cell entry pathway of these different HCV particle subpopulations, which likely occurs by regulated spatiotemporal processes involving several cell surface molecules. One of these molecules is the scavenger receptor BI (SR-BI), a receptor for high density lipoprotein that can bind to the HCV glycoprotein E2. By studying the entry properties of infectious virus subpopulations differing in their buoyant densities, we show that these HCV particles utilize SR-BI in a manifold manner. First, SR-BI mediates primary attachment of HCV particles of intermediate density to cells. These initial interactions involve apolipoproteins, such as apolipoprotein E, present on the surface of HCV particles, but not the E2 glycoprotein, suggesting that lipoprotein components in the virion act as host-derived ligands for important entry factors such as SR-BI. Second, we found that in contrast to this initial attachment, SR-BI mediates entry of HCV particles independent of their buoyant density. This function of SR-BI does not depend on E2/SR-BI interaction but relies on the lipid transfer activity of SR-BI, probably by facilitating entry steps along with other HCV entry co-factors. Finally, our results underscore a third function of SR-BI governed by specific residues in hypervariable region 1 of E2 leading to enhanced cell entry and depending on SR-BI ability to bind to E2.  

5.1130           Complete Genome Sequence of a Tenth Human Polyomavirus

Buck, C.B., Phan, G.Q., Raiji, M.T., Murphy, P.M., McDermott, D.H. and McBride, A.A.

1. Virol., 86(19), 10887 (2012)

  Nine polyomavirus (PyV) species are known to productively infect humans. The circular DNA genomes of PyVs are readily detectable using rolling circle amplification (RCA). RCA-based analysis of condyloma specimens from a patient with warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome demonstrated the presence of a tenth apparently human-tropic polyomavirus species, which we name HPyV10.  

5.1131           Cyclophilins Facilitate Dissociation of the Human Papillomavirus Type 16 Capsid Protein L1 from the L2/DNA Complex following Virus Entry

Bienkowska-Haba, M., Williams, C., Kim, S.M. Garcea, R.L. and Sapp, M.

1. Virol., 86(18), 9875-9887 (2012)

  Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex. In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles.  

5.1132           Generation of an Adenovirus-Parvovirus Chimera with Enhanced Oncolytic Potential

El-Andaloussi, N., Bonifati, S., Kaufmannm, J.K., Mailly, L., Daeffler, L., Deryckere, F., Nettelbeck, D.M., Rommelaere, J. and Marchini, A.

1. Virol., 86(19), 10418-10431 (2012)

  In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral NS expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs NS expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells.  

5.1133           In Vivo Gene Transfer Strategies to Achieve Partial Correction of von Willebrand Disease

Wang, L., Rosenberg, J.B., De, B.P., Ferris, B., Wang, R., Rivella, S., Kaminsky, S.M. and Crystal, R.G. Human Gene Therapy, 23(6), 576-588 (2012)   von Willebrand disease (VWD), the most common hereditary coagulation disorder, results from mutations in the 52-exon gene for von Willebrand factor (VWF), which encodes an 8.4-kB cDNA. Studies with VWF cDNA plasmids have demonstrated that in vivo gene transfer to the liver will correct the coagulation dysfunction in VWF^−/− mice, but the correction is transient. To develop gene therapy for VWF that would mediate long-term expression of the VWF cDNA in liver, we first evaluated segmental pre-mRNA trans-splicing (SPTS) with two adeno-associated virus (AAV) serotype 8 vectors, each delivering one-half of the VWF cDNA. However, although the two vectors functioned well to generate VWF multimers after infection of cells in vitro, the efficiency of SPTS was insufficient to correct the VWF^−/− mouse in vivo. As an alternative, we assessed the ability of a lentiviral vector to transfer the intact murine VWF cDNA in vivo directly to the neonatal liver of VWF^−/− mice, using generation of VWF multimers, bleeding time, and bleeding volume as efficacy parameters. The VWF lentivirus generated VWF multimers and partially or completely corrected the coagulation defect on a persistent basis in 33% of the treated VWF-deficient mice. On the basis of the concept that partial persistent correction with gene transfer could be beneficial in VWD patients, these observations suggest that lentiviral delivery of VWF cDNA should be explored as a candidate for gene therapy in patients with a severe form of VWD.  

5.1134           Intranasal Vaccination with AAV5 and 9 Vectors Against Human Papillomavirus Type 16 in Rhesus Macaques

Nieto, K., Stahl-Hennig, C., Leuchs, B., Müller, M., Gissmann, L. and Kleinschmidt, J.A. Human Gene THerapy, 23(7), 733-741 (2012)   Cervical cancer is the second most common cancer in women worldwide. Persistent high-risk human papillomavirus (HPV) infection has been identified as the causative event for the development of this type of cancer. Recombinant adeno-associated viruses (rAAVs) are currently being developed and evaluated as vaccine vector. In previous work, we demonstrated that rAAVs administered intranasally in mice induced high titers and long-lasting neutralizing antibodies against HPV type 16 (HPV16). To extend this approach to a more human-related species, we immunized rhesus macaques (Macaca mulatta) with AAVs expressing an HPV16 L1 protein using rAAV5 and 9 vectors in an intranasal prophylactic setting. An rAAV5-L1 vector followed by a boost with rAAV9-L1 induced higher titers of L1-specific serum antibodies than a single rAAV5-L1 immunization. L1-specific antibodies elicited by AAV9 vector neutralized HPV16 pseudovirions and persisted for at least 7 months post immunization. Interestingly, nasal application of rAAV9 was immunogenic even in the presence of high AAV9 antibody titers, allowing reimmunization with the same serotype without prevention of the transgene expression. Two of six animals did not respond to AAV-mediated intranasal vaccination, although they were not tolerant, as both developed antibodies after intramuscular vaccination with HPV16 virus-like particles. These data clearly show the efficacy of an intranasal immunization using rAAV9-L1 vectors without the need of an adjuvant. We conclude from our results that rAAV9 vector is a promising candidate for a noninvasive nasal vaccination strategy.  

5.1135           Rapid Transgene Expression in Multiple Precursor Cell Types of Adult Rat Subventricular Zone Mediated by Adeno-Associated Type 1 Vectors

Bockstael, O., Melas, C., Pythoud, C., Levivier, M., McCarty, D., Samulski, R.J., De Witte, O. and Tenenbaum, L. Human Gene Therapy, 23(7), 742-753 (2012)   The adult rat brain subventricular zone (SVZ) contains proliferative precursors that migrate to the olfactory bulb (OB) and differentiate into mature neurons. Recruitment of precursors constitutes a potential avenue for brain repair. We have investigated the kinetics and cellular specificity of transgene expression mediated by AAV2/1 vectors (i.e., adeno-associated virus type 2 pseudotyped with AAV1 capsid) in the SVZ. Self-complementary (sc) and single-stranded (ss) AAV2/1 vectors mediated efficient GFP expression, respectively, at 17 and 24 hr postinjection. Transgene expression was efficient in all the rapidly proliferating cells types, that is, Mash1⁺ precursors (30% of the GFP⁺ cells), Dlx2⁺ neuronal progenitors (55%), Olig2⁺ oligodendrocyte progenitors (35%), and doublecortin-positive (Dcx⁺) migrating cells (40%), but not in the slowly proliferating glial fibrillary acidic protein-positive (GFAP⁺) neural stem cell pool (5%). Because cell cycle arrest by wild-type and recombinant AAV has been described in primary cultures, we examined SVZ proliferative activity after vector injection. Indeed, cell proliferation was reduced immediately after vector injection but was normal after 1 month. In contrast, migration and differentiation of GFP⁺ precursors were unaltered. Indeed, the proportion of Dcx⁺ cells was similar in the injected and contralateral hemispheres. Furthermore, 1 month after vector injection into the SVZ, GFP⁺ cells, found, as expected, in the OB granular cell layer, were mature GABAergic neurons. In conclusion, the rapid and efficient transgene expression in SVZ neural precursors mediated by scAAV2/1 vectors underlines their potential usefulness for brain repair via recruitment of immature cells. The observed transient precursor proliferation inhibition, not affecting their migration and differentiation, will likely not compromise this strategy.  

5.1136           Correction of Brain Oligodendrocytes by AAVrh.10 Intracerebral Gene Therapy in Metachromatic Leukodystrophy Mice

Piguet, F., Sondhi, D., Piraud, M., Fouquet, F., Hackett, N.R., Ahouansou, O., Vanier, M-T., Bieche, I., Aubourg, P., Crystal, R.G., Cartier, N. and Sevin, C. Human Gene Therapy, 23(8), 903-914 (2012)   Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder characterized by accumulation of sulfatides in glial cells and neurons, the result of an inherited deficiency of arylsulfatase A (ARSA; EC 3.1.6.8) and myelin degeneration in the central and peripheral nervous systems. No effective treatment is currently available for the most frequent late infantile (LI) form of MLD, which results in rapid neurological degradation and early death after the onset of clinical manifestations. To potentially arrest or reverse disease progression, ARSA enzyme must be rapidly delivered to brain oligodendrocytes of patients with LI MLD. We previously showed that brain gene therapy with adeno-associated virus serotype 5 (AAV5) driving the expression of human ARSA cDNA under the control of the murine phosphoglycerate kinase (PGK) promoter alleviated most long-term disease manifestations in MLD mice. Herein, we evaluated the short-term effects of AAVrh.10 driving the expression of human ARSA cDNA under the control of the cytomegalovirus/β-actin hybrid (CAG/cu) promoter in 8-month-old MLD mice that already show marked sulfatide accumulation and brain pathology. Within 2 months, and in contrast to results with the AAV5-PGK-ARSA vector, a single intrastriatal injection of AAVrh.10cuARSA resulted in correction of brain sulfatide storage, accumulation of specific sulfatide species in oligodendrocytes, and associated brain pathology in the injected hemisphere. Better potency of the AAVrh.10cuARSA vector was mediated by higher neuronal and oligodendrocyte transduction, axonal transport of the AAVrh.10 vector and ARSA enzyme, as well as higher CAG/cu promoter driven expression of ARSA enzyme. These results strongly support the use of AAVrh.10cuARSA vector for intracerebral gene therapy in rapidly progressing early-onset forms of MLD.  

5.1137           Canine corneal fibroblast and myofibroblast transduction with AAV5

Bosiack, A.P., Giuliano, E.A., Gupta, R and Mohan, R.R. Vet. Ophthalmol., 15(5), 291-298 (2012)   Objective  The aims of this study were (1) to determine the efficacy of adeno-associated vector serotype 5 (AAV5) for delivering gene therapy to canine corneal fibroblasts (CCFs) and myofibroblasts (CCMs) using enhanced green fluorescent protein (GFP) marker gene and (2) to evaluate the cytotoxicity of AAV5 to CCFs and CCMs using an in vitro model. Methods  Healthy donor canine corneas were used to generate primary CCFs by growing cultures in minimal essential medium supplemented with 10% fetal bovine serum. Canine corneal myofibroblasts were produced by growing cultures in serum-free medium containing transforming growth factor β1 (1 ng/mL). An AAV5 titer (6.5 × 10¹² μg/mL) expressing GFP under control of hybrid cytomegalovirus + chicken β-actin promoters (AAV5-gfp) was used to transduce CCF and CCM cultures. Delivered gene expression in CCFs and CCMs was quantified using immunocytochemistry, fluorescent microscopy, and real-time PCR. Transduction efficacy of the AAV5 vector was determined by counting DAPI-stained nuclei and EGFP-positive cells in culture. Phase-contrast microscopy, trypan blue, and dUTP nick end labeling (TUNEL) assays were used to determine the toxicity and safety of AAV5 in this canine corneal model. Results  Topical AAV5 application successfully transduced a significant population of CCFs (42.8%; P < 0.01) and CCMs (28%; P < 0.01). Tested AAV5 did not affect CCF or CCM phenotype or cellular viability and did not cause significant cell death. Conclusions  The tested AAV5 is an effective and safe vector for canine corneal gene therapy in this in vitro model. In vivo studies are warranted.  

5.1138           Concentration and Recovery of Viruses from Water: A Comprehensive Review

Ikner, L.A., Gerba, C.P. and Bright, K.R. Food Environ. Virol., 4(2), 41-67 (2012)   Enteric viruses are a cause of waterborne disease worldwide, and low numbers in drinking water can present a significant risk of infection. Because the numbers are often quite low, large volumes (100–1,000 L) of water are usually processed. The VIRADEL method using microporous filters is most commonly used today for this purpose. Negatively charged filters require the addition of multivalent salts and acidification of the water sample to effect virus adsorption, which can make large-volume sampling difficult. Positively charged filters require no preconditioning of samples, and are able to concentrate viruses from water over a greater pH range than electronegative filters. The most widely used electropositive filter is the Virosorb 1MDS; however, the Environmental Protection Agency has added the positively charged NanoCeram filters to their proposed Method 1615. Ultrafilters concentrate viruses based on size exclusion rather than electrokinetics, but are impractical for field sampling or processing of turbid water. Elution (recovery) of viruses from filters following concentration is performed with organic (e.g., beef extract) or inorganic solutions (e.g., sodium polyphosphates). Eluates are then reconcentrated to decrease the sample volume to enhance detection methods (e.g., cell culture infectivity assays and molecular detection techniques). While the majority of available filters have demonstrated high virus retention efficiencies, the methods to elute and reconcentrate viruses have met with varying degrees of success due to the biological variability of viruses present in water.  

5.1139           Structural investigations of a Podoviridae streptococcus phage C1, implications for the mechanism of viral entry

Aksyuk, A.A., Bowman, V.D., Kaufmann, B., Fields, C., Klose, T., Holdaway. H.A., Fischetti, V.A. and Rossmann, M.G. PNAS, 109(35), 14001-14006 (2012)   The Podoviridae phage C1 was one of the earliest isolated bacteriophages and the first virus documented to be active against streptococci. The icosahedral and asymmetric reconstructions of the virus were calculated using cryo-electron microscopy. The capsid protein has an HK97 fold arranged into a T = 4 icosahedral lattice. The C1 tail is terminated with a φ29-like knob, surrounded by a skirt of twelve long appendages with novel morphology. Several C1 structural proteins have been identified, including a candidate for an appendage. The crystal structure of the knob has an N-terminal domain with a fold observed previously in tube forming proteins of Siphoviridae and Myoviridae phages. The structure of C1 suggests the mechanisms by which the virus digests the cell wall and ejects its genome. Although there is little sequence similarity to other phages, conservation of the structural proteins demonstrates a common origin of the head and tail, but more recent evolution of the appendages.  

5.1140           Novel Approaches to Deliver Molecular Therapeutics in Cardiac Disease Using Adeno-Associated Virus Vectors

Rapti, K., Hajjar, R.J. and Weber, T. Molecular and Translational Med., 391-458 (2012)   Cardiac diseases are the leading cause of mortality in the Western World. Despite significant process in the treatment of cardiovascular diseases, curative treatments remain elusive. In recent years, gene therapy for the treatment of cardiac diseases has emerged as a promising and conceptually novel treatment paradigm. Of all the vectors used for cardiac gene delivery, adeno-associated virus (AAV)-based vectors are the most promising. This is due in part to their nonpathogenic nature, the comparatively low immunogenicity, and their ability to transduce efficiently many of the cell types of the cardiovascular system, in particular cardiomyocytes, resulting in long-term, high-level transgene expression. Here we review the recent development in the field of AAV gene delivery for cardiac diseases. We will discuss the tropism of the naturally occurring AAV serotypes as well as approaches to drive expression exclusively in specific tissues and cell types using transductional, transcriptional, and posttranscriptional approaches. We will examine the recent advances in large-scale AAV vector production and discuss the immune responses against both the vector and the transgene and what challenges this poses for the successful use of AAV vectors in cardiac gene therapy. We will compare the different approaches to deliver AAV vectors to the heart and will assess the potential of promising gene targets for the treatment of cardiac diseases. Finally, we will describe the status of the promising clinical trials that are currently ongoing in the field of cardiac gene therapy.  

5.1141           Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication

Baird, N.L., York, J. and Nunberg, J.H.

1. Virol., 86(20), 11301-11310 (2012)

  Arenaviruses are responsible for acute hemorrhagic fevers with high mortality and pose significant threats to public health and biodefense. These enveloped negative-sense RNA viruses replicate in the cell cytoplasm and express four proteins. To better understand how these proteins insinuate themselves into cellular processes to orchestrate productive viral replication, we have identified and characterized novel cytosolic structures involved in arenavirus replication and transcription. In cells infected with the nonpathogenic Tacaribe virus or the attenuated Candid#1 strain of Junín virus, we find that newly synthesized viral RNAs localize to cytosolic puncta containing the nucleoprotein (N) of the virus. Density gradient centrifugation studies reveal that these replication-transcription complexes (RTCs) are associated with cellular membranes and contain full-length genomic- and antigenomic-sense RNAs. Viral mRNAs segregate at a higher buoyant density and are likewise scant in immunopurified RTCs, consistent with their translation on bulk cellular ribosomes. In addition, confocal microscopy analysis reveals that RTCs contain the lipid phosphatidylinositol-4-phosphate and proteins involved in cellular mRNA metabolism, including the large and small ribosomal subunit proteins L10a and S6, the stress granule protein G3BP1, and a subset of translation initiation factors. Elucidating the structure and function of RTCs will enhance our understanding of virus-cell interactions that promote arenavirus replication and mitigate against host cell immunity. This knowledge may lead to novel intervention strategies to limit viral virulence and pathogenesis.  

5.1142           Impact of capsid modifications by selected peptide ligands on recombinant adeno-associated virus serotype 2-mediated gene transduction

Naumer, M., Popa-Wagner, R. and Kleinschmidt, J.A.

1. Gen. Virol., 93(10), 2131-2141 (2012)

  Vectors based on adeno-associated virus serotype 2 (AAV2) belong to today’s most promising and most frequently used viral vectors in human gene therapy. Like in many other vector systems, the broad but non-specific tropism limits their use for certain cell types or tissues. One approach to screen for transduction-improved vectors is the selection of random peptide libraries displayed directly on the AAV2 capsid. Although the AAV2 library system has been widely applied for the successful selection of improved gene therapy vectors, it remains unknown which steps of the transduction process are most affected and therefore critical for the selection of targeting peptides. Attachment to the cell surface is the first essential step of AAV-mediated gene transduction; however, our experiments challenge the conventional belief that enhanced gene transfer is equivalent to more efficient cell binding of recombinant AAV2 vectors. A comparison of the various steps of gene transfer by vectors carrying a wild-type AAV2 capsid or displaying two exemplary peptide ligands selected from AAV2 random libraries on different human tumour cell lines demonstrated strong alterations in cell binding, cellular uptake, as well as intracellular processing of these vectors. Combined, our results suggest that entry and post-entry events are decisive for the selection of the peptides NDVRSAN and GPQGKNS rather than their cell binding efficiency.  

5.1143           Characterization of a rat model of Huntington’s disease based on targeted expression of mutant huntingtin in the forebrain using adeno-associated viral vectors

Gabery, S., Sajjad, M.U., Hult, S., Soylu, R., Kirik, D. and Petersen, Å. Eur. J. Neurosci., 36(6), 2789-2800 (2012)   Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (htt) gene. Neuropathology is most severe in the striatum and cerebral cortex. As mutant htt is ubiquitously expressed, it has not been possible to establish clear structure-to-function relationships for the clinical aspects. In the present study, we have injected recombinant adeno-associated viral vectors of serotype 5 (rAAV5) expressing an 853-amino-acid fragment of htt with either 79 (mutant) or 18 (wild-type) glutamines (Q) in the dorsal striatum of neonatal rats to achieve expression of htt in the forebrain. Rats were followed for 6 months and compared with control rats. Neuropathological assessment showed long-term expression of the green fluorescent protein (GFP) transgene (used as a marker protein) and accumulation of htt inclusions in the cerebral cortex with the rAAV5-htt-79Q vectors. We estimated that around 10% of NeuN-positive cells in the cerebral cortex and 2% of DARPP-32 neurons in the striatum were targeted with the GFP-expressing vector. Formation of intracellular htt inclusions was not associated with neuronal loss, gliosis or microglia activation and did not lead to altered motor activity or changes in body weight. However, the same mutant htt vector caused orexin loss in the hypothalamus – another area known to be affected in HD. In conclusion, our results demonstrate that widespread forebrain expression of mutant htt can be achieved using rAAV5-vectors and suggest that this technique can be further explored to study region-specific effects of mutant htt or other disease-causing genes in the brain.  

5.1144           The identity of the cell adhesive protein substrate affects the efficiency of adeno-associated virus reverse transduction

McConnell, K.I., Gomez, E.J. and Suh, J. Acta Biomaterialia, 8, 4073-4079 (2012)   Delivering genes from surfaces, called substrate-mediated gene delivery or reverse transduction, is a useful method to achieve spatial localization of gene delivery. We tested the compatibility of adeno-associated virus (AAV) vectors with various cell adhesive proteins to mediate gene delivery from surfaces. Our studies demonstrate that AAV vectors can be successfully adsorbed on collagen I, elastin, and laminin substrates leading to robust gene delivery to overlying cells. Notably, AAV immobilization on laminin yields the highest efficiency of gene expression. This increased gene expression cannot be explained by increases in the levels of virus deposition, transcriptional activity of cells, or virus vector uptake into cells. Further refinement of our knowledge of AAV interactions with extracellular matrix proteins may have important implications in a variety of applications ranging from tissue engineering to in vivo gene therapy.  

5.1145           Activation of specific interneurons improves V1 feature selectivity and visual perception

Lee, S-H., Kwan, A.C., Zhang, S., Phoumthipphavong, V., Flannery, J.G., Masmanidis, S.C., Taniguchi, H., Huang, Z.J., Zhang, F., Boyden, E.S., Deisseroth, K. and Dan, Y. Nature, 488, 379-383 (2012)   Inhibitory interneurons are essential components of the neural circuits underlying various brain functions. In the neocortex, a large diversity of GABA (γ-aminobutyric acid) interneurons has been identified on the basis of their morphology, molecular markers, biophysical properties and innervation pattern^{1, 2, 3}. However, how the activity of each subtype of interneurons contributes to sensory processing remains unclear. Here we show that optogenetic activation of parvalbumin-positive (PV⁺) interneurons in the mouse primary visual cortex (V1) sharpens neuronal feature selectivity and improves perceptual discrimination. Using multichannel recording with silicon probes^{4, 5} and channelrhodopsin-2 (ChR2)-mediated optical activation⁶, we found that increased spiking of PV⁺ interneurons markedly sharpened orientation tuning and enhanced direction selectivity of nearby neurons. These effects were caused by the activation of inhibitory neurons rather than a decreased spiking of excitatory neurons, as archaerhodopsin-3 (Arch)-mediated optical silencing⁷ of calcium/calmodulin-dependent protein kinase IIα (CAMKIIα)-positive excitatory neurons caused no significant change in V1 stimulus selectivity. Moreover, the improved selectivity specifically required PV⁺ neuron activation, as activating somatostatin or vasointestinal peptide interneurons had no significant effect. Notably, PV⁺ neuron activation in awake mice caused a significant improvement in their orientation discrimination, mirroring the sharpened V1 orientation tuning. Together, these results provide the first demonstration that visual coding and perception can be improved by increased spiking of a specific subtype of cortical inhibitory interneurons.  

5.1146           Decreased expression of synapse-related genes and loss of synapses in major depressive disorder

Kang, H.J., Voleti, B., Hajszan, T., Rajkowska, G., Stockmeier, C.A., Licznerski, P., Lepack, A., Majik, M.S., Jeong, L.S., Banasr, M., Son, H. and Duman, R.S. Nature Medicine, 18(9), 1413-1417 (2012)   Previous imaging and postmortem studies have reported a lower brain volume and a smaller size and density of neurons in the dorsolateral prefrontal cortex (dlPFC) of subjects with major depressive disorder (MDD)^{1, 2}. These findings suggest that synapse number and function are decreased in the dlPFC of patients with MDD. However, there has been no direct evidence reported for synapse loss in MDD, and the gene expression alterations underlying these effects have not been identified. Here we use microarray gene profiling and electron microscopic stereology to reveal lower expression of synaptic-function–related genes (CALM2, SYN1, RAB3A, RAB4B and TUBB4) in the dlPFC of subjects with MDD and a corresponding lower number of synapses. We also identify a transcriptional repressor, GATA1, expression of which is higher in MDD and that, when expressed in PFC neurons, is sufficient to decrease the expression of synapse-related genes, cause loss of dendritic spines and dendrites, and produce depressive behavior in rat models of depression.  

5.1147           Restoration of Hearing in the VGLUT3 Knockout Mouse Using Virally Mediated Gene Therapy

Akil, O., Seal, R.P., Burke, K., Wang, C., Alemi, A., During, M., Edwards, R.H. and Lustig, R. Neuron, 75(2), 283-293 (2012)   Mice lacking the vesicular glutamate transporter-3 (VGLUT3) are congenitally deaf due to loss of glutamate release at the inner hair cell afferent synapse. Cochlear delivery of VGLUT3 using adeno-associated virus type 1 (AAV1) leads to transgene expression in only inner hair cells (IHCs), despite broader viral uptake. Within 2 weeks of AAV1-VGLUT3 delivery, auditory brainstem response (ABR) thresholds normalize, along with partial rescue of the startle response. Lastly, we demonstrate partial reversal of the morphologic changes seen within the afferent IHC ribbon synapse. These findings represent a successful restoration of hearing by gene replacement in mice, which is a significant advance toward gene therapy of human deafness.  

5.1148           Limitations of Encapsidation of Recombinant Self-Complementary Adeno-Associated Viral Genomes in Different Serotype Capsids and Their Quantitation

Wang, Y., Ling, C., Song, L., Wang, L., Aslanidi, G.V., Tan, M., Ling, C. and Srivastava, A. Human Gene Therapy Methods, 23(4), 225-233 82012)   We previously reported that self-complementary adeno-associated virus (scAAV) type 2 genomes of up to 3.3 kb can be successfully encapsidated into AAV2 serotype capsids. Here we report that such oversized AAV2 genomes fail to undergo packaging in other AAV serotype capsids, such as AAV1, AAV3, AAV6, and AAV8, as determined by Southern blot analyses of the vector genomes, although hybridization signals on quantitative DNA slot-blots could still be obtained. Recently, it has been reported that quantitative real-time PCR assays may result in substantial differences in determining titers of scAAV vectors depending on the distance between the primer sets and the terminal hairpin structure in the scAAV genomes. We also observed that the vector titers determined by the standard DNA slot-blot assays were highly dependent on the specific probe being used, with probes hybridizing to the ends of viral genomes being significantly overrepresented compared with the probes hybridizing close to the middle of the viral genomes. These differences among various probes were not observed using Southern blot assays. This overestimation of titer is a systemic error during scAAV genome quantification, regardless of viral genome sequences and capsid serotypes. Furthermore, different serotypes capsid and modification of capsid sequence may affect the ability of packaging intact, full-length AAV genomes. Although the discrepancy is modest with wild-type serotype capsid and short viral genomes, the measured titer could be as much as fivefold different with capsid mutant vectors and large genomes. Thus, based on our data, we suggest that Southern blot analyses should be performed routinely to more accurately determine the titers of recombinant AAV vectors. At the very least, the use of probes/primers hybridizing close to the mutant inverted terminal repeat in scAAV genomes is recommended to avoid possible overestimation of vector titers.  

5.1149           Detection of antibodies to HPV vaccine types using a multiplexed immunoassay

Panicker, G., Rajbhandari, I. and Unger, E. FASEB J., 26, 577.8 (2012)   Measurement of HPV antibodies in unvaccinated individuals has been used as measure of lifetime exposure to HPV. With the implementation of HPV vaccines, reliable assays are needed to evaluate the impact of altered dosing schemes or of new vaccine formulations. An ideal assay platform would allow multiplex type-specific detection with minimal sample requirement. We used the Meso Scale Discovery (MSD) electrochemiluminescence ECL based detection platform to develop a multiplex direct- virus-like particles (VLP) assay. HPV 6, 11, 16, and 18 VLPs were produced in mammalian cell-culture and purified using Optiprep™ gradient followed by agarose gel filtration. MSD prepared the plates in the 7-spot/well format, using the purified VLPs (4 spots) and PBS pH 7.4 as blank (one spot). We used four different coating conditions, varying VLP concentrations with and without bovine serum albumin (BSA). Results were evaluated using a set of 17 sera, WHO International Standard for HPV 16, and an optimized ELISA protocol for MSD plates on the SI6000 imager. Three point titrations and the parallel line method was used to calculate antibody titers. Stability of the plates was evaluated by comparing results after varying times of storage. The use of BSA in spot-coating allowed for increased stability of the VLPs on the plate as indicated by similar RLUs on plates tested 1 month apart. The intra and inter-assay CVs between PLL values calculated for each type was less than 8% and 17% respectively. No cross-reactivity was observed between HPV types. The MSD platform shows promise for simultaneous quantitation of the antibody responses to several HPV types in a high- throughput manner. Funding for this project was provided by the American Recovery and Reinvestment Act.    

5.1150           Dissociation of porcine reproductive and respiratory syndrome virus neutralization from antibodies specific to major envelope protein surface epitopes

Li, J. and Murtaugh, M.P. Virology, 433, 367-376 (2012)   Glycoprotein 5 (GP5) and membrane (M) protein are the major proteins in the envelope of porcine reproductive and respiratory syndrome virus (PRRSV). Although viral neutralization epitopes are reported in GP5 and M of type 2 PRRSV, their significance as targets of porcine humoral immunity is not well described. Thus, we constructed recombinant polypeptides containing ectodomain neutralization epitopes to examine their involvement in porcine antibody neutralization and antiviral immunity. PRRSV infection elicited ectodomain-specific antibodies, whose titers did not correlate with the neutralizing antibody (NA) response. Ectodomain-specific antibodies from PRRSV-neutralizing serum bound virus but did not neutralize infectivity. Furthermore, immunization of pigs with ectodomain polypeptides raised specific antibodies and provided partial protection without a detectable NA response. Finally the polypeptides did not block infection of porcine macrophages. These results suggest that the GP5/M ectodomain peptide epitopes are accessible for host antibody recognition, but are not associated with antibody-mediated virus neutralization.  

5.1151           Murine skin and vaginal mucosa are similarly susceptible to infection by pseudovirions of different papillomavirus classifications and species

Handisurya, A., Day, P.M., Thompson, C.D., Buck, C.B., Kwak, K., Roden, R.B.S., Lowy, D.R. and Schiller, J.T. Virology, 433, 385-394 (2012)   Depending upon viral genotype, productive papillomavirus infection and disease display preferential tropism for cutaneous or mucosal stratified squamous epithelia, although the mechanisms are unclear. To investigate papillomavirus entry tropism, we used reporter pseudovirions based on various cutaneous and mucosal papillomavirus species, including the recently identified murine papillomavirus. Pseudovirus transduction of BALB/c mice was examined using an improved murine skin infection protocol and a previously developed cervicovaginal challenge model. In the skin, HPV5, HPV6, HPV16, BPV1 and MusPV1 pseudovirions preferentially transduced keratinocytes at sites of trauma, similar to the genital tract. Skin infection, visualized by in vivo imaging using a luciferase reporter gene, peaked between days 2–3 and rapidly diminished for all pseudovirion types. Murine cutaneous and genital tissues were similarily permissive for pseudovirions of HPV types 5, 6, 8, 16, 18, 26, 44, 45, 51, 58 and animal papillomaviruses BPV1 and MusPV1, implying that papillomavirus' tissue and host tropism is governed primarily by post-entry regulatory events in the mouse.  

5.1152           Use of an in vivo animal model for assessing the role of integrin α6β4 and Syndecan-1 in early steps in papillomavirus infection

Huang, H-S. and Lambert, P.F. Virology, 433, 395-400 (2012)   Human papillomaviruses (HPV) are small DNA tumor viruses. HPV infection requires entry of virions into epithelial host cells that support the viral life cycle. Here, we used an in vivo mouse model, in which HPV pseudoviruses (PVs) are scored for their ability to transduce reporter genes, to test the role of various cellular proteins in entry. We initially investigated the role of integrin α₆β₄ in mediating early steps of HPV infection. Deficiency of integrin α₆β₄ is modestly but significantly suppressed reporter-gene transduction by PVs in conditional integrin β₄ knockout mice. We also investigated the role of syndecan 1, a heparin sulfate proteoglycan (HSPG) for its role in HPV infection. We did not see a significant reduction in reporter-gene transduction by PVs in syndecan-1 null mice. This indicates that this HSPG is not essential for early steps in HPV infection, but does not discount a need of other HSPGs in mediating HPV infection.  

5.1153           Testing of Novel Dengue Virus 2 Vaccines in African Green Monkeys: Safety, Immunogenicity, and Efficacy

Smith, K., Nanda, K., McCarl, V., Spears, C.J., Piper, A., Ribeiro, M., Quiles, M., Briggs, C., Thomas, G.S., Thomas, M.E., Brown, D.T. and Hernandez, R. Am. J. Trop. Med. Hyg., 87(4), 743-753 (2012)   The immunogenicity and safety of three novel host-range vaccines containing deletions in the transmembrane domain of dengue virus serotype 2 (DV2) E glycoprotein were evaluated in African green monkeys. The shorter transmembrane domains are capable of functionally spanning an insect but not a mammalian cell membrane, resulting in production of viral mutants that have reduced infectivity in mammalian hosts but efficient growth in insect cells. Groups of four monkeys received one dose each of test vaccine candidate with no booster immunization. After immunization, levels of viremia produced by each vaccine were determined by infectious center assay. Vaccine recipient immune response to wild-type DV2 challenge was measured on Day 57 by enzyme-linked immunosorbent assay and plaque reduction neutralization test. Two vaccines, DV2ΔGVII and DV2G460P, generated neutralizing antibody in the range of 700–900 50% plaque reduction neutralization test units. All three vaccine strains decreased the length of viremia by at least two days. No safety concerns were identified.  

5.1154           Pseudomonas aeruginosa Keratitis in Mice: Effects of Topical Bacteriophage KPP12 Administration

Fukuda, K., Ishida, W., Uchiyama, J., Rashel, M., Kato, S-i., Morita, T., Muraoka, A., Sumi, T., Matsuzaki, S., Daibata, M. and Fukushima, A. PloS One, 7(10), e47742 (2012)   The therapeutic effects of bacteriophage (phage) KPP12 in Pseudomonas aeruginosa keratitis were investigated in mice. Morphological analysis showed that phage KPP12 is a member of the family Myoviridae, morphotype A1, and DNA sequence analysis revealed that phage KPP12 is similar to PB1-like viruses. Analysis of the phage KPP12 genome did not identify any genes related to drug resistance, pathogenicity or lysogenicity, and so phage KPP12 may be a good candidate for therapeutic. KPP12 showed a broad host range for P. aeruginosa strains isolated from clinical ophthalmic infections. Inoculation of the scarified cornea with P. aeruginosa caused severe keratitis and eventual corneal perforation. Subsequent single-dose administration of KPP12 eye-drops significantly improved disease outcome, and preserved the structural integrity and transparency of the infected cornea. KPP12 treatment resulted in the suppression of neutrophil infiltration and greatly enhanced bacterial clearance in the infected cornea. These results indicate that bacteriophage eye-drops may be a novel adjunctive or alternative therapeutic agent for the treatment of infectious keratitis secondary to antibiotic-resistant bacteria.  

5.1155           Cryo-Electron Tomography of Rubella Virus

Battisti, A.J., Yoder, J.D., Plevka, P., Winkler, D.C., Prasad, V.M., Kuhn, R.J., Frey, T.K., Steven, A.C. and Rossmann, M.G.

1. Virol., 86(20), 11078-11085 (2012)

  Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging tick-borne virus of the Bunyaviridae family that is responsible for a fatal human disease for which preventative or therapeutic measures do not exist. We solved the crystal structure of the CCHFV strain Baghdad-12 nucleocapsid protein (N), a potential therapeutic target, at a resolution of 2.1 Å. N comprises a large globular domain composed of both N- and C-terminal sequences, likely involved in RNA binding, and a protruding arm domain with a conserved DEVD caspase-3 cleavage site at its apex. Alignment of our structure with that of the recently reported N protein from strain YL04057 shows a close correspondence of all folds but significant transposition of the arm through a rotation of 180 degrees and a translation of 40 Å. These observations suggest a structural flexibility that may provide the basis for switching between alternative N protein conformations during important functions such as RNA binding and oligomerization. Our structure reveals surfaces likely involved in RNA binding and oligomerization, and functionally critical residues within these domains were identified using a minigenome system able to recapitulate CCHFV-specific RNA synthesis in cells. Caspase-3 cleaves the polypeptide chain at the exposed DEVD motif; however, the cleaved N protein remains an intact unit, likely due to the intimate association of N- and C-terminal fragments in the globular domain. Structural alignment with existing N proteins reveals that the closest CCHFV relative is not another bunyavirus but the arenavirus Lassa virus instead, suggesting that current segmented negative-strand RNA virus taxonomy may need revision.  

5.1156           Evidence for pH-Dependent Protease Activity in the Adeno-Associated Virus Capsid

Salganik, M., Venkatakrishnan, B., Bennett, A., Lins, B., Yarbrough, J., Muzyczka, N., Agbandje-McKenna, M. and McKenna, R.

1. Virol., 86(21), 11877-11885 (2012)

  Incubation of highly purified adeno-associated virus (AAV) capsids in vitro at pH 5.5 induced significant autocleavage of capsid proteins at several amino acid positions. No autocleavage was seen at pH 7.5. Examination of other AAV serotypes showed at least two different pH-induced cleavage patterns, suggesting that different serotypes have evolved alternative protease cleavage sites. In contrast, incubation of AAV serotypes with an external protease substrate showed that purified AAV capsid preparations have robust protease activity at neutral pH but not at pH 5.5, opposite to what is seen with capsid protein autocleavage. Several lines of evidence suggested that protease activity is inherent in AAV capsids and is not due to contaminating proteins. Control virus preparations showed no protease activity on external substrates, and filtrates of AAV virus preparations also showed no protease activity contaminating the capsids. Further, N-terminal Edman sequencing identified unique autocleavage sites in AAV1 and AAV9, and mutagenesis of amino acids adjacent to these sites eliminated cleavage. Finally, mutation of an amino acid in AAV2 (E563A) that is in a conserved pH-sensitive structural region eliminated protease activity on an external substrate but did not seem to affect autocleavage. Taken together, our data suggested that AAV capsids have one or more protease active sites that are sensitive to pH induction. Further, it appears that acidic pHs comparable to those seen in late endosomes induce a structural change in the capsid that induces autolytic protease activity. The pH-dependent protease activity may have a role in viral infection.  

5.1157           Reconstitution of the Entire Hepatitis C Virus Life Cycle in Nonhepatic Cells

Da Costa, D., Turek, M., Felmlee, D.J., Girardi, E., Pfeffer, S., Long, G., Bartenschlager, R., Zeisel, M.B. and Baumert, T.F.

1. Virol., 86(21), 11919-11925 (2012)

  Hepatitis C virus (HCV) is a human hepatotropic virus, but the relevant host factors restricting HCV infection to hepatocytes are only partially understood. We demonstrate that exogenous expression of defined host factors reconstituted the entire HCV life cycle in human nonhepatic 293T cells. This study shows robust HCV entry, RNA replication, and production of infectious virus in human nonhepatic cells and highlights key host factors required for liver tropism of HCV.  

5.1158           Maintenance of the Flip Sequence Orientation of the Ears in the Parvoviral Left-End Hairpin Is a Nonessential Consequence of the Critical Asymmetry in the Hairpin Stem

Li, L., Cotmore, S.F. and Tattersall, P.

1. Virol., 86(22), 12187-12197 (2012)

  Parvoviral terminal hairpins are essential for viral DNA amplification but are also implicated in multiple additional steps in the viral life cycle. The palindromes at the two ends of the minute virus of mice (MVM) genome are dissimilar and are processed by different resolution mechanisms that selectively direct encapsidation of predominantly negative-sense progeny genomes and conserve a single Flip sequence orientation at the 3′ (left) end of such progeny. The sequence and predicted structure of these 3′ hairpins are highly conserved within the genus Parvovirus, exemplified by the 121-nucleotide left-end sequence of MVM, which folds into a Y-shaped hairpin containing small internal palindromes that form the “ears” of the Y. To explore the potential role(s) of this hairpin in the viral life cycle, we constructed infectious clones with the ear sequences either inverted, to give the antiparallel Flop orientation, or with multiple transversions, conserving their base composition but changing their sequence. These were compared with a “bubble” mutant, designed to activate the normally silent origin in the inboard arm of the hairpin, thus potentially rendering symmetric the otherwise asymmetric junction resolution mechanism that drives maintenance of Flip. This mutant exhibited a major defect in viral duplex and single-strand DNA replication, characterized by the accumulation of covalently closed turnaround forms of the left end, and was rapidly supplanted by revertants that restored asymmetry. In contrast, both sequence and orientation changes in the hairpin ears were tolerated, suggesting that maintaining the Flip orientation of these structures is a consequence of, but not the reason for, asymmetric left-end processing.  

5.1159           Gene therapy restores vision and delays degeneration in the CNGB1−/− mouse model of retinitis pigmentosa

Koch, S., Sothilingam, V., Garrido, M.G., Tanimoto, N., Becirovic, E., Koch, F., Seide, C., Beck, S.C., Seeliger, M.W., Biel, M., Mühlfriedel, R. and Michalakis, S. Hum. Mol. Genet., 21(20), 4486-4496 (2012)   Retinitis pigmentosa (RP) is a group of genetically heterogeneous, severe retinal diseases commonly leading to legal blindness. Mutations in the CNGB1a subunit of the rod cyclic nucleotide-gated (CNG) channel have been found to cause RP in patients. Here, we demonstrate the efficacy of gene therapy as a potential treatment for RP by means of recombinant adeno-associated viral (AAV) vectors in the CNGB1 knockout (CNGB1^−/−) mouse model. To enable efficient packaging and rod-specific expression of the relatively large CNGB1a cDNA (∼4 kb), we used an AAV expression cassette with a short rod-specific promoter and short regulatory elements. After injection of therapeutic AAVs into the subretinal space of 2-week-old CNGB1^−/− mice, we assessed the restoration of the visual system by analyzing (i) CNG channel expression and localization, (ii) retinal function and morphology and (iii) vision-guided behavior. We found that the treatment not only led to expression of full-length CNGB1a, but also restored normal levels of the previously degraded CNGA1 subunit of the rod CNG channel. Both proteins co-localized in rod outer segments and formed regular CNG channel complexes within the treated area of the CNGB1^−/− retina, leading to significant morphological preservation and a delay of retinal degeneration. In the electroretinographic analysis, we also observed restoration of rod-driven light responses. Finally, treated CNGB1^−/− mice performed significantly better than untreated mice in a rod-dependent vision-guided behavior test. In summary, this work provides a proof-of-concept for the treatment of rod channelopathy-associated RP by AAV-mediated gene replacement.  

5.1160           Inhibition of HIV-1 Particle Assembly by 2′,3′-Cyclic-Nucleotide 3′-Phosphodiesterase

Wilson, S.J., Schoggins, J.W., Zang, T., Kutluay, S.B., Jouvenet, N., Alim, M.A., Bitzegelo, J., Rice, C.M. and Bieniasz, P.D. Cell Host & Microbe, 12(4), 585-597 (2012)   The expression of hundreds of interferon-stimulated genes (ISGs) causes the cellular antiviral state in which the replication of many viruses, including HIV-1, is attenuated. We conducted a screen for ISGs that inhibit HIV-1 virion production and found that 2 ,3 -cyclic-nucleotide 3 -phosphodiesterase (CNP), a membrane-associated protein with unknown function in mammals has this property. CNP binds to the structural protein Gag and blocks HIV-1 particle assembly after Gag and viral RNA have associated with the plasma membrane. Several primate lentiviruses are CNP-sensitive, and CNP sensitivity/resistance is determined by a single, naturally dimorphic, codon (E/K40) in the matrix domain of Gag. Like other antiretroviral proteins, CNP displays interspecies variation in antiviral activity. Mice encode an inactive CNP variant and a single amino acid difference in murine versus human CNP determines Gag binding and antiviral activity. Some cell types express high levels of CNP and we speculate that CNP evolved to restrict lentivirus replication therein.  

5.1161           Probing the Early Temporal and Spatial Interaction of the Sindbis Virus Capsid and E2 Proteins with Reverse Genetics

Snyder, J.E., Berrios, C.J., Edwards, T.J., Jose, J., Perera, R and Kuhn, R.J.

1. Virol., 86(22), 12372-12383 (2012)

  A 7-Å cryoelectron microscopy-based reconstruction of Sindbis virus (SINV) was recently generated. Fitting the crystal structure of the SINV capsid protein (Cp) into the density map revealed that the F2-G2 loop of the Cp was shifted away from cytoplasmic domain of E2 (cdE2) in the 7-Å reconstruction relative to its position in the Cp crystal structure. Furthermore, the reconstruction demonstrated that residue E395 in region I of the cytoplasmic domain of the E2 envelope protein (cdE2-RI) and K252 of Cp, part of the Cp F2-G2 loop, formed a putative salt bridge in the virion. We generated amino acid substitutions at residues K250 and K252 of the SINV Cp and explored the resulting phenotypes. In the context of cells infected with wild-type or mutant virus, reversing the charge of these two residues resulted in the appearance of Cp aggregates around cytopathic vacuole type I (CPV-I) structures, the absence of nucleocapsid (NC) formation, and a lack of virus particle release in the infected mammalian cell. However, expressing the same Cp mutants in the cell without the envelope proteins or expressing and purifying the mutants from an Escherichia coli expression system and assembling in vitro yielded NC assembly in all cases. In addition, second-site mutations within cdE2 restored NC assembly but not release of infectious particles. Our data suggest an early temporal and spatial interaction between cdE2-RI and the Cp F2-G2 loop that, when ablated, leads to the absence of NC assembly. This interaction also appears to be important for budding of virus particles.  

5.1162           Molecular pathways for glucose homeostasis, insulin signaling and autophagy in hepatitis C virus induced insulin resistance in a cellular model

Das, G.C. and Hollinger, F.B. Virology, 434, 5-17 (2012)   Chronic HCV infection induces insulin resistance (IR). We studied this in a persistently infected cell line with defects in glucose homeostasis resulting from the phosphorylation of glycogen synthase (GS Ser641) and GS kinase isoform 3β (GSK 3βSer9). Reversal of these effects in cells cured of HCV with interferon supports viral specificity. Insulin signaling was disrupted by IRS-1 Ser312 phosphorylation and dysregulation of the Akt pathway. In infected cells, active autophagy was revealed by the formation of LC3 puncta or by increased levels (50–200%) of the markers Beclin 1 and conjugated Atg5/Atg12. Inhibition of autophagy by 3-methyl-adenine (3-MA) reduced Beclin1 levels, inhibited IRS-1 Ser312 or GS Ser641 phosphorylation and decreased viral load. Furthermore, IRS-1 Ser312 and Beclin1 were co-immunoprecipitated suggesting that they interact. It thus appears that HCV infection disturbs glucose homeostasis or insulin signaling to induce IR and also elicits autophagy that may contribute to this process.  

5.1163           A pyrophosphatase activity associated with purified HIV-1 particles

Ducloux, C., Mougel, M., Goldschmidt, V., Didierlaurent, L., Marquet, R. and Isel, C. Biochimie, 94, 2498-2507 (2012)   Treatment of HIV-1 with nucleoside reverse transcription inhibitors leads to the emergence of resistance mutations in the reverse transcriptase (RT) gene. Resistance to 3′-azido-3′-deoxythymidine (AZT) and to a lesser extent to 2′-3′-didehydro-2′-3′-dideoxythymidine is mediated by phosphorolytic excision of the chain terminator. Wild-type RT excises AZT by pyrophosphorolysis, while thymidine-associated resistance mutations in RT (TAMs) favour ATP as the donor substrate. However, in vitro, resistant RT still uses pyrophosphate more efficiently than ATP. We performed in vitro (−) strong-stop DNA synthesis experiments, with wild-type and AZT-resistant HIV-1 RTs, in the presence of physiologically relevant pyrophosphate and/or ATP concentrations and found that in the presence of pyrophosphate, ATP and AZTTP, TAMs do not enhance in vitro (−) strong-stop DNA synthesis. We hypothesized that utilisation of ATP in vivo is driven by intrinsic low pyrophosphate concentrations within the reverse transcription complex, which could be explained by the packaging of a cellular pyrophosphatase. We showed that over-expressed flagged-pyrophosphatase was associated with HIV-1 viral-like particles. In addition, we demonstrated that when HIV-1 particles were purified in order to avoid cellular microvesicle contamination, a pyrophosphatase activity was specifically associated to them. The presence of a pyrophosphatase activity in close proximity to the reverse transcription complex is most likely advantageous to the virus, even in the absence of any drug pressure.  

5.1164           Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice

Garriga-Canut, M., Agustin-Pavoh, C., Herrmann, F., Sanchez, A., Dierssen, M., Fillat, C. and Isalan, M. PNAS, 109(45), E136-E145 (2012)   Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by expanded CAG repeats in the huntingtin (HTT) gene. Although several palliative treatments are available, there is currently no cure and patients generally die 10–15 y after diagnosis. Several promising approaches for HD therapy are currently in development, including RNAi and antisense analogs. We developed a complementary strategy to test repression of mutant HTT with zinc finger proteins (ZFPs) in an HD model. We tested a “molecular tape measure” approach, using long artificial ZFP chains, designed to bind longer CAG repeats more strongly than shorter repeats. After optimization, stable ZFP expression in a model HD cell line reduced chromosomal expression of the mutant gene at both the protein and mRNA levels (95% and 78% reduction, respectively). This was achieved chromosomally in the context of endogenous mouse HTT genes, with variable CAG-repeat lengths. Shorter wild-type alleles, other genomic CAG-repeat genes, and neighboring genes were unaffected. In vivo, striatal adeno-associated virus viral delivery in R6/2 mice was efficient and revealed dose-dependent repression of mutant HTT in the brain (up to 60%). Furthermore, zinc finger repression was tested at several levels, resulting in protein aggregate reduction, reduced decline in rotarod performance, and alleviation of clasping in R6/2 mice, establishing a proof-of-principle for synthetic transcription factor repressors in the brain.  

5.1165           Exosomal cell-to-cell transmission of alpha synuclein oligomers

Danzer, K., Kranich, L.R., Ruf, W.P., Cagsal-getkin, O., Winslow, A.R., Zhu, L., Vanderburg, C.R. and McLean, P.J. Molecular Neurodegeneration, 7, 42-60 (2012)   Background Aggregation of alpha-synuclein (αsyn) and resulting cytotoxicity is a hallmark of sporadic and familial Parkinson’s disease (PD) as well as dementia with Lewy bodies, with recent evidence implicating oligomeric and pre-fibrillar forms of αsyn as the pathogenic species. Recent in vitro studies support the idea of transcellular spread of extracellular, secreted αsyn across membranes. The aim of this study is to characterize the transcellular spread of αsyn oligomers and determine their extracellular location. Results Using a novel protein fragment complementation assay where αsyn is fused to non-bioluminescent amino-or carboxy-terminus fragments of humanized Gaussia Luciferase we demonstrate here that αsyn oligomers can be found in at least two extracellular fractions: either associated with exosomes or free. Exosome-associated αsyn oligomers are more likely to be taken up by recipient cells and can induce more toxicity compared to free αsyn oligomers. Specifically, we determine that αsyn oligomers are present on both the outside as well as inside of exosomes. Notably, the pathway of secretion of αsyn oligomers is strongly influenced by autophagic activity. Conclusions Our data suggest that αsyn may be secreted via different secretory pathways. We hypothesize that exosome-mediated release of αsyn oligomers is a mechanism whereby cells clear toxic αsyn oligomers when autophagic mechanisms fail to be sufficient. Preventing the early events in αsyn exosomal release and uptake by inducing autophagy may be a novel approach to halt disease spreading in PD and other synucleinopathies.  

5.1166           Avian Adeno-Associated Virus Vector Efficiently Transduces Neurons in the Embryonic and Post-Embryonic Chicken Brain

Matsui, R., Tanabe, Y. and Watanabe, D. PloS One, 7(11), e48730 (2012)   The domestic chicken is an attractive model system to explore the development and function of brain circuits. Electroporation-mediated and retrovirus (including lentivirus) vector-mediated gene transfer techniques have been widely used to introduce genetic material into chicken cells. However, it is still challenging to efficiently transduce chicken postmitotic neurons without harming the cells. To overcome this problem, we searched for a virus vector suitable for gene transfer into chicken neurons, and report here a novel recombinant virus vector derived from avian adeno-associated virus (A3V). A3V vector efficiently transduces neuronal cells, but not non-neuronal cells in the brain. A single A3V injection into a postembryonic chick brain allows gene expression selectively in neuronal cells within 24 hrs. Such rapid and neuron-specific gene transduction raises the possibility that A3V vector can be utilized for studies of memory formation in filial imprinting, which occurs during the early postnatal days. A3V injection into the neural tube near the ear vesicle at early embryonic stage resulted in persistent and robust gene expression until E20.5 in the auditory brainstem. We further devised an A3V-mediated tetracycline (Tet) dependent gene expression system as a tool for studying the auditory circuit, consisting of the nucleus magnocellularis (NM) and nucleus laminaris (NL), that primarily computes interaural time differences (ITDs). Using this Tet system, we can transduce NM neurons without affecting NL neurons. Thus, the A3V technology complements current gene transfer techniques in chicken studies and will contribute to better understanding of the functional organization of neural circuits.  

5.1167           Chapter four – Analysis of Virus Entry and Cellular Membrane Dynamics by Single Particle Tracking

Ewers, H. and Schelhaas, M. Methods in Enzymol., 506, 63-80 (2012)   Viruses have evolved to mimic cellular ligands in order to gain access to their host cells for replication. Since viruses are simple in structure, they rely on host cells for all their transportation needs. Following single virus particles during the initial phase of infection, that is, virus entry into target cells, can reveal crucial information on the mechanism of pathogen infections and likewise cellular transport and membrane dynamics. Here, we give an overview on how to fluorescently label virus particles for live cell microscopy, and on how virus entry can be analyzed by single particle tracking experiments. Highlighted are strategies, on how to chemically introduce fluorophores into virions, and on how to extract quantitative information from live cell data.  

5.1168           Family – Reoviridae

Int. Committee on Taxonomy of Viruses Virus Taxonomy, 541-637 (2012)   This chapter focuses on Reoviridae family that has two subfamilies, Spinareovirinae and Sedoreovirinae. The member genuses of this family include Orthoreovirus, Aquareovirus, Oryzavirus, Orbivirus, Rotavirus, and Seadornavirus. The virus particles of members of the family, collectively called reoviruses, have icosahedral symmetry but may appear spherical in shape. The protein capsid is organized as one, two, or three concentric layers of capsid proteins, which surround the linear dsRNA segments of the viral genome, with an overall diameter of 60–80 nm. The subfamily Spinareovirinae contains viruses that have relatively large spikes or turrets situated at the 12 icosahedral vertices of either the virus or core particle. The subfamily Sedoreovirinae includes viruses that do not have large surface projections on their virions or core particles, thus giving them an almost spherical or smooth appearance. The innermost protein layer of reovirus particles has an internal diameter of approximately 50–60 nm and surrounds the 9, 10, 11, or 12 linear dsRNA genome segments. In the smooth-cored genera, the enzymatically active minor proteins of the virion are attached to the inner surface of the central space at the five-fold axes of symmetry. These include the RNA-dependent RNA polymerase, NTPase, helicase, and capping, and transmethylase enzymes. Particles of some genera can leave infected cells by budding or can bud into the endoplasmic reticulum during morphogenesis, acquiring an envelope derived from cellular membranes. Mature virions lack a lipid envelope, and depending on the genus, a myristyl residue may be covalently attached to one of the virion proteins. Coltiviruses, rotaviruses, and orbiviruses have an intermediate in virus morphogenesis or release, which may have a lipid envelope that is subsequently lost or removed.  

5.1169           Family – Flaviviridae

Int. Committee on Taxonomy of Virus Virus Taxonomy, 1003-1020 (2012)   This chapter focuses on Flaviviridae family, whose member genuses are Flavivirus, Pestivirus, and Hepacivirus. The virions are 40–60 nm in diameter, spherical in shape, and contain a lipid envelope. The capsid is composed of a single protein and the envelope contains two or three virus-encoded membrane proteins. The genomes are positive sense ssRNA of approximately 11, 12.3 and 9.6 kb for members of the genera Flavivirus, Pestivirus, and Hepacivirus, respectively. The virions of members of the family have a single, small basic capsid (C) and two or three membrane-associated envelope proteins. The nonstructural proteins contain sequence motifs characteristic of a serine protease, RNA helicase, and RdRp that are encoded at similar locations along the genome in all genera. The lipids present in virions are derived from host cell membranes and make up 17% of the total virion weight in the case of flaviviruses. The virions contain carbohydrates in the form of glycolipids and glycoproteins, and the genomic RNA of all members of the family has a similar organization, which is the viral mRNA found in infected cells. The virion assembly, including acquisition of a glycoprotein-containing lipid envelope, occurs by budding through intracellular membranes, and the viral particles are transported in cytoplasmic vesicles through the secretory pathway before they are released by exocytosis.  

5.1170           Random Insertion of mCherry Into VP3 Domain of Adeno-associated Virus Yields Fluorescent Capsids With no Loss of Infectivity

Judd, J., Wei, F., Nguyen, P.Q., Tartaglia, L.J., Agbandje-McKenna, M., Silberg, J.J. and Suh, J. Molecular Therapy-Nucleic Acids, 1, e54, (2012)   Adeno-associated virus (AAV)-derived vectors are promising gene delivery systems, and a number of design strategies have been pursued to improve their performance. For example, genetic insertion of proteins into the capsid may be used to achieve vector retargeting, reduced immunogenicity, or to track vector transport. Unfortunately, rational approaches to genetic insertion have experienced limited success due to the unpredictable context-dependent nature of protein folding and the complexity of the capsid’s macroassembly. We report the construction and use of a frame-enriched DNase-based random insertion library based on AAV2 cap, called pAAV2_RaPID (Random Peptide Insertion by DNase). The fluorescent mCherry protein was inserted randomly throughout the AAV2 capsid and the library was selected for fluorescent and infectious variants. A capsid site was identified in VP3 that can tolerate the large protein insertion. In contrast to previous efforts to incorporate fluorescent proteins into the AAV2 capsid, the isolated mCherry mutant maintains native infectivity while displaying robust fluorescence. Collectively, these results demonstrate that the pAAV2_RaPID platform library can be used to create fully infectious AAV vectors carrying large functional protein domains on the capsid.  

5.1171           Potent Inhibition of Late Stages of Hepadnavirus Replication by a Modified Cell Penetrating Peptide

Abdul, F., Ndeboko, B., Buronfoss, T., Zoulim, F., Kann, M., Nielsen, P.E. and Cova, L. PloS One, 7(11), e48721 (2012)   Cationic cell-penetrating peptides (CPPs) and their lipid domain-conjugates (CatLip) are agents for the delivery of (uncharged) biologically active molecules into the cell. Using infection and transfection assays we surprisingly discovered that CatLip peptides were able to inhibit replication of Duck Hepatitis B Virus (DHBV), a reference model for human HBV. Amongst twelve CatLip peptides we identified Deca-(Arg)₈ having a particularly potent antiviral activity, leading to a drastic inhibition of viral particle secretion without detectable toxicity. Inhibition of virion secretion was correlated with a dose-dependent increase in intracellular viral DNA. Deca-(Arg)₈ peptide did neither interfere with DHBV entry, nor with formation of mature nucleocapsids nor with their travelling to the nucleus. Instead, Deca-(Arg)₈ caused envelope protein accumulation in large clusters as revealed by confocal laser scanning microscopy indicating severe structural changes of preS/S. Sucrose gradient analysis of supernatants from Deca-(Arg)₈-treated cells showed unaffected naked viral nucleocapsids release, which was concomitant with a complete arrest of virion and surface protein-containing subviral particle secretion. This is the first report showing that a CPP is able to drastically block hepadnaviral release from infected cells by altering late stages of viral morphogenesis via interference with enveloped particle formation, without affecting naked nucleocapsid egress, thus giving a view inside the mode of inhibition. Deca-(Arg)₈ may be a useful tool for elucidating the hepadnaviral secretory pathway, which is not yet fully understood. Moreover we provide the first evidence that a modified CPP displays a novel antiviral mechanism targeting another step of viral life cycle compared to what has been so far described for other enveloped viruses.  

5.1172           Effects of downstream processing on structural integrity and immunogenicity in the manufacture of papillomavirus type 16 L1 virus-like particles

Chang, D.Y., Kim, H.J. and Kim, H-J. Biotechnol. Bioprosess Eng., 17(4), 755-763 (2012)   There is increasing demand for virus-like particles (VLPs) as a platform for prophylactic vaccine production. However, little attention has been paid to how downstream processing affects the structure and immunogenicity of the VLPs. In this study, we compared three methods of purifying human papillomavirus type 16 (HPV16) VLPs, each including the same cation-exchange chromatography (CEC) step. Method T-1 uses both ammonium sulfate precipitation (ASP) and a step to remove precipitated contaminating proteins (SRPC) prior to CEC, while T-2 uses only the SRPC step prior to CEC and T-3 includes neither step. We compared the structural integrity and immunogenicity of the HPV16 VLPs resulting from these three methods. All three preparations were highly pure. However, the final yields of the VLPs obtained with T-2 were 1.5 and 2 fold higher than with T-1 and T-3, respectively. With respect to structural integrity, T-1 and T-2 HPV16 VLPs had smaller hydrodynamic diameters and higher reactivity towards monoclonal anti-HPV16 neutralizing antibodies than T-3 VLPs, indicating higher potentials of T-1 and T-2 VLPs for eliciting anti-HPV16 neutralizing antibodies. Moreover, it was confirmed that the T-1 and T-2 HPV16 VLPs elicit anti-HPV16 neutralizing antibodies more efficiently than T-3 HPV16 VLPs do in mice immunizations: the abilities for eliciting neutralizing antibodies were in the order T-2 VLP > T-1 VLP > T-3 VLP. We conclude that the process design for purifying HPV VLPs is a critical determinant of the quality of the final product.  

5.1173           A Vector–Host System to Fingerprint Virus Tropism

Hillestad, M.L., Guenzel, A.J., Nath, K.A. and Barry, M.A. Human Gene Therapy, 23(10), 1116-1126 (2012)   Reporter genes are important tools for assessing vector pharmacology in vivo. Although useful, current systems are limited by (1) the need to generate a new vector for each different reporter, (2) the inability to package reporter genes in small vectors, and (3) variations in reporter gene feedback due to variations in cell-to-cell vector copy number. To circumvent these problems, we have used Cre recombinase as a “cat's paw” to activate reporter genes embedded in transgenic mice. The small Cre gene was introduced into self-complementary adeno-associated viral (scAAV) vectors with limited packaging capacity. Injection of scAAV-Cre vectors into mice with loxP-inactivated luciferase enabled in vivo imaging distributions comparable to the signal observed after AAV-luciferase injection. When injected into mT/mG mice, AAV-Cre converted ubiquitous expression of red fluorescent protein (RFP) to green fluorescent protein (GFP) expression only where the vectors transduced cells. Injection into F₁ hybrid luciferase and mT/mG mice enabled simultaneous three-reporter tracking. This system was able to discriminate cell-specific transduction in all organs tested, with particular usefulness for detecting AAV serotype-specific transduction in the liver, kidney, and muscle. Given that F₁ mice bear exactly one copy of luciferase and one copy of RFP-GFP, each reporter gene is either “on” or “off” in a cell. The Cre system therefore provides a unique quantum method to quantify vector delivery that can be applied when vector capacity is limited.  

5.1174           Retrograde Gene Delivery to Hypoglossal Motoneurons Using Adeno-Associated Virus Serotype 9

ElMallah, M.K., Falk, D.J., Lane, M.A., Conlon, T.J., Lee, K-Z., Shafi, N.I., reier, P.J., Byrne, B.J. and Fuller, D.D. Human Gene Therapy Methods, 23(2), 148-156 (2012)   Retrograde viral transport (i.e., muscle to motoneuron) enables targeted gene delivery to specific motor pools. Recombinant adeno-associated virus serotype 9 (AAV9) robustly infects motoneurons, but the retrograde transport capabilities of AAV9 have not been systematically evaluated. Accordingly, we evaluated the retrograde transduction efficiency of AAV9 after direct tongue injection in 129SVE mice as well as a mouse model that displays neuromuscular pathology (Gaa^−/−). Hypoglossal (XII) motoneurons were histologically evaluated 8 weeks after tongue injection with AAV9 encoding green fluorescent protein (GFP) with expression driven by the chicken β-actin promoter (1×10¹¹ vector genomes). On average, GFP expression was detected in 234±43 XII motoneurons 8 weeks after AAV9-GFP tongue injection. In contrast, tongue injection with a highly efficient retrograde anatomical tracer (cholera toxin β subunit, CT-β) resulted in infection of 818±88 XII motoneurons per mouse. The retrograde transduction efficiency of AAV9 was similar between the 129SVE mice and those with neuromuscular disease (Gaa^−/−). Routine hematoxylin and eosin staining and cluster of differentiation (CD) immunostaining for T cells (CD3) indicated no persistent inflammation within the tongue or XII nucleus after AAV9 injection. Additional experiments indicated no adverse effects of AAV9 on the pattern of breathing. We conclude that AAV9 can retrogradely infect a significant portion of a given motoneuron pool in normal and dystrophic mice, and that its transduction efficiency is approximately 30% of what can be achieved with CT-β.  

5.1175           Long-Term Expression and Safety of Administration of AAVrh.10hCLN2 to the Brain of Rats and Nonhuman Primates for the Treatment of Late Infantile Neuronal Ceroid Lipofuscinosis

Sondhi, D., Johnson, L., Purpura, K., Monette, S., Souweidane, M.M., Kaplitt, M.G., Kosofsky, B., Yohay, K., Ballon, D., Dyke, J., Kaminsky, S.M., Hackett, N.R. and Crystal, R.G. Human Gene Therapy Mehods, 23(5), 324-335 (2012)   Late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal, lysosomal storage disorder caused by mutations in the CLN2 gene, results in a deficiency of tripeptidyl-peptidase I (TPP-I) activity in neurons. Our prior studies showed that delivery of the human CLN2 cDNA directly to the CNS, using an adeno-associated virus serotype 2 (AAV2) vector, is safe in children with LINCL. As a second-generation strategy, we have demonstrated that AAVrh.10hCLN2, a rhesus-derived AAV vector, mediates wide distribution of TPP-I through the CNS in a murine model. This study tests the hypothesis that direct administration of AAVrh.10hCLN2 to the CNS of rats and nonhuman primates at doses scalable to humans has an acceptable safety profile and mediates significant CLN2 expression in the CNS. A dose of 10¹¹ genome copies (GC) was administered bilaterally to the striatum of Sprague Dawley rats with sacrifice at 7 and 90 days with no significant impact except for mild vector-related histopathological changes at the site of vector administration. A dose of 1.8×10¹² GC of AAVrh.10hCLN2 was administered to the CNS of 8 African green monkeys. The vector-treated monkeys did not differ from controls in any safety parameter except for mild to moderate white matter edema and inflammation localized to the administration sites of the vector. There were no clinical sequelae to these localized findings. TPP-I activity was >2 SD over background in 31.7±8.1% of brain at 90 days. These findings establish the dose and safety profile for human clinical studies for the treatment of LINCL with AAVrh.10hCLN2.  

5.1176           Role of the SP2 Domain and Its Proteolytic Cleavage in HIV-1 Structural Maturation and Infectivity

De Marco, A., Heuser, A-M., Glass, B., Kräusslich, H-G., Müller, B. and Briggs, J.A.G.

1. Virol., 86(24), 13708-13716 (2012)

  HIV-1 buds as an immature, noninfectious virion. Proteolysis of its main structural component, Gag, is required for morphological maturation and infectivity and leads to release of four functional domains and the spacer peptides SP1 and SP2. The N-terminal cleavages of Gag and the separation of SP1 from CA are all essential for viral infectivity, while the roles of the two C-terminal cleavages and the role of SP2, separating the NC and p6 domains, are less well defined. We have analyzed HIV-1 variants with defective cleavage at either or both sites flanking SP2, or largely lacking SP2, regarding virus production, infectivity, and structural maturation. Neither the presence nor the proteolytic processing of SP2 was required for particle release. Viral infectivity was almost abolished when both cleavage sites were defective and severely reduced when the fast cleavage site between SP2 and p6 was defective. This correlated with an increased proportion of irregular core structures observed by cryo-electron tomography, although processing of CA was unaffected. Mutation of the slow cleavage site between NC and SP2 or deletion of most of SP2 had only a minor effect on infectivity and did not induce major alterations in mature core morphology. We speculate that not only separation of NC and p6 but also the processing kinetics in this region are essential for successful maturation, while SP2 itself is dispensable.  

5.1177           Incorporation of Antigens into Viral Capsids Augments Immunogenicity of Adeno-Associated Virus Vector-Based Vaccines

Rybniker, J., Nowag, A., Janicki, H., Demant, K., Hartmann, P. and Büning, H.

1. Virol., 86(24), 13800-13804 (2012)

  Genetic modification of adeno-associated virus (AAV) capsids has previously been exploited to redirect viral tropism. Here we demonstrate that engineering of AAV capsids as scaffolds for antigen display augments antigen-specific immunogenicity. Combining antigen display with vector-mediated overexpression resulted in a single-shot prime-boost vaccine. This new class of vaccines induced immune responses significantly faster and an IgG antibody pool of higher avidity than conventional vectors, highlighting the potency of capsid modification in vaccine development.  

5.1178           The production and immunogenicity of human papillomavirus type 58 virus-like particles produced in Saccharomyces cerevisiae

Kwag, H-L., Kim, H.J., Chang, D.Y. and Kim, H-J.

1. Microbiol., 50(5), 813-820 (2012)

  Human papillomavirus (HPV) is the cause of most cases of cervical cancer. HPV type 58 (HPV58) is the second most frequent cause of cervical cancer and high-grade squamous intraepithelial lesions (HSIL) in Asia and South / Central America, respectively. However, there is no vaccine against HPV58, although there are commercially available vaccines against HPV16 and 18. In this study, we produced HPV58 L1 protein from Saccharomyces cerevisiae, and investigated its immunogenicity. We first determined the optimum period of culture for obtaining HPV58 L1. We found that a considerable portion of the HPV58 L1 resulting from 48 h culture cannot be recovered by purification, while the HPV58 L1 resulting from 144 h culture is recovered efficiently: the yield of HPV58 L1 finally recovered from 144 h culture was 2.3 times higher than that from 48 h culture, although the production level of L1 protein from 144 h culture was lower than that from 48 h culture. These results indicate that the proportion of functional L1 protein from 144 h-cultured cells is significantly higher than that of 48 h-cultured cells. The HPV58 L1 purified from the 144 h culture was correctly assembled into structures similar to naturally occurring HPV virions. Immunization with the HPV58 L1 efficiently elicited anti-HPV58 neutralizing antibodies and antigen-specific CD4+ and CD8+ T cell proliferations, without the need for adjuvant. Our findings provide a convenient method for obtaining substantial amounts of highly immunogenic HPV58 L1 from S. cerevisiae.  

5.1179           Combination small molecule PPT1 mimetic and CNS-directed gene therapy as a treatment for infantile neuronal ceroid lipofuscinosis

Roberts, M.S., Macauley, S.L., Wong, A.M., Yilmas, D., Hohm, S., Cooper, J.D. and Sands, M.S.

1. Inherit. Metab. Dis., 35(5), 847-857 (2012)

  Infantile neuronal ceroid lipofuscinosis (INCL) is a profoundly neurodegenerative disease of children caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). There is currently no effective therapy for this invariably fatal disease. To date, preclinical experiments using single treatments have resulted in incremental clinical improvements. Therefore, we determined the efficacy of CNS-directed AAV2/5-mediated gene therapy alone and in combination with the systemic delivery of the lysosomotropic PPT1 mimetic phosphocysteamine. Since CNS-directed gene therapy provides relatively high levels of PPT1 activity to specific regions of the brain, we hypothesized that phosphocysteamine would complement that activity in regions expressing subtherapeutic levels of the enzyme. Results indicate that CNS-directed gene therapy alone provided the greatest improvements in biochemical and histological measures as well as motor function and life span. Phosphocysteamine alone resulted in only minor improvements in motor function and no increase in lifespan. Interestingly, phosphocysteamine did not increase the biochemical and histological response when combined with AAV2/5-mediated gene therapy, but it did result in an additional improvement in motor function. These data suggest that a CNS-directed gene therapy approach provides significant clinical benefit, and the addition of the small molecule PPT1 mimetic can further increase that response.  

5.1180           Stability studies of HIV-1 Pr55gag virus-like particles made in insect cells after storage in various formulation media

Lynch, A., Meyers, A.E., Williamson and Rybicki, E.P. Virology J., 9:210, (2012)   Background HIV-1 Pr55^gag virus-like particles (VLPs) expressed by baculovirus in insect cells are considered to be a very promising HIV-1 vaccine candidate, as they have been shown to elicit broad cellular immune responses when tested in animals, particularly when used as a boost to DNA or BCG vaccines. However, it is important for the VLPs to retain their structure for them to be fully functional and effective. The medium in which the VLPs are formulated and the temperature at which they are stored are two important factors affecting their stability. Findings We describe the screening of 3 different readily available formulation media (sorbitol, sucrose and trehalose) for their ability to stabilise HIV-1 Pr55^gag VLPs during prolonged storage. Transmission electron microscopy (TEM) was done on VLPs stored at two different concentrations of the media at three different temperatures (4°C, –20°C and −70°C) over different time periods, and the appearance of the VLPs was compared. VLPs stored in 15% trehalose at −70°C retained their original appearance the most effectively over a period of 12 months. VLPs stored in 5% trehalose, sorbitol or sucrose were not all intact even after 1 month storage at the temperatures tested. In addition, we showed that VLPs stored under these conditions were able to be frozen and re-thawed twice before showing changes in their appearance. Conclusions Although the inclusion of other analytical tools are essential to validate these preliminary findings, storage in 15% trehalose at −70°C for 12 months is most effective in retaining VLP stability.  

5.1181           Immunogenicity of Bivalent Human Papillomavirus DNA Vaccine Using Human Endogenous Retrovirus Envelope-Coated Baculoviral Vectors in Mice and Pigs

Lee, H-J., Hur, Y-K., Cho, Y-D., Kim, M-G., Lee, H-T., Oh, Y-K. and Kim, Y.B. PloS One, 7(11), e50296 (2012)   Human papillomavirus is known to be the major pathogen of cervical cancer. Here, we report the efficacy of a bivalent human papillomavirus type 16 and 18 DNA vaccine system following repeated dosing in mice and pigs using a recombinant baculovirus bearing human endogenous retrovirus envelope protein (AcHERV) as a vector. The intramuscular administration of AcHERV-based HPV16L1 and HPV18L1 DNA vaccines induced antigen-specific serum IgG, vaginal IgA, and neutralizing antibodies to levels comparable to those achieved using the commercially marketed vaccine Cervarix. Similar to Cervarix, AcHERV-based bivalent vaccinations completely blocked subsequent vaginal challenge with HPV type-specific pseudovirions. However, AcHERV-based bivalent vaccinations induced significantly higher cell-mediated immune responses than Cervarix, promoting 4.5- (HPV16L1) and 3.9-(HPV18L1) fold higher interferon-γ production in splenocytes upon stimulation with antigen type-specific pseudovirions. Repeated dosing did not affect the immunogenicity of AcHERV DNA vaccines. Three sequential immunizations with AcHERV-HP18L1 DNA vaccine followed by three repeated dosing with AcHERV-HP16L1 over 11 weeks induced an initial production of anti-HPV18L1 antibody followed by subsequent induction of anti-HPV16L1 antibody. Finally, AcHERV-based bivalent DNA vaccination induced antigen-specific serum IgG immune responses in pigs. These results support the further development of AcHERV as a bivalent human papillomavirus DNA vaccine system for use in preventing the viral infection as well as treating the infected women by inducing both humoral and cell-mediated immune responses. Moreover, the possibility of repeated dosing indicates the utility of AcHERV system for reusable vectors of other viral pathogen vaccines.  

5.1182           Quantification of AAV Particle Titers by Infrared Fluorescence Scanning of Coomassie-Stained Sodium Dodecyl Sulfate–Polyacrylamide Gels

Kohlbrenner, E., Henckaerts, E., Rapti, K., Gordon, R.E., Linden, R.M., Hajjar, R.J. and Weber, T. Human Gene Therapy Methods, 23, 198-203 (2012)   Adeno-associated virus (AAV)-based vectors have gained increasing attention as gene delivery vehicles in basic and preclinical studies as well as in human gene therapy trials. Especially for the latter two—for both safety and therapeutic efficacy reasons—a detailed characterization of all relevant parameters of the vector preparation is essential. Two important parameters that are routinely used to analyze recombinant AAV vectors are (1) the titer of viral particles containing a (recombinant) viral genome and (2) the purity of the vector preparation, most commonly assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by silver staining. An important, third parameter, the titer of total viral particles, that is, the combined titer of both genome-containing and empty viral capsids, is rarely determined. Here, we describe a simple and inexpensive method that allows the simultaneous assessment of both vector purity and the determination of the total viral particle titer. This method, which was validated by comparison with established methods to determine viral particle titers, is based on the fact that Coomassie Brilliant Blue, when bound to proteins, fluoresces in the infrared spectrum. Viral samples are separated by SDS–PAGE followed by Coomassie Brilliant Blue staining and gel analysis with an infrared laser-scanning device. In combination with a protein standard, our method allows the rapid and accurate determination of viral particle titers simultaneously with the assessment of vector purity.  

5.1183           Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Hepatitis C Virus Replication

Romero-Brey, I., Marz, A., Chiramel, A., Lee, J-Y., Chlanda, P., Haselman, U., Santarella-Mellwig, R., Habermanan, A., Hoppe, S., Kallis, S., Walther, P., Antony, C., Krijnse-Locker, J. and Bartenschlager, R. PloS One, 8(12), e1003056 (2012)   All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.  

5.1184           Misregulation of human sortilin splicing leads to the generation of a nonfunctional progranulin receptor

Prudencio, M., Jansen-West, K.R., Lee, W.C., Gendron, T.F., Zhang, Y-J., Xu, Y-F., Gass, J., Stuani, C., Stetler, C., Rademakers, R., Dickson, D.W., Buratti, E. and Petrucelli, L. PNAS, 109(52), 21510-21515 (2012)   Sortilin 1 regulates the levels of brain progranulin (PGRN), a neurotrophic growth factor that, when deficient, is linked to cases of frontotemporal lobar degeneration with TAR DNA-binding protein-43 (TDP-43)–positive inclusions (FTLD-TDP). We identified a specific splicing enhancer element that regulates the inclusion of a sortilin exon cassette (termed Ex17b) not normally present in the mature sortilin mRNA. This enhancer element is consistently present in sortilin RNA of mice and other species but absent in primates, which carry a premature stop codon within the Ex17b sequence. In the absence of TDP-43, which acts as a regulatory inhibitor, Ex17b is included in the sortilin mRNA. In humans, in contrast to mice, the inclusion of Ex17b in sortilin mRNA generates a truncated, nonfunctional, extracellularly released protein that binds to but does not internalize PGRN, essentially acting as a decoy receptor. Based on these results, we propose a potential mechanism linking misregulation of sortilin splicing with altered PGRN metabolism.  

5.1185           Interplay between Basic Residues of Hepatitis C Virus Glycoprotein E2 with Viral Receptors, Neutralizing Antibodies and Lipoproteins

Koutsoudakis, G., Dragum, J., Perez-del-Pulgar, S., Cato-Llrena, M., Mensa, L., Crrespo, XG., Gonzalez, P., Navasa, M. and Forns, X. PloS One, 7(12), e52651 (2012)   Positively-charged amino acids are located at specific positions in the envelope glycoprotein E2 of the hepatitis C virus (HCV): two histidines (H) and four arginines (R) in two conserved WHY and one RGERCDLEDRDR motifs, respectively. Additionally, the E2 hypervariable region 1 (HVR1) is rich in basic amino acids. To investigate the role(s) of these residues in HCV entry, we subjected to comparative infection and sedimentation analysis cell culture-produced (HCVcc, genotype 2a) wild type virus, a panel of alanine single-site mutants and a HVR1-deletion variant. Initially, we analyzed the effects of these mutations on E2-heparan sulfate (HS) interactions. The positive milieu of the HVR1, formulated by its basic amino acids (key residues the conserved H³⁸⁶ and R⁴⁰⁸), and the two highly conserved basic residues H⁴⁸⁸ and R⁶⁴⁸ contributed to E2-HS interactions. Mutations in these residues did not alter the HCVcc-CD81 entry, but they modified the HCVcc-scavenger receptor class B type I (SR-BI) dependent entry and the neutralization by anti-E2 or patients IgG. Finally, separation by density gradients revealed that mutant viruses abolished partially or completely the infectivity of low density particles, which are believed to be associated with lipoproteins. This study shows that there exists a complex interplay between the basic amino acids located in HVR1 and other conserved E2 motifs with the HS, the SR-BI, and neutralizing antibodies and suggests that HCV-associated lipoproteins are implicated in these interactions.  

5.1186           Antitumoral activity of parvovirus-mediated IL-2 and MCP-3/CCL7 delivery into human pancreatic cancer: implication of leucocyte recruitment

Dempe, S., Lavie, M., Struyf, S., Bhat, R., Verbeke, H., Paschek, S., Berghmans, N., Geibig, R., Rommelaere, J., Van Damme, J. and Dinsart, C. Cancer Immunol. Immunother., 61(11), 2113-2123 (2012)   Pancreatic ductal adenocarcinoma (PDAC) represents the fourth leading cause of cancer-related death in western countries. The patients are often diagnosed in advanced metastatic stages, and the prognosis remains extremely poor with an overall 5-year survival rate less than 5 %. Currently, novel therapeutic strategies are being pursued to combat PDAC, including oncolytic viruses, either in their natural forms or armed with immunostimulatory molecules. Natural killer cells are critical players against tumours and infected cells. Recently, we showed that IL-2-activated human NK cells displayed killing activity against PDAC cells, which could further be enhanced through the infection of PDAC cells with the rodent parvovirus H-1PV. In this study, the therapeutic efficacy of parvovirus-mediated delivery of three distinct cyto/chemokines (Il-2, MCP-3/CCL7 and IP-10/CXCL10) was evaluated in xenograft models of human PDAC. We show here that activated NK and monocytic cells were found to be recruited by PDAC tumours upon infection with parvoviruses armed with IL-2 or the chemokine MCP-3/CCL7, resulting in a strong anti-tumour response.  

5.1187           Adeno-associated virus serotype 9 administered systemically after reperfusion preferentially targets cardiomyocytes in the infarct border zone with pharmacodynamics suitable for the attenuation of left ventricular remodeling

Konkalmatt, P.R., Wang, F., Piras, B.A., Xu, Y., O’Connor, D.M., Beyers, R.J., Epstein, F.H., Annex, B.H., Hossack, J.A. and French, B.A.

1. Gene Med., 14(9-10), 609-620 (2012)

  Background Adeno-associated virus serotype 9 (AAV9) vectors provide efficient and uniform gene expression to normal myocardium following systemic administration, with kinetics that approach steady-state within 2–3 weeks. However, as a result of the delayed onset of gene expression, AAV vectors have not previously been administered intravenously after reperfusion for post-infarct gene therapy applications. The present study evaluated the therapeutic potential of post-myocardial infarction gene delivery using intravenous AAV9. Methods AAV9 vectors expressing firefly luciferase, enhanced green fluorescent protein (eGFP) or extracellular superoxide dismutase genes from the cardiac troponin-T (cTnT) promoter (AcTnTLuc, AcTnTeGFP, AcTnTEcSOD) were employed. AcTnTLuc was administered intravenously at 10 min and at 1, 2 and 3 days post-ischemia/reperfusion (IR), and the kinetics of luciferase expression were assessed with bioluminescence imaging. AcTnTeGFP was used to evaluate the distribution of eGFP expression. High-resolution echocardiography was used to evaluate the effects of AcTnTEcSOD on left ventricular (LV) remodeling when injected 10 min post-IR. Results Compared to sham animals, luciferase expression at 2 days after vector administration was elevated by four-, 24-, 210- and 213-fold in groups injected at 10 min, 1 day, 2 days and 3 days post-IR, respectively. The expression of cTnT-driven eGFP was strongest in cardiomyocytes bordering the infarct zone. In the efficacy study of EcSOD, post-infarct LV end-systolic and end-diastolic volumes at days 14 and 28 were significantly smaller in the EcSOD group compared to the control. Conclusions Systemic administration of AAV9 vectors after IR both elevates and accelerates gene expression that preferentially targets cardiomyocytes in the border zone with pharmacodynamics suitable for the attenuation of LV remodeling.  

5.1188           Incorporation of Host Complement Regulatory Proteins into Newcastle Disease Virus Enhances Complement Evasion

Biswas, M., Johnson, J.B., Kumar, S.R.P., Parks, G.D. and Subbiah, E.

1. Virol., 86(23), 12708-12716 (2012)

  Newcastle disease virus (NDV), an avian paramyxovirus, is inherently tumor selective and is currently being considered as a clinical oncolytic virus and vaccine vector. In this study, we analyzed the effect of complement on the neutralization of NDV purified from embryonated chicken eggs, a common source for virus production. Fresh normal human serum (NHS) neutralized NDV by multiple pathways of complement activation, independent of neutralizing antibodies. Neutralization was associated with C3 deposition and the activation of C2, C3, C4, and C5 components. Interestingly, NDV grown in mammalian cell lines was resistant to complement neutralization by NHS. To confirm whether the incorporation of regulators of complement activity (RCA) into the viral envelope afforded complement resistance, we grew NDV in CHO cells stably transfected with CD46 or HeLa cells, which strongly express CD46 and CD55. NDV grown in RCA-expressing cells was resistant to complement by incorporating CD46 and CD55 on virions. Mammalian CD46 and CD55 molecules on virions exhibited homologous restriction, since chicken sera devoid of neutralizing antibodies to NDV were able to effectively neutralize these virions. The incorporation of chicken RCA into NDV produced in embryonated eggs similarly provided species specificity toward chicken sera.  

5.1189           An Adeno-Associated Virus-Based Intracellular Sensor of Pathological Nuclear Factor-κB Activation for Disease-Inducible Gene Transfer

Chtarto, A., Bockstael, O., Bebara, E., Vermoesen, K., Melas, C., Pythoud, C., Levivier, M., De Witte, O., Luthi-Carter, R., Clinkers, R. and Tenenbaum, L. PloS One, 8(1), e53156 (2013)   Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.  

5.1190           Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors

Balakrishnan, B., Sen, D., Hareendran, S., Roshini, V., David, S., Srivastava, A. and Jayandharan, G.R. PloS One, 8(1), e53845 (2013)   The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold) and PERK (up to 8 fold) genes 12–48 hours after infection with self-complementary (sc)AAV2 but less prominent with single-stranded (ss)AAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold) while AAV6 vectors induced a significant increase on all the three major UPR pathways [6–16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (~1.5–2 fold) in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively). However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin) during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.  

5.1191           Flavivirus infection from mosquitoes in vitro reveals cell entry at the plasma membrane

Vancini, R., Kramer, L.D., Ribeiro, M., Hernandez, R. and Brown, D. Virology, 435(2), 406-414 (2013)   Dengue and West Nile viruses are enveloped RNA viruses that belong to genus Flavivirus (family Flaviviridae) and are considered important mosquito-borne viral pathogenic agents worldwide. A potential target for intervention strategies is the virus cell entry mechanism. Previous studies of flavivirus entry have focused on the effects of biochemical and molecular inhibitors on viral entry leading to controversial conclusions suggesting that the process is dependent upon endocytosis and low pH mediated membrane fusion. In this study we analyzed the early events in the infection process by means of electron microscopy and immuno-gold labeling of viral particles during cell entry, and used as a new approach for infecting cells with viruses obtained directly from mosquitoes. The results show that Dengue and West Nile viruses may infect cells by a mechanism that involves direct penetration of the host cell plasma membrane as proposed for alphaviruses.  

5.1192           Heat shock protein 90AB1 and hyperthermia rescue infectivity of HIV with defective cores

Joshi, P., Sloan, B., Torbett, B.E. and Stoddart, C.A. Virology, 436(1), 162-172 (2013)   We previously showed that reduced infectivity of HIV with incompletely processed capsid-spacer protein 1 (CA-SP1) is rescued by cellular activation or increased expression of HSP90AB1, a member of the cytosolic heat shock protein 90 family. Here we show that HSP90AB1 is present in HIV virions and that HSP90AB1, but not nonfunctional mutated HSP90AB1_E42A+D88A, restores infectivity to HIV with mutations in CA that alter core stability. Further, the CA mutants were hypersensitive to pharmacological inhibition of HSP90AB1. In agreement with Roesch et al. (2012), we found that culturing HIV at 39.5 °C enhanced viral infectivity up to 30-fold in human peripheral blood mononuclear cells (p=0.002) and rescued CA-mutant infectivity in nonactivated cells, concurrent with elevated expression of HSP90AB1 during hyperthermia. In sum, the transdominant effect of HSP90AB1 on CA-mutant HIV infectivity suggests a potential role for this class of cellular chaperones in HIV core stability and uncoating.  

5.1193           Comprehensive Mutational Analysis Reveals p6Gag Phosphorylation To Be Dispensable for HIV-1 Morphogenesis and Replication

Radestock, B., Morales, I., Rahman, S.A., Radau, S., Glass, B., Zahedi, R.P., Müller, B. and Kräusslich, H-G.

1. Virol., 87(2), 724-734 (2013)

  The structural polyprotein Gag of human immunodeficiency virus type 1 (HIV-1) is necessary and sufficient for formation of virus-like particles. Its C-terminal p6 domain harbors short peptide motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. p6 has been shown to be the major viral phosphoprotein in HIV-1-infected cells and virions, but the sites and functional relevance of p6 phosphorylation are not clear. Here, we identified phosphorylation of several serine and threonine residues in p6 in purified virus preparations using mass spectrometry. Mutation of individual candidate phosphoacceptor residues had no detectable effect on virus assembly, release, and infectivity, however, suggesting that phosphorylation of single residues may not be functionally relevant. Therefore, a comprehensive mutational analysis was conducted changing all potentially phosphorylatable amino acids in p6, except for a threonine that is part of an essential peptide motif. To avoid confounding changes in the overlapping pol reading frame, mutagenesis was performed in a provirus with genetically uncoupled gag and pol reading frames. An HIV-1 derivative carrying 12 amino acid changes in its p6 region, abolishing all but one potential phosphoacceptor site, showed no impairment of Gag assembly and virus release and displayed only very subtle deficiencies in viral infectivity in T-cell lines and primary lymphocytes. All mutations were stable over 2 weeks of culture in primary cells. Based on these findings, we conclude that phosphorylation of p6 is dispensable for HIV-1 assembly, release, and infectivity in tissue culture.  

5.1194           Oligomeric Properties of Adeno-Associated Virus Rep68 Reflect Its Multifunctionality

Zarate-Perez, F., Mansilla-Soto, J., Bardelli, M., Burgner, J.W., Villamil-Jarauta, M., Kekilli, D., Samso, M., Linden, R.M. and Escalante, C.R.

1. Virol., 87(2), 1232-1241 (2013)

  The adeno-associated virus (AAV) encodes four regulatory proteins called Rep. The large AAV Rep proteins Rep68 and Rep78 are essential factors required in almost every step of the viral life cycle. Structurally, they share two domains: a modified version of the AAA⁺ domain that characterizes the SF3 family of helicases and an N-terminal domain that binds DNA specifically. The combination of these two domains imparts extraordinary multifunctionality to work as initiators of DNA replication and regulators of transcription, in addition to their essential role during site-specific integration. Although most members of the SF3 family form hexameric rings in vitro, the oligomeric nature of Rep68 is unclear due to its propensity to aggregate in solution. We report here a comprehensive study to determine the oligomeric character of Rep68 using a combination of methods that includes sedimentation velocity ultracentrifugation, electron microscopy, and hydrodynamic modeling. We have determined that residue Cys151 induces Rep68 to aggregate in vitro. We show that Rep68 displays a concentration-dependent dynamic oligomeric behavior characterized by the presence of two populations: one with monomers and dimers in slow equilibrium and a second one consisting of a mixture of multiple-ring structures of seven and eight members. The presence of either ATP or ADP induces formation of larger complexes formed by the stacking of multiple rings. Taken together, our results support the idea of a Rep68 molecule that exhibits the flexible oligomeric behavior needed to perform the wide range of functions occurring during the AAV life cycle.  

5.1195           Improved Survival and Reduced Phenotypic Severity Following AAV9/MECP2 Gene Transfer to Neonatal and Juvenile Male Mecp2 Knockout Mice

Gadalla, K.K.E., Bailey, M.E.S., Spike, R.C., Ross, P.D., Woodard, K.T., Kalburgi, S.N., Bachaboina, L., Deng, J.V., West, A.E., Samulski, R.J., Gray, S.J. and Cobb, S.R. Molecular Therapy, 21(1), 18-30 (2013)   Typical Rett syndrome (RTT) is a pediatric disorder caused by loss-of-function mutations in the methyl-CpG binding protein 2 (MECP2) gene. The demonstrated reversibility of RTT-like phenotypes in mice suggests that MECP2 gene replacement is a potential therapeutic option in patients. We report improvements in survival and phenotypic severity in Mecp2-null male mice after neonatal intracranial delivery of a single-stranded (ss) AAV9/chicken β-actin (CBA)-MECP2 vector. Median survival was 16.6 weeks for MECP2-treated versus 9.3 weeks for green fluorescent protein (GFP)-treated mice. ssAAV9/CBA-MECP2–treated mice also showed significant improvement in the phenotype severity score, in locomotor function, and in exploratory activity, as well as a normalization of neuronal nuclear volume in transduced cells. Wild-type (WT) mice receiving neonatal injections of the same ssAAV9/CBA-MECP2 vector did not show any significant deficits, suggesting a tolerance for modest MeCP2 overexpression. To test a MECP2 gene replacement approach in a manner more relevant for human translation, a self-complementary (sc) adeno-associated virus (AAV) vector designed to drive MeCP2 expression from a fragment of the Mecp2 promoter was injected intravenously (IV) into juvenile (4–5 weeks old) Mecp2-null mice. While the brain transduction efficiency in juvenile mice was low (~2–4% of neurons), modest improvements in survival were still observed. These results support the concept of MECP2 gene therapy for RTT.  

5.1196           Displaying High-affinity Ligands on Adeno-associated Viral Vectors Enables Tumor Cell-specific and Safe Gene Transfer

Münch, R.C., Janicki, H., Völker, I., Rasbach, A., Hallek, M., Büning, H.and Buchholz, C.J. Molecular Therapy, 21(1), 109-118 (2013)   Gene transfer vectors derived from the adeno-associated virus (AAV) have recently received increasing attention due to substantial therapeutic benefit in several clinical trials. Nevertheless, their great potential for in vivo gene therapy can only be partially exploited owing to their broad tropism. Current cell surface targeting strategies expanded vector tropism towards transduction of cell types that are inefficiently infected naturally, but failed to restrict or fully re-direct AAV's tropism. Hypothesizing that this limitation can be overcome by equipping natural receptor-blinded AAV vectors with high-affinity ligands, we displayed designed ankyrin repeat proteins (DARPin) as VP2 fusion proteins on AAV capsids ablated for natural primary receptor binding. These second generation targeting vectors demonstrated an as of yet unachieved efficiency to discriminate between target and non-target cells in mono- and mixed cultures. Moreover, DARPin-AAV vectors delivered a suicide gene precisely to tumor tissue and substantially reduced tumor growth without causing fatal liver toxicity. The latter caused death in animals treated with conventional AAV vectors with unmodified capsids, which accumulated in liver tissue and failed to affect tumor growth. This novel targeting platform will be key to translational approaches requiring restricted and cell type-specific in vivo gene delivery.  

5.1197           Embedding siRNA sequences targeting Apolipoprotein B100 in shRNA and miRNA scaffolds results in differential processing and in vivo efficacy

Maczuga, P., Lubelski, J., van Logtenstein, R., Borel, F., Blits, B., Fakkert, E., Costessi, A., Butler, D., van Deventer, S., Petry, H., Koornneef, A. and Konstantinova, P. Molecular Therapy, 21(1), 217-227 (2013)   Overexpression of short hairpin RNA (shRNA) often causes cytotoxicity and using microRNA (miRNA) scaffolds can circumvent this problem. In this study, identically predicted small interfering RNA (siRNA) sequences targeting apolipoprotein B100 (siApoB) were embedded in shRNA (shApoB) or miRNA (miApoB) scaffolds and a direct comparison of the processing and long-term in vivo efficacy was performed. Next generation sequencing of small RNAs originating from shApoB- or miApoB-transfected cells revealed substantial differences in processing, resulting in different siApoB length, 5′ and 3′ cleavage sites and abundance of the guide or passenger strands. Murine liver transduction with adeno-associated virus (AAV) vectors expressing shApoB or miApoB resulted in high levels of siApoB expression associated with strong decrease of plasma ApoB protein and cholesterol. Expression of miApoB from the liver-specific LP1 promoter was restricted to the liver, while the H1 promoter-expressed shApoB was ectopically present. Delivery of 1 × 10¹¹ genome copies AAV-shApoB or AAV-miApoB led to a gradual loss of ApoB and plasma cholesterol inhibition, which was circumvented by delivering a 20-fold lower vector dose. In conclusion, incorporating identical siRNA sequences in shRNA or miRNA scaffolds results in differential processing patterns and in vivo efficacy that may have serious consequences for future RNAi-based therapeutics.  

5.1198           Selective presynaptic enhancement of the prefrontal cortex to nucleus accumbens pathway by cocaine

Suska, A., Lee, B.R., Huang, Y.H., Dong, Y. and Schlüter, O.M. PNAS, 110(2), 713-718 (2013)   The nucleus accumbens (NAc) regulates motivated behavior by, in part, processing excitatory synaptic projections from several brain regions. Among these regions, the prefrontal cortex (PFC) and basolateral amygdala, convey executive control and affective states, respectively. Whereas glutamatergic synaptic transmission within the NAc has been recognized as a primary cellular target for cocaine and other drugs of abuse to induce addiction-related pathophysiological motivational states, the understanding has been thus far limited to drug-induced postsynaptic alterations. It remains elusive whether exposure to cocaine or other drugs of abuse influences presynaptic functions of these excitatory projections, and if so, in which projection pathways. Using optogenetic methods combined with biophysical assays, we demonstrate that the presynaptic release probability (Pr) of the PFC-to-NAc synapses was enhanced after short-term withdrawal (1 d) and long-term (45 d) withdrawal from either noncontingent (i.p. injection) or contingent (self-administration) exposure to cocaine. After long-term withdrawal of contingent drug exposure, the Pr was higher compared with i.p. injected rats. In contrast, within the basolateral amygdala afferents, presynaptic Pr was not significantly altered in any of these experimental conditions. Thus, cocaine-induced procedure- and pathway-specific presynaptic enhancement of excitatory synaptic transmission in the NAc. These results, together with previous findings of cocaine-induced postsynaptic enhancement, suggest an increased PFC-to-NAc shell glutamatergic synaptic transmission after withdrawal from exposure to cocaine. This presynaptic alteration may interact with other cocaine-induced cellular adaptations to shift the functional output of NAc neurons, contributing to the addictive emotional and motivational state.  

5.1199           Characterization of Hepatitis C Virus Recombinants with Chimeric E1/E2 Envelope Proteins and Identification of Single Amino Acids in the E2 Stem Region Important for Entry

Carlsen, T.E., Scheel, T.K.H., Ramirez, S.K.H. and Bukh, J.

1. Virol., 87(3), 1385-1399 (2013)

  The hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexes in vitro. Using genotype 1a/2a JFH1-based recombinants expressing 1a core-NS2, we exchanged E2 with functional isolate sequences of genotypes 1a (alternative isolate), 1b, and 2a. While the 1a-E2 exchange did not impact virus viability, the 2a-E2 recombinant was nonviable. After E2 exchange from three 1b isolates, long delays were observed before spread of infection. For recovered 1b-E2 recombinants, single E2 stem region amino acid changes were identified at residues 706, 707, and 710. In reverse genetic studies, these mutations increased infectivity titers by ∼100-fold, apparently without influencing particle stability or cell binding although introducing slight decrease in particle density. In addition, the 1b-E2 exchange led to a decrease in secreted core protein of 25 to 50%, which was further reduced by the E2 stem region mutations. These findings indicated that compensatory mutations permitted robust infectious virus production, without increasing assembly/release. Studies of E1/E2 heterodimerization showed no differences in intracellular E1/E2 interaction for chimeric constructs with or without E2 stem region mutations. Interestingly, the E2 stem region mutations allowed efficient entry, which was verified in 1a-E1/1b-E2 HCV pseudoparticle assays. A CD81 inhibition assay indicated that the mutations influenced a late step of the HCV entry pathway. Overall, this study identified specific amino acids in the E2 stem region of importance for HCV entry and for production of infectious virus particles.  

5.1200           Disulfide Bonds in Hepatitis C Virus Glycoprotein E1 Control the Assembly and Entry Functions of E2 Glycoprotein

Wahid, A., Helle, F., Descamps, V., Duverlie, G., Penin, F. and Dubuisson, J.

1. Virol, 87(3), 1605-1617 (2013)

  Class II membrane fusion proteins have been described in viruses in which the envelope proteins are derived from a precursor polyprotein containing two transmembrane glycoproteins arranged in tandem. Although the second protein, which carries the membrane fusion function, is in general well characterized, the companion protein, which is a protein chaperone for the folding of the fusion protein, is less well characterized for some viruses, like hepatitis C virus (HCV). To investigate the role of the class II companion glycoprotein E1 of HCV, we chose to target conserved cysteine residues in the protein, and we systematically mutated them in a full-length infectious HCV clone by reverse genetics. All the mutants were infectious, albeit with lower titers than the wild-type virus. The reduced infectivity was in part due to a decrease in viral assembly, as revealed by measurement of intracellular infectivity and by quantification of core protein released from cells transfected with mutant genomes. Analyses of mutated proteins did not show any major defect in folding. However, the mutations reduced virus stability, and they could also affect the density of infectious viral particles. Mutant viruses also showed a defect in cell-to-cell transmission. Finally, our data indicate that HCV glycoprotein E1 can also affect the fusion protein E2 by modulating its recognition by the cellular coreceptor CD81. Therefore, in the context of HCV, our data identify an additional function of a class II companion protein as a molecule that can control the binding capacity of the fusion protein.  

5.1201           Adeno-Associated Viral Vectors for Gene Therapy of Inherited Retinal Degenerations

Flannery, J.G, and Visel, M. Methods in Mol. Biol., 935(7), 351-369 (2013)   Adeno-associated virus (AAV) vectors are in wide use for in vivo gene transfer for the treatment of inherited retinal disease. AAV vectors have been tested in many animal models and have demonstrated ef fi cacy with low toxicity. In this chapter we describe some of the recent methods for small-scale production of these vectors for use in a laboratory setting in volumes and purity appropriate for testing in small and large animals.  

5.1202           Upregulation of reggie-1/flotillin-2 promotes axon regeneration in the rat optic nerve in vivo and neurite growth in vitro

Koch, J.C., Solis, G.P., Bodrikov, V., Michel, U., Haralampieva, D., Shypitsyna, A., Tönges, L., Mathias Bähr, M., Lingor, P. and Stuermer, C.A.O. Neurobiology of Disease, 51, 168-176 (2013)   The ability of fish retinal ganglion cells (RGCs) to regenerate their axons was shown to require the re-expression and function of the two proteins reggie-1 and -2. RGCs in mammals fail to upregulate reggie expression and to regenerate axons after lesion suggesting the possibility that induced upregulation might promote regeneration. In the present study, RGCs in adult rats were induced to express reggie-1 by intravitreal injection of adeno-associated viral vectors (AAV2/1) expressing reggie-1 (AAV.R1-EGFP) 14d prior to optic nerve crush. Four weeks later, GAP-43-positive regenerating axons had crossed the lesion and grown into the nerve at significantly higher numbers and length (up to 5 mm) than the control transduced with AAV.EGFP. Consistently, after transduction with AAV.R1-EGFP as opposed to AAV.EGFP, primary RGCs in vitro grew long axons on chondroitin sulfate proteoglycan (CSPG) and Nogo-A, both glial cell-derived inhibitors of neurite growth, suggesting that reggie-1 can provide neurons with the ability to override inhibitors of neurite growth. This reggie-1-mediated enhancement of growth was reproduced in mouse hippocampal and N2a neurons which generated axons 40–60% longer than their control counterparts. This correlates with the reggie-1-dependent activation of Src and PI3 kinase (PI3K), of the Rho family GTPase Rac1 and downstream effectors such as cofilin. This increased growth also depends on TC10, the GTPase involved in cargo delivery to the growth cone. Thus, the upregulation of reggie-1 in mammalian neurons provides nerve cells with neuron-intrinsic properties required for axon growth and successful regeneration in the adult mammalian CNS.  

5.1203           Long-distance axonal regeneration induced by CNTF gene transfer is impaired by axonal misguidance in the injured adult optic nerve

Pernet, V., Joly, S., Dalkara, D., Jordi, N., Schwarz, O., Christ, F., Schaffer, D.V., Flannery, J.G. and Schwab, M.E. Neurobiology of Disease, 51, 202-213 (2013)   The optic nerve crush injury is a well-accepted model to study the mechanisms of axonal regeneration after trauma in the CNS. The infection of retinal ganglion cells (RGCs) with an adeno-associated virus serotype 2 — ciliary neurotrophic factor (AAV2.CNTF) was previously shown to stimulate axonal regeneration. However, the transfection of axotomized neurons themselves may not be optimal to promote full axonal regeneration in the visual system. Here, we show that the release of CNTF by glial cells is a very powerful stimulus for optic fiber regeneration and RGC survival after optic nerve crush. After 8 weeks, long-distance regeneration of severed optic axons was induced by CNTF until and beyond the optic chiasm. Regenerated axons stayed for at least 6 months in the damaged optic nerve. Strikingly, however, many regenerated axons showed one or several sharp U-turns along their course, suggesting that guidance cues are missing and that long-distance axonal regeneration is limited by the return of the growing axons toward the retina. Even more surprisingly, massive axonal sprouting was observed within the eye, forming a dense plexus of neurites at the inner surface of the retina. These results indicate that massive stimulation of the neuronal growth program can lead to aberrant growth; the absence of local regulatory and guidance factors in the adult, injured optic nerve may therefore represent a major, so far underestimated obstacle to successful axon regeneration.  

5.1204           Local gene delivery of heme oxygenase-1 by adeno-associated virus into osteoarthritic mouse joints exhibiting synovial oxidative stress

Kyostio-Moore, S., Bangari, D.S., Ewing, P., Nambiar, B., Berthelette, P., Sookdeo, C., Hutto, E., Moran, N., Sullivan, J., Matthews, G.L., Scaria, A. and Armentano, D. Osteoarthritis and Cartilage, 21, 358-367 (2013)   Objective To evaluate the role of synovial oxidative stress on joint pathology in a spontaneous mouse model of osteoarthritis (OA) by intra-articular (IA) delivery of recombinant adeno-associated virus (rAAV) expressing anti-oxidant protein heme oxygenase-1 (HO-1). Methods Joint transduction by rAAV vectors was evaluated with serotype 1, 2, 5 and 8 capsids carrying LacZ gene administered by IA injections into STR/ort mice. Transduced cell types were identified by β-galactosidase staining in sectioned joints. Effect of oxidative stress on AAV transduction of primary synoviocytes in vitro was quantitated by fluorescence-activated cell sorting (FACS) analysis. In vivo, the efficacy of rAAV1/HO-1 was tested by IA administration into STR/ort mice followed by histopathological scoring of cartilage. Levels of 3-nitrotyrosine (3-NT) and HO-1 were assessed by immunohistochemistry (IHC) of joint sections. Results Administration of a rAAV1 based vector into OA mouse joints resulted in transduction of the synovium, joint capsule, adipocytes and skeletal muscle while none of the serotypes showed significant cartilage transduction. All OA joints exhibited significantly elevated levels of oxidative stress marker, 3-NT, in the synovium compared to OA-resistant CBA-strain of mice. In vitro studies demonstrated that AAV transgene expression in primary synoviocytes was augmented by oxidative stress induced by H₂O₂ and that a rAAV expressing HO-1 reduced the levels of oxidative stress. In vivo, HO-1 was increased in the synovium of STR/ort mice. However, delivery of rAAV1/HO-1 into OA joints did not reduce cartilage degradation. Conclusions AAV-mediated HO-1 delivery into OA joints during active disease was not sufficient to improve cartilage pathology in this model.  

5.1205           TLR-9 Contributes to the Antiviral Innate Immune Sensing of Rodent Parvoviruses MVMp and H-1PV by Normal Human Immune Cells

Raykov, Z., Grekova, S.P., Hörlein, R., Leuchs, B., Giese, T., Giese, N.A., Rommelaere, J., Zawatzky, R. and Daeffler, L. PloS One, 8(1), e55086 (2013)   The oncotropism of Minute Virus of Mice (MVMp) is partially related to the stimulation of an antiviral response mediated by type-I interferons (IFNs) in normal but not in transformed mouse cells. The present work was undertaken to assess whether the oncotropism displayed against human cells by MVMp and its rat homolog H-1PV also depends on antiviral mechanisms and to identify the pattern recognition receptor (PRR) involved. Despite their low proliferation rate which represents a drawback for parvovirus multiplication, we used human peripheral blood mononuclear cells (hPBMCs) as normal model specifically because all known PRRs are functional in this mixed cell population and moreover because some of its subsets are among the main IFN producers upon infections in mammals. Human transformed models consisted in lines and tumor cells more or less permissive to both parvoviruses. Our results show that irrespective of their permissiveness, transformed cells do not produce IFNs nor develop an antiviral response upon parvovirus infection. However, MVMp- or H-1PV-infected hPBMCs trigger such defense mechanisms despite an absence of parvovirus replication and protein expression, pointing to the viral genome as the activating element. Substantial reduction of an inhibitory oligodeoxynucleotide (iODN) of the latter IFN production identified TLR-9 as a potential PRR for parvoviruses in hPBMCs. However, neither the iODN treatment nor an antibody-induced neutralization of the IFN-triggered effects restored parvovirus multiplication in these cells as expected by their weak proliferation in culture. Finally, given that a TLR-9 activation could also not be observed in parvovirus-infected human lines reported to be endowed with a functional TLR-9 pathway (Namalwa, Raji, and HEK293-TLR9^+/+), our data suggest that transformed human cells do not sense MVMp or H-1PV either because of an absence of PRR expression or an intrinsic, or virus-driven defect in the endosomal sensing of the parvovirus genomes by TLR-9.  

5.1206           Natural variants in the major neutralizing epitope of human papillomavirus minor capsid protein L2

Seitz, H., Schmitt, M., Böhmer, G., Kopp-Schneider, A. and Müller, M. Int. J. Cancer, 132(3), E139-E148 (2013)   The amino terminus of the human papillomavirus minor capsid protein L2 contains a major cross-neutralizing epitope that provides the basis for the development of a broadly protective HPV vaccine. This attainable broad protection would eliminate one of the major drawbacks of the commercial L1-based prophylactic vaccines. In this study, we asked whether there are natural variants of the L2 cross-neutralizing epitope and if these variants provide means for immune escape from vaccine-induced anti-L2 antibodies. For this, we isolated in silico and in vitro, a total of 477 L2 sequences of HPV types 16, 18, 31, 45, 51, 52 and 58. We identified natural L2 epitope variants for HPV 18, 31, 45 and 51. To determine whether these variants escape L2-directed neutralization, we generated pseudovirions encompassing the natural variants and tested these in an in vitro neutralization assay using monoclonal and polyclonal antibodies. Our results indicate that natural variants of the L2 major neutralizing epitope are frequent among two different study populations from Germany and Mongolia and in the GenBank database. Of two identified HPV 31 L2 single amino acid variants, one could be neutralized well, while the other variant was neutralized very poorly. We also observed that single amino acid variants of HPV 18 and 45 are neutralized well while a HPV 18 double variant was neutralized at significantly lower rates, indicating that L2 variants have to be accounted for when developing HPV L2-based prophylactic vaccines.  

5.1207           Genetic identification of C fibres that detect massage-like stroking of hairy skin in vivo

Vrontou, S., Wong, A.M., Rau, K.K., Koerber, H.R. and Anderson, D.J. Nature, 493, 669-673 (2013)   Stroking of the skin produces pleasant sensations that can occur during social interactions with conspecifics, such as grooming¹. Despite numerous physiological studies (reviewed in ref. 2), molecularly defined sensory neurons that detect pleasant stroking of hairy skin^{3, 4} in vivo have not been reported. Previously, we identified a rare population of unmyelinated sensory neurons in mice that express the G-protein-coupled receptor MRGPRB4 (refs 5, 6). These neurons exclusively innervate hairy skin with large terminal arborizations⁷ that resemble the receptive fields of C-tactile (CT) afferents in humans⁸. Unlike other molecularly defined mechanosensory C-fibre subtypes^{9, 10}, MRGPRB4⁺ neurons could not be detectably activated by sensory stimulation of the skin ex vivo. Therefore, we developed a preparation for calcium imaging in the spinal projections of these neurons during stimulation of the periphery in intact mice. Here we show that MRGPRB4⁺ neurons are activated by massage-like stroking of hairy skin, but not by noxious punctate mechanical stimulation. By contrast, a different population of C fibres expressing MRGPRD¹¹ was activated by pinching but not by stroking, consistent with previous physiological and behavioural data^{10, 12}. Pharmacogenetic activation of Mrgprb4-expressing neurons in freely behaving mice promoted conditioned place preference¹³, indicating that such activation is positively reinforcing and/or anxiolytic. These data open the way to understanding the function of MRGPRB4 neurons during natural behaviours, and provide a general approach to the functional characterization of genetically identified subsets of somatosensory neurons in vivo.  

5.1208           Intramuscular scAAV9-SMN Injection Mediates Widespread Gene Delivery to the Spinal Cord and Decreases Disease Severity in SMA Mice

Benkhelifa-Ziyyat, S., Besse, A., Roda, M., Duque, S., Astord, S., Carcenac, R., Marais, T. and Barkats, M. Molecular Therapy, 21(2), 282-290 (2013)   We have recently demonstrated the remarkable efficiency of self-complementary (sc) AAV9 vectors for central nervous system (CNS) gene transfer following intravenous delivery in mice and larger animals. Here, we investigated whether gene delivery to motor neurons (MNs) could also be achieved via intramuscular (i.m.) scAAV9 injection and subsequent retrograde transport along the MNs axons. Unexpectedly, we found that a single injection of scAAV9 into the adult mouse gastrocnemius (GA) mediated widespread MN transduction along the whole spinal cord, without limitation to the MNs connected to the injected muscle. Spinal cord astrocytes and peripheral organs were also transduced, indicating vector spread from the injected muscle to both the CNS and the periphery through release into the blood circulation. Moreover, we showed that i.m. injection of scAAV9 vectors expressing “survival of motor neuron” (Smn) in spinal muscular atrophy (SMA) mice mediated high survival motor neuron (SMN) expression levels at both the CNS and the periphery, and increased the median lifespan from 12 days to 163 days. These findings represent to date the longest extent in survival obtained in SMA mice following i.m. viral vector gene delivery, and might generate a renewed interest in the use of i.m. adeno-associated viruses (AAV) delivery for the development of gene therapy strategies for MN diseases.  

5.1209           Cellular Entry of Human Papillomavirus Type 16 Involves Activation of the Phosphatidylinositol 3-Kinase/Akt/mTOR Pathway and Inhibition of Autophagy

Surviladze, Z., Sterk, R.T., DeHaro, S.A. and Ozbun, M.A.

1. Virol., 87(5), 2508-2517 (2013)

  The mammalian target of rapamycin (mTOR) downstream of phosphatidylinositol 3-kinase (PI3K) in the growth factor receptor (GFR) pathway is a crucial metabolic sensor that integrates growth factor signals in cells. We recently showed that human papillomavirus (HPV) type 16 exposure activates signaling from GFRs in human keratinocytes. Thus, we predicted that the virus would induce the PI3K/mTOR pathway upon interaction with host cells. We detected activation of Akt and mTOR several minutes following exposure of human keratinocytes to HPV type 16 (HPV16) pseudovirions. Activated mTOR induced phosphorylation of the mTOR complex 1 substrates 4E-BP1 and S6K, which led to induction of the functional protein translational machinery. Blockade of epidermal GFR (EGFR) signaling revealed that each of these events is at least partially dependent upon EGFR activation. Importantly, activation of PI3K/Akt/mTOR signaling inhibited autophagy in the early stages of virus-host cell interaction. Biochemical and genetic approaches revealed critical roles for mTOR activation and autophagy suppression in HPV16 early infection events. In summary, the HPV-host cell interaction stimulates the PI3K/Akt/mTOR pathway and inhibits autophagy, and in combination these events benefit virus infection.  

5.1210           Optimization of scAAVIL-1ra In Vitro and In Vivo to Deliver High Levels of Therapeutic Protein for Treatment of Osteoarthritis

Goodrich, L.R., Phillips, J.N., Mcllwraith, C.W., Foti, S.B., Grieger, J.C., Gray, S.J. and Samulski, R.J. Molecular Therapy-Nucleic Acids, 2, e70 (2013)   Osteoarthritis (OA) affects over 40 million people annually. We evaluated interleukin-1 receptor antagonist (IL-1ra) gene transfer in an equine model based on IL-1ra protein therapy which inhibits inflammation through blocking IL-1. Using the self-complementary adeno-associated virus (scAAV)IL-1ra equine gene as a starting construct, we optimized the transgene cassette by analyzing promoters (cytomegalovirus (CMV) versus chicken β-actin hybrid (CBh)), coding sequences (optimized versus unoptimized), vector capsid (serotype 2 versus chimeric capsid), and biological activity in vitro. AAV serotypes 2 and 2.5 CMV scAAVoptIL-1ra were tested in equine joints. We evaluated two doses of scAAVIL-1ra, scAAVGFP, and saline. We developed a novel endoscopy procedure and confirmed vector-derived transgene expression (GFP) in chondrocytes 6 months post-injection. AAVIL-1ra therapeutic protein levels were 200–800 ng/ml of synovial fluid over 23 and 186 days, respectively. No evidence of intra-articular toxicity was detected and no vector genomes were found in contralateral joints based on GFP fluorescence microscopy and quantitative PCR. Finally, we assayed vector-derived IL-1ra activity based on functional assays which supported anti-inflammatory activity of our protein. These studies represent the first large animal intra-articular gene transfer approach with a therapeutic gene using scAAV and demonstrate high levels of protein production over extended time supporting further clinical investigation using scAAV gene therapy for OA.  

5.1211           Virion stiffness regulates immature HIV-1 entry

Pang, H-B., Hevroni, L., Kol, N., Eckert, D.M., Tsvitov, M., Kay, M.S. and Rousso, I. Retrovirology, 10:4 (2013)   Background Human immunodeficiency virus type 1 (HIV-1) undergoes a protease-mediated maturation process that is required for its infectivity. Little is known about how the physical properties of viral particles change during maturation and how these changes affect the viral lifecycle. Using Atomic Force Microscopy (AFM), we previously discovered that HIV undergoes a “stiffness switch”, a dramatic reduction in particle stiffness during maturation that is mediated by the viral Envelope (Env) protein. Results In this study, we show that transmembrane-anchored Env cytoplasmic tail (CT) domain is sufficient to regulate the particle stiffness of immature HIV-1. Using this construct expressed in trans with viral Env lacking the CT domain, we show that increasing particle stiffness reduces viral entry activity in immature virions. A similar effect was also observed for immature HIV-1 pseudovirions containing Env from vesicular stomatitis virus. Conclusions This linkage between particle stiffness and viral entry activity illustrates a novel level of regulation for viral replication, providing the first evidence for a biological role of virion physical properties and suggesting a new inhibitory strategy.  

5.1212           Anxiogenic effects of CGRP within the BNST may be mediated by CRF acting at BNST CRFR1 receptors

Sink, K.S., Chung, A., Ressler, K.J., davis, M. and Walker, D.L. Behavioural Brain Res., 243, 286-293 (2013)   Calcitonin gene-related peptide (CGRP) acting within the bed nucleus of the stria terminalis (BNST) increases anxiety as well as neural activation in anxiety-related structures, and mediates behavioral stress responses. Similar effects have been described following intra-ventricular as well as intra-BNST infusions of the stress-responsive neuropeptide, corticotropin releasing factor (CRF). Interestingly, CGRP-positive terminals within the lateral division of the BNST form perisomatic baskets around neurons that express CRF, suggesting that BNST CGRP could exert its anxiogenic effects by increasing release of CRF from these neurons. With this in mind, the present set of experiments was designed to examine the role of CRFR1 signaling in the anxiogenic effects of CGRP within the BNST and to determine whether CRF from BNST neurons contributes to these effects. Consistent with previous studies, we found that 400 ng CGRP infused bilaterally into the BNST increased the acoustic startle response and induced anxiety-like behavior in the elevated plus maze compared to vehicle. Both of these effects were attenuated by 10 mg/kg PO of the CRFR1 antagonist, GSK876008. GSK876008 alone did not affect startle. An intra-BNST infusion of the CRFR1 antagonist CP376395 (2 μg) also blocked increases in acoustic startle induced by intra-BNST infusion of CGRP, as did virally-mediated siRNA knockdown of CRF expression locally within the BNST. Together, these results suggest that the anxiogenic effects of intra-BNST CGRP may be mediated by CRF from BNST neurons acting at local CRFR1 receptors.  

5.1213           Biophysical and Ultrastructural Characterization of Adeno-Associated Virus Capsid Uncoating and Genome Release

Horowitz, E.D., Rahman, K.S., Bower, B.D., Dismuke, D.J., Falvo, M.R., Griffith, J.D., Harvey, S.C and Asokan, A.

1. Virol., 87(6), 2994-3002 (2013)

  We describe biophysical and ultrastructural differences in genome release from adeno-associated virus (AAV) capsids packaging wild-type DNA, recombinant single-stranded DNA (ssDNA), or dimeric, self-complementary DNA (scDNA) genomes. Atomic force microscopy and electron microscopy (EM) revealed that AAV particles release packaged genomes and undergo marked changes in capsid morphology upon heating in physiological buffer (pH 7.2). When different AAV capsids packaging ss/scDNA varying in length from 72 to 123% of wild-type DNA (3.4 to 5.8 kb) were incrementally heated, the proportion of uncoated AAV capsids decreased with genome length as observed by EM. Genome release was further characterized by a fluorimetric assay, which demonstrated that acidic pH and high osmotic pressure suppress genome release from AAV particles. In addition, fluorimetric analysis corroborated an inverse correlation between packaged genome length and the temperature needed to induce uncoating. Surprisingly, scAAV vectors required significantly higher temperatures to uncoat than their ssDNA-packaging counterparts. However, externalization of VP1 N termini appears to be unaffected by packaged genome length or self-complementarity. Further analysis by tungsten-shadowing EM revealed striking differences in the morphologies of ssDNA and scDNA genomes upon release from intact capsids. Computational modeling and molecular dynamics simulations suggest that the unusual thermal stability of scAAV vectors might arise from partial base pairing and optimal organization of packaged scDNA. Our work further defines the biophysical mechanisms underlying adeno-associated virus uncoating and genome release.  

5.1214           Tetraspanin CD151 Mediates Papillomavirus Type 16 Endocytosis

Scheffer, K.D., Gawlitza, A., Spoden, G.A., Zhang, X.A., Lambert, C., Berditchevski, F. and Florin, L.

1. Virol., 87(6), 3435-3446 (2013)

  Human papillomavirus type 16 (HPV16) is the primary etiologic agent for cervical cancer. The infectious entry of HPV16 into cells occurs via a so-far poorly characterized clathrin- and caveolin-independent endocytic pathway, which involves tetraspanin proteins and actin. In this study, we investigated the specific role of the tetraspanin CD151 in the early steps of HPV16 infection. We show that surface-bound HPV16 moves together with CD151 within the plane of the membrane before they cointernalize into endosomes. Depletion of endogenous CD151 did not affect binding of viral particles to cells but resulted in reduction of HPV16 endocytosis. HPV16 uptake is dependent on the C-terminal cytoplasmic region of CD151 but does not require its tyrosine-based sorting motif. Reexpression of the wild-type CD151 but not mutants affecting integrin functions restored virus internalization in CD151-depleted cells. Accordingly, short interfering RNA (siRNA) gene knockdown experiments confirmed that CD151-associated integrins (i.e., α3β1 and α6β1/4) are involved in HPV16 infection. Furthermore, palmitoylation-deficient CD151 did not support HPV16 cell entry. These data show that complex formation of CD151 with laminin-binding integrins and integration of the complex into tetraspanin-enriched microdomains are critical for HPV16 endocytosis.  

5.1215           Cholinergic signaling in the hippocampus regulates social stress resilience and anxiety- and depression-like behavior

Mineur, Y.S., Obayemi, A., Wigestrand, M.B., Fote, G.M., Calarco, C.A., Li, A.M. and Piciotto, M.R. PNAS, 110(9), 3573-3578 (2013)   Symptoms of depression can be induced in humans through blockade of acetylcholinesterase (AChE) whereas antidepressant-like effects can be produced in animal models and some clinical trials by limiting activity of acetylcholine (ACh) receptors. Thus, ACh signaling could contribute to the etiology of mood regulation. To test this hypothesis, we administered the AChE inhibitor physostigmine to mice and demonstrated an increase in anxiety- and depression-like behaviors that was reversed by administration of nicotinic or muscarinic antagonists. The behavioral effects of physostigmine were also reversed by administration of the selective serotonin reuptake inhibitor fluoxetine. Administration of fluoxetine also increased AChE activity throughout the brain, with the greatest change in the hippocampus. To determine whether cholinergic signaling in the hippocampus could contribute to the systemic effects of cholinergic drugs, we infused physostigmine or virally delivered shRNAs targeting AChE into the hippocampus. Both pharmacological and molecular genetic decreases in hippocampal AChE activity increased anxiety- and depression-like behaviors and decreased resilience to repeated stress in a social defeat paradigm. The behavioral changes due to shRNA-mediated knockdown of AChE were rescued by coinfusion of an shRNA-resistant AChE transgene into the hippocampus and reversed by systemic administration of fluoxetine. These data demonstrate that ACh signaling in the hippocampus promotes behaviors related to anxiety and depression. The sensitivity of these effects to fluoxetine suggests that shRNA-mediated knockdown of hippocampal AChE represents a model for anxiety- and depression-like phenotypes. Furthermore, abnormalities in the cholinergic system may be critical for the etiology of mood disorders and could represent an endophenotype of depression.  

5.1216           AAV-mediated Overexpression of Human α7 Integrin Leads to Histological and Functional Improvement in Dystrophic Mice

Heller, K.N., Montgomery, C.L., Janssen, P.M.L., Clark, K.R., Mendell, J.R. and Rodino-Klapac, R. Molecular Therapy, 21(3), 520-525 (2013)   Duchenne muscular dystrophy (DMD) is a severe muscle disease caused by mutations in the DMD gene, with loss of its gene product, dystrophin. Dystrophin helps link integral membrane proteins to the actin cytoskeleton and stabilizes the sarcolemma during muscle activity. We investigated an alternative therapeutic approach to dystrophin replacement by overexpressing human α7 integrin (ITGA7) using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal and cardiac muscle that links the extracellular matrix (ECM) to the actin skeleton. It is modestly upregulated in DMD muscle and has been proposed to be an important modifier of dystrophic symptoms. We delivered rAAV8.MCK.ITGA7 to the lower limb of mdx mice through isolated limb perfusion (ILP) of the femoral artery. We demonstrated ~50% of fibers in the tibialis anterior (TA) and extensor digitorum longus (EDL) overexpressing α7 integrin at the sarcolemma following AAV gene transfer. The increase in ITGA7 in skeletal muscle significantly protected against loss of force following eccentric contraction-induced injury compared with untreated (contralateral) muscles while specific force following tetanic contraction was unchanged. Reversal of additional dystrophic features included reduced Evans blue dye (EBD) uptake and increased muscle fiber diameter. Taken together, this data shows that rAAV8.MCK.ITGA7 gene transfer stabilizes the sarcolemma potentially preserving mdx muscle from further damage. This therapeutic approach demonstrates promise as a viable treatment for DMD with further implications for other forms of muscular dystrophy.  

5.1217           Trim24-repressed VL30 retrotransposons regulate gene expression by producing noncoding RNA

Herquel, B., Ouararhni, K., Martianov, I., Le Gras, S., Ye, T., Keime, C., Lerouge, T., Jost, B., Cammas, F., Losson, R and Davidson, I. Nature Structural & Mol. Biol., 20(3), 339-346 (2013)   Trim24 (Tif1α) and Trim33 (Tif1γ) interact to form a co-repressor complex that suppresses murine hepatocellular carcinoma. Here we show that Trim24 and Trim33 cooperatively repress retinoic acid receptor–dependent activity of VL30-class endogenous retroviruses (ERVs) in liver. In Trim24-knockout hepatocytes, VL30 derepression leads to accumulation of reverse-transcribed VL30 cDNA in the cytoplasm that correlates with activation of the viral-defense interferon responses mimicking the preneoplastic inflammatory state seen in human liver following exogenous viral infection. Furthermore, upon derepression, VL30 long terminal repeats (LTRs) act as promoter and enhancer elements deregulating expression of neighboring genes and generating enhancer RNAs that are required for LTR enhancer activity in hepatocytes in vivo. These data reinforce the role of the TRIM family of proteins in retroviral restriction and antiviral defense and provide an example of an ERV-derived oncogenic regulatory network  

5.1218           Viral-mediated expression of desmin mutants to create mouse models of myofibrillar myopathy

Joanne, P., Chourbagi, O., Hourde, C., Ferry, A., Butler-Browne, G., Vicart, P., Dumonceaux, J. and Agbulut, O. Skeletal Muscle, 3:4, (2013)   Background The clinical features of myofibrillar myopathies display a wide phenotypic heterogeneity. To this date, no studies have evaluated this parameter due to the absence of pertinent animal models. By studying two mutants of desmin, which induce subtle phenotypic differences in patients, we address this issue using an animal model based on the use of adeno-associated virus (AAV) vectors carrying mutated desmin cDNA. Methods After preparation of the vectors, they were injected directly into the tibialis anterior muscles of C57BL/6 mice to allow expression of wild-type (WT) or mutated (R406W or E413K) desmin. Measurements of maximal force were carried out on the muscle in situ and then the injected muscles were analyzed to determine the structural consequences of the desmin mutations on muscle structure (microscopic observations, histology and immunohistochemistry). Results Injection of AAV carrying WT desmin results in the expression of exogenous desmin in 98% of the muscle fibers without any pathological or functional perturbations. Exogenous WT and endogenous desmin are co-localized and no differences were observed compared to non-injected muscle. Expression of desmin mutants in mouse muscles induce morphological changes of muscle fibers (irregular shape and size) and the appearance of desmin accumulations around the nuclei (for R406W) or in subsarcolemmal regions of fibers (for E413K). These accumulations seem to occur and disrupt the Z-line, and a strong regeneration was observed in muscle expressing the R406W desmin, which is not the case for E413K. Moreover, both mutants of desmin studied here induce a decrease in muscle force generation capacity. Conclusions In this study we show that AAV-mediated expression of desmin mutants in mouse muscles recapitulate the aggregation features, the decrease in contractile function and the morphological changes observed in patients with myofibrillar myopathy. More importantly, our results suggest that the R406W desmin mutant induces a robust muscle regeneration, which is not the case for the E413K mutant. This difference could help to explain the phenotypic differences observed in patients. Our results highlight the heterogeneous pathogenic mechanisms between different desmin mutants and open the way for new advances in the study of myofibrillar myopathies.  

5.1219           High-Efficiency Transduction of Primary Human Hematopoietic Stem Cells and Erythroid Lineage-Restricted Expression by Optimized AAV6 Serotype Vectors In Vitro and in a Murine Xenograft Model In Vivo

Song, L., Li, X., Jayandharan, G.R., Wang, Y., Aslanidi, G.V., Ling, C., Zhong, L., Gao, G., Yoder, M.C., Ling, C., Tan, M. and Srivastava, A. PloS One, 8(3), e58757 (2013)   We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34⁺ cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: β-globin promoter (βp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the β-globin locus control region (HS2-βbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-βp, and increased up to 30-fold and up to 20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34⁺ cells in vitro. Transgene expression from the B19p6 or the HS2-βp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6 in the WT AAV6 capsid, and that from the HS2-βp in the Y705+731F AAV6 capsid, transgene expression from the B19p6 promoter in the Y705+731F AAV6 capsid was significantly higher than that from the HS2-βp, and was detectable up to 12 weeks post-transplantation in primary recipients, and up to 6 additional weeks in secondary transplanted animals. These data demonstrate the feasibility of the use of the novel Y705+731F AAV6-B19p6 vectors for high-efficiency transduction of HSCs as well as expression of the b-globin gene in erythroid progenitor cells for the potential gene therapy of human hemoglobinopathies such as β-thalassemia and sickle cell disease  

5.1220           Optimization of the Capsid of Recombinant Adeno-Associated Virus 2 (AAV2) Vectors: The Final Threshold?

Aslanidi, G.V., Rivers, A.E., Ortiz, L., Song, L., Ling, C., Govindasamy, L., Van Vliet, K., Tan, M., Agbandje-McKenna, M. PloS One, 8(3), e59142 (2013)   The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of AAV2 vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. Removal of several critical tyrosine (Y) and serine (S) residues on the AAV2 capsid has been shown to significantly increase transduction efficiency compared with the wild-type (WT) vectors. In the present study, site-directed mutagenesis of each of the 17 surface-exposed threonine (T) residues was conducted, and the transduction efficiency of four of these mutants, T455V, T491V, T550V, and T659V, was observed to increase up to 4-fold in human HEK293 cells in vitro. The most critical Y, S, and T mutations were subsequently combined, and the quadruple-mutant (Y444+500+730F+T491V) AAV2 vector was identified as the most efficient. This vector increased the transduction efficiency ~24-fold over the WT AAV2 vector, and ~2–3-fold over the previously described triple-mutant (Y444+500+730F) vector in a murine hepatocyte cell line, H2.35, in vitro. Similar results were obtained in murine hepatocytes in vivo following tail vein injection of the Y444+500+730F+T491V scAAV2 vector, and whole-body bioluminescence imaging of C57BL/6 mice. The increase in the transduction efficiency of the Y-T quadruple-mutant over that of the Y triple-mutant correlated with an improved nuclear translocation of the vectors, which exceeded 90%. These observations suggest that further optimization of the AAV2 capsid by targeting amino acid residues involved in phosphorylation may not be possible. This study has thus led to the generation of a novel Y444+500+730F+T491V quadruple-mutant AAV2 vector with potential for use in liver-directed human gene therapy.  

5.1221           Adeno-Associated Virus Serotype 8 Gene Therapy Leads to Significant Lowering of Plasma Cholesterol Levels in Humanized Mouse Models of Homozygous and Heterozygous Familial Hypercholesterolemia

Kassim, S.H., Li, H., Bell, P., Somanathan, S., Lagor, W., Jacobs, F., Billheimer, J., Wilson, J.M. and Rader, D.J. Human Gene Therapy, 24, 19-26 (2013)   Familial hypercholesterolemia (FH) is a life-threatening genetic disease caused by mutations in the gene encoding low-density lipoprotein receptor (LDLR). As a bridge to clinical trials, we generated a “humanized” mouse model lacking LDLR and apolipoprotein B (ApoB) mRNA editing catalytic polypeptide-1 (APOBEC-1) expression and expressing a human ApoB100 transgene in order to permit more authentic simulation of in vivo interactions between the clinical transgene product, human LDLR (hLDLR), and its endogenous ligand, human ApoB100. On a chow diet, the humanized LDLR-deficient mice have substantial hypercholesterolemia and a lipoprotein phenotype more closely resembling human homozygous FH (hoFH) than in previous mouse models of FH. On injection of an adeno-associated virus serotype 8 (AAV8) vector encoding the human LDLR cDNA, significant correction of hypercholesterolemia was realized at doses as low as 1.5×10¹¹ genome copies (GC)/kg. Given that some patients with heterozygous FH (heFH) cannot be adequately treated with current therapy, we then extended our studies to similarly “humanized” mice that were heterozygous for LDLR deficiency, and that have a lipoprotein phenotype resembling heterozygous FH. Injection of AAV8-hLDLR brought about significant reduction in total and LDL cholesterol at doses as low as 5×10¹¹ GC/kg. Collectively, these data demonstrate the safety and efficacy of the liver-specific AAV8-hLDLR vector in the treatment of humanized mice modeling both hoFH and heFH.  

5.1222           Regression of Schwannomas Induced by Adeno-Associated Virus-Mediated Delivery of Caspase-1

Prabhakar, S., Taherian, M., Gianni, D., Conlon, T.J., Fulci, G., Brockmann, J., Stemmer-Rachamimov, A., Sena-Esteves, M., Breakefield, X.O. and Brenner, G.J. Human Gene Therapy, 24, 152-162 (2013)   Schwannomas are tumors formed by proliferation of dedifferentiated Schwann cells. Patients with neurofibromatosis 2 (NF2) and schwannomatosis develop multiple schwannomas in peripheral and cranial nerves. Although benign, these tumors can cause extreme pain and compromise sensory/motor functions, including hearing and vision. At present, surgical resection is the main treatment modality, but it can be problematic because of tumor inaccessibility and risk of nerve damage. We have explored gene therapy for schwannomas, using a model in which immortalized human NF2 schwannoma cells expressing a fluorescent protein and luciferase are implanted in the sciatic nerve of nude mice. Direct injection of an adeno-associated virus (AAV) serotype 1 vector encoding caspase-1 (ICE) under the Schwann-cell specific promoter, P0, leads to regression of these tumors with essentially no vector-mediated neuropathology, and no changes in sensory or motor function. In a related NF2 xenograft model designed to cause measurable pain behavior, the same gene therapy leads to tumor regression and concordant resolution of tumor-associated pain. This AAV1-P0-ICE vector holds promise for clinical treatment of schwannomas by direct intratumoral injection to achieve reduction in tumor size and normalization of neuronal function.  

5.1223           Nuclear α1-Antichymotrypsin Promotes Chromatin Condensation and Inhibits Proliferation of Human Hepatocellular Carcinoma Cells

Santamaria, M., Pardo-Saganta, A., Alvarez-Asiain, L., Di Scala, M., Qian, C., Prieto, J. and Avila, M.A. Gastroenterology, 144, 818-828 (2013)   Background & Aims α1-Antichymotrypsin (α1-ACT), a member of the serpin family (SERPINA3), is an acute-phase protein secreted by hepatocytes in response to cytokines such as oncostatin M. α1-ACT is a protease inhibitor thought to limit tissue damage produced by excessive inflammation-associated proteolysis. However, α1-ACT also is detected in the nuclei of cells, where its activities are unknown. Expression of α1-ACT is down-regulated in human hepatocellular carcinoma (HCC) tissues and cells; we examined its roles in liver regeneration and HCC proliferation. Methods We measured levels of α1-ACT messenger RNA in human HCC samples and healthy liver tissue. We reduced levels of α1-ACT using targeted RNA interference in human HCC (HepG2) and mouse hepatocyte (AML12) cell lines, and overexpressed α1-ACT from lentiviral vectors in Huh7 (HCC) cells and adeno-associated viral vectors in livers of mice. We assessed proliferation, differentiation, and chromatin compaction in cultured cells, and liver regeneration and tumor formation in mice. Results Reducing levels of α1-ACT promoted proliferation of HCC cells in vitro. Oncostatin M up-regulated α1-ACT expression and nuclear translocation, which inhibited HCC cell proliferation and activated differentiation of mouse hepatocytes. We identified amino acids required for α1-ACT nuclear localization, and found that α1-ACT inhibits cell-cycle progression and anchorage-independent proliferation of HCC cells. HCC cells that overexpressed α1-ACT formed smaller tumors in mice than HCC cells that did not express the protein. α1-ACT was observed to self-associate and polymerize in the nuclei of cells; nuclear α1-ACT strongly bound chromatin to promote a condensed state that could prevent cell proliferation. Conclusions α1-ACT localizes to the nuclei of hepatic cells to control chromatin condensation and proliferation. Overexpression of α1-ACT slows the growth of HCC xenograft tumors in nude mice.    

5.1224           A simple procedure to determine the infectivity and host range of viruses infecting anaerobic and hyperthermophilic microorganisms

Gorlas, A. and Geslin, C. Extremophiles, 17(2), 349-355 (2013)   Plaque assay is the method traditionally used to isolate and purify lytic viruses, to determine the viral titer and host range. Whereas most bacterioviruses are either temperate or lytic, the majority of known archeoviruses are not lytic (i.e. they are temperate or chronic). In view of the widespread occurrence of such viruses in extreme environments, we designed an original method, called the inverted spot test, to determine the host range and infectivity of viruses isolated from anaerobic hyperthermophilic and sulfur-reducing microorganisms. Here, we used this approach to prove for the first time the infectivity of Pyrococcus abyssi virus 1 (PAV1) and to confirm the host range of Thermococcus prieurii virus 1 (TPV1), the only two viruses isolated so far from any of the described marine hyperthermophilic archaea (Euryarchaeota phylum, Thermococcales order).  

5.1225           Optical Imaging of HPV Infection in a Murine Model

Kines, R.C., Kobayashi, H., Choyke, P.L. and Bernardo, M.L. Methods in Mol. Biol., 961, 141-149 (2013)   The development of animal models of HPV infection has given investigators a new set of tools to expand basic knowledge of the early events of infection in vivo. The use of HPV pseudovirions, in which the viral genome has been replaced with a reporter pseudogenome, in combination with advanced imaging techniques has facilitated and simplified studies using these models. Herein we provide details for a murine model of cervicovaginal HPV infection in conjunction with several methods for imaging and quantitating the transduced genes, both ex vivo and in vivo.  

5.1226           Arsenic Trioxide Stabilizes Accumulations of Adeno-Associated Virus Virions at the Perinuclear Region, Increasing Transduction In Vitro and In Vivo

Mitchell, A.M., Li, C. and Samulski, R.J.

1. Virol., 87(8), 4571-4583 (2013)

  Interactions with cellular stress pathways are central to the life cycle of many latent viruses. Here, we utilize adeno-associated virus (AAV) as a model to study these interactions, as previous studies have demonstrated that cellular stressors frequently increase transduction of recombinant AAV (rAAV) vectors and may even substitute for helper virus functions. Since several chemotherapeutic drugs are known to increase rAAV transduction, we investigated the effect of arsenic trioxide (As₂O₃), an FDA-approved chemotherapeutic agent with known effects on several other virus life cycles, on the transduction of rAAV. In vitro, As₂O₃ caused a dose-dependent increase in rAAV2 transduction over a broad range of cell lines from various cell types and species (e.g., HEK-293, HeLa, HFF hTERT, C-12, and Cos-1). Mechanistically, As₂O₃ treatment acted to prevent loss of virions from the perinuclear region, which correlated with increased cellular vector genome retention, and was distinguishable from proteasome inhibition. To extend our investigation of the cellular mechanism, we inhibited reactive oxygen species formation and determined that the As₂O₃-mediated increase in rAAV2 transduction was dependent upon production of reactive oxygen species. To further validate our in vitro data, we tested the effect of As₂O₃ on rAAV transduction in vivo and determined that treatment initiated transgene expression as early as 2 days posttransduction and increased reporter expression by up to 10-fold. Moreover, the transduction of several other serotypes of rAAV was also enhanced in vivo, suggesting that As₂O₃ affects a pathway used by several AAV serotypes. In summary, our data support a model wherein As₂O₃ increases rAAV transduction both in vitro and in vivo and maintains perinuclear accumulations of capsids, facilitating productive nuclear trafficking.  

5.1227           A Doubly Fluorescent HIV-1 Reporter Shows that the Majority of Integrated HIV-1 Is Latent Shortly after Infection

Dahabieh, M.S., Ooms, M., Simon, V. and Sadowski, I.

1. Virol., 87(8), 4716-4727 (2013)

  HIV-1 latency poses a major barrier to viral eradication. Canonically, latency is thought to arise from progressive epigenetic silencing of active infections. However, little is known about when and how long terminal repeat (LTR)-silent infections arise since the majority of the current latency models cannot differentiate between initial (LTR-silent) and secondary (progressive silencing) latency. In this study, we constructed and characterized a novel, double-labeled HIV-1 vector (Red-Green-HIV-1 [RGH]) that allows for detection of infected cells independently of LTR activity. Infection of Jurkat T cells and other cell lines with RGH suggests that the majority of integrated proviruses were LTR-silent early postinfection. Furthermore, the LTR-silent infections were transcriptionally competent, as the proviruses could be reactivated by a variety of T cell signaling agonists. Moreover, we used the double-labeled vector system to compare LTRs from seven different subtypes with respect to LTR silencing and reactivation. These experiments indicated that subtype D and F LTRs were more sensitive to silencing, whereas the subtype AE LTR was largely insensitive. Lastly, infection of activated human primary CD4⁺ T cells yielded LTR-silent as well as productive infections. Taken together, our data, generated using the newly developed RGH vector as a sensitive tool to analyze HIV-1 latency on a single-cell level, show that the majority of HIV-1 infections are latent early postinfection.  

5.1228           AAV9.I-1c Delivered via Direct Coronary Infusion in a Porcine Model of Heart Failure Improves Contractility and Mitigates Adverse Remodeling

Fish, K.M., Ladage, D., Kawase, Y., Karakikes, I., Jeong, D., Ly, H., Ishikawa, K., Hadri, L., Tilemann, L., Muller-Ehmsen, J., Samulski, R.J., Kranias, E.G. and Hajjar, R.J. Circ. Heart Fail., 6, 310-317 (2013)   Background—Heart failure is characterized by impaired function and disturbed Ca²⁺ homeostasis. Transgenic increases in inhibitor-1 activity have been shown to improve Ca² cycling and preserve cardiac performance in the failing heart. The aim of this study was to evaluate the effect of activating the inhibitor (I-1c) of protein phosphatase 1 (I-1) through gene transfer on cardiac function in a porcine model of heart failure induced by myocardial infarction. Methods and Results—Myocardial infarction was created by a percutaneous, permanent left anterior descending artery occlusion in Yorkshire Landrace swine (n=16). One month after myocardial infarction, pigs underwent intracoronary delivery of either recombinant adeno-associated virus type 9 carrying I-1c (n=8) or saline (n=6) as control. One month after myocardial infarction was created, animals exhibited severe heart failure demonstrated by decreased ejection fraction (46.4±7.0% versus sham 69.7±8.5%) and impaired (dP/dt)_max and (dP/dt)_min. Intracoronary injection of AAV9.I-1c prevented further deterioration of cardiac function and led to a decrease in scar size. Conclusions—In this preclinical model of heart failure, overexpression of I-1c by intracoronary in vivo gene transfer preserved cardiac function and reduced the scar size.  

5.1229           A role for Sv2c in basal ganglia functions

Dardou, D., Monlezun, S., Foerch, P., Courade, J.P., Cuvelier, L., De Ryck, M and Schiffmann, S.N. Brain Res., 1507, 61-73 (2013)   SV2C is an isoform of the synaptic vesicle 2 protein family that exhibits a particular pattern of brain expression with enriched expression in several basal ganglia nuclei. In the present study, we have investigated SV2C implication in both normal and pathological basal ganglia functioning with a peculiar attention to dopamine neuron containing regions. In SV2C−/− mice, the expression of tyrosine hydroxylase mRNA in midbrain dopaminergic neurons was largely and significantly increased and enkephalin mRNA expression was significantly decreased in the caudate-putamen and accumbens nucleus. The expression of SV2C was studied in two models of dopaminergic denervation (6-OHDA- and MPTP-induced lesions). In dopamine-depleted animals, SV2C mRNA expression was significant increased in the striatum. In order to further understand the role of SV2C, we performed behavioral experiments on SV2C−/− mice and on knock-down mice receiving an injection of adeno-associated virus expressing SV2C miRNA specifically in the ventral midbrain. These modifications of SV2C expression had little or no impact on behavior in open field and elevated plus maze. However, even if complete loss of SV2C had no impact on conditioned place preference induced by cocaine, the specific knock-down of SV2C expression in the dopaminergic neurons completely abolished the development of a CPP while the reaction to an acute drug injection remains similar in these mice compared to control mice. These results showed that SV2C, a poorly functionally characterized protein is strongly involved in normal operation of the basal ganglia network and could be also involved in system adaptation in basal ganglia pathological conditions.  

5.1230           Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates

Gray, S.J., Kalburgi, S.N., McCown, T.J. and Samulski, R.J. Gene Therapy, 20, 450-459 (2013)   Injection of adeno-associated virus (AAV) into the cerebrospinal fluid (CSF) offers a means to achieve widespread transgene delivery to the central nervous system, where the doses can be readily translated from small to large animals. In contrast to studies with other serotypes (AAV2, AAV4 and AAV5) in rodents, we report that a naturally occurring capsid (AAV9) and rationally engineered capsid (AAV2.5) are able to achieve broad transduction throughout the brain and spinal cord parenchyma following a single injection into the CSF (via cisterna magna or lumbar cistern) in non-human primates (NHP). Using either vector at a dose of ~2 × 10¹² vector genome (vg) per 3–6 kg animal, approximately 2% of the entire brain and spinal cord was transduced, covering all regions of the central nervous system (CNS). AAV9 in particular displayed efficient transduction of spinal cord motor neurons. The peripheral organ biodistribution was highly reduced compared with intravascular delivery, and the presence of circulating anti-AAV-neutralizing antibodies up to a 1:128 titer had no inhibitory effect on CNS gene transfer. Intra-CSF delivery effectively translates from rodents to NHPs, which provides encouragement for the use of this approach in humans to treat motor neuron and lysosomal storage diseases.  

5.1231           Activation of the human immune system via toll-like receptors by the oncolytic parvovirus H-1

Sieben, M., Schäfer, P., Dinsart, C., Galle, P.R. and Moehler, M. Int. J. Cancer, 132, 2548-2556 (2013)   This study aimed to investigate the function of toll-like receptors (TLRs) during oncolytic parvovirus H-1 (H-1PV)-induced human immune responses. First, the role of TLRs in the activation of the NFκB transcription factor was characterized; second, the immunologic effects of H-1PV-induced tumor cell lysates (TCL) on human antitumor immune responses were evaluated. A human ex vivo model was used to study immune responses with dendritic cells (DCs). Human embryonic kidney cells (HEK293) transfected to stably express TLRs were used as potential human DC equivalents to further investigate the role of specific TLRs during immune activation. TLR3 and TLR9 were activated by H-1PV infection, which correlated with NFκB translocation to the nucleus and a reduced cytoplasmic IκB expression. Using a TLR-signaling reporter plasmid (pNiFty-Luc), NFκB activity was increased following H-1PV infection. In addition, human DCs coincubated with H-1PV-induced TCL demonstrated increased TLR3 and TLR9 expression. These data suggest that H-1PV-induced TCL stimulate human DCs at least in part through TLR-dependent signaling pathways. Thus, DC maturation occurred through exposure to H-1PV-induced TCL through TLR-signaling leading to NFκB-dependent activation of the adaptive immune system as indicated by the increased expression of CD86, TLR3 and TLR9. Furthermore, the transcription of various cytokines indicates the activation of immune response, therefore the production of the proinflammatory cytokine TNF- was determined. Here, H-1PV-induced TCL significantly enhanced the TNF- level by DCs after coculture. H-1PV oncolytic virotherapy enhances immune priming by different effects on DCs and generates antitumor immunity. These findings potentially offer a new approach to tumor therapy.  

5.1232           Very-Low-Density Lipoprotein (VLDL)-Producing and Hepatitis C Virus-Replicating HepG2 Cells Secrete No More Lipoviroparticles than VLDL-Deficient Huh7.5 Cells

Jammart, B., Michelet, M., Pecheur, E-I., parent, R., bartosch, b., Zoulim, F and Durantel, D.

1. Virol., 87(9), 5065-5080 (2013)

  In the plasma samples of hepatitis C virus (HCV)-infected patients, lipoviroparticles (LVPs), defined as (very-) low-density viral particles immunoprecipitated with anti-β-lipoproteins antibodies are observed. This HCV-lipoprotein association has major implications with respect to our understanding of HCV assembly, secretion, and entry. However, cell culture-grown HCV (HCVcc) virions produced in Huh7 cells, which are deficient for very-low-density lipoprotein (VLDL) secretion, are only associated with and dependent on apolipoprotein E (apoE), not apolipoprotein B (apoB), for assembly and infectivity. In contrast to Huh7, HepG2 cells can be stimulated to produce VLDL by both oleic acid treatment and inhibition of the MEK/extracellular signal-regulated kinase (ERK) pathway but are not permissive for persistent HCV replication. Here, we developed a new HCV cell culture model to study the interaction between HCV and lipoproteins, based on engineered HepG2 cells stably replicating a blasticidin-tagged HCV JFH1 strain (JB). Control Huh7.5-JB as well as HepG2-JB cell lines persistently replicated viral RNA and expressed viral proteins with a subcellular colocalization of double-stranded RNA (dsRNA), core, gpE2, and NS5A compatible with virion assembly. The intracellular RNA replication level was increased in HepG2-JB cells upon dimethyl sulfoxide (DMSO) treatment, MEK/ERK inhibition, and NS5A overexpression to a level similar to that observed in Huh7.5-JB cells. Both cell culture systems produced infectious virions, which were surprisingly biophysically and biochemically similar. They floated at similar densities on gradients, contained mainly apoE but not apoB, and were not neutralized by anti-apoB antibodies. This suggests that there is no correlation between the ability of cells to simultaneously replicate HCV as well as secrete VLDL and their capacity to produce LVPs.  

5.1233           MicroRNA-27a Regulates Lipid Metabolism and Inhibits Hepatitis C Virus Replication in Human Hepatoma Cells

Shirasaki, t., Honda, M., Shimakami, T., Horij, R., Yamashita, T., Sakai, Y., Sakai, A., Okada, H., Watanabe, R., Murakami, S., Yi, M., Lemon, S.M. and Kaneko, S.

1. Virol., 87(9), 5270-5286 (2013)

  The replication and infectivity of the lipotropic hepatitis C virus (HCV) are regulated by cellular lipid status. Among differentially expressed microRNAs (miRNAs), we found that miR-27a was preferentially expressed in HCV-infected liver over hepatitis B virus (HBV)-infected liver. Gene expression profiling of Huh-7.5 cells showed that miR-27a regulates lipid metabolism by targeting the lipid synthetic transcription factor RXRα and the lipid transporter ATP-binding cassette subfamily A member 1. In addition, miR-27a repressed the expression of many lipid metabolism-related genes, including FASN, SREBP1, SREBP2, PPARα, and PPARγ, as well as ApoA1, ApoB100, and ApoE3, which are essential for the production of infectious viral particles. miR-27a repression increased the cellular lipid content, decreased the buoyant density of HCV particles from 1.13 to 1.08 g/cm³, and increased viral replication and infectivity. miR-27a overexpression substantially decreased viral infectivity. Furthermore, miR-27a enhanced in vitro interferon (IFN) signaling, and patients who expressed high levels of miR-27a in the liver showed a more favorable response to pegylated IFN and ribavirin combination therapy. Interestingly, the expression of miR-27a was upregulated by HCV infection and lipid overload through the adipocyte differentiation transcription factor C/EBPα. In turn, upregulated miR-27a repressed HCV infection and lipid storage in cells. Thus, this negative feedback mechanism might contribute to the maintenance of a low viral load and would be beneficial to the virus by allowing it to escape host immune surveillance and establish a persistent chronic HCV infection.  

5.1234           Assessment of Tropism and Effectiveness of New Primate-Derived Hybrid Recombinant AAV Serotypes in the Mouse and Primate Retina

Issa, P.C., De Silva, S.R., Lipinski, D.M., Singh, M.S., Mourravlev, A., You, Q., Barnard, A.R., Hankins, M.W., During, M.J. and MacLaren, R.E. PloS One, 8(4), e60361 (2013)   Adeno-associated viral vectors (AAV) have been shown to be safe in the treatment of retinal degenerations in clinical trials. Thus, improving the efficiency of viral gene delivery has become increasingly important to increase the success of clinical trials. In this study, structural domains of different rAAV serotypes isolated from primate brain were combined to create novel hybrid recombinant AAV serotypes, rAAV2/rec2 and rAAV2/rec3. The efficacy of these novel serotypes were assessed in wild type mice and in two models of retinal degeneration (the Abca4^−/− mouse which is a model for Stargardt disease and in the Pde6b^rd1/rd1 mouse) in vivo, in primate tissue ex-vivo, and in the human-derived SH-SY5Y cell line, using an identical AAV2 expression cassette. We show that these novel hybrid serotypes can transduce retinal tissue in mice and primates efficiently, although no more than AAV2/2 and rAAV2/5 serotypes. Transduction efficiency appeared lower in the Abca4^−/− mouse compared to wild type with all vectors tested, suggesting an effect of specific retinal diseases on the efficiency of gene delivery. Shuffling of AAV capsid domains may have clinical applications for patients who develop T-cell immune responses following AAV gene therapy, as specific peptide antigen sequences could be substituted using this technique prior to vector re-treatments.  

5.1235           Development of a Pseudotyped-Lentiviral-Vector-Based Neutralization Assay for Chikungunya Virus Infection

Kishishita, N., Takeda, N., Anuegoonpipat, A. and Anantapreecha, S.

1. Clin. Microbiol., 51(5), 1389-1395 (2013)

  Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes chikungunya fever in Africa, South Asia, and Southeast Asia. Because the mosquito vector Aedes albopictus is present in habitats across Europe, North America, and East Asia, CHIKV has become a serious worldwide public health concern. Infection with CHIKV typically causes fever, rash, myalgia, and arthralgia. One of the important questions yet to be answered is how the host immune system is involved in the development of this disease. In this study, we prepared a CHIKV-pseudotyped lentiviral vector for use in a safe and convenient neutralization (NT) assay and analyzed its efficacy. The CHIKV-pseudotyped lentiviral vector was prepared by cotransfection with plasmids encoding the CHIKV glycoproteins E3, E2, 6k, and E1, packaging elements, and a luciferase reporter. This alternative to native CHIKV can be safely handled in a biosafety level 2 facility. The NT assay was optimized using sera from CHIKV-immunized mice and then applied to human patient sera. The majority of the serum samples from patients with chikungunya in Thailand showed robust neutralization activities, with titers that were tightly correlated with those determined by a conventional NT assay. Moreover, there was a strong correlation with the CHIKV antibody titers as determined by enzyme-linked immunosorbent assay. Thus, the CHIKV-pseudotyped-lentiviral-vector-based NT assay system is a powerful tool for examining the neutralization activity of patient sera, which will lead to a better understanding of the immune responses involved in CHIKV infection.  

5.1236           Intraganglionic AAV6 Results in Efficient and Long-Term Gene Transfer to Peripheral Sensory Nervous System in Adult Rats

Yu, H., Fischer, G., Ferhatovic, L., Fan, F., Light, A.R., Weihrauch, D., Sapunar, D., Nakai, H., Park, F. and Hogan, Q.H. PloS One, 8(4), e61266 (2013)   We previously demonstrated safe and reliable gene transfer to the dorsal root ganglion (DRG) using a direct microinjection procedure to deliver recombinant adeno-associated virus (AAV) vector. In this study, we proceed to compare the in vivo transduction patterns of self-complementary (sc) AAV6 and AAV8 in the peripheral sensory pathway. A single, direct microinjection of either AAV6 or AAV8 expressing EGFP, at the adjusted titer of 2×10⁹ viral particle per DRG, into the lumbar (L) 4 and L5 DRGs of adult rats resulted in efficient EGFP expression (48±20% for AAV6 and 25±4% for AAV8, mean ± SD) selectively in sensory neurons and their axonal projections 3 weeks after injection, which remained stable for up to 3 months. AAV6 efficiently transfers EGFP to all neuronal size groups without differential neurotropism, while AAV8 predominantly targets large-sized neurons. Neurons transduced with AAV6 penetrate into the spinal dorsal horn (DH) and terminate predominantly in superficial DH laminae, as well as in the dorsal columns and deeper laminae III-V. Only few AAV8-transduced afferents were evident in the superficial laminae, and spinal EGFP was mostly present in the deeper dorsal horn (lamina III-V) and dorsal columns, with substantial projections to the ventral horn. AAV6-mediated EGFP-positive nerve fibers were widely observed in the medial plantar skin of ipsilateral hindpaws. No apparent inflammation, tissue damage, or major pain behaviors were observed for either AAV serotype. Taken together, both AAV6 and AAV8 are efficient and safe vectors for transgene delivery to primary sensory neurons, but they exhibit distinct functional features. Intraganglionic delivery of AAV6 is more uniform and efficient compared to AAV8 in gene transfer to peripheral sensory neurons and their axonal processes.  

5.1237           Phylogenetic Considerations in Designing a Broadly Protective Multimeric L2 Vaccine

Jagu, S., Kwak, K., Schiller, J.T., Lowy, D.R., Kleanthous, H., Kalnin, K., Wang, C., Wang, H-K., Chow, L.T., Huh, W.K., Jaganathan, K.S., Chivukula, S.V. and Roden, R.B.S.

1. Virol., 87(11), 6127-6136 (2013)

  While the oncogenic human papillomavirus (HPV) types with the greatest medical impact are clustered within the α9 and α7 species, a significant fraction of cervical cancers are caused by α5, α6, and α11 viruses. Benign genital warts are caused principally by the α10 viruses HPV6 and HPV11. In an effort to achieve broad protection against both cervical cancer- and genital wart-associated types, we produced at high levels in bacteria a multimeric protein (α11-88x8) fusing eight polypeptides corresponding to a protective domain comprising L2 residues ∼11 to 88 derived from HPV6 (α10), HPV16 (α9), HPV18 (α7), HPV31 (α9), HPV39 (α7), HPV51 (α5), HPV56 (α6), and HPV73 (α11) and a truncated derivative with the last three units deleted (α11-88x5). Mice were immunized three times with α11-88x8 or α11-88x5 adjuvanted with alum or the licensed HPV vaccines and challenged intravaginally with HPV6, HPV16, HPV26, HPV31, HPV33, HPV35, HPV45, HPV51, HPV56, HPV58, or HPV59 pseudovirions. The α11-88x5 and α11-88x8 vaccines induced similarly robust protection against each HPV type tested and indistinguishable HPV16-neutralizing antibody titers. Passive transfer of α11-88x8 antisera was protective. Further, rabbit antisera to α11-88x8 and α11-88x5 similarly neutralized native HPV18 virions. These findings suggest that immunologic competition between units is not a significant issue and that it is not necessary to include a unit of L2 derived from each species to achieve broader protection against diverse medically significant HPV types than is achieved with the licensed HPV vaccines.  

5.1238           Ser129D mutant alpha-synuclein induces earlier motor dysfunction while S129A results in distinctive pathology in a rat model of Parkinson's disease

Febbraro, F., Sahin, G., Farran, A., Soares, S., Jensen, P.H., Kirik, D. and Romero-Ramos, M. Neurobiology of Disease, 56, 47-58 (2013)   Alpha-synuclein phosphorylated at serine 129 (S129) is highly elevated in Parkinson's disease patients where it mainly accumulates in the Lewy bodies. Several groups have studied the role of phosphorylation at the S129 in α-synuclein in a rat model for Parkinson's disease using recombinant adeno-associated viral (rAAV) vectors. The results obtained are inconsistent and accordingly the role of S129 phosphorylation in α-synuclein toxicity remains unclear. This prompted us to re-examine the neuropathological and behavioral effects of the S129 modified α-synuclein species in vivo. For this purpose, we used two mutated forms of human α-synuclein in which the S129 was replaced either with an alanine (S129A), to block phosphorylation, or with an aspartate (S129D), to mimic phosphorylation, and compared them with the wild type α-synuclein. This approach was similar in design to previous studies, however our investigation of dopaminergic degeneration also included performing a detailed study of the α-synuclein induced pathology in the striatum and the analysis of motor deficits. Our results showed that overexpressing S129D or wild type α-synuclein resulted in an accelerated dopaminergic fiber loss as compared with S129A α-synuclein. Furthermore, the motor deficit seen in the group treated with the mutant S129D α-synuclein appeared earlier than the other two forms of α-synuclein. Conversely, S129A α-synuclein showed significantly larger pathological α-synuclein-positive inclusions, and slower dopaminergic fiber loss, when compared to the other two forms of α-synuclein, suggesting a neuroprotective effect of the mutation. When examined at long-term, all three α-synuclein forms resulted in pathological accumulations of α-synuclein in striatal fibers and dopaminergic cell death in the substantia nigra. Our data show that changes in the S129 residue of α-synuclein influence the rate of pathology and neurodegeneration, with an overall deleterious effect of exchanging S129 to a residue mimicking its phosphorylated state.  

5.1239           Chronic intranasal deferoxamine ameliorates motor defects and pathology in the α-synuclein rAAV Parkinson's model

Febbraro, F., Andersen, K.J., Sanchez-Guajardo, V., Tentillier, N. and Romero-Ramos, M. Exp, Neurol., 247, 45-58 (2013)   Parkinson's disease is characterized by neuronal death in the substantia nigra and the presence of intracellular inclusions of α-synuclein in the Lewy bodies. Several lines of data support a role for iron in Parkinson's disease: iron is present in Lewy bodies, iron accumulates in the dopaminergic neurons in the substantia nigra, and Parkinson's disease is correlated with polymorphisms of several genes implicated in iron metabolism. Furthermore, iron can compromise the solubility of α-synuclein through direct interaction and can induce neurotoxicity in vitro. Here, we investigate the possible neuroprotective effect of the iron chelator deferoxamine in vivo to elucidate whether iron chelation can provide meaningful therapy for Parkinson's disease. Hence, we used a Parkinson's disease animal model based on unilateral injection of a recombinant adeno-associated viral vector encoding α-synuclein in the rat midbrain. Rats were treated with a novel deferoxamine delivery approach: 6 mg of the compound was administered intranasally three times a week for 3 or 7 weeks. The behavior of the animals and histopathological changes in the brain were analyzed. Our data show that although intranasal administration of deferoxamine in rats did not protect them from dopaminergic cell death, it did decrease the number of the pathological α-synuclein formations at the terminal level. In addition, this treatment resulted in changes in the immune response and an overall partial improvement in motor behavior. Taken together, our data show that in vivo iron chelation can modulate α-synuclein-induced pathology in the central nervous system. Our data suggest that chronic administration of intranasal deferoxamine may be a valid approach to limiting the mishandling of α-synuclein in the central nervous system observed in Parkinson's disease and slowing disease progression.  

5.1240           Gene delivery of Homer1c rescues spatial learning in a rodent model of cognitive aging

Gerstein, H., Lindstrom, M.J. and Burger, C. Neurobiology of Aging, 34, 1963-1970 (2013)   Homer1c has been shown to play a role in learning and memory. Overexpression of Homer1c in the hippocampus can improve memory in normal rats and can also rescue spatial learning deficits in Homer1 knockout mice. In a previous study, we found that Homer1c mRNA is upregulated after a spatial learning paradigm in aged rats that successfully learn the task, when compared to aged rats that are learning-impaired (AI). This study was designed to validate the role of Homer1c in successful cognitive aging. In this article, we report that gene delivery of Homer1c into the hippocampus of aged learning-impaired rats significantly improves individual performance on an object location memory task. The learning ability of these rats on the Morris Water Maze was also superior to that of AI control rats. In summary, using 2 independent spatial memory tasks, we demonstrate that Homer1c is sufficient to improve the spatial learning deficits in a rodent model of cognitive aging. These results point to Homer1c as a potential therapeutic target for improving age-related cognitive impairment.  

5.1241           Hepatic production of transthyretin L12P leads to intracellular lysosomal aggregates in a new somatic transgenic mouse model

Batista, A.R., Sena-Esteves, M. and Saraiva, M.J. Biochim. Biophys. Acta, 1832, 1183-1193 (2013)   Transthyretin (TTR) is a plasma and cerebrospinal fluid (CSF)-circulating homotetrameric protein. More than 100 point mutations have been identified in the TTR gene and several are related with amyloid diseases. Here we focused our attention in the TTR L12P variant associated with severe peripheral neuropathy and leptomeningeal amyloidosis. By using different cell lines derived from tissues specialized on TTR synthesis, such as the hepatocyte and the choroid plexus expressing WT, V30M, or L12P TTR variants we analyzed secretion, intracellular aggregation and degradation patterns. Also, we used liver-specific AAV gene transfer to assess expression of the L12P variant in vivo. We found the following: (i) decreased secretion with intracellular aggregation of TTR L12P in hepatoma cells relative to WT and V30M variant; this differential property of TTR L12P variant was also observed in mice injected with L12P AAV vector; (ii) differential N-glycosylation pattern of L12P variant in hepatoma cell lysates, conditioned media and mouse sera, which might represent an escape mechanism from ERAD degradation; (iii) intracellular L12P TTR aggregates mainly localized to lysosomes in cultured cells and liver; and (iv) none of the above findings were present in choroid plexus derived cells, suggesting particular secretion/quality control mechanisms that might contribute to leptomeningeal amyloidosis associated with the L12P variant. These observations open new avenues for the treatment of TTR associated leptomeningeal amyloidosis.  

5.1242           Recombinant Adeno-Associated Virus Serotype 6 Efficiently Transduces Primary Human Melanocytes

Sheppard, H.M., Ussher, J.E., Verdon, D., Chen, J., Taylor, J.A. and Dunbar, P.R. PloS One, 8(4), e62753 (2013)   The study of melanocyte biology is important to understand their role in health and disease. However, current methods of gene transfer into melanocytes are limited by safety or efficacy. Recombinant adeno-associated virus (rAAV) has been extensively investigated as a gene therapy vector, is safe and is associated with persistent transgene expression without genome integration. There are twelve serotypes and many capsid variants of rAAV. However, a comparative study to determine which rAAV is most efficient at transducing primary human melanocytes has not been conducted. We therefore sought to determine the optimum rAAV variant for use in the in vitro transduction of primary human melanocytes, which could also be informative to future in vivo studies. We have screened eight variants of rAAV for their ability to transduce primary human melanocytes and identified rAAV6 as the optimal serotype, transducing 7–78% of cells. No increase in transduction was seen with rAAV6 tyrosine capsid mutants. The number of cells expressing the transgene peaked at 6–12 days post-infection, and transduced cells were still detectable at day 28. Therefore rAAV6 should be considered as a non-integrating vector for the transduction of primary human melanocytes.  

5.1243           Pre-immunization with an Intramuscular Injection of AAV9-Human Erythropoietin Vectors Reduces the Vector-Mediated Transduction following Re-Administration in Rat Brain

Yang, C., Yang, W-H., Chen, S-S., Ma, B-F., Li, B., Lu, T., Qu, T-Y., Klein, R.L., Zhao, L-R. and Duan, W-M. PloS One, 8(5), e63876 (2013)   We have recently demonstrated that adeno-associated virus serotype 9 (AAV9)-mediated human erythropoietin (hEPO) gene delivery into the brain protects dopaminergic (DA) neurons in the substantia nigra in a rat model of Parkinson's disease. In the present study, we examined whether pre-exposure to AAV9-hEPO vectors with an intramuscular or intrastriatal injection would reduce AAV9-mediated hEPO transduction in rat brain. We first characterized transgene expression and immune responses against AAV9-hEPO vectors in rat striatum at 4 days, 3 weeks and 6 months, and with doses ranging from 10¹¹ to 10¹³ viral genomes. To sensitize immune system, rats received an injection of AAV9-hEPO into either the muscle or the left striatum, and then sequentially an injection of AAV9-hEPO into the right striatum 3 weeks later. We observed that transgene expression exhibited in a time course and dose dependent manner, and inflammatory and immune responses displayed in a time course manner. Intramuscular, but not intrastriatal injections of AAV9-hEPO resulted in reduced levels of hEPO transduction and increased levels of the major histocompatibility complex (MHC) class I and class II antigen expression in the striatum following AAV9-hEPO re-administration. There were infiltration of the cluster of differentiation 4 (CD4)-and CD8-lymphacytes, and accumulation of activated microglial cells and astrocytes in the virally injected striatum. In addition, the sera from the rats with intramuscular injections of AAV9-hEPO contained greater levels of antibodies against both AAV9 capsid protein and hEPO protein than the other treatment groups. hEPO gene expression was negatively correlated with the levels of circulating antibodies against AAV9 capsid protein. Intramuscular and intrastriatal re-administration of AAV9-hEPO led to increased numbers of red blood cells in peripheral blood. Our results suggest that pre-immunization with an intramuscular injection can lead to the reduction of transgene expression in the striatal re-administration.  

5.1244           Neuroglobin overexpression inhibits oxygen–glucose deprivation-induced mitochondrial permeability transition pore opening in primary cultured mouse cortical neurons

Yu, Z., Liu, N., Li, Y., Xu, J. and Wang, X. Neurobiology of Disease, 56, 95-103 (2013)   Neuroglobin (Ngb) is an endogenous neuroprotective molecule against hypoxic/ischemic brain injury, but the underlying mechanisms remain largely undefined. Our recent study revealed that Ngb can bind to voltage-dependent anion channel (VDAC), a regulator of mitochondria permeability transition (MPT). In this study we examined the role of Ngb in MPT pore (mPTP) opening following oxygen–glucose deprivation (OGD) in primary cultured mouse cortical neurons. Co-immunoprecipitation (Co-IP) and immunocytochemistry showed that the binding between Ngb and VDAC was increased after OGD compared to normoxia, indicating the OGD-enhanced Ngb–VDAC interaction. Ngb overexpression protected primary mouse cortical neurons from OGD-induced neuronal death, to an extent comparable to mPTP opening inhibitor, cyclosporine A (CsA) pretreatment. We further measured the role of Ngb in OGD-induced mPTP opening using Ngb overexpression and knockdown approaches in primary cultured neurons, and recombinant Ngb exposure to isolated mitochondria. Same as CsA pretreatment, Ngb overexpression significantly reduced OGD-induced mPTP opening markers including mitochondria swelling, mitochondrial NAD⁺ release, and cytochrome c (Cyt c) release in primary cultured neurons. Recombinant Ngb incubation significantly reduced OGD-induced NAD⁺ release and Cyt c release from isolated mitochondria. In contrast, Ngb knockdown significantly increased OGD-induced neuron death, and increased OGD-induced mitochondrial NAD⁺ release and Cyt c release as well, and these outcomes could be rescued by CsA pretreatment. In summary, our results demonstrated that Ngb overexpression can inhibit OGD-induced mPTP opening in primary cultured mouse cortical neurons, which may be one of the molecular mechanisms of Ngb's neuroprotection.  

5.1245           Stochastic Model of Tsc1 Lesions in Mouse Brain

Prabhakar, S., Goto, J., Zuang, X., Sena-Esteves, M., Bronson, R., Brockkmann, J., Gianni, D., Wojtkiewicz, G.R., Chen, J.W., Stemmer-Rachamimov, A., Kwiatkowski, D.J. and Brekefield, X.O. PloS One, 8(5), e64224 (2013)   Tuberous sclerosis complex (TSC) is an autosomal dominant disorder due to mutations in either TSC1 or TSC2 that affects many organs with hamartomas and tumors. TSC-associated brain lesions include subependymal nodules, subependymal giant cell astrocytomas and tubers. Neurologic manifestations in TSC comprise a high frequency of mental retardation and developmental disorders including autism, as well as epilepsy. Here, we describe a new mouse model of TSC brain lesions in which complete loss of Tsc1 is achieved in multiple brain cell types in a stochastic pattern. Injection of an adeno-associated virus vector encoding Cre recombinase into the cerebral ventricles of mice homozygous for a Tsc1 conditional allele on the day of birth led to reduced survival, and pathologic findings of enlarged neurons, cortical heterotopias, subependymal nodules, and hydrocephalus. The severity of clinical and pathologic findings as well as survival was shown to be dependent upon the dose and serotype of Cre virus injected. Although several other models of TSC brain disease exist, this model is unique in that the pathology reflects a variety of TSC-associated lesions involving different numbers and types of cells. This model provides a valuable and unique addition for therapeutic assessment.  

5.1246           A pathogenic picornavirus acquires an envelope by hijacking cellular membranes

Feng, Z., Hensley, L., McKnight, K.L., Hu, F., Madden, V., Ping, L., Jeong, H., Walker, C., Lanford, R.E. and Lemon, S.M. Nature, 469, 367-372 (2013)   Animal viruses are broadly categorized structurally by the presence or absence of an envelope composed of a lipid-bilayer membrane¹, attributes that profoundly affect stability, transmission and immune recognition. Among those lacking an envelope, the Picornaviridae are a large and diverse family of positive-strand RNA viruses that includes hepatitis A virus (HAV), an ancient human pathogen that remains a common cause of enterically transmitted hepatitis^{2, 3, 4}. HAV infects in a stealth-like manner and replicates efficiently in the liver⁵. Virus-specific antibodies appear only after 3–4 weeks of infection, and typically herald its resolution^{3, 4}. Although unexplained mechanistically, both anti-HAV antibody and inactivated whole-virus vaccines prevent disease when administered as late as 2 weeks after exposure⁶, when virus replication is well established in the liver⁵. Here we show that HAV released from cells is cloaked in host-derived membranes, thereby protecting the virion from antibody-mediated neutralization. These enveloped viruses (‘eHAV’) resemble exosomes⁷, small vesicles that are increasingly recognized to be important in intercellular communications. They are fully infectious, sensitive to extraction with chloroform, and circulate in the blood of infected humans. Their biogenesis is dependent on host proteins associated with endosomal-sorting complexes required for transport (ESCRT)⁸, namely VPS4B and ALIX. Whereas the hijacking of membranes by HAV facilitates escape from neutralizing antibodies and probably promotes virus spread within the liver, anti-capsid antibodies restrict replication after infection with eHAV, suggesting a possible explanation for prophylaxis after exposure. Membrane hijacking by HAV blurs the classic distinction between ‘enveloped’ and ‘non-enveloped’ viruses and has broad implications for mechanisms of viral egress from infected cells as well as host immune responses.  

5.1247           Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualising and manipulating neuronal circuits in vivo

Kim, J-Y., Ash, R.T., Ceballos-Diaz, C., Levites, Y., Golde, T.E., Smirnakis, S.M. and Jankowsky, J.L. Eur. J. Neurosci., 37, 1203-1220 (2013)   The neonatal intraventricular injection of adeno-associated virus has been shown to transduce neurons widely throughout the brain, but its full potential for experimental neuroscience has not been adequately explored. We report a detailed analysis of the method's versatility with an emphasis on experimental applications where tools for genetic manipulation are currently lacking. Viral injection into the neonatal mouse brain is fast, easy, and accesses regions of the brain including the cerebellum and brainstem that have been difficult to target with other techniques such as electroporation. We show that viral transduction produces an inherently mosaic expression pattern that can be exploited by varying the titer to transduce isolated neurons or densely-packed populations. We demonstrate that the expression of virally-encoded proteins is active much sooner than previously believed, allowing genetic perturbation during critical periods of neuronal plasticity, but is also long-lasting and stable, allowing chronic studies of aging. We harness these features to visualise and manipulate neurons in the hindbrain that have been recalcitrant to approaches commonly applied in the cortex. We show that viral labeling aids the analysis of postnatal dendritic maturation in cerebellar Purkinje neurons by allowing individual cells to be readily distinguished, and then demonstrate that the same sparse labeling allows live in vivo imaging of mature Purkinje neurons at a resolution sufficient for complete analytical reconstruction. Given the rising availability of viral constructs, packaging services, and genetically modified animals, these techniques should facilitate a wide range of experiments into brain development, function, and degeneration.  

5.1248           Analytical technologies for influenza virus-like particle candidate vaccines: challenges and emerging approaches

Thompson, C.M., Petiot, E., Lennaertz, A., Henry, O. and Kamen, A.A. Virol. J., 10:141 (2013)   Influenza virus-like particle vaccines are one of the most promising ways to respond to the threat of future influenza pandemics. VLPs are composed of viral antigens but lack nucleic acids making them non-infectious which limit the risk of recombination with wild-type strains. By taking advantage of the advancements in cell culture technologies, the process from strain identification to manufacturing has the potential to be completed rapidly and easily at large scales. After closely reviewing the current research done on influenza VLPs, it is evident that the development of quantification methods has been consistently overlooked. VLP quantification at all stages of the production process has been left to rely on current influenza quantification methods (i.e. Hemagglutination assay (HA), Single Radial Immunodiffusion assay (SRID), NA enzymatic activity assays, Western blot, Electron Microscopy). These are analytical methods developed decades ago for influenza virions and final bulk influenza vaccines. Although these methods are time-consuming and cumbersome they have been sufficient for the characterization of final purified material. Nevertheless, these analytical methods are impractical for in-line process monitoring because VLP concentration in crude samples generally falls out of the range of detection for these methods. This consequently impedes the development of robust influenza-VLP production and purification processes. Thus, development of functional process analytical techniques, applicable at every stage during production, that are compatible with different production platforms is in great need to assess, optimize and exploit the full potential of novel manufacturing platforms.  

5.1249           Characterization of Cognitive Deficits in Rats Overexpressing Human Alpha-Synuclein in the Ventral Tegmental Area and Medial Septum Using Recombinant Adeno-Associated Viral Vectors

Hall, H., Jewett, M., Landeck, N., Nilsson, N., Schagerlöf, U., Leanza, G. and Kirik, D. PloS One, 8(5), e64844 (2013)   Intraneuronal inclusions containing alpha-synuclein (a-syn) constitute one of the pathological hallmarks of Parkinson's disease (PD) and are accompanied by severe neurodegeneration of A9 dopaminergic neurons located in the substantia nigra. Although to a lesser extent, A10 dopaminergic neurons are also affected. Neurodegeneration of other neuronal populations, such as the cholinergic, serotonergic and noradrenergic cell groups, has also been documented in PD patients. Studies in human post-mortem PD brains and in rodent models suggest that deficits in cholinergic and dopaminergic systems may be associated with the cognitive impairment seen in this disease. Here, we investigated the consequences of targeted overexpression of a-syn in the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways. Rats were injected with recombinant adeno-associated viral vectors encoding for either human wild-type a-syn or green fluorescent protein (GFP) in the ventral tegmental area and the medial septum/vertical limb of the diagonal band of Broca, two regions rich in dopaminergic and cholinergic neurons, respectively. Histopathological analysis showed widespread insoluble a-syn positive inclusions in all major projections areas of the targeted nuclei, including the hippocampus, neocortex, nucleus accumbens and anteromedial striatum. In addition, the rats overexpressing human a-syn displayed an abnormal locomotor response to apomorphine injection and exhibited spatial learning and memory deficits in the Morris water maze task, in the absence of obvious spontaneous locomotor impairment. As losses in dopaminergic and cholinergic immunoreactivity in both the GFP and a-syn expressing animals were mild-to-moderate and did not differ from each other, the behavioral impairments seen in the a-syn overexpressing animals appear to be determined by the long term persisting neuropathology in the surviving neurons rather than by neurodegeneration.  

5.1250           Hypothalamic IGF-I Gene Therapy Prolongs Estrous Cyclicity and Protects Ovarian Structure in Middle-Aged Female Rats

Rodriguez, S.S., Schwerdt, J.I., Barbeito, C.G., Flamini, M.A., Han, Y., Bohn, M.C. and Goya, R.G. Endocrinol., 154(6), 2166-2173 (2013)   There is substantial evidence that age-related ovarian failure in rats is preceded by abnormal responsiveness of the neuroendocrine axis to estrogen positive feedback. Because IGF-I seems to act as a permissive factor for proper GnRH neuronal response to estrogen positive feedback and considering that the hypothalamic content of IGF-I declines in middle-aged (M-A) rats, we assessed the effectiveness of long-term IGF-I gene therapy in the mediobasal hypothalamus (MBH) of M-A female rats to extend regular cyclicity and preserve ovarian structure. We used 3 groups of M-A rats: 1 group of intact animals and 2 groups injected, at 36.2 weeks of age, in the MBH with either a bicistronic recombinant adeno-associated virus (rAAV) harboring the genes for IGF-I and the red fluorescent protein DsRed2, or a control rAAV expressing only DsRed2. Daily vaginal smears were taken throughout the study, which ended at 49.5 weeks of age. We measured serum levels of reproductive hormones and assessed ovarian histology at the end of the study. Although most of the rats injected with the IGF-I rAAV had, on the average, well-preserved estrous cyclicity as well as a generally normal ovarian histology, the intact and control rAAV groups showed a high percentage of acyclic rats at the end of the study and ovaries with numerous enlarged cysts and scarce corpora lutea. Serum LH was higher and hyperprolactinemia lower in the treated animals. These results suggest that overexpression of IGF-I in the MBH prolongs normal ovarian function in M-A female rats.  

5.1251           Bioengineering of AAV2 Capsid at Specific Serine, Threonine, or Lysine Residues Improves Its Transduction Efficiency in Vitro and in Vivo

Gabriel, N., Hareendran, S., Sen, D., Gadkari, R.A., Sudha, G., Selot, R., Hussain, M., Dhaksnamoorthy, R., Samuel, R., Srinivasan, N., Srivastava, A. and Jayandharan, G.R. Human Gene Therapy Methods, 24(2), 80-93 (2013)   We hypothesized that the AAV2 vector is targeted for destruction in the cytoplasm by the host cellular kinase/ubiquitination/proteasomal machinery and that modification of their targets on AAV2 capsid may improve its transduction efficiency. In vitro analysis with pharmacological inhibitors of cellular serine/threonine kinases (protein kinase A, protein kinase C, casein kinase II) showed an increase (20–90%) on AAV2-mediated gene expression. The three-dimensional structure of AAV2 capsid was then analyzed to predict the sites of ubiquitination and phosphorylation. Three phosphodegrons, which are the phosphorylation sites recognized as degradation signals by ubiquitin ligases, were identified. Mutation targets comprising eight serine (S) or seven threonine (T) or nine lysine (K) residues were selected in and around phosphodegrons on the basis of their solvent accessibility, overlap with the receptor binding regions, overlap with interaction interfaces of capsid proteins, and their evolutionary conservation across AAV serotypes. AAV2-EGFP vectors with the wild-type (WT) capsid or mutant capsids (15 S/T→alanine [A] or 9 K→arginine [R] single mutant or 2 double K→R mutants) were then evaluated in vitro. The transduction efficiencies of 11 S/T→A and 7 K→R vectors were significantly higher ( 63–90%) than the AAV2-WT vectors ( 30–40%). Further, hepatic gene transfer of these mutant vectors in vivo resulted in higher vector copy numbers (up to 4.9-fold) and transgene expression (up to 14-fold) than observed from the AAV2-WT vector. One of the mutant vectors, S489A, generated 8-fold fewer antibodies that could be cross-neutralized by AAV2-WT. This study thus demonstrates the feasibility of the use of these novel AAV2 capsid mutant vectors in hepatic gene therapy.  

5.1252           Targeted Modifications in Adeno-Associated Virus Serotype 8 Capsid Improves Its Hepatic Gene Transfer Efficiency In Vivo

Sen, D., Gadkari, R.A., Sudha, G., Gabriel, N., Kumar, Y.S., Selot, R., Samuel, R., Rajalingam, S., Ramya, V., Nair, S.C., Srinivasan, N., Srivastava, A. and Jayandharan, G.R. Human Gene Therapy Methods, 24(2), 104-116 (2013)   Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host–cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T→Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S→A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels ( 9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h.FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h.FIX:Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B.  

5.1253           The Conserved Set of Host Proteins Incorporated into HIV-1 Virions Suggests a Common Egress Pathway in Multiple Cell Types

Linde, M.E., Colquhoun, D.R., Mohien, C.U., Kole, T., Aquino, V., Cotter, R., Edwards, N., Hildreth, J.E.K. and Graham, D.R.

1. Proteome Res., 12(5), 2045-2054 (2013)

  HIV-1 incorporates a large array of host proteins into virions. Determining the host protein composition in HIV virions has technical difficulties, including copurification of microvesicles. We developed an alternative purification technique using cholesterol that differentially modulates the density of virions and microvesicles (density modification, DM) allowing for high-yield virion purification that is essential for tandem mass spectrometric and quantitative proteomic (iTRAQ) analysis. DM purified virions were analyzed using iTRAQ and validated against Optiprep (60% iodixanol) purified virions. We were able to characterize host protein incorporation in DM-purified HIV particles derived from CD4+ T-cell lines; we compared this data set to a reprocessed data set of monocyte-derived macrophages (MDM) derived HIV-1 using the same bioinformatics pipeline. Seventy-nine clustered proteins were shared between the MDM derived and T-cell derived data set. These clusters included an extensive collection of actin isoforms, HLA proteins, chaperones, and a handful of other proteins, many of which have previously been documented to interact with viral proteins. Other proteins of note were ERM proteins, the dynamin domain containing protein EH4, a phosphodiesterase, and cyclophilin A. As these proteins are incorporated in virions produced in both cell types, we hypothesize that these proteins may have direct interactions with viral proteins or may be important in the viral life cycle. Additionally, identified common set proteins are predicted to interact with >1000 related human proteins. Many of these secondary interacting proteins are reported to be incorporated into virions, including ERM proteins and adhesion molecules. Thus, only a few direct interactions between host and viral proteins may dictate the host protein composition in virions. Ultimately, interaction and expression differences in host proteins between cell types may drive virion phenotypic diversity, despite conserved viral protein–host protein interactions between cell types.  

5.1254           Cloak and dagger

Hofer, U. Nature Reviews Microbiol., 11(6), 360-361 (2013)   Hepatitis A virus (HAV), a common cause of food-borne hepatitis, is a member of the Picornaviridae family and, as such, has been classified as a non-enveloped virus. A new study by Feng et al. challenges this view of HAV and finds that virions circulating in the blood are enveloped by host cell-derived membranes.  

5.1255           Hypothalamic κ-Opioid Receptor Modulates the Orexigenic Effect of Ghrelin

Romero-Pico, A., Vazquez, M.J., Gonzalez-Touceda, D., Folgueira, C., Skibicka, K.P., Alvarez-Crespo, M., Van Gestel, M.A., Velasquez, D.A., Schwarzer, C., Herzog, H., Lopez, M., Adan, R.A., Diskson, S.L., Dieguez, C. and Nogueiras, R. Neuropsychopharmacol., 38(7), 1296-1307 (2013)   The opioid system is well recognized as an important regulator of appetite and energy balance. We now hypothesized that the hypothalamic opioid system might modulate the orexigenic effect of ghrelin. Using pharmacological and gene silencing approaches, we demonstrate that ghrelin utilizes a hypothalamic κ-opioid receptor (KOR) pathway to increase food intake in rats. Pharmacological blockade of KOR decreases the acute orexigenic effect of ghrelin. Inhibition of KOR expression in the hypothalamic arcuate nucleus is sufficient to blunt ghrelin-induced food intake. By contrast, the specific inhibition of KOR expression in the ventral tegmental area does not affect central ghrelin-induced feeding. This new pathway is independent of ghrelin-induced AMP-activated protein kinase activation, but modulates the levels of the transcription factors and orexigenic neuropeptides triggered by ghrelin to finally stimulate feeding. Our novel data implicate hypothalamic KOR signaling in the orexigenic action of ghrelin.  

5.1256           TFEB-mediated autophagy rescues midbrain dopamine neurons from α-synuclein toxicity

Decressac, M., Mattson, B., Weikop, P., Lundblad, M., Jakobsson, J. and Björklund, A. PNAS, 110(19), E1817-E1826 (2013)   The aggregation of α-synuclein plays a major role in Parkinson disease (PD) pathogenesis. Recent evidence suggests that defects in the autophagy-mediated clearance of α-synuclein contribute to the progressive loss of nigral dopamine neurons. Using an in vivo model of α-synuclein toxicity, we show that the PD-like neurodegenerative changes induced by excess cellular levels of α-synuclein in nigral dopamine neurons are closely linked to a progressive decline in markers of lysosome function, accompanied by cytoplasmic retention of transcription factor EB (TFEB), a major transcriptional regulator of the autophagy-lysosome pathway. The changes in lysosomal function, observed in the rat model as well as in human PD midbrain, were reversed by overexpression of TFEB, which afforded robust neuroprotection via the clearance of α-synuclein oligomers, and were aggravated by microRNA-128–mediated repression of TFEB in both A9 and A10 dopamine neurons. Delayed activation of TFEB function through inhibition of mammalian target of rapamycin blocked α-synuclein induced neurodegeneration and further disease progression. The results provide a mechanistic link between α-synuclein toxicity and impaired TFEB function, and highlight TFEB as a key player in the induction of α-synuclein–induced toxicity and PD pathogenesis, thus identifying TFEB as a promising target for therapies aimed at neuroprotection and disease modification in PD.  

5.1257           1150 HEPATITIS C VIRUS LIVER TRANSPLANTATION ESCAPE VARIANT IS CHARACTERIZED BY BOTH ENHANCED TRIGLYCERIDE-RICH LIPOPROTEIN ASSOCIATION AND SENSITIVITY TO apoE ANTIBODIES

Felmlee, D.J., Fauvelle, C., Heydmann, L., Hiet, M.S., Fofana, I., Bartenschlager, R., Stoll-Keller, F., Zeisel, M.B., Fafi-Kremer, S. and Baumert, T.F:

1. Hepatol., 58, S409-S566 (2013)

  A unique feature of hepatitis C virus (HCV) is that virions circulate in plasma associated with host triglyceride-rich lipoproteins (TRL), though the functional impact of this association remains undefined. Virus/lipoprotein complex formation may facilitate HCV escape from host neutralizing antibodies by concealing epitopes, although the mechanisms are unknown. We previously demonstrated that escape from antibody-mediated neutralization is a key factor for viral persistence in acute liver graft infection and chronic infection (Fafi-Kremer et al. J Exp Med 2010; Fofana et al. Gastroenterology 2012). Aiming to address the role of HCVTRL associations for viral evasion, we investigated the role of lipoprotein association using an HCVcc chimera expressing the structural proteins of a well characterized post-transplantation escape variant (VL). Fractionation of this virus by iodixanol density gradients and assaying by limiting dilution assay revealed two distinct subpopulations with more than half of infectious virions in densities below 1.05 g/mL. This density profile, which is unique compared to both Jc1 and JFH1, was further investigated for differential sensitivity to neutralizing antibodies from patient serum. Remarkably, the low-density subpopulation was capable of escape, while the high-density subpopulation was highly sensitive to neutralization. Introduction of residue exchange F447L, a mutation identified from a highly neutralized pre-transplant variant, resulted in an increase in the proportion of the low-densit subpopulation. However modification of this residue to alanine shifted the virus to a high-density distribution. Since these variants display altered receptor usage for late steps of HCV entry and use lipoproteins differently, we further investigated apolipoprotein E (apoE) usage. A monoclonal antibody recognizing the LDLreceptor binding domain of apoE was significantly more effective in neutralizing the VL variant than the F447L variant, suggesting a functional role for apoE in the observed phenotype. These findings demonstrate that HCV-TRL associations contribute to viral evasion in acute liver graft infection and identify a potential genetic element within HCV envelope glycoprotein E2 that contributes to lipoprotein association. Taken together, these findings are highly relevant for the development of strategies to prevent liver graft infection and prophylactic B cell vaccines. *DJF was supported by the EASL Dame Sheila Sherlock Postdoctoral Fellowship.  

5.1258           1175 HEPATITIS C VIRUS PROPAGATION IN HUMAN CD4+ AND CD8+ T LYMPHOCYTES

Skardasi, G. and  Michalak T.I.

1. Hepatol., 58, S477-S478 (2013)

  Introduction and Aim: Accumulated molecular and clinical evidence indicate that immune cells can support replication of hepatitis C virus (HCV). To investigate the ability of patient-derived, wild-type HCV to infect CD4+ and CD8+ T lymphocytes and to assess properties of the virions produced, we employed a previously established in vitro HCV replication system in which normal human T cells served as targets. Methods: Plasma of a patient chronically infected with HCV genotype 1, carrying viral load of 1.2x106 copies/ml that was prescreened for its infectivity towards total T cells, served as inoculum to infect normal human CD4+ and CD8+ T cell subsets (>97% pure by flow cytometry). The cells were pre-stimulated with phytohemagglutinin (PHA; 5 mg/ml), exposed to HCV and cultured under alternating stimulation with PHA and/or interleukin-2 (IL-2) for 14 days post-infection, as reported (JGV 2006; 87:3577; Hepatology 2008;49:1431; JVI 2012;86:3723). HCVRNA positive (genomic) and negative (replicative) strands were detected by strand-specific RT-PCR followed by nucleic acid hybridization (RTPCR/ NAH). Intracellular HCV NS5a and core proteins were identified by confocal microscopy. Released HCVRNA-reactive particles were examined by sucrose and iodixanol gradient ultracentrifugations. Clonal sequencing of the HCV 5_-UTR region served to compare the HCV virions harboured by inoculum and in in vitro infected cells. Results: HCVRNA positive and replicative strands, as well as NS5a and core proteins were detected in both CD4+ and CD8+ T cells after infection. Up to 1.2% cells were found NS5a protein positive. HCVRNA-reactive particles displaying distinct sedimentation velocity and buoyant density occurred in inoculums and culture supernatants from CD4+ and CD8+ T cells exposed to this inoculum. Clonal sequencing revealed different HCV variants in infected cells compared to plasma used as inoculum. Conclusions: Patient-derived, wild-type HCV can infect and establish productive replication in normal human both CD4+ and CD8+ T cells as evidenced by detection of HCVRNA replicative strand and intracellular expression of NS5a and core proteins. De novo HCV infection of the T cell subsets was confirmed by identification of unique variants in infected cells and HCVRNAreactive particles with distinct physical properties in cell culture supernatants.  

5.1259           Nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and RNA polymerization

  Ariza, A., Tanner, S.J., Walter, C.T., Dent, K.C., Shepherd, D.A., Wu, W., Matthews, S.V., Hiscox, J.A., Green, T.J., Luo, M., Elliott, R.M., Fooks, A.R., Ashcroft, A.E., Stonehouse, N.J., Ranson, N.A., Barr, J.N. and Edwards, T.A. Nucleic Acids Res., 41(11), 5912-5926 (2013)   All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N–RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75 Å, respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into which RNA is deeply sequestered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetramers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses.  

5.1260           Progressive Multifocal Leukoencephalopathy-Associated Mutations in the JC Polyomavirus Capsid Disrupt Lactoseries Tetrasaccharide c Binding

Maginnis, M.S., Ströh, L.J., Gee, G.V. et al mBio, 4(3), e00247-13 (2013)   The human JC polyomavirus (JCPyV) is the causative agent of the fatal, demyelinating disease progressive multifocal leukoencephalopathy (PML). The Mad-1 prototype strain of JCPyV uses the glycan lactoseries tetrasaccharide c (LSTc) and serotonin receptor 5-HT_2A to attach to and enter into host cells, respectively. Specific residues in the viral capsid protein VP1 are responsible for direct interactions with the α2,6-linked sialic acid of LSTc. Viral isolates from individuals with PML often contain mutations in the sialic acid-binding pocket of VP1 that are hypothesized to arise from positive selection. We reconstituted these mutations in the Mad-1 strain of JCPyV and found that they were not capable of growth. The mutations were then introduced into recombinant VP1 and reconstituted as pentamers in order to conduct binding studies and structural analyses. VP1 pentamers carrying PML-associated mutations were not capable of binding to permissive cells. High-resolution structure determination revealed that these pentamers are well folded but no longer bind to LSTc due to steric clashes in the sialic acid-binding site. Reconstitution of the mutations into JCPyV pseudoviruses allowed us to directly quantify the infectivity of the mutants in several cell lines. The JCPyV pseudoviruses with PML-associated mutations were not infectious, nor were they able to engage sialic acid as measured by hemagglutination of human red blood cells. These results demonstrate that viruses from PML patients with single point mutations in VP1 disrupt binding to sialic acid motifs and render these viruses noninfectious. IMPORTANCE Infection with human JC polyomavirus (JCPyV) is common and asymptomatic in healthy individuals, but during immunosuppression, JCPyV can spread from the kidney to the central nervous system (CNS) and cause a fatal, demyelinating disease, progressive multifocal leukoencephalopathy (PML). Individuals infected with HIV, those who have AIDS, or those receiving immunomodulatory therapies for autoimmune diseases are at serious risk for PML. Recent reports have demonstrated that viral isolates from PML patients often have distinct changes within the major capsid protein. Our structural-functional approach highlights that these mutations result in abolished engagement of the carbohydrate receptor motif LSTc that is necessary for infection. Viruses with PML-associated mutations are not infectious in glial cells, suggesting that they may play an alternative role in PML pathogenesis.  

5.1261           Parvovirus evades interferon-dependent viral control in primary mouse embryonic fibroblasts

Mattei, L.M., Cotmore, S.F., Tattersall, P. and Iwasaki, A. Virology, 442, 20-27 (2013)   Engagement of innate viral sensors elicits a robust antiviral program via the induction of type I interferons (IFNs). Innate defense mechanisms against ssDNA viruses are not well defined. Here, we examine type I IFN induction and effectiveness in controlling a ssDNA virus. Using mouse embryonic fibroblasts (MEFs), we found that a murine parvovirus, minute virus of mice (MVMp), induced a delayed but significant IFN response. MEFs deficient in mitochondrial antiviral signaling protein (MAVS) mounted a wild-type IFN response to MVMp infection, indicating that RIG-I-dependent RNA intermediate recognition is not required for innate sensing of this virus. However, MVMp-induced IFNs, as well recombinant type I IFNs, were unable to inhibit viral replication. Finally, MVMp infected cells became unresponsive to Poly (I:C) stimulation. Together, these data suggest that the MVMp efficiently evades antiviral immune mechanisms imposed by type I IFNs, which may in part explain their efficient transmission between mice.  

5.1262           Stbd1 is highly elevated in skeletal muscle of Pompe disease mice but suppression of its expression does not affect lysosomal glycogen accumulation

Yi, H., Fredrickson, K.B., Das, S., Kishnani, P.S. and Sun, B. Mol. Gen. Metab., 109, 312-314 (2013)   Previous studies strongly suggest that starch binding domain containing protein 1 (Stbd1) plays an important role in intracellular glycogen trafficking into lysosomes. We report here that Stbd1 expression is markedly increased in skeletal muscles but not in heart and liver of GAA-KO mice. An AAV2/9 vector expressing a Stbd1-specific shRNA effectively suppressed Stbd1 expression but did not alter lysosomal glycogen accumulation in the affected tissues of GAA-KO mice. Our results indicate that inhibition of Stbd1 does not appear to be an effective therapeutic approach for Pompe disease.  

5.1263           Viral transfer of the genetically-encoded chloride indicator Clomeleon into ChAT/Cre retinae to study chloride dynamics in "starburst" amacrine cells

Grau, T., Michalakis, S. and Euler, T. Invest. Ophthalmol. Vis. Sci., 54, E-abstract 2504 (2013)   Purpose:Starburst amacrine cells have been shown to play an important role for the retinal computation of visual motion direction (direction selectivity, DS), with their dendrites providing direction-selective input to DS ganglion cells. It was proposed that differential distribution of chloride importers and exporters along starburst cell dendrites lead to the formation of an asymmetrical distribution of chloride that contributes to the dendritic DS computation in starburst cells (Gavrikov et al., 2003, 2006). Here we aimed at generating a model that will allow investigating the physiological distribution of chloride and its potential functional role in starburst amacrine cells. Methods:A Cre-recombinase dependent viral expression construct, pAAV-Flex-Clomeleon, was designed by insertion of PCR-amplified Clomeleon complementary DNA into pAAV-Flex-Arch-GFP vector (Plasmid #22222, Addgene) by replacement of the Arch-GFP sequence. Cloning and mutagenesis were performed by standard techniques and confirmed by DNA sequencing. Recombinant adeno-associated virus (AAV) particles were produced by triple calcium phosphate transfection of 293T cells with pAdDeltaF6, pAAV2/7Y732F and pAAV-Flex-Clomeleon plasmids followed by iodixanol-gradient purification and ion exchange chromatography on a HiTrap Q Sepharose column (Michalakis et al., 2010). Light-Cycler technology (Roche Applied Science) was used to determine genomic recombinant AAV titers. AAVs carrying a reversed and double-floxed Clomeleon were delivered into the vitreous of 21 day old ChAT/Cre mice. Three weeks post-injection ChAT/Cre retinae were removed and Clomeleon expression detected by two-photon microscopy. Results:Using recombinant adeno-associated viruses Clomeleon, a fluorescent chloride indicator protein (Kuner & Augustin, 2000), can be successfully delivered and expressed in starburst amacrine cells of the mouse retina. Preliminary imaging data suggests that Clomeleon is functional in the targeted cells. Conclusions:The chloride biosensor Clomeleon can be functionally and selectively expressed in starburst amacrine cells using a combination of ChAT/Cre mice and recombinant AAV vectors.  

5.1264           Myelin Membrane Assembly Is Driven by a Phase Transition of Myelin Basic Proteins Into a Cohesive Protein Meshwork

Aggarwal, S., Snaidero, N., Pähler, G., Frey, S., Sanchez, P., Zweckstetter, M., Janshoff, A., Schneider, A., Well, M-T., Schaap, I.A.t., Görlich, D. and Simons, M. PloS Biology, 11(6), e1001577 (2013)   Rapid conduction of nerve impulses requires coating of axons by myelin. To function as an electrical insulator, myelin is generated as a tightly packed, lipid-rich multilayered membrane sheath. Knowledge about the mechanisms that govern myelin membrane biogenesis is required to understand myelin disassembly as it occurs in diseases such as multiple sclerosis. Here, we show that myelin basic protein drives myelin biogenesis using weak forces arising from its inherent capacity to phase separate. The association of myelin basic protein molecules to the inner leaflet of the membrane bilayer induces a phase transition into a cohesive mesh-like protein network. The formation of this protein network shares features with amyloid fibril formation. The process is driven by phenylalanine-mediated hydrophobic and amyloid-like interactions that provide the molecular basis for protein extrusion and myelin membrane zippering. These findings uncover a physicochemical mechanism of how a cytosolic protein regulates the morphology of a complex membrane architecture. These results provide a key mechanism in myelin membrane biogenesis with implications for disabling demyelinating diseases of the central nervous system.  

5.1265           Capsid Serotype and Timing of Injection Determines AAV Transduction in the Neonatal Mice Brain

Chakrabarty, P., Rosario, A., Cruz, P., Siemienski, Z., Ceballos-Diaz, C., Crosby, K., Jansen, K., Borchelt, D.R., Kim, J-Y., Jankowsky, J.L., Golde, T.E. and levites, Y. PloS One, 8(6), e67680 (2013)   Adeno-associated virus (AAV) mediated gene expression is a powerful tool for gene therapy and preclinical studies. A comprehensive analysis of CNS cell type tropism, expression levels and biodistribution of different capsid serotypes has not yet been undertaken in neonatal rodents. Our previous studies show that intracerebroventricular injection with AAV2/1 on neonatal day P0 results in widespread CNS expression but the biodistribution is limited if injected beyond neonatal day P1. To extend these observations we explored the effect of timing of injection on tropism and biodistribution of six commonly used pseudotyped AAVs delivered in the cerebral ventricles of neonatal mice. We demonstrate that AAV2/8 and 2/9 resulted in the most widespread biodistribution in the brain. Most serotypes showed varying biodistribution depending on the day of injection. Injection on neonatal day P0 resulted in mostly neuronal transduction, whereas administration in later periods of development (24–84 hours postnatal) resulted in more non-neuronal transduction. AAV2/5 showed widespread transduction of astrocytes irrespective of the time of injection. None of the serotypes tested showed any microglial transduction. This study demonstrates that both capsid serotype and timing of injection influence the regional and cell-type distribution of AAV in neonatal rodents, and emphasizes the utility of pseudotyped AAV vectors for translational gene therapy paradigms.  

5.1266           AAV2 production with optimized N/P ratio and PEI-mediated transfection results in low toxicity and high titer for in vitro and in vivo applications

Huang, X., Hartley, A-V., Yin, Y., Herskowitz, J.H., Lah, J.J. and Ressler, K.J.

1. Virol. Methods, 193, 270-277 (2013)

  The adeno-associated virus (AAV) is one of the most useful viral vectors for gene delivery for both in vivo and in vitro applications. A variety of methods have been established to produce and characterize recombinant AAV (rAAV) vectors; however most methods are quite cumbersome and obtaining consistently high titer can be problematic. This protocol describes a triple-plasmid co-transfection approach with 25 kDa linear polyethylenimine (PEI) in 293T cells for the production of AAV serotype 2. Seventy-two hours post-transfection, supernatant and cells were harvested and purified by a discontinuous iodixanol density gradient ultracentrifugation, then dialyzed and concentrated with an Amicon 15 100,000 MWCO concentration unit. To optimize the protocol for AAV2 production using PEI, various N/P ratios and DNA amounts were compared. We found that an N/P ratio of 40 coupled with 1.05 μg DNA per ml of media (21 μg DNA/15 cm dish) was found to produce the highest yields for viral replication and assembly measured multiple ways. The infectious units, as determined by serial dilution, were between 1 × 10⁸ and 2 × 10⁹ IU/ml. The genomic titer of the viral stock was determined by qPCR and ranged from 2 × 10¹² to 6 × 10¹³ VG/ml. These viral vectors showed high expression both in vivo within the brain and in vitro in cell culture. The use of linear 25 kDa polyethylenamine PEI as a transfection reagent is a simple, more cost-effective, and stable means of high-throughput production of high-titer AAV serotype 2. The use of PEI also eliminates the need to change cell medium post-transfection, lowering cost and workload, while producing high-titer, efficacious AAV2 vectors for routine gene transfer.  

5.1267           Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo

Song, L. et al Cytotherapy, 15, 986-998 (2013)   Background aims Although recombinant adeno-associated virus serotype 2 (AAV2) vectors have gained attention because of their safety and efficacy in numerous phase I/II clinical trials, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low. Only a few additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed. Methods We evaluated the transduction efficiency of all available AAV serotype vectors (AAV1 through AAV10) in primary mouse, cynomolgus monkey and human HSCs. The transduction efficiency of the optimized AAV vectors was also evaluated in human HSCs in a murine xenograft model in vivo. Results We observed that although there are only six amino acid differences between AAV1 and AAV6, AAV1, but not AAV6, transduced mouse HSCs well, whereas AAV6, but not AAV1, transduced human HSCs well. None of the 10 serotypes transduced cynomolgus monkey HSCs in vitro. We also evaluated the transduction efficiency of AAV6 vectors containing mutations in surface-exposed tyrosine residues. We observed that tyrosine (Y) to phenylalanine (F) point mutations in residues 445, 705 and 731 led to a significant increase in transgene expression in human HSCs in vitro and in a mouse xenograft model in vivo. Conclusions These studies suggest that the tyrosine-mutant AAV6 serotype vectors are the most promising vectors for transducing human HSCs and that it is possible to increase further the transduction efficiency of these vectors for their potential use in HSC-based gene therapy in humans.  

5.1268           Unbiased Approach for Virus Detection in Skin Lesions

Bzhalava, D., Johansson, H.,Ekström, J., Faust, H., Möller, B., Eklund, C., Nordin, P., Stenquist, B., Paoli, J., Persson, B., Forslund, O. and Dillner, J. PloS One, 8(6), e65953 (2013)   To assess presence of virus DNA in skin lesions, swab samples from 82 squamous cell carcinomas of the skin (SCCs), 60 actinic keratoses (AKs), paraffin-embedded biopsies from 28 SCCs and 72 kerathoacanthomas (KAs) and fresh-frozen biopsies from 92 KAs, 85 SCCs and 92 AKs were analyzed by high throughput sequencing (HTS) using 454 or Ion Torrent technology. We found total of 4,284 viral reads, out of which 4,168 were Human Papillomavirus (HPV)-related, belonging to 15 known (HPV8, HPV12, HPV20, HPV36, HPV38, HPV45, HPV57, HPV59, HPV104, HPV105, HPV107, HPV109, HPV124, HPV138, HPV147), four previously described putative (HPV 915 F 06 007 FD1, FA73, FA101, SE42) and two putatively new HPV types (SE46, SE47). SE42 was cloned, sequenced, designated as HPV155 and found to have 76% similarity to the most closely related known HPV type. In conclusion, an unbiased approach for viral DNA detection in skin tumors has found that, although some new putative HPVs were found, known HPV types constituted most of the viral DNA.  

5.1269           Gene Transfer of Mutant Mouse Cholinesterase Provides High Lifetime Expression and Reduced Cocaine Responses with No Evident Toxicity

Geng, L., Gao, Y., Chen, X., Hou, S., Zhan, C-G., Radic, Z., Parks, R.J., Russell, S.J., Pham, L. and Brimijoin, S. PloS One, 8(6), e67446 (2013)   Gene transfer of a human cocaine hydrolase (hCocH) derived from butyrylcholinesterase (BChE) by 5 mutations (A199S/F227A/S287G/A328W/Y332G) has shown promise in animal studies for treatment of cocaine addiction. To predict the physiological fate and immunogenicity of this enzyme in humans, a comparable enzyme was created and tested in a conspecific host. Thus, similar mutations (A199S/S227A/S287G/A328W/Y332G) were introduced into mouse BChE to obtain a mouse CocH (mCocH). The cDNA was incorporated into viral vectors based on: a) serotype-5 helper-dependent adenovirus (hdAD) with ApoE promoter, and b) serotype-8 adeno-associated virus with CMV promoter (AAV-CMV) or multiple promoter and enhancer elements (AAV-VIP). Experiments on substrate kinetics of purified mCocH expressed in HEK293T cells showed 30-fold higher activity (U/mg) with ³H-cocaine and 25% lower activity with butyrylthiocholine, compared with wild type BChE. In mice given modest doses of AAV-CMV-mCocH vector (0.7 or 3×10¹¹ particles) plasma hydrolase activity rose 10-fold above control for over one year with no observed immune response. Under the same conditions, transduction of the human counterpart continued less than 2 months and antibodies to hCocH were readily detected. The advanced AAV-VIP-mCocH vector generated a dose-dependent rise in plasma cocaine hydrolase activity from 20-fold (10¹⁰ particles) to 20,000 fold (10¹³ particles), while the hdAD vector (1.7×10¹² particles) yielded a 300,000-fold increase. Neither vector caused adverse reactions such as motor weakness, elevated liver enzymes, or disturbance in spontaneous activity. Furthermore, treatment with high dose hdAD-ApoE-mCocH vector (1.7×10¹² particles) prevented locomotor abnormalities, other behavioral signs, and release of hepatic alanine amino transferase after a cocaine dose fatal to most control mice (120 mg/kg). This outcome suggests that viral gene transfer can yield clinically effective cocaine hydrolase expression for lengthy periods without immune reactions or cholinergic dysfunction, while blocking toxicity from drug overdose.  

5.1270           The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Does Not Replicate in Syrian Hamsters

De Wit, E., Prescott, J., Baseier, L., Bushmaker, T., Thomas, T., Lackemeyer, M.G., Martellaro, C., Milne-Price, S., Haddock, E., Haagmans, B.L., Feldmann, H. and Munster, V.J. PloS One, 8(7), e69127 (2013)   In 2012 a novel coronavirus, MERS-CoV, associated with severe respiratory disease emerged in the Arabian Peninsula. To date, 55 human cases have been reported, including 31 fatal cases. Several of the cases were likely a result of human-to-human transmission. The emergence of this novel coronavirus prompts the need for a small animal model to study the pathogenesis of this virus and to test the efficacy of potential intervention strategies. In this study we explored the use of Syrian hamsters as a small animal disease model, using intratracheal inoculation and inoculation via aerosol. Clinical signs of disease, virus replication, histological lesions, cytokine upregulation nor seroconversion were observed in any of the inoculated animals, indicating that MERS-CoV does not replicate in Syrian hamsters.  

5.1271           Optogenetic Inhibition of Synaptic Release with Chromophore-Assisted Light Inactivation (CALI)

Lin, J.Y., Sann, S.B., Zhou, K., Nabavi, S., Prouix, C.D., Malinow, R., Jin, Y. and Tsien, R.Y. Neuron, 79(2), 241-253 (2013)   Optogenetic techniques provide effective ways of manipulating the functions of selected neurons with light. In the current study, we engineered an optogenetic technique that directly inhibits neurotransmitter release. We used a genetically encoded singlet oxygen generator, miniSOG, to conduct chromophore assisted light inactivation (CALI) of synaptic proteins. Fusions of miniSOG to VAMP2 and synaptophysin enabled disruption of presynaptic vesicular release upon illumination with blue light. In cultured neurons and hippocampal organotypic slices, synaptic release was reduced up to 100%. Such inhibition lasted >1 hr and had minimal effects on membrane electrical properties. When miniSOG-VAMP2 was expressed panneuronally in Caenorhabditis elegans, movement of the worms was reduced after illumination, and paralysis was often observed. The movement of the worms recovered overnight. We name this technique Inhibition of Synapses with CALI (InSynC). InSynC is a powerful way to silence genetically specified synapses with light in a spatially and temporally precise manner.  

5.1272           IL12-Mediated Liver Inflammation Reduces the Formation of AAV Transcriptionally Active Forms but Has No Effect over Preexisting AAV Transgene Expression

Gil-Farina, I., Di Scala, M., Vanrell, L., Olagüe, C., Vales, A., High, K.A., Prieto, J., Mingozzi, F and Gonzales-Aseguinolaza, G. PloS One, 8(7), e67748 (2013)   Recombinant adenoassociated viral vectors (rAAV) have proven to be excellent candidates for gene therapy clinical applications. Recent results showed that cellular immunity to AAV represents a major challenge facing the clinical use of systemic administration of these vectors. Interestingly, no preclinical animal model has previously fully reproduced the clinical findings. The aim of the present work was to enhance the T cell immune response against AAV capsid in mice by the administration of a rAAV expressing the immunostimulatory cytokine IL-12. Our results indicate that although IL-12 expression enhanced the AAV capsid-specific immune response it failed to eliminate transduced hepatocytes and long-term expression was achieved. We found that AAV-mediated transgene expression is altered by IL-12-induced liver inflammation. However, IL-12 expression has no effect over preexisting AAV-mediated transgene expression. IL-12 down-regulates AAV mediated transgene expression via induction of IFN-γ production by NK and T cells, but without altering the transduction efficiency measured by viral genomes. Our results indicate that liver inflammation affects the formation of transcriptionally active AAV vector genomes through an unknown mechanism that can be avoided by the use of DNA-demethylating or anti-inflammatory agents.  

5.1273           The Pathological Phenotypes of Human TDP-43 Transgenic Mouse Models Are Independent of Downregulation of Mouse Tdp-43

Xu, Y-F., Prudencio, M., Hubbard, J.M., Tong, J., Whitelaw, E.C., Jansen-West, K., Stetler, C., Cao, X., Song, J. and Zhang, Y-J. PloS One, 8(7), e69864 (2013)   Tar DNA binding protein 43 (TDP-43) is the major component of pathological deposits in frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and in amyotrophic lateral sclerosis (ALS). It has been reported that TDP-43 transgenic mouse models expressing human TDP-43 wild-type or ALS-associated mutations recapitulate certain ALS and FTLD pathological phenotypes. Of note, expression of human TDP-43 (hTDP-43) reduces the levels of mouse Tdp-43 (mTdp-43). However, it remained unclear whether the mechanisms through which TDP-43 induces ALS or FTLD-like pathologies resulted from a reduction in mTdp-43, an increase in hTDP-43, or a combination of both. In elucidating the role of mTdp-43 and hTDP-43 in hTDP-43 transgenic mice, we observed that reduction of mTdp-43 in non-transgenic mice by intraventricular brain injection of AAV1-shTardbp leads to a dramatic increase in the levels of splicing variants of mouse sortilin 1 and translin. However, the levels of these two abnormal splicing variants are not increased in hTDP-43 transgenic mice despite significant downregulation of mTdp-43 in these mice. Moreover, further downregulation of mTdp-43 in hTDP-43 hemizygous mice, which are asymptomatic, to the levels equivalent to that of mTdp-43 in hTDP-43 homozygous mice does not induce the pathological phenotypes observed in the homozygous mice. Lastly, the number of dendritic spines and the RNA levels of TDP-43 RNA targets critical for synapse formation and function are significantly decreased in symptomatic homozygous mice. Together, our findings indicate that mTdp-43 downregulation does not lead to a loss of function mechanism or account for the pathological phenotypes observed in hTDP-43 homozygous mice because hTDP-43 compensates for the reduction, and associated functions of mTdp-43. Rather, expression of hTDP-43 beyond a certain threshold leads to abnormal metabolism of TDP-43 RNA targets critical for neuronal structure and function, which might be responsible for the ALS or FTLD-like pathologies observed in homozygous hTDP-43 transgenic mice  

5.1274           Continuous DOPA synthesis from a single AAV: dosing and efficacy in models of Parkinson's disease

Cederfjäll, E., Nilsson, N., Sahin, g., Chu, Y., Nikitidou, E., Björklund, T., Kordower, J.H. and Kirik, D. Scientific Reports, 3, 2157 (2013)   We used a single adeno-associated viral (AAV) vector co-expressing tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1) to investigate the relationship between vector dose, and the magnitude and rate of recovery in hemi-parkinsonian rats. Intrastriatal injections of >1E10 genomic copies (gc) of TH-GCH1 vector resulted in complete recovery in drug-naïve behavior tests. Lower vector dose gave partial to no functional improvement. Stereological quantification revealed no striatal NeuN+ cell loss in any of the groups, whereas a TH-GCH1 dose of >1E11 gc resulted in cell loss in globus pallidus. Thus, a TH-GCH1 dose of 1E10 gc gave complete recovery without causing neuronal loss. Safety and efficacy was also studied in non-human primates where the control vector resulted in co-expression of the transgenes in caudate-putamen. In the TH-GCH1 group, GCH1 expression was robust but TH was not detectable. Moreover, TH-GCH1 treatment did not result in functional improvement in non-human primates.  

5.1275           Repair of Mybpc3 mRNA by 5′-trans-splicing in a Mouse Model of Hypertrophic Cardiomyopathy

Mearin, G., Stimpel, D., Krämer, E., Geertz, B., Braren, I., Gedicke-Hornung, C., Precigout, G., Müller, O.J., Katus, H.A., Eschenhagen, T., Voit, T., Garcia, L., Lorain, S. and Varrier, L. Mol. Therapy-Nucleic Acids, 2, e102 (2013)   RNA trans-splicing has been explored as a therapeutic option for a variety of genetic diseases, but not for cardiac genetic disease. Hypertrophic cardiomyopathy (HCM) is an autosomal-dominant disease, characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction. MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C) is frequently mutated. We evaluated the 5′-trans-splicing strategy in a mouse model of HCM carrying a Mybpc3 mutation. 5′-trans-splicing was induced between two independently transcribed molecules, the mutant endogenous Mypbc3 pre-mRNA and an engineered pre-trans-splicing molecule (PTM) carrying a FLAG-tagged wild-type (WT) Mybpc3 cDNA sequence. PTMs were packaged into adeno-associated virus (AAV) for transduction of cultured cardiac myocytes and the heart in vivo. Full-length repaired Mybpc3 mRNA represented up to 66% of total Mybpc3 transcripts in cardiac myocytes and 0.14% in the heart. Repaired cMyBP-C protein was detected by immunoprecipitation in cells and in vivo and exhibited correct incorporation into the sarcomere in cardiac myocytes. This study provides (i) the first evidence of successful 5′-trans-splicing in vivo and (ii) proof-of-concept of mRNA repair in the most prevalent cardiac genetic disease. Since current therapeutic options for HCM only alleviate symptoms, these findings open new horizons for causal therapy of the severe forms of the disease.  

5.1276           Adeno-associated Virus-mediated, Mifepristone-regulated Transgene Expression in the Brain

Maddalena, A., Tereshchenko, J., Bähr, M. and Küger, S. Mol. Therapy-Nucleic Acids, 2, e106 (2013)   Gene therapy, in its current configuration, is irreversible and does not allow control over transgene expression in case of side effects. Only few regulated vector systems are available, and none of these has reached clinical applicability yet. The mifepristone (Mfp)-regulated Gene Switch (GS) system is characterized by promising features such as being composed of mainly human components and an approved small-molecule drug as an inducer. However, it has not yet been evaluated in adeno-associated virus (AAV) vectors, neither has it been tested for applicability in viral vectors in the central nervous system (CNS). Here, we demonstrate that the GS system can be used successfully in AAV vectors in the brain, and that short-term induced glial cell line-derived neurotrophic factor (GDNF) expression prevented neurodegeneration in a rodent model of Parkinson’s disease (PD). We also demonstrate repeated responsiveness to the inducer Mfp and absence of immunological tissue reactions in the rat brain. Human equivalent dosages of Mfp used in this study were lower than those used safely for treatment of psychiatric threats, indicating that the inducer could be safely applied in patients. Our results suggest that the GS system in AAV vectors is well suited for further development towards clinical applicability.  

5.1277           Misguidance and modulation of axonal regeneration by Stat3 and Rho/ROCK signaling in the transparent optic nerve

Pernet, V., Joly, S., Jordi, N., Dalkare, D., Guzik-Kornacka, A., Flannary, J.G. and Schwab, M.E. Cell Death and Disease, 4, e734 (2013)   The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological blockade of ROCK, a key signaling component for myelin-associated growth inhibitors, reduced axonal U-turns and potentiated AAV2.Stat3-ca-induced regeneration. Similar results were obtained after the sustained delivery of CNTF in the axotomized retina. These results show the important role of Stat3 in the activation of the neuronal growth program for regeneration, and they reveal that axonal misguidance is a key limiting factor that can affect long-distance regeneration and target interaction after trauma in the CNS. The correction of axonal misguidance was associated with improved long-distance axon regeneration in the injured adult CNS.  

5.1278           Behaviour-dependent recruitment of long-range projection neurons in somatosensory cortex

Chen, J.L., Carta, S., Soldado-Magraner, J., Schneider, B.L. and Helmchen, F. Nature, 499, 336.340 (2013)   In the mammalian neocortex, segregated processing streams are thought to be important for forming sensory representations of the environment^{1, 2}, but how local information in primary sensory cortex is transmitted to other distant cortical areas during behaviour is unclear. Here we show task-dependent activation of distinct, largely non-overlapping long-range projection neurons in the whisker region of primary somatosensory cortex (S1) in awake, behaving mice. Using two-photon calcium imaging, we monitored neuronal activity in anatomically identified S1 neurons projecting to secondary somatosensory (S2) or primary motor (M1) cortex in mice using their whiskers to perform a texture-discrimination task or a task that required them to detect the presence of an object at a certain location. Whisking-related cells were found among S2-projecting (S2P) but not M1-projecting (M1P) neurons. A higher fraction of S2P than M1P neurons showed touch-related responses during texture discrimination, whereas a higher fraction of M1P than S2P neurons showed touch-related responses during the detection task. In both tasks, S2P and M1P neurons could discriminate similarly between trials producing different behavioural decisions. However, in trials producing the same decision, S2P neurons performed better at discriminating texture, whereas M1P neurons were better at discriminating location. Sensory stimulus features alone were not sufficient to elicit these differences, suggesting that selective transmission of S1 information to S2 and M1 is driven by behaviour.  

5.1279           Biological and biochemical characterization of HIV-1 Gag/dgp41 virus-like particles expressed in Nicotiana benthamiana

Kessans, S.A., Linhart, M.D., Matoba, N. and Mor, T. Plant Biotech. J., 11, 681-690 (2013)   The transmembrane HIV-1 envelope protein gp41 has been shown to play critical roles in the viral mucosal transmission and infection of CD4+ cells. Gag is a structural protein configuring the enveloped viral particles and has been suggested to constitute a target of the cellular immunity that may control viral load. We hypothesized that HIV enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the membrane proximal external, transmembrane and cytoplasmic domains (dgp41) could be expressed in plants. To this end, plant-optimized HIV-1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a Tobamovirus-based expression system or a combination of both. Our results of biophysical, biochemical and electron microscopy characterization demonstrates that plant cells could support not only the formation of enveloped HIV-1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These findings provide further impetus for the journey towards a broadly efficacious and inexpensive subunit vaccine against HIV-1.  

5.1280           Rescue of cardiomyopathy through U7snRNA-mediated exon skipping in Mybpc3-targeted knock-in mice

Gedicke-Hornung, C., Behrens-Gawlik, V., Reischmann, S., Geertz, B., Stimpel, D., Weinberger, F., Schlossarek, S., Precigout, G., Braren, I., Eschenhagen, T., Mearini, G., Lorain, S., Voit, T., Dreyfus, P.A., Garcia, L. and Carrier, L. EMBO Mol. Med., 5, 1060-1077 (2013)   Exon skipping mediated by antisense oligoribonucleotides (AON) is a promising therapeutic approach for genetic disorders, but has not yet been evaluated for cardiac diseases. We investigated the feasibility and efficacy of viral-mediated AON transfer in a Mybpc3-targeted knock-in (KI) mouse model of hypertrophic cardiomyopathy (HCM). KI mice carry a homozygous G>A transition in exon 6, which results in three different aberrant mRNAs. We identified an alternative variant (Var-4) deleted of exons 5–6 in wild-type and KI mice. To enhance its expression and suppress aberrant mRNAs we designed AON-5 and AON-6 that mask splicing enhancer motifs in exons 5 and 6. AONs were inserted into modified U7 small nuclear RNA and packaged in adeno-associated virus (AAV-U7-AON-5+6). Transduction of cardiac myocytes or systemic administration of AAV-U7-AON-5+6 increased Var-4 mRNA/protein levels and reduced aberrant mRNAs. Injection of newborn KI mice abolished cardiac dysfunction and prevented left ventricular hypertrophy. Although the therapeutic effect was transient and therefore requires optimization to be maintained over an extended period, this proof-of-concept study paves the way towards a causal therapy of HCM.  

5.1281           Merkel Cell Polyomavirus Large T Antigen Disrupts Host Genomic Integrity and Inhibits Cellular Proliferation

Li, J., Wang, X., Diaz, J., Tsang, S.H., Buck, C.B. and You, J.

1. Virol., 87(16), 9173-9188 (2013)

  Clonal integration of Merkel cell polyomavirus (MCV) DNA into the host genome has been observed in at least 80% of Merkel cell carcinoma (MCC). The integrated viral genome typically carries mutations that truncate the C-terminal DNA binding and helicase domains of the MCV large T antigen (LT), suggesting a selective pressure to remove this MCV LT region during tumor development. In this study, we show that MCV infection leads to the activation of host DNA damage responses (DDR). This activity was mapped to the C-terminal helicase-containing region of the MCV LT. The MCV LT-activated DNA damage kinases, in turn, led to enhanced p53 phosphorylation, upregulation of p53 downstream target genes, and cell cycle arrest. Compared to the N-terminal MCV LT fragment that is usually preserved in mutants isolated from MCC tumors, full-length MCV LT shows a decreased potential to support cellular proliferation, focus formation, and anchorage-independent cell growth. These apparently antitumorigenic effects can be reversed by a dominant-negative p53 inhibitor. Our results demonstrate that MCV LT-induced DDR activates p53 pathway, leading to the inhibition of cellular proliferation. This study reveals a key difference between MCV LT and simian vacuolating virus 40 LT, which activates a DDR but inhibits p53 function. This study also explains, in part, why truncation mutations that remove the MCV LT C-terminal region are necessary for the oncogenic progression of MCV-associated cancers.  

5.1282           Arteriogenic therapy based on simultaneous delivery of VEGF-A and FGF4 genes improves the recovery from acute limb ischemia

Jazwa, A., Tomczyk, M., Taha, H.M., Hytonen, E., Stoszko, M., Zentilin, L., Giacca, M., Yla-Herttuala, S., Emanueli, C., Jozkowicz, A. and Dulak, J. Vascular Cell, 5:13 (2013)   Background Gene therapy stimulating the growth of blood vessels is considered for the treatment of peripheral and myocardial ischemia. Here we aimed to achieve angiogenic synergism between vascular endothelial growth factor-A (VEGF-A, VEGF) and fibroblast growth factor 4 (FGF4) in murine normoperfused and ischemic limb muscles. Methods Adeno-associated viral vectors (AAVs) carrying β-galactosidase gene (AAV-LacZ), VEGF-A (AAV-VEGF-A) or two angiogenic genes (AAV-FGF4-IRES-VEGF-A) were injected into the normo-perfused adductor muscles of C57Bl/6 mice. Moreover, in a different experiment, mice were subjected to unilateral hindlimb ischemia by femoral artery ligation followed by intramuscular injections of AAV-LacZ, AAV-VEGF-A or AAV-FGF4-IRES-VEGF-A below the site of ligation. Post-ischemic blood flow recovery was assessed sequentially by color laser Doppler. Mice were monitored for 28 days. Results VEGF-A delivered alone (AAV-VEGF-A) or in combination with FGF4 (AAV-FGF4-IRES-VEGF-A) increased the number of capillaries in normo-perfused hindlimbs when compared to AAV-LacZ. Simultaneous overexpression of both agents (VEGF-A and FGF4) stimulated the capillary wall remodeling in the non-ischemic model. Moreover, AAV-FGF4-IRES-VEGF-A faster restored the post-ischemic foot blood flow and decreased the incidence of toe necrosis in comparison to AAV-LacZ. Conclusions Synergy between VEGF-A and FGF4 to produce stable and functional blood vessels may be considered a promising option in cardiovascular gene therapy.  

5.1283           UPF1 Is Crucial for the Infectivity of Human Immunodeficiency Virus Type 1 Progeny Virions

Serquina, A.K.P., Das, S.R., Popova, E., Ojelaba, O.A, Roy, C.K. and Göttlinger, H.G.

1. Virol., 87(16), 8853-8861 (2013)

  The SF1 helicase MOV10 is an antiviral factor that is incorporated into human immunodeficiency virus type 1 (HIV-1) virions. We now report that HIV-1 virions also incorporate UPF1, which belongs to the same SF1 helicase subfamily as MOV10 and functions in the nonsense-mediated decay (NMD) pathway. Unlike ectopic MOV10, the overexpression of UPF1 does not impair the infectivity of HIV-1 progeny virions. However, UPF1 becomes a potent inhibitor of HIV-1 progeny virion infectivity when residues required for its helicase activity are mutated. In contrast, equivalent mutations abolish the antiviral activity of MOV10. Importantly, cells depleted of endogenous UPF1, but not of another NMD core component, produce HIV-1 virions of substantially lower specific infectivity. The defect is at the level of reverse transcription, the same stage of the HIV-1 life cycle inhibited by ectopic MOV10. Thus, whereas ectopic MOV10 restricts HIV-1 replication, the related UPF1 helicase functions as a cofactor at an early postentry step.  

5.1284           Functional Characterization of the Alphavirus TF Protein

Snyder, J.E., Kulcsar, K., Schultz, K.L.W., Riley, C.P., Neary, J.T., Marr, S., Jose, J., Griffin, D.E. and Kuhn, R.J.

1. Virol., 85(11), 8511-8523 (2013)

Alphavirus dogma has long dictated the production of a discrete set of structural proteins during infection of a cell: capsid, pE2, 6K, and E1. However, bioinformatic analyses of alphavirus genomes (A. E. Firth, B. Y. Chung, M. N. Fleeton, and J. F. Atkins, Virol. J. 5:108, 2008) suggested that a ribosomal frameshifting event occurs during translation of the alphavirus structural polyprotein. Specifically, a frameshift event is suggested to occur during translation of the 6K gene, yielding production of a novel protein, termed transframe (TF), comprised of a C-terminal extension of the 6K protein in the −1 open reading frame (ORF). Here, we validate the findings of Firth and colleagues with respect to the production of the TF protein and begin to characterize the function of TF. Using a mass spectrometry-based approach, we identified TF in purified preparations of both Sindbis and Chikungunya virus particles. We next constructed a panel of Sindbis virus mutants with mutations which alter the production, size, or sequence of TF. We demonstrate that TF is not absolutely required in culture, although disrupting TF production leads to a decrease in virus particle release in both mammalian and insect cells. In a mouse neuropathogenesis model, mortality was <15% in animals infected with the TF mutants, whereas mortality was 95% in animals infected with the wild-type virus. Using a variety of additional assays, we demonstrate that TF retains ion-channel activity analogous to that of 6K and that lack of production of TF does not affect genome replication, particle infectivity, or envelope protein transit to the cell surface. The TF protein therefore represents a previously uncharacterized factor important for alphavirus assembly.  

5.1285           Neurotransmitter-Triggered Transfer of Exosomes Mediates Oligodendrocyte–Neuron Communication

Frühbeis, C., Fröhlich, D., Kuo, W.P., Amphornat, J., Thilemann, S., Saab, A.S., Kirchhoff, F., Möbius, W., Goebbels, S., Nave, K-A., Schneider, A., Simons, M., Klugmann, M., Trotter, J., Krämer-Albers, E-M.   Reciprocal interactions between neurons and oligodendrocytes are not only crucial for myelination, but also for long-term survival of axons. Degeneration of axons occurs in several human myelin diseases, however the molecular mechanisms of axon-glia communication maintaining axon integrity are poorly understood. Here, we describe the signal-mediated transfer of exosomes from oligodendrocytes to neurons. These endosome-derived vesicles are secreted by oligodendrocytes and carry specific protein and RNA cargo. We show that activity-dependent release of the neurotransmitter glutamate triggers oligodendroglial exosome secretion mediated by Ca²⁺ entry through oligodendroglial NMDA and AMPA receptors. In turn, neurons internalize the released exosomes by endocytosis. Injection of oligodendroglia-derived exosomes into the mouse brain results in functional retrieval of exosome cargo in neurons. Supply of cultured neurons with oligodendroglial exosomes improves neuronal viability under conditions of cell stress. These findings indicate that oligodendroglial exosomes participate in a novel mode of bidirectional neuron-glia communication contributing to neuronal integrity.  

5.1286           Capsid Antibodies to Different Adeno-Associated Virus Serotypes Bind Common Regions

Gurda, B.L., DiMattia, M.A., Miller, E.B., Bennett, A., McKenna, R., Weichert, W.S., Nelson, C.D., Chen, W-j., Muzycka, N., Olson, N.H., Sinkovits, R.S., Chiorini, J.A., Solotutkhin, S., Kozyreva, O.G., Samulski, R.J., Baker, T.S., Parrish, C.R. and Agbandje-McKenna, M.

1. Virol., 87(16), 9111-9124 (2013)

  Interactions between viruses and the host antibody immune response are critical in the development and control of disease, and antibodies are also known to interfere with the efficacy of viral vector-based gene delivery. The adeno-associated viruses (AAVs) being developed as vectors for corrective human gene delivery have shown promise in clinical trials, but preexisting antibodies are detrimental to successful outcomes. However, the antigenic epitopes on AAV capsids remain poorly characterized. Cryo-electron microscopy and three-dimensional image reconstruction were used to define the locations of epitopes to which monoclonal fragment antibodies (Fabs) against AAV1, AAV2, AAV5, and AAV6 bind. Pseudoatomic modeling showed that, in each serotype, Fabs bound to a limited number of sites near the protrusions surrounding the 3-fold axes of the T=1 icosahedral capsids. For the closely related AAV1 and AAV6, a common Fab exhibited substoichiometric binding, with one Fab bound, on average, between two of the three protrusions as a consequence of steric crowding. The other AAV Fabs saturated the capsid and bound to the walls of all 60 protrusions, with the footprint for the AAV5 antibody extending toward the 5-fold axis. The angle of incidence for each bound Fab on the AAVs varied and resulted in significant differences in how much of each viral capsid surface was occluded beyond the Fab footprints. The AAV-antibody interactions showed a common set of footprints that overlapped some known receptor-binding sites and transduction determinants, thus suggesting potential mechanisms for virus neutralization by the antibodies.  

5.1287           Permissivity of Primary Human Hepatocytes and Different Hepatoma Cell Lines to Cell Culture Adapted Hepatitis C Virus

Helle, F., Brochot, E., Fournier, C., Descamps, V., Izquuierdo, L., Hoffmann, T.W., Morel, V., Herpe, Y-E., Bengrine, A., Belouzard, S., Wychowski, C., Dubuisson, J., Francois, C., Regimbeau, M., Castelain, S. and Duverlie, G. PloS One, 8(8), e70809 (2013)   Significant progress has been made in Hepatitis C virus (HCV) culture since the JFH1 strain cloning. However, developing efficient and physiologically relevant culture systems for all viral genotypes remains an important goal. In this work, we aimed at producing a high titer JFH1 derived virus to test different hepatic cells’ permissivity. To this end, we performed successive infections and obtained a JFH1 derived virus reaching high titers. Six potential adaptive mutations were identified (I599V in E2, R1373Q and M1611T in NS3, S2364P and C2441S in NS5A and R2523K in NS5B) and the effect of these mutations on HCV replication and infectious particle production was investigated. This cell culture adapted virus enabled us to efficiently infect primary human hepatocytes, as demonstrated using the RFP-NLS-IPS reporter protein and intracellular HCV RNA quantification. However, the induction of a strong type III interferon response in these cells was responsible for HCV inhibition. The disruption of this innate immune response led to a strong infection enhancement and permitted the detection of viral protein expression by western blotting as well as progeny virus production. This cell culture adapted virus also enabled us to easily compare the permissivity of seven hepatoma cell lines. In particular, we demonstrated that HuH-7, HepG2-CD81, PLC/PRF/5 and Hep3B cells were permissive to HCV entry, replication and secretion even if the efficiency was very low in PLC/PRF/5 and Hep3B cells. In contrast, we did not observe any infection of SNU-182, SNU-398 and SNU-449 hepatoma cells. Using iodixanol density gradients, we also demonstrated that the density profiles of HCV particles produced by PLC/PRF/5 and Hep3B cells were different from that of HuH-7 and HepG2-CD81 derived virions. These results will help the development of a physiologically relevant culture system for HCV patient isolates.  

5.1288           Susceptibility of Human Placenta Derived Mesenchymal Stromal/Stem Cells to Human Herpesviruses Infection

Avanzi, S., Leoni, v., Rotola, A., Alviano, F., Solimando, L., Lanzoni, G., Bonsi, L., Di Luca, D., Marchionni, C., Alvisi, G. and Ripalti, A. PloS One, 8(8), e71412 (2013)   Fetal membranes (FM) derived mesenchymal stromal/stem cells (MSCs) are higher in number, expansion and differentiation abilities compared with those obtained from adult tissues, including bone marrow. Upon systemic administration, ex vivo expanded FM-MSCs preferentially home to damaged tissues promoting regenerative processes through their unique biological properties. These characteristics together with their immune-privileged nature and immune suppressive activity, a low infection rate and young age of placenta compared to other sources of SCs make FM-MSCs an attractive target for cell-based therapy and a valuable tool in regenerative medicine, currently being evaluated in clinical trials. In the present study we investigated the permissivity of FM-MSCs to all members of the human Herpesviridae family, an issue which is relevant to their purification, propagation, conservation and therapeutic use, as well as to their potential role in the vertical transmission of viral agents to the fetus and to their potential viral vector-mediated genetic modification. We present here evidence that FM-MSCs are fully permissive to infection with Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Varicella zoster virus (VZV), and Human Cytomegalovirus (HCMV), but not with Epstein-Barr virus (EBV), Human Herpesvirus-6, 7 and 8 (HHV-6, 7, 8) although these viruses are capable of entering FM-MSCs and transient, limited viral gene expression occurs. Our findings therefore strongly suggest that FM-MSCs should be screened for the presence of herpesviruses before xenotransplantation. In addition, they suggest that herpesviruses may be indicated as viral vectors for gene expression in MSCs both in gene therapy applications and in the selective induction of differentiation.  

5.1289           Rab18 Binds to Hepatitis C Virus NS5A and Promotes Interaction between Sites of Viral Replication and Lipid Droplets

Salloum, S., Wang, H., Ferguson, C., Parton, R.G. and Tai, A.W. PloS Pathogens, 9(8), e1003513 (2013)   Hepatitis C virus (HCV) is a single-stranded RNA virus that replicates on endoplasmic reticulum-derived membranes. HCV particle assembly is dependent on the association of core protein with cellular lipid droplets (LDs). However, it remains uncertain whether HCV assembly occurs at the LD membrane itself or at closely associated ER membranes. Furthermore, it is not known how the HCV replication complex and progeny genomes physically associate with the presumed sites of virion assembly at or near LDs. Using an unbiased proteomic strategy, we have found that Rab18 interacts with the HCV nonstructural protein NS5A. Rab18 associates with LDs and is believed to promote physical interaction between LDs and ER membranes. Active (GTP-bound) forms of Rab18 bind more strongly to NS5A than a constitutively GDP-bound mutant. NS5A colocalizes with Rab18-positive LDs in HCV-infected cells, and Rab18 appears to promote the physical association of NS5A and other replicase components with LDs. Modulation of Rab18 affects genome replication and possibly also the production of infectious virions. Our results support a model in which specific interactions between viral and cellular proteins may promote the physical interaction between membranous HCV replication foci and lipid droplets.  

5.1290           Evidence suggesting that HCV p7 protects E2 glycoprotein from premature degradation during virus production

Atoom, A.M., Jones, D.M. and Russell, R.S. Virus Res., 176, 199-210 (2013)   The hepatitis C virus (HCV) genome encodes a 63 amino acid (aa) protein, p7, which is located between the structural and non-structural proteins. p7 localizes to endoplasmic reticulum membranes and is composed of two transmembrane domains (TM1 and TM2) and a cytoplasmic loop. While its exact role is unknown, p7 is crucial for assembly and/or release of infectious virus production in cell culture, as well as infectivity in chimpanzees. The contribution of p7 to the HCV life cycle may result from at least two distinct roles. Firstly, several studies have shown that p7 acts as an ion channel, the functionality of which is critical for infection. Secondly, p7 interacts with NS2 in a manner that may regulate the targeting of other structural proteins during the assembly process. In this study, we observed that mutations in TM1 and the cytoplasmic loop of p7 decreased infectious virus production in a single-cycle virus production assay. Analysis of intra- and extracellular virus titers indicated that p7 functions at a stage prior to generation of infectious particles. These effects were not due to altered RNA replication since no effects on levels of NS3 or NS5A protein were observed, and were not a consequence of altered recruitment of core protein to lipid droplets. Similarly, these mutations seemingly did not prevent nucleocapsid oligomerization. Importantly, we found that an alanine triplet substitution including the two basic residues of the cytoplasmic loop, which is integral to p7 ion channel function, significantly reduced E2 glycoprotein levels. A time course experiment tracking E2 levels indicated that E2 was degraded over time, as opposed to being synthesized in reduced quantities. The results of this study provide strong evidence that one of the functions of p7 is to protect HCV glycoproteins from premature degradation during virion morphogenesis.  

5.1291           Opposing actions of hippocampus TNFα receptors on limbic seizure susceptibility

Weinberg, M.S., Blake, B.L: and McCown, T.J. Exp. Neurol., 247, 429-437 (2013)   Resected epileptic tissues exhibit elements of chronic neuroinflammation that include elevated TNFα and increased TNFα receptor activation, but the seizure related consequences of chronic TNFα expression remain unknown. Twenty four hours after acute limbic seizures the rat hippocampus exhibited a rapid upregulation of TNFR1, but a simultaneous downregulation of TNFR2. These limbic seizures also evoked significant increases in measures of neuroinflammation and caused significant neuronal cell death in both the hilus and CA3 of the hippocampus. In order to mimic a state of chronic TNFα exposure, adeno-associated viral vectors were packaged with a TNF receptor 1 (TNFR1) specific agonist, human TNFα, or a TNF receptor 1/2 agonist, rat TNFα. Subsequently, chronic hippocampal overexpression of either TNFR ligand caused microglial activation and blood–brain barrier compromise, a pattern similar to limbic seizure-induced neuroinflammation. However, no evidence was found for neuronal cell death or spontaneous seizure activity. Thus, chronic, in vivo TNFα expression and the subsequent neuroinflammation alone did not cause cell death or elicit seizure activity. In contrast, chronic hippocampal activation of TNFR1 alone significantly increased limbic seizure sensitivity in both amygdala kainic acid and electrical amygdala kindling models, while chronic activation of both TNFR1 and TNFR2 significantly attenuated the amygdala kindling rate. With regard to endogenous TNFα, chronic hippocampal expression of a TNFα decoy receptor significantly reduced seizure-induced cell death in the hippocampus, but did not alter seizure susceptibility. These findings suggest that blockade of endogenous TNFα could attenuate seizure related neuropathology, while selective activation of TNFR2 could exert beneficial therapeutic effects on in vivo seizure sensitivity.  

5.1292           Distinct host cell fates for human malignant melanoma targeted by oncolytic rodent parvoviruses

Vollmers, E.M. and Tattersall, P. Virology, 446, 37-48 (2013)   The rodent parvoviruses are known to be oncoselective, and lytically infect many transformed human cells. Because current therapeutic regimens for metastatic melanoma have low response rates and have little effect on improving survival, this disease is a prime candidate for novel approaches to therapy, including oncolytic parvoviruses. Screening of low-passage, patient-derived melanoma cell lines for multiplicity-dependent killing by a panel of five rodent parvoviruses identified LuIII as the most melanoma-lytic. This property was mapped to the LuIII capsid gene, and an efficiently melanoma tropic chimeric virus shown to undergo three types of interaction with primary human melanoma cells: (1) complete lysis of cultures infected at very low multiplicities; (2) acute killing resulting from viral protein synthesis and DNA replication, without concomitant expansion of the infection, due to failure to export progeny virions efficiently; or (3) complete resistance that operates at an intracellular step following virion uptake, but preceding viral transcription.  

5.1293           Presynaptic Neurexin-3 Alternative Splicing trans-Synaptically Controls Postsynaptic AMPA Receptor Trafficking

Aoto, J., Martinelli, D.C., Malenka, R.C., Tabuchi, K. and Südof, T.C. Cell, 154(1), 75-88 (2013)   Neurexins are essential presynaptic cell adhesion molecules that are linked to schizophrenia and autism and are subject to extensive alternative splicing. Here, we used a genetic approach to test the physiological significance of neurexin alternative splicing. We generated knockin mice in which alternatively spliced sequence #4 (SS4) of neuexin-3 is constitutively included but can be selectively excised by cre-recombination. SS4 of neurexin-3 was chosen because it is highly regulated and controls neurexin binding to neuroligins, LRRTMs, and other ligands. Unexpectedly, constitutive inclusion of SS4 in presynaptic neurexin-3 decreased postsynaptic AMPA, but not NMDA receptor levels, and enhanced postsynaptic AMPA receptor endocytosis. Moreover, constitutive inclusion of SS4 in presynaptic neurexin-3 abrogated postsynaptic AMPA receptor recruitment during NMDA receptor-dependent LTP. These phenotypes were fully rescued by constitutive excision of SS4 in neurexin-3. Thus, alternative splicing of presynaptic neurexin-3 controls postsynaptic AMPA receptor trafficking, revealing an unanticipated alternative splicing mechanism for trans-synaptic regulation of synaptic strength and long-term plasticity.  

5.1294           Cardiac-Selective Expression of Extracellular Superoxide Dismutase After Systemic Injection of Adeno-Associated Virus 9 Protects the Heart Against Post–Myocardial Infarction Left Ventricular Remodeling

Konkalmatt, P.R., Beyers, R.J., O’Connor, D.M., Xu, Y., Seaman, M.E.and French, B.A. Circ. Cardiovasc. Imaging, 6(3), 478-486 (2013)   Background—Cardiac magnetic resonance imaging has not been used previously to document the attenuation of left ventricular (LV) remodeling after systemic gene delivery. We hypothesized that targeted expression of extracellular superoxide dismutase (EcSOD) via the cardiac troponin-T promoter would protect the mouse heart against both myocardial infarction (MI) and subsequent LV remodeling. Methods and Results—Using reporter genes, we first compared the specificity, time course, magnitude, and distribution of gene expression from adeno-associated virus (AAV) 1, 2, 6, 8, and 9 after intravenous injection. The troponin-T promoter restricted gene expression largely to the heart for all AAV serotypes tested. AAV1, 6, 8, and 9 provided early-onset gene expression that approached steady-state levels within 2 weeks. Gene expression was highest with AAV9, which required only 3.15×10¹¹ viral genomes per mouse to achieve an 84% transduction rate. AAV9-mediated, cardiac-selective gene expression elevated EcSOD enzyme activity in heart by 5.6-fold (P=0.015), which helped protect the heart against both acute MI and subsequent LV remodeling. In acute MI, infarct size in EcSOD-treated mice was reduced by 40% compared with controls (P=0.035). In addition, we found that cardiac-selective expression of EcSOD increased myocardial capillary fractional area and decreased neutrophil infiltration after MI. In a separate study of LV remodeling, after a 60-minute coronary occlusion, cardiac magnetic resonance imaging revealed that LV volumes at days 7 and 28 post-MI were significantly lower in the EcSOD group compared with controls.  

5.1295           scAAV-mediated gene transfer of interleukin-1-receptor antagonist to synovium and articular cartilage in large mammalian joints

Watson, R.S., Broome, T.A., Levings, P.P., Rice, B.L., Kay, J.D., Smith, A.D., Gouze, A.D., Gouze, J-N., Dacanay, E.A., Hauswirth, W.W., Nickerson, D.M., Dark, M.U., Colahan, P.T. and Ghivizzani, S.C. Gene Therapy, 20(6), 670-677 82013)   With the long-term goal of developing a gene-based treatment for osteoarthritis (OA), we performed studies to evaluate the equine joint as a model for adeno-associated virus (AAV)-mediated gene transfer to large, weight-bearing human joints. A self-complementary AAV2 vector containing the coding regions for human interleukin-1-receptor antagonist (hIL-1Ra) or green fluorescent protein was packaged in AAV capsid serotypes 1, 2, 5, 8 and 9. Following infection of human and equine synovial fibroblasts in culture, we found that both were only receptive to transduction with AAV1, 2 and 5. For these serotypes, however, transgene expression from the equine cells was consistently at least 10-fold higher. Analyses of AAV surface receptor molecules and intracellular trafficking of vector genomes implicate enhanced viral uptake by the equine cells. Following delivery of 1 × 10¹¹ vector genomes of serotypes 2, 5 and 8 into the forelimb joints of the horse, all three enabled hIL-1Ra expression at biologically relevant levels and effectively transduced the same cell types, primarily synovial fibroblasts and, to a lesser degree, chondrocytes in articular cartilage. These results provide optimism that AAV vectors can be effectively adapted for gene delivery to large human joints affected by OA.  

5.1296            Modulation of feeding by chronic rAAV expression of a relaxin-3 peptide agonist in rat hypothalamus

Ganella, D.E., Callander, G.E., Ma, S., Bye, C.R., Gundlach, A.L. and Bathgate, R.A.D. Gene Therapy, 20(7), 703-716 (2013)   Relaxin-3 is a neuropeptide that is abundantly expressed by discrete brainstem neuron populations that broadly innervate forebrain areas rich in the relaxin-3 G-protein-coupled-receptor, RXFP3. Acute and subchronic central administration of synthetic relaxin-3 or an RXFP3-selective agonist peptide, R3/I5, increase feeding and body weight in rats. Intrahypothalamic injection of relaxin-3 also increases feeding. In this study, we developed a recombinant adeno-associated virus 1/2 (rAAV1/2) vector that drives expression and constitutive secretion of bioactive R3/I5 and assessed the effect of intrahypothalamic injections on daily food intake and body weight gain in adult male rats over 8 weeks. In vitro testing revealed that the vector rAAV1/2-fibronectin (FIB)-R3/I5 directs the constitutive secretion of bioactive R3/I5 peptide. Bilateral injection of rAAV1/2-FIB-R3/I5 vector into the paraventricular nucleus produced an increase in daily food intake and body weight gain (P<0.01, ~23%, respectively), relative to control treatment. In a separate cohort of rats, quantitative polymerase chain reaction analysis of hypothalamic mRNA revealed strong expression of R3/I5 transgene at 3 months post-rAAV1/2-FIB-R3/I5 infusion. Levels of mRNA transcripts for the relaxin-3 receptor RXFP3, the hypothalamic ‘feeding’ peptides neuropeptide Y, AgRP and POMC, and the reproductive hormone, GnRH, were all similar to control, whereas vasopressin and oxytocin (OT) mRNA levels were reduced by ~25% (P=0.051) and ~50% (P<0.005), respectively, in rAAV1/2-FIB-R3/I5-treated rats (at 12 weeks, n=9/8 rats per group). These data demonstrate for the first time that R3/I5 is effective in modulating feeding in the rat by chronic hypothalamic RXFP3 activation and suggest a potential underlying mechanism involving altered OT signalling. Importantly, there was no desensitization of the feeding response over the treatment period and no apparent deleterious health effects, indicating that targeting the relaxin-3–RXFP3 system may be an effective long-term therapy for eating disorders.  

5.1297           Lack of myotubularin (MTM1) leads to muscle hypotrophy through unbalanced regulation of the autophagy and ubiquitin-proteasome pathways

Al-Qusairi. M., Prokic, I., Amoasii, L., Kretz, C., Messaddeq, N., Mandel, J-L. and Laporte, J. FASEB J., 27, 3384-3394 (2013)   Mutations in the phosphoinositide phosphatase myotubularin (MTM1) results in X-linked myotubular/centronuclear myopathy (XLMTM), characterized by a severe decrease in muscle mass and strength in patients and murine models. However, the molecular mechanism involved in the muscle hypotrophy is unclear. Here we show that the IGF1R/Akt pathway is affected in Mtm1-deficient murine muscles, characterized by an increase in IGF1 receptor and Akt levels in both the presymptomatic and symptomatic phases. Moreover, up-regulation of atrogenes was observed in the presymptomatic phase of the myopathy, supporting overactivation of the ubiquitin-proteasome pathway. In parallel, the autophagy machinery was affected as indicated by the increase in the number of autophagosomes and of autophagy markers, such as LC3 and P62. However, phosphorylation of FOXO3a and mTOR were abnormal at late but not at early stages of the disease, suggesting that myotubularin acts both upstream in the IGF1R/Akt pathway and downstream on the balance between the autophagy and ubiquitin-proteasome pathways in vivo. Adeno-associated virus-mediated delivery of Mtm1 into Mtm1-null muscles rescued muscle mass and normalized the expression levels of IGF1 receptor, the ubiquitin-proteasome pathway, and autophagy markers. These data support the hypothesis that the unbalanced regulation of the ubiquitin proteasome pathway and the autophagy machinery is a primary cause of the XLMTM pathogenesis.—Al-Qusairi, L., Prokic, I., Amoasii, L., Kretz, C., Messaddeq, N., Mandel, J.-L., Laporte, J. Lack of myotubularin (MTM1) leads to muscle hypotrophy through unbalanced regulation of the autophagy and ubiquitin-proteasome pathways.  

5.1298           PD-L1/B7-H1 Regulates the Survival but Not the Function of CD8+ T Cells in Herpes Simplex Virus Type 1 Latently Infected Trigeminal Ganglia

Jeon, S., St. Leger, A. J., Cherpes, T.L:, Sheridan, B.S. and Hendricks, R.L:

1. Immunol., 190(12), 6277-6286 (2013)

  HSV type 1 (HSV-1)–specific CD8⁺ T cells provide immunosurveillance of trigeminal ganglion (TG) neurons that harbor latent HSV-1. In C57BL/6 mice, the TG-resident CD8⁺ T cells are HSV specific and maintain a 1:1 ratio of cells recognizing an immunodominant epitope on viral glycoprotein B (gB_498–505-Tet⁺) and cells reactive to subdominant epitopes (gB-Tet⁻). The gB-Tet⁻ CD8⁺ T cells maintain their frequency in TG by balancing a higher rate of proliferation with a correspondingly higher rate of apoptosis. The increased apoptosis is associated with higher expression of programmed death-1 (PD-1) on gB-Tet⁻ CD8⁺ T cells and the interaction with PD-1 ligand (PD-L1/B7-H1). IFN-γ regulated expression of the PD-1 ligand (PD-L1/B7-H1) on neurons bearing higher copies of latent viral genome. In latently infected TG of B7-H1^−/− mice, the number and frequency of PD-1⁺ gB-Tet⁻ CD8⁺ T cells increases dramatically, but gB-Tet⁻ CD8⁺ T cells remain largely nonfunctional and do not provide increased protection from HSV-1 reactivation in ex vivo cultures of latently infected TG. Unlike observations in some chronic infection models, B7-H1 blockade did not increase the function of exhausted gB-Tet⁻ CD8 T cells in latently infected TG.  

5.1299           Recombinant AAV9-TLK1B Administration Ameliorates Fractionated Radiation-Induced Xerostomia

Srinivasan, P., Shanmugam, T., Dayton, R.D., Palaniyandi, S., Abreo, F., Caldito, G., Klein, R.L.and Sunavala-Dossabhoy, G. Human Gene Therapy, 24(6), 604-612 82013)   Salivary glands are highly susceptible to radiation, and patients with head and neck cancer treated with radiotherapy invariably suffer from its distressing side effect, salivary hypofunction. The reduction in saliva disrupts oral functions, and significantly impairs oral health. Previously, we demonstrated that adenoviral-mediated expression of Tousled-like kinase 1B (TLK1B) in rat submandibular glands preserves salivary function after single-dose ionizing radiation. To achieve long-term transgene expression for protection of salivary gland function against fractionated radiation, this study examines the usefulness of recombinant adeno-associated viral vector for TLK1B delivery. Lactated Ringers or AAV2/9 with either TLK1B or GFP expression cassette were retroductally delivered to rat submandibular salivary glands (10¹¹ vg/gland), and animals were exposed, or not, to 20 Gy in eight fractions of 2.5 Gy/day. AAV2/9 transduced predominantly the ductal cells, including the convoluted granular tubules of the submandibular glands. Transgene expression after virus delivery could be detected within 5 weeks, and stable gene expression was observed till the end of study. Pilocarpine-stimulated saliva output measured at 8 weeks after completion of radiation demonstrated >10-fold reduction in salivary flow in saline- and AAV2/9-GFP-treated animals compared with the respective nonirradiated groups (90.8% and 92.5% reduction in salivary flow, respectively). Importantly, there was no decrease in stimulated salivary output after irradiation in animals that were pretreated with AAV2/9-TLK1B (121.5% increase in salivary flow; p<0.01). Salivary gland histology was better preserved after irradiation in TLK1B-treated group, though not significantly, compared with control groups. Single preemptive delivery of AAV2/9-TLK1B averts salivary dysfunction resulting from fractionated radiation. Although AAV2/9 transduces mostly the ductal cells of the gland, their protection against radiation assists in preserving submandibular gland function. AAV2/9-TLK1B treatment could prove beneficial in attenuating xerostomia in patients with head and neck cancer undergoing radiotherapy.    

5.1300           Direct and Retrograde Transduction of Nigral Neurons with AAV6, 8, and 9 and Intraneuronal Persistence of Viral Particles

Löw, K., Aebischer, P. and Schneider, B.L. Human Gene Therapy, 24(6), 613-629 (2013)   Recombinant adeno-associated viral (AAV) vectors of serotypes 6, 8, and 9 were characterized as tools for gene delivery to dopaminergic neurons in the substantia nigra for future gene therapeutic applications in Parkinson's disease. While vectors of all three serotypes transduced nigral dopaminergic neurons with equal efficiency when directly injected to the substantia nigra, AAV6 was clearly superior to AAV8 and AAV9 for retrograde transduction of nigral neurons after striatal delivery. For sequential transduction of nigral dopaminergic neurons, the combination of AAV9 with AAV6 proved to be more powerful than AAV8 with AAV6 or repeated AAV6 administration. Surprisingly, single-stranded viral genomes persisted in nigral dopaminergic neurons within cell bodies and axon terminals in the striatum, and intact assembled AAV capsid was enriched in nuclei of nigral neurons, 4 weeks after virus injections to the substantia nigra. 6-Hydroxydopamine (6-OHDA)–induced degeneration of dopaminergic neurons in the substantia nigra reduced the number of viral genomes in the striatum, in line with viral genome persistence in axon terminals. However, 6-OHDA–induced axonal degeneration did not induce any transsynaptic spread of AAV infection in the striatum. Therefore, the potential presence of viral particles in axons may not represent an important safety issue for AAV gene therapy applications in neurodegenerative diseases.  

5.1301           Ultrastructural analysis of hepatitis C virus particles

Catanese, M.T., Uryu, K., Kopp, M., Edwards, T.J., Andrus, L., Rice, W.J., Silvestry, M., Kuhn, R.J. and Rice, C.M. PNAS, 110(23), 9505-9510 (2013)   Hepatitis C virus (HCV) is a major cause of chronic liver disease, with an estimated 170 million people infected worldwide. Low yields, poor stability, and inefficient binding to conventional EM grids have posed significant challenges to the purification and structural analysis of HCV. In this report, we generated an infectious HCV genome with an affinity tag fused to the E2 envelope glycoprotein. Using affinity grids, previously described to isolate proteins and macromolecular complexes for single-particle EM, we were able to purify enveloped particles directly from cell culture media. This approach allowed for rapid in situ purification of virions and increased particle density that were instrumental for cryo-EM and cryoelectron tomography (cryo-ET). Moreover, it enabled ultrastructural analysis of virions produced by primary human hepatocytes. HCV appears to be the most structurally irregular member of the Flaviviridae family. Particles are spherical, with spike-like projections, and heterogeneous in size ranging from 40 to 100 nm in diameter. Exosomes, although isolated from unfractionated culture media, were absent in highly infectious, purified virus preparations. Cryo-ET studies provided low-resolution 3D structural information of highly infectious virions. In addition to apolipoprotein (apo)E, HCV particles also incorporate apoB and apoA-I. In general, host apolipoproteins were more readily accessible to antibody labeling than HCV glycoproteins, suggesting either lower abundance or masking by host proteins.  

5.1302           Pathological hypertrophy amelioration by PRAS40-mediated inhibition of mTORC1

Völkers, M., Toko, H., Doroudgar, S., Din, S., Quijada, P., Joyo, A.Y., Ornelas, L., Joyo, E., Thuerauf, D.J., Konstandin, M.H., Gude, N., Glembotski, C.C. and Sussman, M.A. PNAS, 110(31), 12661-12666 (2013)   Mechanistic target of rapamycin complex 1 (mTORC1), necessary for cellular growth, is regulated by intracellular signaling mediating inhibition of mTORC1 activation. Among mTORC1 regulatory binding partners, the role of Proline Rich AKT Substrate of 40 kDa (PRAS40) in controlling mTORC1 activity and cellular growth in response to pathological and physiological stress in the heart has never been addressed. This report shows PRAS40 is regulated by AKT in cardiomyocytes and that AKT-driven phosphorylation relieves the inhibitory function of PRAS40. PRAS40 overexpression in vitro blocks mTORC1 in cardiomyocytes and decreases pathological growth. Cardiomyocyte-specific overexpression in vivo blunts pathological remodeling after pressure overload and preserves cardiac function. Inhibition of mTORC1 by PRAS40 preferentially promotes protective mTORC2 signaling in chronic diseased myocardium. In contrast, strong PRAS40 phosphorylation by AKT allows for physiological hypertrophy both in vitro and in vivo, whereas cardiomyocyte-specific overexpression of a PRAS40 mutant lacking capacity for AKT-phosphorylation inhibits physiological growth in vivo, demonstrating that AKT-mediated PRAS40 phosphorylation is necessary for induction of physiological hypertrophy. Therefore, PRAS40 phosphorylation acts as a molecular switch allowing mTORC1 activation during physiological growth, opening up unique possibilities for therapeutic regulation of the mTORC1 complex to mitigate pathologic myocardial hypertrophy by PRAS40.  

5.1303           Astrocyte-derived ATP modulates depressive-like behaviors

Cao, X. et al Nature Med., 19(6), 773-777 (2013)   Major depressive disorder (MDD) is a cause of disability that affects approximately 16% of the world's population¹; however, little is known regarding the underlying biology of this disorder. Animal studies, postmortem brain analyses and imaging studies of patients with depression have implicated glial dysfunction in MDD pathophysiology^{2, 3, 4, 5, 6, 7}. However, the molecular mechanisms through which astrocytes modulate depressive behaviors are largely uncharacterized. Here, we identified ATP as a key factor involved in astrocytic modulation of depressive-like behavior in adult mice. We observed low ATP abundance in the brains of mice that were susceptible to chronic social defeat. Furthermore, we found that the administration of ATP induced a rapid antidepressant-like effect in these mice. Both a lack of inositol 1,4,5-trisphosphate receptor type 2 and transgenic blockage of vesicular gliotransmission induced deficiencies in astrocytic ATP release, causing depressive-like behaviors that could be rescued via the administration of ATP. Using transgenic mice that express a G_q G protein–coupled receptor only in astrocytes to enable selective activation of astrocytic Ca²⁺ signaling, we found that stimulating endogenous ATP release from astrocytes induced antidepressant-like effects in mouse models of depression. Moreover, we found that P2X2 receptors in the medial prefrontal cortex mediated the antidepressant-like effects of ATP. These results highlight astrocytic ATP release as a biological mechanism of MDD.  

5.1304           Mapping the Structural Determinants Responsible for Enhanced T Cell Activation to the Immunogenic Adeno-Associated Virus Capsid from Isolate Rhesus 32.33

Mays, L.E., Wang, L., Tenney, R., Bell, P., Nam, H-J., Lin, J., Gurda, B., Van Vliet, K., Mikals, K., Agbandje-McKenna, M. and Wilson, J.M.

1. Virol., 87(17), 9473-9485 (2013)

  Avoiding activation of immunity to vector-encoded proteins is critical to the safe and effective use of adeno-associated viral (AAV) vectors for gene therapy. While commonly used serotypes, such as AAV serotypes 1, 2, 7, 8, and 9, are often associated with minimal and/or dysfunctional CD8⁺ T cell responses in mice, the threshold for immune activation appears to be lower in higher-order species. We have modeled this discrepancy within the mouse by identifying two capsid variants with differential immune activation profiles: AAV serotype 8 (AAV8) and a hybrid between natural rhesus isolates AAVrh32 and AAVrh33 (AAVrh32.33). Here, we aimed to characterize the structural determinants of the AAVrh32.33 capsid that augment cellular immunity to vector-encoded proteins or those of AAV8 that may induce tolerance. We hypothesized that the structural domain responsible for differential immune activation could be mapped to surface-exposed regions of the capsid, such as hypervariable regions (HVRs) I to IX of VP3. To test this, a series of hybrid AAV capsids was constructed by swapping domains between AAV8 and AAVrh32.33. By comparing their ability to generate transgene-specific T cells in vivo versus the stability of transgene expression in the muscle, we confirmed that the functional domain lies within the VP3 portion of the capsid. Our studies were able to exclude the regions of VP3 which are not sufficient for augmenting the cellular immune response, notably, HVRs I, II, and V. We have also identified HVR IV as a region of interest in conferring the efficiency and stability of muscle transduction to AAVrh32.33.  

5.1305           In Vivo–Directed Evolution of a New Adeno-Associated Virus for Therapeutic Outer Retinal Gene Delivery from the Vitreous

Dalkara, D., Byrne, L.C., Klimczak, R.R., Visel, M., Yin, L., merigan, W.H., Flannary, J.G. and Schaffer, D.V. Sci. Transl. Med., 5, 189ra76 (2013)   Inherited retinal degenerative diseases are a clinically promising focus of adeno-associated virus (AAV)–mediated gene therapy. These diseases arise from pathogenic mutations in mRNA transcripts expressed in the eye’s photoreceptor cells or retinal pigment epithelium (RPE), leading to cell death and structural deterioration. Because current gene delivery methods require an injurious subretinal injection to reach the photoreceptors or RPE and transduce just a fraction of the retina, they are suitable only for the treatment of rare degenerative diseases in which retinal structures remain intact. To address the need for broadly applicable gene delivery approaches, we implemented in vivo–directed evolution to engineer AAV variants that deliver the gene cargo to the outer retina after injection into the eye’s easily accessible vitreous humor. This approach has general implications for situations in which dense tissue penetration poses a barrier for gene delivery. A resulting AAV variant mediated widespread delivery to the outer retina and rescued the disease phenotypes of X-linked retinoschisis and Leber’s congenital amaurosis in corresponding mouse models. Furthermore, it enabled transduction of primate photoreceptors from the vitreous, expanding its therapeutic promise.  

5.1306           Oncosuppressive Suicide Gene Virotherapy “PVH1-yCD/5-FC” for Pancreatic Peritoneal Carcinomatosis Treatment: NFκB and Akt/PI3K Involvement

Rejiba, S., Bigand, C., parmentier, C., Masmoudi, A. and Hajri, A. PloS One, 8(8), e70594 (2013)   Peritoneal carcinomatosis is common in advanced pancreatic cancer. Despite current standard treatment, patients with this disease until recently were considered incurable. Cancer gene therapy using oncolytic viruses have generated much interest over the past few years. Here, we investigated a new gene directed enzyme prodrug therapy (GDEPT) approach for an oncosuppressive virotherapy strategy using parvovirus H1 (PV-H1) which preferentially replicates and kills malignant cells. Although, PV-H1 is not potent enough to destroy tumors, it represents an attractive vector for cancer gene therapy. We therefore sought to determine whether the suicide gene/prodrug system, yCD/5-FC could be rationally combined to PV-H1 augmenting its intrinsic oncolytic activity for pancreatic cancer prevention and treatment. We showed that the engineered recombinant parvovirus rPVH1-yCD with 5-FC treatment increased significantly the intrinsic cytotoxic effect and resulted in potent induction of apoptosis and tumor growth inhibition in chemosensitive and chemoresistant cells. Additionally, the suicide gene-expressing PV-H1 infection reduced significantly the constitutive activities of NFκB and Akt/PI3K. Combination of their pharmacological inhibitors (MG132 and LY294002) with rPVH1-yCD/5-FC resulted in substantial increase of antitumor activity. In vivo, high and sustained expression of NS1 and yCD was observed in the disseminated tumor nodules and absent in normal tissues. Treatment of mice bearing intraperitoneal pancreatic carcinomatosis with rPVH1-yCD/5-FC resulted in a drastic inhibition of tumor cell spreading and subsequent increase in long-term survival. Together, the presented data show the improved oncolytic activity of wPV-H1 by yCD/5-FC and thus provides valuable effective and promising virotherapy strategy for prevention of tumor recurrence and treatment. In the light of this study, the suicide gene parvovirotherapy approach represents a new weapon in the war against pancreatic cancer. Moreover, these preliminary accomplishments are opening new field for future development of new combined targeted therapies to have a meaningful impact on advanced cancer.  

5.1307           Cervicovaginal secretions protect from human papillomavirus infection: Effects of vaginal douching

Chu, T-Y., Chang, Y-C. and Ding, D-C. Taiwanese J. Obstetrics & Gynecol., 52, 241-245 (2013)   Objective Cervicovaginal secretions (CVSs) are reported to protect against human papillomavirus (HPV) infection. Although vaginal douching is known to clear both viral inoculants and CVSs, its effect on CVSs in women with HPV infection is unknown. Materials and Methods The in vitro HPV pseudovirus infection system was used to test the protective activity of CVSs against HPV infection in samples collected before and after vaginal douching. To simulate different time points of vaginal douching in relation to viral exposure, the cell CVS reconstitute was washed after different viral exposure durations. Results In the CVSs of premenopausal and postmenopausal women who did not perform douching, the CVSs inhibited HPV infection by 56.7 ± 1.8% and 53.6 ± 2.5%, respectively; in women who had performed douching, the CVSs inhibited HPV infection by only 31.2 ± 7.1%, which was significantly lower (p < 0.01). Cell washing effectively cleared 60–90% of the infectious load with the greatest activity occurring within 30 minutes after inoculation. In the presence of CVSs, a sustained inhibition of HPV infection existed for up to 8 hours after HPV exposure, and cell washing increased the clearance to up to 82–93% of the infectious load. Conclusion This study confirms the protective activity of CVSs against HPV infection regardless of age. In this in vitro study, the net effect of douching was found to be beneficial.    

5.1308           The strange and critical intersection of hepatitis C and lipoprotein metabolism: “C-zing” the oil

Caldwell, S., Hoehn, K.L. and Hahn, Y.S. Hepatology, 57(5), 1684-1687 (2013)   No abstract available  

5.1309           The antimalarial ferroquine is an inhibitor of hepatitis C virus

Vausselin, T., Calland, N., Belouzard, S., Decamps, V., Douam, F., helle, F., Francois, C., Lavillette, D., Duverlie, G., Wahid, A., Feneant, L., Cocquerel, L., Guerardel, Y., Wychowski, C., Biot, C. and Dubuisson, J. Hepatology, 58(1), 86-97 (2013)   Hepatitis C virus (HCV) is a major cause of chronic liver disease. Despite recent success in improving anti-HCV therapy, additional progress is still needed to develop cheaper and interferon (IFN)-free treatments. Here, we report that ferroquine (FQ), an antimalarial ferrocenic analog of chloroquine, is a novel inhibitor of HCV. FQ potently inhibited HCV infection of hepatoma cell lines by affecting an early step of the viral life cycle. The antiviral activity of FQ on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition to its effect on HCV entry, FQ also inhibited HCV RNA replication, albeit at a higher concentration. We also showed that FQ has no effect on viral assembly and virion secretion. Using a binding assay at 4°C, we showed that FQ does not prevent attachment of the virus to the cell surface. Furthermore, virus internalization was not affected by FQ, whereas the fusion process was impaired in the presence of FQ as shown in a cell-cell fusion assay. Finally, virus with resistance to FQ was selected by sequential passage in the presence of the drug, and resistance was shown to be conferred by a single mutation in E1 glycoprotein (S327A). By inhibiting cell-free virus transmission using a neutralizing antibody, we also showed that FQ inhibits HCV cell-to-cell spread between neighboring cells. Combinations of FQ with IFN, or an inhibitor of HCV NS3/4A protease, also resulted in additive to synergistic activity. Conclusion: FQ is a novel, interesting anti-HCV molecule that could be used in combination with other direct-acting antivirals.  

5.1310           BK Polyomavirus Genotypes Represent Distinct Serotypes with Distinct Entry Tropism

Pastrana, D.V., Ray, U., Magaldi, T.G., Schowalter, R.M., Cuburu, N. and Buck, C.B.

1. Virol., 87(18), 10105-10113 (2013)

  BK polyomavirus (BKV) causes significant urinary tract pathogenesis in immunosuppressed individuals, including kidney and bone marrow transplant recipients. It is currently unclear whether BKV-neutralizing antibodies can moderate or prevent BKV disease. We developed reporter pseudoviruses based on seven divergent BKV isolates and performed neutralization assays on sera from healthy human subjects. The results demonstrate that BKV genotypes I, II, III, and IV are fully distinct serotypes. While nearly all healthy subjects had BKV genotype I-neutralizing antibodies, a majority of subjects did not detectably neutralize genotype III or IV. Surprisingly, BKV subgenotypes Ib1 and Ib2 can behave as fully distinct serotypes. This difference is governed by as few as two residues adjacent to the cellular glycan receptor-binding site on the virion surface. Serological analysis of mice given virus-like particle (VLP)-based BKV vaccines confirmed these findings. Mice administered a multivalent VLP vaccine showed high-titer serum antibody responses that potently cross-neutralized all tested BKV genotypes. Interestingly, each of the neutralization serotypes bound a distinct spectrum of cell surface receptors, suggesting a possible connection between escape from recognition by neutralizing antibodies and cellular attachment mechanisms. The finding implies that different BKV genotypes have different cellular tropisms and pathogenic potentials in vivo. Individuals who are infected with one BKV serotype may remain humorally vulnerable to other BKV serotypes after implementation of T cell immunosuppression. Thus, prevaccinating organ transplant recipients with a multivalent BKV VLP vaccine might reduce the risk of developing posttransplant BKV disease.  

5.1311           Oncolytic Vesicular Stomatitis Virus in an Immunocompetent Model of MUC1-Positive or MUC1-Null Pancreatic Ductal Adenocarcinoma

Hastie, E., Besmer, D.M., Shah, N.R., Murphy, A.M., Moerdyk-Schauwecker, M., Molestina, C., Da Roy, L., Curry, J.M., Mukherjee, P. and Grdzelishvili, V.Z. J.Virol., 87(18), 10283-10294 (2013)   Vesicular stomatitis virus (VSV) is a promising oncolytic agent against various malignancies. Here, for the first time, we tested VSV in vitro and in vivo in a clinically relevant, immunocompetent mouse model of pancreatic ductal adenocarcinoma (PDA). Our system allows the study of virotherapy against PDA in the context of overexpression (80% of PDA patients) or no expression of human mucin 1 (MUC1), a major marker for poor prognosis in patients. In vitro, we tested three VSV recombinants, wild-type VSV, VSV-green fluorescent protein (VSV-GFP), and a safe oncolytic VSV-ΔM51-GFP, against five mouse PDA cell lines that either expressed human MUC1 or were MUC1 null. All viruses demonstrated significant oncolytic abilities independent of MUC1 expression, although VSV-ΔM51-GFP was somewhat less effective in two PDA cell lines. In vivo administration of VSV-ΔM51-GFP resulted in significant reduction of tumor growth for tested mouse PDA xenografts (+MUC1 or MUC1 null), and antitumor efficacy was further improved when the virus was combined with the chemotherapeutic drug gemcitabine. The antitumor effect was transient in all tested groups. The developed system can be used to study therapies involving various oncolytic viruses and chemotherapeutics, with the goal of inducing tumor-specific immunity while preventing premature virus clearance.  

5.1312           The Merkel Cell Polyomavirus Minor Capsid Protein

Schowalter, R.M. and Buck, C.B. PloS Pathogens, 9(8), e1003558 (2013)   The surface of polyomavirus virions is composed of pentameric knobs of the major capsid protein, VP1. In previously studied polyomavirus species, such as SV40, two interior capsid proteins, VP2 and VP3, emerge from the virion to play important roles during the infectious entry process. Translation of the VP3 protein initiates at a highly conserved Met-Ala-Leu motif within the VP2 open reading frame. Phylogenetic analyses indicate that Merkel cell polyomavirus (MCV or MCPyV) is a member of a divergent clade of polyomaviruses that lack the conserved VP3 N-terminal motif. Consistent with this observation, we show that VP3 is not detectable in MCV-infected cells, VP3 is not found in native MCV virions, and mutation of possible alternative VP3-initiating methionine codons did not significantly affect MCV infectivity in culture. In contrast, VP2 knockout resulted in a >100-fold decrease in native MCV infectivity, despite normal virion assembly, viral DNA packaging, and cell attachment. Although pseudovirus-based experiments confirmed that VP2 plays an essential role for infection of some cell lines, other cell lines were readily transduced by pseudovirions lacking VP2. In cell lines where VP2 was needed for efficient infectious entry, the presence of a conserved myristoyl modification on the N-terminus of VP2 was important for its function. The results show that a single minor capsid protein, VP2, facilitates a post-attachment stage of MCV infectious entry into some, but not all, cell types.  

5.1313           Pseudotyped adeno-associated viral vectors for gene transfer in dermal fibroblasts: implications for wound-healing applications

Balaji, S., King, A., Dhamija, Y., Le, L.D., Shaaban, A.F., Crombleholme, T.M. and Keswani, S.

1. Surg. Res., 184, 691-698 (2013)

  Background Cell-specific gene transfer and sustained transgene expression are goals of cutaneous gene therapy. Pseudotyping strategy with adeno-associated viral (AAV) vectors has the potential to confer unique cellular tropism and transduction efficiency. We hypothesize that pseudotyped AAV vectors have differential tropism and transduction efficiency under normal and wound conditions in dermal fibroblasts. Materials and methods We packaged AAV2 genome with green fluorescent protein reporter in capsids of other serotypes, AAV5, AAV7, and AAV8, producing pseudotyped vectors AAV2/5, AAV2/7, and AAV2/8, respectively. Murine and human dermal fibroblasts were transduced by the different pseudotypes for 24 h at multiplicities of infection 10², 10³, 10⁴, and 10⁵. We assessed transduction efficiency at days 3 and 7. Experiments were repeated in a simulated wound environment by adding 10 ng/mL platelet-derived growth factor-B to culture media. Results Transduction efficiency of the pseudotyped AAV vectors was dose dependent. Multiplicity of infection 10⁵ resulted in significantly higher gene transfer. Under normal culture conditions, the pseudotyping strategy conferred differential transduction of dermal fibroblasts, with significantly enhanced transduction of murine cells by AAV2/5 and AAV2/8 compared with AAV2/2. Adeno-associated virus 2/8 was more efficacious in transducing human cells. Under wound conditions, transduction efficiency of AAV2/2, 2/5, and 2/8 was significantly lower in murine fibroblasts. At day 3 under wound conditions, all vectors demonstrated similar transduction efficiency, but by day 7, the three pseudotyped vectors transduced significantly more murine cells compared with AAV2/2. However, in human cells, there was no significant difference in the transduction efficiency of each pseudotype between normal and wound conditions at both 3 and 7 d. Conclusions The AAV pseudotyping strategy represents a gene transfer technology that can result in differential transduction of dermal fibroblasts. The differences in transduction efficiency in murine and human dermal fibroblasts in both the normal and wound environment highlight issues with translatability of gene transfer techniques. These data provide a template for using pseudotyped AAV vectors in cutaneous applications.  

5.1314           Obesity Promotes Liver Carcinogenesis via Mcl-1 Stabilization Independent of IL-6Rα Signaling

Gruber, S., Straub, B.K., Ackermann, P.J., Wunderlich, C.M., Mauer, J., Seeger, J.M., Büring, H., Heukamp, L., Kashkar, H., Schirmacher, P., Brüning, J.C. and Wunderlich, F.T. Cell Reports, 4, 669-680 82013)   Obesity increases the incidence of hepatocellular carcinoma (HCC) development in part through the activation of obesity-associated proinflammatory signaling. Here, we show that in lean mice, abrogation of IL-6Rα signaling protects against diethylnitrosamine (DEN)-induced HCC development. HCC protection occurs via Mcl-1 destabilization, thus promoting hepatocyte apoptosis. IL-6 regulates Mcl-1 stability via the inhibition of PP-1α expression, promoting GSK-3β inactivation. In addition, IL-6 suppresses expression of the Mcl-1 E3 ligase (Mule). Consequently, IL-6Rα deficiency activates PP-1α and Mule expression, resulting in increased Mcl-1 turnover and protection against HCC development. In contrast, in obesity, inhibition of PP-1α and Mule expression, leading to Mcl-1 stabilization, occurs independently of IL-6 signaling. Collectively, this study provides evidence that obesity inhibits hepatocyte apoptosis through Mcl-1 stabilization independent of IL-6 signaling, thus promoting liver carcinogenesis.  

5.1315           Global gene expression profiling of pancreatic islets in mice during streptozotocin-induced β-cell damage and pancreatic Glp-1 gene therapy

Tonne, J.M., Sakuma, T., Deeds, M.C., Munoz-Gomez, M., Barry, M.A., Kudva, Y.C. and Ikeda, Y. Dis.Model Mech., 6(5), 1236-1245 (2013)   Streptozotocin (STZ), a glucosamine-nitrosourea compound, has potent genotoxic effects on pancreatic β-cells and is frequently used to induce diabetes in experimental animals. Glucagon-like peptide-1 (GLP-1) has β-cell protective effects and is known to preserve β-cells from STZ treatment. In this study, we analyzed the mechanisms of STZ-induced diabetes and GLP-1-mediated β-cell protection in STZ-treated mice. At 1 week after multiple low-dose STZ administrations, pancreatic β-cells showed impaired insulin expression, while maintaining expression of nuclear Nkx6.1. This was accompanied by significant upregulation of p53-responsive genes in islets, including a mediator of cell cycle arrest, p21 (also known as Waf1 and Cip1). STZ treatment also suppressed expression of a wide range of genes linked with key β-cell functions or diabetes development, such as G6pc2, Slc2a2 (Glut2), Slc30a8, Neurod1, Ucn3, Gad1, Isl1, Foxa2, Vdr, Pdx1, Fkbp1b and Abcc8, suggesting global β-cell defects in STZ-treated islets. The Tmem229B, Prss53 and Ttc28 genes were highly expressed in untreated islets and strongly suppressed by STZ, suggesting their potential roles in β-cell function. When a pancreas-targeted adeno-associated virus (AAV) vector was employed for long-term Glp-1 gene delivery, pancreatic GLP-1 expression protected mice from STZ-induced diabetes through preservation of the β-cell mass. Despite its potent β-cell protective effects, however, pancreatic GLP-1 overexpression showed limited effects on the global gene expression profiles in the islets. Network analysis identified the programmed-cell-death-associated pathways as the most relevant network in Glp-1 gene therapy. Upon pancreatic GLP-1 expression, upregulation of Cxcl13 and Nptx2 was observed in STZ-damaged islets, but not in untreated normal islets. Given the pro-β-cell-survival effects of Cxcl12 (Sdf-1) in inducing GLP-1 production in α-cells, pancreatic GLP-1-mediated Cxcl13 induction might also play a crucial role in maintaining the integrity of β-cells in damaged islets.  

5.1316           Parvoviral Left-End Hairpin Ears Are Essential during Infection for Establishing a Functional Intranuclear Transcription Template and for Efficient Progeny Genome Encapsidation

Li, L., Cotmore, S.F. and Tattersall, P.

1. Virol., 87(19), 10501-10514 (2013)

  The 121-nucleotide left-end telomere of Minute Virus of Mice (MVM) can be folded into a Y-shaped hairpin with short axial ears that are highly conserved within genus Parvovirus. To explore their potential role(s) during infection, we constructed infectious plasmid clones that lacked one or other ear. Although these were nonviable when transfected into A9 cells, excision of the viral genome and DNA amplification appeared normal, and viral transcripts and proteins were expressed, but progeny virion production was minimal, supporting the idea of a potential role for the ears in genome packaging. To circumvent the absence of progeny that confounded further analysis of these mutants, plasmids were transfected into 293T cells both with and without an adenovirus helper construct, generating single bursts of progeny. These virions bound to A9 cells and were internalized but failed to initiate viral transcription, protein expression, or DNA replication. No defects in mutant virion stability or function could be detected in vitro. Significantly, mutant capsid gene expression and DNA replication could be rescued by coinfection with wild-type virions carrying a replication-competent, capsid-gene-replacement vector. To pinpoint where such complementation occurred, prior transfection of plasmids expressing only MVM nonstructural proteins was explored. NS1 alone, but not NS2, rescued transcription and protein expression from both P4 and P38 promoters, whereas NS1 molecules deleted for their C-terminal transactivation domain did not. These results suggest that the mutant virions reach the nucleus, uncoat, and are converted to duplex DNA but require an intact left-end hairpin structure to form the initiating transcription complex.  

5.1317           Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

Sulli, C., Banik, S.S.R., Schilling, J., Moser, A., Xiang, X., Payne, R., Wanless, A., Willis, S.H., Paes, X., Rucker, J.B. and Doranz, B.J.

1. Virol., 87(19), 10679-10686 (2013)

  The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.  

5.1318           Chimeric Cyanovirin-MPER Recombinantly Engineered Proteins Cause Cell-Free Virolysis of HIV-1

Contarino, M., Bastian, A.R., Venkat, R., Sundaram, R.V.K., McFadden, K., Duffy, C., Gangupomu, V., Baker, M., Abrams, C. and Chaiken, I. Antimicrob. Agents Chemother., 57(10), 4743-4750 (2013)   Human immunodeficiency virus (HIV) is the primary etiologic agent responsible for the AIDS pandemic. In this work, we used a chimeric recombinant protein strategy to test the possibility of irreversibly destroying the HIV-1 virion using an agent that simultaneously binds the Env protein and viral membrane. We constructed a fusion of the lectin cyanovirin-N (CVN) and the gp41 membrane-proximal external region (MPER) peptide with a variable-length (Gly₄Ser)_x linker (where x is 4 or 8) between the C terminus of the former and N terminus of the latter. The His-tagged recombinant proteins, expressed in BL21(DE3)pLysS cells and purified by immobilized metal affinity chromatography followed by gel filtration, were found to display a nanomolar efficacy in blocking BaL-pseudotyped HIV-1 infection of HOS.T4.R5 cells. This antiviral activity was HIV-1 specific, since it did not inhibit cell infection by vesicular stomatitis virus (VSV) or amphotropic-murine leukemia virus. Importantly, the chimeric proteins were found to release intraviral p24 protein from both BaL-pseudotyped HIV-1 and fully infectious BaL HIV-1 in a dose-dependent manner in the absence of host cells. The addition of either MPER or CVN was found to outcompete this virolytic effect, indicating that both components of the chimera are required for virolysis. The finding that engaging the Env protein spike and membrane using a chimeric ligand can destabilize the virus and lead to inactivation opens up a means to investigate virus particle metastability and to evaluate this approach for inactivation at the earliest stages of exposure to virus and before host cell encounter.      

5.1319           Social reward requires coordinated activity of nucleus accumbens oxytocin and serotonin

Dölen, G., Darvishzadeh, A., Huang, K.W. and Malenka, R.C. Nature, 501, 179-184 (2013)   Social behaviours in species as diverse as honey bees and humans promote group survival but often come at some cost to the individual. Although reinforcement of adaptive social interactions is ostensibly required for the evolutionary persistence of these behaviours, the neural mechanisms by which social reward is encoded by the brain are largely unknown. Here we demonstrate that in mice oxytocin acts as a social reinforcement signal within the nucleus accumbens core, where it elicits a presynaptically expressed long-term depression of excitatory synaptic transmission in medium spiny neurons. Although the nucleus accumbens receives oxytocin-receptor-containing inputs from several brain regions, genetic deletion of these receptors specifically from dorsal raphe nucleus, which provides serotonergic (5-hydroxytryptamine; 5-HT) innervation to the nucleus accumbens, abolishes the reinforcing properties of social interaction. Furthermore, oxytocin-induced synaptic plasticity requires activation of nucleus accumbens 5-HT1B receptors, the blockade of which prevents social reward. These results demonstrate that the rewarding properties of social interaction in mice require the coordinated activity of oxytocin and 5-HT in the nucleus accumbens, a mechanistic insight with implications for understanding the pathogenesis of social dysfunction in neuropsychiatric disorders such as autism.  

5.1320           Systemic Delivery of shRNA by AAV9 Provides Highly Efficient Knockdown of Ubiquitously Expressed GFP in Mouse Heart, but Not Liver

Bryan A. Piras, Daniel M. O’Connor, Brent A. French PloS One, 8(9), e75894 (2013)   AAV9 is a powerful gene delivery vehicle capable of providing long-term gene expression in a variety of cell types, particularly cardiomyocytes. The use of AAV-delivery for RNA interference is an intense area of research, but a comprehensive analysis of knockdown in cardiac and liver tissues after systemic delivery of AAV9 has yet to be reported. We sought to address this question by using AAV9 to deliver a short-hairpin RNA targeting the enhanced green fluorescent protein (GFP) in transgenic mice that constitutively overexpress GFP in all tissues. The expression cassette was initially tested in vitro and we demonstrated a 61% reduction in mRNA and a 90% reduction in GFP protein in dual-transfected 293 cells. Next, the expression cassette was packaged as single-stranded genomes in AAV9 capsids to test cardiac GFP knockdown with several doses ranging from 1.8×10¹⁰ to 1.8×10¹¹ viral genomes per mouse and a dose-dependent response was obtained. We then analyzed GFP expression in both heart and liver after delivery of 4.4×10¹¹ viral genomes per mouse. We found that while cardiac knockdown was highly efficient, with a 77% reduction in GFP mRNA and a 71% reduction in protein versus control-treated mice, there was no change in liver expression. This was despite a 4.5-fold greater number of viral genomes in the liver than in the heart. This study demonstrates that single-stranded AAV9 vectors expressing shRNA can be used to achieve highly efficient cardiac-selective knockdown of GFP expression that is sustained for at least 7 weeks after the systemic injection of 8 day old mice, with no change in liver expression and no evidence of liver damage despite high viral genome presence in the liver.  

5.1321           AAV-Mediated, Optogenetic Ablation of Müller Glia Leads to Structural and Functional Changes in the Mouse Retina

Byrne, L.C., Khalid, F., Lee, T., Zin, E.A., Greenberg, K.P., Visel, M., Schaffer, D.V. and Flannery, J.G. PloS One, 8(9), e76075 (2013)   Müller glia, the primary glial cell in the retina, provide structural and metabolic support for neurons and are essential for retinal integrity. Müller cells are closely involved in many retinal degenerative diseases, including macular telangiectasia type 2, in which impairment of central vision may be linked to a primary defect in Müller glia. Here, we used an engineered, Müller-specific variant of AAV, called ShH10, to deliver a photo-inducibly toxic protein, KillerRed, to Müller cells in the mouse retina. We characterized the results of specific ablation of these cells on visual function and retinal structure. ShH10-KillerRed expression was obtained following intravitreal injection and eyes were then irradiated with green light to induce toxicity. Induction of KillerRed led to loss of Müller cells and a concomitant decrease of Müller cell markers glutamine synthetase and cellular retinaldehyde-binding protein, reduction of rhodopsin and cone opsin, and upregulation of glial fibrillary acidic protein. Loss of Müller cells also resulted in retinal disorganization, including thinning of the outer nuclear layer and the photoreceptor inner and outer segments. High resolution imaging of thin sections revealed displacement of photoreceptors from the ONL, formation of rosette-like structures and the presence of phagocytic cells. Furthermore, Müller cell ablation resulted in increased area and volume of retinal blood vessels, as well as the formation of tortuous blood vessels and vascular leakage. Electrophysiologic measures demonstrated reduced retinal function, evident in decreased photopic and scotopic electroretinogram amplitudes. These results show that loss of Müller cells can cause progressive retinal degenerative disease, and suggest that AAV delivery of an inducibly toxic protein in Müller cells may be useful to create large animal models of retinal dystrophies.  

5.1322           miR-7a alleviates the maintenance of neuropathic pain through regulation of neuronal excitability

Sakai, A., Saitow, F., Miyake, N., Miyake, K., Shimada, T. and Suzuki, H. Brain, 136, 2738-2750 (2013)   Neuronal damage in the somatosensory system causes intractable chronic neuropathic pain. Plastic changes in sensory neuron excitability are considered the cellular basis of persistent pain. Non-coding microRNAs modulate specific gene translation to impact on diverse cellular functions and their dysregulation causes various diseases. However, their significance in adult neuronal functions and disorders is still poorly understood. Here, we show that miR-7a is a key functional RNA sustaining the late phase of neuropathic pain through regulation of neuronal excitability in rats. In the late phase of neuropathic pain, microarray analysis identified miR-7a as the most robustly decreased microRNA in the injured dorsal root ganglion. Moreover, local induction of miR-7a, using an adeno-associated virus vector, in sensory neurons of injured dorsal root ganglion, suppressed established neuropathic pain. In contrast, miR-7a overexpression had no effect on acute physiological or inflammatory pain. Furthermore, miR-7a downregulation was sufficient to cause pain-related behaviours in intact rats. miR-7a targeted the β2 subunit of the voltage-gated sodium channel, and decreased miR-7a associated with neuropathic pain caused increased β2 subunit protein expression, independent of messenger RNA levels. Consistently, miR-7a overexpression in primary sensory neurons of injured dorsal root ganglion suppressed increased β2 subunit expression and normalized long-lasting hyperexcitability of nociceptive neurons. These findings demonstrate miR-7a downregulation is causally involved in maintenance of neuropathic pain through regulation of neuronal excitability, and miR-7a replenishment offers a novel therapeutic strategy specific for chronic neuropathic pain.  

5.1323           Subretinal Gene Therapy of Mice With Bardet-Biedl Syndrome Type 1

Seo, S., Mullins, R.E., Dumitrescu, A.V., Bhattarai, S., Gratie, D., Wang, K., Stone, E.M., Sheffield, V. and Drack, A.V. Invest. Ophthalmol. Vis.Sci., 54, 6118-6132 (2013)   Purpose. To study safety and efficacy of subretinal adeno-associated virus (AAV) vector AAV-Bbs1 injection for treatment of a mouse model of Bardet-Biedl syndrome type 1 (BBS1). Methods. Constructs containing a wild-type (WT) Bbs1 gene with and without a FLAG tag in AAV2/5 vectors were generated. Viral genomes were delivered by subretinal injection to right eyes and sham injections to left eyes at postnatal day 30 (P30) to P60. Transgene expression and BBSome reconstitution were evaluated by immunohistochemistry and Western blotting following sucrose gradient ultracentrifugation. Retinal function was analyzed by electroretinogram (ERG) and structure by optical coherence tomography (OCT). Histology and immunohistochemistry were performed on selected eyes. Results. Expression of FLAG-tagged Bbs1 was demonstrated in photoreceptor cells using antibody directed against the FLAG tag. Coinjection of AAV-GFP demonstrated transduction of 24% to 32% of the retina. Western blotting demonstrated BBS1 protein expression and reconstitution of the BBSome. ERG dark-adapted bright flash b-wave amplitudes were higher in AAV-Bbs1–injected eyes than in sham-injected fellow eyes in more than 50% of 19 animals. Anti-rhodopsin staining demonstrated improved localization of rhodopsin in AAV-Bbs1–treated eyes. WT retinas injected with AAV-Bbs1 with or without a FLAG tag showed outer retinal degeneration on ERG, OCT, and histology. Conclusions. In a knock-in model of BBS1, subretinal delivery of AAV-Bbs1 rescues BBSome formation and rhodopsin localization, and shows a trend toward improved ERG. BBS is challenging to treat with gene therapy due to the stoichiometry of the BBSome protein complex and overexpression toxicity.  

5.1324           Mutations in hepatitis C virus p7 reduce both the egress and infectivity of assembled particles via impaired proton channel function

Bentham, M.J., Foster, T.L., McCormick, C. and Griffin, S.

1. Gen. Virol., 94, 2236-2248 (2013)

  Hepatitis C virus (HCV) p7 protein is critical for the efficient production of infectious virions in culture. p7 undergoes genotype-specific protein–protein interactions as well as displaying channel-forming activity, making it unclear whether the phenotypes of deleterious p7 mutations result from the disruption of one or both of these functions. Here, we showed that proton channel activity alone, provided in trans by either influenza virus M2 or genotype 1b HCV p7, was both necessary and sufficient to restore infectious particle production to genotype 2a HCV (JFH-1 isolate) carrying deleterious p7 alanine substitutions within the p7 dibasic loop (R33A, R35A), and the N-terminal trans-membrane region (N15 : C16 : H17/AAA). Both mutations markedly reduced mature p7 abundance, with those in the dibasic loop also significantly reducing levels of mature E2 and NS2. Interestingly, whilst M2 and genotype 1b p7 restored the same level of intracellular infectivity as JFH-1 p7, supplementing with the isogenic protein led to a further increase in secreted infectivity, suggesting a late-acting role for genotype-specific p7 protein interactions. Finally, cells infected by viruses carrying p7 mutations contained non-infectious core-containing particles with densities equivalent to WT HCV, indicating a requirement for p7 proton channel activity in conferring an infectious phenotype to virions.  

5.1325           Broadening the Repertoire of Functional Herpes Simplex Virus Type 1–Specific CD8+ T Cells Reduces Viral Reactivation from Latency in Sensory Ganglia

St. Leger, A.J., Jeon, S. and Hendricks, R.L.

1. Immunol., 191, 2258-2265 (2013)

  A large proportion of the world population harbors HSV type 1 (HSV-1) in a latent state in their trigeminal ganglia (TG). TG-resident CD8⁺ T cells appear important for preventing HSV-1 reactivation from latency and recurrent herpetic disease. In C57BL/6J mice, half of these cells are specific for an immunodominant epitope on HSV-1 glycoprotein B, whereas the other half are specific for 18 subdominant epitopes. In this study, we show that the CD8⁺ T cell dominance hierarchy in the TG established during acute infection is maintained during latency. However, CD8⁺ T cells specific for subdominant epitopes lose functionality, whereas those specific for the immunodominant epitope exhibit increased functionality in latently infected TG. Furthermore, we show that IL-10 produced by 16.4 ± 2.8% of TG-resident CD4⁺ T cells maintains the immunodominance hierarchy in part through selective inhibition of subdominant CD8⁺ T cell proliferation. Upon systemic anti–IL-10R Ab treatment, we observed a significant expansion of functional subdominant CD8⁺ T cells, resulting in significantly improved protection from viral reactivation. In fact, systemic anti–IL-10R Ab treatment prevented viral reactivation in up to 50% of treated mice. Our results not only demonstrate that HSV-1 reactivation from latency can be prevented by expanding the repertoire of functional TG-resident CD8⁺ T cells, but also that IL-10R blockade might have therapeutic potential to reduce or eliminate recurrent herpetic disease.  

5.1326           A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles

Thuenemann, E.C., Meyers, A.E., Verwey, J., Rybicki, E.P. and Lomonossoff, G.P. Plant Biotech. J., 11, 839-846 (2013)   Plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant-based production and assembly of Bluetongue virus-like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus–based HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV-8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways.  

5.1327           Generation of a Hypomorphic Model of Propionic Acidemia Amenable to Gene Therapy Testing

Guenzel, A.J., Hofherr, S.E., Hillestad, M., Barry, M., Weaver, E., Venezia, S., Kraus, J.P., Matern, D. and Barry, M.A. Molecular Therapy, 21(7), 1316-1323 (2013)   Propionic acidemia (PA) is a recessive genetic disease that results in an inability to metabolize certain amino acids and odd-chain fatty acids. Current treatment involves restricting consumption of these substrates or liver transplantation. Deletion of the Pcca gene in mice mimics the most severe forms of the human disease. Pcca⁻ mice die within 36 hours of birth, making it difficult to test intravenous systemic therapies in them. We generated an adult hypomorphic model of PA in Pcca⁻ mice using a transgene bearing an A138T mutant of the human PCCA protein. Pcca^−/−(A138T) mice have 2% of wild-type PCC activity, survive to adulthood, and have elevations in propionyl-carnitine, methylcitrate, glycine, alanine, lysine, ammonia, and markers associated with cardiomyopathy similar to those in patients with PA. This adult model allowed gene therapy testing by intravenous injection with adenovirus serotype 5 (Ad5) and adeno-associated virus 2/8 (AAV8) vectors. Ad5-mediated more rapid increases in PCCA protein and propionyl-CoA carboxylase (PCC) activity in the liver than AAV8 and both vectors reduced propionylcarnitine and methylcitrate levels. Phenotypic correction was transient with first generation Ad whereas AAV8-mediated long-lasting effects. These data suggest that this PA model may be a useful platform for optimizing systemic intravenous therapies for PA.  

5.1328           Muscle-Directed Anti-Aβ Single-Chain Antibody Delivery via AAV1 Reduces Cerebral Aβ Load in an Alzheimer’s Disease Mouse Model

Yang, J., Pattanyak, A., Song, M., Kou, J., taguchi, H., Paul, S., Ponnazhagan, S., Lalonde, R. and fukuchi, K-i.

1. Mol. Neurosci., 49(2), 277-288 (2013)

  We previously reported that anti-amyloid-beta (Aβ) single-chain antibody (scFv59) brain delivery via recombinant adeno-associated virus (rAAV) was effective in reducing cerebral Aβ load in an Alzheimer’s disease (AD) mouse model without inducing inflammation. Here, we investigated the prophylactic effects and mechanism of a muscle-directed gene therapy modality in an AD mouse model. We injected rAAV serotype 1 encoding scFv59 into the right thigh muscles of 3-month-old mice. Nine months later, high levels of scFv59 expression were confirmed in the thigh muscles by both immunoblotting and immunohistochemistry. As controls, model mice were similarly injected with rAAV1 encoding antihuman immunodeficiency virus Gag antibody (scFvGag). AAV1-mediated scFv59 gene delivery was effective in decreasing Aβ deposits in the brain. Compared with the scFvGag group, levels of Aβ in cerebrospinal fluid (CSF) decreased significantly while Aβ in serum tended to increase in the scFv59 group. AAV1-mediated scFv59 gene delivery may alter the equilibrium of Aβ between the blood and brain, resulting in an increased efflux of Aβ from the brain owing to antibody-mediated sequestration/clearance of peripheral Aβ. Our results suggest that muscle-directed scFv59 delivery via rAAV1 may be a prophylactic option for AD and that levels of CSF Aβ may be used to evaluate the efficacy of anti-Aβ immunotherapy.  

5.1329           Enhancing the Utility of Adeno-Associated Virus Gene Transfer through Inducible Tissue-Specific Expression free access

Chen, S-J., Johnston, J., Sandhu, A., Bish, L.T., Hovhannisyan, R., Jno-Charles, O., Sweeney, H.L. and Wilson, J.M. Human Gene Therapy Methods, 24(4), 270-278 (2013)   The ability to regulate both the timing and specificity of gene expression mediated by viral vectors will be important in maximizing its utility. We describe the development of an adeno-associated virus (AAV)-based vector with tissue-specific gene regulation, using the ARGENT dimerizer-inducible system. This two-vector system based on AAV serotype 9 consists of one vector encoding a combination of reporter genes from which expression is directed by a ubiquitous, inducible promoter and a second vector encoding transcription factor domains under the control of either a heart- or liver-specific promoter, which are activated with a small molecule. Administration of the vectors via either systemic or intrapericardial injection demonstrated that the vector system is capable of mediating gene expression that is tissue specific, regulatable, and reproducible over induction cycles. Somatic gene transfer in vivo is being considered in therapeutic applications, although its most substantial value will be in basic applications such as target validation and development of animal models.  

5.1330           Postentry Processing of Recombinant Adeno-Associated Virus Type 1 and Transduction of the Ferret Lung Are Altered by a Factor in Airway Secretions no access

Yan, Z., Sun, X., Evans, I.A., Tyler, S.R., Song, Y., Liu, X., Sui, H. and Engelhardt, J.F. Human Gene Therapy, 24(9), 786-796 (2013)   We recently created a cystic fibrosis ferret model that acquires neonatal lung infection. To develop lung gene therapies for this model, we evaluated recombinant adeno-associated virus (rAAV)-mediated gene transfer to the neonatal ferret lung. Unlike in vitro ferret airway epithelial (FAE) cells, in vivo infection of the ferret lung with rAAV1 required proteasome inhibitors to achieve efficient airway transduction. We hypothesized that differences in transduction between these two systems were because of an in vivo secreted factor that alter the transduction biology of rAAV1. Indeed, treatment of rAAV1 with ferret airway secretory fluid (ASF) strongly inhibited rAAV1, but not rAAV2, transduction of primary FAE and HeLa cells. Properties of the ASF inhibitory factor included a strong affinity for the AAV1 capsid, heat-stability, negative charge, and sensitivity to endoproteinase Glu-C. ASF-treated rAAV1 dramatically inhibited apical transduction of FAE ALI cultures (512-fold), while only reducing viral entry by 55-fold, suggesting that postentry processing of virus was influenced by the inhibitor factor. Proteasome inhibitors rescued transduction in the presence of ASF ( 1600-fold) without effecting virus internalization, while proteasome inhibitors only enhanced transduction 45-fold in the absence of ASF. These findings demonstrate that a factor in lung secretions can influence intracellular processing of rAAV1 in a proteasome-dependent fashion.  

5.1331           Adeno-associated virus serotype 9 efficiently targets ischemic skeletal muscle following systemic delivery

Katwal, A.B., Konkalmatt, P.R., Piras, B.A., Hazarika, S., Li, S.S., Lye, R.J., Sanders, J.M., Ferrante, E.A., Yan, Z., Annex, B.H. and French, B.A. Gene Therapy, 20(9), 930-938 (2013)   Targeting therapeutic gene expression to the skeletal muscle following intravenous (IV) administration is an attractive strategy for treating peripheral arterial disease (PAD), except that vector access to the ischemic limb could be a limiting factor. As adeno-associated virus serotype 9 (AAV-9) transduces skeletal muscle at high efficiency following systemic delivery, we employed AAV-9 vectors bearing luciferase or enhanced green fluorescent protein (eGFP) reporter genes to test the hypothesis that increased desialylation of cell-surface glycans secondary to hindlimb ischemia (HLI) might help offset the reduction in tissue perfusion that occurs in mouse models of PAD. The utility of the creatine kinase-based (CK6) promoter for restricting gene expression to the skeletal muscle was also examined by comparing it with the cytomegalovirus (CMV) promoter after systemic administration following surgically induced HLI. Despite reduced blood flow to the ischemic limbs, CK6 promoter-driven luciferase activities in the ischemic gastrocnemius (GA) muscles were ~34-, ~28- and ~150-fold higher than in the fully perfused contralateral GA, heart and liver, respectively, 10 days after IV administration. Furthermore, luciferase activity from the CK6 promoter in the ischemic GA muscles was ~twofold higher than with CMV, while in the liver CK6-driven activity was ~42-fold lower than with CMV, demonstrating that the specificity of ischemic skeletal muscle transduction can be further improved with the muscle-specific promoters. Studies with Evans blue dye and fluorescently labeled lectins revealed that vascular permeability and desialylation of the cell-surface glycans were increased in the ischemic hindlimbs. Furthermore, AAV9/CK6/Luc vector genome copy numbers were ~sixfold higher in the ischemic muscle compared with the non-ischemic muscle in the HLI model, whereas this trend was reversed when the same genome was packaged in the AAV-1 capsid (which binds sialylated, as opposed to desialylated glycans), further underscoring the importance of desialylation in the ischemic enhancement of transduction displayed by AAV-9. Taken together, these findings suggest two complementary mechanisms contributing to the preferential transduction of ischemic muscle by AAV-9: increased vascular permeability and desialylation. In conclusion, ischemic muscle is preferentially targeted following systemic administration of AAV-9 in a mouse model of HLI. Unmasking of the primary AAV-9 receptor as a result of ischemia may contribute importantly to this effect.  

5.1332           Purification of white spot syndrome virus by iodixanol density gradient centrifugation

Dantas-Lima, J.J., Corteel, M., Corneliussen, M., Bossier, P., Sorgeloos, P. and Nauwynck, H.J.

1. Fish. Dis., 36, 841-851 (2013)

  Up to now, only a few brief procedures for purifying white spot syndrome virus (WSSV) have been described. They were mainly based on sucrose, NaBr and CsCl density gradient centrifugation. This work describes for the first time the purification of WSSV through iodixanol density gradients, using virus isolated from infected tissues and haemolymph of Penaeus vannamei (Boone). The purification from tissues included a concentration step by centrifugation (2.5 h at 60 000 g) onto a 50% iodixanol cushion and a purification step by centrifugation (3 h at 80 000 g) through a discontinuous iodixanol gradient (phosphate-buffered saline, 5%, 10%, 15% and 20%). The purification from infected haemolymph enclosed a dialysis step with a membrane of 1 000 kDa (18 h) and a purification step through the earlier iodixanol gradient. The gradients were collected in fractions and analysed. The number of particles, infectivity titre (in vivo), total protein and viral protein content were evaluated. The purification from infected tissues gave WSSV suspensions with a very high infectivity and an acceptable purity, while virus purified from haemolymph had a high infectivity and a very high purity. Additionally, it was observed that WSSV has an unusually low buoyant density and that it is very sensitive to high external pressures.  

5.1333           Capacity of wild-type and chemokine-armed parvovirus H-1PV for inhibiting neo-angiogenesis

Lavie, M., Struyf, S., Stroh-Deged, A., Rommelaere, J., Van Damme, J. and Dinsart, C. Virology, 447, 221-232 (2013)   Anti-angiogenic therapy has been recognized as a powerful potential strategy for impeding the growth of various tumors. However no major therapeutic effects have been observed to date, mainly because of the emergence of several resistance mechanisms. Among novel strategies to target tumor vasculature, some oncolytic viruses open up new prospects. In this context, we addressed the question whether the rodent parvovirus H-1PV can target endothelial cells. We show that cultures of human normal (HUVEC) and immortalized (KS-IMM) endothelial cells sustain an abortive viral cycle upon infection with H-1PV and are sensitive to H-1PV cytotoxicity. H-1PV significantly inhibits infected KS-IMM tumor growth. This effect may be traced back by the virus ability to both kill proliferating endothelial cells and inhibit VEGF production Recombinant H-1PV vectors can also transduce tumor cells with chemokines endowed with anti-angiogenesis properties, and warrant further validation for the treatment of highly vascularized tumors.  

5.1334           High-Throughput Pseudovirion-Based Neutralization Assay for Analysis of Natural and Vaccine-Induced Antibodies against Human Papillomaviruses

Sehr, P., Rubio, I., Seitz, H., Putzker, K., Ribeiro-Müller, L., Pawlita, M. and Müller, M. PloS One, 8(10), e75677 (2013)   A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA) with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV) 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV) of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA) based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R² = 0.7) was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.  

5.1335           NK-cell-dependent killing of colon carcinoma cells is mediated by natural cytotoxicity receptors (NCRs) and stimulated by parvovirus infection of target cells

Bhat, R. and Rommelaere, J. BMC Cancer 13:367 (2013)   Background Investigating how the immune system functions during malignancies is crucial to developing novel therapeutic strategies. Natural killer (NK) cells, an important component of the innate immune system, play a vital role in immune defense against tumors and virus-infected cells. The poor survival rate in colon cancer makes it particularly important to develop novel therapeutic strategies. Oncolytic viruses, in addition to lysing tumor cells, may have the potential to augment antitumor immune responses. In the present study, we investigate the role of NK cells and how parvovirus H-1PV can modulate NK-cell mediated immune responses against colon carcinoma. Methods Human NK cells were isolated from the blood of healthy donors. The cytotoxicity and antibody-mediated inhibition of NK cells were measured in chromium release assays. Phenotypic assessment of colon cancer and dendritic cells was done by FACS. The statistical significance of the results was calculated with Student’s t test (*p <0.05; **, p < 0.01; ***, p < 0.001). Results We show that IL-2-activated human NK cells can effectively kill colon carcinoma cells. Killing of colon carcinoma cells by NK cells was further enhanced upon infection of the former cells with parvovirus H-1PV. H-1PV has potent oncolytic activity against various tumors, yet its direct killing effect on colon carcinoma cells is limited. The cytotoxicity of NK cells towards colon carcinoma cells, both mock- and H-1PV-infected, was found to be mostly mediated by a combination of natural cytotoxicity receptors (NCRs), namely NKp30, 44, and 46. Colon carcinoma cells displayed low to moderate expression of NK cell ligands, and this expression was modulated upon H-1PV infection. Lysates of H-1PV-infected colon carcinoma cells were found to increase MHC class II expression on dendritic cells. Conclusions Altogether, these data suggest that IL-2-activated NK cells actively kill colon carcinoma cells and that this killing is mediated by several natural cytotoxicity receptors (NCRs) in combination. Additionally, in association with parvovirus H-1PV, IL-2-activated NK cells have the potential to boost immune responses against colon cancer.  

5.1336           Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice

Machida, A., Kuwahara, H., Mayra, a., Kubodera, T., Hirai, T., Sunaga, F., Tajiri, M., Hirai, Y., Shimada, T., Mizusawa, H. and Yokota, T. Molecular Pain, 9:36 (2013)   Background There is considerable interest in inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although short interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge, especially by systemic administration. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) by using short hairpin RNA–expressing single-stranded adeno-associated virus 9 (ssAAV9-shRNA). Results Intraperitoneal administration of ssAAV9-shRNA to neonatal mice resulted in highly effective and specific silencing of a target gene in DRG. We observed an approximately 80% reduction in target mRNA in the DRG, and 74.7% suppression of the protein was confirmed by Western blot analysis. There were no major side effects, and the suppression effect lasted for more than three months after the injection of ssAAV9-shRNA. Conclusions Although we previously showed substantial inhibition of target gene expression in DRG via intrathecal ssAAV9-shRNA administration, here we succeeded in inhibiting target gene expression in DRG neurons via intraperitoneal injection of ssAAV9-shRNA. AAV9-mediated delivery of shRNA will pave the way for creating animal models for investigating the molecular biology of the mechanisms of pain and sensory ganglionopathies.  

5.1337           Encapsidation of Host-Derived Factors Correlates with Enhanced Infectivity of Sindbis Virus

Sokoloski, K.J., Snyder, A.J., Liu, N.H., Hayes, C.A., Mukhopadhyay, S. and Hardy, R.W.

1. Virol., 87(22), 12216-12226 (2013)

  The genus Alphavirus consists of a group of enveloped, single-stranded RNA viruses, many of which are transmitted by arthropods to a wide range of vertebrate host species. Here we report that Sindbis virus (SINV) produced from a representative mammalian cell line consists of at least two unique particle subpopulations, separable on the basis of virion density. In contrast, mosquito-derived SINV consists of a homogeneous population of particles. Our findings indicate that the denser particle subpopulation, SINV^Heavy, is more infectious on a per-particle basis than SINV^Light. SINV produced in mosquito cell lines (SINV^C6/36) exhibited particle-to-PFU ratios similar to those observed for SINV^Heavy. In mammalian cells, viral RNA was synthesized and accumulated more rapidly following infection with SINV^Heavy or SINV^C6/36 than following infection with SINV^Light, due partly to enhanced translation of viral genomic RNA early in infection. Analysis of the individual particle subpopulations indicated that SINV^Heavy and SINV^C6/36 contain host-derived factors whose presence correlates with the enhanced translation, RNA synthesis, and infectivity observed for these particles.    

5.1338           Adeno-Associated Virus Mediated Delivery of a Non-Membrane Targeted Human Soluble CD59 Attenuates Some Aspects of Diabetic Retinopathy in Mice

Adhi, M., Cashman, S.M. and Kumar-Singh, R. PloS One, 8(10), e79661 (2013)   Diabetic retinopathy is the leading cause of visual dysfunction in working adults and is attributed to retinal vascular and neural cell damage. Recent studies have described elevated levels of membrane attack complex (MAC) and reduced levels of membrane associated complement regulators including CD55 and CD59 in the retina of diabetic retinopathy patients as well as in animal models of this disease. We have previously described the development of a soluble membrane-independent form of CD59 (sCD59) that when delivered via a gene therapy approach using an adeno-associated virus vector (AAV2/8-sCD59) to the eyes of mice, can block MAC deposition and choroidal neovascularization. Here, we examine AAV2/8-sCD59 mediated attenuation of MAC deposition and ensuing complement mediated damage to the retina of mice following streptozotocin (STZ) induced diabetes. We observed a 60% reduction in leakage of retinal blood vessels in diabetic eyes pre-injected with AAV2/8-sCD59 relative to negative control virus injected diabetic eyes. AAV2/8-sCD59 injected eyes also exhibited protection from non-perfusion of retinal blood vessels. In addition, a 200% reduction in retinal ganglion cell apoptosis and a 40% reduction in MAC deposition were documented in diabetic eyes pre-injected with AAV2/8-sCD59 relative to diabetic eyes pre-injected with the control virus. This is the first study characterizing a viral gene therapy intervention that targets MAC in a model of diabetic retinopathy. Use of AAV2/8-sCD59 warrants further exploration as a potential therapy for advanced stages of diabetic retinopathy.  

5.1339           Antimicrobial Peptide LL-37 Deteriorate Infectivity of Hepatitis C Virus

Kato, T., Sugiyama, N:, Murayama, A., Matsumura, T., Shiina, M., Asabe, S., Wakita, T. and Imawari, M. Hepatology, 58(4), 443A-444A  (2013)   Although recent studies indicate that supplementation of vitamin D significantly improves a sustained viral response by IFNbased therapy to chronic hepatitis C, detailed mechanisms for the role of vitamin D are not fully elucidated. Previously, we demonstrated that the metabolite of vitamin D, 25-hydroxyvitamin D, has the direct anti-viral effect against hepatitis C virus (HCV) targeting infectious virus production. Since vitamin D is known to be multi-functional, we reasoned that other anti-viral functions of vitamin D, especially through immunomodulatory activity, should be considered. The production of cathelicidin, one of antimicrobial peptides, has been demonstrated to be a part of the vitamin D-dependent antimicrobial pathway in the innate immunity. Cathelicidin is known to kill or inhibit the growth of microbial pathogens including mycobacteria and viruses directly. In this study, by use of HCV JFH-1-based cell culture system, we aimed to clarify the anti-HCV effects of the human cathelicidin, LL-37, produced by monocytes or macrophages in vitamin D dependent manner. HuH-7 cells were treated with LL-37 at the concentration of 10 μg/mL for 1h and infected cell-culture generated HCV (HCVcc) at a multiplicity of  infection of 0.5. HCV infection and production were estimated by measuring the intra- and extra-cellular HCV core antigen (Ag). By treatment of LL-37, intra- and extra-cellular HCV core Ag were reduced to about 30% as compared with untreated control. The cell viability assessed by WST-8 assay was not affected by this treatment. To see the effects on HCV replication, JFH-1 subgenomic replicon RNA transfected cells were treated with LL-37 in various concentrations. However, the inhibition of subgenomic replicon replication by LL-37 treatment was not detected. Next, to clarify the effects on HCV infection, HCVcc was treated with LL-37 at multiple concentrations in 37°C for 1h and infected into naïve HuH-7 cells after purification. We found that the infectivity titer was diminished dose-dependently. The iodixanol gradient analysis revealed that the peak fraction of infectivity titer was disappeared by LL-37 treatment. In conclusion, the vitamin D associated antimicrobial peptide LL-37 deteriorated the infectivity of HCV. In addition to the direct anti-HCV effect of 25-hydroxyvitamin D, this anti-HCV effect of LL-37 might contribute to the improved efficacy of IFN-based therapy by supplementation of vitamin D.  

5.1340           Immunogenic assessment of plant-produced human papillomavirus type 16 L1/L2 chimaeras

Pineo, C.B., Hitzeroth, I.I. and Rybicki, E.P Plant Biotech. J., 11, 964-975 (2013)   Cervical cancer is caused by infection with human papillomaviruses (HPV) and is a global concern, particularly in developing countries, which have ~80% of the burden. HPV L1 virus-like particle (VLP) type–restricted vaccines prevent new infections and associated disease. However, their high cost has limited their application, and cytological screening programmes are still required to detect malignant lesions associated with the nonvaccine types. Thus, there is an urgent need for cheap second-generation HPV vaccines that protect against multiple types. The objective of this study was to express novel HPV-16 L1-based chimaeras, containing cross-protective epitopes from the L2 minor capsid protein, in tobacco plants. These L1/L2 chimaeras contained epitope sequences derived from HPV-16 L2 amino acid 108–120, 56–81 or 17–36 substituted into the C-terminal helix 4 (h4) region of L1 from amino acid 414. All chimaeras were expressed in Nicotiana benthamiana via an Agrobacterium-mediated transient system and targeted to chloroplasts. The chimaeras were highly expressed with yields of ~1.2 g/kg plant tissue; however, they assembled differently, indicating that the length and nature of the L2 epitope affect VLP assembly. The chimaera containing L2 amino acids 108–120 was the most successful candidate vaccine. It assembled into small VLPs and elicited anti-L1 and anti-L2 responses in mice, and antisera neutralized homologous HPV-16 and heterologous HPV-52 pseudovirions. The other chimaeras predominantly assembled into capsomeres and other aggregates and elicited weaker humoral immune responses, demonstrating the importance of VLP assembly for the immunogenicity of candidate vaccines.  

5.1341           Heparin increases the infectivity of Human Papillomavirus Type 16 independent of cell surface proteoglycans and induces L1 epitope exposure

Cerqueira, C., Liu, Y., Kühling, L., Chai, W., Hafezi, W., van Kuppevelt, T.H., Kühn, K.E., Feizi, T and Schelhaas, M.   Human Papillomaviruses (HPVs) are the etiological agents of cervical cancer, and HPV-16 is the most prevalent type. Several HPVs require heparan sulfate proteoglycans (HSPGs) for cell binding. Here, we analyse the phenomenon that preincubation of HPV-16 with increasing concentrations of heparin results in partial restoration rather than more efficient inhibition of infection. While corroborating that the HSPGs are cell-binding receptors for HPV-16, heparin-preincubated virus bound to the extracellular matrix (ECM) via laminin-332. Furthermore, the interaction of virions with heparin, a representative of the highly sulfated S-domains of heparan sulfate (HS) chains of HSPGs, allowed HPV-16 infection in the absence of cell surface HSPGs. Therefore, we concluded that specific glycan moieties but not specific HSPG protein backbones are required for infection. The increased binding of an epitope-specific antibody to the viral capsid after heparin binding suggested that initial conformational changes in the HPV-16 virion occur during infection by interaction with‘heparin-like’ domains of cellular HSPGs. We propose that HS sequences with specific sulfation patterns are required to facilitate HPV-16 infection.  

5.1342           Short Hairpin RNA against PTEN Enhances Regenerative Growth of Corticospinal Tract Axons after Spinal Cord Injury

Zukor, K., Belin, S., Wang, C., Keelan, N., Wang, X. and He, Z.

1. Neurosci., 33(39), 15350-15361 (2013)

  Developing approaches to promote the regeneration of descending supraspinal axons represents an ideal strategy for rebuilding neuronal circuits to improve functional recovery after spinal cord injury (SCI). Our previous studies demonstrated that genetic deletion of phosphatase and tensin homolog (PTEN) in mouse corticospinal neurons reactivates their regenerative capacity, resulting in significant regeneration of corticospinal tract (CST) axons after SCI. However, it is unknown whether nongenetic methods of suppressing PTEN have similar effects and how regenerating axons interact with the extrinsic environment. Herein, we show that suppressing PTEN expression with short-hairpin RNA (shRNA) promotes the regeneration of injured CST axons, and these axons form anatomical synapses in appropriate areas of the cord caudal to the lesion. Importantly, this model of increased CST regrowth enables the analysis of extrinsic regulators of CST regeneration in vivo. We find that regenerating axons avoid dense clusters of fibroblasts and macrophages in the lesion, suggesting that these cell types might be key inhibitors of axon regeneration. Furthermore, most regenerating axons cross the lesion in association with astrocytes, indicating that these cells might be important for providing a permissive bridge for axon regeneration. Lineage analysis reveals that these bridge-forming astrocytes are not derived from ependymal stem cells within the spinal cord, suggesting that they are more likely derived from a subset of mature astrocytes. Overall, this study reveals insights into the critical extrinsic and intrinsic regulators of axon regeneration and establishes shRNA as a viable means to manipulate these regulators and translate findings into other mammalian models.      

5.1343           Imaging the response of the retina to electrical stimulation with genetically encoded calcium indicators

Weitz, A.C., Behrend, M.R., Lee, N.S., Klein, R.L., Chiodo, V.A., Hauswirth, W.W., Humayun, M.S., Weiland, J.D. and Chow, R.H.

1. Neurophysiol., 109(7), 1979-1988 (2013)

  Epiretinal implants for the blind are designed to stimulate surviving retinal neurons, thus bypassing the diseased photoreceptor layer. Single-unit or multielectrode recordings from isolated animal retina are commonly used to inform the design of these implants. However, such electrical recordings provide limited information about the spatial patterns of retinal activation. Calcium imaging overcomes this limitation, as imaging enables high spatial resolution mapping of retinal ganglion cell (RGC) activity as well as simultaneous recording from hundreds of RGCs. Prior experiments in amphibian retina have demonstrated proof of principle, yet experiments in mammalian retina have been hindered by the inability to load calcium indicators into mature mammalian RGCs. Here, we report a method for labeling the majority of ganglion cells in adult rat retina with genetically encoded calcium indicators, specifically GCaMP3 and GCaMP5G. Intravitreal injection of an adeno-associated viral vector targets ∼85% of ganglion cells with high specificity. Because of the large fluorescence signals provided by the GCaMP sensors, we can now for the first time visualize the response of the retina to electrical stimulation in real-time. Imaging transduced retinas mounted on multielectrode arrays reveals how stimulus pulse shape can dramatically affect the spatial extent of RGC activation, which has clear implications in prosthetic applications. Our method can be easily adapted to work with other fluorescent indicator proteins in both wild-type and transgenic mammals.  

5.1344           Tracing Inputs to Inhibitory or Excitatory Neurons of Mouse and Cat Visual Cortex with a Targeted Rabies Virus

Liu, Y-J., Ehrengruber, M.U., Negwer, M., Shao, H-J., Ceting, A.H. and Lyon, D.C. Current Biol., 223(18), 1746-1755 (2013)   Background: Cortical inhibition plays a critical role in controlling and modulating cortical excitation, and a more detailed understanding of the neuronal circuits contributing to each will provide more insight into their roles in complex cortical computations. Traditional neuronal tracers lack a means for easily distinguishing between circuits of inhibitory and excitatory neurons. To overcome this limitation, we have developed a technique for retrogradely labeling inputs to local clusters of inhibitory or excitatory neurons, but not both, using neurotropic adenoassociated and lentiviral vectors, cell-type-specific promoters, and a modified rabies virus. Results: Applied to primary visual cortex (V1) in mouse, the cell-type-specific tracing technique labeled thousands of presynaptically connected neurons and revealed that the dominant source of input to inhibitory and excitatory neurons is local in origin. Neurons in other visual areas are also labeled; the percentage of these intercortical inputs to excitatory neurons is somewhat higher ( 20%) than to inhibitory neurons (<10%), suggesting that intercortical connections have less direct control over inhibition. The inputs to inhibitory neurons were also traced in cat V1, and when aligned with the orientation preference map revealed for the first time that long-range inputs to inhibitory neurons are well tuned to orientation. Conclusions: These novel findings for inhibitory and excitatory circuits in the visual cortex demonstrate the efficacy of our new technique and its ability to work across species, including larger-brained mammals such as the cat. This paves the way for a better understanding of the roles of specific cell types in higher-order perceptual and cognitive processes.  

5.1345           Maturation of silent synapses in amygdala-accumbens projection contributes to incubation of cocaine craving

Lee, B.R. et al Nature Neurosci., 16(11), 1644-1651 (2013)   In rat models of drug relapse and craving, cue-induced cocaine seeking progressively increases after withdrawal from the drug. This 'incubation of cocaine craving' is partially mediated by time-dependent adaptations at glutamatergic synapses in nucleus accumbens (NAc). However, the circuit-level adaptations mediating this plasticity remain elusive. We studied silent synapses, often regarded as immature synapses that express stable NMDA receptors with AMPA receptors being either absent or labile, in the projection from the basolateral amygdala to the NAc in incubation of cocaine craving. Silent synapses were detected in this projection during early withdrawal from cocaine. As the withdrawal period progressed, these silent synapses became unsilenced, a process that involved synaptic insertion of calcium-permeable AMPA receptors (CP-AMPARs). In vivo optogenetic stimulation–induced downregulation of CP-AMPARs at amygdala-to-NAc synapses, which re-silenced some of the previously silent synapses after prolonged withdrawal, decreased incubation of cocaine craving. Our findings indicate that silent synapse–based reorganization of the amygdala-to-NAc projection is critical for persistent cocaine craving and relapse after withdrawal.  

5.1346           ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation

Lin, J.Y., Knutsen, P.M., Muller, A., Kleinfeld, D. and Tsien, R.Y. Nature Neurosci., 16(10), 1499-1508 (2013)   Channelrhodopsins (ChRs) are used to optogenetically depolarize neurons. We engineered a variant of ChR, denoted red-activatable ChR (ReaChR), that is optimally excited with orange to red light (λ ~590–630 nm) and offers improved membrane trafficking, higher photocurrents and faster kinetics compared to existing red-shifted ChRs. Red light is less scattered by tissue and is absorbed less by blood than the blue to green wavelengths that are required by other ChR variants. We used ReaChR expressed in the vibrissa motor cortex to drive spiking and vibrissa motion in awake mice when excited with red light through intact skull. Precise vibrissa movements were evoked by expressing ReaChR in the facial motor nucleus in the brainstem and illumination with red light through the external auditory canal. Thus, ReaChR enables transcranial optical activation of neurons in deep brain structures without the need to surgically thin the skull, form a transcranial window or implant optical fibers.  

5.1347           The immune complex CTA1-DD/IgG adjuvant specifically targets connective tissue mast cells through FcγRIIIA and augments anti-HPV immunity after nasal immunization

Fang, Y., Zhang, T., Lidell, L., Xu, X., Lycke, N. and Xiang, Z. Mucosal Immunol., 6(6), 1168-1178 (2013)   We have previously reported that CTA1-DD/IgG immune complexes augment antibody responses in a mast cell–dependent manner following intranasal (IN) immunizations. However, from a safety perspective, mast cell activation could preclude clinical use. Therefore, we have extended these studies and demonstrate that CTA1-DD/IgG immune complexes administered IN did not trigger an anaphylactic reaction. Importantly, CTA1-DD/IgE immune complexes did not activate mast cells. Interestingly, only connective tissue, but not mucosal, mast cells could be activated by CTA1-DD/IgG immune complexes. This effect was mediated by FcγRIIIA, only expressed on connective tissue mast cells, and found in the nasal submucosa. FcγRIIIA-deficient mice had compromised responses to immunization adjuvanted by CTA1-DD/IgG. Proof-of-concept studies revealed that IN immunized mice with human papillomavirus (HPV) type 16 L1 virus-like particles (VLP) and CTA1-DD/IgG immune complexes demonstrated strong and sustained specific antibody titers in serum and vaginal secretions. From a mast cell perspective, CTA1-DD/IgG immune complexes appear to be safe and effective mucosal adjuvants.  

5.1348           NADH-dehydrogenase Type-2 Suppresses Irreversible Visual Loss and Neurodegeneration in the EAE Animal Model of MS

Talla, V., Yu, H., Chou, T-H., Porciatti, V., Chiodo, V., Boye, S.L., Hauswirth, W.W., Lewin, A.S. and Guy, J. Molecular Therapy, 21(10), 1876-1888 (2013)   To address mitochondrial dysfunction that mediates irreversible visual loss and neurodegeneration of the optic nerve in the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis (MS), mice sensitized for EAE were vitreally injected with self-complementary adenoassociated virus (scAAV) containing the NADH-dehydrogenase type-2 (NDI1) complex I gene that quickly expressed in mitochondria of almost all retinal ganglion cells (RGCs). Visual function assessed by pattern electroretinograms (PERGs) reduced by half in EAE showed no significant reductions with NDI1. Serial optical coherence tomography (OCT) revealed significant inner retinal thinning with EAE that was suppressed by NDI1. Although complex I activity reduced 80% in EAE was not improved by NDI1, in vivo fluorescent probes indicated mitochondrial oxidative stress and apoptosis of the EAE retina were reduced by NDI1. Finally, the 42% loss of axons in the EAE optic nerve was ameliorated by NDI1. Targeting the dysfunctional complex I of EAE responsible for loss of respiration, mitochondrial oxidative stress and apoptosis may be a novel approach to address neuronal and axonal loss responsible for permanent disability that is unaltered by current disease modifying drugs for MS that target inflammation.  

5.1349           Exposure to cocaine regulates inhibitory synaptic transmission from the ventral tegmental area to the nucleus accumbens

Ishikawa, M., Otaka, M., Neumann, P.A., Wang, Z., Cook, J.M., Schlüter, O.M., Dong, Y. and Huang, Y.H.

1. Physiol., 591(19), 4827-4841 (2013)

  Synaptic projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) make up the backbone of the brain reward pathway, a neural circuit that mediates behavioural responses elicited by natural rewards as well as by cocaine and other drugs of abuse. In addition to the well-known modulatory dopaminergic projection, the VTA also provides fast excitatory and inhibitory synaptic input to the NAc, directly regulating NAc medium spiny neurons (MSNs). However, the cellular nature of VTA-to-NAc fast synaptic transmission and its roles in drug-induced adaptations are not well understood. Using viral-mediated in vivo expression of channelrhodopsin 2, the present study dissected fast excitatory and inhibitory synaptic transmission from the VTA to NAc MSNs in rats. Our results suggest that, following repeated exposure to cocaine (15 mg kg⁻¹ day⁻¹ × 5 days, i.p., 1 or 21 day withdrawal), a presynaptic enhancement of excitatory transmission and suppression of inhibitory transmission occurred at different withdrawal time points at VTA-to-NAc core synapses. In contrast, no postsynaptic alterations were detected at either type of synapse. These results suggest that changes in VTA-to-NAc fast excitatory and inhibitory synaptic transmissions may contribute to cocaine-induced alteration of the brain reward circuitry.  

5.1350           The efficacy of combination therapy using adeno-associated virus-mediated co-expression of apoptin and interleukin-24 on hepatocellular carcinoma

Yuan, L., Zhao, H., Zhang, L. and Liu, X. Tumor Biol., 34(5), 3027-3034 (2013)   Multigene-based combination therapy is an effective practice in cancer gene therapy. Apoptin is a chicken anemia virus-derived, p53-independent, Bcl-2-insensitive apoptotic protein with the ability to specifically induce apoptosis in various human tumor cells. Interleukin-24 (IL-24) displays ubiquitous antitumor property and tumor-specific killing activity. Adeno-associated virus (AAV) is a promising gene delivery vehicle due to its advantage of low pathogenicity and long-term gene expression. In this study, we assessed the efficacy of combination therapy using AAV-mediated co-expression of apoptin and interleukin-24 on hepatocellular carcinoma in vitro and in vivo. Our results showed that AAV-mediated co-expression of IL-24 and apoptin significantly suppressed the growth and induced the apoptosis of HepG2 cells in vitro. Furthermore, AAV-mediated combined treatment of IL-24 and apoptin significantly suppressed tumor growth and induced apoptosis of tumor cells in xenograft nude mice. These data suggest that AAV vectors that co-express apoptin and IL-24 have great potential in cancer gene therapy.  

5.1351           Parvoviruses Cause Nuclear Envelope Breakdown by Activating Key Enzymes of Mitosis

Porwal, M., Cohen, S., Snoussi, K., Popa-Wagner, R., Anderson, F., Dugot-Senant, N., Wodrich, H., Dinsart, C., Kleinschmidt, J.A., Pante, N. and Kann, M. PloS Pathogens, 9(10), e1003671 (2013)   Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis.  

5.1352           Sp100 Provides Intrinsic Immunity against Human Papillomavirus Infection

Stepp, W.H., Meyers, J.M. and Mcbride, A.A. mBio, 4(6), e00845-13 (2013)   Most DNA viruses associate with, and reorganize, nuclear domain 10 (ND10) bodies upon entry into the host nucleus. In this study, we examine the roles of the ND10 components PML, Sp100, and Daxx in the establishment of human papillomavirus type 18 (HPV18) infection of primary human keratinocytes. HPV18 DNA or HPV18 quasivirus was introduced into primary human keratinocytes depleted of each ND10 protein by small interfering RNA technology, and genome establishment was determined by using a quantitative immortalization assay and measurements of viral transcription and DNA replication. Keratinocyte depletion of Sp100 resulted in a substantial increase in the number of HPV18-immortalized colonies and a corresponding increase in viral transcription and DNA replication. However, Sp100 repressed viral transcription and replication only during the initial stages of viral establishment, suggesting that Sp100 acts as a repressor of incoming HPV DNA. IMPORTANCE The intrinsic immune system provides a first-line defense against invading pathogens. Host cells contain nuclear bodies (ND10) that are important for antiviral defense, yet many DNA viruses localize here upon cell entry. However, viruses also disrupt, reorganize, and modify individual components of the bodies. In this study, we show that one of the ND10 components, Sp100, limits the infection of human skin cells by human papillomavirus (HPV). HPVs are important pathogens that cause many types of infection of the cutaneous and mucosal epithelium and are the causative agents of several human cancers. Understanding how host cells counteract HPV infection could provide insight into antimicrobial therapies that could limit initial infection.  

5.1353           Identification of TRAPPC8 as a Host Factor Required for Human Papillomavirus Cell Entry

Ishii, Y., Nakahara, T., Kataoka, M., Kusumoto-Matsuo, R., Mori, S., Takeuchi, T. and Kukimoto, I. PloS One, 8(11), e80297 (2013)   Human papillomavirus (HPV) is a non-enveloped virus composed of a circular DNA genome and two capsid proteins, L1 and L2. Multiple interactions between its capsid proteins and host cellular proteins are required for infectious HPV entry, including cell attachment and internalization, intracellular trafficking and viral genome transfer into the nucleus. Using two variants of HPV type 51, the Ma and Nu strains, we have previously reported that MaL2 is required for efficient pseudovirus (PsV) transduction. However, the cellular factors that confer this L2 dependency have not yet been identified. Here we report that the transport protein particle complex subunit 8 (TRAPPC8) specifically interacts with MaL2. TRAPPC8 knockdown in HeLa cells yielded reduced levels of reporter gene expression when inoculated with HPV51Ma, HPV16, and HPV31 PsVs. TRAPPC8 knockdown in HaCaT cells also showed reduced susceptibility to infection with authentic HPV31 virions, indicating that TRAPPC8 plays a crucial role in native HPV infection. Immunofluorescence microscopy revealed that the central region of TRAPPC8 was exposed on the cell surface and colocalized with inoculated PsVs. The entry of Ma, Nu, and L2-lacking PsVs into cells was equally impaired in TRAPPC8 knockdown HeLa cells, suggesting that TRAPPC8-dependent endocytosis plays an important role in HPV entry that is independent of L2 interaction. Finally, expression of GFP-fused L2 that can also interact with TRAPPC8 induced dispersal of the Golgi stack structure in HeLa cells, a phenotype also observed by TRAPPC8 knockdown. These results suggest that during viral intracellular trafficking, binding of L2 to TRAPPC8 inhibits its function resulting in Golgi destabilization, a process that may assist HPV genome escape from the trans-Golgi network.  

5.1354           Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics

Rayaprolu, V., Kruse, S., Kant, R., Venkatakrishnan, B., Movahed, N., Brooke, D., Lins, B., Bennett, A., Pottter, T., McKenna, R., Agbandje-McKenna, M. and Brother, B.

1. Virol., 87(24), 13150-13160 (2013)

  Icosahedral viral capsids are obligated to perform a thermodynamic balancing act. Capsids must be stable enough to protect the genome until a suitable host cell is encountered yet be poised to bind receptor, initiate cell entry, navigate the cellular milieu, and release their genome in the appropriate replication compartment. In this study, serotypes of adeno-associated virus (AAV), AAV1, AAV2, AAV5, and AAV8, were compared with respect to the physical properties of their capsids that influence thermodynamic stability. Thermal stability measurements using differential scanning fluorimetry, differential scanning calorimetry, and electron microscopy showed that capsid melting temperatures differed by more than 20°C between the least and most stable serotypes, AAV2 and AAV5, respectively. Limited proteolysis and peptide mass mapping of intact particles were used to investigate capsid protein dynamics. Active hot spots mapped to the region surrounding the 3-fold axis of symmetry for all serotypes. Cleavages also mapped to the unique region of VP1 which contains a phospholipase domain, indicating transient exposure on the surface of the capsid. Data on the biophysical properties of the different AAV serotypes are important for understanding cellular trafficking and is critical to their production, storage, and use for gene therapy. The distinct differences reported here provide direction for future studies on entry and vector production.  

5.1355           Characterization of Mus musculus Papillomavirus 1 Infection In Situ Reveals an Unusual Pattern of Late Gene Expression and Capsid Protein Localization

Handisurya, A., Day, P.M., Thompson, C.D., Buck, C.B., Pang, Y-Y.S., Lowy, D.R. and Schiller, J.T.

1. Virol., 87(24), 13214-13225 (2013)

  Full-length genomic DNA of the recently identified laboratory mouse papillomavirus 1 (MusPV1) was synthesized in vitro and was used to establish and characterize a mouse model of papillomavirus pathobiology. MusPV1 DNA, whether naked or encapsidated by MusPV1 or human papillomavirus 16 (HPV 16) capsids, efficiently induced the outgrowth of papillomas as early as 3 weeks after application to abraded skin on the muzzles and tails of athymic NCr nude mice. High concentrations of virions were extracted from homogenized papillomatous tissues and were serially passaged for >10 generations. Neutralization by L1 antisera confirmed that infectious transmission was capsid mediated. Unexpectedly, the skin of the murine back was much less susceptible to virion-induced papillomas than the muzzle or tail. Although reporter pseudovirions readily transduced the skin of the back, infection with native MusPV1 resulted in less viral genome amplification and gene expression on the back, including reduced expression of the L1 protein and very low expression of the L2 protein, results that imply skin region-specific control of postentry aspects of the viral life cycle. Unexpectedly, L1 protein on the back was predominantly cytoplasmic, while on the tail the abundant L1 was cytoplasmic in the lower epithelial layers and nuclear in the upper layers. Nuclear localization of L1 occurred only in cells that coexpressed the minor capsid protein, L2. The pattern of L1 protein staining in the infected epithelium suggests that L1 expression occurs earlier in the MusPV1 life cycle than in the life cycle of high-risk HPV and that virion assembly is regulated by a previously undescribed mechanism.  

5.1356           Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells

Cribbs, A.P., Kennedy, A., Gregory, B. and Brennan, F.M. BMC Biotechnology, 13:98 (2013)   Background Lentiviral vectors have emerged as efficient vehicles for transgene delivery in both dividing and non-dividing cells. A number of different modifications in vector design have increased biosafety and transgene expression. However, despite these advances, the transduction of primary human T cells is still challenging and methods to achieve efficient gene transfer are often expensive and time-consuming. Results Here we present a simple optimised protocol for the generation and transduction of lentivirus in primary human CD45RA⁺ T cells. We show that generation of high-titre lentivirus with improved primary T cell transduction is dependent upon optimised ultracentrifuge speed during viral concentration. Moreover, we demonstrate that transduction efficiency can be increased with simple modifications to the culturing conditions. Overall, a transduction efficiency of up to 89% in primary human CD45RA⁺ cells is achievable when these modifications are used in conjunction. Conclusion The optimised protocol described here is easy to implement and should facilitate the production of high-titre lentivirus with superior transduction efficiency in primary human T cells without the need for further purification methods.  

5.1357           Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines

Börner, K., Niopek, D., Cotugna, G., Kaldenbach, M., pankert, T., Willemsen, J., Zhang, X., Schürmann, N., Mockenhaupt, S., Serva, A., Hiet, M-S., Wiedtke, E., Castoldi, M., Starkuviene, V., Erfle, H., Gilbert, D.F., bartenschlager, R., Boutros, M., Binder, M., Streetz, K., Kräusslich, H-G. and Grimm, D. Nucleic Acids Res., 41(21), e199 (2013)   As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.  

5.1358           Pseudovirus mimics cell entry and trafficking of the human polyomavirus JCPyV

Gee, G.V., O’Hara, B.A., Derdowski, A. and Atwood, W.J. Virus Res., 178, 281-286 (2013)   The normally asymptomatic human polyomavirus, JCPyV, is the causative agent of a rare but fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). Individuals at risk for developing PML include those with AIDS, with other underlying immunosuppressive diseases, and in patients treated with immunomodulatory regimens. Drugs to prevent viral reactivation in the setting of immunosuppression or immunomodulation could be used to sustain lives. Development of such drugs has been impeded by the difficulty of growing and studying the virus. We sought to develop a more efficient method for screening drugs that inhibit viral infection. Pseudovirus models have been developed which may be of use in pharmaceutical research. The use of pseudoviruses as models for viral infection is dependent on them using similar pathways for infection as virus. We screened known inhibitors of viral entry for their ability to block pseudovirus infection. Here we show that the pseudovirus based on the human polyomavirus JCPyV recapitulates virus binding, entry and trafficking. This system can be used for high-throughput screening of antiviral drugs.  

5.1359           Inactivation of Hepatitis C Virus Infectivity by Human Breast Milk

Pfaender, S., Heyden, J., Friesland, M., Ciesek, S., Ejaz, A., Steinmann, J., Steinmann, J., Malarski, A., Stoiber, H., Tsiavaliaris, G., Bader, W., jahreis, G., Pietschmann, T. and Steinmann, E. Journal of Infectious Diseases, 208, 1943-1952 (2013)   Background. Hepatitis C virus (HCV) is spread through direct contact with blood, although alternative routes of transmission may contribute to the global burden. Perinatal infection occurs in up to 5% of HCV-infected mothers, and presence of HCV RNA in breast milk has been reported. We investigated the influence of breast milk on HCV infectiousness. Methods/Results. Human breast milk reduced HCV infectivity in a dose-dependent manner. This effect was species-specific because milk from various animals did not inhibit HCV infection. Treatment of HCV with human breast milk did not compromise integrity of viral RNA or capsids but destroyed the lipid envelope. Fractionation of breast milk revealed that the antiviral activity is present in the cream fraction containing the fat. Proteolytic digestion of milk proteins had no influence on its antiviral activity, whereas prolonged storage at 4°C increased antiviral activity. Notably, pretreatment with a lipase inhibitor ablated the antiviral activity and specific free fatty acids of breast milk were antiviral. Conclusions. The antiviral activity of breast milk is linked to endogenous lipase-dependent generation of free fatty acids, which destroy the viral lipid envelope. Therefore, nursing by HCV-positive mothers is unlikely to play a major role in vertical transmission.  

5.1360           Additional Glycosylation Within a Specific Hypervariable Region of Subtype 3a of Hepatitis C Virus Protects Against Virus Neutralization

Anjum, S., Wahid, A., Afzal, M.S., Albecka, A., Alsaleh, K., Ahmad, T., Baumert, T.F., Wychowski, C., Qadri, I., Penin, F. and Dubuisson, J.

1. Infectious Diseases, 208, 1888-1897 (2013)

  Background. The envelope glycoprotein E2 of hepatitis C virus (HCV) contains several hypervariable regions. Interestingly, 2 regions of intragenotypic hypervariability within E2 have been described as being specific to HCV subtype 3a. Based on their amino acid position in E2, they were named HVR495 and HVR575. Here, we further investigated these regions in order to better understand their role in HCV infection. Methods. Sequences of HCV envelope glycoproteins from Pakistani patients infected with subtype 3a were cloned and compared with other subtype 3a sequences. The entry functions and the sensitivity to antibody neutralization of selected HCV glycoprotein sequences were tested in the HCV pseudotyped particles (HCVpp) system. In addition, the cell-cultured HCV system (HCVcc) was also used to confirm some of the data obtained with the HCVpp system. Results. We observed interesting new features within HVR495 and HVR575 for several subtype 3a isolates. Indeed, changes in glycosylation sites were observed with the appearance of a new glycosylation site within HVR495. Importantly, HCVpp and HCVcc that contained this new HVR495 glycosylation site were less sensitive to antibody neutralization. Conclusions. We identified a new glycosylation site within the HVR495 region of HCV subtype 3a that has a protective effect against antibody neutralization.  

5.1361           Morphological and Behavioral Impact of AAV2/5-Mediated Overexpression of Human Wildtype Alpha-Synuclein in the Rat Nigrostriatal System

Gombash, S.E., Manfredsson, F.P., kemp, C.J., Kuhn, N.C., Fleming, S.M., Egan, A.E., Grant, L.M., Ciucci, M.R., MacKeigan, J.P. and Sortwell, C.E. Plos One, 8(11), e81426 (2013)   The discovery of the involvement of alpha-synuclein (α-syn) in Parkinson’s disease (PD) pathogenesis has resulted in the development and use of viral vector-mediated α-syn overexpression rodent models. The goal of these series of experiments was to characterize the neurodegeneration and functional deficits resulting from injection of recombinant adeno-associated virus (rAAV) serotype 2/5-expressing human wildtype α-syn in the rat substantia nigra (SN). Rats were unilaterally injected into two sites in the SN with either rAAV2/5-expressing green fluorescent protein (GFP, 1.2 x 10¹³) or varying titers (2.2 x 10¹², 1.0 x 10¹³, 5.9 x 10¹³, or 1.0 x 10¹⁴) of rAAV2/5-α-syn. Cohorts of rats were euthanized 4, 8, or 12 weeks following vector injection. The severity of tyrosine hydroxylase immunoreactive (THir) neuron death in the SN pars compacta (SNpc) was dependent on vector titer. An identical magnitude of nigrostriatal degeneration (60-70% SNpc THir neuron degeneration and 40-50% loss of striatal TH expression) was observed four weeks following 1.0 x 10¹⁴ titer rAAV2/5-α-syn injection and 8 weeks following 1.0 x 10¹³ titer rAAV2/5-α-syn injection. THir neuron degeneration was relatively uniform throughout the rostral-caudal axis of the SNpc. Despite equivalent nigrostriatal degeneration between the 1.0 x 10¹³ and 1.0 x 10¹⁴ rAAV2/5-α-syn groups, functional impairment in the cylinder test and the adjusting steps task was only observed in rats with the longer 8 week duration of α-syn expression. Motor impairment in the cylinder task was highly correlated to striatal TH loss. Further, 8 weeks following 5.9 x 10¹³ rAAV2/5-α-syn injection deficits in ultrasonic vocalizations were observed. In conclusion, our rAAV2/5-α-syn overexpression model demonstrates robust nigrostriatal α-syn overexpression, induces significant nigrostriatal degeneration that is both vector and duration dependent and under specific parameters can result in motor impairment that directly relates to the level of striatal TH denervation.    

5.1362           Ex vivo intracoronary gene transfer of adeno-associated virus 2 leads to superior transduction over serotypes 8 and 9 in rat heart transplants

Raissadati, A., Jokinen, J.J., Syrjälä, S.O., Keränen, M.A.I., Krebs, R., Tuuminen, R., Arnaudova, R., Rouvinen, E., Anismov, A., Soronen, J., Pajusola, K., Alitalo, K., Nykänen, A.I. and Lemström, K. Transplant. Int., 26(11), 1126-1137 (2013)   Heart transplant gene therapy requires vectors with long-lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno-associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar–Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR-, RT-PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies.    

5.1363           The Role of Apoptosis in Immune Hyporesponsiveness Following AAV8 Liver Gene Transfer

Faust, S.M., Bell, P., Zhu, Y., Sanmiguel, J. and Wilson, J.M. Molecular Therapy, 21(12), 2227-2235 (2013)   Gene therapy provides a significant opportunity to treat a variety of inherited and acquired diseases. However, adverse immune responses toward the adeno-associated virus (AAV) antigens may limit its success. The mechanisms responsible for immunity or tolerance toward AAV-encoded transgene products remain poorly defined. Studies in mice demonstrate that AAV2/8 gene transfer to liver is associated with immunological hyporesponsiveness toward both AAV vector and antigenic transgene product. To evaluate the role of activation-induced cell death (AICD) and cytokine withdrawal (intrinsic cell death) in the deletion of mature T lymphocytes, we compared immunological responses in hepatic AAV2/8 transfer in murine recipients lacking the Fas receptor, and recipients overexpressing Bcl-xL, to WT murine counterparts. Prolonged transgene expression was dependent on both Fas signaling and Bcl-xL–regulated apoptosis in T cells. Abrogation of intrinsic cell death enhanced Th1 responses, whereas AICD functioned to limit neutralizing antibody production toward AAV2/8. In addition, immune hyporesponsiveness and stable transgene expression was dependent on upregulation of FasL expression on transduced hepatocytes and a corresponding apoptosis of infiltrating Fas (+) cells. These data provide evidence that both AICD and apoptosis due to cytokine withdrawal of lymphocytes are essential for immune hyporesponsiveness toward hepatic AAV2/8-encoded transgene product in the setting of liver gene transfer.      

5.1364           Adeno-Associated Viral Vector Serotype 5 Poorly Transduces Liver in Rat Models

Montenegro-Miranda, P.S., Paneda, A., ten Bloemmendaal, L., Duijst, S., de Waart, D.R., Gonzalez Aseguinolaza, G. and Bosma, P.J. PloS One, 8(12), e82597 (2013)   Preclinical studies in mice and non-human primates showed that AAV serotype 5 provides efficient liver transduction and as such seems a promising vector for liver directed gene therapy. An advantage of AAV5 compared to serotype 8 already shown to provide efficient correction in a phase 1 trial in patients suffering from hemophilia B, is its lower seroprevalence in the general population. Our goal is liver directed gene therapy for Crigler-Najjar syndrome type I, inherited severe unconjugated hyperbilirubinemia caused by UGT1A1 deficiency. In a relevant animal model, the Gunn rat, we compared the efficacy of AAV 5 and 8 to that of AAV1 previously shown to be effective. Ferrying a construct driving hepatocyte specific expression of UGT1A1, both AAV8 and AAV1 provided an efficient correction of hyperbilirubinemia. In contrast to these two and to other animal models AAV5 failed to provide any correction. To clarify whether this unexpected finding was due to the rat model used or due to a problem with AAV5, the efficacy of this serotype was compared in a mouse and two additional rat strains. Administration of an AAV5 vector expressing luciferase under the control of a liver specific promoter confirmed that this serotype poorly performed in rat liver, rendering it not suitable for proof of concept studies in this species.  

5.1365           Interactions of peptide triazole thiols with Env gp120 induce irreversible breakdown and inactivation of HIV-1 virions

Bastian, A.R., Contarino, M., Bailey, L.D., Aneja, R., Moreira, D.R.M., Freedman, K., McFadden, K., Duffy, C., Emileh, A., Leslie, G., Jacobson, J.M., Hoxie, J.A. and Chaiken, I. Retrovirology, 10:153, (2013)   Background We examined the underlying mechanism of action of the peptide triazole thiol, KR13 that has been shown previously to specifically bind gp120, block cell receptor site interactions and potently inhibit HIV-1 infectivity. Results KR13, the sulfhydryl blocked KR13b and its parent non-sulfhydryl peptide triazole, HNG156, induced gp120 shedding but only KR13 induced p24 capsid protein release. The resulting virion post virolysis had an altered morphology, contained no gp120, but retained gp41 that bound to neutralizing gp41 antibodies. Remarkably, HIV-1 p24 release by KR13 was inhibited by enfuvirtide, which blocks formation of the gp41 6-helix bundle during membrane fusion, while no inhibition of p24 release occurred for enfuvirtide-resistant virus. KR13 thus appears to induce structural changes in gp41 normally associated with membrane fusion and cell entry. The HIV-1 p24 release induced by KR13 was observed in several clades of HIV-1 as well as in fully infectious HIV-1 virions. Conclusions The antiviral activity of KR13 and its ability to inactivate virions prior to target cell engagement suggest that peptide triazole thiols could be highly effective in inhibiting HIV transmission across mucosal barriers and provide a novel probe to understand biochemical signals within envelope that are involved in membrane fusion.  

5.1366           A Novel Method for the Quantification of Adeno-Associated Virus Vectors for RNA Interference Applications Using Quantitative Polymerase Chain Reaction and Purified Genomic Adeno-Associated Virus DNA as a Standardn.

Wagner, A., Röhrs, V., Kedzierski, R., Fechner, H. and kurreck, J. Human Gene Therapy Methods, 24, 355-363 (2013)   Recombinant adeno-associated virus (rAAV) vectors are promising tools in gene therapy, but accurate quantification of the vector dose remains a critical issue for their successful application. We therefore aimed at the precise determination of the titer of self-complementary AAV (scAAV) vectors to improve the reliability of RNA interference (RNAi)-mediated knockdown approaches. Vector titers were initially determined by quantitative polymerase chain reaction (qPCR) using four primer sets targeting different regions within the AAV vector genome (VG) and either coiled or linearized plasmid standards. Despite very low variability between replicates in each assay, these quantification experiments revealed up to 20-fold variation in vector titers. Therefore, we developed a novel approach for the reproducible determination of titers of scAAV vectors based on the use of purified genomic vector DNA as a standard (scAAV_Std). Consistent results were obtained in qPCR assays using the four primer sets mentioned above. RNAi-mediated silencing of human cyclophilin B (hCycB) by short hairpin RNA-expressing scAAV vectors was investigated in HeLa cells using two independent vector preparations. We found that the required vector titers for efficient knockdown differed by a factor of 3.5 between both preparations. Hence, we also investigated the number of internalized scAAV vectors, termed transduction units (TUs). TUs were determined by qPCR applying the scAAV_Std. Very similar values for 80% hCycB knockdown were obtained for the two AAV vector preparations. Thus, only the determination of TUs, rather than vector concentration, allows for reproducible results in functional analyses using AAV vectors.  

5.1367           Adeno-Associated Virus Enhances Wild-Type and Oncolytic Adenovirus Spread

Laborda, E., Puig-Saus, C., Cascallo, M., Chillon, M. and Alemany, R. Human Gene Therapy Methods, 24, 373-380 (2013)   The contamination of adenovirus (Ad) stocks with adeno-associated viruses (AAV) is usually unnoticed, and it has been associated with lower Ad yields upon large-scale production. During Ad propagation, AAV contamination needs to be detected routinely by polymerase chain reaction without symptomatic suspicion. In this study, we describe that the coinfection of either Ad wild type 5 or oncolytic Ad with AAV results in a large-plaque phenotype associated with an accelerated release of Ad from coinfected cells. This accelerated release was accompanied with the expected decrease in Ad yields in two out of three cell lines tested. Despite this lower Ad yield, coinfection with AAV accelerated cell death and enhanced the cytotoxicity mediated by Ad propagation. Intratumoral coinjection of Ad and AAV in two xenograft tumor models improved antitumor activity and mouse survival. Therefore, we conclude that accidental or intentional AAV coinfection has important implications for Ad-mediated virotherapy.  

5.1368           Transduced Wild-Type but Not P301S Mutated Human Tau Shows Hyperphosphorylation in Transgenic Mice Overexpressing A30P Mutated Human Alpha-Synuclein

Oksman, M., Wisman, L.A., Jiang, H., Miettinen, P., Kirik, D. and Tanila, H. Neurogenerative Dis., 12, 91-102 (2013)   Neuropathological and cell culture studies suggest that tau and α-synuclein pathologies may promote each other. To study the relevance and functional implications of these findings in vivo, we transduced hippocampal neurons of wild-type or human A30P α-synuclein transgenic mice with wild-type or P301S mutated human tau using an adeno-associated virus vector. Green fluorescent protein transduction was used as a control. We assessed spontaneous exploratory activity, anxiety and spatial learning and memory 11 weeks after the transduction and perfused the mice for histology. The transduced tau was mainly found in axon terminals and largely restricted within the hippocampi. In addition, neurons around the injection site showed cytoplasmic staining for human tau in both wild-type and A30P mice. Of these tau-positive neurons, 44% in A30P mice but only 3% in wild-type mice receiving human wild-type tau transduction formed paired helical filament-1 (PHF-1)-positive cytoplasmic densities. In contrast, only 1% of tau-positive neurons were also PHF-1 positive after transduction with P301S tau in mice of either genotype. Transduction of P301S tau reduced swimming speed but otherwise tau transduction had no significant behavioral consequences. Cytoplasmic PHF-1 densities were associated with poor spatial memory in wild-type mice but slightly improved memory in A30P mice, indicating that also tau hyperphosphorylation does not necessarily compromise neural functions. These data demonstrate that α-synuclein promotes tau hyperphosphorylation depending on the amino acids on the 301 site.  

5.1369           Biodistribution of AAV8 Vectors Expressing Human Low-Density Lipoprotein Receptor in a Mouse Model of Homozygous Familial Hypercholesterolemia

Chen, S-J. et al Human Gene Therapy Clin. Develop., 24(4), 154-160 (2013)   Recombinant adeno-associated viral vectors based on serotype 8 (AAV8) transduce liver with superior tropism following intravenous (IV) administration. Previous studies conducted by our lab demonstrated that AAV8-mediated transfer of the human low-density lipoprotein receptor (LDLR) gene driven by a strong liver-specific promoter (thyroxin-binding globulin [TBG]) leads to high level and persistent gene expression in the liver. The approach proved efficacious in reducing plasma cholesterol levels and resulted in the regression of atherosclerotic lesions in a murine model of homozygous familial hypercholesterolemia (hoFH). Prior to advancing this vector, called AAV8.TBG.hLDLR, to the clinic, we set out to investigate vector biodistribution in an hoFH mouse model following IV vector administration to assess the safety profile of this investigational agent. Although AAV genomes were present in all organs at all time points tested (up to 180 days), vector genomes were sequestered mainly in the liver, which contained levels of vector 3 logs higher than that found in other organs. In both sexes, the level of AAV genomes gradually declined and appeared to stabilize 90 days post vector administration in most organs although vector genomes remained high in liver. Vector loads in the circulating blood were high and close to those in liver at the early time point (day 3) but rapidly decreased to a level close to the limit of quantification of the assay. The results of this vector biodistribution study further support a proposed clinical trial to evaluate AAV8 gene therapy for hoFH patients.   Evaluating efficacy of bacteriophage therapy against Staphylococcus aaureus infections using a silworm larval infection system Takemura-Uchiyama, I., Uchiyama, J., Kato, S-I., Inoue, T., Ujihara, T., Ohara, N., Daibata, M. and Matsuzaki, S. FEMS Microbiol. Lett., 347(1), 52-60 (2013)   Silkworm larva has recently been recognized as an alternative model animal for higher mammals to evaluate the effects of antibiotics. In this study, we examined the efficacy of the bacteriophage (phage) therapy, which harnesses phages as antibacterial agents, against Staphylococcus aureus infections, using the silkworm larval infection model. Two newly isolated staphylococcal phages, S25-3 and S13′, were used as therapeutic phage candidates. They were assigned to two different lytic phage genera, Twort-like and AHJD-like viruses, based on their morphologies and the N-terminal amino acid sequences of the major capsid proteins. Both had a broad host range and strong lytic activity and showed preservative quality. Administration of these phages alone caused no adverse effects in the silkworm larvae. Moreover, the viruses showed life-prolonging effects in the silkworm larval infection model 10 min, 6 h, 12 h, and 24 h following infection. Such phage effects in the silkworm larval model were almost paralleled to the therapeutic efficacies in mouse models. These results suggest that phages S25-3 and S13′ are eligible as therapeutic candidates and that the silkworm larval model is valid for the evaluation of phage therapy as well as mouse models.  

5.1370           Hepatitis A virus: Host interactions, molecular epidemiology and evolution

Vaughan, G., Goncalves Rossi, L.M., Forbi, J.C., de Paula, V.S., Purdy, M.A., Xia, G. and Khudyakov, Y.E. Infection, Genetics and Evolution, 21, 227-243 (2014)   Infection with hepatitis A virus (HAV) is the commonest viral cause of liver disease and presents an important public health problem worldwide. Several unique HAV properties and molecular mechanisms of its interaction with host were recently discovered and should aid in clarifying the pathogenesis of hepatitis A. Genetic characterization of HAV strains have resulted in the identification of different genotypes and subtypes, which exhibit a characteristic worldwide distribution. Shifts in HAV endemicity occurring in different parts of the world, introduction of genetically diverse strains from geographically distant regions, genotype displacement observed in some countries and population expansion detected in the last decades of the 20th century using phylogenetic analysis are important factors contributing to the complex dynamics of HAV infections worldwide. Strong selection pressures, some of which, like usage of deoptimized codons, are unique to HAV, limit genetic variability of the virus. Analysis of subgenomic regions has been proven useful for outbreak investigations. However, sharing short sequences among epidemiologically unrelated strains indicates that specific identification of HAV strains for molecular surveillance can be achieved only using whole-genome sequences. Here, we present up-to-date information on the HAV molecular epidemiology and evolution, and highlight the most relevant features of the HAV-host interactions.  

5.1371           Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants

Kawano, M., Morikawa, K., Suda, T., Ohno, N., Matsushita, S., Akatsuka, t., Handa, H. and Matsui, M. Virology, 448, 159-167 (2014)   Virus-like particles (VLPs) are a promising vaccine platform due to the safety and efficiency. However, it is still unclear whether polyomavirus-based VLPs are useful for this purpose. Here, we attempted to evaluate the potential of polyomavirus VLPs for the antiviral vaccine using simian virus 40 (SV40). We constructed chimeric SV40-VLPs carrying an HLA-A^⁎02:01-restricted, cytotoxic T lymphocyte (CTL) epitope derived from influenza A virus. HLA-A^⁎02:01-transgenic mice were then immunized with the chimeric SV40-VLPs. The chimeric SV40-VLPs effectively induced influenza-specific CTLs and heterosubtypic protection against influenza A viruses without the need of adjuvants. Because DNase I treatment of the chimeric SV40-VLPs did not disrupt CTL induction, the intrinsic adjuvant property may not result from DNA contaminants in the VLP preparation. In addition, immunization with the chimeric SV40-VLPs generated long-lasting memory CTLs. We here propose that the chimeric SV40-VLPs harboring an epitope may be a promising CTL-based vaccine platform with self-adjuvant properties.  

5.1372           Orsay virus utilizes ribosomal frameshifting to express a novel protein that is incorporated into virions

Jiang, H., Franz, C.J., Wu, G., Renshaw, H., Zhao, G., Firth, A.E. and Wang, D. Virology, 450-451, 213-221 (2014)   Orsay virus is the first identified virus that is capable of naturally infecting Caenorhabditis elegans. Although it is most closely related to nodaviruses, Orsay virus differs from nodaviruses in its genome organization. In particular, the Orsay virus RNA2 segment encodes a putative novel protein of unknown function, termed delta, which is absent from all known nodaviruses. Here we present evidence that Orsay virus utilizes a ribosomal frameshifting strategy to express a novel fusion protein from the viral capsid (alpha) and delta ORFs. Moreover, the fusion protein was detected in purified virus fractions, demonstrating that it is most likely incorporated into Orsay virions. Furthermore, N-terminal sequencing of both the fusion protein and the capsid protein demonstrated that these proteins must be translated from a non-canonical initiation site. While the function of the alpha–delta fusion remains cryptic, these studies provide novel insights into the fundamental properties of this new clade of viruses.  

5.1373           Production of adeno-associated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery

Chalal, P.S., Schulze, E., Tran, r., Montes, J. and karmen, A.A.

1. Virol. Methods, 196, 163-173 (2014)

  Adeno-associated virus (AAV) is being used successfully in gene therapy. Different serotypes of AAV target specific organs and tissues with high efficiency. There exists an increasing demand to manufacture various AAV serotypes in large quantities for pre-clinical and clinical trials. A generic and scalable method has been described in this study to efficiently produce AAV serotypes (AAV1-9) by transfection of a fully characterized cGMP HEK293SF cell line grown in suspension and serum-free medium. First, the production parameters were evaluated using AAV2 as a model serotype. Second, all nine AAV serotypes were produced successfully with yields of 10¹³ Vg/L cell culture. Subsequently, AAV2 and AAV6 serotypes were produced in 3-L controlled bioreactors where productions yielded up to 10¹³ Vg/L similar to the yields obtained in shake-flasks. For example, for AAV2 10¹³ Vg/L cell culture (6.8 × 10¹¹ IVP/L) were measured between 48 and 64 h post transfection (hpt). During this period, the average cell specific AAV2 yields of 6800 Vg per cell and 460 IVP per cell were obtained with a Vg to IVP ratio of less than 20. Successful operations in bioreactors demonstrated the potential for scale-up and industrialization of this generic process for manufacturing AAV serotypes efficiently.  

5.1374           Novel AAV-DJ Capsid Tyrosine Mutants with Enhanced Transgene Expression in a Pancreatic Cancer Cell Line

Batchu, R., Gruzdyn, O.V., Kung, S.t., Weaver, D.W. and Gruber, S.A.

1. Surg. Res., 186(2), 637-638 (2014)

  Introduction Bioengineered recombinant AAV (rAAV) vectors are preferred for gene therapy due their lack of pathogenicity and wide range of infectivity. However, 2 major challenges still remain: low transduction efficiency and cytoplasmic degradation during intracellular trafficking to the nucleus. The recent development of AAV-DJ, a synthetic capsid derived from AAV-2/8/9 with multi-organ high transduction efficiency and low immunogenicity, as well as the introduction of tyrosine (Y) to phenylalanine (F) capsid mutants preventing intracellular degradation and enhancing transgene expression, address these drawbacks. We hypothesized that combining these approaches by Y to F substitution of surface-exposed residues on rAAV-DJ capsids would create a novel vector demonstrating both enhanced transduction and gene expression. Methods Protein Database (PDB) files were generated by ESyPred3D Web Server 1.0. Positions of the surface-exposed Y residues were visualized and graphically presented using the program YASARA. Site-directed mutagenesis was outsourced to Mutagenex Inc. rAAV was purified via triple transfection of AAV-293 cells followed by iodixanol gradient ultracentrifugation and heparin column chromatography. Transductions were carried out in triplicate 6-well plates and cells were visualized by fluorescence microscopy. Results The molecular 3-dimensional structure of AAV-DJ was generated by prediction of secondary structure elements using PDB files. Visual inspection identified a total of 17 tyrosine residues that are at least partially surface-exposed, of which 8 are fully exposed (Fig. 1A). Due to their close proximity and potential ease of primer design, we selected the Y443, Y445, and Y446 sites for Y to F mutagenesis and generated individual mutants as well as a novel, triple-mutant vector comprised of all 3 (YF443.6). To compare the ability of these vectors to transduce human pancreatic cancer (PANC-1) and embryonic kidney (HEK-293) cells with that of parental AAV-DJ, we produced viral particles with enhanced green fluorescent protein (EGFP) and assessed transgene expression by immunofluorescence. We found that expression levels of the triple mutant significantly exceeded those of parental AAV-DJ in PANC-1 cells (Fig. 1B). In addition, expression levels of 3 of the 4 mutants significantly exceeded those of parental AAV-DJ in HEK-293 cells, with the triple mutant showing the highest transgene expression. Conclusions We combined the structural determinants of the AAV-DJ capsid providing enhanced transduction with introduction of Y to F mutations to generate a novel rAAV hybrid mutant vector with high levels of transgene expression. This vector may prove useful as part of gene therapy protocols for pancreatic cancer.  

5.1375           Epstein-Barr virus-mediated transformation of B cells induces global chromatin changes independent to the acquisition of proliferation

Hernando, H., Islam, A.B.M.M.K., Rodriguez-Ubreva, J., Forne, I., Ciudad, L., Imhof, A., Shannon-Lowe, C. and Ballestar, E. Nucleic Acids Res., 42(1), 249-263 (2014)   Epstein–Barr virus (EBV) infects and transforms human primary B cells inducing indefinite proliferation. To investigate the potential participation of chromatin mechanisms during the EBV-mediated transformation of resting B cells we performed an analysis of global changes in histone modifications. We observed a remarkable decrease and redistribution of heterochromatin marks including H4K20me3, H3K27me3 and H3K9me3. Loss of H4K20me3 and H3K9me3 occurred at constitutive heterochromatin repeats. For H3K27me3 and H3K9me3, comparison of ChIP-seq data revealed a decrease in these marks in thousands of genes, including clusters of HOX and ZNF genes, respectively. Moreover, DNase-seq data comparison between resting and EBV-transformed B cells revealed increased endonuclease accessibility in thousands of genomic sites. We observed that both loss of H3K27me3 and increased accessibility are associated with transcriptional activation. These changes only occurred in B cells transformed with EBV and not in those stimulated to proliferate with CD40L/IL-4, despite their similarities in the cell pathways involved and proliferation rates. In fact, B cells infected with EBNA-2 deficient EBV, which have much lower proliferation rates, displayed similar decreases for heterochromatic histone marks. Our study describes a novel phenomenon related to transformation of B cells, and highlights its independence of the pure acquisition of proliferation.  

5.1376           A Small-Molecule Inhibitor of Hepatitis C Virus Infectivity

Bush, C.O. et al Antimicrob. Agents Chemother., 58(1), 386-396 (2014)   One of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates across a variety of patient populations. Such a regimen will likely require a combination of at least two distinct direct-acting antivirals (DAAs). Combining two or more DAAs with different resistance profiles increases the number of mutations required for viral breakthrough. Currently, most DAAs inhibit HCV replication. We recently reported that the combination of two distinct classes of HCV inhibitors, entry inhibitors and replication inhibitors, prolonged reductions in extracellular HCV in persistently infected cells. We therefore sought to identify new inhibitors targeting aspects of the HCV replication cycle other than RNA replication. We report here the discovery of the first small-molecule HCV infectivity inhibitor, GS-563253, also called HCV infectivity inhibitor 1 (HCV II-1). HCV II-1 is a substituted tetrahydroquinoline that selectively inhibits genotype 1 and 2 HCVs with low-nanomolar 50% effective concentrations. It was identified through a high-throughput screen and subsequent chemical optimization. HCV II-1 only permits the production and release of noninfectious HCV particles from cells. Moreover, infectious HCV is rapidly inactivated in its presence. HCV II-1 resistance mutations map to HCV E2. In addition, HCV-II prevents HCV endosomal fusion, suggesting that it either locks the viral envelope in its prefusion state or promotes a viral envelope conformation change incapable of fusion. Importantly, the discovery of HCV II-1 opens up a new class of HCV inhibitors that prolong viral suppression by HCV replication inhibitors in persistently infected cell cultures.  

5.1377           LIM Homeobox 8 (Lhx8) Is a Key Regulator of the Cholinergic Neuronal Function via a Tropomyosin Receptor Kinase A (TrkA)-mediated Positive Feedback Loop

Tomioka, T., Shimazaki, T., Yamauchi, T., Oki, T., Ohgoh, T. and Okano, H.

1. Biol. Chem., 289(2), 1000-1010 (82014)

  Basal forebrain cholinergic neurons play an important role in cognitive functions such as learning and memory, and they are affected in several neurodegenerative diseases, including Alzheimer disease and Down syndrome. Despite their functional importance, the molecular mechanisms of functional maturation and maintenance of these cholinergic neurons after the differentiation stage have not been fully elucidated. This study demonstrates that the LIM homeobox 8 (Lhx8) transcription factor regulates cholinergic function in rat septal cholinergic neurons in primary cultures from E18.5 embryos and in the adult brain. Lhx8 expression modulated tropomyosin receptor kinase A (TrkA) expression in septal cholinergic neurons in vitro and in vivo, resulting in regulated acetylcholine release as an index of cholinergic function. In addition, Lhx8 expression and function were regulated by nerve growth factor (NGF), and the effect of NGF was potentiated by Lhx8-induced TrkA expression. Together, our findings suggest that positive feedback regulation between Lhx8, TrkA, and NGF is an important regulatory mechanism for cholinergic functions of the septum.  

5.1378           HPV16 infection of HaCaTs is dependent on β4 integrin, and α6 integrin processing

Aksoy, P., Abban, C.Y., Kiashka, E., Quang, W. and Memeses, P.I. Virology, 449, 45-52 (2014)   Our understanding of human papillomavirus (HPV) is still evolving. To further study the field, our laboratory has focused on determining the role of integrins in the initial steps of viral endocytosis into HaCaT cells. Our and others' previous findings have shown that α6 is necessary for infection. Here we show that α3 and β1 were dispensable, and we identified integrin α6β4 complex as necessary for infection in HaCaTs. β4 knock down resulted in a significant decrease in HPV16 PsV infection and perhaps most importantly resulted in defective post-translational α6 processing. We showed that the unprocessed α6 does not localize to the cell surface. We propose that the α6β4 complex is necessary for the formation of an endocytic complex that results in the signaling transduction events necessary for initial endocytosis.  

5.1379           S151A δ-sarcoglycan mutation causes a mild phenotype of cardiomyopathy in mice

Rutschow, D., Bauer, R., Göhringer, C., Bekeredjian, R., Schinkel, S., Straub, V., Koenen, M., Weichenhan, D., Katus, H.A. and Müller, O.J. Eur. J. Hum. Genet., 22, 119-125 (2014)   So far, the role of mutations in the δ-sarcogylcan (Sgcd) gene in causing autosomal dominant dilated cardiomyopathy (DCM) remains inconclusive. A p.S151A missense mutation in exon 6 of the Sgcd gene was reported to cause severe isolated autosomal dominant DCM without affecting skeletal muscle. This is controversial to our previous findings in a large consanguineous family where this p.S151A mutation showed no relevance for cardiac disease. In this study, the potential of the p.S151A mutation to cause DCM was investigated by using two different approaches: (1) engineering and characterization of heterozygous knock-in (S151A-) mice carrying the p.S151A mutation and (2) evaluation of the potential of adeno-associated virus (AAV) 9-based cardiac-specific transfer of p.S151A-mutated Sgcd cDNA to rescue the cardiac phenotype in Sgcd-deficient (Sgcd-null) mice as it has been demonstrated for intact, wild-type Sgcd cDNA. Heterozygous S151A knock-in mice developed a rather mild phenotype of cardiomyopathy. Increased heart to body weight suggests cardiac enlargement in 1-year-old S151A knock-in mice. However, at this age cardiac function, assessed by echocardiography, is maintained and histopathology completely absent. Myocardial expression of p.S151A cDNA, similar to intact Sgcd cDNA, restores cardiac function, although not being able to prevent myocardial histopathology in Sgcd-null mice completely. Our results suggest that the p.S151A mutation causes a mild, subclinical phenotype of cardiomyopathy, which is prone to be overseen in patients carrying such sequence variants. Furthermore, this study shows the suitability of an AAV-mediated cardiac gene transfer approach to analyze whether a sequence variant is a disease-causing mutation.  

5.1380           Sustained relief of neuropathic pain by AAV-targeted expression of CBD3 peptide in rat dorsal root ganglion

Fischer, G., Pan, B., Vilceanu, D., Hogan, Q.H. and Yu, H. Gene Therapy, 21, 44-51 (2014)   The Ca²⁺ channel-binding domain 3 (CBD3) peptide, derived from the collapsin response mediator protein 2 (CRMP-2), is a recently discovered voltage-gated Ca²⁺ channel (VGCC) blocker with a preference for Ca_V2.2. Rodent administration of CBD3 conjugated to cell penetrating motif TAT (TAT-CBD3) has been shown to reduce pain behavior in inflammatory and neuropathic pain models. However, TAT-CBD3 analgesia has limitations, including short half-life, lack of cellular specificity and undesired potential off-site effects. We hypothesized that these issues could be addressed by expressing CBD3 encoded by high-expression vectors in primary sensory neurons. We constructed an adeno-associated viral (AAV) vector expressing recombinant fluorescent CBD3 peptide and injected it into lumbar dorsal root ganglia (DRGs) of rats before spared nerve injury (SNI). We show that selective expression of enhanced green fluorescent protein (EGFP)-CBD3 in lumbar 4 (L4) and L5 DRG neurons and their axonal projections results in effective attenuation of nerve injury-induced neuropathic pain in the SNI model. We conclude that AAV-encoded CBD3 delivered to peripheral sensory neurons through DRG injection may be a valuable approach for exploring the role of presynaptic VGCCs and long-term modulation of neurotransmission, and may also be considered for development as a gene therapy strategy to treat chronic neuropathic pain.    

5.1381           Virally expressed connexin26 restores gap junction function in the cochlea of conditional Gjb2 knockout mice

Yu, Q., Wang, Y., Chang, Q., Wang, J., Gong, S., Li, H. and Lin, X. Gene Therapy, 21, 71-80 (2014)   Mutations in GJB2, which codes for the gap junction (GJ) protein connexin26 (Cx26), are the most common causes of human nonsyndromic hereditary deafness. We inoculated modified adeno-associated viral (AAV) vectors into the scala media of early postnatal conditional Gjb2 knockout mice to drive exogenous Cx26 expression. We found extensive virally expressed Cx26 in cells lining the scala media, and intercellular GJ network was re-established in the organ of Corti of mutant mouse cochlea. Widespread ectopic Cx26 expression neither formed ectopic GJs nor affected normal hearing thresholds in wild-type (WT) mice, suggesting that autonomous cellular mechanisms regulate proper membrane trafficking of exogenously expressed Cx26 and govern the functional manifestation of them. Functional recovery of GJ-mediated coupling among the supporting cells was observed. We found that both cell death in the organ of Corti and degeneration of spiral ganglion neurons in the cochlea of mutant mice were substantially reduced, although auditory brainstem responses did not show significant hearing improvement. This is the first report demonstrating that virally mediated gene therapy restored extensive GJ intercellular network among cochlear non-sensory cells in vivo. Such a treatment performed at early postnatal stages resulted in a partial rescue of disease phenotypes in the cochlea of the mutant mice.  

5.1382           Oncolytic effects of parvovirus H-1 in medulloblastoma are associated with repression of master regulators of early neurogenesis

Lacroix, J., Schlund, F., Leuchs, B., Adolph, K., Sturm, D., Bender, S., Hielscher, T., Pfister, S.M., Witt, O., Rommelaere, J., Schlehofer, J.R. and Witt, H. Int. J. Cancer, 134(3), 703-716 (2014)   Based on extensive pre-clinical studies, the oncolytic parvovirus H-1 (H-1PV) is currently applied to patients with recurrent glioblastoma in a phase I/IIa clinical trial (ParvOryx01, NCT01301430). Cure rates of about 40% in pediatric high-risk medulloblastoma (MB) patients also indicate the need of new therapeutic approaches. In order to prepare a future application of oncolytic parvovirotherapy to MB, the present study preclinically evaluates the cytotoxic efficacy of H-1PV on MB cells in vitro and characterizes cellular target genes involved in this effect. Six MB cell lines were analyzed by whole genome oligonucleotide microarrays after treatment and the results were matched to known molecular and cytogenetic risk factors. In contrast to non-transformed infant astrocytes and neurons, in five out of six MB cell lines lytic H-1PV infection and efficient viral replication could be demonstrated. The cytotoxic effects induced by H-1PV were observed at LD50s below 0.05 p. f. u. per cell indicating high susceptibility. Gene expression patterns in the responsive MB cell lines allowed the identification of candidate target genes mediating the cytotoxic effects of H-1PV. H-1PV induced down-regulation of key regulators of early neurogenesis shown to confer poor prognosis in MB such as ZIC1, FOXG1B, MYC, and NFIA. In MB cell lines with genomic amplification of MYC, expression of MYC was the single gene most significantly repressed after H-1PV infection. H-1PV virotherapy may be a promising treatment approach for MB since it targets genes of functional relevance and induces cell death at very low titers of input virus.  

5.1383           The role of Homer1c in metabotropic glutamate receptor-dependent long-term potentiation

O’Riordan, K., Gerstein, H., Hullinger, R. and Burger, C. Hippocampus, 24, 1-6 (2014)   Group I metabotropic glutamate receptors (mGluR1/5) play a role in synaptic plasticity and they demonstrate direct interactions with the neuronal Homer1c protein. We have previously shown that Homer1c can restore the plasticity deficits in Homer1 knockout mice (H1-KO). Here, we investigated the role of Homer1c in mGluR-dependent synaptic plasticity in wild-type mice, H1-KO, and H1-KO mice overexpressing Homer1c (KO+H1c). We used a form of plasticity induced by activation of mGluR1/5 that transforms short-term potentiaion (STP) induced by a subthreshold theta burst stimulation into long-term potentiation (LTP). We have shown that although acute hippocampal slices from wild-type animals can induce LTP using this stimulation protocol, H1-KO only show STP. Gene delivery of Homer1c into the hippocampus of H1-KO mice rescued LTP to wild-type levels. This form of synaptic plasticity was dependent on mGluR5 but not mGluR1 activation both in wild-type mice and in KO+H1c. mGluR1/5-dependent LTP was blocked with inhibitors of the MEK-ERK and PI3K-mTOR pathways in KO+H1c mice. Moreover, blocking Homer1c–mGluR5 interactions prevented the maintenance of LTP in acute hippocampal slices from KO+H1c. These data indicate that Homer1c–mGluR5 interactions are necessary for mGluR-dependent LTP, and that mGluR1/5-dependent LTP involves PI3K and ERK activation.  

5.1384           Correlation of cell surface marker expression with African swine fever virus infection

Litgow, P., Takamatsu, H., Werling, D., Dixon, L. and Chapman, D. Vet. Microbiol., 168, 413-419 (2014)   The expression of surface markers on African swine fever virus (ASFV) infected cells was evaluated to assess their involvement in infection. Previous findings indicated CD163 expression was correlated with ASFV susceptibility. However, in this study the expression of porcine CD163 on cell lines did not increase the infection rate of these cells indicating other factors are likely to be important in determining susceptibility to infection. On adherent porcine bone marrow (pBM) cells the expression of CD45 was strongly correlated with infection. CD163 and CD203a expression correlated at intermediate levels with infection, indicating cells expressing these markers could become infected but were not preferentially infected by the virus. Most of the cells expressing MHCII were infected, indicating that they may be preferentially infected although expression of MHCII was not essential for infection and a large percentage of the infected cells were MHCII negative. CD16 showed a marked decrease in expression following infection and significantly lower levels of infected cells were shown to express CD16. Altogether these results suggest CD163 may be involved in ASFV infection but it may not be essential; the results also highlight the importance of other cell markers which requiring further investigation.  

5.1385           Chronic Treatment with Novel Small Molecule Hsp90 Inhibitors Rescues Striatal Dopamine Levels but Not α-Synuclein-Induced Neuronal Cell Loss

McFarland, N.R., Dimant, H., Kibuuka, L., Ebrahimi-Fakhari, D., Desjardins, C.A., Danzer, K.M., Danzer, M., Fan, Z., Schwarzschild, M.A., Hirst, W. and McLean, P.J. PloS One, 9(1), e86048 (2014)   Hsp90 inhibitors such as geldanamycin potently induce Hsp70 and reduce cytotoxicity due to α-synuclein expression, although their use has been limited due to toxicity, brain permeability, and drug design. We recently described the effects of a novel class of potent, small molecule Hsp90 inhibitors in cells overexpressing α-synuclein. Screening yielded several candidate compounds that significantly reduced α-synuclein oligomer formation and cytotoxicity associated with Hsp70 induction. In this study we examined whether chronic treatment with candidate Hsp90 inhibitors could protect against α-synuclein toxicity in a rat model of parkinsonism. Rats were injected unilaterally in the substantia nigra with AAV8 expressing human α-synuclein and then treated with drug for approximately 8 weeks by oral gavage. Chronic treatment with SNX-0723 or the more potent, SNX-9114 failed to reduce dopaminergic toxicity in the substantia nigra compared to vehicle. However, SNX-9114 significantly increased striatal dopamine content suggesting a positive neuromodulatory effect on striatal terminals. Treatment was generally well tolerated, but higher dose SNX-0723 (6–10 mg/kg) resulted in systemic toxicity, weight loss, and early death. Although still limited by potential toxicity, Hsp90 inhibitors tested herein demonstrate oral efficacy and possible beneficial effects on dopamine production in a vertebrate model of parkinsonism that warrant further study.  

5.1386           Efficient lysis of epithelial ovarian cancer cells by MAGE-A3-induced cytotoxic T lymphocytes using rAAV-6 capsid mutant vector

Batchu, R.B., Gruzdyn, O.V., Moreno-Bost, A.M., Szmania, S., Jayandharan, G., Srivastava, A., Kolli, B.K., Weaver, D.W., van Rhee, F. and Grubert, S.A. Vaccine, 32, 938-943 (2014)   MAGE-A3 is highly expressed in epithelial ovarian cancer (EOC), making it a promising candidate for immunotherapy. We investigated whether dendritic cells (DCs) transduced with a rAAV-6 capsid mutant vector Y445F could elicit effective MAGE-A3-specific anti-tumor cytotoxic T lymphocyte (CTL) responses in vitro. MAGE-A3 was cloned and rAAV-6-MAGE-A3 purified, followed by proviral genome detection using real-time PCR. Immunofluorescence detection of rAAV-6-Y445F-MAGE-A3-transduced DCs demonstrated 60% transduction efficiency. Fluorescent in situ hybridization analysis confirmed chromosomal integration of rAAV vectors. Flow cytometric analysis of transduced DCs showed unaltered expression of critical monocyte-derived surface molecules with retention of allo-stimulatory activity. Co-culture of autologous T lymphocytes with MAGE-A3-expressing DCs produced CTLs that secreted IFN-γ, and efficiently killed MAGE-A3+ EOC cells. This form of rAAV-based DC immunotherapy, either alone or more likely in combination with other immune-enhancing protocols, may prove useful in the clinical setting for management of EOC.  

5.1387           Profiling of Glycan Receptors for Minute Virus of Mice in Permissive Cell Lines Towards Understanding the Mechanism of Cell Recognition

Halder, S., Cotmore, S., Heimburg-Molinaro, j., Smith, D.F., Cummings, R.D., Chen, X., Trollope, A.J., North, S.J., Haslam, S.M., Dell, A., Tattersall, P., McKenna, R and Agbandje-mcKenna. PloS One, 9(1), e86909 (2014)   The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3′SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3′SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3′SIA-Le^X identified in a previous glycan microarray screen.  

5.1388           Adeno-Associated Virus Capsid Proteins May Play a Role in Transcription and Second-Strand Synthesis of Recombinant Genomes

Salganik, M., Aydemir, F., Nam, H-J., McKenna, R., Agbandje-Mckenna, M. and Muzyczka, N.

1. Virol., 88(2), 1071-1079 (2014)

  A group of four interacting amino acids in adeno-associated virus type 8 (AAV8) called the pH quartet has been shown to undergo a structural change when subjected to acidic pH comparable to that seen in endosomal compartments. We examined the phenotypes of mutants with mutations in these amino acids as well as several nearby residues in the background of AAV2. We found that three of the mutations in this region (Y704A, E562A, and E564A) produce normal titers of mature capsids but are extremely defective for transduction (>10⁷-fold). The remaining mutants were also defective for transduction, but the defect in these mutants (E563A, E561A, H526A, and R389A) is not as severe (3- to 22-fold). Two other mutants (Y700A and Y730A) were found to be defective for virus assembly. One of the extremely defective mutants (Y704A) was found to enter the cell, traffic to the nucleus, and uncoat its DNA nearly as efficiently as the wild type. This suggested that some step after nuclear entry and uncoating was defective. To see if the extremely defective mutants were impaired in second-strand synthesis, the Y704A, E562A, and E564A mutants containing self-complementary DNA were compared with virus containing single-stranded genomes. Two of the mutants (Y704A and E564A) showed 1-log and 3-log improvements in infectivity, respectively, while the third mutant (E562A) showed no change. This suggested that inhibition of second-strand synthesis was responsible for some but not most of the defect in these mutants. Comparison of Y704A mRNA synthesis with that of the wild-type capsid showed that accumulation of steady-state mRNA in the Y704A mutant was reduced 450-fold, even though equal genome numbers were uncoated. Our experiments have identified a novel capsid function. They suggest that AAV capsids may play a role in the initiation of both second-strand synthesis and transcription of the input genome.  

5.1389           Apolipoprotein E Codetermines Tissue Tropism of Hepatitis C Virus and Is Crucial for Viral Cell-to-Cell Transmission by Contributing to a Postenvelopment Step of Assembly

Hueging, K., Doepke, M., Vieyres, G., Bankwitz, D., Frentzen, A., Doerrbacker, J., Gumz, F., Haid, S., Wölk, B., Kaderali, L and Pietschmann, T.

1. Virol., 88(3), 1433-1446 (2014)

  Hepatitis C virus (HCV) predominantly infects human hepatocytes, although extrahepatic virus reservoirs are being discussed. Infection of cells is initiated via cell-free and direct cell-to-cell transmission routes. Cell type-specific determinants of HCV entry and RNA replication have been reported. Moreover, several host factors required for synthesis and secretion of lipoproteins from liver cells, in part expressed in tissue-specific fashion, have been implicated in HCV assembly. However, the minimal cell type-specific requirements for HCV assembly have remained elusive. Here we report that production of HCV trans-complemented particles (HCV_TCP) from nonliver cells depends on ectopic expression of apolipoprotein E (ApoE). For efficient virus production by full-length HCV genomes, microRNA 122 (miR-122)-mediated enhancement of RNA replication is additionally required. Typical properties of cell culture-grown HCV (HCVcc) particles from ApoE-expressing nonliver cells are comparable to those of virions derived from human hepatoma cells, although specific infectivity of virions is modestly reduced. Thus, apolipoprotein B (ApoB), microsomal triglyceride transfer protein (MTTP), and apolipoprotein C1 (ApoC1), previously implicated in HCV assembly, are dispensable for production of infectious HCV. In the absence of ApoE, release of core protein from infected cells is reduced, and production of extracellular as well as intracellular infectivity is ablated. Since envelopment of capsids was not impaired, we conclude that ApoE acts after capsid envelopment but prior to secretion of infectious HCV. Remarkably, the lack of ApoE also abrogated direct HCV cell-to-cell transmission. These findings highlight ApoE as a host factor codetermining HCV tissue tropism due to its involvement in a late assembly step and viral cell-to-cell transmission.    

5.1390           AAV-mediated gene delivery in Dp71-null mouse model with compromised barriers

Vacca, O., Darche, M., Schaffer, D.V., Flannery, J.G., Sahel, J-A., Rendon, A. and Dalkara, D. GLIA, 62(3), 468-476 (2014)   Formation and maintenance of the blood–retinal barrier (BRB) is required for proper vision and breaching of this barrier contributes to the pathology in a wide variety of retinal conditions such as retinal detachment and diabetic retinopathy. Dystrophin Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells, its absence has been related to BRB permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels. Dp71-null mouse is thus an excellent model to approach the study of retinal pathologies showing blood–retinal barrier permeability. We aimed to investigate the participation of Müller cells in the BRB and in the inner limiting membrane of Dp71-null mice compared with wild-type mice in order to understand how these barriers work in this model of permeable BRB. To this aim, we used an Adeno-associated virus (AAV) variant, ShH10-GFP, engineered to target Müller cells specifically. ShH10 coding GFP was introduced by intravitreal injection and Müller cell transduction was studied in Dp71-null mice in comparison to wild-type animals. We show that Müller cell transduction follows a significantly different pattern in Dp71-null mice indicating changes in viral cell-surface receptors as well as differences in the permeability of the inner limiting membrane in this mouse line. However, the compromised BRB of the Dp71-null mice does not lead to virus leakage into the bloodstream when the virus is injected intravitreally – an important consideration for AAV-mediated retinal gene therapy.  

5.1391           Peek-a-boo: membrane hijacking and the pathogenesis of viral hepatitis

Feng, Z. and Lemon, S.M. Trends in Microbiol., 22(2), 59-64 (2014)   Historically, animal viruses have been classified on the basis of the presence or absence of an envelope – an external lipid bilayer membrane typically carrying one or more viral glycoproteins. However, growing evidence indicates that some ‘non-enveloped’ viruses circulate in the blood of infected individuals enveloped in host-derived membranes that provide protection from neutralizing antibodies. In this opinion article, we discuss this novel strategy for virus survival and consider how it contributes to the pathogenesis of acute viral hepatitis. The acquisition of an envelope by non-enveloped viruses profoundly influences their interaction with the host at both the cellular and system level and challenges how we think about vaccine protection against these infections.  

5.1392           Tannins from Hamamelis virginiana Bark Extract: Characterization and Improvement of the Antiviral Efficacy against Influenza A Virus and Human Papillomavirus

Theisen, L.L., Erdelmeier, C.A.J., Spoden, XG.A., Boukhallouk, F., Sausy, A., Florin, L. and Muller, C.P. PloS One, 9(1), e88062 (2014)   Antiviral activity has been demonstrated for different tannin-rich plant extracts. Since tannins of different classes and molecular weights are often found together in plant extracts and may differ in their antiviral activity, we have compared the effect against influenza A virus (IAV) of Hamamelis virginiana L. bark extract, fractions enriched in tannins of different molecular weights and individual tannins of defined structures, including pseudotannins. We demonstrate antiviral activity of the bark extract against different IAV strains, including the recently emerged H7N9, and show for the first time that a tannin-rich extract inhibits human papillomavirus (HPV) type 16 infection. As the best performing antiviral candidate, we identified a highly potent fraction against both IAV and HPV, enriched in high molecular weight condensed tannins by ultrafiltration, a simple, reproducible and easily upscalable method. This ultrafiltration concentrate and the bark extract inhibited early and, to a minor extent, later steps in the IAV life cycle and tannin-dependently inhibited HPV attachment. We observed interesting mechanistic differences between tannin structures: High molecular weight tannin containing extracts and tannic acid (1702 g/mol) inhibited both IAV receptor binding and neuraminidase activity. In contrast, low molecular weight compounds (<500 g/mol) such as gallic acid, epigallocatechin gallate or hamamelitannin inhibited neuraminidase but not hemagglutination. Average molecular weight of the compounds seemed to positively correlate with receptor binding (but not neuraminidase) inhibition. In general, neuraminidase inhibition seemed to contribute little to the antiviral activity. Importantly, antiviral use of the ultrafiltration fraction enriched in high molecular weight condensed tannins and, to a lesser extent, the unfractionated bark extract was preferable over individual isolated compounds. These results are of interest for developing and improving plant-based antivirals.    

5.1393           Bioluminescence-Based Monitoring of Virus Vector-Mediated Gene Transfer in Mice

Maguire, C.A. Methods in Mol. Biol., 1098, 197-209 (2014)   In vivo bioluminescence imaging (BLI) is a powerful technology that gives information on biological processes in living animals over multiple time points. Importantly BLI can also yield anatomical localization of signal which can provide important information when performing biodistribution studies of different macromolecules. This is of particular interest for gene therapy vectors such as adeno-associated virus (AAV) vectors in which knowledge of in vivo gene expression profiles help characterize what target tissues or organs the vector may be useful for. It can also be utilized to assess novel vector systems for their ability to overcome specific in vivo barriers of effective gene therapy. Here we describe BLI of AAV-encoded firefly luciferase (Fluc) expression in mice after intravascular delivery. This protocol can be amended for use with different virus vectors (e.g., lentivirus, adenovirus) as well as nonviral gene delivery (e.g., plasmid DNA, liposomes).  

5.1394           Adeno-Associated Virus-Based Vectors

Dutheil, N. and Bezard, E. Neuromethods, 82, 27-49 (2014)   Viral vectors based on recombinant adeno-associated virus have gained increasing interest over the last two decades as promising delivery vehicles in gene therapy. This enthusiasm is based on their ability to infect a broad range of tissues including proliferative and quiescent cells, to establish long-term expression in vitro and in vivo, combine to an excellent safety profile, as they are replication deficient, poorly immunogenic, and have not been associated to any disease. This chapter provides detailed protocols for small-scale production and purification of adeno-associated vectors and currently used methods for the titration and quality controls of these vectors.  

5.1395           Application of Viral Vectors to Motor Neuron Disorders

Dirren, E. and Schneider, B.L. Neuromethods, 82, 221-242 (2014)   Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are characterized by the progressive loss of motor neurons in the spinal cord and primary motor cortex. Subsequent paralysis of skeletal muscles leads to variable degrees of motor impairment and is inevitably fatal in ALS and type I SMA. A genetic cause has been defined for some of these conditions including SMA. Therefore, motor neuron disorders could become prime targets for gene therapy provided efficient tools can be designed to specifically target widely distributed motor neurons. Here, the application of viral vectors with a neuronal tropism is reviewed in the context of gene delivery to spinal lower motor neurons. The preparation of adeno-associated vector suspensions for motor neuron infection is described. Finally, we emphasize the use of intramuscular and intracerebroventricular delivery of adeno-associated vectors for the specific targeting of motor neurons.  

5.1396           Role of the vector genome and underlying factor IX mutation in immune responses to AAV gene therapy for hemophilia B

Rogers, G.L., Martino, A.T., Zolotukhin, I., Ertl, H.C.L. and Herzog, R.W.

1. Translational Med., 12:25 (2014)

  Background Self-complementary adeno-associated virus (scAAV) vectors have become a desirable vector for therapeutic gene transfer due to their ability to produce greater levels of transgene than single-stranded AAV (ssAAV). However, recent reports have suggested that scAAV vectors are more immunogenic than ssAAV. In this study, we investigated the effects of a self-complementary genome during gene therapy with a therapeutic protein, human factor IX (hF.IX). Methods Hemophilia B mice were injected intramuscularly with ss or scAAV1 vectors expressing hF.IX. The outcome of gene transfer was assessed, including transgene expression as well as antibody and CD8⁺ T cell responses to hF.IX. Results Self-complementary AAV1 vectors induced similar antibody responses (which eliminated systemic hF.IX expression) but stronger CD8⁺ T cell responses to hF.IX relative to ssAAV1 in mice with F9 gene deletion. As a result, hF.IX-expressing muscle fibers were effectively eliminated in scAAV-treated mice. In contrast, mice with F9 nonsense mutation (late stop codon) lacked antibody or T cell responses, thus showing long-term expression regardless of the vector genome. Conclusions The nature of the AAV genome can impact the CD8⁺ T cell response to the therapeutic transgene product. In mice with endogenous hF.IX expression, however, this enhanced immunogenicity did not break tolerance to hF.IX, suggesting that the underlying mutation is a more important risk factor for transgene-specific immunity than the molecular form of the AAV genome.  

5.1397           Pharmacologically controlled, discontinuous GDNF gene therapy restores motor function in a rat model of Parkinson's disease

Tereshchenko, J., Maddalena, A., Bähr, M. and Kügler, S. Neurobiology of Aging, 65, 35-42 (2014)   Neurotrophic factors have raised hopes to be able to cure symptoms and to prevent progressive neurodegeneration in devastating neurological diseases. Gene therapy by means of viral vectors can overcome the hurdle of targeted delivery, but its current configuration is irreversible and thus much less controllable than that of classical pharmacotherapies. We thus aimed at developing a strategy allowing for both curative and controllable neurotrophic factor expression. Therefore, the short-term, intermittent and reversible expression of a neutrophic factor was evaluated for therapeutic efficacy in a slowly progressive animal model of Parkinson's disease (PD). We demonstrate that short-term induced expression of glial cell line derived neurotrophic factor (GDNF) is sufficient to provide i) substantial protection of nigral dopaminergic neurons from degeneration and ii) restoration of dopamine supply and motor behaviour in the partial striatal 6-OHDA model PD. These neurorestorative effects of GDNF lasted several weeks beyond the time of its expression. Later on, therapeutic efficacy ceased, but was restored by a second short induction of GDNF expression, demonstrating that monthly application of the inducing drug mifepristone was sufficient to maintain neuroprotective and neurorestorative GDNF levels. These findings suggest that forthcoming gene therapies for PD or other neurodegenerative disorders can be designed in a way that low frequency application of an approved drug can provide controllable and therapeutically efficient levels of GDNF or other neurotrophic factors. Neurotrophic factor expression can be withdrawn in case of off-target effects or sufficient clinical benefit, a feature that may eventually increase the acceptance of gene therapy for less advanced patients, which may profit better from such approaches.  

5.1398           An AAV Vector-Mediated Gene Delivery Approach Facilitates Reconstitution of Functional Human CD8+ T Cells in Mice

Huang, J., Li, X., Coelho-dos-Reis, J.G:A., Wilson, J.M. and  Tsuji, M. PloS One, 9(2), e88205 (2014)   In the present study, a novel adeno-associated virus (AAV) vector-mediated gene delivery approach was taken to improve the reconstitution of functional CD8⁺ T cells in humanized mice, thereby mimicking the human immune system (HIS). Human genes encoding HLA-A2 and selected human cytokines (A2/hucytokines) were introduced to an immune-deficient mouse model [NOD/SCID/IL2rγ^null (NSG) mice] using AAV serotype 9 (AAV9) vectors, followed by transplantation of human hematopoietic stem cells. NSG mice transduced with AAV9 encoding A2/hucytokines resulted in higher levels of reconstitution of human CD45⁺ cells compared to NSG mice transduced with AAV9 encoding HLA-A2 alone or HLA-A2-transgenic NSG mice. Furthermore, this group of HIS mice also mounted the highest level of antigen-specific A2-restricted human CD8⁺ T-cell response upon vaccination with recombinant adenoviruses expressing human malaria and HIV antigens. Finally, the human CD8⁺ T-cell response induced in human malaria vaccine-immunized HIS mice was shown to be functional by displaying cytotoxic activity against hepatocytes that express the human malaria antigen in the context of A2 molecules. Taken together, our data show that AAV vector-mediated gene delivery is a simple and efficient method to transfer multiple human genes to immune-deficient mice, thus facilitating successful reconstitution of HIS in mice. The HIS mice generated in this study should ultimately allow us to swiftly evaluate the T-cell immunogenicity of various human vaccine candidates in a pre-clinical setting.    

5.1399           Intrathecal administration of IGF-I by AAVrh10 improves sensory and motor deficits in a mouse model of diabetic neuropathy

Homs, J., Pages, G., Ariza, L., Casas, C., Chillon, M., Navarro, X. and Bosch, A. Molecular Therapy-Methods & Clinical Development, 1:7 (2014)   Different adeno-associated virus (AAV) serotypes efficiently transduce neurons from central and peripheral nervous systems through various administration routes. Direct administration of the vectors to the cerebrospinal fluid (CSF) could be an efficient and safe strategy. Here, we show that lumbar puncture of a nonhuman AAV leads to wide and stable distribution of the vector along the spinal cord in adult mice. AAVrh10 efficiently and specifically infects neurons, both in dorsal root ganglia (60% total sensory neurons) and in the spinal cord (up to one-third of α-motor neurons). As a proof of concept, we demonstrate the efficacy of AAVrh10 in a mouse model of diabetic neuropathy, in which intrathecal delivery of the vector coding for insulin-like growth factor (IGF-I) favored the release of the therapeutic protein into the CSF through its expression by sensory and motor neurons. IGF-I–treated diabetic animals showed increased vascular endothelial growth factor expression, activation of Akt/PI3K pathway, and stimulated nerve regeneration and myelination in injured limbs. Moreover, we achieved restoration of nerve conduction velocities in both sensory and motor nerves by AAVrh10, whereas we reached only sensory nerve improvement with AAV1. Our results indicate that intrathecal injection of AAVrh10 is a promising tool to design gene therapy approaches for sensorimotor diseases.  

5.1400           Adipose tissue insulin receptor knockdown via a new primate-derived hybrid recombinant AAV serotype

Liu, X., Magee, D., Wang, C., McMurphy, T., Slater, A., During, M. and Cao, L. Molecular Therapy-Methods & Clinical Development, 1:8 (2014)   Adipose tissue plays an essential role in metabolic homeostasis and holds promise as an alternative depot organ in gene therapy. However, efficient methods of gene transfer into adipose tissue in vivo have yet to be established. Here, we assessed the transduction efficiency to fat depots by a family of novel engineered hybrid capsid serotypes (Rec1~4) recombinant adeno-associated viral (AAV) vectors in comparison with natural serotypes AAV1, AAV8, and AAV9. Rec2 serotype led to widespread transduction in both brown fat and white fat with the highest efficiency among the seven serotypes tested. As a proof-of-efficacy, Rec2 serotype was used to deliver Cre recombinase to adipose tissues of insulin receptor floxed animals. Insulin receptor knockdown led to decreased fat pad mass and morphological and molecular changes in the targeted depot. These novel hybrid AAV vectors can serve as powerful tools to genetically manipulate adipose tissue and provide valuable vehicles to gene therapy targeting adipose tissue.  

5.1401           A novel gene delivery method transduces porcine pancreatic duct epithelial cells

Griffin, M.A., Restrepo, M.S., Abu-El-Haija, M., Wallen, T., Buchanan, E., Rokhlina, T., Chen, Y.H., McCray, P.B., Davidson, B.L., Divekar, A. and Uc, A. Gene Therapy, 21, 123-130 (2014)   Gene therapy offers the possibility to treat pancreatic disease in cystic fibrosis (CF), caused by mutations in the CF transmembrane conductance regulator (CFTR) gene; however, gene transfer to the pancreas is untested in humans. The pancreatic disease phenotype is very similar between humans and pigs with CF; thus, CF pigs create an excellent opportunity to study gene transfer to the pancreas. There are no studies showing efficient transduction of pig pancreas with gene-transfer vectors. Our objective is to develop a safe and efficient method to transduce wild-type (WT) porcine pancreatic ducts that express CFTR. We catheterized the umbilical artery of WT newborn pigs and delivered an adeno-associated virus serotype 9 vector expressing green-fluorescent protein (AAV9CMV.sceGFP) or vehicle to the celiac artery, the vessel that supplies major branches to the pancreas. This technique resulted in stable and dose-dependent transduction of pancreatic duct epithelial cells that expressed CFTR. Intravenous (IV) injection of AAV9CMV.sceGFP did not transduce the pancreas. Our technique offers an opportunity to deliver the CFTR gene to the pancreas of CF pigs. The celiac artery can be accessed via the umbilical artery in newborns and via the femoral artery at older ages—delivery approaches that can be translated to humans.  

5.1402           Suppression of Langerhans cell activation is conserved amongst human papillomavirus α and β genotypes, but not a µ genotype

Da Silva, D.M., Movius, C.A., Raff, A.B., Brand, H.E., Skeate, J.G., Wong, M.K. and Kast, W.M. Virology, 452-453, 279-286 (2014)   Human papillomavirus (HPV) has evolved mechanisms that allow it to evade the human immune system. Studies have shown HPV-mediated suppression of activation of Langerhans cells (LC) is a key mechanism through which HPV16 evades initial immune surveillance. However, it has not been established whether high- and low-risk mucosal and cutaneous HPV genotypes share a common mechanism of immune suppression. Here, we demonstrate that LC exposed to capsids of HPV types 18, 31, 45, 11, (alpha-papillomaviruses) and HPV5 (beta-papillomavirus) similarly suppress LC activation, including lack of costimulatory molecule expression, lack of cytokine and chemokine secretion, lack of migration, and deregulated cellular signaling. In contrast, HPV1 (mu-papillomavirus) induced costimulatory molecule and cytokine upregulation, but LC migration and cellular signaling was suppressed. These results suggest that alpha and beta HPV genotypes, and partially a mu genotype, share a conserved mechanism of immune escape that enables these viruses to remain undetected in the absence of other inflammatory events.  

5.1403           Rift Valley Fever Virus Incorporates the 78 kDa Glycoprotein into Virions Matured in Mosquito C6/36 Cells

Weingartl, H.M., Zhang, S., Marszal, P., McGreevy, A. and Burton, L. PloS One, 9(1), e87385 (2014)   Rift Valley fever virus (RVFV), genus Phlebovirus, family Bunyaviridae is a zoonotic arthropod-borne virus able to transition between distant host species, causing potentially severe disease in humans and ruminants. Viral proteins are encoded by three genomic segments, with the medium M segment coding for four proteins: nonstructural NSm protein, two glycoproteins Gn and Gc and large 78 kDa glycoprotein (LGp) of unknown function. Goat anti-RVFV polyclonal antibody and mouse monoclonal antibody, generated against a polypeptide unique to the LGp within the RVFV proteome, detected this protein in gradient purified RVFV ZH501 virions harvested from mosquito C6/36 cells but not in virions harvested from the mammalian Vero E6 cells. The incorporation of LGp into the mosquito cell line - matured virions was confirmed by immune-electron microscopy. The LGp was incorporated into the virions immediately during the first passage in C6/36 cells of Vero E6 derived virus. Our data indicate that LGp is a structural protein in C6/36 mosquito cell generated virions. The protein may aid the transmission from the mosquitoes to the ruminant host, with a possible role in replication of RVFV in the mosquito host. To our knowledge, this is a first report of different protein composition between virions formed in insect C6/36 versus mammalian Vero E6 cells.  

5.1404           Zonisamide Attenuates α-Synuclein Neurotoxicity by an Aggregation-Independent Mechanism in a Rat Model of Familial Parkinson’s Disease

Arawaka, S., Fukushima, S., Sato, H., Sasaki, A., Koga, K., Koyama, S. and Kato, T. PloS One, 9(2), e89076 (2014)   The anti-epileptic agent zonisamide (ZNS) has been shown to exert protective effects in neurotoxin-based mouse models of Parkinson disease. However, it is unknown whether ZNS can attenuate toxicity of familial Parkinson’s disease-causing gene products. In this study, we investigated the effects of ZNS on neurodegeneration induced by expression of A53T α-synuclein in the rat substantia nigra using a recombinant adeno-associated virus vector. Expression of A53T α-synuclein yielded severe loss of nigral dopamine neurons and striatal dopamine nerve terminals from 2 weeks to 4 weeks after viral injection. Oral administration of ZNS (40 mg/kg/day) significantly delayed the pace of degeneration at 4 weeks after viral injection as compared with the vehicle group. This effect lasted until 8 weeks after viral injection, the final point of observation. ZNS treatment had no impact on the survival of nigrostriatal dopamine neurons in rats expressing green fluorescent protein. Quantification of striatal Ser129-phosphorylated α-synuclein-positive aggregates showed that these aggregates rapidly formed from 2 weeks to 4 weeks after viral injection. This increase was closely correlated with loss of nigrostriatal dopamine neurons. However, ZNS treatment failed to alter the number of all striatal Ser129-phosphorylated α-synuclein-positive aggregates, including small dot-like and large round structures. The number of these aggregates was almost constant at 4 weeks and 8 weeks after viral injection, although ZNS persistently prevented loss of nigrostriatal dopamine neurons during this period. Also, ZNS treatment did not affect the number of striatal aggregates larger than 10 µm in diameter. These data show that ZNS attenuates α-synuclein-induced toxicity in a manner that is independent of the formation and maturation of α-synuclein aggregates in an in vivo model of familial Parkinson’s disease, suggesting that ZNS may protect nigrostriatal dopamine neurons by modulating cellular damage or a cell death pathway commonly caused by neurotoxins and α-synuclein.  

5.1405           Protective Vaccination against Papillomavirus-Induced Skin Tumors under Immunocompetent and Immunosuppressive Conditions: A Preclinical Study Using a Natural Outbred Animal Model

Vinzon, S.E., Braspenning-Wesch, I., Müller, M., Geisller, E.K., Nindl, I., Gröne, H-J., Schäfer, K. and Rösl, F. PloS Pathogens, 10(2), e1003924 (2014)   Certain cutaneous human papillomaviruses (HPVs), which are ubiquitous and acquired early during childhood, can cause a variety of skin tumors and are likely involved in the development of non-melanoma skin cancer, especially in immunosuppressed patients. Hence, the burden of these clinical manifestations demands for a prophylactic approach. To evaluate whether protective efficacy of a vaccine is potentially translatable to patients, we used the rodent Mastomys coucha that is naturally infected with Mastomys natalensis papillomavirus (MnPV). This skin type papillomavirus induces not only benign skin tumours, such as papillomas and keratoacanthomas, but also squamous cell carcinomas, thereby allowing a straightforward read-out for successful vaccination in a small immunocompetent laboratory animal. Here, we examined the efficacy of a virus-like particle (VLP)-based vaccine on either previously or newly established infections. VLPs raise a strong and long-lasting neutralizing antibody response that confers protection even under systemic long-term cyclosporine A treatment. Remarkably, the vaccine completely prevents the appearance of benign as well as malignant skin tumors. Protection involves the maintenance of a low viral load in the skin by an antibody-dependent prevention of virus spread. Our results provide first evidence that VLPs elicit an effective immune response in the skin under immunocompetent and immunosuppressed conditions in an outbred animal model, irrespective of the infection status at the time of vaccination. These findings provide the basis for the clinical development of potent vaccination strategies against cutaneous HPV infections and HPV-induced tumors, especially in patients awaiting organ transplantation.  

5.1406           Studies of inactivation mechanism of non-enveloped icosahedral virus by a visible ultrashort pulsed laser

Tsen, S-W.D., Kingsley, D.H., Poweleit, C., Achilefu, S., Soroka, D.S., Wu, T.C. and Tsen, K-T. Virol. J., 11:20 (2014)   Background Low-power ultrashort pulsed (USP) lasers operating at wavelengths of 425 nm and near infrared region have been shown to effectively inactivate viruses such as human immunodeficiency virus (HIV), M13 bacteriophage, and murine cytomegalovirus (MCMV). It was shown previously that non-enveloped, helical viruses such as M13 bacteriophage, were inactivated by a USP laser through an impulsive stimulated Raman scattering (ISRS) process. Recently, enveloped virus like MCMV has been shown to be inactivated by a USP laser via protein aggregation induced by an ISRS process. However, the inactivation mechanism for a clinically important class of viruses – non-enveloped, icosahedral viruses remains unknown. Results and discussions We have ruled out the following four possible inactivation mechanisms for non-enveloped, icosahedral viruses, namely, (1) inactivation due to ultraviolet C (UVC) photons produced by non-linear optical process of the intense, fundamental laser beam at 425 nm; (2) inactivation caused by thermal heating generated by the direct laser absorption/heating of the virion; (3) inactivation resulting from a one-photon absorption process via chromophores such as porphyrin molecules, or indicator dyes, potentially producing reactive oxygen or other species; (4) inactivation by the USP lasers in which the extremely intense laser pulse produces shock wave-like vibrations upon impact with the viral particle. We present data which support that the inactivation mechanism for non-enveloped, icosahedral viruses is the impulsive stimulated Raman scattering process. Real-time PCR experiments show that, within the amplicon size of 273 bp tested, there is no damage on the genome of MNV-1 caused by the USP laser irradiation. Conclusion We conclude that our model non-enveloped virus, MNV-1, is inactivated by the ISRS process. These studies provide fundamental knowledge on photon-virus interactions on femtosecond time scales. From the analysis of the transmission electron microscope (TEM) images of viral particles before and after USP laser irradiation, the locations of weak structural links on the capsid of MNV-1 were revealed. This important information will greatly aid our understanding of the structure of non-enveloped, icosahedral viruses. We envision that this non-invasive, efficient viral eradication method will find applications in the disinfection of pharmaceuticals, biologicals and blood products in the near future.    

5.1407           Host DNA Damage Response Factors Localize to Merkel Cell Polyomavirus DNA Replication Sites To Support Efficient Viral DNA Replication

Tsang, S.H., Wang, X., Li, J., Buck, C.B. and You, J.

1. Virol., 88(6), 3285-3297 (2014)

  Accumulating evidence indicates a role for Merkel cell polyomavirus (MCPyV) in the development of Merkel cell carcinoma (MCC), making MCPyV the first polyomavirus to be clearly associated with human cancer. With the high prevalence of MCPyV infection and the increasing amount of MCC diagnosis, there is a need to better understand the virus and its oncogenic potential. In this study, we examined the relationship between the host DNA damage response (DDR) and MCPyV replication. We found that components of the ATM- and ATR-mediated DDR pathways accumulate in MCPyV large T antigen (LT)-positive nuclear foci in cells infected with native MCPyV virions. To further study MCPyV replication, we employed our previously established system, in which recombinant MCPyV episomal DNA is autonomously replicated in cultured cells. Similar to native MCPyV infection, where both MCPyV origin and LT are present, the host DDR machinery colocalized with LT in distinct nuclear foci. Immunofluorescence in situ hybridization and bromodeoxyuridine (BrdU) incorporation analysis showed that these DDR proteins and MCPyV LT in fact colocalized at the actively replicating MCPyV replication complexes, which were absent when a replication-defective LT mutant or an MCPyV-origin mutant was introduced in place of wild-type LT or wild-type viral origin. Inhibition of DDR kinases using chemical inhibitors and ATR/ATM small interfering RNA (siRNA) knockdown reduced MCPyV DNA replication without significantly affecting LT expression or the host cell cycle. This study demonstrates that these host DDR factors are important for MCPyV DNA replication, providing new insight into the host machinery involved in the MCPyV life cycle.  

5.1408           Adeno-associated viral serotypes produce differing titers and differentially transduce neurons within the rat basal and lateral amygdala

Holehonnur, R., Luong, J.A., Chaturvedi, D., Ho, A., Lella, S., Hosek, M. and Ploski, J.E. BMC Neurosci., 15:28 (2014)   Background In recent years, there has been an increased interest in using recombinant adeno-associated viruses (AAV) to make localized genetic manipulations within the rodent brain. Differing serotypes of AAV possess divergent capsid protein sequences and these variations greatly influence each serotype’s ability to transduce particular cell types and brain regions. We therefore aimed to determine the AAV serotype that is optimal for targeting neurons within the Basal and Lateral Amygdala (BLA) since the transduction efficiency of AAV has not been previously examined within the BLA. This region is desirable to genetically manipulate due to its role in emotion, learning & memory, and numerous psychiatric disorders. We accomplished this by screening 9 different AAV serotypes (AAV2/1, AAV2/2, AAV2/5, AAV2/7, AAV2/8, AAV2/9, AAV2/rh10, AAV2/DJ and AAV2/DJ8) designed to express red fluorescent protein (RFP) under the regulation of an alpha Ca2+/calmodulin-dependent protein kinase II promoter (αCaMKII). Results We determined that these serotypes produce differing amounts of virus under standard laboratory production. Notably AAV2/2 consistently produced the lowest titers compared to the other serotypes examined. These nine serotypes were bilaterally infused into the rat BLA at the highest titers achieved for each serotype and at a normalized titer of 7.8E + 11 GC/ml. Twenty one days following viral infusion the degree of transduction was quantitated throughout the amygdala. These viruses exhibited differential transduction of neurons within the BLA. AAV2/7 exhibited a trend toward having the highest efficiency of transduction and AAV2/5 exhibited significantly lower transduction efficiency as compared to the serotypes examined. AAV2/5′s decreased ability to transduce BLA neurons correlates with its significantly different capsid protein sequences as compared to the other serotypes examined. Conclusions For laboratories producing their own recombinant adeno-associated viruses, the use of AAV2/2 is likely less desirable since AAV2/2 produces significantly lower titers than many other serotypes of AAV. Numerous AAV serotypes appear to efficiently transduce BLA neurons, with the exception of AAV2/5. Taking into consideration the ability of certain serotypes to achieve high titers and transduce BLA neurons well, in our hands AAV2/DJ8 and AAV2/9 appear to be ideal serotypes to use when targeting neurons within the BLA.  

5.1409           Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

Spannaus, R. and Bodem, J. Virology, 454-455, 145-156 (2014)   In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2׳ and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies. The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation.  

5.1410           The effects of polymorphisms on human gene targeting

Deyle, D.R., Li, L.B., Ren, g. and Russell, D.W. Nucleic Acids Res., 42(5), 3119-3124 (2014)   DNA mismatches that occur between vector homology arms and chromosomal target sequences reduce gene targeting frequencies in several species; however, this has not been reported in human cells. Here we demonstrate that even a single mismatched base pair can significantly decrease human gene targeting frequencies. In addition, we show that homology arm polymorphisms can be used to direct allele-specific targeting or to improve unfavorable vector designs that introduce deletions.  

5.1411           Hepatic transforming growth factor-β 1 stimulated clone-22 D1 controls systemic cholesterol metabolism

Jäger, J., Greiner, V., Strzoda, D., Seibert, O., Niopek, K., Sijmonsma, T.P., Schäfer, M., Jones, A., De Gula, R., martigoni, M., Dallinga-Thie, G.M., Diaz, M.B., Hofman, T.G. and Herzig, S. Mol. Metabolism, 3, 155-166 (2014)   Disturbances in lipid homeostasis are hallmarks of severe metabolic disorders and their long-term complications, including obesity, diabetes, and atherosclerosis. Whereas elevation of triglyceride (TG)-rich very-low-density lipoproteins (VLDL) has been identified as a risk factor for cardiovascular complications, high-density lipoprotein (HDL)-associated cholesterol confers atheroprotection under obese and/or diabetic conditions. Here we show that hepatocyte-specific deficiency of transcription factor transforming growth factor β 1-stimulated clone (TSC) 22 D1 led to a substantial reduction in HDL levels in both wild-type and obese mice, mediated through the transcriptional down-regulation of the HDL formation pathway in liver. Indeed, overexpression of TSC22D1 promoted high levels of HDL cholesterol in healthy animals, and hepatic expression of TSC22D1 was found to be aberrantly regulated in disease models of opposing energy availability. The hepatic TSC22D1 transcription factor complex may thus represent an attractive target in HDL raising strategies in obesity/diabetes-related dyslipidemia and atheroprotection.  

5.1412           AAV vector-mediated secretion of chondroitinase provides a sensitive tracer for axonal arborisations

Alves, J.N., Muir, E.M., Andrews, M.R., Ward, A., Michelmore, N., Dasgupta, D., Verhaagen, J., Moloney, E.B., Keynes, R.J., Fawcett, J.W. and Rogers, J.H.

1. Neuroscience Methods, 227, 107-120 (2014)

  As part of a project to express chondroitinase ABC (ChABC) in neurons of the central nervous system, we have inserted a modified ChABC gene into an adeno-associated viral (AAV) vector and injected it into the vibrissal motor cortex in adult rats to determine the extent and distribution of expression of the enzyme. A similar vector for expression of green fluorescent protein (GFP) was injected into the same location. For each vector, two versions with minor differences were used, giving similar results. After 4 weeks, the brains were stained to show GFP and products of chondroitinase digestion. Chondroitinase was widely expressed, and the AAV-ChABC and AAV-GFP vectors gave similar expression patterns in many respects, consistent with the known projections from the directly transduced neurons in vibrissal motor cortex and adjacent cingulate cortex. In addition, diffusion of vector to deeper neuronal populations led to labelling of remote projection fields which was much more extensive with AAV-ChABC than with AAV-GFP. The most notable of these populations are inferred to be neurons of cortical layer 6, projecting widely in the thalamus, and neurons of the anterior pole of the hippocampus, projecting through most of the hippocampus. We conclude that, whereas GFP does not label the thinnest axonal branches of some neuronal types, chondroitinase is efficiently secreted from these arborisations and enables their extent to be sensitively visualised. After 12 weeks, chondroitinase expression was undiminished.  

5.1413           AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

Tenney, R.M., Bell, C.L. and Wilson, J.M. Virology, 454-455, 227-236 (2014)   Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8׳s robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII & IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype.  

5.1414           Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons

Choi, J-H., Yu, N-K., Baek, G-C., Bakes, J., Seo, D., Nam, H.J., Baek, S.H., Lim, C-S., Lee, Y-S. and Kaang, B-K. Mol. Brain., 7:17, (2014)   Adeno-associated virus (AAV) vectors can deliver transgenes to diverse cell types and are therefore useful for basic research and gene therapy. Although AAV has many advantages over other viral vectors, its relatively small packaging capacity limits its use for delivering large genes. The available transgene size is further limited by the existence of additional elements in the expression cassette without which the gene expression level becomes much lower. By using alternative combinations of shorter elements, we generated a series of AAV expression cassettes and systematically evaluated their expression efficiency in neurons to maximize the transgene size available within the AAV packaging capacity while not compromising the transgene expression. We found that the newly developed smaller expression cassette shows comparable expression efficiency with an efficient vector generally used for strong gene expression. This new expression cassette will allow us to package larger transgenes without compromising expression efficiency.  

5.1415           Vertebrate Cone Opsins Enable Sustained and Highly Sensitive Rapid Control of Gi/o Signaling in Anxiety Circuitry

Masseck, O.A., Spoida, K., Dalkara, D., Maejima, T., Rubelowski, J.M., Wallhorn, L., Deneris, E.S. and Herlitze, S. Neuron, 81(6), 1263-1273 (2014)   G protein-coupled receptors (GPCRs) coupling to G_i/o signaling pathways are involved in the control of important physiological functions, which are difficult to investigate because of the limitation of tools to control the signaling pathway with precise kinetics and specificity. We established two vertebrate cone opsins, short- and long-wavelength opsin, for long-lasting and repetitive activation of G_i/o signaling pathways in vitro and in vivo. We demonstrate for both opsins the repetitive fast, membrane-delimited, ultra light-sensitive, and wavelength-dependent activation of the G_i/o pathway in HEK cells. We also show repetitive control of G_i/o pathway activation in 5-HT_1A receptor domains in the dorsal raphe nucleus (DRN) in brain slices and in vivo, which is sufficient to modulate anxiety behavior in mice. Thus, vertebrate cone opsins represent a class of tools for understanding the role of G_i/o-coupled GPCRs in health and disease.        

5.1416           Up-Regulation of the ATP-Binding Cassette Transporter A1 Inhibits Hepatitis C Virus Infection

Bocchetta, S., Maillard, P., Yamamoto, M., Gondeau, C., Douam, F., Lebreton, S., Lagaye, S., Pol, S., Helle, F., Plengpanich, W., Guerin, M., Bourgine, M., Michel, M.L., Lavillette, D., Roingeard, P., le Goff, W. and Budkowska, A. PloS One, 9(3), e92140 (2014)   Hepatitis C virus (HCV) establishes infection using host lipid metabolism pathways that are thus considered potential targets for indirect anti-HCV strategies. HCV enters the cell via clathrin-dependent endocytosis, interacting with several receptors, and virus-cell fusion, which depends on acidic pH and the integrity of cholesterol-rich domains of the hepatocyte membrane. The ATP-binding Cassette Transporter A1 (ABCA1) mediates cholesterol efflux from hepatocytes to extracellular Apolipoprotein A1 and moves cholesterol within cell membranes. Furthermore, it generates high-density lipoprotein (HDL) particles. HDL protects against arteriosclerosis and cardiovascular disease. We show that the up-regulation of ABCA1 gene expression and its cholesterol efflux function in Huh7.5 hepatoma cells, using the liver X receptor (LXR) agonist GW3965, impairs HCV infection and decreases levels of virus produced. ABCA1-stimulation inhibited HCV cell entry, acting on virus-host cell fusion, but had no impact on virus attachment, replication, or assembly/secretion. It did not affect infectivity or properties of virus particles produced. Silencing of the ABCA1 gene and reduction of the specific cholesterol efflux function counteracted the inhibitory effect of the GW3965 on HCV infection, providing evidence for a key role of ABCA1 in this process. Impaired virus-cell entry correlated with the reorganisation of cholesterol-rich membrane microdomains (lipid rafts). The inhibitory effect could be reversed by an exogenous cholesterol supply, indicating that restriction of HCV infection was induced by changes of cholesterol content/distribution in membrane regions essential for virus-cell fusion. Stimulation of ABCA1 expression by GW3965 inhibited HCV infection of both human primary hepatocytes and isolated human liver slices. This study reveals that pharmacological stimulation of the ABCA1-dependent cholesterol efflux pathway disrupts membrane cholesterol homeostasis, leading to the inhibition of virus–cell fusion and thus HCV cell entry. Therefore besides other beneficial roles, ABCA1 might represent a potential target for HCV therapy.  

5.1417           Biochemical and physiological improvement in a mouse model of Smith–Lemli–Opitz syndrome (SLOS) following gene transfer with AAV vectors

Ying, L., Matabosch, X., Serra, M., Watson, B., Shackleton, C. and Watson, G. Molecular Genetics and Metabolism Reports,1, 103-113 (2014)   Smith–Lemli–Opitz syndrome (SLOS) is an inborn error of cholesterol synthesis resulting from a defect in 7-dehydrocholesterol reductase (DHCR7), the enzyme that produces cholesterol from its immediate precursor 7-dehydrocholesterol. Current therapy employing dietary cholesterol is inadequate. As SLOS is caused by a defect in a single gene, restoring enzyme functionality through gene therapy may be a direct approach for treating this debilitating disorder. In the present study, we first packaged a human DHCR7 construct into adeno-associated virus (AAV) vectors having either type-2 (AAV2) or type-8 (AAV2/8) capsid, and administered treatment to juvenile mice. While a positive response (assessed by increases in serum and liver cholesterol) was seen in both groups, the improvement was greater in the AAV2/8–DHCR7 treated mice. Newborn mice were then treated with AAV2/8–DHCR7 and these mice, compared to mice treated as juveniles, showed higher DHCR7 mRNA expression in liver and a greater improvement in serum and liver cholesterol levels. Systemic treatment did not affect brain cholesterol in any of the experimental groups. Both juvenile and newborn treatments with AAV2/8–DHCR7 resulted in increased rates of weight gain indicating that gene transfer had a positive physiological effect.  

5.1418           Recombinant Adeno-Associated Virus Utilizes Host Cell Nuclear Import Machinery To Enter the Nucleus

Nicolson, S.C. and Samulski, R.J.

1. Virol., 88(8), 4132-4144 (2014)

  Recombinant adeno-associated viral (rAAV) vectors have garnered much promise in gene therapy applications. However, widespread clinical use has been limited by transduction efficiency. Previous studies suggested that the majority of rAAV accumulates in the perinuclear region of cells, presumably unable to traffic into the nucleus. rAAV nuclear translocation remains ill-defined; therefore, we performed microscopy, genetic, and biochemical analyses in vitro in order to understand this mechanism. Lectin blockade of the nuclear pore complex (NPC) resulted in inhibition of nuclear rAAV2. Visualization of fluorescently labeled particles revealed that rAAV2 localized to importin-β-dense regions of cells in late trafficking steps. Additionally, small interfering RNA (siRNA) knockdown of importin-β partially inhibited rAAV2 nuclear translocation and inhibited transduction by 50 to 70%. Furthermore, coimmunopreciptation (co-IP) analysis revealed that capsid proteins from rAAV2 could interact with importin-β and that this interaction was sensitive to the small GTPase Ran. More importantly, mutations to key basic regions in the rAAV2 capsid severely inhibited interactions with importin-β. We tested several other serotypes and found that the extent of importin-β interaction varied, suggesting that different serotypes may utilize alternative import proteins for nuclear translocation. Co-IP and siRNA analyses were used to investigate the role of other karyopherins, and the results suggested that rAAV2 may utilize multiple import proteins for nuclear entry. Taken together, our results suggest that rAAV2 interacts with importin-β alone or in complex with other karyopherins and enters the nucleus via the NPC. These results may lend insight into the design of novel AAV vectors that have an enhanced nuclear entry capability and transduction potential.  

5.1419           A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca2+ Uptake into the Sarcoplasmic Reticulum

Grössl, T., Hammer, E., Bien-Möller, S., Geisler, A., Pinkert, S., Röger, C., Poller, W., Kurreck, J., Völker, U., Vetter, R. and Fechner, H. PloS One, 9(3), e92188 (2014)   In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB) expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr) improves both impaired SERCA2a controlled Ca²⁺ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr) directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr) from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM) over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca²⁺ uptake into sarcoplasmic reticulum (SR) vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca²⁺ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.  

5.1420           A Cytosolic Chaperone Complexes with Dynamic Membrane J-Proteins and Mobilizes a Nonenveloped Virus out of the Endoplasmic Reticulum

Walczak, C.P., Ravindran, M.S., Inoue, T. and Tsai, B. PloS Pathogens, 10(3), e1004007 (2014)   Nonenveloped viruses undergo conformational changes that enable them to bind to, disrupt, and penetrate a biological membrane leading to successful infection. We assessed whether cytosolic factors play any role in the endoplasmic reticulum (ER) membrane penetration of the nonenveloped SV40. We find the cytosolic SGTA-Hsc70 complex interacts with the ER transmembrane J-proteins DnaJB14 (B14) and DnaJB12 (B12), two cellular factors previously implicated in SV40 infection. SGTA binds directly to SV40 and completes ER membrane penetration. During ER-to-cytosol transport of SV40, SGTA disengages from B14 and B12. Concomitant with this, SV40 triggers B14 and B12 to reorganize into discrete foci within the ER membrane. B14 must retain its ability to form foci and interact with SGTA-Hsc70 to promote SV40 infection. Our results identify a novel role for a cytosolic chaperone in the membrane penetration of a nonenveloped virus and raise the possibility that the SV40-induced foci represent cytosol entry sites.  

5.1421           Triple Trans-Splicing Adeno-Associated Virus Vectors Capable of Transferring the Coding Sequence for Full-Length Dystrophin Protein into Dystrophic Mice full access

Koo, T., Popplewell, L., Athanasopoulos, T. and Dickson, G. Human Gene Therapy, 25(2), 98-108 (2014)   Recombinant adeno-associated virus (rAAV) vectors have been shown to permit very efficient widespread transgene expression in skeletal muscle after systemic delivery, making these increasingly attractive as vectors for Duchenne muscular dystrophy (DMD) gene therapy. DMD is a severe muscle-wasting disorder caused by DMD gene mutations leading to complete loss of dystrophin protein. One of the major issues associated with delivery of the DMD gene, as a therapeutic approach for DMD, is its large open reading frame (ORF; 11.1 kb). A series of truncated microdystrophin cDNAs (delivered via a single AAV) and minidystrophin cDNAs (delivered via dual-AAV trans-spliced/overlapping reconstitution) have thus been extensively tested in DMD animal models. However, critical rod and hinge domains of dystrophin required for interaction with components of the dystrophin-associated protein complex, such as neuronal nitric oxide synthase, syntrophin, and dystrobrevin, are missing; these dystrophin domains may still need to be incorporated to increase dystrophin functionality and stabilize membrane rigidity. Full-length DMD gene delivery using AAV vectors remains elusive because of the limited single-AAV packaging capacity (4.7 kb). Here we developed a novel method for the delivery of the full-length DMD coding sequence to skeletal muscles in dystrophic mdx mice using a triple-AAV trans-splicing vector system. We report for the first time that three independent AAV vectors carrying “in tandem” sequential exonic parts of the human DMD coding sequence enable the expression of the full-length protein as a result of trans-splicing events cojoining three vectors via their inverted terminal repeat sequences. This method of triple-AAV-mediated trans-splicing could be applicable to the delivery of any large therapeutic gene (≥11 kb ORF) into postmitotic tissues (muscles or neurons) for the treatment of various inherited metabolic and genetic diseases.  

5.1422           Intracerebroventricular Injection of Adeno-Associated Virus 6 and 9 Vectors for Cell Type–Specific Transgene Expression in the Spinal Cord full access

Dirren, E., Towne, C.L., Setola, V., Redmond Jr., D.E., Schneider, B.L. and Aebischer, P. Human Gene Therapy, 25(2), 109-120 (2014)   In the context of motoneuron diseases, gene delivery as an experimental or therapeutic approach is hindered by the challenge to specifically target cell populations that are widely distributed along the spinal cord. Further complicating the task, transgenes often need to be delivered to motoneurons and/or glial cells to address the non-cell-autonomous mechanisms involved in disease pathogenesis. Intracerebroventricular (ICV) injection of recombinant adeno-associated viruses (AAVs) in newborn mice allows distributing viral vectors throughout the central nervous system while limiting undesired transduction of peripheral organs. Here, we show that by combining the appropriate set of AAV serotype and promoter, specific transgene expression can be achieved in either motoneurons or astrocytes along the whole mouse spinal cord. ICV injection of recombinant AAV6 with the cytomegalovirus (cmv) promoter preferentially targets motoneurons, whereas AAV9 particles combined with the astrocyte-specific gfaABC₁D promoter lead to significant transgene expression selectively targeted to astrocytes. Importantly, ICV coinjection of both AAV6-cmv and AAV9-gfaABC₁D results in segregated expression of two different transgenes in motoneurons and astrocytes, respectively. Relevance of viral vector delivery via the cerebrospinal fluid was further investigated in young nonhuman primates. Intracisternal injection of recombinant AAV6-cmv led to robust cervical transduction of motoneurons, highlighting the potential of this approach for gene therapy and modeling of motoneuron diseases.  

5.1423           Human iPSC models of neuronal ceroid lipofuscinosis capture distinct effects of TPP1 and CLN3 mutations on the endocytic pathway

Lojewski, X. et al Hum. Mol. Genet., 23(8), 2005-( (2014)   Neuronal ceroid lipofuscinosis (NCL) comprises ∼13 genetically distinct lysosomal disorders primarily affecting the central nervous system. Here we report successful reprograming of patient fibroblasts into induced pluripotent stem cells (iPSCs) for the two most common NCL subtypes: classic late-infantile NCL, caused by TPP1(CLN2) mutation, and juvenile NCL, caused by CLN3 mutation. CLN2/TPP1- and CLN3-iPSCs displayed overlapping but distinct biochemical and morphological abnormalities within the endosomal–lysosomal system. In neuronal derivatives, further abnormalities were observed in mitochondria, Golgi and endoplasmic reticulum. While lysosomal storage was undetectable in iPSCs, progressive disease subtype-specific storage material was evident upon neural differentiation and was rescued by reintroducing the non-mutated NCL proteins. In proof-of-concept studies, we further documented differential effects of potential small molecule TPP1 activity inducers. Fenofibrate and gemfibrozil, previously reported to induce TPP1 activity in control cells, failed to increase TPP1 activity in patient iPSC-derived neural progenitor cells. Conversely, nonsense suppression by PTC124 resulted in both an increase of TPP1 activity and attenuation of neuropathology in patient iPSC-derived neural progenitor cells. This study therefore documents the high value of this powerful new set of tools for improved drug screening and for investigating early mechanisms driving NCL pathogenesis.  

5.1424           Mutant Ataxin-3 with an Abnormally Expanded Polyglutamine Chain Disrupts Dendritic Development and Metabotropic Glutamate Receptor Signaling in Mouse Cerebellar Purkinje Cells

Konno, A., Shuvaev, A.N., Miyake, N., Miyake, K., Iizuka, A., Matsuura, S., Huda, F., Nakamura, K., Yanagi, S., Shimada, T. and Hirai, H. Cerebellum., 13(1), 29-41 (2014)   Spinocerebellar ataxia type 3 (SCA3) is caused by the abnormal expansion of CAG repeats within the ataxin-3 gene. Previously, we generated transgenic mice (SCA3 mice) that express a truncated form of ataxin-3 containing abnormally expanded CAG repeats specifically in cerebellar Purkinje cells (PCs). Here, we further characterize these SCA3 mice. Whole-cell patch-clamp analysis of PCs from advanced-stage SCA3 mice revealed a significant decrease in membrane capacitance due to poor dendritic arborization and the complete absence of metabotropic glutamate receptor subtype1 (mGluR1)-mediated retrograde suppression of synaptic transmission at parallel fiber terminals, with an overall preservation of AMPA receptor-mediated fast synaptic transmission. Because these cerebellar phenotypes are reminiscent of retinoic acid receptor-related orphan receptor α (RORα)-defective staggerer mice, we examined the levels of RORα in the SCA3 mouse cerebellum by immunohistochemistry and found a marked reduction of RORα in the nuclei of SCA3 mouse PCs. To confirm that the defects in SCA3 mice were caused by postnatal deposition of mutant ataxin-3 in PCs, not by genome disruption via transgene insertion, we tried to reduce the accumulation of mutant ataxin-3 in developing PCs by viral vector-mediated expression of CRAG, a molecule that facilitates the degradation of stress proteins. Concomitant with the removal of mutant ataxin-3, CRAG-expressing PCs had greater numbers of differentiated dendrites compared to non-transduced PCs and exhibited retrograde suppression of synaptic transmission following mGluR1 activation. These results suggest that postnatal nuclear accumulation of mutant ataxin-3 disrupts dendritic differentiation and mGluR-signaling in SCA3 mouse PCs, and this disruption may be caused by a defect in a RORα-driven transcription pathway.  

5.1425           shRNA-induced saturation of the microRNA pathway in the rat brain

van Gestel, M.A., van Erp, S., Sanders, L.E., Brans, M.A.D., Luijendijk, M.C.M., Merkestein, M., Pasterkamp, R.J. and Adan, R.A.H. Gene Therapy, 21, 205-211 (2014)   RNA interference (RNAi) is a powerful strategy for unraveling gene function and for drug target validation, but exogenous expression of short hairpin RNAs (shRNAs) has been associated with severe side effects. These may be caused by saturation of the microRNA pathway. This study shows degenerative changes in cell morphology and intrusion of blood vessels after transduction of the ventromedial hypothalamus (VMH) of rats with a shRNA expressing adeno-associated viral (AAV) vector. To investigate whether saturation of the microRNA pathway has a role in the observed side effects, expression of neuronal microRNA miR-124 was used as a marker. Neurons transduced with the AAV vector carrying the shRNA displayed a decrease in miR-124 expression. The decreased expression was unrelated to shRNA sequence or target and observed as early as 1 week after injection. In conclusion, this study shows that the tissue response after AAV-directed expression of a shRNA to the VMH is likely to be caused by shRNA-induced saturation of the microRNA pathway. We recommend controlling for miR-124 expression when using RNAi as a tool for studying (loss of) gene function in the brain as phenotypic effects caused by saturation of the RNAi pathway might mask true effects of specific downregulation of the shRNA target.  

5.1426           Repression of the Proapoptotic Cellular BIK/NBK Gene by Epstein-Barr Virus Antagonizes Transforming Growth Factor β1-Induced B-Cell Apoptosis

Campion, E.M., Hakimjavadi, R., Loughran, S.T., Phelan, S., Smith, S.M., D’Souza, B.N., Tierney, R.J., Bell, A.I., Cahill, P.A. and Walls, D.

1. Virol., 88(9), 5001-5013 (2014)

  The Epstein-Barr virus (EBV) establishes a lifelong latent infection in humans. EBV infection of primary B cells causes cell activation and proliferation, a process driven by the viral latency III gene expression program, which includes EBV nuclear proteins (EBNAs), latent membrane proteins, and untranslated RNAs, including microRNAs. Some latently infected cells enter the long-lived memory B-cell compartment and express only EBNA1 transiently (Lat I) or no EBV protein at all (Lat 0). Targeting the molecular machinery that controls B-cell fate decisions, including the Bcl-2 family of apoptosis-regulating proteins, is crucial to the EBV cycle of infection. Here, we show that BIK (also known as NBK), which encodes a proapoptotic “sensitizer” protein, is repressed by the EBNA2-driven Lat III program but not the Lat I program. BIK repression occurred soon after infection of primary B cells by EBV but not by a recombinant EBV in which the EBNA2 gene had been knocked out. Ectopic BIK induced apoptosis in Lat III cells by a mechanism dependent on its BH3 domain and the activation of caspases. We show that EBNA2 represses BIK in EBV-negative B-cell lymphoma-derived cell lines and that this host-virus interaction can inhibit the proapoptotic effect of transforming growth factor β1 (TGF-β1), a key physiological mediator of B-cell homeostasis. Reduced levels of TGF-β1-associated regulatory SMAD proteins were bound to the BIK promoter in response to EBV Lat III or ectopic EBNA2. These data are evidence of an additional mechanism used by EBV to promote B-cell survival, namely, the transcriptional repression of the BH3-only sensitizer BIK.  

5.1427           Local overexpression of the myostatin propeptide increases glucose transporter expression and enhances skeletal muscle glucose disposal

Cleasby, M.E., Jarmin, S., Eilers, W., Elashry, M., Andersen, D.K., Dickson, G. and Foster, K. Am. J. Physiol. Endocrinol. Metab., 306, E814-E823 (2014)   Insulin resistance (IR) in skeletal muscle is a prerequisite for type 2 diabetes and is often associated with obesity. IR also develops alongside muscle atrophy in older individuals in sarcopenic obesity. The molecular defects that underpin this syndrome are not well characterized, and there is no licensed treatment. Deletion of the transforming growth factor-β family member myostatin, or sequestration of the active peptide by overexpression of the myostatin propeptide/latency-associated peptide (ProMyo) results in both muscle hypertrophy and reduced obesity and IR. We aimed to establish whether local myostatin inhibition would have a paracrine/autocrine effect to enhance glucose disposal beyond that simply generated by increased muscle mass, and the mechanisms involved. We directly injected adeno-associated virus expressing ProMyo in right tibialis cranialis/extensor digitorum longus muscles of rats and saline in left muscles and compared the effects after 17 days. Both test muscles were increased in size (by 7 and 11%) and showed increased radiolabeled 2-deoxyglucose uptake (26 and 47%) and glycogen storage (28 and 41%) per unit mass during an intraperitoneal glucose tolerance test. This was likely mediated through increased membrane protein levels of GLUT1 (19% higher) and GLUT4 (63% higher). Interestingly, phosphorylation of phosphoinositol 3-kinase signaling intermediates and AMP-activated kinase was slightly decreased, possibly because of reduced expression of insulin-like growth factor-I in these muscles. Thus, myostatin inhibition has direct effects to enhance glucose disposal in muscle beyond that expected of hypertrophy alone, and this approach may offer potential for the therapy of IR syndromes.  

5.1428           AAV-Mediated Gene Transfer of the Obesity-Associated Gene Etv5 in Rat Midbrain Does Not Affect Energy Balance or Motivated Behavior

Boender, A.J., Koning, N.A., van den Heuvel, J.K., Luijendijk, M.C.M., van Rozen, A.J., la Fleur, S.E. and Adan, R.A.H. PloS One, 9(4), e94159 (2014)   Several genome-wide association studies have implicated the transcription factor E-twenty- six version 5 (Etv5) in the regulation of body mass index. Further substantiating the role of Etv5 in feeding behavior are the findings that targeted disruption of Etv5 in mice leads to decreased body weight gain and that expression of Etv5 is decreased in the ventral tegmental area and substantia nigra pars compacta (VTA/SNpc) after food restriction. As Etv5 has been suggested to influence dopaminergic neurotransmission by driving the expression of genes that are responsible for the synthesis and release of dopamine, we investigated if expression levels of Etv5 are dependent on nutritional state and subsequently influence the expression levels of tyrosine hydroxylase. While it was shown that Etv5 expression in the VTA/SNpc increases after central administration of leptin and that Etv5 was able to drive expression of tyrosine hydroxylase in vitro, AAV-mediated gene transfer of Etv5 into the VTA/SNpc of rats did not alter expression of tyrosine hydroxylase in vivo. Moreover, AAV-mediated gene transfer of Etv5 in the VTA/SNpc did not affect measures of energy balance or performances in a progressive ratio schedule. Thus, these data do not support a role for increased expression of Etv5 in the VTA/SNpc in the regulation of feeding behavior.    

5.1429           Inhibition by Cellular Vacuolar ATPase Impairs Human Papillomavirus Uncoating and Infection

Müller, K.H., Spoden, G.A., Scheffer, K.D., Brunnhöfer, R., De Brabander, J.K., Maier, M.E., Florin, L. and Muller, C.P. Antimicrob. Agents Chemother., 58(5), 2905-2911 (2014)   Several viruses, including human papillomaviruses, depend on endosomal acidification for successful infection. Hence, the multisubunit enzyme vacuolar ATPase (V-ATPase), which is mainly responsible for endosome acidification in the cell, represents an attractive target for antiviral strategies. In the present study, we show that V-ATPase is required for human papillomavirus (HPV) infection and that uncoating/disassembly but not endocytosis is affected by V-ATPase inhibition. The infection inhibitory potencies of saliphenylhalamide, a proven V-ATPase inhibitor, and its derivatives, as well as those of other V-ATPase inhibitors, were analyzed on different HPV types in relevant cell lines. Variation in the selectivity indices among V-ATPase inhibitors was high, while variation for the same inhibitor against different HPV subtypes was low, indicating that broad-spectrum anti-HPV activity can be provided.  

5.1430           Coxsackievirus B Exits the Host Cell in Shed Microvesicles Displaying Autophagosomal Markers

Robinson, S.M., Tsueng, G., Sin, J., Mangale, V., Rahawi, S., McIntyre, L.L., Williams, W., Kha, N., Cruz, C., Hancock, B.M., Nguyen, D.P., Sayen, M.R., Hilton, B.J., Doran, K.S., Segali, A.M., Wolkowicz, R., Cornell, C.T., Whitton, J.L., Gottlieb, R.A. and Feuer, R. PloS Pathogens, 10(4), e1004045 (2014)   Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, “fluorescent timer” protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. “Fluorescent timer” protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of “fluorescent timer” protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. “Fluorescent timer” protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured “fluorescent timer” protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.  

5.1431           Engineered retroviral virus-like particles for receptor targeting

Vorackova, I., Ulbrich, P., Diehl, W.E. and Ruml, T. Arch. Virol., 159, 677-688 (2014)   Retroviral gag proteins, as well as fragments minimally containing the capsid (CA) and nucleocapsid (NC) subunits of Gag, are able to spontaneously assemble into virus-like particles (VLPs). This occurs in mammalian and bacterial cells as well as in in vitro systems. In every circumstance, nucleic acids are incorporated into the forming particles. Here, we took advantage of an in vitro system for the generation of non-enveloped Mason-Pfizer monkey virus (M-PMV) VLPs derived from a self-assembling CA-NC subunit of Gag. These VLPs were modified through N-terminal extension of CA-NC with short oligopeptides that, after the assembly process, were exposed on the surface of VLPs. The employed N-terminal modifications allowed specific interaction with target cells expressing prostate-specific membrane antigen. Using this system, we were able to incorporate selected siRNA into forming VLPs and deliver it into the cytosol of target cells. In comparison with other viral vectors designed for targeted transgene delivery, this M-PMV VLP system represents the lowest risk of generating virus-associated pathology, as the VLPs do not contain any viral coding sequences and are formed in a cell-free system.  

5.1432           Enhanced Gene Targeting of Adult and Pluripotent Stem Cells Using Evolved Adeno-associated Virus

Bartel, M.A. and Schaffer, D.V. Methods in Mol. Biol., 1114, 169-179 (2014)   Efficient approaches for the precise genetic engineering of stem cells can enhance both basic and applied stem cell research. Adeno-associated virus (AAV) vectors have demonstrated high-efficiency gene delivery and gene targeting to numerous cell types, and AAV vectors developed specifically for gene delivery to stem cells have further increased gene targeting frequency compared to plasmid construct techniques. This chapter details the production and purification techniques necessary to generate adeno-associated viral vectors for use in high-efficiency gene targeting of adult or pluripotent stem cell applications. Culture conditions used to achieve high gene targeting frequencies in rat neural stem cells and human pluripotent stem cells are also described.  

5.1433           Herpes Simplex Virus Growth, Preparation, and Assay

Marconi, P. and Manservigi, R. Methods in Mol. Biol., 1144, 19-29 (2014)   In order to study the biology of herpes simplex virus or to use it as a vector in gene therapy, it is necessary to grow the virus and to prepare virus stocks. Many different protocols are available from different research groups working with herpes simplex virus type 1 or 2 (HSV-1 or HSV-2). This chapter describes the procedures used in our laboratory.  

5.1434           The Acyclic Retinoid Peretinoin Inhibits Hepatitis C Virus Replication and Infectious Virus Release in Vitro

Shimakami, T., Honda, M., Shirasaki, T., Takabatake, R., Liu, F., Murai, K., Shiomoto, T., Funaki, M., Yamane, D., Murakami, S., Lemon, S.M. and Kaneko, S. Scientific Reports, 4:4688 (2014)   Clinical studies suggest that the oral acyclic retinoid Peretinoin may reduce the recurrence of hepatocellular carcinoma (HCC) following surgical ablation of primary tumours. Since hepatitis C virus (HCV) infection is a major cause of HCC, we assessed whether Peretinoin and other retinoids have any effect on HCV infection. For this purpose, we measured the effects of several retinoids on the replication of genotype 1a, 1b, and 2a HCV in vitro. Peretinoin inhibited RNA replication for all genotypes and showed the strongest antiviral effect among the retinoids tested. Furthermore, it reduced infectious virus release by 80–90% without affecting virus assembly. These effects could be due to reduced signalling from lipid droplets, triglyceride abundance, and the expression of mature sterol regulatory element-binding protein 1c and fatty acid synthase. These negative effects of Peretinoin on HCV infection may be beneficial in addition to its potential for HCC chemoprevention in HCV-infected patients.  

5.1435           Proteomic Analysis of Glycine Receptor β Subunit (GlyRβ)-interacting Proteins: EVIDENCE FOR SYNDAPIN I REGULATING SYNAPTIC GLYCINE RECEPTORS

Del Pinto, I., Koch, D., Schemm, R., Qualmann, B., Betz, H. and Paarmann, I.

1. Biol. Chem., 289(16), 11396-11409 (2014)

  Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their β subunits (GlyRβ) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRβ, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378–455 of the large intracellular loop of GlyRβ as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRβ that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRβ in vitro and colocalized with GlyRβ upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRβ. Deletion and point mutation analysis disclosed that SdpI binding to GlyRβ is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs.  

5.1436           HIV-1 Nef Inhibits Protease Activity and Its Absence Alters Protein Content of Mature Viral Particles

Mendonca, L.M., Poeys, S.C., Abreu, C.M., Tanuri, A. and Costa, L.J. PloS One, 9(4), e95352 (2014)   Nef is an important player for viral infectivity and AIDS progression, but the mechanisms involved are not completely understood. It was previously demonstrated that Nef interacts with GagPol through p6*-Protease region. Because p6* and Protease are involved in processing, we explored the effect of Nef on viral Protease activity and virion assembly. Using in vitro assays, we observed that Nef is highly capable of inhibiting Protease activity. The IC50 for nef-deficient viruses in drug susceptibility assays were 1.7- to 3.5-fold higher than the wild-type counterpart varying with the type of the Protease inhibitor used. Indicating that, in the absence of Nef, Protease is less sensitive to Protease inhibitors. We compared the protein content between wild-type and nef-deficient mature viral particles by gradient sedimentation and observed up to 2.7-fold reduction in the Integrase levels in nef-deficient mature particles. This difference in levels of Integrase correlated with the difference in infectivity levels of wild type and nef-deficient viral progeny. In addition, an overall decrease in the production of mature particles was detected in nef-deficient viruses. Collectively, our data support the hypothesis that the decreased infectivity typical of nef-deficient viruses is due to an abnormal function of the viral Protease, which is in turn associated with less mature particles being produced and the loss of Integrase content in these particles, and these results may characterize Nef as a regulator of viral Protease activity.  

5.1437           Complementary Induction of Immunogenic Cell Death by Oncolytic Parvovirus H-1PV and Gemcitabine in Pancreatic Cancer

Angelova, A.L., grekova, S.P., Heller, A., Kuhlmann, O., Soyka, E., Giese, T., Aprahamian, M., Bour, G., Rüffer, S., Cziepluch, C., Daeffler, L., Rommelaere, J., Werner, J., Raykov, Z. and Giese, N.A:

1. Virol., 88(10), 5263-5276 (2014)

  Novel therapies employing oncolytic viruses have emerged as promising anticancer modalities. The cure of particularly aggressive malignancies requires induction of immunogenic cell death (ICD), coupling oncolysis with immune responses via calreticulin, ATP, and high-mobility group box protein B1 (HMGB1) release from dying tumor cells. The present study shows that in human pancreatic cancer cells (pancreatic ductal adenocarcinoma [PDAC] cells; n = 4), oncolytic parvovirus H-1 (H-1PV) activated multiple interconnected death pathways but failed to induce calreticulin exposure or ATP release. In contrast, H-1PV elevated extracellular HMGB1 levels by 4.0 ± 0.5 times (58% ± 9% of total content; up to 100 ng/ml) in all infected cultures, whether nondying, necrotic, or apoptotic. An alternative secretory route allowed H-1PV to overcome the failure of gemcitabine to trigger HMGB1 release, without impeding cytotoxicity or other ICD activities of the standard PDAC medication. Such broad resistance of H-1PV-induced HMGB1 release to apoptotic blockage coincided with but was uncoupled from an autocrine interleukin-1β (IL-1β) loop. That and the pattern of viral determinants maintained in gemcitabine-treated cells suggested the activation of an inflammasome/caspase 1 (CASP1) platform alongside DNA detachment and/or nuclear exclusion of HMGB1 during early stages of the viral life cycle. We concluded that H-1PV infection of PDAC cells is signaled through secretion of the alarmin HMGB1 and, besides its own oncolytic effect, might convert drug-induced apoptosis into an ICD process. A transient arrest of cells in the cyclin A1-rich S phase would suffice to support compatibility of proliferation-dependent H-1PV with cytotoxic regimens. These properties warrant incorporation of the oncolytic virus H-1PV, which is not pathogenic in humans, into multimodal anticancer treatments.  

5.1438           Novel Permissive Cell Lines for Complete Propagation of Hepatitis C Virus

Shiokawa, M., Fukuhara, T., Ono, C., yamamoto, S., Okamoto, T., Watanabe, N., Wakita, T. and Matsura, Y.

1. Virol., 88(10), 5578-5594 (2014)

  Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. Although the HCV life cycle has been clarified by studying laboratory strains of HCV derived from the genotype 2a JFH-1 strain (cell culture-adapted HCV [HCVcc]), the mechanisms of particle formation have not been elucidated. Recently, we showed that exogenous expression of a liver-specific microRNA, miR-122, in nonhepatic cell lines facilitates efficient replication but not particle production of HCVcc, suggesting that liver-specific host factors are required for infectious particle formation. In this study, we screened human cancer cell lines for expression of the liver-specific α-fetoprotein by using a cDNA array database and identified liver-derived JHH-4 cells and stomach-derived FU97 cells, which express liver-specific host factors comparable to Huh7 cells. These cell lines permit not only replication of HCV RNA but also particle formation upon infection with HCVcc, suggesting that hepatic differentiation participates in the expression of liver-specific host factors required for HCV propagation. HCV inhibitors targeting host and viral factors exhibited different antiviral efficacies between Huh7 and FU97 cells. Furthermore, FU97 cells exhibited higher susceptibility for propagation of HCVcc derived from the JFH-2 strain than Huh7 cells. These results suggest that hepatic differentiation participates in the expression of liver-specific host factors required for complete propagation of HCV.  

5.1439           HIV-1 Interacts with Human Endogenous Retrovirus K (HML-2) Envelopes Derived from Human Primary Lymphocytes

Brinzevich, D., Young, G.R., Sebra, R., Ayllon, J., Maio, S.M., Deikus, G., Chen, B.K., Fernandez-Sesma, A., Simon, V. and Mulder, L.C.F.

1. Virol., 88(11), 6213-6223 (2014)

  Human endogenous retroviruses (HERVs) are viruses that have colonized the germ line and spread through vertical passage. Only the more recently acquired HERVs, such as the HERV-K (HML-2) group, maintain coding open reading frames. Expression of HERV-Ks has been linked to different pathological conditions, including HIV infection, but our knowledge on which specific HERV-Ks are expressed in primary lymphocytes currently is very limited. To identify the most expressed HERV-Ks in an unbiased manner, we analyzed their expression patterns in peripheral blood lymphocytes using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing. We observe that three HERV-Ks (KII, K102, and K18) constitute over 90% of the total HERV-K expression in primary human lymphocytes of five different donors. We also show experimentally that two of these HERV-K env sequences (K18 and K102) retain their ability to produce full-length and posttranslationally processed envelope proteins in cell culture. We show that HERV-K18 Env can be incorporated into HIV-1 but not simian immunodeficiency virus (SIV) particles. Moreover, HERV-K18 Env incorporation into HIV-1 virions is dependent on HIV-1 matrix. Taken together, we generated high-resolution HERV-K expression profiles specific for activated human lymphocytes. We found that one of the most abundantly expressed HERV-K envelopes not only makes a full-length protein but also specifically interacts with HIV-1. Our findings raise the possibility that these endogenous retroviral Env proteins could directly influence HIV-1 replication.  

5.1440           Myelin Basic Protein Cleaves Cell Adhesion Molecule L1 and Promotes Neuritogenesis and Cell Survival

Lutz, D., Loers, G., Kleene, R., Oezen, I.O., Kataria, H., Katagihallimath, N., Braren, I., Harauz, G. and Schacher, M.

1. Biol. Chem., 289(19), 13503-13518 (2024)

  The cell adhesion molecule L1 is a Lewis^x-carrying glycoprotein that plays important roles in the developing and adult nervous system. Here we show that myelin basic protein (MBP) binds to L1 in a Lewis^x-dependent manner. Furthermore, we demonstrate that MBP is released by murine cerebellar neurons as a sumoylated dynamin-containing protein upon L1 stimulation and that this MBP cleaves L1 as a serine protease in the L1 extracellular domain at Arg⁶⁸⁷ yielding a transmembrane fragment that promotes neurite outgrowth and neuronal survival in cell culture. L1-induced neurite outgrowth and neuronal survival are reduced in MBP-deficient cerebellar neurons and in wild-type cerebellar neurons in the presence of an MBP antibody or L1 peptide containing the MBP cleavage site. Genetic ablation of MBP in shiverer mice and mutagenesis of the proteolytically active site in MBP or of the MBP cleavage site within L1 as well as serine protease inhibitors and an L1 peptide containing the MBP cleavage site abolish generation of the L1 fragment. Our findings provide evidence for novel functions of MBP in the nervous system.    

5.1441           Interleukin-17 Retinotoxicity Is Prevented by Gene Transfer of a Soluble Interleukin-17 Receptor Acting as a Cytokine Blocker: Implications for Age-Related Macular Degeneration

Ardeljan, D., Wang, Y., Park, S., Shen, D., Chu, X.K., Yu, C-R., Abu-Asab, M., Tuo, J., Eberhart, C.G., Olsen, T.W., Mullins, R.F., White, G., Eadsworth, S., Scaria, A. and Chan, C-C. PloS One, 9(4), e95900 (2014)   Age-related macular degeneration (AMD) is a common yet complex retinal degeneration that causes irreversible central blindness in the elderly. Pathology is widely believed to follow loss of retinal pigment epithelium (RPE) and photoreceptor degeneration. Here we report aberrant expression of interleukin-17A (IL17A) and the receptor IL17RC in the macula of AMD patients. In vitro, IL17A induces RPE cell death characterized by the accumulation of cytoplasmic lipids and autophagosomes with subsequent activation of pro-apoptotic Caspase-3 and Caspase-9. This pathology is reduced by siRNA knockdown of IL17RC. IL17-dependent retinal degeneration in a mouse model of focal retinal degeneration can be prevented by gene therapy with adeno-associated virus vector encoding soluble IL17 receptor. This intervention rescues RPE and photoreceptors in a MAPK-dependent process. The IL17 pathway plays a key role in RPE and photoreceptor degeneration and could hold therapeutic potential in AMD.    

5.1442           Immunogenicity of a Trivalent Human Papillomavirus L1 DNA-Encapsidated, Non-Replicable Baculovirus Nanovaccine

Cho, H., Lee, H-J., Heo, Y-K., Cho, Y., Gwon, Y-D., Kim, M-G., Park, K.H., Oh, Y-K. and Kim, Y.B. PloS One, 9(4), e95961 (2014)   Previously, we developed a non-replicating recombinant baculovirus coated with human endogenous retrovirus envelope protein (AcHERV) for enhanced cellular delivery of human papillomavirus (HPV) 16L1 DNA. Here, we report the immunogenicity of an AcHERV-based multivalent HPV nanovaccine in which the L1 segments of HPV 16, 18, and 58 genes were inserted into a single baculovirus genome of AcHERV. To test whether gene expression levels were affected by the order of HPV L1 gene insertion, we compared the efficacy of bivalent AcHERV vaccines with the HPV 16L1 gene inserted ahead of the 18L1 gene (AcHERV-HP16/18L1) with that of AcHERV with the HPV 18L1 gene inserted ahead of the 16L1 gene (AcHERV-HP18/16L1). Regardless of the order, the bivalent AcHERV DNA vaccines retained the immunogenicity of monovalent AcHERV-HP16L1 and AcHERV-HP18L1 DNA vaccines. Moreover, the immunogenicity of bivalent AcHERV-HP16/18L1 was not significantly different from that of AcHERV-HP18/16L1. In challenge tests, both bivalent vaccines provided complete protection against HPV 16 and 18 pseudotype viruses. Extending these results, we found that a trivalent AcHERV nanovaccine encoding HPV 16L1, 18L1, and 58L1 genes (AcHERV-HP16/18/58L1) provided high levels of humoral and cellular immunogenicity against all three subtypes. Moreover, mice immunized with the trivalent AcHERV-based nanovaccine were protected from challenge with HPV 16, 18, and 58 pseudotype viruses. These results suggest that trivalent AcHERV-HPV16/18/58L1 could serve as a potential prophylactic baculoviral nanovaccine against concurrent infection with HPV 16, 18, and 58.  

5.1443           Harnessing High Density Lipoproteins to Block Transforming Growth Factor Beta and to Inhibit the Growth of Liver Tumor Metastases

Medina-Echeverz, J., Fioravanti, J., Diaz-Valdes, N., Frank, K., Aranda, F., Gomar, C., Ardaiz, N., Dotor, J., Umansky, V., Prieto, J. and Berraondo, P. PloS One, 9(5), e96799 (2014)   Transforming growth factor β (TGF-β) is a powerful promoter of cancer progression and a key target for antitumor therapy. As cancer cells exhibit active cholesterol metabolism, high density lipoproteins (HDLs) appear as an attractive delivery system for anticancer TGFβ-inhibitory molecules. We constructed a plasmid encoding a potent TGF-β-blocking peptide (P144) linked to apolipoprotein A-I (ApoA-I) through a flexible linker (pApoLinkerP144). The ApoLinkerP144 sequence was then incorporated into a hepatotropic adeno-associated vector (AAVApoLinkerP144). The aim was to induce hepatocytes to produce HDLs containing a modified ApoA-I capable of blocking TGF-β. We observed that transduction of the murine liver with pApoLinkerP144 led to the appearance of a fraction of circulating HDL containing the fusion protein. These HDLs were able to attenuate TGF-β signaling in the liver and to enhance IL-12 -mediated IFN-γ production. Treatment of liver metastasis of MC38 colorectal cancer with AAVApoLinkerP144 resulted in a significant reduction of tumor growth and enhanced expression of IFN-γ and GM-CSF in cancerous tissue. ApoLinkerP144 also delayed MC38 liver metastasis in Rag2^−/−IL2rγ^−/− immunodeficient mice. This effect was associated with downregulation of TGF-β target genes essential for metastatic niche conditioning. Finally, in a subset of ret transgenic mice, a model of aggressive spontaneous metastatic melanoma, AAVApoLinkerP144 delayed tumor growth in association with increased CD8⁺ T cell numbers in regional lymph nodes. In conclusion, modification of HDLs to transport TGF-β-blocking molecules is a novel and promising approach to inhibit the growth of liver metastases by immunological and non-immunological mechanisms.  

5.1444           Impact of Inhibitors and L2 Antibodies upon the Infectivity of Diverse Alpha and Beta Human Papillomavirus Types

Kwak, K., Jiang, R., Wang, J.W., jagu, S., Kirnbauer, R. and Roden, R.B.S. PloS One, 9(5), e97232 (2014)   The licensed human papillomavirus (HPV) vaccines elicit type-restricted immunity but do not target cutaneous HPV types of the beta genus that are associated with non-melanoma skin cancer in immune-compromised patients, and it is unclear if these diverse types share a common mechanism of infection. Residues 11-88 of minor capsid protein L2 contain cross-protective epitopes, and vaccination with concatamers of this region derived from as many as eight alpha HPV (L2 α11-88x8) is being developed as an alternative prophylactic vaccine with potentially broader efficacy. There is also interest in developing broadly protective topical microbicides, such as carrageenan or heparin that block HPV receptor interactions, or small molecule inhibitors of infection. Here we have examined several inhibitors of HPV infection and antisera to L2 α11-88x8 for their breadth of activity against infection by 34 HPV types from within both the alpha and beta families using pseudovirions (PsV) carrying a luciferase reporter as surrogates for native virus. We observed that both heparin and carrageenan prevented infection by mucosatropic HPV types, but surprisingly PsV of several epidermotropic alpha4 and beta HPV types exhibited increased infectivity especially at low inhibitor concentrations. Furin and γ-secretase inhibitors and L2 α11-88x8 antiserum blocked infection by all HPV PsV types tested. These findings suggest that the distinct tropism of mucosal and cutaneous HPV may reflect distinct cell surface receptor interactions, but a common uptake mechanism dependent upon furin and γ-secretase proteolytic activities. Carrageenan, which is being tested as a vaginal microbicide, broadly inhibited infection by the high-risk mucosatropic HPV PsV, but not most skin tropic alpha and beta HPV. Vaccination with an L2 multimer derived exclusively from alpha papillomavirus sequences induced antibodies that broadly neutralized PsV of all 34 HPVs from within both the alpha and beta families, suggesting each displays conserved L2 neutralizing epitopes.  

5.1445           Intraneural convection enhanced delivery of AAVrh20 for targeting primary sensory neurons

Pleticha, J., Jeng-Singh, C., Rezek, R., Zaibak, M. and Beutler, A.S. Mol. Cell. Neurosci., 60, 72-80 (2014)   Gene therapy using adeno-associated virus (AAV) is an attractive strategy to treat disorders of the peripheral nervous system (PNS), such as chronic pain or peripheral neuropathies. Although intrathecal (IT) administration of AAV has been the standard in the field for targeting the PNS, it lacks anatomical specificity and results in wide rostro-caudal distribution of the vector. An alternative approach is to deliver AAV directly to the peripheral nerve axon. The present study employed convection-enhanced delivery (CED) of a novel AAV serotype, AAVrh20, expressing enhanced green fluorescent protein (EGFP) into rat sciatic nerve investigating its efficacy, anatomical selectivity, and safety, compared to the IT route. Intraneural CED resulted in transduction confined to the ipsilateral L4 and L5 DRG while IT administration led to promiscuous DRG transduction encompassing the entire lumbar region bilaterally. The transduction rate for intraneural AAV administration was similar to IT delivery (24% for L4 and 31.5% for L5 DRG versus 50% for L4 and 19.5% for L5 DRG). The use of hyperosmotic diluent did not further improve the transduction efficiency. AAVrh20 was superior to reference serotypes previously described to be most active for each route. Intraneural CED of AAV was associated with transient allodynia that resolved spontaneously. These findings establish intraneural CED as an alternative to IT administration for AAV mediated gene transfer to the PNS and, based on a reference rodent model, suggest AAVrh20 as a superior serotype for targeting the PNS.  

5.1446           Live Attenuated Tetravalent Dengue Virus Host Range Vaccine Is Immunogenic in African Green Monkeys following a Single Vaccination

Briggs, C.M., Smith, K.M., Piper, A., Huitt, E., Spears, C.J., Quiles, M., Ribeiro, M., Thomas, M.E., Brown, D.T. and hernandez, R.

1. Virol., 88(12), 6729-6742 (2014)

  The causative agent of dengue fever, dengue virus (DENV), is transmitted by mosquitoes, and as distribution of these insects has expanded, so has dengue-related disease. DENV is a member of the Flaviviridae family and has 4 distinct serotypes (DENV-1, -2, -3, and -4). No lasting cross protection is afforded to heterologous serotypes following infection by any one of the individual serotypes. The presence of nonneutralizing antibodies to one serotype can facilitate the occurrence of more-severe dengue hemorrhagic fever through immune enhancement upon infection with a second serotype. For this reason, the development of a safe, tetravalent vaccine to produce a balanced immune response to all four serotypes is critical. We have developed a novel approach to produce safe and effective live-attenuated vaccines for DENV and other insect-borne viruses. Host range (HR) mutants of each DENV serotype were created by truncating transmembrane domain 1 of the E protein and selecting for strains of DENV that replicated well in insect cells but not mammalian cells. These vaccine strains were tested for immunogenicity in African green monkeys (AGMs). No vaccine-related adverse events occurred. The vaccine strains were confirmed to be attenuated in vivo by infectious center assay (ICA). Analysis by 50% plaque reduction neutralization test (PRNT₅₀) established that by day 62 postvaccination, 100% of animals seroconverted to DENV-1, -2, -3, and -4. Additionally, the DENV HR tetravalent vaccine (HR-Tet) showed a tetravalent anamnestic immune response in 100% (16/16) of AGMs after challenge with wild-type (WT) DENV strains.  

5.1447           Production and characterization of high-titer serum-free cell culture grown hepatitis C virus particles of genotype 1–6

Mathiesen, C.K., Jensen, T.B., Prentoe, J., Krarup, H., Nicosia, A., Law, M., Bukh, J. and Gottwein, J.M. Virology, 458-459, 190-208 (2014)   Recently, cell culture systems producing hepatitis C virus particles (HCVcc) were developed. Establishment of serum-free culture conditions is expected to facilitate development of a whole-virus inactivated HCV vaccine. We describe generation of genotype 1–6 serum-free HCVcc (sf-HCVcc) from Huh7.5 hepatoma cells cultured in adenovirus expression medium. Compared to HCVcc, sf-HCVcc showed 0.6–2.1 log₁₀ higher infectivity titers (4.7–6.2 log₁₀ Focus Forming Units/mL), possibly due to increased release and specific infectivity of sf-HCVcc. In contrast to HCVcc, sf-HCVcc had a homogeneous single-peak density profile. Entry of sf-HCVcc depended on HCV co-receptors CD81, LDLr, and SR-BI, and clathrin-mediated endocytosis. HCVcc and sf-HCVcc were neutralized similarly by chronic-phase patient sera and by human monoclonal antibodies targeting conformational epitopes. Thus, we developed serum-free culture systems producing high-titer single-density sf-HCVcc, showing similar biological properties as HCVcc. This methodology has the potential to advance HCV vaccine development and to facilitate biophysical studies of HCV.  

5.1448           Recombinant Adeno-Associated Virus: Efficient Transduction of the Rat VMH and Clearance from Blood

Van Gestel, M.A., Boender, A.J., de Vrind, V.A.J., Garner, K.M., Luijendijk, M.C.M. and Adan, R.A.H. PloS One, 9(5), e97639 (2014)   To promote the efficient and safe application of adeno-associated virus (AAV) vectors as a gene transfer tool in the central nervous system (CNS), transduction efficiency and clearance were studied for serotypes commonly used to transfect distinct areas of the brain. As AAV2 was shown to transduce only small volumes in several brain regions, this study compares the transduction efficiency of three AAV pseudotyped vectors, namely AAV2/1, AAV2/5 and AAV2/8, in the ventromedial nucleus of the hypothalamus (VMH). No difference was found between AAV2/1 and AAV2/5 in transduction efficiency. Both AAV2/1 and AAV2/5 achieved a higher transduction rate than AAV2/8. One hour after virus administration to the brain, no viral particles could be traced in blood, indicating that no or negligible numbers of virions crossed the blood-brain barrier. In order to investigate survival of AAV in blood, clearance was determined following systemic AAV administration. The half-life of AAV2/1, AAV2/2, AAV2/5 and AAV2/8 was calculated by determining virus clearance rates from blood after systemic injection. The half-life of AAV2/2 was 4.2 minutes, which was significantly lower than the half-lives of AAV2/1, AAV2/5 and AAV2/8. With a half-life of more than 11 hours, AAV2/8 particles remained detectable in blood significantly longer than AAV2/5. We conclude that application of AAV in the CNS is relatively safe as no AAV particles are detectable in blood after injection into the brain. With a half-life of 1.67 hours of AAV2/5, a systemic injection with 1×10⁹ genomic copies of AAV would be fully cleared from blood after 2 days.  

5.1449           Single Strain Isolation Method for Cell Culture-Adapted Hepatitis C Virus by End-Point Dilution and Infection

Sugiyama, N., Murayama, A., Suzuki, R., Watanabe, N., Shiina, M., Liang, T.J., Wakita, t. and Kato, t. PloS One, 9(5), e98168 (2014)   The hepatitis C virus (HCV) culture system has enabled us to clarify the HCV life cycle and essential host factors for propagation. However, the virus production level of wild-type JFH-1 (JFH-1/wt) is limited, and this leads to difficulties in performing experiments that require higher viral concentrations. As the cell culture-adapted JFH-1 has been reported to have robust virus production, some mutations in the viral genome may play a role in the efficiency of virus production. In this study, we obtained cell culture-adapted virus by passage of full-length JFH-1 RNA-transfected Huh-7.5.1 cells. The obtained virus produced 3 log-fold more progeny viruses as compared with JFH-1/wt. Several mutations were identified as being responsible for robust virus production, but, on reverse-genetics analysis, the production levels of JFH-1 with these mutations did not reach the level of cell culture-adapted virus. By using the single strain isolation method by end-point dilution and infection, we isolated two strains with additional mutations, and found that these strains have the ability to produce more progeny viruses. On reverse-genetics analysis, the strains with these additional mutations were able to produce robust progeny viruses at comparable levels as cell culture-adapted JFH-1 virus. The strategy used in this study will be useful for identifying strains with unique characteristics, such as robust virus production, from a diverse population, and for determining the responsible mutations for these characteristics.  

5.1450           AAV-Dominant Negative Tumor Necrosis Factor (DN-TNF) Gene Transfer to the Striatum Does Not Rescue Medium Spiny Neurons in the YAC128 Mouse Model of Huntington's Disease

Alto, L.T., Chen, X., Ruhn, K.A., Trevino, I. and Tansey, M.G. PloS One, 9(5), e96544 (2014)   CNS inflammation is a hallmark of neurodegenerative disease, and recent studies suggest that the inflammatory response may contribute to neuronal demise. In particular, increased tumor necrosis factor (TNF) signaling is implicated in the pathology of both Parkinson's disease (PD) and Alzheimer's disease (AD). We have previously shown that localized gene delivery of dominant negative TNF to the degenerating brain region can limit pathology in animal models of PD and AD. TNF is upregulated in Huntington's disease (HD), like in PD and AD, but it is unknown whether TNF signaling contributes to neuronal degeneration in HD. We used in vivo gene delivery to test whether selective reduction of soluble TNF signaling could attenuate medium spiny neuron (MSN) degeneration in the YAC128 transgenic (TG) mouse model of Huntington's disease (HD). AAV vectors encoding cDNA for dominant-negative tumor necrosis factor (DN-TNF) or GFP (control) were injected into the striatum of young adult wild type WT and YAC128 TG mice and achieved 30–50% target coverage. Expression of dominant negative TNF protein was confirmed immunohistologically and biochemically and was maintained as mice aged to one year, but declined significantly over time. However, the extent of striatal DN-TNF gene transfer achieved in our studies was not sufficient to achieve robust effects on neuroinflammation, rescue degenerating MSNs or improve motor function in treated mice. Our findings suggest that alternative drug delivery strategies should be explored to determine whether greater target coverage by DN-TNF protein might afford some level of neuroprotection against HD-like pathology and/or that soluble TNF signaling may not be the primary driver of striatal neuroinflammation and MSN loss in YAC128 TG mice.  

5.1451           SARS-CoV envelope protein palmitoylation or nucleocapid association is not required for promoting virus-like particle production

Tseng, Y-T., Wang, S-M., Huang, K-J. and Wang, C-T.

1. Biomedical Sci., 21:34 (2014)

  Background Coronavirus membrane (M) proteins are capable of interacting with nucleocapsid (N) and envelope (E) proteins. Severe acute respiratory syndrome coronavirus (SARS-CoV) M co-expression with either N or E is sufficient for producing virus-like particles (VLPs), although at a lower level compared to M, N and E co-expression. Whether E can release from cells or E/N interaction exists so as to contribute to enhanced VLP production is unknown. It also remains to be determined whether E palmitoylation or disulfide bond formation plays a role in SARS-CoV virus assembly. Results SARS-CoV N is released from cells through an association with E protein-containing vesicles. Further analysis suggests that domains involved in E/N interaction are largely located in both carboxyl-terminal regions. Changing all three E cysteine residues to alanines did not exert negative effects on E release, E association with N, or E enhancement of VLP production, suggesting that E palmitoylation modification or disulfide bond formation is not required for SARS-CoV virus assembly. We found that removal of the last E carboxyl-terminal residue markedly affected E release, N association, and VLP incorporation, but did not significantly compromise the contribution of E to efficient VLP production. Conclusions The independence of the SARS-CoV E enhancement effect on VLP production from its viral packaging capacity suggests a distinct SARS-CoV E role in virus assembly.  

5.1452           ROCK2 is a major regulator of axonal degeneration, neuronal death and axonal regeneration in the CNS

Koch, J.C., Tönges, L., Barski, E., Michel, U., Bähr, M. and Lingor, P. Cell Death and Disease, 5, e1225 (2014)   The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the central nervous system. Several studies using pharmacological Rho-associated protein kinase (ROCK) inhibitors have shown positive effects on neurite regeneration and suggest additional pro-survival effects in neurons. However, as none of these drugs is completely target specific, it remains unclear how these effects are mediated and whether ROCK is really the most relevant target of the pathway. To answer these questions, we generated adeno-associated viral vectors to specifically downregulate ROCK2 and LIM domain kinase (LIMK)-1 in rat retinal ganglion cells (RGCs) in vitro and in vivo. We show here that specific knockdown of ROCK2 and LIMK1 equally enhanced neurite outgrowth of RGCs on inhibitory substrates and both induced substantial neuronal regeneration over distances of more than 5 mm after rat optic nerve crush (ONC) in vivo. However, only knockdown of ROCK2 but not LIMK1 increased survival of RGCs after optic nerve axotomy. Moreover, knockdown of ROCK2 attenuated axonal degeneration of the proximal axon after ONC assessed by in vivo live imaging. Mechanistically, we demonstrate here that knockdown of ROCK2 resulted in decreased intraneuronal activity of calpain and caspase 3, whereas levels of pAkt and collapsin response mediator protein 2 and autophagic flux were increased. Taken together, our data characterize ROCK2 as a specific therapeutic target in neurodegenerative diseases and demonstrate new downstream effects of ROCK2 including axonal degeneration, apoptosis and autophagy.  

5.1453           Specific tools for targeting and expression in Müller glial cells

Pellisier, L.P., Hoek, R.M., Vos, R.M., Aartsen, W.M., Klimczak, R.R., Hoyng, S.A., Flannery, J.G. and Wijnholds, J. Molecular Therapy – Methods & Clinical Development, 1:14009 (2014)   Despite their physiological roles, Müller glial cells are involved directly or indirectly in retinal disease pathogenesis and are an interesting target for therapeutic approaches for retinal diseases and regeneration such as CRB1 inherited retinal dystrophies. In this study, we characterized the efficiency of adeno-associated virus (AAV) capsid variants and different promoters to drive protein expression in Müller glial cells. ShH10Y and AAV9 were the most powerful capsids to infect mouse Müller glial cells. Retinaldehyde-binding protein 1 (RLBP1) promoter was the most powerful promoter to transduce Müller glial cells. ShH10Y capsids and RLBP1 promoter targeted human Müller glial cells in vitro. We also developed and tested smaller promoters to express the large CRB1 gene via AAV vectors. Minimal cytomegalovirus (CMV) promoter allowed expression of full-length CRB1 protein in Müller glial cells. In summary, ShH10Y and AAV9 capsids, and RLBP1 or minimal CMV promoters are of interest as specific tools to target and express in mouse or human Müller glial cells.  

5.1454           Kidney-specific expression of GFP by in-utero delivery of pseudotyped adeno-associated virus 9

Piconi, J.L., Muff-Luett, A., Wu, D., Bunchman, E., Schaefer, F. and Brophy, P.D. Molecular Therapy – Methods & Clinical Developmen, 1:14014 (2014)   Gene therapy targeting of kidneys has been largely unsuccessful. Recently, a recombinant adeno-associated virus (rAAV) vector was used to target adult mouse kidneys. Our hypothesis is that a pseudotyped rAAV 2/9 vector can produce fetal kidney-specific expression of the green fluorescent protein (GFP) gene following maternal tail vein injection of pregnant mice. Pregnant mice were treated with rAAV2/9 vectors with either the ubiquitous cytomegalovirus promoter or the minimal NPHS1 promoter to drive kidney-specific expression of GFP. Kidneys from dams and pups were analyzed for vector DNA, gene expression, and protein. Vector DNA was identified in kidney tissue out to 12 weeks at low but stable levels, with levels higher in dams than that in pups. Robust GFP expression was identified in the kidneys of both dams and pups treated with the cytomegalovirus (CMV)-enhanced green fluorescent protein (eGFP) vector. When treated with the NPHS1-eGFP vector, dams and pups showed expression of GFP only in kidneys, localized to the glomeruli. An 80-fold increase in GFP mRNA expression in dams and a nearly 12-fold increase in pups was found out to 12 weeks of life. Selective targeting of the fetal kidney with a gene therapy vector was achieved by utilizing the pseudotyped rAAV 2/9 vector containing the NPHS1 promoter.  

5.1455           Calcineurin Downregulation in the Amygdala Is Sufficient to Induce Anxiety-like and Depression-like Behaviors in C57BL/6J Male Mice

Mineur, Y.S., Taylor, S.R. and Picciotto, M.R. Biol. Psychiatry, 75, 991-998 (2014)   Background The calcium-dependent phosphatase calcineurin is highly expressed in the amygdala, a brain area important for behaviors related to mood disorders and anxiety. Organ transplant patients are administered the calcineurin inhibitor cyclosporine A (CsA) chronically and demonstrate an increased incidence of anxiety and mood disorders. It is therefore important to determine whether chronic blockade of calcineurin may contribute to symptoms of anxiety and depression in these patients. Methods Pharmacological (CSA) and viral-mediated gene transfer (adeno-associated viral expression of short hairpin RNA [shRNA]) approaches were used to inhibit calcineurin activity systemically or selectively in the amygdala of the mouse brain to determine the role of calcineurin in behaviors related to anxiety and depression. Results Systemic inhibition of calcineurin activity with CsA or local downregulation of calcineurin levels in the amygdala using adeno-associated viral-delivered shRNAs targeting calcineurin B increased measures of anxiety-like behavior in the elevated plus maze, the light/dark box, and the open field test. A decrease in locomotor activity was also observed in mice treated systemically with CsA. In the forced swim model of depression-like behavior, both systemic CsA treatment and shRNA-mediated calcineurin blockade in the amygdala significantly increased immobility. Conclusions Taken together, these data demonstrate that decreasing calcineurin activity in the amygdala increases anxiety-like behaviors and to some extent depression-like behaviors. These studies suggest that chronic administration of CsA to organ transplant patients could have significant effects on anxiety and mood and this should be recognized as a potential clinical consequence of treatment to prevent transplant rejection.  

5.1456           Quantitative, noninvasive, in vivo longitudinal monitoring of gene expression in the brain by co-AAV transduction with a PET reporter gene

Yoon, S.Y., Gay-Antaki, C., Ponde, D.E., Poptani, H., Vite, C.H. and Wolfe, J.H. Molecular Therapy – Methods & Clinical Development, 1:14016 (2014)   In vivo imaging of vector transgene expression would be particularly valuable for repetitive monitoring of therapy in the brain, where invasive tissue sampling is contraindicated. We evaluated adeno-associated virus vector expression of a dopamine-2 receptor (D2R) mutant (D2R80A) by positron emission tomography in the brains of mice and cats. D2R80A is inactivated for intracellular signaling and binds subphysiologic amounts of the radioactive [18F]-fallypride analog of dopamine. The [18F]-fallypride signal bound to D2R80A in the injection site was normalized to the signal from endogenous D2R in the striatum and showed stable levels of expression within individual animals. A separate adeno-associated virus type 1 vector with identical gene expression control elements, expressing green fluorescent protein or a therapeutic gene, was coinjected with the D2R80A vector at equal doses into specific sites. Both transgenes had similar levels of gene expression by immunohistochemistry, in situ hybridization, and quantitative PCR assays, demonstrating that D2R80A is a faithful surrogate measure for expression of a gene of interest. This dual vector approach allows the D2R80A gene to be used with any therapeutic gene and to be injected into a single site for monitoring while the therapeutic gene can be distributed more widely as needed in each disease.  

5.1457           HIV-1 Nef Is Transferred from Expressing T Cells to Hepatocytic Cells through Conduits and Enhances HCV Replication

Park, I-W., Fan, Y., Luo, X., Ryou, M., Liu, J., Green, L. and He, J.J. PloS One, 9(6), e99545 (2014)   HIV-1 infection enhances HCV replication and as a consequence accelerates HCV-mediated hepatocellular carcinoma (HCC). However, the precise molecular mechanism by which this takes place is currently unknown. Our data showed that infectious HIV-1 failed to replicate in human hepatocytic cell lines. No discernible virus replication was observed, even when the cell lines transfected with HIV-1 proviral DNA were co-cultured with Jurkat T cells, indicating that the problem of liver deterioration in the co-infected patient is not due to the replication of HIV-1 in the hepatocytes of the HCV infected host. Instead, HIV-1 Nef protein was transferred from nef-expressing T cells to hepatocytic cells through conduits, wherein up to 16% (average 10%) of the cells harbored the transferred Nef, when the hepatocytic cells were co-cultured with nef-expressing Jurkat cells for 24 h. Further, Nef altered the size and numbers of lipid droplets (LD), and consistently up-regulated HCV replication by 1.5~2.5 fold in the target subgenomic replicon cells, which is remarkable in relation to the initially indolent viral replication. Nef also dramatically augmented reactive oxygen species (ROS) production and enhanced ethanol-mediated up-regulation of HCV replication so as to accelerate HCC. Taken together, these data indicate that HIV-1 Nef is a critical element in accelerating progression of liver pathogenesis via enhancing HCV replication and coordinating modulation of key intra- and extra-cellular molecules for liver decay.  

5.1458           Roles for Human Papillomavirus Type 16 L1 Cysteine Residues 161, 229, and 379 in Genome Encapsidation and Capsid Stability

Ryndock, E.J., Conway, M.J., alam, S., Gul, S., Murad, S., Christensen, N.D. and Myers, C. PloS One, 9(6), e99488 (2014)   Human papillomavirus (HPV) capsids are formed through a network of inter- and intra-pentameric hydrophobic interactions and disulfide bonds. 72 pentamers of the major capsid protein, L1, and an unknown amount of the minor capsid protein, L2, form the structure of the capsid. There are 12 conserved L1 cysteine residues in HPV16. While C175, C185, and C428 have been implicated in the formation of a critical inter-pentameric disulfide bond, no structural or functional roles have been firmly attributed to any of the other conserved cysteine residues. Here, we show that substitution of cysteine residues C161, C229, and C379 for serine hinders the accumulation of endonuclease-resistant genomes as virions mature within stratifying and differentiating human epithelial tissue. C229S mutant virions form, but are non-infectious. These studies add detail to the differentiation-dependent assembly and maturation that occur during the HPV16 life cycle in human tissue.  

5.1459           A dedicated circuit links direction-selective retinal ganglion cells to the primary visual cortex

Cruz-Martin, A., El-Danaf, R.N., Osakada, F., Sriram, B., Dhande, O.S., Nguyen, P.L., Callaway, E.M., Ghosh, A. and Huberman, A.D. Nature, 507, 358-361 (2014)   How specific features in the environment are represented within the brain is an important unanswered question in neuroscience. A subset of retinal neurons, called direction-selective ganglion cells (DSGCs), are specialized for detecting motion along specific axes of the visual field¹. Despite extensive study of the retinal circuitry that endows DSGCs with their unique tuning properties^{2, 3}, their downstream circuitry in the brain and thus their contribution to visual processing has remained unclear. In mice, several different types of DSGCs connect to the dorsal lateral geniculate nucleus (dLGN)^{4, 5, 6}, the visual thalamic structure that harbours cortical relay neurons. Whether direction-selective information computed at the level of the retina is routed to cortical circuits and integrated with other visual channels, however, is unknown. Here we show that there is a di-synaptic circuit linking DSGCs with the superficial layers of the primary visual cortex (V1) by using viral trans-synaptic circuit mapping^{7, 8} and functional imaging of visually driven calcium signals in thalamocortical axons. This circuit pools information from several types of DSGCs, converges in a specialized subdivision of the dLGN, and delivers direction-tuned and orientation-tuned signals to superficial V1. Notably, this circuit is anatomically segregated from the retino-geniculo-cortical pathway carrying non-direction-tuned visual information to deeper layers of V1, such as layer 4. Thus, the mouse harbours several functionally specialized, parallel retino-geniculo-cortical pathways, one of which originates with retinal DSGCs and delivers direction- and orientation-tuned information specifically to the superficial layers of the primary visual cortex. These data provide evidence that direction and orientation selectivity of some V1 neurons may be influenced by the activation of DSGCs.      

5.1460           Inhibition of miR-25 improves cardiac contractility in the failing heart

Wahlquist, C., Jeong, D., Rojas-Munoz, A., Kho, C., Lee, A., Mitsuyama, S., van Mil, A., park, W.J., Sluijer, J.P.G., Doevendans, P.A.F., Hajjar, R.J. and Mercola, M. Nature, 508, 531-535 (2014)   Heart failure is characterized by a debilitating decline in cardiac function¹, and recent clinical trial results indicate that improving the contractility of heart muscle cells by boosting intracellular calcium handling might be an effective therapy^{2, 3}. MicroRNAs (miRNAs) are dysregulated in heart failure^{4, 5} but whether they control contractility or constitute therapeutic targets remains speculative. Using high-throughput functional screening of the human microRNAome, here we identify miRNAs that suppress intracellular calcium handling in heart muscle by interacting with messenger RNA encoding the sarcoplasmic reticulum calcium uptake pump SERCA2a (also known as ATP2A2). Of 875 miRNAs tested, miR-25 potently delayed calcium uptake kinetics in cardiomyocytes in vitro and was upregulated in heart failure, both in mice and humans. Whereas adeno-associated virus 9 (AAV9)-mediated overexpression of miR-25 in vivo resulted in a significant loss of contractile function, injection of an antisense oligonucleotide (antagomiR) against miR-25 markedly halted established heart failure in a mouse model, improving cardiac function and survival relative to a control antagomiR oligonucleotide. These data reveal that increased expression of endogenous miR-25 contributes to declining cardiac function during heart failure and suggest that it might be targeted therapeutically to restore function.  

5.1461           Omega-3 fatty acids and/or fluvastatin in hepatitis C prior non-responders to combination antiviral therapy – a pilot randomised clinical trial

Sheridan, D.A., Bridge, S.H., Crossey, M.M.E., Felmlee, D.J., Fenwick, F.I., Thomas, H.C., Neely, R.D.G., Taylor-Robinson, S.D. and Bassendine, M.F. Liver Int., 34(5), 737-747 (2014)   Background & Aims Hepatitis C virus (HCV) utilises cholesterol and lipoprotein metabolism for replication and infectivity. Statins and omega-3 (n–3) polyunsaturated fatty acids (PUFA) have been shown to have antiviral properties in vitro. This open label pilot study evaluated the efficacy of fluvastatin (Lescol^® 40–80 mg) and n-3 PUFA (Omacor^®1 g and 2–4 g) on HCV-RNA and lipoviral particles (LVP) in difficult to treat prior non-responders. Methods Patients (n = 60) were randomly allocated in a factorial design to: no active drug; low-dose n-3 PUFA; high-dose n-3 PUFA; fluvastatin; low-dose n-3 PUFA + fluvastatin; or high-dose n-3 PUFA + fluvastatin. 50/60 completed study drugs for 12 weeks and followed up to week 24. Comparison was made between fluvastatin (n = 24) vs no fluvastatin (n = 26) and n-3 PUFA high-dose (n = 17) vs low-dose (n = 17) vs none (n = 16). The primary outcomes were change in total HCV-RNA, LVP and ALT at week 12 compared with baseline. Secondary outcome was change in interferon-gamma-inducible protein-10 (IP10) as a measure of interferon activation. Results 35% had compensated cirrhosis and 45% were prior null responders. There was no significant change in total HCV RNA, LVP, non-LVP or LVP ratio in patients receiving fluvastatin or n-3 PUFAs. ALT was not significantly different in those treated with fluvastatin or n-3 PUFAs. 12 weeks of low-dose n-3 PUFA decreased median IP10 concentration by −39 pg/ml (−111, 7.0 pg/ml Q1–Q3). Conclusions Fluvastatin and n-3 PUFAs have no effect on plasma HCV-RNA or LVP. The effect of low-dose n-3 PUFA on IP10 warrants further prospective evaluation as a supplemental therapy to enhance interferon sensitivity.  

5.1462           Tropism-modified AAV Vectors Overcome Barriers to Successful Cutaneous Therapy

Sallach, J., Di Pasquale, G., Larcher, F., Niehoff, N., Rübsam, M., Huber, A., Chiorini, J., Almarza, D., eming, S.A., Ulus, H., Nishimura, S., Hacker, U.T., Hallek, M., Niessen, C.M. and Büning, H. Molecular Therapy, 22(5), 929-939 (2014)   Autologous human keratinocytes (HK) forming sheet grafts are approved as skin substitutes. Genetic engineering of HK represents a promising technique to improve engraftment and survival of transplants. Although efficacious in keratinocyte-directed gene transfer, retro-/lentiviral vectors may raise safety concerns when applied in regenerative medicine. We therefore optimized adeno-associated viral (AAV) vectors of the serotype 2, characterized by an excellent safety profile, but lacking natural tropism for HK, through capsid engineering. Peptides, selected by AAV peptide display, engaged novel receptors that increased cell entry efficiency by up to 2,500-fold. The novel targeting vectors transduced HK with high efficiency and a remarkable specificity even in mixed cultures of HK and feeder cells. Moreover, differentiated keratinocytes in organotypic airlifted three-dimensional cultures were transduced following topical vector application. By exploiting comparative gene analysis we further succeeded in identifying αvβ8 integrin as a target receptor thus solving a major challenge of directed evolution approaches and describing a promising candidate receptor for cutaneous gene therapy.  

5.1463           Experimental Evolution of an Oncolytic Vesicular Stomatitis Virus with Increased Selectivity for p53-Deficient Cells

Garijo, R., Hernandez-Alonso, P., Rivas, C., Diallo, J-S. and Sanjuan, R.

PloS One, 9(7), e102365 (2014)

  Experimental evolution has been used for various biotechnological applications including protein and microbial cell engineering, but less commonly in the field of oncolytic virotherapy. Here, we sought to adapt a rapidly evolving RNA virus to cells deficient for the tumor suppressor gene p53, a hallmark of cancer cells. To achieve this goal, we established four independent evolution lines of the vesicular stomatitis virus (VSV) in p53-knockout mouse embryonic fibroblasts (p53−/− MEFs) under conditions favoring the action of natural selection. We found that some evolved viruses showed increased fitness and cytotoxicity in p53−/− cells but not in isogenic p53+/+ cells, indicating gene-specific adaptation. However, full-length sequencing revealed no obvious or previously described genetic changes associated with oncolytic activity. Half-maximal effective dose (EC₅₀) assays in mouse p53-positive colon cancer (CT26) and p53-deficient breast cancer (4T1) cells indicated that the evolved viruses were more effective against 4T1 cells than the parental virus or a reference oncolytic VSV (MΔ51), but showed no increased efficacy against CT26 cells. In vivo assays using 4T1 syngeneic tumor models showed that one of the evolved lines significantly delayed tumor growth compared to mice treated with the parental virus or untreated controls, and was able to induce transient tumor suppression. Our results show that RNA viruses can be specifically adapted typical cancer features such as p53 inactivation, and illustrate the usefulness of experimental evolution for oncolytic virotherapy.  

5.1464           Overexpression of the Astrocyte Glutamate Transporter GLT1 Exacerbates Phrenic Motor Neuron Degeneration, Diaphragm Compromise, and Forelimb Motor Dysfunction following Cervical Contusion Spinal Cord Injury

Li, K., Nicaise, C., Sannie, D., Hala, Ts.J., Javed, E., Parker, J.L., Putatunda, r., Regan, K.A., Suain, V., Brion, J-P., Rhoderick, F., Wright, M.C., Poulsen, D.J. and Lepore, A.C.

1. Neurosci., 34(22), 7622-7638 (2014)

  A major portion of spinal cord injury (SCI) cases affect midcervical levels, the location of the phrenic motor neuron (PhMN) pool that innervates the diaphragm. While initial trauma is uncontrollable, a valuable opportunity exists in the hours to days following SCI for preventing PhMN loss and consequent respiratory dysfunction that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxic cell death due to dysregulation of extracellular glutamate homeostasis. GLT1, mainly expressed by astrocytes, is responsible for the vast majority of functional uptake of extracellular glutamate in the CNS, particularly in spinal cord. We found that, in bacterial artificial chromosome-GLT1-enhanced green fluorescent protein reporter mice following unilateral midcervical (C4) contusion SCI, numbers of GLT1-expressing astrocytes in ventral horn and total intraspinal GLT1 protein expression were reduced soon after injury and the decrease persisted for ≥6 weeks. We used intraspinal delivery of adeno-associated virus type 8 (AAV8)-Gfa2 vector to rat cervical spinal cord ventral horn for targeting focal astrocyte GLT1 overexpression in areas of PhMN loss. Intraspinal delivery of AAV8-Gfa2-GLT1 resulted in transduction primarily of GFAP⁺ astrocytes that persisted for ≥6 weeks postinjury, as well as increased intraspinal GLT1 protein expression. Surprisingly, we found that astrocyte-targeted GLT1 overexpression increased lesion size, PhMN loss, phrenic nerve axonal degeneration, and diaphragm neuromuscular junction denervation, and resulted in reduced functional diaphragm innervation as assessed by phrenic nerve-diaphragm compound muscle action potential recordings. These results demonstrate that GLT1 overexpression via intraspinal AAV-Gfa2-GLT1 delivery exacerbates neuronal damage and increases respiratory impairment following cervical SCI.  

5.1465           Actin scaffolding by clathrin heavy chain is required for skeletal muscle sarcomere organization

Vassilopoulos, S., Gentil, C., Laine, J., Buclez, P-O., Franck, A., Ferry, A., Precigout, G., Roth, r., Heuser, J.E., Brodsky, F.M., Garcia, L., Bonne, G., Voit, T., Pietri-Rouxel, F. and Bitoun, M.

1. Cell Biol., 205(3), 377-393 (2014)

  The ubiquitous clathrin heavy chain (CHC), the main component of clathrin-coated vesicles, is well characterized for its role in intracellular membrane traffic and endocytosis from the plasma membrane (PM). Here, we demonstrate that in skeletal muscle CHC regulates the formation and maintenance of PM–sarcomere attachment sites also known as costameres. We show that clathrin forms large coated lattices associated with actin filaments and the muscle-specific isoform of α-actinin at the PM of differentiated myotubes. Depletion of CHC in myotubes induced a loss of actin and α-actinin sarcomeric organization, whereas CHC depletion in vivo induced a loss of contractile force due to the detachment of sarcomeres from the PM. Our results suggest that CHC contributes to the formation and maintenance of the contractile apparatus through interactions with costameric proteins and highlight an unconventional role for CHC in skeletal muscle that may be relevant to pathophysiology of neuromuscular disorders.  

5.1466           Targeted ablation of Crb2 in photoreceptor cells induces retinitis pigmentosa

Alves, C.H., Pellissier, L.P., Vos, R.M., garrido, M.G., SSothilingam, V., Seide, C., Beck, S.C., Klooster, J., Furukawa, T., Flannery, J.G., Verhaagen, J., Seeliger, M.W. and Wijnholds, J. Hum. Mol. Genet., 23(13), 3384-3401 (2014)   In humans, the Crumbs homolog-1 (CRB1) gene is mutated in autosomal recessive Leber congenital amaurosis and early-onset retinitis pigmentosa. In mammals, the Crumbs family is composed of: CRB1, CRB2, CRB3A and CRB3B. Recently, we showed that removal of mouse Crb2 from retinal progenitor cells, and consequent removal from Müller glial and photoreceptor cells, results in severe and progressive retinal degeneration with concomitant loss of retinal function that mimics retinitis pigmentosa due to mutations in the CRB1 gene. Here, we studied the effects of cell-type-specific loss of CRB2 from the developing mouse retina using targeted conditional deletion of Crb2 in photoreceptors or Müller cells. We analyzed the consequences of targeted loss of CRB2 in the adult mouse retina using adeno-associated viral vectors encoding Cre recombinase and short hairpin RNA against Crb2. In vivo retinal imaging by means of optical coherence tomography on retinas lacking CRB2 in photoreceptors showed progressive thinning of the photoreceptor layer and cellular mislocalization. Electroretinogram recordings under scotopic conditions showed severe attenuation of the a-wave, confirming the degeneration of photoreceptors. Retinas lacking CRB2 in developing photoreceptors showed early onset of abnormal lamination, whereas retinas lacking CRB2 in developing Müller cells showed late onset retinal disorganization. Our data suggest that in the developing retina, CRB2 has redundant functions in Müller glial cells, while CRB2 has essential functions in photoreceptors. Our data suggest that short-term loss of CRB2 in adult mouse photoreceptors, but not in Müller glial cells, causes sporadic loss of adhesion between photoreceptors and Müller cells.  

5.1467           CCBE1 Enhances Lymphangiogenesis via A Disintegrin and Metalloprotease With Thrombospondin Motifs-3–Mediated Vascular Endothelial Growth Factor-C Activation

Jeltsch, M., Jha, S.K., Tvorogoy, D., Anisimov, A., Leppänen, V-M., Holopainen, T., Kivelä, R., Ortega, S., Kärpanen, T. and Alitalo, K. Circulation, 129(19), 1962-1971 (2014)   Background—Hennekam lymphangiectasia–lymphedema syndrome (Online Mendelian Inheritance in Man 235510) is a rare autosomal recessive disease, which is associated with mutations in the CCBE1 gene. Because of the striking phenotypic similarity of embryos lacking either the Ccbe1 gene or the lymphangiogenic growth factor Vegfc gene, we searched for collagen- and calcium-binding epidermal growth factor domains 1 (CCBE1) interactions with the vascular endothelial growth factor-C (VEGF-C) growth factor signaling pathway, which is critical in embryonic and adult lymphangiogenesis. Methods and Results—By analyzing VEGF-C produced by CCBE1-transfected cells, we found that, whereas CCBE1 itself does not process VEGF-C, it promotes proteolytic cleavage of the otherwise poorly active 29/31-kDa form of VEGF-C by the A disintegrin and metalloprotease with thrombospondin motifs-3 protease, resulting in the mature 21/23-kDa form of VEGF-C, which induces increased VEGF-C receptor signaling. Adeno-associated viral vector–mediated transduction of CCBE1 into mouse skeletal muscle enhanced lymphangiogenesis and angiogenesis induced by adeno-associated viral vector–VEGF-C. Conclusions—These results identify A disintegrin and metalloprotease with thrombospondin motifs-3 as a VEGF-C–activating protease and reveal a novel type of regulation of a vascular growth factor by a protein that enhances its proteolytic cleavage and activation. The results suggest that CCBE1 is a potential therapeutic tool for the modulation of lymphangiogenesis and angiogenesis in a variety of diseases that involve the lymphatic system, such as lymphedema or lymphatic metastasis.    

5.1468           Turmeric curcumin inhibits entry of all hepatitis C virus genotypes into human liver cells

Anggakusuma, Colpitts, C.C., Schang, L.M., Rachmawati, H., Frentzen, A., Pfaender, S., Behrendt, P., Brown, R.J.P., Bankwitz, D., Steinmann, J., Ott, M., Meuleman, P., Rice, C.M., Ploss, A., Pietschmann, T. and Steinmann, E. Gut, 63, 1137-1149 (2014)   Objective Hepatitis C virus (HCV) infection causes severe liver disease and affects more than 160 million individuals worldwide. People undergoing liver organ transplantation face universal re-infection of the graft. Therefore, affordable antiviral strategies targeting the early stages of infection are urgently needed to prevent the recurrence of HCV infection. The aim of the study was to determine the potency of turmeric curcumin as an HCV entry inhibitor. Design The antiviral activity of curcumin and its derivatives was evaluated using HCV pseudo-particles (HCVpp) and cell-culture-derived HCV (HCVcc) in hepatoma cell lines and primary human hepatocytes. The mechanism of action was dissected using R18-labelled virions and a membrane fluidity assay. Results Curcumin treatment had no effect on HCV RNA replication or viral assembly/release. However, co-incubation of HCV with curcumin potently inhibited entry of all major HCV genotypes. Similar antiviral activities were also exerted by other curcumin derivatives but not by tetrahydrocurcumin, suggesting the importance of α,β-unsaturated ketone groups for the antiviral activity. Expression levels of known HCV receptors were unaltered, while pretreating the virus with the compound reduced viral infectivity without viral lysis. Membrane fluidity experiments indicated that curcumin affected the fluidity of the HCV envelope resulting in impairment of viral binding and fusion. Curcumin has also been found to inhibit cell-to-cell transmission and to be effective in combination with other antiviral agents. Conclusions Turmeric curcumin inhibits HCV entry independently of the genotype and in primary human hepatocytes by affecting membrane fluidity thereby impairing virus binding and fusion.  

5.1469           Partial Correction of the CNS Lysosomal Storage Defect in a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis by Neonatal CNS Administration of an Adeno-Associated Virus Serotype rh.10 Vector Expressing the Human CLN3 Gene

Sondhi, D., Scott, E.C., Chen, A., Hackett, N.R., Wong, Kubiak, A., Nelvagal, H.R., Pearse, Y., Cotman, S.L., Cooper, J.D. and Crystal, R.G. Human Gene Therapy, 25(3), 223-239 (2014)   Juvenile neuronal ceroid lipofuscinosis (JNCL or CLN3 disease) is an autosomal recessive lysosomal storage disease resulting from mutations in the CLN3 gene that encodes a lysosomal membrane protein. The disease primarily affects the brain with widespread intralysosomal accumulation of autofluorescent material and fibrillary gliosis, as well as the loss of specific neuronal populations. As an experimental treatment for the CNS manifestations of JNCL, we have developed a serotype rh.10 adeno-associated virus vector expressing the human CLN3 cDNA (AAVrh.10hCLN3). We hypothesized that administration of AAVrh.10hCLN3 to the Cln3^Δex7/8 knock-in mouse model of JNCL would reverse the lysosomal storage defect, as well as have a therapeutic effect on gliosis and neuron loss. Newborn Cln3^Δex7/8 mice were administered 3×10¹⁰ genome copies of AAVrh.10hCLN3 to the brain, with control groups including untreated Cln3^Δex7/8 mice and wild-type littermate mice. After 18 months, CLN3 transgene expression was detected in various locations throughout the brain, particularly in the hippocampus and deep anterior cortical regions. Changes in the CNS neuronal lysosomal accumulation of storage material were assessed by immunodetection of subunit C of ATP synthase, luxol fast blue staining, and periodic acid-Schiff staining. For all parameters, Cln3^Δex7/8 mice exhibited abnormal lysosomal accumulation, but AAVrh.10hCLN3 administration resulted in significant reductions in storage material burden. There was also a significant decrease in gliosis in AAVrh.10hCLN3-treated Cln3^Δex7/8 mice, and a trend toward improved neuron counts, compared with their untreated counterparts. These data demonstrate that AAVrh.10 delivery of a wild-type cDNA to the CNS is not harmful and instead provides a partial correction of the neurological lysosomal storage defect of a disease caused by a lysosomal membrane protein, indicating that this may be an effective therapeutic strategy for JNCL and other diseases in this category.  

5.1470           Absolute Determination of Single-Stranded and Self-Complementary Adeno-Associated Viral Vector Genome Titers by Droplet Digital PCR

Lock, M., Alvira, M.R., Chen, S-J. and Wilson, J.M. Human Gene Therapy Methods, 25(2), 115-125 (2014)   Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer–probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution analysis in inhibitory tissues.  

5.1471           Effect of bortezomib on the efficacy of AAV9.SERCA2a treatment to preserve cardiac function in a rat pressure-overload model of heart failure

Chanine, A.H., Nonnenmacher, M., Kohlbrenner, E., Jin, D., Kovacic, J.C., Akar, F.G., Hajjar, R.J. and Weber, T. Gene Therapy, 21(4), 379-386 (2014)   Adeno-associated virus (AAV)-based vectors are promising vehicles for therapeutic gene delivery, including for the treatment for heart failure. It has been demonstrated for each of the AAV serotypes 1 through 8 that inhibition of the proteasome results in increased transduction efficiencies. For AAV9, however, the effect of proteasome inhibitors on in vivo transduction has until now not been evaluated. Here we demonstrate, in a well-established rodent heart failure model, that concurrent treatment with the proteasome inhibitor bortezomib does not enhance the efficacy of AAV9.SERCA2a to improve cardiac function as examined by echocardiography and pressure volume analysis. Western blot analysis of SERCA2a protein and reverse transcription-PCR of SERCA2a mRNA demonstrated that bortezomib had no effect on either endogenous rat SERCA2a levels nor on expression levels of human SERCA2a delivered by AAV9.SERCA2a. Similarly, the number of AAV9 genomes in heart samples was unaffected by bortezomib treatment. Interestingly, whereas transduction of HeLa cells and neonatal rat cardiomyocytes by AAV9 was stimulated by bortezomib, transduction of adult rat cardiomyocytes was inhibited. These results indicate an organ/cell-type-specific effect of proteasome inhibition on AAV9 transduction. A future detailed analysis of the underlying molecular mechanisms promises to facilitate the development of improved AAV vectors.  

5.1472           Long-term correction of biochemical and neurological abnormalities in MLD mice model by neonatal systemic injection of an AAV serotype 9 vector

Miyake, N., Miyake, K., Asakawa, N., Yamamoto, M. and Shimada, T. Gene Therapy, 21(4), 427-433 (2014)   As both the immune system and the blood–brain barrier (BBB) are likely to be developmentally immature in the perinatal period, neonatal gene transfer may be useful for the treatment of lysosomal storage disease (LSD) with neurological involvements such as metachromatic leukodystrophy (MLD). In this experiment, we examined the feasibility of single-strand adeno-associated viral serotype-9 (ssAAV9)-mediated systemic neonatal gene therapy of MLD mice. ssAAV9 vector expressing human arylsulfatase A (ASA) and green fluorescent protein (GFP) (ssAAV9/ASA) was injected into the jugular vein of newborn MLD mice. High levels of ASA expression were observed in the muscle and heart for at least 15 months. ASA was continuously secreted into plasma without development of antibodies against ASA. Global gene transfer into the brain and spinal cord (SC), across the BBB, and long-term ASA expression in the central nervous system were detected in treated mice. Significant inhibition of the accumulation of sulfatide (Sulf) in the brain and cervical SC was confirmed by Alcian blue staining and biochemical analysis of the Sulf content. In a behavior test, treated mice showed a greater ability to traverse narrow balance beams than untreated mice. These data clearly demonstrate that MLD mice model can be effectively treated through neonatal systemic injection of ssAAV9/ASA.  

5.1473           Tunable Protease-Activatable Virus Nanonodes

Judd, J., Ho, M.L., Tiwari, A., Gomez, E.J., Dempsey, C., Van Vliet, K., Igoshin, O.A., Silberg, J.J., Agbandje-McKenna, M. and Suh, J. ACSNano, 8(5), 4740-4746 (2014)   We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus–receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery.  

5.1474           HIV Virions as Nanoscopic Test Tubes for Probing Oligomerization of the Integrase Enzyme

Borrenberghs, D., Thys, W., Rocha, S., Demeulemeester, J., Weydert, C., Dedecker, P., Hofkens, J., Debyser, Z. and Hendrix, J. ACS Nano, 8(4), 3531-3545 (2014)   Employing viruses as nanoscopic lipid-enveloped test tubes allows the miniaturization of protein–protein interaction (PPI) assays while preserving the physiological environment necessary for particular biological processes. Applied to the study of the human immunodeficiency virus type 1 (HIV-1), viral biology and pathology can also be investigated in novel ways, both in vitro as well as in infected cells. In this work we report on an experimental strategy that makes use of engineered HIV-1 viral particles, to allow for probing PPIs of the HIV-1 integrase (IN) inside viruses with single-molecule Förster resonance energy transfer (FRET) using fluorescent proteins (FP). We show that infectious fluorescently labeled viruses can be obtained and that the quantity of labels can be accurately measured and controlled inside individual viral particles. We demonstrate, with proper control experiments, the formation of IN oligomers in single viral particles and inside viral complexes in infected cells. Finally, we show a clear effect on IN oligomerization of small molecule inhibitors of interactions of IN with its natural human cofactor LEDGF/p75, corroborating that IN oligomer enhancing drugs are active already at the level of the virus and strongly suggesting the presence of a dynamic, enhanceable equilibrium between the IN dimer and tetramer in viral particles. Although applied to the HIV-1 IN enzyme, our methodology for utilizing HIV virions as nanoscopic test tubes for probing PPIs is generic, i.e., other PPIs targeted into the HIV-1, or PPIs targeted into other viruses, can potentially be studied with a similar strategy.  

5.1475           Long-Term Overexpression of Human Wild-Type and T240R Mutant Parkin in Rat Substantia Nigra Induces Progressive Dopaminergic Neurodegeneration

Van Rompuy, A-S., Lobbestael, E., Van der Perren, A., Van den Haute, C. and Baekelandt, V.

1. Neuropathol. Exp. Neurol., 73(2), 159-174 (2014)

  Mutations in the parkin gene are the most common cause of early-onset autosomal recessive Parkinson disease (PD). The pathogenic mechanisms of how parkin mutations lead to the development of PD are not fully understood. Studies of cell cultures and of Drosophila have suggested a dominant negative effect for the clinical parkin mutant T240R. Conversely, the neuroprotective capacity of parkin has been widely reported; this suggests that the parkin protein may have a potential therapeutic role in PD. Here, we aimed to develop a novel genetic rodent model of PD by overexpression of T240R-parkin and human wild-type parkin as a control in the dopaminergic neurons of adult rats using adeno-associated viral vectors (rAAV2/8). Surprisingly, we found that overexpression not only of T240R-parkin but also of human wild-type parkin induced progressive and dose-dependent dopaminergic cell death in rats, starting from 8 weeks after injection. This degeneration was specific for parkin because similar overexpressionof enhanced green fluorescent protein did not lead to nigral degeneration. Our results warrant caution to the development of therapeutic strategies for PD based on overexpression of parkin or enhancing parkin activity because this might be deleterious for dopaminergic neurons in the long-term.  

5.1476           Reversible Nerve Damage and Corneal Pathology in Murine Herpes Simplex Stromal Keratitis

Yun, H., Rowe, A.M., Latrop, K.L., Harvey, S.A.K. and Hendricks, R.L.

1. Virol., 88(14), 7870-7880 (2014)

  Herpes simplex virus type 1 (HSV-1) shedding from sensory neurons can trigger recurrent bouts of herpes stromal keratitis (HSK), an inflammatory response that leads to progressive corneal scarring and blindness. A mouse model of HSK is often used to delineate immunopathogenic mechanisms and bears many of the characteristics of human disease, but it tends to be more chronic and severe than human HSK. Loss of blink reflex (BR) in human HSK is common and due to a dramatic retraction of corneal sensory nerve termini in the epithelium and the nerve plexus at the epithelial/stromal interface. However, the relationship between loss of BR due to nerve damage and corneal pathology associated with HSK remains largely unexplored. Here, we show a similar retraction of corneal nerves in mice with HSK. Indeed, we show that much of the HSK-associated corneal inflammation in mice is actually attributable to damage to the corneal nerves and accompanying loss of BR and can be prevented or ameliorated by tarsorrhaphy (suturing eyelids closed), a clinical procedure commonly used to prevent corneal exposure and desiccation. In addition, we show that HSK-associated nerve retraction, loss of BR, and severe pathology all are reversible and regulated by CD4⁺ T cells. Thus, defining immunopathogenic mechanisms of HSK in the mouse model will necessitate distinguishing mechanisms associated with the immunopathologic response to the virus from those associated with loss of corneal sensation. Based on our findings, investigation of a possible contribution of nerve damage and BR loss to human HSK also appears warranted.  

5.1477           The Nucleocapsid Domain of Gag Is Dispensable for Actin Incorporation into HIV-1 and for Association of Viral Budding Sites with Cortical F-Actin

Stauffer, S., Rahman, S.A., de marco, A., Carlson, L-A., Glass, B., Oberwinkler, H., Herold, N., Briggs, J.A.G., Müller, B., Grünewald, K and Kräusslich, H-G.

1. Virol., 88(14), 7893-7903 (2014)

  Actin and actin-binding proteins are incorporated into HIV-1 particles, and F-actin has been suggested to bind the NC domain in HIV-1 Gag. Furthermore, F-actin has been frequently observed in the vicinity of HIV-1 budding sites by cryo-electron tomography (cET). Filamentous structures emanating from viral buds and suggested to correspond to actin filaments have been observed by atomic force microscopy. To determine whether the NC domain of Gag is required for actin association with viral buds and for actin incorporation into HIV-1, we performed comparative analyses of virus-like particles (VLPs) obtained by expression of wild-type HIV-1 Gag or a Gag variant where the entire NC domain had been replaced by a dimerizing leucine zipper [Gag(LZ)]. The latter protein yielded efficient production of VLPs with near-wild-type assembly kinetics and size and exhibited a regular immature Gag lattice. Typical HIV-1 budding sites were detected by using cET in cells expressing either Gag or Gag(LZ), and no difference was observed regarding the association of buds with the F-actin network. Furthermore, actin was equally incorporated into wild-type HIV-1 and Gag- or Gag(LZ)-derived VLPs, with less actin per particle observed than had been reported previously. Incorporation appeared to correlate with the relative intracellular actin concentration, suggesting an uptake of cytosol rather than a specific recruitment of actin. Thus, the NC domain in HIV-1 Gag does not appear to have a role in actin recruitment or actin incorporation into HIV-1 particles.  

5.1478           Experimental phage therapy against lethal lung-derived septicemia caused by Staphylococcus aureus in mice

Takemura-uchiyama, I., Uchiyama, J., osanai, M., Morimoto, N., Asagiri, T., Ujihara, T., Daibata, M., Sugiura, t and Matsuzaki, S. Microbes and Infection, 16, 512-517 (2014)   Nosocomial respiratory infections caused by methicillin-resistant Staphylococcus aureus (MRSA) can progress to lethal systemic infections. Bacteriophage (phage) therapy is expected to be effective against these critical infections. Previously, phage S13′ was proposed as a potential therapeutic phage. We here examined phage treatment in a mouse model of lung-derived septicemia using phage S13′. Intraperitoneal phage administration at 6 h postinfection reduced the severity of infection and rescued the infected mice. Phage S13′ can efficiently lyse hospital-acquired MRSA strains causing pneumonia-associated bacteremia in vitro. Thus, phage therapy may be a possible therapeutic intervention in staphylococcal lung-derived septicemia.    

5.1479           Naturally enveloped AAV vectors for shielding neutralizing antibodies and robust gene delivery in vivo

György, B., Fitzpatrick, Z., Crommentuijn, M.H.W., Mu, D. and Maguire, C.A. Biomaterials, 35, 7598-7609 (2014)   Recently adeno-associated virus (AAV) became the first clinically approved gene therapy product in the western world. To develop AAV for future clinical application in a widespread patient base, particularly in therapies which require intravenous (i.v.) administration of vector, the virus must be able to evade pre-existing antibodies to the wild type virus. Here we demonstrate that in mice, AAV vectors associated with extracellular vesicles (EVs) can evade human anti-AAV neutralizing antibodies. We observed different antibody evasion and gene transfer abilities with populations of EVs isolated by different centrifugal forces. EV-associated AAV vector (ev-AAV) was up to 136-fold more resistant over a range of neutralizing antibody concentrations relative to standard AAV vector in vitro. Importantly in mice, at a concentration of passively transferred human antibodies which decreased i.v. administered standard AAV transduction of brain by 80%, transduction of ev-AAV transduction was not reduced and was 4000-fold higher. Finally, we show that expressing a brain targeting peptide on the EV surface allowed significant enhancement of transduction compared to untargeted ev-AAV. Using ev-AAV represents an effective, clinically relevant approach to evade human neutralizing anti-AAV antibodies after systemic administration of vector.  

5.1480           Physiologic and metabolic safety of butyrylcholinesterase gene therapy in mice

Murthy, V., Gao, Y., Geng, L., LeBrasseur, N.K., White, T.A., parks, R.J. and Brimijoin, S. Vaccine, 32, 4155-4162 (2014)   In continuing efforts to develop gene transfer of human butyrylcholinesterase (BChE) as therapy for cocaine addiction, we conducted wide-ranging studies of physiological and metabolic safety. For that purpose, mice were given injections of adeno-associated virus (AAV) vector or helper-dependent adenoviral (hdAD) vector encoding human or mouse BChE mutated for optimal cocaine hydrolysis. Age-matched controls received saline or AAV-luciferase control vector. At times when transduced BChE was abundant, physiologic and metabolic parameters in conscious animals were evaluated by non-invasive Echo-MRI and an automated “Comprehensive Laboratory Animal Monitoring System” (CLAMS). Despite high vector doses (up to 10¹³ particles per mouse) and high levels of transgene protein in the plasma (∼1500-fold above baseline), the CLAMS apparatus revealed no adverse physiologic or metabolic effects. Likewise, body composition determined by Echo-MRI, and glucose tolerance remained normal. A CLAMS study of vector-treated mice given 40 mg/kg cocaine showed none of the physiologic and metabolic fluctuations exhibited in controls. We conclude that neither the tested vectors nor great excesses of circulating BChE affect general physiology directly, while they protect mice from disturbance by cocaine. Hence, viral gene transfer of BChE appears benign and worth exploring as a therapy for cocaine abuse and possibly other disorders as well.  

5.1481           Novel AAV-Based Rat Model of Forebrain Synucleinopathy Shows Extensive Pathologies and Progressive Loss of Cholinergic Interneurons

Aldrin-Kirk, P., Davidsson, M., Holmqvist, S., Li, J-Y. and Björklund, T. PloS One, 9(7), e100869 (2014)   Synucleinopathies, characterized by intracellular aggregation of α-synuclein protein, share a number of features in pathology and disease progression. However, the vulnerable cell population differs significantly between the disorders, despite being caused by the same protein. While the vulnerability of dopamine cells in the substantia nigra to α-synuclein over-expression, and its link to Parkinson's disease, is well studied, animal models recapitulating the cortical degeneration in dementia with Lewy-bodies (DLB) are much less mature. The aim of this study was to develop a first rat model of widespread progressive synucleinopathy throughout the forebrain using adeno-associated viral (AAV) vector mediated gene delivery. Through bilateral injection of an AAV6 vector expressing human wild-type α-synuclein into the forebrain of neonatal rats, we were able to achieve widespread, robust α-synuclein expression with preferential expression in the frontal cortex. These animals displayed a progressive emergence of hyper-locomotion and dysregulated response to the dopaminergic agonist apomorphine. The animals receiving the α-synuclein vector displayed significant α-synuclein pathology including intra-cellular inclusion bodies, axonal pathology and elevated levels of phosphorylated α-synuclein, accompanied by significant loss of cortical neurons and a progressive reduction in both cortical and striatal ChAT positive interneurons. Furthermore, we found evidence of α-synuclein sequestered by IBA-1 positive microglia, which was coupled with a distinct change in morphology. In areas of most prominent pathology, the total α-synuclein levels were increased to, on average, two-fold, which is similar to the levels observed in patients with SNCA gene triplication, associated with cortical Lewy body pathology. This study provides a novel rat model of progressive cortical synucleinopathy, showing for the first time that cholinergic interneurons are vulnerable to α-synuclein over-expression. This animal model provides a powerful new tool for studies of neuronal degeneration in conditions of widespread cortical α-synuclein pathology, such as DLB, as well an attractive model for the exploration of novel biomarkers.  

5.1482           The odd one out: Bacillus ACT bacteriophage CP-51 exhibits unusual properties compared to related Spounavirinae W.Ph. and Bastille

Klummp, J., Schmuki, M., Sozhamannan, S., Beyer, W., Fouts, D.E., Bernbach, V., Calendar, R. and Loessner, M.J. Virology, 462-463, 299-308 (2014)   The Bacillus ACT group includes three important pathogenic species of Bacillus: anthracis, cereus and thuringiensis. We characterized three virulent bacteriophages, Bastille, W.Ph. and CP-51, that infect various strains of these three species. We have determined the complete genome sequences of CP-51, W.Ph. and Bastille, and their physical genome structures. The CP-51 genome sequence could only be obtained using a combination of conventional and second and third next generation sequencing technologies – illustrating the problems associated with sequencing highly modified DNA. We present evidence that the generalized transduction facilitated by CP-51 is independent of a specific genome structure, but likely due to sporadic packaging errors of the terminase. There is clear correlation of the genetic and morphological features of these phages validating their placement in the Spounavirinae subfamily (SPO1-related phages) of the Myoviridae. This study also provides tools for the development of phage-based diagnostics/therapeutics for this group of pathogens.  

5.1483           Targeting cells with single vectors using multiple-feature Boolean logic

Fenno, L.E. et al Nature Methods, 11(7), 763-772 (2014)   Precisely defining the roles of specific cell types is an intriguing frontier in the study of intact biological systems and has stimulated the rapid development of genetically encoded tools for observation and control. However, targeting these tools with adequate specificity remains challenging: most cell types are best defined by the intersection of two or more features such as active promoter elements, location and connectivity. Here we have combined engineered introns with specific recombinases to achieve expression of genetically encoded tools that is conditional upon multiple cell-type features, using Boolean logical operations all governed by a single versatile vector. We used this approach to target intersectionally specified populations of inhibitory interneurons in mammalian hippocampus and neurons of the ventral tegmental area defined by both genetic and wiring properties. This flexible and modular approach may expand the application of genetically encoded interventional and observational tools for intact-systems biology.Targeting cells with single vectors using multiple  

5.1484           Optimization and validation of a high throughput method for detecting neutralizing antibodies against human papillomavirus (HPV) based on pseudovirons

Nie, J., Huang, W., Wu, X. and Wang, Y.

1. Med. Virol., 86(9), 1542-1555 (2014)

  The pseudoviron-based neutralization assay is accepted as the gold standard to evaluate the functional humoral immune response against HPV. The goal of this study was to develop and optimize a human papillomavirus (HPV) neutralization assay using HPV pseudovirons with Gaussia luciferase (Gluc) as the reporter gene. For this purpose, high-titers Gluc pseudovirons were generated by cotransfecting 293TT cells with HPV structural genes and Gluc expressing plasmids. Six types of neutralizing monoclonal antibodies, vaccines immunized serum samples and WHO international antibody standard were used to validate the new developed assay. The ideal circumstances of the assay were identified for cell counts (30,000/well for 96-well plate), pseudoviron inoculating size (100 times RLU above background) and incubation time (72 hr). The sensitivity of the Gluc assay was comparable to secreted alkaline phosphatase (SEAP) assay and higher than the green florescent protein (GFP) assay. The non-specific background for different types of sample was significantly different (rabbit sera > human sera > mouse sera, P < 0.01). The non-specific neutralization effects were not attributed to IgG antibody. The cutoff value for this assay was determined as 50% inhibition at a dilution of 1:40. Without requirements of sample dilution and different incubation times at different temperature before processing, the detection time was shortened from more than 90 min to less than 5 min for a 96-well plate compared with the SEAP-based assay. With the advantages of short detection time and easy-to-use procedure, the newly developed assay is more suitable for large sero-epidemiological studies or clinical trials and more amenable to automation.  

5.1485           Tailored Vaccines Targeting the Elderly Using Whole Inactivated Influenza Vaccines Bearing Cytokine Immunomodulators

Khan, T., Heffron, C.L., High, K.P. and Roberts, P.C.

1. Interferon & Cytokine Res., 34(2), 129-139 (2014)

  Influenza and its complications disproportionately affect the elderly, leading to high morbidity and mortality in this ever-increasing population. Despite widespread vaccination efforts, the current influenza vaccines are less effective in the elderly; hence newer vaccine strategies are needed to improve their efficacy in this age group. We have previously shown that co-presentation of cytokines on the surface of inactivated influenza virus particles affords better protection from lethal homotypic viral challenge in young adult mice than conventional non-adjuvanted whole inactivated vaccine. Here, we determined the efficacy of these vaccine formulations in Balb/c mice “aged” to 17 months (“aged mice”) along with the addition of a membrane-bound interleukin-12 (IL-12) vaccine formulation. Our investigations found that a single low-dose intramuscular vaccination with inactivated whole influenza vaccine co-presenting IL-12 was sufficient to provide enhanced protection from subsequent influenza challenge as compared with non-adjuvanted whole inactivated vaccine. Our results indicate that incorporation of cytokines such as IL-12 in a membrane-bound formulation in whole inactivated vaccine may provide a means to lower the vaccine dose while eliciting enhanced protective responses in the elderly, an age group that responds poorly to current vaccination regimens.  

5.1486           Nuclear receptor 4 group A member 1 determines hepatitis C virus entry efficiency through the regulation of cellular receptor and apolipoprotein E expression

Zhu, W., Pei, R., Jin, R., Hu, X., Zhou, Y., Wang, Y., Wu, C., Lu, M. and Chen, X.

1. Gen. Virol., 95, 1510-1521 (2014)

  Orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) is a transcription factor stimulated by many factors and plays pivotal roles in metabolism, proliferation and apoptosis. In this study, the expression of NR4A1 in Huh7.5.1 cells was significantly upregulated by hepatitis C virus (HCV) infection. The silencing of NR4A1 inhibited the entry of HCV and reduced the specific infectivity of secreted HCV particles but had only minor or no effect on the genome replication and translation, virion assembly and virus release steps of the virus life cycle. Further experiments demonstrated that the silencing of NR4A1 affected virus entry through pan-downregulation of the expression of HCV receptors scavenger receptor BI, occludin, claudin-1 and epidermal growth factor receptor but not CD81. The reduced specific infectivity of HCV in the knockdown cells was due to decreased apolipoprotein E (ApoE) expression. These results explain the delayed spread of HCV in NR4A1 knockdown Huh7.5.1 cells. Thus, NR4A1 plays a role in HCV replication through regulating the expression of HCV receptors and ApoE, and facilitates HCV entry and spread.  

5.1487           Intracerebral Administration of Adeno-Associated Viral Vector Serotype rh.10 Carrying Human SGSH and SUMF1 cDNAs in Children with Mucopolysaccharidosis Type IIIA Disease: Results of a Phase I/II Trial

Tardieu, M. et al Human Gene Therapy, 25(6), 506-516 (2014)   Mucopolysaccharidosis type IIIA is a severe degenerative disease caused by an autosomal recessive defect of a gene encoding a lysosomal heparan-N-sulfamidase, the N-sulfoglycosamine sulfohydrolase (SGSH), the catalytic site of which is activated by a sulfatase-modifying factor (SUMF1). Four children (Patients 1–3, aged between 5.5 and 6 years; Patient 4 aged 2 years 8 months) received intracerebral injections of an adeno-associated viral vector serotype rh.10-SGSH-IRES-SUMF1 vector in a phase I/II clinical trial. All children were able to walk, but their cognitive abilities were abnormal and had declined (Patients 1–3). Patients 1–3 presented with brain atrophy. The therapeutic vector was delivered in a frameless stereotaxic device, at a dose of 7.2×10¹¹ viral genomes/patient simultaneously via 12 needles as deposits of 60 μl over a period of 2 hr. The vector was delivered bilaterally to the white matter anterior, medial, and posterior to the basal ganglia. Immunosuppressive treatment (mycophenolate mofetil and tacrolimus) was initiated 15 days before surgery and maintained for 8 weeks (mycophenolate mofetil) or throughout follow-up (tacrolimus, with progressive dose reduction) to prevent elimination of transduced cells. Safety data collected from inclusion, during the neurosurgery period and over the year of follow-up, showed good tolerance, absence of adverse events related to the injected product, no increase in the number of infectious events, and no biological sign of toxicity related to immunosuppressive drugs. Efficacy analysis was necessarily preliminary in this phase I/II trial on four children, in the absence of validated surrogate markers. Brain atrophy evaluated by magnetic resonance imaging seemed to be stable in Patients 1 and 3 but tended to increase in Patients 2 and 4. Neuropsychological evaluations suggested a possible although moderate improvement in behavior, attention, and sleep in Patients 1–3. The youngest patient was the most likely to display neurocognitive benefit.  

5.1488           Retinoschisin gene therapy in photoreceptors, Müller glia or all retinal cells in the Rs1h−/− mouse

Byrne, L.C., Öztürk, B.E., Lee, t., Fortuny, C., Visel, M., Dalkara, D., Schaffer, D.V. and Flannery, J.G. Gene Therapy, 21, 585-592 (2014)   X-linked retinoschisis, a disease characterized by splitting of the retina, is caused by mutations in the retinoschisin gene, which encodes a putative secreted cell adhesion protein. Currently, there is no effective treatment for retinoschisis, though viral vector-mediated gene replacement therapies offer promise. We used intravitreal delivery of three different AAV vectors to target delivery of the RS1 gene to Müller glia, photoreceptors or multiple cell types throughout the retina. Müller glia radially span the entire retina, are accessible from the vitreous, and remain intact throughout progression of the disease. However, photoreceptors, not glia, normally secrete retinoschisin. We compared the efficacy of rescue mediated by retinoschisin secretion from these specific subtypes of retinal cells in the Rs1h−/− mouse model of retinoschisis. Our results indicate that all three vectors deliver the RS1 gene, and that several cell types can secrete retinoschisin, leading to transport of the protein across the retina. The greatest long-term rescue was observed when photoreceptors produce retinoschisin. Similar rescue was observed with photoreceptor-specific or generalized expression, although photoreceptor secretion may contribute to rescue in the latter case. These results collectively point to the importance of cell targeting and appropriate vector choice in the success of retinal gene therapies.  

5.1489           Autism-Associated Neuroligin-3 Mutations Commonly Impair Striatal Circuits to Boost Repetitive Behaviors

Rothwell, P.E., Fuccillo, M.V., Maxeiner, S., hayton, S.J., Gokce, O., Lim, B.K., Fowler, S.C., Malenka, R.C. and Südhof, T.C. Cell, 158(1), 198-212 (2014)   In humans, neuroligin-3 mutations are associated with autism, whereas in mice, the corresponding mutations produce robust synaptic and behavioral changes. However, different neuroligin-3 mutations cause largely distinct phenotypes in mice, and no causal relationship links a specific synaptic dysfunction to a behavioral change. Using rotarod motor learning as a proxy for acquired repetitive behaviors in mice, we found that different neuroligin-3 mutations uniformly enhanced formation of repetitive motor routines. Surprisingly, neuroligin-3 mutations caused this phenotype not via changes in the cerebellum or dorsal striatum but via a selective synaptic impairment in the nucleus accumbens/ventral striatum. Here, neuroligin-3 mutations increased rotarod learning by specifically impeding synaptic inhibition onto D1-dopamine receptor-expressing but not D2-dopamine receptor-expressing medium spiny neurons. Our data thus suggest that different autism-associated neuroligin-3 mutations cause a common increase in acquired repetitive behaviors by impairing a specific striatal synapse and thereby provide a plausible circuit substrate for autism pathophysiology.  

5.1490           Modulation of Triglyceride and Cholesterol Ester Synthesis Impairs Assembly of Infectious Hepatitis C Virus

Liefhebber, J.M.P., Hague, C.V., Zhang, Q., Wakelam, M.J.O. and McLauchlan, J.

1. Biol. Chem., 289(31), 21276-21288 (2014)

  In hepatitis C virus infection, replication of the viral genome and virion assembly are linked to cellular metabolic processes. In particular, lipid droplets, which store principally triacylglycerides (TAGs) and cholesterol esters (CEs), have been implicated in production of infectious virus. Here, we examine the effect on productive infection of triacsin C and YIC-C8-434, which inhibit synthesis of TAGs and CEs by targeting long-chain acyl-CoA synthetase and acyl-CoA:cholesterol acyltransferase, respectively. Our results present high resolution data on the acylglycerol and cholesterol ester species that were affected by the compounds. Moreover, triacsin C, which blocks both triglyceride and cholesterol ester synthesis, cleared most of the lipid droplets in cells. By contrast, YIC-C8-434, which only abrogates production of cholesterol esters, induced an increase in size of droplets. Although both compounds slightly reduced viral RNA synthesis, they significantly impaired assembly of infectious virions in infected cells. In the case of triacsin C, reduced stability of the viral core protein, which forms the virion nucleocapsid and is targeted to the surface of lipid droplets, correlated with lower virion assembly. In addition, the virus particles that were released from cells had reduced specific infectivity. YIC-C8-434 did not alter the association of core with lipid droplets but appeared to decrease production of infectious virus particles, suggesting a block in virion assembly. Thus, the compounds have antiviral properties, indicating that targeting synthesis of lipids stored in lipid droplets might be an option for therapeutic intervention in treating chronic hepatitis C virus infection.  

5.1491           Maturation of the Human Papillomavirus 16 Capsid

Cardone, G., Moyer, A.L., Cheng, N. et al mBio, 5(4), e01104 (2014)   Papillomaviruses are a family of nonenveloped DNA viruses that infect the skin or mucosa of their vertebrate hosts. The viral life cycle is closely tied to the differentiation of infected keratinocytes. Papillomavirus virions are released into the environment through a process known as desquamation, in which keratinocytes lose structural integrity prior to being shed from the surface of the skin. During this process, virions are exposed to an increasingly oxidative environment, leading to their stabilization through the formation of disulfide cross-links between neighboring molecules of the major capsid protein, L1. We used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV16 capsids assembled in mammalian cells and exposed to an oxidizing environment after cell lysis. Initially, the virion is a loosely connected procapsid that, under in vitro conditions, condenses over several hours into the more familiar 60-nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by ~5% in diameter, its pentameric capsomers change in structure (most markedly in the axial region), and the interaction surfaces between adjacent capsomers are consolidated. A C175S mutant that cannot achieve normal inter-L1 disulfide cross-links shows maturation-related shrinkage but does not achieve the fully condensed 60-nm form. Pseudoatomic modeling based on a 9-Å resolution reconstruction of fully mature capsids revealed C-terminal disulfide-stabilized “suspended bridges” that form intercapsomeric cross-links. The data suggest a model in which procapsids exist in a range of dynamic intermediates that can be locked into increasingly mature configurations by disulfide cross-linking, possibly through a Brownian ratchet mechanism.  

5.1492           Virus Particle Release from Glycosphingolipid-Enriched Microdomains Is Essential for Dendritic Cell-Mediated Capture and Transfer of HIV-1 and Henipavirus

Akiyama, H., Miller, C., patel, H.V., Hatch, S.C., Archer, J., Ramirez, N-G.P. and Gummuluru, S.

1. Virol., 88(16), 8813-8825 (2014)

  Human immunodeficiency virus type 1 (HIV-1) exploits dendritic cells (DCs) to promote its transmission to T cells. We recently reported that the capture of HIV-1 by mature dendritic cells (MDCs) is mediated by an interaction between the glycosphingolipid (GSL) GM3 on virus particles and CD169/Siglec-1 on MDCs. Since HIV-1 preferentially buds from GSL-enriched lipid microdomains on the plasma membrane, we hypothesized that the virus assembly and budding site determines the ability of HIV-1 to interact with MDCs. In support of this hypothesis, mutations in the N-terminal basic domain (29/31KE) or deletion of the membrane-targeting domain of the HIV-1 matrix (MA) protein that altered the virus assembly and budding site to CD63⁺/Lamp-1-positive intracellular compartments resulted in lower levels of virion incorporation of GM3 and attenuation of virus capture by MDCs. Furthermore, MDC-mediated capture and transmission of MA mutant viruses to T cells were decreased, suggesting that HIV-1 acquires GSLs via budding from the plasma membrane to access the MDC-dependent trans infection pathway. Interestingly, MDC-mediated capture of Nipah and Hendra virus (recently emerged zoonotic paramyxoviruses) M (matrix) protein-derived virus-like particles that bud from GSL-enriched plasma membrane microdomains was also dependent on interactions between virion-incorporated GSLs and CD169. Moreover, capture and transfer of Nipah virus envelope glycoprotein-pseudotyped lentivirus particles by MDCs were severely attenuated upon depletion of GSLs from virus particles. These results suggest that GSL incorporation into virions is critical for the interaction of diverse enveloped RNA viruses with DCs and that the GSL-CD169 recognition nexus might be a conserved viral mechanism of parasitization of DC functions for systemic virus dissemination.  

5.1493           Locking and Blocking the Viral Landscape of an Alphavirus with Neutralizing Antibodies

Porta, J., Jose, J., Roehrig, J.T., Blair, C.D., Kuhn, R.J. and Rossmann, M.G.

1. Virol., 88(17), 9616-9623 (2014)

  Alphaviruses are serious, sometimes lethal human pathogens that belong to the family Togaviridae. The structures of human Venezuelan equine encephalitis virus (VEEV), an alphavirus, in complex with two strongly neutralizing antibody Fab fragments (F5 and 3B4C-4) have been determined using a combination of cryo-electron microscopy and homology modeling. We characterize these monoclonal antibody Fab fragments, which are known to abrogate VEEV infectivity by binding to the E2 (envelope) surface glycoprotein. Both of these antibody Fab fragments cross-link the surface E2 glycoproteins and therefore probably inhibit infectivity by blocking the conformational changes that are required for making the virus fusogenic. The F5 Fab fragment cross-links E2 proteins within one trimeric spike, whereas the 3B4C-4 Fab fragment cross-links E2 proteins from neighboring spikes. Furthermore, F5 probably blocks the receptor-binding site, whereas 3B4C-4 sterically hinders the exposure of the fusion loop at the end of the E2 B-domain.  

5.1494           Cellular Proteins Associated with the Interior and Exterior of Vesicular Stomatitis Virus Virions

Moerdyk-Schauwecker, M., Hwang, S-l. and Grdzelishvili, V.Z. PloS One, 9(8), e104688 (2014)   Virus particles (virions) often contain not only virus-encoded but also host-encoded proteins. Some of these host proteins are enclosed within the virion structure, while others, in the case of enveloped viruses, are embedded in the host-derived membrane. While many of these host protein incorporations are likely accidental, some may play a role in virus infectivity, replication and/or immunoreactivity in the next host. Host protein incorporations may be especially important in therapeutic applications where large numbers of virus particles are administered. Vesicular stomatitis virus (VSV) is the prototypic rhabdovirus and a candidate vaccine, gene therapy and oncolytic vector. Using mass spectrometry, we previously examined cell type dependent host protein content of VSV virions using intact (“whole”) virions purified from three cell lines originating from different species. Here we aimed to determine the localization of host proteins within the VSV virions by analyzing: i) whole VSV virions; and ii) whole VSV virions treated with Proteinase K to remove all proteins outside the viral envelope. A total of 257 proteins were identified, with 181 identified in whole virions and 183 identified in Proteinase K treated virions. Most of these proteins have not been previously shown to be associated with VSV. Functional enrichment analysis indicated the most overrepresented categories were proteins associated with vesicles, vesicle-mediated transport and protein localization. Using western blotting, the presence of several host proteins, including some not previously shown in association with VSV (such as Yes1, Prl1 and Ddx3y), was confirmed and their relative quantities in various virion fractions determined. Our study provides a valuable inventory of virion-associated host proteins for further investigation of their roles in the replication cycle, pathogenesis and immunoreactivity of VSV.  

5.1495           Dual Transgene Expression in Murine Cerebellar Purkinje Neurons by Viral Transduction In Vivo

Bosch, M.K., Nerbonne, J.M. and Ornitz, D.M. PloS One, 9(8), e104062 (2014)   Viral-vector mediated gene transfer to cerebellar Purkinje neurons in vivo is a promising avenue for gene therapy of cerebellar ataxias and for genetic manipulation in functional studies of animal models of cerebellar disease. Here, we report the results of experiments designed to identify efficient methods for viral transduction of adult murine Purkinje neurons in vivo. For these analyses, several lentiviral and an adeno-associated virus (AAV), serotype 1, vector with various promoter combinations were generated and compared for in situ transduction efficiency, assayed by fluorescent reporter protein expression in Purkinje neurons. Additional experiments were also conducted to identify the optimal experimental strategy for co-expression of two proteins in individual Purkinje neurons. Of the viruses tested, AAV1 with a CAG promoter exhibited the highest specificity for Purkinje neurons. To deliver two proteins to the same Purkinje neuron, several methods were tested, including: an internal ribosome entry site (IRES), a 2A sequence, a dual promoter vector, and co-injection of two viruses. Efficient expression of both proteins in the same Purkinje neuron was only achieved by co-injecting two AAV1-CAG viruses. We found that use of an AAV1-CAG virus outperformed similar lentivirus vectors and that co-injection of two AAV1-CAG viruses could be used to efficiently deliver two proteins to the same Purkinje neuron in adult mice. AAV1 with a CAG promoter is highly efficient and selective at transducing adult cerebellar Purkinje neurons and two AAV-CAG viruses can be used to efficiently express two proteins in the same neuron in vivo.  

5.1496           Strain-Specific Properties and T Cells Regulate the Susceptibility to Papilloma Induction by Mus musculus Papillomavirus 1

Handisurya, A., Day, P.M., Thompson, C.D., Bonelli, M., Lowy, D.R. and Schiller, J.T. PloS Pathogens, 10(8), e1004314 (2014)   The immunocytes that regulate papillomavirus infection and lesion development in humans and animals remain largely undefined. We found that immunocompetent mice with varying H-2 haplotypes displayed asymptomatic skin infection that produced L1 when challenged with 6×10¹⁰ MusPV1 virions, the recently identified domestic mouse papillomavirus (also designated “MmuPV1”), but were uniformly resistant to MusPV1-induced papillomatosis. Broad immunosuppression with cyclosporin A resulted in variable induction of papillomas after experimental infection with a similar dose, from robust in Cr:ORL SENCAR to none in C57BL/6 mice, with lesional outgrowth correlating with early viral gene expression and partly with reported strain-specific susceptibility to chemical carcinogens, but not with H-2 haplotype. Challenge with 1×10¹² virions in the absence of immunosuppression induced small transient papillomas in Cr:ORL SENCAR but not in C57BL/6 mice. Antibody-induced depletion of CD3⁺ T cells permitted efficient virus replication and papilloma formation in both strains, providing experimental proof for the crucial role of T cells in controlling papillomavirus infection and associated disease. In Cr:ORL SENCAR mice, immunodepletion of either CD4⁺ or CD8⁺ T cells was sufficient for efficient infection and papillomatosis, although deletion of one subset did not inhibit the recruitment of the other subset to the infected epithelium. Thus, the functional cooperation of CD4⁺ and CD8⁺ T cells is required to protect this strain. In contrast, C57BL/6 mice required depletion of both CD4⁺ and CD8⁺ T cells for infection and papillomatosis, and separate CD4 knock-out and CD8 knock-out C57BL/6 were also resistant. Thus, in C57BL/6 mice, either CD4⁺ or CD8⁺ T cell-independent mechanisms exist that can protect this particular strain from MusPV1-associated disease. These findings may help to explain the diversity of pathological outcomes in immunocompetent humans after infection with a specific human papillomavirus genotype.  

5.1497           Identification of Conserved Residues in Hepatitis C Virus Envelope Glycoprotein E2 That Modulate Virus Dependence on CD81 and SRB1 Entry Factors

Lavie, M., Sarrazin, S., Montserret, R., Descamps, V., Baumert, T.F., Duverlie, G., Seron, K., Penin, F. and Dubuisson, J.

1. Virol., 88(18), 10584-10597 (2014)

  In spite of the high variability of its sequence, hepatitis C virus (HCV) envelope glycoprotein E2 contains several conserved regions. In this study, we explored the structural and functional features of the highly conserved E2 segment from amino acid (aa) 502 to 520, which had been proposed as a fusion peptide and shown to strongly overlap a potential conserved neutralizing epitope. For this purpose, we used reverse genetics to introduce point mutations within this region, and we characterized the phenotypes of these mutants in the light of the recently published structure of E2. The functional analyses showed that their phenotypes are in agreement with the positions of the corresponding residues in the E2 crystal structure. In contrast, our data ruled out the involvement of this region in membrane fusion, and they indicate that alternative conformations would be necessary to expose the potential neutralizing epitope present in this segment. Of particular interest, we identified three specific mutations (Y507L, V514A, and V515A) located within this neutralizing epitope which only mildly reduced infectivity and showed no assembly defect. These mutations modulated HCV dependence on the viral receptor SRB1, and/or they also modulated virion sensitivity to neutralizing antibodies. Importantly, their characterization also showed that amino acids Y507, V514, and V515 contribute to E2 interaction with HCV receptor CD81. In conclusion, our data show that the highly conserved E2 segment from aa 502 to 520 plays a key role in cell entry by influencing the association of the viral particle with coreceptors and neutralizing antibodies.  

5.1498           Characterization of the Regulatory Mechanisms of Activating Transcription Factor 3 by Hypertrophic Stimuli in Rat Cardiomyocytes

Koivisto, E., Acosta, A.J., Moilanen, A-M., Tokola, H., Aro, J., Pennanen, H., Säkkinen, H., Kaikkonen, L., Ruskoaho, H. and Rysä, J. PloS One, 9(8), e105168 (2014)   Aims Activating transcription factor 3 (ATF3) is a stress-activated immediate early gene suggested to have both detrimental and cardioprotective role in the heart. Here we studied the mechanisms of ATF3 activation by hypertrophic stimuli and ATF3 downstream targets in rat cardiomyocytes. Methods and Results When neonatal rat cardiomyocytes were exposed to endothelin-1 (ET-1, 100 nM) and mechanical stretching in vitro, maximal increase in ATF3 expression occurred at 1 hour. Inhibition of extracellular signal-regulated kinase (ERK) by PD98059 decreased ET-1– and stretch–induced increase of ATF3 protein but not ATF3 mRNA levels, whereas protein kinase A (PKA) inhibitor H89 attenuated both ATF3 mRNA transcription and protein expression in response to ET-1 and stretch. To characterize further the regulatory mechanisms upstream of ATF3, p38 mitogen-activated protein kinase (MAPK) signaling was investigated using a gain-of-function approach. Adenoviral overexpression of p38α, but not p38β, increased ATF3 mRNA and protein levels as well as DNA binding activity. To investigate the role of ATF3 in hypertrophic process, we overexpressed ATF3 by adenovirus-mediated gene transfer. In vitro, ATF3 gene delivery attenuated the mRNA transcription of interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1), and enhanced nuclear factor-κB (NF-κB) and Nkx-2.5 DNA binding activities. Reduced PAI-1 expression was also detected in vivo in adult rat heart by direct intramyocardial adenovirus-mediated ATF3 gene delivery. Conclusions These data demonstrate that ATF3 activation by ET-1 and mechanical stretch is partly mediated through ERK and cAMP-PKA pathways, whereas p38 MAPK pathway is involved in ATF3 activation exclusively through p38α isoform. ATF3 activation caused induction of modulators of the inflammatory response NF-κB and Nkx-2.5, as well as attenuation of pro-fibrotic and pro-inflammatory proteins IL-6 and PAI-1, suggesting cardioprotective role for ATF3 in the heart.  

5.1499           Hypothalamic prolyl endopeptidase (PREP) regulates pancreatic insulin and glucagon secretion in mice

Kim, D.J., Toda, C., D’Agostino, G., Zeiss, C.J., DiLeone, R.J., Elsworth, J.D., Kibbey, R.G., Chan, O., Harvey, B.K., Richie, C.T., Savolainen, M., Myöhännen, T., Jeong, J.K. and Diano, S. PNAS, 111(32), 11876-11881 (2014)   Prolyl endopeptidase (PREP) has been implicated in neuronal functions. Here we report that hypothalamic PREP is predominantly expressed in the ventromedial nucleus (VMH), where it regulates glucose-induced neuronal activation. PREP knockdown mice (Prep^gt/gt) exhibited glucose intolerance, decreased fasting insulin, increased fasting glucagon levels, and reduced glucose-induced insulin secretion compared with wild-type controls. Consistent with this, central infusion of a specific PREP inhibitor, S17092, impaired glucose tolerance and decreased insulin levels in wild-type mice. Arguing further for a central mode of action of PREP, isolated pancreatic islets showed no difference in glucose-induced insulin release between Prep^gt/gt and wild-type mice. Furthermore, hyperinsulinemic euglycemic clamp studies showed no difference between Prep^gt/gt and wild-type control mice. Central PREP regulation of insulin and glucagon secretion appears to be mediated by the autonomic nervous system because Prep^gt/gt mice have elevated sympathetic outflow and norepinephrine levels in the pancreas, and propranolol treatment reversed glucose intolerance in these mice. Finally, re-expression of PREP by bilateral VMH injection of adeno-associated virus–PREP reversed the glucose-intolerant phenotype of the Prep^gt/gt mice. Taken together, our results unmask a previously unknown player in central regulation of glucose metabolism and pancreatic function.  

5.1500           Overcoming the Cystic Fibrosis Sputum Barrier to Leading Adeno-associated Virus Gene Therapy Vectors

Schuster, B.S., Kim, A.J., Kays, J.C., Kanzawa, M.M., Guggino, W.B., Boyle, M.P., Rowe, S.M., Muzyczka, N., Suk, J.S. and Hanes, J. Molecular Therapy, 22(8), 1484-1493 (2014)   Gene therapy has not yet improved cystic fibrosis (CF) patient lung function in human trials, despite promising preclinical studies. In the human CF lung, inhaled gene vectors must penetrate the viscoelastic secretions coating the airways to reach target cells in the underlying epithelium. We investigated whether CF sputum acts as a barrier to leading adeno-associated virus (AAV) gene vectors, including AAV2, the only serotype tested in CF clinical trials, and AAV1, a leading candidate for future trials. Using multiple particle tracking, we found that sputum strongly impeded diffusion of AAV, regardless of serotype, by adhesive interactions and steric obstruction. Approximately 50% of AAV vectors diffused >1,000-fold more slowly in sputum than in water, with large patient-to-patient variation. We thus tested two strategies to improve AAV diffusion in sputum. We showed that an AAV2 mutant engineered to have reduced heparin binding diffused twice as fast as AAV2 on average, presumably because of reduced adhesion to sputum. We also discovered that the mucolytic N-acetylcysteine could markedly enhance AAV diffusion by altering the sputum microstructure. These studies underscore that sputum is a major barrier to CF gene delivery, and offer strategies for increasing AAV penetration through sputum to improve clinical outcomes.  

5.1501           Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation

Yamane, D. et al Nature Med., 20(8), 927-935 (2014)   Oxidative tissue injury often accompanies viral infection, yet there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase-2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals in vitro, suggesting critical regulation of the conformation of the NS3-4A protease and the NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to transmembrane and membrane-proximal residues within these proteins and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain of HCV. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.  

5.1502           Preclinical Studies on Neurobehavioral and Neuromuscular Effects of Cocaine Hydrolase Gene Therapy in Mice

Murthy, V., Gao, Y., Geng, L., LeBrasseur, N., White, T. and Brimijoin, S.

1. Mol. Neurosci., 53(3), 409-416 (2014)

  Cocaine hydrolase gene transfer of mutated human butyrylcholinesterase (BChE) is evolving as a promising therapy for cocaine addiction. BChE levels after gene transfer can be 1,500-fold above those in untreated mice, making this enzyme the second most abundant plasma protein. Because mutated BChE is approximately 70 % as efficient in hydrolyzing acetylcholine as wild-type enzyme, it is important to examine the impact on cholinergic function. Here, we focused on memory and cognition (Stone T-maze), basic neuromuscular function (treadmill endurance and grip strength), and coordination (Rotarod). BALB/c mice were given adeno-associated virus vector or helper-dependent adenoviral vector encoding mouse or human BChE optimized for cocaine. Age-matched controls received saline or luciferase vector. Despite high doses (up to 10¹³ particles per mouse) and high transgene expression (1,000-fold above baseline), no deleterious effects of vector treatment were seen in neurobehavioral functions. The vector-treated mice performed as saline-treated and luciferase controls in maze studies and strength tests, and their Rotarod and treadmill performance decreased less with age. Thus, neither the viral vectors nor the large excess of BChE caused observable toxic effects on the motor and cognitive systems investigated. This outcome justifies further steps toward an eventual clinical trial of vector-based gene transfer for cocaine abuse.  

5.1503           Incorporation of primary patient-derived glycoproteins into authentic infectious hepatitis C virus particles

Doerrbecker, J., Friesland, M., Riebesehl, N., Ginkel, C., Behrendt, P., Brown, R.J.P., Ciesek, S., Wedemeyer, H., Sarrazin, C., Kaderali, L., Pietschmann, T. and Steinmann, E. Hepatology, 60(2), 508-520 (2014)   The Japanese fulminant hepatitis-1 (JFH1)-based hepatitis C virus (HCV) infection system has permitted analysis of the complete viral replication cycle in vitro. However, lack of robust infection systems for primary, patient-derived isolates limits systematic functional studies of viral intrahost variation and vaccine development. Therefore, we aimed at developing cell culture models for incorporation of primary patient-derived glycoproteins into infectious HCV particles for in-depth mechanistic studies of envelope gene function. To this end, we first constructed a packaging cell line expressing core, p7, and NS2 based on the highly infectious Jc1 genotype (GT) 2a chimeric genome. We show that this packaging cell line can be transfected with HCV replicons encoding cognate Jc1-derived glycoprotein genes for production of single-round infectious particles by way of trans-complementation. Testing replicons expressing representative envelope protein genes from all major HCV genotypes, we observed that virus production occurred in a genotype- and isolate-dependent fashion. Importantly, primary GT 2 patient-derived glycoproteins were efficiently incorporated into infectious particles. Moreover, replacement of J6 (GT 2a) core, p7, and NS2 with GT 1a-derived H77 proteins allowed production of infectious HCV particles with GT 1 patient-derived glycoproteins. Notably, adaptive mutations known to enhance virus production from GT 1a-2a chimeric genomes further increased virus release. Finally, virus particles with primary patient-derived E1-E2 proteins possessed biophysical properties comparable to Jc1 HCVcc particles, used CD81 for cell entry, were associated with ApoE and could be neutralized by immune sera. Conclusion: This work describes cell culture systems for production of infectious HCV particles with primary envelope protein genes from GT 1 and GT 2-infected patients, thus opening up new opportunities to dissect envelope gene function in an individualized fashion.  

5.1504           Engineering a Light-Regulated GABAA Receptor for Optical Control of Neural Inhibition

Lin, W-C., Davenport, C.M., Mourot, A., Vytla, D., Smith, C.M., Medeiros, K.A., Chambers, J.J. and Kramer, R.H. ACS Chem. Biol., 9(7), 1414-1419 (2014)   Optogenetics has become an emerging technique for neuroscience investigations owing to the great spatiotemporal precision and the target selectivity it provides. Here we extend the optogenetic strategy to GABA_A receptors (GABA_ARs), the major mediators of inhibitory neurotransmission in the brain. We generated a light-regulated GABA_A receptor (LiGABAR) by conjugating a photoswitchable tethered ligand (PTL) onto a mutant receptor containing the cysteine-substituted α1-subunit. The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500 and 380 nm light, respectively, resulting in photoswitchable receptor antagonism. In hippocampal neurons, this LiGABAR enabled a robust photoregulation of inhibitory postsynaptic currents. Moreover, it allowed reversible photocontrol over neuron excitation in response to presynaptic stimulation. LiGABAR thus provides a powerful means for functional and mechanistic investigations of GABA_AR-mediated neural inhibition.  

5.1505           Rewiring Host Lipid Metabolism by Large Viruses Determines the Fate of Emiliania huxleyi, a Bloom-Forming Alga in the Ocean

Rosenwasser, S., Mausz, M.A., Schatz, D., Sheyn, U., Malitsky, S., Aharoni, A., Weinstock, E., Tzfadia, O., Ben-Dor, S., Feldmesser, E., Pohnert, G. and Vardi, A. Plant Cell, 26(6), 2689-2707 (2014)   Marine viruses are major ecological and evolutionary drivers of microbial food webs regulating the fate of carbon in the ocean. We combined transcriptomic and metabolomic analyses to explore the cellular pathways mediating the interaction between the bloom-forming coccolithophore Emiliania huxleyi and its specific coccolithoviruses (E. huxleyi virus [EhV]). We show that EhV induces profound transcriptome remodeling targeted toward fatty acid synthesis to support viral assembly. A metabolic shift toward production of viral-derived sphingolipids was detected during infection and coincided with downregulation of host de novo sphingolipid genes and induction of the viral-encoded homologous pathway. The depletion of host-specific sterols during lytic infection and their detection in purified virions revealed their novel role in viral life cycle. We identify an essential function of the mevalonate-isoprenoid branch of sterol biosynthesis during infection and propose its downregulation as an antiviral mechanism. We demonstrate how viral replication depends on the hijacking of host lipid metabolism during the chemical “arms race” in the ocean.  

5.1506           Efficient transduction and optogenetic stimulation of retinal bipolar cells by a synthetic adeno-associated virus capsid and promoter

Cronin, T., Vandenberghe, L.H., Hantz, P., Juttner, J., Reimann, A., Kacso, A-E., Huckfeldt, R.M., Busskamp, V., Kohler, H., Lagali, P.S., Roska, B. and Bennett, J. EMBO Mol. Med., 6(9), 1175-1190 (2014)   In this report, we describe the development of a modified adeno‐associated virus (AAV) capsid and promoter for transduction of retinal ON‐bipolar cells. The bipolar cells, which are post‐synaptic to the photoreceptors, are important retinal targets for both basic and preclinical research. In particular, a therapeutic strategy under investigation for advanced forms of blindness involves using optogenetic molecules to render ON‐bipolar cells light‐sensitive. Currently, delivery of adequate levels of gene expression is a limiting step for this approach. The synthetic AAV capsid and promoter described here achieves high level of optogenetic transgene expression in ON‐bipolar cells. This evokes high‐frequency (~100 Hz) spiking responses in ganglion cells of previously blind, rd1, mice. Our vector is a promising vehicle for further development toward potential clinical use.  

5.1507           Isolation of Protein-Associated Circular DNA from Healthy Cattle Serum

Funk, M., Gunst, K., Lucansky, V., Müller, H., zur Hausen, H. and de Villiers, E-M. Genome Announc., 2(4), e00846 (2014)   Three replication-competent single-stranded DNA molecules sharing nucleotide similarity to transmissible spongiform encephalopathy (TSE)-associated isolate Sphinx 2.36 were isolated from healthy bovine serum.  

5.1508           Transition from an M1 to a mixed neuroinflammatory phenotype increases amyloid deposition in APP/PS1 transgenic mice

Weekman, E.M., Sudduth, T.L., Abner, E.L., Popa, G.J., Mendenhall, M.D., Brothers, H.M., Braun, K., Greenstein, A. and Wilcock, D.M.

1. Neuroinflammation, 11:127 (2014)

  Background The polarization to different neuroinflammatory phenotypes has been described in early Alzheimer’s disease, yet the impact of these phenotypes on amyloid-beta (Aβ) pathology remains unknown. Short-term studies show that induction of an M1 neuroinflammatory phenotype reduces Aβ, but long-term studies have not been performed that track the neuroinflammatory phenotype.   Methods Wild-type and APP/PS1 transgenic mice aged 3 to 4 months received a bilateral intracranial injection of adeno-associated viral (AAV) vectors expressing IFNγ or green fluorescent protein in the frontal cortex and hippocampus. Mice were sacrificed 4 or 6 months post-injection. ELISA measurements were used for IFNγ protein levels and biochemical levels of Aβ. The neuroinflammatory phenotype was determined through quantitative PCR. Microglia, astrocytes, and Aβ levels were assessed with immunohistochemistry.   Results AAV expressing IFNγ induced an M1 neuroinflammatory phenotype at 4 months and a mixed phenotype along with an increase in Aβ at 6 months. Microglial staining was increased at 6 months and astrocyte staining was decreased at 4 and 6 months in mice receiving AAV expressing IFNγ.   Conclusions Expression of IFNγ through AAV successfully induced an M1 phenotype at 4 months that transitioned to a mixed phenotype by 6 months. This transition also appeared with an increase in amyloid burden suggesting that a mixed phenotype, or enhanced expression of M2a and M2c markers, could contribute to increasing amyloid burden and disease progression.  

5.1509           Complementation for an essential ancillary non-structural protein function across parvovirus genera

Mihaylov, I.S., Cotmore, S.F. and Tattersall, P. Virology, 468-470 , 226-237 (2014)   Parvoviruses encode a small number of ancillary proteins that differ substantially between genera. Within the genus Protoparvovirus, minute virus of mice (MVM) encodes three isoforms of its ancillary protein NS2, while human bocavirus 1 (HBoV1), in the genus Bocaparvovirus, encodes an NP1 protein that is unrelated in primary sequence to MVM NS2. To search for functional overlap between NS2 and NP1, we generated murine A9 cell populations that inducibly express HBoV1 NP1. These were used to test whether NP1 expression could complement specific defects resulting from depletion of MVM NS2 isoforms. NP1 induction had little impact on cell viability or cell cycle progression in uninfected cells, and was unable to complement late defects in MVM virion production associated with low NS2 levels. However, NP1 did relocate to MVM replication centers, and supports both the normal expansion of these foci and overcomes the early paralysis of DNA replication in NS2-null infections.    

5.1510           Adeno-Associated Virus Type 2 Wild-Type and Vector-Mediated Genomic Integration Profiles of Human Diploid Fibroblasts Analyzed by Third-Generation PacBio DNA Sequencing

Hüser, D., Gogol-Döring, A., Chen, W. and Heilborn, R.

1. Virol., 88(19), 11253-11263 (2014)

  Genome-wide analysis of adeno-associated virus (AAV) type 2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic sites, including AAVS1 on chromosome 19q13.42. Multiple GAGY/C repeats, resembling consensus AAV Rep-binding sites are preferred, whereas rep-deficient AAV vectors (rAAV) regularly show a random integration profile. This study is the first study to analyze wild-type AAV integration in diploid human fibroblasts. Applying high-throughput third-generation PacBio-based DNA sequencing, integration profiles of wild-type AAV and rAAV are compared side by side. Bioinformatic analysis reveals that both wild-type AAV and rAAV prefer open chromatin regions. Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hot spots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hot spots that correlates with variant hot spot preferences. DNase-Seq patterns of these sites in human tissues, including liver, muscle, heart, brain, skin, and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats.  

5.1511           Auto-associative heparin nanoassemblies: A biomimetic platform against the heparan sulfate-dependent viruses HSV-1, HSV-2, HPV-16 and RSV

Lembo, D., Donalisio, M., Laine, C., cagno, V., Civra, A., Bianchini, E.P., Zeghbib, N. and Bouchemal, K. Eur. J. Pharmaceut. and Biopharmaceut., 88, 275-282 (2014)   A new, simple and green method was developed for the manufacturing of heparin nanoassemblies active against the heparan sulfate-dependent viruses HSV-1, HSV-2, HPV-16 and RSV. These nanoassemblies were obtained by the auto-association of O-palmitoyl-heparin and α-cyclodextrin in water. The synthesized O-palmitoyl-heparin derivatives mixed with α-cyclodextrin resulted in the formation of crystalline hexagonal nanoassemblies as observed by transmission electron microscopy. The nanoassembly mean hydrodynamic diameters were modulated from 340 to 659 nm depending on the type and the initial concentration of O-palmitoyl-heparin or α-cyclodextrin. The antiviral activity of the nanoassemblies was not affected by the concentration of the components. However, the method of the synthesis of O-palmitoyl-heparin affected the antiviral activity of the formulations. We showed that reduced antiviral activity is correlated with lower sulfation degree and anticoagulant activity.  

5.1512           Distinct transduction profiles in the CNS via three injection routes of AAV9 and the application to generation of a neurodegenerative mouse model

Huda, F., Konno, A., matsuzaki, Y., Goenawan, H., Miyake, K., Shimada, T. and Hirai, H. Molecular Therapy – Methods & Clinical Development, 1:14032 (2014)   Using single-stranded adeno-associated virus serotype 9 (ssAAV9) vectors containing the neuron-specific synapsin-I promoter, we examined whether different administration routes (direct cerebellar cortical (DC), intrathecal (IT) and intravenous (IV) injections) could elicit specific transduction profiles in the CNS. The DC injection route robustly and exclusively transduced the whole cerebellum, whereas the IT injection route primarily transduced the cerebellar lobules 9 and 10 close to the injection site and the spinal cord. An IV injection in neonatal mice weakly and homogenously transduced broad CNS areas. In the cerebellar cortex, the DC and IT injection routes transduced all neuron types, whereas the IV injection route primarily transduced Purkinje cells. To verify the usefulness of this method, we generated a mouse model of spinocerebellar ataxia type 1 (SCA1). Mice that received a DC injection of the ssAAV9 vector expressing mutant ATXN1, a protein responsible for SCA1, showed the intranuclear aggregation of mutant ATXN1 in Purkinje cells, significant atrophy of the Purkinje cell dendrites and progressive motor deficits, which are characteristics of SCA1. Thus, ssAAV9-mediated transduction areas, levels, and cell types change depending on the route of injection. Moreover, this approach can be used for the generation of different mouse models of CNS/neurodegenerative diseases.      

5.1513           A simplified purification protocol for recombinant adeno-associated virus vectors

Potter, M., Lins, B., Mietzsch, M., Heilbronn, R., Van Vliet, K., Chipman, P., Agbandje-McKenna, M., Cleaver, B.D., Clement, N., Byrne, B.J. and Zolotukhin, S. Molecular Therapy – Methods & Clincal Development, 1:14034 (2014)   We describe a new rapid, low cost, and scalable method for purification of various recombinant adeno-associated viruses (rAAVs) from the lysates of producer cells of either mammalian or insect origin. The method takes advantage of two general biochemical properties of all characterized AAV serotypes: (i) low isoelectric point of a capsid and (ii) relative biological stability of the viral particle in the acidic environment. A simple and rapid clarification of cell lysate toremove the bulk of proteins and DNA is accomplished by utilizing inexpensive off-the-shelf reagents such as sodium citrate and citric acid. After the low-speed centrifugation step, the supernatant is subjected to cation exchange chromatography via sulfopropyl (SP) column. The eluted virus may then be further concentrated by either centrifugal spin devices or tangential flow filtration yielding material of high titer and Good Manufacturing Practice (GMP) grade biochemical purity. The protocol is validated for rAAV serotypes 2, 8, and 9. The described method makes rAAV vector technology readily available for the low budget research laboratories and could be easily adapted for a large scale GMP production format.  

5.1514           Counteracting Effects of Cellular Notch and Epstein-Barr Virus EBNA2: Implications for Stromal Effects on Virus-Host Interactions

Rowe, M., Raithatha, S. and Shannon-Lowe, C.

1. Virol., 88(20), 12065-12076 (2014)

  A number of diverse environmental cues have been linked to B lymphocyte differentiation and activation. One such cue, Notch-2, may be particularly relevant to the biology of infection with Epstein-Barr virus (EBV), which colonizes the B cell compartment. Activated Notch and EBV nuclear antigen 2 (EBNA2) both function as transcriptional activators by virtue of their interactions with the transcription factor RBP-Jκ. Although EBNA2 and activated Notch appear to have partially overlapping functions, we now report that activated Notch counteracts a crucial EBNA2 function both in newly infected primary B cells and in lymphoblastoid cell lines (LCLs). EBNA2 is directly responsible for the initiation of transcription of the majority of EBV proteins associated with type III latency, leading to the outgrowth of LCLs. One of the key proteins driving this outgrowth is latent membrane protein 1 (LMP1), which is regulated by an EBNA2-responsive element within its ED-L1 promoter. Activation of Notch-2 via Delta-like ligand 1 inhibits EBNA2-mediated initiation of LMP1 transcription. Furthermore, ligated Notch-2 also efficiently turns off LMP1 expression from the ED-L1 promoter in LCLs already expressing LMP1. Modulation of EBV gene expression by Notch was not confined to EBNA2-dependent events. Activated Notch-2 also inhibited EBV entry into the lytic cycle in a B cell non-Hodgkin's lymphoma line by upregulating the cellular transcription factor Zeb2, which represses the transcription of BZLF1. These results support the concept that in vivo, cumulative signals from the microenvironment downregulate EBV gene expression in B cells to the latency 0 gene expression profile observed in B cells entering the peripheral blood.  

5.1515           Insulin-like growth factor 2 reverses memory and synaptic deficits in APP transgenic mice

Pascual-Lucas, M., da Silva, S.V., Di Scala, M., Garcia-Barroso, C., Gonzalez-Aseguinolaza, G., Mulle, C., Alberini, C.M., Cuadrado-Tejedor, M. and Garcia-Osta, A. EMBO Mol. Med., 6(10), 1246-1262 (2014)   Insulin‐like growth factor 2 (IGF2) was recently found to play a critical role in memory consolidation in rats and mice, and hippocampal or systemic administration of recombinant IGF2 enhances memory. Here, using a gene therapy‐based approach with adeno‐associated virus (AAV), we show that IGF2 overexpression in the hippocampus of aged wild‐type mice enhances memory and promotes dendritic spine formation. Furthermore, we report that IGF2 expression decreases in the hippocampus of patients with Alzheimer's disease, and this leads us to hypothesize that increased IGF2 levels may be beneficial for treating the disease. Thus, we used the AAV system to deliver IGF2 or IGF1 into the hippocampus of the APP mouse model Tg2576 and demonstrate that IGF2 and insulin‐like growth factor 1 (IGF1) rescue behavioural deficits, promote dendritic spine formation and restore normal hippocampal excitatory synaptic transmission. The brains of Tg2576 mice that overexpress IGF2 but not IGF1 also show a significant reduction in amyloid levels. This reduction probably occurs through an interaction with the IGF2 receptor (IGF2R). Hence, IGF2 and, to a lesser extent, IGF1 may be effective treatments for Alzheimer's disease.  

5.1516           Novel in vitro models for assembly of VLDL and low-density hepatitis C virus particles

Andreo, U., Scull, M.A., De Jong, Y.P., Ramanan, V., Flatley, B., Schwartz, R.E., Ng, S., Chen, A.A. and   Fisher, E.A. Hepatology, 60, Duppl. 1, 1050A, abstract 1770 (20014)   Hepatitis C virus (HCV) is the leading cause of chronic liver diseases and the most common indication for liver transplantation in the US. HCV derived from infected patients is characterized by a lower buoyant density and higher specific infectivity than in vitro-derived virus likely due HCV association with very low-density lipoproteins (VLDL) in vivo. This interaction is thought to occur during virus assembly and might impact virus susceptibility to neutralization and receptor usage. To enable better characterization of highly infectious HCV particles, we sought to identify a human in vitro model that was competent for VLDL secretion and very low density HCV production. To establish an in vivo reference, we first analyzed the buoyant density of infectious HCV particles derived from immunodeficient human liver chimeric (Fah-/-) mice infected with J6/JFH1. Similar to the uPA human chimeric mouse model, but unlike HCV produced by Huh-7 derived cell lines, we show that higher infectivity is recovered from the lower density fractions following ultracentrifugation on an isopicnic iodixanol gradient. We hypothesized that culture systems with emergent cellular properties (eg. polarization or differentiation status) will give rise to more “in vivo” like lipoproteins and viral particles. Therefore, we used 3D liver ‘organoids’ consisting of Huh-7.5 human hepatoma cells in coculture with fibroblast stromal cells, encapsulated in a fully defined polyethylene glycol (PEG)-based matrix as well as hepatocyte-like cells (iHLC) derived from induced pluripotent stem cells and showed that both secrete more apoB-containing lipoproteins of VLDL density than Huh-7.5 cells cultured in 2D format. Also, these two in vitro models are susceptible to HCV infection and the buoyant density distribution of the particles, is comparable to in vivo HCV particles produced in human liver chimeric mice. Using a commercial quantitative PCR array (RT2 Profiler™ PCR Array, Qiagen), we identified several genes that are differentially regulated in 3D-organoids as compared to 2D cultures. Of particular interest, apoCIII is upregulated in both 3D-organoids and iHLCs compared to Huh-7.5 cells cultured as in 2D. ApoCIII is a VLDL-associated apolipoprotein, well known as a lipoprotein lipase inhibitor and recently implicated in VLDL biogenesis in rat and mouse models. 3D-organoids and iHLCs now offer a human in vitro platform to investigate the role of apoCIII in VLDL biogenesis and HCV production and provide a source of very low-density HCV particles for functional studies.  

5.1517           Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice

Wein, N: et al Nature Med., 20(9), 992-1000 (2014)   Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject–derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5′ exons of DMD.  

5.1518           CAPON-nNOS coupling can serve as a target for developing new anxiolytics

Zhu, L-J. et al Nature Med., 20(9), 1050-1054 (2014)   Anxiety disorders are highly prevalent psychiatric diseases^{1, 2}. There is need for a deeper understanding of anxiety control mechanisms in the mammalian brain and for development of new anxiolytic agents. Here we report that the coupling between neuronal nitric oxide synthase (nNOS) and its carboxy-terminal PDZ ligand (CAPON) can serve as a target for developing new anxiolytic agents. Augmenting nNOS-CAPON interaction in the hippocampus of mice by overexpressing full-length CAPON gave rise to anxiogenic-like behaviors, whereas dissociating CAPON from nNOS by overexpressing CAPON-125C or CAPON-20C (the C-terminal 125 or 20 amino acids of CAPON) or delivering Tat-CAPON-12C (a peptide comprising Tat and the 12 C-terminal amino acids of CAPON) in the hippocampus of mice produced anxiolytic-like effects. Mice subjected to chronic mild stress (CMS) displayed a substantial increase in nNOS-CAPON coupling in the hippocampus and a consequent anxiogenic-like phenotype. Disrupting nNOS-CAPON coupling reversed the CMS-induced anxiogenic-like behaviors. Moreover, small-molecule blockers of nNOS-CAPON binding rapidly produced anxiolytic-like effects. Dexamethasone-induced ras protein 1 (Dexras1)–extracellular signal–regulated kinase (ERK) signaling was involved in the behavioral effects of nNOS-CAPON association. Thus, nNOS-CAPON association contributes to the modulation of anxiety-related behaviors via regulating Dexras1-ERK signaling and can serve as a target for developing potential anxiolytics.  

5.1519           The N-Terminal Domain of NLRC5 Confers Transcriptional Activity for MHC Class I and II Gene Expression

Neerinex, A., Jakobshagen, K., Utermöhlen, O., Büning, H., Steimle, V. and Kufer, T.A.

1. Immunol., 193(6), 3090-3100 (2014)

  Ag presentation to CD4⁺ and CD8⁺ T cells depends on MHC class II and MHC class I molecules, respectively. One important regulatory factor of this process is the transcriptional regulation of MHC gene expression. It is well established that MHC class II transcription relies on the NLR protein CIITA. Recently, another NLR protein, NLRC5, was shown to drive MHC class I expression. The molecular mechanisms of the function of NLRC5 however remain largely elusive. In this study, we present a detailed functional study of the domains of NLRC5 revealing that the N-terminal domain of human NLRC5 has intrinsic transcriptional activity. Domain swapping experiments between NLRC5 and CIITA showed that this domain contributes to MHC class I and MHC class II gene expression with a bias for activation of MHC class I promoters. Delivery of this construct by adeno-associated viral vectors upregulated MHC class I and MHC class II expression in human cells and enhanced lysis of melanoma cells by CD8⁺ cytotoxic T cells in vitro. Taken together, this work provides novel insight into the function of NLRC5 and CIITA in MHC gene regulation.  

5.1520           Intracellular sensing of complement C3 activates cell autonomous immunity

Tam, J.C.H. et al Science, 345:6201, 1134 (2014)   Intracellular pathogens, which include viruses and some bacteria, typically disseminate through extracellular fluids before entering their target cells and beginning replication. While in the extracellular environment, pathogens can be intercepted by humoral immunity or by professional immune cells. However, immune surveillance is not always sufficient to prevent infection, and all cells need innate mechanisms to detect and disable pathogens.  

5.1521           Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing

Yang, B., Treweek, J.B., Kulkarni, R.P., Deverman, B.E., Chen, C-K., Lubeck, E., Shah, S., Cai, L. and Gradinaru, V. Cell, 158(4), 945-958 (2014)   Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies.  

5.1522           Immune Responses in Macaques to a Prototype Recombinant Adenovirus Live Oral Human Papillomavirus 16 Vaccine

Berg, M.G., Adams, R.J., Gammbhira, R., Siracusa, M.C., Scott, A.L., Roden, R.B.S. and ketner, G. Clin. Vaccine Imunol., 21(9), 1224-1231 (2014)   Immunization with human papillomavirus (HPV) L1 virus-like particles (VLPs) prevents infection with HPV. However, the expense and logistical demands of current VLP vaccines will limit their widespread use in resource-limited settings, where most HPV-induced cervical cancer occurs. Live oral adenovirus vaccines have properties that are well-suited for use in such settings. We have described a live recombinant adenovirus vaccine prototype that produces abundant HPV16 L1 protein from the adenovirus major late transcriptional unit and directs the assembly of HPV16 VLPs in tissue culture. Recombinant-derived VLPs potently elicit neutralizing antibodies in mice. Here, we characterize the immune response to the recombinant after dual oral and intranasal immunization of pigtail macaques, in which the virus replicates as it would in immunized humans. The immunization of macaques induced vigorous humoral responses to adenovirus capsid and nonstructural proteins, although, surprisingly, not against HPV L1. In contrast, immunization elicited strong T-cell responses to HPV VLPs as well as adenovirus virions. T-cell responses arose immediately after the primary immunization and were boosted by a second immunization with recombinant virus. T-cell immunity contributes to protection against a wide variety of pathogens, including many viruses. The induction of a strong cellular response by the recombinant indicates that live adenovirus recombinants have potential as vaccines for those agents. These studies encourage and will inform the continued development of viable recombinant adenovirus vaccines.  

5.1523           Rapid and highly efficient inducible cardiac gene knockout in adult mice using AAV-mediated expression of Cre recombinase

Werfel, S., Jungmann, A., Lehmann, L., Ksienzyk, J., Bekeredjian, R., Kaya, Z., Leuchs, B., Nordheim, A., Backs, J., Engelhardt, S., Katus, H.A. and Müller, O.J. Cardiovasc. Res., 104, 15-23 (2014)   Aims Inducible gene targeting in mice using the Cre/LoxP system has become a valuable tool to analyse the roles of specific genes in the adult heart. However, the commonly used Myh6-MerCreMer system requires time-consuming breeding schedules and is potentially associated with cardiac side effects, which may result in transient cardiac dysfunction. The aim of our study was to establish a rapid and simple system for cardiac gene inactivation in conditional knockout mice by gene transfer of a Cre recombinase gene using adeno-associated viral vectors of serotype 9 (AAV9). Methods and results AAV9 vectors expressing Cre under the control of a human cardiac troponin T promoter (AAV-TnT-Cre) enabled a highly efficient Cre/LoxP switching in cardiomyocytes 2 weeks after injection into 5- to 6-week-old ROSA26-LacZ reporter mice. Recombination efficiency was at least as high as observed with the Myh6-MerCreMer system. No adverse side effects were detected upon application of AAV-TnT-Cre. As proof of principle, we studied AAV-TnT-Cre in a conditional knockout model (Srf-flex1 mice) to deplete the myocardium of the transcription factor serum response factor (SRF). Four weeks after AAV-TnT-Cre injection, a strong decrease in the cardiac expression of SRF mRNA and protein was observed. Furthermore, mice developed a severe cardiac dysfunction with increased interstitial fibrosis in accordance with the central role of SRF for the expression of contractile and calcium trafficking proteins in the heart. Conclusions AAV9-mediated expression of Cre is a promising approach for rapid and efficient conditional cardiac gene knockout in adult mice.  

5.1524           Apolipoprotein E Likely Contributes to a Maturation Step of Infectious Hepatitis C Virus Particles and Interacts with Viral Envelope Glycoproteins

Lee, J-Y., Acosta, E.G., Stoeck, I.K., Long, G., Hiet, M-S., Mueller, B., fackler, O.T., kallis, S. and Bartenschlager, O.T.

1. Virol., 88(21), 12422-12437 (2014)

  The assembly of infectious hepatitis C virus (HCV) particles is tightly linked to components of the very-low-density lipoprotein (VLDL) pathway. We and others have shown that apolipoprotein E (ApoE) plays a major role in production of infectious HCV particles. However, the mechanism by which ApoE contributes to virion assembly/release and how it gets associated with the HCV particle is poorly understood. We found that knockdown of ApoE reduces titers of infectious intra- and extracellular HCV but not of the related dengue virus. ApoE depletion also reduced amounts of extracellular HCV core protein without affecting intracellular core amounts. Moreover, we found that ApoE depletion affected neither formation of nucleocapsids nor their envelopment, suggesting that ApoE acts at a late step of assembly, such as particle maturation and infectivity. Importantly, we demonstrate that ApoE interacts with the HCV envelope glycoproteins, most notably E2. This interaction did not require any other viral proteins and depended on the transmembrane domain of E2 that also was required for recruitment of HCV envelope glycoproteins to detergent-resistant membrane fractions. These results suggest that ApoE plays an important role in HCV particle maturation, presumably by direct interaction with viral envelope glycoproteins.  

5.1525           Intra-arterial delivery of AAV vectors to the mouse brain after mannitol mediated blood brain barrier disruption

Foley, C.P., Rubin, D.G., Santillan, A., Sondhi, D., Dyke, J.P., Gobin, Y.P., Crystal, R.G. and ballon, D.J. Journal of Controlled Release, 196, 71-78 (2014)   The delivery of therapeutics to neural tissue is greatly hindered by the blood brain barrier (BBB). Direct local delivery via diffusive release from degradable implants or direct intra-cerebral injection can bypass the BBB and obtain high concentrations of the therapeutic in the targeted tissue, however the total volume of tissue that can be treated using these techniques is limited. One treatment modality that can potentially access large volumes of neural tissue in a single treatment is intra-arterial (IA) injection after osmotic blood brain barrier disruption. In this technique, the therapeutic of interest is injected directly into the arteries that feed the target tissue after the blood brain barrier has been disrupted by exposure to a hyperosmolar mannitol solution, permitting the transluminal transport of the therapy. In this work we used contrast enhanced magnetic resonance imaging (MRI) studies of IA injections in mice to establish parameters that allow for extensive and reproducible BBB disruption. We found that the volume but not the flow rate of the mannitol injection has a significant effect on the degree of disruption. To determine whether the degree of disruption that we observed with this method was sufficient for delivery of nanoscale therapeutics, we performed IA injections of an adeno-associated viral vector containing the CLN2 gene (AAVrh.10CLN2), which is mutated in the lysosomal storage disorder Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL). We demonstrated that IA injection of AAVrh.10CLN2 after BBB disruption can achieve widespread transgene production in the mouse brain after a single administration. Further, we showed that there exists a minimum threshold of BBB disruption necessary to permit the AAV.rh10 vector to pass into the brain parenchyma from the vascular system. These results suggest that IA administration may be used to obtain widespread delivery of nanoscale therapeutics throughout the murine brain after a single administration.  

5.1526           A Beneficiary Role for Neuraminidase in Influenza Virus Penetration through the Respiratory Mucus

Yang, X., Steukers, L., Forier, K., Xiong, R., Braeckmans, K., Van Reeth, K. and Nauwynck, H. PloS One, 9(10), e110026 (2014)   Swine influenza virus (SIV) has a strong tropism for pig respiratory mucosa, which consists of a mucus layer, epithelium, basement membrane and lamina propria. Sialic acids present on the epithelial surface have long been considered to be determinants of influenza virus tropism. However, mucus which is also rich in sialic acids may serve as the first barrier of selection. It was investigated how influenza virus interacts with the mucus to infect epithelial cells. Two techniques were applied to track SIV H1N1 in porcine mucus. The microscopic diffusion of SIV particles in the mucus was analyzed by single particle tracking (SPT), and the macroscopic penetration of SIV through mucus was studied by a virus in-capsule-mucus penetration system, followed by visualizing the translocation of the virions with time by immunofluorescence staining. Furthermore, the effects of neuraminidase on SIV getting through or binding to the mucus were studied by using zanamivir, a neuraminidase inhibitor (NAI), and Arthrobacter ureafaciens neuraminidase. The distribution of the diffusion coefficient shows that 70% of SIV particles were entrapped, while the rest diffused freely in the mucus. Additionally, SIV penetrated the porcine mucus with time, reaching a depth of 65 µm at 30 min post virus addition, 2 fold of that at 2 min. Both the microscopic diffusion and macroscopic penetration were largely diminished by NAI, while were clearly increased by the effect of exogenous neuraminidase. Moreover, the exogenous neuraminidase sufficiently prevented the binding of SIV to mucus which was reversely enhanced by effect of NAI. These findings clearly show that the neuraminidase helps SIV move through the mucus, which is important for the virus to reach and infect epithelial cells and eventually become shed into the lumen of the respiratory tract.  

5.1527           Human Coronavirus NL63 Utilizes Heparan Sulfate Proteoglycans for Attachment to Target Cells

Milewska, A., Zarebski, M., Nowak, P., Stozek, K., Potempa, J. and Pyrc. K.

1. Virol., 88(22), 13221-13230 (2014)

  Human coronavirus NL63 (HCoV-NL63) is an alphacoronavirus that was first identified in 2004 in the nasopharyngeal aspirate from a 7-month-old patient with a respiratory tract infection. Previous studies showed that HCoV-NL63 and the genetically distant severe acute respiratory syndrome (SARS)-CoV employ the same receptor for host cell entry, angiotensin-converting enzyme 2 (ACE2), but it is largely unclear whether ACE2 interactions are sufficient to allow HCoV-NL63 binding to cells. The present study showed that directed expression of angiotensin-converting enzyme 2 (ACE2) on cells previously resistant to HCoV-NL63 renders them susceptible, showing that ACE2 protein acts as a functional receptor and that its expression is required for infection. However, comparative analysis showed that directed expression or selective scission of the ACE2 protein had no measurable effect on virus adhesion. In contrast, binding of HCoV-NL63 to heparan sulfates was required for viral attachment and infection of target cells, showing that these molecules serve as attachment receptors for HCoV-NL63.  

5.1528           Genome of brown tide virus (AaV), the little giant of the Megaviridae, elucidates NCLDV genome expansion and host–virus coevolution

Moniruzzaman, M., LeCleir, G.R., Brown, C.M., Gobler, C.J., Bidle, K.D., Wilson, W.H. and Wilhelm, S.W.

Virology, 466-467, 60-70 (2014)

Aureococcus anophagefferens causes economically and ecologically destructive “brown tides” in the United States, China and South Africa. Here we report the 370,920 bp genomic sequence of AaV, a virus capable of infecting and lysing A. anophagefferens. AaV is a member of the nucleocytoplasmic large DNA virus (NCLDV) group, harboring 377 putative coding sequences and 8 tRNAs. Despite being an algal virus, AaV shows no phylogenetic affinity to the Phycodnaviridae family, to which most algae-infecting viruses belong. Core gene phylogenies, shared gene content and genome-wide similarities suggest AaV is the smallest member of the emerging clade “Megaviridae”. The genomic architecture of AaV demonstrates that the ancestral virus had an even smaller genome, which expanded through gene duplication and assimilation of genes from diverse sources including the host itself – some of which probably modulate important host processes. AaV also harbors a number of genes exclusive to phycodnaviruses – reinforcing the hypothesis that Phycodna- and Mimiviridae share a common ancestor.

 

5.1529           Characteristics of Memory B Cells Elicited by a Highly Efficacious HPV Vaccine in Subjects with No Pre-existing Immunity

Scherer, E.M., Smith, R.A., Simonich, C.A., Niyonzima, N., Carter, J.J. and Galloway, D.A. PloS Pathogens, 10(10), e1004461 (2014)   Licensed human papillomavirus (HPV) vaccines provide near complete protection against the types of HPV that most commonly cause anogenital and oropharyngeal cancers (HPV 16 and 18) when administered to individuals naive to these types. These vaccines, like most other prophylactic vaccines, appear to protect by generating antibodies. However, almost nothing is known about the immunological memory that forms following HPV vaccination, which is required for long-term immunity. Here, we have identified and isolated HPV 16-specific memory B cells from female adolescents and young women who received the quadrivalent HPV vaccine in the absence of pre-existing immunity, using fluorescently conjugated HPV 16 pseudoviruses to label antigen receptors on the surface of memory B cells. Antibodies cloned and expressed from these singly sorted HPV 16-pseudovirus labeled memory B cells were predominantly IgG (>IgA>IgM), utilized diverse variable genes, and potently neutralized HPV 16 pseudoviruses in vitro despite possessing only average levels of somatic mutation. These findings suggest that the quadrivalent HPV vaccine provides an excellent model for studying the development of B cell memory; and, in the context of what is known about memory B cells elicited by influenza vaccination/infection, HIV-1 infection, or tetanus toxoid vaccination, indicates that extensive somatic hypermutation is not required to achieve potent vaccine-specific neutralizing antibody responses.  

5.1530           The cooperative function of arginine residues in the Prototype Foamy Virus Gag C-terminus mediates viral and cellular RNA encapsidation

Hamann, M.V., Müllers, E., Reh, J., Stanke, N., Effantin, G., Weissenhorn, W. and Lindemann, D. Retrovirology, 11:87 (2014)   Background One unique feature of the foamy virus (FV) capsid protein Gag is the absence of Cys-His motifs, which in orthoretroviruses are irreplaceable for multitude functions including viral RNA genome recognition and packaging. Instead, FV Gag contains glycine-arginine-rich (GR) sequences at its C-terminus. In case of prototype FV (PFV) these are historically grouped in three boxes, which have been shown to play essential functions in genome reverse transcription, virion infectivity and particle morphogenesis. Additional functions for RNA packaging and Pol encapsidation were suggested, but have not been conclusively addressed. Results Here we show that released wild type PFV particles, like orthoretroviruses, contain various cellular RNAs in addition to viral genome. Unlike orthoretroviruses, the content of selected cellular RNAs in capsids of PFV vector particles was not altered by viral genome encapsidation. Deletion of individual GR boxes had only minor negative effects (2 to 4-fold) on viral and cellular RNA encapsidation over a wide range of cellular Gag to viral genome ratios examined. Only the concurrent deletion of all three PFV Gag GR boxes, or the substitution of multiple arginine residues residing in the C-terminal GR box region by alanine, abolished both viral and cellular RNA encapsidation (>50 to >3,000-fold reduced), independent of the viral production system used. Consequently, those mutants also lacked detectable amounts of encapsidated Pol and were non-infectious. In contrast, particle release was reduced to a much lower extent (3 to 20-fold). Conclusions Taken together, our data provides the first identification of a full-length PFV Gag mutant devoid in genome packaging and the first report of cellular RNA encapsidation into PFV particles. Our results suggest that the cooperative action of C-terminal clustered positively charged residues, present in all FV Gag proteins, is the main viral protein determinant for viral and cellular RNA encapsidation. The viral genome independent efficiency of cellular RNA encapsidation suggests differential packaging mechanisms for both types of RNAs. Finally, this study indicates that analogous to orthoretroviruses, Gag – nucleic acid interactions are required for FV capsid assembly and efficient particle release.  

5.1531           Structure and immune recognition of trimeric pre-fusion HIV-1 Env

Pancera, M. et al Nature, 514, 455-461 (2014)   The human immunodeficiency virus type 1 (HIV-1) envelope (Env) spike, comprising three gp120 and three gp41 subunits, is a conformational machine that facilitates HIV-1 entry by rearranging from a mature unliganded state, through receptor-bound intermediates, to a post-fusion state. As the sole viral antigen on the HIV-1 virion surface, Env is both the target of neutralizing antibodies and a focus of vaccine efforts. Here we report the structure at 3.5 Å resolution for an HIV-1 Env trimer captured in a mature closed state by antibodies PGT122 and 35O22. This structure reveals the pre-fusion conformation of gp41, indicates rearrangements needed for fusion activation, and defines parameters of immune evasion and immune recognition. Pre-fusion gp41 encircles amino- and carboxy-terminal strands of gp120 with four helices that form a membrane-proximal collar, fastened by insertion of a fusion peptide-proximal methionine into a gp41-tryptophan clasp. Spike rearrangements required for entry involve opening the clasp and expelling the termini. N-linked glycosylation and sequence-variable regions cover the pre-fusion closed spike; we used chronic cohorts to map the prevalence and location of effective HIV-1-neutralizing responses, which were distinguished by their recognition of N-linked glycan and tolerance for epitope-sequence variation.  

5.1532           Conserved and host-specific features of influenza virion architecture

Hutchinson, E.C., Charles, P.D., Hester, S.S., Thomas, B., Trudgian, D., Martinez-Alonso, M. and Fodor, E. Nature Communications, 5:4816 (2014)   Viruses use virions to spread between hosts, and virion composition is therefore the primary determinant of viral transmissibility and immunogenicity. However, the virions of many viruses are complex and pleomorphic, making them difficult to analyse in detail. Here we address this by identifying and quantifying virion proteins with mass spectrometry, producing a complete and quantified model of the hundreds of host-encoded and viral proteins that make up the pleomorphic virions of influenza viruses. We show that a conserved influenza virion architecture is maintained across diverse combinations of virus and host. This ‘core’ architecture, which includes substantial quantities of host proteins as well as the viral protein ​NS1, is elaborated with abundant host-dependent features. As a result, influenza virions produced by mammalian and avian hosts have distinct protein compositions. Finally, we note that influenza virions share an underlying protein composition with exosomes, suggesting that influenza virions form by subverting microvesicle production.      

5.1533           Liver-directed gene therapy corrects cardiovascular lesions in feline mucopolysaccharidosis type I

Hinderer, C., Bell, P., Gurda, B.L., Wang, Q., Louboutin, J-P., Zhu, Y., Bagel, J., O’Donnell, P., Sikora, T., Ruane, T., Wang, P., Haskins, M.E. and Wilson, J.M. PNAS, 111(41), 14894-14899 (2014)   Patients with mucopolysaccharidosis type I (MPS I), a genetic deficiency of the lysosomal enzyme α-l-iduronidase (IDUA), exhibit accumulation of glycosaminoglycans in tissues, with resulting diverse clinical manifestations including neurological, ocular, skeletal, and cardiac disease. MPS I is currently treated with hematopoietic stem cell transplantation or weekly enzyme infusions, but these therapies have significant drawbacks for patient safety and quality of life and do not effectively address some of the most critical clinical sequelae, such as life-threatening cardiac valve involvement. Using the naturally occurring feline model of MPS I, we tested liver-directed gene therapy as a means of achieving long-term systemic IDUA reconstitution. We treated four MPS I cats at 3–5 mo of age with an adeno-associated virus serotype 8 vector expressing feline IDUA from a liver-specific promoter. We observed sustained serum enzyme activity for 6 mo at ∼30% of normal levels in one animal, and in excess of normal levels in three animals. Remarkably, treated animals not only demonstrated reductions in glycosaminoglycan storage in most tissues, but most also exhibited complete resolution of aortic valve lesions, an effect that has not been previously observed in this animal model or in MPS I patients treated with current therapies. These data point to clinically meaningful benefits of the robust enzyme expression achieved with hepatic gene transfer that extend beyond the economic and quality of life advantages over lifelong enzyme infusions.  

5.1534           Optogenetic activation of presynaptic inputs in lateral amygdala forms associative fear memory

Kwon, J-T., nakajima, R., Kim, H-S. et al Learn. Mem., 21, 627-633 (2014)   In Pavlovian fear conditioning, the lateral amygdala (LA) has been highlighted as a key brain site for association between sensory cues and aversive stimuli. However, learning-related changes are also found in upstream sensory regions such as thalamus and cortex. To isolate the essential neural circuit components for fear memory association, we tested whether direct activation of presynaptic sensory inputs in LA, without the participation of upstream activity, is sufficient to form fear memory in mice. Photostimulation of axonal projections from the two main auditory brain regions, the medial geniculate nucleus of the thalamus and the secondary auditory cortex, was paired with aversive footshock. Twenty-four hours later the same photostimulation induced robust conditioned freezing and this fear memory formation was disrupted when glutamatergic synaptic transmission was locally blocked in the LA. Therefore, our results prove for the first time that synapses between sensory input areas and the LA, previously implicated as a crucial brain site for fear memory formation, actually are sufficient to serve as a conditioned stimulus. Our results strongly support the idea that the LA may be sufficient to encode and store associations between neutral cue and aversive stimuli during natural fear conditioning as a critical part of a broad fear memory engram.  

5.1535           Efficiency of Protease-Activatable Virus Nanonodes Tuned Through Incorporation of Wild-Type Capsid Subunits

Ho, M.L., Judd, J., Kuypers, B.E., yamagami, M., Wong, F.F. and Suh, J. Cell. Mol. Bioeng., 7(3), 334-343 (2014)   Virus nanonodes, a tunable multi-input protease-responsive gene delivery platform, was recently built by exploiting the self-assembly property of adeno-associated virus capsids. Upon detection of specific inputs (e.g., matrix metalloproteinases—MMPs), the engineered viruses output gene delivery to targeted cells. The first generation protease-activatable viruses (PAVs) displayed the desired protease-activated cellular receptor binding and transduction behaviors. However, the less than wild type (WT) level of gene delivery achieved by the prototype viruses has left room for improvement. In this report, we have devised a method to tackle this efficiency problem. Specifically, by controlling the ratio of WT to protease-activatable subunits in the assembled 60-mer virus capsid, we can easily increase the level of overall transduction achieved by the PAVs. Since a number of MMPs are overexpressed in a vast range of human pathologies, including cancer and cardiovascular disease, the protease-sensing viruses may find broad clinical use in future gene therapy applications.  

5.1536           Successful anti-scavenger receptor class B type I (SR-BI) monoclonal antibody therapy in humanized mice after challenge with HCV variants with in vitro resistance to SR-BI-targeting agents

Vercauteren, K., Van Den Eede, N., Mesalam, A.A., Beloizard, S., Catanese, M.T., Bankwitz, D., Wong-Staal, F., Cortese, R., Dubuisson, J., Rice, C., Pietschmann, T., Leroux-Roels, G., Nicosia, A. and Meuleman, P. Hepatology, 60, 1508-1518 (2014)   Hepatitis C virus (HCV)-induced endstage liver disease is currently a major indication for liver transplantation. After transplantation the donor liver inevitably becomes infected with the circulating virus. Monoclonal antibodies (mAbs) against the HCV coreceptor scavenger receptor class B type I (SR-BI) inhibit HCV infection of different genotypes, both in cell culture and in humanized mice. Anti-SR-BI mAb therapy is successful even when initiated several days after HCV exposure, supporting its potential applicability to prevent HCV reinfection of liver allografts. However, HCV variants with reduced SR-BI dependency have been described in the literature, which could potentially limit the use of SR-BI targeting therapy. In this study we show, both in a preventative and postexposure setting, that humanized mice infected with HCV variants exhibiting increased in vitro resistance to SR-BI-targeting molecules remain responsive to anti-SR-BI mAb therapy in vivo. A 2-week antibody therapy readily cleared HCV RNA from the circulation of infected humanized mice. We found no evidence supporting increased SR-BI-receptor dependency of viral particles isolated from humanized mice compared to cell culture-produced virus. However, we observed that, unlike wild-type virus, the in vitro infectivity of the resistant variants was inhibited by both human high density lipoprotein (HDL) and very low density lipoprotein (VLDL). The combination of mAb1671 with these lipoproteins further increased the antiviral effect. Conclusion: HCV variants that are less dependent on SR-BI in vitro can still be efficiently blocked by an anti-SR-BI mAb in humanized mice. Since these variants are also more susceptible to neutralization by anti-HCV envelope antibodies, their chance of emerging during anti-SR-BI therapy is severely reduced. Our data indicate that anti-SR-BI receptor therapy could be an effective way to prevent HCV infection in a liver transplant setting.  

5.1537           Induced Maturation of Human Immunodeficiency Virus

Mattei, S., Anders, M., Konvalinka, J., Krässlich, H-G., Briggs, A.G. and Müller, B.

1. Virol., 88(23)

  HIV-1 assembles at the plasma membrane of virus-producing cells as an immature, noninfectious particle. Processing of the Gag and Gag-Pol polyproteins by the viral protease (PR) activates the viral enzymes and results in dramatic structural rearrangements within the virion—termed maturation—that are a prerequisite for infectivity. Despite its fundamental importance for viral replication, little is currently known about the regulation of proteolysis and about the dynamics and structural intermediates of maturation. This is due mainly to the fact that HIV-1 release and maturation occur asynchronously both at the level of individual cells and at the level of particle release from a single cell. Here, we report a method to synchronize HIV-1 proteolysis in vitro based on protease inhibitor (PI) washout from purified immature virions, thereby temporally uncoupling virus assembly and maturation. Drug washout resulted in the induction of proteolysis with cleavage efficiencies correlating with the off-rate of the respective PR-PI complex. Proteolysis of Gag was nearly complete and yielded the correct products with an optimal half-life (t_1/2) of ∼5 h, but viral infectivity was not recovered. Failure to gain infectivity following PI washout may be explained by the observed formation of aberrant viral capsids and/or by pronounced defects in processing of the reverse transcriptase (RT) heterodimer associated with a lack of RT activity. Based on our results, we hypothesize that both the polyprotein processing dynamics and the tight temporal coupling of immature particle assembly and PR activation are essential for correct polyprotein processing and morphological maturation and thus for HIV-1 infectivity.  

5.1538           Copackaging of Multiple Adeno-Associated Viral Vectors in a Single Production Step

Doerfler, P.A., Byrne, B.J. and Clement, N. Human Gene Therapy Methods, 25, 269-276 (2014)   Limiting factors in large preclinical and clinical studies utilizing adeno-associated virus (AAV) for gene therapy are focused on the restrictive packaging capacity, the overall yields, and the versatility of the production methods for single AAV vector production. Furthermore, applications where multiple vectors are needed to provide long expression cassettes, whether because of long cDNA sequences or the need of different regulatory elements, require that each vector be packaged and characterized separately, directly affecting labor and cost associated with such manufacturing strategies. To overcome these limitations, we propose a novel method of vector production that allows for the packaging of multiple expression cassettes in a single transfection step. Here we combined two expression cassettes in predetermined ratios before transfection and empirically demonstrate that the output vector recapitulates the predicted ratios. Titration by quantitative polymerase chain reaction of AAV vector genome copies using shared or unique genetic elements allowed for delineation of the individual vector contribution to the total preparation that showed the predicted differential packaging outcomes. By copackaging green fluorescent protein (GFP) and mCherry constructs, we demonstrate that both vector genome and infectious titers reiterated the ratios utilized to produce the constructs by transfection. Copackaged therapeutic constructs that only differ in transcriptional elements produced a heterogeneous vector population of both constructs in the predefined ratios. This study shows feasibility and reproducibility of a method that allows for two constructs, differing in either transgene or transcription elements, to be efficiently copackaged and characterized simultaneously, reducing cost of manufacturing and release testing.  

5.1539           AAV-mediated persistent bevacizumab therapy suppresses tumor growth of ovarian cancer

Xie, Y., Hicks, M.J., Kaminsky, S.M., Moore, M.A.S., Crystal, R.G. and Rafii, A. Gynecologic Oncology, 135,325-332 (2014)   Rationale Anti-angiogenesis therapies such as bevacizumab, the monoclonal antibody to vascular endothelial growth factor (VEGF), have been used against ovarian cancer, but transient and low peritoneal drug levels are likely a factor in treatment failure. We hypothesized that a single administration of adeno-associated virus (AAV)-mediated intraperitoneal expression of bevacizumab would direct persistent expression and suppress growth and metastasis of ovarian cancer. Methods AAVrh.10BevMab, a rhesus serotype 10 adeno-associated viral vector coding for bevacizumab, was evaluated for the capacity of a single intraperitoneal administration to persistently suppress peritoneal tumor growth in an intraperitoneal model of ovarian carcinomatosis with human ovarian cancer cells in nude immunodeficient mice. Results The data demonstrates that AAVrh10.BevMab mediates persistent and high levels of bevacizumab in the peritoneal cavity following a single intraperitoneal administration in mice. In AAVrh10.BevMab treated A2780 human ovarian cancer-bearing mice, tumor growth was significantly suppressed (p < 0.05) and the area of blood vessels in the tumor was decreased (p < 0.04). Survival of mice with A2780 xenografts or SK-OV3 xenografts was greatly prolonged in the presence of AAVrh10.BevMab (p < 0.001). Administration of AAVrh10.BevMab 4 days after A2780-luciferase cell implantation reduced tumor growth (p < 0.01) and increased mouse survival (p < 0.0001). Combination of AAVrh10.BevMab with cytotoxic reagents paclitaxel or topotecan proved to be more effective in increasing survival than treatment with cytotoxic reagent alone. Conclusion A single administration of AAVrh10.BevMab provides sustained and high local expression of bevacizumab in the peritoneal cavity, and significantly suppresses peritoneal carcinomatosis and increases survival in an ovarian cancer murine model.  

5.1540           Hijacking of an autophagy-like process is critical for the life cycle of a DNA virus infecting oceanic algal blooms

Schatz, D., Shemi, A., Rosenwasser, S., Sabanay, H., Wolf, S.G., Ben-Dor, S. and Vardi, A. New Phytologist, 204, 854-863 (2014)   Marine photosynthetic microorganisms are the basis of marine food webs and are responsible for nearly 50% of the global primary production. Emiliania huxleyi forms massive oceanic blooms that are routinely terminated by large double-stranded DNA coccolithoviruses. The cellular mechanisms that govern the replication cycle of these giant viruses are largely unknown. We used diverse techniques, including fluorescence microscopy, transmission electron microscopy, cryoelectron tomography, immunolabeling and biochemical methodologies to investigate the role of autophagy in host–virus interactions. Hallmarks of autophagy are induced during the lytic phase of E. huxleyi viral infection, concomitant with up-regulation of autophagy-related genes (ATG genes). Pretreatment of the infected cells with an autophagy inhibitor causes a major reduction in the production of extracellular viral particles, without reducing viral DNA replication within the cell. The host-encoded Atg8 protein was detected within purified virions, demonstrating the pivotal role of the autophagy-like process in viral assembly and egress. We show that autophagy, which is classically considered as a defense mechanism, is essential for viral propagation and for facilitating a high burst size. This cellular mechanism may have a major impact on the fate of the viral-infected blooms, and therefore on the cycling of nutrients within the marine ecosystem.  

5.1541           FTO knockdown in rat ventromedial hypothalamus does not affect energy balance

Van Gestel, M.A., Sanders, L.E., de Jong, J.W., Luijendijk, M.C. and Adan, R.A. Physiological Reports, 2(12), e12152 (2014)   Single nucleotide polymorphisms (SNPs) clustered in the first intron of the fat mass and obesity‐associated (FTO) gene has been associated with obesity. FTO expression is ubiquitous, with particularly high levels in the hypothalamic area of the brain. To investigate the region‐specific role of FTO, AAV technology was applied to knockdown FTO in the ventromedial hypothalamus (VMH). No effect of FTO knockdown was observed on bodyweight or parameters of energy balance. Animals were exposed twice to an overnight fast, followed by a high‐fat high‐sucrose (HFHS) diet for 1 week. FTO knockdown did not result in a different response to the diets. A region‐specific role for FTO in the VMH in the regulation of energy balance could not be found.  

5.1542           Exosome-associated hepatitis C virus in cell cultures and patient plasma

Liu, Z., Zhang, X., Yu, Q. and He, J.J: Biochem. Biophys. Res. Comm., 455, 218-222 (2014)   Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell–cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV than exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission.  

5.1543           Inhibition of pathogenic non-enveloped viruses by 25-hydroxycholesterol and 27-hydroxycholesterol

Civra, A., Cagno, V., Donalisio, M., Biasi, F., Leonardizzu, G., Poli, G and Lembo, D. Scientific Reports, 4:7487 (2014)   Recent studies reported a broad but selective antiviral activity of 25-hydroxycholesterol (25HC) against enveloped viruses, being apparently inactive against non-enveloped viruses. Here we show that 25HC is endowed with a marked antiviral activity against three pathogenic non-enveloped viruses, i.e. human papillomavirus-16 (HPV-16), human rotavirus (HRoV), and human rhinovirus (HRhV), thus significantly expanding its broad antiviral spectrum, so far recognized to be limited to viruses with envelope. Moreover, here we disclose the remarkable antiviral activity of another oxysterol of physiological origin, i.e. 27-hydroxycholesterol (27HC), against HPV-16, HRoV and HRhV. We have also identified a much weaker antiviral activity of other oxysterols of pathophysiological relevance, i.e 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol. These findings suggest that appropriate modulation of endogenous production of oxysterols might be a primary host strategy to counteract a broad panel of viral infections. Moreover, 25HC and 27HC could be considered for new therapeutic strategies against HPV-16, HRoV and HRhV.  

5.1544           Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna

Hinderer, C., Bell, P., Vite, C.H., Louboutin, J-P., Grant, R., Bote, E., Yu, H., Pukenas, B., Hurst, r. and Wilson, J.M: Molecular Therapy - Methods & Clinical Development, 1:14051 (2014)   Adeno-associated virus serotype 9 (AAV9) vectors have recently been shown to transduce cells throughout the central nervous system of nonhuman primates when injected into the cerebrospinal fluid (CSF), a finding which could lead to a minimally invasive approach to treat genetic and acquired diseases affecting the entire CNS. We characterized the transduction efficiency of two routes of vector administration into the CSF of cynomolgus macaques—lumbar puncture, which is typically used in clinical practice, and suboccipital puncture, which is more commonly used in veterinary medicine. We found that delivery of vector into the cisterna magna via suboccipital puncture is up to 100-fold more efficient for achieving gene transfer to the brain. In addition, we evaluated the inflammatory response to AAV9-mediated GFP expression in the nonhuman primate CNS. We found that while CSF lymphocyte counts increased following gene transfer, there were no clinical or histological signs of immune toxicity. Together these data indicate that delivery of AAV9 into the cisterna magna is an effective method for achieving gene transfer in the CNS, and suggest that adapting this uncommon injection method for human trials could vastly increase the efficiency of gene delivery.  

5.1545           Type I interferon rapidly restricts infectious hepatitis C virus particle genesis

Meredith, L.W., Farquhar, M.J., Tarr, A.W. and Mckeating, J.A: Hepatology, 60, 1891-1901 (2014)   Interferon-alpha (IFNα) has been used to treat chronic hepatitis C virus (HCV) infection for over 20 years with varying efficacy, depending on the infecting viral genotype. The mechanism of action of IFNα is not fully understood, but is thought to target multiple stages of the HCV lifecycle, inhibiting viral transcription and translation leading to a degradation of viral RNA and protein expression in the infected cell. IFNα induces the expression of an array of interferon-stimulated genes within minutes of receptor engagement; however, the impact of these early responses on the viral lifecycle are unknown. We demonstrate that IFNα inhibits the genesis of infectious extracellular HCV particles within 2 hours of treating infected cells, with minimal effect on the intracellular viral burden. Importantly, this short duration of IFNα treatment of infected cells significantly reduced cell-free and cell-to-cell dissemination. The secreted viral particles showed no apparent change in protein content or density, demonstrating that IFNα inhibits particle infectivity but not secretion rates. To investigate whether particles released from IFNα-treated cells have a reduced capacity to establish infection we used HCV lentiviral pseudotypes (HCVpp) and demonstrated a defect in cell entry. Using a panel of monoclonal antibodies targeting the E2 glycoprotein, we demonstrate that IFNα alters glycoprotein conformation and receptor utilization. Conclusion: These observations show a previously unreported and rapid effect of IFNα on HCV particle infectivity that inhibits de novo infection events. Evasion of this response may be a contributing factor in whether a patient achieves early or rapid virological response, a key indicator of progression to sustained virological response or clearance of viral infection.  

5.1546           Mechanism and treatment for learning and memory deficits in mouse models of Noonan syndrome

Lee, Y-S., Ehninger, D., Zhou, M., Oh, J-Y., kang, M., Kwak, C., Ryu, H-H., Butz, D., Araki, T., Cai, Y., Balaji, J., Sano, Y., Nam, C.I., Kim, H.K., Kaang, B-k., Burger, C., Neel, B.G. and Silva, A.J. Nature Neuroscience, 17(12), 1736-1743 (2014)   In Noonan syndrome (NS) 30–50% of subjects show cognitive deficits of unknown etiology and with no known treatment. Here, we report that knock-in mice expressing either of two NS-associated mutations in Ptpn11, which encodes the nonreceptor protein tyrosine phosphatase Shp2, show hippocampal-dependent impairments in spatial learning and deficits in hippocampal long-term potentiation (LTP). In addition, viral overexpression of an NS-associated allele PTPN11^D61G in adult mouse hippocampus results in increased baseline excitatory synaptic function and deficits in LTP and spatial learning, which can be reversed by a mitogen-activated protein kinase kinase (MEK) inhibitor. Furthermore, brief treatment with lovastatin reduces activation of the GTPase Ras–extracellular signal-related kinase (Erk) pathway in the brain and normalizes deficits in LTP and learning in adult Ptpn11^D61G/+ mice. Our results demonstrate that increased basal Erk activity and corresponding baseline increases in excitatory synaptic function are responsible for the LTP impairments and, consequently, the learning deficits in mouse models of NS. These data also suggest that lovastatin or MEK inhibitors may be useful for treating the cognitive deficits in NS.  

5.1547           VTA CRF neurons mediate the aversive effects of nicotine withdrawal and promote intake escalation

Grieder, T. et al Nature Neuroscience, 17(12), 1751-1758 (2014)   Dopaminergic neurons in the ventral tegmental area (VTA) are well known for mediating the positive reinforcing effects of drugs of abuse. Here we identify in rodents and humans a population of VTA dopaminergic neurons expressing corticotropin-releasing factor (CRF). We provide further evidence in rodents that chronic nicotine exposure upregulates Crh mRNA (encoding CRF) in dopaminergic neurons of the posterior VTA, activates local CRF₁ receptors and blocks nicotine-induced activation of transient GABAergic input to dopaminergic neurons. Local downregulation of Crh mRNA and specific pharmacological blockade of CRF₁ receptors in the VTA reversed the effect of nicotine on GABAergic input to dopaminergic neurons, prevented the aversive effects of nicotine withdrawal and limited the escalation of nicotine intake. These results link the brain reward and stress systems in the same brain region to signaling of the negative motivational effects of nicotine withdrawal.  

5.1548           Mybpc3 gene therapy for neonatal cardiomyopathy enables long-term disease prevention in mice

Mearini, G. et al Nature Communications, 5:5515 (2014)   Homozygous or compound heterozygous frameshift mutations in ​MYBPC3 encoding ​cardiac myosin-binding protein C (​cMyBP-C) cause neonatal hypertrophic cardiomyopathy (HCM), which rapidly evolves into systolic heart failure and death within the first year of life. Here we show successful long-term ​Mybpc3 gene therapy in homozygous ​Mybpc3-targeted knock-in (KI) mice, which genetically mimic these human neonatal cardiomyopathies. A single systemic administration of adeno-associated virus (AAV9)-​Mybpc3 in 1-day-old KI mice prevents the development of cardiac hypertrophy and dysfunction for the observation period of 34 weeks and increases ​Mybpc3 messenger RNA (mRNA) and ​cMyBP-C protein levels in a dose-dependent manner. Importantly, ​Mybpc3 gene therapy unexpectedly also suppresses accumulation of mutant mRNAs. This study reports the first successful long-term gene therapy of HCM with correction of both haploinsufficiency and production of poison peptides. In the absence of alternative treatment options except heart transplantation, gene therapy could become a realistic treatment option for severe neonatal HCM.  

5.1549           Adeno-associated virus-RNAi of GlyRα1 and characterization of its synapse-specific inhibition in OFF alpha transient retinal ganglion cells

Zhang, C., Rompani, S.B., Roska, B. and McCall, M.A.

1. Neurophysiol., 112(12), 3125-3137 (2014)

  In the central nervous system, inhibition shapes neuronal excitation. In spinal cord glycinergic inhibition predominates, whereas GABAergic inhibition predominates in the brain. The retina uses GABA and glycine in approximately equal proportions. Glycinergic crossover inhibition, initiated in the On retinal pathway, controls glutamate release from presynaptic OFF cone bipolar cells (CBCs) and directly shapes temporal response properties of OFF retinal ganglion cells (RGCs). In the retina, four glycine receptor (GlyR) α-subunit isoforms are expressed in different sublaminae and their synaptic currents differ in decay kinetics. GlyRα1, expressed in both On and Off sublaminae of the inner plexiform layer, could be the glycinergic isoform that mediates On-to-Off crossover inhibition. However, subunit-selective glycine contributions remain unknown because we lack selective antagonists or cell class-specific subunit knockouts. To examine the role of GlyRα1 in direct inhibition in mature RGCs, we used retrogradely transported adeno-associated virus (AAV) that performed RNAi and eliminated almost all glycinergic spontaneous and visually evoked responses in PV5 (OFFα_Transient) RGCs. Comparisons of responses in PV5 RGCs infected with AAV-scrambled-short hairpin RNA (shRNA) or AAV-Glra1-shRNA confirm a role for GlyRα1 in crossover inhibition in cone-driven circuits. Our results also define a role for direct GlyRα1 inhibition in setting the resting membrane potential of PV5 RGCs. The absence of GlyRα1 input unmasked a serial and a direct feedforward GABA_Aergic modulation in PV5 RGCs, reflecting a complex interaction between glycinergic and GABA_Aergic inhibition.  

5.1550           Adenosine kinase, glutamine synthetase and EAAT2 as gene therapy targets for temporal lobe epilepsy

Young, D., Fong, D.M., Lawlor, P.A., Wu, A., Mouravlev, A., McRae, M., Glass, M., Dragunow, M. and During, M.J. Gene Therapy, 21, 1029-1040 (2014)   Astrocytes are an attractive cell target for gene therapy, but the validation of new therapeutic candidates is needed. We determined whether adeno-associated viral (AAV) vector-mediated overexpression of glutamine synthetase (GS) or excitatory amino-acid transporter 2 (EAAT2), or expression of microRNA targeting adenosine kinase (miR-ADK) in hippocampal astrocytes in the rat brain could modulate susceptibility to kainate-induced seizures and neuronal cell loss. Transgene expression was found predominantly in astrocytes following direct injection of glial-targeting AAV9 vectors by 3 weeks postinjection. ADK expression in miR-ADK vector-injected rats was reduced by 94–96% and was associated with an ~50% reduction in the duration of kainate-induced seizures and greater protection of dentate hilar neurons but not CA3 neurons compared with miR-control vector-injected rats. In contrast, infusion of AAV-GS and EAAT2 vectors did not afford any protection against seizures or neuronal damage as the level of transcriptional activity of the glial fibrillary acidic promoter was too low to drive any significant increase in transgenic GS or EAAT2 relative to the high endogenous levels of these proteins. Our findings support ADK as a prime therapeutic target for gene therapy of temporal lobe epilepsy and suggest that alternative approaches including the use of stronger glial promoters are needed to increase transgenic GS and EAAT2 expression to levels that may be required to affect seizure induction and propagation.  

5.1551           Gene therapy approach to FAP: in vivo influence of T119M in TTR deposition in a transgenic V30M mouse model

Batista, A.R., Gianni, D., Ventosa, M., Coelho, A.V., Almeida, M.R., Sena-Esteves, M. and Saraiva, M.J. Gene Therapy, 21, 1041-1050 (2014)   Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disorder characterized by extracellular deposition of amyloid fibrils composed by mutated transthyretin (TTR) mainly in the peripheral nervous system. At present, liver transplantation is still the standard treatment to halt the progression of clinical symptoms in FAP, but new therapeutic strategies are emerging, including the use of TTR stabilizers. Here we propose to establish a new gene therapy approach using adeno-associated virus (AAV) vectors to deliver the trans-suppressor TTR T119M variant to the liver of transgenic TTR V30M mice at different ages. This TTR variant is known for its ability to stabilize the tetrameric protein. Analysis of the gastrointestinal tract of AAV-treated animals revealed a significant reduction in deposition of TTR non-fibrillar aggregates in as much as 34% in stomach and 30% in colon, as well as decreased levels of biomarkers associated with TTR deposition, namely the endoplasmic reticulum stress marker BiP and the extracellular matrix protein MMP-9. Moreover, we showed with different studies that our approach leads to an increase in tetrameric and more stable forms of TTR, in favor of destabilized monomers. Altogether our data suggest the possibility to use this gene therapy approach in a prophylactic manner to prevent FAP pathology.  

5.1552           Pre-existing immunity to adeno-associated virus (AAV)2 limits transgene expression following intracerebral AAV2-based gene delivery in a 6-hydroxydopamine model of Parkinson's disease

Janelidze, S., Nordström, U., Kügler, S. and Brundin, P.

1. Gene Med., 16, 300-308 (2014)

  Background Adeno-associated virus (AAV) vectors are used to deliver potentially therapeutic genes in clinical trials in Parkinson's disease (PD). Pre-existing immunity to AAV and a local neuroinflammatory response might negatively affect the efficacy of such AAV-mediated gene delivery. Methods We pre-immunized rats with wild-type AAV-2. Three months later, we created PD-like lesions by intrastriatal injections of 6-hydroxydopamine (6-OHDA) in 50% of the animals. One month later, we injected AAV2 vector expressing enhanced green fluorescent protein (eGFP) in the striatum. Using immunohistochemistry, we assessed eGFP expression, microglia activation and CD8 T cell infiltration. We also measured AAV-2 specific neutralizing antibody titers in the serum. Results The number of striatal cells transduced with AAV2 vector expressing eGFP was reduced by 71% in rats pre-immunized with wild-type AAV2 compared to non-immunized animals. We detected elevated numbers of OX6⁺ activated microglia in the striatum and circulating AAV2-specific neutralizing antibodies in pre-immunized rats. We also observed that the intrastriatal 6-OHDA injection promoted CD8⁺ T cell infiltration and enhanced microglia activation. Nevertheless, the 6-OHDA lesion did not alter AAV2-mediated expression of eGFP in either pre-immunized or non-immunized rats. Conclusions Our findings indicate that intracerebral AAV2-based gene therapy is compromised in rats with pre-existing immunity to AAV2. By contrast, a local neuroinflammatory response, caused by intrastriatal a 6-OHDA injection, does not affect viral vector-mediated transgene expression. Our results emphasize the importance of monitoring circulating AAV-specific neutralizing antibodies in patients undergoing intracerebral gene therapy using AAV vectors  

5.1553           IFITM Proteins Incorporated into HIV-1 Virions Impair Viral Fusion and Spread

Compton, A.A., Bruel, T., Porrot, F., Mallet, A., Sachse, M., Euvrard, M., Liang, C., Casartelli, N. and Schwartz, O. Cell Host & Microbe, 16, 736-747 (2014)   The interferon-induced transmembrane (IFITM) proteins protect cells from diverse virus infections by inhibiting virus-cell fusion. IFITM proteins also inhibit HIV-1 replication through mechanisms only partially understood. We show that when expressed in uninfected lymphocytes, IFITM proteins exert protective effects during cell-free virus infection, but this restriction can be overcome upon HIV-1 cell-to-cell spread. However, when present in virus-producing lymphocytes, IFITM proteins colocalize with viral Env and Gag proteins and incorporate into nascent HIV-1 virions to limit entry into new target cells. IFITM in viral membranes is associated with impaired virion fusion, offering additional and more potent defense against virus spread. Thus, IFITM proteins act additively in both productively infected cells and uninfected target cells to inhibit HIV-1 spread, potentially conferring these proteins with greater breadth and potency against enveloped viruses.  

5.1554           Intermediate Heparan Sulfate Binding During HPV-16 Infection in HaCaTs

Kumar, A., Jacob, T., Abban, C.Y.. and Meneses, P. Am. J. Therapeutics, 21, 331-342 (2014)   Human papillomavirus (HPV) is the most prevalent sexually transmitted disease in the United States and can cause cancer with persistent infection. The most common cancer caused by HPV is cervical carcinoma with an average of 12,000 cases reported every year in the United States. Worldwide, over 500,000 cases of cervical cancer are reported yearly with over 250,000 deaths attributed to the disease. Although much is known about the serious health risks associated with HPV infection, there is still much to be discovered about how HPV binds and enters target cells. Understanding is required on how HPV infections will lead to strategies and therapies for reducing the number of infections and HPV-related diseases, including cancers. The HPV viral particle is composed of 2 viral proteins, L1 and L2. Data suggest that binding of the viral capsid to cells is dependent on the L1 protein. We hypothesize that this initial binding to a heparan sulfate is composed of 2 independent events: the first results in a structural change that exposes a hidden portion of the L1 protein leading to a second binding event on the heparan sulfate. Our experiments tested if this “hidden” portion of L1 is necessary for infection and explored the nature of this binding. We generated a peptide with the sequence of the “hidden” portion of L1. Infection of HaCaT cells in the presence of this peptide is highly reduced. Our results suggest that the binding of the L1 C-terminal domain is dependent on amino acid sequence and is necessary for infection.  

5.1555           Rods in daylight act as relay cells for cone-driven horizontal cell–mediated surround inhibition

Szikra, T., Trenholm, S., Drinnenberg, A., Jüttner, J., Raics, Z., farrow, K., Biel, M., Awatramani, G., Clark, D.A., Sahel, J-A. and da Siveira, R.A. Nature Neuroscience, 17(12), 1728-1735 (2014)   Vertebrate vision relies on two types of photoreceptors, rods and cones, which signal increments in light intensity with graded hyperpolarizations. Rods operate in the lower range of light intensities while cones operate at brighter intensities. The receptive fields of both photoreceptors exhibit antagonistic center-surround organization. Here we show that at bright light levels, mouse rods act as relay cells for cone-driven horizontal cell–mediated surround inhibition. In response to large, bright stimuli that activate their surrounds, rods depolarize. Rod depolarization increases with stimulus size, and its action spectrum matches that of cones. Rod responses at high light levels are abolished in mice with nonfunctional cones and when horizontal cells are reversibly inactivated. Rod depolarization is conveyed to the inner retina via postsynaptic circuit elements, namely the rod bipolar cells. Our results show that the retinal circuitry repurposes rods, when they are not directly sensing light, to relay cone-driven surround inhibition.  

5.1556           Hepatic Farnesoid X-Receptor Isoforms α2 and α4 Differentially Modulate Bile Salt and Lipoprotein Metabolism in Mice

Boesjes, M., Bloks, V.W., Hageman, J., Bos, TS., van Dijk, T.H., havinga, R., Wolters, H., Jonker, J.W., Kuipers, F. and Groen, A.K. PloS One, 9(2), e115028 (2014)   The nuclear receptor FXR acts as an intracellular bile salt sensor that regulates synthesis and transport of bile salts within their enterohepatic circulation. In addition, FXR is involved in control of a variety of crucial metabolic pathways. Four FXR splice variants are known, i.e. FXRα1-4. Although these isoforms show differences in spatial and temporal expression patterns as well as in transcriptional activity, the physiological relevance hereof has remained elusive. We have evaluated specific roles of hepatic FXRα2 and FXRα4 by stably expressing these isoforms using liver-specific self-complementary adeno-associated viral vectors in total body FXR knock-out mice. The hepatic gene expression profile of the FXR knock-out mice was largely normalized by both isoforms. Yet, differential effects were also apparent; FXRα2 was more effective in reducing elevated HDL levels and transrepressed hepatic expression of Cyp8b1, the regulator of cholate synthesis. The latter coincided with a switch in hydrophobicity of the bile salt pool. Furthermore, FXRα2-transduction caused an increased neutral sterol excretion compared to FXRα4 without affecting intestinal cholesterol absorption. Our data show, for the first time, that hepatic FXRα2 and FXRα4 differentially modulate bile salt and lipoprotein metabolism in mice.  

5.1557           Alternative Splicing Coupled Nonsense-Mediated Decay Generates Neuronal Cell Type-Specific Expression of SLM Proteins

Traunmüller, L., Bornmann, C. and Scheiffele, P.

1. Neurosci., 34(50), 16755-16761 (2014)

  The unique physiological and morphological properties of neuronal populations are crucial for the appropriate functioning of neuronal circuits. Alternative splicing represents an attractive mechanism for generating cell type-specific molecular repertoires that steer neuronal development and function. However, the mechanisms that link neuronal identity to alternative splicing programs are poorly understood. We report that cell type-specific, mutually exclusive expression of two alternative splicing regulators, SLM1 and SLM2, in the mouse hippocampus is achieved by a cross-repression mechanism. Deletion of SLM2 in vivo modifies alternative splicing of its paralog Slm1 and stabilizes its mRNA, resulting in expression of SLM1 in previously SLM2-expressing cells. Despite this ectopic upregulation of SLM1, loss of SLM2 severely disrupts the alternative splicing regulation of Nrxn1, Nrxn2, and Nrxn3, highlighting that the two SLM paralogs have partially divergent functions. Our study uncovers a hierarchical, SLM2-dependent mechanism for establishing cell type-specific expression of neuronal splicing regulators in vivo.  

5.1558           Unexpected patterns of Epstein–Barr virus transcription revealed by a High throughput PCR array for absolute quantification of viral mRNA

Tierney, R.J., Shannon-Lowe, C.D., Fitzsimmons, L., Bell, A.I. and Rowe, M. Virology, 474, 117-130 (2015)   We have validated a flexible, high-throughput and relatively inexpensive RT-QPCR array platform for absolute quantification of Epstein–Barr virus transcripts in different latent and lytic infection states. Several novel observations are reported. First, during infection of normal B cells, Wp-initiated latent gene transcripts remain far more abundant following activation of the Cp promoter than was hitherto suspected. Second, EBNA1 transcript levels are remarkably low in all forms of latency, typically ranging from 1 to 10 transcripts per cell. EBNA3A, -3B and -3C transcripts are likewise very low in Latency III, typically at levels similar to or less than EBNA1 transcripts. Thirdly, a subset of lytic gene transcripts is detectable in Burkitt lymphoma lines at low levels, including: BILF1, which has oncogenic properties, and the poorly characterized LF1, LF2 and LF3 genes. Analysis of seven African BL biopsies confirmed this transcription profile but additionally revealed significant expression of LMP2 transcripts.  

5.1559           AAV.shRNA-mediated downregulation of ROCK2 attenuates degeneration of dopaminergic neurons in toxin-induced models of Parkinson's disease in vitro and in vivo

Saal, K-A., Koch, J.C., tatenhorst, L., Szego, E.M., Toledo Ribas, V., Michel, U., Bähr, M., Tönges, L. and Lingor, P. Neurobiology of Disease, 73, 150-162 (2015)   Parkinson's disease (PD) is a neurodegenerative disorder with prominent neuronal cell death in the substantia nigra (SN) and other parts of the brain. Previous studies in models of traumatic and neurodegenerative CNS disease showed that pharmacological inhibition of Rho-associated kinase (ROCK), a molecule involved in inhibitory signaling in the CNS, by small-molecule inhibitors improves neuronal survival and increases regeneration. Most small-molecule inhibitors, however, offer only limited target specificity and also inhibit other kinases, including both ROCK isoforms. To establish the role of the predominantly brain-expressed ROCK2 isoform in models of regeneration and PD, we used adeno-associated viral vectors (AAV) to specifically knockdown ROCK2 in neurons. Rat primary midbrain neurons (PMN) were transduced with AAV expressing short-hairpin-RNA (shRNA) against ROCK2 and LIM-domain kinase 1 (LIMK1), one of the downstream targets of ROCK2. While knock-down of ROCK2 and LIMK1 both enhanced neurite regeneration in a traumatic scratch lesion model, only ROCK2-shRNA protected PMN against 1-methyl-4-phenylpyridinium (MPP⁺) toxicity. Moreover, AAV.ROCK2-shRNA increased levels of the pro-survival markers Bcl-2 and phospho-Erk1. In vivo, AAV.ROCK2-shRNA vectors were injected into the ipsilateral SN and a unilateral 6-OHDA striatal lesion was performed. After four weeks, behavioral, immunohistochemical and biochemical alterations were investigated. Downregulation of ROCK2 protected dopaminergic neurons in the SN from 6-OHDA-induced degeneration and resulted in significantly increased TH-positive neuron numbers. This effect, however, was confined to nigral neuronal somata as striatal terminal density, dopamine and metabolite levels were not significantly preserved. Interestingly, motor behavior was improved in the ROCK2-shRNA treated animals compared to control after four weeks. Our studies thus confirm ROCK2 as a promising therapeutic target in models of PD and demonstrate that neuron-specific inhibition of ROCK2 promotes survival of lesioned dopaminergic neurons.  

5.1560           The role of parkin in the differential susceptibility of tuberoinfundibular and nigrostriatal dopamine neurons to acute toxicant exposure

Benskey, M.J., Manfredsson, F.P., Lookingland, K.J. and Goudreau, J.L. Neurotoxicology, 46, 1-11 (2015)   Parkinson disease causes degeneration of nigrostriatal dopamine (DA) neurons, while tuberoinfundibular DA neurons remain unaffected. A similar pattern is observed following exposure to 1-methy-4-phenyl-1,2,3,6-tetrahydropyradine (MPTP). The mechanism of tuberoinfundibular neuronal recovery from MPTP is associated with up-regulation of parkin protein. Here we tested if parkin mediates tuberoinfundibular neuronal recovery from MPTP by knocking-down parkin in tuberoinfundibular neurons using recombinant adeno-associated virus (rAAV), expressing a short hairpin RNA (shRNA) directed toward parkin. Following knockdown, axon terminal DA and tyrosine hydroxylase (TH) concentrations were analyzed 24 h post-MPTP administration. rAAV-shRNA-mediated knockdown of endogenous parkin rendered tuberoinfundibular neurons susceptible to MPTP induced terminal DA loss, but not TH loss, within 24 h post-MPTP. To determine if the neuroprotective benefits of parkin up-regulation could be translated to nigrostriatal neurons, rAAV expressing human parkin was injected into the substantia nigra of mice and axon terminal DA and TH concentrations were analyzed 24 h post-MPTP. Nigral parkin over-expression prevented loss of TH in the axon terminals and soma of nigrostriatal neurons, but had no effect on terminal DA loss within 24 h post-MPTP. These data show that parkin is necessary for the recovery of terminal DA concentrations within tuberoinfundibular neurons following acute MPTP administration, and parkin can rescue MPTP-induced decreases in TH within nigrostriatal neurons.  

5.1561           Use of tangential flow filtration for improving detection of viral adventitious agents in cell substrates

Furtak, V.A., Dabrazhynetskay, A., Volokhov, D.V. and Chizhikov, V. Biologicals, 43, 23-30 (2015)   In this study, we assessed the feasibility of tangential flow filtration (TFF) for primary concentration of viral adventitious agents (AAs) from large volumes of cell substrate-derived samples, such as cell-free Chinese hamster ovary (CHO) culture supernatants (500 mL) and CHO cell lysates (50 mL), prior to virus detection in them by nucleic acid-based methods (i.e., qPCR and massively parallel sequencing (MPS). The study was conducted using the samples spiked with four model DNA viruses (bovine herpesvirus type 4, human adenovirus type 5, simian polyomavirus SV-40, and bovine parvovirus). The results showed that the combined TFF/MPS approach enables reliable detection of as low as 1000 genome equivalents (GE) of each of the four viruses spiked into the cell substrate samples. The final achieved sensitivities of 2 GE/mL for cell culture supernatant and 20 GE/mL for cell lysate make this approach more sensitive than virus-specific PCR and qPCR assays. The study results allowed us to propose that TFF might be useful and valuable method for simple and rapid concentration of potential AAs in cell substrate samples prior to AAs detection by conventional in vivo, in vitro, or molecular methods.  

5.1562           Dual role of Src kinase in governing neuronal survival

Hossain, M.I., Hoque, A., Lessene, G., Kamaruddin, M.A., Chu, P.W.Y., Ng, I.H.W., Irtegun, S., Ng, D.C.H., Bogoyevitvh, M.A., Burgess, A.W., Hill, A.F. and Cheng, H-C. Brain Res., 1594, 1-14 (2015)   Background Src-family kinases (SFKs) are involved in neuronal survival and their aberrant regulation contributes to neuronal death. However, how they control neuronal survival and death remains unclear. Objective To define the effect of inhibition of Src activity and expression on neuronal survival. Results In agreement with our previous findings, we demonstrated that Src was cleaved by calpain to form a 52-kDa truncated fragment in neurons undergoing excitotoxic cell death, and expression of the recombinant truncated Src fragment induced neuronal death. The data confirm that the neurotoxic signaling pathways are intact in the neurons we used for our study. To define the functional role of neuronal SFKs, we treated these neurons with SFK inhibitors and discovered that the treatment induced cell death, suggesting that the catalytic activity of one or more of the neuronal SFKs is critical to neuronal survival. Using small hairpin RNAs that suppress Src expression, we demonstrated that Src is indispensable to neuronal survival. Additionally, we found that neuronal death induced by expression of the neurotoxic truncated Src mutant, treatment of SFK inhibitors or knock-down of Src expression caused inhibition of the neuroprotective protein kinases Erk1/2, or Akt. Conclusions Src is critical to both neuronal survival and death. Intact Src sustains neuronal survival. However, in the excitotoxic condition, calpain cleavage of Src generates a neurotoxic truncated Src fragment. Both intact Src and the neurotoxic truncated Src fragment exert their biological actions by controlling the activities of neuroprotective protein kinases.  

5.1563           Topical Herpes Simplex Virus 2 (HSV-2) Vaccination with Human Papillomavirus Vectors Expressing gB/gD Ectodomains Induces Genital-Tissue-Resident Memory CD8+ T Cells and Reduces Genital Disease and Viral Shedding after HSV-2 Challenge

Cuburu, N., Wang, K., Goodman, K.N., Pang, Y.Y., Thompson, C.D., Lowy, D.R., Cohen, J.I. and Schiller, J.T.

1. Virol., 89(1), 83-96 (2015)

  No herpes simplex virus 2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular (i.m.) injection of HSV-2 gB and gD in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) or gD (gDsec), but not PsV expressing a cytoplasmic or membrane-bound form, induced circulating and intravaginal-tissue-resident memory CD8⁺ T cells that were able to secrete gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) as well as moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec and HPV-gDsec (HPV-gBsec/gDsec) vaccines conferred longer survival after vaginal challenge with HSV-2 than immunization with HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with a reduced severity of genital lesions and lower levels of viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with a soluble truncated gD protein (gD2t) in alum and monophosphoryl lipid A (MPL) elicited high neutralizing antibody titers and improved survival but did not reduce genital lesions and viral shedding. Vaccination combining ivag HPV-gBsec/gDsec and i.m. gD2t-alum-MPL improved survival and reduced genital lesions and viral shedding. Finally, high levels of circulating HSV-2-specific CD8⁺ T cells, but not serum antibodies, correlated with reduced viral shedding. Taken together, our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection.  

5.1564           Insight into the Mechanism of Inhibition of Adeno-Associated Virus by the Mre11/Rad50/Nbs1 Complex

Lentz, T.B. and Samulski, R.J.

1. Virol., 89(1), 181-194 (2015)

  Adeno-associated virus (AAV) is a dependent virus of the family Parvoviridae. The gene expression and replication of AAV and derived recombinant AAV (rAAV) vectors are severely limited (>10-fold) by the cellular DNA damage-sensing complex made up of Mre11, Rad50, and Nbs1 (MRN). The AAV genome does not encode the means to circumvent this block to productive infection but relies on coinfecting helper virus to do so. Using adenovirus helper proteins E1B55k and E4orf6, which enhance the transduction of AAV via degradation of MRN, we investigated the mechanism through which this DNA damage complex inhibits gene expression from rAAV. We tested the substrate specificity of inhibition and the contribution of different functions of the MRN complex. Our results demonstrate that both single- and double-stranded rAAV vectors are inhibited by MRN, which is in contrast to the predominant model that inhibition is the result of a block to second-strand synthesis. Exploring the contribution of known functions of MRN, we found that inhibition of rAAV does not require downstream DNA damage response factors, including signaling kinases ATM and ATR. The nuclease domain of Mre11 appears to play only a minor role in inhibition, while the DNA binding domain makes a greater contribution. Additionally, mutation of the inverted terminal repeat of the rAAV genome, which has been proposed to be the signal for interaction with MRN, is tolerated by the mechanism of inhibition. These results articulate a model of inhibition of gene expression in which physical interaction is more important than enzymatic activity and several key downstream damage repair factors are dispensable.  

5.1565           APOBEC3A Functions as a Restriction Factor of Human Papillomavirus

Warren, C.J., Xu, T., Guo, K., Griffin, L.M., Westrich, J.A., Lee, D., lambert, P.F., Santiago, M.L. and Pyeon, D.

1. Virol., 89(1), 688-702 (2015)

  Human papillomaviruses (HPVs) are small DNA viruses causally associated with benign warts and multiple cancers, including cervical and head-and-neck cancers. While the vast majority of people are exposed to HPV, most instances of infection are cleared naturally. However, the intrinsic host defense mechanisms that block the early establishment of HPV infections remain mysterious. Several antiviral cytidine deaminases of the human APOBEC3 (hA3) family have been identified as potent viral DNA mutators. While editing of HPV genomes in benign and premalignant cervical lesions has been demonstrated, it remains unclear whether hA3 proteins can directly inhibit HPV infection. Interestingly, recent studies revealed that HPV-positive cervical and head-and-neck cancers exhibited higher rates of hA3 mutation signatures than most HPV-negative cancers. Here, we report that hA3A and hA3B expression levels are highly upregulated in HPV-positive keratinocytes and cervical tissues in early stages of cancer progression, potentially through a mechanism involving the HPV E7 oncoprotein. HPV16 virions assembled in the presence of hA3A, but not in the presence of hA3B or hA3C, have significantly decreased infectivity compared to HPV virions assembled without hA3A or with a catalytically inactive mutant, hA3A/E72Q. Importantly, hA3A knockdown in human keratinocytes results in a significant increase in HPV infectivity. Collectively, our findings suggest that hA3A acts as a restriction factor against HPV infection, but the induction of this restriction mechanism by HPV may come at a cost to the host by promoting cancer mutagenesis.  

5.1566           Enhanced Transgene Expression from Recombinant Single-Stranded D-Sequence-Substituted Adeno-Associated Virus Vectors in Human Cell Lines In Vitro and in Murine Hepatocytes In Vivo

Ling, C., Wang, Y., Lu, Y., Wang, L., Jayandharan, G.R., Aslanidi, G.A., Li, B., Cheng, B., Ma, W., Lentz, T., Ling, C., Xiao, X., Samulski, R.J., Muzyczka, N. and Srivastava, A.

1. Virol., 89(2), 952-961 (2015)

  We have previously reported that the removal of a 20-nucleotide sequence, termed the D sequence, from both ends of the inverted terminal repeats (ITRs) in the adeno-associated virus serotype 2 (AAV2) genome significantly impairs rescue, replication, and encapsidation of the viral genomes (X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Mol Biol 250:573–580, 1995; X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Virol 70:1668–1677, 1996). Here we describe that replacement of only one D sequence in either ITR restores each of these functions, but DNA strands of only single polarity are encapsidated in mature progeny virions. Since most commonly used recombinant AAV vectors contain a single-stranded DNA (ssDNA), which is transcriptionally inactive, efficient transgene expression from AAV vectors is dependent upon viral second-strand DNA synthesis. We have also identified a transcription suppressor sequence in one of the D sequences, which shares homology with the binding site for the cellular NF-κB-repressing factor (NRF). The removal of this D sequence from, and replacement with a sequence containing putative binding sites for transcription factors in, single-stranded AAV (ssAAV) vectors significantly augments transgene expression both in human cell lines in vitro and in murine hepatocytes in vivo. The development of these genome-modified ssAAV vectors has implications not only for the basic biology of AAV but also for the optimal use of these vectors in human gene therapy.  

5.1567           Mechanism of Multivalent Nanoparticle Encounter with HIV-1 for Potency Enhancement of Peptide Triazole Virus Inactivation

Bastian, A.R., Nangarlia, A., Bailey, L.D., Holmes, A., Sundaram, R.V.K., Ang, C., Moreira, D.R.M., Freedman, K., Duffy, C., Contarino, M., Abrams, C., Root, M. and Chaiken, I.

1. Biol. Chem., 290(1), 529-543 (2015)

  Entry of HIV-1 into host cells remains a compelling yet elusive target for developing agents to prevent infection. A peptide triazole (PT) class of entry inhibitor has previously been shown to bind to HIV-1 gp120, suppress interactions of the Env protein at host cell receptor binding sites, inhibit cell infection, and cause envelope spike protein breakdown, including gp120 shedding and, for some variants, virus membrane lysis. We found that gold nanoparticle-conjugated forms of peptide triazoles (AuNP-PT) exhibit substantially more potent antiviral effects against HIV-1 than corresponding peptide triazoles alone. Here, we sought to reveal the mechanism of potency enhancement underlying nanoparticle conjugate function. We found that altering the physical properties of the nanoparticle conjugate, by increasing the AuNP diameter and/or the density of PT conjugated on the AuNP surface, enhanced potency of infection inhibition to impressive picomolar levels. Further, compared with unconjugated PT, AuNP-PT was less susceptible to reduction of antiviral potency when the density of PT-competent Env spikes on the virus was reduced by incorporating a peptide-resistant mutant gp120. We conclude that potency enhancement of virolytic activity and corresponding irreversible HIV-1 inactivation of PTs upon AuNP conjugation derives from multivalent contact between the nanoconjugates and metastable Env spikes on the HIV-1 virus. The findings reveal that multispike engagement can exploit the metastability built into virus the envelope to irreversibly inactivate HIV-1 and provide a conceptual platform to design nanoparticle-based antiviral agents for HIV-1 specifically and putatively for metastable enveloped viruses generally.  

5.1568           The adipocyte differentiation protein APMAP is an endogenous suppressor of Aβ production in the brain

Mosser, S., Alattia, J-R., Dimitrov, M., Matz, A., Pascual, J., Schneider, B.L. and Fraering, P.c. Hum. Mol. Genet., 24(2), 371-382 (2015)   The deposition of amyloid-beta (Aβ) aggregates in the brain is a major pathological hallmark of Alzheimer's disease (AD). Aβ is generated from the cleavage of C-terminal fragments of the amyloid precursor protein (APP-CTFs) by γ-secretase, an intramembrane-cleaving protease with multiple substrates, including the Notch receptors. Endogenous modulation of γ-secretase is pointed to be implicated in the sporadic, age-dependent form of AD. Moreover, specifically modulating Aβ production has become a priority for the safe treatment of AD because the inhibition of γ-secretase results in adverse effects that are related to impaired Notch cleavage. Here, we report the identification of the adipocyte differentiation protein APMAP as a novel endogenous suppressor of Aβ generation. We found that APMAP interacts physically with γ-secretase and its substrate APP. In cells, the partial depletion of APMAP drastically increased the levels of APP-CTFs, as well as uniquely affecting their stability, with the consequence being increased secretion of Aβ. In wild-type and APP/ presenilin 1 transgenic mice, partial adeno-associated virus-mediated APMAP knockdown in the hippocampus increased Aβ production by ∼20 and ∼55%, respectively. Together, our data demonstrate that APMAP is a negative regulator of Aβ production through its interaction with APP and γ-secretase. All observed APMAP phenotypes can be explained by an impaired degradation of APP-CTFs, likely caused by an altered substrate transport capacity to the lysosomal/autophagic system.  

5.1569           Syntaxin 5-Dependent Retrograde Transport to the trans-Golgi Network Is Required for Adeno-Associated Virus Transduction

Nonnenmacher, M.E., Cintrat, J-C., Gillet, D. and Weber, T.

1. Virol., 89(3), 1673-1687 (2015)

  Intracellular transport of recombinant adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasm, allowing subsequent nuclear import and the initiation of gene expression, remain to be elucidated fully. Others and we previously showed that a significant proportion of viral particles are transported to the Golgi apparatus and that Golgi apparatus disruption caused by the drug brefeldin A efficiently blocks AAV serotype 2 (AAV2) transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of transport to the Golgi apparatus for AAV transduction remains to be established definitively. Here, we show that AAV2 trafficking toward the trans-Golgi network (TGN) and the Golgi apparatus correlates with transduction efficiency and relies on a nonclassical retrograde transport pathway that is independent of the retromer complex, late endosomes, and recycling endosomes. AAV2 transduction is unaffected by the knockdown of syntaxins 6 and 16, which are two major effectors in the retrograde transport of both exogenous and endogenous cargo. On the other hand, inhibition of syntaxin 5 function by small interfering RNA silencing or treatment with cyclized Retro-2 strongly decreases AAV2 transduction and transport to the Golgi apparatus. This inhibition of transduction is observed with several AAV serotypes and a number of primary and immortalized cells. Together, our data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi apparatus is a broadly conserved feature of AAV trafficking that appears to be independent of the identity of the receptors used for viral attachment.  

5.1570           Adeno-Associated Virus Serotype 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity

Tseng, Y-S., Gurda, B.L., Chipman, P., McKenna, R., Afione, S., Chiorini, J.A., Muzyczka, N., Olson, N.H., Baker, T.S., Kleinschmidt, J. and Agbandje-McKenna, M.

1. Virol., 89(3), 1794-1808 (2015)

  The clinical utility of the adeno-associated virus (AAV) gene delivery system has been validated by the regulatory approval of an AAV serotype 1 (AAV1) vector for the treatment of lipoprotein lipase deficiency. However, neutralization from preexisting antibodies is detrimental to AAV transduction efficiency. Hence, mapping of AAV antigenic sites and engineering of neutralization-escaping vectors are important for improving clinical efficacy. We report the structures of four AAV-monoclonal antibody fragment complexes, AAV1-ADK1a, AAV1-ADK1b, AAV5-ADK5a, and AAV5-ADK5b, determined by cryo-electron microscopy and image reconstruction to a resolution of ∼11 to 12 Å. Pseudoatomic modeling mapped the ADK1a epitope to the protrusions surrounding the icosahedral 3-fold axis and the ADK1b and ADK5a epitopes, which overlap, to the wall between depressions at the 2- and 5-fold axes (2/5-fold wall), and the ADK5b epitope spans both the 5-fold axis-facing wall of the 3-fold protrusion and portions of the 2/5-fold wall of the capsid. Combined with the six antigenic sites previously elucidated for different AAV serotypes through structural approaches, including AAV1 and AAV5, this study identified two common AAV epitopes: one on the 3-fold protrusions and one on the 2/5-fold wall. These epitopes coincide with regions with the highest sequence and structure diversity between AAV serotypes and correspond to regions determining receptor recognition and transduction phenotypes. Significantly, these locations overlap the two dominant epitopes reported for autonomous parvoviruses. Thus, rather than the amino acid sequence alone, the antigenic sites of parvoviruses appear to be dictated by structural features evolved to enable specific infectious functions.  

5.1571           Perineuronal net digestion with chondroitinase restores memory in mice with tau pathology

Yang, S., Cacquevel, M., Saksida, L.M., Bussey, T.J., Schneider, B.L., Aebischer, P., Melani, R., Pizzorusso, T., Fawcett, J.W. and Grazia Spillantini, M. Exp. Neurol., 265, 48-58 (2015)   Alzheimer's disease is the most prevalent tauopathy and cause of dementia. We investigate the hypothesis that reactivation of plasticity can restore function in the presence of neuronal damage resulting from tauopathy. We investigated two models with tau hyperphosphorylation, aggregation and neurodegeneration: a transgenic mouse model in which the mutant P301S tau is expressed in neurons (Tg P301S), and a model in which an adeno-associated virus expressing P301S tau (AAV-P301S) was injected in the perirhinal cortex, a region critical for object recognition (OR) memory. Both models show profound loss of OR memory despite only 15% neuronal loss in the Tg P301S and 26% in AAV-P301S-injected mice. Recordings from perirhinal cortex slices of 3 month-old P301S transgenic mice showed a diminution in synaptic transmission following temporal stimulation. Chondroitinase ABC (ChABC) can reactivate plasticity and affect memory through actions on perineuronal nets. ChABC was injected into the perirhinal cortex and animals were tested for OR memory 1 week later, demonstrating restoration of OR memory to normal levels. Synaptic transmission indicated by fEPSP amplitude was restored to control levels following ChABC treatment. ChABC did not affect the progression of neurodegenerative tauopathy. These findings suggest that increasing plasticity by manipulation of perineuronal nets offers a novel therapeutic approach to the treatment of memory loss in neurodegenerative disorders.  

5.1572           SKI-1/S1P inhibitor PF-429242 impairs the onset of HCV infection

Blancet, M., Sureau, C., Guevin, C., Seidah, N.GX. and labonte, P. Antiviral Res., 115, 94-104 (2015)   Worldwide, approximately 170 million individuals are afflicted with chronic hepatitis C virus (HCV) infection. To prevent the development of inherent diseases such as cirrhosis and hepatocellular carcinoma, tremendous efforts have been made, leading to the development of promising new treatments. However, their efficiency is still dependent on the viral genotype. Additionally, these treatments that target the virus directly can trigger the emergence of resistant variants. In a previous study, we have demonstrated that a long-term (72 h) inhibition of SKI-1/S1P, a master lipogenic pathway regulator through activation of SREBP, resulted in impaired HCV genome replication and infectious virion secretion. In the present study, we sought to investigate the antiviral effect of the SKI-1/S1P small molecule inhibitor PF-429242 at the early steps of the HCV lifecycle. Our results indicate a very potent antiviral effect of the inhibitor early in the viral lifecycle and that the overall action of the compound relies on two different contributions. The first one is SREBP/SKI-1/S1P dependent and involves LDLR and NPC1L1 proteins, while the second one is SREBP independent. Overall, our study confirms that SKI-1/S1P is a relevant target to impair HCV infection and that PF-429242 could be a promising candidate in the field of HCV infection treatment.  

5.1573           Induction of an Embryonic Mouse Innate Immune Response following Inoculation In Utero with Minute Virus of Mice

Rostovsky, I. and Davis, C.

1. Virol.,89(4), 2182-2191 (2015)

  We used an embryonic-infection model system to show that MVMp, the prototypic minute virus of mice (MVM) serotype and a member of the genus Protoparvovirus, triggers a comprehensive innate immune response in the developing mouse embryo. Direct inoculation of the midtrimester embryo in utero with MVMp results in a widespread, productive infection. During a 96-h infection course, embryonic beta interferon (IFN-β) and IFN-γ transcription were induced 90- and 60-fold, respectively. IFN-β levels correlated with the embryo viral burden, while IFN-γ levels first increased and then decreased. Production of proinflammatory cytokines, interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α), also increased, but by smaller amounts, approximately 7-fold each. We observed increased levels of downstream antiviral effector molecules, PKR and phosphorylated STAT2. Finally, we showed that there is an immune cell response to the virus infection. Infected tissues in the embryo exhibited an increased density of mature leukocytes compared to the same tissues in uninfected embryos. The responses we observed were almost completely restricted to the infected embryos. Uninfected littermates routinely exhibited small increases in innate immune components that rarely reached statistical significance compared to negative controls. Similarly, the placentae of infected embryos did not show any significant increase in transcription of innate immune cytokines. Since the placenta has both embryonic and maternal components, we suggest there is minimal involvement of the dam in the response to infection.  

5.1574           Analysis of Human T-Cell Leukemia Virus Type 1 Particles by Using Cryo-Electron Tomography

Cao, S., Maldonado, J.O., Grigsby, I.F., Mansky, L.M. and Zhang, W.

1. Virol., 89(4), 2430-2435 (2015)

  The particle structure of human T-cell leukemia virus type 1 (HTLV-1) is poorly characterized. Here, we have used cryo-electron tomography to analyze HTLV-1 particle morphology. Particles produced from MT-2 cells were polymorphic, roughly spherical, and varied in size. Capsid cores, when present, were typically poorly defined polyhedral structures with at least one curved region contacting the inner face of the viral membrane. Most of the particles observed lacked a defined capsid core, which likely impacts HTLV-1 particle infectivity.  

5.1575           Effect of Galectins on Viral Transmission

Ouellet, M., St-Pierre, C., Tremblay, M.J: and Sato, S. Methods in Mol. Biol., 1207, 397-420 82015)   Recent reports suggest that some galectins bind to enveloped viruses. They include influenza virus, human immunodeficiency virus-1 (HIV-1), human T-cell leukemia virus-1 (HTLV-1), and Nipah virus. It is also suggested that the interaction between viruses and galectins influences viral attachment to their susceptible cells, affecting the viral infectivity. Our work suggests that galectin-1 increases the infectivity of HIV-1 and HTVL-1. Indeed, galectin-1 promotes the initial adsorption of HIV-1 to CD4⁺ cells through its binding to viral envelope gp120 and facilitates HIV-1 infection in a manner that is dependent on its recognition of β-galactoside residues. Thus, as galectin-1 can be considered as a pattern recognition receptor, HIV-1 exploits this host factor to promote its transmission or replication. In this chapter, we describe methods used to investigate this potential role of galectins in HIV-1 infection as a case in point for future studies on galectin–virus interactions.  

5.1576           HPV Binding Assay to Laminin-332/Integrin α6β4 on Human Keratinocytes

Brendle, S.A. and Christensen, N.D. Methods in Mol. Biol., 1249, 53-66 (2015)   Human papillomaviruses (HPVs) have been shown to bind to Laminin-332 (Ln-332) on the extracellular matrix (ECM) secreted by human keratinocytes. The assay described here is an important tool to study HPV receptor binding to the ECM. The assay can also be modified to study the receptors required for HPV infection and for binding to tissues. We previously showed that Ln-332 is essential for the binding of HPV11 to human keratinocytes and that infectious entry of HPV11 requires α6β4 integrin for the transfer of HPV11 from ECM to host cells (Culp et al., J Virol 80:8940–8950, 2006). We also demonstrated that several of the high-risk HPV types (16, 18, 31 and 45) bind to Ln-332 and/or other components of the ECM in vitro (Broutian et al., J Gen Virol 91:531–540, 2010). The exact binding and internalization mechanism(s) for HPV are still under investigation. A better understanding of these mechanisms will aid in the design of therapeutics against HPVs and ultimately help prevent many cancers. In this chapter, we describe the HPV binding assay to Ln-332/integrin α6β4 on human keratinocytes (ECM). We also present data and suggestions for modifying the assay for testing the specificity of HPV for receptors (by blocking receptors) and binding to human tissues (basement membrane, BM) in order to study binding mechanisms.  

5.1577           Mouse Model of Cervicovaginal Papillomavirus Infection

Cuburu, N., Cerio, R.J., Thompson, C.D. and Day, P.M. Methods in Mol. Biol., 1249, 365-379 (2015)   Virtually all cervical cancers are caused by human papillomavirus infections. The efficient assembly of pseudovirus (PsV) particles incorporating a plasmid expressing a reporter gene has been an invaluable tool in the development of in vitro neutralization assays and in studies of the early mechanisms of viral entry in vitro. Here, we describe a mouse model of human papillomavirus PsV infection of the cervicovaginal epithelium that recapitulates the early events of papillomavirus infection in vivo.  

5.1578           Comparative Antiatherogenic Effects of Intravenous AAV8- and AAV2-Mediated ApoA-IMilano Gene Transfer in Hypercholesterolemic Mice

Tian, F., Wang, L., Arias, A., Yang, M., Sharifi, B. and Shah, P.K.

1. Cardiovasc. Pharmacol. Ther., 20(1), 66-75 (2015)

  Apolipoprotein A-IMilano (ApoA-IM), a naturally occurring Arg₁₇₃ to Cys mutant of ApoA-I, has been shown to reduce atherosclerosis in animal models and in a small phase 2 human trial. We have shown superior atheroprotective effects of ApoA-IM gene compared with wild-type ApoA-I gene using transplantation of retrovirally transduced bone marrow in ApoA-I/ApoE null mice. In this study, we compared the antiatherogenic efficacy of ApoA-IM gene transfer using Recombinant adeno-associated virus (rAAV) 2 or rAAV8 as vectors in ApoA-I/ApoE null mice. Mice received a single intravenous injection of 1.2 × 10¹² vector genomes of AAV2 or AAV8 vectors expressing ApoA-IM or control empty vectors (12 mice/group). Circulating levels of ApoA-IM were higher in recipients of AAV8 compared with AAV2 at 4, 12, and 20 weeks postinjection. Qualitative polymerase chain reaction analysis of RNA collected from different tissues showed that the AAV8-mediated gene transfer resulted in a more efficient transgene expression in the heart, brain, liver, lung, spleen, and kidney of the recipient mice compared with AAV2. Intravenous AAV8-ApoA-IM injection reduced atherosclerosis in the whole aorta (P < .01), aortic sinuses (P < .05), and brachiocephalic arteries (P < .05) compared with the vector control, whereas there was no statistically significant reduction in atherosclerosis in mice receiving intravenous AAV2-ApoA-IM. The ApoA-IM gene was expressed in the aortic tissue of mice receiving AAV8 ApoA-IM but not in those receiving AAV2 ApoA-IM. Immunostaining showed that compared with the vector control, there was reduced macrophage content in the brachiocephalic (P < .05) and aortic sinus plaques (P < .05) of AAV8 ApoA-IM recipients but not in the recipients of AAV2 ApoA-IM. Thus, intravenous injection of AAV8 is more effective than intravenous injection of AAV2 in the expression of ApoA-IM gene. These data provide support for the potential feasibility of this approach for atheroprotection in humans.  

5.1579           Structural basis for serotonergic regulation of neural circuits in the mouse olfactory bulb

Suzuki, Y., Kiyokage, E., Sohn, J., Hioki, H. and Toida, K.

1. Comp. Neurol., 523(2), 262-280 (2015)

  Olfactory processing is well known to be regulated by centrifugal afferents from other brain regions, such as noradrenergic, acetylcholinergic, and serotonergic neurons. Serotonergic neurons widely innervate and regulate the functions of various brain regions. In the present study, we focused on serotonergic regulation of the olfactory bulb (OB), one of the most structurally and functionally well-defined brain regions. Visualization of a single neuron among abundant and dense fibers is essential to characterize and understand neuronal circuits. We accomplished this visualization by successfully labeling and reconstructing serotonin (5-hydroxytryptamine: 5-HT) neurons by infection with sindbis and adeno-associated virus into dorsal raphe nuclei (DRN) of mice. 5-HT synapses were analyzed by correlative confocal laser microscopy and serial-electron microscopy (EM) study. To further characterize 5-HT neuronal and network function, we analyzed whether glutamate was released from 5-HT synaptic terminals using immuno-EM. Our results are the first visualizations of complete 5-HT neurons and fibers projecting from DRN to the OB with bifurcations. We found that a single 5-HT axon can form synaptic contacts to both type 1 and 2 periglomerular cells within a single glomerulus. Through immunolabeling, we also identified vesicular glutamate transporter 3 in 5-HT neurons terminals, indicating possible glutamatergic transmission. Our present study strongly implicates the involvement of brain regions such as the DRN in regulation of the elaborate mechanisms of olfactory processing. We further provide a structure basis of the network for coordinating or linking olfactory encoding with other neural systems, with special attention to serotonergic regulation.  

5.1580           Recombinant adenoassociated virus 2/5-mediated gene transfer is reduced in the aged rat midbrain

Polinski, N.K., Gombash, S.E., Manfredsson, F.P., Lipton, J.W., Kemp, C.J., Cole-Strauss, A., Kanaan, N.M., Steece-Collier, K., Kuhn, N.C., Wohlgenant, S.L. and Sortwell, C.E. Neurobiol. of Aging, 36, 1110-1120 (2015)   Clinical trials are examining the efficacy of viral vector-mediated gene delivery for treating Parkinson's disease. Although viral vector strategies have been successful in preclinical studies, to date clinical trials have disappointed. This may be because of the fact that preclinical studies fail to account for aging. Aging is the single greatest risk factor for developing Parkinson's disease and age alters cellular processes utilized by viral vectors. We hypothesized that the aged brain would be relatively resistant to transduction when compared with the young adult. We examined recombinant adeno-associated virus 2/5-mediated green fluorescent protein (rAAV2/5 GFP) expression in the young adult and aged rat nigrostriatal system. GFP overexpression was produced in both age groups. However, following rAAV2/5 GFP injection to the substantia nigra aged rats displayed 40%–60% less GFP protein in the striatum, regardless of rat strain or duration of expression. Furthermore, aged rats exhibited 40% fewer cells expressing GFP and 4-fold less GFP messenger RNA. rAAV2/5-mediated gene transfer is compromised in the aged rat midbrain, with deficiencies in early steps of transduction leading to significantly less messenger RNA and protein expression.  

5.1581           Functional analysis of porcine reproductive and respiratory syndrome virus N-glycans in infection of permissive cells

Li, J. and Murtagh, M.P. Virology, 477, 82-88 (2015)   The role of envelope protein-linked N-glycans in porcine reproductive and respiratory syndrome virus (PRRSV) infection of permissive cells was examined. N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) oligomer-specific lectins bound to PRRSV and blocked virus attachment, resulting in reduced viral infection. However, addition of GlcNAc oligomers and LacNAc to cell culture together with PRRSV did not block infection. Removal or alteration of envelope protein-linked N-glycans also did not affect virus infection, indicating that PRRSV N-glycans are not required for virus infection. These findings show that steric hindrance of glycans on the PRRSV envelope by lectins or, presumably, other space-filling molecules, may interfere nonspecifically with infection by blocking protein interactions with cell surface receptors. Glycans themselves appear not to be required for infection of permissive cells, but may have important roles in avoidance of host immunity and in protein structure, intracellular virion growth and assembly.  

5.1582           Marinesco-Sjögren syndrome protein SIL1 regulates motor neuron subtype-selective ER stress in ALS

De L’Etang, A.F., Maharjan, N., Brana, M.C., Ruegsegger, C., Rehmann, R., Goswami, A., Roos, A., Troost, D., Schneider, B.L., Weis, J. and Saxene, S. Nature Neurosci., 18(2), 227-238 (2015)   Mechanisms underlying motor neuron subtype–selective endoplasmic reticulum (ER) stress and associated axonal pathology in amyotrophic lateral sclerosis (ALS) remain unclear. Here we show that the molecular environment of the ER between motor neuron subtypes is distinct, with characteristic signatures. We identify cochaperone SIL1, mutated in Marinesco-Sjögren syndrome (MSS), as being robustly expressed in disease-resistant slow motor neurons but not in ER stress–prone fast-fatigable motor neurons. In a mouse model of MSS, we demonstrate impaired ER homeostasis in motor neurons in response to loss of SIL1 function. Loss of a single functional Sil1 allele in an ALS mouse model (SOD1-G93A) enhanced ER stress and exacerbated ALS pathology. In SOD1-G93A mice, SIL1 levels were progressively and selectively reduced in vulnerable fast-fatigable motor neurons. Mechanistically, reduction in SIL1 levels was associated with lowered excitability of fast-fatigable motor neurons, further influencing expression of specific ER chaperones. Adeno-associated virus–mediated delivery of SIL1 to familial ALS motor neurons restored ER homeostasis, delayed muscle denervation and prolonged survival.  

5.1583           microRNA‐379 couples glucocorticoid hormones to dysfunctional lipid homeostasis

de Guia, R.M. et al EMBO J., 34(3), 344-360 (2015)   In mammals, glucocorticoids (GCs) and their intracellular receptor, the glucocorticoid receptor (GR), represent critical checkpoints in the endocrine control of energy homeostasis. Indeed, aberrant GC action is linked to severe metabolic stress conditions as seen in Cushing's syndrome, GC therapy and certain components of the Metabolic Syndrome, including obesity and insulin resistance. Here, we identify the hepatic induction of the mammalian conserved microRNA (miR)‐379/410 genomic cluster as a key component of GC/GR‐driven metabolic dysfunction. Particularly, miR‐379 was up‐regulated in mouse models of hyperglucocorticoidemia and obesity as well as human liver in a GC/GR‐dependent manner. Hepatocyte‐specific silencing of miR‐379 substantially reduced circulating very‐low‐density lipoprotein (VLDL)‐associated triglyceride (TG) levels in healthy mice and normalized aberrant lipid profiles in metabolically challenged animals, mediated through miR‐379 effects on key receptors in hepatic TG re‐uptake. As hepatic miR‐379 levels were also correlated with GC and TG levels in human obese patients, the identification of a GC/GR‐controlled miRNA cluster not only defines a novel layer of hormone‐dependent metabolic control but also paves the way to alternative miRNA‐based therapeutic approaches in metabolic dysfunction.  

5.1584           Sorting of small infectious virus particles by flow virometry reveals distinct infectivity profiles

Gaudin, R. and barteneva, N.S. Nature Communications, 6:6022 (2015)   The nature and concentration of lipids and proteins at the surface of viruses are essential parameters for determining particle infectiveness. Historically, averaged bulk analysis of viral particles has been the primary method to quantitatively investigate these parameters, though this neglects heterogeneity within populations. Here we analyse the properties of Junin virus particles using a sensitive flow virometry assay and further sort virions while conserving their infectiveness. This method allows us to characterize the relationship between infectivity, virus size and RNA content and to compare particles secreted by Vero cells with those from physiologically relevant human primary macrophages. Our study highlights significant differences in particle infectivity according to its nature, the type of producer cells and the lipid membrane composition at the budding site. Together, our results present the flow virometry assay as a powerful and versatile tool to define virus particle profiles.  

5.1585           Microglia constitute a barrier that prevents neurotoxic protofibrillar ​Aβ42 hotspots around plaques

Condello, C., Yuan, P., Schain, A. and Grutzendler, J. Nature Communications, 6:6176 (2015)   In Alzheimer’s disease (AD), ​β-amyloid (​Aβ) plaques are tightly enveloped by microglia processes, but the significance of this phenomenon is unknown. Here we show that microglia constitute a barrier with profound impact on plaque composition and toxicity. Using high-resolution confocal and in vivo two-photon imaging in AD mouse models, we demonstrate that this barrier prevents outward plaque expansion and leads to compact plaque microregions with low ​Aβ42 affinity. Areas uncovered by microglia are less compact but have high ​Aβ42 affinity, leading to the formation of protofibrillar ​Aβ42 hotspots that are associated with more severe axonal dystrophy. In ageing, microglia coverage is reduced leading to enlarged protofibrillar ​Aβ42 hotspots and more severe neuritic dystrophy. ​CX3CR1 gene deletion or anti-​Aβ immunotherapy causes expansion of microglia coverage and reduced neuritic dystrophy. Failure of the microglia barrier and the accumulation of neurotoxic protofibrillar ​Aβ hotspots may constitute novel therapeutic and clinical imaging targets for AD.  

5.1586           The Epstein-Barr Virus BamHI C Promoter Is Not Essential for B Cell Immortalization In Vitro, but It Greatly Enhances B Cell Growth Transformation

Tierney, R.J., nagra, J., Rowe, M., Bell, A.I. and Rickinson, A.B.

1. Virol., 89(5), 2483-2493 (2015)

  Epstein-Barr virus (EBV) infection of B cells leads to the sequential activation of two viral promoters, Wp and Cp, resulting in the expression of six EBV nuclear antigens (EBNAs) and the viral Bcl2 homologue BHRF1. The viral transactivator EBNA2 is required for this switch from Wp to Cp usage during the initial stages of infection. EBNA2-dependent Cp transcription is mediated by the EBNA2 response element (E2RE), a region that contains at least two binding sites for cellular factors; one of these sites, CBF1, interacts with RBP-JK, which then recruits EBNA2 to the transcription initiation complex. Here we demonstrate that the B cell-specific transcription factor BSAP/Pax5 binds to a second site, CBF2, in the E2RE. Deletion of the E2RE in the context of a recombinant virus greatly diminished levels of Cp-initiated transcripts during the initial stages of infection but did not affect the levels of Wp-initiated transcripts or EBNA mRNAs. Consistent with this finding, viruses deleted for the E2RE were not markedly impaired in their ability to induce B cell transformation in vitro. In contrast, a larger deletion of the entire Cp region did reduce EBNA mRNA levels early after infection and subsequently almost completely ablated lymphoblastoid cell line (LCL) outgrowth. Notably, however, rare LCLs could be established following infection with Cp-deleted viruses, and these were indistinguishable from wild-type-derived LCLs in terms of steady-state EBV gene transcription. These data indicate that, unlike Wp, Cp is dispensable for the virus' growth-transforming activity.  

5.1587           Alpha-Defensin HD5 Inhibits Furin Cleavage of Human Papillomavirus 16 L2 To Block Infection

Wiens, M.E. and Smith, J.G.

1. Virol., 89(5), 2866-2874 (2015)

  Human papillomavirus (HPV) is a significant oncogenic virus, but the innate immune response to HPV is poorly understood. Human α-defensin 5 (HD5) is an innate immune effector peptide secreted by epithelial cells in the genitourinary tract. HD5 is broadly antimicrobial, exhibiting potent antiviral activity against HPV at physiologic concentrations; however, the specific mechanism of HD5-mediated inhibition against HPV is unknown. During infection, the HPV capsid undergoes several critical cell-mediated viral protein processing steps, including unfolding and cleavage of the minor capsid protein L2 by host cyclophilin B and furin. Using HPV16 pseudovirus, we show that HD5 interacts directly with the virus and inhibits the furin-mediated cleavage of L2 at the cell surface during infection at a step downstream of the cyclophilin B-mediated unfolding of L2. Importantly, HD5 does not affect the enzymatic activity of furin directly. Thus, our data support a model in which HD5 prevents furin from accessing L2 by occluding the furin cleavage site via direct binding to the viral capsid.  

5.1588           The L1 protein of human papilloma virus 16 expressed by a fowlpox virus recombinant can assemble into virus-like particles in mammalian cell lines but elicits a non-neutralising humoral response

Bissa, M., Zanotto, C., Pacchiono, S., Volonte, L., Venuti, A., Lembo, D., De Giuli Morghen, C. and Radaelli, A. Antviral Res., 116, 67-75 (2015)   Human papilloma virus (HPV)-16 is the prevalent genotype associated with cervical tumours. Virus-like-particle (VLP)-based vaccines have proven to be effective in limiting new infections of high-risk HPVs, but their high cost has hampered their use, especially in the poor developing countries. Avipox-based recombinants are replication-restricted to avian species and represent efficient and safe vectors also for immunocompromised hosts, as they can elicit a complete immune response. A new fowlpox virus recombinant encoding HPV-L1 (FP_L1) was engineered and evaluated side-by-side with a FP recombinant co-expressing L1 and green fluorescent protein (FP_L1GFP) for correct expression of L1 in vitro in different cell lines, as confirmed by Western blotting, immunofluorescence, real-time PCR, and electron microscopy. Mice were also immunised to determine its immunogenicity. Here, we demonstrate that the FP_L1 recombinant better expresses L1 in the absence of GFP, correctly assembles structured capsomers into VLPs, and elicits an immune response in a preclinical animal model. To our knowledge, this is the first report of HPV VLPs assembled in eukaryotic cells using an avipox recombinant.  

5.1589           Development and evaluation of multiplexed immunoassay for detection of antibodies to HPV vaccine types

Panicker, XG., Rajbhandari, I., Gurbaxani, B.M., Querec, ST.D. and Unger e.r.

1. Immunol. Methods, 417, 107-114 (2015)

  Reliable antibody based-assays are needed to evaluate the immunogenicity of current vaccines, impact of altered dosing schemes or of new vaccine formulations. An ideal assay platform would allow multiplex type-specific detection with minimal sample requirement. We used the Meso Scale Discovery (MSD) electrochemiluminescence based detection platform to develop a multiplex direct virus-like particle (VLP) ELISA to detect antibodies to HPV 6, 11, 16, and 18 with a protocol developed for detection using the SI 6000 imager (M4ELISA). MSD prepared the plates in the 7-spot/well format, using the purified VLPs (4 spots) and PBS + BSA pH 7.4 (3 blank spots). Three-point titrations and the parallel line method were used to calculate antibody levels. Dynamic range, precision, and stability of pre-printed plates were determined using a panel of previously characterized sera. Cut-off values using children's sera were established using 99% RLU limits based on the 4-parameter Johnson Su best fit curve. Results of the M4ELISA were compared to competitive Luminex Immunoassay (cLIA) on n = 4454 sera from a predominantly unvaccinated cohort. Using a VLP coating concentration of 80 μg/ml with BSA provided the most robust RLU signal for all types. The dynamic range of the assay was about 1000 fold, with assay variability under 25% for each of the four vaccine types. Long-term stability of the plates extended to about 7 months from the time plates was received in the laboratory after printing. There was moderate agreement (κ = 0.38–0.54) between M4ELISA and cLIA, with antibody detection for each of the 4 types more frequent with M4ELISA. Quantitative analysis however showed a good correlation between concordant samples by both assays (ρ ≥ 0.6). The MSD platform shows promise for simultaneous quantitation of the antibody responses to four HPV vaccine types in a high-throughput manner.  

5.1590           Direct Binding of Retromer to Human Papillomavirus Type 16 Minor Capsid Protein L2 Mediates Endosome Exit during Viral Infection

Popa, A., Zhang, W., Harrison, M.S., Goodner, K., Kazakow, T., Goodwin, E.C., Lipovsky, A., Burd, C.G. and DiMaio, D. PloS Pathogens, 11(2), e1004699 (2015)   Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.  

5.1591           Complex I Subunit Gene Therapy With NDUFA6 Ameliorates Neurodegeneration in EAE

Talla, V., Koilkonda, R., Porciatti, V., Chiodo, V., Boye, S.L., Hauswirth, W.W. and Guy, J. Invest. Ophthalmol. Vis. Sci., 56(2), 1129-1140 (2015)   Purpose. To address the permanent disability induced by mitochondrial dysfunction in experimental autoimmune encephalomyelitis (EAE). Methods. Mice sensitized for EAE were rescued by intravitreal injection of adeno-associated viral vector serotype 2 with the complex I subunit gene scAAV-NDUFA6Flag. Controls were injected with a mitochondrially targeted red fluorescent protein (scAAV-COX8-cherry). Another group received scAAV-COX8-cherry, but was not sensitized for EAE. Serial pattern electroretinograms (PERGs) and optical coherent tomography (OCT) evaluated visual function and structure of the retina at 1, 3, and 6 months post injection (MPI). Treated mice were killed 6 MPI for histopathology. Immunodetection of cleaved caspase 3 gauged apoptosis. Complex I activity was assessed spectrophotometrically. Expression of NDUFA6Flag in the retina and optic nerve were evaluated between 1 week to 1 month post injection by RT-PCR, immunofluorescence and immunoblotting. Results. Reverse transcription-PCR and immunoblotting confirmed NDUFA6Flag overexpression with immunoprecipitation and blue native PAGE showing integration into murine complex I. Overexpression of NDUFA6Flag in the visual system of EAE mice rescued retinal complex I activity completely, axonal loss by 73%, and retinal ganglion cell (RGC) loss by 88%, RGC apoptosis by 66%, and restored the 33% loss of complex I activity in EAE to normal levels; thereby, preventing loss of vision indicated by the 43% reduction in the PERG amplitudes of EAE mice. Conclusions. NDUFA6 gene therapy provided long-term suppression of neurodegeneration in the EAE animal model suggesting that it may also ameliorate the mitochondrial dysfunction associated with permanent disability in optic neuritis and MS patients.  

5.1592           N-glycosylation profiling of porcine reproductive and respiratory syndrome virus envelope glycoprotein 5

Li, J., tao, S., Orlando, SR. and Murtaugh, M.P. Virology, 478, 86-98 (2015)   Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-sense ssRNA virus whose envelope contains four glycoproteins and three nonglycosylated proteins. Glycans of major envelope glycoprotein 5 (GP5) are proposed as important for virus assembly and entry into permissive cells. Structural characterization of GP5 glycans would facilitate the mechanistic understanding of these processes. Thus, we purified the PRRSV type 2 prototype strain, VR2332, and analyzed the virion-associated glycans by both biochemical and mass spectrometric methods. Endoglycosidase digestion showed that GP5 was the primary protein substrate, and that the carbohydrate moieties were primarily complex-type N-glycans. Mass spectrometric analysis (HPLC-ESI-MS/MS) of GP5 N-glycans revealed an abundance of N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) oligomers in addition to sialic acids. GlcNAc and LacNAc accessibility to ligands was confirmed by lectin co-precipitation. Our findings help to explain PRRSV infection of cells lacking sialoadhesin and provide a glycan database to facilitate molecular structural studies of PRRSV.  

5.1593           Carbonic Anhydrase-8 Regulates Inflammatory Pain by Inhibiting the ITPR1-Cytosolic Free Calcium Pathway

Zhuang, G.Z. et al PloS One, 10(3), e0118273 (2015)   Calcium dysregulation is causally linked with various forms of neuropathology including seizure disorders, multiple sclerosis, Huntington’s disease, Alzheimer’s, spinal cerebellar ataxia (SCA) and chronic pain. Carbonic anhydrase-8 (Car8) is an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1), which regulates intracellular calcium release fundamental to critical cellular functions including neuronal excitability, neurite outgrowth, neurotransmitter release, mitochondrial energy production and cell fate. In this report we test the hypothesis that Car8 regulation of ITPR1 and cytoplasmic free calcium release is critical to nociception and pain behaviors. We show Car8 null mutant mice (MT) exhibit mechanical allodynia and thermal hyperalgesia. Dorsal root ganglia (DRG) from MT also demonstrate increased steady-state ITPR1 phosphorylation (pITPR1) and cytoplasmic free calcium release. Overexpression of Car8 wildtype protein in MT nociceptors complements Car8 deficiency, down regulates pITPR1 and abolishes thermal and mechanical hypersensitivity. We also show that Car8 nociceptor overexpression alleviates chronic inflammatory pain. Finally, inflammation results in downregulation of DRG Car8 that is associated with increased pITPR1 expression relative to ITPR1, suggesting a possible mechanism of acute hypersensitivity. Our findings indicate Car8 regulates the ITPR1-cytosolic free calcium pathway that is critical to nociception, inflammatory pain and possibly other neuropathological states. Car8 and ITPR1 represent new therapeutic targets for chronic pain.  

5.1594           Effectiveness of gene delivery systems for pluripotent and differentiated cells

Rapti, K., Stillitano, F., Karakikes, I., Nonnenmacher, M., Weber, T., Hulot, J-S. and Hajjar, R.J. Molecular Therapy – Methods & Clinical Development, 2:14067 (2015)   Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) assert a great future for the cardiovascular diseases, both to study them and to explore therapies. However, a comprehensive assessment of the viral vectors used to modify these cells is lacking. In this study, we aimed to compare the transduction efficiency of recombinant adeno-associated vectors (AAV), adenoviruses and lentiviral vectors in hESC, hiPSC, and the derived cardiomyocytes. In undifferentiated cells, adenoviral and lentiviral vectors were superior, whereas in differentiated cells AAV surpassed at least lentiviral vectors. We also tested four AAV serotypes, 1, 2, 6, and 9, of which 2 and 6 were superior in their transduction efficiency. Interestingly, we observed that AAVs severely diminished the viability of undifferentiated cells, an effect mediated by induction of cell cycle arrest genes and apoptosis. Furthermore, we show that the transduction efficiency of the different viral vectors correlates with the abundance of their respective receptors. Finally, adenoviral delivery of the calcium-transporting ATPase SERCA2a to hESC and hiPSC-derived cardiomyocytes successfully resulted in faster calcium reuptake. In conclusion, adenoviral vectors prove to be efficient for both differentiated and undifferentiated lines, whereas lentiviral vectors are more applicable to undifferentiated cells and AAVs to differentiated cells.  

5.1595           Determining the role of IL-4 induced neuroinflammation in microglial activity and amyloid-β using BV2 microglial cells and APP/PS1 transgenic mice

Latta, C.H., Sudduth, T.L., Weekman, E.M., Brothers, H.M., Abner, E.L., Popa, G.J., Mendenhall, M.D., XGonzalez-Oregon, F., Braun, K. and Wilcock, D.M.

1. Neuroinflammation, 12:41 (2015)

  Background Microglia are considered the resident immune cells of the central nervous system (CNS). In response to harmful stimuli, an inflammatory reaction ensues in which microglia are activated in a sequenced spectrum of pro- and antiinflammatory phenotypes that are akin to the well-characterized polarization states of peripheral macrophages. A “classically” activated M1 phenotype is known to eradicate toxicity. The transition to an “alternatively” activated M2 phenotype encompasses neuroprotection and repair. In recent years, inflammation has been considered an accompanying pathology in response to the accumulation of extracellular amyloid-β (Aβ) in Alzheimer’s disease (AD). This study aimed to drive an M2a-biased immune phenotype with IL-4 in vitro and in vivo and to determine the subsequent effects on microglial activation and Aβ pathology. Methods In vitro, exogenous IL-4 was applied to BV2 microglial cell cultures to evaluate the temporal progression of microglial responses. In vivo, intracranial injections of an adeno-associate-virus (AAV) viral vector were performed to assess long-term expression of IL-4 in the frontal cortex and hippocampus of Aβ-depositing, APP/PS1 transgenic mice. Quantitative real-time PCR was used to assess the fold change in expression of biomarkers representing each of the microglial phenotypes in both the animal tissue and the BV2 cells. ELISAs quantified IL-4 expression and Aβ levels. Histological staining permitted quantification of microglial and astrocytic activity. Results Both in vitro and in vivo models showed an enhanced M2a phenotype, and the in vivo model revealed a trend toward a decreased trend in Aβ deposition. Conclusions In summary, this study offers insight into the therapeutic potential of microglial immune response in AD.  

5.1596           Overexpression of cystatin C in synovium does not reduce synovitis or cartilage degradation in established osteoarthritis

Kyostio-Moore, S., Piraino, S., Berthelette, P., Moran, N., Serriello, J., Bendele, A., Sookdeo, C., Nambiar, B., Ewing, P., Armentano, D. and Matthews, G.L. Arthritis Research & Therapy, 17:5 (2015)   Introduction Cathepsin K (catK) expression is increased in cartilage, bone and synovium during osteoarthritis (OA). To study the role of catK expression and elevated cathepsin activity in the synovium on cartilage destruction in established OA, we overexpressed cystatin C (cysC), a natural cysteine protease inhibitor, in the synovium of rabbit OA joints. Methods The ability of cysC to inhibit activity of cathepsins in rabbit OA synovium lysates was tested in vitro using protease activity assay. In vivo, the tissue localization of recombinant adeno-associated virus (rAAV) with LacZ gene after intra-articular injection was determined by β-galactosidase staining of rabbit joints 4 weeks later. To inhibit cathepsin activity in the synovium, a rAAV2-encoding cysC was delivered intra-articularly into rabbit joints 4 weeks after OA was induced by anterior cruciate ligament transection (ACLT). Seven weeks postinjection, endogenous catK and cysC levels as well as the vector-derived cysC expression in the synovium of normal and OA joints were examined by RNA quantification. Synovial cathepsin activity and catK, catB and catL protein levels were determined by activity and Western blot analyses, respectively. Synovitis and cartilage degradation were evaluated by histopathological scoring. Results In vitro, the ability of cysC to efficiently inhibit activity of purified catK and OA-induced cathepsins in rabbit synovial lysates was demonstrated. In vivo, the intra-articular delivery of rAAV2/LacZ showed transduction of mostly synovium. Induction of OA in rabbit joints resulted in fourfold increase in catK mRNA compared to sham controls while no change was detected in endogenous cysC mRNA levels in the synovium. Protein levels for catK, catB and catL were also increased in the synovium with a concomitant fourfold increase in cathepsin activity. Joints treated with rAAV2/cysC showed both detection of vector genomes and vector-derived cysC transcripts in the synovium. Production of functional cysC by the vector was demonstrated by complete block of cathepsin activity in the synovium. However, this did not decrease synovitis, bone sclerosis or progression of cartilage degradation. Conclusions Increased production of natural cathepsin inhibitor, cysC, in OA synovium does not alleviate synovitis or cartilage pathology during a preexisting OA.  

5.1597           Characterization of Hepatitis C Virus Interaction with Heparan Sulfate Proteoglycans

Xu, Y., Martinez, P., Sweron, K., Luo, G., Allain, F., Dubuisson, J. and Belouzard, S.

1. Virol., 89(7), 3846-3858 (2015)

  Hepatitis C virus (HCV) entry involves binding to cell surface heparan sulfate (HS) structures. However, due to the lipoprotein-like structure of HCV, the exact contribution of virion components to this interaction remains controversial. Here, we investigated the relative contribution of HCV envelope proteins and apolipoprotein E in the HS-binding step. Deletion of hypervariable region 1, a region previously proposed to be involved in HS binding, did not alter HCV virion binding to HS, indicating that this region is not involved in this interaction in the context of a viral infection. Patient sera and monoclonal antibodies recognizing different regions of HCV envelope glycoproteins were also used in a pulldown assay with beads coated with heparin, a close HS structural homologue. Although isolated HCV envelope glycoproteins could interact with heparin, none of these antibodies was able to interfere with the virion-heparin interaction, strongly suggesting that at the virion surface, HCV envelope glycoproteins are not accessible for HS binding. In contrast, results from kinetic studies, heparin pulldown experiments, and inhibition experiments with anti-apolipoprotein E antibodies indicated that this apolipoprotein plays a major role in HCV-HS interaction. Finally, characterization of the HS structural determinants required for HCV infection by silencing of the enzymes involved in the HS biosynthesis pathway and by competition with modified heparin indicated that N- and 6-O-sulfation but not 2-O-sulfation is required for HCV infection and that the minimum HS oligosaccharide length required for HCV infection is a decasaccharide. Together, these data indicate that HCV hijacks apolipoprotein E to initiate its interaction with specific HS structures.  

5.1598           T160-phosphorylated CDK2 defines threshold for HGF-dependent proliferation in primary hepatocytes

Mueller, S., Huard, J., Waldow, K., Huang,X., D'Alessandro, L.A., Bohl, S.,Börner, K., Grimm, D., Klamt, S., Klingmüller, U. and Schilling, M. Mol. Sys.Biol., 11:795 (2015)   Liver regeneration is a tightly controlled process mainly achieved by proliferation of usually quiescent hepatocytes. The specific molecular mechanisms ensuring cell division only in response to proliferative signals such as hepatocyte growth factor (HGF) are not fully understood. Here, we combined quantitative time‐resolved analysis of primary mouse hepatocyte proliferation at the single cell and at the population level with mathematical modeling. We showed that numerous G1/S transition components are activated upon hepatocyte isolation whereas DNA replication only occurs upon additional HGF stimulation. In response to HGF, Cyclin:CDK complex formation was increased, p21 rather than p27 was regulated, and Rb expression was enhanced. Quantification of protein levels at the restriction point showed an excess of CDK2 over CDK4 and limiting amounts of the transcription factor E2F‐1. Analysis with our mathematical model revealed that T160 phosphorylation of CDK2 correlated best with growth factor‐dependent proliferation, which we validated experimentally on both the population and the single cell level. In conclusion, we identified CDK2 phosphorylation as a gate‐keeping mechanism to maintain hepatocyte quiescence in the absence of HGF.  

5.1599           PCSK9, apolipoprotein E and lipoviral particles in chronic hepatitis C genotype 3: Evidence for genotype-specific regulation of lipoprotein metabolism

Bridge, S.H., Sheridan, D.A., Felmlee, D.J., Crossey, M.M.E., Fenwick, F.I., Lanyon, C.V., Dubuc; G., Seidah, N.G., Davignon, J., Thomas, H.C., Taylor-Robinson, S.D., Toms, G.L., Neely, R.D.G. and Bassendine, M.F.

1. Hepatol., 62, 763-770 (2015)

  & Aims Hepatitis C virus (HCV) associates with lipoproteins to form “lipoviral particles” (LVPs) that can facilitate viral entry into hepatocytes. Initial attachment occurs via heparan sulphate proteoglycans and low-density lipoprotein receptor (LDLR); CD81 then mediates a post-attachment event. Proprotein convertase subtilisin kexin type 9 (PCSK9) enhances the degradation of the LDLR and modulates liver CD81 levels. We measured LVP and PCSK9 in patients chronically infected with HCV genotype (G)3. PCSK9 concentrations were also measured in HCV-G1 to indirectly examine the role of LDLR in LVP clearance. Methods HCV RNA, LVP (d <1.07 g/ml) and non-LVP (d >1.07 g/ml) fractions, were quantified in patients with HCV-G3 (n = 39) by real time RT-PCR and LVP ratios (LVPr; LVP/(LVP + non-LVP)) were calculated. Insulin resistance (IR) was assessed using the homeostasis model assessment of IR (HOMA-IR). Plasma PCSK9 concentrations were measured by ELISA in HCV-G3 and HCV-G1 (n = 51). Results In HCV-G3 LVP load correlated inversely with HDL-C (r = −0.421; p = 0.008), and apoE (r = −0.428; p = 0.013). The LVPr varied more than 35-fold (median 0.286; range 0.027 to 0.969); PCSK9 was the strongest negative predictor of LVPr (R² = 16.2%; p = 0.012). HOMA-IR was not associated with LVP load or LVPr. PCSK9 concentrations were significantly lower in HCV-G3 compared to HCV-G1 (p <0.001). PCSK9 did not correlate with LDL-C in HCV-G3 or G1. Conclusions The inverse correlation of LVP with apoE in HCV-G3, compared to the reverse in HCV-G1 suggests HCV genotype-specific differences in apoE mediated viral entry. Lower PCSK9 and LDL concentrations imply upregulated LDLR activity in HCV-G3.  

5.1600           Hamburger polyomaviruses

Peretti, A., FitzGerald, P.C., Bliskovsky, V., Buck, C.B. and Pastrana, D.V.

1. Gen. Virol., 96, 833-839 (2015)

  Epidemiological studies have suggested that consumption of beef may correlate with an increased risk of colorectal cancer. One hypothesis to explain this proposed link might be the presence of a carcinogenic infectious agent capable of withstanding cooking. Polyomaviruses are a ubiquitous family of thermostable non-enveloped DNA viruses that are known to be carcinogenic. Using virion enrichment, rolling circle amplification (RCA) and next-generation sequencing, we searched for polyomaviruses in meat samples purchased from several supermarkets. Ground beef samples were found to contain three polyomavirus species. One species, bovine polyomavirus 1 (BoPyV1), was originally discovered as a contaminant in laboratory FCS. A previously unknown species, BoPyV2, occupies the same clade as human Merkel cell polyomavirus and raccoon polyomavirus, both of which are carcinogenic in their native hosts. A third species, BoPyV3, is related to human polyomaviruses 6 and 7. Examples of additional DNA virus families, including herpesviruses, adenoviruses, circoviruses and gyroviruses were also detected either in ground beef samples or in comparison samples of ground pork and ground chicken. The results suggest that the virion enrichment/RCA approach is suitable for random detection of essentially any DNA virus with a detergent-stable capsid. It will be important for future studies to address the possibility that animal viruses commonly found in food might be associated with disease.  

5.1601           Aerosol-Mediated Delivery of AAV2/6-IκBα Attenuates Lipopolysaccharide-Induced Acute Lung Injury in Rats

MacLoughlin, R.J., Higgins, B.D., Devaney, J., O’Toole, D., Laffey, J.G. and O’Brien, T. Human Gene Therapy, 26(1), 36-46 (2015)   Inhibition of the proinflammatory transcription factor NF-κB has previously been shown to attenuate the inflammatory response in tissue after injury. However, the feasibility and efficacy of aerosolized adeno-associated viral (AAV) vector-delivered transgenes to inhibit the NF-κB pathway are less clear. Initial studies optimized the AAV vector for delivery of transgenes to the pulmonary epithelium. The effect of repeated nebulization on the integrity and transduction efficacy of the AAV vector was then examined. Subsequent in vivo studies examined the efficacy of aerosolized rAAV2/6 overexpressing the NF-κB inhibitor IκBα in a rodent endotoxin-induced lung injury model. Initial in vitro investigations indicated that rAAV2/6 was the most effective vector to transduce the lung epithelium, and maintained its integrity and transduction efficacy after repeated nebulization. In our in vivo studies, animals that received aerosolized rAAV2/6-IκBα demonstrated a significant increase in total IκBα levels in lung tissue relative to null vector-treated animals. Aerosolized rAAV2/6-IκBα attenuated endotoxin-induced bronchoalveolar lavage-detected neutrophilia, interleukin-6 and cytokine-induced neutrophil chemoattractant-1 levels, as well as total protein content, and decreased histologic indices of injury. These results demonstrate that aerosolized AAV vectors encoding human IκBα significantly attenuate endotoxin-mediated lung injury and may be a potential therapeutic candidate in the treatment of acute lung injury.  

5.1602           Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

Münch, R.C., Muth, A., Muik, A., Friedel, T., Schmatz, J., Dreier, B., Trkola, A., Plückthun, A., Büning, H. and Buchholz, C.J. Nature Communications, 6:6246 (2015)   We describe receptor-targeted adeno-associated viral (AAV) vectors that allow genetic modification of rare cell types ex vivo and in vivo while showing no detectable off-targeting. Displaying designed ankyrin repeat proteins (DARPins) on the viral capsid and carefully depleting DARPin-deficient particles, AAV vectors were made specific for ​Her2/​neu, ​EpCAM or ​CD4. A single intravenous administration of vector targeted to the tumour antigen ​Her2/​neu was sufficient to track 75% of all tumour sites and to extend survival longer than the cytostatic antibody Herceptin. ​CD4-targeted AAVs hit human ​CD4-positive cells present in spleen of a humanized mouse model, while CD8-positive cells as well as liver or other off-target organs remained unmodified. Mimicking conditions of circulating tumour cells, ​EpCAM-AAV detected single tumour cells in human blood opening the avenue for tumour stem cell tracking. Thus, the approach developed here delivers genes to target cell types of choice with antibody-like specificity.  

5.1603           Development of a Rapid, Robust, and Universal PicoGreen-Based Method to Titer Adeno-Associated Vectors

Piedra, J., Ontiveros, M., Miravet, S., Penalva, C., Monfar, M. and Chillon, M. Human Gene Therapy Methods, 26(1), 35-42 (2015)   Recombinant adeno-associated viruses (rAAVs) are promising vectors in preclinical and clinical assays for the treatment of diseases with gene therapy strategies. Recent technological advances in amplification and purification have allowed the production of highly purified rAAV vector preparations. Although quantitative polymerase chain reaction (qPCR) is the current method of choice for titrating rAAV genomes, it shows high variability. In this work, we report a rapid and robust rAAV titration method based on the quantitation of encapsidated DNA with the fluorescent dye PicoGreen^®. This method allows detection from 3×10¹⁰ viral genome/ml up to 2.4×10¹³ viral genome/ml in a linear range. Contrasted with dot blot or qPCR, the PicoGreen-based assay has less intra- and interassay variability. Moreover, quantitation is rapid, does not require specific primers or probes, and is independent of the rAAV pseudotype analyzed. In summary, development of this universal rAAV-titering method may have substantive implications in rAAV technology.  

5.1604           Catalytic Immunoglobulin Gene Delivery in a Mouse Model of Alzheimer’s Disease: Prophylactic and Therapeutic Applications

Kou, J., yang, J., Lim, J-E., Pattanayak, A., Song, M., Planque, S., Paul, S. and Fukuchi, K-i. Mol. Neurobiol., 51, 43-56 (2015)   Accumulation of amyloid beta-peptide (Aβ) in the brain is hypothesized to be a causal event leading to dementia in Alzheimer’s disease (AD). Aβ vaccination removes Aβ deposits from the brain. Aβ immunotherapy, however, may cause T cell- and/or Fc-receptor-mediated brain inflammation and relocate parenchymal Aβ deposits to blood vessels leading to cerebral hemorrhages. Because catalytic antibodies do not form stable immune complexes and Aβ fragments produced by catalytic antibodies are less likely to form aggregates, Aβ-specific catalytic antibodies may have safer therapeutic profiles than reversibly-binding anti-Aβ antibodies. Additionally, catalytic antibodies may remove Aβ more efficiently than binding antibodies because a single catalytic antibody can hydrolyze thousands of Aβ molecules. We previously isolated Aβ-specific catalytic antibody, IgV_L5D3, with strong Aβ-hydrolyzing activity. Here, we evaluated the prophylactic and therapeutic efficacy of brain-targeted IgV_L5D3 gene delivery via recombinant adeno-associated virus serotype 9 (rAAV9) in an AD mouse model. One single injection of rAAV9-IgV_L5D3 into the right ventricle of AD model mice yielded widespread, high expression of IgV_L5D3 in the unilateral hemisphere. IgV_L5D3 expression was readily detectable in the contralateral hemisphere but to a much lesser extent. IgV_L5D3 expression was also confirmed in the cerebrospinal fluid. Prophylactic and therapeutic injection of rAAV9-IgV_L5D3 reduced Aβ load in the ipsilateral hippocampus of AD model mice. No evidence of hemorrhages, increased vascular amyloid deposits, increased proinflammatory cytokines, or infiltrating T-cells in the brains was found in the experimental animals. AAV9-mediated anti-Aβ catalytic antibody brain delivery can be prophylactic and therapeutic options for AD.    

5.1605           Reduced hepatic lipid content in Pten-haplodeficient mice because of enhanced AKT2/PKBβ activation in skeletal muscle

Schultze, S.M., Diethrich, M., Hynx, D., Geier, A., Niessen, M., Spinas, G.A., Hemmings, B.A. and Tschopp, O. Liver Int., 35(4), 1354-1366 (2015)   Background & Aims Non-alcoholic fatty liver disease (NAFLD) is a major health problem and occurs frequently in the context of metabolic syndrome and type 2 diabetes mellitus. Hepatocyte-specific Pten-deficiency in mice was shown previously to result in hepatic steatosis due to hyperactivated AKT2. However, the role of peripheral insulin-sensitive tissues on PTEN- and AKT2-dependent accumulation of hepatic lipids has not been addressed. Methods Effects of systemically perturbed PTEN/AKT2 signalling on hepatic lipid content were studied in Pten-haplodeficient (Pten^+/−/Akt2^+/+) mice and Pten-haplodeficient mice lacking Akt2 (Pten^+/−/Akt2^−/−). The liver and skeletal muscle were characterized by histology and/or analysis of insulin signalling. To assess the effects of AKT2 activity in skeletal muscle on hepatic lipid content, AKT2 mutants were expressed in skeletal muscle of Pten^+/+/Akt2^+/+ and Pten^+/−/Akt2^+/+ mice using adeno-associated virus 8. Results Pten^+/−/Akt2^+/+ mice were found to have a more than 2-fold reduction in hepatic lipid content, at a level similar to that observed in Pten^+/−/Akt2^−/− mice. Insulin signalling in the livers of Pten^+/−/Akt2^+/+ mice was enhanced, indicating that extrahepatic factors prevent lipid accumulation. The skeletal muscle of Pten^+/−/Akt2^+/+ mice also showed enhanced insulin signalling. Skeletal muscle-specific expression of constitutively active AKT2 reduced hepatic lipid content in Pten^+/+/Akt2^+/+ mice, and dominant negative AKT2 led to an increase in accumulation of hepatic lipids in both Pten^+/+/Akt2^+/+ and Pten^+/−/Akt2^+/+ mice. Conclusion Our results demonstrate that AKT2 activity in skeletal muscle critically affects lipid accumulation in the livers of Pten^+/+/Akt2^+/+ and Pten^+/−/Akt2^+/+ mice, and emphasize the role of skeletal muscle in the pathology of NAFLD.  

5.1606           Small-molecule inhibitors of JC polyomavirus infection

Yatawara, A., Gaidos, G., Rupasinghe, C.N., O’Hara, B.A., Pellegrini, M., Atwood, W.J. and Mierke, D.F.

1. Peptide Science, 21(3), 236-242 (2015)

  The JC polyomavirus (JCPyV) infects approximately 50% of the human population. In healthy individuals, the infection remains dormant and asymptomatic, but in immuno-suppressed patients, it can cause progressive multifocal leukoencephalopathy (PML), a potentially fatal demyelinating disease. Currently, there are no drugs against JCPyV infection nor for the treatment of PML. Here, we report the development of small-molecule inhibitors of JCPyV that target the initial interaction between the virus and host cell and thereby block viral entry. Utilizing a combination of computational and NMR-based screening techniques, we target the LSTc tetrasaccharide binding site within the VP1 pentameric coat protein of JCPyV. Four of the compounds from the screen effectively block viral infection in our in vitro assays using SVG-A cells. For the most potent compound, we used saturation transfer difference NMR to determine the mode of binding to purified pentamers of JCPyV VP1. Collectively, these results demonstrate the viability of this class of compounds for eventual development of JCPyV-antiviral therapeutics.  

5.1607           Vaccination with Human Papillomavirus Pseudovirus-Encapsidated Plasmids Targeted to Skin Using Microneedles

Kines, R.C., Zarnitsyn, V., Johnson, T.R., Pang, Y-Y.S., Corbett, K.S., Nicewonger, J.D., Gangopadhyay, A., Chen, M., Liu, J., Prausnitz, M.R., Schiller, J.T. and Graham, B.S. PloS One, 10(3), e120797 (2015)   Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.  

5.1608           The Endoplasmic Reticulum Membrane J Protein C18 Executes a Distinct Role in Promoting Simian Virus 40 Membrane Penetration

Bagchi, P., Walczak, C.P. and Tsai, B.

1. Virol., 89(8), 4058-4068 (2015)

  The nonenveloped simian virus 40 (SV40) hijacks the three endoplasmic reticulum (ER) membrane-bound J proteins B12, B14, and C18 to escape from the ER into the cytosol en route to successful infection. How C18 controls SV40 ER-to-cytosol membrane penetration is the least understood of these processes. We previously found that SV40 triggers B12 and B14 to reorganize into discrete puncta in the ER membrane called foci, structures postulated to represent the cytosol entry site (C. P. Walczak, M. S. Ravindran, T. Inoue, and B. Tsai, PLoS Pathog 10:e1004007, 2014). We now find that SV40 also recruits C18 to the virus-induced B12/B14 foci. Importantly, the C18 foci harbor membrane penetration-competent SV40, further implicating this structure as the membrane penetration site. Consistent with this, a mutant SV40 that cannot penetrate the ER membrane and promote infection fails to induce C18 foci. C18 also regulates the recruitment of B12/B14 into the foci. In contrast to B14, C18's cytosolic Hsc70-binding J domain, but not the lumenal domain, is essential for its targeting to the foci; this J domain likewise is necessary to support SV40 infection. Knockdown-rescue experiments reveal that C18 executes a role that is not redundant with those of B12/B14 during SV40 infection. Collectively, our data illuminate C18's contribution to SV40 ER membrane penetration, strengthening the idea that SV40-triggered foci are critical for cytosol entry.  

5.1609           Sublingual Immunization of Trivalent Human Papillomavirus DNA Vaccine in Baculovirus Nanovector for Protection against Vaginal Challenge

Lee, H-J., Cho, H., Kim, M-G., Heo, Y-K., Cho, Y., Gwon, Y-D., Park, K.H., Jim, H., Kim, J., Oh, Y-K., Kim, Y.B. PloS One, 10(3), e0119408 (2015)   Here, we report the immunogenicity of a sublingually delivered, trivalent human papillomavirus (HPV) DNA vaccine encapsidated in a human endogenous retrovirus (HERV) envelope-coated, nonreplicable, baculovirus nanovector. The HERV envelope-coated, nonreplicable, baculovirus-based DNA vaccine, encoding HPV16L1, -18L1 and -58L1 (AcHERV-triHPV), was constructed and sublingually administered to mice without adjuvant. Following sublingual (SL) administration, AcHERV-triHPV was absorbed and distributed throughout the body. At 15 minutes and 1 day post-dose, the distribution of AcHERV-triHPV to the lung was higher than that to other tissues. At 30 days post-dose, the levels of AcHERV-triHPV had diminished throughout the body. Six weeks after the first of three doses, 1×10⁸ copies of SL AcHERV-triHPV induced HPV type-specific serum IgG and neutralizing antibodies to a degree comparable to that of IM immunization with 1×10⁹ copies. AcHERV-triHPV induced HPV type-specific vaginal IgA titers in a dose-dependent manner. SL immunization with 1×10¹⁰ copies of AcHERV-triHPV induced Th1 and Th2 cellular responses comparable to IM immunization with 1×10⁹ copies. Molecular imaging revealed that SL AcHERV-triHPV in mice provided complete protection against vaginal challenge with HPV16, HPV18, and HPV58 pseudoviruses. These results support the potential of SL immunization using multivalent DNA vaccine in baculovirus nanovector for induction of mucosal, systemic, and cellular immune responses.  

5.1610           A Chimeric 18L1-45RG1 Virus-Like Particle Vaccine Cross-Protects against Oncogenic Alpha-7 Human Papillomavirus Types

Huber, B., Schellenbacher, C., Jindra, C., Fink, D., Shafti-Keramat, S. and Kirnbauer, R. PloS One, 10(3), e120152 (2015)   Persistent infection with oncogenic human papillomaviruses (HPV) types causes all cervical and a subset of other anogenital and oropharyngeal carcinomas. Four high-risk (hr) mucosal types HPV16, 18, 45, or 59 cause almost all cervical adenocarcinomas (AC), a subset of cervical cancer (CxC). Although the incidence of cervical squamous cell carcinoma (SCC) has dramatically decreased following introduction of Papanicolaou (PAP) screening, the proportion of AC has relatively increased. Cervical SCC arise mainly from the ectocervix, whereas AC originate primarily from the endocervical canal, which is less accessible to obtain viable PAP smears. Licensed (bivalent and quadrivalent) HPV vaccines comprise virus-like particles (VLP) of the most important hr HPV16 and 18, self-assembled from the major capsid protein L1. Due to mainly type-restricted efficacy, both vaccines do not target 13 additional hr mucosal types causing 30% of CxC. The papillomavirus genus alpha species 7 (α7) includes a group of hr types of which HPV18, 45, 59 are proportionally overrepresented in cervical AC and only partially (HPV18) targeted by current vaccines. To target these types, we generated a chimeric vaccine antigen that consists of a cross-neutralizing epitope (homologue of HPV16 RG1) of the L2 minor capsid protein of HPV45 genetically inserted into a surface loop of HPV18 L1 VLP (18L1-45RG1). Vaccination of NZW rabbits with 18L1-45RG1 VLP plus alum-MPL adjuvant induced high-titer neutralizing antibodies against homologous HPV18, that cross-neutralized non-cognate hr α7 types HPV39, 45, 68, but not HPV59, and low risk HPV70 in vitro, and induced a robust L1-specific cellular immune response. Passive immunization protected mice against experimental vaginal challenge with pseudovirions of HPV18, 39, 45 and 68, but not HPV59 or the distantly related α9 type HPV16. 18L1-45RG1 VLP might be combined with our previously described 16L1-16RG1 VLP to develop a second generation bivalent vaccine with extended spectrum against hr HPV.  

5.1611           Specific gene expression in mouse cortical astrocytes is mediated by a 1740bp-GFAP promoter-driven combined adeno-associated virus

Meng, X., Yang, F., Ouyang, T., Liu, B., Wu, C. and Jiang, W. Neuroscience Lett., 593, 45-50 (2015)   We sought to demonstrate the in vivo transduction efficiency and tropism range in astrocytes of a combined-serotype adeno associated virus (AAV_2/5/7/8/9). To control expression of enhanced green fluorescent protein (EGFP), a 1740bp glial fibrillary acidic protein (GFAP) promoter was obtained and ligated into vectors of each AAV serotype (2/5/7/8/9). Purified AAVs were then injected into the somatosensory cortex of C57BL/6J mice. Cell-type specific antibodies and subsequent immunofluorescence were used to identify astrocytes (GFAP), neurons (neuronal nuclear antigen, NeuN), microglia (ionized calcium-binding adapter molecule 1, Iba1), and oligodendrocytes (myelin basic protein, MBP), whereby, EGFP expression was measured in each cell type at 1–4 weeks post-injection. Our results indicated that the majority of astrocytes expressed EGFP, while only a small number of neurons expressed EGFP. Both microglia and oligodendrocytes lacked EGFP expression after viral injection. Quantitative analyses revealed that the percentage of EGFP-positive astrocytes was about 98% after viral injection, while the EGFP-positive neuronal percentage was less than 2%. Thus, this study shows that using a combined-serotype AAV carrying a 1740bp GFAP promoter results in successful, cell-type specific infection of the central nervous system, with robust gene expression in murine astrocytes.  

5.1612           HIV-1 IN/Pol recruits LEDGF/p75 into viral particles

Desimmie, B.A:, Weyden, C., Schrijvers, R., Vets, S., Demeulemeester, J., Proost, P., Paron, I., De Rijck, J., Mast, J., Bannert, N., Gijsbers, R., Christ, F. and Debyser, Z. Retrovirology, 12:16 (2015)   Background The dynamic interaction between HIV and its host governs the replication of the virus and the study of the virus-host interplay is key to understand the viral lifecycle. The host factor lens epithelium-derived growth factor (LEDGF/p75) tethers the HIV preintegration complex to the chromatin through a direct interaction with integrase (IN). Small molecules that bind the LEDGF/p75 binding pocket of the HIV IN dimer (LEDGINs) block HIV replication through a multimodal mechanism impacting early and late stage replication including HIV maturation. Furthermore, LEDGF/p75 has been identified as a Pol interaction partner. This raised the question whether LEDGF/p75 besides acting as a molecular tether in the target cell, also affects late steps of HIV replication. Results LEDGF/p75 is recruited into HIV-1 particles through direct interaction with the viral IN (or Pol polyprotein) and is a substrate for HIV-1 protease. Incubation in the presence of HIV-1 protease inhibitors resulted in detection of full-length LEDGF/p75 in purified viral particles. We also demonstrate that inhibition of LEDGF/p75-IN interaction by specific mutants or LEDGINs precludes incorporation of LEDGF/p75 in virions, underscoring the specificity of the uptake. LEDGF/p75 depletion did however not result in altered LEDGIN potency. Conclusion Together, these results provide evidence for an IN/Pol mediated uptake of LEDGF/p75 in viral particles and a specific cleavage by HIV protease. Understanding of the possible role of LEDGF/p75 or its cleavage fragments in the viral particle awaits further experimentation.  

5.1613           Alcohol Metabolism by Oral Streptococci and Interaction with Human Papillomavirus Leads to Malignant Transformation of Oral Keratinocytes

Tao, L., Pavlova, S.I., Gasparovich, S.R., Jin, L. and Schwartz, J. Advances in Exp. Med. and Biol., 815, 239-264 (2015)   Poor oral hygiene, ethanol consumption, and human papillomavirus (HPV) are associated with oral and esophageal cancers. However, the mechanism is not fully known. This study examines alcohol metabolism in Streptococcus and its interaction with HPV-16 in the malignant transformation of oral keratinocytes. The acetaldehyde-producing strain Streptococcus gordonii V2016 was analyzed for adh genes and activities of alcohol and aldehyde dehydrogenases. Streptococcus attachment to immortalized HPV-16 infected human oral keratinocytes, HOK (HPV/HOK-16B), human oral buccal keratinocytes, and foreskin keratinocytes was studied. Acetaldehyde, malondialdehyde, DNA damage, and abnormal proliferation among keratinocytes were also quantified. We found that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB, and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol, and ethanol, respectively. S. gordonii V2016 did not show a detectable aldehyde dehydrogenase. AdhE is the major alcohol dehydrogenase in S. gordonii. Acetaldehyde and malondialdehyde production from permissible Streptococcus species significantly increased the bacterial attachment to keratinocytes, which was associated with an enhanced expression of furin to facilitate HPV infection and several malignant phenotypes including acetaldehyde adduct formation, abnormal proliferation, and enhanced migration through integrin-coated basement membrane by HPV-infected oral keratinocytes. Therefore, expression of multiple alcohol dehydrogenases with no functional aldehyde dehydrogenase contributes to excessive production of acetaldehyde from ethanol by oral streptococci. Oral Streptococcus species and HPV may cooperate to transform oral keratinocytes after ethanol exposure. These results suggest a significant clinical interaction, but further validation is warranted.  

5.1614           Neuron-to-neuron α-synuclein propagation in vivo is independent of neuronal injury

Ulusoy, A., Musgrove, R.E., Rusconi, R., Klinkenberg, M., Helwig, M., Schneider, A. and Di Monte, D.A. Acta Neuropathologica Communications, 3:13 (2015)   Introduction Interneuronal propagation of α-synuclein has been demonstrated in a variety of experimental models and may be involved in disease progression during the course of human synucleinopathies. The aim of this study was to assess the role that neuronal injury or, vice versa, cell integrity could have in facilitating interneuronal α-synuclein transfer and consequent protein spreading in an in vivo animal model. Results Viral vectors carrying the DNA for human α-synuclein were injected into the rat vagus nerve to trigger protein overexpression in the medulla oblongata and consequent spreading of human α-synuclein toward pons, midbrain and forebrain. Two vector preparations sharing the same viral construct were manufactured using identical procedures with the exception of methods for their purification. They were also injected at concentrations that induced comparable levels of α-synuclein transduction/overexpression in the medulla oblongata. α-Synuclein load was associated with damage (at 6 weeks post injection) and death (at 12 weeks) of medullary neurons after treatment with only one of the two vector preparations. Of note, neuronal injury and degeneration was accompanied by a substantial reduction of caudo-rostral propagation of human α-synuclein. Conclusions Interneuronal α-synuclein transfer, which underlies protein spreading from the medulla oblongata to more rostral brain regions in this rat model, is not a mere consequence of passive release from damaged or dead neurons. Neuronal injury and degeneration did not exacerbate α-synuclein propagation. In fact, data suggest that cell-to-cell passage of α-synuclein may be particularly efficient between intact, relatively healthy neurons.  

5.1615           Opposing effects of viral mediated brain expression of apolipoprotein E2 (apoE2) and apoE4 on apoE lipidation and Aβ metabolism in apoE4-targeted replacement mice

Hu, J., Liu, C-C., Chen, X-F., Zhang, Y-w., Xu, H. and Bu, G. Mol. Neurodegeneration, 10:6 (2015)   Background Human apolipoprotein E (apoE) exists in three major isoforms: apoE2, apoE3 and apoE4. In the brain, apoE is produced mostly by astrocytes and transports cholesterol to neurons via apoE receptors. Among the gene alleles encoding the three isoforms, the APOE4 allele is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD), whereas APOE2 is protective. ApoE4 confers a gain of toxic function, a loss of neuroprotective function or a combination of both in AD pathogenesis. Given that therapeutic impacts of modulating apoE expression may be isoform-dependent, we sought to investigate the relationship between overexpressing apoE isoform and apoE-related functions in apoE-targeted replacement (TR) mice. Specifically, apoE isoform expression driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was built into an adeno-associated virus serotype 8 (AAV8) vector and injected into the ventricles of postnatal day 2 (P2) apoE3-TR or apoE4-TR mice. Upon confirmation of apoE isoform expression, effects on apoE lipidation and the levels of amyloid-β (Aβ) in the brain were assessed. Results AAV8-GFAP-apoE isoforms were specifically expressed in astrocytes throughout all brain regions, which led to overall increased apoE levels in the brain. Viral mediated overexpression of apoE4 in the apoE4-TR background increased poorly-lipidated apoE lipoprotein particles and decreased apoE-associated cholesterol in apoE4-TR mice. Conversely, apoE2 overexpression in apoE4-TR mice enhanced apoE lipidation and associated cholesterol. Furthermore, overexpression of apoE4 elevated the levels of endogenous Aβ, whereas apoE2 overexpression trended to lower endogenous Aβ. Conclusions Overexpression of apoE isoforms induces differential effects in the apoE4-TR background: apoE4 decreases apoE lipidation and enhances Aβ accumulation, whereas apoE2 has the opposite effects. Our findings suggest that increasing apoE2 in APOE4 carriers is a beneficial strategy to treat AD, whereas increasing apoE4 in APOE4 carriers is likely harmful. We have also established novel methods to express apoE isoforms in mouse brain to study apoE-related pathways in AD and related dementia.  

5.1616           A Mutant Tat Protein Inhibits HIV-1 Reverse Transcription by Targeting the Reverse Transcription Complex

Lin, M-H., Apolloni, A., Cutillas, V., Sivakumaran, H., Martin, S., Li, D., Wei, T., Wang, R., Jin, H., Spann, K. and Harrich, D.

1. Virol., 89(9), 4827-4836 (2015)

  Previously, we reported that a mutant of Tat referred to as Nullbasic inhibits HIV-1 reverse transcription although the mechanism of action is unknown. Here we show that Nullbasic is a reverse transcriptase (RT) binding protein that targets the reverse transcription complex rather than directly inhibiting RT activity. An interaction between Nullbasic and RT was observed by using coimmunoprecipitation and pulldown assays, and a direct interaction was measured by using a biolayer interferometry assay. Mixtures of recombinant 6×His-RT and Nullbasic-FLAG-V5-6×His at molar ratios of up to 1:20,000 did not inhibit RT activity in standard homopolymer primer template assays. An analysis of virus made by cells that coexpressed Nullbasic showed that Nullbasic copurified with virus particles, indicating that it was a virion protein. In addition, analysis of reverse transcription complexes (RTCs) isolated from cells infected with wild type or Nullbasic-treated HIV-1 showed that Nullbasic reduced the levels of viral DNA in RTC fractions. In addition, a shift in the distribution of viral DNA and CAp24 to less-dense non-RTC fractions was observed, indicating that RTC activity from Nullbasic-treated virus was impaired. Further analysis showed that viral cores isolated from Nullbasic-treated HIV undergo increased disassembly in vitro compared to untreated HIV-1. To our knowledge, this is the first description of an antiviral protein that inhibits reverse transcription by targeting the RTC and affecting core stability.  

5.1617           3D Imaging of Axons in Transparent Spinal Cords from Rodents and Nonhuman Primates

Soderblom, C., Lee, D-H., Dawood, A., carballosa, M., Santamaria, A.J., Benavides, F.D., Jergova, S., Grumles, R.M., Thomas, C.K., Park, K.K., Guest, J.D., Lemmon, V.P., Lee, J.K. and Tsoulfas, P. eNeuro, 2(2), 2001-15 (2015)   The histological assessment of spinal cord tissue in three dimensions has previously been very time consuming and prone to errors of interpretation. Advances in tissue clearing have significantly improved visualization of fluorescently labelled axons. While recent proof-of-concept studies have been performed with transgenic mice in which axons were prelabeled with GFP, investigating axonal regeneration requires stringent axonal tracing methods as well as the use of animal models in which transgenic axonal labeling is not available. Using rodent models of spinal cord injury, we labeled axon tracts of interest using both adeno-associated virus and chemical tracers and performed tetrahydrofuran-based tissue clearing to image multiple axon types in spinal cords using light sheet and confocal microscopy. Using this approach, we investigated the relationships between axons and scar-forming cells at the injury site as well as connections between sensory axons and motor pools in the spinal cord. In addition, we used these methods to trace axons in nonhuman primates. This reproducible and adaptable virus-based approach can be combined with transgenic mice or with chemical-based tract-tracing methods, providing scientists with flexibility in obtaining axonal trajectory information from transparent tissue. Significance Statement: Recent advances in tissue clearing techniques have provided a promising method of visualizing axonal trajectories with unprecedented accuracy and speed. While previous studies have utilized transgenic labeling in mice, the use of virus or chemical neuronal tracers will provide additional spatiotemporal control as well as the ability to use animal models in which transgenic axonal labeling is not available. We used adeno-associated viruses (AAVs) and chemical tracers and performed tetrahydrofuran-based tissue clearing to image multiple axon types in the rodent and nonhuman primate spinal cord using light sheet and confocal microscopy. This approach will provide scientists with a simple and flexible method of obtaining axonal trajectory information from transparent tissue.  

5.1618           GLT1 overexpression in SOD1G93A mouse cervical spinal cord does not preserve diaphragm function or extend disease

Li, K., Hala, T.J., Seetharam, S., Poulsen, D.J., Wright, M.C. and Lepore, A.C. Neurobiology of Disease, 78, 12-23 (2015)   Amyotrophic lateral sclerosis (ALS) is characterized by relatively rapid degeneration of both upper and lower motor neurons, with death normally occurring 2–5 years following diagnosis primarily due to respiratory paralysis resulting from phrenic motor neuron (PhMN) loss and consequent diaphragm denervation. In ALS, cellular abnormalities are not limited to MNs. For example, decreased levels and aberrant functioning of the major central nervous system (CNS) glutamate transporter, GLT1, occur in spinal cord and motor cortex astrocytes of both humans with ALS and in SOD1^G93A rodents, a widely studied ALS animal model. This results in dysregulation of extracellular glutamate homeostasis and consequent glutamate excitotoxicity, a primary mechanism responsible for MN loss in ALS animal models and in the human disease. Given these observations of GLT1 dysfunction in areas of MN loss, as well as the importance of testing therapeutic strategies for preserving PhMNs in ALS, we evaluated intraspinal delivery of an adeno-associated virus type 8 (AAV8)–Gfa2 vector to the cervical spinal cord ventral horn of SOD1^G93A ALS mice for focally restoring intraspinal GLT1 expression. AAV8 was specifically injected into the ventral horn bilaterally throughout the cervical enlargement at 110 days of age, a clinically-relevant time point coinciding with phenotypic/symptomatic disease onset. Intraspinal delivery of AAV8–Gfa2–GLT1 resulted in robust transduction primarily of GFAP⁺ astrocytes that persisted until disease endstage, as well as a 2–3-fold increase in total intraspinal GLT1 protein expression in the ventral horn. Despite this robust level of astrocyte transduction and GLT1 elevation, GLT1 overexpression did not protect PhMNs, preserve histological PhMN innervation of the diaphragm NMJ, or prevent decline in diaphragmatic respiratory function as assessed by phrenic nerve–diaphragm compound muscle action potential (CMAP) recordings compared to control AAV8–Gfa2–eGFP injected mice. In addition, AAV–Gfa2–GLT1 did not delay forelimb disease onset, extend disease duration (i.e. time from either forelimb or hindlimb disease onsets to endstage) or prolong overall animal survival. These findings suggest that focal restoration of GLT1 expression in astrocytes of the cervical spinal cord using AAV delivery is not an effective therapy for ALS.  

5.1619           SOD1 silencing in motoneurons or glia rescues neuromuscular function in ALS mice

Dirren, E., Aebischer, J., Rochat, C., Towne, C., Schneider, B.L. and Aebischer, P. Annals of Clin. Translat. Neurol., 2(2), 167-184 (2015)   Objective Amyotrophic lateral sclerosis is an incurable disorder mainly characterized by motoneuron degeneration. Mutations in the superoxide dismutase 1 (SOD1) gene account for 20% of familial forms of the disease. Mutant SOD1 exerts multiple pathogenic effects through the gain of toxic properties in both neurons and glial cells. Here, we compare AAV-based gene therapy suppressing expression of mutant SOD1 in either motoneurons or astrocytes. Methods AAV vectors encoding microRNA against human SOD1 were administered to ^G93ASOD1 mice either by intracerebroventricular injections in pups or by lumbar intrathecal injections in adults. Vector systems were designed to suppress SOD1 expression predominantly in either spinal motoneurons or astrocytes. Electrophysiological and behavioral tests were performed on treated animals to evaluate disease progression. Results Following vector injection in ^G93ASOD1 pups, efficient silencing of SOD1 expression was achieved in motoneurons and/or astrocytes. Most complete protection of motor units was obtained when targeting human SOD1 predominantly in motoneurons. Suppressing SOD1 mainly in astrocytes led to preserved muscle innervation despite only partial protection of spinal motoneurons. In both cases, injection in pups led to full recovery of neuromuscular function and significantly prolonged survival. Vector injections in adult mice also achieved significant protection of neuromuscular function, which was highest when motoneurons were targeted. Interpretation These results suggest that AAV-mediated SOD1 silencing is an effective approach to prevent motoneuron degeneration caused by SOD1 mutation. AAV vectors suppressing SOD1 in motoneurons delay disease onset and show effective neuroprotection. On the other hand, AAV-based SOD1 silencing in astrocytes rescues neuromuscular function following initial denervation.  

5.1620           Characterization of HTT Inclusion Size, Location, and Timing in the zQ175 Mouse Model of Huntington´s Disease: An In Vivo High-Content Imaging Study

Carty, N., Berson, N., Tillack, K., Thiede, C., Scholz, D., Kottig, K., Sedaghat, Y., Gabrysiak, C., Yohrling, G., von der kammer, H., Ebneth, A., mack, V., Munoz-Sanjuan, I. and Kwak, S. PloS One, 10(4), e123527 (2015)   Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Major pathological hallmarks of HD include inclusions of mutant huntingtin (mHTT) protein, loss of neurons predominantly in the caudate nucleus, and atrophy of multiple brain regions. However, the early sequence of histological events that manifest in region- and cell-specific manner has not been well characterized. Here we use a high-content histological approach to precisely monitor changes in HTT expression and characterize deposition dynamics of mHTT protein inclusion bodies in the recently characterized zQ175 knock-in mouse line. We carried out an automated multi-parameter quantitative analysis of individual cortical and striatal cells in tissue slices from mice aged 2–12 months and confirmed biochemical reports of an age-associated increase in mHTT inclusions in this model. We also found distinct regional and subregional dynamics for inclusion number, size and distribution with subcellular resolution. We used viral-mediated suppression of total HTT in the striatum of zQ175 mice as an example of a therapeutically-relevant but heterogeneously transducing strategy to demonstrate successful application of this platform to quantitatively assess target engagement and outcome on a cellular basis.  

5.1621           Ser129 phosphorylation of endogenous α-synuclein induced by overexpression of polo-like kinases 2 and 3 in nigral dopamine neurons is not detrimental to their survival and function

Buck, K., Landeck, N., Ulusoy, A., Majbour, N.K., El-Agnaf, O.M.A. and Kirik, D. Neurobiology of Disease, 78, 100-114 (2015)   Phosphorylation of the α-synuclein (α-syn) protein at Ser129 [P(S129)-α-syn] was found to be the most abundant form in intracellular inclusions in brains from Parkinson's disease (PD) patients. This finding suggests that P(S129)-α-syn plays a central role in the pathogenesis of PD. However, it is at present unclear whether P(S129)-α-syn is pathogenic driving the neurodegenerative process. Rodent studies using neither the phosphomimics of human α-syn nor co-expression of human wild-type α-syn and kinases phosphorylating α-syn at Ser129 gave consistent results. One major concern in interpreting these findings is that human α-syn was expressed above physiological levels inducing neurodegeneration in rat nigral neurons. In order to exclude this confounding factor, we took a different approach and increased the phosphorylation level of endogenous α-syn. For this purpose, we took advantage of recombinant adeno-associated viral (rAAV) vectors to deliver polo-like kinase (PLK) 2 or PLK3 in the substantia nigra and investigated whether increased levels of P(S129)-α-syn compromised the function and survival of nigral dopaminergic neurons. Interestingly, we observed that hyperphosphorylated α-syn did not induce nigral dopaminergic cell death, as assessed at 1 and 4 months. Furthermore, histological analysis did not show any accumulation of α-syn protein or formation of inclusions. Using in vivo microdialysis, we found that the only measurable functional alteration was the depolarisation-induced release of dopamine, while the in vivo synthesis rate of DOPA and dopamine baseline release remained unaltered. Taken together, our results suggest that phosphorylation of α-syn at Ser129 does not confer a toxic gain of function per se.  

5.1622           Extracellular vesicle sorting of α-Synuclein is regulated by sumoylation

Kunadt, M. et al Acta Neuropathol., 129, 695-713 (2015)   Extracellular α-Synuclein has been implicated in interneuronal propagation of disease pathology in Parkinson’s Disease. How α-Synuclein is released into the extracellular space is still unclear. Here, we show that α-Synuclein is present in extracellular vesicles in the central nervous system. We find that sorting of α-Synuclein in extracellular vesicles is regulated by sumoylation and that sumoylation acts as a sorting factor for targeting of both, cytosolic and transmembrane proteins, to extracellular vesicles. We provide evidence that the SUMO-dependent sorting utilizes the endosomal sorting complex required for transport (ESCRT) by interaction with phosphoinositols. Ubiquitination of cargo proteins is so far the only known determinant for ESCRT-dependent sorting into the extracellular vesicle pathway. Our study reveals a function of SUMO protein modification as a Ubiquitin-independent ESCRT sorting signal, regulating the extracellular vesicle release of α-Synuclein. We deciphered in detail the molecular mechanism which directs α-Synuclein into extracellular vesicles which is of highest relevance for the understanding of Parkinson’s disease pathogenesis and progression at the molecular level. We furthermore propose that sumo-dependent sorting constitutes a mechanism with more general implications for cell biology.  

5.1623           High Capsid–Genome Correlation Facilitates Creation of AAV Libraries for Directed Evolution

Nonnenmacher, M., van Bakel, H., Hajjar, R.J: and Weber, T. Molecular Therapy, 23(4), 675-682 (2015)   Directed evolution of adeno-associated virus (AAV) through successive rounds of phenotypic selection is a powerful method to isolate variants with improved properties from large libraries of capsid mutants. Importantly, AAV libraries used for directed evolution are based on the “natural” AAV genome organization where the capsid proteins are encoded in cis from replicating genomes. This is necessary to allow the recovery of the capsid DNA after each step of phenotypic selection. For directed evolution to be used successfully, it is essential to minimize the random mixing of capsomers and the encapsidation of nonmatching viral genomes during the production of the viral libraries. Here, we demonstrate that multiple AAV capsid variants expressed from Rep/Cap containing viral genomes result in near-homogeneous capsids that display an unexpectedly high capsid–DNA correlation. Next-generation sequencing of AAV progeny generated by bulk transfection of a semi-random peptide library showed a strong counter-selection of capsid variants encoding premature stop codons, which further supports a strong capsid–genome identity correlation. Overall, our observations demonstrate that production of “natural” AAVs results in low capsid mosaicism and high capsid–genome correlation. These unique properties allow the production of highly diverse AAV libraries in a one-step procedure with a minimal loss in phenotype–genotype correlation.  

5.1624           Microtubule disruption synergizes with oncolytic virotherapy by inhibiting interferon translation and potentiating bystander killing

Arulanandam, R. et al Nature Communications, 6:6410 (2015)   In this study, we show that several microtubule-destabilizing agents used for decades for treatment of cancer and other diseases also sensitize cancer cells to oncolytic rhabdoviruses and improve therapeutic outcomes in resistant murine cancer models. Drug-induced microtubule destabilization leads to superior viral spread in cancer cells by disrupting type I IFN mRNA translation, leading to decreased IFN protein expression and secretion. Furthermore, microtubule-destabilizing agents specifically promote cancer cell death following stimulation by a subset of infection-induced cytokines, thereby increasing viral bystander effects. This study reveals a previously unappreciated role for microtubule structures in the regulation of the innate cellular antiviral response and demonstrates that unexpected combinations of approved chemotherapeutics and biological agents can lead to improved therapeutic outcomes.    

5.1625           A retrovirus packages nascent host noncoding RNAs from a novel surveillance pathway

Eckwahl, M.J., Sim, S., Smith, D., Telesnitsky, A. and Wolin, S.L. Genes & Dev. 29(6), 646-657 (2015)   Although all retroviruses recruit host cell RNAs into virions, both the spectrum of RNAs encapsidated and the mechanisms by which they are recruited remain largely unknown. Here, we used high-throughput sequencing to obtain a comprehensive description of the RNAs packaged by a model retrovirus, murine leukemia virus. The major encapsidated host RNAs are noncoding RNAs (ncRNAs) and members of the VL30 class of endogenous retroviruses. Remarkably, although Moloney leukemia virus (MLV) assembles in the cytoplasm, precursors to specific tRNAs, small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs) are all enriched in virions. Consistent with their cytoplasmic recruitment, packaging of both pre-tRNAs and U6 snRNA requires the nuclear export receptor Exportin-5. Adenylated and uridylated forms of these RNAs accumulate in cells and virions when the cytoplasmic exoribonuclease DIS3L2 and subunits of the RNA exosome are depleted. Together, our data reveal that MLV recruits RNAs from a novel host cell surveillance pathway in which unprocessed and unneeded nuclear ncRNAs are exported to the cytoplasm for degradation.  

5.1626           Commercially Available Immunoglobulins Contain Virus Neutralizing Antibodies Against All Major Genotypes of Polyomavirus BK

Randhawa, P., Pastrana, D.V., Zeng, G., Huang, Y., Shapiro, R., Sood, R., Puttarajappa, C., Berger, M., Hariharan, S. and Buck, C.B. Am. J. Transplant., 15(4), 1014-1020 (2015)   Neutralizing antibodies (NAbs) form the basis of immunotherapeutic strategies against many important human viral infections. Accordingly, we studied the prevalence, titer, genotype-specificity, and mechanism of action of anti-polyomavirus BK (BKV) NAbs in commercially available human immune globulin (IG) preparations designed for intravenous (IV) use. Pseudovirions (PsV) of genotypes Ia, Ib2, Ic, II, III, and IV were generated by co-transfecting a reporter plasmid encoding luciferase and expression plasmids containing synthetic codon-modified VP1, VP2, and VP3 capsid protein genes into 293TT cells. NAbs were measured using luminometry. All IG preparations neutralized all BKV genotypes, with mean EC50 titers as high as 254 899 for genotype Ia and 6,666 for genotype IV. Neutralizing titers against genotypes II and III were higher than expected, adding to growing evidence that infections with these genotypes are more common than currently appreciated. Batch to batch variation in different lots of IG was within the limits of experimental error. Antibody mediated virus neutralizing was dose dependent, modestly enhanced by complement, genotype-specific, and achieved without effect on viral aggregation, capsid morphology, elution, or host cell release. IG contains potent NAbs capable of neutralizing all major BKV genotypes. Clinical trials based on sound pharmacokinetic principles are needed to explore prophylactic and therapeutic applications of these anti-viral effects, until effective small molecule inhibitors of BKV replication can be developed.  

5.1627           Herpes Simplex Virus Type 1 (HSV-1 )-Derived Recombinant Vectors  for Gene Transfer  and Gene Therapy

Marconi, P., Fraefel, C. and Epstein, A.L. Methods in Mol. Biol., 1254, 269-293 (2015)   Herpes simplex virus type 1 (HSV-1 ) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153-kilobase pair (kbp) double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid , the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis . Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes the approach most commonly used to prepare recombinant HSV-1 vectors through homologous recombination , either in eukaryotic cells or in bacteria.  

5.1628           Gene Therapy for Huntington’s Disease

Wu, A., Fong, D.M. and Young, D. Neuromethods, 98, 121-151 (2015)   Huntington’s disease (HD) is an inherited autosomal dominant neurodegenerative disease characterized by loss of motor control, cognitive decline, and psychiatric manifestations. The underlying genetic cause of HD is a mutation in the huntingtin gene resulting in an expanded polyglutamine tract in huntingtin protein that confers a toxic gain of function. Abnormal intranuclear protein inclusions and the progressive degeneration of medium spiny neurons in the striatum as well as other brain areas at later stages are key neuropathological features of the disease. Gene therapy is an attractive therapeutic option for HD. Therapeutic strategies have primarily centered on neuroprotective and/or neuroregenerative approaches to prevent or ameliorate the extent of striatal neuron loss through the overexpression of neurotrophic factors which boost the resilience of neurons to the toxic effects of mutant huntingtin. More recently, attention has turned to gene silencing or intrabody approaches, powerful approaches that aim to mitigate the pathogenic effects of mutant huntingtin. Promising results have been shown in the evaluation of several of these strategies in rodent and non-human primate models of HD, and gene delivery technology has advanced to the stage where opportunities for long-term therapeutic intervention can be realized. In this chapter, we review the main gene therapy strategies for HD followed by a description of the methods used in our laboratory for the packaging of adeno-associated viral (AAV) vectors for therapeutic gene delivery, methods for AAV vector delivery into the rodent brain, and behavioral tests used for the assessment of functional deficits/recovery in rat models of HD.  

5.1629           A Human Monoclonal IgG That Binds Aβ Assemblies and Diverse Amyloids Exhibits Anti-Amyloid Activities In Vitro and In Vivo

Levites, Y. et al

1. Neurosci., 35(16), 6265-6276 (2015)

  Alzheimer's disease (AD) and familial Danish dementia (FDD) are degenerative neurological diseases characterized by amyloid pathology. Normal human sera contain IgG antibodies that specifically bind diverse preamyloid and amyloid proteins and have shown therapeutic potential in vitro and in vivo. We cloned one of these antibodies, 3H3, from memory B cells of a healthy individual using a hybridoma method. 3H3 is an affinity-matured IgG that binds a pan-amyloid epitope, recognizing both Aβ and λ Ig light chain (LC) amyloids, which are associated with AD and primary amyloidosis, respectively. The pan-amyloid-binding properties of 3H3 were demonstrated using ELISA, immunohistochemical studies, and competition binding assays. Functional studies showed that 3H3 inhibits both Aβ and LC amyloid formation in vitro and abrogates disruption of hippocampal synaptic plasticity by AD-patient-derived soluble Aβ in vivo. A 3H3 single-chain variable fragment (scFv) retained the binding specificity of the 3H3 IgG and, when expressed in the brains of transgenic mice using an adeno-associated virus (AAV) vector, decreased parenchymal Aβ amyloid deposition in TgCRND8 mice and ADan (Danish Amyloid) cerebral amyloid angiopathy in the mouse model of FDD. These data indicate that naturally occurring human IgGs can recognize a conformational, amyloid-specific epitope and have potent anti-amyloid activities, providing a rationale to test their potential as antibody therapeutics for diverse neurological and other amyloid diseases.  

5.1630           WDR12, a Member of Nucleolar PeBoW-Complex, Is Up-Regulated in Failing Hearts and Causes Deterioration of Cardiac Function

Moilanen, A-M. et al PloS One, 10(4), e124907 (2015)   Aims In a recent genome-wide association study, WD-repeat domain 12 (WDR12) was associated with early-onset myocardial infarction (MI). However, the function of WDR12 in the heart is unknown. Methods and Results We characterized cardiac expression of WDR12, used adenovirus-mediated WDR12 gene delivery to examine effects of WDR12 on left ventricular (LV) remodeling, and analyzed relationship between MI associated WDR12 allele and cardiac function in human subjects. LV WDR12 protein levels were increased in patients with dilated cardiomyopathy and rats post-infarction. In normal adult rat hearts, WDR12 gene delivery into the anterior wall of the LV decreased interventricular septum diastolic and systolic thickness and increased the diastolic and systolic diameters of the LV. Moreover, LV ejection fraction (9.1%, P<0.05) and fractional shortening (12.2%, P<0.05) were declined. The adverse effects of WDR12 gene delivery on cardiac function were associated with decreased cellular proliferation, activation of p38 mitogen–activated protein kinase (MAPK)/heat shock protein (HSP) 27 pathway, and increased protein levels of Block of proliferation 1 (BOP1), essential for ribosome biogenesis. Post-infarction WDR12 gene delivery decreased E/A ratio (32%, P<0.05) suggesting worsening of diastolic function. In human subjects, MI associated WDR12 allele was associated significantly with diastolic dysfunction and left atrial size. Conclusions WDR12 triggers distinct deterioration of cardiac function in adult rat heart and the MI associated WDR12 variant is associated with diastolic dysfunction in human subjects.  

5.1631           Radixin regulates synaptic GABAA receptor density and is essential for reversal learning and short-term memory

Hausrat, T.J: et al Nature Communications, 6:6872 (2015)   Neurotransmitter receptor density is a major variable in regulating synaptic strength. Receptors rapidly exchange between synapses and intracellular storage pools through endocytic recycling. In addition, lateral diffusion and confinement exchanges surface membrane receptors between synaptic and extrasynaptic sites. However, the signals that regulate this transition are currently unknown. GABA_A receptors containing α5-subunits (GABA_AR-α5) concentrate extrasynaptically through radixin (Rdx)-mediated anchorage at the actin cytoskeleton. Here we report a novel mechanism that regulates adjustable plasma membrane receptor pools in the control of synaptic receptor density. RhoA/ROCK signalling regulates an activity-dependent Rdx phosphorylation switch that uncouples GABA_AR-α5 from its extrasynaptic anchor, thereby enriching synaptic receptor numbers. Thus, the unphosphorylated form of Rdx alters mIPSCs. Rdx gene knockout impairs reversal learning and short-term memory, and Rdx phosphorylation in wild-type mice exhibits experience-dependent changes when exposed to novel environments. Our data suggest an additional mode of synaptic plasticity, in which extrasynaptic receptor reservoirs supply synaptic GABA_ARs.  

5.1632           Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy

Karakikes, I. et al Nature Communications, 6:6955 (2015)   A number of genetic mutations is associated with cardiomyopathies. A mutation in the coding region of the phospholamban (PLN) gene (R14del) is identified in families with hereditary heart failure. Heterozygous patients exhibit left ventricular dilation and ventricular arrhythmias. Here we generate induced pluripotent stem cells (iPSCs) from a patient harbouring the PLN R14del mutation and differentiate them into cardiomyocytes (iPSC-CMs). We find that the PLN R14del mutation induces Ca²⁺ handling abnormalities, electrical instability, abnormal cytoplasmic distribution of PLN protein and increases expression of molecular markers of cardiac hypertrophy in iPSC-CMs. Gene correction using transcription activator-like effector nucleases (TALENs) ameliorates the R14del-associated disease phenotypes in iPSC-CMs. In addition, we show that knocking down the endogenous PLN and simultaneously expressing a codon-optimized PLN gene reverses the disease phenotype in vitro. Our findings offer novel strategies for targeting the pathogenic mutations associated with cardiomyopathies.  

5.1633           Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization

Guan, J., Bywaters, S.M., Brendle, S.A., Lee, H., Ashley, R.E., Makhov, A.M., Conway, J.F. and Christensen, N.D. Virology, 483, 253-263 (2015)   Cryo-electron microscopy (cryo-EM) was used to solve the structures of human papillomavirus type 16 (HPV16) complexed with fragments of antibody (Fab) from three different neutralizing monoclonals (mAbs): H16.1A, H16.14J, and H263.A2. The structure-function analysis revealed predominantly monovalent binding of each Fab with capsid interactions that involved multiple loops from symmetry related copies of the major capsid protein. The residues identified in each Fab-virus interface map to a conformational groove on the surface of the capsomer. In addition to the known involvement of the FG and HI loops, the DE loop was also found to constitute the core of each epitope. Surprisingly, the epitope mapping also identified minor contributions by EF and BC loops. Complementary immunological assays included mAb and Fab neutralization. The specific binding characteristics of mAbs correlated with different neutralizing behaviors in pre- and post-attachment neutralization assays.  

5.1634           Rod-Derived Cone Viability Factor Promotes Cone Survival by Stimulating Aerobic Glycolysis

Ait-Ali, N., Fridlich, R., Sahel, J-A. and Leveillard, T. Cell, 161, 817-832 (2015)   Rod-derived cone viability factor (RdCVF) is an inactive thioredoxin secreted by rod photoreceptors that protects cones from degeneration. Because the secondary loss of cones in retinitis pigmentosa (RP) leads to blindness, the administration of RdCVF is a promising therapy for this untreatable neurodegenerative disease. Here, we investigated the mechanism underlying the protective role of RdCVF in RP. We show that RdCVF acts through binding to Basigin-1 (BSG1), a transmembrane protein expressed specifically by photoreceptors. BSG1 binds to the glucose transporter GLUT1, resulting in increased glucose entry into cones. Increased glucose promotes cone survival by stimulation of aerobic glycolysis. Moreover, a missense mutation of RdCVF results in its inability to bind to BSG1, stimulate glucose uptake, and prevent secondary cone death in a model of RP. Our data uncover an entirely novel mechanism of neuroprotection through the stimulation of glucose metabolism.  

5.1635           Genetic Deletion of the Transcriptional Repressor NFIL3 Enhances Axon Growth In Vitro but Not Axonal Repair In Vivo

Van der Kallen, L.R., eggers, R., Ehlert, E.M., Verhaagen, J., Smit, A.B. and van Kesteren, R.E. PloS One, 10(5), e0127163 (2015)   Axonal regeneration after injury requires the coordinated expression of genes in injured neurons. We previously showed that either reducing expression or blocking function of the transcriptional repressor NFIL3 activates transcription of regeneration-associated genes Arg1 and Gap43 and strongly promotes axon outgrowth in vitro. Here we tested whether genetic deletion or dominant-negative inhibition of NFIL3 could promote axon regeneration and functional recovery after peripheral nerve lesion in vivo. Contrary to our expectations, we observed no changes in the expression of regeneration-associated genes and a significant delay in functional recovery following genetic deletion of Nfil3. When NFIL3 function was inhibited specifically in dorsal root ganglia prior to sciatic nerve injury, we observed a decrease in regenerative axon growth into the distal nerve segment rather than an increase. Finally, we show that deletion of Nfil3 changes sciatic nerve lesion-induced expression in dorsal root ganglia of genes that are not typically involved in regeneration, including several olfactory receptors and developmental transcription factors. Together our findings show that removal of NFIL3 in vivo does not recapitulate the regeneration-promoting effects that were previously observed in vitro, indicating that in vivo transcriptional control of regeneration is probably more complex and more robust against perturbation than in vitro data may suggest.  

5.1636           Impact of the MRN Complex on Adeno-Associated Virus Integration and Replication during Coinfection with Herpes Simplex Virus 1

Millet, R., Jolinon, N., Nguyen, X-N., Berger, G., Cimarelli, A., Greco, A., Bertrand, P., Odenthal, M., Büning, H. and Salvetti, A.

1. Virol., 89(13), 6824-6834 (2015)

  Adeno-associated virus (AAV) is a helper-dependent parvovirus that requires coinfection with adenovirus (AdV) or herpes simplex virus 1 (HSV-1) to replicate. In the absence of the helper virus, AAV can persist in an episomal or integrated form. Previous studies have analyzed the DNA damage response (DDR) induced upon AAV replication to understand how it controls AAV replication. In particular, it was shown that the Mre11-Rad50-Nbs1 (MRN) complex, a major player of the DDR induced by double-stranded DNA breaks and stalled replication forks, could negatively regulate AdV and AAV replication during coinfection. In contrast, MRN favors HSV-1 replication and is recruited to AAV replication compartments that are induced in the presence of HSV-1. In this study, we examined the role of MRN during AAV replication induced by HSV-1. Our results indicated that knockdown of MRN significantly reduced AAV DNA replication after coinfection with wild-type (wt) HSV-1 or HSV-1 with the polymerase deleted. This effect was specific to wt AAV, since it did not occur with recombinant AAV vectors. Positive regulation of AAV replication by MRN was dependent on its DNA tethering activity but did not require its nuclease activities. Importantly, knockdown of MRN also negatively regulated AAV integration within the human AAVS1 site, both in the presence and in the absence of HSV-1. Altogether, this work identifies a new function of MRN during integration of the AAV genome and demonstrates that this DNA repair complex positively regulates AAV replication in the presence of HSV-1.    

5.1637           Critical assessment of influenza VLP production in Sf9 and HEK293 expression systems

Thompson, C.M., Petiot, E., Mullick, A., Aucoin, M.G., henry, O. and Kamen, A.A. BMC Biotechnology, 15:31 (2015)   Background Each year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus’ fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen’s eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers. Methods In this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles. Results For the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles. Conclusions This study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine.  

5.1638           Survival benefit and phenotypic improvement by hamartin gene therapyin a tuberous sclerosis mouse brain model

Prabhakar, S., Zhang, X., Goto, J., Han, S., Lai, C., Bronson, R., Sena-Esteves, M., Ramesh, V., Stemmer-Rachmimov, A., Kwiatkowski, D.J. and Breakfield, X.O. Neurobiology of Disease, 82, 22-31 (2015)   We examined the potential benefit of gene therapy in a mouse model of tuberous sclerosis complex (TSC) in which there is embryonic loss of Tsc1 (hamartin) in brain neurons. An adeno-associated virus (AAV) vector (serotype rh8) expressing a tagged form of hamartin was injected into the cerebral ventricles of newborn pups with the genotype Tsc1^cc (homozygous for a conditional floxed Tsc1 allele) SynI-cre⁺, in which Tsc1 is lost selectively in neurons starting at embryonic day 12. Vector-treated Tsc1^ccSynIcre⁺ mice showed a marked improvement in survival from a mean of 22 days in non-injected mice to 52 days in AAV hamartin vector-injected mice, with improved weight gain and motor behavior in the latter. Pathologic studies showed normalization of neuron size and a decrease in markers of mTOR activation in treated as compared to untreated mutant littermates. Hence, we show that gene replacement in the brain is an effective therapeutic approach in this mouse model of TSC1. Our strategy for gene therapy has the advantages that therapy can be achieved from a single application, as compared to repeated treatment with drugs, and that AAV vectors have been found to have minimal to no toxicity in clinical trials for other neurologic conditions. Although there are many additional issues to be addressed, our studies support gene therapy as a useful approach in TSC patients.  

5.1639           High energetic excitons in carbon nanotubes directly probe charge-carriers

Vullhorst, D., Mitchell, R.M., Keating, C., Roychowdhury, S., Karavanova, I., Tao-Cheng, J-H. and Buonanno, A. Nature Communications, 5:9681 (2015)   Theory predicts peculiar features for excited-state dynamics in one dimension (1D) that are difficult to be observed experimentally. Single-walled carbon nanotubes (SWNTs) are an excellent approximation to 1D quantum confinement, due to their very high aspect ratio and low density of defects. Here we use ultrafast optical spectroscopy to probe photogenerated charge-carriers in (6,5) semiconducting SWNTs. We identify the transient energy shift of the highly polarizable S₃₃ transition as a sensitive fingerprint of charge-carriers in SWNTs. By measuring the coherent phonon amplitude profile we obtain a precise estimate of the Stark-shift and discuss the binding energy of the S₃₃ excitonic transition. From this, we infer that charge-carriers are formed instantaneously (<50 fs) even upon pumping the first exciton, S₁₁. The decay of the photogenerated charge-carrier population is well described by a model for geminate recombination in 1D.  

5.1640           Scalable Downstream Strategies for Purification of Recombinant Adeno- Associated Virus Vectors in Light of the Properties

Qu, W., Wang, M., Wu, Y. and Xu, R. Current Pharmaceutical Biotechnology, 16(8), 684-695 (2015)   Recombinant adeno-associated virus (rAAV) vector is one of the promising delivery tools for gene therapy. Currently, hundreds of clinical trials are performed but the major barrier for clinical application is the absence of any ideal large scale production technique to obtain sufficient and highly pure rAAV vector. The large scale production technique includes upstream and downstream processing. The upstream processing is a vector package step and the downstream processing is a vector purification step. For large scale downstream processing, the scientists need to recover rAAV from dozens of liters of cell lysate or medium, and a variety of purification strategies have been developed but not comprehensively compared till now. Consequently, this review will evaluate the scalable downstream purification strategies systematically, especially those based on the physicochemical properties of AAV virus, and attempt to find better scalable downstream strategies for rAAV vectors.  

5.1641           Induction of Neuron-Specific Degradation of Coenzyme A Models Pantothenate Kinase-Associated Neurodegeneration by Reducing Motor Coordination in Mice

Shumar, S.A., Fagone, P., Alfonso-Pecchio, A., Gray, J.T., rehg, JE., jackowsky, S. and Leonardi, R. PloS One, 10(6), e130013 (2015)   Pantothenate kinase-associated neurodegeneration, PKAN, is an inherited disorder characterized by progressive impairment in motor coordination and caused by mutations in PANK2, a human gene that encodes one of four pantothenate kinase (PanK) isoforms. PanK initiates the synthesis of coenzyme A (CoA), an essential cofactor that plays a key role in energy metabolism and lipid synthesis. Most of the mutations in PANK2 reduce or abolish the activity of the enzyme. This evidence has led to the hypothesis that lower CoA might be the underlying cause of the neurodegeneration in PKAN patients; however, no mouse model of the disease is currently available to investigate the connection between neuronal CoA levels and neurodegeneration. Indeed, genetic and/or dietary manipulations aimed at reducing whole-body CoA synthesis have not produced a desirable PKAN model, and this has greatly hindered the discovery of a treatment for the disease.  

5.1642           Adenoassociated Virus Serotype 9-Mediated Gene Therapy for X-Linked Adrenoleukodystrophy

Gong, Y., Mu, D., Prabhakar, S., Moser, A., Musolino, P., Ren, J., Breakfield, X.O., Maguire, C.A. and Eichler, F.S. Molecular Therapy, 23(5), 824-834 (2015)   X-linked adrenoleukodystrophy (X-ALD) is a devastating neurological disorder caused by mutations in the ABCD1 gene that encodes a peroxisomal ATP-binding cassette transporter (ABCD1) responsible for transport of CoA-activated very long-chain fatty acids (VLCFA) into the peroxisome for degradation. We used recombinant adenoassociated virus serotype 9 (rAAV9) vector for delivery of the human ABCD1 gene (ABCD1) to mouse central nervous system (CNS). In vitro, efficient delivery of ABCD1 gene was achieved in primary mixed brain glial cells from Abcd1−/− mice as well as X-ALD patient fibroblasts. Importantly, human ABCD1 localized to the peroxisome, and AAV-ABCD1 transduction showed a dose-dependent effect in reducing VLCFA. In vivo, AAV9-ABCD1 was delivered to Abcd1−/− mouse CNS by either stereotactic intracerebroventricular (ICV) or intravenous (IV) injections. Astrocytes, microglia and neurons were the major target cell types following ICV injection, while IV injection also delivered to microvascular endothelial cells and oligodendrocytes. IV injection also yielded high transduction of the adrenal gland. Importantly, IV injection of AAV9-ABCD1 reduced VLCFA in mouse brain and spinal cord. We conclude that AAV9-mediated ABCD1 gene transfer is able to reach target cells in the nervous system and adrenal gland as well as reduce VLCFA in culture and a mouse model of X-ALD.  

5.1643           Intracellular FGF14 (iFGF14) Is Required for Spontaneous and Evoked Firing in Cerebellar Purkinje Neurons and for Motor Coordination and Balance

Bosch, M.K., Carraquillo, Y., Ransdell, J.L., kanakamedala, A., Ornitz, D.M. and Nerbonne, J.M.

1. Neurosci., 35(17), 6752-6769 (2015)

  Mutations in FGF14, which encodes intracellular fibroblast growth factor 14 (iFGF14), have been linked to spinocerebellar ataxia (SCA27). In addition, mice lacking Fgf14 (Fgf14^−/−) exhibit an ataxia phenotype resembling SCA27, accompanied by marked changes in the excitability of cerebellar granule and Purkinje neurons. It is not known, however, whether these phenotypes result from defects in neuronal development or if they reflect a physiological requirement for iFGF14 in the adult cerebellum. Here, we demonstrate that the acute and selective Fgf14-targeted short hairpin RNA (shRNA)-mediated in vivo “knock-down” of iFGF14 in adult Purkinje neurons attenuates spontaneous and evoked action potential firing without measurably affecting the expression or localization of voltage-gated Na⁺ (Nav) channels at Purkinje neuron axon initial segments. The selective shRNA-mediated in vivo “knock-down” of iFGF14 in adult Purkinje neurons also impairs motor coordination and balance. Repetitive firing can be restored in Fgf14-targeted shRNA-expressing Purkinje neurons, as well as in Fgf14^−/− Purkinje neurons, by prior membrane hyperpolarization, suggesting that the iFGF14-mediated regulation of the excitability of mature Purkinje neurons depends on membrane potential. Further experiments revealed that the loss of iFGF14 results in a marked hyperpolarizing shift in the voltage dependence of steady-state inactivation of the Nav currents in adult Purkinje neurons. We also show here that expressing iFGF14 selectively in adult Fgf14^−/− Purkinje neurons rescues spontaneous firing and improves motor performance. Together, these results demonstrate that iFGF14 is required for spontaneous and evoked action potential firing in adult Purkinje neurons, thereby controlling the output of these cells and the regulation of motor coordination and balance.  

5.1644           Opposing Role for Egr3 in Nucleus Accumbens Cell Subtypes in Cocaine Action

Chandra, R., Francis, T.C., Konkalmatt, P., Amgalan, A., Gancarz, A.M., Dietz, D.M. and Lobo, M.K.

1. Neurosci., 35(20), 7927-7937 (2015)

  An imbalance in molecular signaling cascades and transcriptional regulation in nucleus accumbens (NAc) medium spiny neuron (MSN) subtypes, those enriched in dopamine D1 versus D2 receptors, is implicated in the behavioral responses to psychostimulants. To provide further insight into the molecular mechanisms occurring in MSN subtypes by cocaine, we examined the transcription factor early growth response 3 (Egr3). We evaluated Egr3 because it is a target of critical cocaine-mediated signaling pathways and because Egr3-binding sites are found on promoters of key cocaine-associated molecules. We first used a RiboTag approach to obtain ribosome-associated transcriptomes from each MSN subtype and found that repeated cocaine administration induced Egr3 ribosome-associated mRNA in NAc D1-MSNs while reducing Egr3 in D2-MSNs. Using Cre-inducible adeno-associated viruses combined with D1-Cre and D2-Cre mouse lines, we observed that Egr3 overexpression in D1-MSNs enhances rewarding and locomotor responses to cocaine, whereas overexpression in D2-MSNs blunts these behaviors. miRNA knock-down of Egr3 in MSN subtypes produced opposite behavioral responses from those observed with overexpression. Finally, we found that repeated cocaine administration altered Egr3 binding to promoters of genes that are important for cocaine-mediated cellular and behavioral plasticity. Genes with increased Egr3 binding to promoters, Camk2α, CREB, FosB, Nr4a2, and Sirt1, displayed increased mRNA in D1-MSNs and, in some cases, a reduction in D2-MSNs. Histone and the DNA methylation enzymes G9a and Dnmt3a displayed reduced Egr3 binding to their promoters and reduced mRNA in D1-MSNs. Our study provides novel insight into an opposing role of Egr3 in select NAc MSN subtypes in cocaine action.  

5.1645           Human Polyomavirus 7–Associated Pruritic Rash and Viremia in Transplant Recipients

Ho, J.et al Journal of Infectious Disease, 211(10), 1560-1565 (2015)   Human polyomavirus 7 (HPyV7) is one of 11 HPyVs recently discovered through genomic sequencing technologies. Two lung transplant recipients receiving immunosuppressive therapy developed pruritic, brown plaques on the trunk and extremities showing a distinctive epidermal hyperplasia with virus-laden keratinocytes containing densely packed 36–45-nm icosahedral capsids. Rolling circle amplification and gradient centrifugation testing were positive for encapsidated HPyV7 DNA in skin and peripheral blood specimens from both patients, and HPyV7 early and capsid proteins were abundantly expressed in affected tissues. We describe for the first time that HPyV7 is associated with novel pathogenicity in some immunosuppressed individuals.  

5.1646           Gene therapy into photoreceptors and Müller glial cells restores retinal structure and function in CRB1 retinitis pigmentosa mouse models

Pellissier, L.P., Quinn, P.M., Alves, C.H., Vos, R.M., Klooster, J., Flannery, J.G., Heimel, A. and Wijnholds, J. Hum. Mol. Genet., 24(11), 3104-3118 (2015)   Mutations in the Crumbs-homologue-1 (CRB1) gene lead to severe recessive inherited retinal dystrophies. Gene transfer therapy is the most promising cure for retinal dystrophies and has primarily been applied for recessive null conditions via a viral gene expression vector transferring a cDNA encoding an enzyme or channel protein, and targeting expression to one cell type. Therapy for the human CRB1 disease will be more complex, as CRB1 is a structural and signaling transmembrane protein present in three cell classes: Müller glia, cone and rod photoreceptors. In this study, we applied CRB1 and CRB2 gene therapy vectors in Crb1-retinitis pigmentosa mouse models at mid-stage disease. We tested if CRB expression restricted to Müller glial cells or photoreceptors or co-expression in both is required to recover retinal function. We show that targeting both Müller glial cells and photoreceptors with CRB2 ameliorated retinal function and structure in Crb1 mouse models. Surprisingly, targeting a single cell type or all cell types with CRB1 reduced retinal function. We show here the first pre-clinical studies for CRB1-related eye disorders using CRB2 vectors and initial elucidation of the cellular mechanisms underlying CRB1 function.  

5.1647           Long-Term Sex-Biased Correction of Circulating Propionic Acidemia Disease Markers by Adeno-Associated Virus Vectors No Access

Guenzel, A.J., Collard, R., Kraus, J.P., Matern, D. and barry, M.A. Human Gene Therapy, 26(3), 153-160 (2015)   Propionic acidemia (PA) occurs because of mutations in the PCCA or PCCB genes encoding the two subunits of propionyl-CoA carboxylase, a pivotal enzyme in the breakdown of certain amino acids and odd-chain fatty acids. There is no cure for PA, but dietary protein restriction and liver transplantation can attenuate its symptoms. We show here that a single intravenous injection of adeno-associated virus 2/8 (AAV8) or AAVrh10 expressing PCCA into PA hypomorphic mice decreased systemic propionylcarnitine and methyl citrate for up to 1.5 years. However, long-term phenotypic correction was always better in male mice. AAV-mediated PCCA expression was similar in most tissues in males and females at early time points and differed only in the liver. Over 1.5 years, luciferase and PCCA expression remained elevated in cardiac tissue for both sexes. In contrast, transgene expression in the liver and skeletal muscles of female, but not male, mice waned—suggesting that these tissues were major sinks for systemic phenotypic correction. These data indicate that single systemic intravenous therapy by AAV vectors can mediate long-term phenotype correction for PA. However, tissue-specific loss of expression in females reduces efficacy when compared with males. Whether similar sex-biased AAV effects occur in human gene therapy remains to be determined.  

5.1648           Induction and functional significance of the heme oxygenase system in pathological shear stress in vivo

Kang, L., Hillestad, M.L., grande, J.P., Croatt, A.J., Barry, M.A., Farrugia, G., Katusic, Z.S., and nath, K.A. Am. J. Physiol. Heart Circ. Physiol., 308(11), H1402-H1413 (2015)   The present study examined the heme oxygenase (HO) system in an in vivo murine model of pathological shear stress induced by partial carotid artery ligation. In this model, along with upregulation of vasculopathic genes, HO-1 is induced in the endothelium and adventitia, whereas HO-2 is mainly upregulated in the endothelium. Within minutes of ligation, NF-κB, a transcription factor that upregulates vasculopathic genes and HO-1, is activated. Failure to express either HO-1 or HO-2 exaggerates the reduction in carotid blood flow and exacerbates vascular injury. After artery ligation, comparable induction of HO-2 occurred in HO-1^+/+ and HO-1^−/− mice, whereas HO-1 induction was exaggerated in HO-2^−/− mice compared with HO-2^+/+ mice. Upregulation of HO-1 by an adeno-associated viral vector increased vascular HO-1 expression and HO activity and augmented blood flow in both ligated and contralateral carotid arteries. Acute inhibition of HO activity decreased flow in the ligated carotid artery, whereas a product of HO, carbon monoxide (CO), delivered by CO-releasing molecule-3, increased carotid blood flow. In conclusion, in the partial carotid artery ligation model of pathological shear stress, this study provides the first demonstration of 1) upregulation and vasoprotective effects of HO-1 and HO-2 and the vasorelaxant effects of CO as well as 2) vascular upregulation of HO-1 in vivo by an adeno-associated viral vector that is attended by a salutary vascular response. Induction of HO-1 may reside in NF-κB activation, and, along with induced HO-2, such upregulation of HO-1 provides a countervailing vasoprotective response in pathological shear stress in vivo.  

5.1649           Restoring the ON Switch in Blind Retinas: Opto-mGluR6, a Next-Generation, Cell-Tailored Optogenetic Tool

Van Wyk, M., Pielecka-Fortuna, J., Löwel, S. and Kleinlogel, S. PloS Biology, 13(5), e11002143 (2015)   Photoreceptor degeneration is one of the most prevalent causes of blindness. Despite photoreceptor loss, the inner retina and central visual pathways remain intact over an extended time period, which has led to creative optogenetic approaches to restore light sensitivity in the surviving inner retina. The major drawbacks of all optogenetic tools recently developed and tested in mouse models are their low light sensitivity and lack of physiological compatibility. Here we introduce a next-generation optogenetic tool, Opto-mGluR6, designed for retinal ON-bipolar cells, which overcomes these limitations. We show that Opto-mGluR6, a chimeric protein consisting of the intracellular domains of the ON-bipolar cell–specific metabotropic glutamate receptor mGluR6 and the light-sensing domains of melanopsin, reliably recovers vision at the retinal, cortical, and behavioral levels under moderate daylight illumination.  

5.1650           A coding-independent function of an alternative Ube3a transcript during neuronal development

Valluy, J., Bicker, S., Aksoy-Aksel, A., lackinger, M., Sumer, S., Fiore, R., Wüst, T., Seffer, D., metge, f., Dieterich, C., Wöhr, M., Schwarting, R. and Schratt, G. Nature Neurosci., 18(5), 666-673 (2015)   The E3 ubiquitin ligase Ube3a is an important regulator of activity-dependent synapse development and plasticity. Ube3a mutations cause Angelman syndrome and have been associated with autism spectrum disorders (ASD). However, the biological significance of alternative Ube3a transcripts generated in mammalian neurons remains unknown. We report here that Ube3a1 RNA, a transcript that encodes a truncated Ube3a protein lacking catalytic activity, prevents exuberant dendrite growth and promotes spine maturation in rat hippocampal neurons. Surprisingly, Ube3a1 RNA function was independent of its coding sequence but instead required a unique 3′ untranslated region and an intact microRNA pathway. Ube3a1 RNA knockdown increased activity of the plasticity-regulating miR-134, suggesting that Ube3a1 RNA acts as a dendritic competing endogenous RNA. Accordingly, the dendrite-growth-promoting effect of Ube3a1 RNA knockdown in vivo is abolished in mice lacking miR-134. Taken together, our results define a noncoding function of an alternative Ube3a transcript in dendritic protein synthesis, with potential implications for Angelman syndrome and ASD.  

5.1651           Progressive maturation of silent synapses governs the duration of a critical period

Huang, X., Stodieck, S.K., Goetze, b., Cui, L., Wong, M.H., Wenzel, C., Hosang, L., Dong, Y., Löwel, S. and Schlüter, O.M. PNAS, 112, E3131-E3140 (2015)   During critical periods, all cortical neural circuits are refined to optimize their functional properties. The prevailing notion is that the balance between excitation and inhibition determines the onset and closure of critical periods. In contrast, we show that maturation of silent glutamatergic synapses onto principal neurons was sufficient to govern the duration of the critical period for ocular dominance plasticity in the visual cortex of mice. Specifically, postsynaptic density protein-95 (PSD-95) was absolutely required for experience-dependent maturation of silent synapses, and its absence before the onset of critical periods resulted in lifelong juvenile ocular dominance plasticity. Loss of PSD-95 in the visual cortex after the closure of the critical period reinstated silent synapses, resulting in reopening of juvenile-like ocular dominance plasticity. Additionally, silent synapse-based ocular dominance plasticity was largely independent of the inhibitory tone, whose developmental maturation was independent of PSD-95. Moreover, glutamatergic synaptic transmission onto parvalbumin-positive interneurons was unaltered in PSD-95 KO mice. These findings reveal not only that PSD-95–dependent silent synapse maturation in visual cortical principal neurons terminates the critical period for ocular dominance plasticity but also indicate that, in general, once silent synapses are consolidated in any neural circuit, initial experience-dependent functional optimization and critical periods end.      

5.1652           Primary possum macrophage cultures support the growth of a nidovirus associated with wobbly possum disease

Giles, J.C., Perrott, M.R. and Dunowska, M.

1. Virol. Methods, 222, 66-71 (2015)

  The objective of the study was to establish a system for isolation of a recently described, thus far uncultured, marsupial nidovirus associated with a neurological disease of possums, termed wobbly possum disease (WPD). Primary cultures of possum macrophages were established from livers of adult Australian brushtail possums (Trichosurus vulpecula). High viral copy numbers (up to 6.9 × 10⁸/mL of cell lysate) were detected in infected cell culture lysates from up to the 5th passage of the virus, indicating that the putative WPD virus (WPDV) was replicating in cultured cells. A purified virus stock with a density of 1.09 g/mL was prepared using iodixanol density gradient ultracentrifugation. Virus-like particles approximately 60 nm in diameter were observed using electron microscopy in negatively stained preparations of the purified virus. The one-step growth curve of WPDV in macrophage cultures showed the highest increase in intracellular viral RNA between 6 and 12 h post-infection. Maximum levels of cell-associated viral RNA were detected at 24 h post-infection, followed by a decline. Levels of extracellular RNA increased starting at 9 h post-infection, with maximum levels detected at 48 h post-infection. The establishment of the in vitro system to culture WPDV will facilitate further characterisation of this novel nidovirus.  

5.1653           Development of a highly thermostable, adjuvanted human papillomavirus vaccine

Hassett, K.J., Meinerz, N.M., Semmelmann, F., Cousins, M.C., garcea, R.L. and Randolph, T.W. Eur. J. Pharmaceut. Biopharmaceut., 94, 220-228 (2015)   A major impediment to economical, worldwide vaccine distribution is the requirement for a “cold chain” to preserve antigenicity. We addressed this problem using a model human papillomavirus (HPV) vaccine stabilized by immobilizing HPV16 L1 capsomeres, i.e., pentameric subunits of the virus capsid, within organic glasses formed by lyophilization. Lyophilized glass and liquid vaccine formulations were incubated at 50 °C for 12 weeks, and then analyzed for retention of capsomere conformational integrity and the ability to elicit neutralizing antibody responses after immunization of BALB/c mice. Capsomeres in glassy-state vaccines retained tertiary and quaternary structure, and critical conformational epitopes. Moreover, glassy formulations adjuvanted with aluminum hydroxide or aluminum hydroxide and glycopyranoside lipid A were not only as immunogenic as the commercially available HPV vaccine Cervarix®, but also retained complete neutralizing immunogenicity after high-temperature storage. The thermal stability of such adjuvanted vaccine powder preparations may thus eliminate the need for the cold chain.  

5.1654           Development and rescue of human familial hypercholesterolaemia in a xenograft mouse model

Bissig-Choisat, B. et al Nature Communications, 6:7339 82015)   Diseases of lipid metabolism are a major cause of human morbidity, but no animal model entirely recapitulates human lipoprotein metabolism. Here we develop a xenograft mouse model using hepatocytes from a patient with familial hypercholesterolaemia caused by loss-of-function mutations in the low-density lipoprotein receptor (LDLR). Like familial hypercholesterolaemia patients, our familial hypercholesterolaemia liver chimeric mice develop hypercholesterolaemia and a ’humanized‘ serum profile, including expression of the emerging drug targets cholesteryl ester transfer protein and apolipoprotein (a), for which no genes exist in mice. We go on to replace the missing LDLR in familial hypercholesterolaemia liver chimeric mice using an adeno-associated virus 9-based gene therapy and restore normal lipoprotein profiles after administration of a single dose. Our study marks the first time a human metabolic disease is induced in an experimental animal model by human hepatocyte transplantation and treated by gene therapy. Such xenograft platforms offer the ability to validate human experimental therapies and may foster their rapid translation into the clinic.  

5.1655           Nigral overexpression of alpha-synuclein in the absence of parkin enhances alpha-synuclein phosphorylation but does not modulate dopaminergic neurodegeneration

Van Rompuy, A-S., Oliveras-Salva, M., Van der Perren, A., Corti, O., Van den Haute, C. and baekelandt, V. Mol. Neurodegeneration, 10:23 (2015)   Background Alpha-synuclein is a key protein in the pathogenesis of Parkinson’s disease. Mutations in the parkin gene are the most common cause of early-onset autosomal recessive Parkinson’s disease, probably through a loss-of-function mechanism. However, the molecular mechanism by which loss of parkin function leads to the development of the disease and the role of alpha-synuclein in parkin-associated Parkinson’s disease is still not elucidated. Conflicting results were reported about the effect of the absence of parkin on alpha-synuclein-mediated neurotoxicity using a transgenic approach. In this study, we investigated the effect of loss of parkin on alpha-synuclein neuropathology and toxicity in adult rodent brain using viral vectors. Therefore, we overexpressed human wild type alpha-synuclein in the substantia nigra of parkin knockout and wild type mice using two different doses of recombinant adeno-associated viral vectors. Results No difference was observed in nigral dopaminergic cell loss between the parkin knockout mice and wild type mice up to 16 weeks after viral vector injection. However, the level of alpha-synuclein phosphorylated at serine residue 129 in the substantia nigra was significantly increased in the parkin knockout mice compared to the wild type mice while the total expression level of alpha-synuclein was similar in both groups. The increased alpha-synuclein phosphorylation was confirmed in a parkin knockdown cell line. Conclusions These findings support a functional relationship between parkin and alpha-synuclein phosphorylation in rodent brain.  

5.1656           Peptide Triazole Inactivators of HIV-1 Utilize a Conserved Two-Cavity Binding Site at the Junction of the Inner and Outer Domains of Env gp120

Aneja, R., Rashad, A.A., Li, H., Sundaram, R.V.K., Duffy, C., Bailey, L.D. and Chaiken, I.

1. Medicinal Chem., 58(9), 3843-3858 (2015)

  We used coordinated mutagenesis, synthetic design, and flexible docking to investigate the structural mechanism of Env gp120 encounter by peptide triazole (PT) inactivators of HIV-1. Prior results demonstrated that the PT class of inhibitors suppresses binding at both CD4 and coreceptor sites on Env and triggers gp120 shedding, leading to cell-independent irreversible virus inactivation. Despite these enticing anti-HIV-1 phenotypes, structural understanding of the PT–gp120 binding mechanism has been incomplete. Here we found that PT engages two inhibitor ring moieties at the junction between the inner and outer domains of the gp120 protein. The results demonstrate how combined occupancy of two gp120 cavities can coordinately suppress both receptor and coreceptor binding and conformationally entrap the protein in a destabilized state. The two-cavity model has common features with small molecule gp120 inhibitor binding sites and provides a guide for further design of peptidomimetic HIV-1 inactivators based on the PT pharmacophore.  

5.1657           Co-administration with DNA encoding papillomavirus capsid proteins enhances the antitumor effects generated by therapeutic HPV DNA vaccination

Yang, B., Yang, A., Peng, S., Pang, X., Roden, R.B.S., Wu, T-C. and Hung, C-F. Cell & Bioscience, 5:35 (2015)   Background DNA vaccines have emerged as attractive candidates for the control of human papillomavirus (HPV)-associated malignancies. However, DNA vaccines suffer from limited immunogenicity and thus strategies to enhance DNA vaccine potency are needed. We have previously demonstrated that for DNA vaccines encoding HPV-16 E7 antigen (CRT/E7) linkage with calreticulin (CRT) linked enhances both the E7-specific CD8⁺ T cell immune responses and antitumor effects against E7-expressing tumors. In the current study, we aim to introduce an approach to elicit potent CD4⁺ T cell help for the enhancement of antigen-specific CD8⁺ T cell immune responses generated by CRT/E7 DNA vaccination by using co-administration of a DNA vector expressing papillomavirus major and minor capsid antigens, L1 and L2. Result We showed that co-administration of vectors containing codon-optimized bovine papillomavirus type 1 (BPV-1) L1 and L2 in combination with DNA vaccines could elicit enhanced antigen-specific CD8⁺ in both CRT/E7 and ovalbumin (OVA) antigenic systems. We also demonstrated that co-administration of vectors expressing BPV-1 L1 and/or L2 DNA with CRT/E7 DNA led to the generation of L1/L2-specific CD4⁺ T cell immune responses and L1-specific neutralizing antibodies. Furthermore, we showed that co-administration with DNA encoding BPV1 L1 significantly enhances the therapeutic antitumor effects generated by CRT/E7 DNA vaccination. In addition, the observed enhancement of CD8⁺ T cell immune responses by DNA encoding L1 and L2 was also found to extend to HPV-16 L1/L2 system. Conclusion Our strategy elicits both potent neutralizing antibody and therapeutic responses and may potentially be extended to other antigenic systems beyond papillomavirus for the control of infection and/or cancer.  

5.1658           The effects of reference genes in qRT-PCR assays for determining the immune response of bovine cells (MDBK) infected with the Bovine Viral Diarrhea Virus 1 (BVDV-1)

Fredericksen, F., Delgado, F., Cabrera, C., Yanez, A., Gonzalo, C., Villalba, M. and Olavarria, V.H. Gene, 569, 95-103 (2015)   The bovine viral diarrhea virus (BVDV) causes significant economic losses to the dairy industry worldwide, and understanding its infection mechanisms would be extremely useful in designing new and efficient treatments. Due to the limited number of specific antibodies against bovine proteins, differential gene expression analyses are vital for researching host immune responses to viral infection. qRT-PCR provides a sensitive platform to conduct such gene expression analyses, but suitable housekeeping genes are needed for accurate transcript normalization. The present study assessed nine reference genes in bovine kidney cells under conditions of BVDV-1 infection, incubation with pathogen-associated molecular patterns, and co-incubation with BAY117085, a pharmacological inhibitor of the NF-κB signaling pathway. Analyses of Ct values using the BestKeeper and Normfinder programs ranked CD81, RPL4, and GAPDH as the most reliable reference genes. This determination of a stable set of reference genes in this culture system will facilitate analyses of expression levels for genes of interest.  

5.1659           Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration

Campbell, G.R., Rawat, P., Bruckman, R.S. and Spector, S.A. PloS Pathogens, 11(6), e1005018 (2015)   HIV Nef acts as an anti-autophagic maturation factor through interaction with beclin-1 (BECN1). We report that exposure of macrophages to infectious or non-infectious purified HIV induces toll-like receptor 8 (TLR8) and BECN1 dependent dephosphorylation and nuclear translocation of TFEB and that this correlates with an increase in autophagy markers. RNA interference for ATG13, TFEB, TLR8, or BECN1 inhibits this HIV-induced autophagy. However, once HIV establishes a productive infection, TFEB phosphorylation and cytoplasmic sequestration are increased resulting in decreased autophagy markers. Moreover, by 7 d post-infection, autophagy levels are similar to mock infected controls. Conversely, although Nef deleted HIV similarly induces TFEB dephosphorylation and nuclear localization, and increases autophagy, these levels remain elevated during continued productive infection. Thus, the interaction between HIV and TLR8 serves as a signal for autophagy induction that is dependent upon the dephosphorylation and nuclear translocation of TFEB. During permissive infection, Nef binds BECN1 resulting in mammalian target of rapamycin (MTOR) activation, TFEB phosphorylation and cytosolic sequestration, and the inhibition of autophagy. To our knowledge, this is the first report of a virus modulating TFEB localization and helps to explain how HIV modulates autophagy to promote its own replication and cell survival.  

5.1660           Naturally Occurring Capsid Protein Variants of Human Papillomavirus Genotype 31 Represent a Single L1 Serotype

Bissett, S.L., Godi, A., Fleury, M.J.J., Touze, A., Cocuzza, C. and Beddows, S.

1. Virol., 89(15), 7748-7757 (2015)

  We investigated naturally occurring variation within the major (L1) and minor (L2) capsid proteins of oncogenic human papillomavirus (HPV) genotype 31 (HPV31) to determine the impact on capsid antigenicity. L1L2 pseudoviruses (PsVs) representing the three HPV31 variant lineages, variant lineages A, B, and C, exhibited comparable particle-to-infectivity ratios and morphologies. Lineage-specific L1L2 PsVs demonstrated subtle differences in susceptibility to neutralization by antibodies elicited following vaccination or preclinical L1 virus-like particle (VLP) immunization or by monoclonal antibodies; however, these differences were generally of a low magnitude. These data indicate that the diagnostic lineage-specific single nucleotide polymorphisms within the HPV31 capsid genes have a limited effect on L1 antibody-mediated neutralization and that the three HPV31 variant lineages belong to a single L1 serotype. These data contribute to our understanding of HPV L1 variant antigenicity.    

5.1661           Adaptive Mutations Enhance Assembly and Cell-to-Cell Transmission of a High-Titer Hepatitis C Virus Genotype 5a Core-NS2 JFH1-Based Recombinant

Mathiesen, C.K., Prentoe, J., Meredith, L.W., Jensen, T.B., Krarup, H., McKeating, J.A., Gottwein, J.M. and Bukh, J.

1. Virol., 89(15), 7758-7775 (2015)

  Recombinant hepatitis C virus (HCV) clones propagated in human hepatoma cell cultures yield relatively low infectivity titers. Here, we adapted the JFH1-based Core-NS2 recombinant SA13/JFH1_{C3405G,A3696G} (termed SA13/JFH1_orig), of the poorly characterized genotype 5a, to Huh7.5 cells, yielding a virus with greatly improved spread kinetics and an infectivity titer of 6.7 log₁₀ focus-forming units (FFU)/ml. We identified several putative adaptive amino acid changes. In head-to-head infections at fixed multiplicities of infection, one SA13/JFH1_orig mutant termed SA13/JFH1_Core-NS5B, containing 13 amino acid changes (R114W and V187A [Core]; V235L [E1]; T385P [E2]; L782V [p7]; Y900C [NS2]; N2034D, E2238G, V2252A, L2266P, and I2340T [NS5A]; A2500S and V2841A [NS5B]), displayed fitness comparable to that of the polyclonal high-titer adapted virus. Single-cycle virus production assays in CD81-deficient Huh7-derived cells demonstrated that these changes did not affect replication but increased HCV assembly and specific infectivity as early as 24 h posttransfection. Infectious coculture assays in Huh7.5 cells showed a significant increase in cell-to-cell transmission for SA13/JFH1_Core-NS5B viruses as well as viruses with only p7 and nonstructural protein mutations. Interestingly, the E2 hypervariable region 1 (HVR1) mutation T385P caused (i) increased sensitivity to neutralizing patient IgG and human monoclonal antibodies AR3A and AR4A and (ii) increased accessibility of the CD81 binding site without affecting the usage of CD81 and SR-BI. We finally demonstrated that SA13/JFH1_orig and SA13/JFH1_Core-NS5B, with and without the E2 mutation T385P, displayed similar biophysical properties following iodixanol gradient ultracentrifugation. This study has implications for investigations requiring high virus concentrations, such as studies of HCV particle composition and development of whole-virus vaccine antigens.  

5.1662           Development of an intein-mediated split–Cas9 system for gene therapy

Truong, D-J.J., Kühner, K., Kühn, R., Werfel, S., Engelhardt, s., Wurst, W. and Ortiz, O. Nucleic Acids Res., 43(13), 6450-6458 (2015)   Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split–Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split–Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.  

5.1663           Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome

Chang, Q., Wang, J., Kim, Y., Zhou, B., Wang, Y., Li, H. and Lin, X. EMBO Mol. Med., 7(8), 1077-1086 (2015)   Mutations in the potassium channel subunit KCNQ1 cause the human severe congenital deafness Jervell and Lange‐Nielsen (JLN) syndrome. We applied a gene therapy approach in a mouse model of JLN syndrome (Kcnq1^−/− mice) to prevent the development of deafness in the adult stage. A modified adeno‐associated virus construct carrying a Kcnq1 expression cassette was injected postnatally (P0–P2) into the endolymph, which resulted in Kcnq1 expression in most cochlear marginal cells where native Kcnq1 is exclusively expressed. We also found that extensive ectopic virally mediated Kcnq1 transgene expression did not affect normal cochlear functions. Examination of cochlear morphology showed that the collapse of the Reissner's membrane and degeneration of hair cells (HCs) and cells in the spiral ganglia were corrected in Kcnq1^−/− mice. Electrophysiological tests showed normal endocochlear potential in treated ears. In addition, auditory brainstem responses showed significant hearing preservation in the injected ears, ranging from 20 dB improvement to complete correction of the deafness phenotype. Our results demonstrate the first successful gene therapy treatment for gene defects specifically affecting the function of the stria vascularis, which is a major site affected by genetic mutations in inherited hearing loss.  

5.1664           New Insights into the Understanding of Hepatitis C Virus Entry and Cell-to-Cell Transmission by Using the Ionophore Monensin A

Feneant, L. et al

1. Virol., 89(16), 8346-8364 (2015)

  In our study, we characterized the effect of monensin, an ionophore that is known to raise the intracellular pH, on the hepatitis C virus (HCV) life cycle. We showed that monensin inhibits HCV entry in a pangenotypic and dose-dependent manner. Monensin induces an alkalization of intracellular organelles, leading to an inhibition of the fusion step between viral and cellular membranes. Interestingly, we demonstrated that HCV cell-to-cell transmission is dependent on the vesicular pH. Using the selective pressure of monensin, we selected a monensin-resistant virus which has evolved to use a new entry route that is partially pH and clathrin independent. Characterization of this mutant led to the identification of two mutations in envelope proteins, the Y297H mutation in E1 and the I399T mutation in hypervariable region 1 (HVR1) of E2, which confer resistance to monensin and thus allow HCV to use a pH-independent entry route. Interestingly, the I399T mutation introduces an N-glycosylation site within HVR1 and increases the density of virions and their sensitivity to neutralization with anti-apolipoprotein E (anti-ApoE) antibodies, suggesting that this mutation likely induces conformational changes in HVR1 that in turn modulate the association with ApoE. Strikingly, the I399T mutation dramatically reduces HCV cell-to-cell spread. In summary, we identified a mutation in HVR1 that overcomes the vesicular pH dependence, modifies the biophysical properties of particles, and drastically reduces cell-to-cell transmission, indicating that the regulation by HVR1 of particle association with ApoE might control the pH dependence of cell-free and cell-to-cell transmission. Thus, HVR1 and ApoE are critical regulators of HCV propagation.  

5.1665           ERdj5 Reductase Cooperates with Protein Disulfide Isomerase To Promote Simian Virus 40 Endoplasmic Reticulum Membrane Translocation

Inoue, T., Dosey, A., Herbstman, J.F., Ravindran, M.S., Skiniotis, G. and Tsai, B.

1. Virol., 89(17), 8897-8908 (2015)

  The nonenveloped polyomavirus (PyV) simian virus 40 (SV40) traffics from the cell surface to the endoplasmic reticulum (ER), where it penetrates the ER membrane to reach the cytosol before mobilizing into the nucleus to cause infection. Prior to ER membrane penetration, ER lumenal factors impart structural rearrangements to the virus, generating a translocation-competent virion capable of crossing the ER membrane. Here we identify ERdj5 as an ER enzyme that reduces SV40's disulfide bonds, a reaction important for its ER membrane transport and infection. ERdj5 also mediates human BK PyV infection. This enzyme cooperates with protein disulfide isomerase (PDI), a redox chaperone previously implicated in the unfolding of SV40, to fully stimulate membrane penetration. Negative-stain electron microscopy of ER-localized SV40 suggests that ERdj5 and PDI impart structural rearrangements to the virus. These conformational changes enable SV40 to engage BAP31, an ER membrane protein essential for supporting membrane penetration of the virus. Uncoupling of SV40 from BAP31 traps the virus in ER subdomains called foci, which likely serve as depots from where SV40 gains access to the cytosol. Our study thus pinpoints two ER lumenal factors that coordinately prime SV40 for ER membrane translocation and establishes a functional connection between lumenal and membrane events driving this process.  

5.1666           Liver-directed gene therapy of chronic hepadnavirus infection using interferon alpha tethered to apolipoprotein A-I

Berraondo, P. et al

1. Hepatol., 63, 329-336 (2015)

  Background & Aims Current hepatitis B virus (HBV) management is challenging as treatment with nucleos(t)ide analogues needs to be maintained indefinitely and because interferon (IFN)-α therapy is associated with considerable toxicity. Previously, we showed that linking IFNα to apolipoprotein A-I generates a molecule (IA) with distinct antiviral and immunostimulatory activities which lacks the hematological toxicity of IFNα. Methods Here, we analyse the antiviral potential of an adeno-associated vector encoding IFNα fused to apolipoprotein A-I (AAV-IA) in comparison to a vector encoding only IFNα (AAV-IFN) in two animal models of chronic hepadnavirus infection. Results In HBV transgenic mice, we found that both vectors induced marked reductions in serum and liver HBV DNA and in hepatic HBV RNA but AAV-IFN caused lethal pancytopenia. Woodchucks with chronic hepatitis virus (WHV) infection that were treated by intrahepatic injection of vectors encoding the woodchuck sequences (AAV-wIFN or AAV-wIA), experienced only a slight reduction of viremia which was associated with hematological toxicity and high mortality when using AAV-wIFN, while AAV-wIA was well tolerated. However, when we tested AAV-wIA or a control vector encoding woodchuck apolipoprotein A-I (AAV-wApo) in combination with entecavir, we found that AAV-wApo-treated animals exhibited an immediate rebound of viral load upon entecavir withdrawal while, in AAV-wIA-treated woodchucks, viremia and antigenemia remained at low levels for several weeks following entecavir interruption. Conclusions Treatment with AAV-IA is safe and elicits antiviral effects in animal models with difficult to treat chronic hepadnavirus infection. AAV-IA in combination with nucleos(t)ide analogues represents a promising approach for the treatment of HBV infection in highly viremic patients.  

5.1667           Induction and characterization of a replication competent cervid endogenous gammaretrovirus (CrERV) from mule deer cells

Fabryova, H., Hron, T., kabickova, H., Poss, M. and Elleder, D. Virology, 485, 96-103 (2015)   Endogenous retroviruses (ERVs) were acquired during evolution of their host organisms after infection and mendelian inheritance in the germline by their exogenous counterparts. The ERVs can spread in the host genome and in some cases they affect the host phenotype. The cervid endogenous gammaretrovirus (CrERV) is one of only a few well-defined examples of evolutionarily recent invasion of mammalian genome by retroviruses. Thousands of insertionally polymorphic CrERV integration sites have been detected in wild ranging mule deer (Odocoileus hemionus) host populations. Here, we describe for the first time induction of replication competent CrERV by cocultivation of deer and human cells. We characterize the physical properties and tropism of the induced virus. The genomic sequence of the induced virus is phylogenetically related to the evolutionarily young endogenous CrERVs described so far. We also describe the level of replication block of CrERV on deer cells and its capacity to establish superinfection interference.  

5.1668           VPS35 in Dopamine Neurons Is Required for Endosome-to-Golgi Retrieval of Lamp2a, a Receptor of Chaperone-Mediated Autophagy That Is Critical for α-Synuclein Degradation and Prevention of Pathogenesis of Parkinson's Disease

Tang, F-L., Erion, J.R., Tian, Y., Liu, W., Yin, D-M., Ye, J., Tang, B., Mei, L and Xiong, W-C.

1. Neurosci., 35(29), 10613-10628 (2015)

  Vacuolar protein sorting-35 (VPS35) is essential for endosome-to-Golgi retrieval of membrane proteins. Mutations in the VPS35 gene have been identified in patients with autosomal dominant PD. However, it remains poorly understood if and how VPS35 deficiency or mutation contributes to PD pathogenesis. Here we provide evidence that links VPS35 deficiency to PD-like neuropathology. VPS35 was expressed in mouse dopamine (DA) neurons in substantia nigra pars compacta (SNpc) and STR (striatum)—regions that are PD vulnerable. VPS35-deficient mice exhibited PD-relevant deficits including accumulation of α-synuclein in SNpc-DA neurons, loss of DA transmitter and DA neurons in SNpc and STR, and impairment of locomotor behavior. Further mechanical studies showed that VPS35-deficient DA neurons or DA neurons expressing PD-linked VPS35 mutant (D620N) had impaired endosome-to-Golgi retrieval of lysosome-associated membrane glycoprotein 2a (Lamp2a) and accelerated Lamp2a degradation. Expression of Lamp2a in VPS35-deficient DA neurons reduced α-synuclein, supporting the view for Lamp2a as a receptor of chaperone-mediated autophagy to be critical for α-synuclein degradation. These results suggest that VPS35 deficiency or mutation promotes PD pathogenesis and reveals a crucial pathway, VPS35-Lamp2a-α-synuclein, to prevent PD pathogenesis.  

5.1669           Function and Circuitry of VIP+ Interneurons in the Mouse Retina

Park, S.J.H., Borghuis, B.G., Rahmani, P., Zeng, Q., Kim, I-J. and Demb, J.B.

1. Neurosci., 35(30), 10685-10700 (2015)

  Visual processing in the retina depends on coordinated signaling by interneurons. Photoreceptor signals are relayed to ∼20 ganglion cell types through a dozen excitatory bipolar interneurons, each responsive to light increments (ON) or decrements (OFF). ON and OFF bipolar cell pathways become tuned through specific connections with inhibitory interneurons: horizontal and amacrine cells. A major obstacle for understanding retinal circuitry is the unknown function of most of the ∼30–40 amacrine cell types, each of which synapses onto a subset of bipolar cell terminals, ganglion cell dendrites, and other amacrine cells. Here, we used a transgenic mouse line in which vasoactive intestinal polypeptide-expressing (VIP⁺) GABAergic interneurons express Cre recombinase. Targeted whole-cell recordings of fluorescently labeled VIP⁺ cells revealed three predominant types: wide-field bistratified and narrow-field monostratified cells with somas in the inner nuclear layer (INL) and medium-field monostratified cells with somas in the ganglion cell layer (GCL). Bistratified INL cells integrated excitation and inhibition driven by both ON and OFF pathways with little spatial tuning. Narrow-field INL cells integrated excitation driven by the ON pathway and inhibition driven by both pathways, with pronounced hyperpolarizations at light offset. Monostratified GCL cells integrated excitation and inhibition driven by the ON pathway and showed center-surround spatial tuning. Optogenetic experiments showed that, collectively, VIP⁺ cells made strong connections with OFF δ, ON-OFF direction-selective, and W3 ganglion cells but weak, inconsistent connections with ON and OFF α cells. Revealing VIP⁺ cell morphologies, receptive fields and synaptic connections advances our understanding of their role in visual processing.  

5.1670           Cardiac myosin binding protein C regulates postnatal myocyte cytokinesis

Jiang, J., Burgon, P.G., Wakimoto, H., Onoue, K., Gorham, J.M., O’Meara, C.C., Fomovsky, G., McConnell, B.K., Lee, R.T., Seidman, J.G. and Seidman, C.E. PNAS, 112(29), 9046-9051 (2015)   Homozygous cardiac myosin binding protein C-deficient (Mybpc^t/t) mice develop dramatic cardiac dilation shortly after birth; heart size increases almost twofold. We have investigated the mechanism of cardiac enlargement in these hearts. Throughout embryogenesis myocytes undergo cell division while maintaining the capacity to pump blood by rapidly disassembling and reforming myofibrillar components of the sarcomere throughout cell cycle progression. Shortly after birth, myocyte cell division ceases. Cardiac MYBPC is a thick filament protein that regulates sarcomere organization and rigidity. We demonstrate that many Mybpc^t/t myocytes undergo an additional round of cell division within 10 d postbirth compared with their wild-type counterparts, leading to increased numbers of mononuclear myocytes. Short-hairpin RNA knockdown of Mybpc3 mRNA in wild-type mice similarly extended the postnatal window of myocyte proliferation. However, adult Mybpc^t/t myocytes are unable to fully regenerate the myocardium after injury. MYBPC has unexpected inhibitory functions during postnatal myocyte cytokinesis and cell cycle progression. We suggest that human patients with homozygous MYBPC3-null mutations develop dilated cardiomyopathy, coupled with myocyte hyperplasia (increased cell number), as observed in Mybpc^t/t mice. Human patients, with heterozygous truncating MYBPC3 mutations, like mice with similar mutations, have hypertrophic cardiomyopathy. However, the mechanism leading to hypertrophic cardiomyopathy in heterozygous MYBPC3^+/− individuals is myocyte hypertrophy (increased cell size), whereas the mechanism leading to cardiac dilation in homozygous Mybpc3^−/− mice is primarily myocyte hyperplasia.  

5.1671           Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer’s disease amyloid plaques

Gowrishankar, S., Yuan, P., Wu, Y., Schrag, M., paradise, S., Grutzendler, J., De Camilli, P. and Ferguson, S.M. PNAS, 112(28), E3699-E3708 (2015)   Through a comprehensive analysis of organellar markers in mouse models of Alzheimer’s disease, we document a massive accumulation of lysosome-like organelles at amyloid plaques and establish that the majority of these organelles reside within swollen axons that contact the amyloid deposits. This close spatial relationship between axonal lysosome accumulation and extracellular amyloid aggregates was observed from the earliest stages of β-amyloid deposition. Notably, we discovered that lysosomes that accumulate in such axons are lacking in multiple soluble luminal proteases and thus are predicted to be unable to efficiently degrade proteinaceous cargos. Of relevance to Alzheimer’s disease, β-secretase (BACE1), the protein that initiates amyloidogenic processing of the amyloid precursor protein and which is a substrate for these proteases, builds up at these sites. Furthermore, through a comparison between the axonal lysosome accumulations at amyloid plaques and neuronal lysosomes of the wild-type brain, we identified a similar, naturally occurring population of lysosome-like organelles in neuronal processes that is also defined by its low luminal protease content. In conjunction with emerging evidence that the lysosomal maturation of endosomes and autophagosomes is coupled to their retrograde transport, our results suggest that extracellular β-amyloid deposits cause a local impairment in the retrograde axonal transport of lysosome precursors, leading to their accumulation and a blockade in their further maturation. This study both advances understanding of Alzheimer’s disease brain pathology and provides new insights into the subcellular organization of neuronal lysosomes that may have broader relevance to other neurodegenerative diseases with a lysosomal component to their pathology.  

5.1672           Alleviation of off-target effects from vector-encoded shRNAs via codelivered RNA decoys

Mockenhaupt, S., Grosse, S., Rupp, D., bartenschlager, R. and Grimm, D. PNAS, 112(30), E4007-E4016 (2015)   Exogenous RNAi triggers such as shRNAs ideally exert their activities exclusively via the antisense strand that binds and silences designated target mRNAs. However, in principle, the sense strand also possesses silencing capacity that may contribute to adverse RNAi side effects including off-target gene regulation. Here, we address this concern with a novel strategy that reduces sense strand activity of vector-encoded shRNAs via codelivery of inhibitory tough decoy (TuD) RNAs. Using various shRNAs for proof of concept, we validate that coexpression of TuDs can sequester and inactivate shRNA sense strands in human cells selectively without affecting desired antisense activities from the same shRNAs. Moreover, we show how coexpressed TuDs can alleviate shRNA-mediated perturbation of global gene expression by specifically de-repressing off-target transcripts carrying seed matches to the shRNA sense strand. Our combination of shRNA and TuD in a single bicistronic gene transfer vector derived from Adeno-associated virus (AAV) enables a wide range of applications, including gene therapies. To this end, we engineered our constructs in a modular fashion and identified simple hairpin design rules permitting adaptation to preexisting or new shRNAs. Finally, we demonstrate the power of our vectors for combinatorial RNAi strategies by showing robust suppression of hepatitis C virus (HCV) with an AAV expressing a bifunctional TuD against an anti-HCV shRNA sense strand and an HCV-related cellular miRNA. The data and tools reported here represent an important step toward the next generation of RNAi triggers with increased specificity and thus ultimately safety in humans.  

5.1673           The Agmatine-Containing Poly(Amidoamine) Polymer AGMA1 Binds Cell Surface Heparan Sulfates and Prevents Attachment of Mucosal Human Papillomaviruses

Cagno, V., Donalisio; M., Bugatti, A., Civra, A., Cavalli, R., Ranucci, E., Ferruti, P., Rusnati, M. and Lembo, D. Antimicrob. Agents Chemother., 59(9), 5250-5259 (2015)   The agmatine-containing poly(amidoamine) polymer AGMA1 was recently shown to inhibit the infectivity of several viruses, including human papillomavirus 16 (HPV-16), that exploit cell surface heparan sulfate proteoglycans (HSPGs) as attachment receptors. The aim of this work was to assess the antiviral activity of AGMA1 and its spectrum of activity against a panel of low-risk and high-risk HPVs and to elucidate its mechanism of action. AGMA1 was found to be a potent inhibitor of mucosal HPV types (i.e., types 16, 31, 45, and 6) in pseudovirus-based neutralization assays. The 50% inhibitory concentration was between 0.34 μg/ml and 0.73 μg/ml, and no evidence of cytotoxicity was observed. AGMA1 interacted with immobilized heparin and with cellular heparan sulfates, exerting its antiviral action by preventing virus attachment to the cell surface. The findings from this study indicate that AGMA1 is a leading candidate compound for further development as an active ingredient of a topical microbicide against HPV and other sexually transmitted viral infections.  

5.1674           In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector

Zinn, E., Pacouret, S., Khaychuk, V., Turunen, H.T., Carvalho, L.S., Andres-Mateos, E., Shah, S., Shelke, r., maurer, A.C., Plovie, E., Xiao, R. and Vandenberghe, L.H. Cell Reports, 12, 1056-1068 (2015)   Adeno-associated virus (AAV) vectors have emerged as a gene-delivery platform with demonstrated safety and efficacy in a handful of clinical trials for monogenic disorders. However, limitations of the current generation vectors often prevent broader application of AAV gene therapy. Efforts to engineer AAV vectors have been hampered by a limited understanding of the structure-function relationship of the complex multimeric icosahedral architecture of the particle. To develop additional reagents pertinent to further our insight into AAVs, we inferred evolutionary intermediates of the viral capsid using ancestral sequence reconstruction. In-silico-derived sequences were synthesized de novo and characterized for biological properties relevant to clinical applications. This effort led to the generation of nine functional putative ancestral AAVs and the identification of Anc80, the predicted ancestor of the widely studied AAV serotypes 1, 2, 8, and 9, as a highly potent in vivo gene therapy vector for targeting liver, muscle, and retina.  

5.1675           Immunogenic virus-like particles continuously expressed in mammalian cells as a veterinary rabies vaccine candidate

Fontana, D., Kratje, r., Etcheverrigaray, M. and Prieto, C. Vaccine, 33, 4238-4246 (2015)   Rabies is one of the most lethal infectious diseases in the world, with a mortality approaching 100%. There are between 60,000 and 70,000 reported annual deaths, but this is probably an underestimation. Despite the fact that there are vaccines available for rabies, there is a real need of developing more efficacious and cheaper vaccines. This is particularly true for veterinary vaccines because dogs are still the main vector for rabies transmission to human beings. In a previous work, we described the development and characterization of rabies virus-like particles (RV-VLPs) expressed in HEK293 cells. We showed that RV-VLPs are able to induce a specific antibodies response. In this work, we show that VLPs are able to protect mice against virus challenge. Furthermore, we developed a VLPs expressing HEK-293 clone (sP2E5) that grows in serum free medium (SFM) reaching high cell densities. sP2E5 was cultured in perfusion mode in a 5 L bioreactor for 20 days, and the RV-VLPs produced were capable of triggering a protective immune response without the need of concentration or adjuvant addition. Further, these VLPs are able to induce the production of rabies virus neutralizing antibodies. These results demonstrate that RV-VLPs are a promising rabies vaccine candidate.  

5.1676           Delivery of the 7-dehydrocholesterol reductase gene to the central nervous system using adeno-associated virus vector in a mouse model of Smith-Lemli-Opitz Syndrome

Pasta, S., Akhile, O., Tabron, D., Ting, F., Shackleton, C. And Watson, G. Molecular genetics and Metabolism Reports, 4, 92-98 (2015)   Smith Lemli Opitz syndrome (SLOS) is an inherited malformation and mental retardation metabolic disorder with no cure. Mutations in the last enzyme of the cholesterol biosynthetic pathway, 7-dehydrocholesterol reductase (DHCR7), lead to cholesterol insufficiency and accumulation of its dehyrdocholesterol precursors, and contribute to its pathogenesis. The central nervous system (CNS) constitutes a major pathophysiological component of this disorder and remains unamenable to dietary cholesterol therapy due to the impenetrability of the blood brain barrier (BBB). The goal of this study was to restore sterol homeostasis in the CNS. To bypass the BBB, gene therapy using an adeno-associated virus (AAV-8) vector carrying a functional copy of the DHCR7 gene was administered by intrathecal (IT) injection directly into the cerebrospinal fluid of newborn mice. Two months post-treatment, vector DNA and DHCR7 expression was observed in the brain and a corresponding improvement of sterol levels seen in the brain and spinal cord. Interestingly, sterol levels in the peripheral nervous system also showed a similar improvement. This study shows that IT gene therapy can have a positive biochemical effect on sterol homeostasis in the central and peripheral nervous systems in a SLOS animal model. A single dose delivered three days after birth had a sustained effect into adulthood, eight weeks post-treatment. These observations pave the way for further studies to understand the effect of biochemical improvement of sterol levels on neuronal function, to provide a greater understanding of neuronal cholesterol homeostasis, and to develop potential therapies.  

5.1677           Alpha-Synuclein affects neurite morphology, autophagy, vesicle transport and axonal degeneration in CNS neurons

Koch, J.C., Bitow, F., Haack, J., d’Hedouville, Z., Zhang, J-N., Tönges, L., Michel, U., Oliveira, L.M.A., Jovin, T.M., Liman, J., tatenhorst, L., Bähr, M and Lingor, P. Cell Death and Disease, 6, e1811 (2015)   Many neuropathological and experimental studies suggest that the degeneration of dopaminergic terminals and axons precedes the demise of dopaminergic neurons in the substantia nigra, which finally results in the clinical symptoms of Parkinson disease (PD). The mechanisms underlying this early axonal degeneration are, however, still poorly understood. Here, we examined the effects of overexpression of human wildtype alpha-synuclein (αSyn-WT), a protein associated with PD, and its mutant variants αSyn-A30P and -A53T on neurite morphology and functional parameters in rat primary midbrain neurons (PMN). Moreover, axonal degeneration after overexpression of αSyn-WT and -A30P was analyzed by live imaging in the rat optic nerve in vivo. We found that overexpression of αSyn-WT and of its mutants A30P and A53T impaired neurite outgrowth of PMN and affected neurite branching assessed by Sholl analysis in a variant-dependent manner. Surprisingly, the number of primary neurites per neuron was increased in neurons transfected with αSyn. Axonal vesicle transport was examined by live imaging of PMN co-transfected with EGFP-labeled synaptophysin. Overexpression of all αSyn variants significantly decreased the number of motile vesicles and decelerated vesicle transport compared with control. Macroautophagic flux in PMN was enhanced by αSyn-WT and -A53T but not by αSyn-A30P. Correspondingly, colocalization of αSyn and the autophagy marker LC3 was reduced for αSyn-A30P compared with the other αSyn variants. The number of mitochondria colocalizing with LC3 as a marker for mitophagy did not differ among the groups. In the rat optic nerve, both αSyn-WT and -A30P accelerated kinetics of acute axonal degeneration following crush lesion as analyzed by in vivo live imaging. We conclude that αSyn overexpression impairs neurite outgrowth and augments axonal degeneration, whereas axonal vesicle transport and autophagy are severely altered.  

5.1678           Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors

Florencio, G.D., Precigout, G., Beley, C., Buclez, P-O., Garcia, L and Benchaouir, R. Molecular Therapy-Methods and Clinical Development, 2, 15024 (2015)   Recombinant adeno-associated viruses (rAAV) are promising candidates for gene therapy approaches. The last two decades were particularly fruitful in terms of processes applied in the production and purification of this type of gene transfer vectors. This rapid technological evolution led to better yields and higher levels of vector purity. Recently, some reports showed that rAAV produced by transient tri-transfection method in adherent human embryonic kidney 293 cells can be harvested directly from supernatant, leading to easier and faster purification compared to classical virus extraction from cell pellets. Here, we compare these approaches with new vector recovery method using small quantity of detergent at the initial clarification step to treat the whole transfected cell culture. Coupled with tangential flow filtration and iodixanol-based isopycnic density gradient, this new method significantly increases rAAV yields and conserves high vector purity. Moreover, this approach leads to the reduction of the total process duration. Finally, the vectors maintain their functionality, showing unexpected higher in vitro and in vivo transduction efficacies. This new development in rAAV downstream process once more demonstrates the great capacity of these vectors to easily accommodate to large panel of methods, able to furthermore ameliorate their safety, functionality, and scalability.  

5.1679           Identification of the miRNA targetome in hippocampal neurons using RIP-seq

Malmevik, J., Petri, R., Klussendorf, T., Knauff, P., Åkerblom, M., Johansson, J., Soneji, S. and Jakobsson, J. Scientific Reports, 5:12609 (2015)   MicroRNAs (miRNAs) are key players in the regulation of neuronal processes by targeting a large network of target messenger RNAs (mRNAs). However, the identity and function of mRNAs targeted by miRNAs in specific cells of the brain are largely unknown. Here, we established an adeno-associated viral vector (AAV)-based neuron-specific Argonaute2:GFP-RNA immunoprecipitation followed by high-throughput sequencing to analyse the regulatory role of miRNAs in mouse hippocampal neurons. Using this approach, we identified more than two thousand miRNA targets in hippocampal neurons, regulating essential neuronal features such as cell signalling, transcription and axon guidance. Furthermore, we found that stable inhibition of the highly expressed miR-124 and miR-125 in hippocampal neurons led to significant but distinct changes in the AGO2 binding of target mRNAs, resulting in subsequent upregulation of numerous miRNA target genes. These findings greatly enhance our understanding of the miRNA targetome in hippocampal neurons.  

5.1680           An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses

Schreiber, C.A., Sakuma, T., Izumiya, Y., Holditch, S.J., Hickey, R.D., Bressin, R.K., Basu, U., Koide, K., Asokan, A. and Ikeda, Y. PloS Pathogens, 11(8), e1005082 (2015)   Adeno-associated viruses (AAV) have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors.  

5.1681           A Non-enveloped Virus Hijacks Host Disaggregation Machinery to Translocate across the Endoplasmic Reticulum Membrane

Ravindran, M.S., bagchi, P., Inoue, T. and Tsai, B. PloS Pathogens, 11(8), e1005086 (2015)   Mammalian cytosolic Hsp110 family, in concert with the Hsc70:J-protein complex, functions as a disaggregation machinery to rectify protein misfolding problems. Here we uncover a novel role of this machinery in driving membrane translocation during viral entry. The non-enveloped virus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol, a critical infection step. Combining biochemical, cell-based, and imaging approaches, we find that the Hsp110 family member Hsp105 associates with the ER membrane J-protein B14. Here Hsp105 cooperates with Hsc70 and extracts the membrane-penetrating SV40 into the cytosol, potentially by disassembling the membrane-embedded virus. Hence the energy provided by the Hsc70-dependent Hsp105 disaggregation machinery can be harnessed to catalyze a membrane translocation event.  

5.1682           Evidence that the endosomal sorting complex required for transport-II (ESCRT-II) is required for efficient human immunodeficiency virus-1 (HIV-1) production

Meng, B., Ip, N.C.Y., Prestwood, L.J., Abbink, T.E. and Lever, A.M.L. Retrovirology, 12:72 (2015)   Background Egress of a number of different virus species from infected cells depends on proteins of the endosomal sorting complexes required for transport (ESCRT) pathway. HIV has also hijacked this system to bud viruses outward from the cell surface. How ESCRT-I activates ESCRT-III in this process remains unclear with conflicting published evidence for the requirement of ESCRT-II which fulfils this role in other systems. We investigated the role of ESCRT-II using knockdown mediated by siRNA and shRNA, mutants which prevent ESCRT-I/ESCRT-II interaction and a CRISPR/Cas9 EAP45 knockout cell line. Results Depletion or elimination of ESCRT-II components from an HIV infected cell produces two distinct effects. The overall production of HIV-1 Gag is reduced leading to a diminished amount of intracellular virion protein. In addition depletion of ESCRT-II produces an effect similar to that seen when ESCRT-I and -III components are depleted, that of a delayed Gag p26 to p24 +p2 cleavage associated with a reduction in export of virion particles and a visible reduction in budding efficiency in virus producing cells. Mutants that interfere with ESCRT-I interacting with ESCRT-II similarly reduce virus export. The export defect is independent of the decrease in overall Gag production. Using a mutant virus which cannot use the ALIX mediated export pathway exacerbates the decrease in virus export seen when ESCRT-II is depleted. ESCRT-II knockdown does not lead to complete elimination of virus release suggesting that the late domain role of ESCRT-II is required for optimal efficiency of viral budding but that there are additional pathways that the virus can employ to facilitate this. Conclusion ESCRT-II contributes to efficient HIV virion production and export by more than one pathway; both by a transcriptional or post transcriptional mechanism and also by facilitating efficient virus export from the cell through interactions with other ESCRT components.  

5.1683           Lack of infectivity of HBV in feces from patients with chronic hepatitis B virus infection, and infection using chimeric mice

Komatsu, H., Inui, A., Murano, T., Tsunoda, T., Sogo, T. and Fujisawa, T. BMC Res. Notes, 8:366 (2015)   Background Body fluids such as saliva and tears from patients with hepatitis B virus (HBV) infection are known as infectious agents. The infectivity of feces from patients with HBV infection has not been established. The aim of this study was to determine whether feces from HBV carriers can be a source of HBV infection. Methods Thirty-three children and 17 adults (ages 0–49 years, median age 13 years) who were chronically infected with HBV were enrolled. The levels of HBV DNA in the feces from these patients were quantified by real-time PCR, and the levels of fecal HBsAg were measured. Isolated human hepatocytes from chimeric mice with humanized livers were co-cultured with serum, tears and feces from the HBV carriers. Four chimeric mice were inoculated intravenously with sterilized feces from HBV carriers. Results HBV DNA was detected in the feces of 37 (74 %) of the 50 patients. The fecal HBV DNA levels ranged from 2.8 to 8.4 log copies/mL (mean ± SD  =  5.6 ± 1.2 log copies/mL). A significant correlation was observed in the levels of HBV DNA between serum and feces (r  =  0.54, p < 0.05). Of the 13 HBV carries, 7 (54 %) were positive for fecal HBsAg. The fecal HBsAg levels ranged from 0.06 to 1.0 IU/mL (median 0.28 IU/mL). Immunogold electron microscopy showed Dane particles in feces. HBV DNA was detected in the human hepatocytes co-cultured with serum and tears, but not in those co-cultured with feces. HBV DNA was not detected in the serum of the chimeric mice after oral or intravenous inoculation with sterilized fecal samples, which contained 5 log copies/mL of HBV DNA levels. Conclusions Although the positive rate of fecal HBV DNA was high, the fecal HBsAg levels were extremely low. The chimeric mice were not infected with HBV after oral or intravenous inoculation with sterilized fecal samples. Therefore, feces from HBV carriers seem not to serve as an infectious vehicle for the transmission of HBV.  

5.1684           Lack of additive role of ageing in nigrostriatal neurodegeneration triggered by α-synuclein overexpression

Bourdenx, M. et al Acta Neuropathologica Communications, 3:46 (2015)   ntroduction Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons as well as the presence of proteinaceous inclusions named Lewy bodies. α-synuclein (α-syn) is a major constituent of Lewy bodies, and the first disease-causing protein characterized in PD. Several α-syn-based animal models of PD have been developed to investigate the pathophysiology of PD, but none of them recapitulate the full picture of the disease. Ageing is the most compelling and major risk factor for developing PD but its impact on α-syn toxicity remains however unexplored. In this study, we developed and exploited a recombinant adeno-associated viral (AAV) vector of serotype 9 overexpressing mutated α-syn to elucidate the influence of ageing on the dynamics of PD-related neurodegeneration associated with α-syn pathology in different mammalian species. Results Identical AAV pseudotype 2/9 vectors carrying the DNA for human mutant p.A53T α-syn were injected into the substantia nigra to induce neurodegeneration and synucleinopathy in mice, rats and monkeys. Rats were used first to validate the ability of this serotype to replicate α-syn pathology and second to investigate the relationship between the kinetics of α-syn-induced nigrostriatal degeneration and the progressive onset of motor dysfunctions, strikingly reminiscent of the impairments observed in PD patients. In mice, AAV2/9-hα-syn injection into the substantia nigra was associated with accumulation of α-syn and phosphorylated hα-syn, regardless of mouse strain. However, phenotypic mutants with either accelerated senescence or resistance to senescence did not display differential susceptibility to hα-syn overexpression. Of note, p-α-syn levels correlated with nigrostriatal degeneration in mice. In monkeys, hα-syn-induced degeneration of the nigrostriatal pathway was not affected by the age of the animals. Unlike mice, monkeys did not exhibit correlations between levels of phosphorylated α-syn and neurodegeneration. Conclusions In conclusion, AAV2/9-mediated hα-syn induces robust nigrostriatal neurodegeneration in mice, rats and monkeys, allowing translational comparisons among species. Ageing, however, neither exacerbated nigrostriatal neurodegeneration nor α-syn pathology per se. Our unprecedented multi-species investigation thus favours the multiple-hit hypothesis for PD wherein ageing would merely be an aggravating, additive, factor superimposed upon an independent disease process.  

5.1685           Viral expression of ALS-linked ubiquilin-2 mutants causes inclusion pathology and behavioral deficits in mice

Caballos-Diaz, C., Rosario, A.M., park, H-J., Chakrabarty, P., Sacino, A., Cruz, P.E., Siemienski, Z., Lara, N., Moran, C., Ravelo, N., Golde, T.E. and McFarland, N.R. Molecular Neurodegeneration, 10:25 (2015)   Background UBQLN2 mutations have recently been associated with familial forms of amyotrophic lateral sclerosis (ALS) and ALS-dementia. UBQLN2 encodes for ubiquilin-2, a member of the ubiquitin-like protein family which facilitates delivery of ubiquitinated proteins to the proteasome for degradation. To study the potential role of ubiquilin-2 in ALS, we used recombinant adeno-associated viral (rAAV) vectors to express UBQLN2 and three of the identified ALS-linked mutants (P497H, P497S, and P506T) in primary neuroglial cultures and in developing neonatal mouse brains. Results In primary cultures rAAV2/8-mediated expression of UBQLN2 mutants resulted in inclusion bodies and insoluble aggregates. Intracerebroventricular injection of FVB mice at post-natal day 0 with rAAV2/8 expressing wild type or mutant UBQLN2 resulted in widespread, sustained expression of ubiquilin-2 in brain. In contrast to wild type, mutant UBQLN2 expression induced significant pathology with large neuronal, cytoplasmic inclusions and ubiquilin-2-positive aggregates in surrounding neuropil. Ubiquilin-2 inclusions co-localized with ubiquitin, p62/SQSTM, optineurin, and occasionally TDP-43, but were negative for α-synuclein, neurofilament, tau, and FUS. Mutant UBLQN2 expression also resulted in Thioflavin-S-positive inclusions/aggregates. Mice expressing mutant forms of UBQLN2 variably developed a motor phenotype at 3–4 months, including nonspecific clasping and rotarod deficits. Conclusions These findings demonstrate that UBQLN2 mutants (P497H, P497S, and P506T) induce proteinopathy and cause behavioral deficits, supporting a “toxic” gain-of-function, which may contribute to ALS pathology. These data establish also that our rAAV model can be used to rapidly assess the pathological consequences of various UBQLN2 mutations and provides an agile system to further interrogate the molecular mechanisms of ubiquilins in neurodegeneration.  

5.1686           Fluorescent Calcium Indicator Protein Expression in the Mouse Brain Using Recombinant Adeno-Associated Viruses

Heindorf, M. and Hasan, M.T. Cold Spring Harbor Protocols, pdb.prot087635 (2015)   One method for gene delivery and long-term fluorescent calcium indicator protein (FCIP) expression in mammalian neurons in vivo involves the introduction of FCIPs via recombinant adeno-associated virus (rAAV) vectors using constitutive and cell type-specific promoters. This protocol describes the use of rAAVs to express FCIPs in the brain for imaging. Human embryonic kidney 293 cells are first transfected using calcium phosphate. rAAV is then prepared using either an iodixanol gradient or a heparin column. After the virus is purified, its quality is assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, estimation of genomic and functional virus titers by quantitative polymerase chain reaction, and expression in dissociated neurons. Mice are injected with rAAV using a stereotactic instrument and can be imaged ∼3 wk later.  

5.1687           RNA and Nucleocapsid Are Dispensable for Mature HIV-1 Capsid Assembly

Mattel, S., Flemming, A., Anders-Össwein, M., Kräusslich, H-G., Briggs, J.A. and Müller, B.

1. Virol., 89(19), 9739-9747 (2015)

  Human immunodeficiency virus type 1 (HIV-1) is released from infected cells in an immature, noninfectious form in which the structural polyprotein Gag is arranged in a hexameric lattice, forming an incomplete spherical shell. Maturation to the infectious form is mediated by the viral protease, which cleaves Gag at five sites, releasing the CA (capsid) protein, which forms a conical capsid encasing the condensed RNA genome. The pathway of this structural rearrangement is currently not understood, and it is unclear how cone assembly is initiated. RNA represents an integral structural component of retroviruses, and the viral nucleoprotein core has previously been proposed to nucleate mature capsid assembly. We addressed this hypothesis by replacing the RNA-binding NC (nucleocapsid) domain of HIV-1 Gag and the adjacent spacer peptide 2 (SP2) by a leucine zipper (LZ) protein-protein interaction domain [Gag(LZ)] in the viral context. We found that Gag(LZ)-carrying virus [HIV(LZ)] was efficiently released and viral polyproteins were proteolytically processed, though with reduced efficiency. Cryo-electron tomography revealed that the particles lacked a condensed nucleoprotein and contained an increased proportion of aberrant core morphologies caused either by the absence of RNA or by altered Gag processing. Nevertheless, a significant proportion of HIV(LZ) particles contained mature capsids with the wild-type morphology. These results clearly demonstrate that the nucleoprotein complex is dispensable as a nucleator for mature HIV-1 capsid assembly in the viral context.  

5.1688           Polyphenols Inhibit Hepatitis C Virus Entry by a New Mechanism of Action

Calland, N. et al

1. Virol., 89(19), 10053-10063 (2015)

  Despite the validation of direct-acting antivirals for hepatitis C treatment, the discovery of new compounds with different modes of action may still be of importance for the treatment of special patient populations. We recently identified a natural molecule, epigallocatechin-3-gallate (EGCG), as an inhibitor of hepatitis C virus (HCV) targeting the viral particle. The aim of this work was to discover new natural compounds with higher anti-HCV activity than that of EGCG and determine their mode of action. Eight natural molecules with structure similarity to EGCG were selected. HCV JFH1 in cell culture and HCV pseudoparticle systems were used to determine the antiviral activity and mechanism of action of the compounds. We identified delphinidin, a polyphenol belonging to the anthocyanidin family, as a new inhibitor of HCV entry. Delphinidin inhibits HCV entry in a pangenotypic manner by acting directly on the viral particle and impairing its attachment to the cell surface. Importantly, it is also active against HCV in primary human hepatocytes, with no apparent cytotoxicity and in combination with interferon and boceprevir in cell culture. Different approaches showed that neither aggregation nor destruction of the particle occurred. Cryo-transmission electron microscopy observations of HCV pseudoparticles treated with delphinidin or EGCG showed a bulge on particles that was not observed under control conditions. In conclusion, EGCG and delphinidin inhibit HCV entry by a new mechanism, i.e., alteration of the viral particle structure that impairs its attachment to the cell surface.  

5.1689           Rational Design and Engineering of a Modified Adeno-Associated Virus (AAV1)-Based Vector System for Enhanced Retrograde Gene Delivery

Davis, A.S., Federici, TS., Ray, W.C., Boulis, W.C., O’Connor, D., Clark, K.R. and Bartlett, J. Neurosurgery, 76(2), 216-225 (2015)   BACKGROUND: After injection into muscle and peripheral nerves, a variety of viral vectors undergo retrograde transport to lower motor neurons. However, because of its attractive safety profile and durable gene expression, adeno-associated virus (AAV) remains the only vector to have been applied to the human nervous system for the treatment of neurodegenerative disease. Nonetheless, only a very small fraction of intramuscularly injected AAV vector arrives at the spinal cord. OBJECTIVE: To engineer a novel AAV vector by inserting a neuronal targeting peptide (Tet1), with binding properties similar to those of tetanus toxin, into the AAV1 capsid. METHODS: Integral to this approach was the use of structure-based design to increase the effectiveness of functional capsid engineering. This approach allowed the optimization of scaffolding regions for effective display of the foreign epitope while minimizing disruption of the native capsid structure. We also validated an approach by which low-titer tropism-modified AAV vectors can be rescued by particle mosaicism with unmodified capsid proteins. RESULTS: Importantly, our rationally engineered AAV1-based vectors exhibited markedly enhanced transduction of cultured motor neurons, diminished transduction of nontarget cells, and markedly superior retrograde delivery compared with unmodified AAV1 vector. CONCLUSION: This approach promises a significant advancement in the rational engineering of AAV vectors for diseases of the nervous system and other organs.  

5.1690           Septal Glucagon-Like Peptide 1 Receptor Expression Determines Suppression of Cocaine-Induced Behavior

Harasta, A.E., Power, J.M., von Jonquieres, G., karl, T., Drucker, D.J., Housley, G.D., Schneider, M. and Klugmann, M. Neuropsychopharmacology, 40(8), 1969-1978 (2015)   Glucagon-like peptide 1 (GLP-1) and its receptor GLP-1R are a key component of the satiety signaling system, and long-acting GLP-1 analogs have been approved for the treatment of type-2 diabetes mellitus. Previous reports demonstrate that GLP-1 regulates glucose homeostasis alongside the rewarding effects of food. Both palatable food and illicit drugs activate brain reward circuitries, and pharmacological studies suggest that central nervous system GLP-1 signaling holds potential for the treatment of addiction. However, the role of endogenous GLP-1 in the attenuation of reward-oriented behavior, and the essential domains of the mesolimbic system mediating these beneficial effects, are largely unknown. We hypothesized that the central regions of highest Glp-1r gene activity are essential in mediating responses to drugs of abuse. Here, we show that Glp-1r-deficient (Glp-1r^−/−) mice have greatly augmented cocaine-induced locomotor responses and enhanced conditional place preference compared with wild-type (Glp-1r^+/+) controls. Employing mRNA in situ hybridization we located peak Glp-1r mRNA expression in GABAergic neurons of the dorsal lateral septum, an anatomical site with a crucial function in reward perception. Whole-cell patch-clamp recordings of dorsal lateral septum neurons revealed that genetic Glp-1r ablation leads to increased excitability of these cells. Viral vector-mediated Glp-1r gene delivery to the dorsal lateral septum of Glp-1r^−/− animals reduced cocaine-induced locomotion and conditional place preference to wild-type levels. This site-specific genetic complementation did not affect the anxiogenic phenotype observed in Glp-1r^−/− controls. These data reveal a novel role of GLP-1R in dorsal lateral septum function driving behavioral responses to cocaine.  

5.1691           Distinct circuit-dependent functions of presynaptic neurexin-3 at GABAergic and glutamatergic synapses

Aoto, J., Földy, C., Ilcus, S.M.C., Tabuchi, K. and Südhof, T.C. Nature Neurosci., 18(7), 997-1007 (2015)   α- and β-neurexins are presynaptic cell-adhesion molecules whose general importance for synaptic transmission is well documented. The specific functions of neurexins, however, remain largely unknown because no conditional neurexin knockouts are available and targeting all α- and β-neurexins produced by a particular gene is challenging. Using newly generated constitutive and conditional knockout mice that target all neurexin-3α and neurexin-3β isoforms, we found that neurexin-3 was differentially required for distinct synaptic functions in different brain regions. Specifically, we found that, in cultured neurons and acute slices of the hippocampus, extracellular sequences of presynaptic neurexin-3 mediated trans-synaptic regulation of postsynaptic AMPA receptors. In cultured neurons and acute slices of the olfactory bulb, however, intracellular sequences of presynaptic neurexin-3 were selectively required for GABA release. Thus, our data indicate that neurexin-3 performs distinct essential pre- or postsynaptic functions in different brain regions by distinct mechanisms.  

5.1692           Pathway-specific reorganization of projection neurons in somatosensory cortex during learning

Chen, J.L., Margolis, D.J., Stankov, A., Sumanovski, L.T., Schneider, B.L. and Helmchen, F. Nature Neurosci., 18(8), 1101-1108 (2015)   In the mammalian brain, sensory cortices exhibit plasticity during task learning, but how this alters information transferred between connected cortical areas remains unknown. We found that divergent subpopulations of cortico-cortical neurons in mouse whisker primary somatosensory cortex (S1) undergo functional changes reflecting learned behavior. We chronically imaged activity of S1 neurons projecting to secondary somatosensory (S2) or primary motor (M1) cortex in mice learning a texture discrimination task. Mice adopted an active whisking strategy that enhanced texture-related whisker kinematics, correlating with task performance. M1-projecting neurons reliably encoded basic kinematics features, and an additional subset of touch-related neurons was recruited that persisted past training. The number of S2-projecting touch neurons remained constant, but improved their discrimination of trial types through reorganization while developing activity patterns capable of discriminating the animal's decision. We propose that learning-related changes in S1 enhance sensory representations in a pathway-specific manner, providing downstream areas with task-relevant information for behavior.  

5.1693           Cardiac AAV9 Gene Delivery Strategies in Adult Canines: Assessment by Long-term Serial SPECT Imaging of Sodium Iodide Symporter Expression

Moulay, G., Ohtani, T., Ogut, O., Guenzel, A., Behfar, A., Zakeri, R., haines, P., Storlie, J., Bowen, L., Pham, L., Kaye, D., Sandhu, G., O’Connor, M., Russell, S. and Redfield, M. Molecular Therapy, 23(7), 1211-1221 (2015)   Heart failure is a leading cause of morbidity and mortality, and cardiac gene delivery has the potential to provide novel therapeutic approaches. Adeno-associated virus serotype 9 (AAV9) transduces the rodent heart efficiently, but cardiotropism, immune tolerance, and optimal delivery strategies in large animals are unclear. In this study, an AAV9 vector encoding canine sodium iodide symporter (NIS) was administered to adult immunocompetent dogs via epicardial injection, coronary infusion without and with cardiac recirculation, or endocardial injection via a novel catheter with curved needle and both end- and side-holes. As NIS mediates cellular uptake of clinical radioisotopes, expression was tracked by single-photon emission computerized tomography (SPECT) imaging in addition to Western blot and immunohistochemistry. Direct epicardial or endocardial injection resulted in strong cardiac expression, whereas expression after intracoronary infusion or cardiac recirculation was undetectable. A threshold myocardial injection dose that provides robust nonimmunogenic expression was identified. The extent of transmural myocardial expression was greater with the novel catheter versus straight end-hole needle delivery. Furthermore, the authors demonstrate that cardiac NIS reporter gene expression and duration can be quantified using serial noninvasive SPECT imaging up to 1 year after vector administration. These data are relevant to efforts to develop cardiac gene delivery as heart failure therapy.  

5.1694           Neonatal Systemic AAV Induces Tolerance to CNS Gene Therapy in MPS I Dogs and Nonhuman Primates

Hinderer, C. et al Molecular Therapy, 23(8), 1298-1307 (2015)   The potential host immune response to a nonself protein poses a fundamental challenge for gene therapies targeting recessive diseases. We demonstrate in both dogs and nonhuman primates that liver-directed gene transfer using an adeno-associated virus (AAV) vector in neonates induces a persistent state of immunological tolerance to the transgene product, substantially improving the efficacy of subsequent vector administration targeting the central nervous system (CNS). We applied this approach to a canine model of mucopolysaccharidosis type I (MPS I), a progressive neuropathic lysosomal storage disease caused by deficient activity of the enzyme α-l-iduronidase (IDUA). MPS I dogs treated systemically in the first week of life with a vector expressing canine IDUA did not develop antibodies against the enzyme and exhibited robust expression in the CNS upon intrathecal AAV delivery at 1 month of age, resulting in complete correction of brain storage lesions. Newborn rhesus monkeys treated systemically with AAV vector expressing human IDUA developed tolerance to the transgene, resulting in high cerebrospinal fluid (CSF) IDUA expression and no antibody induction after subsequent CNS gene therapy. These findings suggest that inducing tolerance to the transgene product during a critical period in immunological development can improve the efficacy and safety of gene therapy.  

5.1695           CNTF Gene Therapy Confers Lifelong Neuroprotection in a Mouse Model of Human Retinitis Pigmentosa

Lipinski, D.M., barnard, A.R., Singh, M.S., martin, C., Lee, E.J., Davies, W.I. and MacLaren, R.E. Molecular Therapy, 23(8), 1308-1319 (2015)   The long-term outcome of neuroprotection as a therapeutic strategy for preventing cell death in neurodegenerative disorders remains unknown, primarily due to slow disease progression and the inherent difficulty of assessing neuronal survival in vivo. Employing a murine model of retinal disease, we demonstrate that ciliary neurotrophic factor (CNTF) confers life-long protection against photoreceptor degeneration. Repetitive retinal imaging allowed the survival of intrinsically fluorescent cone photoreceptors to be quantified in vivo. Imaging of the visual cortex and assessment of visually-evoked behavioral responses demonstrated that surviving cones retain function and signal correctly to the brain. The mechanisms underlying CNTF-mediated neuroprotection were explored through transcriptome analysis, revealing widespread upregulation of proteolysis inhibitors, which may prevent cellular/extracellular matrix degradation and complement activation in neurodegenerative diseases. These findings provide insights into potential novel therapeutic avenues for diseases such as retinitis pigmentosa and amyotrophic lateral sclerosis, for which CNTF has been evaluated unsuccessfully in clinical trials.  

5.1696           Reprogramming Immune Response With Capsid-Optimized AAV6 Vectors for Immunotherapy of Cancer

Pandya, M., Britt, K., Hoffman, B., Ling, C. and Aslanidi, G.V.

1. Immunother., 38(7), 292-298 (2015)

  In the current studies we generated novel capsid-optimized adeno-associated virus (AAV) serotype 6 (AAV6) vectors expressing a tumor-associated antigen, and assessed their ability to activate a protective T-cell response in an animal model. First, we showed that specific mutations in the AAV6 capsid increase the transduction efficiency of these vectors in mouse bone marrow–derived dendritic cells in vitro for approximately 5-fold compared with the wild-type (WT) AAV6 vectors. Next, we evaluated the ability of the mutant AAV6 vectors to initiate specific T-cell clone proliferation in vivo. Our data indicate that the intramuscular administration of AAV6-S663V+T492V vectors expressing ovalbumin (OVA) led to a strong activation (approximately 9%) of specific T cells in peripheral blood compared with AAV6-WT treated animals (<1%). These OVA-specific T cells have a superior killing ability against mouse prostate cancer cell line RM1 stably expressing the OVA antigen when propagated in vitro. Finally, we evaluated the ability of capsid-optimized AAV6-S663V+T492V vectors to initiate a protective anticancer immune response in vivo. Our results document the suppression of subcutaneous tumor growth in animals immunized with AAV6-S663V+T492V vectors expressing prostatic acid phosphatase (PAP) for approximately 4 weeks in comparison with 1 week and 2 weeks for the negative controls, AAV6-EGFP, and AAV6-WT-PAP treated mice, respectively. These studies suggest that successful inhibition of tumor growth in an animal model would set the stage for potential clinical application of the capsid-optimized AAV6-S663V+T492V vectors.  

5.1697           Adeno-associated virus mediated delivery of an engineered protein that combines the complement inhibitory properties of CD46, CD55 and CD59

Leadererm, D., Cashsman, S.M. and Kumar-Singh, R.

1. Gene Med., 17(6-7), 101-115 (2015)

  Background A variety of disorders are associated with the activation of complement. CD46, CD55 and CD59 are the major membrane associated regulators of complement on human cells. Previously, we have found that independent expression of CD55, CD46 or CD59 through gene transfer protects murine tissues against human complement mediated attack. In the present study, we investigated the potential of combining the complement regulatory properties of CD46, CD55 and CD59 into single gene products expressed from an adeno-associated virus (AAV) vector in a soluble non-membrane anchored form. Methods Minigenes encoding the complement regulatory domains from CD46, CD55 and CD59 (SACT) or CD55 and CD59 (DTAC) were cloned into an AAV vector. The specific regulatory activity of each component of SACT and DTAC was measured in vitro. The recombinant AAV vectors were injected into the peritoneum of mice and the efficacy of the transgene products for being able to protect murine liver vasculature against human complement, specifically the membrane attack complex (MAC), was measured. Results SACT and DTAC exhibited properties similar to CD46, CD55 and CD59 or CD55 and CD59, respectively, in vitro. AAV mediated delivery of SACT or DTAC protected murine liver vasculature from human MAC deposition by 63.2% and 56.7%, respectively. Conclusions When delivered to mice in vivo via an AAV vector, SACT and DTAC are capable of limiting human complement mediated damage. SACT and DTAC merit further study as potential therapies for complement mediated disorders when delivered via a gene therapy approach.  

5.1698           AAV-mediated expression of BAG1 and ROCK2-shRNA promote neuronal survival and axonal sprouting in a rat model of rubrospinal tract injury

Challlagundla, M., Koch, J.C., Ribas, V.T., Michel, U., Kügler, S., Ostendorf, T., Bradke, F., Müller, H.W., Bähr, M. and Lingor, P.

1. Neurochem., 134, 261-275 (2015)

  A lesion to the rat rubrospinal tract is a model for traumatic spinal cord lesions and results in atrophy of the red nucleus neurons, axonal dieback, and locomotor deficits. In this study, we used adeno-associated virus (AAV)-mediated over-expression of BAG1 and ROCK2-shRNA in the red nucleus to trace [by co-expression of enhanced green fluorescent protein (EGFP)] and treat the rubrospinal tract after unilateral dorsal hemisection. We investigated the effects of targeted gene therapy on neuronal survival, axonal sprouting of the rubrospinal tract, and motor recovery 12 weeks after unilateral dorsal hemisection at Th₈ in rats. In addition to the evaluation of BAG1 and ROCK2 as therapeutic targets in spinal cord injury, we aimed to demonstrate the feasibility and the limits of an AAV-mediated protein over-expression versus AAV.shRNA-mediated down-regulation in this traumatic CNS lesion model. Our results demonstrate that BAG1 and ROCK2-shRNA both promote neuronal survival of red nucleus neurons and enhance axonal sprouting proximal to the lesion. Understanding the mechanisms involved in neuronal survival and axonal regeneration after spinal cord injury (SCI) is pivotal for the development of new therapies. We showed that over-expression of BAG1 (Bcl-2-associated athanogene-1) and down-regulation of ROCK2 (Rho-associated protein kinase) improve neuronal survival and axonal sprouting after SCI. Our results imply that BAG1 and ROCK2 represent interesting molecular targets that can be used in future therapeutic strategies for the treatment of SCI. AAV = adeno-associated virus.  

5.1699           Convergence of lemniscal and local excitatory inputs on large GABAergic tectothalamic neurons

Ito, T., Hioki, H., Sohn, J., Okamoto, S., Kaneko, T., Iino, S. and Oliver, D.L:

1. Comp. Neurol., 523,2277-2296 (2015)

  Large GABAergic (LG) neurons form a distinct cell type in the inferior colliculus (IC), identified by the presence of dense VGLUT2-containing axosomatic terminals. Although some of the axosomatic terminals originate from local and commissural IC neurons, it has been unclear whether LG neurons also receive axosomatic inputs from the lower auditory brainstem nuclei, i.e., cochlear nuclei (CN), superior olivary complex (SOC), and nuclei of the lateral lemniscus (NLL). In this study we injected recombinant viral tracers that force infected cells to express GFP in a Golgi-like manner into the lower auditory brainstem nuclei to determine whether these nuclei directly innervate LG cell somata. Labeled axons from CN, SOC, and NLL terminated as excitatory axosomatic endings, identified by colabeling of GFP and VGLUT2, on single LG neurons in the IC. Each excitatory axon made only a few axosomatic contacts on each LG neuron. Inputs to a single LG cell are unlikely to be from a single brainstem nucleus, since lesions of individual nuclei failed to eliminate most VGLUT2-positive terminals on the LG neurons. The estimated number of inputs on a single LG cell body was almost proportional to the surface area of the cell body. Double injections of different viruses into IC and a brainstem nucleus showed that LG neurons received inputs from both. These results demonstrated that both ascending and intrinsic sources converge on the LG somata to control inhibitory tectothalamic projections.  

5.1700           Polar freshwater cyanophage S-EIV1 represents a new widespread evolutionary lineage of phages

Chenard, C., Chan, A.M., Vincent, W.F. and Suttle, C.A. ISME J., 9(9), 2046-2058 (2015)   Cyanobacteria are often the dominant phototrophs in polar freshwater communities; yet, the phages that infect them remain unknown. Here, we present a genomic and morphological characterization of cyanophage S-EIV1 that was isolated from freshwaters on Ellesmere Island (Nunavut, High Arctic Canada), and which infects the polar Synechococcus sp., strain PCCC-A2c. S-EIV1 represents a newly discovered evolutionary lineage of bacteriophages whose representatives are widespread in aquatic systems. Among the 130 predicted open reading frames (ORFs) there is no recognizable similarity to genes that encode structural proteins other than the large terminase subunit and a distant viral morphogenesis protein, indicating that the genes encoding the structural proteins of S-EIV1 are distinct from other viruses. As well, only 19 predicted coding sequences on the 79 178 bp circularly permuted genome have homology with genes encoding proteins of known function. Although S-EIV1 is divergent from other sequenced phage isolates, it shares synteny with phage genes captured on a fosmid from the deep-chlorophyll maximum in the Mediterranean Sea, as well as with an incision element in the genome of Anabaena variabilis (ATCC 29413). Sequence recruitment of metagenomic data indicates that S-EIV1-like viruses are cosmopolitan and abundant in a wide range of aquatic systems, suggesting they have an important ecological role.  

5.1701           Reward and Toxicity of Cocaine Metabolites Generated by Cocaine Hydrolase

Murthy, V., Geng, L., gao, Y., Zhang, B., Miller, J.D., Reyes, S. and Brimijoin, S. Cell. Mol. Neurobiol., 35(6), 819-826 (2015)   Butyrylcholinesterase (BChE) gene therapy is emerging as a promising concept for treatment of cocaine addiction. BChE levels after gene transfer can rise 1000-fold above those in untreated mice, making this enzyme the second most abundant plasma protein. For months or years, gene transfer of a BChE mutated into a cocaine hydrolase (CocH) can maintain enzyme levels that destroy cocaine within seconds after appearance in the blood stream, allowing little to reach the brain. Rapid enzyme action causes a sharp rise in plasma levels of two cocaine metabolites, benzoic acid (BA) and ecgonine methyl ester (EME), a smooth muscle relaxant that is mildly hypotensive and, at best, only weakly rewarding. The present study, utilizing Balb/c mice, tested reward effects and cardiovascular effects of administering EME and BA together at molar levels equivalent to those generated by a given dose of cocaine. Reward was evaluated by conditioned place preference. In this paradigm, cocaine (20 mg/kg) induced a robust positive response but the equivalent combined dose of EME + BA failed to induce either place preference or aversion. Likewise, mice that had undergone gene transfer with mouse CocH (mCocH) showed no place preference or aversion after repeated treatments with a near-lethal 80 mg/kg cocaine dose. Furthermore, a single administration of that same high cocaine dose failed to affect blood pressure as measured using the noninvasive tail-cuff method. These observations confirm that the drug metabolites generated after CocH gene transfer therapy are safe even after a dose of cocaine that would ordinarily be lethal.  

5.1702           VPS35 Deficiency or Mutation Causes Dopaminergic Neuronal Loss by Impairing Mitochondrial Fusion and Function

Tang, F-L., Liu, W., Hu, J-X., Erion, J.R., Ye, J., Mei, L. and Xiong, W-C. Cell Reports, 12, 1631-1643 (2015)   Vacuolar protein sorting-35 (VPS35) is a retromer component for endosomal trafficking. Mutations of VPS35 have been linked to familial Parkinson’s disease (PD). Here, we show that specific deletion of the VPS35 gene in dopamine (DA) neurons resulted in PD-like deficits, including loss of DA neurons and accumulation of α-synuclein. Intriguingly, mitochondria became fragmented and dysfunctional in VPS35-deficient DA neurons, phenotypes that could be restored by expressing VPS35 wild-type, but not PD-linked mutant. Concomitantly, VPS35 deficiency or mutation increased mitochondrial E3 ubiquitin ligase 1 (MUL1) and, thus, led to mitofusin 2 (MFN2) degradation and mitochondrial fragmentation. Suppression of MUL1 expression ameliorated MFN2 reduction and DA neuron loss but not α-synuclein accumulation. These results provide a cellular mechanism for VPS35 dysfunction in mitochondrial impairment and PD pathogenesis.  

5.1703           NPY Y2 receptors in the central amygdala reduce cued but not contextual fear

Verma, D., Wood, J., lach, g., Mietzsch, M., Weger, S., Heilbronn, R., Herzog, H., Bonaventure, P., Sperk, G. and Tasan, R.O. Neuropharmacology, 99, 665-674 (2015)   The amygdala is fundamental for associative fear and extinction learning. Recently, also the central nucleus of the amygdala (CEA) has emerged as a site of plasticity actively controlling efferent connections to downstream effector brain areas. Although synaptic transmission is primarily mediated by glutamate and GABA, neuropeptides critically influence the overall response. While neuropeptide Y (NPY) acting via postsynaptic Y1 receptors exerts an important anxiolytic and fear-reducing action, the role of the predominantly presynaptic Y2 receptors is less defined. To investigate the role of Y2 receptors in the CEA we employed viral-vector mediated over-expression of the Y2 selective agonist NPY_3-36 in fear conditioning and extinction experiments. NPY_3-36 over-expression in the CEA resulted in reduced fear expression during fear acquisition and recall. Interestingly, this effect was blocked by intraperitoneal injection of a brain-penetrant Y2 receptor antagonist. Furthermore, over-expression of NPY_3-36 in the CEA also reduced fear expression during fear extinction of CS-induced but not context-related fear. Again, fear extinction appeared delayed by peripheral injection of a Y2 receptor antagonist JNJ-31020028. Importantly, mice with over-expression of NPY_3-36 in the CEA also displayed reduced spontaneous recovery and reinstatement, suggesting that Y2 receptor activation supports a permanent suppression of fear. Local deletion of Y2 receptors in the CEA, on the other hand, increased the expression of CS-induced freezing during fear recall and fear extinction. Thus, NPY inhibits fear learning and promotes cued extinction by reducing fear expression also via activation of presynaptic Y2 receptors on CEA neurons.  

5.1704           Viral delivery of shRNA to amygdala neurons leads to neurotoxicity and deficits in Pavlovian fear conditioning

De Solis, C.A., Holehonnur, R., Banerjee, A., Luong, J.A., Lella, S.K., Ho, A., pahlavan, B. and Ploski, J.E. Neurobiology of Learning and Memory, 124, 34-47 (2015)   The use of viral vector technology to deliver short hairpin RNAs (shRNAs) to cells of the nervous system of many model organisms has been widely utilized by neuroscientists to study the influence of genes on behavior. However, there have been numerous reports that delivering shRNAs to the nervous system can lead to neurotoxicity. Here we report the results of a series of experiments where adeno-associated viruses (AAV), that were engineered to express shRNAs designed to target known plasticity associated genes (i.e. Arc, Egr1 and GluN2A) or control shRNAs that were designed not to target any rat gene product for depletion, were delivered to the rat basal and lateral nuclei of the amygdala (BLA), and auditory Pavlovian fear conditioning was examined. In our first set of experiments we found that animals that received AAV (3.16E13–1E13 GC/mL; 1 μl/side), designed to knockdown Arc (shArc), or control shRNAs targeting either luciferase (shLuc), or nothing (shCntrl), exhibited impaired fear conditioning compared to animals that received viruses that did not express shRNAs. Notably, animals that received shArc did not exhibit differences in fear conditioning compared to animals that received control shRNAs despite gene knockdown of Arc. Viruses designed to harbor shRNAs did not induce obvious morphological changes to the cells/tissue of the BLA at any dose of virus tested, but at the highest dose of shRNA virus examined (3.16E13 GC/mL; 1 μl/side), a significant increase in microglia activation occurred as measured by an increase in IBA1 immunoreactivity. In our final set of experiments we infused viruses into the BLA at a titer of (1.60E+12 GC/mL; 1 μl/side), designed to express shArc, shLuc, shCntrl or shRNAs designed to target Egr1 (shEgr1), or GluN2A (shGluN2A), or no shRNA, and found that all groups exhibited impaired fear conditioning compared to the group which received a virus that did not express an shRNA. The shEgr1 and shGluN2A groups exhibited gene knockdown of Egr1 and GluN2A compared to the other groups examined respectively, but Arc was not knocked down in the shArc group under these conditions. Differences in fear conditioning among the shLuc, shCntrl, shArc and shEgr1 groups were not detected under these circumstances; however, the shGluN2A group exhibited significantly impaired fear conditioning compared to most of the groups, indicating that gene specific deficits in fear conditioning could be observed utilizing viral mediated delivery of shRNA. Collectively, these data indicate that viral mediated shRNA expression was toxic to neurons in vivo, under all viral titers examined and this toxicity in some cases may be masking gene specific changes in learning. Therefore, the use of this technology in behavioral neuroscience warrants a heightened level of careful consideration and potential methods to alleviate shRNA induced toxicity are discussed.  

5.1705           The ADAR1 editing enzyme is encapsidated into HIV-1 virions

Orecchini, E., Federico, M., Doria, M., Arenaccio, C., Giuliani, E., Ciafre, S.A. and Michienzi, A. Virology, 485, 475-480 (2015)   Adenosine deaminase acting on RNA1 (ADAR1) was previously reported to affect HIV-1 replication. We report data showing that ADAR1 interacts with the HIV-1 p55 Gag protein, the major structural protein of the immature virus capsid. Furthermore, we found that the endogenous ADAR1 is incorporated into virions purified from the supernatant of primary HIV-1-infected CD4⁺ T lymphocytes. Additional experiments demonstrated that the expression of the p55 Gag protein is sufficient for ADAR1 incorporation into virus-like particles (VLPs). Overall, our data originally support the evidence that ADAR1 can be part of the cell protein array uploaded in HIV-1 particles.  

5.1706           Corticotropin-Releasing Hormone Receptor Type 1 (CRHR1) Clustering with MAGUKs Is Mediated via Its C-Terminal PDZ Binding Motif

Bender, J., Engeholm, M., Ederer, M.S., Breu, J., Møller, T.C., Michalakis, S., Rasko, T., Wanker, E.E., Biel, M., Martinez, K.L., Wurst, W. and Deussing, J.M. PloS One, 10(9), e0136768 (2015)   The corticotropin-releasing hormone receptor type 1 (CRHR1) plays an important role in orchestrating neuroendocrine, behavioral, and autonomic responses to stress. To identify molecules capable of directly modulating CRHR1 signaling, we performed a yeast-two-hybrid screen using the C-terminal intracellular tail of the receptor as bait. We identified several members of the membrane-associated guanylate kinase (MAGUK) family: postsynaptic density protein 95 (PSD95), synapse-associated protein 97 (SAP97), SAP102 and membrane associated guanylate kinase, WW and PDZ domain containing 2 (MAGI2). CRHR1 is co-expressed with the identified MAGUKs and with the additionally investigated PSD93 in neurons of the adult mouse brain and in primary hippocampal neurons, supporting the probability of a physiological interaction in vivo. The C-terminal PDZ (PSD-95, discs large, zona occludens 1) binding motif of CRHR1 is essential for its physical interaction with MAGUKs, as revealed by the CRHR1-STAVA mutant, which harbors a functionally impaired PDZ binding motif. The imitation of a phosphorylation at Thr413 within the PDZ binding motif also disrupted the interaction with MAGUKs. In contrast, distinct PDZ domains within the identified MAGUKs are involved in the interactions. Expression of CRHR1 in primary neurons demonstrated its localization throughout the neuronal plasma membrane, including the excitatory post synapse, where the receptor co-localized with PSD95 and SAP97. The co-expression of CRHR1 and respective interacting MAGUKs in HEK293 cells resulted in a clustered subcellular co-localization which required an intact PDZ binding motif. In conclusion, our study characterized the PDZ binding motif-mediated interaction of CRHR1 with multiple MAGUKs, which directly affects receptor function.  

5.1707           A Systematic Approach to Novel Virus Discovery in Emerging Infectious Disease Outbreaks

Sridhar, S., To, K.K.W., Chan, J.F.W., lau, S.K.P., Woo, P.C.Y. and Yuen, K-Y.

1. Mol. Diagnostics, 17(3), 230-241 (2015)

  The discovery of novel viruses is of great importance to human health—both in the setting of emerging infectious disease outbreaks and in disease syndromes of unknown etiology. Despite the recent proliferation of many efficient virus discovery methods, careful selection of a combination of methods is important to demonstrate a novel virus, its clinical associations, and its relevance in a timely manner. The identification of a patient or an outbreak with distinctive clinical features and negative routine microbiological workup is often the starting point for virus hunting. This review appraises the roles of culture, electron microscopy, and nucleic acid detection–based methods in optimizing virus discovery. Cell culture is generally slow but may yield viable virus. Although the choice of cell line often involves trial and error, it may be guided by the clinical syndrome. Electron microscopy is insensitive but fast, and may provide morphological clues to choice of cell line or consensus primers for nucleic acid detection. Consensus primer PCR can be used to detect viruses that are closely related to known virus families. Random primer amplification and high-throughput sequencing can catch any virus genome but cannot yield an infectious virion for testing Koch postulates. A systematic approach that incorporates carefully chosen combinations of virus detection techniques is required for successful virus discovery.  

5.1708           Unique Roles of TLR9- and MyD88-Dependent and -Independent Pathways in Adaptive Immune Responses to AAV-Mediated Gene Transfer

Rogers, G.L., Suzuki, M., Zolotukhin, I., Markusic, D.M., Morel, L.M., Lee, B., Ertl, H.J. and Herzog, R.W.

1. Innate Immun., 7(3), 302-314 (2015)

  The immune system represents a significant barrier to successful gene therapy with adeno-associated viral (AAV) vectors. In particular, adaptive immune responses to the viral capsid or the transgene product are of concern. The sensing of AAV by toll-like receptors (TLRs) TLR2 and TLR9 has been suggested to play a role in innate immunity to the virus and may also shape subsequent adaptive immune responses. Here, we investigated the functions of TLR2, TLR9 and the downstream signaling adaptor MyD88 in antibody and CD8+ T-cell responses. Antibody formation against the transgene product occurred largely independently of TLR signaling following gene transfer with AAV1 or AAV2 vectors, whereas loss of signaling through the TLR9-MyD88 pathway substantially reduced CD8+ T-cell responses. In contrast, MyD88 (but neither of the TLRs) regulated antibody responses to capsid. B cell-intrinsic MyD88 was required for the formation of anti-capsid IgG2c independently of vector serotype or route of administration. However, MyD88^-/- mice instead produced anti-capsid IgG1 that emerged with delayed kinetics but nonetheless completely prevented in vivo readministration. We conclude that there are distinct roles for TLR9 and MyD88 in promoting adaptive immune responses to AAV-mediated gene transfer and that there are redundant MyD88-dependent and MyD88-independent mechanisms that stimulate neutralizing antibody formation against AAV.  

5.1709           Photo-activatable Cre recombinase regulates gene expression in vivo

Schindler, S.E., McCall, J.G., Yan, P., Hyrc, K.L., Li, M., Tucker, C.L., Lee, J-M., Bruchas, M.R. and Diamond, M.I. Scientific Reports, 5:13627 (2015)   Techniques allowing precise spatial and temporal control of gene expression in the brain are needed. Herein we describe optogenetic approaches using a photo-activatable Cre recombinase (PA-Cre) to stably modify gene expression in the mouse brain. Blue light illumination for 12 hours via optical fibers activated PA-Cre in the hippocampus, a deep brain structure. Two-photon illumination through a thinned skull window for 100 minutes activated PA-Cre within a sub-millimeter region of cortex. Light activation of PA-Cre may allow permanent gene modification with improved spatiotemporal precision compared to standard methods.  

5.1710           Comparative Analysis of Cesium Chloride- and Iodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications

Strobel, B., Miller, F.D., Rist, W. and Lamla, T. Human Gene Therapy Methods, 26(4), 147-157 (2015)   Cesium chloride (CsCl)- and iodixanol-based density gradients represent the core step in most protocols for serotype-independent adeno-associated virus (AAV) purification established to date. However, despite controversial reports about the purity and bioactivity of AAV vectors derived from each of these protocols, systematic comparisons of state-of-the-art variants of these methods are sparse. To define exact conditions for such a comparison, we first fractionated both gradients to analyze the distribution of intact, bioactive AAVs and contaminants, respectively. Moreover, we tested four different polishing methods (ultrafiltration, size-exclusion chromatography, hollow-fiber tangential flow filtration, and polyethylene glycol precipitation) implemented after the iodixanol gradient for their ability to deplete iodixanol and protein contaminations. Last, we conducted a side-by-side comparison of the CsCl and iodixanol/ultrafiltration protocol. Our results demonstrate that iodixanol-purified AAV preparations show higher vector purity but harbor more (∼20%) empty particles as compared with CsCl-purified vectors (<1%). Using mass spectrometry, we analyzed prominent protein impurities in the AAV vector product, thereby identifying known and new, possibly AAV-interacting proteins as major contaminants. Thus, our study not only provides a helpful guide for the many laboratories entering the AAV field, but also builds a basis for further investigation of cellular processes involved in AAV vector assembly and trafficking.  

5.1711           Adeno-Associated Virus at 50: A Golden Anniversary of Discovery, Research, and Gene Therapy Success—A Personal Perspective

Hastie, E. and Samulski, R.J. Human Gene Therapy, 26(5), 257-265 (2015)   Fifty years after the discovery of adeno-associated virus (AAV) and more than 30 years after the first gene transfer experiment was conducted, dozens of gene therapy clinical trials are in progress, one vector is approved for use in Europe, and breakthroughs in virus modification and disease modeling are paving the way for a revolution in the treatment of rare diseases, cancer, as well as HIV. This review will provide a historical perspective on the progression of AAV for gene therapy from discovery to the clinic, focusing on contributions from the Samulski lab regarding basic science and cloning of AAV, optimized large-scale production of vectors, preclinical large animal studies and safety data, vector modifications for improved efficacy, and successful clinical applications.  

5.1712           Functional and Biochemical Characterization of Hepatitis C Virus (HCV) Particles Produced in a Humanized Liver Mouse Model

Calattini, S., Fusil, F., Mancip, J., Thi, V.L.D., Granier, C., Gadot, N., Scoazec, J-Y., Zeisel, M.B., Baumert, T.F., Lavillette, D., Dreux, M. and Cosset, F-L.

1. Biol. Chem., 290(38), 23173-23187 (2015)

  Lipoprotein components are crucial factors for hepatitis C virus (HCV) assembly and entry. As hepatoma cells producing cell culture-derived HCV (HCVcc) particles are impaired in some aspects of lipoprotein metabolism, it is of upmost interest to biochemically and functionally characterize the in vivo produced viral particles, particularly regarding how lipoprotein components modulate HCV entry by lipid transfer receptors such as scavenger receptor BI (SR-BI). Sera from HCVcc-infected liver humanized FRG mice were separated by density gradients. Viral subpopulations, termed HCVfrg particles, were characterized for their physical properties, apolipoprotein association, and infectivity. We demonstrate that, in contrast to the widely spread distribution of apolipoproteins across the different HCVcc subpopulations, the most infectious HCVfrg particles are highly enriched in apoE, suggesting that such apolipoprotein enrichment plays a role for entry of in vivo derived infectious particles likely via usage of apolipoprotein receptors. Consistent with this salient feature, we further reveal previously undefined functionalities of SR-BI in promoting entry of in vivo produced HCV. First, unlike HCVcc, SR-BI is a particularly limiting factor for entry of HCVfrg subpopulations of very low density. Second, HCVfrg entry involves SR-BI lipid transfer activity but not its capacity to bind to the viral glycoprotein E2. In conclusion, we demonstrate that composition and biophysical properties of the different subpopulations of in vivo produced HCVfrg particles modulate their levels of infectivity and receptor usage, hereby featuring divergences with in vitro produced HCVcc particles and highlighting the powerfulness of this in vivo model for the functional study of the interplay between HCV and liver components.  

5.1713           Critical role of the neural pathway from the intermediate medial mesopallium to the intermediate hyperpallium apicale in filial imprinting of domestic chicks (Gallus gallus domesticus)

Aoki, N., Yamaguchi, S., Kitajima, T., Takehara, A., Katagiri-Nakagawa, S., Matsui, r., Watanabe, D., Matsushima, T. and Homma, K.J. Neuroscience, 308, 115-124 82015)   Filial imprinting in precocial birds is a useful model for studying early learning and cognitive development, as it is characterized by a well-defined sensitive or critical period. We recently showed that the thyroid hormone 3,5,3′-triiodothyronine (T₃) determines the onset of the sensitive period. Moreover, exogenous injection of T₃ into the intermediate medial mesopallium (IMM) region (analogous to the associative cortex in mammals) enables imprinting even on post-hatch day 4 or 6 when the sensitive period has been terminated. However, the neural mechanisms downstream from T₃ action in the IMM region remain elusive. Here, we analyzed the functional involvement of the intermediate hyperpallium apicale (IMHA) in T₃ action. Bilateral excitotoxic ablation of the IMHA prevented imprinting in newly hatched chicks, and also suppressed the recovery of the sensitive period by systemic intra-venous or localized intra-IMM injection of T₃ in day-4 chicks. In contrast to the effect in the IMM, direct injection of T₃ into the IMHA did not enable imprinting in day-4 chicks. Moreover, bilateral ablation of IMHA after imprinting training impaired recall. These results suggest that the IMHA is critical for memory acquisition downstream following T₃ action in the IMM and further, that it receives and retains information stored in the IMM for recall. Furthermore, both an avian adeno-associated viral construct containing an anterograde tracer (wheat-germ agglutinin) and a retrograde tracer (cholera toxin subunit B) revealed neural connections from the IMM to the IMHA. Taken together, our findings suggest that hierarchical processes from the primary area (IMM) to the secondary area (IMHA) are required for imprinting.  

5.1714           Sinorhizobium meliloti Phage {Phi}M9 Defines a New Group of T4 Superfamily Phages with Unusual Genomic Features but a Common T=16 Capsid

Johnson, M.C., Tatum, K.B., Lynn, J.S., Brewer, T.E., Lu, S., Washburn, B.K., Stroupe, M.E. and Jones, K.M.

1. Virol., 89(21), 10945-10958 (2015)

  Relatively little is known about the phages that infect agriculturally important nitrogen-fixing rhizobial bacteria. Here we report the genome and cryo-electron microscopy structure of the Sinorhizobium meliloti-infecting T4 superfamily phage ΦM9. This phage and its close relative Rhizobium phage vB_RleM_P10VF define a new group of T4 superfamily phages. These phages are distinctly different from the recently characterized cyanophage-like S. meliloti phages of the ΦM12 group. Structurally, ΦM9 has a T=16 capsid formed from repeating units of an extended gp23-like subunit that assemble through interactions between one subunit and the adjacent E-loop insertion domain. Though genetically very distant from the cyanophages, the ΦM9 capsid closely resembles that of the T4 superfamily cyanophage Syn9. ΦM9 also has the same T=16 capsid architecture as the very distant phage SPO1 and the herpesviruses. Despite their overall lack of similarity at the genomic and structural levels, ΦM9 and S. meliloti phage ΦM12 have a small number of open reading frames in common that appear to encode structural proteins involved in interaction with the host and which may have been acquired by horizontal transfer. These proteins are predicted to encode tail baseplate proteins, tail fibers, tail fiber assembly proteins, and glycanases that cleave host exopolysaccharide.  

5.1715           A Molecular Approach Designed to Limit the Replication of Mature DENV2 in Host Cells

Raheel, U., Jamal, M. and Zaidi, N.U.S.S. Viral Immunol., 28(7), 378-384 (2015)   Dengue virus (DENV) is an arthropod-borne virus, which belongs to the Flaviviridae family, and completes its life cycle in two hosts: humans and mosquitoes. For DENV maturation, the surface pre-membrane (prM) protein is cleaved to form a mature membrane protein (M) by furin, which is a cellular enzyme subsequently releasing the mature virus from the host dendritic cell. The objective of the current study was to inhibit mature DENV isotype 2 (DENV2) by RNA-interference in a Vero-81 cell line. Mature DENV2 was propagated in and isolated from U937 cells expressing dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin. Maturation of DENV2 was confirmed by Western blot analysis, where virus stock lacking prM was considered mature. Inhibition studies were carried out by transfection of Vero-81 cells with six synthetic siRNAs along with a control siRNA. Reduction in cellular DENV2 was observed also by focus-reduction assay, immunofluorescence assay (IFA), and real-time quantitative polymerase chain reaction (RT-qPCR). Cells transfected with DENV2SsiRNA2, which was targeting the structural region M of mature DENV2, was able to reduce DENV2 titer by up to 85% in focus reduction assays. A significant reduction in mature DENV2 RNA load was observed by RT-qPCR, confirming the previous findings. IFA also revealed reduced levels of cellular DENV2. These results demonstrated that mature DENV2 can be effectively inhibited by synthetic siRNA targeting the structural region of the genome. Mature DENV2 can be successfully inhibited by siRNAs, and specifically high knock-down efficiency is observed by siRNAs against M region of mature DENV2. This study shows that M represents a potential target for RNAi based inhibitory approaches.  

5.1716           Effective Gene Delivery to Valvular Interstitial Cells Using Adeno-Associated Virus Serotypes 2 and 3

Wong, F.F., Ho, M.L., Yamagami, M., Lam, M.T., Grande-Allen, K.J. and Suh, J. Tissue Engineering: Part C, 21(8), 808-815 (2015)   Currently, curative therapies for heart valve diseases do not exist, thus motivating the need for new therapeutics, regenerative and tissue-engineered valves, and further basic research into pathological mechanisms. For studying valve diseases and developing valve therapies, effective methods to manipulate gene expression in primary valvular interstitial cells (VICs), which promote calcification in disease, would be valuable. Unfortunately, there is little information reported about effective gene delivery methods for VICs. Adeno-associated virus (AAV) is a clinically proven gene delivery vector capable of transducing many cell types and tissues, but has not yet been reported to infect valvular cells. In this study, AAV serotypes 1–9 were tested for their ability to deliver a green fluorescent protein (GFP) reporter into VICs in vitro. Flow cytometry results indicate AAV2 and AAV3 are capable of transducing VICs more efficiently than other serotypes. Furthermore, transduction efficiencies can be optimized by increasing the multiplicity of infection (MOI) and using self-complementary, double-stranded genomes, yielding up to 98% successfully transduced cells. Transduction of VICs by AAV2 or AAV3 in the presence of competing soluble heparin significantly reduces delivery efficiencies, suggesting heparan sulfate proteoglycans act as the primary VIC receptors of these two serotypes. Overall, this study establishes AAV2 and AAV3 as efficient gene delivery vehicles for primary VICs. Such effective delivery vectors for valve cells may be broadly useful for numerous applications, including the study of valvular cell biology, development of valve disease therapies, and regulation of genes for tissue engineering heart valves.  

5.1717           JC polyomavirus mutants escape antibody-mediated neutralization

Ray, U., Cinque, P., gerevini, S., Longo, V., Lazzarin, A., Schippling, S., martin, R., Buck, C.B. and Pastrana, D.V. Science Translational Medicine, 7(306), 306ra151 (2015)   JC polyomavirus (JCV) can be found in the urinary tract in most adults, resulting in a persistent but asymptomatic infection. However, in immunocompromised individuals, JCV opportunistically infects the brain, resulting in the debilitating and frequently fatal disease progressive multifocal leukoencephalopathy (PML). No treatments are currently available for PML, but two papers now identify and exploit a gap in the immune response to JCV. Ray et al. report that JCV strains found in the cerebrospinal fluid of PML patients have mutations that prevent antibody neutralization and that these blind spots can be overcome with vaccination. Jelcic et al. suggest that broadly neutralizing antibodies derived from a patient who recovered from PML may fill this gap.  

5.1718           Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template

Sather, B.D. et al Science Translational Medicine, 7(307), 307ra156 (2015)   Newer gene-editing methods hold promise for correcting human disease but so far have been hampered by low efficiencies when used in primary cells. To address this issue, Sather et al. have devised a more effective way to both disrupt and replace the CCR5 locus in human T cells, a procedure that has already been shown to improve HIV clearance. Serotype 6 of an adeno-associated viral vector worked particularly well for delivery of megaTAL nucleases and homologous donor templates to primary human T cells, achieving efficient gene-editing rates and little toxicity. The megaTALs generate homology-directed repair (rather than previous efforts, which induce nonhomologous end-joining repair) and so was used for both deletion and accurate replacement of the CCR5 locus. The authors demonstrate that chimeric antigen receptors and an HIV fusion inhibitor inserted into the CCR5 locus ameliorate HIV infection in mice and show that their approach also works in CD34⁺ hematopoietic precursor cells.  

5.1719           Gene Augmentation Therapy Restores Retinal Function and Visual Behavior in a Sheep Model of CNGA3 Achromatopsia

Banin, E., Gootwine, E., Obolensky, A., Ezra-Elia, r., Ejzenberg, A., Zelinger, L., Honig, H., Rosov, A., Yamin, E., Sharon, D., Averbukh, E., Hauswirth, W.W. and Ofri, R. Gene Therapy, 23(9), 1423-1433 (2015)   Achromatopsia is a hereditary form of day blindness caused by cone photoreceptor dysfunction. Affected patients suffer from congenital color blindness, photosensitivity, and low visual acuity. Mutations in the CNGA3 gene are a major cause of achromatopsia, and a sheep model of this disease was recently characterized by our group. Here, we report that unilateral subretinal delivery of an adeno-associated virus serotype 5 (AAV5) vector carrying either the mouse or the human intact CNGA3 gene under the control of the red/green opsin promoter results in long-term recovery of visual function in CNGA3-mutant sheep. Treated animals demonstrated shorter maze passage times and a reduced number of collisions with obstacles compared with their pretreatment status, with values close to those of unaffected sheep. This effect was abolished when the treated eye was patched. Electroretinography (ERG) showed marked improvement in cone function. Retinal expression of the transfected human and mouse CNGA3 genes at the mRNA level was shown by polymerase chain reaction (PCR), and cone-specific expression of CNGA3 protein was demonstrated by immunohistochemisrty. The rescue effect has so far been maintained for over 3 years in the first-treated animals, with no obvious ocular or systemic side effects. The results support future application of subretinal AAV5-mediated gene-augmentation therapy in CNGA3 achromatopsia patients.  

5.1720           Viruses transfer the antiviral second messenger cGAMP between cells

Bridgeman, A., Maelfait, J., Davenne, T., Partridge, T., Peng, Y., mayer, A., Dong, T., Kaever, V., Borrow, P. and Rehwinkel, J. Science, 349(6253), 1228-1232 (2015)   Cyclic GMP–AMP synthase (cGAS) detects cytosolic DNA during virus infection and induces an antiviral state. cGAS signals by synthesis of a second messenger, cyclic GMP-AMP (cGAMP), which activates stimulator of interferon genes (STING). We show that cGAMP is incorporated into viral particles, including lentivirus and herpesvirus virions, when these are produced in cGAS-expressing cells. Virions transferred cGAMP to newly infected cells and triggered a STING-dependent antiviral program. These effects were independent of exosomes and viral nucleic acids. Our results reveal a way by which a signal for innate immunity is transferred between cells, potentially accelerating and broadening antiviral responses. Moreover, infection of dendritic cells with cGAMP-loaded lentiviruses enhanced their activation. Loading viral vectors with cGAMP therefore holds promise for vaccine development.  

5.1721           Assembly and Purification of Polyomavirus-Like Particles from Plants

Catrice, E.V.B. and Sainsbury, F. Mol. Biotechnol., 57, 904-913 (2015)   Polyomaviruses are small DNA viruses that have a history of use in biotechnology. The capsids of a number of species have been developed into experimental prophylactic and therapeutic virus-like particle (VLP) vaccines. In order to explore plants as a host for the expression and purification of polyomavirus-like particles, we have transiently expressed the major capsid protein, VP1, in Nicotiana benthamiana leaves. Deletion of a polybasic motif from the N-terminal region of VP1 resulted in increased expression as well as reduced necrosis of leaf tissue, which was associated with differences in subcellular localisation and reduced DNA binding by the deletion variant (ΔVP1). Self-assembled VLPs were recovered from tissue expressing both wild-type VP1 and ΔVP1 by density gradient ultracentrifugation. VLPs composed of ΔVP1 were more homogenous than wtVPLs and, unlike the latter, did not encapsidate nucleic acid. Such homogenous, empty VLPs are of great interest in biotechnology and nanotechnology. In addition, we show that both MPyV VLP variants assembled in plants can be produced with encapsidated foreign protein. Thus, this study demonstrates the utility of plant-based expression of polyomavirus-like particles and the suitability of this host for further developments in polyomavirus-based technologies.  

5.1722           Single-Cell Analysis of B Cell/Antibody Cross-Reactivity Using a Novel Multicolor FluoroSpot Assay

Hadjilaou, A., Green, A.M., Coloma, J. and Harris, E.

1. Immunol., 195(7), 3490-3496 (2015)

  Dengue is a major public health problem globally. It is caused by four antigenically distinct serotypes of dengue virus (DENV1–4), and although serotype-specific and strongly neutralizing cross-reactive immune responses against the four DENV serotypes are thought to be protective, subneutralizing Abs can contribute to increased disease severity upon secondary infection with a different DENV serotype. Understanding the breadth of the immune response in natural DENV infections and in vaccinees is crucial for determining the correlates of protection or disease severity. Transformation of B cell populations to generate mAbs and ELISPOT assays have been used to determine B cell and Ab specificity to DENV; however, both methods have technical limitations. We therefore modified the conventional ELISPOT to develop a Quad-Color FluoroSpot to provide a means of examining B cell/Ab serotype specificity and cross-reactivity on a single-cell basis. Abs secreted by B cells are captured by an Fc-specific Ab on a filter plate. Subsequently, standardized concentrations of all four DENV serotypes are added to allow equal stoichiometry for Ag binding. After washing, the spots, representing individual B cells, are visualized using four fluorescently labeled DENV serotype-specific detection mAbs. This method can be used to better understand the breadth and magnitude of B cell responses following primary and secondary DENV infection or vaccination and their role as immune correlates of protection from subsequent DENV infections. Furthermore, the Quad-Color FluoroSpot assay can be applied to other diseases caused by multiple pathogen serotypes in which determining the serotype or subtype-specific B cell response is important.  

5.1723           Development of Recombinant Adeno-Associated Virus Serotype 2/8 Carrying Kringle Domains of Human Plasminogen for Sustained Expression and Cancer Therapy No Access

Kuo, C-H., Chang, B-I., Lee, F-T., Chen, P-K., lee, J-S., Shi, G-Y. and Wu, H-L. Human Gene Therapy, 26(9), 603-613 (2015)   Angiostatin and other plasminogen derivatives exhibit antitumor activities directly or indirectly, have demonstrated promising anticancer effects in preclinical studies, but have mostly failed in clinical trials partly due to their short serum half-lives. Our previous studies demonstrated that recombinant human plasminogen kringle 1–5 (K1–5) has superior antitumor activity compared with angiostatin. In addition, optimization of recombinant K1–5 with three amino acid substitutions enhances its antitumor effect. The current study was thus undertaken to evaluate prolonged expression of optimized K1–5 as cancer gene therapy. The recombinant adeno-associated virus (AAV) vector was used to express a secreted form of the optimized K1–5 (AAV-sK15tm) to improve its pharmacokinetic profile, which was considered to be the hurdle in angiostatin treatment of cancer. We successfully generated high-titer recombinant AAV vectors and observed sustained transgene expression for 567 days after a single injection of virus. The treated animals did not display any visible signs of abnormalities and showed normal serum biochemistry. The therapeutic potential of this treatment modality was demonstrated by both a strong inhibition of lung metastasis in the mouse B16F10 melanoma model and significant growth retardation of Lewis lung carcinoma xenografts in C57BL/6N mice as well as human A2058 melanoma xenografts in NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice. Taken together, our results suggested that AAV-sK15tm produced long-term suppressive effects on cancer growth in vivo and should warrant serious consideration for clinical development.  

5.1724           ‘Ménage à trois’: a selfish genetic element uses a virus to propagate within Thermotogales

Lossouarn, J., Nesbø, C.L., Mercier, C., Zhaxybayeva, O., Johnson, M.S., Charchuck, R., Farasin, J., Bienvenu, N., Baudoux, A-C., Michoud, G., Jebbar, M. and Geslin, C. Environment. Microbiol., 17(9), 3278-3288 (2015)   Prokaryotic viruses play a major role in the microbial ecology and evolution. However, the virosphere associated with deep-sea hydrothermal ecosystems remains largely unexplored. Numerous instances of lateral gene transfer have contributed to the complex and incongruent evolutionary history of Thermotogales, an order well represented in deep-sea hydrothermal vents. The presence of clustered regularly interspaced short palindromic repeats (CRISPR) loci has been reported in all Thermotogales genomes, suggesting that these bacteria have been exposed to viral infections that could have mediated gene exchange. In this study, we isolated and characterized the first virus infecting bacteria from the order Thermotogales, Marinitoga piezophila virus 1 (MPV1). The host, Marinitoga piezophila is a thermophilic, anaerobic and piezophilic bacterium isolated from a deep-sea hydrothermal chimney. MPV1 is a temperate Siphoviridae-like virus with a 43.7 kb genome. Surprisingly, we found that MPV1 virions carry not only the viral DNA but preferentially package a plasmid of 13.3 kb (pMP1) also carried by M. piezophila. This ‘ménage à trois’ highlights potential relevance of selfish genetic elements in facilitating lateral gene transfer in the deep-sea biosphere.  

5.1725           The caveolin–cavin system plays a conserved and critical role in mechanoprotection of skeletal muscle

Lo, H.P. et al

1. Cell Biol., 210(5), 833-849 (2015)

  Dysfunction of caveolae is involved in human muscle disease, although the underlying molecular mechanisms remain unclear. In this paper, we have functionally characterized mouse and zebrafish models of caveolae-associated muscle disease. Using electron tomography, we quantitatively defined the unique three-dimensional membrane architecture of the mature muscle surface. Caveolae occupied around 50% of the sarcolemmal area predominantly assembled into multilobed rosettes. These rosettes were preferentially disassembled in response to increased membrane tension. Caveola-deficient cavin-1^−/− muscle fibers showed a striking loss of sarcolemmal organization, aberrant T-tubule structures, and increased sensitivity to membrane tension, which was rescued by muscle-specific Cavin-1 reexpression. In vivo imaging of live zebrafish embryos revealed that loss of muscle-specific Cavin-1 or expression of a dystrophy-associated Caveolin-3 mutant both led to sarcolemmal damage but only in response to vigorous muscle activity. Our findings define a conserved and critical role in mechanoprotection for the unique membrane architecture generated by the caveolin–cavin system.  

5.1726           Acute human herpesvirus-6A infection of human mesothelial cells modulates HLA molecules

Caselli, E., Campioni, D., Cavazzini, F., Gentili, V., Bortolotti, D., Caneo, A., Di Luca, D. and Rizzo, R. Arch. Virol., 160(9), 2141-2149 (2015)   Human herpesvirus 6A (HHV-6A) causes ubiquitous infections and has been associated with several diseases in immunosuppressed and immune dysregulated individuals. Although considered a lymphotropic virus, HHV-6A has the potential to infect many cell types, inducing important alterations in the infected cell. In our search for additional potential targets for HHV-6A infection, we analyzed the susceptibility of human mesothelial cells to viral infection. HHV-6A infection was performed and analyzed on primary human mesothelial cells isolated from serous cavity fluid, infected in vitro with a cell-free HHV-6A inoculum. The results demonstrated that mesothelial cells are susceptible to in vitro HHV-6A infection, and more importantly, that the virus induces an alteration of HLA expression on the cell surface, inducing HLA class II and HLA-G de novo expression. Since mesothelial cells play a pivotal role in many processes, including inflammation and antigen presentation, we speculate that, in vivo, this virus-induced perturbation might be correlated to alterations in mesothelium functions.  

5.1727           Transcytosis in the blood–cerebrospinal fluid barrier of the mouse brain with an engineered receptor/ligand system

Mendez-Gomez, H., Galera-Prat, A., meyers, C., Chen, W., Singh, J., Carrion-Vazquez, M. and Muzyczka, N. Molecular Therapy-Methods & Clinical Development, 2, 15037 (2015)   Crossing the blood–brain and the blood–cerebrospinal fluid barriers (BCSFB) is one of the fundamental challenges in the development of new therapeutic molecules for brain disorders because these barriers prevent entry of most drugs from the blood into the brain. However, some large molecules, like the protein transferrin, cross these barriers using a specific receptor that transports them into the brain. Based on this mechanism, we engineered a receptor/ligand system to overcome the brain barriers by combining the human transferrin receptor with the cohesin domain from Clostridium thermocellum, and we tested the hybrid receptor in the choroid plexus of the mouse brain with a dockerin ligand. By expressing our receptor in choroidal ependymocytes, which are part of the BCSFB, we found that our systemically administrated ligand was able to bind to the receptor and accumulate in ependymocytes, where some of the ligand was transported from the blood side to the brain side.  

5.1728           Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells

Strobel, B., Klauser, B., hartig, J.S., lamla, T., Gantner, F. and Kreuz, S. Molecular Therapy,23(10), 1582-1591 (2015)   Cytotoxicity of transgenes carried by adeno-associated virus (AAV) vectors might be desired, for instance, in oncolytic virotherapy or occur unexpectedly in exploratory research when studying sparsely characterized genes. To date, most AAV-based studies use constitutively active promoters (e.g., the CMV promoter) to drive transgene expression, which often hampers efficient AAV production due to cytotoxic, antiproliferative, or unknown transgene effects interfering with producer cell performance. Therefore, we explored artificial riboswitches as novel tools to control transgene expression during AAV production in mammalian cells. Our results demonstrate that the guanine-responsive GuaM8HDV aptazyme efficiently attenuates transgene expression and associated detrimental effects, thereby boosting AAV vector yields up to 23-fold after a single addition of guanine. Importantly, riboswitch-harboring vectors preserved their ability to express functional transgene at high levels in the absence of ligand, as demonstrated in a mouse model of AAV-TGFβ1-induced pulmonary fibrosis. Thus, our study provides the first application-ready biotechnological system-based on aptazymes, which should enable high viral vector yields largely independent of the transgene used. Moreover, the RNA-intrinsic, small-molecule regulatable mode of action of riboswitches provides key advantages over conventional transcription factor–based regulatory systems. Therefore, such riboswitch vectors might be ultimately applied to temporally control therapeutic transgene expression in vivo.  

5.1729           Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk

Wagner, S., Thresher, R., Bland, R. and Laible, G. Scientific Reports, 5:15115 (2015)   Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5 g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland.  

5.1730           Inhibition of development of experimental abdominal aortic aneurysm by c-jun N-terminal protein kinase inhibitor combined with lysyl oxidase gene modified smooth muscle progenitor cells

Chen, F., Zhang, Z. and Zhu, X. Eur. J. Pharmacol., 766, 114-121 (2015)   Chronic inflammation, imbalance between the extracellular matrix synthesis and degradation, and loss of vascular smooth muscle cells (SMCs) contribute to the development of abdominal aortic aneurysm (AAA). The purpose of this study was to investigate the effect of the therapy with periaortic incubation of c-Jun N-terminal protein kinase inhibitor SP600125 infused from an osmotic pump and subadventitial injection of lysyl oxidase (LOX) gene modified autologous smooth muscle progenitor cells (SPCs) on treatment of AAA in a rabbit model. Obvious dilation of the abdominal aorta in the control group was caused by periaortic incubation of calcium chloride and elastase. But the progression of aortic dilation was significantly decreased after the treatment with SP600125 and LOX gene modified SPCs compared to the treatment with phosphate-buffered saline. This therapy could inhibit matrix metalloproteinases expression, enhance elastin synthesis, improve preservation of elastic laminar integrity, benefit SPCs survival and restore SMCs population. It seemed that this method might provide a novel therapeutic strategy to treat AAA.  

5.1731           Cytopathic BVDV-1 strain induces immune marker production in bovine cells through the NF-κB signaling pathway

Fredericksen, F., Carrasco, G., Villalba, M. and Olavarria, V.H. Mol. Immunol., 68, 213-222 (2015)   The bovine viral diarrhea virus (BVDV-1) is a pathogen responsible for high economic losses in the cattle industry worldwide. This virus has the capacity to modulate the immune system of several higher vertebrates, but there is little information available on the cell infection mechanism. To further investigate the effects of BVDV-1 on the activation of the immune response, the Madin-Darby bovine kidney cell line was infected with the cytopathic CH001 field isolate of BVDV-1, and the proinflammatory and antiviral cytokine expression profiles were analyzed. The results showed that BVDV-1 was able to induce the production of BCL3, IL-1β, IL-8, IL-15, IL-18, Mx-1, IRF-1, and IRF-7 in a way similar to polyinosinic–polycytidylic acid. Interestingly, all BVDV-1 activities were blocked by pharmacological inhibitors of the NF-κB signaling pathway. These results, together with in silico analyses showing the presence of several regulatory consensus target motifs, suggest that BVDV-1 regulates gene expression in bovines through the activation of several key transcription factors. Collectively, these data identified BVDV-1 as a viral regulator of immune marker expression, even from early infection. Additionally, this is the first report to find BVDV-1 modulating the activation of cytokine production and transcriptions factors mainly through the NF-κB pathway in vertebrates.  

5.1732           Establishment of an in vitro equine papillomavirus type 2 (EcPV2) neutralization assay and a VLP-based vaccine for protection of equids against EcPV2-associated genital tumors

Schellenbacher, C., Shafti-Keramat, S., Huber, B., Fink, D., Brandt, S. and Kirnbauer, R. Virology, 486, 284-290 (2015)   The consistent and specific presence of Equus caballus papillomavirus type 2 (EcPV2) DNA and mRNA in equine genital squamous cell carcinoma (gSCC) is suggestive of an etiological role in tumor development. To further validate this concept, EcPV2-neutralizing serum antibody titers were determined by an EcPV2 pseudovirion (PsV) neutralization assay. Furthermore, an EcPV2 L1 virus-like particle (VLP)-based vaccine was generated and its prophylactic efficacy evaluated in vivo. All 6/6 gSCC-affected, but only 3/20 tumor-free age-matched animals revealed EcPV2-neutralizing serum antibody titers by PsV assay. Vaccination of NZW rabbits and BalbC mice with EcPV2 L1 VLP using Freund׳s or alum respectively as adjuvant induced high-titer neutralizing serum antibodies (1600–12,800). Passive transfer with rabbit EcPV2–VLP immune sera completely protected mice from experimental vaginal EcPV2 PsV infection. These findings support the impact of EcPV2 in equine gSCC development and recommend EcPV2 L1 VLP as prophylactic vaccine against EcPV2 infection and associated disease in equids.  

5.1733           Alcohol increases the production of hepatitis C virus (HCV) lipo-viro-particles in primary human hepatocytes

Pene, V., Hernandez, C., Bløanc, E., Aoudjehane, L., Le Grand, B., Carpentier, A., Meritet, J.F., Conti, F., Calmus, Y., Rouach, H., Podevin, P. and Rosenberg, A.R. Hepatology, 62(S1), 219A-221A (2015)   BACKGROUND & AIM: Alcoholism is a major aggravating factor of HCV infection, yet the molecular mechanisms of this comorbidity remain elusive. Clinical studies found higher viral loads in patients with chronic excessive alcohol consumption. However, the so-called “viral load” reflects a wide heterogeneity of HCV particles, the most infectious ones being those of lowest buoyant density due to their association with verylow-density lipoproteins (VLDL, the apolipoprotein B-containing triglyceride-rich lipoproteins secreted by hepatocytes) in hybrid structures termed lipo-viro-particles (LVP). With the aim of investigating the impact of alcohol on HCV infectious cycle, we took hepatocytes (PHH), which, contrary to the widely used hepatocarcinoma-derived Huh-7 sublines, retain the liver metabolism of ethanol and secrete authentic VLDL and LVP. METHODS: PHH were infected with the HCV strain JFH1 or a chimeric virus derived thereof and treated with increasing doses of ethanol (0-100 mM) for 2 weeks. Cultures were monitored for HCV genome replication (negative strand RT-qPCR), viral load (clinically used test), production of infectious virus (titration by focus-formation assay), and VLDL secretion (apolipoprotein B ELISA). The density of viral particles was assessed by isopycnic iodixanol ultracentrifugation. RESULTS: Ethanol exposure of HCV-infected PHH caused a time- and dose-dependent increase in the viral load, comparable to that reported in clinical studies. Most strikingly, it caused an even greater increase in the infectious titer but did not significantly affect the viral genome replication, thus pointing to an effect on virus morphogenesis. This effect was not seen in Huh-7.5.1 cells treated in parallel, suggesting that it involves the liver metabolism of ethanol. The higher specific infectivity of HCV particles produced by PHH in the presence of ethanol correlated with lower mean buoyant density, consistent with triglyceride enrichment. Finally, in either HCV-infected or naïve PHH, addition of ethanol caused a time-dependent increase in VLDL secretion. CONCLUSION: This study in the relevant PHH model reveals that ethanol via its metabolites increases the production of HCV particles of lowest buoyant density and highest infectivity, i.e., LVP, most likely due to the impact of ethanol on triglyceride metabolism that results in increased VLDL secretion. Drugs targeting this host metabolic pathway may be useful in difficult-to-treat alcoholic patients, often poorly compliant with therapy and therefore at risk for resistance if treated by direct antivirals only. VP & CH: equal contribution.  

5.1734           High-accuracy biodistribution analysis of adeno-associated virus variants by double barcode sequencing

Marsic, D., Mendez-Gopmez, H. and Zolotukhin, S. Molecular Therapy-Methods & Clinical Development, 2:15041 (2015)   Biodistribution analysis is a key step in the evaluation of adeno-associated virus (AAV) capsid variants, whether natural isolates or produced by rational design or directed evolution. Indeed, when screening candidate vectors, accurate knowledge about which tissues are infected and how efficiently is essential. We describe the design, validation, and application of a new vector, pTR-UF50-BC, encoding a bioluminescent protein, a fluorescent protein and a DNA barcode, which can be used to visualize localization of transduction at the organism, organ, tissue, or cellular levels. In addition, by linking capsid variants to different barcoded versions of the vector and amplifying the barcode region from various tissue samples using barcoded primers, biodistribution of viral genomes can be analyzed with high accuracy and efficiency.  

5.1735           Corticospinal Motor Neurons Are Susceptible to Increased ER Stress and Display Profound Degeneration in the Absence of UCHL1 Function

Jara, J.H., genc, b., Cox, G.A., Bohn, M.C., Roos, R.P., macklis, J.D., Ulupinar, E and Özdinler, P.H. Cereb. Cortex, 25, 4259-4272 (2015)   Corticospinal motor neurons (CSMN) receive, integrate, and relay cerebral cortex's input toward spinal targets to initiate and modulate voluntary movement. CSMN degeneration is central for numerous motor neuron disorders and neurodegenerative diseases. Previously, 5 patients with mutations in the ubiquitin carboxy-terminal hydrolase-L1 (UCHL1) gene were reported to have neurodegeneration and motor neuron dysfunction with upper motor neuron involvement. To investigate the role of UCHL1 on CSMN health and stability, we used both in vivo and in vitro approaches, and took advantage of the Uchl1^nm3419 (UCHL1^−/−) mice, which lack all UCHL1 function. We report a unique role of UCHL1 in maintaining CSMN viability and cellular integrity. CSMN show early, selective, progressive, and profound cell loss in the absence of UCHL1. CSMN degeneration, evident even at pre-symptomatic stages by disintegration of the apical dendrite and spine loss, is mediated via increased ER stress. These findings bring a novel understanding to the basis of CSMN vulnerability, and suggest UCHL1^−/− mice as a tool to study CSMN pathology.  

5.1736           Rhinovirus stimulated IFN-α production: how important are plasmacytoid DCs, monocytes and endosomal pH?

Xi, Y., Finlayson, A., White, O.J., carroll, M.L. and Upham, J.W. Clinical & Translational Immunology, 4, e46 (2015)   Human rhinovirus (HRV) infection is a major cause of asthma exacerbations, which appears to be linked to a defective innate immune response to infection. Although the type I interferons (IFN-α and IFN-β) have a critical role in protecting against most viral infections, the cells responsible for IFN production in response to HRV and the relative importance of pattern recognition receptors located in endosomes has not been fully elucidated. In the current study we demonstrate that, using intracellular flow cytometry, >90% of the IFN-α-producing cells in human blood mononuclear cells following HRV16 exposure are plasmacytoid dendritic cells, whereas monocytes and myeloid dendritic cells contribute only 10% and <1%, respectively, of the IFN-α production. Bafilomycin and chloroquine, agents that inhibit the function of endosomal toll-like receptors (TLRs), significantly reduced the capacity of TLR3-, TLR7- and TLR-9-stimulated cells to produce IFN-α and the IFN-induced chemokine CXCL10 (IP-10). In contrast, only bafilomycin (but not chloroquine) effectively suppressed HRV16-stimulated IFN-α and IP-10 production, whereas neither bafilomycin or chloroquine inhibited HRV16-stimulated interleukin-6 release. Attempts to block IFN-α production with commercially available TLR-specific oligonucleotides were unsuccessful due to major ‘off-target’ effects. These findings suggest that among circulating haemopoietic cells, plasmacytoid dendritic cells and TLRs located within endosomes are critical for inducing efficient IFN-I production in response to HRVs.    

5.1737           Retargeting Oncolytic Vesicular Stomatitis Virus to Human T-Cell Lymphotropic Virus Type 1-Associated Adult T-Cell Leukemia

Betancourt, D., Ramos, J.C. and barber, G.N.

1. Virol., 89(23), 11786-11800 (2015)

  Adult T cell leukemia/lymphoma (ATL) is an aggressive cancer of CD4/CD25⁺ T lymphocytes, the etiological agent of which is human T-cell lymphotropic virus type 1 (HTLV-1). ATL is highly refractory to current therapies, making the development of new treatments a high priority. Oncolytic viruses such as vesicular stomatitis virus (VSV) are being considered as anticancer agents since they readily infect transformed cells compared to normal cells, the former appearing to exhibit defective innate immune responses. Here, we have evaluated the efficacy and safety of a recombinant VSV that has been retargeted to specifically infect and replicate in transformed CD4⁺ cells. This was achieved by replacing the single VSV glycoprotein (G) with human immunodeficiency virus type 1 (HIV-1) gp160 to create a hybrid fusion protein, gp160G. The resultant virus, VSV-gp160G, was found to only target cells expressing CD4 and retained robust oncolytic activity against HTLV-1 actuated ATL cells. VSV-gp160G was further noted to be highly attenuated and did not replicate efficiently in or induce significant cell death of primary CD4⁺ T cells. Accordingly, VSV-gp160G did not elicit any evidence of neurotoxicity even in severely immunocompromised animals such as NOD/Shi-scid, IL-2Rγ-c-null (NSG) mice. Importantly, VSV-gp160G effectively exerted potent oncolytic activity in patient-derived ATL transplanted into NSG mice and facilitated a significant survival benefit. Our data indicate that VSV-gp160G exerts potent oncolytic efficacy against CD4⁺ malignant cells and either alone or in conjunction with established therapies may provide an effective treatment in patients displaying ATL.  

5.1738           Response of Mammalian Macrophages to Challenge with the Chlorovirus Acanthocystis turfacea Chlorella Virus 1

Petro, T.M., Agarkova, I.V., Zhou, Y., Yolken, R.H., Van Etten, J.L. and Dunigan, D.D.

1. Virol., 89(23), 12096-12107 (2015)

  It was recently reported that 44% of the oropharyngeal samples from the healthy humans in a study cohort had DNA sequences similar to that of the chlorovirus ATCV-1 (Acanthocystis turfacea chlorella virus 1, family Phycodnaviridae) and that these study subjects had decreases in visual processing and visual motor speed compared with individuals in whom no virus was detected. Moreover, mice inoculated orally with ATCV-1 developed immune responses to ATCV-1 proteins and had decreases in certain cognitive domains. Because heightened interleukin-6 (IL-6), nitric oxide (NO), and ERK mitogen-activated protein (MAP) kinase activation from macrophages are linked to cognitive impairments, we evaluated cellular responses and viral PFU counts in murine RAW264.7 cells and primary macrophages after exposure to ATCV-1 in vitro for up to 72 h after a virus challenge. Approximately 8% of the ATCV-1 inoculum was associated with macrophages after 1 h, and the percentage increased 2- to 3-fold over 72 h. Immunoblot assays with rabbit anti-ATCV-1 antibody detected a 55-kDa protein consistent with the viral capsid protein from 1 to 72 h and increasing de novo synthesis of a previously unidentified 17-kDa protein beginning at 24 h. Emergence of the 17-kDa protein did not occur and persistence of the 55-kDa protein declined over time when cells were exposed to heat-inactivated ATCV-1. Moreover, starting at 24 h, RAW264.7 cells exhibited cytopathic effects, annexin V staining, and cleaved caspase 3. Activation of ERK MAP kinases occurred in these cells by 30 min postchallenge, which preceded the expression of IL-6 and NO. Therefore, ATCV-1 persistence in and induction of inflammatory factors by these macrophages may contribute to declines in the cognitive abilities of mice and humans.  

5.1739           Vpr Enhances Tumor Necrosis Factor Production by HIV-1-Infected T Cells

Roesch, F., Richard, L., Rua, R., Porrot, F., Casartelli, N. and Schwartz, O.

1. Virol., 89(23), 12118-12130 (2015)

  The HIV-1 accessory protein Vpr displays different activities potentially impacting viral replication, including the arrest of the cell cycle in the G₂ phase and the stimulation of apoptosis and DNA damage response pathways. Vpr also modulates cytokine production by infected cells, but this property remains partly characterized. Here, we investigated the effect of Vpr on the production of the proinflammatory cytokine tumor necrosis factor (TNF). We report that Vpr significantly increases TNF secretion by infected lymphocytes. De novo production of Vpr is required for this effect. Vpr mutants known to be defective for G₂ cell cycle arrest induce lower levels of TNF secretion, suggesting a link between these two functions. Silencing experiments and the use of chemical inhibitors further implicated the cellular proteins DDB1 and TAK1 in this activity of Vpr. TNF secreted by HIV-1-infected cells triggers NF-κB activity in bystander cells and allows viral reactivation in a model of latently infected cells. Thus, the stimulation of the proinflammatory pathway by Vpr may impact HIV-1 replication in vivo.  

5.1740           Determinants Involved in Hepatitis C Virus and GB Virus B Primate Host Restriction

Marnata, C. et al

1. Virol., 89(23), 12131-12144 (2015)

  Hepatitis C virus (HCV) only infects humans and chimpanzees, while GB virus B (GBV-B), another hepatotropic hepacivirus, infects small New World primates (tamarins and marmosets). In an effort to develop an immunocompetent small primate model for HCV infection to study HCV pathogenesis and vaccine approaches, we investigated the HCV life cycle step(s) that may be restricted in small primate hepatocytes. First, we found that replication-competent, genome-length chimeric HCV RNAs encoding GBV-B structural proteins in place of equivalent HCV sequences designed to allow entry into simian hepatocytes failed to induce viremia in tamarins following intrahepatic inoculation, nor did they lead to progeny virus in permissive, transfected human Huh7.5 hepatoma cells upon serial passage. This likely reflected the disruption of interactions between distantly related structural and nonstructural proteins that are essential for virion production, whereas such cross talk could be restored in similarly designed HCV intergenotypic recombinants via adaptive mutations in NS3 protease or helicase domains. Next, HCV entry into small primate hepatocytes was examined directly using HCV-pseudotyped retroviral particles (HCV-pp). HCV-pp efficiently infected tamarin hepatic cell lines and primary marmoset hepatocyte cultures through the use of the simian CD81 ortholog as a coreceptor, indicating that HCV entry is not restricted in small New World primate hepatocytes. Furthermore, we observed genomic replication and modest virus secretion following infection of primary marmoset hepatocyte cultures with a highly cell culture-adapted HCV strain. Thus, HCV can successfully complete its life cycle in primary simian hepatocytes, suggesting the possibility of adapting some HCV strains to small primate hosts.  

5.1741           Identification of an adeno-associated virus binding epitope for AVB sepharose affinity resin

Wang, Q., Lock, M., Prongay, A.J., Alvira, M.R., Petkov, B. and Wilson, J.M. Molecular Therapy-Methods & Clinical Development, 2:15040 (2015)   Recent successes of adeno-associated virus (AAV)–based gene therapy have created a demand for large-scale AAV vector manufacturing and purification techniques for use in clinical trials and beyond. During the development of purification protocols for rh.10, hu.37, AAV8, rh.64R1, AAV3B, and AAV9 vectors, based on a widely used affinity resin, AVB sepharose (GE), we found that, under the same conditions, different serotypes have different affinities to the resin, with AAV3B binding the best and AAV9 the poorest. Further analysis revealed a surface-exposed residue (amino acid number 665 in AAV8 VP1 numbering) differs between the high-affinity AAV serotypes (serine in AAV3B, rh.10, and hu.37) and the low-affinity ones (asparagine in AAV8, rh.64R1, and AAV9). The residue locates within a surface-exposed, variable epitope flanked by highly conserved residues. The substitution of the epitope in AAV8, rh.64R1, and AAV9 with the corresponding epitope of AAV3B (SPAKFA) resulted in greatly increased affinity to AVB sepharose with no reduction in the vectors’ in vitro potency. The presence of the newly identified AVB-binding epitope will be useful for affinity resin selection for the purification of novel AAV serotypes. It also suggests the possibility of vector engineering to yield a universal affinity chromatography purification method for multiple AAV serotypes.  

5.1742           Superinfection exclusion is absent during acute Junin virus infection of Vero and A549 cells

Gaudin, R. and Kirchhausen, T. Scientific Reports, 5, 15990 (2015)   Many viruses have evolved strategies of so-called “superinfection exclusion” to prevent re-infection of a cell that the same virus has already infected. Although Old World arenavirus infection results in down-regulation of its viral receptor and thus superinfection exclusion, whether New World arenaviruses have evolved such a mechanism remains unclear. Here we show that acute infection by the New World Junin virus (JUNV) failed to down-regulate the transferrin receptor and did not induce superinfection exclusion. We observed that Vero cells infected by a first round of JUNV (Candid1 strain) preserve an ability to , 5553-5563 (2015)developed a dual infection assay with the wild-type Candid1 JUNV and a recombinant JUNV-GFP virus to discriminate between first and second infections at the transcriptional and translational levels. We found that Vero and A549 cells already infected by JUNV were fully competent to transcribe viral RNA from a second round of infection. Furthermore, flow cytometry analysis of viral protein expression indicated that viral translation was normal, regardless of whether cells were previously infected or not. We conclude that in acutely infected cells, Junin virus lacks a superinfection exclusion mechanism.  

5.1743           Durable immunity to oncogenic human papillomaviruses elicited by adjuvanted recombinant Adeno-associated virus-like particle immunogen displaying L2 17–36 epitopes

Jagu, S., karanam, B., Wang, J.W., Zayed, H., Weghofer, M., Brendle, S.A., Balogh, K.K., Tossi, K.P., Roden, R.B.S. and Christensen, N.D. Vaccine, 33, 5553-5563 (2015)   Vaccination with the minor capsid protein L2, notably the 17–36 neutralizing epitope, induces broadly protective antibodies, although the neutralizing titers attained in serum are substantially lower than for the licensed L1 VLP vaccines. Here we examine the impact of other less reactogenic adjuvants upon the induction of durable neutralizing serum antibody responses and protective immunity after vaccination with HPV16 and HPV31 L2 amino acids 17–36 inserted at positions 587 and 453 of VP3, respectively, for surface display on Adeno-Associated Virus 2-like particles [AAVLP (HPV16/31L2)]. Mice were vaccinated three times subcutaneously with AAVLP (HPV16/31L2) at two week intervals at several doses either alone or formulated with alum, alum and MPL, RIBI adjuvant or Cervarix. The use of adjuvant with AAVLP (HPV16/31L2) was necessary in mice for the induction of L2-specific neutralizing antibody and protection against vaginal challenge with HPV16. While use of alum was sufficient to elicit durable protection (>3 months after the final immunization), antibody titers were increased by addition of MPL and RIBI adjuvants. To determine the breadth of immunity, rabbits were immunized three times with AAVLP (HPV16/31L2) either alone, formulated with alum ± MPL, or RIBI adjuvants, and after serum collection, the animals were concurrently challenged with HPV16/31/35/39/45/58/59 quasivirions or cottontail rabbit papillomavirus (CRPV) at 6 or 12 months post-immunization. Strong protection against all HPV types was observed at both 6 and 12 months post-immunization, including robust protection in rabbits receiving the vaccine without adjuvant. In summary, vaccination with AAVLP presenting HPV L2 17–36 epitopes at two sites on their surface induced cross-neutralizing serum antibody, immunity against HPV16 in the genital tract, and long-term protection against skin challenge with the 7 most common oncogenic HPV types when using a clinically relevant adjuvant.  

5.1744           Depletion of microglia and inhibition of exosome synthesis halt tau propagation

Asai, H., Ikezu, S., Tsunoda, S., Medalla, M., Luebke, J., Haydar, T., Wolozin, B., Butovsky, O., Kügler, S. and Ikezu, T. Nature Neurosci., 18(11), 1584-1594 (2015)   Accumulation of pathological tau protein is a major hallmark of Alzheimer's disease. Tau protein spreads from the entorhinal cortex to the hippocampal region early in the disease. Microglia, the primary phagocytes in the brain, are positively correlated with tau pathology, but their involvement in tau propagation is unknown. We developed an adeno-associated virus–based model exhibiting rapid tau propagation from the entorhinal cortex to the dentate gyrus in 4 weeks. We found that depleting microglia dramatically suppressed the propagation of tau and reduced excitability in the dentate gyrus in this mouse model. Moreover, we demonstrate that microglia spread tau via exosome secretion, and inhibiting exosome synthesis significantly reduced tau propagation in vitro and in vivo. These data suggest that microglia and exosomes contribute to the progression of tauopathy and that the exosome secretion pathway may be a therapeutic target.  

5.1745           Tau deposition drives neuropathological, inflammatory and behavioral abnormalities independently of neuronal loss in a novel mouse model

Cook, C. et al Hum. Mol. Genet., 24(21), 6198-6212 (2015)   Aberrant tau protein accumulation drives neurofibrillary tangle (NFT) formation in several neurodegenerative diseases. Currently, efforts to elucidate pathogenic mechanisms and assess the efficacy of therapeutic targets are limited by constraints of existing models of tauopathy. In order to generate a more versatile mouse model of tauopathy, somatic brain transgenesis was utilized to deliver adeno-associated virus serotype 1 (AAV1) encoding human mutant P301L-tau compared with GFP control. At 6 months of age, we observed widespread human tau expression with concomitant accumulation of hyperphosphorylated and abnormally folded proteinase K resistant tau. However, no overt neuronal loss was observed, though significant abnormalities were noted in the postsynaptic scaffolding protein PSD95. Neurofibrillary pathology was also detected with Gallyas silver stain and Thioflavin-S, and electron microscopy revealed the deposition of closely packed filaments. In addition to classic markers of tauopathy, significant neuroinflammation and extensive gliosis were detected in AAV1-Tau^P301L mice. This model also recapitulates the behavioral phenotype characteristic of mouse models of tauopathy, including abnormalities in exploration, anxiety, and learning and memory. These findings indicate that biochemical and neuropathological hallmarks of tauopathies are accurately conserved and are independent of cell death in this novel AAV-based model of tauopathy, which offers exceptional versatility and speed in comparison with existing transgenic models. Therefore, we anticipate this approach will facilitate the identification and validation of genetic modifiers of disease, as well as accelerate preclinical assessment of potential therapeutic targets.  

5.1746           SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef

Usami, Y., Wu, Y. and Göttlinger, H.G. Nature, 526, 218-223 (2015)   HIV-1 Nef and the unrelated mouse leukaemia virus glycosylated Gag (glycoGag) strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of both SERINC3 and SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivity. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4⁺ T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS.  

5.1747           Cardiac RKIP induces a beneficial β-adrenoceptor–dependent positive inotropy

Schmid, E. et al Nature Med., 21(11), 1298-1306 (2015)   In heart failure therapy, it is generally assumed that attempts to produce a long-term increase in cardiac contractile force are almost always accompanied by structural and functional damage. Here we show that modest overexpression of the Raf kinase inhibitor protein (RKIP), encoded by Pebp1 in mice, produces a well-tolerated, persistent increase in cardiac contractility that is mediated by the β₁-adrenoceptor (β₁AR). This result is unexpected, as β₁AR activation, a major driver of cardiac contractility, usually has long-term adverse effects. RKIP overexpression achieves this tolerance via simultaneous activation of the β₂AR subtype. Analogously, RKIP deficiency exaggerates pressure overload–induced cardiac failure. We find that RKIP expression is upregulated in mouse and human heart failure, indicative of an adaptive role for RKIP. Pebp1 gene transfer in a mouse model of heart failure has beneficial effects, suggesting a new therapeutic strategy for heart failure therapy.  

5.1748           Macrocyclic Envelope Glycoprotein Antagonists that Irreversibly Inactivate HIV-1 before Host Cell Encounter

Rashad, A.A., Sundaram, R.V.K., Aneja, R., Duffy, C. and Chaiken, I.

1. Med. Chem., 58(18), 7603-7608 (2015)

  We derived macrocyclic HIV-1 antagonists as a new class of peptidomimetic drug leads. Cyclic peptide triazoles (cPTs) retained the gp120 inhibitory and virus-inactivating signature of parent PTs, arguing that cyclization locked an active conformation. The six-residue cPT 9 (AAR029b) exhibited submicromolar antiviral potencies in inhibiting cell infection and triggering gp120 shedding that causes irreversible virion inactivation. Importantly, cPTs were stable to trypsin and chymotrypsin compared to substantial susceptibility of corresponding linear PTs.  

5.1749           Knockout of Lmod2 results in shorter thin filaments followed by dilated cardiomyopathy and juvenile lethality

Pappas, C.T., Mayfield, R.M., Henderson, C., Jamilpour, N., Cover, C., Hernandez, Z., Hutchinson, K.R., Chu, M., Nam, K-H., Valdez, J.M., Wong, P.K., granzier, H.L. and Gregorio, C.C. PNAS, 112(44), 13573-1z3578 (2015)   Leiomodin 2 (Lmod2) is an actin-binding protein that has been implicated in the regulation of striated muscle thin filament assembly; its physiological function has yet to be studied. We found that knockout of Lmod2 in mice results in abnormally short thin filaments in the heart. We also discovered that Lmod2 functions to elongate thin filaments by promoting actin assembly and dynamics at thin filament pointed ends. Lmod2-KO mice die as juveniles with hearts displaying contractile dysfunction and ventricular chamber enlargement consistent with dilated cardiomyopathy. Lmod2-null cardiomyocytes produce less contractile force than wild type when plated on micropillar arrays. Introduction of GFP-Lmod2 via adeno-associated viral transduction elongates thin filaments and rescues structural and functional defects observed in Lmod2-KO mice, extending their lifespan to adulthood. Thus, to our knowledge, Lmod2 is the first identified mammalian protein that functions to elongate actin filaments in the heart; it is essential for cardiac thin filaments to reach a mature length and is required for efficient contractile force and proper heart function during development.  

5.1750           Involvement of nucleophosmin (NPM1/B23) in assembly of infectious HPV16 capsids

Day, P.M., Thompson, C.D., Pang, Y.Y., Lowy, D.R. and Schiller, J.T. Papillomavirus Res., 1, 74-89 (2015)   We report that during assembly of HPV16 pseudovirus (PsV) the minor capsid protein, L2, interacts with the host nucleolar protein nucleophosmin (NPM1/B23). Exogenously-expressed L2 colocalized with NPM1, a complex containing both proteins, could be immunoprecipitated, and L2 could redirect to the nucleus NPM1 that was pharmacologically or genetically restricted to the cytoplasm. Coexpression of the major capsid protein, L1, prevented both the colocalization and the biochemical association, and L1 pentamers could displace L2 from L2/NPM1 complexes attached to a nuclear matrix. HPV16 PsV that was produced in a cell line with reduced NPM1 levels had significantly lower infectivity compared to PsV produced in the parental cell line, although the PsV preparations had comparable L1 and L2 ratios and levels of encapsidated DNA. The PsV produced in NPM1-deficient cells showed increased trypsin sensitivity and exhibited decreased L2 levels during endocytosis. These results suggest a critical role for NPM1 in establishing the correct interactions between L2 and L1 during HPV capsid assembly. A decrease in cellular levels of NPM1 results in the formation of seemingly normal, but unstable, capsids that result in a premature loss of L2, thus inhibiting successful infection. No role for NPM1 in HPV infectious entry was found.  

5.1751           A Novel Virus-Like Particle Based Vaccine Platform Displaying the Placental Malaria Antigen VAR2CSA

Thrane, S., Janitzek, C.M., Agerbæk, M.Ø., Ditlev, S.B., Resende, M., Nielsen, M.A., Theander, T.G., Salanti, A.and Sander, A.F. PloS One, 10(11), e0143071 (2015)   Placental malaria caused by Plasmodium falciparum is a major cause of mortality and severe morbidity. Clinical testing of a soluble protein-based vaccine containing the parasite ligand, VAR2CSA, has been initiated. VAR2CSA binds to the human receptor chondroitin sulphate A (CSA) and is responsible for sequestration of Plasmodium falciparum infected erythrocytes in the placenta. It is imperative that a vaccine against malaria in pregnancy, if administered to women before they become pregnant, can induce a strong and long lasting immune response. While most soluble protein-based vaccines have failed during clinical testing, virus-like particle (VLP) based vaccines (e.g., the licensed human papillomavirus vaccines) have demonstrated high efficacy, suggesting that the spatial assembly of the vaccine antigen is a critical parameter for inducing an optimal long-lasting protective immune response. We have developed a VLP vaccine display platform by identifying regions of the HPV16 L1 coat protein where a biotin acceptor site (AviTag^TM) can be inserted without compromising VLP-assembly. Subsequent biotinylation of Avi-L1 VLPs allow us to anchor monovalent streptavidin (mSA)-fused proteins to the biotin, thereby obtaining a dense and repetitive VLP-display of the vaccine antigen. The mSA-VAR2CSA antigen was delivered on the Avi-L1 VLP platform and tested in C57BL/6 mice in comparison to two soluble protein-based vaccines consisting of naked VAR2CSA and mSA-VAR2CSA. The mSA-VAR2CSA Avi-L1 VLP and soluble mSA-VAR2CSA vaccines induced higher antibody titers than the soluble naked VAR2CSA vaccine after three immunizations. The VAR2CSA Avi-L1 VLP vaccine induced statistically significantly higher endpoint titres compared to the soluble mSA-VAR2CSA vaccine, after 1^st and 2^nd immunization; however, this difference was not statistically significant after 3^rd immunization. Importantly, the VLP-VAR2CSA induced antibodies were functional in inhibiting the binding of parasites to CSA. This study demonstrates that the described Avi-L1 VLP-platform may serve as a versatile system for facilitating optimal VLP-display of large and complex vaccine antigens.  

5.1752           The SUMOylation Pathway Restricts Gene Transduction by Adeno-Associated Viruses

Hölscher, C., Sonntag, F., Henrich, K., Chen, Q., beneke, J., Matula, P., Rohr, K., Kaderali, L., Beil, N., Erfle, H., Kleinschmidt, J. and Müller, M. PloS Pathogens, 11(12), e1005281 (2015)   Adeno-associated viruses are members of the genus dependoviruses of the parvoviridae family. AAV vectors are considered promising vectors for gene therapy and genetic vaccination as they can be easily produced, are highly stable and non-pathogenic. Nevertheless, transduction of cells in vitro and in vivo by AAV in the absence of a helper virus is comparatively inefficient requiring high multiplicity of infection. Several bottlenecks for AAV transduction have previously been described, including release from endosomes, nuclear transport and conversion of the single stranded DNA into a double stranded molecule. We hypothesized that the bottlenecks in AAV transduction are, in part, due to the presence of host cell restriction factors acting directly or indirectly on the AAV-mediated gene transduction. In order to identify such factors we performed a whole genome siRNA screen which identified a number of putative genes interfering with AAV gene transduction. A number of factors, yielding the highest scores, were identified as members of the SUMOylation pathway. We identified Ubc9, the E2 conjugating enzyme as well as Sae1 and Sae2, enzymes responsible for activating E1, as factors involved in restricting AAV. The restriction effect, mediated by these factors, was validated and reproduced independently. Our data indicate that SUMOylation targets entry of AAV capsids and not downstream processes of uncoating, including DNA single strand conversion or DNA damage signaling. We suggest that transiently targeting SUMOylation will enhance application of AAV in vitro and in vivo.  

5.1753           Human immune system mice immunized with Plasmodium falciparum circumsporozoite protein induce protective human humoral immunity against malaria

Huang, J. et al

1. Immunol. Methods, 427, 42-50 (2015)

  In this study, we developed human immune system (HIS) mice that possess functional human CD4 + T cells and B cells, named HIS-CD4/B mice. HIS-CD4/B mice were generated by first introducing HLA class II genes, including DR1 and DR4, along with genes encoding various human cytokines and human B cell activation factor (BAFF) to NSG mice by adeno-associated virus serotype 9 (AAV9) vectors, followed by engrafting human hematopoietic stem cells (HSCs). HIS-CD4/B mice, in which the reconstitution of human CD4 + T and B cells resembles to that of humans, produced a significant level of human IgG against Plasmodium falciparum circumsporozoite (PfCS) protein upon immunization. CD4 + T cells in HIS-CD4/B mice, which possess central and effector memory phenotypes like those in humans, are functional, since PfCS protein-specific human CD4 + T cells secreting IFN-γ and IL-2 were detected in immunized HIS-CD4/B mice. Lastly, PfCS protein-immunized HIS-CD4/B mice were protected from in vivo challenge with transgenic P. berghei sporozoites expressing the PfCS protein. The immune sera collected from protected HIS-CD4/B mice reacted against transgenic P. berghei sporozoites expressing the PfCS protein and also inhibited the parasite invasion into hepatocytes in vitro. Taken together, these studies show that our HIS-CD4/B mice could mount protective human anti-malaria immunity, consisting of human IgG and human CD4 + T cell responses both specific for a human malaria antigen.  

5.1754           Differential myofiber-type transduction preference of adeno-associated virus serotypes 6 and 9

Riaz, M., Raz, Y., Moloney, E.B., van Putten, M., krom, Y.D., van der maarel, S.M., Verhaagen, J. and Raz, V. Skeletal Muscle, 5:37 (2015)   Background Gene therapy strategies are promising therapeutic options for monogenic muscular dystrophies, with several currently underways. The adeno-associated viral (AAV) vector is among the most effective gene delivery systems. However, transduction efficiency in skeletal muscles varies between AAV serotypes, with the underlying factors poorly understood. We hypothesized that myofiber-specific tropism differs between AAV serotypes. Methods We developed a quantitative histology procedure and generated myofiber pattern maps for four myosin heavy chain (MyHC) isotypes. We compared myofiber pattern maps between AAV6 or AAV9 injected tibialis anterior muscle in mice. We correlated MyHC expression with AAV-derived green fluorescence protein (GFP) expression using statistical models. Results We found that MyHC-2x expressing myofibers display a significantly higher preference for AAV transduction, whereas MyHC-2b expressing myofibers negatively correlated with AAV transduction. In addition, we show that AAV9-mediated transduction is enriched in myofibers expressing MyHC-1 and MyHC-1/2a. Moreover, AAV9-mediated transduction can predominantly be predicted by the expression of MyHC isotypes. In contrast, AAV6 transduction can be predicted by myofiber size but not by myofiber types. Conclusions Our findings identify differences between AAV6 and AAV9 for myofiber-type preferences, which could be an underlying factor for mosaic transduction of skeletal muscle. Adjusting AAV serotype for specific muscle conditions can therefore improve transduction efficacy in clinical applications.  

5.1755           Identification and characterization of human nucleus pulposus cell specific serotypes of adeno-associated virus for gene therapeutic approaches of intervertebral disc disorders

Meern, D.S. and Thome, C. BMC Musculosketal Disorders, 16:341 (2015)   Background Intervertebral disc (IVD) disorders are often accompanied by painful inflammatory and immunopathological processes. Nucleus pulposus (NP) cells play a pivotal role in maintenance of IVD by organizing the expression of anabolic, catabolic, anti-catabolic and inflammatory cytokines. Human NP cells have been targeted by gene therapeutic approaches using lentiviral or adenoviral systems that could be critical due to genome incorporation or immunological side effects. Adeno-associated viruses (AAVs), which do not express any viral gene and are not linked with any known disease in humans, are attractive gene delivery vectors. However, their lack of specific tissue tropism and preexisting immune response are main problems for therapeutic applications. Heretofore, AAVs have not been studied in human IVD research. Therefore, we attempted to identify NP cell specific AAV serotype by targeting human NP cells with different self-complementary AAV (scAAV) serotypes. Identification and characterization of the proper serotype is crucial to establish less immunogenic and safer gene therapeutic approaches of IVD disorders. Methods Preoperative magnetic resonance imaging (MRI) was used for grading of IVD degeneration. NP cells were isolated, cultured with low-glucose and transduced with green fluorescent protein (GFP) packing scAAV serotypes (scAAV1-8) in a dose-dependent manner. scAAV titers were determined by quantitative polymerase chain reaction (qPCR). Transduction efficiencies were determined by fluorescence microscopy and fluorescence-activated cell sorting within 48 days of post-transduction. The 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine NP cell viability. Three-dimensional (3D) cell culture and enzyme-linked immunosorbant assay (ELISA) were performed to examine the expression levels of inflammatory, catabolic and matrix proteins in NP cells. Results scAAV6, scAAV2 and scAAV3 showed high and prolonged transgene GFP expressions with transdution efficiencies of 98.6 %, 91.5 % and 89.6 % respectively (p ≤ 0.002). Unlike scAAV6, the serotypes scAAV2 and scAAV3 declined the viability of NP cells by about 25 % and 10 % respectively (p ≤ 0.001). Moreover, scAAV6 did not affect the expression of the inflammatory, catabolic and matrix proteins. Conclusions As original primary research evaluating AAVs in degenerative human IVDs, this study identified scAAV6 as a proper serotype for high, stable and non-immunogenic target gene expression in human NP cells. The data could be very important to design efficient and safer gene therapeutic approaches of IVD disorders.  

5.1756           Interneuronal DISC1 regulates NRG1-ErbB4 signalling and excitatory–inhibitory synapse formation in the mature cortex

Seshadri, s., Faust, T., Ishizuka, K., Delevich, K., Chung, Y., Kim, S-H., Cowles, M., Niwa, M., Jaaro-Peled, H., Tomoda, T., Lai, C., Anton, E.S., Li, B. and Sawa, A. Nature Communications, 6:10118 (2015)   Neuregulin-1 (NRG1) and its receptor ErbB4 influence several processes of neurodevelopment, but the mechanisms regulating this signalling in the mature brain are not well known. DISC1 is a multifunctional scaffold protein that mediates many cellular processes. Here we present a functional relationship between DISC1 and NRG1-ErbB4 signalling in mature cortical interneurons. By cell type-specific gene modulation in vitro and in vivo including in a mutant DISC1 mouse model, we demonstrate that DISC1 inhibits NRG1-induced ErbB4 activation and signalling. This effect is likely mediated by competitive inhibition of binding of ErbB4 to PSD95. Finally, we show that interneuronal DISC1 affects NRG1-ErbB4-mediated phenotypes in the fast spiking interneuron-pyramidal neuron circuit. Post-mortem brain analyses and some genetic studies have reported interneuronal deficits and involvement of the DISC1, NRG1 and ErbB4 genes in schizophrenia, respectively. Our results suggest a mechanism by which cross-talk between DISC1 and NRG1-ErbB4 signalling may contribute to these deficits.  

5.1757           Optogenetic acidification of synaptic vesicles and lysosomes

Rost, B.R., Schneider, F., Grauel, M.K., Wozny, C., Bentz, C., Blessing, A., Rosenmund, T., Jentsch, T.J., Schmitz, D., hegemann, P. and Rosenmund, C. Nature Neuroscience, 18(12), 1845-1852 (2015)   Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes.  

5.1758           Comparative Study of Liver Gene Transfer With AAV Vectors Based on Natural and Engineered AAV Capsids

Wang, L., Bell, P., Somanathan, S., Wang, Q., He, Z., Yu, H., McMenamin, D., Goode, ST., Calcedo, R and Wilson, J.M. Molecular Therapy, 23(12), 1877-1887 (2015)   Vectors based on the clade E family member adeno-associated virus (AAV) serotype 8 have shown promise in patients with hemophilia B and have emerged as best in class for human liver gene therapies. We conducted a thorough evaluation of liver-directed gene therapy using vectors based on several natural and engineered capsids including the clade E AAVrh10 and the largely uncharacterized and phylogenically distinct AAV3B. Included in this study was a putatively superior hepatotropic capsid, AAVLK03, which is very similar to AAV3B. Vectors based on these capsids were benchmarked against AAV8 and AAV2 in a number of in vitro and in vivo model systems including C57BL/6 mice, immune-deficient mice that are partially repopulated with human hepatocytes, and nonhuman primates. Our studies in nonhuman primates and human hepatocytes demonstrated high level transduction of the clade E-derived vectors and equally high transduction with vectors based on AAV3B. In contrast to previous reports, AAVLK03 vectors are not superior to either AAV3B or AAV8. Vectors based on AAV3B should be considered for liver-directed gene therapy when administered following, or before, treatment with the serologically distinct clade E vectors.  

5.1759           Systemic Vascular Transduction by Capsid Mutant Adeno-Associated Virus After Intravenous Injection

Lipinski, D.M., Reid, C.A., Boye, S.L., Peterson, J.J., Qi, X., Boye, S.E., Boulton, M.E. and Hauswirth, W.W. Human Gene Therapy, 26(11), 767-776 (2015)   The ability to effectively deliver genetic material to vascular endothelial cells remains one of the greatest unmet challenges facing the development of gene therapies to prevent diseases with underlying vascular etiology, such as diabetes, atherosclerosis, and age-related macular degeneration. Herein, we assess the effectiveness of an rAAV2-based capsid mutant vector (Y272F, Y444F, Y500F, Y730F, T491V; termed QuadYF+TV) with strong endothelial cell tropism at transducing the vasculature after systemic administration. Intravenous injection of QuadYF+TV resulted in widespread transduction throughout the vasculature of several major organ systems, as assessed by in vivo bioluminescence imaging and postmortem histology. Robust transduction of lung tissue was observed in QuadYF+TV-injected mice, indicating a role for intravenous gene delivery in the treatment of chronic diseases presenting with pulmonary complications, such as α₁-antitrypsin deficiency. The QuadYF+TV vector cross-reacted strongly with AAV2 neutralizing antibodies, however, indicating that a targeted delivery strategy may be required to maximize clinical translatability.  

5.1760           Re-Opening the Critical Window for Estrogen Therapy

Bean, L.A., Kumar, A., Rani, A., Guidi, M., Rosario, A.M., Cruz, P.E., Golde, T.E. and Foster, T.C.

1. Neuroscience, 35(49), 16077-16093 (2015)

  A decline in estradiol (E2)-mediated cognitive benefits denotes a critical window for the therapeutic effects of E2, but the mechanism for closing of the critical window is unknown. We hypothesized that upregulating the expression of estrogen receptor α (ERα) or estrogen receptor β (ERβ) in the hippocampus of aged animals would restore the therapeutic potential of E2 treatments and rejuvenate E2-induced hippocampal plasticity. Female rats (15 months) were ovariectomized, and, 14 weeks later, adeno-associated viral vectors were used to express ERα, ERβ, or green fluorescent protein (GFP) in the CA1 region of the dorsal hippocampus. Animals were subsequently treated for 5 weeks with cyclic injections of 17β-estradiol-3-benzoate (EB, 10 μg) or oil vehicle. Spatial memory was examined 48 h after EB/oil treatment. EB treatment in the GFP (GFP + EB) and ERβ (ERβ + EB) groups failed to improve episodic spatial memory relative to oil-treated animals, indicating closing of the critical window. Expression of ERβ failed to improve cognition and was associated with a modest learning impairment. Cognitive benefits were specific to animals expressing ERα that received EB treatment (ERα + EB), such that memory was improved relative to ERα + oil and GFP + EB. Similarly, ERα + EB animals exhibited enhanced NMDAR-mediated synaptic transmission compared with the ERα + oil and GFP + EB groups. This is the first demonstration that the window for E2-mediated benefits on cognition and hippocampal E2 responsiveness can be reinstated by increased expression of ERα.  

5.1761           AAV ancestral reconstruction library enables selection of broadly infectious viral variants

Santiago-Oritz, J., Ojala, D.S., Westesson, O., Weinstein, J.R., Wong, S.Y., Steinsapir, A., Kumar, S., Holmes, I. and Schaffer, D.V. Gene Therapy, 22(12), 934-946 (2015)   Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. However, enhanced vectors are required to extend these landmark successes to other indications and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties and to gain insights into AAV’s evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes and, in general, ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not use sialic acids, galactose or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19- to 31-fold higher gene expression in the muscle compared with AAV1, a clinically used serotype for muscle delivery, highlighting their promise for gene therapy.  

5.1762           Site-Directed Mutagenesis of Surface-Exposed Lysine Residues Leads to Improved Transduction by AAV2, But Not AAV8, Vectors in Murine Hepatocytes In Vivo

Li, B., Ma, W., Ling, C., Van Vliet, K., Huang, L-Y., Agbandje-Mckenna, M., Srivastava, A. and Aslanidi, G.V. Human Gene Therapy Methods, 26(6), 211-220 (2015)   The ubiquitin–proteasome pathway plays a critical role in the intracellular trafficking of recombinant adeno-associated virus 2 (AAV2) vectors, which negatively impacts the transduction efficiency of these vectors. Because ubiquitination occurs on lysine (K) residues, we performed site-directed mutagenesis where we replaced each of 10 surface-exposed K residues (K258, K490, K507, K527, K532, K544, K549, K556, K665, and K706) with glutamic acid (E) because of similarity of size and lack of recognition by modifying enzymes. The transduction efficiency of K490E, K544E, K549E, and K556E scAAV2 vectors increased in HeLa cells in vitro up to 5-fold compared with wild-type (WT) AAV2 vectors, with the K556E mutant being the most efficient. Intravenous delivery of WT and K-mutant ssAAV2 vectors further corroborated these results in murine hepatocytes in vivo. Because AAV8 vectors transduce murine hepatocytes exceedingly well, and because some of the surface-exposed K residues are conserved between these serotypes, we generated and tested two single mutants (K547E and K569E), and one double-mutant (K547 + 569E) AAV8 vector. However, no significant increase in the transduction efficiency of any of these mutant AAV8 vectors was observed in murine hepatocytes in vivo. These studies suggest that although targeting the surface-exposed K residues is yet another strategy to improve the transduction efficiency of AAV vectors, phenotypic outcome is serotype specific.  

5.1763           Disulfide Sensitivity in the Env Protein Underlies Lytic Inactivation of HIV-1 by Peptide Triazole Thiols

Bailey, L.D., Sundaram, R.V.K., Li, H., Duffy, C., Aneja, R., Bastian, A.R., Holmes, A.P., Kamanna, K., Rashad, A.A. and Chaiken, I. ACS Chem. Biol., 10(12), 2861-2873 (2015)   We investigated the mode of action underlying lytic inactivation of HIV-1 virions by peptide triazole thiol (PTT), in particular the relationship between gp120 disulfides and the C-terminal cysteine-SH required for virolysis. Obligate PTT dimer obtained by PTT SH cross-linking and PTTs with serially truncated linkers between pharmacophore isoleucine–ferrocenyltriazole-proline–tryptophan and cysteine-SH were synthesized. PTT variants showed loss of lytic activity but not binding and infection inhibition upon SH blockade. A disproportionate loss of lysis activity vs binding and infection inhibition was observed upon linker truncation. Molecular docking of PTT onto gp120 argued that, with sufficient linker length, the peptide SH could approach and disrupt several alternative gp120 disulfides. Inhibition of lysis by gp120 mAb 2G12, which binds at the base of the V3 loop, as well as disulfide mutational effects, argued that PTT-induced disruption of the gp120 disulfide cluster at the base of the V3 loop is an important step in lytic inactivation of HIV-1. Further, PTT-induced lysis was enhanced after treating virus with reducing agents dithiothreitol and tris (2-carboxyethyl)phosphine. Overall, the results are consistent with the view that the binding of PTT positions the peptide SH group to interfere with conserved disulfides clustered proximal to the CD4 binding site in gp120, leading to disulfide exchange in gp120 and possibly gp41, rearrangement of the Env spike, and ultimately disruption of the viral membrane. The dependence of lysis activity on thiol–disulfide interaction may be related to intrinsic disulfide exchange susceptibility in gp120 that has been reported previously to play a role in HIV-1 cell infection.  

5.1764           In vitro and in vivo comparability assessment of an AAVvector manufactured by triple transfection in HEK293 cells or in the baculovirus expression system

Steel, M., Woodburn, K., Kniffin, T., Gebretsadik, K., Chan, J., Vijay, S., Chen, H., Chalberg, T.W. and GAsmi, M. Human Gene Therapy, 26, A2-A28 (2015)   AVA-101 is a recombinant, replication-defective adeno-associated serotype 2 viral vector encoding the soluble form of the VEGF receptor type 1 that is currently being evaluated in clinical studies for the treatment for neovascular age-related macular degeneration. For a Ph1/2a study, AVA-101 was manufactured by triple transfection in HEK293 cells and subsequently purified by ion exchange chromatography and iodixanol ultracentrifugation. To improve scalability and cGMP suitability for late stage development, the upstream process was switched to the baculovirus expression system and the downstream purification to chromatography- based system compatible with biopharamaceutical industry manufacturing technologies. In addition, the formulation was modified to improve biocompatibility with the container closure system and infusion device. Vectors manufactured in both processes were compared using qualified analytical assays for titer (qPCR), infectivity (TCID50), transgene expression (transduction/ELISA), identity (Western blot) and purity (SDS/PAGE-Silverstain_). The ratio of empty-to-full vectors by TEM was also evaluated. Vectors biodistribution and potential toxicity effects upon subretinal administration in nonhuman primates were also assessed. Analytical results show that vectors produced by both manufacturing processes exhibit similar characteristics. When observed, differences were within assay variability. Both vectors were well tolerated with no differences observed in safety and biodistribution in animals. These results support the use of the new AVA- 101 manufacturing process for further clinical development.  

5.1765           Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors

Dias-Florencio, G., Precigout, G., Beley, C., Buclez, P., Garcia, L. and Benchaouir, R. Human Gene Therapy, 26, A2-A18 (2015)   Recombinant adeno-associated viruses (rAAV) are promising candidates for gene therapy approaches. Rapid technological evolution in the last two decades led to advances in processes applied in the production and purification of rAAV resulting in better yields and higher levels of vector purity. Recently, some reports showed that rAAV produced by transient tri-transfection method can be harvested directly from supernatant, leading to easier and faster purification compared to classical virus extraction from cell pellets. We compare these approaches with new vector recovery method using small quantity of detergent at the initial clarification step to treat the whole transfected cell culture. Coupled with tangential flow filtration and iodixanol-based isopycnic density gradient, this new method significantly increases rAAV yields and conserves high vector purity. Moreover, this approach leads to the reduction of the total process cost and duration. Finally, the vectors maintain their functionality, showing unexpected higher in vitro and in vivo transduction efficacies. This new development in rAAV downstream process once more demonstrates the great capacity of these vectors to easily accommodate to large panel of methods, able to furthermore ameliorate their safety, functionality and scalability. [Work accepted for publication in Molecular Therapy –Methods & Clinical Development, 2015 may 26].  

5.1766           Standardized large-scale H-1PV production process with efficient quality and quantity monitoring

Leuchs, B., Roscher, M., Müller, M., Kürschner, K. and Rommelaere, J.

1. Virol. Methods, 229, 48-59 (2016)

  The promising anticancer properties of rodent protoparvoviruses, notably H-1PV, have led to their clinical testing. This makes it necessary to produce highly pure, well-characterized virus batches in sufficient quantity. The present work focused on developing standardized production, purification, and characterization procedures as a basis for exploiting H-1PV both preclinically and in clinical trials for anticancer virotherapy. Two infection and two virus purification strategies were tested and the resulting virus preparations compared for their purity and full-, infectious-, and empty-particle contents. The adopted production process, which involves culturing and infecting NB-324K cells in 10-layer CellSTACK^® chambers (1 × 10³ infectious units per infected cell), is simple, scalable, and reproducible. Downstream processing to eliminate contaminating DNA and protein includes DNAse treatment, filtration, and two Iodixanol density-gradient centrifugations, the first gradient being a step gradient and the second, either a step (1 × 10¹⁰ PFU/ml) or a continuous gradient (3 × 10¹¹ PFU/ml). A procedure was also developed for obtaining infectious particle-free preparations of empty virions for research purposes: cesium chloride density gradient centrifugation followed by UV irradiation (1 × 10¹⁴ physical particles/ml). For quick, sensitive determination of physical particles (and hence, particle-to-infectivity ratios), a “Capsid-ELISA” was developed, based on a novel monoclonal antibody that specifically targets assembled capsids.  

5.1767           Evaluation of the maturation of individual Dengue virions with flow virometry

Zicari, S., Arakelyan, A., Fitzgerald, W., Zaitseva, e., Chernomordik, L.V., Margolis, L. and Grivel, J-C. Virology, 488, 20-27 (2016)   High-throughput techniques are needed to analyze individual virions to understand how viral heterogeneity translates into pathogenesis since in bulk analysis the individual characteristics of virions are lost. Individual Dengue virions (DENV) undergo a maturation that involves a proteolytic cleavage of prM precursor into virion-associated M protein. Here, using a new nanoparticle-based technology, “flow virometry”, we compared the maturation of individual DENV produced by BHK-21 and LoVo cells. The latter lacks the furin-protease that mediates prM cleavage. We found that prM is present on about 50% of DENV particles produced in BHK-21 cells and about 85% of DENV virions produced in LoVo, indicating an increase in the fraction of not fully matured virions. Flow virometry allows us to quantify the number of fully mature particles in DENV preparations and proves to be a useful method for studying heterogeneity of the surface proteins of various viruses.  

5.1768           Detection of treatment-resistant infectious HIV after genome-directed antiviral endonuclease therapy

De Silva Feelixge, H.S., Stone, D., Pietz, H.L., Roychoudhury, P., Greninger, A.L., Schiffer, J.T., Aubert, M., Jerome, K.R. Antiviral Res., 126, 90-98 (2016)   Incurable chronic viral infections are a major cause of morbidity and mortality worldwide. One potential approach to cure persistent viral infections is via the use of targeted endonucleases. Nevertheless, a potential concern for endonuclease-based antiviral therapies is the emergence of treatment resistance. Here we detect for the first time an endonuclease-resistant infectious virus that is found with high frequency after antiviral endonuclease therapy. While testing the activity of HIV pol-specific zinc finger nucleases (ZFNs) alone or in combination with three prime repair exonuclease 2 (Trex2), we identified a treatment-resistant and infectious mutant virus that was derived from a ZFN-mediated disruption of reverse transcriptase (RT). Although gene disruption of HIV protease, RT and integrase could inhibit viral replication, a chance single amino acid insertion within the thumb domain of RT produced a virus that could actively replicate. The endonuclease-resistant virus could replicate in primary CD4⁺ T cells, but remained susceptible to treatment with antiretroviral RT inhibitors. When secondary ZFN-derived mutations were introduced into the mutant virus's RT or integrase domains, replication could be abolished. Our observations suggest that caution should be exercised during endonuclease-based antiviral therapies; however, combination endonuclease therapies may prevent the emergence of resistance.  

5.1769           Transient Bluetongue virus serotype 8 capsid protein expression in Nicotiana benthamiana

Van Zyl, A.R., Meyers, A.E. and Rybicki, E.P. Biotechnol. Reports, 9, 15-24 (2016)   Bluetongue virus (BTV) causes severe disease in domestic and wild ruminants, and has recently caused several outbreaks in Europe. Current vaccines include live-attenuated and inactivated viruses; while these are effective, there is risk of reversion to virulence by mutation or reassortment with wild type viruses. Subunit or virus-like particle (VLP) vaccines are safer options: VLP vaccines produced in insect cells by expression of the four BTV capsid proteins are protective against challenge; however, this is a costly production method. We investigated production of BTV VLPs in plants via Agrobacterium-mediated transient expression, an inexpensive production system very well suited to developing country use. Leaves infiltrated with recombinant pEAQ-HT vectors separately encoding the four BTV-8 capsid proteins produced more proteins than recombinant pTRA vectors. Plant expression using the pEAQ-HT vector resulted in both BTV-8 core-like particles (CLPs) and VLPs; differentially controlling the concentration of infiltrated bacteria significantly influenced yield of the VLPs. In situ localisation of assembled particles was investigated by using transmission electron microscopy (TEM) and it was shown that a mixed population of core-like particles (CLPs, consisting of VP3 and VP7) and VLPs were present as paracrystalline arrays in the cytoplasm of plant cells co-expressing all four capsid proteins.  

5.1770           Obesity, diabetes, and leptin resistance promote tau pathology in a mouse model of disease

Platt, T.L., Beckett, TS.L., Kohler, K., Niedowicz, D.M. and Murphym M.P. Neuroscience, 315, 162-174 (2016)   Obesity and type 2 diabetes mellitus (T2DM) convey an increased risk for developing dementia. The microtubule-associated protein tau is implicated in neurodegenerative disease by undergoing hyperphosphorylation and aggregation, leading to cytotoxicity and neurodegeneration. Enzymes involved in the regulation of tau phosphorylation, such as GSK3β, are tightly associated with pathways found to be dysregulated in T2DM. We have shown previously that leptin-resistant mice, which develop obesity and a diabetic phenotype, display elevated levels of tau phosphorylation. Here we show cells cultured with leptin, an adipokine shown to have neuroprotective effects, reduces tau phosphorylation. To explore how this mechanism works in vivo we transduced an existing diabetic mouse line (Lepr^db/db) with a tau mutant (tau^P301L) via adeno-associated virus (AAV). The resulting phenotype included a striking increase in tau phosphorylation and the number of neurofibrillary tangles (NFTs) found within the hippocampus. We conclude that leptin resistance-induced obesity and diabetes accelerates the development of tau pathology. This model of metabolic dysfunction and tauopathy provides a new system in which to explore the mechanisms underlying the ways in which leptin resistance and diabetes influence development of tau pathology, and may ultimately be related to the development of NFTs.  

5.1771           Budded baculovirus particle structure revisited

Wang, Q., Bossch, B-J., Vlak, J.M., van Oers, M-M., Rottier, P.J. and van Lent, J.W.M.

1. Invertebrate Pathol., 134, 15-22 (2016)

  Baculoviruses are a group of enveloped, double-stranded DNA insect viruses with budded (BV) and occlusion-derived (ODV) virions produced during their infection cycle. BVs are commonly described as rod shaped particles with a high apical density of protein extensions (spikes) on the lipid envelope surface. However, due to the fragility of BVs the conventional purification and electron microscopy (EM) staining methods considerably distort the native viral structure. Here, we use cryo-EM analysis to reveal the near-native morphology of two intensively studied baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), as models for BVs carrying GP64 and F as envelope fusion protein on the surface. The now well-preserved AcMNPV and SeMNPV BV particles have a remarkable elongated, ovoid shape leaving a large, lateral space between nucleocapsid (NC) and envelope. Consistent with previous findings the NC has a distinctive cap and base structure interacting tightly with the envelope. This tight interaction may explain the partial retaining of the envelope on both ends of the NC and the disappearance of the remainder of the BV envelope in the negative-staining EM images. Cryo-EM also reveals that the viral envelope contains two layers with a total thickness of ≈6–7 nm, which is significantly thicker than a usual biological membrane (<4 nm) as measured by X-ray scanning. Most spikes are densely clustered at the two apical ends of the virion although some envelope proteins are also found more sparsely on the lateral regions. The spikes on the surface of AcMNPV BVs appear distinctly different from those of SeMNPV. Based on our observations we propose a new near-native structural model of baculovirus BVs.  

5.1772           Manufacturing of recombinant adeno-associated viral vectors: new technologies are welcome

Ayuso, E. Molecular Therapy – Methods& Clinical development, 3:15049 (2016)   Recombinant adeno-associated viral vectors (rAAV) are probably the most powerful tools for in vivo gene delivery. Encouraging preclinical data have been followed by successful gene therapy clinical trials including Leber’s congenital amaurosis type 2 (refs. 1,​2,​3), hemophilia B,4,5 and recently choroideremia.6 These results together with the market authorization of Glybera, an AAV-based product for the treatment of lipoprotein lipase deficiency,7,8 has prompted skeptical investors and biotechnology and pharmaceuticals companies to move into this field. Nonetheless, a major bottleneck to translate these new approaches into the clinic is the manufacturing of rAAV in accordance with current good manufacturing practices (cGMP), requiring costly and timely inefficient protocols. The development of a cGMP-compatible process can be tedious depending on the AAV serotype, vector transgene, and total dose required to launch a phase 1 clinical trial. A recent study by Grieger et al.9 published in Molecular Therapy addresses such challenges for large scale manufacturing of rAAV, providing a flexible protocol based on triple transfection of HEK293 cells in suspension and a purification process that combines ultracentrifugation and ion exchange chromatography. This protocol was validated for multiple serotypes (AAV 1–6, 8, and 9), carrying either single stranded or self-complementary vector genomes with postpurification yields of >1013 vector genomes per liter of culture and a purity suitable for clinical use.  

5.1773           Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A

Zayas, M., Long, G., Madan, V. and Bartenschlager, R. PloS Pathogens, 12(1), e1005376 (2016)   Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles.  

5.1774           Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system

Barroso-Chinea, P., Cruz-Muros, I., Afonso-Oramas, D., Castro-Hernandez, J., Salas-Hernandez, J., Chtarto, A., Luis-Ravelo, D., Humbert-Claude, M., Tenenbaum, L. and Gonzalez-Hernandez, T. Neurobiology of Disease, 88, 44-54 (2016)   The dopamine (DA) transporter (DAT) is a plasma membrane glycoprotein expressed in dopaminergic (DA-) cells that takes back DA into presynaptic neurons after its release. DAT dysfunction has been involved in different neuro-psychiatric disorders including Parkinson's disease (PD). On the other hand, numerous studies support that the glial cell line-derived neurotrophic factor (GDNF) has a protective effect on DA-cells. However, studies in rodents show that prolonged GDNF over-expression may cause a tyrosine hydroxylase (TH, the limiting enzyme in DA synthesis) decline. The evidence of TH down-regulation suggests that another player in DA handling, DAT, may also be regulated by prolonged GDNF over-expression, and the possibility that this effect is induced at GDNF expression levels lower than those inducing TH down-regulation. This issue was investigated here using intrastriatal injections of a tetracycline-inducible adeno-associated viral vector expressing human GDNF cDNA (AAV-tetON-GDNF) in rats, and doxycycline (DOX; 0.01, 0.03, 0.5 and 3 mg/ml) in the drinking water during 5 weeks. We found that 3 mg/ml DOX promotes an increase in striatal GDNF expression of 12 × basal GDNF levels and both DA uptake decrease and TH down-regulation in its native and Ser40 phosphorylated forms. However, 0.5 mg/ml DOX promotes a GDNF expression increase of 3 × basal GDNF levels with DA uptake decrease but not TH down-regulation. The use of western-blot under non-reducing conditions, co-immunoprecipitation and in situ proximity ligation assay revealed that the DA uptake decrease is associated with the formation of DAT dimers and an increase in DAT–α-synuclein interactions, without changes in total DAT levels or its compartmental distribution. In conclusion, at appropriate GDNF transduction levels, DA uptake is regulated through DAT protein–protein interactions without interfering with DA synthesis.  

5.1775           Remote and reversible inhibition of neurons and circuits by small molecule induced potassium channel stabilization

Auffenberg, E., Jurik, A., mattusch, C., Stoffel, R., genewsky, A., Namendorf, C., Schmid, R.M., Rammes, G., Biel, M., Uhr, M., Moosmang, S., Michalakis, S., Wotjak, C.T. and Thoeringer, C.K. Scientific Reports, 6:19293 (2016)   Manipulating the function of neurons and circuits that translate electrical and chemical signals into behavior represents a major challenges in neuroscience. In addition to optogenetic methods using light-activatable channels, pharmacogenetic methods with ligand induced modulation of cell signaling and excitability have been developed. However, they are largely based on ectopic expression of exogenous or chimera proteins. Now, we describe the remote and reversible expression of a Kir2.1 type potassium channel using the chemogenetic technique of small molecule induced protein stabilization. Based on shield1-mediated shedding of a destabilizing domain fused to a protein of interest and inhibition of protein degradation, this principle has been adopted for biomedicine, but not in neuroscience so far. Here, we apply this chemogenetic approach in brain research for the first time in order to control a potassium channel in a remote and reversible manner. We could show that shield1-mediated ectopic Kir2.1 stabilization induces neuronal silencing in vitro and in vivo in the mouse brain. We also validated this novel pharmacogenetic method in different neurobehavioral paradigms.The DD-Kir2.1 may complement the existing portfolio of pharmaco- and optogenetic techniques for specific neuron manipulation, but it may also provide an example for future applications of this principle in neuroscience research.  

5.1776           Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation

Guo, Y., Wang, W., Sun, Y., Ma, C., Wang, X., Wang, X., Liu, P., Shen, S., Li, B., Lin, J., Deng, F., Wang, H. and Lou, Z.

1. Virol., 90(2), 1048-1061 (2016)

  Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NP_core) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NP_core exclude the N- and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NP_core are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses.  

5.1777           A Cell-Free Assembly System for Generating Infectious Human Papillomavirus 16 Capsids Implicates a Size Discrimination Mechanism for Preferential Viral Genome Packaging

Cerquira, C., Pang, Y-Y.S., Day, P.M., Thompson, C.D., Buck, C.B., Lowy, D.R. and Schiller, J.T.

1. Virol., 90(2), 1096-1107 (2016)

  We have established a cell-free in vitro system to study human papillomavirus type 16 (HPV16) assembly, a poorly understood process. L1/L2 capsomers, obtained from the disassembly of virus-like particles (VLPs), were incubated with nuclear extracts to provide access to the range of cellular proteins that would be available during assembly within the host cell. Incorporation of a reporter plasmid “pseudogenome” was dependent on the presence of both nuclear extract and ATP. Unexpectedly, L1/L2 VLPs that were not disassembled prior to incubation with a reassembly mixture containing nuclear extract also encapsidated a reporter plasmid. As with HPV pseudoviruses (PsV) generated intracellularly, infection by cell-free particles assembled in vitro required the presence of L2 and was susceptible to the same biochemical inhibitors, implying the cell-free assembled particles use the infectious pathway previously described for HPV16 produced in cell culture. Using biochemical and electron microscopy analyses, we observed that, in the presence of nuclear extract, intact VLPs partially disassemble, providing a mechanistic explanation to how the exogenous plasmid was packaged by these particles. Further, we provide evidence that capsids containing an <8-kb pseudogenome are resistant to the disassembly/reassembly reaction. Our results suggest a novel size discrimination mechanism for papillomavirus genome packaging in which particles undergo iterative rounds of disassembly/reassembly, seemingly sampling DNA until a suitably sized DNA is encountered, resulting in the formation of a stable virion structure.  

5.1778           Glutathione peroxidase 4 is reversibly induced by HCV to control lipid peroxidation and to increase virion infectivity

Brault, C., Levy, P., Duponchel, S., Michelet, M., Salle, A., Pecheur, E-I., Plissonnier, M-L., Parent, r., Vericel, E., Ivanov, A.V., Demir, M., Steffen, H-M., Odenthal, M., Zoulim, F. and Bartosh, B. Gut, 65, 144.154 (2016)   Objective Inflammation and oxidative stress drive disease progression in chronic hepatitis C (CHC) towards hepatocellular carcinoma. HCV is known to increase intracellular levels of reactive oxygen species (ROS), but how it eliminates ROS is less well known. The role of the ROS scavenger glutathione peroxidase 4 (GPx4), induced by HCV, in the viral life cycle was analysed. Design The study was performed using a replicative in vitro HCV infection model and liver biopsies derived from two different CHC patient cohorts. Results A screen for HCV-induced peroxide scavengers identified GPx4 as a host factor required for HCV infection. The physiological role of GPx4 is the elimination of lipid peroxides from membranes or lipoproteins. GPx4-silencing reduced the specific infectivity of HCV by up to 10-fold. Loss of infectivity correlated with 70% reduced fusogenic activity of virions in liposome fusion assays. NS5A was identified as the protein that mediates GPx4 induction in a phosphatidylinositol-3-kinase-dependent manner. Levels of GPx4 mRNA were found increased in vitro and in CHC compared with control liver biopsies. Upon successful viral eradication, GPx4 transcript levels returned to baseline in vitro and also in the liver of patients. Conclusions HCV induces oxidative stress but controls it tightly by inducing ROS scavengers. Among these, GPx4 plays an essential role in the HCV life cycle. Modulating oxidative stress in CHC by specifically targeting GPx4 may lower specific infectivity of virions and prevent hepatocarcinogenesis, especially in patients who remain difficult to be treated in the new era of interferon-free regimens.  

5.1779           Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies

Fauvelle, C., Felmlee, D.J. et al Gastroenterology, 150(1), 206-217 (2016)   Background & Aims Efforts to develop an effective vaccine against hepatitis C virus (HCV) have been hindered by the propensity of the virus to evade host immune responses. HCV particles in serum and in cell culture associate with lipoproteins, which contribute to viral entry. Lipoprotein association has also been proposed to mediate viral evasion of the humoral immune response, though the mechanisms are poorly defined. Methods We used small interfering RNAs to reduce levels of apolipoprotein E (apoE) in cell culture−derived HCV−producing Huh7.5-derived hepatoma cells and confirmed its depletion by immunoblot analyses of purified viral particles. Before infection of naïve hepatoma cells, we exposed cell culture−derived HCV strains of different genotypes, subtypes, and variants to serum and polyclonal and monoclonal antibodies isolated from patients with chronic HCV infection. We analyzed the interaction of apoE with viral envelope glycoprotein E2 and HCV virions by immunoprecipitation. Results Through loss-of-function studies on patient-derived HCV variants of several genotypes and subtypes, we found that the HCV particle apoE allows the virus to avoid neutralization by patient-derived antibodies. Functional studies with human monoclonal antiviral antibodies showed that conformational epitopes of envelope glycoprotein E2 domains B and C were exposed after depletion of apoE. The level and conformation of virion-associated apoE affected the ability of the virus to escape neutralization by antibodies. Conclusions In cell-infection studies, we found that HCV-associated apoE helps the virus avoid neutralization by antibodies against HCV isolated from chronically infected patients. This method of immune evasion poses a challenge for the development of HCV vaccines.  

5.1780           A targeted functional RNA interference screen uncovers glypican 5 as an entry factor for hepatitis B and D viruses

Verrier, E.R. et al Hepatology, 63(1), 35-48 (2016)   Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcinoma worldwide. Efficient therapeutic approaches for cure are absent. Sharing the same envelope proteins, hepatitis B virus and hepatitis delta virus use the sodium/taurocholate cotransporting polypeptide (a bile acid transporter) as a receptor to enter hepatocytes. However, the detailed mechanisms of the viral entry process are still poorly understood. Here, we established a high-throughput infectious cell culture model enabling functional genomics of hepatitis delta virus entry and infection. Using a targeted RNA interference entry screen, we identified glypican 5 as a common host cell entry factor for hepatitis B and delta viruses. Conclusion: These findings advance our understanding of virus cell entry and open new avenues for curative therapies. As glypicans have been shown to play a role in the control of cell division and growth regulation, virus–glypican 5 interactions may also play a role in the pathogenesis of virus-induced liver disease and cancer.  

5.1781           Human papillomavirus capsids preferentially bind and infect tumor cells

Kines, R.C., Cerio, R.J., Roberts, J.N., Thompson, C.D., de Los Pinos, E., Lowy, D.R. and Schiller, J.T. Int. J. Cancer, 138(4), 901-911 (2016)   We previously determined that human papillomavirus (HPV) virus-like particles (VLPs) and pseudovirions (PsV) did not, respectively, bind to or infect intact epithelium of the cervicovaginal tract. However, they strongly bound heparan sulfate proteoglycans (HSPG) on the basement membrane of disrupted epithelium and infected the keratinocytes that subsequently entered the disrupted site. We here report that HPV capsids (VLP and PsV) have the same restricted tropism for a wide variety of disrupted epithelial and mesothelial tissues, whereas intact tissues remain resistant to binding. However, the HPV capsids directly bind and infect most tumor-derived cell lines in vitro and have analogous tumor-specific properties in vivo, after local or intravenous injection, using orthotopic models for human ovarian and lung cancer, respectively. The pseudovirions also specifically infected implanted primary human ovarian tumors. Heparin and ι-carrageenan blocked binding and infection of all tumor lines tested, implying that tumor cell binding is HSPG-dependent. A survey using a panel of modified heparins indicates that N-sulfation and, to a lesser degree, O-6 sulfation of the surface HSPG on the tumors are important for HPV binding. Therefore, it appears that tumor cells consistently evolve HSPG modification patterns that mimic the pattern normally found on the basement membrane but not on the apical surfaces of normal epithelial or mesothelial cells. Consequently, appropriately modified HPV VLPs and/or PsV could be useful reagents to detect and potentially treat a remarkably broad spectrum of cancers.    

5.1782           In Vivo Evidence for a Lactate Gradient from Astrocytes to Neurons

Mächler, P. et al Cell Metabolism, 23, 94-102 (2016)   Investigating lactate dynamics in brain tissue is challenging, partly because in vivo data at cellular resolution are not available. We monitored lactate in cortical astrocytes and neurons of mice using the genetically encoded FRET sensor Laconic in combination with two-photon microscopy. An intravenous lactate injection rapidly increased the Laconic signal in both astrocytes and neurons, demonstrating high lactate permeability across tissue. The signal increase was significantly smaller in astrocytes, pointing to higher basal lactate levels in these cells, confirmed by a one-point calibration protocol. Trans-acceleration of the monocarboxylate transporter with pyruvate was able to reduce intracellular lactate in astrocytes but not in neurons. Collectively, these data provide in vivo evidence for a lactate gradient from astrocytes to neurons. This gradient is a prerequisite for a carrier-mediated lactate flux from astrocytes to neurons and thus supports the astrocyte-neuron lactate shuttle model, in which astrocyte-derived lactate acts as an energy substrate for neurons.  

5.1783           Viral gene transfer of APPsα rescues synaptic failure in an Alzheimer’s disease mouse model

Fol, R., Braudeau, J., Ledewig, S., Abel, T., Weyer, S.W., Poederer, J-P., Brod, F., Audrain, M., Bemelsmann, A-P., Buchholz, C.J., Korte, M., cartier, N. and Müller, U.C. Acta Neuropathol., 131(2), 247-266 (2016)   Alzheimer’s disease (AD) is characterized by synaptic failure, dendritic and axonal atrophy, neuronal death and progressive loss of cognitive functions. It is commonly assumed that these deficits arise due to β-amyloid accumulation and plaque deposition. However, increasing evidence indicates that loss of physiological APP functions mediated predominantly by neurotrophic APPsα produced in the non-amyloidogenic α-secretase pathway may contribute to AD pathogenesis. Upregulation of APPsα production via induction of α-secretase might, however, be problematic as this may also affect substrates implicated in tumorigenesis. Here, we used a gene therapy approach to directly overexpress APPsα in the brain using AAV-mediated gene transfer and explored its potential to rescue structural, electrophysiological and behavioral deficits in APP/PS1∆E9 AD model mice. Sustained APPsα overexpression in aged mice with already preexisting pathology and amyloidosis restored synaptic plasticity and partially rescued spine density deficits. Importantly, AAV-APPsα treatment also resulted in a functional rescue of spatial reference memory in the Morris water maze. Moreover, we demonstrate a significant reduction of soluble Aβ species and plaque load. In addition, APPsα induced the recruitment of microglia with a ramified morphology into the vicinity of plaques and upregulated IDE and TREM2 expression suggesting enhanced plaque clearance. Collectively, these data indicate that APPsα can mitigate synaptic and cognitive deficits, despite established pathology. Increasing APPsα may therefore be of therapeutic relevance for AD.  

5.1784           p38γ and δ promote heart hypertrophy by targeting the mTOR-inhibitory protein DEPTOR for degradation

Gonzalez-Teran, B., Lopez, J.A., Rodriguez, E., Leiva, L., Martinez-martinez, S., Bernal, J.A., Jimenez-Borreguero, L.J., Redondo, J.M., Vazquez, J. and Sabio, G. Nature Communications, 7:10477 (2016)   Disrupted organ growth leads to disease development. Hypertrophy underlies postnatal heart growth and is triggered after stress, but the molecular mechanisms involved in these processes are largely unknown. Here we show that cardiac activation of p38γ and p38δ increases during postnatal development and by hypertrophy-inducing stimuli. p38γ/δ promote cardiac hypertrophy by phosphorylating the mTORC1 and mTORC2 inhibitor DEPTOR, which leads to its degradation and mTOR activation. Hearts from mice lacking one or both kinases are below normal size, have high levels of DEPTOR, low activity of the mTOR pathway and reduced protein synthesis. The phenotype of p38γ/δ^−/− mice is reverted by overactivation of mTOR with amino acids, shRNA-mediated knockdown of Deptor, or cardiomyocyte overexpression of active p38γ and p38δ. Moreover, in WT mice, heart weight is reduced by cardiac overexpression of DEPTOR. Our results demonstrate that p38γ/δ control heart growth by modulating mTOR pathway through DEPTOR phosphorylation and subsequent degradation.    

5.1785           Distinct cognitive effects and underlying transcriptome changes upon inhibition of individual

miRNAs in hippocampal neuron

Malmevik, J., Petri, R., Knauff, P., Brattås, P.L., Åkerblom, M. and Jakobsson, J. Scientific Reports, 6:19879 (2016)   MicroRNAs (miRNA) are small, non-coding RNAs mediating post-transcriptional regulation of gene expression. miRNAs have recently been implicated in hippocampus-dependent functions such as learning and memory, although the roles of individual miRNAs in these processes remain largely unknown. Here, we achieved stable inhibition using AAV-delivered miRNA sponges of individual, highly expressed and brain-enriched miRNAs; miR-124, miR-9 and miR-34, in hippocampal neurons. Molecular and cognitive studies revealed a role for miR-124 in learning and memory. Inhibition of miR-124 resulted in an enhanced spatial learning and working memory capacity, potentially through altered levels of genes linked to synaptic plasticity and neuronal transmission. In contrast, inhibition of miR-9 or miR-34 led to a decreased capacity of spatial learning and of reference memory, respectively. On a molecular level, miR-9 inhibition resulted in altered expression of genes related to cell adhesion, endocytosis and cell death, while miR-34 inhibition caused transcriptome changes linked to neuroactive ligand-receptor transduction and cell communication. In summary, this study establishes distinct roles for individual miRNAs in hippocampal function.  

5.1786           Production of Virus-Like Particles for Vaccination

Thompson, C.M., Aucoin, M.G. and Kamen, A.A. Methods in Mol. Biol., 1350, 299-315 (2016)   The ability to make a large variety of virus-like particles (VLPs) has been successfully achieved in the baculovirus expression vector system (BEVS)/insect cell system. The production and scale-up of these particles, which are mostly sought as vaccine candidates, are currently being addressed. Furthermore, these VLPs are being investigated as delivery agents for use as therapeutics. The use of host insect cells allows mass production of VLPs in a proven scalable system.  

5.1787           Preferred transduction with AAV8 and AAV9 via thalamic administration in the MPS IIIB model: A comparison of four rAAV serotypes

Gilkes, J.A., Bloom, M.D. and Heldermon, C.D. Molecular Genetics and Metabolism Reports, 6, 48-54 (2016)   Sanfilippo syndrome type B (MPS IIIB) is a lysosomal storage disease caused by a deficiency of N-acetyl-glucosaminidase (NAGLU) activity. Since early therapeutic intervention is likely to yield the most efficacious results, we sought to determine the possible therapeutic utility of rAAV in early gene therapy based interventions. Currently, the application of recombinant adeno-associated virus (AAV) vectors is one of the most widely used gene transfer systems, and represents a promising approach in the treatment of MPS IIIB. From a translational standpoint, a minimally invasive, yet highly efficient method of vector administration is ideal. The thalamus is thought to be the switchboard for signal relay in the central nervous system (CNS) and therefore represents an attractive target. To identify an optimal AAV vector for early therapeutic intervention, and establish whether thalamic administration represents a feasible therapeutic approach, we performed a comprehensive assessment of transduction and biodistribution profiles of four green fluorescent protein (GFP) bearing rAAV serotypes, -5, -8, -9 and -rh10, administered bilaterally into the thalamus. Of the four serotypes compared, AAV8 and -9 proved superior to AAV5 and -rh10 both in biodistribution and transduction efficiency profiles. Genotype differences in transduction efficiency and biodistribution patterns were also observed. Importantly, we conclude that AAV8 and to a lesser extent, AAV9 represent preferable candidates for early gene therapy based intervention in the treatment of MPS IIIB. We also highlight the feasibility of thalamic rAAV administration, and conclude that this method results in moderate rAAV biodistribution with limited treatment capacity, thus suggesting a need for alternate methods of vector delivery.  

5.1788           Proof-of-concept: neonatal intravenous injection of adeno-associated virus vectors results in successful transduction of myenteric and submucosal neurons in the mouse small and large intestine

Buckinx, R., Van Remoortel, S., Gijsbers, R., Waddington, S.N. and Timmermans, J.P. Neurogastroenterology & Motility, 28, 299-305 (2016)   Background Despite the success of viral vector technology in the transduction of the central nervous system in both preclinical research and gene therapy, its potential in neurogastroenterological research remains largely unexploited. This study asked whether and to what extent myenteric and submucosal neurons in the ileum and distal colon of the mouse were transduced after neonatal systemic delivery of recombinant adeno-associated viral vectors (AAVs). Methods Mice were intravenously injected at postnatal day one with AAV pseudotypes AAV8 or AAV9 carrying a cassette encoding enhanced green fluorescent protein (eGFP) as a reporter under the control of a cytomegalovirus promoter. At postnatal day 35, transduction of the myenteric and submucosal plexuses of the ileum and distal colon was evaluated in whole-mount preparations, using immunohistochemistry to neurochemically identify transduced enteric neurons. Key Results The pseudotypes AAV8 and AAV9 showed equal potential in transducing the enteric nervous system (ENS), with 25–30% of the neurons expressing eGFP. However, the percentage of eGFP-expressing colonic submucosal neurons was significantly lower. Neurochemical analysis showed that all enteric neuron subtypes, but not glia, expressed the reporter protein. Intrinsic sensory neurons were most efficiently transduced as nearly 80% of calcitonin gene-related peptide-positive neurons expressed the transgene. Conclusions & Inferences The pseudotypes AAV8 and AAV9 can be employed for gene delivery to both the myenteric and the submucosal plexus, although the transduction efficiency in the latter is region-dependent. These findings open perspectives for novel preclinical applications aimed at manipulating and imaging the ENS in the short term, and in gene therapy in the longer term.  

5.1789           Genetic and pharmacological evidence that endogenous nociceptin/orphanin FQ contributes to dopamine cell loss in Parkinson's disease

Arcuri, L., Viaro, SR., Bido, S., Longo, F., Calcagno, M., Fernagut, P-O., Zaveri, N.T., Calo, G., Bezard, E. and Morari, M. Neurobiology of Disease, 89, 55-64 (2016)   To investigate whether the endogenous neuropeptide nociceptin/orphanin FQ (N/OFQ) contributes to the death of dopamine neurons in Parkinson's disease, we undertook a genetic and a pharmacological approach using NOP receptor knockout (NOP^−/−) mice, and the selective and potent small molecule NOP receptor antagonist (−)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111). Stereological unbiased methods were used to estimate the total number of dopamine neurons in the substantia nigra of i) NOP^−/− mice acutely treated with the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), ii) naïve mice subacutely treated with MPTP, alone or in combination with SB-612111, iii) rats injected with a recombinant adeno-associated viral (AAV) vector overexpressing human mutant p.A53T α-synuclein, treated with vehicle or SB-612111. NOP^−/− mice showed a 50% greater amount of nigral dopamine neurons spared in response to acute MPTP compared to controls, which was associated with a milder motor impairment. SB-612111, given 4 days after MPTP treatment to mimic the clinical condition, prevented the loss of nigral dopamine neurons and striatal dopaminergic terminals caused by subacute MPTP. SB-612111, administered a week after the AAV injections in a clinically-driven protocol, also increased by 50% both the number of spared nigral dopamine neurons and striatal dopamine terminals, and prevented accompanying motor deficits induced by α-synuclein. We conclude that endogenous N/OFQ contributes to dopamine neuron loss in pathogenic and etiologic models of Parkinson's disease through NOP receptor-mediated mechanisms. NOP receptor antagonists might prove effective as disease-modifying agents in Parkinson's disease, through the rescue of degenerating nigral dopamine neurons and/or the protection of the healthy ones.  

5.1790           Production of Human papillomavirus pseudovirions in plants and their use in pseudovirion-based neutralisation assays in mammalian cells

Lamprecth, R., Kennedy, P., Huddy, S.M., Bethke, S., Hendrikse, M., Hitzeroth, I. and Rybicki, E.P. Scientific Reports, 6:20431 (2016)   Human papillomaviruses (HPV) cause cervical cancer and have recently also been implicated in mouth, laryngeal and anogenital cancers. There are three commercially available prophylactic vaccines that show good efficacy; however, efforts to develop second-generation vaccines that are more affordable, stable and elicit a wider spectrum of cross-neutralising immunity are still ongoing. Testing antisera elicited by current and candidate HPV vaccines for neutralizing antibodies is done using a HPV pseudovirion (PsV)-based neutralisation assay (PBNA). PsVs are produced by transfection of mammalian cell cultures with plasmids expressing L1 and L2 capsid proteins, and a reporter gene plasmid, a highly expensive process. We investigated making HPV-16 PsVs in plants, in order to develop a cheaper alternative. The secreted embryonic alkaline phosphatase (SEAP) reporter gene and promoter were cloned into a geminivirus-derived plant expression vector, in order to produce circular dsDNA replicons. This was co-introduced into Nicotiana benthamiana plants with vectors expressing L1 and L2 via agroinfiltration, and presumptive PsVs were purified. The PsVs contained DNA, and could be successfully used for PBNA with anti-HPV antibodies. This is the first demonstration of the production of mammalian pseudovirions in plants, and the first demonstration of the potential of plants to make DNA vaccines.  

5.1791           Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1)

Tomono, T., Hirai, Y., Okada, H., Adachi, K., Ishii, A., Shimada, T., Onodera, M., Tamaoka, A. and Okada, T. Molecular Therapy – Methods & Clinical Development, 3:15058 (2016)   Recombinant adeno-associated virus (rAAV) is an attractive tool for gene transfer and shows potential for use in human gene therapies. The current methods for the production and purification of rAAV from the transfected cell lysate are mainly based on cesium chloride and iodixanol density ultracentrifugation, although those are not scalable. Meanwhile, chromatography-based systems are more scalable. Therefore, in this study, we developed a novel method for the production and purification of rAAV serotype 1 (rAAV1) from serum-free culture supernatant based on ion-exchange and gel-filtration chromatography to obtain highly purified products with an ultracentrifugation-free technique towards Good Manufacturing Practice (GMP) production. The purified rAAV1 displayed three clear and sharp bands (VP1, VP2, and VP3) following sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and more than 90% of rAAV1 particles contained fully packaged viral genomes according to negative-stain electron micrographic analysis. Consequently, the resultant genomic titer of the purified rAAV1 was 3.63 × 1013 v.g./ml (the total titer was 4.17 × 1013 v.g.) from the 4 × 109 HEK293 cells. This novel chromatography-based method will facilitate scale-up of manufacturing for clinical applications in gene therapy.  

5.1792           The aetiology of wobbly possum disease: Reproduction of the disease with purified nidovirus

Giles, J., Perrott, M., Roe, w. and Dunowska, M. Virology, 491, 20-26 (2016)   The objective of this study was to investigate a role of a recently discovered marsupial nidovirus in the development of a neurological disease, termed wobbly possum disease (WPD), in the Australian brushtail possum (Trichosurus vulpecula). Four possums received 1 mL of a standard inoculum that had been prepared from tissues of WPD-affected possums, 4 possums received 1.8 mL (1×10⁶ TCID₅₀) of a cell lysate from inoculated cultures, and 4 possums received 1 mL (×10⁷ TCID₅₀) of a purified WPD isolate. All but one possum that received infectious inocula developed neurological disease and histopathological lesions characteristic for WPD. High levels of viral RNA were detected in livers from all possums that received infectious inocula, but not from control possums. Altogether, our data provide strong experimental evidence for the causative involvement of WPD virus in development of a neurological disease in infected animals.  

5.1793           AAV9-mediated central nervous system–targeted gene delivery via cisterna magna route in mice

Lukashchuk, V., lewis, K.E., Coldicott, I., Grierson, A.J. and Azzouz, M. Molecular Therapy-methods & Clinical development, 3:15055 (2016)   Current barriers to the use of adeno-associated virus serotype 9 (AAV9) in clinical trials for treating neurological disorders are its high expression in many off-target tissues such as liver and heart, and lack of cell specificity within the central nervous system (CNS) when using ubiquitous promoters such as human cytomegalovirus (CMV) or chicken-β-actin hybrid (CAG). To enhance targeting the transgene expression in CNS cells, self-complementary (sc) AAV9 vectors, scAAV9-GFP vectors carrying neuronal Hb9 and synapsin 1, and nonspecific CMV and CAG promoters were constructed. We demonstrate that synapsin 1 and Hb9 promoters exclusively targeted neurons in vitro, although their strengths were up to 10-fold lower than that of CMV. In vivo analyses of mouse tissue after scAAV9-GFP vector delivery via the cisterna magna revealed a significant advantage of synapsin 1 promoter over both Hb9 variants in targeting neurons throughout the brain, since Hb9 promoters were driving gene expression mainly within the motor-related areas of the brain stem. In summary, this study demonstrates that cisterna magna administration is a safe alternative to intracranial or intracerebroventricular vector delivery route using scAAV9, and introduces a novel utility of the Hb9 promoter for the targeted gene expression for both in vivo and in vitro applications.  

5.1794           Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain

Deverman, B.E., pravdo, P.L., Simpson, B., Kumar, S.R., Chan, K.Y., Banerjee, A., Wu, W-L., yang, B., Huber, N., Pasca, S.P. and Gradinaru, V. Nature Biotechnology, 34(2), 204-209 (2016)   Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer^{1, 2, 3, 4, 5, 6}. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics^{7, 8, 9, 10, 11, 12, 13}. Here we describe a capsid selection method, called Cre recombination–based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9 (refs. 14,15,16,17), and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications.  

5.1795           Massively parallel cis-regulatory analysis in the mammalian central nervous system

Shen, S.Q., Myers, C.A., Hughes, A.E.O., Byrne, L.C., Flannery, J.G. and Corbo, J.C. Genome Res., 26, 238-255 (2016)   Cis-regulatory elements (CREs, e.g., promoters and enhancers) regulate gene expression, and variants within CREs can modulate disease risk. Next-generation sequencing has enabled the rapid generation of genomic data that predict the locations of CREs, but a bottleneck lies in functionally interpreting these data. To address this issue, massively parallel reporter assays (MPRAs) have emerged, in which barcoded reporter libraries are introduced into cells, and the resulting barcoded transcripts are quantified by next-generation sequencing. Thus far, MPRAs have been largely restricted to assaying short CREs in a limited repertoire of cultured cell types. Here, we present two advances that extend the biological relevance and applicability of MPRAs. First, we adapt exome capture technology to instead capture candidate CREs, thereby tiling across the targeted regions and markedly increasing the length of CREs that can be readily assayed. Second, we package the library into adeno-associated virus (AAV), thereby allowing delivery to target organs in vivo. As a proof of concept, we introduce a capture library of about 46,000 constructs, corresponding to roughly 3500 DNase I hypersensitive (DHS) sites, into the mouse retina by ex vivo plasmid electroporation and into the mouse cerebral cortex by in vivo AAV injection. We demonstrate tissue-specific cis-regulatory activity of DHSs and provide examples of high-resolution truncation mutation analysis for multiplex parsing of CREs. Our approach should enable massively parallel functional analysis of a wide range of CREs in any organ or species that can be infected by AAV, such as nonhuman primates and human stem cell–derived organoids.  

5.1796           Rationally engineered Troponin C modulates in vivo cardiac function and performance in health and disease

Shettigar, V. et al Nature Communications, 7:10794 (2016)   Treatment for heart disease, the leading cause of death in the world, has progressed little for several decades. Here we develop a protein engineering approach to directly tune in vivo cardiac contractility by tailoring the ability of the heart to respond to the Ca²⁺ signal. Promisingly, our smartly formulated Ca²⁺-sensitizing TnC (L48Q) enhances heart function without any adverse effects that are commonly observed with positive inotropes. In a myocardial infarction (MI) model of heart failure, expression of TnC L48Q before the MI preserves cardiac function and performance. Moreover, expression of TnC L48Q after the MI therapeutically enhances cardiac function and performance, without compromising survival. We demonstrate engineering TnC can specifically and precisely modulate cardiac contractility that when combined with gene therapy can be employed as a therapeutic strategy for heart disease.  

5.1797           Viral vectors for gene therapy and gene modification approaches

Merten, O-W. and Gaillet, B. Biochemical Engineering Journal, 108,98-115 (2016)   Presently, viral vectors are widespread biological products, some of which have already been commercialized. They are derived from viruses modified to render them apt for gene transfer and safe for clinical purposes. They have several applications including the treatment of rare and acquired diseases as well as vaccination. This review briefly presents the four viral vectors mainly used currently (adenoviral, adeno-associated viral, γ-retroviral, and lentiviral vectors), as well as their biology and manufacturing issues. Furthermore, their applications in gene therapy/gene addition and protein transfer approaches are described.  

5.1798           Overexpression of Homer1a in the basal and lateral amygdala impairs fear conditioning and induces an autism-like social impairment

Banerjee, A., Luong, J.A., Ho, A., Saib, A.O. and Ploski, J.E. Molecular Autism, 7:16 (2016)   Background Autism spectrum disorders (ASDs) represent a heterogeneous group of disorders with a wide range of behavioral impairments including social and communication deficits. Apart from these core symptoms, a significant number of ASD individuals display higher levels of anxiety, and some studies indicate that a subset of ASD individuals have a reduced ability to be fear conditioned. Deciphering the molecular basis of ASD has been considerably challenging and it currently remains poorly understood. In this study we examined the molecular basis of autism-like impairments in an environmentally induced animal model of ASD, where pregnant rats are exposed to the known teratogen, valproic acid (VPA), on day 12.5 of gestation and the subsequent progeny exhibit ASD-like symptoms. We focused our analysis on the basal and lateral nucleus of the amygdala (BLA), a region of the brain found to be associated with ASD pathology. Methods We performed whole genome gene expression analysis on the BLA using DNA microarrays to examine differences in gene expression within the amygdala of VPA-exposed animals. We validated one VPA-dysregulated candidate gene (Homer1a) using both quantitative PCR (qRT-PCR) and western blot. Finally, we overexpressed Homer1a within the basal and lateral amygdala of naïve animals utilizing adeno-associated viruses (AAV) and subsequently examined these animals in a battery of behavioral tests associated with ASD, including auditory fear conditioning, social interaction and open field. Results Our microarray data indicated that Homer1a was one of the genes which exhibited a significant upregulation within the amygdala. We observed an increase in Homer1a messenger RNA (mRNA) and protein in multiple cohorts of VPA-exposed animals indicating that dysregulation of Homer1a levels might underlie some of the symptoms exhibited by VPA-exposed animals. To test this hypothesis, we overexpressed Homer1a within BLA neurons utilizing a viral-mediated approach and found that overexpression of Homer1a impaired auditory fear conditioning and reduced social interaction, while having no influence on open-field behavior. Conclusions This study indicates that dysregulation of amygdala Homer1a might contribute to some autism-like symptoms induced by VPA exposure. These findings are interesting in part because Homer1a influences the functioning of Shank3, metabotropic glutamate receptors (mGluR5), and Homer1, and these proteins have previously been associated with ASD, indicating that these differing models of ASD may have a similar molecular basis.  

5.1799           Efficient Gene Suppression in Dorsal Root Ganglia and Spinal Cord Using Adeno-Associated Virus Vectors Encoding Short-Hairpin RNA

Enomoto, M., Hirai, T., Kaburagi, H. and Yokota, T. Methods in Mol. Biol., 1364, 277-290 (2016)   RNA interference is a powerful tool used to induce loss-of-function phenotypes through post-transcriptional gene silencing. Small interfering RNA (siRNA) molecules have been used to target the central nervous system (CNS) and are expected to have clinical utility against refractory neurodegenerative diseases. However, siRNA is characterized by low transduction efficiency, insufficient inhibition of gene expression, and short duration of therapeutic effects, and is thus not ideal for treatment of neural tissues and diseases. To address these problems, viral delivery of short-hairpin RNA (shRNA) expression cassettes that support more efficient and long-lasting transduction into target tissues is expected to be a promising delivery tool. Various types of gene therapy vectors have been developed, such as adenovirus, adeno-associated virus (AAV), herpes simplex virus and lentivirus; however, AAV is particularly advantageous because of its relative lack of immunogenicity and lack of chromosomal integration. In human clinical trials, recombinant AAV vectors are relatively safe and well-tolerated. In particular, serotype 9 of AAV (AAV9) vectors show the highest tropism for neural tissue and can cross the blood–brain barrier, and we have shown that intrathecal delivery of AAV9 yields relatively high gene transduction into dorsal root ganglia or spinal cord. This chapter describes how to successfully use AAV vectors encoding shRNA in vivo, particularly for RNA interference in the central and peripheral nervous system.  

5.1800           Deletion of a Predicted β-Sheet Domain within the Amino Terminus of Herpes Simplex Virus Glycoprotein K Conserved among Alphaherpesviruses Prevents Virus Entry into Neuronal Axons

Jambunathan, N., Charles, A-S., Subramanian, r., Saied, A.A., Naderi, M., Rider, P., Brylinski, M., Chouljenko, V.N. and Kousoulas, K.G.   We have shown previously that herpes simplex virus 1 (HSV-1) lacking expression of the entire glycoprotein K (gK) or expressing gK with a 38-amino-acid deletion (gKΔ31–68 mutation) failed to infect ganglionic neurons after ocular infection of mice. We constructed a new model for the predicted three-dimensional structure of gK, revealing that the gKΔ31–68 mutation spans a well-defined β-sheet structure within the amino terminus of gK, which is conserved among alphaherpesviruses. The HSV-1(McKrae) gKΔ31–68 virus was tested for the ability to enter into ganglionic neuronal axons in cell culture of explanted rat ganglia using a novel virus entry proximity ligation assay (VEPLA). In this assay, cell surface-bound virions were detected by the colocalization of gD and its cognate receptor nectin-1 on infected neuronal surfaces. Capsids that have entered into the cytoplasm were detected by the colocalization of the virion tegument protein UL37, with dynein required for loading of virion capsids onto microtubules for retrograde transport to the nucleus. HSV-1(McKrae) gKΔ31–68 attached to cell surfaces of Vero cells and ganglionic axons in cell culture as efficiently as wild-type HSV-1(McKrae). However, unlike the wild-type virus, the mutant virus failed to enter into the axoplasm of ganglionic neurons. This work suggests that the amino terminus of gK is a critical determinant for entry into neuronal axons and may serve similar conserved functions for other alphaherpesviruses.  

5.1801           Cocaine Hydrolase Gene Transfer Demonstrates Cardiac Safety and Efficacy against Cocaine-Induced QT Prolongation in Mice

Murthy, V., Reyes, S., Geng, L., Gao, Y. and Brimijoin, S.

1. Pharmacol. Exp. Ther., 356, 730-725 (2016)

  Cocaine addiction is associated with devastating medical consequences, including cardiotoxicity and risk-conferring prolongation of the QT interval. Viral gene transfer of cocaine hydrolase engineered from butyrylcholinesterase offers therapeutic promise for treatment-seeking drug users. Although previous preclinical studies have demonstrated benefits of this strategy without signs of toxicity, the specific cardiac safety and efficacy of engineered butyrylcholinesterase viral delivery remains unknown. Here, telemetric recording of electrocardiograms from awake, unrestrained mice receiving a course of moderately large cocaine doses (30 mg/kg, twice daily for 3 weeks) revealed protection against a 2-fold prolongation of the QT interval conferred by pretreatment with cocaine hydrolase vector. By itself, this prophylactic treatment did not affect QT interval duration or cardiac structure, demonstrating that viral delivery of cocaine hydrolase has no intrinsic cardiac toxicity and, on the contrary, actively protects against cocaine-induced QT prolongation.  

5.1802           Proteomics of HCV virions reveals an essential role for the nucleoporin Nup98 in virus morphogenesis

Lussignol, M., Kopp, M., Molloy, K., Vizcay-Barrena, G., Fleck, R.A., Dorner, M., Bell, K.L., Chait, B.T., Rice, C.M. and Catanese, M.T. PNAS, 113(9), 2484-2489 (2016)   Hepatitis C virus (HCV) is a unique enveloped virus that assembles as a hybrid lipoviral particle by tightly interacting with host lipoproteins. As a result, HCV virions display a characteristic low buoyant density and a deceiving coat, with host-derived apolipoproteins masking viral epitopes. We previously described methods to produce high-titer preparations of HCV particles with tagged envelope glycoproteins that enabled ultrastructural analysis of affinity-purified virions. Here, we performed proteomics studies of HCV isolated from culture media of infected hepatoma cells to define viral and host-encoded proteins associated with mature virions. Using two different affinity purification protocols, we detected four viral and 46 human cellular proteins specifically copurifying with extracellular HCV virions. We determined the C terminus of the mature capsid protein and reproducibly detected low levels of the viral nonstructural protein, NS3. Functional characterization of virion-associated host factors by RNAi identified cellular proteins with either proviral or antiviral roles. In particular, we discovered a novel interaction between HCV capsid protein and the nucleoporin Nup98 at cytosolic lipid droplets that is important for HCV propagation. These results provide the first comprehensive view to our knowledge of the protein composition of HCV and new insights into the complex virus–host interactions underlying HCV infection.  

5.1803           Loss of HCN1 enhances disease progression in mouse models of CNG channel-linked retinitis pigmentosa and achromatopsia

Schön, C., Asteriti, S., Koch, S., Sothilingam, V., garcia-garrido, M., tanimoto, N., Herms, J., Seeliger, M.W., Cangiano, L., Biel, M. and Michalakis, S. Hum. Mol. Genet., 25(6), 1165-1175 (2016)   Most inherited blinding diseases are characterized by compromised retinal function and progressive degeneration of photoreceptors. However, the factors that affect the life span of photoreceptors in such degenerative retinal diseases are rather poorly understood. Here, we explore the role of hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1) in this context. HCN1 is known to adjust retinal function under mesopic conditions, and although it is expressed at high levels in rod and cone photoreceptor inner segments, no association with any retinal disorder has yet been found. We investigated the effects of an additional genetic deletion of HCN1 on the function and survival of photoreceptors in a mouse model of CNGB1-linked retinitis pigmentosa (RP). We found that the absence of HCN1 in Cngb1 knockout (KO) mice exacerbated photoreceptor degeneration. The deleterious effect was reduced by expression of HCN1 using a viral vector. Moreover, pharmacological inhibition of HCN1 also enhanced rod degeneration in Cngb1 KO mice. Patch-clamp recordings revealed that the membrane potentials of Cngb1 KO and Cngb1/Hcn1 double-KO rods were both significantly depolarized. We also found evidence for altered calcium homeostasis and increased activation of the protease calpain in Cngb1/Hcn1 double-KO mice. Finally, the deletion of HCN1 also exacerbated degeneration of cone photoreceptors in a mouse model of CNGA3-linked achromatopsia. Our results identify HCN1 as a major modifier of photoreceptor degeneration and suggest that pharmacological inhibition of HCN channels may enhance disease progression in RP and achromatopsia patients.  

5.1804           Long noncoding RNA Chast promotes cardiac remodeling

Viereck, J. et al Science Translational Medicine, 8(326), 326ra22 (2016)   Recent studies highlighted long noncoding RNAs (lncRNAs) to play an important role in cardiac development. However, understanding of lncRNAs in cardiac diseases is still limited. Global lncRNA expression profiling indicated that several lncRNA transcripts are deregulated during pressure overload–induced cardiac hypertrophy in mice. Using stringent selection criteria, we identified Chast (cardiac hypertrophy–associated transcript) as a potential lncRNA candidate that influences cardiomyocyte hypertrophy. Cell fractionation experiments indicated that Chast is specifically up-regulated in cardiomyocytes in vivo in transverse aortic constriction (TAC)–operated mice. In accordance, CHAST homolog in humans was significantly up-regulated in hypertrophic heart tissue from aortic stenosis patients and in human embryonic stem cell–derived cardiomyocytes upon hypertrophic stimuli. Viral-based overexpression of Chast was sufficient to induce cardiomyocyte hypertrophy in vitro and in vivo. GapmeR-mediated silencing of Chast both prevented and attenuated TAC-induced pathological cardiac remodeling with no early signs on toxicological side effects. Mechanistically, Chast negatively regulated Pleckstrin homology domain–containing protein family M member 1 (opposite strand of Chast), impeding cardiomyocyte autophagy and driving hypertrophy. These results indicate that Chast can be a potential target to prevent cardiac remodeling and highlight a general role of lncRNAs in heart diseases.  

5.1805           Impact of HIV-1 Membrane Cholesterol on Cell-Independent Lytic Inactivation and Cellular Infectivity

Sandaram, R.V.K., Li, H., Bailey, L., Rashad, A.A., Aneja, R., Weiss, K., Huynh, J., Bastian, A.R., Papazoglou, E., Abrams, C., Wrenn, S. and Chaiken, I. Biochemistry, 55, 447-458 (2016)   Peptide triazole thiols (PTTs) have been found previously to bind to HIV-1 Env spike gp120 and cause irreversible virus inactivation by shedding gp120 and lytically releasing luminal capsid protein p24. Since the virions remain visually intact, lysis appears to occur via limited membrane destabilization. To better understand the PTT-triggered membrane transformation involved, we investigated the role of envelope cholesterol on p24 release by measuring the effect of cholesterol depletion using methyl beta-cyclodextrin (MβCD). An unexpected bell-shaped response of PTT-induced lysis to [MβCD] was observed, involving lysis enhancement at low [MβCD] vs loss of function at high [MβCD]. The impact of cholesterol depletion on PTT-induced lysis was reversed by adding exogenous cholesterol and other sterols that support membrane rafts, while sterols that do not support rafts induced only limited reversal. Cholesterol depletion appears to cause a reduced energy barrier to lysis as judged by decreased temperature dependence with MβCD. Enhancement/replenishment responses to [MβCD] also were observed for HIV-1 infectivity, consistent with a similar energy barrier effect in the membrane transformation of virus cell fusion. Overall, the results argue that cholesterol in the HIV-1 envelope is important for balancing virus stability and membrane transformation, and that partial depletion, while increasing infectivity, also makes the virus more fragile. The results also reinforce the argument that the lytic inactivation and infectivity processes are mechanistically related and that membrane transformations occurring during lysis can provide an experimental window to investigate membrane and protein factors important for HIV-1 cell entry.  

5.1806           Light-Activated Nuclear Translocation of Adeno-Associated Virus Nanoparticles Using Phytochrome B for Enhanced, Tunable, and Spatially Programmable Gene Delivery

Gomez, E.J., Gerhardt, K., Judd, J., tabor, J.J. and Suh, J. ASC Nano, 10, 225-237 (2016)   Gene delivery vectors that are activated by external stimuli may allow improved control over the location and the degree of gene expression in target populations of cells. Light is an attractive stimulus because it does not cross-react with cellular signaling networks, has negligible toxicity, is noninvasive, and can be applied in space and time with unparalleled precision. We used the previously engineered red (R)/far-red (FR) light-switchable protein phytochrome B (PhyB) and its R light dependent interaction partner phytochrome interacting factor 6 (PIF6) from Arabidopsis thaliana to engineer an adeno-associated virus (AAV) platform whose gene delivery efficiency is controlled by light. Upon exposure to R light, AAV engineered to display PIF6 motifs on the capsid bind to PhyB tagged with a nuclear localization sequence (NLS), resulting in significantly increased translocation of viruses into the host cell nucleus and overall gene delivery efficiency. By modulating the ratio of R to FR light, the gene delivery efficiency can be tuned to as little as 35% or over 600% of the unengineered AAV. We also demonstrate spatial control of gene delivery using projected patterns of codelivered R and FR light. Overall, our successful use of light-switchable proteins in virus capsid engineering extends these important optogenetic tools into the adjacent realm of nucleic acid delivery and enables enhanced, tunable, and spatially controllable regulation of viral gene delivery. Our current light-triggered viral gene delivery prototype may be broadly useful for genetic manipulation of cells ex vivo or in vivo in transgenic model organisms, with the ultimate prospect of achieving dose- and site-specific gene expression profiles for either therapeutic (e.g., regenerative medicine) or fundamental discovery research efforts.  

5.1807           Reduced Antiviral Interferon Production in Poorly Controlled Asthma Is Associated With Neutrophilic Inflammation and High-Dose Inhaled Corticosteroids

Simpson, J.L., Carroll, M., Yang, I.A., Reynolds, P.N., Hodge, S., James, A.L., Gibson, P.G. and Upham, J.W. Chest, 149(3), 704-713 (2016)   Background Asthma is a heterogeneous chronic inflammatory disease in which host defense against respiratory viruses such as human rhinovirus (HRV) may be abnormal. This is a matter of some controversy, with some investigators reporting reduced type I interferon (IFN) synthesis and others suggesting that type I IFN synthesis is relatively normal in asthma. Objective The objective of this study was to examine the responsiveness of circulating mononuclear cells to HRV in a large cohort of participants with poorly controlled asthma and determine whether IFN-α and IFN-β synthesis varies across different inflammatory phenotypes. Methods Eligible adults with asthma (n = 86) underwent clinical assessment, sputum induction, and blood sampling. Asthma inflammatory subtypes were defined by sputum cell count, and supernatant assessed for IL-1β. Peripheral blood mononuclear cells (PBMCs) were exposed to HRV serotype 1b, and IFN-α and IFN-β release was measured by enzyme-linked immunosorbent assay. Results Participants (mean age, 59 years; atopy, 76%) had suboptimal asthma control (mean asthma control questionnaire 6, 1.7). In those with neutrophilic asthma (n = 12), HRV1b-stimulated PBMCs produced significantly less IFN-α than PBMCs from participants with eosinophilic (n = 35) and paucigranulocytic asthma (n = 35). Sputum neutrophil proportion and the dose of inhaled corticosteroids were independent predictors of reduced IFN-α production after HRV1b exposure. Conclusions Antiviral type I IFN production is impaired in those with neutrophilic airway inflammation and in those prescribed high doses of inhaled corticosteroids. Our study is an important step toward identifying those with poorly controlled asthma who might respond best to inhaled IFN therapy during exacerbations.  

5.1808           Small-Scale Recombinant Adeno-Associated Virus Purification

Burger, C. and Nash, K.R. Methods in Mol. Biol., 1382, 95-106 (2016)   Recombinant adeno-associated virus (rAAV) vectors have become increasingly popular in research and clinical trials due to their efficient gene transfer and long-term expression in tissues including brain. In addition, rAAV has demonstrated an impressive safety profile in gene therapy trials. The emergence of rAAV serotypes with different cell tropisms and distribution properties has allowed scientists to tailor serotypes to specific experimental needs. AAV does not have a cytopathic effect; therefore, purification methods require extraction of the viral vector from the cell. This involves gradient ultracentrifugation of the cellular extract sometimes followed by chromatography. This chapter describes a small-scale production method for rAAV purification from ten to twenty 15 cm plates of human embryonic kidney-derived 293B cells (HEK 293) cells that can yield approximately 300 μl of a 5 × 10¹² to 1 × 10¹³ genome copies/ml viral preparation final concentration.  

5.1809           A transducible nuclear/nucleolar protein, mLLP, regulates neuronal morphogenesis and synaptic transmission

Yu, N-K. et al Scientific Reports, 6:22892 (2016)   Cell-permeable proteins are emerging as unconventional regulators of signal transduction and providing a potential for therapeutic applications. However, only a few of them are identified and studied in detail. We identify a novel cell-permeable protein, mouse LLP homolog (mLLP), and uncover its roles in regulating neural development. We found that mLLP is strongly expressed in developing nervous system and that mLLP knockdown or overexpression during maturation of cultured neurons affected the neuronal growth and synaptic transmission. Interestingly, extracellular addition of mLLP protein enhanced dendritic arborization, demonstrating the non-cell-autonomous effect of mLLP. Moreover, mLLP interacts with CCCTC-binding factor (CTCF) as well as transcriptional machineries and modulates gene expression involved in neuronal growth. Together, these results illustrate the characteristics and roles of previously unknown cell-permeable protein mLLP in modulating neural development.  

5.1810           Altering Tropism of rAAV by Directed Evolution

Marsic, D. and Zolotukhin, S. Methods in Mol. Biol., 1382, 151-173 (2016)   evolution represents an attractive approach to derive AAV capsid variants capable of selectively infect specific tissue or cell targets. It involves the generation of an initial library of high complexity followed by cycles of selection during which the library is progressively enriched for target-specific variants. Each selection cycle consists of the following: reconstitution of complete AAV genomes within plasmid molecules; production of virions for which each particular capsid variant is matched with the particular capsid gene encoding it; recovery of capsid gene sequences from target tissue after systemic administration. Prevalent variants are then analyzed and evaluated.  

5.1811           Promotion of mitochondrial biogenesis by necdin protects neurons against mitochondrial insults

Hasegawa, K., Yasuda, T., Shirashi, C., Fujiwara, K., Przedborski, S., Mochizuki, H. and Yoshikawa, K. Nature Communications, 7:10943 (2016)   Neurons rely heavily on mitochondria for their function and survival. Mitochondrial dysfunction contributes to the pathogenesis of neurodegenerative diseases such as Parkinson’s disease. PGC-1α is a master regulator of mitochondrial biogenesis and function. Here we identify necdin as a potent PGC-1α stabilizer that promotes mitochondrial biogenesis via PGC-1α in mammalian neurons. Expression of genes encoding mitochondria-specific proteins decreases significantly in necdin-null cortical neurons, where mitochondrial function and expression of the PGC-1α protein are reduced. Necdin strongly stabilizes PGC-1α by inhibiting its ubiquitin-dependent degradation. Forced expression of necdin enhances mitochondrial function in primary cortical neurons and human SH-SY5Y neuroblastoma cells to prevent mitochondrial respiratory chain inhibitor-induced degeneration. Moreover, overexpression of necdin in the substantia nigra in vivo of adult mice protects dopaminergic neurons against degeneration in experimental Parkinson’s disease. These data reveal that necdin promotes mitochondrial biogenesis through stabilization of endogenous PGC-1α to exert neuroprotection against mitochondrial insults.  

5.1812           Efficacy and safety of myocardial gene transfer of adenovirus, adeno-associated virus and lentivirus vectors in the mouse heart

Merentie, M.,M., Lottonen-Raikaslehto, L., parviainen, V., Huusko, J., Pikkarainen, S., Mendel, M., Laham-Karam, N., Kärjä, V., Rissanen, R., Hedman, M. and Ylä-Herttuala, S. Gene Therapy, 23(3), 296-305 (2016)   Gene therapy is a promising new treatment option for cardiac diseases. For finding the most suitable and safe vector for cardiac gene transfer, we delivered adenovirus (AdV), adeno-associated virus (AAV) and lentivirus (LeV) vectors into the mouse heart with sophisticated closed-chest echocardiography-guided intramyocardial injection method for comparing them with regards to transduction efficiency, myocardial damage, effects on the left ventricular function and electrocardiography (ECG). AdV had the highest transduction efficiency in cardiomyocytes followed by AAV2 and AAV9, and the lowest efficiency was seen with LeV. The local myocardial inflammation and fibrosis in the left ventricle (LV) was proportional to transduction efficiency. AdV caused LV dilatation and systolic dysfunction. Neither of the locally injected AAV serotypes impaired the LV systolic function, but AAV9 caused diastolic dysfunction to some extent. LeV did not affect the cardiac function. We also studied systemic delivery of AAV9, which led to transduction of cardiomyocytes throughout the myocardium. However, also diffuse fibrosis was present leading to significantly impaired LV systolic and diastolic function and pathological ECG changes. Compared with widely used AdV vector, AAV2, AAV9 and LeV were less effective in transducing cardiomyocytes but also less harmful. Local administration of AAV9 was safer and more efficient compared with systemic administration.  

5.1813           Viral infection of the marine alga Emiliania huxleyi triggers lipidome remodeling and induces the production of highly saturated triacylglycerol

Malitsky, S., Ziv, C., Rosenwasser, S., Zheng, S., Schatz, D., Porat, Z., Ben-Dor, S., Aharoni, A. and Vardi, A. New Phytologist, 210(1), 8-96 (2016)  

• Viruses that infect marine photosynthetic microorganisms are major ecological and evolutionary drivers of microbial food webs, estimated to turn over more than a quarter of the total photosynthetically fixed carbon. Viral infection of the bloom-forming microalga Emiliania huxleyi induces the rapid remodeling of host primary metabolism, targeted towards fatty acid metabolism.
• We applied a liquid chromatography-mass spectrometry (LC-MS)-based lipidomics approach combined with imaging flow cytometry and gene expression profiling to explore the impact of viral-induced metabolic reprogramming on lipid composition.
• Lytic viral infection led to remodeling of the cellular lipidome, by predominantly inducing the biosynthesis of highly saturated triacylglycerols (TAGs), coupled with a significant accumulation of neutral lipids within lipid droplets. Furthermore, TAGs were found to be a major component (77%) of the lipidome of isolated virions. Interestingly, viral-induced TAGs were significantly more saturated than TAGs produced under nitrogen starvation.
• This study highlights TAGs as major products of the viral-induced metabolic reprogramming during the host–virus interaction and indicates a selective mode of membrane recruitment during viral assembly, possibly by budding of the virus from specialized subcellular compartments. These findings provide novel insights into the role of viruses infecting microalgae in regulating metabolism and energy transfer in the marine environment and suggest their possible biotechnological application in biofuel production.

 

5.1814           Platelet-derived Growth Factor-B Protects Rat Cardiac Allografts From Ischemia-reperfusion Injury

Tuuminen, R., Dashkevich, A., Keränen, M.I., Raissadati, A., Krebs, R., Jokinen, J.J., Arnaudova, R., Rouvinen, E., Ylä-Herttuala, S., Nykänen, A.I. and Lemström, K.B. Transplantation, 100(2), 303-313 (2016)   Background: Microvascular dysfunction and cardiomyocyte injury are hallmarks of ischemia-reperfusion injury (IRI) after heart transplantation. Platelet-derived growth factors (PDGF) have an ambiguous role in this deleterious cascade. On one hand, PDGF may exert vascular stabilizing and antiapoptotic actions through endothelial-pericyte and endothelial-cardiomyocyte crosstalk in the heart; and on the other hand, PDGF signaling mediates neointimal formation and exacerbates chronic rejection in cardiac allografts. The balance between these potentially harmful and beneficial actions determines the final outcome of cardiac allografts. Methods and Results: We transplanted cardiac allografts from Dark Agouti rat and Balb mouse donors to fully major histocompatibility complex-mismatched Wistar Furth rat or C57 mouse recipients with a clinically relevant 2-hour cold ischemia and 1-hour warm ischemia. Ex vivo intracoronary delivery of adenovirus-mediated gene transfer of recombinant human PDGF-BB upregulated messenger RNA expression of anti-mesenchymal transition and survival factors BMP-7 and Bcl-2 and preserved capillary density in rat cardiac allografts at day 10. In mouse cardiac allografts PDGF receptor-β, but not -α intragraft messenger RNA levels were reduced and capillary protein localization was lost during IRI. The PDGF receptor tyrosine kinase inhibitor imatinib mesylate and a monoclonal antibody against PDGF receptor-α enhanced myocardial damage evidenced by serum cardiac troponin T release in the rat and mouse cardiac allografts 6 hours after reperfusion, respectively. Moreover, imatinib mesylate enhanced rat cardiac allograft vasculopathy, cardiac fibrosis, and late allograft loss at day 56. Conclusions: Our results suggest that PDGF-B signaling may play a role in endothelial and cardiomyocyte recovery from IRI after heart transplantation.  

5.1815           Identification of an Alternative Splicing Product of the Otx2 Gene Expressed in the Neural Retina and Retinal Pigmented Epithelial Cells

Kole, C., Berdugo, N., Da Silva, C., Ait-Ali, N., Millet-Puel, G., pagan, D., Blond, F., poidevin, L., Ripp, R., Fontaine, V., Wincker, P., Zack, D.J., Sahel, J-A., Poch, O. and Leveillard, T. PloS One, 11(3), e0150758 (2016)   To investigate the complexity of alternative splicing in the retina, we sequenced and analyzed a total of 115,706 clones from normalized cDNA libraries from mouse neural retina (66,217) and rat retinal pigmented epithelium (49,489). Based upon clustering the cDNAs and mapping them with their respective genomes, the estimated numbers of genes were 9,134 for the mouse neural retina and 12,050 for the rat retinal pigmented epithelium libraries. This unique collection of retinal of messenger RNAs is maintained and accessible through a web-base server to the whole community of retinal biologists for further functional characterization. The analysis revealed 3,248 and 3,202 alternative splice events for mouse neural retina and rat retinal pigmented epithelium, respectively. We focused on transcription factors involved in vision. Among the six candidates suitable for functional analysis, we selected Otx2S, a novel variant of the Otx2 gene with a deletion within the homeodomain sequence. Otx2S is expressed in both the neural retina and retinal pigmented epithelium, and encodes a protein that is targeted to the nucleus. OTX2S exerts transdominant activity on the tyrosinase promoter when tested in the physiological environment of primary RPE cells. By overexpressing OTX2S in primary RPE cells using an adeno associated viral vector, we identified 10 genes whose expression is positively regulated by OTX2S. We find that OTX2S is able to bind to the chromatin at the promoter of the retinal dehydrogenase 10 (RDH10) gene.  

5.1816           Continuous Collection of Adeno-Associated Virus from Producer Cell Medium Significantly Increases Total Viral Yield No Access

Benskey, M., Sandoval, I.M. and Manfredsson, F.P. Human Gene Therapy Methods, 27(1), 32-45 (2016)   The ability to efficiently produce large amounts of high-titer recombinant adeno-associated virus (AAV) is a prerequisite to the continued success of AAV as a gene therapy tool targeted toward large-animal preclinical studies or human clinical therapeutics. Current manufacturing procedures necessitate laborious and time-consuming purification procedures to obtain AAV particles of sufficient titer and purity for these demanding biomedical applications. The finding that AAV can be harvested and purified from producer cell medium may represent an efficient alternative to purifying AAV from cellular lysates. Here we sought to determine the maximum duration of time, and frequency within which AAV can be harvested from producer cell medium, in order to maximize the yield obtained from a single transfection preparation. Human embryonic kidney 293T cells were transfected with polyethylenimine to produce AAV2/5 expressing green fluorescent protein (GFP), and cellular medium was harvested every 2 days until a maximum duration of 19 days posttransfection. AAV2/5-GFP was released into producer cell medium at a steady state until 7 days posttransfection, at which time titers dropped dramatically. Harvesting medium every two days resulted in the maximum yield of AAV from a single preparation, and the cumulative yield of AAV harvested from the producer cell medium was 4-fold higher than the yield obtained from a traditional purification of AAV from cellular lysates. The AAV2/5 harvested from medium within the 7-day collection time-course mediated high levels of transduction in vivo, comparable to AAV2/5 harvested from cellular lysates. AAV purified from cell lysates showed increasing amounts of empty particles at 5 and 7 days posttransfection, whereas AAV purified from cell medium did not show an increase in the amount of empty particles throughout the 7-day time course. Finally, we extended these findings to AAV2/9, demonstrating that a comparable ratio of AAV2/9 particles are also released for up to 7 days posttransfection.  

5.1817           Quantitative and semi-quantitative measurements of axonal degeneration in tissue and primary neuron cultures

Kneynsberg, A., Collier, T.J., Manfredsson, F.P. and Kanaan, N.M.

1. Neurosci. Methods, 266, 32-41 (2016)

  Background Axon viability is critical for maintaining neural connectivity, which is central to neural functionality. Many neurodegenerative diseases (e.g., Parkinson’s disease (PD) and Alzheimer’s disease) appear to involve extensive axonal degeneration that often precedes somatic loss in affected neural populations. Axonal degeneration involves a number of intracellular pathways and characteristic changes in axon morphology (i.e., swelling, fragmentation, and loss). New method We describe a relatively simple set of methods to quantify the axonal degeneration using the 6-hydroxydopamine neurotoxin model of PD in rats and a colchicine-induced model in primary rat neurons. Specifically, approaches are described that use the spaceballs stereological probe for tissue sections and petrimetrics stereological probe for cultured neurons, and image analysis techniques in both tissue sections and cultured neurons. Results These methods provide a mechanism for obtaining quantitative and semi-quantitative data to track the extent of axonal degeneration and may prove useful as outcome measures in studies aimed at preventing or slowing axonal degeneration in disease models. Comparison with existing methods Existing methods of quantification of axonal degeneration use densitometry and manual counts of axonal projections, but they do not utilize the random, unbiased systematic sampling approaches that are characteristic of stereological methods. The ImageJ thresholding analyses described here provide a descriptive method for quantifying the state of axonal degeneration. Conclusions These methods provide an efficient and effective means to quantify the extent and state of axonal degeneration in animal tissue and cultured neurons and can be used in other models for the same purposes.  

5.1818           The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles

Ziegler, C.M., eisenbauer, P., Bruce, E.A., Weir, M.E., King, B.R., Klaus, J.P., Krementsov, D.N., Shirley, D.J., Ballit, B.A. and Botten, J. PloS Pathogens, 12(3), e1005501 (2016)   Arenaviruses cause severe diseases in humans but establish asymptomatic, lifelong infections in rodent reservoirs. Persistently-infected rodents harbor high levels of defective interfering (DI) particles, which are thought to be important for establishing persistence and mitigating virus-induced cytopathic effect. Little is known about what drives the production of DI particles. We show that neither the PPXY late domain encoded within the lymphocytic choriomeningitis virus (LCMV) matrix protein nor a functional endosomal sorting complex transport (ESCRT) pathway is absolutely required for the generation of standard infectious virus particles. In contrast, DI particle release critically requires the PPXY late domain and is ESCRT-dependent. Additionally, the terminal tyrosine in the PPXY motif is reversibly phosphorylated and our findings indicate that this posttranslational modification may regulate DI particle formation. Thus we have uncovered a new role for the PPXY late domain and a possible mechanism for its regulation.  

5.1819           A regulatable AAV vector mediating GDNF biological effects at clinically-approved sub-antimicrobial doxycycline doses

Chtarto, A. et al Meolecular Therapy-Methods & Clinical Development,5:16027 (2016)   Preclinical and clinical data stress the importance of pharmacologically-controlling glial cell line-derived neurotrophic factor (GDNF) intracerebral administration to treat PD. The main challenge is finding a combination of a genetic switch and a drug which, when administered at a clinically-approved dose, reaches the brain in sufficient amounts to induce a therapeutic effect. We describe a highly-sensitive doxycycline-inducible adeno-associated virus (AAV) vector. This vector allowed for the first time a longitudinal analysis of inducible transgene expression in the brain using bioluminescence imaging. To evaluate the dose range of GDNF biological activity, the inducible AAV vector (8.0 × 109 viral genomes) was injected in the rat striatum at four delivery sites and increasing doxycycline doses administered orally. ERK/Akt signaling activation as well as tyrosine hydroxylase downregulation, a consequence of long-term GDNF treatment, were induced at plasmatic doxycycline concentrations of 140 and 320 ng/ml respectively, which are known not to increase antibiotic-resistant microorganisms in patients. In these conditions, GDNF covered the majority of the striatum. No behavioral abnormalities or weight loss were observed. Motor asymmetry resulting from unilateral GDNF treatment only appeared with a 2.5-fold higher vector and a 13-fold higher inducer doses. Our data suggest that using the herein-described inducible AAV vector, biological effects of GDNF can be obtained in response to sub-antimicrobial doxycycline doses.  

5.1820           Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes

Chamorro, c., Mencia, A., Almarza, D., Duarte, B., Büning, H., Sallach, J., Hausser, I., Del Rio, M., Larcher, F. and Murillas, R. Molecular Therapy-Nucleic Acids, 5, e307 (2016)   Clonal gene therapy protocols based on the precise manipulation of epidermal stem cells require highly efficient gene-editing molecular tools. We have combined adeno-associated virus (AAV)-mediated delivery of donor template DNA with transcription activator-like nucleases (TALE) expressed by adenoviral vectors to address the correction of the c.6527insC mutation in the COL7A1 gene, causing recessive dystrophic epidermolysis bullosa in a high percentage of Spanish patients. After transduction with these viral vectors, high frequencies of homology-directed repair were found in clones of keratinocytes derived from a recessive dystrophic epidermolysis bullosa (RDEB) patient homozygous for the c.6527insC mutation. Gene-edited clones recovered the expression of the COL7A1 transcript and collagen VII protein at physiological levels. In addition, treatment of patient keratinocytes with TALE nucleases in the absence of a donor template DNA resulted in nonhomologous end joining (NHEJ)-mediated indel generation in the vicinity of the c.6527insC mutation site in a large proportion of keratinocyte clones. A subset of these indels restored the reading frame of COL7A1 and resulted in abundant, supraphysiological expression levels of mutant or truncated collagen VII protein. Keratinocyte clones corrected both by homology-directed repair (HDR) or NHEJ were used to regenerate skin displaying collagen VII in the dermo-epidermal junction.  

5.1821           Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy

Freyermuth, F. et al Nature Communications, 7:11067 (2016)   Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.  

5.1822           Self-Complementary Adeno-Associated Virus Vectors Improve Transduction Efficiency of Corneal Endothelial Cells

Gruenert, A.K., Czugala, M., Mueller, C., Schmeer, M., Schleef, M., Kruse, F.E. and Fuchsluger, T.A. PloS One, 11(3), e152589 (2016)   Transplantation of a donor cornea to restore vision is the most frequently performed transplantation in the world. Corneal endothelial cells (CEC) are crucial for the outcome of a graft as they maintain corneal transparency and avoid graft failure due to corneal opaqueness. Given the characteristic of being a monolayer and in direct contact with culture medium during cultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches prior to transplantation. Recombinant adeno-associated virus 2 (rAAV2) vectors represent a promising tool for gene therapy of CEC. However, high vector titers are needed to achieve sufficient gene expression. One of the rate-limiting steps for transgene expression is the conversion of single-stranded (ss-) DNA vector genome into double-stranded (ds-) DNA. This step can be bypassed by using self-complementary (sc-) AAV2 vectors. Aim of this study was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors in CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein (GFP) as transgene were used. Both in CEC and in donor corneas, transduction with scAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. In most cases, only half the vector titer of scAAV2 was required for equal or higher gene expression rates than those of ssAAV2. In human donor corneas, GFP expression was 64.7±11.3% (scAAV) and 38.0±8.6% (ssAAV) (p<0.001), respectively. Furthermore, transduced cells maintained their viability and showed regular morphology. Working together with regulatory authorities, a translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of corneal allografts during cultivation and transplantation could therefore become more realistic.  

5.1823           New Structural Insights into the Genome and Minor Capsid Proteins of BK Polyomavirus using Cryo-Electron Microscopy

Hurdiss, D.L., Morgan, E.L., Thompson, R.F., Prescott, E.L., Panou, M.M., Macdonald, A. and Ranson, N.A. Structure, 24, 528-536 (2016)   BK polyomavirus is the causative agent of several diseases in transplant patients and the immunosuppressed. In order to better understand the structure and life cycle of BK, we produced infectious virions and VP1-only virus-like particles in cell culture, and determined their three-dimensional structures using cryo-electron microscopy (EM) and single-particle image processing. The resulting 7.6-Å resolution structure of BK and 9.1-Å resolution of the virus-like particles are the highest-resolution cryo-EM structures of any polyomavirus. These structures confirm that the architecture of the major structural protein components of these human polyomaviruses are similar to previous structures from other hosts, but give new insight into the location and role of the enigmatic minor structural proteins, VP2 and VP3. We also observe two shells of electron density, which we attribute to a structurally ordered part of the viral genome, and discrete contacts between this density and both VP1 and the minor capsid proteins.  

5.1824           Intracranial AAV-sTRAIL combined with lanatoside C prolongs survival in an orthotopic xenograft mouse model of invasive glioblastoma

Crommentuijn, M.H.W., Maguire, C.A., Niers, J.M., Vandertop, W.P., Badr, C.E., Würdinger, T. and Tannous, B.A. Mol. Oncol., 10, 625-634 (2016)   Glioblastoma (GBM) is the most common malignant brain tumor in adults. We designed an adeno-associated virus (AAV) vector for intracranial delivery of secreted, soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) to GBM tumors in mice and combined it with the TRAIL-sensitizing cardiac glycoside, lanatoside C (lan C). We applied this combined therapy to two different GBM models using human U87 glioma cells and primary patient-derived GBM neural spheres in culture and in orthotopic GBM xenograft models in mice. In U87 cells, conditioned medium from AAV2-sTRAIL expressing cells combined with lan C induced 80% cell death. Similarly, lan C sensitized primary GBM spheres to sTRAIL causing over 90% cell death. In mice bearing intracranial U87 tumors treated with AAVrh.8-sTRAIL, administration of lan C caused a decrease in tumor-associated Fluc signal, while tumor size increased within days of stopping the treatment. Another round of lan C treatment re-sensitized GBM tumor to sTRAIL-induced cell death. AAVrh.8-sTRAIL treatment alone and combined with lanatoside C resulted in a significant decrease in tumor growth and longer survival of mice bearing orthotopic invasive GBM brain tumors. In summary, AAV-sTRAIL combined with lanatoside C induced cell death in U87 glioma cells and patient-derived GBM neural spheres in culture and in vivo leading to an increased in overall mice survival.  

5.1825           Human liver chimeric mice as a new model of chronic hepatitis E virus infection and preclinical drug evaluation

Allweis, L., Gass, S., Giersch, K., Groth, A., Kah, J., Volz, T., Rapp, G., Schöbel, A., Lohse, A.W., Polywka, S., Pischke, S., herker, E., Dandri, M. and Lütgehetmann, M.

1. Hepatol., 64, 1033-1040 (2016)

  Background & Aims Hepatitis E virus (HEV) is a major cause of acute hepatitis as well as chronic infection in immunocompromised individuals; however, in vivo infection models are limited. The aim of this study was to establish a small animal model to improve our understanding of HEV replication mechanisms and permit the development of effective therapeutics. Methods UPA/SCID/beige mice repopulated with primary human hepatocytes were used for infection experiments with HEV genotype (GT) 1 and 3. Virological parameters were determined at the serological and intrahepatic level by real time PCR, immunohistochemistry and RNA in situ hybridization. Results Establishment of HEV infection was achieved after intravenous injection of stool-derived virions and following co-housing with HEV-infected animals but not via inoculation of serum-derived HEV. GT 1 infection resulted in a rapid rise of viremia and high stable titres in serum, liver, bile and faeces of infected mice for more than 25 weeks. In contrast, viremia in GT 3 infected mice developed more slowly and displayed lower titres in all analysed tissues as compared to GT 1. HEV-infected human hepatocytes could be visualized using HEV ORF2 and ORF3 specific antibodies and HEV RNA in situ hybridization probes. Finally, six-week administration of ribavirin led to a strong reduction of viral replication in the serum and liver of GT 1 infected mice. Conclusion We established an efficient model of HEV infection to test the efficacy of antiviral agents and to exploit mechanisms of HEV replication and interaction with human hepatocytes in vivo.  

5.1826           Microglia-specific targeting by novel capsid-modified AAV6 vectors

Rosario, A.K. et al Molecular Therapy-Methods & Clinical Devepment, 3:16026 (2016)   Recombinant adeno-associated viruses (rAAV) have been widely used in gene therapy applications for central nervous system diseases. Though rAAV can efficiently target neurons and astrocytes in mouse brains, microglia, the immune cells of the brain, are refractile to rAAV. To identify AAV capsids with microglia-specific transduction properties, we initially screened the most commonly used serotypes, AAV1–9 and rh10, on primary mouse microglia cultures. While these capsids were not permissive, we then tested the microglial targeting properties of a newly characterized set of modified rAAV6 capsid variants with high tropism for monocytes. Indeed, these newly characterized rAAV6 capsid variants, specially a triply mutated Y731F/Y705F/T492V form, carrying a self-complementary genome and microglia-specific promoters (F4/80 or CD68) could efficiently and selectively transduce microglia in vitro. Delivery of these constructs in mice brains resulted in microglia-specific expression of green fluorescent protein, albeit at modest levels. We further show that CD68 promoter–driven expression of the inflammatory cytokine, interleukin-6, using this capsid variant leads to increased astrogliosis in the brains of wild-type mice. Our study describes the first instance of AAV-targeted microglial gene expression leading to functional modulation of the innate immune system in mice brains. This provides the rationale for utilizing these unique capsid/promoter combinations for microglia-specific gene targeting for modeling or functional studies.  

5.1827           Purification of Virus-Like Particles (VLPs) from Plants

Van Zyl, A.R. and Hitzeroth, I.I. Methods in Mol. Biol., 1404, 569-579 (2016)   Viral coat proteins expressed in plants often form virus-like particles (VLPs) which are good vaccine candidates as they are safe and highly immunogenic and can be easily purified. The VLPs can be purified by rate-zonal density centrifugation which is based on the size of the VLP or they can be purified by isopycnic centrifugation which is a fast and simple method and results in isolation of VLPs with the same density. Details on how to apply both rate-zonal and isopycnic centrifugation for VLP purification from plants are provided in this chapter.  

5.1828           Development of Rabies Virus-Like Particles for Vaccine Applications: Production, Characterization, and Protection Studies

Fontana, D., Etcheverrigaray, M., Kratje, R. and Prieto, C. Methods in Mol. Biol., 1403, 155-166 (2016)   Rabies is a viral infection of the central nervous system for which vaccination is the only treatment possible. Besides preexposure, vaccination is highly recommended for people living in endemic areas, veterinarians, and laboratory workers. Our group has developed rabies virus-like particles (RV-VLPs) with immunogenic features expressed in mammalian cells for vaccine applications. In this chapter the methods to obtain and characterize a stable HEK293 cell line expressing RV-VLPs are detailed. Further, analytical ultracentrifugation steps to purify the obtained VLPs are developed, as well as western blot, dynamic light scattering, and immunogold electron microscopy to analyze the size, distribution, shape, and antigenic conformation of the purified particles. Finally, immunization protocols are described to study the immunogenicity of RV-VLPs.  

5.1829           Brief wide-field photostimuli evoke and modulate oscillatory reverberating activity in cortical networks

Pulizzi, R., Musumeci, G., Van den Haute, C., Van De Vijver, S., Baekelandt, V. & Giugliano, M. Scientific Reports, 6:24701 (2016)   Cell assemblies manipulation by optogenetics is pivotal to advance neuroscience and neuroengineering. In in vivo applications, photostimulation often broadly addresses a population of cells simultaneously, leading to feed-forward and to reverberating responses in recurrent microcircuits. The former arise from direct activation of targets downstream, and are straightforward to interpret. The latter are consequence of feedback connectivity and may reflect a variety of time-scales and complex dynamical properties. We investigated wide-field photostimulation in cortical networks in vitro, employing substrate-integrated microelectrode arrays and long-term cultured neuronal networks. We characterized the effect of brief light pulses, while restricting the expression of channelrhodopsin to principal neurons. We evoked robust reverberating responses, oscillating in the physiological gamma frequency range, and found that such a frequency could be reliably manipulated varying the light pulse duration, not its intensity. By pharmacology, mathematical modelling, and intracellular recordings, we conclude that gamma oscillations likely emerge as in vivo from the excitatory-inhibitory interplay and that, unexpectedly, the light stimuli transiently facilitate excitatory synaptic transmission. Of relevance for in vitro models of (dys)functional cortical microcircuitry and in vivo manipulations of cell assemblies, we give for the first time evidence of network-level consequences of the alteration of synaptic physiology by optogenetics.  

5.1830           Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-Associated Virus Vectors

Boye, S.L., Bennett, A., Scalabrino, M.L., McVullough, K.T., Van Vliet, K., Choudhury, S., Ruan, Q., Peterson, J., Agbandje-McKenna, M. and Boye, S.E.

1. Virol., 90(8), 4215-4231 (2016)

  Adeno-associated viruses (AAVs) currently are being developed to efficiently transduce the retina following noninvasive, intravitreal (Ivt) injection. However, a major barrier encountered by intravitreally delivered AAVs is the inner limiting membrane (ILM), a basement membrane rich in heparan sulfate (HS) proteoglycan. The goal of this study was to determine the impact of HS binding on retinal transduction by Ivt-delivered AAVs. The heparin affinities of AAV2-based tyrosine-to-phenylalanine (Y-F) and threonine-to-valine (T-V) capsid mutants, designed to avoid proteasomal degradation during cellular trafficking, were established. In addition, the impact of grafting HS binding residues onto AAV1, AAV5, and AAV8(Y733F) as well as ablation of HS binding by AAV2-based vectors on retinal transduction was investigated. Finally, the potential relationship between thermal stability of AAV2-based capsids and Ivt-mediated transduction was explored. The results show that the Y-F and T-V AAV2 capsid mutants bind heparin but with slightly reduced affinity relative to that of AAV2. The grafting of HS binding increased Ivt transduction by AAV1 but not by AAV5 or AAV8(Y733F). The substitution of any canonical HS binding residues ablated Ivt-mediated transduction by AAV2-based vectors. However, these same HS variant vectors displayed efficient retinal transduction when delivered subretinally. Notably, a variant devoid of canonical HS binding residues, AAV2(4pMut)ΔHS, was remarkably efficient at transducing photoreceptors. The disparate AAV phenotypes indicate that HS binding, while critical for AAV2-based vectors, is not the sole determinant for transduction via the Ivt route. Finally, Y-F and T-V mutations alter capsid stability, with a potential relationship existing between stability and improvements in retinal transduction by Ivt injection.  

5.1831           Distinct Entry Mechanisms for Nonenveloped and Quasi-Enveloped Hepatitis E Viruses

Yin, X., Ambardekar, C., Lu, Y. and Feng, Z.

1. Virol., 90(8), 4232-4242 (2016)

  The hepatitis E virus (HEV) sheds into feces as nonenveloped virions but circulates in the blood in a membrane-associated, quasi-enveloped form (eHEV). Since the eHEV virions lack viral proteins on the surface, we investigated the entry mechanism for eHEV. We found that compared to nonenveloped HEV virions, eHEV attachment to the cell was much less efficient, requiring a longer inoculation time to reach its maximal infectivity. A survey of cellular internalization pathways identified clathrin-mediated endocytosis as the main route for eHEV entry. Unlike nonenveloped HEV virions, eHEV entry requires Rab5 and Rab7, small GTPases involved in endosomal trafficking, and blocking endosomal acidification abrogated eHEV infectivity. However, low pH alone was not sufficient for eHEV uncoating, suggesting that additional steps are required for entry. Supporting this concept, eHEV infectivity was substantially reduced in cells depleted of Niemann-Pick disease type C1, a lysosomal protein required for cholesterol extraction from lipid, or in cells treated with an inhibitor of lysosomal acid lipase. These data support a model in which the quasi-envelope is degraded within the lysosome prior to virus uncoating, a potentially novel mechanism for virus entry.  

5.1832           Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras

Kuo, L., Hurst-hess, K.R., Koetzner, C.A. and Masters, P.S.

1. Virol., 90(9), 4357-4368 (2016)

  The coronavirus membrane (M) protein is the central actor in virion morphogenesis. M organizes the components of the viral membrane, and interactions of M with itself and with the nucleocapsid (N) protein drive virus assembly and budding. In order to further define M-M and M-N interactions, we constructed mutants of the model coronavirus mouse hepatitis virus (MHV) in which all or part of the M protein was replaced by its phylogenetically divergent counterpart from severe acute respiratory syndrome coronavirus (SARS-CoV). We were able to obtain viable chimeras containing the entire SARS-CoV M protein as well as mutants with intramolecular substitutions that partitioned M protein at the boundaries between the ectodomain, transmembrane domains, or endodomain. Our results show that the carboxy-terminal domain of N protein, N3, is necessary and sufficient for interaction with M protein. However, despite some previous genetic and biochemical evidence that mapped interactions with N to the carboxy terminus of M, it was not possible to define a short linear region of M protein sufficient for assembly with N. Thus, interactions with N protein likely involve multiple linearly discontiguous regions of the M endodomain. The SARS-CoV M chimera exhibited a conditional growth defect that was partially suppressed by mutations in the envelope (E) protein. Moreover, virions of the M chimera were markedly deficient in spike (S) protein incorporation. These findings suggest that the interactions of M protein with both E and S protein are more complex than previously thought.  

5.1833           Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor

Chou, Y-y., Cuevaas, C., Carocci, M., Stubbs, S.H., Ma, M., Cureton, D.K., Chao, L., Evesson, F., He, K., Yang, P.L., Whelan, S.P., Ross, S.R., Kirchhausen, T. and Gaudin, R.

1. Virol., 90(9), 4494-4510 (2016)

  Virus entry into cells is a multistep process that often requires the subversion of subcellular machineries. A more complete understanding of these steps is necessary to develop new antiviral strategies. While studying the potential role of the actin network and one of its master regulators, the small GTPase Cdc42, during Junin virus (JUNV) entry, we serendipitously uncovered the small molecule ZCL278, reported to inhibit Cdc42 function as an entry inhibitor for JUNV and for vesicular stomatitis virus, lymphocytic choriomeningitis virus, and dengue virus but not for the nonenveloped poliovirus. Although ZCL278 did not interfere with JUNV attachment to the cell surface or virus particle internalization into host cells, it prevented the release of JUNV ribonucleoprotein cores into the cytosol and decreased pH-mediated viral fusion with host membranes. We also identified SVG-A astroglial cell-derived cells to be highly permissive for JUNV infection and generated new cell lines expressing fluorescently tagged Rab5c or Rab7a or lacking Cdc42 using clustered regularly interspaced short palindromic repeat (CRISPR)-caspase 9 (Cas9) gene-editing strategies. Aided by these tools, we uncovered that perturbations in the actin cytoskeleton or Cdc42 activity minimally affect JUNV entry, suggesting that the inhibitory effect of ZCL278 is not mediated by ZCL278 interfering with the activity of Cdc42. Instead, ZCL278 appears to redistribute viral particles from endosomal to lysosomal compartments. ZCL278 also inhibited JUNV replication in a mouse model, and no toxicity was detected. Together, our data suggest the unexpected antiviral activity of ZCL278 and highlight its potential for use in the development of valuable new tools to study the intracellular trafficking of pathogens.  

5.1834           Nucleic Acid Binding by Mason-Pfizer Monkey Virus CA Promotes Virus Assembly and Genome Packaging

Füzik, T., Pichalova, R., Schur, F.K.M., Strohalmova, K., Krizova, I., Hadravova, R., Rumlova, M., Briggs, J.A., Ulbrich, P and Rumi, T.

1. Virol., 90(9), 4593-4603 (2016)

  The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle.  

5.1835           Conditional deletion of L1CAM in human neurons impairs both axonal and dendritic arborization and action potential generation

Patzke, C., Acuna, C., Giam, L.R., Wernig, M. and Südhof, T.C.

1. Exp. Med., 213(4), 499-515 (2016)

  Hundreds of L1CAM gene mutations have been shown to be associated with congenital hydrocephalus, severe intellectual disability, aphasia, and motor symptoms. How such mutations impair neuronal function, however, remains unclear. Here, we generated human embryonic stem (ES) cells carrying a conditional L1CAM loss-of-function mutation and produced precisely matching control and L1CAM-deficient neurons from these ES cells. In analyzing two independent conditionally mutant ES cell clones, we found that deletion of L1CAM dramatically impaired axonal elongation and, to a lesser extent, dendritic arborization. Unexpectedly, we also detected an ∼20–50% and ∼20–30% decrease, respectively, in the levels of ankyrinG and ankyrinB protein, and observed that the size and intensity of ankyrinG staining in the axon initial segment was significantly reduced. Overexpression of wild-type L1CAM, but not of the L1CAM point mutants R1166X and S1224L, rescued the decrease in ankyrin levels. Importantly, we found that the L1CAM mutation selectively decreased activity-dependent Na⁺-currents, altered neuronal excitability, and caused impairments in action potential (AP) generation. Thus, our results suggest that the clinical presentations of L1CAM mutations in human patients could be accounted for, at least in part, by cell-autonomous changes in the functional development of neurons, such that neurons are unable to develop normal axons and dendrites and to generate normal APs.  

5.1836           Cardiac Stim1 Silencing Impairs Adaptive Hypertrophy and Promotes Heart Failure Through Inactivation of mTORC2/Akt Signaling

Benard, L., Oh, J.G., Cacheux, M., Lee, A., Nonnenmacher, M., Matasic, D.S., Kphlbrenner, E., Kho, C., Pavoine, C., Hajjar, R.J. and Hulot, J-S. Circulation, 133, 1458-1471 (2016)   Background—Stromal interaction molecule 1 (STIM1) is a dynamic calcium signal transducer implicated in hypertrophic growth of cardiomyocytes. STIM1 is thought to act as an initiator of cardiac hypertrophic response at the level of the sarcolemma, but the pathways underpinning this effect have not been examined. Methods and Results—To determine the mechanistic role of STIM1 in cardiac hypertrophy and during the transition to heart failure, we manipulated STIM1 expression in mice cardiomyocytes by using in vivo gene delivery of specific short hairpin RNAs. In 3 different models, we found that Stim1 silencing prevents the development of pressure overload–induced hypertrophy but also reverses preestablished cardiac hypertrophy. Reduction in STIM1 expression promoted a rapid transition to heart failure. We further showed that Stim1 silencing resulted in enhanced activity of the antihypertrophic and proapoptotic GSK-3β molecule. Pharmacological inhibition of glycogen synthase kinase-3 was sufficient to reverse the cardiac phenotype observed after Stim1 silencing. At the level of ventricular myocytes, Stim1 silencing or inhibition abrogated the capacity for phosphorylation of Akt^S473, a hydrophobic motif of Akt that is directly phosphorylated by mTOR complex 2. We found that Stim1 silencing directly impaired mTOR complex 2 kinase activity, which was supported by a direct interaction between STIM1 and Rictor, a specific component of mTOR complex 2. Conclusions—These data support a model whereby STIM1 is critical to deactivate a key negative regulator of cardiac hypertrophy. In cardiomyocytes, STIM1 acts by tuning Akt kinase activity through activation of mTOR complex 2, which further results in repression of GSK-3β activity.  

5.1837           Comparative Effects of Diet-Induced Lipid Lowering Versus Lipid Lowering Along With Apo A-I Milano Gene Therapy on Regression of Atherosclerosis

Wang, L., Tian, F., Arias, A., Yang, M., Sharifi, B.G. and Shah, P.K.

1. Cardiovasc. Pharmacol. Therapeut.21(3), 320-328 (2016)

  Apolipoprotein A-1 (Apo A-I) Milano, a naturally occurring Arg₁₇₃ to Cys mutant of Apo A-1, has been shown to reduce atherosclerosis in animal models and in a small phase 2 human trial. We have shown the superior atheroprotective effects of Apo A-I Milano (Apo A-IM) gene compared to wild-type Apo A-I gene using transplantation of retrovirally transduced bone marrow in Apo A-I/Apo E null mice. In this study, we compared the effect of dietary lipid lowering versus lipid lowering plus Apo A-IM gene transfer using recombinant adeno-associated virus (rAAV) 8 as vectors on atherosclerosis regression in Apo A-I/Apo E null mice. All mice were fed a high-cholesterol diet from age of 6 weeks until week 20, and at 20 weeks, 10 mice were euthanized to determine the extent of atherosclerosis. After 20 weeks, an additional 20 mice were placed on either a low-cholesterol diet plus empty rAAV (n = 10) to serve as controls or low-cholesterol diet plus 1 single intravenous injection of 1.2 × 10¹² vector genomes of adeno-associated virus (AAV) 8 vectors expressing Apo A-IM (n = 10). At the 40 week time point, intravenous AAV8 Apo A-IM recipients showed a significant regression of atherosclerosis in the whole aorta (P < .01), aortic sinuses (P < .05), and brachiocephalic arteries (P < .05) compared to 20-week-old mice, whereas low-cholesterol diet plus empty vector control group showed no significant regression in lesion size. Immunostaining showed that compared to the 20-week-old mice, there was a significantly reduced macrophage content in the brachiocephalic (P < .05) and aortic sinus plaques (P < .05) of AAV8 Apo A-IM recipients. These data show that although dietary-mediated cholesterol lowering halts progression of atherosclerosis, it does not induce regression, whereas combination of low-cholesterol diet and AAV8 mediated Apo A-I Milano gene therapy induces rapid and significant regression of atherosclerosis in mice. These data provide support for the potential feasibility of this approach for atherosclerosis regression.  

5.1838           Copackaged AAV9 Vectors Promote Simultaneous Immune Tolerance and Phenotypic Correction of Pompe DiseaseNo Access

Doerfler, P.A., Todd, A.G., Clement, N., Falk, D.J., Nayak, S., Herzog, R.W. and Byrne, B.J: Human Gene Therapy, 27(1), 43-59 (2016)   Pompe disease is a progressive neuromuscular disorder caused by lysosomal accumulation of glycogen from a deficiency in acid alpha-glucosidase (GAA). Replacement of the missing enzyme is available by repeated protein infusions; however, efficacy is limited by immune response and inability to restore enzymatic function in the central nervous system. An alternative therapeutic option is adeno-associated virus (AAV)-mediated gene therapy, which results in widespread gene transfer and prolonged transgene expression. Both enzyme replacement therapy (ERT) and gene therapy can elicit anti-GAA immune reactions that dampen their effectiveness and pose life-threatening risks to patient safety. To modulate the immune responses related to gene therapy, we show that a human codon-optimized GAA (coGAA) driven by a liver-specific promoter (LSP) using AAV9 is capable of promoting immune tolerance in a Gaa^−/− mouse model. Copackaging AAV9-LSP-coGAA with the tissue-restricted desmin promoter (AAV9-DES-coGAA) demonstrates the necessary cell autonomous expression in cardiac muscle, skeletal muscle, peripheral nerve, and the spinal cord. Simultaneous high-level expression in liver led to the expansion of GAA-specific regulatory T-cells (T_regs) and induction of immune tolerance. Transfer of T_regs into naïve recipients prevented pathogenic allergic reactions after repeated ERT challenges. Copackaged AAV9 also attenuated preexisting humoral and cellular immune responses, which enhanced the biochemical correction. Our data present a therapeutic design in which simultaneous administration of two copackaged AAV constructs may provide therapeutic benefit and resolve immune reactions in the treatment of multisystem disorders.  

5.1839           Noninvasive Imaging Reveals Stable Transgene Expression in Mouse Airways After Delivery of a Nonintegrating Recombinant Adeno-Associated Viral VectorNo Access

Vidovic, D., Gijsbers, R., Quiles-Jimenez, A., Dooley, J., Van den haute, C., Van der Perren, A., Liston, A., Baekelandt, V., Debyser, Z. and Carlon, M.S. Human Gene Therapy, 27(1), 60-71 (2016)   Gene therapy holds promise to cure a wide range of genetic and acquired diseases. Recent successes in recombinant adeno-associated viral vector (rAAV)-based gene therapy in the clinic for hereditary disorders such as Leber's congenital amaurosis and hemophilia B encouraged us to reexplore an rAAV approach for pulmonary gene transfer. Only limited clinical successes have been achieved for airway gene transfer so far, underscoring the need for further preclinical development of rAAV-based gene therapy for pulmonary disorders. We sought to determine the preclinical potential of an airway-tropic serotype, rAAV2/5, encoding reporter genes when delivered to mouse airways. Although several groups have assessed the stability of gene transfer using a nonintegrating rAAV in mouse airways, long-term stability for more than a year has not been reported. Additionally, an extensive quantitative analysis of the specific cell types targeted by rAAV2/5 using cell-specific markers is lacking. We obtained sustained gene expression in upper and lower airways up to 15 months after vector administration, a substantial proportion of the lifespan of a laboratory mouse. In addition, we demonstrated that readministration of rAAV2/5 to the airways is feasible and increases gene expression 14 months after primary vector administration, despite the presence of circulating neutralizing antibodies. Finally, identification of transduced cell types revealed different subpopulations being targeted by rAAV2/5, with 64% of β-galactosidase-positive cells being ciliated cells, 34% club cells in the conducting airways, and 75% alveolar type II cells in the alveoli at 1 month postinjection. This underscores the therapeutic potential of a nonintegrating rAAV vector to develop a gene therapeutic drug for a variety of pulmonary disorders, such as cystic fibrosis, primary ciliary dyskinesia, and surfactant deficiencies.  

5.1840           Cone-Specific Promoters for Gene Therapy of Achromatopsia and Other Retinal DiseasesNo Access

Ye, G-J., Budzynski, E., Sonnentag, P., Nork, T.M., Sheibani, N., Gurel, Z., Boye, S.L., Peterson, J.J., Boye, S.E., Hauswirth, W.W. and Chulay, J.D. Human Gene Therapy, 27(1), 72-82 (2016)   Adeno-associated viral (AAV) vectors containing cone-specific promoters have rescued cone photoreceptor function in mouse and dog models of achromatopsia, but cone-specific promoters have not been optimized for use in primates. Using AAV vectors administered by subretinal injection, we evaluated a series of promoters based on the human L-opsin promoter, or a chimeric human cone transducin promoter, for their ability to drive gene expression of green fluorescent protein (GFP) in mice and nonhuman primates. Each of these promoters directed high-level GFP expression in mouse photoreceptors. In primates, subretinal injection of an AAV-GFP vector containing a 1.7-kb L-opsin promoter (PR1.7) achieved strong and specific GFP expression in all cone photoreceptors and was more efficient than a vector containing the 2.1-kb L-opsin promoter that was used in AAV vectors that rescued cone function in mouse and dog models of achromatopsia. A chimeric cone transducin promoter that directed strong GFP expression in mouse and dog cone photoreceptors was unable to drive GFP expression in primate cones. An AAV vector expressing a human CNGB3 gene driven by the PR1.7 promoter rescued cone function in the mouse model of achromatopsia. These results have informed the design of an AAV vector for treatment of patients with achromatopsia.  

5.1841           Exosome-associated AAV vector as a robust and convenient neuroscience tool

Hudry, E., Martin, C., Gandhi, S., György, B., Scheffer, D.I., Mu, D., >Merkel, S.F., Mingozzi, F., Fitzpatrick, Z., Dimant, H., Masek, M., Ragan, T., Tan, S., Brisson, A.R., Ramirez, S.H., Hyman, B.T. and Maguire, C.A. Gene Therapy, 23, 380-392 (2016)   Adeno-associated virus (AAV) vectors are showing promise in gene therapy trials and have proven to be extremely efficient biological tools in basic neuroscience research. One major limitation to their widespread use in the neuroscience laboratory is the cost, labor, skill and time-intense purification process of AAV. We have recently shown that AAV can associate with exosomes (exo-AAV) when the vector is isolated from conditioned media of producer cells, and the exo-AAV is more resistant to neutralizing anti-AAV antibodies compared with standard AAV. Here, we demonstrate that simple pelleting of exo-AAV from media via ultracentrifugation results in high-titer vector preparations capable of efficient transduction of central nervous system (CNS) cells after systemic injection in mice. We observed that exo-AAV is more efficient at gene delivery to the brain at low vector doses relative to conventional AAV, even when derived from a serotype that does not normally efficiently cross the blood–brain barrier. Similar cell types were transduced by exo-AAV and conventionally purified vector. Importantly, no cellular toxicity was noted in exo-AAV-transduced cells. We demonstrated the utility and robustness of exo-AAV-mediated gene delivery by detecting direct GFP fluorescence after systemic injection, allowing three-dimensional reconstruction of transduced Purkinje cells in the cerebellum using ex vivo serial two-photon tomography. The ease of isolation combined with the high efficiency of transgene expression in the CNS, may enable the widespread use of exo-AAV as a neuroscience research tool. Furthermore, the ability of exo-AAV to evade neutralizing antibodies while still transducing CNS after peripheral delivery is clinically relevant.  

5.1842           Retinal lipid and glucose metabolism dictates angiogenesis through the lipid sensor Ffar1

Joyal, J-S. et al Nature Med., 22(4), 439-445 (2016)   Tissues with high metabolic rates often use lipids, as well as glucose, for energy, conferring a survival advantage during feast and famine¹. Current dogma suggests that high-energy–consuming photoreceptors depend on glucose^{2, 3}. Here we show that the retina also uses fatty acid β-oxidation for energy. Moreover, we identify a lipid sensor, free fatty acid receptor 1 (Ffar1), that curbs glucose uptake when fatty acids are available. Very-low-density lipoprotein receptor (Vldlr), which is present in photoreceptors⁴ and is expressed in other tissues with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acid^{5, 6}. In the retinas of Vldlr^−/− mice with low fatty acid uptake⁶ but high circulating lipid levels, we found that Ffar1 suppresses expression of the glucose transporter Glut1. Impaired glucose entry into photoreceptors results in a dual (lipid and glucose) fuel shortage and a reduction in the levels of the Krebs cycle intermediate α-ketoglutarate (α-KG). Low α-KG levels promotes stabilization of hypoxia-induced factor 1a (Hif1a) and secretion of vascular endothelial growth factor A (Vegfa) by starved Vldlr^−/− photoreceptors, leading to neovascularization. The aberrant vessels in the Vldlr^−/− retinas, which invade normally avascular photoreceptors, are reminiscent of the vascular defects in retinal angiomatous proliferation, a subset of neovascular age-related macular degeneration (AMD)⁷, which is associated with high vitreous VEGFA levels in humans. Dysregulated lipid and glucose photoreceptor energy metabolism may therefore be a driving force in macular telangiectasia, neovascular AMD and other retinal diseases.  

5.1843           Adeno-associated virus–delivered artificial microRNA extends survival and delays paralysis in an amyotrophic lateral sclerosis mouse model

Stoica, L., Todeasa, S.H., Cabrera, G.T., Salameh, J.S., ElMallah, M.K., Mueller, C., Brown Jr, R.H. and Sena-Esteves, M. Ann. Neurol., 79(4), 687-700 (2016)   Objective Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, paralysis, and death within 5 years of diagnosis. About 10% of cases are inherited, of which 20% are due to mutations in the superoxide dismutase 1 (SOD1) gene. Riluzole, the only US Food and Drug Administration–approved ALS drug, prolongs survival by only a few months. Experiments in transgenic ALS mouse models have shown decreasing levels of mutant SOD1 protein as a potential therapeutic approach. We sought to develop an efficient adeno-associated virus (AAV)-mediated RNAi gene therapy for ALS. Methods A single-stranded AAV9 vector encoding an artificial microRNA against human SOD1 was injected into the cerebral lateral ventricles of neonatal SOD1^G93A mice, and impact on disease progression and survival was assessed. Results This therapy extended median survival by 50% and delayed hindlimb paralysis, with animals remaining ambulatory until the humane endpoint, which was due to rapid body weight loss. AAV9-treated SOD1^G93A mice showed reduction of mutant human SOD1 mRNA levels in upper and lower motor neurons and significant improvements in multiple parameters including the numbers of spinal motor neurons, diameter of ventral root axons, and extent of neuroinflammation in the SOD1^G93A spinal cord. Mice also showed previously unexplored changes in pulmonary function, with AAV9-treated SOD1^G93A mice displaying a phenotype reminiscent of patient pathophysiology. Interpretation These studies clearly demonstrate that an AAV9-delivered SOD1-specific artificial microRNA is an effective and translatable therapeutic approach for ALS.  

5.1844           Testing anti-HIV activity of antiretroviral agents in vitro using flow cytometry analysis of CEM-GFP cells infected with transfection-derived HIV-1 NL4-3

Frezza, C., Grelli, S., Federico, M., Marino-Merlo, F., Mastino, A. and Macchi, B.

1. Med. Virol., 88(6), 979-986 (2016)

  An assay, specifically optimized to evaluate the anti-HIV activity of antiretrovirals by flow cytometry analysis, is described. As widely used anti-HIV agents, zidovudine (AZT), abacavir (ABC), 2′,3′-dideoxyinosine (DDI), lamivudine (3TC), nevirapine (NVP), and efavirenz (EFV), and as drugs of recent approval raltegravir (RAL), etravirine (ETR), and rilpivirine (RPV), were utilized as reference drugs. HIV-1 NL4-3 virus was prepared by transfection of HEK293T cells with purified plasmid DNA and quantified by p24 antigen-capture assay. For infection, CEM-GFP cells were exposed to vehicle or to several concentrations of the drugs for 2 hr at 37°C before HIV-1 NL4-3 was added to each sample. The adsorption was prolonged for 3 hr at 37°C. After 72 hr of incubation, HIV-induced GFP expression in infected CEM-GFP cells was assessed by flow cytometry analysis and expressed as % positive cells. For comparison, p24 production in supernatants was assessed by a commercial ELISA kit. On the basis of IC₅₀ values, the anti-HIV activity, as assayed by this method, was EFV > 3TC > AZT > NVP > DDI > ABC and ETR > RPV > RAL. The comparison between the IC₅₀ values calculated through flow cytometry and p24 production revealed overlapping results, showing that the optimized protocol of CEM-GFP infection with HIV NL4-3 is a suitable method to perform quantitative, rapid and low-expensive screening tests to evaluate the in vitro effect of new candidate anti-HIV drugs.  

5.1845           Subunit-selective N-Methyl-D-aspartate (NMDA) Receptor Signaling through Brefeldin A-resistant Arf Guanine Nucleotide Exchange Factors BRAG1 and BRAG2 during Synapse Maturation

Elagabani, M.N., Brisevac, D., Kintscher, M., Pohle, J., Köhr, G., Schmitz, D. and Kornau, H-C.

1. Biol. Chem., 291(17), 9105-9118 (2016)

  The maturation of glutamatergic synapses in the CNS is regulated by NMDA receptors (NMDARs) that gradually change from a GluN2B- to a GluN2A-dominated subunit composition during postnatal development. Here we show that NMDARs control the activity of the small GTPase ADP-ribosylation factor 6 (Arf6) by consecutively recruiting two related brefeldin A-resistant Arf guanine nucleotide exchange factors, BRAG1 and BRAG2, in a GluN2 subunit-dependent manner. In young cortical cultures, GluN2B and BRAG1 tonically activated Arf6. In mature cultures, Arf6 was activated through GluN2A and BRAG2 upon NMDA treatment, whereas the tonic Arf6 activation was not detectable any longer. This shift in Arf6 regulation and the associated drop in Arf6 activity were reversed by a knockdown of BRAG2. Given their sequential recruitment during development, we examined whether BRAG1 and BRAG2 influence synaptic currents in hippocampal CA1 pyramidal neurons using patch clamp recordings in acute slices from mice at different ages. The number of AMPA receptor (AMPAR) miniature events was reduced by depletion of BRAG1 but not by depletion of BRAG2 during the first 2 weeks after birth. In contrast, depletion of BRAG2 during postnatal weeks 4 and 5 reduced the number of AMPAR miniature events and compromised the quantal sizes of both AMPAR and NMDAR currents evoked at Schaffer collateral synapses. We conclude that both Arf6 activation through GluN2B-BRAG1 during early development and the transition from BRAG1- to BRAG2-dependent Arf6 signaling induced by the GluN2 subunit switch are critical for the development of mature glutamatergic synapses.  

5.1846           Bacterial superglue enables easy development of efficient virus-like particle based vaccines

Thrane, S. et al

1. Nanobiotechnology, 14:30, (2016)

  Background Virus-like particles (VLPs) represent a significant advance in the development of subunit vaccines, combining high safety and efficacy. Their particulate nature and dense repetitive subunit organization makes them ideal scaffolds for display of vaccine antigens. Traditional approaches for VLP-based antigen display require labor-intensive trial-and-error optimization, and often fail to generate dense antigen display. Here we utilize the split-intein (SpyTag/SpyCatcher) conjugation system to generate stable isopeptide bound antigen-VLP complexes by simply mixing of the antigen and VLP components. Results Genetic fusion of SpyTag or SpyCatcher to the N-terminus and/or C-terminus of the Acinetobacter phage AP205 capsid protein resulted in formation of stable, nonaggregated VLPs expressing one SpyCatcher, one SpyTag or two SpyTags per capsid protein. Mixing of spy-VLPs with eleven different vaccine antigens fused to SpyCatcher or SpyTag resulted in formation of antigen-VLP complexes with coupling efficiencies (% occupancy of total VLP binding sites) ranging from 22–88 %. In mice, spy-VLP vaccines presenting the malaria proteins Pfs25 or VAR2CSA markedly increased antibody titer, affinity, longevity and functional efficacy compared to corresponding vaccines employing monomeric proteins. The spy-VLP vaccines also effectively broke B cell self-tolerance and induced potent and durable antibody responses upon vaccination with cancer or allergy-associated self-antigens (PD-L1, CTLA-4 and IL-5). Conclusions The spy-VLP system constitutes a versatile and rapid method to develop highly immunogenic VLP-based vaccines. Our data provide proof-of-concept for the technology’s ability to present complex vaccine antigens to the immune system and elicit robust functional antibody responses as well as to efficiently break B cell self-tolerance. The spy-VLP-system may serve as a generic tool for the cost-effective development of effective VLP-vaccines against both infectious- and non-communicable diseases and could facilitate rapid and unbiased screening of vaccine candidate antigens.  

5.1847           Inhibition of Heat Shock Protein 90 Prevents HIV Rebound

Joshi, P., Maidji, E. and Stoddart, C.A.

1. Biol. Chem., 291(19), 10332-10346 (2016)

  HIV evades eradication because transcriptionally dormant proviral genomes persist in long-lived reservoirs of resting CD4⁺ T cells and myeloid cells, which are the source of viral rebound after cessation of antiretroviral therapy. Dormant HIV genomes readily produce infectious virus upon cellular activation because host transcription factors activated specifically by cell stress and heat shock mediate full-length HIV transcription. The molecular chaperone heat shock protein 90 (Hsp90) is overexpressed during heat shock and activates inducible cellular transcription factors. Here we show that heat shock accelerates HIV transcription through induction of Hsp90 activity, which activates essential HIV-specific cellular transcription factors (NF-κB, NFAT, and STAT5), and that inhibition of Hsp90 greatly reduces gene expression mediated by these factors. More importantly, we show that Hsp90 controls virus transcription in vivo by specific Hsp90 inhibitors in clinical development, tanespimycin (17-(allylamino)-17-demethoxygeldanamycin) and AUY922, which durably prevented viral rebound in HIV-infected humanized NOD scid IL-2Rγ^−/− bone marrow-liver-thymus mice up to 11 weeks after treatment cessation. Despite the absence of rebound viremia, we were able to recover infectious HIV from PBMC with heat shock. Replication-competent virus was detected in spleen cells from these nonviremic Hsp90 inhibitor-treated mice, indicating the presence of a tissue reservoir of persistent infection. Our novel findings provide in vivo evidence that inhibition of Hsp90 activity prevents HIV gene expression in replication-competent cellular reservoirs that would typically cause rebound in plasma viremia after antiretroviral therapy cessation. Alternating or supplementing Hsp90 inhibitors with current antiretroviral therapy regimens could conceivably suppress rebound viremia from persistent HIV reservoirs.  

5.1848           Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors

Ling, C., Yin, Z., Li, J., Zhang, D., Aslanidi, G. and Srivastava, A. Molecular Therapy-Methods in Clin. Develop., 3:16029 (2016)   Although recombinant adeno-associated virus serotype 3 (AAV3) vectors were largely ignored previously, owing to their poor transduction efficiency in most cells and tissues examined, our initial observation of the selective tropism of AAV3 serotype vectors for human liver cancer cell lines and primary human hepatocytes has led to renewed interest in this serotype. AAV3 vectors and their variants have recently proven to be extremely efficient in targeting human and nonhuman primate hepatocytes in vitro as well as in vivo. In the present studies, we wished to evaluate the relative contributions of the cis-acting inverted terminal repeats (ITRs) from AAV3 (ITR3), as well as the trans-acting Rep proteins from AAV3 (Rep3) in the AAV3 vector production and transduction. To this end, we utilized two helper plasmids: pAAVr2c3, which carries rep2 and cap3 genes, and pAAVr3c3, which carries rep3 and cap3 genes. The combined use of AAV3 ITRs, AAV3 Rep proteins, and AAV3 capsids led to the production of recombinant vectors, AAV3-Rep3/ITR3, with up to approximately two to fourfold higher titers than AAV3-Rep2/ITR2 vectors produced using AAV2 ITRs, AAV2 Rep proteins, and AAV3 capsids. We also observed that the transduction efficiency of Rep3/ITR3 AAV3 vectors was approximately fourfold higher than that of Rep2/ITR2 AAV3 vectors in human hepatocellular carcinoma cell lines in vitro. The transduction efficiency of Rep3/ITR3 vectors was increased by ~10-fold, when AAV3 capsids containing mutations in two surface-exposed residues (serine 663 and threonine 492) were used to generate a S663V+T492V double-mutant AAV3 vector. The Rep3/ITR3 AAV3 vectors also transduced human liver tumors in vivo approximately twofold more efficiently than those generated with Rep2/ITR2. Our data suggest that the transduction efficiency of AAV3 vectors can be significantly improved both using homologous Rep proteins and ITRs as well as by capsid optimization. Thus, the combined use of homologous Rep proteins, ITRs, and capsids should also lead to more efficacious other AAV serotype vectors for their optimal use in human gene therapy.  

5.1849           Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system

Buclez, P-O., Florencio, G.D.F., Relizani, K., Beley, C., Garcia, L. and Benchaouir, R. Molecular Therapy-Methods & Clinical Development, 3:16035 (2016)   Recombinant adeno-associated viruses (rAAV) are largely used for gene transfer in research, preclinical developments, and clinical trials. Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. Upstream processes able to produce large amounts of rAAV were developed, particularly those using baculovirus expression vector system. In parallel, downstream processes present a large panel of purification methods, often including multiple and time consuming steps. Here, we show that simple tangential flow filtration, coupled with an optimized iodixanol-based isopycnic density gradient, is sufficient to purify several liters of crude lysate produced by baculovirus expression vector system in only one working day, leading to high titers and good purity of rAAV products. Moreover, we show that the viral vectors retain their in vitro and in vivo functionalities. Our results demonstrate that simple, rapid, and relatively low-cost methods can easily be implemented for obtaining a high-quality grade of gene therapy products based on rAAV technology.  

5.1850           Characterization of the Adeno-Associated Virus 1 and 6 Sialic Acid Binding Site

Huang, L-Y., Patel, A., Ng, R., Miller, E.B., halder, S., McKenna, R., Asokan, A. and Agbandje-McKenna, M.

1. Virol., 90(11), 5219-5230 (2016)

  The adeno-associated viruses (AAVs), which are being developed as gene delivery vectors, display differential cell surface glycan binding and subsequent tissue tropisms. For AAV serotype 1 (AAV1), the first viral vector approved as a gene therapy treatment, and its closely related AAV6, sialic acid (SIA) serves as their primary cellular surface receptor. Toward characterizing the SIA binding site(s), the structure of the AAV1-SIA complex was determined by X-ray crystallography to 3.0 Å. Density consistent with SIA was observed in a pocket located at the base of capsid protrusions surrounding icosahedral 3-fold axes. Site-directed mutagenesis substitution of the amino acids forming this pocket with structurally equivalent residues from AAV2, a heparan sulfate binding serotype, followed by cell binding and transduction assays, further mapped the critical residues conferring SIA binding to AAV1 and AAV6. For both viruses five of the six binding pocket residues mutated (N447S, V473D, N500E, T502S, and W503A) abolished SIA binding, whereas S472R increased binding. All six mutations abolished or decreased transduction by at least 50% in AAV1. Surprisingly, the T502S substitution did not affect transduction efficiency of wild-type AAV6. Furthermore, three of the AAV1 SIA binding site mutants—S472R, V473D, and N500E—escaped recognition by the anti-AAV1 capsid antibody ADK1a. These observations demonstrate that common key capsid surface residues dictate both virus binding and entry processes, as well as antigenic reactivity. This study identifies an important functional capsid surface “hot spot” dictating receptor attachment, transduction efficiency, and antigenicity which could prove useful for vector engineering.  

5.1851           Late Maturation Steps Preceding Selective Nuclear Export and Egress of Progeny Parvovirus

Wolfisberg, R., Kempf, C. and Ros, C.

1. Virol., 90(11), 5462-5474 (2016)

  Although the mechanism is not well understood, growing evidence indicates that the nonenveloped parvovirus minute virus of mice (MVM) may actively egress before passive release through cell lysis. We have dissected the late maturation steps of the intranuclear progeny with the aims of confirming the existence of active prelytic egress and identifying critical capsid rearrangements required to initiate the process. By performing anion-exchange chromatography (AEX), we separated intranuclear progeny particles by their net surface charges. Apart from empty capsids (EC), two distinct populations of full capsids (FC) arose in the nuclei of infected cells. The earliest population of FC to appear was infectious but, like EC, could not be actively exported from the nucleus. Further maturation of this early population, involving the phosphorylation of surface residues, gave rise to a second, late population with nuclear export potential. While capsid surface phosphorylation was strictly associated with nuclear export capacity, mutational analysis revealed that the phosphoserine-rich N terminus of VP2 (N-VP2) was dispensable, although it contributed to passive release. The reverse situation was observed for the incoming particles, which were dephosphorylated in the endosomes. Our results confirm the existence of active prelytic egress and reveal a late phosphorylation event occurring in the nucleus as a selective factor for initiating the process.  

5.1852           Interferon Regulator Factor 8 (IRF8) Limits Ocular Pathology during HSV-1 Infection by Restraining the Activation and Expansion of CD8+ T Cells

Sun, L., St. Leger, A.J., Yu, C-R., He, C., Maahdi, R., Chan, C., Wang, H., Morse III, H.C. and Egwuagu, C.E. PloS One, 11(5), e0155420 (2016)   Interferon Regulatory Factor-8 (IRF8) is constitutively expressed in monocytes and B cell lineages and plays important roles in immunity to pathogens and cancer. Although IRF8 expression is induced in activated T cells, the functional relevance of IRF8 in T cell-mediated immunity is not well understood. In this study, we used mice with targeted deletion of Irf8 in T-cells (IRF8KO) to investigate the role of IRF8 in T cell-mediated responses during herpes simplex virus 1 (HSV-1) infection of the eye. In contrast to wild type mice, HSV-1-infected IRF8KO mice mounted a more robust anti-HSV-1 immune response, which included marked expansion of HSV-1-specific CD8+ T cells, increased infiltration of inflammatory cells into the cornea and trigeminal ganglia (TG) and enhanced elimination of virus within the trigeminal ganglion. However, the consequence of the enhanced immunological response was the development of ocular inflammation, limbitis, and neutrophilic infiltration into the cornea of HSV-1-infected IRF8KO mice. Surprisingly, we observed a marked increase in virus-specific memory precursor effector cells (MPEC) in IRF8KO mice, suggesting that IRF8 might play a role in regulating the differentiation of effector CD8+ T cells to the memory phenotype. Together, our data suggest that IRF8 might play a role in restraining excess lymphocyte proliferation. Thus, modulating IRF8 levels in T cells can be exploited therapeutically to prevent immune-mediated ocular pathology during autoimmune and infectious diseases of the eye.  

5.1853           First-in-class small molecule potentiators of cancer virotherapy

Dornan, M.H. et al Scientific Reports, 6:26786 (2016)   The use of engineered viral strains such as gene therapy vectors and oncolytic viruses (OV) to selectively destroy cancer cells is poised to make a major impact in the clinic and revolutionize cancer therapy. In particular, several studies have shown that OV therapy is safe and well tolerated in humans and can infect a broad range of cancers. Yet in clinical studies OV therapy has highly variable response rates. The heterogeneous nature of tumors is widely accepted to be a major obstacle for OV therapeutics and highlights a need for strategies to improve viral replication efficacy. Here, we describe the development of a new class of small molecules for selectively enhancing OV replication in cancer tissue. Medicinal chemistry studies led to the identification of compounds that enhance multiple OVs and gene therapy vectors. Lead compounds increase OV growth up to 2000-fold in vitro and demonstrate remarkable selectivity for cancer cells over normal tissue ex vivo and in vivo. These small molecules also demonstrate enhanced stability with reduced electrophilicity and are highly tolerated in animals. This pharmacoviral approach expands the scope of OVs to include resistant tumors, further potentiating this transformative therapy. It is easily foreseeable that this approach can be applied to therapeutically enhance other attenuated viral vectors.  

5.1854           Successful disabling of the 5′ UTR of HCV using adeno-associated viral vectors to deliver modular multimeric primary microRNA mimics

Bourhill, T., Arbuthnot, P. and Ely, A.

1. Virol. Methods, 235, 26-33 (2016)

  Chronic hepatitis C virus (HCV) infection is a major health concern and is strongly associated with cirrhosis, hepatocellular carcinoma and liver-related mortality. The HCV genome is the template for both protein translation and viral replication and, being RNA, is amenable to direct genetic silencing by RNA interference (RNAi). HCV is a highly mutable virus and is capable of escaping RNAi-mediated silencing. This has highlighted the importance of developing RNAi-based therapy that simultaneously targets multiple regions of the HCV genome. To develop a multi-targeting RNAi activator, a novel approach for the generation of anti-HCV gene therapy was investigated. Five artificial primary miRNA (pri-miR) were each designed to mimic the naturally occurring monomeric pri-miR-31. Potent knockdown of an HCV reporter was seen with four of the five constructs and were processed according to the intended design. The design of the individual pri-miR mimics enabled the modular assembly into multimeric mimics of any possible conformation. Consequently the four potent pri-miR mimics were used to generate polycistronic cassettes, which showed impressive silencing of an HCV target. To further their application as a gene therapy, recombinant adeno-associated viral (rAAV) vectors that express the polycistronic pri-miR mimics were generated. All AAV-delivered anti-HCV pri-miR mimics significantly knocked down the expression of an HCV target and showed inhibition of HCV replicon replication. Here we describe a protocol for the generation of therapeutic rAAVs that express modular polycistronic pri-miR cassettes allowing for rapid alteration and generation of tailored therapeutic constructs against HCV.  

5.1855           Role of Ultraviolet Radiation in Papillomavirus-Induced Disease

Uberoi, A., Yoshida, S., Frazer, I.H., Pitot, H.C. and lambert, P.F. PloS One, 12(5), e1005664 (2016)   Human papillomaviruses are causally associated with 5% of human cancers. The recent discovery of a papillomavirus (MmuPV1) that infects laboratory mice provides unique opportunities to study the life cycle and pathogenesis of papillomaviruses in the context of a genetically manipulatable host organism. To date, MmuPV1-induced disease has been found largely to be restricted to severely immunodeficient strains of mice. In this study, we report that ultraviolet radiation (UVR), specifically UVB spectra, causes wild-type strains of mice to become highly susceptible to MmuPV1-induced disease. MmuPV1-infected mice treated with UVB develop warts that progress to squamous cell carcinoma. Our studies further indicate that UVB induces systemic immunosuppression in mice that correlates with susceptibility to MmuPV1-associated disease. These findings provide new insight into how MmuPV1 can be used to study the life cycle of papillomaviruses and their role in carcinogenesis, the role of host immunity in controlling papillomavirus-associated pathogenesis, and a basis for understanding in part the role of UVR in promoting HPV infection in humans.  

5.1856           A brain microvasculature endothelial cell-specific viral vector with the potential to treat

neurovascular and neurological diseases

Köbelin, J., Dogbevia, G., Michelfelder, S., Ridder, D.A., Hunger, A., Wenzel, J., Seismann, H., Lampe, M., Bannach, J., Pasparakis, M., Kleinschmidt, J.A., Schwaninger, M. and Trepel, M. EMBO Mol. Med., 8(6), 609-625 (2016)   Gene therapy critically relies on vectors that combine high transduction efficiency with a high degree of target specificity and that can be administered through a safe intravenous route. The lack of suitable vectors, especially for gene therapy of brain disorders, represents a major obstacle. Therefore, we applied an in vivo screening system of random ligand libraries displayed on adeno‐associated viral capsids to select brain‐targeted vectors for the treatment of neurovascular diseases. We identified a capsid variant showing an unprecedented degree of specificity and long‐lasting transduction efficiency for brain microvasculature endothelial cells as the primary target of selection. A therapeutic vector based on this selected viral capsid was used to markedly attenuate the severe cerebrovascular pathology of mice with incontinentia pigmenti after a single intravenous injection. Furthermore, the versatility of this selection system will make it possible to select ligands for additional in vivo targets without requiring previous identification of potential target‐specific receptors.  

5.1857           HTCC: Broad Range Inhibitor of Coronavirus Entry

Milewska, A., Kaminski, K., Ciejka, J., Kosowizc, K., Zeglen, S., Wojarski, J., Nowakowska, M., Szczubialka, K. and Pyrc, K. PloS One, 11(6), e0156552 (2016)   To date, six human coronaviruses have been known, all of which are associated with respiratory infections in humans. With the exception of the highly pathogenic SARS and MERS coronaviruses, human coronaviruses (HCoV-NL63, HCoV-OC43, HCoV-229E, and HCoV-HKU1) circulate worldwide and typically cause the common cold. In most cases, infection with these viruses does not lead to severe disease, although acute infections in infants, the elderly, and immunocompromised patients may progress to severe disease requiring hospitalization. Importantly, no drugs against human coronaviruses exist, and only supportive therapy is available. Previously, we proposed the cationically modified chitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and its hydrophobically-modified derivative (HM-HTCC) as potent inhibitors of the coronavirus HCoV-NL63. Here, we show that HTCC inhibits interaction of a virus with its receptor and thus blocks the entry. Further, we demonstrate that HTCC polymers with different degrees of substitution act as effective inhibitors of all low-pathogenic human coronaviruses.  

5.1858           Intracerebral adeno-associated virus gene delivery of apolipoprotein E2 markedly reduces brain amyloid pathology in Alzheimer's disease mouse models

Zhao, L., Gottesdiener, A.J., Parmar, M., Li, M., Kaminsky, S.M., Chiuchiolo, M.J., Sondhi, D., Sullivan, P.M., Holtzman, D.M., Crystal, R.G. and Paul, S.M. Neurobiology of Aging, 44, 159-172 (2016)   The common apolipoprotein E alleles (ε4, ε3, and ε2) are important genetic risk factors for late-onset Alzheimer's disease, with the ε4 allele increasing risk and reducing the age of onset and the ε2 allele decreasing risk and markedly delaying the age of onset. Preclinical and clinical studies have shown that apolipoprotein E (APOE) genotype also predicts the timing and amount of brain amyloid-β (Aβ) peptide deposition and amyloid burden (ε4 > ε3 > ε2). Using several administration protocols, we now report that direct intracerebral adeno-associated virus (AAV)–mediated delivery of APOE2 markedly reduces brain soluble (including oligomeric) and insoluble Aβ levels as well as amyloid burden in 2 mouse models of brain amyloidosis whose pathology is dependent on either the expression of murine Apoe or more importantly on human APOE4. The efficacy of APOE2 to reduce brain Aβ burden in either model, however, was highly dependent on brain APOE2 levels and the amount of pre-existing Aβ and amyloid deposition. We further demonstrate that a widespread reduction of brain Aβ burden can be achieved through a single injection of vector via intrathalamic delivery of AAV expressing APOE2 gene. Our results demonstrate that AAV gene delivery of APOE2 using an AAV vector rescues the detrimental effects of APOE4 on brain amyloid pathology and may represent a viable therapeutic approach for treating or preventing Alzheimer's disease especially if sufficient brain APOE2 levels can be achieved early in the course of the disease.  

5.1859           Structure of the T4 baseplate and its function in triggering sheath contraction

Taylor, N.M.I., Prokhorov, N.S., Guerrero-Ferreira, R.C., Shneider, M.M., Browning, C., Goldie, K.N., Stahlberg, H. and Leiman, P.G. Nature 533 (7603), 346-352 (2016)   Several systems, including contractile tail bacteriophages, the type VI secretion system and R-type pyocins, use a multiprotein tubular apparatus to attach to and penetrate host cell membranes. This macromolecular machine resembles a stretched, coiled spring (or sheath) wound around a rigid tube with a spike-shaped protein at its tip. A baseplate structure, which is arguably the most complex part of this assembly, relays the contraction signal to the sheath. Here we present the atomic structure of the approximately 6-megadalton bacteriophage T4 baseplate in its pre- and post-host attachment states and explain the events that lead to sheath contraction in atomic detail. We establish the identity and function of a minimal set of components that is conserved in all contractile injection systems and show that the triggering mechanism is universally conserved.  

5.1860           A Preclinical Study Evaluating AAVrh10-Based Gene Therapy for Sanfilippo Syndrome No Access

Winner, L.K., Beard, H., Hassiotis, S., Lau, A.A., Luck, A.J., Hopwood, J.J. and Hemsley, K.M. Mucopolysaccharidosis type IIIA (MPS IIIA) is predominantly a disorder of the central nervous system, caused by a deficiency of sulfamidase (SGSH) with subsequent storage of heparan sulfate-derived oligosaccharides. No widely available therapy exists, and for this reason, a mouse model has been utilized to carry out a preclinical assessment of the benefit of intraparenchymal administration of a gene vector (AAVrh10-SGSH-IRES-SUMF1) into presymptomatic MPS IIIA mice. The outcome has been assessed with time, measuring primary and secondary storage material, neuroinflammation, and intracellular inclusions, all of which appear as the disease progresses. The vector resulted in predominantly ipsilateral distribution of SGSH, with substantially less detected in the contralateral hemisphere. Vector-derived SGSH enzyme improved heparan sulfate catabolism, reduced microglial activation, and, after a time delay, ameliorated GM3 ganglioside accumulation and halted ubiquitin-positive lesion formation in regions local to, or connected by projections to, the injection site. Improvements were not observed in regions of the brain distant from, or lacking connections with, the injection site. Intraparenchymal gene vector administration therefore has therapeutic potential provided that multiple brain regions are targeted with vector, in order to achieve widespread enzyme distribution and correction of disease pathology.  

5.1861           Re-silencing of silent synapses unmasks anti-relapse effects of environmental enrichment

Ma, Y-Y-. Wang, X., Huang, Y., marie, H., Nestler, E.J., Schlüter, O.M. and Dong, Y. PNAS, 113(18), 5089-5094 (2016)   Environmental enrichment (EE) has long been postulated as a behavioral treatment for drug addiction based on its preventive effects in animal models: rodents experiencing prior EE exhibit increased resistance to establishing drug taking and seeking. However, the therapeutic effects of EE, namely, the effects of EE when applied after drug exposure, are often marginal and transient. Using incubation of cue-induced cocaine craving, a rat relapse model depicting progressive intensification of cocaine seeking after withdrawal from cocaine self-administration, our present study reveals that after cocaine withdrawal, in vivo circuit-specific long-term depression (LTD) unmasks the therapeutic power of EE to achieve long-lasting anti-relapse effects. Specifically, our previous results show that cocaine self-administration generates AMPA receptor (AMPAR)-silent excitatory synapses within the basolateral amygdala (BLA) to nucleus accumbens (NAc) projection, and maturation of these silent synapses via recruiting calcium-permeable (CP) AMPARs contributes to incubation of cocaine craving. Here, we show that after cocaine withdrawal and maturation of silent synapses, the BLA-to-NAc projection became highly resistant to EE. However, optogenetic LTD applied to this projection in vivo transiently re-silenced these silent synapses by removing CP-AMPARs. During this transient window, application of EE resulted in the insertion of nonCP-AMPARs, thereby remodeling the “incubated” BLA-to-NAc projection. Consequently, incubation of cocaine craving was decreased persistently. These results reveal a mechanistic basis through which the persistent anti-relapse effects of EE can be unleashed after drug withdrawal.  

5.1862           Structure of faustovirus, a large dsDNA virus

Klose, T., Reteno, D.G., Benamar, S., Hollerbach, A., Colson, P., La Scola, B and Rossmann, M.G. PNAS, 113(22), 6206-6211 (2016)   Many viruses protect their genome with a combination of a protein shell with or without a membrane layer. Here we describe the structure of faustovirus, the first DNA virus (to our knowledge) that has been found to use two protein shells to encapsidate and protect its genome. The crystal structure of the major capsid protein, in combination with cryo-electron microscopy structures of two different maturation stages of the virus, shows that the outer virus shell is composed of a double jelly-roll protein that can be found in many double-stranded DNA viruses. The structure of the repeating hexameric unit of the inner shell is different from all other known capsid proteins. In addition to the unique architecture, the region of the genome that encodes the major capsid protein stretches over 17,000 bp and contains a large number of introns and exons. This complexity might help the virus to rapidly adapt to new environments or hosts.  

5.1863           Widespread Central Nervous System Gene Transfer and Silencing After Systemic Delivery of Novel AAV-AS Vector

Choudhury, S.R. et al Moleculaar Therapy, 24(4), 726-735 (2016)   Effective gene delivery to the central nervous system (CNS) is vital for development of novel gene therapies for neurological diseases. Adeno-associated virus (AAV) vectors have emerged as an effective platform for in vivo gene transfer, but overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. Here, we investigated the possibility of improving CNS transduction of existing AAV capsids by genetically fusing peptides to the N-terminus of VP2 capsid protein. A novel vector AAV-AS, generated by the insertion of a poly-alanine peptide, is capable of extensive gene transfer throughout the CNS after systemic administration in adult mice. AAV-AS is 6- and 15-fold more efficient than AAV9 in spinal cord and cerebrum, respectively. The neuronal transduction profile varies across brain regions but is particularly high in the striatum where AAV-AS transduces 36% of striatal neurons. Widespread neuronal gene transfer was also documented in cat brain and spinal cord. A single intravenous injection of an AAV-AS vector encoding an artificial microRNA targeting huntingtin (Htt) resulted in 33–50% knockdown of Htt across multiple CNS structures in adult mice. This novel AAV-AS vector is a promising platform to develop new gene therapies for neurodegenerative disorders.  

5.1864           Involvement of serotonin 2C receptor RNA editing in accumbal neuropeptide Y expression and behavioural despair

Aoki, M., Watanabe, Y., Yoshimoto, K., Tsujimura, A., Yamamoto, T., Kanamura, N. and Tanaka, M. Eur. J. Neurosci., 43(9), 1219-1228 (2016)   Serotonin 2C receptors (5-HT_2CRs) are widely expressed in the central nervous system, and are associated with various neurological disorders. 5-HT_2CR mRNA undergoes adenosine-to-inosine RNA editing at five sites within its coding sequence, resulting in expression of 24 different isoforms. Several edited isoforms show reduced activity, suggesting that RNA editing modulates serotonergic systems in the brain with causative relevance to neuropsychiatric disorders. Transgenic mice solely expressing the non-edited 5-HT_2CR INI-isoform (INI) or the fully edited VGV-isoform exhibit various phenotypes including metabolic abnormalities, aggressive behaviour, anxiety-like behaviour, and depression-like behaviour. Here, we examined the behavioural phenotype and molecular changes of INI mice on a C57BL/6J background. INI mice showed an enhanced behavioural despair in the forced swimming test, elevated sensitivity to the tricyclic antidepressant desipramine, and significantly decreased serotonin in the nucleus accumbens (NAc), amygdala, and striatum. They also showed reduced expression of neuropeptide Y (NPY) mRNA in the NAc. In addition, by stereotactic injection of adeno-associated virus encoding NPY into the NAc, we demonstrated that accumbal NPY overexpression relieved behavioural despair. Our results suggest that accumbal NPY expression may be regulated by 5-HT_2CR RNA editing, and its impairment may be linked to mood disorders.  

5.1865           A Schisandra-Derived Compound Schizandronic Acid Inhibits Entry of Pan-HCV Genotypes into Human Hepatocytes

Qian, X-J., Zhang, X-L., Zhao, P., Jin, Y-S., Chen, H-S., Xu, Q-Q-., Ren, H., Zhu, S-Y., Tang, H-L., Zhu, Y-Z. and Qi, Z-T. Scientific Reports, 6: 27268 (2016)   Despite recent progress in the development of hepatitis C virus (HCV) inhibitors, cost-effective antiviral drugs, especially among the patients receiving liver transplantations, are still awaited. Schisandra is a traditional medicinal herb used to treat a range of liver disorders including hepatitis for thousands of years in China. To isolate the bioactive compounds of schisandra for the treatment of HCV infection, we screened a schisandra-extracts library and identified a tetracyclic triterpenoid, schizandronic acid (SZA), as a novel HCV entry inhibitor. Our findings suggested that SZA potently inhibited pan-HCV genotype entry into hepatoma cells and primary human hepatocytes without interfering virus binding on cell surface or internalization. However, virion-cell fusion process was impaired in the presence of SZA, along with the increased host membrane fluidity. We also found that SZA inhibited the spread of HCV to the neighboring cells, and combinations of SZA with interferon or telaprevir resulted in additive synergistic effect against HCV. Additionally, SZA diminished the establishment of HCV infection in vivo. The SZA target is different from conventional direct-acting antiviral agents, therefore, SZA is a potential therapeutic compound for the development of effective HCV entry inhibitors, especially for patients who need to prevent HCV reinfection during the course of liver transplantations.  

5.1866           Rotavirus replication and the role of cellular lipid droplets: New therapeutic targets?

Lever, A. and Desselberger, U.

1. Formasan Medical Association, 115, 389-394 (2016)

  Rotaviruses (RVs) are a major cause of acute gastroenteritis in infants and young children worldwide. These viruses infect the villous epithelium of the small intestine. Part of their replication occurs in cytoplasmic inclusion bodies termed viroplasms. Viroplasms and the lipid droplets (LDs) of cellular organelles are known to interact both physically and functionally. Compounds interfering with the homoeostasis of LDs significantly decrease the production of infectious RV progeny. There is considerable scope for more detailed exploration of such compounds as potential antiviral agents for a disease for which at present no specific therapy exists.  

5.1867           A Molecular-Level Account of the Antigenic Hantaviral Surface

Li, S., Rissanen, I., Zeltina, A., Hepojoki, J., Raghwani, J., Harlos, K., Pybus, O.G., Huiskonen, J.T. and Bowden, T.A. Cell Reports, 15, 959-967 (2016)   Hantaviruses, a geographically diverse group of zoonotic pathogens, initiate cell infection through the concerted action of Gn and Gc viral surface glycoproteins. Here, we describe the high-resolution crystal structure of the antigenic ectodomain of Gn from Puumala hantavirus (PUUV), a causative agent of hemorrhagic fever with renal syndrome. Fitting of PUUV Gn into an electron cryomicroscopy reconstruction of intact Gn-Gc spike complexes from the closely related but non-pathogenic Tula hantavirus localized Gn tetramers to the membrane-distal surface of the virion. The accuracy of the fitting was corroborated by epitope mapping and genetic analysis of available PUUV sequences. Interestingly, Gn exhibits greater non-synonymous sequence diversity than the less accessible Gc, supporting a role of the host humoral immune response in exerting selective pressure on the virus surface. The fold of PUUV Gn is likely to be widely conserved across hantaviruses.  

5.1868           In Vivo Hepatic Reprogramming of Myofibroblasts with AAV Vectors as a Therapeutic Strategy for Liver Fibrosis

Milad Rezvani, Regina Español-Suñer, Yann Malato, Laure Dumont, Andrew A. Grimm, Eike Kienle, Julia G. Bindman, Ellen Wiedtke, Bernadette Y. Hsu, Syed J. Naqvi, Robert F. Schwabe, Carlos U. Corvera, Dirk Grimm, Holger Willenbring Cell Stem Cell, 18(6), 809-816 (2016)   Willenbring and colleagues establish in vivo reprogramming of myofibroblasts into hepatocytes as a potential therapeutic strategy for liver fibrosis that addresses its main outcome-determining factors: insufficient hepatocyte function and collagen accumulation. Their use of AAV vectors to deliver the reprogramming factors supports future clinical translation.  

5.1869           Identifying the Target Cells and Mechanisms of Merkel Cell Polyomavirus Infection

Liu,W., Yang, R., Payne, A.S., Schowalter, R.M., Spurgeon, M., Lambert, P.F. Xu, W., Buck,C.B. and   You, J. Cell Host & Microbe, 19(6), 775-787 (82016)   Merkel cell polyomavirus (MCPyV) infection can lead to Merkel cell carcinoma, a lethal skin cancer. Liu et al. identify dermal fibroblasts as the target of productive MCPyV infection in human skin. This study establishes a cell culture model and identifies a kinase inhibitor as a potential therapeutic agent against MCPyV.  

5.1870           Inflammation-induced reversible switch of the neuron-specific enolase promoter from Purkinje

neurons to Bergmann glia

Sawada, Y., Konno, A., Nagaoka, J. and Hirai, H. Scientific Reports, 6:27758 (2016)   Neuron-specific enolase (NSE) is a glycolytic isoenzyme found in mature neurons and cells of neuronal origin. Injecting adeno-associated virus serotype 9 (AAV9) vectors carrying the NSE promoter into the cerebellar cortex is likely to cause the specific transduction of neuronal cells, such as Purkinje cells (PCs) and interneurons, but not Bergmann glia (BG). However, we found BG-predominant transduction without PC transduction along a traumatic needle tract for viral injection. The enhancement of neuroinflammation by the co-application of lipopolysaccharide (LPS) with AAV9 significantly expanded the BG-predominant area concurrently with the potentiated microglial activation. The BG-predominant transduction was gradually replaced by the PC-predominant transduction as the neuroinflammation dissipated. Experiments using glioma cell cultures revealed significant activation of the NSE promoter due to glucose deprivation, suggesting that intracellularly stored glycogen is metabolized through the glycolytic pathway for energy. Activation of the glycolytic enzyme promoter in BG concurrently with inactivation in PC may have pathophysiological significance for the production of lactate in activated BG and the utilization of lactate, which is provided by the BG-PC lactate shuttle, as a primary energy resource in injured PCs.  

5.1871           Ultramicroscopy as a novel tool to unravel the tropism of AAV gene therapy vectors in the brain

Alves, S., Bode, J., Bemelmans, A-P., von Kalle, C., Cartier, N. and Tews, B. Scientific Reports, 6:28272 (2016)   Recombinant adeno-associated viral (AAV) vectors have advanced to the vanguard of gene therapy. Numerous naturally occurring serotypes have been used to target cells in various tissues. There is a strong need for fast and dynamic methods which efficiently unravel viral tropism in whole organs. Ultramicroscopy (UM) is a novel fluorescence microscopy technique that images optically cleared undissected specimens, achieving good resolutions at high penetration depths while being non-destructive. UM was applied to obtain high-resolution 3D analysis of AAV transduction in adult mouse brains, especially in the hippocampus, a region of interest for Alzheimer’s disease therapy. We separately or simultaneously compared transduction efficacies for commonly used serotypes (AAV9 and AAVrh10) using fluorescent reporter expression. We provide a detailed comparative and quantitative analysis of the transduction profiles. UM allowed a rapid analysis of marker fluorescence expression in neurons with intact projections deep inside the brain, in defined anatomical structures. Major hippocampal neuronal transduction was observed with both vectors, with slightly better efficacy for AAV9 in UM. Glial response and synaptic marker expression did not change post transduction.We propose UM as a novel valuable complementary tool to efficiently and simultaneously unravel tropism of different viruses in a single non-dissected adult rodent brain.  

5.1872           Characterization of the Inflammasome in Human Kupffer Cells in Response to Synthetic Agonists and Pathogens

Zannetti, C. et al

1. Immunol., 197(1), 356-367 (2016)

  The liver is the largest gland in the human body and functions as an innate immune organ. Liver macrophages called Kupffer cells (KC) constitute the largest group of macrophages in the human body. Innate immune responses involving KC represent the first line of defense against pathogens in the liver. Human monocyte-derived macrophages have been used to characterize inflammasome responses that lead to the release of the proinflammatory cytokines IL-1β and IL-18, but it has not yet been determined whether human KC contain functional inflammasomes. We show, to our knowledge for the first time, that KC express genes and proteins that make up several different inflammasome complexes. Moreover, activation of KC in response to the absent in melanoma 2 (AIM2) inflammasome led to the production of IL-1β and IL-18, which activated IL-8 transcription and hepatic NK cell activity, respectively. Other inflammasome responses were also activated in response to selected bacteria and viruses. However, hepatitis B virus inhibited the AIM2 inflammasome by reducing the mRNA stability of IFN regulatory factor 7, which regulated AIM2 transcription. These data demonstrate the production of IL-1β and IL-18 in KC, suggesting that KC contain functional inflammasomes that could be important players in the innate immune response following certain infections of the liver. We think our findings could potentially aid therapeutic approaches against chronic liver diseases that activate the inflammasome.  

5.1873           Systemically administered AAV9-sTRAIL combats invasive glioblastoma in a patient-derived orthotopic xenograft model

Crommentuijn, M.H.W., Kantar, R., Noske, D.P., Vandertorp, W.P., Badr, C.E., Würdinger, T., Maguire, C.A. and Tannous, B.A. Molecular Therapy-Oncolytics, 3:16017 (2016)   Adeno-associated virus (AAV) vectors expressing tumoricidal genes injected directly into brain tumors have shown some promise, however, invasive tumor cells are relatively unaffected. Systemic injection of AAV9 vectors provides widespread delivery to the brain and potentially the tumor/microenvironment. Here we assessed AAV9 for potential glioblastoma therapy using two different promoters driving the expression of the secreted anti-cancer agent sTRAIL as a transgene model; the ubiquitously active chicken β-actin (CBA) promoter and the neuron-specific enolase (NSE) promoter to restrict expression in brain. Intravenous injection of AAV9 vectors encoding a bioluminescent reporter showed similar distribution patterns, although the NSE promoter yielded 100-fold lower expression in the abdomen (liver), with the brain-to-liver expression ratio remaining the same. The main cell types targeted by the CBA promoter were astrocytes, neurons and endothelial cells, while expression by NSE promoter mostly occurred in neurons. Intravenous administration of either AAV9-CBA-sTRAIL or AAV9-NSE-sTRAIL vectors to mice bearing intracranial patient-derived glioblastoma xenografts led to a slower tumor growth and significantly increased survival, with the CBA promoter having higher efficacy. To our knowledge, this is the first report showing the potential of systemic injection of AAV9 vector encoding a therapeutic gene for the treatment of brain tumors.  

5.1874           AAV Natural Infection Induces Broad Cross-Neutralizing Antibody Responses to Multiple AAV Serotypes in Chimpanzees

Calcedo, R. and Wilson, J.M. Human Gene Therapy Clinical Development, 27(2), 79-82 (2016)   Cross-sectional studies of primates have revealed that natural neutralizing antibody (NAb) responses to adeno-associated viruses (AAV) span multiple serotypes. This differs from the phenotype of the NAb response to an AAV vector delivered to seronegative nonhuman primates that is typically restricted to the administered AAV serotype. To better understand the mechanism by which natural AAV infections result in broad NAb responses, we conducted a longitudinal study spanning 10 years in which we evaluated serum-circulating AAV NAb levels in captive-housed chimpanzees. In a cohort of 25 chimpanzees we identified 3 distinct groups of animals: those that never seroconverted to AAV (naïve), those that were persistently seropositive (chronic), and those that seroconverted during the 10-year period (acute). For the chronic group we found a broad seroresponse characterized by NAbs reacting to multiple AAV serotypes. A similar cross-neutralization pattern of NAbs was observed in the acute group. These data support our hypothesis that a single natural infection with AAV induces a broadly cross-reactive NAb response to multiple AAV serotypes.  

5.1875           Cyclooxygenase inhibition targets neurons to prevent early behavioural decline in Alzheimer’s disease model mice

Woodling, N. et al Brain, 139, 2063-2081 (2016)   Identifying preventive targets for Alzheimer’s disease is a central challenge of modern medicine. Non-steroidal anti-inflammatory drugs, which inhibit the cyclooxygenase enzymes COX-1 and COX-2, reduce the risk of developing Alzheimer’s disease in normal ageing populations. This preventive effect coincides with an extended preclinical phase that spans years to decades before onset of cognitive decline. In the brain, COX-2 is induced in neurons in response to excitatory synaptic activity and in glial cells in response to inflammation. To identify mechanisms underlying prevention of cognitive decline by anti-inflammatory drugs, we first identified an early object memory deficit in APP_Swe-PS1_ΔE9 mice that preceded previously identified spatial memory deficits in this model. We modelled prevention of this memory deficit with ibuprofen, and found that ibuprofen prevented memory impairment without producing any measurable changes in amyloid-β accumulation or glial inflammation. Instead, ibuprofen modulated hippocampal gene expression in pathways involved in neuronal plasticity and increased levels of norepinephrine and dopamine. The gene most highly downregulated by ibuprofen was neuronal tryptophan 2,3-dioxygenase (Tdo2), which encodes an enzyme that metabolizes tryptophan to kynurenine. TDO2 expression was increased by neuronal COX-2 activity, and overexpression of hippocampal TDO2 produced behavioural deficits. Moreover, pharmacological TDO2 inhibition prevented behavioural deficits in APP_Swe-PS1_ΔE9 mice. Taken together, these data demonstrate broad effects of cyclooxygenase inhibition on multiple neuronal pathways that counteract the neurotoxic effects of early accumulating amyloid-β oligomers.  

5.1876           Dengue virus NS1 enhances viral replication and pro-inflammatory cytokine production in human dendritic cells

Alayli, F. and Scholle, F. Virology, 496, 227-236 (2016)   Dengue virus (DV) has become the most prevalent arthropod borne virus due to globalization and climate change. It targets dendritic cells during infection and leads to production of pro-inflammatory cytokines and chemokines. Several DV non-structural proteins (NS) modulate activation of human dendritic cells. We investigated the effect of DV NS1 on human monocyte-derived dendritic cells (mo-DCs) during dengue infection. NS1 is secreted into the serum of infected individuals where it interacts with various immune mediators and cell types. We purified secreted DV1 NS1 from supernatants of 293T cells that over-express the protein. Upon incubation with mo-DCs, we observed NS1 uptake and enhancement of early DV1 replication. As a consequence, mo-DCs that were pre-exposed to NS1 produced more pro-inflammatory cytokines in response to subsequent DV infection compared to DCs exposed to heat-inactivated NS1 (HNS1). Therefore the presence of exogenous NS1 is able to modulate dengue infection in mo-DCs.  

5.1877           Transduction of interleukin-10 through renal artery attenuates vascular neointimal proliferation and infiltration of immune cells in rat renal allograft

Xie, J., Li, X:, Meng, D., Liang, Q., Wang, X., Wang, L., Wang, R., Xiang, M. and Chen, S. Immunol. Letters, 176, 105-113 (2016)   Renal transplantation is the treatment of choice for end-stage renal failure. Although acute rejection is not a major issue anymore, chronic rejection, especially vascular rejection, is still a major factor that might lead to allograft dysfunction on the long term. The role of the local immune-regulating cytokine interleukin-10 (IL-10) in chronic renal allograft is unclear. Many clinical observations showed that local IL-10 level was negatively related to kidney allograft function. It is unknown this negative relationship was the result of immunostimulatory property or insufficient immunosuppression property of local IL-10. We performed ex vivo transduction before transplantation through artery of the renal allograft using adeno-associated viral vectors carrying IL-10 gene. Twelve weeks after transplantation, we found intrarenal IL-10 gene transduction significantly inhibited arterial neointimal proliferation, the number of occluded intrarenal artery, interstitial fibrosis, peritubular capillary congestion and glomerular inflammation in renal allografts compared to control allografts receiving PBS or vectors carrying YFP. IL-10 transduction increased serum IL-10 level at 4 weeks but not at 8 and 12 weeks. Renal IL-10 level increased while serum creatinine decreased significantly in IL-10 group at 12 weeks compared to PBS or YFP controls. Immunohistochemical staining showed unchanged total T cells (CD3) and B cells (CD45R/B220), decreased cytotoxic T cells (CD8), macrophages (CD68) and increased CD4+ and FoxP3+ cells in IL-10 group. In summary, intrarenal IL-10 inhibited the allograft rejection while modulated immune response.  

5.1878           LATS-YAP/TAZ controls lineage specification by regulating TGFβ signaling and Hnf4α expression during liver development

Lee, D-H., Park, J.O., Kim, T-S., Kim, S-K., Kim, T-h., Kim, M-c., Park, G.S., Kim, J-H., Kuninaka, S., Olson, E.N., Saya, H., Kim, S-Y., Lee, H. and Lim, D-S. Nature Communications, 7:11961 (2016)   The Hippo pathway regulates the self-renewal and differentiation of various adult stem cells, but its role in cell fate determination and differentiation during liver development remains unclear. Here we report that the Hippo pathway controls liver cell lineage specification and proliferation separately from Notch signalling, using mice and primary hepatoblasts with liver-specific knockout of Lats1 and Lats2 kinase, the direct upstream regulators of YAP and TAZ. During and after liver development, the activation of YAP/TAZ induced by loss of Lats1/2 forces hepatoblasts or hepatocytes to commit to the biliary epithelial cell (BEC) lineage. It increases BEC and fibroblast proliferation by up-regulating TGFβ signalling, but suppresses hepatoblast to hepatocyte differentiation by repressing Hnf4α expression. Notably, oncogenic YAP/TAZ activation in hepatocytes induces massive p53-dependent cell senescence/death. Together, our results reveal that YAP/TAZ activity levels govern liver cell differentiation and proliferation in a context-dependent manner.  

5.1879           CUEDC2 modulates cardiomyocyte oxidative capacity by regulating GPX1 stability

Jian, Z., Liang, B., Pan, X., Xu, G., Guo, S-S., Li, T., Zhou, T., Xiao, Y-B. and Li, A-L. EMBO Mol. Med., 8(7), 813-829 (2016)   The irreversible loss of cardiomyocytes due to oxidative stress is the main cause of heart dysfunction following ischemia/reperfusion (I/R) injury and ageing‐induced cardiomyopathy. Here, we report that CUEDC2, a CUE domain‐containing protein, plays a critical role in oxidative stress‐induced cardiac injury. Cuedc2^−/− cardiomyocytes exhibited a greater resistance to oxidative stress‐induced cell death. Loss of CUEDC2 enhanced the antioxidant capacity of cardiomyocytes, promoted reactive oxygen species (ROS) scavenging, and subsequently inhibited the redox‐dependent activation of signaling pathways. Notably, CUEDC2 promoted E3 ubiquitin ligases tripartite motif‐containing 33 (TRIM33)‐mediated the antioxidant enzyme, glutathione peroxidase 1 (GPX1) ubiquitination, and proteasome‐dependent degradation. Ablation of CUEDC2 upregulated the protein level of GPX1 in the heart significantly. Strikingly, in vivo, the infarct size of Cuedc2^−/− heart was significantly decreased after I/R injury, and aged Cuedc2^−/− mice preserved better heart function as the overall ROS levels in their hearts were significantly lower. Our results demonstrated a novel role of CUEDC2 in cardiomyocyte death regulation. Manipulating CUEDC2 level might be an attractive therapeutic strategy for promoting cardiomyocyte survival following oxidative stress‐induced cardiac injury.  

5.1880           High-efficiency transduction and specific expression of ChR2opt for optogenetic manipulation of primary cortical neurons mediated by recombinant adeno-associated viruses

Jin, L., Lange, W., Kempmann, A., Maybeck, V., Günther, A., Gruteser, N., Baumann, A. and Offenhäuser, A.

1. Biotechnol., 233, 171-180 (2016)

  In recent years, optogenetic approaches have significantly advanced the experimental repertoire of cellular and functional neuroscience. Yet, precise and reliable methods for specific expression of optogenetic tools remain challenging. In this work, we studied the transduction efficiency of seven different adeno-associated virus (AAV) serotypes in primary cortical neurons and revealed recombinant (r) AAV6 to be the most efficient for constructs under control of the cytomegalovirus (CMV) promoter. To further specify expression of the transgene, we exchanged the CMV promoter for the human synapsin (hSyn) promoter. In primary cortical-glial mixed cultures transduced with hSyn promoter-containing rAAVs, expression of ChR2opt (a Channelrhodopsin-2 variant) was limited to neurons. In these neurons action potentials could be reliably elicited upon laser stimulation (473 nm). The use of rAAV serotype alone to restrict expression to neurons results in a lower transduction efficiency than the use of a broader transducing serotype with specificity conferred via a restrictive promoter. Cells transduced with the hSyn driven gene expression were able to elicit action potentials with more spatially and temporally accurate illumination than neurons electrofected with the CMV driven construct. The hSyn promoter is particularly suited to use in AAVs due to its small size. These results demonstrate that rAAVs are versatile tools to mediate specific and efficient transduction as well as functional and stable expression of transgenes in primary cortical neurons.  

5.1881           Large scale production of a mammalian cell derived quadrivalent hepatitis C virus like particle vaccine

Earnest-Silveira, L., Christiansen, D., Herrmann, S., Ralph, S.A., Das, S., GOwans, E.J. and Torresi, J.

1. Virol Methods, 236, 87-92 (2016)

  A method for the large-scale production of a quadrivalent mammalian cell derived hepatitis C virus-like particles (HCV VLPs) is described. The HCV core E1 and E2 coding sequences of genotype 1a, 1b, 2a or 3a were co-expressed in Huh7 cell factories using a recombinant adenoviral expression system. The structural proteins self-assembled into VLPs that were purified from Huh7 cell lysates by iodixanol ultracentrifugation and Stirred cell ultrafiltration. Electron microscopy, revealed VLPs of the different genotypes that are morphologically similar. Our results show that it is possible to produce large quantities of individual HCV genotype VLPs with relative ease thus making this approach an alternative for the manufacture of a quadrivalent mammalian cell derived HCV VLP vaccine.  

5.1882           Rotavirus Replication: the Role of Lipid Droplets

Cheung, W., Gaunt, E., Lever, A. and Desselberger, U. Viral Gastroenteritis, 175-187 (2016)   As is the case for all viruses, their replication depends on the interaction of viral components with cellular organelles and proteins. Here the role of the cellular organelles lipid droplets for rotavirus replication is reviewed. Newly formed rotavirus viroplasms interact with lipid droplets during the replication cycle, as shown by confocal microscopy, fluorescence resonance energy transfer, equilibrium ultracentrifugation of rotavirus-infected cell extracts, and lipid analyses. Disturbance of the cellular lipid droplet homoeostasis with chemical compounds inducing lipolysis or blockage of fatty acid biosynthesis was shown to reduce the number and size of viroplasms, the amount of newly synthesized rotavirus dsRNA and the infectivity of viral progeny. Thus, rotavirus has joined the growing list of viruses interacting with lipid droplets during their replication, opening a new area for search of antivirals.  

5.1883           Superior In vivo Transduction of Human Hepatocytes Using Engineered AAV3 Capsid

Vercauteren, K., Hoffman, B.E., Zolotukhin, I., Keeler, G.D., Xiao, J.W., Basner-Tschakarjan, E., High, K.A., Ertl, H.C.J., Rice, C.M., Srivastava, A., de Jong, Y.P. and Herzog, R.W. Molecular Therapy, 24(6), 1042-1049 (2016)   Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in “humanized” mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (~3-times, ~8-times, and ~80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.  

5.1884           Pulmonary Targeting of Adeno-associated Viral Vectors by Next-generation Sequencing-guided Screening of Random Capsid Displayed Peptide Libraries

Köbelin, J., Sieber, T., Michelfelder, S., Lunding, L., Spies, E., Hunger, A., Alawi, M., Rapti, K., Indenbirken, D., Müller, O.J., Pasqualini, R., Arap, W., Kleinschmidt, J.A. and Trepel, M. Molecular Therapy, 24(6), 1050-1061 (2016)   Vectors mediating strong, durable, and tissue-specific transgene expression are mandatory for safe and effective gene therapy. In settings requiring systemic vector administration, the availability of suited vectors is extremely limited. Here, we present a strategy to select vectors with true specificity for a target tissue from random peptide libraries displayed on adeno-associated virus (AAV) by screening the library under circulation conditions in a murine model. Guiding the in vivo screening by next-generation sequencing, we were able to monitor the selection kinetics and to determine the right time point to discontinue the screening process. The establishment of different rating scores enabled us to identify the most specifically enriched AAV capsid candidates. As proof of concept, a capsid variant was selected that specifically and very efficiently delivers genes to the endothelium of the pulmonary vasculature after intravenous administration. This technical approach of selecting target-specific vectors in vivo is applicable to any given tissue of interest and therefore has broad implications in translational research and medicine.  

5.1885           Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons

Tabet, R. et al PNAS, 113(26), E3619-E3628 (2016)   Fragile X syndrome (FXS) is caused by the absence of the Fragile X Mental Retardation Protein (FMRP) in neurons. In the mouse, the lack of FMRP is associated with an excessive translation of hundreds of neuronal proteins, notably including postsynaptic proteins. This local protein synthesis deregulation is proposed to underlie the observed defects of glutamatergic synapse maturation and function and to affect preferentially the hundreds of mRNA species that were reported to bind to FMRP. How FMRP impacts synaptic protein translation and which mRNAs are most important for the pathology remain unclear. Here we show by cross-linking immunoprecipitation in cortical neurons that FMRP is mostly associated with one unique mRNA: diacylglycerol kinase kappa (Dgkκ), a master regulator that controls the switch between diacylglycerol and phosphatidic acid signaling pathways. The absence of FMRP in neurons abolishes group 1 metabotropic glutamate receptor-dependent DGK activity combined with a loss of Dgkκ expression. The reduction of Dgkκ in neurons is sufficient to cause dendritic spine abnormalities, synaptic plasticity alterations, and behavior disorders similar to those observed in the FXS mouse model. Overexpression of Dgkκ in neurons is able to rescue the dendritic spine defects of the Fragile X Mental Retardation 1 gene KO neurons. Together, these data suggest that Dgkκ deregulation contributes to FXS pathology and support a model where FMRP, by controlling the translation of Dgkκ, indirectly controls synaptic proteins translation and membrane properties by impacting lipid signaling in dendritic spine.  

5.1886           Expression of an Activated Integrin Promotes Long-Distance Sensory Axon Regeneration in the Spinal Cord

Cheah, M., Andrews, M.R., Chew, D.J., Moloney, E.B., Verhaagen, J., Fässler, R. and Fawcett, J.W.

1. Neurosci., 36(27), 7283-7297 (2016)

  After CNS injury, axon regeneration is blocked by an inhibitory environment consisting of the highly upregulated tenascin-C and chondroitin sulfate proteoglycans (CSPGs). Tenascin-C promotes growth of axons if they express a tenascin-binding integrin, particularly α9β1. Additionally, integrins can be inactivated by CSPGs, and this inhibition can be overcome by the presence of a β1-binding integrin activator, kindlin-1. We examined the synergistic effect of α9 integrin and kindlin-1 on sensory axon regeneration in adult rat spinal cord after dorsal root crush and adeno-associated virus transgene expression in dorsal root ganglia. After 12 weeks, axons from C6–C7 dorsal root ganglia regenerated through the tenascin-C-rich dorsal root entry zone into the dorsal column up to C1 level and above (>25 mm axon length) through a normal pathway. Animals also showed anatomical and electrophysiological evidence of reconnection to the dorsal horn and behavioral recovery in mechanical pressure, thermal pain, and ladder-walking tasks. Expression of α9 integrin or kindlin-1 alone promoted much less regeneration and recovery.  

5.1887           Hepatitis E Virus (HEV) ORF2 Antigen Levels Differentiate Between Acute and Chronic HEV Infection

Behrendt, P., Bremer, B., Todt, D., Brown, R.J.P., heim, A., Manus, M.P., Steinmann, E. and Wedemeyer, H.

1. Infectious Diseases, 214(3), 361-368 (2016)

  Background. Hepatitis E virus (HEV) genotype 3 infections are frequent in Europe and North America, with acute and chronic courses described in the literature. HEV RNA detection by real-time polymerase chain reaction (PCR) is the gold standard for diagnosis. Recently, an anti-HEV antigen (Ag)–specific enzyme-linked immunosorbent assay (ELISA) directed against the HEV capsid became commercially available. The effectiveness of anti-HEV Ag–specific ELISA at detecting HEV genotype 3 infections remains undefined. Methods. The performance of anti-HEV Ag–ELISA was compared with that of real-time PCR, using sera from a cohort of acutely infected individuals, in addition to a cohort of chronically infected patients undergoing ribavirin therapy. Furthermore, virion properties were evaluated by density fractionation. Results. Anti-HEV Ag–specific ELISA was less sensitive than real-time PCR at detection of HEV infection. Anti-HEV Ag–specific ELISA revealed significantly higher HEV Ag in chronically infected individuals as compared to acutely infected patients, with high sensitivity and specificity to distinguish acute from chronic HEV infection. Of note, HEV Ag remained detectable for >100 days after HEV RNA clearance in ribavirin-treated patients with chronic HEV. Density gradients revealed the presence of membrane-associated virions in the sera, with a different distribution as compared to HEV RNA. Conclusions. The anti-HEV Ag–specific ELISA is less sensitive than HEV RNA real-time PCR but represents a useful tool to discriminate chronic from acute infection.  

5.1888           Pharmacodynamics of anti-HIV gene therapy using viral vectors and targeted endonucleases

Roychoudhury, P., De Silva Feelixge, H.S., Pietz, H.L., Stone, D., Jerome, K.R. and Schiffer, J.T.

1. Antimicrob. Chemother., 71(8), 2089-2099 (2016)

  Objectives A promising curative approach for HIV is to use designer endonucleases that bind and cleave specific target sequences within latent genomes, resulting in mutations that render the virus replication incompetent. We developed a mathematical model to describe the expression and activity of endonucleases delivered to HIV-infected cells using engineered viral vectors in order to guide dose selection and predict therapeutic outcomes. Methods We developed a mechanistic model that predicts the number of transgene copies expressed at a given dose in individual target cells from fluorescence of a reporter gene. We fitted the model to flow cytometry datasets to determine the optimal vector serotype, promoter and dose required to achieve maximum expression. Results We showed that our model provides a more accurate measure of transduction efficiency compared with gating-based methods, which underestimate the percentage of cells expressing reporter genes. We identified that gene expression follows a sigmoid dose–response relationship and that the level of gene expression saturation depends on vector serotype and promoter. We also demonstrated that significant bottlenecks exist at the level of viral uptake and gene expression: only ∼1 in 220 added vectors enter a cell and, of these, depending on the dose and promoter used, between 1 in 15 and 1 in 1500 express transgene. Conclusions Our model provides a quantitative method of dose selection and optimization that can be readily applied to a wide range of other gene therapy applications. Reducing bottlenecks in delivery will be key to reducing the number of doses required for a functional cure.    

5.1889           Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver

Sun, T., Yi, H., Yang, C., Kishani, P.S. and Sun, B.

1. Biol. Chem., 291(32), 16479-16484 (2016)

  A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function.  

5.1890           Mutants at the 2-Fold Interface of Adeno-associated Virus Type 2 (AAV2) Structural Proteins Suggest a Role in Viral Transcription for AAV Capsids

Aydemir, F., Salganik, M., Resztak, J., Singh, J., Bennett, A., Agbandje-McKenna, M. and Muzyczka, N.

1. Virol., 90(16), 7196-7204 (2016)

  We previously reported that an amino acid substitution, Y704A, near the 2-fold interface of adeno-associated virus (AAV) was defective for transcription of the packaged genome (M. Salganik, F. Aydemir, H. J. Nam, R. McKenna, M. Agbandje-McKenna, and N. Muzyczka, J Virol 88:1071–1079, 2013, doi: http://dx.doi.org/10.1128/JVI.02093-13). In this report, we have characterized the defect in 6 additional capsid mutants located in a region ∼30 Å in diameter on the surface of the AAV type 2 (AAV2) capsid near the 2-fold interface. These mutants, which are highly conserved among primate serotypes, displayed a severe defect (3 to 6 logs) in infectivity. All of the mutants accumulated significant levels of uncoated DNA in the nucleus, but none of the mutants were able to accumulate significant amounts of genomic mRNA postinfection. In addition, wild-type (wt) capsids that were bound to the conformational antibody A20, which is known to bind the capsid surface in the region of the mutants, were also defective for transcription. In all cases, the mutant virus particles, as well as the antibody-bound wild-type capsids, were able to enter the cell, travel to the nucleus, uncoat, and synthesize a second strand but were unable to transcribe their genomes. Taken together, the phenotype of these mutants provides compelling evidence that the AAV capsid plays a role in the transcription of its genome, and the mutants map this functional region on the surface of the capsid near the 2-fold interface. This appears to be the first example of a viral structural protein that is also involved in the transcription of the viral genome that it delivers to the nucleus.  

5.1891           Hepatitis C virus suppresses Hepatocyte Nuclear Factor 4 alpha, a key regulator of hepatocellular carcinoma

Vallianou, I., Dafou, D., Vassilaki, N., mavromara, P.and Hadzopoulou-Cladaras, M. Int. J. Biochem. Cell Biol., 78, 315-326 (2016)   Hepatitis C Virus (HCV) infection presents with a disturbed lipid profile and can evolve to hepatic steatosis and hepatocellular carcinoma (HCC). Hepatocyte Nuclear Factor 4 alpha (HNF4α) is the most abundant transcription factor in the liver, a key regulator of hepatic lipid metabolism and a critical determinant of Epithelial to Mesenchymal Transition and hepatic development. We have previously shown that transient inhibition of HNF4α initiates transformation of immortalized hepatocytes through a feedback loop consisting of miR-24, IL6 receptor (IL6R), STAT3, miR-124 and miR-629, suggesting a central role of HNF4α in HCC. However, the role of HNF4α in Hepatitis C Virus (HCV)-related hepatocarcinoma has not been evaluated and remains controversial. In this study, we provide strong evidence suggesting that HCV downregulates HNF4α expression at both transcriptional and translational levels. The observed decrease of HNF4α expression correlated with the downregulation of its downstream targets, HNF1α and MTP. Ectopic overexpression of HCV proteins also exhibited an inhibitory effect on HNF4α levels. The inhibition of HNF4α expression by HCV appeared to be mediated at transcriptional level as HCV proteins suppressed HNF4α gene promoter activity. HCV also up-regulated IL6R, activated STAT3 protein phosphorylation and altered the expression of acute phase genes. Furthermore, as HCV triggered the loss of HNF4α a consequent change of miR-24, miR-629 or miR-124 was observed. Our findings demonstrated that HCV-related HCC could be mediated through HNF4α-microRNA deregulation implying a possible role of HNF4α in HCV hepatocarcinogenesis. HCV inhibition of HNF4α could be sustained to promote HCC.  

5.1892           Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells

Langout-Astrie, C.J., yang, Z., Polisetti, S.M., Welsbie, D.S., Hauswirth, W.W., Zack, D.J., Merbs, S.L. and Enke, R.A. Exp. Eye Res., 151, 61-67 (2016)   Targeted expression of Cre recombinase in murine retinal ganglion cells (RGCs) by viral vector is an effective strategy for creating tissue-specific gene knockouts for investigation of genetic contribution to RGC degeneration associated with optic neuropathies. Here we characterize dosage, efficacy and toxicity for sufficient intravitreal delivery of a capsid mutant Adeno-associated virus 2 (AAV2) vector encoding Cre recombinase. Wild type and Rosa26 (R26) LacZ mice were intravitreally injected with capsid mutant AAV2 viral vectors. Murine eyes were harvested at intervals ranging from 2 weeks to 15 weeks post-injection and were assayed for viral transduction, transgene expression and RGC survival. 109 vector genomes (vg) were sufficient for effective in vivo targeting of murine ganglion cell layer (GCL) retinal neurons. Transgene expression was observed as early as 2 weeks post-injection of viral vectors and persisted to 11 weeks. Early expression of Cre had no significant effect on RGC survival, while significant RGC loss was detected beginning 5 weeks post-injection. Early expression of viral Cre recombinase was robust, well-tolerated and predominantly found in GCL neurons suggesting this strategy can be effective in short-term RGC-specific mutation studies in experimental glaucoma models such as optic nerve crush and transection experiments. RGC degeneration with Cre expression for more than 4 weeks suggests that Cre toxicity is a limiting factor for targeted mutation strategies in RGCs.  

5.1893           Neuregulin-1 promotes functional improvement by enhancing collateral sprouting in SOD1G93A ALS mice and after partial muscle denervation

Mancuso, R. et al Neurobiology of Disease, 95, 168-178 (2016)   Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motoneurons, which is preceded by loss of neuromuscular connections in a “dying back” process. Neuregulin-1 (Nrg1) is a neurotrophic factor essential for the development and maintenance of neuromuscular junctions, and Nrg1 receptor ErbB4 loss-of-function mutations have been reported as causative for ALS. Our main goal was to investigate the role of Nrg1 type I (Nrg1-I) in SOD1^G93A mice muscles. We overexpressed Nrg1-I by means of an adeno-associated viral (AAV) vector, and investigated its effect by means of neurophysiological techniques assessing neuromuscular function, as well as molecular approaches (RT-PCR, western blot, immunohistochemistry, ELISA) to determine the mechanisms underlying Nrg1-I action. AAV-Nrg1-I intramuscular administration promoted motor axon collateral sprouting by acting on terminal Schwann cells, preventing denervation of the injected muscles through Akt and ERK1/2 pathways. We further used a model of muscle partial denervation by transecting the L4 spinal nerve. AAV-Nrg1-I intramuscular injection enhanced muscle reinnervation by collateral sprouting, whereas administration of lapatinib (ErbB receptor inhibitor) completely blocked it. We demonstrated that Nrg1-I plays a crucial role in the collateral reinnervation process, opening a new window for developing novel ALS therapies for functional recovery rather than preservation.  

5.1894           A novel roseobacter phage possesses features of podoviruses, siphoviruses, prophages and gene transfer agents

Zhan, Y., Huang, S., Voget, S., Simon, M. and Chen, F. Scientific Reports, 6:30372 (2016)   Bacteria in the Roseobacter lineage have been studied extensively due to their significant biogeochemical roles in the marine ecosystem. However, our knowledge on bacteriophage which infects the Roseobacter clade is still very limited. Here, we report a new bacteriophage, phage DSS3Φ8, which infects marine roseobacter Ruegeria pomeroyi DSS-3. DSS3Φ8 is a lytic siphovirus. Genomic analysis showed that DSS3Φ8 is most closely related to a group of siphoviruses, CbK-like phages, which infect freshwater bacterium Caulobacter crescentus. DSS3Φ8 contains a smaller capsid and has a reduced genome size (146 kb) compared to the CbK-like phages (205–279 kb). DSS3Φ8 contains the DNA polymerase gene which is closely related to T7-like podoviruses. DSS3Φ8 also contains the integrase and repressor genes, indicating its potential to involve in lysogenic cycle. In addition, four GTA (gene transfer agent) genes were identified in the DSS3Φ8 genome. Genomic analysis suggests that DSS3Φ8 is a highly mosaic phage that inherits the genetic features from siphoviruses, podoviruses, prophages and GTAs. This is the first report of CbK-like phages infecting marine bacteria. We believe phage isolation is still a powerful tool that can lead to discovery of new phages and help interpret the overwhelming unknown sequences in the viral metagenomics.  

5.1895           Small GTPases Rab8a and Rab11a Are Dispensable for Rhodopsin Transport in Mouse Photoreceptors

Ying, G., gerstner, C.D., Frederick, J.M., Boye, S.L., Hauswirth, W.W. and Baehr, W. PloS One, 11(8), e0161236 (2016)   Rab11a and Rab8a are ubiquitous small GTPases shown as required for rhodopsin transport in Xenopus laevis and zebrafish photoreceptors by dominant negative (dn) disruption of function. Here, we generated retina-specific Rab11a (retRab11a) and Rab8a (retRab8a) single and double knockout mice to explore the consequences in mouse photoreceptors. Rhodopsin and other outer segment (OS) membrane proteins targeted correctly to OS and electroretinogram (ERG) responses in all three mutant mouse lines were indistinguishable from wild-type (WT). Further, AAV (adeno-associated virus)-mediated expression of dnRab11b in retRab11a-/- retina, or expression of dnRab8b in retRab8a-/- retina did not cause OS protein mislocalization. Finally, a retRab8a-/- retina injected at one month of age with AAVs expressing dnRab11a, dnRab11b, dnRab8b, and dnRab10 (four dn viruses on Rab8a-/- background) and harvested three months later exhibited normal OS protein localization. In contrast to results obtained with dnRab GTPases in Xenopus and zebrafish, mouse Rab11a and Rab8a are dispensable for proper rhodopsin and outer segment membrane protein targeting. Absence of phenotype after expression of four dn Rab GTPases in a Rab8a-/- retina suggests that Rab8b and Rab11b paralogs maybe dispensable as well. Our data thus demonstrate significant interspecies variation in photoreceptor membrane protein and rhodopsin trafficking.  

5.1896           Lmx1a and Lmx1b regulate mitochondrial functions and survival of adult midbrain dopaminergic neurons

Doucet-Beaupre, H. et al PNAS, 113(30), E4387-E4396 (2016)   The LIM-homeodomain transcription factors Lmx1a and Lmx1b play critical roles during the development of midbrain dopaminergic progenitors, but their functions in the adult brain remain poorly understood. We show here that sustained expression of Lmx1a and Lmx1b is required for the survival of adult midbrain dopaminergic neurons. Strikingly, inactivation of Lmx1a and Lmx1b recreates cellular features observed in Parkinson’s disease. We found that Lmx1a/b control the expression of key genes involved in mitochondrial functions, and their ablation results in impaired respiratory chain activity, increased oxidative stress, and mitochondrial DNA damage. Lmx1a/b deficiency caused axonal pathology characterized by α-synuclein⁺ inclusions, followed by a progressive loss of dopaminergic neurons. These results reveal the key role of these transcription factors beyond the early developmental stages and provide mechanistic links between mitochondrial dysfunctions, α-synuclein aggregation, and the survival of dopaminergic neurons.  

5.1897           Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination

Braten, O. et al PNAS, 113(32), E4639-E4647 (2016)   The “canonical” proteasomal degradation signal is a substrate-anchored polyubiquitin chain. However, a handful of proteins were shown to be targeted following monoubiquitination. In this study, we established—in both human and yeast cells—a systematic approach for the identification of monoubiquitination-dependent proteasomal substrates. The cellular wild-type polymerizable ubiquitin was replaced with ubiquitin that cannot form chains. Using proteomic analysis, we screened for substrates that are nevertheless degraded under these conditions compared with those that are stabilized, and therefore require polyubiquitination for their degradation. For randomly sampled representative substrates, we confirmed that their cellular stability is in agreement with our screening prediction. Importantly, the two groups display unique features: monoubiquitinated substrates are smaller than the polyubiquitinated ones, are enriched in specific pathways, and, in humans, are structurally less disordered. We suggest that monoubiquitination-dependent degradation is more widespread than assumed previously, and plays key roles in various cellular processes.  

5.1898           Cocaine-Induced Synaptic Alterations in Thalamus to Nucleus Accumbens Projection

Neumann, P.A., Wang, Y., Yan, Y., Wang, Y., Ishikawa, M., Cui, R., Huang, Y.H., Sesack, S.R., Schlüter, O.M. and Dong, Y. Neuropsychopharmacol., 41(9), 2399-2410 (2016)   Exposure to cocaine induces addiction-associated behaviors partially through remodeling neurocircuits in the nucleus accumbens (NAc). The paraventricular nucleus of thalamus (PVT), which projects to the NAc monosynaptically, is activated by cocaine exposure and has been implicated in several cocaine-induced emotional and motivational states. Here we show that disrupting synaptic transmission of select PVT neurons with tetanus toxin activated via retrograde trans-synaptic transport of cre from NAc efferents decreased cocaine self-administration in rats. This projection underwent complex adaptations after self-administration of cocaine (0.75 mg/kg/infusion; 2 h/d × 5 d, 1d overnight training). Specifically, 1d after cocaine self-administration, we observed increased levels of AMPA receptor (AMPAR)-silent glutamatergic synapses in this projection, accompanied by a decreased ratio of AMPAR-to-NMDA receptor (NMDAR)-mediated EPSCs. Furthermore, the decay kinetics of NMDAR EPSCs was significantly prolonged, suggesting insertion of new GluN2B-containing NMDARs to PVT-to-NAc synapses. After 45-d withdrawal, silent synapses within this projection returned to the basal levels, accompanied by a return of the AMPAR/NMDAR ratio and NMDAR decay kinetics to the basal levels. In amygdala and infralimbic prefrontal cortical projections to the NAc, a portion of cocaine-generated silent synapses becomes unsilenced by recruiting calcium-permeable AMPARs (CP-AMPARs) after drug withdrawal. However, the sensitivity of PVT-to-NAc synapses to CP-AMPAR-selective antagonists was not changed after withdrawal, suggesting that CP-AMPAR trafficking is not involved in the evolution of cocaine-generated silent synapses within this projection. Meanwhile, the release probability of PVT-to-NAc synapses was increased after short- and long-term cocaine withdrawal. These results reveal complex and profound alterations at PVT-to-NAc synapses after cocaine exposure and withdrawal.  

5.1899           In Vivo Selection Yields AAV-B1 Capsid for Central Nervous System and Muscle Gene Therapy

Choudhury, S.R. et al Molecular Therapay, 24(7), 1247-1257 (2016)   Adeno-associated viral (AAV) vectors have shown promise as a platform for gene therapy of neurological disorders. Achieving global gene delivery to the central nervous system (CNS) is key for development of effective therapies for many of these diseases. Here we report the isolation of a novel CNS tropic AAV capsid, AAV-B1, after a single round of in vivo selection from an AAV capsid library. Systemic injection of AAV-B1 vector in adult mice and cat resulted in widespread gene transfer throughout the CNS with transduction of multiple neuronal subpopulations. In addition, AAV-B1 transduces muscle, β-cells, pulmonary alveoli, and retinal vasculature at high efficiency. This vector is more efficient than AAV9 for gene delivery to mouse brain, spinal cord, muscle, pancreas, and lung. Together with reduced sensitivity to neutralization by antibodies in pooled human sera, the broad transduction profile of AAV-B1 represents an important improvement over AAV9 for CNS gene therapy.  

5.1900           Development of Optimized AAV Serotype Vectors for High-Efficiency Transduction at Further Reduced Doses No Access

Ling, C., Li, B., Ma, W.M. and Srivastava, A. Human Gene Therapy Methods, 27(4), 143-149 (2016)   We have described the development of capsid-modified next-generation AAV vectors for both AAV2 and AAV3 serotypes, in which specific surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues on viral capsids were modified to achieve high-efficiency transduction at lower doses. We have also described the development of genome-modified AAV vectors, in which the transcriptionally inactive, single-stranded AAV genome was modified to achieve improved transgene expression. Here, we describe that combination of capsid modifications and genome modifications leads to the generation of optimized AAV serotype vectors, which transduce cells and tissues more efficiently, both in vitro and in vivo, at ∼20–30-fold reduced doses. These studies have significant implications in the potential use of the optimized AAV serotype vectors in human gene therapy.  

5.1901           Evaluation of an Optimized Injection System for Retinal Gene Therapy in Human Patients No Access

Fischer, M.D., Hickey, D., Singh, M.S. and Maclaren, R.E. Human Gene Therapy Methods, 27(4), 150-158 (2016)   Many retinal gene therapy clinical trials require subretinal injections of small volumes of adeno-associated viral (AAV) vector solutions in patients with retinal dystrophies, using equipment not specifically designed for this purpose. We therefore evaluated an optimized injection system in order to identify variables that might influence the rate of injection and final dose of vector delivered. An optimized injection system was assembled with a 41G polytetrafluoroethylene tip for retinal gene therapy. Flow rate was recorded at relevant infusion pressures (2–22 psi [14–152 kPa]), different target pressures (0.02–30 mm Hg [0.003–4 kPa]) and temperatures (18°C vs. 36°C) using a semiautomated Accurus^® Surgical System. Retention of AAV2/8 and AAV2/8^Y733F vector was quantified after simulating loading/injection with or without 0.001% Pluronic^® F-68 (PF-68). The optimized injection system provided a linear flow rate (μl/s)-to-infusion pressure (psi) relationship (y = 0.62x; r² = 0.99), independent of temperature and pressure changes relevant for intraocular surgery (18–36°C, 0.02–30 mm Hg). Differences in length of 41G polytetrafluoroethylene tips caused significant variation in flow rate (p < 0.001). Use of PF-68 significantly (p < 0.001) reduced loss of vector genomes in the injection system by 55% (AAV2/8) and 52% (AAV2/8^Y733F). A customized subretinal injection system assembled using equipment currently available in the operating room can deliver a controlled volume of vector at a fixed rate across a range of possible clinical parameters encountered in vitreoretinal surgery. The inclusion of 0.001% PF-68 had a significant effect on the final dose of vector genomes delivered. The described technique is currently used successfully in a clinical trial.  

5.1902           Resolving Adeno-Associated Viral Particle Diversity With Charge Detection Mass Spectrometry

Pierson, E.E., Keifer, D.Z., Asokan, A. and Jarrold, M.F. Anal. Chem., 88(13), 6718-6725 (2016)   Recombinant adeno-associated viruses (AAVs) are promising vectors for human gene therapy. However, current methods for evaluating AAV particle populations and vector purity are inefficient and low resolution. Here, we show that charge detection mass spectrometry (CDMS) can resolve capsids that contain the entire vector genome from those that contain partial genomes and from empty capsids. Measurements were performed for both single-stranded and self-complementary genomes. The self-complementary AAV vector preparation appears to contain particles with partially truncated genomes averaging at half the genome length. Comparison to results from electron microscopy with manual particle counting shows that CDMS has no significant mass discrimination in the relevant mass range (after a correction for the ion velocity is taken into account). Empty AAV capsids are intrinsically heterogeneous, and capsids from different sources have slightly different masses. However, the average masses of both the empty and full capsids are in close agreement with expected values. Mass differences between the empty and full capsids for both single-stranded and self-complementary AAV vectors indicate that the genomes are largely packaged without counterions.  

5.1903           Antimicrobial peptide LL-37 attenuates infection of hepatitis C virus

Matsumura, T., Sugiyama, N., Murayama, A., Yamada, N., Shiina, M., Asabe, S., Wakita, T., Imawari, M. and Kato, T. Hepatol. Res., 46(8), 924-932 (2016)   Aim Although recent studies indicate that supplementation with vitamin D (VD) potentiates a sustained viral response by interferon-based therapy to chronic hepatitis C, detailed mechanisms are not fully defined. The production of cathelicidin, an antimicrobial peptide, has been demonstrated to be part of the VD-dependent antimicrobial pathway in innate immunity. Cathelicidin is known to directly kill or inhibit the growth of microbial pathogens including mycobacteria and viruses. Methods We used a hepatitis C virus (HCV) cell culture system to clarify the anti-HCV effects of the human cathelicidin, LL-37. HuH-7 cells were administrated with LL-37 and infected with cell culture-generated HCV (HCVcc). HCV propagation was estimated by measuring the level of HCV core antigen (Ag). Results Treatment with LL-37 resulted in decreased intra- and extracellular levels of HCV core Ag, suggesting inhibition of HCV propagation. To assess the effects of LL-37 on HCV replication, JFH-1 subgenomic replicon RNA-transfected cells were treated with LL-37. However, inhibition of HCV replication was not detected by this assay. To clarify the effects on HCV infection, we treated HCVcc with LL-37 and removed the antimicrobial peptide prior to use of the virus in infection. This exposure of HCVcc to LL-37 diminished the infectivity titers in a dose-dependent fashion. Iodixanol density gradient analysis revealed that the peak fraction of infectivity titer was eliminated by LL-37 treatment. Conclusion The VD-associated antimicrobial peptide LL-37 attenuated the infectivity of HCV. This anti-HCV effect of LL-37 may explain the contribution of VD to the improved efficacy of interferon-based therapy.  

5.1904           Analysis of the human immunodeficiency virus-1 RNA packageome

Eckwahl, M.J., Arnion, H., Kharytonchyk, S., Zang, T., Bieniasz, P.D., Telesnitsky, A. and Wolin, S.L. RNA, 22, 1228-1238 (2016)   All retroviruses package cellular RNAs into virions. Studies of murine leukemia virus (MLV) revealed that the major host cell RNAs encapsidated by this simple retrovirus were LTR retrotransposons and noncoding RNAs (ncRNAs). Several classes of ncRNAs appeared to be packaged by MLV shortly after synthesis, as precursors to tRNAs, small nuclear RNAs, and small nucleolar RNAs were all enriched in virions. To determine the extent to which the human immunodeficiency virus (HIV-1) packages similar RNAs, we used high-throughput sequencing to characterize the RNAs within infectious HIV-1 virions produced in CEM-SS T lymphoblastoid cells. We report that the most abundant cellular RNAs in HIV-1 virions are 7SL RNA and transcripts from numerous divergent and truncated members of the long interspersed element (LINE) and short interspersed element (SINE) families of retrotransposons. We also detected precursors to several tRNAs and small nuclear RNAs as well as transcripts derived from the ribosomal DNA (rDNA) intergenic spacers. We show that packaging of a pre-tRNA requires the nuclear export receptor Exportin 5, indicating that HIV-1 recruits at least some newly made ncRNAs in the cytoplasm. Together, our work identifies the set of RNAs packaged by HIV-1 and reveals that early steps in HIV-1 assembly intersect with host cell ncRNA biogenesis pathways.  

5.1905           MicroRNA-511 Binds to FKBP5 mRNA, Which Encodes a Chaperone Protein, and Regulates Neuronal Differentiation

Zheng, D., Sabbagh, J.J., Blair, L.J., Darling, A.L., Wen, X. and Dickey, C.A.

1. Biol. Chem., 291(34), 17897-17906 (2016)

  Single nucleotide polymorphisms in the FKBP5 gene increase the expression of the FKBP51 protein and have been associated with increased risk for neuropsychiatric disorders such as major depression and post-traumatic stress disorder. Moreover, levels of FKBP51 are increased with aging and in Alzheimer disease, potentially contributing to disease pathogenesis. However, aside from its glucocorticoid responsiveness, little is known about what regulates FKBP5. In recent years, non-coding RNAs, and in particular microRNAs, have been shown to modulate disease-related genes and processes. The current study sought to investigate which miRNAs could target and functionally regulate FKBP5. Following in silico data mining and initial target expression validation, miR-511 was found to suppress FKBP5 mRNA and protein levels. Using luciferase p-miR-Report constructs and RNA pulldown assays, we confirmed that miR-511 bound directly to the 3′-UTR of FKBP5, validating the predicted gene-microRNA interaction. miR-511 suppressed glucocorticoid-induced up-regulation of FKBP51 in cells and primary neurons, demonstrating functional, disease-relevant control of the protein. Consistent with a regulator of FKBP5, miR-511 expression in the mouse brain decreased with age but increased following chronic glucocorticoid treatment. Analysis of the predicted target genes of miR-511 revealed that neurogenesis, neuronal development, and neuronal differentiation were likely controlled by these genes. Accordingly, miR-511 increased neuronal differentiation in cells and enhanced neuronal development in primary neurons. Collectively, these findings show that miR-511 is a functional regulator of FKBP5 and can contribute to neuronal differentiation.  

5.1906           A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

Landeck, N., Hall, H., Ardah, M.T., Majbour, N.K., El-Agnaf, O.M.A., Halliday, G. and Kirik, D. Molecular Neurodegeneration, 11:16 (2016)   Background Alpha-synuclein (asyn) has been shown to play an important role in the neuropathology of Parkinson’s disease (PD). In the diseased brain, classic intraneuronal inclusions called Lewy bodies contain abnormal formations of asyn protein which is mostly phosphorylated at serine 129 (pS129 asyn). This suggests that post-translational modifications may play a role in the pathogenic process. To date, several uniplex assays have been developed in order to quantify asyn not only in the brain but also in cerebrospinal fluid and blood samples in order to correlate asyn levels to disease severity and progression. Notably, only four assays have been established to measure pS129 asyn specifically and none provide simultaneous readout of the total and pS129 species. Therefore, we developed a sensitive high-throughput duplex assay quantifying total and pS129 human asyn (h-asyn) in the same well hence improving accuracy as well as saving time, consumables and samples. Results Using our newly established duplex assay we measured total and pS129 h-asyn in vitro showing that polo-like kinase 2 (PLK2) can phosphorylate asyn up to 41 % in HEK293 cells and in vivo the same kinase phosphorylated h-asyn up to 17 % in rat ventral midbrain neurons. Interestingly, no increase in phosphorylation was observed when PLK2 and h-asyn were co-expressed in rat striatal neurons. Furthermore, using this assay we investigated h-asyn levels in brain tissue samples from patients with PD as well as PD dementia and found significant differences in pS129 h-asyn levels not only between disease tissue and healthy control samples but also between the two distinct disease states especially in hippocampal tissue samples. Conclusions These results demonstrate that our duplex assay for simultaneous quantification is a useful tool to study h-asyn phosphorylation events in biospecimens and will be helpful in studies investigating the precise causative link between post-translational modification of h-asyn and PD pathology.  

5.1907           Immunomodulatory effects of exosomes produced by virus-infected cells

Petrik, J. Transfusion and Apheresis Science, 55, 84-91 (2016)   Viruses have developed a spectrum of ways to modify cellular pathways to hijack the cell machinery for the synthesis of their nucleic acid and proteins. Similarly, they use intracellular vesicular mechanisms of trafficking for their assembly and eventual release, with a number of viruses acquiring their envelope from internal or plasma cell membranes. There is an increasing number of reports on viral exploitation of cell secretome pathways to avoid recognition and stimulation of the immune response. Extracellular vesicles (EV) containing viral particles have been shown to shield viruses after exiting the host cell, in some cases challenging the boundaries between viral groups traditionally characterised as enveloped and non-enveloped. Apart from viral particles, EV can spread the virus also carrying viral genome and can modify the target cells through their cargo of virus-coded miRNAs and proteins as well as selectively packaged cellular mRNAs, miRNAs, proteins and lipids, differing in composition and quantities from the cell of origin.  

5.1908           DNA Minicircle Technology Improves Purity of Adeno-associated Viral Vector Preparations

Schnödt, M., Schmeer, M., kracher, B., Krüsemann, C., Espinosa, L.E., Grünert, A., Fuchsluger, T., Rischmüller, M. , Schleef, M. and Büning, H. Molecular Therapy – Nucleic Acids, 5, e355 (2016)   Adeno-associated viral (AAV) vectors are considered as one of the most promising delivery systems in human gene therapy. In addition, AAV vectors are frequently applied tools in preclinical and basic research. Despite this success, manufacturing pure AAV vector preparations remains a difficult task. While empty capsids can be removed from vector preparations owing to their lower density, state-of-the-art purification strategies as of yet failed to remove antibiotic resistance genes or other plasmid backbone sequences. Here, we report the development of minicircle (MC) constructs to replace AAV vector and helper plasmids for production of both, single-stranded (ss) and self-complementary (sc) AAV vectors. As bacterial backbone sequences are removed during MC production, encapsidation of prokaryotic plasmid backbone sequences is avoided. This is of particular importance for scAAV vector preparations, which contained an unproportionally high amount of plasmid backbone sequences (up to 26.1% versus up to 2.9% (ssAAV)). Replacing standard packaging plasmids by MC constructs not only allowed to reduce these contaminations below quantification limit, but in addition improved transduction efficiencies of scAAV preparations up to 30-fold. Thus, MC technology offers an easy to implement modification of standard AAV packaging protocols that significantly improves the quality of AAV vector preparations.      

5.1909           The extreme N-terminus of TDP-43 mediates the cytoplasmic aggregation of TDP-43 and associated toxicity in vivo

Sasaguri, H., Chew, J., Xu, Y-F., Gendron, T.F., Garrett, A., Lee, C.W., Jansen-West, K., Bauer, P.O., Perkerson, E.A., Tong, J., Stetler, C. and Zhang, Y-J. Brain Res., 1647, 57-64 (2016)   Inclusions of Tar DNA- binding protein 43 (TDP-43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP). Pathological TDP-43 exhibits the disease-specific biochemical signatures, which include its ubiquitination, phosphorylation and truncation. Recently, we demonstrated that the extreme N-terminus of TDP-43 regulates formation of abnormal cytoplasmic TDP-43 aggregation in cultured cells and primary neurons. However, it remained unclear whether this N-terminal domain mediates TDP-43 aggregation and the associated toxicity in vivo. To investigate this, we expressed a GFP-tagged TDP-43 with a nuclear localization signal mutation (GFP-TDP-43_NLSm) and a truncated form without the extreme N-terminus (GFP-TDP-43_10-414-NLSm) by adeno-associated viral (AAV) vectors in mouse primary cortical neurons and murine central nervous system. Compared to neurons containing GFP alone, expression of GFP-TDP-43_NLSm resulted in the formation of ubiquitin-positive cytoplasmic inclusions and activation of caspase-3, an indicator of cell death. Moreover, mice expressing GFP-TDP-43_NLSm proteins show reactive gliosis and develop neurological abnormalities. However, by deletion of TDP-43's extreme N-terminus, these pathological alterations can be abrogated. Together, our study provides further evidence confirming the critical role of the extreme N-terminus of TDP-43 in regulating protein structure as well as mediating toxicity associated with its aggregation.  

5.1910           The microRNA cluster miR-183/96/182 contributes to long-term memory in a protein phosphatase 1-dependent manner

Woldemichael, B.T., Jawaid, A., Kremer, E.A., Gaur, N., Krol, J., Marchais, A. and Mansuy, I.M. Nature Communications, 7:12594 (2016)   Memory formation is a complex cognitive function regulated by coordinated synaptic and nuclear processes in neurons. In mammals, it is controlled by multiple molecular activators and suppressors, including the key signalling regulator, protein phosphatase 1 (PP1). Here, we show that memory control by PP1 involves the miR-183/96/182 cluster and its selective regulation during memory formation. Inhibiting nuclear PP1 in the mouse brain, or training on an object recognition task similarly increases miR-183/96/182 expression in the hippocampus. Mimicking this increase by miR-183/96/182 overexpression enhances object memory, while knocking-down endogenous miR-183/96/182 impairs it. This effect involves the modulation of several plasticity-related genes, with HDAC9 identified as an important functional target. Further, PP1 controls miR-183/96/182 in a transcription-independent manner through the processing of their precursors. These findings provide novel evidence for a role of miRNAs in memory formation and suggest the implication of PP1 in miRNAs processing in the adult brain.  

5.1911           Distinct Particle Morphologies Revealed through Comparative Parallel Analyses of Retrovirus-Like Particles

Martin, J.L., Cao, S., Maldonado, J.O., Zhang, W. and Mansky, L.M.

1. Virol., 90(18), 8074-8084 (2016)

  The Gag protein is the main retroviral structural protein, and its expression alone is usually sufficient for production of virus-like particles (VLPs). In this study, we sought to investigate—in parallel comparative analyses—Gag cellular distribution, VLP size, and basic morphological features using Gag expression constructs (Gag or Gag-YFP, where YFP is yellow fluorescent protein) created from all representative retroviral genera: Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus, and Spumavirus. We analyzed Gag cellular distribution by confocal microscopy, VLP budding by thin-section transmission electron microscopy (TEM), and general morphological features of the VLPs by cryogenic transmission electron microscopy (cryo-TEM). Punctate Gag was observed near the plasma membrane for all Gag constructs tested except for the representative Beta- and Epsilonretrovirus Gag proteins. This is the first report of Epsilonretrovirus Gag localizing to the nucleus of HeLa cells. While VLPs were not produced by the representative Beta- and Epsilonretrovirus Gag proteins, the other Gag proteins produced VLPs as confirmed by TEM, and morphological differences were observed by cryo-TEM. In particular, we observed Deltaretrovirus-like particles with flat regions of electron density that did not follow viral membrane curvature, Lentivirus-like particles with a narrow range and consistent electron density, suggesting a tightly packed Gag lattice, and Spumavirus-like particles with large envelope protein spikes and no visible electron density associated with a Gag lattice. Taken together, these parallel comparative analyses demonstrate for the first time the distinct morphological features that exist among retrovirus-like particles. Investigation of these differences will provide greater insights into the retroviral assembly pathway.  

5.1912           Immunogenicity of next-generation HPV vaccines in non-human primates: Measles-vectored HPV vaccine versus Pichia pastoris recombinant protein vaccine

Gupta, G., Giannino, V., Rishi, N. and Glueck, R. Vaccine, 34, 4724-4731 (2016)   Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide. HPVs are oncogenic small double-stranded DNA viruses that are the primary causal agent of cervical cancer and other types of cancers, including in the anus, oropharynx, vagina, vulva, and penis. Prophylactic vaccination against HPV is an attractive strategy for preventing cervical cancer and some other types of cancers. However, there are few safe and effective vaccines against HPV infections. Current first-generation commercial HPV vaccines are expensive to produce and deliver. The goal of this study was to develop an alternate potent HPV recombinant L1-based vaccines by producing HPV virus-like particles into a vaccine that is currently used worldwide. Live attenuated measles virus (MV) vaccines have a well-established safety and efficacy record, and recombinant MV (rMV) produced by reverse genetics may be useful for generating candidate HPV vaccines to meet the needs of the developing world. We studied in non-human primate rMV-vectored HPV vaccine in parallel with a classical alum adjuvant recombinant HPV16L1 and 18L1 protein vaccine produced in Pichia pastoris. A combined prime-boost approach using both vaccines was evaluated, as well as immune interference due to pre-existing immunity against the MV. The humoral immune response induced by the MV, Pichia-expressed vaccine, and their combination as priming and boosting approaches was found to elicit HPV16L1 and 18L1 specific total IgG and neutralizing antibody titres. Pre-existing antibodies against measles did not prevent the immune response against HPV16L1 and 18L1.  

5.1913           Viral Vector-Based Dissection of Marmoset GFAP Promoter in Mouse and Marmoset Brains

Shinohara, Y., Konno, A., Takahashi, N., Matsuzaki, Y., Kishi, S. and Hirai, H. PloS One, 11(8), e0162023 (2016)   Adeno-associated virus (AAV) vectors are small in diameter, diffuse easily in the brain, and represent a highly efficient means by which to transfer a transgene to the brain of a large animal. A major demerit of AAV vectors is their limited accommodation capacity for transgenes. Thus, a compact promoter is useful when delivering large transgenes via AAV vectors. In the present study, we aimed to identify the shortest astrocyte-specific GFAP promoter region that could be used for AAV-vector-mediated transgene expression in the marmoset brain. The 2.0-kb promoter region upstream of the GFAP gene was cloned from the marmoset genome, and short promoters (1.6 kb, 1.4 kb, 0.6 kb, 0.3 kb and 0.2 kb) were obtained by progressively deleting the original 2.0-kb promoter from the 5’ end. The short promoters were screened in the mouse cerebellum in terms of their strength and astrocyte specificity. We found that the 0.3-kb promoter maintained 40% of the strength of the original 2.0-kb promoter, and approximately 90% of its astrocyte specificity. These properties were superior to those of the 1.4-kb, 0.6-kb (20% promoter strength) and 0.2-kb (70% astrocyte specificity) promoters. Then, we verified whether the 0.3-kb GFAP promoter retained astrocyte specificity in the marmoset cerebral cortex. Injection of viral vectors carrying the 0.3-kb marmoset GFAP promoter specifically transduced astrocytes in both the cerebral cortex and cerebellar cortex of the marmoset. These results suggest that the compact 0.3-kb promoter region serves as an astrocyte-specific promoter in the marmoset brain, which permits us to express a large gene by AAV vectors that have a limited accommodation capacity.  

5.1914           The CD63-Syntenin-1 Complex Controls Post-Endocytic Trafficking of Oncogenic Human Papillomaviruses

Grässel, L., Fast, L.A., Scheffer, K.D., Boukhallouk, F., Spoden, G.A., Tenzer, S., Boller, K., Bago, R., Rajesh, S., Overduin, M., Berditchevski, F. and Florin, L. Scientific Reports, 6:32337 (2016)   Human papillomaviruses enter host cells via a clathrin-independent endocytic pathway involving tetraspanin proteins. However, post-endocytic trafficking required for virus capsid disassembly remains unclear. Here we demonstrate that the early trafficking pathway of internalised HPV particles involves tetraspanin CD63, syntenin-1 and ESCRT-associated adaptor protein ALIX. Following internalisation, viral particles are found in CD63-positive endosomes recruiting syntenin-1, a CD63-interacting adaptor protein. Electron microscopy and immunofluorescence experiments indicate that the CD63-syntenin-1 complex controls delivery of internalised viral particles to multivesicular endosomes. Accordingly, infectivity of high-risk HPV types 16, 18 and 31 as well as disassembly and post-uncoating processing of viral particles was markedly suppressed in CD63 or syntenin-1 depleted cells. Our analyses also present the syntenin-1 interacting protein ALIX as critical for HPV infection and CD63-syntenin-1-ALIX complex formation as a prerequisite for intracellular transport enabling viral capsid disassembly. Thus, our results identify the CD63-syntenin-1-ALIX complex as a key regulatory component in post-endocytic HPV trafficking.  

5.1915           Identification of a New Benzimidazole Derivative as an Antiviral against Hepatitis C Virus

Vausselin, T. et al

1. Virol., 90(19), 8422-8434 (2016)

  Aminoquinolines and piperazines, linked or not, have been used successfully to treat malaria, and some molecules of this family also exhibit antiviral properties. Here we tested several derivatives of 4-aminoquinolines and piperazines for their activity against hepatitis C virus (HCV). We screened 11 molecules from three different families of compounds, and we identified anti-HCV activity in cell culture for six of them. Of these, we selected a compound (B5) that is currently ending clinical phase I evaluation for neurodegenerative diseases. In hepatoma cells, B5 inhibited HCV infection in a pangenotypic and dose-dependent manner, and its antiviral activity was confirmed in primary hepatocytes. B5 also inhibited infection by pseudoparticles expressing HCV envelope glycoproteins E1 and E2, and we demonstrated that it affects a postattachment stage of the entry step. Virus with resistance to B5 was selected by sequential passage in the presence of the drug, and reverse genetics experiments indicated that resistance was conferred mainly by a single mutation in the putative fusion peptide of E1 envelope glycoprotein (F291I). Furthermore, analyses of the effects of other closely related compounds on the B5-resistant mutant suggest that B5 shares a mode of action with other 4-aminoquinoline-based molecules. Finally, mice with humanized liver that were treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle.  

5.1916           Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing

Trochet, D., Prudhon, B., Jollet, A., Lorain, S. and Bitoun, M. Molecular Therapy-Nucleic Acids, 5, e362 (2016)   Dynamin 2 (DNM2) is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the Dnm2-mRNA in vitro and in vivo in mice. We show that classical 3′-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5′-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated Dnm2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development.  

5.1917           Apolipoprotein(a) inhibits hepatitis C virus entry

Oliveira, C. et al

1. Clin. Virol., abstract 40, S82-S83 (2016)

  In the last two decades, the development of different cell-based models has greatly contributed to improve the knowledge of HCV life cycle. However, it is still impossible to grow primary HCV isolates from each genotype in cell culture. This would open new perspectives to investigate viral determinants responsible for the different natural course and treatment outcome of hepatitis C as well as to develop a vaccine. In this study we hypothesized that this hindrance could be due to the presence of inhibitory factors in patient serum. Combining polyethylene glycol precipitation, iodixanol gradient and size exclusion chromatography, we obtained a purified fraction enriched in inhibitory factors from a pool of HCV seronegative serums. Mass spectrometry analysis of this fraction identified apolipoprotein(a) (apo(a)) as a potential inhibitor of the early step of HCV life cycle. Apo(a) consists of ten kringle IV-like domains (KIV), one kringle V-like domain (KV) and a protease-like domain that are homologous to plasminogen domains. Each of the ten KIV domains is present in a single copy with the exception of KIV type 2 (KIV 2), which is encoded in a variable number of tandemly repeated copies by the apo(a) gene, which gives rise to several apo(a) size isoforms in the human population. In addition, in human serum, apo(a) covalently links to the Apolipoprotein B component of a low density lipoprotein via a disulfide bridge to form a lipoprotein(a). The inhibitory effect of apo(a) on HCV entry was confirmed using a recombinant virus derived from the JFH1 strain and supernatant of cells transfected with plasmids expressing apo(a) as well as purified recombinant isoforms of apo(a). Our results also suggest that the larger the protein is, the better the inhibition is. We are currently testing several deletion mutants of apo(a) to identify critical domains for the inhibitory activity and to decipher the mechanism of inhibition. Altogether, our results identify apo(a) as an additional component of the lipid metabolism modulating HCV infection.  

5.1918           HIV-1 Tat-shortened neurite outgrowth through regulation of microRNA-132 and its target gene expression

Rahimian, P. and He, J.J.

1. Neuroinflammation, 13:247 (2016)

  Background Synaptodendritic damage is a pathological hallmark of HIV-associated neurocognitive disorders, and HIV-1 Tat protein is known to cause such injury in the central nervous system. In this study, we aimed to determine the molecular mechanisms of Tat-induced neurite shortening, specifically the roles of miR-132, an important regulator of neurite morphogenesis in this process. Methods The relationship between Tat expression and miR-132 expression was first determined using reverse transcription quantitative PCR (qRT-PCR) in Tat-transfected astrocytes and neurons, astrocytes from Tat-transgenic mice, and HIV-infected astrocytes. qRT-PCR and Western blotting were performed to determine Tat effects on expression of miR-132 target genes methyl CpG-binding protein 2, Rho GTPase activator p250GAP, and brain-derived neurotrophic factor. Exosomes were isolated from Tat-expressing astrocytes, and exosomal microRNA (miRNA) uptake into neurons was studied using miRNA labeling and flow cytometry. The lactate dehydrogenase release was used to determine the cytotoxicity, while immunostaining was used to determine neurite lengths and synapse formation. Tat basic domain deletion mutant and miR-132 mimic and inhibitor were used to determine the specificity of the relationship between Tat and miR-132 and its effects on astrocytes and neurons and the underlying mechanisms of Tat-induced miR-132 expression. Results Tat significantly induced miR-132 expression, ensuing down-regulation of miR-132 target genes in astrocytes and neurons. miR-132 induction was associated with phosphorylation of cAMP response element-binding protein and required the basic domain of Tat. miRNA-132 induction had no effects on astrocyte activation or survival but was involved in the direct neurotoxicity of Tat. miR-132 was present in astrocyte-derived exosomes and was taken up by neurons, causing neurite shortening. Conclusions Tat-induced miR-132 expression contributes to both direct and astrocyte-mediated Tat neurotoxicity and supports the important roles of miR-132 in controlling neurite outgrowth.  

5.1919           Chronic Kappa opioid receptor activation modulates NR2B: Implication in treatment resistant depression

Dogra, S., Kumar, A., Umrao, D., Sahasrabuddhe, A.A. and Yadav, P.N. Scientific Reports, 6:33401 (2016)   Psychotomimetic and prodepressive effect by kappa opioid receptor (KOR) activation in rodents and human is widely known. Significantly, recent clinical investigations demonstrated the salutary effects of KOR antagonists in patients with treatment resistant depression, indicating essential role of KOR signaling in refractory depression. This study was undertaken to reveal the molecular determinant of KOR mediated depression and antidepressant response of KOR antagonist. We observed that chronic KOR activation by U50488, a selective KOR agonist, significantly increased depression like symptoms (behavioral despair, anhedonia and sociability) in C57BL/6J mice, which were blocked by KOR antagonist norBNI and antidepressant imipramine, but not by fluoxetine or citalopram. Further, chronic KOR activation increased phosphorylation of NR2B subunit of NMDA at tyrosine 1472 (pNR2B NMDA) in the hippocampus, but not in the cortex. Similar to behavioral effects norBNI and imipramine, but not SSRIs, blocked NR2B phosphorylation. Moreover, KOR induced depression like behaviors were reversed by NR2B selective inhibitor Ro 25-6981. Mechanistic studies in primary cultured neurons and brain tissues using genetic and pharmacological approaches revealed that stimulation of KOR modulates several molecular correlates of depression. Thus, these findings elucidate molecular mechanism of KOR signaling in treatment resistant depression like behaviors in mice.  

5.1920           Comparison of human papillomavirus type 16 replication in tonsil and foreskin epithelia

Israr, M., Biryukov, J., Ryndock, E.J., Alam, S. and Meyers, C. Virology, 499, 82-90 (2016)   Human papillomavirus (HPV) is well recognized as a causative agent for anogenital and oropharyngeal cancers, however, the biology of HPV infection at different mucosal locations, specifically the oral cavity, is not well understood. Importantly, it has yet to be determined if oral tissues are permissive for HPV infection and replication. We investigated for the first time the titers, infectivity, and maturation of HPV16 in oral epithelial versus genital epithelial tissue. We show that infectious HPV16 virions can be produced in oral tissue. This demonstrates, for the first time, that infectious virus could be spread via the oral cavity. HPV16 derived from oral tissue utilize a tissue-spanning redox gradient that facilitates the maturation of virions over time. Maturation is manifested by virion stability and increased susceptibility to neutralization with anti-HPV16 L1 antibodies. However, susceptibility to neutralization by anti-HPV16 L2 specific antibodies decreases during the maturation of HPV16 virions in oral tissue.  

5.1921           Deimmunization for gene therapy: host matching of synthetic zinc finger constructs enables long-term mutant Huntingtin repression in mice

Agustin-Pavon, C., Mielcarek, M., Garriga-Canut, M. and Isalan, M. Mol. Neurodegeneration, 11:64 (2016)   Background Synthetic zinc finger (ZF) proteins can be targeted to desired DNA sequences and are useful tools for gene therapy. We recently developed a ZF transcription repressor (ZF-KOX1) able to bind to expanded DNA CAG-repeats in the huntingtin (HTT) gene, which are found in Huntington’s disease (HD). This ZF acutely repressed mutant HTT expression in a mouse model of HD and delayed neurological symptoms (clasping) for up to 3 weeks. In the present work, we sought to develop a long-term single-injection gene therapy approach in the brain. Method Since non-self proteins can elicit immune and inflammatory responses, we designed a host-matched analogue of ZF-KOX1 (called mZF-KRAB), to treat mice more safely in combination with rAAV vector delivery. We also tested a neuron-specific enolase promoter (pNSE), which has been reported as enabling long-term transgene expression, to see whether HTT repression could be observed for up to 6 months after AAV injection in the brain. Results After rAAV vector delivery, we found that non-self proteins induce significant inflammatory responses in the brain, in agreement with previous studies. Specifically, microglial cells were activated at 4 and 6 weeks after treatment with non-host-matched ZF-KOX1 or GFP, respectively, and this was accompanied by a moderate neuronal loss. In contrast, the host-matched mZF-KRAB did not provoke these effects. Nonetheless, we found that using a pCAG promoter (CMV early enhancer element and the chicken β-actin promoter) led to a strong reduction in ZF expression by 6 weeks after injection. We therefore tested a new non-viral promoter to see whether the host-adapted ZF expression could be sustained for a longer time. Vectorising mZF-KRAB with a promoter-enhancer from neuron-specific enolase (Eno2, rat) resulted in up to 77 % repression of mutant HTT in whole brain, 3 weeks after bilateral intraventricular injection of 10¹⁰ virions. Importantly, repressions of 48 % and 23 % were still detected after 12 and 24 weeks, respectively, indicating that longer term effects are possible. Conclusion Host-adapted ZF-AAV constructs displayed a reduced toxicity and a non-viral pNSE promoter improved long-term ZF protein expression and target gene repression. The optimized constructs presented here have potential for treating HD.  

5.1922           Linear biocompatible glyco-polyamidoamines as dual action mode virus infection inhibitors with potential as broad-spectrum microbicides for sexually transmitted diseases

Mauro, N., Ferruti, P., Ranucci, E., Manfredi, A., Berzi, A., Clerici, M., Cagno, V., limbo, D., Palmioli, A. and Sattin, S. Scientific Reports, 6:33393 (2016)   The initial steps of viral infections are mediated by interactions between viral proteins and cellular receptors. Blocking the latter with high-affinity ligands may inhibit infection. DC-SIGN, a C-type lectin receptor expressed by immature dendritic cells and macrophages, mediates human immunodeficiency virus (HIV) infection by recognizing mannose clusters on the HIV-1 gp120 envelope glycoprotein. Mannosylated glycodendrimers act as HIV entry inhibitors thanks to their ability to block this receptor. Previously, an amphoteric, but prevailingly cationic polyamidoamine named AGMA1 proved effective as infection inhibitor for several heparan sulfate proteoglycan-dependent viruses, such as human papilloma virus HPV-16 and herpes simplex virus HSV-2. An amphoteric, but prevailingly anionic PAA named ISA23 proved inactive. It was speculated that the substitution of mannosylated units for a limited percentage of AGMA1 repeating units, while imparting anti-HIV activity, would preserve the fundamentals of its HPV-16 and HSV-2 infection inhibitory activity. In this work, four biocompatible linear PAAs carrying different amounts of mannosyl-triazolyl pendants, Man-ISA7, Man-ISA14, Man-AGMA6.5 and Man-AGMA14.5, were prepared by reaction of 2-(azidoethyl)-α-D-mannopyranoside and differently propargyl-substituted AGMA1 and ISA23. All mannosylated PAAs inhibited HIV infection. Both Man-AGMA6.5 and Man-AGMA14.5 maintained the HPV-16 and HSV-2 activity of the parent polymer, proving broad-spectrum, dual action mode virus infection inhibitors.  

5.1923           Quantitative Lipid Droplet Proteome Analysis Identifies Annexin A3 as a Cofactor for HCV Particle Production

Rösch, K., Kwiatkowski, M., Hofman, S. et al

Cell Reports, 16, 3219-3231 (2016)

Lipid droplets are vital to hepatitis C virus (HCV) infection as the putative sites of virion assembly, but morphogenesis and egress of virions remain ill defined. We performed quantitative lipid droplet proteome analysis of HCV-infected cells to identify co-factors of that process. Our results demonstrate that HCV disconnects lipid droplets from their metabolic function. Annexin A3 (ANXA3), a protein enriched in lipid droplet fractions, strongly impacted HCV replication and was characterized further: ANXA3 is recruited to lipid-rich fractions in HCV-infected cells by the viral core and NS5A proteins. ANXA3 knockdown does not affect HCV RNA replication but severely impairs virion production with lower specific infectivity and higher density of secreted virions. ANXA3 is essential for the interaction of viral envelope E2 with apolipoprotein E (ApoE) and for trafficking, but not lipidation, of ApoE in HCV-infected cells. Thus, we identified ANXA3 as a regulator of HCV maturation and egress.

 

5.1924           Functional organization of the HIV lipid envelope

Huarte, N., Carravilla, P., Cruz, A., Lorizate, M., Nieto-garai, J.A., Kräusslich, H-G., Perez-Gil, J., Requejo-Isidro, J. and Nieva, J.L. Scientific Reports, 6:34190 (2016)   The chemical composition of the human immunodeficiency virus type 1 (HIV-1) membrane is critical for fusion and entry into target cells, suggesting that preservation of a functional lipid bilayer organization may be required for efficient infection. HIV-1 acquires its envelope from the host cell plasma membrane at sites enriched in raft-type lipids. Furthermore, infectious particles display aminophospholipids on their surface, indicative of dissipation of the inter-leaflet lipid asymmetry metabolically generated at cellular membranes. By combining two-photon excited Laurdan fluorescence imaging and atomic force microscopy, we have obtained unprecedented insights into the phase state of membranes reconstituted from viral lipids (i.e., extracted from infectious HIV-1 particles), established the role played by the different specimens in the mixtures, and characterized the effects of membrane-active virucidal agents on membrane organization. In determining the molecular basis underlying lipid packing and lateral heterogeneity of the HIV-1 membrane, our results may help develop compounds with antiviral activity acting by perturbing the functional organization of the lipid envelope.  

5.1925           Wnt Regulates Proliferation and Neurogenic Potential of Müller Glial Cells via a Lin28/let-7 miRNA-Dependent Pathway in Adult Mammalian Retinas

Yao, K., Qiu, S., Tian, L., Snider, W.D., Flannery, J.G., Schaffer, D.V. and Chen, B. Cell Reports, 17, 165-178 (2016)   In cold-blooded vertebrates such as zebrafish, Müller glial cells (MGs) readily proliferate to replenish lost retinal neurons. In mammals, however, MGs lack regenerative capability as they do not spontaneously re-enter the cell cycle unless the retina is injured. Here, we show that gene transfer of β-catenin in adult mouse retinas activates Wnt signaling and MG proliferation without retinal injury. Upstream of Wnt, deletion of GSK3β stabilizes β-catenin and activates MG proliferation. Downstream of Wnt, β-catenin binds to the Lin28 promoter and activates transcription. Deletion of Lin28 abolishes β-catenin-mediated effects on MG proliferation, and Lin28 gene transfer stimulates MG proliferation. We further demonstrate that let-7 miRNAs are critically involved in Wnt/Lin28-regulated MG proliferation. Intriguingly, a subset of cell-cycle-reactivated MGs express markers for amacrine cells. Together, these results reveal a key role of Wnt-Lin28-let7 miRNA signaling in regulating proliferation and neurogenic potential of MGs in the adult mammalian retina.  

5.1926           Potential for cellular stress response to hepatic factor VIII expression from AAV vector

Zolotukhin, I., Markusic, D.M., palaschak, B., Hoffman, B.E., Srikanthan, M.A. and Herzog, R.W. Mol. Therapy-Methods & Clinical Development, 3:16063 (2016)   Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII) or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII) and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity.  

5.1927           Prokineticin-2 upregulation during neuronal injury mediates a compensatory protective response against dopaminergic neuronal degeneration

Gordon, R., Neal, M.L., Luo, J., Langley, M.R., Harischandra, D.S., Panicker, N., Charli, A., Jin, H., Anantharam, V., Woodruff, T.M., Zhou, Q-Y., kanthasamy, A. and Kanthasamy, A. Nature Communications, 7:12932 (2016)   Prokineticin-2 (PK2), a recently discovered secreted protein, regulates important physiological functions including olfactory biogenesis and circadian rhythms in the CNS. Interestingly, although PK2 expression is low in the nigral system, its receptors are constitutively expressed on nigrostriatal neurons. Herein, we demonstrate that PK2 expression is highly induced in nigral dopaminergic neurons during early stages of degeneration in multiple models of Parkinson’s disease (PD), including PK2 reporter mice and MitoPark mice. Functional studies demonstrate that PK2 promotes mitochondrial biogenesis and activates ERK and Akt survival signalling pathways, thereby driving neuroprotection. Importantly, PK2 overexpression is protective whereas PK2 receptor antagonism exacerbates dopaminergic degeneration in experimental PD. Furthermore, PK2 expression increased in surviving nigral dopaminergic neurons from PD brains, indicating that PK2 upregulation is clinically relevant to human PD. Collectively, our results identify a paradigm for compensatory neuroprotective PK2 signalling in nigral dopaminergic neurons that could have important therapeutic implications for PD.  

5.1928           Anti-Epidermal Growth Factor Receptor Gene Therapy for Glioblastoma

Hicks, M.J., Chiuchiolo, M.J:, Ballon, D., Dyke, J.P., Aronowitz, E., Funato, K., Tabar, V., Havlicek, D., Fan, F., Sondhi, D., Kaminsky, S.M. and Crystal, R.G. PloS One, 11(10), e0162978 (2016)   Glioblastoma multiforme (GBM) is the most common and aggressive primary intracranial brain tumor in adults with a mean survival of 14 to 15 months. Aberrant activation of the epidermal growth factor receptor (EGFR) plays a significant role in GBM progression, with amplification or overexpression of EGFR in 60% of GBM tumors. To target EGFR expressed by GBM, we have developed a strategy to deliver the coding sequence for cetuximab, an anti-EGFR antibody, directly to the CNS using an adeno-associated virus serotype rh.10 gene transfer vector. The data demonstrates that single, local delivery of an anti-EGFR antibody by an AAVrh.10 vector coding for cetuximab (AAVrh.10Cetmab) reduces GBM tumor growth and increases survival in xenograft mouse models of a human GBM EGFR-expressing cell line and patient-derived GBM. AAVrh10.CetMab-treated mice displayed a reduction in cachexia, a significant decrease in tumor volume and a prolonged survival following therapy. Adeno-associated-directed delivery of a gene encoding a therapeutic anti-EGFR monoclonal antibody may be an effective strategy to treat GBM.  

5.1929           N-acetylaspartate supports the energetic demands of developmental myelination via oligodendroglial aspartoacylase

Francis, J.S., Wojtas, I., markov, V., Gray, S.J., McCown, T.J., Samulski, R.J., Bilanuik, L.T., Wang, D.J., De Vivo, D.C., Janson, C.G. and Leone, P: Neurobiology of Disease, 96, 323-334 (2916)   Breakdown of neuro-glial N-acetyl-aspartate (NAA) metabolism results in the failure of developmental myelination, manifest in the congenital pediatric leukodystrophy Canavan disease caused by mutations to the sole NAA catabolizing enzyme aspartoacylase. Canavan disease is a major point of focus for efforts to define NAA function, with available evidence suggesting NAA serves as an acetyl donor for fatty acid synthesis during myelination. Elevated NAA is a diagnostic hallmark of Canavan disease, which contrasts with a broad spectrum of alternative neurodegenerative contexts in which levels of NAA are inversely proportional to pathological progression. Recently generated data in the nur7 mouse model of Canavan disease suggests loss of aspartoacylase function results in compromised energetic integrity prior to oligodendrocyte death, abnormalities in myelin content, spongiform degeneration, and motor deficit. The present study utilized a next-generation “oligotropic” adeno-associated virus vector (AAV-Olig001) to quantitatively assess the impact of aspartoacylase reconstitution on developmental myelination. AAV-Olig001-aspartoacylase promoted normalization of NAA, increased bioavailable acetyl-CoA, and restored energetic balance within a window of postnatal development preceding gross histopathology and deteriorating motor function. Long-term effects included increased oligodendrocyte numbers, a global increase in myelination, reversal of vacuolation, and rescue of motor function. Effects on brain energy observed following AAV-Olig001-aspartoacylase gene therapy are shown to be consistent with a metabolic profile observed in mild cases of Canavan disease, implicating NAA in the maintenance of energetic integrity during myelination via oligodendroglial aspartoacylase.  

5.1930           Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging

Lauren M. Drouin, Bridget Lins, Maria Janssen, Antonette Bennett, Paul Chipman, Robert McKenna, Weijun Chen, Nicholas Muzyczka, Giovanni Cardone, Timothy S. Baker, and Mavis Agbandje-McKenna

1. Virol., 90(19), 8542-8551 (2016)

  The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the βA strand region under the icosahedral 2-fold axis rather than antiparallel to the βB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency.  

5.1931           Heat Shock Protein 70 Family Members Interact with Crimean-Congo Hemorrhagic Fever Virus and Hazara Virus Nucleocapsid Proteins and Perform a Functional Role in the Nairovirus Replication Cycle

Rebecca Surtees, Stuart D. Dowall, Amelia Shaw, Stuart Armstrong, Roger Hewson, Miles W. Carroll, Jamel Mankouri, Thomas A. Edwards, Julian A. Hiscox, and John N. Barr

1. Virol., 90(20), 9305-9316 (2016)

  The Nairovirus genus of the Bunyaviridae family contains serious human and animal pathogens classified within multiple serogroups and species. Of these serogroups, the Crimean-Congo hemorrhagic fever virus (CCHFV) serogroup comprises sole members CCHFV and Hazara virus (HAZV). CCHFV is an emerging zoonotic virus that causes often-fatal hemorrhagic fever in infected humans for which preventative or therapeutic strategies are not available. In contrast, HAZV is nonpathogenic to humans and thus represents an excellent model to study aspects of CCHFV biology under conditions of more-accessible biological containment. The three RNA segments that form the nairovirus genome are encapsidated by the viral nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes that are substrates for RNA synthesis and packaging into virus particles. We used quantitative proteomics to identify cellular interaction partners of CCHFV N and identified robust interactions with cellular chaperones. These interactions were validated using immunological methods, and the specific interaction between native CCHFV N and cellular chaperones of the HSP70 family was confirmed during live CCHFV infection. Using infectious HAZV, we showed for the first time that the nairovirus N-HSP70 association was maintained within both infected cells and virus particles, where N is assembled as RNPs. Reduction of active HSP70 levels in cells by the use of small-molecule inhibitors significantly reduced HAZV titers, and a model for chaperone function in the context of high genetic variability is proposed. These results suggest that chaperones of the HSP70 family are required for nairovirus replication and thus represent a genetically stable cellular therapeutic target for preventing nairovirus-mediated disease.  

5.1932           Human Cathelicidin Compensates for the Role of Apolipoproteins in Hepatitis C Virus Infectious Particle Formation

Puig-Basagoiti, F., Fukuharaa, T., Tamura, T., Ono., C., Uemura, K., Kawachi, Y., Yamamoto, S., Mori, H., Kurihara, T., Okamoto, T., Aizaki, H. and Matsuura, Y.

1. Virol., 90(19), 8464-8477 (2016)

  Exchangeable apolipoproteins (ApoA, -C, and -E) have been shown to redundantly participate in the formation of infectious hepatitis C virus (HCV) particles during the assembly process, although their precise role in the viral life cycle is not well understood. Recently, it was shown that the exogenous expression of only short sequences containing amphipathic α-helices from various apolipoproteins is sufficient to restore the formation of infectious HCV particles in ApoB and ApoE double-gene-knockout Huh7 (BE-KO) cells. In this study, through the expression of a small library of human secretory proteins containing amphipathic α-helix structures, we identified the human cathelicidin antimicrobial peptide (CAMP), the only known member of the cathelicidin family of antimicrobial peptides (AMPs) in humans and expressed mainly in bone marrow and leukocytes. We showed that CAMP is able to rescue HCV infectious particle formation in BE-KO cells. In addition, we revealed that the LL-37 domain in CAMP containing amphipathic α-helices is crucial for the compensation of infectivity in BE-KO cells, and the expression of CAMP in nonhepatic 293T cells expressing claudin 1 and microRNA miR-122 confers complete propagation of HCV. These results suggest the possibility of extrahepatic propagation of HCV in cells with low-level or no expression of apolipoproteins but expressing secretory proteins containing amphipathic α-helices such as CAMP.  

5.1933           Neglected but Important Role of Apolipoprotein E Exchange in Hepatitis C Virus Infection

Yang, Z., Wang, X., Chi, X., Zhao, F., Guo, J., Ma, P., Zhong, J., Niu, J., Pan, X. and Long, G.

1. Virol., 90(21), 9632-9643 (2016)

  Hepatitis C virus (HCV) is a major cause of chronic liver disease, infecting approximately 170 million people worldwide. HCV assembly is tightly associated with the lipoprotein pathway. Exchangeable apolipoprotein E (apoE) is incorporated on infectious HCV virions and is important for infectious HCV virion morphogenesis and entry. Moreover, the virion apoE level is positively correlated with its ability to escape E2 antibody neutralization. However, the role of apoE exchange in the HCV life cycle is unclear. In this study, the relationship between apoE expression and cell permissiveness to HCV infection was assessed by infecting apoE knockdown and derived apoE rescue cell lines with HCV. Exchange of apoE between lipoproteins and HCV lipoviral particles (LVPs) was evaluated by immunoprecipitation, infectivity testing, and viral genome quantification. Cell and heparin column binding assays were applied to determine the attachment efficiency of LVPs with different levels of incorporated apoE. The results showed that cell permissiveness for HCV infection was determined by exogenous apoE-associated lipoproteins. Furthermore, apoE exchange did occur between HCV LVPs and lipoproteins, which was important to maintain a high apoE level on LVPs. Lipid-free apoE was capable of enhancing HCV infectivity for apoE knockdown cells but not apoE rescue cells. A higher apoE level on LVPs conferred more efficient LVP attachment to both the cell surface and heparin beads. This study revealed that exogenous apoE-incorporating lipoproteins from uninfected hepatocytes safeguarded the apoE level of LVPs for more efficient attachment during HCV infection.  

5.1934           Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories

Fridmann-Sirkis, Y., Milrot, E., Mutsafi, Y., Ben-Dor, S., levin, Y., Savidor, A., kartvelishvily, E. and Minsky, A.

1. Virol., 90(21), 10039-10047 (2016)

  The recent discovery of multiple giant double-stranded DNA (dsDNA) viruses blurred the consensual distinction between viruses and cells due to their size, as well as to their structural and genetic complexity. A dramatic feature revealed by these viruses as well as by many positive-strand RNA viruses is their ability to rapidly form elaborate intracellular organelles, termed “viral factories,” where viral progeny are continuously generated. Here we report the first isolation of viral factories at progressive postinfection time points. The isolated factories were subjected to mass spectrometry-based proteomics, bioinformatics, and imaging analyses. These analyses revealed that numerous viral proteins are present in the factories but not in mature virions, thus implying that multiple and diverse proteins are required to promote the efficiency of viral factories as “production lines” of viral progeny. Moreover, our results highlight the dynamic and highly complex nature of viral factories, provide new and general insights into viral infection, and substantiate the intriguing notion that viral factories may represent the living state of viruses.  

5.1935           Differential effects of peripheral and brain tumor necrosis factor on inflammation, sickness, emotional behavior and memory in mice

Klaus, F., Paterna, J-C., Marzorati, E., Sigrist, H., Götze, L., Schwendener, S., Beramini, G., Jehli, E., Azzinnari, D., Fuertig, R., Fontana, A., Seifritz, E. and Pryce, C.R. Brain, Behavior, and Immunity, 58, 310-326 (2016)   Tumor necrosis factor alpha (TNF) is increased in depression and clinical-trial evidence indicates that blocking peripheral TNF has some antidepressant efficacy. In rodents, peripheral or intracerebroventricular TNF results in sickness e.g. reduced body weight, altered emotional behavior and impaired memory. However, the underlying pathways and responsible brain regions are poorly understood. The aim of this mouse study was to increase understanding by comparing the effects of sustained increases in TNF in the circulation, in brain regions impacted by increased circulating TNF, or specific brain regions. Increased peripheral TNF achieved by repeated daily injection (IP-TNF) or osmotic pump resulted in decreased body weight, decreased saccharin (reward) consumption, and increased memory of an aversive conditioned stimulus. These effects co-occurred with increased plasma interleukin-6 and increased IP-derived TNF in brain peri-ventricular regions. An adenovirus-associated viral TNF vector (AAV-TNF) was constructed, brain injection of which resulted in dose-dependent, sustained and region-specific TNF expression, and was without effect on blood cytokine levels. Lateral ventricle AAV-TNF yielded increased TNF in the same brain regions as IP-TNF. In contrast to IP-TNF it was without effect on body weight, saccharin consumption and fear memory, although it did increase anxiety. Hippocampal AAV-TNF led to decreased body weight. It increased conditioning to but not subsequent memory of an aversive context, suggesting impaired consolidation; it also increased anxiety. Amygdala AAV-TNF was without effect on body weight and aversive stimulus learning-memory, but reduced saccharin consumption and increased anxiety. This study adds significantly to the evidence that both peripheral and brain region-specific increases in TNF lead to both sickness and depression- and anxiety disorder-relevant behavior and do so via different pathways. It thereby highlights the complexity in terms of indirect and direct pathways via which increased TNF can act and which need to be taken into account when considering it as a therapeutic target.  

5.1936           High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells by AAV6

Ling, C., Bhukhai, K., Yin, Z., Tan, M., Yoder, M.C., leboulch, P., payen, E. and Srivastava, A. Scientific Reports, 6:35495 (2016)   We have reported that of the 10 commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem/progenitor cells (HSPCs). However, the transduction efficiency of the wild-type (WT) AAV6 vector varies greatly in HSPCs from different donors. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced less efficiently with the WT AAV6 vectors. The first strategy involved modifications of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705 + Y731F + T492V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density. The combined use of these strategies resulted in transduction efficiency exceeding ~90% in HSPCs at significantly reduced vector doses. Our studies have significant implications in the optimal use of capsid-optimized AAV6 vectors in genome editing in HSPCs.  

5.1937           How can rAAV-α-synuclein and the fibril α-synuclein models advance our understanding of Parkinson's disease?

Volpicelli-Daley, L.A., Kirik, D., Stoyka, L.E., Standaert, D.G. and Harms, A.S.

1. Neurochem., 139 (Suppl. 1), 131-155 (2016)

  Animal models of Parkinson's disease (PD) are important for understanding the mechanisms of the disease and can contribute to developing and validating novel therapeutics. Ideally, these models should replicate the cardinal features of PD, such as progressive neurodegeneration of catecholaminergic neurons and motor defects. Many current PD models emphasize pathological forms of α-synuclein, based on findings that autosomal dominant mutations in α-synuclein and duplications/triplications of the SNCA gene cause PD. In addition, Lewy bodies and Lewy neurites, primarily composed of α-synuclein, represent the predominant pathological characteristics of PD. These inclusions have defined features, such as insolubility in non-ionic detergent, hyperphosphorylation, proteinase K sensitivity, a filamentous appearance by electron microscopy, and β-sheet structure. Furthermore, it has become clear that Lewy bodies and Lewy neurites are found throughout the peripheral and central nervous system, and could account not only for motor symptoms, but also for non-motor symptoms of the disease. The goal of this review is to describe two new α-synuclein-based models: the recombinant adeno-associated viral vector-α-synuclein model and the α-synuclein fibril model. An advantage of both models is that they do not require extensive crossbreeding of rodents transgenic for α-synuclein with other rodents transgenic for genes of interest to study the impact of such genes on PD-related pathology and phenotypes. In addition, abnormal α-synuclein can be expressed in brain regions relevant for disease. Here, we discuss the features of each model, how each model has contributed thus far to our understanding of PD, and the advantages and potential caveats of each model.  

5.1938           A paper-based immunoassay to determine HPV vaccination status at the point-of-care

Grant, B.D., Smith, C.A., Castle, P.E., Scheurer, M.E. and Richards-Kortum, R. Vaccine, 34, 5656-5663 (2016)   Objective To develop and evaluate a paper-based point-of-care HPV serology test to determine if an individual has received two or more HPV immunizations. Methods The paper-based immunoassay was constructed using a nitrocellulose lateral flow strip with adsorbed HPV16 virus-like particles serving as the capturing moiety. Three capture zones containing virus-like particles were placed in series to allow for visual discrimination between high and low HPV16 plasma antibody concentrations. A plasma separation membrane was used to allow whole blood to be applied directly to the assay. All reagents were dried on glass fiber pads during device fabrication and were rehydrated with buffer at the time of use. A pilot study consisting of 35 subjects with a history of zero, one, two or three HPV vaccines was conducted to evaluate the immunoassay. The completed paper-based immunoassays were scanned for visual interpretation by three researchers who were blinded to the true results and separately evaluated quantitatively using MATLAB. Results For the 28 tests valid for analysis, fifteen subjects reported receiving two or more HPV vaccines, three reported receiving one, and ten reported having no HPV vaccinations. The paper-based immunoassays for all fifteen subjects who reported having received two or more HPV vaccines were judged positive by all researchers. Twelve of the thirteen tests from individuals reporting one or zero vaccinations were deemed negative by all observers. One test from an unvaccinated individual was judged positive by two out of three reviewers. Quantitatively, all tests were correctly separated between the two groups. Conclusions We successfully designed and tested a HPV serology test amenable to the point-of-care. The device showed promising results in a pilot study for discriminating between those who received two or more HPV vaccinations and those who did not. Furthermore, this device offers a platform for producing other semi-quantitative point-of-care serological tests.  

5.1939           A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons

Tervo, D.G.R., Hwang, B-Y., Viswanathan, S., Looger, L.L., Schaffer, D.V. and Karpova, A.Y. Neuron, 92, 372-382 (2016)   Efficient retrograde access to projection neurons for the delivery of sensors and effectors constitutes an important and enabling capability for neural circuit dissection. Such an approach would also be useful for gene therapy, including the treatment of neurodegenerative disorders characterized by pathological spread through functionally connected and highly distributed networks. Viral vectors, in particular, are powerful gene delivery vehicles for the nervous system, but all available tools suffer from inefficient retrograde transport or limited clinical potential. To address this need, we applied in vivo directed evolution to engineer potent retrograde functionality into the capsid of adeno-associated virus (AAV), a vector that has shown promise in neuroscience research and the clinic. A newly evolved variant, rAAV2-retro, permits robust retrograde access to projection neurons with efficiency comparable to classical synthetic retrograde tracers and enables sufficient sensor/effector expression for functional circuit interrogation and in vivo genome editing in targeted neuronal populations.  

5.1940           BAR Proteins PSTPIP1/2 Regulate Podosome Dynamics and the Resorption Activity of Osteoclasts

Sztacho, M., Segeletz, S., Sanchez-Fernandez, M.A., Czupalia, C., Niehage, C. and Hoflack, B. PloS One, 11(10), e0164829 (2016)   Bone resorption in vertebrates relies on the ability of osteoclasts to assemble F-actin-rich podosomes that condense into podosomal belts, forming sealing zones. Sealing zones segregate bone-facing ruffled membranes from other membrane domains, and disassemble when osteoclasts migrate to new areas. How podosome/sealing zone dynamics is regulated remains unknown. We illustrate the essential role of the membrane scaffolding F-BAR-Proline-Serine-Threonine Phosphatase Interacting Proteins (PSTPIP) 1 and 2 in this process. Whereas PSTPIP2 regulates podosome assembly, PSTPIP1 regulates their disassembly. PSTPIP1 recruits, through its F-BAR domain, the protein tyrosine phosphatase non-receptor type 6 (PTPN6) that de-phosphophorylates the phosphatidylinositol 5-phosphatases SHIP1/2 bound to the SH3 domain of PSTPIP1. Depletion of any component of this complex prevents sealing zone disassembly and increases osteoclast activity. Thus, our results illustrate the importance of BAR domain proteins in podosome structure and dynamics, and identify a new PSTPIP1/PTPN6/SHIP1/2-dependent negative feedback mechanism that counterbalances Src and PI(3,4,5)P3 signalling to control osteoclast cell polarity and activity during bone resorption.  

5.1941           In vitro inhibition of human papillomavirus following use of a carrageenan-containing vaginal gel

Novetsky, A.P., Keller, M.J., Gradissimo, A., Chen, Z., Morgan, S.L., Xue, X., Stricker, H.D., Fernandez-Romero, J.A., Burk, R. and Einstein, M.H. Gynecol. Oncol., 143, 313-318 (2016)   Objective To assess in vitro efficacy of Divine 9, a carrageenan-based vaginal lubricant that is being studied as a microbicide to inhibit HPV16 pseudovirus (PsV) infection. Methods Sexually active US women between 19 and 35 years without prior HPV vaccination or cervical intraepithelial neoplasia were instructed to use Divine 9 vaginally with an applicator either before sex only or before and after intercourse. Women who applied a single dose of gel returned for cervicovaginal lavage (CVL) collection 1, 4 or 8–12 h after intercourse versus those who applied gel before and after intercourse returned 1, 4 or 8–12 h after the second gel dose. Carrageenan concentrations were assessed using an ELISA assay and the inhibitory activity was assessed using a PsV-based neutralization assay against HPV16 infection. Carrageenan concentrations and the percentage of PsV16 inhibition were compared using the Wilcoxon rank sum test. Results Thirteen women were enrolled and thirty specimens from different time-points were assessed. 87% of CVL samples had detectable carrageenans with levels decreasing over time from intercourse. 93% of CVL samples had detectable PsV16 inhibition with median inhibition of 97.5%. PsV16 inhibition decreased over time, but remained high, with median inhibition of 98.1%, 97.4% and 83.4% at 1, 4 and 8–12 h, respectively. Higher carrageenan concentrations were associated with higher levels of PsV16 inhibition (rho = 0.69). Conclusions This is the first report of a human study investigating in vitro HPV inhibition of a carrageenan-based vaginal lubricant with CVL collected after sexual intercourse. We demonstrate excellent efficacy in preventing PsV16 infection.  

5.1942           Chapter 4 Proteomic Studies of HIV-1

Graham, D.R.M. Proteomic Studies of HIV-1, 39-58 (2016)   The application of proteomics has become routine in many fields allowing for the simultaneous measurement of proteins representing a vast array of different processes in tissues, cells, and biological fluids. The application of proteomics to the study of viruses remains a challenge due to many factors including the limited sample, sample preparation requirements, and specialized bioinformatics that have to be applied to studying viruses like HIV-1. Here we detail an extensive overview of all of the sample preparation strategies as they apply to HIV-1, a primer in the understanding of mass spectrometry (MS) as it applies to HIV-1 proteomic and basic bioinformatic approaches that need to be taken to ensure success. We also introduce new and emerging technologies that will allow for the application of HIV-1 proteomics to the clinic for potential clinical applications. Finally, we describe alternative approaches to mass spectrometry and discuss strategies that can be used for systems biology approaches to the study of HIV-1.  

5.1943           Genetic Disruption of Circadian Rhythms in the Suprachiasmatic Nucleus Causes Helplessness, Behavioral Despair, and Anxiety-like Behavior in Mice

Landgraf, D., Long, J.E., Prouix, C.D., Barandas, R., Malinow, R. and Welsh, D.K. Biological Psychiatry, 80(11), 827-835 (2016)   Background Major depressive disorder is associated with disturbed circadian rhythms. To investigate the causal relationship between mood disorders and circadian clock disruption, previous studies in animal models have employed light/dark manipulations, global mutations of clock genes, or brain area lesions. However, light can impact mood by noncircadian mechanisms; clock genes have pleiotropic, clock-independent functions; and brain lesions not only disrupt cellular circadian rhythms but also destroy cells and eliminate important neuronal connections, including light reception pathways. Thus, a definitive causal role for functioning circadian clocks in mood regulation has not been established. Methods We stereotactically injected viral vectors encoding short hairpin RNA to knock down expression of the essential clock gene Bmal1 into the brain’s master circadian pacemaker, the suprachiasmatic nucleus (SCN). Results In these SCN-specific Bmal1-knockdown (SCN-Bmal1-KD) mice, circadian rhythms were greatly attenuated in the SCN, while the mice were maintained in a standard light/dark cycle, SCN neurons remained intact, and neuronal connections were undisturbed, including photic inputs. In the learned helplessness paradigm, the SCN-Bmal1-KD mice were slower to escape, even before exposure to inescapable stress. They also spent more time immobile in the tail suspension test and less time in the lighted section of a light/dark box. The SCN-Bmal1-KD mice also showed greater weight gain, an abnormal circadian pattern of corticosterone, and an attenuated increase of corticosterone in response to stress. Conclusions Disrupting SCN circadian rhythms is sufficient to cause helplessness, behavioral despair, and anxiety-like behavior in mice, establishing SCN-Bmal1-KD mice as a new animal model of depression.  

5.1944           A multifunctional AAV–CRISPR–Cas9 and its host response

Zhew, W.L., Tabebordbar, M., Cheng, J.K.W., Mali, P., Wu, E.Y., Ng, A.H.M., Zhu, K., Wagers, A.J. and Church, G.M. Nature Methods, 13(10), 868-874 (2016)   CRISPR–Cas9 delivery by adeno-associated virus (AAV) holds promise for gene therapy but faces critical barriers on account of its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV–split-Cas9, a multifunctional platform customizable for genome editing, transcriptional regulation, and other previously impracticable applications of AAV–CRISPR–Cas9. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV–CRISPR–Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce extensive cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics.  

5.1945           Alpha-1 Antitrypsin Gene Therapy Ameliorates Bone Loss in Ovariectomy-Induced Osteoporosis Mouse ModelNo Access

Akbar, M.A., Cao, J.J., Lu, Y., nardo, D., Chen, M-J., Elshika, A.S., Ahamed, R., Brantly, M., Shannon Holliday, L. and Song, S. Human Gene Therapy, 27(9), 679-686 (2016)   Osteoporosis is a major healthcare burden affecting mostly postmenopausal women characterized by compromised bone strength and increased risk of fragility fracture. Although pathogenesis of this disease is complex, elevated proinflammatory cytokine production is clearly involved in bone loss at menopause. Therefore, anti-inflammatory strategies hold a great potential for the prevention of postmenopausal osteoporosis. In this study, we investigated the effect of gene therapy of recombinant adeno-associated virus (rAAV)–mediated human alpha-1 antitrypsin (hAAT), a multifunctional protein that has anti-inflammatory property, on bone loss in an ovariectomy-induced osteoporosis mouse model. Adult ovariectomized (OVX) mice were intraperitoneally (i.p.) injected with hAAT (protein therapy), rAAV8-CB-hAAT (gene therapy), or phosphate buffer saline (PBS). Age-matched and sham-operated animals were used as controls. Eight weeks after the treatment, animals were sacrificed and bone-related biomarkers and vertebral bone structure were evaluated. Results showed that hAAT gene therapy significantly decreased serum IL-6 level and receptor activator of NF-κB (RANK) gene expression in bone. Importantly, hAAT gene therapy increased bone volume/total volume and decreased structure model index (SMI) compared to PBS injection in OVX mice. These results demonstrate that hAAT gene therapy by rAAV vector efficiently mitigates bone loss possibly through inhibition of proinflammatory cytokine IL-6 and RANK gene expression. Considering the safety profile of hAAT and rAAV vector in humans, our results provide a new alternative for the treatment of osteoporosis.  

5.1946           Adeno-Associated Viral Vectors Transduce Mature Human Adipocytes in Three-Dimensional Slice CulturesNo Access

Kallendrusch, S., Schopow, N., Stadler, S.C., Büning, H. and Hacker, U.T. Human Gene Therapy Methods, 27(5), 171-173 (2016)   Adipose tissue plays a pivotal role, both in the regulation of energy homeostasis and as an endocrine organ. Consequently, adipose tissue dysfunction is closely related to insulin resistance, morbid obesity, and metabolic syndrome. To study molecular mechanisms and to develop novel therapeutic strategies, techniques are required to genetically modify mature adipocytes. Here, we report on adeno-associated viral (AAV) vectors as a versatile tool to transduce human mature adipocytes in organotypic three-dimensional tissue cultures.    

5.1947           Characterization and Complete Genome Sequences of Three N4-Like Roseobacter Phages Isolated from the South China Sea

Li, B., Zhang, Si., Long, L. And Huang, S. Curr. Microbiol., 73(3), 409-418 (2016)   Three bacteriophages (RD-1410W1-01, RD-1410Ws-07, and DS-1410Ws-06) were isolated from the surface water of Sanya Bay, northern South China Sea, on two marine bacteria type strains of the Roseobacter lineage. These phages have an isometric head and a short tail, morphologically belonging to the Podoviridae family. Two of these phages can infect four of seven marine roseobacter strains tested and the other one can infect three of them, showing relatively broader host ranges compared to known N4-like roseophages. One-step growth curves showed that these phages have similar short latent periods (1–2 h) but highly variable burst sizes (27–341 pfu cell⁻¹). Their complete genomes show high level of similarities to known N4-like roseophages in terms of genome size, G + C content, gene content, and arrangement. The morphological and genomic features of these phages indicate that they belong to the N4likevirus genus. Moreover, comparative genomic analysis based on 43 N4-like phages (10 roseobacter phages and 33 phages infecting other lineages of bacteria) revealed a core genome of 18 genes shared by all the 43 phages and 38 genes shared by all the ten roseophages. The 38 core genes of N4-like roseophages nearly make up 70 % of each genome in length. Phylogenetic analysis based on the concatenated core gene products showed that our phage isolates represent two new phyletic branches, suggesting the broad genetic diversity of marine N4-like roseophages remains.  

5.1948           Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina

Sengupta, A.,, Chaffiol, A., mace, E., Caplette, R., Desrosiers, M., lampic, M., Forster, V., Marre, O., Lin, J.Y., Sahel, J-A., Picaud, S., Dalkara, D. and Duebel, J. EMBO Mol. Med., 8(11), 1248-1264 (2016)   Targeting the photosensitive ion channel channelrhodopsin‐2 (ChR2) to the retinal circuitry downstream of photoreceptors holds promise in treating vision loss caused by retinal degeneration. However, the high intensity of blue light necessary to activate channelrhodopsin‐2 exceeds the safety threshold of retinal illumination because of its strong potential to induce photochemical damage. In contrast, the damage potential of red‐shifted light is vastly lower than that of blue light. Here, we show that a red‐shifted channelrhodopsin (ReaChR), delivered by AAV injections in blind rd1 mice, enables restoration of light responses at the retinal, cortical, and behavioral levels, using orange light at intensities below the safety threshold for the human retina. We further show that postmortem macaque retinae infected with AAV‐ReaChR can respond with spike trains to orange light at safe intensities. Finally, to directly address the question of translatability to human subjects, we demonstrate for the first time, AAV‐ and lentivirus‐mediated optogenetic spike responses in ganglion cells of the postmortem human retina.  

5.1949           Fusion of Human Fetal Mesenchymal Stem Cells with “Degenerating” Cerebellar Neurons in Spinocerebellar Ataxia Type 1 Model Mice

Huda, F., Fan, Y., Suzuki, M., Konno, A., matsuzaki, Y., Takahashi, N., Chan, J.K.Y. and Hirai, H. PloS One, 11(11), e0164202 (2016)  

Mesenchymal stem cells (MSCs) migrate to damaged tissues, where they participate in tissue repair. Human fetal MSCs (hfMSCs), compared with adult MSCs, have higher proliferation rates, a greater differentiation capacity and longer telomeres with reduced senescence. Therefore, transplantation of quality controlled hfMSCs is a promising therapeutic intervention. Previous studies have shown that intravenous or intracortical injections of MSCs result in the emergence of binucleated cerebellar Purkinje cells (PCs) containing an MSC-derived marker protein in mice, thus suggesting a fusion event. However, transdifferentiation of MSCs into PCs or transfer of a marker protein from an MSC to a PC cannot be ruled out. In this study, we unequivocally demonstrated the fusion of hfMSCs with murine PCs through a tetracycline-regulated (Tet-off) system with or without a Cre-dependent genetic inversion switch (flip-excision; FLEx). In the FLEx-Tet system, we performed intra-cerebellar injection of viral vectors expressing tetracycline transactivator (tTA) and Cre recombinase into either non-symptomatic (4-week-old) or clearly symptomatic (6–8-month-old) spinocerebellar ataxia type 1 (SCA1) mice. Then, the mice received an injection of 50,000 genetically engineered hfMSCs that expressed GFP only in the presence of Cre recombinase and tTA. We observed a significant emergence of GFP-expressing PCs and interneurons in symptomatic, but not non-symptomatic, SCA1 mice 2 weeks after the MSC injection. These results, together with the results obtained using age-matched wild-type mice, led us to conclude that hfMSCs have the potential to preferentially fuse with degenerating PCs and interneurons but not with healthy neurons.

 

5.1950           Adeno-Associated Virus–Mediated Delivery of CRISPR–Cas Systems for Genome Engineering in Mammalian Cells

Gaj, T. and Schaffer, D.V. Cold Spring Harb. Protoc., pdb.prot086868 (2016)   The CRISPR–Cas9 system has emerged as a highly versatile platform for introducing targeted genome modifications into mammalian cells and model organisms. However, fully capitalizing on the therapeutic potential for this system requires its safe and efficient delivery into relevant cell types. Adeno-associated virus (AAV) vectors are a clinically promising class of engineered gene-delivery vehicles capable of safely infecting a broad range of dividing and nondividing cell types, while also serving as a highly effective donor template for homology-directed repair. Together, CRISPR–Cas9 and AAV technologies have the potential to accelerate both basic research and clinical applications of genome engineering. Here, we present a step-by-step protocol for AAV-mediated delivery of CRISPR–Cas systems into mammalian cells. Procedures are given for the preparation of high-titer virus capable of achieving a diverse range of genetic modifications, including gene knockout and integration.  

5.1951           An open-hardware platform for optogenetics and photobiology

Gerhardt, K.P., Olson, E.J., Castillo-Hair, S.M., Hartsough, L.A., landry, B.P., Ekness, F., Yokoo, R., Gomez, E.J., Ramakrishnan, P., Suh, J., Savage, D. and Tabor, J.J.

Scientific Reports, 6:35363 (2016)

In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments.

 

5.1952           Cutthroat Trout Virus-Towards a Virus Model to Support Hepatitis E Research

Von Nordheim, M., Boinay, M., leisi, R., Kempf, C. And Ros, C. Viruses, 8(10), E 289 (2016)   Cutthroat trout virus (CTV) is a non-pathogenic fish virus belonging to the Hepeviridae family, and it is distantly related to hepatitis E virus (HEV). Here, we report the development of an efficient cell culture system where CTV can consistently replicate to titers never observed before with a hepevirus. By using the rainbow trout gill (RTGill-W1) cell line, CTV reaches 1010 geq/mL intracellularly and 10⁸ geq/mL extracellularly within 5-6 days in culture. We additionally established a qPCR system to investigate CTV infectivity, and developed a specific antibody directed against the viral capsid protein encoded by ORF2. With these methods, we were able to follow the progressive accumulation of viral RNA and the capsid protein, and their intracellular distribution during virus replication. Virus progeny purified through iodixanol density gradients indicated-that similar to HEV-CTV produced in cell culture is also lipid-associated. The lack of an efficient cell culture system has greatly impeded studies with HEV, a major human pathogen that causes hepatitis worldwide. Although several cell culture systems have recently been established, the replication efficiency of HEV is not robust enough to allow studies on different aspects of the virus replication cycle. Therefore, a surrogate virus that can replicate easily and efficiently in cultured cells would be helpful to boost research studies with hepeviruses. Due to its similarities, but also its key differences to HEV, CTV represents a promising tool to elucidate aspects of the replication cycle of Hepeviridae in general, and HEV in particular.  

5.1953           Minicircle HBV cccDNA with a Gaussia luciferase reporter for investigating HBV cccDNA biology and developing cccDNA-targeting drugs

Li, F., Cheng, L., Murphy, C.M., Reszka-Blanco, N.J:, Wu, Y., Chi, L., Hu, J. and Su, L. Scientific Reports, 6:36483 (2016)   Chronic Hepatitis B Virus (HBV) infection is generally not curable with current anti-viral drugs. Virus rebounds after stopping treatment from the stable HBV covalently-closed-circular DNA (cccDNA). The development of drugs that directly target cccDNA is hampered by the lack of robust HBV cccDNA models. We report here a novel HBV cccDNA technology that will meet the need. We engineered a minicircle HBV cccDNA with a Gaussia Luciferase reporter (mcHBV-GLuc cccDNA), which serves as a surrogate to measure cccDNA activity. The mcHBV-GLuc cccDNA was easily produced in bacteria, and it formed minichromosomes as HBV cccDNA episome DNA does when it was transfected into human hepatocytes. Compared to non-HBV minicircle plasmids, mcHBV-GLuc cccDNA showed persistent HBV-GLuc activity and HBx-dependent gene expression. Importantly, the mcHBV-GLuc cccDNA showed resistance to interferons (IFN) treatment, indicating its unique similarity to HBV cccDNA that is usually resistant to long-term IFN treatment in chronic HBV patients. Most importantly, GLuc illuminates cccDNA as a surrogate of cccDNA activity, providing a very sensitive and quick method to detect trace amount of cccDNA. The mcHBV-GLuc cccDNA model is independent of HBV infection, and will be valuable for investigating HBV cccDNA biology and for developing cccDNA-targeting drugs.  

5.1954           Bacterial superglue generates a full-length circumsporozoite protein virus-like particle vaccine capable of inducing high and durable antibody responses

Janitzek, C.M., Matondo, S., Thrane, S., Nielsen, M.A., kavishe, R., Mwakalinga, S.B., Theander, T.G., Salanti, A. and Sander, A.F. Malar. J., 15:545 (2016)   Background Malaria, caused by Plasmodium falciparum, continues to have a devastating impact on global health, emphasizing the great need for a malaria vaccine. The circumsporozoite protein (CSP) is an attractive target for a malaria vaccine, and forms a major component of RTS,S, the most clinically advanced malaria vaccine. The clinical efficacy of RTS,S has been moderate, yet has demonstrated the viability of a CSP-based malaria vaccine. In this study, a vaccine comprised of the full-length CSP antigen presented on a virus-like particle (VLP) is produced using a split-intein conjugation system (SpyTag/SpyCatcher) and the immunogenicity is tested in mice. Methods Full-length 3d7 CSP protein was genetically fused at the C-terminus to SpyCatcher. The CSP-SpyCatcher antigen was then covalently attached (via the SpyTag/SpyCatcher interaction) to Acinetobacter phage AP205 VLPs which were modified to display one SpyTag per VLP subunit. To evaluate the VLP-display effect, the immunogenicity of the VLP vaccine was tested in mice and compared to a control vaccine containing AP205 VLPs plus unconjugated CSP. Results Full-length CSP was conjugated at high density (an average of 112 CSP molecules per VLP) to AP205 SpyTag-VLPs. Vaccination of mice with the CSP Spy-VLP vaccine resulted in significantly increased antibody titres over a course of 7 months as compared to the control group (2.6-fold higher at 7 months after immunization). Furthermore, the CSP Spy-VLP vaccine appears to stimulate production of IgG2a antibodies, which has been linked with a more efficient clearing of intracellular parasite infection. Conclusion This study demonstrates that the high-density display of CSP on SpyTag-VLPs, significantly increases the level and quality of the vaccine-induced humoral response, compared to a control vaccine consisting of soluble CSP plus AP205 VLPs. The SpyTag-VLP platform utilized in this study constitutes a versatile and rapid method to develop highly immunogenic vaccines. It might serve as a generic tool for the cost-effective development of effective VLP-vaccines, e.g., against malaria.  

5.1955           Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry

Borrenberghs, D., Dirix, L., De Wit, F., Rocha, S., Blokken, J., De Houwer, S., Gijsbers, R., Christ, F., Hofkens, J., Hendrix, J. and Debyser, Z. Scientific Reports, 6:36485 (2016)   Nuclear entry is a selective, dynamic process granting the HIV-1 pre-integration complex (PIC) access to the chromatin. Classical analysis of nuclear entry of heterogeneous viral particles only yields averaged information. We now have employed single-virus fluorescence methods to follow the fate of single viral pre-integration complexes (PICs) during infection by visualizing HIV-1 integrase (IN). Nuclear entry is associated with a reduction in the number of IN molecules in the complexes while the interaction with LEDGF/p75 enhances IN oligomerization in the nucleus. Addition of LEDGINs, small molecule inhibitors of the IN-LEDGF/p75 interaction, during virus production, prematurely stabilizes a higher-order IN multimeric state, resulting in stable IN multimers resistant to a reduction in IN content and defective for nuclear entry. This suggests that a stringent size restriction determines nuclear pore entry. Taken together, this work demonstrates the power of single-virus imaging providing crucial insights in HIV replication and enabling mechanism-of-action studies.  

5.1956           The long non-coding RNA Morrbid regulates Bim and short-lived myeloid cell lifespan

Kotzin, J.J. et al

Nature, 537(7619), 239-243 (2016)

Neutrophils, eosinophils and ‘classical’ monocytes collectively account for about 70% of human blood leukocytes and are among the shortest-lived cells in the body1, 2. Precise regulation of the lifespan of these myeloid cells is critical to maintain protective immune responses and minimize the deleterious consequences of prolonged inflammation1, 2. However, how the lifespan of these cells is strictly controlled remains largely unknown. Here we identify a long non-coding RNA that we termed Morrbid, which tightly controls the survival of neutrophils, eosinophils and classical monocytes in response to pro-survival cytokines in mice. To control the lifespan of these cells, Morrbid regulates the transcription of the neighbouring pro-apoptotic gene, Bcl2l11 (also known as Bim), by promoting the enrichment of the PRC2 complex at the Bcl2l11 promoter to maintain this gene in a poised state. Notably, Morrbid regulates this process in cis, enabling allele-specific control of Bcl2l11 transcription. Thus, in these highly inflammatory cells, changes in Morrbid levels provide a locus-specific regulatory mechanism that allows rapid control of apoptosis in response to extracellular pro-survival signals. As MORRBID is present in humans and dysregulated in individuals with hypereosinophilic syndrome, this long non-coding RNA may represent a potential therapeutic target for inflammatory disorders characterized by aberrant short-lived myeloid cell lifespan.

 

5.1957           Cytokine-Like 1 Regulates Cardiac Fibrosis via Modulation of TGF-β Signaling

Kim, J., Kim, J., Lee, S.H., Kepreotis, S.V., Yoo, J., Chun, J-S., Hajjar, R.J., Jeong, D. and Park, W.J. PloS One, 11(11), e0166480 (2016)   Cytokine-like 1 (Cytl1) is a secreted protein that is involved in diverse biological processes. A comparative modeling study indicated that Cytl1 is structurally and functionally similar to monocyte chemoattractant protein 1 (MCP-1). As MCP-1 plays an important role in cardiac fibrosis (CF) and heart failure (HF), we investigated the role of Cytl1 in a mouse model of CF and HF. Cytl1 was upregulated in the failing mouse heart. Pressure overload-induced CF was significantly attenuated in cytl1 knock-out (KO) mice compared to that from wild-type (WT) mice. By contrast, adeno-associated virus (AAV)-mediated overexpression of cytl1 alone led to the development of CF in vivo. The endothelial-mesenchymal transition (EndMT) and the transdifferentiation of fibroblasts (FBs) to myofibroblasts (MFBs) have been suggested to contribute considerably to CF. Adenovirus-mediated overexpression of cytl1 was sufficient to induce these two critical CF-related processes in vitro, which were completely abrogated by co-treatment with SB-431542, an antagonist of TGF-β receptor 1. Cytl1 induced the expression of TGF-β2 both in vivo and in vitro. Antagonizing the receptor for MCP-1, C-C chemokine receptor type 2 (CCR2), with CAS 445479-97-0 did not block the pro-fibrotic activity of Cytl1 in vitro. Collectively, our data suggest that Cytl1 plays an essential role in CF likely through activating the TGF-β-SMAD signaling pathway. Although the receptor for Cyt1l remains to be identified, Cytl1 provides a novel platform for the development of anti-CF therapies.  

5.1958           Hepatitis C Virus Is Released via a Noncanonical Secretory Route

Bayer, K., Banning, C., Bruss, V., Wiltzer-bach, L. and Schindler, M.

1. Virol., 90(23), 10558-10573 (2016)

  We analyzed hepatitis C virus (HCV) morphogenesis using viral genomes encoding a mCherry-tagged E1 glycoprotein. HCV-E1-mCherry polyprotein expression, intracellular localization, and replication kinetics were comparable to those of untagged HCV, and E1-mCherry-tagged viral particles were assembled and released into cell culture supernatants. Expression and localization of structural E1 and nonstructural NS5A followed a temporospatial pattern with a succinct decrease in the number of replication complexes and the appearance of E1-mCherry punctae. Interaction of the structural proteins E1, Core, and E2 increased at E1-mCherry punctae in a time-dependent manner, indicating that E1-mCherry punctae represent assembled or assembling virions. E1-mCherry did not colocalize with Golgi markers. Furthermore, the bulk of viral glycoproteins within released particles revealed an EndoH-sensitive glycosylation pattern, indicating an absence of viral glycoprotein processing by the Golgi apparatus. In contrast, HCV-E1-mCherry trafficked with Rab9-positive compartments and inhibition of endosomes specifically suppressed HCV release. Our data suggest that assembled HCV particles are released via a noncanonical secretory route involving the endosomal compartment.  

5.1959           The {gamma}134.5 Neurovirulence Gene of Herpes Simplex Virus 1 Modifies the Exosome Secretion Profile in Epithelial Cells

Heikkilä, O., Ryödl, E. and Hukkanen, V.

1. Virol., 90(23), 10981-10984 (2016)

  Recently, it has been demonstrated that herpes simplex virus 1 (HSV-1)-infected cells secrete exosomes that deliver to uninfected cells the innate immune sensor STING and viral RNAs (1, 2). Here, we report for the first time that the deletion of the viral γ₁34.5 neurovirulence gene affects HSV-induced exosome secretion. HSV-1, lacking both copies of the γ₁34.5 neurovirulence gene, is a proficient gene therapy vector backbone for use in the nervous system. Although the γ₁34.5 mutants are nonneurovirulent, they can still spread in different cell types of the nervous system (3, 4). However, the possibility that exosomes copurify with HSV preparations raises concern about the vector purity and reproducibility in gene therapy use.  

5.1960           Molecular basis for the formation of ribonucleoprotein complex of Crimean-Congo hemorrhagic fever virus

Wang, X., Li, B., Guo, Y., Shen, S., Zhao, L., Zhang, P., Sun, Y., Sui, S-F., Deng, F. and Lou, Z.

1. Struct. Biol., 196, 455-465 (2016)

  Negative-sense single-strand RNA (-ssRNA) viruses comprise a large family of pathogens that cause severe human infectious diseases. All -ssRNA viruses encode a nucleocapsid protein (NP) to encapsidate the viral genome, which, together with polymerase, forms a ribonucleoprotein complex (RNP) that is packaged into virions and acts as the template for viral replication and transcription. In our previous work, we solved the monomeric structure of NP encoded by Crimean-Congo hemorrhagic fever virus (CCHFV), which belongs to the Nairovirus genus within the Bunyaviridae family, and revealed its unusual endonuclease activity. However, the mechanism of CCHFV RNP formation remains unclear, due to the difficulty in reconstructing the oligomeric CCHFV NP-RNA complex. Here, we identified and isolated the oligomeric CCHFV NP-RNA complex that formed in expression cells. Sequencing of RNA extracted from the complex revealed sequence specificity and suggested a potential encapsidation signal facilitating the association between NP and viral genome. A cryo-EM reconstruction revealed the ring-shaped architecture of the CCHFV NP-RNA oligomer, thus defining the interaction between the head and stalk domains that results in NP multimerization. This structure also suggested a modified gating mechanism for viral genome encapsidation, in which both the head and stalk domains participate in RNA binding. This work provides insight into the distinct mechanism underlying CCHFV RNP formation compared to other -ssRNA viruses.  

5.1961           Brd4 Activates Early Viral Transcription upon Human Papillomavirus 18 Infection of Primary Keratinocytes

McKinney, C.C., Kim, M.J., Chen, D. and McBride, A.A. mBio, 7(6), e01644-16 (2016)   Human papillomaviruses (HPVs) replicate in the cutaneous and mucosal epithelia, and the infectious cycle is synchronous with the differentiation program of the host keratinocytes. The virus initially infects dividing cells in the lower layers of the epithelium, where it establishes a persistent infection. The viral genome is maintained as a low-copy-number, extrachromosomal element in these proliferating cells but switches to the late stage of the life cycle in differentiated cells. The cellular chromatin adaptor protein Brd4 is involved in several stages and processes of the viral life cycle. In concert with the viral transcriptional regulator E2, Brd4 can repress transcription from the early viral promoter. Brd4 and E2 form a complex with the viral genome that associates with host chromosomes to partition the viral genome in dividing cells; Brd4 also localizes to active sites of productive HPV DNA replication. However, because of the difficulties in producing HPV viral particles, the role of Brd4 in modulating viral transcription and replication at the initial stage of infection is unclear. In this study, we have used an HPV18 quasivirus-based genome delivery system to assess the role of Brd4 in the initial infectivity of primary human keratinocytes. We show that, upon infection of primary human keratinocytes with HPV18 quasivirus, Brd4 activates viral transcription and replication. Furthermore, this activation is independent of the functional interaction between Brd4 and the HPV18 E2 protein.  

5.1962           The Intracellular Cholesterol Transport Inhibitor U18666A Inhibits the Exosome-Dependent Release of Mature Hepatitis C Virus

Elgner, F., Ren, H., Medvedev, R., Ploen, D., Himmelsbach, K., Boller, K. and Hildt, E.

1. Virol., 90(24), 1181-1196 (2016)

  Hepatitis C virus (HCV) particles are described as lipoviroparticles which are released similarly to very-low-density lipoproteins (VLDLs). However, the release mechanism is still poorly understood; the canonical endoplasmic reticulum-Golgi intermediate compartment (ERGIC) pathway as well as endosome-dependent release has been proposed. Recently, the role of exosomes in the transmission of HCV has been reported. Only a minor fraction of the de novo-synthesized lipoviroparticles is released by the infected cell. To investigate the relevance of multivesicular bodies (MVBs) for viral morphogenesis and release, the MVB inhibitor U18666A was used. Intracellular trafficking was analyzed by confocal microscopy and electron microscopy. Moreover, an mCherry-tagged HCV variant was used. Conditions were established that enable U18666A-dependent inhibition of MVBs without affecting viral replication. Under these conditions, significant inhibition of the HCV release was observed. The assembly of viral particles is not affected. In U18666A-treated cells, intact infectious viral particles accumulate in CD63-positive exosomal structures and large dysfunctional lysosomal structures (multilamellar bodies). These retained particles possess a lower density, reflecting a misloading with lipids. Our data indicate that at least a fraction of HCV particles leaves the cell via the endosomal pathway. Endosomes facilitate the sorting of HCV particles for release or degradation.  

5.1963           Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus

Hirai-Yuki, A., Hensley, L., Whitmire, J.K. and lemon, S.M. mBio, 7(6), e1998-16 (2016)   Hepatitis A virus (HAV) is an unusual picornavirus that is released from cells cloaked in host-derived membranes. These quasi-enveloped virions (eHAV) are the only particle type circulating in blood during infection, whereas only nonenveloped virions are shed in feces. The reason for this is uncertain. Hepatocytes, the only cell type known to support HAV replication in vivo, are highly polarized epithelial cells with basolateral membranes facing onto hepatic (blood) sinusoids and apical membranes abutting biliary canaliculi from which bile is secreted to the gut. To assess whether eHAV and nonenveloped virus egress from cells via vectorially distinct pathways, we studied infected polarized cultures of Caco-2 and HepG2-N6 cells. Most (>99%) progeny virions were released apically from Caco-2 cells, whereas basolateral (64%) versus apical (36%) release was more balanced with HepG2-N6 cells. Both apically and basolaterally released virions were predominantly enveloped, with no suggestion of differential vectorial release of eHAV versus naked virions. Basolateral to apical transcytosis of either particle type was minimal (<0.02%/h) in HepG2-N6 cells, arguing against this as a mechanism for differences in membrane envelopment of serum versus fecal virus. High concentrations of human bile acids converted eHAV to nonenveloped virions, whereas virus present in bile from HAV-infected Ifnar1^−/− Ifngr1^−/− and Mavs^−/− mice banded over a range of densities extending from that of eHAV to that of nonenveloped virions. We conclude that nonenveloped virions shed in feces are derived from eHAV released across the canalicular membrane and stripped of membranes by the detergent action of bile acids within the proximal biliary canaliculus.  

5.1964           Novel recombinant papillomavirus genomes expressing selectable genes

Van Doorslaer, K., Porter, S., McKinney, C., Stepp, W.H. and McBride, A. Scientific Reports, 6:37782 (2016)   Papillomaviruses infect and replicate in keratinocytes, but viral proteins are initially expressed at low levels and there is no effective and quantitative method to determine the efficiency of infection on a cell-to-cell basis. Here we describe human papillomavirus (HPV) genomes that express marker proteins (antibiotic resistance genes and Green Fluorescent Protein), and can be used to elucidate early stages in HPV infection of primary keratinocytes. To generate these recombinant genomes, the late region of the oncogenic HPV18 genome was replaced by CpG free marker genes. Insertion of these exogenous genes did not affect early replication, and had only minimal effects on early viral transcription. When introduced into primary keratinocytes, the recombinant marker genomes gave rise to drug-resistant keratinocyte colonies and cell lines, which maintained the extrachromosomal recombinant genome long-term. Furthermore, the HPV18 “marker” genomes could be packaged into viral particles (quasivirions) and used to infect primary human keratinocytes in culture. This resulted in the outgrowth of drug-resistant keratinocyte colonies containing replicating HPV18 genomes. In summary, we describe HPV18 marker genomes that can be used to quantitatively investigate many aspects of the viral life cycle.  

5.1965           Recognition of extremophilic archaeal viruses by eukaryotic cells: a promising nanoplatform from the third domain of life

Uldahl, K.B., Wu, L., hall, A., papathanasiou, P., Peng, X. and Moghimi, S.M. Scientific Reports, 6:37966 (2016)   Viruses from the third domain of life, Archaea, exhibit unusual features including extreme stability that allow their survival in harsh environments. In addition, these species have never been reported to integrate into human or any other eukaryotic genomes, and could thus serve for exploration of novel medical nanoplatforms. Here, we selected two archaeal viruses Sulfolobus monocaudavirus 1 (SMV1) and Sulfolobus spindle shaped virus 2 (SSV2) owing to their unique spindle shape, hyperthermostable and acid-resistant nature and studied their interaction with mammalian cells. Accordingly, we followed viral uptake, intracellular trafficking and cell viability in human endothelial cells of brain (hCMEC/D3 cells) and umbilical vein (HUVEC) origin. Whereas SMV1 is efficiently internalized into both types of human cells, SSV2 differentiates between HUVECs and hCMEC/D3 cells, thus opening a path for selective cell targeting. On internalization, both viruses localize to the lysosomal compartments. Neither SMV1, nor SSV2 induced any detrimental effect on cell morphology, plasma membrane and mitochondrial functionality. This is the first study demonstrating recognition of archaeal viruses by eukaryotic cells which provides good basis for future exploration of archaeal viruses in bioengineering and development of multifunctional vectors.  

5.1966           Transcriptional activity of novel ALDH1L1 promoters in the rat brain following AAV vector-mediated gene transfer

Mudannayake, J.M:, Mouravlev, A., Fong, D.M. and Young, D. Mol. Therapy-Methods & Clin. Development, 3, 16075 (2016)   Aldehyde dehydrogenase family 1, member L1 (ALDH1L1) is a recently characterized pan-astrocytic marker that is more homogenously expressed throughout the brain than the classic astrocytic marker, glial fibrillary acidic protein. We generated putative promoter sequence variants of the rat ALDH1L1 gene for use in adeno-associated viral vector-mediated gene transfer, with an aim to achieve selective regulation of transgene expression in astrocytes in the rat brain. Unexpectedly, ALDH1L1 promoter variants mediated transcriptional activity exclusively in neurons in the substantia nigra pars compacta as assessed by luciferase reporter expression at 3 weeks postvector infusion. This selectivity for neurons in the substantia nigra pars compacta also persisted in the context of adeno-associated viral serotype 5, 8 or 9 vector-mediated gene delivery. An in vivo promoter comparison showed the highest performing ALDH1L1 promoter variant mediated higher transgene expression than the neuronal-specific synapsin 1 and tyrosine hydroxylase promoters. The ALDH1L1 promoter was also transcriptionally active in dentate granule neurons following intrahippocampal adeno-associated viral vector infusion, whereas transgene expression was detected in both striatal neurons and astrocytes following vector infusion into the striatum. Our results demonstrate the potential suitability of the ALDH1L1 promoter as a new tool in the development of gene therapy and disease modelling applications.  

5.1967           Tailored transgene expression to specific cell types in the central nervous system after peripheral injection with AAV9

Dashkoff, J., Lerner, E.P., truong, N., Klickstein, J.A., Fan, Z., Mu, D., Maguire, D.M., Hyman, B. and Hudry, E. Mol. Ther.-methods &Clin. Development, 3, 16081 (2016)   The capacity of certain adeno-associated virus (AAV) vectors to cross the blood–brain barrier after intravenous delivery offers a unique opportunity for noninvasive brain delivery. However, without a well-tailored system, the use of a peripheral route injection may lead to undesirable transgene expression in nontarget cells or organs. To refine this approach, the present study characterizes the transduction profiles of new self-complementary AAV9 (scAAV9) expressing the green fluorescent protein (GFP) either under an astrocyte (glial fibrillary acidic (GFA) protein) or neuronal (Synapsin (Syn)) promoter, after intravenous injection of adult mice (2 × 1013 vg/kg). ScAAV9-GFA-GFP and scAAV9-Syn-GFP robustly transduce astrocytes (11%) and neurons (17%), respectively, without aberrant expression leakage. Interestingly, while the percentages of GFP-positive astrocytes with scAAV9-GFA-GFP are similar to the performances observed with scAAV9-CBA-GFP (broadly active promoter), significant higher percentages of neurons express GFP with scAAV9-Syn-GFP. GFP-positive excitatory as well as inhibitory neurons are observed, as well as motor neurons in the spinal cord. Additionally, both activated (GFAP-positive) and resting astrocytes (GFAP-negative) express the reporter gene after scAAV9-GFA-GFP injection. These data thoroughly characterize the gene expression specificity of AAVs fitted with neuronal and astrocyte-selective promoters after intravenous delivery, which will prove useful for central nervous system (CNS) gene therapy approaches in which peripheral expression of transgene is a concern.  

5.1968           Impact of age and vector construct on striatal and nigral transgene expression

Polinski, N.K., Manfredsson, F.P., Benskey, M.J., Fischer, D.L., Kemp, C.J., Steece-Collier, K., Sandoval, I.M., Paumier, K.L. and Sortwell, C.E. Mol. Ther.-Methods &Clin. Development, 3, 16082 (2016)   Therapeutic protein delivery using viral vectors has shown promise in preclinical models of Parkinson’s disease (PD) but clinical trial success remains elusive. This may partially be due to a failure to include advanced age as a covariate despite aging being the primary risk factor for PD. We investigated transgene expression following intracerebral injections of recombinant adeno-associated virus pseudotypes 2/2 (rAAV2/2), 2/5 (rAAV2/5), 2/9 (rAAV2/9), and lentivirus (LV) expressing green fluorescent protein (GFP) in aged versus young adult rats. Both rAAV2/2 and rAAV2/5 yielded lower GFP expression following injection to either the aged substantia nigra or striatum. rAAV2/9-mediated GFP expression was deficient in the aged striatonigral system but displayed identical transgene expression between ages in the nigrostriatal system. Young and aged rats displayed equivalent GFP levels following LV injection to the striatonigral system but LV-delivered GFP was deficient in delivering GFP to the aged nigrostriatal system. Notably, age-related transgene expression deficiencies revealed by protein quantitation were poorly predicted by GFP-immunoreactive cell counts. Further, in situ hybridization for the viral CβA promoter revealed surprisingly limited tropism for astrocytes compared to neurons. Our results demonstrate that aging is a critical covariate to consider when designing gene therapy approaches for PD.  

5.1969           Dynamics of antigen presentation to transgene product-specific CD4+ T cells and of Treg induction upon hepatic AAV gene transfer

Perrin, G.Q., Zolotukhin, I., Sherman, A., Biswas, M., de Jong, Y.P., terhorst, C., Davidoff, A.M. and Herzog, R.W. Mol. Ther.- Methods & Clin. Development, 3, 16083 (2016)   The tolerogenic hepatic microenvironment impedes clearance of viral infections but is an advantage in viral vector gene transfer, which often results in immune tolerance induction to transgene products. Although the underlying tolerance mechanism has been extensively studied, our understanding of antigen presentation to transgene product-specific CD4+ T cells remains limited. To address this, we administered hepatotropic adeno-associated virus (AAV8) vector expressing cytoplasmic ovalbumin (OVA) into wt mice followed by adoptive transfer of transgenic OVA-specific T cells. We find that that the liver-draining lymph nodes (celiac and portal) are the major sites of MHC II presentation of the virally encoded antigen, as judged by in vivo proliferation of DO11.10 CD4+ T cells (requiring professional antigen-presenting cells, e.g., macrophages) and CD4+CD25+FoxP3+ Treg induction. Antigen presentation in the liver itself contributes to activation of CD4+ T cells egressing from the liver. Hepatic-induced Treg rapidly disseminate through the systemic circulation. By contrast, a secreted OVA transgene product is presented in multiple organs, and OVA-specific Treg emerge in both the thymus and periphery. In summary, liver draining lymph nodes play an integral role in hepatic antigen presentation and peripheral Treg induction, which results in systemic regulation of the response to viral gene products.  

5.1970           Cell-Type-Specific Optical Recording of Membrane Voltage Dynamics in Freely Moving Mice

Marshall, J.D., Li, Z., Zhang, Y., Gong, Y., St-Pierre, F., Lin, M.Z. and Schnitzer, M.J: Cell, 167, 1650-1662 (2016)   Electrophysiological field potential dynamics are of fundamental interest in basic and clinical neuroscience, but how specific cell types shape these dynamics in the live brain is poorly understood. To empower mechanistic studies, we created an optical technique, TEMPO, that records the aggregate trans-membrane voltage dynamics of genetically specified neurons in freely behaving mice. TEMPO has >10-fold greater sensitivity than prior fiber-optic techniques and attains the noise minimum set by quantum mechanical photon shot noise. After validating TEMPO’s capacity to track established oscillations in the delta, theta, and gamma frequency bands, we compared the D1- and D2-dopamine-receptor-expressing striatal medium spiny neurons (MSNs), which are interspersed and electrically indistinguishable. Unexpectedly, MSN population dynamics exhibited two distinct coherent states that were commonly indiscernible in electrical recordings and involved synchronized hyperpolarizations across both MSN subtypes. Overall, TEMPO allows the deconstruction of normal and pathologic neurophysiological states into trans-membrane voltage activity patterns of specific cell types.    

5.1971           Anti-hIgE gene therapy of peanut-induced anaphylaxis in a humanized murine model of peanut allergy

Pagovich, O.E., Wang, B., Chiuchiolo, M.J., Kaminsky, S.M., Sondhi, D., Jose, C.L., Price, C.C., Brooks, S.F., Mesey, J.G. and Crystal, R.G.

1. Allergy Clin. Immunol., 138(6), 1652-1662e7 (2016)

  Background Peanuts are the most common food to provoke fatal or near-fatal anaphylactic reactions. Treatment with an anti-hIgE mAb is efficacious but requires frequent parenteral administration. Objective Based on the knowledge that peanut allergy is mediated by peanut-specific IgE, we hypothesized that a single administration of an adeno-associated virus (AAV) gene transfer vector encoding for anti-hIgE would protect against repeated peanut exposure in the host with peanut allergy. Methods We developed a novel humanized murine model of peanut allergy that recapitulates the human anaphylactic response to peanuts in NOD-scid IL2Rgamma^null mice transferred with blood mononuclear cells from donors with peanut allergy and then sensitized with peanut extract. As therapy, we constructed an adeno-associated rh.10 serotype vector coding for a full-length, high-affinity, anti-hIgE antibody derived from the Fab fragment of the anti-hIgE mAb omalizumab (AAVrh.10anti-hIgE). In the reconstituted mice peanut-specific IgE was induced by peanut sensitization and hypersensitivity, and reactions were provoked by feeding peanuts to mice with symptoms similar to those of human subjects with peanut allergy. Results A single administration of AAVrh.10anti-hIgE vector expressed persistent levels of anti-hIgE. The anti-hIgE vector, administered either before sensitization or after peanut sensitization and manifestation of the peanut-induced phenotype, blocked IgE-mediated alterations in peanut-induced histamine release, anaphylaxis scores, locomotor activity, and free IgE levels and protected animals from death caused by anaphylaxis. Conclusion If this degree of persistent efficacy translates to human subjects, AAVrh.10anti-hIgE could be an effective 1-time preventative therapy for peanut allergy and possibly other severe, IgE-mediated allergies.  

5.1972           Intratumoral Immunization by p19Arf and Interferon-β Gene Transfer in a Heterotopic Mouse Model of Lung Carcinoma

Catani, J.P.P., Medrano, R.F.V., Hunger, A., Del Valle, P., Adjemian, S., Zanatta, d.b., Kroener, G., Costanzi-Strauss, E. and Strauss, B.E. Translational Oncol., 9(6), 565-574 (2016)   Therapeutic strategies that act by eliciting and enhancing antitumor immunity have been clinically validated as an effective treatment modality but may benefit from the induction of both cell death and immune activation as primary stimuli. Using our AdRGD-PG adenovector platform, we show here for the first time that in situ gene transfer of p19Arf and interferon-β (IFNβ) in the LLC1 mouse model of lung carcinoma acts as an immunotherapy. Although p19Arf is sufficient to induce cell death, only its pairing with IFNβ significantly induced markers of immunogenic cell death. In situ gene therapy with IFNβ, either alone or in combination with p19Arf, could retard tumor progression, but only the combined treatment was associated with a protective immune response. Specifically in the case of combined intratumoral gene transfer, we identified 167 differentially expressed genes when using microarray to evaluate tumors that were treated in vivo and confirmed the activation of CCL3, CXCL3, IL1α, IL1β, CD274, and OSM, involved in immune response and chemotaxis. Histologic evaluation revealed significant tumor infiltration by neutrophils, whereas functional depletion of granulocytes ablated the antitumor effect of our approach. The association of in situ gene therapy with cisplatin resulted in synergistic elimination of tumor progression. In all, in situ gene transfer with p19Arf and IFNβ acts as an immunotherapy involving recruitment of neutrophils, a desirable but previously untested outcome, and this approach may be allied with chemotherapy, thus providing significant antitumor activity and warranting further development for the treatment of lung carcinoma.  

5.1973           Tollip, an early regulator of the acute inflammatory response in the substantia nigra

Humbert-Claude, M., Duc, D., Dwir, D., Thieren, L., Sandström von Tobel, J., Begka, C., Legueux, F., Velin, D., Maillard, M.H., Do, K.Q., Monnet-Tschudi, F. and Trenenbaum, L.

1. Neuroinflammation, 13:303 (2016)

  Background Tollip is a ubiquitously expressed protein, originally described as a modulator of the IL-1R/TLR-NF-κB signaling pathways. Although this property has been well characterized in peripheral cells, and despite some evidence of its expression in the central nervous system, the role of Tollip in neuroinflammation remains poorly understood. The present study sought to explore the implication of Tollip in inflammation in the substantia nigra pars compacta, the structure affected in Parkinson’s disease. Methods We first investigated Tollip distribution in the midbrain by immunohistochemistry. Then, we addressed TLR4-mediated response by intra-nigral injections of lipopolysaccharide (LPS), a TLR4 agonist, on inflammatory markers in Tollip knockout (KO) and wild-type (WT) mice. Results We report an unexpectedly high Tollip immunostaining in dopaminergic neurons of the mice brain. Second, intra-nigral injection of LPS led to increased susceptibility to neuroinflammation in Tollip KO compared to Tollip WT mice. This was demonstrated by a significant increase of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and interferon gamma (IFN-γ) messenger RNA (mRNA) in the midbrain of Tollip KO mice upon LPS injection. Consistently, brain rAAV viral vector transduction with a nuclear factor kappa B (NF-κB)-inducible reporter gene confirmed increased NF-κB activation in Tollip KO mice. Lastly, Tollip KO mice displayed higher inducible NO synthase (iNOS) production, both at the messenger and protein level when compared to LPS-injected WT mice. Tollip deletion also aggravated LPS-induced oxidative and nitrosative damages, as indicated by an increase of 8-oxo-2′-deoxyguanosine and nitrotyrosine immunostaining, respectively. Conclusions Altogether, these findings highlight a critical role of Tollip in the early phase of TLR4-mediated neuroinflammation. As brain inflammation is known to contribute to Parkinson’s disease, Tollip may be a potential target for neuroprotection.  

5.1974           Viral Vector-Based Targeting of miR-21 in Cardiac Nonmyocyte Cells Reduces Pathologic Remodeling of the Heart

Ramanujam, D., Sassi, Y., Laggerbauer, B. and Wngelhardt, S. Molecular Therapy, 24(11), 1939-1948 (2016)   Systemic inhibition of miR-21 has proven effective against myocardial fibrosis and dysfunction, while studies in cardiac myocytes suggested a protective role in this cell type. Considering potential implications for therapy, we aimed to determine the cell fraction where miR-21 exerts its pathological activity. We developed a viral vector-based strategy for gene targeting of nonmyocyte cardiac cells in vivo and compared global to cardiac myocyte-specific and nonmyocyte-specific deletion of miR-21 in chronic left ventricular pressure overload. Murine moloney virus and serotype 9 of adeno-associated virus were engineered to encode improved Cre recombinase for genetic deletion in miR-21^fl/fl mice. Pericardial injection of murine moloney virus-improved Cre recombinase to neonates achieved highly selective genetic ablation of miR-21 in nonmyocyte cardiac cells, identified as cardiac fibroblasts and endothelial cells. Upon left ventricular pressure overload, cardiac function was only preserved in mice with miR-21 deficiency in nonmyocyte cardiac cells, but not in mice with global or cardiac myocyte-specific ablation. Our data demonstrate that miR-21 exerts its pathologic activity directly in cardiac nonmyocytes and encourage further development of antimiR-21 therapy toward cellular tropism.  

5.1975           CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells

Dever, D.P. et al Nature, 539(7629), 384-389 (2016)   The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.  

5.1976           MeCP2 and histone deacetylases 1 and 2 in dorsal striatum collectively suppress repetitive behaviors

Mahgoub, M., Adachi, M., Suzuki, K., Liu, X., Kavali, E.T., Chahrour, M.H. and Monteggia, L.M. Nature Neuroscience, 19(11), 1506-1512 (2016)   Class I histone deacetylases (HDACs) Hdac1 and Hdac2 can associate together in protein complexes with transcriptional factors such as methyl-CpG-binding protein 2 (MeCP2). Given their high degree of sequence identity, we examined whether Hdac1 and Hdac2 were functionally redundant in mature mouse brain. We demonstrate that postnatal forebrain-specific deletion of both Hdac1 and Hdac2 in mice impacts neuronal survival and results in an excessive grooming phenotype caused by dysregulation of Sap90/Psd95-associated protein 3 (Sapap3; also known as Dlgap3) in striatum. Moreover, Hdac1- and Hdac2-dependent regulation of Sapap3 expression requires MECP2, the gene involved in the pathophysiology of Rett syndrome. We show that postnatal forebrain-specific deletion of Mecp2 causes excessive grooming, which is rescued by restoring striatal Sapap3 expression. Our results provide new insight into the upstream regulation of Sapap3 and establish the essential role of striatal Hdac1, Hdac2 and MeCP2 for suppression of repetitive behaviors.  

5.1977           Organization of long-range inputs and outputs of frontal cortex for top-down control

Zhang, S., Xu, M., Chang, W-C., Ma, C., Do, J.P.H., Jeong, D., Lei, T., Fan, J.L. and Dan, Y. Nature Neuroscience, 19(12), 1733-1742 (2016)   Long-range projections from the frontal cortex are known to modulate sensory processing in multiple modalities. Although the mouse has become an increasingly important animal model for studying the circuit basis of behavior, the functional organization of its frontal cortical long-range connectivity remains poorly characterized. Here we used virus-assisted circuit mapping to identify the brain networks for top-down modulation of visual, somatosensory and auditory processing. The visual cortex is reciprocally connected to the anterior cingulate area, whereas the somatosensory and auditory cortices are connected to the primary and secondary motor cortices. Anterograde and retrograde tracing identified the cortical and subcortical structures belonging to each network. Furthermore, using new viral techniques to target subpopulations of frontal neurons projecting to the visual cortex versus the superior colliculus, we identified two distinct subnetworks within the visual network. These findings provide an anatomical foundation for understanding the brain mechanisms underlying top-down control of behavior.  

5.1978           A viral strategy for targeting and manipulating interneurons across vertebrate species

Dimidschstein, J. et al Nature Neuroscience, 19(12), 1743-1749 (2016)   A fundamental impediment to understanding the brain is the availability of inexpensive and robust methods for targeting and manipulating specific neuronal populations. The need to overcome this barrier is pressing because there are considerable anatomical, physiological, cognitive and behavioral differences between mice and higher mammalian species in which it is difficult to specifically target and manipulate genetically defined functional cell types. In particular, it is unclear the degree to which insights from mouse models can shed light on the neural mechanisms that mediate cognitive functions in higher species, including humans. Here we describe a novel recombinant adeno-associated virus that restricts gene expression to GABAergic interneurons within the telencephalon. We demonstrate that the viral expression is specific and robust, allowing for morphological visualization, activity monitoring and functional manipulation of interneurons in both mice and non-genetically tractable species, thus opening the possibility to study GABAergic function in virtually any vertebrate species.      

5.1979           Single residue AAV capsid mutation improves transduction of photoreceptors in the Abca4−/− mouse and bipolar cells in the rd1 mouse and human retina ex vivo

De Silva, S.R., Issa, P.C., Singh, M.S., Lipinski, D.M., Barnea-Cramer, A.O., Walker, N.J., barnard, A.R., Hankins, M.W. and MacLaren, R.E. Gene Therapy, 23, 767-774 (2016)   Gene therapy using adeno-associated viral (AAV) vectors for the treatment of retinal degenerations has shown safety and efficacy in clinical trials. However, very high levels of vector expression may be necessary for the treatment of conditions such as Stargardt disease where a dual vector approach is potentially needed, or in optogenetic strategies for end-stage degeneration in order to achieve maximal light sensitivity. In this study, we assessed two vectors with single capsid mutations, rAAV2/2(Y444F) and rAAV2/8(Y733F) in their ability to transduce retina in the Abca4^−/− and rd1 mouse models of retinal degeneration. We noted significantly increased photoreceptor transduction using rAAV2/8(Y733F) in the Abca4^−/− mouse, in contrast to previous work where vectors tested in this model have shown low levels of photoreceptor transduction. Bipolar cell transduction was achieved following subretinal delivery of both vectors in the rd1 mouse, and via intravitreal delivery of rAAV2/2(Y444F). The successful use of rAAV2/8(Y733F) to target bipolar cells was further validated on human tissue using an ex vivo culture system of retinal explants. Capsid mutant AAV vectors transduce human retinal cells and may be particularly suited to treat retinal degenerations in which high levels of transgene expression are required.    

5.1980           Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism

Powell, S.K., Khan, N., parker, C.L., Samulski, R.J., Matsushima, G., Gray, S.J. and McCown, T.J. Gene Therapy, 23, 807-814 (2016)   No adeno-associated virus (AAV) capsid has been described in the literature to exhibit a primary oligodendrocyte tropism when a constitutive promoter drives gene expression, which is a significant barrier for efficient in vivo oligodendrocyte gene transfer. The vast majority of AAV vectors, such as AAV1, 2, 5, 6, 8 or 9, exhibit a dominant neuronal tropism in the central nervous system. However, a novel AAV capsid (Olig001) generated using capsid shuffling and directed evolution was recovered after rat intravenous delivery and subsequent capsid clone rescue, which exhibited a >95% tropism for striatal oligodendrocytes after rat intracranial infusion where a constitutive promoter drove gene expression. Olig001 contains a chimeric mixture of AAV1, 2, 6, 8 and 9, but unlike these parental serotypes after intravenous administration Olig001 has very low affinity for peripheral organs, especially the liver. Furthermore, in mixed glial cell cultures, Olig001 exhibits a 9-fold greater binding when compared with AAV8. This novel oligodendrocyte-preferring AAV vector exhibits characteristics that are a marked departure from previously described AAV serotypes.  

5.1981           Somatic Therapy of a Mouse SMA Model with a U7 snRNA Gene Correcting SMN2 Splicing

Odermatt, P., Trüb, J., Furrer, L., Fricker, R., Marti, A. and Schümperli, D. Molecular Therapy, 24(10), 1797-1805 (2016)   Spinal Muscular Atrophy is due to the loss of SMN1 gene function. The duplicate gene SMN2 produces some, but not enough, SMN protein because most transcripts lack exon 7. Thus, promoting the inclusion of this exon is a therapeutic option. We show that a somatic gene therapy using the gene for a modified U7 RNA which stimulates this splicing has a profound and persistent therapeutic effect on the phenotype of a severe Spinal Muscular Atrophy mouse model. To this end, the U7 gene and vector and the production of pure, highly concentrated self-complementary (sc) adenovirus-associated virus 9 vector particles were optimized. Introduction of the functional vector into motoneurons of newborn Spinal Muscular Atrophy mice by intracerebroventricular injection led to a highly significant, dose-dependent increase in life span and improvement of muscle functions. Besides the central nervous system, the therapeutic U7 RNA was expressed in the heart and liver which may additionally have contributed to the observed therapeutic efficacy. This approach provides an additional therapeutic option for Spinal Muscular Atrophy and could also be adapted to treat other diseases of the central nervous system with regulatory small RNA genes.  

5.1982           Insulin Therapy Improves Adeno-Associated Virus Transduction of Liver and Skeletal Muscle in Mice and Cultured Cells No Access

Carrig, S., Bujjiga, E., Wopat, M.J: and martino, A.T. Human Gene Therapy, 27(11), 892-905 (2016)   Adeno-associated virus (AAV) gene transfer is a promising treatment for genetic abnormalities. Optimal AAV vectors are showing success in clinical trials. Gene transfer to skeletal muscle and liver is being explored as a potential therapy for some conditions, that is, α₁-antitrypsin (AAT) disorder and hemophilia B. Exploring approaches that enhance transduction of liver and skeletal muscle, using these vectors, is beneficial for gene therapy. Regulating hormones as an approach to improve AAV transduction is largely unexplored. In this study we tested whether insulin therapy improves liver and skeletal muscle gene transfer. In vitro studies demonstrated that the temporary coadministration (2, 8, and 24 hr) of insulin significantly improves AAV2-CMV-LacZ transduction of cultured liver cells and differentiated myofibers, but not of lung cells. In addition, there was a dose response related to this improved transduction. Interestingly, when insulin was not coadministered with the virus but given 24 hr afterward, there was no increase in the transgene product. Insulin receptor gene (INSR) expression levels were increased 5- to 13-fold in cultured liver cells and differentiated myofibers when compared with lung cells. Similar INSR gene expression profiles occurred in mouse tissues. Insulin therapy was performed in mice, using a subcutaneously implanted insulin pellet or a high-carbohydrate diet. Insulin treatment began just before intramuscular delivery of AAV1-CMV-schFIX or liver-directed delivery of AAV8-CMV-schFIX and continued for 28 days. Both insulin augmentation therapies improved skeletal muscle- and liver-directed gene transduction in mice as seen by a 3.0- to 4.5-fold increase in human factor IX (hFIX) levels. The improvement was observed even after the insulin therapy ended. Monitoring insulin showed that insulin levels increased during the brief period of rAAV delivery and during the entire insulin augmentation period (28 days). This study demonstrates that AAV transduction of liver or skeletal muscle can be improved by insulin therapy  

5.1983           Delivery of an Adeno-Associated Virus Vector into Cerebrospinal Fluid Attenuates Central Nervous System Disease in Mucopolysaccharidosis Type II Mice No Access

Hinderer, C., katz, N., Louboutin, J-P-. Bell, P., Yu, H., Nayal, M., Kozarsky, K., O’Brien, W.T., Goode, T. and Wilson, J.M. Human Gene Therapy, 27(11), 906-915 (2016)   Mucopolysaccharidosis type II (MPS II) is a rare X-linked genetic disorder caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS), leading to impaired catabolism of ubiquitous polysaccharides and abnormal accumulation of these undegraded substrates in the lysosome. Like many lysosomal storage diseases, MPS II is characterized by both somatic and central nervous system (CNS) involvement. Intravenous enzyme replacement therapy can improve somatic manifestations of MPS II, but systemic IDS does not cross the blood–brain barrier and therefore cannot address CNS disease. In this study, an adeno-associated virus serotype 9 vector carrying the IDS gene was injected into the cerebrospinal fluid (CSF) of IDS deficient mice, a model of MPS II. Treated mice exhibited dose-dependent IDS expression and resolution of brain storage lesions, as well as improvement in long-term memory in a novel object recognition test. These findings suggest that delivery of adeno-associated virus vectors into CSF could serve as a platform for efficient, long-term enzyme delivery to the CNS, potentially addressing this critical unmet need for patients with MPS II and many related lysosomal enzyme deficiencies.  

5.1984           The Brain-Enriched MicroRNA miR-9-3p Regulates Synaptic Plasticity and Memory

Sim, S-E. et al

1. Neuroscience., 36(33), 8641-8652 (2016)

  MicroRNAs (miRNAs) are small, noncoding RNAs that posttranscriptionally regulate gene expression in many tissues. Although a number of brain-enriched miRNAs have been identified, only a few specific miRNAs have been revealed as critical regulators of synaptic plasticity, learning, and memory. miR-9-5p/3p are brain-enriched miRNAs known to regulate development and their changes have been implicated in several neurological disorders, yet their role in mature neurons in mice is largely unknown. Here, we report that inhibition of miR-9-3p, but not miR-9-5p, impaired hippocampal long-term potentiation (LTP) without affecting basal synaptic transmission. Moreover, inhibition of miR-9-3p in the hippocampus resulted in learning and memory deficits. Furthermore, miR-9-3p inhibition increased the expression of the LTP-related genes Dmd and SAP97, the expression levels of which are negatively correlated with LTP. These results suggest that miR-9-3p-mediated gene regulation plays important roles in synaptic plasticity and hippocampus-dependent memory.  

5.1985           FOXO3a regulates BNIP3 and modulates mitochondrial calcium, dynamics, and function in cardiac stress

Chaanine, A.H., Kohlbrenner, E., Gamb, S.I., Guenzel, A.J., Klaus, K., Fayyaz, A.U., Nair, K.S., Hajjar, R.J., and Redfield, M.M. Am. J. Physiol. Heart Circ. Physiol., 311, H1540-H1559 (2016)   The forkhead box O3a (FOXO3a) transcription factor has been shown to regulate glucose metabolism, muscle atrophy, and cell death in postmitotic cells. Its role in regulation of mitochondrial and myocardial function is not well studied. Based on previous work, we hypothesized that FOXO3a, through BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3), modulates mitochondrial morphology and function in heart failure (HF). We modulated the FOXO3a-BNIP3 pathway in normal and phenylephrine (PE)-stressed adult cardiomyocytes (ACM) in vitro and developed a cardiotropic adeno-associated virus serotype 9 encoding dominant-negative FOXO3a (AAV9.dn-FX3a) for gene delivery in a rat model of HF with preserved ejection fraction (HFpEF). We found that FOXO3a upregulates BNIP3 expression in normal and PE-stressed ACM, with subsequent increases in mitochondrial Ca²⁺, leading to decreased mitochondrial membrane potential, mitochondrial fragmentation, and apoptosis. Whereas dn-FX3a attenuated the increase in BNIP3 expression and its consequences in PE-stressed ACM, AAV9.dn-FX3a delivery in an experimental model of HFpEF decreased BNIP3 expression, reversed adverse left ventricular remodeling, and improved left ventricular systolic and, particularly, diastolic function, with improvements in mitochondrial structure and function. Moreover, AAV9.dn-FX3a restored phospholamban phosphorylation at S16 and enhanced dynamin-related protein 1 phosphorylation at S637. Furthermore, FOXO3a upregulates maladaptive genes involved in mitochondrial apoptosis, autophagy, and cardiac atrophy. We conclude that FOXO3a activation in cardiac stress is maladaptive, in that it modulates Ca²⁺ cycling, Ca²⁺ homeostasis, and mitochondrial dynamics and function. Our results suggest an important role of FOXO3a in HF, making it an attractive potential therapeutic target.  

5.1986           Insight into the mechanisms of enhanced retinal transduction by the engineered AAV2 capsid variant -7m8

Khabou, H., Desrosiers, M., Wineckler, C., Fouquet, S., Auregan, G., Bemelmans, A-P., Sahel, J-A. and Dalkara, D. Biotechnology and Bioengineering, 113(12), 2712-2724 (2016)   Recently, we described a modified AAV2 vector—AAV2-7m8—having a capsid-displayed peptide insertion of 10 amino acids with enhanced retinal transduction properties. The insertion of the peptide referred to as 7m8 is responsible for high-level gene delivery into deep layers of the retina when virus is delivered into the eye's vitreous. Here, we further characterize AAV2-7m8 mediated gene delivery to neural tissue and investigate the mechanisms by which the inserted peptide provides better transduction away from the injection site. First, in order to understand if the peptide exerts its effect on its own or in conjunction with the neighboring amino acids, we inserted the 7m8 peptide at equivalent positions on three other AAV capsids, AAV5, AAV8, and AAV9, and evaluated its effect on their infectivity. Intravitreal delivery of these peptide insertion vectors revealed that only AAV9 benefited from 7m8 insertion in the context of the retina. We then investigated AAV2-7m8 and AAV9-7m8 properties in the brain, to better evaluate the spread and efficacy of viral transduction in view of the peptide insertion. While 7m8 insertion led to higher intensity gene expression, the spread of gene expression remained unchanged compared to the parental serotypes. Our results indicate that the 7m8 peptide insertion acts by increasing efficacy of cellular entry, with little effect on the spread of viral particles in neural tissue. The effects of peptide insertion are capsid and tissue dependent, highlighting the importance of the microenvironment in gene delivery using AAV.  

5.1987           Impact of reducing and oxidizing agents on the infectivity of Qβ phage and the overall structure of its capsid

Loison, P., Majou, D., Gelhaye, E., Boudaud, N. and Gantzer, C. FEMS Microbiol. Ecol., 92(11), fiw153 (2016)   Qβ phages infect Escherichia coli in the human gut by recognizing F-pili as receptors. Infection therefore occurs under reducing conditions induced by physiological agents (e.g. glutathione) or the intestinal bacterial flora. After excretion in the environment, phage particles are exposed to oxidizing conditions and sometimes disinfection. If inactivation does not occur, the phage may infect new hosts in the human gut through the oral route. During such a life cycle, we demonstrated that, outside the human gut, cysteines of the major protein capsid of Qβ phage form disulfide bonds. Disinfection with NaClO does not allow overoxidation to occur. Such oxidation induces inactivation rather by irreversible damage to the minor proteins. In the presence of glutathione, most disulfide bonds are reduced, which slightly increases the capacity of the phage to infect E. coli in vitro. Such reduction is reversible and barely alters infectivity of the phage. Reduction of all disulfide bonds by dithiothreitol leads to complete capsid destabilization. These data provide new insights into how the phages are impacted by oxidizing-reducing conditions outside their host cell and raises the possibility of the intervention of the redox during life cycle of the phage.  

5.1988           Gene therapy for Ebola virus infections based on AAV vectors and Zmapp antibody cocktail

Robert, M.A., Nassouri, N., Chahala, P.S., Venne, M.H., Kamen, A., Kobinger, G. and Gaillet, R.G. Human Gene Therapy, 27, A42, abstract OR54 (2016)   The Zmapp cocktail contains chimeric neutralizing antibodies (c13C6, c2G4 and c4G7) against Ebola virus. It is among the most promising experimental approaches for treating Ebola infections. However, because of high doses required of purified antibodies, its use on large populations poses a manufacturing challenge and is of economic concern in developing countries. To address these potential issues, recombinant vectors derived from adeno-associated virus (rAAVs) are very attractive. They 1) can be produced in large quantity, 2) permit long-term expression thus, reducing the number of treatments, 3) are highly stable to storage conditions and 4) are efficiently administered intranasally. Our main goals are to develop a treatment based on rAAVs to deliver genes coding for Zmapp antibodies and to scale-up the manufacturing at reasonable costs. In this study, three rAAVs (serotypes 9 and DJ) expressing one of the three antibodies were produced in shake flasks (200 mL) and in WAVE bioreactors (10 L). rAAVs were produced by transfection using our patented cGMP compatible HEK293 cell line in suspension culture without serum. Light and heavy chains were expressed under the same expression cassette by using a 2a peptide and furin cleavage sequences. rAAVs productions were either directly purified by an iodixanol step-gradient or concentrated first by tangential flow filtration. Titers were obtained by qPCR. Antibodies produced in transduced HEK293 and CHO cells were characterized by western blot, LC-MS/MS and a functional assay. The efficacy of rAAV-c2G4 for preventing Ebola infections is currently under evaluation in a mouse model challenged with the virus.  

5.1989           Pre-clinical optimization of AAV gene therapy in a feline model of GM1 gangliosidosis

Gray-Edwards, H.L., Gross, A.L., Randle, A.N., Taylor, A., Brunson, B.L., Murdock, B., Stoica, L., Todessa, S., Lata, J., Sena-Esteves, M. and martin, D.R. Human Gene Therapy, 27, A50, abstract P016 (2016)   A deficiency of lysosomal b-galactosidase (bgal) causes the rapidly progressive and fatal neurologic disease, GM1 gangliosidosis, for which no treatment exists. Adeno-associated viral (AAV) gene therapy is effective in GM1 cats, with a greater than 6 fold increase in lifespan after bilateral injection of the thalamus and deep cerebellar nuclei. Delivery routes and AAV purification methods were tested to maximize safety and efficacy for upcoming clinical trials. To avoid injection of the cerebellum and improve cortical distribution, 3 cerebrospinal fluid (CSF)-based routes were tested: cisterna magna (CM), bilateral intracerebroventricular (ICV), or lumbar cistern (LC). GM1 cats were treated with 1e12 vector genomes/kg body weight with AAVrh10 expressing a feline bgal cDNA. After LC injection,  enzyme was limited to the spinal cord, where it reached a maximum of 0.7-fold normal in the lumbar region. CM or ICV delivery restored bgal activity to 0.5–1.4 fold normal in the spinal cord and up to 0.7-fold normal in the cerebellum and caudal cerebrum, with no statistical difference between CM and ICV routes. A long-standing question regarding the influence of AAV production methods was tested by injecting the thalamus and lateral ventricle of GM1 cats with vector purified by cesium chloride or iodixanol centrifugation. All cats showed salutary effects of treatment, and clinical disease progression between groups will be presented. Based on the results of this study, injection of the thalamus and CSF (CM or ICV) should prove beneficial in clinical trials. Combining CM and ICV injection could further enhance therapeutic effect.  

5.1990           Large scale purification of Adeno-associated virus (AAV) with continuous flow ultracentrifugation

Chen, H., Merino, S. and Ho, C.Y. Human Gene Therapy, 27, A160, abstract P375 (2016)   Adeno-associated virus (AAV) vectors have gained more and more attention in the field of gene therapy research. So far AAV vectors have usually been purified through either density gradient ultracentrifugation in small volumes centrifuge tubes or column chromatography. Though these purification methods have their unique benefits, there is still a need for technology that can process large volume of lysate with high AAV recovery rate. We reasoned that continuous flow ultracentrifugation could meet these requirements. We tested the AlfaWassermann’s AWPromatix 1000TM, a research scale continuous flow ultracentrifuge, as a proof of concept for AAV vector purification. In the initial experiments, we tested cesium chloride (CsCl) solution as density gradient media for AAV vector purification but found out that CsCl solution was not stable enough to form a linear gradient even when sucrose was added to increase its viscosity for AAV purification.We then tested iodixanol solution as density gradient media and got satisfactory purification of AAV vectors. Our results indicate that we can obtain nearpurified AAV vectors in a single-step of centrifugation with AAV recovery rate exceeding 50%. Further experiments indicate that minor impurities associated with the purified AAV vectors could be removed by adding salts to the iodixanol solution such as CsCl to increase the ionic strength of the density gradient. The data presented here indicate that continuous flow centrifugation can be used for large scale purification of AAV vectors and it should provide an additional tool to facilitate the translation from research to the clinic.  

5.1991           High Efficiency CRISPR/Cas9-mediated Gene Editing in Primary Human T-cells Using Mutant Adenoviral E4orf6/E1b55k “Helper” Proteins

Gwiazda, K.S., Grier, A.E., Sahni, J., Burleigh, S.M., martin, U., yang, J.G., Popp, N.A., Krutein, M.C., Khan, I.F., Jacoby, K., Jensen, N.C., Rawlings, D.J. and Scharenberg, A.M. Molecular Therapy, 24(9), 1570-1580 (2016)   Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components—Cas9, guide RNAs and recombination templates—to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.  

5.1992           Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector

Grieger, J.C., Soltys, S.M. and Samulski, R.J. Molecular Therapy, 24(2), 287-297 (2016)   Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1 × 10⁵ vector genome containing particles (vg)/cell or greater than 1 × 10¹⁴ vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1–6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (>90% full particles), provides postpurification yields (>1 × 10¹³ vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered into patients. In addition, we report a minimum of a fivefold increase in overall vector production by implementing a perfusion method that entails harvesting rAAV from the culture media at numerous time-points post-transfection.  

5.1993           Silent IL2RG Gene Editing in Human Pluripotent Stem Cells

Li, L.B., Ma, C., Awong, G., Kennedy, M., Gornalusse, G., keller, G., Kaufman, D.S. and Russell, D.W. Molecular Therapy, 24(3), 582-591 (2016)   Many applications of pluripotent stem cells (PSCs) require efficient editing of silent chromosomal genes. Here, we show that a major limitation in isolating edited clones is silencing of the selectable marker cassette after homologous recombination and that this can be overcome by using a ubiquitous chromatin opening element (UCOE) promoter-driven transgene. We use this strategy to edit the silent IL2RG locus in human PSCs with a recombinant adeno-associated virus (rAAV)-targeting vector in the absence of potentially genotoxic, site-specific nucleases and show that IL2RG is required for natural killer and T-cell differentiation of human PSCs. Insertion of an active UCOE promoter into a silent locus altered the histone modification and cytosine methylation pattern of surrounding chromatin, but these changes resolved when the UCOE promoter was removed. This same approach could be used to correct IL2RG mutations in X-linked severe combined immunodeficiency patient-derived induced PSCs (iPSCs), to prevent graft versus host disease in regenerative medicine applications, or to edit other silent genes.  

5.1994           34. Development and Validation of Identity and Homogeneity Assays for AAV Preparations

Pacouret, S. et al Moleculara Therapy, 24, Supplement 1, S15 (2016)   Adeno-associated virus (AAV) vectors have emerged as key clinical candidates for gene therapy. Yet, the efficiency and safety of these 20-25 nm biological nanoparticles remain difficult to harmonize across pre-clinical studies due to the limitations of current analytical tools. The presence of residual DNA, protein contaminants, empty particles and VP subunits resulting from incomplete capsid assembly are variables that can strongly modulate the reliability of in vivo data and that, therefore, need to be closely monitored in AAV research laboratories. In this work, 70 AAV preparations, obtained with various production (baculo/Sf9 and triple transfection system) and purification (iodixanol gradient and double cesium-chloride gradient) techniques were analyzed using a thermal shift assay based on the fluorescent dye Sypro^® Orange. The fluorescence fingerprint obtained did not only allow to discriminate various AAV serotypes based on their capsid melting temperatures, but also enabled to probe the homogeneity and purity of AAV vector preparations, investigated in parallel using dynamic light scattering (DLS) and polyacrylamide gel electrophoresis. In particular, a double fluorescence transition indicated the presence of capsid-associated protein contaminants whereas a high initial fluorescence background correlated with the presence of free protein contaminants and capsid subunits, possibly resulting from capsid degradation during vector purification or storage. The variability, sensitivity and precision of this assay were further investigated in two different AAV research laboratories. This simple, fast (analysis of 94 preps in ~6 hrs) and low-cost assay emerges as a relevant tool for characterization of AAV vector preparations and will help to increase the reliability of in vivo gene transfer studies.  

5.1995           97. Large Scale Purification of AAV with Continuous Flow Ultracentrifugation

Chen, H., Marino, S. and Ho, C.Y. Molecular Therapy, 24, Supplement 1, S42 (2016)   Since its first approval in Europe as gene therapy drug in human use in 2012, adeno-associated viral (AAV) vectors have gained more and more attentions in the field for gene therapy research. So far AAV vectors have usually been purified through either density gradient ultracentrifugation in small volume centrifuge tubes or column chromatography. Though these purification methods have their unique benefits, there is still a need for technology that can process large volume of lysate with high AAV recovery rate. We reasoned that continuous flow ultracentrifugation could meet these requirements. We tested the Alfa Wasserman's AW Promatix 1000™, a research scale continuous flow ultracentrifuge, as a prove of concept for AAV vector purification. In the initial experiments, we tested cesium chloride (CsCl) solution as density gradient media for AAV vector purification but found out that CsCl solution was not stable enough to form a linear gradient even when sucrose was added to increase its viscosity for AAV purification. We then tested iodixanol solution as density gradient media and got satisfactory purification of AAV vectors. Our results indicate that we can obtain near-purified AAV vectors in a single-step of centrifugation with AAV recovery rate exceeding 50%. Further experiments indicate that minor impurities associated with the purified AAV vectors could be removed by adding salts to the iodixanol solution such as CsCl to increase the ionic strength of the density gradient. The data presented here indicate that continuous flow ultracentrifugation can be used for large scale purification of AAV vectors and it should provide an additional tool to facilitate the translation from research to the clinic.  

5.1996           254. New Chimeric Gene Therapy Vectors Based on Four Different Mammalian Bocaviruses

Fakhiri, J., Schneider, M., Kailasan, S., Meister, M., McKenna, M.A., Yan, Z., Qui, J. and Grimm, D. Moleculara Therapy, 24, Supplement 1, S100 (2016)   Parvoviruses have long been developed as safe, efficient and versatile DNA delivery vectors for gene therapy applications. In particular adeno-associated viral (AAV) vectors have emerged as lead candidates owing to their apathogenicity and amenability to genetic modifications. Yet, a drawback is their limited cargo capacity of 4.9 kb, which is insufficient to accommodate larger genes. Intriguingly, it was shown recently that oversized single-stranded AAV genomes can be packaged into capsids of human bocavirus 1 (HboV1, also a parvovirus), yielding HboV1/AAV chimeras that specifically and efficiently transduce human airway epithelia (HAE). Motivated by this pioneering work, we aimed to expand the repertoire of HboV/AAV chimeras by vectorizing four additional primate bocaviruses known to infect the gastrointestinal (GI) tract. We thus assembled and cloned the VP1/VP2 ORFs from three human variants (HboV2-4) and gorilla bocavirus into a helper plasmid derived from HboV1, carrying the genes required for HboV1 replication and packaging. To assess viral particle assembly, HEK293T cells were transfected with three plasmids: (i) one of our new HboV helpers; (ii) a self-complementary AAV-YFP vector; and (iii) pDG, a plasmid encoding all genes for AAV packaging and replication. In all cases, correct expression of VP1/VP2 proteins was confirmed by Western blot analyses of cell lysates. Also, following large-scale production and iodixanol gradient purification, qPCR analyses of the 40% phase showed the presence of DNase-resistant particles for all five bocaviral serotypes. Most importantly, titration of these particles revealed comparable quantities, demonstrating that expression of NS and NP1 proteins from HboV1 supports assembly of the four other bocaviruses. Analysis in primary HAE showed YFP transgene expression for all chimeric vectors except for HboV2/AAV, congruent with prior detection of the cognate wild-type viruses in nasopharyngeal aspirates. Further in line with epidemiological data, we noted a marked difference in infectivity, from 15% for HboV1, to below 1% for the others. In looming experiments, the new vectors will be studied in primary epithelial cells from the GI tract, which are the putative natural target cells for HboV2-4 and gorilla bocavirus. Interestingly, infectivity of all chimeras could be boosted by adding proteasome inhibitors, reminiscent of data with AAV. Hence, to enhance escape from the proteasome degradation pathway and improve transduction, we mutated surface tyrosines in the VP2 protein of HboV1. Functional assessment of the resulting mutants is currently ongoing. Collectively, the large capacity, unique cell specificities and ability to cross-package AAV DNA make this novel vector set highly attractive for human gene therapy applications. In the future, it should be moreover rewarding to attempt molecular evolution of bocavirus capsids, taking advantage of the profound experience with AAV vectors.  

5.1997           292. Towards Large-Scale Manufacturing of Adeno-Associated Virus by Transient Transfection of HEK293 Suspension Cells in a Stirred Tank Bioreactor Using Serum-Free Medium

Chalai, P., Schulze, E.A., Bernier, A., Lanthier, S., Coulombe, N., Kamen, A. and Gilbert, R. Molecular Therapy, 24, Supplement 1, S117 (2016)   Adeno-Associated Virus (AAV) vectors showing safety profile in phase I clinical trials and its ability to transduce gene expression in various tissues have made it a vector of choice for gene delivery. There are different modes of AAV vector production and each has advantages and disadvantages. Here we demonstrated that the production of AAV by transient transfection in a serum-free medium using NRC's patented cGMP compliant human embryonic kidney HEK293 cell line (clone HEK293SF-3F6) adapted for growth in suspension can be readily scaled-up in stirred tank bioreactors. We employed triple-plasmid / polyethylenimine (PEI) based transient transfection technique. As a proof of concept, we demonstrated that nine serotypes of AAV (AAV-1 to AAV-9) encoding GFP can be produced by our cell line HEK293SF with yields of about 1E+13 genome-containing particles per liter (Vg/L). Depending on the serotypes 4-30% of AAV is present in the supernatant of the cell culture at 48hpt. The presence of plasmids and plasmid polyplexes that were not taken up by the cells or were not brought into the cell nucleus were removed by Iodixanol-ultracentrifugation method and Benzonase treatment before analyzing by real-time PCR. About 25% loss in genome containing viral particle counts were observed by Iodixanol purification method based on infectivity assay. Productions of AAV2 and AAV6 encoding GFP were demonstrated in 3L stirred tank bioreactors. Purification scheme was based on column chromatography - a scalable process. Different chromatography media, such as cation exchanger, anion exchanger and hydrophobic interaction chromatography, were tested with each AAV serotypes for their ability to adsorb and elute efficiently. The purification scheme was then adopted by integrating best chromatography medium and sequence dependent upon the AAV serotype in use. We demonstrated the purification scheme for AAV2 based on ion-exchange and hydrophobic interaction chromatography steps. The SDS-PAGE showed the purity of the final product and the presence of three capsid proteins VP1, VP2 and VP3 on Western blot corresponding to the only three bands present in the final product on SDS-PAGE. To extend the storage life of AAV we explored lyophilization technique to study the stability of AAV2 and AAV6 under lyophilized conditions. The AAV2 and AAV6 were stable for over 40 weeks based on infectivity assay. We demonstrated the scalability of the process up to 45L. Productions tested in 20 and 500 mL cultures in shake flasks were scaled up in 2 and 45L cultures (in 3- and 60-L stirred tank bioreactors, respectively). The volumetric yields and purification recoveries were comparable at all of these production scale levels demonstrating scalability of transient transfection at even larger scale is possible to generate material necessary for dosages required for gene therapy application.  

5.1998           555. Development of a Post-Exposure Treatment for Ebola Virus Infections Based on AAV Vectors and Zmapp Antibody Cocktail

Robert, M-A., Kamen, A., Kobinger, G., Gilbert, R. and Gaillet, B. Molecular Therapy, 24, Supplement 1, S222 (2016)   The recent Ebola outbreak in West Africa has been the deadliest in the history. To prevent future recurrence of such outbreak, better treatments and effective vaccines against Ebola virus are desirable. Among such promising treatments, the Zmapp cocktail containing neutralizing antibodies (13C6, 2G4 and 4G7) has successfully treated some patients. However, the feasibility of using it on large populations especially in developing countries is questionable. To address this potential issue, we propose to employ recombinant vectors derived from adeno-associated virus (rAAV). There are several advantages of using rAAV: because of 1) their safety profile; 2) only one injection (or a few) would be required; 3) the high stability of lyophilized rAAVs at ambient temperature and; 4) the panel of available serotypes. Because of these interesting features, we are currently developing a treatment based on three rAAVs to deliver the genes for the Zmapp cocktail of antibodies. We have already produced at small scale a rAAV expressing the 2G4 antibody. The DNA sequences for the heavy chain and light chains were codon-optimized for better expression in humans and were designed to be expressed from the same gene. A strong promoter (CAG) resistant to silencing in vivo was chosen to drive gene expression of the antibody. The rAAV were produced by transfection using our patented cGMP compatible HEK293 cell line. The production was performed in suspension culture in the absence of serum. Secretion of 2G4 antibody by rAAV transduced cells (HEK293 and CHO cells) was confirmed. The results demonstrated that rAAV-CAG-2G4 was functional and allowed for the correct assembly of the heavy and light chains of 2G4. Purification of 200 mL of rAAV-CAG-2G4 production was performed by ultracentrifugation on an iodixanol density-step gradient. Two other rAAVs coding 13C6 and 4G7 antibodies are in the processed of being constructed and produced in a similar manner. We are also in the process of comparing the efficacy of two serotypes of AAV (9 and DJ) in mice by intranasal delivery. Using the best serotype, the rAAVs will be produced and purified from a starting suspension culture of 20 L. Their efficacy for treating Ebola infections will then be evaluated in a mouse model infected by the virus.  

5.1999           594. Exosome-Associated AAV Enhances Retinal Transduction Following Intravitreal Injection

György, B., Wassmer, S., Carvalho, L., Maguire, C. and Vandenbeerghe, L.H. Molecular Therapy, 24, Supplement 1, S235 (2016)   Introduction Adeno-associated virus (AAV) has been shown to be associated with cell derived exosomes (exo-AAV). Exo-AAV outperforms regular AAV in transduction efficacy and evades neutralizing anti-AAV antibodies. Here we investigate the retinal transduction profile of exo-AAV vectors when administered intravitreally in BL6 adult mice. Methods Exo-AAV vectors were isolated from the culture media of triple-transfected 293T cells (AAV2 rep/cap, GFP transgene and adenovirus helper plasmid) by differential centrifugation. Regular AAV vectors were isolated from the cell lysate by iodixanol density gradient ultracentrifugation. To assess the transduction ability of the regular AAV2 and exo-AAV2 , we performed intravitreal injections into mice (n= 12 eyes, 1×10¹² VG/mL), using AAV2 as the control (n=14 eyes, 1×10¹² VG/mL). Fundus imaging was performed at 2 and 4 weeks post-injection, at which point the eyes were collected for immunohistological processing. Results We found that the exo-AAV2 showed an early onset of robust GFP expression via fundus imaging at 2 weeks post injection, as compared to regular AAV2. This was further enhanced by larger spread and brightness of GFP expression at 4 weeks. Histological processing of retinal sections shows GFP expression after exo-AAV2 expression in the nerve fiber layer, retinal ganglion cell layer, inner nuclear layer, outer nuclear layer and photoreceptor inner and outer segments. Staining with ganglion cell and bipolar cell markers show co-localization of GFP in these cell types. AAV2 injected animals show GFP expression mostly restricted to the nerve fiber and ganglion cell layers. Conclusion Intravitreal injections are an ideal delivery route to the retina in humans as it is minimally invasive and performed routinely in the clinic. Repeated pre-clinical work by others and us using AAV2 shows that an intravitreal injection is limited to targeting the nerve fiber layer (consequently, optic nerve) and retinal ganglion cells. Here, we demonstrate that exo-AAV2 targets all cell layers of the retina at robust levels and successfully targets retinal bipolar cells. As a result, an intravitreal injection of exo-AAV with a cell specific promoter may be the vector of choice for future gene therapy.  

5.2000           Clinical Improvement of Alpha-mannosidosis Cat Following a Single Cisterna Magna Infusion of AAV1

Yoon, S.Y., Bagel, J.H., O’Donnell, P.A., Vite, C.H. and Wolfe, J.H. Molecular Therapy, 24(1), 26-33 (2016)   Lysosomal storage diseases (LSDs) are debilitating neurometabolic disorders for most of which long-term effective therapies have not been developed. Gene therapy is a potential treatment but a critical barrier to treating the brain is the need for global correction. We tested the efficacy of cisterna magna infusion of adeno-associated virus type 1 (AAV1) expressing feline alpha-mannosidase gene in the postsymptomatic alpha-mannosidosis (AMD) cat, a homologue of the human disease. Lysosomal alpha-mannosidase (MANB) activity in the cerebrospinal fluid (CSF) and serum were increased above the control values in untreated AMD cats. Clinical neurological signs were delayed in onset and reduced in severity. The lifespan of the treated cats was significantly extended. Postmortem histopathology showed resolution of lysosomal storage lesions throughout the brain. MANB activity in brain tissue was significantly above the levels of untreated tissues. The results demonstrate that a single cisterna magna injection of AAV1 into the CSF can mediate widespread neuronal transduction of the brain and meaningful clinical improvement. Thus, cisterna magna gene delivery by AAV1 appears to be a viable strategy for treatment of the whole brain in AMD and should be applicable to many of the neurotropic LSDs as well as other neurogenetic disorders.  

5.2001           In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery

Garanto, A., Chung, D.C., Duijkers, L., Corral-Serrano, J.C., Messchaert, M., Xiao, R., Bennett, J., Vandenberghe, L.H. and Collin, R.W.J. Hum. Mol. Genet., 25(12), 2552-2563 (2016)   Leber congenital amaurosis (LCA) is a severe disorder resulting in visual impairment usually starting in the first year of life. The most frequent genetic cause of LCA is an intronic mutation in CEP290 (c.2991 + 1655A > G) that creates a cryptic splice donor site resulting in the insertion of a pseudoexon (exon X) into CEP290 mRNA. Previously, we showed that naked antisense oligonucleotides (AONs) effectively restored normal CEP290 splicing in patient-derived lymphoblastoid cells. We here explore the therapeutic potential of naked and adeno-associated virus (AAV)-packaged AONs in vitro and in vivo. In both cases, AON delivery fully restored CEP290 pre-mRNA splicing, significantly increased CEP290 protein levels and rescued a ciliary phenotype present in patient-derived fibroblast cells. Moreover, administration of naked and AAV-packaged AONs to the retina of a humanized mutant Cep290 mouse model, carrying the intronic mutation, showed a statistically significant reduction of exon X-containing Cep290 transcripts, without compromising the retinal structure. Together, our data highlight the tremendous therapeutic prospective of AONs for the treatment of not only CEP290-associated LCA but potentially many other subtypes of retinal dystrophy caused by splicing mutations.  

5.2002           AAV-mediated gene therapy in Dystrophin-Dp71 deficient mouse leads to blood-retinal barrier restoration and oedema reabsorption

Vacca, O., Charles-Messance, H., El mathari, B., Sene, A., Barbe, P., Fouquet, S., Aragon, J., Darche, M., Giocanti-Auregan, A., Paques, M., Sahel, J-A., Tadayoni, R., Montanez, C., Dalkara, D. and Rendon, A. Hum. Mol. Genet., 25(14), 3070-3079 (2016)   Dystrophin-Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells that provide a mechanical link at the Müller cell membrane by direct binding to actin and a transmembrane protein complex. Its absence has been related to blood-retinal barrier (BRB) permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels (1). We have previously shown that the adeno-associated virus (AAV) variant, ShH10, transduces Müller cells in the Dp71-null mouse retina efficiently and specifically (2,3). Here, we use ShH10 to restore Dp71 expression in Müller cells of Dp71 deficient mouse to study molecular and functional effects of this restoration in an adult mouse displaying retinal permeability. We show that strong and specific expression of exogenous Dp71 in Müller cells leads to correct localization of Dp71 protein restoring all protein interactions in order to re-establish a proper functional BRB and retina homeostasis thus preventing retina from oedema. This study is the basis for the development of new therapeutic strategies in dealing with diseases with BRB breakdown and macular oedema such as diabetic retinopathy (DR).  

5.2003           The Effect of Heat on the Physicochemical Properties of Bacteriophage MS2

Brie, A., Bertrand, I., Meo, M., Boudaud, N. and gantzeer, C. Food Environ. Virol., 8(4), 251-261 (2016)   The differences in physicochemical characteristics between infectious and non-infectious viral particles are poorly known. Even for heat, which is known as one of the most efficient treatments to inactivate enteric viruses, the global inactivation mechanisms have not been described yet. Such knowledge would help distinguish between both types of particles and therefore clarify the interpretation of the presence of viral genomes in food after heat treatment. In this study, we examined in particular the differences in electrostatic charge and hydrophobicity between the two particle types. MS2 phage, a common surrogate for enteric viruses, was used as a model virus. The heat-induced inactivation process of the infectious phages caused hydrophobic domains to be transiently exposed and their charge to become less negative. The particles also became progressively permeable to small molecules such as SYPRO Orange dye. The presence of non-infectious phage particles in which the genome was not accessible to RNases has been clearly demonstrated. These observations were done for MS2 phages exposed to a temperature of 60 °C. When exposed to a temperature higher than their critical temperature (72 °C), the particles were disrupted and the genome became available for RNases. At lower temperatures, 60 °C in this study, the transient expression of hydrophobic domains of remaining infectious phages appeared as an interesting parameter for improving their specific detection.  

5.2004           Lytic Inactivation of Human Immunodeficiency Virus by Dual Engagement of gp120 and gp41 Domains in the Virus Env Protein Trimer

Paarajuli, B., Acharya, K., Yu, R., Ngo, B., Rashad, A.A., Abrams, C.F. and Chaiken, I.M. Biochemistry, 55(44), 6100-6114 (2016)   We recently reported the discovery of a recombinant chimera, denoted DAVEI (dual-acting virucidal entry inhibitor), which is able to selectively cause specific and potent lytic inactivation of both pseudotyped and fully infectious human immunodeficiency virus (HIV-1) virions. The chimera is composed of the lectin cyanovirin-N (CVN) fused to the 20-residue membrane-proximal external region (MPER) of HIV-1 gp41. Because the Env gp120-binding CVN domain on its own is not lytic, we sought here to determine how the MPER_(DAVEI) domain is able to endow the chimera with virolytic activity. We used a protein engineering strategy to identify molecular determinants of MPER_(DAVEI) that are important for function. Recombinant mutagenesis and truncation demonstrated that the MPER_(DAVEI) domain could be significantly minimized without loss of function. The dependence of lysis on specific MPER sequences of DAVEI, determination of minimal linker length, and competition by a simplified MPER surrogate peptide suggested that the MPER domain of DAVEI interacts with the Env spike trimer, likely with the gp41 region. This conclusion was further supported by observations from binding of the biotinylated MPER surrogate peptide to Env protein expressed on cells, monoclonal antibody competition, a direct binding enzyme-linked immunosorbent assay on viruses with varying numbers of trimeric spikes on their surfaces, and comparison of maximal interdomain spacing in DAVEI to that in high-resolution structures of Env. The finding that MPER_(DAVEI) in CVN–MPER linker sequences can be minimized without loss of virolytic function provides an improved experimental path for constructing size-minimized DAVEI chimeras and molecular tools for determining how simultaneous engagement of gp120 and gp41 by these chimeras can disrupt the metastable virus Env spike.  

5.2005           Microbatch Mixing: “Shaken not Stirred”, a Method for Macromolecular Microcrystal Production for Serial Crystallography

Mahon, B.P., Kurian, J.J., Lomelino, C.L., Smith, I.R., Socorro, L., Bennett, A., Hendon, A.M., Chipman, P., Savin, D.A., Agbandje-McKenna, M. and Mckenna, R. Crys. Growth Des., 16(11), 6214-6221 (2016)   advances of serial crystallography techniques at synchrotron and X-ray free electron laser facilities have made possible the acquisition of useable data sets to determine 3-dimensional structures of macromolecules from micro- to nanosized crystals. In addition, the same technological hallmarks have contributed significantly to the field of time-resolved crystallography. However, the production of usable crystalline slurries for serial crystallographic experiments has been one of the limiting factors and contributes to an alternative sample “bottleneck” in crystal growth. In this study, we propose a method: labeled microbatch mixing (MBM), which has the capability to produce large quantities of microcrystals of macromolecules suitable for serial crystallographic experiments. This is shown to be successful for producing lysozyme, carbonic anhydrase, and adeno-associated virus crystals. MBM takes advantage of secondary nucleation induced by mixing via the application of steady agitation during the crystallization process. This leads to excessive nucleation, resulting in large quantities of well-diffracting microcrystals. MBM therefore presents a method that can potentially be applied to a range of macromolecules and a possible simple protocol to produce microcrystals for serial crystallographic experiments.  

5.2006           Allele-specific regulation of mutant Huntingtin by Wig1, a downstream target of p53

Kim, S-H., Shahani, N., Bae, B-II, Sbodio, J.I., Chung, Y., Nakaso, K., Paul, B.D. and Sawa, A. Hum. Mol. Genet., 25(12), 2514-2524 (2016)   p53 has been implicated in the pathophysiology of Huntington’s disease (HD). Nonetheless, the molecular mechanism of how p53 may play a unique role in the pathology remains elusive. To address this question at the molecular and cellular biology levels, we initially screened differentially expressed molecules specifically dependent on p53 in a HD animal model. Among the candidate molecules, wild-type p53-induced gene 1 (Wig1) is markedly upregulated in the cerebral cortex of HD patients. Wig1 preferentially upregulates the level of mutant Huntingtin (Htt) compared with wild-type Htt. This allele-specific characteristic of Wig1 is likely to be explained by higher affinity binding to mutant Htt transcripts than normal counterpart for the stabilization. Knockdown of Wig1 level significantly ameliorates mutant Htt-elicited cytotoxicity and aggregate formation. Together, we propose that Wig1, a key p53 downstream molecule in HD condition, play an important role in stabilizing mutant Htt mRNA and thereby accelerating HD pathology in the mHtt-p53-Wig1 positive feedback manner.  

5.2007           Maximum levels of hepatitis C virus lipoviral particles are associated with early and persistent infection

Sheridan, D.A., Hajarizadeh, B., Fenwick, F.I., Matthews, G.V., Appligate, T., Douglas, M., Neely, D., Askew, B., Dore, G.J., Lloyd, A.R., George, J., Bassendine, M.F. and Grebely, J. Liver Int., 36(12), 1774-1782 (2016)   Background & Aims Hepatitis C virus (HCV) is bound to plasma lipoproteins and circulates as an infectious lipoviral particle (LVP). Experimental evidence indicates that LVPs have decreased susceptibility to antibody-mediated neutralisation and higher infectivity. This study tested the hypothesis that LVPs are required to establish persistent infection, and conversely, low levels of LVP in recent HCV infection increase the probability of spontaneous HCV clearance. Methods LVP in non-fasting plasma was measured using the concentration of HCV RNA bound to large >100 nm sized lipoproteins after ex vivo addition of a lipid emulsion, that represented the maximum concentration of LVP (maxi-LVP). This method correlated with LVP in fasting plasma measured using iodixanol density gradient ultracentrifugation. Maxi-LVP was measured in a cohort of 180 HCV participants with recent HCV infection and detectable HCV RNA from the Australian Trial in Acute Hepatitis C (ATAHC) and Hepatitis C Incidence and Transmission Study in prison (HITS-p) cohorts. Results Spontaneous clearance occurred in 15% (27 of 180) of individuals. In adjusted analyses, low plasma maxi-LVP level was independently associated with spontaneous HCV clearance (≤827 IU/ml; adjusted odds ratio 3.98, 95% CI: 1.02, 15.51, P = 0.047), after adjusting for interferon lambda-3 rs8099917 genotype, estimated duration of HCV infection and total HCV RNA level. Conclusions Maxi-LVP is a biomarker for the maximum concentration of LVP in non-fasting samples. Low maxi-LVP level is an independent predictor of spontaneous clearance of acute HCV.  

5.2008           Natural mutations in IFITM3 modulate post‐translational regulation and toggle antiviral specificity

Compton, A.A., Roy, N., Porrot, F., Billet, A., Casartelli, N., Yount, J.S., Liang, C. and Schwartz, O. EMBO Reports, 17(11), 1657-1671 (2016)   The interferon‐induced transmembrane (IFITM) proteins protect host cells from diverse virus infections. IFITM proteins also incorporate into HIV‐1 virions and inhibit virus fusion and cell‐to‐cell spread, with IFITM3 showing the greatest potency. Here, we report that amino‐terminal mutants of IFITM3 preventing ubiquitination and endocytosis are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV‐1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino‐terminal mutations that modify protein localization and function. This suggests that “runaway” IFITM3 variants could be selected for altered antiviral activity. Furthermore, we show that adaptations in IFITM3 result in a trade‐off in antiviral specificity, as variants exhibiting enhanced activity against HIV‐1 poorly restrict influenza A virus. Overall, we provide the first experimental evidence that diversification of IFITM3 genes may boost the antiviral coverage of host cells and provide selective functional advantages.  

5.2009           Role of Tetra Amino Acid Motif Properties on the Function of Protease-Activatable Viral Vectors

Robinson, T.M., Judd, J., Ho, M.L. and Suh, J. ACS Biomater. Sci. Eng., 2(11), 2026-2033 (2016)   Protease-activatable viruses (PAV) based on adeno-associated virus have previously been generated for gene delivery to pathological sites characterized by elevated extracellular proteases. “Peptide locks”, composed of a tetra-aspartic acid motif flanked by protease cleavage sequences, were inserted into the virus capsid to inhibit virus-host cell receptor binding and transduction. In the presence of proteases, the peptide locks are cleaved off the capsid, restoring the virus’ ability to bind cells and deliver cargo. Although promising, questions remained regarding how the peptide locks prevented cell binding. In particular, it was unclear if the tetra-amino acid (4AA) motif blocks receptor binding via electrostatic repulsion or steric obstruction. To explore this question, we generated a panel of PAVs with lock designs incorporating altered 4AA motifs, each wielding various chemical properties (negative, positive, uncharged polar, and hydrophobic) and characterized the resultant PAV candidates. Notably, all mutants display reduced receptor binding and decreased transduction efficiency in the absence of proteases, suggesting simple electrostatics between heparin and the D4 motif do not play an exclusive role in obstructing virus-receptor binding. Even small hydrophobic (A4) and uncharged polar (SGGS) motifs confer a reduction in heparin binding compared to the wild type. Furthermore, both uncharged polar N4 and Q4 mutants (comparable in size to the D4 and E4 motifs respectively, but lacking the negative charge) demonstrate partial ablation of heparin binding. Collectively, these results support a possible dual mechanism of PAV lock operation, where steric hindrance and electrostatics make nonredundant contributions to the disruption of virus-receptor interactions. Finally, because of high virus titer production and superior capsid stability, only the negatively charged 4AA motifs remain viable design choices for PAV construction. Future studies probing the structure–function relationship of PAVs will further expand its promise as a gene delivery vector able to target diseased tissues exhibiting elevated extracellular proteases.   An open-hardware platform for optogenetics and photobiology Gerhardt, K.P., Olson, E.J., Castillo-Hair, S.M., Hartsough, L.A., landry, B.P., Ekness, F., Yokoo, R., Gomez, E.J., Ramakrishnan, P., Suh, J., Savage, D. and Tabor, J.J. Scientific Reports, 6:35363 (2016)   In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments.   Intracranial AAV-sTRAIL combined with lanatoside C prolongs survival in an orthotopic xenograft mouse model of invasive glioblastoma Crommentuijn, M.H.W., Maguire, C.S., Niers, J.M., Vandertop, W.P., Badr, C.E., Würdinger, T. and Tannous, B.A. Mol. Oncol., 10, 625-634 (2016)   Glioblastoma (GBM) is the most common malignant brain tumor in adults. We designed an adeno-associated virus (AAV) vector for intracranial delivery of secreted, soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) to GBM tumors in mice and combined it with the TRAIL-sensitizing cardiac glycoside, lanatoside C (lan C). We applied this combined therapy to two different GBM models using human U87 glioma cells and primary patient-derived GBM neural spheres in culture and in orthotopic GBM xenograft models in mice. In U87 cells, conditioned medium from AAV2-sTRAIL expressing cells combined with lan C induced 80% cell death. Similarly, lan C sensitized primary GBM spheres to sTRAIL causing over 90% cell death. In mice bearing intracranial U87 tumors treated with AAVrh.8-sTRAIL, administration of lan C caused a decrease in tumor-associated Fluc signal, while tumor size increased within days of stopping the treatment. Another round of lan C treatment re-sensitized GBM tumor to sTRAIL-induced cell death. AAVrh.8-sTRAIL treatment alone and combined with lanatoside C resulted in a significant decrease in tumor growth and longer survival of mice bearing orthotopic invasive GBM brain tumors. In summary, AAV-sTRAIL combined with lanatoside C induced cell death in U87 glioma cells and patient-derived GBM neural spheres in culture and in vivo leading to an increased in overall mice survival.  

5.2010           A generic viral dynamic model to systematically characterize the interaction between oncolytic virus kinetics and tumor growth

Titze, M.I., Frank, J., Ehrhardt, M., Smola, S., Graf, N. and Lehr, T. Eur. J. Pharmaceut. Sci., 97, 38-46 (2017)   Oncolytic viruses (OV) represent an encouraging new therapeutic concept for treatment of human cancers. OVs specifically replicate in tumor cells and initiate cell lysis whilst tumor cells act as endogenous bioreactors for virus amplification. This complex bidirectional interaction between tumor and oncolytic virus hampers the establishment of a straight dose-concentration-effect relation. We aimed to develop a generic mathematical pharmacokinetic/pharmacodynamics (PK/PD) model to characterize the relationship between tumor cell growth and kinetics of different OVs. U87 glioblastoma cell growth and titer of Newcastle disease virus (NDV), reovirus (RV) and parvovirus (PV) were systematically determined in vitro. PK/PD analyses were performed using non-linear mixed effects modeling. A viral dynamic model (VDM) with a common structure for the three different OVs was developed which simultaneously described tumor growth and virus replication. Virus specific parameters enabled a comparison of the kinetics and tumor killing efficacy of each OV. The long-term interactions of tumor cells with NDV and RV were simulated to predict tumor reoccurrence. Various treatment scenarios (single and multiple dosing with same OV, co-infection with different OVs and combination with hypothetical cytotoxic compounds) were simulated and ranked for efficacy using a newly developed treatment rating score. The developed VDM serves as flexible tool for the systematic cross-characterization of tumor-virus relationships and supports preselection of the most promising treatment regimens for follow-up in vivo analyses.  

5.2011           Interaction between subclinical doses of the Parkinson’s disease associated gene, α-synuclein, and the pesticide, rotenone, precipitates motor dysfunction and nigrostriatal neurodegeneration in rats

Naughton, C., O’Toole, D., Kirik, D. and Dowd, E. Behavioural Brain Res., 316, 160-168 (2017)   In most patients, Parkinson’s disease is thought to emerge after a lifetime of exposure to, and interaction between, various genetic and environmental risk factors. One of the key genetic factors linked to this condition is α-synuclein, and the α-synuclein protein is pathologically associated with idiopathic cases. However, α-synuclein pathology is also present in presymptomatic, clinically “normal” individuals suggesting that environmental factors, such as Parkinson’s disease-linked agricultural pesticides, may be required to precipitate Parkinson’s disease in these individuals. In this context, the aim of this study was to assess the behavioural and neuropathological impact of exposing rats with a subclinical load of α-synuclein to subclinical doses of the organic pesticide, rotenone. Rats were randomly assigned to two groups for intra-nigral infusion of AAV_2/5-GFP or AAV_2/5-α-synuclein. Post viral motor function was assessed at 8, 10 and 12 weeks in the Corridor, Stepping and Whisker tests of lateralised motor function. At week 12, animals were performance-matched to receive a subsequent intra-striatal challenge of the organic pesticide rotenone (or its vehicle) to yield four final groups (Control, Rotenone, AAV_2/5-α-synuclein and Combined). Behavioural testing resumed one week after rotenone surgery and continued for 5 weeks. We found that, when administered alone, neither intra-nigral AAV-α-synuclein nor intra-striatal rotenone caused sufficient nigrostriatal neurodegeneration to induce a significant motor impairment in their own right. However, when these were administered sequentially to the same rats, the interaction between the two Parkinsonian challenges significantly exacerbated nigrostriatal neurodegeneration which precipitated a pronounced impairment in motor function. These results indicate that exposing rats with a subclinical α-synuclein-induced pathology to the pesticide, rotenone, profoundly exacerbates their Parkinsonian neuropathology and dysfunction, and highlights the potential importance of this interaction in the etiology of, and in driving the pathogenesis of Parkinson’s disease.  

5.2012           A Single Vector Platform for High-Level Gene Transduction of Central Neurons: Adeno-Associated Virus Vector Equipped with the Tet-Off System

Sohn, J., Takahashi, M., Okamoto, s., Ishida, Y., Furuta, T. and Hioki, H. PloS One, 12(1), e0169611 (2017)   Visualization of neurons is indispensable for the investigation of neuronal circuits in the central nervous system. Virus vectors have been widely used for labeling particular subsets of neurons, and the adeno-associated virus (AAV) vector has gained popularity as a tool for gene transfer. Here, we developed a single AAV vector Tet-Off platform, AAV-SynTetOff, to improve the gene-transduction efficiency, specifically in neurons. The platform is composed of regulator and response elements in a single AAV genome. After infection of Neuro-2a cells with the AAV-SynTetOff vector, the transduction efficiency of green fluorescent protein (GFP) was increased by approximately 2- and 15-fold relative to the conventional AAV vector with the human cytomegalovirus (CMV) or human synapsin I (SYN) promoter, respectively. We then injected the AAV vectors into the mouse neostriatum. GFP expression in the neostriatal neurons infected with the AAV-SynTetOff vector was approximately 40-times higher than that with the CMV or SYN promoter. By adding a membrane-targeting signal to GFP, the axon fibers of neostriatal neurons were clearly visualized. In contrast, by attaching somatodendritic membrane-targeting signals to GFP, axon fiber labeling was mostly suppressed. Furthermore, we prepared the AAV-SynTetOff vector, which simultaneously expressed somatodendritic membrane-targeted GFP and membrane-targeted red fluorescent protein (RFP). After injection of the vector into the neostriatum, the cell bodies and dendrites of neostriatal neurons were labeled with both GFP and RFP, whereas the axons in the projection sites were labeled only with RFP. Finally, we applied this vector to vasoactive intestinal polypeptide-positive (VIP+) neocortical neurons, one of the subclasses of inhibitory neurons in the neocortex, in layer 2/3 of the mouse primary somatosensory cortex. The results revealed the differential distribution of the somatodendritic and axonal structures at the population level. The AAV-SynTetOff vector developed in the present study exhibits strong fluorescence labeling and has promising applications in neuronal imaging.  

5.2013           The Role of DCT in HPV16 Infection of HaCaTs

Aksoy, P., Meneses, P.I. PloS One, 12(1), e0170158 (2017)   Persistent infection with high-risk human papillomavirus (HPV) genotype is a major factor leading to many human cancers. Mechanisms of HPV entry into host cells and genome trafficking towards the nucleus are incompletely understood. Dopachrome tautomerase (DCT) was identified as a cellular gene required for HPV infection in HeLa cells on a siRNA screen study. Here, we confirm that DCT knockdown significantly decreases HPV infection in the human keratinocyte HaCaT cells as was observed in HeLas. We investigated the effects of DCT knockdown and found that DCT depletion caused increased reactive oxygen species (ROS) levels, DNA damage and altered cell cycle in HaCaT cells. We observed increased viral DNA localization at the endoplasmic reticulum but an overall decrease in infection in DCT knockdown cells. This observation suggests that viral DNA might be retained in the ER due to altered cell cycle, and viral particles are incapable of further movement towards the nucleus in DCT knockdown cells.  

5.2014           Silencing Genes in the Heart

Fechner, H., Vetter, R., Kurreck, j. and Poller, W. Methods in Mol. Biol., 1521, 17-39 (2017)   Silencing of cardiac genes by RNA interference (RNAi) has developed into a powerful new method to treat cardiac diseases. Small interfering (si)RNAs are the inducers of RNAi, but cultured primary cardiomyocytes and heart are highly resistant to siRNA transfection. This can be overcome by delivery of small hairpin (sh)RNAs or artificial microRNA (amiRNAs) by cardiotropic adeno-associated virus (AAV) vectors. Here we describe as example of the silencing of a cardiac gene, the generation and cloning of shRNA, and amiRNAs directed against the cardiac protein phospholamban. We further describe the generation of AAV shuttle plasmids with self complementary vector genomes, the production of AAV vectors in roller bottles, and their purification via iodixanol gradient centrifugation and concentration with filter systems. Finally we describe the preparation of primary neonatal rat cardiomyocytes (PNRC), the transduction of PNRC with AAV vectors, and the maintenance of the transduced cell culture.  

5.2015           Production and Characterization of Vectors Based on the Cardiotropic AAV Serotype 9

Kohlbrenner, E. and Weber, T. Methods in Mol. Biol., 1521, 91-107 (2017)   Vectors based on adeno-associated virus serotype 9 (AAV9) efficiently transduce cardiomyocytes in both rodents and large animal models upon either systemic or regional vector delivery. In this chapter, we describe the most widely used production and purification method of AAV9. This production approach does not depend on the use of a helpervirus but instead on transient transfection of HEK293T cells with a plasmid containing the recombinant AAV genome and a second plasmid encoding the AAV9 capsid proteins, the AAV Rep proteins and the adenoviral helper functions. The recombinant AAV is then purified by iodixanol density gradient centrifugation. This chapter also describes in detail the characterization and quality control methods required for assuring high quality vector preparations, which is of particular importance for experiments in large animal models.  

5.2016           Alternative Polyadenylation of Human Bocavirus at Its 3' End Is Regulated by Multiple Elements and Affects Capsid Expression

Hao, S., Zhang, J., Chen, Z., Xu, H., Wang, H. and Guan, W.

1. Virol., 91(3), e02026-16 (2017)

  Alternative processing of human bocavirus (HBoV) P5 promoter-transcribed RNA is critical for generating the structural and nonstructural protein-encoding mRNA transcripts. The regulatory mechanism by which HBoV RNA transcripts are polyadenylated at proximal [(pA)p] or distal [(pA)d] polyadenylation sites is still unclear. We constructed a recombinant HBoV infectious clone to study the alternative polyadenylation regulation of HBoV. Surprisingly, in addition to the reported distal polyadenylation site, (pA)d, a novel distal polyadenylation site, (pA)d2, which is located in the right-end hairpin (REH), was identified during infectious clone transfection or recombinant virus infection. (pA)d2 does not contain typical hexanucleotide polyadenylation signal, upstream elements (USE), or downstream elements (DSE) according to sequence analysis. Further study showed that HBoV nonstructural protein NS1, REH, and cis elements of (pA)d were necessary and sufficient for efficient polyadenylation at (pA)d2. The distance and sequences between (pA)d and (pA)d2 also played a key role in the regulation of polyadenylation at (pA)d2. Finally, we demonstrated that efficient polyadenylation at (pA)d2 resulted in increased HBoV capsid mRNA transcripts and protein translation. Thus, our study revealed that all the bocaviruses have distal poly(A) signals on the right-end palindromic terminus, and alternative polyadenylation at the HBoV 3′ end regulates its capsid expression.  

5.2017           Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity

Bogerd, H.P., kennedy, E.M., Whisnant, A.W. and Cullen, B.R. mBio, 8(1), e02125-16 (2017)   Analysis of the incorporation of cellular microRNAs (miRNAs) into highly purified HIV-1 virions revealed that this largely, but not entirely, mirrored the level of miRNA expression in the producer CD4⁺ T cells. Specifically, of the 58 cellular miRNAs detected at significant levels in the producer cells, only 5 were found in virions at a level 2- to 4-fold higher than that predicted on the basis of random cytoplasmic sampling. Of note, these included two miRNAs, miR-155 and miR-92a, that were reported previously to at least weakly bind HIV-1 transcripts. To test whether miRNA binding to the HIV-1 genome can induce virion incorporation, artificial miRNA target sites were introduced into the viral genome and a 10- to 40-fold increase in the packaging of the cognate miRNAs into virions was then observed, leading to the recruitment of up to 1.6 miRNA copies per virion. Importantly, this high level of incorporation significantly inhibited HIV-1 virion infectivity. These results suggest that target sites for cellular miRNAs can inhibit RNA virus replication at two distinct steps, i.e., during infection and during viral gene expression, thus explaining why a range of different RNA viruses appear to have evolved to avoid cellular miRNA binding to their genome.  

5.2018           α-Defensin HD5 Inhibits Human Papillomavirus 16 Infection via Capsid Stabilization and Redirection to the Lysosome

Wiens, M.E. and Smith, J.G. mBio, 8(1), e02304-16 (2017)   α-Defensins are an important class of abundant innate immune effectors that are potently antiviral against a number of nonenveloped viral pathogens; however, a common mechanism to explain their ability to block infection by these unrelated viruses is lacking. We previously found that human defensin 5 (HD5) blocks a critical host-mediated proteolytic processing step required for human papillomavirus (HPV) infection. Here, we show that bypassing the requirement for this cleavage failed to abrogate HD5 inhibition. Instead, HD5 altered HPV trafficking in the cell. In the presence of an inhibitory concentration of HD5, HPV was internalized and reached the early endosome. The internalized capsid became permeable to antibodies and proteases; however, HD5 prevented dissociation of the viral capsid from the genome, reduced viral trafficking to the trans-Golgi network, redirected the incoming viral particle to the lysosome, and accelerated the degradation of internalized capsid proteins. This mechanism is equivalent to the mechanism by which HD5 inhibits human adenovirus. Thus, our data support capsid stabilization and redirection to the lysosome during infection as a general antiviral mechanism of α-defensins against nonenveloped viruses.  

5.2019           Radioiodinated Capsids Facilitate In Vivo Non-Invasive Tracking of Adeno-Associated Gene Transfer Vectors

Kothari, P. et al Scientific Reports, 7:39594 (2017)   Viral vector mediated gene therapy has become commonplace in clinical trials for a wide range of inherited disorders. Successful gene transfer depends on a number of factors, of which tissue tropism is among the most important. To date, definitive mapping of the spatial and temporal distribution of viral vectors in vivo has generally required postmortem examination of tissue. Here we present two methods for radiolabeling adeno-associated virus (AAV), one of the most commonly used viral vectors for gene therapy trials, and demonstrate their potential usefulness in the development of surrogate markers for vector delivery during the first week after administration. Specifically, we labeled adeno-associated virus serotype 10 expressing the coding sequences for the CLN2 gene implicated in late infantile neuronal ceroid lipofuscinosis with iodine-124. Using direct (Iodogen) and indirect (modified Bolton-Hunter) methods, we observed the vector in the murine brain for up to one week using positron emission tomography. Capsid radioiodination of viral vectors enables non-invasive, whole body, in vivo evaluation of spatial and temporal vector distribution that should inform methods for efficacious gene therapy over a broad range of applications.    

5.2020           Experience-Dependent Equilibration of AMPAR-Mediated Synaptic Transmission during the Critical Period

Han, K-S., Cooke, S.F. and Xu, W. Cell Reports, 18, 892-904 (2017)   Experience-dependent synapse refinement is essential for functional optimization of neural circuits. However, how sensory experience sculpts excitatory synaptic transmission is poorly understood. Here, we show that despite substantial remodeling of synaptic connectivity, AMPAR-mediated synaptic transmission remains at equilibrium during the critical period in the mouse primary visual cortex. The maintenance of this equilibrium requires neurogranin (Ng), a postsynaptic calmodulin-binding protein important for synaptic plasticity. With normal visual experience, loss of Ng decreased AMPAR-positive synapse numbers, prevented AMPAR-silent synapse maturation, and increased spine elimination. Importantly, visual deprivation halted synapse loss caused by loss of Ng, revealing that Ng coordinates experience-dependent AMPAR-silent synapse conversion to AMPAR-active synapses and synapse elimination. Loss of Ng also led to sensitized long-term synaptic depression (LTD) and impaired visually guided behavior. Our synaptic interrogation reveals that experience-dependent coordination of AMPAR-silent synapse conversion and synapse elimination hinges upon Ng-dependent mechanisms for constructive synaptic refinement during the critical period.  

5.2021           Novel Mutant AAV2 Rep Proteins Support AAV2 Replication without Blocking HSV-1 Helpervirus Replication

Seyffert, M., Glauser, D.L., Schraner, E.M., de Oliveira, A-P., Mansilla-Soto, J., Vigt, B., Büning, H., Linden, R.M., Ackermann, M. and Fraefel, C. PloS One, 12(1), e0170908 (2017)   As their names imply, parvoviruses of the genus Dependovirus rely for their efficient replication on the concurrent presence of a helpervirus, such as herpesvirus, adenovirus, or papilloma virus. Adeno-associated virus 2 (AAV2) is such an example, which in turn can efficiently inhibit the replication of each helpervirus by distinct mechanisms. In a previous study we have shown that expression of the AAV2 rep gene is not compatible with efficient replication of herpes simplex virus 1 (HSV-1). In particular, the combined DNA-binding and ATPase/helicase activities of the Rep68/78 proteins have been shown to exert opposite effects on the replication of AAV2 and HSV-1. While essential for AAV2 DNA replication these protein activities account for the Rep-mediated inhibition of HSV-1 replication. Here, we describe a novel Rep mutant (Rep-D371Y), which displayed an unexpected phenotype. Rep-D371Y did not block HSV-1 replication, but still supported efficient AAV2 replication, at least when a double-stranded AAV2 genome template was used. We also found that the capacity of Rep-D371Y to induce apoptosis and a Rep-specific DNA damage response was significantly reduced compared to wild-type Rep. These findings suggest that AAV2 Rep-helicase subdomains exert diverging activities, which contribute to distinct steps of the AAV2 life cycle. More important, the novel AAV2 mutant Rep-D371Y may allow deciphering yet unsolved activities of the AAV2 Rep proteins such as DNA second-strand synthesis, genomic integration or packaging, which all involve the Rep-helicase activity.  

5.2022           ApoE2, ApoE3, and ApoE4 Differentially Stimulate APP Transcription and Aβ Secretion

Huang, Y-W.A., Zhou, B., Wernig, M., Südof, T.C. Cell, 168, 427-441 (2017)   Human apolipoprotein E (ApoE) apolipoprotein is primarily expressed in three isoforms (ApoE2, ApoE3, and ApoE4) that differ only by two residues. ApoE4 constitutes the most important genetic risk factor for Alzheimer’s disease (AD), ApoE3 is neutral, and ApoE2 is protective. How ApoE isoforms influence AD pathogenesis, however, remains unclear. Using ES-cell-derived human neurons, we show that ApoE secreted by glia stimulates neuronal Aβ production with an ApoE4 > ApoE3 > ApoE2 potency rank order. We demonstrate that ApoE binding to ApoE receptors activates dual leucine-zipper kinase (DLK), a MAP-kinase kinase kinase that then activates MKK7 and ERK1/2 MAP kinases. Activated ERK1/2 induces cFos phosphorylation, stimulating the transcription factor AP-1, which in turn enhances transcription of amyloid-β precursor protein (APP) and thereby increases amyloid-β levels. This molecular mechanism also regulates APP transcription in mice in vivo. Our data describe a novel signal transduction pathway in neurons whereby ApoE activates a non-canonical MAP kinase cascade that enhances APP transcription and amyloid-β synthesis.    

5.2023           Cutaneous HPV8 and MmuPV1 E6 Proteins Target the NOTCH and TGF-β Tumor Suppressors to Inhibit Differentiation and Sustain Keratinocyte Proliferation

Meyers, J.M., Uberoi, M., Lambert, P.F. and munger, K. PloS Pathogens, 13(1), e1006171 (2017)   Cutaneous beta-papillomaviruses are associated with non-melanoma skin cancers that arise in patients who suffer from a rare genetic disorder, Epidermodysplasia verruciformis (EV) or after immunosuppression following organ transplantation. Recent studies have shown that the E6 proteins of the cancer associated beta human papillomavirus (HPV) 5 and HPV8 inhibit NOTCH and TGF-β signaling. However, it is unclear whether disruption of these pathways may contribute to cutaneous HPV pathogenesis and carcinogenesis. A recently identified papillomavirus, MmuPV1, infects laboratory mouse strains and causes cutaneous skin warts that can progress to squamous cell carcinoma. To determine whether MmuPV1 may be an appropriate model to mechanistically dissect the molecular contributions of cutaneous HPV infections to skin carcinogenesis, we investigated whether MmuPV1 E6 shares biological and biochemical activities with HPV8 E6. We report that the HPV8 and MmuPV1 E6 proteins share the ability to bind to the MAML1 and SMAD2/SMAD3 transcriptional cofactors of NOTCH and TGF-beta signaling, respectively. Moreover, we demonstrate that these cutaneous papillomavirus E6 proteins inhibit these two tumor suppressor pathways and that this ability is linked to delayed differentiation and sustained proliferation of differentiating keratinocytes. Furthermore, we demonstrate that the ability of MmuPV1 E6 to bind MAML1 is necessary for papilloma formation in experimentally infected mice. Our results, therefore, suggest that experimental MmuPV1 infection in mice will be a robust and useful experimental system to model key aspects of cutaneous HPV infection, pathogenesis and carcinogenesis.  

5.2024           Inclusion of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances AAV2-Driven Transduction of Mouse and Human Retina

Patricio, M.I., Barnard, A.R., Orlans, H.O., McClements, M.E. and Maclaren, R.E. Molecular Therapy – Nucleic Acids, 6, 198-208 (2017)   The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) has been included in the transgene cassette of adeno-associated virus (AAV) in several gene therapy clinical trials, including those for inherited retinal diseases. However, the extent to which WPRE increases transgene expression in the retina is still unclear. To address this question, AAV2 vectors containing a reporter gene with and without WPRE were initially compared in vitro and subsequently in vivo by subretinal delivery in mice. In both instances, the presence of WPRE led to significantly higher levels of transgene expression as measured by fundus fluorescence, western blot, and immunohistochemistry. The two vectors were further compared in human retinal explants derived from patients undergoing clinically indicated retinectomy, where again the presence of WPRE resulted in an enhancement of reporter gene expression. Finally, an analogous approach using a transgene currently employed in a clinical trial for choroideremia delivered similar results both in vitro and in vivo, confirming that the WPRE effect is transgene independent. Our data fully support the inclusion of WPRE in ongoing and future AAV retinal gene therapy trials, where it may allow a therapeutic effect to be achieved at an overall lower dose of vector.  

5.2025           Apelin-36 Modulates Blood Glucose and Body Weight Independently of Canonical APJ Receptor Signaling

Galon-Tilleman, H., yang, H., Bednarek, M.A., Spurlock, S.M., Paavola, K.J., Ko, B., To, C., Luo, J., Tian, H., Jermutus, L., Grimsby, J., Rondinone, C.M., Konkar, A. and Kaplan, D.D.

1. Bio. Chem., 292(5), 1925-1933 (2017)

  Apelin-36 was discovered as the endogenous ligand for the previously orphan receptor APJ. Apelin-36 has been linked to two major types of biological activities: cardiovascular (stimulation of cardiac contractility and suppression of blood pressure) and metabolic (improving glucose homeostasis and lowering body weight). It has been assumed that both of these activities are modulated through APJ. Here, we demonstrate that the metabolic activity of apelin-36 can be separated from canonical APJ activation. We developed a series of apelin-36 variants in which evolutionarily conserved residues were mutated, and evaluated their ability to modulate glucose homeostasis and body weight in chronic mouse models. We found that apelin-36(L28A) retains full metabolic activity, but is 100-fold impaired in its ability to activate APJ. In contrast to its full metabolic activity, apelin-36(L28A) lost the ability to suppress blood pressure in spontaneously hypertensive rats (SHR). We took advantage of these findings to develop a longer-acting variant of apelin-36 that could modulate glucose homeostasis without impacting blood pressure (or activating APJ). Apelin-36-[L28C(30kDa-PEG)] is 10,000-fold less potent than apelin-36 at activating the APJ receptor but retains its ability to significantly lower blood glucose and improve glucose tolerance in diet-induced obese mice. Apelin-36-[L28C(30kDa-PEG)] provides a starting point for the development of diabetes therapeutics that are devoid of the blood pressure effects associated with canonical APJ activation.  

5.2026           Protective Effects of Human and Mouse Soluble Scavenger-Like CD6 Lymphocyte Receptor in a Lethal Model of Polymicrobial Sepsis

Martinez-Florensa, M., Consuegra-Fernandez, M., Aranda, F., Armiger-Borras, N., Di Scala, M., Carrasco, E., Pachon, J., Villa, J., Gonzalez-Aseguinolaza, G. and Lozano, F. Antimicrob. Agents Chemother., 61(1), e01391-16 (2017)   Sepsis still constitutes an unmet clinical need, which could benefit from novel adjunctive strategies to conventional antibiotic therapy. The soluble form of the scavenger-like human CD6 lymphocyte receptor (shCD6) binds to key pathogenic components from Gram-positive and -negative bacteria and shows time- and dose-dependent efficacy in mouse models of monobacterial sepsis. The objective of the present work was to demonstrate the effectiveness of infusing mouse and human sCD6 by different systemic routes, either alone or as adjunctive therapy to gold standard antibiotics, in a lethal model of polymicrobial sepsis. To this end, C57BL/6 mice undergoing high-grade septic shock induced by cecal ligation and puncture (CLP; ≥90% lethality) were infused via the intraperitoneal (i.p.) or intravenous (i.v.) route with shCD6 at different doses and time points, either alone or in combination with imipenem/cilastatin (I/C) at a dose of 33 mg/kg of body weight every 8 h. Significantly reduced mortality and proinflammatory cytokine levels were observed by i.p. infusion of a single shCD6 dose (1.25 mg/kg) 1 h pre- or post-CLP. When using the i.v. route, mice survival was significantly extended by starting shCD6 infusion at later time points post-CLP (up to 6 h after CLP). Significant adjunctive effects on mouse survival were observed by i.p. or i.v. infusion of shCD6 in combination with i.p. I/C post-CLP. Similar results were obtained in mice expressing high sustained levels (5 to 10 μg/ml) of mouse sCD6 in serum by means of transduction with hepatotropic adeno-associated virus (AAV). Taken together, the data support the conserved antibacterial effects of human and mouse sCD6 and their use as adjunctive therapy in experimental models of complex and severe polymicrobial sepsis.  

5.2027           Selective molecular impairment of spontaneous neurotransmission modulates synaptic efficacy

Crawford, D.C., Ramirez, D.M.O., Trauterman, B., Monteggia, L.M. and Kavalali, E.T. Nature Communications, 8:14436 (2017)   Recent studies suggest that stimulus-evoked and spontaneous neurotransmitter release processes are mechanistically distinct. Here we targeted the non-canonical synaptic vesicle SNAREs Vps10p-tail-interactor-1a (vti1a) and vesicle-associated membrane protein 7 (VAMP7) to specifically inhibit spontaneous release events and probe whether these events signal independently of evoked release to the postsynaptic neuron. We found that loss of vti1a and VAMP7 impairs spontaneous high-frequency glutamate release and augments unitary event amplitudes by reducing postsynaptic eukaryotic elongation factor 2 kinase (eEF2K) activity subsequent to the reduction in N-methyl-D-aspartate receptor (NMDAR) activity. Presynaptic, but not postsynaptic, loss of vti1a and VAMP7 occludes NMDAR antagonist-induced synaptic potentiation in an intact circuit, confirming the role of these vesicular SNAREs in setting synaptic strength. Collectively, these results demonstrate that spontaneous neurotransmission signals independently of stimulus-evoked release and highlight its role as a key regulator of postsynaptic efficacy.  

5.2028           Spinal or supraspinal phosphorylation deficiency at the MOR C-terminus does not affect morphine tolerance in vivo

Kibaly, C., Lin, H-Y., Loh, H.H. and law, P-Y. Pharmacol. Res., 119, 153-168 (2017)   The development of tolerance to morphine, one of the most potent analgesics, in the management of chronic pain is a significant clinical problem and its mechanisms are poorly understood. Morphine exerts its pharmacological effects via the μ-opioid receptor (MOR). Tolerance is highly connected to G-protein-coupled receptors (GPCR) phosphorylation and desensitization increase. Because morphine desensitization previously has been shown to be MOR phosphorylation- and ß-arrestin2-independent (in contrast to agonists such as fentanyl), we examined the contribution of phosphorylation of the entire C-terminus to the development of antinociceptive tolerance to the partial (morphine) and full (fentanyl) MOR agonists in vivo. In MOR knockout (MORKO) mice, we delivered via lentivirus the genes encoding the wild-type MOR (WTMOR) or a phosphorylation-deficient MOR (Cterm(-S/T)MOR) in which all of the serine and threonine residues were mutated to alanine into the ventrolateral periaqueductal grey matter (vlPAG) or lumbar spinal cord (SC), structures that are involved in nociception. We compared the analgesic ED₅₀ in WTMOR- and Cterm(-S/T)MOR-expressing MORKO mice before and after morphine or fentanyl tolerance was induced. Morphine acute antinociception was partially restored in WTMOR- or Cterm(-S/T)MOR-transferred MORKO mice. Fentanyl acute antinociception was observed only in MORKO mice with the transgenes expressed in the SC. Morphine antinociceptive tolerance was not affected by expressing Cterm(-S/T)MOR in the vlPAG or SC of MORKO mice. Fentanyl-induced tolerance in MORKO mice expressing WTMOR or Cterm(-S/T)MOR, is greater than morphine-induced tolerance. Thus, MOR C-terminus phosphorylation does not appear to be critical for morphine tolerance in vivo.  

5.2029           Smac mimetics synergize with immune checkpoint inhibitors to promote tumour immunity against glioblastoma

Beug, S.T. et al Nature Communications, 8:14278 (2017)   Small-molecule inhibitor of apoptosis (IAP) antagonists, called Smac mimetic compounds (SMCs), sensitize tumours to TNF-α-induced killing while simultaneously blocking TNF-α growth-promoting activities. SMCs also regulate several immunomodulatory properties within immune cells. We report that SMCs synergize with innate immune stimulants and immune checkpoint inhibitor biologics to produce durable cures in mouse models of glioblastoma in which single agent therapy is ineffective. The complementation of activities between these classes of therapeutics is dependent on cytotoxic T-cell activity and is associated with a reduction in immunosuppressive T-cells. Notably, the synergistic effect is dependent on type I IFN and TNF-α signalling. Furthermore, our results implicate an important role for TNF-α-producing cytotoxic T-cells in mediating the anti-cancer effects of immune checkpoint inhibitors when combined with SMCs. Overall, this combinatorial approach could be highly effective in clinical application as it allows for cooperative and complimentary mechanisms in the immune cell-mediated death of cancer cells.  

5.2030           Self-complementary adeno-associated virus serotype 6 mediated knockdown of ADAMTS4 induces long-term and effective enhancement of aggrecan in degenerative human nucleus pulposus cells: A new therapeutic approach for intervertebral disc disorders

Mern, D.S., Tschugg, A., hartmann, S.and Thome, C. PloS One, 12(2), e0172182 (2017)   Inhibition of intervertebral disc (IVD) degeneration, which is often accompanied by painful inflammatory and immunopathological processes, is challenging. Current IVD gene therapeutic approaches are based on adenoviral gene delivery systems, which are limited by immune reactions to their viral proteins. Their applications in IVDs near to sensitive neural structure could provoke toxicity and immunological side-effects with neurological deficits. Self-complementary adeno-associated virus (scAAV) vectors, which do not express any viral gene and are not linked with any known disease in humans, are attractive therapeutic gene delivery vectors in degenerative IVDs. However, scAAV-based silencing of catabolic or inflammatory factor has not yet been investigated in human IVD cells. Therefore, we used scAAV6, the most suitable serotype for transduction of human nucleus pulposus (NP) cells, to knockdown the major catabolic gene (ADAMTS4) of IVD degeneration. IVD degeneration grades were determined by preoperative magnetic resonance imaging. Lumbar NP tissues of degeneration grade III were removed from 12 patients by nucleotomy. NP cells were isolated and cultured with low-glucose. Titre of recombinant scAAV6 vectors targeting ADAMTS4, transduction efficiencies, transduction units, cell viabilities and expression levels of target genes were analysed using quantitative PCR, fluorescence microscopy, fluorescence-activated cell sorting, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assays, quantitative reverse transcription PCR, western blot and enzyme-linked immunosorbent assays during 48 days of post-transduction. Transduction efficiencies between 98.2% and 37.4% and transduction units between 611 and 245 TU/cell were verified during 48 days of post-transduction (p<0.001). scAAV6-mediated knockdown of ADAMTS4 with maximum 87.7% and minimum 40.1% was confirmed on day 8 and 48 with enhanced the level of aggrecan 48.5% and 30.2% respectively (p<0.001). scAAV6-mediated knockdown of ADAMTS4 showed no impact on cell viability and expression levels of other inflammatory catabolic proteins. Thus, our results are promising and may help to design long-term and less immunogenic gene therapeutic approaches in IVD disorders, which usually need prolonged therapeutic period between weeks and months.    

5.2031           In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni

Kim, E., Koo, T., Park, S.W., Kim, D., Kim, K., Cho, H-Y., Song, D.W., Lee, K.L., Jung, M.H., Kim, S., Kim, J.H., Kim, J.H. and Kim, J-S. Nature Communications, 8:14500 (2017)   Several CRISPR-Cas9 orthologues have been used for genome editing. Here, we present the smallest Cas9 orthologue characterized to date, derived from Campylobacter jejuni (CjCas9), for efficient genome editing in vivo. After determining protospacer-adjacent motif (PAM) sequences and optimizing single-guide RNA (sgRNA) length, we package the CjCas9 gene, its sgRNA sequence, and a marker gene in an all-in-one adeno-associated virus (AAV) vector and produce the resulting virus at a high titer. CjCas9 is highly specific, cleaving only a limited number of sites in the human or mouse genome. CjCas9, delivered via AAV, induces targeted mutations at high frequencies in mouse muscle cells or retinal pigment epithelium (RPE) cells. Furthermore, CjCas9 targeted to the Vegfa or Hif1a gene in RPE cells reduces the size of laser-induced choroidal neovascularization, suggesting that in vivo genome editing with CjCas9 is a new option for the treatment of age-related macular degeneration.  

5.2032           rAAV8-733-Mediated Gene Transfer of CHIP/Stub-1 Prevents Hippocampal Neuronal Death in Experimental Brain Ischemia

Cabral-Miranda, F., Nicoloso-Simoes, E., Adao-Novaes, J., Chiodo, V., hauswirth, W.W., Linden, R., Barreto Chiarini, L. and Petrs-Silva, H. Molecular Therapy, 25(2), 392-400 (2017)   Brain ischemia is a major cause of adult disability and death, and it represents a worldwide health problem with significant economic burden for modern society. The identification of the molecular pathways activated after brain ischemia, together with efficient technologies of gene delivery to the CNS, may lead to novel treatments based on gene therapy. Recombinant adeno-associated virus (rAAV) is an effective platform for gene transfer to the CNS. Here, we used a serotype 8 rAAV bearing the Y733F mutation (rAAV8-733) to overexpress co-chaperone E3 ligase CHIP (also known as Stub-1) in rat hippocampal neurons, both in an oxygen and glucose deprivation model in vitro and in a four-vessel occlusion model of ischemia in vivo. We show that CHIP overexpression prevented neuronal degeneration in both cases and led to a decrease of both eIF2α (serine 51) and AKT (serine 473) phosphorylation, as well as reduced amounts of ubiquitinated proteins following hypoxia or ischemia. These data add to current knowledge of ischemia-related signaling in the brain and suggest that gene therapy based on the role of CHIP in proteostasis may provide a new venue for brain ischemia treatment.  

5.2033           Rescue of Hearing by Gene Delivery to Inner-Ear Hair Cells Using Exosome-Associated AAV

György, B., Sage, C., Indzhykulian, A.A. et al Molecular Therapy, 25(2), 379-391 (2017)   Adeno-associated virus (AAV) is a safe and effective vector for gene therapy for retinal disorders. Gene therapy for hearing disorders is not as advanced, in part because gene delivery to sensory hair cells of the inner ear is inefficient. Although AAV transduces the inner hair cells of the mouse cochlea, outer hair cells remain refractory to transduction. Here, we demonstrate that a vector, exosome-associated AAV (exo-AAV), is a potent carrier of transgenes to all inner ear hair cells. Exo-AAV1-GFP is more efficient than conventional AAV1-GFP, both in mouse cochlear explants in vitro and with direct cochlear injection in vivo. Exo-AAV shows no toxicity in vivo, as assayed by tests of auditory and vestibular function. Finally, exo-AAV1 gene therapy partially rescues hearing in a mouse model of hereditary deafness (lipoma HMGIC fusion partner-like 5/tetraspan membrane protein of hair cell stereocilia [Lhfpl5/Tmhs^−/−]). Exo-AAV is a powerful gene delivery system for hair cell research and may be useful for gene therapy for deafness.  

5.2034           Different Modes of Visual Integration in the Lateral Geniculate Nucleus Revealed by Single-Cell-Initiated Transsynaptic Tracing

Rompani, S.B., Müllner, F.E., Wanner, A., Zhang, C., Roth, C.N., Yonehara, K. and Roska, B. Neuron, 93, 767-776 (2017)   The thalamus receives sensory input from different circuits in the periphery. How these sensory channels are integrated at the level of single thalamic cells is not well understood. We performed targeted single-cell-initiated transsynaptic tracing to label the retinal ganglion cells that provide input to individual principal cells in the mouse lateral geniculate nucleus (LGN). We identified three modes of sensory integration by single LGN cells. In the first, 1–5 ganglion cells of mostly the same type converged from one eye, indicating a relay mode. In the second, 6–36 ganglion cells of different types converged from one eye, revealing a combination mode. In the third, up to 91 ganglion cells converged from both eyes, revealing a binocular combination mode in which functionally specialized ipsilateral inputs joined broadly distributed contralateral inputs. Thus, the LGN employs at least three modes of visual input integration, each exhibiting different degrees of specialization.  

5.2035           AAV-Nrf2 Promotes Protection and Recovery in Animal Models of Oxidative Stress

Liang, K.J., Woodward, K.T., Weaver, M.A., Gaylor, J.P., Weiss, E.R. and Samulski, R.J. Molecular Therapy, 25(3), 765-779 (2017)   NRF2 is a transcription factor that drives antioxidant gene expression in multiple organ systems. We hypothesized that Nrf2 overexpression could be therapeutically applied toward diseases in which redox homeostasis is disrupted. In this study, adeno-associated virus (AAV)-Nrf2 was tested in a mouse model of acute acetaminophen-induced liver toxicity and successfully conferred protection from hepatotoxicity, validating the vector design and early onset of NRF2-mediated protection. Furthermore, therapeutic potential of AAV-Nrf2 in chronic disease also was tested in a light-induced mouse model of age-related macular degeneration. Adult BALB/c mice were intravitreally injected with AAV-Nrf2 and subject to light damage following injection. Retinal thickness and function were monitored following light damage using optical coherence tomography and electroretinography, respectively. By 3 months post-damage, injected eyes had greater retinal thickness compared to uninjected controls. At 1 month post-damage, AAV-Nrf2 injection facilitated full functional recovery from light damage. Our results suggest a therapeutic potential for Nrf2 overexpression in acute and long-term capacities in multiple organ systems, opening up doors for combination gene therapy where replacement gene therapy requires additional therapeutic support to prevent further degeneration.  

5.2036           Purification of foamy viral particles

Spannaous, R., Miller, C., Lindemann, D. and Bodem, J. Virology, 506, 28-33 (2017)   Foamy viruses are non-pathogenic retroviruses and represent a tool for vector development. For gene therapy applications and for analyses of viral protein composition infectious particles need to be purified, which has been difficult for foamy viruses in the past. Here, we describe a novel, simple, and fast purification method for prototype foamy viruses with high purity using size exclusion and affinity chromatography. More than 99,9% of the contaminating proteins were removed. The purified viruses were used to determine the amount of the incorporated Pol protein relative to Gag. The determined Gag to Pol PR-RT ratio of 30:1 confirmed previous studies suggesting FV virions encapsidate fewer number of Pol molecules than orthoretroviruses.  

5.2037           α-Synuclein binds and sequesters PIKE-L into Lewy bodies, triggering dopaminergic cell death via AMPK hyperactivation

Kang, S.S., Zhang, Z., Liu, X., Manfredsson, F., He, L., Iuvone, M., Cao, X., Sun, Y.E., Jin, L. and Ye, K. PNAS, 114(5), 1183-1188 (2017)   The abnormal aggregation of fibrillar α-synuclein in Lewy bodies plays a critical role in the pathogenesis of Parkinson’s disease. However, the molecular mechanisms regulating α-synuclein pathological effects are incompletely understood. Here we show that α-synuclein binds phosphoinositide-3 kinase enhancer L (PIKE-L) in a phosphorylation-dependent manner and sequesters it in Lewy bodies, leading to dopaminergic cell death via AMP-activated protein kinase (AMPK) hyperactivation. α-Synuclein interacts with PIKE-L, an AMPK inhibitory binding partner, and this action is increased by S129 phosphorylation through AMPK and is decreased by Y125 phosphorylation via Src family kinase Fyn. A pleckstrin homology (PH) domain in PIKE-L directly binds α-synuclein and antagonizes its aggregation. Accordingly, PIKE-L overexpression decreases dopaminergic cell death elicited by 1-methyl-4-phenylpyridinium (MPP⁺), whereas PIKE-L knockdown elevates α-synuclein oligomerization and cell death. The overexpression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or α-synuclein induces greater dopaminergic cell loss and more severe motor defects in PIKE-KO and Fyn-KO mice than in wild-type mice, and these effects are attenuated by the expression of dominant-negative AMPK. Hence, our findings demonstrate that α-synuclein neutralizes PIKE-L’s neuroprotective actions in synucleinopathies, triggering dopaminergic neuronal death by hyperactivating AMPK.  

5.2038           In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus

Dai, X., Li, Z.,, Lai, M., Shu, S., Du, Y., Zhou, Z.H. and Sun, R. Nature 541, 112-116 (2017)   Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid^{1, 2, 3}, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome^{4, 5, 6, 7}; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host ‘sex pilus’ (F-pilus)⁸; it was the first fully sequenced organism⁹ and is a model system for studies of translational gene regulation^{10, 11}, RNA–protein interactions^{12, 13, 14}, and RNA virus assembly^{15, 16, 17}. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice^{18, 19}. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection⁸, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem–loops, and identified three conserved motifs of RNA–coat protein interactions among 15 of these stem–loops with diverse sequences. The stem–loop at the 3′ end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome–capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA–capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.  

5.2039           Trafficking of adeno-associated virus vectors across a model of the blood–brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells

Merkel, S.F., Andrews, A.M., Lutton, E.M., Mu, D., Hudry, E., Hyman, B.T., Maguire, C.A. and Ramirez, S.H.

1. Neurochem., 140(2), 216-230 (2017)

  Developing therapies for central nervous system (CNS) diseases is exceedingly difficult because of the blood–brain barrier (BBB). Notably, emerging technologies may provide promising new options for the treatment of CNS disorders. Adeno-associated virus serotype 9 (AAV9) has been shown to transduce cells in the CNS following intravascular administration in rodents, cats, pigs, and non-human primates. These results suggest that AAV9 is capable of crossing the BBB. However, mechanisms that govern AAV9 transendothelial trafficking at the BBB remain unknown. Furthermore, possibilities that AAV9 may transduce brain endothelial cells or affect BBB integrity still require investigation. Using primary human brain microvascular endothelial cells as a model of the human BBB, we performed transduction and transendothelial trafficking assays comparing AAV9 to AAV2, a serotype that does not cross the BBB or transduce endothelial cells effectively in vivo. Results of our in vitro studies indicate that AAV9 penetrates brain microvascular endothelial cells barriers more effectively than AAV2, but has reduced transduction efficiency. In addition, our data suggest that (i) AAV9 penetrates endothelial barriers through an active, cell-mediated process, and (ii) AAV9 fails to disrupt indicators of BBB integrity such as transendothelial electrical resistance, tight junction protein expression/localization, and inflammatory activation status. Overall, this report shows how human brain endothelial cells configured in BBB models can be utilized for evaluating transendothelial movement and transduction kinetics of various AAV capsids. Importantly, the use of a human in vitro BBB model can provide import insight into the possible effects that candidate AVV gene therapy vectors may have on the status of BBB integrity.  

5.2040           Spinal cord stimulation improves forelimb use in an alpha-synuclein animal model of Parkinson's disease

Brys, I., Bobela, W., Schneider, B.L., Aebischer, P and Fuentes, R. Int. J. Neurosci., 127(1), 28-36 (2017)   Neuromodulation by spinal cord stimulation has been proposed as a symptomatic treatment for Parkinson's disease. We tested the chronic effects of spinal cord stimulation in a progressive model of Parkinson's based on overexpression of alpha-synuclein in the substantia nigra. Adult Sprague Dawley rats received unilateral injections of adeno-associated virus serotype 6 (AAV6) in the substantia nigra to express alpha-synuclein. Locomotion and forepaw use of the rats were evaluated during the next 10 weeks. Starting on week 6, a group of AAV6-injected rats received spinal cord stimulation once a week. At the end of the experiment, tyrosine hydroxylase and alpha-synuclein immunostaining were performed. Rats with unilateral alpha-synuclein expression showed a significant decrease in the use of the contralateral forepaw, which was mildly but significantly reverted by spinal cord stimulation applied once a week from the 6th to the 10th week after the AAV6 injection. Long-term spinal cord stimulation proved to be effective to suppress or delay motor symptoms in a sustained and progressive model of Parkinson's and might become an alternative, less invasive neuromodulation option to treat this disease.  

5.2041           Efficient Gene Delivery and Expression in Pancreas and Pancreatic Tumors by Capsid-Optimized AAV8 Vectors

Chen, M., Maeng, K., Nawab, A., francois, R.A., Bray, J.K., Reinhard, M.K., Boye, S.L., Hauswirth, W.W., Kaye, F.J., Aslanidi, G., Srivastava, A. and Zajac-Kaye, M. Human Gene Therapy Methods, 28(1), 49-59 (2017)   Despite efforts to use adeno-associated viral (AAV) vector–mediated gene therapy for treatment of pancreatic ductal adenocarcinoma (PDAC), transduction efficiency remains a limiting factor and thus improvement of AAV delivery would significantly facilitate the treatment of this malignancy. Site-directed mutagenesis of specific tyrosine (Y) residues to phenylalanine (F) on the surface of various AAV serotype capsids has been reported as a method for enhancing gene transfer efficiencies. In the present studies, we determine whether Y-to-F mutations could also enhance AAV8 gene transfer in the pancreas to facilitate gene therapy for PDAC. Three different Y-to-F mutant vectors (a single-mutant, Y733F; a double-mutant, Y447F+Y733F; and a triple-mutant, Y275F+Y447F+Y733F) and wild-type AAV8 (WT-AAV8) were administered by intraperitoneal or tail-vein routes to Kras^G12D+/−, Kras^G12D+/−/Pten^+/−, and wild-type mice. The transduction efficiency of these vectors expressing the mCherry reporter gene was evaluated 2 weeks post administration in pancreas or PDAC and correlated with viral genome copy numbers. Our comparative and quantitative analyses of the transduction profiles demonstrated that the Y-to-F double-mutant exhibited the highest mCherry expression in pancreatic tissues (range 45–70%) compared with WT-AAV8 (7%; p < 0.01). We also detected a 7-fold higher level of vector genome copy numbers in normal pancreas following transduction with the double-mutant AAV8 compared with WT-AAV8 (10,285 vs. 1,500 vector copies/μg DNA respectively, p < 0.05). In addition, we observed that intraperitoneal injection of the double-mutant AAV8 led to a 15-fold enhanced transduction efficiency as compared to WT-AAV8 in mouse PDAC, with a corresponding ∼14-fold increase in vector genome copy numbers (26,575 vs. 2,165 copies/μg DNA respectively, p < 0.05). These findings indicate that the Y447+Y733F-AAV8 leads to a significant enhancement of transduction efficiency in both normal and malignant pancreatic tissues, suggesting the potential use of this vector in targeting pancreatic diseases in general, and PDAC in particular.  

5.2042           AAV Delivery of Endothelin-1 shRNA Attenuates Cold-Induced Hypertension

Chen, P.G-F. and Sun, Z. Human Gene Therapy, 28(2), 190-199 (2017)   Cold temperatures are associated with increased prevalence of hypertension. Cold exposure increases endothelin-1 (ET1) production. The purpose of this study is to determine whether upregulation of ET1 contributes to cold-induced hypertension (CIH). In vivo RNAi silencing of the ET1 gene was achieved by adeno-associated virus 2 (AAV2) delivery of ET1 short-hairpin small interfering RNA (ET1-shRNA). Four groups of male rats were used. Three groups were given AAV.ET1-shRNA, AAV.SC-shRNA (scrambled shRNA), and phosphate-buffered saline (PBS), respectively, before exposure to a moderately cold environment (6.7 ± 2°C), while the last group was given PBS and kept at room temperature (warm, 24 ± 2°C) and served as a control. We found that systolic blood pressure of the PBS-treated and SC-shRNA–treated groups increased significantly within 2 weeks of exposure to cold, reached a peak level (145 ± 4.8 mmHg) by 6 weeks, and remained elevated thereafter. By contrast, blood pressure of the ET1-shRNA-treated group did not increase, suggesting that silencing of ET1 prevented the development of CIH. Animals were euthanized after 10 weeks of exposure to cold. Cold exposure significantly increased the left ventricle (LV) surface area and LV weight in cold-exposed rats, suggesting LV hypertrophy. Superoxide production in the heart was increased by cold exposure. Interestingly, ET1-shRNA prevented cold-induced superoxide production and cardiac hypertrophy. ELISA assay indicated that ET1-shRNA abolished the cold-induced upregulation of ET1 levels, indicating effective silencing of ET1. In conclusion, upregulation of ET1 plays a critical role in the pathogenesis of CIH and cardiac hypertrophy. AAV delivery of ET1-shRNA is an effective therapeutic strategy for cold-related cardiovascular disease.  

5.2043           Toxic effects of human and rodent variants of alpha-synuclein in vivo

Landeck, N., Buck, K. and Kirik, D. Eur. J. Neurosci., 45(4), 536-547 (2017)   In Parkinson's disease, abnormal alpha-synuclein (asyn) accumulation leads to the formation of soluble oligomeric species thought to be toxic to cells as well as intraneuronal inclusions. To date, the precise mechanisms leading to aggregation of asyn in the brain is not well-understood. Previous studies in yeast, drosophila, and transgenic mice suggested that a non-A beta component depleted version of human asyn [h-asyn(D70-83)] or human beta-synuclein (h-bsyn), naturally lacking this centrally located hydrophobic region, are less prone to form aggregates in vitro and are expected to be less toxic compared to h-asyn in vivo, although not all experimental studies unequivocally support the latter view. To address this outstanding issue, we directly compared the neurotoxicity of human asyn against that of h-asyn(D70-83), h-bsyn as well as rat asyn using an adeno-associated viral vector to express these proteins in a dose-response study where the vector load was varied over two orders of magnitude. By quantifying the neurodegeneration of rat substantia nigra dopamine neurons here we show that h-asyn, h-bsyn, and h-asyn(D70-83) display comparable neurotoxicity across the vector doses tested. On the other hand, rat asyn and GFP control vectors displayed a different profile, where no detectable neurodegeneration was seen except at the highest vector titer. Thus, the two main conclusions of our study are that (i) deletion of the central hydrophobic region in h-asyn is not sufficient to alter its neurotoxic properties and (ii) expression of the widely used GFP control protein can cause measurable neurodegeneration at high titers.  

5.2044           miR-15b mediates oxaliplatin-induced chronic neuropathic pain through BACE1 down-regulation

Ito, N., Sakai, A., Miyaki, N., maruyama, M., Iwasaki, H., Miyake, K., Okada, T., Sakamoto, A. and Suzuki, H. Br. J. Pharmacol., 174(5), 386-395 (2017)   Background and Purpose Although oxaliplatin is an effective anti-cancer platinum compound, it can cause painful chronic neuropathy, and its molecular mechanisms are poorly understood. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression in a sequence-specific manner. Although miRNAs have been increasingly recognized as important modulators in a variety of pain conditions, their involvement in chemotherapy-induced neuropathic pain is unknown. Experimental Approach Oxaliplatin-induced chronic neuropathic pain was induced in rats by i.p. injections of oxaliplatin (2 mg·kg⁻¹) for five consecutive days. The expression levels of miR-15b and β-site amyloid precursor protein-cleaving enzyme 1 (BACE1 also known as β-secretase 1) were examined in the dorsal root ganglion (DRG). To examine the function of miR-15b, an adeno-associated viral vector encoding miR-15b was injected into the DRG in vivo. Key Results Among the miRNAs examined in the DRG in the late phase of oxaliplatin-induced neuropathic pain, miR-15b was most robustly increased. Our in vitro assay results determined that BACE1 was a target of miR-15b. BACE1 and miR-15b were co-expressed in putative myelinated and unmyelinated DRG neurons. Overexpression of miR-15b in DRG neurons caused mechanical allodynia in association with reduced expression of BACE1. Consistent with these results, a BACE1 inhibitor dose-dependently induced significant mechanical allodynia. Conclusions and Implications These findings suggest that miR-15b contributes to oxaliplatin-induced chronic neuropathic pain at least in part through the down-regulation of BACE1.  

5.2045           Empty Adeno-Associated Virus Capsids: Contaminant or Natural Decoy?

Flotte, T.R. Human Gene Therapy, 28(2), 147-148 (2017)   No abstract available.  

5.2046           Physicochemical characterization and immunological properties of Pichia pastoris based HPV16L1 and 18L1 virus like particles

Gupta, G., Glueck, R. and Rishi, N. Biologicals, 46, 11-22 82017)   There continues to be an urgent need for cost-effective prophylaxis for HPV-associated cancers in socio-economically underdeveloped nations. Presently HPV vaccines, which are commercially available, are adjuvanted virus-like particles (VLPs) expressed from various recombinant expression systems. They have been characterized by different methods as safe, pure, and potent HPV vaccine antigens. We cloned and expressed L1 proteins of HPV16 & 18 in Pichia pastoris and tested their immunogenicity. We observed that HPVL1 proteins (16L1 and 18L1) are expressed in Pichia pastoris at high levels. Critical physicochemical parameters of these HPV recombinant L1 proteins were characterized by SDS PAGE, western blotting, peptide mapping, glycosylation pattern, mass spectrometry, host cell DNA and protein analysis, electron microscopy, and immunogenicity analysis. These data establish a blueprint of HPV recombinant protein antigens for standardizing & developing an alternative high-quality, cost-effective vaccine for HPV as well as similar recombinant protein-based vaccines.  

5.2047           Scalable Production of AAV Vectors

Szarek, E. and Hung, J. Genetic Engineering & Biotech. News, 37(2), 22-23 (2017)   Vectors derived from adeno-associated virus (AAV) provide promising gene delivery vehicles that can be used effectively in large-scale productions for preclinical target identification/validation studies, or used in large animal models and clinical trials of human gene therapy. Why is AAV one of the most promising viral gene transfer vectors? Notably, recombinant adeno-associated virus (rAAV) vectors come in different serotypes (AAV 1–9), each with different tissue tropisms. rAAV provides a high rate of gene transfer efficiency, long-term gene expression, and natural replication deficiency. It is nonpathogenic and does not have the capability of altering biological properties upon integration of the host cell. However, achieving preclinical efficacy testing, especially in large animal models and toxicology studies, requires vector quantities that simply cannot be produced in a laboratory setting or in most research-grade vector core facilities. Current methods for transfection require use of adherent HEK 293 cell cultures, expanded by preparing multiple culture plates. Ideally, a single large-scale suspension culture would be a replacement for multiple culture plates. In this tutorial, we examine some of the currently available schemes used in generating rAAV from suspension cultures, and describe what it takes to achieve scalable rAAV production.  

5.2048           Pre-clinical Assessment of C134, a Chimeric Oncolytic Herpes Simplex Virus, in Mice and Non-human Primates

Cassady, K.A., Bauer, D.F., Roth, J., Chambers, M.R., Shoeb, T., Coleman, J., Prichard, M., Gillespie, G.Y. and Markert, J.M. Molecular Therapy:Oncolytics, 5, 1-10 (2017)   Oncolytic herpes simplex virus (oHSV) type I constructs are investigational anti-neoplastic agents for a variety of malignancies, including malignant glioma. Clinical trials to date have supported the safety of these agents even when directly administered in the CNS. Traditional pre-clinical US Food and Drug Administration (FDA) toxicity studies for these agents have included the use of two species, generally including murine and primate studies. Recently, the FDA has decreased its requirement of non-human primates as an animal model for ethical reasons, especially for established viral systems where there are good alternative model systems. Here we present data demonstrating the safety of C134, a chimeric oHSV construct, in CBA mice as well as in a limited number of the HSV-sensitive non-human primate Aotus nancymaae as a proposed agent for clinical trials. These data, along with the previously conducted clinical trials of oHSV constructs, support the use of the CBA mouse model as sufficient for the pre-clinical toxicity studies of this agent. We summarize our experience with different HSV recombinants and differences between them using multiple assays to assess neurovirulence, as well as our experience with C134 in a limited number of A. nancymaae.  

5.2049           The mechanism of sirtuin 2–mediated exacerbation of alpha-synuclein toxicity in models of Parkinson disease

De Oliveira, R.M. et al PloS Biology, 15(3), e000374 (2017)   Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation and toxicity, demonstrating the potential therapeutic value of sirtuin 2 inhibition in synucleinopathies.  

5.2050           Tyrosine-mutated AAV2-mediated shRNA silencing of PTEN promotes axon regeneration of adult optic nerve

Huang, Z., Hu, Z., Xie, P. and Liu, Q. PloS One, 12(3), e0174096 (2017)   Activating PI3K/AKT/mTOR signaling pathway via deleting phosphatase and tensin homolog (PTEN) has been confirmed to enhance intrinsic growth capacity of neurons to facilitate the axons regeneration of central nervous system after injury. Considering conditional gene deletion is currently not available in clinical practice, we exploited capsid residue tyrosine 444 to phenylalanine mutated single-stranded adeno-associated virus serotype 2 (AAV2) as a vector delivering short hairpin RNA to silence PTEN to promote retinal ganglion cells (RGCs) survival and axons regeneration in adult rat optic nerve axotomy paradigm. We found that mutant AAV2 displayed higher infection efficiency to RGCs and Müller cells by intravitreal injection, mediated PTEN suppression, resulted in much more RGCs survival and more robust axons regeneration compared with wild type AAV2, due to the different extent of the mTOR complex-1 activation and glutamate aspartate transporter (GLAST) regulation. These results suggest that high efficiency AAV2-mediated PTEN knockdown represents a practicable therapeutic strategy for optic neuropathy.  

5.2051           A Novel Inhibitor IDPP Interferes with Entry and Egress of HCV by Targeting Glycoprotein E1 in a Genotype-Specific Manner

Lee, M., Yang, J., Jo, E., Lee, J-Y., Kim, H-Y., Bartenschlager, R., Shin, E-C., Bae, Y-S. and Windisch, M.P. Scientific Reports, 7:44676 (2017)   Despite recent advances in curing chronic hepatitis C (CHC), the high economic burden to therapy, viral drug resistance, difficult to treat hepatitis C virus (HCV) genotypes and patient groups are still of concern. To address this unmet medical needs, we devised strategies to identify novel viral interventions through target-free high-throughput screening of small molecules utilizing a phenotypic-based HCV infection assay. Thereby, a very potent (EC50 46 ± 26 pM) iminodipyridinopyrimidine (IDPP) drug candidate was selected, and confirmed in primary human hepatocytes (EC50 0.5 nM). IDPP mainly targets a post-attachment step of HCV without affecting endosomal acidification, prevents the secretion of infectious particles and viral cell-to-cell spread. The putative molecular target of IDPP is glycoprotein E1, as revealed by selection for viral drug resistance (Gly-257-Arg). IDPP was synergistic in combination with FDA-approved HCV drugs and inhibited pre-existing resistant HCV strains induced by today’s therapies. Interestingly, IDPP exclusively inhibited HCV genotype 2. However, we identified the genotype-specificity determining region in E1 and generated HCV genotype 1 susceptible to IDPP by changing one amino acid in E1 (Gln-257-Gly). Together, our results indicate an opportunity to provide an alternative treatment option for CHC and will shed light on the poorly understood function of HCV glycoprotein E1.  

5.2052           Identification of Novel Functions for Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly

Haddad, J.G., Rouille, Y., hanoulle, X., Descamps, V., Hamze, M., Dabboussi, F., Baumert, T.F., Duverlie, G., Lavie, M. and Dubuisson, J.

1. Virol., 91(8), e00048-17 (2017)

  Hepatitis C virus (HCV) envelope glycoprotein complex is composed of E1 and E2 subunits. E2 is the receptor-binding protein as well as the major target of neutralizing antibodies, whereas the functions of E1 remain poorly defined. Here, we took advantage of the recently published structure of the N-terminal region of the E1 ectodomain to interrogate the functions of this glycoprotein by mutating residues within this 79-amino-acid region in the context of an infectious clone. The phenotypes of the mutants were characterized to determine the effects of the mutations on virus entry, replication, and assembly. Furthermore, biochemical approaches were also used to characterize the folding and assembly of E1E2 heterodimers. Thirteen out of 19 mutations led to viral attenuation or inactivation. Interestingly, two attenuated mutants, T213A and I262A, were less dependent on claudin-1 for cellular entry in Huh-7 cells. Instead, these viruses relied on claudin-6, indicating a shift in receptor dependence for these two mutants in the target cell line. An unexpected phenotype was also observed for mutant D263A which was no longer infectious but still showed a good level of core protein secretion. Furthermore, genomic RNA was absent from these noninfectious viral particles, indicating that the D263A mutation leads to the assembly and release of viral particles devoid of genomic RNA. Finally, a change in subcellular colocalization between HCV RNA and E1 was observed for the D263A mutant. This unique observation highlights for the first time cross talk between HCV glycoprotein E1 and the genomic RNA during HCV morphogenesis.  

5.2053           PABPN1 gene therapy for oculopharyngeal muscular dystrophy

Malerba, A. et al Nature Communications, 8:14848 (2017)   Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset muscle disorder characterized by ptosis, swallowing difficulties, proximal limb weakness and nuclear aggregates in skeletal muscles. OPMD is caused by a trinucleotide repeat expansion in the PABPN1 gene that results in an N-terminal expanded polyalanine tract in polyA-binding protein nuclear 1 (PABPN1). Here we show that the treatment of a mouse model of OPMD with an adeno-associated virus-based gene therapy combining complete knockdown of endogenous PABPN1 and its replacement by a wild-type PABPN1 substantially reduces the amount of insoluble aggregates, decreases muscle fibrosis, reverts muscle strength to the level of healthy muscles and normalizes the muscle transcriptome. The efficacy of the combined treatment is further confirmed in cells derived from OPMD patients. These results pave the way towards a gene replacement approach for OPMD treatment.  

5.2054           Exosome-associated AAV2 vector mediates robust gene delivery into the murine retina upon intravitreal injection

Wassmer, S.J., Carvalho, L.S., György, B., Vandenberghe, L.H. and Maguire, C.A. Scientific Reports, 7:45329 (2017)   Widespread gene transfer to the retina is challenging as it requires vector systems to overcome physical and biochemical barriers to enter and diffuse throughout retinal tissue. We investigated whether exosome-associated adeno-associated virus, (exo-AAV) enabled broad retinal targeting following intravitreal (IVT) injection, as exosomes have been shown to traverse biological barriers and mediate widespread distribution upon systemic injection. We packaged an AAV genome encoding green fluorescent protein (GFP) into conventional AAV2 and exo-AAV2 vectors. Vectors were IVT injected into the eyes of adult mice. GFP expression was noninvasively monitored by fundus imaging and retinal expression was analyzed 4 weeks post-injection by qRT-PCR and histology. Exo-AAV2 outperformed conventional AAV2 in GFP expression based on fundus image analysis and qRT-PCR. Exo-AAV2 demonstrated deeper penetration in the retina, efficiently reaching the inner nuclear and outer plexiform, and to a lesser extent the outer nuclear layer. Cell targets were ganglion cells, bipolar cells, Müller cells, and photoreceptors. Exo-AAV2 serves as a robust gene delivery tool for murine retina, and the simplicity of production and isolation should make it widely applicable to basic research of the eye.  

5.2055           Cochlear gene therapy with ancestral AAV in adult mice: complete transduction of inner hair cells without cochlear dysfunction

Suzuki, J., Hashimoto, K., Xiao, R., Vandenberghe, L.H. and Liberman, M.C. Scientific Reports, 7:45524 (2017)   The use of viral vectors for inner ear gene therapy is receiving increased attention for treatment of genetic hearing disorders. Most animal studies to date have injected viral suspensions into neonatal ears, via the round window membrane. Achieving transduction of hair cells, or sensory neurons, throughout the cochlea has proven difficult, and no studies have been able to efficiently transduce sensory cells in adult ears while maintaining normal cochlear function. Here, we show, for the first time, successful transduction of all inner hair cells and the majority of outer hair cells in an adult cochlea via virus injection into the posterior semicircular canal. We used a “designer” AAV, AAV2/Anc80L65, in which the main capsid proteins approximate the ancestral sequence state of AAV1, 2, 8, and 9. Our injections also transduced ~10% of spiral ganglion cells and a much larger fraction of their satellite cells. In the vestibular sensory epithelia, the virus transduced large numbers of hair cells and virtually all the supporting cells, along with close to half of the vestibular ganglion cells. We conclude that this viral vector and this delivery route hold great promise for gene therapy applications in both cochlear and vestibular sense organs.  

5.2056           The MVMp P4 promoter is a host cell-type range determinant in vivo

Meir, C., Mincberg, M., Rostovsky, I., Tal, S., Vollmers, E.M., Levi, A., Tattersall, P. And Davis, C. Virology, 506, 141-151 (2017)   The protoparvovirus early promoters, e.g. P4 of Minute Virus of Mice (MVM), play a critical role during infection. Initial P4 activity depends on the host transcription machinery only. Since this is cell-type dependent, it is hypothesized that P4 is a host cell-type range determinant. Yet host range determinants have mapped mostly to capsid, never P4. Here we test the hypothesis using the mouse embryo as a model system. Disruption of the CRE element of P4 drastically decreased infection levels without altering range. However, when we swapped promoter elements of MVM P4 with those from equivalent regions of the closely related H1 virus, we observed elimination of infection in fibroblasts and chondrocytes and the acquisition of infection in skeletal muscle. We conclude that P4 is a host range determinant and a target for modifying the productive infection potential of the virus - an important consideration in adapting these viruses for oncotherapy.      

5.2057           Sensitive luminescent reporter viruses reveal appreciable release of hepatitis C virus NS5A protein into the extracellular environment

Eyre, N.S., Aloia, A.L., Joyce, M.A., Chulanetra, M., Tyrrell, D.L. and Beard, M.R. Virology, 507, 20-31 (2017)   The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis.  

5.2058           Human polyomavirus 6 and 7 are associated with pruritic and dyskeratotic dermatoses

Nguyen, K.D., Lee, E.E., Yue, Y., Stork, J., Pock, L., North, J.P., Vandergriff, T., Cockerell, C., Hosler, G.A., Pastrana, D.V., Buck, C.B. and Wang, R.C.

1. Am. Acad. Dermatol., 76(5), 932-940e3 (2017)

  Background Human polyomavirus (HPyV)6 and HPyV7 are shed chronically from human skin. HPyV7, but not HPyV6, has been linked to a pruritic skin eruption of immunosuppression. Objective We determined whether biopsy specimens showing a characteristic pattern of dyskeratosis and parakeratosis might be associated with polyomavirus infection. Methods We screened biopsy specimens showing “peacock plumage” histology by polymerase chain reaction for HPyVs. Cases positive for HPyV6 or HPyV7 were then analyzed by immunohistochemistry, electron microscopy, immunofluorescence, quantitative polymerase chain reaction, and complete sequencing, including unbiased, next-generation sequencing. Results We identified 3 additional cases of HPyV6 or HPyV7 skin infections. Expression of T antigen and viral capsid was abundant in lesional skin. Dual immunofluorescence staining experiments confirmed that HPyV7 primarily infects keratinocytes. High viral loads in lesional skin compared with normal-appearing skin and the identification of intact virions by both electron microscopy and next-generation sequencing support a role for active viral infections in these skin diseases. Limitation This was a small case series of archived materials. Conclusion We have found that HPyV6 and HPyV7 are associated with rare, pruritic skin eruptions with a distinctive histologic pattern and describe this entity as “HPyV6- and HPyV7-associated pruritic and dyskeratotic dermatoses.”  

5.2059           Intranasal immunization of pigs with porcine reproductive and respiratory syndrome virus-like particles plus 2′, 3′-cGAMP VacciGrade™ adjuvant exacerbates viremia after virus challenge

Van Noort, A., Nelsen, A., Pillatzki, A.E., Diel, D.G., Li, F., Nelson, E. and Wang, X. Virol. J., 14:76 (2017)   Background Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure in pregnant sows and acute respiratory disease in young pigs. It is a leading infectious agent of swine respiratory complex, which has significant negative economic impact on the swine industry. Commercial markets currently offer both live attenuated and killed vaccines; however, increasing controversy exists about their efficacy providing complete protection. Virus-like particles (VLPs) possess many desirable features of a potent vaccine candidate and have been proven to be highly immunogenic and protective against virus infections. Here we explored the efficacy of PRRSV VLPs together with the use of a novel 2′, 3′-cGAMP VacciGrade™ adjuvant. Methods Animals were immunized twice intranasally with phosphate buffered saline (PBS), PRRSV VLPs, or PRRSV VLPs plus 2′, 3′-cGAMP VacciGrade™ at 2 weeks apart. Animals were challenged with PRRSV-23983 at 2 weeks post the second immunization. PRRSV specific antibody response and cytokines were measured. Viremia, clinical signs, and histological lesions were evaluated. Results PRRSV N protein specific antibody was detected in all animals at day 10 after challenge, but no significant difference was observed among the vaccinated and control groups. Surprisingly, a significantly higher viremia was observed in the VLPs and VLPs plus the adjuvant groups compared to the control group. The increased viremia is correlated with a higher interferon-α induction in the serum of the VLPs and the VLPs plus the adjuvant groups. Conclusions Intranasal immunizations of pigs with PRRSV VLPs and VLPs plus the 2′, 3′-cGAMP VacciGrade™ adjuvant exacerbates viremia. A higher level of interferon-α production, but not interferon-γ and IL-10, is correlated with enhanced virus replication. Overall, PRRSV VLPs and PRRSV VLPs plus the adjuvant fail to provide protection against PRRSV challenge. Different dose of VLPs and alternative route of vaccination such as intramuscular injection should be explored in the future studies to fully assess the feasibility of such a vaccine platform for PRRSV control and prevention.  

5.2060           Transduction Profile of the Marmoset Central Nervous System Using Adeno-Associated Virus Serotype 9 Vectors

Matsuzaki, Y., Konno, A., Mukai, R., Hondo, F., Hirato, M., Yoshimoto, Y. and Hirai, H. Mol. Neurobiol., 54(3), 1745-1758 (2017)   The common marmoset is a small New World primate that has attracted remarkable attention as a potential experimental animal link between rodents and humans. Adeno-associated virus (AAV) vector-mediated expression of a disease-causing gene or a potential therapeutic gene in the brain may allow the construction of a marmoset model of a brain disorder or an exploration of the possibility of gene therapy. To gain more insights into AAV vector-mediated transduction profiles in the marmoset central nervous system (CNS), we delivered AAV serotype 9 (AAV9) vectors expressing GFP to the cisterna magna or the cerebellar cortex. Intracisternally injected AAV9 vectors expanded in the CNS according to the cerebrospinal fluid (CSF) flow, by retrograde transport through neuronal axons or via intermediary transcytosis, resulting in diffuse and global transduction within the CNS. In contrast, cerebellar parenchymal injection intensely transduced a more limited area, including the cerebellar cortex and cerebellar afferents, such as neurons of the pontine nuclei, vestibular nucleus and inferior olivary nucleus. In the spinal cord, both administration routes resulted in labeling of the dorsal column and spinocerebellar tracts, presumably by retrograde transport from the medulla oblongata and cerebellum, respectively. Motor neurons and dorsal root ganglia were also transduced, possibly by diffusion of the vector down the subarachnoid space along the cord. Thus, these two administration routes led to distinct transduction patterns in the marmoset CNS, which could be utilized to generate different disease animal models and to deliver therapeutic genes for the treatment of diseases affecting distinct brain areas.  

5.2061           A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear

Landegger, L.D., Pan, B., Askew, C., Wassmer, S.J., Gluck, S.D., Galvin, A., Taylor, R., Forge, A., Stankovic, K.M., Holt, J.R. and Vandenberghe, L.H. Nature Biotech.,35(3), 280-284 (2017)   Efforts to develop gene therapies for hearing loss have been hampered by the lack of safe, efficient, and clinically relevant delivery modalities^{1, 2}. Here we demonstrate the safety and efficiency of Anc80L65, a rationally designed synthetic vector³, for transgene delivery to the mouse cochlea. Ex vivo transduction of mouse organotypic explants identified Anc80L65 from a set of other adeno-associated virus (AAV) vectors as a potent vector for the cochlear cell targets. Round window membrane injection resulted in highly efficient transduction of inner and outer hair cells in mice, a substantial improvement over conventional AAV vectors. Anc80L65 round window injection was well tolerated, as indicated by sensory cell function, hearing and vestibular function, and immunologic parameters. The ability of Anc80L65 to target outer hair cells at high rates, a requirement for restoration of complex auditory function, may enable future gene therapies for hearing and balance disorders.  

5.2062           Cutting Edge: Innate Immune Augmenting Vesicular Stomatitis Virus Expressing Zika Virus Proteins Confers Protective Immunity

Betancourt, D., de Queiroz, N.M.G.P., Xia, T., Ahn, J. and Baarber, G.N.

1. Immunol., 198(8), 3023-3028 (2017)

  Zika virus (ZIKV) has become a serious public health concern because of its link to brain damage in developing human fetuses. Recombinant vesicular stomatitis virus (rVSV) was shown to be a highly effective and safe vector for the delivery of foreign immunogens for vaccine purposes. In this study, we generated rVSVs (wild-type and attenuated VSV with mutated matrix protein [VSVm] versions) that express either the full length ZIKV envelope protein (ZENV) alone or include the ZENV precursor to the membrane protein upstream of the envelope protein, and our rVSV-ZIKV constructs showed efficient immunogenicity in murine models. We also demonstrated maternal protective immunity in challenged newborn mice born to female mice vaccinated with VSVm-ZENV containing the transmembrane domain. Our data indicate that rVSVm may be a suitable strategy for the design of effective vaccines against ZIKV.  

5.2063           Probing the Link among Genomic Cargo, Contact Mechanics, and Nanoindentation in Recombinant Adeno-Associated Virus 2

Zeng, C., Moller-Tank, S., Asokan, A. And Dragnea, B.

1. Phys. Chem. B., 121(8), 1843-1853 (2017)

  Recombinant adeno-associated virus (AAV) is a promising gene therapy vector. To make progress in this direction, the relationship between the characteristics of the genomic cargo and the capsid stability must be understood in detail. The goal of this study is to determine the role of the packaged vector genome in the response of AAV particles to mechanical compression and adhesion to a substrate. Specifically, we used atomic force microscopy to compare the mechanical properties of empty AAV serotype 2 (AAV2) capsids and AAV2 vectors packaging single-stranded DNA or self-complementary DNA. We found that all species underwent partial deformation upon adsorption from buffer on an atomically flat graphite surface. Upon adsorption, a preferred orientation toward the twofold symmetry axis on the capsid, relative to the substrate, was observed. The magnitude of the bias depended on the cargo type, indicating that the interfacial properties may be influenced by cargo. All particles showed a significant relative strain before rupture. Different from interfacial interactions, which were clearly cargo-dependent, the elastic response to directional stress was largely dominated by the capsid properties. Nevertheless, small differences between particles laden with different cargo were measurable; scAAV vectors were the most resilient to external compression. We also show how elastic constant and rupture force data sets can be analyzed according a multivariate conditional probability approach to determine the genome content on the basis of a database of mechanical properties acquired from nanoindentation assays. Implications for understanding how recombinant AAV capsid–genome interactions can affect vector stability and effectiveness of gene therapy applications are discussed.  

5.2064           Hepatitis C virus infection propagates through interactions between Syndecan-1 and CD81 and impacts the hepatocyte glycocalyx

Grigorov, B., Reungoat, E., dit Maurin, A.G., Varbanov, M., Blaising, J., Michelet, M., Manuel, R., Parent, R., Bartosch, B., Zoulim, F., Ruggiero, F. and Pecheur, E-I. Cell. Microbiol., 19, e12711 (2017)   The hepatitis C virus (HCV) infects hepatocytes after binding to heparan sulfate proteoglycans, in particular Syndecan-1, followed by recognition of the tetraspanin CD81 and other receptors. Heparan sulfate proteoglycans are found in a specific microenvironment coating the hepatocyte surface called the glycocalyx and are receptors for extracellular matrix proteins, cytokines, growth factors, lipoproteins, and infectious agents. We investigated the mutual influence of HCV infection on the glycocalyx and revealed new links between Syndecan-1 and CD81. Hepatocyte infection by HCV was inhibited after knocking down Syndecan-1 or Xylosyltransferase 2, a key enzyme of Syndecan-1 biosynthesis. Simultaneous knockdown of Syndecan-1 and CD81 strongly inhibited infection, suggesting their cooperative action. At early infection stages, Syndecan-1 and virions colocalized at the plasma membrane and were internalized in endosomes. Direct interactions between Syndecan-1 and CD81 were revealed in primary and transformed hepatocytes by immunoprecipitation and proximity ligation assays. Expression of Syndecan-1 and Xylosyltransferase 2 was altered within days post-infection, and the remaining Syndecan-1 pool colocalized poorly with CD81. The data indicate a profound reshuffling of the hepatocyte glycocalyx during HCV infection, possibly required for establishing optimal conditions of viral propagation.  

5.2065           Reduced Potency and Incomplete Neutralization of Broadly Neutralizing Antibodies against Cell-to-Cell Transmission of HIV-1 with Transmitted Founder Envs

Li, H., Zony, C., Chen, P. and Chen, B.K.

1. Virol., 91(9), e02425-16 (2017)

  Broadly neutralizing antibodies (bNAbs) have been isolated from HIV-1 patients and can potently block infection of a wide spectrum of HIV-1 subtypes. These antibodies define common epitopes shared by many viral isolates. While bNAbs potently antagonize infection with cell-free virus, inhibition of HIV-1 transmission from infected to uninfected CD4⁺ T cells through virological synapses (VS) has been found to require greater amounts of antibody. In this study, we examined two well-studied molecular clones and two transmitted/founder (T/F) clones for their sensitivities to a panel of bNAbs in cell-free and cell-to-cell infection assays. We observed resistance of cell-to-cell transmission to antibody neutralization that was reflected not only by reductions of antibody potency but also by decreases in maximum neutralization capacity relative to the levels seen with cell-free infections. BNAbs targeting different epitopes exhibited incomplete neutralization against cell-associated virus with T/F Envs, which was not observed with the cell-free form of the same virus. We further identified the membrane-proximal internal tyrosine-based sorting motif as a determinant that can affect the incomplete neutralization of these T/F clones in cell-to-cell infection. These findings indicate that the signal that affects surface expression and/or internalization of Env from the plasma membrane can modulate the presentation of neutralizing epitopes on infected cells. These results highlight that a fraction of virus can escape from high concentrations of antibody through cell-to-cell infection while remaining sensitive to neutralization in cell-free infection. The ability to fully inhibit cell-to-cell transmission may represent an important consideration in the development of antibodies for treatment or prophylaxis.  

5.2066           Targeting cell surface HIV-1 Env protein to suppress infectious virus formation

Bastian, A.R., Ang, C.G., Kamanna, K., Shaheen, F., Huang, Y-H., McFadden, K., Duffy, C., Bailey, L.D., Sundaram, R.V.K. and Chaiken, I. Virus Res., 235, 33-36 (2017)   HIV-1 Env protein is essential for host cell entry, and targeting Env remains an important antiretroviral strategy. We previously found that a peptide triazole thiol KR13 and its gold nanoparticle conjugate AuNP-KR13 directly and irreversibly inactivate the virus by targeting the Env protein, leading to virus gp120 shedding, membrane disruption and p24 capsid protein release. Here, we examined the consequences of targeting cell-surface Env with the virus inactivators. We found that both agents led to formation of non-infectious virus from transiently transfected HEK293T cells. The budded non-infectious viruses lacked Env gp120 but contained gp41. Importantly, budded virions also retained the capsid protein p24, in stark contrast to p24 leakage from viruses directly treated by these agents and arguing that the agents led to deformed viruses by transforming the cells at a stage before virus budding. We found that the Env inactivators caused gp120 shedding from the transiently transfected HEK293T cells as well as non-producer CHO-K1-gp160 cells. Additionally, AuNP-KR13 was cytotoxic against the virus-producing HEK293T and CHO-K1-gp160 cells, but not untransfected HEK293T or unmodified CHO-K1 cells. The results obtained reinforce the argument that cell-surface HIV-1 Env is metastable, as on virus particles, and provides a conformationally vulnerable target for virus suppression and infectious cell inactivation.  

5.2067           In vivo dynamics of AAV-mediated gene delivery to sensory neurons of the trigeminal ganglia

Dang, C.H., Aubert, M., De Silva Feelixge, H.S., Diem, K., Loprieno, M.A., Roychoudhury, P., Stone, D. and Jerome, K.R. Scientific Reports, 7:927 (2017)   The ability to genetically manipulate trigeminal ganglion (TG) neurons would be useful in the study of the craniofacial nervous system and latent alphaherpesvirus infections. We investigated adeno-associated virus (AAV) vectors for gene delivery to the TG after intradermal whiskerpad delivery in mice. We demonstrated that AAV vectors of serotypes 1, 7, 8, and 9 trafficked from the whiskerpad into TG neurons and expressed transgenes within cell bodies and axons of sensory neurons in all three branches of the TG. Gene expression was highest with AAV1, and steadily increased over time up to day 28. Both constitutive and neuronal-specific promoters were able to drive transgene expression in TG neurons. Levels of vector genomes in the TG increased with input dose, and multiple transgenes could be co-delivered to TG neurons by separate AAV vectors. In conclusion, AAV1 vectors are suitable for gene delivery to TG sensory neurons following intradermal whiskerpad injection.  

5.2068           Sustained inhibition of hepatitis B virus replication in vivo after systemic injection of AAVs encoding artificial antiviral primary micro RNAs

Maepa, M.B., Ely, A., Grayson, W. and Arbuthnot, P. Molecular Therapy – Nucleic Acids, 7, 190-199 ()2017)   Chronic infection with hepatitis B virus (HBV) remains a problem of global significance and improving available treatment is important to prevent life-threatening complications arising in persistently infected individuals. HBV is susceptible to silencing by exogenous artificial intermediates of the RNA interference (RNAi) pathway. However, toxicity of Pol III cassettes and short duration of silencing by effectors of the RNAi pathway may limit anti-HBV therapeutic utility. To advance RNAi-based HBV gene silencing, mono- and trimeric artificial primary microRNAs (pri-miRs) derived from pri-miR-31 were placed under control of the liver-specific modified murine transthyretin promoter. The sequences, which target the X sequence of HBV, were incorporated into recombinant hepatotropic self-complementary adeno-associated viruses (scAAVs). Systemic intravenous injection of the vectors into HBV transgenic mice at a dose of 1 × 10¹¹ per animal effected significant suppression of markers of HBV replication for at least 32 weeks. The pri-miRs were processed according to the intended design, and intrahepatic antiviral guide sequences were detectable for 40 weeks after the injection. There was no evidence of toxicity, and innate immunostimulation was not detectable following the injections. This efficacy is an improvement on previously reported RNAi-based inhibition of HBV replication and is important to clinical translation of the technology.  

5.2069           Sema3f Protects Against Subretinal Neovascularization In Vivo

Sun, Y. et al EBioMedicine, 18, 281-287 (2017)   Pathological neovascularization of the outer retina is the hallmark of neovascular age-related macular degeneration (nAMD). Building on our previous observations that semaphorin 3F (Sema3f) is expressed in the outer retina and demonstrates anti-angiogenic potential, we have investigated whether Sema3f can be used to protect against subretinal neovascularization in two mouse models. Both in the very low-density lipid-receptor knockout (Vldlr^−/−) model of spontaneous subretinal neovascularization as well as in the mouse model of laser-induced choroidal neovascularization (CNV), we found protective effects of Sema3f against the formation of pathologic neovascularization. In the Vldlr^−/− model, AAV-induced overexpression of Sema3f reduced the size of pathologic neovascularization by 56%. In the laser-induced CNV model, intravitreally injected Sema3f reduced pathologic neovascularization by 30%. Combined, these results provide the first evidence from two distinct in vivo models for a use of Sema3f in protecting the outer retina against subretinal neovascularization.  

5.2070           Role of cleavage at the core-E1 junction of hepatitis C virus polyprotein in viral morphogenesis

Pene, V., Lemasson, M., harper, F., Pierron, G. and Rosenberg, R. PloS One, 12(4), e0175810 (2017)   In hepatitis C virus (HCV) polyprotein sequence, core protein terminates with E1 envelope signal peptide. Cleavage by signal peptidase (SP) separates E1 from the complete form of core protein, anchored in the endoplasmic reticulum (ER) membrane by the signal peptide. Subsequent cleavage of the signal peptide by signal-peptide peptidase (SPP) releases the mature form of core protein, which preferentially relocates to lipid droplets. Both of these cleavages are required for the HCV infectious cycle, supporting the idea that HCV assembly begins at the surface of lipid droplets, yet SPP-catalyzed cleavage is dispensable for initiation of budding in the ER. Here we have addressed at what step(s) of the HCV infectious cycle SP-catalyzed cleavage at the core-E1 junction is required. Taking advantage of the sole system that has allowed visualization of HCV budding events in the ER lumen of mammalian cells, we showed that, unexpectedly, mutations abolishing this cleavage did not prevent but instead tended to promote the initiation of viral budding. Moreover, even though no viral particles were released from Huh-7 cells transfected with a full-length HCV genome bearing these mutations, intracellular viral particles containing core protein protected by a membrane envelope were formed. These were visualized by electron microscopy as capsid-containing particles with a diameter of about 70 nm and 40 nm before and after delipidation, respectively, comparable to intracellular wild-type particle precursors except that they were non-infectious. Thus, our results show that SP-catalyzed cleavage is dispensable for HCV budding per se, but is required for the viral particles to acquire their infectivity and secretion. These data support the idea that HCV assembly occurs in concert with budding at the ER membrane. Furthermore, capsid-containing particles did not accumulate in the absence of SP-catalyzed cleavage, suggesting the quality of newly formed viral particles is controlled before secretion.  

5.2071           Interferon Gamma Prevents Infectious Entry of Human Papillomavirus 16 via an L2-Dependent Mechanism

Day, P.M., Thompson, C.D., Lowy, D.R. and Schiller, J.T.

1. Virol., 91(10), e00168-17 (2017)

  In this study, we report that gamma interferon (IFN-γ) treatment, but not IFN-α, -β, or -λ treatment, dramatically decreased infection of human papillomavirus 16 (HPV16) pseudovirus (PsV). In a survey of 20 additional HPV and animal papillomavirus types, we found that many, but not all, PsV types were also inhibited by IFN-γ. Microscopic and biochemical analyses of HPV16 PsV determined that the antiviral effect was exerted at the level of endosomal processing of the incoming capsid and depended on the JAK2/STAT1 pathway. In contrast to infection in the absence of IFN-γ, where L1 proteolytic products are produced during endosomal capsid processing and L2/DNA complexes segregate from L1 in the late endosome and travel to the nucleus, IFN-γ treatment led to decreased L1 proteolysis and retention of L2 and the viral genome in the late endosome/lysosome. PsV sensitivity or resistance to IFN-γ treatment was mapped to the L2 protein, as determined with infectious hybrid PsV, in which the L1 protein was derived from an IFN-γ-sensitive HPV type and the L2 protein from an IFN-γ-insensitive type or vice versa.  

5.2072           A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles

Meador, L.R., Kessans, S.A., Kilbourne, J., Kibler, K.V., pantaleo, G., Rodetiguez, M.E., Blattman, J.N., Jacobs, B.L. and Mor, T.S. Virology, 507, 242-256 (2017)   Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viral vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1.  

5.2073           Improved MECP2 Gene Therapy Extends the Survival of MeCP2-Null Mice without Apparent Toxicity after Intracisternal Delivery

Sinnett, S.E., Hector, R.D., Gadalla, K.K.E., Heindel, C., Chen, D., Zaric, V., Bailey, M.E.S., Cobb, S.R. and Gray, S.J. Molecular Therapy – Methods & Clin.Development, 5, 106-115 (2017)   Intravenous administration of adeno-associated virus serotype 9 (AAV9)/hMECP2 has been shown to extend the lifespan of Mecp2^−/y mice, but this delivery route induces liver toxicity in wild-type (WT) mice. To reduce peripheral transgene expression, we explored the safety and efficacy of AAV9/hMECP2 injected into the cisterna magna (ICM). AAV9/hMECP2 (1 × 10¹² viral genomes [vg]; ICM) extended Mecp2^−/y survival but aggravated hindlimb clasping and abnormal gait phenotypes. In WT mice, 1 × 10¹² vg of AAV9/hMECP2 induced clasping and abnormal gait. A lower dose mitigated these adverse phenotypes but failed to extend survival of Mecp2^−/y mice. Thus, ICM delivery of this vector is impractical as a treatment for Rett syndrome (RTT). To improve the safety of MeCP2 gene therapy, the gene expression cassette was modified to include more endogenous regulatory elements believed to modulate MeCP2 expression in vivo. In Mecp2^−/y mice, ICM injection of the modified vector extended lifespan and was well tolerated by the liver but did not rescue RTT behavioral phenotypes. In WT mice, these same doses of the modified vector had no adverse effects on survival or neurological phenotypes. In summary, we identified limitations of the original vector and demonstrated that an improved vector design extends Mecp2^−/y survival, without apparent toxicity.    

5.2074           Efficient Production of Papillomavirus Gene Delivery Vectors in Defined In Vitro Reactions

Cerqueira, C., Thompson, C.D., Day, P.M., Pang, Y-Y.S., Lowy, D.R. and Schiller, J.T. Molecular Therapy – Methods & Clin. Development, 5, 165-179 (2017)   Papillomavirus capsids can package a wide variety of nonviral DNA plasmids and deliver the packaged genetic material to cells, making them attractive candidates for targeted gene delivery vehicles. However, the papillomavirus vectors generated by current methods are unlikely to be suitable for clinical applications. We have developed a chemically defined, cell-free, papillomavirus-based vector production system that allows the incorporation of purified plasmid DNA (pseudogenome) into high-titer papillomavirus L1/L2 capsids. We investigated the incorporation of several DNA forms into a variety of different papillomavirus types, including human and animal types. Our results show that papillomavirus capsids can package and transduce linear or circular DNA under defined conditions. Packaging and transduction efficiencies were surprisingly variable across capsid types, DNA forms, and assembly reaction conditions. The pseudoviruses produced by these methods are sensitive to the same entry inhibitors as cell-derived pseudovirions, including neutralizing antibodies and heparin. The papillomavirus vector production systems developed in this study generated as high as 10¹¹ infectious units/mg of L1. The pseudoviruses were infectious both in vitro and in vivo and should be compatible with good manufacturing practice (GMP) requirements.  

5.2075           Tau association with synaptic vesicles causes presynaptic dysfunction

Zhou, L. et al Nature Communications, 8:15295 (2017)   Tau is implicated in more than 20 neurodegenerative diseases, including Alzheimer’s disease. Under pathological conditions, Tau dissociates from axonal microtubules and missorts to pre- and postsynaptic terminals. Patients suffer from early synaptic dysfunction prior to Tau aggregate formation, but the underlying mechanism is unclear. Here we show that pathogenic Tau binds to synaptic vesicles via its N-terminal domain and interferes with presynaptic functions, including synaptic vesicle mobility and release rate, lowering neurotransmission in fly and rat neurons. Pathological Tau mutants lacking the vesicle binding domain still localize to the presynaptic compartment but do not impair synaptic function in fly neurons. Moreover, an exogenously applied membrane-permeable peptide that competes for Tau-vesicle binding suppresses Tau-induced synaptic toxicity in rat neurons. Our work uncovers a presynaptic role of Tau that may be part of the early pathology in various Tauopathies and could be exploited therapeutically.  

5.2076           ISCA1 is essential for mitochondrial Fe4S4 biogenesis in vivo

Beilschmidt, L.K., de Choudens, S.O., Fouirnier, M., Sanakis, I., Hograindleur, M-A-., Clemancey, M., Blondin, G., Schmucker, S., Eisenmann, A., Weiss, A., Koebel, P., Messaddeq, N., Puccio, H. and Martelli, A. Nature Communications, 8:15124 (2017)   Mammalian A-type proteins, ISCA1 and ISCA2, are evolutionarily conserved proteins involved in iron–sulfur cluster (Fe–S) biogenesis. Recently, it was shown that ISCA1 and ISCA2 form a heterocomplex that is implicated in the maturation of mitochondrial Fe4S4 proteins. Here we report that mouse ISCA1 and ISCA2 are Fe2S2-containing proteins that combine all features of Fe–S carrier proteins. We use biochemical, spectroscopic and in vivo approaches to demonstrate that despite forming a complex, ISCA1 and ISCA2 establish discrete interactions with components of the late Fe–S machinery. Surprisingly, knockdown experiments in mouse skeletal muscle and in primary cultures of neurons suggest that ISCA1, but not ISCA2, is required for mitochondrial Fe4S4 proteins biogenesis. Collectively, our data suggest that cellular processes with different requirements for ISCA1, ISCA2 and ISCA1–ISCA2 complex seem to exist.  

5.2077           Global Representations of Goal-Directed Behavior in Distinct Cell Types of Mouse Neocortex

William E. Allen, Isaac V. Kauvar, Michael Z. Chen, Ethan B. Richman, Samuel J. Yang, Ken Chan, Viviana Gradinaru, Benjamin E. Deverman, Liqun Luo , Karl Deisseroth Neuron, 94, 891-907 (2017)   The successful planning and execution of adaptive behaviors in mammals may require long-range coordination of neural networks throughout cerebral cortex. The neuronal implementation of signals that could orchestrate cortex-wide activity remains unclear. Here, we develop and apply methods for cortex-wide Ca²⁺ imaging in mice performing decision-making behavior and identify a global cortical representation of task engagement encoded in the activity dynamics of both single cells and superficial neuropil distributed across the majority of dorsal cortex. The activity of multiple molecularly defined cell types was found to reflect this representation with type-specific dynamics. Focal optogenetic inhibition tiled across cortex revealed a crucial role for frontal cortex in triggering this cortex-wide phenomenon; local inhibition of this region blocked both the cortex-wide response to task-initiating cues and the voluntary behavior. These findings reveal cell-type-specific processes in cortex for globally representing goal-directed behavior and identify a major cortical node that gates the global broadcast of task-related information.  

5.2078           Exploiting the kinesin-1 molecular motor to generate a virus membrane penetration site

Ravindran, M.S., Engelke, M.F., Verhey, K.J. and Tsai, B. Nature Communications, 8:15496 (2017)   Viruses exploit cellular machineries to penetrate a host membrane and cause infection, a process that remains enigmatic for non-enveloped viruses. Here we probe how the non-enveloped polyomavirus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol, a crucial infection step. We find that the microtubule-based motor kinesin-1 is recruited to the ER membrane by binding to the transmembrane J-protein B14. Strikingly, this motor facilitates SV40 ER-to-cytosol transport by constructing a penetration site on the ER membrane called a ‘focus’. Neither kinesin-2, kinesin-3 nor kinesin-5 promotes foci formation or infection. The specific use of kinesin-1 is due to its unique ability to select posttranslationally modified microtubules for cargo transport and thereby spatially restrict focus formation to the perinucleus. These findings support the idea of a ‘tubulin code’ for motor-dependent trafficking and establish a distinct kinesin-1 function in which a motor is exploited to create a viral membrane penetration site.  

5.2079           SGTA-Dependent Regulation of Hsc70 Promotes Cytosol Entry of Simian Virus 40 from the Endoplasmic Reticulum

Dupzyk, A., Williams, J.M., Bagchi, P., Inoue, T. and Tsai, B.

1. Virol., 91(12), e00232-17 (2017)

  Membrane penetration by nonenveloped viruses remains enigmatic. In the case of the nonenveloped polyomavirus simian virus 40 (SV40), the virus penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol and then traffics to the nucleus to cause infection. We previously demonstrated that the cytosolic Hsc70-SGTA-Hsp105 complex is tethered to the ER membrane, where Hsp105 and SGTA facilitate the extraction of SV40 from the ER and transport of the virus into the cytosol. We now find that Hsc70 also ejects SV40 from the ER into the cytosol in a step regulated by SGTA. Although SGTA's N-terminal domain, which mediates homodimerization and recruits cellular adaptors, is dispensable during ER-to-cytosol transport of SV40, this domain appears to exert an unexpected post-ER membrane translocation function during SV40 entry. Our study thus establishes a critical function of Hsc70 within the Hsc70-SGTA-Hsp105 complex in promoting SV40 ER-to-cytosol membrane penetration and unveils a role of SGTA in controlling this step.  

5.2080           Reverse Transcription Mechanically Initiates HIV-1 Capsid Disassembly

Rankovic, S., Varadarajan, J., Ramelho, R., Aiken, C. and Rousso, I.

1. Virol., 91(12), e00289-17 (2017)

  The HIV-1 core consists of the viral genomic RNA and several viral proteins encased within a conical capsid. After cell entry, the core disassembles in a process termed uncoating. Although HIV-1 uncoating has been linked to reverse transcription of the viral genome in target cells, the mechanism by which uncoating is initiated is unknown. Using time-lapse atomic force microscopy, we analyzed the morphology and physical properties of isolated HIV-1 cores during the course of reverse transcription in vitro. We found that, during an early stage of reverse transcription the pressure inside the capsid increases, reaching a maximum after 7 h. High-resolution mechanical mapping reveals the formation of a stiff coiled filamentous structure underneath the capsid surface. Subsequently, this coiled structure disappears, the stiffness of the capsid drops precipitously to a value below that of a pre-reverse transcription core, and the capsid undergoes partial or complete rupture near the narrow end of the conical structure. We propose that the transcription of the relatively flexible single-stranded RNA into a more rigid filamentous structure elevates the pressure within the core, which triggers the initiation of capsid disassembly.  

5.2081           Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles

Oliveira, C. et al Hepatology, 65(6), 1851-1864 (2017)   The development of different cell culture models has greatly contributed to increased understanding of the hepatitis C virus (HCV) life cycle. However, it is still challenging to grow HCV clinical isolates in cell culture. If overcome, this would open new perspectives to study HCV biology, including drug-resistant variants emerging with new antiviral therapies. In this study we hypothesized that this hurdle could be due to the presence of inhibitory factors in patient serum. Combining polyethylene glycol precipitation, iodixanol gradient, and size-exclusion chromatography, we obtained from HCV-seronegative sera a purified fraction enriched in inhibitory factors. Mass spectrometric analysis identified apolipoprotein(a) (apo[a]) as a potential inhibitor of HCV entry. Apo(a) consists of 10 kringle IV domains (KIVs), one kringle V domain, and an inactive protease domain. The 10 KIVs are present in a single copy with the exception of KIV type 2 (KIV₂), which is encoded in a variable number of tandemly repeated copies, giving rise to numerous apo(a) size isoforms. In addition, apo(a) covalently links to the apolipoprotein B component of a low-density lipoprotein through a disulfide bridge to form lipoprotein(a). Using a recombinant virus derived from the JFH1 strain, we confirmed that plasma-derived and recombinant lipoprotein(a) as well as purified recombinant apo(a) variants were able to specifically inhibit HCV by interacting with infectious particles. Our results also suggest that small isoforms are less inhibitory than the large ones. Finally, we observed that the lipoprotein moiety of HCV lipoviroparticles was essential for inhibition, whereas functional lysine-binding sites in KIV₇, KIV₈, and KIV₁₀ were not required. Conclusions: Our results identify apo(a) as an additional component of the lipid metabolism modulating HCV infection.  

5.2082           Evaluation of MYBPC3 trans-Splicing and Gene Replacement as Therapeutic Options in Human iPSC-Derived Cardiomyocytes

Prondzynski, M., Krämer, E., Laufer, S.D., Shibamiya, A., Pless, O., Flenner, F., Müller, O.J., Münch, J., Redwood, c., Hansen, A., patten, M., Eschenhagen, T., Mearini, G. and Carrier, L. Molecular Therapy – Nucleic Acids, 7, 475-486 (2017)   Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of hypertrophic cardiomyopathy (HCM) carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited ∼50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 5′ or 3′ pre-trans-splicing molecules represented ∼1% of total MYBPC3 transcripts in healthy hiPSC-CMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in ∼2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1) the feasibility of trans-splicing, although with low efficiency, and (2) efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation.  

5.2083           Description of a novel multiplex avidity assay for evaluating HPV antibodies

Brady, A.M., Unger, E.R. and Panicker, G.

1. Immunol. Methods, 47, 31-36 (2017)

  Limited data exists regarding antibody avidity for human papillomavirus (HPV). We describe development of a multiplex electrochemiluminescent avidity ELISA for four HPV types (HPV 6, 11, 16, 18) by adding a dissociating step to our established multiplex HPV VLP ELISA. Initial experiments exploring ammonium thiocyanate, sodium thiocyanate and guanidine hydrochloride (GuHCl) as dissociating agents identified GuHCl as most promising. Dissociation conditions with GuHCl were varied (concentration, incubation time, temperature) to select conditions with minimal impact on VLP integrity as measured with monoclonal antibodies to conformational epitopes. Avidity index (AI) was calculated based on a standard curve as ratio of bound IgG in GuHCl treated versus untreated sample. To evaluate our assay we determined AI in sera with known HPV titers. We selected 32 residual anonymized sera from individuals with a wide range of titers for HPV6, 11, 16, and 18. AIs were similar across multiple dilutions of serum within the assay's dynamic range and were reproducible with two plate lots. This assay will aid in understanding HPV antibody avidity and maturation in response to natural infection and varying vaccine schedules. This is the first report of a VLP-based multiplexed avidity ELISA that evaluates assay parameters for all nine HPV vaccine types.  

5.2084           Improving the malaria transmission-blocking activity of a Plasmodium falciparum 48/45 based vaccine antigen by SpyTag/SpyCatcher mediated virus-like display

Singh, S., Thrane, S., Janitzek, C.M., Nielsen, M.A., Theander, T.G., Theisen, M., Salanti, A. and Sander, A.F. Vaccine, 35, 3726-3732 (2017)   Malaria is a devastating disease caused by Plasmodium parasites, resulting in almost 0.5 million deaths per year. The Pfs48/45 protein exposed on the P. falciparum sexual stages is one of the most advanced antigen candidates for a transmission-blocking (TB) vaccine in the clinical pipeline. However, it remains essential to identify an optimal vaccine formulation that can facilitate induction of a long-lasting TB anti-Pfs48/45 response. Here we report on the development and evaluation of two Pfs48/45-based virus-like particle (VLP) vaccines generated using the AP205 SpyTag/Catcher VLP system. Two different recombinant proteins (SpyCatcher-R0.6C and SpyCatcher-6C), comprising the Pfs48/45-6C region, were covalently attached to the surface of Spy-tagged Acinetobacter phage AP205 VLPs. Resulting Pfs48/45-VLP complexes appeared as non-aggregated particles of ∼30 nm, each displaying an average of 216 (R0.6C) or 291 (6C) copies of the antigens. Both R0.6C and 6C VLP conjugates were strongly reactive with a monoclonal antibody (mAb45.1) targeting a conformational TB Pfs48/45 epitope, suggesting that the TB epitope is accessible for immune recognition on the particles. To select the most suitable vaccine formulation for downstream clinical studies the two VLP vaccines were tested in CD1 mice using different adjuvant formulations. The study demonstrates that VLP-display of R0.6C and 6C significantly increases antigen immunogenicity when using Montanide ISA 720 VG as extrinsic adjuvant.  

5.2085           Efficient production of recombinant adeno-associated viral vector, serotype DJ/8, carrying the GFP gene

Hashimoto, H., Mizushima, T., Chijiwa, T., Nakamura, M. and Suemizu, H. Virus Res., 238, 63-68 (2017)   The purpose of this study was to establish an efficient method for the preparation of an adeno-associated viral (AAV), serotype DJ/8, carrying the GFP gene (AAV-DJ/8-GFP). We compared the yields of AAV-DJ/8 vector, which were produced by three different combination methods, consisting of two plasmid DNA transfection methods (lipofectamine and calcium phosphate co-precipitation; CaPi) and two virus DNA purification methods (iodixanol and cesium chloride; CsCl). The results showed that the highest yield of AAV-DJ/8-GFP vector was accomplished with the combination method of lipofectamine transfection and iodixanol purification. The viral protein expression levels and the transduction efficacy in HEK293 and CHO cells were not different among four different combination methods for AAV-DJ/8-GFP vectors. We confirmed that the AAV-DJ/8-GFP vector could transduce to human and murine hepatocyte-derived cell lines. These results show that AAV-DJ/8-GFP, purified by the combination of lipofectamine and iodixanol, produces an efficient yield without altering the characteristics of protein expression and AAV gene transduction.  

5.2086           Tau interactome mapping based identification of Otub1 as Tau deubiquitinase involved in accumulation of pathological Tau forms in vitro and in vivo

Wang, P., Joberty, G., Buist, A., Vanoosthuyse, A., Stancu, I-C., Vasconcelos, B., Pierrot, N., Faelth-Savitski, M., Kienlen-Campard, P., Octave, J-N., Bantscheff, M., Drewes, G., Moeschars, D. and Dewachter, I. Acta Neuropathol, 133, 731-749 (2017)   Dysregulated proteostasis is a key feature of a variety of neurodegenerative disorders. In Alzheimer’s disease (AD), progression of symptoms closely correlates with spatiotemporal progression of Tau aggregation, with “early” oligomeric Tau forms rather than mature neurofibrillary tangles (NFTs) considered to be pathogenetic culprits. The ubiquitin–proteasome system (UPS) controls degradation of soluble normal and abnormally folded cytosolic proteins. The UPS is affected in AD and is identified by genomewide association study (GWAS) as a risk pathway for AD. The UPS is determined by balanced regulation of ubiquitination and deubiquitination. In this work, we performed isobaric tags for relative and absolute quantitation (iTRAQ)-based Tau interactome mapping to gain unbiased insight into Tau pathophysiology and to identify novel Tau-directed therapeutic targets. Focusing on Tau deubiquitination, we here identify Otub1 as a Tau-deubiquitinating enzyme. Otub1 directly affected Lys48-linked Tau deubiquitination, impairing Tau degradation, dependent on its catalytically active cysteine, but independent of its noncanonical pathway modulated by its N-terminal domain in primary neurons. Otub1 strongly increased AT8-positive Tau and oligomeric Tau forms and increased Tau-seeded Tau aggregation in primary neurons. Finally, we demonstrated that expression of Otub1 but not its catalytically inactive form induced pathological Tau forms after 2 months in Tau transgenic mice in vivo, including AT8-positive Tau and oligomeric Tau forms. Taken together, we here identified Otub1 as a Tau deubiquitinase in vitro and in vivo, involved in formation of pathological Tau forms, including small soluble oligomeric forms. Otub1 and particularly Otub1 inhibitors, currently under development for cancer therapies, may therefore yield interesting novel therapeutic avenues for Tauopathies and AD.  

5.2087           A MicroRNA124 Target Sequence Restores Astrocyte Specificity of gfaABC1D-Driven Transgene Expression in AAV-Mediated Gene Transfer

Taschenberger, G., Tereshchenko, J. and Kügler, S. Molecular Therapy – Nucleic Acids, 8, 13-25 (2017)   Experimentally restricting transgene expression exclusively to astrocytes has proven difficult. Using adeno-associated-virus-mediated gene transfer, we assessed two commonly used glial fibrillary acidic protein promoters: the full-length version gfa2 (2,210-bp human glial fibrillary acidic protein [GFAP] promoter) and the truncated variant gfaABC₁D (681-bp GFAP promoter). The capacity to drive efficient, but also cell-type specific, expression of the EGFP in astrocytes was tested both in vitro in rat primary cortical cultures as well as in vivo in the rat striatum. We observed an efficient, but not entirely astrocyte-specific, gfa2-driven reporter expression. gfaABC₁D exhibited a weaker activity, and most importantly, off-target, neuronal expression of the transgene occurred in a larger fraction of cells. Therefore, we explored the potential of a microRNA (miR)-specific target-sequence-based approach for abolishing off-target expression. When miR124 target sequences were incorporated into the 3′ UTR, neuronal gene expression was effectively silenced. However, unexpectedly, the insertion of an additional sequence in the 3′ UTR clearly diminished transgene expression. In conclusion, the gfaABC₁D promoter on its own is not sufficient to specifically target transgene expression to astrocytes and is not well suited for AAV-based gene targeting, even if short promoter sequences are required. The combination with a miR de-targeting sequence represents a promising experimental strategy that eliminates off-target, neuronal expression.  

5.2088           Expression of P301L-hTau in mouse MEC induces hippocampus-dependent memory deficit

Liu, X., Zeng, K., Li, M., Wang, Q., Liu, R., zhang, B., Wang, J-Z., Shu, X. and Wang, X. Scientific Reports, 7:3914 (2017)   Intracellular accumulation of abnormally phosphorylated tau in different types of neurons is a pathological characteristic of Alzheimer’s disease (AD). While tau modification and associated neuronal loss and hypometabolism start in the entorhinal cortex (EC) in early AD patients, the mechanism by which mutant P301L hTau leads to dementia is not fully elucidated. Here, we studied the effects of P301L hTau transduction in the medial EC (MEC) of mice on tau phosphorylation and accumulation, and cognitive deficit. We found that the exogenous mutant tau protein was restricted in MEC without spreading to other brain regions at one month after transduction. Interestingly, expression of the mutant tau in MEC induces endogenous tau hyperphosphorylation and accumulation in hippocampus and cortex, and inhibits neuronal activity with attenuated PP-DG synapse plasticity, leading to hippocampus-dependent memory deficit with intact olfactory function. These findings suggest a novel neuropathological mechanism of early AD, which is initiated by tau accumulation in MEC, and demonstrate a tau pathological model of early stage AD.  

5.2089           An R-CaMP1.07 reporter mouse for cell-type-specific expression of a sensitive red fluorescent calcium indicator

Bethge, P., Carta, S., Lorenzo, D.A., Egolf, L., Goniotaki, D., Madisen, L., Voigt, F.F., Chen, J.L., Schneider, B., Ohkura, M., Nakai, J., Zeng, H., Aguzzi, A. and Helmchen, F. PloS One, 12(6), e0179460 (2017)   Genetically encoded calcium indicators (GECIs) enable imaging of in vivo brain cell activity with high sensitivity and specificity. In contrast to viral infection or in utero electroporation, indicator expression in transgenic reporter lines is induced noninvasively, reliably, and homogenously. Recently, Cre/tTA-dependent reporter mice were introduced, which provide high-level expression of green fluorescent GECIs in a cell-type-specific and inducible manner when crossed with Cre and tTA driver mice. Here, we generated and characterized the first red-shifted GECI reporter line of this type using R-CaMP1.07, a red fluorescent indicator that is efficiently two-photon excited above 1000 nm. By crossing the new R-CaMP1.07 reporter line to Cre lines driving layer-specific expression in neocortex we demonstrate its high fidelity for reporting action potential firing in vivo, long-term stability over months, and versatile use for functional imaging of excitatory neurons across all cortical layers, especially in the previously difficult to access layers 4 and 6.  

5.2090           Disparate Contributions of Human Retrovirus Capsid Subdomains to Gag-Gag Oligomerization, Virus Morphology, and Particle Biogenesis

Martin, J.L., Mendonca, L.M., Angert, I., Mueller, J.D., Zhang, W. and Mansky, L.M.

1. Virol., 91(14), e00298-17 (2017)

  The capsid domain (CA) of the retroviral Gag protein is a primary determinant of Gag oligomerization, which is a critical step for immature Gag lattice formation and virus particle budding. Although the human immunodeficiency virus type 1 (HIV-1) CA carboxy-terminal domain (CTD) is essential for CA-CA interactions, the CA CTD has been suggested to be largely dispensable for human T-cell leukemia virus type 1 (HTLV-1) particle biogenesis. To more clearly define the roles of the HTLV-1 CA amino-terminal domain (NTD) and CA CTD in particle biogenesis, we generated and analyzed a panel of Gag proteins with chimeric HIV-1/HTLV-1 CA domains. Subcellular distribution and protein expression levels indicated that Gag proteins with a chimeric HIV-1 CA NTD/HTLV-1 CA CTD did not result in Gag oligomerization regardless of the parent Gag background. Furthermore, chimeric Gag proteins with the HTLV-1 CA NTD produced particles phenotypically similar to HTLV-1 immature particles, highlighting the importance of the HTLV-1 CA NTD in HTLV-1 immature particle morphology. Taken together, these observations support the conclusion that the HTLV-1 CA NTD can functionally replace the HIV-1 CA CTD, but the HIV-1 CA NTD cannot replace the HTLV-1 CA CTD, indicating that the HTLV-1 CA subdomains provide distinct contributions to Gag-Gag oligomerization, particle morphology, and biogenesis. Furthermore, we have shown for the first time that HIV-1 and HTLV-1 Gag domains outside the CA (e.g., matrix and nucleocapsid) impact Gag oligomerization as well as immature particle size and morphology.  

5.2091           Dense Array of Spikes on HIV-1 Virion Particles

Stano, A., leaman, D.P., Kim, A.S., Zhang, L., Autin, L., Ingale, J., Gift, S.K., Truong, J., Wyatt, R., Olson, A.J. and Zwick, M.B.

1. Virol., 91(14), e000415-17 (2017)

  HIV-1 is rare among viruses for having a low number of envelope glycoprotein (Env) spikes per virion, i.e., ∼7 to 14. This exceptional feature has been associated with avoidance of humoral immunity, i.e., B cell activation and antibody neutralization. Virus-like particles (VLPs) with increased density of Env are being pursued for vaccine development; however, these typically require protein engineering that alters Env structure. Here, we used instead a strategy that targets the producer cell. We employed fluorescence-activated cell sorting (FACS) to sort for cells that are recognized by trimer cross-reactive broadly neutralizing antibody (bnAb) and not by nonneutralizing antibodies. Following multiple iterations of FACS, cells and progeny virions were shown to display higher levels of antigenically correct Env in a manner that correlated between cells and cognate virions (P = 0.027). High-Env VLPs, or hVLPs, were shown to be monodisperse and to display more than a 10-fold increase in spikes per particle by electron microscopy (average, 127 spikes; range, 90 to 214 spikes). Sequencing revealed a partial truncation in the C-terminal tail of Env that had emerged in the sort; however, iterative rounds of “cell factory” selection were required for the high-Env phenotype. hVLPs showed greater infectivity than standard pseudovirions but largely similar neutralization sensitivity. Importantly, hVLPs also showed superior activation of Env-specific B cells. Hence, high-Env HIV-1 virions, obtained through selection of producer cells, represent an adaptable platform for vaccine design and should aid in the study of native Env.  

5.2092           The mechanism of sirtuin 2–mediated exacerbation of alpha-synuclein toxicity in models of Parkinson disease

De Oliveira, R.M. et al PloS Biology, 15(3), e2000374 (2017)   Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation and toxicity, demonstrating the potential therapeutic value of sirtuin 2 inhibition in synucleinopathies.  

5.2093           Neonatal AAV delivery of alpha-synuclein induces pathology in the adult mouse brain

Delenclos, M., Faroqi, A.H., Yue, M., Kurti, A., Castanedes-Casey, M., Rousseau, L., Phillips, V., Dickson, D.W., Fryer, J.D. and McLean, P.J. Acta Neuropathol. Commun., 5:51 (2017)   Abnormal accumulation of alpha-synuclein (αsyn) is a pathological hallmark of Lewy body related disorders such as Parkinson’s disease and Dementia with Lewy body disease. During the past two decades, a myriad of animal models have been developed to mimic pathological features of synucleinopathies by over-expressing human αsyn. Although different strategies have been used, most models have little or no reliable and predictive phenotype. Novel animal models are a valuable tool for understanding neuronal pathology and to facilitate development of new therapeutics for these diseases. Here, we report the development and characterization of a novel model in which mice rapidly express wild-type αsyn via somatic brain transgenesis mediated by adeno-associated virus (AAV). At 1, 3, and 6 months of age following intracerebroventricular (ICV) injection, mice were subjected to a battery of behavioral tests followed by pathological analyses of the brains. Remarkably, significant levels of αsyn expression are detected throughout the brain as early as 1 month old, including olfactory bulb, hippocampus, thalamic regions and midbrain. Immunostaining with a phospho-αsyn (pS129) specific antibody reveals abundant pS129 expression in specific regions. Also, pathologic αsyn is detected using the disease specific antibody 5G4. However, this model did not recapitulate behavioral phenotypes characteristic of rodent models of synucleinopathies. In fact no deficits in motor function or cognition were observed at 3 or 6 months of age. Taken together, these findings show that transduction of neonatal mouse with AAV-αsyn can successfully lead to rapid, whole brain transduction of wild-type human αsyn, but increased levels of wildtype αsyn do not induce behavior changes at an early time point (6 months), despite pathological changes in several neurons populations as early as 1 month.  

5.2094           Novel oligodendroglial alpha synuclein viral vector models of multiple system atrophy: studies in rodents and nonhuman primates

Mandel, R.J., Marmion, D.J., Kirik, D., Chu, Y., heindel, C., McCown, T., Gray, S.J. and Kordower, J.H. Acta Neuropathol. Commun., 5:47 (2017)   Multiple system atrophy (MSA) is a horrible and unrelenting neurodegenerative disorder with an uncertain etiology and pathophysiology. MSA is a unique proteinopathy in which alpha-synuclein (α-syn) accumulates preferentially in oligodendroglia rather than neurons. Glial cytoplasmic inclusions (GCIs) of α-syn are thought to elicit changes in oligodendrocyte function, such as reduced neurotrophic support and demyelination, leading to neurodegeneration. To date, only a murine model using one of three promoters exist to study this disease. We sought to develop novel rat and nonhuman primate (NHP) models of MSA by overexpressing α-syn in oligodendroglia using a novel oligotrophic adeno-associated virus (AAV) vector, Olig001. To establish tropism, rats received intrastriatal injections of Olig001 expressing GFP. Histological analysis showed widespread expression of GFP throughout the striatum and corpus callosum with >95% of GFP+ cells co-localizing with oligodendroglia and little to no expression in neurons or astrocytes. We next tested the efficacy of this vector in rhesus macaques with intrastriatal injections of Olig001 expressing GFP. As in rats, we observed a large number of GFP+ cells in gray matter and white matter tracts of the striatum and the corpus callosum, with 90–94% of GFP+ cells co-localizing with an oligodendroglial marker. To evaluate the potential of our vector to elicit MSA-like pathology in NHPs, we injected rhesus macaques intrastriatally with Olig001 expressing the α-syn transgene. Histological analysis 3-months after injection demonstrated widespread α-syn expression throughout the striatum as determined by LB509 and phosphorylated serine-129 α-syn immunoreactivity, all of which displayed as tropism similar to that seen with GFP. As in MSA, Olig001-α-syn GCIs in our model were resistant to proteinase K digestion and caused microglial activation. Critically, demyelination was observed in the white matter tracts of the corpus callosum and striatum of Olig001-α-syn but not Olig001-GFP injected animals, similar to the human disease. These data support the concept that this vector can provide novel rodent and nonhuman primate models of MSA.  

5.2095           A nonenveloped virus with a lipid envelope: hepatitis A virus as used in virus-reduction studies

Kapsch, A-M., farcet, M.R., Antoine, G. and Kreil, T.R. Transfusion, 57(6), 1433-1439 (2017)   BACKGROUND Recently, a quasi-lipid–enveloped (LE) form of the traditionally nonlipid-enveloped (NLE) hepatitis A virus (HAV) was described in human serum and cell culture-derived HAV stocks. This discovery challenges the understanding of HAV reduction in virus clearance studies of plasma products, which were performed under the premise of an NLE nature of this virus. Here, the presence of LE particles in HAV stocks used for reduction studies was verified, and the hypothesis that LE and NLE particles might contribute to the differential heat sensitivity of HAV variants during heat treatment of human serum albumin was evaluated. STUDY DESIGN AND METHODS Cell culture lysates and supernatants of two cytopathic HAV variants, HM175/18f and HM175/24a, were characterized for their LE and NLE particle content by isopycnic gradient centrifugation. The obtained fractions were characterized for relative infectivity and then subjected to heat treatment (58.0 ± 1.0°C for 590 ± 10 minutes) in 12.5% human serum albumin to investigate their respective heat sensitivity. RESULTS Preparations of the two HAV variants contained either LE particles (HM175/24a) or LE and NLE particles (HM175/18f) with equivalent specific infectivity. For HM175/18f, heat sensitivity of LE and NLE fractions did not differ significantly, and inactivation of the whole virus stock was identical to the NLE particle inactivation profile, whereas the HM175/24a variant was more heat sensitive. CONCLUSION The results indicate that, in heat-treatment studies, the LE or NLE HAV phenotype is less important than the choice of HAV variant, and the most heat-resistant HM175/18f should be used.  

5.2096           Protein composition of the hepatitis A virus quasi-envelope

McKnight, K.L., Xie, L., Gonzalez-Lopez, O., Rivera-Serrano, E.E., Chen, X. and Lemon, S.M. PNAS, 114(25), 6587-6592 (2017)   The Picornaviridae are a diverse family of RNA viruses including many pathogens of medical and veterinary importance. Classically considered “nonenveloped,” recent studies show that some picornaviruses, notably hepatitis A virus (HAV; genus Hepatovirus) and some members of the Enterovirus genus, are released from cells nonlytically in membranous vesicles. To better understand the biogenesis of quasi-enveloped HAV (eHAV) virions, we conducted a quantitative proteomics analysis of eHAV purified from cell-culture supernatant fluids by isopycnic ultracentrifugation. Amino acid-coded mass tagging (AACT) with stable isotopes followed by tandem mass spectrometry sequencing and AACT quantitation of peptides provided unambiguous identification of proteins associated with eHAV versus unrelated extracellular vesicles with similar buoyant density. Multiple peptides were identified from HAV capsid proteins (53.7% coverage), but none from nonstructural proteins, indicating capsids are packaged as cargo into eHAV vesicles via a highly specific sorting process. Other eHAV-associated proteins (n = 105) were significantly enriched for components of the endolysosomal system (>60%, P < 0.001) and included many common exosome-associated proteins such as the tetraspanin CD9 and dipeptidyl peptidase 4 (DPP4) along with multiple endosomal sorting complex required for transport III (ESCRT-III)-associated proteins. Immunoprecipitation confirmed that DPP4 is displayed on the surface of eHAV produced in cell culture or present in sera from humans with acute hepatitis A. No LC3-related peptides were identified by mass spectrometry. RNAi depletion studies confirmed that ESCRT-III proteins, particularly CHMP2A, function in eHAV biogenesis. In addition to identifying surface markers of eHAV vesicles, the results support an exosome-like mechanism of eHAV egress involving endosomal budding of HAV capsids into multivesicular bodies.  

5.2097           Multi-virion infectious units arise from free viral particles in an enveloped virus

Cuevas, J.M., Duran-Moreno,, M. and Sanjuan, R. Nature Microbiol., 21:17078 (2017)   Many animal viruses are enveloped in a lipid bilayer taken up from cellular membranes. Because viral surface proteins bind to these membranes to initiate infection, we hypothesized that free virions may also be capable of interacting with the envelopes of other virions extracellularly. Here, we demonstrate this hypothesis in the vesicular stomatitis virus (VSV), a prototypic negative-strand RNA virus composed of an internal ribonucleocapsid, a matrix protein and an external envelope¹. Using microscopy, dynamic light scattering, differential centrifugation and flow cytometry, we show that free viral particles can spontaneously aggregate into multi-virion infectious units. We also show that, following establishment of these contacts, different viral genetic variants are co-transmitted to the same target cell. Furthermore, virion–virion binding can determine key aspects of viral fitness such as antibody escape. In purified virions, this process is driven by protein–lipid interactions probably involving the VSV surface glycoprotein and phosphatidylserine. Whereas we found that multi-virion complexes occurred unfrequently in standard cell cultures, they were abundant in other fluids such as saliva, a natural VSV shedding route². Our findings contrast with the commonly accepted perception of virions as passive propagules and show the ability of enveloped viruses to establish collective infectious units, which could in turn facilitate the evolution of virus–virus interactions and of social-like traits³.  

5.2098           Alternative Start Sites Downstream of Non-Sense Mutations Drive Antigen Presentation and Tolerance Induction to C-Terminal Epitopes

Ashley, S.N., Somanathan, S., Hinderer, C., Arias, M., McMenamin, D., Draper, C. and Wilson, J.M.

1. Immunol., 198, 4581-4587 (2017)

  CTL responses to the transgene product remain an active area of concern for the gene therapy field. A patient’s underlying genetic mutation may influence the qualitative nature of these potentially destructive T cell responses. Individuals with a mutation that introduces a premature termination codon (PTC) that prevents synthesis of the full-length peptide are considered more likely to mount a transgene-specific T cell response because of a lack of immune tolerance to C-terminal epitopes as a consequence of absent endogenous Ag presentation. In this article, we demonstrate that a human ornithine transcarbamylase gene containing various PTC-inducing non-sense mutations is able to generate and present epitopes downstream of the termination codon. Generation of these epitopes occurs primarily from alternative translation start sites downstream of the stop codon. Furthermore, we show that expression of these genes from adeno-associated virus vectors in C57BL/6 mice is able to induce peripheral tolerance to epitopes downstream of the PTC. These results suggest that, despite the lack of full-length endogenous protein, patients with PTC-inducing non-sense mutations may still present T cell epitopes downstream of the premature termination site that may render the subject tolerant to wild-type transgene products.  

5.2099           Inflammatory signals from photoreceptor modulate pathological retinal angiogenesis via c-Fos

Sun, Y., Lin, Z., Liu, C-H., Liegl, R., Fredrick, T.W., Meng, S.S., Burnim, S.B., Wang, Z., Akula, J.D., Pu, W.T., Chen, J. and Smith, L.E.H.

1. Exp. Med., 214(6), 1753-1767 (2017)

  Pathological neovessels growing into the normally avascular photoreceptors cause vision loss in many eye diseases, such as age-related macular degeneration and macular telangiectasia. Ocular neovascularization is strongly associated with inflammation, but the source of inflammatory signals and the mechanisms by which these signals regulate the disruption of avascular privilege in photoreceptors are unknown. In this study, we found that c-Fos, a master inflammatory regulator, was increased in photoreceptors in a model of pathological blood vessels invading photoreceptors: the very low-density lipoprotein receptor–deficient (Vldlr^−/−) mouse. Increased c-Fos induced inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor (TNF), leading to activation of signal transducer and activator of transcription 3 (STAT3) and increased TNFα–induced protein 3 (TNFAIP3) in Vldlr^−/− photoreceptors. IL-6 activated the STAT3/vascular endothelial growth factor A (VEGFA) pathway directly, and elevated TNFAIP3 suppressed SOCS3 (suppressor of cytokine signaling 3)–activated STAT3/VEGFA indirectly. Inhibition of c-Fos using photoreceptor-specific AAV (adeno-associated virus)-hRK (human rhodopsin kinase)–sh_c-fos or a chemical inhibitor substantially reduced the pathological neovascularization and rescued visual function in Vldlr^−/− mice. These findings suggested that the photoreceptor c-Fos controls blood vessel growth into the normally avascular photoreceptor layer through the inflammatory signal–induced STAT3/VEGFA pathway.  

5.2100           Improving the Quality of Adeno-Associated Viral Vector Preparations: The Challenge of Product-Related Impurities free access

Schnödt, M. and Büning, H. Human Gene Therapy Methods, 28(3), 101-108 (2017)   Adeno-associated viral (AAV) vectors have emerged as one of the most popular gene transfer systems in both research and clinical gene therapy. As AAV vectors are derived from a stealth, nonpathogenic virus and lack active integrase activity, these vectors are frequently applied for in vivo gene therapy of liver, muscle, and other postmitotic tissues. Although long-term transgene expression from AAV vector episomes is reported from these tissues, the episomal nature of AAV—once regarded as disadvantage—has become an attractive feature for gene-editing approaches targeting proliferating cells. In response to the high demand, AAV vector production is receiving special attention. Besides particle yields and biological activity, the most important concern is improving vector purity. The most difficult task in this regard is removal of defective particles, that is, capsids that are either empty or contain DNA other than the full-length vector genomes. Herein, we characterize and discuss these so-called product-related impurities, methods for their detection, as well as strategies to avoid or reduce their formation.  

5.2101           Systemic Correction of Murine Glycogen Storage Disease Type IV by an AAV-Mediated Gene Therapy

Yi, H., Zhang, Q., Brooks, E.D., Yang, C., Thurberg, B.L., Kishnani, P.S. and Sun, B. Human Gene Therapy, 28(3), 286-294 (2017)   Deficiency of glycogen branching enzyme (GBE) causes glycogen storage disease type IV (GSD IV), which is characterized by the accumulation of a less branched, poorly soluble form of glycogen called polyglucosan (PG) in multiple tissues. This study evaluates the efficacy of gene therapy with an adeno-associated viral (AAV) vector in a mouse model of adult form of GSD IV (Gbe1^ys/ys). An AAV serotype 9 (AAV9) vector containing a human GBE expression cassette (AAV-GBE) was intravenously injected into 14-day-old Gbe1^ys/ys mice at a dose of 5 × 10¹¹ vector genomes per mouse. Mice were euthanized at 3 and 9 months of age. In the AAV-treated mice at 3 months of age, GBE enzyme activity was highly elevated in heart, which is consistent with the high copy number of the viral vector genome detected. GBE activity also increased significantly in skeletal muscles and the brain, but not in the liver. The glycogen content was reduced to wild-type levels in muscles and significantly reduced in the liver and brain. At 9 months of age, though GBE activity was only significantly elevated in the heart, glycogen levels were significantly reduced in the liver, brain, and skeletal muscles of the AAV-treated mice. In addition, the AAV treatment resulted in an overall decrease in plasma activities of alanine transaminase, aspartate transaminase, and creatine kinase, and a significant increase in fasting plasma glucose concentration at 9 months of age. This suggests an alleviation of damage and improvement of function in the liver and muscles by the AAV treatment. This study demonstrated a long-term benefit of a systemic injection of an AAV-GBE vector in Gbe1^ys/ys mice.  

5.2102           Adeno-Associated Virus Vectors and Stem Cells: Friends or Foes?

Brown, N., Song, L., Kollu, N.R. and Hirsch, M.L. Human Gene Therapy, 28(6), 450-463 (2017)   The infusion of healthy stem cells into a patient—termed “stem-cell therapy”—has shown great promise for the treatment of genetic and non-genetic diseases, including mucopolysaccharidosis type 1, Parkinson's disease, multiple sclerosis, numerous immunodeficiency disorders, and aplastic anemia. Stem cells for cell therapy can be collected from the patient (autologous) or collected from another “healthy” individual (allogeneic). The use of allogenic stem cells is accompanied with the potentially fatal risk that the transplanted donor T cells will reject the patient's cells—a process termed “graft-versus-host disease.” Therefore, the use of autologous stem cells is preferred, at least from the immunological perspective. However, an obvious drawback is that inherently as “self,” they contain the disease mutation. As such, autologous cells for use in cell therapies often require genetic “correction” (i.e., gene addition or editing) prior to cell infusion and therefore the requirement for some form of nucleic acid delivery, which sets the stage for the AAV controversy discussed herein. Despite being the most clinically applied gene delivery context to date, unlike other more concerning integrating and non-integrating vectors such as retroviruses and adenovirus, those based on adeno-associated virus (AAV) have not been employed in the clinic. Furthermore, published data regarding AAV vector transduction of stem cells are inconsistent in regards to vector transduction efficiency, while the pendulum swings far in the other direction with demonstrations of AAV vector–induced toxicity in undifferentiated cells. The variation present in the literature examining the transduction efficiency of AAV vectors in stem cells may be due to numerous factors, including inconsistencies in stem-cell collection, cell culture, vector preparation, and/or transduction conditions. This review summarizes the controversy surrounding AAV vector transduction of stem cells, hopefully setting the stage for future elucidation and eventual therapeutic applications.    

5.2103           Direct Intracranial Injection of AAVrh8 Encoding Monkey β-N-Acetylhexosaminidase Causes Neurotoxicity in the Primate Brain

Golebiowski, D. et al Human Gene Therapy, 28(6), 510-522 (2017)   GM2 gangliosidoses, including Tay–Sachs disease and Sandhoff disease, are lysosomal storage disorders caused by deficiencies in β-N-acetylhexosaminidase (Hex). Patients are afflicted primarily with progressive central nervous system (CNS) dysfunction. Studies in mice, cats, and sheep have indicated safety and widespread distribution of Hex in the CNS after intracranial vector infusion of AAVrh8 vectors encoding species-specific Hex α- or β-subunits at a 1:1 ratio. Here, a safety study was conducted in cynomolgus macaques (cm), modeling previous animal studies, with bilateral infusion in the thalamus as well as in left lateral ventricle of AAVrh8 vectors encoding cm Hex α- and β-subunits. Three doses (3.2 × 10¹² vg [n = 3]; 3.2 × 10¹¹ vg [n = 2]; or 1.1 × 10¹¹ vg [n = 2]) were tested, with controls infused with vehicle (n = 1) or transgene empty AAVrh8 vector at the highest dose (n = 2). Most monkeys receiving AAVrh8-cmHexα/β developed dyskinesias, ataxia, and loss of dexterity, with higher dose animals eventually becoming apathetic. Time to onset of symptoms was dose dependent, with the highest-dose cohort producing symptoms within a month of infusion. One monkey in the lowest-dose cohort was behaviorally asymptomatic but had magnetic resonance imaging abnormalities in the thalami. Histopathology was similar in all monkeys injected with AAVrh8-cmHexα/β, showing severe white and gray matter necrosis along the injection track, reactive vasculature, and the presence of neurons with granular eosinophilic material. Lesions were minimal to absent in both control cohorts. Despite cellular loss, a dramatic increase in Hex activity was measured in the thalamus, and none of the animals presented with antibody titers against Hex. The high overexpression of Hex protein is likely to blame for this negative outcome, and this study demonstrates the variations in safety profiles of AAVrh8-Hexα/β intracranial injection among different species, despite encoding for self-proteins.  

5.2104           Improved gene delivery to adult mouse spinal cord through the use of engineered hybrid adeno-associated viral serotypes

Siu, J.J., Queen, N:J., Huang, W., Yin, F.Q., Liu, X., Wang, C., McTigue, D.M. and Cao, L. Gene Therapy, 24(6), 361-369 (2017)   Adeno-associated viral (AAV) vectors are often used in gene therapy for neurological disorders because of its safety profile and promising results in clinical trials. One challenge to AAV gene therapy is effective transduction of large numbers of the appropriate cell type, which can be overcome by modulating the viral capsid through DNA shuffling. Our previous study demonstrates that Rec2, among a family of novel engineered hybrid capsid serotypes (Rec1~4) transduces adipose tissue with far superior efficiency than naturally occurring AAV serotypes. Here we assessed the transduction of adult spinal cord at two different doses of AAV vectors expressing green fluorescent protein (2 × 10⁹ or 4 × 10⁸ viral particles) via intraparenchymal injection at the thoracic vertebral level T9. In comparison with an equal dose of the currently preferable AAV9 serotype, Rec3 serotype transduced a broader region of the spinal cord up to ~1.5 cm longitudinally and displayed higher transgene expression and increased maximal transduction rates of astrocytes at either dose and neurons at the lower dose. These novel engineered hybrid vectors could provide powerful tools at lower production costs to manipulate gene expression in the spinal cord for mechanistic studies or provide potent vehicles for gene therapy delivery, such as neurotrophins, to the spinal cord.  

5.2105           Engineering Recombinant Virus-like Nanoparticles from Plants for Cellular Delivery

Brillault, L., Jutras, P.V., Dashti, N., Thuenemann, E.C., Morgan, G., Lomonossoff, G.P., landsberg, M.J. and Sainbury, F. ACS Nano, 11(4), 3476-3484 (2017)   Understanding capsid assembly following recombinant expression of viral structural proteins is critical to the design and modification of virus-like nanoparticles for biomedical and nanotechnology applications. Here, we use plant-based transient expression of the Bluetongue virus (BTV) structural proteins, VP3 and VP7, to obtain high yields of empty and green fluorescent protein (GFP)-encapsidating core-like particles (CLPs) from leaves. Single-particle cryo-electron microscopy of both types of particles revealed considerable differences in CLP structure compared to the crystal structure of infection-derived CLPs; in contrast, the two recombinant CLPs have an identical external structure. Using this insight, we exploited the unencumbered pore at the 5-fold axis of symmetry and the absence of encapsidated RNA to label the interior of empty CLPs with a fluorescent bioconjugate. CLPs containing 120 GFP molecules and those containing approximately 150 dye molecules were both shown to bind human integrin via a naturally occurring Arg-Gly-Asp motif found on an exposed loop of the VP7 trimeric spike. Furthermore, fluorescently labeled CLPs were shown to interact with a cell line overexpressing the surface receptor. Thus, BTV CLPs present themselves as a useful tool in targeted cargo delivery. These results highlight the importance of detailed structural analysis of VNPs in validating their molecular organization and the value of such analyses in aiding their design and further modification.  

5.2106           Achieving the Promise of Therapeutic Extracellular Vesicles: The Devil is in Details of Therapeutic Loading

Sutaria, D.S., Badawi, M., Phelps, M.A. and Schmittgen, T.D. Pharm. Res., 34(5), 1053-1066 (2017)   Extracellular vesicles (EVs) represent a class of cell secreted organelles which naturally contain biomolecular cargo such as miRNA, mRNA and proteins. EVs mediate intercellular communication, enabling the transfer of functional nucleic acids from the cell of origin to the recipient cells. In addition, EVs make an attractive delivery vehicle for therapeutics owing to their increased stability in circulation, biocompatibility, low immunogenicity and toxicity profiles. EVs can also be engineered to display targeting moieties on their surfaces which enables targeting to desired tissues, organs or cells. While much has been learned on the role of EVs as cell communicators, the field of therapeutic EV application is currently under development. Critical to the future success of EV delivery system is the description of methods by which therapeutics can be successfully and efficiently loaded within the EVs. Two methods of loading of EVs with therapeutic cargo exist, endogenous and exogenous loading. We have therefore focused this review on describing the various published approaches for loading EVs with therapeutics.  

5.2107           5-HT2C Receptor Knockdown in the Amygdala Inhibits Neuropathic-Pain-Related Plasticity and Behaviors

Ji, G., Zhang, W., Mahimainathan, L., Narasimhan, M., Kiritoshi, T., Fan, X., Wang, J., Green, T. and Neugebauer, V.

1. Neurosci., 37(6), 1378-1393 (2017)

  Neuroplasticity in the amygdala drives pain-related behaviors. The central nucleus (CeA) serves major amygdala output functions and can generate emotional-affective behaviors and modulate nocifensive responses. The CeA receives excitatory and inhibitory inputs from the basolateral nucleus (BLA) and serotonin receptor subtype 5-HT_2CR in the BLA, but not CeA, has been implicated anxiogenic behaviors and anxiety disorders. Here, we tested the hypothesis that 5-HT_2CR in the BLA plays a critical role in CeA plasticity and neuropathic pain behaviors in the rat spinal nerve ligation (SNL) model. Local 5-HT_2CR knockdown in the BLA with stereotaxic injection of 5-HT_2CR shRNA AAV vector decreased vocalizations and anxiety- and depression-like behaviors and increased sensory thresholds of SNL rats, but had no effect in sham controls. Extracellular single-unit recordings of CeA neurons in anesthetized rats showed that 5-HT_2CR knockdown blocked the increase in neuronal activity (increased responsiveness, irregular spike firing, and increased burst activity) in SNL rats. At the synaptic level, 5-HT_2CR knockdown blocked the increase in excitatory transmission from BLA to CeA recorded in brain slices from SNL rats using whole-cell patch-clamp conditions. Inhibitory transmission was decreased by 5-HT_2CR knockdown in control and SNL conditions to a similar degree. The findings can be explained by immunohistochemical data showing increased expression of 5-HT_2CR in non-GABAergic BLA cells in SNL rats. The results suggest that increased 5-HT_2CR in the BLA contributes to neuropathic-pain-related amygdala plasticity by driving synaptic excitation of CeA neurons. As a rescue strategy, 5-HT_2CR knockdown in the BLA inhibits neuropathic-pain-related behaviors.  

5.2108           Structural Similarities between Neuregulin 1–3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons

Vullhorst, D., Ahmad, T., karavanova, I., Keating, C. and Buonanno, A.

1. Neurosci., 37(21), 5232-5249 (2017)

  The Neuregulin (NRG) family of ErbB ligands is comprised of numerous variants originating from the use of different genes, alternative promoters, and splice variants. NRGs have generally been thought to be transported to axons and presynaptic terminals where they signal via ErbB3/4 receptors in paracrine or juxtacrine mode. However, we recently demonstrated that unprocessed pro-NRG2 accumulates on cell bodies and proximal dendrites, and that NMDAR activity is required for shedding of its ectodomain by metalloproteinases. Here we systematically investigated the subcellular distribution and processing of major NRG isoforms in rat hippocampal neurons. We show that NRG1 isotypes I and II, which like NRG2 are single-pass transmembrane proteins with an Ig-like domain, share the same subcellular distribution and ectodomain shedding properties. We furthermore show that NRG3, like CRD-NRG1, is a dual-pass transmembrane protein that harbors a second transmembrane domain near its amino terminus. Both NRG3 and CRD-NRG1 cluster on axons through juxtacrine interactions with ErbB4 present on GABAergic interneurons. Interestingly, although single-pass NRGs accumulate as unprocessed proforms, axonal puncta of CRD-NRG1 and NRG3 are comprised of processed protein. Mutations of CRD-NRG1 and NRG3 that render them resistant to BACE cleavage, as well as BACE inhibition, result in the loss of axonal puncta and in the accumulation of unprocessed proforms in neuronal soma. Together, these results define two groups of NRGs with distinct membrane topologies and fundamentally different targeting and processing properties in central neurons. The implications of this functional diversity for the regulation of neuronal processes by the NRG/ErbB pathway are discussed.  

5.2109           AAV-mediated delivery of optogenetic constructs to the macaque brain triggers humoral immune responses

Mendoza, S.D., El-Shamayleh, Y. and Horwitz, G.D.

1. Neurophysiol., 117(5), 2004-2013 (2017)

  Gene delivery to the primate central nervous system via recombinant adeno-associated viral vectors (AAV) allows neurophysiologists to control and observe neural activity precisely. A current limitation of this approach is variability in vector transduction efficiency. Low levels of transduction can foil experimental manipulations, prompting vector readministration. The ability to make multiple vector injections into the same animal, even in cases where successful vector transduction has already been achieved, is also desirable. However, vector readministration has consequences for humoral immunity and gene delivery that depend on vector dosage and route of administration in complex ways. As part of optogenetic experiments in rhesus monkeys, we analyzed blood sera collected before and after AAV injections into the brain and quantified neutralizing antibodies to AAV using an in vitro assay. We found that injections of AAV1 and AAV9 vectors elevated neutralizing antibody titers consistently. These immune responses were specific to the serotype injected and were long lasting. These results demonstrate that optogenetic manipulations in monkeys trigger immune responses to AAV capsids, suggesting that vector readministration may have a higher likelihood of success by avoiding serotypes injected previously  

5.2110           Plasmacytoid and conventional dendritic cells cooperate in crosspriming AAV capsid-specific CD8+ T cells

Rogers, G.L., Shirley, J.L., Zolotukhin, I., Kumar, S.R.P., Sherman, A., Perrin, G.Q., Hoffman, B.E., Srivastava, A., Basner-Tschakarjan, E., Wallet, M.A., Terhorst, C., Biswas, M. and Herzog, R.W. Blood, 129(24), 3184-3195 (2017)   Adeno-associated virus (AAV) is a replication-deficient parvovirus that is extensively used as a gene therapy vector. CD8⁺ T-cell responses against the AAV capsid protein can, however, affect therapeutic efficacy. Little is known about the in vivo mechanism that leads to the crosspriming of CD8⁺ T cells against the input viral capsid antigen. In this study, we report that the Toll-like receptor 9 (TLR9)–MyD88 pattern-recognition receptor pathway is uniquely capable of initiating this response. By contrast, the absence of TLR2, STING, or the addition of TLR4 agonist has no effect. Surprisingly, both conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) are required for the crosspriming of capsid-specific CD8⁺ T cells, whereas other antigen-presenting cells are not involved. TLR9 signaling is specifically essential in pDCs but not in cDCs, indicating that sensing of the viral genome by pDCs activates cDCs in trans to cross-present capsid antigen during CD8⁺ T-cell activation. Cross-presentation and crosspriming depend not only on TLR9, but also on interferon type I signaling, and both mechanisms can be inhibited by administering specific molecules to prevent induction of capsid-specific CD8⁺ T cells. Thus, these outcomes directly point to therapeutic interventions and demonstrate that innate immune blockade can eliminate unwanted immune responses in gene therapy.  

5.2111           Safety and Efficacy Evaluation of rAAV2tYF-PR1.7-hCNGA3 Vector Delivered by Subretinal Injection in CNGA3 Mutant Achromatopsia Sheep free access

Gootwine, E., Ofri, R., Banin, E., Obolensky, A., Averbukh, E., Ezra-Elia, R., Ross, M., Honig, H., Rosov, A., Yamin, E., Ye, G-j., Knop, D.R., Robinson, P.M., Chulay, J.D. and Shearman, M.S. Human Gene Therapy Clin. Develop., 28(2), 96-107 (2017)   Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector expressing the human CNGA3 gene designated AGTC-402 (rAAV2tYF-PR1.7-hCNGA3) for the treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. The results are herein reported of a study evaluating safety and efficacy of AGTC-402 in CNGA3-deficient sheep. Thirteen day–blind sheep divided into three groups of four or five animals each received a subretinal injection of an AAV vector expressing a CNGA3 gene in a volume of 500 μL in the right eye. Two groups (n = 9) received either a lower or higher dose of the AGTC-402 vector, and one efficacy control group (n = 4) received a vector similar in design to one previously shown to rescue cone photoreceptor responses in the day-blind sheep model (rAAV5-PR2.1-hCNGA3). The left eye of each animal received a subretinal injection of 500 μL of vehicle (n = 4) or was untreated (n = 9). Subretinal injections were generally well tolerated and not associated with systemic toxicity. Most animals had mild to moderate conjunctival hyperemia, chemosis, and subconjunctival hemorrhage immediately after surgery that generally resolved by postoperative day 7. Two animals treated with the higher dose of AGTC-402 and three of the efficacy control group animals had microscopic findings of outer retinal atrophy with or without inflammatory cells in the retina and choroid that were procedural and/or test-article related. All vector-treated eyes showed improved cone-mediated electroretinography responses with no change in rod-mediated electroretinography responses. Behavioral maze testing under photopic conditions showed significantly improved navigation times and reduced numbers of obstacle collisions in all vector-treated eyes compared to their contralateral control eyes or pre-dose results in the treated eyes. These results support the use of AGTC-402 in clinical studies in patients with achromatopsia caused by CNGA3 mutations, with careful evaluation for possible inflammatory and/or toxic effects.  

5.2112           Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1

Franzoso, F.D., Seyffert, M., Vogel, R., Yakimovich, A., de Andrade Pereira, B., Meier, A.F., Sutter, S.O., Tobler, K., Vogt, B., Greber, U.F., Büning, H., Ackermann, M. and Fraefel, C. J: Virol., 91(15), e00357-17 (2017)   Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G₂-phase cells, while HSV-1 DNA replication is restricted to G₁ phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G₂-phase cells, suggesting that the preference for S/G₂ phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G₂-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate.  

5.2113           Polymorphic Nature of Human T-Cell Leukemia Virus Type 1 Particle Cores as Revealed through Characterization of a Chronically Infected Cell Line

Meissner, M.E., Mendonca, L.M., Zhang, W. and Mansky, L.M.

1. Virol., 91(16), e00369-17 (2017)

  Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 cell-to-cell transmission is dependent on the release of infectious virus particles into the virological synapse. The HTLV-1 particle structure is still poorly understood, and previous studies analyzed viruses produced by transformed lymphocytic cell lines chronically infected with HTLV-1, particularly the MT-2 cell line, which harbors truncated proviruses and expresses aberrant forms of the Gag protein. In this study, we demonstrate that the chronically infected SP cell line harbors a relatively low number of proviruses, making it a more promising experimental system for the study of the HTLV-1 particle structure. We first identified the genomic sites of integration and characterized the genetic structure of the gag region in each provirus. We also determined that despite encoding a truncated Gag protein, only the full-length Gag protein was incorporated into virus particles. Cryo-transmission electron microscopy analyses of the purified virus particles revealed three classes of particles based upon capsid core morphology: complete cores, incomplete cores, and particles without distinct electron densities that would correlate with the capsid region of a core structure. Observed cores were generally polygonal, and virus particles were on average 115 nm in diameter. These data corroborate particle morphologies previously observed for MT-2 cells and provide evidence that the known poor infectivity of HTLV-1 particles may correlate with HTLV-1 particle populations containing few virus particles possessing a complete capsid core structure.  

5.2114           Extracellular Interactions between Hepatitis C Virus and Secreted Apolipoprotein E

Li, Z., Li, Y., Bi, Y., Zhang, H., Tao, Y., Li, Q., Con, W. and Dong, S. J: Virol., 91(15), e02227-16 (2017)   Interactions between hepatitis C virus (HCV) and lipoproteins in humans play an important role in the efficient establishment of chronic infection. Apolipoprotein E (ApoE) on the HCV envelope mediates virus attachment to host cells as well as immune evasion. This interaction is thought to occur in hepatocytes, as ApoE plays dual functions in HCV assembly and maturation as well as cell attachment. In the present study, we found that secreted ApoE (sApoE) can also bind to viral particles via its C-terminal domain after HCV is released from the cell. Furthermore, the binding affinity of interactions between the sApoE N terminus and cell surface receptors affected HCV infectivity in a dose-dependent manner. The extracellular binding of sApoE to HCV is dependent on HCV envelope proteins, and recombinant HCV envelope proteins are also able to bind to sApoE. These results suggest that extracellular interactions between HCV and sApoE may potentially complicate vaccine development and studies of viral pathogenesis.  

5.2115           Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants

Lam, A.M., Ren, S., Espiritu, C., Kelly, M., Lau, V., Zheng, L., Hartman, G.D., Flores, O.A. and Klumpp, K. Antimicrob. Agents Chemother., 61(8), e00680-17 (2017)   The hepatitis B virus (HBV) core protein serves multiple essential functions in the viral life cycle, and antiviral agents that target the core protein are being developed. Capsid assembly modulators (CAMs) are compounds that target core and misdirect capsid assembly, resulting in the suppression of HBV replication and virion production. Besides HBV DNA, circulating HBV RNA has been detected in patient serum and can be associated with the treatment response. Here we studied the effect of HBV CAMs on the production of extracellular HBV RNA using infected HepaRG cells and primary human hepatocytes. Representative compounds from the sulfonamide carboxamide and heteroaryldihydropyrimidine series of CAMs were evaluated and compared to nucleos(t)ide analogs as inhibitors of the viral polymerase. The results showed that CAMs blocked extracellular HBV RNA with efficiencies similar to those with which they blocked pregenomic RNA (pgRNA) encapsidation, HBV DNA replication, and Dane particle production. Nucleos(t)ide analogs inhibited viral replication and virion production but not encapsidation or production of extracellular HBV RNA. Profiling of HBV RNA from both culture supernatants and patient serum showed that extracellular viral RNA consisted of pgRNA and spliced pgRNA variants with an internal deletion(s) but still retained the sequences at both the 5′ and 3′ ends. Similar variants were detected in the supernatants of infected cells with and without nucleos(t)ide analog treatment. Overall, our data demonstrate that HBV CAMs represent direct antiviral agents with a profile differentiated from that of nucleos(t)ide analogs, including the inhibition of extracellular pgRNA and spliced pgRNA.  

5.2116           Development of a Novel Virus-Like Particle Vaccine Platform That Mimics the Immature Form of Alphavirus

Urakami, A., Sakurai, A., Ishikawa, M., Yap, M.L., Flores-garcia, Y., Haseda, Y., Aoshi, T., Zavala, F.P., Rossmann, M.G., Kuno, S., Ueno, R. and Akahata, W. Clin. Vaccine Immunol., 24(7), e00090-17 (2017)   Virus-like particles (VLPs) are noninfectious multiprotein structures that are engineered to self-assemble from viral structural proteins. Here, we developed a novel VLP-based vaccine platform utilizing VLPs from the chikungunya virus. We identified two regions within the envelope protein, a structural component of chikungunya, where foreign antigens can be inserted without compromising VLP structure. Our VLP displays 480 copious copies of an inserted antigen on the VLP surface in a highly symmetric manner and is thus capable of inducing strong immune responses against any inserted antigen. Furthermore, by mimicking the structure of the immature form of the virus, we altered our VLP's in vivo dynamics and enhanced its immunogenicity. We used the circumsporozoite protein (CSP) of the Plasmodium falciparum malaria parasite as an antigen and demonstrated that our VLP-based vaccine elicits strong immune responses against CSP in animals. The sera from immunized monkeys protected mice from malaria infection. Likewise, mice vaccinated with P. yoelii CSP-containing VLPs were protected from an infectious sporozoite challenge. Hence, our uniquely engineered VLP platform can serve as a blueprint for the development of vaccines against other pathogens and diseases.  

5.2117           MicroRNA cluster miR-17-92 regulates multiple functionally related voltage-gated potassium channels in chronic neuropathic pain

Sakai, A., Saitow, F., maruyama, M., Miyake, N., Miyake, K., Shimada, T., Okada, T. AND Suzuki, H. Nature Communications, 8:16079 (2017)   miR-17-92 is a microRNA cluster with six distinct members. Here, we show that the miR-17-92 cluster and its individual members modulate chronic neuropathic pain. All cluster members are persistently upregulated in primary sensory neurons after nerve injury. Overexpression of miR-18a, miR-19a, miR-19b and miR-92a cluster members elicits mechanical allodynia in rats, while their blockade alleviates mechanical allodynia in a rat model of neuropathic pain. Plausible targets for the miR-17-92 cluster include genes encoding numerous voltage-gated potassium channels and their modulatory subunits. Single-cell analysis reveals extensive co-expression of miR-17-92 cluster and its predicted targets in primary sensory neurons. miR-17-92 downregulates the expression of potassium channels, and reduced outward potassium currents, in particular A-type currents. Combined application of potassium channel modulators synergistically alleviates mechanical allodynia induced by nerve injury or miR-17-92 overexpression. miR-17-92 cluster appears to cooperatively regulate the function of multiple voltage-gated potassium channel subunits, perpetuating mechanical allodynia.  

5.2118           Entorhinal tau pathology disrupts hippocampal-prefrontal oscillatory coupling during associative learning

Tanninen, S.E., Nouriziabari, B., Morrisey, M.D., Bakir, R., Dayton, R.D., Klein, R.L., Takehara-Nishiuchi, K. Neurobiol. Aging, 58, 151-162 (2017)   A neural signature of asymptomatic preclinical Alzheimer's disease (AD) is disrupted connectivity between brain regions; however, its underlying mechanisms remain unknown. Here, we tested whether a preclinical pathologic feature, tau aggregation in the entorhinal cortex (EC) is sufficient to disrupt the coordination of local field potentials (LFPs) between its efferent regions. P301L-mutant human tau or green fluorescent protein (GFP) was virally overexpressed in the EC of adult rats. LFPs were recorded from the dorsal hippocampus and prelimbic medial prefrontal cortex while the rats underwent trace eyeblink conditioning where they learned to associate 2 stimuli separated by a short time interval. In GFP-expressing rats, the 2 regions strengthened phase-phase and amplitude-amplitude couplings of theta and gamma oscillations during the interval separating the paired stimuli. Despite normal memory acquisition, this learning-related, inter-region oscillatory coupling was attenuated in the tau-expressing rats while prefrontal phase-amplitude theta-gamma cross-frequency coupling was elevated. Thus, EC tau aggregation caused aberrant long-range circuit activity during associative learning, identifying a culprit for the neural signature of preclinical AD stages.  

5.2119           TFEB-mediated activation of the lysosome-autophagy system affects the transduction efficiency of adeno-associated virus 2

Popp, L., Gomez, E., Orji, W., Ho, M., Suh, J. And Segatori, L. Virology, 510, 1-8 (2017)   Adeno-associated virus (AAV)-mediated gene transfer is an appealing therapeutic option due to AAV's safety profile. Effective delivery of AAV's genetic cargo to the nucleus, however, requires evasion of host cell barriers, including cellular clearance mechanisms mediated by the lysosome-autophagy system. We used AAV serotype 2 to monitor the autophagic response to cellular internalization of AAV and to characterize the effect of AAV-induced activation of autophagy on transgene expression. We found AAV2 internalization to induce activation of transcription factor EB, a master regulator of autophagy and lysosomal biogenesis, and upregulation of the lysosome-autophagy system. We showed that AAV2-induced activation of autophagy parallels a reduction in transgene expression, but also an increase in autophagic clearance of protein aggregates. These results can inform the design of AAV vectors with autophagy-modulating properties for applications ranging from the design of efficient gene delivery vectors to the treatment of diseases characterized by accumulation of autophagic cargo.  

5.2120           The function of DNA binding protein nucleophosmin in AAV replication

Satkunanathan, S., Thorpe, R. and Zhao, Y. Virology, 510, 46-54 (2017)   Adeno-associated viruses (AAV) contain minimal viral proteins necessary for their replication. During virus assembly, AAV acquire, inherently and submissively, various cellular proteins. Our previous studies identified the association of AAV vectors with the DNA binding protein nucleophosmin (NPM1). Nucleophosmin has been reported to enhance AAV infection by mobilizing AAV capsids into and out of the nucleolus, indicating the importance of NPM1 in the AAV life cycle; however the role of NPM1 in AAV production remains unknown. In this study, we systematically investigated NPM1 function on AAV production using NPM1 knockdown cells and revealing for the first time the presence of G-quadruplex DNA sequences (GQRS) in the AAV genome, the synergistic NPM1-GQRS function in AAV production and the significant enhancement of NPM1 gene knockdown on AAV vector production. Understanding the role of cellular proteins in the AAV life cycle will greatly facilitate high titre production of AAV vectors for clinical use.  

5.2121           Cryo-EM maps reveal five-fold channel structures and their modification by gatekeeper mutations in the parvovirus minute virus of mice (MVM) capsid

Subramanian, S., Organtini, L.J., Grossman, A., Domeier, P.P., Cifuente, J.O., Makhov, A.M., Conway, J.F., D’Abramo Jr., A., Cotmore, S.F., Tattersall, P. and Hafenstein, S. Virology, 510, 216-223 (2017)   In minute virus of mice (MVM) capsids, icosahedral five-fold channels serve as portals mediating genome packaging, genome release, and the phased extrusion of viral peptides. Previous studies suggest that residues L172 and V40 are essential for channel function. The structures of MVMi wildtype, and mutant L172T and V40A virus-like particles (VLPs) were solved from cryo-EM data. Two constriction points, termed the mid-gate and inner-gate, were observed in the channels of wildtype particles, involving residues L172 and V40 respectively. While the mid-gate of V40A VLPs appeared normal, in L172T adjacent channel walls were altered, and in both mutants there was major disruption of the inner-gate, demonstrating that direct L172:V40 bonding is essential for its structural integrity. In wildtype particles, residues from the N-termini of VP2 map into claw-like densities positioned below the channel opening, which become disordered in the mutants, implicating both L172 and V40 in the organization of VP2 N-termini.  

5.2122           Rationally Engineered AAV Capsids Improve Transduction and Volumetric Spread in the CNS

Kanaan, N.M., Sellnow, R.C., Boye, S.L., Coberly, B., Bennett, A., Agbandje-McKenna, M., Sortwell, C.E., Hauswirth, W.W., Boye, S.E. and Manfredsson, F.P: Molecular Therapy – Nucleic Acids, 8, 184-197 (2017)   Adeno-associated virus (AAV) is the most common vector for clinical gene therapy of the CNS. This popularity originates from a high safety record and the longevity of transgene expression in neurons. Nevertheless, clinical efficacy for CNS indications is lacking, and one reason for this is the relatively limited spread and transduction efficacy in large regions of the human brain. Using rationally designed modifications of the capsid, novel AAV capsids have been generated that improve intracellular processing and result in increased transgene expression. Here, we sought to improve AAV-mediated neuronal transduction to minimize the existing limitations of CNS gene therapy. We investigated the efficacy of CNS transduction using a variety of tyrosine and threonine capsid mutants based on AAV2, AAV5, and AAV8 capsids, as well as AAV2 mutants incapable of binding heparan sulfate (HS). We found that mutating several tyrosine residues on the AAV2 capsid significantly enhanced neuronal transduction in the striatum and hippocampus, and the ablation of HS binding also increased the volumetric spread of the vector. Interestingly, the analogous tyrosine substitutions on AAV5 and AAV8 capsids did not improve the efficacy of these serotypes. Our results demonstrate that the efficacy of CNS gene transfer can be significantly improved with minor changes to the AAV capsid and that the effect is serotype specific.  

5.2123           Anti-adenoviral Artificial MicroRNAs Expressed from AAV9 Vectors Inhibit Human Adenovirus Infection in Immunosuppressed Syrian Hamsters

Schaar, K., Geisler, A., Kraus, M., Pinkert, S., Pryshliak, M., Spencer, J.F., Tollefson, A.E., Ying, B., Kurreck, J., Wold, W.S., Klopfleisch, R., Toth, K. and Fechner, H. Molecular Therapy – Nucleic Acids, 8, 300-316 (2017)   Infections of immunocompromised patients with human adenoviruses (hAd) can develop into life-threatening conditions, whereas drugs with anti-adenoviral efficiency are not clinically approved and have limited efficacy. Small double-stranded RNAs that induce RNAi represent a new class of promising anti-adenoviral therapeutics. However, as yet, their efficiency to treat hAd5 infections has only been investigated in vitro. In this study, we analyzed artificial microRNAs (amiRs) delivered by self-complementary adeno-associated virus (scAAV) vectors for treatment of hAd5 infections in immunosuppressed Syrian hamsters. In vitro evaluation of amiRs targeting the E1A, pTP, IVa2, and hexon genes of hAd5 revealed that two scAAV vectors containing three copies of amiR-pTP and three copies of amiR-E1A, or six copies of amiR-pTP, efficiently inhibited hAd5 replication and improved the viability of hAd5-infected cells. Prophylactic application of amiR-pTP/amiR-E1A- and amiR-pTP-expressing scAAV9 vectors, respectively, to immunosuppressed Syrian hamsters resulted in the reduction of hAd5 levels in the liver of up to two orders of magnitude and in reduction of liver damage. Concomitant application of the vectors also resulted in a decrease of hepatic hAd5 infection. No side effects were observed. These data demonstrate anti-adenoviral RNAi as a promising new approach to combat hAd5 infection.  

5.2124           CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors

Bak, R.O. and Porteus, M.H. Cell Reports, 20, 750-756 (2017)   The CRISPR/Cas9 system has recently been shown to facilitate high levels of precise genome editing using adeno-associated viral (AAV) vectors to serve as donor template DNA during homologous recombination (HR). However, the maximum AAV packaging capacity of ∼4.5 kb limits the donor size. Here, we overcome this constraint by showing that two co-transduced AAV vectors can serve as donors during consecutive HR events for the integration of large transgenes. Importantly, the method involves a single-step procedure applicable to primary cells with relevance to therapeutic genome editing. We use the methodology in primary human T cells and CD34⁺ hematopoietic stem and progenitor cells to site-specifically integrate an expression cassette that, as a single donor vector, would otherwise amount to a total of 6.5 kb. This approach now provides an efficient way to integrate large transgene cassettes into the genomes of primary human cells using HR-mediated genome editing with AAV vectors.  

5.2125           Distinct Ventral Pallidal Neural Populations Mediate Separate Symptoms of Depression

Knowland, D., Lilascharoen, V., Pham, C., Shin, S., Wang, E.H-J. and Lim, B.K. Cell, 170, 284-297 (2017)   Major depressive disorder (MDD) patients display a common but often variable set of symptoms making successful, sustained treatment difficult to achieve. Separate depressive symptoms may be encoded by differential changes in distinct circuits in the brain, yet how discrete circuits underlie behavioral subsets of depression and how they adapt in response to stress has not been addressed. We identify two discrete circuits of parvalbumin-positive (PV) neurons in the ventral pallidum (VP) projecting to either the lateral habenula or ventral tegmental area contributing to depression. We find that these populations undergo different electrophysiological adaptations in response to social defeat stress, which are normalized by antidepressant treatment. Furthermore, manipulation of each population mediates either social withdrawal or behavioral despair, but not both. We propose that distinct components of the VP PV circuit can subserve related, yet separate depressive-like phenotypes in mice, which could ultimately provide a platform for symptom-specific treatments of depression.  

5.2126           Selective Optogenetic Control of Purkinje Cells in Monkey Cerebellum

El-Shamayleh, Y., Kojima, Y.K., Soetedjo, R. and Horwitz, G.D. Neuron, 95, 51-62 (2017)   Purkinje cells of the primate cerebellum play critical but poorly understood roles in the execution of coordinated, accurate movements. Elucidating these roles has been hampered by a lack of techniques for manipulating spiking activity in these cells selectively—a problem common to most cell types in non-transgenic animals. To overcome this obstacle, we constructed AAV vectors carrying the channelrhodopsin-2 (ChR2) gene under the control of a 1 kb L7/Pcp2 promoter. We injected these vectors into the cerebellar cortex of rhesus macaques and tested vector efficacy in three ways. Immunohistochemical analyses confirmed selective ChR2 expression in Purkinje cells. Neurophysiological recordings confirmed robust optogenetic activation. Optical stimulation of the oculomotor vermis caused saccade dysmetria. Our results demonstrate the utility of AAV–L7–ChR2 for revealing the contributions of Purkinje cells to circuit function and behavior, and they attest to the feasibility of promoter-based, targeted, genetic manipulations in primates.  

5.2127           BDNF over-expression induces striatal serotonin fiber sprouting and increases the susceptibility to l-DOPA-induced dyskinesia in 6-OHDA-lesioned rats

Tronci, E., Mapolitano, F., Munoz, A., Fidalgo, C., Rossi, F., Björklund, A., Usiello, A. and Carta, M. Exp. Neurol., 297, 73-81 (2017)   In addition to its role in neuronal survival, the brain neurotrophic factor (BDNF) has been shown to influence serotonin transmission and synaptic plasticity, events strongly implicated in the appearance of l-DOPA-induced dyskinesia (LID), a motor complication occurring in parkinsonian patients after long-term treatment with the dopamine precursor.   In order to evaluate a possible influence of BDNF in the appearance of LID, 6-OHDA-lesioned rats received a striatal injection of different concentrations of an adeno-associated viral (AAV) vector over-expressing either BDNF or GFP, as control vector. Eight weeks later, animals started to receive a daily treatment with l-DOPA (4–6 mg/kg plus benserazide 4–6 mg/kg, s.c.) or saline, and dyskinesias, as well as l-DOPA-induced rotations, were evaluated at several time-points. Moreover, molecular changes in striatal D1 receptor-dependent cAMP/PKA and ERK/mTORC signaling pathways, as well as, sprouting of striatal serotonin axons, were measured. Results showed that the AAV-BDNF vector injection induced striatal over-expression of BDNF, as well as striatal and pallidal serotonin axon hyperinnervation. Moreover, rats that over-expressed BDNF were more prone to develop LID and l-DOPA-induced rotations, compared to the GFP-treated control group. Finally, rats that over-expressed BDNF showed increased levels of striatal D1R-dependent signaling phospho-proteins in response to l-DOPA administration. This study suggests that BDNF over-expression, by inducing changes in pre-synaptic serotonin axonal trophism, is able to exacerbate maladaptive responses to l-DOPA administration.  

5.2128           Codon-Optimized RPGR Improves Stability and Efficacy of AAV8 Gene Therapy in Two Mouse Models of X-Linked Retinitis Pigmentosa

Fischer, M.D., McClements, M.E., Martinez-Fernandez de la Camara, C., Bellingrath, J-S., Dauletbekov, D., Ramsden, S.C., Hickey, D.G., barbard, A.R. and Maclaren, R.E.   X-linked retinitis pigmentosa (XLRP) is generally a severe form of retinitis pigmentosa, a neurodegenerative, blinding disorder of the retina. 70% of XLRP cases are due to mutations in the retina-specific isoform of the gene encoding retinitis pigmentosa GTPase regulator (RPGR^ORF15). Despite successful RPGR^ORF15 gene replacement with adeno-associated viral (AAV) vectors being established in a number of animal models of XLRP, progression to human trials has not yet been possible. The inherent sequence instability in the purine-rich region of RPGR^ORF15 (which contains highly repetitive nucleotide sequences) leads to unpredictable recombination errors during viral vector cloning. While deleted RPGR may show some efficacy in animal models, which have milder disease, the therapeutic effect of a mutated RPGR variant in patients with XLRP cannot be predicted. Here, we describe an optimized gene replacement therapy for human XLRP disease using an AAV8 vector that reliably and consistently produces the full-length correct RPGR protein. The glutamylation pattern in the RPGR protein derived from the codon-optimized sequence is indistinguishable from the wild-type variant, implying that codon optimization does not significantly alter post-translational modification. The codon-optimized sequence has superior stability and expression levels in vitro. Significantly, when delivered by AAV8 vector and driven by the rhodopsin kinase promoter, the codon-optimized RPGR rescues the disease phenotype in two relevant animal models (Rpgr^−/y and C57BL/6J^Rd9/Boc) and shows good safety in C57BL6/J wild-type mice. This work provides the basis for clinical trial development to treat patients with XLRP caused by RPGR mutations.  

5.2129           Activity-Dependent Gating of Parvalbumin Interneuron Function by the Perineuronal Net Protein Brevican

Favuzzi, E., Marques-Smith., Deogracias, R. et al Neuron, 95, 639-655 (2017)   Activity-dependent neuronal plasticity is a fundamental mechanism through which the nervous system adapts to sensory experience. Several lines of evidence suggest that parvalbumin (PV+) interneurons are essential in this process, but the molecular mechanisms underlying the influence of experience on interneuron plasticity remain poorly understood. Perineuronal nets (PNNs) enwrapping PV+ cells are long-standing candidates for playing such a role, yet their precise contribution has remained elusive. We show that the PNN protein Brevican is a critical regulator of interneuron plasticity. We find that Brevican simultaneously controls cellular and synaptic forms of plasticity in PV+ cells by regulating the localization of potassium channels and AMPA receptors, respectively. By modulating Brevican levels, experience introduces precise molecular and cellular modifications in PV+ cells that are required for learning and memory. These findings uncover a molecular program through which a PNN protein facilitates appropriate behavioral responses to experience by dynamically gating PV+ interneuron function.  

5.2130           ssAAVs containing cassettes encoding SaCas9 and guides targeting hepatitis B virus inactivate replication of the virus in cultured cells

Scott, T., Moyo, B., Nicholson, S., Maepa, M.B., Watashi, K., Ely, A., Weinberg, M.S. and Arbuthnot, P. Scientific Reports, 7:7401 (2017)   Management of infection with hepatitis B virus (HBV) remains a global health problem. Persistence of stable covalently closed circular DNA (cccDNA) during HBV replication is responsible for modest curative efficacy of currently licensed drugs. Novel gene editing technologies, such as those based on CRISPR/Cas9, provide the means for permanently disabling cccDNA. However, efficient delivery of antiviral sequences to infected hepatocytes is challenging. A limiting factor is the large size of sequences encoding Cas9 from Streptococcus pyogenes, and resultant incompatibility with the popular single stranded adeno-associated viral vectors (ssAAVs). We thus explored the utility of ssAAVs for delivery of engineered CRISPR/Cas9 of Staphylococcus aureus (Sa), which is encoded by shorter DNA sequences. Short guide RNAs (sgRNAs) were designed with cognates in the S open reading frame of HBV and incorporated into AAVs that also encoded SaCas9. Intended targeted mutation of HBV DNA was observed after transduction of cells with the all-in-one vectors. Efficacy against HBV-infected hNTCP-HepG2 cells indicated that inactivation of cccDNA was successful. Analysis of likely off-target mutagenesis revealed no unintended sequence changes. Use of ssAAVs to deliver all components required to disable cccDNA by SaCas9 is novel and the technology has curative potential for HBV infection.  

5.2131           Mitochondrial division inhibitor-1 is neuroprotective in the A53T-α-synuclein rat model of Parkinson’s disease

Bido, S., Soria, F.N., Fan, R.Z., Bezard, E. and Tieu, K. Scientific Reports, 7:7495 (2017)   Alpha-synuclein (α-syn) is involved in both familial and sporadic Parkinson’s disease (PD). One of the proposed pathogenic mechanisms of α-syn mutations is mitochondrial dysfunction. However, it is not entirely clear the impact of impaired mitochondrial dynamics induced by α-syn on neurodegeneration and whether targeting this pathway has therapeutic potential. In this study we evaluated whether inhibition of mitochondrial fission is neuroprotective against α-syn overexpression in vivo. To accomplish this goal, we overexpressed human A53T-α- synuclein (hA53T-α-syn) in the rat nigrostriatal pathway, with or without treatment using the small molecule Mitochondrial Division Inhibitor-1 (mdivi-1), a putative inhibitor of the mitochondrial fission Dynamin-Related Protein-1 (Drp1). We show here that mdivi-1 reduced neurodegeneration, α-syn aggregates and normalized motor function. Mechanistically, mdivi-1 reduced mitochondrial fragmentation, mitochondrial dysfunction and oxidative stress. These in vivo results support the negative role of mutant α-syn in mitochondrial function and indicate that mdivi-1 has a high therapeutic potential for PD.  

5.2132           Non-clinical safety and efficacy of a recombinant AAV2/8 vector administered intravenously for treatment of mucopolysaccharidosis type VI

Ferla, R., Alliegro, M., Marteau, J-B., Dell’Anno, M., Nusco, E., Pouillot, S., Galimberti, S., Valsecchi, M.G., Zuliani, V. and Auricchio, A. Molecular Therapy: Methods & Clin. Development, 6,143-158 (2017)   In vivo gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. We recently demonstrated that AAV8-mediated liver gene transfer is effective in animal models of mucopolysaccharidosis type VI (MPS VI), a rare lysosomal storage disease that is caused by arylsulfatase B (ARSB) deficiency. In preparing for a first-in-human trial, we performed non-clinical studies to assess the safety of intravenous administrations of AAV2/8.TBG.hARSB produced under good manufacturing practice-like conditions. No toxicity was observed in AAV-treated mice, except for a transient increase in alanine aminotransferase in females and thyroid epithelial hypertrophy. AAV2/8.TBG.hARSB biodistribution and expression confirmed the liver as the main site of both infection and transduction. Shedding and breeding studies suggest that the risk of both horizontal and germline transmission is minimal. An AAV dose-response study in MPS VI mice was performed to define the range of doses to be used in the clinical study. Overall, these data support the non-clinical safety and efficacy of AAV2/8.TBG.hARSB and pave the way for a phase I/II clinical trial based on intravascular infusions of AAV8 in patients with MPS VI.  

5.2133           Minimal Purkinje Cell-Specific PCP2/L7 Promoter Virally Available for Rodents and Non-human Primates

Nitta, K., Matsuzaki, Y., Konno, A. and Hirai, H. Molecular Therapy: Methods in Clin. Development, 6, 159-170 (2017)   Cell-type-specific promoters in combination with viral vectors and gene-editing technology permit efficient gene manipulation in specific cell populations. Cerebellar Purkinje cells play a pivotal role in cerebellar functions. Although the Purkinje cell-specific L7 promoter is widely used for the generation of transgenic mice, it remains unsuitable for viral vectors because of its large size (3 kb) and exceedingly weak promoter activity. Here, we found that the 0.8-kb region (named here as L7-6) upstream of the transcription initiation codon in the first exon was alone sufficient as a Purkinje cell-specific promoter, presenting a far stronger promoter activity over the original 3-kb L7 promoter with a sustained significant specificity to Purkinje cells. Intravenous injection of adeno-associated virus vectors that are highly permeable to the blood-brain barrier confirmed the Purkinje cell specificity of the L7-6 in the CNS. The features of the L7-6 were also preserved in the marmoset, a non-human primate. The high sequence homology of the L7-6 among mouse, marmoset, and human suggests the preservation of the promoter strength and Purkinje cell specificity features also in humans. These findings suggest that L7-6 will facilitate the cerebellar research targeting the pathophysiology and gene therapy of cerebellar disorders.  

5.2134           Host-derived apolipoproteins play comparable roles with viral secretory proteins Erns and NS1 in the infectious particle formation of Flaviviridae

Fukuhara, T. et al PloS Pathogens, 13(6), e1006475 (2017)   Amphipathic α-helices of exchangeable apolipoproteins have shown to play crucial roles in the formation of infectious hepatitis C virus (HCV) particles through the interaction with viral particles. Among the Flaviviridae members, pestivirus and flavivirus possess a viral structural protein E^rns or a non-structural protein 1 (NS1) as secretory glycoproteins, respectively, while Hepacivirus including HCV has no secretory glycoprotein. In case of pestivirus replication, the C-terminal long amphipathic α-helices of E^rns are important for anchoring to viral membrane. Here we show that host-derived apolipoproteins play functional roles similar to those of virally encoded E^rns and NS1 in the formation of infectious particles. We examined whether E^rns and NS1 could compensate for the role of apolipoproteins in particle formation of HCV in apolipoprotein B (ApoB) and ApoE double-knockout Huh7 (BE-KO), and non-hepatic 293T cells. We found that exogenous expression of either E^rns or NS1 rescued infectious particle formation of HCV in the BE-KO and 293T cells. In addition, expression of apolipoproteins or NS1 partially rescued the production of infectious pestivirus particles in cells upon electroporation with an E^rns-deleted non-infectious RNA. As with exchangeable apolipoproteins, the C-terminal amphipathic α-helices of E^rns play the functional roles in the formation of infectious HCV or pestivirus particles. These results strongly suggest that the host- and virus-derived secretory glycoproteins have overlapping roles in the viral life cycle of Flaviviridae, especially in the maturation of infectious particles, while E^rns and NS1 also participate in replication complex formation and viral entry, respectively. Considering the abundant hepatic expression and liver-specific propagation of these apolipoproteins, HCV might have evolved to utilize them in the formation of infectious particles through deletion of a secretory viral glycoprotein gene.  

5.2135           Adeno-associated viral serotypes differentially transduce inhibitory neurons within the rat amygdala

De Solis, C.A., Hosek, M.P., Holehonnur, R., Ho, A., Luong, A.B.J.A., Jones, L.E., Chaturvedi, D. and Ploski, J.E. Brain Res., 1672, 148-162 (2017)   Recombinant adeno-associated viruses (AAV) are frequently used to make localized genetic manipulations within the rodent brain. It is accepted that the different viral serotypes possess differing affinities for particular cell types, but it is not clear how these properties affect their ability to transduce specific neuronal cell sub-types. Here, we examined ten AAV serotypes for their ability to transduce neurons within the rat basal and lateral nuclei of the amygdala (BLA) and the central nucleus of the amygdala (CeA). AAV2 based viral genomes designed to express either green fluorescent protein (GFP) from a glutamate decarboxylase (GAD65) promoter or the far-red fluorescent protein (E2-Crimson) from a phosphate-activated glutaminase (PAG) promoter were created and pseudotyped as AAV2/1, AAV2/4, AAV2/5, AAV2/6, AAV2/7, AAV 2/8, AAV2/9, AAV2/rh10, AAV2/DJ and AAV2/DJ8. These viruses were infused into the BLA and CeA at equal titers and twenty-one days later tissue within the amygdala was examined for viral transduction efficiency. These serotypes transduced neurons with similar efficiency, except for AAV4 and AAV5, which exhibited significantly less efficient neuronal transduction. Notably, AAV4 and AAV5 possess the most divergent capsid protein sequences compared to the other commonly available serotypes. We found that the Gad65-GFP virus did not exclusively express GFP within inhibitory neurons, as assessed by fluorescent in situ hybridization (FISH), but when this virus was used to transduce CeA neurons, the majority of the neurons that expressed GFP were in fact inhibitory neurons and this was likely due to the fact that this nucleus contains a very high percentage of inhibitory neurons.  

5.2136           Maturation of secreted HCV particles by incorporation of secreted ApoE protects from antibodies by enhancing infectivity

Bankwitz, D., Doepke, M., Hueging, K., Weller, R., Bruening, J., Behrendt, P., Lee, J-Y., Vondran, F.W.R., Manns, M.P., Bartenschlager, R. and Pietschmann, T.

1. Hepatol., 67, 480-489 (2017)

  Background & Aims Hepatitis C virus (HCV) evades humoral immunity and establishes chronic infections. Virus particles circulate in complex with lipoproteins facilitating antibody escape. Apolipoprotein E (ApoE) is essential for intracellular HCV assembly and for HCV cell entry. We aimed to explore if ApoE released from non-infected cells interacts with and modulates secreted HCV particles. Methods ApoE secreted from non-infected cells was incubated with HCV from primary human hepatocytes or Huh-7.5 cells. Co-immunoprecipitation, viral infectivity and neutralization experiments were conducted. Results Physiological levels of secreted ApoE (10–60 µg/ml) enhanced the infectivity of HCV up to 8-fold across all genotypes, which indirectly decreased virus neutralization by antibodies targeting E1 or E2 up to 10-fold. Infection enhancement was observed for particles produced in primary human hepatocytes and Huh-7.5 cells. Selective depletion of ApoE ablated infection enhancement. Addition of HA-tagged ApoE to HCV particles permitted co-precipitation of HCV virions. Serum ApoE levels ranged between 10–60 µg/ml, which is ca 100-fold higher than in Huh-7.5 conditioned cell culture fluids. Serum-derived HCV particles carried much higher amounts of ApoE than cell culture-derived HCV particles. Serum ApoE levels correlated with efficiency of co-precipitation of HCV upon exogenous addition of HA-ApoE. ApoE-dependent infection enhancement was independent of the hypervariable region 1 and SR-B1, but was dependent on heparan sulfate proteoglycans (HSPGs). Conclusions Physiological quantities of secreted ApoE stimulate HCV infection and increase antibody escape, by incorporating into virus particles and enhancing particle interactions with cellular HSPGs. Thus, secreted particles undergo ApoE-dependent maturation to enhance infectivity and to facilitate evasion from neutralizing antibodies.  

5.2137           Primary sensory neuron-specific interference of TRPV1 signaling by adeno-associated virus-encoded TRPV1 peptide aptamer attenuates neuropathic pain

Xiang, H., Liu, Z., Wang, F., Xu, H., Roberts, C., Fischer, G., Stucky, C.L., Dean, C., Pan, B., Hogan, Q.H. and Yu, H. Mol. Pain, 13, 1-8 (2017)   Background TRPV1 (transient receptor potential vanilloid subfamily member 1) is a pain signaling channel highly expressed in primary sensory neurons. Attempts for analgesia by systemic TRPV1 blockade produce undesirable side effects, such as hyperthermia and impaired heat pain sensation. One approach for TRPV1 analgesia is to target TRPV1 along the peripheral sensory pathway. Results For functional blockade of TRPV1 signaling, we constructed an adeno-associated virus (AAV) vector expressing a recombinant TRPV1 interfering peptide aptamer, derived from a 38mer tetrameric assembly domain (TAD), encompassing residues 735 to 772 of rat TRPV1, fused to the C-terminus of enhanced green fluorescent protein (EGFP). AAV-targeted sensory neurons expressing EGFP-TAD after vector injection into the dorsal root ganglia (DRG) revealed decreased inward calcium current and diminished intracellular calcium accumulation in response to capsaicin, compared to neurons of naïve or expressing EGFP alone. To examine the potential for treating neuropathic pain, AAV-EGFP-TAD was injected into fourth and fifth lumbar (L) DRGs of rats subjected to neuropathic pain by tibial nerve injury (TNI). Results showed that AAV-directed selective expression of EGFP-TAD in L4/L5 DRG neuron somata, and their peripheral and central axonal projections can limit TNI-induced neuropathic pain behavior, including hypersensitivity to heat and, to a less extent, mechanical stimulation. Conclusion Selective inhibition of TRPV1 activity in primary sensory neurons by DRG delivery of AAV-encoded analgesic interfering peptide aptamers is efficacious in attenuation of neuropathic pain. With further improvements of vector constructs and in vivo application, this approach might have the potential to develop as an alternative gene therapy strategy to treat chronic pain, especially heat hypersensitivity, without complications due to systemic TRPV1 blockade.  

5.2138           Transforming Growth Factor‐β Receptor III is a Potential Regulator of Ischemia‐Induced Cardiomyocyte Apoptosis

Sun, F. et al

J. Am. Heart Assoc., 6, e005357 (2017)

Background Myocardial infarction (MI) is often accompanied by cardiomyocyte apoptosis, which decreases heart function and leads to an increased risk of heart failure. The aim of this study was to examine the effects of transforming growth factor‐β receptor III (TGFβR3) on cardiomyocyte apoptosis during MI.

Methods and Results An MI mouse model was established by left anterior descending coronary artery ligation. Cell viability, apoptosis, TGFβR3, and mitogen‐activated protein kinase signaling were assessed by methylthiazolyldiphenyl‐tetrazolium bromide assay, terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling assay, immunofluorescence, electron microscopy, and Western blotting. Our results demonstrated that TGFβR3 expression in the border region of the heart was dynamically changed during MI. After stimulation with H2O2, TGFβR3 overexpression in cardiomyocytes led to increased cell apoptosis and activation of p38 signaling, whereas TGFβR3 knockdown had the opposite effect. ERK1/2 and JNK1/2 signaling was not altered by TGFβR3 modulation, and p38 inhibitor (SB203580) reduced the effect of TGFβR3 on apoptosis, suggesting that p38 has a nonredundant function in activating apoptosis. Consistent with the in vitro observations, cardiac TGFβR3 transgenic mice showed augmented cardiomyocyte apoptosis, enlarged infarct size, increased injury, and enhanced p38 signaling upon MI. Conversely, cardiac loss of function of TGFβR3 by adeno‐associated viral vector serotype 9–TGFβR3 short hairpin RNA attenuated the effects of MI in mice.

Conclusions TGFβR3 promotes apoptosis of cardiomyocytes via a p38 pathway–associated mechanism, and loss of TGFβR3 reduces MI injury, which suggests that TGFβR3 may serve as a novel therapeutic target for MI.

     

5.2139           Lentiviral Delivery of miR-133b Improves Functional Recovery After Spinal Cord Injury in Mice

Theis, T., Yoo, M., park, C.S., Chen, J., Kügler, S., Gibbs, K.M. and Schachner, M Mol. Neurobiol., 54(6), 4659-4671 (2017)   Based on the observation that microRNA (miRNA) 133b enhances regeneration after spinal cord injury in the adult zebrafish, we investigated whether this miRNA would be beneficial in a mammalian system in vitro and in vivo. We found that infection of cultured neurons with miR-133b promotes neurite outgrowth in vitro on an inhibitory substrate consisting of mixed chondroitin sulfate proteoglycans, when compared to infection with green fluorescent protein (GFP) for control. In vivo, viral infection of the injured adult mouse spinal cord at the time of injury at and in the vicinity of the lesion site enhanced expression of miR-133b. Measurements of locomotor recovery by Basso Mouse Scale (BMS) showed improvement of recovery starting at 4 weeks after injury and virus injection. This improvement was associated with downregulation of the expression levels of Ras homolog gene family member A (RhoA), chondroitin sulfate proteoglycans, and microglia/macrophage marker in the spinal cord as assayed 6 weeks after injury. Potential inhibitory molecules carrying consensus sequences for binding of miR-133b were identified in silico and verified in a reporter assay in vitro showing reductions in expression of RhoA, xylosyltransferase 1 (Xylt1), ephrin receptor A7 (Epha7), and purinergic receptor P2X ligand-gated ion channel 4 (P2RX4). These results encourage targeting miR-133 for therapy.  

5.2140           Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery

Gaj, T., Staahl, B.T., Rodrigues, G.C., Limsirichai, P., Ekman, F.K., Doudna, J.A. and Schaffer, D.V. Nucleic Acids Res., 45(11), e98 (2017)   Realizing the full potential of genome editing requires the development of efficient and broadly applicable methods for delivering programmable nucleases and donor templates for homology-directed repair (HDR). The RNA-guided Cas9 endonuclease can be introduced into cells as a purified protein in complex with a single guide RNA (sgRNA). Such ribonucleoproteins (RNPs) can facilitate the high-fidelity introduction of single-base substitutions via HDR following co-delivery with a single-stranded DNA oligonucleotide. However, combining RNPs with transgene-containing donor templates for targeted gene addition has proven challenging, which in turn has limited the capabilities of the RNP-mediated genome editing toolbox. Here, we demonstrate that combining RNP delivery with naturally recombinogenic adeno-associated virus (AAV) donor vectors enables site-specific gene insertion by homology-directed genome editing. Compared to conventional plasmid-based expression vectors and donor templates, we show that combining RNP and AAV donor delivery increases the efficiency of gene addition by up to 12-fold, enabling the creation of lineage reporters that can be used to track the conversion of striatal neurons from human fibroblasts in real time. These results thus illustrate the potential for unifying nuclease protein delivery with AAV donor vectors for homology-directed genome editing.  

5.2141           Accelerated atherosclerosis development in C57Bl6 mice by overexpressing AAV-mediated PCSK9 and partial carotid ligation

Kumar, S., Kang, D-W., Rezvan, A. and Jo, H. Lab. Invest., 97(8), 935-945 (2017)   Studying the role of a particular gene in atherosclerosis typically requires a time-consuming and often difficult process of generating double knockouts or transgenics on ApoE^−/− or LDL receptor (LDLR)^−/− background. Recently, it was reported that adeno-associated-virus-8 (AAV8)-mediated overexpression of PCSK9 (AAV8-PCSK9) rapidly induced hyperlipidemia. However, using this method in C57BL6 wild-type (C57) mice, it took ~3 months to develop atherosclerosis. Our partial carotid ligation model is used to rapidly develop atherosclerosis by inducing disturbed flow in the left common carotid artery within 2 weeks in ApoE^−/− or LDLR^−/− mice. Here, we combined these two approaches to develop an accelerated model of atherosclerosis in C57 mice. C57 mice were injected with AAV9-PCSK9 or AAV9-luciferase (control) and high-fat diet was initiated. A week later, partial ligation was performed. Compared to the control, AAV-PCSK9 led to elevated serum PCSK9, hypercholesterolemia, and rapid atherosclerosis development within 3 weeks as determined by gross plaque imaging, and staining with Oil-Red-O, Movat’s pentachrome, and CD45 antibody. These plaque lesions were comparable to the atherosclerotic lesions that have been previously observed in ApoE^−/− or LDLR^−/− mice that were subjected to partial carotid ligation and high-fat diet. Next, we tested whether our method can be utilized to rapidly determine the role of a particular gene in atherosclerosis. Using eNOS^−/− and NOX1^−/y mice on C57 background, we found that the eNOS^−/− mice developed more advanced lesions, while the NOX1^−/y mice developed less atherosclerotic lesions as compared to the C57 controls. These results are consistent with the previous findings using double knockouts (eNOS^−/−_ApoE^−/− and NOX1^−/y_ApoE^−/−). AAV9-PCSK9 injection followed by partial carotid ligation is an effective and time-saving approach to rapidly induce atherosclerosis. This accelerated model is well-suited to quickly determine the role of gene(s) interest without generating double or triple knockouts.  

5.2142           Motor deficits and beta oscillations are dissociable in an alpha-synuclein model of Parkinson's disease

Brys, I., Nunes, J. and Fuentes, R. Eur. J. Neurosci., 46(3), 1906-1917 (2017)   Parkinson's disease (PD) is a neurodegenerative disorder characterised by progressive motor symptoms resulting from chronic loss of dopaminergic neurons in the nigrostriatal pathway. The over expression of the protein alpha-synuclein in the substantia nigra has been used to induce progressive dopaminergic neuronal loss and to reproduce key histopathological and temporal features of PD in animal models. However, the neurophysiological aspects of the alpha-synuclein PD model have been poorly characterised. Hereby, we performed chronic in vivo electrophysiological recordings in the corticostriatal circuit of rats injected with viral vector to over express alpha-synuclein in the right substantia nigra. Our model, previously shown to exhibit mild motor deficits, presented moderate dopaminergic cell loss but did not present prominent local field potential oscillations in the beta frequency range (11–30 Hz), considered a hallmark of PD, during the 9 weeks after onset of alpha-synuclein over expression. Spinal cord stimulation, a potential PD symptomatic therapy, was applied regularly from sixth to ninth week after alpha-synuclein over expression onset and had an inhibitory effect on the firing rate of corticostriatal neurons in both control and alpha-synuclein hemispheres. Dopamine synthesis inhibition at the end of the experiment resulted in severe parkinsonian symptoms such as akinesia and increased beta and high-frequency (>90 Hz) oscillations. These results suggest that the alpha-synuclein PD model with moderate level of dopaminergic depletion does not reproduce the prominent corticostriatal beta oscillatory activity associated to parkinsonian conditions.  

5.2143           The Alphabet Soup of HIV Reservoir Markers

Sharaf, R and Li, J.Z. Curr. HIV/AIDS Rep., 14, 72-81 (2017)   Purpose of Review Despite the success of antiretroviral therapy in suppressing HIV, life-long therapy is required to avoid HIV reactivation from long-lived viral reservoirs. Currently, there is intense interest in searching for therapeutic interventions that can purge the viral reservoir to achieve complete remission in HIV patients off antiretroviral therapy. The evaluation of such interventions relies on our ability to accurately and precisely measure the true size of the viral reservoir. In this review, we assess the most commonly used HIV reservoir assays, as a clear understanding of the strengths and weaknesses of each is vital for the accurate interpretation of results and for the development of improved assays. Recent Findings The quantification of intracellular or plasma HIV RNA or DNA levels remains the most commonly used tests for the characterization of the viral reservoir. While cost-effective and high-throughput, these assays are not able to differentiate between replication-competent or defective fractions or quantify the number of infected cells. Viral outgrowth assays provide a lower bound for the fraction of cells that can produce infectious virus, but these assays are laborious, expensive and substantially underestimate the potential reservoir of replication-competent provirus. Newer assays are now available that seek to overcome some of these problems, including full-length proviral sequencing, inducible HIV RNA assays, ultrasensitive p24 assays and murine adoptive transfer techniques. Summary The development and evaluation of strategies for HIV remission rely upon our ability to accurately and precisely quantify the size of the remaining viral reservoir. At this time, all current HIV reservoir assays have drawbacks such that combinations of assays are generally needed to gain a more comprehensive view of the viral reservoir. The development of novel, rapid, high-throughput assays that can sensitively quantify the levels of the replication-competent HIV reservoir is still needed.  

5.2144           Covalent coupling of high-affinity ligands to the surface of viral vector particles by protein trans-splicing mediates cell type-specific gene transfer

Muik, A., Reul, J., friedel, T., Muth, A., Hartmann, K.P., Schneider, I.C., Münch, R.C. and Buchholz, C.J. Biomaterials, 144, 84-94  (2017)   We have established a novel approach for the covalent coupling of large polypeptides to the surface of fully assembled adeno-associated viral gene transfer vector (AAV) particles via split-intein mediated protein-trans-splicing (PTS). This way, we achieved selective gene transfer to distinct cell types. Single-chain variable fragments (scFvs) or designed ankyrin repeat proteins (DARPins), exhibiting high-affinity binding to cell surface receptors selectively expressed on the surface of target cells, were coupled to AAV particles harboring mutations in the capsid proteins which ablate natural receptor usage. Both, the AAV capsid protein VP2 and multiple separately produced targeting ligands recognizing Her2/neu, EpCAM, CD133 or CD30 were genetically fused with complementary split-intein domains. Optimized coupling conditions led to an effective conjugation of each targeting ligand to the universal AAV capsid and translated into specific gene transfer into target receptor-positive cell types in vitro and in vivo. Interestingly, PTS-based AAVs exhibited significantly less gene transfer into target receptor-negative cells than AAVs displaying the same targeting ligand but coupled genetically. Another important consequence of the PTS technology is the possibility to now display scFvs or other antibody-derived domain formats harboring disulfide-bonds in a functionally active form on the surface of AAV particles. Hence, the custom combination of a universal AAV vector particle and targeting ligands of various formats allows for an unprecedented flexibility in the generation of gene transfer vectors targeted to distinct cell types.  

5.2145           The host-cell restriction factor SERINC5 restricts HIV-1 infectivity without altering the lipid composition and organization of viral particles

Trautz, B., Wiedemann, H., Lüchtenborg, C., Pierini, V., Kranich, J., Glass, B., Kräusslich, H-G., Brocker, T., Pizzato, M., Ruggieri, A., Brügger, B. and Fackler, O.T.

1. Biol. Chem., 292(33), 13702-13713 (2017)

  The host-cell restriction factor SERINC5 potently suppresses the infectivity of HIV, type 1 (HIV-1) particles, and is counteracted by the viral pathogenesis factor Nef. However, the molecular mechanism by which SERINC5 restricts HIV-1 particle infectivity is still unclear. Because SERINC proteins have been suggested to facilitate the incorporation of serine during the biosynthesis of membrane lipids and because lipid composition of HIV particles is a major determinant of the infectious potential of the particles, we tested whether SERINC5-mediated restriction of HIV particle infectivity involves alterations of membrane lipid composition. We produced and purified HIV-1 particles from SERINC5293T cells with very low endogenous SERINC5 levels under conditions in which ectopically expressed SERINC5 restricts HIV-1 infectivity and is antagonized by Nef and analyzed both virions and producer cells with quantitative lipid MS. SERINC5 restriction and Nef antagonism were not associated with significant alterations in steady-state lipid composition of producer cells and HIV particles. Sphingosine metabolism kinetics were also unaltered by SERINC5 expression. Moreover, the levels of phosphatidylserine on the surface of HIV-1 particles, which may trigger uptake into non-productive internalization pathways in target cells, did not change upon expression of SERINC5 or Nef. Finally, saturating the phosphatidylserine-binding sites on HIV target cells did not affect SERINC5 restriction or Nef antagonism. These results demonstrate that the restriction of HIV-1 particle infectivity by SERINC5 does not depend on alterations in lipid composition and organization of HIV-1 particles and suggest that channeling serine into lipid biosynthesis may not be a cardinal cellular function of SERINC5.  

5.2146           Smac mimetics and oncolytic viruses synergize in driving anticancer T-cell responses through complementary mechanisms

Kim, D-S. et al Nature Communications, 8:344 (2017)   Second mitochondrial activator of caspase (Smac)-mimetic compounds and oncolytic viruses were developed to kill cancer cells directly. However, Smac-mimetic compound and oncolytic virus therapies also modulate host immune responses in ways we hypothesized would complement one another in promoting anticancer T-cell immunity. We show that Smac-mimetic compound and oncolytic virus therapies synergize in driving CD8⁺ T-cell responses toward tumors through distinct activities. Smac-mimetic compound treatment with LCL161 reinvigorates exhausted CD8⁺ T cells within immunosuppressed tumors by targeting tumor-associated macrophages for M1-like polarization. Oncolytic virus treatment with vesicular stomatitis virus (VSV^ΔM51) promotes CD8⁺ T-cell accumulation within tumors and CD8⁺ T-cell activation within the tumor-draining lymph node. When combined, LCL161 and VSV^ΔM51 therapy engenders CD8⁺ T-cell-mediated tumor control in several aggressive mouse models of cancer. Smac-mimetic compound and oncolytic virus therapies are both in clinical development and their combination therapy represents a promising approach for promoting anticancer T-cell immunity.  

5.2147           Entry and Release of Hepatitis C Virus in Polarized Human Hepatocytes

Belouzard, S., Danneels, A., Feneant, L., Seron, K., Rouille, Y. and Dubuisson, J.

1. Virol., 91(18), e00478-17 (2017)

  Hepatitis C virus (HCV) primarily infects hepatocytes, which are highly polarized cells. The relevance of cell polarity in the HCV life cycle has been addressed only in distantly related models and remains poorly understood. Although polarized epithelial cells have a rather simple morphology with a basolateral and an apical domain, hepatocytes exhibit complex polarization structures. However, it has been reported that some selected polarized HepG2 cell clones can exhibit a honeycomb pattern of distribution of the tight-junction proteins typical of columnar polarized epithelia, which can be used as a simple model to study the role of cell polarization in viral infection of hepatocytes. To obtain similar clones, HepG2 cells expressing CD81 (HepG2-CD81) were used, and clones were isolated by limiting dilutions. Two clones exhibiting a simple columnar polarization capacity when grown on a semipermeable support were isolated and characterized. To test the polarity of HCV entry and release, our polarized HepG2-CD81 clones were infected with cell culture-derived HCV. Our data indicate that HCV binds equally to both sides of the cells, but productive infection occurs mainly when the virus is added at the basolateral domain. Furthermore, we also observed that HCV virions are released from the basolateral domain of the cells. Finally, when polarized cells were treated with oleic acid and U0126, a MEK inhibitor, to promote lipoprotein secretion, a higher proportion of infectious viral particles of lower density were secreted. This cell culture system provides an excellent model to investigate the influence of cell polarization on the HCV life cycle.  

5.2148           Trans-dissemination of exosomes from HIV-1-infected cells fosters both HIV-1 trans-infection in resting CD4+ T lymphocytes and reactivation of the HIV-1 reservoir

Chiozzini, C., Arenaccio, C., Olivetta, E., Anticoli, S., Manfredi, F., Ferrantelli, F., d’Ettorre, G., Scietroma, I., Andreotti, m. and Federico, M. Arch. Virol., 162(9), 2565-2577 (2017)   Intact HIV-1 and exosomes can be internalized by dendritic cells (DCs) through a common pathway leading to their transmission to CD4⁺ T lymphocytes by means of mechanisms defined as trans-infection and trans-dissemination, respectively. We previously reported that exosomes from HIV-1-infected cells activate both uninfected quiescent CD4⁺ T lymphocytes, which become permissive to HIV-1, and latently infected cells, with release of HIV-1 particles. However, nothing is known about the effects of trans-dissemination of exosomes produced by HIV-1-infected cells on uninfected or latently HIV-1-infected CD4⁺ T lymphocytes. Here, we report that trans-dissemination of exosomes from HIV-1-infected cells induces cell activation in resting CD4⁺ T lymphocytes, which appears stronger with mature than immature DCs. Using purified preparations of both HIV-1 and exosomes, we observed that mDC-mediated trans-dissemination of exosomes from HIV-1-infected cells to resting CD4⁺ T lymphocytes induces efficient trans-infection and HIV-1 expression in target cells. Most relevant, when both mDCs and CD4⁺ T lymphocytes were isolated from combination anti-retroviral therapy (ART)-treated HIV-1-infected patients, trans-dissemination of exosomes from HIV-1-infected cells led to HIV-1 reactivation from the viral reservoir. In sum, our data suggest a role of exosome trans-dissemination in both HIV-1 spread in the infected host and reactivation of the HIV-1 reservoir.  

5.2149           Induction of Oxidants Distinguishes Susceptibility of Prostate Carcinoma Cell Lines to p53 Gene Transfer Mediated by an Improved Adenoviral VectorNo Access

Tamura, R.E., Hunger, A., Fernandes, D.C., Laurindo, F.R., Costanzi-Strauss, E. and Strauss, B.E. Human Gene Therapy, 28(8), 639-653 (2017)   Previously, the authors developed an adenoviral vector, Ad-PG, where transgene expression is regulated by a p53-responsive promoter. When used to transfer the p53 cDNA, a positive feedback mechanism is established. In the present study, a critical comparison is performed between Ad-PGp53 and AdRGD-PGp53, where the RGD motif was incorporated in the adenoviral fiber protein. AdRGD-PGp53 provided superior transgene expression levels and resulted in the killing of prostate carcinoma cell lines DU145 and PC3. In vitro, this effect was associated with increased production of cytoplasmic and mitochondrial oxidants, DNA damage as revealed by detection of phosphorylated H2AX, as well as cell death consistent with apoptosis. Differential gene expression of key mediators of reactive oxygen species pathways was also observed. Specifically, it was noted that induction of known p53-target genes Sestrin2 and PIG3, as well as a novel target, NOX1, occurred in PC3 cells only when transduced with the improved vector, AdRGD-PGp53. The participation of NOX1 was confirmed upon its inhibition using a specific peptide, resulting in reduced cell death. In situ gene therapy also resulted in significantly improved inhibition of tumor progression consistent with oxidant-induced DNA damage only when treated with the novel AdRGD-PGp53 vector. The study shows that the improved adenovirus overcomes limitations associated with other p53-expressing vectors and induces oxidant-mediating killing, thus supporting its further development for cancer gene therapy.  

5.2150           miRNA-mediated post-transcriptional silencing of transgenes leads to increased adeno-associated viral vector yield and targeting specificity

Reid, C.A., Boye, S.L., Hauswirth, W.W. and Lipinski, D.M. Gene Therapy, 24(8), 462-469 (2017)   The production of high-titer recombinant adeno-associated virus (rAAV) vector is essential for treatment of genetic diseases affecting the retina and choroid, where anatomical constraints may limit injectable volumes. Problematically, cytotoxicity arising from overexpression of the transgene during vector production frequently leads to a reduction in vector yield. Herein, we evaluate the use of microRNA (miRNA)-mediated silencing to limit overexpression of cytotoxic transgenes during packaging as a method of increasing vector yield. We examined if post-transcriptional regulation of transgenes during packaging via miRNA technology would lead to increased rAAV yields. Our results demonstrate that silencing of cytotoxic transgenes during production resulted in up to a 22-fold increase in vector yield. The inclusion of organ-specific miRNA sequences improved biosafety by limiting off-target expression following systemic rAAV administration. The small size (22–23 bp) of the target site allows for the inclusion of multiple copies into the vector with minimal impact on coding capacity. Taken together, our results suggest that inclusion of miRNA target sites into the 3′-untranslated region of the AAV cassette allow for silencing of cytotoxic transgenes during vector production leading to improved vector yield, in addition to increasing targeting specificity without reliance on cell-specific promoters.  

5.2151           Optimization of design and production strategies for novel adeno-associated viral display peptide libraries

Körbelin, J., Hunger, A., Alawi, M., Sieber, T., Binder, M. and Trepel, M. Gene Therapy, 24(8), 470-481 (2017)   Libraries displaying random peptides on the surface of adeno-associated virus (AAV) are powerful tools for the generation of target-specific gene therapy vectors. However, for unknown reasons the success rate of AAV library screenings is variable and the influence of the production procedure has not been thoroughly evaluated. During library screenings, the capsid variants with the most favorable tropism are enriched over several selection rounds on a target of choice and identified by subsequent sequencing of the encapsidated viral genomes encoding the library capsids with targeting peptide insertions. Thus, a high capsid-genome correlation is crucial to obtain the correct information about the selected capsid variants. Producing AAV libraries by a two-step protocol with pseudotyped library transfer shuttles has been proposed as one way to ensure such a correlation. Here we show that AAV2 libraries produced by such a protocol via transfer shuttles display an unexpected additional bias in the amino-acid composition which confers increased heparin affinity and thus similarity to wildtype AAV2 tropism. This bias may fundamentally impair the intended use of AAV libraries, discouraging the use of transfer shuttles for the production of AAV libraries in the future.  

5.2152           Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems

Chan, K.Y., Jang, M.J., Yoo, B.B., Greenbaum, A., Ravi, N., Wu, W-L., Sanchez-Guardado, L., Lois, c., Mazmanian, S.K., Deverman, B.E. and Gradinaru, V. Nature Neurosci., 20(8), 1172-1179 (2017)   Adeno-associated viruses (AAVs) are commonly used for in vivo gene transfer. Nevertheless, AAVs that provide efficient transduction across specific organs or cell populations are needed. Here, we describe AAV-PHP.eB and AAV-PHP.S, capsids that efficiently transduce the central and peripheral nervous systems, respectively. In the adult mouse, intravenous administration of 1 × 10¹¹ vector genomes (vg) of AAV-PHP.eB transduced 69% of cortical and 55% of striatal neurons, while 1 × 10¹² vg of AAV-PHP.S transduced 82% of dorsal root ganglion neurons, as well as cardiac and enteric neurons. The efficiency of these vectors facilitates robust cotransduction and stochastic, multicolor labeling for individual cell morphology studies. To support such efforts, we provide methods for labeling a tunable fraction of cells without compromising color diversity. Furthermore, when used with cell-type-specific promoters and enhancers, these AAVs enable efficient and targetable genetic modification of cells throughout the nervous system of transgenic and non-transgenic animals.  

5.2153           Correction of a splicing defect in a mouse model of congenital muscular dystrophy type 1A using a homology-directed-repair-independent mechanism

Kemaladewi, D.  et al

Nature Med., 23(8), 984-989 (2017)   Splice-site defects account for about 10% of pathogenic mutations that cause Mendelian diseases¹. Prevalence is higher in neuromuscular disorders (NMDs)², owing to the unusually large size and multi-exonic nature of genes encoding muscle structural proteins. Therapeutic genome editing to correct disease-causing splice-site mutations has been accomplished only through the homology-directed repair pathway^{3, 4, 5}, which is extremely inefficient in postmitotic tissues such as skeletal muscle⁶. Here we describe a strategy using nonhomologous end-joining (NHEJ) to correct a pathogenic splice-site mutation. As a proof of principle, we focus on congenital muscular dystrophy type 1A (MDC1A), which is characterized by severe muscle wasting and paralysis⁷. Specifically, we correct a splice-site mutation that causes the exclusion of exon 2 from Lama2 mRNA and the truncation of Lama2 protein in the dy^2J/dy^2J mouse model of MDC1A⁸. Through systemic delivery of adeno-associated virus (AAV) carrying clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 genome-editing components, we simultaneously excise an intronic region containing the mutation and create a functional donor splice site through NHEJ. This strategy leads to the inclusion of exon 2 in the Lama2 transcript and restoration of full-length Lama2 protein. Treated dy^2J/dy^2J mice display substantial improvement in muscle histopathology and function without signs of paralysis.  

5.2154           HEV and transfusion-recipient risk

Izopet, J., Lhomme, S., Chapuy-Regaud, S., Mansuy, J.M., Kamar, N. and Avravanel, F. Transfusion Clinique et Biologique, 24, 176-181 (2017)   HEV infections are mainly food- and water-borne but transfusion-transmission has occurred in both developing and developed countries. The infection is usually asymptomatic but it can lead to fulminant hepatitis in patients with underlying liver disease and pregnant women living in developing countries. It also causes chronic hepatitis E, with progressive fibrosis and cirrhosis, in approximately 60% of immunocompromised patients infected with HEV genotype 3. The risk of a transfusion-transmitted HEV infection is linked to the frequency of viremia in blood donors, the donor virus load and the volume of plasma in the final transfused blood component. Several developed countries have adopted measures to improve blood safety based on the epidemiology of HEV.  

5.2155           Interaction of extremophilic archaeal viruses with human and mouse complement system and viral biodistribution in mice

Wu, L., Buch Uldahl, K., Chen, F., Benasutti, H., Logvinski, D., Vu, V., Banda, N.B., Peng, X., Simberg, D. and Moghimi, S.M. Mol. Immunol., 90, 273-279 (2017)   Archaeal viruses offer exceptional biophysical properties for modification and exploration of their potential in bionanotechnology, bioengineering and nanotherapeutic developments. However, the interaction of archaeal viruses with elements of the innate immune system has not been explored, which is a necessary prerequisite if their potential for biomedical applications to be realized. Here we show complement activation through lectin (via direct binding of MBL/MASPs) and alternative pathways by two extremophilic archaeal viruses (Sulfolobus monocaudavirus 1 and Sulfolobus spindle-shaped virus 2) in human serum. We further show some differences in initiation of complement activation pathways between these viruses. Since, Sulfolobus monocaudavirus 1 was capable of directly triggering the alternative pathway, we also demonstrate that the complement regulator factor H has no affinity for the viral surface, but factor H deposition is purely C3-dependent. This suggests that unlike some virulent pathogens Sulfolobus monocaudavirus 1 does not acquire factor H for protection. Complement activation with Sulfolobus monocaudavirus 1 also proceeds in murine sera through MBL-A/C as well as factor D-dependent manner, but C3 deficiency has no overall effect on viral clearance by organs of the reticuloendothelial system on intravenous injection. However, splenic deposition was significantly higher in C3 knockout animals compared with the corresponding wild type mice. We discuss the potential application of these viruses in biomedicine in relation to their complement activating properties.  

5.2156           Vaccine-induced immune responses against both Gag and Env improve control of simian immunodeficiency virus replication in rectally challenged rhesus macaques

Martins, M.A. et al PloS Patahogens, 13(7), e1006529 (2017)   The ability to control lentivirus replication may be determined, in part, by the extent to which individual viral proteins are targeted by the immune system. Consequently, defining the antigens that elicit the most protective immune responses may facilitate the design of effective HIV-1 vaccines. Here we vaccinated four groups of rhesus macaques with a heterologous vector prime/boost/boost/boost (PBBB) regimen expressing the following simian immunodeficiency virus (SIV) genes: env, gag, vif, rev, tat, and nef (Group 1); env, vif, rev, tat, and nef (Group 2); gag, vif, rev, tat, and nef (Group 3); or vif, rev, tat, and nef (Group 4). Following repeated intrarectal challenges with a marginal dose of the neutralization-resistant SIVmac239 clone, vaccinees in Groups 1–3 became infected at similar rates compared to control animals. Unexpectedly, vaccinees in Group 4 became infected at a slower pace than the other animals, although this difference was not statistically significant. Group 1 exhibited the best post-acquisition virologic control of SIV infection, with significant reductions in both peak and chronic phase viremia. Indeed, 5/8 Group 1 vaccinees had viral loads of less than 2,000 vRNA copies/mL of plasma in the chronic phase. Vaccine regimens that did not contain gag (Group 2), env (Group 3), or both of these inserts (Group 4) were largely ineffective at decreasing viremia. Thus, vaccine-induced immune responses against both Gag and Env appeared to maximize control of immunodeficiency virus replication. Collectively, these findings are relevant for HIV-1 vaccine design as they provide additional insights into which of the lentiviral proteins might serve as the best vaccine immunogens.  

5.2157           TIM1 (HAVCR1) Is Not Essential for Cellular Entry of Either Quasi-enveloped or Naked Hepatitis A Virions

Das, A., Hirai-Yuki, A., Gonzalez-Lopez, O., Rhein, B., Moller-Tank, S., Brouilette, R., Hensley, L., Misumi, I., Lovell, W., Cullen, J.M., Whitmire, J.K., Maury, W. and Lemon, S.M. mBio, 8(5), e00969-17 (2017)   Receptor molecules play key roles in the cellular entry of picornaviruses, and TIM1 (HAVCR1) is widely accepted to be the receptor for hepatitis A virus (HAV), an unusual, hepatotropic human picornavirus. However, its identification as the hepatovirus receptor predated the discovery that hepatoviruses undergo nonlytic release from infected cells as membrane-cloaked, quasi-enveloped HAV (eHAV) virions that enter cells via a pathway distinct from naked, nonenveloped virions. We thus revisited the role of TIM1 in hepatovirus entry, examining both adherence and infection/replication in cells with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-engineered TIM1 knockout. Cell culture-derived, gradient-purified eHAV bound Huh-7.5 human hepatoma cells less efficiently than naked HAV at 4°C, but eliminating TIM1 expression caused no difference in adherence of either form of HAV, nor any impact on infection and replication in these cells. In contrast, TIM1-deficient Vero cells showed a modest reduction in quasi-enveloped eHAV (but not naked HAV) attachment and replication. Thus, TIM1 facilitates quasi-enveloped eHAV entry in Vero cells, most likely by binding phosphatidylserine (PtdSer) residues on the eHAV membrane. Both Tim1^−/− Ifnar1^−/− and Tim4^−/− Ifnar1^−/− double-knockout mice were susceptible to infection upon intravenous challenge with infected liver homogenate, with fecal HAV shedding and serum alanine aminotransferase (ALT) elevations similar to those in Ifnar1^−/− mice. However, intrahepatic HAV RNA and ALT elevations were modestly reduced in Tim1^−/−Ifnar1^−/− mice compared to Ifnar1^−/− mice challenged with a lower titer of gradient-purified HAV or eHAV. We conclude that TIM1 is not an essential hepatovirus entry factor, although its PtdSer-binding activity may contribute to the spread of quasi-enveloped virus and liver injury in mice. Receptor molecules play key roles in the cellular entry of picornaviruses, and TIM1 (HAVCR1) is widely accepted to be the receptor for hepatitis A virus (HAV), an unusual, hepatotropic human picornavirus. However, its identification as the hepatovirus receptor predated the discovery that hepatoviruses undergo nonlytic release from infected cells as membrane-cloaked, quasi-enveloped HAV (eHAV) virions that enter cells via a pathway distinct from naked, nonenveloped virions. We thus revisited the role of TIM1 in hepatovirus entry, examining both adherence and infection/replication in cells with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-engineered TIM1 knockout. Cell culture-derived, gradient-purified eHAV bound Huh-7.5 human hepatoma cells less efficiently than naked HAV at 4°C, but eliminating TIM1 expression caused no difference in adherence of either form of HAV, nor any impact on infection and replication in these cells. In contrast, TIM1-deficient Vero cells showed a modest reduction in quasi-enveloped eHAV (but not naked HAV) attachment and replication. Thus, TIM1 facilitates quasi-enveloped eHAV entry in Vero cells, most likely by binding phosphatidylserine (PtdSer) residues on the eHAV membrane. Both Tim1−/− Ifnar1−/− and Tim4−/− Ifnar1−/− double-knockout mice were susceptible to infection upon intravenous challenge with infected liver homogenate, with fecal HAV shedding and serum alanine aminotransferase (ALT) elevations similar to those in Ifnar1−/− mice. However, intrahepatic HAV RNA and ALT elevations were modestly reduced in Tim1−/−Ifnar1−/− mice compared to Ifnar1−/− mice challenged with a lower titer of gradient-purified HAV or eHAV. We conclude that TIM1 is not an essential hepatovirus entry factor, although its PtdSer-binding activity may contribute to the spread of quasi-enveloped virus and liver injury in mice.  

5.2158           Analysis of Hepatitis C Virus Particle Heterogeneity in Immunodeficient Human Liver Chimeric fah-/- Mice

Andreo, U., de Jong, Y.P., Scull, M.A., Xiao, J.W., Vercauteren, K., Quirk, C., Mommersteeg, M.C., Bergaya, S., Menon, A., Fisher, E.A. and Rice, C.M. Cell. Mol. Gastroenterol. Hepatol., 4, 405-417 (2017)   Background & Aims Hepatitis C virus (HCV) is a leading cause of chronic liver diseases and the most common indication for liver transplantation in the United States. HCV particles in the blood of infected patients are characterized by heterogeneous buoyant densities, likely owing to HCV association with lipoproteins. However, clinical isolates are not infectious in vitro and the relative infectivity of the particles with respect to their buoyant density therefore cannot be determined, pointing to the need for better in vivo model systems. Methods To analyze the evolution of the buoyant density of in vivo–derived infectious HCV particles over time, we infected immunodeficient human liver chimeric fumaryl acetoacetate hydrolase-/- mice with J6/JFH1 and performed ultracentrifugation of infectious mouse sera on isopicnic iodixanol gradients. We also evaluated the impact of a high sucrose diet, which has been shown to increase very-low-density lipoprotein secretion by the liver in rodents, on lipoprotein and HCV particle characteristics. Results Similar to the severe combined immunodeficiency disease/Albumin-urokinase plasminogen activator human liver chimeric mouse model, density fractionation of infectious mouse serum showed higher infectivity in the low-density fractions early after infection. However, over the course of the infection, viral particle heterogeneity increased and the overall in vitro infectivity diminished without loss of the human liver graft over time. In mice provided with a sucrose-rich diet we observed a minor shift in HCV infectivity toward lower density that correlated with a redistribution of triglycerides and cholesterol among lipoproteins. Conclusions Our work indicates that the heterogeneity in buoyant density of infectious HCV particles evolves over the course of infection and can be influenced by diet.  

5.2159           Technical considerations for the use of CRISPR/Cas9 in hematology research

Gundry, M.C., Dever, D.P., Yudovich, D., Bauer, D.E., Haas, S., Wilkinson, A.C. and Singbrant, S. Exp. Hematol., 54, 4-11 (2017)   The hematopoietic system is responsible for transporting oxygen and nutrients, fighting infections, and repairing tissue damage. Hematopoietic system dysfunction therefore causes a range of serious health consequences. Lifelong hematopoiesis is maintained by repopulating multipotent hematopoietic stem cells (HSCs) that replenish shorter-lived, mature blood cell types. A prokaryotic mechanism of immunity, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system, has been recently “repurposed” to mutate mammalian genomes efficiently and in a sequence-specific manner. The application of this genome-editing technology to hematology has afforded new approaches for functional genomics and even the prospect of “correcting” dysfunctional HSCs in the treatment of serious genetic hematological diseases. In this Perspective, we provide an overview of three recent CRISPR/Cas9 methods in hematology: gene disruption, gene targeting, and saturating mutagenesis. We also summarize the technical considerations and advice provided during the May 2017 International Society of Experimental Hematology New Investigator Committee webinar on the same topic. The mammalian hematopoietic system plays an essential role in health and disease, carrying oxygen and nutrients around the body, fighting infection, and helping to repair tissue damage. A small number of multipotent hematopoietic stem cells (HSCs) are responsible for the lifelong balanced production of mature blood cells [1]. Under homeostasis, HSCs are located in the bone marrow and maintained in a long-term quiescent state [2]. However, after stress or bone marrow transplantation, hematopoietic stem and progenitor cells (HSPCs) can be activated to expand and reestablish homeostasis [1,3,4]. The ability of HSCs to repopulate efficiently and drive long-term blood formation after transplantation makes them an attractive and widely used tool in many clinical settings [5]. The ability to genetically modify HSCs has therefore been a long-standing desire in clinical hematology, and one that would also represent a powerful tool for basic hematological research. The field of genome editing has been recently revolutionized by the introduction of engineered nucleases, with the promise of a controlled genomic engineering approach [6]. Zinc finger nucleases, transcription activator-like effector-based nucleases, and in particular, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology have been developed to introduce precise genomic alterations [7–12]. The CRISPR/Cas9 technology was adapted from a prokaryotic immune system and includes, among others, the endonuclease Cas9 and a single-guide RNA (sgRNA), which targets the Cas9 in a desired region of genome through Watson–Crick base pairing (Fig. 1). CRISPR/Cas9-induced DNA double-strand breaks can be repaired by the error-prone non-homologous end-joining (NHEJ) pathway, which frequently results in the introduction of insertions or deletions (indels). Alternatively, homologous recombination (HR) can be exploited to introduce precise genomic modifications using homologous DNA donor templates. The field of hematology has made great progress with CRISPR/Cas9 applications, and although studies describing efficient protocols for gene disruption and HR-mediated gene-targeting approaches in various hematopoietic cell types have been published [13–18], no reviews describe the technical considerations for the use of CRISPR/Cas9 in hematology research.  

5.2160           Envelope glycoprotein mobility on HIV-1 particles depends on the virus maturation state

Chojnacki, J., Waithe, D., Carravilla, P., Huarte, N., Galiani, S., Enderlein, J. and Eggeling, C. Nature Communications, 8:545 (2017)   Human immunodeficiency virus type 1 (HIV-1) assembles as immature particles, which require the proteolytic cleavage of structural polyprotein Gag and the clustering of envelope glycoprotein Env for infectivity. The details of mechanisms underlying Env clustering remain unknown. Here, we determine molecular dynamics of Env on the surface of individual HIV-1 particles using scanning fluorescence correlation spectroscopy on a super-resolution STED microscope. We find that Env undergoes a maturation-induced increase in mobility, highlighting diffusion as one cause for Env clustering. This mobility increase is dependent on Gag-interacting Env tail but not on changes in viral envelope lipid order. Diffusion of Env and other envelope incorporated proteins in mature HIV-1 is two orders of magnitude slower than in the plasma membrane, indicating that HIV-1 envelope is intrinsically a low mobility environment, mainly due to its general high lipid order. Our results provide insights into dynamic properties of proteins on the surface of individual virus particles.  

5.2161           Characterization of the lipid envelope of exosome encapsulated HEV particles protected from the immune response

Chapuy-Regaud, S., Dubois, M., Plisson-Chastang, C., Bonnefois, T., Lhomme, S., Bertrand-Michel, J., You, B., Simoneau, S., Gleizes, P-E., Flan, B., Abravanel, F. and Izopet, J. Biochimie, 141, 70-79 (2017)   The hepatitis E virus (HEV) is the most common cause of acute hepatitis worldwide. Although HEV is a small, naked RNA virus, HEV particles become associated with lipids in the blood of infected patients and in the supernatant of culture systems. The egress of these particles from cells implies the exocytosis pathway but the question of the role of the resulting HEV RNA containing exosomes and the nature of the lipids they contain has not been fully addressed. We determined the lipid proportions of exosomes from uninfected and HEV-infected cells and their role in HEV spreading. We cultured a suitable HEV strain on HepG2/C3A cells and analyzed the population of exosomes containing HEV RNA using lipidomics methods and electron microscopy. We also quantified HEV infectivity using an infectivity endpoint method based on HEV RNA quantification to calculate the tissue culture infectious dose 50. Exosomes produced by HEV-infected HepG2/C3A cells contained encapsidated HEV RNA. These HEV RNA-containing exosomes were infectious but ten times less than stools. HEV from stools, but not exosome-associated HEV from culture supernatant, was neutralized by anti-HEV antibodies in a dose-dependent manner. HEV infection did not influence the morphology or lipid proportions of the bulk of exosomes. These exosomes contained significantly more cholesterol, phosphatidylserine, sphingomyelin and ceramides than the parent cells, but less phosphoinositides and polyunsaturated fatty acids. Exosomes play a major role in HEV egress but HEV infection does not modify the characteristics of the bulk of exosomes produced by infected cells. PS and cholesterol enriched in these vesicles could then be critical for HEV entry. HEV particles in exosomes are protected from the immune response which could lead to the wide circulation of HEV in its host.  

5.2162           Generation of Efficient miRNA Inhibitors Using Tough Decoy Constructs

Yoo, J., Hajjar, J. and Jeong, D. Methods in Mol. Biol., 1521, 41-53 (2017)   Over the last decade a previously unappreciated mechanism of gene regulation has been uncovered that is mediated by a large class of small noncoding RNAs known as microRNAs (miRNAs), and this mechanism is utilized by organisms ranging from plants to humans. MiRNAs are important downregulators of gene expression and are seen to be dysregulated in disease development. Thus inhibition of aberrantly upregulated miRNAs as a therapeutic approach has become a promising field. Many models of miRNA inhibitors currently exist, with decoy models being the most successful in current research. A promising inhibition model is the tough decoy (TuD) RNAs inhibitor, which uses antisense sequences to bind to target miRNAs, preventing them from binding to their endogenous targets. Since the TuD inhibitors have the ability to be successfully used in vitro and in vivo studies, this is a covetable inhibition method. In this chapter, we introduce how to design and generate miRNA tough decoy inhibitors with an adeno-associated viral construct. TuD inhibitors will have two miRNA binding sites. The TuD will include stem sequences, a miRNA binding site, and linkers. In vitro validation experiments to confirm the effectiveness of the TuD to inhibit miRNA are described. We also propose some practical approaches for making a TuD for miRNA of interest. We hope this chapter facilitates readers to create a simpler method to generate TuD that can be used for miRNA loss of function studies.  

5.2163           Gene Delivery for the Generation of Bioartificial Pacemaker

Chan, P.K.W. and Li, R.A. Methods in Mol. Biol., 1521, 293-306 (2017)   Electronic pacemakers have been used in patients with heart rhythm disorders for device-supported pacing. While effective, there are such shortcomings as limited battery life, permanent implantation of catheters, the lack of autonomic neurohumoral responses, and risks of lead dislodging. Here we describe protocols for establishing porcine models of sick sinus syndrome and complete heart block, and the generation of bioartificial pacemaker by delivering a strategically engineered form of hyperpolarization-activated cyclic nucleotide-gated pacemaker channel protein via somatic gene transfer to convert atrial or ventricular muscle cardiomyocytes into nodal-like cells that rhythmically fire action potentials.  

5.2164           Cell-Based Measurement of Neutralizing Antibodies Against Adeno-Associated Virus (AAV)

Jungmann, A., Müller, O. and Rapti, K. Methods in Mol. Biol., 1521, 109-126 (2017)   In recent years gene therapy using adeno-associated viral (AAV) vectors to treat cardiac disease has seen an unprecedented surge, owing to its safety, low immunogenicity relative to other vectors and high and long-term transduction efficiency. This field has also been hampered by the presence of preexisting neutralizing antibodies, not only in patients participating in clinical trials but also in preclinical large animal models. These conflicting circumstances have generated the need for a simple, efficient, and fast assay to screen subjects for the presence of neutralizing antibodies, or lack thereof, in order for them to be included in gene therapy trials.  

5.2165           Mef2C restrains microglial inflammatory response and is lost in brain ageing in an IFN-I-dependent manner

Deczkowska, A. et al Nature Communications, 8:717 (2017)   During ageing, microglia acquire a phenotype that may negatively affect brain function. Here we show that ageing microglial phenotype is largely imposed by interferon type I (IFN-I) chronically present in aged brain milieu. Overexpression of IFN-β in the CNS of adult wild-type mice, but not of mice lacking IFN-I receptor on their microglia, induces an ageing-like transcriptional microglial signature, and impairs cognitive performance. Furthermore, we demonstrate that age-related IFN-I milieu downregulates microglial myocyte-specific enhancer factor 2C (Mef2C). Immune challenge in mice lacking Mef2C in microglia results in an exaggerated microglial response and has an adverse effect on mice behaviour. Overall, our data indicate that the chronic presence of IFN-I in the brain microenvironment, which negatively affects cognitive function, is mediated via modulation of microglial activity. These findings may shed new light on other neurological conditions characterized by elevated IFN-I signalling in the brain.  

5.2166           Chemical optimization of macrocyclic HIV-1 inactivators for improving potency and increasing the structural diversity at the triazole ring

Rashad, A.A., Chaiken, I. et al Organic & Biomolecular Chem., 15(37), 7717-7980 (2017)   HIV-1 entry inhibition remains an urgent need for AIDS drug discovery and development. We previously reported the discovery of cyclic peptide triazoles (cPTs) that retain the HIV-1 irreversible inactivation functions of the parent linear peptides (PTs) and have massively increased proteolytic resistance. Here, in an initial structure–activity relationship investigation, we evaluated the effects of variations in key structural and functional components of the cPT scaffold in order to produce a platform for developing next-generation cPTs. Some structural elements, including stereochemistry around the cyclization residues and Ile and Trp side chains in the gp120-binding pharmacophore, exhibited relatively low tolerance for change, reflecting the importance of these components for function. In contrast, in the pharmacophore-central triazole position, the ferrocene moiety could be successfully replaced with smaller aromatic rings, where a p-methyl-phenyl methylene moiety gave cPT 24 with an IC₅₀ value of 180 nM. Based on the observed activity of the biphenyl moiety when installed on the triazole ring (cPT 23, IC₅₀ ∼ 269 nM), we further developed a new on-resin synthetic method to easily access the bi-aryl system during cPT synthesis, in good yields. A thiophene-containing cPT AAR029N2 (36) showed enhanced entropically favored binding to Env gp120 and improved antiviral activity (IC₅₀ ∼ 100 nM) compared to the ferrocene-containing analogue. This study thus provides a crucial expansion of chemical space in the pharmacophore to use as a starting point, along with other allowable structural changes, to guide future optimization and minimization for this important class of HIV-1 killing agents.  

5.2167           A directed evolution approach to select for novel Adeno-associated virus capsids on an HIV-1 producer T cell line

Wooley, D.P., Sharma, P., Weinstein, J.R., Narayan, P.K.L., Schaffer, D.V. and Excoffon, K.J.D.A.

1. Virol. Methods, 250, 47-54 (2017)

  A directed evolution approach was used to select for Adeno-associated virus (AAV) capsids that would exhibit more tropism toward an HIV-1 producer T cell line with the long-term goal of developing improved gene transfer vectors. A library of AAV variants was used to infect H9 T cells previously infected or uninfected by HIV-1 followed by AAV amplification with wild-type adenovirus. Six rounds of biological selection were performed, including negative selection and diversification after round three. The H9 T cells were successfully infected with all three wild-type viruses (AAV, adenovirus, and HIV-1). Four AAV cap mutants best representing the small number of variants emerging after six rounds of selection were chosen for further study. These mutant capsids were used to package an AAV vector and subsequently used to infect H9 cells that were previously infected or uninfected by HIV-1. A quantitative polymerase chain reaction assay was performed to measure cell-associated AAV genomes. Two of the four cap mutants showed a significant increase in the amount of cell-associated genomes as compared to wild-type AAV2. This study shows that directed evolution can be performed successfully to select for mutants with improved tropism for a T cell line in the presence of HIV-1.  

5.2168           Rab5B determines HBV release pathways by promoting transport of LHBs from ER to MVB

Inoue, J. et al Hepatology, 66(1), Suppl., Abstract 1491, 149-1185 (2017)   Background/aim: It has been shown that hepatitis B virus (HBV) utilizes functions of the vesicle trafficking system for the life cycle, and that multivesicular body (MVB) is required for the envelopment of the core particle. The small GTPase Rab proteins are known as molecular switches in vesicle trafficking, and we previously showed that knockdown of Rab5B significantly increased the release of infectious HBV particles. In this study, we aimed to clarify the roles of Rab5 proteins in the HBV life cycle. Methods: Using HepG2.2.15 cells stably expressing HBV particle, Rab5A, B, or C was depleted by siRNA-mediated knockdown and the changes in released viral particles were observed. The intracellular viral protein and HBV DNA levels were determined with western/Southern blotting and protein localization was analyzed using confocal immunofluorescent microscopy. The organelles in the cells were fractionated with density-gradient centrifugation using OptiPrep. Results: After the Rab5B depletion, LHBs, which is essential for the infectivity of viral particles, were significantly accumulated in the endoplasmic reticulum (ER). The effect was rescued by the overexpression of wild type Rab5B. The results from confocal microscopy showed that the transport of LHBs from ER to MVB might be inhibited by Rab5B knockdown. The density-gradient centrifucentrifugation showed that the levels of LHBs in the ER fraction were increased, whereas those in the endosome fraction were decreased. Interestingly, the intracellular HBV DNA level was lowered after Rab5B knockdown, while the release of infectious HBV particles was enhanced. These might suggest that LHBs accumulated in ER, not only in MVB, is utilized for the formation of the envelope. The fine co-localization of core proteins and HBs proteins in ER after Rab5B depletion supports this notion. Therefore, there might be two pathways of HBV envelopment and release: MVB-dependent and MVB-independent. Which pathway is utilized more might be determined by Rab5B.  

5.2169           The cardiac microenvironment uses non-canonical WNT signaling to activate monocytes after myocardial infarction

Meyer, I.S., Jungmann, A., Dieterich, C., Zhang, M., Lasitschka, F., Werkmeister, S., Haas, J., Müller, O.J., Boutros, M., Nahrendorf, m., katus, H.A., Hardt, S.E. and Leuschner, F. EMBO Mol. Med., 9(9), 1279-1293 (2017)   A disturbed inflammatory response following myocardial infarction (MI) is associated with poor prognosis and increased tissue damage. Monocytes are key players in healing after MI, but little is known about the role of the cardiac niche in monocyte activation. This study investigated microenvironment-dependent changes in inflammatory monocytes after MI. RNA sequencing analysis of murine Ly6C^high monocytes on day 3 after MI revealed differential regulation depending on location. Notably, the local environment strongly impacted components of the WNT signaling cascade. Analysis of WNT modulators revealed a strong upregulation of WNT Inhibitory Factor 1 (WIF1) in cardiomyocytes—but not fibroblasts or endothelial cells—upon hypoxia. Compared to wild-type (WT) littermates, WIF1 knockout mice showed severe adverse remodeling marked by increased scar size and reduced ejection fraction 4 weeks after MI. While FACS analysis on day 1 after MI revealed no differences in neutrophil numbers, the hearts of WIF1 knockouts contained significantly more inflammatory monocytes than hearts from WT animals. Next, we induced AAV-mediated cardiomyocyte-specific WIF1 overexpression, which attenuated the monocyte response and improved cardiac function after MI, as compared to control-AAV-treated animals. Finally, WIF1 overexpression in isolated cardiomyocytes limited the activation of non-canonical WNT signaling and led to reduced IL-1β and IL-6 expression in monocytes/macrophages. Taken together, we investigated the cardiac microenvironment's interaction with recruited monocytes after MI and identified a novel mechanism of monocyte activation. The local initiation of non-canonical WNT signaling shifts the accumulating myeloid cells toward a pro-inflammatory state and impacts healing after myocardial infarction.  

5.2170           Interference with the production of infectious viral particles and bimodal inhibition of replication are broadly conserved antiviral properties of IFITMs

Tartour, K. et al PloS Pathogens, 13(9), e1006610 (2017)   IFITMs are broad antiviral factors that block incoming virions in endosomal vesicles, protecting target cells from infection. In the case of HIV-1, we and others reported the existence of an additional antiviral mechanism through which IFITMs lead to the production of virions of reduced infectivity. However, whether this second mechanism of inhibition is unique to HIV or extends to other viruses is currently unknown. To address this question, we have analyzed the susceptibility of a broad spectrum of viruses to the negative imprinting of the virion particles infectivity by IFITMs. The results we have gathered indicate that this second antiviral property of IFITMs extends well beyond HIV and we were able to identify viruses susceptible to the three IFITMs altogether (HIV-1, SIV, MLV, MPMV, VSV, MeV, EBOV, WNV), as well as viruses that displayed a member-specific susceptibility (EBV, DUGV), or were resistant to all IFITMs (HCV, RVFV, MOPV, AAV). The swapping of genetic elements between resistant and susceptible viruses allowed us to point to specificities in the viral mode of assembly, rather than glycoproteins as dominant factors of susceptibility. However, we also show that, contrarily to X4-, R5-tropic HIV-1 envelopes confer resistance against IFITM3, suggesting that viral receptors add an additional layer of complexity in the IFITMs-HIV interplay. Lastly, we show that the overall antiviral effects ascribed to IFITMs during spreading infections, are the result of a bimodal inhibition in which IFITMs act both by protecting target cells from incoming viruses and in driving the production of virions of reduced infectivity. Overall, our study reports for the first time that the negative imprinting of the virion particles infectivity is a conserved antiviral property of IFITMs and establishes IFITMs as a paradigm of restriction factor capable of interfering with two distinct phases of a virus life cycle.  

5.2171           Pentagalloylglucose, a highly bioavailable polyphenolic compound present in Cortex moutan, efficiently blocks hepatitis C virus entry

Behrendt, P. et al Antiviral Res., 147, 19-28 (2017)   Approximately 142 million people worldwide are infected with hepatitis C virus (HCV). Although potent direct acting antivirals are available, high costs limit access to treatment. Chronic hepatitis C virus infection remains a major cause of orthotopic liver transplantation. Moreover, re-infection of the graft occurs regularly. Antivirals derived from natural sources might be an alternative and cost-effective option to complement therapy regimens for global control of hepatitis C virus infection. We tested the antiviral properties of a mixture of different Chinese herbs/roots named Zhi Bai Di Huang Wan (ZBDHW) and its individual components on HCV. One of the ZBDHW components, Penta-O-Galloyl-Glucose (PGG), was further analyzed for its mode of action in vitro, its antiviral activity in primary human hepatocytes as well as for its bioavailability and hepatotoxicity in mice. ZBDHW, its component Cortex Moutan and the compound PGG efficiently block entry of HCV of all major genotypes and also of the related flavivirus Zika virus. PGG does not disrupt HCV virion integrity and acts primarily during virus attachment. PGG shows an additive effect when combined with the well characterized HCV inhibitor Daclatasvir. Analysis of bioavailability in mice revealed plasma levels above tissue culture IC₅₀ after a single intraperitoneal injection. In conclusion, PGG is a pangenotypic HCV entry inhibitor with high bioavailability. The low cost and wide availability of this compound make it a promising candidate for HCV combination therapies, and also emerging human pathogenic flaviviruses like ZIKV.  

5.2172           Extracellular lipid-free apolipoprotein E inhibits HCV replication and induces ABCG1-dependent cholesterol efflux

Crouchet, E., Lefevre, m., Verrier, E.R., Oudot, M.A., Baumert, T.F. and Schuster, C. Gut, 66, 896-907 (2017)   Objective The HCV life cycle and the lipid metabolism are inextricably intertwined. In the blood, HCV virions are associated with lipoproteins, forming lipoviroparticles (LVPs), which are the most infectious form of the virus. Apolipoprotein E (apoE), a key LVP component, plays an essential role in HCV entry, assembly and egress. ApoE is also a cell host factor involved in lipoprotein homeostasis. Although the majority of apoE is associated with lipoproteins, a lipid-free (LF) form exists in blood. However, the role of LF-apoE in both lipid metabolism and HCV life cycle is poorly understood. Design In this study, using the cell culture-derived HCV model system in human hepatoma Huh7.5.1 cells and primary human hepatocytes (PHH), we investigated the effect of LF-apoE on the early steps of HCV life cycle and on the lipid metabolism of hepatic cells. Results A dose-dependent decrease in HCV replication was observed when Huh7.5.1 cells and PHH were treated with increasing amounts of LF-apoE. We showed that LF-apoE acts on HCV replication independently of previously described apoE receptors. We observed that LF-apoE induced a marked hepatic cholesterol efflux via the ATP-binding cassette subfamily G member 1 (ABCG1) protein that in turn inhibits HCV replication. LF-apoE also increases both apolipoprotein AI and high-density lipoprotein production. Conclusions Our findings highlight a new mechanism in lipid metabolism regulation and interaction of the lipid metabolism with the HCV life cycle, which may be important for viral pathogenesis and might also be explored for antiviral therapy.  

5.2173           Study of hepatitis E virus infection of genotype 1 and 3 in mice with humanised liver

Sayed, I.M. et al Gut, 66, 920-929 (2017)   Objective The hepatitis E virus (HEV) is responsible for approximately 20 million infections per year worldwide. Although most infected people can spontaneously clear an HEV infection, immune-compromised individuals may evolve towards chronicity. Chronic HEV infection can be cured using ribavirin, but viral isolates with low ribavirin sensitivity have recently been identified. Although some HEV isolates can be cultured in vitro, in vivo studies are essentially limited to primates and pigs. Since the use of these animals is hampered by financial, practical and/or ethical concerns, we evaluated if human liver chimeric mice could serve as an alternative. Design Humanised mice were inoculated with different HEV-containing preparations. Results Chronic HEV infection was observed after intrasplenic injection of cell culture-derived HEV, a filtered chimpanzee stool suspension and a patient-derived stool suspension. The viral load was significantly higher in the stool compared with the plasma. Overall, the viral titre in genotype 3-infected mice was lower than that in genotype 1-infected mice. Analysis of liver tissue of infected mice showed the presence of viral RNA and protein, and alterations in host gene expression. Intrasplenic injection of HEV-positive patient plasma and oral inoculation of filtered stool suspensions did not result in robust infection. Finally, we validated our model for the evaluation of novel antiviral compounds against HEV using ribavirin. Conclusions Human liver chimeric mice can be infected with HEV of different genotypes. This small animal model will be a valuable tool for the in vivo study of HEV infection and the evaluation of novel antiviral molecules.  

5.2174           C9orf72 expansion disrupts ATM-mediated chromosomal break repair

Walker, C. et al Nature Neurosci., 20(9), 1225-1235 (2017)   Hexanucleotide repeat expansions represent the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, though the mechanisms by which such expansions cause neurodegeneration are poorly understood. We report elevated levels of DNA–RNA hybrids (R-loops) and double strand breaks in rat neurons, human cells and C9orf72 ALS patient spinal cord tissues. Accumulation of endogenous DNA damage is concomitant with defective ATM-mediated DNA repair signaling and accumulation of protein-linked DNA breaks. We reveal that defective ATM-mediated DNA repair is a consequence of P62 accumulation, which impairs H2A ubiquitylation and perturbs ATM signaling. Virus-mediated expression of C9orf72-related RNA and dipeptide repeats in the mouse central nervous system increases double strand breaks and ATM defects and triggers neurodegeneration. These findings identify R-loops, double strand breaks and defective ATM-mediated repair as pathological consequences of C9orf72 expansions and suggest that C9orf72-linked neurodegeneration is driven at least partly by genomic instability.  

5.2175           Prevention of Neurocognitive Deficiency in Mucopolysaccharidosis Type II Mice by Central Nervous System–Directed, AAV9-Mediated Iduronate Sulfatase Gene Transfer

Laoharawee, K., Podetz-Pedersen, K.M., Nguyen, T.T., Evenstar, L.B., Kitto, K.F., Nan, Z., Fairbanks, C.A., Low, W.C., Kozarsky, K.F. and Mclvor, R.S. Human Gene Therapy, 28(8), 626-638 (2017)   Mucopolysaccharidosis type II (MPS II; Hunter syndrome) is a rare X-linked recessive lysosomal disorder caused by defective iduronate-2-sulfatase (IDS), resulting in accumulation of heparan sulfate and dermatan sulfate glycosaminoglycans (GAGs). Enzyme replacement is the only Food and Drug Administration–approved therapy available for MPS II, but it is expensive and does not improve neurologic outcomes in MPS II patients. This study evaluated the effectiveness of adeno-associated virus (AAV) vector encoding human IDS delivered intracerebroventricularly in a murine model of MPS II. Supraphysiological levels of IDS were observed in the circulation (160-fold higher than wild type) for at least 28 weeks post injection and in most tested peripheral organs (up to 270-fold) at 10 months post injection. In contrast, only low levels of IDS were observed (7–40% of wild type) in all areas of the brain. Sustained IDS expression had a profound effect on normalization of GAG in all tested tissues and on prevention of hepatomegaly. Additionally, sustained IDS expression in the central nervous system (CNS) had a prominent effect in preventing neurocognitive deficit in MPS II mice treated at 2 months of age. This study demonstrates that CNS-directed, AAV9 mediated gene transfer is a potentially effective treatment for Hunter syndrome, as well as other monogenic disorders with neurologic involvement.  

5.2176           Repulsive Guidance Molecule a (RGMa) Induces Neuropathological and Behavioral Changes That Closely Resemble Parkinson's Disease

Korecka, J.A., Moloney, E.B., Eggers, R., Hobo, B., Scheffer, S., Ras-Verloop, N., Pasterkamp, R.J., Swaab, D.F., Smit, A.B., van kesteren, R.E., Bossers, K. and Verhaagen, J.

1. Neurosci., 37(39), 9361-9379 (2017)

  Repulsive guidance molecule member a (RGMa) is a membrane-associated or released guidance molecule that is involved in axon guidance, cell patterning, and cell survival. In our previous work, we showed that RGMa is significantly upregulated in the substantia nigra of patients with Parkinson's disease. Here we demonstrate the expression of RGMa in midbrain human dopaminergic (DA) neurons. To investigate whether RGMa might model aspects of the neuropathology of Parkinson's disease in mouse, we targeted RGMa to adult midbrain dopaminergic neurons using adeno-associated viral vectors. Overexpression of RGMa resulted in a progressive movement disorder, including motor coordination and imbalance, which is typical for a loss of DA release in the striatum. In line with this, RGMa induced selective degeneration of dopaminergic neurons in the substantia nigra (SN) and affected the integrity of the nigrostriatal system. The degeneration of dopaminergic neurons was accompanied by a strong microglia and astrocyte activation. The behavioral, molecular, and anatomical changes induced by RGMa in mice are remarkably similar to the clinical and neuropathological hallmarks of Parkinson's disease. Our data indicate that dysregulation of RGMa plays an important role in the pathology of Parkinson's disease, and antibody-mediated functional interference with RGMa may be a disease modifying treatment option.  

5.2177           Optimization of Retinal Gene Therapy for X-Linked Retinitis Pigmentosa Due to RPGR Mutations

Beltran, W.A. et al Molecular Therapy, 25(8), 1866-1880 (2017)   X-linked retinitis pigmentosa (XLRP) caused by mutations in the RPGR gene is an early onset and severe cause of blindness. Successful proof-of-concept studies in a canine model have recently shown that development of a corrective gene therapy for RPGR-XLRP may now be an attainable goal. In preparation for a future clinical trial, we have here optimized the therapeutic AAV vector construct by showing that GRK1 (rather than IRBP) is a more efficient promoter for targeting gene expression to both rods and cones in non-human primates. Two transgenes were used in RPGR mutant (XLPRA2) dogs under the control of the GRK1 promoter. First was the previously developed stabilized human RPGR (hRPGRstb). Second was a new full-length stabilized and codon-optimized human RPGR (hRPGRco). Long-term (>2 years) studies with an AAV2/5 vector carrying hRPGRstb under control of the GRK1 promoter showed rescue of rods and cones from degeneration and retention of vision. Shorter term (3 months) studies demonstrated comparable preservation of photoreceptors in canine eyes treated with an AAV2/5 vector carrying either transgene under the control of the GRK1 promoter. These results provide the critical molecular components (GRK1 promoter, hRPGRco transgene) to now construct a therapeutic viral vector optimized for RPGR-XLRP patients.  

5.2178           AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations

Pacouret, S. et al Molecular Therapy, 25(6), 1375-1386 (2017)   Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.  

5.2179           Palmitoylation Contributes to Membrane Curvature in Influenza A Virus Assembly and Hemagglutinin-Mediated Membrane Fusion

Chlanda, P., Mekhedov, E., Waters, H., Sodt, A.-, Schwartz, C., nair, V., Blank, P.S. and Zimmerberg, J.

1. Virol., 91(21), e00947-17 (2017)

  The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. Deacylation interrupted HA-M1 interactions since deacylated mutant HA failed to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild-type HA, deacylated HA is correlated with released particles with flat envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strain-independent failure of fusion pore enlargement among H2 (A/Japan/305/57), H3 (A/Aichi/2/68), and H3 (A/Udorn/72) viruses. In contradistinction, acylation made no difference in the low-pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded, as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins which help lower the energetic barrier to pore expansion.  

5.2180           Specific disruption of contextual memory recall by sparse additional activity in the dentate gyrus

Cho, H-Y., Kim, M. and Haan, J-H. Neurobiology of Learning and Memory, 145, 190-198 (2017)   The dentate gyrus (DG) of the hippocampus is essential for contextual and spatial memory processing. While lesion or silencing of the DG impairs contextual memory encoding and recall, overly activated DG also prevents proper memory retrieval. Abnormally elevated activity in the DG is repeatedly reported in amnesic mild cognitive impairment (aMCI) patients or aged adults. Although the correlation between memory failure and abnormally active hippocampus is clear, their causal relationship or the underlying nature of such interfering activity is not well understood. Using optogenetics aided by a carefully controlled adeno-associated virus infection system, we were able to examine the differential effects of abnormally activated hippocampus on mice motor behavior and memory function, depending on the extent of the stimulation. Optogenetic stimulation of massive proportion of dorsal DG cells resulted in memory retrieval impairment, but also induced increase in general locomotion. Random additional activity in a sparse population of dorsal DG neurons, however, interfered with contextual memory recall without inducing hyperactivity. Our findings thus establish the causal role of elevated DG activity on memory recall failure, suggesting such aberrant DG activity may contribute to amnesic symptoms in aMCI patients and aged adults.  

5.2181           Motifs in the tau protein that control binding to microtubules and aggregation determine pathological effects

Lathuiliere, A., Valdes, P., papin, S., Cacquevel, M., Maclachlan, C., Knott, G.W., Muhs, A., Paganetti, P. and Schneider, B.L. Scientific Reports, 7:13556 (2017)   Tau pathology is associated with cognitive decline in Alzheimer’s disease, and missense tau mutations cause frontotemporal dementia. Hyperphosphorylation and misfolding of tau are considered critical steps leading to tauopathies. Here, we determine how motifs controlling conformational changes in the microtubule-binding domain determine tau pathology in vivo. Human tau was overexpressed in the adult mouse forebrain to compare variants carrying residues that modulate tau propensity to acquire a β-sheet conformation. The P301S mutation linked to frontotemporal dementia causes tau aggregation and rapidly progressing motor deficits. By comparison, wild-type tau becomes heavily hyperphosphorylated, and induces behavioral impairments that do not progress over time. However, the behavioral defects caused by wild-type tau can be suppressed when β-sheet breaking proline residues are introduced in the microtubule-binding domain of tau. This modification facilitates tau interaction with microtubules, as shown by lower levels of phosphorylation, and by the enhanced protective effects of mutated tau against the severing of the cytoskeleton in neurons exposed to vinblastine. Altogether, motifs that are critical for tau conformation determine interaction with microtubules and subsequent pathological modifications, including phosphorylation and aggregation.    

5.2182           Reducing sarcolipin expression mitigates Duchenne muscular dystrophy and associated cardiomyopathy in mice

Voit, A., Patel, V., Pachon, R., Shah, V., Bakhutma, M., Kohlbrenner, E., McArdle, J.J., Dell’Italia, L.J., Mendell, J.R., Xie, L-H., Hajjar, R.J., Duan, D., Fraidenraich, D. and Babu, G.J. Nature Communications, 8:1068 (2017)   Sarcolipin (SLN) is an inhibitor of the sarco/endoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA) and is abnormally elevated in the muscle of Duchenne muscular dystrophy (DMD) patients and animal models. Here we show that reducing SLN levels ameliorates dystrophic pathology in the severe dystrophin/utrophin double mutant (mdx:utr^−/−) mouse model of DMD. Germline inactivation of one allele of the SLN gene normalizes SLN expression, restores SERCA function, mitigates skeletal muscle and cardiac pathology, improves muscle regeneration, and extends the lifespan. To translate our findings into a therapeutic strategy, we knock down SLN expression in 1-month old mdx:utr^−/− mice via adeno-associated virus (AAV) 9-mediated RNA interference. The AAV treatment markedly reduces SLN expression, attenuates muscle pathology and improves diaphragm, skeletal muscle and cardiac function. Taken together, our findings suggest that SLN reduction is a promising therapeutic approach for DMD.  

5.2183           A New Promoter Allows Optogenetic Vision Restoration with Enhanced Sensitivity in Macaque Retina

Chaffiol., A. et al Molecular Therapy, 25(11), 2546-2560 (2017)   The majority of inherited retinal degenerations converge on the phenotype of photoreceptor cell death. Second- and third-order neurons are spared in these diseases, making it possible to restore retinal light responses using optogenetics. Viral expression of channelrhodopsin in the third-order neurons under ubiquitous promoters was previously shown to restore visual function, albeit at light intensities above illumination safety thresholds. Here, we report (to our knowledge, for the first time) activation of macaque retinas, up to 6 months post-injection, using channelrhodopsin-Ca²⁺-permeable channelrhodopsin (CatCh) at safe light intensities. High-level CatCh expression was achieved due to a new promoter based on the regulatory region of the gamma-synuclein gene (SNCG) allowing strong expression in ganglion cells across species. Our promoter, in combination with clinically proven adeno-associated virus 2 (AAV2), provides CatCh expression in peri-foveolar ganglion cells responding robustly to light under the illumination safety thresholds for the human eye. On the contrary, the threshold of activation and the proportion of unresponsive cells were much higher when a ubiquitous promoter (cytomegalovirus [CMV]) was used to express CatCh. The results of our study suggest that the inclusion of optimized promoters is key in the path to clinical translation of optogenetics.  

5.2184           Characterization of the Quasi-Enveloped Hepatitis E Virus Particles Released by the Cellular Exosomal Pathway

Nagashima, S., Takahashi, T., Tanggis, Nishizawa, T., Nishiyama, T., Primadharsini, P.P. and Okamoto, H.

1. Virol., 91(22), e00822-17 (2017)

  Our previous studies demonstrated that membrane-associated hepatitis E virus (HEV) particles—now considered “quasi-enveloped particles”—are present in the multivesicular body with intraluminal vesicles (exosomes) in infected cells and that the release of HEV virions is related to the exosomal pathway. In this study, we characterized exosomes purified from the culture supernatants of HEV-infected PLC/PRF/5 cells. Purified CD63-, CD9-, or CD81-positive exosomes derived from the culture supernatants of HEV-infected cells that had been cultivated in serum-free medium were found to contain HEV RNA and the viral capsid (ORF2) and ORF3 proteins, as determined by reverse transcription-PCR (RT-PCR) and Western blotting, respectively. Furthermore, immunoelectron microscopy, with or without prior detergent and protease treatment, revealed the presence of virus-like particles in the exosome fraction. These particles were 39.6 ± 1.0 nm in diameter and were covered with a lipid membrane. After treatment with detergent and protease, the diameter of these virus-like particles was 26.9 ± 0.9 nm, and the treated particles became accessible with an anti-HEV ORF2 monoclonal antibody (MAb). The HEV particles in the exosome fraction were capable of infecting naive PLC/PRF/5 cells but were not neutralized by an anti-HEV ORF2 MAb which efficiently neutralizes nonenveloped HEV particles in cell culture. These results indicate that the membrane-wrapped HEV particles released by the exosomal pathway are copurified with the exosomes in the exosome fraction and suggest that the capsids of HEV particles are individually covered by lipid membranes resembling those of exosomes, similar to enveloped viruses.  

5.2185           Treatment of hypertension by increasing impaired endothelial TRPV4-KCa2.3 interaction

He, D. et al EMBO Mol. Med., 9(11), 1491-1503 (2017)   The currently available antihypertensive agents have undesirable adverse effects due to systemically altering target activity including receptors, channels, and enzymes. These effects, such as loss of potassium ions induced by diuretics, bronchospasm by beta-blockers, constipation by Ca²⁺ channel blockers, and dry cough by ACEI, lead to non-compliance with therapies (Moser, 1990). Here, based on new hypertension mechanisms, we explored a new antihypertensive approach. We report that transient receptor potential vanilloid 4 (TRPV4) interacts with Ca²⁺-activated potassium channel 3 (KCa2.3) in endothelial cells (ECs) from small resistance arteries of normotensive humans, while ECs from hypertensive patients show a reduced interaction between TRPV4 and KCa2.3. Murine hypertension models, induced by high-salt diet, N(G)-nitro-l-arginine intake, or angiotensin II delivery, showed decreased TRPV4-KCa2.3 interaction in ECs. Perturbation of the TRPV4-KCa2.3 interaction in mouse ECs by overexpressing full-length KCa2.3 or defective KCa2.3 had hypotensive or hypertensive effects, respectively. Next, we developed a small-molecule drug, JNc-440, which showed affinity for both TRPV4 and KCa2.3. JNc-440 significantly strengthened the TRPV4-KCa2.3 interaction in ECs, enhanced vasodilation, and exerted antihypertensive effects in mice. Importantly, JNc-440 specifically targeted the impaired TRPV4-KCa2.3 interaction in ECs but did not systemically activate TRPV4 and KCa2.3. Together, our data highlight the importance of impaired endothelial TRPV4-KCa2.3 coupling in the progression of hypertension and suggest a novel approach for antihypertensive drug development.  

5.2186           Src-dependent phosphorylation of μ-opioid receptor at Tyr336 modulates opiate withdrawal

Zhang, L., Kibaly, C., Wang, Y-J., Xu, C., Song, K.Y., Mcgarrah, P.W., Loh, H.H., Liu, J-G. and Law, P-Y. EMBO Mol. Med., 9(11), 1521-1536 (2017)   Opiate withdrawal/negative reinforcement has been implicated as one of the mechanisms for the progression from impulsive to compulsive drug use. Increase in the intracellular cAMP level and protein kinase A (PKA) activities within the neurocircuitry of addiction has been a leading hypothesis for opiate addiction. This increase requires the phosphorylation of μ-opioid receptor (MOR) at Tyr³³⁶ by Src after prolonged opiate treatment in vitro. Here, we report that the Src-mediated MOR phosphorylation at Tyr³³⁶ is a prerequisite for opiate withdrawal in mice. We observed the recruitment of Src in the vicinity of MOR and an increase in phosphorylated Tyr³³⁶ (pY336) levels during naloxone-precipitated withdrawal. The intracerebroventricular or stereotaxic injection of a Src inhibitor (AZD0530), or Src shRNA viruses attenuated pY336 levels, and several somatic withdrawal signs. This was also observed in Fyn^−/− mice. The stereotaxic injection of wild-type MOR, but not mutant (Y336F) MOR, lentiviruses into the locus coeruleus of MOR^−/− mice restored somatic withdrawal jumping. Regulating pY336 levels during withdrawal might be a future target for drug development to prevent opiate addictive behaviors.  

5.2187           Site Specific Modification of Adeno-Associated Virus Enables Both Fluorescent Imaging of Viral Particles and Characterization of the Capsid Interactome

Chandran, J.S., Sharp, P.S:, karyka, E., da Conceicao Aves-Cruzeiro, J.M., Coldicott, I., Castelli, L., Hautbergue, G., Collins, M.O. and Azzouz, M. Scientific Reports, 7:14766 (2017)   Adeno-associated viruses (AAVs) are attractive gene therapy vectors due to their low toxicity, high stability, and rare integration into the host genome. Expressing ligands on the viral capsid can re-target AAVs to new cell types, but limited sites have been identified on the capsid that tolerate a peptide insertion. Here, we incorporated a site-specific tetracysteine sequence into the AAV serotype 9 (AAV9) capsid, to permit labelling of viral particles with either a fluorescent dye or biotin. We demonstrate that fluorescently labelled particles are detectable in vitro, and explore the utility of the method in vivo in mice with time-lapse imaging. We exploit the biotinylated viral particles to generate two distinct AAV interactomes, and identify several functional classes of proteins that are highly represented: actin/cytoskeletal protein binding, RNA binding, RNA splicing/processing, chromatin modifying, intracellular trafficking and RNA transport proteins. To examine the biological relevance of the capsid interactome, we modulated the expression of two proteins from the interactomes prior to AAV transduction. Blocking integrin αVβ6 receptor function reduced AAV9 transduction, while reducing histone deacetylase 4 (HDAC4) expression enhanced AAV transduction. Our method demonstrates a strategy for inserting motifs into the AAV capsid without compromising viral titer or infectivity.  

5.2188           Cardiac myocyte miR-29 promotes pathological remodeling of the heart by activating Wnt signaling

Sassi, Y. et al Nature Communications, 8:1614 (2017)   Chronic cardiac stress induces pathologic hypertrophy and fibrosis of the myocardium. The microRNA-29 (miR-29) family has been found to prevent excess collagen expression in various organs, particularly through its function in fibroblasts. Here, we show that miR-29 promotes pathologic hypertrophy of cardiac myocytes and overall cardiac dysfunction. In a mouse model of cardiac pressure overload, global genetic deletion of miR-29 or antimiR-29 infusion prevents cardiac hypertrophy and fibrosis and improves cardiac function. Targeted deletion of miR-29 in cardiac myocytes in vivo also prevents cardiac hypertrophy and fibrosis, indicating that the function of miR-29 in cardiac myocytes dominates over that in non-myocyte cell types. Mechanistically, we found cardiac myocyte miR-29 to de-repress Wnt signaling by directly targeting four pathway factors. Our data suggests that, cell- or tissue-specific antimiR-29 delivery may have therapeutic value for pathological cardiac remodeling and fibrosis.  

5.2189           Cis-regulatory landscapes of four cell types of the retina

Hartl, D., Krebs, A.R., Jüttner, J., Roska, B. and Schübeler, D. Nucelic Acids Res., 45(20), 11607-11621 (2017)   The retina is composed of ∼50 cell-types with specific functions for the process of vision. Identification of the cis-regulatory elements active in retinal cell-types is key to elucidate the networks controlling this diversity. Here, we combined transcriptome and epigenome profiling to map the regulatory landscape of four cell-types isolated from mouse retinas including rod and cone photoreceptors as well as rare inter-neuron populations such as horizontal and starburst amacrine cells. Integration of this information reveals sequence determinants and candidate transcription factors for controlling cellular specialization. Additionally, we refined parallel reporter assays to enable studying the transcriptional activity of large collection of sequences in individual cell-types isolated from a tissue. We provide proof of concept for this approach and its scalability by characterizing the transcriptional capacity of several hundred putative regulatory sequences within individual retinal cell-types. This generates a catalogue of cis-regulatory regions active in retinal cell types and we further demonstrate their utility as potential resource for cellular tagging and manipulation.  

5.2190           Identification of liver-specific enhancer–promoter activity in the 3′ untranslated region of the wild-type AAV2 genome

Logan, G.J. et al Nature Genetics, 49(8), 1267-1273 (2017)   Vectors based on adeno-associated virus type 2 (AAV2) are powerful tools for gene transfer and genome editing applications1,2. The level of interest in this system has recently surged in response to reports of therapeutic efficacy in human clinical trials, most notably for those in patients with hemophilia B (ref. 3). Understandably, a recent report drawing an association between AAV2 integration events and human hepatocellular carcinoma (HCC)4 has generated controversy about the causal or incidental nature of this association and the implications for AAV vector safety5,6,7,8,9. Here we describe and functionally characterize a previously unknown liver-specific enhancer–promoter element in the wild-type AAV2 genome that is found between the stop codon of the cap gene, which encodes proteins that form the capsid, and the right-hand inverted terminal repeat. This 124-nt sequence is within the 163-nt common insertion region of the AAV genome, which has been implicated in the dysregulation of known HCC driver genes4 and thus offers added insight into the possible link between AAV integration events and the multifactorial pathogenesis of HCC10.  

5.2191           Asparagine endopeptidase cleaves α-synuclein and mediates pathologic activities in Parkinson's disease

Zhang, Z. et al Nature Struct. Mol. Biol., 24(8), 632-642 (2017)   Aggregated forms of α-synuclein play a crucial role in the pathogenesis of synucleinopathies such as Parkinson's disease (PD). However, the molecular mechanisms underlying the pathogenic effects of α-synuclein are not completely understood. Here we show that asparagine endopeptidase (AEP) cleaves human α-synuclein, triggers its aggregation and escalates its neurotoxicity, thus leading to dopaminergic neuronal loss and motor impairments in a mouse model. AEP is activated and cleaves human α-synuclein at N103 in an age-dependent manner. AEP is highly activated in human brains with PD, and it fragments α-synuclein, which is found aggregated in Lewy bodies. Overexpression of the AEP-cleaved α-synuclein1–103 fragment in the substantia nigra induces both dopaminergic neuronal loss and movement defects in mice. In contrast, inhibition of AEP-mediated cleavage of α-synuclein (wild type and A53T mutant) diminishes α-synuclein's pathologic effects. Together, these findings support AEP's role as a key mediator of α-synuclein-related etiopathological effects in PD.  

5.2192           Postsynaptic adhesion GPCR latrophilin-2 mediates target recognition in entorhinal-hippocampal synapse assembly

Anderson, G.R., Maxeiner, S., Sando, R., Tsetsenius, T., Malenka, R.C. and Südhof, T.C:

1. Cell Biol., 216(11), 3831-3846 (2017)

  Synapse assembly likely requires postsynaptic target recognition by incoming presynaptic afferents. Using newly generated conditional knock-in and knockout mice, we show in this study that latrophilin-2 (Lphn2), a cell-adhesion G protein–coupled receptor and presumptive α-latrotoxin receptor, controls the numbers of a specific subset of synapses in CA1-region hippocampal neurons, suggesting that Lphn2 acts as a synaptic target-recognition molecule. In cultured hippocampal neurons, Lphn2 maintained synapse numbers via a postsynaptic instead of a presynaptic mechanism, which was surprising given its presumptive role as an α-latrotoxin receptor. In CA1-region neurons in vivo, Lphn2 was specifically targeted to dendritic spines in the stratum lacunosum-moleculare, which form synapses with presynaptic entorhinal cortex afferents. In this study, postsynaptic deletion of Lphn2 selectively decreased spine numbers and impaired synaptic inputs from entorhinal but not Schaffer-collateral afferents. Behaviorally, loss of Lphn2 from the CA1 region increased spatial memory retention but decreased learning of sequential spatial memory tasks. Thus, Lphn2 appears to control synapse numbers in the entorhinal cortex/CA1 region circuit by acting as a domain-specific postsynaptic target-recognition molecule.  

5.2193           Nucleus accumbens feedforward inhibition circuit promotes cocaine self-administration

Yu, J., Yan, Y., Li, K-L., Wang, Y., Huang, Y.H., Urban, N.N., Nestler, E.J., Schlüter, O:M. and Dong, Y. PNAS, 114(41), E750-E759 (2017)   The basolateral amygdala (BLA) sends excitatory projections to the nucleus accumbens (NAc) and regulates motivated behaviors partially by activating NAc medium spiny neurons (MSNs). Here, we characterized a feedforward inhibition circuit, through which BLA-evoked activation of NAc shell (NAcSh) MSNs was fine-tuned by GABAergic monosynaptic innervation from adjacent fast-spiking interneurons (FSIs). Specifically, BLA-to-NAcSh projections predominantly innervated NAcSh FSIs compared with MSNs and triggered action potentials in FSIs preceding BLA-mediated activation of MSNs. Due to these anatomical and temporal properties, activation of the BLA-to-NAcSh projection resulted in a rapid FSI-mediated inhibition of MSNs, timing-contingently dictating BLA-evoked activation of MSNs. Cocaine self-administration selectively and persistently up-regulated the presynaptic release probability of BLA-to-FSI synapses, entailing enhanced FSI-mediated feedforward inhibition of MSNs upon BLA activation. Experimentally enhancing the BLA-to-FSI transmission in vivo expedited the acquisition of cocaine self-administration. These results reveal a previously unidentified role of an FSI-embedded circuit in regulating NAc-based drug seeking and taking.  

5.2194           Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9

Pinto, B.S., Saxena, T., Oliveira, R., Xia, G., Swanson, M.S. and Wang, E:T. Molecular Cell, 68(3), 479-490 (2017)   Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs endothelial corneal dystrophy, and C9orf72-ALS/FTD. Reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that deactivated Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences; targeting the non-template strand was more effective. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, and C9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.  

5.2195           SMV1, an extremely stable thermophilic virus platform for nanoparticle trafficking in the mammalian GI tract

Uldahl, K:B., Walk, S.T., Olshefsky, S.C., Young, M.J. and peng, X.

1. Appl. Microbiol., 123(5), 1286-1297 (2017)

  Aims Analysis of the stability and safety of Sulfolobus monocaudavirus 1 (SMV1) during passage through the mammalian GI tract. Methods and Results A major challenge of using nano-vectors to target gut microbiome is their survival during passage through the extremely acidic and proteolytic environment of the mammalian GI tract. Here, we investigated the thermo-acidophilic archaeal virus SMV1 as a candidate therapeutic nano-vector for the distal mammalian GI tract microbiome. We investigated the anatomical distribution, vector stability and immunogenicity of this virus following oral ingestion in mice and compared these traits to the more classically used Inovirus vector M13KE. We found that SMV1 particles were highly stable under both simulated GI tract conditions (in vitro) and in mice (in vivo). Moreover, SMV1 could not be detected in tissues outside the GI tract and it elicited a nearly undetectable inflammatory response. Finally, we used human intestinal organoids (HIOs) to show that labelled SMV1 did not invade or otherwise perturb the human GI tract epithelium. Conclusion Sulfolobus monocaudavirus 1 appeared stable and safe during passage though the mammalian GI tract.  

5.2196           Protocol for Efficient Generation and Characterization of Adeno-Associated Viral Vectors

Jungmann, A., Leuchs, B., Rommelaere, J., Katus, H.A. and Müller, O.J. Hum. Gene Ther. Methods, 28(5), 235-246 (2017)   Adeno-associated virus vectors are a powerful tool for gene transfer approaches. We have established a simple and fast plasmid-based production system for achieving high adeno-associated virus titers within 6 working days. The same procedure can be used for all serotypes and thus allows direct comparability of different serotypes. In this protocol we describe a step-by-step procedure that results in well-characterized vectors suitable for both in vitro approaches and preclinical studies.  

5.2197           Downregulation of Human Endogenous Retrovirus Type K (HERV-K) Viral env RNA in Pancreatic Cancer Cells Decreases Cell Proliferation and Tumor Growth

Li, M., Radvanyi, L., Yin, B., Li, J., Chivukula, R., Lin, K., Lu, Y., Shen, J.J., Chang, D.Z., Li, D., Johanning, G.L. and Wang-Johanning, F. Clin. Cancer Res., 23(19), 5892-5911 (2017)   Purpose: We investigated the role of the human endogenous retrovirus type K (HERV-K) envelope (env) gene in pancreatic cancer. Experimental Design: shRNA was employed to knockdown (KD) the expression of HERV-K in pancreatic cancer cells. Results: HERV-K env expression was detected in seven pancreatic cancer cell lines and in 80% of pancreatic cancer patient biopsies, but not in two normal pancreatic cell lines or uninvolved normal tissues. A new HERV-K splice variant was discovered in several pancreatic cancer cell lines. Reverse transcriptase activity and virus-like particles were observed in culture media supernatant obtained from Panc-1 and Panc-2 cells. HERV-K viral RNA levels and anti-HERV-K antibody titers were significantly higher in pancreatic cancer patient sera (N = 106) than in normal donor sera (N = 40). Importantly, the in vitro and in vivo growth rates of three pancreatic cancer cell lines were significantly reduced after HERV-K KD by shRNA targeting HERV-K env, and there was reduced metastasis to lung after treatment. RNA-Seq results revealed changes in gene expression after HERV-K env KD, including RAS and TP53. Furthermore, downregulation of HERV-K Env protein expression by shRNA also resulted in decreased expression of RAS, p-ERK, p-RSK, and p-AKT in several pancreatic cancer cells or tumors. Conclusions: These results demonstrate that HERV-K influences signal transduction via the RAS–ERK–RSK pathway in pancreatic cancer. Our data highlight the potentially important role of HERV-K in tumorigenesis and progression of pancreatic cancer, and indicate that HERV-K viral proteins may be attractive biomarkers and/or tumor-associated antigens, as well as potentially useful targets for detection, diagnosis, and immunotherapy of pancreatic cancer.  

5.2198           Autophagy protects pancreatic beta cell mass and function in the setting of a high-fat and high-glucose diet

Sheng, Q., Xiao, X., Prasadan, K., Chen, C., Ming, Y., Fusco, J., Gangopadhyay, N.N., Ricks, D. and Gittes, G.K. Scientific Reports, 7:16348 (2017)   Autophagy is a major regulator of pancreatic beta cell homeostasis. Altered autophagic activity has been implicated in the beta cells of patients with type 2 diabetes, and in the beta cells of obese diabetic rodents. Here, we show that autophagy was induced in beta cells by either a high-fat diet or a combined high-fat and high-glucose diet, but not by high-glucose alone. However, a high-glucose intake alone did increase beta cell mass and insulin secretion moderately. Depletion of Atg7, a necessary component of the autophagy pathway, in beta cells by pancreatic intra-ductal AAV8-shAtg7 infusion in C57BL/6 mice, resulted in decreased beta cell mass, impaired glucose tolerance, defective insulin secretion, and increased apoptosis when a combined high-fat and high-glucose diet was given, seemingly due to suppression of autophagy. Taken together, our findings suggest that the autophagy pathway may act as a protective mechanism in pancreatic beta cells during a high-calorie diet.  

5.2199           Subthalamic Nucleus Deep Brain Stimulation Does Not Modify the Functional Deficits or Axonopathy Induced by Nigrostriatal α-Synuclein Overexpression

Fischer, D.L., manfredsson, F.P., Kemp, C.J., Cole-Strauss, A., Lipton, J.W., Duffy, M.F., Polinsky, N.K., Steece-Collier, K., Collier, T.J., Gombash, S.E., Buhlinger, D.J. and Sortwell, C.E. Scientific Reports, 7:16356 (2017)   Subthalamic nucleus deep brain stimulation (STN DBS) protects dopaminergic neurons of the substantia nigra pars compacta (SNpc) against 6-OHDA and MPTP. We evaluated STN DBS in a parkinsonian model that displays α-synuclein pathology using unilateral, intranigral injections of recombinant adeno-associated virus pseudotype 2/5 to overexpress wildtype human α-synuclein (rAAV2/5 α-syn). A low titer of rAAV2/5 α-syn results in progressive forelimb asymmetry, loss of striatal dopaminergic terminal density and modest loss of SNpc dopamine neurons after eight weeks, corresponding to robust human-Snca expression and no effect on rat-Snca, Th, Bdnf or Trk2. α-syn overexpression increased phosphorylation of ribosomal protein S6 (p-rpS6) in SNpc neurons, a readout of trkB activation. Rats received intranigral injections of rAAV2/5 α-syn and three weeks later received four weeks of STN DBS or electrode implantation that remained inactive. STN DBS did not protect against α-syn-mediated deficits in forelimb akinesia, striatal denervation or loss of SNpc neuron, nor did STN DBS elevate p-rpS6 levels further. ON stimulation, forelimb asymmetry was exacerbated, indicating α-syn overexpression-mediated neurotransmission deficits. These results demonstrate that STN DBS does not protect the nigrostriatal system against α-syn overexpression-mediated toxicity. Whether STN DBS can be protective in other models of synucleinopathy is unknown.  

5.2200           Full-Length Isoforms of Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Accumulate in the Cytoplasm of Cells Undergoing the Lytic Cycle of Replication

Garrigues, H.J., Howard, K., Barcy, S., Ikoma, M., Moses, A., Deutsch, G.H., Wu, D., Ueda, K. and Rose, T.M:

1. Virol., 91(24), e01532-17 (2017)

  The latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) performs a variety of functions to establish and maintain KSHV latency. During latency, LANA localizes to discrete punctate spots in the nucleus, where it tethers viral episomes to cellular chromatin and interacts with nuclear components to regulate cellular and viral gene expression. Using highly sensitive tyramide signal amplification, we determined that LANA localizes to the cytoplasm in different cell types undergoing the lytic cycle of replication after de novo primary infection and after spontaneous, tetradecanoyl phorbol acetate-, or open reading frame 50 (ORF50)/replication transactivator (RTA)-induced activation. We confirmed the presence of cytoplasmic LANA in a subset of cells in lytically active multicentric Castleman disease lesions. The induction of cellular migration by scratch-wounding confluent cell cultures, culturing under subconfluent conditions, or induction of cell differentiation in primary cultures upregulated the number of cells permissive for primary lytic KSHV infection. The induction of lytic replication was characterized by high-level expression of cytoplasmic LANA and nuclear ORF59, a marker of lytic replication. Subcellular fractionation studies revealed the presence of multiple isoforms of LANA in the cytoplasm of ORF50/RTA-activated Vero cells undergoing primary infection. Mass spectrometry analysis demonstrated that cytoplasmic LANA isoforms were full length, containing the N-terminal nuclear localization signal. These results suggest that trafficking of LANA to different subcellular locations is a regulated phenomenon, which allows LANA to interact with cellular components in different compartments during both the latent and the replicative stages of the KSHV life cycle.  

5.2201           The full transcription map of mouse papillomavirus type 1 (MmuPV1) in mouse wart tissues

Xue, X-Y., Majerciak, V., Uberoi, A., Kim, B-H., Gotte, D., Chen, X., Cam, M., lambert, P.F. and Zheng, Z-M. PloS Pathogens, 13(11), e1006715 (2017)   Mouse papillomavirus type 1 (MmuPV1) provides, for the first time, the opportunity to study infection and pathogenesis of papillomaviruses in the context of laboratory mice. In this report, we define the transcriptome of MmuPV1 genome present in papillomas arising in experimentally infected mice using a combination of RNA-seq, PacBio Iso-seq, 5’ RACE, 3’ RACE, primer-walking RT-PCR, RNase protection, Northern blot and in situ hybridization analyses. We demonstrate that the MmuPV1 genome is transcribed unidirectionally from five major promoters (P) or transcription start sites (TSS) and polyadenylates its transcripts at two major polyadenylation (pA) sites. We designate the P₇₅₀₃, P₃₆₀ and P₈₅₉ as “early” promoters because they give rise to transcripts mostly utilizing the polyadenylation signal at nt 3844 and therefore can only encode early genes, and P₇₁₀₇ and P₅₃₃ as “late” promoters because they give rise to transcripts utilizing polyadenylation signals at either nt 3844 or nt 7047, the latter being able to encode late, capsid proteins. MmuPV1 genome contains five splice donor sites and three acceptor sites that produce thirty-six RNA isoforms deduced to express seven predicted early gene products (E6, E7, E1, E1^M1, E1^M2, E2 and E8^E2) and three predicted late gene products (E1^E4, L2 and L1). The majority of the viral early transcripts are spliced once from nt 757 to 3139, while viral late transcripts, which are predicted to encode L1, are spliced twice, first from nt 7243 to either nt 3139 (P₇₁₀₇) or nt 757 to 3139 (P₅₃₃) and second from nt 3431 to nt 5372. Thirteen of these viral transcripts were detectable by Northern blot analysis, with the P₅₃₃-derived late E1^E4 transcripts being the most abundant. The late transcripts could be detected in highly differentiated keratinocytes of MmuPV1-infected tissues as early as ten days after MmuPV1 inoculation and correlated with detection of L1 protein and viral DNA amplification. In mature warts, detection of L1 was also found in more poorly differentiated cells, as previously reported. Subclinical infections were also observed. The comprehensive transcription map of MmuPV1 generated in this study provides further evidence that MmuPV1 is similar to high-risk cutaneous beta human papillomaviruses. The knowledge revealed will facilitate the use of MmuPV1 as an animal virus model for understanding of human papillomavirus gene expression, pathogenesis and immunology.  

5.2202           Molecular Therapy of Melanocortin-4-Receptor Obesity by an Autoregulatory BDNF Vector

Siu, J.J., Queen, N.J., Liu, X., Huang, W., Murphy, T.M. and Cao, L. Molecular Therapy – Methods & Clinical Development, 7, 83-95 (2017)   Mutations in the melanocortin-4-receptor (MC4R) comprise the most common monogenic form of severe early-onset obesity, and conventional treatments are either ineffective long-term or contraindicated. Immediately downstream of MC4R—in the pathway for regulating energy balance—is brain-derived neurotrophic factor (BDNF). Our previous studies show that adeno-associated virus (AAV)-mediated hypothalamic BDNF gene transfer alleviates obesity and diabetes in both diet-induced and genetic models. To facilitate clinical translation, we developed a built-in autoregulatory system to control therapeutic gene expression mimicking the body’s natural feedback systems. This autoregulatory approach leads to a sustainable plateau of body weight after substantial weight loss is achieved. Here, we examined the efficacy and safety of autoregulatory BDNF gene therapy in Mc4r heterozygous mice, which best resemble MC4R obese patients. Mc4r heterozygous mice were treated with either autoregulatory BDNF vector or YFP control and monitored for 30 weeks. BDNF gene therapy prevented the development of obesity and metabolic syndromes characterized by decreasing body weight and adiposity, suppressing food intake, alleviating hyperleptinemia and hyperinsulinemia, improving glucose and insulin tolerance, and increasing energy expenditure, without adverse cardiovascular function or behavioral disturbances. These safety and efficacy data provide preclinical evidence that BDNF gene therapy is a compelling treatment option for MC4R-deficient obese patients.      

5.2203           Structure, proteome and genome of Sinorhizobium meliloti phage ΦM5: A virus with LUZ24-like morphology and a highly mosaic genome

Johjnson, M., Sena-Lelez, M., Washburn, B.K., Platt, G.N., Lu, S., Brewer, T.E., Lynn, J.S., Stroupe, M.E. and Jones, K.M.

1. Struct. Biol., 200, 343-359 (2017)

  Bacteriophages of nitrogen-fixing rhizobial bacteria are revealing a wealth of novel structures, diverse enzyme combinations and genomic features. Here we report the cryo-EM structure of the phage capsid at 4.9–5.7 Å-resolution, the phage particle proteome, and the genome of the Sinorhizobium meliloti-infecting Podovirus ΦM5. This is the first structure of a phage with a capsid and capsid-associated structural proteins related to those of the LUZ24-like viruses that infect Pseudomonas aeruginosa. Like many other Podoviruses, ΦM5 is a T = 7 icosahedron with a smooth capsid and short, relatively featureless tail. Nonetheless, this group is phylogenetically quite distinct from Podoviruses of the well-characterized T7, P22, and epsilon 15 supergroups. Structurally, a distinct bridge of density that appears unique to ΦM5 reaches down the body of the coat protein to the extended loop that interacts with the next monomer in a hexamer, perhaps stabilizing the mature capsid. Further, the predicted tail fibers of ΦM5 are quite different from those of enteric bacteria phages, but have domains in common with other rhizophages. Genomically, ΦM5 is highly mosaic. The ΦM5 genome is 44,005 bp with 357 bp direct terminal repeats (DTRs) and 58 unique ORFs. Surprisingly, the capsid structural module, the tail module, the DNA-packaging terminase, the DNA replication module and the integrase each appear to be from a different lineage. One of the most unusual features of ΦM5 is its terminase whose large subunit is quite different from previously-described short-DTR-generating packaging machines and does not fit into any of the established phylogenetic groups.  

5.2204           Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques

Burwitz, B.J. et al Nature Communications, 8:2146 (2017)   Hepatitis B virus (HBV) is a major global health concern, and the development of curative therapeutics is urgently needed. Such efforts are impeded by the lack of a physiologically relevant, pre-clinical animal model of HBV infection. Here, we report that expression of the HBV entry receptor, human sodium-taurocholate cotransporting polypeptide (hNTCP), on macaque primary hepatocytes facilitates HBV infection in vitro, where all replicative intermediates including covalently closed circular DNA (cccDNA) are present. Furthermore, viral vector-mediated expression of hNTCP on hepatocytes in vivo renders rhesus macaques permissive to HBV infection. These in vivo macaque HBV infections are characterized by longitudinal HBV DNA in serum, and detection of HBV DNA, RNA, and HBV core antigen (HBcAg) in hepatocytes. Together, these results show that expressing hNTCP on macaque hepatocytes renders them susceptible to HBV infection, thereby establishing a physiologically relevant model of HBV infection to study immune clearance and test therapeutic and curative approaches.  

5.2205           Hypothalamus Specific Re-Introduction of SNORD116 into Otherwise Snord116 Deficient Mice Increased Energy Expenditure

Qi, Y., Purtell, L., Fu, M., Zhang, L., Zolotukhin, S., Campell, L. and Herzog, H.

1. Neuroendocrinol., 29(10), e12457 (2017)

  The Snord116 gene cluster has been recognised as a critical contributor to the Prader–Willi syndrome (PWS), with mice lacking Snord116 displaying many classical PWS phenotypes, including low postnatal body weight, reduced bone mass and increased food intake. However, these mice do not develop obesity as a result of increased energy expenditure. To understand the physiological function of SNORD116 better and potentially rescue the altered metabolism of Snord116^−/− mice, we used an adeno-associated viral (AAV) approach to reintroduce the product of the Snord116 gene into the hypothalamus in Snord116^−/− mice at different ages. The results obtained show that mid-hypothalamic re-introduction of SNORD116 in 6-week-old Snord116^−/− mice leads to significantly reduced body weight and weight gain, which is associated with elevated energy expenditure. Importantly, when the intervention targets other areas such as the anterior region of the hypothalamus or the reintroduction occurs in older mice, the positive effects on energy expenditure are diminished. These data indicate that the metabolic symptoms of PWS develop gradually and the Snord116 gene plays a critical role during this process. Furthermore, when we investigated the consequences of SNORD116 re-introduction under conditions of thermoneutrality where the mild cold stress influences are avoided, we also observed a significant increase in energy expenditure. In conclusion, the rescue of mid-hypothalamic Snord116 deficiency in young Snord116 germline deletion mice increases energy expenditure, providing fundamental information contributing to potential virus-mediated genetic therapy in PWS.  

5.2206           Lysophosphatidylcholine acyltransferase 1 is downregulated by hepatitis C virus: impact on production of lipo-viro-particles

Beilstein, F., lemasson, M., Pene, v., Rainteau, D., Demignot, S. and Rosenberg, A.R. Gut, 66(12), 2160-2169 (2017)   Objective HCV is intimately linked with the liver lipid metabolism, devoted to the efflux of triacylglycerols stored in lipid droplets (LDs) in the form of triacylglycerol-rich very-low-density lipoproteins (VLDLs): (i) the most infectious HCV particles are those of lowest density due to association with triacylglycerol-rich lipoproteins and (ii) HCV-infected patients frequently develop hepatic steatosis (increased triacylglycerol storage). The recent identification of lysophosphatidylcholine acyltransferase 1 (LPCAT1) as an LD phospholipid-remodelling enzyme prompted us to investigate its role in liver lipid metabolism and HCV infectious cycle. Design Huh-7.5.1 cells and primary human hepatocytes (PHHs) were infected with JFH1-HCV. LPCAT1 depletion was achieved by RNA interference. Cells were monitored for LPCAT1 expression, lipid metabolism and HCV production and infectivity. The density of viral particles was assessed by isopycnic ultracentrifugation. Results Upon HCV infection, both Huh-7.5.1 cells and PHH had decreased levels of LPCAT1 transcript and protein, consistent with transcriptional downregulation. LPCAT1 depletion in either naive or infected Huh-7.5.1 cells resulted in altered lipid metabolism characterised by LD remodelling, increased triacylglycerol storage and increased secretion of VLDL. In infected Huh-7.5.1 cells or PHH, LPCAT1 depletion increased production of the viral particles of lowest density and highest infectivity. Conclusions We have identified LPCAT1 as a modulator of liver lipid metabolism downregulated by HCV, which appears as a viral strategy to increase the triacylglycerol content and hence infectivity of viral particles. Targeting this metabolic pathway may represent an attractive therapeutic approach to reduce both the viral titre and hepatic steatosis.  

5.2207           Epigenetic editing of the Dlg4/PSD95 gene improves cognition in aged and Alzheimer’s disease mice

Bustos, F. et al Brain, 140(12), 3252-3268 (2017)   The Dlg4 gene encodes for post-synaptic density protein 95 (PSD95), a major synaptic protein that clusters glutamate receptors and is critical for plasticity. PSD95 levels are diminished in ageing and neurodegenerative disorders, including Alzheimer’s disease and Huntington’s disease. The epigenetic mechanisms that (dys)regulate transcription of Dlg4/PSD95, or other plasticity genes, are largely unknown, limiting the development of targeted epigenome therapy. We analysed the Dlg4/PSD95 epigenetic landscape in hippocampal tissue and designed a Dlg4/PSD95 gene-targeting strategy: a Dlg4/PSD95 zinc finger DNA-binding domain was engineered and fused to effector domains to either repress (G9a, Suvdel76, SKD) or activate (VP64) transcription, generating artificial transcription factors or epigenetic editors (methylating H3K9). These epi-editors altered critical histone marks and subsequently Dlg4/PSD95 expression, which, importantly, impacted several hippocampal neuron plasticity processes. Intriguingly, transduction of the artificial transcription factor PSD95-VP64 rescued memory deficits in aged and Alzheimer’s disease mice. Conclusively, this work validates PSD95 as a key player in memory and establishes epigenetic editing as a potential therapy to treat human neurological disorders.  

5.2208           Communication via extracellular vesicles enhances viral infection of a cosmopolitan alga

Schatz, D., Rosenwasser, S., Malitsky, S., Wolf, S.G., Feldmesser, E. and Vardi, A. Nature Microbiol., 2(11), 1485-1492 82017)   Communication between microorganisms in the marine environment has immense ecological impact by mediating trophic-level interactions and thus determining community structure1. Extracellular vesicles (EVs) are produced by bacteria2,3, archaea4, protists5 and metazoans, and can mediate pathogenicity6 or act as vectors for intercellular communication. However, little is known about the involvement of EVs in microbial interactions in the marine environment7. Here we investigated the signalling role of EVs produced during interactions between the cosmopolitan alga Emiliania huxleyi and its specific virus (EhV, Phycodnaviridae)8, which leads to the demise of these large-scale oceanic blooms9,10. We found that EVs are highly produced during viral infection or when bystander cells are exposed to infochemicals derived from infected cells. These vesicles have a unique lipid composition that differs from that of viruses and their infected host cells, and their cargo is composed of specific small RNAs that are predicted to target sphingolipid metabolism and cell-cycle pathways. EVs can be internalized by E. huxleyi cells, which consequently leads to a faster viral infection dynamic. EVs can also prolong EhV half-life in the extracellular milieu. We propose that EVs are exploited by viruses to sustain efficient infectivity and propagation across E. huxleyi blooms. As these algal blooms have an immense impact on the cycling of carbon and other nutrients11,12, this mode of cell–cell communication may influence the fate of the blooms and, consequently, the composition and flow of nutrients in marine microbial food webs.  

5.2209           Adeno-associated virus-mediated delivery of genes to mouse spermatogonial stem cells

Watanabe, S., Kanatsu-Shinohara, M., Ogonuki, N., Matoba, S., Ogura, A. and Shinohara, T. Biol. Reprod., 96(1), 221-231 (2017)   Spermatogenesis is a complicated process that originates from spermatogonial stem cells (SSCs), which have self-renewal activity. Because SSCs are the only stem cells in the body that transmit genetic information to the next generation, they are an attractive target for germline modification. Although several virus vectors have been successfully used to transduce SSCs, cell toxicity or insertional mutagenesis of the transgene has limited their usage. Adeno-associated virus (AAV) is unique among virus vectors because of its target specificity and low toxicity in somatic cells, and clinical trials have shown that it has promise for gene therapy. However, there are conflicting reports on the possibility of germline integration of AAV into the genome of male germ cells, including SSCs. Here, we examined the usefulness of AAV vectors for exploring germline gene modification in SSCs. AAV1 infected cultured SSCs without apparent toxicity. Moreover, SSCs that were infected in fresh testis cells generated normal appearing spermatogenic colonies after spermatogonial transplantation. A microinsemination experiment produced offspring that underwent excision of the floxed target gene by AAV1-mediated Cre expression. Analysis of the offspring DNA showed no evidence of AAV integration, suggesting a low risk of germline integration by AAV infection. Although more extensive experiments are required to assess the risk of germline integration, our results show that AAV1 is useful for genetic manipulation of SSCs, and gene transduction by AAV will provide a useful approach to overcome potential problems associated with previous virus vector-mediated gene transduction.  

5.2210           The amino-terminus of the hepatitis C virus (HCV) p7 viroporin and its cleavage from glycoprotein E2-p7 precursor determine specific infectivity and secretion levels of HCV particle types

Denolly, S., Mialon, C., Bourlet, T., Amirache, F., penin, F., Lindenbach, B., Boson, B. and Cosset, F-L. PloS Pathogens, 13(12), e1006774 (2017)   Viroporins are small transmembrane proteins with ion channel activities modulating properties of intracellular membranes that have diverse proviral functions. Hepatitis C virus (HCV) encodes a viroporin, p7, acting during assembly, envelopment and secretion of viral particles (VP). HCV p7 is released from the viral polyprotein through cleavage at E2-p7 and p7-NS2 junctions by signal peptidase, but also exists as an E2p7 precursor, of poorly defined properties. Here, we found that ectopic p7 expression in HCVcc-infected cells reduced secretion of particle-associated E2 glycoproteins. Using biochemical assays, we show that p7 dose-dependently slows down the ER-to-Golgi traffic, leading to intracellular retention of E2, which suggested that timely E2p7 cleavage and p7 liberation are critical events to control E2 levels. By studying HCV mutants with accelerated E2p7 processing, we demonstrate that E2p7 cleavage controls E2 intracellular expression and secretion levels of nucleocapsid-free subviral particles and infectious virions. In addition, our imaging data reveal that, following p7 liberation, the amino-terminus of p7 is exposed towards the cytosol and coordinates the encounter between NS5A and NS2-based assembly sites loaded with E1E2 glycoproteins, which subsequently leads to nucleocapsid envelopment. We identify punctual mutants at p7 membrane interface that, by abrogating NS2/NS5A interaction, are defective for transmission of infectivity owing to decreased secretion of core and RNA and to increased secretion of non/partially-enveloped particles. Altogether, our results indicate that the retarded E2p7 precursor cleavage is essential to regulate the intracellular and secreted levels of E2 through p7-mediated modulation of the cell secretory pathway and to unmask critical novel assembly functions located at p7 amino-terminus.  

5.2211           Targeted delivery of AAV-transduced mesenchymal stromal cells to hepatic tissue for ex vivo gene therapy

Gabriel, N., Samule, R. and Jayandharan, G.R.

1. Tissue Eng. Regen. Med., 11(5), 1354-1364 (2017)

  Adeno-associated virus (AAV)-mediated gene therapy holds great promise if challenges related to vector neutralization by pre-existing antibodies are circumvented. The use of autologous or allogeneic cells to shield the vector might offer the possibility of successful gene transfer in such a situation. In the present study, we evaluated the feasibility of AAV-transduced mesenchymal stromal cells (MSCs) as a vehicle for hepatic gene transfer in a murine liver injury model. In our initial studies to determine the most suitable vector, we observed that AAV1 (91%) and AAV6 (72%) serotypes are highly efficient in transducing MSCs. Subsequently, we generated a transient liver injury model to analyse the efficacy of MSCs homing to the liver, as well as their hepatic gene transfer efficiency; our data show that administration of acetaminophen (500 mg/kg) served as a cue for the homing of MSCs to the liver. Furthermore, sex-mismatched transplantation of AAV1-infected MSCs demonstrated a 3.5-fold (day 7) and 2.2-fold (day 28) higher hepatic gene transfer efficiency. To further corroborate this, we estimated the donor cell Y chromosome copies in the liver of recipient female mice. Our data revealed a 12.7-fold increase in average genome copies of male MSCs in the livers of recipient mice with injury compared to control, 60 days after transplantation. However, in vivo administration of AAV-transduced MSCs in the presence of neutralization antibodies (intravenous immunoglobulin, IVIG) was not beneficial. This is possibly due to the clearance of transplanted MSCs by circulating IVIG and underscores the need to develop suitable in vivo models to study such a mode of gene transfer.  

5.2212           Structural studies of Chikungunya virus maturation

Yap, M.L., Klose, T., Urakami, A., hasan, S.S., Akahata, W. and Rossmann, M.G. PNAS, 114(52), 13703-13707 (2017)   Cleavage of the alphavirus precursor glycoprotein p62 into the E2 and E3 glycoproteins before assembly with the nucleocapsid is the key to producing fusion-competent mature spikes on alphaviruses. Here we present a cryo-EM, 6.8-Å resolution structure of an “immature” Chikungunya virus in which the cleavage site has been mutated to inhibit proteolysis. The spikes in the immature virus have a larger radius and are less compact than in the mature virus. Furthermore, domains B on the E2 glycoproteins have less freedom of movement in the immature virus, keeping the fusion loops protected under domain B. In addition, the nucleocapsid of the immature virus is more compact than in the mature virus, protecting a conserved ribosome-binding site in the capsid protein from exposure. These differences suggest that the posttranslational processing of the spikes and nucleocapsid is necessary to produce infectious virus.  

5.2213           Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells

Kontratov, O., Marsic, D., Crosson, S.M., Mendez-Gomez, H.R., Moskalenko, O., Mietzsch, M., Heilbronn, R., Allison, J.R., Green, K.B., Agbandje-McKenna, M. Molecular Therapy, 25(12), 2661-2675 (2017)   The major drawback of the Baculovirus/Sf9 system for recombinant adeno-associated viral (rAAV) manufacturing is that most of the Bac-derived rAAV vector serotypes, with few exceptions, demonstrate altered capsid compositions and lower biological potencies. Here, we describe a new insect cell-based production platform utilizing attenuated Kozak sequence and a leaky ribosome scanning to achieve a serotype-specific modulation of AAV capsid proteins stoichiometry. By way of example, rAAV5 and rAAV9 were produced and comprehensively characterized side by side with HEK293-derived vectors. A mass spectrometry analysis documented a 3-fold increase in both viral protein (VP)1 and VP2 capsid protein content compared with human cell-derived vectors. Furthermore, we conducted an extensive analysis of encapsidated single-stranded viral DNA using next-generation sequencing and show a 6-fold reduction in collaterally packaged contaminating DNA for rAAV5 produced in insect cells. Consequently, the re-designed rAAVs demonstrated significantly higher biological potencies, even in a comparison with HEK293-manufactured rAAVs mediating, in the case of rAAV5, 4-fold higher transduction of brain tissues in mice. Thus, the described system yields rAAV vectors of superior infectivity and higher genetic identity providing a scalable platform for good manufacturing practice (GMP)-grade vector production.  

5.2214           AAV-PHP.B-Mediated Global-Scale Expression in the Mouse Nervous System Enables GBA1 Gene Therapy for Wide Protection from Synucleinopathy

Morabito, G. et al Molecular Therapy, 25(12), 2727-2742 (2017)   The lack of technology for direct global-scale targeting of the adult mouse nervous system has hindered research on brain processing and dysfunctions. Currently, gene transfer is normally achieved by intraparenchymal viral injections, but these injections target a restricted brain area. Herein, we demonstrated that intravenous delivery of adeno-associated virus (AAV)-PHP.B viral particles permeated and diffused throughout the neural parenchyma, targeting both the central and the peripheral nervous system in a global pattern. We then established multiple procedures of viral transduction to control gene expression or inactivate gene function exclusively in the adult nervous system and assessed the underlying behavioral effects. Building on these results, we established an effective gene therapy strategy to counteract the widespread accumulation of α-synuclein deposits throughout the forebrain in a mouse model of synucleinopathy. Transduction of A53T-SCNA transgenic mice with AAV-PHP.B-GBA1 restored physiological levels of the enzyme, reduced α-synuclein pathology, and produced significant behavioral recovery. Finally, we provided evidence that AAV-PHP.B brain penetration does not lead to evident dysfunctions in blood-brain barrier integrity or permeability. Altogether, the AAV-PHP.B viral platform enables non-invasive, widespread, and long-lasting global neural expression of therapeutic genes, such as GBA1, providing an invaluable approach to treat neurodegenerative diseases with diffuse brain pathology such as synucleinopathies.  

5.2215           A microRNA screen reveals that elevated hepatic ectodysplasin A expression contributes to obesity-induced insulin resistance in skeletal muscle

Awazawa, M., Gabel, p., Tsaousidou, E., Nolte, H., Krüger, M., Schmitz, J., Ackermann, P.J., Brandt, C., Altmüller, J., Motameny, S., Wunderlich, F.T., Kornfeld, J-W., Blüther, M. and Brüning, J. Nature Med., 23(12), 1466-1473 (2017)   Over 40% of microRNAs (miRNAs) are located in introns of protein-coding genes, and many of these intronic miRNAs are co-regulated with their host genes1,2. In such cases of co-regulation, the products of host genes and their intronic miRNAs can cooperate to coordinately regulate biologically important pathways3,4. Therefore, we screened intronic miRNAs dysregulated in the livers of mouse models of obesity to identify previously uncharacterized protein-coding host genes that may contribute to the pathogenesis of obesity-associated insulin resistance and type 2 diabetes mellitus. Our approach revealed that expression of both the gene encoding ectodysplasin A (Eda), the causal gene in X-linked hypohidrotic ectodermal dysplasia (XLHED)5, and its intronic miRNA, miR-676, was increased in the livers of obese mice. Moreover, hepatic EDA expression is increased in obese human subjects and reduced upon weight loss, and its hepatic expression correlates with systemic insulin resistance. We also found that reducing miR-676 expression in db/db mice increases the expression of proteins involved in fatty acid oxidation and reduces the expression of inflammatory signaling components in the liver. Further, we found that Eda expression in mouse liver is controlled via PPARγ and RXR-α, increases in circulation under conditions of obesity, and promotes JNK activation and inhibitory serine phosphorylation of IRS1 in skeletal muscle. In accordance with these findings, gain- and loss-of-function approaches reveal that liver-derived EDA regulates systemic glucose metabolism, suggesting that EDA is a hepatokine that can contribute to impaired skeletal muscle insulin sensitivity in obesity.  

5.2216           Tropism of engineered and evolved recombinant AAV serotypes in the rd1 mouse and ex vivo primate retina

Hickey, D.G., Edwards, T.L., Barnard, A.R., Singh, M.S., de Silba, S.R., McClements, M.E., Flannery, J.G., Hankins, M.W. and MacLaren, R E. Gene Therapy, 24, 787-800 (2017)   There is much debate on the adeno-associated virus (AAV) serotype that best targets specific retinal cell types and the route of surgical delivery—intravitreal or subretinal. This study compared three of the most efficacious AAV vectors known to date in a mouse model of retinal degeneration (rd1 mouse) and macaque and human retinal explants. Green fluorescent protein (GFP) driven by a ubiquitous promoter was packaged into three AAV capsids: AAV2/8(Y733F), AAV2/2(quad Y-F) and AAV2/2(7m8). Overall, AAV2/2(7m8) transduced the largest area of retina and resulted in the highest level of GFP expression, followed by AAV2/2(quad Y-F) and AAV2/8(Y733F). AAV2/2(7m8) and AAV2/2(quad Y-F) both resulted in similar patterns of transduction whether they were injected intravitreally or subretinally. AAV2/8(Y733F) transduced a significantly smaller area of retina when injected intravitreally compared with subretinally. Retinal ganglion cells, horizontal cells and retinal pigment epithelium expressed relatively high levels of GFP in the mouse retina, whereas amacrine cells expressed low levels of GFP and bipolar cells were infrequently transduced. Cone cells were the most frequently transduced cell type in macaque retina explants, whereas Müller cells were the predominant transduced cell type in human retinal explants. Of the AAV serotypes tested, AAV2/2(7m8) was the most effective at transducing a range of cell types in degenerate mouse retina and macaque and human retinal explants.  

5.2217           In utero delivery of rAAV2/9 induces neuronal expression of the transgene in the brain: towards new models of Parkinson’s disease

Chansel-Debordeaux, L., Bourdenx, M., Dovero, S., Grouthier, V., Dutheil, N., Expana, A., Groc, L., Jimenz, C., Bezard, E. and Dehay, B. Gene Therapy, 24, 801-809 (2017)   Animal models are essential tools for basic pathophysiological research as well as validation of therapeutic strategies for curing human diseases. However, technical difficulties associated with classical transgenesis approaches in rodent species higher than Mus musculus have prevented this long-awaited development. The availability of viral-mediated gene delivery systems in the past few years has stimulated the production of viruses with unique characteristics. For example, the recombinant adeno-associated virus serotype 9 (rAAV2/9) crosses the blood–brain barrier, is capable of transducing developing cells and neurons after intravenous injection and mediates long-term transduction. Whilst post-natal delivery is technically straightforward, in utero delivery bears the potential of achieving gene transduction in neurons at embryonic stages during which the target area is undergoing development. To test this possibility, we injected rAAV2/9 carrying either A53T mutant human α-synuclein or green fluorescent protein, intracerebroventricularly in rats at embryonic day 16.5. We observed neuronal transgene expression in most regions of the brain at 1 and 3 months after birth. This proof-of-concept experiment introduces a new opportunity to model brain diseases in rats.   A plasmid from an Antarctic haloarchaeon uses specialized membrane vesicles to disseminate and infect plasmid-free cells Erdmann, S., Tschitschko, B., Zhong, L., Raftery, M.J. and Cavicchioli, R Nature Microbiol.,2,1446-1455 (2017)   The major difference between viruses and plasmids is the mechanism of transferring their genomic information between host cells. Here, we describe the archaeal plasmid pR1SE from an Antarctic species of haloarchaea that transfers via a mechanism similar to a virus. pR1SE encodes proteins that are found in regularly shaped membrane vesicles, and the vesicles enclose the plasmid DNA. The released vesicles are capable of infecting a plasmid-free strain, which then gains the ability to produce plasmid-containing vesicles. pR1SE can integrate and replicate as part of the host genome, resolve out with fragments of host DNA incorporated or portions of the plasmid left behind, form vesicles and transfer to new hosts. The pR1SE mechanism of transfer of DNA could represent the predecessor of a strategy used by viruses to pass on their genomic DNA and fulfil roles in gene exchange, supporting a strong evolutionary connection between plasmids and viruses.   CASAAV: A CRISPR‐Based Platform for Rapid Dissection of Gene Function In Vivo VanDusen, N.J., Guo, Y., Gu, W. and Pu, W.T. Current Protocols in Mol. Biol., 120(1), 31.11.1-31.11.14 (2017)   In vivo loss‐of‐function studies are currently limited by the need for appropriate conditional knockout alleles. CRISPR/Cas9 is a powerful tool commonly used to induce loss‐of‐function mutations in vitro. However, CRISPR components have been difficult to deploy in vivo. To address this problem, we developed the CASAAV (CRISPR/Cas9/AAV‐based somatic mutagenesis) platform, in which recombinant adeno‐associated virus (AAV) is used to deliver tandem guide RNAs and Cre recombinase to Cre‐dependent Cas9‐P2A‐GFP mice. Because Cre is under the control of a tissue‐specific promoter, this system allows temporally controlled, cell type‐selective knockout of virtually any gene to be obtained within a month using only one mouse line. Here, we focus on gene disruption in cardiomyocytes, but the system could easily be adapted to inactivate genes in other cell types transduced by AAV.    

5.2218           N-Myc Downstream-Regulated Gene 1 Restricts Hepatitis C Virus Propagation by Regulating Lipid Droplet Biogenesis and Viral Assembly

Schweitzer, C.J., Zhang, F., Boyer, A., Valdez, K., Cam, M. and Liang, J.

1. Virol., 92(2), e01166-17 (2018)

  Host cells harbor various intrinsic mechanisms to restrict viral infections as a first line of antiviral defense. Viruses have evolved various countermeasures against these antiviral mechanisms. Here we show that N-Myc downstream-regulated gene 1 (NDRG1) limits productive hepatitis C virus (HCV) infection by inhibiting viral assembly. Interestingly, HCV infection downregulates NDRG1 protein and mRNA expression. The loss of NDRG1 increases the size and number of lipid droplets, which are the sites of HCV assembly. HCV suppresses NDRG1 expression by upregulating MYC, which directly inhibits the transcription of NDRG1. The upregulation of MYC also leads to the reduced expression of the NDRG1-specific kinase serum/glucocorticoid-regulated kinase 1 (SGK1), resulting in a markedly diminished phosphorylation of NDRG1. The knockdown of MYC during HCV infection rescues NDRG1 expression and phosphorylation, suggesting that MYC regulates NDRG1 at both the transcriptional and posttranslational levels. Overall, our results suggest that NDRG1 restricts HCV assembly by limiting lipid droplet formation. HCV counteracts this intrinsic antiviral mechanism by downregulating NDRG1 via a MYC-dependent mechanism.  

5.2219           Genotype 2 Strains of Porcine Reproductive and Respiratory Syndrome Virus Dysregulate Alveolar Macrophage Cytokine Production via the Unfolded Protein Response

Chen, W-Y., Schnitzlein, W.M., Calzada-Nova, G. and Zuckermann, F.A.

1. Virol., 92(2), e01251-17 (2018)

  Porcine reproductive and respiratory syndrome virus (PRRSV) infects alveolar macrophages (AMϕ), causing dysregulated alpha interferon (IFN-α) and tumor necrosis factor alpha (TNF-α) production through a mechanism(s) yet to be resolved. Here, we show that AMϕ infected with PRRSV secreted a reduced quantity of IFN-α following exposure of the cell to synthetic double-stranded RNA (dsRNA). This reduction did not correlate with reduced IFNA1 gene transcription. Rather, it coincided with two events that occurred late during infection and that were indicative of translational attenuation, specifically, the activation of eukaryotic translation initiation factor 2α (eIF2α) and the appearance of stress granules. Notably, the typical rapid production of TNF-α by AMϕ exposed to lipopolysaccharide (LPS) was suppressed or enhanced by PRRSV, depending on when the LPS exposure occurred after virus infection. If exposure was delayed until 6 h postinfection (hpi) so that the development of the cytokine response coincided with the time in which phosphorylation of eIF2α by the stress sensor PERK (protein kinase RNA [PKR]-like ER kinase) occurred, inhibition of TNF-α production was observed. However, if LPS exposure occurred at 2 hpi, prior to a detectable onset of eIF2α phosphorylation, a synergistic response was observed due to the earlier NF-κB activation via the stress sensor IRE1α (inositol-requiring kinase 1α). These results suggest that the asynchronous actions of two branches of the unfolded protein response (UPR), namely, IRE1α, and PERK, activated by ER stress resulting from the virus infection, are associated with enhancement or suppression of TNF-α production, respectively.  

5.2220           Characterization of Recombinant Flaviviridae Viruses Possessing a Small Reporter Tag

Tamura, T. et al

1. Virol., 92(2), e01582-17 (2018)

  The family Flaviviridae consists of four genera, Flavivirus, Pestivirus, Pegivirus, and Hepacivirus, and comprises important pathogens of human and animals. Although the construction of recombinant viruses carrying reporter genes encoding fluorescent and bioluminescent proteins has been reported, the stable insertion of foreign genes into viral genomes retaining infectivity remains difficult. Here, we applied the 11-amino-acid subunit derived from NanoLuc luciferase to the engineering of the Flaviviridae viruses and then examined the biological characteristics of the viruses. We successfully generated recombinant viruses carrying the split-luciferase gene, including dengue virus, Japanese encephalitis virus, hepatitis C virus (HCV), and bovine viral diarrhea virus. The stability of the viruses was confirmed by five rounds of serial passages in the respective susceptible cell lines. The propagation of the recombinant luciferase viruses in each cell line was comparable to that of the parental viruses. By using a purified counterpart luciferase protein, this split-luciferase assay can be applicable in various cell lines, even when it is difficult to transduce the counterpart gene. The efficacy of antiviral reagents against the recombinant viruses could be monitored by the reduction of luciferase expression, which was correlated with that of viral RNA, and the recombinant HCV was also useful to examine viral dynamics in vivo. Taken together, our findings indicate that the recombinant Flaviviridae viruses possessing the split NanoLuc luciferase gene generated here provide powerful tools to understand viral life cycle and pathogenesis and a robust platform to develop novel antivirals against Flaviviridae viruses.  

5.2221           Authentic Patient-Derived Hepatitis C Virus Infects and Productively Replicates in Primary CD4+ and CD8+ T Lymphocytes In Vitro

Skardasi, G., Chen, A.Y. and Michalak, T.I.

1. Virol., 92(3), e01790-17 (2018)

  Accumulated evidence indicates that immune cells can support the replication of hepatitis C virus (HCV) in infected patients and in culture. However, there is a scarcity of data on the degree to which individual immune cell types support HCV propagation and on characteristics of virus assembly. We investigated the ability of authentic, patient-derived HCV to infect in vitro two closely related but functionally distinct immune cell types, CD4⁺ and CD8⁺ T lymphocytes, and assessed the properties of the virus produced by these cells. The HCV replication system in intermittently mitogen-stimulated T cells was adapted to infect primary human CD4⁺ or CD8⁺ T lymphocytes. HCV replicated in both cell types although at significantly higher levels in CD4⁺ than in CD8⁺ T cells. Thus, the HCV RNA replicative (negative) strand was detected in CD4⁺ and CD8⁺ cells at estimated mean levels ± standard errors of the means of 6.7 × 10² ± 3.8 × 10² and 1.2 × 10² ± 0.8 × 10² copies/μg RNA, respectively (P < 0.0001). Intracellular HCV NS5a and/or core proteins were identified in 0.9% of CD4⁺ and in 1.2% of CD8⁺ T cells. Double staining for NS5a and T cell type-specific markers confirmed that transcriptionally competent virus replicated in both cell types. Furthermore, an HCV-specific protease inhibitor, telaprevir, inhibited infection in both CD4⁺ and CD8⁺ cells. The emergence of unique HCV variants and the release of HCV RNA-reactive particles with biophysical properties different from those of virions in plasma inocula suggested that distinct viral particles were assembled, and therefore, they may contribute to the pool of circulating virus in infected patients.  

5.2222           Entry of Human Coronavirus NL63 into the Cell

Milewska, A., Nowak, P., Owczarek, K., Szczepanski, A., Zaregski, M., Joang, A., Berniak, K., Wojarski, J., Zeglen, S., Baster, Z., Rajfur, Z. and Purc, K.

1. Virol., 92(3), e01933-17 (2018)

  The first steps of human coronavirus NL63 (HCoV-NL63) infection were previously described. The virus binds to target cells by use of heparan sulfate proteoglycans and interacts with the ACE2 protein. Subsequent events, including virus internalization and trafficking, remain to be elucidated. In this study, we mapped the process of HCoV-NL63 entry into the LLC-Mk2 cell line and ex vivo three-dimensional (3D) tracheobronchial tissue. Using a variety of techniques, we have shown that HCoV-NL63 virions require endocytosis for successful entry into the LLC-MK2 cells, and interaction between the virus and the ACE2 molecule triggers recruitment of clathrin. Subsequent vesicle scission by dynamin results in virus internalization, and the newly formed vesicle passes the actin cortex, which requires active cytoskeleton rearrangement. Finally, acidification of the endosomal microenvironment is required for successful fusion and release of the viral genome into the cytoplasm. For 3D tracheobronchial tissue cultures, we also observed that the virus enters the cell by clathrin-mediated endocytosis, but we obtained results suggesting that this pathway may be bypassed.  

5.2223           Overexpression of parkin protects retinal ganglion cells in experimental glaucoma

Dai, Y., Hu, X. and Sun, X. Cell Death & Disease, 9:88 (2018)   Glaucoma is a leading cause of irreversible blindness and characterized by progressive damage of retinal ganglion cells (RGCs). Growing evidences have linked impaired mitophagy with neurodegenerative diseases, while the E3 ubiquitin ligase parkin may play a key role. However, the pathophysiological relationship between parkin and glaucoma remains largely unknown. Using chronic hypertensive glaucoma rats induced by translimbal laser photocoagulation, we show here that the protein level of parkin and its downstream optineurin proteins were increased in hypertensive retinas. The ratio of LC3-II to LC3-I, the number of mitophagosomes, and unhealthy mitochondria were increased in hypertensive optic nerves. Overexpression of parkin by viral vectors increased RGC survival in glaucomatous rats in vivo and under excitotoxicity in vitro. It also promoted optineurin expression and improved mitochondrial health. In parkin-overexpressed glaucomatous rats, the ratio of LC3-II to LC3-I, LAMP1 level, and the number of mitophagosomes in optic nerve were decreased at 3 days, yet increased at 2 weeks following intraocular pressure (IOP) elevation. These findings demonstrate that dysfunction of mitophagy exist in RGCs of glaucomatous rats. Overexpression of parkin exerted a significant protective effect on RGCs and partially restored dysfunction of mitophagy in response to cumulative IOP elevation.  

5.2224           RFX1 and RFX3 Transcription Factors Interact with the D Sequence of Adeno-Associated Virus Inverted Terminal Repeat and Regulate AAV Transduction

Julien, L., Chassagne, J., Peccate, C., Lorain, S., Pietri-Rouxel, F., Danos, O. and Benkhelifa-Ziiyyat, S. Scientific Reports, 8:210 (2018)   Adeno-associated virus (AAV) transduction efficiency depends on the way in which cellular proteins process viral genomes in the nucleus. In this study, we have investigated the binding of nuclear proteins to the double stranded D (dsD) sequence of the AAV inverted terminal repeat (ITRs) by electromobility shift assay. We present here several lines of evidence that transcription factors belonging to the RFX protein family bind specifically and selectively to AAV2 and AAV1 dsD sequences. Using supershift experiments, we characterize complexes containing RFX1 homodimers and RFX1/RFX3 heterodimers. Following transduction of HEK-293 cells, the AAV genome can be pulled-down by RFX1 and RFX3 antibodies. Moreover, our data suggest that RFX proteins which interact with transcriptional enhancers of several mammalian DNA viruses, can act as regulators of AAV mediated transgene expression.  

5.2225           Neuroprotective Drug for Nerve Trauma Revealed Using Artificial Intelligence

Romeo-Guitart, D., Fores, J., Herrando-grabulosa, M., Valls, R., Leiva-Rodriguez, T., Galea, E., Gonzalez-Perez, F., Navarro, X., Petegnief, V., Bosch, A., Coma, M., Mas, J.M. and Casas, C. Scientific Reports, 8:1879 (2018)   Here we used a systems biology approach and artificial intelligence to identify a neuroprotective agent for the treatment of peripheral nerve root avulsion. Based on accumulated knowledge of the neurodegenerative and neuroprotective processes that occur in motoneurons after root avulsion, we built up protein networks and converted them into mathematical models. Unbiased proteomic data from our preclinical models were used for machine learning algorithms and for restrictions to be imposed on mathematical solutions. Solutions allowed us to identify combinations of repurposed drugs as potential neuroprotective agents and we validated them in our preclinical models. The best one, NeuroHeal, neuroprotected motoneurons, exerted anti-inflammatory properties and promoted functional locomotor recovery. NeuroHeal endorsed the activation of Sirtuin 1, which was essential for its neuroprotective effect. These results support the value of network-centric approaches for drug discovery and demonstrate the efficacy of NeuroHeal as adjuvant treatment with surgical repair for nervous system trauma.      

5.2226           Pharmacogenetic stimulation of neuronal activity increases myelination in an axon-specific manner

Mitew, S., Gobius, I., Fenion, L.R., McDougall, S.J., Hawkes, D., Xing, Y.L., Bujalka, H., Gundlach, A.L., Richards, L.J., Kilpatrick, T.J.., merson, T.D. and Emery, B. Nature Communications, 9:306 (2018)   Mounting evidence suggests that neuronal activity influences myelination, potentially allowing for experience-driven modulation of neural circuitry. The degree to which neuronal activity is capable of regulating myelination at the individual axon level is unclear. Here we demonstrate that stimulation of somatosensory axons in the mouse brain increases proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) within the underlying white matter. Stimulated axons display an increased probability of being myelinated compared to neighboring non-stimulated axons, in addition to being ensheathed with thicker myelin. Conversely, attenuating neuronal firing reduces axonal myelination in a selective activity-dependent manner. Our findings reveal that the process of selecting axons for myelination is strongly influenced by the relative activity of individual axons within a population. These observed cellular changes are consistent with the emerging concept that adaptive myelination is a key mechanism for the fine-tuning of neuronal circuitry in the mammalian CNS.  

5.2227           Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses

Yoon, Y., Wang, D., Tai, P.W.L., Riley, J., Gao, G.. and Rivera-Perez, J.A. Nature Communications, 9:412 (2018)   Recent advances using CRISPR-Cas9 approaches have dramatically enhanced the ease for genetic manipulation in rodents. Notwithstanding, the methods to deliver nucleic acids into pre-implantation embryos have hardly changed since the original description of mouse transgenesis more than 30 years ago. Here we report a novel strategy to generate genetically modified mice by transduction of CRISPR-Cas9 components into pre-implantation mouse embryos via recombinant adeno-associated viruses (rAAVs). Using this approach, we efficiently generated a variety of targeted mutations in explanted embryos, including indel events produced by non-homologous end joining and tailored mutations using homology-directed repair. We also achieved gene modification in vivo by direct delivery of rAAV particles into the oviduct of pregnant females. Our approach greatly simplifies the generation of genetically modified mice and, more importantly, opens the door for streamlined gene editing in other mammalian species.  

5.2228           Chemogenetic modulation of cholinergic interneurons reveals their regulating role on the direct and indirect output pathways from the striatum

Aldrin-Kirk, P., Heuer, A., Ottosson, D., Davidsson, M., Mattsson, B. and Björklund, T. Neurobiology of Disease, 109, 148-162 (2018)   The intricate balance between dopaminergic and cholinergic neurotransmission in the striatum has been thoroughly difficult to characterize. It was initially described as a seesaw with a competing function of dopamine versus acetylcholine. Recent technical advances however, have brought this view into question suggesting that the two systems work rather in concert with the cholinergic interneurons (ChIs) driving dopamine release. In this study, we have utilized two transgenic Cre-driver rat lines, a choline acetyl transferase ChAT-Cre transgenic rat and a novel double-transgenic tyrosine hydroxylase TH-Cre/ChAT-Cre rat to further elucidate the role of striatal ChIs in normal motor function and in Parkinson's disease. Here we show that selective and reversible activation of ChIs using chemogenetic (DREADD) receptors increases locomotor function in intact rats and potentiate the therapeutic effect of L-DOPA in the rats with lesions of the nigral dopamine system. However, the potentiation of the L-DOPA effect is accompanied by an aggravation of L-DOPA induced dyskinesias (LIDs). These LIDs appear to be driven primarily through the indirect striato-pallidal pathway since the same effect can be induced by the D2 agonist Quinpirole. Taken together, the results highlight the intricate regulation of balance between the two output pathways from the striatum orchestrated by the ChIs.  

5.2229           Fusion of Anthopleurin-B to AAV2 increases specificity of cardiac gene transfer

Finet, J.E., Wan, X. and Donahue, J.K. Virology, 513, 43-51 (2018)   AAV-mediated gene therapy has become a promising therapeutic strategy for chronic diseases. Its clinical utilization, however, is limited by the potential risk of off-target effects. In this work we attempt to overcome this challenge, hypothesizing that cardiac ion channel-specific ligands could be fused onto the AAV capsid, and narrow its tropism to cardiac myocytes. We successfully fused the cardiac sodium channel (Na_v1.5)-binding toxin Anthopleurin-B onto the AAV2 capsid without compromising virus integrity, and demonstrated increased specificity of cardiomyocyte attachment. Although virus attachment to Na_v1.5 did not supersede the natural heparan-mediated virus binding, heparan-binding ablated vectors carrying Anthopleurin-B eliminated hepatic and other extracardiac gene transfer, while preserving cardiac myocyte gene transfer. Virus binding to the cardiac sodium channel transiently decreased sodium current density, but did not cause any arrhythmias. Our findings expand the knowledge of attachment, infectivity, and intracellular processing of AAV vectors, and present an alternative strategy for vector retargeting.  

5.2230           Otx2-Genetically Modified Retinal Pigment Epithelial Cells Rescue Photoreceptors after Transplantation

Kole, C. et al Molecular Therapy, 26(1), 219-237 (2018)   Inherited retinal degenerations are blinding diseases characterized by the loss of photoreceptors. Their extreme genetic heterogeneity complicates treatment by gene therapy. This has motivated broader strategies for transplantation of healthy retinal pigmented epithelium to protect photoreceptors independently of the gene causing the disease. The limited clinical benefit for visual function reported up to now is mainly due to dedifferentiation of the transplanted cells that undergo an epithelial-mesenchymal transition. We have studied this mechanism in vitro and revealed the role of the homeogene OTX2 in preventing dedifferentiation through the regulation of target genes. We have overexpressed OTX2 in retinal pigmented epithelial cells before their transplantation in the eye of a model of retinitis pigmentosa carrying a mutation in Mertk, a gene specifically expressed by retinal pigmented epithelial cells. OTX2 increases significantly the protection of photoreceptors as seen by histological and functional analyses. We observed that the beneficial effect of OTX2 is non-cell autonomous, and it is at least partly mediated by unidentified trophic factors. Transplantation of OTX2-genetically modified cells may be medically effective for other retinal diseases involving the retinal pigmented epithelium as age-related macular degeneration.  

5.2231           Gene Therapy-Induced Antigen-Specific Tregs Inhibit Neuro-inflammation and Reverse Disease in a Mouse Model of Multiple Sclerosis

Keeler, G.D., Kumar, S., Palaschak, B., Silverberg, E.L., Markusic, D.M., Jones, N.T. and Hoffman, B.E. Molecular Therapy, 26(1), 173-183 (2018)   The devastating neurodegenerative disease multiple sclerosis (MS) could substantially benefit from an adeno-associated virus (AAV) immunotherapy designed to restore a robust and durable antigen-specific tolerance. However, developing a sufficiently potent and lasting immune-regulatory therapy that can intervene in ongoing disease is a major challenge and has thus been elusive. We addressed this problem by developing a highly effective and robust tolerance-inducing in vivo gene therapy. Using a pre-clinical animal model, we designed a liver-targeting gene transfer vector that expresses full-length myelin oligodendrocyte glycoprotein (MOG) in hepatocytes. We show that by harnessing the tolerogenic nature of the liver, this powerful gene immunotherapy restores immune tolerance by inducing functional MOG-specific regulatory T cells (Tregs) in vivo, independent of major histocompatibility complex (MHC) restrictions. We demonstrate that mice treated prophylactically are protected from developing disease and neurological deficits. More importantly, we demonstrate that when given to mice with preexisting disease, ranging from mild neurological deficits to severe paralysis, the gene immunotherapy abrogated CNS inflammation and significantly reversed clinical symptoms of disease. This specialized approach for inducing antigen-specific immune tolerance has significant therapeutic potential for treating MS and other autoimmune disorders.  

5.2232           Novel expression of immunogenic foot-and-mouth disease virus-like particles in Nicotiana benthamiana

Veeparan, V.P., van Zyl, A.R., Wigdorovitz, A., Rybicki, E.P. and Meyers, A.E. Virus Res., 244, 213-217 (2018)   Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals and is endemic in Africa, parts of South America and southern Asia. The causative agent, FMD virus (FMDV) is a member of the genus Aphthovirus, family Picornaviridae. Vaccines currently used against FMDV are chemically inactivated virus strains which are produced under high-level biocontainment facilities, thus raising their cost. The development of recombinant FMDV vaccines has focused predominantly on FMDV virus-like particle (VLP) subunit vaccines for which promising results have been achieved. These VLPs are attractive candidates because they avoid the use of live virus in production facilities, but conserve the complete repertoire of conformational epitopes of the virus. Recombinant FMDV VLPs are formed by the expression and assembly of the three structural proteins VP0, VP1 and VP3. This can be attained by co-expression of the three individual structural capsid proteins or by co-expression of the viral capsid precursor P1-2A together with the viral protease 3C. The latter proteolytically cleaves P1-2A into the respective structural proteins. These VLPS are produced in mammalian or insect cell culture systems, which are expensive and can be easily contaminated. Plants, such as Nicotiana benthamiana, potentially provide a more cost-effective and very highly scalable platform for recombinant protein and VLP production. In this study, P1-2A was transiently expressed in N. benthamiana alone, without the 3C protease. Surprisingly, there was efficient processing of the P1-2A polyprotein into its component structural proteins, and subsequent assembly into VLPs. The yield was ∼0.030 μg per gram of fresh leaf material. Partially purified VLPs were preliminarily tested for immunogenicity in mice and shown to stimulate the production of FMDV-specific antibodies. This study, has important implications for simplifying the production and expression of potential vaccine candidates against FMDV in plants, in the absence of 3C expression.  

5.2233           Confirmation of specificity of reactivity in a solid phase ELISA for the detection of hepatitis E viral antigen improves utility of the assay

Ankcorn, M.J., Ijaz, S., Haywood, B., Neuberger, J., Elsharkawy, A.M., Maggs, J. and Tedder, R.S

1. Virol. Methods, 252, 42-48 (2018)

  Genotype 3 hepatitis E virus (HEV) can lead to persistent infections in immunocompromised hosts. A recently available commercial assay for the detection of HEV antigen (HEV-Ag ELISA, Wantai diagnostics) may enable the study of HEV-Ag dynamics in such persistent infections, however currently there is no confirmatory test available. We generated a putative neutralising reagent from a pool of four convalescent blood donor samples and explored neutralising activity against HEV antigens from clinical samples, HEV tissue-culture and virus-like particles. Using this neutralisation method we were able to differentiate true reactivity from non-specific reactivity in plasma, stool and urine samples. This could also facilitate the introduction of HEV-Ag detection as a screening assay or the study of HEV-Ag in different body fluids.  

5.2234           Intravenous administration of the adeno-associated virus-PHP.B capsid fails to upregulate transduction efficiency in the marmoset brain

Matsuzaki, Y., Konno, A., Mochizuki, R., Shinohara, Y., Nitta, K., Okada, Y. and Hirai, H. Neuroscience Letters, 665, 182-188 (2018)   Intravenous administration of adeno-associated virus (AAV)-PHP.B, a capsid variant of AAV9 containing seven amino acid insertions, results in a greater permeability of the blood brain barrier (BBB) than standard AAV9 in mice, leading to highly efficient and global transduction of the central nervous system (CNS). The present study aimed to examine whether the enhanced BBB penetrance of AAV-PHP.B observed in mice also occurs in non-human primates. Thus, a young adult (age, 1.6 years) and an old adult (age, 7.2 years) marmoset received an intravenous injection of AAV-PHP.B expressing enhanced green fluorescent protein (EGFP) under the control of the constitutive CBh promoter (a hybrid of cytomegalovirus early enhancer and chicken β-actin promoter). Age-matched control marmosets were treated with standard AAV9-capsid vectors. The animals were sacrificed 6 weeks after the viral injection. Based on the results, only limited transduction of neurons (0–2%) and astrocytes (0.1–2.5%) was observed in both AAV-PHP.B- and AAV9-treated marmosets. One noticeable difference between AAV-PHP.B and AAV9 was the marked transduction of the peripheral dorsal root ganglia neurons. Indeed, the soma and axons in the projection from the spinal cord to the nucleus cuneatus in the medulla oblongata were strongly labeled with EGFP by AAV-PHP.B. Thus, except for the peripheral dorsal root ganglia neurons, the AAV-PHP.B transduction efficiency in the CNS of marmosets was comparable to that of AAV9 vectors.  

5.2235           Guanylin and uroguanylin mRNA expression is increased following Roux-en-Y gastric bypass, but guanylins do not play a significant role in body weight regulation and glycemic control

Fernandez-Cohen, M.L. et al Peptides, 101, 32-43 (2018)   Aim To determine whether intestinal expression of guanylate cyclase activator 2A (GUCA2A) and guanylate cyclase activator 2B (GUCA2B) genes is regulated in obese humans following Roux-en-Y gastric bypass (RYGB), and to evaluate the corresponding guanylin (GN) and uroguanylin (UGN) peptides for potentially contributing to the beneficial metabolic effects of RYGB. Methods Enteroendocrine cells were harvested peri- and post-RYGB, and GUCA2A/GUCA2B mRNA expression was compared. GN, UGN and their prohormones (proGN, proUGN) were administered subcutaneously in normal-weight mice to evaluate effects on food intake and glucose regulation. The effect of pro-UGN or UGN overexpression, using adeno-associated virus (AAV) vectors, was assessed in diet-induced obese (DIO) mice. Intracerebroventricular administration of GN and UGN was performed in rats for assessment of putative centrally mediated effects on food intake. GN and UGN, as well as their prohormones, were evaluated for effects on glucose-stimulated insulin secretion (GSIS) in rat pancreatic islets and perfused rat pancreas. Results GUCA2A and GUCA2B mRNA expression was significantly upregulated in enteroendocrine cells after RYGB. Peripheral administration of guanylins or prohormones did not influence food intake, oral glucose tolerance, and GSIS. Central administration of GN and UGN did not affect food intake in rats. Chronic AVV-mediated overexpression of UGN and proUGN had no effect on body weight or glucose homeostasis in DIO mice. Conclusion GN and UGN, as well as their prohormones, do not seem to play a significant role in body weight regulation and glycemic control, suggesting that guanylin-family peptides do not show promise as targets for the treatment of obesity or diabetes.  

5.2236           The traditional use of Vachellia nilotica for sexually transmitted diseases is substantiated by the antiviral activity of its bark extract against sexually transmitted viruses

Donaliso, M., Cagno, V., Civra, A., Gibellini, D., Musumeci, G., Ritta, M., Ghosh, M. and Lembo, D.

1. Ehnopharmacol., 213, 403-408 (2018)

  Ethnopharmacological relevance Vachellia (Acacia) nilotica and other plants of this genus have been used in traditional medicine of Asian and African countries to treat many disorders, including sexually transmitted diseases, but few studies were performed to validate their anti-microbial and anti-viral activity against sexually transmitted infections. Aim of the study The present study was undertaken to explore whether the ethnomedical use of V.nilotica to treat genital lesions is substantiated by its antiviral activity against the human immunodeficiency virus (HIV), the herpes simplex virus (HSV) and the human papillomavirus (HPV). Materials and methods The antiviral activity of V.nilotica was tested in vitro by virus-specific inhibition assays using HSV-2 strains, sensible or resistant to acyclovir, HIV-1IIIb strain and HPV-16 pseudovirion (PsV). The potential mode of action of extract against HSV-2 and HPV-16 was further investigated by virus inactivation and time-of-addition assays on cell cultures. Results V.nilotica chloroform, methanolic and water bark extracts exerted antiviral activity against HSV-2 and HPV-16 PsV infections; among these, methanolic extract showed the best EC50s with values of 4.71 and 1.80 µg/ml against HSV-2 and HPV-16, respectively, and it was also active against an acyclovir-resistant HSV-2 strain with an EC50 of 6.71 µg/ml. By contrast, no suppression of HIV infection was observed. Investigation of the mechanism of action revealed that the methanolic extract directly inactivated the infectivity of the HPV-16 particles, whereas a partial virus inactivation and interference with virus attachment (EC50 of 2.74 µg/ml) were both found to contribute to the anti-HSV-2 activity. Conclusions These results support the traditional use of V.nilotica applied externally for the treatment of genital lesions. Further work remains to be done in order to identify the bioactive components.  

5.2237           Neurogranin in the nucleus accumbens regulates NMDA receptor tolerance and motivation for ethanol seeking

Reker, A.N., Oliveros, A., Sullivan III, J.M., Nahar, L., Hinton, D.J., Kim, T., Bruner, C. R., Choi, D-S., Goeders, N.E. and Nam, H.W. Neuropharmacol., 131, 58-67 (2018)   Dysfunction of N-methyl-d-aspartate receptor (NMDAR) signaling in the nucleus accumbens (NAc) has been implicated in the pathophysiology of alcohol use disorders (AUD). Neurogranin (Ng), a calmodulin-binding protein, is exclusively expressed in the post-synapse, and mediates NMDAR driven synaptic plasticity by regulating the calcium-calmodulin (Ca²⁺-CaM) pathway. To study the functional role of Ng in AUD, we administrated behavior tests including Pavlovian instrument transfer (PIT), operant conditioning, and rotarod test using Ng null mice (Ng^–/– mice). We used adeno-associated virus (AAV)-mediated Ng expression and pharmacological manipulation to validate behavioral responses in Ng^–/– mice. The results from our multidisciplinary approaches demonstrated that deficit of Ng increases tolerance to NMDAR inhibition and elicit faster cue reactivity during PIT without changes in ethanol reward. Operant conditioning results demonstrated that Ng^–/– mice self-administered significantly more ethanol and displayed reduced sensitivity to aversive motivation. We identified that ethanol exposure decreases mGluR5 (metabotropic glutamate receptor 5) expression in the NAc of Ng^–/– mice and pharmacological inhibition of mGluR5 reverses NMDAR desensitization in Ng^–/– mice. Together these findings specifically suggest that accumbal Ng plays an essential role in the counterbalance between NMDAR and mGluR5 signaling; which alters NMDAR resistance, and thereby altering aversive motivation for ethanol and may ultimately contribute to susceptibility for alcohol addiction.  

5.2238           Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in Normal and Pancreatic Cancer Mice

Quirin, K.A., Kwon, J.J., Alioufi, A., Factora, T., Temm, C.J., Jacobsen, M., Sandusky, G.E., Shontz, K., Chicoine, L.G., Clark, K.R., Mendell, J.T., Korc, M. and Kota, J. Molecular Therapy – Methods in Clin. Development., 8, 8-20 (2018)   Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 10¹² viral genomes (vg). Intraductal delivery of 1 × 10¹¹ vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 10¹¹ vg. In a Kras^G12D-driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.  

5.2239           Drd3 Signaling in the Lateral Septum Mediates Early Life Stress-Induced Social Dysfunction

Shin, S., Pribiag, H., Lilascharoen, V., Knowland, D., Wang, X-Y. and Lim, B.K. Neuron, 97, 195-208 (2018)   Early life stress (ELS) in the form of child abuse/neglect is associated with an increased risk of developing social dysfunction in adulthood. Little is known, however, about the neural substrates or the neuromodulatory signaling that govern ELS-induced social dysfunction. Here, we show that ELS-induced downregulation of dopamine receptor 3 (Drd3) signaling and its corresponding effects on neural activity in the lateral septum (LS) are both necessary and sufficient to cause social abnormalities in adulthood. Using in vivo Ca²⁺ imaging, we found that Drd3-expressing-LS (Drd3^LS) neurons in animals exposed to ELS show blunted activity in response to social stimuli. In addition, optogenetic activation of Drd3^LS neurons rescues ELS-induced social impairments. Furthermore, pharmacological treatment with a Drd3 agonist, which increases Drd3^LS neuronal activity, normalizes the social dysfunctions of ELS mice. Thus, we identify Drd3 in the LS as a critical mediator and potential therapeutic target for the social abnormalities caused by ELS.  

5.2240           Photoreceptor glucose metabolism determines normal retinal vascular growth

Fu, Z. et al EMBO Mol. Med., 10(1), 76-90 (2018)   The neural cells and factors determining normal vascular growth are not well defined even though vision-threatening neovessel growth, a major cause of blindness in retinopathy of prematurity (ROP) (and diabetic retinopathy), is driven by delayed normal vascular growth. We here examined whether hyperglycemia and low adiponectin (APN) levels delayed normal retinal vascularization, driven primarily by dysregulated photoreceptor metabolism. In premature infants, low APN levels correlated with hyperglycemia and delayed retinal vascular formation. Experimentally in a neonatal mouse model of postnatal hyperglycemia modeling early ROP, hyperglycemia caused photoreceptor dysfunction and delayed neurovascular maturation associated with changes in the APN pathway; recombinant mouse APN or APN receptor agonist AdipoRon treatment normalized vascular growth. APN deficiency decreased retinal mitochondrial metabolic enzyme levels particularly in photoreceptors, suppressed retinal vascular development, and decreased photoreceptor platelet-derived growth factor (Pdgfb). APN pathway activation reversed these effects. Blockade of mitochondrial respiration abolished AdipoRon-induced Pdgfb increase in photoreceptors. Photoreceptor knockdown of Pdgfb delayed retinal vascular formation. Stimulation of the APN pathway might prevent hyperglycemia-associated retinal abnormalities and suppress phase I ROP in premature infants.  

5.2241           Immunogenicity of plant-produced African horse sickness virus-like particles: implications for a novel vaccine

Dennis, S.J., Meyers, A.E., Guthrie, A.J., Hitzeroth, I.I: and Rybicki, E.P. Plant Biotechnol.J., 16, 442-450 (2018)   African horse sickness (AHS) is a debilitating and often fatal viral disease affecting horses in much of Africa, caused by the dsRNA orbivirus African horse sickness virus (AHSV). Vaccination remains the single most effective weapon in combatting AHS, as there is no treatment for the disease apart from good animal husbandry. However, the only commercially available vaccine is a live-attenuated version of the virus (LAV). The threat of outbreaks of the disease outside its endemic region and the fact that the LAV is not licensed for use elsewhere in the world, have spurred attempts to develop an alternative safer, yet cost-effective recombinant vaccine. Here, we report the plant-based production of a virus-like particle (VLP) AHSV serotype five candidate vaccine by Agrobacterium tumefaciens-mediated transient expression of all four capsid proteins in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant expression vector system. The production process is fast and simple, scalable, economically viable, and most importantly, guinea pig antiserum raised against the vaccine was shown to neutralize live virus in cell-based assays. To our knowledge, this is the first report of AHSV VLPs produced in plants, which has important implications for the containment of, and fight against the spread of, this deadly disease.  

5.2242           Prophylactic immunization with human papillomavirus vaccines induces oral immunity in mice

Ahn, J., Peng, S., Hung, C-F., Roden, B.S.and Best, S.R. Laryngoscope, 128, E16-E20 (2018)   Objective Although it has been shown that prophylactic vaccination can induce genital immunity, there is inadequate information on human papillomavirus (HPV) vaccine-induced oral immunity, which is of particular interest due to HPV-associated oropharyngeal malignancies and recurrent respiratory papillomatosis. Therefore, we assessed the efficacy of various HPV vaccines against oral HPV pseudovirus (PsV) infection in mice. Study Design Preclinical scientific investigation. Methods C57BL/6 mice were vaccinated three times at 2-week intervals with either Gardasil (Merck, Kenilworth, NJ) (50 µL intramuscular injection) or a candidate pan-HPV L2 vaccine with alum adjuvant (25 µg subcutaneous injection). Additional mice were immunized with passive transfer of either Gardasil (Merck) human antisera or nonimmunized sera (100 µL intraperitoneal injection). All vaccinated and naïve control mice were then challenged with HPV16 E6E7 luciferase PsV in the oral mucosa. Visualization of HPV PsV infection was monitored through in vivo luciferase imaging. Results Oral luciferase-expressing HPV16 PsV infection was not detected in Gardasil (Merck), L2 vaccine, and Gardasil (Merck) antisera-immunized mice, whereas robust luciferase expression was observed in all control mice. An in vitro neutralization assay from sera of Gardasil-vaccinated (Merck) mice confirmed that vaccine efficacy was due to neutralizing antibodies. Conclusion Oral HPV16 PsV infection in mice was completely prevented with all methods of prophylactic HPV immunization. These findings provide preliminary evidence that human vaccines induce protection against oral HPV infection, which has significant public health implications for HPV-associated oropharyngeal malignancies.  

5.2243           Advancements in the design and scalable production of viral gene transfer vectors

Sharon, D. and Kamen, A. Biotechnol. Bioengineering, 115(1), 25-40 (2018)   The last 10 years have seen a rapid expansion in the use of viral gene transfer vectors, with approved therapies and late stage clinical trials underway for the treatment of genetic disorders, and multiple forms of cancer, as well as prevention of infectious diseases through vaccination. With this increased interest and widespread adoption of viral vectors by clinicians and biopharmaceutical industries, there is an imperative to engineer safer and more efficacious vectors, and develop robust, scalable and cost-effective production platforms for industrialization. This review will focus on major innovations in viral vector design and production systems for three of the most widely used viral vectors: Adenovirus, Adeno-Associated Virus, and Lentivirus.  

5.2244           A role for domain I of the hepatitis C virus NS5A protein in virus assembly

Yin, C., Goonawardane, N., Stewart, H. and Harris, M. PloS Pathogens, 14(1), e1006834 (2018)   The NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and assembly. NS5A comprises three domains, of these domain I is believed to be involved exclusively in genome replication. In contrast, domains II and III are required for the production of infectious virus particles and are largely dispensable for genome replication. Domain I is highly conserved between HCV and related hepaciviruses, and is highly structured, exhibiting different dimeric conformations. To investigate the functions of domain I in more detail, we conducted a mutagenic study of 12 absolutely conserved and surface-exposed residues within the context of a JFH-1-derived sub-genomic replicon and infectious virus. Whilst most of these abrogated genome replication, three mutants (P35A, V67A and P145A) retained the ability to replicate but showed defects in virus assembly. P35A exhibited a modest reduction in infectivity, however V67A and P145A produced no infectious virus. Using a combination of density gradient fractionation, biochemical analysis and high resolution confocal microscopy we demonstrate that V67A and P145A disrupted the localisation of NS5A to lipid droplets. In addition, the localisation and size of lipid droplets in cells infected with these two mutants were perturbed compared to wildtype HCV. Biophysical analysis revealed that V67A and P145A abrogated the ability of purified domain I to dimerize and resulted in an increased affinity of binding to HCV 3’UTR RNA. Taken together, we propose that domain I of NS5A plays multiple roles in assembly, binding nascent genomic RNA and transporting it to lipid droplets where it is transferred to Core. Domain I also contributes to a change in lipid droplet morphology, increasing their size. This study reveals novel functions of NS5A domain I in assembly of infectious HCV and provides new perspectives on the virus lifecycle.  

5.2245           Chimeric HCMV/HSV-1 and Δγ134.5 oncolytic herpes simplex virus elicit immune mediated antigliomal effect and antitumor memory12

Ghonime, M.G., Jackson, J., Shah, A., Roth, J., Li, M., Saunders, U., Coleman, J., Gillespie, G.Y., Markert, J.M. and Cassady, K.A. Translational Oncol., 11(1), 86-93 (2018)   Malignant gliomas are the most common primary brain tumor and are characterized by rapid and highly invasive growth. Because of their poor prognosis, new therapeutic strategies are needed. Oncolytic virotherapy (OV) is a promising strategy for treating cancer that incorporates both direct viral replication mediated and immune mediated mechanisms to kill tumor cells. C134 is a next generation Δγ₁34.5 oHSV-1 with improved intratumoral viral replication. It remains safe in the CNS environment by inducing early IFN signaling which restricts its replication in non-malignant cells. We sought to identify how C134 performed in an immunocompetent tumor model that restricts its replication advantage over first generation viruses. To achieve this we identified tumors that have intact IFN signaling responses that restrict C134 and first generation virus replication similarly. Our results show that both viruses elicit a T cell mediated anti-tumor effect and improved animal survival but that subtle difference exist between the viruses effect on median survival despite equivalent in vivo viral replication. To further investigate this we examined the anti-tumor activity in immunodeficient mice and in syngeneic models with re-challenge. These studies show that the T cell response is integral to C134 replication independent anti-tumor response and that OV therapy elicits a durable and circulating anti-tumor memory. The studies also show that repeated intratumoral administration can extend both OV anti-tumor effects and induce durable anti-tumor memory that is superior to tumor antigen exposure alone.  

5.2246           In Vivo Electrochemical Studies of Optogenetic Control of Glutamate Signaling Measured Using Enzyme-Based Ceramic Microelectrode Arrays

Burmeister, J.J., Pomerleau, F., Quintero, J.E., Huettl, P., Ai, Y., jakobsson, J., Lundblad, M., Heuer, A., Slevin, J.T. and Gerhardt, G.A. Neuromethods, 130, 327-351 (2018)   Direct electrochemical measurements of glutamate release in vivo were combined with optogenetics in order to examine light-induced control of glutamate neurotransmission in the rodent brain. Self-referenced recordings of glutamate using ceramic-based microelectrode arrays (MEAs) in hippocampus and frontal cortex demonstrated precise optical control of light-induced glutamate release through channelrhodopsin (ChR2) expression in both rat hippocampus and frontal cortex. Although the virus was only injected unilaterally, bilateral and rostro-caudal expression was observed in slice imaging, indicating diffusion and active transport of the viral particles. Methodology for the optogenetic control of glutamate signaling in the rat brain is thoroughly explained with special attention paid to MEA enzyme coating and cleaning for the benefit of other investigators. These data support that optogenetic control of glutamate signaling is robust with certain advantages as compared to other methods to modulate the in vivo control of glutamate signaling.  

5.2247           High-efficiency transduction of spinal cord motor neurons by intrauterine delivery of integration-deficient lentiviral vectors

Ahmed, S.G., Waddington, S.N., Boza-Moran, M.G. and Yanez-Munoz, R.J.

1. Controlled Release, 273, 99-107 (2018)

  Integration-deficient lentiviral vectors (IDLVs) are promising gene delivery tools that retain the high transduction efficiency of standard lentiviral vectors, yet fail to integrate as proviruses and are instead converted into episomal circles. These episomes are metabolically stable and support long-term expression of transgenes in non-dividing cells, exhibiting a decreased risk of insertional mutagenesis. We have embarked on an extensive study to compare the transduction efficiency of IDLVs pseudotyped with different envelopes (vesicular stomatitis, Rabies, Mokola and Ross River viral envelopes) and self-complementary adeno-associated viral vectors, serotype-9 (scAAV-9) in spinal cord tissues after intraspinal injection of mouse embryos (E16). Our results indicate that IDLVs can transduce motor neurons (MNs) at extremely high efficiency regardless of the envelope pseudotype while scAAV9 mediates gene delivery to ~ 40% of spinal cord motor neurons, with other non-neuronal cells also transduced. Long-term expression studies revealed stable gene expression at 7 months post-injection. Taken together, the results of this study indicate that IDLVs may be efficient tools for in utero cord transduction in therapeutic strategies such as for treatment of inherited early childhood neurodegenerative diseases.  

5.2248           Induction of alpha-synuclein pathology in the enteric nervous system of the rat and non-human primate results in gastrointestinal dysmotility and transient CNS pathology

Manfredsson, F.P., Luk, K.C., Benskey, M.J., gezer, A., Garcia, J., Kuhn, N.C., Sandoval, I.M., Patterson, J.R., O’Mara, A., Yonkers, R. and Kordower, J.H. Neurobiology of Disease, 112, 106-118 (2018)   Alpha-Synuclein (α-syn) is by far the most highly vetted pathogenic and therapeutic target in Parkinson's disease. Aggregated α-syn is present in sporadic Parkinson's disease, both in the central nervous system (CNS) and peripheral nervous system (PNS). The enteric division of the PNS is of particular interest because 1) gastric dysfunction is a key clinical manifestation of Parkinson's disease, and 2) Lewy pathology in myenteric and submucosal neurons of the enteric nervous system (ENS) has been referred to as stage zero in the Braak pathological staging of Parkinson's disease. The presence of Lewy pathology in the ENS and the fact that patients often experience enteric dysfunction before the onset of motor symptoms has led to the hypothesis that α-syn pathology starts in the periphery, after which it spreads to the CNS via interconnected neural pathways. Here we sought to directly test this hypothesis in rodents and non-human primates (NHP) using two distinct models of α-syn pathology: the α-syn viral overexpression model and the preformed fibril (PFF) model. Subjects (rat and NHP) received targeted enteric injections of PFFs or adeno-associated virus overexpressing the Parkinson's disease associated A53T α-syn mutant. Rats were evaluated for colonic motility monthly and sacrificed at 1, 6, or 12 months, whereas NHPs were sacrificed 12 months following inoculation, after which the time course and spread of pathology was examined in all animals. Rats exhibited a transient GI phenotype that resolved after four months. Minor α-syn pathology was observed in the brainstem (dorsal motor nucleus of the vagus and locus coeruleus) 1 month after PFF injections; however, no pathology was observed at later time points (nor in saline or monomer treated animals). Similarly, a histopathological analysis of the NHP brains revealed no pathology despite the presence of robust α-syn pathology throughout the ENS which persisted for the entirety of the study (12 months). Our study shows that induction of α-syn pathology in the ENS is sufficient to induce GI dysfunction. Moreover, our data suggest that sustained spread of α-syn pathology from the periphery to the CNS and subsequent propagation is a rare event, and that the presence of enteric α-syn pathology and dysfunction may represent an epiphenomenon.  

5.2249           Merkel Cell Polyomavirus Infection of Animal Dermal Fibroblasts

Liu, W., Krump, N.A., MacDonald, M. and You, J.

1. Virol., 92(4), e01610-17 (2018)

  Merkel cell polyomavirus (MCPyV) is the first polyomavirus to be associated with human cancer. Mechanistic studies attempting to fully elucidate MCPyV's oncogenic mechanisms have been hampered by the lack of animal models for MCPyV infection. In this study, we examined the ability of MCPyV-GFP pseudovirus (containing a green fluorescent protein [GFP] reporter construct), MCPyV recombinant virions, and several MCPyV chimeric viruses to infect dermal fibroblasts isolated from various model animals, including mouse (Mus musculus), rabbit (Oryctolagus cuniculus), rat (Rattus norvegicus), chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), patas monkey (Erythrocebus patas), common woolly monkey (Lagothrix lagotricha), red-chested mustached tamarin (Saguinus labiatus), and tree shrew (Tupaia belangeri). We found that MCPyV-GFP pseudovirus was able to enter the dermal fibroblasts of all species tested. Chimpanzee dermal fibroblasts were the only type that supported vigorous MCPyV gene expression and viral replication, and they did so to a level beyond that of human dermal fibroblasts. We further demonstrated that both human and chimpanzee dermal fibroblasts produce infectious MCPyV virions that can successfully infect new cells. In addition, rat dermal fibroblasts supported robust MCPyV large T antigen expression after infection with an MCPyV chimeric virus in which the entire enhancer region of the MCPyV early promoter has been replaced with the simian virus 40 (SV40) analog. Our results suggest that viral transcription and/or replication events represent the major hurdle for MCPyV cross-species transmission. The capacity of rat dermal fibroblasts to support MCPyV early gene expression suggests that the rat is a candidate model organism for studying viral oncogene function during Merkel cell carcinoma (MCC) oncogenic progression.  

5.2250           Minor Capsid Protein L2 Polytope Induces Broad Protection against Oncogenic and Mucosal Human Papillomaviruses

Pouyanfard, S., Spagnoli, G., Bulli, L., Balz, K., yang, F., odenwald, C., Seitz, h., Mariz, F.C., Bolchi, A., Ottonello, S. and Müller, M.

1. Virol., 92(4), e01930-17 (2018)

  The amino terminus of the human papillomavirus (HPV) minor capsid protein L2 contains a major cross-neutralization epitope which provides the basis for the development of a broadly protecting HPV vaccine. A wide range of protection against different HPV types would eliminate one of the major drawbacks of the commercial, L1-based prophylactic vaccines. Previously, we have reported that insertion of the L2 epitope into a scaffold composed of bacterial thioredoxin protein generates a potent antigen inducing comprehensive protection against different animal and human papillomaviruses. We also reported, however, that although protection is broad, some oncogenic HPV types escape the neutralizing antibody response, if L2 epitopes from single HPV types are used as immunogen. We were able to compensate for this by applying a mix of thioredoxin proteins carrying L2 epitopes from HPV16, -31, and -51. As the development of a cost-efficient HPV prophylactic vaccines is one of our objectives, this approach is not feasible as it requires the development of multiple good manufacturing production processes in combination with a complex vaccine formulation. Here, we report the development of a thermostable thioredoxin-based single-peptide vaccine carrying an L2 polytope of up to 11 different HPV types. The L2 polytope antigens have excellent abilities in respect to broadness of protection and robustness of induced immune responses. To further increase immunogenicity, we fused the thioredoxin L2 polytope antigen with a heptamerization domain. In the final vaccine design, we achieve protective responses against all 14 oncogenic HPV types that we have analyzed plus the low-risk HPVs 6 and 11 and a number of cutaneous HPVs.  

5.2251           Ubiquitination of the Cytoplasmic Domain of Influenza A Virus M2 Protein Is Crucial for Production of Infectious Virus Particles

Su, W-S., Yu, W-Y., Huang, S-H. and Lai, M.M.C.

1. Virol., 92(4), e01972-17 (2018)

  Virus replication is mediated by interactions between the virus and host. Here, we demonstrate that influenza A virus membrane protein 2 (M2) can be ubiquitinated. The lysine residue at position 78, which is located in the cytoplasmic domain of M2, is essential for M2 ubiquitination. An M2-K78R (Lys78→Arg78) mutant, which produces ubiquitination-deficient M2, showed a severe defect in the production of infectious virus particles. M2-K78R mutant progeny contained more hemagglutinin (HA) proteins, less viral RNAs, and less internal viral proteins, including M1 and NP, than the wild-type virus. Furthermore, most of the M2-K78R mutant viral particles lacked viral ribonucleoproteins upon examination by electron microscopy and exhibited slightly lower densities. We also found that mutant M2 colocalized with the M1 protein to a lesser extent than for the wild-type virus. These findings may account for the reduced incorporation of viral ribonucleoprotein into virions. By blocking the second round of virus infection, we showed that the M2 ubiquitination-defective mutant exhibited normal levels of virus replication during the first round of infection, thereby proving that M2 ubiquitination is involved in the virus production step. Finally, we found that the M2-K78R mutant virus induced autophagy and apoptosis earlier than did the wild-type virus. Collectively, these results suggest that M2 ubiquitination plays an important role in infectious virus production by coordinating the efficient packaging of the viral genome into virus particles and the timing of virus-induced cell death.  

5.2252           Bunyavirus requirement for endosomal K+ reveals new roles of cellular ion channels during infection

Hover, S., Foster, B., Fontana, J., Kohl, A., Goldstein, S.A.N., Barr, J.N. and Mankouri, J. PloS Pathogens, 14(1), e1006845 (2018)   In order to multiply and cause disease a virus must transport its genome from outside the cell into the cytosol, most commonly achieved through the endocytic network. Endosomes transport virus particles to specific cellular destinations and viruses exploit the changing environment of maturing endocytic vesicles as triggers to mediate genome release. Previously we demonstrated that several bunyaviruses, which comprise the largest family of negative sense RNA viruses, require the activity of cellular potassium (K⁺) channels to cause productive infection. Specifically, we demonstrated a surprising role for K⁺ channels during virus endosomal trafficking. In this study, we have used the prototype bunyavirus, Bunyamwera virus (BUNV), as a tool to understand why K⁺ channels are required for progression of these viruses through the endocytic network. We report three major findings: First, the production of a dual fluorescently labelled bunyavirus to visualize virus trafficking in live cells. Second, we show that BUNV traffics through endosomes containing high [K⁺] and that these K⁺ ions influence the infectivity of virions. Third, we show that K⁺ channel inhibition can alter the distribution of K⁺ across the endosomal system and arrest virus trafficking in endosomes. These data suggest high endosomal [K⁺] is a critical cue that is required for virus infection, and is controlled by cellular K⁺ channels resident within the endosome network. This highlights cellular K⁺ channels as druggable targets to impede virus entry, infection and disease.    

5.2253           Small Scale Production of Recombinant Adeno-Associated Viral Vectors for Gene Delivery to the Nervous System

Verhaagen, J., Hobo, B., Ehlert, E.M.E., Eggers, R., Korecka, J.A., Hoyng, S.A., Attwell, C.L., Harvey, A.R. and Mason, M.R.J. Methods in Mol. Biol., 1715, 3-17 (2018)   Adeno-associated viral vectors have numerous applications in neuroscience, including the study of gene function in health and disease, targeting of light-sensitive proteins to anatomically distinct sets of neurons to manipulate neuronal activity (optogenetics), and the delivery of fluorescent protein to study anatomical connectivity in the brain. Moreover several phase I/II clinical trials for gene therapy of eye and brain diseases with adeno-associated viral vectors have shown that these vectors are well tolerated by human patients. In this chapter we describe a detailed protocol for the small scale production of recombinant adeno-associated viral vectors. This protocol can be executed by investigators with experience in cell culture and molecular biological techniques in any well-equipped molecular neurobiology laboratory. With this protocol we typically obtain research batches of 100–200 μL that range in titer from 5 × 10¹² to 2 × 10¹³ genomic copies/mL.  

5.2254           Small and Micro-Scale Recombinant Adeno-Associated Virus Production and Purification for Ocular Gene Therapy Applications

Reid, C.A. and Lipinski, D.M. Methods in Mol. Biol., 1715, 19-31 (2018)   Over the past two decades recombinant adeno-associated virus (rAAV) vectors have emerged as the gold standard for transferring genetic material to cells of the retina. The ability to effectively produce small batches of rAAV vector at high enough purity for in vitro and in vivo applications in a cost-effective manner is paramount. This is particularly the case when conducting preclinical experiments to screen novel serotypes, promoters or transgenes, where production of numerous vector batches is required. Current vector production methods often produce large quantities of vector, limiting the cost-effectiveness and practicality of such screening experiments, which often require only small volumes of vector to carry out. Herein, we describe a method to produce high titer (10¹²–10¹³ vector genomes (vg)/mL) rAAV vector on small (~100 μL) or micro (~15 μL) scale for in vitro and in vivo applications.  

5.2255           Design and Development of AAV-based Gene Supplementation Therapies for Achromatopsia and Retinitis Pigmentosa

Schön, C., Becirovic, e., Biel, M. and Michalakis, S. Methods in Mol. Biol., 1715, 33-46 (2018)   Achromatopsia (ACHM) and retinitis pigmentosa (RP) are inherited disorders caused by mutations in cone and rod photoreceptor-specific genes, respectively. ACHM strongly impairs daylight vision, whereas RP initially affects night vision and daylight vision at later stages. Currently, gene supplementation therapies utilizing recombinant adeno-associated virus (rAAV) vectors are being developed for various forms of ACHM and RP. In this chapter, we describe the procedure of designing and developing specific and efficient rAAV vectors for cone- and rod-specific gene supplementation.  

5.2256           AAV Serotype Testing on Cultured Human Donor Retinal Explants

Buck, T.M., Pellissier, L.P., Vos, R.M., van Dijk, E.H.C., Boon, C.J.F. and Wijnholds, J. Methods in Mol. Biol., 1715, 275-288 (2018)   This protocol details on a screening method for infectivity and tropism of different serotypes of adeno-associated viruses (AAVs) on human retinal explants with cell-type specific or ubiquitous green fluorescent protein (GFP) expression vectors. Eyes from deceased adult human donors are enucleated and the retinas are isolated. Each retina is punched into eight to ten 6-mm equal pieces. Whatman™ paper punches are placed on the retinas and the stack is transferred onto 24-well culture inserts with the photoreceptors facing the membrane. AAVs are applied on the retinal explant punches to allow transduction for 48 h. Retinas are nourished by a serum-free Neurobasal®-A based medium composition that allows extended culturing of explants containing photoreceptor inner and outer segments. The protocols include quality control measurements and histological staining for retina cells. The cost and time effective procedure permits AAV transgene expression assays, RNAi knockdown, and pharmacological intervention on human retinas for 21 days ex vivo.    

5.2257           Ultrafiltered recombinant AAV8 vector can be safely administered in vivo and efficiently transduces liver

Kleven, M.D., Gomes, M.M., Wortham, A.M., Enns, C.A. and Kahl, A.

PloS One, 13(4), e0194728 (2018)

Viral vectors are extensively purified for use in biomedical research, in order to separate biologically active virus particles and to eliminate production related impurities that are assumed to be detrimental to the host. For recombinant adeno-associated virus (rAAV) vectors this is typically accomplished using density gradient-based methods, which are tedious and require specialized ultracentrifugation equipment. In order to streamline the preparation of rAAV vectors for pilot and small animal studies, we recently devised a simple ultrafiltration approach that permits rapid virus concentration and partial removal of production-related impurities. Here we show that systemic administration of such rapidly prepared (RP) rAAV8 vectors in mice is safe and efficiently transduces the liver. Across a range of doses, delivery of RP rAAV8-CMV-eGFP vector induced enhanced green fluorescent protein (eGFP) expression in liver that was comparable to that obtained from a conventional iodixanol gradient-purified (IP) vector. Surprisingly, no liver inflammation or systemic cytokine induction was detected in RP rAAV injected animals, revealing that residual impurities in the viral vector preparation are not deleterious to the host. Together, these data demonstrate that partially purified rAAV vector can be safely and effectively administered in vivo. The speed and versatility of the RP method and lack of need for cumbersome density gradients or expensive ultracentrifuge equipment will enable more widespread use of RP prepared rAAV vectors, such as for pilot liver gene transfer studies.

 

5.2258           A Hitchhiker’s Guide to the Selection of Viral Vectors for Optogenetic Studies

Thompson, K.R. and Towne, C. Neuromethods, 133, 1-23 (2018)   The very first article to describe optogenetics in neural systems used viruses as delivery vectors (Boyden et al., Nat Neurosci 8(9):1263–1268, 2005). Since then, viral-mediated gene delivery has become the method of choice for opsin expression in the field. There are many classes of viruses, each with unique attributes that can be taken advantage of to serve specific experimental needs. For example, precise cellular targeting can be achieved by exploiting the propensity of different vectors to transduce specific cell types. Distinct anatomical inputs or outputs to defined regions can be identified and manipulated by choosing vectors for opsin expression with retrograde or anterograde trafficking abilities. Some vectors also have the capability to spread between synaptically connected neurons, and this holds great potential for the determination of structure–function relationships across complex networks. Here we review the major viral vector types used in optogenetic studies and offer a detailed protocol for the production of adeno-associated virus, which has become the most popular vector for optogenetic applications. This chapter is intended to provide an understanding of basic principles in vectorology and to serve as a user’s guide to aid in the selection of appropriate vector. The engineering of recombinant viruses promises to expand the level of experimental precision and control, and may one day even lead to effective optogenetic therapies.  

5.2259           CRISPR/Cas9 genome editing in human hematopoietic stem cells

Bak, R.O., Dever, D.P. and Porteus, M.H. Nature Protocols, 13(2), 358-376 (2018)   Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene–function relationships and potentially transform curative hematological gene and cell therapies. However, there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein, we provide a detailed protocol for the production, enrichment, and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry. Using this protocol, researchers can introduce single-nucleotide changes into the genome or longer gene cassettes with the precision of genome editing. Along with our troubleshooting and optimization guidelines, researchers can use this protocol to streamline HSC genome editing at any locus of interest. The in vitro HSC-targeting protocol and analyses can be completed in 3 weeks, and the long-term in vivo HSC engraftment analyses in immunodeficient mice can be achieved in 16 weeks. This protocol enables manipulation of genes for investigation of gene functions during hematopoiesis, as well as for the correction of genetic mutations in HSC transplantation–based therapies for diseases such as sickle cell disease, β-thalassemia, and primary immunodeficiencies.  

5.2260           A major lineage of non-tailed dsDNA viruses as unrecognized killers of marine bacteria

Kauffman, K.M., Hussain, F.A., Yang, J., Arevalo, P., Brown, J.M., Chang, W.K., Vanlnsbergher, D., Elsherbini, J., Sharma, R.S., Cutler, M.B., Kelly, L. and Polz, M.F. Nature, 554, 118-122 (2018)   The most abundant viruses on Earth are thought to be double-stranded DNA (dsDNA) viruses that infect bacteria¹. However, tailed bacterial dsDNA viruses (Caudovirales), which dominate sequence and culture collections, are not representative of the environmental diversity of viruses^2,3. In fact, non-tailed viruses often dominate ocean samples numerically⁴, raising the fundamental question of the nature of these viruses. Here we characterize a group of marine dsDNA non-tailed viruses with short 10-kb genomes isolated during a study that quantified the diversity of viruses infecting Vibrionaceae bacteria. These viruses, which we propose to name the Autolykiviridae, represent a novel family within the ancient lineage of double jelly roll (DJR) capsid viruses. Ecologically, members of the Autolykiviridae have a broad host range, killing on average 34 hosts in four Vibrio species, in contrast to tailed viruses which kill on average only two hosts in one species. Biochemical and physical characterization of autolykiviruses reveals multiple virion features that cause systematic loss of DJR viruses in sequencing and culture-based studies, and we describe simple procedural adjustments to recover them. We identify DJR viruses in the genomes of diverse major bacterial and archaeal phyla, and in marine water column and sediment metagenomes, and find that their diversity greatly exceeds the diversity that is currently captured by the three recognized families of such viruses. Overall, these data suggest that viruses of the non-tailed dsDNA DJR lineage are important but often overlooked predators of bacteria and archaea that impose fundamentally different predation and gene transfer regimes on microbial systems than on tailed viruses, which form the basis of all environmental models of bacteria–virus interactions.  

5.2261           Glial fibrillary acidic protein promoter determines transgene expression in satellite glial cells following intraganglionic adeno-associated virus delivery in adult rats

Xiang, H., Xu, H., Fan, F., Shin, S-M., Hogan, Q. and Yu, H.

1. Neuro. Res., 96(3), 436-448 (2018)

  Recombinant adeno-associated viral (AAV)-mediated therapeutic gene transfer to dorsal root ganglia (DRG) is an effective and safe tool for treating chronic pain. However, AAV with various constitutively active promoters leads to transgene expression predominantly to neurons, while glial cells are refractory to AAV transduction in the peripheral nervous system. The present study evaluated whether in vivo satellite glial cell (SGC) transduction in the DRG can be enhanced by the SGC-specific GFAP promoter and by using shH10 and shH19, which are engineered capsid variants with Müller glia-prone transduction. Titer-matched AAV6 (as control), AAVshH10, and AAVshH19, all encoding the EGFP driven by the constitutively active CMV promoter, as well as AAV6-EGFP and AAVshH10-EGFP driven by a GFAP promoter (AAV6-GFAP-EGFP and AAVshH10-GFAP-EGFP), were injected into DRG of adult male rats. Neurotropism of gene expression was determined and compared by immunohistochemistry. Results showed that injection of AAV6- and AAVshH10-GFAP-EGFP induces robust EGFP expression selectively in SGCs, whereas injection of either AAVshH10-CMV-EGFP or AAVshH19-CMV-EGFP into DRG resulted in a similar in vivo transduction profile to AAV6-CMV-EGFP, all showing efficient transduction of sensory neurons without significant transduction of glial cell populations. Coinjection of AAV6-CMV-mCherry and AAV6-GFAP-EGFP induces transgene expression in neurons and SGCs separately. This report, together with our prior studies, demonstrates that the GFAP promoter rather than capsid tropism determines selective gene expression in SGCs following intraganglionic AAV delivery in adult rats. A dual AAV system, one with GFAP promoter and the other with CMV promoter, can efficiently express transgenes selectively in neurons versus SGCs.  

5.2262           Nestin expression is dynamically regulated in cardiomyocytes during embryogenesis

Hertig, V., Matos-Nieves, A., Garg, V., Villeneuve, L., Mamarbachi, M., Caland, L., Calderone, A.

1. Cell Physiol., 233(4), 3218-3229 (2018)

  The transcriptional factors implicated in the expression of the intermediate filament protein nestin in cardiomyocytes during embryogenesis remain undefined. In the heart of 9,5–10,5 day embryonic mice, nestin staining was detected in atrial and ventricular cardiomyocytes and a subpopulation co-expressed Tbx5. At later stages of development, nestin immunoreactivity in cardiomyocytes gradually diminished and was absent in the heart of 17,5 day embryonic mice. In the heart of wild type 11,5 day embryonic mice, 54 ± 7% of the trabeculae expressed nestin and the percentage was significantly increased in the hearts of Tbx5^+/− and Gata4^+/− embryos. The cell cycle protein Ki67 and transcriptional coactivator Yap-1 were still prevalent in the nucleus of nestin⁽⁺⁾-cardiomyocytes identified in the heart of Tbx5^+/− and Gata4^+/− embryonic mice. Phorbol 12,13-dibutyrate treatment of neonatal rat ventricular cardiomyocytes increased Yap-1 phosphorylation and co-administration of the p38 MAPK inhibitor SB203580 led to significant dephosphorylation. Antagonism of dephosphorylated Yap-1 signalling with verteporfin inhibited phorbol 12,13-dibutyrate/SB203580-mediated nestin expression and BrdU incorporation of neonatal cardiomyocytes. Nestin depletion with an AAV9 containing a shRNA directed against the intermediate filament protein significantly reduced the number of neonatal cardiomyocytes that re-entered the cell cycle. These findings demonstrate that Tbx5- and Gata4-dependent events negatively regulate nestin expression in cardiomyocytes during embryogenesis. By contrast, dephosphorylated Yap-1 acting via upregulation of the intermediate filament protein nestin plays a seminal role in the cell cycle re-entry of cardiomyocytes. Based on these data, an analogous role of Yap-1 may be prevalent in the heart of Tbx5^+/− and Gata4^+/− mice.  

5.2263           Synthetic AAV/CRISPR vectors for blocking HIV-1 expression in persistently infected astrocytes

Kunze, C., Börner, k., Kienle, E., Orschmann, T., Rusha, E., Schneider, M., Radivojkov-Blagojevic, M., Drukker, m., Desbordes, S., Grimm, D. and Brack-Werner, R. Glia, 66(2), 413-427 (2018)   Astrocytes, the most abundant cells in the mammalian brain, perform key functions and are involved in several neurodegenerative diseases. The human immunodeficiency virus (HIV) can persist in astrocytes, contributing to the HIV burden and neurological dysfunctions in infected individuals. While a comprehensive approach to HIV cure must include the targeting of HIV-1 in astrocytes, dedicated tools for this purpose are still lacking. Here we report a novel Adeno-associated virus-based vector (AAV9P1) with a synthetic surface peptide for transduction of astrocytes. Analysis of AAV9P1 transduction efficiencies with single brain cell populations, including primary human brain cells, as well as human brain organoids demonstrated that AAV9P1 targeted terminally differentiated human astrocytes much more efficiently than neurons. We then investigated whether AAV9P1 can be used to deliver HIV-inhibitory genes to astrocytes. To this end we generated AAV9P1 vectors containing genes for HIV-1 proviral editing by CRISPR/Cas9. Latently HIV-1 infected astrocytes transduced with these vectors showed significantly diminished reactivation of proviruses, compared with untransduced cultures. Sequence analysis identified mutations/deletions in key HIV-1 transcriptional control regions. We conclude that AAV9P1 is a promising tool for gene delivery to astrocytes and may facilitate inactivation/destruction of persisting HIV-1 proviruses in astrocyte reservoirs.  

5.2264           MS2 and Qβ bacteriophages reveal the contribution of surface hydrophobicity on the mobility of non-enveloped icosahedral viruses in SDS-based capillary zone electrophoresis

Sautrey, G., Brie, A., Gantzer, C. and Walcarius, A. Electrophoresis, 39(2), 377-385 (2018)   SDS is commonly employed as BGE additive in CZE analysis of non-enveloped icosahedral viruses. But the way by which SDS interacts with the surface of such viruses remains to date poorly known, making complicate to understand their behavior during a run. In this article, two related bacteriophages, MS2 and Qβ, are used as model to investigate the migration mechanism of non-enveloped icosahedral viruses in SDS-based CZE. Both phages are characterized by similar size and surface charge but significantly different surface hydrophobicity (Qβ > MS2, where ‘>’ means ‘more hydrophobic than’). By comparing their electrophoretic mobility in the presence or not of SDS on both sides of the CMC, we show that surface hydrophobicity of phages is a key factor influencing their mobility and that SDS-virus association is driven by hydrophobic interactions at the surface of virions. The CZE analyses of heated MS2 particles, which over-express hydrophobic domains at their surface, confirm this finding. The correlations between the present results and others from the literature suggest that the proposed mechanism might not be exclusive to the bacteriophages examined here.  

5.2265           Multi-modal Potentiation of Oncolytic Virotherapy by Vanadium Compounds

Selman, M., Rousso, C., Bergeron, A., Son, H.H., Krishnan, R., El-Sayes, N.A., Varette, O., Chen, A., Le Boeuf, F., Tzelepis, F., Bell, J.C., Crans, D.C. and Diallo, J-S. Molecular Therapy, 26(1), 56-69 (2018)   Oncolytic viruses (OV) are an emerging class of anticancer bio-therapeutics that induce antitumor immunity through selective replication in tumor cells. However, the efficacy of OVs as single agents remains limited. We introduce a strategy that boosts the therapeutic efficacy of OVs by combining their activity with immuno-modulating, small molecule protein tyrosine phosphatase inhibitors. We report that vanadium-based phosphatase inhibitors enhance OV infection in vitro and ex vivo, in resistant tumor cell lines. Furthermore, vanadium compounds increase antitumor efficacy in combination with OV in several syngeneic tumor models, leading to systemic and durable responses, even in models otherwise refractory to OV and drug alone. Mechanistically, this involves subverting the antiviral type I IFN response toward a death-inducing and pro-inflammatory type II IFN response, leading to improved OV spread, increased bystander killing of cancer cells, and enhanced antitumor immune stimulation. Overall, we showcase a new ability of vanadium compounds to simultaneously maximize viral oncolysis and systemic anticancer immunity, offering new avenues for the development of improved immunotherapy strategies.  

5.2266           In Vivo Selection of a Computationally Designed SCHEMA AAV Library Yields a Novel Variant for Infection of Adult Neural Stem Cells in the SVZ

Ojala, D.S., Sun, S., Santiago-Ortiz, J.L., Shapiro, M.G., Romero, P.A. and Scaffer, D.V. Molecular Therapy, 26(1), 304-319 (2018)   Directed evolution continues to expand the capabilities of complex biomolecules for a range of applications, such as adeno-associated virus vectors for gene therapy; however, advances in library design and selection strategies are key to develop variants that overcome barriers to clinical translation. To address this need, we applied structure-guided SCHEMA recombination of the multimeric adeno-associated virus (AAV) capsid to generate a highly diversified chimeric library with minimal structural disruption. A stringent in vivo Cre-dependent selection strategy was implemented to identify variants that transduce adult neural stem cells (NSCs) in the subventricular zone. A novel variant, SCH9, infected 60% of NSCs and mediated 24-fold higher GFP expression and a 12-fold greater transduction volume than AAV9. SCH9 utilizes both galactose and heparan sulfate as cell surface receptors and exhibits increased resistance to neutralizing antibodies. These results establish the SCHEMA library as a valuable tool for directed evolution and SCH9 as an effective gene delivery vector to investigate subventricular NSCs.  

5.2267           Oligonucleotide conjugated multi-functional adeno-associated viruses

Katrekar, D., Moreno, A.M., Chen, G., Worlikar, A. and Mali, P. Scientific Reports, 8:3589 (82018)   Recombinant adeno-associated viruses (AAVs) are among the most commonly used vehicles for in vivo gene delivery. However, their tropism is limited, and additionally their efficacy can be negatively affected by prevalence of neutralizing antibodies in sera. Methodologies to systematically engineer AAV capsid properties would thus be of great relevance. In this regard, we develop here multi-functional AAVs by engineering precision tethering of oligonucleotides onto the AAV surface, and thereby enabling a spectrum of nucleic-acid programmable functionalities. Towards this, we engineered genetically encoded incorporation of unnatural amino acids (UAA) bearing bio-orthogonal chemical handles onto capsid proteins. Via these we enabled site-specific coupling of oligonucleotides onto the AAV capsid surface using facile click chemistry. The resulting oligo-AAVs could be sequence specifically labeled, and also patterned in 2D using DNA array substrates. Additionally, we utilized these oligo conjugations to engineer viral shielding by lipid-based cloaks that efficaciously protected the AAV particles from neutralizing serum. We confirmed these ‘cloaked AAVs’ retained full functionality via their ability to transduce a range of cell types, and also enable robust delivery of CRISPR-Cas9 effectors. Taken together, we anticipate this programmable oligo-AAV system will have broad utility in synthetic biology and AAV engineering applications.  

5.2268           Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells

Hung, K.L., Meitlis, I., Hale, M., Chen, C-Y., Singh, S., jackson, S.W., Miao, C.H., Khan, I.F., Rawlings, D.J. and James, R.G. Molecular Therapy, 26(2), 456-467 (2018)   The ability to engineer primary human B cells to differentiate into long-lived plasma cells and secrete a de novo protein may allow the creation of novel plasma cell therapies for protein deficiency diseases and other clinical applications. We initially developed methods for efficient genome editing of primary B cells isolated from peripheral blood. By delivering CRISPR/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes under conditions of rapid B cell expansion, we achieved site-specific gene disruption at multiple loci in primary human B cells (with editing rates of up to 94%). We used this method to alter ex vivo plasma cell differentiation by disrupting developmental regulatory genes. Next, we co-delivered RNPs with either a single-stranded DNA oligonucleotide or adeno-associated viruses containing homologous repair templates. Using either delivery method, we achieved targeted sequence integration at high efficiency (up to 40%) via homology-directed repair. This method enabled us to engineer plasma cells to secrete factor IX (FIX) or B cell activating factor (BAFF) at high levels. Finally, we show that introduction of BAFF into plasma cells promotes their engraftment into immunodeficient mice. Our results highlight the utility of genome editing in studying human B cell biology and demonstrate a novel strategy for modifying human plasma cells to secrete therapeutic proteins.  

5.2269           A conserved Eph family receptor-binding motif on the gH/gL complex of Kaposi’s sarcoma-associated herpesvirus and rhesus monkey rhadinovirus

Grosskopf, A.K., Ensseer, A., Neipel, F., Jungnickl, D., Schlagowski, S., Desrosiers, R.C. and Hahn, A.S. PloS Pathogens, 14(2), e1006912 (2018)   Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus associated with Kaposi’s sarcoma and two B-cell malignancies. The rhesus monkey rhadinovirus (RRV) is a virus of nonhuman primates that is closely related to KSHV. Eph family receptor tyrosine kinases (Ephs) are cellular receptors for the gH/gL glycoprotein complexes of both KSHV and RRV. Through sequence analysis and mutational screens, we identified conserved residues in the N-terminal domain of KSHV and RRV glycoprotein H that are critical for Eph-binding in vitro. Homology-based structural predictions of the KSHV and RRV gH/gL complexes based on the Epstein-Barr-Virus gH/gL crystal structure located these amino acids in a beta-hairpin on gH, which is likely stabilized by gL and is optimally positioned for protein-protein interactions. Guided by these predictions, we generated recombinant RRV and KSHV strains mutated in the conserved motif as well as an RRV gL null mutant. Inhibition experiments using these mutants confirmed that disruption of the identified Eph-interaction motif or of gL expression resulted in complete detargeting from Ephs. However, all mutants were infectious on all cell types tested, exhibiting normal attachment but a reduction in infectivity of up to one log order of magnitude. While Eph-binding-negative RRV mutants were replication-competent on fibroblasts, their infectivity was comparatively more reduced on endothelial cells with a substantial subpopulation of endothelial cells remaining resistant to infection. Together, this provides evidence for a cell type-specific use of Ephs by RRV. Furthermore, our results demonstrate that gL is dispensable for infection by RRV. Its deletion caused a reduction in infectivity similar to that observed after mutation of Eph-binding residues in gH. Our findings would be compatible with an ability of KSHV and RRV to use other, less efficient entry mediators in lieu of Ephs, although these host factors may not be uniformly expressed by all cells.  

5.2270           The Amino Terminus of Herpes Simplex Virus 1 Glycoprotein K (gK) Is Required for gB Binding to Akt, Release of Intracellular Calcium, and Fusion of the Viral Envelope with Plasma Membranes

Musarrat, F., Jambunathan, N., Rider, P.J., Chouljenko, V.N. and Kousoulas, K.G.

1. Virol., 92(6), e01842-17 (2018)

  Previously, we have shown that the amino terminus of glycoprotein K (gK) binds to the amino terminus of gB and that deletion of the amino-terminal 38 amino acids of gK prevents herpes simplex virus 1 (HSV-1) infection of mouse trigeminal ganglia after ocular infection and virus entry into neuronal axons. Recently, it has been shown that gB binds to Akt during virus entry and induces Akt phosphorylation and intracellular calcium release. Proximity ligation and two-way immunoprecipitation assays using monoclonal antibodies against gB and Akt-1 phosphorylated at S473 [Akt-1(S473)] confirmed that HSV-1(McKrae) gB interacted with Akt-1(S473) during virus entry into human neuroblastoma (SK-N-SH) cells and induced the release of intracellular calcium. In contrast, the gB specified by HSV-1(McKrae) gKΔ31-68, lacking the amino-terminal 38 amino acids of gK, failed to interact with Akt-1(S473) and induce intracellular calcium release. The Akt inhibitor miltefosine inhibited the entry of McKrae but not the gKΔ31-68 mutant into SK-N-SH cells. Importantly, the entry of the gKΔ31-68 mutant but not McKrae into SK-N-SH cells treated with the endocytosis inhibitors pitstop-2 and dynasore hydrate was significantly inhibited, indicating that McKrae gKΔ31-68 entered via endocytosis. These results suggest that the amino terminus of gK functions to regulate the fusion of the viral envelope with cellular plasma membranes.  

5.2271           Actin-Dependent Nonlytic Rotavirus Exit and Infectious Virus Morphogenetic Pathway in Nonpolarized Cells

Trejo-Cerro, O., Eichwald, C., Schraner, E.M., Silva-Ayala, D., Lopez, S. and Arias, C.F.

1. Virol., 92(6), e2076-17 (2018)

  During the late stages of rotavirus morphogenesis, the surface proteins VP4 and VP7 are assembled onto the previously structured double-layered virus particles to yield a triple-layered, mature infectious virus. The current model for the assembly of the outer capsid is that it occurs within the lumen of the endoplasmic reticulum. However, it has been shown that VP4 and infectious virus associate with lipid rafts, suggesting that the final assembly of the rotavirus spike protein VP4 involves a post-endoplasmic reticulum event. In this work, we found that the actin inhibitor jasplakinolide blocks the cell egress of rotavirus from nonpolarized MA104 cells at early times of infection, when there is still no evidence of cell lysis. These findings contrast with the traditional assumption that rotavirus is released from nonpolarized cells by a nonspecific mechanism when the cell integrity is lost. Inspection of the virus present in the extracellular medium by use of density flotation gradients revealed that a fraction of the released virus is associated with low-density membranous structures. Furthermore, the intracellular localization of VP4, its interaction with lipid rafts, and its targeting to the cell surface were shown to be prevented by jasplakinolide, implying a role for actin in these processes. Finally, the VP4 present at the plasma membrane was shown to be incorporated into the extracellular infectious virus, suggesting the existence of a novel pathway for the assembly of the rotavirus spike protein.  

5.2272           A new cell culture model to genetically dissect the complete human papillomavirus life cycle

Bienkowska-Haba, M., Luszczek, W., Myers, J.E., Keiffer, T.R., DiGiuseppe, S., Polk, P., Bodily, J.M., Scott, R.S. and Sapp, M. PloS Pathogens, 14(3), e1006846 (2018)   Herein, we describe a novel infection model that achieves highly efficient infection of primary keratinocytes with human papillomavirus type 16 (HPV16). This cell culture model does not depend on immortalization and is amenable to extensive genetic analyses. In monolayer cell culture, the early but not late promoter was active and yielded a spliced viral transcript pattern similar to HPV16-immortalized keratinocytes. However, relative levels of the E8^E2 transcript increased over time post infection suggesting the expression of this viral repressor is regulated independently of other early proteins and that it may be important for the shift from the establishment to the maintenance phase of the viral life cycle. Both the early and the late promoter were strongly activated when infected cells were subjected to differentiation by growth in methylcellulose. When grown as organotypic raft cultures, HPV16-infected cells expressed late E1^E4 and L1 proteins and replication foci were detected, suggesting that they supported the completion of the viral life cycle. As a proof of principle that the infection system may be used for genetic dissection of viral factors, we analyzed E1, E6 and E7 translation termination linker mutant virus for establishment of infection and genome maintenance. E1 but not E6 and E7 was essential to establish infection. Furthermore, E6 but not E7 was required for episomal genome maintenance. Primary keratinocytes infected with wild type HPV16 immortalized, whereas keratinocytes infected with E6 and E7 knockout virus began to senesce 25 to 35 days post infection. The novel infection model provides a powerful genetic tool to study the role of viral proteins throughout the viral life cycle but especially for immediate early events and enables us to compare low- and high-risk HPV types in the context of infection.  

5.2273           The Impact of the CD9 Tetraspanin on Lentivirus Infectivity and Exosome Secretion

Böker, K.O., Lemus-Diaz, n., Ferreira, R.r., Schiller, L., Schneider, S. and Gruber, J. Molecular Therapy, 26(2), 634-647 (2018)   Efficient transduction tools are a hallmark for both research and therapy development. Here, we introduce new insights into the generation of lentiviral vectors with improved performance by utilizing producer cells with increased production rates of extracellular vesicles through CD9 overexpression. Most human cells secrete small vesicles from their surface (microvesicles) or intraluminal endosome-derived membranes (exosomes). In particular, enhanced levels of the tetraspanin CD9 result in significantly increased numbers of extracellular vesicles with exosome-like features that were secreted from four different human cell lines. Intriguingly, exosomes and their biogenesis route display similarities to lentivirus and we examined the impact of CD9 expression on release and infectivity of recombinant lentiviral vectors. Although the titers of released viral particles were not increased upon production in high CD9 cells, we observed improved performance in terms of both speed and efficiency of lentiviral gene delivery into numerous human cell lines, including HEK293, HeLa, SH-SY5Y, as well as B and T lymphocytes. Here, we demonstrate that enhanced CD9 enables lentiviral transduction in the absence of any pseudotyping viral glycoprotein or fusogenic molecule. Our findings indicate an important role of CD9 for lentiviral vector and exosome biogenesis and point out a remarkable function of this tetraspanin in membrane fusion, viral infectivity, and exosome-mediated horizontal information transfer.  

5.2274           Nephron segment-specific gene expression using AAV vectors

Asico, L.D., Cuevas, S., Ma, X., Jose, P.A., Armando, I. and Konkalmatt, P.R. Biochem. Biophys. Res. Comm., 497, 19-24 (2018)   AAV9 vector provides efficient gene transfer in all segments of the renal nephron, with minimum expression in non-renal cells, when administered retrogradely via the ureter. It is important to restrict the transgene expression to the desired cell type within the kidney, so that the physiological endpoints represent the function of the transgene expressed in that specific cell type within kidney. We hypothesized that segment-specific gene expression within the kidney can be accomplished using the highly efficient AAV9 vectors carrying the promoters of genes that are expressed exclusively in the desired segment of the nephron in combination with administration by retrograde infusion into the kidney via the ureter. We constructed AAV vectors carrying eGFP under the control of: kidney-specific cadherin (KSPC) gene promoter for expression in the entire nephron; Na⁺/glucose co-transporter (SGLT2) gene promoter for expression in the S1 and S2 segments of the proximal tubule; sodium, potassium, 2 chloride co-transporter (NKCC2) gene promoter for expression in the thick ascending limb of Henle's loop (TALH); E-cadherin (ECAD) gene promoter for expression in the collecting duct (CD); and cytomegalovirus (CMV) early promoter that provides expression in most of the mammalian cells, as control. We tested the specificity of the promoter constructs in vitro for cell type-specific expression in mouse kidney cells in primary culture, followed by retrograde infusion of the AAV vectors via the ureter in the mouse. Our data show that AAV9 vector, in combination with the segment-specific promoters administered by retrograde infusion via the ureter, provides renal nephron segment-specific gene expression.  

5.2275           Allele-specific silencing therapy for Dynamin 2-related dominant centronuclear myopathy

Trochet, D., Prudhon, B., Beuvin, M., Peccate, C., Lorain, S., Julien, L., Benkhelifa-Ziyyat, S., Rabai, A., Mamchaoui, K., Ferry, A., Laporte, J., Guicheney, P., Vassilopoulos, S. and Bitoun, M. EMBO Mol. Med., 10(2), 239-253 (2018)   Rapid advances in allele-specific silencing by RNA interference established a strategy of choice to cure dominant inherited diseases by targeting mutant alleles. We used this strategy for autosomal-dominant centronuclear myopathy (CNM), a rare neuromuscular disorder without available treatment due to heterozygous mutations in the DNM2 gene encoding Dynamin 2. Allele-specific siRNA sequences were developed in order to specifically knock down the human and murine DNM2-mRNA harbouring the p.R465W mutation without affecting the wild-type allele. Functional restoration was achieved in muscle from a knock-in mouse model and in patient-derived fibroblasts, both expressing the most frequently encountered mutation in patients. Restoring either muscle force in a CNM mouse model or DNM2 function in patient-derived cells is an essential breakthrough towards future gene-based therapy for dominant centronuclear myopathy.  

5.2276           Hepatitis E virus replication and interferon responses in human placental cells

Knegendorf, L. et al Hepatol. Comm., 2(2), 173-187 (2018)   Hepatitis E virus (HEV) is a member of the genus Orthohepevirus in the family Hepeviridae and the causative agent of hepatitis E in humans. HEV is a major health problem in developing countries, causing mortality rates up to 25% in pregnant women. However, these cases are mainly reported for HEV genotype (gt)1, while gt3 infections are usually associated with subclinical courses of disease. The pathogenic mechanisms of adverse maternal and fetal outcome during pregnancy in HEV-infected pregnant women remain elusive. In this study, we observed that HEV is capable of completing the full viral life cycle in placental-derived cells (JEG-3). Following transfection of JEG-3 cells, HEV replication of both HEV gts could be observed. Furthermore, determination of extracellular and intracellular viral capsid levels, infectivity, and biophysical properties revealed production of HEV infectious particles with similar characteristics as in liver-derived cells. Viral entry was analyzed by infection of target cells and detection of either viral RNA or staining for viral capsid protein by immunofluorescence. HEV gt1 and gt3 were efficiently inhibited by ribavirin in placental as well as in human hepatoma cells. In contrast, interferon-α sensitivity was lower in the placental cells compared to liver cells for gt1 but not gt3 HEV. Simultaneous determination of interferon-stimulated gene expression levels demonstrated an efficient HEV-dependent restriction in JEG-3. Conclusion: We showed differential tissue-specific host responses to HEV genotypes, adding to our understanding of the mechanisms contributing to fatal outcomes of HEV infections during pregnancy. Using this cell-culture system, new therapeutic options for HEV during pregnancy can be identified and evaluated.    

5.2277           Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery

Giannelli, S.G., Luoni, M., Castoldi, V., Massimino, L., Cabassi, T., Angeloni,. D., Demontis, G.C., Leocani, L., Andreazzoli, m. and Broccoli, V. Hum. Mol. Genet., 27(5), 761-779 (2018)   P23H is the most common mutation in the RHODOPSIN (RHO) gene leading to a dominant form of retinitis pigmentosa (RP), a rod photoreceptor degeneration that invariably causes vision loss. Specific disruption of the disease P23H RHO mutant while preserving the wild-type (WT) functional allele would be an invaluable therapy for this disease. However, various technologies tested in the past failed to achieve effective changes and consequently therapeutic benefits. We validated a CRISPR/Cas9 strategy to specifically inactivate the P23H RHO mutant, while preserving the WT allele in vitro. We, then, translated this approach in vivo by delivering the CRISPR/Cas9 components in murine Rho^+/P23H mutant retinae. Targeted retinae presented a high rate of cleavage in the P23H but not WT Rho allele. This gene manipulation was sufficient to slow photoreceptor degeneration and improve retinal functions. To improve the translational potential of our approach, we tested intravitreal delivery of this system by means of adeno-associated viruses (AAVs). To this purpose, the employment of the AAV9-PHP.B resulted the most effective in disrupting the P23H Rho mutant. Finally, this approach was translated successfully in human cells engineered with the homozygous P23H RHO gene mutation. Overall, this is a significant proof-of-concept that gene allele specific targeting by CRISPR/Cas9 technology is specific and efficient and represents an unprecedented tool for treating RP and more broadly dominant genetic human disorders affecting the eye, as well as other tissues.  

5.2278           miR-25 Tough Decoy Enhances Cardiac Function in Heart Failure

Jeong, D., Yoo, J., Lee, p., kepreotis, S.V., Lee, A., Wahlquist, C., Brown, B.D., Kho, C., Mercola, M. and Hajjar, R.J. Molecular Therapy, 26(3), 718-729 (2018)   MicroRNAs are promising therapeutic targets, because their inhibition has the potential to normalize gene expression in diseased states. Recently, our group found that miR-25 is a key SERCA2a regulating microRNA, and we showed that multiple injections of antagomirs against miR-25 enhance cardiac contractility and function through SERCA2a restoration in a murine heart failure model. However, for clinical application, a more stable suppressor of miR-25 would be desirable. Tough Decoy (TuD) inhibitors are emerging as a highly effective method for microRNA inhibition due to their resistance to endonucleolytic degradation, high miRNA binding affinity, and efficient delivery. We generated a miR-25 TuD inhibitor and subcloned it into a cardiotropic AAV9 vector to evaluate its efficacy. The AAV9 TuD showed selective inhibition of miR-25 in vitro cardiomyoblast culture. In vivo, AAV9-miR-25 TuD delivered to the murine pressure-overload heart failure model selectively decreased expression of miR-25, increased levels of SERCA2a protein, and ameliorated cardiac dysfunction and fibrosis. Our data indicate that miR-25 TuD is an effective long-term suppressor of miR-25 and a promising therapeutic candidate to treat heart failure.  

5.2279           Non-invasive detection of adeno-associated viral gene transfer using a genetically encoded CEST-MRI reporter gene in the murine heart

Meier, S., Gilad, A.A., Brandon, J.A., Qian, C., Gao, E., Abisambra, J.F. and Vandsburger, M. Scientific Reports, 8:4638 (2018)   Research into gene therapy for heart failure has gained renewed interest as a result of improved safety and availability of adeno-associated viral vectors (AAV). While magnetic resonance imaging (MRI) is standard for functional assessment of gene therapy outcomes, quantitation of gene transfer/expression relies upon tissue biopsy, fluorescence or nuclear imaging. Imaging of gene expression through the use of genetically encoded chemical exchange saturation transfer (CEST)-MRI reporter genes could be combined with clinical cardiac MRI methods to comprehensively probe therapeutic gene expression and subsequent outcomes. The CEST-MRI reporter gene Lysine Rich Protein (LRP) was cloned into an AAV9 vector and either administered systemically via tail vein injection or directly injected into the left ventricular free wall of mice. Longitudinal in vivo CEST-MRI performed at days 15 and 45 after direct injection or at 1, 60 and 90 days after systemic injection revealed robust CEST contrast in myocardium that was later confirmed to express LRP by immunostaining. Ventricular structure and function were not impacted by expression of LRP in either study arm. The ability to quantify and link therapeutic gene expression to functional outcomes can provide rich data for further development of gene therapy for heart failure.  

5.2280           Pharmacology of Recombinant Adeno-associated Virus Production

Penaud-Budloo, M., Francois, A., Clement, N. and Ayuso, E. Molecular Therapy – Methods & Clin. Development, 8, 166-180 (2018)   Recombinant adeno-associated viral (rAAV) vectors have been used in more than 150 clinical trials with a good safety profile and significant clinical benefit in many genetic diseases. In addition, due to their ability to infect non-dividing and dividing cells and to serve as efficient substrate for homologous recombination, rAAVs are being used as a tool for gene-editing approaches. However, manufacturing of these vectors at high quantities and fulfilling current good manufacturing practices (GMP) is still a challenge, and several technological platforms are competing for this niche. Herein, we will describe the most commonly used upstream methods to produce rAAVs, paying particular attention to the starting materials (input) used in each platform and which related impurities can be expected in final products (output). The most commonly found impurities in rAAV stocks include defective particles (i.e., AAV capsids that do contain the therapeutic gene or are not infectious), residual proteins from host cells and helper viruses (adenovirus, herpes simplex virus, or baculoviruses), and illegitimate DNA from plasmids, cells, or helper viruses that may be encapsidated into rAAV particles. Given the role that impurities may play in immunotoxicity, this article reviews the impurities inherently associated with each manufacturing platform.  

5.2281           An Adeno-Associated Virus-Based Toolkit for Preferential Targeting and Manipulating Quiescent Neural Stem Cells in the Adult Hippocampus

Crowther, A.J., Lim, S-A., Asrican, B., Albright, B.H., Wooten, J., Yeh, C-Y., Bao, H., Cerri, D.H., Hu, J., Shih, Y-Y.I, Asokan, A. and Song, J. Stem Cell Reports, 10, 1146-1159 (2018)   Quiescent neural stem cells (qNSCs) with radial morphology are the only proven source of new neurons in the adult mammalian brain. Our understanding of the roles of newly generated neurons depends on the ability to target and manipulate adult qNSCs. Although various strategies have been developed to target and manipulate adult hippocampal qNSCs, they often suffer from prolonged breeding, low recombination efficiency, and non-specific labeling. Therefore, developing a readily manufactured viral vector that allows flexible packaging and robust expression of various transgenes in qNSCs is a pressing need. Here, we report a recombinant adeno-associated virus serotype 4 (rAAV4)-based toolkit that preferentially targets hippocampal qNSCs and allows for lineage tracing, functional analyses, and activity manipulation of adult qNSCs. Importantly, targeting qNSCs in a non-Cre-dependent fashion opens the possibility for studying qNSCs in less genetically tractable animal species and may have translational impact in gene therapy by preferentially targeting qNSCs.  

5.2282           Dopamine Secretion Is Mediated by Sparse Active Zone-like Release Sites

Liu, C., Kershberg, L., Wang, J., Schneeberger, S. and Kaeser, P.S. Cell, 172, 706-718 (2018)   Dopamine controls essential brain functions through volume transmission. Different from fast synaptic transmission, where neurotransmitter release and receptor activation are tightly coupled by an active zone, dopamine transmission is widespread and may not necessitate these organized release sites. Here, we determine whether striatal dopamine secretion employs specialized machinery for release. Using super resolution microscopy, we identified co-clustering of the active zone scaffolding proteins bassoon, RIM and ELKS in ∼30% of dopamine varicosities. Conditional RIM knockout disrupted this scaffold and, unexpectedly, abolished dopamine release, while ELKS knockout had no effect. Optogenetic experiments revealed that dopamine release was fast and had a high release probability, indicating the presence of protein scaffolds for coupling Ca²⁺ influx to vesicle fusion. Hence, dopamine secretion is mediated by sparse, mechanistically specialized active zone-like release sites. This architecture supports spatially and temporally precise coding for dopamine and provides molecular machinery for regulation.  

5.2283           Therapeutic Efficacy of Vectored PGT121 Gene Delivery in HIV-1-Infected Humanized Mice

Badamchi-Zadeh, A., Tartaglia, L.J., Abbink, P., Bricault, C.A., Liu, P-T., Boyd, M., Kirilova, M., Mercado, N.B., Nanayakkara, O.S., Vrbanac, V.B., Tager, A.M., Larocca, R.A., Seaman, M.S. and Barouch, D.H.

1. Virol., 92(7), e02213-17 (2018)

  Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies. However, administration of purified bNAbs poses challenges in resource-poor settings, where the HIV-1 disease burden is greatest. In vivo vector-based production of bNAbs represents an alternative strategy. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121 in wild-type and immunocompromised C57BL/6 mice as well as in HIV-1-infected bone marrow-liver-thymus (BLT) humanized mice. Ad5.PGT121 and AAV1.PGT121 produced functional antibody in vivo. Ad5.PGT121 produced PGT121 rapidly within 6 h, whereas AAV1.PGT121 produced detectable PGT121 in serum by 72 h. Serum PGT121 levels were rapidly reduced by the generation of anti-PGT121 antibodies in immunocompetent mice but were durably maintained in immunocompromised mice. In HIV-1-infected BLT humanized mice, Ad5.PGT121 resulted in a greater reduction of viral loads than did AAV1.PGT121. Ad5.PGT121 also led to more-sustained virologic control than purified PGT121 IgG. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice. Further evaluation of vector delivery of HIV-1 bNAbs is warranted, although approaches to prevent the generation of antiantibody responses may also be required.  

5.2284           An Alternate Route for Adeno-associated Virus (AAV) Entry Independent of AAV Receptor

Dudek, A.M., Pillay, S., Puschnik, A.S., Nagamine, C.M., Cheng, F., Qiu, J., Carette, J.E. and Vandenberghe, L.H.

1. Virol., 92(7), e02213-17 (2018)

  Determinants and mechanisms of cell attachment and entry steer adeno-associated virus (AAV) in its utility as a gene therapy vector. Thus far, a systematic assessment of how diverse AAV serotypes engage their proteinaceous receptor AAVR (KIAA0319L) to establish transduction has been lacking, despite potential implications for cell and tissue tropism. Here, a large set of human and simian AAVs as well as in silico-reconstructed ancestral AAV capsids were interrogated for AAVR usage. We identified a distinct AAV capsid lineage comprised of AAV4 and AAVrh32.33 that can bind and transduce cells in the absence of AAVR, independent of the multiplicity of infection. Virus overlay assays and rescue experiments in nonpermissive cells demonstrate that these AAVs are unable to bind to or use the AAVR protein for entry. Further evidence for a distinct entry pathway was observed in vivo, as AAVR knockout mice were equally as permissive to transduction by AAVrh32.33 as wild-type mice upon systemic injection. We interestingly observe that some AAV capsids undergo a low level of transduction in the absence of AAVR, both in vitro and in vivo, suggesting that some capsids may have a multimodal entry pathway. In aggregate, our results demonstrate that AAVR usage is conserved among all primate AAVs except for those of the AAV4 lineage, and a non-AAVR pathway may be available to other serotypes. This work furthers our understanding of the entry of AAV, a vector system of broad utility in gene therapy.  

5.2285           An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation

Ingmarsdotter, C.K., Zeng, J., Long, Z., Lever, A.M.L. and Kenyon, J.C. Retrovirol., 15:25 (2018)   Background NSC260594, a quinolinium derivative from the NCI diversity set II compound library, was previously identified in a target-based assay as an inhibitor of the interaction between the HIV-1 (ψ) stem-loop 3 (SL3) RNA and Gag. This compound was shown to exhibit potent antiviral activity. Here, the effects of this compound on individual stages of the viral lifecycle were examined by qRT-PCR, ELISA and Western blot, to see if its actions were specific to the viral packaging stage. The structural effects of NSC260594 binding to the HIV-1 gRNA were also examined by SHAPE and dimerization assays. Results Treatment of cells with NSC260594 did not reduce the number of integration events of incoming virus, and treatment of virus producing cells did not affect the level of intracellular Gag protein or viral particle release as determined by immunoblot. However, NSC260594 reduced the incorporation of gRNA into virions by up to 82%, without affecting levels of gRNA inside the cell. This reduction in packaging correlated closely with the reduction in infectivity of the released viral particles. To establish the structural effects of NSC260594 on the HIV-1 gRNA, we performed SHAPE analyses to pinpoint RNA structural changes. NSC260594 had a stabilizing effect on the wild type RNA that was not confined to SL3, but that was propagated across the structure. A packaging mutant lacking SL3 did not show this effect. Conclusions NSC260594 acts as a specific inhibitor of HIV-1 RNA packaging. No other viral functions are affected. Its action involves preventing the interaction of Gag with SL3 by stabilizing this small RNA stem-loop which then leads to stabilization of the global packaging signal region (psi or ψ). This confirms data, previously only shown in analyses of isolated SL3 oligonucleotides, that SL3 is structurally labile in the presence of Gag and that this is critical for the complete psi region to be able to adopt different conformations. Since replication is otherwise unaffected by NSC260594 the flexibility of SL3 appears to be a unique requirement for genome encapsidation and identifies this process as a highly specific drug target. This study is proof of principle that development of a new class of antiretroviral drugs that specifically target viral packaging by binding to the viral genomic RNA is achievable.  

5.2286           The Lipase Activity of Phospholipase D2 is Responsible for Nigral Neurodegeneration in a Rat Model of Parkinson’s Disease

Mendez-Gomez, H.R., Singh, J., Myers, C., Chen, W., Gorbatyuk, O.S. and Muzyczka, N. Neuroscience, 377, 174-183 (2018)   Phospholipase D2 (PLD2), an enzyme involved in vesicle trafficking and membrane signaling, interacts with α-synuclein, a protein known to contribute in the development of Parkinson disease (PD). We previously reported that PLD2 overexpression in rat substantia nigra pars compacta (SNc) causes a rapid neurodegeneration of dopamine neurons, and that α-synuclein suppresses PLD2-induced nigral degeneration (Gorbatyuk et al., 2010). Here, we report that PLD2 toxicity is due to its lipase activity. Overexpression of a catalytically inactive mutant (K758R) of PLD2 prevents the loss of dopaminergic neurons in the SNc and does not show signs of toxicity after 10 weeks of overexpression. Further, mutant K758R does not affect dopamine levels in the striatum. In contrast, mutants that prevent PLD2 interaction with dynamin or growth factor receptor bound protein 2 (Grb2) but retained lipase activity, continued to show rapid neurodegeneration. These findings suggest that neither the interaction of PLD2 with dynamin, which has a role in vesicle trafficking, nor the PLD2 interaction with Grb2, which has multiple roles in cell cycle control, chemotaxis and activation of tyrosine kinase complexes, are the primary cause of neurodegeneration. Instead, the synthesis of phosphatidic acid (the product of PLD2), which is a second messenger in multiple cellular pathways, appears to be the key to PLD2 induced neurodegeneration. The fact that α-synuclein is a regulator of PLD2 activity suggests that regulation of PLD2 activity could be important in the progression of PD.  

5.2287           AAV6 K531 serves a dual function in selective receptor and antibody ADK6 recognition

Bennett, A.D., Wong, K., Lewis, J., Tseng, Y-S., Smith, J.K., Chipman, P., Mckenna, R., Samulski, R.J., Kleinschjmidt, J. and Agbandje-McKenna, M. Virology, 518, 369-376 (2018)   Adeno-associated viruses (AAVs) are being developed as vectors for the treatment of genetic disorders. However, pre-existing antibodies present a significant limitation to achieving optimal efficacy for the AAV gene delivery system. Efforts aimed at engineering vectors with the ability to evade the immune response include identification of residues on the virus capsid important for these interactions and changing them. Here K531 is identified as the determinant of monoclonal antibody ADK6 recognition by AAV6, and not the closely related AAV1. The AAV6-ADK6 complex structure was determined by cryo-electron microscopy and the footprint confirmed by cell-based assays. The ADK6 footprint overlaps previously identified AAV antigenic regions and neutralizes by blocking essential cell surface glycan attachment sites. This study thus expands the available repertoire of AAV-antibody information that can guide the design of host immune escaping AAV vectors able to maintain capsid functionality.  

5.2288           AAV-9 mediated phosphatase-1 inhibitor-1 overexpression improves cardiac contractility in unchallenged mice but is deleterious in pressure-overload

Schwab, D.M., Tileman, L., Bauer, R., Heckmann, M., Jungmann, A., Wagner, M., Burgis, J., Vettel, C., Katus, H.A., El-Armouche, A. and Müller, O.J. Gene Therapy, 25, 13-19 (2018)   The downregulation of β-adrenergic receptors (β-AR) and decreased cAMP-dependent protein kinase activity in failing hearts results in decreased phosphorylation and inactivation of phosphatase-inhibitor-1 (I-1), a distal amplifier element of β-adrenergic signaling, leading to increased protein phosphatase 1 activity and dephosphorylation of key phosphoproteins, including phospholamban. Downregulated and hypophosphorylated I-1 likely contributes to β-AR desensitization; therefore its modulation is a promising approach in heart failure treatment. Aim of our study was to assess the effects of adeno-associated virus serotype 9 (AAV9) - mediated cardiac-specific expression of constitutively active inhibitor-1 (I-1c) and to investigate whether I-1c is able to attenuate the development of heart failure in mice subjected to transverse aortic constriction (TAC). 6-8 week old C57BL/6 N wild-type mice were subjected to banding of the transverse aorta (TAC). Two days later 2.8 × 1012 AAV-9 vector particles harbouring I-1c cDNA under transcriptional control of a human troponin T-promoter (AAV9/I-1c) were intravenously injected into the tail vein of these mice (n=12). AAV9 containing a Renilla luciferase reporter (AAV9/hRluc) was used as a control vector (n=12). Echocardiographic analyses were performed weekly to evaluate cardiac morphology and function. 4 weeks after TAC pressure- volume measurements were performed and animals were sacrificed for histological and molecular analyses. Both groups exhibited progressive contractile dysfunction and myocardial remodeling. Surprisingly, echocardiographic assessment and histological analyses showed significantly increased left ventricular hypertrophy in AAV9/I-1c treated mice compared to AAV9/hRluc treated controls as well as reduced contractility. Pressure-volume loops revealed significantly impaired contractility after AAV9/I-1c treatment. At the molecular level, hearts of AAV9/I-1c treated TAC mice showed a hyperphosphorylation of the SR Ca2+-ATPase inhibitor phospholamban. In contrast, expression of AAV9/I-1c in unchallenged animals resulted in selective enhancement of phospholamban phosphorylation and augmented cardiac contractility. Our data suggest that AAV9-mediated cardiac-specific overexpression of I-1c, previously associated with enhanced calcium cycling, improves cardiac contractile function in unchallenged animals but failed to protect against cardiac remodeling induced by hemodynamic stress questioning the use of I-1c as a potential strategy to treat heart failure in conditions with increased afterload.  

5.2289           A Workflow for In Vivo Evaluation of Candidate Inputs and Outputs for Cell Classifier Gene Circuits

Dastor, M., Schreiber, J., Prochazka, L., Angelici, B., Kleinert, J., Klebba, I., Doshi, J., Shen, L. and Benenson, Y. ACS Synth. Biol., 7, 474-489 (2018)   Cell classifier gene circuits that integrate multiple molecular inputs to restrict the expression of therapeutic outputs to cancer cells have the potential to result in efficacious and safe cancer therapies. Preclinical translation of the hitherto developments requires creating the conditions where the animal model, the delivery platform, in vivo expression levels of the inputs, and the efficacy of the output, all come together to enable detailed evaluation of the fully assembled circuits. Here we show an integrated workflow that addresses these issues and builds the framework for preclinical classifier studies using the design framework of microRNA (miRNA, miR)-based classifier gene circuits. Specifically, we employ HCT-116 colorectal cancer cell xenograft in an experimental mouse metastatic liver tumor model together with Adeno-associated virus (AAV) vector delivery platform. Novel engineered AAV-based constructs are used to validate in vivo the candidate inputs miR-122 and miR-7 and, separately, the cytotoxic output HSV-TK/ganciclovir. We show that while the data are largely consistent with expectations, crucial insights are gained that could not have been obtained in vitro. The results highlight the importance of detailed stepwise interrogation of the experimental parameters as a necessary step toward clinical translation of synthetic gene circuits.  

5.2290           Systemic IGF-1 gene delivery by rAAV9 improves spontaneous autoimmune peripheral polyneuropathy (SAPP)

Gao, T., Bogdanova, N., Ghauri, S., Zhang, G., Lin, J. and Sheikh, K. Scientific Reports, 8:5408 (2018)   Spontaneous autoimmune peripheral polyneuropathy (SAPP) is a mouse model of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) in non-obese diabetic (NOD) mice null for costimulatory molecule, B7-2 gene (B7-2−/−). SAPP is a chronic progressive and multifocal inflammatory and demyelinating polyneuropathy of spontaneous onset with secondary axonal degeneration. Insulin-like growth factor 1(IGF-1) is a pleiotropic factor with neuroprotective, regenerative, and anti-inflammatory effects with extensive experience in its preclinical and clinical use. Systemic delivery of recombinant adeno-associated virus serotype 9 (rAAV9) provides robust and widespread gene transfer to central and peripheral nervous systems making it suitable for gene delivery in neurological diseases. A significant proportion of patients with inflammatory neuropathies like CIDP do not respond to current clinical therapies and there is a need for new treatments. In this study, we examined the efficacy IGF-1 gene therapy by systemic delivery with rAAV9 in SAPP model. The rAAV9 construct also contained a reporter gene to monitor the surrogate expression of IGF-1. We found significant improvement in neuropathic disease after systemic delivery of rAAV9/IGF-1 gene at presymptomatic and symptomatic stages of SAPP model. These findings support that IGF-1 treatment (including gene therapy) is a viable therapeutic option in immune neuropathies such as CIDP.  

5.2291           Hepatitis E virus lifecycle and identification of 3 forms of the ORF2 capsid protein

Montpellier, C. et al

1. Hepatol., 68, Suppl. 1, abstract SAT-386 (2018)

  Background and Aims: Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. Approximately 2 billion people live in areas endemic for HEV and are at risk of infection. The HEV genome encodes 3 proteins, including the ORF2 capsid protein. Detailed analyses of the HEV lifecycle has been hampered by the lack of an efficient viral culture system. Methods:We performed studies with gt3 HEV cell culture-produced particles (HEVcc) and patient blood and stool samples. Samples were fractionated on iodixanol gradients and cushions. Infectivity assays were performed in vitro and in human liver chimeric mice. Proteins were analyzed by biochemical and proteomic approaches. Infectious particles were analyzed by transmission electron microscopy. HEV antigen levels were measured with the Wantai ELISA. Results: We developed an efficient cell culture system and isolated HEV particles that were infectious in vitro and in vivo. Using transmission electron microscopy, we defined the ultrastructure of HEVcc and particles from patient sera and stool samples. We also identified the precise sequence of the infectious particle-associated ORF2 capsid protein. In cultured cells and in samples from patients, HEV produced 3 forms of the ORF2 capsid protein: infectious/intracellular ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c). The ORF2i protein associated with infectious particles, whereas the ORF2g and ORF2c proteins were massively secreted glycoproteins not associated with infectious particles. ORF2g and ORF2c were the most abundant antigens detected in sera from patients. Conclusions: We developed a cell culture system and characterized HEV particles; we identified 3 ORF2 capsid proteins (ORF2i, ORF2g, and ORFc). These findings will advance our understanding of the HEV lifecycle and improve diagnosis.  

5.2292           Interaction between Toll-Like Receptor 9-CpG Oligodeoxynucleotides and Hepatitis B Virus Virions Leads to Entry Inhibition in Hepatocytes and Reduction of Alpha Interferon Production by Plasmacytoid Dendritic Cells

Aillot, L., Bonnin, M., Ait-Goughoulte, M., Bendriss-Vermare, N., Maadadi, S., Dimier, L., Subic, M., Scholtes, C., Najera, I., Zoulin, F., Lucifora, J. and Durantel, D. Antimicrob. Agents Chemother., 62(4), e01741-17 (2018)   We previously reported that Toll-like receptor 9 (TLR9)-CpG oligonucleotides could inhibit the establishment of hepatitis B virus (HBV) infections in hepatocytes. Our aim was to uncover the underlying mechanisms of this inhibition. HepaRG cells, RPMI-B lymphoblastoma cells, and primary plasmacytoid dendritic cells (pDCs) exposed to HBV and TLR9 ligands/agonists in various configurations were used. We observed an inhibition of HBV infection upon TLR9 stimulations only when agonist was applied during inoculation. This inhibition was independent of interleukin-6 (IL-6)/interferon-inducible protein 10 (IP-10) production as well as of TLR9 expression in hepatocytes. We further demonstrated an entry inhibition mechanism by showing a noncovalent binding of TLR9 agonist to HBV particles. Besides inhibiting HBV entry into hepatocytes, this biophysical interaction between HBV virions and TLR9 agonist was responsible for a reduction of alpha interferon (IFN-α) expression by pDCs. Interestingly, subviral particles composed of only HBsAg were able to genuinely inhibit the TLR9 pathway, without titrating TLR9 ligands. To conclude, our data suggest that synthetic TLR9-CpG oligonucleotides can strongly inhibit HBV entry by “coating” HBV virions and thereby preventing their interaction with cellular receptor. This titration effect of TLR9 agonist is also artifactually responsible for the inhibition of TLR9 engagement in pDCs, whereas a genuine inhibition of this innate pathway was confirmed with HBsAg subviral particles.  

5.2293           Superinfection Exclusion between Two High-Risk Human Papillomavirus Types during a Coinfection

Biryukov, J. and Meyers, C.

1. Virol., 92(8), e01993-17 (2018)

  Superinfection exclusion is a common phenomenon whereby a single cell is unable to be infected by two types of the same pathogen. Superinfection exclusion has been described for various viruses, including vaccinia virus, measles virus, hepatitis C virus, influenza A virus, and human immunodeficiency virus. Additionally, the mechanism of exclusion has been observed at various steps of the viral life cycle, including attachment, entry, viral genomic replication, transcription, and exocytosis. Human papillomavirus (HPV) is the causative agent of cervical cancer. Recent epidemiological studies indicate that up to 50% women who are HPV positive (HPV⁺) are infected with more than one HPV type. However, no mechanism of superinfection exclusion has ever been identified for HPV. Here, we show that superinfection exclusion exists during a HPV coinfection and that it occurs on the cell surface during the attachment/entry phase of the viral life cycle. Additionally, we are able to show that the minor capsid protein L2 plays a role in this exclusion. This study shows, for the first time, that superinfection exclusion occurs during HPV coinfections and describes a potential molecular mechanism through which it occurs.  

5.2294           Gram scale preparation of clozapine N-oxide (CNO), a synthetic small molecule actuator for muscarinic acetylcholine DREADDs

Van der Peet, P.L., Gunawan, C., Abdul-Ridha, A., Ma, S., Scott, D.J., Gundlach, A.L., Bathgate, R.A.D., White, J.M. and Williams, S.J. MethodsX, 5, 257-267 (2018)   Chemogenetics uses engineered proteins that are controlled by small molecule actuators, allowing in vivo functional studies of proteins with temporal and dose control, and include Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). One major class of DREADDs are mutated muscarinic receptors that are unresponsive to acetylcholine, and are activated by administration of clozapine N-oxide (CNO). However, CNO is available in only small amounts and large scale studies involving animals and multiple cohorts are prohibitively expensive for many investigators. The precursor, clozapine, is also expensive when purchased from specialist suppliers. Here we report:

A simple extraction method of clozapine from commercial tablets;

A simple preparation of CNO from clozapine, and for the first time its single-crystal X-ray structure; and

That the CNO prepared by this method specifically activates the DREADD receptor hM3Dq in vivo. This method provides large quantities of CNO suitable for large-scale DREADD applications that is identical to commercial material.  

5.2295           Evaluation of a method to measure HHV-6B infection in vitro based on cell size

Becerra-Artiles, A., Santoro, T. and Stern, L.J. Virology J., 15:4 (2018)   Background Human herpesvirus 6 (HHV-6A and HHV-6B) infection of cell cultures can be measured by different methods, including immunofluorescence microscopy, flow cytometry, or quantification of virus DNA by qPCR. These methods are reliable and sensitive but require long processing times and can be costly. Another method used in the field relies on the identification of enlarged cells in the culture; this method requires little sample processing and is relatively fast. However, visual inspection of cell cultures can be subjective and it can be difficult to establish clear criteria to decide if a cell is enlarged. To overcome these issues, we explored a method to monitor HHV-6B infections based on the systematic and objective measurement of the size of cells using an imaging-based automated cell counter. Results The size of cells in non-infected and HHV-6B-infected cultures was measured at different times post-infection. The relatively narrow size distribution observed for non-infected cultures contrasted with the broader distributions observed in infected cultures. The average size of cultures shifted towards higher values after infection, and the differences were significant for cultures infected with relatively high doses of virus and/or screened at longer times post-infection. Correlation analysis showed that the trend observed for average size was similar to the trend observed for two other methods to measure infection: amount of virus DNA in supernatant and the percentage of cells expressing a viral antigen. In order to determine the performance of the size-based method in differentiating non-infected and infected cells, receiver operating characteristic (ROC) curves were used to analyze the data. Analysis using size of individual cells showed a moderate performance in detecting infected cells (area under the curve (AUC) ~ 0.80-0.87), while analysis using the average size of cells showed a very good performance in detecting infected cultures (AUC ~ 0.99). Conclusions The size-based method proved to be useful in monitoring HHV-6B infections for cultures where a substantial fraction of cells were infected and when monitored at longer times post-infection, with the advantage of being relatively fast and easy. It is a convenient method for monitoring virus production in-vitro and bulk infection of cells.  

5.2296           Single Particle Imaging of Polarized Hepatoma Organoids upon Hepatitis C Virus Infection Reveals an Ordered and Sequential Entry Process

Baktash, Y., Madhav, A., Coller, K.E. and Randall, G. Cell Host & Microbe, 23, 382-394 (2018)   Hepatitis C virus (HCV) enters hepatocytes via various entry factors, including scavenger receptor BI (SR-B1), cluster of differentiation 81 (CD81), epidermal growth factor receptor (EGFR), claudin-1 (CLDN1), and occludin (OCLN). As CLDN1 and OCLN are not readily accessible due to their tight junctional localization, HCV likely accesses them by either disrupting cellular polarity or migrating to the tight junction. In this study, we image HCV entry into a three-dimensional polarized hepatoma system and reveal that the virus sequentially engages these entry factors through actin-dependent mechanisms. HCV initially localizes with the early entry factors SR-B1, CD81, and EGFR at the basolateral membrane and then accumulates at the tight junction in an actin-dependent manner. HCV associates with CLDN1 and then OCLN at the tight junction and is internalized via clathrin-mediated endocytosis by an active process requiring EGFR. Thus, HCV uses a dynamic and multi-step process to engage and enter host cells.  

5.2297           LEAP2 Is an Endogenous Antagonist of the Ghrelin Receptor

Ge, X., Yang, H., Benarek, M.A. et al Cell Metab., 27, 461-469 (2018)   Ghrelin, an appetite-stimulatory hormone secreted by the stomach, was discovered as a ligand for the growth hormone secretagogue receptor (GHSR). Through GHSR, ghrelin stimulates growth hormone (GH) secretion, a function that evolved to protect against starvation-induced hypoglycemia. Though the biology mediated by ghrelin has been described in great detail, regulation of ghrelin action is poorly understood. Here, we report the discovery of liver-expressed antimicrobial peptide 2 (LEAP2) as an endogenous antagonist of GHSR. LEAP2 is produced in the liver and small intestine, and its secretion is suppressed by fasting. LEAP2 fully inhibits GHSR activation by ghrelin and blocks the major effects of ghrelin in vivo, including food intake, GH release, and maintenance of viable glucose levels during chronic caloric restriction. In contrast, neutralizing antibodies that block endogenous LEAP2 function enhance ghrelin action in vivo. Our findings reveal a mechanism for fine-tuning ghrelin action in response to changing environmental conditions.  

5.2298           Intramuscular Adeno-Associated Virus–Mediated Expression of Monoclonal Antibodies Provides 100% Protection Against Ebola Virus Infection in Mice

Van Lieshout, L.P., Soule, G., Sorensen, D., Frost, K.L., He, S., Tierney, K., Safronetz, D., Booth, S.A., Kobinger, G.P., Qiu, X. and Wootton, S.K.

1. Infect. Dis., 217(6), 916-925 (2018)

  The 2013–2016 West Africa outbreak demonstrated the epidemic potential of Ebola virus and highlighted the need for counter strategies. Monoclonal antibody (mAb)–based therapies hold promise as treatment options for Ebola virus infections. However, production of clinical-grade mAbs is labor intensive, and immunity is short lived. Conversely, adeno-associated virus (AAV)–mediated mAb gene transfer provides the host with a genetic blueprint to manufacture mAbs in vivo, leading to steady release of antibody over many months. Here we demonstrate that AAV-mediated expression of nonneutralizing mAb 5D2 or 7C9 confers 100% protection against mouse-adapted Ebola virus infection, while neutralizing mAb 2G4 was 83% protective. A 2-component cocktail, AAV-2G4/AAV-5D2, provided complete protection when administered 7 days prior to challenge and was partially protective with a 3-day lead time. Finally, AAV-mAb therapies provided sustained protection from challenge 5 months following AAV administration. AAV-mAb may be a viable alternative strategy for vaccination against emerging infectious diseases.  

5.2299           Metallothioneins are neuroprotective agents in lysosomal storage disorders

Cavalca, E., Cesani, M., Gifford, J.C., Sena-Esteves, M., Terreni, M.R., Leoncini, G., Peviani, M. and Biffi, A. Ann. Neurol., 83(2), 418-432 (2018)   Objective Lysosomal storage disorders (LSDs) are a broad class of inherited metabolic diseases caused by the defective activity of lysosomal enzymes. Central nervous system (CNS) manifestations are present in roughly 50% of LSD patients and represent an unmet medical need for them. We explored the therapeutic potential of metallothioneins (MTs), a newly identified family of proteins with reported neuroprotective roles, in the murine models of two LSDs with CNS involvement. Methods MT‐1 overexpressing transgenic mice (MTtg) were crossed with the murine models of Batten and Krabbe diseases. Changes in the survival and manifestations of the disease in the MTtg setting were assessed. In addition, we analyzed the therapeutic effects of MT‐1 CNS gene delivery in one of these LSD models. Results Constitutive expression of MT‐1 exerted favorable phenotypic effects in both LSD models. MT‐LSD mice showed a 5% to 10% increase in survival and slower disease progression as compared to not‐transgenic LSD mice. Rescue of Purkinje cells from degeneration and apoptosis was also observed in the MT‐LSD models. This phenotypic amelioration was accompanied by a modulation of the disease‐associated activated inflammatory microglia phenotype, and by a reduction of oxidative stress. Importantly, for the clinical translation of our findings, the very same effects were obtained when MTs were delivered to brains by systemic AAV gene transfer. Interpretation MTs can be considered novel therapeutic agents (and targets) in LSDs and potentiate the effects of approaches aiming at correction of the disease‐causing enzyme deficiency in the CNS.  

5.2300           Two viruses, MCV1 and MCV2, which infect Marinitoga bacteria isolated from deep‐sea hydrothermal vents: functional and genomic analysis

Mercier, C., Lossouarn, J., Nesbø, C.L., haverkamp, T.H.A., Baudoux, A.C., Jebbar, M., Bienvenu, N., Thiroux, S., Dupont, S. and Geslin, C. Environ. Microbiol., 20(2), 577-587 (2018)   Viruses represent a driving force in the evolution of microorganisms including those thriving in extreme environments. However, our knowledge of the viral diversity associated to microorganisms inhabiting the deep‐sea hydrothermal vents remains limited. The phylum of Thermotogae, including thermophilic bacteria, is well represented in this environment. Only one virus was described in this phylum, MPV1 carried by Marinitoga piezophila. In this study, we report on the functional and genomic characterization of two new bacterioviruses that infect bacteria from the Marinitoga genus. Marinitoga camini virus 1 and 2 (MCV1 and MCV2) are temperate siphoviruses with a linear dsDNA genome of 53.4 kb and 50.5 kb respectively. Here, we present a comparative genomic analysis of the MCV1 and MCV2 viral genomes with that of MPV1. The results indicate that even if the host strains come from geographically distant sites, their genomes share numerous similarities. Interestingly, heavy metals did not induce viral production, instead the host of MCV1 produced membrane vesicles. This study highlights interaction of mobile genetic elements (MGE) with their hosts and the importance of including hosts‐MGEs' relationships in ecological studies.  

5.2301           Single Intramuscular Injection of AAV-shRNA Reduces DNM2 and Prevents Myotubular Myopathy in Mice

Tasfaout, H., Lionello, V.M., Kretz, C., Koebel, P., Messaddeq, N., Bitz, D., Laporte, J. and Cowling, B.S. Molecular Therapy, 26(4), 1082-1092 (2018)   Myotubular myopathy, or X-linked centronuclear myopathy, is a severe muscle disorder representing a significant burden for patients and their families. It is clinically characterized by neonatal and severe muscle weakness and atrophy. Mutations in the myotubularin (MTM1) gene cause myotubular myopathy, and no specific curative treatment is available. We previously found that dynamin 2 (DNM2) is upregulated in both Mtm1 knockout and patient muscle samples, whereas its reduction through antisense oligonucleotides rescues the clinical and histopathological features of this myopathy in mice. Here, we propose a novel approach targeting Dnm2 mRNA. We screened and validated in vitro and in vivo several short hairpin RNA (shRNA) sequences that efficiently target Dnm2 mRNA. A single intramuscular injection of AAV-shDnm2 resulted in long-term reduction of DNM2 protein level and restored muscle force, mass, histology, and myofiber ultrastructure and prevented molecular defects linked to the disease. Our results demonstrate a robust DNM2 knockdown and provide an alternative strategy based on reduction of DNM2 to treat myotubular myopathy.  

5.2302           Evaluation of a method to measure HHV-6B infection in vitro based on cell size

Becerra-Artiles, A., Santoro, T. and Stern, L.J.

Virology J., 15:4 (2018)

Background

Human herpesvirus 6 (HHV-6A and HHV-6B) infection of cell cultures can be measured by different methods, including immunofluorescence microscopy, flow cytometry, or quantification of virus DNA by qPCR. These methods are reliable and sensitive but require long processing times and can be costly. Another method used in the field relies on the identification of enlarged cells in the culture; this method requires little sample processing and is relatively fast. However, visual inspection of cell cultures can be subjective and it can be difficult to establish clear criteria to decide if a cell is enlarged. To overcome these issues, we explored a method to monitor HHV-6B infections based on the systematic and objective measurement of the size of cells using an imaging-based automated cell counter.

Results

The size of cells in non-infected and HHV-6B-infected cultures was measured at different times post-infection. The relatively narrow size distribution observed for non-infected cultures contrasted with the broader distributions observed in infected cultures. The average size of cultures shifted towards higher values after infection, and the differences were significant for cultures infected with relatively high doses of virus and/or screened at longer times post-infection. Correlation analysis showed that the trend observed for average size was similar to the trend observed for two other methods to measure infection: amount of virus DNA in supernatant and the percentage of cells expressing a viral antigen. In order to determine the performance of the size-based method in differentiating non-infected and infected cells, receiver operating characteristic (ROC) curves were used to analyze the data. Analysis using size of individual cells showed a moderate performance in detecting infected cells (area under the curve (AUC) ~ 0.80-0.87), while analysis using the average size of cells showed a very good performance in detecting infected cultures (AUC ~ 0.99).

Conclusions

The size-based method proved to be useful in monitoring HHV-6B infections for cultures where a substantial fraction of cells were infected and when monitored at longer times post-infection, with the advantage of being relatively fast and easy. It is a convenient method for monitoring virus production in-vitro and bulk infection of cells.

 

5.2303           Enhanced Production of Exosome-Associated AAV by Overexpression of the Tetraspanin CD9

Schiller, L.T., Lemus-Diaz, N., Ferreira, R.R., Böker, K.O. and Gruber, J. Molecular Therapy – Methods & Clinical Development, 9, 278-287 (2018)   Research on cell-free vesicles revealed a multitude of characteristics, in particular of microvesicles and exosomes, that range from their potential as biomarkers to a function in horizontal transfer of genetic information from cell to cell and also include supportive functions in viral infection. Exosome-associated adeno-associated viruses (exo-AAVs) are of particular interest for the past couple of years, since they introduced a new source of highly potent recombinant AAVs with improved features, including accelerated transduction rates and more efficient immune escape. However, key factors like the mode of action, efficiency of production or engineering of exo-AAVs remain elusive to a large extent. Here, we used the established system of CD9 over-expression to boost the exosome output of AAV producing HEK-AAV cells. The CD9-powered high-exosome environment was established during exo-AAV1 production, and we could demonstrate that the yield of exo-AAVs dramatically increased when compared to standard exo-AAVs. Furthermore, we report that exo-AAV-CD9_GFP was more efficient in transduction of cells in the same titer ranges as standard exo-AAVs. Our results provide a technological approach for the generation of exo-AAVs with superior performance.  

5.2304           The Biological Activity of AAV Vectors for Choroideremia Gene Therapy Can Be Measured by In Vitro Prenylation of RAB6A

Patricio, M.I., Barnard, A.R., Cox, C.I., Blue, C. and Maclaren, R.E. Molecular Therapy – Methods & Clinical Development, 9, 288-295 (2018)   Choroideremia (CHM) is a rare, X-linked recessive retinal dystrophy caused by mutations in the CHM gene. CHM is ubiquitously expressed in human cells and encodes Rab escort protein 1 (REP1). REP1 plays a key role in intracellular trafficking through the prenylation of Rab GTPases, a reaction that can be reproduced in vitro. With recent advances in adeno-associated virus (AAV) gene therapy for CHM showing gene replacement to be a promising approach, an assay to assess the biological activity of the vectors is of the uttermost importance. Here we sought to compare the response of two Rab proteins, RAB27A and RAB6A, to the incorporation of a biotinylated lipid donor in a prenylation reaction in vitro. First, we found the expression of REP1 to be proportional to the amount of recombinant AAV (rAAV)2/2-REP1 used to transduce the cells. Second, prenylation of RAB6A appeared to be more sensitive to REP1 protein expression than prenylation of RAB27A. Moreover, the method was reproducible in other cell lines. These results support the further development of a prenylation reaction using a biotinylated lipid donor and RAB6A to assess the biological activity of AAV vectors for CHM gene therapy.  

5.2305           HAVCR1 (CD365) and Its Mouse Ortholog Are Functional Hepatitis A Virus (HAV) Cellular Receptors That Mediate HAV Infection

Costafreda, M.I. and Kaplan, G.

1. Virol., 92(9), e2065-17 (2018)

  The hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), classified as CD365, was initially discovered as an HAV cellular receptor using an expression cloning strategy. Due to the lack of HAV receptor-negative replication-competent cells, it was not possible to fully prove that HAVCR1 was a functional HAV receptor. However, biochemistry, classical virology, and epidemiology studies further supported the functional role of HAVCR1 as an HAV receptor. Here, we show that an anti-HAVCR1 monoclonal antibody that protected African green monkey kidney (AGMK) cells against HAV infection only partially protected monkey Vero E6 cells and human hepatoma Huh7 cells, indicating that these two cell lines express alternative yet unidentified HAV receptors. Therefore, we focused our work on AGMK cells to further characterize the function of HAVCR1 as an HAV receptor. Advances in clustered regularly interspaced short palindromic repeat/Cas9 technology allowed us to knock out the monkey ortholog of HAVCR1 in AGMK cells. The resulting AGMK HAVCR1 knockout (KO) cells lost susceptibility to HAV infection, including HAV-free viral particles (vpHAV) and exosomes purified from HAV-infected cells (exo-HAV). Transfection of HAVCR1 cDNA into AGMK HAVCR1 KO cells restored susceptibility to vpHAV and exo-HAV infection. Furthermore, transfection of the mouse ortholog of HAVCR1, mHavcr1, also restored the susceptibility of AGMK HAVCR1 KO cells to HAV infection. Taken together, our data clearly show that HAVCR1 and mHavcr1 are functional HAV receptors that mediate HAV infection. This work paves the way for the identification of alternative HAV receptors to gain a complete understanding of their interplay with HAVCR1 in the cell entry and pathogenic processes of HAV.  

5.2306           TRPV1 SUMOylation regulates nociceptive signaling in models of inflammatory pain

Wang, Y., Guo, Y., Tian, Q., Dong, Q. Et al Nature Communications, 9, 1529 (2018)   Although TRPV1 channels represent a key player of noxious heat sensation, the precise mechanisms for thermal hyperalgesia remain unknown. We report here that conditional knockout of deSUMOylation enzyme, SENP1, in mouse dorsal root ganglion (DRG) neurons exacerbated thermal hyperalgesia in both carrageenan- and Complete Freund’s adjuvant-induced inflammation models. TRPV1 is SUMOylated at a C-terminal Lys residue (K822), which specifically enhances the channel sensitivity to stimulation by heat, but not capsaicin, protons or voltage. TRPV1 SUMOylation is decreased by SENP1 but upregulated upon peripheral inflammation. More importantly, the reduced ability of TRPV1 knockout mice to develop inflammatory thermal hyperalgesia was rescued by viral infection of lumbar 3/4 DRG neurons of wild-type TRPV1, but not its SUMOylation-deficient mutant, K822R. These data suggest that TRPV1 SUMOylation is essential for the development of inflammatory thermal hyperalgesia, through a mechanism that involves sensitization of the channel response specifically to thermal stimulation.  

5.2307           Simultaneous Assessment of Clearance, Metabolism, Induction, and Drug-Drug Interaction Potential Using a Long-Term In Vitro Liver Model for a Novel Hepatitis B Virus Inhibitor

Kratochwill, N.A., Triyatni, M., Mueller, M.B., Klammers, F. et al

1. Pharmacol. Exp. Ther., 365, 237-248 (2018)

  Long-term in vitro liver models are now widely explored for human hepatic metabolic clearance prediction, enzyme phenotyping, cross-species metabolism, comparison of low clearance drugs, and induction studies. Here, we present studies using a long-term liver model, which show how metabolism and active transport, drug-drug interactions, and enzyme induction in healthy and diseased states, such as hepatitis B virus (HBV) infection, may be assessed in a single test system to enable effective data integration for physiologically based pharmacokinetic (PBPK) modeling. The approach is exemplified in the case of (3S)-4-[[(4R)-4-(2-Chloro-4-fluorophenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic acid RO6889678, a novel inhibitor of HBV with a complex absorption, distribution, metabolism, and excretion (ADME) profile. RO6889678 showed an intracellular enrichment of 78-fold in hepatocytes, with an apparent intrinsic clearance of 5.2 µl/min per mg protein and uptake and biliary clearances of 2.6 and 1.6 µl/min per mg protein, respectively. When apparent intrinsic clearance was incorporated into a PBPK model, the simulated oral human profiles were in good agreement with observed data at low doses but were underestimated at high doses due to unexpected overproportional increases in exposure with dose. In addition, the induction potential of RO6889678 on cytochrome P450 (P450) enzymes and transporters at steady state was assessed and cotreatment with ritonavir revealed a complex drug-drug interaction with concurrent P450 inhibition and moderate UDP-glucuronosyltransferase induction. Furthermore, we report on the first evaluation of in vitro pharmacokinetics studies using HBV-infected HepatoPac cocultures. Thus, long-term liver models have great potential as translational research tools exploring pharmacokinetics of novel drugs in vitro in health and disease  

5.2308           Evaluation of Intrathecal Routes of Administration for Adeno-Associated Viral Vectors in Large Animals

Hinderer, C., Bell, P., katz, N., Vite, C.H., Louboutin, J-P., Bote, E., Yu, H., Zhu, Y., Casal, M.L., Bagel, J., O’Donnell, P., Wang, p., Haskins, M.E., Goode, T. and Wilson, J.M. Human Gene Therapy, 29(1), 15-24 (2018)   Delivery of adeno-associated viral (AAV) vectors into the cerebrospinal fluid (CSF) can achieve gene transfer to cells throughout the brain and spinal cord, potentially making many neurological diseases tractable gene therapy targets. Identifying the optimal route of CSF access for intrathecal AAV delivery will be a critical step in translating this approach to clinical practice. We previously demonstrated that vector injection into the cisterna magna is a safe and effective method for intrathecal AAV delivery in nonhuman primates; however, this procedure is not commonly used in clinical practice. More routine methods of administration into the CSF are now being explored, including intracerebroventricular (ICV) injection and injection through a lumbar puncture. In this study, we compared ICV and intracisternal (IC) AAV administration in dogs. We also evaluated vector administration via lumbar puncture in nonhuman primates, with some animals placed in the Trendelenburg position after injection, a maneuver that has been suggested to improve cranial distribution of vector. In the dog study, ICV and IC vector administration resulted in similarly efficient transduction throughout the brain and spinal cord. However, animals in the ICV cohort developed encephalitis associated with a T-cell response to the transgene product, a phenomenon that was not observed in the IC cohort. In the nonhuman primate study, transduction efficiency was not improved by placing animals in the Trendelenburg position after injection. These findings illustrate important limitations of commonly used methods for CSF access in the context of AAV delivery, and will be important for informing the selection of a route of administration for first-in-human studies.  

5.2309           Development of In Vivo Imaging Tools for Investigating Astrocyte Activation in Epileptogenesis

Kostoula, C., Pascente, R., Ravizza, T., McCown, T., Schoch, S., Vezzani, A., Becker, A.J. and van Loo, K.M.J. Mol. Neurobiol., 55(5), 463-472 (2018)   Insights into the dynamic changes in molecular processes occurring in the brain during epileptogenesis can substantially improve our understanding of their pathogenetic relevance. In this context, neuroinflammation is a potential mechanism of epileptogenesis which has recently been investigated in animal models by MRI or PET molecular imaging. Here, we developed an alternative and complementary molecular imaging strategy by designing a serotype 8 recombinant adeno-associated virus (AAV8) harboring promoter fragments of the GFAP or IL-1β promoter and a luciferase reporter gene. Mice were injected intrahippocampally with rAAV8 and treated with intracortical kainic acid to induce status epilepticus (SE) and hence epileptogenesis. In vivo bioluminescence imaging combined with immunohistochemistry revealed a significant activation of the GFAP promoter 24 h and 3 days after kainate-induced SE. For IL-1β, we identified the promoter region required for studying cell-specific induction of the promoter in longitudinal studies. We conclude that the GFAP promoter fragment represents a useful tool for monitoring the in vivo activation of astrocytes with an inflammatory phenotype during epileptogenesis, or under other pathophysiological conditions.    

5.2310           Nontoxic, double-deletion-mutant rabies viral vectors for retrograde targeting of projection neurons

Chatterjee, S., Sullivan, H.A., MacLennan, B., Xu, R., Hou, Y.Y., Lavin, T. et al Nature Neurosci., 21, 638-646 (2018)   Recombinant rabies viral vectors have proven useful for applications including retrograde targeting of projection neurons and monosynaptic tracing, but their cytotoxicity has limited their use to short-term experiments. Here we introduce a new class of double-deletion-mutant rabies viral vectors that left transduced cells alive and healthy indefinitely. Deletion of the viral polymerase gene abolished cytotoxicity and reduced transgene expression to trace levels but left vectors still able to retrogradely infect projection neurons and express recombinases, allowing downstream expression of other transgene products such as fluorophores and calcium indicators. The morphology of retrogradely targeted cells appeared unperturbed at 1 year postinjection. Whole-cell patch-clamp recordings showed no physiological abnormalities at 8 weeks. Longitudinal two-photon structural and functional imaging in vivo, tracking thousands of individual neurons for up to 4 months, showed that transduced neurons did not die but retained stable visual response properties even at the longest time points imaged.  

5.2311           Effect of apoptosis‐associated speck‐like protein containing a caspase recruitment domain on vaccine efficacy: Overcoming the effects of its deficiency with aluminum hydroxide adjuvant

Lee, D-K., Lee, E-Y., Kim, R-H., Kwak, H-W., Kim, J.Y., Kim, H., kang, K-W., Lee, S-M., Park, J-H., Chang, J. and Nam, J-H. Microbiol. Immunol., 62(3), 176-186 (2018)   Host factors such as nutritional status and immune cell state are important for vaccine efficacy. Inflammasome activation may be important for triggering vaccine‐induced humoral and cell‐mediated immune responses. Formulations with alum as a typical adjuvant to overcome the effects of host factors have recently been shown to induce inflammasome activation, which augments vaccine efficacy. Apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC) is one of the main components of inflammasomes, but it is not clear whether ASC affects the vaccine‐induced immune response. Herein, we used two types of vaccines: inactivated influenza vaccine not formulated with alum, and HPV vaccine formulated with alum. We gave the vaccines to ASC knockout (ASC^−/−) mice to investigate the role of ASC in vaccine efficacy. Influenza vaccine‐immunized ASC^−/− mice did not show antibody titers in week 2 after the first vaccination. After boosting, the antibody titer in ASC^−/− mice was about half that in wild type (WT) mice. Furthermore, a cytotoxic T‐lymphocyte response against influenza vaccine was not induced in ASC^−/− mice. Therefore, vaccinated ASC^−/− mice did not show effective protection against viral challenge. ASC^−/− mice immunized with alum‐formulated HPV vaccine showed similar antibody titers and T‐cell proliferation compared with immunized WT mice. However, the HPV vaccine without alum induced up to threefold lower titers of HPV‐specific antibody titers in ASC^−/− mice compared with those in WT mice. These findings suggest that alum in vaccine can overcome the ASC‐deficient condition.  

5.2312           Restricted HIV-1 Env glycan engagement by lectin-reengineered DAVEI protein chimera is sufficient for lytic inactivation of the virus

Parajuli, B., Acharya, k., Bach, H.C., parajuli, B., Zhang, S., Smith III, A.B., Abrams, C.F. and Chaiken, I. Biochem. J., 475, 931-957 (2018)   We previously reported a first-generation recombinant DAVEI construct, a dual action virus entry inhibitor composed of cyanovirin-N (CVN) fused to a membrane proximal external region or its derivative peptide Trp3. DAVEI exhibits potent and irreversible inactivation of HIV-1 (human immunodeficiency virus) viruses by dual engagement of gp120 and gp41. However, the promiscuity of CVN to associate with multiple glycosylation sites in gp120 and its multivalency limit current understanding of the molecular arrangement of the DAVEI molecules on trimeric spike. Here, we constructed and investigated the virolytic function of second-generation DAVEI molecules using a simpler lectin, microvirin (MVN). MVN is a monovalent lectin with a single glycan-binding site in gp120, is structurally similar to CVN and exhibits no toxicity or mitogenicity, both of which are liabilities with CVN. We found that, like CVN-DAVEI-L2-3Trp (peptide sequence DKWASLWNW), MVN-DAVEI2-3Trp exploits a similar mechanism of action for inducing HIV-1 lytic inactivation, but by more selective gp120 glycan engagement. By sequence redesign, we significantly increased the potency of MVN-DAVEI2-3Trp protein. Unlike CVN-DAVEI2-3Trp, re-engineered MVN-DAVEI2-3Trp(Q81K/M83R) virolytic activity and its interaction with gp120 were both competed by 2G12 antibody. That the lectin domain in DAVEIs can utilize MVN without loss of virolytic function argues that restricted HIV-1 Env (envelope glycoprotein) glycan engagement is sufficient for virolysis. It also shows that DAVEI lectin multivalent binding with gp120 is not required for virolysis. MVN-DAVEI2-3Trp(Q81K/M83R) provides an improved tool to elucidate productive molecular arrangements of Env-DAVEI enabling virolysis and also opens the way to form DAVEI fusions made up of gp120-binding small molecules linked to Trp3 peptide.  

5.2313           A novel triple mutant AAV6 capsid induces rapid and potent transgene expression in the muscle and respiratory tract of mice

Van Lieshout, L.P., Domm, J.M., Rindler, T.N., Frost, K.L., Sorensen, D.L., Meddina, S.J., Booth, S.A., Bridges, J.P. and Wootton, S.K. Molecular Therapy – Methods & Clin. Develop., 9, 323-329 (2018)   Gene therapy for the treatment of genetic disorders has demonstrated considerable therapeutic success in clinical trials. Among the most effective and commonly used gene delivery vectors are those based on adeno-associated virus (AAV). Despite these advances in clinical gene therapy, further improvements in AAV vector properties such as rapid intracellular processing and transgene expression, targeted transduction of therapeutically relevant cell types, and longevity of transgene expression, will render extension of such successes to many other human diseases. Engineering of AAV capsids continues to evolve the specificity and efficiency of AAV-mediated gene transfer. Here, we describe a triple AAV6 mutant, termed AAV6.2FF, containing F129L, Y445F, and Y731F mutations. AAV6.2FF yielded 10-fold greater transgene expression in lung than AAV6 after 21 days. Additionally, this novel capsid demonstrated 101-fold and 49-fold increased transgene expression in the muscle and lungs, respectively, 24 hr post vector delivery when compared with the parental AAV6. Furthermore, AAV6.2FF retains heparin sulfate binding capacity and displays a 10-fold increase in resistance to pooled immunoglobulin neutralization in vitro. The rapid and potent expression mediated by AAV6.2FF is ideally suited to applications such as vectored immunoprophylaxis, in which rapid transgene expression is vital for use during an outbreak response scenario.  

5.2314           Attenuation of the Niemann-Pick type C2 disease phenotype by intracisternal administration of an AAVrh.10 vector expressing Npc2

Markmann, S., Christie-Reid, J.J., Rosenberg, J.B., De, B.P., Kaminsky, S.M., Crystal, R.G. and Sondhi, D. Exp. Neurol., 306, 25-33 (2018)   Niemann-Pick type C2 (NPC2) disease is a rare, neurodegenerative disorder caused by mutations in the NPC2 gene, leading to lysosomal accumulation of unesterified cholesterol and other lipids. It is characterized by hepatosplenomegaly, liver dysfunction and severe neurological manifestations, resulting in early death. There is no effective therapy for NPC2 disease. Here, we evaluated the effectiveness of an adeno-associated virus (AAV), serotype rh.10 gene transfer vector expressing the mouse Npc2 gene (AAVrh.10-mNpc2-HA, HA tagged to facilitate analysis) to treat the disease in an Npc2−/− mouse model. A single intracisternal administration of the AAVrh.10-mNpc2-HA to 6 week old Npc2−/− mice mediated vector DNA, transgene mRNA and protein expression in brain and other organs. Compared to untreated Npc2−/− mice, AAV-treated Npc2−/− mice demonstrated amelioration of disease pathology in the brain, reduced lysosomal storage, reduced Purkinje cell death, decreased gliosis, and improved performance in behavioral tasks. Treatment-related reduction in serum disease markers was detected early and this effect persisted. Liver and spleen pathology were improved with significant reduction of liver cholesterol and sphingomyelin levels in treated Npc2−/− mice. Finally, administration of AAVrh.10-mNpc2-HA significantly extended life-span. Taken together, these data demonstrate the benefit of a one-time intracisternal administration of AAVrh.10-mNpc2-HA as a life-long treatment for NPC2 disease.  

5.2315           Efficient Enrichment of Gene-Modified Primary T Cells via CCR5-Targeted Integration of Mutant Dihydrofolate Reductase

Paul, B., Romano Ibarra, G.S., Hubbard, N., Einhaus, T., Astrakhan, A., Rawlings, D.J., Kiem, H-P., and Peterson, C.W. Molecular Therapy – Methods Clin. Develop., 9, 347-357 (2018)   Targeted gene therapy strategies utilizing homology-driven repair (HDR) allow for greater control over transgene integration site, copy number, and expression—significant advantages over traditional vector-mediated gene therapy with random genome integration. However, the relatively low efficiency of HDR-based strategies limits their clinical application. Here, we used HDR to knock in a mutant dihydrofolate reductase (mDHFR) selection gene at the gene-edited CCR5 locus in primary human CD4⁺ T cells and selected for mDHFR-modified cells in the presence of methotrexate (MTX). Cells were transfected with CCR5-megaTAL nuclease mRNA and transduced with adeno-associated virus containing an mDHFR donor template flanked by CCR5 homology arms, leading to up to 40% targeted gene insertion. Clinically relevant concentrations of MTX led to a greater than 5-fold enrichment for mDHFR-modified cells, which maintained a diverse TCR repertoire over the course of expansion and drug selection. Our results demonstrate that mDHFR/MTX-based selection can be used to enrich for gene-modified T cells ex vivo, paving the way for analogous approaches to increase the percentage of HIV-resistant, autologous CD4⁺ T cells infused into HIV⁺ patients, and/or for in vivo selection of gene-edited T cells for the treatment of cancer.  

5.2316           Type A viral hepatitis: A summary and update on the molecular virology, epidemiology, pathogenesis and prevention

Lemon, S.M., Ott, J., Van Damme, P. and Shouval, D.

J. Hepatol., 68(1), 167-184 (2018)

Although epidemic jaundice was well known to physicians of antiquity, it is only in recent years that medical science has begun to unravel the origins of hepatitis A virus (HAV) and the unique pathobiology underlying acute hepatitis A in humans. Improvements in sanitation and the successful development of highly efficacious vaccines have markedly reduced the worldwide occurence of this enterically-transmitted infection over the past quarter century, yet the virus persists in vulnerable populations and those without HAV immunity and remains a common cause of food-borne disease outbreaks in economically-advantaged societies. Reductions in HAV incidence have led to increases in the median age at which infection occurs, often resulting in more severe disease in affected persons and paradoxical increases in disease burden in some developing nations. Here, we summarize recent advances in the molecular virology and epidemiology of HAV, an atypical member of the Picornaviridae family, survey what is known of the pathogenesis of hepatitis A in humans and the host-pathogen interactions that typify the infection. The article also reviews medical and public health aspects of HAV vaccination and disease prevention.

 

5.2317           A Novel Triple-Mutant AAV6 Capsid Induces Rapid and Potent Transgene Expression in the Muscle and Respiratory Tract of Mice

Van Lieshout, L.P., Domm, J.M., Rindler, T.N., Frost, K.L., Sorensen, D.L., Medina, S.J., Booth, S.A., Bridges, J.P. and Wooton, S.K. Molecular Therapy – Methods Clin. Develop., 9, 323-329 (2018)   Gene therapy for the treatment of genetic disorders has demonstrated considerable therapeutic success in clinical trials. Among the most effective and commonly used gene delivery vectors are those based on adeno-associated virus (AAV). Despite these advances in clinical gene therapy, further improvements in AAV vector properties such as rapid intracellular processing and transgene expression, targeted transduction of therapeutically relevant cell types, and longevity of transgene expression, will render extension of such successes to many other human diseases. Engineering of AAV capsids continues to evolve the specificity and efficiency of AAV-mediated gene transfer. Here, we describe a triple AAV6 mutant, termed AAV6.2FF, containing F129L, Y445F, and Y731F mutations. AAV6.2FF yielded 10-fold greater transgene expression in lung than AAV6 after 21 days. Additionally, this novel capsid demonstrated 101-fold and 49-fold increased transgene expression in the muscle and lungs, respectively, 24 hr post vector delivery when compared with the parental AAV6. Furthermore, AAV6.2FF retains heparin sulfate binding capacity and displays a 10-fold increase in resistance to pooled immunoglobulin neutralization in vitro. The rapid and potent expression mediated by AAV6.2FF is ideally suited to applications such as vectored immunoprophylaxis, in which rapid transgene expression is vital for use during an outbreak response scenario.  

5.2318           Liposome Lipid-Based Formulation Has the Least Influence on rAAV Transduction Compared to Other Transfection Agents

Guo, P., Yu, C., Wang, Q.,  Zhang, R., Meng, X. and Fang, Y. Molecular Therapy – Mthods Clin. Develop., 9, 367-375 (2018)   Recombinant adeno-associated virus (rAAV) vectors are considered ideal vehicles for human gene therapy. Meanwhile, non-viral strategies, such as transfection agents (TAs), have also shown promise to deliver genetic materials, such as siRNA. Transduction with the rAAV vector is performed concurrently with transfection with plasmid DNA or RNA. In the present study, we report that various TAs inhibited rAAV-mediated transgene expression at diverse levels. Overall, cationic polymers and dendrimers dramatically blocked rAAV transduction, while lipid-based liposomes displayed the least effect. The inhibitory effect was dependent on the dose of TAs and the timing of infection, suggesting that the early stages of viral infection were involved. In addition, the present results indicate that the transgene expression of rAAV vectors was significantly increased by liposome-mediated transfection with adenoviral helper genes. At the same time, this was dramatically inhibited by liposome-mediated transfection with the trichosanthin gene encoding a type I ribosome-inactivating protein isolated from traditional Chinese medicine. Furthermore, liposomes also have little effect on rAAV-mediated transgene expression in vivo. Taken together, these findings suggest liposome as the best choice of TAs, which should be used in combination with rAAV-mediated gene therapy.

5.2319           Nuclease-free Adeno-Associated Virus-Mediated Il2rg Gene Editing in X-SCID Mice

Hiramoto, T., Li, B.Li, Funk, S.E., Hirata, R.K. and Russell, D.W. Molecular Therapy, 26(5), 1255-1265 (2018)   X-linked severe combined immunodeficiency (X-SCID) has been successfully treated by hematopoietic stem cell (HSC) transduction with retroviral vectors expressing the interleukin-2 receptor subunit gamma gene (IL2RG), but several patients developed malignancies due to vector integration near cellular oncogenes. This adverse side effect could in principle be avoided by accurate IL2RG gene editing with a vector that does not contain a functional promoter or IL2RG gene. Here, we show that adeno-associated virus (AAV) gene editing vectors can insert a partial Il2rg cDNA at the endogenous Il2rg locus in X-SCID murine bone marrow cells and that these ex vivo-edited cells repopulate transplant recipients and produce CD4⁺ and CD8⁺ T cells. Circulating, edited lymphocytes increased over time and appeared in secondary transplant recipients, demonstrating successful editing in long-term repopulating cells. Random vector integration events were nearly undetectable, and malignant transformation of the transplanted cells was not observed. Similar editing frequencies were observed in human hematopoietic cells. Our results demonstrate that therapeutically relevant HSC gene editing can be achieved by AAV vectors in the absence of site-specific nucleases and suggest that this may be a safe and effective therapy for hematopoietic diseases where in vivo selection can increase edited cell numbers.  

5.2320           CRISPR-LbCpf1 prevents choroidal neovascularization in a mouse model of age-related macular degeneration

Koo, T., park, S.W., Jo, D.H., Kim, D., Kim, J.H., Cho, H-Y., Kim, J., Kim, J.H. and Kim, J-S. Nature Communications, 9:1855 (2018)   LbCpf1, derived from Lachnospiraceae bacterium ND2006, is a CRISPR RNA-guided endonuclease and holds promise for therapeutic applications. Here we show that LbCpf1 can be used for therapeutic gene editing in a mouse model of age-related macular degeneration (AMD). The intravitreal delivery of LbCpf1, targeted to two angiogenesis-associated genes encoding vascular endothelial growth factor A (Vegfa) and hypoxia inducing factor 1a (Hif1a), using adeno-associated virus, led to efficient gene disruption with no apparent off-target effects in the retina and retinal pigment epithelium (RPE) cells. Importantly, LbCpf1 targeted to Vegfa or Hif1a in RPE cells reduced the area of laser-induced choroidal neovascularization as efficiently as aflibercept, an anti-VEGF drug currently used in the clinic, without inducing cone dysfunction. Unlike aflibercept, LbCpf1 targeted to Vegfa or Hif1a achieved a long-term therapeutic effect on CNV, potentially avoiding repetitive injections. Taken together, these results indicate that LbCpf1-mediated in vivo genome editing to ablate pathologic angiogenesis provides an effective strategy for the treatment of AMD and other neovascularization-associated diseases.  

5.2321           Evaluation of Dried Blood Spots and Oral Fluids as Alternatives to Serum for Human Papillomavirus Antibody Surveillance

Louie, K.S., Dalel, J., Reuter, C., Bissett, S.L., Kleeman, M., Ashdown-Barr, L., Banwait, R., Godi, A., Sasieni, P. and Beddows, S. mSphere, 3(3), e00043-18 (2018)   Human papillomavirus (HPV) vaccination elicits high-titer genotype-specific antibody responses that are associated with a reduced risk of cervical disease caused by vaccine-incorporated genotypes. Our objective was to evaluate dried blood spots (DBSs) and oral mucosal transudate (OMT) as alternative samples to serum to confirm HPV vaccine antibody status. A study was carried out to evaluate the feasibility of detecting HPV16 and HPV18 antibodies in OMT, DBSs, and sera among women who self-reported being unvaccinated or fully vaccinated with the HPV vaccine. Serum had the highest sensitivity (100%) for detection of antibodies against both HPV16 and HPV18 but the lowest specificity, due to the detection of natural infection antibodies in 16% of unvaccinated women. Conversely, DBSs and OMT had lower sensitivity (96% and 82%, respectively) but high specificity (98%). We confirmed that these antibodies were functional (i.e., neutralizing) and that their detection was quantitatively reproducible and well correlated between sample types when normalized to IgG content. DBSs and OMT are appropriate alternative sample types for HPV vaccine surveillance. These alternative sample types warrant consideration for the purposes of cervical screening, diagnosis, and management, but more work will be needed to establish the stringent parameters required for such application.  

5.2322           DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus

Zhou, X-H., Bi, Z-X., Guo, X-J., Zhang, Z., Zhao, Y., Wang, M., Zhu, Y-L., Jie, H-Y., Yu, Y., Hung, T. and Lu, Z-Z.

1. Virol. Methods, 257, 85-92 (2018)

  Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coli BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix. E. coli competent cells were transformed with the assembled product, and plasmids (pKFAV4) were extracted and confirmed to contain viral genome by restriction analysis and sequencing. Virus was successfully rescued from linear pKFAV4-transfected chicken LMH cells. This approach was further verified in cloning of human adenovirus 5 genome. Our results indicated that DNA assembly technique simplified the construction of infectious clone of adenovirus, suggesting its possible application in virus traditional or reverse genetics.  

5.2323           Extracellular Conformational Changes in the Capsid of Human Papillomaviruses Contribute to Asynchronous Uptake into Host Cells

Becker, M., Greune, L., Schmidt, M.A. and Schelhaas, M.

1. Virol., 92(11), e02106-17 (2018)

  Human papillomavirus 16 (HPV16) is the leading cause of cervical cancer. For initial infection, HPV16 utilizes a novel endocytic pathway for host cell entry. Unique among viruses, uptake occurs asynchronously over a protracted period of time, with half-times between 9 and 12 h. To trigger endocytic uptake, the virus particles need to undergo a series of structural modifications after initial binding to heparan sulfate proteoglycans (HSPGs). These changes involve proteolytic cleavage of the major capsid protein L1 by kallikrein-8 (KLK8), exposure of the N terminus of the minor capsid protein L2 by cyclophilins, and cleavage of this N terminus by furin. Overall, the structural changes are thought to facilitate the engagement of an elusive secondary receptor for internalization. Here, we addressed whether structural changes are the rate-limiting steps during infectious internalization of HPV16 by using structurally primed HPV16 particles. Our findings indicate that the structural modifications mediated by cyclophilins and furin, which lead to exposure and cleavage, respectively, of the L2 N terminus contribute to the slow and asynchronous internalization kinetics, whereas conformational changes elicited by HSPG binding and KLK8 cleavage did not. However, these structural modifications accounted for only 30 to 50% of the delay in internalization. Therefore, we propose that limited internalization receptor availability for engagement of HPV16 causes slow and asynchronous internalization in addition to rate-limiting structural changes in the viral capsid.  

5.2324           Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting

Charlesworth, C.T., Camarena, J., Cromer, M.K., Vaidyanathan, S., Bak, R.O., Carte, J.M., Potter, J., Dever, D.P. and Porteus, M.H. Molecular Therapy – Nucleic Acids, 12, 89-104 (2018)   Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the β-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70% of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 10⁵ cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of β-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.  

5.2325           ATG5 overexpression is neuroprotective and attenuates cytoskeletal and vesicle-trafficking alterations in axotomized motoneurons

Leiva-Rodriguez, T., Romeo-Guitart, D., Mormolejo-Martinez-Artesero, S., Herrando-Grabulosa, M., Bossch, A., Fores, J. and Casas, C. Cell Death & Disease, 9:626 (2018)   Injured neurons should engage endogenous mechanisms of self-protection to limit neurodegeneration. Enhancing efficacy of these mechanisms or correcting dysfunctional pathways may be a successful strategy for inducing neuroprotection. Spinal motoneurons retrogradely degenerate after proximal axotomy due to mechanical detachment (avulsion) of the nerve roots, and this limits recovery of nervous system function in patients after this type of trauma. In a previously reported proteomic analysis, we demonstrated that autophagy is a key endogenous mechanism that may allow motoneuron survival and regeneration after distal axotomy and suture of the nerve. Herein, we show that autophagy flux is dysfunctional or blocked in degenerated motoneurons after root avulsion. We also found that there were abnormalities in anterograde/retrograde motor proteins, key secretory pathway factors, and lysosome function. Further, LAMP1 protein was missorted and underglycosylated as well as the proton pump v-ATPase. In vitro modeling revealed how sequential disruptions in these systems likely lead to neurodegeneration. In vivo, we observed that cytoskeletal alterations, induced by a single injection of nocodazole, were sufficient to promote neurodegeneration of avulsed motoneurons. Besides, only pre-treatment with rapamycin, but not post-treatment, neuroprotected after nerve root avulsion. In agreement, overexpressing ATG5 in injured motoneurons led to neuroprotection and attenuation of cytoskeletal and trafficking-related abnormalities. These discoveries serve as proof of concept for autophagy-target therapy to halting the progression of neurodegenerative processes.  

5.2326           The Neuropeptide Tac2 Controls a Distributed Brain State Induced by Chronic Social Isolation Stress

Zelikowsky, M., Hui, M., Karigo, T., Choe, A., Yang, B., Blanco, M., Beadle, K., Gradinaru, V., Deverman, B.E. and Anderson, D.J. Cell, 173(5), 1265-1279 (2018)   Chronic social isolation causes severe psychological effects in humans, but their neural bases remain poorly understood. 2 weeks (but not 24 hr) of social isolation stress (SIS) caused multiple behavioral changes in mice and induced brain-wide upregulation of the neuropeptide tachykinin 2 (Tac2)/neurokinin B (NkB). Systemic administration of an Nk3R antagonist prevented virtually all of the behavioral effects of chronic SIS. Conversely, enhancing NkB expression and release phenocopied SIS in group-housed mice, promoting aggression and converting stimulus-locked defensive behaviors to persistent responses. Multiplexed analysis of Tac2/NkB function in multiple brain areas revealed dissociable, region-specific requirements for both the peptide and its receptor in different SIS-induced behavioral changes. Thus, Tac2 coordinates a pleiotropic brain state caused by SIS via a distributed mode of action. These data reveal the profound effects of prolonged social isolation on brain chemistry and function and suggest potential new therapeutic applications for Nk3R antagonists.  

5.2327           Helper-free Production of Laboratory Grade AAV and Purification by Iodixanol Density Gradient Centrifugation

Crosson, S.M., Dib, P., Smith, J.K. and Zolotukhin, S. Molecular Therapy – Methods & Clin. Develop., 10, 1-7 (2018)   Adeno-associated virus (AAV) is one of the most promising gene therapy vectors and is widely used as a gene delivery vehicle for basic research. As AAV continues to become the vector of choice, it is increasingly important for new researchers to have access to a simplified production and purification protocol for laboratory grade recombinant AAV. Here we report a detailed protocol for serotype independent production of AAV using a helper-free HEK293 cell system followed by iodixanol gradient purification, a method described earlier.¹ While the core principals of this mammalian AAV production system are unchanged, there have been significant advancements in the production and purification procedure that serve to boost yield, maximize efficiency, and increase the purity of AAV preps. Using this protocol, we are able to constantly obtain high quantities of laboratory grade AAV particles (>5 × 10¹² vg) in a week’s time, largely independent of serotype.  

5.2328           Segmentation of the rabies virus genome

Hidaka, Y., Lim, C-K., Takayama-Ito, M., Park, C-H., Kimitsuki, K., Shiwa, N., Inoue, K-i, and Itou, T.

Virus Res., 252, 68-75 (2018)

  We established a system for the recovery of a segmented recombinant rabies virus, the virus genome RNA of which was divided into two parts: segment 1 encoding the nucleoprotein, phosphoprotein, matrix protein, and glycoprotein genes, and segment 2 encoding the large RNA-dependent RNA polymerase gene. The morphology of the segmented recombinant rabies virus was bullet-like in shape with a length of approximately 130 nm, which is shorter than the 200-nm long non-segmented recombinant rabies virus. The segmented recombinant rabies virus was maintained for at least 18 passages. The virus multiplication rate of the segmented recombinant rabies virus was lower than that of the non-segmented recombinant rabies virus during the passages, and the relative amounts of virus genome RNAs for segment 1 and segment 2 differed in the supernatant of the segmented recombinant rabies virus infected cells. These results suggest that the segmented recombinant rabies virus packages either segment 1 or segment 2 into each virus particle. Thus, co-infection with segmented recombinant rabies virus particles packaging segment 1 or segment 2 may be necessary for the production of progeny virus.  

5.2329           Dynein Engages and Disassembles Cytosol-Localized Simian Virus 40 To Promote Infection

Ravindran, M.S., Spriggs, C.C., Verhey, K.-J. and Tsai, B.

1. Virol., 92(12), e00353-18 (2018)

  During entry, polyomavirus (PyV) is endocytosed and sorts to the endoplasmic reticulum (ER), where it penetrates the ER membrane to reach the cytosol. From the cytosol, the virus moves to the nucleus to cause infection. How PyV is transported from the cytosol into the nucleus, a crucial infection step, is unclear. We found that upon reaching the cytosol, the archetypal PyV simian virus 40 (SV40) recruits the cytoplasmic dynein motor, which disassembles the viral particle. This reaction enables the resulting disassembled virus to enter the nucleus to promote infection. Our findings reveal how a cytosolic motor can be hijacked to impart conformational changes to a viral particle, a process essential for successful infection.  

5.2330           Isolation of a natural DNA virus of Drosophila melanogaster, and characterisation of host resistance and immune responses

Palmer, W.H., Medd, N.C., Beard, P.M. and Obbard, D.J. PloS Pathogens, 14(6), e1007050 (2019)   Drosophila melanogaster has played a key role in our understanding of invertebrate immunity. However, both functional and evolutionary studies of host-virus interaction in Drosophila have been limited by a dearth of native virus isolates. In particular, despite a long history of virus research, DNA viruses of D. melanogaster have only recently been described, and none have been available for experimental study. Here we report the isolation and comprehensive characterisation of Kallithea virus, a large double-stranded DNA virus, and the first DNA virus to have been reported from wild populations of D. melanogaster. We find that Kallithea virus infection is costly for adult flies, reaching high titres in both sexes and disproportionately reducing survival in males, and movement and late fecundity in females. Using the Drosophila Genetic Reference Panel, we quantify host genetic variance for virus-induced mortality and viral titre and identify candidate host genes that may underlie this variation, including Cdc42-interacting protein 4. Using full transcriptome sequencing of infected males and females, we examine the transcriptional response of flies to Kallithea virus infection and describe differential regulation of virus-responsive genes. This work establishes Kallithea virus as a new tractable model to study the natural interaction between D. melanogaster and DNA viruses, and we hope it will serve as a basis for future studies of immune responses to DNA viruses in insects.  

5.2331           Rhodanine derivatives as potent anti-HIV and anti-HSV microbicides

Tintori, C., Iovenitti, G., Ceresola, E.R., Ferrarese, R., Zamperini, C., Brai, A., Poli, G., Dreassi, E., Cagno, V., Lembo, D., Canducci, F. and Botta, M. PloS One, 13(6), e0198478 (2018)   Although highly active antiretroviral therapies (HAART) remarkably increased life expectancy of HIV positive people, the rate of novel HIV-1 infections worldwide still represent a major concern. In this context, pre-exposure prophylaxis (PrEP) approaches such as vaginal microbicide gels topically releasing antiretroviral drugs, showed to have a striking impact in limiting HIV-1 spread. Nevertheless, the co-presence of other genital infections, particularly those due to HSV-1 or 2, constitute a serious drawback that strongly limits the efficacy of PrEP approaches. For this reason, combinations of different compounds with mixed antiviral and antiretroviral activity are thoroughly investigated Here we report the synthesis and the biological evaluation of a novel series of rhodanine derivatives, which showed to inhibit both HIV-1 and HSV-1/2 replication at nanomolar concentration, and were found to be active also on acyclovir resistant HSV-2 strains. The compounds showed a considerable reduction of activity in presence of serum due to a high binding to serum albumin, as determined through in vitro ADME evaluations. However, the most promising compound of the series maintained a considerable activity in gel formulation, with an EC₅₀ comparable to that obtained for the reference drug tenofovir. Moreover, the series of compounds showed pharmacokinetic properties suitable for topical formulation, thus suggesting that the novel rhodanine derivatives could represent effective agents to be used as dual anti HIV/HSV microbicides in PrEP approaches.  

5.2332           Functional Rescue of Dystrophin Deficiency in Mice Caused by Frameshift Mutations Using Campylobacter jejuni Cas9

Koo, T., Lu-Nguyen, N., Malerba, A., Kim, E., Kim, D., Caappellari, O., Cho, H-Y., Dickson, G., Poppwell, L. and Kim, J-S. Molecular Therapy, 26(6), 1529-1538 (2018)   Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle-wasting disease caused by mutations in the DMD gene. In 51% of DMD cases, a reading frame is disrupted because of deletion of several exons. Here, we show that CjCas9 derived from Campylobacter jejuni can be used as a gene-editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. Herein, we used Cas9 derived from S. pyogenes to generate Dmd knockout mice with a frameshift mutation in Dmd gene. Then, we expressed CjCas9, its single-guide RNA, and the EGFP gene in the tibialis anterior muscle of the Dmd knockout mice using an all-in-one adeno-associated virus (AAV) vector. CjCas9 cleaved the target site in the Dmd gene efficiently in vivo and induced small insertions or deletions at the target site. This treatment resulted in conversion of the disrupted Dmd reading frame from out of frame to in frame, leading to the expression of dystrophin in the sarcolemma. Importantly, muscle strength was enhanced in the CjCas9-treated muscles, without off-target mutations, indicating high efficiency and specificity of CjCas9. This work suggests that in vivo DMD frame correction, mediated by CjCas9, has great potential for the treatment of DMD and other neuromuscular diseases.  

5.2333           Sustained AAV9-mediated expression of a non-self protein in the CNS of non-human primates after immunomodulation

Ramsingh, A., Gray, S.J., Reilly, A., Koday, M., Bratt, D., Koday, M.T., Murmane, R., Smedley, J., Hu, Y., Messer, A. and Fuller, D.H. PloS One, 13(6), e0198154 (2018)   A critical issue in transgene delivery studies is immune reactivity to the transgene- encoded protein and its impact on sustained gene expression. Here, we test the hypothesis that immunomodulation by rapamycin can decrease immune reactivity after intrathecal AAV9 delivery of a transgene (GFP) in non-human primates, resulting in sustained GFP expression in the CNS. We show that rapamycin treatment clearly reduced the overall immunogenicity of the AAV9/GFP vector by lowering GFP- and AAV9-specific antibody responses, and decreasing T cell responses including cytokine and cytolytic effector responses. Spinal cord GFP protein expression was sustained for twelve weeks, with no toxicity. Immune correlates of robust transgene expression include negligible GFP-specific CD4 and CD8 T cell responses, absence of GFP-specific IFN-γ producing T cells, and absence of GFP-specific cytotoxic T cells, which support the hypothesis that decreased T cell reactivity results in sustained transgene expression. These data strongly support the use of modest doses of rapamycin to modulate immune responses for intrathecal gene therapies, and potentially a much wider range of viral vector-based therapeutics.  

5.2334           Overexpression of TFEB Drives a Pleiotropic Neurotrophic Effect and Prevents Parkinson’s Disease-Related Neurodegeneration

Torra, A., parent, A., Cuadros, T., Rodriguez-Galvan, B., Ruiz-Bronchal, E., Ballabio, A., Bortolozzi, A., Vila, m. and Bove, J. Molecular Therapy, 26(6), 1552-1567 (2018)   The possible implication of transcription factor EB (TFEB) as a therapeutic target in Parkinson’s disease has gained momentum since it was discovered that TFEB controls lysosomal biogenesis and autophagy and that its activation might counteract lysosomal impairment and protein aggregation. However, the majority of putative direct targets of TFEB described to date is linked to a range of biological processes that are not related to the lysosomal-autophagic system. Here, we assessed the effect of overexpressing TFEB with an adeno-associated viral vector in mouse substantia nigra dopaminergic neurons. We demonstrate that TFEB overexpression drives a previously unknown bona fide neurotrophic effect, giving rise to cell growth, higher tyrosine hydroxylase levels, and increased dopamine release in the striatum. TFEB overexpression induces the activation of the mitogen-activated protein kinase 1/3 (MAPK1/3) and AKT pro-survival pathways, phosphorylation of mTORC1 effectors 4E-binding protein 1 (4E-BP1) and S6 kinase B1 (S6K1), and increased protein synthesis. We show that TFEB overexpression prevents dopaminergic cell loss and counteracts atrophy and the associated protein synthesis decline in the MPTP mouse model of Parkinson’s disease. Our results suggest that increasing TFEB activity might prevent neuronal death and restore neuronal function in Parkinson’s disease and other neurodegenerative diseases through different mechanisms.  

5.2335           Gene Transfer of Engineered Calmodulin Alleviates Ventricular Arrhythmias in a Calsequestrin‐Associated Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia

Liu, B., Walton, S.D., Ho, H-T., Belevych, A.E. et al

1. Am. Heart.Assoc., 7, e008155 (2018)

  Background Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic syndrome characterized by sudden death. There are several genetic forms of CPVT associated with mutations in genes encoding the cardiac ryanodine receptor (RyR2) and its auxiliary proteins including calsequestrin (CASQ2) and calmodulin (CaM). It has been suggested that impairment of the ability of RyR2 to stay closed (ie, refractory) during diastole may be a common mechanism for these diseases. Here, we explore the possibility of engineering CaM variants that normalize abbreviated RyR2 refractoriness for subsequent viral‐mediated delivery to alleviate arrhythmias in non–CaM‐related CPVT. Methods and Results To that end, we have designed a CaM protein (GSH‐M37Q; dubbed as therapeutic CaM or T‐CaM) that exhibited a slowed N‐terminal Ca dissociation rate and prolonged RyR2 refractoriness in permeabilized myocytes derived from CPVT mice carrying the CASQ2 mutation R33Q. This T‐CaM was introduced to the heart of R33Q mice through recombinant adeno‐associated viral vector serotype 9. Eight weeks postinfection, we performed confocal microscopy to assess Ca handling and recorded surface ECGs to assess susceptibility to arrhythmias in vivo. During catecholamine stimulation with isoproterenol, T‐CaM reduced isoproterenol‐promoted diastolic Ca waves in isolated CPVT cardiomyocytes. Importantly, T‐CaM exposure abolished ventricular tachycardia in CPVT mice challenged with catecholamines. Conclusions Our results suggest that gene transfer of T‐CaM by adeno‐associated viral vector serotype 9 improves myocyte Ca handling and alleviates arrhythmias in a calsequestrin‐associated CPVT model, thus supporting the potential of a CaM‐based antiarrhythmic approach as a therapeutic avenue for genetically distinct forms of CPVT.  

5.2336           Loss of Tmem106b is unable to ameliorate frontotemporal dementia-like phenotypes in an AAV mouse model of C9ORF72-repeat induced toxicity

Nicholson, A.M., Zhou, X., Perkerson, R.B., Parsons, T.M., Chew, J., Brooks, M. et al Acta Neuropathol. Comm., 6:42 (2018)   Loss-of-function mutations in progranulin (GRN) and a non-coding (GGGGCC)_n hexanucleotide repeat expansions in C9ORF72 are the two most common genetic causes of frontotemporal lobar degeneration with aggregates of TAR DNA binding protein 43 (FTLD-TDP). TMEM106B encodes a type II transmembrane protein with unknown function. Genetic variants in TMEM106B associated with reduced TMEM106B levels have been identified as disease modifiers in individuals with GRN mutations and C9ORF72 expansions. Recently, loss of Tmem106b has been reported to protect the FTLD-like phenotypes in Grn−/− mice. Here, we generated Tmem106b−/− mice and examined whether loss of Tmem106b could rescue FTLD-like phenotypes in an AAV mouse model of C9ORF72-repeat induced toxicity. Our results showed that neither partial nor complete loss of Tmem106b was able to rescue behavioral deficits induced by the expression of (GGGGCC)₆₆ repeats (66R). Loss of Tmem106b also failed to ameliorate 66R-induced RNA foci, dipeptide repeat protein formation and pTDP-43 pathological burden. We further found that complete loss of Tmem106b increased astrogliosis, even in the absence of 66R, and failed to rescue 66R-induced neuronal cell loss, whereas partial loss of Tmem106b significantly rescued the neuronal cell loss but not neuroinflammation induced by 66R. Finally, we showed that overexpression of 66R did not alter expression of Tmem106b and other lysosomal genes in vivo, and subsequent analyses in vitro found that transiently knocking down C9ORF72, but not overexpression of 66R, significantly increased TMEM106B and other lysosomal proteins. In summary, reducing Tmem106b levels failed to rescue FTLD-like phenotypes in a mouse model mimicking the toxic gain-of-functions associated with overexpression of 66R. Combined with the observation that loss of C9ORF72 and not 66R overexpression was associated with increased levels of TMEM106B, this work suggests that the protective TMEM106B haplotype may exert its effect in expansion carriers by counteracting lysosomal dysfunction resulting from a loss of C9ORF72.  

5.2337           Distinct in vivo roles of secreted APP ectodomain variants APPsα and APPsβ in regulation of spine density, synaptic plasticity, and cognition

Richter, M.C., Ludewig, S., Winschel, A., Abel, T., Bold, C., Salzburger, L.R., Klein, S., Han, k., Weyer, S.W., Fritz, A-K., Laube, B., Wolfer, D.P., Buchholz, C.J., Korte, M. and Müller, U. EMBO J., 37, e98335 (2018)   Increasing evidence suggests that synaptic functions of the amyloid precursor protein (APP), which is key to Alzheimer pathogenesis, may be carried out by its secreted ectodomain (APPs). The specific roles of APPsα and APPsβ fragments, generated by non‐amyloidogenic or amyloidogenic APP processing, respectively, remain however unclear. Here, we expressed APPsα or APPsβ in the adult brain of conditional double knockout mice (cDKO) lacking APP and the related APLP2. APPsα efficiently rescued deficits in spine density, synaptic plasticity (LTP and PPF), and spatial reference memory of cDKO mice. In contrast, APPsβ failed to show any detectable effects on synaptic plasticity and spine density. The C‐terminal 16 amino acids of APPsα (lacking in APPsβ) proved sufficient to facilitate LTP in a mechanism that depends on functional nicotinic α7‐nAChRs. Further, APPsα showed high‐affinity, allosteric potentiation of heterologously expressed α7‐nAChRs in oocytes. Collectively, we identified α7‐nAChRs as a crucial physiological receptor specific for APPsα and show distinct in vivo roles for APPsα versus APPsβ. This implies that reduced levels of APPsα that might occur during Alzheimer pathogenesis cannot be compensated by APPsβ.  

5.2338           Widespread transduction of astrocytes and neurons in the mouse central nervous system after systemic delivery of a self-complementary AAV-PHP.B vector

Rincon, M.Y., de Vin, F., Duque, S.I., Fripont, S., Castaldo, S.A., Bouhuijzen-Wenger, J. and Holt, M.G. Gene Therapy, 25(2), 83-92 (2018)   Until recently, adeno-associated virus 9 (AAV9) was considered the AAV serotype most effective in crossing the blood–brain barrier (BBB) and transducing cells of the central nervous system (CNS), following systemic injection. However, a newly engineered capsid, AAV-PHP.B, is reported to cross the BBB at even higher efficiency. We investigated how much we could boost CNS transgene expression by using AAV-PHP.B carrying a self-complementary (sc) genome. To allow comparison, 6 weeks old C57BL/6 mice received intravenous injections of scAAV2/9-GFP or scAAV2/PHP.B-GFP at equivalent doses. Three weeks postinjection, transgene expression was assessed in brain and spinal cord. We consistently observed more widespread CNS transduction and higher levels of transgene expression when using the scAAV2/PHP.B-GFP vector. In particular, we observed an unprecedented level of astrocyte transduction in the cortex, when using a ubiquitous CBA promoter. In comparison, neuronal transduction was much lower than previously reported. However, strong neuronal expression (including spinal motor neurons) was observed when the human synapsin promoter was used. These findings constitute the first reported use of an AAV-PHP.B capsid, encapsulating a scAAV genome, for gene transfer in adult mice. Our results underscore the potential of this AAV construct as a platform for safer and more efficacious gene therapy vectors for the CNS.    

5.2339           PD-L1/B7-H1 Inhibits Viral Clearance by Macrophages in HSV-1–Infected Corneas

Jeon, S., Rowe, A.M., Carroll, K.L., Harvey, S.A.K. and Hendricks, R.L.

1. Immunology, 200(11), 3711-3719 (2018)

  Immune privilege helps protect the cornea from damaging inflammation but can also impair pathogen clearance from this mucosal surface. Programmed death-ligand 1 (PD-L1 or B7-H1) contributes to corneal immune privilege by inhibiting the function of a variety of immune cells. We asked whether programmed death-1 (PD-1)/PD-L1 interaction regulates HSV-1 clearance from infected corneas. We show that PD-L1 is constitutively expressed in the corneal epithelium and is upregulated upon HSV-1 corneal infection, with peak expression on CD45⁺ cells NK cells, dendritic cells, neutrophils, and macrophages and CD45⁻ corneal epithelial cells at 4 d postinfection (dpi). As early as 1 dpi, HSV-1–infected corneas of B7-H1^−/− mice as compared with wild-type mice showed increased chemokine expression and this correlated with increased migration of inflammatory cells into the viral lesions and decreased HSV-1 corneal titers. Local PD-L1 blockade caused a similar increase in viral clearance, suggesting a local effect of PD-1/PD-L1 in the cornea. The enhanced HSV-1 clearance at 2 dpi resulting from PD-1/PD-L1 blockade is mediated primarily by a monocyte/macrophage population. Studies in bone marrow chimeras demonstrated enhanced viral clearance when PD-L1 was absent only from nonhematopoietic cells. We conclude that PD-L1 expression on corneal cells negatively impacts the ability of the innate immune system to clear HSV-1 from infected corneas.  

5.2340           Metastatic cancers promote cachexia through ZIP14 upregulation in skeletal muscle

Wang, G., Biswas, A.K., Ma, W., Kandpal, M., Coker, C. et al Nature Med., 24, 770-781 (2018)   Patients with metastatic cancer experience a severe loss of skeletal muscle mass and function known as cachexia. Cachexia is associated with poor prognosis and accelerated death in patients with cancer, yet its underlying mechanisms remain poorly understood. Here, we identify the metal-ion transporter ZRT- and IRT-like protein 14 (ZIP14) as a critical mediator of cancer-induced cachexia. ZIP14 is upregulated in cachectic muscles of mice and in patients with metastatic cancer and can be induced by TNF-α and TGF-β cytokines. Strikingly, germline ablation or muscle-specific depletion of Zip14 markedly reduces muscle atrophy in metastatic cancer models. We find that ZIP14-mediated zinc uptake in muscle progenitor cells represses the expression of MyoD and Mef2c and blocks muscle-cell differentiation. Importantly, ZIP14-mediated zinc accumulation in differentiated muscle cells induces myosin heavy chain loss. These results highlight a previously unrecognized role for altered zinc homeostasis in metastatic cancer–induced muscle wasting and implicate ZIP14 as a therapeutic target for its treatment.  

5.2341           Dimethyl fumarate potentiates oncolytic virotherapy through NF-κB inhibition

Selman, M., Ou, P., Rousso, C., Bergeron, A., Krishnan, R., Pikor, L., Chen, A., Keller, B.A., Ilkow, C., Bell, J.C. and Diallo, J-S. Sci. Transl. Med., 10(425), eaao1613 (2018)   Resistance to oncolytic virotherapy is frequently associated with failure of tumor cells to get infected by the virus. Dimethyl fumarate (DMF), a common treatment for psoriasis and multiple sclerosis, also has anticancer properties. We show that DMF and various fumaric and maleic acid esters (FMAEs) enhance viral infection of cancer cell lines as well as human tumor biopsies with several oncolytic viruses (OVs), improving therapeutic outcomes in resistant syngeneic and xenograft tumor models. This results in durable responses, even in models otherwise refractory to OV and drug monotherapies. The ability of DMF to enhance viral spread results from its ability to inhibit type I interferon (IFN) production and response, which is associated with its blockade of nuclear translocation of the transcription factor nuclear factor κB (NF-κB). This study demonstrates that unconventional application of U.S. Food and Drug Administration–approved drugs and biological agents can result in improved anticancer therapeutic outcomes.  

5.2342           Non-human papillomaviruses for gene delivery in vitro and in vivo

Bayer, L., Gümpel, J., Hause, G., Müller, M. and Grunwald, T. PloS One, 13(6), e0198996 (2018)   Papillomavirus capsids are known to have the ability to package DNA plasmids and deliver them both in vitro and in vivo. Of all known papillomavirus types, human papillomaviruses (HPVs) are by far the most intensely studied. Although HPVs work well as gene transfer vectors, their use is limited as most individuals are exposed to this virus either through a HPV vaccination or natural infection. To circumvent these constraints, we produced pseudovirions (PsVs) of ten non-human papillomavirus types and tested their transduction efficiencies in vitro. PsVs based on Macaca fascicularis papillomavirus-11 and Puma concolor papillomavirus-1 were further tested in vivo. Intramuscular transduction by PsVs led to months-long expression of a reporter plasmid, indicating that PsVs have potential as gene delivery vectors.  

5.2343           Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy

Ryu, S-M., Koo, T., Kim, K., Lim, K., Baek, G., Kim, S-T., Kim, H.S., Kim, D-e., Lee, H., Chung, E. and Kim, J-S. Nature Biotech., 36(6), 536-539 (2018)   Adenine base editors (ABEs) composed of an engineered adenine deaminase and the Streptococcus pyogenes Cas9 nickase enable adenine-to-guanine (A-to-G) single-nucleotide substitutions in a guide RNA (gRNA)-dependent manner. Here we demonstrate application of this technology in mouse embryos and adult mice. We also show that long gRNAs enable adenine editing at positions one or two bases upstream of the window that is accessible with standard single guide RNAs (sgRNAs). We introduced the Himalayan point mutation in the Tyr gene by microinjecting ABE mRNA and an extended gRNA into mouse embryos, obtaining Tyr mutant mice with an albino phenotype. Furthermore, we delivered the split ABE gene, using trans-splicing adeno-associated viral vectors, to muscle cells in a mouse model of Duchenne muscular dystrophy to correct a nonsense mutation in the Dmd gene, demonstrating the therapeutic potential of base editing in adult animals.  

5.2344           Gene Transfer of ZMapp Antibodies Mediated by Recombinant Adeno-Associated Virus Protects Against Ebola Infections

Robert, M-A., Nassoury, N., Chahal, P.S., Venne, M-H., Racine, T., Qiu, X., Kobinger, G., Kamen, A., Gilbert, R and Gaillet, B. Human Gene Therapy, 29(4), 452-456 (2018)   Vectored delivery of the ZMapp antibody cocktail (c2G4, c4G7, and c13C6) by using recombinant adeno-associated viruses (rAAVs) could be useful for preventive immunization against Ebola virus infections because rAAVs can generate long-term antibody expression. Three rAAVs (serotype 9) encoding chimeric ZMapp antibodies were produced by triple-plasmid transfection up to 10 L-scale in WAVE bioreactors using HEK293 cells grown in suspension/serum-free conditions. Efficacy of AAV-c2G4 via intravenous (i.v.), intramuscular (i.m.), and intranasal (i.n.) routes of administration was evaluated in mice with two different doses of 2.7 × 1010 and 13.0 × 1010 vector genomes (vg). The best protective efficacies after Ebola challenge were obtained with the i.v. and i.m. routes. Serum concentrations of ZMapp antibodies positively correlated with survivability. Efficacy of the rAAV-ZMapp cocktail was then evaluated at a higher dose of 30.0 × 1010 vg. It conferred a more robust protection (90% i.v. and 60% i.m.) than rAAV-c4G7 (30%) and rAAV-c13C6 (70%), both administered separately at the same dose. Delivery of rAAV-c2G4 alone achieved up to 100% protection (100% i.v. and 90% i.m.) at the same dose. In conclusion, the preventive treatment was effective in mice. However, no advantage was observed for using the rAAV-ZMapp cocktail in comparison to the utilization of the single rAAV-c2G4.  

5.2345           An Inducible Promoter Responsive to Different Porphyrinogenic Stimuli Improves Gene Therapy Vectors for Acute Intermittent Porphyria

Serrano-Mendioroz, I., Sampedro, A., Alegre, M., de Salamanca, R.E., Berraondo, P. and Fontanellas, A. Human Gene Therapy, 29(4), 480-491 (2018)   Porphobilinogen deaminase (PBGD) gene therapy represents a promising therapeutic option for acute intermittent porphyria (AIP) patients suffering recurrent acute attacks. A first-in-human Phase I clinical trial confirmed the safety and tolerability of adeno-associated virus (AAV)-AAT-PBGD gene therapy, but higher doses and/or more efficient vectors are needed to achieve therapeutic expression of the transgene. This study assayed the insertion into the promoter of a short enhancer element able to induce transgene expression during exposure to endogenous and exogenous stimuli related to the pathology of the disease. The inclusion in tandem of two elements of the minimal functional sequence of human δ-aminolevulinic acid synthase drug-responsive enhancing sequence (ADRES) positioned upstream of the promoter strongly induced transgene expression in the presence of estrogens, starvation, and certain drugs known to trigger attacks in porphyria patients. The inclusion of two ADRES motives in an AAV vector improved therapeutic efficacy, reducing 10-fold the effective dose in AIP mice. In conclusion, the inclusion of specific enhancer elements in the promoter of gene therapy vectors for AIP was able to overexpress the therapeutic transgene when it is most needed, at the time when porphyrinogenic factors increase the demand for hepatic heme and precipitate acute porphyria attacks.  

5.2346           Chapter 2 – Targeting Transgene and RNA Interference-Based Gene Silencing Sequences to Astrocytes Using Viral Vector-Mediated Approaches

Fong, D.M., Wu, A.and Young, D. Gene Therapy in Neurological Disorders, 41-61 (2018)   Astrocytes play a key role in the pathogenesis of many neurodegenerative diseases. Their abundance in brain regions affected by neurodegeneration makes them an attractive cell target for central nervous system (CNS) gene therapy. In this chapter, strategies to target transgene expression as well as RNA interference-based gene silencing sequences to astrocytes using adeno-associated viral (AAV) and lentiviral vector are discussed. Methods for the generation of the knockdown plasmids as well as protocols for AAV vector packaging and vector infusion are presented.  

5.2347           Chapter 6 – Prophylactic and Therapeutic Applications of Catalytic Immunoglobulin Gene Delivery in a Mouse Model of Alzheimer’s Disease

Fukuchi, K-I., Yang, J., Kou, J., Song, M., Lalonde, R., Planque, S.A. and Paul, S. Gene Therapy in Neurological Disorders, 139-161 (2018)   The amyloid hypothesis has been the main theory to explain the etiology of Alzheimer’s disease (AD) and provided potentially preventive and therapeutic targets. Although Aβ-immunotherapy holds promise for AD, its vascular complications reduce the efficacy and safety. We produced an adeno-associated virus vector encoding Aβ-specific catalytic antibody, rAAV9-IgV_L5D3, and evaluated the prophylactic and therapeutic efficacy and safety of rAAV9-IgV_L5D3 brain delivery in an AD mouse model. One single injection of rAAV9-IgV_L5D3 into the right ventricle achieved widespread, high expression of IgV_L5D3 in the right hippocampus and cerebrospinal fluid but less expression in the left hemisphere. The prophylactic and therapeutic application of rAAV9-IgV_L5D3 reduced Aβ load in the right hippocampus. The therapeutic application improved long-term but not short-term memory on the Morris water maze without increasing cerebral amyloid angiopathy, microhemorrhage, and inflammation. Brain-targeted gene delivery of Aβ-specific catalytic antibody can be a safer and more effective approach for AD prevention and treatment than conventional anti-Aβ antibodies.  

5.2348           Chapter 13 – Expressing Full-Length Dystrophin Using Adeno-Associated Virus

Kodippili, K. and Duan, D. Gene Therapy in Neurological Disorders, 259-276 (2018)   Adeno-associated virus (AAV) mediated gene replacement therapy holds great promise in treating inherited muscle diseases such as Duchenne muscular dystrophy (DMD). DMD is the most common lethal muscle disorder in children and is caused by mutations in the dystrophin gene. The full-length dystrophin coding sequence is approximately 11.2 kb. However, the maximal carrying capacity of the AAV vector is only 5 kb. To overcome this gene delivery conundrum, we developed a creative tri-AAV vector strategy. Specifically, we fragmented the full-length dystrophin cDNA expression cassette into three sections and engineered unique recombination signals for each section. Modified dystrophin cDNA fragments were independently packaged into three AAV vectors. Co-delivery of these vectors into dystrophin-deficient mice resulted in full-length dystrophin expression. Similar strategies can be used to deliver other large therapeutic genes.  

5.2349           Characterization of BK Polyomaviruses from Kidney Transplant Recipients Suggests a Role for APOBEC3 in Driving In-Host Virus Evolution

Peretti, A., Geoghegan, E.M., pastrana, D.V., Smola, S., Feld, P. Et al Cell Host & Microbe, 23(5), 628-635 (2018)   BK polyomavirus (BKV) frequently causes nephropathy (BKVN) in kidney transplant recipients (KTRs). BKV has also been implicated in the etiology of bladder and kidney cancers. We characterized BKV variants from two KTRs who developed BKVN followed by renal carcinoma. Both patients showed a swarm of BKV sequence variants encoding non-silent mutations in surface loops of the viral major capsid protein. The temporal appearance and disappearance of these mutations highlights the intra-patient evolution of BKV. Some of the observed mutations conferred resistance to antibody-mediated neutralization. The mutations also modified the spectrum of receptor glycans engaged by BKV during host cell entry. Intriguingly, all observed mutations were consistent with DNA damage caused by antiviral APOBEC3 cytosine deaminases. Moreover, APOBEC3 expression was evident upon immunohistochemical analysis of renal biopsies from KTRs. These results provide a snapshot of in-host BKV evolution and suggest that APOBEC3 may drive BKV mutagenesis in vivo.  

5.2350           Targeting protein and peptide therapeutics to the heart via tannic acid modification

Shin, M., Lee, H-A., Lee, M., Shin, Y., Song, J-J., Kang, S-W. et al Nature Biomed. Engineering, 2, 304-317 (2018)   Systemic injection into blood vessels is the most common method of drug administration. However, targeting drugs to the heart is challenging, owing to its dynamic mechanical motions and large cardiac output. Here, we show that the modification of protein and peptide therapeutics with tannic acid—a flavonoid found in plants that adheres to extracellular matrices, elastins and collagens—improves their ability to specifically target heart tissue. Tannic-acid-modified (TANNylated) proteins do not adsorb on endothelial glycocalyx layers in blood vessels, yet they penetrate the endothelium to thermodynamically bind to myocardium extracellular matrix before being internalized by myoblasts. In a rat model of myocardial ischaemia-reperfusion injury, TANNylated basic fibroblast growth factor significantly reduced infarct size and increased cardiac function. TANNylation of systemically injected therapeutic proteins, peptides or viruses may enhance the treatment of heart diseases.  

5.2351           A novel serological assay for influenza based on DiD fluorescence dequenching that is free from observer bias and potentially automatable – A proof of concept study

Makarkov, A.I., Patel, A.R., Bainov, V. and Ward, B.J. Vaccine, 36, 4485-4493 (2018)   Background Serum hemagglutination inhibition (HAI) and microneutralization (MN) antibodies are often used as a correlate of protection for influenza. However, these manual assays are labor-intensive and difficult to standardize due to variability in biologic reagents used and subjective interpretation of the results. Methods Sera with known HAI and MN titers were used to assess a novel test based on the inhibition of fluorescence ‘dequenching’. Whole influenza virions (A/California/07/2009 (H1N1), A/Hong Kong/4801/2014 (H3N2) and B/Brisbane/60/2008) labelled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) were exposed to serial dilutions of serum and mixed with turkey red blood cells followed by acidification of the media (pH 5.0–5.5). The H1N1 and B/Brisbane strains were high hemagglutinating while the H3N2 strain had low hemagglutinating activity. In some experiments, labelled virions were subjected to repetitive freeze-thaw cycles prior to use in the assay. Results In the absence of detectable HAI/MN antibodies, there were consistent and substantial increases from baseline DiD fluorescence upon acidification. Sera with known high titer HAI/MN antibodies reduced or completely prevented DiD dequenching at low dilutions with progressive increases in fluorescence at higher dilutions, which permitted a reproducible assignment of an antibody ‘titer’ based on baseline and acidified DiD fluorescence values. The ‘titers’ measured by the DiD dequenching assay were highly correlated with HAI/MN results for the H1N1 and B strains (Spearman’s correlation coefficients (r_s) 0.874 to 0.946, p < 10⁻⁷ to 10⁻³⁵). Correlations with HAI/MN titres for the low-hemagglutinating H3N2 strain tested were lower but remained statistically significant (r_s 0.547–0.551, p < 0.004). Freeze-thawing of the DiD pre-stained virus stocks had no significant impact on the results of the assay. Conclusions The DiD dequenching assay may be a labour-saving and more objective alternative to the classic serologies. This novel assay could theoretically be standardized across laboratories using pre-stained virions and has the potential to be fully automated.  

5.2352           Distributed hepatocytes expressing telomerase repopulate the liver in homeostasis and injury

Lin, S., Nascimento, E.M., Gajera, C.R., Chen, L., Neuhöfer, P., Garbuzov, A., Wang, S. and Artandi, S.E. Nature, 556, 244-248 (2018)   Hepatocytes are replenished gradually during homeostasis and robustly after liver injury1, 2. In adults, new hepatocytes originate from the existing hepatocyte pool3,4,5,6,7,8, but the cellular source of renewing hepatocytes remains unclear. Telomerase is expressed in many stem cell populations, and mutations in telomerase pathway genes have been linked to liver diseases9,10,11. Here we identify a subset of hepatocytes that expresses high levels of telomerase and show that this hepatocyte subset repopulates the liver during homeostasis and injury. Using lineage tracing from the telomerase reverse transcriptase (Tert) locus in mice, we demonstrate that rare hepatocytes with high telomerase expression (TERTHigh hepatocytes) are distributed throughout the liver lobule. During homeostasis, these cells regenerate hepatocytes in all lobular zones, and both self-renew and differentiate to yield expanding hepatocyte clones that eventually dominate the liver. In response to injury, the repopulating activity of TERTHigh hepatocytes is accelerated and their progeny cross zonal boundaries. RNA sequencing shows that metabolic genes are downregulated in TERTHigh hepatocytes, indicating that metabolic activity and repopulating activity may be segregated within the hepatocyte lineage. Genetic ablation of TERTHigh hepatocytes combined with chemical injury causes a marked increase in stellate cell activation and fibrosis. These results provide support for a ‘distributed model’ of hepatocyte renewal in which a subset of hepatocytes dispersed throughout the lobule clonally expands to maintain liver mass.  

5.2353           AAVvector-mediated in vivo reprogramming into pluripotency

Senis, E., Mosteiro, L., Wilkening, S., Wiedtke, E., Nowrouzi, A., Afzal, S., Fronza, R., Landerer, H., Abad, M., Niopek, D., Schmidt, M., Serrano, M. and Grimm, D. Nature Communications, 9:2651 (2018)   In vivo reprogramming of somatic cells into induced pluripotent stem cells (iPSC) holds vast potential for basic research and regenerative medicine. However, it remains hampered by a need for vectors to express reprogramming factors (Oct-3/4, Klf4, Sox2, c-Myc; OKSM) in selected organs. Here, we report OKSM delivery vectors based on pseudotyped Adeno-associated virus (AAV). Using the AAV-DJ capsid, we could robustly reprogram mouse embryonic fibroblasts with low vector doses. Swapping to AAV8 permitted to efficiently reprogram somatic cells in adult mice by intravenous vector delivery, evidenced by hepatic or extra-hepatic teratomas and iPSC in the blood. Notably, we accomplished full in vivo reprogramming without c-Myc. Most iPSC generated in vitro or in vivo showed transcriptionally silent, intronic or intergenic vector integration, likely reflecting the increased host genome accessibility during reprogramming. Our approach crucially advances in vivo reprogramming technology, and concurrently facilitates investigations into the mechanisms and consequences of AAV persistence.  

5.2354           Anterior cingulate cortex and its input to the basolateral amygdala control innate fear response

Jhang, J., Lee, H., kang, M.S., Lee, H-S., Park, H. and Han, J-H. Nature Communications, 9:2744 (2018)   Prefrontal brain areas are implicated in the control of fear behavior. However, how prefrontal circuits control fear response to innate threat is poorly understood. Here, we show that the anterior cingulate cortex (ACC) and its input to the basolateral nucleus of amygdala (BLA) contribute to innate fear response to a predator odor in mice. Optogenetic inactivation of the ACC enhances freezing response to fox urine without affecting conditioned freezing. Conversely, ACC stimulation robustly inhibits both innate and conditioned freezing. Circuit tracing and slice patch recordings demonstrate a monosynaptic glutamatergic connectivity of ACC-BLA but no or very sparse ACC input to the central amygdala. Finally, our optogenetic manipulations of the ACC-BLA projection suggest its inhibitory control of innate freezing response to predator odors. Together, our results reveal the role of the ACC and its projection to BLA in innate fear response to olfactory threat stimulus.  

5.2355           Exchange of functional domains between a bacterial conjugative relaxase and the integrase of the human adeno-associated virus

Agündez, L., Zarate-Perez, F., Meier, A.F., Bardelli, M., Llosa, M., Escalente, C.R., Linden, R.M. and Henckaerts, E. PloS One, 13(7), e0200841 (2018)   Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase. The purified chimeric protein retained oligomerization properties and DNA helicase activities similar to Rep68, while its DNA binding specificity and cleaving-joining activity at oriT was similar to TrwC. Interestingly, the chimeric protein could catalyse site-specific integration in bacteria with an efficiency comparable to that of TrwC, while the HUH domain of TrwC alone was unable to catalyze this reaction, implying that the Rep68 C-terminal helicase domain is complementing the TrwC HUH domain to achieve site-specific integration into TrwC targets in bacteria. Our results illustrate how HUH domains could have acquired through evolution other domains in order to attain new roles, contributing to the functional flexibility observed in this protein superfamily.  

5.2356           A Recombinant Baculovirus Efficiently Generates Recombinant Adeno-Associated Virus Vectors in Cultured Insect Cells and Larvae

Wu, Y., Jiang, L., Geng, H., Yang, T., Han, Z., He, X., Lin, K. and Xu, F. Molecular Therapy – Methods in Clin. Develop., 10, 38-47 (2018)   Current large-scale recombinant adeno-associated virus (rAAV) production systems based on the baculovirus expression vector (BEV) remain complicated and cost-intensive, and they lack versatility and flexibility. Here we present a novel recombinant baculovirus integrated with all packaging elements for the production of rAAV. To optimize BEV construction, ribosome leaky-scanning mechanism was used to express AAV Rep and Cap proteins downstream of the PH and P10 promoters in the pFast.Bac.Dual vector, respectively, and the rAAV genome was inserted between the two promoters. The yields of rAAV2, rAAV8, and rAAV9 derived from the BEV-infected Sf9 cells exceeded 105 vector genomes (VG) per cell. The BEV was shown to be stable and showed no apparent decrease of rAAV yield after at least four serial passages. The rAAVs derived from the new Bac system displayed high-quality and high-transduction activity. Additionally, rAAV2 could be efficiently generated from BEV-infected beet armyworm larvae at a per-larvae yield of 2.75 ± 1.66 × 1010 VG. The rAAV2 derived from larvae showed a structure similar to the rAAV2 derived from HEK293 cells, and it also displayed high-transduction activity. In summary, the novel BEV is ideally suitable for large-scale rAAV production. Further, this study exploits a potential cost-efficient platform for rAAV production in insect larvae.  

5.2357           An Isolated Limb Infusion Method Allows for Broad Distribution of rAAVrh74.MCK.GALGT2 to Leg Skeletal Muscles in the Rhesus Macaque

Xu, R., Jia, Y., Zygmunt, D.A., Cramer, M.L., Crowe, K.E. et al Molecular Therapy – Methods in Clin. Develop., 10, 89-104 (2018)   Recombinant adeno-associated virus (rAAV)rh74.MCK.GALGT2 is a muscle-specific gene therapy that is being developed to treat forms of muscular dystrophy. Here we report on an isolated limb infusion technique in a non-human primate model, where hindlimb blood flow is transiently isolated using balloon catheters to concentrate vector in targeted leg muscles. A bilateral dose of 2.5 × 10¹³ vector genomes (vg)/kg/limb was sufficient to induce GALGT2-induced glycosylation in 10%–60% of skeletal myofibers in all leg muscles examined. There was a 19-fold ± 6-fold average limb-wide increase in vector genomes per microgram genomic DNA at a bilateral dose of 2.5 × 10¹³ vg/kg/limb compared with a bilateral dose of 6 × 10¹² vg/kg/limb. A unilateral dose of 6 × 10¹³ vg/kg/limb showed a 12- ± 3-fold increase in treated limb muscles compared to contralateral untreated limb muscles, which received vector only after release into the systemic circulation from the treated limb. Variability in AAV biodistribution between different segments of the same muscle was 125% ± 18% for any given dose, while variability between the same muscle for any given treatment dose was 45% ± 7%. These experiments demonstrate that treatment of muscles throughout the leg with rAAVrh74.MCK.GALGT2 can be accomplished safely using an isolated limb infusion technique, where balloon catheters transiently isolate the limb vasculature, but that intra- and inter-muscle transduction variability is a significant issue.  

5.2358           Combination of IAP Antagonists and TNF-α-Armed Oncolytic Viruses Induce Tumor Vascular Shutdown and Tumor Regression

Beug, S.T., Pichette, S.J., St-Jean, M., Holbrook, J., Walker, D.E., LaCasse, E.C. and Korneluk, R.G. Molecular Therapy – Oncolytics, 10, 28-39 (2018)   Smac mimetic compounds (SMCs) are anti-cancer drugs that antagonize Inhibitor of Apoptosis proteins, which consequently sensitize cancer cells to death in the presence of proinflammatory ligands such as tumor necrosis factor alpha (TNF-α). SMCs synergize with the attenuated oncolytic vesicular stomatitis virus (VSVΔ51) by eliciting an innate immune response, which is dependent on the endogenous production of TNF-α and type I interferon. To improve on this SMC-mediated synergistic response, we generated TNF-α-armed VSVΔ51 to produce elevated levels of this death ligand. Due to ectopic expression of TNF-α from infected cells, a lower viral dose of TNF-α-armed VSVΔ51 combined with treatment of the SMC LCL161 was sufficient to improve the survival rate compared to LCL161 and unarmed VSVΔ51 co-therapy. This improved response is attributed to a bystander effect whereby the spread of TNF-α from infected cells leads to the death of uninfected cells in the presence of LCL161. In addition, the treatments induced vascular collapse in solid tumors with a concomitant increase of tumor cell death, revealing another mechanism by which cytokine-armed VSVΔ51 in combination with LCL161 can kill tumor cells. Our studies demonstrate the potential for cytokine-engineered oncolytic virus and SMCs as a new combination immunotherapy for cancer treatment.  

5.2359           Critical Role of the Human T-Cell Leukemia Virus Type 1 Capsid N-Terminal Domain for Gag-Gag Interactions and Virus Particle Assembly

Martin, J.L., Mendonca, L.M., marusinec, R., Zuczek, J., Angert, I., Blower, R.J., Mueller, J.D., Perilla, J.R., Zhang, W. and Mansky, L.M.

1. Virol., 92(14), e00333-18 (2018)

  The retroviral Gag protein is the main structural protein responsible for virus particle assembly and release. Like human immunodeficiency virus type 1 (HIV-1) Gag, human T-cell leukemia virus type 1 (HTLV-1) has a structurally conserved capsid (CA) domain, including a _-hairpin turn and a centralized coiled-coil-like structure of six _ helices in the CA amino-terminal domain (NTD), as well as four _-helices in the CA carboxy-terminal domain (CTD). CA drives Gag oligomerization, which is critical for both immature Gag lattice formation and particle production. The HIV-1 CA CTD has previously been shown to be a primary determinant for CA-CA interactions, and while both the HTLV-1 CA NTD and CTD have been implicated in Gag-Gag interactions, our recent observations have implicated the HTLV-1 CA NTD as encoding key determinants that dictate particle morphology. Here, we have conducted alanine-scanning mutagenesis in the HTLV-1 CA NTD nucleotide-encoding sequences spanning the loop regionsand amino acids at the beginning and ends of _-helices due to their structural dissimilarity from the HIV-1 CA NTD structure. We analyzed both Gag subcellular distribution and efficiency of particle production for these mutants. We discovered several important residues (i.e., M17, Q47/F48, and Y61). Modeling implicated that these residues reside at the dimer interface (i.e., M17 and Y61) or at the trimer interface (i.e., Q47/F48). Taken together, these observations highlight the critical role of the HTLV-1 CA NTD in Gag-Gag interactions and particle assembly, which is, to the best of our knowledge, in contrast to HIV-1 and other retroviruses.  

5.2360           Bag2 Is a Component of a Cytosolic Extraction Machinery That Promotes Membrane Penetration of a Nonenveloped Virus

Dupzyk, A. and Tsai, B.

1. Virol., 92(15), e00607-18 (2018)

  During entry, the nonenveloped polyomavirus (PyV) simian virus 40 (SV40) traffics from the cell surface to the endoplasmic reticulum (ER), where it penetrates the ER membrane to reach the cytosol; the virus is then transported into the nucleus to cause infection. Although a coherent understanding of SV40's host entry is emerging, how the virus is ejected from the ER into the cytosol remains mysterious. Our previous analyses revealed that the cytosolic Hsc70-SGTA-Hsp105 complex binds to SV40 and extracts it from the ER into the cytosol. We now report that the nucleotide exchange factor (NEF) Bag2 stimulates SV40 release from Hsc70, thereby enabling successful virus arrival at the cytosol, which leads to infection. Hsp105, another NEF of Hsc70, displays a function overlapping that of Bag2, underscoring the importance of this release reaction. Our findings identify a new component of an extraction machinery essential during membrane penetration of a nonenveloped virus and provide further mechanistic insights into this process.  

5.2361           Mapping and Engineering Functional Domains of the Assembly-Activating Protein of Adeno-associated Viruses

Tse, L.V., Moller-Tank, S., Meganck, R.M. and Asokan, A.

1. Virol., 92(14), e00393-18 (2018)

  Adeno-associated viruses (AAVs) encode a unique assembly-activating protein (AAP) within their genomes that is essential for capsid assembly. Studies to date have focused on establishing the role of AAP as a chaperone that mediates the stability, nucleolar transport, and assembly of AAV capsid proteins. Here, we map structure-function correlates of AAP using secondary structure analysis, followed by deletion and substitutional mutagenesis of specific domains, namely, the N-terminal hydrophobic region (HR), conserved core (CC), proline-rich region (PRR), threonine/serine-rich region (T/S), and basic region (BR). First, we establish that the centrally located PRR and T/S are flexible linker domains that can either be deleted completely or replaced by heterologous functional domains that enable ancillary functions such as fluorescent imaging or increased AAP stability. We also demonstrate that the C-terminal BR domains can be substituted with heterologous nuclear or nucleolar localization sequences that display various abilities to support AAV capsid assembly. Further, by replacing the BR domain with immunoglobulin (IgG) Fc domains, we assessed AAP complexation with AAV capsid subunits and demonstrate that the hydrophobic region (HR) and the conserved core (CC) in the AAP N terminus are the sole determinants for viral protein (VP) recognition. However, VP recognition alone is not sufficient for capsid assembly. Our study sheds light on the modular structure-function correlates of AAP and provides multiple approaches to engineer AAP that might prove useful toward understanding and controlling AAV capsid assembly.  

5.2362           In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation

Moreno, A.M., Fu, J. Zhu, J., katrekar, D., Shih, Y-R.V., marlett, J., Cabotaje, j., tat, J., Naughton, J., Lisowski, L., Varghese, S., Zhang, K. and mali, P. Molecular Therapy, 26(7), 1818-1827 (2018)   Development of efficacious in vivo delivery platforms for CRISPR-Cas9-based epigenome engineering will be critical to enable the ability to target human diseases without permanent modification of the genome. Toward this, we utilized split-Cas9 systems to develop a modular adeno-associated viral (AAV) vector platform for CRISPR-Cas9 delivery to enable the full spectrum of targeted in situ gene regulation functionalities, demonstrating robust transcriptional repression (up to 80%) and activation (up to 6-fold) of target genes in cell culture and mice. We also applied our platform for targeted in vivo gene-repression-mediated gene therapy for retinitis pigmentosa. Specifically, we engineered targeted repression of Nrl, a master regulator of rod photoreceptor determination, and demonstrated Nrl knockdown mediates in situ reprogramming of rod cells into cone-like cells that are resistant to retinitis pigmentosa-specific mutations, with concomitant prevention of secondary cone loss. Furthermore, we benchmarked our results from Nrl knockdown with those from in vivo Nrl knockout via gene editing. Taken together, our AAV-CRISPR-Cas9 platform for in vivo epigenome engineering enables a robust approach to target disease in a genomically scarless and potentially reversible manner.  

5.2363           Viral RNA load and histological changes in tissues following experimental infection with an arterivirus of possums (wobbly possum disease virus)

Giles, J., Perrott, M., Roe, W., Shrestha, K., Aberdein, D., Morel, P. and Dunowska, M. Virology, 522, 73-80 (2018)   Tissues from Australian brushtail possums (Trichosurus vulpecula) that had been experimentally infected with wobbly possum disease (WPD) virus (WPDV) were examined to elucidate pathogenesis of WPDV infection. Mononuclear inflammatory cell infiltrates were present in livers, kidneys, salivary glands and brains of WPD-affected possums. Specific staining was detected by immunohistochemistry within macrophages in the livers and kidneys, and undefined cell types in the brains. The highest viral RNA load was found in macrophage-rich tissues. The detection of viral RNA in the salivary gland, serum, kidney, bladder and urine is compatible with transmission via close physical contact during encounters such as fighting or grooming, or by contact with an environment that has been contaminated with saliva or urine. Levels of viral RNA remained high in all tissues tested throughout the study, suggesting that on-going virus replication and evasion of the immune responses may be important in the pathogenesis of disease.  

5.2364           Evaluation of bias induced by viral enrichment and random amplification protocols in metagenomic surveys of saliva DNA viruses

Parras-Molto, M., Rodriguez-Galet, A., Suarez-Rodriguez, P. and Lopez-Bueno, A. Microbiome, 6:119 (2018)   Background Viruses are key players regulating microbial ecosystems. Exploration of viral assemblages is now possible thanks to the development of metagenomics, the most powerful tool available for studying viral ecology and discovering new viruses. Unfortunately, several sources of bias lead to the misrepresentation of certain viruses within metagenomics workflows, hindering the shift from merely descriptive studies towards quantitative comparisons of communities. Therefore, benchmark studies on virus enrichment and random amplification protocols are required to better understand the sources of bias. Results We assessed the bias introduced by viral enrichment on mock assemblages composed of seven DNA viruses, and the bias from random amplification methods on human saliva DNA viromes, using qPCR and deep sequencing, respectively. While iodixanol cushions and 0.45 μm filtration preserved the original composition of nuclease-protected viral genomes, low-force centrifugation and 0.22 μm filtration removed large viruses. Comparison of unamplified and randomly amplified saliva viromes revealed that multiple displacement amplification (MDA) induced stochastic bias from picograms of DNA template. However, the type of bias shifted to systematic using 1 ng, with only a marginal influence by amplification time. Systematic bias consisted of over-amplification of small circular genomes, and under-amplification of those with extreme GC content, a negative bias that was shared with the PCR-based sequence-independent, single-primer amplification (SISPA) method. MDA based on random priming provided by a DNA primase activity slightly outperformed those based on random hexamers and SISPA, which may reflect differences in ability to handle sequences with extreme GC content. SISPA viromes showed uneven coverage profiles, with high coverage peaks in regions with low linguistic sequence complexity. Despite misrepresentation of certain viruses after random amplification, ordination plots based on dissimilarities among contig profiles showed perfect overlapping of related amplified and unamplified saliva viromes and strong separation from unrelated saliva viromes. This result suggests that random amplification bias has a minor impact on beta diversity studies. Conclusions Benchmark analyses of mock and natural communities of viruses improve understanding and mitigate bias in metagenomics surveys. Bias induced by random amplification methods has only a minor impact on beta diversity studies of human saliva viromes.  

5.2365           Ancestral Adeno-Associated Virus Vector Delivery of Opsins to Spiral Ganglion Neurons: Implications for Optogenetic Cochlear Implants

Duarte, M.J., Kanumuri, V.V., landegger, L.D., Tarabichi, O., Sinha, S., Meng, X., Hight, A.E., Kozin, E.D., Stankovic, k., Brown, M.C. and Lee, D.J. Molecular Therapy, 26(8), 1931-1939 (2018)   Optogenetics is a transformative technology based on light-sensitive microbial proteins, known as opsins, that enable precise modulation of neuronal activity with pulsed radiant energy. Optogenetics has been proposed as a means to improve auditory implant outcomes by reducing channel interaction and increasing electrode density, but the introduction of opsins into cochlear spiral ganglion neurons (SGNs) in vivo has been challenging. Here we test opsin delivery using a synthetically developed ancestral adeno-associated virus (AAV) vector called Anc80L65. Wild-type C57BL/6 mouse pups were injected via the round window of cochlea with Anc80L65 carrying opsin Chronos under the control of a CAG promoter. Following an incubation of 6–22 weeks, pulsed blue light was delivered to cochlear SGNs via a cochleosotomy approach and flexible optical fiber. Optically evoked auditory brainstem responses (oABRs) and multiunit activity in inferior colliculus (IC) were observed. Post-experiment cochlear histology demonstrated opsin expression in SGNs (mean = 74%), with an even distribution of opsin along the cochlear basal/apical gradient. This study is the first to describe robust SGN transduction, opsin expression, and optically evoked auditory electrophysiology in neonatal mice. Ultimately, this work may provide the basis for a new generation of cochlear implant based on light.  

5.2366           HCV NS5A dimer interface residues regulate HCV replication by controlling its self-interaction, hyperphosphorylation, subcellular localization and interaction with cyclophilin A

Shanmugam, S., Nichols, A.K., Saravanabalaji, D., Welsch, C. and Yi, M. PloS Pathogens, 14(7), e1007177 (2018)   The HCV NS5A protein plays multiple roles during viral replication, including viral genome replication and virus particle assembly. The crystal structures of the NS5A N-terminal domain indicated the potential existence of the NS5A dimers formed via at least two or more distinct dimeric interfaces. However, it is unknown whether these different forms of NS5A dimers are involved in its numerous functions. To address this question, we mutated the residues lining the two different NS5A dimer interfaces and determined their effects on NS5A self-interaction, NS5A-cyclophilin A (CypA) interaction, HCV RNA replication and infectious virus production. We found that the mutations targeting either of two dimeric interfaces disrupted the NS5A self-interaction in cells. The NS5A dimer-interrupting mutations also inhibited both viral RNA replication and infectious virus production with some genotypic differences. We also determined that reduced NS5A self-interaction was associated with altered NS5A-CypA interaction, NS5A hyperphosphorylation and NS5A subcellular localization, providing the mechanistic bases for the role of NS5A self-interaction in multiple steps of HCV replication. The NS5A oligomers formed via different interfaces are likely its functional form, since the residues at two different dimeric interfaces played similar roles in different aspects of NS5A functions and, consequently, HCV replication. In conclusion, this study provides novel insight into the functional significance of NS5A self-interaction in different steps of the HCV replication, potentially, in the form of oligomers formed via multiple dimeric interfaces.  

5.2367           Regulation of striatal cells and goal-directed behavior by cerebellar outputs

Xiao, L., Bornmann, C., Hatstatt-Burkle, L. and Scheiffele, P. Nature Communications, 9:3133 (2018)   The cerebellum integrates descending motor commands and sensory information to generate predictions and detect errors during ongoing behaviors. Cerebellar computation has been proposed to control motor but also non-motor behaviors, including reward expectation and cognitive flexibility. However, the organization and functional contribution of cerebellar output channels are incompletely understood. Here, we elaborate the cell-type specificity of a broad connectivity matrix from the deep cerebellar nuclei (DCN) to the dorsal striatum in mice. Cerebello-striatal connections arise from all deep cerebellar subnuclei and are relayed through intralaminar thalamic nuclei (ILN). In the dorsal striatum, these connections target medium spiny neurons, but also ChAT-positive interneurons, a class of tonically active interneurons implicated in shifting and updating behavioral strategies. Chemogenetic silencing of cerebello-striatal connectivity modifies function of striatal ChAT-positive interneurons. We propose that cerebello-striatal connections relay cerebellar computation to striatal circuits for goal-directed behaviors.  

5.2368           Development of an inducible anti-VEGF rAAV gene therapy strategy for the treatment of wet AMD

Reid, C.A., Nettesheim, E.R., Connor, T.B. and Lipinski, D.M. Scientific Reports, 8:11763 (2018)   Vascular endothelial growth factor (VEGF) is a key mediator in the development and progression of choroidal neovascularization (CNV) in patients with wet age-related macular degeneration (AMD). As a consequence, current treatment strategies typically focus on the administration of anti-VEGF agents, such as Aflibercept (Eylea), that inhibit VEGF function. While this approach is largely successful at counteracting CNV progression, the treatment can require repetitive (i.e. monthly) intravitreal injections of the anti-VEGF agent throughout the patient’s lifetime, imposing a substantial financial and medical burden on the patient. Moreover, repetitive injection of anti-VEGF agents over a period of years may encourage progression of retinal and choroidal atrophy in patients with AMD, leading to a decrease in visual acuity. Herein, we have developed a single-injection recombinant adeno-associated virus (rAAV)-based gene therapy treatment for wet AMD that prevents CNV formation through inducible over-expression of Eylea. First, we demonstrate that by incorporating riboswitch elements into the rAAV expression cassette allows protein expression levels to be modulated in vivo through oral supplementation on an activating ligand (e.g. tetracycline). We subsequently utilized this technology to modulate the intraocular concentration of Eylea following rAAV delivery, leading to nearly complete (p = 0.0008) inhibition of clinically significant CNV lesions in an established mouse model of wet AMD. The results shown in this study pave the way for the development of a personalized gene therapy strategy for the treatment of wet AMD that is substantially less invasive and more clinically adaptable than the current treatment paradigm of repetitive bolus injections of anti-VEGF agents.  

5.2369           Dysregulation of NAD+ Metabolism Induces a Schwann Cell Dedifferentiation Program

Sasaki, Y., Hackett, A.R., Kim, S., Strickland, A. and Milbrandt, J.

1. Neurosci., 38(29), 6546-6562 (2018)

  The Schwann cell (SC) is the major component of the peripheral nervous system (PNS) that provides metabolic and functional support for peripheral axons. The emerging roles of SC mitochondrial function for PNS development and axonal stability indicate the importance of SC metabolism in nerve function and in peripheral neuropathies associated with metabolic disorders. Nicotinamide adenine dinucleotide (NAD⁺) is a crucial molecule in the regulation of cellular metabolism and redox homeostasis. Here, we investigated the roles of NAD⁺ metabolism in SC functions in vivo by mutating NAMPT, the rate-limiting enzyme of NAD⁺ biosynthesis, specifically in SCs. NAMPT SC knock-out male and female mice (NAMPT SCKO mice) had delayed SC maturation in development and developed hypomyelinating peripheral neuropathy without axon degeneration or decreased SC survival. JUN, a master regulator of SC dedifferentiation, is elevated in NAMPT SCKO SCs, suggesting that decreased NAD⁺ levels cause them to arrest at an immature stage. Nicotinic acid administration rescues the NAD⁺ decline and reverses the SC maturation defect and the development of peripheral neuropathy, indicating the central role of NAD⁺ in PNS development. Upon nicotinic acid withdrawal in adulthood, NAMPT SCKO mice showed rapid and severe peripheral neuropathy and activation of ERK/MEK/JUN signaling, which in turn promotes SC dedifferentiation. These data demonstrate the importance of NAD⁺ metabolism in SC maturation and nerve development and maintenance and suggest that altered SC NAD⁺ metabolism could underlie neuropathies associated with diabetes and aging.  

5.2370           Defining the impact of melanopsin missense polymorphisms using in vivo functional rescue

Rodgers, J., Highes, S., Pothecary, C.A., Brown, L.A., Hickey, D.G., Peirson, S.N. and Hankins, M.W. Hum. Mol. Genet., 27(15), 2589-2603 (2018)   Melanopsin (OPN4) is an opsin photopigment expressed within intrinsically photosensitive retinal ganglion cells (ipRGCs) that mediate non-image forming (NIF) responses to light. Two single-nucleotide polymorphisms (SNPs) in human melanopsin (hOPN4), Pro10Leu and Thr394Ile, have recently been associated with abnormal NIF responses to light, including seasonal affective disorder. It has been suggested these behavioural changes are due to altered melanopsin signalling. However, there is currently no direct evidence to support this. Here we have used ipRGC-specific delivery of hOPN4 wild-type (WT), Pro10Leu or Thr394Ile adeno-associated viruses (AAV) to determine the functional consequences of hOPN4 SNPs on melanopsin-driven light responses and associated behaviours. Immunohistochemistry confirmed hOPN4 AAVs exclusively transduced mouse ipRGCs. Behavioural phenotyping performed before and after AAV injection demonstrated that both hOPN4 Pro10Leu and Thr394Ile could functionally rescue pupillary light responses and circadian photoentrainment in Opn4^−/− mice, with no differences in NIF behaviours detected for animals expressing either SNP compared to hOPN4 WT. Multi-electrode array recordings revealed that ipRGCs expressing hOPN4 Thr394Ile exhibit melanopsin-driven light responses with significantly attenuated response amplitude, decreased sensitivity and faster offset kinetics compared to hOPN4 WT. IpRGCs expressing hOpn4 Pro10Leu also showed reduced response amplitude. Collectively these data suggest Thr394Ile and Pro10Leu may be functionally significant SNPs, which result in altered melanopsin signalling. To our knowledge, this study provides the first direct evidence for the effects of hOPN4 polymorphisms on melanopsin-driven light responses and NIF behaviours in vivo, providing further insight into the role of these SNPs in melanopsin function and human physiology.  

5.2371           HIV-1 gag recruits PACSIN2 to promote virus spreading

Popov, S., Popova, E., Inoue, M., Wu, Y. and Göttlinger, H. PNAS, 115(27), 7093-7098 (2018)   The p2b domain of Rous sarcoma virus (RSV) Gag and the p6 domain of HIV-1 Gag contain late assembly (L) domains that engage the ESCRT membrane fission machinery and are essential for virus release. We now show that the PPXY-type RSV L domain specifically recruits the BAR domain protein PACSIN2 into virus-like particles (VLP), in addition to the NEDD4-like ubiquitin ligase ITCH and ESCRT pathway components such as TSG101. PACSIN2, which has been implicated in the remodeling of cellular membranes and the actin cytoskeleton, is also recruited by HIV-1 p6 independent of its ability to engage the ESCRT factors TSG101 or ALIX. Moreover, PACSIN2 is robustly recruited by NEDD4-2s, a NEDD4-like ubiquitin ligase capable of rescuing HIV-1 budding defects. The NEDD4-2s–induced incorporation of PACSIN2 into VLP correlated with the formation of Gag-ubiquitin conjugates, indicating that PACSIN2 binds ubiquitin. Although PACSIN2 was not required for a single cycle of HIV-1 replication after infection with cell-free virus, HIV-1 spreading was nevertheless severely impaired in T cell lines and primary human peripheral blood mononuclear cells depleted of PACSIN2. HIV-1 spreading could be restored by reintroduction of wild-type PACSIN2, but not of a SH3 domain mutant unable to interact with the actin polymerization regulators WASP and N-WASP. Overall, our observations indicate that PACSIN2 promotes the cell-to-cell spreading of HIV-1 by connecting Gag to the actin cytoskeleton.  

5.2372           In Vivo Potency Assay for Adeno-Associated Virus–Based Gene Therapy Vectors Using AAVrh.10 as an Example

De, B.P., Chen, A., Salami, C.O., Van de Graaf, B., Rosenberg, J.B., pagovich, O.E., Sondhi, D., Crystal, R.G. and Kaminsky, S.M. Human Gene Therapy Methods, 29(3), 146-155 (2018)   The development of a drug product requires rigorous methods of characterization and quality control to assure drug potency. Gene therapy products, a relatively new strategy for drug design with very few licensed examples, represent a unique challenge for the measure of potency. Unlike traditional drugs, potency for a gene therapeutic is a tally of the measures of multiple steps, including infectivity, transcription, translation, protein modifications, proper localization of the protein product, and protein function. This is particularly challenging for products based on the adeno-associated virus (AAV) platform, which has poor in vitro infectivity, limiting the sensitivity and thus the usefulness of cell-based assays. A rigorous in vivo assay has been established that separately evaluates infection, transcription, and resulting protein levels with specifications for each based on real time polymerase chain reaction (DNA and RNA) and standard protein assays. For an acceptance criterion, an administered vector must have vector DNA, transgene mRNA, and transgene expressed protein each concurrently meet individual specifications or the production lot fails. Using the AAVrh.10 serotype as a model vector and three different transgenes as examples, the assay is based on intravenous administration of the vector to male mice. At 2 weeks, the harvested liver is homogenized and assessed for vector genome levels (to assess for vector delivery), mRNA (to assess vector infectivity and transcription), and protein in the liver or serum (to assess protein expression). For all AAV vectors, the assay is robust and reproducible: vector DNA (linearity 10²–10⁹ copies, coefficient of variation) intra-assay <0.8%, inter-assay <0.5%; mRNA intra-assay <3.3%, inter-assay <3.4%. The reproducibility of the assay for transgene expressed protein is product specific. This in vivo potency assay is a strategy for characterization and a quantitative lot release test, providing a path forward to meet regulatory drug requirements for any AAV gene therapy vectors.  

5.2373           Biology of the Adrenal Gland Cortex Obviates Effective Use of Adeno-Associated Virus Vectors to Treat Hereditary Adrenal Disorders

Markmann, S., De, B.P., Reid, J., Jose, C.L., Rosenberg, J.B., Leopold, P.L., Kaminsky, S.M., Sondhi, D., Pagovich, O. and Crystal, R.G. Human Gene Therapy, 29(4), 403-412 (2018)   Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder occurring in 1:10,000 to 1:20,000 live births. In >95% of the cases, CAH results from mutations in the CYP21A2 gene, encoding the adrenal steroid enzyme 21-hydroxylase (21OH). Cardinal phenotypic features of CAH include genital ambiguity and sexual precocity, and in severe cases, neonatal salt loss and death. Current standard of care consists of lifelong oral steroid replacement to reverse the cortisol deficiency. Although significant advances in the treatment of CAH have been made, the burden of a lifelong therapeutic intervention is not ideal for quality of life. Gene therapy for CAH by adeno-associated virus (AAV) vectors has been shown to efficiently transduce the adrenal cortex, restoring normal steroidogenesis in the short term. However, adrenocortical cells are continuously renewed by stem cells located at the adrenal capsule, which differentiate as they centripetally migrate towards the adrenal medulla where they undergo apoptosis. In this context, we hypothesized that AAV-mediated genetic correction of the adrenal cortex will work short term but will eventually lead to a loss of correction. To test this hypothesis, we administered intravenously an AAV serotype rh.10 gene transfer vector (AAVrh.10-21OH-HA) to 21-hydroxylase deficient mice (21OH^−/−). The data demonstrates that a single intravenous administration efficiently transduces adrenocortical cells leading to 21OH-HA expression and restoration of normal steroidogenesis. However, the duration of therapeutic efficacy lasted for only 8 weeks, accompanied by loss of 21OH-HA expression in the adrenal gland. Analysis in immunodeficient mice confirmed that the disappearance of transgene expression was not due to an antiviral/transgene immune response. Taken together, these results demonstrate that a single treatment with an adeno-associated viral vector expressing a functional copy of the mutated gene can only transiently treat adrenocortical hereditary disorders and that strategies to genetically modify the adrenocortical stem cells population will likely be required.  

5.2374           Aldolase promotes the development of cardiac hypertrophy by targeting AMPK signaling

Li, Y., Zhang, D., Kong, L., Shi, H., Tian, X., Gao, L., Liu, Y., Wu, L., Du, B., Huang, Z., Liang, C., Wang, Z., Yao, R. and Zhang, Y. Exp. Cell Res., 370, 78-86 (2018)   Metabolic dysfunction is a hallmark of cardiac hypertrophy and heart failure. During cardiac failure, the metabolism of cardiomyocyte switches from fatty acid oxidation to glycolysis. However, the roles of key metabolic enzymes in cardiac hypertrophy are not understood fully. Here in the present work, we identified Aldolase A (AldoA) as a core regulator of cardiac hypertrophy. The mRNA and protein levels of AldoA were significantly up-regulated in transverse aortic constriction (TAC)- and isoproterenol (ISO)-induced hypertrophic mouse hearts. Overexpression of AldoA in cardiomyocytes promoted ISO-induced cardiomyocyte hypertrophy, whereas AldoA knockdown repressed cardiomyocyte hypertrophy. In addition, adeno-associated virus 9 (AAV9)-mediated in vivo knockdown of AldoA in the hearts rescued ISO-induced decrease in cardiac ejection fraction and fractional shortening and repressed cardiac hypertrophy. Mechanism study revealed that AldoA repressed the activation of AMP-dependent protein kinase (AMPK) signaling in a liver kinase B1 (LKB1)-dependent and AMP-independent manner. Inactivation of AMPK is a core mechanism underlying AldoA-mediated promotion of ISO-induced cardiomyocyte hypertrophy. By contrast, activation of AMPK with metformin and AICAR blocked AldoA function during cardiomyocyte hypertrophy. In summary, our data support the notion that AldoA-AMPK axis is a core regulatory signaling sensing energetic status and participates in cardiac hypertrophy.  

5.2375           Direct Visualization of the Conformational Dynamics of Single Influenza Hemagglutinin Trimers

Das, D.K., Govindan, R., Nikic-Spiegel, I., krammer, F., Lemke, E.A. and Monro, J.B.

Cell, 174, 926-937 (2018)

Influenza hemagglutinin (HA) is the canonical type I viral envelope glycoprotein and provides a template for the membrane-fusion mechanisms of numerous viruses. The current model of HA-mediated membrane fusion describes a static “spring-loaded” fusion domain (HA2) at neutral pH. Acidic pH triggers a singular irreversible conformational rearrangement in HA2 that fuses viral and cellular membranes. Here, using single-molecule Förster resonance energy transfer (smFRET)-imaging, we directly visualized pH-triggered conformational changes of HA trimers on the viral surface. Our analyses reveal reversible exchange between the pre-fusion and two intermediate conformations of HA2. Acidification of pH and receptor binding shifts the dynamic equilibrium of HA2 in favor of forward progression along the membrane-fusion reaction coordinate. Interaction with the target membrane promotes irreversible transition of HA2 to the post-fusion state. The reversibility of HA2 conformation may protect against transition to the post-fusion state prior to arrival at the target membrane.

5.2376           Transient expression of heat- and acid-resistant foot-and-mouth disease virus P1-2A mutants in Nicotiana benthamiana

Veerapen, V.P., van Zyl, A.R., Rybicki, E.P. and Meyers, A.E. Virus Res., 256, 45-49 (2018)   Recombinant foot-and-mouth disease virus-like particles (VLPs) can be expressed in a number of expression systems including plants. However, yields in plants have formerly been shown to be low, possibly due to their acid and/or heat lability, previously shown to affect VLP yields produced in other systems. This work describes the introduction of mutations into the FMDV structural protein-encoding gene (P1-2A) which have been previously shown to increase acid and thermostability. VLPs expressed in plants using the mutant constructs had negative rather than positive effects on yield and temperature and acid stability compared to the control.  

5.2377           Therapeutic efficacy of regulable GDNF expression for Huntington's and Parkinson's disease by a high-induction, background-free “GeneSwitch” vector

Cheng, S., Tereschenko, J., Zimmer, V., Vachey, G., Pythoud, C., Rey, M., Liefhebber, J., Raina, A., Streit, F., Mazur, A., Bähr, M., Konstantinova, p., Dwglon, N. and Kügler, S. Exp. Neurol., 309, 79-90 (2018)   Gene therapy is currently an irreversible approach, without possibilities to fine-tune or halt the expression of a therapeutic gene product. Especially when expressing neurotrophic factors to treat neurodegenerative disorders, options to regulate transgene expression levels might be beneficial. We thus developed an advanced single-genome inducible AAV vector for expression of GDNF, under control of the approved small molecule drug mifepristone. In the rat brain, GDNF expression can be induced over a wide range up to three hundred-fold over endogenous background, and completely returns to baseline within 3–4 weeks. When applied with appropriate serotype and titre, the vector is absolutely free of any non-induced background expression. In the BACHD model of Huntington's disease we demonstrate that the vector can be kept in a continuous ON-state for extended periods of time. In a model of Parkinson's disease we demonstrate that repeated short-term expression of GDNF restores motor capabilities in 6-OHDA-lesioned rats. We also report on sex-dependent pharmacodynamics of mifepristone in the rodent brain. Taken together, we show that wide-range and high-level induction, background-free, fully reversible and therapeutically active GDNF expression can be achieved under tight pharmacological control by this novel AAV - “Gene Switch” vector.  

5.2378           A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells

Vakulskas, C.A., Dever, D.P., Rettig, G.R., Turk, R. Jacobi, A.M. et al Nature Med., 24, 1216-1224 (2018)   Translation of the CRISPR–Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications.  

5.2379           Hippocampal PPARα is a novel therapeutic target for depression and mediates the antidepressant actions of fluoxetine in mice

Song, L., Wang, H., Wang, Y-J., Wang, J-L., Zhu, Q., Wu, F., Zhang, W. and Jiang, B. Br.J. Pharmacol., 175, 2968-2987 (2018)   Background and Purpose Developing novel pharmacological targets beyond the monoaminergic system is now a popular strategy for treating depression. PPARα is a nuclear receptor protein that functions as a transcription factor,‐regulating gene expression. We have previously reported that both WY14643 and fenofibrate, two pharmacological agonists of PPARα, have antidepressant‐like effects in mice, implying that PPARα is a potential antidepressant target. Experimental Approach We first used various biotechnological methods to evaluate the effects of chronic stress and fluoxetine on hippocampal PPARα. The viral‐mediated genetic approach was then employed to explore whether hippocampal PPARα was an antidepressant target. PPARα inhibitors, PPARα‐knockout (KO) mice and PPARα‐knockdown (KD) mice were further used to determine the role of PPARα in the antidepressant effects of fluoxetine. Key Results Chronic stress significantly decreased mRNA and protein levels of PPARα in the hippocampus, but not other regions, and also fully reduced the recruitment of hippocampal PPARα to the cAMP response element‐binding (CREB) promoter. Genetic overexpression of hippocampal PPARα induced significant antidepressant‐like actions in mice by promoting CREB‐mediated biosynthesis of brain‐derived neurotrophic factor. Moreover, fluoxetine notably restored the stress‐induced negative effects on hippocampal PPARα. Using PPARα antagonists fully blocked the antidepressant effects of fluoxetine in mice, and similarly, both PPARα‐KO and PPARα‐KD abolished the effects of fluoxetine. Besides, PPARα‐KO and PPARα‐KD aggravated depression in mice. Conclusions and Implications Hippocampal PPARα is a potential novel antidepressant target that mediates the antidepressant actions of fluoxetine in mice.  

5.2380           N6-Methyladenosine–binding proteins suppress HIV-1 infectivity and viral production

Lu, W., Timuru, N., St. Gelais, C., Koneru, P.C., Liu, C., Kvaratskhelia, M., He, C. and Wu, L.

1. Biol. Chem., 293(34), 12992-13005 (2018)

  The internal N⁶-methyladenosine (m⁶A) modification of cellular mRNA regulates post-transcriptional gene expression. The YTH domain family proteins (YTHDF1–3 or Y1–3) bind to m⁶A-modified cellular mRNAs and modulate their metabolism and processing, thereby affecting cellular protein translation. We previously reported that HIV-1 RNA contains the m⁶A modification and that Y1–3 proteins inhibit HIV-1 infection by decreasing HIV-1 reverse transcription activity. Here, we investigated the mechanisms of Y1–3–mediated inhibition of HIV-1 infection in target cells and the effect of Y1–3 on viral production levels in virus-producing cells. We found that Y1–3 protein overexpression in HIV-1 target cells decreases viral genomic RNA (gRNA) levels and inhibits both early and late reverse transcription. Purified recombinant Y1–3 proteins preferentially bound to the m⁶A-modified 5′ leader sequence of gRNA compared with its unmodified RNA counterpart, consistent with the strong binding of Y1–3 proteins to HIV-1 gRNA in infected cells. HIV-1 mutants with two altered m⁶A modification sites in the 5′ leader sequence of gRNA exhibited significantly lower infectivity than WT, replication-competent HIV-1, confirming that these sites alter viral infection. HIV-1 produced from cells in which endogenous Y1, Y3, or Y1–3 proteins were knocked down singly or together had increased viral infectivity compared with HIV-1 produced in control cells. Interestingly, we found that Y1–3 proteins and HIV-1 Gag protein formed a complex with RNA in HIV-1–producing cells. Overall, these results indicate that Y1–3 proteins inhibit HIV-1 infection and provide new insights into the mechanisms by which the m⁶A modification of HIV-1 RNA affects viral replication.  

5.2381           Production and characterization of a novel HPV anti-L2 monoclonal antibody panel

Bywaters, S.M., Brendle, S.A., Biryukov, J., Wang, J.W., Walston, J., Milici, J., Roden, R.B., Meyers, C. and Christensen, N.D. Virology, 524, 106-113 (2018)   The major capsid protein of HPV, L1, assembles into pentamers that form a T = 7 icosahedral particle, but the location of the co-assembled minor capsid protein, L2, remains controversial. Several researchers have developed useful monoclonal antibodies targeting L2, but most react with linear epitopes toward the N-terminus. As a means to better define the virus capsid and better assess the localization and exposure of L2 epitopes in the context of assembled HPV, we have developed a panel of 30 monoclonal antibodies (mAbs) which target the N-terminus of L2 amino acids 11–200, previously defined as a broadly protective immunogen. Select mAbs were processed with enzymes and anti-L2 Fabs were generated. These new mAb/Fab probes will be beneficial in future studies to unravel the placement of L2 and to help better define the role of L2 in the HPV lifecycle and the nature of the broadly protective epitopes.  

5.2382           Design of a Novel Gene Therapy Construct to Achieve Sustained Brain-Derived Neurotrophic Factor Signaling in Neurons

Osborne, A., Wang, A.X.Z., Tassoni, A., Widdowson, P.S. and Martin, K.R. Human Gene Ther., 29(7), 828-841 (2018)   Brain-derived neurotrophic factor (BDNF) acting through the tropomyosin-related receptor-B (TrkB) is an important signaling system for the maintenance and survival of neurons. Gene therapy using either recombinant adeno-associated virus (AAV) or lentiviral vectors can provide sustained delivery of BDNF to tissues where reduced BDNF signaling is hypothesized to contribute to disease pathophysiology. However, elevation in BDNF at target sites has been shown to lead to a downregulation of TrkB receptors, thereby reducing the effect of chronic BDNF delivery over time. A novel gene sequence has been designed coding both the ligand (BDNF) and the TrkB receptor in a single transgene separated by a short viral-2A sequence. The single transgene is efficiently processed intracellularly in vitro and in vivo to yield the two mature proteins, which are then independently transported to their final cellular locations: TrkB receptors to the cell surface, and BDNF contained within secretory vesicles. To accommodate the coding sequences of both BDNF and TrkB receptors within the narrow confines of the AAV vectors (4.7 kb pairs), the coding region for the pro-domain of BDNF was removed and the signal peptide sequence modified to improve production, intracellular transport, and secretion of mature BDNF (mBDNF). Intracellular processing and efficacy was shown in HEK293 cells and SH-SY5Y neuroblastoma cells using plasmid DNA and after incorporating the TrkB-2A-mBDNF into an AAV2 vector. Increased BDNF/TrkB-mediated intracellular signaling pathways were observed after AAV2 vector transfection while increased TrkB phosphorylation could be detected in combination with neuroprotection from hydrogen peroxide–induced oxidative stress. Correct processing was also shown in vivo in mouse retinal ganglion cells after AAV2 vector administration to the eye. This novel construct is currently being investigated for its efficacy in animal models to determine its potential to progress to human clinical studies in the future.  

5.2383           A Calsequestrin Cis-Regulatory Motif Coupled to a Cardiac Troponin T Promoter Improves Cardiac Adeno-Associated Virus Serotype 9 Transduction Specificity

Chamberlain, K., Riyad, J.M., garnett, T., Kohlbrenner, E., Mookerjee, A., Elmastour, F., Benard, L., Chen, J., VandenDriessche, T., Chuah, M.K., Marian, A.J., Hajjar, R., Gurha, P. and Weber, T. Human Gene Ther., 29(8), 927-937 (2018)   Adeno-associated virus serotype 9 (AAV9) is an efficient vector for gene transfer to the myocardium. However, the use of ubiquitous promoters, such as the cytomegalovirus (CMV) promoter, can result in expression of the transgene in organs other than the heart. This study tested if the efficiency and specificity of cardiac transcription from a chicken cardiac troponin T (TnT) promoter could be further increased by incorporating a cardiomyocyte-specific transcriptional cis-regulatory motif from human calsequestrin 2 (CS-CRM4) into the expression cassette (Enh.TnT). The efficiency of luciferase expression from the TnT and Enh.TnT constructs was compared to expression of luciferase under the control of the CMV promoter in both adult and neonatal mice. Overall, expression levels of luciferase in the heart were similar in mice injected with AAV9.TnT.Luc, AAV9.Enh.TnT.Luc and AAV9.CMV.Luc. In contrast, expression levels of luciferase activity in nontarget organs, including the liver and muscle, was lower in mice injected with the AAV9.TnT.Luc compared to AAV9.CMV.Luc and was negligible with AAV9.Enh.TnT. In neonates, in organs other than the heart, luciferase expression levels were too low to be quantified for all constructs. Taken together, the data show that the AAV9 Enh.TnT constructs drives high levels of expression of the transgene in the myocardium, with insignificant expression in other organs. These properties reduce the risks associated with the AAV9-mediated expression of the therapeutic protein of interest in nontarget organs. The excellent cardiac specificity should allow for the use of higher vector doses than are currently used, which might be essential to achieve the levels of transgene expression necessary for therapeutic benefits. Taken together, the findings suggest that the Enh.TnT transcription unit is a potentially attractive tool for clinical cardiac gene therapy in adults.  

5.2384           Systemic Gene Delivery by Single-Dose Intracardiac Administration of scAAV2/9 and scAAV2/rh10 Variants in Newborn Rats

Chansel-Debordeaux, L., Bourdenx, M., Dutheil, N., Dovero, S., Canron, M-H., Jimenez, C., Bezard, E. and Dehay, B. Human Gene Ther. Methods, 29(4), 189-199 (2018)   Recombinant adeno-associated virus serotype 9 (rAAV2/9) and pseudotype rhesus-10 (rAAV2/rh10) are used for gene delivery, especially into the central nervous system. Both serotypes cross the blood–brain barrier and mediate stable long-term transduction in dividing and nondividing cells. Among possible routes of administration, intracardiac injection holds the potential for widespread vector diffusion associated with a relatively simple approach. In this study adopting the intracardiac route, we compare the cell-specific tropism and transfection efficacy of a panel of engineered rAAV2/9 and rAAV2/rh10 vectors encoding the enhanced green fluorescent protein. We observed transduction in the brain and peripherally, with a predominant neuronal tropism while the various serotypes achieved different expression patterns.  

5.2385           Bifunctional Chimera That Coordinately Targets Human Immunodeficiency Virus 1 Envelope gp120 and the Host-Cell CCR5 Coreceptor at the Virus–Cell Interface

Rashad, A.A., Song, L-R., Holmes, A.P., Acharya, K., Zhang, S., Wang, Z-L., Gary, E., Xie, X., Pirrone, V., Kutzler, M.A., Long, Y-Q. and Chaiken, I.

1. Med. Chem., 61(11), 5020-5033 (2018)

  To address the urgent need for new agents to reduce the global occurrence and spread of AIDS, we investigated the underlying hypothesis that antagonists of the HIV-1 envelope (Env) gp120 protein and the host-cell coreceptor (CoR) protein can be covalently joined into bifunctional synergistic combinations with improved antiviral capabilities. A synthetic protocol was established to covalently combine a CCR5 small-molecule antagonist and a gp120 peptide triazole antagonist to form the bifunctional chimera. Importantly, the chimeric inhibitor preserved the specific targeting properties of the two separate chimera components and, at the same time, exhibited low to subnanomolar potencies in inhibiting cell infection by different pseudoviruses, which were substantially greater than those of a noncovalent mixture of the individual components. The results demonstrate that targeting the virus–cell interface with a single molecule can result in improved potencies and also the introduction of new phenotypes to the chimeric inhibitor, such as the irreversible inactivation of HIV-1.  

5.2386           Purification and Proteomics of Influenza Virions

Hutchinson, E.C. and Stegmann, M. Methods Mol. Biol., 1836, 89-120 (2018)   This chapter describes a basic workflow for analyzing the protein composition of influenza virions. In order to obtain suitable material, the chapter describes how to concentrate influenza virions from the growth media of infected cells and to purify them by ultracentrifugation through a density gradient. This approach is also suitable for purifying influenza virions from the allantoic fluid of embryonated chicken eggs. As a small quantity of microvesicles are co-purified with virions, optional steps are included to increase the stringency of purification by enriching material with viral receptor binding and cleaving activity. Material purified in this way can be used for a variety of downstream applications, including proteomics. As a detailed example of this, the chapter also describes a standard workflow for analyzing the protein composition of concentrated virions by liquid chromatography and tandem mass spectrometry.  

5.2387           Deciphering Virus Entry with Fluorescently Labeled Viral Particles

Hoffmann, A.B., Mazelier, m., Leger, P. and Lozach, P-Y. Methods Mol. Biol., 1836, 159-183 (2018)   To infect host cells, viruses have to gain access to the intracellular compartment. The infection process starts with the attachment of viruses to the cell surface. Then a complex series of events, highly dynamic, tightly intricate, and often hard to investigate, follows. This includes virus displacement at the plasma membrane, binding to receptors, signaling, internalization, and release of the viral genome and material into the cytosol. In the past decades, the emergence of sensitive, accurate fluorescence-based technologies has opened new perspectives of investigations in the field. Visualization of single viral particles in fixed and living cells as well as quantification of each virus entry step has been made possible. Here we describe the procedure to fluorescently label viral particles. We also illustrate how to use this powerful tool to decipher the entry of viruses with the most recent fluorescence-based techniques such as high-speed confocal and total internal reflection microscopy, flow cytometry, and fluorimetry.  

5.2388           Human papillomavirus type 16 antagonizes IRF6 regulation of IL-1β

Alnouze, M., Rochefort, P., Parroche, P., Roblot, G., Tout, I., Briat, F., Zannetti, C. et al PloS Pathogens, 14(8), e1007158 (2018)   Human papillomavirus type 16 (HPV16) and other oncoviruses have been shown to block innate immune responses and to persist in the host. However, to avoid viral persistence, the immune response attempts to clear the infection. IL-1β is a powerful cytokine produced when viral motifs are sensed by innate receptors that are members of the inflammasome family. Whether oncoviruses such as HPV16 can activate the inflammasome pathway remains unknown. Here, we show that infection of human keratinocytes with HPV16 induced the secretion of IL-1β. Yet, upon expression of the viral early genes, IL-1β transcription was blocked. We went on to show that expression of the viral oncoprotein E6 in human keratinocytes inhibited IRF6 transcription which we revealed regulated IL-1β promoter activity. Preventing E6 expression using siRNA, or using E6 mutants that prevented degradation of p53, showed that p53 regulated IRF6 transcription. HPV16 abrogation of p53 binding to the IRF6 promoter was shown by ChIP in tissues from patients with cervical cancer. Thus E6 inhibition of IRF6 is an escape strategy used by HPV16 to block the production IL-1β. Our findings reveal a struggle between oncoviral persistence and host immunity; which is centered on IL-1β regulation.  

5.2389           Restoration of vision after de novo genesis of rod photoreceptors in mammalian retinas

Yao, K., Qiu, S., Wang, Y.V., Park, S.J.H., Mohns, E.J., Mehta, B., Liu, X., Chang, B., Zenisek, D., Crair, M.C:, Demb, J.B. and Chen, B. Nature, 560, 484-488 (2018) In zebrafish, Müller glia (MG) are a source of retinal stem cells that can replenish damaged retinal neurons and restore vision1. In mammals, however, MG do not spontaneously re-enter the cell cycle to generate a population of stem or progenitor cells that differentiate into retinal neurons. Nevertheless, the regenerative machinery may exist in the mammalian retina, as retinal injury can stimulate MG proliferation followed by limited neurogenesis2,3,4,5,6,7. Therefore, there is still a fundamental question regarding whether MG-derived regeneration can be exploited to restore vision in mammalian retinas. Gene transfer of β-catenin stimulates MG proliferation in the absence of injury in mouse retinas8. Here we report that following gene transfer of β-catenin, cell-cycle-reactivated MG can be reprogrammed to generate rod photoreceptors by subsequent gene transfer of transcription factors essential for rod cell fate specification and determination. MG-derived rods restored visual responses in Gnat1rd17Gnat2cpfl3 double mutant mice, a model of congenital blindness9,10, throughout the visual pathway from the retina to the primary visual cortex. Together, our results provide evidence of vision restoration after de novo MG-derived genesis of rod photoreceptors in mammalian retinas.  

5.2390           G2019S LRRK2 mutation facilitates α-synuclein neuropathology in aged mice

Novello, S., Arcuri, L., Dovero, S., Dutheil, N., Shimshek, D.R., Bezaard, e. and Morari, M. Neurobiology of Disease, 120, 21-33 (2018)   Fibrillization of α-synuclein is instrumental for the development of Parkinson's disease (PD), thus modulating this process can have profound impact on disease initiation/progression. Here, the impact of the p.G2019S mutation of leucine-rich repeat kinase 2 (LRRK2), which is most frequently associated with familial and sporadic PD, on α-synuclein pathology was investigated. G2019S knock-in mice and wild-type controls were injected with a recombinant adeno-associated viral vector serotype 2/9 (AAV2/9) overexpressing human mutant p.A53T α-synuclein (AAV2/9-hα-syn). Control animals were injected with AAV2/9 carrying green fluorescent protein. Motor behavior, transgene expression, α-syn and pSer129 α-syn load, number of nigral dopamine neurons and density of striatal dopaminergic terminals were evaluated. To investigate the effect of aging, experiments were performed in 3- and 12-month-old mice, evaluated 20 and 12 weeks after virus injection, respectively. hα-syn overexpression induced progressive motor deficits, loss of nigral dopaminergic neurons and striatal terminals, and appearance of proteinase K-resistant aggregates of pSer129 α-syn in both young and old mice. Although no genotype difference was observed in 3-month-old mice, degeneration of nigral dopaminergic neurons was higher in 12-month-old G2019S knock-in mice compared with age-matched wild-type controls (−55% vs −39%, respectively). Consistently, a two-fold higher load of pSer129 α-syn aggregates was found in 12-month-old G2019S knock-in mice. We conclude that G2019S LRRK2 facilitates α-synucleinopathy and degeneration of nigral dopaminergic neurons, and that aging is a major determinant of this effect.  

5.2391           Cell-Penetrating Peptide Mediates Intracellular Membrane Passage of Human Papillomavirus L2 Protein to Trigger Retrograde Trafficking

Zhang, P., da Siva, G.M., Deatherage, C., Burd, C. and DiMaio, D. Cell, 174(6), 1465-1476 (2018)   Cell-penetrating peptides (CPPs) are short protein segments that can transport cargos into cells. Although CPPs are widely studied as potential drug delivery tools, their role in normal cell physiology is poorly understood. Early during infection, the L2 capsid protein of human papillomaviruses binds retromer, a cytoplasmic trafficking factor required for delivery of the incoming non-enveloped virus into the retrograde transport pathway. Here, we show that the C terminus of HPV L2 proteins contains a conserved cationic CPP that drives passage of a segment of the L2 protein through the endosomal membrane into the cytoplasm, where it binds retromer, thereby sorting the virus into the retrograde pathway for transport to the trans-Golgi network. These experiments define the cell-autonomous biological role of a CPP in its natural context and reveal how a luminal viral protein engages an essential cytoplasmic entry factor.  

5.2392           Sub-2 Å Ewald curvature corrected structure of an AAV2 capsid variant

Tan, Y.Z., Aiyer, S., Mietzsch, M., Hull, J.A., Mckenna, R., Grieger, j., Samulski, R.J., Baker, T.S., Agbandje-Mckenna, M. and Lyumkis, D. Nature Communications, 9:3628 (2018)   Single-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities. Here, we exploit several technical advances to improve resolution, including per-particle contrast transfer function (CTF) refinement and correction for Ewald sphere curvature. The latter is demonstrated with several experimental samples and should become more standard as resolutions increase or at lower microscope accelerating voltages. The combined application of the described methods to micrographs recorded on a Titan Krios enables structure determination at ~1.86-Å resolution of an adeno-associated virus serotype 2 variant (AAV2), an important gene-delivery vehicle. The resulting structural details provide an improved model for understanding the biology of AAV that will guide future vector development for gene therapy.  

5.2393           More than Rotating Flagella: Lipopolysaccharide as a Secondary Receptor for Flagellotropic Phage 7-7-1

Gonzalez, F., Helm, R.F., Broadway, K.M. and Scharf, B.E.

1. Bacteriol., 200(19), e0063-18 (2018)

  Bacteriophage 7-7-1, a member of the family Myoviridae, infects the soil bacterium Agrobacterium sp. strain H13-3. Infection requires attachment to actively rotating bacterial flagellar filaments, with flagellar number, length, and rotation speed being important determinants for infection efficiency. To identify the secondary receptor(s) on the cell surface, we isolated motile, phage-resistant Agrobacterium sp. H13-3 transposon mutants. Transposon insertion sites were pinpointed using arbitrary primed PCR and bioinformatics analyses. Three genes were recognized, whose corresponding proteins had the following computationally predicted functions: AGROH133_07337, a glycosyltransferase; AGROH133_13050, a UDP-glucose 4-epimerase; and AGROH133_08824, an integral cytoplasmic membrane protein. The first two gene products are part of the lipopolysaccharide (LPS) synthesis pathway, while the last is predicted to be a relatively small (13.4-kDa) cytosolic membrane protein with up to four transmembrane helices. The phenotypes of the transposon mutants were verified by complementation and site-directed mutagenesis. Additional characterization of motile, phage-resistant mutants is also described. Given these findings, we propose a model for Agrobacterium sp. H13-3 infection by bacteriophage 7-7-1 where the phage initially attaches to the flagellar filament and is propelled down toward the cell surface by clockwise flagellar rotation. The phage then attaches to and degrades the LPS to reach the outer membrane and ejects its DNA into the host using its syringe-like contractile tail. We hypothesize that the integral membrane protein plays an important role in events following viral DNA ejection or in LPS processing and/or deployment. The proposed two-step attachment mechanism may be conserved among other flagellotropic phages infecting Gram-negative bacteria.  

5.2394           Hepatitis E Virus Lifecycle and Identification of 3 Forms of the ORF2 Capsid Protein

Montpellier, C., Wychowski, C. et al Gastroenterology, 154(1), 211-223 (2018)   Background & Aims Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. Approximately 2 billion people live in areas endemic for HEV and are at risk of infection. The HEV genome encodes 3 proteins, including the ORF2 capsid protein. Detailed analyses of the HEV life cycle has been hampered by the lack of an efficient viral culture system. Methods We performed studies with gt3 HEV cell culture–produced particles and patient blood and stool samples. Samples were fractionated on iodixanol gradients and cushions. Infectivity assays were performed in vitro and in human liver chimeric mice. Proteins were analyzed by biochemical and proteomic approaches. Infectious particles were analyzed by transmission electron microscopy. HEV antigen levels were measured with the Wantaï enzyme-linked immunosorbent assay. Results We developed an efficient cell culture system and isolated HEV particles that were infectious in vitro and in vivo. Using transmission electron microscopy, we defined the ultrastructure of HEV cell culture–produced particles and particles from patient sera and stool samples. We also identified the precise sequence of the infectious particle-associated ORF2 capsid protein. In cultured cells and in samples from patients, HEV produced 3 forms of the ORF2 capsid protein: infectious/intracellular ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c). The ORF2i protein associated with infectious particles, whereas the ORF2g and ORF2c proteins were massively secreted glycoproteins not associated with infectious particles. ORF2g and ORF2c were the most abundant antigens detected in sera from patients. Conclusions We developed a cell culture system and characterized HEV particles; we identified 3 ORF2 capsid proteins (ORF2i, ORF2g, and ORFc). These findings will advance our understanding of the HEV life cycle and improve diagnosis.  

5.2395           Functional analyses of Pericentrin and Syne-2 interaction in ciliogenesis

Falk, N., Kessler, K., Schramm, S-F., Boldt, K., Becirovic, E., Michalakis, S., Regus-Leidig, H., Noegel, A.A., Ueffing, M., Thiel, C.T., Roepman, R., Brandstätter, J.H. and Giessl, A.

1. Cell Sci., 131, jcs218487 (2018)

  Pericentrin (Pcnt) is a multifunctional scaffold protein and mutations in the human PCNT gene are associated with several diseases, including ciliopathies. Pcnt plays a crucial role in ciliary development in olfactory receptor neurons, but its function in the photoreceptor-connecting cilium is unknown. We downregulated Pcnt in the retina ex vivo and in vivo via a virus-based RNA interference approach to study Pcnt function in photoreceptors. ShRNA-mediated knockdown of Pcnt impaired the development of the connecting cilium and the outer segment of photoreceptors, and caused a nuclear migration defect. In protein interaction screens, we found that the outer nuclear membrane protein Syne-2 (also known as Nesprin-2) is an interaction partner of Pcnt in photoreceptors. Syne-2 is important for positioning murine photoreceptor cell nuclei and for centrosomal migration during early ciliogenesis. CRISPR/Cas9-mediated knockout of Syne-2 in cell culture led to an overexpression and mislocalization of Pcnt and to ciliogenesis defects. Our findings suggest that the Pcnt–Syne-2 complex is important for ciliogenesis and outer segment formation during retinal development and plays a role in nuclear migration.  

5.2396           Methods for Enrichment and Sequencing of Oral Viral Assemblages: Saliva, Oral Mucosa, and Dental Plaque Viromes

Parrs-Molto, M. and Lopez-Bueno, A. Methods in Mol. Biol., 1838, 143-161 (2018)   The oral cavity is a major portal of entry for human pathogens including viruses. However, metagenomics has revealed that highly personalized and time-persistent bacteriophage assemblages dominate this habitat. Most oral bacteriophages follow lysogenic life cycles, deploying complex strategies to manage bacterial homeostasis. Although bacterial dysbiosis underlies common oral pathologies such as caries and periodontitis, the cause of these bacteria replacements remains obscure, and it is theorized that bacteriophages play an important role. The enormous sensitivity of metagenomics coupled with next-generation sequencing has made technically feasible to address the putative role of bacteriophages in oral dysbiosis and represents a valuable tool to discover new human viruses. This chapter proposes a workflow that consists of a simple viral enrichment protocol, two alternative random amplification methods, and next-generation sequencing to access virome composition in three oral environments: supragingival plaque, saliva, and mucosa. These protocols circumvent some well-known sources of bias, providing genomic information about DNA and RNA viral communities with minimal contamination from human and bacterial sources.  

5.2397           AAVrh.10-Mediated APOE2 Central Nervous System Gene Therapy for APOE4-Associated Alzheimer's Disease

Rosenberg, J.B., Kaplitt, M.G., De, B.P., Chen, A., Flagiello, T., Salami, C. et al Human Gene Ther. Clin Develop., 29(1), 24-47 (2018)   Alzheimer's disease (AD) is a progressive degenerative neurological disorder affecting nearly one in nine elderly people in the United States. Population studies have shown that an inheritance of the apolipoprotein E (APOE) variant APOE4 allele increases the risk of developing AD, whereas APOE2 homozygotes are protected from late-onset AD. It was hypothesized that expression of the “protective” APOE2 variant by genetic modification of the central nervous system (CNS) of APOE4 homozygotes could reverse or prevent progressive neurologic damage. To assess the CNS distribution and safety of APOE2 gene therapy for AD in a large-animal model, intraparenchymal, intracisternal, and intraventricular routes of delivery to the CNS of nonhuman primates of AAVrh.10hAPOE2-HA, an AAVrh.10 serotype coding for an HA-tagged human APOE2 cDNA sequence, were evaluated. To evaluate the route of delivery that achieves the widest extent of APOE2 expression in the CNS, the expression of APOE2 in the CNS was evaluated 2 months following vector administration for APOE2 DNA, mRNA, and protein. Finally, using conventional toxicology assays, the safety of the best route of delivery was assessed. The data demonstrated that while all three routes are capable of mediating ApoE2 expression in AD relevant regions, intracisternal delivery of AAVrh.10hAPOE2-HA safely mediated wide distribution of ApoE2 with the least invasive surgical intervention, thus providing the optimal strategy to deliver vector-mediated human APOE2 to the CNS.  

5.2398           Tissue-dependent expression and translation of circular RNAs with recombinant AAV vectors in vivo

Meganck, R.M., Borchardt, E.K., Castelllanos Rivera, R.M., Scalabrino, M.L., Wilusz, J.E., marzluff, W.F. and Asokan, A. Molecular Therapy – Nucleic Acids, 13, 89-98 (2018)   Circular RNAs (circRNAs) are long-lived, covalently closed RNAs that are abundantly expressed and evolutionarily conserved across eukaryotes. Possible functions ranging from microRNA (miRNA) and RNA binding protein sponges to regulators of transcription and translation have been proposed. Here we describe the design and characterization of recombinant adeno-associated viral (AAV) vectors packaging transgene cassettes containing intronic sequences that promote backsplicing to generate circularized RNA transcripts. Using a split GFP transgene, we demonstrate the capacity of vectors containing different flanking intronic sequences to efficiently drive persistent circRNA formation in vitro. Further, translation from circRNA is efficiently driven by an internal ribosomal entry site (IRES). Upon injecting AAV vectors encoding circRNA in mice, we observed robust transgene expression in the heart, but low transduction in the liver for the intronic elements tested. Expression in the murine brain was restricted to astrocytes following systemic or intracranial administration, while intravitreal injection in the eye yielded robust transgene expression across multiple retinal cell layers. These results highlight the potential for exploiting AAV-based circRNA expression to study circRNA function and tissue-specific regulation in animal models, as well as development of therapeutic platforms using this approach.  

5.2399           The impact of chlorine and heat on the infectivity and physicochemical properties of bacteriophage MS2

Brie, A., Gantzer, C., Boudaud, N. and Bertrand, I. FEMS Microbiol. Ecol., 94(8), fiy106 (2018)   Enteric viruses and bacteriophages are exposed to various inactivating factors outside their host, and among them chlorine and heat are the most commonly used sanitizer in water industry and treatment in the food industry, respectively. Using MS2 phages as models for enteric viruses, we investigated the impact of free chlorine and heat on their physicochemical properties. Free chlorine was first evaluated alone. No increase in either capsid permeability or hydrophobicity was observed. The negative surface charge slightly increased suggesting molecular changes in the capsid. However, a weakening of the capsid by chlorine was suggested by differential scanning fluorimetry. This phenomenon was confirmed when chlorination was followed by a heat treatment. Indeed, an increase in the inactivation of MS2 phages and the permeability of their capsids to RNases was observed. More interestingly, an increase in the expression of hydrophobic domains at the phage surface was observed, but only for phages remaining infectious. The chlorine-caused weakening of the capsid suggested that, for an optimal use, the oxidant should be followed by heat. The increased permeability to RNases and the expression of hydrophobic domains may contribute to the development or improvement of molecular methods specific for infectious enteric viruses.  

5.2400           AAV9 intracerebroventricular gene therapy improves lifespan, locomotor function and pathology in a mouse model of Niemann–Pick type C1 disease

Hughes, M.P., Smith, D.A., Morris, L., Fletcher, C., Colaco, A., Huebecker, M., Tordo, J., Palomar, N., Massaro, G., Henckaerts, E., Waddington, S.N., Platt, F.M. and Rahim, A.A. Hum. Mol. Genet., 27(17), 3079-3098 (2018)   Niemann–Pick type C disease (NP-C) is a fatal neurodegenerative lysosomal storage disorder. It is caused in 95% of cases by a mutation in the NPC1 gene that encodes NPC1, an integral transmembrane protein localized to the limiting membrane of the lysosome. There is no cure for NP-C but there is a disease-modifying drug (miglustat) that slows disease progression but with associated side effects. Here, we demonstrate in a well-characterized mouse model of NP-C that a single administration of AAV-mediated gene therapy to the brain can significantly extend lifespan, improve quality of life, prevent or ameliorate neurodegeneration, reduce biochemical pathology and normalize or improve various indices of motor function. Over-expression of human NPC1 does not cause adverse effects in the brain and correctly localizes to late endosomal/lysosomal compartments. Furthermore, we directly compare gene therapy to licensed miglustat. Even at a low dose, gene therapy has all the benefits of miglustat but without adverse effects. On the basis of these findings and on-going ascendency of the field, we propose intracerebroventricular gene therapy as a potential therapeutic option for clinical use in NP-C.  

5.2401           Neuroprotection of retinal ganglion cells by a novel gene therapy construct that achieves sustained enhancement of brain-derived neurotrophic factor/tropomyosin-related kinase receptor-B signaling

Osborne, A., Khatib, T.Z., Songra, L., Barber, A.C., Hall, K., Kong, Y.X., Widdowson, P.S. and Martin, K.R. Cell Death & Disease, 9:1007 (2018)   Previous studies have demonstrated that intravitreal delivery of brain-derived neurotrophic factor (BDNF) by injection of recombinant protein or by gene therapy can alleviate retinal ganglion cell (RGC) loss after optic nerve injury. BDNF gene therapy can improve RGC survival in experimental models of glaucoma, the leading cause of irreversible blindness worldwide. However, the therapeutic efficacy of BDNF supplementation alone is time limited at least in part due to BDNF receptor downregulation. Tropomyosin-related receptor kinase-B (TrkB) downregulation has been reported in many neurological diseases including glaucoma, potentially limiting the effect of sustained or repeated BDNF delivery.   Here, we characterize a novel adeno-associated virus (AAV) gene therapy (AAV2 TrkB-2A-mBDNF) that not only increases BDNF production but also improves long-term neuroprotective signaling by increasing expression of the BDNF receptor (TrkB) within the inner retina. This approach leads to significant and sustained elevation of survival signaling pathways ERK and AKT within RGCs over 6 months and avoids the receptor downregulation which we observe with treatment with AAV2 BDNF alone. We validate the neuroprotective efficacy of AAV2 TrkB-2A-mBDNF in a mouse model of optic nerve injury, where it outperforms conventional AAV2 BDNF or AAV2 TrkB therapy, before showing powerful proof of concept neuroprotection of RGCs and axons in a rat model of chronic intraocular pressure (IOP) elevation. We also show that there are no adverse effects of the vector on retinal structure or function as assessed by histology and electroretinography in young or aged animals. Further studies are underway to explore the potential of this vector as a candidate for progression into clinical studies to protect RGCs in patients with glaucoma and progressive visual loss despite conventional IOP-lowering treatment.  

5.2402           PF74 Reinforces the HIV-1 Capsid To Impair Reverse Transcription-Induced Uncoating

Rankovic, S., Ramalho, R., Aiken, C. and Rousso, I.

1. Virol., 92(20), e00845-18 (2018)

  The RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed in a cone-shaped capsid shell that disassembles following cell entry via a process known as uncoating. During HIV-1 infection, the capsid is important for reverse transcription and entry of the virus into the target cell nucleus. The small molecule PF74 inhibits HIV-1 infection at early stages by binding to the capsid and perturbing uncoating. However, the mechanism by which PF74 alters capsid stability and reduces viral infection is presently unknown. Here, we show, using atomic force microscopy (AFM), that binding of PF74 to recombinant capsid-like assemblies and to HIV-1 isolated cores stabilizes the capsid in a concentration-dependent manner. At a PF74 concentration of 10 μM, the mechanical stability of the core is increased to a level similar to that of the intrinsically hyperstable capsid mutant E45A. PF74 also prevented the complete disassembly of HIV-1 cores normally observed during 24 h of reverse transcription. Specifically, cores treated with PF74 only partially disassembled: the main body of the capsid remained intact and stiff, and a cap-like structure dissociated from the narrow end of the core. Moreover, the internal coiled structure that was observed to form during reverse transcription in vitro persisted throughout the duration of the measurement (∼24 h). Our results provide direct evidence that PF74 directly stabilizes the HIV-1 capsid lattice, thereby permitting reverse transcription while interfering with a late step in uncoating.  

5.2403           Functional Study of the C-Terminal Part of the Hepatitis C Virus E1 Ectodomain

Moustafa, E.I., Haddad, J.G., Linna, L., Hanoulle, X., Descamps, V., Mesalam, A.A., Baumert, T.F., Duverlie, G., Meuleman, P., Dubuisson, J. and Lavie, M.

1. Virol., 92(20), e00939-18 (2018)

  In the hepatitis C virus (HCV) envelope glycoproteins E1 and E2, which form a heterodimer, E2 is the receptor binding protein and the major target of neutralizing antibodies, whereas the function of E1 remains less characterized. To investigate E1 functions, we generated a series of mutants in the conserved residues of the C-terminal region of the E1 ectodomain in the context of an infectious clone. We focused our analyses on two regions of interest. The first region is located in the middle of the E1 glycoprotein (between amino acid [aa] 270 and aa 291), which contains a conserved hydrophobic sequence and was proposed to constitute a putative fusion peptide. The second series of mutants was generated in the region from aa 314 to aa 342 (the aa314-342 region), which has been shown to contain two α helices (α2 and α3) by nuclear magnetic resonance studies. Of the 22 generated mutants, 20 were either attenuated or noninfectious. Several mutations modulated the virus's dependence on claudin-1 and the scavenger receptor BI coreceptors for entry. Most of the mutations in the putative fusion peptide region affected virus assembly. Conversely, mutations in the α-helix aa 315 to 324 (315-324) residues M318, W320, D321, and M322 resulted in a complete loss of infectivity without any impact on E1E2 folding and on viral assembly. Further characterization of the W320A mutant in the HCVpp model indicated that the loss of infectivity was due to a defect in viral entry. Together, these results support a role for E1 in modulating HCV interaction with its coreceptors and in HCV assembly. They also highlight the involvement of α-helix 315-324 in a late step of HCV entry.  

5.2404           Mapping an Adeno-associated Virus 9-Specific Neutralizing Epitope To Develop Next-Generation Gene Delivery Vectors

Giles, A.R., Govindasamy, L., Somanathan, S. and Wilson, J.M.

1. Virol., 92(20), e01011-18 (2018)

  Recent clinical trials have demonstrated the potential of adeno-associated virus (AAV)-based vectors for treating rare diseases. However, significant barriers remain for the translation of these vectors into widely available therapies. In particular, exposure to the AAV capsid can generate an immune response of neutralizing antibodies. One approach to overcome this response is to map the AAV-specific neutralizing epitopes and rationally design an AAV capsid able to evade neutralization. To accomplish this, we isolated a monoclonal antibody against AAV9 following immunization of BALB/c mice and hybridoma screening. This antibody, PAV9.1, is specific for intact AAV9 capsids and has a high neutralizing titer of >1:160,000. We used cryo-electron microscopy to reconstruct PAV9.1 in complex with AAV9. We then mapped its epitope to the 3-fold axis of symmetry on the capsid, specifically to residues 496-NNN-498 and 588-QAQAQT-592. Capsid mutagenesis demonstrated that even a single amino acid substitution within this epitope markedly reduced binding and neutralization by PAV9.1. In addition, in vivo studies showed that mutations in the PAV9.1 epitope conferred a “liver-detargeting” phenotype to the mutant vectors, unlike AAV9, indicating that the residues involved in PAV9.1 interactions are also responsible for AAV9 tropism. However, we observed minimal changes in binding and neutralizing titer when we tested these mutant vectors for evasion of polyclonal sera from mice, macaques, or humans previously exposed to AAV. Taken together, these studies demonstrate the complexity of incorporating mapped neutralizing epitopes and previously identified functional motifs into the design of novel capsids able to evade immune response.  

5.2405           Longitudinal In Vivo Monitoring of the CNS Demonstrates the Efficacy of Gene Therapy in a Sheep Model of CLN5 Batten Disease

Mitchell, N.L., Russell, K.N., Wellby, M.P., Wicky, H.E., Schoderboeck, L., Barrell, G.K., Melzer, T.R., Gray, S.J., Hughes, S. and palmer, D.N. Molecular Therapy, 26(10), 2366-2378 (2018)   Neuronal ceroid lipofuscinoses (NCLs; Batten disease) are neurodegenerative lysosomal storage diseases predominantly affecting children. Single administration of brain-directed lentiviral or recombinant single-stranded adeno-associated virus 9 (ssAAV9) vectors expressing ovine CLN5 into six pre-clinically affected sheep with a naturally occurring CLN5 NCL resulted in long-term disease attenuation. Treatment efficacy was demonstrated by non-invasive longitudinal in vivo monitoring developed to align with assessments used in human medicine. The treated sheep retained neurological and cognitive function, and one ssAAV9-treated animal has been retained and is now 57 months old, almost triple the lifespan of untreated CLN5-affected sheep. The onset of visual deficits was much delayed. Computed tomography and MRI showed that brain structures and volumes remained stable. Because gene therapy in humans is more likely to begin after clinical diagnosis, self-complementary AAV9-CLN5 was injected into the brain ventricles of four 7-month-old affected sheep already showing early clinical signs in a second trial. This also halted disease progression beyond their natural lifespan. These findings demonstrate the efficacy of CLN5 gene therapy, using three different vector platforms, in a large animal model and, thus, the prognosis for human translation.  

5.2406           Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells

Cromer, M.K., Vaidyanathan, S., Ryan, D.E., Curry, B., Lucas, A.B., Camarena, J., Kaushik, M., Hay, S.R., Martin, R.M., Steinfeld, I., Bak, R.O., Dever, D.P., Hendel, A., Bruhn, L. and Porteus, M.H. Molecular Therapy, 26(10), 2431-2442 (2018)   Genome-editing technologies are currently being translated to the clinic. However, cellular effects of the editing machinery have yet to be fully elucidated. Here, we performed global microarray-based gene expression measurements on human CD34⁺ hematopoietic stem and progenitor cells that underwent editing. We probed effects of the entire editing process as well as each component individually, including electroporation, Cas9 (mRNA or protein) with chemically modified sgRNA, and AAV6 transduction. We identified differentially expressed genes relative to control treatments, which displayed enrichment for particular biological processes. All editing machinery components elicited immune, stress, and apoptotic responses. Cas9 mRNA invoked the greatest amount of transcriptional change, eliciting a distinct viral response and global transcriptional downregulation, particularly of metabolic and cell cycle processes. Electroporation also induced significant transcriptional change, with notable downregulation of metabolic processes. Surprisingly, AAV6 evoked no detectable viral response. We also found Cas9/sgRNA ribonucleoprotein treatment to be well tolerated, in spite of eliciting a DNA damage signature. Overall, this data establishes a benchmark for cellular tolerance of CRISPR/Cas9-AAV6-based genome editing, ensuring that the clinical protocol is as safe and efficient as possible.  

5.2407           Novel engineered, membrane-localized variants of vascular endothelial growth factor (VEGF) protect retinal ganglion cells: a proof-of-concept study

Shen, J., Xiao, R., Bair, J., Wang, F., Vandenberghe, L.H., Dartt, D., Baranov, P. and Ng, Y.S.E. Cell Death & Disease, 9:1018 (2018)   Endogenous vascular endothelial growth factor (VEGF-A) can protect retinal ganglion cells (RGC) from stress-induced cell death in ocular hypertensive glaucoma. To exploit the neuroprotective function of VEGF-A for therapeutic application in ocular disorders such as glaucoma while minimizing unwanted vascular side effects, we engineered two novel VEGF variants, eVEGF-38 and eVEGF-53. These variants of the diffusible VEGF-A isoform VEGF121 are expressed as dimeric concatamers and remain tethered to the cell membrane, thus restricting the effects of the engineered VEGF to the cells expressing the protein. For comparison, we tested a Myc-tagged version of VEGF189, an isoform that binds tightly to the extracellular matrix and heparan sulfate proteoglycans at the cell surface, supporting only autocrine and localized juxtacrine signaling. In human retinal endothelial cells (hREC), expression of eVEGF-38, eVEGF-53, or VEGF189 increased VEGFR2 phosphorylation without increasing expression of pro-inflammatory markers, relative to VEGF165 protein and vector controls. AAV2-mediated transduction of eVEGF-38, eVEGF-53, or VEGF189 into primary mouse RGC promoted synaptogenesis and increased the average total length of neurites and axons per RGC by ~ 12-fold, an increase that was mediated by VEGFR2 and PI3K/AKT signaling. Expression of eVEGF-38 in primary RGC enhanced expression of genes associated with neuritogenesis, axon outgrowth, axon guidance, and cell survival. Transduction of primary RGC with any of the membrane-associated VEGF constructs increased survival both under normal culture conditions and in the presence of the cytotoxic chemicals H2O2 (via VEGFR2/PI3K/AKT signaling) and N-methyl-d-aspartate (via reduced Ca2+ influx). Moreover, RGC number was increased in mouse embryonic stem cell-derived retinal organoid cultures transduced with the eVEGF-53 construct. The novel, engineered VEGF variants eVEGF-38 and eVEGF-53 show promise as potential therapeutics for retinal RGC neuroprotection when delivered using a gene therapy approach.  

5.2408           Interaction between noradrenergic and cholinergic signaling in amygdala regulates anxiety- and depression-related behaviors in mice

Mineur, Y.S., Cahuzac, E.L., Mose, T.N., Bentham, M.P., Plantenga, M.E., Thompson, D.C. and Picciotto, M.R. Neuropsychopharmacol., 43, 2118-2125 (2018)   Medications that target the noradrenergic system are important therapeutics for depression and anxiety disorders. More recently, clinical studies have shown that the α2-noradrenergic receptor (α2AR) agonist guanfacine can decrease stress-induced smoking relapse during acute abstinence, suggesting that targeting the noradrenergic system may aid in smoking cessation through effects on stress pathways in the brain. Acetylcholine (ACh), like the nicotine in tobacco, acts at nicotinic acetylcholine receptors (nAChRs) to regulate behaviors related to anxiety and depression. We therefore investigated interactions between guanfacine and ACh signaling in tests of anxiolytic and antidepressant efficacy in female and male C57BL/6J mice, focusing on the amygdala as a potential site of noradrenergic/cholinergic interaction. The antidepressant-like effects of guanfacine were blocked by shRNA-mediated knockdown of α2AR in amygdala. Knockdown of the high-affinity β2 nAChR subunit in amygdala also prevented antidepressant-like effects of guanfacine, suggesting that these behavioral effects require ACh signaling through β2-containing nAChRs in this brain area. Ablation of NE terminals prevented the anxiolytic- and antidepressant-like effects of the nicotinic partial agonist cytisine, whereas administration of the cholinesterase antagonist physostigmine induced a depression-like phenotype that was not altered by knocking down α2AR in the amygdala. These studies suggest that ACh and NE have opposing actions on behaviors related to anxiety and depression and that cholinergic signaling through β2-containing nAChRs and noradrenergic signaling through α2a receptors in neurons of the amygdala are critical for regulation of these behaviors.  

5.2409           Use of Whole-Genome Sequencing of Adenovirus in Immunocompromised Pediatric Patients to Identify Nosocomial Transmission and Mixed-Genotype Infection

Houldcraft, C.J., Roy, S., Morfopoulou, S., Margetts, B.K., Depledge, D.P., Cudini, J., Shah, D., Brown, J.R., Romero, E.Y., Williams, R., Cloutman-Green, E., Rao, K., Standing, J.F., Hartley, J.C. and Breuer, J.

1. Infect. Dis., 218, 1261-1271 (2018)

  Background Adenoviruses are significant pathogens for the immunocompromised, arising from primary infection or reinfection. Serotyping is insufficient to support nosocomial transmission investigations. We investigate whether whole-genome sequencing (WGS) provides clinically relevant information on transmission among patients in a pediatric tertiary hospital. Methods We developed a target-enriched adenovirus WGS technique for clinical samples and retrospectively sequenced 107 adenovirus-positive residual diagnostic samples, including viremias (>5 × 10⁴ copies/mL), from 37 patients collected January 2011–March 2016. Whole-genome sequencing was used to determine genotype and for phylogenetic analysis. Results Adenovirus sequences were recovered from 105 of 107 samples. Full genome sequences were recovered from all 20 nonspecies C samples and from 36 of 85 species C viruses, with partial genome sequences recovered from the rest. Whole-genome phylogenetic analysis suggested linkage of 3 genotype A31 cases and uncovered an unsuspected epidemiological link to an A31 infection first detected on the same ward 4 years earlier. In 9 samples from 1 patient who died, we identified a mixed genotype adenovirus infection. Conclusions Adenovirus WGS from clinical samples is possible and useful for genotyping and molecular epidemiology. Whole-genome sequencing identified likely nosocomial transmission with greater resolution than conventional genotyping and distinguished between adenovirus disease due to single or multiple genotypes.  

5.2410           Cofilin-1 phosphorylation catalyzed by ERK1/2 alters cardiac actin dynamics in dilated cardiomyopathy caused by lamin A/C gene mutation

Chatzifrangkesou, M., Yadin, d., Marais, T., Chardonnet, S., Cohen-Tannoudji, M. et al Hum. Mol. Genet., 27(17), 3060-3078 (2018)   Hyper-activation of extracellular signal-regulated kinase (ERK) 1/2 contributes to heart dysfunction in cardiomyopathy caused by mutations in the lamin A/C gene (LMNA cardiomyopathy). The mechanism of how this affects cardiac function is unknown. We show that active phosphorylated ERK1/2 directly binds to and catalyzes the phosphorylation of the actin depolymerizing factor cofilin-1 on Thr25. Cofilin-1 becomes active and disassembles actin filaments in a large array of cellular and animal models of LMNA cardiomyopathy. In vivo expression of cofilin-1, phosphorylated on Thr25 by endogenous ERK1/2 signaling, leads to alterations in left ventricular function and cardiac actin. These results demonstrate a novel role for cofilin-1 on actin dynamics in cardiac muscle and provide a rationale on how increased ERK1/2 signaling leads to LMNA cardiomyopathy.  

5.2411           Serotype survey of AAV gene delivery via subconjunctival injection in mice

Song, L., llanga, T., Conatser, L.M., Zaric, V., Gilger, B.C.and Hirsch, M.L Gene Therapy, 25, 402-414 (2018)   AAV gene therapy approaches in the posterior eye resulted in the first FDA-approved gene therapy-based drug. However, application of AAV vectorology to the anterior eye has yet to enter even a Phase I trial. Furthermore, the simple and safe subconjunctival injection has been relatively unexplored in regard to AAV vector transduction. To determine the utility of this route for the treatment of various ocular disorders, a survey of gene delivery via natural AAV serotypes was performed and correlated to reported cellular attachment factors. AAV serotypes packaged with a self-complementary reporter were administered via subconjunctival injection to WT mice. Subconjunctival injection of AAV vectors was without incidence; however, vector shedding in tears was noted weeks following administration. AAV transduction was serotype dependent in anterior segment tissues including the eye lid, conjunctiva, and cornea, as well as the periocular tissues including muscle. Transgene product in the cornea was highest for AAV6 and AAV8, however, their corneal restriction was remarkably different; AAV6 appeared restricted to the endothelium layer while AAV8 efficiently transduced the stromal layer. Reported AAV cellular receptors were not well correlated to vector transduction; although, in some cases they were conserved among mouse and human ocular tissues. Subconjunctival administration of particular AAV serotypes may be a simple and safe targeted gene delivery route for ocular surface, muscular, corneal, and optic nerve diseases.  

5.2412           Activation of GABAergic Neurons in the Nucleus Accumbens Mediates the Expression of Cocaine-Associated Memory

Zhang, T., Deyama, S., Domoto, M., Wada, S., Yanagida, J., Sasase, H., Hinoi, E., Nishitani, N., Nagayasu, K., Kaneko, S. and Kaneda, K. Biol. Pharm. Bull., 41(7), 1084-1088 (2018)   Cocaine-associated environmental cues elicit craving and relapse to cocaine use by recalling the rewarding memory of cocaine. However, the neuronal mechanisms underlying the expression of cocaine-associated memory are not fully understood. Here, we investigated the possible contribution of γ-aminobutyrate (GABA)ergic neurons in the nucleus accumbens (NAc), a key brain region associated with the rewarding and reinforcing effects of cocaine, to the expression of cocaine-associated memory using the conditioned place preference (CPP) paradigm combined with designer receptors exclusively activated by designer drugs (DREADD) technology. The inhibitory DREADD hM4Di was selectively expressed in NAc GABAergic neurons of vesicular GABA transporter-Cre (vGAT-Cre) mice by infusing adeno-associated virus (AAV) vectors. Ex vivo electrophysiological recordings revealed that bath application of clozapine-N-oxide (CNO) significantly hyperpolarized membrane potentials and reduced the number of spikes induced by depolarizing current injections in hM4Di-positive NAc neurons. Additionally, systemic CNO injections into cocaine-conditioned mice 30 min before posttest session significantly reduced CPP scores compared to saline-injected mice. These results indicate that chemogenetic inhibition of NAc GABAergic neurons attenuated the expression of cocaine CPP, suggesting that NAc GABAergic neuronal activation is required for the environmental context-induced expression of cocaine-associated memory.  

5.2413           Self-complementary and tyrosine-mutant rAAV vectors enhance transduction in cystic fibrosis bronchial epithelial cells

Lopes-Pacheco, M., Kitoko, J.Z., Morales, M.M., Petrs-Silva, H. and Rocco, P.R.M. Exp. Cell Res., 372, 99-107 (2018)   Recombinant adeno-associated virus (rAAV) vector platforms have shown considerable therapeutic success in gene therapy for inherited disorders. In cystic fibrosis (CF), administration of first-generation rAAV2 was safe, but clinical benefits were not clearly demonstrated. Therefore, next-generation vectors that overcome rate-limiting steps in rAAV transduction are needed to obtain successful gene therapy for this devastating disease. In this study, we evaluated the effects of single-strand or self-complementary (sc) rAAV vectors containing single or multiple tyrosine-to-phenylalanine (Y-F) mutations in capsid surface-exposed residues on serotypes 2, 8 or 9. For this purpose, CF bronchial epithelial (CFBE) cells were transduced with rAAV vectors, and the transgene expression of enhanced green fluorescence protein (eGFP) was analyzed at different time points. The effects of vectors on the cell viability, host cell cycle and in association with co-adjuvant drugs that modulate intracellular vector trafficking were also investigated. Six rAAV vectors demonstrated greater percentage of eGFP⁺ cells compared to their counterparts at days 4, 7 and 10 post-transduction: rAAV2 Y(272,444,500,730)F, with 1.95-, 3.5- and 3.06-fold increases; rAAV2 Y(252,272,444,500,704,730)F, with 1.65-, 2.12-, and 2-fold increases; scrAAV2 WT, with 1.69-, 2.68-, and 2.32-fold increases; scrAAV8 Y773F, with 57-, 6.06-, and 7-fold increases; scrAAV9 WT, with 7.47-, 4.64-, and 3.66-fold increases; and scrAAV9 Y446F, with 8.39-, 4.62-, and 4.4-fold increases. At days 15, 20, and 30 post-transduction, these vectors still demonstrated higher transgene expression than transfected cells. Although the percentage of eGFP⁺ cells reduced during the time-course analysis, the delta mean fluorescence intensity increased. These vectors also led to increased percentage of cells in G₁-phase without eliciting any cytotoxicity. Prior administration of bortezomib or genistein did not increase eGFP expression in cells transduced with either rAAV2 Y(272,444,500,730)F or rAAV2 Y(252,272,444,500,704,730)F. In conclusion, self-complementary and tyrosine capsid mutations on rAAV serotypes 2, 8, and 9 led to more efficient transduction than their counterparts in CFBE cells by overcoming the intracellular trafficking and second-strand DNA synthesis limitations.  

5.2414           Light Control of the Tet Gene Expression System in Mammalian Cells

Yamada, M., Suzuki, Y., Nagasaki, S.C., Okuno, H. and Imayoshi, I. Cell Reports, 25(2), 487-500 (2018)   Gene expression and its network structure are dynamically altered in multicellular systems during morphological, functional, and pathological changes. To precisely analyze the functional roles of dynamic gene expression changes, tools that manipulate gene expression at fine spatiotemporal resolution are needed. The tetracycline (Tet)-controlled gene expression system is a reliable drug-inducible method, and it is used widely in many mammalian cultured cells and model organisms. Here, we develop a photoactivatable (PA)-Tet-OFF/ON system for precise temporal control of gene expression at single-cell resolution. By integrating the cryptochrome 2-cryptochrome-interacting basic helix-loop-helix 1 (Cry2-CIB1) light-inducible binding switch, expression of the gene of interest is tightly regulated under the control of light illumination and drug application in our PA-Tet-OFF/ON system. This system has a large dynamic range of downstream gene expression and rapid activation/deactivation kinetics. We also demonstrate the optogenetic regulation of exogenous gene expression in vivo, such as in developing and adult mouse brains.  

5.2415           γ-Secretase promotes membrane insertion of the human papillomavirus L2 capsid protein during virus infection

Inoue, T., Zhang, P., Zhang, W., Goodner-Bingham, K., Dupzyk, A., DiMaio, D. and Tsai, B.

1. Cell Biol., 217(10), 3545-3559 (2018)

  Despite their importance as human pathogens, entry of human papillomaviruses (HPVs) into cells is poorly understood. The transmembrane protease γ-secretase executes a crucial function during the early stages of HPV infection, but the role of γ-secretase in infection and the identity of its critical substrate are unknown. Here we demonstrate that γ-secretase harbors a previously uncharacterized chaperone function, promoting low pH–dependent insertion of the HPV L2 capsid protein into endosomal membranes. Upon membrane insertion, L2 recruits the cytosolic retromer, which enables the L2 viral genome complex to enter the retrograde transport pathway and traffic to the Golgi en route for infection. Although a small fraction of membrane-inserted L2 is also cleaved by γ-secretase, this proteolytic event appears dispensable for HPV infection. Our findings demonstrate that γ-secretase is endowed with an activity that can promote membrane insertion of L2, thereby targeting the virus to the productive infectious pathway.  

5.2416           An attenuated replication-competent chikungunya virus with a fluorescently tagged envelope

Jin, J., Sherman, M.B., Chafets, D., Dinglasan, N., Lu, K., Lee, T-H., Carlson, L-A., Muench, M.O. and Simmons, G. PloS Neglected Tropical Dis. 12(7), e0006693 (2018)   Background Chikungunya virus (CHIKV) is the most common alphavirus infecting humans worldwide, causing acute and chronically debilitating arthralgia at a great economic expense. Methodology/Principal findings To facilitate our study of CHIKV, we generated a mCherry tagged replication-competent chimeric virus, CHIKV 37997-mCherry. Single particle cryoEM demonstrated icosahedral organization of the chimeric virus and the display of mCherry proteins on virus surface. CHIKV 37997-mCherry is attenuated in both IFNαR knockout and wild-type mice. Strong anti-CHIKV and anti-mCherry antibody responses were induced in CHIKV 37997-mCherry infected mice. Conclusions/Significance Our work suggests that chimeric alphaviruses displaying foreign antigen can serve as vaccines against both aphaviruses and other pathogens and diseases.  

5.2417           High-resolution structures of HIV-1 Gag cleavage mutants determine structural switch for virus maturation

Mattei, S., Tan, A., Glass, B., Müller, B., Kräusslich, H-G. and Briggs, J.A.G. PNAS, 115(40), E9401-E9410 (2018)   HIV-1 maturation occurs via multiple proteolytic cleavages of the Gag polyprotein, causing rearrangement of the virus particle required for infectivity. Cleavage results in beta-hairpin formation at the N terminus of the CA (capsid) protein and loss of a six-helix bundle formed by the C terminus of CA and the neighboring SP1 peptide. How individual cleavages contribute to changes in protein structure and interactions, and how the mature, conical capsid forms, are poorly understood. Here, we employed cryoelectron tomography to determine morphology and high-resolution CA lattice structures for HIV-1 derivatives in which Gag cleavage sites are mutated. These analyses prompt us to revise current models for the crucial maturation switch. Unlike previously proposed, cleavage on either terminus of CA was sufficient, in principle, for lattice maturation, while complete processing was needed for conical capsid formation. We conclude that destabilization of the six-helix bundle, rather than beta-hairpin formation, represents the main determinant of structural maturation.  

5.2418           Primary Effect of SERCA2a Gene Transfer on Conduction Reserve in Chronic Myocardial Infarction

Motloch, L.J., Cacheux, m., Ishikawa, K., Xie, C., Hu, J., Aguero, J., Fish, K-M-, Hajjar, R.J. and Akar, F.G.

1. Am. Heart Assoc., 7:e009598 (2018)

  Background SERCA2a gene transfer (GT) improves mechano‐electrical function in animal models of nonischemic heart failure Whether SERCA2a GT reverses pre‐established remodeling at an advanced stage of ischemic heart failure is unclear. We sought to uncover the electrophysiological effects of adeno‐associated virus serotype 1.SERCA2a GT following myocardial infarction (MI). Methods and Results Pigs developed mechanical dysfunction 1 month after anterior MI, at which point they received intracoronary adeno‐associated virus serotype 1.SERCA2a (MI+SERCA2a) or saline (MI) and were maintained for 2 months. Age‐matched naive pigs served as controls (Control). In vivo ECG‐and‐hemodynamic properties were assessed before and after dobutamine stress. The electrophysiological substrate was measured using optical action potential (AP) mapping in controls, MI, and MI+SERCA2a preparations. In vivo ECG measurements revealed comparable QT durations between groups. In contrast, prolonged QRS duration and increased frequency of R′ waves were present in MI but not MI+SERCA2a pigs relative to controls. SERCA2a GT reduced in in vivo arrhythmias in response to dobutamine. Ex vivo preparations from MI but not MI+SERCA2a or control pigs were prone to pacing‐induced ventricular tachycardia and fibrillation. Underlying these arrhythmias was pronounced conduction velocity slowing in MI versus MI+SERCA2a at elevated rates leading to ventricular tachycardia and fibrillation. Reduced susceptibility to ventricular tachycardia and fibrillation in MI+SERCA2a pigs was not related to hemodynamic function, contractile reserve, fibrosis, or the expression of Cx43 and Nav1.5. Rather, SERCA2a GT decreased phosphoactive CAMKII‐delta levels by >50%, leading to improved excitability at fast rates. Conclusions SERCA2a GT increases conduction velocity reserve, likely by preventing CAMKII overactivation. Our findings suggest a primary effect of SERCA2a GT on myocardial excitability, independent of altered mechanical function.  

5.2419           Kindlin-1 Regulates Astrocyte Activation and Pain Sensitivity in Rats With Neuropathic Pain

Zhao, B., Pan, Y., Xu, H. and Song, X. Reg. Anesth. Pain Med., 43, 547-553 (2018)   Background and Objectives Astrocyte activation has been implicated in the pathogenesis of neuropathic pain, but the involvement of kindlin-1 in astrocyte activation and neuropathic pain has not yet been illustrated. Using a chronic constriction injury (CCI) rat model of neuropathic pain, we investigated the expression levels of kindlin-1 during neuropathic pain and the influences of kindlin-1 on regulating pain sensitivity. Methods Neuropathic pain was induced in rats by CCI of the sciatic nerve. Rats were randomly assigned to 4 groups: sham operation, CCI, CCI + kindlin-1 short hairpin RNA (shRNA), and CCI + kindlin-1 groups. Animals in the CCI + kindling-1 shRNA and CCI + kindlin-1 groups were given kindlin-1 shRNA or kindlin-1 virus infection to reduce or overexpress kindlin-1, respectively. Kindlin-1 expression was persistently increased in rats 10 days after CCI. A large proportion of glial fibrillary acidic protein (GFAP)–positive astrocytes expressed kindlin-1 in spinal cord tissues of rats after CCI. Results Compared with the sham operation group, CCI animals exhibited increased GFAP expression and GFAP-positive astrocytes in the spinal cord. Down-regulation of kindlin-1 reduced the up-regulation of GFAP in the spinal cord, whereas overexpression of kindlin-1 promoted elevation of GFAP levels. Kindlin-1 silencing elevated the mechanical and thermal pain thresholds of CCI rats (P < 0.05). However, overexpression of kindlin-1 aggravated CCI-induced pain sensitivity. Conclusions Kindlin-1 may regulate pain sensitivity by affecting activated astrocytes in the spinal cord. Inhibition of kindlin-1 may provide a novel paradigm for the management of neuropathic pain.  

5.2420           IL-15 regulates susceptibility of CD4+ T cells to HIV infection

Manganaro, L., Hong, P., Hernandez, M.M., Argyle, D., Mulder, L.C.F., Potla, U., Diaz-Griffero, F., Lee, B., Fernandez-Sesma, A. and Simon, V. PNAS, 115(41), E9659-E9667 (2018)   HIV integrates into the host genome to create a persistent viral reservoir. Stimulation of CD4⁺ memory T lymphocytes with common γc-chain cytokines renders these cells more susceptible to HIV infection, making them a key component of the reservoir itself. IL-15 is up-regulated during primary HIV infection, a time when the HIV reservoir established. Therefore, we investigated the molecular and cellular impact of IL-15 on CD4⁺ T-cell infection. We found that IL-15 stimulation induces SAM domain and HD domain-containing protein 1 (SAMHD1) phosphorylation due to cell cycle entry, relieving an early block to infection. Perturbation of the pathways downstream of IL-15 receptor (IL-15R) indicated that SAMHD1 phosphorylation after IL-15 stimulation is JAK dependent. Treating CD4⁺ T cells with Ruxolitinib, an inhibitor of JAK1 and JAK2, effectively blocked IL-15–induced SAMHD1 phosphorylation and protected CD4⁺ T cells from HIV infection. Using high-resolution single-cell immune profiling using mass cytometry by TOF (CyTOF), we found that IL-15 stimulation altered the composition of CD4⁺ T-cell memory populations by increasing proliferation of memory CD4⁺ T cells, including CD4⁺ T memory stem cells (T_SCM). IL-15–stimulated CD4⁺ T_SCM, harboring phosphorylated SAMHD1, were preferentially infected. We propose that IL-15 plays a pivotal role in creating a self-renewing, persistent HIV reservoir by facilitating infection of CD4⁺ T cells with stem cell-like properties. Time-limited interventions with JAK1 inhibitors, such as Ruxolitinib, should prevent the inactivation of the endogenous restriction factor SAMHD1 and protect this long-lived CD4⁺ T-memory cell population from HIV infection.  

5.2421           A central amygdala to zona incerta projection is required for acquisition and remote recall of conditioned fear memory

Zhou, M., Liu, Z., Melin, M.D., Ng, Y.H., Xu, W. and Südhof, T.C. Nature Neurosci., 21, 1515-1519 (2018)   The formation and retrieval of conditioned fear memories critically depend on the amygdala. Here we identify an inhibitory projection from somatostatin-positive neurons in the central amygdala to parvalbumin-positive neurons in the zona incerta that is required for both recent and remote fear memories. Thus, the amygdala inhibitory input to parvalbumin-positive neurons in the zona incerta, a nucleus not previously implicated in fear memory, is an essential component of the fear memory circuitry.  

5.2422           Collective Infection of Cells by Viral Aggregates Promotes Early Viral Proliferation and Reveals a Cellular-Level Allee Effect

Andreu-Moreno, I. and Sanjuan, R. Current Biol., 28, 3212-3219 (2018)   In addition to the conventional release of free, individual virions, virus dispersal can involve multi-virion assemblies that collectively infect cells. However, the implications of collective infection for viral fitness remain largely unexplored. Using vesicular stomatitis virus, here, we compare the fitness of free versus saliva-aggregated viral particles. We find that aggregation has a positive effect on early progeny production, conferring a fitness advantage relative to equal numbers of free particles in most cell types. The advantage of aggregation resides, at least partially, in increasing the cellular multiplicity of infection. In mouse embryonic fibroblasts, the per capita, short-term viral progeny production peaked for a dose of ca. three infectious particles per cell. This reveals an Allee effect restricting early viral proliferation at the cellular level, which should select for dispersal in groups. We find that genetic complementation between deleterious mutants is probably not the mechanism underlying the fitness advantage of collective infection. Instead, this advantage is cell type dependent and correlates with cellular permissivity to the virus, as well as with the ability of host cells to mount an antiviral innate immune response.  

5.2423           Safety and immunogenicity of plant-produced African horse sickness virus-like particles in horses

Dennis, S.J., O’Kennedy, M.M., Rutkowska, D., Tsekoa, T., Lourens, C.W., Hitzeroth, I.I., Meyers, A.E. and Rybicki, E.P. Vet. Res., 49:105 (2018)   African horse sickness (AHS) is caused by multiple serotypes of the dsRNA AHSV and is a major scourge of domestic equids in Africa. While there are well established commercial live attenuated vaccines produced in South Africa, risks associated with these have encouraged attempts to develop new and safer recombinant vaccines. Previously, we reported on the immunogenicity of a plant-produced AHS serotype 5 virus-like particle (VLP) vaccine, which stimulated high titres of AHS serotype 5-specific neutralizing antibodies in guinea pigs. Here, we report a similar response to the vaccine in horses. This is the first report demonstrating the safety and immunogenicity of plant-produced AHS VLPs in horses.  

5.2424           Infectivity of adeno-associated virus serotypes in mouse testis

Rajasekaran, S., Thatte, J., Periasamy, J., javali, A., Jayaram, M., Sen, D., Krishnagopal, A., Jayandaran, G.R. and Sambasivan, R. BMC Biotech., 18:70 (2018)   Background Recombinant adeno-associated viruses (AAVs) are emerging as favoured transgene delivery vectors for both research applications and gene therapy. In this context, a thorough investigation of the potential of various AAV serotypes to transduce specific cell types is valuable. Here, we rigorously tested the infectivity of a number of AAV serotypes in murine testis by direct testicular injection. Results We report the tropism of serotypes AAV2, 5, 8, 9 and AAVrh10 in mouse testis. We reveal unique infectivity of AAV2 and AAV9, which preferentially target intertubular testosterone-producing Leydig cells. Remarkably, AAV2 TM, a mutant for capsid designed to increase transduction, displayed a dramatic alteration in tropism; it infiltrated seminiferous tubules unlike wildtype AAV2 and transduced Sertoli cells. However, none of the AAVs tested infected spermatogonial cells. Conclusions In spite of direct testicular injection, none of the tested AAVs appeared to infect sperm progenitors as assayed by reporter expression. This lends support to the current view that AAVs are safe gene-therapy vehicles. However, testing the presence of rAAV genomic DNA in germ cells is necessary to assess the risk of individual serotypes.  

5.2425           13 - Use of adeno-associated viruses to inhibit Glutaredoxin-1 in mouse skeletal muscle

Perez, B.F., Kimura, T., Tsukahara, Y., Ido, Y., Pimentel, D., Adachi, T., Bachschmid, M. and Matsui, R. Free Radical Biol. Med., 128, Suppl. 1, 25 (2018)   Glutaredoxin-1 (Glrx) is a cytosolic enzyme that reverses glutathione (GSH) adducts of protein thiols generated through oxidative post-translational modifications. GSH-adducts regulate cellular signaling and transcription factors. We previously found that after femoral artery ligation Glrx knockout mice improved blood flow recovery in relation to activation of hypoxia-inducible factor (HIF)-1α via GSH adducts, indicating Glrx as an anti-angiogenic enzyme. Objective We hypothesized that local Glrx gene inhibition might improve poor blood flow recovery after ischemia in aging or diabetes. To inhibit Glrx gene expression in vitro and in vivo, we developed an improved protocol to produce adeno-associated viruses (AAVs) with a bicistronic expression system coding for Glrx shRNA (shGlrx) and mVenus, a yellow fluorescent protein. Methods Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation, aqueous two-phase partitioning, and an iodixanol gradient, which resulted in AAVs with adequate titer and purity for in vivo use. We tested the AAVs on C2C12 skeletal muscle cells and mouse gastrocnemius muscles. Results Quiescent confluent C2C12 cells transduced with a shGlrx-containing AAV exhibited a decrease of ~75% in the Glrx mRNA and protein levels after 4 days. Intramuscular injection (4.4 x 10¹² viral genome/50 μl) of AAV-shGlrx transduced mVenus expression in skeletal muscle efficiently, but non-injected muscle, heart, or liver showed no mVenus expression. However, 4 weeks after intramuscular injection, Glrx protein levels remained unchanged compared to control-AAV injected or non-injected muscle, and inflammatory markers were increased. Six weeks after virus injection, Glrx expression in the muscle decreased 80% at the mRNA and 50% at the protein level. Conclusion Intramuscular injection of AAV-shGlrx resulted in muscle-specific attenuation of Glrx expression after at least 6 weeks. AAV intramuscular injection caused local inflammation in the early stage which may counteract the effects of shGlrx. (R01HL133013, R03AG051857, R01DK103750).  

5.2426           Potassium channel-based optogenetic silencing

Sierra, Y.A.B., Rost, B.R., Pofahl, M., Fernandes, A.M., Kopton, R.A., Moser, S., Holtkamp, D. et al Nature Communications, 9:4611 (2018)   Optogenetics enables manipulation of biological processes with light at high spatio-temporal resolution to control the behavior of cells, networks, or even whole animals. In contrast to the performance of excitatory rhodopsins, the effectiveness of inhibitory optogenetic tools is still insufficient. Here we report a two-component optical silencer system comprising photoactivated adenylyl cyclases (PACs) and the small cyclic nucleotide-gated potassium channel SthK. Activation of this ‘PAC-K’ silencer by brief pulses of low-intensity blue light causes robust and reversible silencing of cardiomyocyte excitation and neuronal firing. In vivo expression of PAC-K in mouse and zebrafish neurons is well tolerated, where blue light inhibits neuronal activity and blocks motor responses. In combination with red-light absorbing channelrhodopsins, the distinct action spectra of PACs allow independent bimodal control of neuronal activity. PAC-K represents a reliable optogenetic silencer with intrinsic amplification for sustained potassium-mediated hyperpolarization, conferring high operational light sensitivity to the cells of interest.  

5.2427           Dorsal BNST α2A-Adrenergic Receptors Produce HCN-Dependent Excitatory Actions That Initiate Anxiogenic Behaviors

Harris, N.A., Isaac, A.T., Günther, A., Merkel, K., Melchior, J., Xu, M., Eguakun, E. et al

1. Neurosci., 38(42), 8922-8942 (2018)

  Stress is a precipitating agent in neuropsychiatric disease and initiates relapse to drug-seeking behavior in addicted patients. Targeting the stress system in protracted abstinence from drugs of abuse with anxiolytics may be an effective treatment modality for substance use disorders. α_2A-adrenergic receptors (α_2A-ARs) in extended amygdala structures play key roles in dampening stress responses. Contrary to early thinking, α_2A-ARs are expressed at non-noradrenergic sites in the brain. These non-noradrenergic α_2A-ARs play important roles in stress responses, but their cellular mechanisms of action are unclear. In humans, the α_2A-AR agonist guanfacine reduces overall craving and uncouples craving from stress, yet minimally affects relapse, potentially due to competing actions in the brain. Here, we show that heteroceptor α_2A-ARs postsynaptically enhance dorsal bed nucleus of the stria terminalis (dBNST) neuronal activity in mice of both sexes. This effect is mediated by hyperpolarization-activated cyclic nucleotide-gated cation channels because inhibition of these channels is necessary and sufficient for excitatory actions. Finally, this excitatory action is mimicked by clozapine-N-oxide activation of the G_i-coupled DREADD hM4Di in dBNST neurons and its activation elicits anxiety-like behavior in the elevated plus maze. Together, these data provide a framework for elucidating cell-specific actions of GPCR signaling and provide a potential mechanism whereby competing anxiogenic and anxiolytic actions of guanfacine may affect its clinical utility in the treatment of addiction.  

5.2428           In vivo quantification of glial activation in minipigs overexpressing human α‐synuclein

Lillethorup, T.P., Glud, A.N., landeck, N., Alstrup, A.K.O., jakobzen, A., Vang, K., Doudet, D.J., Brooks, D.J., Kirik, D., Hinz, R., Sørensen, J.C. and Landau, A.M. Synapse, 72(12), e22060 (2018)   Parkinson’s disease is characterized by a progressive loss of substantia nigra (SN) dopaminergic neurons and the formation of Lewy bodies containing accumulated alpha‐synuclein (α‐syn). The pathology of Parkinson’s disease is associated with neuroinflammatory microglial activation, which may contribute to the ongoing neurodegeneration. This study investigates the in vivo microglial and dopaminergic response to overexpression of α‐syn. We used positron emission tomography (PET) and the 18 kDa translocator protein radioligand, [¹¹C](R)PK11195, to image brain microglial activation and (+)‐α‐[¹¹C]dihydrotetrabenazine ([¹¹C]DTBZ), to measure vesicular monoamine transporter 2 (VMAT2) availability in Göttingen minipigs following injection with recombinant adeno‐associated virus (rAAV) vectors expressing either mutant A53T α‐syn or green fluorescent protein (GFP) into the SN (4 rAAV‐α‐syn, 4 rAAV‐GFP, 5 non‐injected control minipigs). We performed motor symptom assessment and immunohistochemical examination of tyrosine hydroxylase (TH) and transgene expression. Expression of GFP and α‐syn was observed at the SN injection site and in the striatum. We observed no motor symptoms or changes in striatal [¹¹C]DTBZ binding potential in vivo or striatal or SN TH staining in vitro between the groups. The mean [¹¹C](R)PK11195 total volume of distribution was significantly higher in the basal ganglia and cortical areas of the α‐syn group than the control animals. We conclude that mutant α‐syn expression in the SN resulted in microglial activation in multiple sub‐ and cortical regions, while it did not affect TH stains or VMAT2 availability. Our data suggest that microglial activation constitutes an early response to accumulation of α‐syn in the absence of dopamine neuron degeneration.  

5.2429           Neuraminidase‐mediated desialylation augments AAV9‐mediated gene expression in skeletal muscle

Zhu, H., Wang, T., Lye, R.J., French, B.A. and Annex, B.H.

1. Gene Med., 20(9), e3049 (2018)

  Background Following systemic delivery, AAV9‐mediated gene expression is significantly increased in ischemic versus non‐ischemic muscle, suggesting that AAV9 is an attractive vector for treating peripheral arterial disease. Potential mechanisms underlying ischemia‐augmented expression include: (i) increased vascular permeability and (ii) “unmasking” of endogenous AAV9 receptors. In the present study, we aimed to reconstitute the ischemic induction of AAV9 in vivo, using local injection of histamine (to increase vascular permeability) and neuraminidase (to desialylate cell surface glycans). Methods Bioassays were performed to optimize the effects of histamine and neuraminidase after intramuscular injection. Histamine and/or neuraminidase were then injected intramuscularly shortly before intravenous injection of an AAV9 vector expressing luciferase. Luciferase expression was serially assessed with bioluminescence imaging. At the end of the study, tissues were harvested for assays of luciferase activity and AAV9 genome copy number aiming to assess AAV‐mediated gene expression and transduction, respectively. Results Intramuscular injection of either neuraminidase or neuraminidase plus histamine significantly increased both transduction and gene expression, whereas histamine alone had little effect. Pre‐injection with neuraminidase increased AAV9‐mediated gene delivery by four‐ to nine‐fold and luciferase activity by 60–100‐fold. Luciferase activity in neuraminidase‐injected muscle was > 100‐fold higher than in any off‐target tissue (including heart, liver and brain). Conclusions The ischemic induction of AAV9‐mediated gene expression in muscle can largely be reconstituted by pre‐injecting neuraminidase intranmuscularly. This strategy may prove useful in future human gene therapy protocols as a quick and efficient means to selectively target systemically injected AAV9 to localized regions of muscle, thus decreasing the potential for adverse effects in off‐target tissues.  

5.2430           Novel phage–host interactions and evolution as revealed by a cyanomyovirus isolated from an estuarine environment

Xu, Y., Zhang, R., Wang, N., Cai, L., Tong, Y., Sun, Q., Chen, F. and Jiao, N. Environmental Microbiol., 20(8), 2974-2989 (2018)   Cyanophages are thought to affect the community structure, population dynamics, metabolic activity and evolution of picocyanobacteria and to impact the biogeochemical cycling in aquatic ecosystems. Here, we report an estuarine Synechococcus phage, S‐CBWM1, which represents a novel viral lineage and exhibits interesting genetic features related to phage–host interactions and evolution. S‐CBWM1 encapsidates four virion‐associated proteins related to cellular metabolic regulation. Several novel auxiliary metabolic genes related to multidrug efflux, cell wall and capsule synthesis or modifications were also identified. In addition, the presence of the largest number of tRNA genes hitherto found in a phage genome may contribute to the translation efficiency of unique genes. These genomic and proteomic features of S‐CBWM1 suggested phage–host interactions involved in adaptation to eutrophic estuarine environments. Phylogenetic and metagenomic analysis of the polγ gene in the S‐CBWM1 genome provided new insights into the evolutionary path of mitochondrial DNA polymerase gamma. The S‐CBWM1 psbA contains two group I introns, representing the first instance of multiple introns within psbA from phage. The isolation of S‐CBWM1 reveals that estuarine ecosystems contain evolutionarily novel cyanophages that drive unique phage–host interactions.  

5.2431           Redundant Late Domain Functions of Tandem VP2 YPX3L Motifs in Nonlytic Cellular Egress of Quasi-enveloped Hepatitis A Virus

Gonzalez-Lopez, O., Rivera-Serrano, E.E., Hu, F., Hensley, L., McKnight, K.L., Ren, J., Sturat, D., Fry, E.E. and Lemon, S.M.

1. Virol., 92(23), e01308-18 (2018)

  The quasi-envelopment of hepatitis A virus (HAV) capsids in exosome-like virions (eHAV) is an important but incompletely understood aspect of the hepatovirus life cycle. This process is driven by recruitment of newly assembled capsids to endosomal vesicles into which they bud to form multivesicular bodies with intraluminal vesicles that are later released at the plasma membrane as eHAV. The endosomal sorting complexes required for transport (ESCRT) are key to this process, as is the ESCRT-III-associated protein, ALIX, which also contributes to membrane budding of conventional enveloped viruses. YPX_1or3L late domains in the structural proteins of these viruses mediate interactions with ALIX, and two such domains exist in the HAV VP2 capsid protein. Mutational studies of these domains are confounded by the fact that the Tyr residues (important for interactions of YPX_1or3L peptides with ALIX) are required for efficient capsid assembly. However, single Leu-to-Ala substitutions within either VP2 YPX₃L motif (L1-A and L2-A mutants) were well tolerated, albeit associated with significantly reduced eHAV release. In contrast, simultaneous substitutions in both motifs (L1,2-A) eliminated virus release but did not inhibit assembly of infectious intracellular particles. Immunoprecipitation experiments suggested that the loss of eHAV release was associated with a loss of ALIX recruitment. Collectively, these data indicate that HAV YPX₃L motifs function as redundant late domains during quasi-envelopment and viral release. Since these motifs present little solvent-accessible area in the crystal structure of the naked extracellular capsid, the capsid structure may be substantially different during quasi-envelopment.  

5.2432           High density Huh7.5 cell hollow fiber bioreactor culture for high-yield production of hepatitis C virus and studies of antivirals

Pihl, A.F., Offersgaard, A.F., mathiesen, C.K., Prentoe, J., Fahnøe, U., Krarup, H., Bukh, J. and Gottwein, J.M. Scientific Reports, 8:17505 (2018)   Chronic hepatitis C virus (HCV) infection poses a serious global public health burden. Despite the recent development of effective treatments there is a large unmet need for a prophylactic vaccine. Further, antiviral resistance might compromise treatment efficiency in the future. HCV cell culture systems are typically based on Huh7 and derived hepatoma cell lines cultured in monolayers. However, efficient high cell density culture systems for high-yield HCV production and studies of antivirals are lacking. We established a system based on Huh7.5 cells cultured in a hollow fiber bioreactor in the presence or absence of bovine serum. Using an adapted chimeric genotype 5a virus, we achieved peak HCV infectivity and RNA titers of 7.6 log₁₀ FFU/mL and 10.4 log₁₀ IU/mL, respectively. Bioreactor derived HCV showed high genetic stability, as well as buoyant density, sensitivity to neutralizing antibodies AR3A and AR4A, and dependency on HCV co-receptors CD81 and SR-BI comparable to that of HCV produced in monolayer cell cultures. Using the bioreactor platform, treatment with the NS5A inhibitor daclatasvir resulted in HCV escape mediated by the NS5A resistance substitution Y93H. In conclusion, we established an efficient high cell density HCV culture system with implications for studies of antivirals and vaccine development.  

5.2433           TM6SF2 Promotes Lipidation and Secretion of Hepatitis C Virus in Infected Hepatocytes

Boyer, A., Park, S.B., de Boer, Y.S., Li, Q. and Liang, T.J:. Gastroenterology, 155, 1923-1935 (2018)   Background & Aims Hepatitis C virus (HCV) co-opts the very-low-density lipoprotein pathway for morphogenesis, maturation, and secretion, and circulates as lipoviroparticles (LVPs). We investigated the functions and underlying mechanisms of the lipid-associated TM6SF2 protein in modulating LVP formation and the HCV life cycle. Methods We knocked down or overexpressed TM6SF2 in hepatic cells and examined HCV infection, measuring viral RNA and protein levels and infectious LVP titers. The density of secreted LVPs was evaluated by iodixanol gradient assay. We measured levels and patterns of TM6SF2 in liver biopsies from 73 patients with chronic hepatitis C, livers of HCV-infected humanized Alb-uPA/SCID/beige mice, and HCV-infected Huh7.5.1 cells. Results TM6SF2 knockdown in hepatocytes reduced viral RNA and infectious viral particle secretion without affecting HCV genome replication, translation, or assembly. Overexpression of TM6SF2 reduced intracellular levels of HCV RNA and infectious LVPs, and conversely increased their levels in the culture supernatants. In HCV-infected cells, TM6SF2 overexpression resulted in production of more infectious LVPs in the lower-density fractions of supernatant. HCV infection increased TM6SF2 expression in cultured cells, humanized livers of mice, and liver tissues of HCV patients. TM6SF2 messenger RNA levels correlated positively with HCV RNA levels in liver biopsies from patients. SREBF2 appears to mediate the ability of HCV to increase the expression of TM6SF2 in hepatic cells. Conclusions In studies of cells, mice and human liver tissues, we found TM6SF2 is required for maturation, lipidation, and secretion of infectious LVPs. HCV, in turn, up-regulates expression of TM6SF2 to facilitate productive infection.  

5.2434           Investigating the neuroprotective effect of AAV-mediated β-synuclein overexpression in a transgenic model of synucleinopathy

Sargent, D., Betemps, D., Drouyers, M., Verchere, J., Gaillard, d., Arsac, J-N., Lakdar, l., Salvetti, A. and Baron, T. Scientific Reports, 8:17563 (2018)   Parkinson’s disease (PD) and multiple system atrophy (MSA) are neurodegenerative diseases characterized by inclusions mainly composed of α-synuclein (α-syn) aggregates. The objective of this study was to investigate if β-synuclein (β-syn) overexpression could have beneficial effects by inhibiting the aggregation of α-syn. The M83 transgenic mouse is a model of synucleinopathy, which develops severe motor symptoms associated with aggregation of α-syn. M83 neonate or adult mice were injected with adeno-associated virus vectors carrying the human β-syn gene (AAVβ-syn) or green fluorescent protein gene (AAVGFP) using different injection sites. The M83 disease was - or not - accelerated using extracts of M83 brains injected with brain extract from mouse (M83) or human (MSA) origins. AAV vectors expression was confirmed using Western blot and ELISA technics. AAV mediated β-syn overexpression did not delay the disease onset or reduce the α-syn phosphorylated at serine 129 levels detected by ELISA, regardless of the AAV injection route and the inoculation of brain extracts. Instead, a proteinase-K resistant β-syn staining was detected by immunohistochemistry, specifically in sick M83 mice overexpressing β-syn after inoculation of AAVβ-syn. This study indicated for the first time that viral vector-mediated β-syn overexpression could form aggregates in a model of synucleinopathy.  

5.2435           Elevated Interleukin-38 Level Associates with Clinical Response to Atorvastatin in Patients with Hyperlipidemia

Yang, N., Song, Y., Dong, B., Li, Y., Kou, L., Yang, J. and Qin, Q. Cell. Physiol. Biochem., 49(2), 653-661 (2018)   Background/Aims: Hyperlipidemia is a risk factor for various cardiovascular and metabolic disorders. And it is tightly related to chronic inflammation. Interleukin-38 (IL-38) represents a new member of anti-inflammatory cytokines. Thus we studied the important role of IL-38 in hyperlipidemia development and treatment. Methods: The mRNA level of IL-38 in PBMCs (peripheral blood mononuclear cells) and serum IL-38 levels in hyperlipidemia patients and healthy controls were measured by real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunoassay (ELISA). The hyperlipidemia patients were further divided into two groups (Sensitive and Resistant Group) according to their clinical response to Atorvastatin therapy. Finally, the effects of IL-38 on hyperlipidemia was evaluated in the mice model. Results: Data showed that the IL-38 mRNA and serum protein levels were higher in patients with hyperlipidemia compared with healthy controls. And the IL-38 mRNA and serum protein levels were higher in patients sensitive to Atorvastatin therapy than the resistant group. In vitro, IL-38 inhibited the production of IL-6, IL-1β and CRP in PBMCs of patients with hyperlipidemia. In the mice model of hyperlipidemia, IL-38 was also elevated during the hyperlipidemia development. Furthermore, the IL-38 over-expressed by adeno-associated virus significantly inhibited the hyperlipidemia development, inflammatory factor secretion and also the atherosclerosis process. Conclusion: Thus our data showed that IL-38 might present protective effects on hyperlipidemia treatment.  

5.2436           Interleukin-35 Expression in Non-Small Cell Lung Cancer is Associated with Tumor Progression

Sun, M., Zheng, X., Meng, Q., Dong, Y., Zhang, G., Rao, D., An, X., Yang, Z., Pan, L. and Zhang, S. Cell. Physiol. Biochem., 51(4), 1839-1851 (2018)   Background/Aims: Lung cancer continues to be the leading cause of cancer related deaths worldwide due to its high incidence, malignant behavior and lack of major advancements in treatment strategy. The occurrence and development of lung cancer is closely related to inflammation. Thus, we conducted the present study to investigate the effects of IL-35 (Interleukin 35), a newly identified anti-inflammatory factor, on non-small cell lung cancer (NSCLC), which accounts for about 85% of all lung cancers. Methods: We first evaluated the IL-35 expression in 384 pairs of NSCLC samples and their adjacent normal mucosa by realtime PCR, ELISA (Enzyme-linked immunoassay) and tissue microarrays. Then the role of IL-35 on patient survival rates, cancer progression and their sensitivity to chemotherapy drugs were assessed. Results: IL-35 was barely expressed in the NSCLC tissues but highly expressed in the adjacent normal tissues. The down-regulation of IL-35 was significantly correlated with the results of American Joint Committee on Cancer stage, differentiation and it was also shown to be an independent prognostic indicator of disease-free survival and overall survival for patients with NSCLC. Overexpression of IL-35 in NSCLC cells suppressed cell migration, invasion, proliferation, colony formation through suppressing β-catenin. IL-35 inhibited NSCLC formation in the mice model and sensitize the cancer cells to chemotherapy drugs. Conclusion: Our results showed that IL-35 plays an inhibitory role in NSCLC development and function as a novel prognostic indicator and a potential therapeutic target.  

5.2437           Shortening the Half-Life of Cas9 Maintains Its Gene Editing Ability and Reduces Neuronal Toxicity

Yang, S., Li, S.and Li, X-J. Cell Reports, 25, 2653-2659 (2018)   Virus-mediated expression of CRISPR/Cas9 is commonly used for genome editing in animal brains to model or treat neurological diseases, but the potential neurotoxicity of overexpressing bacterial Cas9 in the mammalian brain remains unknown. Through RNA sequencing (RNA-seq) analysis, we find that virus-mediated expression of Cas9 influences the expression of genes involved in neuronal functions. Reducing Abnormalities in synaptic inhibition play a critical role in psychiatric disorders, and accordingly, it is essential to understand the molecular mechanisms linking components of the inhibitory postsynapse to psychiatrically relevant neural circuits and behaviors. Here we study the role of IgSF9b, an adhesion protein that has been associated with affective disorders, in the amygdala anxiety circuitry. We show that deletion of IgSF9b normalizes anxiety-related behaviors and neural processing in mice lacking the synapse organizer Neuroligin-2 (Nlgn2), which was proposed to complex with IgSF9b. This normalization occurs through differential effects of Nlgn2 and IgSF9b at inhibitory synapses in the basal and centromedial amygdala (CeM), respectively. Moreover, deletion of IgSF9b in the CeM of adult Nlgn2 knockout mice has a prominent anxiolytic effect. Our data place IgSF9b as a key regulator of inhibition in the amygdala and indicate that IgSF9b-expressing synapses in the CeM may represent a target for anxiolytic therapies.the genome editing capacity of Cas9 but significantly alleviates neurotoxicity. Thus, modification of Cas9 by shortening its half-life can help develop CRISPR/Cas9-based therapeutic approaches for treating neurological disorders.  

5.2438           Tailoring the AAV2 capsid vector for bone-targeting

Almeciga-Diaz, C.J., Montano, A.M., barrera, L.A. and Tomatsu, S. Pediatric Res., 84, 545-551 (2018)   Background Targeting specific tissues remains a major challenge to the promise of gene therapy. For example, several strategies have failed to target adeno-associated virus 2 (AAV2) vectors, to bone. We have evaluated in vitro and in vivo the affinity of an AAV2 vector to bone matrix, hydroxyapatite (HA) to treat Mucopolysacccharidosis IVA. Methods To increase vector affinity to HA, an aspartic acid octapeptide (D8) was inserted immediately after the N-terminal region of the VP2 capsid protein. The modified vector had physical titers and transduction efficiencies comparable to the unmodified vector. Results The bone-targeting vector had significantly higher HA affinity and vector genome copies in bone than the unmodified vector. The modified vector was also released from HA, and its enzyme activity in bone, 3 months post infusion, was 4.7-fold higher than the unmodified vector. Conclusion Inserting a bone-targeting peptide into the vector capsid increases gene delivery and expression in the bone without decreasing enzyme expression. This approach could be a novel strategy to treat systemic bone diseases.  

5.2439           IgSF9b regulates anxiety behaviors through effects on centromedial amygdala inhibitory synapses

Babaev, O., Cruces-Solis, H., Chatain, C.P., hammer, M., Wenger, S., Ali, H., Karalis, N., de Hoz, L., Schlüter, O.M., Yanagawa, Y., Ehrenreich, H., Taschenberger, H., Brose, N. and Krueger-Burg, D. Nature Communications, 9:5400 (2018)   Abnormalities in synaptic inhibition play a critical role in psychiatric disorders, and accordingly, it is essential to understand the molecular mechanisms linking components of the inhibitory postsynapse to psychiatrically relevant neural circuits and behaviors. Here we study the role of IgSF9b, an adhesion protein that has been associated with affective disorders, in the amygdala anxiety circuitry. We show that deletion of IgSF9b normalizes anxiety-related behaviors and neural processing in mice lacking the synapse organizer Neuroligin-2 (Nlgn2), which was proposed to complex with IgSF9b. This normalization occurs through differential effects of Nlgn2 and IgSF9b at inhibitory synapses in the basal and centromedial amygdala (CeM), respectively. Moreover, deletion of IgSF9b in the CeM of adult Nlgn2 knockout mice has a prominent anxiolytic effect. Our data place IgSF9b as a key regulator of inhibition in the amygdala and indicate that IgSF9b-expressing synapses in the CeM may represent a target for anxiolytic therapies.  

5.2440           CRISPR/Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I

Zabaleta, N., Barberia, M., Martin-Higueras, C., Zapata-Linares, N. et al Nature Communications, 9:5454 (2018)   CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1−/− mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites.  

5.2441           Cryo-EM Structures of Eastern Equine Encephalitis Virus Reveal Mechanisms of Virus Disassembly and Antibody Neutralization

Hasan, S.S., Sun, C., Kim, A.S., Diamond, M.S., Klimstra, W.B. and Rossmann, M.G. Cell Reports, 25, 3136-3147 (2018)   Alphaviruses are enveloped pathogens that cause arthritis and encephalitis. Here, we report a 4.4-Å cryoelectron microscopy (cryo-EM) structure of eastern equine encephalitis virus (EEEV), an alphavirus that causes fatal encephalitis in humans. Our analysis provides insights into viral entry into host cells. The envelope protein E2 showed a binding site for the cellular attachment factor heparan sulfate. The presence of a cryptic E2 glycan suggests how EEEV escapes surveillance by lectin-expressing myeloid lineage cells, which are sentinels of the immune system. A mechanism for nucleocapsid core release and disassembly upon viral entry was inferred based on pH changes and capsid dissociation from envelope proteins. The EEEV capsid structure showed a viral RNA genome binding site adjacent to a ribosome binding site for viral genome translation following genome release. Using five Fab-EEEV complexes derived from neutralizing antibodies, our investigation provides insights into EEEV host cell interactions and protective epitopes relevant to vaccine design.  

5.2442           An opposing function of paralogs in balancing developmental synapse maturation

Favaro, P.D., Huang, X., Hosang, L., Stodieck, S., Cui, L., Liu, Y., Engelhardt, K-A., Schmitz, F., Dong, Y., Löwel, S. and Schluter, O.M. PloS Biology, 16(12), e2006838 (2018)   The disc-large (DLG)–membrane-associated guanylate kinase (MAGUK) family of proteins forms a central signaling hub of the glutamate receptor complex. Among this family, some proteins regulate developmental maturation of glutamatergic synapses, a process vulnerable to aberrations, which may lead to neurodevelopmental disorders. As is typical for paralogs, the DLG-MAGUK proteins postsynaptic density (PSD)-95 and PSD-93 share similar functional domains and were previously thought to regulate glutamatergic synapses similarly. Here, we show that they play opposing roles in glutamatergic synapse maturation. Specifically, PSD-95 promoted, whereas PSD-93 inhibited maturation of immature α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid–type glutamate receptor (AMPAR)–silent synapses in mouse cortex during development. Furthermore, through experience-dependent regulation of its protein levels, PSD-93 directly inhibited PSD-95’s promoting effect on silent synapse maturation in the visual cortex. The concerted function of these two paralogs governed the critical period of juvenile ocular dominance plasticity (jODP), and fine-tuned visual perception during development. In contrast to the silent synapse–based mechanism of adjusting visual perception, visual acuity improved by different mechanisms. Thus, by controlling the pace of silent synapse maturation, the opposing but properly balanced actions of PSD-93 and PSD-95 are essential for fine-tuning cortical networks for receptive field integration during developmental critical periods, and imply aberrations in either direction of this process as potential causes for neurodevelopmental disorders.  

5.2443           Metagenomic Discovery of 83 New Human Papillomavirus Types in Patients with Immunodeficiency

Pastrana, D.V., Peretti, A., Welch, N.L., Borgogna, C., Olivero, C., Badolato, R. et al mSphere, 3(6), e00645-18 (2018)   Several immunodeficiencies are associated with high susceptibility to persistent and progressive human papillomavirus (HPV) infection leading to a wide range of cutaneous and mucosal lesions. However, the HPV types most commonly associated with such clinical manifestations in these patients have not been systematically defined. Here, we used virion enrichment, rolling circle amplification, and deep sequencing to identify circular DNA viruses present in skin swabs and/or wart biopsy samples from 48 patients with rare genetic immunodeficiencies, including patients with warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, or epidermodysplasia verruciformis (EV). Their profiles were compared with the profiles of swabs from 14 healthy adults and warts from 6 immunologically normal children. Individual patients were typically infected with multiple HPV types; up to 26 different types were isolated from a single patient (multiple anatomical sites, one time point). Among these, we identified the complete genomes of 83 previously unknown HPV types and 35 incomplete genomes representing possible additional new types. HPV types in the genus Gammapapillomavirus were common in WHIM patients, whereas EV patients mainly shed HPVs from the genus Betapapillomavirus. Preliminary evidence based on three WHIM patients treated with plerixafor, a leukocyte mobilizing agent, suggest that longer-term therapy may correlate with decreased HPV diversity and increased predominance of HPV types associated with childhood skin warts.  

5.2444           Comparative features of infections of two Massachusetts (Mass) infectious bronchitis virus (IBV) variants isolated from Western Canadian layer flocks

Amarasiinghe, A., De Silva Senapathi, U., Abdul-Cader, M.S., Popowich, S., Marshall, F., Cork, S.C., van der Meer, F., Gomis, S. and Abdul-Careem, M.F. BMC Vet. Res., 14:391 (2018)   Background Infectious bronchitis virus (IBV) is one of the leading causes of mortality and morbidity in chickens. There are numerous serotypes and variants, which do not confer cross protection resulting in failure of currently used IBV vaccines. Although variant IBV isolates with major genetic differences have been subjected to comparative studies, it is unknown whether minor genetic differences in IBV variants within a serotype are different in terms of pathogenesis and eliciting host responses. Two Massachusetts (Mass) variant IBV isolates recovered from commercial layer flocks in the Western Canadian provinces of Alberta (AB) and Saskatchewan (SK) were compared genetically and evaluated for their pathogenicity, tissue distribution and ability to recruit and replicate in macrophages. Results Although whole genome sequencing of these two Mass IBV isolates showed low similarity with the M41 vaccinal strain, they had an identical nucleotide sequence at open reading frames (ORFs) 3a, 3b, envelop (E), matrix (M), 5a and 5b. The rest of the ORFs of these 2 IBV isolates showed 99.9% nucleotide similarity. However, upon experimental infection, we found that the IBV isolate originating from AB was different to the one that originated in SK due to higher tracheal lesion scores and lower lung viral replication and lower genome loads in cecal tonsils. Nevertheless, both IBV isolates elicited host responses characterized by significant macrophage recruitment to the respiratory tract and there was evidence that both IBV isolates replicated within tracheal and lung macrophages. Conclusions Overall, this study shows that Mass variant IBV isolates, although possessing minor genetic variations, can lead to significant differences in pathogenicity in young chickens. Further studies are required to investigate the pathogenicity of these two Mass variant IBV isolates in laying hens.  

5.2445           Impact of Naturally Occurring Variation in the Human Papillomavirus 58 Capsid Proteins on Recognition by Type-Specific Neutralizing Antibodies

Godi, A., Martinelli, M., Haque, M., Li, S., Zhao, Q., Xia, N., Cocuzza, C.E. and Beddows, S.

1. Infect. Dis., 218, 1611-1621 (2018)

  Background Naturally occurring variants of human papillomavirus (HPV) 58 have been defined as lineages and sublineages but little is known about the impact of this diversity on protein function. We investigated the impact of variation within the major (L1) and minor (L2) capsid proteins of HPV58 on susceptibility to neutralizing antibodies. Methods Pseudovirus (PsV) representing A1, A2, A3, B1, B2, C, D1, and D2 variants were evaluated for their susceptibility to antibodies elicited during natural infection, preclinical antisera generated against virus-like particles, and monoclonal antibodies (MAbs). Results Lineage C PsV demonstrated a decreased sensitivity to antibodies raised against lineage A antigens. Exchange of the DE, FG, and/or HI loops between sublineage A1 and lineage C demonstrated that residues within all 3 loops were essential for the differential sensitivity to natural infection antibodies, with slightly different requirements for the animal antisera and MAbs. Comparison between the HPV58 A1 L1 pentamer crystal structure and an HPV58 C homology model indicated that these differences in neutralization sensitivity were likely due to subtle epitope sequence changes rather that major structural alterations. Conclusions These data improve our understanding of the impact of natural variation on HPV58 capsid antigenicity and raise the possibility of lineage-specific serotypes.  

5.2446           Structure and architecture of immature and mature murine leukemia virus capsids

Qu, K., Glass, B., Dolezal, m., Schur, F.K.M., Murciano, B., Rein, A., Rumlova, M., Rumi, T., Kräusslich, H-G. and Briggs, J.A.G. PNAS, 115(50), E11751-E11760 (2018)   Retroviruses assemble and bud from infected cells in an immature form and require proteolytic maturation for infectivity. The CA (capsid) domains of the Gag polyproteins assemble a protein lattice as a truncated sphere in the immature virion. Proteolytic cleavage of Gag induces dramatic structural rearrangements; a subset of cleaved CA subsequently assembles into the mature core, whose architecture varies among retroviruses. Murine leukemia virus (MLV) is the prototypical γ-retrovirus and serves as the basis of retroviral vectors, but the structure of the MLV CA layer is unknown. Here we have combined X-ray crystallography with cryoelectron tomography to determine the structures of immature and mature MLV CA layers within authentic viral particles. This reveals the structural changes associated with maturation, and, by comparison with HIV-1, uncovers conserved and variable features. In contrast to HIV-1, most MLV CA is used for assembly of the mature core, which adopts variable, multilayered morphologies and does not form a closed structure. Unlike in HIV-1, there is similarity between protein–protein interfaces in the immature MLV CA layer and those in the mature CA layer, and structural maturation of MLV could be achieved through domain rotations that largely maintain hexameric interactions. Nevertheless, the dramatic architectural change on maturation indicates that extensive disassembly and reassembly are required for mature core growth. The core morphology suggests that wrapping of the genome in CA sheets may be sufficient to protect the MLV ribonucleoprotein during cell entry.  

5.2447           Abstract 16378: Robust Cardiac Gene Delivery and Evasion of Neutralizing Antibodies by Extracellular Vesicle-Associated AAV Vectors

Adamiak, M., Liang, Y. Mathiyalagan, P. Agarwal, M, Jha, D. Kohlbrenner, E. Chepurko, E Jeong, D. Ceholski, D. Dubois, N. Hajjar, R. and  Sahoo, S. Circulation, 138, Suppl.1, abstract 16378 (2018)   Introduction: Due to their excellent safety profile in clinical trials, adeno-associated virus (AAV) has become the vector of choice for gene delivery to the myocardium. However, pre-existing immunity to AAV from naturally present neutralizing antibodies (NAbs, present in >60% of the human population) significantly limits the potential candidates for the AAV-based gene therapy. NAbs bind to AAV and block its infection, greatly reducing transduction and clinical efficacy. Thus, development of novel AAV-based vectors that can circumvent the effect of NAbs is essential for clinical administration of AAVs. Objectives: We investigated the effectiveness of extracellular vesicle (EV)-associated AAV (EV-AAV) in resisting NAbs and enhancing vector transduction in the myocardium. Methods and Results: We isolated EV-AAV from the conditioned medium of infected 293 cells using a multi-step iodixanol density gradient ultracentrifugation. Electron microscopy, flow cytometry, bioluminescence imaging and echocardiography were used to quantify AAV-mediated gene delivery and transduction efficiency. EV-AAV6-mCherry and EV-AAV9-FLuc were more resistant to antibody-mediated neutralization than conventional AAV and induced significantly higher mCherry or firefly luciferase (FLuc) expression both in vitro and in vivo. To test the therapeutic efficacy, EV-AAV9-SERCA2a or AAV9-SERCA2a were injected intramyocardially in post-MI mice preinjected with human NAbs (IVIg). Remarkably, EV-AAV9-SERCA2a outperformed standard AAV, significantly improving cardiac function both in the presence and absence of NAbs (%EF 57.4 ± 4.7 vs. 29.2± 2.3, respectively; 6 weeks after surgery). Conclusion: EV-AAV highly outperforms conventional AAV in delivering beneficial genes, such as SERCA2a. The use of naturally enveloped AAV vectors represents a promising approach to evade NAbs, and to facilitate the clinical translation of AAV-based gene therapies to a larger human population.      

5.2448           Reprogramming the Activatable Peptide Display Function of Adeno-Associated Virus Nanoparticles

Thadani, N.N., Dempsey, C., Zhao, J., Vasquez, S.M. and Suh, J. ACS Nano, 12, 1445-1454 (2018)   We harnessed an intrinsic activatable peptide display behavior shared by several parvoviruses, including the adeno-associated virus (AAV), in order to design protein-based nanodevices that can carry out an exogenous functional output in response to stimulus detection. Specifically, we generated truncated viral capsid subunits that, when combined with native capsid components into mosaic capsids, can perform robust activatable peptide display. By modulating the ratio of subunits in the mosaic capsid, properties of the activatable peptide display function can be optimized. Interestingly, the truncated subunits can form homomeric capsids not observed in nature, but at the price of losing the ability to carry out activatable peptide display. Collectively, our results demonstrate the importance of capsid mosaicism when activatable peptide display is desired and help explain why the wild-type AAV capsid exists as a mosaic of different subunits. This proof-of-concept study illustrates a strategy for reprogramming a particular conformational output behavior of AAV in pursuit of the long-term vision of creating stimulus-responsive nanodevices.  

5.2449           Reverse transduction can improve efficiency of AAV vectors in transduction‐resistant cells

Lee, E.J., Robinson, T.M., Tabor, J.J., Mikos, A.G. and Suh, J. Biotechnol. Bioeng., 115(12), 3042-3049 (2018)   Reverse transduction, also known as substrate‐mediated gene delivery, is a strategy in which viral vectors are first coated onto a surface that subsequently comes into contact with mammalian cells. The cells internalize the surface‐attached vectors, resulting in transgene expression. We hypothesized that forcing the interaction between cells and adeno‐associated virus (AAV) vectors through a reverse transduction format would increase in vitro gene delivery efficiencies of the vectors in transduction‐resistant cells. We tested this hypothesis by comparing the gene delivery efficiencies of three AAV serotypes using either standard or reverse transduction approaches. Our study reveals reverse transduction of AAV7 and AAV9 can significantly improve their delivery efficiencies. In contrast, AAV2 does not perform better under the reverse transduction format. Interestingly, increased vector uptake by cells does not provide a complete explanation for the increased transduction efficiency. Our findings offer a simple and practical method for improving transduction outcomes in vitro in cell types less permissive to a particular AAV vector.  

5.2450           SMAC Mimetics Induce Autophagy-Dependent Apoptosis of HIV-1-Infected Resting Memory CD4+ T Cells

Campbell, G.R., Bruckman, R., Chu, Y-L., Trout, R.N. and Spector, S.A. Cell Host & Microbe, 24(5), 689-702 (2018)   Long-lived resting memory CD4+ T cells (T CM) are a major reservoir of latent HIV infection. We hypothesized that latent HIV-T CM cells are maintained by aberrant expression of cell survival factors, including XIAP, BIRC2/cIAP1, and beclin-1. DIABLO/SMAC mimetics are therapeutic agents that compromise cell survival by hijacking host apoptotic machinery. We found that DIABLO/SMAC mimetics (birinapant, GDC-0152, and embelin) selectively kill HIV-T CM without increasing virus production or targeting uninfected T CM. Treatment of HIV-T CM with DIABLO/SMAC mimetics promoted XIAP and BIRC2 degradation, which triggered autophagy and the formation of a cell death complex consisting of pro-apoptotic (FADD, RIPK1, RIPK3, and caspase 8) and autophagy (ATG5, ATG7, and SQSTM1) proteins. Genetic or pharmacological inhibition of autophagy induction, but not autophagy-mediated degradation, abrogated this interaction and subsequent cell death. Our findings identify a mechanism whereby DIABLO/SMAC mimetics exploit autophagy and apoptotic machinery to selectively induce killing of HIV-T CM without viral reactivation while sparing uninfected cells.  

5.2451           Ultrafast optogenetic stimulation of the auditory pathway by targeting‐optimized Chronos

Keppeler, D., Merino, M., de la Morena, D.L., Bali, B., Huet, A.T., Gehrt, A., Wrobel, C., Subramanian, S., Dombrowski, T., Wolf, F., Rankovic, V., Neef, A. and Moser, T. EMBO J., 37, e99649 (2018)   Optogenetic tools, providing non‐invasive control over selected cells, have the potential to revolutionize sensory prostheses for humans. Optogenetic stimulation of spiral ganglion neurons (SGNs) in the ear provides a future alternative to electrical stimulation used in cochlear implants. However, most channelrhodopsins do not support the high temporal fidelity pertinent to auditory coding because they require milliseconds to close after light‐off. Here, we biophysically characterized the fast channelrhodopsin Chronos and revealed a deactivation time constant of less than a millisecond at body temperature. In order to enhance neural expression, we improved its trafficking to the plasma membrane (Chronos‐ES/TS). Following efficient transduction of SGNs using early postnatal injection of the adeno‐associated virus AAV‐PHP.B into the mouse cochlea, fiber‐based optical stimulation elicited optical auditory brainstem responses (oABR) with minimal latencies of 1 ms, thresholds of 5 μJ and 100 μs per pulse, and sizable amplitudes even at 1,000 Hz of stimulation. Recordings from single SGNs demonstrated good temporal precision of light‐evoked spiking. In conclusion, efficient virus‐mediated expression of targeting‐optimized Chronos‐ES/TS achieves ultrafast optogenetic control of neurons.  

5.2452           Molecular basis for the acid-initiated uncoating of human enterovirus D68

Liu, Y., Sheng, J., van Vliet, A.L.W., Buda, G., van Kuppervald, F.J.M. and Rossmann, M.G. PNAS, 115(52), E12209-E12217 (2018)   Enterovirus D68 (EV-D68) belongs to a group of enteroviruses that contain a single positive-sense RNA genome surrounded by an icosahedral capsid. Like common cold viruses, EV-D68 mainly causes respiratory infections and is acid-labile. The molecular mechanism by which the acid-sensitive EV-D68 virions uncoat and deliver their genome into a host cell is unknown. Using cryoelectron microscopy (cryo-EM), we have determined the structures of the full native virion and an uncoating intermediate [the A (altered) particle] of EV-D68 at 2.2- and 2.7-Å resolution, respectively. These structures showed that acid treatment of EV-D68 leads to particle expansion, externalization of the viral protein VP1 N termini from the capsid interior, and formation of pores around the icosahedral twofold axes through which the viral RNA can exit. Moreover, because of the low stability of EV-D68, cryo-EM analyses of a mixed population of particles at neutral pH and following acid treatment demonstrated the involvement of multiple structural intermediates during virus uncoating. Among these, a previously undescribed state, the expanded 1 (“E1”) particle, shows a majority of internal regions (e.g., the VP1 N termini) to be ordered as in the full native virion. Thus, the E1 particle acts as an intermediate in the transition from full native virions to A particles. Together, the present work delineates the pathway of EV-D68 uncoating and provides the molecular basis for the acid lability of EV-D68 and of the related common cold viruses.  

5.2453           Enhancing atrial‐specific gene expression using a calsequestrin cis‐regulatory module 4 with a sarcolipin promoter

Yoo, J., Kohlbrenner, E., Kim, O., Hajjar, R.J. and Jeong, D.

1. Gene Med., 20, e3060 (2018)

  Background Cardiac gene therapy using the adeno‐associated virus serotype 9 vector is widely used because of its efficient transduction. However, the promoters used to drive expression often cause off‐target localization. To overcome this, studies have applied cardiac‐specific promoters, although expression is debilitated compared to that of ubiquitous promoters. To address these issues in the context of atrial‐specific gene expression, an enhancer calsequestrin cis‐regulatory module 4 (CRM4) and the highly atrial‐specific promoter sarcolipin were combined to enhance expression and minimize off tissue expression. Methods To observe expression and bio‐distribution, constructs were generated using two different reporter genes: luciferase and enhanced green fluorescent protein (EGFP). The ubiquitous cytomegalovirus (CMV), sarcolipin (SLN) and CRM4 combined with sarcolipin (CRM4.SLN) were compared and analyzed using the luciferase assay, western blotting, a quantitative polymerase chain reaction and fluorescence imaging. Results The CMV promoter containing vectors showed the strongest expression in vitro and in vivo. However, the module SLN combination showed enhanced atrial expression and a minimized off‐target effect even when compared with the individual SLN promoter. Conclusions For gene therapy involving atrial gene transfer, the CRM4.SLN combination is a promising alternative to the use of the CMV promoter. CRM4.SLN had significant atrial expression and minimized extra‐atrial expression.  

5.2454           Adeno‐Associated Virus Production, Purification, and Titering

Chen, Y.H., Keiser, M.S. and Davidson, B.L. Current Protocols in Mouse Biology, 8(4), e56 (2018)   Adeno‐associated virus (AAV) vectors are exemplary tools for studying gene function in vivo and are particularly favorable for transferring genes of interest into brain tissues. They have shown great promise as a gene therapy vector for preclinical and clinical applications. However, the ability to use this tool is often hampered because the viruses themselves are not readily available. Many methods have been developed for AAV production. Here, we describe a simple method for small‐ to medium‐scale (10¹²‐10¹³ viral particles) production of AAV based on Polyethylenimine Max (PEI Max)–mediated triple transfection of HEK 293 cells and purification with iodixanol gradient ultracentrifugation. These methods will provide users with ample material of sufficient quality for performing in vivo gene transfer.  

5.2455           Dosage Thresholds and Influence of Transgene Cassette in Adeno-Associated Virus–Related Toxicity

Khabou, H., Cordeau, C., Pacot, L., Fisson, S. and Dalkara, D. Human Gene Therapy, 29(11), 1235-1241 (2018)   Today, there are >500 published studies and 40 clinical trials to treat retinal disorders using gene therapy. The great majority of them rely on the use of adeno-associated virus vectors (AAV) for therapeutic gene delivery. Thus far, AAVs have an excellent safety profile in the clinic. Nevertheless, it is known that AAV-mediated gene delivery leads to toxicity at higher input doses in experimental gene therapy. This study reveals the factors that contribute to retinal toxicity after subretinal administration of AAV vectors in wild-type mice. The study shows that alongside the input dose, the nature of the transgene and the cells mediating the expression determine the extent of toxicity. Importantly, the study shows that AAV vectors encoding green fluorescent protein (GFP) used as controls in experimental gene therapy are toxic at doses as low as 5 × 10⁹ vg, confounding the observed therapeutic effect in gene therapy paradigms. Altogether, the data show the importance of reducing input doses while increasing transgene expression levels via the use of more efficient capsids and promoters in order to avoid side effects in AAV-mediated gene therapy. Furthermore, the toxicity observed with AAV-GFP vectors imply a reinterpretation of previous gene therapy studies where the therapeutic effect was measured in relation to this control.  

5.2456           Codon-Optimization of Wild-Type Adeno-Associated Virus Capsid Sequences Enhances DNA Family Shuffling while Conserving Functionality

Cabanes-Creus, M., Ginn, S.L., Amaya, A.K., Liao, S.H.Y., Westhaus, A. et al Molecular Therapy – Methods & Clin. Develop., 12, 71-84 (2019)   Adeno-associated virus (AAV) vectors have become one of the most widely used gene transfer tools in human gene therapy. Considerable effort is currently being focused on AAV capsid engineering strategies with the aim of developing novel variants with enhanced tropism for specific human cell types, decreased human seroreactivity, and increased manufacturability. Selection strategies based on directed evolution rely on the generation of highly variable AAV capsid libraries using methods such as DNA-family shuffling, a technique reliant on stretches of high DNA sequence identity between input parental capsid sequences. This identity dependence for reassembly of shuffled capsids is inherently limiting and results in decreased shuffling efficiency as the phylogenetic distance between parental AAV capsids increases. To overcome this limitation, we have developed a novel codon-optimization algorithm that exploits evolutionarily defined codon usage at each amino acid residue in the parental sequences. This method increases average sequence identity between capsids, while enhancing the probability of retaining capsid functionality, and facilitates incorporation of phylogenetically distant serotypes into the DNA-shuffled libraries. This technology will help accelerate the discovery of an increasingly powerful repertoire of AAV capsid variants for cell-type and disease-specific applications.  

5.2457           Gene Transfer with AAV9-PHP.B Rescues Hearing in a Mouse Model of Usher Syndrome 3A and Transduces Hair Cells in a Non-human Primate

Gyorgy, B., Meijer, E.J., Ivanchenko, M.V., Tenneson, K., Emond, F. et al Molecular Therapy – Methods & Clin. Develop., 13, 1-13 (2019)   Hereditary hearing loss often results from mutation of genes expressed by cochlear hair cells. Gene addition using AAV vectors has shown some efficacy in mouse models, but clinical application requires two additional advances. First, new AAV capsids must mediate efficient transgene expression in both inner and outer hair cells of the cochlea. Second, to have the best chance of clinical translation, these new vectors must also transduce hair cells in non-human primates. Here, we show that an AAV9 capsid variant, PHP.B, produces efficient transgene expression of a GFP reporter in both inner and outer hair cells of neonatal mice. We show also that AAV9-PHP.B mediates almost complete transduction of inner and outer HCs in a non-human primate. In a mouse model of Usher syndrome type 3A deafness (gene CLRN1), we use AAV9-PHP.B encoding Clrn1 to partially rescue hearing. Thus, we have identified a vector with promise for clinical treatment of hereditary hearing disorders, and we demonstrate, for the first time, viral transduction of the inner ear of a primate with an AAV vector.  

5.2458           Decreased circulating ErbB4 ectodomain fragments as a read-out of impaired signaling function in amyotrophic lateral sclerosis

Lopez-Font, I., Sogorb-Esteve, A., Javier-Torrent, M., Brinkmalm, G. et al Neurobiol. Dis., 124, 428-438 (2019)   ErbB4 is a transmembrane receptor tyrosine kinase that binds to neuregulins to activate signaling. Proteolytic cleavage of ErbB4 results in release of soluble fragments of ErbB4 into the interstitial fluid. Disruption of the neuregulin-ErbB4 pathway has been suggested to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). This study assesses whether soluble proteolytic fragments of the ErbB4 ectodomain (ecto-ErbB4) can be detected in cerebrospinal fluid (CSF) and plasma, and if the levels are altered in ALS. Immunoprecipitation combined with mass spectrometry or western blotting analyses confirmed the presence of ecto-ErbB4 in human CSF. Several anti-ErbB4-reactive bands, including a 55 kDa fragment, were detected in CSF. The bands were generated in the presence of neuregulin-1 (Nrg1) and were absent in plasma from ErbB4 knockout mice. Ecto-ErbB4 levels were decreased in CSF from ALS patients (n = 20) and ALS with concomitant frontotemporal dementia patients (n = 10), compared to age-matched controls (n = 13). A similar decrease was found for the short ecto-ErbB4 fragments in plasma of the same subjects. Likewise, the 55-kDa ecto-ErbB4 fragments were decreased in the plasma of the two transgenic mouse models of ALS (SOD1^G93A and TDP-43^A315T). Intracellular ErbB4 fragments were decreased in the frontal cortex from SOD1^G93A mice, indicating a reduction in Nrg-dependent induction of ErbB4 proteolytic processing, and suggesting impaired signaling. Accordingly, overexpression of Nrg1 induced by an adeno-associated viral vector increased the levels of the ecto-ErbB4 fragment in the SOD1^G93A mice. We conclude that the determination of circulating ecto-ErbB4 fragments could be a tool to evaluate the impairment of the ErbB4 pathway and may be a useful biomarker in ALS.  

5.2459           PKG1α oxidation negatively regulates food seeking behaviour and reward

Durafford, C., Huckstepp, R.T.R., Braren, I., Fernandes, C., Brock, O., Delogu, A., Prysyazhna, O., Burgoyne, J. and Eaton, P. Redox Biol., 21, 101007 (2019)   Genes that are highly conserved in food seeking behaviour, such as protein kinase G (PKG), are of interest because of their potential role in the global obesity epidemic. PKG1α can be activated by binding of cyclic guanosine monophosphate (cGMP) or oxidant-induced interprotein disulfide bond formation between the two subunits of this homodimeric kinase. PKG1α activation by cGMP plays a role in reward and addiction through its actions in the ventral tegmental area (VTA) of the brain. ‘Redox dead’ C42S PKG1α knock-in (KI) mice, which are fully deficient in oxidant-induced disulfide-PKG1α formation, display increased food seeking and reward behaviour compared to wild-type (WT) littermates. Rewarding monoamines such as dopamine, which are released during feeding, are metabolised by monoamine oxidase to generate hydrogen peroxide that was shown to mediate PKG1α oxidation. Indeed, inhibition of monoamine oxidase, which prevents it producing hydrogen peroxide, attenuated PKG1α oxidation and increased sucrose preference in WT, but not KI mice. The deficient reward phenotype of the KI mice was rescued by expressing WT kinase that can form the disulfide state in the VTA using an adeno-associated virus, consistent with PKG1α oxidation providing a break on feeding behaviour. In conclusion, disulfide-PKG1α in VTA neurons acts as a negative regulator of feeding and therefore may provide a novel therapeutic target for obesity.  

5.2460           Synapse-Selective Control of Cortical Maturation and Plasticity by Parvalbumin-Autonomous Action of SynCAM 1

Ribic, A., Crair, M.C. and Biederer, T. Cell Reports, 26, 381-393 (2019)   Cortical plasticity peaks early in life and tapers in adulthood, as exemplified in the primary visual cortex (V1), wherein brief loss of vision in one eye reduces cortical responses to inputs from that eye during the critical period but not in adulthood. The synaptic locus of cortical plasticity and the cell-autonomous synaptic factors determining critical periods remain unclear. We here demonstrate that the immunoglobulin protein Synaptic Cell Adhesion Molecule 1 (SynCAM 1/Cadm1) is regulated by visual experience and limits V1 plasticity. Loss of SynCAM 1 selectively reduces the number of thalamocortical inputs onto parvalbumin (PV⁺) interneurons, impairing the maturation of feedforward inhibition in V1. SynCAM 1 acts in PV⁺ interneurons to actively restrict cortical plasticity, and brief PV⁺-specific knockdown of SynCAM 1 in adult visual cortex restores juvenile-like plasticity. These results identify a synapse-specific, cell-autonomous mechanism for thalamocortical visual circuit maturation and closure of the visual critical period.  

5.2461           Characterization of a quasi-enveloped, fast replicating hepevirus from fish and its use as hepatitis E virus surrogate

Bochud, M., Schäfer, W., Roth, N.J. and Ros, C.

1. Virol. Methods, 263, 111-119 (2019)

  Hepatitis E virus (HEV) is an emerging concern for the safety of plasma-derived medicinal products. The lack of an efficient cell culture system hampers the studies on HEV biology as well as validation studies to test the capacity of virus reduction steps to clear HEV. Hence, a surrogate hepevirus that can efficiently replicate in cell culture is needed. Cutthroat trout virus (CTV) is a non-pathogenic fish hepevirus, which can replicate in cell culture to high titers. Under interferon inhibition, CTV replication reached up to 5 × 10⁷ genome equivalents per μL in 4-5 days. The intracellular CTV progeny was already lipid-associated, suggesting that the envelope is acquired from intracellular membranes. Transmission electron microscopy of purified quasi-enveloped virus revealed exosome-like structures with an average size of 40 nm, in contrast to 27–34 nm for the non-enveloped virus. The quasi-enveloped virus was significantly less infectious than the non-enveloped virus. Assays based on quantitative RT-PCR, immunofluorescence and immunocytochemistry were established to evaluate virus inactivation. Cold ethanol fractionation removed 3.0 log of CTV and pasteurization of human albumin inactivated more than 3.7 log to below the limit of detection. Similar to HEV, virus replication was promoted in the presence of 17β-estradiol, an effect that can contribute to the understanding of the exacerbated virulence of HEV in pregnant women. These results together reveal substantial similarities between the human and fish HEV and validate CTV as a practical virus model to use in some applications for evaluating the HEV reduction capacity of biological manufacturing process steps.  

5.2462           Toward genome editing in X-linked RP—development of a mouse model with specific treatment relevant features

Schlegel, J., Hoffmann, J. Röll, D., Müller, B., Günther, S., Zhang, W., Janise, A., Vössing, C., Fühler, B., Neidhardt, J., Khanna, H., Lorenz, B. and Stieger, K. Translational Res., 203, 57-72 (2019)   Genome editing represents a powerful tool to treat inherited disorders. Highly specific endonucleases induce a DNA double strand break near the mutant site, which is subsequently repaired by cellular DNA repair mechanisms that involve the presence of a wild type template DNA. In vivo applications of this strategy are still rare, in part due to the absence of appropriate animal models carrying human disease mutations and knowledge of the efficient targeting of endonucleases. Here we report the generation and characterization of a new mouse model for X-linked retinitis pigmentosa (XLRP) carrying a point mutation in the mutational hotspot exon ORF15 of the RPGR gene as well as a recognition site for the homing endonuclease I-SceI. Presence of the genomic modifications was verified at the RNA and protein levels. The mutant protein was observed at low levels. Optical coherence tomography studies revealed a slowly progressive retinal degeneration with photoreceptor loss starting at 9 months of age, paralleling the onset of functional deficits as seen in the electroretinogram. Early changes to the outer retinal bands can be used as biomarker during treatment applications. We further show for the first time efficient targeting using the I-SceI enzyme at the genomic locus in a proof of concept in photoreceptors following adeno-associated virus mediated gene transfer in vivo. Taken together, our studies not only provide a human-XLRP disease model but also act as a platform to design genome editing technology for retinal degenerative diseases using the currently available endonucleases.  

5.2463           Pharmacological enhancement of retinoid-related orphan receptor α function mitigates spinocerebellar ataxia type 3 pathology

Watanave, M., Hoshino, C., Konno, A., Fukuzaki, Y., matsuzaki, Y., Ishitani, T. and Hirai, H. Neurobiol. Dis., 121, 263-273 (2019)   Cerebellar Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex, and damage to PCs results in motor deficits. Spinocerebellar ataxia type 3 (SCA3, also known as Machado–Joseph disease), a hereditary neurodegenerative disease, is caused by an abnormal expansion of the polyglutamine tract in the causative ATXN3 protein. SCA3 affects a wide range of cells in the central nervous system, including those in the cerebellum. To unravel SCA3 pathology, we used adeno-associated virus serotype 9 (AAV9) vectors to express full-length ATXN3 with an abnormally expanded 89 polyglutamine stretch (ATXN3[Q89]) in cerebellar neurons of mature wild-type mice. Mice expressing ATXN3[Q89] exhibited motor impairment in a manner dependent on the viral titer. Immunohistochemistry of the cerebellum showed ubiquitinated nuclear aggregates in PCs; degeneration of PC dendrites; and a significant decrease in multiple proteins including retinoid-related orphan receptor α (RORα), a transcription factor, and type 1 metabotropic glutamate receptor (mGluR1) signaling molecules. Patch clamp analysis of ATXN3[Q89]-expressing PCs revealed marked defects in mGluR1 signaling. Notably, the emergence of behavioral, morphological, and functional defects was inhibited by a single injection of SR1078, an RORα/γ agonist. These results suggest that RORα plays a key role in mutant ATXN3-mediated aberrant phenotypes and that the pharmacological enhancement of RORα could function as a method for therapeutic intervention in SCA3.  

5.2464           Diet-dependent function of the extracellular matrix proteoglycan Lumican in obesity and glucose homeostasis

Wolff, G., Taranko, A.E., Meln, L., Weinmann, K., Sijmonsma, T. et al Mol. Metabolism, 19, 97-106 (2019)   Objective Extracellular matrix remodeling is required for adipose expansion under increased caloric intake. In turn, inhibited expandability due to aberrant collagen deposition promotes insulin resistance and progression towards the metabolic syndrome. An emerging role for the small leucine-rich proteoglycan Lumican in metabolically driven nonalcoholic fatty liver disease sparks an interest in further understanding its role in diet-induced obesity and metabolic complications. Methods Whole body ablation of Lumican (Lum^−/−) gene and adeno-associated virus-mediated over-expression were used in combination with control or high fat diet to assess energy balance, glucose homeostasis as well as adipose tissue health and remodeling. Results Lumican was found to be particularly enriched in the stromal cells isolated from murine gonadal white adipose tissue. Likewise murine and human visceral fat showed a robust increase in Lumican as compared to fat from the subcutaneous depot. Lumican null female mice exhibited moderately increased fat mass, decreased insulin sensitivity and increased liver triglycerides in a diet-dependent manner. These changes coincided with inflammation in adipose tissue and no overt effects in adipose expandability, i.e. adipocyte formation and hypertrophy. Lumican over-expression in visceral fat and liver resulted in improved insulin sensitivity and glucose clearance. Conclusions These data indicate that Lumican may represent a functional link between the extracellular matrix, glucose homeostasis, and features of the metabolic syndrome.  

5.2465           Impairment of chaperone-mediated autophagy affects neuronal homeostasis through altered expression of DJ-1 and CRMP-2 proteins

Brekk, O.R., Makridakis, M., Mavroeidi, P., Vlahou, A., Xilouri, M. and Stefanis, L: Mol. Cell. Neurosci., 95, 1-12 (2019)   Chaperone-mediated autophagy (CMA) is a substrate-specific mode of lysosomal proteolysis, with multiple lines of evidence connecting its dysfunction to both ageing and disease. We have recently shown that CMA impairment through knock-down of the lysosomal receptor LAMP2A is detrimental to neuronal viability in vivo; however, it is not clear which subset of proteins regulated by the CMA pathway mediate such changes. In this study, we have manipulated CMA function through alterations of LAMP2A abundance in primary rat cortical neurons, to identify potential changes to the neuronal proteome occurring prior to neurotoxic effects. We have identified a list of proteins with significant, >2-fold change in abundance following our manipulations, of which PARK7/DJ-1 – an anti-oxidant implicated in hereditary forms of Parkinson's Disease (PD), and DPYSL2/CRMP-2 – a microtubule-binding phosphoprotein involved in schizophrenia pathogenesis – were both found to have measurable effects on neuronal homeostasis and phenotype. Taken together, this study describes alterations in the abundance of neuronal proteins involved in neuropsychiatric disorders upon CMA manipulation, and suggests that such alterations may in part be responsible for the neurodegeneration observed upon CMA impairment in vivo.  

5.2466           Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins

Mangeot, P.E:, Risson, V., Fusil, F., Marnet, A., Laurent, E., Blin, J. et al Nature Communications, 10:45 (2019)   Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Here, we use engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades are also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.  

5.2467           HEPATITIS E VIRUS GENOTYPE 3 AND CAPSID PROTEIN IN URINE OF INFECTED SOLID-ORGAN-TRANSPLANT RECIPIENTS

Marion, O., Caaapelli, N., Lhomme, S., Dubois, M., Pucelle, M., Abravanel, F., Kamar, N. and Izopet, J. Transplant. Int., 32(S1), abstract 076 (2019)   Introduction: Hepatitis E virus genotype 3 (HEV3) is responsible for acute and chronic hepatitis in solid-organ-transplant recipients. HEV was recently found in the urine of some acutely and chronically genotype 4-infected patients. We investigated the urinary excretion of HEV3 by solid-organ-transplant recipients during the acute phase of infection. We also assessed the values of urinary and serum HEV antigen (Ag) concentrations as indicators of the development of a chronic infection. Methods: We examined the urinary excretion of HEV3 by 24 consecutive solid-organ-transplant recipients at the acute phase of HEV hepatitis and characterized the excreted virus. Results: Urinary HEV RNA was detected in half of the patients (12 out of the 24) and urinary HEV Ag was detected in all but one (96%). The density of RNA-containing HEV particles in urine was low (1.11–1.12 g/cm3), corresponding to lipid-associated virions. The urinary HEV Ag and HEV RNA sedimented independently in iodixanol density gradients, suggesting that most of the HEV Ag was not associated with infectious virions. The urinary HEV RNA/Ag detected was not associated with impaired kidney function or de novo proteinuria. Finally, there was more HEV Ag in the serum at the acute phase of HEV infection in solid-organ-transplant recipien ts whose infection became chronic. Conclusions: HEV is frequently excreted in the urine of HEV3-infected solid- organ-transplant recipients without impairing renal function. The excreted urinary particles has a lipid envelope. Urinary HEV Ag was a sensitive indicator of HEV infection as was HEV Ag in serum. Acute phase serum HEV Ag was a good prognostic marker for the development of a chronic HEV infection.  

5.2468           Synaptotagmin-3 drives AMPA receptor endocytosis, depression of synapse strength, and forgetting

Awasthi, A., Ramachandran, B., Ahmed, S., Benito, E., Shinoda, Y., Nitzan, N., Heukamp, A.a, Rannio, S., Martens, H., Barth, J., Burk, K., Wang, Y.T., Fischer, A. and Dean, C. Science, 363, 44 (2019)  

Memories are stored as molecular and cellular changes in the brain. Synapses, the nodes of connection between neurons, can store memories by virtue of their ability to tune the efficacy of communication between neurons. This property of synaptic plasticity makes it possible for the brain to store and retrieve memories—to replay patterns of electrical activity that occurred during an important event. Forgetting leads to the inability to retrieve memories by making them latent or decaying them below any useful quality. However, what determines whether a memory is forgotten? A mechanism is the regulation of neurotransmitter receptor numbers on the postsynaptic plasma membrane. These receptors mediate synaptic transmission by transducing presynaptically released neurotransmitters into an electrical signal. Neuronal activity strengthens synapses by inserting receptors or weakens synapses by removing receptors from the postsynaptic membrane. Receptor trafficking is controlled through calcium influx into the neuron; however, the calcium sensors mediating this control are not known.

 

5.2469           Regulation of dopamine neurotransmission from serotonergic neurons by ectopic expression of the dopamine D2 autoreceptor blocks levodopa-induced dyskinesia

Sellnow, R., Newman, J.H., Chaaaaambers, N., West, A.R., Steece-Collier, K., Sandoval, I., Benskey, M.J., Bishop, C. and Manfredsson, F.P: Acta Neuropathol. Commun., 7:8 (2019)   Levodopa-induced dyskinesias (LID) are a prevalent side effect of chronic treatment with levodopa (L-DOPA) for the motor symptoms of Parkinson’s disease (PD). It has long been hypothesized that serotonergic neurons of the dorsal raphe nucleus (DRN) are capable of L-DOPA uptake and dysregulated release of dopamine (DA), and that this “false neurotransmission” phenomenon is a main contributor to LID development. Indeed, many preclinical studies have demonstrated LID management with serotonin receptor agonist treatment, but unfortunately, promising preclinical data has not been translated in large-scale clinical trials. Importantly, while there is an abundance of convincing clinical and preclinical evidence supporting a role of maladaptive serotonergic neurotransmission in LID expression, there is no direct evidence that dysregulated DA release from serotonergic neurons impacts LID formation. In this study, we ectopically expressed the DA autoreceptor D2R_s (or GFP) in the DRN of 6-hydroxydopamine (6-OHDA) lesioned rats. No negative impact on the therapeutic efficacy of L-DOPA was seen with rAAV-D2R_s therapy. However, D2R_s treated animals, when subjected to a LID-inducing dose regimen of L-DOPA, remained completely resistant to LID, even at high doses. Moreover, the same subjects remained resistant to LID formation when treated with direct DA receptor agonists, suggesting D2R_s activity in the DRN blocked dyskinesogenic L-DOPA priming of striatal neurons. In vivo microdialysis confirmed that DA efflux in the striatum was reduced with rAAV-D2R_s treatment, providing explicit evidence that abnormal DA release from DRN neurons can affect LID. This is the first direct evidence of dopaminergic neurotransmission in DRN neurons and its modulation with rAAV-D2R_s gene therapy confirms the serotonin hypothesis in LID, demonstrating that regulation of serotonergic neurons achieved with a gene therapy approach offers a novel and potent antidyskinetic therapy.  

5.2470           Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated Virus

Jo, D.H., Koo, T., Cho, C.S., Kim, J.H., Kim, J-S. and Kim, J.H. Molecular Therapy, 27(1), 130-136 (2019)   Genome editing with CRISPR systems provides an unprecedented opportunity to modulate cellular responses in pathological conditions by inactivating undruggable targets, such as transcription factors. Previously, we demonstrated that the smallest Cas9 ortholog characterized to date, from Campylobacter jejuni (CjCas9) targeted to Hif1a and delivered in an adeno-associated virus (AAV) vector, effectively suppressed pathological choroidal neovascularization in the mouse retina. Before implementation of CjCas9 as an in vivo therapeutic modality, it is essential to investigate the long-term effects of target gene disruption via AAV-mediated delivery of CjCas9 in vivo. In this study, histologic and electroretinographic analyses demonstrated that CjCas9 targeted to Hif1a did not induce any definite toxicity in the retina, although the target gene was mutated with a frequency ranging from 45% to 79% in retinal or retinal pigment epithelial cells. Importantly, at 14 months after injection, no indels were detected at potential off-target sites identified using Digenome-seq and Cas-OFFinder, suggesting that long-term expression of CjCas9 does not aggravate off-target effects. Taken together, our results show that intravitreal injection of AAV encoding CjCas9 targeted to Hif1a effectively induced and maintained mutations in retinal tissues for more than 1 year and did not affect retinal histologic integrity or functions.  

5.2471           Induction and Suppression of NF-κB Signalling by a DNA Virus of Drosophila

Palmer, W.H., Joosten, J., Overheul, G.J., Jansen, P.W., Vermeulen, M., Obbard, D.J. and Van Rij, R.P.

1. Virol., 93(3), e01443-18 (2019)

  Interactions between the insect immune system and RNA viruses have been extensively studied in Drosophila, in which RNA interference, NF-κB, and JAK-STAT pathways underlie antiviral immunity. In response to RNA interference, insect viruses have convergently evolved suppressors of this pathway that act by diverse mechanisms to permit viral replication. However, interactions between the insect immune system and DNA viruses have received less attention, primarily because few Drosophila-infecting DNA virus isolates are available. In this study, we used a recently isolated DNA virus of Drosophila melanogaster, Kallithea virus (KV; family Nudiviridae), to probe known antiviral immune responses and virus evasion tactics in the context of DNA virus infection. We found that fly mutants for RNA interference and immune deficiency (Imd), but not Toll, pathways are more susceptible to Kallithea virus infection. We identified the Kallithea virus-encoded protein gp83 as a potent inhibitor of Toll signalling, suggesting that Toll mediates antiviral defense against Kallithea virus infection but that it is suppressed by the virus. We found that Kallithea virus gp83 inhibits Toll signalling through the regulation of NF-κB transcription factors. Furthermore, we found that gp83 of the closely related Drosophila innubila nudivirus (DiNV) suppresses D. melanogaster Toll signalling, suggesting an evolutionarily conserved function of Toll in defense against DNA viruses. Together, these results provide a broad description of known antiviral pathways in the context of DNA virus infection and identify the first Toll pathway inhibitor in a Drosophila virus, extending the known diversity of insect virus-encoded immune inhibitors.  

5.2472           Noninvasive optical activation of Flp recombinase for genetic manipulation in deep mouse brain regions

Jung, H., Kim. S-W., Kim, M., Honmg, J., Yu, D., Kim, J.H., Lee, Y., Kim, S., Woo, D., Shin, H-S., Park, B.O. and Heo, W.D. Nature Communications, 10:314 (2019)   Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits. Here, we develop a highly light-sensitive and efficient photoactivatable Flp recombinase (PA-Flp) that is suitable for genetic manipulation in vivo. The highly light-sensitive property of PA-Flp is ideal for activation in deep mouse brain regions by illumination with a noninvasive light-emitting diode. In addition, PA-Flp can be extended to the Cre-lox system through a viral vector as Flp-dependent Cre expression platform, thereby activating both Flp and Cre. Finally, we demonstrate that PA-Flp–dependent, Cre-mediated Cav3.1 silencing in the medial septum increases object-exploration behavior in mice. Thus, PA-Flp is a noninvasive, highly efficient, and easy-to-use optogenetic module that offers a side-effect-free and expandable genetic manipulation tool for neuroscience research.  

5.2473           AAV2/8 anti-angiogenic gene therapy using single chain antibodies inhibits murine choroidal neovascularization

Hughes, C.P., O’Flynn, N.M.J., gatherer, M., McClements, M.E., Scott, J.A., MacLaren, R.E., Goverdhan, S., Glennie, M.J. and Lotery, A.J. Molecular Therapy – Methods & Clin. Develop., 13, 86-98 (2019)   While anti-angiogenic therapies for wet age-related macular degeneration (AMD) are effective for many patients, they require multiple injections and are expensive and prone to complications. Gene therapy could be an elegant solution for this problem by providing a long-term source of anti-angiogenic proteins after a single administration. Another potential issue with current therapeutic proteins containing a fragment crystallizable (Fc) domain (such as whole antibodies like bevacizumab) is the induction of an unwanted immune response. In wet AMD, a low level of inflammation is already present, so to avoid exacerbation of disease by the therapeutic protein, we propose single-chain fragment variable (scFv) antibodies, which lack the Fc domain, as a safer alternative. To investigate the feasibility of this, anti-vascular endothelial growth factor (VEGF)-blocking antibodies in two formats were produced and tested in vitro and in vivo. The scFv transgene was then cloned into an adeno-associated virus (AAV) vector. A therapeutic effect in a mouse model of choroidal neovascularization (CNV) was demonstrated with antibodies in both scFv and immunoglobulin G1 (IgG1) formats (p < 0.04). Importantly, the scFv anti-VEGF antibody expressed from an AAV vector also had a significant beneficial effect (p = 0.02), providing valuable preclinical data for future translation to the clinic.  

5.2474           Zika Virus Dependence on Host Hsp70 Provides a Protective Strategy against Infection and Disease

Taguwa, S., Yeh, M-T., Rainbolt, K., Nyak, A., Shao, H., Gestwicki, J.E., Andino, R. and Frydman, J. Cell Reports, 26, 906-920 (2019)   The spread of mosquito-borne Zika virus (ZIKV), which causes neurological disorders and microcephaly, highlights the need for countermeasures against sudden viral epidemics. Here, we tested the concept that drugs targeting host proteostasis provide effective antivirals. We show that different cytosolic Hsp70 isoforms are recruited to ZIKV-induced compartments and are required for virus replication at pre- and post-entry steps. Drugs targeting Hsp70 significantly reduce replication of different ZIKV strains in human and mosquito cells, including human neural stem cells and a placental trophoblast cell line, at doses without appreciable toxicity to the host cell. By targeting several ZIKV functions, including entry, establishment of active replication complexes, and capsid assembly, Hsp70 inhibitors are refractory to the emergence of drug-resistant virus. Importantly, these drugs protected mouse models from ZIKV infection, reducing viremia, mortality, and disease symptoms. Hsp70 inhibitors are thus attractive candidates for ZIKV therapeutics with the added benefit of a broad spectrum of action.  

5.2475           HDAC inhibitor valproic acid protects heart function through Foxm1 pathway after acute myocardial infarction

Tian, S., Lei, I., Gao, W., Liu, L., Guo, Y., Creech, J., Herron, T.J., Xian, S., Ma, P.X., Chen, E., Li, Y., Alam, H.B. and Wang, Z. EBioMed., 39, 83-94 (2019)   Background Epigenetic histone acetylation is a major event controlling cell functions, such as metabolism, differentiation and repair. Here, we aim to determine whether Valproic acid (VPA), a FDA approved inhibitor of histone deacetylation for bipolar disease, could protect heart against myocardial infarction (MI) injury and elucidate key molecular pathways. Methods VPA was administrated to MI rats at different time points, onset and after MI injury. Echocardiography, histology, serum biology assays, and gene expression, inhibition, and over-expression were performed to characterize the systolic function, infarct size, gene and signaling pathways. Findings VPA treatment reduced the infarct size by ~50% and preserved the systolic function of heart after acute MI in rats. Even 60 min after infarction, VPA treatment significantly decreased infarct size. Furthermore, long-term treatment of VPA markedly improved myocardial performance. VPA regulated gene expression essential for cell survival and anti-inflammatory response. Consequently, oxidative stress and cell death were notably reduced after VPA treatment. Moreover, Foxm1 was identified as a potential key target of VPA. Overexpression of Foxm1 provided similar heart protective effect to VPA treatment. Particularly, both VPA treatment and Foxm1 over-expression repressed inflammatory response after MI for heart protection. In contrast, inhibition of Foxm1 activity abolished the cardiac protective effect of VPA. VPA mediated CM protection through Foxm1 upregulation was also identified in a human ESC derived CM hypoxia/reperfusion system. Interpretation VPA treatments significantly reduce cardiac damage after MI and the cardioprotective effect of VPA is likely mediated via Foxm1 pathway.  

5.2476           In vivo proximity proteomics of nascent synapses reveals a novel regulator of cytoskeleton-mediated synaptic maturation

Spence, E.F., Dube, S., Uezu, A., Locke, M., Soderblom, E.J. and Soderling, S. Nature Communications, 10:386 (2019)   Excitatory synapse formation during development involves the complex orchestration of both structural and functional alterations at the postsynapse. However, the molecular mechanisms that underlie excitatory synaptogenesis are only partially resolved, in part because the internal machinery of developing synapses is largely unknown. To address this, we apply a chemicogenetic approach, in vivo biotin identification (iBioID), to discover aspects of the proteome of nascent synapses. This approach uncovered sixty proteins, including a previously uncharacterized protein, CARMIL3, which interacts in vivo with the synaptic cytoskeletal regulator proteins SrGAP3 (or WRP) and actin capping protein. Using new CRISPR-based approaches, we validate that endogenous CARMIL3 is localized to developing synapses where it facilitates the recruitment of capping protein and is required for spine structural maturation and AMPAR recruitment associated with synapse unsilencing. Together these proteomic and functional studies reveal a previously unknown mechanism important for excitatory synapse development in the developing perinatal brain.  

5.2477           Neuropilin-1-mediated pruning of corticospinal tract fibers is required for motor recovery after spinal cord injury

Nakanishi, T., Fujita, Y. and Yamashita, T. Cell Death & Disease, 10:67 (2019)   Following incomplete spinal cord injury (SCI), reorganization of the corticospinal tract (CST) contributes to spontaneous motor recovery. Axotomized CST fibers form collaterals and make synapses with interneurons, followed by pruning of excess fibers. Although axonal pruning is involved in refinement of neural circuits, its molecular mechanisms and functional roles remain poorly understood. To address these questions, we performed dorsal hemisections of mouse thoracic spinal cord. We observed that Neuropilin-1 (Nrp1) mRNA was upregulated in layer 5 pyramidal neurons in the motor cortex 14 days after SCI, when the pruning occurred. Nrp1 knockdown using adeno-associated virus (AAV) vector encoding Nrp1 shRNA in the hindlimb motor area impaired the pruning of collaterals after SCI. Nrp1 knockout by injecting AAV vector encoding Cre recombinase into Nrp1 floxed mice also suppressed axonal pruning. Propriospinal neurons, interneurons that connect CST and motoneurons, expressed Semaphorin 3A (Sema3A), the ligand of Nrp1. Furthermore, the genetic deletion of Nrp1 specifically in the hindlimb motor area suppressed the recovery of skilled movement at 21 and 28 days after SCI. The present findings demonstrate that the pruning of collaterals mediated by Nrp1 is required for motor recovery after SCI, and suggest that refinement of the neuronal network facilitates motor recovery.  

5.2478           Enhanced phosphorylation of T153 in soluble tau is a defining biochemical feature of the A152T tau risk variant

Carlomagno, Y., Chung, D-e.C., Yue, M., Kurti, A., Avendano, N.M.et al Acta Neuropathol. Commun., 7:10 (2019)   Pathogenic mutations in the tau gene (microtubule associated protein tau, MAPT) are linked to the onset of tauopathy, but the A152T variant is unique in acting as a risk factor for a range of disorders including Alzheimer’s disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and dementia with Lewy bodies (DLB). In order to provide insight into the mechanism by which A152T modulates disease risk, we developed a novel mouse model utilizing somatic brain transgenesis with adeno-associated virus (AAV) to drive tau expression in vivo, and validated the model by confirming the distinct biochemical features of A152T tau in postmortem brain tissue from human carriers. Specifically, Tau^A152T-AAV mice exhibited increased tau phosphorylation that unlike animals expressing the pathogenic P301L mutation remained localized to the soluble fraction. To investigate the possibility that the A152T variant might alter the phosphorylation state of tau on T152 or the neighboring T153 residue, we generated a novel antibody that revealed significant accumulation of soluble tau species that were hyperphosphorylated on T153 (pT153) in Tau^A152T-AAV mice, which were absent the soluble fraction of Tau^P301L-AAV mice. Providing new insight into the role of A152T in modifying risk of tauopathy, as well as validating the Tau^A152T-AAV model, we demonstrate that the presence of soluble pT153-positive tau species in human postmortem brain tissue differentiates A152T carriers from noncarriers, independent of disease classification. These results implicate both phosphorylation of T153 and an altered solubility profile in the mechanism by which A152T modulates disease risk.  

5.2479           Impact of the Assembly-Activating Protein on Molecular Evolution of Synthetic Adeno-Associated Virus Capsids

Herrmann, A-K., Grosse, S., Börner, K., Krämer, C., Wiedtke, E., Gunkel, M. and Grimm, D. Human Gene Therapy, 30(1), 21-35 (2019)   Over the last decade, the role of the assembly-activating protein (AAP) has begun to be dissected for the formation of adeno-associated virus (AAV) capsids based on different viral serotypes. Recently, the authors' group has specifically studied AAP's relevance during production of AAV gene therapy vectors in mammalian or insect cells, and AAP was found to be essential for capsid protein stabilization and generation of functional vector particles. Here, the lingering question is additionally addressed of whether molecular AAV evolution via DNA family shuffling of viral capsid genes would perturb AAP functionality due to concurrent and inadvertent recombination of the AAP open reading frame. To this end, a battery of complementary experiments was conducted in which: (1) the ability of chimeric AAP from AAVDJ, a hybrid of serotypes 2, 8, and 9, was tested to rescue AAP knockouts in the three parental serotypes; (2) the functionality of 60 chimeric AAPs extracted from five shuffled, unselected capsid libraries was measured; (3) whether production of different shuffled libraries, 10 wild-type serotypes or 25 individual chimeric capsids, can be enhanced by overexpression of AAP cocktails was assessed; and (4) the activity of 12 chimeric AAPs isolated from a shuffled library that was iteratively selected in vivo in mouse livers was studied. Collectively, the data demonstrate a remarkable tolerance of AAP for recombination via DNA family shuffling, evidenced by the findings that (1) all chimeric AAPs studied here retained at least partial activity, even in cases where the cognate hybrid capsid may be non-functional, and that (2) ectopic AAP overexpression did not enhance production of shuffled AAV chimeras or libraries, implying that the inherently encoded hybrid AAP variants are sufficiently active. Together, this work provides compelling evidence that AAP is not rate limiting during AAV capsid shuffling and thereby relieves a major concern in the field of AAV vector evolution.  

5.2480           Systemic Delivery of AAVB1-GAA Clears Glycogen and Prolongs Survival in a Mouse Model of Pompe Disease

Keeleer, A.M., Zieger, M., Todeasa, S.H., McCall, A.L., Gifford, J.C., Birsak, S., Choudhury, S.R., Byrne, B.J., Sena-Esteves, M. and ElMallah, M.K. Human Gene Therapy, 30(1), 57-68 (2019)   Pompe disease is an autosomal recessive glycogen storage disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA deficiency results in systemic lysosomal glycogen accumulation and cellular disruption in muscle and the central nervous system (CNS). Adeno-associated virus (AAV) gene therapy is ideal for Pompe disease, since a single systemic injection may correct both muscle and CNS pathologies. Using the Pompe mouse (B6;129-Gaa^Tm1Rabn/J), this study sought to explore if AAVB1, a newly engineered vector with a high affinity for muscle and CNS, reduces systemic weakness and improves survival in adult mice. Three-month-old Gaa^–/– animals were injected with either AAVB1 or AAV9 vectors expressing GAA and tissues were harvested 6 months later. Both AAV vectors prolonged survival. AAVB1-treated animals had a robust weight gain compared to the AAV9-treated group. Vector genome levels, GAA enzyme activity, and histological analysis indicated that both vectors transduced the heart efficiently, leading to glycogen clearance, and transduced the diaphragm and CNS at comparable levels. AAVB1-treated mice had higher GAA activity and greater glycogen clearance in the tongue. Finally, AAVB1-treated animals showed improved respiratory function comparable to wild-type animals. In conclusion, AAVB1-GAA offers a promising therapeutic option for the treatment of muscle and CNS in Pompe disease.  

5.2481           Use of a Silkworm Larva Model in Phage Therapy Experiments

Uchiyama, J., Takemura-Uchiyama, I. and Matsuzaki, S. Methods in Mol. Biol., 1898, 173-181 (2019)   Antibiotic-resistant bacteria can cause intractable infections in humans and animals, with damaging effects to health care and economics. Phage therapy is considered a possible alternative to chemotherapy for treating infections, but still requires laborious in vivo experiments before its introduction into society and its further development. Recently, silkworm larvae have been recognized as highly convenient and useful model animals, and an alternative to higher animals. We describe the procedure for experimental phage therapy to treat Staphylococcus aureus infections in silkworm larvae.  

5.2482           Production and Purification of Cell Culture Hepatitis C Virus

De la Fuente, C. and Catanese, M.T: Methods in Mol. Biol., 1911, 105-119 (2019)   Hepatitis C virus (HCV) is a peculiar member of the Flaviviridae family, with features in between an enveloped virus and a human lipoprotein and, consequently, unusual biophysical properties that made its production and purification rather challenging. Here we describe methods to generate HCV stocks in cell culture by electroporating in vitro transcribed viral RNA into permissive cell lines as well as downstream concentration and purification strategies.  

5.2483           Live Cell Imaging of Hepatitis C Virus Trafficking in Hepatocytes

Baktash, Y. and Randall, G. Methods in Mol. Biol., 1911, 263-274 (2019)   Standard fixed cell confocal microscopy is inherently limited in visualizing dynamic processes involving two- and three-dimensional movement. To overcome these limitations, live cell imaging approaches have been developed to study hepatitis C virus (HCV) entry, replicase protein trafficking, virion assembly, and egress. These studies have relied on fluorescent labeling of viral proteins by epitope tag insertion, genome labeling via nucleophilic dyes, or using lipophilic dyes to label the virion envelope. In this method review, we describe two approaches to study HCV virion trafficking in live cells. Lipophilic labeling of the envelope allows for study of the early events (through virion uncoating/fusion) in the HCV lifecycle. Tetracysteine (TC) tag insertion into the capsid protein permits study of virion assembly and capsid trafficking via binding of a fluorogenic biarsenical dye.  

5.2484           Genome-wide association study of cervical cancer suggests a role for ARRDC3 gene in human papillomavirus infection

Takeuchi, F., Kukimoto, I., Li, Z., Li, S., Li, N., Hu, Z. et al Hum. Mol. Genet., 28(2), 341-348 (2019)   The development of cervical cancer is initiated by human papillomavirus (HPV) infection and involves both viral and host genetic factors. Genome-wide association studies (GWAS) of cervical cancer have identified associations in the HLA locus and two loci outside HLA, but the principal genes that control infection and pathogenesis have not been identified. In the present study, we performed GWAS of cervical cancer in East Asian populations, involving 2609 cases and 4712 controls in the discovery stage and 1461 cases and 3295 controls in the follow-up stage. We identified novel-significant associations at 5q14 with the lead single nucleotide polymorphism (SNP) rs59661306 (P = 2.4 × 10^–11) and at 7p11 with the lead SNP rs7457728 (P = 1.2 × 10^–8). In 5q14, the chromatin region of the GWAS-significant SNPs was found to be in contact with the promoter of the ARRDC3 (arrestin domain-containing 3) gene. In our functional studies, ARRDC3 knockdown in HeLa cells caused significant reductions in both cell growth and susceptibility to HPV16 pseudovirion infection, suggesting that ARRDC3 is involved in the infectious entry of HPV into the cell. Our study advances the understanding of host genes that are responsible for cervical cancer susceptibility and guides future research on HPV infection and cancer development.  

5.2485           Novel chimeric gene therapy vectors based on Adeno-associated virus (AAV) and four different mammalian bocaviruses (BoV)

Fakhiri, J., Schneider, M.A:, Puschhof, J., Stanifer, M., Schildgen, V., Holderbach, S., Voss, Y., El Andari, J.,Schildgen, O., Boulant, s., Meister, M., Clevers, H., Yan, Z., Qiu, J. and Grimm, D. Molecular Therapy – Clin. Develop., 12, 202-222 (2019)   Parvoviruses are highly attractive templates for the engineering of safe, efficient, and specific gene therapy vectors, as best exemplified by adeno-associated virus (AAV). Another candidate that currently garners increasing attention is human bocavirus 1 (HBoV1). Notably, HBoV1 capsids can cross-package recombinant (r)AAV2 genomes, yielding rAAV2/HBoV1 chimeras that specifically transduce polarized human airway epithelia (pHAEs). Here, we largely expanded the repertoire of rAAV/BoV chimeras, by assembling packaging plasmids encoding the capsid genes of four additional primate bocaviruses, HBoV2–4 and GBoV (Gorilla BoV). Capsid protein expression and efficient rAAV cross-packaging were validated by immunoblotting and qPCR, respectively. Interestingly, not only HBoV1 but also HBoV4 and GBoV transduced pHAEs as well as primary human lung organoids. Flow cytometry analysis of pHAEs revealed distinct cellular specificities between the BoV isolates, with HBoV1 targeting ciliated, club, and KRT5+ basal cells, whereas HBoV4 showed a preference for KRT5+ basal cells. Surprisingly, primary human hepatocytes, skeletal muscle cells, and T cells were also highly amenable to rAAV/BoV transduction. Finally, we adapted our pipeline for AAV capsid gene shuffling to all five BoV isolates. Collectively, our chimeric rAAV/BoV vectors and bocaviral capsid library represent valuable new resources to dissect BoV biology and to breed unique gene therapy vectors.  

5.2486           Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes

Capelli, N., Marion, O., Dubois, M., Allart, S., Bertrand-Michel, J., Lhomme, S., Abravanel, F., Izopet, J. and Chapuy-Regaud, S.   Hepatitis E virus (HEV) is a common cause of acute viral hepatitis worldwide. Most HEV infections are asymptomatic, but immunocompromised patients infected with HEV genotype 3 (HEV3), HEV4, or HEV7 may develop chronic infections. The HEV particles in stools are naked (nHEV), while those in the serum and culture supernatants (eHEV) are associated with lipids. Hepatocytes are polarized epithelial cells that have basolateral (oriented toward the blood) and apical (oriented toward the bile) exosomal pathways. We isolated a subclone, F2, from the human hepatocarcinoma cell line HepG2/C3A that grew as a polarized monolayer culture and had better HEV production than HepG2/C3A cells. F2 cells cultured on semipermeable collagen inserts and infected basolaterally with nHEV3 released 94.6% of virus particles apically, those infected with eHEV3 released 96.8% apically, and eHEV1-infected cells released 99.3% apically. Transcytosis was not involved. Density gradient centrifugation and NP-40 treatment showed that HEV particles released both apically and basolaterally were lipid associated. The apically released HEV3 and HEV1 particles were six and nine times more infectious than those released basolaterally, respectively. Confocal microscopy indicated that the open reading frame 2 (ORF2) capsid protein colocalized apically with ORF3 virus protein, the apical marker DPP4, and the recycling endosome GTPase Rab27a. The amounts of soluble glycosylated ORF2 secreted apically and basolaterally were similar. These polarized-hepatocyte data suggest that infectious HEV particles are mainly released into bile, while the small fraction released into blood could spread HEV throughout the host.  

5.2487           In vivo transduction of ETV2 improves cardiac function and induces vascular regeneration following myocardial infarction

Lee, S., Lee, D.H., park, B-W., Kim, R., Hoang, A.D., Woo, S-K., Xiong, W., Lee, Y.J., Ban K. and Park, H-J. Exp. Mol. Med., 51:13 (2019)   Vascular regeneration in ischemic hearts has been considered a target for new therapeutic strategies. It has been reported that ETV2 is essential for vascular development, injury-induced neovascularization and direct cell reprogramming of non-endothelial cells into endothelial cells. Thus, the objective of this study was to explore the therapeutic potential of ETV2 in murine models of myocardial infarction in vivo. Direct myocardial delivery of lentiviral ETV2 into rodents undergoing myocardial infarction dramatically upregulated the expression of markers for angiogenesis as well as anti-fibrosis and anti-inflammatory factors in vivo. Consistent with these findings, echocardiography showed significantly improved cardiac function in hearts with induced myocardial infarction upon ETV2 injection compared to that in the control virus-injected group as determined by enhanced ejection fraction and fractional shortening. In addition, ETV2-injected hearts were protected against massive fibrosis with a remarkable increase in capillary density. Interestingly, major fractions of capillaries were stained positive for ETV2. In addition, ECs infected with ETV2 showed enhanced proliferation, suggesting a direct role of ETV2 in vascular regeneration in diseased hearts. Furthermore, culture media from ETV2-overexpressing cardiac fibroblasts promoted endothelial cell migration based on scratch assay. Importantly, intramyocardial injection of the adeno-associated virus form of ETV2 into rat hearts with induced myocardial infarction designed for clinical applicability consistently resulted in significant augmentation of cardiac function. We provide compelling evidence that ETV2 has a robust effect on vascular regeneration and enhanced cardiac repair after myocardial infarction, highlighting a potential therapeutic function of ETV2 as an efficient means to treat failing hearts.  

5.2488           Intravenous Injection of an AAV-PHP.B Vector Encoding Human Acid α-Glucosidase Rescues Both Muscle and CNS Defects in Murine Pompe Disease

Lim, J-A., Yi, H., Gao, F., Raben, N., Kishnani, P.S. and Sun, B. Molecular Therapy – Methods Clin. Develop., 12, 233-245 (2019)   Pompe disease, a severe and often fatal neuromuscular disorder, is caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). The disease is characterized by the accumulation of excess glycogen in the heart, skeletal muscle, and CNS. Currently approved enzyme replacement therapy or experimental adeno-associated virus (AAV)-mediated gene therapy has little effect on CNS correction. Here we demonstrate that a newly developed AAV-PHP.B vector can robustly transduce both the CNS and skeletal muscles in GAA-knockout (GAAKO) mice. A single intravenous injection of an AAV-PHP.B vector expressing human GAA under the control of cytomegalovirus (CMV) enhancer-chicken β-actin (CB) promoter into 2-week-old GAAKO mice resulted in widespread GAA expression in the affected tissues. Glycogen contents were reduced to wild-type levels in the brain and heart, and they were significantly decreased in skeletal muscle by the AAV treatment. The histological assay showed no visible glycogen in any region of the brain and spinal cord of AAV-treated mice. In this study, we describe a set of behavioral tests that can detect early neurological deficits linked to extensive lysosomal glycogen accumulation in the CNS of untreated GAAKO mice. Furthermore, we demonstrate that the therapy can help prevent the development of these abnormalities.  

5.2489           Longer Inactivating Sequence in Peptide Lock Improves Performance of Synthetic Protease-Activatable Adeno-Associated Virus

Chen, M.Y., Robinson, T.M. and Suh, J. ACS Synth. Biol., 8(1), 91-98 (2019)   Adeno-associated viruses (AAVs) are promising gene therapy vectors but may exhibit off-target delivery due to broad tissue tropism. We recently developed a synthetic protease-activatable AAV vector, named provector, that transduces cells preferentially in environments rich in matrix metalloproteinases (MMPs) which are elevated in a variety of diseases, including various cancers and heart diseases. The provector displays peptide locks made up of MMP recognition sites flanking an inactivating sequence (IS) composed of four aspartic acid residues (D4). When present, the IS prevents AAV from binding cell receptors and no transduction occurs (OFF state). High levels of MMPs cleave the recognition sequences and release the IS from the capsid surface, restoring cell receptor binding (ON state). The AAV9 provector prototype is not optimal as it displays baseline OFF transduction at 5–10% of that of the wild-type capsid, which can lead to off-target delivery. We hypothesized that changes to the IS may decrease OFF state transduction. We created a provector panel with IS of lengths 0 (D0) to 10 (D10) aspartic acid residues and characterized this panel in vitro. Notably, we find that the D10 provector has an OFF transduction of less than 1% of wild-type capsid and an ON/OFF transduction ratio of 27, the best outcome achieved for any provector thus far. In summary, our results enable us to define new design rules for the provector platform, specifically that (1) the IS is necessary for provector locking and (2) increasing the number of aspartic acid residues in this sequence improves locking.  

5.2490           A Robust and All-Inclusive Pipeline for Shuffling of Adeno-Associated Viruses

Hermann, A-K., Bender, C., Kienle, E., Grosse, S., El Andari, J., Botta, J., Schümann, N., Wiedtke, E., Niopek, D. and Grimm, D. ACs Synth. Biol., 8(1), 194-206 (2019)   Adeno-associated viruses (AAV) are attractive templates for engineering of synthetic gene delivery vectors. A particularly powerful technology for breeding of novel vectors with improved properties is DNA family shuffling, i.e., generation of chimeric capsids by homology-driven DNA recombination. Here, to make AAV DNA shuffling available to a wider community, we present a robust experimental and bioinformatical pipeline comprising: (i) standardized and partially codon-optimized plasmids carrying 12 different AAV capsid genes; (ii) a scalable protocol including troubleshooting guide for viral library production; and (iii) the freely available software SALANTO for comprehensive analysis of chimeric AAV DNA and protein sequences. Moreover, we describe a set of 12 premade and ready-to-use AAV libraries. Finally, we demonstrate the usefulness of DNA barcoding technology to trace AAV capsid libraries within a complex mixture. Our protocols and resources facilitate the implementation and tailoring of AAV evolution technology in any laboratory interested in customized viral gene transfer.  

5.2491           Systemic AAV vectors for widespread and targeted gene delivery in rodents

Challis, R.C., Kumar, S.R., Chan, K.Y., Challis, C., Beadle, K., Jang, M.J., Kim, H.M., Rajendran, P.S., Tompkins, J.D., Shivkumar, K., Deverman, B.E. and Gradinaru, V. Nature Protocols, 14(2), 379-414 (2019)   We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.  

5.2492           Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10

Maeder, M., Stefanidakis, M., Wilson, C.J., Baral, R., Barrera, L.A. et al Nature Medicine, 25(2), 229-233 (2019)   Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.  

5.2493           Vti1b promotes TRPV1 sensitization during inflammatory pain

Sondermann, J.R., Barry, A.M., Jahn, O., Michel, N., Abdelaziz, R., Kügler, S., Gomez-Varela, D. and Schnidt, M. Pain, 160(2), 508-527 82019)   Sensitization of the transient receptor potential ion channel vanilloid 1 (TRPV1) is critically involved in inflammatory pain. To date, manifold signaling cascades have been shown to converge onto TRPV1 and enhance its sensitization. However, many of them also play a role for nociceptive pain, which limits their utility as targets for therapeutic intervention. Here, we show that the vesicle transport through interaction with t-SNAREs homolog 1B (Vti1b) protein promotes TRPV1 sensitization upon inflammation in cell culture but leaves normal functioning of TRPV1 intact. Importantly, the effect of Vti1b can be recapitulated in vivo: Virus-mediated knockdown of Vti1b in sensory neurons attenuated thermal hypersensitivity during inflammatory pain without affecting mechanical hypersensitivity or capsaicin-induced nociceptive pain. Interestingly, TRPV1 and Vti1b are localized in close vicinity as indicated by proximity ligation assays and are likely to bind to each other, either directly or indirectly, as suggested by coimmunoprecipitations. Moreover, using a mass spectrometry–based quantitative interactomics approach, we show that Vti1b is less abundant in TRPV1 protein complexes during inflammatory conditions compared with controls. Alongside, we identify numerous novel and pain state-dependent binding partners of native TRPV1 in dorsal root ganglia. These data represent a unique resource on the dynamics of the TRPV1 interactome and facilitate mechanistic insights into TRPV1 regulation. We propose that inflammation-related differences in the TRPV1 interactome identified here could be exploited to specifically target inflammatory pain in the future.  

5.2494           Lymphocyte-Derived Exosomal MicroRNAs Promote Pancreatic β Cell Death and May Contribute to Type 1 Diabetes Development

Guay, C., Kruit, J.K., Rome, S., Romero, P., Dotta, F. and Regazzi, R. Cell Metabolism, 29(2), 348-361 (2019)   Type 1 diabetes is an autoimmune disease initiated by the invasion of pancreatic islets by immune cells that selectively kill the β cells. We found that rodent and human T lymphocytes release exosomes containing the microRNAs (miRNAs) miR-142-3p, miR-142-5p, and miR-155, which can be transferred in active form to β cells favoring apoptosis. Inactivation of these miRNAs in recipient β cells prevents exosome-mediated apoptosis and protects non-obese diabetic (NOD) mice from diabetes development. Islets from protected NOD mice display higher insulin levels, lower insulitis scores, and reduced inflammation. Looking at the mechanisms underlying exosome action, we found that T lymphocyte exosomes trigger apoptosis and the expression of genes involved in chemokine signaling, including Ccl2, Ccl7, and Cxcl10, exclusively in β cells. The induction of these genes may promote the recruitment of immune cells and exacerbate β cell death during the autoimmune attack. Our data point to exosomal-miRNA transfer as a communication mode between immune and insulin-secreting cells.  

5.2495           AAV-Mediated Gene Delivery to the Mouse Liver

Cunningham, S.C. and Alexander, I.E. Methods in Mol. Biol., 1937, 213-219 (2019)   The liver is an attractive target for gene therapy due to the high incidence of liver disease phenotypes. Adeno-associated viral vectors (AAV) are currently the most popular gene delivery system for targeting the liver, reflecting high transduction efficiency in vivo and the availability of a toolkit of multiple different capsids with high liver tropism. While AAV vectors confer stable gene transfer in the relatively quiescent adult liver, the predominantly episomal nature of AAV vector genomes results in less stable expression in the growing liver as a consequence of episome clearance during hepatocellular replication. This is an important consideration in experimental design involving young animals, particularly mice, where liver growth is rapid. Given the immense value of murine models for dissecting disease pathophysiology, experimental therapeutics and vector development, this technical manuscript focuses on AAV-mediated transduction of the mouse liver. Xenograft models, in which chimeric mouse-human livers can be established, are also amenable to AAV-mediated gene transfer and have proven to be powerful tools for in vivo selection and characterization of novel human-specific capsids. While yet to be confirmed, such models have the potential to more accurately predict transduction efficiency of clinical candidate vectors than nonhuman primate models.  

5.2496           AAV Production Using Baculovirus Expression Vector System

Sandro, Q., Relizani, K. and Benchaouir, R. Methods in Mol. Biol., 1937, 91-99 (2019)   Gene transfer and gene therapy are powerful approaches for many biological research applications and promising avenues for the treatment of many genetic or cancer diseases. The most efficient gene transfer tools are currently derived from viruses. Among them, the recombinant adeno-associated viruses (AAVs) are vectors of choice for many fundamental and therapeutic applications. The increasing number of clinical trials involving AAVs demonstrates the need to implement production and purification processes to meet the quantitative and qualitative demands of regulatory agencies for the use of these vectors in clinical trials. In this context, the rise of production levels on an industrial scale appeared essential. The introduction, in 2002, of an AAV process using a baculovirus expression vector system (BEVS) has circumvented this technological lock. The advantage of BEVS in expanding the AAV production in insect cells has been to switch the process to bioreactor systems, which are the ideal equipment for scaling up. We describe here a method for producing AAV vectors using the BEVS which can be easily used by research laboratories wishing to overcome the difficulties associated with the scaling up of production levels. The method provides sufficient quantities of AAV vectors to initiate preclinical projects in large animal models or for research projects where a single batch of vectors will consolidate the repeatability and reproducibility of in vitro and especially in vivo experimental approaches.  

5.2497           Multimodal Production of Adeno-Associated Virus

Sandoval, I.M., Kuhn, N.M. and Manfredsson, F.P. Methods in Mol. Biol., 1937, 101-124 (2019)   Adeno-associated virus (AAV) is an increasingly popular tool in the research laboratory, and use of this viral vector clinically is occurring at an accelerated pace. Nevertheless, despite its popularity, AAV is a relatively cumbersome virus to produce; however, significant efforts have been invested to develop, optimize, and simplify methodology that allows the generation of high-quality AAV with significantly increased production yields. Here we describe multiple modalities for production and purification of AAV particles produced in HEK293 cell cultures using an iodixanol density gradient. We include two methods adapted for harvesting virus from the culture media: tangential flow filtration (TFF) and polyethylene glycol precipitation (PEGylation). Moreover, we also describe the protocol for anion exchange chromatography, which can be used after the iodixanol gradient as an additional purification step. Last, we provide various protocols for determining virus titer.  

5.2498           Perilipin-2 is critical for efficient lipoprotein and hepatitis C virus particle production

Lassen, S., Grüttner, C., Nguyen-Dinh, V. and Herker, E.

1. Cell Sci., 132, jcs217042 (2019)

  In hepatocytes, PLIN2 is the major protein coating lipid droplets (LDs), an organelle the hepatitis C virus (HCV) hijacks for virion morphogenesis. We investigated the consequences of PLIN2 deficiency on LDs and on HCV infection. Knockdown of PLIN2 did not affect LD homeostasis, likely due to compensation by PLIN3, but severely impaired HCV particle production. PLIN2-knockdown cells had slightly larger LDs with altered protein composition, enhanced local lipase activity and higher β-oxidation capacity. Electron micrographs showed that, after PLIN2 knockdown, LDs and HCV-induced vesicular structures were tightly surrounded by ER-derived double-membrane sacs. Strikingly, the LD access for HCV core and NS5A proteins was restricted in PLIN2-deficient cells, which correlated with reduced formation of intracellular HCV particles that were less infectious and of higher density, indicating defects in maturation. PLIN2 depletion also reduced protein levels and secretion of ApoE due to lysosomal degradation, but did not affect the density of ApoE-containing lipoproteins. However, ApoE overexpression in PLIN2-deficient cells did not restore HCV spreading. Thus, PLIN2 expression is required for trafficking of core and NS5A proteins to LDs, and for formation of functional low-density HCV particles prior to ApoE incorporation.  

5.2499           Role of the PRC2-Six1-miR-25 signaling axis in heart failure

Oh, J.G., Jang, S.P., Yoo, J., Lee, M-A., Lee, S.H., Lim, T., Jeong, E., Kho, C., Kook, H., Hajjar, R.J., Park, W.J. and Jeong, D.

1. Mol. Cell. Cardiol., 129, 58-68 (2019)

  The reduced expression of cardiac sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA2a) is a hallmark of heart failure. We previously showed that miR-25 is a crucial transcriptional regulator of SERCA2a in the heart. However, the precise mechanism of cardiac miR-25 regulation is largely unknown. Literatures suggested that miR-25 is regulated by the transcriptional co-factor, sine oculis homeobox homolog 1 (Six1), which in turn is epigenetically regulated by polycomb repressive complex 2 (PRC 2) in cardiac progenitor cells. Therefore, we aimed to investigate whether Six1 and PRC2 are indeed involved in the regulation of the miR-25 level in the setting of heart failure. Six1 was up-regulated in the failing hearts of humans and mice. Overexpression of Six1 led to adverse cardiac remodeling, whereas knock-down of Six1 attenuated pressure overload-induced cardiac dysfunction. The adverse effects of Six1 were ameliorated by knock-down of miR-25. The epigenetic repression on the Six1 promoter by PRC2 was significantly reduced in failing hearts. Epigenetic repression of Six1 is relieved through a reduction of PRC2 activity in heart failure. Six1 up-regulates miR-25, which is followed by reduction of cardiac SERCA2a expression. Collectively, these data showed that the PRC2-Six1-miR-25 signaling axis is involved in heart failure. Our finding introduces new insight into potential treatments of heart failure.  

5.2500           Standardized, Scalable, and Timely Flexible Adeno-Associated Virus Vector Production Using Frozen High-Density HEK-293 Cell Stocks and CELLdiscs

Strobel, B., Zuckschwendt, K., Zimmermann, G., Mayer, C., Eytner, R., Rechsteiner, P., Kreuz, S. and Lamla, T. Human Gene Therapy Methods, 30(1), 23-33 (2019)   Adeno-associated virus (AAV) vectors currently represent the most attractive platform for viral gene therapy and are also valuable research tools to study gene function or establish disease models. Consequently, many academic labs, core facilities, and biotech/pharma companies meanwhile produce AAVs for research and early clinical development. Whereas fast, universal protocols for vector purification (downstream processing) are available, AAV production using adherent HEK-293 cells still requires time-consuming passaging and extensive culture expansion before transfection. Moreover, most scalable culture platforms require special equipment or extensive method development. To tackle these limitations in upstream processing, this study evaluated frozen high-density cell stocks as a ready-to-seed source of producer cells, and further investigated the multilayered CELLdisc culture system for upscaling. The results demonstrate equal AAV productivity using frozen cell stock–derived cultures compared to conventionally cultured cells, as well as scalability using CELLdiscs. Thus, by directly seeding freshly thawed cells into CELLdiscs, AAV production can be easily upscaled and efficiently standardized to low-passage, high-viability cells in a timely flexible manner, potentially dismissing time-consuming routine cell culture work. In conjunction with a further optimized iodixanol protocol, this process enabled supply to a large-animal study with two high-yield AAV2 capsid variant batches (0.6–1.2 × 10¹⁵ vector genomes) in as little as 4 weeks.  

5.2501           Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Fripont, S., Marneffe, C., Marino, M., Rincon, M. and Holt, M.G.

1. Vis. Exp., 143, e58960 (82019)

  Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer (≥1 x 10¹³ vector genomes/mL) and a high purity, ready for in vitro or in vivo use.    

5.2502           Adeno-associated virus 9-mediated RNA interference targeting SOCS3 alleviates diastolic heart failure in rats

Gao, J., Chen, Y., Zhou, J., Liu, Y. and Su, P. Gene, 697, 111-18 (2019)   Objective To explore the effect of adeno-associated virus 9-mediated RNA interference targeting SOCS3 (AAV9-SOCS3 siRNA) on the treatment of diastolic heart failure (DHF). Method A rat DHF model was established, and cardiac function and hemodynamic changes were measured. HE, Sirius red and TUNEL staining were applied to observe the pathological changes in the myocardium. Immunoblotting and immunohistochemical staining were utilized to detect SOCS3 expression. The expression levels of various factors, including fibrosis-related factors (collagen I, collagen II, α-SMA and TGF-β), inflammatory-related factors (IL-1β, IL-6, TNF-α, p-p65 and ICAM-1) and factors related to the JAK/STAT signal pathway were analyzed by immunoblotting and/or qPCR. The serum levels of IL-1β, IL-6, and TNF-α were measured using ELISA. Results SOCS3 expression was significantly downregulated in the DHF rat model by SOCS3 siRNA delivery. In the successfully established DHF rat model, cardiac function was clearly decreased, and cardiomyocyte apoptosis and myocardial fibrosis were significantly increased. These changes were ameliorated by treatment with AAV9-SOCS3 siRNA. The expression levels of p-JAK2 and p-STAT3 were significantly upregulated in the AAV9-SOCS3 siRNA group compared with the sham and AAV9-siRNA control groups, indicating that SOCS3 is a negative regulator of this signaling pathway. The expression levels of collagen I/III, α-SMA and TGF-β were also decreased at both the mRNA and protein levels. In addition, the serum and myocardial tissue expression levels of inflammatory-related factors, such as IL-6, IL-1β, and TNF-α, were also reduced by the administration of AAV9-SOCS3 siRNA compared with the AAV9-siRNA control. Conclusions SOCS3 gene silencing by AAV9-SOCS3 siRNA administration in a DHF rat model significantly reduced myocardial fibrosis and the inflammatory response and improved heart function. Therefore, this treatment is a potential therapeutic method for treating DHF.  

5.2503           Development of a liver‐specific Tet‐off AAV8 vector for improved safety of insulin gene therapy for diabetes

Gan, S.U., Fu, Z., Sia, K.C., Kon, O.L., Calne, R. and Lee, K.O.

1. Gene Med., 21, e3067 (2019)

  Background Diabetes mellitus is caused by a partial or complete lack of insulin production in the body. We have previously shown that a single injection of an adeno‐associated virus serotype 8 (AAV8) vector carrying a modified and codon optimized human insulin gene induced hepatic production of insulin and corrected streptozotocin (STZ)‐induced diabetes in mice for more than 1 year. Insulin production was constitutive, analogous to long‐acting insulin therapy. Methods We have developed a single AAV8 vector with a Tet‐Off regulatable system as a safety mechanism to turn off insulin secretion should hypoglycaemia develop in vector‐treated diabetic mice. We first transfected HepG2 cells or freshly isolated rat hepatocytes in vitro with the Tet‐Off system (pAAV‐Tetoff_bidir‐Alb‐luc) regulating a luciferase reporter gene. We subsequently incorporated a furin‐cleavable codon‐optimised human proinsulin cDNA into pAAV‐Tetoff_bidir backbone to form the doxycycline inducible pAAV‐Tetoff_bidir‐Alb‐hINSco. Results Using STZ‐induced diabetic mice, we were able to switch off insulin secretion repeatedly with doxycycline administration, and showed full restoration of insulin secretion on withdrawing doxycycline. Conclusions The present study provides proof of concept that, under circumstances when inappropriate basal insulin secretion is a safety concern, insulin secretion from AAV8 gene therapy can be turned off reversibly with doxycycline.    

5.2504           HCN4 knockdown in dorsal hippocampus promotes anxiety‐like behavior in mice

Gunther, A., Luczak, V., Gruteser, N., Abel, T. and Baumann, A. Genes, Brain and Behavior, 18, e12550 (2019)   Hyperpolarization‐activated and cyclic nucleotide‐gated (HCN) channels mediate the I_h current in the murine hippocampus. Disruption of the I_h current by knockout of HCN1, HCN2 or tetratricopeptide repeat‐containing Rab8b‐interacting protein has been shown to affect physiological processes such as synaptic integration and maintenance of resting membrane potentials as well as several behaviors in mice, including depressive‐like and anxiety‐like behaviors. However, the potential involvement of the HCN4 isoform in these processes is unknown. Here, we assessed the contribution of the HCN4 isoform to neuronal processing and hippocampus‐based behaviors in mice. We show that HCN4 is expressed in various regions of the hippocampus, with distinct expression patterns that partially overlapped with other HCN isoforms. For behavioral analysis, we specifically modulated HCN4 expression by injecting recombinant adeno‐associated viral (rAAV) vectors mediating expression of short hairpin RNA against hcn4 (shHcn4) into the dorsal hippocampus of mice. HCN4 knockdown produced no effect on contextual fear conditioning or spatial memory. However, a pronounced anxiogenic effect was evident in mice treated with shHcn4 compared to control littermates. Our findings suggest that HCN4 specifically contributes to anxiety‐like behaviors in mice.  

5.2505           A dual‐AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock‐out mice

Al-Moyed, H., Cepeda, A., Jung, SY., Moser, T., Kügler, S. and Reisinger, E. EMBO Mol. Med., 11(1), e9396 (2019)   Normal hearing and synaptic transmission at afferent auditory inner hair cell (IHC) synapses require otoferlin. Deafness DFNB9, caused by mutations in the OTOF gene encoding otoferlin, might be treated by transferring wild‐type otoferlin cDNA into IHCs, which is difficult due to the large size of this transgene. In this study, we generated two adeno‐associated viruses (AAVs), each containing half of the otoferlin cDNA. Co‐injecting these dual‐AAV2/6 half‐vectors into the cochleae of 6‐ to 7‐day‐old otoferlin knock‐out (Otof^−/−) mice led to the expression of full‐length otoferlin in up to 50% of IHCs. In the cochlea, otoferlin was selectively expressed in auditory hair cells. Dual‐AAV transduction of Otof^−/− IHCs fully restored fast exocytosis, while otoferlin‐dependent vesicle replenishment reached 35–50% of wild‐type levels. The loss of 40% of synaptic ribbons in these IHCs could not be prevented, indicating a role of otoferlin in early synapse maturation. Acoustic clicks evoked auditory brainstem responses with thresholds of 40–60 dB. Therefore, we propose that gene delivery mediated by dual‐AAV vectors might be suitable to treat deafness forms caused by mutations in large genes such as OTOF.  

5.2506           Impact of pre-existing dengue immunity on human antibody and memory B cell responses to Zika

Andrade, P., Gimblet-Ochieng, C., Modirian, F., Collins, M., Cardenas, M., Katzelnick, L.C., Montoya, M., Michlmayr, D., Kuan, G., Balmaseda, A., Coloma, J., de Silva, A.M. and Harris, E. Nature Communications, 10, 938 (2019)   Little is known about enduring memory B cell (MBC) responses to Zika virus (ZIKV) and their relationship with circulating antibodies. Here we comprehensively assess MBC frequency and specificity alongside serum binding and neutralizing antibody responses to ZIKV ~2 weeks and ~8 months postinfection in 31 pediatric subjects with 0, 1 or >1 prior infections with the related dengue virus (DENV). ZIKV infection elicits a robust type-specific MBC response, and the majority of late convalescent anti-ZIKV serum neutralizing activity is attributable to ZIKV-specific antibodies. The number of prior DENV infections does not influence type-specific or cross-reactive MBC responses, although ZIKV has the highest cross-reactivity with DENV3. DENV cross-reactive MBCs expanded by ZIKV infection decline in number and proportion by late convalescence. Finally, ZIKV induces greater cross-reactivity in the MBC pool than in serum antibodies. Our data suggest immunity to DENV only modestly shapes breadth and magnitude of enduring ZIKV antibody responses.  

5.2507           Ligand Coupling to the AAV Capsid for Cell-Specific Gene Transfer

Reul, J., Muik, A. and Buchholz, C.J. Methods in Mol. Biol., 1950, 35-50 (2019)   Cell entry of AAV vectors is initiated by contacting the cell surface attachment receptor. This process can be rationally engineered through mutating the contact residues on the AAV capsid and covalently coupling targeting ligands to the capsid surface that exhibit high affinity for a cell surface protein of choice. This way, selective gene delivery to target-receptor positive cell types has been achieved. Two methods for coupling targeting ligands to the AAV capsid can be distinguished. Genetic coupling is achieved through expressing fusion proteins composed of the capsid protein VP2 and the targeting ligand in packaging cells. Biochemical coupling involves split-intein-mediated protein trans-splicing between the mutated AAV capsid and the targeting ligand. While genetic coupling is restricted to designed ankyrin repeat proteins as targeting ligand, biochemical coupling tolerates single-chain antibody fragments as well.  

5.2508           Peripheral AAV Injection for Retrograde Transduction of Dorsal Root and Trigeminal Ganglia

Bloom, B.C., Watson, Z.L. and Neumann, D.M. Methods in Mol. Biol., 1950, 237-247 (2019)   Adeno-associated Virus (AAV) vectors are useful vehicles for delivering transgenes to a number of different tissues and organs in vivo. To date, most of these applications deliver the vectors to their target by either infusion into the bloodstream or direct injection into the target tissue. Recently there has been progress in delivering AAV vectors to neurons of the peripheral nervous system (PNS) following application of vectors to the peripheral epithelium, such as the skin or eye. This delivery only requires treatment of the epithelium to access the underlying nerve termini, and following treatment the vectors are transported retrogradely to the cell bodies of these neurons in the ganglia, such as dorsal root ganglia (DRG) or trigeminal ganglia (TG). Here we describe the methodology for highly efficient transduction of mouse DRG and rabbit TG following application of AAV vectors to the foot, or to the cornea, respectively.  

5.2509           AAV Vectors for Efficient Gene Delivery to Rodent Hearts

Lopez-Gordo, E., Kohlbrenner, E., Katz, M.G. and Weber, T. Methods in Mol. Biol., 1950, 311-332 (2019)   Currently, gene therapy is one of the most promising fields in biomedicine, with great therapeutic potential for an array of inherited and acquired diseases. Adeno-associated viral (AAV) vectors have emerged as promising tools to deliver selectively a therapeutic payload to target organs, including the heart. In this chapter, we describe the production and quality control of recombinant AAV (rAAV) vectors of the serotype 9, the most cardiotropic AAV serotype when delivered systemically in rodents. We also describe the systemic administration of rAAV vectors and the local delivery of rAAV vectors by direct intramyocardial injection. Taken together, the methods described in this chapter will allow the reader to deliver efficiently therapeutic genes to the rodent heart, both globally and regionally.  

5.2510           rAAV-Mediated Gene Delivery to Adipose Tissue

Huang, W., Queen, N.J. and Cao, L. Methods in Mol. Biol., 1950, 389-405 (2019)   Recombinant adeno-associated virus (rAAV) vectors are attractive vehicles for gene therapy. Yet, it is challenging to genetically manipulate adipose tissue in adults due to the low transduction efficiency of naturally occurring AAV serotypes. We recently demonstrated that a novel engineered hybrid serotype Rec2 achieves high transduction of adipose tissue that is superior to naturally occurring serotypes via direct injection to adipose depots. Furthermore, the administration route influences the tropism and efficacy of Rec2 vector: oral administration transduces interscapular brown fat, while intraperitoneal injection preferentially targets visceral fat. Multiple in vivo studies by our lab and others have demonstrated that Rec2 vector provides a powerful tool to genetically manipulate adipose tissue for basic research and potential gene therapies of genetic and acquired diseases. Here we provide detailed protocols for AAV production and delivery to adipose tissue by direct injection, oral administration, and intraperitoneal injection.  

5.2511           Aberrant deposition of stress granule-resident proteins linked to C9orf72-associated TDP-43 proteinopathy

Chew, J., Cook, C., Gendron, T.F., Jansen-West, K., del Rosso, G., Daughrity, L.M., Castanedes-Casey, M., Kurti, A., Stankowski, J.N., Disney, M.D:, Rothstein, J.D., Dickson, D.W., Fryer, J.D., Zhang, Y.J. and Petrucelli, L. Mol. Neurodegeneration, 14:9 (2019)   Background A G₄C₂ hexanucleotide repeat expansion in the noncoding region of C9orf72 is the major genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). Putative disease mechanisms underlying c9FTD/ALS include toxicity from sense G₄C₂ and antisense G₂C₄ repeat-containing RNA, and from dipeptide repeat (DPR) proteins unconventionally translated from these RNA products. Methods Intracerebroventricular injections with adeno-associated virus (AAV) encoding 2 or 149 G₄C₂ repeats were performed on postnatal day 0, followed by assessment of behavioral and neuropathological phenotypes. Results Relative to control mice, gliosis and neurodegeneration accompanied by cognitive and motor deficits were observed in (G₄C₂)₁₄₉ mice by 6 months of age. Recapitulating key pathological hallmarks, we also demonstrate that sense and antisense RNA foci, inclusions of poly(GA), poly(GP), poly(GR), poly(PR), and poly(PA) DPR proteins, and inclusions of endogenous phosphorylated TDP-43 (pTDP-43) developed in (G₄C₂)₁₄₉ mice but not control (G₄C₂)₂ mice. Notably, proteins that play a role in the regulation of stress granules – RNA-protein assemblies that form in response to translational inhibition and that have been implicated in c9FTD/ALS pathogenesis – were mislocalized in (G₄C₂)₁₄₉ mice as early as 3 months of age. Specifically, we observed the abnormal deposition of stress granule components within inclusions immunopositive for poly(GR) and pTDP-43, as well as evidence of nucleocytoplasmic transport defects. Conclusions Our in vivo model of c9FTD/ALS is the first to robustly recapitulate hallmark features derived from both sense and antisense C9orf72 repeat-associated transcripts complete with neurodegeneration and behavioral impairments. More importantly, the early appearance of persistent pathological stress granules prior to significant pTDP-43 deposition implicates an aberrant stress granule response as a key disease mechanism driving TDP-43 proteinopathy in c9FTD/ALS.  

5.2512           Hepatitis E virus genotype 3 and capsid protein in the blood and urine of immunocompromised patients

Marion, O., Capelli, N., Lhomme, S., Dubois, M., Pucelle, M., Abravanel, F., kamar, N.a nd Izopet, J.

1. Infection, 78, 232-240 (2019)

  Objectives Hepatitis E virus genotype 3 (HEV3) is responsible for acute and chronic liver disease in solid organ transplant (SOT) recipients. HEV was recently found in the urine of some acutely and chronically genotype 4-infected patients. Methods We examined the urinary excretion of HEV3 by 24 consecutive SOT recipients at the acute phase of HEV hepatitis and characterized the excreted virus. Results Urinary HEV RNA was detected in 12 (50%) of the 24 transplanted patients diagnosed with HEV hepatitis. Urinary HEV antigen (Ag) was detected in all but one of the patients (96%). The density of RNA-containing HEV particles in urine was low (1.11-1.12 g/cm³), corresponding to lipid-associated virions. The urinary HEV RNA/Ag detected was not associated with impaired kidney function or de novo proteinuria. Finally, there was more HEV Ag in the serum at the acute phase of HEV infection in SOT recipients whose infection became chronic. Conclusions HEV3 excreted via the urine of SOT recipients at the acute phase of HEV hepatitis has a lipid envelope. Renal function was not impaired. While urinary HEV Ag was a sensitive indicator of HEV infection, only acute phase serum HEV Ag indicated the development of a chronic infection.  

5.2513           Delivery of Glucosylceramidase Beta Gene Using AAV9 Vector Therapy as a Treatment Strategy in Mouse Models of Gaucher Disease

Du, S., Qu, H., Cui, R., Jiang, N., Zhang, M., Li, X., Ma, J., Zhang, J. and Ma, D. Human Gene Therapy, 30(2), 155-167 (2019)   Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the GBA gene. Enzyme replacement treatment is the most effective therapy available for type 1 GD patients, but it is very expensive and does not improve neurologic outcomes in type 2 and 3 GD patients. This study evaluated the effectiveness of an adeno-associated virus 9 (AAV9) vector expressing the Gba gene delivered systemically in GD mouse models. To detect the therapeutic effects of the AAV9-mediated Gba transfer on the systemic symptoms of GD, an inducible whole-body Gba knockout mouse was developed in which tamoxifen effectively induced whole-body Gba gene deletion, and the mice displayed systemic symptoms of GD. The AAV9-CMV-Gba vector, with the expression of Gba driven by the universal CMV promoter, restored GCase activity in multiple organs and prolonged the lifespan in tamoxifen-induced GD mice after intravenous injection. Mice with brain-specific Gba deletion were also included in this study as a model of neuropathic GD (nGD) and injected intraperitoneally on postnatal day 5 with the AAV9-SYN-Gba vector; this improved the GCase activity, ameliorated the neuropathological changes and extended the mean lifespan two-fold. This study demonstrates that AAV9-mediated gene transfer is a potentially effective treatment for GD.  

5.2514           Glutathione S-transferases promote proinflammatory astrocyte-microglia communication during brain inflammation

Kano, S-i., Choi, E.Y., Dohi, E., Agarwal, S., Chang, D.J., Wilson, A.M., Lo, B.D., Rose, I.V.L., Gonzalez, S., Imai, T. and Sawa, A. Science Signaling, 12, eaar2124 (201)   Astrocytes and microglia play critical roles in brain inflammation. Here, we report that glutathione S-transferases (GSTs), particularly GSTM1, promote proinflammatory signaling in astrocytes and contribute to astrocyte-mediated microglia activation during brain inflammation. In vivo, astrocyte-specific knockdown of GSTM1 in the prefrontal cortex attenuated microglia activation in brain inflammation induced by systemic injection of lipopolysaccharides (LPS). Knocking down GSTM1 in astrocytes also attenuated LPS-induced production of the proinflammatory cytokine tumor necrosis factor–α (TNF-α) by microglia when the two cell types were cocultured. In astrocytes, GSTM1 was required for the activation of nuclear factor κB (NF-κB) and the production of proinflammatory mediators, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and C-C motif chemokine ligand 2 (CCL2), both of which enhance microglia activation. Our study suggests that GSTs play a proinflammatory role in priming astrocytes and enhancing microglia activation in a microglia-astrocyte positive feedback loop during brain inflammation.  

5.2515           Heterochromatin anomalies and double-stranded RNA accumulation underlie C9orf72 poly(PR) toxicity

Zhang, Y-J., Guo, L., Gonzales, P.K., Gendron, T.F., Wu, Y. et al Science, 363(6428), eaav2606 (2019)   INTRODUCTION Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are fatal neurodegenerative diseases that share clinical and neuropathological features. Furthermore, the most common genetic cause of both FTD and ALS is a GGGGCC (G₄C₂) repeat expansion in the C9orf72 gene. This repeat expansion leads to several abnormalities, including C9orf72 haploinsufficiency, the accumulation of repeat RNA, and the production of five aggregation-prone proteins composed of repeating dipeptides. However, the contribution of these abnormalities to disease pathogenesis remains unresolved. RATIONALE Among the five dipeptide repeat proteins nonconventionally translated from expanded G₄C₂ repeats, proline-arginine (PR) repeat proteins [poly(PR) proteins] have proven especially toxic in various model systems. Their involvement in C9orf72-associated FTD and ALS (c9FTD/ALS) has nevertheless been questioned because poly(PR) pathology is relatively infrequent in c9FTD/ALS patient brains. Postmortem tissues, however, represent end-stage disease and do not necessarily reflect early events in the disease process. Therefore, we generated mice that express poly(PR) in the brain to evaluate the temporal consequences of its expression in a mammalian in vivo model. More specifically, we engineered mice to express green fluorescent protein (GFP)–conjugated (PR)₅₀ (a 50-repeat PR protein) or GFP via intracerebroventricular administration of adeno-associated viral vectors and then performed behavioral, pathological, and transcriptomic characterizations of poly(PR) mice in comparison with control GFP mice. RESULTS We found that ~60% of poly(PR)-expressing mice died by 4 weeks of age and had significantly decreased brain and body weights at death compared with age-matched GFP control mice. Poly(PR) mice that escaped premature death developed motor and memory impairments, likely as a consequence of their progressive brain atrophy, neuron loss, loss of poly(PR)-positive cells, and gliosis. In investigating the mechanisms by which poly(PR) caused neurodegeneration and functional deficits, we found that poly(PR) localized to heterochromatin (highly condensed regions of transcriptionally silent chromatin) and caused abnormal histone H3 methylation, features that we also detected in brain tissues from patients with c9FTD/ALS. Additionally, we observed aberrations in nuclear lamins and heterochromatin protein 1α (HP1α), key proteins that maintain heterochromatin structure and regulate gene silencing. Nuclear lamina invaginations and decreased HP1α protein expression were seen in poly(PR)-positive cells in poly(PR) mice, and in vitro studies demonstrated that poly(PR) disrupted HP1α liquid phases. Because poly(PR)-induced histone H3 posttranslational modifications, lamin invaginations, and decreased HP1α levels could profoundly affect gene expression, we compared transcriptome profiles between control and poly(PR) mice. As well as analyzing differentially expressed genes, we examined repetitive element expression given that repetitive DNA sequences make up a large portion of heterochromatin and that repetitive elements are substantially up-regulated in the brains of c9FTD/ALS patients. Whereas the majority of differentially expressed genes in poly(PR) mice were down-regulated, repetitive elements were markedly up-regulated, and this up-regulation was accompanied by the accumulation of double-stranded RNA. Furthermore, we confirmed that HP1α depletion caused double-stranded RNA accumulation in human induced pluripotent stem cell–derived neurons and decreased their survival. CONCLUSION Our studies provide compelling evidence that, by disrupting HP1α liquid phases, interacting with heterochromatin, and eliciting aberrant histone posttranslational modifications, poly(PR) adversely influences heterochromatin structure. Consequently, repetitive element expression is induced and double-stranded RNA accumulates, contributing to the neurodegeneration seen in patients with c9FTD/ALS. Rescuing histone methylation, lamin, and HP1α abnormalities and/or inhibiting abnormal repetitive element expression may represent promising therapeutic strategies for treating c9FTD/ALS.  

5.2516           Gene therapy targeting SARM1 blocks pathological axon degeneration in mice

Geisler, S., Huang, S.X., Strickland, A., Doan, R.A., Summers, D.W., mao, X., park, J., DiAntonio, A. and Milbrandt, J.

1. Exp. Med., 216(2), 294-303 (2019)

  Axonal degeneration (AxD) following nerve injury, chemotherapy, and in several neurological disorders is an active process driven by SARM1, an injury-activated NADase. Axons of SARM1-null mice exhibit greatly delayed AxD after transection and in models of neurological disease, suggesting that inhibiting SARM1 is a promising strategy to reduce pathological AxD. Unfortunately, no drugs exist to target SARM1. We, therefore, developed SARM1 dominant-negatives that potently block AxD in cellular models of axotomy and neuropathy. To assess efficacy in vivo, we used adeno-associated virus–mediated expression of the most potent SARM1 dominant-negative and nerve transection as a model of severe AxD. While axons of vehicle-treated mice degenerate rapidly, axons of mice expressing SARM1 dominant-negative can remain intact for >10 d after transection, similar to the protection observed in SARM1-null mice. We thus developed a novel in vivo gene therapeutic to block pathological axon degeneration by inhibiting SARM1, an approach that may be applied clinically to treat manifold neurodegenerative diseases characterized by axon loss.  

5.2517           Viability RT-qPCR Combined with Sodium Deoxycholate Pre-treatment for Selective Quantification of Infectious Viruses in Drinking Water Samples

Canh, V.D., Kasuga, I., Furumai, H. and Katayama, H. Food and Environmental Virol., 11(1), 40-51 (2019)   The presence of pathogenic viruses in drinking water is a major public health concern. Although viability RT-qPCR methods were developed to quantify infectious viruses, they may not always reflect viral infectivity, therefore leading to false-positive results. In this study, sodium deoxycholate (SD) pre-treatment was used to improve the efficiency of viability RT-qPCR methods with respect to exclusive quantification of infectious viruses. The ability of SD pre-treatment to enhance the penetration of three viability markers, namely, ethidium monoazide (EMA, 100 µM), propidium monoazide (PMA, 100 µM), and cis-dichlorodiammineplatinum (CDDP, 1000 µM), into heat-treated (90 °C for 1 min) Aichi virus at various concentrations (0.01–0.5%) was evaluated. The optimal SD concentration was found to be 0.1% for all markers. EMA/PMA/CDDP-RT-qPCR with 0.1% SD pre-treatment was significantly more effective than without SD pre-treatment in determining AiV inactivation after heat (50, 60, 70, 80, or 90 °C for 1 min) or chlorine treatment (1 mgCl₂/L for 1, 2, 5, or 10 min). Among the viability RT-qPCR methods tested, CDDP-RT-qPCR with SD pre-treatment (SD-CDDP-RT-qPCR) was the most effective in reflecting viral infectivity. Performance testing of SD-CDDP-RT-qPCR in concentrated drinking water samples did not reveal any significant effects of SD-CDDP treatment. Thus, SD-CDDP-RT-qPCR could be a useful tool for monitoring infectious virus presence in drinking water.  

5.2518           A Human Papillomavirus-Independent Cervical Cancer Animal Model Reveals Unconventional Mechanisms of Cervical Carcinogenesis

He, C., Xiangmin, L., Huang, C., Rueda, B.R., Davis, J.S. and Wang, C. Cell Reports, 26, 2636-2650 (2019)   HPV infections are common in healthy women and only rarely cause cervical cancer, suggesting that individual genetic susceptibility may play a critical role in the establishment of persistent HPV infection and the development of cervical cancer. Here, we provide convincing in vitro and in vivo evidence showing that differential expression and activation of YAP1 oncogene determine individual susceptibility to HPV infection and cervical carcinogenesis. We found that hyperactivation of YAP1 in mouse cervical epithelium was sufficient to induce invasive cervical cancer. Cervical epithelial cell-specific HPV16 E6/E7 and YAP1 double-knockin mouse model demonstrated that high-risk HPV synergized with hyperactivated YAP1 to promote the initiation and progression of cervical cancer. Our mechanistic studies indicated that hyperactivation of YAP1 in cervical epithelial cells facilitated HPV infection by increasing the putative HPV receptor molecules and disrupting host cell innate immunity. Our finding reveals an unconventional mechanism for cervical carcinogenesis.  

5.2519           ACTN2 mutations cause “Multiple structured Core Disease” (MsCD)

Lonage, X., Romero, M.B., Grosgogeat, C.A., Malfatti, E., Donkervoort, S. et al Acta Neuropathologica, 137(3), 501-519 (2019)   The identification of genes implicated in myopathies is essential for diagnosis and for revealing novel therapeutic targets. Here we characterize a novel subclass of congenital myopathy at the morphological, molecular, and functional level. Through exome sequencing, we identified de novo ACTN2 mutations, a missense and a deletion, in two unrelated patients presenting with progressive early-onset muscle weakness and respiratory involvement. Morphological and ultrastructural analyses of muscle biopsies revealed a distinctive pattern with the presence of muscle fibers containing small structured cores and jagged Z-lines. Deeper analysis of the missense mutation revealed mutant alpha-actinin-2 properly localized to the Z-line in differentiating myotubes and its level was not altered in muscle biopsy. Modelling of the disease in zebrafish and mice by exogenous expression of mutated alpha-actinin-2 recapitulated the abnormal muscle function and structure seen in the patients. Motor deficits were noted in zebrafish, and muscle force was impaired in isolated muscles from AAV-transduced mice. In both models, sarcomeric disorganization was evident, while expression of wild-type alpha-actinin-2 did not result in muscle anomalies. The murine muscles injected with mutant ACTN2 displayed cores and Z-line defects. Dominant ACTN2 mutations were previously associated with cardiomyopathies, and our data demonstrate that specific mutations in the well-known Z-line regulator alpha-actinin-2 can cause a skeletal muscle disorder.  

5.2520           miR-124 dosage regulates prefrontal cortex function by dopaminergic modulation

Kozuka, T., Omari, Y., Watanabe, S., Tarusawa, E., Yamamoto, H., Chaya, T., Furuhashi, M., Moritta, M., Sato, T., Hirose, S., Ohkawa, Y., Yoshimura, Y., Hikida, T. and Furukawa, T. Scientific Reports, 9:3445 (2019)   MicroRNA-124 (miR-124) is evolutionarily highly conserved among species and one of the most abundantly expressed miRNAs in the developing and mature central nervous system (CNS). Previous studies reported that miR-124 plays a role in CNS development, such as neuronal differentiation, maturation, and survival. However, the role of miR-124 in normal brain function has not yet been revealed. Here, we subjected miR-124-1+/− mice, to a comprehensive behavioral battery. We found that miR-124-1+/− mice showed impaired prepulse inhibition (PPI), methamphetamine-induced hyperactivity, and social deficits. Whole cell recordings using prefrontal cortex (PFC) slices showed enhanced synaptic transmission in layer 5 pyramidal cells in the miR-124-1+/− PFC. Based on the results of behavioral and electrophysiological analysis, we focused on genes involved in the dopaminergic system and identified a significant increase of Drd2 expression level in the miR-124-1+/− PFC. Overexpression or knockdown of Drd2 in the control or miR-124-1+/− PFC demonstrates that aberrant Drd2 signaling leads to impaired PPI. Furthermore, we identified that expression of glucocorticoid receptor gene Nr3c1, which enhances Drd2 expression, increased in the miR-124-1+/− PFC. Taken together, the current study suggests that miR-124 dosage modulates PFC function through repressing the Drd2 pathway, suggesting a critical role of miR-124 in normal PFC function.    

5.2521           Vector uncoating limits adeno-associated viral vector-mediated transduction of human dendritic cells and vector immunogenicity

Rossi, A., Dupaty, L., Aillot, L., Zhang, L., Gallien, C., Hallek, M., Odenthal, M., Adriouch, S., Salvetti, A. and Büning, H. Scientific Reports, 9:3631 (2019)   AAV vectors poorly transduce Dendritic cells (DC), a feature invoked to explain AAV’s low immunogenicity. However, the reason for this non-permissiveness remained elusive. Here, we performed an in-depth analysis using human monocyte-derived immature DC (iDC) as model. iDC internalized AAV vectors of various serotypes, but even the most efficient serotype failed to transduce iDC above background. Since AAV vectors reached the cell nucleus, we hypothesized that AAV’s intracellular processing occurs suboptimal. On this basis, we screened an AAV peptide display library for capsid variants more suitable for DC transduction and identified the I/VSS family which transduced DC with efficiencies of up to 38%. This property correlated with an improved vector uncoating. To determine the consequence of this novel feature for AAV’s in vivo performance, we engineered one of the lead candidates to express a cytoplasmic form of ovalbumin, a highly immunogenic model antigen, and assayed transduction efficiency as well as immunogenicity. The capsid variant clearly outperformed the parental serotype in muscle transduction and in inducing antigen-specific humoral and T cell responses as well as anti-capsid CD8+ T cells. Hence, vector uncoating represents a major barrier hampering AAV vector-mediated transduction of DC and impacts on its use as vaccine platform.  

5.2522           Infectious Entry of Merkel Cell Polyomavirus

Becker, M., Dominguez, M., Greune, L., Soria-Martinez, L., Pfleiderer, M.M., Schowalter, R., Buck, C.B., Blaum, B.S., Schmidt, M.A. and Schelhaas, M.

1. Virol., 93(6), e02004-18 (2019)

  Merkel cell polyomavirus (MCPyV) is a small, nonenveloped tumor virus associated with an aggressive form of skin cancer, Merkel cell carcinoma (MCC). MCPyV infections are highly prevalent in the human population, with MCPyV virions being continuously shed from human skin. However, the precise host cell tropism(s) of MCPyV remains unclear: MCPyV is able to replicate within a subset of dermal fibroblasts, but MCPyV DNA has also been detected in a variety of other tissues. However, MCPyV appears different from other polyomaviruses, as it requires sulfated polysaccharides, such as heparan sulfates and/or chondroitin sulfates, for initial attachment. Like other polyomaviruses, MCPyV engages sialic acid as a (co)receptor. To explore the infectious entry process of MCPyV, we analyzed the cell biological determinants of MCPyV entry into A549 cells, a highly transducible lung carcinoma cell line, in comparison to well-studied simian virus 40 and a number of other viruses. Our results indicate that MCPyV enters cells via caveolar/lipid raft-mediated endocytosis but not macropinocytosis, clathrin-mediated endocytosis, or glycosphingolipid-enriched carriers. The viruses were internalized in small endocytic pits that led the virus to endosomes and from there to the endoplasmic reticulum (ER). Similar to other polyomaviruses, trafficking required microtubular transport, acidification of endosomes, and a functional redox environment. To our surprise, the virus was found to acquire a membrane envelope within endosomes, a phenomenon not reported for other viruses. Only minor amounts of viruses reached the ER, while the majority was retained in endosomal compartments, suggesting that endosome-to-ER trafficking is a bottleneck during infectious entry.  

5.2523           RNA-Binding Motif Protein 24 (RBM24) Is Involved in Pregenomic RNA Packaging by Mediating Interaction between Hepatitis B Virus Polymerase and the Epsilon Element

Yao, Y., Yang, B., Chen, Y., Wang, H., Hu, X., Zhou, Y., Gao, X., Lu, M., Niu, J., Wen, Z., Wu, C. and Chen, X.

1. Virol., 93(6), e02161-18 (2019)

  Encapsidation of pregenomic RNA (pgRNA) is a crucial step in hepatitis B virus (HBV) replication. Binding by viral polymerase (Pol) to the epsilon stem-loop (ε) on the 5′-terminal region (TR) of pgRNA is required for pgRNA packaging. However, the detailed mechanism is not well understood. RNA-binding motif protein 24 (RBM24) inhibits core translation by binding to the 5′-TR of pgRNA. Here, we demonstrate that RBM24 is also involved in pgRNA packaging. RBM24 directly binds to the lower bulge of ε via RNA recognition submotifs (RNPs). RBM24 also interacts with Pol in an RNA-independent manner. The alanine-rich domain (ARD) of RBM24 and the reverse transcriptase (RT) domain of Pol are essential for binding between RBM24 and Pol. In addition, overexpression of RBM24 increases Pol-ε interaction, whereas RBM24 knockdown decreases the interaction. RBM24 was able to rescue binding between ε and mutant Pol lacking ε-binding activity, further showing that RBM24 mediates the interaction between Pol and ε by forming a Pol-RBM24-ε complex. Finally, RBM24 significantly promotes the packaging efficiency of pgRNA. In conclusion, RBM24 mediates Pol-ε interaction and formation of a Pol-RBM24-ε complex, which inhibits translation of pgRNA and results in pgRNA packing into capsids/virions for reverse transcription and DNA synthesis.  

5.2524           A Photoactivatable Botulinum Neurotoxin for Inducible Control of Neurotransmission

Liu, Q., Sinnen, B.L., Boxer, E.E., Aoto, J., Tucker, C.L. and Kennedy, M.J. Neuron, 101, 863-875 (2019)   Regulated secretion is critical for diverse biological processes ranging from immune and endocrine signaling to synaptic transmission. Botulinum and tetanus neurotoxins, which specifically proteolyze vesicle fusion proteins involved in regulated secretion, have been widely used as experimental tools to block these processes. Genetic expression of these toxins in the nervous system has been a powerful approach for disrupting neurotransmitter release within defined circuitry, but their current utility in the brain and elsewhere remains limited by lack of spatial and temporal control. Here we engineered botulinum neurotoxin B so that it can be activated with blue light. We demonstrate the utility of this approach for inducibly disrupting excitatory neurotransmission, providing a first-in-class optogenetic tool for persistent, light-triggered synaptic inhibition. In addition to blocking neurotransmitter release, this approach will have broad utility for conditionally disrupting regulated secretion of diverse bioactive molecules, including neuropeptides, neuromodulators, hormones, and immune molecules.  

5.2525           Brain tyrosinase overexpression implicates age-dependent neuromelanin production in Parkinson’s disease pathogenesis

Carballo-Carbajal, I., Laguna, A., Romero-Gimenez, J., Cuadros, T., Bove, J. et al Nature Communications, 10:973 (2019)   In Parkinson’s disease (PD) there is a selective degeneration of neuromelanin-containing neurons, especially substantia nigra dopaminergic neurons. In humans, neuromelanin accumulates with age, the latter being the main risk factor for PD. The contribution of neuromelanin to PD pathogenesis remains unknown because, unlike humans, common laboratory animals lack neuromelanin. Synthesis of peripheral melanins is mediated by tyrosinase, an enzyme also present at low levels in the brain. Here we report that overexpression of human tyrosinase in rat substantia nigra results in age-dependent production of human-like neuromelanin within nigral dopaminergic neurons, up to levels reached in elderly humans. In these animals, intracellular neuromelanin accumulation above a specific threshold is associated to an age-dependent PD phenotype, including hypokinesia, Lewy body-like formation and nigrostriatal neurodegeneration. Enhancing lysosomal proteostasis reduces intracellular neuromelanin and prevents neurodegeneration in tyrosinase-overexpressing animals. Our results suggest that intracellular neuromelanin levels may set the threshold for the initiation of PD.  

5.2526           Tau exhibits unique seeding properties in globular glial tauopathy

Chung, D-e.C., Carlomagno, Y., Cook, C.N., Jansen-West, K., Daughrity, L., Lewis-Tuffin, L.J., Castanedes-Casey, M., DeTure, M., Dickson, D.W. and Petrucelli, L. Acta Neuropathologica Comm., 7:36 (2019)   Tauopathies are neurodegenerative disorders characterized by aggregation of microtubule associated tau protein in neurons and glia. They are clinically and pathologically heterogeneous depending on the isoform of tau protein that accumulates (three or four 31-to-32-amino-acid repeats [3R or 4R] in the microtubule binding domain), as well as the cellular and neuroanatomical distribution of tau pathology. Growing evidence suggests that distinct tau conformers may contribute to the characteristic features of various tauopathies. Globular glial tauopathy (GGT) is a rare 4R tauopathy with globular cytoplasmic inclusions within neurons and glial cells. Given the unique cellular distribution and morphology of tau pathology in GGT, we sought to determine if tau species in GGT had distinctive biological properties. To address this question, we performed seeding analyses with postmortem brain tissues using a commercial tau biosensor cell line. We found that brain lysates from GGT cases had significantly higher seeding competency than other tauopathies, including corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and Alzheimer’s disease (AD). The robust seeding activity of GGT brain lysates was independent of phosphorylated tau burden and diminished upon removal of tau from samples, suggesting that seeding properties were indeed mediated by tau in the lysates. In addition, cellular inclusions in the tau biosensor cell line induced by GGT had a distinct, globular morphology that was markedly different from inclusions induced by other tauopathies, further highlighting the unique nature of tau species in GGT. Characterization of different tau species in GGT showed that detergent-insoluble, fibril-like tau contained the highest seeding activity, as reflected in its ability to increase tau aggregation in primary glial cultures. Taken together, our data suggest that unique seeding properties differentiate GGT-tau from other tauopathies, which provides new insight into pathogenic heterogeneity of primary neurodegenerative tauopathies.  

5.2527           Oral administration of the cannabigerol derivative VCE-003.2 promotes subventricular zone neurogenesis and protects against mutant huntingtin-induced neurodegeneration

Aguareles, J., Paraiso-Luna, J., Palomares, B., Bajo-Graneras, R., Navarrete, C., Ruiz-Calvo, A., Garcia-Rincon, D., Garcia-Taboada, E., Guzman, M., Munoz, E. and Galve-Ropoth, I. Translational Neurodegeneration, 8:9 (2019)   Background The administration of certain cannabinoids provides neuroprotection in models of neurodegenerative diseases by acting through various cellular and molecular mechanisms. Many cannabinoid actions in the nervous system are mediated by CB₁ receptors, which can elicit psychotropic effects, but other targets devoid of psychotropic activity, including CB₂ and nuclear PPARγ receptors, can also be the target of specific cannabinoids. Methods We investigated the pro-neurogenic potential of the synthetic cannabigerol derivative, VCE-003.2, in striatal neurodegeneration by using adeno-associated viral expression of mutant huntingtin in vivo and mouse embryonic stem cell differentiation in vitro. Results Oral administration of VCE-003.2 protected striatal medium spiny neurons from mutant huntingtin-induced damage, attenuated neuroinflammation and improved motor performance. VCE-003.2 bioavailability was characterized and the potential undesired side effects were evaluated by analyzing hepatotoxicity after chronic treatment. VCE-003.2 promoted subventricular zone progenitor mobilization, increased doublecortin-positive migrating neuroblasts towards the injured area, and enhanced effective neurogenesis. Moreover, we demonstrated the proneurogenic activity of VCE-003.2 in embryonic stem cells. VCE-003.2 was able to increase neuroblast formation and striatal-like CTIP2-mediated neurogenesis. Conclusions The cannabigerol derivative VCE-003.2 improves subventricular zone-derived neurogenesis in response to mutant huntingtin-induced neurodegeneration, and is neuroprotective by oral administration.  

5.2528           rAAVrh74.MCK.GALGT2 Protects against Loss of Hemodynamic Function in the Aging mdx Mouse Heart

Xu, R., Jia, Y., Zygmunt, D.A. and Martin, P.T. Molecular Therapy, 27(3), 636-649 (2019)   Dilated cardiomyopathy is a common cause of death in patients with Duchenne muscular dystrophy (DMD). Gene therapies for DMD must, therefore, have a therapeutic impact in cardiac as well as skeletal muscles. Our previous studies have shown that GALGT2 overexpression in mdx skeletal muscles can prevent muscle damage. Here we have tested whether rAAVrh74.MCK.GALGT2 gene therapy in mdxcardiac muscle can prevent the loss of heart function. Treatment of mdx hearts with rAAVrh74.MCK.GALGT2 1 day after birth did not negatively alter hemodynamicfunction, tested at 3 months of age, and it prevented early left ventricular remodeling and expression of fibrotic gene markers. Intravenous treatment of mdxmice with rAAVrh74.MCK.GALGT2 at 2 months of age significantly improved stroke volume and cardiac output compared to mock-treated mice analyzed at 17 months, both at rest and after stimulation with dobutamine. rAAVrh74.MCK.GALGT2treatment of mdx heart correlated with increased glycosylation of α-dystroglycan with the CT glycan and increased utrophin protein expression. These data provide the first demonstration that GALGT2 overexpression can inhibit the loss of cardiac function in the dystrophin-deficient heart and, thus, may benefit both cardiac and skeletal muscles in DMD patients.  

5.2529           Protease-Activatable Adeno-Associated Virus Vector for Gene Delivery to Damaged Heart Tissue

Guenther, C.M., Brun, M.J., Bennett, A.D., Ho, M.L., Chen, W., Zhu, B., Lam, M. et al Molecular Therapy, 27(3), 611-622 (2019)   Adeno-associated virus (AAV) has emerged as a promising gene delivery vector because of its non-pathogenicity, simple structure and genome, and low immunogenicity compared to other viruses. However, its adoption as a safe and effective delivery vector for certain diseases relies on altering its tropism to deliver transgenes to desired cell populations. To this end, we have developed a protease-activatable AAV vector, named provector, that responds to elevated extracellular protease activity commonly found in diseased tissue microenvironments. The AAV9-based provector is initially inactive, but then it can be switched on by matrix metalloproteinases (MMP)-2 and -9. Cryo-electron microscopy and image reconstruction reveal that the provector capsid is structurally similar to that of AAV9, with a flexible peptide insertion at the top of the 3-fold protrusions. In an in vivo model of myocardial infarction (MI), the provector is able to deliver transgenes site specifically to high-MMP-activity regions of the damaged heart, with concomitant decreased delivery to many off-target organs, including the liver. The AAV provector may be useful in the future for enhanced delivery of transgenes to sites of cardiac damage.  

5.2530           A serum protein factor mediates maturation and apoB-association of HCV particles in the extracellular milieu

Denolly, S., Granier, C., Fontaine, N., Bourlot, T., Guerin, N. and Cosset, F-L.

1. Hepatol., 70, 626-638 (2019)

  Background & Aims In the sera of infected patients, hepatitis C virus (HCV) particles display heterogeneous forms with low-buoyant densities (<1.08), underscoring their lipidation via association with apoB-containing lipoproteins, which was proposed to occur during assembly or secretion from infected hepatocytes. However, the mechanisms inducing this association remain poorly-defined and most cell culture grown HCV (HCVcc) particles exhibit higher density (>1.08) and poor/no association with apoB. We aimed to elucidate the mechanisms of lipidation and to produce HCVcc particles resembling those in infected sera. Methods We produced HCVcc particles of Jc1 or H77 strains from Huh-7.5 hepatoma cells cultured in standard conditions (10%-fetal calf serum) vs. in serum-free or human serum conditions before comparing their density profiles to patient-derived virus. We also characterized wild-type and Jc1/H77 hypervariable region 1 (HVR1)-swapped mutant HCVcc particles produced in serum-free media and incubated with different serum types or with purified lipoproteins. Results Compared to serum-free or fetal calf serum conditions, production with human serum redistributed most HCVcc infectious particles to low density (<1.08) or very-low density (<1.04) ranges. In addition, short-time incubation with human serum was sufficient to shift HCVcc physical particles to low-density fractions, in time- and dose-dependent manners, which increased their specific infectivity, promoted apoB-association and induced neutralization-resistance. Moreover, compared to Jc1, we detected higher levels of H77 HCVcc infectious particles in very-low-density fractions, which could unambiguously be attributed to strain-specific features of the HVR1 sequence. Finally, all 3 lipoprotein classes, i.e., very-low-density, low-density and high-density lipoproteins, could synergistically induce low-density shift of HCV particles; yet, this required additional non-lipid serum factor(s) that include albumin. Conclusions The association of HCV particles with lipids may occur in the extracellular milieu. The lipidation level depends on serum composition as well as on HVR1-specific properties. These simple culture conditions allow production of infectious HCV particles resembling those of chronically-infected patients.  

5.2531           Disease-associated mutations of claudin-19 disrupt retinal neurogenesis and visual function

Wang, S-B., Xu, T., Peng, S., Singh, D., Ghiassi-Nejad, M., Adelman, R.A. and Rizzolo, L.J. Communications Biology, 2:113 (2019)   Mutations of claudin-19 cause Familial Hypomagnesaemia and Hypercalciuria, Nephrocalcinosis with Ocular Involvement. To study the ocular disease without the complications of the kidney disease, naturally occurring point mutations of human CLDN19 were recreated in human induced pluripotent cells or overexpressed in the retinae of newborn mice. In human induced pluripotent cells, we show that the mutation affects retinal neurogenesis and maturation of retinal pigment epithelium (RPE). In mice, the mutations diminish the P1 wave of the electroretinogram, activate apoptosis in the outer nuclear layer, and alter the morphology of bipolar cells. If mice are given 9-cis-retinal to counter the loss of retinal isomerase, the P1 wave is partially restored. The ARPE19 cell line fails to express claudin-19. Exogenous expression of wild type, but not mutant claudin-19, increases the expression of RPE signature genes. Mutated claudin-19 affects multiple stages of RPE and retinal differentiation through its effects on multiple functions of the RPE.  

5.2532           Chloroviruses Lure Hosts through Long-Distance Chemical Signaling

Dunigan, D.D., Al.Sammak, M., Al-Ameeli, Z., Agarkova, I.V., DeLong, J.P. and Van Etten, J.L.

J. Virol., 93(7), e01688-18 (2019)

Chloroviruses exist in aquatic systems around the planet and they infect certain eukaryotic green algae that are mutualistic endosymbionts in a variety of protists and metazoans. Natural chlorovirus populations are seasonally dynamic, but the precise temporal changes in these populations and the mechanisms that underlie them have heretofore been unclear. We recently reported the novel concept that predator/prey-mediated virus activation regulates chlorovirus population dynamics, and in the current study, we demonstrate virus-packaged chemotactic modulation of prey behavior.

 

5.2533           The Polar Region of the HIV-1 Envelope Protein Determines Viral Fusion and Infectivity by Stabilizing the gp120-gp41 Association

Lu, W., Chen, S., Yu, J., Behrens, R., Wiggins, j., Sherer, N., Liu, S-L., Xiong, Y., Xiang, S-H. and Wu, L.

1. Virol., 93(7), e02128-18 (2019)

  HIV-1 enters cells through binding between viral envelope glycoprotein (Env) and cellular receptors to initiate virus and cell fusion. HIV-1 Env precursor (gp160) is cleaved into two units noncovalently bound to form a trimer on virions, including a surface unit (gp120) and a transmembrane unit (gp41) responsible for virus binding and membrane fusion, respectively. The polar region (PR) at the N terminus of gp41 comprises 17 residues, including 7 polar amino acids. Previous studies suggested that the PR contributes to HIV-1 membrane fusion and infectivity; however, the precise role of the PR in Env-mediated viral entry and the underlying mechanisms remain unknown. Here, we show that the PR is critical for HIV-1 fusion and infectivity by stabilizing Env trimers. Through analyzing the PR sequences of 57,645 HIV-1 isolates, we performed targeted mutagenesis and functional studies of three highly conserved polar residues in the PR (S532P, T534A, and T536A) which have not been characterized previously. We found that single or combined mutations of these three residues abolished or significantly decreased HIV-1 infectivity without affecting viral production. These PR mutations abolished or significantly reduced HIV-1 fusion with target cells and also Env-mediated cell-cell fusion. Three PR mutations containing S532P substantially reduced gp120 and gp41 association, Env trimer stability, and increased gp120 shedding. Furthermore, S532A mutation significantly reduced HIV-1 infectivity and fusogenicity but not Env expression and cleavage. Our findings suggest that the PR of gp41, particularly the key residue S532, is structurally essential for maintaining HIV-1 Env trimer, viral fusogenicity, and infectivity.

5.2534           AAV cis-regulatory sequences are correlated with ocular toxicity

Xiong, W., Wu, D.M., Xue, Y., Wang, S.K., Xhung, M.J., Ji, X., Rana, P., Zhao, S.R., Mai, S. and Cepko, C.L. PNAS, 116(12), 5785-5794 (2019)   Adeno-associated viral vectors (AAVs) have become popular for gene therapy, given their many advantages, including their reduced inflammatory profile compared with that of other viruses. However, even in areas of immune privilege such as the eye, AAV vectors are capable of eliciting host-cell responses. To investigate the effects of such responses on several ocular cell types, we tested multiple AAV genome structures and capsid types using subretinal injections in mice. Assays of morphology, inflammation, and physiology were performed. Pathological effects on photoreceptors and the retinal pigment epithelium (RPE) were observed. Müller glia and microglia were activated, and the proinflammatory cytokines TNF-α and IL-1β were up-regulated. There was a strong correlation between cis-regulatory sequences and toxicity. AAVs with any one of three broadly active promoters, or an RPE-specific promoter, were toxic, while AAVs with four different photoreceptor-specific promoters were not toxic at the highest doses tested. There was little correlation between toxicity and transgene, capsid type, preparation method, or cellular contaminants within a preparation. The toxic effect was dose-dependent, with the RPE being more sensitive than photoreceptors. Our results suggest that ocular AAV toxicity is associated with certain AAV cis-regulatory sequences and/or their activity and that retinal damage occurs due to responses by the RPE and/or microglia. By applying multiple, sensitive assays of toxicity, AAV vectors can be designed so that they can be used safely at high dose, potentially providing greater therapeutic efficacy.      

5.2535           Therapeutic FGF19 promotes HDL biogenesis and transhepatic cholesterol efflux to prevent atherosclerosis

Zhou, M., Learned, R.M., Rossi, S.J., Tian, H., DePaoli, A.M. and Ling, L.

1. Lipid Res., 60, 550-565 (2019)

  Fibroblast growth factor (FGF)19, an endocrine hormone produced in the gut, acts in the liver to control bile acid synthesis. NGM282, an engineered FGF19 analog, is currently in clinical development for treating nonalcoholic steatohepatitis. However, the molecular mechanisms that integrate FGF19 with cholesterol metabolic pathways are incompletely understood. Here, we report that FGF19 and NGM282 promote HDL biogenesis and cholesterol efflux from the liver by selectively modulating LXR signaling while ameliorating hepatic steatosis. We further identify ABCA1 and FGF receptor 4 as mediators of this effect, and that administration of a HMG-CoA reductase inhibitor or a blocking antibody against proprotein convertase subtilisin/kexin type 9 abolished FGF19-associated elevations in total cholesterol, HDL cholesterol (HDL-C), and LDL cholesterol in db/db mice. Moreover, we show that a constitutively active MEK1, but not a constitutively active STAT3, mimics the effect of FGF19 and NGM282 on cholesterol change. In dyslipidemic Apoe^−/− mice fed a Western diet, treatment with NGM282 dramatically reduced atherosclerotic lesion area in aortas. Administration of NGM282 to healthy volunteers for 7 days resulted in a 26% increase in HDL-C levels compared with placebo. These findings outline a previously unrecognized role for FGF19 in the homeostatic control of cholesterol and may have direct impact on the clinical development of FGF19 analogs.  

5.2536           A proof-of-concept study for the design of a VLP-based combinatorial HPV and placental malaria vaccine

Janitzek, C.M., Peabody, J., Thrane, S., Carlsen, P.H.R., Theander, T.G., Salanti, A., Chackerian, B., Nielsen, M.A. and Sander, A.F. Scientific Reports, 9:5260 (2019)   In Africa, cervical cancer and placental malaria (PM) are a major public health concern. There is currently no available PM vaccine and the marketed Human Papillomavirus (HPV) vaccines are prohibitively expensive. The idea of a combinatorial HPV and PM vaccine is attractive because the target population for vaccination against both diseases, adolescent girls, would be overlapping in Sub-Saharan Africa. Here we demonstrate proof-of-concept for a combinatorial vaccine utilizing the AP205 capsid-based virus-like particle (VLP) designed to simultaneously display two clinically relevant antigens (the HPV RG1 epitope and the VAR2CSA PM antigen). Three distinct combinatorial VLPs were produced displaying one, two or five concatenated RG1 epitopes without obstructing the VLP’s capacity to form. Co-display of VAR2CSA was achieved through a split-protein Tag/Catcher interaction without hampering the vaccine stability. Vaccination with the combinatorial vaccine(s) was able to reduce HPV infection in vivo and induce anti-VAR2CSA IgG antibodies, which inhibited binding between native VAR2CSA expressed on infected red blood cells and chondroitin sulfate A in an in vitro binding-inhibition assay. These results show that the Tag/Catcher AP205 VLP system can be exploited to make a combinatorial vaccine capable of eliciting antibodies with dual specificity.  

5.2537           A Prime-Pull-Amplify Vaccination Strategy To Maximize Induction of Circulating and Genital-Resident Intraepithelial CD8+ Memory T Cells

Cuburu, N., Kim, R., Guittard, G.C., Thompson, C.D., Day, P.M., Hamm, D.E., Pang, Y-Y.S., Graham, B.S., Lowy, J. and Schiller, J.T.

1. Immunol., 202, 1250-1264 (2019)

  Recent insight into the mechanisms of induction of tissue-resident memory (T_RM) CD8⁺ T cells (CD8⁺ T_RM) enables the development of novel vaccine strategies against sexually transmitted infections. To maximize both systemic and genital intraepithelial CD8⁺ T cells against vaccine Ags, we assessed combinations of i.m. and intravaginal routes in heterologous prime-boost immunization regimens with unrelated viral vectors. Only i.m. prime followed by intravaginal boost induced concomitant strong systemic and intraepithelial genital-resident CD8⁺ T cell responses. Intravaginal boost with vectors expressing vaccine Ags was far superior to intravaginal instillation of CXCR3 chemokine receptor ligands or TLR 3, 7, and 9 agonists to recruit and increase the pool of cervicovaginal CD8⁺ T_RM. Transient Ag presentation increased trafficking of cognate and bystander circulating activated, but not naive, CD8⁺ T cells into the genital tract and induced in situ proliferation and differentiation of cognate CD8⁺ T_RM. Secondary genital CD8⁺ T_RM were induced in the absence of CD4⁺ T cell help and shared a similar TCR repertoire with systemic CD8⁺ T cells. This prime-pull-amplify approach elicited systemic and genital CD8⁺ T cell responses against high-risk human papillomavirus type 16 E7 oncoprotein and conferred CD8-mediated protection to a vaccinia virus genital challenge. These results underscore the importance of the delivery route of nonreplicating vectors in prime-boost immunization to shape the tissue distribution of CD8⁺ T cell responses. In this context, the importance of local Ag presentation to elicit genital CD8⁺ T_RM provides a rationale to develop novel vaccines against sexually transmitted infections and to treat human papillomavirus neoplasia  

5.2538           The RNA-Binding Protein PUM2 Impairs Mitochondrial Dynamics and Mitophagy During Aging

D’Amico, D., Mottis, A., Potenza, F., Knott, G., Schneider, B.L. and Auwerx, J. Molecular Cell, 73(4), 775-787 (2019)   Little information is available about how post-transcriptional mechanisms regulate the aging process. Here, we show that the RNA-binding protein Pumilio2 (PUM2), which is a translation repressor, is induced upon aging and acts as a negative regulator of lifespan and mitochondrial homeostasis. Multi-omics and cross-species analyses of PUM2 function show that it inhibits the translation of the mRNA encoding for the mitochondrial fission factor ( Mff), thereby impairing mitochondrial fission and mitophagy. This mechanism is conserved in C. elegans by the PUM2 ortholog PUF-8. puf-8 knock-down in old nematodes and Pum2 CRISPR/Cas9-mediated knockout in the muscles of elderly mice enhances mitochondrial fission and mitophagy in both models, hence improving mitochondrial quality control and tissue homeostasis. Our data reveal how a PUM2-mediated layer of post-transcriptional regulation links altered Mff translation to mitochondrial dynamics and mitophagy, thereby mediating age-related mitochondrial dysfunctions.  

5.2539           In vivo RNA editing of point mutations via RNA-guided adenosine deaminases

Katrekar, D., Chen, G., Meluzzi, D., Ganesh, A., Worlikar, A., Shih, Y-R., Varghese, S. and Mali, P. Nature Methods, 16, 239-242 (2019)   We present in vivo sequence-specific RNA base editing via adenosine deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). To achieve this, we systematically engineered adRNAs to harness ADARs, and comprehensively evaluated the specificity and activity of the toolsets in vitro and in vivo via two mouse models of human disease. We anticipate that this platform will enable tunable and reversible engineering of cellular RNAs for diverse applications.  

5.2540           Development of a CRISPR/Cas9-based therapy for Hutchinson–Gilford progeria syndrome

Santiago-Fernandez, O., Osorio, F.G., Quesada, V., Rodriguez, F., Basso, S., Maeso, D., Rolas, L., Barkaway, A., Nourshargh, S., Folgueras, A.R., Freije, J.M. and Lopez-Otin, C. Nature Medicine, 25, 423-426 (2019)   CRISPR/Cas9-based therapies hold considerable promise for the treatment of genetic diseases. Among these, Hutchinson–Gilford progeria syndrome, caused by a point mutation in the LMNA gene, stands out as a potential candidate. Here, we explore the efficacy of a CRISPR/Cas9-based approach that reverts several alterations in Hutchinson–Gilford progeria syndrome cells and mice by introducing frameshift mutations in the LMNA gene.  

5.2541           Excitatory neuron–specific SHP2-ERK signaling network regulates synaptic plasticity and memory

Ryu., H-H-. Kim, T., Kim, J-W., kang, M., Park, P., Kim, Y.G.et al Sci. Signal., 12(571), eaau5755 (2019)   Mutations in RAS signaling pathway components cause diverse neurodevelopmental disorders, collectively called RASopathies. Previous studies have suggested that dysregulation in RAS–extracellular signal–regulated kinase (ERK) activation is restricted to distinct cell types in different RASopathies. Some cases of Noonan syndrome (NS) are associated with gain-of-function mutations in the phosphatase SHP2 (encoded by PTPN11); however, SHP2 is abundant in multiple cell types, so it is unclear which cell type(s) contribute to NS phenotypes. Here, we found that expressing the NS-associated mutant SHP2^D61G in excitatory, but not inhibitory, hippocampal neurons increased ERK signaling and impaired both long-term potentiation (LTP) and spatial memory in mice, although endogenous SHP2 was expressed in both neuronal types. Transcriptomic analyses revealed that the genes encoding SHP2-interacting proteins that are critical for ERK activation, such as GAB1 and GRB2, were enriched in excitatory neurons. Accordingly, expressing a dominant-negative mutant of GAB1, which reduced its interaction with SHP2^D61G, selectively in excitatory neurons, reversed SHP2^D61G-mediated deficits. Moreover, ectopic expression of GAB1 and GRB2 together with SHP2^D61G in inhibitory neurons resulted in ERK activation. These results demonstrate that RAS-ERK signaling networks are notably different between excitatory and inhibitory neurons, accounting for the cell type–specific pathophysiology of NS and perhaps other RASopathies.  

5.2542           Hypothalamic gene transfer of BDNF promotes healthy aging in mice

McMurphy, T., Huang, W., Liu, X., Siu, J.J., Queen, N.J., Xiao, R. and Cao, L. Aging Cell, 18, e12846 (2019)   The aging process and age‐related diseases all involve perturbed energy adaption and impaired ability to cope with adversity. Brain‐derived neurotrophic factor (BDNF) in the hypothalamus plays important role in regulation of energy balance. Our previous studies show that recombinant adeno‐associated virus (AAV)‐mediated hypothalamic BDNF gene transfer alleviates obesity, diabetes, and metabolic syndromes in both diet‐induced and genetic models. Here we examined the efficacy and safety of a built‐in autoregulatory system to control transgene BDNF expression mimicking the body's natural feedback systems in middle‐aged mice. Twelve‐month‐old mice were treated with either autoregulatory BDNF vector or yellow fluorescence protein (YFP) control, maintained on normal diet, and monitored for 28 weeks. BDNF gene transfer prevented the development of aging‐associated metabolic declines characterized by: preventing aging‐associated weight gain, reducing adiposity, reversing the decline of brown fat activity, increasing adiponectin while reducing leptin and insulin in circulation, improving glucose tolerance, increasing energy expenditure, alleviating hepatic steatosis, and suppressing inflammatory genes in the hypothalamus and adipose tissues. Moreover, BDNF treatment reduced anxiety‐like and depression‐like behaviors. These safety and efficacy data provide evidence that hypothalamic BDNF is a target for promoting healthy aging.  

5.2543           Cideb controls sterol‐regulated ER export of SREBP/SCAP by promoting cargo loading at ER exit sites

Su, L., Zhou, L., Chen, F-J., Wang, H., Qian, H., Sheng, Y. et al EMBO J., 38(7), e100156 (2019)   SREBPs are master regulators of lipid homeostasis and undergo sterol‐regulated export from ER to Golgi apparatus for processing and activation via COPII‐coated vesicles. While COPII recognizes SREBP through its escort protein SCAP, factor(s) specifically promoting SREBP/SCAP loading to the COPII machinery remains unknown. Here, we show that the ER/lipid droplet‐associated protein Cideb selectively promotes the loading of SREBP/SCAP into COPII vesicles. Sterol deprivation releases SCAP from Insig and enhances ER export of SREBP/SCAP by inducing SCAP‐Cideb interaction, thereby modulating sterol sensitivity. Moreover, Cideb binds to the guanine nucleotide exchange factor Sec12 to enrich SCAP/SREBP at ER exit sites, where assembling of COPII complex initiates. Loss of Cideb inhibits the cargo loading of SREBP/SCAP, reduces SREBP activation, and alleviates diet‐induced hepatic steatosis. Our data point to a linchpin role of Cideb in regulated ER export of SREBP and lipid homeostasis.  

5.2544           Prime-Boost Immunizations with DNA, Modified Vaccinia Virus Ankara, and Protein-Based Vaccines Elicit Robust HIV-1 Tier 2 Neutralizing Antibodies against the CAP256 Superinfecting Virus

Van Diepen, M.T., Chapman, R., Douglass, N., Galant, S., Moore, P.L., margolin, E., Ximba, P., Morris, L., Rybicki, E.P. and Williamson, A-L.

1. Virol., 93(8), e02155-18 (2019)

  A vaccine regimen that elicits broadly neutralizing antibodies (bNAbs) is a major goal in HIV-1 vaccine research. In this study, we assessed the immunogenicity of the CAP256 superinfecting viral envelope (CAP256 SU) protein delivered by modified vaccinia virus Ankara (MVA) and DNA vaccines in different prime-boost combinations followed by a soluble protein (P) boost. The envelope protein (Env) contained a flexible glycine linker and I559P mutation. Trimer-specific bNAbs PGT145, PG16, and CAP256 VRC26_08 efficiently bound to the membrane-bound CAP256 envelope expressed on the surface of cells transfected or infected with the DNA and MVA vaccines. The vaccines were tested in two different vaccination regimens in rabbits. Both regimens elicited autologous tier 2 neutralizing antibodies (NAbs) and high-titer binding antibodies to the matching CAP256 Env and CAP256 V1V2 loop scaffold. The immunogenicity of DNA and MVA vaccines expressing membrane-bound Env alone was compared to that of Env stabilized in a more native-like conformation on the surface of Gag virus-like particles (VLPs). The inclusion of Gag in the DNA and MVA vaccines resulted in earlier development of tier 2 NAbs for both vaccination regimens. In addition, a higher proportion of the rabbits primed with DNA and MVA vaccines that included Gag developed tier 2 NAbs than did those primed with vaccine expressing Env alone. Previously, these DNA and MVA vaccines expressing subtype C mosaic HIV-1 Gag were shown to elicit strong T cell responses in mice. Here we show that when the CAP256 SU envelope protein is included, these vaccines elicit autologous tier 2 NAbs.  

5.2545           Characterization and epidemiological survey of porcine sapelovirus in China

Li, Y., Du, L., Jin, T., Cheng, Y., Zhang, X., Jiao, S., Huang, T., Zhang, Y., Yan, Y., Gu, J. and Zhou, J. Vet. Microbiol., 232, 13-21 (2019)   Porcine sapelovirus (PSV) is a causative agent of acute diarrhoea, respiratory distress, reproductive failure, and polioencephalomyelitis in swine. Here, we report the isolation, genomic sequence, and biological characterization of PSV isolated from pig diarrhoeal samples. In our study, two PSV strains were identified with a diameter of approximately 25 nm, and their full genomes were 7564 nucleotides in length. We named the strains PSV-JXXY-a2 and PSV-JXXY-c. Phylogenetic analysis showed that the two virus isolates were classified into the China cluster. Moreover, the PSV-JXXY-a2 strain could be inactivated quickly at 54℃ and adapted to grow on different cell lines of porcine, human, and baby hamster origin. Pathogenicity investigation showed that the isolated PSV could infect neonatal piglets efficiently and caused diarrhoea in piglets. Further epidemiological investigation revealed a high prevalence of PSV in pig herds, and the PSV-positive rates in pigs with diarrhoea were much higher than in asymptomatic samples in China. Together, our findings demonstrate that PSV-JXXY-a2 is pathogenic to neonatal piglets and advance knowledge on the prevalence of PSV infection.  

5.2546           Induction of neutralizing antibodies by human papillomavirus vaccine generated in mammalian cells

Wu, X., Ma, X., Li, Y., Xu, Y., Zhang, n., Xu, S., Nawaz, W. and Wu, Z. Antibody Theraapeutics, 2(2), 45-53 (2019)   Background Cervical cancer caused by human papillomavirus (HPV) infections is one of the most common cancers affecting women worldwide. Current preventative HPV vaccines on the market are composed of HPV L1 protein produced either in the yeast such as Gardasil or in the insect cells such as Cervarix. The duration of efficacy and cross-protection remain highly desirable for the improvement of current prophylactic HPV vaccine. Given that HPV carries out infection and replicates in mammalian cells, L2 protein, which is not included in the current licensed vaccines, is included in the third generation of HPV vaccine in pursuing of providing broader prevention. We hypothesize that a virus-like particle (VLP) consisting of HPV L1 plus L2 proteins generated in mammalian cells will present conformations more closely to native HPV, thus it will provide more durable and broader efficacy of prevention. Methods We took advantage of 293TT cells to produce VLP containing L1 and L2 proteins of HPV16 and HPV18, respectively. Results VLP particles of uniformed size and morphology were observed, and potent and broadly neutralizing antibodies were induced in mice and rabbits. In addition, compared to bivalent HPV vaccine of Cervarix, our HPV L1-L2 VLPs elicited higher titer of anti-sera, and the anti-sera also presented comparable neutralization potency against HPV16 and HPV18 infections even a much less potent adjuvant was used in our case. Conclusion Our VLPs were capable of eliciting stronger and more broadly neutralizing activities against various HPV subtypes and were potential candidate HPV vaccines.  

5.2547           Gene correction for SCID-X1 in long-term hematopoietic stem cells

Pavel-Dinu, M., Wiebking, V., Dejene, B.T., Srifa, W. Mmantri, S. et al Nature Communications, 10:1634 (2019)   Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34⁺ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.  

5.2548           An Asymmetric Opening of HIV-1 Envelope Mediates Antibody-Dependent Cellular Cytotoxicity

Alsahafi, N., Bakouche, N., Kazemi, M., Rouiller, I., Finzi, A. and Munro, J.B. Cell Host & Microbe, 25(4), 578-587 (2019)   The HIV-1 envelope glycoprotein (Env) (gp120-gp41) ₃ is the target for neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC). HIV-1 Env is flexible, sampling different conformational states. Before engaging CD4, Env adopts a closed conformation (State 1) that is largely antibody resistant. CD4 binding induces an intermediate state (State 2), followed by an open conformation (State 3) that is susceptible to engagement by antibodies that recognize otherwise occluded epitopes. We investigate conformational changes in Env that induce ADCC in the presence of a small-molecule CD4-mimetic compound (CD4mc). We uncover an asymmetric Env conformation (State 2A) recognized by antibodies targeting the conserved gp120 inner domain and mediating ADCC. Sera from HIV+ individuals contain these antibodies, which can stabilize Env State 2A in combination with CD4mc. Additionally, triggering State 2A on HIV-infected primary CD4 ⁺ T cells exposes epitopes that induce ADCC. Strategies that induce this Env conformation may represent approaches to fight HIV-1 infection.  

5.2549           Rapid and Simple Screening of CRISPR Guide RNAs (gRNAs) in Cultured Cells Using Adeno-Associated Viral (AAV) Vectors

Fakhiri, J., Nickl, M. and Grimm, D. Methods in Mol. Biol., 1961, 111-126 (2019)   Genome editing reagents including the recently introduced CRISPR/Cas9 system have become established and widely used molecular tools to answer fundamental biological questions and to target and treat genetic diseases. The CRISPR system, originally derived from bacteria and archaea, can be delivered into cells using different techniques, comprising (1) transfection of mRNA or plasmid DNA, (2) electroporation of plasmid DNA or the Cas9 protein in a complex with a g(uide)RNA, or (3) use of nonviral or viral vectors. Among the latter, Adeno-associated viruses (AAVs) are particularly attractive owing to many favorable traits: (1) their apathogenicity and episomal persistence, (2) the ease of virus production and purification, (3) the safe handling under lowest biosafety level 1 conditions, and (4) the availability of numerous natural serotypes and synthetic capsid variants with distinct cell specificities. Here, we describe a fast and simple protocol for small-scale packaging of CRISPR/Cas9 components into AAV vectors. To showcase its potential, we employ this method for screening of gRNAs targeting the murine miR-122 locus in Hepa1–6 cells (using AAV serotype 6, AAV6) or the 5′LTR of the human immunodeficiency virus (HIV) in HeLaP4-NLtr cells (using a synthetic AAV9 variant). We furthermore provide a detailed protocol for large-scale production of purified AAV/CRISPR vector stocks that permit higher cleavage efficiencies in vitro and are suitable for direct in vivo applications.  

5.2550           IncRNA UCA1-Mediated Cdc42 Signaling Promotes Oncolytic Vaccinia Virus Cell-to-Cell Spread in Ovarian Cancer

Horita, K., Kurosaki, H., Nakatake, M., Kuwano, N., oishi, T., Itamochi, H., Sato, S., Kono, H., ito, M., Hasegawa, K., harada, T. and Nakamura, T. Molecular Therapy – Oncolytics, 13, 35-48 (2019)   Oncolytic vaccinia virus (OVV) has demonstrated appropriate safety profiles for clinical development. Although designed to kill cancer cells efficiently, OVV sensitivity varies in individual cancers, and predictive biomarkers of therapeutic responses have not been identified. Here we found that OVV was much more efficient in KFTX paclitaxel-resistant ovarian cancer cells compared to that in KFlow paclitaxel-sensitive cells. Microarray analysis identified long non-coding RNA urothelial carcinoma-associated 1 (UCA1) upregulation, which contributed to both enhanced paclitaxel resistance and OVV spread. In addition, UCA1 expression correlated with efficient OVV spread in other ovarian cell lines and primary cancer cell cultures. When host pathways underlying OVV spread were analyzed, differences were detected in the activation of the Rho GTPase Cdc42, suggesting that filopodia formation enhances OVV cell-to-cell spread and tumor migration. Moreover, we established a clinically relevant mouse model of peritoneal metastasis using KFTX or KFlow cells. Paclitaxel exerted anti-tumor effects on KFlow, but not KFTX, tumors. In mice bearing KFTX cells after paclitaxel failure, OVV treatment induced the regression of residual tumors and improved survival. Our findings demonstrated that UCA1 promotes OVV cell-to-cell spread in ovarian cancer, resulting in enhanced therapeutic outcome.  

5.2551           Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies

Rajendran, P., Challis, R.C., Fowlkes, C.C., Hanna, P., Tompkins, J.D. et al Nature Communications, 10:1944 (2019)   Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.  

5.2552           Near physiological spectral selectivity of cochlear optogenetics

Dieter, A., Duque-Afonso, C.J., Rankovic, V., Jeschke, M. and Moser, T. Nature Communications, 10:1962 (2019)   Cochlear implants (CIs) electrically stimulate spiral ganglion neurons (SGNs) and partially restore hearing to half a million CI users. However, wide current spread from intracochlear electrodes limits spatial selectivity (i.e. spectral resolution) of electrical CIs. Optogenetic stimulation might become an alternative, since light can be confined in space, promising artificial sound encoding with increased spectral selectivity. Here we compare spectral selectivity of optogenetic, electric, and acoustic stimulation by multi-channel recordings in the inferior colliculus (IC) of gerbils. When projecting light onto tonotopically distinct SGNs, we observe corresponding tonotopically ordered IC activity. An activity-based comparison reveals that spectral selectivity of optogenetic stimulation is indistinguishable from acoustic stimulation for modest intensities. Moreover, optogenetic stimulation outperforms bipolar electric stimulation at medium and high intensities and monopolar electric stimulation at all intensities. In conclusion, we demonstrate better spectral selectivity of optogenetic over electric SGN stimulation, suggesting the potential for improved hearing restoration by optical CIs.  

5.2553           Bile-duct proliferation as an unexpected side-effect after AAV2-LDLR gene transfer to rabbit liver

Hytönen, E., Laurema, A., Kankkonen, H., Miyanohara, A., Kärjä, V., Hujo, M., laham-Karam, N., and Ylä-Herttuala, S. Scientific Reports, 9:6934 (2019)   Familial hypercholesterolemia (FH) is an inherited disease of lipoprotein metabolism caused by a defect in the LDL receptor (LDLR) leading to severe hypercholesterolemia, and associated with an increased risk of coronary heart disease and myocardial infarction. We have developed a gene therapy protocol for FH using AAV2, AAV9 and lentiviral vectors and tested safety and efficacy in LDL receptor deficient Watanabe Heritable Hyperlipidemic rabbits. We show that LV-LDLR produced a significant long-lasting decrease in total serum cholesterol whereas AAV9-LDLR resulted only in a transient decrease and AAV2-LDLR failed to reduce serum cholesterol levels. A significant pathological side effect, bile-duct proliferation, was seen in the liver of AAV2-LDLR rabbits associated with an increased expression of Cyr61 matricellular protein. Special attention should be given to liver changes in gene therapy applications when genes affecting cholesterol and lipoprotein metabolism are used for therapy.  

5.2554           Linking YAP to Müller Glia Quiescence Exit in the Degenerative Retina

Hamon, A., Garcia-Garcia, D., Ail, D., Locker, M., Roger, J.E. and Perron, M. Cell Reports, 27, 1712-1725 (2019)   Contrasting with fish or amphibian, retinal regeneration from Müller glia is largely limited in mammals. In our quest toward the identification of molecular cues that may boost their stemness potential, we investigated the involvement of the Hippo pathway effector YAP (Yes-associated protein), which is upregulated in Müller cells following retinal injury. Conditional Yap deletion in mouse Müller cells prevents cell-cycle gene upregulation that normally accompanies reactive gliosis upon photoreceptor cell death. We further show that, in Xenopus, a species endowed with efficient regenerative capacity, YAP is required for their injury-dependent proliferative response. In the mouse retina, where Müller cells do not spontaneously proliferate, YAP overactivation is sufficient to induce their reprogramming into highly proliferative cells. Overall, we unravel a pivotal role for YAP in tuning Müller cell proliferative response to injury and highlight a YAP-EGFR (epidermal growth factor receptor) axis by which Müller cells exit their quiescence state, a critical step toward regeneration.  

5.2555           Enveloped viruses distinct from HBV induce dissemination of hepatitis D virus in vivo

Perez-vargas, J., Amirache, F., Boson, B., Mialon, C., Freitas, N., Sureau, C., Fusil, F. and Cosset, F-L. Nature Communications, 10:2098 (2019)   Hepatitis D virus (HDV) doesn’t encode envelope proteins for packaging of its ribonucleoprotein (RNP) and typically relies on the surface glycoproteins (GPs) from hepatitis B virus (HBV) for virion assembly, envelopment and cellular transmission. HDV RNA genome can efficiently replicate in different tissues and species, raising the possibility that it evolved, and/or is still able to transmit, independently of HBV. Here we show that alternative, HBV-unrelated viruses can act as helper viruses for HDV. In vitro, envelope GPs from several virus genera, including vesiculovirus, flavivirus and hepacivirus, can package HDV RNPs, allowing efficient egress of HDV particles in the extracellular milieu of co-infected cells and subsequent entry into cells expressing the relevant receptors. Furthermore, HCV can propagate HDV infection in the liver of co-infected humanized mice for several months. Further work is necessary to evaluate whether HDV is currently transmitted by HBV-unrelated viruses in humans.  

5.2556           Tbx6 induces cardiomyocyte proliferation in postnatal and adult mouse hearts

Haginiwa, S., Sadahiro, T., Kojima, H., Isomi, M., Tamura, F., Kurosu, S., Tani, H., Muraoka, N., Miyake, N., Miyake, K., Fukuda, K. and Ieda, M. Biochem. Biophys. Res. Comm., 513, 1041-1047 (2019)   Cardiovascular disease is a leading cause of death worldwide. Mammalian cardiomyocytes (CMs) proliferate during embryonic development, whereas they largely lose their regenerative capacity after birth. Defined factors expressed in cardiac progenitors or embryonic CMs may activate the cell cycle and induce CM proliferation in postnatal and adult hearts. Here, we report that the overexpression of Tbx6, enriched in the cardiac mesoderm (progenitor cells), induces CM proliferation in postnatal and adult mouse hearts. By screening 24 factors enriched in cardiac progenitors or embryonic CMs, we found that only Tbx6 could induce CM proliferation in primary cultured postnatal rat CMs. Intriguingly, it did not induce the proliferation of cardiac fibroblasts. We next generated a recombinant adeno-associated virus serotype 9 vector encoding Tbx6 (AAV9-Tbx6) for transduction into mouse CMs in vivo. The subcutaneous injection of AAV9-Tbx6 into neonatal mice induced CM proliferation in postnatal and adult mouse hearts. Mechanistically, Tbx6 overexpression upregulated multiple cell cycle activators including Aurkb, Mki67, Ccna1, and Ccnb2 and suppressed the tumor suppressor Rb1. Thus, Tbx6 promotes CM proliferation in postnatal and adult mouse hearts by modifying the expression of cell cycle regulators.  

5.2557           Epilepsy Gene Therapy Using an Engineered Potassium Channel

Snowball, A., Chabrol., E., Wykes, R.C., Shekh-Ahmad, T., Cornford, J.H., Lieb, A., Hughes, M.P., Massaro, G., Rahim, A.A., Hashemi, K.S., Kullmann, D.M., Walker, M.C. and Schorge, S.

1. Neurosci., 39(16), 3159-3169 (2019)

  Refractory focal epilepsy is a devastating disease for which there is frequently no effective treatment. Gene therapy represents a promising alternative, but treating epilepsy in this way involves irreversible changes to brain tissue, so vector design must be carefully optimized to guarantee safety without compromising efficacy. We set out to develop an epilepsy gene therapy vector optimized for clinical translation. The gene encoding the voltage-gated potassium channel Kv1.1, KCNA1, was codon optimized for human expression and mutated to accelerate the recovery of the channels from inactivation. For improved safety, this engineered potassium channel (EKC) gene was packaged into a nonintegrating lentiviral vector under the control of a cell type-specific CAMK2A promoter. In a blinded, randomized, placebo-controlled preclinical trial, the EKC lentivector robustly reduced seizure frequency in a male rat model of focal neocortical epilepsy characterized by discrete spontaneous seizures. When packaged into an adeno-associated viral vector (AAV2/9), the EKC gene was also effective at suppressing seizures in a male rat model of temporal lobe epilepsy. This demonstration of efficacy in a clinically relevant setting, combined with the improved safety conferred by cell type-specific expression and integration-deficient delivery, identify EKC gene therapy as being ready for clinical translation in the treatment of refractory focal epilepsy.  

5.2558           The GPI-Linked Protein LY6A Drives AAV-PHP.B Transport across the Blood-Brain Barrier

Hordeaux, J., Yuan, Y., Clark, P.M., Wang, Q., martino, R.A., Sims, J.J., Bell, P., Raymond, A., Stanford, W.L. and Wilson, J.M. Molecular Therapy, 27(5), 912-921 (2019)   Efficient delivery of gene therapy vectors across the blood-brain barrier (BBB) is the holy grail of neurological disease therapies. A variant of the neurotropic vector adeno-associated virus (AAV) serotype 9, called AAV-PHP.B, was shown to very efficiently deliver transgenes across the BBB in C57BL/6J mice. Based on our recent observation that this phenotype is mouse strain dependent, we used whole-exome sequencing-based genetics to map this phenotype to a specific haplotype of lymphocyte antigen 6 complex, locus A (Ly6a) (stem cell antigen-1 [Sca-1]), which encodes a glycosylphosphatidylinositol (GPI)-anchored protein whose function had been thought to be limited to the biology of hematopoiesis. Additional biochemical and genetic studies definitively linked high BBB transport to the binding of AAV-PHP.B with LY6A (SCA-1). These studies identify, for the first time, a ligand for this GPI-anchored protein and suggest a role for it in BBB transport that could be hijacked by viruses in natural infections or by gene therapy vectors to treat neurological diseases.  

5.2559           BIRC5 is a target for molecular imaging and detection of human pancreatic cancer

Liu, S-H., Hong, Y., Markowiak, S., Sanchez, R., Creeden, J., Nemunaitis, J., Kalinoski, A., Willey, J., Erhardt, P., Lee, J., van Dam, M. and Brunicardi, F.C. Cancer Lett., 457, 10-19 (2019)   Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer mortality with a dismal overall survival rate and an urgent need for detection of minute tumors. Current diagnostic modalities have high sensitivity and specificity for larger tumors, but not for minute PDAC. In this study, we test the feasibility of a precision diagnostic platform for detecting and localizing minute human PDAC in mice. This platform includes: 1) defining BIRC5 as an early PDAC-upregulated gene and utilizing an enhanced BIRC5 super-promoter to drive expression of dual Gaussia luciferase (GLuc) and sr39 thymidine kinase (sr39TK) reporter genes exponentially and specifically in PDAC; 2) utilizing a genetically-engineered AAV₂^RGD to ensure targeted delivery of GLuc and sr39TK specifically to PDAC; 3) using serologic GLuc and sr39TK microPET/CT imaging to detect and localize minute human PDAC in mice. The study demonstrates feasibility of a precision diagnostic platform using an integrated technology through a multiple-stage amplification strategy of dual reporter genes to enhance the specificity and sensitivity of detection and localization of minute PDAC tumors and currently undetectable disease.  

5.2560           A simplified roller bottle platform for the production of a new generation VLPs rabies vaccine for veterinary applications

Fontana, D., Marsili, F., garay, E., Battagliotti, J., Etcheverrigaray, M., Kratje, R. and Prieto, C. Comp. Immunol. Microbiol. Infect. Dis., 65, 70-75 (2019)   Rabies is a neglected disease with an estimated annual mortality of 55,000 human deaths, affecting mainly low-income countries. Over 95% of these cases result from virus transmission through the bite of infected dogs and for this reason there is a real need for a cheap and effective rabies veterinary vaccine to be used in mass vaccination campaigns. In this work, we describe the establishment of a simple platform for the production of a virus-like particles based rabies vaccine using mammalian cells and roller bottles as culture system. Adherent cells were cultured during more than 15 days and VLPs were continuously produced and secreted to the culture supernatant. Immunogenicity and protective efficacy of VLPs were tested through rabies virus neutralizing antibody test and NIH potency test. These viral particles induced high titer of long lasting neutralizing antibodies and protected mice against active virus challenge. Therefore, this development represents a promising platform for the production of a new generation and virus-free rabies vaccine candidate for veterinary applications.      

5.2561           TIM1 (HAVCR1): an Essential “Receptor” or an “Accessory Attachment Factor” for Hepatitis A Virus?

Das, A., Maury, W. and Lemon, S.M.

1. Virol., 93(11), e01793-18 (2019)

  In their recent report, Costafreda and Kaplan (1) claim that CRISPR/Cas9 knockout of TIM1 (also called HAVCR1) renders the GL37 clone of African green monkey kidney (AGMK) cells resistant to infection with hepatitis A virus (HAV). They concluded that TIM1 “is required for infection” and is a “functional receptor” for HAV, as Kaplan and colleagues suggested many years ago (2). This surprised us, as we had previously found that both naked and quasi-enveloped HAV (eHAV) virions (3) readily infect Vero (and Huh-7.5) cells in which TIM1 expression was knocked out by CRISPR/Cas9 gene editing (4). We observed that the binding of quasi-enveloped virions to Vero cells was reduced (but not eliminated) at 4°C by TIM1 knockout, suggesting that TIM1 acts as an accessory attachment factor by binding phosphatidylserine (Ptd-Ser) on the eHAV quasi-envelope surface (5, 6). However, eHAV infection proceeded efficiently at 37°C, and we noted no differences in either binding or infection with naked HAV (4). We suggested to Dr. Kaplan that we exchange our knockout cell lines to resolve these conflicting observations but were told that his cells were no longer available due to a freezer malfunction. We thus generated a new TIM1 knockout cell line (GL37-KO) using GL37 cells obtained from Dr. Kaplan in 2012 and one of the single-guide RNAs (sgRNAs) described by Costafreda and Kaplan that target exon 2 (ACACTGCCCTGCCGCTACAA) (1) (Fig. 1A) cloned into the lenticrisprV2 plasmid (Addgene catalog number 52961) (4). Lentivirus-transduced GL37 cells were expanded in media containing puromycin for 3 weeks, as described previously (4). DNA sequencing of surviving cells showed 100% identity to the Chlorocebus aethiops genome (GenBank accession number X98252.1) and confirmed disruption of the TIM1 sequence by small indels at codon 38, leading to a frameshift within exon 2 (Fig. 1A). Cell surface staining with antibodies to human TIM1 confirmed the loss of TIM1 expression in the GL37-KO cells compared to its expression in control cells (GL37-Ctrl) transduced with a nontargeting sgRNA (AACCTACGGGCTACGATACG) (Fig. 1B).  

5.2562           Reply to Das et al., “TIM1 (HAVCR1): an Essential ‘Receptor’ or an ‘Accessory Attachment Factor’ for Hepatitis A Virus?”

Costafreda, M. and Kaplan, G.

1. Virol., 93(11), e02040-18 (2019)

  We have recently shown that HAVCR1 is a functional hepatitis A virus (HAV) cellular receptor in clone GL37 of African green monkey kidney (GL37) cells that mediates cell entry of viral particles (vpHAV) and exosomes produced in HAV-infected cells (exo-HAV or eHAV) (1). We also showed that HAVCR1 is the predominant HAV receptor in GL37 cells, whereas alternative, yet-unidentified HAV receptors are coexpressed with HAVCR1 in other primate cells, such as Vero E6 and Huh7 cells (1). The knockout (KO) of HAVCR1 using CRISPR/Cas9 technology significantly reduced the susceptibility of GL37 cells to HAV infection, which was regained upon transfection of HAVCR1 cDNA (1), supporting the notion that resistance to HAV infection in these GL37 HAVCR1 KO cells is due to the lack of HAV receptors rather than other factors. Taken together with our previous work showing the direct interaction of HAV with HAVCR1 in cellular and cell-free systems (2–10), our data clearly indicated that HAVCR1 is indeed a functional HAV receptor of vpHAV and exo-HAV. For the letter to the editor of Das et al. (11), the Lemon lab and Maury lab (Lemon/Maury labs) tried to reproduce our results using GL37 cells and one of the sgRNAs that we described in our paper (1) but used a flawed experimental design that led them to conclude incorrectly that HAVCR1 was not a functional vpHAV receptor. The Lemon/Maury lab data clearly show that the susceptibility of vpHAV is reduced significantly in their GL37 HAVCR1 KO cells compared to that in wild-type GL37 (GL37 wt) cells (see Fig. 1D and E in reference 11). However, the HAV background level in their GL37 HAVCR1 KO cells was ∼200 times higher than in ours (1), which is likely due to a combination of factors, including the CRISPR/Cas9 strategy used to generate their HAVCR1 KO cells (Fig. 1A), the short vpHAV adsorption times, and the high multiplicities of infection used in their experiments. The Lemon/Maury labs produced their GL37 HAVCR1 KO cells using a single sgRNA cloned into a lentivirus vector to mediate a single cut in exon 2 of the HAVCR1 gene, selected the pool of puromycin-resistant transduced cells, verified that heterogeneous small insertions and deletions (indels) were introduced in exon 2, and used the uncloned heterogeneous cell population to analyze the function of HAVCR1 as an HAV receptor. This simple and fast procedure resulted in the selection of a pool of heterogeneous puromycin-resistant cells consisting mainly of HAVCR1 KO cells but also most likely containing cells with various degrees of susceptibility to vpHAV infection due to (i) small in-frame indels that did not prevent the HAV receptor function of HAVCR1, (ii) off-target events that conferred puromycin resistance but did not knock out HAVCR1, and (iii) expression of different levels of alternative HAV receptors that are not predominant in AGMK GL37 cells compared to in Vero and Huh7 cells (1), which would significantly increase the background level of HAV infection in their heterogenous pool of puromycin-resistant cells. To circumvent the problems of using a heterogeneous pool of cells to evaluate HAVCR1 as a functional HAV receptor, we (i) utilized double sgRNAs to produce large and well-defined out-of-frame deletions of 100 and 101 nucleotides in the HAVCR1 gene and (ii) single-cell cloned transfectants to generate a homogeneous HAVCR1 KO cell clone highly resistant to vpHAV and exo-HAV infection (1). The Lemon/Maury labs also argued that vpHAV did not bind to HAVCR1 at the cell surface by comparing levels of binding to GL37 wt and GL37 HAVCR1 KO cells but used a suboptimal adsorption time of 2 h, which limited binding of vpHAV to HAVCR1 (Fig. 1B and C), in contrast to the 12 h used in our experiments, which significantly increases binding of vpHAV to GL37 wt but not GL37 HAVCR1 KO cells. To rebut their additional suggestions, we show in Fig. 1D and E that the low background levels of HAV produced in HAVCR1 KO cells did not spread to the remaining HAVCR1 KO cells in the monolayer, as expected from the knockout of a functional viral receptor but not an accessory attachment factor. In our recent paper (1), we showed that treatment of purified naked vpHAV with 1% Sarkosyl, an ionic detergent, did not affect their infectivity in our GL37 HAVCR1 KO cells transfected with HAVCR1 cDNA, indicating that lipids are not required for the infection of naked vpHAV mediated by HAVCR1. Here, we extracted lipids from exo-HAV using 1% Sarkosyl and purified the naked HAV particles in iodixanol gradients (Fig. 1F). The infectivity of these naked HAV particles in GL37 HAVCR1 KO cells transfected with HAVCR1 cDNA was similar to that of the untreated exo-HAV cells (Fig. 1G), further showing that lipids are not required for the HAVCR1-mediated infection of naked HAV particles. In summary, we showed that HAVCR1 plays an essential role as an HAV functional receptor of vpHAV and exo-HAV in GL37 cells by using a double sgRNA CRISPR/Cas9 knockout strategy and single-cell cloning of the KO cells, which resulted in the isolation of a GL37 HAVCR1 KO clone resistant to HAV infection with a low background of susceptibility to HAV infection. In contrast, the Lemon/Maury labs used a single sgRNA strategy to select a heterogeneous pool of cells with a high background of susceptibility to HAV infection as well as comparatively short HAV incubation times that obscured their data analysis.  

5.2563           Modulation of Sialic Acid Dependence Influences the Central Nervous System Transduction Profile of Adeno-associated Viruses

Albright, B.H., Simon, K.E., Pillai, M., Devlin, G.W. and Asokan, A.

1. Virol., 93(11), e00332-19 (2019)

  Central nervous system (CNS) transduction by systemically administered recombinant adeno-associated viral (AAV) vectors requires crossing the blood-brain barrier (BBB). We recently mapped a structural footprint on the AAVrh.10 capsid, which, when grafted onto the AAV1 capsid (AAV1RX), enables viral transport across the BBB; however, the underlying mechanisms remain unknown. Here, we establish through structural modeling that this footprint overlaps in part the sialic acid (SIA) footprint on AAV1. We hypothesized that altered SIA-capsid interactions may influence the ability of AAV1RX to transduce the CNS. Using AAV1 variants with altered SIA footprints, we map functional attributes of these capsids to their relative SIA dependence. Specifically, capsids with ablated SIA binding can penetrate and transduce the CNS with low to moderate efficiency. In contrast, AAV1 shows strong SIA dependency and does not transduce the CNS after systemic administration and, instead, transduces the vasculature and the liver. The AAV1RX variant, which shows an intermediate SIA binding phenotype, effectively enters the brain parenchyma and transduces neurons at levels comparable to the level of AAVrh.10. In corollary, the reciprocal swap of the AAV1RX footprint onto AAVrh.10 (AAVRX1) attenuated CNS transduction relative to that of AAVrh.10. We conclude that the composition of residues within the capsid variable region 1 (VR1) of AAV1 and AAVrh.10 profoundly influences tropism, with altered SIA interactions playing a partial role in this phenotype. Further, we postulate a Goldilocks model, wherein optimal glycan interactions can influence the CNS transduction profile of AAV capsids.  

5.2564           Memory Decline and Its Reversal in Aging and Neurodegeneration Involve miR-183/96/182 Biogenesis

Jawaid, A., Woldemichael, B.T., Kremer, E.A., Laferriere, F., Gaur, N., Afroz, T., Polymenidou, M. and Mansuy, I.M. Mol. Neurobiol., 56(5), 3451-3462 (2019)   Aging is characterized by progressive memory decline that can lead to dementia when associated with neurodegeneration. Here, we show in mice that aging-related memory decline involves defective biogenesis of microRNAs (miRNAs), in particular miR-183/96/182 cluster, resulting from increased protein phosphatase 1 (PP1) and altered receptor SMAD (R-SMAD) signaling. Correction of the defect by miR-183/96/182 overexpression in hippocampus or by environmental enrichment that normalizes PP1 activity restores memory in aged animals. Regulation of miR-183/96/182 biogenesis is shown to involve the neurodegeneration-related RNA-binding proteins TDP-43 and FUS. Similar alterations in miR-183/96/182, PP1, and R-SMADs are observed in the brains of patients with amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD), two neurodegenerative diseases with pathological aggregation of TDP-43. Overall, these results identify new mechanistic links between miR-183/96/182, PP1, TDP-43, and FUS in age-related memory deficits and their reversal.  

5.2565           Effects of Neutralizing Antibody Production on AAV-PHP.B-Mediated Transduction of the Mouse Central Nervous System

Shinohara, Y., Konno, A., Nita, K., Matsuzaki, Y., Yasui, H., Suwa, J., Hiromura, K. and Hirai, H. Mol. Neurobiol., 56(5), 4203-4214 (2019)   Adeno-associated virus (AAV)-PHP.B, a capsid variant of AAV serotype 9, is highly permeable to the blood-brain barrier. A major obstacle to the systemic use of AAV-PHP.B is the generation of neutralizing antibodies (NAbs); however, temporal profiles of NAb production after exposure to AAV-PHP.B, and the influence on later AAV-PHP.B administration, remains unknown. To address these, AAV-PHP.Bs expressing either GFP or mCherry by neuron-specific or astrocyte-specific promoters were intravenously administered to mice at various intervals, and brain expression was examined. Injection of two AAV-PHP.Bs, separated temporally, showed that as little as a 1-day interval between injections resulted in a significant decrease in expression of the second transgene, with a complete loss of expression after 7 days, paralleling an increase in serum NAb titers. Brain parenchymal injection was explored to circumvent the presence of NAbs. Mice systemically pre-treated with an AAV-PHP.B were injected intra-cerebrally with an AAV-PHP.B expressing GFP. After 2 weeks, marked GFP expression in the cerebellum was evident, showing that pre-existing NAbs did not affect the AAV-PHP.B directly injected into the brain. In contrast, reversing the injection order, i.e., cerebellar injection followed by systemic injection, completely eliminated expression of the second transgene. We confirmed that intra-cerebellar injection produced NAbs in the serum, but not in the cerebrospinal fluid (CSF). Our results indicate that the preclusion of brain transduction by a second AAV-PHP.B administration begins from the first day following systemic injection and is established within 1 week. Serum NAbs can be avoided by directly injecting AAV-PHP.Bs into brain tissue.  

5.2566           Clathrin plaques and associated actin anchor intermediate filaments in skeletal muscle

Franck, A., Laine, J., Moulay, G., Lemerle, E., Trichet, M., Gentil, C., Benkhelifa-Ziyyat, S., Lacene, E., Bul. M.T., Brochier, G., Guicheney, P., Romero, N., Bitoun, M and Vassilopoulos, S. Mol. Biol. Cell, 30(5), 579-590 (2019)   Clathrin plaques are stable features of the plasma membrane observed in several cell types. They are abundant in muscle, where they localize at costameres that link the contractile apparatus to the sarcolemma and connect the sarcolemma to the basal lamina. Here, we show that clathrin plaques and surrounding branched actin filaments form microdomains that anchor a three-dimensional desmin intermediate filament (IF) web. Depletion of clathrin plaque and branched actin components causes accumulation of desmin tangles in the cytoplasm. We show that dynamin 2, whose mutations cause centronuclear myopathy (CNM), regulates both clathrin plaques and surrounding branched actin filaments, while CNM-causing mutations lead to desmin disorganization in a CNM mouse model and patient biopsies. Our results suggest a novel paradigm in cell biology, wherein clathrin plaques act as platforms capable of recruiting branched cortical actin, which in turn anchors IFs, both essential for striated muscle formation and function.In muscle, clathrin plaques form microdomains that anchor a three-dimensional desmin intermediate filament web. Depletion of clathrin plaques, dynamin 2, and branched actin cause accumulation of desmin tangles in the cytoplasm, while centronuclear myopathy–causing dynamin 2 mutations lead to desmin disorganization in mice and humans.  

5.2567           Glycogen Synthase Kinase-3α Promotes Fatty Acid Uptake and Lipotoxic Cardiomyopathy

Nakamura, M., Liu, T., Husain, S., Tian, B., Li, H. and Sadoshima, J. Cell Metabolism, 29(5), 1119-1134 (2019)   Obesity induces lipotoxic cardiomyopathy, a condition in which lipid accumulation in cardiomyocytes causes cardiac dysfunction. Here, we show that glycogen synthase kinase-3α (GSK-3α) mediates lipid accumulation in the heart. Fatty acids (FAs) upregulate GSK-3α, which phosphorylates PPARα at Ser280 in the ligand-binding domain (LBD). This modification ligand independently enhances transcription of a subset of PPARα targets, selectively stimulating FA uptake and storage, but not oxidation, thereby promoting lipid accumulation. Constitutively active GSK-3α, but not GSK-3β, was sufficient to drive PPARα signaling, while cardiac-specific knockdown of GSK-3α, but not GSK-3β, or replacement of PPARα Ser280 with Ala conferred resistance to lipotoxicity in the heart. Fibrates, PPARα ligands, inhibited phosphorylation of PPARα at Ser280 by inhibiting the interaction of GSK-3α with the LBD of PPARα, thereby reversing lipotoxic cardiomyopathy. These results suggest that GSK-3α promotes lipid anabolism through PPARα-Ser280 phosphorylation, which underlies the development of lipotoxic cardiomyopathy in the context of obesity.  

5.2568           Calcium Channel Subunit α2δ4 Is Regulated by Early Growth Response 1 and Facilitates Epileptogenesis

Vam Loo, K.M:J., Rummel, C.K., Pitsch, J., Müller, J.A., Bikbaev, A.F., Martinez-Chavez, E., Blaess, S., Dietrich, D., Heine, M., Becker, A.J. and Schoch, S.

1. Neurosci., 39(17), 3187-3175 (2019)

  Transient brain insults, including status epilepticus (SE), can trigger a period of epileptogenesis during which functional and structural reorganization of neuronal networks occurs resulting in the onset of focal epileptic seizures. In recent years, mechanisms that regulate the dynamic transcription of individual genes during epileptogenesis and thereby contribute to the development of a hyperexcitable neuronal network have been elucidated. Our own results have shown early growth response 1 (Egr1) to transiently increase expression of the T-type voltage-dependent Ca²⁺ channel (VDCC) subunit Ca_V3.2, a key proepileptogenic protein. However, epileptogenesis involves complex and dynamic transcriptomic alterations; and so far, our understanding of the transcriptional control mechanism of gene regulatory networks that act in the same processes is limited. Here, we have analyzed whether Egr1 acts as a key transcriptional regulator for genes contributing to the development of hyperexcitability during epileptogenesis. We found Egr1 to drive the expression of the VDCC subunit α2δ4, which was augmented early and persistently after pilocarpine-induced SE. Furthermore, we show that increasing levels of α2δ4 in the CA1 region of the hippocampus elevate seizure susceptibility of mice by slightly decreasing local network activity. Interestingly, we also detected increased expression levels of Egr1 and α2δ4 in human hippocampal biopsies obtained from epilepsy surgery. In conclusion, Egr1 controls the abundance of the VDCC subunits Ca_V3.2 and α2δ4, which act synergistically in epileptogenesis, and thereby contributes to a seizure-induced “transcriptional Ca²⁺ channelopathy.”  

5.2569           A Quantitative Chloride Channel Conductance Assay for Efficacy Testing of AAV.BEST1

Wood, S.R., McClements, M.E., Martinez-Fernandez de la Camara, C., Patricio, M.I., Uggenti, C., Sekaran, S., Barnard, A.R., Manson, F.D. and MacLaren, R.E. Human Gene Therapy Methods, 30(2), 44-52 (2019   Mutations in the human BEST1 gene are responsible for a number of distinct retinal disorders known as bestrophinopathies, for which there are no current treatments. The protein product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE) where it localizes to the basolateral membrane and acts as a Ca²⁺-activated chloride channel. Recent studies have shown successful BEST1-mediated gene transfer to the RPE, indicating human clinical trials of BEST1 gene therapy may be on the horizon. A critical aspect of such trials is the ability to assess the efficacy of vector prior to patient administration. Here, an assay is presented that enables the quantitative assessment of AAV-mediated BEST1 chloride conductance as a measure of vector efficacy. Expression of BEST1 following transduction of HEK293 cells with AAV.BEST1 vectors was confirmed by liquid chromatography, Western blot, and immunocytochemistry. Whole-cell patch-clamp showed increased chloride conductance in BEST1-transduced cells compared to sham-transduced and untransduced controls. Exogenous chloride current correlated to BEST1 expression level, with an enhanced AAV.BEST1.WPRE vector providing higher expression levels of BEST1 and increases in chloride conductance. This study presents in vitro electrophysical quantification of bestrophin-1 following AAV-mediated gene transfer, providing vital functional data on an AAV gene therapy product that will support a future application for regulatory approval.  

5.2570           Distinct Roles of Direct Transduction Versus Exposure to the Tumor Secretome on Murine Endothelial Cells After Melanoma Gene Therapy with Interferon-β and p19Arf

De Luna Vieira, I., Tamura, R.E., Hunger, a. and Strauss, B.E.

1. Interferon & Cytokine Res., 39(4), 246-258 (2019)

  Tumor vasculature plays a central role in tumor progression, making it an attractive therapeutic target. In this study, we explore the antiangiogenic potential of our melanoma gene therapy approach combining interferon β (IFNβ) and p19Arf gene transfer. Since these proteins are modulators of tumor vasculature, we explore the impact of IFNβ and p19Arf gene transfer on murine endothelial cells (tEnd). Adenovirus-mediated gene transfer of p19Arf to tEnd cells inhibited proliferation, tube formation, migration, and led to increased expression of genes related to the p53 cell death pathway, yet IFNβ gene transfer had no significant impact on tEnd viability. Alternatively, tEnd cells were exposed to the factors generated by transduced B16 (mouse melanoma) cells using either coculture or conditioned medium. In either case, transduction of B16 cells with the IFNβ vector, whether alone or in combination with p19Arf, resulted in endothelial cell death. Strikingly, treatment of tEnd cells with recombinant IFNβ did not induce death, demonstrating that additional factors produced by B16 cells contributed to the demise of tEnd cells. In this work, we have shown that our melanoma gene therapy strategy produces desirable negative effects on endothelial cells, possibly correlating with antiangiogenic activity.  

5.2571           Vesicular Stomatitis Virus-Based Vaccine Protects Mice against Crimean-Congo Hemorrhagic Fever

Rodriguez, S.E., Cross, R.W., Fenton, K.A., Bente, D.A., Mire, C.E. and Geisbert, T.W. Scientific Reports, 9:7755 (2019)   Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne bunyavirus, can cause a life-threatening hemorrhagic syndrome in humans but not in its animal host. The virus is widely distributed throughout southeastern Europe, the Middle East, Africa, and Asia. Disease management has proven difficult and there are no broadly licensed vaccines or therapeutics. Recombinant vesicular stomatitis viruses (rVSV) expressing foreign glycoproteins (GP) have shown promise as experimental vaccines for several viral hemorrhagic fevers. Here, we developed and assessed a replication competent rVSV vector expressing the CCHFV glycoprotein precursor (GPC), which encodes CCHFV structural glycoproteins. This construct drives strong expression of CCHFV-GP, in vitro. Using these vectors, we vaccinated STAT-1 knock-out mice, an animal model for CCHFV. The vector was tolerated and 100% efficacious against challenge from a clinical strain of CCHFV. Anti-CCHFV-GP IgG and neutralizing antibody titers were observed in surviving animals. This study demonstrates that a rVSV expressing only the CCHFV-GP has the potential to serve as a replication competent vaccine platform against CCHF infections.  

5.2572           Genetic silencing of striatal CaV1.3 prevents and ameliorates levodopa dyskinesia

Steece-Collier, K., Stancati, J.A., Collier, N.J., Sandoval, I.M., Mercado, N.M., Sortwell, C.E., Collier, T.J. and Manfredsson, F.P. Movement Disorders, 34(5), 697-707 (2019)   Background Levodopa‐induced dyskinesias are an often debilitating side effect of levodopa therapy in Parkinson's disease. Although up to 90% of individuals with PD develop this side effect, uniformly effective and well‐tolerated antidyskinetic treatment remains a significant unmet need. The pathognomonic loss of striatal dopamine in PD results in dysregulation and disinhibition of striatal CaV1.3 calcium channels, leading to synaptopathology that appears to be involved in levodopa‐induced dyskinesias. Although there are clinically available drugs that can inhibit CaV1.3 channels, they are not adequately potent and have only partial and transient impact on levodopa‐induced dyskinesias. Methods To provide unequivocal target validation, free of pharmacological limitations, we developed a CaV1.3 shRNA to provide high‐potency, target‐selective, mRNA‐level silencing of striatal CaV1.3 channels and examined its ability to impact levodopa‐induced dyskinesias in severely parkinsonian rats. Results We demonstrate that vector‐mediated silencing of striatal CaV1.3 expression in severely parkinsonian rats prior to the introduction of levodopa can uniformly and completely prevent induction of levodopa‐induced dyskinesias, and this antidyskinetic benefit persists long term and with high‐dose levodopa. In addition, this approach is capable of ameliorating preexisting severe levodopa‐induced dyskinesias. Importantly, motoric responses to low‐dose levodopa remained intact in the presence of striatal CaV1.3 silencing, indicating preservation of levodopa benefit without dyskinesia liability. Discussion The current data provide some of the most profound antidyskinetic benefit reported to date and suggest that genetic silencing of striatal CaV1.3 channels has the potential to transform treatment of individuals with PD by allowing maintenance of motor benefit of levodopa in the absence of the debilitating levodopa‐induced dyskinesia side effect.  

5.2573           Structurally defined signaling in neuro‐glia units in the enteric nervous system

Boesmans, W., Hao, M.M., Fung, C., Li, Z., Van den Haute, C., tack, J., Pachnis, V. and Vanden Berghe, P. Glia, 67, 1167-1178 (2019)   Coordination of gastrointestinal function relies on joint efforts of enteric neurons and glia, whose crosstalk is vital for the integration of their activity. To investigate the signaling mechanisms and to delineate the spatial aspects of enteric neuron‐to‐glia communication within enteric ganglia we developed a method to stimulate single enteric neurons while monitoring the activity of neighboring enteric glial cells. We combined cytosolic calcium uncaging of individual enteric neurons with calcium imaging of enteric glial cells expressing a genetically encoded calcium indicator and demonstrate that enteric neurons signal to enteric glial cells through pannexins using paracrine purinergic pathways. Sparse labeling of enteric neurons and high‐resolution analysis of the structural relation between neuronal cell bodies, varicose release sites and enteric glia uncovered that this form of neuron‐to‐glia communication is contained between the cell body of an enteric neuron and its surrounding enteric glial cells. Our results reveal the spatial and functional foundation of neuro‐glia units as an operational cellular assembly in the enteric nervous system.  

5.2574           Chimaeric Rift Valley Fever Virus‐Like Particle Vaccine Candidate Production in Nicotiana benthamiana

Mbewana, S., Meyers, A.E. and Rybicki, E.P. Biotech. J., 14(4), 1800238 (2019)   Rift Valley fever virus (RVFV) is an emerging mosquito‐borne virus and hemorrhagic fever agent, which causes abortion storms in farmed small ruminants and potentially causes miscarriages in humans. Although live‐attenuated vaccines are available for animals, they can only be used in endemic areas and there are currently no commercially available vaccines for humans. Here the authors describe the production of chimaeric RVFV virus‐like particles transiently expressed in Nicotiana benthamiana by Agrobacterium tumefaciens‐mediated gene transfer. The glycoprotein (Gn) gene is modified by removing its ectodomain (Gne) and fusing it to the transmembrane domain and cytosolic tail‐encoding region of avian influenza H5N1 hemagglutinin. This is expressed transiently in N. benthamiana with purified protein yields calculated to be ≈57 mg kg⁻¹ fresh weight. Transmission electron microscopy shows putative chimaeric RVFV Gne‐HA particles of 49–60 nm which are immunogenic, eliciting Gn‐specific antibody responses in vaccinated mice without the use of adjuvant. To our knowledge, this is the first demonstration of the synthesis of Gne‐HA chimaeric RVFV VLPs and the first demonstration of a detectable yield of RVFV Gn in plants.  

5.2575           Pharmacokinetic stability of macrocyclic peptide triazole HIV‐1 inactivators alone and in liposomes

Aneja, R., Grigoletto, A., Nangarlia, A., Rashad, A.A., Wrenn, S., Jacobson, J.M., Pasut, G. and Chaiken, I.

1. Pep. Sci., 25(4), e3155 (2019)

  Previously, we reported the discovery of macrocyclic peptide triazoles (cPTs) that bind to HIV‐1 Env gp120, inhibit virus cell infection with nanomolar potencies, and cause irreversible virion inactivation. Given the appealing virus‐killing activity of cPTs and resistance to protease cleavage observed in vitro, we here investigated in vivo pharmacokinetics of the cPT AAR029b. AAR029b was investigated both alone and encapsulated in a PEGylated liposome formulation that was designed to slowly release inhibitor. Pharmacokinetic analysis in rats showed that the half‐life of FITC‐AAR029b was substantial both alone and liposome‐encapsulated, 2.92 and 8.87 hours, respectively. Importantly, liposome‐encapsulated FITC‐AAR029b exhibited a 15‐fold reduced clearance rate from serum compared with the free FITC‐cPT. This work thus demonstrated both the in vivo stability of cPT alone and the extent of pharmacokinetic enhancement via liposome encapsulation. The results obtained open the way to further develop cPTs as long‐acting HIV‐1 inactivators against HIV‐1 infection.  

5.2576           Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET

Lu, M., Ma, X., Castillo-Menendez, L.R., Gorman, J., Alssahafi, N. et al Nature, 568, 415-419 (2019)   The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic1^,2^,3^,4^,5^,6^,7^,8. Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3)8^,9^,10. It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers)5^,11^,12^,13^,14^,15^,16^,17^,18. Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1_JR-FL strain in complex with the antibody PGT15119. Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pre-triggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies—and thus of interest for the design of immunogens—remains unknown.  

5.2577           Social evolution of innate immunity evasion in a virus

Domingo-Calap, P., Segredo-Otera, E., Duran-Moreno, M. and Sanjuan, R. Nature Microbiol., 4, 1006-1013 (2019)   Antiviral immunity has been studied extensively from the perspective of virus−cell interactions, yet the role of virus−virus interactions remains poorly addressed. Here, we demonstrate that viral escape from interferon (IFN)-based innate immunity is a social process in which IFN-stimulating viruses determine the fitness of neighbouring viruses. We propose a general and simple social evolution framework to analyse how natural selection acts on IFN shutdown and validate it in cell cultures and mice infected with vesicular stomatitis virus. Furthermore, we find that IFN shutdown is costly because it reduces short-term viral progeny production, thus fulfilling the definition of an altruistic trait. Hence, in well-mixed populations, the IFN-blocking wild-type virus is susceptible to invasion by IFN-stimulating variants and spatial structure consequently determines whether IFN shutdown can evolve. Our findings reveal that fundamental social evolution rules govern viral innate immunity evasion and virulence and suggest possible antiviral interventions.  

5.2578           Safety and efficacy evaluations of an adeno-associated virus variant for preparing IL10-secreting human neural stem cell-based therapeutics

Cho, M., Jung, K., Kim, S-H., Kim, I-S., Kim, M., Shin, M., Lee, H., Park, K.I. and Jang, J-H. Gene Therapy, 26, 135-150 (2019)   Gene therapy technologies are inevitably required to boost the therapeutic performance of cell therapies; thus, validating the efficacy of gene carriers specifically used for preparing cellular therapeutics is a prerequisite for evaluating the therapeutic capabilities of gene and cell combinatorial therapies. Herein, the efficacy of a recombinant adeno-associated virus derivative (rAAVr3.45) was examined to evaluate its potential as a gene carrier for genetically manipulating interleukin-10 (IL10)-secreting human neural stem cells (hNSCs) that can potentially treat ischemic injuries or neurological disorders. Safety issues that could arise during the virus preparation or viral infection were investigated; no replication-competent AAVs were detected in the final cell suspensions, transgene expression was mostly transient, and no severe interference on endogenous gene expression by viral infection occurred. IL10 secretion from hNSCs infected by rAAVr3.45 encoding IL10 did not alter the transcriptional profile of any gene by more than threefold, but the exogenously boosted IL10 was sufficient to provoke immunomodulatory effects in an ischemic brain injury animal model, thereby accelerating the recovery of neurological deficits and the reduction of brain infarction volume. This study presents evidence that rAAVr3.45 can be potentially used as a gene carrier to prepare stem cell therapeutics.  

5.2579           Adeno-associated virus vector as a platform for gene therapy delivery

Wang, D., Tai, P.L.W. and Gao, G. Nature Reviews Drug Discovery, 18, 358-378 (2019)   Adeno-associated virus (AAV) vectors are the leading platform for gene delivery for the treatment of a variety of human diseases. Recent advances in developing clinically desirable AAV capsids, optimizing genome designs and harnessing revolutionary biotechnologies have contributed substantially to the growth of the gene therapy field. Preclinical and clinical successes in AAV-mediated gene replacement, gene silencing and gene editing have helped AAV gain popularity as the ideal therapeutic vector, with two AAV-based therapeutics gaining regulatory approval in Europe or the United States. Continued study of AAV biology and increased understanding of the associated therapeutic challenges and limitations will build the foundation for future clinical success.  

5.2580           Extensive Epitranscriptomic Methylation of A and C Residues on Murine Leukemia Virus Transcripts Enhances Viral Gene Expression

Courtney, D.G., Chalem, A., Bogerd, H.P., Law, B.A., Kennedy, E.M., Holley, C.L. and Cullen, B.R. mBio, 10(3), e01209-19 (2019)   While it has been known for several years that viral RNAs are subject to the addition of several distinct covalent modifications to individual nucleotides, collectively referred to as epitranscriptomic modifications, the effect of these editing events on viral gene expression has been controversial. Here, we report the purification of murine leukemia virus (MLV) genomic RNA to homogeneity and show that this viral RNA contains levels of N⁶-methyladenosine (m⁶A), 5-methylcytosine (m⁵C), and 2′O-methylated (Nm) ribonucleotides that are an order of magnitude higher than detected on bulk cellular mRNAs. Mapping of m⁶A and m⁵C residues on MLV transcripts identified multiple discrete editing sites and allowed the construction of MLV variants bearing silent mutations that removed a subset of these sites. Analysis of the replication potential of these mutants revealed a modest but significant attenuation in viral replication in 3T3 cells in culture. Consistent with a positive role for m⁶A and m⁵C in viral replication, we also demonstrate that overexpression of the key m⁶A reader protein YTHDF2 enhances MLV replication, while downregulation of the m⁵C writer NSUN2 inhibits MLV replication.  

5.2581           Liver-directed gene therapy results in long-term correction of progressive familial intrahepatic cholestasis type 3 in mice

Aronson, S.J., Bakker, R.S., Shi, X., Beuers, U., Paulusma, C.C. and Bosma, P.J.

1. Hepatol., 71, 153-162 (2019)

  Background & Aims Progressive familial intrahepatic cholestasis type 3 (PFIC3), for which there are limited therapeutic options, often leads to end-stage liver disease before adulthood due to impaired ABCB4-dependent phospholipid transport to bile. Using adeno-associated virus serotype 8 (AAV8)-mediated gene therapy, we aimed to restore the phospholipid content in bile to levels that prevent liver damage, thereby enabling stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3. Methods Ten-week-old Abcb4^−/− mice received a single dose of AAV8-hABCB4 (n = 10) or AAV8-GFP (n = 7) under control of a liver specific promoter via tail vein injection. Animals were sacrificed either 10 or 26 weeks after vector administration to assess transgene persistence, after being challenged with a 0.1% cholate diet for 2 weeks. Periodic evaluation of plasma cholestatic markers was performed and bile duct cannulation enabled analysis of biliary phospholipids. Liver fibrosis and the Ki67 proliferation index were assessed by immunohistochemistry. Results Stable transgene expression was achieved in all animals that received AAV8-hABCB4 up to 26 weeks after administration. AAV8-hABCB4 expression restored biliary phospholipid excretion, increasing the phospholipid and cholesterol content in bile to levels that ameliorate liver damage. This resulted in normalization of the plasma cholestatic markers, alkaline phosphatase and bilirubin. In addition, AAV8-hABCB4 prevented progressive liver fibrosis and reduced hepatocyte proliferation for the duration of the study. Conclusion Liver-directed gene therapy provides stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3. Translational studies that verify the clinical feasibility of this approach are warranted.  

5.2582           LC/MS analysis and deep sequencing reveal the accurate RNA composition in the HIV-1 virion

Simonova, A., Svojanovska, B., Trylcova, J., Hubalek, M., Moracik, O., Zavrel, M., Pavova, M., Hodek, J., Weber, j., Cvacka, J., Paces, J. and Cahova, H. Scientific Reports, 9:8697 (2019)   The mechanism of action of various viruses has been the primary focus of many studies. Yet, the data on RNA modifications in any type of virus are scarce. Methods for the sensitive analysis of RNA modifications have been developed only recently and they have not been applied to viruses. In particular, the RNA composition of HIV-1 virions has never been determined with sufficiently exact methods. Here, we reveal that the RNA of HIV-1 virions contains surprisingly high amount of the 1-methyladenosine. We are the first to use a liquid chromatography-mass spectrometry analysis (LC/MS) of virion RNA, which we combined with m¹A profiling and deep sequencing. We found that m¹A was present in the tRNA, but not in the genomic HIV-1 RNA and the abundant 7SL RNA. We were able to calculate that an HIV-1 virion contains per 2 copies of genomic RNA and 14 copies of 7SL RNA also 770 copies of tRNA, which is approximately 10 times more than thus far expected. These new insights into the composition of the HIV-1 virion can help in future studies to identify the role of nonprimer tRNAs in retroviruses. Moreover, we present a promising new tool for studying the compositions of virions.  

5.2583           Spatiotemporal Coupling of the Hepatitis C Virus Replication Cycle by Creating a Lipid Droplet- Proximal Membranous Replication Compartment

Lee, J-Y., Cortese, M., Haselmann, U., Schwab, Y., Ruggieri, A. and Bartenschlager, R. Cell Reports, 27, 3602-3617 (2019)   The hepatitis C virus (HCV) is a major cause of chronic liver disease, affecting around 71 million people worldwide. Viral RNA replication occurs in a membranous compartment composed of double-membrane vesicles (DMVs), whereas virus particles are thought to form by budding into the endoplasmic reticulum (ER). It is unknown how these steps are orchestrated in space and time. Here, we established an imaging system to visualize HCV structural and replicase proteins in live cells and with high resolution. We determined the conditions for the recruitment of viral proteins to putative assembly sites and studied the dynamics of this event and the underlying ultrastructure. Most notable was the selective recruitment of ER membranes around lipid droplets where structural proteins and the viral replicase colocalize. Moreover, ER membranes wrapping lipid droplets were decorated with double membrane vesicles, providing a topological map of how HCV might coordinate the steps of viral replication and virion assembly.  

5.2584           Therapeutic potential of AAV9-S15D-RLC gene delivery in humanized MYL2 mouse model of HCM

Yadav, S., Yuan, C-C., Kazmierczak, K., Liang, J., Huang, W., Takeuchi, L.M., Kanashiro-Takeuchi, R.M. and Szczesna-Cordary, D.

1. Mol. Med., 97(7), 1033-1047 (2019)

  Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disorder characterized by ventricular hypertrophy, myofibrillar disarray, and fibrosis, and is primarily caused by mutations in sarcomeric genes. With no definitive cure for HCM, there is an urgent need for the development of novel preventive and reparative therapies. This study is focused on aspartic acid-to-valine (D166V) mutation in the myosin regulatory light chain, RLC (MYL2 gene), associated with a malignant form of HCM. Since myosin RLC phosphorylation is critical for normal cardiac function, we aimed to exploit this post-translational modification via phosphomimetic-RLC gene therapy. We hypothesized that mimicking/modulating cardiac RLC phosphorylation in non-phosphorylatable D166V myocardium would improve heart function of HCM-D166V mice. Adeno-associated virus, serotype-9 (AAV9) was used to deliver phosphomimetic human RLC variant with serine-to-aspartic acid substitution at Ser15-RLC phosphorylation site (S15D-RLC) into the hearts of humanized HCM-D166V mice. Improvement of heart function was monitored by echocardiography, invasive hemodynamics (PV-loops) and muscle contractile mechanics. A significant increase in cardiac output and stroke work and a decrease in relaxation constant, Tau, shown to be prolonged in HCM mice, were observed in AAV- vs. PBS-injected HCM mice. Strain analysis showed enhanced myocardial longitudinal shortening in AAV-treated vs. control mice. In addition, increased maximal contractile force was observed in skinned papillary muscles from AAV-injected HCM hearts. Our data suggest that myosin RLC phosphorylation may have important translational implications for the treatment of RLC mutations-induced HCM and possibly play a role in other disease settings accompanied by depressed Ser15-RLC phosphorylation.  

5.2585           Contributions of mTOR Activation-Mediated Upregulation of Synapsin II and Neurite Outgrowth to Hyperalgesia in STZ-Induced Diabetic Rats

He, W-y., Zhang, b., Zhao, W-c., He, J., Zhang, L., Xiong, Q-m., Wang, J. and Wang, H-b. ACS Chem. Neurosci., 10, 2385-2396 (2019)   Painful diabetic neuropathy (PDN) is among the common complications in diabetes mellitus (DM), with its underlying mechanisms largely unknown. Synapsin II is primarily expressed in the spinal dorsal horn, and its upregulation mediates a superfluous release of glutamate and a deficiency of GABAergic interneuron synaptic transmission, which is directly implicated in the facilitation of pain signals in the hyperalgesic nociceptive response. Recently, synapsin II has been revealed to be associated with the modulation of neurite outgrowth, whereas the process of this neuronal structural neuroplasticity following neuronal hyperexcitability still remains unclear. In this study, we found that under conditions of elevated glucose, TNF-α induced the activation of mTOR, mediating the upregulation of synapsin II and neurite outgrowth in dorsal horn neurons. In vivo, we demonstrated that mTOR and synapsin II were upregulated and coexpressed in the spinal dorsal horn neurons in rats with streptozotocin (STZ)-induced diabetes. Furthermore, the intrathecal administration of the mTOR inhibitor rapamycin or synapsin II shRNA significantly diminished the expression of synapsin II, effectively mitigating hyperalgesia in PDN rats. We are the first to discover that in STZ-induced diabetic rats the activation of mTOR mediates the upregulation of synapsin II and neurite outgrowth, both contributing to hyperalgesia. These findings may benefit the clinical therapy of PDN by provision of a novel target.  

5.2586           Combination Suicide Gene Delivery with an Adeno-Associated Virus Vector Encoding Inducible Caspase-9 and a Chemical Inducer of Dimerization Is Effective in a Xenotransplantation Model of Hepatocellular Carcinoma

Khan, N., Bammidi, S., Chattopadhyay, S. and Jayandharan, G.R. Bioconjugate Chem., 30, 1754-1762 (2019)   Current treatment approaches for hepatocellular carcinoma (HCC) have a narrow therapeutic index and alternate modes of treatment are thus required. We have utilized a gene delivery vector containing inducible caspase 9 (iCasp9) gene, which is a synthetic analogue based on the mammalian caspase 9 and fused to a human FK506 binding protein that allows its conditional dimerization to a synthetic, small molecule [chemical inducer of dimerization, AP20187] and results in target cell apoptosis. In our studies, we have tested these synthetic vectors based on an adeno-associated virus platform for their potential anti-tumorigenic effect in human HCC cells in vitro and in a HCC tumor model developed in nude mice. Our data demonstrates that the iCasp9-AP20187 bioconjugate is able to trigger terminal effectors of cellular apoptosis and presents a viable approach for the potential treatment of HCC.  

5.2587           Interleukin-37 sensitize the elderly type 2 diabetic patients to insulin therapy through suppressing the gut microbiota dysbiosis

Li, T., Li, H., Li, W., Chen, S., Feng, T., Jiao, W., Wu, C., Dong, J., Li, Y., Li, S., Feng, m. and Wei, X. Mol. Immunol., 112, 322-329 (2019)   Objective The morbidity and prevalence of type 2 diabetes mellitus (DM) are increasing in the elderly population. Interleukin 37 (IL-37) play important roles in anti-inflammatory and anti-bacteria immune responses, but its role in the development of type 2 DM in the elderly is unclear. Therefore, we investigated whether IL-37 is associated with type 2 DM in the elderly and the underlying mechanism. Methods Hospitalized patients (aged 65–95 years) with recently diagnosed type 2 diabetes mellitus were studied retrospectively and compared with healthy subjects without glucose metabolism abnormalities. A diabetic mouse model was established by feeding ob/ob mice (C57BL/6) a high-fat, carbohydrate-free diet. Plasma glucose and insulin levels were determined by glucose oxidase assay and radioimmunoassay, respectively. The IL-37 expression level was determined by real-time PCR, western blot and ELISA (Enzyme-linked immunoassay). Results Statistic analysis showed that the IL-37 level was significantly associated with type 2 DM and insulin resistance in the elderly. The patients were then divided into insulin therapy sensitive and resistant group according to their response to insulin therapy. Data showed that the IL-37 was highly expressed in the insulin therapy sensitive group. And this was related to the less severe gut microbiota dysbiosis. In the mice model, overexpressing the IL-37 could suppress the gut microbiota dysbiosis and also the diabetes development. Conclusion Thus our results showed that higher IL-37 was associated with increased insulin sensitive in elderly type 2 DM patients through suppressing the gut microbiota dysbiosis.  

5.2588           CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection

Chen, S., Sun, S., Moonen, D., Lee, c., Lee, A.Y-L, Schaffer, D.V. and He, L. Cell Reports, 27(13), 3780-3789.e4 (2019)   Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging, conditional knockout, site-specific transgene insertion, and gene replacement; however, existing methods to generate such animals remain laborious and costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP electroporation and AAV donor infection), combining adeno-associated virus (AAV)-mediated HDR donor delivery with Cas9/sgRNA RNP electroporation to engineer large site-specific modifications in the mouse genome with high efficiency and throughput. We successfully targeted a 774 bp fluorescent reporter, a 2.1 kb CreERT2 driver, and a 3.3 kb expression cassette into endogenous loci in both embryos and live mice. CRISPR-READI is applicable to most widely used knockin schemes requiring donor lengths within the 4.9 kb AAV packaging capacity. Altogether, CRISPR-READI is an efficient, high-throughput, microinjection-free approach for sophisticated mouse genome engineering with potential applications in other mammalian species.  

5.2589           Hypoxia-induced human deoxyribonuclease I is a cellular restriction factor of hepatitis B virus

Hallez, C., Li, X., Suspene, R., Thiers, V., Bouzidi, M.S., Dorobantu, C.M., Lucansky, V., Wain-Hobson, S., Gaudin, R. and Vartanian, J-P. Nature Microbiol., 4, 1196-1207 (2019)   Numerous human APOBEC3 cytidine deaminases have proven to be, inter alia, host cell restriction factors for retroviruses and hepadnaviruses. Although they can bind to genomic RNA and become encapsidated, they are only catalytically active on single-stranded DNA. As there are many cellular deoxyribonucleases (DNases), we hypothesized that a parallel could be struck between APOBEC3 and DNases. For human hepatitis B virus (HBV), we show that DNase I can considerably reduce the virion genome copy number from a variety of transfected or infected cells. DNASE1 is overexpressed and encapsidated in HBV particles in vitro in hypoxic environments and in vivo in cirrhotic patient livers as well as in the serum of infected patients. The use of CoCl₂ and dimethyloxalylglycine, mimetic agents used to induce hypoxia by inhibiting prolyl hydroxylase enzymes that stabilize hypoxia-inducible factor (HIF)-1α, showed that the formation of HIF-1α/HIF-1β heterodimers results in the induction of DNASE1. Indeed, transfection with HIF-1α and HIF-1β expression constructs upregulated DNASE1. These findings suggest that human DNase I can impact HBV replication through the catabolism of the DNA genome within the capsid. The activity of DNases in general may explain in part the high frequency of empty or ‘light’ hepatitis B virions observed in vivo.  

5.2590           Hypervariable region 1 and N-linked glycans of hepatitis C regulate virion neutralization by modulating envelope conformations

Prentoe, J., Velazquez-Moctezuma, R., Augestad, E.H., Galli, A., Wang, R., Law, m., Alter, H. and Bukh, J. PNAS, 116(20), 10039-10047 (2019)   Hepatitis C virus (HCV) is a pervasive human pathogen for which a vaccine is urgently needed. Vaccine development relies on inducing neutralizing antibodies (NAbs), but HCV employs complex mechanisms to evade neutralization that remain incompletely understood. Here, we discover a unique interplay between two separate molecular features of HCV envelope protein E2, the hypervariable region 1 and N-linked glycans, that protect HCV from NAbs. Furthermore, we find that they share a mechanism in which they broadly modulate NAb epitope availability by influencing stability of closed and open envelope protein conformations (e.g., envelope breathing) and virus entry dependency on scavenger receptor BI. The apparent importance of structural dynamics in understanding HCV NAb evasion and entry have important implications for rational vaccine design.  

5.2591           Robust CTCF-Based Chromatin Architecture Underpins Epigenetic Changes in the Heart Failure Stress–Gene Response

Lee, D.P., Tan, W.L.W., Anene-Nzelu, C.G., Lee, C.J.M., li, P.Y. et al Circulation, 139(16), 1937-1956 (2019)   Background: The human genome folds in 3 dimensions to form thousands of chromatin loops inside the nucleus, encasing genes and cis-regulatory elements for accurate gene expression control. Physical tethers of loops are anchored by the DNA-binding protein CTCF and the cohesin ring complex. Because heart failure is characterized by hallmark gene expression changes, it was recently reported that substantial CTCF-related chromatin reorganization underpins the myocardial stress–gene response, paralleled by chromatin domain boundary changes observed in CTCF knockout. Methods: We undertook an independent and orthogonal analysis of chromatin organization with mouse pressure-overload model of myocardial stress (transverse aortic constriction) and cardiomyocyte-specific knockout of Ctcf. We also downloaded published data sets of similar cardiac mouse models and subjected them to independent reanalysis. Results: We found that the cardiomyocyte chromatin architecture remains broadly stable in transverse aortic constriction hearts, whereas Ctcf knockout resulted in ≈99% abolition of global chromatin loops. Disease gene expression changes correlated instead with differential histone H3K27-acetylation enrichment at their respective proximal and distal interacting genomic enhancers confined within these static chromatin structures. Moreover, coregulated genes were mapped out as interconnected gene sets on the basis of their multigene 3D interactions. Conclusions: This work reveals a more stable genome-wide chromatin framework than previously described. Myocardial stress–gene transcription responds instead through H3K27-acetylation enhancer enrichment dynamics and gene networks of coregulation. Robust and intact CTCF looping is required for the induction of a rapid and accurate stress response.  

5.2592           Enhancement of Adeno-Associated Virus-Mediated Gene Therapy Using Hydroxychloroquine in Murine and Human Tissues

Chandler, L.C., Barnard, A.R., Caddy, S.L., Patricio, M.I., McClements, M.E., Fu, H., Rada, C., MacLaren, R.E. and Xue, K. Molecular Therapy - Methods & Clin. Develop., 14, 77-89 (2019)   The therapeutic effects of gene therapy using adeno-associated virus (AAV) vectors are dependent on the efficacy of viral transduction. Currently, we have reached the safe limits of AAV vector dose, beyond which damaging inflammatory responses are seen. To improve the efficacy of AAV transduction, we treated mouse embryonic fibroblasts, primate retinal pigment epithelial cells, and human retinal explants with hydroxychloroquine (HCQ) 1 h prior to transduction with an AAV2 vector encoding GFP driven by a ubiquitous CAG promoter. This led to a consistent increase in GFP expression, up to 3-fold, compared with vector alone. Comparing subretinal injections of AAV2.CAG.GFP vector alone versus co-injection with 18.75 μM HCQ in paired eyes in mice, mean GFP expression was 4.6-fold higher in retinae co-treated with HCQ without retinal toxicity. A comparative 5.9-fold effect was seen with an AAV8(Y733F).GRK1.GFP vector containing the photoreceptor-specific rhodopsin kinase promoter. While the mechanism of action remains to be fully elucidated, our data suggest that a single pulse of adjunctive HCQ could safely improve AAV transduction in vivo, thus providing a novel strategy for enhancing the clinical effects of gene therapy.  

5.2593           CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites

Jayavaradhan, R., Pilis, D.M., Goodman, M., Zhang, F., Zhang, Y., Andreassen, P.R. and malik, P. Nature Communications, 10:2866 (2019)   Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPR-Cas9 by homology-dependent repair (HDR) is limited by the competing error-prone non-homologous end-joining (NHEJ) DNA repair pathway. Here, we define a safer and efficient system that promotes HDR-based precise genome editing, while reducing NHEJ locally, only at CRISPR-Cas9-induced DSBs. We fused a dominant-negative mutant of 53BP1, DN1S, to Cas9 nucleases, and the resulting Cas9-DN1S fusion proteins significantly block NHEJ events specifically at Cas9 cut sites and improve HDR frequency; HDR frequency reached 86% in K562 cells. Cas9-DN1S protein maintains this effect in different human cell types, including leukocyte adhesion deficiency (LAD) patient-derived immortalized B lymphocytes, where nearly 70% of alleles were repaired by HDR and 7% by NHEJ. Our CRISPR-Cas9-DN1S system is clinically relevant to improve the efficiencies of precise gene correction/insertion, significantly reducing error-prone NHEJ events at the nuclease cleavage site, while avoiding the unwanted effects of global NHEJ inhibition.  

5.2594           Soluble Heparin Binding Epidermal Growth Factor-Like Growth Factor Is a Regulator of GALGT2 Expression and GALGT2-Dependent Muscle and Neuromuscular Phenotypes

Cramer, M.L., Xu, R. and Martin, P.T. Mol. Cell. Biol., 39(14), e00140-19 (2019)   GALGT2 (also B4GALNT2) encodes a glycosyltransferase that is normally confined to the neuromuscular and myotendinous junction in adult skeletal muscle. GALGT2 overexpression in muscle can inhibit muscular dystrophy in mouse models of the disease by inducing the overexpression of surrogate muscle proteins, including utrophin, agrin, laminins, and integrins. Despite its well-documented biological properties, little is known about the endogenous regulation of muscle GALGT2 expression. Here, we demonstrate that epidermal growth factor receptor (EGFR) ligands can activate the human GALGT2 promoter. Overexpression of one such ligand, soluble heparin-binding EGF-like growth factor (sHB-EGF), also stimulated mouse muscle Galgt2 gene expression and expression of GALGT2-inducible surrogate muscle genes. Deletion analysis of the GALGT2 promoter identified a 45-bp region containing a TFAP4-binding site that was required for sHB-EGF activation. sHB-EGF increased TFAP4 binding to this site in muscle cells and increased endogenous Tfap4 gene expression. sHB-EGF also increased muscle EGFR protein expression and activated EGFR-Akt signaling. sHB-EGF expression was concentrated at the neuromuscular junction, and Hbegf deletion reduced Galgt2-dependent synaptic glycosylation. Hbegf deletion also mimicked Galgt2-dependent neuromuscular and muscular dystrophy phenotypes. These data demonstrate that sHB-EGF is an endogenous regulator of muscle Galgt2 gene expression and can mimic Galgt2-dependent muscle phenotypes.  

5.2595           Identifying Synaptic Proteins by In Vivo BioID from Mouse Brain

Ueze, A. and Soderling, S. Methods in Mol. Biol., 2008, 107-119 (2019)   Two anatomically and functionally distinct types of synapses are present in the central nervous system, excitatory synapses, and inhibitory synapses. Purification and analysis of the protein complex at the excitatory postsynapses have led to fundamental insights into neurobiology. In contrast, the biochemical purification and analysis of the inhibitory postsynaptic density have been largely intractable. The recently developed method called BioID employs the biotin ligase mutant, BirA*, fused to a bait protein to label and capture proximal proteins. We adapted the BioID approach to enable in vivo BioID, or iBioID of inhibitory synaptic complexes in the mouse brain. This protocol describes the iBioID method to allow synaptic bait proteins to target synaptic complexes, label, and purify biotinylated proteins from the mouse brain. This technique can be easily adapted to target other substructures in vivo that have been difficult to purify and analyze in the past.  

5.2596           A virus-like particle vaccine prevents equine encephalitis virus infection in nonhuman primates

Ko, S-Y., Akahata, W., Yang, E.S., Kong, W-P., Bruke, C.W., Honnold, S.P. et al Sci. Transl. Med., 11, eaav3113 (2019)   Western, Eastern, and Venezuelan equine encephalitis viruses (WEEV, EEEV, and VEEV, respectively) are important mosquito-borne agents that pose public health and bioterrorism threats. Despite considerable advances in understanding alphavirus replication, there are currently no available effective vaccines or antiviral treatments against these highly lethal pathogens. To develop a potential countermeasure for viral encephalitis, we generated a trivalent, or three-component, EEV vaccine composed of virus-like particles (VLPs). Monovalent VLPs elicited neutralizing antibody responses and protected mice and nonhuman primates (NHPs) against homologous challenges, but they were not cross-protective. In contrast, NHPs immunized with trivalent VLPs were completely protected against aerosol challenge by each of these three EEVs. Passive transfer of IgG from immunized NHPs protected mice against aerosolized EEV challenge, demonstrating that the mechanism of protection was humoral. Because they are replication incompetent, these trivalent VLPs represent a potentially safe and effective vaccine that can protect against diverse encephalitis viruses.  

5.2597           In vivo outcome of homology-directed repair at the HBB gene in HSC using alternative donor template delivery methods

Pattabhi, S., Lotti, S.N., Berger, M.P., Singh, S., Lux, C.T., Jacoby, K.L., Lee, C., Negre, O., Scharenberg, A.M. and Rawlings, D.J: Molecular Therapy – Nucleic Acids, 17, 277-288 (2019)   Gene editing following designer nuclease cleavage in the presence of a DNA donor template can revert mutations in disease-causing genes. For optimal benefit, reversion of the point mutation in HBB leading to sickle cell disease (SCD) would permit precise homology-directed repair (HDR) while concurrently limiting on-target non-homologous end joining (NHEJ)-based HBB disruption. In this study, we directly compared the relative efficiency of co-delivery of a novel CRISPR/Cas9 ribonucleoprotein targeting HBB in association with recombinant adeno-associated virus 6 (rAAV6) versus single-stranded oligodeoxynucleotides (ssODNs) to introduce the sickle mutation (GTC or GTG; encoding E6V) or a silent change (GAA; encoding E6optE) in human CD34⁺ mobilized peripheral blood stem cells (mPBSCs) derived from healthy donors. In vitro, rAAV6 outperformed ssODN donor template delivery and mediated greater HDR correction, leading to both higher HDR rates and a higher HDR:NHEJ ratio. In contrast, at 12–14 weeks post-transplant into recipient, immunodeficient, NOD, B6, SCID Il2rγ^−/− Kit(W41/W41) (NBSGW) mice, a ∼6-fold higher proportion of ssODN-modified cells persisted in vivo compared to recipients of rAAV6-modified mPBSCs. Together, our findings highlight that methodology for donor template delivery markedly impacts long-term persistence of HBB gene-modified mPBSCs, and they suggest that the ssODN platform is likely to be most amenable to direct clinical translation.  

5.2598           AAV-Mediated Expression of Broadly Neutralizing and Vaccine-like Antibodies Targeting the HIV-1 Envelope V2 Region

Van den Berg, F.T., Makoah, N.A., Ali, S.A., Scott, T.A., Mapengo, R.E. et al Molecular Therapy – Methods in Clin. Develop., 14, 100-112 (2019)   HIV-1 infection continues to be a global health challenge and a vaccine is urgently needed. Broadly neutralizing antibodies (bNAbs) are considered essential as they inhibit multiple HIV-1 strains, but they are difficult to elicit by conventional immunization. In contrast, non-neutralizing antibodies that correlated with reduced risk of infection in the RV144 HIV vaccine trial are relatively easy to induce, but responses are not durable. To overcome these obstacles, adeno-associated virus (AAV) vectors were used to provide long-term expression of antibodies targeting the V2 region of the HIV-1 envelope protein, including the potent CAP256-VRC26.25 bNAb, as well as non-neutralizing CAP228 antibodies that resemble those elicited by vaccination. AAVs mediated effective antibody expression in cell culture and immunocompetent mice. Mean concentrations of human immunoglobulin G (IgG) in mouse sera increased rapidly following a single AAV injection, reaching 8–60 μg/mL for CAP256 antibodies and 44–220 μg/mL for CAP228 antibodies over 24 weeks, but antibody concentrations varied for individual mice. Secreted antibodies collected from serum retained the expected binding and neutralizing activity. The vectors generated here are, therefore, suitable for the delivery of V2-targeting HIV antibodies, and they could be used in a vectored immunoprophylaxis (VIP) approach to sustain the level of antibody expression required to prevent HIV infection.  

5.2599           Reducing off target viral delivery in ovarian cancer gene therapy using a protease-activated AAV2 vector platform

Tong, J.G., Evans, A.C., Ho, M.L., Guenther, C.M., Brun, M.J., Judd, J., Wu, E. and Suh, J.

1. Controlled Release, 307, 292-301 (2019)

  Gene therapy is a promising strategy for treating metastatic epithelial ovarian cancer (EOC). However, efficient vector targeting to tumors is difficult and off-target effects can be severely detrimental. Most vector targeting approaches rely on surface receptors overexpressed on some subpopulation of cancer cells. Unfortunately, there is no universally expressed cell surface biomarker for tumor cells. As an alternative, we developed an adeno-associated virus (AAV) based “Provector” whose cellular transduction can be activated by extracellular proteases, such as matrix metalloproteinases (MMP) that are overexpressed in the tumor microenvironments of the most aggressive forms of EOC. In a non-tumor bearing mouse model, the Provector demonstrates efficient de-targeting of healthy tissues, especially the liver, where viral delivery is <1% of AAV2. In an orthotopic HeyA8 tumor model of EOC, the Provector maintains decreased off-target delivery in the liver and other tissues but with no loss in tumor delivery. Notably, approximately 10% of the injected Provector is still detected in the blood at 24 h while >99% of injected AAV2 has been cleared from the blood by 1 h. Furthermore, mouse serum raised against the Provector is 16-fold less able to neutralize Provector transduction compared to AAV2 serum neutralizing AAV2 transduction (1:200 vs 1:3200 serum dilution, respectively). Thus, the Provector appears to generate less neutralizing antibodies than AAV2. Importantly, serum against AAV2 does not neutralize the Provector as well as AAV2, suggesting that pre-existing antibodies against AAV2 would not negate the clinical application of Provectors. Taken together, we present an EOC gene delivery vector platform based on AAV with decreased off-target delivery without loss of on-target specificity, and greater immunological stealth over the traditional AAV2 gene delivery vector.  

5.2600           Deficiency in BDNF/TrkB Neurotrophic Activity Stimulates δ-Secretase by Upregulating C/EBPβ in Alzheimer’s Disease

Wang, Z-H., Xiang, J., Liu, X., Wu, S., Wang, J-Z. and Ye, K. Cell Reports, 28, 655-669 (2019)   BDNF/TrkB neurotrophic signaling regulates neuronal development, differentiation, and survival, and deficient BDNF/TrkB activity underlies neurodegeneration in Alzheimer’s disease (AD). However, exactly how BDNF/TrkB participates in AD pathology remains unclear. Here, we show that deprivation of BDNF/TrkB increases inflammatory cytokines and activates the JAK2/STAT3 pathway, resulting in the upregulation of transcription factor C/EBPβ. This, in turn, results in increased expression of δ-secretase, leading to both APP and Tau fragmentation by δ-secretase and neuronal loss, which can be blocked by expression of STAT3 Y705F, knockdown of C/EBPβ, or the δ-secretase enzymatic-dead C189S mutant. Inhibition of this pathological cascade can also rescue impaired synaptic plasticity and cognitive dysfunctions. Importantly, reduction in BDNF/TrkB neurotrophic signaling is inversely coupled with an increase in JAK2/STAT3, C/EBPβ, and δ-secretase escalation in human AD brains. Therefore, our findings provide a mechanistic link between BDNF/TrkB reduction, C/EBPβ upregulation, δ-secretase activity, and Aβ and Tau alterations in murine brains.  

5.2601           Standard screening methods underreport AAV-mediated transduction and gene editing

Lang, J.F., Toulmin, S.A., Brida, K.L., Eisenlohr, L.C. and Davidson, B.L. Nature Communications, 10:3415 (2019)   Conventional methods to discern adeno-associated virus (AAV) vector transduction patterns are based on high, stable expression of a reporter gene. As a consequence, conventionally described tropisms omit cell types that undergo transient transduction, or have low but undetectable levels of reporter expression. This creates a blind spot for AAV-based genome editing applications because only minimal transgene expression is required for activity. Here, we use editing-reporter mice to fill this void. Our approach sensitively captures both high and low transgene expression from AAV vectors. Using AAV8 and other serotypes, we demonstrate the superiority of the approach in a side-by-side comparison with traditional methods, demonstrate numerous, previously unknown sites of AAV targeting, and better predict the gene editing footprint after AAV-CRISPR delivery. We anticipate that this system, which captures the full spectrum of transduction patterns from AAV vectors in vivo, will be foundational to current and emerging AAV technologies.  

5.2602           Kaposi Sarcoma-Associated Herpesvirus Glycoprotein H Is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types

Muniraju, M., Mutsvunguma, L.Z., Foley, J., Escalante, G.M., Rodriguez, E., nabiee, R., Totonchy, J., Mulama, D.H., Nyagol., J., Wussow, F., barasa, A.K., Brehm, M. and Ogembo, J.G:

1. Virol., 93(16), e00630-19 (2019)

  Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and is the causative infectious agent of Kaposi sarcoma and two malignancies of B cell origin. To date, there is no licensed KSHV vaccine. Development of an effective vaccine against KSHV continues to be limited by a poor understanding of how the virus initiates acute primary infection in vivo in diverse human cell types. The role of glycoprotein H (gH) in herpesvirus entry mechanisms remains largely unresolved. To characterize the requirement for KSHV gH in the viral life cycle and in determination of cell tropism, we generated and characterized a mutant KSHV in which expression of gH was abrogated. Using a bacterial artificial chromosome containing a complete recombinant KSHV genome and recombinant DNA technology, we inserted stop codons into the gH coding region. We used electron microscopy to reveal that the gH-null mutant virus assembled and exited from cells normally, compared to wild-type virus. Using purified virions, we assessed infectivity of the gH-null mutant in diverse mammalian cell types in vitro. Unlike wild-type virus or a gH-containing revertant, the gH-null mutant was unable to infect any of the epithelial, endothelial, or fibroblast cell types tested. However, its ability to infect B cells was equivocal and remains to be investigated in vivo due to generally poor infectivity in vitro. Together, these results suggest that gH is critical for KSHV infection of highly permissive cell types, including epithelial, endothelial, and fibroblast cells.  

5.2603           Design of reverse transcriptase–specific nucleosides to visualize early steps of HIV-1 replication by click labeling

De Wit, F., Pillalamarri, S.R., Sebastian-Martin, A., Venkatesham, A., Van Aerschot, A. and Debyser, Z.

1. Biol. Chem., 294(31), 11862-11875 (2019)

  Only a small portion of human immunodeficiency virus type 1 (HIV-1) particles entering the host cell results in productive infection, emphasizing the importance of identifying the functional virus population. Because integration of viral DNA (vDNA) is required for productive infection, efficient vDNA detection is crucial. Here, we use click chemistry to label viruses with integrase coupled to eGFP (HIV_IN-eGFP) and visualize vDNA. Because click labeling with 5-ethynyl-2′-deoxyuridine is hampered by intense background staining of the host nucleus, we opted for developing HIV-1 reverse transcriptase (RT)-specific 2′-deoxynucleoside analogs that contain a clickable triple bond. We synthesized seven propargylated 2′-deoxynucleosides and tested them for lack of cytotoxicity and viral replication inhibition, RT-specific primer extension and incorporation kinetics in vitro, and the capacity to stain HIV-1 DNA. The triphosphate of analog A5 was specifically incorporated by HIV-1 RT, but no vDNA staining was detected during infection. Analog A3 was incorporated in vitro by HIV-1 RT and human DNA polymerase γ and did enable specific HIV-1 DNA labeling. Additionally, A3 supported mitochondria-specific DNA labeling, in line with the in vitro findings. After obtaining proof-of-principle of RT-specific DNA labeling reported here, further chemical refinement is necessary to develop even more efficient HIV-1 DNA labels without background staining of the nucleus or mitochondria.  

5.2604           Plasmacytoid dendritic cells orchestrate innate and adaptive anti-tumor immunity induced by oncolytic coxsackievirus A21

Müller, L.M.E., Holmes, M., Michael, J.L., Scott, G.B., West, E.J., Scott, K.J. et al

1. Immunother. Cancer, 7:164 (2019)

  Background The oncolytic virus, coxsackievirus A21 (CVA21), has shown promise as a single agent in several clinical trials and is now being tested in combination with immune checkpoint blockade. Combination therapies offer the best chance of disease control; however, the design of successful combination strategies requires a deeper understanding of the mechanisms underpinning CVA21 efficacy, in particular, the role of CVA21 anti-tumor immunity. Therefore, this study aimed to examine the ability of CVA21 to induce human anti-tumor immunity, and identify the cellular mechanism responsible. Methods This study utilized peripheral blood mononuclear cells from i) healthy donors, ii) Acute Myeloid Leukemia (AML) patients, and iii) patients taking part in the STORM clinical trial, who received intravenous CVA21; patients receiving intravenous CVA21 were consented separately in accordance with local institutional ethics review and approval. Collectively, these blood samples were used to characterize the development of innate and adaptive anti-tumor immune responses following CVA21 treatment. Results An Initial characterization of peripheral blood mononuclear cells, collected from cancer patients following intravenous infusion of CVA21, confirmed that CVA21 activated immune effector cells in patients. Next, using hematological disease models which were sensitive (Multiple Myeloma; MM) or resistant (AML) to CVA21-direct oncolysis, we demonstrated that CVA21 stimulated potent anti-tumor immune responses, including: 1) cytokine-mediated bystander killing; 2) enhanced natural killer cell-mediated cellular cytotoxicity; and 3) priming of tumor-specific cytotoxic T lymphocytes, with specificity towards known tumor-associated antigens. Importantly, immune-mediated killing of both MM and AML, despite AML cells being resistant to CVA21-direct oncolysis, was observed. Upon further examination of the cellular mechanisms responsible for CVA21-induced anti-tumor immunity we have identified the importance of type I IFN for NK cell activation, and demonstrated that both ICAM-1 and plasmacytoid dendritic cells were key mediators of this response. Conclusion This work supports the development of CVA21 as an immunotherapeutic agent for the treatment of both AML and MM. Additionally, the data presented provides an important insight into the mechanisms of CVA21-mediated immunotherapy to aid the development of clinical biomarkers to predict response and rationalize future drug combinations.  

5.2605           Gene therapy for C1 esterase inhibitor deficiency in a Murine Model of Hereditary angioedema

Qiu, T., Chiuchiolo, M.J., Whaley, A.S., Russo, A.R., Sondhi, D., Kaminsky, S.M., Crystal, R.G. and Pagovich, O.E: Allergy, 74(6), 1081-1089 (82019)   Background Hereditary angioedema (HAE) is a life‐threatening, autosomal dominant disorder characterized by unpredictable, episodic swelling of the face, upper airway, oropharynx, extremities, genitalia, and gastrointestinal tract. Almost all cases of HAE are caused by mutations in the SERPING1 gene resulting in a deficiency in functional plasma C1 esterase inhibitor (C1EI), a serine protease inhibitor that normally inhibits proteases in the contact, complement, and fibrinolytic systems. Current treatment of HAE includes long‐term prophylaxis with attenuated androgens or human plasma‐derived C1EI and management of acute attacks with human plasma‐derived or recombinant C1EI, bradykinin, and kallikrein inhibitors, each of which requires repeated administration. As an approach to effectively treat HAE with a single treatment, we hypothesized that a one‐time intravenous administration of an adeno‐associated virus (AAV) gene transfer vector expressing the genetic sequence of the normal human C1 esterase inhibitor (AAVrh.10hC1EI) would provide sustained circulating C1EI levels sufficient to prevent angioedema episodes. Methods To study the efficacy of AAVrh.10hC1EI, we used CRISPR/Cas9 technology to create a heterozygote C1EI‐deficient mouse model (S63±) that shares characteristics associated with HAE in humans including decreased plasma C1EI and C4 levels. Phenotypically, these mice have increased vascular permeability of skin and internal organs. Results Systemic administration of AAVrh.10hC1EI to the S63± mice resulted in sustained human C1EI activity levels above the predicted therapeutic levels and correction of the vascular leak in skin and internal organs. Conclusion A single treatment with AAVrh.10hC1EI has the potential to provide long‐term protection from angioedema attacks in affected individuals.  

5.2606           A pipeline for rapidly generating genetically engineered mouse models of pancreatic cancer using in vivo CRISPR-Cas9-mediated somatic recombination

Ideno, N., Yamaguchi, H., Okumura, T., Huang, J., Brun, M.J., Ho, M.L., Suh, J., Gupta, S., Maitra, A. and Ghosh, B. Lab. Invest., 99(8), 1233-1244 (2019)   Genetically engineered mouse models (GEMMs) that recapitulate the major genetic drivers in pancreatic ductal adenocarcinoma (PDAC) have provided unprecedented insights into the pathogenesis of this lethal neoplasm. Nonetheless, generating an autochthonous model is an expensive, time consuming and labor intensive process, particularly when tissue specific expression or deletion of compound alleles are involved. In addition, many of the current PDAC GEMMs cause embryonic, pancreas-wide activation or loss of driver alleles, neither of which reflects the cognate human disease scenario. The advent of CRISPR/Cas9 based gene editing can potentially circumvent many of the aforementioned shortcomings of conventional breeding schema, but ensuring the efficiency of gene editing in vivo remains a challenge. Here we have developed a pipeline for generating PDAC GEMMs of complex genotypes with high efficiency using a single “workhorse” mouse strain expressing Cas9 in the adult pancreas under a p48 promoter. Using adeno-associated virus (AAV) mediated delivery of multiplexed guide RNAs (sgRNAs) to the adult murine pancreas of p48-Cre; LSL-Cas9 mice, we confirm our ability to express an oncogenic Kras ^G12D allele through homology-directed repair (HDR), in conjunction with CRISPR-induced disruption of cooperating alleles (Trp53, Lkb1 and Arid1A). The resulting GEMMs demonstrate a spectrum of precursor lesions (pancreatic intraepithelial neoplasia [PanIN] or Intraductal papillary mucinous neoplasm [IPMN] with eventual progression to PDAC. Next generation sequencing of the resulting murine PDAC confirms HDR of oncogenic Kras^G12D allele at the endogenous locus, and insertion deletion (“indel”) and frameshift mutations of targeted tumor suppressor alleles. By using a single “workhorse” mouse strain and optimal AAV serotype for in vivo gene editing with combination of driver alleles, we present a facile autochthonous platform for interrogation of the PDAC genome.  

5.2607           Pharmacological doses of melatonin impede cognitive decline in tau‐related Alzheimer models, once tauopathy is initiated, by restoring the autophagic flux

Luengo, E., Buendia, I., Fernandez-Mendivil, C., Trigo-Alonso, P., Negredo, P., Michalska, P., Hernandez-Garcia, B., Sanchez-Ramos, C., Bernal, J.A., Ikezu, T., Leon, R. and Lopez, M.G.

1. Pineal Res., 67, e12578 (2019)

  Alterations in autophagy are increasingly being recognized in the pathogenesis of proteinopathies like Alzheimer's disease (AD). This study was conducted to evaluate whether melatonin treatment could provide beneficial effects in an Alzheimer model related to tauopathy by improving the autophagic flux and, thereby, prevent cognitive decline. The injection of AAV‐hTau^P301L viral vectors and treatment/injection with okadaic acid were used to achieve mouse and human ex vivo, and in vivo tau‐related models. Melatonin (10 μmol/L) impeded oxidative stress, tau hyperphosphorylation, and cell death by restoring autophagy flux in the ex vivo models. In the in vivo studies, intracerebroventricular injection of AAV‐hTau^P301L increased oxidative stress, neuroinflammation, and tau hyperphosphorylation in the hippocampus 7 days after the injection, without inducing cognitive impairment; however, when animals were maintained for 28 days, cognitive decline was apparent. Interestingly, late melatonin treatment (10 mg/kg), starting once the alterations mentioned above were established (from day 7 to day 28), reduced oxidative stress, neuroinflammation, tau hyperphosphorylation, and caspase‐3 activation; these observations correlated with restoration of the autophagy flux and memory improvement. This study highlights the importance of autophagic dysregulation in tauopathy and how administration of pharmacological doses of melatonin, once tauopathy is initiated, can restore the autophagy flux, reduce proteinopathy, and prevent cognitive decline. We therefore propose exogenous melatonin supplementation or the development of melatonin derivatives to improve autophagy flux for the treatment of proteinopathies like AD.  

5.2608           Hepatitis E Virus (HEV) Open Reading Frame 2 Antigen Kinetics in Human-Liver Chimeric Mice and Its Impact on HEV Diagnosis

Sayed, I.M., Verhoye, L., Montpellier, C., Abravanel, F., Izopet, J., Cocquerrel, L. and Meuleman, P.

J. Infect. Dis., 220, 811-819 (2019)

  Background Hepatitis E virus infection (HEV) is an emerging problem in developed countries. Diagnosis of HEV     infection is based on the detection of HEV-specific antibodies, viral RNA, and/or antigen (Ag).        Humanized mice were previously reported as a model for the study of HEV infection, but published data were focused on the quantification of viral RNA. However, the kinetics of HEV Ag expression during infection remains poorly understood. Methods Plasma specimens and suspensions of fecal specimens from HEV-infected and ribavirin-treated        humanized mice were analyzed using HEV antigen–specific enzyme-linked immunosorbent assay, reverse transcription–quantitative polymerase chain reaction analysis, density gradient analysis, and Western blotting. Result Open reading frame 2 (ORF2) Ag was detected in both plasma and stool from HEV-infected mice, and levels increased over time. Contrary to HEV RNA, ORF2 Ag levels were higher in mouse plasma than in stool. Interestingly, ORF2 was detected in plasma from mice that tested negative for HEV RNA in plasma but positive for HEV RNA in stool and was detected after viral clearance in mice that were treated with ribavirin. Plasma density gradient analysis revealed the presence of the noninfectious glycosylated form of ORF2. Conclusion ORF2 Ag can be used as a marker of active HEV infection and for assessment of the effect of antiviral therapy, especially when fecal samples are not available or molecular diagnostic tests are not accessible.  

5.2609           Deletion of Class II ADP-Ribosylation Factors in Mice Causes Tremor by the Nav1.6 Loss in Cerebellar Purkinje Cell Axon Initial Segments

Hosoi, N., Shibasaki, K., Hosono, M., Konno, A., Shinoda, Y., Kiyonari, H., Inoue, K., Muramatsu, S-i., Ishizaki, Y., Hirai, H., Furuichi, T. and Saddakata, T.

1. Neurosci., 39(32), 6339-6353 (2019)

  ADP-ribosylation factors (ARFs) are a family of small monomeric GTPases comprising six members categorized into three classes: class I (ARF1, 2, and 3), class II (ARF4 and 5), and class III (ARF6). In contrast to class I and III ARFs, which are the key regulators in vesicular membrane trafficking, the cellular function of class II ARFs remains unclear. In the present study, we generated class II ARF-deficient mice and found that ARF4^+/−/ARF5^−/− mice exhibited essential tremor (ET)-like behaviors. In vivo electrophysiological recordings revealed that ARF4^+/−/ARF5^−/− mice of both sexes exhibited abnormal brain activity when moving, raising the possibility of abnormal cerebellar excitability. Slice patch-clamp experiments demonstrated the reduced excitability of the cerebellar Purkinje cells (PCs) in ARF4^+/−/ARF5^−/− mice. Immunohistochemical and electrophysiological analyses revealed a severe and selective decrease of pore-forming voltage-dependent Na⁺ channel subunit Nav1.6, important for maintaining repetitive action potential firing, in the axon initial segment (AIS) of PCs. Importantly, this decrease in Nav1.6 protein localized in the AIS and the consequent tremors in ARF4^+/−/ARF5^−/− mice could be alleviated by the PC-specific expression of ARF5 using adeno-associated virus vectors. Together, our data demonstrate that the decreased expression of the class II ARF proteins in ARF4^+/−/ARF5^−/− mice, leading to a haploinsufficiency of ARF4 in the absence of ARF5, impairs the localization of Nav1.6 to the AIS and hence reduces the membrane excitability in PCs, resulting in the ET-like movement disorder. We suggest that class II ARFs function in localizing specific proteins, such as Nav1.6, to the AIS.  

5.2610           TLR9-Activating CpG-B ODN but Not TLR7 Agonists Triggers Antibody Formation to Factor IX in Muscle Gene Transfer

Butterfield, J.S.S., Biswas, M., Shirley, J.L., Kumar, S.R.P., Sherman, A., Terhorst, C., Ling, C. and Herzog, R.W. Hum. Gen. Ther. Methods, 30(3), 81-92 (2019)   Innate immune signals that promote B cell responses in gene transfer are generally ill-defined. In this study, we evaluate the effect of activating endosomal Toll-like receptors 7, 8, and 9 (TLR7, TLR7/8, and TLR9) on antibody formation during muscle-directed gene therapy with adeno-associated virus (AAV) vectors. We examined whether activation of endosomal TLRs, by adenine analog CL264 (TLR7 agonist), imidazolquinolone compound R848 (TLR7/8 agonist), or class B CpG oligodeoxynucleotides ODN1826 (TLR9 agonist), could augment antibody formation upon intramuscular administration of AAV1 expressing human clotting factor IX (AAV1-hFIX) in mice. The TLR9 agonist robustly enhanced antibody formation by the 1st week, thus initially eliminating systemic hFIX expression. By contrast, the TLR7 and TLR7/8 agonists did not markedly promote antibody formation, or significantly reduce circulating hFIX. We concurrently investigated the effects of these TLR agonists during muscle gene transfer on mature B cells and dendritic cells (DCs) in the draining lymph nodes including conventional DCs (CD11b⁺ or CD8α⁺ cDCs), monocyte-derived dendritic cells (moDCs), and plasmacytoid dendritic cells (pDCs). Only TLR9 stimulation caused a striking increase in the frequency of moDCs within 24 h. The TLR7/8 and TLR9 agonists activated pDCs, both subsets of cDCs, and mature B cells, whereas the TLR7 agonist had only mild effects on these cells. Thus, these TLR ligands have distinct effects on DCs and mature B cells, yet only the TLR9 agonist enhanced the humoral immune response against AAV-expressed hFIX. These new findings indicate a unique ability of certain TLR9 agonists to stimulate B cell responses in muscle gene transfer through enrichment of moDCs.  

5.2611           An AAV Dual Vector Strategy Ameliorates the Stargardt Phenotype in Adult Abca4−/− Mice

McClements, M., barnard, A.R., Singh, M.S., Issa, P.C., Jiang, Z., Radu, R.A. and Maclaren, R.E. Hum. Gen. Ther., 30(5), 590-600 (2019)   The recent approval in the United States of the first adeno-associated viral (AAV) vector for the treatment of an inherited retinal degeneration validates this approach for the treatment of many other diseases. A major limiting factor continues to be the size restriction of the AAV transgene at under 5 kb. Stargardt disease is the most prevalent form of recessively inherited blindness and is caused by mutations in ABCA4, the gene that codes for ATP-binding cassette transporter protein family member 4, which has a coding sequence length of 6.8 kb. Dual vector approaches increase the capacity of AAV gene therapy, but at the cost of substantially reduced levels of target protein, which may be insufficient to achieve a therapeutic effect. Here we show that the efficacy of recombination of dual vectors is dependent on the length of DNA overlap between two transgenes. With optimized recombination, full-length ABCA4 protein is expressed in the photoreceptor outer segments of Abca4^−/− mice at levels sufficient to reduce bisretinoid formation and correct the autofluorescent phenotype. These observations support a dual vector approach in future clinical trials using AAV gene therapy to treat Stargardt disease.  

5.2612           Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans

Jüttner, J., Szabo, A., Gross-Scherf, B., Morikawa, R.K., Rompani, S.B. et al Nature Neurosci., 22, 1345-1356 (2019)   Targeting genes to specific neuronal or glial cell types is valuable for both understanding and repairing brain circuits. Adeno-associated viruses (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is an unsolved problem. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that a number of these AAVs specifically target expression to neuronal and glial cell types in the mouse and non-human primate retina in vivo and in the human retina in vitro. We demonstrate applications for recording and stimulation, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.  

5.2613           Inhibition of Semaphorin3A Promotes Ocular Dominance Plasticity in the Adult Rat Visual Cortex

Boggio, E., Ehlert, E.M., Lupori, L., Moloney, E.B., De Winter, F., Vander Kool, C.W., Baroncelli, L., Mercollari, V., Blits, B., Fawcett, J.W., Verhaagen, J. and Pizzoeusso, T. Mol. Neurobiol., 56(9), 5987-5997 (2019)   Perineuronal nets (PNNs) are condensed structures in the extracellular matrix that mainly surround GABA-ergic parvalbumin-positive interneurons in the adult brain. Previous studies revealed a parallel between PNN formation and the closure of the critical period. Moreover, ocular dominance plasticity is enhanced in response to PNN manipulations in adult animals. However, the mechanisms through which perineuronal nets modulate plasticity are still poorly understood. Recent work indicated that perineuronal nets may convey molecular signals by binding and storing proteins with important roles in cellular communication. Here we report that semaphorin3A (Sema3A), a chemorepulsive axon guidance cue known to bind to important perineuronal net components, is necessary to dampen ocular dominance plasticity in adult rats. First, we showed that the accumulation of Sema3A in PNNs in the visual cortex correlates with critical period closure, following the same time course of perineuronal nets maturation. Second, the accumulation of Sema3A in perineuronal nets was significantly reduced by rearing animals in the dark in the absence of any visual experience. Finally, we developed and characterized a tool to interfere with Sema3A signaling by means of AAV-mediated expression of receptor bodies, soluble proteins formed by the extracellular domain of the endogenous Sema3A receptor (neuropilin1) fused to a human IgG Fc fragment. By using this tool to antagonize Sema3A signaling in the adult rat visual cortex, we found that the specific inhibition of Sema3A promoted ocular dominance plasticity. Thus, Sema3A accumulates in perineuronal nets in an experience-dependent manner and its presence in the mature visual cortex inhibits plasticity.  

5.2614           Wolfberry‐Derived Zeaxanthin Dipalmitate Attenuates Ethanol‐Induced Hepatic Damage

Gao, H., Lv, Y., Liu, Y., Li, J., Wang, X., Zhou, Z., Tipoe, G.L., Ouyang, S., Guo, Y., Zhang, J., Hao, X., Li, W., Koike, K., So, K-F. and Xiao, J. Mol. Nutr. Food Res., 63, 1801339 (2019)   Scope Besides abstinence and nutritional support, there is no proven clinical treatment for patients with alcoholic fatty liver disease (AFLD). Here, the therapeutic effects and mechanisms of action of wolfberry‐derived zeaxanthin dipalmitate (ZD) on AFLD models are demonstrated. Methods and results The hepatoprotective effects of ZD are evaluated in vitro and in vivo. Direct interacting receptors of ZD on cell membranes are identified by liver‐specific knockdown and biophysical measurements. Downstream signaling pathways are delineated using molecular and cellular biological methods. It is demonstrated that ZD attenuates hepatocyte and whole‐liver injury in ethanol‐treated cells (dose: 1 µm) and a chronic binge AFLD rat model (dose: 10 mg kg^–1), respectively. The direct targets of ZD on the cell membrane include receptor P2X7 and adiponectin receptor 1 (adipoR1). Signals from P2X7 and adipoR1 modulate the phosphatidylinositide 3‐kinase‐Akt and/or AMP‐activated protein kinase‐FoxO3a pathways, to restore mitochondrial autophagy (mitophagy) functions suppressed by ethanol intoxication. In addition, ZD alleviates hepatic inflammation partially via the inhibition of Nod‐like receptor 3 inflammasome, whose activation is a direct consequence of suppressed mitophagy. Liver‐specific inhibition of receptors or mitophagy significantly impairs the beneficial effects of ZD. Conclusions ZD is an effective and promising agent for the potential treatment of AFLD.  

5.2615           The smallest ssDNA phage infecting a marine bacterium

Zhan, Y. and Chen, F. Environmental Microbiol., 21(6), 1916-1928 (2019)   In the marine environment, only a few lytic single‐stranded DNA (ssDNA) phages have been isolated and characterized, despite the fact that diverse ssDNA bacteriophages have been discovered via metagenomic studies. In this study, we isolated and characterized a new ssDNA phage, vB_RpoMi‐Mini, which infects a marine bacterium Ruegeria pomeroyi DSS‐3. With a genome size of 4248 bp and only four putative open reading frames (ORF), vB_RpoMi‐Mini becomes the smallest ssDNA phage among the known ssDNA phage isolates and represents the DNA bacteriophage with the least number of ORFs. Genome‐wide analysis reveals that bacteriophage Mini is distantly related to the known ssDNA phages and belongs to an unclassified ssDNA phage within the Microviridae family. The presence of peptidase in vB_RpoMi‐Mini genome further implies that horizontal gene transfer could be an important driving force in the evolution of ssDNA phages. Bacteriophage Mini seems to have lost the spike protein commonly seen in ssDNA phages, suggesting that ssDNA phage can be more diverse than previously thought. Metagenomic analysis indicates that Mini‐like phages are widely distributed in the environments. The discovery of vB_RpoMi‐Mini expands our understanding of ssDNA phages in nature, and also indicates our dearth of knowledge regarding of ssDNA phages.  

5.2616           LARP1 binding to hepatitis C virus particles is correlated with intracellular retention of viral infectivity

Plissonnier, M-L., Cottarel, J., Piver, E., Kullolli, M., Centonze, F.G., Pitteri, S., Farhan, H., Meunier, J-C., Zoulim, F. and Parent, R. Virus Res., 271, 197679 (2019)   Hepatitis C virus (HCV) virions contain a subset of host liver cells proteome often composed of interesting virus-interacting factors. A proteomic analysis performed on double gradient-purified clinical HCV highlighted the translation regulator LARP1 on these virions. This finding was validated using post-virion capture and immunoelectron microscopy, as well as immunoprecipitation applied to in vitro (Huh7.5 liver cells) grown (Gt2a, JFH1 strain) and patient-derived (Gt1a) HCV particles. Upon HCV infection of Huh7.5 cells, we observed a drastic transfer of LARP1 to lipid droplets, inducing colocalization with core proteins. RNAi-mediated depletion of LARP1 using the C911 control approach decreased extracellular infectivity of HCV Gt1a (H77), Gt2a (JFH1), and Gt3a (S52 chimeric strain), yet increased their intracellular infectivity. This latter effect was unrelated to changes in the hepatocyte secretory pathway, as evidenced using a functional RUSH assay. These results indicate that LARP1 binds to HCV, an event associated with retention of intracellular infectivity.  

5.2617           Efficient Inhibition of Human Papillomavirus Infection by L2 Minor Capsid-Derived Lipopeptide

Yan, H., Foo, S-S., Chen, W., Yoo, J-S., Shin, W-J., Wu, C. and Jung, J.U. mBio, 10(4), e01834-19 (2019)   The amino (N)-terminal region of human papillomavirus (HPV) minor capsid protein (L2) is a highly conserved region which is essential for establishing viral infection. Despite its importance in viral infectivity, the role of the HPV N-terminal domain has yet to be fully characterized. Using fine mapping analysis, we identified a 36-amino-acid (aa) peptide sequence of the L2 N terminus, termed L2N, that is critical for HPV infection. Ectopic expression of L2N with the transmembrane sequence on the target cell surface conferred resistance to HPV infection. Additionally, L2N peptide with chemical or enzymatic lipidation at the carboxyl (C) terminus efficiently abrogated HPV infection in target cells. Among the synthetic L2N lipopeptides, a stearoylated lipopeptide spanning aa 13 to 46 (13-46st) exhibited the most potent anti-HPV activity, with a half-maximal inhibitory concentration (IC₅₀) of ∼200 pM. Furthermore, we demonstrated that the 13-46st lipopeptide inhibited HPV entry by blocking trans-Golgi network retrograde trafficking of virion particles, leading to rapid degradation. Fundamentally, the inhibitory effect of L2N lipopeptides appeared to be evolutionarily conserved, as they showed cross-type inhibition among various papillomaviruses. In conclusion, our findings provide new insights into the critical role of the L2N sequence in the HPV entry mechanism and identify the therapeutic potential of L2N lipopeptide as an effective anti-HPV agent.  

5.2618           Affinity chromatography for vaccines manufacturing: Finally ready for prime time?

Zhao, M., Vanderluis, M., Stout, J., Haupts, U., Sanders, M. and jacquemart, R. Vaccine, 37, 5491-5503 (2019)   Affinity chromatography is among the most powerful separation techniques, achieving the finest separation with high yields even in the most challenging feed streams. Incorporating affinity chromatography in vaccine purification has long been attempted by researchers to improve unit yield and purity with the secondary goal of reducing the number of downstream process operations. Despite the success in laboratory-scale proof of concept, implementation of this technique in pilot or cGMP manufacturing has rarely been realised due to technical and economic challenges in design and manufacturing of ideal ligands as well as availability of high-productivity chromatography media. This paper reviews evolving technologies in engineered ligands and chromatography media that are encouraging companies to re-visit the possible use of affinity chromatography in larger scale vaccine purification. It is postulated that commercial-scale implementation of high throughput single-use affinity chromatography can significantly simplify process architecture, improve productivity and flexibility, and reduce cost of goods.  

5.2619           Impact of repeated pressurization on virus removal by reverse osmosis membranes for household water treatment

Torii, S., Hashimoto, T., Do, A.T., Furamai, H. and katayama, H. Environ. Sci. Water Res. Technol., 5, 910-919 (2019)   Reverse osmosis (RO) membranes are commoditized and available for household water treatment (HWT) in areas where access to safe water is limited. The RO membranes for HWT (residential RO) are typically operated intermittently without a cleaning process. This suggests that a unique mechanism of membrane deterioration, membrane oxidation, one of the main causes of RO membrane deterioration in industrial settings (desalination and wastewater reclamation), is not involved. Furthermore, the intermittent operation provides repeated shear stress on the membrane surface. This study aimed to evaluate the impact of repeated pressurization on virus (bacteriophage MS2 and φX-174) removal by residential RO and to determine the location of integrity loss. We repeatedly pressurized and de-pressurized spiral-wound residential RO membranes for up to 10 000 cycles, while periodically evaluating virus removal. E. coli removal was also determined after 10 000 cycles. Moreover, these membranes were examined for virus and E. coli removal in a flat-sheet configuration. For the first 3000–4000 cycles, φX-174 removal was maintained at approximately 4 log₁₀ (99.99%), and then dramatically decreased. After 10 000 cycles, even E. coli leaked from the membrane. The deterioration of virus removal in a flat-sheet configuration indicates integrity loss at the membrane surface. Therefore, repeated pressurization deteriorated the virus removal performance of residential RO. The number of times that the RO membrane can be pressurized should be included as a criterion to determine the frequency of membrane replacement in residential RO.  

5.2620           GluA4-targeted AAV vectors deliver genes selectively to interneurons while relying on the AAV receptor for entry

Hartmann, J., Thalheimer, F.B., Höpfner, F., Kerzel, T., Khodosevich, K., Garcia-Gonzalez, D., Monyer, H., Diester, I., Büning, H., Carette, J.E., Fries, p. and Buchholz, C.J. Molecular Therapy – Methods & Clin Develop., 14, 252-260 (2019)   Selective gene delivery into subtypes of interneurons remains an important challenge in vector development. Adeno-associated virus (AAV) vector particles are especially promising for intracerebral injections. For cell entry, AAV2 particles are supposed to attach to heparan-sulfate proteoglycans (HSPGs) followed by endocytosis via the AAV receptor (AAVR). Here, we assessed engineered AAV particles deficient in HSPG attachment but competent in recognizing the glutamate receptor 4 (GluA4, also known as GluRD or GRIA4) through a displayed GluA4-specific DARPin (designed ankyrin repeat protein). When injected into the mouse brain, histological evaluation revealed that in various regions, more than 90% of the transduced cells were interneurons, mainly of the parvalbumin-positive subtype. Although part of the selectivity was mediated by the DARPin, the chosen spleen focus-forming virus (SFFV) promoter had contributed as well. Further analysis revealed that the DARPin mediated selective attachment to GluA4-positive cells, whereas gene delivery required expression of AAVR. Our data suggest that cell selectivity of AAV particles can be modified rationally and efficiently through DARPins, but expression of the AAV entry receptor remains essential.  

5.2621           Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene Expression

Courtney, D.G., Tsai, K., Bogerd, H.P., Swanstrom, R., Holley, C.L. and Cullen, B.R: Cell Host & Microbe, 26, 217-227 (2019)   How the covalent modification of mRNA ribonucleotides, termed epitranscriptomic modifications, alters mRNA function remains unclear. One issue has been the difficulty of quantifying these modifications. Using purified HIV-1 genomic RNA, we show that this RNA bears more epitranscriptomic modifications than the average cellular mRNA, with 5-methylcytosine (m⁵C) and 2′O-methyl modifications being particularly prevalent. The methyltransferase NSUN2 serves as the primary writer for m⁵C on HIV-1 RNAs. NSUN2 inactivation inhibits not only m⁵C addition to HIV-1 transcripts but also viral replication. This inhibition results from reduced HIV-1 protein, but not mRNA, expression, which in turn correlates with reduced ribosome binding to viral mRNAs. In addition, loss of m⁵C dysregulates the alternative splicing of viral RNAs. These data identify m⁵C as a post-transcriptional regulator of both splicing and function of HIV-1 mRNA, thereby affecting directly viral gene expression.  

5.2622           AAV-ie enables safe and efficient gene transfer to inner ear cells

Tan, F., Chu, C., Qi, J., Li, W., You, D., Li, K., Chen, X., Zhao, W. et al Nature Communications, 10:3733 (2019   Hearing loss is the most common sensory disorder. While gene therapy has emerged as a promising treatment of inherited diseases like hearing loss, it is dependent on the identification of gene delivery vectors. Adeno-associated virus (AAV) vector-mediated gene therapy has been approved in the US for treating a rare inherited eye disease but no safe and efficient vectors have been identified that can target the diverse types of inner ear cells. Here, we identify an AAV variant, AAV-inner ear (AAV-ie), for gene delivery in mouse inner ear. Our results show that AAV-ie transduces the cochlear supporting cells (SCs) with high efficiency, representing a vast improvement over conventional AAV serotypes. Furthermore, after AAV-ie-mediated transfer of the Atoh1 gene, we find that many SCs trans-differentiated into new HCs. Our results suggest that AAV-ie is a useful tool for the cochlear gene therapy and for investigating the mechanism of HC regeneration.  

5.2623           Microglia affect α-synuclein cell-to-cell transfer in a mouse model of Parkinson’s disease

George, S., Rey, N.L., Tyson, T., Esquibel, C., Myerdirk, L., Schulz, E., Pierce, S., Burmeister, A.R., Madaj, Z., Steiner, J.A., galvis, M.L.E., Brundin, L. and Brundin, P. Mol. Neurodegeneration, 14:34 (2019)   Background Cell-to-cell propagation of α-synuclein (α-syn) aggregates is thought to contribute to the pathogenesis of Parkinson’s disease (PD) and underlie the spread of α-syn neuropathology. Increased pro-inflammatory cytokine levels and activated microglia are present in PD and activated microglia can promote α-syn aggregation. However, it is unclear how microglia influence α-syn cell-to-cell transfer. Methods We developed a clinically relevant mouse model to monitor α-syn prion-like propagation between cells; we transplanted wild-type mouse embryonic midbrain neurons into a mouse striatum overexpressing human α-syn (huα-syn) following adeno-associated viral injection into the substantia nigra. In this system, we depleted or activated microglial cells and determined the effects on the transfer of huα-syn from host nigrostriatal neurons into the implanted dopaminergic neurons, using the presence of huα-syn within the grafted cells as a readout. Results First, we compared α-syn cell-to-cell transfer between host mice with a normal number of microglia to mice in which we had pharmacologically ablated 80% of the microglia from the grafted striatum. With fewer host microglia, we observed increased accumulation of huα-syn in grafted dopaminergic neurons. Second, we assessed the transfer of α-syn into grafted neurons in the context of microglia activated by one of two stimuli, lipopolysaccharide (LPS) or interleukin-4 (IL-4). LPS exposure led to a strong activation of microglial cells (as determined by microglia morphology, cytokine production and an upregulation in genes involved in the inflammatory response in the LPS-injected mice by RNA sequencing analysis). LPS-injected mice had significantly higher amounts of huα-syn in grafted neurons. In contrast, injection of IL-4 did not change the proportion of grafted dopamine neurons that contained huα-syn relative to controls. As expected, RNA sequencing analysis on striatal tissue revealed differential gene expression between LPS and IL-4-injected mice; with the genes upregulated in tissue from mice injected with LPS including several of those involved in an inflammatory response. Conclusions The absence or the hyperstimulation of microglia affected α-syn transfer in the brain. Our results suggest that under resting, non-inflammatory conditions, microglia modulate the transfer of α-syn. Pharmacological regulation of neuroinflammation could represent a future avenue for limiting the spread of PD neuropathology.  

5.2624           Divergent engagements between adeno-associated viruses with their cellular receptor AAVR

Zhang, R., Xu, G., Cao, L., Sun, Z., He, Y., Cui, M., Sun, Y., Li, S. et al Nature Communications, 10:3760 (2019)   Adeno-associated virus (AAV) receptor (AAVR) is an essential receptor for the entry of multiple AAV serotypes with divergent rules; however, the mechanism remains unclear. Here, we determine the structures of the AAV1-AAVR and AAV5-AAVR complexes, revealing the molecular details by which PKD1 recognizes AAV5 and PKD2 is solely engaged with AAV1. PKD2 lies on the plateau region of the AAV1 capsid. However, the AAV5-AAVR interface is strikingly different, in which PKD1 is bound at the opposite side of the spike of the AAV5 capsid than the PKD2-interacting region of AAV1. Residues in strands F/G and the CD loop of PKD1 interact directly with AAV5, whereas residues in strands B/C/E and the BC loop of PKD2 make contact with AAV1. These findings further the understanding of the distinct mechanisms by which AAVR recognizes various AAV serotypes and provide an example of a single receptor engaging multiple viral serotypes with divergent rules.  

5.2625           Plug-and-Play Protein Modification Using Homology-Independent Universal Genome Engineering

Gao, Y., Hisey, E., Bradshaw, T.W.A., Xiang, Y., Diao, Y. and Soderling, S.H. Neuron, 103, 583-597 (2019)   Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the “plug-and-play” nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.  

5.2626           Intrathecal Adeno-Associated Viral Vector-Mediated Gene Delivery for Adrenomyeloneuropathy

Gong, Y., Berenson, A., Laheji, F., Gao, G., Wang, D., Ng, C., Volak, A., Kok, R., Kreouzis, V., Dijkstra, I.M., Kemp, S., Maguire, C.A. and Eichler, F. Human Gene Therapy, 30(5), 544-555 (2019)   Mutations in the gene encoding the peroxisomal ATP-binding cassette transporter (ABCD1) cause elevations in very long-chain fatty acids (VLCFAs) and the neurodegenerative disease adrenoleukodystrophy (ALD). In most adults, this manifests as the spinal cord axonopathy adrenomyeloneuropathy (AMN). A challenge in virus-based gene therapy in AMN is how to achieve functional gene correction to the entire spinal cord while minimizing leakage into the systemic circulation, which could contribute to toxicity. In the present study, we used an osmotic pump to deliver adeno-associated viral (AAV) vector into the lumbar cerebrospinal fluid space in mice. We report that slow intrathecal delivery of recombinant AAV serotype 9 (rAAV9) achieves efficient gene transfer across the spinal cord and dorsal root ganglia as demonstrated with two different transgenes, GFP and ABCD1. In the Abcd1^–/– mouse, gene correction after continuous rAAV9-CBA-hABCD1 delivery led to a 20% decrease in VLCFA levels in spinal cord compared with controls. The major cell types transduced were astrocytes, vascular endothelial cells, and neurons. Importantly, rAAV9 delivered intrathecally by osmotic pump, in contrast to bolus injection, reduced systemic leakage into peripheral organs, particularly liver and heart tissue.  

5.2627           Somatic Gene Editing of GUCY2D by AAV-CRISPR/Cas9 Alters Retinal Structure and Function in Mouse and Macaque

McCullough, K.T., Boye, S.L., Fajardo, D., Calabro, K., Peterson, J.J., Strang, C.E., et al Human Gene Therapy, 30(5), 571-589 (2019)   Mutations in GUCY2D, the gene encoding retinal guanylate cyclase-1 (retGC1), are the leading cause of autosomal dominant cone–rod dystrophy (CORD6). Significant progress toward clinical application of gene replacement therapy for Leber congenital amaurosis (LCA) due to recessive mutations in GUCY2D (LCA1) has been made, but a different approach is needed to treat CORD6 where gain of function mutations cause dysfunction and dystrophy. The CRISPR/Cas9 gene editing system efficiently disrupts genes at desired loci, enabling complete gene knockout or homology directed repair. Here, adeno-associated virus (AAV)-delivered CRISPR/Cas9 was used specifically to edit/disrupt this gene's early coding sequence in mouse and macaque photoreceptors in vivo, thereby knocking out retGC1 expression and demonstrably altering retinal function and structure. Neither preexisting nor induced Cas9-specific T-cell responses resulted in ocular inflammation in macaques, nor did it limit GUCY2D editing. The results show, for the first time, the ability to perform somatic gene editing in primates using AAV-CRISPR/Cas9 and demonstrate the viability this approach for treating inherited retinal diseases in general and CORD6 in particular.  

5.2628           Taste Receptor Cells in Mice Express Receptors for the Hormone Adiponectin

Crosson, S.M., marques, A.M., Dib, P., Dotosn, C.D., Munger, S.D. and Zolotukhin, S. Chemical Senses, 44(6), 409-422 (2019)   The metabolic hormone adiponectin is secreted into the circulation by adipocytes and mediates key biological functions, including insulin sensitivity, adipocyte development, and fatty acid oxidation. Adiponectin is also abundant in saliva, where its functions are poorly understood. Here we report that murine taste receptor cells (TRCs) express specific adiponectin receptors and may be a target for salivary adiponectin. This is supported by the presence of all three known adiponectin receptors in transcriptomic data obtained by RNA-seq analysis of purified circumvallate (CV) taste buds. As well, immunohistochemical analysis of murine CV papillae showed that two adiponectin receptors, ADIPOR1 and T-cadherin, are localized to subsets of TRCs. Immunofluorescence for T-cadherin was primarily co-localized with the Type 2 TRC marker phospholipase C β2, suggesting that adiponectin signaling could impact sweet, bitter, or umami taste signaling. However, adiponectin null mice showed no differences in behavioral lick responsiveness compared with wild-type controls in brief-access lick testing. AAV-mediated overexpression of adiponectin in the salivary glands of adiponectin null mice did result in a small but significant increase in behavioral lick responsiveness to the fat emulsion Intralipid. Together, these results suggest that salivary adiponectin can affect TRC function, although its impact on taste responsiveness and peripheral taste coding remains unclear.  

5.2629           CRISPR-Cas9-Mediated Genome Editing Increases Lifespan and Improves Motor Deficits in a Huntington’s Disease Mouse Model

Ekman, F.K., Ojala, D.S., Adil, M.M., Lopez, P.A., Schaffer, D.V. and Gaj, T. Molecular Therapy-Nucleic Acids, 17, 829-839 (2019)   Huntington’s disease (HD) is a currently incurable and, ultimately, fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion within exon 1 of the huntingtin (HTT) gene, which results in the production of a mutant protein that forms inclusions and selectively destroys neurons in the striatum and other adjacent structures. The RNA-guided Cas9 endonuclease from CRISPR-Cas9 systems is a versatile technology for inducing DNA double-strand breaks that can stimulate the introduction of frameshift-inducing mutations and permanently disable mutant gene function. Here, we show that the Cas9 nuclease from Staphylococcus aureus, a small Cas9 ortholog that can be packaged alongside a single guide RNA into a single adeno-associated virus (AAV) vector, can be used to disrupt the expression of the mutant HTT gene in the R6/2 mouse model of HD following its in vivo delivery to the striatum. Specifically, we found that CRISPR-Cas9-mediated disruption of the mutant HTT gene resulted in a ∼50% decrease in neuronal inclusions and significantly improved lifespan and certain motor deficits. These results thus illustrate the potential for CRISPR-Cas9 technology to treat HD and other autosomal dominant neurodegenerative disorders caused by a trinucleotide repeat expansion via in vivo genome editing.  

5.2630           Neurite orientation dispersion and density imaging reveals white matter and hippocampal microstructure changes produced by Interleukin-6 in the TgCRND8 mouse model of amyloidosis

Colon-Perez, L.M., Ibanez, K.R., Suarez, M., Torroella, K., Acuna, K., Ofori, E., Levites, Y., Vaillancourt, D.E., Golde, T.E., Chakrabarty, P. and Febo, M. NeuroImage, 202, 116138 (2019)   Extracellular β-amyloid (Aβ) plaque deposits and inflammatory immune activation are thought to alter various aspects of tissue microstructure, such as extracellular free water, fractional anisotropy and diffusivity, as well as the density and geometric arrangement of axonal processes. Quantifying these microstructural changes in Alzheimer’s disease and related neurodegenerative dementias could serve to monitor or predict disease course. In the present study we used high-field diffusion magnetic resonance imaging (dMRI) to investigate the effects of Aβ and inflammatory interleukin-6 (IL6), alone or in combination, on in vivo tissue microstructure in the TgCRND8 mouse model of Alzheimer’s-type Aβ deposition. TgCRND8 and non-transgenic (nTg) mice expressing brain-targeted IL6 or enhanced glial fibrillary protein (EGFP controls) were scanned at 8 months of age using a 2-shell, 54-gradient direction dMRI sequence at 11.1 T. Images were processed using the diffusion tensor imaging (DTI) model or the neurite orientation dispersion and density imaging (NODDI) model. DTI and NODDI processing in TgCRND8 mice revealed a microstructure pattern in white matter (WM) and hippocampus consistent with radial and longitudinal diffusivity deficits along with an increase in density and geometric complexity of axonal and dendritic processes. This included reduced FA, mean, axial and radial diffusivity, and increased orientation dispersion (ODI) and intracellular volume fraction (ICVF) measured in WM and hippocampus. IL6 produced a ‘protective-like’ effect on WM FA in TgCRND8 mice, observed as an increased FA that counteracted a reduction in FA observed with endogenous Aβ production and accumulation. In addition, we found that ICVF and ODI had an inverse relationship with the functional connectome clustering coefficient. The relationship between NODDI and graph theory metrics suggests that currently unknown microstructure alterations in WM and hippocampus are associated with diminished functional network organization in the brain.  

5.2631           Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I

Gomez-Ospina, N., Scharenberg, S.G., Mostrel, N., Bak, R.O., Mantri, S., Quadros, R.M. et al Nature Communications, 10:4045 (2019)   Lysosomal enzyme deficiencies comprise a large group of genetic disorders that generally lack effective treatments. A potential treatment approach is to engineer the patient’s own hematopoietic system to express high levels of the deficient enzyme, thereby correcting the biochemical defect and halting disease progression. Here, we present an efficient ex vivo genome editing approach using CRISPR-Cas9 that targets the lysosomal enzyme iduronidase to the CCR5 safe harbor locus in human CD34+ hematopoietic stem and progenitor cells. The modified cells secrete supra-endogenous enzyme levels, maintain long-term repopulation and multi-lineage differentiation potential, and can improve biochemical and phenotypic abnormalities in an immunocompromised mouse model of Mucopolysaccharidosis type I. These studies provide support for the development of genome-edited CD34+ hematopoietic stem and progenitor cells as a potential treatment for Mucopolysaccharidosis type I. The safe harbor approach constitutes a flexible platform for the expression of lysosomal enzymes making it applicable to other lysosomal storage disorders.  

5.2632           Long-term Therapeutic Efficacy of Intravenous AAV-Mediated Hamartin Replacement in Mouse Model of Tuberous Sclerosis Type 1

Prabhakar, S., Cheah, P.S, Zhang, X., Zinter, M., Gianatasio, M., Hudry, E., Bronson, R.T., Kwiatkowski, D.J., Stemmer-Rachamimov, A., Maguire, C.A., Sena-Esteves, M., Tannous, B.A. and Breakfield, X.O. Molecular Therapy-Methods & Clin. Develop., 15, 18-26 (2019)   Tuberous sclerosis complex (TSC) is a tumor suppressor syndrome caused by mutations in TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins act as a complex that inhibits mTOR-mediated cell growth and proliferation. Loss of either protein leads to overgrowth in many organs, including subependymal nodules, subependymal giant cell astrocytomas and cortical tubers in human brain. Neurologic manifestations in TSC include intellectual disability, autism, hydrocephalus and epilepsy. In a stochastic mouse model of TSC1 brain lesions, complete loss of Tsc1 is achieved in homozygous Tsc1-floxed mice in a subpopulation of neural cells in the brain by intracerebroventricular (ICV) injection at birth of an adeno-associated virus vector (AAV) encoding Cre recombinase. This results in median survival of 38 days and brain pathology, including subependymal lesions and enlargement of neuronal cells. Remarkably, when these mice were injected intravenously on day 21 with an AAV9 vector encoding hamartin, most survived at least up to 429 days in apparently healthy condition with marked reduction in brain pathology. Thus, a single intravenous administration of an AAV vector encoding hamartin restored protein function in enough cells in the brain to extend lifespan in this TSC1 mouse model.  

5.2633           The duck EB66® cell substrate reveals a novel retrotransposon

Perugi, F., Freslon-Evain, C., Batard, L., Guillet, P. and Schwamborn, K. Biologicals, 61, 22-31 (2019)   During the establishment of the duck EB66® cell line as a new cell substrate for vaccine production in industry, a very low level of reverse transcriptase (RT) activity was detected in the culture supernatant by product-enhanced RT assay but a whole battery of tests failed to evidence infectious particles. Results from extensive biochemical and physical testing demonstrated that RT activity was associated to an intracellular, non-enveloped and dense structure different from an infectious retroviruses. In silico analysis of Anas platyrhynchos genome revealed that the most likely candidates for encoding a ribonucleoprotein (RNP)-associated RT were nine copies of chicken repeat 1 (CR1)-like elements, belonging to the non-long terminal repeat retrotransposons. The presence of the full length Anas platyrhynchos chicken repeat 1-like sequence (APCR1) was confirmed in EB66® cells and the related ribonucleic acid was present in the RT-containing fraction of EB66® cells.  

5.2634           Plant-produced Bluetongue chimaeric VLP vaccine candidates elicit serotype-specific immunity in sheep

Mokoena, N.B., Moetlhoa, B., Rutkowska, D.A., Mamputha, S., Dibakwane, V.S., Tsekoa, T.L. and O’Kennedy, M.M. Vaccine, 37, 6068-6075 (2019)   Bluetongue (BT) is a hemorrhagic non-contagious, biting midge-transmitted disease of wild and domestic ruminants that is caused by bluetongue virus (BTV). Annual vaccination plays a pivotal role in BT disease control in endemic regions. Due to safety concerns of the current BTV multivalent live attenuated vaccine (LAV), a safe efficacious new generation subunit vaccine such as a plant-produced BT virus-like particle (VLP) vaccine is imperative. Previously, homogenous BTV serotype 8 (BTV-8) VLPs were successfully produced in Nicotiana benthamiana plants and provided protective immunity in sheep. In this study, combinations of BTV capsid proteins from more than one serotype were expressed and assembled to form chimaeric BTV-3 and BTV-4 VLPs in N. benthamiana plants. The assembled homogenous BTV-8, as well as chimaeric BTV-3 and chimaeric BTV-4 VLP serotypes, were confirmed by SDS-PAGE, Transmission Electron microscopy (TEM) and protein confirmation using liquid chromatography-mass spectrometry (LC-MS/MS) based peptide sequencing. As VP2 is the major determinant eliciting protective immunity, the percentage coverage and number of unique VP2 peptides detected in assembled chimaeric BT VLPs were used as a guide to assemble the most appropriate chimaeric combinations. Both plant-produced chimaeric BTV-3 and BTV-4 VLPs were able to induce long-lasting serotype-specific neutralizing antibodies equivalent to the monovalent LAV controls. Antibody levels remained high to the end of the trial. Combinations of homogenous and chimaeric BT VLPs have great potential as a safe, effective multivalent vaccine with the ability to distinguish between vaccinated and infected individuals (DIVA) due to the absence of non-structural proteins.  

5.2635           Membrane Protein of Human Coronavirus NL63 Is Responsible for Interaction with the Adhesion Receptor

Naskalska, A., Dabrowska. A., Szczepanski, A., Milewska, A., Jasik, K.P. and Pyrc, K.

1. Virol., 93(19), e00355-19 (2019)

  Human coronavirus NL63 (HCoV-NL63) is a common respiratory virus that causes moderately severe infections. We have previously shown that the virus uses heparan sulfate proteoglycans (HSPGs) as the initial attachment factors, facilitating viral entry into the cell. In the present study, we show that the membrane protein (M) of HCoV-NL63 mediates this attachment. Using viruslike particles lacking the spike (S) protein, we demonstrate that binding to the cell is not S protein dependent. Furthermore, we mapped the M protein site responsible for the interaction with HSPG and confirmed its relevance using a viable virus. Importantly, in silico analysis of the region responsible for HSPG binding in different clinical isolates and the Amsterdam I strain did not exhibit any signs of cell culture adaptation

5.2636           CCN1–Yes-Associated Protein Feedback Loop Regulates Physiological and Pathological Angiogenesis

Lee, S., Ahad, A., Luu, M., Moon, S., Caesar, J., Cardosa, W.V., Grant, M.B. and Chaqour, B. Mol. Cell. Biol., 39(18), e00107-19 (2019)   Cellular communication network factor 1 (CCN1) is a dynamically expressed, matricellular protein required for vascular development and tissue repair. The CCN1 gene is a presumed target of Yes-associated protein (YAP), a transcriptional coactivator that regulates cell growth and organ size. Herein, we demonstrate that the CCN1 promoter is indeed a direct genomic target of YAP in endothelial cells (ECs) of new blood vessel sprouts and that YAP deficiency in mice downregulates CCN1 and alters cytoskeletal and mitogenic gene expression. Interestingly, CCN1 overexpression in cultured ECs inactivates YAP in a negative feedback and causes its nuclear exclusion. Accordingly, EC-specific deletion of the CCN1 gene in mice mimics a YAP gain-of-function phenotype, characterized by EC hyperproliferation and blood vessel enlargement. CCN1 brings about its effect by providing cells with a soft compliant matrix that creates YAP-repressive cytoskeletal states. Concordantly, pharmacological inhibition of cell stiffness recapitulates the CCN1 deletion vascular phenotype. Furthermore, adeno-associated virus-mediated expression of CCN1 reversed the pathology of YAP hyperactivation and the subsequent aberrant growth of blood vessels in mice with ischemic retinopathy. Our studies unravel a new paradigm of functional interaction between CCN1 and YAP and underscore the significance of their interplay in the pathogenesis of neovascular diseases.  

5.2637           Trehalose promotes the survival of random-pattern skin flaps by TFEB mediated autophagy enhancement

Wu, H., Chen, H., Zheng, Z., Li, J., Ding, J., Juang, Z., Jia, C., Shen, Z., Bao, G., Wu, L., Al Mamun, A., Xu, H., Gao, W. and Zhou, K. Cell Death & Disease, 10:483 (2019)   Random-pattern skin flaps are commonly used and valuable tools in reconstructive surgery, however, post-operative random skin flap necrosis remains a major and common complication. Previous studies have suggested that activating autophagy, a major pathway for degradation of intracellular waste, may improve flap survival. In this study, we investigated whether trehalose, a novel and potent autophagy activator, improves random skin flap viability. Our results demonstrated that trehalose significantly improves viability, augments blood flow, and decreases tissue edema. Furthermore, we found that trehalose leads to increased angiogenesis, decreased apoptosis, and reduced oxidative stress. Using immunohistochestry and western blot, we demonstrated that trehalose augments autophagy, and that inhibition of autophagy augmentation using 3MA significantly blunted the aforementioned benefits of trehalose therapy. Mechanistically, we showed that trehalose’s autophagy augmentation is mediated by activation and nuclear translocation of TFEB, which may be due to inhibition of Akt and activation of the AMPK-SKP2-CARM1 signaling pathway. Altogether, our results established that trehalose is a potent agent capable for significantly increasing random-pattern skin flap survival by augmenting autophagy and subsequently promoting angiogenesis, reducing oxidative stress, and inhibiting cell death.  

5.2638           Astroglial dysfunctions drive aberrant synaptogenesis and social behavioral deficits in mice with neonatal exposure to lengthy general anesthesia

Zhou, B., Chen, l., Liao, P., Huang, L., Chen, Z., Liao, D., Yang, L., Wang, j., Yu, G., Wang, L., Zhang, J., Zuo, Y., Liu, J. and Jiang, R. PloS Biology, 17(8), e3000086 (2019)   Lengthy use of general anesthetics (GAs) causes neurobehavioral deficits in the developing brain, which has raised significant clinical concerns such that the United States Food and Drug Administration (FDA) is warning on the use of GAs in children younger than 3 years. However, the molecular and cellular mechanisms for GAs-induced neurotoxicity remain largely unknown. Here, we report that sevoflurane (Sevo), a commonly used GA in pediatrics, caused compromised astrocyte morphogenesis spatiotemporally correlated to synaptic overgrowth, with reduced synaptic function in developing cortex in a regional-, exposure-length-, and age-specific manner. Sevo disrupted astrocyte Ca²⁺ homeostasis both acutely and chronically, which led to the down-regulation of Ezrin, an actin-binding membrane-bound protein, which we found was critically involved in astrocyte morphogenesis in vivo. Importantly, overexpression of astrocyte Ezrin rescued astrocytic and neuronal dysfunctions and fully corrected deficits in social behaviors in developing mice with lengthy Sevo exposure. Our data uncover that, in addition to neurons, astrocytes may represent important targets for GAs to exert toxic effects and that astrocyte morphological integrity is crucial for synaptogenesis and neurological behaviors.  

5.2639           Production of adeno-associated virus vectors for in vitro and in vivo applications

Kimura, T., Ferran, B ., Tsukahara, Y., Shang, Q., Desai, S., Fedoce, A., Pimentel, D.R., Luptak, I., Adachi, T., Ido, Y., matsui, R. and Bachschmid, M.M. Scientific Reports, 9:3601 (2019)   Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Of note, we achieved titers of 10¹⁰–10¹¹ viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. Our study provides an improved protocol for a more economical and efficient purified AAV preparation.  

5.2640           Circular RNA CircFndc3b modulates cardiac repair after myocardial infarction via FUS/VEGF-A axis

Naga, V., Garikipati, N.S., Verma, S.K., Cheng, Z., Liang, D., Truongcao, M.M., Cimini, M., Yue, Y., Huang, G. et al Nature Communications, 10:4317 (2019)   Circular RNAs are generated from many protein-coding genes, but their role in cardiovascular health and disease states remains unknown. Here we report identification of circRNA transcripts that are differentially expressed in post myocardial infarction (MI) mouse hearts including circFndc3b which is significantly down-regulated in the post-MI hearts. Notably, the human circFndc3b ortholog is also significantly down-regulated in cardiac tissues of ischemic cardiomyopathy patients. Overexpression of circFndc3b in cardiac endothelial cells increases vascular endothelial growth factor-A expression and enhances their angiogenic activity and reduces cardiomyocytes and endothelial cell apoptosis. Adeno-associated virus 9 -mediated cardiac overexpression of circFndc3b in post-MI hearts reduces cardiomyocyte apoptosis, enhances neovascularization and improves left ventricular functions. Mechanistically, circFndc3b interacts with the RNA binding protein Fused in Sarcoma to regulate VEGF expression and signaling. These findings highlight a physiological role for circRNAs in cardiac repair and indicate that modulation of circFndc3b expression may represent a potential strategy to promote cardiac function and remodeling after MI.  

5.2641           Correction: Permissivity of Primary Human Hepatocytes and Different Hepatoma Cell Lines to Cell Culture Adapted Hepatitis C Virus

Helle, F., Brochot, E., Fournier, C., Descamps, V., Izquierdo, L., Hoffmann, T.W. et al PloS One, 14(9), e0223022 (2019)   The cell line HepG2-CD81 appears incorrectly throughout the article [1]. The correct cell line is HuH-7. This correction is in line with the correction for Belouzard et al., “Entry and release of hepatitis C virus in polarized human hepatocytes,” [2] whose authors provided the cell line for analysis. Results concerning the HepG2-CD81 permissivity to HCV (Figure 6 in [1]) and the HCV virions production by HepG2-CD81 cells (Figure 7 in [1]) must be interpreted as permissivity of a HuH-7 clone to HCV and HCV virions production by a HuH-7 clone, respectively. There are errors in Fig 6, “Infection of hepatoma cell lines with cell culture adapted HCV”, and Fig 7, “Profiles of density of HCV produced in different hepatoma cell lines”, which reference the HepG2-CD81 cell line. In addition, there are errors in the captions for Figs 6 and 7, which reference the HepG2-CD81 cell line. Please see the corrected Figs 6 and 7, and their corrected captions, here.  

5.2642           Distinct Mechanisms for Visual and Motor-Related Astrocyte Responses in Mouse Visual Cortex

Slezak, M., Kandler, S., Van veldhoven, P.P., Van den Haute, C., Bonin, V. and Holt, M.G. Current Biol., 29, 3120-3127 (2019)   Astrocytes are a major cell type in the mammalian nervous system, are in close proximity to neurons, and show rich Ca²⁺ activity thought to mediate cellular outputs. Astrocytes show activity linked to sensory [1, 2] and motor [3, 4] events, reflecting local neural activity and brain-wide neuromodulatory inputs. Sensory responses are highly variable [5, 6, 7, 8, 9, 10], which may reflect interactions between distinct input types [6, 7, 9]. However, the diversity of inputs generating astrocyte activity, particularly during sensory stimulation and behavior, is not fully understood [11, 12]. Using a combination of Ca²⁺ imaging, a treadmill assay, and visual stimulation, we examined the properties of astrocyte activity in mouse visual cortex associated with motor or sensory events. Consistent with previous work, motor activity activated astrocytes across the cortex with little specificity, reflecting a diffuse neuromodulatory mechanism. In contrast, moving visual stimuli generated specific activity patterns that reflected the stimulus' trajectory within the visual field, precisely as one would predict if astrocytes reported local neural activity. Visual responses depended strongly on behavioral state, with astrocytes showing high amplitude Ca²⁺ transients during locomotion and little activity during stillness. Furthermore, the amplitudes of visual responses were highly correlated with pupil size, suggesting a role of arousal. Interestingly, while depletion of cortical noradrenaline abolished locomotor responses, visual responses were only reduced in amplitude and their spatiotemporal organization remained intact, suggesting two distinct types of inputs underlie visual responses. We conclude that cortical astrocytes integrate local sensory information and behavioral state, suggesting a role in information processing.  

5.2643           The Protein Phosphatase 1 Complex Is a Direct Target of AKT that Links Insulin Signaling to Hepatic Glycogen Deposition

Li, Q., Zhao, Q., Zhang, J., Chen, Y., Xu, L. and Li, P. Cell Reports, 28, 3406-3422 (2019)   Insulin-stimulated hepatic glycogen synthesis is central to glucose homeostasis. Here, we show that PPP1R3G, a regulatory subunit of protein phosphatase 1 (PP1), is directly phosphorylated by AKT. PPP1R3G phosphorylation fluctuates with fasting-refeeding cycle and is required for insulin-stimulated dephosphorylation, i.e., activation of glycogen synthase (GS) in hepatocytes. In this study, we demonstrate that knockdown of PPP1R3G significantly inhibits insulin response. The introduction of wild-type PPP1R3G, and not phosphorylation-defective mutants, increases hepatic glycogen deposition, blood glucose clearance, and insulin sensitivity in vivo. Mechanistically, phosphorylated PPP1R3G displays increased binding for, and promotes dephosphorylation of, phospho-GS. Furthermore, PPP1R3B, another regulatory subunit of PP1, binds to the dephosphorylated GS, thereby relaying insulin stimulation to hepatic glycogen deposition. Importantly, this PP1-mediated signaling cascade is independent of GSK3. Therefore, we reveal a regulatory axis consisting of insulin/AKT/PPP1R3G/PPP1R3B that operates in parallel to the GSK3-dependent pathway, controlling glycogen synthesis and glucose homeostasis in insulin signaling.  

5.2644           Efficient CRISPR/Cas9-Mediated Gene Knockin in Mouse Hematopoietic Stem and Progenitor Cells

Tran, N.T., Sommermann, T., Graf, R., Kühn, R., Rajewsky, K. and Chu, V.T. Cell Reports, 28, 3510-3522 (2019)   Mutations accumulating in hematopoietic stem and progenitor cells (HSPCs) during development can cause severe hematological disorders. Modeling these mutations in mice is essential for understanding their functional consequences. Here, we describe an efficient CRISPR/Cas9-based system to knock in and repair genes in mouse HSPCs. CRISPR/Cas9 ribonucleoproteins, in combination with recombinant adeno-associated virus (rAAV)-DJ donor templates, led to gene knockin efficiencies of up to 30% in the Lmnb1 and Actb loci of mouse HSPCs in vitro. The targeted HSPCs engraft and reconstitute all immune cell lineages in the recipient mice. Using this approach, we corrected a neomycin-disrupted Rag2 gene. The Rag2-corrected HSPCs restore B and T cell development in vivo, confirming the functionality of the approach. Our method provides an efficient strategy to study gene function in the hematopoietic system and model hematological disorders in vivo, without the need for germline mutagenesis.    

5.2645           Inducing Different Neuronal Subtypes from Astrocytes in the Injured Mouse Cerebral Cortex

Mattugini, N., Bocchi, R., Scheuss, V., Russo, G.L., Torper, O., Lao, C.L. and Götz, M. Neuron, 103, 1086-1096 (2019)   Astrocytes are particularly promising candidates for reprogramming into neurons, as they maintain some of the original patterning information from their radial glial ancestors. However, to which extent the position of astrocytes influences the fate of reprogrammed neurons remains unknown. To elucidate this, we performed stab wound injury covering an entire neocortical column, including the gray matter (GM) and white matter (WM), and targeted local reactive astrocytes via injecting FLEx switch (Cre-On) adeno-associated viral (AAV) vectors into mGFAP-Cre mice. Single proneural factors were not sufficient for adequate reprogramming, although their combination with the nuclear receptor-related 1 protein (Nurr1) improved reprogramming efficiency. Nurr1 and Neurogenin 2 (Ngn2) resulted in high-efficiency reprogramming of targeted astrocytes into neurons that develop lamina-specific hallmarks, including the appropriate long-distance axonal projections. Surprisingly, in the WM, we did not observe any reprogrammed neurons, thereby unveiling a crucial role of region- and layer-specific differences in astrocyte reprogramming.  

5.2646           Gene manipulation in liver ductal organoids by optimized recombinant adeno-associated virus vectors

Wei, J., Ran, G., Wang, X., Jiang, N., Liang, J., Lin, X., Ling, C. and Zhao, B.

1. Biol. Chem., 294(38), 14096-14104 (2019)

  Understanding the mechanism of how liver ductal cells (cholangiocytes) differentiate into hepatocytes would permit liver-regenerative medicine. Emerging liver ductal organoids provide an ex vivo system to investigate cholangiocyte-to-hepatocyte differentiation. However, as current gene manipulation methods require organoid dissociation into single cells and have only low efficiency, it is difficult to dissect specific gene functions in these organoids. Here we developed the adeno-associated virus (AAV) vector AAV-DJ as a powerful tool to transduce mouse and human liver ductal organoids. Via AAV-DJ–mediated up- or down-regulation of target genes, we successfully manipulated cholangiocyte-to-hepatocyte differentiation. We induced differentiation by overexpressing the hepatocyte-specifying regulator hepatocyte nuclear factor 4α (HNF4α) and blocked differentiation by stimulating Notch signaling or interfering with Smad signaling. Further screening for transcriptional factors critical for cholangiocyte-to-hepatocyte differentiation identified HOP homeobox (HOPX), T-box 15 (TBX15), and transcription factor CP2-like 1 (TFCP2L1) as master regulators. We conclude that this highly efficient and convenient gene manipulation system we developed could facilitate investigation into genes involved in cell lineage transitions and enable application of engineered organoids in regenerative medicine.  

5.2647           Achieving tight control of a photoactivatable Cre recombinase gene switch: new design strategies and functional characterization in mammalian cells and rodent

Meador, k., Wysoczynski, C.L., Norris, A.J., Aoto, j., Bruchas, M.R. and Tucker, C.L. Nucleic Acids Res., 47(17), e97 (2019)   A common mechanism for inducibly controlling protein function relies on reconstitution of split protein fragments using chemical or light-induced dimerization domains. A protein is split into fragments that are inactive on their own, but can be reconstituted after dimerization. As many split proteins retain affinity for their complementary half, maintaining low activity in the absence of an inducer remains a challenge. Here, we systematically explore methods to achieve tight regulation of inducible proteins that are effective despite variation in protein expression level. We characterize a previously developed split Cre recombinase (PA-Cre2.0) that is reconstituted upon light-induced CRY2-CIB1 dimerization, in cultured cells and in vivo in rodent brain. In culture, PA-Cre2.0 shows low background and high induced activity over a wide range of expression levels, while in vivo the system also shows low background and sensitive response to brief light inputs. The consistent activity stems from fragment compartmentalization that shifts localization toward the cytosol. Extending this work, we exploit nuclear compartmentalization to generate light-and-chemical regulated versions of Cre recombinase. This work demonstrates in vivo functionality of PA-Cre2.0, describes new approaches to achieve tight inducible control of Cre DNA recombinase, and provides general guidelines for further engineering and application of split protein fragments.    

5.2648           Production of rVSV-ZEBOV in serum-free suspension culture of HEK 293SF cells

Gelinas, J-F., Azizi, H., Kiesslich, S., Lanthier, S., Perdersen, J., Cchahal, PS., Ansorge, S., Kobinger, G., Gilbert, R.and Kamen, A.A. Vaccine, 37, 6624-6632 (2019)   Ebola virus disease is an urgent international priority. Promising results for several vaccine candidates have been reported in non-human primate studies and clinical trials with the most promising being the rVSV-ZEBOV vaccine. In this study, we sought to produce rVSV-ZEBOV in HEK 293SF cells in suspension and serum-free media. The purpose of this study was to establish a process using the HEK 293SF production platform, optimise the production titre, demonstrate scalability and the efficiency of the generated material to elicit an immune reaction in an animal model. Critical process parameters were evaluated to maximize production yield and process robustness and the following operating conditions: 1–2 × 10⁶ cells/mL grown in HyClone HyCell TransFx-H media infected at an MOI of 0.001 with a temperature shift to 34 °C during the production phase and a harvest of the product after 48 h. Using these conditions, scalability in a 3.5 L controlled bioreactor was shown reaching a titre of 1.19 × 10⁸ TCID₅₀/mL at the peak of production, the equivalent of 4165 doses of vaccine per litre. The produced virus was shown to be thermostable in the culture media and, when concentrated, purified and administered to mice, demonstrated the ability to induce a ZEBOV-specific immune response.  

5.2649           Interhemispheric Connectivity Potentiates the Basolateral Amygdalae and Regulates Social Interaction and Memory

Huang, T-N., Hsu, T-T., Lin, M-H., Tao, M-H., Lin, J.Y. and Hsueh, Y-P. Cell Reports, 29, 34-48 (2019)   Impaired interhemispheric connectivity is commonly found in various psychiatric disorders, although how interhemispheric connectivity regulates brain function remains elusive. Here, we use the mouse amygdala, a brain region that is critical for social interaction and fear memory, as a model to demonstrate that contralateral connectivity intensifies the synaptic response of basolateral amygdalae (BLA) and regulates amygdala-dependent behaviors. Retrograde tracing and c-FOS expression indicate that contralateral afferents widely innervate BLA non-randomly and that some BLA neurons innervate both contralateral BLA and the ipsilateral central amygdala (CeA). Our optogenetic and electrophysiological studies further suggest that contralateral BLA input results in the synaptic facilitation of BLA neurons, thereby intensifying the responses to cortical and thalamic stimulations. Finally, pharmacological inhibition and chemogenetic disconnection demonstrate that BLA contralateral facilitation is required for social interaction and memory. Our study suggests that interhemispheric connectivity potentiates the synaptic dynamics of BLA neurons and is critical for the full activation and functionality of amygdalae.  

5.2650           Conformational Changes and Nuclear Entry of Porcine Circovirus without Disassembly

Wang, H., Zhang, K., Lin, C., Zhou, J., Jin, Y., Dong, W., Gu, J. and Zhou, J.

1. Virol., 93(20), e00824-19 (2019)

  A relatively stable and flexible capsid is critical to the viral life cycle. However, the capsid dynamics and cytosol trafficking of porcine circovirus type 2 (PCV2) during its infectious cycle are poorly understood. Here, we report the structural stability and conformation flexibility of PCV2 virions by genome labeling and the use of three monoclonal antibodies (MAbs) against the native capsid of PCV2. Genome labeling showed that the infectivity of the PCV2 virion was not affected by conjugation with deoxy-5-ethynylcytidine (EdC). Heat stability experiments indicated that PCV2 capsids started to disassemble at 65°C, causing binding incompetence for all antibodies, and the viral genome was released without capsid disassembly upon heating at 60°C. Antibody binding experiments with PCV2 showed that residues 186 to 192 were concealed in the early endosomes of epithelial PK-15 and monocytic 3D4/31 cells with or without chloroquine treatment and then exposed in PK-15 cytosol and the 3D4/31 nucleus. Viral propagation and localization experiments showed that PCV2 replication and cytosol trafficking were not significantly affected by microtubule depolymerization in monocytic 3D4/31 cells treated with nocodazole. These findings demonstrated that nuclear targeting of viral capsids involved conformational changes, the PCV2 genome was released from the assembled capsid, and the transit of PCV2 particles was independent of microtubules in 3D4/31 cells.  

5.2651           Neuromodulator Signaling Bidirectionally Controls Vesicle Numbers in Human Synapses

Patzke, C., Brockmann, M.M., Dai, J., Acuna, C., Rosenmund, C. and Südhof, T.C. Cell, 179, 498-513 (2019)   Neuromodulators bind to pre- and postsynaptic G protein-coupled receptors (GPCRs), are able to quickly change intracellular cyclic AMP (cAMP) and Ca²⁺ levels, and are thought to play important roles in neuropsychiatric and neurodegenerative diseases. Here, we discovered in human neurons an unanticipated presynaptic mechanism that acutely changes synaptic ultrastructure and regulates synaptic communication. Activation of neuromodulator receptors bidirectionally controlled synaptic vesicle numbers within nerve terminals. This control correlated with changes in the levels of cAMP-dependent protein kinase A-mediated phosphorylation of synapsin-1. Using a conditional deletion approach, we reveal that the neuromodulator-induced control of synaptic vesicle numbers was largely dependent on synapsin-1. We propose a mechanism whereby non-phosphorylated synapsin-1 “latches” synaptic vesicles to presynaptic clusters at the active zone. cAMP-dependent phosphorylation of synapsin-1 then removes the vesicles. cAMP-independent dephosphorylation of synapsin-1 in turn recruits vesicles. Synapsin-1 thereby bidirectionally regulates synaptic vesicle numbers and modifies presynaptic neurotransmitter release as an effector of neuromodulator signaling in human neurons.  

5.2652           Homologous Recombination-Based Genome Editing by Clade F AAVs Is Inefficient in the Absence of a Targeted DNA Break

Rogers, G.I., Chen, H-Y., Morales, H. and Cannon, P.M. Molecular Therapy, 27(10), 1726-1736 (2019)   Adeno-associated virus (AAV) vectors are frequently used as donor templates for genome editing by homologous recombination. Although modification rates are typically under 1%, they are greatly enhanced by targeted double-stranded DNA breaks (DSBs). A recent report described clade F AAVs mediating high-efficiency homologous recombination-based editing in the absence of DSBs. The clade F vectors included AAV9 and a series isolated from human hematopoietic stem and progenitor cells (HSPCs). We evaluated these vectors by packaging homology donors into AAV9 and an AAVHSC capsid and examining their ability to insert GFP at the CCR5 and AAVS1 loci in human HSPCs and cell lines. As a control, we used AAV6, which effectively edits HSPCs but only when combined with a targeted DSB. Each AAV vector promoted GFP insertion in the presence of matched CCR5 or AAVS1 zinc-finger nucleases (ZFNs), but none supported detectable editing in the absence of the nucleases. Rates of editing with ZFNs correlated with transduction efficiencies for each vector, implying no differences in the ability of donor sequences delivered by the different vectors to direct genome editing. Our results, therefore, do not support that clade F AAVs can perform high-efficiency genome editing in the absence of a DSB.  

5.2653           Restoration of visual function by transplantation of optogenetically engineered photoreceptors

Garita-Hernandez, m., Llampic, M., Chaffiol, A., Guibbal, L., Routet, F. et al Nature Communications, 10:4524 (2019)   A major challenge in the treatment of retinal degenerative diseases, with the transplantation of replacement photoreceptors, is the difficulty in inducing the grafted cells to grow and maintain light sensitive outer segments in the host retina, which depends on proper interaction with the underlying retinal pigment epithelium (RPE). Here, for an RPE-independent treatment approach, we introduce a hyperpolarizing microbial opsin into photoreceptor precursors from newborn mice, and transplant them into blind mice lacking the photoreceptor layer. These optogenetically-transformed photoreceptors are light responsive and their transplantation leads to the recovery of visual function, as shown by ganglion cell recordings and behavioral tests. Subsequently, we generate cone photoreceptors from human induced pluripotent stem cells, expressing the chloride pump Jaws. After transplantation into blind mice, we observe light-driven responses at the photoreceptor and ganglion cell levels. These results demonstrate that structural and functional retinal repair is possible by combining stem cell therapy and optogenetics.  

5.2654           Transient Receptor Potential V Channels Are Essential for Glucose Sensing by Aldolase and AMPK

Li, M., Zhang, C-S., Zong, Y., Xie, X-S., Hardie, D.G. and Lin, S-C. Cell Metabolism, 30, 508-524 (2019)   Fructose-1,6-bisphosphate (FBP) aldolase links sensing of declining glucose availability to AMPK activation via the lysosomal pathway. However, how aldolase transmits lack of occupancy by FBP to AMPK activation remains unclear. Here, we show that FBP-unoccupied aldolase interacts with and inhibits endoplasmic reticulum (ER)-localized transient receptor potential channel subfamily V, inhibiting calcium release in low glucose. The decrease of calcium at contact sites between ER and lysosome renders the inhibited TRPV accessible to bind the lysosomal v-ATPase that then recruits AXIN:LKB1 to activate AMPK independently of AMP. Genetic depletion of TRPVs blocks glucose starvation-induced AMPK activation in cells and liver of mice, and in nematodes, indicative of physical requirement of TRPVs. Pharmacological inhibition of TRPVs activates AMPK and elevates NAD⁺ levels in aged muscles, rejuvenating the animals’ running capacity. Our study elucidates that TRPVs relay the FBP-free status of aldolase to the reconfiguration of v-ATPase, leading to AMPK activation in low glucose.  

5.2655           Gene Therapy Correction of Aldehyde Dehydrogenase 2 Deficiency

Matsumura, Y., Stiles, K.M., Reid, J., Frenk, E.Z., Cronin, S., Pagovich, O.E. and Crystal, R.G. Molecular Therapy-Methods Clin. Develop., 15, 72-82 (2019)   Aldehyde dehydrogenase 2 (ALDH2) deficiency causes “Asian flush syndrome,” presenting as alcohol-induced facial flushing, tachycardia, nausea, and headaches. One of the most common hereditary enzyme deficiencies, it affects 35-40% of East Asians and 8% of the world population. ALDH2 is the key enzyme in ethanol metabolism; with ethanol challenge the common ALDH2*2 (E487K) mutation results in accumulation of toxic acetaldehyde, ALDH2*2 heterozygotes have increased risk for upper digestive tract cancers, compounded by smoking and drinking alcohol. We hypothesized that a one-time administration of an adeno-associated virus (AAV) gene transfer vector expressing the human ALDH2 coding sequence (AAVrh.10hALDH2) would correct the deficiency state. AAVrh.10hALDH2 was administered intravenously to Aldh2 knockout (Aldh2-/-) and Aldh2 E487K knockin homozygous (Aldh2E487K+/+) mice. Following acute ethanol ingestion, untreated ALDH2-deficient mice had elevated acetaldehyde levels and performed poorly in behavioral tests. In contrast, treated Aldh2-/- and Aldh2E487K+/+ mice had lower serum acetaldehyde levels and improved behavior. Thus, in vivo AAV-mediated ALDH2 therapy may reverse the deficiency state in ALDH2*2 individuals, eliminating the Asian flush syndrome and reducing the risk for associated disorders.  

5.2656           Targeting adeno-associated virus vectors for local delivery to fractures and systemic delivery to the skeleton

Lee, L.R., Peacock, L., Lisowski, D., Munns, C.F. and Schindeler, A. Molecular Therapy-Methods Clin. Develop., 15, 101-111 (2019)   A panel of 18 recombinant adeno-associated virus (rAAV) variants, both natural and engineered, constitutively expressing Cre recombinase under the cytomegalovirus early enhancer/chicken β actin (CAG) promoter, were screened for their ability to transduce bone in Ai9 fluorescent reporter mice. Transgenic Cre-induced tdTomato expression served as a measure of transduction efficiency and alkaline phosphatase (AP) activity as an osteoblastic marker. Single injections of AAV8, AAV9, and AAV-DJ into midshaft tibial fractures yielded robust tdTomato expression in the callus. Next, the bone cell-specific promoters Sp7 and Col2.3 were tested to restrict Cre expression in an alternate model of systemic delivery by intravenous injection. Although CAG promoter constructs packaged into AAV8 produced high levels of tdTomato in the bone, liver, heart, spleen, and kidney, bone-specific promoter constructs restricted Cre expression to osseous tissues. AAV variants were further tested in vitro in a human osteoblast cell line (hFOB1.19), measuring GFP reporter expression by flow cytometry after 72 h. AAV2, AAV5, and AAV-DJ showed the highest transduction efficiency. In summary, we produced AAV vectors for selective and high-efficiency in vivo gene delivery to murine bone. The AAV8-Sp7-Cre vector has significant practical applications for inducing gene deletion postnatally in floxed mouse models.  

5.2657           Removal of Endotoxin from rAAV Samples Using a Simple Detergent-Based Protocol

Kondratova, L., Kondratov, O., Ragheb, R. and Zolotukhin, S. Molecular Therapy-Methods Clin. Develop., 15, 112-119 (2019)   Endotoxin is the most common contaminant found in protein samples. Even a small amount of endotoxin can induce strong allergic reaction and death of a host organism. Endotoxin is also often detected in recombinant adeno-associated virus (rAAV) stocks prepared in research laboratories using off-the-shelf reagents; purifying rAAV stocks from endotoxin using commercial reagents sometimes results in significant titer loss. The problem is exacerbated due to the recently expanded diversity of rAAV serotypes and capsid variants, which, due to their variable capsid surface charge, display differential affinity toward endotoxin. In this paper, we describe a simple universal protocol of purifying vector stocks irrespective of AAV serotype. The protocol is based on subjecting endotoxin-contaminated rAAV to mild detergent treatment, followed by repeated buffer-exchange washing and concentrating viral stock by low-speed centrifugation. Multiple assays were employed to test the physical and biological equivalency of the viral stocks before and after purification. The described protocol has been routinely utilized to purify vector stocks contaminated at levels as high as >1,000 endotoxin units (EU)/mL to produce viral vectors with practically undetectable levels of endotoxin (<2.5 EU/mL), with the titer’s recovery in the range of 50%–100%.  

5.2658           Biodistribution of adeno-associated virus type 2 with mutations in the capsid that contribute to heparan sulfate proteoglycan binding

Gorbatyuk, O.S:, Warrington Jr., K.H., Gorbatyuk, M.S., Zolotukhin, I., Lewin, A.S. and Muzyczka, N. Virus Res., 274, 197771, (2019)   We compared the phenotypes of three mutant AAV2 viruses containing mutations in arginine amino acids (R585, R588 and R484) previously shown to be involved in AAV2 heparan sulfate binding. The transduction efficiencies of wild type and mutant viruses were determined in the eye, the brain and peripheral organs following subretinal, striatal and intravenous injection, respectively, in mice and rats. We found that each of the three mutants (the single mutant R585A; the double mutant R585, 588A; and the triple mutant R585, 588, 484A) had a unique phenotype compared to wt and each other. R585A was completely defective for transducing peripheral organs via intravenous injection, suggesting that R585A may be useful for targeting peripheral organs by substitution of peptide ligands in the capsid surface. In the brain, all three mutants displayed widespread transduction, with the double mutant R585, 588A displaying the greatest spread and the greatest number of transduced neurons. The double mutant was also extremely efficient for retrograde transport, while the triple mutant was almost completely defective for retrograde transport. This suggested that R484 may be directly involved in interaction with the transport machinery. Finally, the double mutant also displayed improved transduction of the eye compared to wild type and the other mutants.  

5.2659           AAV gene therapy utilizing glycosylation-independent lysosomal targeting tagged GAA in the hypoglossal motor system of Pompe mice

Doyle, B.M., Turner, S.M.F., Sunshine, M.D., Doerfler, P.A., Poirier, A.F., Vaught, L.A., Jorgensen,

M.L.,Falk, D.J., Byrne, B.J. and Fuller, D.D.

Molecular Therapy-Methods Clin. Develop., 15, 194-203 (2019)

  Pompe disease is caused by mutations in the gene encoding the lysosomal glycogen metabolizing enzyme, acid-alpha glucosidase (GAA). Tongue myofibers and hypoglossal motoneurons appear particularly susceptible in Pompe disease. Here we used intramuscular delivery of adeno-associated virus serotype 9 (AAV9) for targeted delivery of an enhanced form of GAA to tongue myofibers and motoneurons in 6-mo old Pompe (Gaa^-/-) mice. We hypothesized that addition of a glycosylation-independent lysosomal targeting tag to the protein would result in enhanced expression in tongue (hypoglossal) motoneurons when compared to the untagged GAA. Mice received an injection into the base of the tongue with AAV9 encoding either the tagged or untagged enzyme; tissues were harvested 4 months later. Both AAV9 constructs effectively drove GAA expression in lingual myofibers and hypoglossal motoneurons. However, mice treated with the AAV9 construct encoding the modified acid-alpha glucosidase enzyme had a >200% increase in the number GAA positive motoneurons as compared to the untagged GAA (p<0.008). Our results confirm that tongue delivery of AAV9 encoding GAA can effectively target tongue myofibers and associated motoneurons in Pompe (Gaa^-/-) mice and indicate that the effectiveness of this approach can be improved by addition of the glycosylation-independent lysosomal targeting tag.  

5.2660           Nanobody-enhanced targeting of AAV gene therapy vectors

Eichhoff, A.M., Börner, K., Albrecht, B., Scäfer, W., Baum, N., Haag, F., Körbelin, J., Trepel, M., Braren, D., Adriouch, S. and Koch-Nolte, F. Molecular Therapy-Methods Clin. Develop., 15, 211-220 (2019)   A limiting factor for the use of AAV as vectors in gene therapy is the broad tropism of AAV serotypes, i.e. the parallel infection of several cell types. Nanobodies are single immunoglobulin variable domains from heavy chain antibodies that naturally occur in camelids. Their small size and high solubility allows easy reformatting into fusion proteins. Here we show that a membrane protein-specific nanobody can be inserted into a surface loop of the VP1 capsid protein of AAV2. Using three structurally distinct membrane proteins - a multispan ion channel, a single-span transmembrane protein, and a GPI-anchored ecto-enzyme - we show that this strategy can dramatically enhance the transduction of specific target cells by recombinant AAV2. Moreover we show that the nanobody VP1 fusion of AAV2 can be incorporated into the capsids of AAV1, AAV8, and AAV9, and thereby effectively redirect the target specificity of other AAV serotypes. Nanobody-mediated targeting provides a highly efficient AAV targeting strategy that is likely to open up new avenues for genetic engineering of cells.  

5.2661           De Novo Isolation & Affinity Maturation of yeast-displayed Virion-binding human fibronectin domains by flow cytometric screening against Virions

Heinzelman, P., Low, A., Simeon, R., Wright, G.A. and Chen, Z.

1. Biol. Eng., 13:76 (2019)

  Background The promise of biopharmaceuticals comprising one or more binding domains motivates the development of novel methods for de novo isolation and affinity maturation of virion-binding domains. Identifying avenues for overcoming the challenges associated with using virions as screening reagents is paramount given the difficulties associated with obtaining high-purity virus-associated proteins that retain the conformation exhibited on the virion surface. Results Fluorescence activated cell sorting (FACS) of 1.5 × 10⁷ clones taken from a naïve yeast surface-displayed human fibronectin domain (Fn3) against whole virions yielded two unique binders to Zika virions. Construction and FACS of site-directed binding loop mutant libraries based on one of these binders yielded multiple progeny clones with enhanced Zika-binding affinities. These affinity-matured clones bound Zika virions with low double- or single-digit nanomolar affinity in ELISA assays, and expressed well as soluble proteins in E. coli shake flask culture, with post-purification yields exceeding 10 mg/L. Conclusions FACS of a yeast-displayed binding domain library is an efficient method for de novo isolation of virion-binding domains. Affinities of isolated virion-binding clones are readily enhanced via FACS screening of mutant progeny libraries. Given that most binding domains are compatible with yeast display, the approach taken in this work may be broadly utilized for generating virion-binding domains against many different viruses for use in passive immunotherapy and the prevention of viral infection.  

5.2662           AAVR-Displaying Interfaces: Serotype-Independent Adeno-Associated Virus Capture and Local Delivery Systems

Kim, S-H., Lee, S., Lee, H., Cho, M., Schaffer, D.V. and Jang, J-H. Molecular Therapy-Nucleic Acids, 18, 432-443 (2019)   Interfacing gene delivery vehicles with biomaterials has the potential to play a key role in diversifying gene transfer capabilities, including localized, patterned, and controlled delivery. However, strategies for modifying biomaterials to interact with delivery vectors must be redesigned whenever new delivery vehicles and applications are explored. We have developed a vector-independent biomaterial platform capable of interacting with various adeno-associated viral (AAV) serotypes. A water-soluble, cysteine-tagged, recombinant protein version of the recently discovered multi-AAV serotype receptor (AAVR), referred to as cys-AAVR, was conjugated to maleimide-displaying polycaprolactone (PCL) materials using click chemistry. The resulting cys-AAVR-PCL system bound to a broad range of therapeutically relevant AAV serotypes, thereby providing a platform capable of modulating the delivery of all AAV serotypes. Intramuscular injection of cys-AAVR-PCL microspheres with bound AAV vectors resulted in localized and sustained gene delivery as well as reduced spread to off-target organs compared to a vector solution. This cys-AAVR-PCL system is thus an effective approach for biomaterial-based AAV gene delivery for a broad range of therapeutic applications.  

5.2663           Treatment of Experimental Autoimmune Encephalomyelitis by Sustained Delivery of Low-Dose IFN-α

Vasquez, m., Consuegra-Fernandez, M., Aranda, F., Jimenez, A., Tenesaca, S., Fernandez-Sendin, M., Gomar, C., Claudia, N.A., De Train, A., Caseres, N., Lasarte, J.J., Lozano, F. and Berraondo, P.

1. Immunol., 203, 696-704 (2019)

  Multiple sclerosis (MS) is a chronic autoimmune disease with no curative treatment. The immune regulatory properties of type I IFNs have led to the approval of IFN-β for the treatment of relapsing-remitting MS. However, there is still an unmet need to improve the tolerability and efficacy of this therapy. In this work, we evaluated the sustained delivery of IFN-α1, either alone or fused to apolipoprotein A-1 by means of an adeno-associated viral (AAV) system in the mouse model of myelin oligodendrocyte glycoprotein–induced experimental autoimmune encephalomyelitis. These in vivo experiments demonstrated the prophylactic and therapeutic efficacy of the AAV–IFN-α or AAV–IFN-α fused to apolipoprotein A-1 vectors in experimental autoimmune encephalomyelitis, even at low doses devoid of hematological or neurologic toxicity. The sustained delivery of such low-dose IFN-α resulted in immunomodulatory effects, consisting of proinflammatory monocyte and T regulatory cell expansion. Moreover, encephalitogenic T lymphocytes from IFN-α–treated mice re-exposed to the myelin oligodendrocyte glycoprotein peptide in vitro showed a reduced proliferative response and cytokine (IL-17A and IFN-γ) production, in addition to upregulation of immunosuppressive molecules, such as IL-10, IDO, or PD-1. In conclusion, the results of the present work support the potential of sustained delivery of low-dose IFN-α for the treatment of MS and likely other T cell–dependent chronic autoimmune disorders.  

5.2664           A reference map of murine cardiac transcription factor chromatin occupancy identifies dynamic and conserved enhancers

Akerberg, B.N., Gu, F., VanDusen, N.J., Zhang, X., Dong, R., Li, K., Zhang, B. et al Nature Communications, 104907 (2019)   Mapping the chromatin occupancy of transcription factors (TFs) is a key step in deciphering developmental transcriptional programs. Here we use biotinylated knockin alleles of seven key cardiac TFs (GATA4, NKX2-5, MEF2A, MEF2C, SRF, TBX5, TEAD1) to sensitively and reproducibly map their genome-wide occupancy in the fetal and adult mouse heart. These maps show that TF occupancy is dynamic between developmental stages and that multiple TFs often collaboratively occupy the same chromatin region through indirect cooperativity. Multi-TF regions exhibit features of functional regulatory elements, including evolutionary conservation, chromatin accessibility, and activity in transcriptional enhancer assays. H3K27ac, a feature of many enhancers, incompletely overlaps multi-TF regions, and multi-TF regions lacking H3K27ac retain conservation and enhancer activity. TEAD1 is a core component of the cardiac transcriptional network, co-occupying cardiac regulatory regions and controlling cardiomyocyte-specific gene functions. Our study provides a resource for deciphering the cardiac transcriptional regulatory network and gaining insights into the molecular mechanisms governing heart development.  

5.2665           Therapeutic Effects of Intravitreously Administered Bacteriophage in a Mouse Model of Endophthalmitis Caused by Vancomycin-Sensitive or -Resistant Enterococcus faecalis

Kishimoto, T., Ishida, W., Fukuda, K., Nakajima, I., Suzuki, T., Uchiyama, J., Matsuzaki, S., Todokoro, D., Daibata, M. and Fukushima, A. Antimicrob. Agents Chemother., 63(11), e1088-19 (2019)   Endophthalmitis due to infection with Enterococcus spp. progresses rapidly and often results in substantial and irreversible vision loss. Given that the frequency of this condition caused by vancomycin-resistant Enterococcus faecalis has been increasing, the development of novel therapeutics is urgently required. We have demonstrated the therapeutic potential of bacteriophage ΦEF24C-P2 in a mouse model of endophthalmitis caused by vancomycin-sensitive (EF24) or vancomycin-resistant (VRE2) strains of E. faecalis. Phage ΦEF24C-P2 induced rapid and pronounced bacterial lysis in turbidity reduction assays with EF24, VRE2, and clinical isolates derived from patients with E. faecalis-related postoperative endophthalmitis. Endophthalmitis was induced in mice by injection of EF24 or VRE2 (1 × 10⁴ cells) into the vitreous. The number of viable bacteria in the eye increased to >1 × 10⁷ CFU, and neutrophil infiltration into the eye was detected as an increase in myeloperoxidase activity at 24 h after infection. A clinical score based on loss of visibility of the fundus as well as the number of viable bacteria and the level of myeloperoxidase activity in the eye were all significantly decreased by intravitreous injection of ΦEF24C-P2 6 h after injection of EF24 or VRE2. Whereas histopathologic analysis revealed massive infiltration of inflammatory cells and retinal detachment in vehicle-treated eyes, the number of these cells was greatly reduced and retinal structural integrity was preserved in phage-treated eyes. Our results thus suggest that intravitreous phage therapy is a potential treatment for endophthalmitis caused by vancomycin-sensitive or -resistant strains of E. faecalis.  

5.2666           Defining the multiplicity and time of infection for the production of Zaire Ebola virus-like particles in the insect cell-baculovirus expression system

Pastor, A.R., Gonzalez-Dominguez, G., Diaz-Salinas, M.A., Ramirez, O.T. and Palomares, L.A. Vaccine, 37, 6962-6969 (2019)   The Ebola virus disease is a public health challenge. To date, the only available treatments are medical support or the emergency administration of experimental drugs. The absence of licensed vaccines against Ebola virus impedes the prevention of infection. Vaccines based on recombinant virus-like particles (VLP) are a promising alternative. The Zaire Ebola virus serotype (ZEBOV) is the most aggressive with the highest mortality rates. Production of ZEBOV-VLP has been accomplished in mammalian and insect cells by the recombinant coexpression of three structural proteins, the glycoprotein (GP), the matrix structural protein VP40, and the nucleocapsid protein (NP). However, specific conditions to manipulate protein concentrations and improve assembly into VLP have not been determined to date. Here, we used a design of experiments (DoE) approach to determine the best MOI and TOI for three recombinant baculoviruses: bac-GP, bac-VP40 and bac-NP, each coding for one of the main structural proteins of ZEBOV. We identified two conditions where the simultaneous expression of the three recombinant proteins was observed. Interestingly, a temporal and stoichiometric interplay between the three structural proteins was observed. VP40 was required for the correct assembly of ZEBOV-VLP. High NP concentrations reduced the accumulation of GP, which has been reported to be necessary for inducing a protective immune response. Electron microscopy showed that the ZEBOV-VLP produced were morphologically similar to the native virus micrographs previously reported in the literature. A strategy for producing ZEBOV in insect cells, which consists in using a high MOI of bac-VP40 and bac-GP, and reducing expression of NP, either by delaying infection or reducing the MOI of bac-NP, was the most adequate for the production of VLP.  

5.2667           In Vivo Dynamics of Reporter Flaviviridae Viruses

Tamura, T., Igarashi, M., Enkhold, B., Suzuki, T., Okamatsu, M., Ono, C., Mori, H., Izumi, T., Sato, A., Fauzyah, Y., Okamoto, T., Sakoda, Y., Fukuhara, T. and Matsuura, Y.

1. Virol., 93(22), e01191-19 (2019)

  Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses, including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo. Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to that of the wild-type virus in vitro. The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus, and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the E^rns or E2 protein also showed propagation comparable to that of the wild-type virus. The recombinant CSFV carrying HiBiT in E^rns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by quantitative PCR (qPCR). Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses and provide a robust platform for the development of novel antivirals.  

5.2668           Machine learning-guided channelrhodopsin engineering enables minimally invasive optogenetics

Bedbrook, C.N., Yang, K.K., Robinson, J.E., mackey, E.D., Gradinaru, V. and Arnold, F.H. Nature Methods, 16, 1176-1184 (2019)   We engineered light-gated channelrhodopsins (ChRs) whose current strength and light sensitivity enable minimally invasive neuronal circuit interrogation. Current ChR tools applied to the mammalian brain require intracranial surgery for transgene delivery and implantation of fiber-optic cables to produce light-dependent activation of a small volume of tissue. To facilitate expansive optogenetics without the need for invasive implants, our engineering approach leverages the substantial literature of ChR variants to train statistical models for the design of high-performance ChRs. With Gaussian process models trained on a limited experimental set of 102 functionally characterized ChRs, we designed high-photocurrent ChRs with high light sensitivity. Three of these, ChRger1–3, enable optogenetic activation of the nervous system via systemic transgene delivery. ChRger2 enables light-induced neuronal excitation without fiber-optic implantation; that is, this opsin enables transcranial optogenetics.    

5.2669           AAV-encoded CaV2.2 peptide aptamer CBD3A6K for primary sensory neuron-targeted treatment of established neuropathic pain

Yu, H., Shin, S.M., Xiang, H., Chao, D., Cai, Y., Xu, H., Khanna, R., Pan, B. and Hogan, Q.H. Gene Therapy, 26, 308-323 (2019)   Transmission of pain signals from primary sensory neurons to secondary neurons of the central nervous system is critically dependent on presynaptic voltage-gated calcium channels. Calcium channel-binding domain 3 (CBD3), derived from the collapsin response mediator protein 2 (CRMP2), is a peptide aptamer that is effective in blocking N-type voltage-gated calcium channel (Ca_V2.2) activity. We previously reported that recombinant adeno-associated virus (AAV)-mediated restricted expression of CBD3 affixed to enhanced green fluorescent protein (EGFP) in primary sensory neurons prevents the development of cutaneous mechanical hypersensitivity in a rat neuropathic pain model. In this study, we tested whether this strategy is effective in treating established pain. We constructed AAV6-EGFP-CBD3A6K (AAV6-CBD3A6K) expressing a fluorescent CBD3A6K (replacing A to K at position 6 of CBD3 peptide), which is an optimized variant of the parental CBD3 peptide that is a more potent blocker of Ca_V2.2. Delivery of AAV6-CBD3A6K into lumbar (L) 4 and 5 dorsal root ganglia (DRG) of rats 2 weeks following tibial nerve injury (TNI) induced transgene expression in neurons of these DRG and their axonal projections, accompanied by attenuation of pain behavior. We additionally observed that the increased Ca_V2.2α1b immunoreactivity in the ipsilateral spinal cord dorsal horn and DRG following TNI was significantly normalized by AAV6-CBD3A6K treatment. Finally, the increased neuronal activity in the ipsilateral dorsal horn that developed after TNI was reduced by AAV6-CBD3A6K treatment. Collectively, these results indicate that DRG-restricted AAV6 delivery of CBD3A6K is an effective analgesic molecular strategy for the treatment of established neuropathic pain.  

5.2670           Pten loss results in inappropriate excitatory connectivity

Skelton, P.D., Frazel, P.W., Lee, D., Suh, H. and Luikart, B.W. Mol. Psychiatry, 24, 1627-1640 (2019)   Pten mutations are associated with autism spectrum disorder. Pten loss of function in neurons increases excitatory synaptic connectivity, contributing to an imbalance between excitation and inhibition. We aimed to determine whether Pten loss results in aberrant connectivity in neural circuits. We compared postnatally generated wild-type and Pten knockout granule neurons integrating into the dentate gyrus using a variety of methods to examine their connectivity. We found that postsynaptic Pten loss provides an advantage to dendritic spines in competition over a limited pool of presynaptic boutons. Retrograde monosynaptic tracing with rabies virus reveals that this results in synaptic contact with more presynaptic partners. Using independently excitable opsins to interrogate multiple inputs onto a single neuron, we found that excess connectivity is established indiscriminately from among glutamatergic afferents. Therefore, Pten loss results in inappropriate connectivity whereby neurons are coupled to a greater number of synaptic partners.  

5.2671           Impaired D2 receptor-dependent dopaminergic transmission in prefrontal cortex of awake mouse model of Parkinson’s disease

Li, M., Xu, H., Chen, G., Sun, S., Wang, Q., Liu, B. et al Brain, 142, 3099-3115 (2019)   The loss-of-function mutation in PARK7/DJ-1 is one of the most common causes of autosomal recessive Parkinson’s disease, and patients carrying PARK7 mutations often exhibit both a progressive movement disorder and emotional impairment, such as anxiety. However, the causes of the emotional symptom accompanying PARK7-associated and other forms of Parkinson’s disease remain largely unexplored. Using two-photon microscopic Ca²⁺ imaging in awake PARK7^−/− and PARK7^+/+ mice, we found that (i) PARK7^−/− neurons in the frontal association cortex showed substantially higher circuit activity recorded as spontaneous somatic Ca²⁺ signals; (ii) both basal and evoked dopamine release remained intact, as determined by both electrochemical dopamine recordings and high performance liquid chromatography in vivo; (iii) D2 receptor expression was significantly decreased in postsynaptic frontal association cortical neurons, and the hyper-neuronal activity were rescued by D2 receptor intervention using either local pharmacology or viral D2 receptor over-expression; and (iv) PARK7^−/− mice showed anxiety-like behaviours that were rescued by either local D2 receptor pharmacology or overexpression. Thus, for first time, we demonstrated a robust D2 receptor-dependent phenotype of individual neurons within the prefrontal cortex circuit in awake parkinsonian mice that linked with anxiety. Our work sheds light on early-onset phenotypes and the mechanisms underlying Parkinson’s disease by imaging brain circuits in an awake mouse model.  

5.2672           Sarcolipin overexpression impairs myogenic differentiation in Duchenne muscular dystrophy

Niranjan, N., Mareedu, S., Tian, Y., Kodippili, K., Fefelova, N., Voit, A., Xie, L-H., Duan, D. and Babu, G.J. Am. J. Physiol. Cell Physiol., 317, C813-C824 (2019)   Reduction in the expression of sarcolipin (SLN), an inhibitor of sarco(endo)plasmic reticulum (SR) Ca²⁺-ATPase (SERCA), ameliorates severe muscular dystrophy in mice. However, the mechanism by which SLN inhibition improves muscle structure remains unclear. Here, we describe the previously unknown function of SLN in muscle differentiation in Duchenne muscular dystrophy (DMD). Overexpression of SLN in C₂C₁₂ resulted in decreased SERCA pump activity, reduced SR Ca²⁺ load, and increased intracellular Ca²⁺ (Ca 2+ i  Cai2+ ) concentration. In addition, SLN overexpression resulted in altered expression of myogenic markers and poor myogenic differentiation. In dystrophin-deficient dog myoblasts and myotubes, SLN expression was significantly high and associated with defective Ca 2+ i  Cai2+ cycling. The dystrophic dog myotubes were less branched and associated with decreased autophagy and increased expression of mitochondrial fusion and fission proteins. Reduction in SLN expression restored these changes and enhanced dystrophic dog myoblast fusion during differentiation. In summary, our data suggest that SLN upregulation is an intrinsic secondary change in dystrophin-deficient myoblasts and could account for the Ca 2+ i  Cai2+ mishandling, which subsequently contributes to poor myogenic differentiation. Accordingly, reducing SLN expression can improve the Ca 2+ i  Cai2+ cycling and differentiation of dystrophic myoblasts. These findings provide cellular-level supports for targeting SLN expression as a therapeutic strategy for DMD.  

5.2673           An Engineered AAV6 Based Vaccine Induces High Cytolytic Anti-Tumor Activity by Directly Targeting Dendritic Cells and Improves Antigen Presentation

Krotova, K., Day, A. and Aslanidi, K. Molecular Therapy-Oncolytics, 15, 166-177 (2019)   We have previously shown that an AAV6-based vaccine generates high levels of antigen-specific CD8⁺ T cells. Further modifications described here led to significantly increased levels of antigen-specific CD8⁺ and CD4⁺ T cells, enhanced formation of memory cells, and superior antigen-specific killing capacity in a murine model. By tracking reporter-gene-positive dendritic cells, we showed that they were directly targeted with modified AAV6 in vivo. Our vaccine’s anti-cancer potential was evaluated with the antigen ovalbumin against a B16F10 melanoma cell line stably expressing ovalbumin. The vaccination showed superior protection in a murine model of metastatic melanoma. The vaccination significantly delayed solid tumor growth but did not completely prevent tumor development. We show that tumors in immunized mice escaped vaccine-induced killing by losing ovalbumin expression. The vaccine induced massive tumor infiltration with NK and CD8⁺ T cells with upregulated PD-1 expression. Thus, a vaccination of a combination of anti-PD-1 antibodies demonstrated significant improvement in the treatment efficacy. To summarize, we showed that a bioengineered AAV6-based vaccine elicits strong and long-lasting cellular and humoral responses against an encoded antigen. To increase AAV vaccine efficiency and mitigate tumor escape through antigen loss, we intended to target several antigens in combination with treatments targeting the tumor microenvironment.  

5.2674           AAV-mediated expression of AP-1 neutralizing RNA decoy oligonucleotides attenuates transplant vasculopathy in mouse aortic allografts

Remes, A., Franz, M., Mohr, F., Weber, A., Rapti, K., Jungmanns, A., Karck, M., Hecker, M., Kallenbach, K., Müller, O.J., Arif, R. and Wagner, A.H. Molecular Therapy-Methods Clin. Develop., 15, 246-256 (2019)   Transplant vasculopathy (TV), characterized by obstructive lesions in affected vessels, represents one of the long-term complications of cardiac transplantation. Activation of the transcription factor activator protein-1 (AP-1) is implicated in smooth muscle cell (SMC) phenotypic switch from contractile to synthetic function, increasing the migration and proliferation rate of these cells. We hypothesize that adeno-associated virus (AAV)-mediated delivery of an RNA hairpin AP-1 decoy oligonucleotide (dON) might effectively ameliorate TV severity in a mouse aortic allograft model. Aortic allografts from DBA/2 mice ex vivo transduced with modified AAV9-SLR carrying a targeting peptide within the capsid surface were transplanted into the infrarenal aorta of C57BL/6 mice. Cyclosporine A (10 mg/kg BW) was administered daily. AP-1 dONs were intracellularly expressed in the graft tissue as small hairpin RNA proved by fluorescent in situ hybridization. Explantation after 30 days and histomorphometric evaluation revealed that AP-1 dON treatment significantly reduced intima-to-media ratio by 41.5% (p < 0.05) in the grafts. In addition, expression of adhesion molecules, cytokines, as well as numbers of proliferative SMCs, matrix metalloproteinase-9-positive cells, and inflammatory cell infiltration were significantly decreased in treated aortic grafts. Our findings demonstrate the feasibility, efficacy, and specificity of the anti-AP-1 RNA dON approach for the treatment of allograft vasculopathy in an animal model. Moreover, the AAV-based approach in general provides the possibility to achieve a prolonged delivery of nucleic-acids-based therapeutics in to the blood vessel wall.  

5.2675           Developing an Anion Exchange Chromatography (AEX) Assay for Determining Empty and Full Capsid Contents in AAV6.2

Wang, C., Mulagapati, S.H.R., Chen, Z., Du, J., Zhao, X., Xi, G., Chen, L., Linke, T., Gao, C., Schmelzer, A.E. and Liu, D. Molecular Therapy-Methods Clin. Develop., 15, 257-263 (2019)   Adeno-associated virus (AAV) vectors are clinically proven gene delivery vehicles that are attracting an increasing amount of attention. Non-genome-containing empty AAV capsids are by-products during AAV production that have been reported to potentially impact AAV product safety and efficacy. Therefore, the presence and amount of empty AAV capsids need to be characterized during process development. Multiple methods have been reported to characterize empty AAV capsid levels, including transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), charge detection mass spectrometry (CDMS), UV spectrophotometry, and measuring capsid and genome copies by ELISA and qPCR. However, these methods may lack adequate accuracy and precision or be challenging to transfer to a quality control (QC) lab due to the difficulty of implementation. In this study, we used AAV serotype 6.2 (AAV6.2) as an example to show the development of a QC-friendly anion exchange chromatography (AEX) assay for the determination of empty and full capsid percentages. The reported assay requires several microliters of material with a minimum titer of 5 × 10¹¹ vg/mL, and it can detect the presence of as low as 2.9% empty capsids in AAV6.2 samples. Additionally, the method is easy to deploy, can be automated, and has been successfully implemented to support testing of various in-process and release samples.  

5.2676           Intra- and extracellular β-amyloid overexpression via adeno-associated virus-mediated gene transfer impairs memory and synaptic plasticity in the hippocampus

Forner, S., Martini, A.C., Prieto, G.A., Dang, C.T., Rodriguez-Ortiz, C.J. et al Scientific Reports, 9:15936 (2019)   Alzheimer’s disease (AD), the most common age-related neurodegenerative disorder, is currently conceptualized as a disease of synaptic failure. Synaptic impairments are robust within the AD brain and better correlate with dementia severity when compared with other pathological features of the disease. Nevertheless, the series of events that promote synaptic failure still remain under debate, as potential triggers such as β-amyloid (Aβ) can vary in size, configuration and cellular location, challenging data interpretation in causation studies. Here we present data obtained using adeno-associated viral (AAV) constructs that drive the expression of oligomeric Aβ either intra or extracellularly. We observed that expression of Aβ in both cellular compartments affect learning and memory, reduce the number of synapses and the expression of synaptic-related proteins, and disrupt chemical long-term potentiation (cLTP). Together, these findings indicate that during the progression AD the early accumulation of Aβ inside neurons is sufficient to promote morphological and functional cellular toxicity, a phenomenon that can be exacerbated by the buildup of Aβ in the brain parenchyma. Moreover, our AAV constructs represent a valuable tool in the investigation of the pathological properties of Aβ oligomers both in vivo and in vitro.  

5.2677           The Hepatokine TSK does not affect brown fat thermogenic capacity, body weight gain, and glucose homeostasis

Mouchlroud, M., Cambre, E., Aldow, M., Caron, A., Jubinville, E., Turcotte, L. et al Mol. Metabolism, 30, 184-191 (2019)   Objectives Hepatokines are proteins secreted by the liver that impact the functions of the liver and various tissues through autocrine, paracrine, and endocrine signaling. Recently, Tsukushi (TSK) was identified as a new hepatokine that is induced by obesity and cold exposure. It was proposed that TSK controls sympathetic innervation and thermogenesis in brown adipose tissue (BAT) and that loss of TSK protects against diet-induced obesity and improves glucose homeostasis. Here we report the impact of deleting and/or overexpressing TSK on BAT thermogenic capacity, body weight regulation, and glucose homeostasis. Methods We measured the expression of thermogenic genes and markers of BAT innervation and activation in TSK-null and TSK-overexpressing mice. Body weight, body temperature, and parameters of glucose homeostasis were also assessed in the context of TSK loss and overexpression. Results The loss of TSK did not affect the thermogenic activation of BAT. We found that TSK-null mice were not protected against the development of obesity and did not show improvement in glucose tolerance. The overexpression of TSK also failed to modulate thermogenesis, body weight gain, and glucose homeostasis in mice. Conclusions TSK is not a significant regulator of BAT thermogenesis and is unlikely to represent an effective target to prevent obesity and improve glucose homeostasis.  

5.2678           TLR9 signaling mediates adaptive immunity following systemic AAV gene therapy

Ashley, S.N., Somanathan, S., Giles, A.R. and Wilson, J.M. Cell. Immunol., 346, 103997 (2019)   An ongoing concern of in vivo gene therapy is adaptive immune responses against the protein product of a transgene, particularly for recessive diseases in which antigens are not presented to lymphocytes during central tolerance induction. Here we show that Toll-like receptor 9 (TLR9) signaling activates T cells against an epitope tagged mitochondria-targeted ornithine transcarbamylase (OTC) following the administration of a systemic adeno-associated virus (AAV) vector. Using a transgenic mouse model system, we demonstrate that TLR9 signaling extrinsic to T cells induces a robust cytotoxic T-cell response against the transgene and results in transgene expression loss. Overall, our results suggest that inflammation mediated by TLR9 signaling and the presence of high affinity transgene-specific T cells is important for the development of adaptive immune responses to transgene products following AAV gene therapy.  

5.2679           Short-Term Local Expression of a PD-L1 Blocking Antibody from a Self-Replicating RNA Vector Induces Potent Antitumor Responses

Ballesteros-Briones, M.C., Martisova, E., Casales, E., Silva-Pilipich, N., Bunuales, m. et al Molecular Therapy, 27(11), 1892-1905 (2019)   Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFV-aPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and safe approach for cancer treatment.  

5.2680           AAV-PHP.B Administration Results in a Differential Pattern of CNS Biodistribution in Non-human Primates Compared with Mice

Liguore, W.A., Domire, J.S:, Button, D., Wang, Y., Dufour, B.D., Srinivasan, S. and McBride, J.L. Molecular Therapy, 27(11), 2018-2037 (2019)   The ability of recombinant adeno-associated virus (AAV) to deliver transgenes to the CNS has allowed for several advancements in the field of gene therapy to treat brain disorders. Although most AAVs do not readily cross the blood-brain barrier and transduce the CNS following peripheral administration, AAV-PHP.B has recently been shown to transduce brains of mice with higher efficiency compared with its parent serotype, AAV9, following injection into the retro-orbital sinus. Here, we extended this foundational work by comparing AAV-PHP.B transduction efficiency in wild-type C57BL/6J mice using four clinically applicable delivery strategies including two intravascular (intra-jugular vein and intra-carotid artery) and two intra-cerebral spinal fluid (CSF) routes (intra-cisterna magna and intra-lateral ventricle). We scaled up these comparisons in a larger-animal model and evaluated transduction efficiency of AAV-PHP.B in the rhesus macaque. We found widespread and largely equal CNS transduction in mice following all four injection strategies, whereas we observed a differential pattern of transduction in macaques with broad cortical and spinal cord transduction seen after intrathecal administration and only very low transduction following intravascular administration. Taken together, these results suggest that AAV-PHP.B may be a useful gene therapy vector for neurological disorders, particularly those stemming from broad cortical or spinal cord neuropathology.  

5.2681           Capsid containing virus like particle vaccine against Zika virus made from a stable cell line

Garg, H., Mehmetoglu-Gurbuz, T., Ruddy, G.M. and Joshi, A. Vaccine, 37, 7123-7131 (2019)   Zika virus infection during pregnancy is associated with severe birth defects including microcephaly in the new born. The lack of specific treatment calls for the development of a safe and effective vaccine for use in pregnant women. We recently tested the efficacy of a Virus Like Particle (VLP) vaccine for Zika virus in mice and found that Capsid-preMembrane-Env (CprME) VLPs generated a better neutralizing antibody response than preMembrane-Env (prME) VLPs. The superiority of CprME VLPs suggested that inclusion of capsid in the vaccine may enhance the immune response. However, production of CprME VLPs requires co-expression of NS2B-3 protease, which creates a major hurdle for generation of stable cell lines. To overcome this limitation, we generated a bicistronic vector that expresses CprME and NS2B-3 using an IRES sequence. This bicistronic expression cassette, in a lentiviral vector, was used to create a stable cell line that constitutively secretes CprME VLPs. The expression of NS2B-3, presence of capsid in the secreted VLPs, efficiency of VLP release, and stability of the cell line was extensively tested. Antigen sparing studies in mice using prME and CprME VLPs, both derived from stable cell lines, confirmed the superiority of CprME VLPs in generation of neutralizing antibody response. Capsid specific antibodies were detected in CprME VLP immunized mice providing mechanistic insights into the superiority of these VLPs. Challenge of CprME VLP immunized mice with Zika PRVABC59 showed complete protection against day 3 viremia further validating the efficacy of the vaccine. Our study is the first to generate a stable cell line secreting Zika CprME VLPs via natural NS2B-3 cleavage, demonstrate incorporation of capsid in CprME VLPs and complete protection in challenge studies. This is a major advancement for the Zika vaccine platform that is safe for use in pregnant women and readily scalable for use in developing countries.  

5.2682           Upregulation of Yy1 Suppresses Dilated Cardiomyopathy caused by Ttn insufficiency

Liao, D., Chen, W., Tan, C.Y., Wong, J.X., Chang, P.S., Tan, L.W., Foo, R. and Jiang, J. Scientific Reports, 9:16330 (2019)   Truncating variants in TTN (TTNtv), coding for the largest structural protein in the sarcomere, contribute to the largest portion of familial and ambulatory dilated cardiomyopathy (DCM). TTN haploinsufficiency caused by TTNtv is suggested as the disease mechanism. However, it is unclear whether TTN insufficiency causes DCM. Moreover, it is unknown whether modulation of downstream pathways serves as a therapeutic strategy for DCM caused by TTN insufficiency. Here, we show that reduction of cardiac Ttn expression by adeno-associated virus mediated shRNA (Ttn shRNA) generated DCM in mouse, demonstrating impaired cardiac performance, enlarged left ventricle (LV) and reduced LV wall thickness. A screen of 10 dysregulated and selected genes identified that Yin Yang 1 (Yy1) significantly suppressed DCM caused by Ttn shRNA. Gene profiling by RNAseq showed Yy1 modulated cell growth related genes. Ttn insufficiency activated cardiomyocyte cell cycle reentry by upregulating of Ccnd1 and Ccnd2. Cardiomyocytes activated by Ttn insufficiency did not advance to S phase by EdU incorporation assay. Yy1 promoted cardiomyocyte cell cycle by further enhancing Ccnd1 and Ccnd2 and increasing DNA replication without undergoing cell division. Importantly, upregulation of Ccnd1 and Ccnd2 suppressed DCM caused by Ttn insufficiency. Our findings demonstrate that DCM caused by Ttn insufficiency can be treated by therapeutically promoting cardiac cell cycle.  

5.2683           Safe and Sustained Expression of Human Iduronidase After Intrathecal Administration of Adeno-Associated Virus Serotype 9 in Infant Rhesus Monkeys

Hordeaux, J., Hinderer, C., Buza, E.L., Louboutin, J-P-. Jahan, T., Bell, P., Chichester, J.A., Tarantal, A.F. and Wilson, J.M. Human Gene Therapy, 30(8), 957-966 (2019)   Many neuropathic diseases cause early, irreversible neurologic deterioration, which warrants therapeutic intervention during the first months of life. In the case of mucopolysaccharidosis type I, a recessive lysosomal storage disorder that results from a deficiency of the lysosomal enzyme α-l-iduronidase (IDUA), one of the most promising treatment approaches is to restore enzyme expression through gene therapy. Specifically, administering pantropic adeno-associated virus (AAV) encoding IDUA into the cerebrospinal fluid (CSF) via suboccipital administration has demonstrated remarkable efficacy in large animals. Preclinical safety studies conducted in adult nonhuman primates supported a positive risk–benefit profile of the procedure while highlighting potential subclinical toxicity to primary sensory neurons located in the dorsal root ganglia (DRG). This study investigated the long-term performance of intrathecal cervical AAV serotype 9 gene transfer of human IDUA administered to 1-month-old rhesus monkeys (N = 4) with half of the animals tolerized to the human transgene at birth via systemic administration of an AAV serotype 8 vector expressing human IDUA from the liver. Sustained expression of the transgene for almost 4 years is reported in all animals. Transduced cells were primarily pyramidal neurons in the cortex and hippocampus, Purkinje cells in the cerebellum, lower motor neurons, and DRG neurons. Both tolerized and non-tolerized animals were robust and maintained transgene expression as measured by immunohistochemical analysis of brain tissue. However, the presence of antibodies in the non-tolerized animals led to a loss of measurable levels of secreted enzyme in the CSF. These results support the safety and efficiency of treating neonatal rhesus monkeys with AAV serotype 9 gene therapy delivered into the CSF.  

5.2684           Alpha-1-Antitrypsin Promoter Improves the Efficacy of an Adeno-Associated Virus Vector for the Treatment of Mitochondrial Neurogastrointestinal Encephalomyopathy

Cabrera-Perez, R., Villa-Julia, F., Hirano, M., Mingozzi, F., Torres-Torronteras, J. and Martl, R. Human Gene Therapy, 30(8), 985-998 (2019)   Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a devastating disease caused by mutations in TYMP, which encodes thymidine phosphorylase (TP). In MNGIE patients, TP dysfunction results in systemic thymidine and deoxyuridine overload, which interferes with mitochondrial DNA replication. Preclinical studies have shown that gene therapy using a lentiviral vector targeted to hematopoietic stem cells or an adeno-associated virus (AAV) vector transcriptionally targeted to liver are feasible approaches to treat MNGIE. Here, we studied the effect of various promoters (thyroxine-binding globulin [TBG], phosphoglycerate kinase [PGK], hybrid liver-specific promoter [HLP], and alpha-1-antitrypsin [AAT]) and DNA configuration (single stranded or self complementary) on expression of the TYMP transgene in the AAV8 serotype in a murine model of MNGIE. All vectors restored liver TP activity and normalized nucleoside homeostasis in mice. However, the liver-specific promoters TBG, HLP, and AAT were more effective than the constitutive PGK promoter, and the self-complementary DNA configuration did not provide any therapeutic advantage over the single-stranded configuration. Among all constructs, only AAV-AAT was effective in all mice treated at the lowest dose (5 × 10¹⁰ vector genomes/kg). As use of the AAT promoter will likely minimize the dose needed to achieve clinical efficacy as compared to the other promoters tested, we propose using the AAT promoter in the vector eventually designed for clinical use.  

5.2685           Capsid Engineering Overcomes Barriers Toward Adeno-Associated Virus Vector-Mediated Transduction of Endothelial Cells

Zhang, L., Rossi, A., Lange, l., Meumann, N., Koitzsch, U. et al Human Gen Therapy, 30(10), 1284-1296 (2019)   Endothelial cells (EC) are targets in gene therapy and regenerative medicine, but they are inefficiently transduced with adeno-associated virus (AAV) vectors of various serotypes. To identify barriers hampering efficient transduction and to develop an optimized AAV variant for EC transduction, we screened an AAV serotype 2-based peptide display library on primary human macrovascular EC. Using a new high-throughput selection and monitoring protocol, we identified a capsid variant, AAV-V_EC, which outperformed the parental serotype as well as first-generation targeting vectors in EC transduction. AAV vector uptake was improved, resulting in significantly higher transgene expression levels from single-stranded vector genomes detectable within a few hours post-transduction. Notably, AAV-V_EC transduced not only proliferating EC but also quiescent EC, although higher particle-per-cell ratios had to be applied. Also, induced pluripotent stem cell-derived endothelial progenitor cells, a novel tool in regenerative medicine and gene therapy, were highly susceptible toward AAV-V_EC transduction. Thus, overcoming barriers by capsid engineering significantly expands the AAV tool kit for a wide range of applications targeting EC.  

5.2686           Development of Versatile and Flexible Sf9 Packaging Cell Line-Dependent OneBac System for Large-Scale Recombinant Adeno-Associated Virus Production

Wu, Y., Mei, T., Jiang, l., Han, Z., Dong, R., Yang, T. and Xu, F. Human Gene Therapy Methods, 30(5), 172-183 (2019)   Recombinant adeno-associated viruses (rAAVs) are excellent vectors for gene delivery. However, current Sf9/Cap-Rep packaging cell line-dependent OneBac systems still lack versatility and flexibility for large-scale production of rAAVs. In this study, we developed an improved OneBac system that includes a novel dual-function baculovirus expression vector (BEV) termed BEV/Cap-(ITR-GOI) that carries both the AAV Cap gene and rAAV genome inverted terminal repeat (ITR) sequences flanking the gene of interest (GOI), a versatile Sf9-GFP/Rep packaging cell line that harbors silent copies of the AAV2 Rep gene that can be expressed after BEV infection, and constitutively expressed green fluorescent protein (GFP) reporter genes to facilitate cell line screening. The BEV/Cap-(ITR-GOI) construct allows flexibility to switch among different Cap gene serotypes using simple BEV reconstruction, and is stable for at least five serial passages. Furthermore, the Sf9-GFP/Rep stable cell line is versatile for production of different rAAV serotypes. The yield levels for rAAV2, rAAV8, and rAAV9 exceeded 10⁵ vector genomes (VG) per cell, which is similar to other currently available large-scale rAAV production systems. The new Bac system-derived rAAVs have biophysical properties similar to HEK293 cell-derived rAAVs, as well as high quality and activity. In summary, the novel Sf9-GFP/Rep packaging cell line-dependent OneBac system can facilitate large-scale rAAV production and rAAV-based gene therapy.  

5.2687           AAV2/DJ-mediated alpha-synuclein overexpression in the rat substantia nigra as early stage model of Parkinson’s disease

Von Hövel, F.F., Rumpel, R., ratzka, A., Schreiner, D. and Grothe, C. Cell Tissue Res., 378(1), 1-14 (2019)   Parkinson’s disease (PD) is pathologically characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and alpha-synucleinopathy. We mimic the disease pathology with overexpression of either the human α-syn wildtype (α-syn-WT) or E46K mutant form (α-syn-E46K) in DA neurons of the SNpc in adult rats using AAV2/DJ as a viral vector for the first time. Transduction efficiency was compared to an equal virus titer expressing the green fluorescent protein (GFP). Motor skills of all animals were evaluated in the cylinder and amphetamine-induced rotation test over a total time period of 12 weeks. Additionally, stereological quantification of DA cells and striatal fiber density measurements were performed every 4 weeks after injection. Rats overexpressing α-syn-WT showed a progressive loss of DA neurons with 40% reduction after 12 weeks accompanied by a greater loss of striatal DA fibers. In contrast, α-syn-E46K led to this reduction after 4 weeks without further progress. Insoluble α-syn positive cytoplasmic inclusions were observed in both groups within DA neurons of the SNpc and VTA. In addition, both α-syn groups developed a characteristic worsening of the rotational behavior over time. However, only the α-syn-WT group reached statistically significant different values in the cylinder test. Summarizing these effects, we established a motor symptom animal model of PD by using AAV2/DJ in the brain for the first time. Thereby, overexpressing of α-syn-E46K mimicked a rather pre-symptomatic stage of the disease, while the α-syn-WT overexpressing animals imitated an early symptomatic stage of PD.  

5.2688           rAAVrh74.MCK.GALGT2 Demonstrates Safety and Widespread Muscle Glycosylation after Intravenous Delivery in C57BL/6J Mice

Zygmunt, D.A., Xu, R., Jia, Y., Ashbrook, A., Menke, C., Shao, G., Yoon, J.H., Hamilton, S., Pisharath, H., Bolon, B. and martin, P.T: Moeclular Therapy-Methods Clin. Develop., 15, 305-319 (2019)   rAAVrh74.MCK.GALGT2 is a surrogate gene therapy that inhibits muscular dystrophy in multiple animal models. Here, we report on a dose-response study of functional muscle GALGT2 expression as well as toxicity and biodistribution studies after systemic intravenous (i.v.) delivery of rAAVrh74.MCK.GALGT2. A dose of 4.3 × 10¹⁴vg/kg (measured with linear DNA standard) resulted in GALGT2-induced glycosylation in the majority of skeletal myofibers throughout the body and in almost all cardiomyocytes, while several lower doses also showed significant muscle glycosylation. No adverse clinical signs or treatment-dependent changes in tissue or organ pathology were noted at 1 or 3 months post-treatment. Blood cell and serum enzyme chemistry measures in treated mice were all within the normal range except for alkaline phosphatase (ALP) activity, which was elevated in serum but not in tissues. Some anti-rAAVrh74 capsid T cell responses were noted at 4 weeks post-treatment, but all such responses were not present at 12 weeks. Using intramuscular delivery, GALGT2-induced muscle glycosylation was increased in Cmah-deficient mice, which have a humanized sialoglycome, relative to wild-type mice, suggesting that use of mice may underestimate GALGT2 activity in human muscle. These data demonstrate safety and high transduction of muscles throughout the body plan with i.v. delivery of rAAVrh74.MCK.GALGT2.  

5.2689           Delivering genes across the blood-brain barrier: LY6A, a novel cellular receptor for AAV-PHP.B capsids

Huang, Q., Chan, K.Y., Tobey, I.G., Chan, Y.A., Poterba, T., Boutros, C.L., Balazs, A.B., Daneman, R., Bloom, J.M., Seed, C. and Deverman, B.E. PloS One, 14(11), e0225206 (2019)   The engineered AAV-PHP.B family of adeno-associated virus efficiently delivers genes throughout the mouse central nervous system. To guide their application across disease models, and to inspire the development of translational gene therapy vectors for targeting neurological diseases in humans, we sought to elucidate the host factors responsible for the CNS tropism of the AAV-PHP.B vectors. Leveraging CNS tropism differences across 13 mouse strains, we systematically determined a set of genetic variants that segregate with the permissivity phenotype, and rapidly identified LY6A as an essential receptor for the AAV-PHP.B vectors. Interfering with LY6A by CRISPR/Cas9-mediated Ly6a disruption or with blocking antibodies reduced transduction of mouse brain endothelial cells by AAV-PHP.eB, while ectopic expression of Ly6a increased AAV-PHP.eB transduction of HEK293T and CHO cells by 30-fold or more. Importantly, we demonstrate that this newly discovered mode of AAV binding and transduction can occur independently of other known AAV receptors. These findings illuminate the previously reported species- and strain-specific tropism characteristics of the AAV-PHP.B vectors and inform ongoing efforts to develop next-generation AAV vehicles for human CNS gene therapy.  

5.2690           Hepatitis B virus-induced modulation of liver macrophage function promotes hepatocyte infection

Faure-Dupuy, S., Delphin, M., Aillot, L., Heikenwälder, M., Durantel, D. and Lucifora, J.

1. Hepatol., 71, 1086-1098 (2019)

  Background & Aims Liver macrophages can be involved in both pathogen clearance and/or pathogenesis. To get further insight on their role during chronic hepatitis B virus (HBV) infections, our aim was to phenotypically and functionally characterize in vivo and ex vivo the interplay between HBV, primary human liver macrophages (PLMs) and primary blood monocytes differentiated into pro-inflammatory or anti-inflammatory macrophages (M1-MDMs or M2-MDMs, respectively). Methods PLMs or primary blood monocytes, either ex vivo differentiated into M1-MDMs or M2-MDMs, were exposed to HBV and their activation followed by ELISA or quantitative reverse transcription PCR (RT-qPCR). Liver biopsies from HBV-infected patients were analysed by RT-qPCR or immunohistochemistry. Viral parameters in HBV-infected primary human hepatocytes and differentiated HepaRG cells were followed by ELISA, qPCR and RT-qPCR analyses. Results HBc protein was present within the macrophages of liver biopsies taken from HBV-infected patients. Macrophages from HBV-infected patients also expressed higher levels of anti-inflammatory macrophage markers than those from non-infected patients. Ex vivo exposure of naive PLMs to HBV led to reduced secretion of pro-inflammatory cytokines. Upon exposure to HBV or HBV-producing cells during differentiation and activation, M1-MDMs secreted less IL-6 and IL-1β, whereas M2-MDMs secreted more IL-10 when exposed to HBV during activation. Finally, cytokines produced by M1-MDMs, but not those produced by HBV-exposed M1-MDMs, decreased HBV infection of hepatocytes. Conclusions Altogether, our data strongly suggest that HBV modulates liver macrophage functions to favour the establishment of infection.    

5.2691           FGF21 augments autophagy in random-pattern skin flaps via AMPK signaling pathways and improves tissue survival

Zhou, K., Chen, H., Lin, J., Xu, H., Wu, H., Bao, G., Li, J., Deng, X., Shui, X., Gao, W., Ding, j., Xiao, J. and Xu, H. Cell Death & Disease, 10:872 (2019)   Random-pattern skin flap is commonly used for surgical tissue reconstruction due to its ease and lack of axial vascular limitation. However, ischemic necrosis is a common complication, especially in distal parts of skin flaps. Previous studies have shown that FGF21 can promote angiogenesis and protect against ischemic cardiovascular disease, but little is known about the effect of FGF21 on flap survival. In this study, using a rat model of random skin flaps, we found that the expression of FGF21 is significantly increased after establishment skin flaps, suggesting that FGF21 may exert a pivotal effect on flap survival. We conducted experiments to elucidate the role of FGF21 in this model. Our results showed that FGF21 directly increased the survival area of skin flaps, blood flow intensity, and mean blood vessel density through enhancing angiogenesis, inhibiting apoptosis, and reducing oxidative stress. Our studies also revealed that FGF21 administration leads to an upregulation of autophagy, and the beneficial effects of FGF21 were reversed by 3-methyladenine (3MA), which is a well-known inhibitor of autophagy, suggesting that autophagy plays a central role in FGF21’s therapeutic benefit on skin flap survival. In our mechanistic investigation, we found that FGF21-induced autophagy enhancement is mediated by the dephosphorylation and nuclear translocation of TFEB; this effect was due to activation of AMPK-FoxO3a-SPK2-CARM1 and AMPK-mTOR signaling pathways. Together, our data provides novel evidence that FGF21 is a potent modulator of autophagy capable of significantly increasing random skin flap viability, and thus may serve as a promising therapy for clinical use.  

5.2692           Selection of an efficient AAV vector for robust CNS transgene expression

Hanlon, K.,Meltzer, J.C., Buzhdygan, T., Cheng, M.J., Sena-Esteves, M., Bennett, R.E. et al Molecular Therapy-Methods Clin. Develop., 15, 320-332 (2019)   Adeno-associated virus (AAV) capsid libraries have generated improved transgene delivery vectors. We designed an AAV library construct, iTransduce, that combines a peptide library on the AAV9 capsid with a Cre cassette to enable sensitive detection of transgene expression. After only two selection rounds of the library delivered intravenously in transgenic mice carrying a Cre-inducible fluorescent protein, we flow-sorted fluorescent cells from brain and DNA sequencing revealed two dominant capsids. One of the capsids, termed AAV-F, mediated transgene expression in the brain cortex over 65-fold (astrocytes) and 171-fold (neurons) higher than the parental AAV9. High transduction efficiency was sex-independent, sustained in two mouse strains (C57BL/6 and BALB/c) making it a highly useful capsid for CNS transduction of mice. Future work in large animal models will test the translation potential of AAV-F.  

5.2693           Homogenous generation of dopaminergic neurons from multiple hiPSC lines by transient expression of transcription factors

Mahajan, S., Raina, A., Fokken, C., Kügler, S. and Bähn, M. Cell Death and Disease, 10:898 (2019)   A major hallmark of Parkinson's disease is loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The pathophysiological mechanisms causing this relatively selective neurodegeneration are poorly understood, and thus experimental systems allowing to study dopaminergic neuron dysfunction are needed. Induced pluripotent stem cells (iPSCs) differentiated toward a dopaminergic neuronal phenotype offer a valuable source to generate human dopaminergic neurons. However, currently available protocols result in a highly variable yield of dopaminergic neurons depending on the source of hiPSCs. We have now developed a protocol based on HBA promoter-driven transient expression of transcription factors by means of adeno-associated viral (AAV) vectors, that allowed to generate very consistent numbers of dopaminergic neurons from four different human iPSC lines. We also demonstrate that AAV vectors expressing reporter genes from a neuron-specific hSyn1 promoter can serve as surrogate markers for maturation of hiPSC-derived dopaminergic neurons. Dopaminergic neurons differentiated by transcription factor expression showed aggravated neurodegeneration through α-synuclein overexpression, but were not sensitive to γ-synuclein overexpression, suggesting that these neurons are well suited to study neurodegeneration in the context of Parkinson’s disease.  

5.2694           Quantification of phosphoinositides reveals strong enrichment of PIP2 in HIV-1 compared to producer cell membranes

Mücksch, F., Citir, M., Lüchtenborg, C., Glass, B., Traynor-Kaplan, A., Schultz, C., Brügger, B. and Kräusslich, H-G. Scientific Reports, 9:17661 (2019)   Human immunodeficiency virus type 1 (HIV-1) acquires its lipid envelope during budding from the plasma membrane of the host cell. Various studies indicated that HIV-1 membranes differ from producer cell plasma membranes, suggesting budding from specialized membrane microdomains. The phosphoinositide PI(4,5)P₂ has been of particular interest since PI(4,5)P₂ is needed to recruit the viral structural polyprotein Gag to the plasma membrane and thus facilitates viral morphogenesis. While there is evidence for an enrichment of PIP₂ in HIV-1, fully quantitative analysis of all phosphoinositides remains technically challenging and therefore has not been reported, yet. Here, we present a comprehensive analysis of the lipid content of HIV-1 and of plasma membranes from infected and non-infected producer cells, resulting in a total of 478 quantified lipid compounds, including molecular species distribution of 25 different lipid classes. Quantitative analyses of phosphoinositides revealed strong enrichment of PIP₂, but also of PIP₃, in the viral compared to the producer cell plasma membrane. We calculated an average of ca. 8,000 PIP₂ molecules per HIV-1 particle, three times more than Gag. We speculate that the high density of PIP₂ at the HIV-1 assembly site is mediated by transient interactions with viral Gag polyproteins, facilitating PIP₂ concentration in this microdomain. These results are consistent with our previous observation that PIP₂ is not only required for recruiting, but also for stably maintaining Gag at the plasma membrane. We believe that this quantitative analysis of the molecular anatomy of the HIV-1 lipid envelope may serve as standard reference for future investigations.  

5.2695           Recombinant virus-like particles presenting IL-33 successfully modify the tumor microenvironment and facilitate antitumor immunity in a model of breast cancer

Feng, X., Liu, H., Chu, X., Sun, P., Huang, W., Liu, C., Yang, X., Sun, W., Bai, H. and Ma, Y. Acta Biomaterialia, 100, 316-325 (2019)   Recently, interleukin (IL)-33 has been closely associated with a variety of clinical cancers. IL-33 presents both protumorigenic, and less frequently, antitumorigenic functions depending on disease conditions. IL-33 signaling appears to be a possible target for the treatment of applicable tumor diseases. This study aimed to develop an effective approach to intervene in IL-33 functioning in tumors and reveal the immunotherapeutic potential of anti-IL-33 active immunization. Recombinant truncated hepatitis B virus core antigen (HBcAg), presenting mature IL-33 molecules on the surface of virus-like particles (VLPs), was prepared and used to immunize BALB/c mice in a model of murine 4T1 breast cancer. The immunization was performed through either a preventive or therapeutic strategy in two separate studies. Anti-IL-33 immunization with VLPs elicited a persistent and highly titrated specific antibody response and significantly suppressed orthotopic tumor growth in the preventive study and lung metastasis in both studies. The underlying mechanisms might include promoting tumor-specific Th1 and CTL-mediated cellular responses and the expression of the effector molecule interferon-γ (IFN-γ), suppressing T-helper type 2 (Th2) responses, and significantly reducing the infiltration of immunosuppressive Treg (regulatory T) cells and myeloid-derived suppressor cells (MDSCs) into tumor tissues in the immunized mice. In conclusion, anti-IL-33 active immunization employing recombinant VLPs as an antigen delivery platform effectively modified the tumor microenvironment and promoted antitumor immunity, indicating the potential of this approach as a new and promising immunotherapeutic strategy for the treatment of cancers where IL-33 plays a definite protumorigenic role.  

5.2696           A single combination gene therapy treats multiple age-related diseases

Davidsohn, N., Pezzone, M., Vernet, A., Graveline, A., Oliver, D., Slomovic, S., Punthambaker, S., Sun, X., Liao, R., Bonventre, J.V. and Church, G.M. PNAS, 116(47), 23505-23511 (2019)   Human and animal longevity is directly bound to their health span. While previous studies have provided evidence supporting this connection, therapeutic implementation of this knowledge has been limited. Traditionally, diseases are researched and treated individually, which ignores the interconnectedness of age-related conditions, necessitates multiple treatments with unrelated substances, and increases the accumulative risk of side effects. In this study, we address and overcome this deadlock by creating adeno-associated virus (AAV)-based antiaging gene therapies for simultaneous treatment of several age-related diseases. We demonstrate the modular and extensible nature of combination gene therapy by testing therapeutic AAV cocktails that confront multiple diseases in a single treatment. We observed that 1 treatment comprising 2 AAV gene therapies was efficacious against all 4 diseases.  

5.2697           Established PABPN1 intranuclear inclusions in OPMD muscle can be efficiently reversed by AAV-mediated knockdown and replacement of mutant expanded PABPN1

Malerba, A., Klein, P., Lu-Nguyen, N., Capppellari, O., Strings-Uformbach, V., Harbaran, S., Roelvink, P., Suhy, D., Trollet, C. and Dickson, G. Hum. Mol. Genet., 28(19), 3301-3308 (2019)   Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant late-onset muscular dystrophy affecting approximately 1:100 000 individuals in Europe. OPMD is mainly characterized by progressive eyelid drooping (ptosis) and dysphagia although muscles of the limbs can also be affected late in life. This muscle disease is due to a trinucleotide repeat expansion in the polyA-binding protein nuclear-1 gene. Patients express a protein with an 11–18 alanine tract that is misfolded and prone to form intranuclear inclusions, which are the hallmark of the disease. Other features of OPMD include muscle fibrosis and atrophy in affected muscles. Currently, no pharmacological treatments are available, and OPMD patients can only be referred to surgeons for cricopharyngeal myotomy or corrective surgery of extraocular muscles to ease ptosis. We recently tested a two-AAV `silence’ and `replace’ vector-based gene therapy treatment in a mouse model of OPMD. We demonstrate here that this gene therapy approach can revert already established insoluble aggregates and partially rescues the muscle from atrophy, which are both crucially important since in most cases OPMD patients already have an established disease when diagnosed. This strategy also prevents the formation of muscle fibrosis and stabilizes the muscle strength to the level of healthy muscles. Furthermore, we show here that similar results can be obtained using a single AAV vector incorporating both the `silence’ and `replace’ cassettes. These results further support the application of a gene therapy approach as a novel treatment for OPMD in humans.  

5.2698           Brain Endothelial Cells Maintain Lactate Homeostasis and Control Adult Hippocampal Neurogenesis

Wang, J., Cui, Y., Yu, Z., Yang, H., Guo, W. and Yang, X. Cell Stem Cell, 25(6), 754-767 (2019)   Increased understanding of the functions of lactate has suggested a close relationship between lactate homeostasis and normal brain activity because of its importance as an energy source and signaling molecule. Here we show that lactate levels affect adult hippocampal neurogenesis. Cerebrovascular-specific deletion of PTEN causes learning and memory deficits and disrupts adult neurogenesis with accompanying lactate accumulation. Consistently, administering lactate to wild-type animals impairs adult hippocampal neurogenesis. The endothelial PTEN/Akt pathway increases monocarboxylic acid transporter 1 (MCT1) expression to enhance lactate transport across the brain endothelium. Moreover, cerebrovascular overexpression of MCT1 or deletion of Akt1 restores MCT1 expression, decreases lactate levels, and normalizes hippocampal neurogenesis and cognitive function in PTEN mutant mice. Together, these findings delineate how the brain endothelium maintains lactate homeostasis and contributes to adult hippocampal neurogenesis and cognitive functions.  

5.2699           A CD33 Antigen-Targeted AAV6 Vector Expressing an Inducible Caspase-9 Suicide Gene Is Therapeutic in a Xenotransplantation Model of Acute Myeloid Leukemia

Khan, N., Bammidi, S. and Jayandharan, G.R. Bioconjugate Chem., 30, 2404-2416 (2019)   Current chemotherapeutic regimens for acute myeloid leukemia (AML) have been modestly effective in patients and are associated with poor long-term survival (<30% at 5 years). Viral vector-based suicide gene therapy is an attractive option, if these vectors can target the AML cells with high specificity and efficiency. In this study, we have developed a receptor-specific adeno-associated virus (AAV) based vector to target the CD33 antigen which is overexpressed in leukemic cells. A targeting peptide was rationally designed from the antigen-binding regions of a CD33 monoclonal antibody. This peptide was further expressed on the capsid of the AAV6 vector, since this serotype was most efficient among AAV1-rh10 vectors to infect the pro-monocytic, human myeloid leukemia cells (U937). AAV6-CD33 vectors expressing a suicide gene, the inducible caspase 9 (iCasp9), and its prodrug AP20187 significantly reduced (∼59%) the viability of U937 cells. To further test its efficacy and specificity in vivo, AAV6-CD33 vectors were administered into a xenotransplantation model of AML in zebrafish through systemic delivery. We observed a significant antileukemic effect with AAV6-CD33 vectors, with a markedly higher survival (100% for AAV6-CD33 vectors vs 15% for mock-treated) and a higher number of TUNEL positive apoptotic cells after systemic vector delivery. Taken together, our work demonstrates the efficacy and translational potential of CD33-targeted AAV6 vectors for cytotoxic gene therapy in AML.  

5.2700           Comprehensive plasma and tissue profiling reveals systemic metabolic alterations in cardiac hypertrophy and failure

Müller, O., Heckmann, M.B., Ding, l., Rapti, K., Rangrez, A.Y., Gerken, T. et al Cardiovasc. Res., 115, 1296-1305 (2019)   Aims Heart failure is characterized by structural and metabolic cardiac remodelling. The aim of the present study is to expand our understanding of the complex metabolic alterations in the transition from pathological hypertrophy to heart failure and exploit the results from a translational perspective. Methods and results Mice were subjected to transverse aortic constriction (TAC) or sham surgery and sacrificed 2 weeks, 4 weeks, or 6 weeks after the procedure. Samples from plasma, liver, skeletal muscle, and heart were collected and analysed using metabolomics. Cardiac samples were also analysed by transcriptional profiling. Progressive alterations of key cardiac metabolic pathways and gene expression patterns indicated impaired mitochondrial function and a metabolic switch during transition to heart failure. Similar to the heart, liver, and skeletal muscle revealed significant metabolic alterations such as depletion of essential fatty acids and glycerolipids in late stages of heart failure. Circulating metabolites, particularly fatty acids, reflected cardiac metabolic defects, and deteriorating heart function. For example, inverse correlation was found between plasma and the heart levels of triacylglycerol (C18:1, C18:2, C18:3), and sphingomyelin (d18:1, C23:0) already at an early stage of heart failure. Interestingly, combining metabolic and transcriptional data from cardiac tissue revealed that decreased carnitine shuttling and transportation preceded mitochondrial dysfunction. We, thus, studied the therapeutic potential of OCTN2 (Organic Cation/Carnitine Transporter 2), an important factor for carnitine transportation. Cardiac overexpression of OCTN2 using an adeno-associated viral vector significantly improved ejection fraction and reduced interstitial fibrosis in mice subjected to TAC. Conclusion Comprehensive plasma and tissue profiling reveals systemic metabolic alterations in heart failure, which can be used for identification of novel biomarkers and potential therapeutic targets.  

5.2701           Corticostriatal Flow of Action Selection Bias

Hwang, E.J., Link, T.D., Hu, Y.Y., O’Neil, K., Lim, B.K. and Komiyama, T. Neuron, 104, 1126-1140 (2019)   The posterior parietal cortex (PPC) performs many functions, including decision making and movement control. It remains unknown which input and output pathways of PPC support different functions. We addressed this issue in mice, focusing on PPC neurons projecting to the dorsal striatum (PPC-STR) and the posterior secondary motor cortex (PPC-pM2). Projection-specific, retrograde labeling showed that PPC-STR and PPC-pM2 represent largely distinct subpopulations, with PPC-STR receiving stronger inputs from association areas and PPC-pM2 receiving stronger sensorimotor inputs. Two-photon calcium imaging during decision making revealed that the PPC-STR population encodes history-dependent choice bias more strongly than PPC-pM2 or general PPC populations. Furthermore, optogenetic inactivation of PPC-STR neurons or their terminals in STR decreased history-dependent bias, while inactivation of PPC-pM2 neurons altered movement kinematics. Therefore, PPC biases action selection through its STR projection while controlling movements through PPC-pM2 neurons. PPC may support multiple functions through parallel subpopulations, each with distinct input-output connectivity.  

5.2702           Plant-produced chimeric virus-like particles - a new generation vaccine against African horse sickness

Rutkowska, D.A., Mokoena, N.B., Tsekoa, T.L., Dibakwane, V.S. and O’Kennedy, M.M. BMC Veterinary Res., 15:432 (2019)   Background African horse sickness (AHS) is a severe arthropod-borne viral disease of equids, with a mortality rate of up to 95% in susceptible naïve horses. Due to safety concerns with the current live, attenuated AHS vaccine, alternate safe and effective vaccination strategies such as virus-like particles (VLPs) are being investigated. Transient plant-based expression systems are a rapid and highly scalable means of producing such African horse sickness virus (AHSV) VLPs for vaccine purposes. Results In this study, we demonstrated that transient co-expression of the four AHSV capsid proteins in agroinfiltrated Nicotiana benthamiana dXT/FT plants not only allowed for the assembly of homogenous AHSV-1 VLPs but also single, double and triple chimeric VLPs, where one capsid protein originated from one AHS serotype and at least one other capsid protein originated from another AHS serotype. Following optimisation of a large scale VLP purification procedure, the safety and immunogenicity of the plant-produced, triple chimeric AHSV-6 VLPs was confirmed in horses, the target species. Conclusions We have successfully shown assembly of single and double chimeric AHSV-7 VLPs, as well as triple chimeric AHSV-6 VLPs, in Nicotiana benthamiana dXT/FT plants. Plant produced chimeric AHSV-6 VLPs were found to be safe for administration into 6 month old foals as well as capable of eliciting a weak neutralizing humoral immune response in these target animals against homologous AHSV virus.  

5.2703           Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse

Zhang, J-P., Cheng, X-X., Zhao, M., Li, G-H., Xu, J. et al Genome Biol., 20:276 (2019)   Background Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%). Results We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the Alb locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target BDDF8 to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8 restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects. Conclusions These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8 at Alb introns after AAV-mediated delivery of editing components.    

5.2704           Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates

Elfarrash, S., Møller Jensen, N., Ferreira, n., Betzer, c., Thevathasan, J.V., Diekmann, R., Adel, m., Omar, N.M., Boraie, M., Gad, S., Ries, J., Kirik, D., Nabavi, S. and Jensen, P.H. Acta Neuropathologica Comm., 7:213 (2019)   Here we describe the use of an organotypic hippocampal slice model for studying α-synuclein aggregation and inter-neuronal spreading initiated by microinjection of pre-formed α-synuclein fibrils (PFFs). PFF injection at dentate gyrus (DG) templates the formation of endogenous α-synuclein aggregates in axons and cell bodies of this region that spread to CA3 and CA1 regions. Aggregates are insoluble and phosphorylated at serine-129, recapitulating Lewy pathology features found in Parkinson’s disease and other synucleinopathies. The model was found to favor anterograde spreading of the aggregates. Furthermore, it allowed development of slices expressing only serine-129 phosphorylation-deficient human α-synuclein (S129G) using an adeno-associated viral (AAV) vector in α-synuclein knockout slices. The processes of aggregation and spreading of α-synuclein were thereby shown to be independent of phosphorylation at serine-129. We provide methods and highlight crucial steps for PFF microinjection and characterization of aggregate formation and spreading. Slices derived from genetically engineered mice or manipulated using viral vectors allow testing of hypotheses on mechanisms involved in the formation of α-synuclein aggregates and their prion-like spreading.  

5.2705           NMDA Receptors Regulate Neuregulin 2 Binding to ER-PM Junctions and Ectodomain Release

Vullhorst, D. and Buonanno, A. Mol. Neurobiol., 56(12), 8345-8363 (2019)   Unprocessed pro-neuregulin 2 (pro-NRG2) accumulates on neuronal cell bodies at junctions between the endoplasmic reticulum and plasma membrane (ER-PM junctions). NMDA receptors (NMDARs) trigger NRG2 ectodomain shedding from these sites followed by activation of ErbB4 receptor tyrosine kinases, and ErbB4 signaling cell-autonomously downregulates intrinsic excitability of GABAergic interneurons by reducing voltage-gated sodium channel currents. NMDARs also promote dispersal of Kv2.1 clusters from ER-PM junctions and cause a hyperpolarizing shift in its voltage-dependent channel activation, suggesting that NRG2/ErbB4 and Kv2.1 work together to regulate intrinsic interneuron excitability in an activity-dependent manner. Here we explored the cellular processes underlying NMDAR-dependent NRG2 shedding in cultured rat hippocampal neurons. We report that NMDARs control shedding by two separate but converging mechanisms. First, NMDA treatment disrupts binding of pro-NRG2 to ER-PM junctions by post-translationally modifying conserved Ser/Thr residues in its intracellular domain. Second, using a mutant NRG2 protein that cannot be modified at these residues and that fails to accumulate at ER-PM junctions, we demonstrate that NMDARs also directly promote NRG2 shedding by ADAM-type metalloproteinases. Using pharmacological and shRNA-mediated knockdown, and metalloproteinase overexpression, we unexpectedly find that ADAM10, but not ADAM17/TACE, is the major NRG2 sheddase acting downstream of NMDAR activation. Together, these findings reveal how NMDARs exert tight control over the NRG2/ErbB4 signaling pathway, and suggest that NRG2 and Kv2.1 are co-regulated components of a shared pathway that responds to elevated extracellular glutamate levels.  

5.2706           Retina transduction by rAAV2 after intravitreal injection: comparison between mouse and rat

Dias, M.S., Araujo, V.G., Vasconcelos, T., Li, Q., Hauswirth, W.W., Linden, R. and Petrs-Silva, H. Gene Therapy, 26, 479-490 (2019)   Adeno-associated virus vectors (rAAV) are currently the most common vehicle used in clinical trials of retinal gene therapy, usually delivered through subretinal injections to target cells of the outer retina. However, targeting the inner retina requires intravitreal injections, a simple and safe procedure, which is effective for transducing the rodent retina, but still of low efficiency in the eyes of primates. We investigated whether adjuvant pharmacological agents may enhance rAAV transduction of the retinas of mouse and rat after intravitreal delivery. Tyrosine kinase inhibitors were highly efficient in mice, especially imatinib and genistein, and promoted transduction even of the outer retina. In rats, however, we report that they were not effective. Even with direct proteasomal inhibition in rats, the effects upon transduction were only minimal and restricted to the inner retina. Even tyrosine capsid mutant rAAVs in rats had a transduction profile similar to wtAAV. Thus, the differences between mouse and rat, in both eye size and the inner limiting membrane, compromise the efficiency of AAV vectors penetration from the vitreous into the retina, and impact the efficacy of strategies developed to enhance intravitreal retinal rAAV transduction. Further improvement of strategies, then are required.  

5.2707           Post‐translational modifications in capsid proteins of recombinant adeno‐associated virus (AAV) 1‐rh10 serotypes

Mary, B., Maurya, S., Arumugam, S., Kumar, V. and Jayandharan, J. FEBS J., 286(24), 4964-4981 (2019)   Post‐translational modifications in viral capsids are known to fine‐tune and regulate several aspects of the infective life cycle of several viruses in the host. Recombinant viruses that are generated in a specific producer cell line are likely to inherit unique post‐translational modifications during intra‐cellular maturation of its capsid proteins. Data on such post‐translational modifications in the capsid of recombinant adeno‐associated virus serotypes (AAV1‐rh10) is limited. We have employed liquid chromatography and mass spectrometry analysis to characterize post‐translational modifications in AAV1‐rh10 capsid protein. Our analysis revealed a total of 52 post‐translational modifications in AAV2‐AAVrh10 capsids, including ubiquitination (17%), glycosylation (36%), phosphorylation (21%), SUMOylation (13%) and acetylation (11%). While AAV1 had no detectable post‐translational modification, at least four AAV serotypes had >7 post‐translational modifications in their capsid protein. About 82% of these post‐translational modifications are novel. A limited validation of AAV2 capsids by MALDI‐TOF and western blot analysis demonstrated minimal glycosylation and ubiquitination of AAV2 capsids. To further validate this, we disrupted a glycosylation site identified in AAV2 capsid (AAV2‐N253Q), which severely compromised its packaging efficiency (~ 100‐fold vs. AAV2 wild‐type vectors). In order to confirm other post‐translational modifications detected such as SUMOylation, mutagenesis of a SUMOylation site(K258Q) in AAV2 was performed. This mutant vector demonstrated reduced levels of SUMO‐1/2/3 proteins and negligible transduction, 2 weeks after ocular gene transfer. Our study underscores the heterogeneity of post‐translational modifications in AAV vectors. The data presented here, should facilitate further studies to understand the biological relevance of post‐translational modifications in AAV life cycle and the development of novel bioengineered AAV vectors for gene therapy applications.  

5.2708           Functional identity of hypothalamic melanocortin neurons depends on Tbx3

Quarta, C., Fisette, A., Xu, Y., Collden, G., Legutko, B., Tseng, Y-T. et al Nature Metabolism, 1, 222-235 (2019)   Heterogeneous populations of hypothalamic neurons orchestrate energy balance via the release of specific signatures of neuropeptides. However, how specific intracellular machinery controls peptidergic identities and function of individual hypothalamic neurons remains largely unknown. The transcription factor T-box 3 (Tbx3) is expressed in hypothalamic neurons sensing and governing energy status, whereas human TBX3 haploinsufficiency has been linked with obesity. Here, we demonstrate that loss of Tbx3 function in hypothalamic neurons causes weight gain and other metabolic disturbances by disrupting both the peptidergic identity and plasticity of Pomc/Cart and Agrp/Npy neurons. These alterations are observed after loss of Tbx3 in both immature hypothalamic neurons and terminally differentiated mouse neurons. We further establish the importance of Tbx3 for body weight regulation in Drosophila melanogaster and show that TBX3 is implicated in the differentiation of human embryonic stem cells into hypothalamic Pomc neurons. Our data indicate that Tbx3 directs the terminal specification of neurons as functional components of the melanocortin system and is required for maintaining their peptidergic identity. In summary, we report the discovery of a key mechanistic process underlying the functional heterogeneity of hypothalamic neurons governing body weight and systemic metabolism.  

5.2709           The lipid-droplet-associated protein ABHD5 protects the heart through proteolysis of HDAC4

Jebessa, Z., Shanmukha, K.D., Dewenter, m., lehmann, L.H., Xu, C. et al Nature Metabolism, 1, 1157-1167 (2019)   Catecholamines stimulate the first step of lipolysis through protein kinase A (PKA)-dependent release of the lipid-droplet-associated protein abhydrolase domain containing 5 (ABHD5) from perilipin to coactivate the lipase adipose triglyceride lipase (ATGL). Here, we unmask a proteolytic and cardioprotective function of ABHD5. ABHD5 acts in vivo and in vitro as a serine protease that cleaves histone deacetylase 4 (HDAC4). Through the production of an amino-terminal polypeptide of HDAC4 (HDAC4-NT), ABHD5 inhibits MEF2-dependent gene expression and thereby controls glucose handling. ABHD5 deficiency leads to neutral-lipid storage disease in mice. Cardiac-specific gene therapy using the gene encoding HDAC4-NT does not protect against intracardiomyocyte lipid accumulation, but strikingly protects against heart failure, thereby challenging the concept of lipotoxicity-induced heart failure. ABHD5 levels are reduced in failing human hearts, and murine transgenic ABHD5 expression protects against pressure-overload-induced heart failure. These findings represent a conceptual advance by connecting lipid with glucose metabolism through HDAC4 proteolysis, and enable new translational approaches to treating cardiometabolic disease.  

5.2710           Rational Engineering and Preclinical Evaluation of Neddylation and SUMOylation Site Modified Adeno-Associated Virus Vectors in Murine Models of Hemophilia B and Leber Congenital Amaurosis

Maurya, S., Mary, B. and Jayandharan, G.R. Hum. Gen. Ther., 30(12), 1461-1476 (2019)   Synthetic engineering of viral vectors such as adeno-associated virus (AAV) is crucial to overcome host transduction barriers observed during clinical gene therapy. We reasoned that exploring the role of cellular ubiquitin-like modifiers (UBLs) such as Neddylation or SUMOylation during AAV transduction could be beneficial. Using a combination of in silico biochemical and molecular engineering strategies, we have studied the impact of these UBLs during AAV2 infection and further developed Neddylation or SUMOylation site–modified AAV vectors and validated them in multiple disease models in vitro and in vivo. Hepatic gene transfer of two novel vectors developed, K105Q (SUMOylation-site mutant) and K665Q (Neddylation-site mutant), demonstrated a significantly improved human coagulation factor (F) IX expression (up to two-fold) in a murine model of hemophilia B. Furthermore, subretinal gene transfer of AAV2-K105Q vector expressing RPE65 gene demonstrated visual correction in a murine model of a retinal degenerative disease (rd12 mice). These vectors did not have any adverse immunogenic events in vivo. Taken together, we demonstrate that gene delivery vectors specifically engineered at UBLs can improve the therapeutic outcome during AAV-mediated ocular or hepatic gene therapy.  

5.2711           The Influence of Murine Genetic Background in Adeno-Associated Virus Transduction of the Mouse Brain

He, T., Itano, S., Earley, L.F., Hall, N.E., Riddick, N., Samulski, R.J. and Li, C. Hum. Gene. Ther. Clin. Develop., 30(4), 169-181 (2019)   Adeno-associated virus (AAV) vectors have become an important tool for delivering therapeutic genes for a wide range of neurological diseases. AAV serotypes possess differential cellular tropism in the central nervous system. Although several AAV serotypes or mutants have been reported to transduce the brain efficiently, conflicting data occur across studies with the use of various rodent strains from different genetic backgrounds. Herein, we performed a systematic comparison of the brain transduction properties among five AAV serotypes (AAV2, 5, 7, 8, and 9) in two common rodent strains (C57BL/6J and FVB/N), following local intrastriatal injection of AAV vectors encoding enhanced green fluorescent protein (EGFP) driven by the CBh promoter. Important differences were found regarding overall cellular tropism and transduction efficiency, including contralateral transduction among the AAV serotypes and between the mouse strains. We have further found loss of NeuN-immunoreactivity and microglial activation from AAV transduction in the different mouse strains. The important strain-specific differences from our study suggest that the genetic background of the mouse may affect AAV serotype transduction properties in the brain. These data can provide valuable information about how to choose an effective AAV vector for clinical application and interpret the data obtained from preclinical studies and clinical trials.   Sevoflurane Exerts an Anti-depressive Action by Blocking the HMGB1/TLR4 Pathway in Unpredictable Chronic Mild Stress Rats Guo, Z., Zhao, F., Wang, Y., Wang, Y., Geng, M., Zhang, Y., Ma, Q. and Xu, X.

1. Mol. Neurosci., 69, 546-556 (2019)

  This study was performed to investigate whether sevoflurane has an anti-depressive effect and to elucidate its underlying mechanism. Unpredictable chronic mild stress (uCMS)–treated rats were used for inducing depressive-like behavior and subsequently treated with sevoflurane. A forced swimming test was conducted with the rats. An ELISA was performed to detect the levels of brain-derived neurotrophic factor (BDNF) and inflammatory cytokines in the hippocampus of the rats. Differentially expressed genes in uCMS and normal rats were analyzed by microarray. qRT-PCR, western blot, and flow cytometry, and gain and loss of function measurements were carried out to determine the association between sevoflurane and the HMGB1/TLR4 pathway. A forced swimming test with uCMS rats exposed to sevoflurane demonstrated that a 2% sevoflurane treatment resulted in an anti-depressive effect. In addition, ELISAs of TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1 beta), IL-6 (interleukin-6), and BDNF supported an effect of sevoflurane on inflammatory cytokines and a neurotrophic factor. HMGB1 was dramatically induced in uCMS rats, and the HMGB1/TLR4 pathway was implicated in sevoflurane exposure. A 2% sevoflurane treatment resulted in a restoration of HMGB1/TLR4 signaling and expression of cytokines and BDNF. HMGB1 overexpression partially prevented the protective effect of 2% SF, suggesting sevoflurane protects uCMS rats.  

5.2712           Targeted viral vector transduction of relaxin-3 neurons in the rat nucleus incertus using a novel cell-type specific promoter

Wykes, A.D., Ma, S., Bathgate, R.A.D.. and Gundlach, A.L. IBRO Reports, 8, 1-10 (2020)   Modern neuroscience utilizes transgenic techniques extensively to study the activity and function of brain neural networks. A key feature of this approach is its compatibility with molecular methods for selective transgene expression in neuronal circuits of interest. Until now, such targeted transgenic approaches have not been applied to the extensive circuitry involving the neuropeptide, relaxin-3. Pharmacological and gene knock-out studies have revealed relaxin-3 signalling modulates interrelated behaviours and cognitive processes, including stress and anxiety, food and alcohol consumption, and spatial and social memory, highlighting the potential of this system as a therapeutic target. In the present study, we aimed to identify a promoter sequence capable of regulating cell-type specific transgene expression from an adeno-associated viral (AAV) vector in relaxin-3 neurons of the rat nucleus incertus (NI). In parallel to relaxin-3 promoter sequences, we also tested an AAV vector containing promoter elements for the tropomyosin receptor kinase A (TrkA) gene, as TrkA is co-expressed with relaxin-3 in rat NI neurons. Stereotaxic injection of an mCherry-expressing AAV vector revealed widespread non-specific TrkA promoter (880 bp) activity in and adjacent to the NI at 8 weeks post-treatment. In contrast, mCherry expression was successfully restricted to relaxin-3 NI neurons with 98% specificity using a 1736 bp relaxin-3 promoter. In addition to detailed anatomical mapping of NI relaxin-3 networks, illustrated here in association with GABAergic medial septum neurons, this method for targeted transgene delivery offers a versatile tool for ongoing preclinical studies of relaxin-3 circuitry.  

5.2713           rAAV2-Retro Enables Extensive and High-Efficient Transduction of Lower Motor Neurons following Intramuscular Injection

Chen, Z., Fan, G., Li, A., Yuan, j. and Xu, T. Molecular Therapy-Methods Clin. Develop., 17, 21-33 (2020)   The motor system controls muscle movement through lower motor neurons in the spinal cord and brainstem. Lower motor neurons are efferent neurons in the central nervous system (CNS) characterized by axonal projections that reach specific targets in the periphery. Lower motor neuron lesions result in the denervation and dysfunction of peripheral skeletal muscle. Great progress has been made to develop therapeutic strategies to transduce lower motor neurons with genes. However, the widespread distribution of lower motor neurons makes their specific, extensive, and efficient transduction a challenge. In this study, we demonstrated that, compared to the other tested recombinant adeno-associated virus (rAAV) serotypes, rAAV2-retro mediated the most efficient retrograde transduction of lower motor neurons in the spinal cord following intramuscular injection in neonatal mice. A single injection of rAAV2-retro in a single muscle enabled the efficient and extensive transduction of lower motor neurons in the spinal cord and brainstem rather than transducing only the lower motor neurons connected to the injected muscle. rAAV2-retro achieved the extensive transduction of lower motor neurons by the cerebrospinal fluid pathway. Our work suggests that gene delivery via the intramuscular injection of rAAV2-retro represents a promising tool in the development of gene therapy strategies for motor neuron diseases.  

5.2714           Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries

Schmit, P.F., Pacouret, S., Zinn, E., Telford, E., Nicolaou, F., Broucque, F., Andres-Mateos, E., Xiao, R., Penaud-Budloo, M., Bouzelha, M., Jaulin, N., Adjali, O., Ayuso, E. and Vandernberghe, L.H. Molecular Therapy-Methods Clin. Develop., 17, 107-121 (2020)   Generation and screening of libraries of adeno-associated virus (AAV) variants have emerged as a powerful method for identifying novel capsids for gene therapy applications. For the majority of libraries, vast population diversity requires multiplexed production, in which a library of inverted terminal repeat (ITR)-containing plasmid variants is transfected together into cells to generate the viral library. This process has the potential to be confounded by cross-packaging and mosaicism, in which particles are comprised of genomes and capsid monomers derived from different library members. Here, we investigate the prevalence of cross-packaging and mosaicism in simplified, minimal libraries using novel assays designed to assess capsid composition and packaging fidelity. We show that AAV library variants are prone to cross-packaging and capsid mosaic formation when produced at high plasmid levels, although to a lesser extent than in a recombinant context. We also provide experimental evidence that dilution of input library DNA significantly increases capsid monomer homogeneity and increases capsid:genome correlation in AAV libraries. Lastly, we determine that similar dilution methods yield higher-quality libraries when used for in vivo screens. Together, these findings quantitatively characterized the prevalence of cross-packaging and mosaicism in AAV libraries and established conditions that minimize related noise in subsequent screens.  

5.2715           AAV-Mediated CRISPR/Cas9 Gene Editing in Murine Phenylketonuria

Richards, D.Y., Winn, S.R., Dudley, S., Nygaard, S., Mighell, T.L., Grompe, M. and Harding, C.O. Molecular Therapy-Metehods Clin. Develop., 17, 234-245 (2020)   Phenylketonuria (PKU) due to recessively inherited phenylalanine hydroxylase (PAH) deficiency results in hyperphenylalaninemia, which is toxic to the central nervous system. Restriction of dietary phenylalanine intake remains the standard of PKU care and prevents the major neurologic manifestations of the disease, yet shortcomings of dietary therapy remain, including poor adherence to a difficult and unpalatable diet, an increased incidence of neuropsychiatric illness, and imperfect neurocognitive outcomes. Gene therapy for PKU is a promising novel approach to promote lifelong neurological protection while allowing unrestricted dietary phenylalanine intake. In this study, liver-tropic recombinant AAV2/8 vectors were used to deliver CRISPR/Cas9 machinery and facilitate correction of the Pah^enu2 allele by homologous recombination. Additionally, a non-homologous end joining (NHEJ) inhibitor, vanillin, was co-administered with the viral drug to promote homology-directed repair (HDR) with the AAV-provided repair template. This combinatorial drug administration allowed for lifelong, permanent correction of the Pah^enu2 allele in a portion of treated hepatocytes of mice with PKU, yielding partial restoration of liver PAH activity, substantial reduction of blood phenylalanine, and prevention of maternal PKU effects during breeding. This work reveals that CRISPR/Cas9 gene editing is a promising tool for permanent PKU gene editing.  

5.2716           A NeuroD1 AAV-Based Gene Therapy for Functional Brain Repair after Ischemic Injury through In Vivo Astrocyte-to-Neuron Conversion

Chen, Y-C., Ma, N-X., Pei, Z-F., Wu, Z., Do-Monte, F.H., Keefe, S. Yellin, E. et al Molecular Therapy, 28(1), 217-234 (2020)   Adult mammalian brains have largely lost neuroregeneration capability except for a few niches. Previous studies have converted glial cells into neurons, but the total number of neurons generated is limited and the therapeutic potential is unclear. Here, we demonstrate that NeuroD1-mediated in situ astrocyte-to-neuron conversion can regenerate a large number of functional new neurons after ischemic injury. Specifically, using NeuroD1 adeno-associated virus (AAV)-based gene therapy, we were able to regenerate one third of the total lost neurons caused by ischemic injury and simultaneously protect another one third of injured neurons, leading to a significant neuronal recovery. RNA sequencing and immunostaining confirmed neuronal recovery after cell conversion at both the mRNA level and protein level. Brain slice recordings found that the astrocyte-converted neurons showed robust action potentials and synaptic responses at 2 months after NeuroD1 expression. Anterograde and retrograde tracing revealed long-range axonal projections from astrocyte-converted neurons to their target regions in a time-dependent manner. Behavioral analyses showed a significant improvement of both motor and cognitive functions after cell conversion. Together, these results demonstrate that in vivo cell conversion technology through NeuroD1-based gene therapy can regenerate a large number of functional new neurons to restore lost neuronal functions after injury.  

5.2717           dCas9-Based Scn1a Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice

Colasante, G., Lignani, G., Brusco, S., Di Berardino, C., Carpenter, J., Giannelli, S. et al Molecular Therapy, 28(1), 235-253 (2020)   Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Na_v1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Na_v1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recovered their firing ability, and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach to DS and other disorders resulting from altered gene dosage.  

5.2718           Viral metagenomic analysis of the cheese surface: A comparative study of rapid procedures for extracting viral particles

Dugat-Bony, E., Lossouarn, J., De Paepe, m., Sarthou, A-S., Fedala, Y., Petit, M-A. and Chailou, S. Food Microbiol., 85, 103278 (2020)   The structure and functioning of microbial communities from fermented foods, including cheese, have been extensively studied during the past decade. However, there is still a lack of information about both the occurrence and the role of viruses in modulating the function of this type of spatially structured and solid ecosystems. Viral metagenomics was recently applied to a wide variety of environmental samples and standardized procedures for recovering viral particles from different type of materials has emerged. In this study, we adapted a procedure originally developed to extract viruses from fecal samples, in order to enable efficient virome analysis of cheese surface. We tested and validated the positive impact of both addition of a filtration step prior to virus concentration and substitution of purification by density gradient ultracentrifugation by a simple chloroform treatment to eliminate membrane vesicles. Viral DNA extracted from the several procedures, as well as a vesicle sample, were sequenced using Illumina paired-end MiSeq technology and the subsequent clusters assembled from the virome were analyzed to assess those belonging to putative phages, plasmid-derived DNA, or even from bacterial chromosomal DNA. The best procedure was then chosen, and used to describe the first cheese surface virome, using Epoisses cheese as example. This study provides the basis of future investigations regarding the ecological importance of viruses in cheese microbial ecosystems.  

5.2719           In experimental challenge with infectious clones of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), MrNV alone can cause mortality in freshwater prawn (Macrobrachium rosenbergii)

Gangnonngiw, W., Bunnontae, M., Phiwsaiya, K., Senapin, S. and Dhar, A.K. Virology, 540, 30-37 (2020)   To overcome the lack of immortal shrimp cell lines for shrimp viral research, we constructed and tested DNA infectious clones of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) often found together in freshwater prawn (M. rosenbergii) exhibiting white tail disease (WTD). Full-length cDNAs of MrNV and XSV genomic RNA were individually inserted into the baculovirus pFastBacDUAL shuttle vector. Individual Sf9 (insect cell line) transfection resulted in production of RNA (RT-PCR) and capsid proteins (immunofluorescence) for both viruses. Presence of respective virions was confirmed by density gradient purification followed by RT-PCR and transmission electron microscopy. Infectivity was by tested in immersion-challenge tests with M. rosenbergii post-larvae (PL) using both semi-purified viruses, individually or combined, and confirmed by histological analysis (morphology and immunofluorescence) and quantitative RT-PCR. Mortality accompanied by WTD lesions occurred with MrNV alone or in combination with XSV but not with XSV alone, despite its replication.  

5.2720           Ex Vivo/In vivo Gene Editing in Hepatocytes Using “All-in-One” CRISPR-Adeno-Associated Virus Vectors with a Self-Linearizing Repair Template

Kroooss, S.A:, Dai, Z., Schmidt, F., Sharma, A.D., Büning, H. and Ott, M. iScience, 23, 100764 (2020)   Adeno-associated virus (AAV)-based vectors are considered efficient and safe gene delivery systems in gene therapy. We combined two guide RNA genes, Cas9, and a self-linearizing repair template in one vector (AIO-SL) to correct fumarylacetoacetate hydrolase (FAH) deficiency in mice. The vector genome of 5.73 kb was packaged into VP2-depleted AAV particles (AAV2/8^ΔVP2), which, however, did not improve cargo capacity. Reprogrammed hepatocytes were treated with AIO-SL.AAV2^ΔVP2 and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8^ΔVP2 likewise led to FAH expression and long-term survival. The AIO-SL vector achieved an ∼6-fold higher degree of template integration than vectors without template self-linearization. Subsequent analysis revealed that AAV8 particles, in contrast to AAV2, incorporate oversized genomes distinctly greater than 5.2 kb. Finally, our AAV8-based vector represents a promising tool for gene editing strategies to correct monogenic liver diseases requiring (large) fragment removal and/or simultaneous sequence replacement.  

5.2721           Intrinsic Differential Scanning Fluorimetry for Fast and Easy Identification of Adeno-Associated Virus Serotypes

Rieser, R., Penaud-Budloo, M., Bouzelha, M., Rossi, A., Menzen, T., Biel, M., Büning, H., Ayuso, E., Winter, G. and Michalakis, S.

1. Pharmaceut. Sci., 109, 854-862 (2020)

  Recombinant adeno-associated virus (AAV) vectors have evolved as the most promising technology for gene therapy due to their good safety profile, high transduction efficacy, and long-term gene expression in non-dividing cells. AAV-based gene therapy holds great promise for treating genetic disorders like inherited blindness, muscular atrophy, or bleeding disorders. Multiple naturally occurring and engineered AAV serotypes exist, which differ in capsid sequence and as a consequence in cellular tropism. Individual AAV capsids differ in thermal stability and have a characteristic melting temperature (T_m), which enables serotype-specific discrimination of AAV vectors. Differential scanning fluorimetry (DSF) combined with a dye-like SYPRO Orange (SO-DSF), which binds to hydrophobic regions of unfolded proteins, has been successfully applied to determine the T_m of AAV capsids. Here, we present DSF measurement of intrinsic fluorescence signal (iDSF) as a simple alternative method for determination of AAV capsid T_m. The study demonstrates that DSF measurement of intrinsic fluorescence signal is a simple, accurate, and rapid alternative to SO-DSF, which enables characterization of AAV capsid stability with excellent precision and without the need of SO or any other dye.  

5.2722           Viral expression of a SERCA2a-activating PLB mutant improves calcium cycling and synchronicity in dilated cardiomyopathic hiPSC-CMs

Stroik, D.R., Ceholski, D.K., Bidwell, P.A., Mleczko, J., Thanel, P.F., Kamdar, F., Autry, J.M., Cornea, R.L. and Thomas, D.D.

1. Mol. Cell. Cardiol., 138, 59-65 (2020)

  There is increasing momentum toward the development of gene therapy for heart failure (HF) that is defined by impaired calcium (Ca²⁺) transport and reduced contractility. We have used FRET (fluorescence resonance energy transfer) between fluorescently-tagged SERCA2a (the cardiac Ca²⁺ pump) and PLB (phospholamban, ventricular peptide inhibitor of SERCA) to test directly the effectiveness of loss-of-inhibition/gain-of-binding (LOI/GOB) PLB mutants (PLB_M) that were engineered to compete with the binding of inhibitory wild-type PLB (PLB_WT). Our therapeutic strategy is to relieve PLB_WT inhibition of SERCA2a by using the reserve adrenergic capacity mediated by PLB to enhance cardiac contractility. Using a FRET assay, we determined that the combination of a LOI PLB mutation (L31A) and a GOB PLB mutation (I40A) results in a novel engineered LOI/GOB PLB_M (L31A/I40A) that effectively competes with PLB_WT binding to cardiac SERCA2a in HEK293-6E cells. We demonstrated that co-expression of PLB_M enhances SERCA Ca-ATPase activity by increasing enzyme Ca²⁺ affinity (1/K_Ca) in PLB_WT-inhibited HEK293 cell homogenates. For an initial assessment of PLB_M physiological effectiveness, we used human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) from a healthy individual. In this system, we observed that adeno-associated virus 2 (rAAV2)-driven expression of PLB_M enhances the amplitude of SR Ca²⁺ release and the rate of SR Ca²⁺ re-uptake. To assess therapeutic potential, we used a hiPSC-CM model of dilated cardiomyopathy (DCM) containing PLB mutation R14del, where we observed that rAAV2-driven expression of PLB_M rescues arrhythmic Ca²⁺ transients and alleviates decreased Ca²⁺ transport. Thus, we propose that PLB_M transgene expression is a promising gene therapy strategy that directly targets the underlying pathophysiology of abnormal Ca²⁺ transport and thus contractility in underlying systolic heart failure.  

5.2723           Influence of cell-penetrating peptides on the activity and stability of virus-based nanoparticles

Vanova, J., Hejtmankova, A., Suchanova, J.Z., Sauerova, P., Forstova, J., Kalbacova, M.H. and Spanielova, H. Int. J. Pharmaceut., 576, 119008 (2020)   Viral nanoparticles represent potential natural versatile platforms for targeted gene and drug delivery. Improving the efficiency of gene transfer mediated by viral vectors could not only enhance their therapeutic potential, but also contribute to understanding the limitations in interactions of nanoparticles with cells and the development of new therapeutic approaches. In this study, four cell-penetrating peptides (CPPs), cationic octaarginine (R8), histidine-rich peptides (LAH4 and KH27K) and fusogenic peptide (FUSO), are investigated for their effect on infection by mouse polyomavirus (MPyV) or on transduction of reporter genes delivered by MPyV or related viral vectors. Peptides noncovalently associated with viral particles enhance gene transfer (with the exception of FUSO). Removal of cellular heparan sulfates by the heparinase does not significantly change the enhancing potential of CPPs. Instead, CPPs influences the physical state of viral particles: R8 slightly destabilizes the intact virus, KH27K induces its aggregation and LAH4 promotes disassembly and aggregation of the particles that massively and rapidly associate with cells. The findings indicate that peptides acting as transduction-enhancing agents of polyomavirus-based nanoparticles modulate their physical state, which can be an important prerequisite for sensitization of cells and determination of the further fate of viral particles inside cells.  

5.2724           In vivo elongation of thin filaments results in heart failure

Mi-Mi, L., Farman, G.P., Mayfiled, R.M., Strom, J., Chu, M., Pappas, C.T. and Gregorio, C.C. PloS One, 15(1), e0226138 (2020)   A novel cardiac-specific transgenic mouse model was generated to identify the physiological consequences of elongated thin filaments during post-natal development in the heart. Remarkably, increasing the expression levels in vivo of just one sarcomeric protein, Lmod2, results in ~10% longer thin filaments (up to 26% longer in some individual sarcomeres) that produce up to 50% less contractile force. Increasing the levels of Lmod2 in vivo (Lmod2-TG) also allows us to probe the contribution of Lmod2 in the progression of cardiac myopathy because Lmod2-TG mice present with a unique cardiomyopathy involving enlarged atrial and ventricular lumens, increased heart mass, disorganized myofibrils and eventually, heart failure. Turning off of Lmod2 transgene expression at postnatal day 3 successfully prevents thin filament elongation, as well as gross morphological and functional disease progression. We show here that Lmod2 has an essential role in regulating cardiac contractile force and function.  

5.2725           Efficacy of a plant‐produced virus‐like particle vaccine in chickens challenged with Influenza A H6N2 virus

Smith, T., O’Kennedy, M.M., Wandrag, D.B.R., Adeyemi, M. and Abolnik, C. Plant Biotechnol. J., 18(2), 502-512 (2020)   The efficacy, safety, speed, scalability and cost‐effectiveness of producing hemagglutinin‐based virus‐like particle (VLP) vaccines in plants are well‐established for human influenza, but untested for the massive poultry influenza vaccine market that remains dominated by traditional egg‐grown oil‐emulsion whole inactivated virus vaccines. For optimal efficacy, a vaccine should be closely antigenically matched to the field strain, requiring that influenza A vaccines be updated regularly. In this study, an H6 subtype VLP transiently expressed in Nicotiana benthamiana was formulated into a vaccine and evaluated for efficacy in chickens against challenge with a heterologous H6N2 virus. A single dose of the plant‐produced H6 VLP vaccine elicited an immune response comparable to two doses of a commercial inactivated H6N2 vaccine, with mean hemagglutination inhibition titres of 9.3 log₂ and 8.8 log₂, respectively. Compared to the non‐vaccinated control, the H6 VLP vaccine significantly reduced the proportion of shedders and the magnitude of viral shedding by >100‐fold in the oropharynx and >6‐fold in the cloaca, and shortened oropharyngeal viral shedding by at least a week. Despite its potency, the cost of the antigenic mismatch between the inactivated H6N2 vaccine and challenge strain was evident not only in this vaccine's failure to reduce viral shedding compared to the non‐vaccinated group, but its apparent exacerbation of oropharyngeal viral shedding until 21 days post‐challenge. We estimate that a kilogram of plant leaf material can produce H6 VLP vaccines sufficient for between 5000 and 30 000 chickens, depending on the effective dose and whether one or two immunizations are administered.  

5.2726           Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

Park, J-H., Burgess, J.D., Faroqi, A.H., DeMeo, N.N., Fiesel, F.C., Springer, W., Delenclos, M. and McLean, P.J. Mol. Neurodegeneration, 15:5 (2020)   Background Misfolding and aggregation of the presynaptic protein alpha-synuclein (αsyn) is a hallmark of Parkinson’s disease (PD) and related synucleinopathies. Although predominantly localized in the cytosol, a body of evidence has shown that αsyn localizes to mitochondria and contributes to the disruption of key mitochondrial processes. Mitochondrial dysfunction is central to the progression of PD and mutations in mitochondrial-associated proteins are found in familial cases of PD. The sirtuins are highly conserved nicotinamide adenine dinucleotide (NAD⁺)-dependent enzymes that play a broad role in cellular metabolism and aging. Interestingly, mitochondrial sirtuin 3 (SIRT3) plays a major role in maintaining mitochondrial function and preventing oxidative stress, and is downregulated in aging and age-associated diseases such as neurodegenerative disorders. Herein, we hypothesize that αsyn is associated with decreased SIRT3 levels contributing to impaired mitochondrial dynamics and biogenesis in PD. Methods The level of mitochondrial SIRT3 was assessed in cells expressing oligomeric αsyn within the cytosolic and mitochondrial-enriched fractions. Mitochondrial integrity, respiration, and health were examined using several markers of mitochondrial dynamics and stress response and by measuring the rate of oxygen consumption (OCR). Our findings were validated in a rodent model of PD as well as in human post-mortem Lewy body disease (LBD) brain tissue. Results Here, we demonstrate that αsyn associates with mitochondria and induces a decrease in mitochondrial SIRT3 levels and mitochondrial biogenesis. We show that SIRT3 downregulation is accompanied by decreased phosphorylation of AMPK and cAMP-response element binding protein (CREB), as well as increased phosphorylation of dynamin-related protein 1 (DRP1), indicative of impaired mitochondrial dynamics. OCR was significantly decreased suggesting a mitochondria respiratory deficit. Interestingly treatment with AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) restores SIRT3 expression, improves mitochondrial function, and decreases αsyn oligomer formation in a SIRT3-dependent manner. Conclusions Together, our findings suggest that pharmacologically increasing SIRT3 levels can counteract αsyn-induced mitochondrial dysfunction by reducing αsyn oligomers and normalizing mitochondrial bioenergetics. These data support a protective role for SIRT3 in PD-associated pathways and contribute significant mechanistic insight into the interplay of SIRT3 and αsyn.  

5.2727           Expansion, in vivo–ex vivo cycling, and genetic manipulation of primary human hepatocytes

Michailidis, E., Vercauteren, K., Mancio-Silva, L., Andrus, l., Jahan, C. et al PNAS, 117(3), 1678-1688 (2020)   Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum. mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.  

5.2728           Robust hepatitis E virus infection and transcriptional response in human hepatocytes

Todt, D., Friesland, M., Moeller, N., Praditya, D., Kinast, V., Brüggemann, Y et al PNAS, 117(3), 1731-1741 (2020)   Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and the leading cause for acute viral hepatitis worldwide. The virus is classified as a member of the genus Orthohepevirus A within the Hepeviridae family. Due to the absence of a robust cell culture model for HEV infection, the analysis of the viral life cycle, the development of effective antivirals and a vaccine is severely limited. In this study, we established a protocol based on the HEV genotype 3 p6 (Kernow C-1) and the human hepatoma cell lines HepG2 and HepG2/C3A with different media conditions to produce intracellular HEV cell culture-derived particles (HEVcc) with viral titers between 10⁵ and 10⁶ FFU/mL. Viral titers could be further enhanced by an HEV variant harboring a mutation in the RNA-dependent RNA polymerase. These HEVcc particles were characterized in density gradients and allowed the trans-complementation of subgenomic reporter HEV replicons. In addition, in vitro produced intracellular-derived particles were infectious in liver-humanized mice with high RNA copy numbers detectable in serum and feces. Efficient infection of primary human and swine hepatocytes using the developed protocol could be observed and was inhibited by ribavirin. Finally, RNA sequencing studies of HEV-infected primary human hepatocytes demonstrated a temporally structured transcriptional defense response. In conclusion, this robust cell culture model of HEV infection provides a powerful tool for studying viral–host interactions that should facilitate the discovery of antiviral drugs for this important zoonotic pathogen.  

5.2729           Infectious cell culture system for concurrent propagation and purification of Megalocytivirus ISKNV and nervous necrosis virus from Asian Sea bass (Lates calcarifer)

Jitrakorn, S., Gangnoongiw, W., Bunnontae, M., Manajt, O., Rattanarojpong, T., Chaivisuthangkura, P., Dong, H.T. and Saksmerpome, V. Aquaculture, 520, 734931 (2020)   Megalocytivirus ISKNV and nervous necrosis virus (NNV) are two major viral pathogens affecting marine fish farms in Asia Pacific region. The present study reports an unexpected discovery of dual infections of both viruses in a single Asian sea bass (Lates calcarifer) farm experiencing ~50% cumulative fish mortality. A commercial Grunt Fin (GF) cell line was able to propagate concurrently two viruses from the clinical samples. Each virus was successfully purified from the co-infected cell culture, and its morphology was visualized by transmission electron microscopy (TEM). Subsequently, two viruses were identified as Megalocytivirus ISKNV and NNV from specific PCR assays using the extracted nucleic acids and sequencing of amplified PCR products. Approximately 10⁴ and 10³ copies of Megalocytivirus ISKNV and NNV were estimated in 200 ng DNA and RNA extracted from the co-infected cells. NNV propagation in GF cells was specifically confirmed using immunofluorescence microscopy. This is the first report for capability of NNV to propagate in the commercial GF cell line. This investigation opens up a promising in vitro model for studying concurrent infections of Megalocytivirus ISKNV and NNV and suggests a possible platform for concurrent production of Megalocytivirus ISKNV and NNV for bivalent vaccine.  

5.2730           Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses

Levy, J.M., Yeh, W-H., Pendse, N., Davis, J.R., Hennessey, E., Butcher, R., Koblan, L.W., Comander, J., Liu, Q. and Liu, D.R.   The success of base editors for the study and treatment of genetic diseases depends on the ability to deliver them in vivo to the relevant cell types. Delivery via adeno-associated viruses (AAVs) is limited by AAV packaging capacity, which precludes the use of full-length base editors. Here, we report the application of dual AAVs for the delivery of split cytosine and adenine base editors that are then reconstituted by trans-splicing inteins. Optimized dual AAVs enable in vivo base editing at therapeutically relevant efficiencies and dosages in the mouse brain (up to 59% of unsorted cortical tissue), liver (38%), retina (38%), heart (20%) and skeletal muscle (9%). We also show that base editing corrects, in mouse brain tissue, a mutation that causes Niemann–Pick disease type C (a neurodegenerative ataxia), slowing down neurodegeneration and increasing lifespan. The optimized delivery vectors should facilitate the efficient introduction of targeted point mutations into multiple tissues of therapeutic interest.  

5.2731           The emergence of transcriptional identity in somatosensory neurons

Sharma, N., Flaherty, K., Lezgiyeva, K., Wagner, D.E., Klein, A.M. and Ginty, D.D: Nature, 577, 392-398 (2020)   More than twelve morphologically and physiologically distinct subtypes of primary somatosensory neuron report salient features of our internal and external environments1^,2^,3^,4. It is unclear how specialized gene expression programs emerge during development to endow these subtypes with their unique properties. To assess the developmental progression of transcriptional maturation of each subtype of principal somatosensory neuron, we generated a transcriptomic atlas of cells traversing the primary somatosensory neuron lineage in mice. Here we show that somatosensory neurogenesis gives rise to neurons in a transcriptionally unspecialized state, characterized by co-expression of transcription factors that become restricted to select subtypes as development proceeds. Single-cell transcriptomic analyses of sensory neurons from mutant mice lacking transcription factors suggest that these broad-to-restricted transcription factors coordinate subtype-specific gene expression programs in subtypes in which their expression is maintained. We also show that neuronal targets are involved in this process; disruption of the prototypic target-derived neurotrophic factor NGF leads to aberrant subtype-restricted patterns of transcription factor expression. Our findings support a model in which cues that emanate from intermediate and final target fields promote neuronal diversification in part by transitioning cells from a transcriptionally unspecialized state to transcriptionally distinct subtypes by modulating the selection of subtype-restricted transcription factors.  

5.2732           Herpes Simplex Virus Growth, Preparation, and Assay

Sutter, S.O., Marconi, P. and Meier, A.F. Methods in Mol. Biol., 2060, 57-72 (2020)   The human herpesvirus family members, in particular herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2), are abundant and extremely contagious viruses with a high seroprevalence in the human population emphasizing the importance of studying their biology. Hence, the propagation and purification of virus stocks constitute a key element in laboratory work.  

5.2733           Engineering HSV-1 Vectors for Gene Therapy

Goins, W.F., Huang, S., Hall, B., Marzulli, M., Cohen, J.B. and Glorioso, J.C: Methods in Mol. Biol., 2060, 73-90 (2020)   Virus vectors have been employed as gene transfer vehicles for various preclinical and clinical gene therapy applications and with the approval of Glybera (Alipogene tiparvovec) as the first gene therapy product as a standard medical treatment (Yla-Herttuala, Mol Ther 20:1831–1832, 2013), gene therapy has reached the status of being a part of standard patient care. Replication-competent herpes simplex virus (HSV) vectors that replicate specifically in actively dividing tumor cells have been used in Phase I–III human trials in patients with glioblastoma multiforme (GBM), a fatal form of brain cancer, and in malignant melanoma. In fact, Imlygic^® (T-VEC, Talimogene laherparepvec, formerly known as OncoVex GM-CSF), displayed efficacy in a recent Phase-III trial when compared to standard GM-CSF treatment alone (Andtbacka et al., J Clin Oncol 31:sLBA9008, 2013), and has since become the first FDA-approved viral gene therapy product used in standard patient care (October 2015) (Pol et al., Oncoimmunology 5:e1115641, 2016). Moreover, increased efficacy was observed when Imlygic^® was combined with checkpoint inhibitory antibodies as a frontline therapy for malignant melanoma (Ribas et al., Cell 170:1109–1119.e1110, 2017; Dummer et al., Cancer Immunol Immunother 66:683–695, 2017). In addition to the replication-competent oncolytic HSV vectors like T-VEC, replication-defective HSV vectors have been employed in Phase I–II human trials and have been explored as delivery vehicles for disorders such as pain, neuropathy and other neurodegenerative conditions. Research during the last decade on the development of HSV vectors has resulted in the engineering of recombinant vectors that are completely replication defective, nontoxic, and capable of long-term transgene expression in neurons. This chapter describes methods for the construction of recombinant genomic HSV vectors based on the HSV-1 replication-defective vector backbones, steps in their purification, and their small-scale production for use in cell culture experiments as well as preclinical animal studies.  

5.2734           Rescue, Purification, and Characterization of a Recombinant HSV Expressing a Transgenic Protein

Vannini, A., Petrovic, B., Gatta, V., Leoni, V., Pepe, S., Menotti, L., Campadelli-Fiume, G. and Gianni, T. Methods in Mol. Biol., 2060, 153-168 (2020)   In the previous chapter, we describe the engineering of a HSV-BAC genome by galK recombineering. Here we describe the procedures to reconstitute, or regenerate, the replicating recombinant virus, and the methods to purify it and characterize it for the correct expression of the transgene. We present the example of R-115, a recombinant expressing murine interleukin 12 (mIL12) from the US1–US2 intergenic region. A specific method for the production of highly purified virions by iodixanol gradient, suitable for in vivo applications, is also detailed.  

5.2735           Virus-Derived Nanoparticles

Dashti, N.H. and Sainsbury, F. Methods in Mol. Biol., 2073, 149-162 (2020)   Capsid-based virus particles are widely engineered as viral nanoparticles and virus-like nanoparticles. The highly organized and uniform capsid structures make them ideal candidates for both in vitro and in vivo applications such as therapeutic delivery vehicles or enzymatic nanoreactors. Viruses have adapted to naturally infect a wide variety of organisms making their production achievable in various expression systems from bacterial to plants. Viral capsids can be modified externally and internally to suit the final application. The wide range of possible applications, ease of production in the system of choice, and customizable modification of viral capsids makes them an attractive choice in the field of nanotechnology. In this chapter we aim to provide a generic protocol for the purification and characterization of virus-derived nanoparticles and methodology for chemically labelling them to monitor their uptake in mammalian cells.  

5.2736           Adeno-Associated Virus as Gene Delivery Vehicle into the Retina

Deng, S. and Oka, K. Methods in Mol. Biol., 2092, 77-90 (2020)   Initially discovered as a contaminant of adenovirus preparations, adeno-associated virus (AAV) has proved one of the most promising viral vectors for human gene therapy. The safety profile of AAV has been well-characterized in vivo studies, and the first gene therapy for patients with vision loss caused by Leber congenital amaurosis or retinitis pigmentosa was approved by the US Food and Drug Administration in 2017. This is an exciting era for investigators working on retina biology and treatments for blindness. In this chapter, we provide detailed methods for laboratory-scale production, purification, and characterization of AAV.  

5.2737           Disruption of the HIV-1 Envelope allosteric network blocks CD4-induced rearrangements

Henderson, R., Lu, M., Zhou, Y., Mu, Z., Parks, R., Han, Q., Hsu, A.L. et al Nature Communications, 11:520 (2020)   The trimeric HIV-1 Envelope protein (Env) mediates viral-host cell fusion via a network of conformational transitions, with allosteric elements in each protomer orchestrating host receptor-induced exposure of the co-receptor binding site and fusion elements. To understand the molecular details of this allostery, here, we introduce Env mutations aimed to prevent CD4-induced rearrangements in the HIV-1 BG505 Env trimer. Binding analysis and single−molecule Förster Resonance Energy Transfer confirm that these mutations prevent CD4-induced transitions of the HIV-1 Env. Structural analysis by single−particle cryo-electron microscopy performed on the BG505 SOSIP mutant Env proteins shows rearrangements in the gp120 topological layer contacts with gp41. Displacement of a conserved tryptophan (W571) from its typical pocket in these Env mutants renders the Env insensitive to CD4 binding. These results reveal the critical function of W571 as a conformational switch in Env allostery and receptor-mediated viral entry and provide insights on Env conformation that are relevant for vaccine design.  

5.2738           Transient cAMP elevation during systems consolidation enhances remote contextual fear memory

Lee, J., Lee, H-R., Kim, J-I., Baek, J., Jang, E-H., Lee, J., Kim, M., Lee, R.U., Kim, S., Park, P. and Kaang, B-K. Neurobiology of Learning and Memory, 169, 107171 (2020)   Memory is stored in our brains over a temporally graded transition. With time, recently formed memories are transformed into remote memories for permanent storage; multiple brain regions, such as the hippocampus and neocortex, participate in this process. In this study, we aimed to understand the molecular mechanism of systems consolidation of memory and to investigate the brain regions that contribute to this regulation. We first carried out a contextual fear memory test using a transgenic mouse line, which expressed exogenously-derived Aplysia octopamine receptors in the forebrain region, such that, in response to octopamine treatment, cyclic adenosine monophosphate (cAMP) levels could be transiently elevated. From this experiment, we revealed that transient elevation of cAMP levels in the forebrain during systems consolidation led to an enhancement in remote fear memory and increased miniature excitatory synaptic currents in layer II/III of the anterior cingulate cortex (ACC). Furthermore, using an adeno-associated-virus-driven DREADD system, we investigated the specific regions in the forebrain that contribute to the regulation of memory transfer into long-term associations. Our results implied that transient elevation of cAMP levels was induced chemogenetically in the ACC, but not in the hippocampus, and showed a significant enhancement of remote memory. This finding suggests that neuronal activation during systems consolidation through the elevation of cAMP levels in the ACC contributes to remote memory enhancement.  

5.2739           A MAFG-lncRNA axis links systemic nutrient abundance to hepatic glucose metabolism

Pradas-Juni et al Nature Communications, 11:644 (2020)   Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease.  

5.2740           Simultaneous downregulation of miR-21 and upregulation of miR-7 has anti-tumor efficacy

Bhere, D., Arghiani, N., Lechtich, E.R., Yao, Y., Alsaab, S., Bei, F., Mation, M.M. and Shah, K. Scientific Reports, 10:1779 (2020)   Dysregulation of miRNA expression has been implicated in cancer. Numerous strategies have been explored to modulate miR but sub-optimal delivery and inability to concurrently target multiple pathways involved in tumor progression have limited their efficacy. In this study, we explored the potential co-modulation of upregulated miR-21 and downregulated miR-7 to enhance therapeutic outcomes in heterogenic tumor types. We first engineered lentiviral (LV) and adeno-associated viral (AAV) vectors that preferentially express anti-sense miR against miR-21(miRzip-21) and show that modulating miR-21 via miRzip extensively targets tumor cell proliferation, migration and invasion in vitro in a broad spectrum of cancer types and has therapeutic efficacy in vivo. Next, we show a significantly increased expression of caspase-mediated apoptosis by simultaneously downregulating miR-21 and upregulating miR-7 in different tumor cells. In vivo co-treatment with AAV-miRzip-21 and AAV-miR-7 in mice bearing malignant brain tumors resulted in significantly decreased tumor burden with a corresponding increase in survival. To our knowledge, this is the first study that demonstrates the therapeutic efficacy of simultaneously upregulating miR-7 and downregulating miR-21 and establishes a roadmap towards clinical translation of modulating miRs for various cancer types.  

5.2741           A cathelicidin-related antimicrobial peptide suppresses cardiac hypertrophy induced by pressure overload by regulating IGFR1/PI3K/AKT and TLR9/AMPKα

Wang, X., Chen, L., Zhao, X., Xiao, L., Yi, S., Kong, Y., Jiang, Y. and Zhang, J. Cell Death & Disease, 11:96 (2020)   Cathelicidin-related antimicrobial peptide (CRAMP), an antimicrobial peptide, was reported to protect against myocardial ischemia/reperfusion injury. However, the effect of CRAMP on pressure overload-induced cardiac hypertrophy was unknown. This study explored the role of CRAMP on cardiac hypertrophy. A cardiac hypertrophy mouse model was induced by aortic banding surgery. Seven days after surgery, mice were given mCRAMP by intraperitoneal injection (8 mg/kg/d) for 7 weeks. Cardiac hypertrophy was evaluated by the hypertrophic response and fibrosis level as well as cardiac function. Mice were also injected with AAV9-shCRAMP to knockdown CRAMP in the mouse heart. CRAMP levels first increased and then reduced in the remodeling heart, as well as in angiotensin II-stimulated endothelial cells but not in cardiomyocytes and fibroblasts. mCRAMP protected against the pressure overload-induced cardiac remodeling process, while CRAMP knockdown accelerated this process. mCRAMP reduced the inflammatory response and oxidative stress in the hypertrophic heart, while mCRAMP deficiency deteriorated the pressure overload-induced inflammatory response and oxidative stress. mCRAMP inhibited the angiotensin II-stimulated hypertrophic response and oxidative stress in neonatal rat cardiomyocytes, but mCRAMP did not help the angiotensin II-induced inflammatory response and oxidative stress in endothelial cells. Mechanistically, we found that mCRAMP suppressed the cardiac hypertrophic response by activating the IGFR1/PI3K/AKT pathway via directly binding to IGFR1. AKT knockout mice completely reversed the anti-hypertrophic effect of mCRAMP but not its anti-oxidative effect. We also found that mCRAMP ameliorated cardiac oxidative stress by activating the TLR9/AMPKa pathway. This was confirmed by a TLR9 knockout mouse experiment, in which a TLR9 knockout partly reversed the anti-hypertrophic effect of mCRAMP and completely counteracted the anti-oxidative effect of mCRAMP. In summary, mCRAMP protected against pressure overload-induced cardiac hypertrophy by activating both the IGFR1/PI3K/AKT and TLR9/AMPKa pathways in cardiomyocytes.  

5.2742           Generation of light-producing somatic-transgenic mice using adeno-associated virus vectors

Karda, R., Rahim, A.A., Wong, A.M.S., Suff, N., Diaz, J.A. et al Scientific Report, 10:2121 (2020)   We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced inflammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models.  

5.2743           Improved cell-specificity of adeno-associated viral vectors for medullary thyroid carcinoma using calcitonin gene regulatory elements

Levy, H.C., Hulvey, D., Adamson-Small, L., Jn-Simon, N., Prima, V., Rivkees, S. and Hobbs, J.A: PloS One, 15(2), e0228005 (2020)   Targeted gene therapy using recombinant adeno-associated virus (rAAV) vectors is a potential therapeutic strategy for treating cancer, and tissue-specific promoters may help with tissue targeting. Medullary thyroid carcinoma (MTC) is a disease of the calcitonin secreting thyroid C cells, and calcitonin is highly expressed in MTC tumors compared to other cells. To target MTC cells, we evaluated an rAAV serotype 2 vector (rAAV2-pM+104-GFP) containing a modified calcitonin/calcitonin gene related peptide promoter (pM+104) and a green fluorescent protein (GFP) reporter gene. In vitro transduction experiments comparing the MTC TT cell line with non-MTC cell lines demonstrated that rAAV2-pM+104-GFP infection yielded significantly (p < 0.05) higher GFP expression in TT cells than in non-MTC cell lines (HEK293 and HeLa), and significantly higher expression than in TT cells infected with the positive control rAAV2-pCBA-GFP vector. The rAAV2-pCBA-GFP control vector included a well-characterized, ubiquitously expresses control promoter, the chicken beta actin promoter with a cytomegalovirus enhancer (pCBA). In vivo experiments using a TT cell xenograft tumor mouse model showed that tumors directly injected with 2 x 10¹⁰ vg of rAAV2-pM+104-GFP vector resulted in GFP expression detected in 21.7% of cells, 48 hours after the injection. Furthermore, GFP expression was significantly higher for rAAV-pM+104-GFP treatments with a longer vector treatment duration and higher vector dose, with up to 52.6% (q < 0.05) GFP cells detected 72 hours after injecting 1x 10¹¹ vg/tumor. These data show that we have developed an rAAV vector with improved selectivity for MTC.  

5.2744           Neuropeptide Y neurons in the nucleus accumbens modulate anxiety-like behavior

Yamada, S., Islam, M.S:, van Kooten, N., Bovee, S., Oh, Y-M., Tsujimura, A., Watanabe, Y. and Tanaka., M. Exp. Neurol., 327, 113216 (2020)   Neuropeptide Y (NPY) is a 36-amino acid neuropeptide that is widely expressed in the central nervous system, including the cerebral cortex, nucleus accumbens (NAc) and hypothalamus. We previously analyzed the behavior of transgenic mice exclusively expressing an unedited RNA isoform of the 5-HT_2C receptor. These mice showed decreased NPY gene expression in the NAc and exhibited behavioral despair, suggesting that NAc NPY neurons may be involved in mood disorder; however, their role in this behavior remained unknown. Therefore, in the present study, we investigated the functional role of NAc NPY neurons in anxiety-like behavior by examining the impact of specific ablation or activation of NAc NPY neurons using NPY-Cre mice and Cre-dependent adeno-associated virus. Diphtheria toxin-mediated ablation of NAc NPY neurons significantly increased anxiety-like behavior in the open field and elevated plus maze tests, compared with before toxin treatment. Moreover, chemogenetic activation of NAc NPY neurons reduced anxiety-like behavior in both behavioral tests compared with control mice. These results suggest that NPY neurons in the NAc are involved in the modulation of anxiety in mice.  

5.2745           Gene therapy for overexpressing Neuregulin 1 type I in skeletal muscles promotes functional improvement in the SOD1G93A ALS mice

Modol-Caballero,  G., Herrando-Grabulosa, M., Garcia-Lareu, B., Solanes, N., Verdes, S., Osta, R., Francos-Quijorna, I., Lopez-Vales, R., Calvo, A.C., Bosch, A. and Navarro, X. Neurobiol. Disease, 137, 104793 (2020)   Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting motoneurons (MNs), with no effective treatment currently available. The molecular mechanisms that are involved in MN death are complex and not fully understood, with partial contributions of surrounding glial cells and skeletal muscle to the disease. Neuregulin 1 (NRG1) is a trophic factor highly expressed in MNs and neuromuscular junctions. Recent studies have suggested a crucial role of the isoform I (NRG1-I) in the collateral reinnervation process in skeletal muscle, and NRG1-III in the preservation of MNs in the spinal cord, opening a window for developing novel therapies for neuromuscular diseases like ALS. In this study, we overexpressed NRG1-I widely in the skeletal muscles of the SOD1^G93A transgenic mouse. The results show that NRG1 gene therapy activated the survival pathways in muscle and spinal cord, increasing the number of surviving MNs and neuromuscular junctions and reducing the astroglial reactivity in the spinal cord of the treated SOD1^G93A mice. Furthermore, NRG1-I overexpression preserved motor function and delayed the onset of clinical disease. In summary, our data indicates that NRG1 plays an important role on MN survival and muscle innervation in ALS, and that viral-mediated overexpression of NRG1 isoforms may be considered as a promising approach for ALS treatment.  

5.2746           Sevoflurane anesthesia-mediated oxidative stress and cognitive impairment in hippocampal neurons of old rats can be ameliorated by expression of brain derived neurotrophic factor

Xu, Z. and Qian, B. Neurosci. Lett., 721, 134785 (2020)   Postoperative cognitive dysfunction in elderly patients has been related to neurodegenerative disorders and mortality. Sevoflurane anesthesia has been implicated in both postoperative cognitive dysfunction and neurotoxicity. Given the advantages of using inhaled anesthetics like sevoflurane, it is important to understand how their usage results in neurotoxicity and subsequently devise ways to circumvent or attenuate the anesthetic-mediated induction in neurotoxicity. We have used an aged rat model to investigate the molecular mechanisms by which sevoflurane inhalation results in neurotoxicity and whether modulation of these molecular mechanisms can inhibit or attenuate neurotoxicity and cognitive learning and memory impairment in these animals. Low- or high-dose of sevoflurane resulted in reactive oxygen species generation, increased NADPH oxidase protein expression, apoptosis and autophagy. Sevoflurane inhalation resulted in significant inhibition of brain derived neurotrophic factor (BDNF) and cognitive impairment. And the activation of PI3K/Akt/mTOR signaling pathways are attenuated in sevoflurane-mediated anesthesia. Adeno-associated virus (AAV)-mediated expression of Bdnf, but not controls EGFP, attenuated sevoflurane-induced oxidative stress and cognitive impairment in the rats. Our results highlight that AAV-mediated gene therapy might offer a potential therapeutic opportunity to treat post-operative cognitive impairment resulting from inhaled anesthetics.  

5.2747           Successful Transduction with AAV Vectors after Selective Depletion of Anti-AAV Antibodies by Immunoadsorption

Orlowski, A., Katz, M.G., Gubara, S.M., Fargnoli, A.S., Fish, K.M. and Weber, T. Molecular Therapy-Methods & Clin. Develop., 16, 192-203 (2020)   Gene therapy with adeno-associated virus (AAV)-based vectors shows great promise for the gene therapeutic treatment of a broad array of diseases. In fact, the treatment of genetic diseases with AAV vectors is currently the only in vivo gene therapy approach that is approved by the US Food and Drug Administration (FDA). Unfortunately, pre-existing antibodies against AAV severely limit the patient population that can potentially benefit from AAV gene therapy, especially if the vector is delivered by intravenous injection. Here, we demonstrate that we can selectively deplete anti-AAV antibodies by hemapheresis combined with AAV9 particles coupled to Sepharose beads. In rats that underwent hemapheresis and immunoadsorption, luciferase expression was dramatically increased in the hearts and fully restored in the livers of these rats. Importantly, our method can be readily adapted for the use in clinical AAV gene therapy.    

5.2748           Hepatic DNAJB9 Drives Anabolic Biasing to Reduce Steatosis and Obesity

Sun, F., Liao, Y., Qu, X., Huang, H., Li, P. and Fu, S. Cell Reports, 30, 1835-1847 (2020)   Nutrients stimulate the anabolic synthesis of proteins and lipids, but selective insulin resistance in obesity biases the anabolic program toward lipogenesis. Here, we report the identification of a DNAJB9-driven program that favors protein synthesis and energy production over lipid accumulation. We show there are two pools of DNAJB9 cochaperone. DNAJB9 in the ER lumen promotes the degradation of the lipogenic transcription factor SREBP1c through ERAD, whereas its counterpart on the ER membrane promotes the assembly of mTORC2 in the cytosol and stimulates the synthesis of proteins and ATP. The expression of Dnajb9 is induced by nutrients and downregulated in the obese mouse liver. Restoration of hepatic DNAJB9 expression effectively improves insulin sensitivity, restores protein synthesis, and suppresses food intake, accompanied by reduced hepatic steatosis and adiposity in multiple mouse models of obesity. Therefore, targeting the anabolic balance may provide a unique opportunity to tackle obesity and diabetes.  

5.2749           GPR108 Is a Highly Conserved AAV Entry Factor

Dudek, A.M., Zabaleta, N., Zinn, E., Pillay, S., Zengel, J., Porter, C., Franceschini, J.S., Estelien, R., Carette, J.E., Zhou, G.L. and Vandenberghe, L.H. Molecular Therapy, 28(2), 367-381 (2020)   Adeno-associated virus (AAV) is a highly promising gene transfer vector, yet major cellular requirements for AAV entry are poorly understood. Using a genome-wide CRISPR screen for entry of evolutionarily divergent serotype AAVrh32.33, we identified GPR108, a member of the G protein-coupled receptor superfamily, as an AAV entry factor. Of greater than 20 divergent AAVs across all AAV clades tested in human cell lines, only AAV5 transduction was unaffected in the GPR108 knockout (KO). GPR108 dependency was further shown in murine and primary cells in vitro. These findings are further validated in vivo, as the Gpr108 KO mouse demonstrates 10- to 100-fold reduced expression for AAV8 and rh32.33 but not AAV5. Mechanistically, both GPR108 N- and C-terminal domains are required for transduction, and on the capsid, a VP1 unique domain that is not conserved on AAV5 can be transferred to confer GPR108 independence onto AAV2 chimeras. In vitro binding and fractionation studies indicate reduced nuclear import and cytosolic accumulation in the absence of GPR108. We thus have identified the second of two AAV entry factors that is conserved between mice and humans relevant both in vitro and in vivo, further providing a mechanistic understanding to the tropism of AAV gene therapy vectors.  

5.2750           Interleukin-38 increases the insulin sensitivity in children with the type 2 diabetes

Liu, Y., Chen, T., Zhou, F., Mu, D. and Liu, S. Int. Immunopharmacol., 82, 106264 (2020)   The prevalence of type 2 diabetes mellitus (DM) is increasing in the children population. It is well known that inflammation contributes to the type 2 DM pathogenesis. Interleukin 38 (IL-38) is one newly identified anti-inflammatory factor. Therefore, we investigated whether the expression level of IL-38 is associated with type 2 DM in the children and the underlying mechanism. Children with recently diagnosed type 2 diabetes mellitus were recruited and studied. The healthy subjects without glucose metabolism abnormalities were used as controls. The IL-38 expression level was determined by quantitative PCR and ELISA (Enzyme-linked immunoassay). Statistic analysis showed that the IL-38 level was significantly associated with type 2 DM and insulin resistance in the children. The patients were then divided into two groups, one group sensitive to insulin therapy while the other resistant to insulin therapy. Data showed that the IL-38 was highly expressed in the group sensitive to insulin therapy. In the mice model, overexpressing the IL-38 could suppress the expression of IL-36, a pro-inflammatory factor, and also the diabetes development. Thus our results showed that higher IL-38 was associated with the increased insulin sensitive in children with type 2 DM and inhibited T2DM development in the mouse model through suppressing the function of IL-36.  

5.2751           REEP5 depletion causes sarco-endoplasmic reticulum vacuolization and cardiac functional defects

Lee, S-H., Hadipour-Lakmehsari, S., Murthy, H.R., Gibb, N., Miyake, T. et al Nature Communications, 11:965 (2020)   The sarco-endoplasmic reticulum (SR/ER) plays an important role in the development and progression of many heart diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. Here, we report a cardiac enriched, SR/ER membrane protein, REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes results in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca²⁺ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants show sensitized cardiac dysfunction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrates cardiac dysfunction. These results demonstrate the critical role of REEP5 in SR/ER organization and function as well as normal heart function and development.  

5.2752           Structure comparison of the chimeric AAV2.7m8 vector with parental AAV2

Bennett, A., Keravala, A., Makal, V., Kurian, J., Belbellaa, B., Aeran, R., Tsseng, Y-S., Sousa, D., Spear, J., Gasmi, M. and Agbandje-McKenna, M.

1. Struct. Biol., 209, 107433 (2020)

  The AAV2.7m8 vector is an engineered capsid with a 10-amino acid insertion in adeno-associated virus (AAV) surface variable region VIII (VR-VIII) resulting in the alteration of an antigenic region of AAV2 and the ability to efficiently transduce retina cells following intravitreal administration. Directed evolution and in vivo screening in the mouse retina isolated this vector. In the present study, we sought to identify the structural differences between a recombinant AAV2.7m8 (rAAV2.7m8) vector packaging a GFP genome and its parental serotype, AAV2, by cryo-electron microscopy (cryo-EM) and image reconstruction. The structures of rAAV2.7m8 and AAV2 were determined to 2.91 and 3.02 Å resolution, respectively. The rAAV2.7m8 amino acid side-chains for residues 219–745 (the last C-terminal residue) were interpretable in the density map with the exception of the 10 inserted amino acids. While observable in a low sigma threshold density, side-chains were only resolved at the base of the insertion, likely due to flexibility at the top of the loop. A comparison to parental AAV2 (ordered from residues 217–735) showed the structures to be similar, except at some side-chains that had different orientations and, in VR-VIII containing the 10 amino acid insertion. VR-VIII is part of an AAV2 antigenic epitope, and the difference is consistent with rAAV2.7m8′s escape from a known AAV2 monoclonal antibody, C37-B. The observations provide valuable insight into the configuration of inserted surface peptides on the AAV capsid and structural differences to be leveraged for future AAV vector rational design, especially for retargeted tropism and antibody escape.  

5.2753           Gene therapy conversion of striatal astrocytes into GABAergic neurons in mouse models of Huntington’s disease

Wu, Z., Parry, M., Hou, X-Y., Liu, M-H., Wang, H., Cain, R., Pei, Z-F., Chen, Y-C., Guo, Z-Y., Abhijeet, S. and Chen, G. Nature Communications, 11:1105 (2020)   Huntington’s disease (HD) is caused by Huntingtin (Htt) gene mutation resulting in the loss of striatal GABAergic neurons and motor functional deficits. We report here an in vivo cell conversion technology to reprogram striatal astrocytes into GABAergic neurons in both R6/2 and YAC128 HD mouse models through AAV-mediated ectopic expression of NeuroD1 and Dlx2 transcription factors. We found that the astrocyte-to-neuron (AtN) conversion rate reached 80% in the striatum and >50% of the converted neurons were DARPP32⁺ medium spiny neurons. The striatal astrocyte-converted neurons showed action potentials and synaptic events, and projected their axons to the targeted globus pallidus and substantia nigra in a time-dependent manner. Behavioral analyses found that NeuroD1 and Dlx2-treated R6/2 mice showed a significant extension of life span and improvement of motor functions. This study demonstrates that in vivo AtN conversion may be a disease-modifying gene therapy to treat HD and other neurodegenerative disorders.  

5.2754           Chapter Thirteen - Production and use of adeno-associated virus vectors as tools for cancer immunotherapy

Fernandez-Sendin, M., Tenesaca, S., Vasquez, M., Aranda, F. and Berraondo, P. Methods in Enzymol., 635, 185-203 (2020)   Recombinant adeno-associated viruses (rAAVs) are attractive tools for research in cancer immunotherapy. A single administration of an AAV vector in tumor mouse models induces a progressive increase in transgene expression which reaches a plateau 1 or 2 weeks after administration. The rAAV is then able to maintain the expression of the immunostimulatory transgene. Thus, the use of these vectors obviates the need for frequent administrations of the therapeutic protein to achieve the antitumor effect. The long-term expression of AAV vectors can be exploited for the evaluation of the antitumor activity of immune-enhancing proteins. Most preclinical studies have focused on the expression of cytokines and on the induction of immune responses elicited by tumor-associated antigens expressed by rAAVs. Notwithstanding, rAAVs may not be suitable for immunostimulatory proteins that require high and/or immediate expression. In this chapter, we review a feasible, reliable and detailed protocol to produce and purify AAV vectors as a tool for cancer immunotherapy strategies.  

5.2755           Hippocampal overexpression of chordin protects against the chronic social defeat stress-induced depressive-like effects in mice

Wang, C-N., Gong, S-N., Guan, w., Wang, J-L., Gao, T-T., Wang, Y., Sun, F. and Jiang, B. Brain Res. Bulletin, 158, 31-39 (2020)   Depression is a serious and worldwide neuropsychiatric disesase, and developing novel antidepressant targets beyond the monoaminergic systems is now popular and necessary. Bone morphogenetic protein (BMP) signals modulate numerous developmental, physiological, and homeostatic processes. The functions of BMPs are also regulated by secreted extracellular antagonists such as chordin and noggin. Chordin has abundant expression in adult brain, and may play critical role in the central nervous system. In this study, the chronic social defeat stress (CSDS) model of depression, various behavioral tests, western blotting, quantitative real-time reverse transcription PCR, immunohistochemistry, recombinant mouse chordin protein and AAV-Chordin-EGFP were together used to explore the role of chordin in the pathogenesis of depression. It was found that CSDS significantly decreased the expression of chordin in the hippocampus but not other related brain regions. Moreover, both pharmacological and genetic overexpression of hippocampal chordin fully protected against the CSDS-induced depressive-like effects in mice. Collectively, hippocampal chordin could be a novel antidepressant target, and this study further highlights the importance of the hippocampal BMP system in the pathophysiology of depression.  

5.2756           Selective EMC subunits act as molecular tethers of intracellular organelles exploited during viral entry

Bagchi, P., Torress, M., Qi, L. and Tsai, B. Nature Communications, 11:1127 (2020)   Although viruses must navigate the complex host endomembrane system to infect cells, the strategies used to achieve this is unclear. During entry, polyomavirus SV40 is sorted from the late endosome (LE) to the endoplasmic reticulum (ER) to cause infection, yet how this is accomplished remains enigmatic. Here we find that EMC4 and EMC7, two ER membrane protein complex (EMC) subunits, support SV40 infection by promoting LE-to-ER targeting of the virus. They do this by engaging LE-associated Rab7, presumably to stabilize contact between the LE and ER. These EMC subunits also bind to the ER-resident fusion machinery component syntaxin18, which is required for SV40-arrival to the ER. Our data suggest that EMC4 and EMC7 act as molecular tethers, inter-connecting two intracellular compartments to enable efficient transport of a virus between these compartments. As LE-to-ER transport of cellular cargos is unclear, our results have broad implications for illuminating inter-organelle cargo transport.  

5.2757           LATS suppresses mTORC1 activity to directly coordinate Hippo and mTORC1 pathways in growth control

Gan, W., Dai, X., Dai, X., Xie, J., Yin, S., Zhu, J. et al Nature Cell Biol., 22, 246-256 (2020)   The Hippo and mammalian target of rapamycin complex 1 (mTORC1) pathways are the two predominant growth-control pathways that dictate proper organ development. We therefore explored potential crosstalk between these two functionally relevant pathways to coordinate their growth-control functions. We found that the LATS1 and LATS2 kinases, the core components of the Hippo pathway, phosphorylate S606 of Raptor, an essential component of mTORC1, to attenuate mTORC1 activation by impairing the interaction of Raptor with Rheb. The phosphomimetic Raptor-S606D knock-in mutant led to a reduction in cell size and proliferation. Compared with Raptor^+/+ mice, Raptor^D/D knock-in mice exhibited smaller livers and hearts, and a significant inhibition of elevation in mTORC1 signalling induced by Nf2 or Lats1 and Lats2 loss. Thus, our study reveals a direct link between the Hippo and mTORC1 pathways to fine-tune organ growth.  

5.2758           Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy

Moretti, A., Fonteyne, L., Giesert, F., Hoppmann, P., Meier, A.B., Bozoglu, T., Baehr, A. et al Nature Med., 26, 207-214 (2020)   Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51–52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca²⁺ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.  

5.2759           Neuronal Trans-differentiation by Transcription Factors Ascl1 and Nurr1: Induction of a Dopaminergic Neurotransmitter Phenotype in Cortical GABAergic Neurons

Raina, A., Mahajani, S., Bähr, M. and Kügler, S. Mol. Neurobiol., 57, 249-260 (2020)   Neurons with a desired neurotransmitter phenotype can be differentiated from induced pluripotent stem cells or from somatic cells only through tedious protocols with relatively low yield. Readily available cortical neurons isolated from embryonic rat brain, which have already undergone a complete neuronal differentiation process, might serve as alternative template source. These cultures consist of 85% glutamatergic and 15% GABAergic neurons, and we attempted to trans-differentiate them into dopaminergic neurons. Transcription factors Nurr1, Lmx1A and Pitx3, essential determinants of a dopaminergic cell fate during CNS development, were not sufficient to induce tyrosine hydroxylase expression in a significant number of cells. Combining Nurr1 with the generic neuronal differentiator and re-programming factor Ascl1, however, resulted in generation of neurons which express dopaminergic markers TH, AADC, VMAT2 and DAT. Only neurons of GABAergic phenotype could be trans-differentiated towards a dopaminergic neurotransmitter phenotype, while for glutamatergic neurons, this process proved to be neurotoxic. Intriguingly, GABAergic neurons isolated from embryonal midbrain could not be trans-differentiated into dopaminergic neurons by Ascl1 and Nurr1. Thus, in principle, post-mitotic embryonal neurons can serve as templates for neurons with a desired neurotransmitter phenotype. However, neurotransmitter phenotype plasticity critically depends on the differentiation history of the template neurons, which can result in relatively low yields of dopaminergic neurons.  

5.2760           AAV-Mediated Expression of Dominant-Negative ULK1 Increases Neuronal Survival and Enhances Motor Performance in the MPTP Mouse Model of Parkinson’s Disease

Balke, D., Tatenhorst, L., Dambeck, V., Rib as, V.T. Vahsen, B.F., Michel, U., Bähr, M. and Lingor, P. Mol. Neurobiol., 57, 685-697 (2020)   Loss of nigrostriatal projections by axonal degeneration is a key early event in Parkinson’s disease (PD) pathophysiology, being accountable for the lack of dopamine in the nigrostriatal system and resulting in motor symptoms such as bradykinesia, rigidity, and tremor. Since autophagy is an important mechanism contributing to axonal degeneration, we aimed to evaluate the effects of competitive autophagy inhibition in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD in vivo. Adeno-associated viral vector (AAV)–mediated overexpression of a dominant-negative form of the unc-51 like autophagy-initiating kinase (ULK1.DN) in the substantia nigra was induced 3 weeks before MPTP treatment. Analysis of motor behavior demonstrated a significant improvement of ULK1.DN expressing mice after MPTP treatment. Immunohistochemical analyses of dopaminergic nigral neurons and nigrostriatal projections revealed a significant protection from MPTP-induced neurotoxicity after ULK1.DN expression. Western blot analysis linked these findings to an activation of mTOR signaling. Taken together, our results indicate that expression of ULK1.DN can attenuate MPTP-induced axonal neurodegeneration, suggesting that ULK1 could be a promising novel target in the treatment of PD.  

5.2761           In Vivo Delivery of Cassettes Encoding Anti-HBV Primary MicroRNAs Using an Ancestral Adeno-Associated Viral Vector

Mnyandu, N., Arbuthnot, P. and Maepa, M.B: Methods in Mol. Biol., 2115, 171-183 (2020)   Chronic hepatitis B, a liver disease resulting from persisting hepatitis B virus (HBV) infection, remains a global health challenge despite the availability of an effective vaccine. Various preclinical studies using adeno-associated viruses (AAVs) to deliver anti-HBV RNA interference (RNAi) activators to mediate long-lasting HBV silencing show promise. Recent positive outcomes observed in clinical trials and the FDA approval of AAV-based drugs further demonstrate the potential of AAVs in antiviral therapeutic development. However, the prevalence of neutralizing antibodies against vectors based on extant AVV capsids limits the application of these vectors in human. The exciting reports on in silico designed and in vitro synthesized ancestral AAV (Anc80L65) with a potential to evade prevailing AAV neutralizing antibodies will significantly contribute to the success of these vectors in humans. Here, we describe methods for production and in vivo characterization of Anc80L65 expressing anti-HBV RNAi activators.  

5.2762           The Emergence of a Stable Neuronal Ensemble from a Wider Pool of Activated Neurons in the Dorsal Medial Prefrontal Cortex during Appetitive Learning in Mice

Brebner, L., Ziminsky, J.J., Margetts-Smith, G., Sieburg, M.C., Reeve, H.M., Nowotny, T., Hirrlinger, J., Heintz, T.G., Lagnado, L., Kato, S., Kobayashi, K., Ramsey, L.A., Hall, C.N., Crombag, H.S. and Koya, E.

1. Neurosci., 40(2), 395-410 (2020)

  Animals selectively respond to environmental cues associated with food reward to optimize nutrient intake. Such appetitive conditioned stimulus–unconditioned stimulus (CS-US) associations are thought to be encoded in select, stable neuronal populations or neuronal ensembles, which undergo physiological modifications during appetitive conditioning. These ensembles in the medial prefrontal cortex (mPFC) control well-established, cue-evoked food seeking, but the mechanisms involved in the genesis of these ensembles are unclear. Here, we used male Fos-GFP mice that express green fluorescent protein (GFP) in recently behaviorally activated neurons, to reveal how dorsal mPFC neurons are recruited and modified to encode CS-US memory representations using an appetitive conditioning task. In the initial conditioning session, animals did not exhibit discriminated, cue-selective food seeking, but did so in later sessions indicating that a CS-US association was established. Using microprism-based in vivo 2-Photon imaging, we revealed that only a minority of neurons activated during the initial session was consistently activated throughout subsequent conditioning sessions and during cue-evoked memory recall. Notably, using ex vivo electrophysiology, we found that neurons activated following the initial session exhibited transient hyperexcitability. Chemogenetically enhancing the excitability of these neurons throughout subsequent conditioning sessions interfered with the development of reliable cue-selective food seeking, indicated by persistent, nondiscriminated performance. We demonstrate how appetitive learning consistently activates a subset of neurons to form a stable neuronal ensemble during the formation of a CS-US association. This ensemble may arise from a pool of hyperexcitable neurons activated during the initial conditioning session.  

5.2763           Optogenetic Modulation of TrkB Signaling in the Mouse Brain

Hong, J. and Heo, W.D.

1. Mol. Biol., 432, 815-827 (2020)

  Optogenetic activation of receptors has advantages compared with chemical or ligand treatment because of its high spatial and temporal precision. Especially in the brain, the use of a genetically encoded light-tunable receptor is superior to direct infusion or systemic drug treatment. We applied light-activatable TrkB receptors in the mouse brain with reduced basal activity by incorporating Cry2PHR mutant, Opto-cytTrkB(E281A). Upon AAV mediated gene delivery, this form was expressed at sufficient levels in the mouse hippocampus (HPC) and medial entorhinal cortex (MEC) retaining normal canonical signal transduction by the blue light stimulus, even by delivery of noninvasive LED light on the mouse head. Within target cells, where its expression was driven by a cell type-specific promoter, Opto-cytTrkB(E281A)-mediated TrkB signaling could be controlled by adjusting light-stimulating conditions. We further demonstrated that Opto-cytTrkB(E281A) could locally induce TrkB signaling in axon terminals in the MEC-HPC. In summary, Opto-cytTrkB(E281A) will be useful for elucidating time- and region-specific roles of TrkB signaling ranging from cellular function to neural circuit mechanisms.  

5.2764           High-mobility group AT-hook 1 promotes cardiac dysfunction in diabetic cardiomyopathy via autophagy inhibition

Wu, Q-Q., Liu, C., Cai, Z., Xie, Q., Hu, T., Duan, M., Wu, H., Yuan, Y. and Tang, Q. Cell Death & Disease, 11:160 (2020)   High-mobility group AT-hook1 (HMGA1, formerly HMG-I/Y), an architectural transcription factor, participates in a number of biological processes. However, its effect on cardiac remodeling (refer to cardiac inflammation, apoptosis and dysfunction) in diabetic cardiomyopathy remains largely indistinct. In this study, we found that HMGA1 was upregulated in diabetic mouse hearts and high-glucose-stimulated cardiomyocytes. Overexpression of HMGA1 accelerated high-glucose-induced cardiomyocyte inflammation and apoptosis, while HMGA1 knockdown relieved inflammation and apoptosis in cardiomyocytes in response to high glucose. Overexpression of HMGA1 in mice heart by adeno-associated virus 9 (AAV9) delivery system deteriorated the inflammatory response, increased apoptosis and accelerated cardiac dysfunction in streptozotocin-induced diabetic mouse model. Knockdown of HMGA1 by AAV9-shHMGA1 in vivo ameliorated cardiac remodeling in diabetic mice. Mechanistically, we found that HMGA1 inhibited the formation rather than the degradation of autophagy by regulating P27/CDK2/mTOR signaling. CDK2 knockdown or P27 overexpression blurred HMGA1 overexpression-induced deteriorating effects in vitro. P27 overexpression in mice heart counteracted HMGA1 overexpression-induced increased cardiac remodeling in diabetic mice. The luciferase reporter experiment confirmed that the regulatory effect of HMGA1 on P27 was mediated by miR-222. In addition, a miR-222 antagomir counteracted HMGA1 overexpression-induced deteriorating effects in vitro. Taken together, our data indicate that HMGA1 aggravates diabetic cardiomyopathy by directly regulating miR-222 promoter activity, which inhibits P27/mTOR-induced autophagy.  

5.2765           HIV protease cleaves the antiviral m6A reader protein YTHDF3 in the viral particle

Jurczyszak, D., Zhang, W., Terry, S.N., Kehrer, T., Bermudez Gonzalez, M.C., McGregor, E., Mulder, L.C.F., Exkwahl, M.J., Pan, T. and Simon, V. PloS Pathogens, 16(2), e1008305 (2020)   N⁶-methyladenosine (m⁶A) is the most abundant HIV RNA modification but the interplay between the m⁶A reader protein YTHDF3 and HIV replication is not well understood. We found that knockout of YTHDF3 in human CD4+ T-cells increases infection supporting the role of YTHDF3 as a restriction factor. Overexpression of the YTHDF3 protein in the producer cells reduces the infectivity of the newly produced viruses. YTHDF3 proteins are incorporated into HIV particles in a nucleocapsid-dependent manner permitting the m⁶A reader protein to limit infection in the new target cell at the step of reverse transcription. Importantly, HIV protease cleaves the virion-incorporated full-length YTHDF3 protein, a process which is blocked by HIV protease inhibitors used to treat HIV infected patients. Mass-spectrometry confirmed the proteolytic processing of YTHDF3 in the virion. Thus, HIV protease cleaves the virion-encapsidated host m⁶A effector protein in addition to the viral polyproteins to ensure optimal infectivity of the mature virion.  

5.2766           Utilizing minimally purified secreted rAAV for rapid and cost-effective manipulation of gene expression in the CNS

Goodwin, M.S., Croft, C.L., Futch, H.S., Ryu, D., Ceballos-Diaz, C., Liu, X. et al Mol. Neurogeneration, 15:15 (2020)   Background Recombinant adeno-associated virus (rAAV) is widely used in the neuroscience field to manipulate gene expression in the nervous system. However, a limitation to the use of rAAV vectors is the time and expense needed to produce them. To overcome this limitation, we evaluated whether unpurified rAAV vectors secreted into the media following scalable PEI transfection of HEK293T cells can be used in lieu of purified rAAV. Methods We packaged rAAV2-EGFP vectors in 30 different wild-type and mutant capsids and subsequently collected the media containing secreted rAAV. Genomic titers of each rAAV vector were assessed and the ability of each unpurified virus to transduce primary mixed neuroglial cultures (PNGCs), organotypic brain slice cultures (BSCs) and the mouse brain was evaluated. Results There was ~ 40-fold wide variance in the average genomic titers of the rAAV2-EGFP vector packaged in the 30 different capsids, ranging from a low ~ 4.7 × 10¹⁰ vector genomes (vg)/mL for rAAV2/5-EGFP to a high of ~ 2.0 × 10¹² vg/mL for a capsid mutant of rAAV2/8-EGFP. In PNGC studies, we observed a wide range of transduction efficiency among the 30 capsids evaluated, with the rAAV2/6-EGFP vector demonstrating the highest overall transduction efficiency. In BSC studies, we observed robust transduction by wild-type capsid vectors rAAV2/6, 2/8 and 2/9, and by capsid mutants of rAAV2/1, 2/6, and 2/8. In the in vivo somatic brain transgenesis (SBT) studies, we found that intra-cerebroventricular injection of media containing unpurified rAAV2-EGFP vectors packaged with select mutant capsids resulted in abundant EGFP positive neurons and astrocytes in the hippocampus and forebrain of non-transgenic mice. We demonstrate that unpurified rAAV can express transgenes at equivalent levels to lysate-purified rAAV both in vitro and in vivo. We also show that unpurified rAAV is sufficient to drive tau pathology in BSC and neuroinflammation in vivo, recapitulating previous studies using purified rAAV. Conclusions Unpurified rAAV vectors secreted into the media can efficiently transduce brain cells in vitro and in vivo, providing a cost-effective way to manipulate gene expression. The use of unpurified virus will greatly reduce costs of exploratory studies and further increase the utility of rAAV vectors for standard laboratory use.  

5.2767           In vivo engineering of lymphocytes after systemic exosome-associated AAV delivery

Breuer, C.B., Hanlon, K.S., Natasan, J-S., Volak, A., Meliani, A., Mingozzi, F., Kleinstiver, B.P., Moon, J.J. and Maguire, C.A. Scientific Reports, 10:4544 (2020)   Ex-vivo gene therapy using stem cells or T cells transduced by retroviral or lentiviral vectors has shown remarkable efficacy in the treatment of immunodeficiencies and cancer. However, the process is expensive, technically challenging, and not readily scalable to large patient populations, particularly in underdeveloped parts of the world. Direct in vivo gene therapy would avoid these issues, and such approaches with adeno-associated virus (AAV) vectors have been shown to be safe and efficacious in clinical trials for diseases affecting differentiated tissues such as the liver and CNS. However, the ability to transduce lymphocytes with AAV in vivo after systemic delivery has not been carefully explored. Here, we show that both standard and exosome-associated preparations of AAV8 vectors can effectively transduce a variety of immune cell populations including CD4⁺ T cells, CD8⁺ T cells, B cells, macrophages, and dendritic cells after systemic delivery in mice. We provide direct evidence of T cell transduction through the detection of AAV genomes and transgene mRNA, and show that intracellular and transmembrane proteins can be expressed. These findings establish the feasibility of AAV-mediated in vivo gene delivery to immune cells which will facilitate both basic and applied research towards the goal of direct in vivo gene immunotherapies.  

5.2768           Determination of parvovirus retention profiles in virus filter membranes using laser scanning microscopy

Leisi, R., Bieri, J., Roth, N.J. and Ros, C.

1. Membrane Sci., 603, 118012 (2020)

  Virus filtration is a highly effective method in the downstream processing of biotherapeutic products to provide effective removal of potential infectious agents based on a size exclusion mechanism. The direct visualization of viruses retained inside the filter membrane represents a valuable tool to get a deeper understanding of the filtration process and to explain observations of virus breakthrough under particular operating conditions. Parvoviruses, which are used as worst-case models in validation studies, were purified and labeled with fluorescent dyes to detect their retention pattern inside the filter membrane using laser scanning microscopy. Critical factors influencing the reproducibility and accuracy of the approach were identified and optimized. The retention profiles revealed detectable differences between viruses, suggesting that the use of bacteriophages or nanoparticles as surrogates is limited in their applicability to accurately predict the behavior of parvoviruses in filter membranes. The established method enables a direct and quantitative analysis of the virus retention profile, adding a valuable tool to the conventional measurement of the viral load reduction to better understand the mechanism underlying the removal of viruses during nanofiltration of biotherapeutic products.  

5.2769           Premature termination codon readthrough upregulates progranulin expression and improves lysosomal function in preclinical models of GRN deficiency

Frew, J., Baradaran-Heravi, A., Balgi, A.D., Wu, X., Yan, T.D., Arns, S., Shidmoossavee, F.S. et al Mol. Neurodegeneration, 15:21 (2020)   Background Frontotemporal lobar degeneration (FTLD) is a devastating and progressive disorder, and a common cause of early onset dementia. Progranulin (PGRN) haploinsufficiency due to autosomal dominant mutations in the progranulin gene (GRN) is an important cause of FTLD (FTLD-GRN), and nearly a quarter of these genetic cases are due to a nonsense mutation. Premature termination codons (PTC) can be therapeutically targeted by compounds allowing readthrough, and aminoglycoside antibiotics are known to be potent PTC readthrough drugs. Restoring endogenous PGRN through PTC readthrough has not previously been explored as a therapeutic intervention in FTLD. Methods We studied whether the aminoglycoside G418 could increase PGRN expression in HEK293 and human induced pluripotent stem cell (hiPSC)-derived neurons bearing the heterozygous S116X, R418X, and R493X pathogenic GRN nonsense mutations. We further tested a novel substituted phthalimide PTC readthrough enhancer in combination with G418 in our cellular models. We next generated a homozygous R493X knock-in hiPSC isogenic line (R493X^−/− KI), assessing whether combination treatment in hiPSC-derived neurons and astrocytes could increase PGRN and ameliorate lysosomal dysfunction relevant to FTLD-GRN. To provide in vivo proof-of-concept of our approach, we measured brain PGRN after intracerebroventricular administration of G418 in mice expressing the V5-tagged GRN nonsense mutation R493X. Results The R418X and R493X mutant GRN cell lines responded to PTC readthrough with G418, and treatments increased PGRN levels in R493X^−/− KI hiPSC-derived neurons and astrocytes. Combining G418 with a PTC readthrough enhancer increased PGRN levels over G418 treatment alone in vitro. PGRN deficiency has been shown to impair lysosomal function, and the mature form of the lysosomal protease cathepsin D is overexpressed in R493X^−/− KI neurons. Increasing PGRN through G418-mediated PTC readthrough normalized this abnormal lysosomal phenotype in R493X^−/− KI neuronal cultures. A single intracerebroventricular injection of G418 induced GRN PTC readthrough in 6-week-old AAV-GRN-R493X-V5 mice. Conclusions Taken together, our findings suggest that PTC readthrough may be a potential therapeutic strategy for FTLD caused by GRN nonsense mutations.  

5.2770           Anti-Phospho-Tau Gene Therapy for Chronic Traumatic Encephalopathy

Sacramento, C.B., Sondhi, D., Rosenberg, J.B., Chen, A., Giordano, S.,Pey, E., Lee, V., Stiles, K.M., Havlicek, D.F., Leopold, P.L., Kaminsky, S.M. and Crystal, R.G. Human Gene Therapy, 31(1-2), 57-69 (2020)   Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disorder caused by repetitive trauma to the central nervous system (CNS) suffered by soldiers, contact sport athletes, and civilians following accident-related trauma. CTE is a CNS tauopathy, with trauma-induced inflammation leading to accumulation of hyperphosphorylated forms of the microtubule-binding protein Tau (pTau), resulting in neurofibrillary tangles and progressive loss of neurons. At present, there are no therapies to treat CTE. We hypothesized that direct CNS administration of an adeno-associated virus (AAV) vector coding for an anti-pTau antibody would generate sufficient levels of anti-pTau in the CNS to suppress pTau accumulation thus interrupting the pathogenic process. Using a serotype AAVrh.10 gene transfer vector coding for a monoclonal antibody directed against pTau, we demonstrate the feasibility of this strategy in a murine CTE model in which pTau accumulation was elicited by repeated traumatic brain injury (TBI) using a closed cortical impact procedure over 5 days. Direct delivery of AAVrh.10 expression vectors coding for either of the two different anti-pTau antibodies to the hippocampus of these TBI mice significantly reduced pTau levels across the CNS. Using doses that can be safely scaled to humans, the data demonstrate that CNS administration of AAVrh.10anti-pTau is effective, providing a new strategy to interrupt the CTE consequences of TBI.  

5.2771           Ly6a Differential Expression in Blood–Brain Barrier Is Responsible for Strain Specific Central Nervous System Transduction Profile of AAV-PHP.B

Batista, a.r., King, O.D., Reardon, C.P., Davis, C., Shankaracharya, Philip, V., Gray-Edwards, H., Aronin, N., Lutz, C., Landers, J. and Sena-Esteves, M. Human Gene Therapy, 31(1-2), 90-102 (2020)   Adeno-associated virus (AAV) gene therapy for neurological diseases was revolutionized by the discovery that AAV9 crosses the blood–brain barrier (BBB) after systemic administration. Transformative results have been documented in various inherited diseases, but overall neuronal transduction efficiency is relatively low. The recent development of AAV-PHP.B with ∼60-fold higher efficiency than AAV9 in transducing the adult mouse brain was the major first step toward acquiring the ability to deliver genes to the majority of cells in the central nervous system (CNS). However, little is known about the mechanism utilized by AAV to cross the BBB, and how it may diverge across species. In this study, we show that AAV-PHP.B is ineffective for systemic CNS gene transfer in the inbred strains BALB/cJ, BALB/cByJ, A/J, NOD/ShiLtJ, NZO/HILtJ, C3H/HeJ, and CBA/J mice, but it is highly potent in C57BL/6J, FVB/NJ, DBA/2J, 129S1/SvImJ, and AKR/J mice and also the outbred strain CD-1. We used the power of classical genetics to uncover the molecular mechanisms AAV-PHP.B engages to transduce CNS at high efficiency, and by quantitative trait locus mapping we identify a 6 Mb region in chromosome 15 with an logarithm of the odds (LOD) score ∼20, including single nucleotide polymorphisms in the coding region of 9 different genes. Comparison of the publicly available data on the genome sequence of 16 different mouse strains, combined with RNA-seq data analysis of brain microcapillary endothelia, led us to conclude that the expression level of Ly6a is likely the determining factor for differential efficacy of AAV-PHP.B in transducing the CNS across different mouse strains.  

5.2772           Systemic Adeno-Associated Virus-Mediated Gene Therapy Prevents the Multiorgan Disorders Associated with Aldehyde Dehydrogenase 2 Deficiency and Chronic Ethanol Ingestion

Matsumura, Y., Li, N., Alwaseem, H., Pagovich, O.E., Crystal, R.G., Greenblatt, M.B. and Stiles, K.M: Human Gene Therapy, 21(3-4), 163-182 (2020)   Aldehyde dehydrogenase type 2 (ALDH2), a key enzyme in ethanol metabolism, processes toxic acetaldehyde to nontoxic acetate. ALDH2 deficiency affects 8% of the world population and 35–45% of East Asians. The ALDH2*2 allele common genetic variant has a glutamic acid-to-lysine substitution at position 487 (E487K) that reduces the oxidizing ability of the enzyme resulting in systemic accumulation of acetaldehyde with ethanol ingestion. With chronic ethanol ingestion, mutations in ALDH2 are associated with a variety of hematological, neurological, and dermatological abnormalities, and an increased risk for esophageal cancer and osteoporosis. Based on our prior studies demonstrating that a one-time administration of an adeno-associated virus (AAV) serotype rh.10 gene transfer vector expressing the human ALDH2 cDNA (AAVrh.10hALDH2) prevents the acute effects of ethanol administration (the “Asian flush syndrome”), we hypothesized that AAVrh.10hALDH2 would also prevent the chronic disorders associated with ALDH2 deficiency and chronic ethanol ingestion. To assess this hypothesis, AAVrh.10hALDH2 (10¹¹ genome copies) was administered intravenously to two models of ALDH2 deficiency, Aldh2 knockout homozygous (Aldh2^−/−) and knockin homozygous (Aldh2^E487K+/+) mice (n = 10 per group). Four weeks after vector administration, mice were given drinking water with 10–15% ethanol for 12 weeks. Strikingly, compared with nonethanol drinking littermates, AAVrh.10hALDH2 administration prevented chronic ethanol-induced serum acetaldehyde accumulation and elevated liver malondialdehyde levels, loss of body weight, reduced hemoglobin levels, reduced performance in locomotor activity tests, accumulation of esophageal DNA damage and DNA adducts, and development of osteopenia. AAVrh.10hALDH2 should be considered as a preventative therapy for the increased risk of chronic disorders associated with ALDH2 deficiency and chronic alcohol exposure.  

5.2773           Pre-arrayed Pan-AAV Peptide Display Libraries for Rapid Single-Round Screening

Börner, K., Kienle, E., Huang, L-Y., Weinmann, J., Sacher, A., Bayer, P. et al Molecular Therapy, 28(4), 1016-1032 (2020)   Display of short peptides on the surface of adeno-associated viruses (AAVs) is a powerful technology for the generation of gene therapy vectors with altered cell specificities and/or transduction efficiencies. Following its extensive prior use in the best characterized AAV serotype 2 (AAV2), recent reports also indicate the potential of other AAV isolates as scaffolds for peptide display. In this study, we systematically explored the respective capacities of 13 different AAV capsid variants to tolerate 27 peptides inserted on the surface followed by production of reporter-encoding vectors. Single-round screening in pre-arrayed 96-well plates permitted rapid and simple identification of superior vectors in >90 cell types, including T cells and primary cells. Notably, vector performance depended not only on the combination of capsid, peptide, and cell type, but also on the position of the inserted peptide and the nature of flanking residues. For optimal data availability and accessibility, all results were assembled in a searchable online database offering multiple output styles. Finally, we established a reverse-transduction pipeline based on vector pre-spotting in 96- or 384-well plates that facilitates high-throughput library panning. Our comprehensive illustration of the vast potential of alternative AAV capsids for peptide display should accelerate their in vivo screening and application as unique gene therapy vectors.  

5.2774           Oncolytic vesicular stomatitis virus–based cellular vaccine improves triple-negative breast cancer outcome by enhancing natural killer and CD8+ T-cell functionality

Niavarani, S-R., Lawson, C., Boudaud, M., Simard, C. and Tai, L-H.

1. Immunother. Cancer, 8, e000465 (2020)

  The occurrence of triple-negative breast cancer (TNBC) is significant, with an estimated 40,000 cases diagnosed in US women each year (American Cancer Society, 2018). TNBC is a vastly heterogeneous disease that are grouped together histologically since they lack hormone and Her-2 receptors. However, TNBC is best considered as an umbrella term, encompassing a wide spectrum of entities with distinct genetic, transcriptional, histological and clinical differences.^1–3 As a group, TNBC is associated with high proliferation, early recurrence and poor survival rates.^{2 4} This aggressive disease is resistant to widely used targeted therapies such as trastuzumab and endocrine therapies, which have been effective at reducing breast cancer mortality. The best chance for survival is early detection, followed by neoadjuvant chemotherapy (NAC) and surgical resection.⁵ Patients with early-stage TNBC have increased rates of pathologic complete response (pCR) after NAC compared with other breast cancer subtypes. Indeed, the best prognostic factor for TNBC is the patient’s response to NAC. However, the increased pCR rates, but worse survival observed in TNBC—termed the triple negative paradox—appears to be driven by higher relapse rates among those patients whose tumors are not eradicated by chemotherapy.^{2 5} There are very limited and often ineffective treatment options for patients with poor prognosis (chemoresistant and late-stage/metastatic) TNBC.  

5.2775           Anatomy of a viral entry platform differentially functionalized by integrins α3 and α6

Finke, J., Mikulicic, S., Loster, A-L., Gawlitza, A., Florin, L. and Lang, T. Scientific Reports, 10:5356 (2020)   During cell invasion, human papillomaviruses use large CD151 patches on the cell surface. Here, we studied whether these patches are defined architectures with features for virus binding and/or internalization. Super-resolution microscopy reveals that the patches are assemblies of closely associated nanoclusters of CD151, integrin α3 and integrin α6. Integrin α6 is required for virus attachment and integrin α3 for endocytosis. We propose that CD151 organizes viral entry platforms with different types of integrin clusters for different functionalities. Since numerous viruses use tetraspanin patches, we speculate that this building principle is a blueprint for cell-surface architectures utilized by viral particles.  

5.2776           Conformational changes in the Ebola virus membrane fusion machine induced by pH, Ca2+, and receptor binding

Das, D.K., Bulow, U., Diehl, W.E., Durham, N.D., Senjobe, F., Chandran, K., Luban, J. and Munro, J.B. PloS Biology, 18(2), e3000626 (2020)   The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, removing the glycan cap and exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. NPC1 binding to cleaved GP1 is required for entry. How this interaction translates to GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a bulk fluorescence dequenching assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca²⁺, and NPC1 binding synergistically induce conformational changes in GP2 and permit virus-liposome lipid mixing. Acidic pH and Ca²⁺ shifted the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. Glycan cap cleavage on GP1 enabled GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the postfusion 6-helix bundle; NPC1 binding further promoted transition to the irreversible conformation. Thus, the glycan cap of GP1 may allosterically protect against inactivation of EBOV by premature triggering of GP2.  

5.2777           AAV6 Vexosomes Mediate Robust Suicide Gene Delivery in a Murine Model of Hepatocellular Carcinoma

Khan, N., Maurya, S., Bammidi, S. and Jayandharan, G. Molecular Therapy-Methods & Clin. Develop., 17, 497-504 (2020)   During recombinant Adeno-associated virus (AAV) production, a proportionately large amount of vectors is released in the culture supernatant, which is often discarded. It has been shown that these vectors often associate with vesiculated structures, such as exosomes. Exosome-associated AAV (vexosomes) represent an additional gene-delivery platform. The efficiency of such vexosomes in suicide gene therapy is unexplored. In the present study, we have generated AAV serotype 6 vexosomes containing an inducible caspase 9 (iCasp9) suicide gene by a differential ultracentrifugation-based protocol. We further tested the cytotoxic potential of these vexosomes in a human hepatocellular carcinoma (HCC) model in vitro and in vivo. The AAV6-iCasp9 containing vexosomes, when primed with a pro-drug (AP20187), demonstrated a significant loss in cell viability (57% ± 8% versus 100% ± 4.8%, p < 0.001) in comparison to mock-treated Huh7 cells. An intratumoral administration of AAV6-iCasp9 vexosomes and AP20187 in a murine xenograft model revealed a 2.3-fold increase in tumor regression in comparison to untreated animals. These findings were further corroborated by histological analysis and apoptosis assays. In conclusion, our data demonstrate the therapeutic potential of AAV6 vexosomes in a xenotransplantation model of HCC. Furthermore, the simplicity in production and isolation of vexosomes should further facilitate its application in other malignancies.  

5.2778           Site-Directed Mutagenesis Improves the Transduction Efficiency of Capsid Library-Derived Recombinant AAV Vectors

Ran, G., Chen, X., Xie, Y., Zheng, Q., Xie, J., Yu, C., Pittman, N., Qi, S., Yu, F-X., Agbandje-McKenna, M., Srivatava, A. and Ling, C. Molecular Therapy-Methods & Clin. Develop., 17, 545-555 (2020)   Recombinant adeno-associated virus (rAAV) vectors selected from capsid libraries present enormous advantages in high selectivity of tissue tropism and their potential use in human gene therapy applications. For example, rAAV-LK03, was used in a gene therapy trial for hemophilia A (ClinicalTrials.gov: NCT03003533). However, high doses in patients resulted in severe adverse events and subsequent loss of factor VIII (FVIII) expression. Thus, additional strategies are needed to enhance the transduction efficiency of capsid library-derived rAAV vectors such that improved clinical efficacy can be achieved at low vector doses. In this study, we characterized two commonly used library-derived rAAV vectors, rAAV-DJ and rAAV-LK03. It was concluded that rAAV-DJ shared similar transport pathways (e.g., cell surface binding, endocytosis-dependent internalization, and cytoplasmic trafficking) with rAAV serotype 2, while rAAV-LK03 and rAAV serotype 3 shared similar transport pathways. We then performed site-directed mutagenesis of surface-exposed tyrosine (Y), serine (S), aspartic acid (D), and tryptophan (W) residues on rAAV-DJ and rAAV-LK03 capsids. Our results demonstrated that rAAV-DJ-S269T and rAAV-LK03-Y705+731F variants had significantly enhanced transduction efficiency compared to wild-type counterparts. Our studies suggest that the strategy of site-directed mutagenesis should be applicable to other non-natural AAV variants for their optimal use in human gene therapy.  

5.2779           Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses

Letko, M., Marzi, A. and Munster, V. Nature Microbiol., 5, 562-569 (2020)   Over the past 20 years, several coronaviruses have crossed the species barrier into humans, causing outbreaks of severe, and often fatal, respiratory illness. Since SARS-CoV was first identified in animal markets, global viromics projects have discovered thousands of coronavirus sequences in diverse animals and geographic regions. Unfortunately, there are few tools available to functionally test these viruses for their ability to infect humans, which has severely hampered efforts to predict the next zoonotic viral outbreak. Here, we developed an approach to rapidly screen lineage B betacoronaviruses, such as SARS-CoV and the recent SARS-CoV-2, for receptor usage and their ability to infect cell types from different species. We show that host protease processing during viral entry is a significant barrier for several lineage B viruses and that bypassing this barrier allows several lineage B viruses to enter human cells through an unknown receptor. We also demonstrate how different lineage B viruses can recombine to gain entry into human cells, and confirm that human ACE2 is the receptor for the recently emerging SARS-CoV-2.  

5.2780           Interference with ERK-dimerization at the nucleocytosolic interface targets pathological ERK1/2 signaling without cardiotoxic side-effects

Tomasovic, A. et al Nature Communications, 11:1733 (2020)   Dysregulation of extracellular signal-regulated kinases (ERK1/2) is linked to several diseases including heart failure, genetic syndromes and cancer. Inhibition of ERK1/2, however, can cause severe cardiac side-effects, precluding its wide therapeutic application. ERK^T188-autophosphorylation was identified to cause pathological cardiac hypertrophy. Here we report that interference with ERK-dimerization, a prerequisite for ERK^T188-phosphorylation, minimizes cardiac hypertrophy without inducing cardiac adverse effects: an ERK-dimerization inhibitory peptide (EDI) prevents ERK^T188-phosphorylation, nuclear ERK1/2-signaling and cardiomyocyte hypertrophy, protecting from pressure-overload-induced heart failure in mice whilst preserving ERK1/2-activity and cytosolic survival signaling. We also examine this alternative ERK1/2-targeting strategy in cancer: indeed, ERK^T188-phosphorylation is strongly upregulated in cancer and EDI efficiently suppresses cancer cell proliferation without causing cardiotoxicity. This powerful cardio-safe strategy of interfering with ERK-dimerization thus combats pathological ERK1/2-signaling in heart and cancer, and may potentially expand therapeutic options for ERK1/2-related diseases, such as heart failure and genetic syndromes.  

5.2781           Development of an In Vitro Biopotency Assay for an AAV8 Hemophilia B Gene Therapy Vector Suitable for Clinical Product Release

Lengler, J., Coulibaly, S., Gruber, B., ilk, R., Mayrhofer, R., Scheiflinger, F., Hoellriegl, W., Falkner, F.G. and Rottensteiner, H. Molecular Therapy-Methods & Clin. Develop., 17, 581-588 (2020)   Gene therapy product release requires reliable and consistent demonstration of biopotency. In hemophilia B vectors, this is usually determined in vivo by measuring the plasma levels of the expressed human factor IX (FIX) transgene product in FIX knockout mice. To circumvent this laborious assay, we developed an in vitro method in which the HepG2 human liver cell line was infected with the vector, and the resulting FIX activity was determined in the conditioned medium using a chromogenic assay. The initial low sensitivity of the assay, particularly toward adeno-associated viral serotype 8 (AAV8), increased approximately 100-fold and allowed linear measurement in a broad range of multiplicities of infection. Statistical parameters indicated high assay repeatability (relative standard deviation (RSD) < 5%) and intra-assay reproducibility (RSD < 20%). To compare the performance of the in vitro and in vivo biopotency assay, we applied statistical analyses including regression techniques and variation decomposition to the results obtained for 25 AAV8-FIX vector lots (BAX 335). These showed a highly significant correlation, with the cell culture-based assay demonstrating less variation than the in vivo test. The in vitro assay thus constitutes a viable alternative to using animals for lot release testing.  

5.2782           Pooled Screens Identify GPR108 and TM9SF2 as Host Cell Factors Critical for AAV Transduction

Meisen, W.H., Nejad, Z.B., Hardy, M., Zhao, H., Oliveiro, O., Wang, S., Hale, C., Ollmann, M.M. and Collins, P.J. Molecular Therapy-methods & Clin. Develop., 17, 601-611 (2020)   Adeno-associated virus (AAV) has been used extensively as a vector for gene therapy. Despite its widespread use, the mechanisms by which AAV enters the cell and is trafficked to the nucleus are poorly understood. In this study, we performed two pooled, genome-wide screens to identify positive and negative factors modulating AAV2 transduction. Genome-wide libraries directed against all human genes with four designs per gene or eight designs per gene were transduced into U-2 OS cells. These pools were transduced with AAV2 encoding EGFP and sorted based on the intensity of EGFP expression. Analysis of enriched and depleted barcodes in the sorted samples identified several genes that putatively decreased AAV2 transduction. A subset of screen hits was validated in flow cytometry and imaging studies. In addition to KIAA0319L (AAVR), we confirmed the role of two genes, GPR108 and TM9SF2, in mediating viral transduction in eight different AAV serotypes. Interestingly, GPR108 displayed serotype selectivity and was not required for AAV5 transduction. Follow-up studies suggested that GPR108 localized primarily to the Golgi, where it may interact with AAV and play a critical role in mediating virus escape or trafficking. Cumulatively, these results expand our understanding of the process of AAV transduction in different cell types and serotypes.  

5.2783           Inhibition of HDAC6 activity protects dopaminergic neurons from alpha-synuclein toxicity

Francelle, L., Outreiro, T.F. and Rappoldi, G.A. Scientific Reports, 10:6064 (2020)   The neuropathological hallmarks of Parkinson’s disease include preferential vulnerability of dopaminergic neurons of the substantia nigra pars compacta, and accumulation of intraneuronal protein inclusions known as Lewy bodies. These inclusions contain, among other proteins, aggregated alpha-synuclein and histone deacetylase 6 (HDAC6). In our study we found that selective inhibition of HDAC6 activity by Tubastatin A has protective effects in a rat model of Parkinson’s disease. We provide evidence that this protection may be due to the activation of chaperone-mediated autophagy through the up-regulation of key members of this pathway. Moreover, Tubastatin A significantly inhibited the expression of a toxic form of alpha-synuclein that is phosphorylated at serine position 129. Tubastatin A treatment also permitted to partially modulate neuroinflammation. Taken together, our study highlights the neuroprotective effects of Tubastatin A in a rat model of Parkinson’s disease and provides mechanistic insight in Tubastatin A-mediated protection against alpha-synuclein toxicity and substantia nigra degeneration. These findings are of potential therapeutic value in Parkinson’s disease and other synucleinopathies.  

5.2784           Golgi-associated BICD adaptors couple ER membrane penetration and disassembly of a viral cargo

Spriggs, C.C., Badeiyan, S., Verhey, K.J., Cianfrocco, M.A. and Tsai, B.

1. Celll. Biol., 219(5), e201908099 (2020)

  During entry, viruses must navigate through the host endomembrane system, penetrate cellular membranes, and undergo capsid disassembly to reach an intracellular destination that supports infection. How these events are coordinated is unclear. Here, we reveal an unexpected function of a cellular motor adaptor that coordinates virus membrane penetration and disassembly. Polyomavirus SV40 traffics to the endoplasmic reticulum (ER) and penetrates a virus-induced structure in the ER membrane called “focus” to reach the cytosol, where it disassembles before nuclear entry to promote infection. We now demonstrate that the ER focus is constructed proximal to the Golgi-associated BICD2 and BICDR1 dynein motor adaptors; this juxtaposition enables the adaptors to directly bind to and disassemble SV40 upon arrival to the cytosol. Our findings demonstrate that positioning of the virus membrane penetration site couples two decisive infection events, cytosol arrival and disassembly, and suggest cargo remodeling as a novel function of dynein adaptors.  

5.2785           T cell infiltration in both human multiple system atrophy and a novel mouse model of the disease

Williams, G.P., Marmion, D.J., Schonhoff, A.M., Kurkuvenaite, A., Won, W-J., Standaert, D.G., Kordower, J.H. and Harms, A.S. Acta Neuropathologica, 139, 855-974 (2020)   Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by abnormal accumulation of alpha-synuclein (α-syn) in oligodendrocytes accompanied by inflammation, demyelination, and subsequent synapse and neuronal loss. Little is known about the mechanisms of neurodegeneration in MSA. However, recent work has highlighted the important role of the immune system to the pathophysiology of other synuclein-related diseases such as Parkinson’s disease. In this study, we investigated postmortem brain tissue from MSA patients and control subjects for evidence of immune activation in the brain. We found a significant increase of HLA-DR⁺ microglia in the putamen and substantia nigra of MSA patient tissue compared to controls, as well as significant increases in CD3⁺, CD4⁺, and CD8⁺ T cells in these same brain regions. To model MSA in vivo, we utilized a viral vector that selectively overexpresses α-syn in oligodendrocytes (Olig001-SYN) with > 95% tropism in the dorsal striatum of mice, resulting in demyelination and neuroinflammation similar to that observed in human MSA. Oligodendrocyte transduction with this vector resulted in a robust inflammatory response, which included increased MHCII expression on central nervous system (CNS) resident microglia, and infiltration of pro-inflammatory monocytes into the CNS. We also observed robust infiltration of CD4 T cells into the CNS and antigen-experienced CD4 T cells in the draining cervical lymph nodes. Importantly, genetic deletion of TCR-β or CD4 T cells attenuated α-syn-induced inflammation and demyelination in vivo. These results suggest that T cell priming and infiltration into the CNS are key mechanisms of disease pathogenesis in MSA, and therapeutics targeting T cells may be disease modifying.  

5.2786           Combined Treatment with Peptide-Conjugated Phosphorodiamidate Morpholino Oligomer-PPMO and AAV-U7 Rescues the Severe DMD Phenotype in Mice

Forand, A., Muchir, A., Mougenot, N., Sevoz-Couche, C., Peccate, C., Lemaitre, M., Izabelle, C., Wood, M., Lorain, S. and Pietri-Rouxel, F. Molecular Therapy-Methods  & Clin. Develop., 17, 695-708 (2020)   Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by an absence of the dystrophin protein, which is essential for muscle fiber integrity. Among the developed therapeutic strategies for DMD, the exon-skipping approach corrects the frameshift and partially restores dystrophin expression. It could be achieved through the use of antisense sequences, such as peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) or the small nuclear RNA-U7 carried by an adeno-associated virus (AAV) vector. AAV-based gene therapy approaches have potential for use in DMD treatment but are subject to a major limitation: loss of the AAV genome, necessitating readministration of the vector, which is not currently possible, due to the immunogenicity of the capsid. The PPMO approach requires repeated administrations and results in only weak cardiac dystrophin expression. Here, we evaluated a combination of PPMO- and AAV-based therapy in a mouse model of severe DMD. Striking benefits of this combined therapy were observed in striated muscles, with marked improvements in heart and diaphragm structure and function, with unrivalled extent of survival, opening novel therapeutic perspectives for patients.  

5.2787           Intra-striatal AAV2.retro administration leads to extensive retrograde transport in the rhesus macaque brain: implications for disease modeling and therapeutic development

Weiss, A.R., Liguore, W.A., Domire, J.S., Button, D. and McBride, J.L. Scientific Reports, 10:6970 (2020)   Recently, AAV2.retro, a new capsid variant capable of efficient retrograde transport in brain, was generated in mice using a directed evolution approach. However, it remains unclear to what degree transport will be recapitulated in the substantially larger and more complex nonhuman primate (NHP) brain. Here, we compared the biodistribution of AAV2.retro with its parent serotype, AAV2, in adult macaques following delivery into the caudate and putamen, brain regions which comprise the striatum. While AAV2 transduction was primarily limited to the injected brain regions, AAV2.retro transduced cells in the striatum and in dozens of cortical and subcortical regions with known striatal afferents. We then evaluated the capability of AAV2.retro to deliver disease-related gene cargo to biologically-relevant NHP brain circuits by packaging a fragment of human mutant HTT, the causative gene mutation in Huntington’s disease. Following intra-striatal delivery, pathological mHTT-positive protein aggregates were distributed widely among cognitive, motor, and limbic cortico-basal ganglia circuits. Together, these studies demonstrate strong retrograde transport of AAV2.retro in NHP brain, highlight its utility in developing novel NHP models of brain disease and suggest its potential for querying circuit function and delivering therapeutic genes in the brain, particularly where treating dysfunctional circuits, versus single brain regions, is warranted.  

5.2788           Aging-regulated anti-apoptotic long non-coding RNA Sarrah augments recovery from acute myocardial infarction

Trembinski, D.J., Bink, D.I., Theodorou, K., Sommer, J., Fischer, A., van Bergen, A. et al Nature Comminucations, 11:2039 (2020)   Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.  

5.2789           Small Alphaherpesvirus Latency-Associated Promoters Drive Efficient and Long-Term Transgene Expression in the CNS

Maturana, C.J., Verpeut, J.L., Pisano, T.J., Dhanerawala, Z.M., Esteves, A., Enquist, L.W. and Engel, E.A. Molecular Therapy-Methods & Clin. Develop., 17, 843-857 (2020)   Recombinant adeno-associated viruses (rAAVs) are used as gene therapy vectors to treat central nervous system (CNS) diseases. Despite their safety and broad tropism, important issues need to be corrected such as the limited payload capacity and the lack of small gene promoters providing long-term, pan-neuronal transgene expression in the CNS. Commonly used gene promoters are relatively large and can be repressed a few months after CNS transduction, risking the long-term performance of single-dose gene therapy applications. We used a whole-CNS screening approach based on systemic delivery of AAV-PHP.eB, iDisco+ tissue-clearing and light-sheet microscopy to identify three small latency-associated promoters (LAPs) from the herpesvirus pseudorabies virus (PRV). These promoters are LAP1 (404 bp), LAP2 (498 bp), and LAP1_2 (880 bp). They drive chronic transcription of the virus-encoded latency-associated transcript (LAT) during productive and latent phases of PRV infection. We observed stable, pan-neuronal transgene transcription and translation from AAV-LAPs in the CNS for 6 months post AAV transduction. In several CNS areas, the number of cells expressing the transgene was higher for LAP2 than the large conventional EF1α promoter (1,264 bp). Our data suggest that the LAPs are suitable candidates for viral vector-based CNS gene therapies requiring chronic transgene expression after one-time viral-vector administration.  

5.2790           Enhanced Transduction of Human Hematopoietic Stem Cells by AAV6 Vectors: Implications in Gene Therapy and Genome Editing

Yang, H., Qing, K., Keeler, G.D., Yin, L., Mietzsch, M., Ling, C., Hoffman, B.E., Agbandje-McKenna, M., Tan, M., Wang, W. and Srivastava, A. Molecular Therapy-Nucleic Acids, 20, 451-458 (2020)   We have reported that of the 10 most commonly used adeno-associated virus (AAV) serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs) in vitro, as well as in vivo. More recently, polyvinyl alcohol (PVA), was reported to be a superior replacement for human serum albumin (HSA) for ex vivo expansion of HSCs. Since HSA has been shown to increase the transduction efficiency of AAV serotype vectors, we evaluated whether PVA could also enhance the transduction efficiency of AAV6 vectors in primary human HSCs. We report here that up to 12-fold enhancement in the transduction efficiency of AAV6 vectors can be achieved in primary human HSCs with PVA. We also demonstrate that the improvement in the transduction efficiency is due to PVA-mediated improved entry and intracellular trafficking of AAV6 vectors in human hematopoietic cells in vitro, as well as in murine hepatocytes in vivo. Taken together, our studies suggest that the use of PVA is an attractive strategy to further improve the efficacy of AAV6 vectors. This has important implications in the optimal use of these vectors in the potential gene therapy and genome editing for human hemoglobinopathies such as β-thalassemia and sickle cell disease.  

5.2791           Hexanucleotide Repeat Expansions in c9FTD/ALS and SCA36 Confer Selective Patterns of Neurodegeneration In Vivo

Todd, T.W., McEachin, Z.T., Chew, J., Bassell, G.J., Rossoll, W. and Petrucelli, L. Cell Reports, 31, 107616 (2020)   A G₄C₂ hexanucleotide repeat expansion in an intron of C9orf72 is the most common cause of frontal temporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). A remarkably similar intronic TG₃C₂ repeat expansion is associated with spinocerebellar ataxia 36 (SCA36). Both expansions are widely expressed, form RNA foci, and can undergo repeat-associated non-ATG (RAN) translation to form similar dipeptide repeat proteins (DPRs). Yet, these diseases result in the degeneration of distinct subsets of neurons. We show that the expression of these repeat expansions in mice is sufficient to recapitulate the unique features of each disease, including this selective neuronal vulnerability. Furthermore, only the G₄C₂ repeat induces the formation of aberrant stress granules and pTDP-43 inclusions. Overall, our results demonstrate that the pathomechanisms responsible for each disease are intrinsic to the individual repeat sequence, highlighting the importance of sequence-specific RNA-mediated toxicity in each disorder.    

5.2792           Extracellular vesicles provide a capsid-free vector for oncolytic adenoviral DNA delivery

Saari, H., Turunen, T., Löhmus, A., Turunen, M., Jalasvuori, M., Butcher, S.J., Ylä-Herttuala, S., Viitala, T., Cerullo, V., Siljander, P.R.M. and Yliperttula, M.

1. Extracellular Vesicles, 9(1), 1747206 (2020)

  Extracellular vesicles (EVs) have been showcased as auspicious candidates for delivering therapeutic cargo, including oncolytic viruses for cancer treatment. Delivery of oncolytic viruses in EVs could provide considerable advantages, hiding the viruses from the immune system and providing alternative entry pathways into cancer cells. Here we describe the formation and viral cargo of EVs secreted by cancer cells infected with an oncolytic adenovirus (IEVs, infected cell-derived EVs) as a function of time after infection. IEVs were secreted already before the lytic release of virions and their structure resembled normally secreted EVs, suggesting that they were not just apoptotic fragments of infected cells. IEVs were able to carry the viral genome and induce infection in other cancer cells. As such, the role of EVs in the life cycle of adenoviruses may be an important part of a successful infection and may also be harnessed for cancer- and gene therapy.  

5.2793           Current challenges in biotherapeutic particles manufacturing

Moleirinho, M.G., Silva, R.J.S., Alves, P.M., Carrondo, J.T. and Peixoto, C. Expert Opinion on Biological Therapy, 20(5), 451-465 (2020)   Introduction: The development of novel complex biotherapeutics led to new challenges in biopharmaceutical industry. The potential of these particles has been demonstrated by the approval of several products, in the different fields of gene therapy, oncolytic therapy, and tumor vaccines. However, their manufacturing still presents challenges related to the high dosages and purity required. Areas covered: The main challenges that biopharmaceutical industry faces today and the most recent developments in the manufacturing of different biotherapeutic particles are reported here. Several unit operations and downstream trains to purify virus, virus-like particles and extracellular vesicles are described. Innovations on the different purification steps are also highlighted with an eye on the implementation of continuous and integrated processes. Expert opinion: Manufacturing platforms that consist of a low number of unit operations, with higher-yielding processes and reduced costs will be highly appreciated by the industry. The pipeline of complex therapeutic particles is expanding and there is a clear need for advanced tools and manufacturing capacity. The use of single-use technologies, as well as continuous integrated operations, are gaining ground in the biopharmaceutical industry and should be supported by more accurate and faster analytical methods.  

5.2794           Systematically optimized BCMA/CS1 bispecific CAR-T cells robustly control heterogeneous multiple myeloma

Zah, E., Nam, E., Bhuvan, V., tran, U., Ji, B.Y., Gosliner, S.B., Wang, X., Brown, C.E. and Chen, Y.Y. Nature Communications, 11:2283 (2020)   Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable clinical efficacy against B-cell malignancies, yet marked vulnerability to antigen escape and tumor relapse exists. Here we report the rational design and optimization of bispecific CAR-T cells with robust activity against heterogeneous multiple myeloma (MM) that is resistant to conventional CAR-T cell therapy targeting B-cell maturation antigen (BCMA). We demonstrate that BCMA/CS1 bispecific CAR-T cells exhibit superior CAR expression and function compared to T cells that co-express individual BCMA and CS1 CARs. Combination therapy with anti–PD-1 antibody further accelerates the rate of initial tumor clearance in vivo, while CAR-T cell treatment alone achieves durable tumor-free survival even upon tumor re-challenge. Taken together, the BCMA/CS1 bispecific CAR presents a promising treatment approach to prevent antigen escape in CAR-T cell therapy against MM, and the vertically integrated optimization process can be used to develop robust cell-based therapy against novel disease targets.  

5.2795           An essential N-terminal serine-rich motif in the AAV VP1 and VP2 subunits that may play a role in viral transcription

Robinson, T., Ho, M.L., Wahlig, B., Gough, V., Banta, A., Gamas, K.R., Kang, B., Lee, E., Chen, W. and Suh, J. Virology, 546, 127-132 (2020)   Adeno-associated virus (AAV) is one of the most researched, clinically utilized gene therapy vectors. Though clinical success has been achieved, transgene delivery and expression may be hindered by cellular and tissue barriers. Understanding the role of receptor binding, entry, endosomal escape, cytoplasmic and nuclear trafficking, capsid uncoating, and viral transcription in therapeutic efficacy is paramount. Previous studies have shown that N-terminal regions of the AAV capsid proteins are responsible for endosomal escape and nuclear trafficking, however the mechanisms remain unknown. We identified a highly-conserved three-residue serine/threonine (S/T) motif in the capsid N-terminus, previously uncharacterized in its role in intracellular trafficking and transduction. Using alanine scanning mutagenesis, we found S155 and the flanking residues, D154 and G158, are essential for AAV2 transduction efficiency. Remarkably, specific capsid mutants show a 5 to 9-fold decrease in viral mRNA transcripts, highlighting a potential role of the S/T motif in transcription of the viral genome.  

5.2796           Translational Feasibility of Lumbar Puncture for Intrathecal AAV Administration

Hinderer, C., katz, N., Dyer, C., Goode, T., Johansson, J., Bell, P., Richman, L., Buza, E. and Wilson, J.M. Molecular Therapy-Methods & Clin. Develop., 17, 969-974 (2020)   Preclinical studies have demonstrated that a single injection of an adeno-associated virus (AAV) vector into the cerebrospinal fluid (CSF) can achieve widespread gene transfer throughout the central nervous system. Successfully translating this approach to humans requires identifying factors that influence AAV distribution in the CSF so that optimal parameters can be replicated in the clinic. In the context of developing a motor neuron-targeted gene therapy for spinal muscular atrophy, we conducted studies in nonhuman primates to evaluate the impact of injection volume on spinal cord transduction after AAV delivery via lumbar puncture. Lumbar injection of an AAVhu68 vector targeted motor neurons throughout the spinal cord, but only in juvenile nonhuman primates administered large injection volumes, equivalent to about half of the total CSF volume. Upon repeating this study with clinically relevant injection volumes and larger animals, we found that lumbar puncture failed to achieve significant transduction of the spinal cord. In contrast, vector administered into the cisterna magna distributed reproducibly throughout the spinal cord in both juvenile and adult animals. These findings highlight the challenges of translating AAV delivery via lumbar puncture to humans and suggest that delivery into the cisterna magna may represent a more feasible alternative.  

5.2797           CRISPR/Cas9-Mediated miR-29b Editing as a Treatment of Different Types of Muscle Atrophy in Mice

Li, J., Wang, L., Hua, X., Tang, H., Chen, R., Yang, T., Das, S. and Xiao, J. Molecular Therapy, 28(5), 1359-1372 (2020)   Muscle atrophy is the loss of skeletal muscle mass and strength in response to diverse catabolic stimuli. At present, no effective treatments except exercise have been shown to reduce muscle atrophy clinically. Here, we report that CRISPR/Cas9-mediated genome editing through local injection into gastrocnemius muscles or tibialis anterior muscle efficiently targets the biogenesis processing sites in pre-miR-29b. In vivo, this CRISPR-based treatment prevented the muscle atrophy induced by angiotensin II (AngII), immobilization, and denervation via activation of the AKT-FOXO3A-mTOR signaling pathway and protected against AngII-induced myocyte apoptosis in mice, leading to significantly increased exercise capacity. Our work establishes CRISPR/Cas9-based gene targeting on miRNA as a potential durable therapy for the treatment of muscle atrophy and expands the strategies available interrogating miRNA function in vivo.  

5.2798           Type I IFN Sensing by cDCs and CD4+ T Cell Help Are Both Requisite for Cross-Priming of AAV Capsid-Specific CD8+ T Cells

Shirley, J.L., Keeler, G.D., Sherman, A., Zolotukhin, I., Markusic, D.M., Hoffman, B.E., Morel, L.M., Wallet, M.A., Terhorst, C. and Herzog, R.W. Molecular Therapy, 28(3), 758-770 (2020)   Adeno-associated virus (AAV) vectors are widely used in clinical gene therapy to correct genetic disease by in vivo gene transfer. Although the vectors are useful, in part because of their limited immunogenicity, immune responses directed at vector components have complicated applications in humans. These include, for instance, innate immune sensing of vector components by plasmacytoid dendritic cells (pDCs), which sense the vector DNA genome via Toll-like receptor 9. Adaptive immune responses employ antigen presentation by conventional dendritic cells (cDCs), which leads to cross-priming of capsid-specific CD8⁺ T cells. In this study, we sought to determine the mechanisms that promote licensing of cDCs, which is requisite for CD8⁺ T cell activation. Blockage of type 1 interferon (T1 IFN) signaling by monoclonal antibody therapy prevented cross-priming. Furthermore, experiments in cell-type-restricted knockout mice showed a specific requirement for the receptor for T1 IFN (IFNaR) in cDCs. In contrast, natural killer (NK) cells are not needed, indicating a direct rather than indirect effect of T1 IFN on cDCs. In addition, co-stimulation by CD4⁺ T cells via CD40-CD40L was required for cross-priming, and blockage of co-stimulation but not of T1 IFN additionally reduced antibody formation against capsid. These mechanistic insights inform the development of targeted immune interventions.  

5.2799           The Effect of CpG Sequences on Capsid-Specific CD8+ T Cell Responses to AAV Vector Gene Transfer

Xiang, Z., Kurupati, R.K., Li, Y., Kuranda, K., Zhou, X., Mingozzi, F., High, K.A. and Ertl, H.C.J. Molelcular Therapy, 28(3), 771-783 (2020)   Transfer of genes by adeno-associated virus (AAV) vectors is benefiting patients with particular genetic defects. Challenges remain by rejection of AAV-transduced cells, which may be caused by CD8⁺ T lymphocytes directed to AAV capsid antigens. Reducing the number of CpG motifs from the genome of AAV vectors reduces expansion of naive T cells directed against an epitope within the capsid. In contrast, AAV capsid-specific memory CD8⁺ T cells respond more vigorously to AAV vectors lacking CpG motifs than to those with CpG motifs presumably reflecting dampening of T cell expansion by cytokines from the innate immune system. Depending on the purification method, AAV vector preparations can contain substantial amounts of empty AAV particles that failed to package the genome. Others have used empty particles as decoys to AAV-neutralizing antibodies. We tested if empty AAV vectors given alone or mixed with genome-containing AAV vectors induce proliferation of naive or memory CD8⁺ T cells directed to an antigen within an AAV capsid. Naive CD8⁺ T cells failed to respond to empty AAV vectors, which in contrast induced expansion of AAV-specific memory CD8⁺ T cells.  

5.2800           Repair of Retinal Degeneration following Ex Vivo Minicircle DNA Gene Therapy and Transplantation of Corrected Photoreceptor Progenitors

Barnea-Cramer, A.O., Singh, M., Fischer, D., De Silva, S., McClements, M.E., Barnard, A.R. and MacLaren, R.E. Molecular Therapy, 28(3), 830-844 (2020)   The authors describe retinal reconstruction and restoration of visual function in heritably blind mice missing the rhodopsin gene using a novel method of ex vivo gene therapy and cell transplantation. Photoreceptor precursors with the same chromosomal genetic mutation were treated ex vivo using minicircle DNA, a non-viral technique that does not present the packaging limitations of adeno-associated virus (AAV) vectors. Following transplantation, genetically modified cells reconstructed a functional retina and supported vision in blind mice harboring the same founder gene mutation. Gene delivery by minicircles showed comparable long-term efficiency to AAV in delivering the missing gene, representing the first non-viral system for robust treatment of photoreceptors. This important proof-of-concept finding provides an innovative convergence of cell and gene therapies for the treatment of hereditary neurodegenerative disease and may be applied in future studies toward ex vivo correction of patient-specific cells to provide an autologous source of tissue to replace lost photoreceptors in inherited retinal blindness. This is the first report using minicircles in photoreceptor progenitors and the first to transplant corrected photoreceptor precursors to restore vision in blind animals.  

5.2801           Role of DJ‐1 in Modulating Glycative Stress in Heart Failure

Shimizu, Y., Nicholson, C.K., Polavarapu, R., Pantner, Y., Husain, A., Naqvi, N., Chin, L-S., Li, L., and Calvert, J.W.

1. Am. Heart. Ass., 9, e014691 (2020)

  Background DJ‐1 is a ubiquitously expressed protein typically associated with the development of early onset Parkinson disease. Recent data suggest that it also plays a role in the cellular response to stress. Here, we sought to determine the role DJ‐1 plays in the development of heart failure. Methods and Results Initial studies found that DJ‐1 deficient mice (DJ‐1 knockout; male; 8–10 weeks of age) exhibited more severe left ventricular cavity dilatation, cardiac dysfunction, hypertrophy, and fibrosis in the setting of ischemia‐reperfusion–induced heart failure when compared with wild‐type littermates. In contrast, the overexpression of the active form of DJ‐1 using a viral vector approach resulted in significant improvements in the severity of heart failure when compared with mice treated with a control virus. Subsequent studies aimed at evaluating the underlying protective mechanisms found that cardiac DJ‐1 reduces the accumulation of advanced glycation end products and activation of the receptor for advanced glycation end products—thus, reducing glycative stress. Conclusions These results indicate that DJ‐1 is an endogenous cytoprotective protein that protects against the development of ischemia‐reperfusion–induced heart failure by reducing glycative stress. Our findings also demonstrate the feasibility of using a gene therapy approach to deliver the active form of DJ‐1 to the heart as a therapeutic strategy to protect against the consequences of ischemic injury, which is a major cause of death in western populations.  

5.2802           Cas9-AAV6-engineered human mesenchymal stromal cells improved cutaneous wound healing in diabetic mice

Srifa, W., Kosaric, N., Amorin, A., Jadi, O., Park, Y., Mantri, S., Camarena, J., Gurtner, G.C. and Porrteus, M. Nature Communications, 11:2470 (2020)   Human mesenchymal stromal cells (hMSCs) are a promising source for engineered cell-based therapies in which genetic engineering could enhance therapeutic efficacy and install novel cellular functions. Here, we describe an optimized Cas9-AAV6-based genome editing tool platform for site-specific mutagenesis and integration of up to more than 3 kilobases of exogenous DNA in the genome of hMSCs derived from the bone marrow, adipose tissue, and umbilical cord blood without altering their ex vivo characteristics. We generate safe harbor-integrated lines of engineered hMSCs and show that engineered luciferase-expressing hMSCs are transiently active in vivo in wound beds of db/db mice. Moreover, we generate PDGF-BB- and VEGFA-hypersecreting hMSC lines as short-term, local wound healing agents with superior therapeutic efficacy over wildtype hMSCs in the diabetic mouse model without replacing resident cells long-term. This study establishes a precise genetic engineering platform for genetic studies of hMSCs and development of engineered hMSC-based therapies.  

5.2803           Long-Term Efficacy of AAV9-U7snRNA-Mediated Exon 51 Skipping in mdx52 Mice

Aupy, P., Zarrouki, F., Sandro, Q., Gastaldi, C., Buclez, P-O., Mamchaoui, K., Garcia, L., Vaillend, C. and Goyenvalle, A. Molecular Therapy-Methods & Clin. Develop., 17, 1037-1047 (2020)   Gene therapy and antisense approaches hold promise for the treatment of Duchenne muscular dystrophy (DMD). The advantages of both therapeutic strategies can be combined by vectorizing antisense sequences into an adeno-associated virus (AAV) vector. We previously reported the efficacy of AAV-U7 small nuclear RNA (U7snRNA)-mediated exon skipping in the mdx mouse, the dys⁻/utr⁻ mouse, and the golden retriever muscular dystrophy (GRMD) dog model. In this study, we examined the therapeutic potential of an AAV-U7snRNA targeting the human DMD exon 51, which could be applicable to 13% of DMD patients. A single injection of AAV9-U7 exon 51 (U7ex51) induces widespread and sustained levels of exon 51 skipping, leading to significant restoration of dystrophin and improvement of the dystrophic phenotype in the mdx52 mouse. However, levels of dystrophin re-expression are lower than the skipping levels, in contrast with previously reported results in the mdx mouse, suggesting that efficacy of exon skipping may vary depending on the targeted exon. Additionally, while low levels of exon skipping were measured in the brain, the dystrophin protein could not be detected, in line with a lack of improvement of their abnormal behavioral fear response. These results thus confirm the high therapeutic potential of the AAV-mediated exon-skipping approach, yet the apparent discrepancies between exon skipping and protein restoration levels suggest some limitations of this experimental model.  

5.2804           Different Serotypes of Adeno-Associated Virus Vector- and Lentivirus-Mediated Tropism in Choroid Plexus by Intracerebroventricular Delivery

Chen, X., He, Y., Tian, Y., Wang, Y., Wu, Z., Lan, T., Wang, H., Cheng, K. and Xie, P. Human Gene Therapy, 31(7-8), 440-447 (2020)   Regulation of gene expression by viral vectors is an effective method for researchers to explore the function of gene products in a target tissue. The choroid plexus (CP) is an important target for gene therapy of neuropsychiatric diseases such as Alzheimer's disease and major depressive disorder. However, viral tropism in CP has not been well studied as a result of limited viral vector applications. To identify CP-specific viral vectors, we intracerebroventricularly administered six different serotypes of adeno-associated virus (AAV) vectors (AAV2/1, AAV2/5, AAV2/8, AAV2/9, AAV2-BR1, and AAV2-PHP.eB) and lentivirus in adult mice. Tropism in CP was compared among these viruses. We found that AAV2/5 and AAV2/8 displayed remarkable infections in CP, while AAV2/1 infected both ependymal cells and cells in the CP. Except for the low infection intensity of AAV2/9 and lentivirus in the CP, no infection intensity was found for CP tissues injected with AAV2-BR1 or AAV2-PHP.eB. Green fluorescence protein expression in the CP after AAV2/5 infection was confirmed by Western blotting. AAV2/5-mediated tropism in epithelial cells of the CP was verified by immunostaining with transthyretin. In this study, we identified for the first time that serotype-specific AAVs 5 and 8 may be robust research tools for intracerebroventricular gene delivery.  

5.2805           Adeno-Associated Virus D-Sequence-Mediated Suppression of Expression of a Human Major Histocompatibility Class II Gene: Implications in the Development of Adeno-Associated Virus Vectors for Modulating Humoral Immune Response

Kwon, H-J., Qing, K., Ponnazhagan, S., Wang, X-S., markusic, D.M., Gupte, S., Boye, S.E. and Srivastava, A. Human Gene Therapy, 31(9-10), 565-574 (2020)   A 20-nt long sequence, termed the D-sequence, in the adeno-associated virus (AAV) inverted terminal repeat was observed to share a partial sequence homology with the X-box in the regulatory region of the human leukocyte antigen DRA (HLA-DRA) promoter of the human major histocompatibility complex class II (MHC-II) genes. The D-sequence was also shown to specifically interact with the regulatory factor binding to the X-box (RFX), binding of which to the X-box is a critical step in the MHC-II gene expression, suggesting that D-sequence might compete for RFX transcription factor binding, thereby suppressing expression from the MHC-II promoter. In DNA-mediated transfection experiments, using a reporter gene under the control of the HLA-DRA promoter, D-sequence oligonucleotides were found to inhibit expression of the reporter gene expression in HeLa and 293 cells by ∼93% and 96%, respectively. No inhibition was observed when nonspecific synthetic oligonucleotides were used. D-sequence oligonucleotides had no effect on expression from the cytomegalovirus immediate-early gene promoter. Interferon-γ-mediated activation of MHC-II gene expression was also inhibited by D-sequence oligonucleotides as well as after infection with either the wild-type AAV or transduction with recombinant AAV vectors. These studies suggest that the D-sequence-mediated downregulation of the MHC-II gene expression may be exploited toward the development of novel AAV vectors capable of dampening the host humoral response, which has important implication in the optimal use of these vectors in human gene therapy.  

5.2806           High-Throughput In Vitro, Ex Vivo, and In Vivo Screen of Adeno-Associated Virus Vectors Based on Physical and Functional Transduction

Westhaus, A., Cabanes-Creus, M., Rybicki, A., Balttazar, G., Navarro, R.G., Zhu, E. et al Human Gene Therapy, 31(9-10), 575-589 (2020)   Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro, ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3′-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants.  

5.2807           The strategic combination of trastuzumab emtansine with oncolytic rhabdoviruses leads to therapeutic synergy

Arulanadam, R., Taha, Z., Garcia, V., Selman, M., Chen, A., Varette, O., Jirovec, A. et al Communications Biol., 3:254 (2020)   We have demonstrated that microtubule destabilizing agents (MDAs) can sensitize tumors to oncolytic vesicular stomatitis virus (VSVΔ51) in various preclinical models of cancer. The clinically approved T-DM1 (Kadcyla®) is an antibody-drug conjugate consisting of HER2-targeting trastuzumab linked to the potent MDA and maytansine derivative DM1. We reveal that combining T-DM1 with VSVΔ51 leads to increased viral spread and tumor killing in trastuzumab-binding, VSVΔ51-resistant cancer cells. In vivo, co-treatment of VSVΔ51 and T-DM1 increased overall survival in HER2-overexpressing, but trastuzumab-refractory, JIMT1 human breast cancer xenografts compared to monotherapies. Furthermore, viral spread in cultured HER2⁺ human ovarian cancer patient-derived ascites samples was enhanced by the combination of VSVΔ51 and T-DM1. Our data using the clinically approved Kadcyla® in combination with VSVΔ51 demonstrates proof of concept that targeted delivery of a viral-sensitizing molecule using an antibody-drug conjugate can enhance oncolytic virus activity and provides rationale for translation of this approach.  

5.2808           In vitro assay for the efficacy assessment of AAV vectors expressing microdystrophin

Danilov, K.A:, Vassilieva, S.G., Polikarpova, A.V., Starikova, A.V., Shmidt, A.A., Galkin, I.I., Tsitriana, A.A., Egorova, T.V., Orlov, S.N. and Kotelevtsev, Y.V: Exp. Cell Res., 392, 112033 (2020)   AAV-delivered microdystrophin genes hold great promise for Duchenne muscular dystrophy (DMD) treatment. It is anticipated that the optimization of engineered dystrophin genes will be required to increase the efficacy and reduce the immunogenicity of transgenic proteins. An in vitro system is required for the efficacy testing of genetically engineered dystrophin genes. We report here on the proof of concept for an in vitro assay based on the assessment of sarcolemma damage after repetitively applied electrical stimuli. The primary cell culture of myoblasts was established from wild-type C57BL/10ScSnJ and dystrophin-deficient mdx mice. The preparation parameters and the differentiation of contractile myotubes were optimized. DAPI and TO-PRO-3 dyes were used to assess myotubular membrane permeability in response to electrical pulse stimulation (EPS). Myotubes derived from mdx mice exhibited a greater increase in membrane damage, as assessed by TO-PRO-3-measured permeability after EPS, than was exhibited by the healthy control myotubes. AAV-DJ particles carrying the microdystrophin gene were used to transduce mdx-derived differentiated myotubes. Microdystrophin delivery ameliorated the disease phenotype and reduced the EPS-induced membrane damage to a level comparable to that of the healthy controls. Thus, the in vitro system was shown to be capable of supporting studies on DMD gene therapy.  

5.2809           IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses

Evnouchidou, I., Chappert, P., Benadda, S., Zucchetti, A., Weimershaus, M., Bens, M., Caillens, V., Koumantou, K D., Lotersztajn, S., van Endert, P., Davoust, J., Guermonprez, P., Hivroz, C., Gross, D.A. and Saveanu, L. Nature Communications, 11:2279 (2020)   T cell receptor (TCR) activation is modulated by mechanisms such as TCR endocytosis, which is thought to terminate TCR signalling. Here we show that, upon internalization, TCR continues to signal from a set of specialized endosomes that are crucial for T cell functions. Mechanistically, TCR ligation leads to clathrin-mediated internalization of the TCR-CD3ζ complex, while maintaining CD3ζ signalling, in endosomal vesicles that contain the insulin responsive aminopeptidase (IRAP) and the SNARE protein Syntaxin 6. Destabilization of this compartment through IRAP deletion enhances plasma membrane expression of the TCR-CD3ζ complex, yet compromises overall CD3ζ signalling; moreover, the integrity of this compartment is also crucial for T cell activation and survival after suboptimal TCR activation, as mice engineered with a T cell-specific deletion of IRAP fail to develop efficient polyclonal anti-tumour responses. Our results thus reveal a previously unappreciated function of IRAP-dependent endosomal TCR signalling in T cell activation.  

5.2810           Single-administration, thermostable human papillomavirus vaccines prepared with atomic layer deposition technology

Garcea, R.L., Meinerz, N.M., Dong, M., Funke, H., Ghazvini, S. and Randolph,, T.W. Npj/Vaccines, 5:45 (2020)   Cold-chain requirements affect worldwide distribution of many vaccines. In addition, vaccines requiring multiple doses impose logistical and financial burdens, as well as patient compliance barriers. To address such limitations, we have developed new technologies to prepare thermostable, single-shot, prime-boost microparticle vaccines. Antigen/adjuvant formulations containing glass-forming polymers and trehalose first are spray-dried to form glassy microparticles that confer thermostability. Atomic layer deposition (ALD) reactions conducted in fluidized beds are then used to coat the microparticles with defined numbers of molecular layers of alumina that modulate the timed release of the internalized antigen and act as adjuvants. We have used a model HPV16 L1 capsomere antigen to evaluate the properties of these technologies. Thermostabilized powders containing HPV16 L1 capsomeres were prepared by spray-drying, coated by ALD with up to 500 molecular layers of alumina, and injected into mice. Antigen distribution was assessed by live-animal IR dye tracking of injected labeled antigen. Antibody responses were measured weekly by ELISA, and neutralizing antibodies were measured by pseudovirus neutralization assays at selected time points. Thermostability was evaluated by measuring antibody responses after incubating ALD-coated antigen powders for one month at 50 °C. Single doses of the ALD-coated vaccine formulations elicited a prime-boost immune response, and produced neutralizing responses and antibody titers that were equivalent or superior to conventional prime-boost doses of liquid formulations. Antibody titers were unaffected by month-long incubation of the formulations at 50 °C. Single-dose, thermostable antigen preparations may overcome current limitations in HPV vaccine delivery as well as being widely applicable to other antigens.  

5.2811           Mesencephalic astrocyte–derived neurotrophic factor is an ER-resident chaperone that protects against reductive stress in the heart

Arrieta, A., Blackwood, E.A., Stauffer, W.T., Domingo, M.S., Bilal, A.S., Thuerauf, D.J., Pentoney, A.N., Aivati, C., Sarakki, A.V., Doroudgar, S. and Glembotski, C.C.

1. Biol. Chem., 295(22), 7566-7583 (2020)

  We have previously demonstrated that ischemia/reperfusion (I/R) impairs endoplasmic reticulum (ER)-based protein folding in the heart and thereby activates an unfolded protein response sensor and effector, activated transcription factor 6α (ATF6). ATF6 then induces mesencephalic astrocyte-derived neurotrophic factor (MANF), an ER-resident protein with no known structural homologs and unclear ER function. To determine MANF's function in the heart in vivo, here we developed a cardiomyocyte-specific MANF-knockdown mouse model. MANF knockdown increased cardiac damage after I/R, which was reversed by AAV9-mediated ectopic MANF expression. Mechanistically, MANF knockdown in cultured neonatal rat ventricular myocytes (NRVMs) impaired protein folding in the ER and cardiomyocyte viability during simulated I/R. However, this was not due to MANF-mediated protection from reactive oxygen species generated during reperfusion. Because I/R impairs oxygen-dependent ER protein disulfide formation and such impairment can be caused by reductive stress in the ER, we examined the effects of the reductive ER stressor DTT. MANF knockdown in NRVMs increased cell death from DTT-mediated reductive ER stress, but not from nonreductive ER stresses caused by thapsigargin-mediated ER Ca²⁺ depletion or tunicamycin-mediated inhibition of ER protein glycosylation. In vitro, recombinant MANF exhibited chaperone activity that depended on its conserved cysteine residues. Moreover, in cells, MANF bound to a model ER protein exhibiting improper disulfide bond formation during reductive ER stress but did not bind to this protein during nonreductive ER stress. We conclude that MANF is an ER chaperone that enhances protein folding and myocyte viability during reductive ER stress.  

5.2812           Enhanced phosphorylation of PERK in primary cultured neurons as an autonomous neuronal response to prion infection

Tanaka, M., Yamasaki, T., Hasebe, R., Suzuki, A. and Horiuchi, M. PloS One, 15(6), e0234147 (2020)   Conversion of cellular prion protein (PrP^C) into the pathogenic isoform of prion protein (PrP^Sc) in neurons is one of the key pathophysiological events in prion diseases. However, the molecular mechanism of neurodegeneration in prion diseases has yet to be fully elucidated because of a lack of suitable experimental models for analyzing neuron-autonomous responses to prion infection. In the present study, we used neuron-enriched primary cultures of cortical and thalamic mouse neurons to analyze autonomous neuronal responses to prion infection. PrP^Sc levels in neurons increased over the time after prion infection; however, no obvious neuronal losses or neurite alterations were observed. Interestingly, a finer analysis of individual neurons co-stained with PrP^Sc and phosphorylated protein kinase RNA-activated-like endoplasmic reticulum (ER) kinase (p-PERK), the early cellular response of the PERK-eukaryotic initiation factor 2 (eIF2α) pathway, demonstrated a positive correlation between the number of PrP^Sc granular stains and p-PERK granular stains, in cortical neurons at 21 dpi. Although the phosphorylation of PERK was enhanced in prion-infected cortical neurons, there was no sign of subsequent translational repression of synaptic protein synthesis or activations of downstream unfolded protein response (UPR) in the PERK-eIF2α pathway. These results suggest that PrP^Sc production in neurons induces ER stress in a neuron-autonomous manner; however, it does not fully activate UPR in prion-infected neurons. Our findings provide insights into the autonomous neuronal responses to prion propagation and the involvement of neuron-non-autonomous factor(s) in the mechanisms of neurodegeneration in prion diseases.  

5.2813           Treatment of a Mouse Model of ALS by In Vivo Base Editing

Lim, C.K.W., Gapinske, M., Brooks, A.K., Woods, W.S., Powell, J.E., Zeballos C., M.A., Winter, J., Perez-Pinera, P. and Gaj, T. Molecular Therapy, 28(3), 1177-1189 (2020)   Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal disorder that can be caused by mutations in the superoxide dismutase 1 (SOD1) gene. Although ALS is currently incurable, CRISPR base editors hold the potential to treat the disease through their ability to create nonsense mutations that can permanently disable the expression of the mutant SOD1 gene. However, the restrictive carrying capacity of adeno-associated virus (AAV) vectors has limited their therapeutic application. In this study, we establish an intein-mediated trans-splicing system that enables in vivo delivery of cytidine base editors (CBEs) consisting of the widely used Cas9 protein from Streptococcus pyogenes. We show that intrathecal injection of dual AAV particles encoding a split-intein CBE engineered to trans-splice and introduce a nonsense-coding substitution into a mutant SOD1 gene prolonged survival and markedly slowed the progression of disease in the G93A-SOD1 mouse model of ALS. Adult animals treated by this split-intein CRISPR base editor had a reduced rate of muscle atrophy, decreased muscle denervation, improved neuromuscular function, and up to 40% fewer SOD1 immunoreactive inclusions at end-stage mice compared to control mice. This work expands the capabilities of single-base editors and demonstrates their potential for gene therapy.  

5.2814           Variability in Cardiac miRNA-122 Level Determines Therapeutic Potential of miRNA-Regulated AAV Vectors

Kraszewska, I., Tomczyk, M., Andrysiak, K., Biniecka, M., Geisler, A., Fechner, H., Zembala, M., Stepniewski, J., Dulak, J. and Jazwa-Kusior, A. Molecular Therapy-Methods & Clin. Develop., 17, 1190-1201 (2020)   Systemically delivered adeno-associated viral vector serotype 9 (AAV9) effectively transduces murine heart, but provides transgene expression also in liver and skeletal muscles. Improvement of the selectivity of transgene expression can be achieved through incorporation of target sites (TSs) for miRNA-122 and miRNA-206 into the 3′ untranslated region (3′ UTR) of the expression cassette. Here, we aimed to generate such miRNA-122- and miRNA-206-regulated AAV9 vector for a therapeutic, heart-specific overexpression of heme oxygenase-1 (HO-1). We successfully validated the vector functionality in murine cell lines corresponding to tissues targeted by AAV9. Next, we evaluated biodistribution of transgene expression following systemic vector delivery to HO-1-deficient mice of mixed C57BL/6J × FVB genetic background. Although AAV genomes were present in the hearts of these animals, HO-1 protein expression was either absent or significantly impaired. We found that miRNA-122, earlier described as liver specific, was present also in the hearts of C57BL/6J × FVB mice. Various levels of miRNA-122 expression were observed in the hearts of other mouse strains, in heart tissues of patients with cardiomyopathy, and in human induced pluripotent stem cell-derived cardiomyocytes in which we also confirmed such posttranscriptional regulation of transgene expression. Our data clearly indicate that therapeutic utilization of miRNA-based regulation strategy needs to consider inter-individual variability.  

5.2815           Novel AAV44.9-Based Vectors Display Exceptional Characteristics for Retinal Gene Therapy

Boye, S., Choudhury, S., Crosson, S., Di Pasquale, G., Afione, S. Mellen, R. et al Molecular Therapy, 28(6), 1464-1478 (2020)   The majority of inherited retinal diseases (IRDs) are caused by mutations in genes expressed in photoreceptors (PRs). The ideal vector to address these conditions is one that transduces PRs in large areas of retina with the smallest volume/lowest titer possible, and efficiently transduces foveal cones, the cells responsible for acute, daylight vision that are often the only remaining area of functional retina in IRDs. The purpose of our study was to evaluate the retinal tropism and potency of a novel capsid, AAV44.9, and rationally designed derivatives thereof. We found that AAV44.9 and AAV44.9(E531D) transduced retinas of subretinally injected (SRI) mice with higher efficiency than did benchmark AAV5- and AAV8-based vectors. In macaques, highly efficient cone and rod transduction was observed following submacular and peripheral SRI. AAV44.9- and AAV44.9(E531D)-mediated GFP fluorescence extended laterally well beyond SRI bleb margins. Notably, extrafoveal injection (i.e., fovea not detached during surgery) led to transduction of up to 98% of foveal cones. AAV44.9(E531D) efficiently transduced parafoveal and perifoveal cones, whereas AAV44.9 did not. AAV44.9(E531D) was also capable of restoring retinal function to a mouse model of IRD. These novel capsids will be useful for addressing IRDs that would benefit from an expansive treatment area.  

5.2816           A Molecular Determinant of West Nile Virus Secretion and Morphology as a Target for Viral Attenuation

Bassel, J., Burlaud-Gaillard, J., Feher, M., Roingeard, P., Rey, F.A. and Pardigon, N.

1. Virol., 94(12), e00086-20 (2020)

  West Nile virus (WNV), a member of the Flavivirus genus and currently one of the most common arboviruses worldwide, is associated with severe neurological disease in humans. Its high potential to reemerge and rapidly disseminate makes it a bona fide global public health problem. The surface membrane glycoprotein (M) has been associated with Flavivirus-induced pathogenesis. Here, we identified a key amino acid residue at position 36 of the M protein whose mutation impacts WNV secretion and promotes viral attenuation. We also identified a compensatory site at position M-43 whose mutation stabilizes M-36 substitution both in vitro and in vivo. Moreover, we found that introduction of the two mutations together confers a full attenuation phenotype and protection against wild-type WNV lethal challenge, eliciting potent neutralizing-antibody production in mice. Our study thus establishes the M protein as a new viral target for rational design of attenuated WNV strains.  

5.2817           Protocol for Efficient CRISPR/Cas9/AAV-Mediated Homologous Recombination in Mouse Hematopoietic Stem and Progenitor Cells

Tran, N.T., Trombke, J., Rajewsky, K., Chu, V.T. STAR Protocols, 1, 100028 (2020)   Mutations that accumulate in self-renewing hematopoietic stem and progenitor cells (HSPCs) can cause severe blood disorders. To model such disorders in mice, we developed a CRISPR/Cas9/adeno-associated virus (AAV)-based system to knock in and repair genes by homologous recombination in mouse HSPCs. Here, we provide a step-by-step protocol to achieve high efficiency of gene knockin in mouse HSPCs, while maintaining engraftment capacity. This approach enables the functional study of hematopoietic disease mutations in vivo, without requiring germline mutagenesis.  

5.2818           Full-length 5'RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum

Stadelmayer, B., Diederichs, A., Chapus, F., Carter, K., Testoni, B. and Zoulim, F.

1. Hepatol., 73, 40-51 (2020)

  Background & Aims Covalently closed circular DNA (cccDNA) is the episomal form of the HBV genome that stably resides in the nucleus of infected hepatocytes. cccDNA is the template for the transcription of 6 major viral RNAs, i.e. preC, pg, preS1/2, S and HBx RNA. All viral transcripts share the same 3' end and are all to various degrees subsets of each other. Especially under infection conditions, it has been difficult to study in depth the transcription of the different viral transcripts. We thus wanted to develop a method with which we could easily detect the full spectrum of viral RNAs in any lab. Methods We set up an HBV full-length 5'RACE (rapid amplification of cDNA ends) method with which we measured and characterized the full spectrum of viral RNAs in cell culture and in chronically infected patients. Results In addition to canonical HBx transcripts coding for full-length X, we identified shorter HBx transcripts potentially coding for short X proteins. We showed that interferon-β treatment leads to a strong reduction of preC and pgRNAs but has only a moderate effect on the other viral transcripts. We found pgRNA, 1 spliced pgRNA variant and a variety of HBx transcripts associated with viral particles generated by HepAD38 cells. The different HBx RNAs are both capped and uncapped. Lastly, we identified 3 major categories of circulating RNA species in patients with chronic HBV infection: pgRNA, spliced pgRNA variants and HBx. Conclusions This HBV full-length 5'RACE method should significantly contribute to the understanding of HBV transcription during the course of infection and therapy and may guide the development of novel therapies aimed at targeting cccDNA.  

5.2819           An effective inactivant based on singlet oxygen-mediated lipid oxidation implicates a new paradigm for broad-spectrum antivirals

Zeng, L., Wang, M-D., Ming, S-L., Li, G-L., Yu, P-W-. Qi, Y.L., Jiang, D-W., Yang, G-Y., Wang, J, and Chu, B-B. Redox Biol., 36, 101601 (2020)   Emerging viral pathogens cause substantial morbidity and pose a severe threat to health worldwide. However, a universal antiviral strategy for producing safe and immunogenic inactivated vaccines is lacking. Here, we report an antiviral strategy using the novel singlet oxygen (¹O₂)-generating agent LJ002 to inactivate enveloped viruses and provide effective protection against viral infection. Our results demonstrated that LJ002 efficiently generated ¹O₂ in solution and living cells. Nevertheless, LJ002 exhibited no signs of acute toxicity in vitro or in vivo. The ¹O₂ produced by LJ002 oxidized lipids in the viral envelope and consequently destroyed the viral membrane structure, thus inhibiting the viral and cell membrane fusion necessary for infection. Moreover, the ¹O₂-based inactivated pseudorabies virus (PRV) vaccine had no effect on the content of the viral surface proteins. Immunization of mice with LJ002-inactiviated PRV vaccine harboring comparable antigen induced more neutralizing antibody responses and efficient protection against PRV infection than conventional formalin-inactivated vaccine. Additionally, LJ002 inactivated a broad spectrum of enveloped viruses. Together, our results may provide a new paradigm of using broad-spectrum, highly effective inactivants functioning through ¹O₂-mediated lipid oxidation for developing antivirals that target the viral membrane fusion process.  

5.2820           Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors

Rumachik, N.G., Malaker, S.A., Poweleit, N., Maynard, L.H., Adams, C.M., Leib, R.D., Cirolia, G., Thomas, D., Stamnes, S., Holt, K., Sinn, P., May, A.P. and Paulk, N.K. Molecular Therapy-Methods & Clin. Develop., 18,  98-118 (2020)   Different approaches are used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Spodoptera frugiperda (Sf9) insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed a controlled comparative production analysis varying only the host cell species but maintaining all other parameters. We characterized differences with multiple analytical approaches: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM (transmission electron cryomicroscopy), denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and functional transduction assessments in vitro and in vivo, including in humanized liver mice. Using these approaches, we have made two major discoveries: (1) rAAV capsids have post-translational modifications (PTMs), including glycosylation, acetylation, phosphorylation, and methylation, and these differ between platforms; and (2) rAAV genomes are methylated during production, and these are also differentially deposited between platforms. Our data show that host cell protein impurities differ between platforms and can have their own PTMs, including potentially immunogenic N-linked glycans. Human-produced rAAVs are more potent than baculovirus-Sf9 vectors in various cell types in vitro (p < 0.05–0.0001), in various mouse tissues in vivo (p < 0.03–0.0001), and in human liver in vivo (p < 0.005). These differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity, as well as cost considerations.  

5.2821           TBC1D5-Catalyzed Cycling of Rab7 Is Required for Retromer-Mediated Human Papillomavirus Trafficking during Virus Entry

Xie, J., Heim, E.N., Crite, m. and DiMaio, D. Cell Reports, 31, 107750 (2020)   During virus entry, human papillomaviruses are sorted by the cellular trafficking complex, called retromer, into the retrograde transport pathway to traffic from the endosome to downstream cellular compartments, but regulation of retromer activity during HPV entry is poorly understood. Here we selected artificial proteins that modulate cellular proteins required for HPV infection and discovered that entry requires TBC1D5, a retromer-associated, Rab7-specific GTPase-activating protein. Binding of retromer to the HPV L2 capsid protein recruits TBC1D5 to retromer at the endosome membrane, which then stimulates hydrolysis of Rab7-GTP to drive retromer disassembly from HPV and delivery of HPV to the retrograde pathway. Although the cellular retromer cargos CIMPR and DMT1-II require only GTP-bound Rab7 for trafficking, HPV trafficking requires cycling between GTP- and GDP-bound Rab7. Thus, ongoing cargo-induced membrane recruitment, assembly, and disassembly of retromer complexes drive HPV trafficking.  

5.2822           Local miRNA-Dependent Translational Control of GABAAR Synthesis during Inhibitory Long-Term Potentiation

Rajgor, D., Purkey, A.M., Sanderson, J.L., Welle, T.M., Garcia, J.D., Dell’Acqua, M.L. and Smith, K.R. Cell Reports, 31, 107785 (2020)   Molecular mechanisms underlying plasticity at brain inhibitory synapses remain poorly characterized. Increased postsynaptic clustering of GABA_A receptors (GABA_ARs) rapidly strengthens inhibition during inhibitory long-term potentiation (iLTP). However, it is unclear how synaptic GABA_AR clustering is maintained to sustain iLTP. Here, we identify a role for miR376c in regulating the translation of mRNAs encoding the synaptic α1 and γ2 GABA_AR subunits, GABRA1 and GABRG2, respectively. Following iLTP induction, transcriptional repression of miR376c is induced through a calcineurin-NFAT-HDAC signaling pathway and promotes increased translation and clustering of synaptic GABA_ARs. This pathway is essential for the long-term expression of iLTP and is blocked by miR376c overexpression, specifically impairing inhibitory synaptic strength. Finally, we show that local de novo synthesis of synaptic GABA_ARs occurs exclusively in dendrites and in a miR376c-dependent manner following iLTP. Together, this work describes a local post-transcriptional mechanism that regulates inhibitory synaptic plasticity via miRNA control of dendritic protein synthesis.  

5.2823           Prdm1 overexpression causes a photoreceptor fate-shift in nascent, but not mature, bipolar cells

Goodson, N.B:, Park, K.U., Silver, J.S., Chiodo, V.A., Hauswirth, W.W., and Brzezinski IV, J.A. Develop. Biol., 464, 111-123 (2020)   The transcription factors Prdm1 (Blimp1) and Vsx2 (Chx10) work downstream of Otx2 to regulate photoreceptor and bipolar cell fates in the developing retina. Mice that lack Vsx2 fail to form bipolar cells while Prdm1 mutants form excess bipolars at the direct expense of photoreceptors. Excess bipolars in Prdm1 mutants appear to derive from rods, suggesting that photoreceptor fate remains mutable for some time after cells become specified. Here we tested whether bipolar cell fate is also plastic during development. To do this, we created a system to conditionally misexpress Prdm1 at different stages of bipolar cell development. We found that Prdm1 blocks bipolar cell formation if expressed before the fate choice decision occurred. When we misexpressed Prdm1 just after the decision to become a bipolar cell was made, some cells were reprogrammed into photoreceptors. In contrast, Prdm1 misexpression in mature bipolar cells did not affect cell fate. We also provide evidence that sustained misexpression of Prdm1 was selectively toxic to photoreceptors. Our data show that bipolar fate is malleable, but only for a short temporal window following fate specification. Prdm1 and Vsx2 act by stabilizing photoreceptor and bipolar fates in developing OTX2+ cells of the retina.  

5.2824           Experimental Variables that Affect Human Hepatocyte AAV Transduction in Liver Chimeric Mice

Zou, C., Vercauteren, K.O.A., Michailidis, E., Kabbani, M., Zoluthkin, I., Quirk, C., Chiriboga, L., Yazicioglu, M., Anguela, X.M., Meuleman, P., High, K.A., Herzog, R.W. and de Jong, Y.P Molecular Therapy-Methods & Clin. Develop., 18, 189-198 (2020)   Adeno-associated virus (AAV) vector serotypes vary in their ability to transduce hepatocytes from different species. Chimeric mouse models harboring human hepatocytes have shown translational promise for liver-directed gene therapies. However, many variables that influence human hepatocyte transduction and transgene expression in such models remain poorly defined. Here, we aimed to test whether three experimental conditions influence AAV transgene expression in immunodeficient, fumaryl-acetoactetate-hydrolase-deficient (Fah^−/−) chimeric mice repopulated with primary human hepatocytes. We examined the effects of the murine liver injury cycle, human donor variability, and vector doses on hepatocyte transduction with various AAV serotypes expressing a green fluorescent protein (GFP). We determined that the timing of AAV vector challenge in the liver injury cycle resulted in up to 7-fold differences in the percentage of GFP expressing human hepatocytes. The GFP+ hepatocyte frequency varied 7-fold between human donors without, however, changing the relative transduction efficiency between serotypes for an individual donor. There was also a clear relationship between AAV vector doses and human hepatocyte transduction and transgene expression. We conclude that several experimental variables substantially affect human hepatocyte transduction in the Fah^−/− chimera model, attention to which may improve reproducibility between findings from different laboratories.  

5.2825           A Novel Gene Therapy Approach for GSD III Using an AAV Vector Encoding a Bacterial Glycogen Debranching Enzyme

Lim, J-A., Choi, S.J., Gao, F., Kishnani, P.S. and Sun, B. Molecular Therapy-Methods & Clin. Develop., 18, 240-249 (2020)   Glycogen storage disease type III (GSD III) is an inherited disorder caused by a deficiency of glycogen debranching enzyme (GDE), which results in the accumulation of abnormal glycogen (limit dextrin) in the cytoplasm of liver, heart, and skeletal muscle cells. Currently, there is no curative treatment for this disease. Gene therapy with adeno-associated virus (AAV) provides an optimal treatment approach for monogenic diseases like GSD III. However, the 4.6 kb human GDE cDNA is too large to be packaged into a single AAV vector due to its small carrying capacity. To overcome this limitation, we tested a new gene therapy approach in GSD IIIa mice using an AAV vector ubiquitously expressing a smaller bacterial GDE, Pullulanase, whose cDNA is 2.2 kb. Intravenous injection of the AAV vector (AAV9-CB-Pull) into 2-week-old GSD IIIa mice blocked glycogen accumulation in both cardiac and skeletal muscles, but not in the liver, accompanied by the improvement of muscle functions. Subsequent treatment with a liver-restricted AAV vector (AAV8-LSP-Pull) reduced liver glycogen content by 75% and reversed hepatic fibrosis while maintaining the effect of AAV9-CB-Pull treatment on heart and skeletal muscle. Our results suggest that AAV-mediated gene therapy with Pullulanase is a possible treatment for GSD III.  

5.2826           Viral delivery of multiple miRNAs promotes retinal ganglion cell survival and functional preservation after optic nerve crush injury

Mead, B., Cullather, E., Nakaya, N., Niu, Y., Kole, C., Ahmed, Z., Tomarev, S. Exp. Eye Res., 197, 108071 (2020)   Bone marrow mesenchymal stem cell (BMSC)-derived small extracellular vesicles (sEV) but not fibroblast sEV provide retinal ganglion cell (RGC) neuroprotection both in vitro and in vivo, with miRNAs playing an essential role. More than 40 miRNAs were more abundant in BMSC-sEV than in fibroblast-sEV. The purpose of this study was to test the in vitro and in vivo neuroprotective and axogenic properties of six candidate miRNAs (miR-26a, miR-17, miR-30c-2, miR-92a, miR-292, and miR-182) that were more abundant in BMSC-sEV than in fibroblast-sEV. Adeno-associated virus 2 (AAV2) expressing a combination of three of the above candidate miRNAs were added to heterogenous adult rat retinal cultures or intravitreally injected into rat eyes one week before optic nerve crush (ONC) injury. Survival and neuritogenesis of βIII-tubulin⁺ RGCs was assessed in vitro, as well as the survival of RBPMS⁺ RGCs and regeneration of their axons in vivo. Retinal nerve fiber layer thickness (RNFL) was measured to assess axonal density whereas positive scotopic threshold response electroretinography amplitudes provided a readout of RGC function. Qualitative retinal expression of PTEN, a target of several of the above miRNAs, was used to confirm successful miRNA activity. AAV2 reliably transduced RGCs in vitro and in vivo. Viral delivery of miRNAs in vitro showed a trend towards neuroprotection but remained insignificant. Delivery of selected combinations of miRNAs (miR-17-5p, miR-30c-2 and miR-92a; miR-92a, miR-292 and miR-182) before ONC provided significant therapeutic benefits according to the above measurable endpoints. However, no single miRNA appeared to be responsible for the effects observed, whilst positive effects observed appeared to coincide with successful qualitative reduction in PTEN immunofluorescence in the retina. Viral delivery of miRNAs provides a possible neuroprotective strategy for injured RGCs that is conducive to therapeutic manipulation.  

5.2827           Engineering monocyte/macrophage−specific glucocerebrosidase expression in human hematopoietic stem cells using genome editing

Scharenberg, S.G., Poletto, E., Lucot, K.L., Colella, P., Sheikali, A., Montine, T.J., Porteus, M.H. and Gomez-Ospina, N. Nature Communications, 11:3327 (2020)   Gaucher disease is a lysosomal storage disorder caused by insufficient glucocerebroside activity. Its hallmark manifestations are attributed to infiltration and inflammation by macrophages. Current therapies for Gaucher disease include life−long intravenous administration of recombinant glucocerebroside and orally-available glucosylceramide synthase inhibitors. An alternative approach is to engineer the patient’s own hematopoietic system to restore glucocerebrosidase expression, thereby replacing the affected cells, and constituting a potential one-time therapy for this disease. Here, we report an efficient CRISPR/Cas9-based approach that targets glucocerebrosidase expression cassettes with a monocyte/macrophage-specific element to the CCR5 safe-harbor locus in human hematopoietic stem and progenitor cells. The targeted cells generate glucocerebroside-expressing macrophages and maintain long-term repopulation and multi-lineage differentiation potential with serial transplantation. The combination of a safe-harbor and a lineage-specific promoter establishes a universal correction strategy and circumvents potential toxicity of ectopic glucocerebrosidase in the stem cells. Furthermore, it constitutes an adaptable platform for other lysosomal enzyme deficiencies.  

5.2828           Berberine Chloride is an Alphavirus Inhibitor That Targets Nucleocapsid Assembly

Wan, J.J., Brown, R.S. and kielian, M. mBio, 11(3), e01382-20 (2020)   Alphaviruses are enveloped positive-sense RNA viruses that can cause serious human illnesses such as polyarthritis and encephalitis. Despite their widespread distribution and medical importance, there are no licensed vaccines or antivirals to combat alphavirus infections. Berberine chloride (BBC) is a pan-alphavirus inhibitor that was previously identified in a replicon-based small-molecule screen. This work showed that BBC inhibits alphavirus replication but also suggested that BBC might have additional effects later in the viral life cycle. Here, we show that BBC has late effects that target the virus nucleocapsid (NC) core. Infected cells treated with BBC late in infection were unable to form stable cytoplasmic NCs or assembly intermediates, as assayed by gradient sedimentation. In vitro studies with recombinant capsid protein (Cp) and purified genomic RNA (gRNA) showed that BBC perturbs core-like particle formation and potentially traps the assembly process in intermediate states. Particles produced from BBC-treated cells were less infectious, despite efficient particle production and only minor decreases in genome packaging. In addition, BBC treatment of free virus particles strongly decreased alphavirus infectivity. In contrast, the infectivity of the negative-sense RNA virus vesicular stomatitis virus was resistant to BBC treatment of infected cells or free virus. Together, our data indicate that BBC alters alphavirus Cp-gRNA interactions and oligomerization and suggest that this may cause defects in NC assembly and in disassembly during subsequent virus entry. Thus, BBC may be considered a novel alphavirus NC assembly inhibitor.  

5.2829           Adeno‐associated virus‐vectored influenza vaccine elicits neutralizing and Fcγ receptor‐activating antibodies

Demminger, D.E., Walz, L., Dietert, K., Hoffmann, H., Planz, O., Bruber, A.D., von Messling, V. and Wolff, T. EMBO Mol. Med., 12:e10938 (2020)   The current seasonal inactivated influenza vaccine protects only against a narrow range of virus strains as it triggers a dominant antibody response toward the hypervariable hemagglutinin (HA ) head region. The discovery of rare broadly protective antibodies against conserved regions in influenza virus proteins has propelled research on distinct antigens and delivery methods to efficiently induce broad immunity toward drifted or shifted virus strains. Here, we report that adeno‐associated virus (AAV ) vectors expressing influenza virus HA or chimeric HA protected mice against homologous and heterologous virus challenges. Unexpectedly, immunization even with wild‐type HA induced antibodies recognizing the HA ‐stalk and activating FcγR‐dependent responses indicating that AAV ‐vectored expression balances HA head‐ and HA stalk‐specific humoral responses. Immunization with AAV ‐HA partially protected also ferrets against a harsh virus challenge. Results from this study provide a rationale for further clinical development of AAV vectors as influenza vaccine platform, which could benefit from their approved use in human gene therapy.  

5.2830           Compromised DNA repair is responsible for diabetes‐associated fibrosis

Kumar, V., Agrawal, R., Pandey, A., Kopf, S., Hoeffgen, M., Kaymak, S., Bandapalli, O.R., Gorbunova, V., Seluanov, A., Mall, M.A., Herzig, S. and Nawroth, P.P. EMBO J., 39:e103477 (2020)   Diabetes‐associated organ fibrosis, marked by elevated cellular senescence, is a growing health concern. Intriguingly, the mechanism underlying this association remained unknown. Moreover, insulin alone can neither reverse organ fibrosis nor the associated secretory phenotype, favoring the exciting notion that thus far unknown mechanisms must be operative. Here, we show that experimental type 1 and type 2 diabetes impairs DNA repair, leading to senescence, inflammatory phenotypes, and ultimately fibrosis. Carbohydrates were found to trigger this cascade by decreasing the NAD ⁺/NADH ratio and NHEJ ‐repair in vitro and in diabetes mouse models. Restoring DNA repair by nuclear over‐expression of phosphomimetic RAGE reduces DNA damage, inflammation, and fibrosis, thereby restoring organ function. Our study provides a novel conceptual framework for understanding diabetic fibrosis on the basis of persistent DNA damage signaling and points to unprecedented approaches to restore DNA repair capacity for resolution of fibrosis in patients with diabetes.  

5.2831           A Small-Molecule-Responsive Riboswitch Enables Conditional Induction of Viral Vector-Mediated Gene Expression in Mice

Strobel, B., Düchs, M.J., Blazevic, D., Rechtsteiner, P., Braun, C., Braun-Kroker, K.S., Schmid, B., Ciossek, T., Gottschling, D., Hartig, J.S. and Kreuz, S. ACS Synth. Biol., 9, 1292-1305 (2020)   Adeno-associated viral (AAV) vector-mediated gene therapy holds great potential for future medical applications. However, to facilitate safer and broader applicability and to enable patient-centric care, therapeutic protein expression should be controllable, ideally by an orally administered drug. The use of protein-based systems is considered rather undesirable, due to potential immunogenicity and the limited coding space of AAV. Ligand-dependent riboswitches, in contrast, are small and characterized by an attractive mode-of-action based on mRNA-self-cleavage, independent of coexpressed foreign protein. While a promising approach, switches available to date have only shown moderate potency in animals. In particular, ON-switches that induce transgene expression upon ligand administration so far have achieved rather disappointing results. Here we present the utilization of the previously described tetracycline-dependent ribozyme K19 for controlling AAV-mediated transgene expression in mice. Using this tool switch, we provide first proof for the feasibility of clinically desired key features, including multiorgan functionality, potent regulation (up to 15-fold induction), reversibility, and the possibility to fine-tune and repeatedly induce expression. The systematic assessment of ligand and reporter protein plasma levels further enabled the characterization of pharmacokinetic-pharmacodynamic relationships. Thus, our results strongly support future efforts to develop engineered riboswitches for applications in clinical gene therapy.  

5.2832           A Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations from Viral DNA Extracts

Guerin, K., Rego, M., Bourges, D., Ersing, I., Haery, L., DeMaio, K.H., Sanders, E., Tasissa, M., Kostman, M., Tilgren, M., Hanley, L.M., Mueller, I., Mitsopoulos, A. and Fan, M. Human Gene Therapy, 31(11-12), 664-678 (2020)   Recombinant adeno-associated virus (rAAV) vectors are increasingly popular gene delivery tools in biological systems. They are safe and lead to high-level, long-term transgene expression. rAAV are available in multiple serotypes, natural or engineered, which enable targeting to a wide array of tissues and cell types. In addition, rAAVs are relatively easily produced in a well-equipped lab or obtained from a viral vector core facility. Unfortunately, there is no standardization of quality control assays beyond titering and purity assessments. Next-generation sequencing (NGS) can be used to identify rAAV preparations. Because the rAAV genome is single stranded, previous studies have assumed that rAAV genomes must be converted to double strands before NGS. We demonstrate that rAAV DNA extracts exist primarily as double-stranded species. We hypothesize that these molecules form from the natural base pairing of complementary [+] and [−] strands after DNA extraction and show that rAAV DNA extracts are sufficient templates for downstream NGS without the labor-intensive double-stranding step. Here, we provide a detailed protocol for the simple and rapid NGS of rAAV genomes from DNA extracts. With this protocol, users can quickly confirm the identity of an rAAV preparation and detect the presence of contaminating rAAV DNA. In addition, we share custom Python scripts that allow users to accurately determine the serotype and detect Cre-independent DNA recombination events in rAAV containing Lox sites within minutes. We have used these scripts to analyze more than 100 rAAV preparations. Although we focused on the detection of cross-contaminating rAAV DNA and recombination events, our Python scripts can be customized to detect other sequences or events, such as reverse packaging of plasmid backbone or DNA from the packaging cell line. We find that the NGS of rAAV DNA extracts, termed viral genome sequencing, is a simple and powerful method for rAAV validation.  

5.2833           Potent human broadly neutralizing antibodies to hepatitis B virus from natural controllers

Hehle, V., Beretta, M., Bourgine, M., Ait-Goughoulte, M., Planchais, C., Morisse, S., Vesin, B. et al

1. Exp, Med., 217(10), e20200840 (2020)

  Rare individuals can naturally clear chronic hepatitis B virus (HBV) infection and acquire protection from reinfection as conferred by vaccination. To examine the protective humoral response against HBV, we cloned and characterized human antibodies specific to the viral surface glycoproteins (HBsAg) from memory B cells of HBV vaccinees and controllers. We found that human HBV antibodies are encoded by a diverse set of immunoglobulin genes and recognize various conformational HBsAg epitopes. Strikingly, HBsAg-specific memory B cells from natural controllers mainly produced neutralizing antibodies able to cross-react with several viral genotypes. Furthermore, monotherapy with the potent broadly neutralizing antibody Bc1.187 suppressed viremia in vivo in HBV mouse models and led to post-therapy control of the infection in a fraction of animals. Thus, human neutralizing HBsAg antibodies appear to play a key role in the spontaneous control of HBV and represent promising immunotherapeutic tools for achieving HBV functional cure in chronically infected humans.  

5.2834           Amelioration of an Inherited Metabolic Liver Disease through Creation of a De Novo Start Codon by Cytidine Base Editing

Yang, L., Wang, L., Huo, Y., Chen, X., Yin, S., Hu, Y., Zhang, X., Zheng, R. et al Molecular Therapy, 28(7), 1673-1683 (2020)   Base editing technology efficiently generates nucleotide conversions without inducing excessive double-strand breaks (DSBs), which makes it a promising approach for genetic disease therapy. In this study, we generated a novel hereditary tyrosinemia type 1 (HT1) mouse model, which contains a start codon mutation in the fumarylacetoacetate hydrolase (Fah) gene by using an adenine base editor (ABE7.10). To investigate the feasibility of base editing for recombinant adeno-associated virus (rAAV)-mediated gene therapy, an intein-split cytosine base editor (BE4max) was developed. BE4max efficiently induced C-to-T conversion and restored the start codon to ameliorate HT1 in mice, but an undesired bystander mutation abolished the effect of on-target editing. To solve this problem, an upstream sequence was targeted to generate a de novo in-frame start codon to initiate the translation of FAH. After treatment, almost all C-to-T conversions created a start codon and restored Fah expression, which efficiently ameliorated the disease without inducing off-target mutations. Our study demonstrated that base editing-mediated creation of de novo functional elements would be an applicable new strategy for genetic disease therapy.  

5.2835           A Synaptic Circuit Required for Acquisition but Not Recall of Social Transmission of Food Preference

Wang, C.Y., Liu, Z., Ng, Y.H. and Südof, T.C. Neuron, 107, 144-157 (2020)   During social transmission of food preference (STFP), the combination of an olfactory sensory input with a social cue induces long-term memory of a food odor. How a social cue produces long-term learning of an olfactory input, however, remains unknown. Here we show that the neurons of the anterior olfactory nucleus (AON), which form abundant synaptic projections onto granule cells in the olfactory bulb (OB), express the synaptogenic molecule C1ql3. Deletion of C1ql3 in the dorsolateral AON impaired synaptic AON→OB connections and abolished acquisition, but not recall, of STFP memory without significantly affecting basal olfaction. Moreover, deletion in granule cells of the OB of Bai3, a postsynaptic GPCR that binds C1ql3, similarly suppressed synaptic transmission at AON→OB projections and abolished acquisition, but not recall, of STFP memory. Thus, synaptic AON→OB connections are selectively required for STFP memory acquisition and are formed by an essential interaction of presynaptic C1ql3 with postsynaptic Bai3.  

5.2836           ESCRT‐II functions by linking to ESCRT‐I in human immunodeficiency virus‐1 budding

Meng, B., lp, N.C.Y., Abbink, T.E.M., Kenyon, J.C. and Lever, A.M.L. Cell. Microbiol., 22, e13161 (2020)   Human immunodeficiency virus (HIV) uses the ESCRT (endosomal sorting complexes required for transport) protein pathway to bud from infected cells. Despite the roles of ESCRT‐I and ‐III in HIV budding being firmly established, participation of ESCRT‐II in this process has been controversial. EAP45 is a critical component of ESCRT‐II. Previously, we utilised a CRISPR‐Cas9 EAP45 knockout cell line to assess the involvement of ESCRT‐II in HIV replication. We demonstrated that the absence of ESCRT‐II impairs HIV budding. Here, we show that virus spread is also defective in physiologically relevant CRISPR/Cas9 EAP45 knockout T cells. We further show reappearance of efficient budding by re‐introduction of EAP45 expression into EAP45 knockout cells. Using expression of selected mutants of EAP45, we dissect the domain requirement responsible for this function. Our data show at the steady state that rescue of budding is only observed in the context of a Gag/Pol, but not a Gag expressor, indicating that the size of cargo determines the usage of ESCRT‐II. EAP45 acts through the YPXL‐ALIX pathway as partial rescue is achieved in a PTAP but not a YPXL mutant virus. Our study clarifies the role of ESCRT‐II in the late stages of HIV replication and reinforces the notion that ESCRT‐II plays an integral part during this process as it does in sorting ubiquitinated cargos and in cytokinesis.  

5.2837           Myostatin inhibition in combination with antisense oligonucleotide therapy improves outcomes in spinal muscular atrophy

Zhou, H., Meng, J., Malerba, A., Catapano, F., Sintusek, P., Jarmin, S., Feng, L., Lu-Nguyen, N., Sun, L., Mariot, V., Dumonceaux, J., Morgan, J.E., Gissen, P., Dickson, G. and Muntoni, F.

1. Cochexia, Sarcopenia and Muscle, 11, 768-782 (2020)

  Background Spinal muscular atrophy (SMA) is caused by genetic defects in the survival motor neuron 1 (SMN1 ) gene that lead to SMN deficiency. Different SMN‐restoring therapies substantially prolong survival and function in transgenic mice of SMA. However, these therapies do not entirely prevent muscle atrophy and restore function completely. To further improve the outcome, we explored the potential of a combinatorial therapy by modulating SMN production and muscle‐enhancing approach as a novel therapeutic strategy for SMA. Methods The experiments were performed in a mouse model of severe SMA. A previously reported 25‐mer morpholino antisense oligomer PMO25 was used to restore SMN expression. The adeno‐associated virus‐mediated expression of myostatin propeptide was used to block the myostatin pathway. Newborn SMA mice were treated with a single subcutaneous injection of 40 μg/g (therapeutic dose) or 10 μg/g (low‐dose) PMO25 on its own or together with systemic delivery of a single dose of adeno‐associated virus‐mediated expression of myostatin propeptide. The multiple effects of myostatin inhibition on survival, skeletal muscle phenotype, motor function, neuromuscular junction maturation, and proprioceptive afferences were evaluated. Results We show that myostatin inhibition acts synergistically with SMN‐restoring antisense therapy in SMA mice treated with the higher therapeutic dose PMO25 (40 μg/g), by increasing not only body weight (21% increase in male mice at Day 40), muscle mass (38% increase), and fibre size (35% increase in tibialis anterior muscle in 3 month female SMA mice), but also motor function and physical performance as measured in hanging wire test (two‐fold increase in time score) and treadmill exercise test (two‐fold increase in running distance). In SMA mice treated with low‐dose PMO25 (10 μg/g), the early application of myostatin inhibition prolongs survival (40% increase), improves neuromuscular junction maturation (50% increase) and innervation (30% increase), and increases both the size of sensory neurons in dorsal root ganglia (60% increase) and the preservation of proprioceptive synapses in the spinal cord (30% increase). Conclusions These data suggest that myostatin inhibition, in addition to the well‐known effect on muscle mass, can also positively influence the sensory neural circuits that may enhance motor neurons function. While the availability of the antisense drug Spinraza for SMA and other SMN‐enhancing therapies has provided unprecedented improvement in SMA patients, there are still unmet needs in these patients. Our study provides further rationale for considering myostatin inhibitors as a therapeutic intervention in SMA patients, in combination with SMN‐restoring drugs.  

5.2838           MicroRNA‐based recombinant AAV vector assembly improves efficiency of suicide gene transfer in a murine model of lymphoma

Khan, N., Cheemadan, S., Saxena, H., Bammidi, S. and Jayandharan, G.R. Cancer Med., 9, 3188-3201 (2020)   Recent success in clinical trials with recombinant Adeno‐associated virus (AAV)‐based gene therapy has redirected efforts in optimizing AAV assembly and production, to improve its potency. We reasoned that inclusion of a small RNA during vector assembly, which specifically alters the phosphorylation status of the packaging cells may be beneficial. We thus employed microRNAs (miR‐431, miR‐636) identified by their ability to bind AAV genome and also dysregulate Mitogen‐activated protein kinase (MAPK) signaling during vector production, by a global transcriptome study in producer cells. A modified vector assembly protocol incorporating a plasmid encoding these microRNAs was developed. AAV2 vectors packaged in the presence of microRNA demonstrated an improved gene transfer potency by 3.7‐fold, in vitro. Furthermore, AAV6 serotype vectors encoding an inducible caspase 9 suicide gene, packaged in the presence of miR‐636, showed a significant tumor regression (~2.2‐fold, P  < .01) in a syngeneic murine model of T‐cell lymphoma. Taken together, we have demonstrated a simple but effective microRNA‐based approach to improve the assembly and potency of suicide gene therapy with AAV vectors.  

5.2839           In Vivo Assessment of Cell Death and Nigrostriatal Pathway Integrity Following Continuous Expression of C3 Transferase

Gupta, R.V., Santiago-Lopez, A.J., Berglund, K., Gross, R.E. and Gutekunst, C-A.N. Neurosci., 442, 183-192 (2020)   The bacterial exoenzyme C3 transferase (C3) irreversibly inhibits RhoA GTPase leading to stimulation of axonal outgrowth in injured neurons. C3 has been used successfully in models of neurotrauma and shows promise as an option to support cell survival and axonal growth of dopaminergic (DA) neurons in Parkinson’s disease (PD) cell therapy. Whether the continuous expression of C3 in DA neurons is well-tolerated is unknown. To assess the potential neurotoxicity of sustained expression of C3 in DA neurons, we generated Cre recombinase-dependent adeno-associated viral vectors (AAV) for targeted C3 delivery to DA neurons of the mouse substantia nigra pars compacta (SNc). The effect of continuous expression of C3 on DA neurons was assessed by immunohistochemistry and compared to that of Enhanced Yellow Fluorescent Protein (EYFP) as negative controls. We did not find significant reduction of tyrosine hydroxylase (TH) expression levels nor the presence of cleaved activated caspase 3. Astrocytic activation as determined by GFAP expression was comparable to EYFP controls. To evaluate the impact of C3 expression on striatal terminals of the nigrostriatal pathway, we compared the rotational behavior of wildtype mice injected unilaterally with either C3 or 6-hydroxydopamine (6-OHDA). Mice injected with C3 exhibited similar ipsiversive rotations to the site of injection in comparison to control mice injected with EYFP and significantly fewer ipsiversive rotations compared to 6-OHDA lesioned mice. Non-significant difference between C3 and EYFP controls in behavioral and histological analyses demonstrate that transduced DA neurons express C3 continuously without apparent adverse effects, supporting the use of C3 in efficacy studies targeting DA neurons.  

5.2840           AAV-Mediated Combination Gene Therapy for Neuropathic Pain: GAD65, GDNF, and IL-10

Kim, D., Kim, K-R., Kwon, Y., Kim, M., Kim, M-J., Sim, Y., Ji, H., Park, J-J., Cho, J-H., Choi, H. and Kim, S. Molecular Therapy-Methods & Clin. Develop., 18, 473-483 (2020)   Neuropathic pain is a chronic pain state characterized by nerve damage, inflammation, and nociceptive neuron hyperactivity. As the underlying pathophysiology is complex, a more effective therapy for neuropathic pain would be one that targets multiple elements. Here, we generated recombinant adeno-associated viruses (AAVs) encoding three therapeutic genes, namely, glutamate decarboxylase 65, glial cell-derived neurotrophic factor, and interleukin-10, with various combinations. The efficacy for pain relief was evaluated in a rat spared nerve injury model of neuropathic pain. The maximal analgesic effect was achieved when the AAVs expressing all three genes were administered to rats with neuropathic pain. The combination of two virus constructs expressing the three genes was named KLS-2031 and evaluated as a potential novel therapeutic for neuropathic pain. Single transforaminal epidural injections of KLS-2031 into the intervertebral foramen to target the appropriate dorsal root ganglion produced notable long-term analgesic effects in female and male rats. Furthermore, KLS-2031 mitigated the neuroinflammation, neuronal cell death, and dorsal root ganglion hyperexcitability induced by the spared nerve injury. These results suggest that KLS-2031 represents a promising therapeutic option for refractory neuropathic pain.  

5.2841           A non-invasive far-red light-induced split-Cre recombinase system for controllable genome engineering in mice

Wu, J., Wang, M., Yang, X., Yi, C., Jiang, J., Yu, Y. and Ye, H. Nature Communications, 11:3708 (2020)   The Cre-loxP recombination system is a powerful tool for genetic manipulation. However, there are widely recognized limitations with chemically inducible Cre-loxP systems, and the UV and blue-light induced systems have phototoxicity and minimal capacity for deep tissue penetration. Here, we develop a far-red light-induced split Cre-loxP system (FISC system) based on a bacteriophytochrome optogenetic system and split-Cre recombinase, enabling optogenetical regulation of genome engineering in vivo solely by utilizing a far-red light (FRL). The FISC system exhibits low background and no detectable photocytotoxicity, while offering efficient FRL-induced DNA recombination. Our in vivo studies showcase the strong organ-penetration capacity of FISC system, markedly outperforming two blue-light-based Cre systems for recombination induction in the liver. Demonstrating its strong clinical relevance, we successfully deploy a FISC system using adeno-associated virus (AAV) delivery. Thus, the FISC system expands the optogenetic toolbox for DNA recombination to achieve spatiotemporally controlled, non-invasive genome engineering in living systems.  

5.2842           Dysregulation of the Synaptic Cytoskeleton in the PFC Drives Neural Circuit Pathology, Leading to Social Dysfunction

Kim, I.H., Kim, N., Courtland, J.L., Yin, H.H. and Soderling, S.H. Cell Reports, 32, 107965 (2020)   Psychiatric disorders are highly heritable pathologies of altered neural circuit functioning. How genetic mutations lead to specific neural circuit abnormalities underlying behavioral disruptions, however, remains unclear. Using circuit-selective transgenic tools and a mouse model of maladaptive social behavior (ArpC3 mutant), we identify a neural circuit mechanism driving dysfunctional social behavior. We demonstrate that circuit-selective knockout (ctKO) of the ArpC3 gene within prefrontal cortical neurons that project to the basolateral amygdala elevates the excitability of the circuit neurons, leading to disruption of socially evoked neural activity and resulting in abnormal social behavior. Optogenetic activation of this circuit in wild-type mice recapitulates the social dysfunction observed in ArpC3 mutant mice. Finally, the maladaptive sociability of ctKO mice is rescued by optogenetically silencing neurons within this circuit. These results highlight a mechanism of how a gene-to-neural circuit interaction drives altered social behavior, a common phenotype of several psychiatric disorders.  

5.2843           Tetraspanin CD9 affects HPV16 infection by modulating ADAM17 activity and the ERK signalling pathway

Mikulicic, S., Fritzen, A., Scheffer, K., Strunk, J., Cabanas, C., Sperrhacke, M., Reiss, K. and Florin, L. Med. Microbiol. Immunol., 209, 461-471 (2020)   Human papillomaviruses (HPV) are causative agents of various tumours such as cervical cancer. HPV binding to the cell surface of keratinocytes leads to virus endocytosis at tetraspanin enriched microdomains. Complex interactions of the capsid proteins with host proteins as well as ADAM17-dependent ERK1/2 signal transduction enable the entry platform assembly of the oncogenic HPV type 16. Here, we studied the importance of tetraspanin CD9, also known as TSPAN29, in HPV16 infection of different epithelial cells. We found that both overexpression and loss of the tetraspanin decreased infection rates in cells with low endogenous CD9 levels, while reduction of CD9 expression in keratinocytes that exhibit high-CD9 protein amounts, led to an increase of infection. Therefore, we concluded that low-CD9 supports infection. Moreover, we found that changes in CD9 amounts affect the shedding of the ADAM17 substrate transforming growth factor alpha (TGFα) and the downstream phosphorylation of ERK. These effects correlate with those on infection rates suggesting that a specific CD9 optimum promotes ADAM17 activity, ERK signalling and virus infection. Together, our findings implicate that CD9 regulates HPV16 infection through the modulation of ADAM17 sheddase activity.  

5.2844           Identification of substances which regulate activity of corticotropin-releasing factor-producing neurons in the paraventricular nucleus of the hypothalamus

Mukai, Y., Nagayama, A., Itoi, K. and Yamanaka, A. Scientific Reports, 10:13639 (2020)   The stress response is a physiological system for adapting to various internal and external stimuli. Corticotropin-releasing factor-producing neurons in the paraventricular nucleus of the hypothalamus (PVN-CRF neurons) are known to play an important role in the stress response as initiators of the hypothalamic–pituitary–adrenal axis. However, the mechanism by which activity of PVN-CRF neurons is regulated by other neurons and bioactive substances remains unclear. Here, we developed a screening method using calcium imaging to identify how physiological substances directly affect the activity of PVN-CRF neurons. We used acute brain slices expressing a genetically encoded calcium indicator in PVN-CRF neurons using CRF-Cre recombinase mice and an adeno-associated viral vector under Cre control. PVN-CRF neurons were divided into ventral and dorsal portions. Bath application of candidate substances revealed 12 substances that increased and 3 that decreased intracellular calcium concentrations. Among these substances, angiotensin II and histamine mainly increased calcium in the ventral portion of the PVN-CRF neurons via AT₁ and H₁ receptors, respectively. Conversely, carbachol mainly increased calcium in the dorsal portion of the PVN-CRF neurons via both nicotinic and muscarinic acetylcholine receptors. Our method provides a precise and reliable means of evaluating the effect of a substance on PVN-CRF neuronal activity.  

5.2845           Targeted gene correction of human hematopoietic stem cells for the treatment of Wiskott - Aldrich Syndrom

Rai, R., Romito, M., Rivers, E., Turchiano, G., Blattner, G., Vetharoy, W., Ladon, D., Andrieux, G., Zhang, F., Zinicola, M., Leon-Rico, D., Santilli, G., Thrasher, A.J. and Cavazza, A. Nature Communications, 11:4034 (2020)   Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency. Although clinical gene therapy approaches using lentiviral vectors have produced encouraging results, full immune and platelet reconstitution is not always achieved. Here we show that a CRISPR/Cas9-based genome editing strategy allows the precise correction of WAS mutations in up to 60% of human hematopoietic stem and progenitor cells (HSPCs), without impairing cell viability and differentiation potential. Delivery of the editing reagents to WAS HSPCs led to full rescue of WASp expression and correction of functional defects in myeloid and lymphoid cells. Primary and secondary transplantation of corrected WAS HSPCs into immunodeficient mice showed persistence of edited cells for up to 26 weeks and efficient targeting of long-term repopulating stem cells. Finally, no major genotoxicity was associated with the gene editing process, paving the way for an alternative, yet highly efficient and safe therapy.  

5.2846           Gene editing and elimination of latent herpes simplex virus in vivo

Aubert, M., Strongin, D.E., Roychoudhury, P., Loprieno, M.A., Haick, A.K. et al Nature Communications, 11:4148 (2020)   We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection.  

5.2847           MICAL2 is essential for myogenic lineage commitment

Giarratana, N., Conti, F., La Rovere, R., Gjsbers, R., Caraj, P., Duelen, R., Vervliet, T. et al Cell Death & Disease, 11:654 (2020)   Contractile myofiber units are mainly composed of thick myosin and thin actin (F-actin) filaments. F-Actin interacts with Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing them and preventing repolymerization. In this study, we found that MICAL2 increases during myogenic differentiation of adult and pluripotent stem cells (PSCs) towards skeletal, smooth and cardiac muscle cells and localizes in the nucleus of acute and chronic regenerating muscle fibers. In vivo delivery of Cas9–Mical2 guide RNA complexes results in muscle actin defects and demonstrates that MICAL2 is essential for skeletal muscle homeostasis and functionality. Conversely, MICAL2 upregulation shows a positive impact on skeletal and cardiac muscle commitments. Taken together these data demonstrate that modulations of MICAL2 have an impact on muscle filament dynamics and its fine-tuned balance is essential for the regeneration of muscle tissues.  

5.2848           Intra-tracheal delivery of AAV6 vectors results in sustained transduction in murine lungs without genomic integration

Colon-Cortes, Y., Hasan, M.A. and Aslanidi, G. Gene:X, 5, 100037 (2020)   Despite the progress made in AAV-based gene therapy targeting different organ systems, lung-targeted gene therapy using AAV vectors has not been effective, mostly due to the poor transduction and un-sustained gene expression in airway epithelium. Furthermore, concerns over possible harmful insertional mutagenesis seen in other cell types, particularly hepatocytes, raised a question about AAV safety. In this study, we evaluate the long-term persistence of this vector in mouse lungs and any possible harmful integration of these vectors into the host genome. AAV6 vectors expressing reporter gene (firefly luciferase) were delivered to the lungs of C57BL/6 mice through intra-tracheal intubation. Despite the large variation among individual animals, most animals had high and sustained luciferase activity with a peak from 2 to 3 weeks post-transduction before a significant decline between 15 and 19 weeks post-transduction. More importantly, even after its decline, most animals maintained detectable luciferase expression for 150 days or more, which was confirmed by post-necropsy qPCR analysis of luciferase gene expression. At the termination point of experiments, an average of one copy of AAV expression cassette per mouse genome was detected. We also found that partial overlaps between the AAV6 expression cassette and the mouse genome were distributed broadly with no apparent systematic preference in any mouse chromosomal map location. In summary, our data suggest that AAV6 mediated long-term gene expression in the lungs with no evidence of genomic integration, and thus, any insertional mutagenesis.  

5.2849           Alternative splicing of clathrin heavy chain contributes to the switch from coated pits to plaques

Moulay, G., Laine, J., Lemaitre, M., Nakamori, M., Nishino, I., Caillol, G., Mamchaoui, K., Julien, L., Dingli, F., Loew, D., Biltoun, M., Leterrier, C., Furling, D. and Vassilopoulos, S.

1. Cell Biol., 219(9), e201912061 (2020)

  Clathrin function directly derives from its coat structure, and while endocytosis is mediated by clathrin-coated pits, large plaques contribute to cell adhesion. Here, we show that the alternative splicing of a single exon of the clathrin heavy chain gene ( CLTC exon 31) helps determine the clathrin coat organization. Direct genetic control was demonstrated by forced CLTC exon 31 skipping in muscle cells that reverses the plasma membrane content from clathrin plaques to pits and by promoting exon inclusion that stimulated flat plaque assembly. Interestingly, mis-splicing of CLTC exon 31 found in the severe congenital form of myotonic dystrophy was associated with reduced plaques in patient myotubes. Moreover, forced exclusion of this exon in WT mice muscle induced structural disorganization and reduced force, highlighting the contribution of this splicing event for the maintenance of tissue homeostasis. This genetic control on clathrin assembly should influence the way we consider how plasticity in clathrin-coated structures is involved in muscle development and maintenance.  

5.2850           Systemic AAV9 gene therapy using the synapsin I promoter rescues a mouse model of neuronopathic Gaucher disease but with limited cross-correction potential to astrocytes

Massaro, G., Hughes, M.P., Whaler, S.M., Wallom, K-L., Priestman, D.A., Platt, F.M., Waddington, S.N. and Rahim, A.A. Hum. Mol. Genet., 29(12), 1933-1949 (2020)   Gaucher disease is caused by mutations in the GBA gene, which encodes for the lysosomal enzyme β-glucocerebrosidase (GCase), resulting in the accumulation of storage material in visceral organs and in some cases the brain of affected patients. While there is a commercially available treatment for the systemic manifestations, neuropathology still remains untreatable. We previously demonstrated that gene therapy represents a feasible therapeutic tool for the treatment of the neuronopathic forms of Gaucher disease (nGD). In order to further enhance the therapeutic affects to the central nervous system, we systemically delivered an adeno-associated virus (AAV) serotype 9 carrying the human GBA gene under control of a neuron-specific promoter to an nGD mouse model. Gene therapy increased the life span of treated animals, rescued the lethal neurodegeneration, normalized the locomotor behavioural defects and ameliorated the visceral pathology. Together, these results provided further indication of gene therapy as a possible effective treatment option for the neuropathic forms of Gaucher disease.  

5.2851           Efficient whole brain transduction by systemic infusion of minimally purified AAV-PHP.eB

Konno, A. and Hirai, H.

1. Neurosci. Methods, 346, 108914 (2020)

  Background Adeno-associated virus (AAV) vectors have excellent properties as gene transfer vehicles. The recent development of AAV-PHP.eB, highly BBB-permeable capsid variant of AAV serotype 9, has opened up systemic application for whole brain transduction. To attain high transduction efficacy, much efforts have been paid to purify AAV vectors using gradient centrifugation or column chromatography. These methods are time-consuming, cost substantially and require expensive equipment. New method We propose a simple purification method for the production of systemically applicable AAV-PHP.eB targeting the brain. The new method, which we named minimal purification (MP) method, requires only 2 steps: removal of cell debris using a syringe filter and concentration using a disposable ultrafiltration device. Results The MP method yielded 2 times more AAV-PHP.eB than the standard ultracentrifuge purification (UCP) method. Intravenous injection of AAV-PHP.eB prepared using the MP method caused robust whole brain transduction without overt toxicity on the liver and kidney. Moreover, we found almost no difference in cellular density and morphology of brain microglia between control mice and mice treated systemically with the MP viral solution, suggesting no influence of the viral injection on brain immunity. Comparison with existing methods The new method, which requires only a benchtop centrifuge and takes only 2–4 h to obtain a ready-to-use viral solution, is much less expensive than the existing UCP method, and can avoid cumbersome and time-consuming purification processes. Conclusions This simplified method further expands the use of AAV vectors in the neuroscience community.  

5.2852           Promoter Orientation within an AAV-CRISPR Vector Affects Cas9 Expression and Gene Editing Efficiency

Fry, L.E., Peddle, C.F., Stevanovic, M., barnaard, A.R., McClements, M.E. and MacLaren, R.E. CRISPR J., 3(4), 276-283 (2020)   Adeno-associated virus (AAV) vectors have been widely adopted for delivery of CRISPR-Cas components, especially for therapeutic gene editing. For a single vector system, both the Cas9 and guide RNA (gRNA) are encoded within a single transgene, usually from separate promoters. Careful design of this bi-cistronic construct is required due to the minimal packaging capacity of AAV. We investigated how placement of the U6 promoter expressing the gRNA on the reverse strand to SaCas9 driven by a cytomegalovirus promoter affected gene editing rates compared to placement on the forward strand. We show that orientation in the reverse direction reduces editing rates from an AAV vector due to reduced transcription of both SaCas9 and guide RNA. This effect was observed only following AAV transduction; it was not seen following plasmid transfection. These results have implications for the design of AAV-CRISPR vectors, and suggest that results from optimizing plasmid transgenes may not translate when delivered via AAV.  

5.2853           KIAA1522 potentiates TNFα-NFκB signaling to antagonize platinum-based chemotherapy in lung adenocarcinoma

Wang, B., Jing, T., Jin, W., Chen, J., Wu, C., Wang, M. and Liu, Y.

1. Exp. Clin. Cancer Res., 39:170 (2020)

  Background The platinum-based chemotherapy is the first-line regimen for the treatment of Non-small cell lung cancer (NSCLC). However, the therapeutic efficiency is largely limited by tenacious chemo-insensitivity that results in inferior prognosis in a cohort of patients. It has been known that KIAA1522 is aberrantly expressed and implicated in several types of solid tumors including NSCLC. Nowadays, knowledge about this gene is quite limited. Here, we aimed to identify the role of KIAA1522 in lung adenocarcinomas, and the molecular events that underlie KIAA1522-mediated chemoresistance to the platinum. Methods Immunohistochemistry were used to detect KIAA1522 expression in clinical NSCLC samples. Then, the survival analyses were performed to assess the link between KIAA1522 expression and overall survival or therapeutic outcome. In vivo depletion of KIAA1522 in adenocarcinoma cells were achieved by adeno-associated virus-mediated sgRNA/Cre delivery into the conditional Kras^G12D/Cas9 expressed mice, which were designated to identify the roles of KIAA1522 in tumorigenesis and/or chemotherapy responses. The effects of KIAA1522 and downstream molecular events were studied by pharmacology in mice model and assays using in vitro cultured cells. The clinical relevance of our findings was examined by data-mining of online datasets from multiple cohorts. Results The clinical evidences reveal that KIAA1522 independently predicts both the overall survival and the outcome of platinum-based chemotherapy in lung adenocarcinomas. By using a Kras^G12D-driven murine lung adenocarcinoma model and performing in vitro assays, we demonstrated that KIAA1522 is a critical positive regulator of lung adenocarcinoma and a modulator of cisplatin response. KIAA1522 potentiates the TNFα-TNFR2-NFκB signaling which in turn intensifies recalcitrance to cisplatin treatment. These results were further manifested by integrative bioinformatic analyses of independent datasets, in which KIAA1522 is tightly associated with the activity of TNFα-NFκB pathway and the cisplatin-resistant gene signatures. More strikingly, overexpression of KIAA1522 counteracts the cisplatin-induced tumor growth arrest in vivo, and this effect can be remarkably diminished by the disruption of NFκB activity. Conclusion High expression of KIAA1522 is turned out to be an indicator of dismal effectiveness of platinum-based therapy in lung adenocarcinomas. KIAA1522 hyperactivates TNFα-NFκB signaling to facilitate resistance to platinum reagents. Targeting NFκB signaling through small molecule inhibitors may be a rational strategy to conquer chemoresistance and synergize platinum-based chemotherapy in KIAA1522 overexpressed lung adenocarcinomas.  

5.2854           Adeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking

Lins-Austin, B., Patel, S., Mietzsch, M., Brooke, D., Bennett, A. et al Viruses, 12, 668 (2020)   Adeno-associated viruses (AAVs) are small, non-pathogenic ssDNA viruses being used as therapeutic gene delivery vectors for the treatment of a variety of monogenic diseases. An obstacle to successful gene delivery is inefficient capsid trafficking through the endo/lysosomal pathway. This study aimed to characterize the AAV capsid stability and dynamics associated with this process for a select number of AAV serotypes, AAV1, AAV2, AAV5, and AAV8, at pHs representative of the early and late endosome, and the lysosome (6.0, 5.5, and 4.0, respectively). All AAV serotypes displayed thermal melt temperatures that varied with pH. The stability of AAV1, AAV2, and AAV8 increased in response to acidic conditions and then decreased at pH 4.0. In contrast, AAV5 demonstrated a consistent decrease in thermostability in response to acidification. Negative-stain EM visualization of liposomes in the presence of capsids at pH 5.5 or when heat shocked showed induced remodeling consistent with the externalization of the PLA₂ domain of VP1u. These observations provide clues to the AAV capsid dynamics that facilitate successful infection. Finally, transduction assays revealed a pH and temperature dependence with low acidity and temperatures > 4 °C as detrimental factors.  

5.2855           Molecular Characterization of Hemorrhagic Enteritis Virus (HEV) Obtained from Clinical Samples in Western Canada 2017–2018

Palomino-Tapia, V., Mitevski, D., Inglis, T., van der Meer, F. and Abdul-Careem, M.F. Viruses, 12, 941 (2020)   Hemorrhagic enteritis virus (HEV) is an immunosuppressive adenovirus that causes an acute clinical disease characterized by hemorrhagic gastroenteritis in 4-week-old turkeys and older. Recurrent incidence of secondary infections (e.g., systemic bacterial infections, cellulitis, and elevated mortality), may be associated with the presence of field-type HEV in Canadian turkey farms. We speculate that field-type HEV and vaccine/vaccine-like strains can be differentiated through analysis of the viral genomes, hexon genes, and the specific virulence factors (e.g., ORF1, E3, and fib knob domain). Nine out of sixteen spleens obtained from cases suspected of immunosuppression by HEV were analyzed. The limited data obtained showed that: (1) field-type HEV circulates in many non-vaccinated western Canadian flocks; (2) field-type HEV circulates in vaccinated flocks with increased recurrent bacterial infections; and (3) the existence of novel point mutations in hexon, ORF1, E3, and specially fib knob domains. This is the first publication showing the circulation of wild-type HEV in HEV-vaccinated flocks in Western Canada, and the usefulness of a novel procedure that allows whole genome sequencing of HEV directly from spleens, without passaging in cell culture or passaging in vivo. Further studies focusing more samples are required to confirm our observations and investigate possible vaccination failure.    

5.2856           Specific Antibodies Induced by Immunization with Hepatitis B Virus-Like Particles Carrying Hepatitis C Virus Envelope Glycoprotein 2 Epitopes Show Differential Neutralization Efficiency

Czarnota, A., Offersgaard, A., Pihl, A.F., Prentoe, J., Bukh, J., Gottwein, K.M., Bienkowska-Szewczyk, K and Grzyb, K. Vaccines, 8, 294 (2020)   Hepatitis C virus (HCV) infection with associated chronic liver diseases is a major health problem worldwide. Here, we designed hepatitis B virus (HBV) small surface antigen (sHBsAg) virus-like particles (VLPs) presenting different epitopes derived from the HCV E2 glycoprotein (residues 412–425, 434–446, 502–520, and 523–535 of isolate H77C). Epitopes were selected based on their amino acid sequence conservation and were previously reported as targets of HCV neutralizing antibodies. Chimeric VLPs obtained in the Leishmania tarentolae expression system, in combination with the adjuvant Addavax, were used to immunize mice. Although all VLPs induced strong humoral responses, only antibodies directed against HCV 412–425 and 523–535 epitopes were able to react with the native E1E2 glycoprotein complexes of different HCV genotypes in ELISA. Neutralization assays against genotype 1–6 cell culture infectious HCV (HCVcc), revealed that only VLPs carrying the 412–425 epitope induced efficient HCV cross-neutralizing antibodies, but with isolate specific variations in efficacy that could not necessarily be explained by differences in epitope sequences. In contrast, antibodies targeting 434–446, 502–520, and 523–535 epitopes were not neutralizing HCVcc, highlighting the importance of conformational antibodies for efficient virus neutralization. Thus, 412–425 remains the most promising linear E2 epitope for further bivalent, rationally designed vaccine research.  

5.2857           Chloroviruses

Van Etten, J.L., Agarkova, I.V. and Dunigan, D.D. Viruses, 12, 20 (2020)   Chloroviruses are large dsDNA, plaque-forming viruses that infect certain chlorella-like green algae; the algae are normally mutualistic endosymbionts of protists and metazoans and are often referred to as zoochlorellae. The viruses are ubiquitous in inland aqueous environments throughout the world and occasionally single types reach titers of thousands of plaque-forming units per ml of native water. The viruses are icosahedral in shape with a spike structure located at one of the vertices. They contain an internal membrane that is required for infectivity. The viral genomes are 290 to 370 kb in size, which encode up to 16 tRNAs and 330 to ~415 proteins, including many not previously seen in viruses. Examples include genes encoding DNA restriction and modification enzymes, hyaluronan and chitin biosynthetic enzymes, polyamine biosynthetic enzymes, ion channel and transport proteins, and enzymes involved in the glycan synthesis of the virus major capsid glycoproteins. The proteins encoded by many of these viruses are often the smallest or among the smallest proteins of their class. Consequently, some of the viral proteins are the subject of intensive biochemical and structural investigation.  

5.2858           Overexpression of Hepatocyte Chemerin-156 Lowers Tumor Burden in a Murine Model of Diethylnitrosamine-Induced Hepatocellular Carcinoma

Haberl, E.M., Pohl, R., Rein-Fishboeck, L., Feder, S., Sinal, C.J., Bruckmann, A., Hoering, M., Krautbauer, S., Liebisch, G. and Buechler, C. Int. J. Mol. Sci., 21, 252 (2020)   The tumor inhibitory potential of the highly active chemerin-156 isoform was described in orthotopic models of hepatocellular carcinoma (HCC). The majority of HCC arises in the fibrotic liver, which was not reproduced in these studies. Here, a potential therapeutic activity of chemerin-156 was evaluated in diethylnitrosamine (DEN)-induced liver cancer, which mimics fibrosis-associated HCC. Mice were infected with adeno-associated virus (AAV) six months after DEN injection to overexpress chemerin-156 in the liver, and animals injected with non-recombinant-AAV served as controls. Three months later, the animals were killed. Both groups were comparable with regard to liver steatosis and fibrosis. Of note, the number of very small tumors was reduced by chemerin-156. Anyhow, the expression of inflammatory and profibrotic genes was similar in larger tumors of control and chemerin-156-AAV-infected animals. Although genes with a role in lipid metabolism, like 3-hydroxy-3-methylglutaryl-coenzym-A--reductase, were overexpressed in tumors of animals with high chemerin-156, total hepatic cholesterol, diacylglycerol and triglyceride levels, and distribution of individual lipid species were normal. Chemerin-156-AAV-infected mice had elevated hepatic and systemic chemerin. Ex vivo activation of the chemerin receptor chemokine-like receptor 1 increased in parallel with serum chemerin, illustrating the biological activity of the recombinant protein. In the tumors, chemerin-155 was the most abundant variant. Chemerin-156 was not detected in tumors of the controls and was hardly found in chemerin-156-AAV infected animals. In conclusion, the present study showed that chemerin-156 overexpression caused a decline in the number of small lesions but did not prevent the growth of pre-existing neoplasms.  

5.2859           AAV-Mediated Gene Delivery to 3D Retinal Organoids Derived from Human Induced Pluripotent Stem Cells

Garita-Hernandez, M., Routet, F., Guibbal, L., Khabou, H., Toualbi, L., Riancho, L., Reichman, S., Duebel, J., Sahel, J-A., Goureau, O. and Dalkara, D. Int. J. Mol. Sci., 21, 994 (2020)   Human induced pluripotent stem cells (hiPSCs) promise a great number of future applications to investigate retinal development, pathophysiology and cell therapies for retinal degenerative diseases. Specific approaches to genetically modulate hiPSC would be valuable for all of these applications. Vectors based on adeno-associated virus (AAV) have shown the ability for gene delivery to retinal organoids derived from hiPSCs. Thus far, little work has been carried out to investigate mechanisms of AAV-mediated gene delivery and the potential advantages of engineered AAVs to genetically modify retinal organoids. In this study, we compared the early transduction efficiency of several recombinant and engineered AAVs in hiPSC-derived RPE cells and retinal organoids in relation to the availability of their cell-surface receptors and as a function of time. The genetic variant AAV2-7m8 had a superior transduction efficiency when applied at day 44 of differentiation on retinal organoids and provided long-lasting expressions for at least 4 weeks after infection without compromising cell viability. All of the capsids we tested transduced the hiPSC-RPE cells, with the AAV2-7m8 variant being the most efficient. Transduction efficiency was correlated with the presence of primary cell-surface receptors on the hiPS-derived organoids. Our study explores some of the mechanisms of cell attachment of AAVs and reports long-term gene expression resulting from gene delivery in retinal organoids.  

5.2860           The Role of Dorsal Raphe Serotonin Neurons in the Balance between Reward and Aversion

Nagai, Y., Takayama, K., Nishitani, N., Andoh, C., Koda, M., Shirakawa, H., Nakagawa, T., Nagayasu, K., Yamanaka, A. and Kaneko, S. Int. J. Mol. Sci., 21, 2160 (2020)   Background: Reward processing is fundamental for animals to survive and reproduce. Many studies have shown the importance of dorsal raphe nucleus (DRN) serotonin (5-HT) neurons in this process, but the strongly correlative link between the activity of DRN 5-HT neurons and rewarding/aversive potency is under debate. Our primary objective was to reveal this link using two different strategies to transduce DRN 5-HT neurons. Methods: For transduction of 5-HT neurons in wildtype mice, adeno-associated virus (AAV) bearing the mouse tryptophan hydroxylase 2 (TPH2) gene promoter was used. For transduction in Tph2-tTA transgenic mice, AAVs bearing the tTA-dependent TetO enhancer were used. To manipulate the activity of 5-HT neurons, optogenetic actuators (CheRiff, eArchT) were expressed by AAVs. For measurement of rewarding/aversive potency, we performed a nose-poke self-stimulation test and conditioned place preference (CPP) test. Results: We found that stimulation of DRN 5-HT neurons and their projections to the ventral tegmental area (VTA) increased the number of nose-pokes in self-stimulation test and CPP scores in both targeting methods. Concomitantly, CPP scores were decreased by inhibition of DRN 5-HT neurons and their projections to VTA. Conclusion: Our findings indicate that the activity of DRN 5-HT neurons projecting to the VTA is a key modulator of balance between reward and aversion.  

5.2861           Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

Jones-Tabah, J., Mohammad, H., Hadj-Youssef, S., Kim, L.E., Martin, R.D., Benaliouad, F., Tanny, J.C., Clarke, P.B.S. and Hebert, T.E. Scientific Reports, 10:14426 (2020)   As with many G protein-coupled receptors (GPCRs), the signalling pathways regulated by the dopamine D1 receptor (D1R) are dynamic, cell type-specific, and can change in the face of disease or drug exposures. In striatal neurons, the D1R activates cAMP/protein kinase A (PKA) signalling. However, in Parkinson’s disease (PD), alterations in this pathway lead to functional upregulation of extracellular regulated kinases 1/2 (ERK1/2), contributing to L-DOPA-induced dyskinesia (LID). In order to detect D1R activation in vivo and to study the progressive dysregulation of D1R signalling in PD and LID, we developed ratiometric fiber-photometry with Förster resonance energy transfer (FRET) biosensors and optically detected PKA and ERK1/2 signalling in freely moving rats. We show that in Parkinsonian animals, D1R signalling through PKA and ERK1/2 is sensitized, but that following chronic treatment with L-DOPA, these pathways become partially desensitized while concurrently D1R activation leads to greater induction of dyskinesia.  

5.2862           Proteomic Profiling of Purified Rabies Virus Particles

Zhang, Y., Wang, Y., Feng, Y., Tu, Z., Lou, Z. and Tu, C. Virologica Sinica, 35, 143-155 (2020)   While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus, these characteristics have been largely neglected in studies on rabies virus (RABV). Here, we purified the RABV virions with good purity and integrity, and analyzed their proteome by nano LC–MS/MS, followed by the confirmation with immunoblot and immuno-electronic microscopy. In addition to the 5 viral proteins, 49 cellular proteins were reproducibly identified to be incorporated into matured RABV virions. Function annotation suggested that 24 of them were likely involved in virus replication. Furthermore, cryo-EM was employed to observe the purified RABV virions, generating high-resolution pictures of the bullet-shaped virion structure of RABV. This study has provided new insights into the host proteins composition in RABV virion and shed the light for further investigation on molecular mechanisms of RABV infection, as well as the discovery of new anti-RABV therapeutics.  

5.2863           Effect of heart ischemia and administration route on biodistribution and transduction efficiency of AAV9 vectors

Garcia-Olloqui, P., Rodriguez-Madoz, J., Di Scala, M., Abizanda, G., Vales, A., Olagüe, C., Iglesias-garcia, O., Larequi, E., Aguado-Alvaro, L.P., Riuz-Villalba, A., Prosper, F., Gonzalez-Aseguinolaza, G. and Pelacho, B.

1. Tissue Eng. Regen. Med., 14, 123-134 (2020)

  Adeno‐associated viruses (AAV) have become one of the most promising tools for gene transfer in clinics. Among all the serotypes, AAV9 has been described as the most efficient for cardiac transduction. In order to achieve optimal therapeutic delivery in heart disease, we have explored AAV9 transduction efficiency in an infarcted heart using different routes of administration and promoters, including a cardiac‐specific one. AAV9 vectors carrying luciferase or green fluorescence protein under the control of the ubiquitous elongation‐factor‐1‐alpha or the cardiac‐specific troponin‐T (TnT) promoters were administered by intramyocardial or intravenous injection, either in healthy or myocardial‐infarcted mice. The transduction efficacy and specificity, the time‐course expression, and the safety of each vector were tested. High transgene expression levels were found in the heart, but not in the liver, of mice receiving AAV‐TnT, which was significantly higher after intramyocardial injection regardless of ischemia‐induction. On the contrary, high hepatic transgene expression levels were detected with the elongation‐factor‐1‐alpha‐promoter, independently of the administration route and heart damage. Moreover, tissue‐specific green fluorescence protein expression was found in cardiomyocytes with the TnT vector, whereas minimal cardiac expression was detected with the ubiquitous one. Interestingly, we found that myocardial infarction greatly increased the transcriptional activity of AAV genomes. Our findings show that the use of cardiac promoters allows for specific and stable cardiac gene expression, which is optimal and robust when intramyocardially injected. Furthermore, our data indicate that the pathological status of the tissue can alter the transcriptional activity of AAV genomes, an aspect that should be carefully evaluated for clinical applications.  

5.2864           Generation of a MOR‐CreER knock‐in mouse line to study cells and neural circuits involved in mu opioid receptor signaling

Okunomiya, T., Hioki, H., Nishimura, C., Yaaawata, S., Imayoshi, I., Kageyama, R., Takahashi, R. and Watanabe, D. Genesis, 58, e23341 (2020)   Mu opioid receptor (MOR) is involved in various brain functions, such as pain modulation, reward processing, and addictive behaviors, and mediates the main pharmacologic effects of morphine and other opioid compounds. To gain genetic access to MOR‐expressing cells, and to study physiological and pathological roles of MOR signaling, we generated a MOR‐CreER knock‐in mouse line, in which the stop codon of the Oprm1 gene was replaced by a DNA fragment encoding a T2A peptide and tamoxifen (Tm)‐inducible Cre recombinase. We show that the MOR‐CreER allele undergoes Tm‐dependent recombination in a discrete subtype of neurons that express MOR in the adult nervous system, including the olfactory bulb, cerebral cortex, striosome compartments in the striatum, hippocampus, amygdala, thalamus, hypothalamus, interpeduncular nucleus, superior and inferior colliculi, periaqueductal gray, parabrachial nuclei, cochlear nucleus, raphe nuclei, pontine and medullary reticular formation, ambiguus nucleus, solitary nucleus, spinal cord, and dorsal root ganglia. The MOR‐CreER mouse line combined with a Cre‐dependent adeno‐associated virus vector enables robust gene manipulation in the MOR‐enriched striosomes. Furthermore, Tm treatment during prenatal development effectively induces Cre‐mediated recombination. Thus, the MOR‐CreER mouse is a powerful tool to study MOR‐expressing cells with conditional gene manipulation in developing and mature neural tissues.  

5.2865           Comprehensive Dual- and Triple-Feature Intersectional Single-Vector Delivery of Diverse Functional Payloads to Cells of Behaving Mammals

Fenno, L.E., Ramakrishnan, C., Kim, Y.S., Pichamoorthy, N., Hong, A.S.O. and Deisseroth, K. Neuron, 107, 836-853 (2020)   The resolution and dimensionality with which biologists can characterize cell types have expanded dramatically in recent years, and intersectional consideration of such features (e.g., multiple gene expression and anatomical parameters) is increasingly understood to be essential. At the same time, genetically targeted technology for writing in and reading out activity patterns for cells in living organisms has enabled causal investigation in physiology and behavior; however, cell-type-specific delivery of these tools (including microbial opsins for optogenetics and genetically encoded Ca²⁺ indicators) has thus far fallen short of versatile targeting to cells jointly defined by many individually selected features. Here, we develop a comprehensive intersectional targeting toolbox including 39 novel vectors for joint-feature-targeted delivery of 13 molecular payloads (including opsins, indicators, and fluorophores), systematic approaches for development and optimization of new intersectional tools, hardware for in vivo monitoring of expression dynamics, and the first versatile single-virus tools (Triplesect) that enable targeting of triply defined cell types.  

5.2866           Functional microRNA screen uncovers O-linked N-acetylglucosamine transferase as a host factor modulating hepatitis C virus morphogenesis and infectivity

Herzog, K., Bandiera, S., Pernot, S., Fauvelle, C., Jühling, F. et al Gut, 69, 380-392 (2020)   Objective Infection of human hepatocytes by the hepatitis C virus (HCV) is a multistep process involving both viral and host factors. microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Given that miRNAs were indicated to regulate between 30% and 75% of all human genes, we aimed to investigate the functional and regulatory role of miRNAs for the HCV life cycle. Design To systematically reveal human miRNAs affecting the HCV life cycle, we performed a two-step functional high-throughput miRNA mimic screen in Huh7.5.1 cells infected with recombinant cell culture-derived HCV. miRNA targeting was then assessed using a combination of computational and functional approaches. Results We uncovered miR-501-3p and miR-619-3p as novel modulators of HCV assembly/release. We discovered that these miRNAs regulate O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) protein expression and identified OGT and O-GlcNAcylation as regulators of HCV morphogenesis and infectivity. Furthermore, increased OGT expression in patient-derived liver tissue was associated with HCV-induced liver disease and cancer. Conclusion miR-501-3p and miR-619-3p and their target OGT are previously undiscovered regulatory host factors for HCV assembly and infectivity. In addition to its effect on HCV morphogenesis, OGT may play a role in HCV-induced liver disease and hepatocarcinogenesis.  

5.2867           Hepatitis E virus replication in human intestinal cells

Marion, O., Lhomme, S., Nayrac, M., Dubois, M., Pucelle, M., Requena, M., Migueres, M., Abravanel, F., Peron, J.M., Carrere, N., Suc, B., Delobel, P., Kamar, N. and Izopet, J. Gut, 69, 901-910 (2020)   Objective Hepatitis E virus (HEV), one of the most common agent of acute hepatitis worldwide, is mainly transmitted enterically, via contaminated water for HEV genotypes 1 (HEV1) and HEV2, or by eating raw or undercooked infected meat for HEV genotype 3 (HEV3) and HEV4. However, little is known about how the ingested HEV reaches the liver or its ability to replicate in intestinal cells. Design We developed human primary cultures of small intestine epithelial cells and intestinal explants obtained from small bowel resections. The epithelial cells were also polarised on transwells. Cells were infected with Kernow-p6 strain or clinically derived virions. Results Primary intestinal cells supported the growth of Kernow-p6 strain and HEV1 and HEV3 clinically derived virions. Polarised enterocytes infected with HEV1 and HEV3 strains released HEV particles vectorially: mostly into the apical compartment with a little basally. Iodixanol density gradient centrifugation of enterocyte-derived HEV virions gave bands at a density of 1.06–1.08 g/cm³, corresponding to that of quasi-enveloped HEV particles. Ribavirin therapy inhibited HEV excretion from the basal surface but not from the apical side of infected human enterocytes. HEV virions also infected intestinal tissue explants. Lastly, HEV RNA and antigen were detected in the intestinal crypts of a chronically infected patient. Conclusion HEV can replicate in intestinal cells and reaches the liver as quasi-enveloped virions.  

5.2868           A Chemogenetic Tool that Enables Functional Neural Circuit Analysis

Ngo, H.B., Melo, M.R., Layfield, S., McDougall, S.J.M., Bathgate, R.A.D. and Allen, A.M. Cell Reports, 32, 108139 (2020)   Chemogenetics enables manipulation of neuronal activity in experimental animals. While providing information about the transduced neuron expressing a ligand-activated molecule, chemogenetics does not provide understanding about the antecedent circuit that drives that neuron’s activity. For current approaches, this is not feasible, because the activating molecules are not genetically encoded. The insect allatostatin/allatostatin receptor system, a highly specific, powerful inhibitory chemogenetic approach, has this advantage, because the ligand, being a peptide, is genetically encoded. We developed viral vector-based systems to express biologically active allatostatin in neurons in vivo and allatostatin receptors in subpopulations of postsynaptic neurons. We demonstrate that activity-dependent release of allatostatin induces inhibition of allatostatin receptor-expressing neurons. We validate the approach in the vagal viscerosensory system where inhibitory, rather than the usual excitatory, viscerosensory input leads to sustained decreases in baroreceptor reflex sensitivity and bodyweight.  

5.2869           Deletion of NoxO1 limits atherosclerosis development in female mice

Buchmann, G.K., Schürmann, C., Warwick, T., Schulz, M.H., Spaeth, M., Müller, O.J., Schröder, K., Jo, H., Weissmann, N. and Brandes, R.P. Redox Biol., 37, 101713 (2020)   Objective Oxidative stress is a risk factor for atherosclerosis. NADPH oxidases of the Nox family produce ROS but their contribution to atherosclerosis development is less clear. Nox2 promotes and Nox4 rather limits atherosclerosis. Although Nox1 with its cytosolic co-factors are largely expressed in epithelial cells, a role for Nox1 for atherosclerosis development was suggested. To further define the role of this homologue, the role of its essential cytosolic cofactor, NoxO1, was determined for atherosclerosis development with the aid of knockout mice. Methods and results Wildtype (WT) and NoxO1 knockout mice were treated with high fat diet and adeno-associated virus (AAV) overexpressing pro-protein convertase subtilisin/kexin type 9 (PCSK9) to induce hepatic low-density lipoprotein (LDL) receptor loss. As a result, massive hypercholesterolemia was induced and spontaneous atherosclerosis developed within three month. Deletion of NoxO1 reduced atherosclerosis formation in brachiocephalic artery and aortic arch in female but not male NoxO1−/− mice as compared to WT littermates. This was associated with a reduced pro-inflammatory cytokine signature in the plasma of female but not male NoxO1−/− mice. MACE-RNAseq of the vessel did not reveal this signature and the expression of the Nox1/NoxO1 system was low to not detectable. Conclusions The scaffolding protein NoxO1 plays some role in atherosclerosis development in female mice probably by attenuating the global inflammatory burden.  

5.2870           Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution

Cowan, C.S., Renner, M., De Gennaro, M., Roma, G., Nigsch, F. and Roska, B. Cell, 182, 1623-1640 (2020)   Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable “developed” state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.  

5.2871           Protocol to Design, Clone, and Validate sgRNAs for In Vivo Reverse Genetic Studies

Hunker, A.C. and Zweifel, L.S. Star Protocols, 1, 100070 (2020)   AAV-CRISPR/Cas9 permits gene mutagenesis in the adult CNS. Current methods determining in vivo on-target mutagenesis have been limited by the ability to isolate virally transduced cells. This protocol optimizes a workflow for the design, cloning, and validation of sgRNAs delivered by AAVs in vivo that can be applied to any target gene in the CNS of rat or mouse model systems and can be adapted to Cre or Flp driver lines using AAV-FLEX-SaCas9-sgRNA or AAV-FLEXfrt-SaCas9-sgRNA, respectively.  

5.2872           Adeno-Associated Virus-Induced Dorsal Root Ganglion Pathology

Hordeaux, J., Buza, E.L., Dyer, C., Goode, T., Mitchell, T.W., Richmann, L. et al Human Gene Therapy, 31(15-16), 808-818 (2020)   The administration of adeno-associated virus (AAV) vectors to nonhuman primates (NHP) via the blood or cerebrospinal fluid (CSF) can lead to dorsal root ganglion (DRG) pathology. The pathology is minimal to moderate in most cases; clinically silent in affected animals; and characterized by mononuclear cell infiltrates, neuronal degeneration, and secondary axonopathy of central and peripheral axons on histopathological analysis. We aggregated data from 33 nonclinical studies in 256 NHP and performed a meta-analysis of the severity of DRG pathology to compare different routes of administration, dose, time course, study conduct, age of the animals, sex, capsid, promoter, capsid purification method, and transgene. DRG pathology was observed in 83% of NHP that were administered AAV through the CSF, and 32% of NHP that received an intravenous (IV) injection. We show that dose and age at injection significantly affected the severity whereas sex had no impact. DRG pathology was minimal at acute time points (i.e., <14 days), similar from one to 5 months post-injection, and was less severe after 6 months. Vector purification method had no impact, and all capsids and promoters that we tested resulted in some DRG pathology. The data presented here from five different capsids, five different promoters, and 20 different transgenes suggest that DRG pathology is almost universal after AAV gene therapy in nonclinical studies using NHP. None of the animals receiving a therapeutic transgene displayed any clinical signs. Incorporation of sensitive techniques such as nerve-conduction velocity testing can show alterations in a minority of animals that correlate with the severity of peripheral nerve axonopathy. Monitoring sensory neuropathies in human central nervous system and high-dose IV clinical studies seems prudent to determine the functional consequences of DRG pathology.  

5.2873           Stress-Induced Mouse Model of the Cardiac Manifestations of Friedreich's Ataxia Corrected by AAV-mediated Gene Therapy

Salami, CV.O., Jackson, K., Jose, C., Alyass, L., Cisse, G-l., De, B.P., Stiles, K.M., Chiuchiolo, M.J., Sondhi, D., Crystal, R.G. and Kaminsky, S.M. Human Gene Therapy, 31(15-16), 819-827 (2020)   Friedreich's ataxia (FA), an autosomal recessive disorder caused by a deficiency in the expression of frataxin (FXN), is characterized by progressive ataxia and hypertrophic cardiomyopathy. Although cardiac dysfunction is the most common cause of mortality in FA, the cardiac disease remains subclinical for most of the clinical course because the neurologic disease limits muscle oxygen demands. Previous FXN knockout mouse models exhibit fatal cardiomyopathy similar to human FA, but in contrast to the human condition, untreated mice become moribund by 2 months of age, unlike humans where the cardiac disease often does not manifest until the third decade. The study was designed to create a mouse model for early FA disease relevant to the time for which a gene therapy would likely be most effective. To generate a cardiac-specific mouse model of FA cardiomyopathy similar to the human disease, we used a cardiac promoter (αMyhc) driving CRE recombinase cardiac-specific excision of FXN exon 4 to generate a mild, cardiac-specific FA model that is normal at rest, but exhibits the cardiac phenotype with stress. The hearts of αMyhc mice had decreased levels of FXN and activity of the mitochondrial complex II/complex IV respiratory chain. At rest, αMyhc mice exhibited normal cardiac function as assessed by echocardiographic assessment of ejection fraction and fractional shortening, but when the heart was stressed chemically with dobutamine, αMyhc mice compared with littermate control mice had a 62% reduction in the stress ejection fraction (p < 2 × 10⁻⁴) and 71% reduction in stress-related fractional shortening (p < 10⁻⁵). When assessing functional cardiac performance using running on an inclined treadmill, αMyhc mice stayed above the midline threefold less than littermate controls (p < 0.02). A one-time intravenous administration of 10¹¹ genome copies of AAVrh.10hFXN, an adeno-associated virus (AAV) serotype rh10 gene transfer vector expressing human FXN, corrected the stress-induced ejection fraction and fractional shortening phenotypes. Treated αMyhc mice exhibited exercise performance on a treadmill indistinguishable from littermate controls (p > 0.07). These αMyhc mice provide an ideal model to study long-term cardiac complications due to FA and AAV-mediated gene therapy correction of stress-induced cardiac phenotypes typical of human FA.  

5.2874           Defining Transcription Regulatory Elements in the Human Frataxin Gene: Implications for Gene Therapy

Li, J., Wang, J., Gonzalez, T.J., Asokan, A., Napierala, J.S. and Napierala, M. Human Gene Therapy, 31(15-16), 839-851 (2020)   Friedreich's ataxia (FRDA) is the most common inherited form of ataxia in humans. It is caused by severe downregulation of frataxin (FXN) expression instigated by hyperexpansion of the GAA repeats located in intron 1 of the FXN gene. Despite numerous studies focused on identifying compounds capable of stimulating FXN expression, current knowledge regarding cis-regulatory elements involved in FXN gene expression is lacking. Using a combination of episomal and genome-integrated constructs, we defined a minimal endogenous promoter sequence required to efficiently drive FXN expression in human cells. We generated 19 constructs varying in length of the DNA sequences upstream and downstream of the ATG start codon. Using transient transfection, we evaluated the capability of these constructs to drive FXN expression. These analyses allowed us to identify a region of the gene indispensable for FXN expression. Subsequently, selected constructs containing the FXN expression control regions of varying lengths were site specifically integrated into the genome of HEK293T and human-induced pluripotent stem cells (iPSCs). FXN expression was detected in iPSCs and persisted after differentiation to neuronal and cardiac cells, indicating lineage independent function of defined regulatory DNA sequences. Finally, based on these results, we generated AAV encoding miniFXN genes and demonstrated in vivo FXN expression in mice. Results of these studies identified FXN sequences necessary to express FXN in human and mouse cells and provided rationale for potential use of endogenous FXN sequence in gene therapy strategies for FRDA.  

5.2875           Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing

Tran, M.T.N., Khalid, M.K.N.M., Wang, Q., Walker, J.K.R., Lidgerwood, G.E., Dilworth, K.L., Lisowski, L., Pebay, A. and Hewitt, A.W. Nature Communications, 11:4871 (2020)   Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.  

5.2876           Myelin replacement triggered by single-cell demyelination in mouse cortex

Snaidero, N., Schifferer, M., Mezydlo, A., Zalc, B., Kerschensteiner, M. and Misgeld, T. Nature Communications, 11:4901 (2020)   Myelin, rather than being a static insulator of axons, is emerging as an active participant in circuit plasticity. This requires precise regulation of oligodendrocyte numbers and myelination patterns. Here, by devising a laser ablation approach of single oligodendrocytes, followed by in vivo imaging and correlated ultrastructural reconstructions, we report that in mouse cortex demyelination as subtle as the loss of a single oligodendrocyte can trigger robust cell replacement and remyelination timed by myelin breakdown. This results in reliable reestablishment of the original myelin pattern along continuously myelinated axons, while in parallel, patchy isolated internodes emerge on previously unmyelinated axons. Therefore, in mammalian cortex, internodes along partially myelinated cortical axons are typically not reestablished, suggesting that the cues that guide patchy myelination are not preserved through cycles of de- and remyelination. In contrast, myelin sheaths forming continuous patterns show remarkable homeostatic resilience and remyelinate with single axon precision.  

5.2877           Anti-Retroviral Protease Inhibitors Regulate Human Papillomavirus 16 Infection of Primary Oral and Cervical Epithelium

Alam, S., Chatterjee, S., Kang, S.D., Milici, J., Biryukov, J., Chen, H. and Meyers, C. Cancers, 12, 2664 (2020)   Epidemiology studies suggest that Human Immunodeficiency Virus (HIV)-infected patients on highly active anti-retroviral therapy (HAART) may be at increased risk of acquiring opportunistic Human Papillomavirus (HPV) infections and developing oral and cervical cancers. Effective HAART usage has improved survival but increased the risk for HPV-associated cancers. In this manuscript, we report that Protease Inhibitors (PI) treatment of three-dimensional tissues derived from primary human gingiva and cervical epithelial cells compromised cell-cell junctions within stratified epithelium and enhanced paracellular permeability of HPV16 to the basal layer for infection, culminating in de novo biosynthesis of progeny HPV16 as determined using 5-Bromo-2′-deoxyuridine (BrdU) labeling of newly synthesized genomes. We propose that HAART/PI represent a novel class of co-factors that modulate HPV infection of the target epithelium. Our in vitro tissue culture model is an important tool to study the mechanistic role of anti-retroviral drugs in promoting HPV infections in HAART-naïve primary epithelium. Changes in subsequent viral load could promote new infections, create HPV reservoirs that increase virus persistence, and increase the risk of oral and cervical cancer development in HIV-positive patients undergoing long-term HAART treatment  

5.2878           Inhibition of MicroRNA 6937 Delays Photoreceptor and Vision Loss in a Mouse Model of Retinitis Pigmentosa

Anasagasti, A., Lara-Lopez, A., Milla-Navarro, S., Escudero-Arraras, L., Rodriguez-Hidalgo, M., Zabaleta, N., Aseguinolaza, G.G., de la Villa, P. and Riuz-Ederra, J. Pharmaceutics, 12, 913 (2020)   Inherited retinal dystrophies (IRDs) are a group of rare retinal conditions, including retinitis pigmentosa (RP), caused by monogenic mutations in 1 out of more than 250 genes. Despite recent advancements in gene therapy, there is still a lack of an effective treatment for this group of retinal conditions. MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNAs that inhibit gene expression. Control of miRNAs-mediated protein expression has been described as a widely used mechanism for post-transcriptional regulation in many physiological and pathological processes in different organs, including the retina. Our main purpose was to test the hypothesis that modulation of a group of miRNAs can protect photoreceptor cells from death in the rd10 mouse model of retinitis pigmentosa. For this, we incorporated modulators of three miRNAs in adeno-associated viruses (AAVs), which were administered through sub-retinal injections. The results obtained indicate that inhibition of the miR-6937-5p slows down the visual deterioration of rd10 mice, reflected by an increased electroretinogram (ERG) wave response under scotopic conditions and significant preservation of the outer nuclear layer thickness. This work contributes to broadening our knowledge on the molecular mechanisms underlying retinitis pigmentosa and supports the development of novel therapeutic approaches for RP based on miRNA modulation.  

5.2879           Pronounced Therapeutic Benefit of a Single Bidirectional AAV Vector Administered Systemically in Sandhoff Mice

Lahey, H.G., Webber, C.J., Golebiowski, D., Izzo, C.M., Horn, E., Taghian, T., Rodriguez, P., Batista, A.R., Ellis, L.E., Hwang, M., martin, D.R., Gray-Edwards, H. and Sena-Esteves, M. Molecular Therapy, 28(10), 2150-2160 (2020)   The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are fatal lysosomal storage disorders caused by mutations in the HEXA and HEXB genes, respectively. These mutations cause dysfunction of the lysosomal enzyme β-N-acetylhexosaminidase A (HexA) and accumulation of GM2 ganglioside (GM2) with ensuing neurodegeneration, and death by 5 years of age. Until recently, the most successful therapy was achieved by intracranial co-delivery of monocistronic adeno-associated viral (AAV) vectors encoding Hex alpha and beta-subunits in animal models of SD. The blood-brain barrier crossing properties of AAV9 enables systemic gene therapy; however, the requirement of co-delivery of two monocistronic AAV vectors to overexpress the heterodimeric HexA protein has prevented the use of this approach. To address this need, we developed multiple AAV constructs encoding simultaneously HEXA and HEXB using AAV9 and AAV-PHP.B and tested their therapeutic efficacy in 4- to 6-week-old SD mice after systemic administration. Survival and biochemical outcomes revealed superiority of the AAV vector design using a bidirectional CBA promoter with equivalent dose-dependent outcomes for both capsids. AAV-treated mice performed normally in tests of motor function, CNS GM2 ganglioside levels were significantly reduced, and survival increased by >4-fold with some animals surviving past 2 years of age.  

5.2880           The E3 ubiquitin ligase HectD3 attenuates cardiac hypertrophy and inflammation in mice

Raangrez, A.Y., Borlepawar, A., Schmiedel, N., Deshpande, A., Remes, A., Kumari, M., Bernt, A., Christen, L., Helbig, A., Jungmann, A., Sossalla, S., Tholey, A., Müller, O.J., Frank, D. and Frey, N. Communications Biology, 3:562 (2020)   Myocardial inflammation has recently been recognized as a distinct feature of cardiac hypertrophy and heart failure. HectD3, a HECT domain containing E3 ubiquitin ligase has previously been investigated in the host defense against infections as well as neuroinflammation; its cardiac function however is still unknown. Here we show that HectD3 simultaneously attenuates Calcineurin-NFAT driven cardiomyocyte hypertrophy and the pro-inflammatory actions of LPS/interferon-γ via its cardiac substrates SUMO2 and Stat1, respectively. AAV9-mediated overexpression of HectD3 in mice in vivo not only reduced cardiac SUMO2/Stat1 levels and pathological hypertrophy but also largely abolished macrophage infiltration and fibrosis induced by pressure overload. Taken together, we describe a novel cardioprotective mechanism involving the ubiquitin ligase HectD3, which links anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and Stat1. In a broader perspective, these findings support the notion that cardiomyocyte growth and inflammation are more intertwined than previously anticipated.  

5.2881           Synapse type-specific proteomic dissection identifies IgSF8 as a hippocampal CA3 microcircuit organizer

Apostolo, N., Smukowski, S.N., Vanderlinden, J., Condomitti, G., Rybakin, V., ten Bos, J., Trobiani, L., Portegies, S., Vennekens, K.M., Gounko, N.V., Cormoletti, D., Wierda, K.D., Savas, J.N. and de Wit, J. Nature Communications, 11:5171 (2020)   Excitatory and inhibitory neurons are connected into microcircuits that generate circuit output. Central in the hippocampal CA3 microcircuit is the mossy fiber (MF) synapse, which provides powerful direct excitatory input and indirect feedforward inhibition to CA3 pyramidal neurons. Here, we dissect its cell-surface protein (CSP) composition to discover novel regulators of MF synaptic connectivity. Proteomic profiling of isolated MF synaptosomes uncovers a rich CSP composition, including many CSPs without synaptic function and several that are uncharacterized. Cell-surface interactome screening identifies IgSF8 as a neuronal receptor enriched in the MF pathway. Presynaptic Igsf8 deletion impairs MF synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition. Consequently, IgSF8 loss impairs excitation/inhibition balance and increases excitability of CA3 pyramidal neurons. Our results provide insight into the CSP landscape and interactome of a specific excitatory synapse and reveal IgSF8 as a critical regulator of CA3 microcircuit connectivity and function.  

5.2882           AAV9-Retro mediates efficient transduction with axon terminal absorption and blood–brain barrier transportation

Lin, K., Zhong, X., Li, L., Ying, M., yang, T., Zhang, Z., He, X. and Xu, F. Molecular Brain, 13:138 (2020)   Recombinant adeno-associated viruses (rAAVs), particularly those that permit efficient gene transfer to neurons from axonal terminals or across the blood–brain barrier, are useful vehicles for structural and functional studies of the neural circuit and for the treatment of many gene-deficient brain diseases that need to compensate for the correct genes in every cell in the whole brain. However, AAVs with these two advantages have not been reported. Here, we describe a new capsid engineering method, which exploits the combination of different capsids and aims to yield a capsid that can provide more alternative routes of administration that are more suitable for the wide-scale transduction of the central nervous system (CNS). A new AAV variant, AAV9-Retro, was developed by inserting the 10-mer peptide fragment from AAV2-Retro into the capsid of AAV9, and the biodistribution properties were evaluated in mice. By intracranial and intravenous injection in the mice, we found that AAV9-Retro can retrogradely infect projection neurons with an efficiency comparable to that of AAV2-Retro and retains the characteristic of AAV9, which can be transported across the nervous system. Our strategy provides a new tool for the manipulation of neural circuits and future preclinical and clinical treatment of some neurological and neurodegenerative disorders.  

5.2883           Capsid-Labelled HIV To Investigate the Role of Capsid during Nuclear Import and Integration

Bönisch, I.Z., Dirix, L., Lemmens, V., Borrenberghs, D., De Wit, F., Vernaillen, F., Rocha, S., Chrisst, F., Hendrix, J., Hofkens, J. and Debyser, Z.

1. Virol., 94(7), e01024-19 (2020)

  The HIV-1 capsid protein performs multiple roles in virus replication both during assembly and particle release and during virus trafficking into the nucleus. In order to decipher the roles of capsid protein during early replication, a reliable method to follow its intracellular distribution is required. To complement existing approaches to track HIV-1 capsid during early infection, we developed an HIV-1 imaging strategy, relying on viruses incorporating enhanced green fluorescent protein (eGFP)-tagged capsid (CA-eGFP) protein and mCherry-tagged integrase (IN-mCherry). Wild-type infectivity and sensitivity to inhibition by PF74 point to the functionality of CA-eGFP-containing complexes. Low numbers of CA-eGFP molecules were located inside the viral core and imported into the nucleus without significant loss in intensity. Less than 5% of particles carrying both CA-eGFP and IN-mCherry retained both labelled proteins after nuclear entry, implying a major uncoating event at the nuclear envelope dissociating IN and CA. Still, 20% of all CA-eGFP-containing complexes were detected in the nucleus. Unlike for IN-mCherry complexes, addition of the integrase inhibitor raltegravir had no effect on CA-eGFP-containing complexes, suggesting that these may be not (yet) competent for integration. Our imaging strategy offers alternative visualization of viral capsid trafficking and helps clarify its potential role during integration.  

5.2884           Coevolution of Adeno-associated Virus Capsid Antigenicity and Tropism through a Structure-Guided Approach

Havlik, L.P., Simon, K.E., Smith, J.K., Klinc, K.A., Tse, L.V., Oh, D.K., Fanous, M.M., Meganck, R.M., Mietzsch, R.M., Kleinschmidt, J., Agbandje-McKenna, M. and Asokan, A.

1. Virol., 94(19), e00976-20 (2020)

  Adeno-associated viruses (AAV) are composed of nonenveloped, icosahedral protein shells that can be adapted to package and deliver recombinant therapeutic DNA. Approaches to engineer recombinant capsids for gene therapy applications have focused on rational design or library-based approaches that can address one or two desirable attributes; however, there is an unmet need to comprehensively improve AAV vector properties. Such cannot be achieved by utilizing sequence data alone but requires harnessing the three-dimensional (3D) structural properties of AAV capsids. Here, we solve the structures of a natural AAV isolate complexed with antibodies using cryo-electron microscopy and harness this structural information to engineer AAV capsid libraries through saturation mutagenesis of different antigenic footprints. Each surface loop was evolved by infectious cycling in the presence of a helper adenovirus to yield a new AAV variant that then serves as a template for evolving the next surface loop. This stepwise process yielded a humanized AAV8 capsid (AAVhum.8) displaying nonnatural surface loops that simultaneously display tropism for human hepatocytes, increased gene transfer efficiency, and neutralizing antibody evasion. Specifically, AAVhum.8 can better evade neutralizing antisera from multiple species than AAV8. Further, AAVhum.8 displays robust transduction in a human liver xenograft mouse model with expanded tropism for both murine and human hepatocytes. This work supports the hypothesis that critical properties, such as AAV capsid antibody evasion and tropism, can be coevolved by combining rational design and library-based evolution for clinical gene therapy.  

5.2885           Neuron type-specific expression of a mutant KRAS impairs hippocampal-dependent learning and memory

Ryu, H-H., Kang, M., Hwang, K-D., Jang, H.B., Kim, S.J. and Lee, Y-S. Scientific Reports, 10:17730 (2020)   KRAS mutations are associated with rare cases of neurodevelopmental disorders that can cause intellectual disabilities. Previous studies showed that mice expressing a mutant KRAS have impaired the development and function of GABAergic inhibitory neurons, which may contribute to behavioural deficits in the mutant mice. However, the underlying cellular mechanisms and the role of excitatory neurons in these behavioural deficits in adults are not fully understood. Herein, we report that neuron type-specific expression of a constitutively active mutant KRAS^G12V in either excitatory or inhibitory neurons resulted in spatial memory deficits in adult mice. In inhibitory neurons, KRAS^G12V induced ERK activation and enhanced GABAergic synaptic transmission. Expressing KRAS^G12V in inhibitory neurons also impaired long-term potentiation in the hippocampal Shaffer-collateral pathway, which could be rescued by picrotoxin treatment. In contrast, KRAS^G12V induced ERK activation and neuronal cell death in excitatory neurons, which might have contributed to the severe behavioural deficits. Our results showed that both excitatory and inhibitory neurons are involved in mutant KRAS-associated learning deficits in adults via distinct cellular mechanisms.  

5.2886           Triose Kinase Controls the Lipogenic Potential of Fructose and Dietary Tolerance

Liu, L., Liao, Y., Chen, Y-G., Xu, S. and Fu, S. Cell Metabolism, 32, 605-618 (2020)   The surge in fructose consumption is a major factor behind the rapid rise of nonalcoholic fatty liver disease in modern society. Through flux and genetic analyses, we demonstrate that fructose is catabolized at a much higher rate than glucose, and triose kinase (TK) couples fructolysis with lipogenesis metabolically and transcriptionally. In the absence of TK, fructose oxidation is accelerated through the activation of aldehyde dehydrogenase (ALDH) and serine biosynthesis, accompanied by increased oxidative stress and fructose aversion. TK is also required by the endogenous fructolysis pathway to drive lipogenesis and hepatic triglyceride accumulation under high-fat diet and leptin-deficient conditions. Intriguingly, a nonsynonymous TK allele (rs2260655_A) segregated during human migration out of Africa behaves as TK null for its inability to rescue fructose toxicity and increase hepatic triglyceride accumulation. Therefore, we posit TK as a metabolic switch controlling the lipogenic potential of fructose and its dietary tolerance.  

5.2887           Correction of Three Prominent Mutations in Mouse and Human Models of Duchenne Muscular Dystrophy by Single-Cut Genome Editing

Min, Y-L., Chemello, F., Li, H., Rodriguez-Caycedo, C., Sanchez-Ortiz, E., Mireault, A.A., McAnally, J.R., Shelton, J.M., Zhang, Y., Bassel-Duby, R. and Olson, E.N. Molecular Therapy, 28(9), 2044-2055 (2020)   Duchenne muscular dystrophy (DMD), one of the most common neuromuscular disorders of children, is caused by the absence of dystrophin protein in striated muscle. Deletions of exons 43, 45, and 52 represent mutational “hotspot” regions in the dystrophin gene. We created three new DMD mouse models harboring deletions of exons 43, 45, and 52 to represent common DMD mutations. To optimize CRISPR-Cas9 genome editing using the single-cut strategy, we identified single guide RNAs (sgRNAs) capable of restoring dystrophin expression by inducing exon skipping and reframing. Intramuscular delivery of AAV9 encoding SpCas9 and selected sgRNAs efficiently restored dystrophin expression in these new mouse models, offering a platform for future studies of dystrophin gene correction therapies. To validate the therapeutic potential of this approach, we identified sgRNAs capable of restoring dystrophin expression by the single-cut strategy in cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) with each of these hotspot deletion mutations. We found that the potential effectiveness of individual sgRNAs in correction of DMD mutations cannot be predicted a priori, highlighting the importance of sgRNA design and testing as a prelude for applying gene editing as a therapeutic strategy for DMD.  

5.2888           Response of human melanoma cell lines to interferon-beta gene transfer mediated by a modified adenoviral vector

David, T.I.P., Cerqueira, O.L.D., Lana, M.G., Medrano, R.F.V., Hunger, A. and Strauss, B.E: Scientific Reports, 10:17893 (2020)   Since melanomas often retain wild type p53, we developed an adenoviral vector, AdRGD-PG, which provides robust transduction and transgene expression in response to p53. Previously, this vector was used for interferon-β gene transfer in mouse models of melanoma, resulting in control of tumor progression, but limited cell killing. Here, the AdRGD-PG-hIFNβ vector encoding the human interferon-β cDNA (hIFNβ) was used to transduce human melanoma cell lines SK-MEL-05 and SK-MEL-147 (both wild type p53). In vitro, cell death was induced in more than 80% of the cells and correlated with elevated annexinV staining and caspase 3/7 activity. Treatment with hIFNβ promoted cell killing in neighboring, non-transduced cells, thus revealing a bystander effect. In situ gene therapy resulted in complete inhibition of tumor progression for SK-MEL-147 when using nude mice with no evidence of hepatotoxicity. However, the response in Nod-Scid mice was less robust. For SK-MEL-05, tumor inhibition was similar in nude and Nod-Scid mice and was less efficient than seen for SK-MEL-147, indicating both cell type and host specific responses. The AdRGD-PG-hIFNβ vector provides extensive killing of human melanoma cells in vitro and a potent anti-tumor effect in vivo. This study provides a critical advance in the development of our melanoma gene therapy approach.  

5.2889           Characterization of hepatic macrophages and evaluation of inflammatory response in heme oxygenase-1 deficient mice exposed to scAAV9 vectors

Tomczyk, M., Kraszewska, I., Maka, R., Waligorska, A., Dulak, j. and Jazwa-Kusior, A. PloS One, 15(10), e240691 (2020)   Adeno-associated viral (AAV) vectors are characterised by low immunogenicity, although humoral and cellular responses may be triggered upon infection. Following systemic administration high levels of vector particles accumulate within the liver. Kupffer cells (KCs) are liver resident macrophages and an important part of the liver innate immune system. Decreased functional activity of KCs can contribute to exaggerated inflammatory response upon antigen exposure. Heme oxygenase-1 (HO-1) deficiency is associated with considerably reduced numbers of KCs. In this study we aimed to investigate the inflammatory responses in liver and to characterise two populations of hepatic macrophages in adult wild type (WT) and HO-1 knockout (KO) mice following systemic administration of one or two doses (separated by 3 months) of self-complementary (sc)AAV9 vectors. At steady state, the livers of HO-1 KO mice contained significantly higher numbers of monocyte-derived macrophages (MDMs), but significantly less KCs than their WT littermates. Three days after re-administration of scAAV9 we observed increased mRNA level of monocyte chemoattractant protein-1 (Mcp-1) in the livers of both WT and HO-1 KO mice, but the protein level and the macrophage infiltration were not affected. Three days after the 1^st and 3 days after the 2^nd vector dose the numbers of AAV genomes in the liver were comparable between both genotypes indicating similar transduction efficiency, but the percentage of transgene-expressing MDMs and KCs was higher in WT than in HO-1 KO mice. In the primary culture, KCs were able to internalize AAV9 particles without induction of TLR9-mediated immune responses, but no transgene expression was observed. In conclusion, in vivo and in vitro cultured KCs have different susceptibility to scAAV9 vectors. Regardless of the presence or absence of HO-1 and initial numbers of KCs in the liver, scAAV9 exhibits a low potential to stimulate inflammatory response at the analysed time points.  

5.2890           Combined prophylactic and therapeutic immune responses against human papillomaviruses induced by a thioredoxin-based L2-E7 nanoparticle vaccine

Zhao, X., Yang, F., Mariz, F., Osen, W., Bolchi, A., Ottonrllo, S. and Müller, M. PloS Pathogens, 16(9), e1008827 (2020)   Global burden of cervical cancer, the most common cause of mortality caused by human papillomavirus (HPV), is expected to increase during the next decade, mainly because current alternatives for HPV vaccination and cervical cancer screening programs are costly to be established in low-and-middle income countries. Recently, we described the development of the broadly protective, thermostable vaccine antigen Trx-8mer-OVX313 based on the insertion of eight different minor capsid protein L2 neutralization epitopes into a thioredoxin scaffold from the hyperthermophilic archaeon Pyrococcus furiosus and conversion of the resulting antigen into a nanoparticle format (median radius ~9 nm) upon fusion with the heptamerizing OVX313 module. Here we evaluated whether the engineered thioredoxin scaffold, in addition to humoral immune responses, can induce CD8⁺ T-cell responses upon incorporation of MHC-I-restricted epitopes. By systematically examining the contribution of individual antigen modules, we demonstrated that B-cell and T-cell epitopes can be combined into a single antigen construct without compromising either immunogenicity. While CD8⁺ T-cell epitopes had no influence on B-cell responses, the L2 polytope (8mer) and OVX313-mediated heptamerization of the final antigen significantly increased CD8⁺ T-cell responses. In a proof-of-concept experiment, we found that vaccinated mice remained tumor-free even after two consecutive tumor challenges, while unvaccinated mice developed tumors. A cost-effective, broadly protective vaccine with both prophylactic and therapeutic properties represents a promising option to overcome the challenges associated with prevention and treatment of HPV-caused diseases.  

5.2891           p120 catenin recruits HPV to γ-secretase to promote virus infection

Harwood, M.C., Dupzyk, A.J., Inoue, T., DiMaio, D. and Tsai, B. PloS Pathogens, 16(10), e1008946 (2020)   During internalization and trafficking, human papillomavirus (HPV) moves from the cell surface to the endosome where the transmembrane protease γ-secretase promotes insertion of the viral L2 capsid protein into the endosome membrane. Protrusion of L2 through the endosome membrane into the cytosol allows the recruitment of cytosolic host factors that target the virus to the Golgi en route for productive infection. How endosome-localized HPV is delivered to γ-secretase, a decisive infection step, is unclear. Here we demonstrate that cytosolic p120 catenin, likely via an unidentified transmembrane protein, interacts with HPV at early time-points during viral internalization and trafficking. In the endosome, p120 is not required for low pH-dependent disassembly of the HPV L1 capsid protein from the incoming virion. Rather, p120 is required for HPV to interact with γ-secretase–an interaction that ensures the virus is transported along a productive route. Our findings clarify an enigmatic HPV infection step and provide critical insights into HPV infection that may lead to new therapeutic strategies against HPV-induced diseases.  

5.2892           Chemically Defined, High-Density Insect Cell-Based Expression System for Scalable AAV Vector Production

Kurasawa, J.H., Park, A., Sowers, C.R., Halpin, R.A., Tovchigreshko, A., Dobson, C.L., Schmelzer, A.E., Gao, C., Wilson, S.D. and Ikeda, Y. Molecular Therapy-Methods Clin. Develop., 19, 330-340 (2020)   The recombinant adeno-associated virus (AAV) vector is one of the most utilized viral vectors in gene therapy due to its robust, long-term in vivo transgene expression and low toxicity. One major hurdle for clinical AAV applications is large-scale manufacturing. In this regard, the baculovirus-based AAV production system is highly attractive due to its scalability and predictable biosafety. Here, we describe a simple method to improve the baculovirus-based AAV production using the ExpiSf Baculovirus Expression System with a chemically defined medium for suspension culture of high-density ExpiSf9 cells. Baculovirus-infected ExpiSf9 cells produced up to 5 × 10¹¹ genome copies of highly purified AAV vectors per 1 mL of suspension culture, which is up to a 19-fold higher yield than the titers we obtained from the conventional Sf9 cell-based system. When mice were administered the same dose of AAV vectors, we saw comparable transduction efficiency and biodistributions between the vectors made in ExpiSf9 and Sf9 cells. Thus, the ExpiSf Baculovirus Expression System would support facile and scalable AAV manufacturing amenable for preclinical and clinical applications.  

5.2893           Cryo-EM structure of eastern equine encephalitis virus in complex with heparan sulfate analogues

Chen, C-L., Hasan, S.S., Klose, T., Sun, Y., Buda, G., Sun, C., Klimstra, W.B., and Rossmann, M.G. PNAS, 117(16), 8890-8899 (2020)   Eastern equine encephalitis virus (EEEV), a mosquito-borne icosahedral alphavirus found mainly in North America, causes human and equine neurotropic infections. EEEV neurovirulence is influenced by the interaction of the viral envelope protein E2 with heparan sulfate (HS) proteoglycans from the host’s plasma membrane during virus entry. Here, we present a 5.8-Å cryoelectron microscopy (cryo-EM) structure of EEEV complexed with the HS analog heparin. “Peripheral” HS binding sites were found to be associated with the base of each of the E2 glycoproteins that form the 60 quasi-threefold spikes (q3) and the 20 sites associated with the icosahedral threefold axes (i3). In addition, there is one HS site at the vertex of each q3 and i3 spike (the “axial” sites). Both the axial and peripheral sites are surrounded by basic residues, suggesting an electrostatic mechanism for HS binding. These residues are highly conserved among EEEV strains, and therefore a change in these residues might be linked to EEEV neurovirulence.  

5.2894           Kv4.1, a Key Ion Channel For Low Frequency Firing of Dentate Granule Cells, Is Crucial for Pattern Separation

Kim, K-R., Lee, S.Y., Yoon, S.H., Kim, Y., Jeong, H-J., Lee, S., Suh, Y.H., kang, J-S., Cho, H., Lee, S-H., Kim, M-H. and Ho, W-K.

1. Neurosci., 40(11), 2200-2214 (2020)

  The dentate gyrus (DG) in the hippocampus may play key roles in remembering distinct episodes through pattern separation, which may be subserved by the sparse firing properties of granule cells (GCs) in the DG. Low intrinsic excitability is characteristic of mature GCs, but ion channel mechanisms are not fully understood. Here, we investigated ionic channel mechanisms for firing frequency regulation in hippocampal GCs using male and female mice, and identified Kv4.1 as a key player. Immunofluorescence analysis showed that Kv4.1 was preferentially expressed in the DG, and its expression level determined by Western blot analysis was higher at 8-week than 3-week-old mice, suggesting a developmental regulation of Kv4.1 expression. With respect to firing frequency, GCs are categorized into two distinctive groups: low-frequency (LF) and high-frequency (HF) firing GCs. Input resistance (R_in) of most LF-GCs is lower than 200 MΩ, suggesting that LF-GCs are fully mature GCs. Kv4.1 channel inhibition by intracellular perfusion of Kv4.1 antibody increased firing rates and gain of the input-output relationship selectively in LF-GCs with no significant effect on resting membrane potential and R_in, but had no effect in HF-GCs. Importantly, mature GCs from mice depleted of Kv4.1 transcripts in the DG showed increased firing frequency, and these mice showed an impairment in contextual discrimination task. Our findings suggest that Kv4.1 expression occurring at late stage of GC maturation is essential for low excitability of DG networks and thereby contributes to pattern separation.  

5.2895           No evidence of SARS-CoV-2 transfusion transmission despite RNA detection in blood donors showing symptoms after donation

Cappy, P., Candotti, D., Sauvage, V., Lucas, Q., Boizeau, L., Gomez, J., Enouf, V., Chabli, L., Pillonel, J., Tiberghien, P., Morel, P. and Laperche, S. Blood, 136(16), 1888-1891 (2020)   Since December 2019, a novel human coronavirus, SARS-CoV-2, has emerged from China where the first cases of COVID-19 were described.^1,2  It is the third highly pathogenic coronavirus introduced in humans from animal reservoirs^3,4  and has spread worldwide, leading to an unprecedented pandemic. By August 2020, more than 20 million cases have been reported worldwide, including almost 0.8 million deaths. France has reported more than 200 000 cases and 30 000 deaths with an epidemic peak at week 14 (30 March-5 April).⁵  SARS-CoV-2 is mostly transmitted through airborne droplets,^6-8  with a reproduction number (R₀) varying between 2.5 and 3.5 before the implementation of control measures.^9-11  COVID-19 incubation is short (5.7-5.9 days)¹²  and mostly asymptomatic or with moderate symptoms, but 20% to 25% of infected individuals develop severe symptoms, some of them needing intensive care.^6,13,14  First reported data suggested the presence of SARS-CoV-2 RNAemia in patients with severe symptoms,^10,11,15  but not in infected asymptomatic individuals.^16,17  However, a Chinese study described 4 of more than 7400 blood donors who had extremely low plasma viral load: 2 remaining asymptomatic and 2 declaring fever after donation.¹⁸  Therefore, the question of transmission of SARS-CoV-2 through transfusion became relevant. In the context of the SARS-CoV-2 pandemic, we relied on the French hemovigilance network to investigate the presence of SARS-CoV-2 RNA in the plasma of blood donors reporting COVID-19–like symptoms after donation or in donations involved in recipient-initiated trace-back studies, whenever a patient develop symptoms related to COVID-19 shortly after a blood transfusion. This investigation was conducted in accordance with the Declaration of Helsinki. We also addressed the issue of the infectivity of positive samples using direct and enriched virus culture. From March 2020, blood donors were asked to report to the French National Blood Service (EFS) any SARS-CoV-2–positive reverse transcriptase-polymerase chain reaction (RT-PCR) result or COVID-19–like symptoms occurring within 15 days after donation (postdonation information [PDI]). Moreover, whenever a patient developed COVID-19–like symptoms within 14 days after a blood transfusion, involved donations were traced back. For each donation meeting 1 of these criteria, the cryopreserved plasma sample from the EFS repository biobank was initially tested for SARS-CoV-2 RNA using SARS-CoV-2 RNA R-GENE assay (BioMérieux, Craponne, France) and then with a SARS-CoV-2 real-time PCR (NRC-rtPCR) set up by the National Reference Center for respiratory viruses (Pasteur Institute).¹⁹  Positive samples were sequenced after amplification by an in-house seminested RT-PCR targeting the RdRp region (Table 1) and tested for total anti–SARS-CoV-2 antibodies using Platelia SARS-CoV-2 Total Ab (Bio-Rad, Marnes-la-Coquette, France). SARS-CoV-2 infectivity was evaluated in plasma retrieved from the plasma units using virus isolation on Vero E6 cells (2 successive 6-day cultures). To improve sensitivity, virions were concentrated by ultracentrifugation from 12 mL plasma or 10.5 mL plasma on iodixanol pad for RT-PCR and viral culture, respectively.  

5.2896           Identification of AAV serotypes for lung gene therapy in human embryonic stem cell-derived lung organoids

Meyer-Berg, H., Yang, L.Z., de Lucas, M.P., Zambrano, A., Hyde, S.C. and Gill, D.R: Stem Cell Research and Therapy, 11:448 (2020)   Gene therapy is being investigated for a range of serious lung diseases, such as cystic fibrosis and emphysema. Recombinant adeno-associated virus (rAAV) is a well-established, safe, viral vector for gene delivery with multiple naturally occurring and artificial serotypes available displaying alternate cell, tissue, and species-specific tropisms. Efficient AAV serotypes for the transduction of the conducting airways have been identified for several species; however, efficient serotypes for human lung parenchyma have not yet been identified. Here, we screened the ability of multiple AAV serotypes to transduce lung bud organoids (LBOs)—a model of human lung parenchyma generated from human embryonic stem cells. Microinjection of LBOs allowed us to model transduction from the luminal surface, similar to dosing via vector inhalation. We identified the naturally occurring rAAV2 and rAAV6 serotypes, along with synthetic rAAV6 variants, as having tropism for the human lung parenchyma. Positive staining of LBOs for surfactant proteins B and C confirmed distal lung identity and suggested the suitability of these vectors for the transduction of alveolar type II cells. Our findings establish LBOs as a new model for pulmonary gene therapy and stress the relevance of LBOs as a viral infection model of the lung parenchyma as relevant in SARS-CoV-2 research.  

5.2897           Modifiers of Adeno-Associated Virus-Mediated Gene Expression in Implication for Serotype-Universal Neutralizing Antibody Assay

Krotova, K. and Aslanidi, G. Human Gene Therapy, 31(19), 1124-1131 (2020)   Adeno-associated virus (AAV)-based gene therapy is undergoing major expansion into clinical practice, with two treatments currently being granted Food and Drug Administration (FDA) approval. However, the presence of pre-existing neutralizing antibodies (NAB) is one of the significant hurdles for the clinical application of AAV vectors that significantly limits the patient population, which benefits from the treatment. A reliable diagnostic to evaluate the patient's seropositivity is required to ensure the effectiveness of the AAV-mediated therapeutic. Here, we describe a simple method for the determination of AAV NAB activity based on our finding that Compound C makes HEK293 cell highly permissive for infection by 10 commonly used AAV serotypes.  

5.2898           Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants

Weinmann, J., Weis, S., Sippel, J., Tulalamba, W., Remes, A., El Andari, J. et al Nature Communications, 11:5432 (2020)   Adeno-associated virus (AAV) forms the basis for several commercial gene therapy products and for countless gene transfer vectors derived from natural or synthetic viral isolates that are under intense preclinical evaluation. Here, we report a versatile pipeline that enables the direct side-by-side comparison of pre-selected AAV capsids in high-throughput and in the same animal, by combining DNA/RNA barcoding with multiplexed next-generation sequencing. For validation, we create three independent libraries comprising 183 different AAV variants including widely used benchmarks and screened them in all major tissues in adult mice. Thereby, we discover a peptide-displaying AAV9 mutant called AAVMYO that exhibits superior efficiency and specificity in the musculature including skeletal muscle, heart and diaphragm following peripheral delivery, and that holds great potential for muscle gene therapy. Our comprehensive methodology is compatible with any capsids, targets and species, and will thus facilitate and accelerate the stratification of optimal AAV vectors for human gene therapy.  

5.2899           Inactivation of RNA and DNA viruses in water by copper and silver ions and their synergistic effect

Soliman, M.Y.M., Meddema, G., Bonilla, B.E., Brouns, S.J.J. and van Halem, D. Water Res. X, 9, 100077 (2020)   Cu and Ag have been used as bactericidal agents since ancient times, yet their antiviral capacity in water remains poorly understood. This study tested the effect of copper (Cu) and silver (Ag) on model RNA and DNA viruses MS2 and PhiX 174 in solution at pH 6–8. Cu caused MS2 inactivation with similar rates at pH 6 and 7 but was inert towards PhiX 174 regardless of pH. Ag inactivated both viruses, causing denaturation of MS2 and loss of capsid spikes in PhiX 174. Ag inactivation rates were pH dependent and increased with increasing pH. At pH 8, 6.5 logs of PhiX were inactivated after 3 h and 3 logs of MS2 after only 10 min. The combined use of Cu and Ag revealed synergy in disinfecting MS2 at pH ≥ 7. Although metal concentrations used were higher than the desired values for drinking water treatment, the results prove a promising potential of Cu and Ag combinations as efficient viricidal agents.  

5.2900           Orsay Virus CP-δ Adopts a Novel β-Bracelet Structural Fold and Incorporates into Virions as a Head Fiber

Guo, Y.R., Fan, Y., ZHzou, Y., Jin, M., Zhang, J.L., Jiang, H., Holt, M.V., Wang, T., Young, N.L., Wang, D., Zhong, W. and Tao, Y.J.

1. Virol., 94(21), e01560-20 (2020)

  Fiber proteins are commonly found in eukaryotic and prokaryotic viruses, where they play important roles in mediating viral attachment and host cell entry. They typically form trimeric structures and are incorporated into virions via noncovalent interactions. Orsay virus, a small RNA virus which specifically infects the laboratory model nematode Caenorhabditis elegans, encodes a fibrous protein δ that can be expressed as a free protein and as a capsid protein-δ (CP-δ) fusion protein. Free δ has previously been demonstrated to facilitate viral exit following intracellular expression; however, the biological significance and prevalence of CP-δ remained relatively unknown. Here, we demonstrate that Orsay CP-δ is covalently incorporated into infectious particles, the first example of any attached viral fibers known to date. The crystal structure of δ(1–101) (a deletion mutant containing the first 101 amino acid [aa] residues of δ) reveals a pentameric, 145-Å long fiber with an N-terminal coiled coil followed by multiple β-bracelet repeats. Electron micrographs of infectious virions depict particle-associated CP-δ fibers with dimensions similar to free δ. The δ proteins from two other nematode viruses, Le Blanc and Santeuil, which both specifically infect Caenorhabditis briggsae, were also found to form fibrous molecules. Recombinant Le Blanc δ was able to block Orsay virus infection in worm culture and vice versa, suggesting these two viruses likely compete for the same cell receptor(s). Thus, we propose that while CP-δ likely mediates host cell attachment for all three nematode viruses, additional downstream factor(s) ultimately determine the host specificity and range of each virus.  

5.2901           Fast Retrograde Access to Projection Neuron Circuits Underlying Vocal Learning in Songbirds

Düring, D.N., Dittrich, F., Rocha, M.D., Kasper, R., Gahr, M. and Hahnloser, R.H.R: Cell Reports, 33, 108364 (2020)   Understanding the structure and function of neural circuits underlying speech and language is a vital step toward better treatments for diseases of these systems. Songbirds, among the few animal orders that share with humans the ability to learn vocalizations from a conspecific, have provided many insights into the neural mechanisms of vocal development. However, research into vocal learning circuits has been hindered by a lack of tools for rapid genetic targeting of specific neuron populations to meet the quick pace of developmental learning. Here, we present a viral tool that enables fast and efficient retrograde access to projection neuron populations. In zebra finches, Bengalese finches, canaries, and mice, we demonstrate fast retrograde labeling of cortical or dopaminergic neurons. We further demonstrate the suitability of our construct for detailed morphological analysis, for in vivo imaging of calcium activity, and for multi-color brainbow labeling.  

5.2902           Restoration of Cingulate Long-Term Depression by Enhancing Non-apoptotic Caspase 3 Alleviates Peripheral Pain Hypersensitivity

Wang, Y-J., Liu, M-G., Wang, J-H., Luo, J-H., Xu, T-L. and Li, X-Y. Cell Reports, 33, 108369 (2020)   Nerve injury in somatosensory pathways may lead to neuropathic pain, which affects the life quality of ∼8% of people. Long-term enhancement of excitatory synaptic transmission along somatosensory pathways contributes to neuropathic pain. Caspase 3 (Casp3) plays a non-apoptotic role in the hippocampus and regulates internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits. Whether Casp3-AMPAR interaction is involved in the maintenance of peripheral hypersensitivity after nerve injury remained unknown. Here, we show that nerve injury suppresses long-term depression (LTD) and downregulates Casp3 in the anterior cingulate cortex (ACC). Interfering with interactions between Casp3 and AMPAR subunits or reducing Casp3 activity in the ACC suppresses LTD induction and causes peripheral hypersensitivity. Overexpression of Casp3 restores LTD and reduces peripheral hypersensitivity after nerve injury. We reveal how Casp3 is involved in the maintenance of peripheral hypersensitivity. Our findings suggest that restoration of LTD via Casp3 provides a therapeutic strategy for neuropathic pain management.  

5.2903           Viral rhodopsins 1 are an unique family of light-gated cation channels

Zabelski, D. et al Nature Communications, 11:5707 (2020)   Phytoplankton is the base of the marine food chain as well as oxygen and carbon cycles and thus plays a global role in climate and ecology. Nucleocytoplasmic Large DNA Viruses that infect phytoplankton organisms and regulate the phytoplankton dynamics encompass genes of rhodopsins of two distinct families. Here, we present a functional and structural characterization of two proteins of viral rhodopsin group 1, OLPVR1 and VirChR1. Functional analysis of VirChR1 shows that it is a highly selective, Na⁺/K⁺-conducting channel and, in contrast to known cation channelrhodopsins, it is impermeable to Ca²⁺ ions. We show that, upon illumination, VirChR1 is able to drive neural firing. The 1.4 Å resolution structure of OLPVR1 reveals remarkable differences from the known channelrhodopsins and a unique ion-conducting pathway. Thus, viral rhodopsins 1 represent a unique, large group of light-gated channels (viral channelrhodopsins, VirChR1s). In nature, VirChR1s likely mediate phototaxis of algae enhancing the host anabolic processes to support virus reproduction, and therefore, might play a major role in global phytoplankton dynamics. Moreover, VirChR1s have unique potential for optogenetics as they lack possibly noxious Ca²⁺ permeability.  

5.2904           In Situ Detection of Adeno-associated Viral Vector Genomes with SABER-FISH

Wang, S.K., Lapan, S.W., Hong, C.M., Krause, T.B. and Cepko, C.L. Molecular Therapy-Methods & Clin. Develop., 19, 376-386 (2020)   Gene therapy with recombinant adeno-associated viral (AAV) vectors is a promising modality for the treatment of a variety of human diseases. Nonetheless, there remain significant gaps in our understanding of AAV vector biology, due in part to the lack of robust methods to track AAV capsids and genomes. In this study, we describe a novel application of signal amplification by exchange reaction fluorescence in situ hybridization (SABER-FISH) that enabled the visualization and quantification of individual AAV genomes after vector administration in mice. These genomes could be seen in retinal cells within 3 h of subretinal AAV delivery, were roughly full length, and correlated with vector expression in both photoreceptors and the retinal pigment epithelium. SABER-FISH readily detected AAV genomes in the liver and muscle following retro-orbital and intramuscular AAV injections, respectively, demonstrating its utility in different tissues. Using SABER-FISH, we also found that retinal microglia, a cell type deemed refractory to AAV transduction, are in fact efficiently infected by multiple AAV serotypes, but appear to degrade AAV genomes prior to nuclear localization. Our findings show that SABER-FISH can be used to visualize AAV genomes in situ, allowing for studies of AAV vector biology and the tracking of transduced cells following vector administration.  

5.2905           Oncolytic Rhabdovirus Vaccine Boosts Chimeric Anti-DEC205 Priming for Effective Cancer Immunotherapy

Tzelepis, F., Birdi, H.K., Jirovec, A., Boscardin, S., de Souza, C.T., Hooshyar, M., Chen, A., Sutherland, K., Parks, R.J., Werier, J. and Diallo, J-S. Molecular Therapy-Oncolytics, 19, 240-252 (2020)   Prime-boost vaccination employing heterologous viral vectors encoding an antigen is an effective strategy to maximize the antigen-specific immune response. Replication-deficient adenovirus serotype 5 (Ad5) is currently being evaluated clinically in North America as a prime in conjunction with oncolytic rhabdovirus Maraba virus (MG1) as a boost. The use of an oncolytic rhabdovirus encoding a tumor antigen elicits a robust anti-cancer immune response and extends survival in murine models of cancer. Given the prevalence of pre-existing immunity to Ad5 globally, we explored the potential use of DEC205-targeted antibodies as an alternative agent to prime antigen-specific responses ahead of boosting with an oncolytic rhabdovirus expressing the same antigen. We found that a prime-boost vaccination strategy, consisting of an anti-DEC205 antibody fused to the model antigen ovalbumin (OVA) as a prime and oncolytic rhabdovirus-OVA as a boost, led to the formation of a robust antigen-specific immune response and improved survival in a B16-OVA tumor model. Overall, our study shows that anti-DEC205 antibodies fused to cancer antigens are effective to prime oncolytic rhabdovirus-boosted cancer antigen responses and may provide an alternative for patients with pre-existing immunity to Ad5 in humans.  

5.2906           Constructing and evaluating caspase-activatable adeno-associated virus vector for gene delivery to the injured heart

Brun, M.J., Song, K., Kang, B., Lueck, C., Chen, W., Thatcher, K., Gao, E., Koch, W.J., Lincoln, J., Rajan, S and Suh, J.

1. Controlled Release, 328, 834-845 (2020)

  Adeno-associated virus (AAV) is a promising vector for gene therapy, but its broad tropism can be detrimental if the transgene being delivered is harmful when expressed ubiquitously in the body, i.e. in non-target tissues. Delivering the transgene of interest to target cells at levels high enough to be therapeutically effective while maintaining safety by minimizing delivery to off-target cells is a prevalent challenge in the field of gene therapy. We have developed a protease activatable vector (provector) platform based on AAV9 that can be injected systemically to deliver therapeutic transgenes site-specifically to diseased cells by responding to extracellular proteases present at the disease site. The provector platform consists of a peptide insertion into the virus capsid which disrupts the virus' ability to bind to cell surface receptors. This peptide contains a blocking motif (aspartic acid residues) flanked on either side by cleavage sequences that are recognized by certain proteases. Exposure to proteases cleaves the peptides off the capsid, activating or “switching ON” the provector. In response to the activation, the provectors regain their ability to bind and transduce cells. Here, we have designed a provector that is activated by cysteine aspartic proteases (caspases), which have roles in inflammation and apoptosis and thus are elevated at sites of diseases such as heart failure, neurodegenerative diseases, and ischemic stroke. This provector demonstrates a 200-fold reduction in transduction ability in the OFF state compared to AAV9, reducing the virus' ability to transduce off-target healthy tissue. Following exposure to and proteolysis by caspase-3, the provector shows a 95-fold increase in transduction compared to the OFF state. The switchable transduction behavior was found to be a direct result of the peptide insertion ablating the ability of the virus to bind to cells. In vivo studies were conducted to characterize the biodistribution, blood circulation time, neutralizing antibody formation, and targeted delivery ability of the caspase-activatable provector in a model of heart failure.  

5.2907           Modification of a Constitutive to Glucose-Responsive Liver-Specific Promoter Resulted in Increased Efficacy of Adeno-Associated Virus Serotype 8-Insulin Gene Therapy of Diabetic Mice

Sia, K.C., Fu, Z.Y., Calne, R.Y., Nathwani, A.C., Lee, K.O. and Gan, S.U. Cells, 9:2474 (2020)   We have previously used a hepatotropic adeno-associated viral (AAV) vector with a modified human insulin gene to treat diabetic mice. The HLP (hybrid liver-specific promoter) used was constitutively active and non-responsive to glucose. In this study, we examined the effects of addition of glucose responsive elements (R3G) and incorporation of a 3′ albumin enhancer (3′iALB) on insulin expression. In comparison with the original promoter, glucose responsiveness was only observed in the modified promoters in vitro with a 36 h lag time before the peak expression. A 50% decrease in the number of viral particles at 5 × 10⁹ vector genome (vg)/mouse was required by AAV8-R3GHLP-hINSco to reduce the blood sugar level to near normoglycemia when compared to the original AAV8-HLP-hINSco that needed 1 × 10¹⁰ vg/mouse. The further inclusion of an 860 base-pairs 3′iALB enhancer component in the 3′ untranslated region increased the in vitro gene expression significantly but this increase was not observed when the packaged virus was systemically injected in vivo. The addition of R3G to the HLP promoter in the AAV8-human insulin vector increased the insulin expression and secretion, thereby lowering the required dosage for basal insulin treatment. This in turn reduces the risk of liver toxicity and cost of vector production.  

5.2908           Evaluation of novel HIV vaccine candidates using recombinant vesicular stomatitis virus vector produced in serum-free Vero cell cultures

Mangion, M., Gelinas, J-F., Gashti, A.B.Z., Azizi, H., Kiesslich, S., Nassoury, N., Chahal, P.S., Kobinger, G., Gilbert, R., garnier, A., Gaillet, B.and Kamen, A. Vaccine, 38, 7949-7955 (2020)   Acquired Immune Deficiency Syndrome (AIDS) in humans is a result of the destruction of the immune system caused by Human Immunodeficiency Virus (HIV) infection. This serious epidemic is still progressing world-wide. Despite advances in treatment, a safe and effective preventive HIV vaccine is desired to combat this disease, and to save millions of lives. However, such a vaccine is not available yet although extensive amounts of resources in research and development have been invested over three decades. In light of the recently approved Ebola virus disease vaccine based on a recombinant vesicular stomatitis virus (rVSV-ZEBOV), we present the results of our work on three novel VSV-vectored HIV vaccine candidates. We describe the design, rescue, production and purification method and evaluate their immunogenicity in mice prior to preclinical studies that will be performed in non-human primates. The production of each of the three candidate vaccines (rVSV-B6-NL4.3Env/SIVtm, rVSV-B6-NL4.3Env/Ebtm and rVSV-B6-A74Env(PN6)/SIVtm) was evaluated in small scale in Vero cells and it was found that production kinetics on Vero cells vary depending on the HIV gp surface protein used. Purified virus preparations complied with the WHO restrictions for the residual DNA and host cell protein contents. Finally, when administered to mice, all three rVSV-HIV vaccine candidates induced an HIV gp140-specific antibody response.    

5.2909           Long non-coding RNA Gm15441 attenuates hepatic inflammasome activation in response to PPARA agonism and fasting

Brocker, C.N., Kim, D., Melia, T., karri, K., Velenosi, T.J., Takahashi, S., Aibara, D., Bonzo, J.A., levi, M., Waxman, D.J. and Gonzales, F.J. Nature Communications, 11:5847 (2020)   Exploring the molecular mechanisms that prevent inflammation during caloric restriction may yield promising therapeutic targets. During fasting, activation of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) promotes the utilization of lipids as an energy source. Herein, we show that ligand activation of PPARα directly upregulates the long non-coding RNA gene Gm15441 through PPARα binding sites within its promoter. Gm15441 expression suppresses its antisense transcript, encoding thioredoxin interacting protein (TXNIP). This, in turn, decreases TXNIP-stimulated NLR family pyrin domain containing 3 (NLRP3) inflammasome activation, caspase-1 (CASP1) cleavage, and proinflammatory interleukin 1β (IL1B) maturation. Gm15441-null mice were developed and shown to be more susceptible to NLRP3 inflammasome activation and to exhibit elevated CASP1 and IL1B cleavage in response to PPARα agonism and fasting. These findings provide evidence for a mechanism by which PPARα attenuates hepatic inflammasome activation in response to metabolic stress through induction of lncRNA Gm15441.  

5.2910           ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

Willems, J., de Jong, A.P.H., Scheefhals, N., Mertens, E., Catsburg, L.A.E., Poorthuis, R., de Winter, F., Verhaagen, J., Meye, F.J. and MacGillavry, H.D: PloS Biology, 18(4), e3000665 (2020)   The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knock-ins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.  

5.2911           A Deoxyribonucleic Acid Decoy Trapping DUX4 for the Treatment of Facioscapulohumeral Muscular Dystrophy

Mariot, V., Joubert, R., Marsollier, A-C., Hourde, C., Voit, T. and Dumonceaux, J. Molecular Therapy-Nucleic Acids, 22, 1191-1199 (2020)   Facioscapulohumeral dystrophy (FSHD) is characterized by a loss of repressive epigenetic marks leading to the aberrant expression of the DUX4 transcription factor. In muscle, DUX4 acts as a poison protein though the induction of multiple downstream genes. So far, there is no therapeutic solution for FSHD. Because DUX4 is a transcription factor, we developed an original therapeutic approach, based on a DNA decoy trapping the DUX4 protein, preventing its binding to genomic DNA and thereby blocking the aberrant activation of DUX4’s transcriptional network. In vitro, transfection of a DUX4 decoy into FSHD myotubes reduced the expression of the DUX4 network genes. In vivo, both double-stand DNA DUX4 decoys and adeno-associated viruses (AAVs) carrying DUX4 binding sites reduced transcriptional activation of genes downstream of DUX4 in a DUX4-expressing mouse model. Our study demonstrates, both in vitro and in vivo, the feasibility of the decoy strategy and opens new avenues of research.  

5.2912           Intraarticular Adeno‐Associated Virus Serotype AAV‐PHP.S–Mediated Chemogenetic Targeting of Knee‐Innervating Dorsal Root Ganglion Neurons Alleviates Inflammatory Pain in Mice

Chakrabarti, S., Pattison, L.A., Doleschall, B., Rickman, R.H., Blake, H., Callejo, G., Hoppenstall, P.A. and St. John Smith, E. Arthritis & Rhematol., 72(10), 1749-1758 (2020)   Objective Joint pain is the major clinical symptom of arthritis that affects millions of people. Controlling the excitability of knee‐innervating dorsal root ganglion (DRG) neurons (knee neurons) could potentially provide pain relief. We undertook this study to evaluate whether the newly engineered adeno‐associated virus (AAV) serotype, AAV‐PHP.S, can deliver functional artificial receptors to control knee neuron excitability following intraarticular knee injection. Methods The AAV‐PHP.S virus, packaged with dTomato fluorescent protein and either excitatory (G_q) or inhibitory (G_i) designer receptors exclusively activated by designer drugs (DREADDs), was injected into the knee joints of adult mice. Labeling of DRG neurons with AAV‐PHP.S from the knee was evaluated using immunohistochemistry. The functionality of G_q‐ and G_i‐DREADDs was evaluated using whole‐cell patch clamp electrophysiology on acutely cultured DRG neurons. Pain behavior in mice was assessed using a digging assay, dynamic weight bearing, and rotarod performance, before and after intraperitoneal administration of the DREADD activator, Compound 21. Results We showed that AAV‐PHP.S can deliver functional genes into ~7% of lumbar DRG neurons when injected into the knee joint in a similar manner to the well‐established retrograde tracer, fast blue. Short‐term activation of AAV‐PHP.S–delivered G_q‐DREADD increased excitability of knee neurons in vitro (P = 0.02 by unpaired t‐test), without inducing overt pain in mice when activated in vivo. By contrast, in vivo G_i‐DREADD activation alleviated digging deficits induced by Freund's complete adjuvant–mediated knee inflammation (P = 0.0002 by repeated‐measures analysis of variance [ANOVA] followed by Holm‐Sidak multiple comparisons test). A concomitant decrease in knee neuron excitability was observed in vitro (P = 0.005 by ANOVA followed by Holm‐Sidak multiple comparisons test). Conclusion We describe an AAV‐mediated chemogenetic approach to specifically control joint pain, which may be utilized in translational arthritic pain research.  

5.2913           Development of Highly Efficient Dual‐AAV Split Adenosine Base Editor for In Vivo Gene Therapy

Chen, Y., Zhi, S., Liu, W., Wen, J., Hu, S., Cao, T., Sun, H., Li, Y., Liu, Y., Liang, P. and Huang, J. Small Methods, 4, 2000309 (2020)   The adenosine base editor (ABE) is able to catalyze A•T to C•G conversion efficiently and precisely in vivo, representing a new method for gene therapy. Adeno associated virus (AAV) is a well‐studied vector for gene delivery in vivo. However, due to the limited loading capacity of AAV vector (≈4800 bp), it is difficult to package ABE (≈5400 bp) into a single AAV. To tackle this problem, ABE can be split into two smaller parts through intein‐mediated protein trans‐splicing. Here, 14 different split sites of nCas9 (Cas9 nickase) in combination with three different inteins (Mxe, Npu, and Rma) are screened through a GFP‐based reporter system to identify novel split‐ABEs. After infecting HEK293T and HeLa cells with dual AAVs, two split‐ABEs (split‐ABE‐Rma573 and split‐ABE‐Rma674) that can edit the target gene efficiently are identified. Furthermore, these dual‐AAV split‐ABEs can effectively disrupt the splicing acceptor of PCSK9 in mouse liver and the splicing donor of NR2E3 in mouse retina through AI‐MAST strategy. This study provides two new split‐ABEs to investigate gene function in vivo and in gene therapy, representing a new method to treat diseases by precisely repairing point mutations or inactivating genes through the AI‐MAST strategy.  

5.2914           Antibody‐enhanced hepatitis E virus nanofiltration during the manufacture of human immunoglobulin

Kapesch, A-M., Farcet, M.R., Wieser, A., Ahmad, M.Q., Miyabayashi, T., Baylis, S.A., Blåumel, J. and Kreil, T.R. Transfusion, 60, 2500-2507 (2020)   Background Circulation of hepatitis E virus (HEV) in areas where plasma is sourced for the manufacture of plasma‐derived medicinal products (PDMPs) has prompted verification of HEV clearance. HEV exists as quasi lipid‐enveloped (LE) and non–lipid‐enveloped (NLE) forms, which might be of relevance for HEV clearance from manufacturing processes of antibody‐containing PDMPs with solvent/detergent (S/D) treatment upstream of further clearance steps. Study Design and Methods Presence of different HEV particles in stocks used in clearance studies was investigated, with nanofilters graded around the assumed HEV particle sizes and by gradient centrifugation. HEV removal by 35‐nm nanofiltration was investigated in the presence or absence of HEV antibodies, in buffer as well as in immunoglobulin (IG) manufacturing process intermediates. Results HEV particles consistent with LE, NLE, and an “intermediate” (IM) phenotype, obtained after S/D treatment, were seen in different HEV stocks. In the absence of HEV antibodies, log reduction factors (LRFs) of 4.0 and 2.5 were obtained by 35‐nm nanofiltration of LE and IM HEV, consistent with the larger and smaller sizes of these phenotypes. Addition of HEV antibodies enhanced IM HEV removal around 1000‐fold (LRF, 5.6). Effective (LRF, >4.8 and >4.0) HEV removal was obtained for the nanofiltration processing step for IG intermediates with varying HEV antibody content. Conclusion HEV spikes used in clearance studies should be carefully selected, as differences in physicochemical properties might affect HEV clearance. Antibody‐mediated enhancement of HEV nanofiltration was demonstrated in IG process intermediates even at low HEV antibody concentration, illustrating the robustness of this manufacturing step.  

5.2915           AAV‐mediated gene transfer of DNase I in the liver of mice with colorectal cancer reduces liver metastasis and restores local innate and adaptive immune response

Xia, Y., He, J., Zhang, h., Wang, H., Tetz, G., Maguire, C.A., Wang, Y., Onuma, A., Genkin, D., Tetz, V., Stepanov, A., Terekhov, S., Ukrainskaya, V., Huang, H. and Tsung, A. Mol. Oncol., 14, 2920-2936 (2020)   Liver metastasis is the main cause of colorectal cancer (CRC)‐related death. Neutrophil extracellular traps (NETs) play important roles in CRC progression. Deoxyribonuclease I (DNase I) has been shown to alter NET function by cleaving DNA strands comprising the NET backbone. Moreover, DNase I displays high antimetastatic activity in multiple tumor models. To circumvent long‐term daily administrations of recombinant DNase I, we have developed an adeno‐associated virus (AAV) gene therapy vector to specifically express DNase I in the liver. In this study, we demonstrate AAV‐mediated DNase I liver gene transfer following a single intravenous injection suppresses the development of liver metastases in a mouse model of CRC liver metastasis. Increased levels of neutrophils and NET formation in tumors are associated with poor prognosis in many patients with advanced cancers. Neutrophil infiltration and NET formation were inhibited in tumor tissues with AAV‐DNase I treatment. This approach restored local immune responses at the tumor site by increasing the percentage of CD8⁺ T cells while keeping CD4⁺ T cells similar between AAV‐DNase I and AAV‐null treatments. Our data suggest that AAV‐mediated DNase I liver gene transfer is a safe and effective modality to inhibit metastasis and represents a novel therapeutic strategy for CRC.  

5.2916           Molecular elevation of insulin receptor signaling improves memory recall in aged Fischer 344 rats

Frazier, H.N., Anderson, K.L., Ghoweri, A.O., Lin, R-L., Hawkinson, T.R., Popa, G.J., Sompol, P., Mendenhall, M.D., Norris, C.M. and Thibault, O. Aging Cell, 19, e13220 (2020)   As demonstrated by increased hippocampal insulin receptor density following learning in animal models and decreased insulin signaling, receptor density, and memory decline in aging and Alzheimer's diseases, numerous studies have emphasized the importance of insulin in learning and memory processes. This has been further supported by work showing that intranasal delivery of insulin can enhance insulin receptor signaling, alter cerebral blood flow, and improve memory recall. Additionally, inhibition of insulin receptor function or expression using molecular techniques has been associated with reduced learning. Here, we sought a different approach to increase insulin receptor activity without the need for administering the ligand. A constitutively active, modified human insulin receptor (IRβ) was delivered to the hippocampus of young (2 months) and aged (18 months) male Fischer 344 rats in vivo. The impact of increasing hippocampal insulin receptor expression was investigated using several outcome measures, including Morris water maze and ambulatory gait performance, immunofluorescence, immunohistochemistry, and Western immunoblotting. In aged animals, the IRβ construct was associated with enhanced performance on the Morris water maze task, suggesting that this receptor was able to improve memory recall. Additionally, in both age‐groups, a reduced stride length was noted in IRβ‐treated animals along with elevated hippocampal insulin receptor levels. These results provide new insights into the potential impact of increasing neuronal insulin signaling in the hippocampus of aged animals and support the efficacy of molecularly elevating insulin receptor activity in vivo in the absence of the ligand to directly study this process.  

5.2917           AAV9‐mediated AIRE gene delivery clears circulating antibodies and tissue T‐cell infiltration in a mouse model of autoimmune polyglandular syndrome type‐1

Almaghrabi, S., Azzouz, M. and Ahnini, R.T. Clin. Transl. Immunol., 99, e1166 (2020)   Objectives Autoimmune polyglandular syndrome type‐1 (APS‐1) is a monogenic recessive disorder characterised by multiple endocrine abnormalities, chronic mucocutaneous candidiasis and high titres of serum autoantibodies. To date, no curative treatment is available; current therapies manage the symptoms rather than treating the cause and have major side effects. APS‐1 is caused by mutations in the autoimmune regulator (AIRE) gene. AIRE mediates central tolerance by directing the ectopic expression of tissue‐specific antigens (TSAs) in medullary thymic epithelial cells, causing the deletion of self‐reactive thymocytes. Therefore, loss‐of‐function mutations in AIRE result in a multisystem autoimmune disease. Because of the monogenic aetiology of APS‐1 and availability of an APS‐1 mouse model, we have explored the option of restoring functional AIRE using adeno‐associated virus serotype 9 (AAV9). Methods The efficacy of AAV9‐AIRE (AAV9 carrying AIRE cDNA) gene therapy was assessed in an APS‐1 mouse model. We performed intrathymic injection of AAV9‐AIRE into APS‐1 mouse model using ultrasound imaging technique to accurately locating the thymus. We evaluated the efficiency of this approach alongside measures of autoimmunity and histology of target tissues. Results Intrathymic injection of AAV9‐AIRE demonstrated high transduction efficiency and restored AIRE expression in the thymus. AIRE gene delivery led to a significant increase in TSA expression, and importantly a significant reduction of serum autoantibodies in treated versus control mice, which fell to near‐undetectable levels by 4 weeks post‐treatment. Furthermore, histological analysis of treated animals showed near‐normal tissue morphology with no lymphocytic infiltrations, a hallmark of untreated Aire‐deficient mice. Conclusion This study has demonstrated the feasibility of AAV9‐AIRE as a vehicle for gene therapy for APS‐1.  

5.2918           Astrocytic reprogramming combined with rehabilitation strategy improves recovery from spinal cord injury

Yang, T., Xing, L., Yu, W., Cai, Y., Cui, S. and Chen, G. FASEB J., 34, 15504-15515 (2020)   After spinal cord injury (SCI), the irreversible loss of neurons and the dense glial scar are two of the leading causes of axon regeneration failure. The adult mammalian spinal cord lacks the ability to spontaneously produce new neurons, making it a key challenge to provide new neurons for spinal cord regeneration. Additionally, the dual role of the glial scar (both inhibitory and protective) makes it difficult to manipulate it for therapeutic purposes. In this study, using a single transcription factor Sry‐related HMG‐box 2 (Sox2) delivered by adeno‐associated virus (AAV), we reprogrammed some of the astrocytes targeted by the viral vectors in the glial scar into neurons in a severe SCI model. We show that this astrocytic reprogramming alone can propel axon regeneration by not only replenishing the lost neurons, but also moderately reducing the density of the glial scar without interrupting its integrity. Beyond that, astrocytic reprogramming can significantly improve functional recovery when combined with running wheel rehabilitation, which provides use‐dependent plasticity. These findings may provide us with a new idea for how to manipulate the glial scar and a promising therapeutic strategy that combines biological intervention with a rehabilitation strategy.  

5.2919           Anti-Aβ antibodies bound to neuritic plaques enhance microglia activity and mitigate tau pathology

Laversenne, V., Nazeeruddin, S., Källstig, E.C., Colin, P., Voize, C. and Schneider, B. Acta Neuropathol. Commun., 8:198 (2020)   The brain pathology of Alzheimer’s disease (AD) is characterized by the misfolding and aggregation of both the amyloid beta (Aβ) peptide and hyperphosphorylated forms of the tau protein. Initial Aβ deposition is considered to trigger a sequence of deleterious events contributing to tau pathology, neuroinflammation and ultimately causing the loss of synapses and neurons. To assess the effect of anti-Aβ immunization in this context, we generated a mouse model by overexpressing the human tau protein in the hippocampus of 5xFAD mice. Aβ plaque deposition combined with human tau overexpression leads to an array of pathological manifestations including the formation of tau-positive dystrophic neurites and accumulation of hyperphosphorylated tau at the level of neuritic plaques. Remarkably, the presence of human tau reduces microglial clustering in proximity to the Aβ plaques, which may affect the barrier role of microglia. In this mouse model, continuous administration of anti-Aβ antibodies enhances the clustering of microglial cells even in the presence of tau. Anti-Aβ immunization increases plaque compaction, reduces the spread of tau in the hippocampal formation and prevents the formation of tau-positive dystrophic neurites. However, the treatment does not significantly reduce tau-induced neurodegeneration in the dentate gyrus. These results highlight that anti-Aβ immunization is able to enhance microglial activity around neuritic plaques, mitigating part of the tau-induced pathological manifestations.  

5.2920           Distinct prefrontal top-down circuits differentially modulate sensorimotor behavior

Huda, R., Sipe, G.O., Breton-Provencher, V., Cruz, G., Pho, G.N., Adam, E., Gunter, L.M., Sullins, A., Wickersham, I.R. and Sur, M. Nature Communications, 11:6007 (2020)   Sensorimotor behaviors require processing of behaviorally relevant sensory cues and the ability to select appropriate responses from a vast behavioral repertoire. Modulation by the prefrontal cortex (PFC) is thought to be key for both processes, but the precise role of specific circuits remains unclear. We examined the sensorimotor function of anatomically distinct outputs from a subdivision of the mouse PFC, the anterior cingulate cortex (ACC). Using a visually guided two-choice behavioral paradigm with multiple cue-response mappings, we dissociated the sensory and motor response components of sensorimotor control. Projection-specific two-photon calcium imaging and optogenetic manipulations show that ACC outputs to the superior colliculus, a key midbrain structure for response selection, principally coordinate specific motor responses. Importantly, ACC outputs exert control by reducing the innate response bias of the superior colliculus. In contrast, ACC outputs to the visual cortex facilitate sensory processing of visual cues. Our results ascribe motor and sensory roles to ACC projections to the superior colliculus and the visual cortex and demonstrate for the first time a circuit motif for PFC function wherein anatomically non-overlapping output pathways coordinate complementary but distinct aspects of visual sensorimotor behavior.  

5.2921           Self-complementarity in adeno-associated virus enhances transduction and gene expression in mouse cochlear tissues

Casey, G., SAskew, C., Brimble, M.A., Samulski, R.J., Davidoff, A.M., Li, C. and Walters, B.J. PloS One, 15(11), e0242599 (2020)   Sensorineural hearing loss is one of the most common disabilities worldwide. Such prevalence necessitates effective tools for studying the molecular workings of cochlear cells. One prominent and effective vector for expressing genes of interest in research models is adeno-associated virus (AAV). However, AAV efficacy in transducing cochlear cells can vary for a number of reasons including serotype, species, and methodology, and oftentimes requires high multiplicity of infection which can damage the sensory cells. Reports in other systems suggest multiple approaches can be used to enhance AAV transduction including self-complementary vector design and pharmacological inhibition of degradation. Here we produced AAV to drive green fluorescent protein (GFP) expression in explanted neonatal mouse cochleae. Treatment with eeyarestatin I, tyrphostin 23, or lipofectamine 2000 did not result in increased transduction, however, self-complementary vector design resulted in significantly more GFP positive cells when compared to single-stranded controls. Similarly, self-complementary AAV2 vectors demonstrated enhanced transduction efficiency compared to single stranded AAV2 when injected via the posterior semicircular canal, in vivo. Self-complementary vectors for AAV1, 8, and 9 serotypes also demonstrated robust GFP transduction in cochlear cells in vivo, though these were not directly compared to single stranded vectors. These findings suggest that second-strand synthesis may be a rate limiting step in AAV transduction of cochlear tissues and that self-complementary AAV can be used to effectively target large numbers of cochlear cells in vitro and in vivo.  

5.2922           Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency

Cao, W., Dong, B., Horling, F., Firrman, J.A., Lengler, J., Klugmann, M. et al Molecular Therapy-Methods & Clin. Develop., 19, 486-495 (2020)   One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an in vitro cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A.  

5.2923           Astrocyte-derived clusterin suppresses amyloid formation in vivo

Wojtas, A.M., Sens, J.P., Kang, S.S., Baker, K.E., Berry, T.J., Kurti, A., Daughrity, L., Jansen-West, K.R., Dickson, D.W., Petrucelli, L., Bu, G., Liu, C-C. and Fryer, J.D. Mol. Neurodegeneration, 15:71 (2020)   Background Accumulation of amyloid-β (Aβ) peptide in the brain is a pathological hallmark of Alzheimer’s disease (AD). The clusterin (CLU) gene confers a risk for AD and CLU is highly upregulated in AD patients, with the common non-coding, protective CLU variants associated with increased expression. Although there is strong evidence implicating CLU in amyloid metabolism, the exact mechanism underlying the CLU involvement in AD is not fully understood or whether physiologic alterations of CLU levels in the brain would be protective. Results We used a gene delivery approach to overexpress CLU in astrocytes, the major source of CLU expression in the brain. We found that CLU overexpression resulted in a significant reduction of total and fibrillar amyloid in both cortex and hippocampus in the APP/PS1 mouse model of AD amyloidosis. CLU overexpression also ameliorated amyloid-associated neurotoxicity and gliosis. To complement these overexpression studies, we also analyzed the effects of haploinsufficiency of Clu using heterozygous (Clu^+/−) mice and control littermates in the APP/PS1 model. CLU reduction led to a substantial increase in the amyloid plaque load in both cortex and hippocampus in APP/PS1; Clu^+/− mice compared to wild-type (APP/PS1; Clu^+/+) littermate controls, with a concomitant increase in neuritic dystrophy and gliosis. Conclusions Thus, both physiologic ~ 30% overexpression or ~ 50% reduction in CLU have substantial impacts on amyloid load and associated pathologies. Our results demonstrate that CLU plays a major role in Aβ accumulation in the brain and suggest that efforts aimed at CLU upregulation via pharmacological or gene delivery approaches offer a promising therapeutic strategy to regulate amyloid pathology.  

5.2924           Clusterin ameliorates tau pathology in vivo by inhibiting fibril formation

Wojtas, A.M., Carlomagno, Y., Sens, J.P., Kang, S.S., Jensen, T.D., Kurti, A. et al Acta Neuropathol. Commun., 8:210 (2020)   The molecular chaperone Clusterin (CLU) impacts the amyloid pathway in Alzheimer’s disease (AD) but its role in tau pathology is unknown. We observed CLU co-localization with tau aggregates in AD and primary tauopathies and CLU levels were upregulated in response to tau accumulation. To further elucidate the effect of CLU on tau pathology, we utilized a gene delivery approach in CLU knock-out (CLU KO) mice to drive expression of tau bearing the P301L mutation. We found that loss of CLU was associated with exacerbated tau pathology and anxiety-like behaviors in our mouse model of tauopathy. Additionally, we found that CLU dramatically inhibited tau fibrilization using an in vitro assay. Together, these results demonstrate that CLU plays a major role in both amyloid and tau pathologies in AD.  

5.2925           A Single Injection of an Optimized Adeno-Associated Viral Vector into Cerebrospinal Fluid Corrects Neurological Disease in a Murine Model of GM1 Gangliosidosis

Hinderer, C., Nosratbakhsh, B., katz, N. and Wilson, J.M. Human Gene Therapy, 31(21), 1169-1177 (2020)   GM1 gangliosidosis is a rare neurodegenerative lysosomal storage disease caused by loss-of-function mutations in the gene encoding beta-galactosidase (β-gal). There are no approved treatments for GM1 gangliosidosis. Previous studies in animal models have demonstrated that adeno-associated viral (AAV) vector-mediated gene transfer to the brain can restore β-gal expression and prevent the onset of neurological signs. We developed an optimized AAV vector expressing human β-gal and evaluated the efficacy of a single intracerebroventricular injection of this vector into the cerebrospinal fluid (CSF) of a murine disease model. The AAV vector administration into the CSF increased β-gal activity in the brain, reduced neuronal lysosomal storage lesions, prevented the onset of neurological signs and gait abnormalities, and increased survival. These findings demonstrate the potential therapeutic activity of this vector and support its subsequent development for the treatment of GM1 gangliosidosis.  

5.2926           An Update of the Virion Proteome of Kaposi Sarcoma-Associated Herpesvirus

Nabiee, R., Syed, B., Castano, J.R., Lalani, R. and Totonchy, J.E: Viruses, 12:1382 (2020)   The virion proteins of Kaposi sarcoma-associated herpesvirus (KSHV) were initially characterized in 2005 in two separate studies that combined the detection of 24 viral proteins and a few cellular components via LC-MS/MS or MALDI-TOF. Despite considerable advances in the sensitivity and specificity of mass spectrometry instrumentation in recent years, leading to significantly higher yields in detections, the KSHV virion proteome has not been revisited. In this study, we have re-examined the protein composition of purified KSHV virions via ultra-high resolution Qq time-of-flight mass spectrometry (UHR-QqTOF). Our results confirm the detection of all previously reported virion proteins, in addition to 17 other viral proteins, some of which have been characterized as virion-associated using other methods, and 10 novel proteins identified as virion-associated for the first time in this study. These results add KSHV ORF9, ORF23, ORF35, ORF48, ORF58, ORF72/vCyclin, K3, K9/vIRF1, K10/vIRF4, and K10.5/vIRF3 to the list of KSHV proteins that can be incorporated into virions. The addition of these proteins to the KSHV virion proteome provides novel and important insight into early events in KSHV infection mediated by virion-associated proteins. Data are available via ProteomeXchange with identifier PXD022626.  

5.2927           Cellular Immune Responses in Rainbow Trout (Onchorhynchus mykiss) Following Vaccination and Challenge Against Salmonid Alphavirus (SAV)

Veenstra, K.A., Hodneland, K., Fischer, S., Takehana, K., Belmonte, R. and Fischer, U. Vaccines, 8:725 (2020)   Viral disease outbreaks remain a significant limiting factor for aquaculture. The majority of licensed vaccines used in the industry are administered as oil-adjuvanted formulations carrying inactivated whole pathogens. Cell-mediated immune responses, in particular those based on virus-specific cytotoxic T-cells (CTLs) to conventional inactivated oil-based vaccines, are largely unexplored. As vaccines cannot be optimized against viral pathogens if knowledge of host cellular immune mechanisms remains unknown, in this study we examined fundamental cell-mediated immune responses after vaccination of rainbow trout with an oil-adjuvanted inactivated vaccine against salmonid alphavirus (SAV) and after infection with SAV. A unique in vitro model system was developed to examine MHC class I restricted CTL responses in a clonal line of rainbow trout. The levels of cell-mediated cytotoxicity were compared to pathology, virus load, specific antibody response, changes in immune cell populations, and mRNA expression. Our results hint that different protective mechanisms are being triggered by infection compared to vaccination. While vaccination itself did not cause a strong cytotoxic or humoral response, subsequent challenge of vaccinated fish resulted in significantly stronger and faster specific cytotoxicity, alongside reduced viral titers and pathology. Hence, testing a vaccine on the capacity to induce cell-mediated cytotoxicity will still require a challenge test. Examination of cellular markers additionally indicates that the initial innate response induced by the vaccine could play an important role in steering adaptive mechanisms.  

5.2928           Preclinical testing of AAV9-PHP.B for transgene expression in the non-human primate cochlea

Ivanchenko, M.V., Hanlon, K.S., Devine, M.K., Tenneson, K., Emond, F., Lafond, J-F., Kenna, M.A., Corey, D.P. and maguire, C.A. Hearing Res., 394, 107930 (2020)   In a number of mouse models of hereditary deafness, therapeutic transgene delivery to the cochlea and vestibular organs using adeno-associated viral vectors (AAVs) has shown striking rescue of hearing and balance. However, only a subset of AAV capsids have shown efficacy in transducing both inner hair cells and outer hair cells, and it is also not clear which of these can be translated to treatment of human inner ear. We recently reported efficient transgene expression of a GFP reporter in a non-human primate cochlea, in both inner and outer hair cells, following injection of the AAV9 capsid variant PHP.B via the round window membrane (RWM). However efficiency was poor at a lower dose. To further define the transduction potential of AAV9-PHP.B, we have performed a dosing study in the cynomolgus monkey and assessed vector-encoded GFP expression. Three animals were injected in both ears and four doses were tested. We describe a transmastoid surgical approach needed to access the RWM of this common primate model. We found that AAV9-PHP.B transduced nearly 100% of both IHCs and OHCs, from base to apex, at the higher doses (3.5 × 10¹¹ and 7 × 10¹¹ vector genomes). However, at lower doses there was a steep reduction in viral transduction. Thus, AAV9-PHP.B efficiently transduces the IHCs and OHCs of nonhuman primates, and should be considered as an AAV capsid for inner ear gene therapy in humans.  

5.2929           Efficient viral transduction in mouse inner ear hair cells with utricle injection and AAV9-PHP.B

Lee, J., Nist-Lund, C., Solanes, P., Goldberg, H., Wu, J., Pan, B., Schneider, B.L. and Holt, J.R. Hearing Res., 394, 107882 (2020)   Viral delivery of exogenous coding sequences into the inner ear has the potential for therapeutic benefit for patients suffering genetic or acquired hearing loss. To devise improved strategies for viral delivery, we investigated two injection techniques, round window membrane injection or a novel utricle injection method, for their ability to safely and efficiently transduce sensory hair cells and neurons of the mouse inner ear. In addition, we evaluated three synthetic AAV vectors (Anc80L65, AAV9-PHP.B, AAV2.7m8) encoding enhanced green fluorescent protein (eGFP) and three promoters (Cmv, Synapsin, Gfap) for their ability to transduce and drive expression in desired cell types. We found the utricle injection method with AAV9-PHP.B and a Cmv promoter was the most efficient combination for driving robust eGFP expression in both inner and outer hair cells. We found eGFP expression levels rose over 3–5 days post-injection, a viral dose of 1.5 × 10⁹ gc yielded half maximal eGFP expression and that the utricle injection method yielded transduced hair cells even when delivered as late as postnatal day 16. Sensory transduction and auditory thresholds were unaltered in injected mice relative to uninjected wild-type controls. Vestibular end organs were also transduced without affecting balance behavior. The Synapsin promoter and the Gfap promoter drove strong eGFP expression in inner ear neurons and supporting cells, respectively. We conclude the AAV9-PHP.B vector and the utricle injection method are well-suited for delivery of exogenous gene constructs into inner ears of mouse models of auditory and vestibular dysfunction.  

5.2930           Chapter Nine - Quasi-enveloped hepatitis virus assembly and release

Feng, Z. Adv. Virus Res., 108, 315-336 (2020)   Hepatitis A virus (HAV) and hepatitis E virus (HEV) infections are the main causes for acute hepatitis worldwide. Both viruses had long been considered as nonenveloped viruses. However, recent work has uncovered that both viruses circulate in the bloodstream as membrane-cloaked, “quasi-enveloped” particles that are, surprisingly, infectious and likely the only form mediating virus spread within the host. The discovery of quasi-enveloped HAV and HEV particles has fundamentally changed the traditional view on the life cycle and pathogenesis of these viruses. However, because HAV and HEV are phylogenetically unrelated and their capsid assembly processes are quite distinct, it is not clear whether they use similar or different mechanisms for envelopment and exit. This review provides an overview of the current knowledge about the assembly and exit processes of HAV and HEV and perspectives for future studies.  

5.2931           CRISPR-Cas9-Mediated ELANE Mutation Correction in Hematopoietic Stem and Progenitor Cells to Treat Severe Congenital Neutropenia

Tran, N.T., Graf, R., Wulf-Goldenberg, A., Stecklum, M., Strauss, G., Kühn, R., Kocks, C., Rajewsky, K. and Chu, V.T. Molecular Therapy, 28(12), 2621-2634 (2020)   Severe congenital neutropenia (SCN) is a monogenic disorder. SCN patients are prone to recurrent life-threatening infections. The main causes of SCN are autosomal dominant mutations in the ELANE gene that lead to a block in neutrophil differentiation. In this study, we use CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)6 as a donor template delivery system to repair the ELANE^L172P mutation in SCN patient-derived hematopoietic stem and progenitor cells (HSPCs). We used a single guide RNA (sgRNA) specifically targeting the mutant allele, and an sgRNA targeting exon 4 of ELANE. Using the latter sgRNA, ∼34% of the known ELANE mutations can in principle be repaired. We achieved gene correction efficiencies of up to 40% (with sgELANE-ex4) and 56% (with sgELANE-L172P) in the SCN patient-derived HSPCs. Gene repair restored neutrophil differentiation in vitro and in vivo upon HSPC transplantation into humanized mice. Mature edited neutrophils expressed normal elastase levels and behaved normally in functional assays. Thus, we provide a proof of principle for using CRISPR-Cas9 to correct ELANE mutations in patient-derived HSPCs, which may translate into gene therapy for SCN.  

5.2932           A comparison of AAV-vector production methods for gene therapy and preclinical assessment

Davidsson, M., negrini, M., Hauser, S., Svanbergsson, A., Lockowandt, M., Tomasello, G., manfredsson, F.P. and Heuer, A. Scientific Reports, 10:22532 (2020)   Adeno Associated Virus (AAV)-mediated gene expression in the brain is widely applied in the preclinical setting to investigate the therapeutic potential of specific molecular targets, characterize various cellular functions, and model central nervous system (CNS) diseases. In therapeutic applications in the clinical setting, gene therapy offers several advantages over traditional pharmacological based therapies, including the ability to directly manipulate disease mechanisms, selectively target disease-afflicted regions, and achieve long-term therapeutic protein expression in the absence of repeated administration of pharmacological agents. Next to the gold-standard iodixanol-based AAV vector production, we recently published a protocol for AAV production based on chloroform-precipitation, which allows for fast in-house production of small quantities of AAV vector without the need for specialized equipment. To validate our recent protocol, we present here a direct side-by-side comparison between vectors produced with either method in a series of in vitro and in vivo assays with a focus on transgene expression, cell loss, and neuroinflammatory responses in the brain. We do not find differences in transduction efficiency nor in any other parameter in our in vivo and in vitro panel of assessment. These results suggest that our novel protocol enables most standardly equipped laboratories to produce small batches of high quality and high titer AAV vectors for their experimental needs.  

5.2933           Real-Time Conformational Dynamics of SARS-CoV-2 Spikes on Virus Particles

Lu, M., Uchil, P.D., Li, W., Kwong, P.D., Blanchard, S.C. and Mothes, W. Cell Host & Microbe, 28(6), 880-891 (2020)   The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) mediates viral entry into cells and is critical for vaccine development against coronavirus disease 2019 (COVID-19). Structural studies have revealed distinct conformations of S, but real-time information that connects these structures is lacking. Here we apply single-molecule fluorescence (Förster) resonance energy transfer (smFRET) imaging to observe conformational dynamics of S on virus particles. Virus-associated S dynamically samples at least four distinct conformational states. In response to human receptor angiotensin-converting enzyme 2 (hACE2), S opens sequentially into the hACE2-bound S conformation through at least one on-path intermediate. Conformational preferences observed upon exposure to convalescent plasma or antibodies suggest mechanisms of neutralization involving either competition with hACE2 for binding to the receptor-binding domain (RBD) or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and conformations for immunogen design.  

5.2934           Disulfide stabilization of human norovirus GI.1 virus-like particles focuses immune response toward blockade epitopes

Verardi, R., Lindesmith, L.C., Tsybovsky, Y., Gorman, J., Chaunag, G-Y., Edwards, C.E., Brewer-Jensen, P.D., Mallory, M.L., Ou, L., Schön, A., Shi, W., Tully, E.S., Goergiou, G., Baric, R.S. and Kwong, P.D. Npj Vaccines, 5:110 (2020)   Human noroviruses are non-enveloped, single-strand RNA viruses that cause pandemic outbreaks of acute gastroenteritis. A bivalent vaccine containing GI.1 and GII.4 virus-like particles (VLPs) has been shown to be safe and highly immunogenic, but its efficacy and durability have been limited. Here, we show that norovirus GI.1 VLPs are unstable and contain a substantial fraction of dissociated VLP components. Broadly reactive, non-neutralizing antibodies isolated from vaccinated donors bound to the dissociated components, but not to the intact VLPs. Engineering of interprotomer disulfide bonds within the shell domain prevented disassembly of the VLPs, while preserving antibody accessibility to blockade epitopes. Without adjuvant, mice immunized with stabilized GI.1 VLPs developed faster blockade antibody titers compared to immunization with wild-type GI.1 VLPs. In addition, immunization with stabilized particles focused immune responses toward surface-exposed epitopes and away from occluded epitopes. Overall, disulfide-stabilized norovirus GI.1 VLPs elicited improved responses over the non-disulfide-stabilized version, suggesting their promise as candidate vaccines.  

5.2935           EGFR-Binding Peptides: From Computational Design towards Tumor-Targeting of Adeno-Associated Virus Capsids

Feiner, R.C., Kemker, I., Krutzke, l., Allmendinger, E., Mandell, D.J., Sewald, n., Kochanek, S. and Müller, K.M. Int. J. Mol. Sci., 21:9535 (2020)   The epidermal growth factor receptor (EGFR) plays a central role in the progression of many solid tumors. We used this validated target to analyze the de novo design of EGFR-binding peptides and their application for the delivery of complex payloads via rational design of a viral vector. Peptides were computationally designed to interact with the EGFR dimerization interface. Two new peptides and a reference (EDA peptide) were chemically synthesized, and their binding ability characterized. Presentation of these peptides in each of the 60 capsid proteins of recombinant adeno-associated viruses (rAAV) via a genetic based loop insertion enabled targeting of EGFR overexpressing tumor cell lines. Furthermore, tissue distribution and tumor xenograft specificity were analyzed with systemic injection in chicken egg chorioallantoic membrane (CAM) assays. Complex correlations between the targeting of the synthetic peptides and the viral vectors to cells and in ovo were observed. Overall, these data demonstrate the potential of computational design in combination with rational capsid modification for viral vector targeting opening new avenues for viral vector delivery and specifically suicide gene therapy.  

5.2936           Improvement of PR8-Derived Recombinant Clade 2.3.4.4c H5N6 Vaccine Strains by Optimization of Internal Genes and H103Y Mutation of Hemagglutinin

An, S-H., Hong, S-M., Son, S-E., Song, J-H., Lee, C-Y., Choi, J-G., Lee, Y-J., Jeong, J-H., Kim, J-B., Song, C-S., Kim, J-H., Choi, K-S. and Kwon, H-J. Vaccines, 8:781 (2020)   Clade 2.3.4.4c H5N6 avian influenza A viruses (AIVs) may have originally adapted to infect chickens and have caused highly pathogenic avian influenza (HPAI) in poultry and human fatalities. Although A/Puerto Rico/8/1934 (H1N1) (PR8)-derived recombinant clade 2.3.4.4c H5N6 vaccine strains have been effective in embryonated chicken eggs-based vaccine production system, they need to be improved in terms of immunogenicity and potential mammalian pathogenicity. We replaced the PB2 gene alone or the PB2 (polymerase basic protein 2), NP (nucleoprotein), M (matrix protein) and NS (non-structural protein) genes together in the PR8 strain with corresponding genes from AIVs with low pathogenicity to remove mammalian pathogenicity and to match CD8+ T cell epitopes with contemporary HPAI viruses, respectively, without loss of viral fitness. Additionally, we tested the effect of the H103Y mutation of hemagglutinin (HA) on antigen productivity, mammalian pathogenicity and heat/acid stability. The replacement of PB2 genes and the H103Y mutation reduced the mammalian pathogenicity but increased the antigen productivity of the recombinant vaccine strains. The H103Y mutation increased heat stability but unexpectedly decreased acid stability, probably resulting in increased activation pH for HA. Interestingly, vaccination with inactivated recombinant virus with replaced NP, M and NS genes halted challenge virus shedding earlier than the recombinant vaccine without internal genes replacement. In conclusion, we successfully generated recombinant clade 2.3.4.4c H5N6 vaccine strains that were less pathogenic to mammals and more productive and heat stable than conventional PR8-derived recombinant strains by optimization of internal genes and the H103Y mutation of HA.  

5.2937           Human Antibodies Protect against Aerosolized Eastern Equine Encephalitis Virus Infection

Williamson, L.E., Gilliland Jr., T., Yadav, P.K., Ohi, M.D., Klimstra, W.B. and Crowe Jr., J.E. Cell, 183(7), 1884-1900 (2020)   Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics.  

5.2938           Liver-expressed Cd302 and Cr1l limit hepatitis C virus cross-species transmission to mice

Brown, R.J.P., Tegtmeyer, B., Sheldon, J., Khera, T., Anggakusuma, Todt, D. et al Sci. Adv., 6:eabd3233 (2020)   Hepatitis C virus (HCV) has no animal reservoir, infecting only humans. To investigate species barrier determinants limiting infection of rodents, murine liver complementary DNA library screening was performed, identifying transmembrane proteins Cd302 and Cr1l as potent restrictors of HCV propagation. Combined ectopic expression in human hepatoma cells impeded HCV uptake and cooperatively mediated transcriptional dysregulation of a noncanonical program of immunity genes. Murine hepatocyte expression of both factors was constitutive and not interferon inducible, while differences in liver expression and the ability to restrict HCV were observed between the murine orthologs and their human counterparts. Genetic ablation of endogenous Cd302 expression in human HCV entry factor transgenic mice increased hepatocyte permissiveness for an adapted HCV strain and dysregulated expression of metabolic process and host defense genes. These findings highlight human-mouse differences in liver-intrinsic antiviral immunity and facilitate the development of next-generation murine models for preclinical testing of HCV vaccine candidates.  

5.2939           Novel self-replicating α-synuclein polymorphs that escape ThT monitoring can spontaneously emerge and acutely spread in neurons

DeGiorgi, F., Laferriere, F., Zinghirino, F., Faggiani, E., Lends, A. et al Sci. Adv., 6:eabc4364 (2020)   The conformational strain diversity characterizing α-synuclein (α-syn) amyloid fibrils is thought to determine the different clinical presentations of neurodegenerative diseases underpinned by a synucleinopathy. Experimentally, various α-syn fibril polymorphs have been obtained from distinct fibrillization conditions by altering the medium constituents and were selected by amyloid monitoring using the probe thioflavin T (ThT). We report that, concurrent with classical ThT-positive products, fibrillization in saline also gives rise to polymorphs invisible to ThT (τ⁻). The generation of τ⁻ fibril polymorphs is stochastic and can skew the apparent fibrillization kinetics revealed by ThT. Their emergence has thus been ignored so far or mistaken for fibrillization inhibitions/failures. They present a yet undescribed atomic organization and show an exacerbated propensity toward self-replication in cortical neurons, and in living mice, their injection into the substantia nigra pars compacta triggers a synucleinopathy that spreads toward the dorsal striatum, the nucleus accumbens, and the insular cortex.  

5.2940           Astrocytes and microglia play orchestrated roles and respect phagocytic territories during neuronal corpse removal in vivo

Damisah, E.C., Hill, R.A., Rai, A., Chen, F., Rothlin, C.V., Ghosh, S. and Grutzendler, J. Sci. Adv., 6:eaba3239 (2020)   Cell death is prevalent throughout life; however, the coordinated interactions and roles of phagocytes during corpse removal in the live brain are poorly understood. We developed photochemical and viral methodologies to induce death in single cells and combined this with intravital optical imaging. This approach allowed us to track multicellular phagocytic interactions with precise spatiotemporal resolution. Astrocytes and microglia engaged with dying neurons in an orchestrated and synchronized fashion. Each glial cell played specialized roles: Astrocyte processes rapidly polarized and engulfed numerous small dendritic apoptotic bodies, while microglia migrated and engulfed the soma and apical dendrites. The relative involvement and phagocytic specialization of each glial cell was plastic and controlled by the receptor tyrosine kinase Mertk. In aging, there was a marked delay in apoptotic cell removal. Thus, a precisely orchestrated response and cross-talk between glial cells during corpse removal may be critical for maintaining brain homeostasis.  

5.2941           Neuronal metabolic rewiring promotes resilience to neurodegeneration caused by mitochondrial dysfunction

Motori, E., Atanassov, I., Kochan, S.M.V., Folz-Donahue, K., Sakthivelu, V., Giavalisco, P., Toni, N., Puyal, J. and Larsson, N.G. Sci. Adv., 6:eaba8271 (2020)   Neurodegeneration in mitochondrial disorders is considered irreversible because of limited metabolic plasticity in neurons, yet the cell-autonomous implications of mitochondrial dysfunction for neuronal metabolism in vivo are poorly understood. Here, we profiled the cell-specific proteome of Purkinje neurons undergoing progressive OXPHOS deficiency caused by disrupted mitochondrial fusion dynamics. We found that mitochondrial dysfunction triggers a profound rewiring of the proteomic landscape, culminating in the sequential activation of precise metabolic programs preceding cell death. Unexpectedly, we identified a marked induction of pyruvate carboxylase (PCx) and other anaplerotic enzymes involved in replenishing tricarboxylic acid cycle intermediates. Suppression of PCx aggravated oxidative stress and neurodegeneration, showing that anaplerosis is protective in OXPHOS-deficient neurons. Restoration of mitochondrial fusion in end-stage degenerating neurons fully reversed these metabolic hallmarks, thereby preventing cell death. Our findings identify a previously unappreciated pathway conferring resilience to mitochondrial dysfunction and show that neurodegeneration can be reversed even at advanced disease stages.  

5.2942           Dynamic Fas signaling network regulates neural stem cell proliferation and memory enhancement

Kim, S., Kim, N., Lee, J., Kim, S., Hong, J., Son, S. and Heo, W.D. Sci. Adv., 6:eaaz9691 (2020)   Activation of Fas (CD95) is observed in various neurological disorders and can lead to both apoptosis and prosurvival outputs, yet how Fas signaling operates dynamically in the hippocampus is poorly understood. The optogenetic dissection of a signaling network can yield molecular-level explanations for cellular responses or fates, including the signaling dysfunctions seen in numerous diseases. Here, we developed an optogenetically activatable Fas that works in a physiologically plausible manner. Fas activation in immature neurons of the dentate gyrus triggered mammalian target of rapamycin (mTOR) activation and subsequent brain-derived neurotrophic factor secretion. Phosphorylation of extracellular signal–regulated kinase (Erk) in neural stem cells was induced under prolonged Fas activation. Repetitive activation of this signaling network yielded proliferation of neural stem cells and a transient increase in spatial working memory in mice. Our results demonstrate a novel Fas signaling network in the dentate gyrus and illuminate its consequences for adult neurogenesis and memory enhancement.  

5.2943           Knockdown of lactate dehydrogenase by adeno‐associated virus‐delivered CRISPR/Cas9 system alleviates primary hyperoxaluria type 1

Zheng, R., Fang, X., Chen, X., Huang, Y., Xu, G., He, L., Li, Y., Niu, X., Yang, l., Wang, L., Li, D. and Geng, H. Clin. Transl. Med., 10, e261 (2020)   Background Primary hyperoxaluria type 1 (PH1) is a rare genetic disorder caused by endogenous overproduction of hepatic oxalate, leading to hyperoxaluria, recurrent calcium oxalate kidney stones, and end‐stage renal disease. Lactate dehydrogenase (LDH) is an ideal target for diminishing oxalate production as it is responsible for glyoxylate to oxalate conversion in the liver, the last step of oxalate metabolism. Here, we investigated the therapeutic efficacy and potential side effects of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to ameliorate PH1 via specifically disrupting the hepatic LDH. Methods Pheochromocytoma (PC12) cells were used to assess the efficacy of cleavage of single‐guide RNAs in vitro. PH1 neonatal rats were injected with a single administration of adeno‐associated virus to deliver the CRISPR/Cas9 system that targeted LDH. Three weeks after injection, a liver biopsy was performed to detect LDH expression, liver injury, and liver metabolomics. Urinary oxalate was regularly monitored, and renal calcium oxalate deposition was evaluated after 4 weeks of 0.5% ethylene glycol challenge. After 6 months of treatment, animals were euthanized, and ex‐liver organs were harvested for toxicity analysis. Results The Ldha gene was specifically knocked out in 20% of the liver cells of PH1 rats in the treatment group, leading to a 50% lower LDH expression than that in the control group. Compared to the control groups, urinary oxalate levels were significantly decreased, and renal calcium oxalate precipitation was largely mitigated in the treatment group throughout the entire 6‐month study period. While no CRISPR/Cas9‐associated off‐target edits or hepatotoxicity were detected, we observed mild metabolic changes in the liver tricarboxylic acid (TCA) and glycolysis pathways. Conclusions CRISPR/Cas9‐mediated LDH disruption may represent an applicable new strategy for alleviating PH1 for its long‐lasting effect and low editorial efficiency requirements.  

5.2944           Striatal Nurr1 Facilitates the Dyskinetic State and Exacerbates Levodopa-Induced Dyskinesia in a Rat Model of Parkinson's Disease

Sellnov, R., Steece-Collier, K., Altwal, F., Dandoval, M.S., Kordower, J.H., Collier, T.J., Sortwell, C.E., West, A.R. and manfredsson, F.P.

1. Neurosci., 40(18), 3691-3675 (2020)

  The transcription factor Nurr1 has been identified to be ectopically induced in the striatum of rodents expressing l-DOPA-induced dyskinesia (LID). In the present study, we sought to characterize Nurr1 as a causative factor in LID expression. We used rAAV2/5 to overexpress Nurr1 or GFP in the parkinsonian striatum of LID-resistant Lewis or LID-prone Fischer-344 (F344) male rats. In a second cohort, rats received the Nurr1 agonist amodiaquine (AQ) together with l-DOPA or ropinirole. All rats received a chronic DA agonist and were evaluated for LID severity. Finally, we performed single-unit recordings and dendritic spine analyses on striatal medium spiny neurons (MSNs) in drug-naïve rAAV-injected male parkinsonian rats. rAAV-GFP injected LID-resistant hemi-parkinsonian Lewis rats displayed mild LID and no induction of striatal Nurr1 despite receiving a high dose of l-DOPA. However, Lewis rats overexpressing Nurr1 developed severe LID. Nurr1 agonism with AQ exacerbated LID in F344 rats. We additionally determined that in l-DOPA-naïve rats striatal rAAV-Nurr1 overexpression (1) increased cortically-evoked firing in a subpopulation of identified striatonigral MSNs, and (2) altered spine density and thin-spine morphology on striatal MSNs; both phenomena mimicking changes seen in dyskinetic rats. Finally, we provide postmortem evidence of Nurr1 expression in striatal neurons of l-DOPA-treated PD patients. Our data demonstrate that ectopic induction of striatal Nurr1 is capable of inducing LID behavior and associated neuropathology, even in resistant subjects. These data support a direct role of Nurr1 in aberrant neuronal plasticity and LID induction, providing a potential novel target for therapeutic development.  

5.2945           Cell Type-Specific Oxidative Stress Genomic Signatures in the Globus Pallidus of Dopamine-Depleted Mice

Lawler, A.J., Brown, A.R., Bouchard, R.S., Toong, N., Kim, Y., Velraj, N., Fox, G., Kleyman, M., kang, B., Gittis, A.H. and Pfenning, A.R.

1. Neurosci., 40(50), 9772-9783 (2020)

  Neuron subtype dysfunction is a key contributor to neurologic disease circuits, but identifying associated gene regulatory pathways is complicated by the molecular complexity of the brain. For example, parvalbumin-expressing (PV⁺) neurons in the external globus pallidus (GPe) are critically involved in the motor deficits of dopamine-depleted mouse models of Parkinson's disease, where cell type-specific optogenetic stimulation of PV⁺ neurons over other neuron populations rescues locomotion. Despite the distinct roles these cell types play in the neural circuit, the molecular correlates remain unknown because of the difficulty of isolating rare neuron subtypes. To address this issue, we developed a new viral affinity purification strategy, Cre-Specific Nuclear Anchored Independent Labeling, to isolate Cre recombinase-expressing (Cre⁺) nuclei from the adult mouse brain. Applying this technology, we performed targeted assessments of the cell type-specific transcriptomic and epigenetic effects of dopamine depletion on PV⁺ and PV^– cells within three brain regions of male and female mice: GPe, striatum, and cortex. We found GPe PV⁺ neuron-specific gene expression changes that suggested increased hypoxia-inducible factor 2α signaling. Consistent with transcriptomic data, regions of open chromatin affected by dopamine depletion within GPe PV⁺ neurons were enriched for hypoxia-inducible factor family binding motifs. The gene expression and epigenomic experiments performed on PV⁺ neurons isolated by Cre-Specific Nuclear Anchored Independent Labeling identified a transcriptional regulatory network mediated by the neuroprotective factor Hif2a as underlying neural circuit differences in response to dopamine depletion.  

5.2946           Therapeutic Breast Reconstruction Using Gene Therapy–Delivered IFNγ Immunotherapy

Davis, C.R., Than, P.A., Khong, S.M.L., Rodrigues, M., Findlay, M.W., Navarrete, D.J., Ghali, S., Vaidya, J.S. and Gurtner, G.C: Mol. Cancer Ther., 19, 697-705 (2020)   After mastectomy, breast reconstruction is increasingly performed using autologous tissue with the aim of improving quality of life. During this procedure, autologous tissue is excised, relocated, and reattached using microvascular anastomoses at the site of the extirpated breast. The period during which the tissue is ex vivo may allow genetic modification without any systemic exposure to the vector. Could such access permit delivery of therapeutic agents using the tissue flap as a vehicle? Such delivery may be more targeted and oncologically efficient than systemic therapy, and avoid systemic complications. The cytokine IFNγ has antitumor effects, and systemic toxicity could be circumvented by localized delivery of the IFNγ gene via gene therapy to autologous tissue used for breast reconstruction, which then releases IFNγ and exerts antitumor effects. In a rat model of loco-regional recurrence (LRR) with MADB-106-Luc and MAD-MB-231-Luc breast cancer cells, autologous tissue was transduced ex vivo with an adeno-associated viral vector encoding IFNγ. The “Therapeutic Reconstruction” released IFNγ at the LRR site and eliminated cancer cells, significantly decreased tumor burden, and increased survival compared with sham reconstruction (P <0.05). Mechanistically, localized IFNγ immunotherapy stimulated M1 macrophages to target cancer cells within the regional confines of the modified tumor environment. This concept of “Therapeutic Breast Reconstruction” using ex vivo gene therapy of autologous tissue offers a new application for immunotherapy in breast cancer with a dual therapeutic effect of both reconstructing the ablative defect and delivering local adjuvant immunotherapy.  

5.2947           Ubqln4 Facilitates Endoplasmic Reticulum-to-Cytosol Escape of a Nonenveloped Virus during Infection

Liu, X. and Tsai, B.

1. Virol., 94(11), e00103-20 (2020)

  The nonenveloped polyomavirus simian virus 40 (SV40) must penetrate the host endoplasmic reticulum (ER) membrane to enter the cytosol in order to promote infection. How this is accomplished is not entirely clear. Here, we demonstrate that the cytosolic chaperone Ubiquilin4 (Ubqln4) binds directly to the ER membrane J proteins B12 and B14. Strategically localized at the ER-cytosol interface, Ubqln4 captures SV40 emerging from the ER, thereby facilitating escape of the virus from the ER into the cytosol, which leads to infection. Strikingly, Ubqln4 engages the J proteins in a J-domain-independent manner, in contrast to the previously reported Hsc70-Hsp105-SGTA-Bag2 cytosolic complex that also mediates SV40 ER-to-cytosol transport. Our results also reveal that the H domain and STI1 motif (1-2) of Ubqln4 support J protein binding, essential for SV40 infection. Together, these data further clarify the molecular basis by which a nonenveloped virus escapes a host membrane during infectious entry.  

5.2948           Impact of Natural or Synthetic Singletons in the Capsid of Human Bocavirus 1 on Particle Infectivity and Immunoreactivity

Fakhiri, J., Linse, K-P., Mietzsch, M., Xu, M., Schneider, M.A., Meister, M., Schildgen, O., Schnitzler, P., Soderlund-Venermo, M., Agbandje-McKenna and Grimm, D.

1. Virol., 94(11), e00170-20 (2020)

  Human bocavirus 1 (HBoV1) is a parvovirus that gathers increasing attention due to its pleiotropic role as a pathogen and emerging vector for human gene therapy. Curiously, albeit a large variety of HBoV1 capsid variants has been isolated from human samples, only one has been studied as a gene transfer vector to date. Here, we analyzed a cohort of HBoV1-positive samples and managed to PCR amplify and sequence 29 distinct HBoV1 capsid variants. These differed from the originally reported HBoV1 reference strain in 32 nucleotides or four amino acids, including a frequent change of threonine to serine at position 590. Interestingly, this T590S mutation was associated with lower viral loads in infected patients. Analysis of the time course of infection in two patients for up to 15 weeks revealed a gradual accumulation of T590S, concurrent with drops in viral loads. Surprisingly, in a recombinant vector context, T590S was beneficial and significantly increased titers compared to that of T590 variants but had no major impact on their transduction ability or immunoreactivity. Additional targeted mutations in the HBoV1 capsid identified several residues that are critical for transduction, capsid assembly, or DNA packaging. Our new findings on the phylogeny, infectivity, and immunoreactivity of HBoV1 capsid variants improve our understanding of bocaviral biology and suggest strategies to enhance HBoV1 gene transfer vectors.  

5.2949           Insights into the Role of Innate Immunity in Cervicovaginal Papillomavirus Infection from Studies Using Gene-Deficient Mice

Scagnolari, C., Cannella, F., Pierangeli, A., Pilgrim, R.M., Antonelli, G., Rowley, D., Wong, M., Best, S., Xing, D., Roden, R.B.S. and Viscidi, R.

1. Virol., 94(12), e00087 (2020)

  We demonstrate that female C57BL/6J mice are susceptible to a transient lower genital tract infection with MmuPV1 mouse papillomavirus and display focal histopathological abnormalities resembling those of human papillomavirus (HPV) infection. We took advantage of strains of genetically deficient mice to study in vivo the role of innate immune signaling in the control of papillomavirus. At 4 months, we sacrificed MmuPV1-infected mice and measured viral 757/3139 spliced transcripts by TaqMan reverse transcription-PCR (RT-PCR), localization of infection by RNAscope in situ hybridization, and histopathological abnormities by hematoxylin and eosin (H&E) staining. Among mice deficient in receptors for pathogen-associated molecular patterns, MyD88^−/− and STING^−/− mice had 1,350 and 80 copies of spliced transcripts/μg RNA, respectively, while no viral expression was detected in MAVS^−/− and Ripk2^−/− mice. Mice deficient in an adaptor molecule, STAT1^−/−, for interferon signaling had 46,000 copies/μg RNA. Among mice with targeted deficiencies in the inflammatory response, interleukin-1 receptor knockout (IL-1R^−/−) and caspase-1^−/− mice had 350 and 30 copies/μg RNA, respectively. Among mice deficient in chemokine receptors, CCR6^−/− mice had 120 copies/μg RNA, while CXCR2^−/− and CXCR3^−/− mice were negative. RNAscope confirmed focal infection in MyD88^−/−, STAT1^−/−, and CCR6^−/− mice but was negative for other gene-deficient mice. Histological abnormalities were seen only in the latter mice. Our findings and the literature support a working model of innate immunity to papillomaviruses involving the activation of a MyD88-dependent pathway and IL-1 receptor signaling, control of viral replication by interferon-stimulated genes, and clearance of virus-transformed dysplastic cells by the action of the CCR6/CCL20 axis.  

5.2950           Secretome Analysis of Cardiomyocytes Identifies PCSK6 (Proprotein Convertase Subtilisin/Kexin Type 6) as a Novel Player in Cardiac Remodeling After Myocardial Infarction

Kuhn, T.C., Knobel, J., Burkert-Rettenmaier, S., Li, X., Meyer, I.S., Jungmann, A., Sicklinger, F., Backs, J., Lasitschka, F., Müller, O.J., Katus, H.A., Krijgsveld, J. and Leuschner, F. Circulation, 141, 1628-1644 (2020)  

Background:

Acute occlusion of a coronary artery results in swift tissue necrosis. Bordering areas of the infarcted myocardium can also experience impaired blood supply and reduced oxygen delivery, leading to altered metabolic and mechanical processes. Although transcriptional changes in hypoxic cardiomyocytes are well studied, little is known about the proteins that are actively secreted from these cells.

Methods:

We established a novel secretome analysis of cardiomyocytes by combining stable isotope labeling and click chemistry with subsequent mass spectrometry analysis. Further functional validation experiments included ELISA measurement of human samples, murine left anterior descending coronary artery ligation, and adeno-associated virus 9–mediated in vivo overexpression in mice.

Results:

The presented approach is feasible for analysis of the secretome of primary cardiomyocytes without serum starvation. A total of 1026 proteins were identified to be secreted within 24 hours, indicating a 5-fold increase in detection compared with former approaches. Among them, a variety of proteins have not yet been explored in the context of cardiovascular pathologies. One of the secreted factors most strongly upregulated upon hypoxia was PCSK6 (proprotein convertase subtilisin/kexin type 6). Validation experiments revealed an increase of PCSK6 on mRNA and protein level in hypoxic cardiomyocytes. PCSK6 expression was elevated in hearts of mice after 3 days of ligation of the left anterior descending artery, a finding confirmed by immunohistochemistry. ELISA measurements in human serum also indicate distinct kinetics for PCSK6 in patients with acute myocardial infarction, with a peak on postinfarction day 3. Transfer of PCSK6-depleted cardiomyocyte secretome resulted in decreased expression of collagen I and III in fibroblasts compared with control treated cells, and small interfering RNA–mediated knockdown of PCSK6 in cardiomyocytes impacted transforming growth factor-β activation and SMAD3 (mothers against decapentaplegic homolog 3) translocation in fibroblasts. An adeno-associated virus 9–mediated, cardiomyocyte-specific overexpression of PCSK6 in mice resulted in increased collagen expression and cardiac fibrosis, as well as decreased left ventricular function, after myocardial infarction.

Conclusions:

A novel mass spectrometry–based approach allows investigation of the secretome of primary cardiomyocytes. Analysis of hypoxia-induced secretion led to the identification of PCSK6 as being crucially involved in cardiac remodeling after acute myocardial infarction.

 

5.2951           Genetic ablation of Cullin-RING E3 ubiquitin ligase 7 restrains pressure overload-induced myocardial fibrosis

Anger, M., Scheufele, F., Ramanujam, D., Meyer, K., Nakajima, H., Filed, L.J., Engelhardt, S. and Sarikas, A. Plos One, 15(12), e0244096 (2020)   Fibrosis is a pathognomonic feature of structural heart disease and counteracted by distinct cardioprotective mechanisms, e.g. activation of the phosphoinositide 3-kinase (PI3K) / AKT pro-survival pathway. The Cullin-RING E3 ubiquitin ligase 7 (CRL7) was identified as negative regulator of PI3K/AKT signalling in skeletal muscle, but its role in the heart remains to be elucidated. Here, we sought to determine whether CRL7 modulates to cardiac fibrosis following pressure overload and dissect its underlying mechanisms. For inactivation of CRL7, the Cullin 7 (Cul7) gene was deleted in cardiac myocytes (CM) by injection of adeno-associated virus subtype 9 (AAV9) vectors encoding codon improved Cre-recombinase (AAV9-CMV-iCre) in Cul7^flox/flox mice. In addition, Myosin Heavy Chain 6 (Myh6; alpha-MHC)-MerCreMer transgenic mice with tamoxifen-induced CM-specific expression of iCre were used as alternate model. After transverse aortic constriction (TAC), causing chronic pressure overload and fibrosis, AAV9-CMV-iCre induced Cul7-/- mice displayed a ~50% reduction of interstitial cardiac fibrosis when compared to Cul7+/+ animals (6.7% vs. 3.4%, p<0.01). Similar results were obtained with Cul7^flox/flox Myh6-Mer-Cre-Mer^Tg(1/0) mice which displayed a ~30% reduction of cardiac fibrosis after TAC when compared to Cul7^+/+ Myh6-Mer-Cre-Mer^Tg(1/0) controls after TAC surgery (12.4% vs. 8.7%, p<0.05). No hemodynamic alterations were observed. AKT^Ser473 phosphorylation was increased 3-fold (p<0.01) in Cul7-/- vs. control mice, together with a ~78% (p<0.001) reduction of TUNEL-positive apoptotic cells three weeks after TAC. In addition, CM-specific expression of a dominant-negative CUL7^1152stop mutant resulted in a 16.3-fold decrease (p<0.001) of in situ end-labelling (ISEL) positive apoptotic cells. Collectively, our data demonstrate that CM-specific ablation of Cul7 restrains myocardial fibrosis and apoptosis upon pressure overload, and introduce CRL7 as a potential target for anti-fibrotic therapeutic strategies of the heart.  

5.2952           Host restriction of emerging high-pathogenic bunyaviruses via MOV10 by targeting viral nucleoprotein and blocking ribonucleoprotein assembly

Mo, Q., Xu, Z., Deng, F., Wang, H., Ning, Y-J. PloS Pathogens, 16(12), e1009129 (2020)   Bunyavirus ribonucleoprotein (RNP) that is assembled by polymerized nucleoproteins (N) coating a viral RNA and associating with a viral polymerase can be both the RNA synthesis machinery and the structural core of virions. Bunyaviral N and RNP thus could be assailable targets for host antiviral defense; however, it remains unclear which and how host factors target N/RNP to restrict bunyaviral infection. By mass spectrometry and protein-interaction analyses, we here show that host protein MOV10 targets the N proteins encoded by a group of emerging high-pathogenic representatives of bunyaviruses including severe fever with thrombocytopenia syndrome virus (SFTSV), one of the most dangerous pathogens listed by World Health Organization, in RNA-independent manner. MOV10 that was further shown to be induced specifically by SFTSV and related bunyaviruses in turn inhibits the bunyaviral replication in infected cells in series of loss/gain-of-function assays. Moreover, animal infection experiments with MOV10 knockdown corroborated the role of MOV10 in restricting SFTSV infection and pathogenicity in vivo. Minigenome assays and additional functional and mechanistic investigations demonstrate that the anti-bunyavirus activity of MOV10 is likely achieved by direct impact on viral RNP machinery but independent of its helicase activity and the cellular interferon pathway. Indeed, by its N-terminus, MOV10 binds to a protruding N-arm domain of N consisting of only 34 amino acids but proving important for N function and blocks N polymerization, N-RNA binding, and N-polymerase interaction, disabling RNP assembly. This study not only advances the understanding of bunyaviral replication and host restriction mechanisms but also presents novel paradigms for both direct antiviral action of MOV10 and host targeting of viral RNP machinery.  

5.2953           Shedding-Resistant HIV-1 Envelope Glycoproteins Adopt Downstream Conformations That Remain Responsive to Conformation-Preferring Ligands

Lu, M., Ma, X., Reichard, N., Terry, D.S., Arthos, J., Smith III, A.B., Sodroski, J.G., Blanchard, S.C. and Mothes, W.

1. Virol., 94(17), e00597-20 (2020)

  The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer of gp120-gp41 heterodimers mediates virus entry into CD4-positive (CD4⁺) cells. Single-molecule fluorescence resonance energy transfer (smFRET) has revealed that native Env on the surface of viruses predominantly exists in a pretriggered conformation (state 1) that is preferentially recognized by many broadly neutralizing antibodies (bNAbs). Env is activated by binding receptor CD4, which drives transitions through a default intermediate conformation (state 2) into the three-CD4-bound open conformation (state 3). The application of smFRET to assess the conformational state of existing Env constructs and ligand complexes recently revealed that all current high-resolution structures correspond to downstream states 2 and 3. The structure of state 1, therefore, remains unknown. We sought to identify conditions whereby HIV-1 Env could be stabilized in the pretriggered state 1 for possible structural characterization. Shedding of gp120, known to severely complicate structural studies, can be prevented by using the uncleaved gp160_JR-FL precursor with alterations in the protease cleavage site (R508S/R511S) or by introducing a disulfide bridge between gp120 and gp41 designated “SOS” (A501C/T605C). smFRET demonstrated that both shedding-preventing modifications shifted the conformational landscape of Env downstream toward states 2 and 3. However, both membrane-bound Env proteins on the surface of intact viruses remained conformationally dynamic, responsive to state-stabilizing ligands, and able to be stabilized in state 1 by specific ligands such as the Bristol-Myers Squibb (BMS) entry inhibitors. The here-described identification of state 1-stabilizing High-mobility group AT-hook 1 promotes cardiac dysfunction in diabetic cardiomyopathy via autophagy inhibitionconditions may enable structural characterization of the state 1 conformation of HIV-1 Env.  

5.2954           SUMOylation Targets Adeno-associated Virus Capsids but Mainly Restricts Transduction by Cellular Mechanisms

Chen, Q., Njenga, R., Leuchs, B., Chiocca, S., Kleinschmidt, J. and Müller, M.

1. Virol., 94(19), e00871-20 (2020)

  Adeno-associated virus (AAV) has proven to be a promising candidate for gene therapy due to its nonpathogenic nature, ease of production, and broad tissue tropism. However, its transduction capabilities are not optimal due to the interaction with various host factors within the cell. In a previous study, we identified members of the small ubiquitin-like modifier (SUMO) pathway as significant restriction factors in AAV gene transduction. In the present study, we explored the scope of this restriction by focusing on the AAV capsid and host cell proteins as targets. We show that during vector production, the capsid protein VP2 becomes SUMOylated, as indicated by deletion and point mutations of VP2 or the obstruction of its N terminus via the addition of a tag. We observed that SUMOylated AAV capsids display higher stability than non-SUMOylated capsids. Prevention of capsid SUMOylation by VP2 mutations did not abolish transduction restriction by SUMOylation; however, it reduced activation of gene transduction by shutdown of the cellular SUMOylation pathway. This indicates a link between capsid SUMOylation and SUMOylation of cellular proteins in restricting gene transduction. Infection with AAV triggers general SUMOylation of cellular proteins. In particular, the DAXX protein, a putative host cell restriction factor that can become SUMOylated, is able to restrict AAV gene transduction by reducing the intracellular No evidence of SARS accumulation of AAV vectors. We also observe that the coexpression of a SUMOylation inhibitor with an AAV2 reporter gene vector increased gene transduction significantly.  

5.2955           Mitochondria-Endoplasmic Reticulum Contacts in Reactive Astrocytes Promote Vascular Remodeling

Göbel, J., Engelhardt, E., Pelzer, P., Conzelmann, K-K., Motori, E. and Bergami, M. Cell Metabolism, 31, 791-808 (2020)   Astrocytes have emerged for playing important roles in brain tissue repair; however, the underlying mechanisms remain poorly understood. We show that acute injury and blood-brain barrier disruption trigger the formation of a prominent mitochondrial-enriched compartment in astrocytic endfeet, which enables vascular remodeling. Integrated imaging approaches revealed that this mitochondrial clustering is part of an adaptive response regulated by fusion dynamics. Astrocyte-specific conditional deletion of Mitofusin 2 (Mfn2) suppressed perivascular mitochondrial clustering and disrupted mitochondria-endoplasmic reticulum (ER) contact sites. Functionally, two-photon imaging experiments showed that these structural changes were mirrored by impaired mitochondrial Ca²⁺ uptake leading to abnormal cytosolic transients within endfeet in vivo. At the tissue level, a compromised vascular complexity in the lesioned area was restored by boosting mitochondrial-ER perivascular tethering in MFN2-deficient astrocytes. These data unmask a crucial role for mitochondrial dynamics in coordinating astrocytic local domains and have important implications for repairing the injured brain.  

5.2956           Structure and flexibility in cortical representations of odour space

Pashkovski, S., Iurilli, G., Chicharro, D., Drummey, K., Franks, K.M., Panzeri, S. and Datta, S.R. Nature, 583, 253-258 (2020)   The cortex organizes sensory information to enable discrimination and generalization1^,2^,3^,4. As systematic representations of chemical odour space have not yet been described in the olfactory cortex, it remains unclear how odour relationships are encoded to place chemically distinct but similar odours, such as lemon and orange, into perceptual categories, such as citrus5^,6^,7. Here, by combining chemoinformatics and multiphoton imaging in the mouse, we show that both the piriform cortex and its sensory inputs from the olfactory bulb represent chemical odour relationships through correlated patterns of activity. However, cortical odour codes differ from those in the bulb: cortex more strongly clusters together representations for related odours, selectively rewrites pairwise odour relationships, and better matches odour perception. The bulb-to-cortex transformation depends on the associative network originating within the piriform cortex, and can be reshaped by passive odour experience. Thus, cortex actively builds a structured representation of chemical odour space that highlights odour relationships; this representation is similar across individuals but remains plastic, suggesting a means through which the olfactory system can assign related odour cues to common and yet personalized percepts.  

5.2957           Glucose metabolism links astroglial mitochondria to cannabinoid effects

Jimenez-Blasco, D., Busquets-Garcia, A., Hebert-Chatelain, E., Serrat, R., Vicente-Gutierez, C. et al Nature, 583, 603-608 (2020)   Astrocytes take up glucose from the bloodstream to provide energy to the brain, thereby allowing neuronal activity and behavioural responses1^,2^,3^,4^,5. By contrast, astrocytes are under neuronal control through specific neurotransmitter receptors5^,6^,7. However, whether the activation of astroglial receptors can directly regulate cellular glucose metabolism to eventually modulate behavioural responses is unclear. Here we show that activation of mouse astroglial type-1 cannabinoid receptors associated with mitochondrial membranes (mtCB₁) hampers the metabolism of glucose and the production of lactate in the brain, resulting in altered neuronal functions and, in turn, impaired behavioural responses in social interaction assays. Specifically, activation of astroglial mtCB₁ receptors reduces the phosphorylation of the mitochondrial complex I subunit NDUFS4, which decreases the stability and activity of complex I. This leads to a reduction in the generation of reactive oxygen species by astrocytes and affects the glycolytic production of lactate through the hypoxia-inducible factor 1 pathway, eventually resulting in neuronal redox stress and impairment of behavioural responses in social interaction assays. Genetic and pharmacological correction of each of these effects abolishes the effect of cannabinoid treatment on the observed behaviour. These findings suggest that mtCB₁ receptor signalling can directly regulate astroglial glucose metabolism to fine-tune neuronal activity and behaviour in mice.  

5.2958           Chemico-genetic discovery of astrocytic control of inhibition in vivo

Takano, T., Wallace, J.T., Baldwin, K.T., Purkey, A.M., Uezu, A., Courtland, J.L., Soderblom, E.J., Shimogori, T., Maness, P.F., Eroglu, C. and Soderling, S.H. Nature, 588, 298-302 (2020)   Perisynaptic astrocytic processes are an integral part of central nervous system synapses1^,2; however, the molecular mechanisms that govern astrocyte–synapse adhesions and how astrocyte contacts control synapse formation and function are largely unknown. Here we use an in vivo chemico-genetic approach that applies a cell-surface fragment complementation strategy, Split-TurboID, and identify a proteome that is enriched at astrocyte–neuron junctions in vivo, which includes neuronal cell adhesion molecule (NRCAM). We find that NRCAM is expressed in cortical astrocytes, localizes to perisynaptic contacts and is required to restrict neuropil infiltration by astrocytic processes. Furthermore, we show that astrocytic NRCAM interacts transcellularly with neuronal NRCAM coupled to gephyrin at inhibitory postsynapses. Depletion of astrocytic NRCAM reduces numbers of inhibitory synapses without altering glutamatergic synaptic density. Moreover, loss of astrocytic NRCAM markedly decreases inhibitory synaptic function, with minor effects on excitation. Thus, our results present a proteomic framework for how astrocytes interface with neurons and reveal how astrocytes control GABAergic synapse formation and function.  

5.2959           Gangliosides are essential endosomal receptors for quasi-enveloped and naked hepatitis A virus

Das, A., Barrientos, R., Shiota, T., Madigan, V., Misumi, I.McKnight, K.L., Sun, L., Li, Z., Meganck, R.M., Li, Y., Kaluzna, E., Asokan, A., Whitmire, J.K., Kapustina, M., Zhang, Q. and Lemon, S.M. Nature Microbiol., 5, 1069-1078 (2020)   The Picornaviridae are a diverse family of positive-strand RNA viruses that includes numerous human and veterinary pathogens1. Among these, hepatitis A virus (HAV), a common cause of acute hepatitis in humans, is unique in that it is hepatotropic and is released from hepatocytes without lysis in small vesicles that resemble exosomes2^,3. These quasi-enveloped virions are infectious and are the only form of virus that can be detected in the blood during acute infection2. By contrast, non-enveloped naked virions are shed in faeces and stripped of membranes by bile salts during passage through the bile ducts to the gut4. How these two distinct types of infectious hepatoviruses enter cells to initiate infection is unclear. Here, we describe a genome-wide forward screen that shows that glucosylceramide synthase and other components of the ganglioside synthetic pathway are crucial host factors that are required for cellular entry by hepatoviruses. We show that gangliosides—preferentially disialogangliosides—function as essential endolysosome receptors that are required for infection by both naked and quasi-enveloped virions. In the absence of gangliosides, both virion types are efficiently internalized through endocytosis, but capsids fail to uncoat and accumulate within LAMP1⁺ endolysosomes. Gangliosides relieve this block, binding to the capsid at low pH and facilitating a late step in entry involving uncoating and delivery of the RNA genome to the cytoplasm. These results reveal an atypical cellular entry pathway for hepatoviruses that is unique among picornaviruses.  

5.2960           Computational design of anti-CRISPR proteins with improved inhibition potency

Mathony, J., Harteveld, Z., Schmelas, C., Upmeier zu Belzen, J., Aschenbenner, S. et al Nature Chem Biol., 16, 725-730 (2020)   Anti-CRISPR (Acr) proteins are powerful tools to control CRISPR–Cas technologies. However, the available Acr repertoire is limited to naturally occurring variants. Here, we applied structure-based design on AcrIIC1, a broad-spectrum CRISPR–Cas9 inhibitor, to improve its efficacy on different targets. We first show that inserting exogenous protein domains into a selected AcrIIC1 surface site dramatically enhances inhibition of Neisseria meningitidis (Nme)Cas9. Then, applying structure-guided design to the Cas9-binding surface, we converted AcrIIC1 into AcrIIC1X, a potent inhibitor of the Staphylococcus aureus (Sau)Cas9, an orthologue widely applied for in vivo genome editing. Finally, to demonstrate the utility of AcrIIC1X for genome engineering applications, we implemented a hepatocyte-specific SauCas9 ON-switch by placing AcrIIC1X expression under regulation of microRNA-122. Our work introduces designer Acrs as important biotechnological tools and provides an innovative strategy to safeguard CRISPR technologies.  

5.2961           Viral manipulation of functionally distinct interneurons in mice, non-human primates and humans

Vormstein-Schneider, D., Lin, J.D., Pelkey, K.A:, Chittajallu, R., Guo, B., Arias-garcia, M.A. et al Nature Neurosci., 23, 1629-1636 (2020)   Recent success in identifying gene-regulatory elements in the context of recombinant adeno-associated virus vectors has enabled cell-type-restricted gene expression. However, within the cerebral cortex these tools are largely limited to broad classes of neurons. To overcome this limitation, we developed a strategy that led to the identification of multiple new enhancers to target functionally distinct neuronal subtypes. By investigating the regulatory landscape of the disease gene Scn1a, we discovered enhancers selective for parvalbumin (PV) and vasoactive intestinal peptide-expressing interneurons. Demonstrating the functional utility of these elements, we show that the PV-specific enhancer allowed for the selective targeting and manipulation of these neurons across vertebrate species, including humans. Finally, we demonstrate that our selection method is generalizable and characterizes additional PV-specific enhancers with exquisite specificity within distinct brain regions. Altogether, these viral tools can be used for cell-type-specific circuit manipulation and hold considerable promise for use in therapeutic interventions.  

5.2962           Metabolic engineering generates a transgene-free safety switch for cell therapy

Wiebking, V., Patterson, J.O., Martin, R., Chanda, M.K., Lee, C.M., Srifa, W., bao, G. and Porteus, M.H. Nature Biotechnol., 38, 1441-1450 (2020)   Safeguard mechanisms can ameliorate the potential risks associated with cell therapies but currently rely on the introduction of transgenes. This limits their application owing to immunogenicity or transgene silencing. We aimed to create a control mechanism for human cells that is not mediated by a transgene. Using genome editing methods, we disrupt uridine monophosphate synthetase (UMPS) in the pyrimidine de novo synthesis pathway in cell lines, pluripotent cells and primary human T cells. We show that this makes proliferation dependent on external uridine and enables us to control cell growth by modulating the uridine supply, both in vitro and in vivo after transplantation in xenograft models. Additionally, disrupting this pathway creates resistance to 5-fluoroorotic acid, which enables positive selection of UMPS-knockout cells. We envision that this approach will add an additional level of safety to cell therapies and therefore enable the development of approaches with higher risks, especially those that are intended for limited treatment durations.  

5.2963           Gene replacement ameliorates deficits in mouse and human models of cyclin-dependent kinase-like 5 disorder

Gao, Y., Irvine, E.E., Eleftheriadou, I., Naranjo, C.J., Hearn-Yeates, F., Bosch, L., GLegola, J.A:, Murdoch, L., Czerniak, A., Meloni, I., Renieri, A. Kinali, M. and Mazarakis, N.D: Brain, 143(3), 811-832 (2020)   Cyclin-dependent kinase-like 5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene. It predominantly affects females who typically present with severe early epileptic encephalopathy, global developmental delay, motor dysfunction, autistic features and sleep disturbances. To develop a gene replacement therapy, we initially characterized the human CDKL5 transcript isoforms expressed in the brain, neuroblastoma cell lines, primary astrocytes and embryonic stem cell-derived cortical interneurons. We found that the isoform 1 and to a lesser extent the isoform 2 were expressed in human brain, and both neuronal and glial cell types. These isoforms were subsequently cloned into recombinant adeno-associated viral (AAV) vector genome and high-titre viral vectors were produced. Intrajugular delivery of green fluorescence protein via AAV vector serotype PHP.B in adult wild-type male mice transduced neurons and astrocytes throughout the brain more efficiently than serotype 9. Cdkl5 knockout male mice treated with isoform 1 via intrajugular injection at age 28–30 days exhibited significant behavioural improvements compared to green fluorescence protein-treated controls (10¹² vg per animal, n = 10 per group) with PHP.B vectors. Brain expression of the isoform 1 transgene was more abundant in hindbrain than forebrain and midbrain. Transgene brain expression was sporadic at the cellular level and most prominent in hippocampal neurons and cerebellar Purkinje cells. Correction of postsynaptic density protein 95 cerebellar misexpression, a major fine cerebellar structural abnormality in Cdkl5 knockout mice, was found in regions of high transgene expression within the cerebellum. AAV vector serotype DJ efficiently transduced CDKL5-mutant human induced pluripotent stem cell-derived neural progenitors, which were subsequently differentiated into mature neurons. When treating CDKL5-mutant neurons, isoform 1 expression led to an increased density of synaptic puncta, while isoform 2 ameliorated the calcium signalling defect compared to green fluorescence protein control, implying distinct functions of these isoforms in neurons. This study provides the first evidence that gene therapy mediated by AAV vectors can be used for treating CDKL5 disorder.  

5.2964           Global CNS correction in a large brain model of human alpha-mannosidosis by intravascular gene therapy

Yoon, S.Y., Hunter, J.E., Chawla, S., Clarke, D.L., Molony, C., O’Donnell, P.A., Bagel, J.H., Kumar, M.J., Poptani, H., Vite, C.H. and Wolfe, J.H. Brain, 143(7), 2058-2072 (2020)   Intravascular injection of certain adeno-associated virus vector serotypes can cross the blood–brain barrier to deliver a gene into the CNS. However, gene distribution has been much more limited within the brains of large animals compared to rodents, rendering this approach suboptimal for treatment of the global brain lesions present in most human neurogenetic diseases. The most commonly used serotype in animal and human studies is 9, which also has the property of being transported via axonal pathways to distal neurons. A small number of other serotypes share this property, three of which were tested intravenously in mice compared to 9. Serotype hu.11 transduced fewer cells in the brain than 9, rh8 was similar to 9, but hu.32 mediated substantially greater transduction than the others throughout the mouse brain. To evaluate the potential for therapeutic application of the hu.32 serotype in a gyrencephalic brain of larger mammals, a hu.32 vector expressing the green fluorescent protein reporter gene was evaluated in the cat. Transduction was widely distributed in the cat brain, including in the cerebral cortex, an important target since mental retardation is an important component of many of the human neurogenetic diseases. The therapeutic potential of a hu.32 serotype vector was evaluated in the cat homologue of the human lysosomal storage disease alpha-mannosidosis, which has globally distributed lysosomal storage lesions in the brain. Treated alpha-mannosidosis cats had reduced severity of neurological signs and extended life spans compared to untreated cats. The extent of therapy was dose dependent and intra-arterial injection was more effective than intravenous delivery. Pre-mortem, non-invasive magnetic resonance spectroscopy and diffusion tensor imaging detected differences between the low and high doses, and showed normalization of grey and white matter imaging parameters at the higher dose. The imaging analysis was corroborated by post-mortem histological analysis, which showed reversal of histopathology throughout the brain with the high dose, intra-arterial treatment. The hu.32 serotype would appear to provide a significant advantage for effective treatment of the gyrencephalic brain by systemic adeno-associated virus delivery in human neurological diseases with widespread brain lesions.  

5.2965           Transduction Efficiency of Adeno-Associated Virus Serotypes After Local Injection in Mouse and Human Skeletal Muscle

Muraine, L., Bensalah, M., Dhiab,k J., Cordova, G., Arandel, L., Marhic, A., Chapart, M., Vasseur, S., Benkhelifa-Ziyyat, S., Bigot, A., Butler-Browne, G., Mouly, V., Negroni, E. and Trollet, C. Human Gene Therapy, 31(3-4), 233-240 (2020)   The adeno-associated virus (AAV) vector is an efficient tool for gene delivery in skeletal muscle. AAV-based therapies show promising results for treatment of various genetic disorders, including muscular dystrophy. These dystrophies represent a heterogeneous group of diseases affecting muscles and typically characterized by progressive skeletal muscle wasting and weakness and the development of fibrosis. The tropism of each AAV serotype has been extensively studied using systemic delivery routes, but very few studies have compared their transduction efficiency through direct intramuscular injection. Yet, in some muscular dystrophies, where only a few muscles are primarily affected, a local intramuscular injection to target these muscles would be the most appropriate route. A comprehensive comparison between different recombinant AAV (rAAV) serotypes is therefore needed. In this study, we investigated the transduction efficiency of rAAV serotypes 1–10 by local injection in skeletal muscle of control C57BL/6 mice. We used a CMV-nls-LacZ reporter cassette allowing nuclear expression of LacZ to easily localize targeted cells. Detection of β-galactosidase activity on muscle cryosections demonstrated that rAAV serotypes 1, 7, 8, 9, and 10 were more efficient than the others, with rAAV9 being the most efficient in mice. Furthermore, using a model of human muscle xenograft in immunodeficient mice, we observed that in human muscle, rAAV8 and rAAV9 had similar transduction efficiency. These findings demonstrate for the first time that the human muscle xenograft can be used to evaluate AAV-based therapeutical approaches in a human context.  

5.2966           Dose Range Finding Studies with Two RPGR Transgenes in a Canine Model of X-Linked Retinitis Pigmentosa Treated with Subretinal Gene Therapy

Song, C., Dufour, V.L., Cideciyan, A.V., Ye, G.J., Swider, M., Newmark, J.A., Timmers, A.M., Robinson, P.M., Knop, D.R., Chulay, J.D., Jacobson, S.G., Aguirre, G.D., Beltran, W.A. and Shearman, M.S. Human gene Therapy, 31(13-14), 743-755 (2020)   Recombinant adeno-associated viral (rAAV) vector-mediated gene therapy is being developed to treat X-linked retinitis pigmentosa (XLRP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. In preparation for a clinical gene therapy trial, we conducted dose range finding (DRF) studies with an AAV2 capsid with three surface tyrosine residues changed to phenylalanine (AAV2tYF) vector administered by subretinal injection in a naturally occurring RPGR-mutant canine model (XLPRA2) to compare two different human RPGR (hRPGR) transgenes and to establish a reasonable starting dose for a clinical trial. Different dose levels of two candidate vectors (0.15 mL at 1.2 × 10¹⁰–3.0 × 10¹² vg/mL of rAAV2tYF-GRK1-hRPGRco or 4 × 10¹⁰–3.0 × 10¹² vg/mL of rAAV2tYF-GRK1-hRPGRstb), 6.0 × 10¹¹ vg/mL rAAV5-GRK1-hRPGRco reference vector or Vehicle were subretinally administered, and the dogs were followed for 8 weeks postdose. Ophthalmic examinations, analyses of retinal structure by in vivo imaging using confocal scanning laser ophthalmoscopy (cSLO)/optical coherence tomography (OCT) in the Lower (4.0 × 10¹⁰ vg/mL) and Lowest (1.2 × 10¹⁰ vg/mL) Doses, immunological responses by cell based assays or enzyme-linked immunosorbent assay, RPGR transgene expression, and reversal of opsin mislocalization by immunohistochemistry were performed. No sustained signs of ocular discomfort or ophthalmic complications were noted in any of the injected eyes except some in the High Dose group (3.0 × 10¹² vg/mL), which showed signs of retinal detachment and inflammation. A change in fundus reflectivity suggestive of a rescue effect was seen in the High, Mid (6.0 × 10¹¹ vg/mL), and Low (1.2 × 10¹¹ vg/mL) Dose groups. cSLO/OCT demonstrated qualitative and quantitative evidence of rescue effect in eyes treated with the Lower Dose. Anti-hRPGR antibodies were absent, but neutralizing antibody titers against AAV2 were detected in all animals dosed with rAAV2tYF in an apparent dose-related pattern. RPGR expression was stronger for rAAV2tYF-GRK1-hRPGRco compared to rAAV2tYF-GRK1-hRPGRstb at all dose levels. Subretinal administration of rAAV2tYF-GRK1-hRPGRco and rAAV2tYF-GRK1-hRPGRstb both corrected rod and cone opsin mislocalization, two early markers of disease in the XLPRA2 canine model of RPGR-XLRP. These results support the selection and use of rAAV2tYF-GRK1-hRPGRco (AGTC-501) and guided the initial doses in clinical studies in patients with XLRP caused by RPGR mutations.    

5.2967           Development of a Clinical Candidate AAV3 Vector for Gene Therapy of Hemophilia B

Brown, H.C., Doering, C.B:, Herzog, R.W., Ling, C., Markusic, D.M., Spencer, H.T., Srivastava, A. and Srivastava, A. Human Gene Therapy, 31(19-20), 1114-1123 (2020)   Although recombinant adeno-associated virus serotype 8 (AAV8) and serotype 5 (AAV5) vectors have shown efficacy in Phase 1 clinical trials for gene therapy of hemophilia B, it has become increasingly clear that these serotypes are not optimal for transducing primary human hepatocytes. We have previously reported that among the 10 most commonly used AAV serotypes, AAV serotype 3 (AAV3) vectors are the most efficient in transducing primary human hepatocytes in vitro as well as in “humanized” mice in vivo, and suggested that AAV3 vectors expressing human coagulation factor IX (hFIX) may be a more efficient alternative for clinical gene therapy of hemophilia B. In the present study, we extended these findings to develop an AAV3 vector incorporating a compact yet powerful liver-directed promoter as well as optimized hFIX cDNA sequence inserted between two AAV3 inverted terminal repeats. When packaged into an AAV3 capsid, this vector yields therapeutic levels of hFIX in hemophilia B and in “humanized” mice in vivo. Together, these studies have resulted in an AAV3 vector predicted to achieve clinical efficacy at reduced vector doses, without the need for immune-suppression, for clinical gene therapy of hemophilia B.  

5.2968           Quantification of Adeno-Associated Virus with Safe Nucleic Acid Dyes

Xu, J., DeVries, S.H. and Zhu, Y. Human Gene Therapy, 31(19-20), 1086-1099 (2020)   Adeno-associated virus (AAV) is the most commonly used viral vector for both biological and gene therapeutic applications. Although many methods have been developed to measure quantity attributes of AAV, they are often technically challenging and time-consuming. Here, we report a method to titer AAV with GelGreen^® dye, a safe green fluorescence nucleic acid dye recently engineered by Biotium company (Fremont, CA). This method, hereinafter referred to as GelGreen method, provides a fast (∼30 min) and reliable strategy for AAV titration. To validate GelGreen method, we measured genome titer of an AAV reference material AAV8RSM and compared our titration results with those determined by Reference Material Working Group (ARMWG). We showed that GelGreen results and capsid enzyme-linked immunosorbent assay results are comparable with each other. We also showed that GelRed^® dye, a red fluorescence dye from Biotium, can be used to directly “visualize” AAV genome titer on a conventional gel imager, presenting an especially direct approach to estimate viral quantity. Finally, we showed that GelGreen and GelRed dyes can also be used to quantify self-complementary AAV (scAAV) and crudely purified AAV samples. In summary, we described a technique to titer AAV by using new generation of safe DNA dyes. This technique is simple, safe, reliable, and cost efficient. It has potential to be broadly applied for quantifying and normalizing AAV viral vectors.  

5.2969           Identification of a glycan cluster in gp120 essential for irreversible HIV-1 lytic inactivation by a lectin-based recombinantly engineered protein conjugate

Parajuli, B., Acharya, K., Nangarlia, A., Zhang, S., parajuli, B., Dick, A., Ngo, B., Abrams, C.F. and Chaiken, I. Biochem. J., 477, 4263-4280 (2020)   We previously discovered a class of recombinant lectin conjugates, denoted lectin DLIs (‘dual-acting lytic inhibitors’) that bind to the HIV-1 envelope (Env) protein trimer and cause both lytic inactivation of HIV-1 virions and cytotoxicity of Env-expressing cells. To facilitate mechanistic investigation of DLI function, we derived the simplified prototype microvirin (MVN)-DLI, containing an MVN domain that binds high-mannose glycans in Env, connected to a DKWASLWNW sequence (denoted ‘Trp3’) derived from the membrane-associated region of gp41. The relatively much stronger affinity of the lectin component than Trp3 argues that the lectin functions to capture Env to enable Trp3 engagement and consequent Env membrane disruption and virolysis. The relatively simplified engagement pattern of MVN with Env opened up the opportunity, pursued here, to use recombinant glycan knockout gp120 variants to identify the precise Env binding site for MVN that drives DLI engagement and lysis. Using mutagenesis combined with a series of biophysical and virological experiments, we identified a restricted set of residues, N262, N332 and N448, all localized in a cluster on the outer domain of gp120, as the essential epitope for MVN binding. By generating these mutations in the corresponding HIV-1 virus, we established that the engagement of this glycan cluster with the lectin domain of MVN*-DLI is the trigger for DLI-derived virus and cell inactivation. Beyond defining the initial encounter step for lytic inactivation, this study provides a guide to further elucidate DLI mechanism, including the stoichiometry of Env trimer required for function, and downstream DLI optimization.  

5.2970           A long noncoding RNA CHAIR protects the heart from pathological stress

Qian, Y., Zhang, M., Zhou, N., Xu, X., Zhang, J., Ding, Q. and Wang, J. Clin. Sci., 134, 1843-1857 (2020)   Mammalian genomes have been found to be extensively transcribed. In addition to classic protein coding genes, a large numbers of long noncoding genes (lncRNAs) have been identified, while their functions, especially in heart diseases, remain to be established. We hypothesized that heart failure progression is controlled by tissue-specific lncRNAs. In the present study, we found that the cardiac-enriched lncRNA 4632428C04Rik, named as cardiomyocyte hypertrophic associated inhibitory RNA (CHAIR), is dynamically regulated during heart development, is expressed at low levels in embryonic hearts and accumulated at high levels in adult hearts. More interestingly, the lncRNA was down-regulated during cardiac hypertrophy and failure both in mice and humans. Importantly, loss of lncRNA CHAIR has no effects on normal hearts, whereas it results in accelerated heart function decline, increased hypertrophy, and exacerbated heart failure in response to stress. In contrast, restoring the expression of lncRNA CHAIR rescued the hearts from hypertrophy and failure. DNMT3A was recruited to CHAIR promoter during heart failure to suppress its expression. Reciprocally, CHAIR interacted with DNMT3A to inhibit its DNA-binding activity. Taken together, our data revealed a new cardioprotective lncRNA that represses heart failure through an epigenetic mechanism.  

5.2971           Enoyl coenzyme A hydratase 1 combats obesity and related metabolic disorders by promoting adipose tissue browning

Mao, X., Huang, D., Rao, C., Du, M., Liang, M., Li, F., Liu, B. and Huang, K. Am. J. Physiol. Endocrinol. Metab., 318, E318-E329 (2020)   Browning of white adipose tissue (WAT) has been recognized as an important strategy for the treatment of obesity, insulin resistance, and diabetes. Enoyl coenzyme A hydratase 1 (ECH1) is a widely known enzyme involved in lipid metabolism. However, whether and how ECH1 is implicated in browning of WAT remain obscure. Adeno-associated, virus-mediated genetic engineering of ECH1 in adipose tissue was used in investigations in mouse models of obesity induced by a high-fat diet (HFD) or browning induced by cold exposure. Metabolic parameters showed that ECH1 overexpression decreased weight gain and improved insulin sensitivity and lipid profile after 8 wk of an HFD. Further work revealed that these changes were associated with enhanced energy expenditure and increased appearance of brown-like adipocytes in inguinal WAT, as verified by a remarkable increase in uncoupling protein 1 and thermogenic gene expression. In vitro, ECH1 induced brown fat-related gene expression in adipocytes differentiated from primary stromal vascular fractions, whereas knockdown of ECH1 reversed this effect. Mechanistically, ECH1 regulated the thermogenic program by inhibiting mammalian target of rapamycin signaling, which may partially explain the potential mechanism for ECH1 regulating adipose browning. In summary, ECH1 may participate in the pathology of obesity by regulating browning of WAT, which probably provides us with a new therapeutic strategy for combating obesity.  

5.2972           MicroRNA-mediated inhibition of transgene expression reduces dorsal root ganglion toxicity by AAV vectors in primates

Hordeaux, J., Buza, E.L., Jeffrey, B., Song, C., Jahan, T., Yuan, Y., Zhu, Y., Bell, P., Li, M., Chichester, J.A., Calcedo, R. and Wilson, J.M. Sci. Transl. Med., 12, eaba9188 (2020)   Delivering adeno-associated virus (AAV) vectors into the central nervous system of nonhuman primates (NHPs) via the blood or cerebral spinal fluid is associated with dorsal root ganglion (DRG) toxicity. Conventional immune-suppression regimens do not prevent this toxicity, possibly because it may be caused by high transduction rates, which can, in turn, cause cellular stress due to an overabundance of the transgene product in target cells. To test this hypothesis and develop an approach to eliminate DRG toxicity, we exploited endogenous expression of microRNA (miR) 183 complex, which is largely restricted to DRG neurons, to specifically down-regulate transgene expression in these cells. We introduced sequence targets for miR183 into the vector genome within the 3′ untranslated region of the corresponding transgene messenger RNA and injected vectors into the cisterna magna of NHPs. Administration of unmodified AAV vectors resulted in robust transduction of target tissues and toxicity in DRG neurons. Consistent with the proposal that immune system activity does not mediate this neuronal toxicity, we found that steroid administration was ineffective in alleviating this pathology. However, including miR183 targets in the vectors reduced transgene expression in, and toxicity of, DRG neurons without affecting transduction elsewhere in the primate’s brain. This approach might be useful in reducing DRG toxicity and the associated morbidity and should facilitate the development of AAV-based gene therapies for many central nervous system diseases.  

5.2973           Slowing late infantile Batten disease by direct brain parenchymal administration of a rh.10 adeno-associated virus expressing CLN2

Sondhi, D., Kaminsky, S.M., hackett, N.R., pagovich, O.E., Rosenberg, J.B., De, B.P. et al Sci. Transl. Med., 12, eabb5413 (2020)   Late infantile Batten disease (CLN2 disease) is an autosomal recessive, neurodegenerative lysosomal storage disease caused by mutations in the CLN2 gene encoding tripeptidyl peptidase 1 (TPP1). We tested intraparenchymal delivery of AAVrh.10hCLN2, a nonhuman serotype rh.10 adeno-associated virus vector encoding human CLN2, in a nonrandomized trial consisting of two arms assessed over 18 months: AAVrh.10hCLN2-treated cohort of 8 children with mild to moderate disease and an untreated, Weill Cornell natural history cohort consisting of 12 children. The treated cohort was also compared to an untreated European natural history cohort of CLN2 disease. The vector was administered through six burr holes directly to 12 sites in the brain without immunosuppression. In an additional safety assessment under a separate protocol, five children with severe CLN2 disease were treated with AAVrh.10hCLN2. The therapy was associated with a variety of expected adverse events, none causing long-term disability. Induction of systemic anti-AAVrh.10 immunity was mild. After therapy, the treated cohort had a 1.3- to 2.6-fold increase in cerebral spinal fluid TPP1. There was a slower loss of gray matter volume in four of seven children by MRI and a 42.4 and 47.5% reduction in the rate of decline of motor and language function, compared to Weill Cornell natural history cohort (P < 0.04) and European natural history cohort (P < 0.0001), respectively. Intraparenchymal brain administration of AAVrh.10hCLN2 slowed the progression of disease in children with CLN2 disease. However, improvements in vector design and delivery strategies will be necessary to halt disease progression using gene therapy.  

5.2974           Distinct evolutionary paths in chronic lymphocytic leukemia during resistance to the graft-versus-leukemia effect

Bachireddy, P., Ennis, C., Nguyen, V.N., Gohil, S.H., Clement, K. et al Sci. Transl. Med., 12, eabb7661 (2020)   Leukemic relapse remains a major barrier to successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) for aggressive hematologic malignancies. The basis for relapse of advanced lymphoid malignancies remains incompletely understood and may involve escape from the graft-versus-leukemia (GvL) effect. We hypothesized that for patients with chronic lymphocytic leukemia (CLL) treated with allo-HSCT, leukemic cell–intrinsic features influence transplant outcomes by directing the evolutionary trajectories of CLL cells. Integrated genetic, transcriptomic, and epigenetic analyses of CLL cells from 10 patients revealed that the clinical kinetics of post-HSCT relapse are shaped by distinct molecular dynamics. Early relapses after allo-HSCT exhibited notable genetic stability; single CLL cell transcriptional analysis demonstrated a cellular heterogeneity that was static over time. In contrast, CLL cells relapsing late after allo-HSCT displayed notable genetic evolution and evidence of neoantigen depletion, consistent with marked single-cell transcriptional shifts that were unique to each patient. We observed a greater rate of epigenetic change for late relapses not seen in early relapses or relapses after chemotherapy alone, suggesting that the selection pressures of the GvL bottleneck are unlike those imposed by chemotherapy. No selective advantage for human leukocyte antigen (HLA) loss was observed, even when present in pretransplant subpopulations. Gain of stem cell modules was a common signature associated with leukemia relapse regardless of posttransplant relapse kinetics. These data elucidate the biological pathways that underlie GvL resistance and posttransplant relapse.  

5.2975           Intratumoral expression of IL-7 and IL-12 using an oncolytic virus increases systemic sensitivity to immune checkpoint blockade

Nakao, S., Arai, Y., Tasaki, M., Yamashita, M., Murakami, R., Kawase, T., Amino, N., Nakatake, M., Kurosaki, H., Mori, M., Takeuchi, M. and Nakamura, T. Sci. Transl. Med., 12, eaxx7992 (2020)   The immune status of the tumor microenvironment is a key indicator in determining the antitumor effectiveness of immunotherapies. Data support the role of activation and expansion of tumor-infiltrating lymphocytes (TILs) in increasing the benefit of immunotherapies in patients with solid tumors. We found that intratumoral injection of a tumor-selective oncolytic vaccinia virus encoding interleukin-7 (IL-7) and IL-12 into tumor-bearing immunocompetent mice activated the inflammatory immune status of previously poorly immunogenic tumors and resulted in complete tumor regression, even in distant tumor deposits. Mice achieving complete tumor regression resisted rechallenge with the same tumor cells, suggesting establishment of long-term tumor-specific immune memory. Combining this virotherapy with anti–programmed cell death-1 (PD-1) or anti–cytotoxic T lymphocyte antigen 4 (CTLA4) antibody further increased the antitumor activity as compared to virotherapy alone, in tumor models unresponsive to either of the checkpoint inhibitor monotherapies. These findings suggest that administration of an oncolytic vaccinia virus carrying genes encoding for IL-7 and IL-12 has antitumor activity in both directly injected and distant noninjected tumors through immune status changes rendering tumors sensitive to immune checkpoint blockade. The benefit of intratumoral IL-7 and IL-12 expression was also observed in humanized mice bearing human cancer cells. These data support further investigation in patients with non-inflamed solid tumors.  

5.2976           Reduction of advanced tau-mediated memory deficits by the MAP kinase p38γ

Ittner, A., Asih, P.R., Tan, A.R.P., Prikas, E., Bertz, J., Stefanoska, K., Lin, Y., Volkerling, A.M., Ke, Y.D., Delerue, F. and Ittner, L.M. Acta Neuropathologica, 140, 279-294 (2020)   Hyperphosphorylation of the neuronal tau protein contributes to Alzheimer’s disease (AD) by promoting tau pathology and neuronal and cognitive deficits. In contrast, we have previously shown that site-specific tau phosphorylation can inhibit toxic signals induced by amyloid-β (Aβ) in mouse models. The post-synaptic mitogen-activated protein (MAP) kinase p38γ mediates this site-specific phosphorylation on tau at Threonine-205 (T205). Using a gene therapeutic approach, we draw on this neuroprotective mechanism to improve memory in two Aβ-dependent mouse models of AD at stages when advanced memory deficits are present. Increasing activity of post-synaptic kinase p38γ that targets T205 in tau reduced memory deficits in symptomatic Aβ-induced AD models. Reconstitution experiments with wildtype human tau or phosphorylation-deficient tauT205A showed that T205 modification is critical for downstream effects of p38γ that prevent memory impairment in APP-transgenic mice. Furthermore, genome editing of the T205 codon in the murine Mapt gene showed that this single side chain in endogenous tau critically modulates memory deficits in APP-transgenic Alzheimer’s mice. Ablating the protective effect of p38γ activity by genetic p38γ deletion in a tau transgenic mouse model that expresses non-pathogenic tau rendered tau toxic and resulted in impaired memory function in the absence of human Aβ. Thus, we propose that modulating neuronal p38γ activity serves as an intrinsic tau-dependent therapeutic approach to augment compromised cognition in advanced dementia.  

5.2977           Therapeutic Role of Neuregulin 1 Type III in SOD1-Linked Amyotrophic Lateral Sclerosis

Modol-Caballero, G., Garcia-Lareu, B., Verdes, S., Ariza, L., Sanchez-Brualla, I., Brocard, F., Bosch, A., Navarro, X. and Herrando-Grabulosa, M. Neurotherapeutics, 17, 1048-1060 (2020)   Amyotrophic lateral sclerosis (ALS) is a devastating motoneuron (Mn) disease without effective cure currently available. Death of MNs in ALS is preceded by failure of neuromuscular junctions and axonal retraction. Neuregulin 1 (NRG1) is a neurotrophic factor highly expressed in MNs and neuromuscular junctions that support axonal and neuromuscular development and maintenance. NRG1 and its ErbB receptors are involved in ALS. Reduced NRG1 expression has been found in ALS patients and in the ALS SOD1^G93A mouse model; however, the expression of the isoforms of NRG1 and its receptors is still controversial. Due to the reduced levels of NRG1 type III (NRG1-III) in the spinal cord of ALS patients, we used gene therapy based on intrathecal administration of adeno-associated virus to overexpress NRG1-III in SOD1^G93A mice. The mice were evaluated from 9 to 16 weeks of age by electrophysiology and rotarod tests. At 16 weeks, samples were harvested for histological and molecular analyses. Our results indicate that overexpression of NRG1-III is able to preserve neuromuscular function of the hindlimbs, improve locomotor performance, increase the number of surviving MNs, and reduce glial reactivity in the treated female SOD1^G93A mice. Furthermore, the NRG1-III/ErbB4 axis appears to regulate MN excitability by modulating the chloride transporter KCC2 and reduces the expression of the MN vulnerability marker MMP-9. However, NRG1-III did not have a significant effect on male mice, indicating relevant sex differences. These findings indicate that increasing NRG1-III at the spinal cord is a promising approach for promoting MN protection and functional improvement in ALS.  

5.2978           Systemic AAV9.LAMP2B injection reverses metabolic and physiologic multiorgan dysfunction in a murine model of Danon disease

Manso, A.M., Hashem, S.I., Nelson, B.C., Gault, E., Soto-Hermida, A., Villarruel, E. et al Sci. Transl. Med., 12, eaxx1744 (2020)   Danon disease (DD) is a rare X-linked autophagic vacuolar myopathy associated with multiorgan dysfunction, including the heart, skeletal muscle, and liver. There are no specific treatments, and most male patients die from advanced heart failure during the second or third decade of life. DD is caused by mutations in the lysosomal-associated membrane protein 2 (LAMP2) gene, a key mediator of autophagy. LAMP2 has three isoforms: LAMP2A, LAMP2B, and LAMP2C. LAMP2B is the predominant isoform expressed in cardiomyocytes. This study evaluates the efficacy of human LAMP2B gene transfer using a recombinant adeno-associated virus 9 carrying human LAMP2B (AAV9.LAMP2B) in a Lamp2 knockout (KO) mouse, a DD model. AAV9.LAMP2B was intravenously injected into 2- and 6-month-old Lamp2 KO male mice to assess efficacy in adolescent and adult phenotypes. Lamp2 KO mice receiving AAV9.LAMP2B demonstrated dose-dependent restoration of human LAMP2B protein in the heart, liver, and skeletal muscle tissue. Impaired autophagic flux, evidenced by increased LC3-II, was abrogated by LAMP2B gene transfer in all tissues in both cohorts. Cardiac function was also improved, and transaminases were reduced in AAV9.LAMP2B-treated KO mice, indicating favorable effects on the heart and liver. Survival was also higher in the older cohort receiving high vector doses. No anti-LAMP2 antibodies were detected in mice that received AAV9.LAMP2B. In summary, LAMP2B gene transfer improves metabolic and physiologic function in a DD murine model, suggesting that a similar therapeutic approach may be effective for treating patients with this highly morbid disease.  

5.2979           Treating Bietti crystalline dystrophy in a high-fat diet-exacerbated murine model using gene therapy

Qu, B., Wu, S., Jiao, G., Zou, X., Li, Z., Guo, L., Sun, X., Huang, C., Sun, Z., Zhang, Y., Li, H., Zhou, Q., Sui, R. and Li, W. Gene Therapy, 27, 370-382 (2020)   Lipid metabolic deficiencies are associated with many genetic disorders. Bietti crystalline dystrophy (BCD), a blindness-causing inherited disorder with changed lipid profiles, is more common in Chinese and Japanese than other populations. Our results reveal that mouse models lacking Cyp4v3 have less physiological and functional changes than those of BCD patients with this gene defect. After the administration of a high-fat diet (HFD), the occurrence of retinal lesions were both accelerated and aggregated in the Cyp4v3^−/− mouse models, implying that changed lipid levels were not only associated factors but also risk factors to BCD patients. Facilitated by the results, we found that the reduced electroretinography waveforms and retinal thickness observed in the HFD-induced mouse models were effectively recovered after subretinal delivery of a human CYP4V2 gene carried by an adeno-associated virus vector, which demonstrates the potential curability of BCD by gene therapy.  

5.2980           A Multiligand Architectural Photosensitizer That Targets Hemagglutinin on Envelope of Influenza Virus for Photodynamic Inactivation

Jeong, H., Lee, J-J., Lee, J. and Na, K. Small, 16, 2000556 (2020)   The efficacy of current antiviral drugs used to treat influenza has been declining because of mutations and resistance of the virus. Herein, a light-sensitive multiligand architecture is developed consisting of chitosan conjugated to a photosensitizer and 6'-sialyllactose (SL) to develop an antiviral agent against influenza with a different mechanism of action (SL-chitosan-Chlorin e6, SCC). Saturation transfer difference-nuclear magnetic resonance determined that the ability of SCC to bind to viral hemagglutinin is stronger than that of the monomeric substance. Virus recognition is confirmed by immunofluorescence and transmission electron microscope imaging. SCC induces viral inactivation by causing permanent membrane damage through its photoactivity. Viral membrane is oxidized by the photoactivity of SCC, thus, the virus membrane collapses. Furthermore, using the plaque reduction assay to evaluate the inhibitory effect of SCC on influenza A and B, it is found that its antiviral effects are 23% and 50% higher than the conventional antiviral drug. Additionally, SCC prevents infection by influenza in 100% of mice subjected to laser irradiation. These results indicate that this photodynamic multiligand structure can overcome the limitations of existing antiviral agents and suggest a pertinent methodology of prophylaxis and treatment by preemptively attacking the virus before it enters the host cell.  

5.2981           Updates in Hepatitis E virus (HEV) field; lessons learned from human liver chimeric mice

Sayed, I.M. and Meuleman, P. Rev. Med. Virol., 30, e2086 (2020)   Hepatitis E virus (HEV) is the most common cause of viral hepatitis globally, and it is an emerging pathogen in developed countries. In vivo studies of HEV have long been hindered due to the lack of an efficient small animal model. Recently, human liver chimeric mice were described as an elegant model to study chronic HEV infection. HEV infection was established in mice with humanized liver that were challenged with stool preparations containing HEV genotype (gt)1 and/or gt3. An increase in viral load and the level of HEV Ag in mouse samples were markers of active infection. Plasma-derived HEV preparations were less infectious. The kinetics of HEV ORF2 Ag during HEV infection and its impact on HEV diagnosis were described in this model. In addition, the nature of HEV particles and HEV ORF2 Ag were characterized. Moreover, humanized mice were used to study the impact of HEV infection on the hepatic innate transcriptome and evaluation of anti-HEV therapies. This review highlights recent advances in the HEV field gathered from well-established experimental mouse models, with an emphasis on this model as a tool for elucidating the course of HEV infection, the study of the HEV life cycle, the interaction of the virus with the host, and the evaluation of new anti-HEV therapies.  

5.2982           Mechanism for enhanced transduction of hematopoietic cells by recombinant adeno-associated virus serotype 6 vectors

Zhong, C., Yu, Q., Jia, W., Yu, X., Yu, D., Yang, M., Wang, L., Ling, C. and Zhu, L. FASEB J., 34, 12379-12391 (2020)   Hematopoietic gene delivery, such as hematopoietic stem/progenitor cells (HSPCs), is a promising treatment for both inherited and acquired diseases, such as hemophilia. Recently, a combined strategy to achieve more than 90% transduction efficiency was documented using recombinant adeno-associated virus serotype 6 (rAAV6) vectors. However, the mechanisms of enhanced vector transduction efficiency in hematopoietic cells are largely unknown. In this manuscript, we first reported that proteasome inhibitors, which are well-known to facilitate rAAV intracellular trafficking in various cell types, are not effective in hematopoietic cells. From the screening of small molecules derived from traditional Chinese medicine, we demonstrated that shikonin, a potential reactive oxygen species (ROS) generator, significantly increased the in vitro and ex vivo transgene expression mediated by rAAV6 vectors in hematopoietic cells, including human cord blood-derived CD34 + HSPCs. Shikonin mainly targeted vector intracellular trafficking, instead of host cell entry or endonuclear single to double strand vector DNA transition, in a vector serotype-dependent manner. Moreover, a ROS scavenger completely prevented the capability of shikonin to enhance rAAV6 vector-mediated transgene expression. Taken together, these studies expand our understanding of rAAV6-mediated transduction in hematopoietic cells and are informative for improving rAAV6-based treatment of blood diseases.  

5.2983           A Versatile Adeno-Associated Viral Vector Cross-Linking Platform Capable of Tuning Cellular Tropisms and Simultaneously Inducing Solid-Phase Gene Delivery

Yoo, S., kang, B., Oh, S., Kim, Y. and jang, J-H. ACS Appl. Bio. Mater., 3, 4747-4857 (2020)   Developing gene carriers with improved affinities for target cells and the simultaneous diversification of their delivery modes will be pivotal for upgrading gene therapy technologies. In this study, a simple and versatile adeno-associated virus (AAV) conjugation platform using the cross-linker 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP) is proposed. Depending on the quantity of the DTSSP molecules, the AAV-DTSSP complexes could either be linked with the relevant biomolecules for altering cellular tropisms or further form a self-assembled AAV-DTSSP pellet capable of mimicking a polymeric gene delivery system. At lower quantities of DTSSP, the AAV-DTSSP complexes were conjugated with aminated l-fucose molecules, whose levels are typically upregulated in pancreatic cancer cells, resulting in enhanced gene delivery efficiencies in pancreatic cancer cells. At higher concentrations of DTSSP, visible solid forms of the AAV-DTSSP pellets were formed, and the AAV pellets demonstrated the capability to induce a localized, sustained gene expression pattern comparable to that of conventional biomaterial-based approaches. Thus, a multipurpose AAV cross-linking platform, which can enable AAV vector systems that are capable of altering cellular tropisms and simultaneously inducing solid-phase delivery, will provide crucial insights into vector design for further upgrading of gene delivery technologies.  

5.2984           Adeno-associated virus characterization for cargo discrimination through nanopore responsiveness

Karawdeniya, B., Bandara, Y.M.N.D.Y., Khan, A.I., Chen, W.T.T., Vu, H-A., Morshed, A., Suh, J., Dutta, P. and Kim, M.J. Nanoscale, 12, 23721-23731 (2020)   Solid-state nanopore (SSN)-based analytical methods have found abundant use in genomics and proteomics with fledgling contributions to virology – a clinically critical field with emphasis on both infectious and designer-drug carriers. Here we demonstrate the ability of SSN to successfully discriminate adeno-associated viruses (AAVs) based on their genetic cargo [double-stranded DNA (AAV_dsDNA), single-stranded DNA (AAV_ssDNA) or none (AAV_empty)], devoid of digestion steps, through nanopore-induced electro-deformation (characterized by relative current change; ΔI/I₀). The deformation order was found to be AAV_empty > AAV_ssDNA > AAV_dsDNA. A deep learning algorithm was developed by integrating support vector machine with an existing neural network, which successfully classified AAVs from SSN resistive-pulses (characteristic of genetic cargo) with >95% accuracy – a potential tool for clinical and biomedical applications. Subsequently, the presence of AAV_empty in spiked AAV_dsDNA was flagged using the ΔI/I₀ distribution characteristics of the two types for mixtures composed of ∼75 : 25% and ∼40 : 60% (in concentration) AAV_empty: AAV_dsDNA.  

5.2985           Production of the Cytokine VEGF-A by CD4+ T and Myeloid Cells Disrupts the Corneal Nerve Landscape and Promotes Herpes Stromal Keratitis

Yun, H., Yee, M.B., Lathrop, K.L., Kinchinton, P.R., Hendricks, R.L. and St. Leger, A.J. Immunity, 53, 1050-1062 (2020)   Herpes simplex virus type 1 (HSV-1)-infected corneas can develop a blinding immunoinflammatory condition called herpes stromal keratitis (HSK), which involves the loss of corneal sensitivity due to retraction of sensory nerves and subsequent hyperinnervation with sympathetic nerves. Increased concentrations of the cytokine VEGF-A in the cornea are associated with HSK severity. Here, we examined the impact of VEGF-A on neurologic changes that underly HSK using a mouse model of HSV-1 corneal infection. Both CD4⁺ T cells and myeloid cells produced pathogenic levels of VEGF-A within HSV-1-infected corneas, and CD4⁺ cell depletion promoted reinnervation of HSK corneas with sensory nerves. In vitro, VEGF-A from infected corneas repressed sensory nerve growth and promoted sympathetic nerve growth. Neutralizing VEGF-A in vivo using bevacizumab inhibited sympathetic innervation, promoted sensory nerve regeneration, and alleviated disease. Thus, VEGF-A can shape the sensory and sympathetic nerve landscape within the cornea, with implications for the treatment of blinding corneal disease.  

5.2986           Primary and Secondary Dengue Virus Infections Elicit Similar Memory B-Cell Responses, but Breadth to Other Serotypes and Cross-Reactivity to Zika Virus Is Higher in Secondary Dengue

Andrade, P., Narvekar, P., Montoya, M., Michlmayr, D., Balmaseda, A. Coleman, J. and Harris, E.

1. Infect. Dis., 222(4), 590-600 (2020)

  Background The 4 antigenically distinct serotypes of dengue virus (DENV) share extensive homology with each other and with the closely related Zika flavivirus (ZIKV). The development of polyclonal memory B cells (MBCs) to the 4 DENV serotypes and ZIKV during DENV infection is not fully understood. Methods In this study, we analyzed polyclonal MBCs at the single-cell level from peripheral blood mononuclear cells collected ~2 weeks or 6–7 months postprimary or postsecondary DENV infection from a pediatric hospital-based study in Nicaragua using a Multi-Color FluoroSpot assay. Results Dengue virus elicits robust type-specific and cross-reactive MBC responses after primary and secondary DENV infection, with a significantly higher cross-reactive response in both. Reactivity to the infecting serotype dominated the total MBC response. Although the frequency and proportion of type-specific and cross-reactive MBCs were comparable between primary and secondary DENV infections, within the cross-reactive response, the breadth of MBC responses against different serotypes was greater after secondary DENV infection. Dengue virus infection also induced cross-reactive MBC responses recognizing ZIKV, particularly after secondary DENV infection. Conclusions Overall, our study sheds light on the polyclonal MBC response to DENV and ZIKV in naive and DENV-preimmune subjects, with important implications for natural infections and vaccine development.  

5.2987           Lack of APP and APLP2 in GABAergic Forebrain Neurons Impairs Synaptic Plasticity and Cognition

Mehr, A., Hick, M., Ludewig, S., Müller, M.,Herrmann, U., von Engelhardt, J., Wolfer, D.P., Korte, M. and Müller, U.C. Cerebral Cortex, 30, 4044-4063 (2020)   Amyloid-β precursor protein (APP) is central to the pathogenesis of Alzheimer’s disease, yet its physiological functions remain incompletely understood. Previous studies had indicated important synaptic functions of APP and the closely related homologue APLP2 in excitatory forebrain neurons for spine density, synaptic plasticity, and behavior. Here, we show that APP is also widely expressed in several interneuron subtypes, both in hippocampus and cortex. To address the functional role of APP in inhibitory neurons, we generated mice with a conditional APP/APLP2 double knockout (cDKO) in GABAergic forebrain neurons using DlxCre mice. These DlxCre cDKO mice exhibit cognitive deficits in hippocampus-dependent spatial learning and memory tasks, as well as impairments in species-typic nesting and burrowing behaviors. Deficits at the behavioral level were associated with altered neuronal morphology and synaptic plasticity Long-Term Potentiation (LTP). Impaired basal synaptic transmission at the Schafer collateral/CA1 pathway, which was associated with altered compound excitatory/inhibitory synaptic currents and reduced action potential firing of CA1 pyramidal cells, points to a disrupted excitation/inhibition balance in DlxCre cDKOs. Together, these impairments may lead to hippocampal dysfunction. Collectively, our data reveal a crucial role of APP family proteins in inhibitory interneurons to maintain functional network activity.  

5.2988           Restoring the natural tropism of AAV2 vectors for human liver

Cabanes-Creus, M., Hallwirth, C.V., Westhaus, A., Ng, B.H., Liao, S.H.Y., Zhu, E. et al Sci. Transl. Med., 12, eaba3312 (2020)   Recent clinical successes in gene therapy applications have intensified interest in using adeno-associated viruses (AAVs) as vectors for therapeutic gene delivery. Although prototypical AAV2 shows robust in vitro transduction of human hepatocyte–derived cell lines, it has not translated into an effective vector for liver-directed gene therapy in vivo. This is consistent with observations made in Fah^−/−/Rag2^−/−/Il2rg^−/− (FRG) mice with humanized livers, showing that AAV2 functions poorly in this xenograft model. Here, we derived naturally hepatotropic AAV capsid sequences from primary human liver samples. We demonstrated that capsid mutations, likely acquired as an unintentional consequence of tissue culture propagation, attenuated the intrinsic human hepatic tropism of natural AAV2 and related human liver AAV isolates. These mutations resulted in amino acid changes that increased binding to heparan sulfate proteoglycan (HSPG), which has been regarded as the primary cellular receptor mediating AAV2 infection of human hepatocytes. Propagation of natural AAV variants in vitro showed tissue culture adaptation with resulting loss of tropism for human hepatocytes. In vivo readaptation of the prototypical AAV2 in FRG mice with a humanized liver resulted in restoration of the intrinsic hepatic tropism of AAV2 through decreased binding to HSPG. Our results challenge the notion that high affinity for HSPG is essential for AAV2 entry into human hepatocytes and suggest that natural AAV capsids of human liver origin are likely to be more effective for liver-targeted gene therapy applications than culture-adapted AAV2.  

5.2989           Hippocampal knockdown of α2 nicotinic or M1 muscarinic acetylcholine receptors in C57BL/6J male mice impairs cued fear conditioning

Mineur, Y.S., Ernsten, C., Islam, A., Maibom, K.L. and Picciotto, M.R. Genes, Brain and Behavior, 19(6), e12677 (2020)   Acetylcholine (ACh) signaling in the hippocampus is important for behaviors related to learning, memory and stress. In this study, we investigated the role of two ACh receptor subtypes previously shown to be involved in fear and anxiety, the M1 mAChR and the α2 nAChR, in mediating the effects of hippocampal ACh on stress-related behaviors. Adeno-associated viral vectors containing short-hairpin RNAs targeting M1 or α2 were infused into the hippocampus of male C57BL/6J mice, and behavior in a number of paradigms related to stress responses and fear learning was evaluated. There were no robust effects of hippocampal M1 mAChR or α2 nAChR knockdown (KD) in the light/dark box, tail suspension, forced swim or novelty-suppressed feeding tests. However, effects on fear learning were observed in both KD groups. Short term learning was intact immediately after training in all groups of mice, but both the M1 and α2 hippocampal knock down resulted in impaired cued fear conditioning 24 h after training. In addition, there was a trend for a deficit in contextual memory the M1 mAChR KD group 24 h after training. These results suggest that α2 nicotinic and M1 muscarinic ACh receptors in the hippocampus contribute to fear learning and could be relevant targets to modify brain circuits involved in stress-induced reactivity to associated cues.  

5.2990           Improved Noninvasive In Vivo Tracking of AAV-9 Gene Therapy Using the Perchlorate-Resistant Sodium Iodide Symporter from Minke Whale

Concilio, S.C., Suksanpaisan, L., Pham, L., Peng, K-W. and Russell, S.J. Molecular Therapy, 29(1), 236-243 (2021)   The sodium iodide symporter (NIS) is widely used as a reporter gene to noninvasively monitor the biodistribution and durability of vector-mediated gene expression via gamma scintigraphy, single-photon emission computed tomography (SPECT), and positron-emission tomography (PET). However, the approach is limited by background signal due to radiotracer uptake by endogenous NIS-expressing tissues. In this study, using the SPECT tracer pertechnetate (^99mTcO₄) and the PET tracer tetrafluoroborate (B¹⁸F₄), in combination with the NIS inhibitor perchlorate, we compared the transport properties of human NIS and minke whale (Balaenoptera acutorostrata scammoni) NIS in vitro and in vivo. Based on its relative resistance to perchlorate, the NIS protein from minke whale appeared to be the superior candidate reporter gene. SPECT and PET imaging studies in nude mice challenged with NIS-encoding adeno-associated virus (AAV)-9 vectors confirmed that minke whale NIS, in contrast to human and endogenous mouse NIS, continues to function as a reliable reporter even when background radiotracer uptake by endogenous NIS is blocked by perchlorate.  

5.2991           Enhanced Efficacy and Increased Long-Term Toxicity of CNS-Directed, AAV-Based Combination Therapy for Krabbe Disease

Li, Y., Miller, C.A., Shea, L.K., Jiang, X., Guzman, M.A., Chandler, R.J., Ramakrishnan, S.M., Smith, S.N., Vendetti, C.P., Vogler, C.A., Ory, D.S., Ley, T.J: and Sands, M.S. Molecular Therapy, 29(2), 691-701 (2021)   Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC) and the progressive accumulation of the toxic metabolite psychosine. We showed previously that central nervous system (CNS)-directed, adeno-associated virus (AAV)2/5-mediated gene therapy synergized with bone marrow transplantation and substrate reduction therapy (SRT) to greatly increase therapeutic efficacy in the murine model of Krabbe disease (Twitcher). However, motor deficits remained largely refractory to treatment. In the current study, we replaced AAV2/5 with an AAV2/9 vector. This single change significantly improved several endpoints primarily associated with motor function. However, nearly all (14/16) of the combination-treated Twitcher mice and all (19/19) of the combination-treated wild-type mice developed hepatocellular carcinoma (HCC). 10 out of 10 tumors analyzed had AAV integrations within the Rian locus. Several animals had additional integrations within or near genes that regulate cell growth or death, are known or potential tumor suppressors, or are associated with poor prognosis in human HCC. Finally, the substrate reduction drug L-cycloserine significantly decreased the level of the pro-apoptotic ceramide 18:0. These data demonstrate the value of AAV-based combination therapy for Krabbe disease. However, they also suggest that other therapies or co-morbidities must be taken into account before AAV-mediated gene therapy is considered for human therapeutic trials.  

5.2992           Anti-tau scFvs Targeted to the Cytoplasm or Secretory Pathway Variably Modify Pathology and Neurodegenerative Phenotypes

Goodwin, M.S., Sinyavskaya, O., Burg, F., O’Neal, V., Ceballos-Diaz, C., Cruz, P.E., Lewis, J., Giasson, B.J., Davies, P., Golde, T.E: and levites, Y. Molecular Therapy, 29(2), 859-872 (2021)   Immunotherapies designed to treat neurodegenerative tauopathies that primarily engage extracellular tau may have limited efficacy as tau is primarily intracellular. We generated tau-targeting single-chain variable fragments (scFvs) and intrabodies (iBs) from the phosphorylated tau-specific antibodies CP13 and PHF1 and the pan-tau antibody Tau5. Recombinant adeno-associated virus (rAAV) was utilized to express these antibody fragments in homozygous JNPL3 P301L tau mice. Two iBs (CP13i, PHF1i) and one scFv (PHF1s) abrogated tau pathology and delayed time to severe hindlimb paralysis. In a second tauopathy model (rTg4510), CP13i and PHF1i reduced tau pathology, but cognate scFvs did not. These data demonstrate that (1) disease-modifying efficacy does not require antibody effector functions, (2) the intracellular targeting of tau with phosphorylated tau-specific iBs is more effective than extracellular targeting with the scFvs, and (3) robust effects on tau pathology before neurodegeneration only resulted in modest disease modification as assessed by delay of severe motor phenotype.  

5.2993           Single and Dual Vector Gene Therapy with AAV9-PHP.B Rescues Hearing in Tmc1 Mutant Mice

Wu, J., Solanes, P., Nist-Lund, C., Spataro, S., Shubinaa-Oleinik, O., Marcovich, I., Goldberg, H., Schneider, B.L. and Holt, J.R. Molecular Therapy, 29(3), 973-988 (2021)   AAV-mediated gene therapy is a promising approach for treating genetic hearing loss. Replacement or editing of the Tmc1 gene, encoding hair cell mechanosensory ion channels, is effective for hearing restoration in mice with some limitations. Efficient rescue of outer hair cell function and lack of hearing recovery with later-stage treatment remain issues to be solved. Exogenous genes delivered with the adeno-associated virus (AAV)9-PHP.B capsid via the utricle transduce both inner and outer hair cells of the mouse cochlea with high efficacy. Here, we demonstrate that AAV9-PHP.B gene therapy can promote hair cell survival and successfully rescues hearing in three distinct mouse models of hearing loss. Tmc1 replacement with AAV9-PHP.B in a Tmc1 knockout mouse rescues hearing and promotes hair cell survival with equal efficacy in inner and outer hair cells. The same treatment in a recessive Tmc1 hearing-loss model, Baringo, partially recovers hearing even with later-stage treatment. Finally, dual delivery of Streptococcus pyogenes Cas9 (SpCas9) and guide RNA (gRNA) in separate AAV9-PHP.B vectors selectively disrupts a dominant Tmc1 allele and preserves hearing in Beethoven mice, a model of dominant, progressive hearing loss. Tmc1-targeted gene therapies using single or dual AAV9-PHP.B vectors offer potent and versatile approaches for treating dominant and recessive deafness.  

5.2994           Increasing the Specificity of AAV-Based Gene Editing through Self-Targeting and Short-Promoter Strategies

Breton, C., Furmanak, T., Avitto, A.N., Smith, A.M., larshaw, C., Yan, H., Greig, J.A: and Wilson, J.M. Molecular Therapy, 29(3), 1047-1056 (2021)   Our group previously used adeno-associated viral vectors (AAVs) to express an engineered meganuclease specific for a sequence in the PCSK9 gene (M2PCSK9), a clinical target for treating coronary heart disease. Upon testing this nuclease in non-human primates, we observed specific editing characterized by several insertions and deletions (indels) in the target sequence as well as indels in similar genomic sequences. We hypothesized that high nuclease expression increases off-target editing. Here, we reduced nuclease expression using two strategies. The first was a self-targeting strategy that involved inserting the M2PCSK9 target sequence into the AAV genome that expresses the nuclease and/or fusing the nuclease to a specific peptide to promote its degradation. The second strategy used a shortened version of the parental promoter to reduce nuclease expression. Mice administered with these second-generation AAV vectors showed reduced PCSK9 expression due to the nuclease on-target activity and reduced off-target activity. All vectors induced a stable reduction of PCSK9 in primates treated with self-targeting and short-promoter AAVs. Compared to the meganuclease-expressing parental AAV vector, we observed a significant reduction in off-target activity. In conclusion, we increased the in vivo nuclease specificity using a clinically relevant strategy that can be applied to other genome-editing nucleases.  

5.2995           Telomerase therapy attenuates cardiotoxic effects of doxorubicin

Chatterjee, S., Hofer, T., Costa, A., Lu, D., batkai, S., Gupta, S.K., Boledani, E., Zweigerdt, R., Megias, D., Streckfuss-Bömeke, K., Brandenberger, C., Thum, T. and Bär, C. Molecular Therapy, 29(4), 1395-1410 (2021)   Doxorubicin is one of the most potent chemotherapeutic agents. However, its clinical use is restricted due to the severe risk of cardiotoxicity, partially attributed to elevated production of reactive oxygen species (ROS). Telomerase canonically maintains telomeres during cell division but is silenced in adult hearts. In non-dividing cells such as cardiomyocytes, telomerase confers pro-survival traits, likely owing to the detoxification of ROS. Therefore, we hypothesized that pharmacological overexpression of telomerase may be used as a therapeutic strategy for the prevention of doxorubicin-induced cardiotoxicity. We used adeno-associated virus (AAV)-mediated gene therapy for long-term expression of telomerase in in vitro and in vivo models of doxorubicin-induced cardiotoxicity. Overexpression of telomerase protected the heart from doxorubicin-mediated apoptosis and rescued cardiac function, which was accompanied by preserved cardiomyocyte size. At the mechanistic level, we observed altered mitochondrial morphology and dynamics in response to telomerase expression. Complementary in vitro experiments confirmed the anti-apoptotic effects of telomerase overexpression in human induced pluripotent stem cell-derived cardiomyocytes after doxorubicin treatment. Strikingly, elevated levels of telomerase translocated to the mitochondria upon doxorubicin treatment, which helped to maintain mitochondrial function. Thus, telomerase gene therapy could be a novel preventive strategy for cardiotoxicity by chemotherapy agents such as the anthracyclines.  

5.2996           Fusogenic oncolytic vaccinia virus enhances systemic antitumor immune response by modulating the tumor microenvironment

Nakatake, M., Kuwano, N., Kaitsurumaru, E., Kurosaki, H. and Nakamura, T. Molecular Therapy, 29(5), 1782-1793 (2021)   Oncolytic viruses induce antitumor immunity following direct viral oncolysis. However, their therapeutic effects are limited in distant untreated tumors because their antitumor function depends on indirect antitumor immunity. Here, we generated a novel fusogenic oncolytic vaccinia virus (FUVAC) and compared its antitumor activity with that of its parental non-fusogenic virus. Compared with the parent, FUVAC exerted the cytopathic effect and induced immunogenic cell death in human and murine cancer cells more efficiently. In a bilateral tumor-bearing syngeneic mouse model, FUVAC administration significantly inhibited tumor growth in both treated and untreated tumors. However, its antitumor effects were completely suppressed by CD8⁺ T cell depletion. Notably, FUVAC reduced the number of tumor-associated immune-suppressive cells in treated tumors, but not in untreated tumors. Mice treated with FUVAC before an immune checkpoint inhibitor (ICI) treatment achieved complete response (CR) in both treated and untreated tumors, whereas ICI alone did not show antitumor activity. Mice achieving CR rejected rechallenge with the same tumor cells, suggesting establishment of a long-term tumor-specific immune memory. Thus, FUVAC improves the tumor immune microenvironment and enhances systemic antitumor immunity, suggesting that, alone and in combination with ICI, it is a novel immune modulator for overcoming oncolytic virus-resistant tumors.  

5.2997           Systemic Treatment of Fabry Disease Using a Novel AAV9 Vector Expressing α-Galactosidase A

Biferi, M.G., Cohen-Tannoudji, M., Garcia-Silva, A., Souto-Rodriguez, O., Vieitez-Gonzalez, I. et al Molecular Therapy-Methods & Clin. Develop., 20, 1-17 (2021)   Fabry disease is a rare X-linked disorder affecting α-galactosidase A, a rate-limiting enzyme in lysosomal catabolism of glycosphingolipids. Current treatments present important limitations, such as low half-life and limited distribution, which gene therapy can overcome. The aim of this work was to test a novel adeno-associated viral vector, serotype 9 (AAV9), ubiquitously expressing human α-galactosidase A to treat Fabry disease (scAAV9-PGK-GLA). The vector was preliminary tested in newborns of a Fabry disease mouse model. 5 months after treatment, α-galactosidase A activity was detectable in the analyzed tissues, including the central nervous system. Moreover, we tested the vector in adult animals of both sexes at two doses and disease stages (presymptomatic and symptomatic) by single intravenous injection. We found that the exogenous α-galactosidase A was active in peripheral tissues as well as the central nervous system and prevented glycosphingolipid accumulation in treated animals up to 5 months following injection. Antibodies against α-galactosidase A were produced in 9 out of 32 treated animals, although enzyme activity in tissues was not significantly affected. These results demonstrate that scAAV9-PGK-GLA can drive widespread and sustained expression of α-galactosidase A, cross the blood brain barrier after systemic delivery, and reduce pathological signs of the Fabry disease mouse model.  

5.2998           CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice

Stone, D., Long, K.R., Loprieno, M.A., De Silva Feelixge, H.S., Kenkel, E.J. et al Molecular Therapy-Methods & Clin. Develop., 20, 258-275 (2021)   Chronic hepatitis B virus (HBV) infection is a major public health problem. New treatment approaches are needed because current treatments do not target covalently closed circular DNA (cccDNA), the template for HBV replication, and rarely clear the virus. We harnessed adeno-associated virus (AAV) vectors and CRISPR-Staphylococcus aureus (Sa)Cas9 to edit the HBV genome in liver-humanized FRG mice chronically infected with HBV and receiving entecavir. Gene editing was detected in livers of five of eight HBV-specific AAV-SaCas9-treated mice, but not control mice, and mice with detectable HBV gene editing showed higher levels of SaCas9 delivery to HBV⁺ human hepatocytes than those without gene editing. HBV-specific AAV-SaCas9 therapy significantly improved survival of human hepatocytes, showed a trend toward decreasing total liver HBV DNA and cccDNA, and was well tolerated. This work provides evidence for the feasibility and safety of in vivo gene editing for chronic HBV infections, and it suggests that with further optimization, this approach may offer a plausible way to treat or even cure chronic HBV infections.  

5.2999           Applying machine learning to predict viral assembly for adeno-associated virus capsid libraries

Marques, A.D., Kummer, M., Kondratov, O., Banerjee, A., Moskalenko, O. and Zolotukhin, S. Molecular Therapy-Methods Clin. Develop., 20, 276-286 (2021)   Machine learning (ML) can aid in novel discoveries in the field of viral gene therapy. Specifically, big data gathered through next-generation sequencing (NGS) of complex capsid libraries is an especially prominent source of lost potential in data analysis and prediction. Furthermore, adeno-associated virus (AAV)-based capsid libraries are becoming increasingly popular as a tool to select candidates for gene therapy vectors. These higher complexity AAV capsid libraries have previously been created and selected in vivo; however, in silico analysis using ML computer algorithms may augment smarter and more robust libraries for selection. In this study, data of AAV capsid libraries gathered before and after viral assembly are used to train ML algorithms. We found that two ML computer algorithms, artificial neural networks (ANNs), and support vector machines (SVMs), can be trained to predict whether unknown capsid variants may assemble into viable virus-like structures. Using the most accurate models constructed, hypothetical mutation patterns in library construction were simulated to suggest the importance of N495, G546, and I554 in AAV2-derived capsids. Finally, two comparative libraries were generated using ML-derived data to biologically validate these findings and demonstrate the predictive power of ML in vector design.  

5.3000           Rapid evolution of blood-brain-barrier-penetrating AAV capsids by RNA-driven biopanning

Nonnenmacher, M., Wang, W., Child, M.A., Ren, X-Q., Huang, C., Ren, A.Z., Tocci, J., Chen, Q., Bittner, K., Tyson, K., Pande, N., Chung, C.H-Y., Paul, S.M. and Hou, J. Molecular Therapy-Methods & Clin. Develop., 20, 366-378 (2021)   Therapeutic payload delivery to the central nervous system (CNS) remains a major challenge in gene therapy. Recent studies using function-driven evolution of adeno-associated virus (AAV) vectors have successfully identified engineered capsids with improved blood-brain barrier (BBB) penetration and CNS tropism in mouse. However, these strategies require transgenic animals and thus are limited to rodents. To address this issue, we developed a directed evolution approach based on recovery of capsid library RNA transcribed from CNS-restricted promoters. This RNA-driven screen platform, termed TRACER (Tropism Redirection of AAV by Cell-type-specific Expression of RNA), was tested in the mouse with AAV9 peptide display libraries and showed rapid emergence of dominant sequences. Ten individual variants were characterized and showed up to 400-fold higher brain transduction over AAV9 following systemic administration. Our results demonstrate that the TRACER platform allows rapid selection of AAV capsids with robust BBB penetration and CNS tropism in non-transgenic animals.  

5.3001           AAV-CRB2 protects against vision loss in an inducible CRB1 retinitis pigmentosa mouse model

Buck, T.M., Vos, R.M., Alves, C.H. and Wijnholds, J. Molecular Therapy-Methods & Clin. Develop., 20, 423-441 (2021)   Loss of Crumbs homolog 1 (CRB1) or CRB2 proteins in Müller cells or photoreceptors in the mouse retina results in a CRB dose-dependent retinal phenotype. In this study, we present a novel Müller cell-specific Crb1^KOCrb2^LowMGC retinitis pigmentosa mouse model (complete loss of CRB1 and reduced levels of CRB2 specifically in Müller cells). The Crb double mutant mice showed deficits in electroretinography, optokinetic head tracking, and retinal morphology. Exposure of retinas to low levels of dl-α-aminoadipate acid induced gliosis and retinal disorganization in Crb1^KOCrb2^LowMGC retinas but not in wild-type or Crb1-deficient retinas. Crb1^KOCrb2^LowMGC mice showed a substantial decrease in inner/outer photoreceptor segment length and optokinetic head-tracking response. Intravitreal application of rAAV vectors expressing human CRB2 (hCRB2) in Müller cells of Crb1^KOCrb2^LowMGC mice subsequently exposed to low levels of dl-α-aminoadipate acid prevented loss of vision, whereas recombinant adeno-associated viral (rAAV) vectors expressing human CRB1 (hCRB1) did not. Both rAAV vectors partially protected the morphology of the retina. The results suggest that hCRB expression in Müller cells is vital for control of retinal cell adhesion at the outer limiting membrane, and that the rAAV-cytomegalovirus (CMV)-hCRB2 vector is more potent than rAAV-minimal CMV (CMVmin)-hCRB1 in protection against loss of vision.    

5.3002           AAV9-mediated gene delivery of MCT1 to oligodendrocytes does not provide a therapeutic benefit in a mouse model of ALS

Eykens, C., Rossaert, E., Duque, S., Rue, L., Bento-Abreu, A. et al Molecular Therapy-Methods & Clin. Develop., 20, 508-519 (2021)   Oligodendrocyte dysfunction has been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by progressive motor neuron loss. The failure of trophic support provided by oligodendrocytes is associated with a concomitant reduction in oligodendroglial monocarboxylate transporter 1 (MCT1) expression and is detrimental for the long-term survival of motor neuron axons. Therefore, we established an adeno-associated virus 9 (AAV9)-based platform by which MCT1 was targeted mostly to white matter oligodendrocytes to investigate whether this approach could provide a therapeutic benefit in the SOD1^G93A mouse model of ALS. Despite good oligodendrocyte transduction and AAV-mediated MCT1 transgene expression, the disease outcome of SOD1^G93A mice was not altered. Our study further increases our current understanding about the complex nature of oligodendrocyte pathology in ALS and provides valuable insights into the future development of therapeutic strategies to efficiently modulate these cells.  

5.3003           Preclinical biodistribution, tropism, and efficacy of oligotropic AAV/Olig001 in a mouse model of congenital white matter disease

Francis, J.S:, Markov, V., Wojtas, I.D., Gray, S., McCown, T., Samulski, R.J., Figueroa, M. and Leone, P. Molecular Therapy-Methods & Clin. Develop., 20, 520-534 (2021)   Recent advances in adeno-associated viral (AAV) capsid variants with novel oligotropism require validation in models of disease in order to be viable candidates for white matter disease gene therapy. We present here an assessment of the biodistribution, tropism, and efficacy of a novel AAV capsid variant (AAV/ Olig001) in a model of Canavan disease. We first define a combination of dose and route of administration of an AAV/Olig001-GFP reporter conducive to widespread CNS oligodendrocyte transduction in acutely symptomatic animals that model the Canavan brain at time of diagnosis. Administration of AAV/Olig001-GFP resulted in >70% oligotropism in all regions of interest except the cerebellum without the need for lineage-specific expression elements. Intracerebroventricular infusion into the cerebrospinal fluid (CSF) was identified as the most appropriate route of administration and employed for delivery of an AAV/Olig001 vector to reconstitute oligodendroglial aspartoacylase (ASPA) in adult Canavan mice, which resulted in a dose-dependent rescue of ASPA activity, motor function, and a near-total reduction in vacuolation. A head-to-head efficacy comparison with astrogliotropic AAV9 highlighted a significant advantage conferred by oligotropic AAV/Olig001 that was independent of overall transduction efficiency. These results support the continued development of AAV/Olig001 for advancement to clinical application to white matter disease.  

5.3004           CRISPR-mediated rapid generation of neural cell-specific knockout mice facilitates research in neurophysiology and pathology

Xiao, D., Zhang, W., Wang, Q., LI, X., Zhang, Y., Rasouli, J., Casella, G., Ciric, B., Curtis, M., Rostami, A. and Zhang, G-X. Molecular Therapy-Methods & Clin. Develop., 20, 755-764 (2021)   Inducible conditional knockout mice are important tools for studying gene function and disease therapy, but their generation is costly and time-consuming. We introduced clustered regularly interspaced short palindromic repeats (CRISPR) and Cre into an LSL-Cas9 transgene-carrying mouse line by using adeno-associated virus (AAV)-PHP.eB to rapidly knockout gene(s) specifically in central nervous system (CNS) cells of adult mice. NeuN in neurons and GFAP in astrocytes were knocked out 2 weeks after an intravenous injection of vector, with an efficiency comparable to that of inducible Cre-loxP conditional knockout. For functional testing, we generated astrocyte-specific Act1 knockout mice, which exhibited a phenotype similar to mice with Cre-loxP-mediated Act1 knockout, in an animal model of multiple sclerosis (MS), an autoimmune disorder of the CNS. With this novel technique, neural cell-specific knockout can be induced rapidly (few weeks) and cost-effectively. Our study provides a new approach to building inducible conditional knockout mice, which would greatly facilitate research on CNS biology and disease.  

5.3005           AAV-S: A versatile capsid variant for transduction of mouse and primate inner ear

Ivanchenko, M.V., Hanlon, K.S., hathaway, D.M., Klein, A.J., peters, C.W., Li, Y., Tamvakologos, P.I., Nammour, J., Maguire, C.A. and Corey, D.P. Molecular Therapy-Methods & Clin. Develop., 21, 382-398 (2021)   Gene therapy strategies using adeno-associated virus (AAV) vectors to treat hereditary deafnesses have shown remarkable efficacy in some mouse models of hearing loss. Even so, there are few AAV capsids that transduce both inner and outer hair cells—the cells that express most deafness genes—and fewer still shown to transduce hair cells efficiently in primates. AAV capsids with robust transduction of inner and outer hair cells in primate cochlea will be needed for most clinical trials. Here, we test a capsid that we previously isolated from a random capsid library, AAV-S, for transduction in mouse and non-human primate inner ear. In both mice and cynomolgus macaques, AAV-S mediates highly efficient reporter gene expression in a variety of cochlear cells, including inner and outer hair cells, fibrocytes, and supporting cells. In a mouse model of Usher syndrome type 3A, AAV-S encoding CLRN1 robustly and durably rescues hearing. Overall, our data indicate that AAV-S is a promising candidate for therapeutic gene delivery to the human inner ear.  

5.3006           Single amino acid insertion allows functional transduction of murine hepatocytes with human liver tropic AAV capsids

Cabanes-Creus, M., Navarro, R.G., Liao, S.H:Y., Baltazar, G., Drouyer, M., Zhu, E., Scott, S., Luong, C., Wilson, L.O.W., Alexander, I.E. and Lisowski, L. Molecular Therapy-Methods & Clin. Develop., 21, 607-620 (82021)   Recent successes in clinical gene therapy applications have intensified the interest in using adeno-associated viruses (AAVs) as vectors for gene delivery into human liver. An inherent intriguing characteristic of AAVs is that vector variants vary substantially in their ability to transduce hepatocytes from different species. This has historically limited the value of preclinical studies using rodent models for predicting the efficiency of AAV vectors in liver-targeted gene therapy clinical studies. In this work, we aimed to investigate the key determinants of the observed differential interspecies transduction abilities among AAV variants. We took advantage of domain swapping strategies between AAV-KP1, a newly identified variant with enhanced murine liver tropism, and AAV3b, which functions poorly in mice. The systematic in vivo comparison of AAV3b/AAV-KP1 chimeric variants allowed us to identify a threonine insertion at position 265 within variable region I (VR-I) as the key residue that confers murine hepatic transduction to human-derived clade B (AAV2-like) and clade C (AAV3b-like) variants. We propose to use this insertion to generate phylogenetically related AAV surrogates in support of toxicology and dosing studies in the murine liver model.  

5.3007           Substantial restoration of night vision in adult mice with congenital stationary night blindness

Varin, J., Bouzidi, N., Gauvain, G., Joffrois, C., Desrosiers, M., Robert, C., De Sousa Diaz, M.M., Neuille, M., Michiels, C., Nassisi, M., Sahel, J-A., Picaud, S., Audo, I., Dalkara, D. and Zeitz, C. Molecular Therapy-Methods & Clin. Develop., 22, 15-25 (2021)   Complete congenital stationary night blindness (cCSNB) due to mutations in TRPM1, GRM6, GPR179, NYX, or leucine-rich repeat immunoglobulin-like transmembrane domain 3 (LRIT3) is an incurable inherited retinal disorder characterized by an ON-bipolar cell (ON-BC) defect. Since the disease is non-degenerative and stable, treatment could theoretically be administrated at any time in life, making it a promising target for gene therapy. Until now, adeno-associated virus (AAV)-mediated therapies lead to significant functional improvements only in newborn cCSNB mice. Here we aimed to restore protein localization and function in adult Lrit3^−/− mice. LRIT3 localizes in the outer plexiform layer and is crucial for TRPM1 localization at the dendritic tips of ON-BCs and the electroretinogram (ERG)-b-wave. AAV2-7m8-Lrit3 intravitreal injections were performed targeting either ON-BCs, photoreceptors (PRs), or both. Protein localization of LRIT3 and TRPM1 at the rod-to-rod BC synapse, functional rescue of scotopic responses, and ON-responses detection at the ganglion cell level were achieved in a few mice when ON-BCs alone or both PRs and ON-BCs, were targeted. More importantly, a significant number of treated adult Lrit3^−/− mice revealed an ERG b-wave recovery under scotopic conditions, improved optomotor responses, and on-time ON-responses at the ganglion cell level when PRs were targeted. Functional rescue was maintained for at least 4 months after treatment.  

5.3008           Outcomes of progranulin gene therapy in the retina are dependent on time and route of delivery

Zin, E.A., Han, D., tran, J., Morisson-Welch, N., Visel, M., Kuronen, M. and Flannery, J.C. Molecular Therapy-Methods & Clin. Develop., 22, 40-51(2021)   Neuronal ceroid lipofuscinosis (NCL) is a family of neurodegenerative diseases caused by mutations to genes related to lysosomal function. One variant, CNL11, is caused by mutations to the gene encoding the protein progranulin, which regulates neuronal lysosomal function. Absence of progranulin causes cerebellar atrophy, seizures, dementia, and vision loss. As progranulin gene therapies targeting the brain are developed, it is advantageous to focus on the retina, as its characteristics are beneficial for gene therapy development: the retina is easily visible through direct imaging, can be assessed through quantitative methods in vivo, and requires smaller amounts of adeno-associated virus (AAV). In this study we characterize the retinal degeneration in a progranulin knockout mouse model of CLN11 and study the effects of gene replacement at different time points. Mice heterologously expressing progranulin showed a reduction in lipofuscin deposits and microglia infiltration. While mice that receive systemic AAV92YF-scCAG-PGRN at post-natal day 3 or 4 show a reduction in retina thinning, mice injected intravitreally at months 1 and 6 with AAV2.7m8-scCAG-PGRN exhibit no improvement, and mice injected at 12 months of age have thinner retinas than do their controls. Thus, delivery of progranulin proves to be time sensitive and dependent on route of administration, requiring early delivery for optimal therapeutic benefit.  

5.3009           Characterizing the cellular immune response to subretinal AAV gene therapy in the murine retina

Chandler, L.C., McClements, M.E., Yusuf, I.H., de la Camara, C., M-F. Molecular Therapy-Methods & Clin. Develop., 22, 52-65 (2021)   Although adeno-associated viral (AAV) vector-mediated retinal gene therapies have demonstrated efficacy, the mechanisms underlying dose-dependent retinal inflammation remain poorly understood. Here, we present a quantitative analysis of cellular immune response to subretinal AAV gene therapy in mice using multicolor flow cytometry with a panel of key immune cell markers. A significant increase in CD45⁺ retinal leukocytes was detected from day 14 post-subretinal injection of an AAV8 vector (1 × 10⁹ genome copies) encoding green fluorescent protein (GFP) driven by a ubiquitous promoter. These predominantly consisted of infiltrating peripheral leukocytes including macrophages, natural killer cells, CD4 and CD8 T cells, and natural killer T cells; no significant change in resident microglia population was detected. This cellular response was persistent at 28 days and suggestive of type 1 cell-mediated effector immunity. High levels (80%) of GFP fluorescence were found in the microglia, implicating their role in viral antigen presentation and peripheral leukocyte recruitment. When compared against AAV.GFP in paired eyes, an equivalent dose of an otherwise identical vector encoding the human therapeutic transgene Rab-escort protein 1 (REP1) elicited a significantly diminished cellular immune response (4.2-fold; p = 0.0221). However, the distribution of immune cell populations remained similar, indicating a common mechanism of AAV-induced immune activation.  

5.3010           Pre-retinal delivery of recombinant adeno-associated virus vector significantly improves retinal transduction efficiency

Zhang, H., Sajdak, B.S., Merriman, D.K., Carroll, J. and Lipinski, D.M. Molecular Therapy-Methods & Clin. Develop., 22, 96-106 (2021)   Intravitreal injection is the most widely used injection technique for ocular gene delivery. However, vector diffusion is attenuated by physical barriers and neutralizing antibodies in the vitreous. The 13-lined ground squirrel (13-LGS), as in humans, has a larger relative vitreous body volume than the more common rodent models such as rats and mice, which would further reduce transduction efficiency with the intravitreal injection route. We report here a “pre-retinal” injection approach that leads to detachment of the posterior hyaloid membrane and delivers vector into the space between vitreous and inner retina. Vectors carrying a ubiquitously expressing mCherry reporter were injected into the deep vitreous or pre-retinal space in adult wild-type 13-LGSs. Then, adeno-associated virus (AAV)-mediated mCherry expression was evaluated with non-invasive imaging, immunofluorescence, and flow cytometry. Compared to deep vitreous delivery, pre-retinal administration achieved pan-retinal gene expression with a lower vector dose volume and significantly increased the number of transduced cone photoreceptors. These results suggest that pre-retinal injection is a promising tool in the development of gene therapy strategies in animal models and is a potential approach for use in human research, particularly in younger individuals with an intact posterior hyaloid membrane and stable vitreous.  

5.3011           Gene supplementation of CYP27A1 in the liver restores bile acid metabolism in a mouse model of cerebrotendinous xanthomatosis

Lumbreras, S., Ricobaraza, A., Baila-rueda, L., Gonzalez-Apariicio, M., Mora-Jimenez, L., Uriarte, I., Bunuales, M., Avila, M.A., Monte, M.J., Marin, J.J.G., Cenarro, A., Gonzalez-Aseguinolaza, G. and Hernandez-Alcoceba, R. Molecular Therapy-Methods & Clin. Develop., 22, 210-221 (2021)   Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive disease caused by mutations in the CYP27A1 gene, encoding the sterol 27-hydroxylase. Disruption of the bile acid biosynthesis pathway and accumulation of toxic precursors such as cholestanol cause chronic diarrhea, bilateral juvenile cataracts, tissue deposition of cholestanol and cholesterol (xanthomas), and progressive motor/neuropsychiatric alterations. We have evaluated the therapeutic potential of adeno-associated virus (AAV) vectors expressing CYP27A1 in a CTX mouse model. We found that a vector equipped with a strong liver-specific promoter (albumin enhancer fused with the α1 anti-trypsin promoter) is well tolerated and shows therapeutic effect at relatively low doses (1.5 × 10¹² viral genomes [vg]/kg), when less than 20% of hepatocytes overexpress the transgene. This vector restored bile acid metabolism and normalized the concentration of most bile acids in plasma. By contrast, standard treatment (oral chenodeoxycholic acid [CDCA]), while reducing cholestanol, did not normalize bile acid composition in plasma and resulted in supra-physiological levels of CDCA and its derivatives. At the transcriptional level, only the vector was able to avoid the induction of xenobiotic-induced pathways in mouse liver. In conclusion, the overexpression of CYP27A1 in a fraction of hepatocytes using AAV vectors is well tolerated and provides full metabolic restoration in Cyp27a1^−/− mice. These features make gene therapy a feasible option for the etiological treatment of CTX patients.  

5.3012           Low endotoxin E. coli strain-derived plasmids reduce rAAV vector-mediated immune responses both in vitro and in vivo

Zhang, Q., Wang, T., Zhu, X., Tian, X., Zhong, C., Ran, G., Xie, Y., Zhaao, B., Zhu, L. and Ling, C. Molelcular Therapy-Methods & Clin. Develop., 22, 293 (2021)   The major challenge of recombinant adeno-associated virus (rAAV) vectors is host immunological barriers. Compared to the neutralizing antibody and the cytotoxic T lymphocyte response, the host immune responses induced by unsatisfactory rAAV manufacturing were largely ignored previously. rAAV vector production usually requires large amounts of plasmid DNAs. The DNA are commonly isolated from the DH5α bacterial strain, which contains lipopolysaccharide (LPS) contamination. LPS, also named endotoxin, in plasmid DNA is intractable, and residual endotoxin in the subsequent rAAV vectors may result in substantial host immune response. Recently, a ClearColi K12 bacterial strain is commercially available, with genetically modified LPS that does not trigger endotoxic response in mammalian cells. Here, we produced rAAV-DJ vectors by plasmids yielded from either DH5α or ClearColi K12 bacterial strains. Our data indicated that the ClearColi K12 strain had satisfactory protection for the rAAV inverted terminal repeat (ITR) sequence. As expected, the ClearColi K12-derived rAAV-DJ vectors had lower endotoxin levels. The physical and biological equivalency of the purified viral stocks were confirmed by electron micrographs, Coomassie blue staining, and transduction assays. Most importantly, the ClearColi K12-derived rAAV-DJ vectors triggered reduced nuclear factor-kappa B (NF-κB) signaling pathway both in cell cultures in vitro and in C57BL/6 mice retinas in vivo. We believe that the use of the ClearColi K12 bacterial strain could eliminate the LPS in the purified vector stock at the source. Our data indicate its promising use in future clinical development.  

5.3013           Optimized pharmacological control over the AAV-Gene-Switch vector for regulable gene therapy

Cheng, S., van Gaalen, M.M., Bähr, M., garea-Rodriguez, E. and Kügler, S. Molecular Therapy-Methods & Clin. Develop., 23, 1-10 (2021)   Gene therapy in its current design is an irreversible process. It cannot be stopped in case of unwanted side effects, nor can expression levels of therapeutics be adjusted to individual patient’s needs. Thus, the Gene-Switch (GS) system for pharmacologically regulable neurotrophic factor expression was established for treatment of parkinsonian patients. Mifepristone, the synthetic steroid used to control transgene expression of the GS vector, is an approved clinical drug. However, pharmacokinetics and -dynamics of mifepristone vary considerably between different experimental animal species and depend on age and gender. In humans, but not in any other species, mifepristone binds to a high-affinity plasma carrier protein. We now demonstrate that the formulation of mifepristone can have robust impact on its ability to activate the GS system. Furthermore, we show that a pharmacological booster, ritonavir (Rtv), robustly enhances the pharmacological effect of mifepristone, and allows it to overcome gender- and species-specific pharmacokinetic and -dynamic issues. Most importantly, we demonstrate that the GS vector can be efficiently controlled by mifepristone in the presence of its human plasma carrier protein, α1-acid glycoprotein, in a “humanized” rat model. Thus, we have substantially improved the applicability of the GS vector toward therapeutic use in patients.    

5.3014           High throughput screening of novel AAV capsids identifies variants for transduction of adult NSCs within the subventricular zone

Kremer, L.P.M., Cerrizuela, S., Dehler, S., Stiehl, T., Weinmann, J., Abendroth, H., Kleber, S., Laure, A., El Andari, J., Anders, S., Marciniaki-Czochra, A.M., Grimm, D. and Martin-Villalba, A. Molecular Therapy-Methods & Clin. Develop., 23, 33-50 (2021)   The adult mammalian brain entails a reservoir of neural stem cells (NSCs) generating glial cells and neurons. However, NSCs become increasingly quiescent with age, which hampers their regenerative capacity. New means are therefore required to genetically modify adult NSCs for re-enabling endogenous brain repair. Recombinant adeno-associated viruses (AAVs) are ideal gene-therapy vectors due to an excellent safety profile and high transduction efficiency. We thus conducted a high-throughput screening of 177 intraventricularly injected barcoded AAV variants profiled by RNA sequencing. Quantification of barcoded AAV mRNAs identified two synthetic capsids, peptide-modified derivative of wild-type AAV9 (AAV9_A2) and peptide-modified derivative of wild-type AAV1 (AAV1_P5), both of which transduce active and quiescent NSCs. Further optimization of AAV1_P5 by judicious selection of the promoter and dose of injected viral genomes enabled labeling of 30%–60% of the NSC compartment, which was validated by fluorescence-activated cell sorting (FACS) analyses and single-cell RNA sequencing. Importantly, transduced NSCs readily produced neurons. The present study identifies AAV variants with a high regional tropism toward the ventricular-subventricular zone (v-SVZ) with high efficiency in targeting adult NSCs, thereby paving the way for preclinical testing of regenerative gene therapy.  

5.3015           Real-time MR tracking of AAV gene therapy with βgal-responsive MR probe in a murine model of GM1-gangliosidosis

Taghian, T., Baatista, A.R., Kamper, S., Caldwell, M., Lilley, L. et al Molecular Therapy-Methods & Clin. Develop., 23, 128-134 (2021)   Transformative results of adeno-associated virus (AAV) gene therapy in patients with spinal muscular atrophy and Leber’s congenital amaurosis led to approval of the first two AAV products in the United States to treat these diseases. These extraordinary results led to a dramatic increase in the number and type of AAV gene-therapy programs. However, the field lacks non-invasive means to assess levels and duration of therapeutic protein function in patients. Here, we describe a new magnetic resonance imaging (MRI) technology for real-time reporting of gene-therapy products in the living animal in the form of an MRI probe that is activated in the presence of therapeutic protein expression. For the first time, we show reliable tracking of enzyme expression after a now in-human clinical trial AAV gene therapy (ClinicalTrials.gov: NTC03952637) encoding lysosomal acid beta-galactosidase (βgal) using a self-immolative βgal-responsive MRI probe. MRI enhancement in AAV-treated enzyme-deficient mice (GLB-1^−/−) correlates with βgal activity in central nervous system and peripheral organs after intracranial or intravenous AAV gene therapy, respectively. With >1,800 gene therapies in phase I/II clinical trials (ClinicalTrials.gov), development of a non-invasive method to track gene expression over time in patients is crucial to the future of the gene-therapy field.  

5.3016           Optimizing AAV2/6 microglial targeting identified enhanced efficiency in the photoreceptor degenerative environment

Maes, M.E., Wügenstein, G.M., Colombo, G., Casado-Polanco, R. and Siegert, S. Molecular Therapy-Methods & Clin. Develop., 23, 210-224 (2021)   Adeno-associated viruses (AAVs) are widely used to deliver genetic material in vivo to distinct cell types such as neurons or glial cells, allowing for targeted manipulation. Transduction of microglia is mostly excluded from this strategy, likely due to the cells’ heterogeneous state upon environmental changes, which makes AAV design challenging. Here, we established the retina as a model system for microglial AAV validation and optimization. First, we show that AAV2/6 transduced microglia in both synaptic layers, where layer preference corresponds to the intravitreal or subretinal delivery method. Surprisingly, we observed significantly enhanced microglial transduction during photoreceptor degeneration. Thus, we modified the AAV6 capsid to reduce heparin binding by introducing four point mutations (K531E, R576Q, K493S, and K459S), resulting in increased microglial transduction in the outer plexiform layer. Finally, to improve microglial-specific transduction, we validated a Cre-dependent transgene delivery cassette for use in combination with the Cx3cr1^CreERT2 mouse line. Together, our results provide a foundation for future studies optimizing AAV-mediated microglia transduction and highlight that environmental conditions influence microglial transduction efficiency  

5.3017           AAV-mediated PEX1 gene augmentation improves visual function in the PEX1-Gly844Asp mouse model for mild Zellweger spectrum disorder

Agryiou, C., Plosa, A., Song, J.Y., Omri, S., Steele, B., Cecyre, B., McDougald, D.S., Di Pietro, E., Bouchard, J-F., Bennett, J., Hacia, J.G., Lachapelle, P. and Braverman, N.E. Molecular Therapy-Methods & Clin. Develop., 23, 225-240 (2021)   Patients with Zellweger spectrum disorder (ZSD) commonly present with vision loss due to mutations in PEX genes required for peroxisome assembly and function. Here, we evaluate PEX1 retinal gene augmentation therapy in a mouse model of mild ZSD bearing the murine equivalent (PEX1-p[Gly844Asp]) of the most common human mutation. Experimental adeno-associated virus 8.cytomegalovirus.human PEX1.hemagglutinin (AAV8.CMV.HsPEX1.HA) and control AAV8.CMV.EGFP vectors were administered by subretinal injection in contralateral eyes of early (5-week-old)- or later (9-week-old)-stage retinopathy cohorts. HsPEX1.HA protein was expressed in the retina with no gross histologic side effects. Peroxisomal metabolic functions, assessed by retinal C26:0 lysophosphatidylcholine (lyso-PC) levels, were partially normalized after therapeutic vector treatment. Full-field flash electroretinogram (ffERG) analyses at 8 weeks post-injection showed a 2-fold improved retinal response in the therapeutic relative to control vector-injected eyes. ffERG improved by 1.6- to 2.5-fold in the therapeutic vector-injected eyes when each cohort reached 25 weeks of age. At 32 weeks of age, the average ffERG response was double in the therapeutic relative to control vector-injected eyes in both cohorts. Optomotor reflex analyses trended toward improvement. These proof-of-concept studies represent the first application of gene augmentation therapy to treat peroxisome biogenesis disorders and support the potential for retinal gene delivery to improve vision in these patients.  

5.3018           Lentiviral and adeno-associated vectors efficiently transduce mouse T lymphocytes when targeted to murine CD8

Michels, A., Frank, A.M., Günther, D.M., Mataei, M., Börner, K., Grimm, D., Hartmann, J. and Buchholz, C.J. Molecular Therapy-Methods & Clin. Develop., 23, 334-347 (2021)   Preclinical studies on gene delivery into mouse lymphocytes are often hampered by insufficient activity of lentiviral (LV) and adeno-associated vectors (AAVs) as well as missing tools for cell type selectivity when considering in vivo gene therapy. Here, we selected designed ankyrin repeat proteins (DARPins) binding to murine CD8. The top-performing DARPin was displayed as targeting ligand on both vector systems. When used on engineered measles virus (MV) glycoproteins, the resulting mCD8-LV transduced CD8+ mouse lymphocytes with near-absolute (>99%) selectivity. Despite its lower functional titer, mCD8-LV achieved 4-fold higher gene delivery to CD8+ cells than conventional VSV-LV when added to whole mouse blood. Addition of mCD8-LV encoding a chimeric antigen receptor (CAR) specific for mouse CD19 to splenocytes resulted in elimination of B lymphocytes and lymphoma cells. For display on AAV, the DARPin was inserted into the GH2-GH3 loop of the AAV2 capsid protein VP1, resulting in a DARPin-targeted AAV we termed DART-AAV. Stocks of mCD8-AAV contained similar genome copies as AAV2 but were >20-fold more active in gene delivery in mouse splenocytes, while exhibiting >99% specificity for CD8+ cells. These results suggest that receptor targeting can overcome blocks in transduction of mouse splenocytes.  

5.3019           Recombinant oncolytic adenovirus expressing a soluble PVR elicits long-term antitumor immune surveillance

Zhang, H., Zhang, Y., Dong, J., Li, B., Xu, C., Wei, M., Wu, J. and Wei, J. Moleculr Therapy-Oncolytics, 20, 12-22 (2021)   Oncolytic virotherapy (OVT) has been suggested to be effective. However, the suppressive effects of checkpoints and insufficient costimulatory signals limit OVT-induced antitumor immune responses. In this study, we constructed a replicative adenovirus, Ad5sPVR, that expresses the soluble extracellular domain of poliovirus receptor (sPVR). We showed that sPVR can bind to both T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) and CD226, and the binding affinity of sPVR to TIGIT is stronger than that of PVR to CD226. In the H22 hepatocellular carcinoma (HCC) ascites model, Ad5sPVR treatment increased the infiltration of CD8+ T cells and the release of interferon (IFN)-γ, exhibiting an antitumor effect with long-term tumor-specific immune surveillance. In line with this, Ad5sPVR also effectively improved antitumor outcomes in solid tumors. In conclusion, while Ad5sPVR plays a role in oncolysis and transforms cold tumors into hot tumors, sPVR expressed by Ad5sPVR can block the PVR/TIGIT checkpoint and activate CD226, thereby greatly improving the efficacy of OVT. This study provides a new way to develop potential oncolytic viral drugs.  

5.3020           Oncolytic vaccinia virus induces a novel phenotype of CD8+ effector T cells characterized by high ICOS expression

Yamashita, M., Tasaki, M., Murakami, R., Arai, Y., Nakamura, T. and Nakao, S. Molecular Therapy-Oncolytics, 20, 422-432 82021)   Characterization of the intratumoral immune status is important for developing immunotherapies and evaluating their antitumor effectiveness. CD8⁺ T cells are one of the most important cell types that directly and indirectly contribute to antitumor efficacy by releasing cytolytic molecules and inflammatory cytokines in the tumor microenvironment. Previously, we engineered a tumor-selective oncolytic vaccinia virus that encodes interleukin-7 (IL-7) and IL-12 and demonstrated its usefulness as an agent for in situ vaccination against tumors, with data showing that antitumor efficacy was reliant upon CD8⁺ T cells recruited by viral treatment. Here, we investigated the phenotypic changes in intratumoral CD8⁺ T cells caused by this oncolytic virus and found increased expression of inducible co-stimulator (ICOS) in PD-1⁻CD8⁺ T cells. Unlike previously reported ICOS⁺CD8⁺ T cells, a subset of ICOS⁺PD-1⁻CD8⁺ T cells showed effector function characterized by granzyme B expression. ICOS expression was induced by the backbone virus, which did not encode any immune transgenes and was independent of upregulation of the type I interferon pathway. Not only did we identify a novel effector cell subset characterized by ICOS expression, but our findings also shed light on a potential unknown aspect of the mechanism of oncolytic vaccinia virotherapy.  

5.3021           Engineering highly efficient backsplicing and translation of synthetic circRNAs

Meganck, R.M., Liu, J., hale, A.E., Simon, K.E., Fanous, M.M., Vincent, H.A., Wilusz, J.E., Moorman, N.J., Marzluff, W.F. and Asokan, A. Molecular Therapy-Nucleic Acids, 23, 821-834 (2021)   Circular RNAs (circRNAs) are highly stable RNA molecules that are attractive templates for expression of therapeutic proteins and non-coding RNAs. In eukaryotes, circRNAs are primarily generated by the spliceosome through backsplicing. Here, we interrogate different molecular elements including intron type and length, Alu repeats, internal ribosome entry sites (IRESs), and exon length essential for circRNA formation and exploit this information to engineer robust backsplicing and circRNA expression. Specifically, we leverage the finding that the downstream intron can tolerate large inserts without affecting splicing to achieve tandem expression of backspliced circRNAs and tRNA intronic circRNAs from the same template. Further, truncation of selected intronic regions markedly increased circRNA formation in different cell types in vitro as well as AAV-mediated circRNA expression in cardiac and skeletal muscle tissue in vivo. We also observed that different IRES elements and exon length influenced circRNA expression and translation, revealing an exonic contribution to splicing, as evidenced by different RNA species produced. Taken together, these data provide new insight into improving the design and expression of synthetic circRNAs. When combined with AAV capsid and promoter technologies, the backsplicing introns and IRES elements constituting this modular platform significantly expand the gene expression toolkit.  

5.3022           BB-301: a silence and replace AAV-based vector for the treatment of oculopharyngeal muscular dystrophy

Strings-Ufombah, V., Malerba, A., Kao, S-C., Harbaran, S., Roth, F., Cappellari, O., Lu-Nguyen, N., Takahashi, K., Mukadam, S., Kilfoil, G., Kloth, C., Roelvink, P., Dickson, G., Trollet, C. and Suhy, D. Molecular Therapy-Nucleic Acids, 24, 67-78 (2021)   Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant disease that results from an alanine expansion in the N-terminal domain of Poly-A Binding Protein Nuclear-1 (PABPN1). We have recently demonstrated that a two-vector gene therapy strategy significantly ameliorated the pathology in a mouse model of OPMD. This approach entailed intramuscular injection of two recombinant adeno-associated viruses (AAVs), one expressing three short hairpin RNAs (shRNAs) to silence both mutant and wild-type PABPN1 and one expressing a codon-optimized version of PABPN1 that is insensitive to RNA interference. Here we report the continued development of this therapeutic strategy by delivering “silence and replace” sequences in a single AAV vector named BB-301. This construct is composed of a modified AAV serotype 9 (AAV9) capsid that expresses a unique single bifunctional construct under the control of the muscle-specific Spc5-12 promoter for the co-expression of both the codon-optimized PABPN1 protein and two small inhibitory RNAs (siRNAs) against PABPN1 modeled into microRNA (miRNA) backbones. A single intramuscular injection of BB-301 results in robust inhibition of mutant PABPN1 and concomitant replacement of the codon-optimized PABPN1 protein. The treatment restores muscle strength and muscle weight to wild-type levels as well as improving other physiological hallmarks of the disease in a mouse model of OPMD.  

5.3023           Cone-shaped HIV-1 capsids are transported through intact nuclear pores

Zila, V., Margiotta, e., Turonova, B., Lusic, M., Kräusslich, H-G. and Beck, M. Cell, 184, 1032-1046 (2021)   Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrated that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, cone-shaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.  

5.3024           Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia

Asarnow, D., Wang, B., Lee, W-H., Cheng, Y., Craik, C.S.a dn Wang, C-I., Cell, 184, 3192-3204 (2021)   Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of the viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Although antibodies that block this interaction are in emergency use as early coronavirus disease 2019 (COVID-19) therapies, the precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that potently block ACE2 binding but exhibit divergent neutralization efficacy against the live virus. Strikingly, these neutralizing antibodies can inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in individuals with COVID-19. As revealed by cryoelectron microscopy, multiple structures of Spike-antibody complexes have distinct binding modes that not only block ACE2 binding but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion with profound implications for COVID-19 pathology and immunity.  

5.3025           GABA-receptive microglia selectively sculpt developing inhibitory circuits

Favuzzi, E., Huang, S., Saldi, G.A., Datta, S.R., Stevens, B. and Fishell, G. Cell, 184, 4048-4063 (2021)   Microglia, the resident immune cells of the brain, have emerged as crucial regulators of synaptic refinement and brain wiring. However, whether the remodeling of distinct synapse types during development is mediated by specialized microglia is unknown. Here, we show that GABA-receptive microglia selectively interact with inhibitory cortical synapses during a critical window of mouse postnatal development. GABA initiates a transcriptional synapse remodeling program within these specialized microglia, which in turn sculpt inhibitory connectivity without impacting excitatory synapses. Ablation of GABA_B receptors within microglia impairs this process and leads to behavioral abnormalities. These findings demonstrate that brain wiring relies on the selective communication between matched neuronal and glial cell types.  

5.3026           Preservation of vision after CaMKII-mediated protection of retinal ganglion cells

Guo, X., Zhou, J., Starr, C., Demb, J.B., Crair, M.C. and Chen, B. Cell, 184, 4299-4314 (2021)   Retinal ganglion cells (RGCs) are the sole output neurons that transmit visual information from the retina to the brain. Diverse insults and pathological states cause degeneration of RGC somas and axons leading to irreversible vision loss. A fundamental question is whether manipulation of a key regulator of RGC survival can protect RGCs from diverse insults and pathological states, and ultimately preserve vision. Here, we report that CaMKII-CREB signaling is compromised after excitotoxic injury to RGC somas or optic nerve injury to RGC axons, and reactivation of this pathway robustly protects RGCs from both injuries. CaMKII activity also promotes RGC survival in the normal retina. Further, reactivation of CaMKII protects RGCs in two glaucoma models where RGCs degenerate from elevated intraocular pressure or genetic deficiency. Last, CaMKII reactivation protects long-distance RGC axon projections in vivo and preserves visual function, from the retina to the visual cortex, and visually guided behavior.  

5.3027           Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species

Tabebordbar, M., Lagerborg, K.A., Stanton, A., Beggs, A.H., Wagers, A.J. and Sabeti, P.C. Cell, 184, 4919-4938 (2021)   Replacing or editing disease-causing mutations holds great promise for treating many human diseases. Yet, delivering therapeutic genetic modifiers to specific cells in vivo has been challenging, particularly in large, anatomically distributed tissues such as skeletal muscle. Here, we establish an in vivo strategy to evolve and stringently select capsid variants of adeno-associated viruses (AAVs) that enable potent delivery to desired tissues. Using this method, we identify a class of RGD motif-containing capsids that transduces muscle with superior efficiency and selectivity after intravenous injection in mice and non-human primates. We demonstrate substantially enhanced potency and therapeutic efficacy of these engineered vectors compared to naturally occurring AAV capsids in two mouse models of genetic muscle disease. The top capsid variants from our selection approach show conserved potency for delivery across a variety of inbred mouse strains, and in cynomolgus macaques and human primary myotubes, with transduction dependent on target cell expressed integrin heterodimers.  

5.3028           Revisiting astrocyte to neuron conversion with lineage tracing in vivo

Wang, L-L., Serrano, C., Zhong, X., Ma, S., Zou, Y. and Zhang, C-L. Cell, 184, 5465-5481 (2021)   In vivo cell fate conversions have emerged as potential regeneration-based therapeutics for injury and disease. Recent studies reported that ectopic expression or knockdown of certain factors can convert resident astrocytes into functional neurons with high efficiency, region specificity, and precise connectivity. However, using stringent lineage tracing in the mouse brain, we show that the presumed astrocyte-converted neurons are actually endogenous neurons. AAV-mediated co-expression of NEUROD1 and a reporter specifically and efficiently induces reporter-labeled neurons. However, these neurons cannot be traced retrospectively to quiescent or reactive astrocytes using lineage-mapping strategies. Instead, through a retrograde labeling approach, our results reveal that endogenous neurons are the source for these viral-reporter-labeled neurons. Similarly, despite efficient knockdown of PTBP1 in vivo, genetically traced resident astrocytes were not converted into neurons. Together, our results highlight the requirement of lineage-tracing strategies, which should be broadly applied to studies of cell fate conversions in vivo.  

5.3029           Bombesin-like peptide recruits disinhibitory cortical circuits and enhances fear memories

Melzer, S., Newmark, E.R., Mizuno, G.O., Levasseur, J., Tian, L. and Sabatini, B.L. Cell, 184, 5622-5634 (2021)   Disinhibitory neurons throughout the mammalian cortex are powerful enhancers of circuit excitability and plasticity. The differential expression of neuropeptide receptors in disinhibitory, inhibitory, and excitatory neurons suggests that each circuit motif may be controlled by distinct neuropeptidergic systems. Here, we reveal that a bombesin-like neuropeptide, gastrin-releasing peptide (GRP), recruits disinhibitory cortical microcircuits through selective targeting and activation of vasoactive intestinal peptide (VIP)-expressing cells. Using a genetically encoded GRP sensor, optogenetic anterograde stimulation, and trans-synaptic tracing, we reveal that GRP regulates VIP cells most likely via extrasynaptic diffusion from several local and long-range sources. In vivo photometry and CRISPR-Cas9-mediated knockout of the GRP receptor (GRPR) in auditory cortex indicate that VIP cells are strongly recruited by novel sounds and aversive shocks, and GRP-GRPR signaling enhances auditory fear memories. Our data establish peptidergic recruitment of selective disinhibitory cortical microcircuits as a mechanism to regulate fear memories.  

5.3030           Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex

Mich, J.K., Graybuck, L.T., Hess, E.E., Lein, E.S., Ting, J.T. and Levi, B.P. Cell Reports, 34, 108754 (2021)   Viral genetic tools that target specific brain cell types could transform basic neuroscience and targeted gene therapy. Here, we use comparative open chromatin analysis to identify thousands of human-neocortical-subclass-specific putative enhancers from across the genome to control gene expression in adeno-associated virus (AAV) vectors. The cellular specificity of reporter expression from enhancer-AAVs is established by molecular profiling after systemic AAV delivery in mouse. Over 30% of enhancer-AAVs produce specific expression in the targeted subclass, including both excitatory and inhibitory subclasses. We present a collection of Parvalbumin (PVALB) enhancer-AAVs that show highly enriched expression not only in cortical PVALB cells but also in some subcortical PVALB populations. Five vectors maintain PVALB-enriched expression in primate neocortex. These results demonstrate how genome-wide open chromatin data mining and cross-species AAV validation can be used to create the next generation of non-species-restricted viral genetic tools.  

5.3031           Optic nerve regeneration screen identifies multiple genes restricting adult neural repair

Lindborg, J.A., Tran, N.M., Chenette, D.M., Kim, I-J., Sanes, J.R. and Strittmatter, S.M. Cell Reports, 34, 108777 (2021)   Adult mammalian central nervous system (CNS) trauma interrupts neural networks and, because axonal regeneration is minimal, neurological deficits persist. Repair via axonal growth is limited by extracellular inhibitors and cell-autonomous factors. Based on results from a screen in vitro, we evaluate nearly 400 genes through a large-scale in vivo regeneration screen. Suppression of 40 genes using viral-driven short hairpin RNAs (shRNAs) promotes retinal ganglion cell (RGC) axon regeneration after optic nerve crush (ONC), and most are validated by separate CRISPR-Cas9 editing experiments. Expression of these axon-regeneration-suppressing genes is not significantly altered by axotomy. Among regeneration-limiting genes, loss of the interleukin 22 (IL-22) cytokine allows an early, yet transient, inflammatory response in the retina after injury. Reduced IL-22 drives concurrent activation of signal transducer and activator of transcription 3 (Stat3) and dual leucine zipper kinase (DLK) pathways and upregulation of multiple neuron-intrinsic regeneration-associated genes (RAGs). Including IL-22, our screen identifies dozens of genes that limit CNS regeneration. Suppression of these genes in the context of axonal damage could support improved neural repair.  

5.3032           Recurrent emergence of SARS-CoV-2 spike deletion H69/V70 and its role in the Alpha variant B.1.1.7

Meng, B., Kemp, S.A., Papa, G., Robertson, D.L., Bailey, D. and Gupta, R.K. Cell Reports, 35, 109292 (2021)   We report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike ΔH69/V70 in multiple independent lineages, often occurring after acquisition of receptor binding motif replacements such as N439K and Y453F, known to increase binding affinity to the ACE2 receptor and confer antibody escape. In vitro, we show that, although ΔH69/V70 itself is not an antibody evasion mechanism, it increases infectivity associated with enhanced incorporation of cleaved spike into virions. ΔH69/V70 is able to partially rescue infectivity of spike proteins that have acquired N439K and Y453F escape mutations by increased spike incorporation. In addition, replacement of the H69 and V70 residues in the Alpha variant B.1.1.7 spike (where ΔH69/V70 occurs naturally) impairs spike incorporation and entry efficiency of the B.1.1.7 spike pseudotyped virus. Alpha variant B.1.1.7 spike mediates faster kinetics of cell-cell fusion than wild-type Wuhan-1 D614G, dependent on ΔH69/V70. Therefore, as ΔH69/V70 compensates for immune escape mutations that impair infectivity, continued surveillance for deletions with functional effects is warranted.  

5.3033           Somatostatin interneurons restrict cell recruitment to retinally driven spontaneous activity in the developing cortex

Leighton, A.H., Cheyne, J.E., Houwen, G.J., Maldonado, P.P., De Winter, F., Levelt, C.N. and Lohmann, C. Cell Reports, 36, 109316 (2021)   During early development, before the eyes open, synaptic refinement of sensory networks depends on activity generated by developing neurons themselves. In the mouse visual system, retinal cells spontaneously depolarize and recruit downstream neurons to bursts of activity, where the number of recruited cells determines the resolution of synaptic retinotopic refinement. Here we show that during the second post-natal week in mouse visual cortex, somatostatin (SST)-expressing interneurons control the recruitment of cells to retinally driven spontaneous activity. Suppressing SST interneurons increases cell participation and allows events to spread farther along the cortex. During the same developmental period, a second type of high-participation, retina-independent event occurs. During these events, cells receive such large excitatory charge that inhibition is overwhelmed and large parts of the cortex participate in each burst. These results reveal a role of SST interneurons in restricting retinally driven activity in the visual cortex, which may contribute to the refinement of retinotopy.  

5.3034           Brain endothelial PTEN/AKT/NEDD4-2/MFSD2A axis regulates blood-brain barrier permeability

Cui, Y., Wang, Y., Song, X., Teng, Y., Wang, J. and Yang, X. Cell Reports, 36, 109327 (2021)   The low level of transcytosis is a unique feature of cerebrovascular endothelial cells (ECs), ensuring restrictive blood-brain barrier (BBB) permeability. Major facilitator superfamily domain-containing 2a (MFSD2A) is a key regulator of the BBB function by suppressing caveolae-mediated transcytosis. However, the mechanisms regulating MFSD2A at the BBB have been barely explored. Here, we show that cerebrovascular EC-specific deletion of Pten (phosphatase and tensin homolog) results in a dramatic increase in vesicular transcytosis by the reduction of MFSD2A, leading to increased transcellular permeability of the BBB. Mechanistically, AKT signaling inhibits E3 ubiquitin ligase NEDD4-2-mediated MFSD2A degradation. Consistently, cerebrovascular Nedd4-2 overexpression decreases MFSD2A levels, increases transcytosis, and impairs BBB permeability, recapitulating the phenotypes of Pten-deficient mice. Furthermore, Akt deletion decreases phosphorylated NEDD4-2 levels, restores MFSD2A levels, and normalizes BBB permeability in Pten-mutant mice. Altogether, our work reveals the essential physiological function of the PTEN/AKT/NEDD4-2/MFSD2A axis in the regulation of BBB permeability.  

5.3035           Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis

Liu, Y., Shukha, Y., Hayek, T., Chen, Y.E. and Rom, O. Cell reports, 36(4), 109420 (2021)   Dysregulated glycine metabolism is emerging as a common denominator in cardiometabolic diseases, but its contribution to atherosclerosis remains unclear. In this study, we demonstrate impaired glycine-oxalate metabolism through alanine-glyoxylate aminotransferase (AGXT) in atherosclerosis. As found in patients with atherosclerosis, the glycine/oxalate ratio is decreased in atherosclerotic mice concomitant with suppression of AGXT. Agxt deletion in apolipoprotein E-deficient (Apoe^−/−) mice decreases the glycine/oxalate ratio and increases atherosclerosis with induction of hepatic pro-atherogenic pathways, predominantly cytokine/chemokine signaling and dysregulated redox homeostasis. Consistently, circulating and aortic C-C motif chemokine ligand 5 (CCL5) and superoxide in lesional macrophages are increased. Similar findings are observed following dietary oxalate overload in Apoe^−/− mice. In macrophages, oxalate induces mitochondrial dysfunction and superoxide accumulation, leading to increased CCL5. Conversely, AGXT overexpression in Apoe^−/− mice increases the glycine/oxalate ratio and decreases aortic superoxide, CCL5, and atherosclerosis. Our findings uncover dysregulated oxalate metabolism via suppressed AGXT as a driver and therapeutic target in atherosclerosis.  

5.3036           Homeostatic control of nuclear-encoded mitochondrial gene expression by the histone variant H2A.Z is essential for neuronal survival

Lowden, C., Boulet, A., Boehler, N.A., Liu, B-h., Leary, S.C. and Cheng, H-Y.M. Cell Reports, 36(11), 109704 (2021)   Histone variants are crucial regulators of chromatin structure and gene transcription, yet their functions within the brain remain largely unexplored. Here, we show that the H2A histone variant H2A.Z is essential for neuronal survival. Mice lacking H2A.Z in GABAergic neurons or Purkinje cells (PCs) present with a progressive cerebellar ataxia accompanied by widespread degeneration of PCs. Ablation of H2A.Z in other neuronal subtypes also triggers cell death. H2A.Z binds to the promoters of key nuclear-encoded mitochondrial genes to regulate their expression and promote organelle function. Bolstering mitochondrial activity genetically or by organelle transplant enhances the survival of H2A.Z-ablated neurons. Changes in bioenergetic status alter H2A.Z occupancy at the promoters of nuclear-encoded mitochondrial genes, an adaptive response essential for cell survival. Our results highlight that H2A.Z fulfills a key, conserved role in neuronal survival by acting as a transcriptional rheostat to regulate the expression of genes critical to mitochondrial function.  

5.3037           Enhancer viruses for combinatorial cell-subclass-specific labeling

Graybuck, L.T., Daigle, T.L., Sedeno-Cortes, A.E., Ting, J.T., Zeng, H. and tasic, B. Neuron, 109, 1449-1464 (2021)   Rapid cell type identification by new genomic single-cell analysis methods has not been met with efficient experimental access to these cell types. To facilitate access to specific neural populations in mouse cortex, we collected chromatin accessibility data from individual cells and identified enhancers specific for cell subclasses and types. When cloned into recombinant adeno-associated viruses (AAVs) and delivered to the brain, these enhancers drive transgene expression in specific cortical cell subclasses. We extensively characterized several enhancer AAVs to show that they label different projection neuron subclasses as well as a homologous neuron subclass in human cortical slices. We also show how coupling enhancer viruses expressing recombinases to a newly generated transgenic mouse, Ai213, enables strong labeling of three different neuronal classes/subclasses in the brain of a single transgenic animal. This approach combines unprecedented flexibility with specificity for investigation of cell types in the mouse brain and beyond.  

5.3038           Subcellular specificity of cannabinoid effects in striatonigral circuits

Soria-Gomez, E., Zottola, A.C.P., Mariani, Y., Baufreton, J., Marsicano, G. and Bellocchio, L. Neuron, 109, 1513-1526 (2021)   Recent advances in neuroscience have positioned brain circuits as key units in controlling behavior, implying that their positive or negative modulation necessarily leads to specific behavioral outcomes. However, emerging evidence suggests that the activation or inhibition of specific brain circuits can actually produce multimodal behavioral outcomes. This study shows that activation of a receptor at different subcellular locations in the same neuronal circuit can determine distinct behaviors. Pharmacological activation of type 1 cannabinoid (CB₁) receptors in the striatonigral circuit elicits both antinociception and catalepsy in mice. The decrease in nociception depends on the activation of plasma membrane-residing CB₁ receptors (pmCB₁), leading to the inhibition of cytosolic PKA activity and substance P release. By contrast, mitochondrial-associated CB₁ receptors (mtCB₁) located at the same terminals mediate cannabinoid-induced catalepsy through the decrease in intra-mitochondrial PKA-dependent cellular respiration and synaptic transmission. Thus, subcellular-specific CB₁ receptor signaling within striatonigral circuits determines multimodal control of behavior.  

5.3039           Ventral pallidum DRD3 potentiates a pallido-habenular circuit driving accumbal dopamine release and cocaine seeking

Pribiag, H., Shin. S., Wang, E.H-J., Lilascharoen, V., Li, Y. and Lim, B.K. Neuron, 109, 2165-2182 (2021)   Drugs of abuse induce persistent remodeling of reward circuit function, a process thought to underlie the emergence of drug craving and relapse to drug use. However, how circuit-specific, drug-induced molecular and cellular plasticity can have distributed effects on the mesolimbic dopamine reward system to facilitate relapse to drug use is not fully elucidated. Here, we demonstrate that dopamine receptor D3 (DRD3)-dependent plasticity in the ventral pallidum (VP) drives potentiation of dopamine release in the nucleus accumbens during relapse to cocaine seeking after abstinence. We show that two distinct VP DRD3⁺ neuronal populations projecting to either the lateral habenula (LHb) or the ventral tegmental area (VTA) display different patterns of activity during drug seeking following abstinence from cocaine self-administration and that selective suppression of elevated activity or DRD3 signaling in the LHb-projecting population reduces drug seeking. Together, our results uncover how circuit-specific DRD3-mediated plasticity contributes to the process of drug relapse.  

5.3040           Specific and behaviorally consequential astrocyte Gq GPCR signaling attenuation in vivo with iβARK

Nagai, J., Bellaford, A., Qu, Z., Golshani, P., Gradinaru, V. and Khakh, B.S. Neuron, 109, 2256-2274 (2021)   Astrocytes respond to neurotransmitters and neuromodulators using G-protein-coupled receptors (GPCRs) to mediate physiological responses. Despite their importance, there has been no method to genetically, specifically, and effectively attenuate astrocyte G_q GPCR pathways to explore consequences of this prevalent signaling mechanism in vivo. We report a 122-residue inhibitory peptide from β-adrenergic receptor kinase 1 (iβARK; and inactive D110A control) to attenuate astrocyte G_q GPCR signaling. iβARK significantly attenuated G_q GPCR Ca²⁺ signaling in brain slices and, in vivo, altered behavioral responses, spared other GPCR responses, and did not alter astrocyte spontaneous Ca²⁺ signals, morphology, electrophysiological properties, or gene expression in the striatum. Furthermore, brain-wide attenuation of astrocyte G_q GPCR signaling with iβARK using PHP.eB adeno-associated viruses (AAVs), when combined with c-Fos mapping, suggested nuclei-specific contributions to behavioral adaptation and spatial memory. iβARK extends the toolkit needed to explore functions of astrocyte G_q GPCR signaling within neural circuits in vivo.  

5.3041           HepaCAM controls astrocyte self-organization and coupling

Baldwin, K.T., Tan, C.X., Strader, S.T., Estevez, R., Ji, R-R. and Eroglu, C. Neuron, 109, 2427-2442 (2021)   Astrocytes extensively infiltrate the neuropil to regulate critical aspects of synaptic development and function. This process is regulated by transcellular interactions between astrocytes and neurons via cell adhesion molecules. How astrocytes coordinate developmental processes among one another to parse out the synaptic neuropil and form non-overlapping territories is unknown. Here we identify a molecular mechanism regulating astrocyte-astrocyte interactions during development to coordinate astrocyte morphogenesis and gap junction coupling. We show that hepaCAM, a disease-linked, astrocyte-enriched cell adhesion molecule, regulates astrocyte competition for territory and morphological complexity in the developing mouse cortex. Furthermore, conditional deletion of Hepacam from developing astrocytes significantly impairs gap junction coupling between astrocytes and disrupts the balance between synaptic excitation and inhibition. Mutations in HEPACAM cause megalencephalic leukoencephalopathy with subcortical cysts in humans. Therefore, our findings suggest that disruption of astrocyte self-organization mechanisms could be an underlying cause of neural pathology.  

5.3042           Anterior thalamic dysfunction underlies cognitive deficits in a subset of neuropsychiatric disease models

Roy, D.S., zhang, Y., Aida, T., McCarroll, S.A., Wickersham, I.R. and Feng, G. Neurom, 109, 2590-2603 (2021)   Neuropsychiatric disorders are often accompanied by cognitive impairments/intellectual disability (ID). It is not clear whether there are converging mechanisms underlying these debilitating impairments. We found that many autism and schizophrenia risk genes are expressed in the anterodorsal subdivision (AD) of anterior thalamic nuclei, which has reciprocal connectivity with learning and memory structures. CRISPR-Cas9 knockdown of multiple risk genes selectively in AD thalamus led to memory deficits. While the AD is necessary for contextual memory encoding, the neighboring anteroventral subdivision (AV) regulates memory specificity. These distinct functions of AD and AV are mediated through their projections to retrosplenial cortex, using differential mechanisms. Furthermore, knockdown of autism and schizophrenia risk genes PTCHD1, YWHAG, or HERC1 from AD led to neuronal hyperexcitability, and normalization of hyperexcitability rescued memory deficits in these models. This study identifies converging cellular to circuit mechanisms underlying cognitive deficits in a subset of neuropsychiatric disease models.  

5.3043           Nicotine inhibits the VTA-to-amygdala dopamine pathway to promote anxiety

Nguyen, C., Mondoloni, S., Le Borgne, T., Mourot, A., Marti, F. and Faure, P. Neuron, 109, 2604-2615 (2021)   Nicotine stimulates dopamine (DA) neurons of the ventral tegmental area (VTA) to establish and maintain reinforcement. Nicotine also induces anxiety through an as yet unknown circuitry. We found that nicotine injection drives opposite functional responses of two distinct populations of VTA DA neurons with anatomically segregated projections: it activates neurons that project to the nucleus accumbens (NAc), whereas it inhibits neurons that project to the amygdala nuclei (Amg). We further show that nicotine mediates anxiety-like behavior by acting on β2-subunit-containing nicotinic acetylcholine receptors of the VTA. Finally, using optogenetics, we bidirectionally manipulate the VTA-NAc and VTA-Amg pathways to dissociate their contributions to anxiety-like behavior. We show that inhibition of VTA-Amg DA neurons mediates anxiety-like behavior, while their activation prevents the anxiogenic effects of nicotine. These distinct subpopulations of VTA DA neurons with opposite responses to nicotine may differentially drive the anxiogenic and the reinforcing effects of nicotine.  

5.3044           Loss of nigral excitation of cholinergic interneurons contributes to parkinsonian motor impairments

Cai, Y., Nielsen, B.E., Boxer, E.E., Aoto, J. and Ford, C.P. Neuron, 109, 1-13 (2021)   Progressive loss of dopamine inputs in Parkinson’s disease leads to imbalances in coordinated signaling of dopamine and acetylcholine (ACh) in the striatum, which is thought to contribute to parkinsonian motor symptoms. As reciprocal interactions between dopamine inputs and cholinergic interneurons (ChIs) control striatal dopamine and ACh transmission, we examined how partial dopamine depletion in an early-stage mouse model for Parkinson’s disease alters nigral regulation of cholinergic activity. We found region-specific alterations in how remaining dopamine inputs regulate cholinergic excitability that differ between the dorsomedial (DMS) and dorsolateral (DLS) striatum. Specifically, we found that dopamine depletion downregulates metabotropic glutamate receptors (mGluR1) on DLS ChIs at synapses where dopamine inputs co-release glutamate, abolishing the ability of dopamine inputs to drive burst firing. This loss underlies parkinsonian motor impairments, as viral rescue of mGluR1 signaling in DLS ChIs was sufficient to restore circuit function and attenuate motor deficits in early-stage parkinsonian mice.  

5.3045           An AAV-based, room-temperature-stable, single-dose COVID-19 vaccine provides durable immunogenicity and protection in non-human primates

Zabaleta, N., Dai, W., Bhatt, U., Wilson, J.M., Le Grand, R. and Vandenberghe, L.H. Cell Host & Microbe, 29, 1437-1453 (2021)   The SARS-CoV-2 pandemic has affected more than 185 million people worldwide resulting in over 4 million deaths. To contain the pandemic, there is a continued need for safe vaccines that provide durable protection at low and scalable doses and can be deployed easily. Here, AAVCOVID-1, an adeno-associated viral (AAV), spike-gene-based vaccine candidate demonstrates potent immunogenicity in mouse and non-human primates following a single injection and confers complete protection from SARS-CoV-2 challenge in macaques. Peak neutralizing antibody titers are sustained at 1 year and complemented by functional memory T cell responses. The AAVCOVID vector has no relevant pre-existing immunity in humans and does not elicit cross-reactivity to common AAVs used in gene therapy. Vector genome persistence and expression wanes following injection. The single low-dose requirement, high-yield manufacturability, and 1-month stability for storage at room temperature may make this technology well suited to support effective immunization campaigns for emerging pathogens on a global scale.  

5.3046           Live imaging of SARS-CoV-2 infection in mice reveals that neutralizing antibodies require Fc function for optimal efficacy

Ullah, I., Prevost, J., Ladinsky, M.S., Finzi, A., Kumar, P. and Uchil, P.D., Immunity, 54, 2143-2158 (2021)   Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.  

5.3047           Intranasal vaccination with a Newcastle disease virus-vectored vaccine protects hamsters from SARS-CoV-2 infection and disease

Warner, B.M., Santry, L.A., Susta, L., Kobasa, D. and Wootton, S.K. iScience, 24, 103219 (2021)   The pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19). Worldwide efforts are being made to develop vaccines to mitigate this pandemic. We engineered two recombinant Newcastle disease virus (NDV) vectors expressing either the full-length SARS-CoV-2 spike protein (NDV-FLS) or a version with a 19 amino acid deletion at the carboxy terminus (NDV-Δ19S). Hamsters receiving two doses (prime-boost) of NDV-FLS developed a robust SARS-CoV-2-neutralizing antibody response, with elimination of infectious virus in the lungs and minimal lung pathology at five days post-challenge. Single-dose vaccination with NDV-FLS significantly reduced SARS-CoV-2 replication in the lungs but only mildly decreased lung inflammation. NDV-Δ19S-treated hamsters had a moderate decrease in SARS-CoV-2 titers in lungs and presented with severe microscopic lesions, suggesting that truncation of the spike protein was a less effective strategy. In summary, NDV-vectored vaccines represent a viable option for protection against COVID-19.  

5.3048           Acute gene inactivation in the adult mouse liver using the CRISPR-Cas9 technology

Wang, X., Xu, B-L. and Chen, X-W. STAR Protocols, 2, 100611 (2021)   Genetic manipulation in mice allows the discovery of gene function and biological mechanisms in vivo. The widely used Cre/LoxP system usually takes months to years especially when starting with the production of floxed alleles of a new gene of interest (GOI). Here, we describe a protocol using the CRISPR-Cas9 system to acutely inactivate the GOI in adult mice. This protocol enables hepatocyte-specific gene editing within 4 weeks in adult mice and avoids compensatory effects of traditional gene inactivation initiated during various developmental stages.  

5.3049           Comparison of the expression and toxicity of AAV2/9 carrying the human A53T α-synuclein gene in presence or absence of WPRE

Sun, X., Yu, X., Zhang, L., Zhao, W., Wang, M., Zhang, Y., Li, X., Gao, R., Breger, L.S., Dovero, S., Porras, G., Fernagut, P-O., Dehay, B., Bezard, E. and Qin, C. Heiyon, 7, e06302 (2021)   Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) is thought to enhance transgene expression of target genes delivered by adeno-associated viral (AAV) vectors. This study assessed the protein expression of α-synuclein, phosphorylated α-synuclein at Serine 129, extent of nigrostriatal degeneration as well as subsequent behavioral deficits induced by unilateral intranigral stereotactic injection in male adult C57BL/6J mice of an AAV2/9 expressing A53T human α-synuclein under the control of the synapsin promoter in presence or absence of the WPRE. The presence of WPRE enabled to achieve greater nigrostriatal degeneration and synucleinopathy which was concomitant with worsened forelimb use asymmetry. This work refines a mouse Parkinson's disease model in which anatomo-pathology is related to behavioral deficits.  

5.3050           Humoral and cell-mediated immune responses to plant-produced African horse sickness virus VP7 quasi-crystals

Fearon, S.H., Dennis, S.J., Hitzeroth, I.I. and Rybicki, E.P. Virus Res., 294, 198284 (2021)   African horse sickness (AHS) is a devastating viral disease affecting equines and has resulted in many disastrous epizootics. To date, no successful therapeutic treatment exists for AHS, and commercially used live-attenuated vaccines have various undesirable side effects. Previous studies have shown that mice inoculated with insoluble African horse sickness virus (AHSV) VP7 crystals are protected from live challenge with a lethal dose of AHSV. This study investigates the humoral and cell-mediated immune responses in guinea-pigs to a safer monovalent vaccine alternative based on AHSV-5 VP7 quasi-crystals produced in plants. Guinea-pigs received prime- and boost-inoculations of between 10 and 50 μg of purified plant-produced AHSV VP7. Western immunoblot analysis of the humoral response showed stimulation of high titres of anti-VP7 antibodies 28 days after the boost-inoculation in sera from three of the five experimental animals. In addition, RNA-seq transcriptome profiling of guinea-pig spleen-derived RNA highlighted thirty significantly (q ≤ 0.05) differentially expressed genes involved in innate and adaptive immunity. Differential expression of genes involved in Th1, Th2 and Th17 cell differentiation suggest a cell-mediated immune response to AHSV-5 VP7. Upregulation of several important cytokines and cytokine receptors were noted, including TNFSF14, CX3CR1, IFNLR1 and IL17RA. Upregulation of IL17RA suggests a Th17 response which has been reported as a key component in AHSV immunity. While further investigation is needed to validate these findings, these results suggest that AHSV-5 VP7 quasi-crystals produced in N. benthamiana are immunogenic and induce both humoral and cell-mediated responses.  

5.3051           A stable platform for the production of virus-like particles pseudotyped with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein

Roy, S., Ghani, K., de Campos-Lima, P.O. and Caruso, M. Virus Res., 295, 198305 (2021)   In this study, we showed that a codon optimized version of the spike (S) protein of SARS-CoV-2 can migrate to the cell membrane. However, efficient production of Moloney murine leukemia (MLV) infectious viral particles was only achieved with stable expression of a shorter S version in C-terminal (ΔS) in MLV Gag-pol expressing cells. As compared to transient transfections, this platform generated viruses with a 1000-fold higher titer. ΔS was 15-times more efficiently incorporated into VLPs as compared to S, and that was not due to steric interference between the cytoplasmic tail and the MLV capsid, as similar differences were also observed with extracellular vesicles. The amount of ΔS incorporated into VLPs released from producer cells was high and estimated at 1.25 μg/mL S2 equivalent (S is comprised of S1 and S2). The resulting VLPs could potentially be used alone or as a boost of other immunization strategies for COVID-19.  

5.3052           Generation of a cost-effective cell line for support of high-throughput isolation of primary human B cells and monoclonal neutralizing antibodies

Whaley, R.E., Ameny, S., Arkatkar, T., Seese, A., Wall, A., Khan, I., Carter, J.J., Scherer, E.M., Rawlings, D.J., Galloway, D.A., McElrath, M.J., Cohen, K.W. and McGuire, A.T.

1. Immunol. Methods, 488, 112901 (2021)

  The isolation of human monoclonal antibodies (mAbs) arising from natural infection with human pathogens has proven to be a powerful technology, facilitating the understanding of the host response to infection at a molecular level. mAbs can reveal sites of vulnerability on pathogens and illuminate the biological function of the antigenic targets. Moreover, mAbs have the potential to be used directly for therapeutic applications such as passive delivery to prevent infection in susceptible target populations, and as treatment of established infection. The isolation of antigen-specific B cells from vaccine trials can also assist in deciphering whether the desired B cells are being targeted by a given vaccine. Several different processes have been developed to isolate mAbs, but all are generally labor-intensive and result in varying degrees of efficiency. Here, we describe the development of a cost-effective feeder cell line that stably expresses CD40-ligand, interleukin-2 and interleukin-21. Sorting of single B cells onto a layer of irradiated feeder cells sustained antibody production that permits functional screening of secreted antibodies in a manner that enables subsequent recovery of B cells for recombinant antibody cloning. As a proof of concept, we show that this approach can be used to isolate B cells that secrete antibodies that neutralize human papilloma virus (HPV) from participants of an HPV vaccine study.  

5.3053           Multiplex immunoassay to measure antibody response to nine HPV vaccine types

Panicker, G., Rajbhandari, I., Pathak, H.N., Brady, A.M. and Unger, E.R.

1. Immunol. Methods, 498, 113136 (2021)

  Well-characterized HPV serology assays are required to evaluate performance of biosimilar candidate vaccines, reduced dosing schedules and novel administration methods. We report characterization of an expanded assay, M9ELISA, that detects antibodies to HPV virus-like particles (VLP) of nine types using direct IgG ELISA on the Meso Scale Discovery (MSD) electrochemiluminescence platform. The method is based on the previously published M4ELISA which detects antibodies to HPV6,11,16, and 18. It has been modified to add detection of antibodies to HPV31,33,45,52 and 58, and to streamline assay and reduce background. The M9ELISA plates were prepared with purified type specific L1 + L2 VLPs coated on 10-spot/well standard MSD microplates. Results of ELISA on three serial dilutions of serum were read on MSD imager, and titers calculated using the parallel line method. Evaluations included dynamic range, assay reproducibility, and stability over time. We compared M9ELISA results to those from a pseudovirion-based neutralization assay in sera from a mixed cohort of unvaccinated and vaccinated individuals (n = ~116) and to competitive Luminex immunoassay (cLIA) results in sera from a predominantly unvaccinated cohort (n = 4426). The linear range of the assay extended over 5 logs, with inter-assay reproducibility coefficient of variation ≤25% for all types. The pre-coated plates were stable for at least 2 years. Spearman correlation of antibody titers showed excellent correlation with PBNA (r = 0.86–0.97) and moderate correlation (r = 0.52–0.68) with cLIA. Thus, the M9ELISA can serve as a useful platform for high-throughput, sensitive and simultaneous quantitation of the antibody responses to nine HPV vaccine types.

5.3054           Selective targeting of striatal parvalbumin-expressing interneurons for transgene delivery

Azevedo, M.D., Sander, S., Jeanneret, C., Olfat, S. and Tenenbaum, L.

1. Neurosci. Methods, 354, 109105 (2021)

  PV^Cre mice--> combined with AAV-FLEX vectors allowed efficient and specific targeting of PV⁺ interneurons in the striatum. However, diffusion of viral particles to the globus pallidus caused massive transduction of PV⁺ projection neurons and subsequent anterograde transport of the transgene product to the subthalamic nucleus and the substantia nigra pars reticulata. Different AAV serotypes (1 and 9) and promoters (CBA and human synapsin) were evaluated. The combination of AAV1, a moderate expression level (human synapsin promoter) and a precise adjustment of the stereotaxic coordinates in the anterior and dorsolateral part of the striatum were necessary to avoid transduction of PV⁺ GP projection neurons. Even in the absence of direct transduction due to diffusion of viral particles, GP PV⁺ projection neurons could be retrogradely transduced via their terminals present in the dorsal striatum. However, in the absence of diffusion, GP-Str PV⁺ projection neurons were poorly or not transduced suggesting that retrograde transduction did not significantly impair the selective targeting of striatal PV⁺ neurons. Finally, a prominent reduction of the number of striatal PV⁺ interneurons (about 50 %) was evidenced in the presence of the Cre recombinase suggesting that functional effects of AAV-mediated transgene expression in PV⁺ striatal interneurons in PV^Cre mice should be analyzed with caution.  

5.3055           Avian adeno-associated virus as an anterograde transsynaptic vector

Ito, T., Ono, M., Matsui, R., Watanabe, D. and Ohmori, H.

1. Neurosci. Methods, 359, 109221 (2021)

  Background Retrograde and anterograde transsynaptic viral vectors are useful tools for studying the input and output organization of neuronal circuitry, respectively. While retrograde transsynaptic viral vectors are widely used, viral vectors that show anterograde transsynaptic transduction are not common. New method We chose recombinant avian adeno-associated virus (A3V) carrying the mCherry gene and injected it into the eyeball, cochlear duct, and midbrain auditory center of chickens. We observed different survival times to examine the virus transcellular transport and the resulting mCherry expression. To confirm the transcellular transduction mode, we co-injected A3V and cholera toxin B subunit. Results Injecting A3V into the eyeball and cochlea labeled neurons in the visual and auditory pathways, respectively. Second-, and third-order labeling occurred approximately two and seven days, respectively, after injection into the midbrain. The distribution of labeled neurons strongly suggests that A3V transport is preferentially anterograde and transduces postsynaptic neurons. Comparison with existing method(s) A3V displays no extrasynaptic leakage and moderate speed of synapse passage, which is better than other viruses previously reported. Compared with AAV1&9, which have been shown to pass one synapse anterogradely, A3V passes several synapses in the anterograde direction. Conclusions A3V would be a good tool to study the topographic organization of projection axons and their target neurons.  

5.3056           Hepatitis E virus persists in the ejaculate of chronically infected men

Horvatis, T., Wissmann, J-E., Johne, R., Lütgehetmann, M., Steinmann, E. and Pischke, S.

1. Hepatol., 75, 55-63 (2021)

  Background & Aims Hepatitis E virus (HEV) infections are prevalent worldwide. Various viruses have been detected in the ejaculate and can outlast the duration of viremia, indicating replication beyond the blood-testis barrier. HEV replication in diverse organs, however, is still widely misunderstood. We aimed to determine the occurrence, features and morphology of HEV in the ejaculate. Methods The presence of HEV in testis was assessed in 12 experimentally HEV-genotype 3-infected pigs. We further tested ejaculate, urine, stool and blood from 3 chronically HEV genotype 3-infected patients and 6 immunocompetent patients with acute HEV infection by HEV-PCR. Morphology and genomic characterization of HEV particles from various human compartments were determined by HEV-PCR, density gradient measurement, immune-electron microscopy and genomic sequencing. Results In 2 of the 3 chronically HEV-infected patients, we observed HEV-RNA (genotype 3c) in seminal plasma and semen with viral loads >2 logs higher than in the serum. Genomic sequencing showed significant differences between viral strains in the ejaculate compared to stool. Under ribavirin-treatment, HEV shedding in the ejaculate continued for >9 months following the end of viremia. Density gradient measurement and immune-electron microscopy characterized (enveloped) HEV particles in the ejaculate as intact. Conclusions The male reproductive system was shown to be a niche of HEV persistence in chronic HEV infection. Surprisingly, sequence analysis revealed distinct genetic HEV variants in the stool and serum, originating from the liver, compared to variants in the ejaculate originating from the male reproductive system. Enveloped HEV particles in the ejaculate did not morphologically differ from serum-derived HEV particles.  

5.3057           Clinical efficacy of a novel, high-sensitivity HBcrAg assay in the management of chronic hepatitis B and HBV reactivation

Inoue, T., Kusumoto, S., Iio, E., Maatsuura, K., Aoyagi, K. and Tanaka, Y.

1. Hepatol., 75, 302-310 (2021)

  Background & Aims A fully automated, novel high-sensitivity hepatitis B core-related antigen assay (iTACT-HBcrAg) has been developed. We demonstrate the clinical utility of iTACT-HBcrAg for monitoring chronic hepatitis B (CHB) and for the early detection of HBV reactivation. Methods After fundamental assessments, the clinical performance of iTACT-HBcrAg was compared with other HBV markers. i) Serial sera, available from 161 HBeAg-negative patients with CHB and persistently undetectable HBV DNA, were measured by iTACT-HBcrAg and a conventional HBcrAg assay (G-HBcrAg). ii) Serial sera from 13 HBV-reactivated patients were measured by iTACT-HBcrAg and an ultra-high-sensitivity HBsAg immune complex transfer-chemiluminescent enzyme immunoassay (lower limit of detection; 0.0005 IU/ml, ICT-CLEIA) to compare HBV DNA detection. iii) To elucidate the various HBcrAg components detected by iTACT-HBcrAg, OptiPrep density gradient centrifugation analysis was performed on sera obtained before and after HBV reactivation. Results The analytical performance of iTACT-HBcrAg was satisfactory. The sensitivity of iTACT-HBcrAg (2.1 Log U/ml) was approximately 10-fold greater than that of G-HBcrAg (2.8 Log U/ml). i) HBcrAg was detectable in the sera of 97.5% (157/161) of patients with CHB by iTACT-HBcrAg, of whom 75.2% (121/161) had ≥2.8 Log U/ml HBcrAg and 22.4% (36/161) had 2.1–2.8 Log U/ml HBcrAg, which was undetectable by G-HBcrAg. ii) 9 and 2 of 13 HBV-reactivated patients were HBcrAg-positive by iTACT-HBcrAg before and at HBV DNA positivity, respectively; 7 and 4 were HBcrAg-positive by iTACT-HBcrAg before and at HBsAg-positivity by ICT-CLEIA, respectively. iii) The HBcrAg detected by iTACT-HBcrAg before HBV reactivation was contained in empty particles (22 KDa precore protein). Conclusions iTACT-HBcrAg could be used to better monitor responses to anti-HBV treatments in HBeAg-negative patients and for the early detection of HBV reactivation. Lay summary A fully automated, novel high-sensitivity hepatitis B core-related antigen assay (iTACT-HBcrAg) has been developed. iTACT-HBcrAg can be used to monitor HBeAg-negative patients with chronic hepatitis B, as well as for the early detection of HBV reactivation. iTACT-HBcrAg could be used as a general marker of disease progression and treatment response.  

5.3058           Targeting Cholesterol Metabolism as Efficient Antiviral Strategy Against the Hepatitis E Virus

Glitscher, M., Martin, D.H., Woytinek, K., Schmidt, B., Tabari, D., Scholl, C., Stingl, J.C., Seelow, E., Choi, M. and Hildt, E. Cell. Mol. Gastroenterol. Hepatol., 12(1), 159-180 (2021)   Background and aims The Hepatitis E virus hijacks the endosomal system for its release. These structures are highly dependent on cholesterol. Hence, this study investigates the impact of HEV on cholesterol-metabolism, the effect of intracellular cholesterol content on HEV-release and the potential of cholesterol-modulators to serve as antivirals. Methods Intracellular cholesterol-content of cells was modulated and impacts on HEV were monitored using qPCR, Western blot, microscopy, virus-titration and density-gradient centrifugation. Blood-lipids and HEV-RNA were routinely quantified in chronically infected patients during follow-up visits. Results In HEV-infected cells, decreased levels of cholesterol are found. In patients, HEV infection decreases serum-lipid concentrations. Importantly, statin treatment herein increases viral titers. Similarly, reduction of intracellular cholesterol via simvastatin treatment increases viral release in vitro. On the contrary, elevating intracellular cholesterol via LDL or 25-hydroxycholesterol strongly reduces viral release due to enhanced lysosomal degradation of HEV. Drug-induced elevation of intracellular cholesterol via fenofibrate or PSC833 impairs HEV release via the same mechanism. Conclusions This study analyses the crosstalk between HEV and intracellular cholesterol. The results highlight the importance of an intact cholesterol homeostasis for HEV-release and thereby identify a potential target for antiviral strategies. Especially fenofibrate is considered a promising novel antiviral against HEV. Beyond this, the study may help clinicians evaluating co-treatments of HEV-infected patients with statins, as this may be counter indicated.  

5.3059           SMN Depleted Mice Offer a Robust and Rapid Onset Model of Nonalcoholic Fatty Liver Disease

Deguise, M-O., Pileggi, C., De Repentigny, Y., Beauvais, A., Tierney, A. et al Cell. Mol. Gastroenterol. Hepatol., 12(1), 354-377e.3 (2021)   Background & Aims Nonalcoholic fatty liver disease (NAFLD) is considered a health epidemic with potential devastating effects on the patients and the healthcare systems. Current preclinical models of NAFLD are invariably imperfect and generally take a long time to develop. A mouse model of survival motor neuron (SMN) depletion (Smn^2B/- mice) was recently shown to develop significant hepatic steatosis in less than 2 weeks from birth. The rapid onset of fatty liver in Smn^2B/- mice provides an opportunity to identify molecular markers of NAFLD. Here, we investigated whether Smn^2B/- mice display typical features of NAFLD/nonalcoholic steatohepatitis (NASH). Methods Biochemical, histologic, electron microscopy, proteomic, and high-resolution respirometry were used. Results The Smn^2B/- mice develop microvesicular steatohepatitis within 2 weeks, a feature prevented by AAV9-SMN gene therapy. Although fibrosis is not overtly apparent in histologic sections of the liver, there is molecular evidence of fibrogenesis and presence of stellate cell activation. The consequent liver damage arises from mitochondrial reactive oxygen species production and results in hepatic dysfunction in protein output, complement, coagulation, iron homeostasis, and insulin-like growth factor-1 metabolism. The NAFLD phenotype is likely due to non-esterified fatty acid overload from peripheral lipolysis subsequent to hyperglucagonemia compounded by reduced muscle use and insulin resistance. Despite the low hepatic mitochondrial content, isolated mitochondria show enhanced β-oxidation, likely as a compensatory response, resulting in the production of reactive oxygen species. In contrast to typical NAFLD/NASH, the Smn^2B/- mice lose weight because of their associated neurological condition (spinal muscular atrophy) and develop hypoglycemia. Conclusions The Smn^2B/- mice represent a good model of microvesicular steatohepatitis. Like other models, it is not representative of the complete NAFLD/NASH spectrum. Nevertheless, it offers a reliable, low-cost, early-onset model that is not dependent on diet to identify molecular players in NAFLD pathogenesis and can serve as one of the very few models of microvesicular steatohepatitis for both adult and pediatric populations.  

5.3060           Temporally multiplexed dual-plane imaging of neural activity with four-dimensional precision

Onda, M., Takeuchi, R.F., Isobe, K., Suzuki, T., Masaki, Y., Morimoto, N. and Osakada, F. Neurosci. Res., 171, 9-18 (2021)   Spatiotemporal patterns of neural activity generate brain functions, such as perception, memory, and behavior. Four-dimensional (4-D: x, y, z, t) analyses of such neural activity will facilitate understanding of brain functions. However, conventional two-photon microscope systems observe single-plane brain tissue alone at a time with cellular resolution. It faces a trade-off between the spatial resolution in the x-, y-, and z-axes and the temporal resolution by a limited point-by-point scan speed. To overcome this trade-off in 4-D imaging, we developed a holographic two-photon microscope for dual-plane imaging. A spatial light modulator (SLM) provided an additional focal plane at a different depth. Temporal multiplexing of split lasers with an optical chopper allowed fast imaging of two different focal planes. We simultaneously recorded the activities of neurons on layers 2/3 and 5 of the cerebral cortex in awake mice in vivo. The present study demonstrated the proof-of-concept of dual-plane two-photon imaging of neural circuits by using the temporally multiplexed SLM-based microscope. The temporally multiplexed holographic microscope, combined with in vivo labeling with genetically encoded probes, enabled 4-D imaging and analysis of neural activities at cellular resolution and physiological timescales. Large-scale 4-D imaging and analysis will facilitate studies of not only the nervous system but also of various biological systems.  

5.3061           Systemic effect of FHL1 on neuromuscular junction and myotube formation via insulin-like growth factor and myostatin signaling pathways

Wu, J., Zhao, K., Du, Z., Chen, Y., Zhang, F., Jiang, W., Zheng, J., Wu, X., Shen, C. and Xiao, X. Biochnm. Biophys. Res. Comm., 537, 125-131 (2021)   Four-and-a-half LIM domain protein 1 (FHL1) is a member of the FHL protein family that serves as a scaffold protein to maintain normal cellular structure and function. Its mutations have been implicated in multiple muscular diseases. These FHL1 related myopathies are characterized by symptoms such as progressive muscle loss, rigid or bent spine, even cardiac or respiratory failure in some patients, which implies pathological problems not only in muscles, but also in the nervous system. Moreover, decreased FHL1 protein level has been found in patients with FHL1 mutations, indicating the protein loss-of-function as a pathological cause of such diseases. These findings suggest the significance of understanding the systemic role of FHL1 in the homeostasis of nervous system and muscle. Here we reported that Fhl1 loss in C2C12 myotubes obscured acetylcholine receptor (AChR) clustering in addition to myotube fusion, which was associated with impaired MuSK phosphorylation. Mechanistically, myostatin-SMAD2/3 signaling was enhanced, whereas IGF-PI3K-AKT signaling was suppressed in Fhl1^−/− C2C12 myotubes. Reversion of these molecular alterations rescued AChR clustering and differentiation deficits. These data outline a systemic regulation of AChR clustering and myotube fusion by FHL1, which may offer clues for mechanism study and development of therapeutic strategies to treat FHL1 related myopathies.  

5.3062           Adaptive mutations promote hepatitis C virus assembly by accelerating core translocation to the endoplasmic reticulum

Zheng, F., Li, N., Xu, Y., Zhou, Y. and Li, Y-P.

1. Biol. Chem., 296, 100018 (2021)

  The envelopment of hepatitis C virus (HCV) is believed to occur primarily in the endoplasmic reticulum (ER)-associated membrane, and the translocation of viral Core protein from lipid droplets (LDs) to the ER is essential for the envelopment of viral particles. However, the factors involved are not completely understood. Herein, we identified eight adaptive mutations that enhanced virus spread and infectivity of genotype 1a clone TNcc in hepatoma Huh7 cells through long-term culture adaptation and reverse genetic study. Of eight mutations, I853V in NS2 and C2865F in NS5B were found to be minimal mutation sets that enabled an increase in virus production without apparently affecting RNA replication, thus suggesting its roles in the post-replication stage of the HCV life cycle. Using a protease K protection and confocal microscopy analysis, we demonstrated that C2865F and the combination of I853V/C2865F enhanced virus envelopment by facilitating Core translocation from the LDs to the ER. Buoyant density analysis revealed that I853V/C2865F contributed to the release of virion with a density of ∼1.10 g/ml. Moreover, we demonstrated that NS5B directly interacted with NS2 at the protease domain and that mutations I853V, C2865F, and I853V/C2865F enhanced the interaction. In addition, C2865F also enhanced the interaction between NS5B and Core. In conclusion, this study demonstrated that adaptive mutations in NS2 and NS5B promoted HCV envelopment by accelerating Core translocation from the LDs to the ER and reinforced the interaction between NS2 and NS5B. The findings facilitate our understanding of the assembly of HCV morphogenesis.  

5.3063           Genetic suppression of the dopamine D3 receptor in striatal D1 cells reduces the development of L-DOPA-induced dyskinesia

Lanza, K., Centner, A., Coyle, M., Del Priore, I., Manfredsson, F.P. and Bishop, C. Exp. Neurol., 336, 113534 (2021)   Parkinson's Disease (PD) is symptomatically managed with L-DOPA but chronic use results in L-DOPA-induced dyskinesia (LID) characterized by abnormal involuntary movements (AIMs). In LID, dopamine D3 receptors (D3R) are upregulated on D1 receptor (D1R)-bearing medium spiny neurons where the can synergistically drive downstream signaling and motor behaviors. Despite evidence implying D1R-D3R cooperativity in LID, the dyskinesiogenic role of D3R has never been directly tested. To this end, we developed a specific cre-dependent microRNA (miRNA) to irreversibly prevent D3R upregulation in D1R striatal cells. D1-Cre rats received unilateral 6-hydroxydopamine lesions. Three weeks later, rats received an adeno-associated virus expressing either D3R miRNA or a scrambled (SCR) miRNA delivered into the striatum. After 4 weeks, rats received chronic L-DOPA (6 mg/kg) or vehicle. AIMs development and motor behaviors were assayed throughout treatment. At the conclusion of the experiment, efficacy and fidelity of the miRNA strategy was analyzed using in situ hybridization (ISH). ISH analyses demonstrated that D1R+/D3R+ cells were upregulated in LID and that the selective D3R miRNA reduced D1R+/D3R+ co-expression. Importantly, silencing of D3R also significantly attenuated LID development without impacting L-DOPA efficacy or other locomotion. These data highlight a dyskinesiogenic role of D3R within D1R cells in LID and highlight aberrant D1R-D3R interactions as targets of LID management.  

5.3064           Viral-based rodent and nonhuman primate models of multiple system atrophy: Fidelity to the human disease

Marmion, D.J., Rutkowski, A.A., Chatterjee, D., Hiller, B.M., Werner, M.H., Bezard, E., Kirik, D., McCown, T. and Jeffrey, S.J.G. Neurobiol. Disease, 148, 105184 (2021)   Multiple system atrophy (MSA) is a rare and extremely debilitating progressive neurodegenerative disease characterized by variable combinations of parkinsonism, cerebellar ataxia, dysautonomia, and pyramidal dysfunction. MSA is a unique synucleinopathy, in which alpha synuclein-rich aggregates are present in the cytoplasm of oligodendroglia. The precise origin of the alpha synuclein (aSyn) found in the glial cytoplasmic inclusions (GCIs) as well the mechanisms of neurodegeneration in MSA remain unclear. Despite this fact, cell and animal models of MSA rely on oligodendroglial overexpression of aSyn. In the present study, we utilized a novel oligotrophic AAV, Olig001, to overexpress aSyn specifically in striatal oligodendrocytes of rats and nonhuman primates in an effort to further characterize our novel viral vector-mediated MSA animal models. Using two cohorts of animals with 10-fold differences in Olig001 vector titers, we show a dose-dependent formation of MSA-like pathology in rats. High titer of Olig001-aSyn in these animals were required to produce the formation of pS129+ and proteinase K resistant aSyn-rich GCIs, demyelination, and neurodegeneration. Using this knowledge, we injected high titer Olig001 in the putamen of cynomolgus macaques. After six months, histological analysis showed that oligodendroglial overexpression of aSyn resulted in the formation of hallmark GCIs throughout the putamen, demyelination, a 44% reduction of striatal neurons and a 12% loss of nigral neurons. Furthermore, a robust inflammatory response similar to MSA was produced in Olig001-aSyn NHPs, including microglial activation, astrogliosis, and a robust infiltration of T cells into the CNS. Taken together, oligodendroglial-specific viral vector-mediated overexpression of aSyn in rats and nonhuman primates faithfully reproduces many of the pathological disease hallmarks found in MSA. Future studies utilizing these large animal models of MSA would prove extremely valuable as a pre-clinical platform to test novel therapeutics that are so desperately needed for MSA.  

5.3065           Current progress and limitations of AAV mediated delivery of protein therapeutic genes and the importance of developing quantitative pharmacokinetic/pharmacodynamic (PK/PD) models

Chowhury, E.A., Meno-Tetang, G.M., Chang, H.Y., Wu, S., Huang, H.W., Jamier, T., Chandran, J. and Shah, D.K. Adv. Drug Delivery Reviews, 170, 214-237 (2021)   While protein therapeutics are one of the most successful class of drug molecules, they are expensive and not suited for treating chronic disorders that require long-term dosing. Adeno-associated virus (AAV) mediated in vivo gene therapy represents a viable alternative, which can deliver the genes of protein therapeutics to produce long-term expression of proteins in target tissues. Ongoing clinical trials and recent regulatory approvals demonstrate great interest in these therapeutics, however, there is a lack of understanding regarding their cellular disposition, whole-body disposition, dose-exposure relationship, exposure-response relationship, and how product quality and immunogenicity affects these important properties. In addition, there is a lack of quantitative studies to support the development of pharmacokinetic-pharmacodynamic models, which can support the discovery, development, and clinical translation of this delivery system. In this review, we have provided a state-of-the-art overview of current progress and limitations related to AAV mediated delivery of protein therapeutic genes, along with our perspective on the steps that need to be taken to improve clinical translation of this therapeutic modality.  

5.3066           S-Allylmercaptocysteine improves alcoholic liver disease partly through a direct modulation of insulin receptor signaling

Luo, P., Zhang, M., Zhang, R., Zhang, H., Liu, Y., Li, W., Sun, X., Yu, Q., Tipoe, G.L. Acta Pharmaceutica Sinica B, 11(3), 668-679 (2021)   Alcoholic liver disease (ALD) causes insulin resistance, lipid metabolism dysfunction, and inflammation. We investigated the protective effects and direct regulating target of S-allylmercaptocysteine (SAMC) from aged garlic on liver cell injury. A chronic ethanol-fed ALD in vivo model (the NIAAA model) was used to test the protective functions of SAMC. It was observed that SAMC (300 mg/kg, by gavage method) effectively ameliorated ALD-induced body weight reduction, steatosis, insulin resistance, and inflammation without affecting the health status of the control mice, as demonstrated by histological, biochemical, and molecular biology assays. By using biophysical assays and molecular docking, we demonstrated that SAMC directly targeted insulin receptor (INSR) protein on the cell membrane and then restored downstream IRS-1/AKT/GSK3β signaling. Liver-specific knock-down in mice and siRNA-mediated knock-down in AML-12 cells of Insr significantly impaired SAMC (250 μmol/L in cells)-mediated protection. Restoration of the IRS-1/AKT signaling partly recovered hepatic injury and further contributed to SAMC's beneficial effects. Continuous administration of AKT agonist and recombinant IGF-1 in combination with SAMC showed hepato-protection in the mice model. Long-term (90-day) administration of SAMC had no obvious adverse effect on healthy mice. We conclude that SAMC is an effective and safe hepato-protective complimentary agent against ALD partly through the direct binding of INSR and partial regulation of the IRS-1/AKT/GSK3β pathway.  

5.3067           Generation of nonhuman primate retinitis pigmentosa model by in situ knockout of RHO in rhesus macaque retina

Li, S., Hu, Y., Li, Y., Hu, M., Wang, W. et al Science Bulletin, 66, 374-385 (2021)   Retinitis pigmentosa (RP) is a form of inherited retinal degenerative diseases that ultimately involves the macula, which is present in primates but not in the rodents. Therefore, creating nonhuman primate (NHP) models of RP is of critical importance to study its mechanism of pathogenesis and to evaluate potential therapeutic options in the future. Here we applied adeno-associated virus (AAV)-delivered CRISPR/SaCas9 technology to knockout the RHO gene in the retinae of the adult rhesus macaque (Macaca mulatta) to investigate the hypothesis whether non-germline mutation of the RHO gene is sufficient to recapitulate RP. Through a series of studies, we were able to demonstrate successful somatic editing of the RHO gene and reduced RHO protein expression. More importantly, the mutant macaque retinae displayed clinical RP phenotypes, including photoreceptor degeneration, retinal thinning, abnormal rod subcellular structures, and reduced photoresponse. Therefore, we suggest somatic editing of the RHO gene is able to phenocopy RP, and the reduced time span in generating NHP mutant accelerates RP research and expands the utility of NHP model for human disease study.  

5.3068           Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling

Singh, R., Kaundal, R.K., Zhao, B., Bouchareb, R. and Lebeche, D. Pharmacological Res., 167, 105414 (2021)   Cardiac fibrosis is characterized by excessive deposition of extracellular matrix proteins and myofibroblast differentiation. Our previous findings have implicated resistin in cardiac fibrosis; however, the molecular mechanisms underlying this process are still unclear. Here we investigated the role of resistin in fibroblast-to-myofibroblast differentiation and elucidated the pathways involved in this process. Fibroblast-to-myofibroblast transdifferentiation was induced with resistin or TGFβ1 in NIH-3T3 and adult cardiac fibroblasts. mRNA and protein expression of fibrotic markers were analyzed by qPCR and immunoblotting. Resistin-knockout mice, challenged with a high-fat diet (HFD) for 20 weeks to stimulate cardiac impairment, were analyzed for cardiac function and fibrosis using histologic and molecular methods. Cardiac fibroblasts stimulated with resistin displayed increased fibroblast-to-myofibroblast conversion, with increased levels of αSma, col1a1, Fn, Ccn2 and Mmp9, with remarkable differences in the actin network appearance. Mechanistically, resistin promotes fibroblast-to-myofibroblast transdifferentiation and fibrogenesis via JAK2/STAT3 and JNK/c-Jun signaling pathways, independent of TGFβ1. Resistin-null mice challenged with HFD showed an improvement in cardiac function and a decrease in tissue fibrosis and reduced mRNA levels of fibrogenic markers. These findings are the first to delineate the role of resistin in the process of cardiac fibroblast-to-myofibroblast differentiation via JAK/STAT3 and JNK/c-Jun pathways, potentially leading to stimulation of cardiac fibrosis.  

5.3069           Global Knockdown of Retinoid-related Orphan Receptor α in Mature Purkinje Cells Reveals Aberrant Cerebellar Phenotypes of Spinocerebellar Ataxia

Yasui, H., Matsuzaki, Y., Konno, A. and Hirai, H. Neuroscience, 462, 328-336 (2021)   Retinoid-related orphan receptor α (RORα) is a transcription factor expressed in a variety of tissues throughout the body. Knockout of RORα leads to various impairments, including defects in cerebellar development, circadian rhythm, lipid metabolism, immune function, and bone development. Previous studies have shown significant reduction of RORα expression in Purkinje cells (PCs) of spinocerebellar ataxia (SCA) type 1 and type 3/MJD (Machado–Joseph disease) model mice. However, it remains unclear to what extent the RORα reduction in PCs is involved in the disease pathology. Here, RORα expression was downregulated specifically in mature mouse PCs by intravenous infusion of blood–brain barrier-permeable adeno-associated virus (AAV), expressing a microRNA against RORα (miR-RORα) under the control of the PC-specific L7-6 promoter. The systemic AAV infusion led to extensive transduction of PCs. The RORα knock-down caused degeneration of PCs including disruption of the PC monolayer alignment and dendrite atrophy. In behavioral experiments, mice expressing miR-RORα showed motor learning deficits, and later, overt cerebellar ataxia. Thus, RORα in mature PCs plays pivotal roles in maintenance of PC dendrites and the monolayer alignment, and consequently, motor learning and motor function. Decrease in RORα expression in PCs could be a primary etiology of the cerebellar symptoms in patients with SCA1 and SCA3/MJD.  

5.3070           Digital CRISPR-based method for the rapid detection and absolute quantification of nucleic acids

Wu, X., Tay, J.K., Goh, C.K., Chan, C., Lee, Y.H., Springs, S.L., Wang, D.Y., Loh, K.S., Lu, T.K. and Yu, H. Biomaterials, 274, 120876 (2021)   Rapid diagnostics of adventitious agents in biopharmaceutical/cell manufacturing release testing and the fight against viral infection have become critical. Quantitative real-time PCR and CRISPR-based methods rapidly detect DNA/RNA in 1 h but suffer from inter-site variability. Absolute quantification of DNA/RNA by methods such as digital PCR reduce this variability but are currently too slow for wider application. Here, we report a RApid DIgital Crispr Approach (RADICA) for absolute quantification of nucleic acids in 40-60 min. Using SARS-CoV-2 as a proof-of-concept target, RADICA allows for absolute quantification with a linear dynamic range of 0.6–2027 copies/μL (R² value > 0.99), high accuracy and low variability, no cross-reactivity to similar targets, and high tolerance to human background DNA. RADICA's versatility is validated against other targets such as Epstein-Barr virus (EBV) from human B cells and patients' serum. RADICA can accurately detect and absolutely quantify EBV DNA with similar dynamic range of 0.5–2100 copies/μL (R² value > 0.98) in 1 h without thermal cycling, providing a 4-fold faster alternative to digital PCR-based detection. RADICA therefore enables rapid and sensitive absolute quantification of nucleic acids which can be widely applied across clinical, research, and biomanufacturing areas.  

5.3071           Blocking endothelial TRPV4-Nox2 interaction helps reduce ROS production and inflammation, and improves vascular function in obese mice

Gao, M., Han, J., Zhu, Y., Tang, C., Liu, L., Xiao, W. and Ma, X.

1. Mol. Cell. Cardiol., 157, 66-76 (2021)

  Obesity induces inflammation and oxidative stress, and ultimately leads to vasodilatory dysfunction in which Transient receptor potential vanilloid type 4 (TRPV4) and Nicotinamide Adenine Dinucleotide Phosphate Oxidase (Nox2) have been reported to be involved. However, little attention has been paid to the role of the TRPV4-Nox2 complex in these problems. The purpose of this study was to figure out the role of the TRPV4-Nox2 complex in obesity-induced inflammation, oxidative stress, and vasodilatory dysfunction. Using fluorescence resonance energy transfer and immunoprecipitation assays, we found enhanced TRPV4 and Nox2 interactions in obese mice. Using q-PCR, fluorescent dye dihydroethidium staining, and myotonic techniques, we found that obesity caused inflammation, oxidative stress, and vasodilatory dysfunction. Using adeno-associated viruses, we found that enhancement or attenuation of TRPV4-Nox2 interaction altered the vaso-function. Based on these findings, we found a small-molecule drug, M12, that interrupted the TRPV4-Nox2 interaction, thereby reducing inflammatory factors and reactive oxygen species production and helping to restore the vasodilatory function. In summary, our results revealed a new mechanism by which obesity-induced inflammation, oxidative stress, and vasodilatory dysfunction is caused by enhanced TRPV4-Nox2 interactions. Using M12 to interrupt the TRPV4-Nox2 interaction may have anti-inflammatory and anti-oxidative stress effects and help restore vasodilatory function and thus provide a new therapeutic approach to obesity.  

5.3072           Intravitreal gene therapy protects against retinal dysfunction and degeneration in sheep with CLN5 Batten disease

Murray, S.J., Russell, K.N., Melzer, T.R., Gray, S.J., Heap, S.J., Palmer, D.N. and Mitchell, N.L. Exp. Eye res., 207, 108600 (2021)   Neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of inherited neurodegenerative diseases primarily affecting children. A common feature across most NCLs is the progressive loss of vision. We performed intravitreal injections of self-complementary AAV9 vectors packaged with either ovine CLN5 or CLN6 into one eye of 3-month-old CLN5^−/− or CLN6^−/− animals, respectively. Electroretinography (ERG) was performed every month following treatment, and retinal histology was assessed post-mortem in the treated compared to untreated eye. In CLN5^−/− animals, ERG amplitudes were normalised in the treated eye whilst the untreated eye declined in a similar manner to CLN5 affected controls. In CLN6^−/− animals, ERG amplitudes in both eyes declined over time although the treated eye showed a slower decline. Post-mortem examination revealed significant attenuation of retinal atrophy and lysosomal storage body accumulation in the treated eye compared with the untreated eye in CLN5^−/− animals. This proof-of-concept study provides the first observation of efficacious intravitreal gene therapy in a large animal model of NCL. In particular, the single administration of AAV9-mediated intravitreal gene therapy can successfully ameliorate retinal deficits in CLN5^−/− sheep. Combining ocular gene therapy with brain-directed therapy presents a promising treatment strategy to be used in future sheep trials aiming to halt neurological and retinal disease in CLN5 Batten disease.  

5.3073           Phenotypic characterization of cell culture-derived hepatitis E virus subjected to different chemical treatments: Application in virus removal via nanofiltration

Ideno, S., Inoue, T., Takahashi, K., Urayama, T., Maeno, H., Takeuchi, K. and Sakai, K.

1. Virol. Methods, 296, 114244 (2021)

  Safety evaluation for the hepatitis E virus (HEV) is required for plasma fractionation products. Plasma-derived HEV (pHEV) is quite unique in that it is associated with a lipid membrane, which, when stripped during manufacturing processes, induces morphological changes in the virus, making it difficult to select proper HEV phenotypes for clearance studies. We developed a convenient system for the preparation of a high titer cell culture-derived HEV (cHEV). In this system, PLC/PRF/5 cells transfected with the wild-type HEV genome generated lipid membrane-associated cHEV for a long period even after cryopreservation. We also examined how this lipid membrane-associated cHEV can be used to verify the robustness of pHEV removal via 19-nm nanofiltration. Sodium-deoxycholate and trypsin (NaDOC/T) treatment not only dissolved lipid but also digested membrane-associated proteins from pHEV and cHEV, making the resulting cHEV particle smaller in size than any pHEV phenotypes generated by ethanol or solvent-detergent treatment in this study. In both 19-nm and 35-nm nanofiltration, cHEV behaved identically to pHEV. These results indicate that cHEV is a useful resource for viral clearance studies in term of availability, and the use of NaDOC/T-treated cHEV ensured robust pHEV removal capacity via 19-nm nanofiltration.  

5.3074           Intramolecular recombination enables the formation of hepatitis B virus (HBV) cccDNA in mice after HBV genome transfer using recombinant AAV vectors

Ko, C., Su, J., Festag, J., Bester, R. and Kosinska, A.D: Antiviral Res., 194, 105140 (2021)   The mouse is not a natural host of hepatitis B virus (HBV) infection and - despite engraftment of hepatocytes with the HBV receptor - does not support formation of HBV covalently closed circular (ccc) DNA serving as a template for viral transcription and permitting persistent infection. In a recent study, cccDNA formation in mouse hepatocytes has been described following an HBV genome delivery by a recombinant, adeno-associated virus vector (rAAV) (Lucifora et al., 2017). The integrity of HBV cccDNA, its origin and functionality, however, remained open. In this study, we investigated the identity, origin, and functionality of cccDNA established in mice infected with rAAV carrying 1.3-fold overlength HBV genomes. We show that replication of HBV genotypes A, B, C and D can be initiated in mouse livers, and that cccDNA derived from all genotypes is detected. Restriction enzyme and exonuclease digestion as well as sequencing analysis of cccDNA amplicons revealed authentic HBV cccDNA without any detectable alteration compared to cccDNA established after HBV infection of human liver cells. Mouse livers transduced with a core protein-deficient HBV using rAAV still supported cccDNA formation demonstrating that the genesis of cccDNA was independent of HBV replication. When mice were infected with an rAAV-HBV1.3 carrying premature stop codons in the 5′ but not in the 3′ core protein open reading frame, the stop codon was partially replaced by the wild-type sequence. This strongly indicated that intramolecular recombination, based on >900 identical base pairs residing at the both ends of the HBV1.3 transgene was the origin of cccDNA formation. Accordingly, we observed a constant loss of cccDNA molecules from mouse livers over time, while HBeAg levels increased over the first two weeks after rAAV-HBV1.3 infection and remained constant thereafter, suggesting a minor contribution of the cccDNA molecules formed to viral transcription and protein expression. In summary, our results provide strong evidence that intramolecular recombination of an overlength, linear HBV genome, but not HBV genome recycling, enables cccDNA formation in rAAV-HBV mouse models.  

5.3075           Hypercholesterolemia risk associated Abca6 does not regulate lipoprotein metabolism in mice or hamster

He, B., Kang, S., Chen, Z., Liu, X., Wang, J., Li, X., Liu, X., Zheng, L., Luo, M. and Wang, Y. BBA-Mol. Cell Biol. Lipids, 1866, 159006 (2021)   Hypercholesterolemia has strong heritability and about 40–60% of hypercholesterolemia is caused by genetic risk factors. A number of monogenic genes have been identified so far for familial hypercholesterolemia (FH). However, in the general population, more than 90% of individuals with LDL cholesterol over 190 mg/dL do not carry known FH mutations. Large scale whole-exome sequencing has identified thousands of variants that are predicted to be loss-of-function (LoF) and each individual has a median of about twenty rare LoF variants and several hundreds more common LoF variants. However, majority of those variants have not been characterized and their functional consequence remains largely unknown. Rs77542162 is a common missense variant in ABCA6 and is strongly associated with hypercholesterolemia in different populations. ABCA6 is a cholesterol responsive gene and has been suggested to play a role in lipid metabolism. However, whether and how rs77542162 and ABCA6 regulate lipoprotein metabolism remain unknown. In current study, we systemically characterized the function of rs77542162 and ABCA6 in cultured cells and in vivo of rodents. We found that Abca6 is specifically expressed on the basolateral surface of hepatocytes in mouse liver. The rs77542162 variant disrupts ABCA6 protein stability and results in loss of functional protein. However, we found no evidence that Abca6 plays a role in lipoprotein metabolism in either normal mice or hypercholesterolemia mice or hamsters. Thus, our results suggest that Abca6 does not regulate lipoprotein metabolism in rodents and highlight the challenge and importance of functional characterization of disease-associated variants in animal models.  

5.3076           Hepatitis A Virus (Picornaviridae)

Dotzauer, A. Encyclopedia of Virology, 2, 362-372 (2021)   Hepatitis A is the most common viral-caused liver disease, whose incidence is associated with hygienic factors. Its age-dependent clinical appearance is heterogeneous varying between normal, prolonged, relapsing and fulminant. Chronic infections are not described, and elimination of the non-cytolytic hepatitis A virus (HAV) is mediated by immunopathogenetic mechanisms. The slow adaptive immune responses against HAV, which spreads fecal-orally, significantly deviate from the average course during viral infections. HAV exhibits several unique properties and mechanisms of its interaction with the host. To evade innate cellular antiviral defense mechanisms, HAV inhibits the induction of both interferon synthesis and apoptosis. There is no specific antiviral treatment, but vaccines are available.  

5.3077           Vesicle-Mediated Transcytosis and Export of Viruses

Rivera-Sarrano, E.E. and Lemon, S.M. Encyclopedia of Virology, 1, 529-541 (2021)   Viruses usurp cellular mechanisms of vesicle-mediated transport to both pass through and gain egress from cells. Transcytosis is a process by which viruses undergo vectorial internalization, transport across the cytoplasm, and release on the opposing side of polarized cells in the absence of productive infection. It allows viruses to traverse defensive barriers posed by layers of epithelial cells to gain access to underlying tissue compartments, ultimately contributing to within-host dissemination and viral pathogenesis. Viruses also utilize vesicle-mediated transport to facilitate the nonlytic release of newly replicated virions, exploiting normal cellular functions involved in secretory autophagy or the biogenesis of exosomes.  

5.3078           Phycodnaviruses (Phycodnaviridae)

Van etten, J.L. and Dunigan, D.D. Encyclopedia of Virology, 4, 687-695 (2021)   Viruses in the family Phycodnaviridae infect aquatic algae and they are present in inland, coastal and marine environments throughout the world, sometimes in very high concentrations. Consequently, phycodnaviruses contribute to microbial composition and diversity, nutrient cycling, carbon flow, and other biogeochemically-important processes in aqueous environments. The viruses are relatively large and have 170–450 kbp double-stranded (ds) genomes that encode ~200 to ~550 proteins and as many as 16 tRNAs. The algal viruses encode many interesting and unexpected proteins but over half of the predicted virus-encoded proteins do not match proteins in the databases and so their functions are unknown.  

5.3079           Suppressor of cytokine signalling-2 controls hepatic gluconeogenesis and hyperglycemia by modulating JAK2/STAT5 signalling pathway

Zhang, X., Zhuang, Y., Qin, T., Chang, M., Ji, X., Wang, N., Zhang, Z., Zhou, H., Wang, Q. and Li, J.Z. Metabolism Clin. Exp., 122, 154823 (2021)   Hepatic gluconeogenesis plays a crucial role in maintaining blood glucose homeostasis in mammals. Globe knockout of suppressor of cytokine signalling-2 (SOCS2), a feedback inhibitor of cytokine signalling, has been shown resistant to high-fat-diet (HFD)-induced hepatic steatosis with impaired glucose tolerance in mice. However, the underlying mechanism of SOCS2 regulates hepatic glucose homeostasis still undefined. In the present study, we demonstrated that the hepatic SOCS2 expression is markedly reduced in fasted C57BL/6 J mice or db/db mice. Moreover, hepatic SOCS2 expression levels are induced by metformin treatment. Ablation of SOCS2 attenuates suppressing effects of metformin on gluconeogenesis in hepatocytes. Gain- and loss-of-function studies indicated that SOCS2 regulates hepatic gluconeogenic genes expression and glucose output by mediating JAK2/STAT5 signalling pathway in db/db mice. Mechanistically, we observed that SOCS2 inactivates STAT5 by attenuating the interaction between JAK2 and STAT5, which in turn reduces hepatic gluconeogenesis. The present study reveals a critical role of SOCS2 in regulating hepatic gluconeogenesis. The inhibitory effect of metformin on gluconeogenesis is mediated, at least in part, by upregulating SOCS2 and therefore reducing hepatic gluconeogenic genes expression. SOCS2 may represent a new therapeutic target for the treatment of diabetes.  

5.3080           Atherosclerosis-associated hepatic secretion of VLDL but not PCSK9 is dependent on cargo receptor protein Surf4

Wang, B., Shen, Y., Zhai, L., xia, X., Gu, H-m. et al

1. Lipid Res., 62, 100091 (2021)

  Plasma LDL is produced from catabolism of VLDL and cleared from circulation mainly via the hepatic LDL receptor (LDLR). Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes LDLR degradation, increasing plasma LDL-C levels. Circulating PCSK9 is mainly secreted by the liver, whereas VLDL is exclusively secreted by hepatocytes. However, the mechanism regulating their secretion is not completely understood. Surfeit 4 (Surf4) is a cargo receptor localized in the ER membrane. It recruits cargos into coat protein complex II vesicles to facilitate their secretion. Here, we investigated the role of Surf4 in VLDL and PCSK9 secretion. We generated Surf4 liver-specific knockout mice and found that knockout of Surf4 did not affect PCSK9 secretion, whereas it significantly reduced plasma levels of cholesterol, triglyceride, and lipid-binding protein apolipoprotein B (apoB). In cultured human hepatocytes, Surf4 coimmunoprecipitated and colocalized with apolipoprotein B100, and Surf4 silencing reduced secretion of apolipoprotein B100. Furthermore, knockdown of Surf4 in LDLR knockout (Ldlr^−/−) mice significantly reduced triglyceride secretion, plasma levels of apoB and non-HDL-C, and the development of atherosclerosis. However, Surf4 liver-specific knockout mice and Surf4 knockdown in Ldlr^−/− mice displayed similar levels of liver lipids and plasma alanine aminotransferase activity as control mice, indicating that inhibition of Surf4 does not cause notable liver damage. Expression of stearoyl-CoA desaturase-1 was also reduced in the liver of these mice, suggesting a reduction in de novo lipogenesis. In summary, hepatic deficiency of Surf4 reduced VLDL secretion and the development of atherosclerosis but did not cause significant hepatic lipid accumulation or liver damage.  

5.3081           Human stem cell-based retina on chip as new translational model for validation of AAV retinal gene therapy vectors

Achberger, K., Cipriano, M., Düchs, M.J., Schön, C., Michelfelder, S. et al Stem Cell Reports, 16, 2242-2256 (2021)   Gene therapies using adeno-associated viruses (AAVs) are among the most promising strategies to treat or even cure hereditary and acquired retinal diseases. However, the development of new efficient AAV vectors is slow and costly, largely because of the lack of suitable non-clinical models. By faithfully recreating structure and function of human tissues, human induced pluripotent stem cell (iPSC)-derived retinal organoids could become an essential part of the test cascade addressing translational aspects. Organ-on-chip (OoC) technology further provides the capability to recapitulate microphysiological tissue environments as well as a precise control over structural and temporal parameters. By employing our recently developed retina on chip that merges organoid and OoC technology, we analyzed the efficacy, kinetics, and cell tropism of seven first- and second-generation AAV vectors. The presented data demonstrate the potential of iPSC-based OoC models as the next generation of screening platforms for future gene therapeutic studies.  

5.3082           Comparative structural, biophysical, and receptor binding study of true type and wild type AAV2

Bennett, A., Hull, J., Jolinon, N., Tordo, J., Moss, K., Binns, E. et al

1. Struct. Biol., 213, 107795 (2021)

  Adeno-associated viruses (AAV) are utilized as gene transfer vectors in the treatment of monogenic disorders. A variant, rationally engineered based on natural AAV2 isolates, designated AAV-True Type (AAV-TT), is highly neurotropic compared to wild type AAV2 in vivo, and vectors based on it, are currently being evaluated for central nervous system applications. AAV-TT differs from AAV2 by 14 amino acids, including R585S and R588T, two residues previously shown to be essential for heparan sulfate binding of AAV2. The capsid structures of AAV-TT and AAV2 visualized by cryo-electron microscopy at 3.4 and 3.0 Å resolution, respectively, highlighted structural perturbations at specific amino acid differences. Differential scanning fluorimetry (DSF) performed at different pH conditions demonstrated that the melting temperature (T_m) of AAV2 was consistently ∼5 °C lower than AAV-TT, but both showed maximal stability at pH 5.5, corresponding to the pH in the late endosome, proposed as required for VP1u externalization to facilitate endosomal escape. Reintroduction of arginines at positions 585 and 588 in AAV-TT caused a reduction in T_m, demonstrating that the lack of basic amino acids at these positions are associated with capsid stability. These results provide structural and thermal annotation of AAV2/AAV-TT residue differences, that account for divergent cell binding, transduction, antigenic reactivity, and transduction of permissive tissues between the two viruses. Specifically, these data indicate that AAV-TT may not utilize a glycan receptor mediated pathway to enter cells and may have lower antigenic properties as compared to AAV2.  

5.3083           Identification of coagulation factor IX variants with enhanced activity through ancestral sequence reconstruction

Knight, K., Coyle, C.W., Raford, C.E., Parker, E.T., Fedanov, A., Shields, J.M. et al Blood Advances, 5(17), 3333-3343 (2021)   Orthologous proteins contain sequence disparity guided by natural selection. In certain cases, species-specific protein functionality predicts pharmacological enhancement, such as greater specific activity or stability. However, immunological barriers generally preclude use of nonhuman proteins as therapeutics, and difficulty exists in the identification of individual sequence determinants among the overall sequence disparity. Ancestral sequence reconstruction (ASR) represents a platform for the prediction and resurrection of ancient gene and protein sequences. Recently, we demonstrated that ASR can be used as a platform to facilitate the identification of therapeutic protein variants with enhanced properties. Specifically, we identified coagulation factor VIII (FVIII) variants with improved specific activity, biosynthesis, stability, and resistance to anti-human FVIII antibody–based inhibition. In the current study, we resurrected a panel of ancient mammalian coagulation factor IX (FIX) variants with the goal of identifying improved pharmaceutical candidates. One variant (An96) demonstrated 12-fold greater FIX activity production than human FIX. Addition of the R338L Padua substitution further increased An96 activity, suggesting independent but additive mechanisms. after adeno-associated virus 2 (AAV2)/8-FIX gene therapy, 10-fold greater plasma FIX activity was observed in hemophilia B mice administered AAV2/8-An96–Padua as compared with AAV2/8-human FIX–Padua. Furthermore, phenotypic correction conferred by the ancestral variant was confirmed using a saphenous vein bleeding challenge and thromboelastography. Collectively, these findings validate the ASR drug discovery platform as well as identify an ancient FIX candidate for pharmaceutical development.  

5.3084           N-terminal serine/threonine motif has diverse and important effects on behavior of multiple AAV serotypes

Chen, M.Y., Chen, W., Tong, J., Ho, M.L. and Suh, J. Virology, 563, 107-113 (2021)   Adeno-associated virus (AAV) is a promising gene therapy vector, but questions remain regarding mechanisms of basic viral functions. We previously showed that a serine/threonine (S/T) triplet motif and its flanking residues, located in the overlapping N-terminus of VP1/VP2 and highly conserved across most AAV serotypes, are critical for viral transcript production in vitro. Here we generate a panel of S/T triplet mutants in AAV serotypes 2, 4, and 9 and characterize their behaviors in vitro and in vivo using next generation sequencing. We show that S/T triplet mutations can significantly hinder some stages of transduction in a serotype-dependent manner in vitro. Interestingly, these defects are largely overcome in C57BL/6 mice, with only one mutant displaying altered behavior in vivo. Taken together, our results identify a short N-terminal capsid motif with diverse roles across several AAV serotypes which better informs engineering efforts to improve AAV as a vector for gene therapy.  

5.3085           VTA MC3R neurons control feeding in an activity- and sex-dependent manner in mice

Dunigan, A.I., Olson, D.P. and Roseberry, A.G. Neuropharmacol., 197, 108746 (2021)   Increasing evidence indicates that the melanocortin and mesolimbic dopamine (DA) systems interact to regulate feeding and body weight. Because melanocortin-3 receptors (MC3R) are highly expressed in the ventral tegmental area (VTA), we tested whether VTA neurons expressing these receptors (VTA MC3R neurons) control feeding and body weight in vivo. We also tested whether there were sex differences in the ability of VTA MC3R neurons to control feeding, as MC3R −/− mice show sex-dependent alterations in reward feeding and DA levels, and there are clear sex differences in multiple DA-dependent behaviors and disorders. Designer receptors exclusively activated by designer drugs (DREADD) were used to acutely activate and inhibit VTA MC3R neurons and changes in food intake and body weight were measured. Acutely altering the activity of VTA MC3R neurons decreased feeding in an activity- and sex-dependent manner, with acute activation decreasing feeding, but only in females, and acute inhibition decreasing feeding, but only in males. These differences did not appear to be due to sex differences in the number of VTA MC3R neurons, the ability of hM3Dq to activate VTA MC3R neurons, or the proportion of VTA MC3R neurons expressing tyrosine hydroxylase (TH). These studies demonstrate an important role for VTA MC3R neurons in the control of feeding and reveal important sex differences in behavior, whereby opposing changes in neuronal activity in male and female mice cause similar changes in behavior.  

5.3086           Monitoring of a progressive functional dopaminergic deficit in the A53T-AAV synuclein rats by combining 6-[18F]fluoro-L-m-tyrosine imaging and motor performances analysis

Becker, G., Michel, A., Bahr, M.A., Mairet-Coello, G., Lemaire, C. et al Neurobiol. Aging, 107, 142-152 (2021)   With the emergence of disease-modifying therapies for Parkinson's disease, reliable longitudinal markers are needed to quantify pathology and demonstrate disease progression. We developed the A53T-AAV rat model of synucleinopathy by combining longitudinal measures over 12 weeks. We first characterized the progression of the motor and dopaminergic deficits. Then, we monitored the disease progression using the [¹⁸F]FMT Positron Emission Tomography (PET) radiotracer. The nigral injection of A53T-AAV led to an increase in phosphorylated α-synuclein on S129, a progressive accumulation of α-synuclein aggregates, and a decrease of dopaminergic function associated with a deterioration of motor activity. The longitudinal monitoring of A53T-AAV rats with [¹⁸F]FMT PET showed a progressive reduction of the K_c outcome parameter in the caudate putamen from the lesioned side. Interestingly, the progressive reduction in the [¹⁸F]FMT PET signal correlated with defects in the stepping test. In conclusion, we established a progressive rat model of α-synuclein pathology which monitors the deficit longitudinally using both the [¹⁸F]FMT PET tracer and behavioral parameters, 2 features that have strong relevance for translational approaches.  

5.3087           Sustainability of neutralising antibodies induced by bivalent or quadrivalent HPV vaccines and correlation with efficacy: a combined follow-up analysis of data from two randomised, double-blind, multicentre, phase 3 trials

Mariz, F.C,m Gray, P., Bender, N., Eriksson, T., Kann, H., Apter, D., Paavonen, J., Pajunen, E., Prager, K.M. et al Lancet Infect. Dis., , 1458-1468 (2021)   Background Quadrivalent and bivalent vaccines against oncogenic human papillomavirus (HPV) are used worldwide with different reported overall efficacies against HPV infections. Although protective concentrations of vaccine-induced antibodies are still not formally defined, we evaluated the sustainability of neutralising antibodies in vaccine trial participants 2–12 years after vaccination and the correlation with reported vaccine efficacy. Methods We did a follow-up analysis of data from the Finnish cohorts of two international, randomised, double-blind, phase 3 trials of HPV vaccines, PATRICIA (bivalent, HPV16 and 18) and FUTURE II (quadrivalent, HPV6, 11, 16, and 18). In 2002 and 2004–05, respectively, Finnish girls aged 16–17 years participated in one of these two trials and consented to health registry follow-up with the Finnish Cancer Registry. The cohorts were also linked with the Finnish Maternity Cohort (FMC) that collects first-trimester serum samples from nearly all pregnant Finnish women, resulting in 2046 post-vaccination serum samples obtained during up to 12 years of follow-up. We obtained serum samples from the FMC-based follow-up of the FUTURE II trial (from the quadrivalent vaccine recipients) and the PATRICIA trial (from corresponding bivalent vaccine recipients who were aligned by follow-up time, and matched by the number of pregnancies). We assessed neutralising antibody concentrations (type-specific seroprevalence) to HPV6, 16, and 18, and cross-neutralising antibody responses to non-vaccine HPV types 31, 33, 45, 52, and 58 from 2 to 12 years after vaccination. Findings Up to Dec 31, 2016, we obtained and analysed 577 serum samples from the quadrivalent vaccine recipients and 568 from the bivalent vaccine recipients. In 681 first-pregnancy serum samples, neutralising antibodies to HPV6, 16, and 18 were generally found up to 12 years after vaccination. However, 51 (15%) of 339 quadrivalent vaccine recipients had no detectable HPV18 neutralising antibodies 2–12 years after vaccination, whereas all 342 corresponding bivalent vaccine recipients had HPV18 neutralising antibodies.. In seropositive quadrivalent vaccine recipients, HPV16 geometric mean titres (GMT) halved by years 5–7 (GMT 3679, 95% CI 2377 to 4708) compared with years 2–4 (6642, 2371 to 13 717). Between 5 and 12 years after vaccination, GMT of neutralising antibodies to HPV16 and 18 were 5·7 times and 12·4 times higher, respectively, in seropositive bivalent vaccine recipients than in the quadrivalent vaccine recipients. Cross-neutralising antibodies to HPV31, 33, 45, 52, and 58 were more prevalent in the bivalent vaccine recipients but, when measurable, sustainable up to 12 years after vaccination with similar GMTs in both vaccine cohorts. Seroprevalence for HPV16, 31, 33, 52, and 58 significantly correlated with vaccine efficacy against persistent HPV infections in the bivalent vaccine recipients only (r_s=0·90, 95% CI 0·09 to 0·99, p=0·037, compared with r_s=0·62, 95% CI –0·58 to 0·97, p=0·27 for the quadrivalent vaccine recipients). Correlation of protection with prevalence of neutralising or cross-neutralising HPV antibodies was not significant in the quadrivalent vaccine recipients.  

5.3088           Chapter Nine - Inhibition of hepatitis C virus by vitamin D

Murayama, A. and kato, T. Vitamines and Hormnes, 117, 227-238 (2021)   Until the development of direct-acting antivirals (DAAs), interferon (IFN)-based therapy had been the primary treatment strategy for patients with chronic hepatitis C, even though this therapy has a therapeutic limitations and considerable side effects. Therefore, many efforts have been made to improve the efficacy of treatment. Several clinical studies have clearly shown that supplementation with vitamin D of IFN-based therapy improves treatment efficacy. To clarify the molecular mechanisms of the effect of vitamin D on IFN-based therapy, several researchers have performed basic research with cell culture models of hepatitis C virus (HCV). Consequently, two vitamin D₃ metabolites, 25-hydroxyvitamin D₃ (25-(OH)D₃) and 1α,25-dihydroxyvitamin D₃ (1α,25-(OH)₂D₃), have been suggested to have anti-HCV effects. 25-(OH)D₃ inhibits HCV production by suppressing infectious virus assembly through reducing apolipoprotein expression, while 1α,25-(OH)₂D₃ inhibits HCV production by modulating IFN signaling and/or inducing various host factors associated with the inhibition of viral genome replication. In addition, an antimicrobial peptide, LL-37, which is known to be partly regulated by vitamin D, was also reported to exhibit an anti-HCV effect by disrupting infectious viral particles directly. In conclusion, vitamin D₃ supplementation improves the response rate of IFN-based therapy via the direct and/or indirect anti-HCV effects of vitamin D₃ metabolites.  

5.3089           AAV-mediated in vivo genome editing in vascular endothelial cells

Wu, W., Yang, Y., Yao, F., Dong, L., Xia, X., Zhang, S. and Lei, H. Methods, 194, 12-17 (2021)   In vivo genome editing meets numerous challenges including efficiency and safety. Here we describe an efficient in vivo genome editing method of delivering CRISPR-Cas9 into vascular endothelial cells with adeno-associated viruses (AAVs). In this system, expression of SpCas9 is driven by a specific endothelial promoter of intercellular adhesion molecule 2 (pICAM2) to restrict this foreign enzyme in vascular endothelial cells, which can be efficiently infected by AAV1. We exemplify this approach by editing VEGFR2 in retinal vascular endothelial cells in a mouse model of oxygen-induced retinopathy, and expect that this simplified protocol can be expanded to other researches on editing endothelial genome in vivo.  

5.3090           AAV manufacturing for clinical use: Insights on current challenges from the upstream process perspective

Dobrowaky, T., Gianni, D., Pieracci, J. and Suh, J. Current Opinion in Biomedical Engineering, 20, 100353 (2021)   As gene therapy is experiencing a second wave of interest, recombinant adeno-associated virus (rAAV) has rapidly emerged as one of the most attractive viral transfer tools, thanks to its favorable safety profile and long-term transgene expression. The recent approval of rAAV molecules for the treatment of congenital amaurosis and spinal muscular atrophy has been paving the road for the development of several rAAV-based therapeutics targeting a continuously increasing range of indications. To support this rapid growth, the field is in high demand for robust, scalable and economically viable manufacturing processes. This review will provide an overview of the most common platforms currently adopted at the manufacturing level for rAAV production, focusing on the challenges and bottlenecks experienced in the upstream process.  

5.3091           46. Characterization of Acute Toxicity after High-Dose Systemic Adeno-Associated Virus in Nonhuman Primates, Including Impact of Vector Characteristics

Hordeaux, J., Song, C., Wielechowski, E., Ramezani, A., Dyeer, C., Buza, E.L. et al. ASGCT Annual Meeting Absract, 29(4), Suppl. 1 1-427(2021)   PA Dose-limiting toxicities have occurred following intravenous administration of high doses of adeno-associated virus (AAV) to target the musculoskeletal or central nervous systems. Acute elevations in liver enzymes and/or reductions in platelets have been observed in most high-dose AAV clinical trials. Although infrequent, severe toxicities have included anemia, renal failure, complement activation, and, in the worst cases, fatal hepatobiliary disease. We previously reported the development of acute thrombocytopenia and transaminitis in most nonhuman primates (NHPs) within days of of receiving high doses of AAV (in the 1x1014 GC/kg range). Some animals recover while some develop a lethal syndrome of coagulopathy, liver failure, hemorrhage, and shock. The acute initial presentation of thrombocytopenia and transaminase elevation is common to NHPs and humans and has not been observed in other species in our experience. Although the progression of the initial acute toxicity may differ in human patients with pre-existing conditions compared to healthy animals, we propose that NHPs can be used as a model to better characterize the acute toxicity of high doses of AAV to reduce risk in clinical trials. We conducted studies aimed at investigating the effect of capsids, prophylactic steroids, and vector purification methods on the incidence and severity of acute toxicity following high-dose AAV systemic administration. We administered ten rhesus macaques (16 months to 4.6 years old with anti-capsid neutralizing titers < 5 and weighing 3-7 kg) with AAV9 or an engineered capsid derived from AAV9 (AAV-PHP.eB) encoding green fluorescent protein at 1 or 2 x 1014 GC/kg. We then monitored for acute toxicity for 2 weeks post-administration. All vectors were produced from transient transfection of adherent HEK293 cells and either purified using affinity column chromatography or an iodixanol gradient. Both purification methods yielded   more than 70% of full capsids. We observed acute toxicity in the majority of animals, regardless of the purification method, characterized by thrombocytopenia, liver enzyme elevation with or without hyperbilirubinemia, and increased coagulation times on day 3 post-administration. Most animals recovered from the initial toxicity except for two AAV-PHP.eB-dosed animals that had to be humanely euthanized due to severe coagulopathy. Prophylactic steroids appeared to help with the recovery from the initial toxicity but did not prevent it from occurring. The toxicity was dose dependent, with AAV9 capsid having a narrow safety margin: 1 x 1014 GC/kg was well tolerated whereas a 2-fold increase led to thrombocytopenia in the majority of animals. Importantly, we observed complement activation concurrent with the thrombocytopenia on day 3 with activation of the alternate pathway and elevation of complement Bb fragment and SC5b-9 membrane attack complex, whereas the classical pathway did not appear to be activated (unchanged C4 and C4a levels). Accordingly, there were no detectable IgM antibodies to the capsid on day 3, suggesting that immune complexes were not the cause of toxicity at 3 days post-administration in NHPs. Collectively, our data suggest that the acute toxicity that occurs after high-dose AAV in NHPs causes thrombocytopenia and liver injury on day 3 post-administration and resolves by day 7 in the majority of animals. This toxicity is capsid dependent, dose dependent, unrelated to a vector purification method, and involves transient activation of the alternate complement pathway.  

5.3092           233. AAV Mediated Apelin Transduction Facilitates Cell Cycle Entry of cTnT-Positive Cells in the Heart

Park, A., McMurphy, T., Choi, S.M., Bao, W., Alfaro, A:, Wang, Q-D., et al ASGCT Annual Meeting Abstracts, 29(4), Suppl. 1, 1-427 (2021)   Aims Terminally differentiated cardiomyocytes do not undergo mitotic division, resulting in minimal cardiac regenerative capacity upon injury. Previous studies have reported that apelin-13 can enhance cardiac repair via enhanced proliferation of myocardial stem cells and/or induction of neovascularization in the injured heart. Here our aim is to test the impact of AAV-mediated apelin gene delivery on the proliferation of cardiac cells in vivo. Methods AAV9 vectors encoding a full length pro-apelin was produced using triple plasmid transfection and purified through iodixanol gradient. 2E11 genome copies of AAV vectors were intravenously administered into C57bl/6 mice (4-8 weeks old). After 5 days of post AAV injection, mice were put under drinking water with BrdU (0.8mg/ml) for 10 days. At 15 days of post AAV injection, mouse hearts were harvested for FACS analysis, immunohistochemistry (IHC) and transcriptomic analysis. Serum samples were also harvested to measure circulating levels of apelin-13 by ELISA. Results AAV administration resulted in a 5-fold increase in apelin-13 levels in vivo when compared to the control group. IHC analysis found increased number of Ki67 (cell proliferation marker)- positive cells in the heart sections. When cardiac cells were dissociated and characterized for BrdU uptake and expression of a cardiomyocyte marker cTnT by FACS, we found BrdU-positive cells were mostly cTnTpositive and increased 5-fold in the AAV-apelin-treated group. RNAseq analysis identified activation of cell cycle and cell division pathways, with marked upregulation of CDK1, KIF14, BUB1B, PLK1, RGCC, TTK, and KNTC1 genes, upon AAV-apelin treatment. Conclusions AAV-mediated pro-apelin gene delivery achieved sustained apelin-13 overexpression in mice, which induced cell cycle entry of cTnT-positive cells in the heart. This strategy may offer a cardiac regenerative therapy for the injured heart  

5.3093           282. Engineered AAV Capsid Variants for Improved Ocular Delivery

Nachtrab, G., Wilson, A., Geddes, C., narayan, N., Batra, R., Gibbs, D. ASGCT Annual Meeting Abstract, 29(4), Suppl. 1, 1-427 (2021)   Retinal cells, specifically photoreceptors (rods and cones), are key targets for AAV-mediated gene therapy to treat a range of inherited retinal diseases (IRDs). However, the transduction of photoreceptor cells and retinal neurons by most naturally-occurring AAV serotypes is limited and is associated with a narrow therapeutic dosing range. Currently, delivery of low-titer AAV vectors is not therapeutically effective. Delivery of high-titer AAV vectors has been shown to drive toxicity in vivo due to immune responses to the capsid and off-target transduction of non-retinal cell types. Here we describe a “bottom-up” rational capsid engineering approach to develop novel AAV serotypes with improved transduction of retinal interneurons and photoreceptor cells, reduced immunogenicity and favorable manufacturing characteristics. Novel AAV2REP-CAP helper plasmids were generated with capsid cDNAs containing mutations in VP1/2 and 3, or chimeric capsid cDNAs generated by domain swapping between naturally-occurring AAV serotypes with favorable retinal and neuronal transduction profiles. Recombinant AAV vectors were generated by triple transfection in HEK293T cells and purified by iodixanol gradient purification before titration by qPCR or ddPCR to assess manufacturability. AAV vectors produced with novel capsid variants were also assessed for neutralizing antibody-responses using in vitro binding assays with samples of human serum. To assess in vivo transduction of retinal interneurons and photoreceptor cells, AAV vectors expressing fluorescent reporters were encapsidated with either novel engineered capsids or capsids previously shown to be effective in the retina and delivered to the sub-retinal space in wild-type mice. Vectors were titer matched, and cell type targeting, vector spread, transduction and immune profiling were assessed histologically at specific time points post injection. We also assessed these engineered ocular capsid variants in retinal organoids and other neuronal tissues. These studies identified rationally engineered novel AAV serotypes that were easily produced to high titer, demonstrate improved transduction of photoreceptors, and had low immunogenicity in vitro and in vivo. These novel serotypes represent optimized AAV vectors suitable for clinical translation of gene therapies for inherited retinal disease.  

5.3094           307. Engineered AAV Capsid Variants for Improved Systemic Delivery to Muscle

Nachtrab, G., Geddes, C., Gutierrez, H., Batra, R., Gibbs, D. ASGCT Annual Meeting Abstracts, 29(4), Suppl. 1, 1-427 (2021)   Muscle is an important target tissue for AAV-mediated gene therapy to treat a range of inherited muscle disorders and neuromuscular diseases (e.g., DMD, DM1). However, the transduction of muscle cells by the current lead AAV serotypes following systemic delivery requires large volumes of high-titer AAV vector and is associated with toxicity in vivo due to immune responses to the capsid and off-target transduction of non-muscle cell types, in particular transduction of the liver. Here we describe a “bottom-up” rational capsid engineering approach to develop novel AAV serotypes with improved transduction of muscle cells, reduced immunogenicity, de-targeting from liver and favorable manufacturing characteristics. Novel AAV2REP-CAP helper plasmids were generated with capsid cDNAs containing mutations in VP1/2 and 3, or chimeric capsid cDNAs generated by domain swapping between naturally occurring AAV serotypes with favorable muscle tropism and reduced hepatocyte transduction profiles. Recombinant AAV vectors were generated by triple transfection in HEK293T cells and purified by iodixanol gradient purification before titration by qPCR or ddPCR to assess manufacturability. AAV vectors produced with novel capsid variants were also assessed for neutralizing antibody-binding assays using samples of human serum. To assess in vivo transduction of muscle cells, AAV vectors expressing fluorescent reporters were encapsidated with either novel engineered capsids or capsids previously shown to be effective in targeting muscle and delivered to directly to the muscle by intramuscular injection (IM) in wild-type mice and subsequently by systemic intravenous (IV delivery). Vectors were titer matched, and cell type specific targeting, vector spread, transduction and immune profiling was assessed histologically in muscle, liver and other tissues at specific time points post injection. We also assessed the transduction profiles and expression characteristics of our engineered capsid variants in muscle, liver and other tissues, including differentiated myocyte cultures in vitro. Together, these studies identified rationally engineered novel AAV serotypes that were easily produced to high titer and demonstrate improved transduction of muscle following either IM or systemic delivery. Novel engineered AAV serotypes also showed reduced liver transduction with low immunogenicity in vitro and in vivo. These novel serotypes represent optimized AAV vectors suitable for clinical translation of gene therapies targeting inherited muscle disorders and neuromuscular diseases.  

5.3095           Muscle follistatin gene delivery increases muscle protein synthesis independent of periodical physical inactivity and fasting

Nissinen, T.A., Hentilä, J., Fachada, V., Lautaoja, J.H., Pasternack, A., Ritvos, O., Kivela, R and Hulmi, J.J. FASEB J, 35(3), e21387 (2021)   Blocking of myostatin and activins effectively counteracts muscle atrophy. However, the potential interaction with physical inactivity and fasting in the regulation of muscle protein synthesis is poorly understood. We used blockade of myostatin and activins by recombinant adeno-associated virus (rAAV)-mediated follistatin (FS288) overexpression in mouse tibialis anterior muscle. To investigate the effects on muscle protein synthesis, muscles were collected 7 days after rAAV-injection in the nighttime or in the daytime representing high and low levels of activity and feeding, respectively, or after overnight fasting, refeeding, or ad libitum feeding. Muscle protein synthesis was increased by FS288 independent of the time of the day or the feeding status. However, the activation of mTORC1 signaling by FS288 was attenuated in the daytime and by overnight fasting. FS288 also increased the amount of mTOR colocalized with lysosomes, but did not alter their localization toward the sarcolemma. This study shows that FS288 gene delivery increases muscle protein synthesis largely independent of diurnal fluctuations in physical activity and food intake or feeding status, overriding the physiological signals. This is important for eg cachectic and sarcopenic patients with reduced physical activity and appetite. The FS288-induced increase in mTORC1 signaling and protein synthesis may be in part driven by increased amount of mTOR colocalized with lysosomes, but not by their localization toward sarcolemma.  

5.3096           Altered theta and beta oscillatory synchrony in a genetic mouse model of pathological anxiety

Cruces-Solis, H., Babaev, O., Ali, H., Chatain, C.P., Mykytiuk, V., Balekoglu, N., Wenger, S. and Krueger-Burg, D. FASEB J, 35(6), e21585 (2021)   While the neural circuits mediating normal, adaptive defensive behaviors have been extensively studied, substantially less is currently known about the network mechanisms by which aberrant, pathological anxiety is encoded in the brain. Here we investigate in mice how deletion of Neuroligin-2 (Nlgn2), an inhibitory synapse-specific adhesion protein that has been associated with pathological anxiety and other psychiatric disorders, alters the communication between key brain regions involved in mediating defensive behaviors. To this end, we performed multi-site simultaneous local field potential (LFP) recordings from the basolateral amygdala (BLA), centromedial amygdala (CeM), bed nucleus of the stria terminalis (BNST), prefrontal cortex (mPFC) and ventral hippocampus (vHPC) in an open field paradigm. We found that LFP power in the vHPC was profoundly increased and was accompanied by an abnormal modulation of the synchrony of theta frequency oscillations particularly in the vHPC-mPFC-BLA circuit. Moreover, deletion of Nlgn2 increased beta and gamma frequency synchrony across the network, and this increase was associated with increased center avoidance. Local deletion of Nlgn2 in the vHPC and BLA revealed that they encode distinct aspects of this avoidance phenotype, with vHPC linked to immobility and BLA linked to a reduction in exploratory activity. Together, our data demonstrate that alterations in long-range functional connectivity link synaptic inhibition to abnormal defensive behaviors, and that both exaggerated activation of normal defensive circuits and recruitment of fundamentally distinct mechanisms contribute to this phenotype. Nlgn2 knockout mice therefore represent a highly relevant model to study the role of inhibitory synaptic transmission in the circuits underlying anxiety disorders.  

5.3097           Advances in Lentivirus Purification

Moreira, A.S., Cavaco, D.G., Faria, T.Q., Alves, P.M., Carrondo, M.J. and Peixoto, C. Biotechnol. J., 16, 2000019 (2021)   Lentiviral vectors (LVs) have been increasingly used as a tool for gene and cell therapies since they can stably integrate the genome in dividing and nondividing cells. LV production and purification processes have evolved substantially over the last decades. However, the increasing demands for higher quantities with more restrictive purity requirements are stimulating the development of novel materials and strategies to supply the market with LV in a cost-effective manner. A detailed review of each downstream process unit operation is performed, limitations, strengths, and potential outcomes being covered. Currently, the majority of large-scale LV manufacturing processes are still based on adherent cell culture, although it is known that the industry is migrating fast to suspension cultures. Regarding the purification strategy, it consists of batch chromatography and membrane technology. Nevertheless, new solutions are being created to improve the current production schemes and expand its clinical use.  

5.3098           Production, Processing, and Characterization of Synthetic AAV Gene Therapy Vectors

El Andari, J. and Grimm, D. Biotechnol. J., 16, 2000025 (2021)   Over the last two decades, gene therapy vectors based on wild-type Adeno-associated viruses (AAV) are safe and efficacious in numerous clinical trials and are translated into three approved gene therapy products. Concomitantly, a large body of preclinical work has illustrated the power and potential of engineered synthetic AAV capsids that often excel in terms of an organ or cell specificity, the efficiency of in vitro or in vivo gene transfer, and/or reactivity with anti-AAV immune responses. In turn, this has created a demand for new, scalable, easy-to-implement, and plug-and-play platform processes that are compatible with the rapidly increasing range of AAV capsid variants. Here, the focus is on recent advances in methodologies for downstream processing and characterization of natural or synthetic AAV vectors, comprising different chromatography techniques and thermostability measurements. To illustrate the breadth of this portfolio, two chimeric capsids are used as representative examples that are derived through forward- or backwards-directed molecular evolution, namely, AAV-DJ and Anc80. Collectively, this ever-expanding arsenal of technologies promises to facilitate the development of the next AAV vector generation derived from synthetic capsids and to accelerate their manufacturing, and to thus boost the field of human gene therapy.  

5.3099           Plant-made dengue virus-like particles produced by co-expression of structural and non-structural proteins induce a humoral immune response in mice

Ponndorf, D., Meshcheriakova, Y., Thuenemann, E:C., Alonso, A.D., Overman, R.Holton, N., Dowall, S., Kennedy, E., Stocks, M., Lomonossoff, G.P. and Peyret, H. Plant Biotechnol. J., 19, 745-756 (2021)   Dengue virus (DENV) is an emerging threat causing an estimated 390 million infections per year. Dengvaxia, the only licensed vaccine, may not be adequately safe in young and seronegative patients; hence, development of a safer, more effective vaccine is of great public health interest. Virus-like particles (VLPs) are a safe and very efficient vaccine strategy, and DENV VLPs have been produced in various expression systems. Here, we describe the production of DENV VLPs in Nicotiana benthamiana using transient expression. The co-expression of DENV structural proteins (SP) and a truncated version of the non-structural proteins (NSPs), lacking NS5 that contains the RNA-dependent RNA polymerase, led to the assembly of DENV VLPs in plants. These VLPs were comparable in appearance and size to VLPs produced in mammalian cells. Contrary to data from other expression systems, expression of the protein complex prM-E was not successful, and strategies used in other expression systems to improve the VLP yield did not result in increased yields in plants but, rather, increased purification difficulties. Immunogenicity assays in BALB/c mice revealed that plant-made DENV1-SP + NSP VLPs led to a higher antibody response in mice compared with DENV-E domain III displayed inside bluetongue virus core-like particles and a DENV-E domain III subunit. These results are consistent with the idea that VLPs could be the optimal approach to creating candidate vaccines against enveloped viruses.  

5.3100           Dopamine promotes the neurodegenerative potential of β-synuclein

Raina, A., Leite, K., Guerin, S., Mahajani, S.U., chakrabarti, K.S., Voll, D., Becker, S., Griesinger, C., Bähr, M. and Kügler, S.

1. Neurochem., 156(5), 674-691 (2021)

  A contribution of α-Synuclein (α-Syn) to etiology of Parkinson´s disease (PD) and Dementia with Lewy bodies (DLB) is currently undisputed, while the impact of the closely related β-Synuclein (β-Syn) on these disorders remains enigmatic. β-Syn has long been considered to be an attenuator of the neurotoxic effects of α-Syn, but in a rodent model of PD β-Syn induced robust neurodegeneration in dopaminergic neurons of the substantia nigra. Given that dopaminergic nigral neurons are selectively vulnerable to neurodegeneration in PD, we now investigated if dopamine can promote the neurodegenerative potential of β-Syn. We show that in cultured rodent and human neurons a dopaminergic neurotransmitter phenotype substantially enhanced β-Syn-induced neurodegeneration, irrespective if dopamine is synthesized within neurons or up-taken from extracellular space. Nuclear magnetic resonance interaction and thioflavin-T incorporation studies demonstrated that dopamine and its oxidized metabolites 3,4-dihydroxyphenylacetaldehyde (DOPAL) and dopaminochrome (DCH) directly interact with β-Syn, thereby enabling structural and functional modifications. Interaction of DCH with β-Syn inhibits its aggregation, which might result in increased levels of neurotoxic oligomeric β-Syn. Since protection of outer mitochondrial membrane integrity prevented the additive neurodegenerative effect of dopamine and β-Syn, such oligomers might act at a mitochondrial level similar to what is suggested for α-Syn. In conclusion, our results suggest that β-Syn can play a significant pathophysiological role in etiology of PD through its interaction with dopamine metabolites and thus should be re-considered as a disease-relevant factor, at least for those symptoms of PD that depend on degeneration of nigral dopaminergic neurons.  

5.3101           Cholesterol in the Viral Membrane is a Molecular Switch Governing HIV-1 Env Clustering

Nieto-Garai, J.A:, Arboleya, A., Otaegi, S., Chojnacki, J., Casas, J., Fabrias, G., Contreras, F-X., Kräusslich, H-G. and Lorizate, M. Advanced Science, 8, 2003468 (2021)   HIV-1 entry requires the redistribution of envelope glycoproteins (Env) into a cluster and the presence of cholesterol (chol) in the viral membrane. However, the molecular mechanisms underlying the specific role of chol in infectivity and the driving force behind Env clustering remain unknown. Here, gp41 is demonstrated to directly interact with chol in the viral membrane via residues 751–854 in the cytoplasmic tail (CT_751–854). Super-resolution stimulated emission depletion (STED) nanoscopy analysis of Env distribution further demonstrates that both truncation of gp41 CT_751–854 and depletion of chol leads to dispersion of Env clusters in the viral membrane and inhibition of virus entry. This work reveals a direct interaction of gp41 CT with chol and indicates that this interaction is an important orchestrator of Env clustering.  

5.3102           Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders

Pavlou, M., Schön, C., Occelli, L.M., Rossi, A., Meumann, N. et al EMBO Mol. Med., 13, e13392 (2021)   Gene therapy using recombinant adeno-associated virus (rAAV) vectors to treat blinding retinal dystrophies has become clinical reality. Therapeutically impactful targeting of photoreceptors still relies on subretinal vector delivery, which detaches the retina and harbours substantial risks of collateral damage, often without achieving widespread photoreceptor transduction. Herein, we report the development of novel engineered rAAV vectors that enable efficient targeting of photoreceptors via less invasive intravitreal administration. A unique in vivo selection procedure was performed, where an AAV2-based peptide-display library was intravenously administered in mice, followed by isolation of vector DNA from target cells after only 24 h. This stringent selection yielded novel vectors, termed AAV2.GL and AAV2.NN, which mediate widespread and high-level retinal transduction after intravitreal injection in mice, dogs and non-human primates. Importantly, both vectors efficiently transduce photoreceptors in human retinal explant cultures. As proof-of-concept, intravitreal Cnga3 delivery using AAV2.GL lead to cone-specific expression of Cnga3 protein and rescued photopic cone responses in the Cnga3^−/− mouse model of achromatopsia. These novel rAAV vectors expand the clinical applicability of gene therapy for blinding human retinal dystrophies.  

5.3103           Activation of the medial preoptic area (MPOA) ameliorates loss of maternal behavior in a Shank2 mouse model for autism

Grabrucker, S., Pagano, J., Schweizer, J., Urrutia-Ruiz, C., Schön, M., Thome, K., Ehret, G., Grabucker, A.M., Zhang, R., Hengerer, B., Bockmann, J., Verpelli, C., Sala, C. and Boeckers, T.M. EMBO J., 40, e104267 (2021)   Impairments in social relationships and awareness are features observed in autism spectrum disorders (ASDs). However, the underlying mechanisms remain poorly understood. Shank2 is a high-confidence ASD candidate gene and localizes primarily to postsynaptic densities (PSDs) of excitatory synapses in the central nervous system (CNS). We show here that loss of Shank2 in mice leads to a lack of social attachment and bonding behavior towards pubs independent of hormonal, cognitive, or sensitive deficits. Shank2^−/− mice display functional changes in nuclei of the social attachment circuit that were most prominent in the medial preoptic area (MPOA) of the hypothalamus. Selective enhancement of MPOA activity by DREADD technology re-established social bonding behavior in Shank2^−/− mice, providing evidence that the identified circuit might be crucial for explaining how social deficits in ASD can arise.  

5.3104           Capsid-like particles decorated with the SARS-CoV-2 receptor-binding domain elicit strong virus neutralization activity

Fougeroux, C. et al Nature Comm., 12:324 (2021)   The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we develop two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. RBD antigens are displayed on AP205 CLPs through a split-protein Tag/Catcher, ensuring unidirectional and high-density display of RBD. Both soluble recombinant RBD and RBD displayed on CLPs bind the ACE2 receptor with nanomolar affinity. Mice are vaccinated with soluble RBD or CLP-displayed RBD, formulated in Squalene-Water-Emulsion. The RBD-CLP vaccines induce higher levels of serum anti-spike antibodies than the soluble RBD vaccines. Remarkably, one injection with our lead RBD-CLP vaccine in mice elicits virus neutralization antibody titers comparable to those found in patients that had recovered from COVID-19. Following booster vaccinations, the virus neutralization titers exceed those measured after natural infection, at serum dilutions above 1:10,000. Thus, the RBD-CLP vaccine is a highly promising candidate for preventing COVID-19.    

5.3105           The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells

Sharma, R., Dever, D.P., Lee, C.M., Azizi, A., Pan, Y., Camarena, J., Köhnke, T., Bao, G., Porteus, M.H. and Majeti, R. Nature Comm., 12:472 (2021)   Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.  

5.3106           Gene therapy via canalostomy approach preserves auditory and vestibular functions in a mouse model of Jervell and Lange-Nielsen syndrome type 2

Wu, X., Zhang, L., Li, Y., Zhang, W., Wang, J., Cai, C. and Lin, X. Nature Comm., 12:697 (2021)   Mutations in voltage-gated potassium channel KCNE1 cause Jervell and Lange-Nielsen syndrome type 2 (JLNS2), resulting in congenital deafness and vestibular dysfunction. We conducted gene therapy by injecting viral vectors using the canalostomy approach in Kcne1^−/− mice to treat both the hearing and vestibular symptoms. Results showed early treatment prevented collapse of the Reissner’s membrane and vestibular wall, retained the normal size of the semicircular canals, and prevented the degeneration of inner ear cells. In a dose-dependent manner, the treatment preserved auditory (16 out of 20 mice) and vestibular (20/20) functions in mice treated with the high-dosage for at least five months. In the low-dosage group, a subgroup of mice (13/20) showed improvements only in the vestibular functions. Results supported that highly efficient transduction is one of the key factors for achieving the efficacy and maintaining the long-term therapeutic effect. Secondary outcomes of treatment included improved birth and litter survival rates. Our results demonstrated that gene therapy via the canalostomy approach, which has been considered to be one of the more feasible delivery methods for human inner ear gene therapy, preserved auditory and vestibular functions in a dose-dependent manner in a mouse model of JLNS2.  

5.3107           Photoactivatable CaMKII induces synaptic plasticity in single synapses

Shibata, A.C.E., Ueda, H.H., Eto, K., Onda, M., Sato, A., Ohba, T., nabekura, J. and Murakoshi, H. Nature Comm., 12:751 (2021)   Optogenetic approaches for studying neuronal functions have proven their utility in the neurosciences. However, optogenetic tools capable of inducing synaptic plasticity at the level of single synapses have been lacking. Here, we engineered a photoactivatable (pa)CaMKII by fusing a light-sensitive domain, LOV2, to CaMKIIα. Blue light or two-photon excitation reversibly activated paCaMKII. Activation in single spines was sufficient to induce structural long-term potentiation (sLTP) in vitro and in vivo. paCaMKII activation was also sufficient for the recruitment of AMPA receptors and functional LTP in single spines. By combining paCaMKII with protein activity imaging by 2-photon FLIM-FRET, we demonstrate that paCaMKII activation in clustered spines induces robust sLTP via a mechanism that involves the actin-regulatory small GTPase, Cdc42. This optogenetic tool for dissecting the function of CaMKII activation (i.e., the sufficiency of CaMKII rather than necessity) and for manipulating synaptic plasticity will find many applications in neuroscience and other fields.  

5.3108           Membrane type 1 matrix metalloproteinase promotes LDL receptor shedding and accelerates the development of atherosclerosis

Alabi, A., Xia, X-D., Gu, H-M., Wang, F., Deng, S-J., Yang, N., Adijiang, A., Douglas, D.N., Kneteman, N.M., Xue, Y., Chen, L., Qin, S., Wang, G. and Zhang, D-W. Nature Comm., 12:1889 (2021)   Plasma low-density lipoprotein (LDL) is primarily cleared by LDL receptor (LDLR). LDLR can be proteolytically cleaved to release its soluble ectodomain (sLDLR) into extracellular milieu. However, the proteinase responsible for LDLR cleavage is unknown. Here we report that membrane type 1-matrix metalloproteinase (MT1-MMP) co-immunoprecipitates and co-localizes with LDLR and promotes LDLR cleavage. Plasma sLDLR and cholesterol levels are reduced while hepatic LDLR is increased in mice lacking hepatic MT1-MMP. Opposite effects are observed when MT1-MMP is overexpressed. MT1-MMP overexpression significantly increases atherosclerotic lesions, while MT1-MMP knockdown significantly reduces cholesteryl ester accumulation in the aortas of apolipoprotein E (apoE) knockout mice. Furthermore, sLDLR is associated with apoB and apoE-containing lipoproteins in mouse and human plasma. Plasma levels of sLDLR are significantly increased in subjects with high plasma LDL cholesterol levels. Thus, we demonstrate that MT1-MMP promotes ectodomain shedding of hepatic LDLR, thereby regulating plasma cholesterol levels and the development of atherosclerosis.  

5.3109           Coding and non-coding roles of MOCCI (C15ORF48) coordinate to regulate host inflammation and immunity

Lee, C.Q., Kerouanton, B., Chothhani, S., Zhang, S., Chen, Y., Mantri, C.K. et al Nature Comm., 12:2130 (2021)   Mito-SEPs are small open reading frame-encoded peptides that localize to the mitochondria to regulate metabolism. Motivated by an intriguing negative association between mito-SEPs and inflammation, here we screen for mito-SEPs that modify inflammatory outcomes and report a mito-SEP named “Modulator of cytochrome C oxidase during Inflammation” (MOCCI) that is upregulated during inflammation and infection to promote host-protective resolution. MOCCI, a paralog of the NDUFA4 subunit of cytochrome C oxidase (Complex IV), replaces NDUFA4 in Complex IV during inflammation to lower mitochondrial membrane potential and reduce ROS production, leading to cyto-protection and dampened immune response. The MOCCI transcript also generates miR-147b, which targets the NDUFA4 mRNA with similar immune dampening effects as MOCCI, but simultaneously enhances RIG-I/MDA-5-mediated viral immunity. Our work uncovers a dual-component pleiotropic regulation of host inflammation and immunity by MOCCI (C15ORF48) for safeguarding the host during infection and inflammation.  

5.3110           Neurexins regulate presynaptic GABAB-receptors at central synapses

Luo, F., Sclip, A., Merrill, S. and Südhof, T:C. Nature Comm., 12:2380 (2021)   Diverse signaling complexes are precisely assembled at the presynaptic active zone for dynamic modulation of synaptic transmission and synaptic plasticity. Presynaptic GABA_B-receptors nucleate critical signaling complexes regulating neurotransmitter release at most synapses. However, the molecular mechanisms underlying assembly of GABA_B-receptor signaling complexes remain unclear. Here we show that neurexins are required for the localization and function of presynaptic GABA_B-receptor signaling complexes. At four model synapses, excitatory calyx of Held synapses in the brainstem, excitatory and inhibitory synapses on hippocampal CA1-region pyramidal neurons, and inhibitory basket cell synapses in the cerebellum, deletion of neurexins rendered neurotransmitter release significantly less sensitive to GABA_B-receptor activation. Moreover, deletion of neurexins caused a loss of GABA_B-receptors from the presynaptic active zone of the calyx synapse. These findings extend the role of neurexins at the presynaptic active zone to enabling GABA_B-receptor signaling, supporting the notion that neurexins function as central organizers of active zone signaling complexes.  

5.3111           Tanc2-mediated mTOR inhibition balances mTORC1/2 signaling in the developing mouse brain and human neurons

Kim, S-G., Lee, S., Kim, Y., Park, J., Woo, D., Kim, D. et al Nature Comm., 12:2695 (2021)   mTOR signaling, involving mTORC1 and mTORC2 complexes, critically regulates neural development and is implicated in various brain disorders. However, we do not fully understand all of the upstream signaling components that can regulate mTOR signaling, especially in neurons. Here, we show a direct, regulated inhibition of mTOR by Tanc2, an adaptor/scaffolding protein with strong neurodevelopmental and psychiatric implications. While Tanc2-null mice show embryonic lethality, Tanc2-haploinsufficient mice survive but display mTORC1/2 hyperactivity accompanying synaptic and behavioral deficits reversed by mTOR-inhibiting rapamycin. Tanc2 interacts with and inhibits mTOR, which is suppressed by mTOR-activating serum or ketamine, a fast-acting antidepressant. Tanc2 and Deptor, also known to inhibit mTORC1/2 minimally affecting neurodevelopment, distinctly inhibit mTOR in early- and late-stage neurons. Lastly, Tanc2 inhibits mTORC1/2 in human neural progenitor cells and neurons. In summary, our findings show that Tanc2 is a mTORC1/2 inhibitor affecting neurodevelopment.  

5.3112           Tissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice

Hunt, C., Hartford, S.A., White, D., Pefanis, E., Hanna, T. et al Nature Comm., 12:2770 (2021)   CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.  

5.3113           Synaptic plasticity-dependent competition rule influences memory formation

Jeong, Y., Cho, H-Y., Kim, M., Oh, J-P., Kang, M.S., Yoo, M., Lee, H-S. and Han, J-H. Nature Comm., 12:3915 (2021)   Memory is supported by a specific collection of neurons distributed in broad brain areas, an engram. Despite recent advances in identifying an engram, how the engram is created during memory formation remains elusive. To explore the relation between a specific pattern of input activity and memory allocation, here we target a sparse subset of neurons in the auditory cortex and thalamus. The synaptic inputs from these neurons to the lateral amygdala (LA) are not potentiated by fear conditioning. Using an optogenetic priming stimulus, we manipulate these synapses to be potentiated by the learning. In this condition, fear memory is preferentially encoded in the manipulated cell ensembles. This change, however, is abolished with optical long-term depression (LTD) delivered shortly after training. Conversely, delivering optical long-term potentiation (LTP) alone shortly after fear conditioning is sufficient to induce the preferential memory encoding. These results suggest a synaptic plasticity-dependent competition rule underlying memory formation.  

5.3114           Oncolytic H-1 parvovirus binds to sialic acid on laminins for cell attachment and entry

Kulkarni, A., Ferreira, T., Bretscher, C., Grewenig, A., El-Andaloussi, N. et al Nature Comm., 12:3834 (2021)   H-1 parvovirus (H-1PV) is a promising anticancer therapy. However, in-depth understanding of its life cycle, including the host cell factors needed for infectivity and oncolysis, is lacking. This understanding may guide the rational design of combination strategies, aid development of more effective viruses, and help identify biomarkers of susceptibility to H-1PV treatment. To identify the host cell factors involved, we carry out siRNA library screening using a druggable genome library. We identify one crucial modulator of H-1PV infection: laminin γ1 (LAMC1). Using loss- and gain-of-function studies, competition experiments, and ELISA, we validate LAMC1 and laminin family members as being essential to H-1PV cell attachment and entry. H-1PV binding to laminins is dependent on their sialic acid moieties and is inhibited by heparin. We show that laminins are differentially expressed in various tumour entities, including glioblastoma. We confirm the expression pattern of laminin γ1 in glioblastoma biopsies by immunohistochemistry. We also provide evidence of a direct correlation between LAMC1 expression levels and H-1PV oncolytic activity in 59 cancer cell lines and in 3D organotypic spheroid cultures with different sensitivities to H-1PV infection. These results support the idea that tumours with elevated levels of γ1 containing laminins are more susceptible to H-1PV-based therapies.  

5.3115           Single-component near-infrared optogenetic systems for gene transcription regulation

Kaberniuk, A.A., Baloban, M., Monakhov, M.V., Shcherbakova, D.M. and Verkhusha, V.V. Nature Comm., 12:3859 (2021)   Near-infrared (NIR) optogenetic systems for transcription regulation are in high demand because NIR light exhibits low phototoxicity, low scattering, and allows combining with probes of visible range. However, available NIR optogenetic systems consist of several protein components of large size and multidomain structure. Here, we engineer single-component NIR systems consisting of evolved photosensory core module of Idiomarina sp. bacterial phytochrome, named iLight, which are smaller and packable in adeno-associated virus. We characterize iLight in vitro and in gene transcription repression in bacterial and gene transcription activation in mammalian cells. Bacterial iLight system shows 115-fold repression of protein production. Comparing to multi-component NIR systems, mammalian iLight system exhibits higher activation of 65-fold in cells and faster 6-fold activation in deep tissues of mice. Neurons transduced with viral-encoded iLight system exhibit 50-fold induction of fluorescent reporter. NIR light-induced neuronal expression of green-light-activatable CheRiff channelrhodopsin causes 20-fold increase of photocurrent and demonstrates efficient spectral multiplexing.  

5.3116           Epigenetic inactivation of the autophagy–lysosomal system in appendix in Parkinson’s disease

Gordevicius, J., Li, P., Marshall, L.L., Killinger, B.A., lang, S., Ensink, E. et al Nature Comm., 12:5234 (2021)   The gastrointestinal tract may be a site of origin for α-synuclein pathology in idiopathic Parkinson’s disease (PD). Disruption of the autophagy-lysosome pathway (ALP) may contribute to α-synuclein aggregation. Here we examined epigenetic alterations in the ALP in the appendix by deep sequencing DNA methylation at 521 ALP genes. We identified aberrant methylation at 928 cytosines affecting 326 ALP genes in the appendix of individuals with PD and widespread hypermethylation that is also seen in the brain of individuals with PD. In mice, we find that DNA methylation changes at ALP genes induced by chronic gut inflammation are greatly exacerbated by α-synuclein pathology. DNA methylation changes at ALP genes induced by synucleinopathy are associated with the ALP abnormalities observed in the appendix of individuals with PD specifically involving lysosomal genes. Our work identifies epigenetic dysregulation of the ALP which may suggest a potential mechanism for accumulation of α-synuclein pathology in idiopathic PD.  

5.3117           A human liver cell-based system modeling a clinical prognostic liver signature for therapeutic discovery

Crouchet, E., Bandiera, S., Fujiwara, N., Li, S., El Sagnire, H. et al Nature Comm., 12:5525 (2021)   Chronic liver disease and hepatocellular carcinoma (HCC) are life-threatening diseases with limited treatment options. The lack of clinically relevant/tractable experimental models hampers therapeutic discovery. Here, we develop a simple and robust human liver cell-based system modeling a clinical prognostic liver signature (PLS) predicting long-term liver disease progression toward HCC. Using the PLS as a readout, followed by validation in nonalcoholic steatohepatitis/fibrosis/HCC animal models and patient-derived liver spheroids, we identify nizatidine, a histamine receptor H2 (HRH2) blocker, for treatment of advanced liver disease and HCC chemoprevention. Moreover, perturbation studies combined with single cell RNA-Seq analyses of patient liver tissues uncover hepatocytes and HRH2⁺, CLEC5A^high, MARCO^low liver macrophages as potential nizatidine targets. The PLS model combined with single cell RNA-Seq of patient tissues enables discovery of urgently needed targets and therapeutics for treatment of advanced liver disease and cancer prevention.  

5.3118           Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

Kim, B., Kim, J., Chun, M., Park, I., Kwak, D., Choi, M., Kim, K. and Choe, H.K. Scientific Reports, 11:2575 (2021)   The mammalian molecular clock is based on a transcription-translation feedback loop (TTFL) comprising the Period1, 2 (Per1, 2), Cryptochrome1, 2 (Cry1, 2), and Brain and Muscle ARNT-Like 1 (Bmal1) genes. The robustness of the TTFL is attributed to genetic redundancy among some essential clock genes, deterring genetic studies on molecular clocks using genome editing targeting single genes. To manipulate multiple clock genes in a streamlined and efficient manner, we developed a CRISPR-Cas9-based single adeno-associated viral (AAV) system targeting the circadian clock (CSAC) for essential clock genes including Pers, Crys, or Bmal1. First, we tested several single guide RNAs (sgRNAs) targeting individual clock genes in silico and validated their efficiency in Neuro2a cells. To target multiple genes, multiplex sgRNA plasmids were constructed using Golden Gate assembly and packaged into AAVs. CSAC efficiency was evident through protein downregulation in vitro and ablated molecular oscillation ex vivo. We also measured the efficiency of CSAC in vivo by assessing circadian rhythms after injecting CSAC into the suprachiasmatic nuclei of Cas9-expressing knock-in mice. Circadian locomotor activity and body temperature rhythms were severely disrupted in these mice, indicating that our CSAC is a simple yet powerful tool for investigating the molecular clock in vivo.  

5.3119           Reduced spontaneous itch in mouse models of cholestasis

Langedijk, J., Boiler, R., Tolenaars, D., ten Bloemendaal, L., Duijst, S., de Waart, D., Beuers, U., Bosma, P. and Elferink, R.O. Scientific Reports, 11:6127 (2021)   Pruritus is one of the most distressing symptoms in cholestatic patients. Plasma autotaxin (ATX) activity correlates with the severity of pruritus in cholestatic patients, but the pathophysiology is unclear. To study pruritus in mice, we measured scratch activity in cholestatic Atp8b1 mutant mice, a model for Progressive Familial Intrahepatic Cholestasis type 1, and wild type mice (WT) with alpha-naphthylisothiocyanate (ANIT)-induced cholestasis. To induce cholestasis, Atp8b1 mutant mice received a diet containing 0.1% cholic acid (CA) and WT mice were treated with ANIT. In these mice ATX was also overexpressed by transduction with AAV-ATX. Scratch activity was measured using an unbiased, electronic assay. Marked cholestasis was accomplished in both Atp8b1 mutant mice on a CA-supplemented diet and in ANIT-treatment in WT mice, but scratch activity was decreased rather than increased while plasma ATX activity was increased. Plasma ATX activity was further increased up to fivefold with AAV-ATX, but this did not induce scratch activity. In contrast to several reports two cholestatic mouse models did not display increased scratch activity as a measure of itch perception. Increasing plasma ATX activity by overexpression also did not lead to increased scratch activity in mice. This questions whether mice are suitable to study cholestatic itch.  

5.3120           Mixture interactions at mammalian olfactory receptors are dependent on the cellular environment

Corey, E:A., Zolotukhin, S., Ache, B.E. and Ukhanov, K. Scientific Reports, 11, 9278 (2021)   Functional characterization of mammalian olfactory receptors (ORs) remains a major challenge to ultimately understanding the olfactory code. Here, we compare the responses of the mouse Olfr73 ectopically expressed in olfactory sensory neurons using AAV gene delivery in vivo and expressed in vitro in cell culture. The response dynamics and concentration-dependence of agonists for the ectopically expressed Olfr73 were similar to those reported for the endogenous Olfr73, however the antagonism previously reported between its cognate agonist and several antagonists was not replicated in vivo. Expressing the OR in vitro reproduced the antagonism reported for short odor pulses, but not for prolonged odor exposure. Our findings suggest that both the cellular environment and the stimulus dynamics shape the functionality of Olfr73 and argue that characterizing ORs in ‘native’ conditions, rather than in vitro, provides a more relevant understanding of ligand-OR interactions.  

5.3121           AAV-mediated YAP expression in cardiac fibroblasts promotes inflammation and increases fibrosis

Francisco, J., Zhang, Y., Nakada, Y., Jeong, J.I., Huang, C-Y., Ivessa, A., Oka, S., Babu, G.J. and Del Re, D.P Scientific Reports, 11:10553 (2021)   Fibrosis is a hallmark of heart disease independent of etiology and is thought to contribute to impaired cardiac dysfunction and development of heart failure. However, the underlying mechanisms that regulate the differentiation of fibroblasts to myofibroblasts and fibrotic responses remain incompletely defined. As a result, effective treatments to mitigate excessive fibrosis are lacking. We recently demonstrated that the Hippo pathway effector Yes-associated protein (YAP) is an important mediator of myofibroblast differentiation and fibrosis in the infarcted heart. Yet, whether YAP activation in cardiac fibroblasts is sufficient to drive fibrosis, and how fibroblast YAP affects myocardial inflammation, a significant component of adverse cardiac remodeling, are largely unknown. In this study, we leveraged adeno-associated virus (AAV) to target cardiac fibroblasts and demonstrate that chronic YAP expression upregulated indices of fibrosis and inflammation in the absence of additional stress. YAP occupied the Ccl2 gene and promoted Ccl2 expression, which was associated with increased macrophage infiltration, pro-inflammatory cytokine expression, collagen deposition, and cardiac dysfunction in mice with cardiac fibroblast-targeted YAP overexpression. These results are consistent with other recent reports and extend our understanding of YAP function in modulating fibrotic and inflammatory responses in the heart.  

5.3122           Efficient gene transfer into zebra finch germline-competent stem cells using an adenoviral vector system

Jung, K.M., Kim, Y.M, Kim, J.L. and Han, J.Y. Scientific Reports, 11:14746 (2021)   Zebra finch is a representative animal model for studying the molecular basis of human disorders of vocal development and communication. Accordingly, various functional studies of zebra finch have knocked down or introduced foreign genes in vivo; however, their germline transmission efficiency is remarkably low. The primordial germ cell (PGC)-mediated method is preferred for avian transgenic studies; however, use of this method is restricted in zebra finch due to the lack of an efficient gene transfer method for the germline. To target primary germ cells that are difficult to transfect and manipulate, an adenovirus-mediated gene transfer system with high efficiency in a wide range of cell types may be useful. Here, we isolated and characterized two types of primary germline-competent stem cells, PGCs and spermatogonial stem cells (SSCs), from embryonic and adult reproductive tissues of zebra finch and demonstrated that genes were most efficiently transferred into these cells using an adenovirus-mediated system. This system was successfully used to generate gene-edited PGCs in vitro. These results are expected to improve transgenic zebra finch production.  

5.3123           Lentiviral and AAV-mediated expression of palivizumab offer protection against Respiratory Syncytial Virus infection

Antepowicz, A., Habib, O., Kirsebom, F., Johansson, C., Gill, D.R. and Hyde, S.C. Scientific Reports, 11:15694 (2021)   Respiratory syncytial virus (RSV) infection is a common cause of hospitalisation in infants and the elderly. Palivizumab prophylaxis is the only approved treatment modality but is costly and only offered to select vulnerable populations. Here, we investigated gene delivery approaches via recombinant adeno-associated virus (rAAV2/8) and simian immunodeficiency virus (rSIV.F/HN) vectors to achieve sustained in vivo production of palivizumab in a murine model. Delivery of palivizumab-expressing vectors 28 days prior to RSV challenge resulted in complete protection from RSV-induced weight loss. This approach offers prophylaxis against RSV infection, allowing for wider use and reduction in treatment costs in vulnerable populations.  

5.3124           A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation

Stepanenko, A.A., Sosnovtseva, A.O., Valikhov, M.P. and Chekhonin, V.P. Scienfitic Reports, 11:16088 (2021)   Two-cycle cesium chloride (2 × CsCl) gradient ultracentrifugation is a conventional approach for purifying recombinant adenoviruses (rAds) for research purposes (gene therapy, vaccines, and oncolytic vectors). However, rAds containing the RGD-4C peptide in the HI loop of the fiber knob domain tend to aggregate during 2 × CsCl gradient ultracentrifugation resulting in a low infectious titer yield or even purification failure. An iodixanol-based purification method preventing aggregation of the RGD4C-modified rAds has been proposed. However, the reason explaining aggregation of the RGD4C-modified rAds during 2 × CsCl but not iodixanol gradient ultracentrifugation has not been revealed. In the present study, we showed that rAds with the RGD-4C peptide in the HI loop but not at the C-terminus of the fiber knob domain were prone to aggregate during 2 × CsCl but not iodixanol gradient ultracentrifugation. The cysteine residues with free thiol groups after the RGD motif within the inserted RGD-4C peptide were responsible for formation of the interparticle disulfide bonds under atmospheric oxygen and aggregation of Ad5-delta-24-RGD4C-based rAds during 2 × CsCl gradient ultracentrifugation, which could be prevented using iodixanol gradient ultracentrifugation, most likely due to antioxidant properties of iodixanol. A cysteine-to-glycine substitution of the cysteine residues with free thiol groups (RGD-2C2G) prevented aggregation during 2 × CsCl gradient purification but in coxsackie and adenovirus receptor (CAR)-low/negative cancer cell lines of human and rodent origin, this reduced cytolytic efficacy to the levels observed for a fiber non-modified control vector. However, both Ad5-delta-24-RGD4C and Ad5-delta-24-RGD2C2G were equally effective in the murine immunocompetent CT-2A glioma model due to a primary role of antitumor immune responses in the therapeutic efficacy of oncolytic virotherapy.  

5.3125           Exploring rare cellular activity in more than one million cells by a transscale scope

Ichimura, T., Kakizuka, T., Horikawa, K., Seiriki, K., Kasai, A., Hashimoto, H., Fujita, K., Watanabe, T.M. and Nagai, T. Scientific Reports, 11:16539 (2021)   In many phenomena of biological systems, not a majority, but a minority of cells act on the entire multicellular system causing drastic changes in the system properties. To understand the mechanisms underlying such phenomena, it is essential to observe the spatiotemporal dynamics of a huge population of cells at sub-cellular resolution, which is difficult with conventional tools such as microscopy and flow cytometry. Here, we describe an imaging system named AMATERAS that enables optical imaging with an over-one-centimeter field-of-view and a-few-micrometer spatial resolution. This trans-scale-scope has a simple configuration, composed of a low-power lens for machine vision and a hundred-megapixel image sensor. We demonstrated its high cell-throughput, capable of simultaneously observing more than one million cells. We applied it to dynamic imaging of calcium ions in HeLa cells and cyclic-adenosine-monophosphate in Dictyostelium discoideum, and successfully detected less than 0.01% of rare cells and observed multicellular events induced by these cells.  

5.3126           DJ-1 attenuates the glycation of mitochondrial complex I and complex III in the post-ischemic heart

Paantner, Y., Polavarapu, R., Chin, L.S., Li, L., Shimizu, Y. and Calvert, J.W. Scientific Reports, 11:19408 (2021)   DJ-1 is a ubiquitously expressed protein that protects cells from stress through its conversion into an active protease. Recent work found that the active form of DJ-1 was induced in the ischemic heart as an endogenous mechanism to attenuate glycative stress—the non-enzymatic glycosylation of proteins. However, specific proteins protected from glycative stress by DJ-1 are not known. Given that mitochondrial electron transport proteins have a propensity for being targets of glycative stress, we investigated if DJ-1 regulates the glycation of Complex I and Complex III after myocardial ischemia–reperfusion (I/R) injury. Initial studies found that DJ-1 localized to the mitochondria and increased its interaction with Complex I and Complex III 3 days after the onset of myocardial I/R injury. Next, we investigated the role DJ-1 plays in modulating glycative stress in the mitochondria. Analysis revealed that compared to wild-type control mice, mitochondria from DJ-1 deficient (DJ-1 KO) hearts showed increased levels of glycative stress following I/R. Additionally, Complex I and Complex III glycation were found to be at higher levels in DJ-1 KO hearts. This corresponded with reduced complex activities, as well as reduced mitochondrial oxygen consumption ant ATP synthesis in the presence of pyruvate and malate. To further determine if DJ-1 influenced the glycation of the complexes, an adenoviral approach was used to over-express the active form of DJ-1(AAV9-DJ1ΔC). Under I/R conditions, the glycation of Complex I and Complex III were attenuated in hearts treated with AAV9-DJ1ΔC. This was accompanied by improvements in complex activities, oxygen consumption, and ATP production. Together, this data suggests that cardiac DJ-1 maintains Complex I and Complex III efficiency and mitochondrial function during the recovery from I/R injury. In elucidating a specific mechanism for DJ-1’s role in the post-ischemic heart, these data break new ground for potential therapeutic strategies using DJ-1 as a target.  

5.3127           Plant-expressed virus-like particles reveal the intricate maturation process of a eukaryotic virus

Castells-Graells, R., Ribeiro, J.R.S., Domitrovic, T., Hesketh, E.L., Scarff, C.A, Johnson, J.E., Ranson, N.A., Lawson, D.M. and Lomonossoff, G.P. Communications Biol., 4:619 (2021)   Many virus capsids undergo exquisitely choreographed maturation processes in their host cells to produce infectious virions, and these remain poorly understood. As a tool for studying virus maturation, we transiently expressed the capsid protein of the insect virus Nudaurelia capensis omega virus (NωV) in Nicotiana benthamiana and were able to purify both immature procapsids and mature capsids from infiltrated leaves by varying the expression time. Cryo-EM analysis of the plant-produced procapsids and mature capsids to 6.6 Å and 2.7 Å resolution, respectively, reveals that in addition to large scale rigid body motions, internal regions of the subunits are extensively remodelled during maturation, creating the active site required for autocatalytic cleavage and infectivity. The mature particles are biologically active in terms of their ability to lyse membranes and have a structure that is essentially identical to authentic virus. The ability to faithfully recapitulate and visualize a complex maturation process in plants, including the autocatalytic cleavage of the capsid protein, has revealed a ~30 Å translation-rotation of the subunits during maturation as well as conformational rearrangements in the N and C-terminal helical regions of each subunit.  

5.3128           The structure of a plant-specific partitivirus capsid reveals a unique coat protein domain architecture with an intrinsically disordered protrusion

Byrne, M., Kashyap, A., Esquirol. L., Ranson, N and Sainsbuury, F. Comminucations Biol., 4:1155 (2021)   Persistent plant viruses may be the most common viruses in wild plants. A growing body of evidence for mutualism between such viruses and their hosts, suggests that they play an important role in ecology and agriculture. Here we present the capsid structure of a plant-specific partitivirus, Pepper cryptic virus 1, at 2.9 Å resolution by Cryo-EM. Structural features, including the T = 1 arrangement of 60 coat protein dimers, are shared with fungal partitiviruses and the picobirnavirus lineage of dsRNA viruses. However, the topology of the capsid is markedly different with protrusions emanating from, and partly comprising, the binding interface of coat protein dimers. We show that a disordered region at the apex of the protrusion is not required for capsid assembly and represents a hypervariable site unique to, and characteristic of, the plant-specific partitiviruses. These results suggest a structural basis for the acquisition of additional functions by partitivirus coat proteins that enables mutualistic relationships with diverse plant hosts.  

5.3129           Il-10 signaling reduces survival in mouse models of synucleinopathy

Cockey, S.G., McFarland, K.N., Koller, E.J., Brooks, M.M.T., De La Cruz, E.G., Cruz, P.E., Ceballos-Diaz, C., Rosario, A.M., Levites, Y.R., Borchelt, D.R., Golde, T.E., Glasson, B.I. and Chakrabarty, P. Npj Parkinson’s Disease, 17:30 (2021)   Parkinson’s disease (PD) and related synucleinopathies are characterized by chronic neuroinflammation leading to the premise that anti-inflammatory therapies could ameliorate synucleinopathy and associated sequelae. To test this idea, we used recombinant adeno-associated viruses (AAV) to express the anti-inflammatory cytokine, Interleukin (Il)-10, in Line M83 transgenic mice that expresses the PD-associated A53T mutant human α-synuclein (αSyn). Contrary to our expectations, we observed that intraspinal Il-10 expression initiated at birth upregulated microgliosis and led to early death in homozygous M83+/+ mice. We further observed that Il-10 preconditioning led to reduced lifespan in the hemizygous M83+/− mice injected with preformed αSyn aggregates in hindlimb muscles. To determine the mechanistic basis for these adverse effects, we took advantage of the I87A variant Il-10 (vIl-10) that has predominantly immunosuppressive properties. Sustained intraspinal expression of vIl-10 in preformed αSyn-aggregate seeded M83+/− mice resulted in earlier death, accelerated αSyn pathology, pronounced microgliosis, and increased apoptosis compared to control mice. AAV-vIl-10 expression robustly induced p62 and neuronal LC3B accumulation in these mice, indicating that Il-10 signaling mediated preconditioning of the neuraxis can potentially exacerbate αSyn accumulation through autophagy dysfunction in the neurons. Together, our data demonstrate unexpected adverse effects of both Il-10 and its immunosuppressive variant, vIl-10, in a mouse model of PD, highlighting the pleiotropic functions of immune mediators and their complex role in non-cell autonomous signaling in neurodegenerative proteinopathies.  

5.3130           α-Synuclein-induced dysregulation of neuronal activity contributes to murine dopamine neuron vulnerability

Dagra, A., Miller, D.R., Lin, M., Gopinath, A., Shaerzadeh, F., Harris, S., Sorrentino, Z.A. et al Npj Parkinson’s Disease, 7:76 (2021)   Pathophysiological damages and loss of function of dopamine neurons precede their demise and contribute to the early phases of Parkinson’s disease. The presence of aberrant intracellular pathological inclusions of the protein α-synuclein within ventral midbrain dopaminergic neurons is one of the cardinal features of Parkinson’s disease. We employed molecular biology, electrophysiology, and live-cell imaging to investigate how excessive α-synuclein expression alters multiple characteristics of dopaminergic neuronal dynamics and dopamine transmission in cultured dopamine neurons conditionally expressing GCaMP6f. We found that overexpression of α-synuclein in mouse (male and female) dopaminergic neurons altered neuronal firing properties, calcium dynamics, dopamine release, protein expression, and morphology. Moreover, prolonged exposure to the D2 receptor agonist, quinpirole, rescues many of the alterations induced by α-synuclein overexpression. These studies demonstrate that α-synuclein dysregulation of neuronal activity contributes to the vulnerability of dopaminergic neurons and that modulation of D2 receptor activity can ameliorate the pathophysiology. These findings provide mechanistic insights into the insidious changes in dopaminergic neuronal activity and neuronal loss that characterize Parkinson’s disease progression with significant therapeutic implications.  

5.3131           Neurog2 directly converts astrocytes into functional neurons in midbrain and spinal cord

Liu, F., Zhang, Y., Chen, F., Yuan, J., Li, S., Han, S., Lu, D., Geng, J., Rao, Z., Sun, L., Xu, J., Shi, Y., Wang, X. and Liu, Y. Cell Death & Disease, 12:225 (2021)   Conversion of astrocytes into neurons in vivo offers an alternative therapeutic approach for neuronal loss after injury or disease. However, not only the efficiency of the conversion of astrocytes into functional neurons by single Neurog2, but also the conundrum that whether Neurog2-induced neuronal cells (Neurog2-iNs) are further functionally integrated into existing matured neural circuits remains unknown. Here, we adopted the AAV(2/8) delivery system to overexpress single factor Neurog2 into astrocytes and found that the majority of astrocytes were successfully converted into neuronal cells in multiple brain regions, including the midbrain and spinal cord. In the midbrain, Neurog2-induced neuronal cells (Neurog2-iNs) exhibit neuronal morphology, mature electrophysiological properties, glutamatergic identity (about 60%), and synapse-like configuration local circuits. In the spinal cord, astrocytes from both the intact and lesioned sources could be converted into functional neurons with ectopic expression of Neurog2 alone. Notably, further evidence from our study also proves that Neurog2-iNs in the intact spinal cord are capable of responding to diverse afferent inputs from dorsal root ganglion (DRG). Together, this study does not merely demonstrate the feasibility of Neurog2 for efficient in vivo reprogramming, it gives an indication for the Neurog2-iNs as a functional and potential factor in cell-replacement therapy.  

5.3132           Long noncoding RNA AVAN promotes antiviral innate immunity by interacting with TRIM25 and enhancing the transcription of FOXO3a

Li, C., Liu, L., Liu, Q., Wang, K., Cheng, S., Zhao, L., Xia, M., Wang, C., Duan, Y. et al Cell Death & Differentiation, 28, 2900-2915 (2021)   Accumulating evidence has shown that long noncoding RNAs (lncRNAs) are involved in several biological processes, including immune responses. However, the role of lncRNAs in antiviral innate immune responses remains largely elusive. Here, we identify an uncharacterized human lncRNA AVAN from influenza A virus (IAV) infected patients, that is significantly upregulated following RNA virus infection. During IAV infection, AVAN play an indispensable role in antiviral immune responses. In vivo, we enforced the expression of AVAN in transgenic mice or adeno-associated virus encoding AVAN delivery system and found that AVAN significantly alleviated IAV virulence and virus replication. Mechanistically, nuclear AVAN positively regulates the transcription of forkhead box O3A (FOXO3a) by associating with its promoter and inducing chromatin remodeling to promote neutrophil chemotaxis. Meanwhile, cytoplasmic AVAN binds directly to the E3 ligase TRIM25 and enhances TRIM25-mediated K63-linked ubiquitination of RIG-I, thereby promoting TRIM25- and RIG-I-mediated antiviral innate immune responses, including the induction of type I interferon and ISGs. Moreover, AVAN binds to the B Box/CCD domain of TRIM25 and 1–200nt of AVAN were the functional moieties. Collectively, our findings highlight the potential clinical implications of human lncRNA AVAN as a key positive regulator of the antiviral innate immune response and a promising target for developing broad antiviral therapeutics.  

5.3133           Functional expression of complement factor I following AAV-mediated gene delivery in the retina of mice and human cells

Dreismann, A.K., McClements, M.E., Barnard, A.R., Orhan, E., Hughes, J.P., Lachmann, P.J. and MacLaren, R.E. Gene Therapy, 28, 265-276 (2021)   Dry age-related macular degeneration (AMD) is characterised by loss of central vision and currently has no approved medical treatment. Dysregulation of the complement system is thought to play an important role in disease pathology and supplementation of Complement Factor I (CFI), a key regulator of the complement system, has the potential to provide a treatment option for AMD. In this study, we demonstrate the generation of AAV constructs carrying the human CFI sequence and expression of CFI in cell lines and in the retina of C57BL/6 J mice. Four codon optimised constructs were compared to the most common human CFI sequence. All constructs expressed CFI protein; however, most codon optimised sequences resulted in significantly reduced CFI secretion compared to the non-optimised CFI sequence. In vivo expression analysis showed that CFI was predominantly expressed in the RPE and photoreceptors. Secreted protein in vitreous humour was demonstrated to be functionally active. The findings presented here have led to the formulation of an AAV-vectored gene therapy product currently being tested in a first-in-human clinical trial in subjects with geographic atrophy secondary to dry AMD (NCT03846193).  

5.3134           Immunogenicity and inflammatory properties of respiratory syncytial virus attachment G protein in cotton rats

Martinez, M.E., Gonzalez, C.C., Huey, D., Peeples, M.E., McCarty, D. and Niewiesk, S. PloS One, 16(2), e0246770 (2021)   Human respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants and young children worldwide. The attachment (G) protein of RSV is synthesized by infected cells in both a membrane bound (mG) and secreted form (sG) and uses a CX3C motif for binding to its cellular receptor. Cell culture and mouse studies suggest that the G protein mimics the cytokine CX3CL1 by binding to CX3CR1 on immune cells, which is thought to cause increased pulmonary inflammation in vivo. However, because these studies have used RSV lacking its G protein gene or blockade of the G protein with a G protein specific monoclonal antibody, the observed reduction in inflammation may be due to reduced virus replication and spread, and not to a direct role for G protein as a viral chemokine. In order to more directly determine the influence of the soluble and the membrane-bound forms of G protein on the immune system independent of its attachment function for the virion, we expressed the G protein in cotton rat lungs using adeno-associated virus (AAV), a vector system which does not itself induce inflammation. We found no increase in pulmonary inflammation as determined by histology and bronchoalveolar lavage after inoculation of AAVs expressing the membrane bound G protein, the secreted G protein or the complete G protein gene which expresses both forms. The long-term low-level expression of AAV-G did, however, result in the induction of non-neutralizing antibodies, CD8 T cells and partial protection from challenge with RSV. Complete protection was accomplished through co-immunization with AAV-G and an AAV expressing cotton rat interferon α.  

5.3135           Short-term treatment of golden retriever muscular dystrophy (GRMD) dogs with rAAVrh74.MHCK7.GALGT2 induces muscle glycosylation and utrophin expression but has no significant effect on muscle strength

Martin, P.T., Zygmunt, D.A., Ashbrook, A., Hamilton, S., Packer, D., Birch, S.M., Bettis, A.K., Balog-Alvarez, C.J., Guo, L-J., Hghiem, P.P.ø and Kornegay, J.N. PloS One, 16(3), e0248721 (2021)   We have examined the effects of intravenous (IV) delivery of rAAVrh74.MHCK7.GALGT2 in the golden retriever muscular dystrophy (GRMD) model of Duchenne Muscular Dystrophy (DMD). After baseline testing, GRMD dogs were treated at 3 months of age and reassessed at 6 months. This 3–6 month age range is a period of rapid disease progression, thus offering a relatively short window to establish treatment efficacy. Measures analyzed included muscle AAV transduction, GALGT2 transgene expression, GALGT2-induced glycosylation, muscle pathology, and muscle function. A total of five dogs were treated, 4 at 2x10¹⁴vg/kg and one at 6x10¹⁴vgkg. The 2x10¹⁴vg/kg dose led to transduction of regions of the heart with 1–3 vector genomes (vg) per nucleus, while most skeletal muscles were transduced with 0.25–0.5vg/nucleus. GALGT2-induced glycosylation paralleled levels of myofiber vg transduction, with about 90% of cardiomyocytes having increased glycosylation versus 20–35% of all myofibers across the skeletal muscles tested. Conclusions from phenotypic testing were limited by the small number of dogs. Treated dogs had less pronounced fibrosis and overall lesion severity when compared to control groups, but surprisingly no significant changes in limb muscle function measures. GALGT2-treated skeletal muscle and heart had elevated levels of utrophin protein expression and GALGT2-induced expression of glycosylated α dystroglycan, providing further evidence of a treatment effect. Serum chemistry, hematology, and cardiac function measures were largely unchanged by treatment. Cumulatively, these data show that short-term intravenous treatment of GRMD dogs with rAAVrh74.MHCK7.GALGT2 at high doses can induce muscle glycosylation and utrophin expression and may be safe over a short 3-month interval, but that such treatments had only modest effects on muscle pathology and did not significantly improve muscle strength.  

5.3136           Non-permissive human conventional CD1c+ dendritic cells enable trans-infection of human primary renal tubular epithelial cells and protect BK polyomavirus from neutralization

Sikorski, M., Coulon, F., Peltier, C., Bradeau, C., Garcia, A. et al PloS Pathogens, 17(2), e1009042 (2021)   The BK polyomavirus (BKPyV) is a ubiquitous human virus that persists in the renourinary epithelium. Immunosuppression can lead to BKPyV reactivation in the first year post-transplantation in kidney transplant recipients (KTRs) and hematopoietic stem cell transplant recipients. In KTRs, persistent DNAemia has been correlated to the occurrence of polyomavirus-associated nephropathy (PVAN) that can lead to graft loss if not properly controlled. Based on recent observations that conventional dendritic cells (cDCs) specifically infiltrate PVAN lesions, we hypothesized that those cells could play a role in BKPyV infection. We first demonstrated that monocyte-derived dendritic cells (MDDCs), an in vitro model for mDCs, captured BKPyV particles through an unconventional GRAF-1 endocytic pathway. Neither BKPyV particles nor BKPyV-infected cells were shown to activate MDDCs. Endocytosed virions were efficiently transmitted to permissive cells and protected from the antibody-mediated neutralization. Finally, we demonstrated that freshly isolated CD1c⁺ mDCs from the blood and kidney parenchyma behaved similarly to MDDCs thus extending our results to cells of clinical relevance. This study sheds light on a potential unprecedented CD1c⁺ mDC involvement in the BKPyV infection as a promoter of viral spreading.  

5.3137           A lysine ring in HIV capsid pores coordinates IP6 to drive mature capsid assembly

Renner, N., Mallery, D.L., Faysal, K.M.R., Peng, W., Jacques, D.A., Böcking, T. and James, L.C. PloS Pathogens, 17(2), e1009164 (2021)   The HIV capsid self-assembles a protective conical shell that simultaneously prevents host sensing whilst permitting the import of nucleotides to drive DNA synthesis. This is accomplished through the construction of dynamic, highly charged pores at the centre of each capsid multimer. The clustering of charges required for dNTP import is strongly destabilising and it is proposed that HIV uses the metabolite IP6 to coordinate the pore during assembly. Here we have investigated the role of inositol phosphates in coordinating a ring of positively charged lysine residues (K25) that forms at the base of the capsid pore. We show that whilst IP5, which can functionally replace IP6, engages an arginine ring (R18) at the top of the pore, the lysine ring simultaneously binds a second IP5 molecule. Dose dependent removal of K25 from the pore severely inhibits HIV infection and concomitantly prevents DNA synthesis. Cryo-tomography reveals that K25A virions have a severe assembly defect that inhibits the formation of mature capsid cones. Monitoring both the kinetics and morphology of capsids assembled in vitro reveals that while mutation K25A can still form tubes, the ability of IP6 to drive assembly of capsid cones has been lost. Finally, in single molecule TIRF microscopy experiments, capsid lattices in permeabilised K25 mutant virions are rapidly lost and cannot be stabilised by IP6. These results suggest that the coordination of IP6 by a second charged ring in mature hexamers drives the assembly of conical capsids capable of reverse transcription and infection.  

5.3138           Human parvovirus B19 interacts with globoside under acidic conditions as an essential step in endocytic trafficking

Bieri, J., Leisi, R., Bircher, C. and Ros, C. PloS Pathogens, 17(4), e1009434 (2021)   The glycosphingolipid (GSL) globoside (Gb4) is essential for parvovirus B19 (B19V) infection. Historically considered the cellular receptor of B19V, the role of Gb4 and its interaction with B19V are controversial. In this study, we applied artificial viral particles, genetically modified cells, and specific competitors to address the interplay between the virus and the GSL. Our findings demonstrate that Gb4 is not involved in the binding or internalization process of the virus into permissive erythroid cells, a function that corresponds to the VP1u cognate receptor. However, Gb4 is essential at a post-internalization step before the delivery of the single-stranded viral DNA into the nucleus. In susceptible erythroid Gb4 knockout cells, incoming viruses were arrested in the endosomal compartment, showing no cytoplasmic spreading of capsids as observed in Gb4-expressing cells. Hemagglutination and binding assays revealed that pH acts as a switch to modulate the affinity between the virus and the GSL. Capsids interact with Gb4 exclusively under acidic conditions and dissociate at neutral pH. Inducing a specific Gb4-mediated attachment to permissive erythroid cells by acidification of the extracellular environment led to a non-infectious uptake of the virus, indicating that low pH-mediated binding to the GSL initiates active membrane processes resulting in vesicle formation. In summary, this study provides mechanistic insight into the interaction of B19V with Gb4. The strict pH-dependent binding to the ubiquitously expressed GSL prevents the redirection of the virus to nonpermissive tissues while promoting the interaction in acidic intracellular compartments as an essential step in infectious endocytic trafficking.  

5.3139           Glycometabolism regulates hepatitis C virus release

Yu, T., Yang, Q., Tian, F., Chang, H., Hu, Z., Yu, B., han, L., Xing, Y., Jiu, Y., He, Y. and Zhong, J. PloS Pathogens, 17(7), e1009746 (2021)   HCV cell-culture system uses hepatoma-derived cell lines for efficient virus propagation. Tumor cells cultured in glucose undergo active aerobic glycolysis, but switch to oxidative phosphorylation for energy production when cultured in galactose. Here, we investigated whether modulation of glycolysis in hepatocytes affects HCV infection. We showed HCV release, but not entry, genome replication or virion assembly, is significantly blocked when cells are cultured in galactose, leading to accumulation of intracellular infectious virions within multivesicular body (MVB). Blockade of the MVB-lysosome fusion or treatment with pro-inflammatory cytokines promotes HCV release in galactose. Furthermore, we found this glycometabolic regulation of HCV release is mediated by MAPK-p38 phosphorylation. Finally, we showed HCV cell-to-cell transmission is not affected by glycometabolism, suggesting that HCV cell-to-supernatant release and cell-to-cell transmission are two mechanistically distinct pathways. In summary, we demonstrated glycometabolism regulates the efficiency and route of HCV release. We proposed HCV may exploit the metabolic state in hepatocytes to favor its spread through the cell-to-cell transmission in vivo to evade immune response.  

5.3140           NK cells eliminate Epstein-Barr virus bound to B cells through a specific antibody-mediated uptake

Alari-Pahissa, e., Ataya, M., Moraitis, I., Campoe-Ruiz, M., Altadill, M., Muntasell, A., Moles, A. and Lopez-Botet, M. PloS Pathogens, 17(8), e1009868 (2021)   Epstein Barr virus (EBV) causes a highly prevalent and lifelong infection contributing to the development of some malignancies. In addition to the key role played by T cells in controlling this pathogen, NK cells mediate cytotoxicity and IFNγ production in response to EBV-infected B cells in lytic cycle, both directly and through antibody (Ab)-dependent activation. We recently described that EBV-specific Ab-dependent NK cell interaction with viral particles (VP) bound to B cells triggered degranulation and TNFα secretion but not B cell lysis nor IFNγ production. In this report we show that NK cell activation under these conditions reduced B cell transformation by EBV. NK cells eliminated VP from the surface of B cells through a specific and active process which required tyrosine kinase activation, actin polymerization and Ca2+, being independent of proteolysis and perforin. VP were displayed at the NK cell surface before being internalized and partially shuttled to early endosomes and lysosomes. VP transfer was encompassed by a trogocytosis process including the EBV receptor CD21, together with CD19 and CD20. Our study reveals a novel facet of the antibody-dependent NK cell mediated response to this viral infection.  

5.3141           Completion of neuronal remodeling prompts myelination along developing motor axon branches

Wang, M., Kleele, T., Xiao, Y., Plucinska, G., Avramopoulos, P., Engelhardt, S., Schwab, M,H., Kneussel, M., Czopka, T., Sherman, D.L., Brophy, P.J., Misgeld, T. and Brill, M.S.

1. Cell Biol., 220(4), e201911114 (2021)

  Neuronal remodeling and myelination are two fundamental processes during neurodevelopment. How they influence each other remains largely unknown, even though their coordinated execution is critical for circuit function and often disrupted in neuropsychiatric disorders. It is unclear whether myelination stabilizes axon branches during remodeling or whether ongoing remodeling delays myelination. By modulating synaptic transmission, cytoskeletal dynamics, and axonal transport in mouse motor axons, we show that local axon remodeling delays myelination onset and node formation. Conversely, glial differentiation does not determine the outcome of axon remodeling. Delayed myelination is not due to a limited supply of structural components of the axon–glial unit but rather is triggered by increased transport of signaling factors that initiate myelination, such as neuregulin. Further, transport of promyelinating signals is regulated via local cytoskeletal maturation related to activity-dependent competition. Our study reveals an axon branch–specific fine-tuning mechanism that locally coordinates axon remodeling and myelination.  

5.3142           Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads

Belicova, L., Repnik, U., Delpierre, J., Gralinska, E., Seifert, S., Valenzuela, J.I. et al

1. Cell Biol., 220(10)

  Lumen morphogenesis results from the interplay between molecular pathways and mechanical forces. In several organs, epithelial cells share their apical surfaces to form a tubular lumen. In the liver, however, hepatocytes share the apical surface only between adjacent cells and form narrow lumina that grow anisotropically, generating a 3D network of bile canaliculi (BC). Here, by studying lumenogenesis in differentiating mouse hepatoblasts in vitro, we discovered that adjacent hepatocytes assemble a pattern of specific extensions of the apical membrane traversing the lumen and ensuring its anisotropic expansion. These previously unrecognized structures form a pattern, reminiscent of the bulkheads of boats, also present in the developing and adult liver. Silencing of Rab35 resulted in loss of apical bulkheads and lumen anisotropy, leading to cyst formation. Strikingly, we could reengineer hepatocyte polarity in embryonic liver tissue, converting BC into epithelial tubes. Our results suggest that apical bulkheads are cell-intrinsic anisotropic mechanical elements that determine the elongation of BC during liver tissue morphogenesis.  

5.3143           Gene therapy for tuberous sclerosis complex type 2 in a mouse model by delivery of AAV9 encoding a condensed form of tuberin

Cheah, P-S., Prabhakar, S., Yellen, D.Y., Beauchamp, R.L., Zhang, X. et al Science Advances, 7, eabb1703 (2021)   Tuberous sclerosis complex (TSC) results from loss of a tumor suppressor gene - TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins formed a complex to inhibit mTORC1-mediated cell growth and proliferation. Loss of either protein leads to overgrowth lesions in many vital organs. Gene therapy was evaluated in a mouse model of TSC2 using an adeno-associated virus (AAV) vector carrying the complementary for a “condensed” form of human tuberin (cTuberin). Functionality of cTuberin was verified in culture. A mouse model of TSC2 was generated by AAV-Cre recombinase disruption of Tsc2-floxed alleles at birth, leading to a shortened lifespan (mean 58 days) and brain pathology consistent with TSC. When these mice were injected intravenously on day 21 with AAV9-cTuberin, the mean survival was extended to 462 days with reduction in brain pathology. This demonstrates the potential of treating life-threatening TSC2 lesions with a single intravenous injection of AAV9-cTuberin.    

5.3144           Prefusion structure of human cytomegalovirus glycoprotein B and structural basis for membrane fusion

Liu, Y., Heim, K.P., Che, Y., Chi, X., Qiu, X., Han, S., Dormitzer, P.R. and Yang, X. Science Advances, 7, eab3178 (2021)   Human cytomegalovirus (HCMV) causes congenital disease with long-term morbidity. HCMV glycoprotein B (gB) transitions irreversibly from a metastable prefusion to a stable postfusion conformation to fuse the viral envelope with a host cell membrane during entry. We stabilized prefusion gB on the virion with a fusion inhibitor and a chemical cross-linker, extracted and purified it, and then determined its structure to 3.6-Å resolution by electron cryomicroscopy. Our results revealed the structural rearrangements that mediate membrane fusion and details of the interactions among the fusion loops, the membrane-proximal region, transmembrane domain, and bound fusion inhibitor that stabilized gB in the prefusion state. The structure rationalizes known gB antigenic sites. By analogy to successful vaccine antigen engineering approaches for other viral pathogens, the high-resolution prefusion gB structure provides a basis to develop stabilized prefusion gB HCMV vaccine antigens.  

5.3145           A stable immature lattice packages IP6 for HIV capsid maturation

Mallery, D.L., Kleinpeter, A.B., Renner, N., Faysal, K.M.R., Novikova, M. et al Science Advances, 7, eabe4716 (2021)   HIV virion assembly begins with the construction of an immature lattice consisting of Gag hexamers. Upon virion release, protease-mediated Gag cleavage leads to a maturation event in which the immature lattice disassembles and the mature capsid assembles. The cellular metabolite inositiol hexakisphosphate (IP₆) and maturation inhibitors (MIs) both bind and stabilize immature Gag hexamers, but whereas IP₆ promotes virus maturation, MIs inhibit it. Here we show that HIV is evolutionarily constrained to maintain an immature lattice stability that ensures IP₆ packaging without preventing maturation. Replication-deficient mutant viruses with reduced IP₆ recruitment display increased infectivity upon treatment with the MI PF46396 (PF96) or the acquisition of second-site compensatory mutations. Both PF96 and second-site mutations stabilise the immature lattice and restore IP₆ incorporation, suggesting that immature lattice stability and IP₆ binding are interdependent. This IP₆ dependence suggests that modifying MIs to compete with IP₆ for Gag hexamer binding could substantially improve MI antiviral potency.  

5.3146           Receptor-ligand supplementation via a self-cleaving 2A peptide–based gene therapy promotes CNS axonal transport with functional recovery

Khatib, T:Z., Osborne, A., Yang, S., Ali, Z., Jia, W., Manyakin, I., Hall, K., Watt, R., Widdowson, P.S. and Martin, K.R. Science Advances, 7, eabd2590 (2021)   Gene replacement approaches are leading to a revolution in the treatment of previously debilitating monogenic neurological conditions. However, the application of gene therapy to complex polygenic conditions has been limited. Down-regulation or dysfunction of receptor expression in the disease state or in the presence of excess ligand has been shown to compromise therapeutic efficacy. Here, we offer evidence that combined overexpression of both brain-derived neurotrophic factor and its receptor, tropomyosin receptor kinase B, is more effective in stimulating axonal transport than either receptor administration or ligand administration alone. We also show efficacy in experimental glaucoma and humanized tauopathy models. Simultaneous administration of a ligand and its receptor by a single gene therapy vector overcomes several problems relating to ligand deficiency and receptor down-regulation that may be relevant to multiple neurodegenerative diseases. This approach shows promise as a strategy to target intrinsic mechanisms to improve neuronal function and facilitate repair.  

5.3147           Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing

Chemello, F., Chai, A.C., Li, H., Rodrigues-Caycedo, C., Sanchez-Ortiz, E., Atmanli, A., Mireault, A.A., Liu, N., Bassel-Duby, R and Olson, E.N. Sciences Advances, 7, eabg4910 (2021)   Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor (ABE) to modify splice donor sites of the dystrophin gene, causing skipping of a common DMD deletion mutation of exon 51 (∆Ex51) in cardiomyocytes derived from human induced pluripotent stem cells, restoring dystrophin expression. Prime editing was also capable of reframing the dystrophin open reading frame in these cardiomyocytes. Intramuscular injection of ∆Ex51 mice with adeno-associated virus serotype-9 encoding ABE components as a split-intein trans-splicing system allowed gene editing and disease correction in vivo. Our findings demonstrate the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome, although improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with DMD.  

5.3148           Hierarchy in sensory processing reflected by innervation balance on cortical interneurons

Ma, G., Liu, Y., Wang, l., Xiao, Z., Song, K., Wang, Y. et al Science Advances, 7, eabf5676 (2021)   Sensory processing is subjected to modulation by behavioral contexts that are often mediated by long-range inputs to cortical interneurons, but their selectivity to different types of interneurons remains largely unknown. Using rabies-virus tracing and optogenetics-assisted recording, we analyzed the long-range connections to various brain regions along the hierarchy of visual processing, including primary visual cortex, medial association cortices, and frontal cortices. We found that hierarchical corticocortical and thalamocortical connectivity is reflected by the relative weights of inputs to parvalbumin-positive (PV⁺) and vasoactive intestinal peptide–positive (VIP⁺) neurons within the conserved local circuit motif, with bottom-up and top-down inputs preferring PV⁺ and VIP⁺ neurons, respectively. Our algorithms based on innervation weights for these two types of local interneurons generated testable predictions of the hierarchical position of many brain areas. These results support the notion that preferential long-range inputs to specific local interneurons are essential for the hierarchical information flow in the brain.  

5.3149           Functional coordination of BET family proteins underlies altered transcription associated with memory impairment in fragile X syndrome

Kim, S-K., Liu, X., Park, J., Um, D., Kilaru, G., Chiang, C-M., Kang, M., Huber, K.M., Kang, K. and Kim, T-K. Science Advances, 7, eabf7346 (2021)   Bromodomain and extraterminal proteins (BET) are epigenetic readers that play critical roles in gene regulation. Pharmacologic inhibition of the bromodomain present in all BET family members is a promising therapeutic strategy for various diseases, but its impact on individual family members has not been well understood. Using a transcriptional induction paradigm in neurons, we have systematically demonstrated that three major BET family proteins (BRD2/3/4) participated in transcription with different recruitment kinetics, interdependency, and sensitivity to a bromodomain inhibitor, JQ1. In a mouse model of fragile X syndrome (FXS), BRD2/3 and BRD4 showed oppositely altered expression and chromatin binding, correlating with transcriptional dysregulation. Acute inhibition of CBP/p300 histone acetyltransferase (HAT) activity restored the altered binding patterns of BRD2 and BRD4 and rescued memory impairment in FXS. Our study emphasizes the importance of understanding the BET coordination controlled by a balanced action between HATs with different substrate specificity.  

5.3150           Spatiotemporally confined red light-controlled gene delivery at single-cell resolution using adeno-associated viral vectors

Hörner, M., Jerez-Longres, C., Hudek, A., Hook, S., Yousefi, O.S., Schamel, W.W.A., Hörner, C., Zurbriggen, M.D., Ye, H., Wagner, H.J. and Weber, W. Science Advances, 7, eabf0797 (2021)   Methodologies for the controlled delivery of genetic information into target cells are of utmost importance for genetic engineering in both fundamental and applied research. However, available methods for efficient gene transfer into user-selected or even single cells suffer from low throughput, the need for complicated equipment, high invasiveness, or side effects by off-target viral uptake. Here, we engineer an adeno-associated viral (AAV) vector system that transfers genetic information into native target cells upon illumination with cell-compatible red light. This OptoAAV system allows adjustable and spatially resolved gene transfer down to single-cell resolution and is compatible with different cell lines and primary cells. Moreover, the sequential application of multiple OptoAAVs enables spatially resolved transduction with different transgenes. The approach presented is likely extendable to other classes of viral vectors and is expected to foster advances in basic and applied genetic research.  

5.3151           Planar cell polarity signaling components are a direct target of β-amyloid–associated degeneration of glutamatergic synapses

Feng, B., Freitas, A.E., Gorodetski, L., Wang, j., Tian, R., Lee, Y.R., Grewal, A.S. and Zou, Y. Science Advances, 7, eabh2307 (2021)   The signaling pathway directly controlling the maintenance of adult glutamatergic synapses has not been well understood. Planar cell polarity (PCP) signaling components were recently shown to play essential roles in the formation of glutamatergic synapses. Here, we show that they are localized in the adult synapses and are essential for their maintenance. Synapse loss at early stages of Alzheimer’s disease is thought to be induced by β-amyloid (Aβ) pathology. We found that oligomeric Aβ binds to Celsr3 and assists Vangl2 in disassembling synapses. Moreover, a Wnt receptor and regulator of PCP signaling, Ryk, is also required for Aβ-induced synapse loss. In the 5XFAD mouse model of Alzheimer’s disease, Ryk conditional knockout or a function-blocking monoclonal Ryk antibody protected synapses and preserved cognitive function. We propose that tipping of the fine balance of Wnt/PCP signaling components in glutamatergic synapses may cause synapse degeneration in neurodegenerative disorders with Aβ pathology.  

5.3152           Invasion of phagocytic Galectin 3 expressing macrophages in the diabetic brain disrupts vascular repair

Mehina, E.M.F., Taylor, S., Boghozian, R., White, E., Choi, S.E., Cheema, M.S., Korbelin, J. and Brown, C.E. Science Advances, 7, eabg2712 (2021)   The cellular events that dictate the repair of damaged vessels in the brain, especially in those with vascular risk factors such as diabetes, is poorly understood. Here, we dissected the role of resident microglia and infiltrative macrophages in determining the repair of ruptured cerebral microvessels. Using in vivo time-lapse imaging, gene expression analysis, and immunohistochemistry, we identified a unique population of phagocytic Galectin 3 (Gal3) expressing macrophages, distinct from resident microglia, which infiltrated and aggregated at the site of injury in diabetic mice and were associated with the elimination of microvessels. Depletion of these infiltrative macrophages in diabetic mice attenuated phagocytic activity and prevented the loss of blood vessels after injury. These findings highlight a previously unknown role for infiltrative Gal3 expressing macrophages in promoting vessel elimination after brain injury and provide impetus for future studies to determine whether depleting these cells can facilitate vascular repair in at risk populations.  

5.3153           Retromer stabilizes transient membrane insertion of L2 capsid protein during retrograde entry of human papillomavirus

Xie, J., Zhang, P., Crite, M., Lindsay, C.V. and DiMaio, D. Science Advances, 7, eabh4276 (2021)   Retromer, a cellular protein trafficking complex, sorts human papillomaviruses (HPVs) into the retrograde pathway for transport of HPV to the nucleus during virus entry. Here, we conducted a protein modulation screen to isolate four artificial transmembrane proteins called traptamers that inhibit different steps of HPV entry. By analyzing cells expressing pairs of traptamers, we ordered the trafficking steps during entry into a coherent pathway. One traptamer stimulates ubiquitination of the L2 capsid protein or associated proteins and diverts incoming virus to the lysosome, whereas the others act downstream by preventing sequential passage of the virus through retrograde compartments. Complex genetic interactions between traptamers revealed that a cell-penetrating peptide (CPP) on L2 mediates transient insertion of L2 into the endosome membrane, which is stabilized by retromer-L2 binding. These results define the retrograde entry route taken by HPV and show that retromer can play a role in CPP-mediated membrane insertion.  

5.3154           Cas9-expressing chickens and pigs as resources for genome editing in livestock

Rieblinger, B., Sid, H., Duda, D., Bozoglu, T., Klinger, R. et al PNAS, 118(10), e2022562118 (2021)   Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.  

5.3155           Nr2e3 is a genetic modifier that rescues retinal degeneration and promotes homeostasis in multiple models of retinitis pigmentosa

Li, S., Datta, S., Brabbit, E., Love, Z., Woytowicz, V., Flattery, K., Capri, J. et al Gene Therapy, 28, 223-241 (2021)   Recent advances in viral vector engineering, as well as an increased understanding of the cellular and molecular mechanism of retinal diseases, have led to the development of novel gene therapy approaches. Furthermore, ease of accessibility and ocular immune privilege makes the retina an ideal target for gene therapies. In this study, the nuclear hormone receptor gene Nr2e3 was evaluated for efficacy as broad-spectrum therapy to attenuate early to intermediate stages of retinal degeneration in five unique mouse models of retinitis pigmentosa (RP). RP is a group of heterogenic inherited retinal diseases associated with over 150 gene mutations, affecting over 1.5 million individuals worldwide. RP varies in age of onset, severity, and rate of progression. In addition, ~40% of RP patients cannot be genetically diagnosed, confounding the ability to develop personalized RP therapies. Remarkably, Nr2e3 administered therapy resulted in reduced retinal degeneration as observed by increase in photoreceptor cells, improved electroretinogram, and a dramatic molecular reset of key transcription factors and associated gene networks. These therapeutic effects improved retinal homeostasis in diseased tissue. Results of this study provide evidence that Nr2e3 can serve as a broad-spectrum therapy to treat multiple forms of RP.  

5.3156           Gene Transfer in Adeno-Associated Virus Seropositive Rhesus Macaques Following Rapamycin Treatment and Subcutaneous Delivery of AAV6, but Not Retargeted AAV6 Vectors

Stone, D., Kenkel, E.J., Loprieno, M.A., Tanaka, M., Harshans, T., De Silva Feelixge, S. et al Human Gene Therapy, 32(1-2), 96-112 (2021)   Adeno-associated virus (AAV) vectors such as AAV6, which shows tropism for primary human CD4⁺ T cells in vitro, are being explored for delivery of anti-HIV therapeutic modalities in vivo. However, pre-existing immunity and sequestration in nontarget organs can significantly hinder their performance. To overcome these challenges, we investigated whether immunosuppression would allow gene delivery by AAV6 or targeted AAV6 derivatives in seropositive rhesus macaques. Animals were immune suppressed with rapamycin before intravenous (IV) or subcutaneous (SC) delivery of AAV, and we monitored vector biodistribution, gene transfer, and safety. Macaques received phosphate-buffered saline, AAV6 alone, or an equal dose of AAV6 and an AAV6-55.2 vector retargeted to CD4 through a direct ankyrin repeat protein (DARPin). AAV6 and AAV6-55.2 vector genomes were found in peripheral blood mononuclear cells and most organs up to 28 days postadministration, with the highest levels seen in liver, spleen, lymph nodes (LNs), and muscle, suggesting that retargeting did not prevent vector sequestration. Despite vector genome detection, gene expression from AAV6-55.2 was not detected in any tissue. SC injection of AAV6 facilitated efficient gene expression in muscle adjacent to the injection site, plus low-level gene expression in spleen, LNs, and liver, whereas gene expression following IV injection of AAV6 was predominantly seen in the spleen. AAV vectors were well tolerated, although elevated liver enzymes were detected in three of four AAV-treated animals 14 days after rapamycin withdrawal. One SC-injected animal had muscle inflammation proximal to the injection site, plus detectable T cell responses against transgene and AAV6 capsid at study finish. Overall, our data suggest that rapamycin treatment may offer a possible strategy to express anti-HIV therapeutics such as broadly neutralizing antibodies from muscle. This study provides important safety and efficacy data that will aid study design for future anti-HIV gene therapies.  

5.3157           Induced Liver Regeneration Enhances CRISPR/Cas9-Mediated Gene Repair in Tyrosinemia Type 1

Zhang, Q-S., Tiyaboonchai, A., Nygaard, S., Baradar, K., Major, A., Balaji, N. and Grompe, M. Human Gene Therapy, 32(5-6), 294-301 (2021)   The efficiency of gene repair by homologous recombination in the liver is enhanced by CRISP/Cas9 incision near the mutation. In this study, we explored interventions designed to further enhance in vivo hepatocyte gene repair in a model of hereditary tyrosinemia. A two-AAV system was employed: one virus carried a Staphylococcus pyogenes Cas9 (SpCas9) expression cassette and the other harbored a U6 promoter-driven sgRNA and a fragment of fumarylacetoacetate hydrolase (Fah) genomic DNA as the homologous recombination donor. In neonatal mice, a gene correction frequency of ∼10.8% of hepatocytes was achieved. The efficiency in adult mice was significantly lower at ∼1.6%. To determine whether hepatocyte replication could enhance the targeting frequency, cell division was induced with thyroid hormone T3. This more than doubled the gene correction efficiency to 3.5% (p < 0.005). To determine whether SpCas9 delivery was rate limiting, the gene repair AAV was administered to SpCas9 transgenic mice. However, this did not significantly enhance gene repair. Finally, we tested whether the Fanconi anemia (FA) DNA repair pathway was important in hepatocyte gene repair. Gene correction frequencies were significantly lower in neonatal mice lacking the FA complementation group A (Fanca) gene. Taken together, we conclude that pharmacological induction of hepatocyte replication along with manipulation of DNA repair pathways could be a useful strategy for enhancing in vivo gene correction.  

5.3158           Linking the Expression of Therapeutic Genes to Unfolded Protein Response: A New Option for Anti-Hepatitis B Virus Gene Therapy

Nistal-Villan, E., Argemi, J., de Jaime-Soguero, A., Ferrero, R., di Scala, M., Rodriguez-Garcia, E., Coll, A., Rius-Rocabert, S., Prieto, J., Gonzalez-Aseguinolaza, G. and Aragon, T. Human Gene Therapy, 32(7-8), 341-348 (2021)   Tight control of transgene expression is key to ensure the efficacy of a wide range of gene therapy interventions, in which the magnitude and duration of gene expression have to be adjusted to therapeutic needs, thereby limiting secondary effects. The development of upgraded strategies to link transgene expression to pathological stress episodes is an unmet need in gene therapy. Here, we propose an expression strategy that associates transgene expression to an intracellular stress coping mechanism, the unfolded protein response. Specifically, we harnessed the cis elements required to sustain the noncanonical splicing of X-box binding protein 1 (XBP1) messenger RNA (mRNA) in response to the dysfunction of the endoplasmic reticulum (ER), a situation commonly known as ER stress, to drive the expression of heterologous genes. Since ER stress features a wide variety of pathological conditions, including viral infections, cancer, or metabolic disorders, this new expression module stimulates the synthesis of therapeutic genes as a response to cellular damage, and ensures their expression only when necessary. Validation of this inducible expression system was performed in vitro and in vivo, and its potential to limit/inhibit viral infections has been shown in proof-of principle experiments.  

5.3159           Prevalence of Adeno-Associated Virus 3 Capsid Binding and Neutralizing Antibodies in Healthy and Hemophilia B Individuals from India

Daniel, H.D-J., Kumar, S., Kannangai, R., Lakshmi, K.M., Agbandje-Mckenna, M., Coleman, K., Srivastava, A., Srivastava, A. and Abraham, A.M. Human Gene Therapy, 32(9-10), 451-457 (2021)   Adeno-associated virus (AAV) vector-based gene therapy offers a new treatment option for individuals with hemophilia. Pre-existing anti-AAV antibodies significantly impact the use of AAV vectors. Even relatively low titers of AAV neutralizing antibodies (NAb) from natural AAV infections against the capsid have been shown to inhibit the transduction of intravenously administered AAV in animal models and were associated with limited efficacy in human trials. This is important for determining the primary eligibility of patients for AAV vector-based gene therapy clinical trials. Current techniques to screen AAV antibodies include AAV capsid enzyme-linked immunosorbent assay (ELISA) for total antibodies and a transduction inhibition assay (TIA) for NAb. This study developed and screened total capsid binding anti-AAV3 antibodies by using ELISA and determined NAb levels by TIA using mCherry flow cytometry in healthy individuals with hemophilia B in India. One hundred and forty-three apparently healthy controls and 92 individuals with hemophilia B were screened. The prevalence of total and NAb in healthy controls was 79.7% and 65%, respectively; the prevalence of total and NAb in patients with hemophilia B for AAV3 was 92.4% and 91.3%, respectively.  

5.3160           Consequences of Mammalian Target of Rapamycin Inhibition on Adeno-Associated Virus Hepatic Transduction Efficacy

Perez-Iturralde, A., Carte, B. and Aldabe, R. Human Gene Therapy, 32(19-20), 1242-1250 (2021)   The efficiency of recombinant adeno-associated virus (AAV) vectors transducing host cells is very low, limiting their therapeutic potential in patients. There are several cellular pathways interacting and interfering with the journey of the AAV from the cell surface to the nucleus, opening the possibility to enhance AAV transduction by modifying these interactions. In this study, we explored the results of AAV hepatic transduction when different mammalian target of rapamycin (mTOR) inhibitors, rapamycin, MLN0128, RapaLink-1, were used in preconditioned juvenile and adult mice. We confirmed rapamycin as an AAV hepatic transduction enhancer in juvenile and adult mice; however, RapaLink-1, a stronger mTOR inhibitor and a clear hepatic autophagy inducer, had no positive effect. Moreover, MLN0128 reduced AAV hepatic transduction. Therefore, our results show a complex interaction between the mTOR pathway and AAV-mediated hepatic transduction and indicate that mTOR inhibition is not a straightforward strategy for improving AAV transduction. More studies are necessary to elucidate the molecular mechanisms involved in the positive and negative effects of mTOR inhibitors on AAV transduction efficiency.  

5.3161           Preclinical Assessment of a Gene-Editing Approach in a Mouse Model of Mitochondrial Neurogastrointestinal Encephalomyopathy

Pares, M., Fornaguera, C., Vila-Julia, F., Oh, S., Fan, S.H.Y., Tam, Y.K., COmes, N., Vidal, F., Marti, R., Borros, S. and Barquinero, J. Human Gene Therapy, 32(19-20), 1210-1223 (2021)   Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare disease caused by recessive mutations in the TYMP gene, which encodes the enzyme thymidine phosphorylase (TP). In this study, the efficient integration of a TYMP transgene into introns of the Tymp and Alb loci of hepatocytes in a murine model of MNGIE was achieved by the coordinated delivery and activity of CRISPR/Cas9 and a TYMP cDNA. CRISPR/Cas9 was delivered either as mRNA using lipid nanoparticle (LNP) or polymeric nanoparticle, respectively, or in an AAV2/8 viral vector; the latter was also used to package the TYMP cDNA. Insertion of the cDNA template downstream of the Tymp and Alb promoters ensured transgene expression. The best in vivo results were obtained using LNP carrying the CRISPR/Cas9 mRNAs. Treated mice showed a consistent long-term (1 year) reduction in plasma nucleoside (thymidine and deoxyuridine) levels that correlated with the presence of TYMP mRNA and functional enzyme in liver cells. In mice with an edited Alb locus, the transgene produced a hybrid Alb-hTP protein that was secreted, with supraphysiological levels of TP activity detected in the plasma. Equivalent results were obtained in mice edited at the Tymp locus. Finally, some degree of gene editing was found in animals treated only with AAV vectors containing the DNA templates, in the absence of nucleases, although there was no impact on plasma nucleoside levels. Overall, these results demonstrate the feasibility of liver-directed genome editing in the long-term correction of MNGIE, with several advantages over other methods.  

5.3162           Dose-Dependent Microdystrophin Expression Enhancement in Cardiac Muscle by a Cardiac-Specific Regulatory Element

Malerba, A., Sidoli, C., Luc-Nguyen, N., Herath, S., Heron, A.L., Abdul-Razak, H., Jarmin, S., VandenDriessche, T., Chuah, M.K., Dickson, G. and Popplewell, L. Human Gene Therapy, 32(19-20), 1138-1146 (2021)   Duchenne muscular dystrophy (DMD) is an X-linked recessive disease that affects 1:5,000 live male births and is characterized by muscle wasting. By the age of 13 years, affected individuals are often wheelchair bound and suffer from respiratory and cardiac failure, which results in premature death. Although the administration of corticosteroids and ventilation can relieve the symptoms and extend the patients' lifespan, currently no cure exists for DMD. Among the different approaches under preclinical and clinical testing, gene therapy, using adeno-associated viral (AAV) vectors, is one of the most promising. In this study, we delivered intravenously AAV9 vectors expressing the microdystrophin MD1 (ΔR4-R23/ΔCT) under control of the synthetic muscle-specific promoter Spc5-12 and assessed the effect of adding a cardiac-specific cis-regulatory module (designated as CS-CRM4) on its expression profile in skeletal and cardiac muscles. Results show that Spc5-12 promoter, in combination with an AAV serotype that has high tropism for the heart, drives high MD1 expression levels in cardiac muscle in mdx mice. The additional regulatory element CS-CRM4 can further improve MD1 expression in cardiac muscles, but its effect is dose dependent and enhancement becomes evident only at lower vector doses.  

5.3163           Optimized Protocol for Accurate Titration of Adeno-Associated Virus Vectors

Suoranta, T., Laham-Karam, N. and Yla-Herttuala, S. Human Gene Therapy, 32(19-20), 1270-1279 82021)   Adeno-associated virus (AAV) is currently the most popular gene delivery vector for in vivo gene therapy. However, variability in titration methods between different laboratories affects the reproducibility of experiments and evaluation of safety and efficacy in clinical trials. We describe an optimized protocol for AAV titration, including quantitative PCR (qPCR) standard preparation and quantitation and treatment of AAV samples before qPCR and droplet digital PCR (ddPCR) titration. During the protocol development, we observed that quantitation of the qPCR standard was dependent on its conformation and that A260-based quantitation overestimated the plasmid copy numbers, introducing significant error. Linearized, free inverted terminal repeat (free-ITR), and supercoiled standards were compared with enhanced green fluorescent protein (EGFP), SV40p(A), and AAV2-ITR qPCR assays and we found that using the AAV2-ITR assay together with either linearized or supercoiled standard led to overestimation of the titers, while EGFP and SV40p(A) assays were more accurate with the linearized standard. Finally, we compared extraction of AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 genomes by heat denaturation, proteinase K treatment, and kit extraction. Kit extraction, which contained proteinase K treatment in denaturing buffer before spin-column purification, significantly increased the titers acquired for all the serotypes in both qPCR and ddPCR. These improvements resulted in an accurate quantitation of the ATCC reference standard and in a robust and reliable protocol for AAV titration.  

5.3164           In Vivo Detection of Extracellular Adenosine Triphosphate in a Mouse Model of Traumatic Brain Injury

Faroqi, A.H., Lim, M.J., Kee, E.C., Lee, J.H., Burgess, J.D., Chen, R., Di Virgilio, F., Delenclos, M. and McLean, P.J.

1. Neurotrauma, 38, 655-664 (2021)

  Traumatic brain injury (TBI) is traditionally characterized by primary and secondary injury phases, both contributing to pathological and morphological changes. The mechanisms of damage and chronic consequences of TBI remain to be fully elucidated, but synaptic homeostasis disturbances and impaired energy metabolism are proposed to be a major contributor. It has been proposed that an increase of extracellular (eATP) adenosine triphosphate (ATP) in the area immediately surrounding impact may play a pivotal role in this sequence of events. After tissue injury, rupture of cell membranes allows release of intracellular ATP into the extracellular space, triggering a cascade of toxic events and inflammation. ATP is a ubiquitous messenger; however, simple and reliable techniques to measure its concentration have proven elusive. Here, we integrate a sensitive bioluminescent eATP sensor known as pmeLUC, with a controlled cortical impact mouse model to monitor eATP changes in a living animal after injury. Using the pmeLUC probe, a rapid increase of eATP is observed proximal to the point of impact within minutes of the injury. This event is significantly attenuated when animals are pretreated with an ATP hydrolyzing agent (apyrase) before surgery, confirming the contribution of eATP. This new eATP reporter could be useful for understanding the role of eATP in the pathogenesis in TBI and may identify a window of opportunity for therapeutic intervention.  

5.3165           SSH1 impedes SQSTM1/p62 flux and MAPT/Tau clearance independent of CFL (cofilin) activation

Fang, C., Woo, J-A.A., Liu, T., Zhao, X., Cazzaro, S., Yan, Y., matlack, J., Kee, T., LePochat, P. and Kang, D.E. Autopaghy, 17(9), 2144-2165 (2021)   Accumulation of toxic protein assemblies and damaged mitochondria are key features of neurodegenerative diseases, which arise in large part from clearance defects in the Macroautophagy/autophagy-lysosome system. The autophagy cargo receptor SQSTM1/p62 plays a major role in the clearance of ubiquitinated cargo through Ser403 phosphorylation by multiple kinases. However, no phosphatase is known to physiologically dephosphorylate SQSTM1 on this activating residue. RNAi-mediated knockdown and overexpression experiments using genetically encoded fluorescent reporters and defined mutant constructs in cell lines, primary neurons, and brains show that SSH1, the canonical CFL (cofilin) phosphatase, mediates the dephosphorylation of phospho-Ser403-SQSTM1, thereby impairing SQSTM1 flux and phospho-MAPT/tau clearance. The inhibitory action of SSH1 on SQSTM1 is fully dependent on SQSTM1 Ser403 phosphorylation status and is separable from SSH1-mediated CFL activation. These findings reveal a unique action of SSH1 on SQSTM1 independent of CFL and implicate an inhibitory role of SSH1 in SQSTM1-mediated clearance of autophagic cargo, including phospho-MAPT/tau.  

5.3166           Dynamics and competition of CRISPR–Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing

Fu, Y-W., Dai, X-Y., Wang, W-T., Yang, Z-X., Zhao, J-J., Zhang, J-P., Wen, W., Zhang, F., Oberg, K.C., Zhang, L., Cheng, T. and Zhang, X-B. Nucleic Acids Res., 49(2). 969-985 (2021)   Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T₅₀ (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.  

5.3167           The evolutionary history of ACE2 usage within the coronavirus subgenus Sarbecovirus

Wells, H.L., Letko, M., Lasso, G., Ssebide, B., Nziza, J., Byarugaba, D.K. et al Virus Ecolution, 7(1), veab007 (2021)   Severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and SARS-CoV-2 are not phylogenetically closely related; however, both use the angiotensin-converting enzyme 2 (ACE2) receptor in humans for cell entry. This is not a universal sarbecovirus trait; for example, many known sarbecoviruses related to SARS-CoV-1 have two deletions in the receptor binding domain of the spike protein that render them incapable of using human ACE2. Here, we report three sequences of a novel sarbecovirus from Rwanda and Uganda that are phylogenetically intermediate to SARS-CoV-1 and SARS-CoV-2 and demonstrate via in vitro studies that they are also unable to utilize human ACE2. Furthermore, we show that the observed pattern of ACE2 usage among sarbecoviruses is best explained by recombination not of SARS-CoV-2, but of SARS-CoV-1 and its relatives. We show that the lineage that includes SARS-CoV-2 is most likely the ancestral ACE2-using lineage, and that recombination with at least one virus from this group conferred ACE2 usage to the lineage including SARS-CoV-1 at some time in the past. We argue that alternative scenarios such as convergent evolution are much less parsimonious; we show that biogeography and patterns of host tropism support the plausibility of a recombination scenario, and we propose a competitive release hypothesis to explain how this recombination event could have occurred and why it is evolutionarily advantageous. The findings provide important insights into the natural history of ACE2 usage for both SARS-CoV-1 and SARS-CoV-2 and a greater understanding of the evolutionary mechanisms that shape zoonotic potential of coronaviruses. This study also underscores the need for increased surveillance for sarbecoviruses in southwestern China, where most ACE2-using viruses have been found to date, as well as other regions such as Africa, where these viruses have only recently been discovered.  

5.3168           Identification of Two Critical Neutralizing Epitopes in the Receptor Binding Domain of Hepatitis B Virus preS1

Yato, K., Onodera, T., Matsuda, M., Moriyama, S., Fujimoto, A., Watashi, K., Aizaki, H. et al

1. Virol., 95(5), e01680-20 (2021)

  Hepatitis B virus (HBV) infection is a major public health problem. Human hepatocytes are infected with HBV via binding between the preS1 region in the large envelope protein of HBV and sodium taurocholate cotransporting polypeptide. Although several monoclonal antibodies (MAbs) that recognize the receptor binding domain in preS1 and neutralize HBV infection have been isolated, details of neutralizing epitopes are not understood. In this study, we generated 13 MAbs targeting the preS1 receptor binding domain from preS1-specific memory B cells derived from DNA-immunized mice. The MAbs were classified into three groups according to the epitope regions, designated epitopes I to III. A virus neutralization assay revealed that MAbs recognizing epitopes I and III neutralized HBV infection, suggesting that these domains are critical epitopes for viral neutralization. In addition, a neutralization assay against multiple genotypes of HBV revealed that epitope I is a semipangenotypic neutralizing epitope, whereas epitope III is a genotype-specific epitope. We also showed that neutralizing MAbs against preS1 could neutralize HBV bearing a vaccine-induced escape mutation. These findings provide insight into novel immunoprophylaxis for the prevention and treatment of HBV infection.  

5.3169           CRISPR/Cas9-Constructed Pseudorabies Virus Mutants Reveal the Importance of UL13 in Alphaherpesvirus Escape from Genome Silencing

Van Cleemput, J., Koyuncu, O.O., laval, K., Engel, E.A. and Enquist, L.W.

1. Virol., 95(6), e02286-20 (2021)

  Latent and recurrent productive infection of long-living cells, such as neurons, enables alphaherpesviruses to persist in their host populations. Still, the viral factors involved in these events remain largely obscure. Using a complementation assay in compartmented primary peripheral nervous system (PNS) neuronal cultures, we previously reported that productive replication of axonally delivered genomes is facilitated by pseudorabies virus (PRV) tegument proteins. Here, we sought to unravel the role of tegument protein UL13 in this escape from silencing. We first constructed four new PRV mutants in the virulent Becker strain using CRISPR/Cas9-mediated gene replacement: (i) PRV Becker defective for UL13 expression (PRV ΔUL13), (ii) PRV where UL13 is fused to eGFP (PRV UL13-eGFP), and two control viruses (iii and iv) PRV where VP16 is fused with mTurquoise at either the N terminus (PRV mTurq-VP16) or the C terminus (PRV VP16-mTurq). Live-cell imaging of PRV capsids showed efficient retrograde transport after axonal infection with PRV UL13-eGFP, although we did not detect dual-color particles. However, immunofluorescence staining of particles in mid-axons indicated that UL13 might be cotransported with PRV capsids in PNS axons. Superinfecting nerve cell bodies with UV-inactivated PRV ΔUL13 failed to efficiently promote escape from genome silencing compared to UV-PRV wild type and UV-PRV UL13-eGFP superinfection. However, UL13 does not act directly in the escape from genome silencing, as adeno-associated virus (AAV)-mediated UL13 expression in neuronal cell bodies was not sufficient to provoke escape from genome silencing. Based on this, we suggest that UL13 may contribute to initiation of productive infection through phosphorylation of other tegument proteins.  

5.3170           Structural Basis for the Shared Neutralization Mechanism of Three Classes of Human Papillomavirus Type 58 Antibodies with Disparate Modes of Binding

He, M., Chi, X., Zha, Z., Li, Y., Chen, J., Huang, Y. et al

1. Virol., 95(7), e01587-20 (2021)

  Human papillomavirus type 58 (HPV58) is associated with cervical cancer and poses a significant health burden worldwide. Although the commercial 9-valent HPV vaccine covers HPV58, the structural and molecular-level neutralization sites of the HPV58 complete virion are not fully understood. Here, we report the high-resolution (∼3.5-Å) structure of the complete HPV58 pseudovirus (PsV58), determined by using cryo-electron microscopy (cryo-EM). Three representative neutralizing monoclonal antibodies (nAbs 5G9, 2H3, and A4B4) were selected through clustering from a panel of nAbs against HPV58. Bypassing the steric hindrance and symmetry mismatch in the HPV Fab-capsid immune complex, we present three different neutralizing epitopes in the PsV58 and show that, despite differences in binding, these nAbs share a neutralization mechanism. These results offer insight into HPV58 genotype specificity and broaden our understanding of HPV58 neutralization sites for antiviral research.  

5.3171           Adeno-associated Virus (AAV) Capsid Chimeras with Enhanced Infectivity Reveal a Core Element in the AAV Genome Critical for both Cell Transduction and Capsid Assembly

Viney, L., Bürckstümmer, T., Eddington, C., Mietzsch, M., Choudhry, M., Henley, T. and Agbandje-McKenna, M.

1. Virol., 95(7), e02023-20 (2021)

  Adeno-associated viruses (AAVs) have attracted significant attention in the field of gene and cell therapy due to their use in highly effective delivery of therapeutic genes into human cells. The ability to generate recombinant AAV (rAAV) vectors comprising unique or substituted protein sequences has led to the development of capsid variants with improved therapeutic properties. Seeking novel AAV vectors capable of enhanced transduction for therapeutic applications, we have developed a series of unique capsid variants termed AAV X-Vivo (AAV-XV) derived from chimeras of AAV12 VP1/2 sequences and the VP3 sequence of AAV6. These AAV variants showed enhanced infection compared to the wild-type parental viruses of human primary T cells, hematopoietic stem cells (HSCs), and neuronal cell lines, and superiority over AAV6 for genomic integration of DNA sequences by AAV alone or in combination with CRISPR gene editing. AAV-XV variants demonstrate transduction efficiency equivalent to AAV6 but at 100-fold-lower multiplicities of infection (MOI), enabling T cell engineering at low AAV doses. The protein coding sequence of these novel AAV chimeras revealed disruptions within the assembly-activating protein (AAP), which likely accounted for the observed lower virus yield. A series of genome alterations, reverting the AAP sequence back to that of wild-type AAV6, had a negative impact on the enhanced transduction seen with AAV-VX, indicating overlapping functions within this sequence for both viral assembly and effective T cell transduction. Our findings show these AAV-XV variants are highly efficient at cell transduction at low doses and demonstrate the importance of the AAP coding region in both viral particle assembly and cell infection.  

5.3172           Localization of the WD Repeat-Containing Protein 5 to the Virion Assembly Compartment Facilitates Human Cytomegalovirus Assembly

Yang, B., Yao, Y., Wu, H., Yang, H., Ma, X-H., Li, D., Wang, X-Z., Huang, S-N. et al

1. Virol., 95(8), e02101-20 (2021)

  We previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WD repeat-containing protein 5 (WDR5) to facilitate capsid nuclear egress. Here, we further show that HCMV infection results in WDR5 localization in a juxtanuclear region and that its localization to this cellular site is associated with viral replication and late viral gene expression. Furthermore, WDR5 accumulated in the virion assembly compartment (vAC) and colocalized with vAC markers of γ-tubulin, early endosomes, and viral vAC marker proteins pp65, pp28, and glycoprotein B (gB). WDR5 coimmunoprecipitated with multiple virion proteins, including major capsid protein (MCP), pp150, pp65, pIRS1, and pTRS1, which may explain WDR5 accumulation in the vAC during infection. WDR5 fractionated with virions in either the presence or absence of Triton X-100 and was present in purified viral particles, suggesting that WDR5 was incorporated into HCMV virions. Thus, WDR5 localized to the vAC and was incorporated into virions, raising the possibility that in addition to capsid nuclear egress, WDR5 could also participate in cytoplasmic HCMV virion morphogenesis.  

5.3173           Respiratory Syncytial Virus Activates Rab5a To Suppress IRF1-Dependent Lambda Interferon Production, Subverting the Antiviral Defense of Airway Epithelial Cells

Mo, S., Tang, W., Xie, J., Chen, S., Ren, L., Zang, N., Xie, X., Deng, Y., Ggao, L. and Liu, E.

1. Virol., 95(8), e02333-20 (2021)

  he limited antiviral options and lack of an effective vaccine against human respiratory syncytial virus (RSV) highlight the need for a novel antiviral therapy. One alternative is to identify and target the host factors required for viral infection. Here, using RNA interference to knock down Rab proteins, we provide multiple lines of evidence that Rab5a is required for RSV infection: (i) Rab5a is upregulated both in RSV A2-infected A549 cells and RSV A2-challenged BALB/c mouse airway epithelial cells at early infection phase; (ii) short hairpin RNA (shRNA)-mediated knockdown of Rab5a is associated with reduced lung pathology in RSV A2-challenged mice; (iii) Rab5a expression is correlated with disease severity of RSV infection of infants. Knockdown of Rab5a increases lambda interferon (IFN-λ) production by mediating interferon regulatory factor 1 (IRF1) nuclear translocation. Our results highlight a new role for Rab5a in RSV infection, such that its depletion inhibits RSV infection by stimulating the endogenous respiratory epithelial antiviral immunity, which suggests that Rab5a is a potential target for novel therapeutics against RSV infection.  

5.3174           A Persistent Giant Algal Virus, with a Unique Morphology, Encodes an Unprecedented Number of Genes Involved in Energy Metabolism

Blanc-Mathieu, R., Dahle, H., Hofgaard, A., Brandt, D., Ban, H., Kalinowski, J., Ogata, H. and Sandaa, R-A.

1. Virol., 95(8), e02446-20 (2021)

  Viruses have long been viewed as entities possessing extremely limited metabolic capacities. Over the last decade, however, this view has been challenged, as metabolic genes have been identified in viruses possessing large genomes and virions—the synthesis of which is energetically demanding. Here, we unveil peculiar phenotypic and genomic features of Prymnesium kappa virus RF01 (PkV RF01), a giant virus of the Mimiviridae family. We found that this virus encodes an unprecedented number of proteins involved in energy metabolism, including all four succinate dehydrogenase (SDH) subunits (A to D), as well as key enzymes in the β-oxidation pathway. The SDHA gene was transcribed upon infection, indicating that the viral SDH is actively used by the virus, potentially to modulate its host’s energy metabolism. We detected orthologous SDHA and SDHB genes in numerous genome fragments from uncultivated marine Mimiviridae viruses, which suggests that the viral SDH is widespread in oceans. PkV RF01 was less virulent than other cultured prymnesioviruses, a phenomenon that may be linked to the metabolic capacity of this virus and is suggestive of relatively long coevolution with its hosts. It also has a unique morphology compared to those of other characterized viruses in the Mimiviridae family. Finally, we found that PkV RF01 is the only alga-infecting Mimiviridae virus encoding two aminoacyl-tRNA synthetases and enzymes corresponding to an entire base excision repair (BER) pathway, as seen in heterotroph-infecting Mimiviridae viruses. These Mimiviridae encoded-enzymes were found to be monophyletic and branching at the root of the eukaryotic tree of life. This placement suggests that the last common ancestor of Mimiviridae was endowed with a large, complex genome prior to the divergence of known extant eukaryotes.  

5.3175           SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their Requirements for Receptor Expression and Proteolytic Activation

Hörnich, B.F., Grosskopf, A.K., Schlagowski, S., Tenbusch, M., Kleine-Weber, H., Neipel, F., Stahl-Hennig, C. and Hahn, A.S.

1. Virol., 95(9), e00002-21 (2021)

  Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S (SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to that of SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector cell-target cell fusion when ACE2 or TMPRSS2 was limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target cell-effector cell fusion was unaltered compared to that of wild-type (wt) SARS2-S, but syncytia remained smaller. Mutation of the S2 cleavage (S2′) site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor bromhexine, unlike the inhibitor camostat, was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S. Paradoxically, bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite ambroxol exhibited inhibitory activity under some conditions. On Calu-3 lung cells, ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend toward weak inhibition of authentic SARS-CoV-2.  

5.3176           HIV-1 Uncoating Occurs via a Series of Rapid Biomechanical Changes in the Core Related to Individual Stages of Reverse Transcription

Rankovic, S., Deshpande, A., Hael, S., Aiken, C. and Rousso, I.

1. Virol., 95(10), e00166-21 (2021)

  The HIV core consists of the viral genome and associated proteins encased by a cone-shaped protein shell, termed the capsid. Successful infection requires reverse transcription of the viral genome and disassembly of the capsid shell within a cell in a process known as uncoating. The integrity of the viral capsid is critical for reverse transcription, yet the viral capsid must be breached to release the nascent viral DNA prior to integration. We employed atomic force microscopy to study the stiffness changes in HIV-1 cores during reverse transcription in vitro in reaction mixtures containing the capsid-stabilizing host metabolite IP₆. Cores exhibited a series of stiffness spikes, with up to three spikes typically occurring between 10 to 30, 40 to 80, and 120 to 160 min after the initiation of reverse transcription. The addition of the reverse transcriptase (RT) inhibitor efavirenz eliminated the appearance of these spikes and the subsequent disassembly of the capsid, establishing that both result from reverse transcription. Using the timed addition of efavirenz and analysis of an RNase H-defective RT mutant, we established that the first stiffness spike requires minus-strand strong stop DNA synthesis, with subsequent spikes requiring later stages of reverse transcription. Additional rapid atomic force microscopy imaging experiments revealed repeated morphological changes in cores that were temporally correlated with the observed stiffness spikes. Our study reveals discrete mechanical changes in the viral core that are likely related to specific stages of reverse transcription. These reverse transcription-induced changes in the capsid progressively remodel the viral core to prime it for temporally accurate uncoating in target cells.  

5.3177           Effects of Altering Heparan Sulfate Proteoglycan Binding and Capsid Hydrophilicity on Retinal Transduction by Adeno-associated Virus

Crosson, S.M., Bennett, A., Fajardo, D., Peterson, J.J., Zhang, H., Li, W., Leahy, M.T., Jennings, C.K., Boyd, R.F., Boye, S.L., Agbandje-McKenna, M. and Boye, S.E.

1. Virol., 95(10), e02440-20 (2021)

  Adeno-associated viruses (AAVs) have recently emerged as the leading vector for retinal gene therapy. However, AAV vectors that are capable of achieving clinically relevant levels of transgene expression and widespread retinal transduction are still an unmet need. Using rationally designed AAV2-based capsid variants, we investigate the role of capsid hydrophilicity and hydrophobicity as it relates to retinal transduction. We show that hydrophilic, single-amino-acid mutations (V387R, W502H, E530K, L583R) in AAV2 negatively impact retinal transduction when heparan sulfate proteoglycan (HSPG) binding remains intact. Conversely, addition of hydrophobic point mutations to an HSPG binding-deficient capsid (AAV2ΔHS) leads to increased retinal transduction in both mouse and macaque. Our top performing vector, AAV2(4pMut)ΔHS, achieved robust rod and cone photoreceptor (PR) transduction in macaque, especially in the fovea, and demonstrates the ability to spread laterally beyond the borders of the subretinal injection (SRI) bleb. This study both evaluates biophysical properties of AAV capsids that influence retinal transduction and assesses the transduction and tropism of a novel capsid variant in a clinically relevant animal model.  

5.3178           Activation of STING Signaling Pathway Effectively Blocks Human Coronavirus Infection

Liu, W., Reyes, H.M., Yang, J.F., Li, Y., Stewart, K.M., Basil, M.C., Lin, S.M., Katzen, J., Morrisey, E.E., Weiss, S.R. and You, J.

1. Virol., 95(12), e00490-21 (2021)

  The COVID-19 pandemic poses a serious global health threat. The rapid global spread of SARS-CoV-2 highlights an urgent need to develop effective therapeutics for blocking SARS-CoV-2 infection and spread. Stimulator of Interferon Genes (STING) is a chief element in host antiviral defense pathways. In this study, we examined the impact of the STING signaling pathway on coronavirus infection using the human coronavirus OC43 (HCoV-OC43) model. We found that HCoV-OC43 infection did not stimulate the STING signaling pathway, but the activation of STING signaling effectively inhibits HCoV-OC43 infection to a much greater extent than that of type I interferons (IFNs). We also discovered that IRF3, the key STING downstream innate immune effector, is essential for this anticoronavirus activity. In addition, we found that the amidobenzimidazole (ABZI)-based human STING agonist diABZI robustly blocks the infection of not only HCoV-OC43 but also SARS-CoV-2. Therefore, our study identifies the STING signaling pathway as a potential therapeutic target that could be exploited for developing broad-spectrum antiviral therapeutics against multiple coronavirus strains in order to face the challenge of future coronavirus outbreaks.  

5.3179           Multiple Viral microRNAs Regulate Interferon Release and Signaling Early during Infection with Epstein-Barr Virus

Bouvet, M., Voigt, S., Tagawa, T., Albanese, M., Chen, Y-F.A., Chen, Y., Fachko, D.N., Pich, D., Göbel, C., Skalsky, R.L. and Hammerschmidt, W. mBio, 12(2), e03440-20 (2021)   Epstein-Barr virus (EBV), a human herpesvirus, encodes 44 microRNAs (miRNAs), which regulate many genes with various functions in EBV-infected cells. Multiple target genes of the EBV miRNAs have been identified, some of which play important roles in adaptive antiviral immune responses. Using EBV mutant derivatives, we identified additional roles of viral miRNAs in governing versatile type I interferon (IFN) responses upon infection of human primary mature B cells. We also found that Epstein-Barr virus-encoded small RNAs (EBERs) and LF2, viral genes with previously reported functions in inducing or regulating IFN-I pathways, had negligible or even contrary effects on secreted IFN-α in our model. Data mining and Ago PAR-CLIP experiments uncovered more than a dozen previously uncharacterized, direct cellular targets of EBV miRNA associated with type I IFN pathways. We also identified indirect targets of EBV miRNAs in B cells, such as TRL7 and TLR9, in the prelatent phase of infection. The presence of epigenetically naive, non-CpG methylated viral DNA was essential to induce IFN-α secretion during EBV infection in a TLR9-dependent manner. In a newly established fusion assay, we verified that EBV virions enter a subset of plasmacytoid dendritic cells (pDCs) and determined that these infected pDCs are the primary producers of IFN-α in EBV-infected peripheral blood mononuclear cells. Our findings document that many EBV-encoded miRNAs regulate type I IFN response in newly EBV infected primary human B cells in the prelatent phase of infection and dampen the acute release of IFN-α in pDCs upon their encounter with EBV.  

5.3180           Two-Component Nanoparticle Vaccine Displaying Glycosylated Spike S1 Domain Induces Neutralizing Antibody Response against SARS-CoV-2 Variants

Van Oosten, L., Altenburg, J.J., Fougeroux, C., Geertsema, C., van den End, F., et al mBio, 12(5), e01813-21 (2021)   Vaccines pave the way out of the SARS-CoV-2 pandemic. Besides mRNA and adenoviral vector vaccines, effective protein-based vaccines are needed for immunization against current and emerging variants. We have developed a virus-like particle (VLP)-based vaccine using the baculovirus-insect cell expression system, a robust production platform known for its scalability, low cost, and safety. Baculoviruses were constructed encoding SARS-CoV-2 spike proteins: full-length S, stabilized secreted S, or the S1 domain. Since subunit S only partially protected mice from SARS-CoV-2 challenge, we produced S1 for conjugation to bacteriophage AP205 VLP nanoparticles using tag/catcher technology. The S1 yield in an insect-cell bioreactor was ∼11 mg/liter, and authentic protein folding, efficient glycosylation, partial trimerization, and ACE2 receptor binding was confirmed. Prime-boost immunization of mice with 0.5 μg S1-VLPs showed potent neutralizing antibody responses against Wuhan and UK/B.1.1.7 SARS-CoV-2 variants. This two-component nanoparticle vaccine can now be further developed to help alleviate the burden of COVID-19.  

5.3181           Histone Modifications in Papillomavirus Virion Minichromosomes

Porter, S.S., Liddle, J.C., Browne, K., Pastrana, D.V., Garcia, B.A., Buck, C.B., Weitzman, M.D. and Mcbride, A.A. mBio, 12(1), e03274-20 (2021)   An unusual feature of papillomaviruses is that their genomes are packaged into virions along with host histones. Viral minichromosomes were visualized as “beads on a string” by electron microscopy in the 1970s but, to date, little is known about the posttranslational modifications of these histones. To investigate this, we analyzed the histone modifications in HPV16/18 quasivirions, wart-derived bovine papillomavirus (BPV1), and wart-derived human papillomavirus type 1 (HPV1) using quantitative mass spectrometry. The chromatin from all three virion samples had abundant posttranslational modifications (acetylation, methylation, and phosphorylation). These histone modifications were verified by acid urea polyacrylamide electrophoresis and immunoblot analysis. Compared to matched host cell controls, the virion minichromosome was enriched in histone modifications associated with active chromatin and depleted for those commonly found in repressed chromatin. We propose that the viral minichromosome acquires specific histone modifications late in infection that are coupled to the mechanisms of viral replication, late gene expression, and encapsidation. We predict that, in turn, these same modifications benefit early stages of infection by helping to evade detection, promoting localization of the viral chromosome to beneficial regions of the nucleus, and promoting early transcription and replication.  

5.3182           Structure-Guided Design of a Synthetic Mimic of an Endothelial Protein C Receptor-Binding PfEMP1 Protein

Barber, N.M., Lau, C.K.Y., Turner, L., Watson, G., Thrane, S., Lusingu, J.P.A., Lavstsen, T. and Higgins, M.K. mSphere, 6(1), e01081-20 (2021)   Structure-guided vaccine design provides a route to elicit a focused immune response against the most functionally important regions of a pathogen surface. This can be achieved by identifying epitopes for neutralizing antibodies through structural methods and recapitulating these epitopes by grafting their core structural features onto smaller scaffolds. In this study, we conducted a modified version of this protocol. We focused on the PfEMP1 protein family found on the surfaces of erythrocytes infected with Plasmodium falciparum. A subset of PfEMP1 proteins bind to endothelial protein C receptor (EPCR), and their expression correlates with development of the symptoms of severe malaria. Structural studies revealed that PfEMP1 molecules present a helix-kinked-helix motif that forms the core of the EPCR-binding site. Using Rosetta-based design, we successfully grafted this motif onto a three-helical bundle scaffold. We show that this synthetic binder interacts with EPCR with nanomolar affinity and adopts the expected structure. We also assessed its ability to bind to antibodies found in immunized animals and in humans from malaria-endemic regions. Finally, we tested the capacity of the synthetic binder to effectively elicit antibodies that prevent EPCR binding and analyzed the degree of cross-reactivity of these antibodies across a diverse repertoire of EPCR-binding PfEMP1 proteins. Despite our synthetic binder adopting the correct structure, we find that it is not as effective as the CIDRα domain on which it is based for inducing adhesion-inhibitory antibodies. This cautions against the rational design of focused immunogens that contain the core features of a ligand-binding site of a protein family, rather than those of a neutralizing antibody epitope.  

5.3183           RanDeL-Seq: a High-Throughput Method to Map Viral cis- and trans-Acting Elements

Notton, T., Glazier, J.J., Saykally, V.R., Thompson, C.E. and Weinberger, L.S. mBio, 12(1), e01724-20 (2021)   It has long been known that noncoding genomic regions can be obligate cis elements acted upon in trans by gene products. In viruses, cis elements regulate gene expression, encapsidation, and other maturation processes, but mapping these elements relies on targeted iterative deletion or laborious prospecting for rare spontaneously occurring mutants. Here, we introduce a method to comprehensively map viral cis and trans elements at single-nucleotide resolution by high-throughput random deletion. Variable-size deletions are randomly generated by transposon integration, excision, and exonuclease chewback and then barcoded for tracking via sequencing (i.e., random deletion library sequencing [RanDeL-seq]). Using RanDeL-seq, we generated and screened >23,000 HIV-1 variants to generate a single-base resolution map of HIV-1’s cis and trans elements. The resulting landscape recapitulated HIV-1’s known cis-acting elements (i.e., long terminal repeat [LTR], Ψ, and Rev response element [RRE]) and, surprisingly, indicated that HIV-1’s central DNA flap (i.e., central polypurine tract [cPPT] to central termination sequence [CTS]) is as critical as the LTR, Ψ, and RRE for long-term passage. Strikingly, RanDeL-seq identified a previously unreported ∼300-bp region downstream of RRE extending to splice acceptor 7 that is equally critical for sustained viral passage. RanDeL-seq was also used to construct and screen a library of >90,000 variants of Zika virus (ZIKV). Unexpectedly, RanDeL-seq indicated that ZIKV’s cis-acting regions are larger than the untranscribed (UTR) termini, encompassing a large fraction of the nonstructural genes. Collectively, RanDeL-seq provides a versatile framework for generating viral deletion mutants, enabling discovery of replication mechanisms and development of novel antiviral therapeutics, particularly for emerging viral infections.  

5.3184           Contributions of the Four Essential Entry Glycoproteins to HSV-1 Tropism and the Selection of Entry Routes

Hilterbrand, A.T., Daly, R.E. and Heldwein, E.E. mBio, 12(2), e00143-21 (2021)   Herpes simplex viruses (HSV-1 and HSV-2) encode up to 16 envelope proteins, four of which are essential for entry. However, whether these four proteins alone are sufficient to dictate the broad cellular tropism of HSV-1 and the selection of different cell type-dependent entry routes is unknown. To begin addressing this, we previously pseudotyped vesicular stomatitis virus (VSV), lacking its native glycoprotein G, with only the four essential entry glycoproteins of HSV-1: gB, gH, gL, and gD. This novel VSVΔG-BHLD pseudotype recapitulated several important features of HSV-1 entry: the requirement for gB, gH, gL, gD, and a cellular receptor and sensitivity to anti-gB and anti-gH/gL neutralizing antibodies. However, due to the use of a single cell type in that study, the tropism of the VSVΔG-BHLD pseudotype was not investigated. Here, we show that the cellular tropism of the pseudotype is severely limited compared to that of wild-type HSV-1 and that its entry pathways differ from the native HSV-1 entry pathways. To test the hypothesis that other HSV-1 envelope proteins may contribute to HSV-1 tropism, we generated a derivative pseudotype containing the HSV-1 glycoprotein C (VSVΔG-BHLD-gC) and observed a gC-dependent increase in entry efficiency in two cell types. We propose that the pseudotyping platform developed here has the potential to uncover functional contributions of HSV-1 envelope proteins to entry in a gain-of-function manner.  

5.3185           Development of a New Reverse Genetics System for Ebola Virus

Gan, T., Zhou, D., Huang, Y., Xiao, S., Ma, Z., Hu, X., Tong, Y., Yan, H. and Zhong, J. mSphere, 6(3), e00235-21 (2021)   Ebola virus (EBOV) is a highly pathogenic negative-stranded RNA virus that has caused several deadly endemics in the past decades. EBOV reverse genetics systems are available for studying live viruses under biosafety level 4 (BSL-4) or subviral particles under BSL-2 conditions. However, these systems all require cotransfection of multiple plasmids expressing viral genome and viral proteins essential for EBOV replication, which is technically challenging and unable to naturally mimic virus propagation using the subviral particle. Here, we established a new EBOV reverse genetics system only requiring transfection of a single viral RNA genome into an engineered cell line that stably expresses viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and large (L) proteins and has been fine-tuned for its superior permissiveness for EBOV replication. Using this system, subviral particles expressing viral VP40, glycoprotein (GP), and VP24 could be produced and continuously propagated and eventually infect the entire cell population. We demonstrated the authentic response of the subviral system to antivirals and uncovered that the VP35 amount is critical for optimal virus replication. Furthermore, we showed that fully infectious virions can be efficiently rescued by delivering the full-length EBOV genome into the same supporting cell, and the efficiency is not affected by genome polarity or virus variant specificity. In summary, our work provides a new tool for studying EBOV under different biosafety levels.  

5.3186           Tau assemblies do not behave like independently acting prion-like particles in mouse neural tissue

Miller, L.V.C., Mukadam, A.S., Durrant, C.S., Vayburd, M.J., Katsinelos, T., Tuck, B.J., Sanford, S., Sheppard, O., Knox, C., Cheng, S., James, L.C., Coleman, M.P. and McEwan, W.A. Acta Neuuropathol. Comm., 9:41 (2021)   A fundamental property of infectious agents is their particulate nature: infectivity arises from independently-acting particles rather than as a result of collective action. Assemblies of the protein tau can exhibit seeding behaviour, potentially underlying the apparent spread of tau aggregation in many neurodegenerative diseases. Here we ask whether tau assemblies share with classical pathogens the characteristic of particulate behaviour. We used organotypic hippocampal slice cultures from P301S tau transgenic mice in order to precisely control the concentration of extracellular tau assemblies in neural tissue. Whilst untreated slices displayed no overt signs of pathology, exposure to recombinant tau assemblies could result in the formation of intraneuronal, hyperphosphorylated tau structures. However, seeding ability of tau assemblies did not titrate in a one-hit manner in neural tissue. The results suggest that seeding behaviour of tau arises at high concentrations, with implications for the interpretation of high-dose intracranial challenge experiments and the possible contribution of seeded aggregation to human disease.  

5.3187           Hsp90 co-chaperones, FKBP52 and Aha1, promote tau pathogenesis in aged wild-type mice

Criado-Marrero, M., Gebru, N.T., Blazier, D.M., Gould, L.A., Baker, J.D., Beaulieu-Abdelahad, D. and Blair, L.J. Acta Neuropathol. Comm., 9:65 (2021)   The microtubule associated protein tau is an intrinsically disordered phosphoprotein that accumulates under pathological conditions leading to formation of neurofibrillary tangles, a hallmark of Alzheimer’s disease (AD). The mechanisms that initiate the accumulation of phospho-tau aggregates and filamentous deposits are largely unknown. In the past, our work and others’ have shown that molecular chaperones play a crucial role in maintaining protein homeostasis and that imbalance in their levels or activity can drive tau pathogenesis. We have found two co-chaperones of the 90 kDa heat shock protein (Hsp90), FK506-binding protein 52 (FKBP52) and the activator of Hsp90 ATPase homolog 1 (Aha1), promote tau aggregation in vitro and in the brains of tau transgenic mice. Based on this, we hypothesized that increased levels of these chaperones could promote tau misfolding and accumulation in the brains of aged wild-type mice. We tested this hypothesis by overexpressing Aha1, FKBP52, or mCherry (control) proteins in the hippocampus of 9-month-old wild-type mice. After 7 months of expression, mice were evaluated for cognitive and pathological changes. Our results show that FKBP52 overexpression impaired spatial reversal learning, while Aha1 overexpression impaired associative learning in aged wild-type mice. FKBP52 and Aha1 overexpression promoted phosphorylation of distinct AD-relevant tau species. Furthermore, FKBP52 activated gliosis and promoted neuronal loss leading to a reduction in hippocampal volume. Glial activation and phospho-tau accumulation were also detected in areas adjacent to the hippocampus, including the entorhinal cortex, suggesting that after initiation these pathologies can propagate through other brain regions. Overall, our findings suggest a role for chaperone imbalance in the initiation of tau accumulation in the aging brain.  

5.3188           Receptor-mediated endocytosis 8 (RME-8)/DNAJC13 is a novel positive modulator of autophagy and stabilizes cellular protein homeostasis

Besemer, A.S., Maus, J., Ax, M.D.A., Stein, A., Vo, S., Freese, C. et al Cell. Mol. Life Sci., 78, 645-660 (2021)   The cellular protein homeostasis (proteostasis) network responds effectively to insults. In a functional screen in C. elegans, we recently identified the gene receptor-mediated endocytosis 8 (rme-8; human ortholog: DNAJC13) as a component of the proteostasis network. Accumulation of aggregation-prone proteins, such as amyloid-β 42 (Aβ), α-synuclein, or mutant Cu/Zn-superoxide dismutase (SOD1), were aggravated upon the knockdown of rme-8/DNAJC13 in C. elegans and in human cell lines, respectively. DNAJC13 is involved in endosomal protein trafficking and associated with the retromer and the WASH complex. As both complexes have been linked to autophagy, we investigated the role of DNAJC13 in this degradative pathway. In knockdown and overexpression experiments, DNAJC13 acts as a positive modulator of autophagy. In contrast, the overexpression of the Parkinson’s disease-associated mutant DNAJC13(N855S) did not enhance autophagy. Reduced DNAJC13 levels affected ATG9A localization at and its transport from the recycling endosome. As a consequence, ATG9A co-localization at LC3B-positive puncta under steady-state and autophagy-induced conditions is impaired. These data demonstrate a novel function of RME-8/DNAJC13 in cellular homeostasis by modulating ATG9A trafficking and autophagy.  

5.3189           The functional characteristics of optogenetic gene therapy for vision restoration

Lindner, M., Gilhooley, M.J., Peirson, S.N., Hughes, S. and Hankins, M.W. Cell. Mol. Life Sci., 78, 1597-1613 (2021)   Optogenetic strategies to restore vision in patients blind from end-stage retinal degenerations aim to render remaining retinal neurons light-sensitive. We present an innovative combination of multi-electrode array recordings together with a complex pattern-generating light source as a toolset to determine the extent to which neural retinal responses to complex light stimuli can be restored following viral delivery of red-shifted channelrhodopsin in the retinally degenerated mouse. Our data indicate that retinal output level spatiotemporal response characteristics achieved by optogenetic gene therapy closely parallel those observed for normal mice but equally reveal important limitations, some of which could be mitigated using bipolar-cell targeted gene-delivery approaches. As clinical trials are commencing, these data provide important new information on the capacity and limitations of channelrhodopsin-based gene therapies. The toolset we established enables comparing optogenetic constructs and stem-cell-based techniques, thereby providing an efficient and sensitive starting point to identify future approaches for vision restoration.  

5.3190           Plaque associated microglia hyper-secrete extracellular vesicles and accelerate tau propagation in a humanized APP mouse model

Clayton, K., Delpech, J.C., herron, S., Iwahara, N., Ericsson, M., Saito, T., Saido, T.C., Ikezu, S. and Ikezu, T. Mol. Neurodegeneration, 16:18 (2021)   Background Recent studies suggest that microglia contribute to tau pathology progression in Alzheimer’s disease. Amyloid plaque accumulation transforms microglia, the primary innate immune cells in the brain, into neurodegenerative microglia (MGnD), which exhibit enhanced phagocytosis of plaques, apoptotic neurons and dystrophic neurites containing aggregated and phosphorylated tau (p-tau). It remains unclear how microglia promote disease progression while actively phagocytosing pathological proteins, therefore ameliorating pathology. Methods Adeno-associated virus expressing P301L tau mutant (AAV-P301L-tau) was stereotaxically injected into the medial entorhinal cortex (MEC) in C57BL/6 (WT) and humanized APP mutant knock-in homozygote (App^NL-G-F) mice at 5 months of age. Mice were fed either chow containing a colony stimulating factor-1 receptor inhibitor (PLX5622) or control chow from 4 to 6 months of age to test the effect of microglia depletion. Animals were tested at 6 months of age for immunofluorescence, biochemistry, and FACS of microglia. In order to monitor microglial extracellular vesicle secretion in vivo, a novel lentiviral EV reporter system was engineered to express mEmerald-CD9 (mE-CD9) specifically in microglia, which was injected into the same region of MEC. Results Expressing P301L tau mutant in the MEC induced tau propagation to the granule cell layer of the hippocampal dentate gyrus, which was significantly exacerbated in App^NL-G-F mice compared to WT control mice. Administration of PLX5622 depleted nearly all microglia in mouse brains and dramatically reduced propagation of p-tau in WT and to a greater extent in App^NL-G-F mice, although it increased plaque burden and plaque-associated p-tau⁺ dystrophic neurites. Plaque-associated MGnD microglia strongly expressed an EV marker, tumor susceptibility gene 101, indicative of heightened synthesis of EVs. Intracortical injection of mE-CD9 lentivirus successfully induced microglia-specific expression of mE-CD9⁺ EV particles, which were significantly enhanced in Mac2⁺ MGnD microglia compared to Mac2⁻ homeostatic microglia. Finally, consecutive intracortical injection of mE-CD9 lentivirus and AAV-P301L-tau into App^NL-G-F mice revealed encapsulation of p-tau in microglia-specific mE-CD9⁺ EVs as determined by super-resolution microscopy and immuno-electron microscopy. Discussion Our findings suggest that MGnD microglia hyper-secrete p-tau⁺ EVs while compacting Aβ plaques and clearing NP tau, which we propose as a novel mechanistic link between amyloid plaque deposition and exacerbation of tau propagation in App^NL-G-F mice.  

5.3191           Modulating innate immune activation states impacts the efficacy of specific Aβ immunotherapy

Levites, Y., Funk, C., Wang, X., Chakrabarty, P., McFarland, K.N: et al Mol. Neurodegeneration, 16:32 (2021)   Introduction Passive immunotherapies targeting Aβ continue to be evaluated as Alzheimer’s disease (AD) therapeutics, but there remains debate over the mechanisms by which these immunotherapies work. Besides the amount of preexisting Aβ deposition and the type of deposit (compact or diffuse), there is little data concerning what factors, independent of those intrinsic to the antibody, might influence efficacy. Here we (i) explored how constitutive priming of the underlying innate activation states by Il10 and Il6 might influence passive Aβ immunotherapy and (ii) evaluated transcriptomic data generated in the AMP-AD initiative to inform how these two cytokines and their receptors’ mRNA levels are altered in human AD and an APP mouse model. Methods rAAV2/1 encoding EGFP, Il6 or Il10 were delivered by somatic brain transgenesis to neonatal (P0) TgCRND8 APP mice. Then, at 2 months of age, the mice were treated bi-weekly with a high-affinity anti-Aβ1–16 mAb5 monoclonal antibody or control mouse IgG until 6 months of age. rAAV mediated transgene expression, amyloid accumulation, Aβ levels and gliosis were assessed. Extensive transcriptomic data was used to evaluate the mRNA expression levels of IL10 and IL6 and their receptors in the postmortem human AD temporal cortex and in the brains of TgCRND8 mice, the later at multiple ages. Results Priming TgCRND8 mice with Il10 increases Aβ loads and blocks efficacy of subsequent mAb5 passive immunotherapy, whereas priming with Il6 priming reduces Aβ loads by itself and subsequent Aβ immunotherapy shows only a slightly additive effect. Transcriptomic data shows that (i) there are significant increases in the mRNA levels of Il6 and Il10 receptors in the TgCRND8 mouse model and temporal cortex of humans with AD and (ii) there is a great deal of variance in individual mouse brain and the human temporal cortex of these interleukins and their receptors. Conclusions The underlying immune activation state can markedly affect the efficacy of passive Aβ immunotherapy. These results have important implications for ongoing human AD immunotherapy trials, as they indicate that underlying immune activation states within the brain, which may be highly variable, may influence the ability for passive immunotherapy to alter Aβ deposition.  

5.3192           Interleukin-9 promotes intestinal barrier injury of sepsis: a translational research

Sun, J-K., Zhou, J., Sun, X-P., Shen, X., Zhu, D-M., Wang, X., Zhou, S-M. and Mu, X-W.

1. Intensive Care, 9:37 (2021)

  Background Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Intestinal mucosal barrier injury is one of the important manifestations of sepsis. Interleukin-9 (IL-9) and IL-9-producing CD4(+) T cells were emerging pro-inflammatory mediators with development of intestinal injury. However, it is unclear whether IL-9 is related to the intestinal barrier injury of sepsis. Methods To investigate the roles of IL-9-producing CD4(+) T cells and IL-9 in the process of barrier injury in sepsis, serum IL-9-producing CD4(+) T cell percentages, IL-9, and D-lactate levels were measured in septic patients and controls. The markers of barrier function in serum and intestinal tissue were also collected in septic rats. Moreover, the barrier injury degree and survival rate of septic rats were also investigated after increasing or interfering with IL-9 expression. Results The serum IL-9-producing CD4(+) T cell percentages, IL-9, and D-lactate levels were significantly higher in septic patients or rats than those in controls. IL-9-producing CD4(+) T cells and IL-9 levels were positively correlated with D-lactate levels and had a high predictive value of 28-day mortality in septic patients. The non-survivors had significantly higher serum T cell percentages, IL-9, and D-lactate levels compared with survivors. In septic rats, IL-9 increased the expression levels of D-lactate, whereas that decreased the expression levels of zonula occludens 1. Moreover, the barrier injury was aggravated or alleviated by increasing or interfering with IL-9 expression, respectively. Survival rate analysis also showed that IL-9 decreased the 14-day survival rate of septic rats. Conclusion IL-9 is closely related to intestinal mucosal barrier injury and mortality in sepsis. IL-9 blockade has the potential to improve the barrier injury in sepsis.  

5.3193           LRRC4 functions as a neuron-protective role in experimental autoimmune encephalomyelitis

Zhang, Y., Li, D., Zeng, Q., Feng, J., Fu, H., Luo, Z., Xiao, B., Yang, H. and Wu, M. Mol. Med., 27:44 (2021)   Background Leucine rich repeat containing 4 (LRRC4), also known as netrin-G ligand-2 (NGL-2), belongs to the superfamily of LRR proteins and serves as a receptor for netrin-G2. LRRC4 regulates the formation of excitatory synapses and promotes axon differentiation. Mutations in LRRC4 occur in Autism Spectrum Disorder (ASD) and intellectual disability. Multiple sclerosis (MS) is a chronic neuroinflammatory disease with spinal cords demyelination and neurodegeneration. Here, we sought to investigate whether LRRC4 is involved in spinal cords neuron-associated diseases. Methods LRRC4 was detected in the CNS of experimental autoimmune encephalomyelitis (EAE) mice by the use of real-time PCR and western blotting. LRRC4^−/− mice were created and immunized with myelin oligodendrocyte glycoprotein peptide (MOG)_35–55. Pathological changes in spinal cords of LRRC4^−/− and WT mice 15 days after immunization were examined by using hematoxylin and eosin (H&E), Luxol Fast Blue (LFB) staining and immunohistochemistry. The number of Th1/Th2/Th17/Treg cells in spleens and blood were measured with flow cytometry. Differential gene expression in the spinal cords from WT and LRRC4^−/− mice was analyzed by using RNA sequencing (RNA-seq). Adeno-associated virus (AAV) vectors were used to overexpress LRRC4 (AAV-LRRC4) and were injected into EAE mice to assess the therapeutic effect of AAV-LRRC4 ectopic expression on EAE. Results We report that LRRC4 is mainly expressed in neuron of spinal cords, and is decreased in the spinal cords of the EAE mice. Knockout of LRRC4 have a disease progression quickened and exacerbated with more severe myelin degeneration and infiltration of leukocytes into the spinal cords. We also first found that Rab7b is high expressed in EAE mice, and the deficiency of LRRC4 induces the elevated NF-κB p65 by up-regulating Rab7b, and up-regulation of IL-6, IFN-γ and down-regulation of TNF-α, results in more severe Th1 immune response in LRRC4^−/− mice. Ectopic expression of LRRC4 alleviates the clinical symptoms of EAE mice and protects the neurons from immune damages. Conclusions We identified a neuroprotective role of LRRC4 in the progression of EAE, which may be used as a potential target for auxiliary support therapeutic treatment of MS.  

5.3194           Development of an AAV9-RNAi-mediated silencing strategy to abrogate TRPM4 expression in the adult heart

Medert, R., Jungmann, A., Hildesbrand, S., Busch, M., Grimm, D., Flockerzi, V., Müller, O.J., Most, P., Schumacher, D. and Freichel, M. Pflügers Arch-Eur. J. Physiol., 473, 533-546 (2021)   The cation channel transient receptor potential melastatin 4 (TRPM4) is a calcium-activated non-selective cation channel and acts in cardiomyocytes as a negative modulator of the L-type Ca²⁺ influx. Global deletion of TRPM4 in the mouse led to increased cardiac contractility under β-adrenergic stimulation. Consequently, cardiomyocyte-specific inactivation of the TRPM4 function appears to be a promising strategy to improve cardiac contractility in heart failure patients. The aim of this study was to develop a gene therapy approach in mice that specifically silences the expression of TRPM4 in cardiomyocytes. First, short hairpin RNA^miR30 (shRNA^miR30) sequences against the TRPM4 mRNA were screened in vitro using lentiviral transduction for a stable expression of the shRNA cassettes. Western blot analysis identified three efficient shRNA^miR30 sequences out of six, which reduced the endogenous TRPM4 protein level by up to 90 ± 6%. Subsequently, the most efficient shRNA^miR30 sequences were delivered into cardiomyocytes of adult mice using adeno-associated virus serotype 9 (AAV9)-mediated gene transfer. Initially, the AAV9 vector particles were administered via the lateral tail vein, which resulted in a downregulation of TRPM4 by 46 ± 2%. Next, various optimization steps were carried out to improve knockdown efficiency in vivo. First, the design of the expression cassette was streamlined for integration in a self-complementary AAV vector backbone for a faster expression. Compared to the application via the lateral tail vein, intravenous application via the retro-orbital sinus has the advantage that the vector solution reaches the heart directly and in a high concentration, and eventually a TRPM4 knockdown efficiency of 90 ± 7% in the heart was accomplished by this approach. By optimization of the shRNA^miR30 constructs and expression cassette as well as the route of AAV9 vector application, a 90% reduction of TRPM4 expression was achieved in the adult mouse heart. In the future, AAV9-RNAi-mediated inactivation of TRPM4 could be a promising strategy to increase cardiac contractility in preclinical animal models of acute and chronic forms of cardiac contractile failure.  

5.3195           CERTL reduces C16 ceramide, amyloid-β levels, and inflammation in a model of Alzheimer’s disease

Civelli, S.M., Luo, Q., Stevens, J.A.A., Giovagnoni, C., van Kruining, D. et al Alzheimer’s Res. Ther., 13:45 (2021)   Background Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer’s disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain. Methods A plasmid expressing CERT_L, the long isoform of CERTs, was used to study the interaction of CERT_L with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERT_L protein was employed to study interaction of CERT_L with amyloid-β (Aβ), Aβ aggregation process in presence of CERT_L, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERT_L was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay. Results Here, we report that CERT_L binds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERT_L, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERT_L in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype. Conclusion Our results demonstrate a crucial role of CERT_L in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases.  

5.3196           GABAergic neuron-specific whole-brain transduction by AAV-PHP.B incorporated with a new GAD65 promoter

Hoshino, C., Konno, A., Hosoi, A., Hosoi, N., Kaneko, R., Mukai, R., Naka, J. and Hirai, H. Mol. Brain., 14:33 (2021)   GABAergic interneurons play a critical role in tuning neural networks in the central nervous system, and their defects are associated with neuropsychiatric disorders. Currently, the mDlx enhancer is solely used for adeno-associated virus (AAV) vector-mediated transgene delivery into cortical interneurons. Here, we developed a new inhibitory neuron-specific promoter (designated as the mGAD65 promoter), with a length of 2.5 kb, from a mouse genome upstream of exon 1 of the Gad2 gene encoding glutamic acid decarboxylase (GAD) 65. Intravenous infusion of blood–brain barrier-penetrating AAV-PHP.B expressing an enhanced green fluorescent protein under the control of the mGAD65 promoter transduced the whole brain in an inhibitory neuron-specific manner. The specificity and efficiency of the mGAD65 promoter for GABAergic interneurons, which was assessed at the motor cortex, were almost identical to or slightly higher than those of the mDlx enhancer. Immunohistochemical analysis revealed that the mGAD65 promoter preferentially transduced parvalbumin (PV)-expressing interneurons. Notably, the mGAD65 promoter transduced chandelier cells more efficiently than the mDlx enhancer and robustly labeled their synaptic boutons, called the cartridge, targeting the axon initial segments of excitatory pyramidal neurons. To test the ability of the mGAD65 promoter to express a functional molecule, we virally expressed G-CaMP, a fluorescent Ca²⁺ indicator, in the motor cortex, and this enabled us to monitor spontaneous and drug-induced Ca²⁺ activity in GABAergic inhibitory neurons. These results suggest that the mGAD65 promoter is useful for AAV-mediated targeting and manipulation of GABAergic neurons with the dominance of cortical PV-expressing neurons, including chandelier cells.  

5.3197           Rabbit haemorrhagic disease virus Lagovirus europaeus/GI.1d strain: genome sequencing, in vivo virus replication kinetics, and viral dose effect

Droillard, C., Lemaitre, E., Amelot, M., Blanchard, Y., Keita, A., Eterradossi, N. and Gall-Recule, G.L. BMC Vet. Res., 17:257 (2021)   Background Rabbit haemorrhagic disease virus Lagovirus europaeus/GI.1d variant (GI.1d/RHDV) was identified in 1990 in France, and until the emergence of the new genotype GI.2, it was the main variant circulating in the country. The early stages of RHDV infection have been described in a few studies of rabbits experimentally infected with earlier strains, but no information was given on the minimum infective dose. We report the genomic and phenotypic characterisation of a GI.1d/RHDV strain collected in 2000 in France (GI.1d/00–21). Results We performed in vivo assays in rabbits to study virus replication kinetics in several tissues at the early stage of infection, and to estimate the minimum infective dose. Four tested doses, negligible (10^− 1 viral genome copies), low (10⁴), high (10⁷) and very high (10¹¹) were quantified using a method combining density gradient centrifugation of the viral particles and an RT-qPCR technique developed to quantify genomic RNA (gRNA). The GI.1d/00–21 genome showed the same genomic organisation as other lagoviruses; however, a substitution in the 5′ untranslated region and a change in the potential p23/2C-like helicase cleavage site were observed. We showed that the liver of one of the two rabbits inoculated via the oral route was infected at 16 h post-infection and all tissues at 39 h post-infection. GI.1d/00–21 induced classical RHD signs (depression) and lesions (haemorrhage and splenomegaly). Although infective dose estimation should be interpreted with caution, the minimum infective dose that infected an inoculated rabbit was lower or equal to 10⁴ gRNA copies, whereas between 10⁴ and 10⁷ gRNA copies were required to also induce mortality. Conclusions These results provide a better understanding of GI.1d/RHDV infection in rabbits. The genome analysis showed a newly observed mutation in the 5′ untranslated region of a lagovirus, whose role remains unknown. The phenotypic analysis showed that the pathogenicity of GI.1d/00–21 and the replication kinetics in infected organs were close to those reported for the original GI.1 strains, and could not alone explain the observed selective advantage of the GI.1d strains. Determining the minimum dose of viral particles required to cause mortality in rabbits is an important input for in vivo studies.  

5.3198           Altered Env conformational dynamics as a mechanism of resistance to peptide-triazole HIV-1 inactivators

Zhang, S., Holmes, A.P., Dick, A., Rashad, A.A., Enriquez-Rodriguez, L., Canziani, G.A., Root, M.J. and Chaiken, I.M. Retrovirology, 18:31 (2021)   Background We previously developed drug-like peptide triazoles (PTs) that target HIV-1 Envelope (Env) gp120, potently inhibit viral entry, and irreversibly inactivate virions. Here, we investigated potential mechanisms of viral escape from this promising class of HIV-1 entry inhibitors. Results HIV-1 resistance to cyclic (AAR029b) and linear (KR13) PTs was obtained by dose escalation in viral passaging experiments. High-level resistance for both inhibitors developed slowly (relative to escape from gp41-targeted C-peptide inhibitor C37) by acquiring mutations in gp120 both within (Val255) and distant to (Ser143) the putative PT binding site. The similarity in the resistance profiles for AAR029b and KR13 suggests that the shared IXW pharmacophore provided the primary pressure for HIV-1 escape. In single-round infectivity studies employing recombinant virus, V255I/S143N double escape mutants reduced PT antiviral potency by 150- to 3900-fold. Curiously, the combined mutations had a much smaller impact on PT binding affinity for monomeric gp120 (four to ninefold). This binding disruption was entirely due to the V255I mutation, which generated few steric clashes with PT in molecular docking. However, this minor effect on PT affinity belied large, offsetting changes to association enthalpy and entropy. The escape mutations had negligible effect on CD4 binding and utilization during entry, but significantly altered both binding thermodynamics and inhibitory potency of the conformationally-specific, anti-CD4i antibody 17b. Moreover, the escape mutations substantially decreased gp120 shedding induced by either soluble CD4 or AAR029b. Conclusions Together, the data suggest that the escape mutations significantly modified the energetic landscape of Env’s prefusogenic state, altering conformational dynamics to hinder PT-induced irreversible inactivation of Env. This work therein reveals a unique mode of virus escape for HIV-1, namely, resistance by altering the intrinsic conformational dynamics of the Env trimer.  

5.3199           Metabolic labeling of enterovirus 71 with quantum dots for the study of virus receptor usage

Ke, X., Li, C., Luo, D., Wang, T., Liu, Y., Tan, Z., Du, M., He, Z., Wang, H., Zheng, Z. and Zhang, Y.

1. Nanobiotechnol., 19:295 (2021)

  Fluorescent labeling and dynamic tracking is a powerful tool for exploring virus infection mechanisms. However, for small-sized viruses, virus tracking studies are usually hindered by a lack of appropriate labeling methods that do not dampen virus yield or infectivity. Here, we report a universal strategy for labeling viruses with chemical dyes and Quantum dots (QDs). Enterovirus 71 (EV71) was produced in a cell line that stably expresses a mutant methionyl-tRNA synthetase (MetRS), which can charge azidonorleucine (ANL) to the methionine sites of viral proteins during translation. Then, the ANL-containing virus was easily labeled with DBCO-AF647 and DBCO-QDs. The labeled virus shows sufficient yield and no obvious decrease in infectivity and can be used for imaging the virus entry process. Using the labeled EV71, different functions of scavenger receptor class B, member 2 (SCARB2), and heparan sulfate (HS) in EV71 infection were comparatively studied. The cell entry process of a strong HS-binding EV71 strain was investigated by real-time dynamic visualization of EV71-QDs in living cells. Taken together, our study described a universal biocompatible virus labeling method, visualized the dynamic viral entry process, and reported details of the receptor usage of EV71.  

5.3200           Immunization with Leishmania tarentolae-derived norovirus virus-like particles elicits high humoral response and stimulates the production of neutralizing antibodies

Panasiuk, M., Zimmer, K., Czarnota, A., Grzyb, K., Narajczyk, M., Peszynska-Sularz, G., Zoledowska, S., Nidzworski, D., Hovhannisyan, L, and Gromadzka, B. Microbial Cell factories, 20:186 (2021)   Background Noroviruses are a major cause of epidemic and sporadic acute non-bacterial gastroenteritis worldwide. Unfortunately, the development of an effective norovirus vaccine has proven difficult and no prophylactic vaccine is currently available. Further research on norovirus vaccine development should be considered an absolute priority and novel vaccine candidates are needed. One of the recent approaches in safe vaccine development is the use of virus-like particles (VLPs). VLP-based vaccines show great immunogenic potential as they mimic the morphology and structure of viral particles without the presence of the virus genome. Results This study is the first report showing successful production of norovirus VLPs in the protozoan Leishmania tarentolae (L. tarentolae) expression system. Protozoan derived vaccine candidate is highly immunogenic and able to not only induce a strong immune response (antibody titer reached 10⁴) but also stimulate the production of neutralizing antibodies confirmed by receptor blocking assay. Antibody titers able to reduce VLP binding to the receptor by > 50% (BT₅₀) were observed for 1:5–1:320 serum dilutions. Conclusions Norovirus VLPs produced in L. tarentolae could be relevant for the development of the norovirus vaccine.  

5.3201           Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion

Wen, W., Quan, Z-J., Li, S-A., Yang, Z-X., Fu, Y-W., Zhang, F., Li, G-H., Zhao, M., Yin, M-D., Xu, J., Zhang, J-P., Cheng, T. and Zhang, X-B. Genome Biol., 22:236 (2021)   Background After repairing double-strand breaks (DSBs) caused by CRISPR-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences. Results We show that large deletions are ubiquitous but are dependent on editing sites and cell types. Human primary T cells display more significant deletions than hematopoietic stem and progenitor cells (HSPCs), whereas we observe low levels in induced pluripotent stem cells (iPSCs). We find that the homology-directed repair (HDR) with single-stranded oligodeoxynucleotides (ssODNs) carrying short homology reduces the deletion damage by almost half, while adeno-associated virus (AAV) donors with long homology reduce large deletions by approximately 80%. In the absence of HDR, the insertion of a short double-stranded ODN by NHEJ reduces deletion indexes by about 60%. Conclusions Timely bridging of broken ends by HDR and NHEJ vastly decreases the unintended consequences of dsDNA cleavage. These strategies can be harnessed in gene editing applications to attenuate unintended outcomes.  

5.3202           IL-7 coupled with IL-12 increases intratumoral T cell clonality, leading to complete regression of non-immunogenic tumors

Tasaki, M., Yamashita, M., Arai, Y., Nakamura, T. and Nakao, S. Cancer Immunol. Immunother., 70, 3557-3571 (2021)   Immune checkpoint inhibitors against PD-1, PD-L1 and CTLA-4 have altered the treatment paradigm for various types of cancers in the past decade. However, they offer clinical benefits to only a subset of patients. Evaluation and identification of an appropriate therapeutic approach to improve intratumoral immune status are needed for better treatment outcomes. We previously demonstrated that intratumoral expression of IL-7 and IL-12 increased tumor-infiltrating lymphocytes in poorly immunogenic tumors, resulting in a higher tumor regression rate than IL-12 alone. However, the mechanism underlying the difference in efficacy with and without IL-7 remains unclear. Here, we identified a previously unknown effect of IL-7 on the T cell receptor (TCR) repertoire of intratumoral CD8⁺ T cells, which is induced in the presence of IL-12. While IL-7 alone increased the diversity of intratumoral CD8⁺ T cells, IL-7 with IL-12 increased a limited number of high-frequency clones, conversely augmenting IL-12 function to increase the clonality. The proportion of mice with multiple high-frequency clones in tumors correlated with that achieving complete tumor regression in efficacy studies. These findings provide a scientific rationale for combining IL-7 and IL-12 in anticancer immunotherapy and unveil a novel IL-7 function on intratumoral TCR repertoire.  

5.3203           A Scalable Manufacturing Approach to Single Dose Vaccination against HPV

Shao, S., Ortega-Rivera, O.A., Ray, S. and Pokorski, J.K. Vaccines, 9:66 (2021)   Human papillomavirus (HPV) is a globally prevalent sexually-transmitted pathogen, responsible for most cases of cervical cancer. HPV vaccination rates remain suboptimal, partly due to the need for multiple doses, leading to a lack of compliance and incomplete protection. To address the drawbacks of current HPV vaccines, we used a scalable manufacturing process to prepare implantable polymer–protein blends for single-administration with sustained delivery. Peptide epitopes from HPV16 capsid protein L2 were conjugated to the virus-like particles derived from bacteriophage Qβ, to enhance their immunogenicity. The HPV-Qβ particles were then encapsulated into poly(lactic-co-glycolic acid) (PLGA) implants, using a benchtop melt-processing system. The implants facilitated the slow and sustained release of HPV-Qβ particles without the loss of nanoparticle integrity, during high temperature melt processing. Mice vaccinated with the implants generated IgG titers comparable to the traditional soluble injections and achieved protection in a pseudovirus neutralization assay. HPV-Qβ implants offer a new vaccination platform; because the melt-processing is so versatile, the technology offers the opportunity for massive upscale into any geometric form factor. Notably, microneedle patches would allow for self-administration in the absence of a healthcare professional, within the developing world. The Qβ technology is highly adaptable, allowing the production of vaccine candidates and their delivery devices for multiple strains or types of viruses.  

5.3204           The Immunogenicity of Capsid-Like Particle Vaccines in Combination with Different Adjuvants Using Different Routes of Administration

Janitzek, C.M., Carlsen, P.H.R., Thrane, S., Khanna, V.M., Jakob, V., Barnier-Quer, C., Collin, N., Theander, T.G., Salanti, A., Nielsen, M.A. and Sander, A.F. Vaccines, 9:131 (2021)   Capsid-like particle (CLP) displays can be used to enhance the immunogenicity of vaccine antigens, but a better understanding of how CLP vaccines are best formulated and delivered is needed. This study compared the humoral immune responses in mice elicited against two different vaccine antigens (a bacterial protein and a viral peptide) delivered on an AP205 CLP platform using six different adjuvant formulations. In comparison to antibody responses obtained after immunization with the unadjuvanted CLP vaccine, three of the adjuvant systems (neutral liposomes/monophosphoryl lipid A/quillaja saponaria 21, squalene-in-water emulsion, and monophosphoryl lipid A) caused significantly increased antibody levels, whereas formulation with the three other adjuvants (aluminum hydroxide, cationic liposomes, and cationic microparticles) resulted in similar or even decreased antibody responses. When delivering the soluble bacterial protein in a squalene-in-water emulsion, 4-log lower IgG levels were obtained compared to when the protein was delivered on CLPs without the adjuvant. The AP205 CLP platform promoted induction of both IgG1 and IgG2 subclasses, which could be skewed towards a higher production of IgG1 (aluminum hydroxide). Compared to other routes, intramuscular administration elicited the highest IgG levels. These results indicate that the effect of the external adjuvant does not always synergize with the adjuvant effect of the CLP display, which underscores the need for empirical testing of different extrinsic adjuvants.  

5.3205           Head-to-Head Comparison of Modular Vaccines Developed Using Different Capsid Virus-Like Particle Backbones and Antigen Conjugation Systems

Fredsgaard, L., Goksøyr, L., Thrane, S., Aves, K-L., Theander, T.G. and Sander, A.F. Vaccines, 9:539 (2021)   Capsid virus-like particles (cVLPs) are used as molecular scaffolds to increase the immunogenicity of displayed antigens. Modular platforms have been developed whereby antigens are attached to the surface of pre-assembled cVLPs. However, it remains unknown to what extent the employed cVLP backbone and conjugation system may influence the immune response elicited against the displayed antigen. Here, we performed a head-to-head comparison of antigen-specific IgG responses elicited by modular cVLP-vaccines differing by their employed cVLP backbone or conjugation system, respectively. Covalent antigen conjugation (i.e., employing the SpyTag/SpyCatcher system) resulted in significantly higher antigen-specific IgG titers compared to when using affinity-based conjugation (i.e., using biotin/streptavidin). The cVLP backbone also influenced the antigen-specific IgG response. Specifically, vaccines based on the bacteriophage AP205 cVLP elicited significantly higher antigen-specific IgG compared to corresponding vaccines using the human papillomavirus major capsid protein (HPV L1) cVLP. In addition, the AP205 cVLP platform mediated induction of antigen-specific IgG with a different subclass profile (i.e., higher IgG2a and IgG2b) compared to HPV L1 cVLP. These results demonstrate that the cVLP backbone and conjugation system can individually affect the IgG response elicited against a displayed antigen. These data will aid the understanding and process of tailoring modular cVLP vaccines to achieve improved immune responses.  

5.3206           Development of a Macrophage-Based ADCC Assay

Uccellini, M.B., Aslam, S., Liu, S.T.H., Alam, F. and Garcia-Sastre, A. Vaccines, 9:660 (2021)   Fc-dependent effector functions are an important determinant of the in vivo potency of therapeutic antibodies. Effector function is determined by the combination of FcRs bound by the antibody and the cell expressing the relevant FcRs, leading to antibody-dependent cellular cytotoxicity (ADCC). A number of ADCC assays have been developed; however, they suffer from limitations in terms of throughput, reproducibility, and in vivo relevance. Existing assays measure NK cell-mediated ADCC activity; however, studies suggest that macrophages mediate the effector function of many antibodies in vivo. Here, we report the development of a macrophage-based ADCC assay that relies on luciferase expression in target cells as a measure of live cell number. In the presence of primary mouse macrophages and specific antibodies, loss of luciferase signal serves as a surrogate for ADCC-dependent killing. We show that the assay functions for a variety of mouse and human isotypes with a model antigen/antibody complex in agreement with the known effector function of the isotypes. We also use this assay to measure the activity of a number of influenza-specific antibodies and show that the assay correlates well with the known in vivo effector functions of these antibodies  

5.3207           Critical Assessment of Purification and Analytical Technologies for Enveloped Viral Vector and Vaccine Processing and Their Current Limitations in Resolving Co-Expressed Extracellular Vesicles

Minh, A.D. and Kamen, A.A. Vaccines, 9:823 (2021)   Viral vectors and viral vaccines are invaluable tools in prevention and treatment of diseases. Many infectious diseases are controlled using vaccines designed from subunits or whole viral structures, whereas other genetic diseases and cancers are being treated by viruses used as vehicles for delivering genetic material in gene therapy or as therapeutic agents in virotherapy protocols. Viral vectors and vaccines are produced in different platforms, from traditional embryonated chicken eggs to more advanced cell cultures. All these expression systems, like most cells and cellular tissues, are known to spontaneously release extracellular vesicles (EVs). EVs share similar sizes, biophysical characteristics and even biogenesis pathways with enveloped viruses, which are currently used as key ingredients in a number of viral vectors and licensed vaccine products. Herein, we review distinctive features and similarities between EVs and enveloped viruses as we revisit the downstream processing steps and analytical technologies currently implemented to produce and document viral vector and vaccine products. Within a context of well-established viral vector and vaccine safety profiles, this review provides insights on the likely presence of EVs in the final formulation of enveloped virus products and discusses the potential to further resolve and document these components.  

5.3208           Immunogenicity and Antiviral Response of Therapeutic Hepatitis B Vaccination in a Mouse Model of HBeAg-Negative, Persistent HBV Infection

Kosinska, A.D., Festag, J., Mück-Häusl, M., Festag, M.M. and Asen, T. Vaccines, 9:841 (2021)   During the natural course of chronic hepatitis B virus (HBV) infection, the hepatitis B e antigen (HBeAg) is typically lost, while the direct transmission of HBeAg-negative HBV may result in fulminant hepatitis B. While the induction of HBV-specific immune responses by therapeutic vaccination is a promising, novel treatment option for chronic hepatitis B, it remains unclear whether a loss of HBeAg may influence its efficacy or tolerability. We therefore generated an adeno-associated virus (AAV)-vector that carries a 1.3-fold overlength HBV genome with a typical stop-codon mutation in the pre-core region and initiates the replication of HBeAg(−) HBV in mouse livers. Infection of C57BL/6 mice established persistent HBeAg(−) HBV-replication without any detectable anti-HBV immunity or liver damage. HBV-carrier mice were immunized with TherVacB, a therapeutic hepatitis B vaccine that uses a particulate HBV S and a core protein for prime vaccination, and a modified vaccinia Ankara (MVA) for boost vaccination. The TherVacB immunization of HBeAg(+) and HBeAg(−) HBV carrier mice resulted in the effective induction of HBV-specific antibodies and the loss of HBsAg but only mild liver damage. Intrahepatic, HBV-specific CD8 T cells induced in HBeAg(−) mice expressed more IFNγ but showed similar cytolytic activity. This indicates that the loss of HBeAg improves the performance of therapeutic vaccination by enhancing non-cytolytic effector functions.  

5.3209           A Crucial Role of ACBD3 Required for Coxsackievirus Infection in Animal Model Developed by AAV-Mediated CRISPR Genome Editing Technique

Shin, H.J., Ku, K.B., Kim, S., Kim, H.S., Kim, Y-S., Kim, B-T., Kim, S-J. and Kim, C. Viruses, 13:237 (2021)   Genetic screens using CRISPR/Cas9 have been exploited to discover host–virus interactions. These screens have identified viral dependencies on host proteins during their life cycle and potential antiviral strategies. The acyl-CoA binding domain containing 3 (ACBD3) was identified as an essential host factor for the Coxsackievirus B3 (CVB3) infection. Other groups have also investigated the role of ACBD3 as a host factor for diverse enteroviruses in cultured cells. However, it has not been tested if ACBD3 is required in the animal model of CVB3 infection. Owing to embryonic lethality, conventional knockout mice were not available for in vivo study. As an alternative approach, we used adeno-associated virus (AAV)-mediated CRISPR genome editing to generate mice that lacked ACBD3 within the pancreas, the major target organ for CVB3. Delivery of sgRNAs using self-complementary (sc) AAV8 efficiently induced a loss-of-function mutation in the pancreas of the Cas9 knock-in mice. Loss of ACBD3 in the pancreas resulted in a 100-fold reduction in the CVB3 titer within the pancreas and a noticeable reduction in viral protein expression. These results indicate a crucial function of ACBD3 in CVB3 infection in vivo. AAV-mediated CRISPR genome editing may be applicable to many in vivo studies on the virus–host interaction and identify a novel target for antiviral therapeutics.  

5.3210           Incorporation of CD55 into the Zika Viral Envelope Contributes to Its Stability against Human Complement

Malekshahi, Z., Bernklau, S., Schiela, B., Koske, I., Banki, Z., Stiasny, K., Harris, C.L., Würzner, R. and Stoiber, H. Viruses, 13:510 (2021)   The rapid spread of the virus in Latin America and the association of the infection with microcephaly in newborns or Guillain–Barré Syndrome in adults prompted the WHO to declare the Zika virus (ZIKV) epidemic to be an international public health emergency in 2016. As the virus was first discovered in monkeys and is spread not only by mosquitos but also from human to human, we investigated the stability to the human complement of ZIKV derived from mosquito (ZIKVInsect), monkey (ZIKVVero), or human cells (ZIKVA549 and ZIKVFibro), respectively. At a low serum concentration (10%), which refers to complement concentrations found on mucosal surfaces, the virus was relatively stable at 37 °C. At higher complement levels (up to 50% serum concentration), ZIKV titers differed significantly depending on the cell line used for the propagation of the virus. While the viral titer of ZIKVInsect decreased about two orders in magnitude, when incubated with human serum, the virus derived from human cells was more resistant to complement-mediated lysis (CML). By virus-capture assay and Western blots, the complement regulator protein CD55 was identified to be incorporated into the viral envelope. Blocking of CD55 by neutralizing Abs significantly increased the sensitivity to human complement. Taken together, these data indicate that the incorporation of CD55 from human cells contributes to the stability of ZIKV against complement-mediated virolysis.  

5.3211           Bombyx mori Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a Bombyx mori Nuclear Polyhedrosis Virus Expression System

Yu, Q., Chang, P., Liu, X., Lü, P., Tang, Q., Guo, Z., Qiu, J., Chen, K. and Yao, Q. Viruses, 13:704 (2021)   Recombinant adeno-associated virus (AAV) vectors have broad application prospects in the field of gene therapy. The establishment of low-cost and large-scale manufacturing is now the general agenda for industry. The baculovirus-insect cell/larva expression system has great potential for these applications due to its scalability and predictable biosafety. To establish a more efficient production system, Bombyx mori pupae were used as a new platform and infected with recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV). The production of a chimeric recombinant adeno-associated virus (rAAV) serotype 2/human bocavirus type-1 (HBoV1) vector was used to evaluate the efficiency of this new baculovirus expression vector (BEV)–insect expression system. For this purpose, we constructed two recombinant BmNPVs, which were named rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP. The yields of rAAV2/HBoV1 derived from the rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP co-infected BmN cells exceeded 2 × 10⁴ vector genomes (VG) per cell. The rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP can express stably for at least five passages. Significantly, rAAV2/HBoV1 could be efficiently generated from BmNPV-infected silkworm larvae and pupae at average yields of 2.52 × 10¹² VG/larva and 4.6 × 10¹² VG/pupa, respectively. However, the vectors produced from the larvae and pupae had a high percentage of empty particles, which suggests that further optimization is required for this platform in the future. Our work shows that silkworm pupae, as an efficient bioreactor, have great potential for application in the production of gene therapy vectors.  

5.3212           Identification of Receptor Binding Proteins in Flagellotropic Agrobacterium Phage 7-7-1

Gonzalez, F. and Scharf, B.E. Viruses, 13:1267 (2021)   The rapid discovery of new and diverse bacteriophages has driven the innovation of approaches aimed at detailing interactions with their bacterial hosts. Previous studies on receptor binding proteins (RBPs) mainly relied on their identification in silico and are based on similarities to well-characterized systems. Thus, novel phage RBPs unlike those currently annotated in genomic and proteomic databases remain largely undiscovered. In this study, we employed a screen to identify RBPs in flagellotropic Agrobacterium phage 7-7-1. Flagellotropic phages utilize bacterial flagella as receptors. The screen identified three candidate RBPs, Gp4, Gp102, and Gp44. Homology modelling predicted that Gp4 is a trimeric, tail associated protein with a central β-barrel, while the structure and function of Gp102 and Gp44 are less obvious. Studies with purified Gp41-247 confirmed its ability to bind and interact with host cells, highlighting the robustness of the RBP screen. We also discovered that Gp41-247 inhibits the growth of host cells in a motility and lipopolysaccharide (LPS) dependent fashion. Hence, our results suggest interactions between Gp41-247, rotating flagellar filaments and host glycans to inhibit host cell growth, which presents an impactful and intriguing focus for future studies.  

5.3213           Mouse Papillomavirus L1 and L2 Are Dispensable for Viral Infection and Persistence at Both Cutaneous and Mucosal Tissues

Brendle, S., Li, J.J., Cladel, N.M., Shearer, D.A., Budgeon, L.R., Balogh, K.K., Atkins, H., Costa-Fujishima, M., Lopez, p., Christensen, N.D., Doorbar, J., Murooka, T.T. and Hu, J. Viruses, 13:1824 (2021)   Papillomavirus L1 and L2, the major and minor capsid proteins, play significant roles in viral assembly, entry, and propagation. In the current study, we investigate the impact of L1 and L2 on viral life cycle and tumor growth with a newly established mouse papillomavirus (MmuPV1) infection model. MmuPV1 L1 knockout, L2 knockout, and L1 plus L2 knockout mutant genomes (designated as L1ATGko-4m, L2ATGko, and L1-L2ATGko respectively) were generated. The mutants were examined for their ability to generate lesions in athymic nude mice. Viral activities were examined by qPCR, immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM) analyses. We demonstrated that viral DNA replication and tumor growth occurred at both cutaneous and mucosal sites infected with each of the mutants. Infections involving L1ATGko-4m, L2ATGko, and L1-L2ATGko mutant genomes generally resulted in smaller tumor sizes compared to infection with the wild type. The L1 protein was absent in L1ATGko-4m and L1-L2ATGko mutant-treated tissues, even though viral transcripts and E4 protein expression were robust. Therefore, L1 is not essential for MmuPV1-induced tumor growth, and this finding parallels our previous observations in the rabbit papillomavirus model. Very few viral particles were detected in L2ATGko mutant-infected tissues. Interestingly, the localization of L1 in lesions induced by L2ATGko was primarily cytoplasmic rather than nuclear. The findings support the hypothesis that the L2 gene influences the expression, location, transport, and assembly of the L1 protein in vivo.  

5.3214           Bioluminescent Optogenetics: A Novel Experimental Therapy to Promote Axon Regeneration after Peripheral Nerve Injury

English, A.W., Berglund, K., Carrasco, D., Goebel, K., Gross, R.E., Isaacson, R., Mistretta, O.C. and Wynans, C. Int. J. Mol. Sci., 22:7217 (2021)   Functional recovery after peripheral nerve injury (PNI) is poor, mainly due to the slow and incomplete regeneration of injured axons. Experimental therapies that increase the excitability of the injured axons have proven remarkably successful in promoting regeneration, but their clinical applicability has been limited. Bioluminescent optogenetics (BL-OG) uses luminopsins, fusion proteins of light-generating luciferase and light-sensing ion channels that could be used to increase neuronal excitability if exposed to a suitable substrate. Excitatory luminopsins were expressed in motoneurons of transgenic mice and in wildtype mice transduced with adeno-associated viral vectors. Intraperitoneal administration of coelenterazine (CTZ), a known luciferase substrate, generated intense bioluminescence in peripheral axons. This bioluminescence increased motoneuron excitability. A single administration of CTZ immediately after sciatic nerve transection and repair markedly enhanced motor axon regeneration. Compound muscle action potentials were 3–4 times larger than controls by 4 weeks after injury. The results observed with transgenic mice were comparable to those of mice in which the luminopsin was expressed using viral vectors. Significantly more motoneurons had successfully reinnervated muscle targets four weeks after nerve injury in BL-OG treated mice than in controls. Bioluminescent optogenetics is a promising therapeutic approach to enhancing axon regeneration after PNI.  

5.3215           Sustained Elevated Blood Pressure Accelerates Atherosclerosis Development in a Preclinical Model of Disease

Gonzalez-Guerra, A., Roche-Molina, M., Garcia-Quintans, N., Sanchez-Ramos, Martin-Perez, D., Lytvyn, M., de Nicolas-Hernandez. J., Rivera-Torres, j., Arroyo, D.F., Sanz-Rosa, D. and Bernal, J.A. Int. J. Mol. Sci., 22:8448 (2021)   The continuous relationship between blood pressure (BP) and cardiovascular events makes the distinction between elevated BP and hypertension based on arbitrary cut-off values for BP. Even mild BP elevations manifesting as high-normal BP have been associated with cardiovascular risk. We hypothesize that persistent elevated BP increases atherosclerotic plaque development. To evaluate this causal link, we developed a new mouse model of elevated BP based on adeno-associated virus (AAV) gene transfer. We constructed AAV vectors to support transfer of the hRenin and hAngiotensinogen genes. A single injection of AAV-Ren/Ang (10¹¹ total viral particles) induced sustained systolic BP increase (130 ± 20 mmHg, vs. 110 ± 15 mmHg in controls; p = 0.05). In ApoE^−/− mice, AAV-induced mild BP elevation caused larger atherosclerotic lesions evaluated by histology (10-fold increase vs. normotensive controls). In this preclinical model, atheroma plaques development was attenuated by BP control with a calcium channel blocker, indicating that a small increase in BP within a physiological range has a substantial impact on plaque development in a preclinical model of atherosclerosis. These data support that non-optimal BP represents a risk for atherosclerosis development. Earlier intervention in elevated BP may prevent or delay morbidity and mortality associated with atherosclerosis.  

5.3216           Functional Availability of ON-Bipolar Cells in the Degenerated Retina: Timing and Longevity of an Optogenetic Gene Therapy

Kralik, J. and Kleinlogel, S. Int. J. Mol. Sci., 22:11515 (2021)   Degenerative diseases of the retina are responsible for the death of photoreceptors and subsequent loss of vision in patients. Nevertheless, the inner retinal layers remain intact over an extended period of time, enabling the restoration of light sensitivity in blind retinas via the expression of optogenetic tools in the remaining retinal cells. The chimeric Opto-mGluR6 protein represents such a tool. With exclusive ON-bipolar cell expression, it combines the light-sensitive domains of melanopsin and the intracellular domains of the metabotropic glutamate receptor 6 (mGluR6), which naturally mediates light responses in these cells. Albeit vision restoration in blind mice by Opto-mGluR6 delivery was previously shown, much is left to be explored in regard to the effects of the timing of the treatment in the degenerated retina. We performed a functional evaluation of Opto-mGluR6-treated murine blind retinas using multi-electrode arrays (MEAs) and observed long-term functional preservation in the treated retinas, as well as successful therapeutical intervention in later stages of degeneration. Moreover, the treatment decreased the inherent retinal hyperactivity of the degenerated retinas to levels undistinguishable from healthy controls. Finally, we observed for the first time micro electroretinograms (mERGs) in optogenetically treated animals, corroborating the origin of Opto-mGluR6 signalling at the level of mGluR6 of ON-bipolar cells.  

5.3217           Erythropoietin Gene Therapy Delays Retinal Degeneration Resulting from Oxidative Stress in the Retinal Pigment Epithelium

Biswal, M.R., Wang, Z., Paulson, R.J., Uddin, R.R., Tong, Y., Zhu, P., Li, H. and Lewin, A.S. Antioxidants, 10:842 (2021)   Erythropoietin (EPO) plays an important role in erythropoiesis by its action in blocking apoptosis of progenitor cells and protects both photoreceptors and retinal ganglion cells from induced or inherited degeneration. A modified form of EPO, EPO-R76E has attenuated erythropoietic activity but is effective in inhibiting apoptosis, oxidative stress, and inflammation in several models of retinal degeneration. In this study, we used recombinant Adeno Associated Virus (AAV) to provide long-term sustained delivery of EPO-R76E and demonstrated its effects in a mouse model of dry-AMD in which retinal degeneration is induced by oxidative stress in the retinal pigment epithelial (RPE) cells. Experimental vector AAV-EPO-R76E and control vector AAV-GFP were packaged into serotype-1 (AAV1) to enable RPE selective expression. RPE oxidative stress-mediated retinal degeneration was induced by exon specific deletion of the protective enzyme MnSOD (encoded by Sod2) by cre/lox mechanism. Experimental mice received subretinal injection of AAV-EPO-R76E in the right eye and AAV-GFP in the left eye. Western blotting of RPE/choroid protein samples from AAV-EPO-R76E injected eyes showed RPE specific EPO expression. Retinal function was monitored by electroretinography (ERG). EPO-R76E over-expression in RPE delayed the retinal degeneration as measured by light microscopy in RPE specific Sod2 knockout mice. Delivery of EPO-R76E vector can be used as a tool to prevent retinal degeneration induced by RPE oxidative stress, which is implicated as a potential cause of Age-Related Macular Degeneration.  

5.3218           Genetic Constructs for the Control of Astrocytes’ Activity

Borodinova, A.A., Balaban, P.M., Bezprozvanny, I.B., Salmina, A.B. and Vlasova, O.L. Cells, 10:1600 (2021)   In the current review, we aim to discuss the principles and the perspectives of using the genetic constructs based on AAV vectors to regulate astrocytes’ activity. Practical applications of optogenetic approaches utilizing different genetically encoded opsins to control astroglia activity were evaluated. The diversity of astrocytic cell-types complicates the rational design of an ideal viral vector for particular experimental goals. Therefore, efficient and sufficient targeting of astrocytes is a multiparametric process that requires a combination of specific AAV serotypes naturally predisposed to transduce astroglia with astrocyte-specific promoters in the AAV cassette. Inadequate combinations may result in off-target neuronal transduction to different degrees. Potentially, these constraints may be bypassed with the latest strategies of generating novel synthetic AAV serotypes with specified properties by rational engineering of AAV capsids or using directed evolution approach by searching within a more specific promoter or its replacement with the unique enhancer sequences characterized using modern molecular techniques (ChIP-seq, scATAC-seq, snATAC-seq) to drive the selective transgene expression in the target population of cells or desired brain regions. Realizing these strategies to restrict expression and to efficiently target astrocytic populations in specific brain regions or across the brain has great potential to enable future studies.  

5.3219           The metabolic signaling of the nucleoredoxin-like 2 gene supports brain function

Jaillard, C., Ouerchtati, F., Clerin, E., Millet-Puel, G., Corsi, M. et al Redox. Biol., 48, 102198 (2021)   The nucleoredoxin gene NXNL2 encodes for two products through alternative splicing, rod-derived cone viability factor-2 (RdCVF2) that mediates neuronal survival and the thioredoxin-related protein (RdCVF2L), an enzyme that regulates the phosphorylation of TAU. To investigate the link between NXNL2 and tauopathies, we studied the Nxnl2 knockout mouse (Nxnl2^−/−). We established the expression pattern of the Nxnl2 gene in the brain using a Nxnl2 reporter mouse line, and characterized the behavior of the Nxnl2^−/− mouse at 2 months of age. Additionally, long term potential recording and metabolomic from hippocampal specimens were collected at 2 months of age. We studied TAU oligomerization, phosphorylation and aggregation in Nxnl2^−/− brain at 18 months of age. Finally, newborn Nxnl2^−/− mice were treated with adeno-associated viral vectors encoding for RdCVF2, RdCVF2L or both and measured the effect of this therapy on long-term potential, glucose metabolism and late-onset tauopathy. Nxnl2^−/− mice at 2 months of age showed severe behavioral deficiency in fear, pain sensitivity, coordination, learning and memory. The Nxnl2^−/− also showed deficits in long-term potentiation, demonstrating that the Nxnl2 gene is involved in regulating brain functions. Dual delivery of RdCVF2 and RdCVF2L in newborn Nxnl2^−/− mice fully correct long-term potentiation through their synergistic action. The expression pattern of the Nxnl2 gene in the brain shows a predominant expression in circumventricular organs, such as the area postrema. Glucose metabolism of the hippocampus of Nxnl2^−/− mice at 2 months of age was reduced, and was not corrected by gene therapy. At 18-month-old Nxnl2^−/− mice showed brain stigmas of tauopathy, such as oligomerization, phosphorylation and aggregation of TAU. This late-onset tauopathy can be prevented, albeit with modest efficacy, by recombinant AAVs administrated to newborn mice. The Nxnl2^−/− mice have memory dysfunction at 2-months that resembles mild-cognitive impairment and at 18-months exhibit tauopathy, resembling to the progression of Alzheimer's disease. We propose the Nxnl2^−/− mouse is a model to study multistage aged related neurodegenerative diseases. The NXNL2 metabolic and redox signaling is a new area of therapeutic research in neurodegenerative diseases.  

5.3220           Impaired glucocorticoid receptor expression in liver disrupts feeding-induced gene expression, glucose uptake, and glycogen storage

Præstholm, S.M., Correia, C.M., Goitea, V.E., Pedersen, T.Å., Fægerman, N.J. and Grøntved, L. Cell Reports, 37, 109938 (2021)   The transition from a fasted to a fed state is associated with extensive transcriptional remodeling in hepatocytes facilitated by hormonal- and nutritional-regulated transcription factors. Here, we use a liver-specific glucocorticoid receptor (GR) knockout (L-GRKO) model to investigate the temporal hepatic expression of GR target genes in response to feeding. Interestingly, in addition to the well-described fasting-regulated genes, we identify a subset of hepatic feeding-induced genes that requires GR for full expression. This includes Gck, which is important for hepatic glucose uptake, utilization, and storage. We show that insulin and glucocorticoids cooperatively regulate hepatic Gck expression in a direct GR-dependent manner by a 4.6 kb upstream GR binding site operating as a Gck enhancer. L-GRKO blunts preprandial and early postprandial Gck expression, which ultimately affects early postprandial hepatic glucose uptake, phosphorylation, and glycogen storage. Thus, GR is positively involved in feeding-induced gene expression and important for postprandial glucose metabolism in the liver.  

5.3221           Flavivirus vaccines: Virus-like particles and single-round infectious particles as promising alternatives

Cuevas-Juarez, E., Pando-Robles, V. and Palomares, L.A. Vaccine, 39, 6990-7000 (2021)   The genus flavivirus of the Flaviridae family includes several human pathogens, like dengue, Zika, Japanese encephalitis, and yellow fever virus. These viruses continue to be a significant threat to human health. Vaccination remains the most useful approach to reduce the impact of flavivirus fever. However, currently available vaccines can induce severe side effects or have low effectiveness. An alternative is the use of recombinant vaccines, of which virus-like particles (VLP) and single-round infectious particles (SRIP) are of especial interest. VLP consist of the virus structural proteins produced in a heterologous system that self-assemble in a structure almost identical to the native virus. They are highly immunogenic and have been effective vaccines for other viruses for over 30 years. SRIP are promising vaccine candidates, as they induce both cellular and humoral responses, as viral proteins are expressed. Here, the state of the art to produce both types of particles and their use as vaccines against flaviviruses are discussed. We summarize the different approaches used for the design and production of flavivirus VLP and SRIP, the evidence for their safety and efficacy, and the main challenges for their use as commercial vaccines.  

5.3222           Protocol for evaluating the role of a gene in protecting mouse retinal ganglion cells

Guo, X., Starr, C., Zhou, J. and Chen, B. STAR Protocols, 2, 100932 (2021)   The degeneration of retinal ganglion cells (RGCs) leads to irreversible vision loss in a variety of pathological states. Here, we describe a protocol to evaluate the role of a gene in protecting mouse RGCs when they sustain injuries from excitotoxicity or axonal damage. This protocol includes the procedures for gene transfer through AAV intravitreal injection, induction of RGC injuries by NMDA-induced excitotoxicity or optic nerve crush, and retina immunohistochemistry to assess RGC survival.  

5.3223           Imprinted lncRNA Dio3os preprograms intergenerational brown fat development and obesity resistance

Chen, Y-T., Yang, Q-Y., Hu, Y., Liu, X-D., de Avila, J.M., Zhu, M-J., Nathanielsz, P.W. and Du, M. Nature Comm., 12:6845 (2021)   Maternal obesity (MO) predisposes offspring to obesity and metabolic disorders but little is known about the contribution of offspring brown adipose tissue (BAT). We find that MO impairs fetal BAT development, which persistently suppresses BAT thermogenesis and primes female offspring to metabolic dysfunction. In fetal BAT, MO enhances expression of Dio3, which encodes deiodinase 3 (D3) to catabolize triiodothyronine (T3), while a maternally imprinted long noncoding RNA, Dio3 antisense RNA (Dio3os), is inhibited, leading to intracellular T3 deficiency and suppression of BAT development. Gain and loss of function shows Dio3os reduces D3 content and enhances BAT thermogenesis, rendering female offspring resistant to high fat diet-induced obesity. Attributing to Dio3os inactivation, its promoter has higher DNA methylation in obese dam oocytes which persists in fetal and adult BAT, uncovering an oocyte origin of intergenerational obesity. Overall, our data uncover key features of Dio3os activation in BAT to prevent intergenerational obesity and metabolic dysfunctions.  

5.3224           Chronic nicotine increases midbrain dopamine neuron activity and biases individual strategies towards reduced exploration in mice

Dongelmans, M., Durand-de Cuttoli, R., Nguyen, C., Come, M., Durante, E.K. et al Nature Comm., 12:6945 (2021)   Long-term exposure to nicotine alters brain circuits and induces profound changes in decision-making strategies, affecting behaviors both related and unrelated to drug seeking and consumption. Using an intracranial self-stimulation reward-based foraging task, we investigated in mice the impact of chronic nicotine on midbrain dopamine neuron activity and its consequence on the trade-off between exploitation and exploration. Model-based and archetypal analysis revealed substantial inter-individual variability in decision-making strategies, with mice passively exposed to nicotine shifting toward a more exploitative profile compared to non-exposed animals. We then mimicked the effect of chronic nicotine on the tonic activity of dopamine neurons using optogenetics, and found that photo-stimulated mice adopted a behavioral phenotype similar to that of mice exposed to chronic nicotine. Our results reveal a key role of tonic midbrain dopamine in the exploration/exploitation trade-off and highlight a potential mechanism by which nicotine affects the exploration/exploitation balance and decision-making.  

5.3225           Treatment of adult metachromatic leukodystrophy model mice using intrathecal administration of type 9 AAV vector encoding arylsulfatase A

Miyake, N., Miyake, K., Sakai, A., Yamamoto, M., Suzuki, H. and Shimada, T. Scientific Reports, 11:20513 (2021)   Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by an arylsulfatase A (ARSA) deficiency and characterized by severe neurological symptoms resulting from demyelination within the central and peripheral nervous systems. We investigated the feasibility and efficacy of intrathecal administration of a type 9 adeno-associated viral vector encoding ARSA (AAV9/ARSA) for the treatment of 6-week-old MLD model mice, which are presymptomatic, and 1-year-old mice, which exhibit neurological abnormalities. Immunohistochemical analysis following AAV9/ARSA administration showed ARSA expression within the brain, with highest activities in the cerebellum and olfactory bulbs. In mice treated at 1 year, alcian blue staining and quantitative analysis revealed significant decreases in stored sulfatide. Behaviorally, mice treated at 1 year showed no improvement in their ability to traverse narrow balance beams as compared to untreated mice. By contrast, MLD mice treated at 6 weeks showed significant decreases in stored sulfatide throughout the entire brain and improved ability to traverse narrow balance beams. These findings suggest intrathecal administration of an AAV9/ARSA vector is a promising approach to treating genetic diseases of the central nervous system, including MLD, though it may be essential to begin therapy before the onset of neurological symptoms.  

5.3226           Efficacy of AAV serotypes to target Schwann cells after intrathecal and intravenous delivery

Kagiava, A., Richter, J., Tryfonos, C., leal-Julia, M., Sargiannidou, I., Christodoulou, C., Bosch, A. and Kleopa, K.A: Scientific Reports, 11:23358 (2021)   To optimize gene delivery to myelinating Schwann cells we compared clinically relevant AAV serotypes and injection routes. AAV9 and AAVrh10 vectors expressing either EGFP or the neuropathy-associated gene GJB1/Connexin32 (Cx32) under a myelin specific promoter were injected intrathecally or intravenously in wild type and Gjb1-null mice, respectively. Vector biodistribution in lumbar roots and sciatic nerves was higher in AAVrh10 injected mice while EGFP and Cx32 expression rates and levels were similar between the two serotypes. A gradient of biodistribution away from the injection site was seen with both intrathecal and intravenous delivery, while similar expression rates were achieved despite higher vector amounts injected intravenously. Quantified immune cells in relevant tissues were similar to non-injected littermates. Overall, AAV9 and AAVrh10 efficiently transduce Schwann cells throughout the peripheral nervous system with both clinically relevant routes of administration, although AAV9 and intrathecal injection may offer a more efficient approach for treating demyelinating neuropathies.  

5.3227           Elabela gene therapy promotes angiogenesis after myocardial infarction

Jin, L., Pan, Y., Li, Q., Li, J. and Wang, Z.

1. Cell. Mol. Med., 25, 8537-8545 (2021)

  This study was aimed at investigating whether Elabela (ELA) gene therapy can promote angiogenesis in the treatment of myocardial infarction (MI). The fusion expression plasmid pAAV-3 × Flag/ELA-32 was successfully constructed using molecular cloning technique. The model of acute MI was established by ligating the left anterior descending coronary artery in mice. Adeno-associated virus serotype 9 (AAV9) was injected into the surrounding myocardium and tail vein immediately after the model was established. AAV was injected again from the tail vein one week later. Compared with the MI+PBS (control) group, the serum N-terminal pro-brain natriuretic peptide (NT-proBNP) concentration, and the values of left ventricular end-diastolic diameter (LVDd) and left ventricular end-systolic diameter (LVDs) of the MI+AAV-ELA (gene therapy) group were significantly decreased, while the value of left ventricular ejection fraction was significantly increased at 2 and 4 weeks after operation. Compared with the control group, the expression of CD105 and vWF and the percentage of CD31- and Ki67–co-positive cells were significantly increased in the gene therapy group. Moreover, the expressions of apelin peptide jejunum (APJ) receptor, vascular endothelial growth factor (VEGF), VEGFR2, Jagged1 and Notch3 in the heart tissue around the infarction were up-regulated in mice with gene therapy. The results suggest that ELA activates VEFG/VEGFR2 and Jagged1/Notch3 pathways through APJ to promote angiogenesis after myocardial infarction. ELA gene therapy may be used in the treatment of ischaemic cardiomyopathy in future.  

5.3228           Adult-born neurons promote cognitive flexibility by improving memory precision and indexing

Berdugo-Vega, G., Lee, C-C., Garthe, A., Kempermann, G. and Calegari, F. Hippocampus, 31, 1068-1079 (2021)   Adult neurogenesis in the hippocampal dentate gyrus (DG) is an extraordinary form of plasticity fundamental for cognitive flexibility. Recent evidence showed that newborn neurons differentially modulate input to the infra- and supra-pyramidal blades of the DG during the processing of spatial and contextual information, respectively. However, how this differential regulation by neurogenesis is translated into different aspects contributing cognitive flexibility is unclear. Here, we increased adult-born neurons by a genetic expansion of neural stem cells and studied their influence during navigational learning. We found that increased neurogenesis improved both memory precision and flexibility. Interestingly, each of these gains was associated with distinct subregional patterns of activity and better separation of memory representations in the DG-CA3 network. Our results highlight the role of adult-born neurons in promoting memory precision and indexing and suggests their anatomical allocation within specific DG-CA3 compartments, together contributing to cognitive flexibility.  

5.3229           Urinary FABP1 is a biomarker for impaired proximal tubular protein reabsorption and is synergistically enhanced by concurrent liver injury

Kawakami, R., Matsui, M., Konno, A., Kaneko, R., Shrestha, S. et al

1. Pathol., 255, 362-373 (2021)

  Urinary fatty acid binding protein 1 (FABP1, also known as liver-type FABP) has been implicated as a biomarker of acute kidney injury (AKI) in humans. However, the precise biological mechanisms underlying its elevation remain elusive. Here, we show that urinary FABP1 primarily reflects impaired protein reabsorption in proximal tubule epithelial cells (PTECs). Bilateral nephrectomy resulted in a marked increase in serum FABP1 levels, suggesting that the kidney is an essential organ for removing serum FABP1. Injected recombinant FABP1 was filtered through the glomeruli and robustly reabsorbed via the apical membrane of PTECs. Urinary FABP1 was significantly elevated in mice devoid of megalin, a giant endocytic receptor for protein reabsorption. Elevation of urinary FABP1 was also observed in patients with Dent disease, a rare genetic disease characterized by defective megalin function in PTECs. Urinary FABP1 levels were exponentially increased following acetaminophen overdose, with both nephrotoxicity and hepatotoxicity observed. FABP1-deficient mice with liver-specific overexpression of FABP1 showed a massive increase in urinary FABP1 levels upon acetaminophen injection, indicating that urinary FABP1 is liver-derived. Lastly, we employed transgenic mice expressing diphtheria toxin receptor (DT-R) either in a hepatocyte- or in a PTEC-specific manner, or both. Upon administration of diphtheria toxin (DT), massive excretion of urinary FABP1 was induced in mice with both kidney and liver injury, while mice with either injury type showed marginal excretion. Collectively, our data demonstrated that intact PTECs have a considerable capacity to reabsorb liver-derived FABP1 through a megalin-mediated mechanism. Thus, urinary FABP1, which is synergistically enhanced by concurrent liver injury, is a biomarker for impaired protein reabsorption in AKI. These findings address the use of urinary FABP1 as a biomarker of histologically injured PTECs that secrete FABP1 into primary urine, and suggest the use of this biomarker to simultaneously monitor impaired tubular reabsorption and liver function.  

5.3230           Neuronal Menin Overexpression Rescues Learning and Memory Phenotype in CA1-Specific α7 nAChRs KD Mice

Batool, S., Akhter, B., Zaidi, J., Visser, F., Petrie, G., Hill, M. and Syed, N. Cells, 10:3286 (2021)   The perturbation of nicotinic cholinergic receptors is thought to underlie many neurodegenerative and neuropsychiatric disorders, such as Alzheimer’s and schizophrenia. We previously identified that the tumor suppressor gene, MEN1, regulates both the expression and synaptic targeting of α7 nAChRs in the mouse hippocampal neurons in vitro. Here we sought to determine whether the α7 nAChRs gene expression reciprocally regulates the expression of menin, the protein encoded by the MEN1 gene, and if this interplay impacts learning and memory. We demonstrate here that α7 nAChRs knockdown (KD) both in in vitro and in vivo, initially upregulated and then subsequently downregulated menin expression. Exogenous expression of menin using an AAV transduction approach rescued α7 nAChRs KD mediated functional and behavioral deficits specifically in hippocampal (CA1) neurons. These effects involved the modulation of the α7 nAChR subunit expression and functional clustering at the synaptic sites. Our data thus demonstrates a novel and important interplay between the MEN1 gene and the α7 nAChRs in regulating hippocampal-dependent learning and memory.  

5.3231           The Small Molecule Alpha-Synuclein Aggregator, FN075, Enhances Alpha-Synuclein Pathology in Subclinical AAV Rat Models

Kelly, R., Caairns, A.G., Åden, J., Almvist, F., Bemelmans, A-P., Brouillet, E., Patton, T., McKernan, D.P. and Dowd, E. Biomolecules, 11:1685 (2021)   Animal models of Parkinson’s disease, in which the human α-synuclein transgene is overexpressed in the nigrostriatal pathway using viral vectors, are widely considered to be the most relevant models of the human condition. However, although highly valid, these models have major limitations related to reliability and variability, with many animals exhibiting pronounced α-synuclein expression failing to demonstrate nigrostriatal neurodegeneration or motor dysfunction. Therefore, the aim of this study was to determine if sequential intra-nigral administration of AAV-α-synuclein followed by the small α-synuclein aggregating molecule, FN075, would enhance or precipitate the associated α-synucleinopathy, nigrostriatal pathology and motor dysfunction in subclinical models. Rats were given unilateral intra-nigral injections of AAV-α-synuclein (either wild-type or A53T mutant) followed four weeks later by a unilateral intra-nigral injection of FN075, after which they underwent behavioral testing for lateralized motor functionality until they were sacrificed for immunohistological assessment at 20 weeks after AAV administration. In line with expectations, both of the AAV vectors induced widespread overexpression of human α-synuclein in the substantia nigra and striatum. Sequential administration of FN075 significantly enhanced the α-synuclein pathology with increased density and accumulation of the pathological form of the protein phosphorylated at serine 129 (pS129-α-synuclein). However, despite this enhanced α-synuclein pathology, FN075 did not precipitate nigrostriatal degeneration or motor dysfunction in these subclinical AAV models. In conclusion, FN075 holds significant promise as an approach to enhancing the α-synuclein pathology in viral overexpression models, but further studies are required to determine if alternative administration regimes for this molecule could improve the reliability and variability in these models.  

5.3232           In Vivo Modelling of Hepatitis B Virus Subgenotype A1 Replication Using Adeno-Associated Viral Vectors

Limani, S.W., Mnyandu, N., Ely, A., Wadee, R., Kramvis, A., Arbuthnot, P. and Maepa, M.B: Viruses, 13:2247 (2021)   The paucity of animal models that simulate the replication of the hepatitis B virus (HBV) is an impediment to advancing new anti-viral treatments. The work reported here employed recombinant adeno-associated viruses (AAVs) to model HBV subgenotype A1 and subgenotype D3 replication in vitro and in vivo. Infection with subgenotype A1 is endemic to parts of sub-Saharan Africa, and it is associated with a high risk of hepatocellular carcinoma. Recombinant AAV serotype 2 (AAV2) and 8 (AAV8) vectors bearing greater-than-genome-length sequences of HBV DNA from subgenotype A1 and D3, were produced. Transduced liver-derived cultured cells produced HBV surface antigen and core antigen. Administration of AAV8 carrying HBV subgenotype A1 genome (AAV8-A1) to mice resulted in the sustained production of HBV replication markers over a six-month period, without elevated inflammatory cytokines, expression of interferon response genes or alanine transaminase activity. Markers of replication were generally higher in animals treated with subgenotype D3 genome-bearing AAVs than in those receiving the subgenotype A1-genome-bearing vectors. To validate the use of the AAV8-A1 murine model for anti-HBV drug development, the efficacy of anti-HBV artificial primary-microRNAs was assessed. Significant silencing of HBV markers was observed over a 6-month period after administering AAVs. These data indicate that AAVs conveniently and safely recapitulate the replication of different HBV subgenotypes, and the vectors may be used to assess antivirals’ potency.  

5.3233           Addiction-Associated Genetic Variants Implicate Brain Cell Type- and Region-Specific Cis-Regulatory Elements in Addiction Neurobiology

Srinivasan, C., Phan, B.N., Lawler, A.J., Ramamurthy, E., Kleyman, M., Brown, A.R., Kaplow, I.M., Wirthlin, M.E. and Pfenning, A.R.

1. Neurosci., 41(43), 9008-9030 (2021)

  Recent large genome-wide association studies have identified multiple confident risk loci linked to addiction-associated behavioral traits. Most genetic variants linked to addiction-associated traits lie in noncoding regions of the genome, likely disrupting cis-regulatory element (CRE) function. CREs tend to be highly cell type-specific and may contribute to the functional development of the neural circuits underlying addiction. Yet, a systematic approach for predicting the impact of risk variants on the CREs of specific cell populations is lacking. To dissect the cell types and brain regions underlying addiction-associated traits, we applied stratified linkage disequilibrium score regression to compare genome-wide association studies to genomic regions collected from human and mouse assays for open chromatin, which is associated with CRE activity. We found enrichment of addiction-associated variants in putative CREs marked by open chromatin in neuronal (NeuN⁺) nuclei collected from multiple prefrontal cortical areas and striatal regions known to play major roles in reward and addiction. To further dissect the cell type-specific basis of addiction-associated traits, we also identified enrichments in human orthologs of open chromatin regions of female and male mouse neuronal subtypes: cortical excitatory, D1, D2, and PV. Last, we developed machine learning models to predict mouse cell type-specific open chromatin, enabling us to further categorize human NeuN⁺ open chromatin regions into cortical excitatory or striatal D1 and D2 neurons and predict the functional impact of addiction-associated genetic variants. Our results suggest that different neuronal subtypes within the reward system play distinct roles in the variety of traits that contribute to addiction.  

5.3234           p38γ and p38δ regulate postnatal cardiac metabolism through glycogen synthase 1

Santamans, A.M., Montalvo-Romeral, V., Mora, A., Lopez, J.A., Gonzalez-Romero, F. et al PloS Biology, 19(11), e3001447 (2021)   During the first weeks of postnatal heart development, cardiomyocytes undergo a major adaptive metabolic shift from glycolytic energy production to fatty acid oxidation. This metabolic change is contemporaneous to the up-regulation and activation of the p38γ and p38δ stress-activated protein kinases in the heart. We demonstrate that p38γ/δ contribute to the early postnatal cardiac metabolic switch through inhibitory phosphorylation of glycogen synthase 1 (GYS1) and glycogen metabolism inactivation. Premature induction of p38γ/δ activation in cardiomyocytes of newborn mice results in an early GYS1 phosphorylation and inhibition of cardiac glycogen production, triggering an early metabolic shift that induces a deficit in cardiomyocyte fuel supply, leading to whole-body metabolic deregulation and maladaptive cardiac pathogenesis. Notably, the adverse effects of forced premature cardiac p38γ/δ activation in neonate mice are prevented by maternal diet supplementation of fatty acids during pregnancy and lactation. These results suggest that diet interventions have a potential for treating human cardiac genetic diseases that affect heart metabolism.  

5.3235           Structure of native HIV-1 cores and their interactions with IP6 and CypA

Ni, T., Zhu, Y., Yang, Z., Xu, C., Chaban, Y., Nesterova, T., Ning, J., Böcking, T., Parker, M.W., Monnie, C., Ahn, J., Perilla, J.R. and Zhang, P. Sci.Adv., 7, eabj5715 (2021)   The viral capsid plays essential roles in HIV replication and is a major platform engaging host factors. To overcome challenges in study native capsid structure, we used the perfringolysin O to perforate the membrane of HIV-1 particles, thus allowing host proteins and small molecules to access the native capsid while improving cryo–electron microscopy image quality. Using cryo–electron tomography and subtomogram averaging, we determined the structures of native capsomers in the presence and absence of inositol hexakisphosphate (IP6) and cyclophilin A and constructed an all-atom model of a complete HIV-1 capsid. Our structures reveal two IP6 binding sites and modes of cyclophilin A interactions. Free energy calculations substantiate the two binding sites at R18 and K25 and further show a prohibitive energy barrier for IP6 to pass through the pentamer. Our results demonstrate that perfringolysin O perforation is a valuable tool for structural analyses of enveloped virus capsids and interactions with host cell factors.  

5.3236           Prickle promotes the formation and maintenance of glutamatergic synapses by stabilizing the intercellular planar cell polarity complex

Ban, Y., Yu, T., Feng, B., Lorentz, C., Wang, X., Baker, C. and Zou, Y. Sci. Adv., 7, eabh2974 (2021)   Whether there exists a common signaling mechanism that assembles all glutamatergic synapses is unknown. We show here that knocking out Prickle1 and Prickle2 reduced the formation of the PSD-95–positive glutamatergic synapses in the hippocampus and medial prefrontal cortex in postnatal development by 70–80%. Prickle1 and Prickle2 double knockout in adulthood lead to the disassembly of 70 to 80% of the postsynaptic-density(PSD)-95–positive glutamatergic synapses. PSD-95–positive glutamatergic synapses in the hippocampus of Prickle2^E8Q/E8Q mice were reduced by 50% at postnatal day 14. Prickle2 promotes synapse formation by antagonizing Vangl2 and stabilizing the intercellular complex of the planar cell polarity (PCP) components, whereas Prickle2 E8Q fails to do so. Coculture experiments show that the asymmetric PCP complexes can determine the presynaptic and postsynaptic polarity. In summary, the PCP components regulate the assembly and maintenance of a large number of glutamatergic synapses and specify the direction of synaptic transmission.  

5.3237           The Multidrug Efflux System AcrABZ-TolC Is Essential for Infection of Salmonella Typhimurium by the Flagellum-Dependent Bacteriophage Chi

Esteves, N.C., Porwollik, S., McClelland, M. and Scharf, B.E.

1. Virol., 95(11), e00394-21 (2021)

  Bacteriophages are the most abundant biological entities in the biosphere. Due to their host specificity and ability to kill bacteria rapidly, bacteriophages have many potential health care applications, including therapy against antibiotic-resistant bacteria. Infection by flagellotropic bacteriophages requires a properly rotating bacterial flagellar filament. The flagella-dependent phage χ (chi) infects serovars of the pathogenic enterobacterium Salmonella enterica. However, cell surface receptors and proteins involved in other stages of χ infection have not been discovered to date. We screened a multigene deletion library of S. enterica serovar Typhimurium by spotting mutants on soft agar plates seeded with bacteriophage χ and monitoring their ability to grow and form a swim ring, a characteristic of bacteriophage-resistant motile mutants. Those multigene deletion regions identified to be important for χ infectivity were further investigated by characterizing the phenotypes of corresponding single-gene deletion mutants. In this way, we identified motile mutants with various degrees of resistance to χ. Deletions in individual genes encoding the AcrABZ-TolC multidrug efflux system drastically reduced infection by bacteriophage χ. Furthermore, an acrABtolC triple deletion strain was fully resistant to χ. Infection was severely reduced but not entirely blocked by the deletion of the gene tig, encoding the molecular chaperone trigger factor. Finally, deletion in genes encoding enzymes involved in the synthesis of the antioxidants glutathione (GSH) and uric acid resulted in reduced infectivity. Our findings begin to elucidate poorly understood processes involved in later stages of flagellotropic bacteriophage infection and inform research aimed at the use of bacteriophages to combat antibiotic-resistant bacterial infections.  

5.3238           Co-opting regulation bypass repair as a gene-correction strategy for monogenic diseases

Hu, J., Bourne, R.A., McGrath, B., Lin, A., Pei, Z. and Cavener, D.R. Molecular Therapy, 29(11), 3274-3292 (2021)   With the development of CRISPR-Cas9-mediated gene-editing technologies, correction of disease-causing mutations has become possible. However, current gene-correction strategies preclude mutation repair in post-mitotic cells of human tissues, and a unique repair strategy must be designed and tested for each and every mutation that may occur in a gene. We have developed a novel gene-correction strategy, co-opting regulation bypass repair (CRBR), which can repair a spectrum of mutations in mitotic or post-mitotic cells and tissues. CRBR utilizes the non-homologous end joining (NHEJ) pathway to insert a coding sequence (CDS) and transcription/translation terminators targeted upstream of any CDS mutation and downstream of the transcriptional promoter. CRBR results in simultaneous co-option of the endogenous regulatory region and bypass of the genetic defect. We validated the CRBR strategy for human gene therapy by rescuing a mouse model of Wolcott-Rallison syndrome (WRS) with permanent neonatal diabetes caused by either a large deletion or a nonsense mutation in the PERK (EIF2AK3) gene. Additionally, we integrated a CRBR GFP-terminator cassette downstream of the human insulin promoter in cadaver pancreatic islets of Langerhans, which resulted in insulin promoter regulated expression of GFP, demonstrating the potential utility of CRBR in human tissue gene repair.  

5.3239           Production and purification of high-titer OrfV for preclinical studies in vaccinology and cancer therapy

Van Vloten, J.P., Minott, J.A., McAusland, T.M., Ingrao, J.C., Santry, L.A., McFadden, G., Petrik, J.J., Bridle, B.W. and Wootton, S.K. Molecular Therapy-Methods Clin. Develop., 23, 434-447 (2021)   Poxviruses have been used extensively as vaccine vectors for human and veterinary medicine and have recently entered the clinical realm as immunotherapies for cancer. We present a comprehensive method for producing high-quality lots of the poxvirus Parapoxvirus ovis (OrfV) for use in preclinical models of vaccinology and cancer therapy. OrfV is produced using a permissive sheep skin-derived cell line and is released from infected cells by repeated freeze-thaw combined with sonication. We present two methods for isolation and purification of bulk virus. Isolated virus is concentrated to high titer using polyethylene glycol to produce the final in vivo-grade product. We also describe methods for quantifying OrfV infectious virions and determining genomic copy number to evaluate virus stocks. The methods herein will provide researchers with the ability to produce high-quality, high-titer OrfV for use in preclinical studies, and support the translation of OrfV-derived technologies into the clinic.  

5.3240           Infection courses, virological features and IFN-α responses of HBV genotypes in cell culture and animal models

Zhang, M., Zhang, Z., Imamura, M., Saito, T., Chayama, K. and Liang, T.J:

1. Hepatol., 75(6), 1335-1345 (2021)

  Background & Aims HBV consists of 9 major genotypes (A to I), 1 minor strain (designated J) and multiple subtypes, which may be associated with different clinical characteristics. As only cell lines expressing genotype D3 have been established, herein, we aimed to establish stable cell lines producing high-titer cell culture-generated HBV (HBVcc) of different genotypes and to explore their infectivity, virological features and responses to treatment. Methods Stable cell lines producing high titers of HBV genotype A2, B2, C1, E, F1b and H were generated by transfecting plasmids containing a replication-competent 1.3x length HBV genome and an antibiotic marker into HepG2 cells that can support HBV replication. Clones with the highest levels of HBV DNA and/or HBeAg were selected and expanded for large-scale purification of HBVcc. HBVcc of different genotypes were tested in cells and a humanized chimeric mouse model. Results HBVcc genotypes were infectious in mouse-passaged primary human hepatocytes (PXB cells) and responded differently to human interferon (IFN)-α with variable kinetics of reduction in HBV DNA, HBeAg and HBsAg. HBVcc of all genotypes were infectious in humanized chimeric mice but with variable kinetics of viremia and viral antigen production. Treatment of infected mice with human IFN-α resulted in modest and variable reductions of viremia and viral antigenemia. HBVcc passaged in humanized chimeric mice (HBVmp) infected PXB cells much more efficiently than that of the original HBVcc viral stock. Conclusions Herein, we generated stable cell lines producing HBV of various genotypes that are infectious in vitro and in vivo. We observe genotype-associated variations in viral antigen production, infection kinetics and responses to human IFN-α treatment in these models.  

5.3241           Amino Acid Polymorphism in Hepatitis B Virus Associated With Functional Cure

Honda, T., Yamada, N., Murayama, A., Shiina, M., Aly, H.H. et al Cell. Mol. Gastroenterol. Hepatol., 12(5), 1583-1598 (2021)   Background & Aims To provide an adequate treatment strategy for chronic hepatitis B, it is essential to know which patients are expected to have a good prognosis and which patients do not require therapeutic intervention. Previously, we identified the substitution of isoleucine to leucine at amino acid 97 (I97L) in the hepatitis B core region as a key predictor among patients with stable hepatitis. In this study, we attempted to identify the point at which I97L affects the hepatitis B virus (HBV) life cycle and to elucidate the underlying mechanisms governing the stabilization of hepatitis. Methods To confirm the clinical features of I97L, we used a cohort of hepatitis B e antigen–negative patients with chronic hepatitis B infected with HBV-I97 wild-type (wt) or HBV-I97L. The effects of I97L on viral characteristics were evaluated by in vitro HBV production and infection systems with the HBV reporter virus and cell culture-generated HBV. Results The ratios of reduction in hepatitis B surface antigen and HBV DNA were higher in patients with HBV-I97L than in those with HBV-I97wt. HBV-I97L exhibited lower infectivity than HBV-I97wt in both infection systems with reporter HBV and cell culture-generated HBV. HBV-I97L virions exhibiting low infectivity primarily contained a single-stranded HBV genome. The lower efficiency of cccDNA synthesis was demonstrated after infection of HBV-I97L or transfection of the molecular clone of HBV-I97L. Conclusions The I97L substitution reduces the level of cccDNA through the generation of immature virions with single-stranded genomes. This I97L-associated low efficiency of cccDNA synthesis may be involved in the stabilization of hepatitis.  

5.3242           Hepatitis B virus polymerase-specific T cell epitopes shift in a mouse model of chronic infection

Hasanpourghadi, M., Novikov, M., Newman, D., Xiang, Z., Zhou, X.Y., Magowan, C. and Ertl, H.C.J. Virology J., 18:242 (2021)   Background Chronic hepatitis B virus (HBV) infection (CHB) is a significant public health problem that could benefit from treatment with immunomodulators. Here we describe a set of therapeutic HBV vaccines that target the internal viral proteins. Methods Vaccines are delivered by chimpanzee adenovirus vectors (AdC) of serotype 6 (AdC6) and 7 (AdC7) used in prime only or prime-boost regimens. The HBV antigens are fused into an early T cell checkpoint inhibitor, herpes simplex virus (HSV) glycoprotein D (gD), which enhances and broadens vaccine-induced cluster of differentiation (CD8)⁺ T cell responses. Results Our results show that the vaccines are immunogenic in mice. They induce potent CD8⁺ T cell responses that recognize multiple epitopes. CD8⁺ T cell responses increase after a boost, although the breadth remains similar. In mice, which carry high sustained loads of HBV particles due to a hepatic infection with an adeno-associated virus (AAV)8 vector expressing the 1.3HBV genome, CD8⁺ T cell responses to the vaccines are attenuated with a marked shift in the CD8⁺ T cells’ epitope recognition profile. Conclusions Our data show that in different stains of mice including those that carry a human major histocompatibility complex (MHC) class I antigen HBV vaccines adjuvanted with a checkpoint inhibitor induce potent and broad HBV-specific CD8⁺ T cell responses and lower but still detectable CD4⁺ T cell responses. CD8⁺ T cell responses are reduced and their epitope specificity changes in mice that are chronically exposed to HBV antigens. Implications for the design of therapeutic HBV vaccines are discussed.  

5.3243           Transplantation of human embryonic stem cells alleviates motor dysfunction in AAV2-Htt171-82Q transfected rat model of Huntington’s disease

Islam, J., So, K.H., KC, E., Moon, H.C., Kim, A., Hyun, S.H., Kim, S.and Park, Y.S. Stem Cell research & Therapy, 12:585 (2021)   Background Human embryonic stem cells (hESCs) transplantation had shown to provide a potential source of cells in neurodegenerative disease studies and lead to behavioral recovery in lentivirus transfected or, toxin-induced Huntington's disease (HD) rodent model. Here, we aimed to observe if transplantation of superparamagnetic iron oxide nanoparticle (SPION)-labeled hESCs could migrate in the neural degenerated area and improve motor dysfunction in an AAV2-Htt171-82Q transfected Huntington rat model. Methods All animals were randomly allocated into three groups at first: HD group, sham group, and control group. After six weeks, the animals of the HD group and sham group were again divided into two subgroups depending on animals receiving either ipsilateral or contralateral hESCs transplantation. We performed cylinder test and stepping test every two weeks after AAV2-Htt171-82Q injection and hESCs transplantation. Stem cell tracking was performed once per two weeks using T2 and T2*-weighted images at 4.7 Tesla MRI. We also performed immunohistochemistry and immunofluorescence staining to detect the presence of hESCs markers, huntingtin protein aggregations, and iron in the striatum. Results After hESCs transplantation, the Htt virus-injected rats exhibited significant behavioral improvement in behavioral tests. SPION labeled hESCs showed migration with hypointense signal in MRI. The cells were positive with βIII-tubulin, GABA, and DARPP32. Conclusion Collectively, our results suggested that hESCs transplantation can be a potential treatment for motor dysfunction of Huntington's disease.  

5.3244           Lunapark-dependent formation of a virus-induced ER exit site contains multi-tubular ER junctions that promote viral ER-to-cytosol escape

Bagchi, P., Liu, X., Cho, W.J. and Tsai, B. Cell Reports, 37, 110077 (2021)   Viruses rearrange host membranes to support different entry steps. Polyomavirus simian virus 40 (SV40) reorganizes the endoplasmic reticulum (ER) membrane to generate focus structures that enable virus ER-to-cytosol escape, a decisive infection step. The molecular architecture of the ER exit site that might illuminate why it is ideally suited for membrane penetration is unknown. Here 3D focused ion beam scanning electron microscopy (FIB-SEM) reconstruction reveals that the ER focus structure consists of multi-tubular ER junctions where SV40 preferentially localizes, suggesting that tubular branch points are virus ER-to-cytosol penetration sites. Functional analysis demonstrates that lunapark—an ER membrane protein that typically stabilizes three-way ER junctions—relocates to the ER foci, where it supports focus formation, leading to SV40 ER escape and infection. Our results reveal how a virus repurposes the activity of an ER membrane protein to form a virus-induced ER substructure required for membrane escape and suggest that ER tubular junctions are vulnerable sites exploited by viruses for membrane penetration.  

5.3245           Neurexin-3 defines synapse- and sex-dependent diversity of GABAergic inhibition in ventral subiculum

Boxer, E.E., Seng, C., Lukacsovich, D., Kennedy, M.J., Földy, C. and Aoto, J. Cell Reports, 37, 110098 (2021)   Ventral subiculum (vSUB) is integral to the regulation of stress and reward; however, the intrinsic connectivity and synaptic properties of the inhibitory local circuit are poorly understood. Neurexin-3 (Nrxn3) is highly expressed in hippocampal inhibitory neurons, but its function at inhibitory synapses has remained elusive. Using slice electrophysiology, imaging, and single-cell RNA sequencing, we identify multiple roles for Nrxn3 at GABAergic parvalbumin (PV) interneuron synapses made onto vSUB regular-spiking (RS) and burst-spiking (BS) principal neurons. Surprisingly, we find that intrinsic connectivity of vSUB and synaptic function of Nrxn3 in vSUB are sexually dimorphic. We reveal that PVs make preferential contact with RS neurons in male mice, but BS neurons in female mice. Furthermore, we determine that despite comparable Nrxn3 isoform expression in male and female PV neurons, Nrxn3 knockout impairs synapse density, postsynaptic strength, and inhibitory postsynaptic current (IPSC) amplitude at PV-RS synapses in males, but enhances presynaptic release and IPSC amplitude in females.  

5.3246           A genetically encoded fluorescent biosensor for extracellular L-lactate

Nasu, Y., Murphy-Royal, C., Wen, Y., Haidey, J.N., Molina, R.S. et al Nature Comm., 12:7058 (2021)  

L-Lactate, traditionally considered a metabolic waste product, is increasingly recognized as an important intercellular energy currency in mammals. To enable investigations of the emerging roles of intercellular shuttling of L-lactate, we now report an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. This biosensor, designated eLACCO1.1, enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.

 

5.3247           GPR180 is a component of TGFβ signalling that promotes thermogenic adipocyte function and mediates the metabolic effects of the adipocyte-secreted factor CTHRC1

Balazova, L., Balaz, M., Horvath, C., Horvath, A., Moser, C. et al Nature Comm., 12:7144 (2021)   Activation of thermogenic brown and beige adipocytes is considered as a strategy to improve metabolic control. Here, we identify GPR180 as a receptor regulating brown and beige adipocyte function and whole-body glucose homeostasis, whose expression in humans is associated with improved metabolic control. We demonstrate that GPR180 is not a GPCR but a component of the TGFβ signalling pathway and regulates the activity of the TGFβ receptor complex through SMAD3 phosphorylation. In addition, using genetic and pharmacological tools, we provide evidence that GPR180 is required to manifest Collagen triple helix repeat containing 1 (CTHRC1) action to regulate brown and beige adipocyte activity and glucose homeostasis. In this work, we show that CTHRC1/GPR180 signalling integrates into the TGFβ signalling as an alternative axis to fine-tune and achieve low-grade activation of the pathway to prevent pathophysiological response while contributing to control of glucose and energy metabolism.  

5.3248           Antiviral Potential of the Antimicrobial Drug Atovaquone against SARS-CoV-2 and Emerging Variants of Concern

Carter-Timofte, M.E., Arulanandam, R., Kurmasheva, N., Fu, K., Laroche, G. et al ACS Infect. Dis., 7, 3034-3051 (2021)   The antimicrobial medication malarone (atovaquone/proguanil) is used as a fixed-dose combination for treating children and adults with uncomplicated malaria or as chemoprophylaxis for preventing malaria in travelers. It is an inexpensive, efficacious, and safe drug frequently prescribed around the world. Following anecdotal evidence from 17 patients in the provinces of Quebec and Ontario, Canada, suggesting that malarone/atovaquone may present some benefits in protecting against COVID-19, we sought to examine its antiviral potential in limiting the replication of SARS-CoV-2 in cellular models of infection. In VeroE6 expressing human TMPRSS2 and human lung Calu-3 epithelial cells, we show that the active compound atovaquone at micromolar concentrations potently inhibits the replication of SARS-CoV-2 and other variants of concern including the alpha, beta, and delta variants. Importantly, atovaquone retained its full antiviral activity in a primary human airway epithelium cell culture model. Mechanistically, we demonstrate that the atovaquone antiviral activity against SARS-CoV-2 is partially dependent on the expression of TMPRSS2 and that the drug can disrupt the interaction of the spike protein with the viral receptor, ACE2. Additionally, spike-mediated membrane fusion was also reduced in the presence of atovaquone. In the United States, two clinical trials of atovaquone administered alone or in combination with azithromycin were initiated in 2020. While we await the results of these trials, our findings in cellular infection models demonstrate that atovaquone is a potent antiviral FDA-approved drug against SARS-CoV-2 and other variants of concern in vitro.  

5.3249           Cell type-selective targeted delivery of a recombinant lysosomal enzyme for enzyme therapies

Baik, A.D., Calafati, P., Zhang, X., Aaron, N.A., Mehra, A., Moller-Tank, S. et al Molecular Therapy, 29(12), 3512-3524 (2021)   Lysosomal diseases are a class of genetic disorders predominantly caused by loss of lysosomal hydrolases, leading to lysosomal and cellular dysfunction. Enzyme replacement therapy (ERT), where recombinant enzyme is given intravenously, internalized by cells, and trafficked to the lysosome, has been applied to treat several lysosomal diseases. However, current ERT regimens do not correct disease phenotypes in all affected organs because the biodistribution of enzyme uptake does not match that of the affected cells that require the enzyme. We present here targeted ERT, an approach that utilizes antibody-enzyme fusion proteins to target the enzyme to specific cell types. The antibody moiety recognizes transmembrane proteins involved in lysosomal trafficking and that are also preferentially expressed in those cells most affected in disease. Using Pompe disease (PD) as an example, we show that targeted ERT is superior to ERT in treating the skeletal muscle phenotypes of PD mice both as a protein replacement therapeutic and as a gene therapy.  

5.3250           The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy

Ran, G., Feng, X-I., Xie, Y-I., Zheng, Q-y., Guo, P-p., Yang, M., Feng, Y-l., Ling, C., Zhu, L-q. and Zhong, C.

1. Integrative Med., 19, 515-525 (2021)

  Objective Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells. Methods A miR122 target (122T) sequence was incorporated into the 3′ untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined. Results The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells. Conclusion HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.  

5.3251           Hemagglutinin Nanoparticulate Vaccine with Controlled Photochemical Immunomodulation for Pathogenic Influenza-Specific Immunity

Jeong, H., Lee, C-S., Lee, J., Lee, J., Hwang, H.S., Lee, M. and Na, K. Adv. Sci., 8, 2100118 (2021)   Recently, viral infectious diseases, including COVID-19 and Influenza, are the subjects of major concerns worldwide. One strategy for addressing these concerns focuses on nasal vaccines, which have great potential for achieving successful immunization via safe, easy, and affordable approaches. However, conventional nasal vaccines have major limitations resulting from fast removal when pass through nasal mucosa and mucociliary clearance hindering their effectiveness. Herein a nanoparticulate vaccine (NanoVac) exhibiting photochemical immunomodulation and constituting a new self-assembled immunization system of a photoactivatable polymeric adjuvant with influenza virus hemagglutinin for efficient nasal delivery and antigen-specific immunity against pathogenic influenza viruses is described. NanoVac increases the residence period of antigens and further enhances by spatiotemporal photochemical modulation in the nasal cavity. As a consequence, photochemical immunomodulation of NanoVacs successfully induces humoral and cellular immune responses followed by stimulation of mature dendritic cells, plasma cells, memory B cells, and CD4⁺ and CD8⁺ T cells, resulting in secretion of antigen-specific immunoglobulins, cytokines, and CD8⁺ T cells. Notably, challenge with influenza virus after nasal immunization with NanoVacs demonstrates robust prevention of viral infection. Thus, this newly designed vaccine system can serve as a promising strategy for developing vaccines that are active against current hazardous pathogen outbreaks and pandemics.  

5.3252           Strategies to package recombinant Adeno-Associated Virus expressing the N-terminal gasdermin domain for tumor treatment

Lu, Y., He, W., Huang, X., He, Y., Gou, X., Liu, X., Hu, Z., Xu, W., Rahman, K., Li, S., Hu, S., Luo, J. and Cao, G. Nature Comm., 12:7155 (2021)  

Pyroptosis induced by the N-terminal gasdermin domain (GSDM^NT) holds great potential for anti-tumor therapy. However, due to the extreme cytoxicity of GSDM^NT, it is challenging to efficiently produce and deliver GSDM^NT into tumor cells. Here, we report the development of two strategies to package recombinant adeno-associated virus (rAAV) expressing GSDM^NT: 1) drive the expression of GSDM^NT by a mammal specific promoter and package the virus in Sf9 insect cells to avoid its expression; 2) co-infect rAAV-Cre to revert and express the double-floxed inverted GSDM^NT. We demonstrate that these rAAVs can induce pyroptosis and prolong survival in preclinical cancer models. The oncolytic-viruses induce pyroptosis and evoke a robust immune-response. In a glioblastoma model, rAAVs temporarily open the blood-brain barrier and recruit tumor infiltrating lymphocytes into the brain. The oncolytic effect is further improved in combination with anti-PD-L1. Together, our strategies efficiently produce and deliver GSDM^NT into tumor cells and successfully induce pyroptosis, which can be exploited for anti-tumor therapy.

 

5.3253           A Transgenic Model Reveals the Role of Klotho in Pancreatic Cancer Development and Paves the Way for New Klotho-Based Therapy

Rubenstein, T.A., Reuveni, I., Hesin, A., Klein-Goldsberg, A., Olauson, H. et al Cancers, 13:6297 (2021)   Klotho is an anti-aging transmembrane protein, which can be shed and can function as a hormone. Accumulating data indicate that klotho is a tumor suppressor in a wide array of malignancies, and designate the subdomain KL1 as the active region of the protein towards this activity. We aimed to study the role of klotho as a tumor suppressor in pancreatic ductal adenocarcinoma (PDAC). Bioinformatics analyses of The Cancer Genome Atlas (TCGA) datasets revealed a correlation between the survival of PDAC patients, levels of klotho expression, and DNA methylation, and demonstrated a unique hypermethylation pattern of klotho in pancreatic tumors. The in vivo effects of klotho and KL1 were examined using three mouse models. Employing a novel genetic model, combining pancreatic klotho knockdown with a mutation in Kras, the lack of klotho contributed to PDAC generation and decreased mousece survival. In a xenograft model, administration of viral particles carrying sKL, a spliced klotho isoform containing the KL1 domain, inhibited pancreatic tumors. Lastly, treatment with soluble sKL prolonged survival of Pdx1-Cre; Kras^G12D/+;Trp53^R172H/+ (KPC) mice, a model known to recapitulate human PDAC. In conclusion, this study provides evidence that klotho is a tumor suppressor in PDAC. Furthermore, these data suggest that the levels of klotho expression and DNA methylation could have prognostic value in PDAC patients, and that administration of exogenous sKL may serve as a novel therapeutic strategy to treat PDAC.  

5.3254           Improved targeting of human CD4+ T cells by nanobody-modified AAV2 gene therapy vectors

Hamann, M.V., Beschorner, N., Vu. X-K., hauber, I., Lange, U.C., Traenkle, B., Kaiser, P.D., Foth, D., Schneider, C., Büning, H., Rothbauer, U. and Hauber, J. PloS One, 16(12), e0261269 (2021)   Adeno-associated viruses (AAV) are considered non-pathogenic in humans, and thus have been developed into powerful vector platforms for in vivo gene therapy. Although the various AAV serotypes display broad tropism, frequently infecting multiple tissues and cell types, vectors for specific and efficient targeting of human CD4⁺ T lymphocytes are largely missing. In fact, a substantial translational bottleneck exists in the field of therapeutic gene transfer that would require in vivo delivery into peripheral disease-related lymphocytes for subsequent genome editing. To solve this issue, capsid modification for retargeting AAV tropism, and in turn improving vector potency, is considered a promising strategy. Here, we genetically modified the minor AAV2 capsid proteins, VP1 and VP2, with a set of novel nanobodies with high-affinity for the human CD4 receptor. These novel vector variants demonstrated improved targeting of human CD4⁺ cells, including primary human peripheral blood mononuclear cells (PBMC) and purified human CD4⁺ T lymphocytes. Thus, the technical approach presented here provides a promising strategy for developing specific gene therapy vectors, particularly targeting disease-related peripheral blood CD4⁺ leukocytes.  

5.3255           Molecular self-avoidance in synaptic neurexin complexes

Wang, C.Y., Trotter, J.H., Liakath-Ali, K.L., Lee, S-J., Liu, X. and Südhof, T.C. Sci. Adv., 7, eabk1924 (2021)   Synapses are thought to be organized by interactions of presynaptic neurexins with postsynaptic ligands, particularly with neuroligins and cerebellins. However, when a neuron forms adjacent pre- and postsynaptic specializations, as in dendrodendritic or axo-axonic synapses, nonfunctional cis neurexin/ligand interactions would be energetically favored. Here, we reveal an organizational principle for preventing synaptic cis interactions (“self-avoidance”). Using dendrodendritic synapses between mitral and granule cells in the olfactory bulb as a paradigm, we show that, owing to its higher binding affinity, cerebellin-1 blocks the cis interaction of neurexins with neuroligins, thereby enabling trans neurexin/neuroligin interaction. In mitral cells, ablating either cerebellin-1 or neuroligins severely impaired granule cell➔mitral cell synapses, as did overexpression of wild-type neurexins but not of mutant neurexins unable to bind to neuroligins. Our data uncover a molecular interaction network that organizes the self-avoidance of nonfunctional neurexin/ligand cis interactions, thus allowing assembly of physiological trans interactions.  

5.3256           Dual-vector gene therapy restores cochlear amplification and auditory sensitivity in a mouse model of DFNB16 hearing loss

Shubina-Oleinik, O., Nist-Lund, C., French, C., Rockowitz, S., Shearer, A.E. and Holt, J.R. Sci. Adv., 7, eabi7629 (2021)   Hearing loss affects an estimated 466 million people worldwide, with a substantial fraction due to genetic causes. Approximately 16% of genetic hearing loss is caused by pathogenic mutations in STRC, a gene that encodes the protein stereocilin. To develop gene therapy strategies for patients with STRC hearing loss, we generated a mouse model with a targeted deletion in the Strc gene. We devised a novel dual-vector approach to circumvent the size limitation of AAV vectors and drive expression of full-length STRC protein. To target outer hair cells, which are difficult to transduce, we used synthetic AAV9-PHP.B vectors for efficient dual-vector transduction. We report robust recovery of exogenous STRC expression in outer hair cells of Strc-deficient mice, recovery of hair bundle morphology, substantially improved cochlear amplification, and enhanced auditory sensitivity. The data raise the prospect that our strategy could benefit ~2.3 million patients worldwide affected by STRC mutations.  

5.3257           A Comparative Study on Delivery of Externally Attached DNA by Papillomavirus VLPs and Pseudoviruses

Brendle, S., Cladel, N., Balogh, K., Alam, S., Christensen, N., Meyers, C. and Hu, J. Vaccines, 9:1501 (2021)   Human papillomavirus (HPV) 16 capsids have been chosen as a DNA delivery vehicle in many studies. Our preliminary studies suggest that HPV58 capsids could be better vehicles than HPV16 capsids to deliver encapsidated DNA in vitro and in vivo. In the current study, we compared HPV16, HPV58, and the cottontail rabbit papillomavirus (CRPV) capsids either as L1/L2 VLPs or pseudoviruses (PSVs) to deliver externally attached GFP-expressing DNA. Both rabbit and human cells were used to test whether there was a species-specific effect. DNA delivery efficiency was determined by quantifying either GFP-expressing cell populations or mean fluorescent intensities (MFI) by flow cytometry. Interestingly, CRPV and 58-VLPs and PSVs were significantly more efficient at delivering attached DNA when compared to 16-VLPs and PSVs. A capsid/DNA ratio of 2:1 showed the highest efficiency for delivering external DNA. The PSVs with papillomavirus DNA genomes also showed higher efficiency than those with irrelevant plasmid DNA. HPV16L1/58L2 hybrid VLPs displayed increased efficiency compared to HPV58L1/16L2 VLPs, suggesting that L2 may play a critical role in the delivery of attached DNA. Additionally, we demonstrated that VLPs increased in vivo infectivity of CRPV DNA in rabbits. We conclude that choosing CRPV or 58 capsids to deliver external DNA could improve DNA uptake in in vitro and in vivo models.  

5.3258           SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion

Miccocheva, P., Kemp, S.A., Dhar, M.S., Papa, G., Meng, B. et al Nature, 599, 114-119 (2021)   The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.  

5.3259           PIAS2-mediated blockade of IFN-β signaling: a basis for sporadic Parkinson disease dementia

Magalhaes, J., Tresse, E., Ejlerkov, P., Hu, E., Liu, Y., Marin, A. et al Molecular Psychiatry, 26, 6083-6099 (2021)   Familial Parkinson disease (PD) is associated with rare genetic mutations, but the etiology in most patients with sporadic (s)PD is largely unknown, and the basis for its progression to dementia (sPDD) is poorly characterized. We have identified that loss of IFNβ or IFNAR1, the receptor for IFNα/β, causes pathological and behavioral changes resembling PDD, prompting us to hypothesize that dysregulated genes in IFNβ-IFNAR signaling pathway predispose one to sPD. By transcriptomic analysis, we found defective neuronal IFNβ-IFNAR signaling, including particularly elevated PIAS2 associated with sPDD. With meta-analysis of GWASs, we identified sequence variants in IFNβ-IFNAR-related genes in sPD patients. Furthermore, sPDD patients expressed higher levels of PIAS2 mRNA and protein in neurons. To determine its function in brain, we overexpressed PIAS2 under a neuronal promoter, alone or with human α-synuclein, in the brains of mice, which caused motor and cognitive impairments and correlated with intraneuronal phosphorylated (p)α-synuclein accumulation and dopaminergic neuron loss. Ectopic expression of neuronal PIAS2 blocked mitophagy, increased the accumulation of senescent mitochondrial and oxidative stress, as evidenced by excessive oxDJ1 and 8OHdG, by inactivating ERK1/2-P53 signaling. Conversely, PIAS2 knockdown rescued the clinicopathological manifestations of PDD in Ifnb^–/– mice on restoring mitochondrial homeostasis, oxidative stress, and pERK1/2-pP53 signaling. The regulation of JAK-STAT2-PIAS2 signaling was crucial for neurite outgrowth and neuronal survival and excitability and thus might prevent cognitive impairments. Our findings provide insights into the progression of sPD and dementia and have implications for new therapeutic approaches.  

5.3260           Widespread, Specific, and Efficient Transgene Expression in Oligodendrocytes After Intracerebral and Intracerebroventricular Delivery of Viral Vectors in Rodent Brain

Peelaerts, W., Brito, F., Van den haute, C., Janer, A.B., Steiner, J.A., Brundin, P. and Baekelandt, V. Human Gene Therapy, 32(11-12), 616-627 (2021)   Several neurodegenerative disorders are characterized by oligodendroglial pathology and myelin loss. Oligodendrogliopathies are a group of rare diseases for which there currently is no therapy. Gene delivery through viral vectors to oligodendrocytes is a potential strategy to deliver therapeutic molecules to oligodendrocytes for disease modification. However, targeting oligodendroglial cells in vivo is challenging due to their widespread distribution in white and gray matter. In this study, we aimed to address several of these difficulties by designing and testing different oligodendroglial targeting vectors in rat and mouse brain, utilizing different promoters, serotypes, and delivery routes. We found that different oligodendroglial promoters (myelin basic protein [MBP], cytomegalovirus-enhanced MBP, and myelin-associated glycoprotein [MAG]) vary considerably in their ability to drive oligodendroglial transgene expression and different viral vector serotypes (rAAV2/7, rAAV2/8, and rAAV2/9) exhibit varying efficacies in transducing oligodendrocytes. Different administration routes through intracerebral or intraventricular injection allow widespread targeting of mature oligodendrocytes. Delivery of rAAV2/9-MAG-GFP into the cerebrospinal fluid results in GFP expression along the entire rostrocaudal axis of the spinal cord. Collectively, these results show that oligodendrocytes can be targeted with high specificity and widespread expression, which will be useful for gene therapeutic interventions or disease modeling purposes.  

5.3261           Neutralizing Antibody Evasion and Transduction with Purified Extracellular Vesicle-Enveloped Adeno-Associated Virus Vectors

Cheng, M., Dietz, l., Gong, Y., Eichler, F., Nammour, J., Ng, C., Grimm, D. and maguire, C.A. Human Gene Therapy, 32(23-24), 1457-1470 (2021)   Adeno-associated virus (AAV) is classified as a nonenveloped DNA virus. However, several years ago, we discovered that in media of packaging cells producing recombinant AAV vectors, AAV capsids can associate with the interior and surface of extracellular vesicles (EVs), sometimes referred to as exosomes. Since then, we and others have demonstrated that exosome-enveloped AAV, exo-AAV, can enhance transduction in vivo as well as evade neutralizing antibodies. While promising, these data were generated with differential centrifugation to pellet the exo-AAV. This method results in a heterogeneous mixture of exo-AAV, coprecipitating proteins, as well as free AAV capsids. To define the properties of exo-AAV more accurately, in this study, we used a density gradient method to purify exo-AAV. We next performed head-to-head comparisons of standard AAV1, differential centrifuged exo-AAV1, and gradient purified exo-AAV1 for antibody evasion and transgene expression in the murine brain. We found purified exo-AAV1 to be more resistant to neutralizing antibodies than the other AAV preparations. Direct intracranial injection of purified exo-AAV1 into mice resulted in robust transduction, which transduced a larger area of brain than standard AAV1. We also identified the recently described membrane-associated accessory protein by mass spectrometry of purified exo-AAV1 preparations. Finally, we used a scalable method, size-exclusion chromatography to isolate exo-AAV1, and demonstrated functional transduction in cultured cells and increased antibody resistance. Together, these data suggest that higher purity exo-AAV will have beneficial characteristics for gene delivery and also may lead to mechanistic insights into the incorporation of AAV into EVs.    

5.3262           HIV-1 Env-Dependent Cell Killing by Bifunctional Small-Molecule/Peptide Conjugates

Gaffney, A., Nangarlia, A., Ang, C.G., Gossert, S., Ahmed, A.A.R., Hossain, M.A., Abrams, C.F., Smith III, A.B. and Chaiken, I. ASC Chem. Biol., 16, 193-204 (2021)   A strategy has been established for the synthesis of a family of bifunctional HIV-1 inhibitor covalent conjugates with the potential to bind simultaneously to both the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein trimeric complex (Env). One component of the conjugates is derived from BNM-III-170, a small-molecule CD4 mimic that binds to gp120. The second component, comprised of the peptide DKWASLWNW (“Trp3”), was derived from the N-terminus of the HIV-1 gp41 Membrane Proximal External Region (MPER) and found previously to bind to the gp41 subunit of Env. The resulting bifunctional conjugates were shown to inhibit virus cell infection with low micromolar potency and to induce lysis of the HIV-1 virion. Crucially, virolysis was found to be dependent on the covalent linkage of the BNM-III-170 and Trp3 domains, as coadministration of a mixture of the un-cross-linked components proved to be nonlytic. However, a significant magnitude of lytic activity was observed in Env-negative and other control pseudoviruses, suggesting parallel mechanisms of action of the conjugates involving Env interaction and direct membrane disruption. Computational modeling suggested strong membrane-binding activity of BNM-III-170, which may underly the nonspecific virolytic effects of the conjugates. To investigate the scope of the membrane effect, cell-based cytotoxicity and membrane permeability assays were performed employing flow cytometry. Here, we observed a dose-dependent and specific cytotoxic effect on HIV-1 Env-expressing cells by the small-molecule bifunctional inhibitor. Most importantly, Env-negative cells were not susceptible to the cytotoxic effect upon exposure to this construct at concentrations where cell-killing effects were observed for Env-positive cells. Computational structural modeling supports a mechanism in which the bifunctional inhibitors bind to the gp120 and gp41 subunits in tandem in open-state Env trimers and induce relative motion of the gp120 subunits consistent with models of Env inactivation. This observation supports the idea that the cell-killing effect of the small-molecule bifunctional inhibitor is due to specific Env conformational triggering. This work lays important groundwork to advance a small-molecule bifunctional inhibitor approach for eliminating Env-expressing infected cells and the eradication of HIV-1.  

5.3263           Inhibiting HTLV-1 Protease: A Viable Antiviral Target

Lockbaum, G.J., Henes, M., Talledge, N., Rusere, L.N., Kosovrasti, K., Nalivaika, E.A., Somsanduran, M., Ali, A., Mansky, L.M., Yilmaz, N.K. and Schiffer, C.A. ACS Chem. Biol., 16, 529-538 (2021)   Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that can cause severe paralytic neurologic disease and immune disorders as well as cancer. An estimated 20 million people worldwide are infected with HTLV-1, with prevalence reaching 30% in some parts of the world. In stark contrast to HIV-1, no direct acting antivirals (DAAs) exist against HTLV-1. The aspartyl protease of HTLV-1 is a dimer similar to that of HIV-1 and processes the viral polyprotein to permit viral maturation. We report that the FDA-approved HIV-1 protease inhibitor darunavir (DRV) inhibits the enzyme with 0.8 μM potency and provides a scaffold for drug design against HTLV-1. Analogs of DRV that we designed and synthesized achieved submicromolar inhibition against HTLV-1 protease and inhibited Gag processing in viral maturation assays and in a chronically HTLV-1 infected cell line. Cocrystal structures of these inhibitors with HTLV-1 protease highlight opportunities for future inhibitor design. Our results show promise toward developing highly potent HTLV-1 protease inhibitors as therapeutic agents against HTLV-1 infections.  

5.3264           Dementia with Lewy bodies—associated ß-synuclein mutations V70M and P123H cause mutation-specific neuropathological lesions

Psol, M., Darvas, S.G., Leite, K., Mahajani, S.U., Bähr, M. and Kügler, S. Hum. Mol. Genet., 30(3-4), 247-264 (2021)   Beta (ß)-synuclein (ß-Syn) has long been considered to be an attenuator for the neuropathological effects caused by the Parkinson’s disease-related alpha (α)-synuclein (α-Syn) protein. However, recent studies demonstrated that overabundant ß-Syn can form aggregates and induce neurodegeneration in central nervous system (CNS) neurons in vitro and in vivo, albeit at a slower pace as compared with α-Syn. Here, we demonstrate that ß-Syn mutants V70M, detected in a sporadic case of dementia with Lewy bodies (DLB), and P123H, detected in a familial case of DLB, robustly aggravate the neurotoxic potential of ß-Syn. Intriguingly, the two mutations trigger mutually exclusive pathways. ß-Syn V70M enhances morphological mitochondrial deterioration and degeneration of dopaminergic and non-dopaminergic neurons, but it has no influence on neuronal network activity. Conversely, ß-Syn P123H silences neuronal network activity, but it does not aggravate neurodegeneration. ß-Syn wild type (WT), V70M and P123H formed proteinase K-resistant intracellular fibrils within neurons, albeit with less stable C-termini as compared with α-Syn. Under cell-free conditions, ß-Syn V70M demonstrated a much slower pace of fibril formation as compared with WT ß-Syn, and P123H fibrils present with a unique phenotype characterized by large numbers of short, truncated fibrils. Thus, it is possible that V70M and P123H cause structural alterations in ß-Syn, which are linked to their distinct neuropathological profiles. The extent of the lesions caused by these neuropathological profiles is almost identical to that of overabundant α-Syn and is thus likely to be directly involved into the etiology of DLB. Overall, this study provides insights into distinct disease mechanisms caused by mutations of ß-Syn.  

5.3265           ALS/FTD-causing mutation in cyclin F causes the dysregulation of SFPQ

Rayner, S.L., Cheng, F., Hogan, A.L., Grima, N., Yang, S. et al Hum. Mol. Genet., 30(11), 971-984 (2021)   Previously, we identified missense mutations in CCNF that are causative of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Hallmark features of these diseases include the build-up of insoluble protein aggregates as well as the mislocalization of proteins such as transactive response DNA binding protein 43 kDa (TDP-43). In recent years, the dysregulation of SFPQ (splicing factor proline and glutamine rich) has also emerged as a pathological hallmark of ALS/FTD. CCNF encodes for the protein cyclin F, a substrate recognition component of an E3 ubiquitin ligase. We have previously shown that ALS/FTD-linked mutations in CCNF cause disruptions to overall protein homeostasis that leads to a build-up of K48-linked ubiquitylated proteins as well as defects in autophagic machinery. To investigate further processes that may be affected by cyclin F, we used a protein-proximity ligation method, known as Biotin Identification (BioID), standard immunoprecipitations and mass spectrometry to identify novel interaction partners of cyclin F and infer further process that may be affected by the ALS/FTD-causing mutation. Results demonstrate that cyclin F closely associates with proteins involved with RNA metabolism as well as a number of RNA-binding proteins previously linked to ALS/FTD, including SFPQ. Notably, the overexpression of cyclin F(S621G) led to the aggregation and altered subcellular distribution of SFPQ in human embryonic kidney (HEK293) cells, while leading to altered degradation in primary neurons. Overall, our data links ALS/FTD-causing mutations in CCNF to converging pathological features of ALS/FTD and provides a link between defective protein degradation systems and the pathological accumulation of a protein involved in RNA processing and metabolism.  

5.3266           Dual targeting of brain region-specific kinases potentiates neurological rescue in Spinocerebellar ataxia type 1

Lee, W-S., Lavery, L., Rousseaux, M.W.C., Rutledge, E.B., Jang, Y. et al EMBO J., 40, e106106 (2021)   A critical question in neurodegeneration is why the accumulation of disease-driving proteins causes selective neuronal loss despite their brain-wide expression. In Spinocerebellar ataxia type 1 (SCA1), accumulation of polyglutamine-expanded Ataxin-1 (ATXN1) causes selective degeneration of cerebellar and brainstem neurons. Previous studies revealed that inhibiting Msk1 reduces phosphorylation of ATXN1 at S776 as well as its levels leading to improved cerebellar function. However, there are no regulators that modulate ATXN1 in the brainstem—the brain region whose pathology is most closely linked to premature death. To identify new regulators of ATXN1, we performed genetic screens and identified a transcription factor-kinase axis (ZBTB7B-RSK3) that regulates ATXN1 levels. Unlike MSK1, RSK3 is highly expressed in the human and mouse brainstems where it regulates Atxn1 by phosphorylating S776. Reducing Rsk3 rescues brainstem-associated pathologies and deficits, and lowering Rsk3 and Msk1 together improves cerebellar and brainstem function in an SCA1 mouse model. Our results demonstrate that selective vulnerability of brain regions in SCA1 is governed by region-specific regulators of ATXN1, and targeting multiple regulators could rescue multiple degenerating brain areas.  

5.3267           Mutations in the IFNγ-JAK-STAT Pathway Causing Resistance to Immune Checkpoint Inhibitors in Melanoma Increase Sensitivity to Oncolytic Virus Treatment

Hguyen, T-T., Ramsey, L., Ahanfeshar-Adams, M., Lajoie, M., Schadendorf, D., Alain, T. and Watson, I.R. Clin. Cancer Res., 27, 3432-3442 (2021)   Purpose: Next-generation sequencing studies and CRISPR-Cas9 screens have established mutations in the IFNγ-JAK-STAT pathway as an immune checkpoint inhibitor (ICI) resistance mechanism in a subset of patients with melanoma. We hypothesized ICI resistance mutations in the IFNγ pathway would simultaneously render melanomas susceptible to oncolytic virus (OV) therapy. Experimental Design: Cytotoxicity experiments were performed with a number of OVs on a matched melanoma cell line pair generated from a baseline biopsy and a progressing lesion with complete JAK2 loss from a patient that relapsed on anti-PD-1 therapy, in melanoma lines following JAK1/2 RNA interference (RNAi) and pharmacologic inhibition and in Jak2 knockout (KO) B16-F10 mouse melanomas. Furthermore, we estimated the frequency of genetic alterations in the IFNγ-JAK-STAT pathway in human melanomas. Results: The melanoma line from an anti-PD-1 progressing lesion was 7- and 22-fold more sensitive to the modified OVs, herpes simplex virus 1 (HSV1-dICP0) and vesicular stomatitis virus (VSV-Δ51), respectively, compared with the line from the baseline biopsy. RNAi, JAK1/2 inhibitor studies, and in vivo studies of Jak2 KOs B16-F10 melanomas revealed a significant increase in VSV-Δ51 sensitivity with JAK/STAT pathway inhibition. Our analysis of The Cancer Genome Atlas data estimated that approximately 11% of ICI-naïve cutaneous melanomas have alterations in IFNγ pathway genes that may confer OV susceptibility. Conclusions: We provide mechanistic support for the use of OVs as a precision medicine strategy for both salvage therapy in ICI-resistant and first-line treatment in melanomas with IFNγ-JAK-STAT pathway mutations. Our study also supports JAK inhibitor–OV combination therapy for treatment-naïve melanomas without IFN signaling defects.  

5.3268           Golgi apparatus-synthesized sulfated glycosaminoglycans mediate polymerization and activation of the cGAMP sensor STING

Fang, R., Jiang, Q., Guan, Y., Gao, P., Zhang, R., Zhao, Z. and Jiang, Z. Immunity, 54, 962-975 (2021)   Activation of the cyclic guanosine monophosphate (GMP)-AMP (cGAMP) sensor STING requires its translocation from the endoplasmic reticulum to the Golgi apparatus and subsequent polymerization. Using a genome-wide CRISPR-Cas9 screen to define factors critical for STING activation in cells, we identified proteins critical for biosynthesis of sulfated glycosaminoglycans (sGAGs) in the Golgi apparatus. Binding of sGAGs promoted STING polymerization through luminal, positively charged, polar residues. These residues are evolutionarily conserved, and selective mutation of specific residues inhibited STING activation. Purified or chemically synthesized sGAGs induced STING polymerization and activation of the kinase TBK1. The chain length and O-linked sulfation of sGAGs directly affected the level of STING polymerization and, therefore, its activation. Reducing the expression of Slc35b2 to inhibit GAG sulfation in mice impaired responses to vaccinia virus infection. Thus, sGAGs in the Golgi apparatus are necessary and sufficient to drive STING polymerization, providing a mechanistic understanding of the requirement for endoplasmic reticulum (ER)-to-Golgi apparatus translocation for STING activation.  

5.3269           4R Tau Modulates Cocaine-Associated Memory through Adult Dorsal Hippocampal Neurogenesis

Li, H., Xu, W., Wang, D., Wang, L., Fang, Q., Wan, X. et al

1. Neurosci., 41(31), 6753-6774 (2021)

  The development, persistence and relapse of drug addiction require drug memory that generally develops with drug administration-paired contextual stimuli. Adult hippocampal neurogenesis (AHN) contributes to cocaine memory formation; however, the underlying mechanism remains unclear. Male mice hippocampal expression of Tau was significantly decreased during the cocaine-associated memory formation. Genetic overexpression of four microtubule-binding repeats Tau (4R Tau) in the mice hippocampus disrupted cocaine memory by suppressing AHN. Furthermore, 4R Tau directly interacted with phosphoinositide 3-kinase (PI3K)-p85 and impaired its nuclear translocation and PI3K-AKT signaling, processes required for hippocampal neuron proliferation. Collectively, 4R Tau modulates cocaine memory formation by disrupting AHN, suggesting a novel mechanism underlying cocaine memory formation and provide a new strategy for the treatment of cocaine addiction.  

5.3270           Dissociated Role of Thalamic and Cortical Input to the Lateral Amygdala for Consolidation of Long-Term Fear Memory

Lee, Y., Oh, J-P. and Han, J-H.

1. Neurosci., 41(46), 9561-9570 (2021)

  Post-encoding coordinated reactivation of memory traces distributed throughout interconnected brain regions is thought to be critical for consolidation of memories. However, little is known about the role of neural circuit pathways during post-learning periods for consolidation of memories. To investigate this question, we optogenetically silenced the inputs from both auditory cortex and thalamus in the lateral amygdala (LA) for 15 min immediately following auditory fear conditioning (FC) and examined its effect on fear memory formation in mice of both sexes. Optogenetic inhibition of both inputs disrupted long-term fear memory formation tested 24 h after FC. This effect was specific such that the same inhibition did not affect short-term memory and context-dependent memory. Moreover, long-term memory was intact if the inputs were inhibited at much later time points after FC (3 h or 1 d after FC), indicating that optical inhibition for 15 min itself does not produce any nonspecific deleterious effect on fear memory retrieval. Selective inhibition of thalamic input was sufficient to impair consolidation of auditory fear memory. In contrast, selective inhibition of cortical input disrupted remote fear memory without affecting recent memory. These results reveal a dissociated role of thalamic and cortical input to the LA during early post-learning periods for consolidation of long-term fear memory.  

5.3271           Context-Specific Function of the Engineered Peptide Domain of PHP.B

Martino, R.A., Fluck III, E.C., Murphy, J., Wang, Q., Hoff, H., Pumroy, R.A., Lee, C.Y., Sims, J.J., Roy, S., Moiseenkova-Bell, V.Y. and Wilson, J.M:

1. Virol., 95(20), e01164-21 (2021)

  One approach to improve the utility of adeno-associated virus (AAV)-based gene therapy is to engineer the AAV capsid to (i) overcome poor transport through tissue barriers and (ii) redirect the broadly tropic AAV to disease-relevant cell types. Peptide- or protein-domain insertions into AAV surface loops can achieve both engineering goals by introducing a new interaction surface on the AAV capsid. However, we understand little about the impact of insertions on capsid structure and the extent to which engineered inserts depend on a specific capsid context to function. Here, we examine insert-capsid interactions for the engineered variant AAV9-PHP.B. The 7-amino-acid peptide insert in AAV9-PHP.B facilitates transport across the murine blood-brain barrier via binding to the receptor Ly6a. When transferred to AAV1, the engineered peptide does not bind Ly6a. Comparative structural analysis of AAV1-PHP.B and AAV9-PHP.B revealed that the inserted 7-amino-acid loop is highly flexible and has remarkably little impact on the surrounding capsid conformation. Our work demonstrates that Ly6a binding requires interactions with both the PHP.B peptide and specific residues from the AAV9 HVR VIII region. An AAV1-based vector that incorporates a larger region of AAV9-PHP.B—including the 7-amino-acid loop and adjacent HVR VIII amino acids—can bind to Ly6a and localize to brain tissue. However, unlike AAV9-PHP.B, this AAV1-based vector does not penetrate the blood-brain barrier. Here we discuss the implications for AAV capsid engineering and the transfer of engineered activities between serotypes.  

5.3272           MicroRNA-145-5p targeting of TRIM2 mediates the apoptosis of retinal ganglion cells via the PI3K/AKT signaling pathway in glaucoma

Wu, K., LI, S., Yang, Q., Zhou, Z., Fu, M., Yang, X., Hao, K., Liu, Y. and Ji, H.

1. Gene Med., 23, e3378 (2021)

  Background There is accumulating evidence to suggest that microRNAs (miRNAs) are associated with the progressive optic neuropathy including glaucoma. Apoptosis of retinal ganglion cells (RGCs) is a hallmark of glaucoma. The present study focused on the effects of miR-145-5p on RGC apoptosis in glaucoma. Methods We established a glaucoma rat model by intraocular injection of N-methyl-d-aspartic acid (NMDA). RGCs were isolated from newborn rats and treated with NMDA. Hematoxylin and eosin staining was performed to detect morphological changes in the retinas of rats. The expression of miR-145-5p and tripartite motif-containing 2 (TRIM2) in RGCs was measured by RT-qPCR. The viability of RGCs was measured by MTT assay. Flow cytometry analysis and TUNEL assays were conducted to assess the apoptosis of RGCs. The interaction between miR-145-5p and TRIM2 was investigated using a luciferase reporter assay. Results Rats injected with NMDA showed a thinner ganglion cell layer (GCL) and inner plexiform layer (IPL) as well as increased expression of miR-145-5p. Silencing of miR-145-5p significantly increased the GCL and IPL in the glaucoma rat model. Moreover, miR-145-5p expression was upregulated in RGCs ex vivo in response to NMDA. Silencing of miR-145-5p promoted cell viability and suppressed apoptosis in NMDA-treated RGCs. Mechanistically, miR-145-5p targeted the TRIM2 3′ untranslated region to suppress its expression. TRIM2 was upregulated in NMDA-treated RGCs and protected RGCs against NMDA-induced apoptosis. Furthermore, miR-145-5p suppressed the PI3K/AKT pathway by downregulating TRIM2 in NMDA-treated RGCs. Conclusions Suppression of miR-145-5p inhibited the apoptosis of RGCs via TRIM2-mediated activation of the PI3K/AKT signaling pathway in NMDA-induced glaucoma.  

5.3273           Gene Editing Rescues In vitro T Cell Development of RAG2-Deficient Induced Pluripotent Stem Cells in an Artificial Thymic Organoid System

Gardner, C.L., Pavel.Dinu, M., Dobbs, K., Bosticardo, M., Reardon, P.K., Lack, J. et al

1. Clin. Immunol., 41, 852-862 (2021)

  Severe combined immune deficiency (SCID) caused by RAG1 or RAG2 deficiency is a genetically determined immune deficiency characterized by the virtual absence of T and B lymphocytes. Unless treated with hematopoietic stem cell transplantation (HSCT), patients with RAG deficiency succumb to severe infections early in life. However, HSCT carries the risk of graft-versus-host disease. Moreover, a high rate of graft failure and poor immune reconstitution have been reported after unconditioned HSCT. Expression of the RAG genes is tightly regulated, and preclinical attempts of gene therapy with heterologous promoters have led to controversial results. Using patient-derived induced pluripotent stem cells (iPSCs) and an in vitro artificial thymic organoid system as a model, here we demonstrate that gene editing rescues the progressive T cell differentiation potential of RAG2-deficient cells to normal levels, with generation of a diversified T cell repertoire. These results suggest that targeted gene editing may represent a novel therapeutic option for correction of this immunodeficiency.  

5.3274           Nrn1 Overexpression Attenuates Retinal Ganglion Cell Apoptosis, Promotes Axonal Regeneration, and Improves Visual Function Following Optic Nerve Crush in Rats

Huang, T., Li, H., Zhang, S., Liu, F., Wang, D. and Xu, J.

1. Mol. Neurosci., 71, 66-79 (2021)

  Neuritin (Nrn1) is a small highly conserved extracellular membrane protein involved in the process of neural cell survival and differentiation, axonal and dendritic growth, and synapse formation and maturation. Previous studies have demonstrated that intravitreal injection of recombinant Nrn1 as a gene therapy could alleviate retinal ganglion cell (RGC) apoptosis and promote optic nerve axon regeneration after optic nerve crush (ONC). However, the mechanism underlying the repairing effect of Nrn1 against optic never injury remains elusive. In this study, a rAAV2-mediated Nrn1 overexpression vector (AAV2-Nrn1) was applied to treat ONC through intravitreal injection for the purpose of further exploring the effect and mechanism of Nrn1 in repairing the injured optic nerve. The results showed that AAV2-Nrn1 was mainly transfected into RGCs without affecting astrocytes. Nrn1 overexpression effectively reduced RGC apoptosis and promoted optic nerve regeneration and visual function restoration as demonstrated by retinal imaging, histopathological analysis, and physiological function detection in vivo following ONC. Immunoblot assay revealed that functional molecules of Nrn1 activated the Akt1 and Stat3 pathways and inhibited the mitochondrial apoptotic pathway. The results of the present study may provide experimental evidence for further application of Nrn1 to the clinical treatment of optic nerve injury.  

5.3275           In vitro functional genetic modification of canine adenovirus type 2 genome by CRISPR/Cas9

Sajib, A.M., Agarwal, P., Patton, D.J., Nance, R.L., Stahr, N.A., Kretzschmar, W.P., Sandey, M. and Smith, B.F. Lab. Invest., 101, 1627-1636 (2021)   Genetically modified oncolytic adenoviruses have been proposed as a vehicle for cancer therapy. However, several concerns, such as toxicity to normal cells and organs, lack of suitable cell surface receptors to allow viral entry to the desired cell type(s), and activation of both innate and adaptive immune systems in patients, restrict the successful clinical application of adenoviral-mediated cancer gene therapy. Successful virotherapy will require efficient transductional and transcriptional targeting to enhance therapeutic efficacy by ensuring targeted adenoviral infection, replication, and/or therapeutic transgene expression. Targeted modification of viral components, such as viral capsid, fiber knob, and the insertion of transgenes for expression, are prerequisites for the necessary transductional and transcriptional targeting of adenovirus. However, the conventional approach to modify the adenoviral genome is complex, time consuming, and expensive. It is dependent on the presence of unique restriction enzyme sites that may or may not be present in the target location. Clustered regularly interspaced short palindromic repeat (CRISPR) along with the RNA-guided nuclease Cas9 (CRISPR/Cas9) is one of the most powerful tools that has been adopted for precise genome editing in a variety of cells and organisms. However, the ability of the CRISPR/Cas9 system to precisely and efficiently make genetic modification, as well as introduce gene replacements, in adenoviral genomes, remains essentially unknown. Herein the ability of in vitro CRISPR/CAS9-mediated editing of the canine adenovirus type 2 (CAV2) genome to promote targeted modification of the viral genome was assessed. To demonstrate the feasibility of this goal, CRISPR/Cas9 has been used to successfully insert the RFP (red fluorescent protein) reporter construct into the CAV2 genome. Initial results demonstrated high efficiency and accuracy for in vitro CRISPR-mediated editing of the large CAV2 genome. Furthermore, this application was expanded, using multiple guide RNAs, to conduct gene replacement in the CAV2 genome by substituting a portion of the E3 gene with a construct designed to express a single chain antibody to canine PD-1. Thus, this work provides a significantly improved and efficient method for targeted editing of adenoviruses to generate altered and potentially therapeutic viral genomes in the shortest possible time.  

5.3276           Leukocyte cell-derived chemotaxin 2 promotes the development of nonalcoholic fatty liver disease through STAT-1 pathway in mice

Wang, J., Chen, Y., Pan, R., Wu, C., Chen, S., Li, L:, Li, Y., Yu, C., Meng, Z-x and Xu, C. Liver Int., 41, 777-787 (2021)   Background Nonalcoholic fatty liver disease (NAFLD), whose pathogenesis remains unelucidated, has become an increasingly prevalent disease globally requiring novel treatment strategies. This study aims to explore the role of leukocyte cell-derived chemotaxin 2 (LECT2), one of the known hepatokines, in the development of NAFLD. Methods The serum LECT2 level was evaluated in patients with NAFLD and male C57BL/6 mice fed a high-fat diet (HFD) for 8 weeks. Tail intravenous injection of adeno-associated virus that contained Lect2 short hairpin RNA or Lect2 overexpression plasmid was administered to mice to inhibit or increase hepatic Lect2 expression. Hepatic steatosis was evaluated by histological staining with haematoxylin and eosin and Oil Red O, and also by quantitative hepatic triglyceride measurements. RNA-seq was performed to discover the specific targets of LECT2 on NAFLD. Results Serum and hepatic LECT2 levels were elevated in NAFLD patients and HFD-fed mice. Inhibition of hepatic Lect2 expression alleviated HFD-induced hepatic steatosis and inflammation, whereas hepatic overexpression of Lect2 aggravated HFD-induced hepatic steatosis and inflammation. RNA-seq and bioinformatical analysis suggested that the signal transducers and activators of transcription-1 (STAT-1) pathway might play an indispensable role in the interaction between LECT2 and NAFLD. A STAT-1 inhibitor could reverse the accumulation of hepatic lipids caused by Lect2 overexpression. Conclusion LECT2 expression is significantly elevated in NAFLD. LECT2 induces the occurrence and development of NAFLD through the STAT-1 pathway. LECT2 may be a potential therapeutic target for NAFLD.  

5.3277           Liver alanine catabolism promotes skeletal muscle atrophy and hyperglycaemia in type 2 diabetes

Okun, J.G., Rusu, P.M., Chan, A.Y., Wu, Y., Yap, Y.W., Sharkie, T. et al Nature Metabolism, 3, 394-409 (2021)   Both obesity and sarcopenia are frequently associated in ageing, and together may promote the progression of related conditions such as diabetes and frailty. However, little is known about the pathophysiological mechanisms underpinning this association. Here we show that systemic alanine metabolism is linked to glycaemic control. We find that expression of alanine aminotransferases is increased in the liver in mice with obesity and diabetes, as well as in humans with type 2 diabetes. Hepatocyte-selective silencing of both alanine aminotransferase enzymes in mice with obesity and diabetes retards hyperglycaemia and reverses skeletal muscle atrophy through restoration of skeletal muscle protein synthesis. Mechanistically, liver alanine catabolism driven by chronic glucocorticoid and glucagon signalling promotes hyperglycaemia and skeletal muscle wasting. We further provide evidence for amino acid–induced metabolic cross-talk between the liver and skeletal muscle in ex vivo experiments. Taken together, we reveal a metabolic inter-tissue cross-talk that links skeletal muscle atrophy and hyperglycaemia in type 2 diabetes.    

5.3278           In vivo base editing rescues Hutchinson–Gilford progeria syndrome in mice

Koblan, L.W., Erdos, M.R., Wilson, C., Cabral, W.A., levy, J.M. et al Nature, 589, 608-614 (2021)   Hutchinson–Gilford progeria syndrome (HGPS or progeria) is typically caused by a dominant-negative C•G-to-T•A mutation (c.1824 C>T; p.G608G) in LMNA, the gene that encodes nuclear lamin A. This mutation causes RNA mis-splicing that produces progerin, a toxic protein that induces rapid ageing and shortens the lifespan of children with progeria to approximately 14 years1^,2^,3^,4. Adenine base editors (ABEs) convert targeted A•T base pairs to G•C base pairs with minimal by-products and without requiring double-strand DNA breaks or donor DNA templates5^,6. Here we describe the use of an ABE to directly correct the pathogenic HGPS mutation in cultured fibroblasts derived from children with progeria and in a mouse model of HGPS. Lentiviral delivery of the ABE to fibroblasts from children with HGPS resulted in 87–91% correction of the pathogenic allele, mitigation of RNA mis-splicing, reduced levels of progerin and correction of nuclear abnormalities. Unbiased off-target DNA and RNA editing analysis did not detect off-target editing in treated patient-derived fibroblasts. In transgenic mice that are homozygous for the human LMNA c.1824 C>T allele, a single retro-orbital injection of adeno-associated virus 9 (AAV9) encoding the ABE resulted in substantial, durable correction of the pathogenic mutation (around 20–60% across various organs six months after injection), restoration of normal RNA splicing and reduction of progerin protein levels. In vivo base editing rescued the vascular pathology of the mice, preserving vascular smooth muscle cell counts and preventing adventitial fibrosis. A single injection of ABE-expressing AAV9 at postnatal day 14 improved vitality and greatly extended the median lifespan of the mice from 215 to 510 days. These findings demonstrate the potential of in vivo base editing as a possible treatment for HGPS and other genetic diseases by directly correcting their root cause.  

5.3279           Neuroprosthetic baroreflex controls haemodynamics after spinal cord injury

Squair, J.W:, Gautier, M., Mahe, L., Soriano, J., Rowald, A., Biochat, A. et al Nature, 590, 308-314 (2021)   Spinal cord injury (SCI) induces haemodynamic instability that threatens survival1^,2^,3, impairs neurological recovery4^,5, increases the risk of cardiovascular disease6^,7, and reduces quality of life8^,9. Haemodynamic instability in this context is due to the interruption of supraspinal efferent commands to sympathetic circuits located in the spinal cord10, which prevents the natural baroreflex from controlling these circuits to adjust peripheral vascular resistance. Epidural electrical stimulation (EES) of the spinal cord has been shown to compensate for interrupted supraspinal commands to motor circuits below the injury11, and restored walking after paralysis12. Here, we leveraged these concepts to develop EES protocols that restored haemodynamic stability after SCI. We established a preclinical model that enabled us to dissect the topology and dynamics of the sympathetic circuits, and to understand how EES can engage these circuits. We incorporated these spatial and temporal features into stimulation protocols to conceive a clinical-grade biomimetic haemodynamic regulator that operates in a closed loop. This ‘neuroprosthetic baroreflex’ controlled haemodynamics for extended periods of time in rodents, non-human primates and humans, after both acute and chronic SCI. We will now conduct clinical trials to turn the neuroprosthetic baroreflex into a commonly available therapy for people with SCI.  

5.3280           Bispecific IgG neutralizes SARS-CoV-2 variants and prevents escape in mice

De Gasparo, R., Pedotti, M., Simonelli, L., Nickl, P., Muecksch, F., Cassaniti, I. et al Nature, 593, 424-428 (2021)   Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191^,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.  

5.3281           GluD1 is a signal transduction device disguised as an ionotropic receptor

Dal, J., Patzke, C., Liaskath-Ali, K., Selgneur, e. and Südhof, T.C. Nature, 595, 261-265 (2021)   Ionotropic glutamate delta receptors 1 (GluD1) and 2 (GluD2) exhibit the molecular architecture of postsynaptic ionotropic glutamate receptors, but assemble into trans-synaptic adhesion complexes by binding to secreted cerebellins that in turn interact with presynaptic neurexins1^,2^,3^,4. It is unclear whether neurexin–cerebellin–GluD1/2 assemblies serve an adhesive synapse-formation function or mediate trans-synaptic signalling. Here we show in hippocampal synapses, that binding of presynaptic neurexin–cerebellin complexes to postsynaptic GluD1 controls glutamate receptor activity without affecting synapse numbers. Specifically, neurexin-1–cerebellin-2 and neurexin-3–cerebellin-2 complexes differentially regulate NMDA (N-methyl-D-aspartate) receptors and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors by activating distinct postsynaptic GluD1 effector signals. Of note, minimal GluD1 and GluD2 constructs containing only their N-terminal cerebellin-binding and C-terminal cytoplasmic domains, joined by an unrelated transmembrane region, fully control the levels of NMDA and AMPA receptors. The distinct signalling specificity of presynaptic neurexin-1 and neurexin-35^,6 is encoded by their alternatively spliced splice site 4 sequences, whereas the regulatory functions of postsynaptic GluD1 are mediated by conserved cytoplasmic sequence motifs spanning 5–13 residues. Thus, GluDs are signalling molecules that regulate NMDA and AMPA receptors by an unexpected transduction mechanism that bypasses their ionotropic receptor architecture and directly converts extracellular neurexin–cerebellin signals into postsynaptic receptor responses.  

5.3282           Mechanical actions of dendritic-spine enlargement on presynaptic exocytosis

Ucar, H., Watanabe, S., Noguchi, J., Morimoto, Y., Jino, Y., Yagishita, S., Takahashi, N. and Kasai, H. Nature, 600, 686-689 (2021)   Synaptic transmission involves cell-to-cell communication at the synaptic junction between two neurons, and chemical and electrical forms of this process have been extensively studied. In the brain, excitatory glutamatergic synapses are often made on dendritic spines that enlarge during learning1^,2^,3^,4^,5. As dendritic spines and the presynaptic terminals are tightly connected with the synaptic cleft6, the enlargement may have mechanical effects on presynaptic functions7. Here we show that fine and transient pushing of the presynaptic boutons with a glass pipette markedly promotes both the evoked release of glutamate and the assembly of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins8^,9^,10^,11^,12—as measured by Förster resonance transfer (FRET) and fluorescence lifetime imaging—in rat slice culture preparations13. Both of these effects persisted for more than 20 minutes. The increased presynaptic FRET was independent of cytosolic calcium (Ca²⁺), but dependent on the assembly of SNARE proteins and actin polymerization in the boutons. Notably, a low hypertonic solution of sucrose (20 mM) had facilitatory effects on both the FRET and the evoked release without inducing spontaneous release, in striking contrast with a high hypertonic sucrose solution (300 mM), which induced exocytosis by itself14. Finally, spine enlargement induced by two-photon glutamate uncaging enhanced the evoked release and the FRET only when the spines pushed the boutons by their elongation. Thus, we have identified a mechanosensory and transduction mechanism15 in the presynaptic boutons, in which the evoked release of glutamate is enhanced for more than 20 min.  

5.3283           AIDA directly connects sympathetic innervation to adaptive thermogenesis by UCP1

Shi, M., Huang, X-Y., Ren, X-Y., Wei, X-Y., Ma, Y., Lin, Z-Z. et al Nature Cell Biol., 23, 268-277 (2021)   The sympathetic nervous system–catecholamine–uncoupling protein 1 (UCP1) axis plays an essential role in non-shivering adaptive thermogenesis. However, whether there exists a direct effector that physically connects catecholamine signalling to UCP1 in response to acute cold is unknown. Here we report that outer mitochondrial membrane-located AIDA is phosphorylated at S161 by the catecholamine-activated protein kinase A (PKA). Phosphorylated AIDA translocates to the intermembrane space, where it binds to and activates the uncoupling activity of UCP1 by promoting cysteine oxidation of UCP1. Adipocyte-specific depletion of AIDA abrogates UCP1-dependent thermogenesis, resulting in hypothermia during acute cold exposure. Re-expression of S161A-AIDA, unlike wild-type AIDA, fails to restore the acute cold response in Aida-knockout mice. The PKA–AIDA–UCP1 axis is highly conserved in mammals, including hibernators. Denervation of the sympathetic postganglionic fibres abolishes cold-induced AIDA-dependent thermogenesis. These findings uncover a direct mechanistic link between sympathetic input and UCP1-mediated adaptive thermogenesis.  

5.3284           Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations

Wang, L., Xue, W., Zhang, H., Gao, R., Qiu, H., Wei, J. et al Nature Cell Biol., 23, 552-563 (2021)   The fusion of CRISPR–Cas9 with cytidine deaminases leads to base editors (BEs) capable of programmable C-to-T editing, which has potential in clinical applications but suffers from off-target (OT) mutations. Here, we used a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, the tBE remains inactive at OT sites with the fusion of a cleavable dCDI, therefore eliminating unintended mutations. When binding at on-target sites, the tBE is transformed to cleave off the dCDI domain and catalyses targeted deamination for precise base editing. After delivery into mice through a dual-adeno-associated virus (AAV) system, the tBE system created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9, resulting in a ~30–40% decrease in total cholesterol. The development of tBE establishes a highly specific base editing system and its in vivo efficacy has potential for therapeutic applications.  

5.3285           Gene replacement of α-globin with β-globin restores hemoglobin balance in β-thalassemia-derived hematopoietic stem and progenitor cells

Cromer, M.K., Camarena, J., Martin, R.M., Lesch, B.J., Vakulskas, C.A. et al Nature Med., 27, 677-687 (2021)   β-Thalassemia pathology is due not only to loss of β-globin (HBB), but also to erythrotoxic accumulation and aggregation of the β-globin-binding partner, α-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in β-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize β-globin:α-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing β-thalassemia.  

5.3286           Anterograde transneuronal tracing and genetic control with engineered yellow fever vaccine YFV-17D

Li, E., Guo, J., Oh, S.J., Luo, Y., Oliveros, H.C., Du, W., Arano, R. et al Nature Methods, 18, 1542-1551 (2021)   Transneuronal viruses are powerful tools for tracing neuronal circuits or delivering genes to specific neurons in the brain. While there are multiple retrograde viruses, few anterograde viruses are available. Further, available anterograde viruses often have limitations such as retrograde transport, high neuronal toxicity or weak signals. We developed an anterograde viral system based on a live attenuated vaccine for yellow fever—YFV-17D. Replication- or packaging-deficient mutants of YFV-17D can be reconstituted in the brain, leading to efficient synapse-specific and anterograde-only transneuronal spreading, which can be controlled to achieve either monosynaptic or polysynaptic tracing. Moreover, inducible transient replication of YFV-17D mutant is sufficient to induce permanent transneuronal genetic modifications without causing neuronal toxicity. The engineered YFV-17D systems can be used to express fluorescent markers, sensors or effectors in downstream neurons, thus providing versatile tools for mapping and functionally controlling neuronal circuits.  

5.3287           Divergent pallidal pathways underlying distinct Parkinsonian behavioral deficits

Lilascharoen, V., Wang, E.H-J., Do, N., Pate, S.C., Tran, A.N., Yoon, C.D:, Choi, J-H., Wang, X-Y., Pribiag, H., Park, Y-G., Chung, K. and Lim, B.K. Nature Neurosci., 24, 504-515 (2021)   The basal ganglia regulate a wide range of behaviors, including motor control and cognitive functions, and are profoundly affected in Parkinson’s disease (PD). However, the functional organization of different basal ganglia nuclei has not been fully elucidated at the circuit level. In this study, we investigated the functional roles of distinct parvalbumin-expressing neuronal populations in the external globus pallidus (GPe-PV) and their contributions to different PD-related behaviors. We demonstrate that substantia nigra pars reticulata (SNr)-projecting GPe-PV neurons and parafascicular thalamus (PF)-projecting GPe-PV neurons are associated with locomotion and reversal learning, respectively. In a mouse model of PD, we found that selective manipulation of the SNr-projecting GPe-PV neurons alleviated locomotor deficit, whereas manipulation of the PF-projecting GPe-PV neurons rescued the impaired reversal learning. Our findings establish the behavioral importance of two distinct GPe-PV neuronal populations and, thereby, provide a new framework for understanding the circuit basis of different behavioral deficits in the Parkinsonian state.  

5.3288           Corticothalamic feedback sculpts visual spatial integration in mouse thalamus

Born, G., Schneider-Soupiadis, F.A:, Erisken, S., Vaiceliunaite, A., Lao, C.L., Mobarhan, M.H., Spacek, M.A., Einevoll, G.T. and Busse, L. Nature Neurosci., 24, 1711-1720 (2021)   En route from the retina to the cortex, visual information passes through the dorsolateral geniculate nucleus (dLGN) of the thalamus, where extensive corticothalamic (CT) feedback has been suggested to modulate spatial processing. How this modulation arises from direct excitatory and indirect inhibitory CT feedback pathways remains enigmatic. Here, we show that in awake mice, retinotopically organized cortical feedback sharpens receptive fields (RFs) and increases surround suppression in the dLGN. Guided by a network model indicating that widespread inhibitory CT feedback is necessary to reproduce these effects, we targeted the visual sector of the thalamic reticular nucleus (visTRN) for recordings. We found that visTRN neurons have large RFs, show little surround suppression and exhibit strong feedback-dependent responses to large stimuli. These features make them an ideal candidate for mediating feedback-enhanced surround suppression in the dLGN. We conclude that cortical feedback sculpts spatial integration in the dLGN, likely via recruitment of neurons in the visTRN.  

5.3289           Use of CRISPR/Cas9-mediated disruption of CNS cell type genes to profile transduction of AAV by neonatal intracerebroventricular delivery in mice

Torregrosa, T., Lehman, S., Hana, S., Marsh, G., Xu, S., Koszka, K., Mastrangelo, N., McVampbell, A., Henderson, C.E. and Lo, S-C. Gene Therapy, 28, 456-468 (2021)   Adeno-associated virus (AAV) transduction efficiency and tropism are conventionally determined by high expression of a fluorescent reporter gene. Emerging data has suggested that such conventional methods may underestimate AAV transduction for cells in which reporter expression from AAV vectors is undetectable. To explore an alternative method that captures AAV transduction in cells in which low expression of a cargo is sufficient for the intended activity, we sought after CRISPR/Cas9-mediated gene disruption. In this study, we use AAV to deliver CRISPR/guide RNA designed to abolish the genes NeuN, GFAP, or MOG expressed specifically in neurons, astrocytes, or oligodendrocytes respectively in the central nervous system (CNS) of mice. Abrogated expression of these cell-type-specific genes can be measured biochemically in CNS subregions and provides quantitative assessment of AAV transduction in these CNS cell types. By using this method, we compared CNS transduction of AAV9, AAV-PHP.B, and AAV-PHP.eB delivered via intracerebroventricular injection (ICV) in neonatal mice. We found both AAV-PHP.B and AAV-PHP.eB resulted in marked disruption of the NeuN gene by CRISPR/Cas9, significantly greater than AAV9 in several brain regions and spinal cord. In contrast, only modest disruption of the GFAP gene and the MOG gene was observed by all three AAV variants. Since the procedure of ICV circumvents the blood–brain barrier, our data suggests that, independent of their ability to cross the blood–brain barrier, AAV-PHP.B variants also exhibit remarkably improved neuronal transduction in the CNS. We anticipate this approach will facilitate profiling of AAV cellular tropism in murine CNS.  

5.3290           Deficiency of Klc2 Induces Low-Frequency Sensorineural Hearing Loss in C57BL/6 J Mice and Human

Fu, X., An, Y., Wang, H., Li, P., Lin, J., Yuan, J., Yue, R., Jin, Y., Gao, J. and Chai, R. Mol. Neurobiol., 58, 4376-4391 (2021)   The transport system in cochlear hair cells (HCs) is important for their function, and the kinesin family of proteins transports numerous cellular cargos via the microtubule network in the cytoplasm. Here, we found that Klc2 (kinesin light chain 2), the light chain of kinesin-1 that mediates cargo binding and regulates kinesin-1 motility, is essential for cochlear function. We generated mice lacking Klc2, and they suffered from low-frequency hearing loss as early as 1 month of age. We demonstrated that deficiency of Klc2 resulted in abnormal transport of mitochondria and the down-regulation of the GABAA receptor family. In addition, whole-genome sequencing (WGS) of patient showed that KLC2 was related to low-frequency hearing in human. Hence, to explore therapeutic approaches, we developed adeno-associated virus containing the Klc2 wide-type cDNA sequence, and Klc2-null mice delivered virus showed apparent recovery, including decreased ABR threshold and reduced out hair cell (OHC) loss. In summary, we show that the kinesin transport system plays an indispensable and special role in cochlear HC function in mice and human and that mitochondrial localization is essential for HC survival.  

5.3291           Biodistribution of Adeno-Associated Virus Serotype 5 Viral Vectors Following Intrathecal Injection

Pflepsen, K.r., Peterson, C.D., Kitto, K.F., Riedl, M.S., McIvor, R.S., Wilcox, G.L., Vulchanova, L. and Fairbanks, C.A. Mol. Pharmaceutcs,18, 3741-3749 (2021)   The pharmacokinetic profile of AAV particles following intrathecal delivery has not yet been clearly defined. The present study evaluated the distribution profile of adeno-associated virus serotype 5 (AAV5) viral vectors following lumbar intrathecal injection in mice. After a single bolus intrathecal injection, viral DNA concentrations in mouse whole blood, spinal cord, and peripheral tissues were determined using quantitative polymerase chain reaction (qPCR). The kinetics of AAV5 vector in whole blood and the concentration over time in spinal and peripheral tissues were analyzed. Distribution of the AAV5 vector to all levels of the spinal cord, dorsal root ganglia, and into systemic circulation occurred rapidly within 30 min following injection. Vector concentration in whole blood reached a maximum 6 h postinjection with a half-life of approximately 12 h. Area under the curve data revealed the highest concentration of vector distributed to dorsal root ganglia tissue. Immunohistochemical analysis revealed AAV5 particle colocalization with the pia mater at the spinal cord and macrophages in the dorsal root ganglia (DRG) 30 min after injection. These results demonstrate the widespread distribution of AAV5 particles through cerebrospinal fluid and preferential targeting of DRG tissue with possible clearance mechanisms via DRG macrophages.  

5.3292           Bluetongue Virus Particles as Nanoreactors for Enzyme Delivery and Cancer Therapy

Thuenemann, E.C., Le, D.H.T., Lomonossoff, G.P. and Steinmetz, N.F: Mol. Pharmaceutics, 18, 1150-1156 (2021)   The side effects of chemotherapy can be reduced by targeting tumor cells with an enzyme (or the corresponding gene) that converts a nontoxic prodrug into a toxic drug inside the tumor cells, also killing the surrounding tumor cells via the bystander effect. Viruses are the most efficient gene delivery vehicles because they have evolved to transfer their own nucleic acids into cells, but their efficiency must be balanced against the risks of infection, the immunogenicity of nucleic acids, and the potential for genomic integration. We therefore tested the effectiveness of genome-free virus-like particles (VLPs) for the delivery of Herpes simplex virus 1 thymidine kinase (HSV1-TK), the most common enzyme used in prodrug conversion therapy. HSV1-TK is typically delivered as a gene, but in the context of VLPs, it must be delivered as a protein. We constructed VLPs and smaller core-like particles (CLPs) based on Bluetongue virus, with HSV1-TK fused to the inner capsid protein VP3. TK-CLPs and TK-VLPs could be produced in large quantities in plants. The TK-VLPs killed human glioblastoma cells efficiently in the presence of ganciclovir, with an IC50 value of 14.8 μM. Conversely, CLPs were ineffective because they remained trapped in the endosomal compartment, in common with many synthetic nanoparticles. VLPs are advantageous because they can escape from endosomes and therefore allow HSV1-TK to access the cytosolic adenosine triphosphate (ATP) required for the phosphorylation of ganciclovir. The VLP delivery strategy of TK protein therefore offers a promising new modality for the treatment of cancer with systemic prodrugs such as ganciclovir.  

5.3293           Exosome-mediated delivery of gene vectors for gene therapy

Duan, L., Xu, L., Xu, X., Qin, Z., Zhou, X., Xiao, Y., Liang, Y. and Xia, J. Nanoscale, 13, 1387-1397 (2021)   Gene vectors are nucleic acids that carry genetic materials or gene editing devices into cells to exert the sustained production of therapeutic proteins or to correct erroneous genes of the cells. However, the cell membrane sets a barrier for the entry of nucleic acid molecules, and nucleic acids are easily degraded or neutralized when they are externally administered into the body. Carriers to encapsulate, protect and deliver nucleic acid molecules therefore are essential for clinical applications of gene therapy. The secreted organelles, exosomes, which naturally mediate the communications between cells, have been engineered to encapsulate and deliver nucleic acids to the desired tissues and cells. The fusion of exosomes with liposomes can increase the loading capacity and also retain the targeting capability of exosomes. Altogether, this review summarizes the most recent designs of exosome-based applications for gene delivery and their future perspectives in gene therapy.  

5.3294           HSV-1 H129-Derived Anterograde Neural Circuit Tracers: Improvements, Production, and Applications

Yang, H., Xiong, F., Song, Y-G., Jiang, H-F., Qin, H-B., Zhou, J., Lu, S., Grieco, S.F., Xu, X., Zeng, W-B., Zhao, F. and Luo, M-H. Neurosci. Bull., 37(5), 701-719 (2021)   Anterograde viral tracers are powerful and essential tools for dissecting the output targets of a brain region of interest. They have been developed from herpes simplex virus 1 (HSV-1) strain H129 (H129), and have been successfully applied to map diverse neural circuits. Initially, the anterograde polysynaptic tracer H129-G4 was used by many groups. We then developed the first monosynaptic tracer, H129-dTK-tdT, which was highly successful, yet improvements are needed. Now, by inserting another tdTomato expression cassette into the H129-dTK-tdT genome, we have created H129-dTK-T2, an updated version of H129-dTK-tdT that has improved labeling intensity. To help scientists produce and apply our H129-derived viral tracers, here we provide the protocol describing our detailed and standardized procedures. Commonly-encountered technical problems and their solutions are also discussed in detail. Broadly, the dissemination of this protocol will greatly support scientists to apply these viral tracers on a large scale.  

5.3295           Specific Expression of Glial-Derived Neurotrophic Factor in Muscles as Gene Therapy Strategy for Amyotrophic Lateral Sclerosis

Modol-Caballero, G., Garcia-Lareu, B., Herrando-Grabulosa, M., Verdes, S., Lopez-Vales, R., Pages, G., Chillon, M., Navarro, X. and Bosch, A. Neurotherapeutics, 18, 1113-1126 (2021)   Glial cell line–derived neurotrophic factor (GDNF) is a powerful neuroprotective growth factor. However, systemic or intrathecal administration of GDNF is associated with side effects. Here, we aimed to avoid this by restricting the transgene expression to the skeletal muscle by gene therapy. To specifically target most skeletal muscles in the mouse model of amyotrophic lateral sclerosis (ALS), SOD1^G93A transgenic mice were intravenously injected with adeno-associated vectors coding for GDNF under the control of the desmin promoter. Treated and control SOD1^G93A mice were evaluated by rotarod and nerve conduction tests from 8 to 20 weeks of age, and then histological and molecular analyses were performed. Muscle-specific GDNF expression delayed the progression of the disease in SOD1^G93A female and male mice by preserving the neuromuscular function; increasing the number of innervated neuromuscular junctions, the survival of spinal motoneurons; and reducing glial reactivity in treated SOD1^G93A mice. These beneficial actions are attributed to a paracrine protective mechanism from the muscle to the motoneurons by GDNF. Importantly, no adverse secondary effects were detected. These results highlight the potential of muscle GDNF-targeted expression for ALS therapy.  

5.3296           Knockdown of Muscle-Specific Ribosomal Protein L3-Like Enhances Muscle Function in Healthy and Dystrophic Mice

Kao, B.R., Malerba, A., Lu-Nguyen, N.B., Harish, P., McCarthy, J.J., Dickson, G. and Popplewell, L.J. Nucleic Acid Therapeutics, 31(6), 457-464 (2021)   Ribosomal protein L3-like (RPL3L) is a poorly characterized ribosomal protein that is exclusively expressed in skeletal and cardiac muscle. RPL3L is also downregulated in Duchenne muscular dystrophy (DMD), suggesting that it may play an important role in muscle biology. In this study, we investigated the role of RPL3L in skeletal muscle of healthy C57 and dystrophic mdx mice. We show that RPL3L is developmentally regulated and that intramuscular adeno-associated virus (AAV)-mediated RPL3L knockdown in the tibialis anterior of C57 and mdx mice results in increased specific force with improved resistance to eccentric contraction induced muscle damage in dystrophic muscles. The mechanism by which RPL3L knockdown improves muscle function remains unclear. Histological observations showed a significant increase in muscle length and decrease in muscle cross-sectional area after RPL3L inhibition suggesting that this ribosomal protein may play a role in myofiber morphology. The endogenous downregulation of RPL3L in DMD may be a protective mechanism that attempts to improve skeletal muscle function and counteract the dystrophic phenotype.      

5.3297           Exploration of p53 plus interferon-beta gene transfer for the sensitization of human colorectal cancer cell lines to cell death

Del Valle, P.R., Mendonca, S.A., Antunes, F., Hunger, A., Tamura, R.E., Zanatta, D.B. and Strauss, B.E. Cancer Biololy and Therapy, 22(4), 301-310 (2021)   While treatments for colorectal cancer continue to improve, some 50% of patients succumb within 5 years, pointing to the need for additional therapeutic options. We have developed a modified non-replicating adenoviral vector for gene transfer, called AdRGD-PG, which offers improved levels of transduction and transgene expression. Here, we employ the p53-responsive PG promoter to drive expression of p53 or human interferon-β (hIFNβ) in human colorectal cancer cell lines HCT116wt (wtp53), HCT116−/- (p53 deficient) and HT29 (mutant p53). The HCT116 cell lines were both easily killed with p53 gene transfer, while combined p53 and hIFNβ cooperated for the induction of HT29 cell death and emission of immunogenic cell death (ICD) markers. Elevated annexinV staining and caspase 3/7 activity point to cell death by a mechanism consistent with apoptosis. P53 gene transfer alone or in combination with hIFNβ sensitized all cell lines to chemotherapy, permitting the application of low drug doses while still achieving significant loss of viability. While endogenous p53 status was not sufficient to predict response to treatment, combined p53 and hIFNβ provided an additive effect in HT29 cells. We propose that this approach may prove effective for the treatment of colorectal cancer, permitting the use of limited drug doses.  

5.3298           Improvement of RG1-VLP vaccine performance in BALB/c mice by substitution of alhydrogel with the next generation polyphosphazene adjuvant PCEP

Valencia, S.M., Zacharia, A., Marin, A., Matthews, R.L., Wu, C-K., Myers, B., Sanders, C., Difilippantonio, S., Kirnbauer, R., Roden, R.B., Pinto, L.A., Shoemaker, R.H., Andrianov, A.K. and Marshall, J.D. Human Vaccines & Immunotherapeutics, 17(8), 2748-2761 (2021)   Current human papillomavirus (HPV) vaccines provide substantial protection against the most common HPV types responsible for oral and anogenital cancers, but many circulating cancer-causing types remain for which vaccine coverage is lacking. In addition, all current HPV vaccines rely on aluminum salt-based adjuvant formulations that function through unclear mechanisms with few substitutes available. In an effort to expand the toolbox of available adjuvants suitable for HPV vaccines, we compared the immunogenicity of the RG1-VLP (virus-like particle) vaccine in BALB/c mice when formulated with either the aluminum hydroxide adjuvant Alhydrogel or the novel polyphosphazene macromolecular adjuvant poly[di (carboxylatoethylphenoxy) phosphazene] (PCEP). PCEP-formulated RG1-VLPs routinely outperformed VLP/Alhydrogel in several measurements of VLP-specific humoral immunity, including consistent improvements in the magnitude of antibody (Ab) responses to both HPV16-L1 and the L2 RG1 epitope as well as neutralizing titers to HPV16 and cross-neutralization of pseudovirion (PsV) types HPV18 and HPV39. Dose-sparing studies indicated that RG1-VLPs could be reduced in dose by 75% and the presence of PCEP ensured activity comparable to a full VLP dose adjuvanted by Alhydrogel. In addition, levels of HPV16-L1 and -L2-specific Abs were achieved after two vaccinations with PCEP as adjuvant that were equivalent to or greater than levels achieved with three vaccinations with Alhydrogel alone, indicating that the presence of PCEP resulted in accelerated immune responses that could allow for a decreased dose schedule. Given the extensive clinical track record of polyphosphazenes, these data suggest that substitution of alum-based adjuvants with PCEP for the RG1-VLP vaccine could lead to rapid seropositivity requiring fewer boosts, the dose-sparing of commercial VLP-based vaccines, and the establishment of longer-lasting humoral responses to HPV.  

5.3299           Differentiated Cells in Prolonged Hypoxia Produce Highly Infectious Native-Like Hepatitis C Virus Particles

Cochard, J., Bull-Maurer, A., Tauber, C., Burlaud-Gaillard, J., Mazurier, F., Meunier, J-C., Roingeard, P. and Chouteau, P. Hepatology, 74(2), 627-640 (2021)   Background and Aims Standard hepatitis C virus (HCV) cell-culture models present an altered lipid metabolism and thus produce lipid-poor lipoviral particles (LVPs). These models are thereby weakly adapted to explore the complete natural viral life cycle. Approach and Results To overcome these limitations, we used an HCV cell-culture model based on both cellular differentiation and sustained hypoxia to better mimic the host-cell environment. The long-term exposure of Huh7.5 cells to DMSO and hypoxia (1% O₂) significantly enhanced the expression of major differentiation markers and the cellular hypoxia adaptive response by contrast with undifferentiated and normoxic (21% O₂) standard conditions. Because hepatocyte-like differentiation and hypoxia are key regulators of intracellular lipid metabolism, we characterized the distribution of lipid droplets (LDs) and demonstrated that experimental cells significantly accumulate larger and more numerous LDs relative to standard cell-culture conditions. An immunocapture (IC) and transmission electron microscopy (TEM) method showed that differentiated and hypoxic Huh7.5 cells produced lipoproteins significantly larger than those produced by standard Huh7.5 cell cultures. The experimental cell culture model is permissive to HCV–Japanese fulminant hepatitis (JFH1) infection and produces very-low-buoyant-density LVPs that are 6-fold more infectious than LVPs formed by standard JFH1-infected Huh7.5 cells. Finally, the IC–TEM approach and antibody-neutralization experiments revealed that LVPs were highly lipidated, had a global ultrastructure and a conformation of the envelope glycoprotein complex E1E2 close to that of the ones circulating in infected individuals. Conclusions This relevant HCV cell culture model thus mimics the complete native intracellular HCV life cycle and, by extension, can be proposed as a model of choice for studies of other hepatotropic viruses.  

5.3300           Orexin (hypocretin) mediates light-dependent fluctuation of hippocampal function in a diurnal rodent

Soler, J.E., Xiong, H., Samad, F., Manfredsson, F.P., Robison, A.J., Nunez, A.A: and Yan, L. Hippocampus, 31, 1104-1114 (2021)   Environmental lighting conditions play a central role in cognitive function, but the underlying mechanisms remain unclear. Utilizing a diurnal rodent model, the Nile grass rat (Arvicanthis niloticus), we previously found that daytime light intensity affects hippocampal function in this species in a manner similar to its effects in humans. Compared to animals housed in a 12:12 h bright light–dark (brLD) cycle, grass rats kept in a 12:12 h dim light–dark (dimLD) cycle showed impaired spatial memory in the Morris water maze (MWM) and reduced CA1 apical dendritic spine density. The present study explored the neural substrates mediating the effects of daylight intensity on hippocampal function focusing on the hypothalamic orexin (hypocretin) system. First, animals housed in dimLD were treated with daily intranasal administration of orexin A peptide over five training days of the MWM task. Compared to vehicle controls, this treatment led to superior spatial memory accompanied by increased phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II α and glutamate receptor 1 within the CA1. To assess the role of hippocampal orexinergic signaling, an adeno-associated viral vector (AAV) expressing an orexin receptor 1 (OX1R) shRNA was injected into the dorsal hippocampus targeting the CA1 of animals housed in brLD. AAV-mediated knockdown of OX1R within the hippocampus resulted in deficits in MWM performance and reduced CA1 apical dendritic spine density. These results are consistent with the view that the hypothalamic orexinergic system underlies the modulatory role of daytime illumination on hippocampal function in diurnal mammals.  

5.3301           Glycerol-3-phosphate biosynthesis regenerates cytosolic NAD+ to alleviate mitochondrial disease

Liu, S., Fu, S., Wang, G., Dong, M., Liu, Q. and Jiang, H. Cell Metabolism, 33, 1974-1987 (2021)   Electron transport chain (ETC) dysfunction or hypoxia causes toxic NADH accumulation. How cells regenerate NAD⁺ under such conditions remains elusive. Here, integrating bioinformatic analysis and experimental validation, we identify glycerol-3-phosphate (Gro3P) biosynthesis as an endogenous NAD⁺-regeneration pathway. Under genetic or pharmacological ETC inhibition, disrupting Gro3P synthesis inhibits yeast proliferation, shortens lifespan of C. elegans, impairs growth of cancer cells in culture and in xenografts, and causes metabolic derangements in mouse liver. Moreover, the Gro3P shuttle selectively regenerates cytosolic NAD⁺ under mitochondrial complex I inhibition; enhancing Gro3P synthesis promotes shuttle activity to restore proliferation of complex I-impaired cells. Mouse brain has much lower levels of Gro3P synthesis enzymes as compared with other organs. Strikingly, enhancing Gro3P synthesis suppresses neuroinflammation and extends lifespan in the Ndufs4^−/− mice. Collectively, our results reveal Gro3P biosynthesis as an evolutionarily conserved coordinator of NADH/NAD⁺ redox homeostasis and present a therapeutic target for mitochondrial complex I diseases.  

5.3302           Turnover of fear engram cells by repeated experience

Cho, H-Y., Shin, W., Lee, H-S., Suh, B., Kim, E. and Han, J-H. Current Biology, 31, 5450-5461 (2021)   A sparse population of neurons active during a learning event has been identified as memory engram cells. However, cells that are recruited to support memory when experience is repeated have been scarcely explored. Evidence from previous studies provides contradictory views. To address these questions, we employed learning-dependent cell labeling in the lateral amygdala (LA) and applied electrophysiological recording, spine imaging, and optogenetic tools to the labeled neurons with or without retraining. We found that engram cells established from original fear learning became dispensable for memory retrieval specifically with relearning, and this correlated with a reduction of synaptic transmission and loss of dendritic spines in these neurons. Despite such decreased connectivity, direct activation of these neurons resulted in fear-memory recall. We further identified that repeated memory was encoded in neurons active during relearning. These results suggest a shift in neuronal ensembles encoding fear memory in the LA by relearning through disconnection of the existing engram neurons established from original experience.  

5.3303           Molecular characterization of a porcine sapelovirus strain isolated in China

Li, N., Tao, J., Li, B., Cheng, J., Shi, Y., Xiahui, S. and Liu, H. Arch. Virol., 166, 2683-2692 (2021)   Porcine sapelovirus (PSV) infections have been associated with a wide spectrum of symptoms, ranging from asymptomatic infection to clinical signs including diarrhoea, pneumonia, reproductive disorders, and polioencephalomyelitis. Although it has a global distribution, there have been relatively few studies on PSV in domestic animals. We isolated a PSV strain, SHCM2019, from faecal specimens from swine, using PK-15 cells. To investigate its molecular characteristics and pathogenicity, the genomic sequence of strain SHCM2019 was analysed, and clinical manifestations and pathological changes occurring after inoculation of neonatal piglets were observed. The virus isolated using PK-15 cells was identified as PSV using RT-PCR, transmission electron microscopy (TEM), and immunofluorescence assay (IFA). Sequencing results showed that the full-length genome of the SHCM2019 strain was 7,567 nucleotides (nt) in length, including a 27-nucleotide poly(A) tail. Phylogenetic analysis demonstrated that this virus was a PSV isolate belonging to the Chinese strain cluster. Recombination analysis indicated that there might be a recombination breakpoint upstream of the 3D region of the genome. Pathogenicity experiments demonstrated that the virus isolate could cause diarrhoea and pneumonia in piglets. In breif, a recombinant PSV strain, SHCM2019, was isolated and shown to be pathogenic. Our results may provide a reference for future research on the pathogenic mechanism and evolutionary characteristics of PSV.  

5.3304           Recombinant adenovirus expressing the fusion protein PD1PVR improves CD8+ T cell-mediated antitumor efficacy with long-term tumor-specific immune surveillance in hepatocellular carcinoma

Zhang, H., Zhang, Y., Dong, J., Zuo, S., Meng, G., Wu, J. and Wei, J. Cell. Oncol., 44, 1243-1255 (2021)   Purpose Treatment-associated upregulation of suppressive checkpoints and a lack of costimulatory signals compromise the antitumor efficacy of oncolytic virus immunotherapy. Therefore, we aimed to identify highly effective therapeutic targets to provide a proof-of-principle for immune checkpoint together with oncolytic virus-mediated viro-immunotherapy for cancer. Methods A fusion protein containing both the extracellular domain of programmed death-1 (PD-1) and the poliovirus receptor (PVR) was designed. Next, the corresponding expression fragment was inserted into the genome of a replication-competent adenovirus to generate Ad5sPD1PVR. The infection, expression, replication and oncolysis of Ad5sPD1PVR were investigated in hepatocellular carcinoma (HCC) cell lines. Immune activation and the antitumor efficacy of Ad5sPD1PVR were examined in HCC tumor models including a humanized immunocompetent mouse model. Results Ad5sPD1PVR effectively infected and replicated in HCC cells and secreted sPD1PVR. In a H22 ascitic HCC mouse model, intraperitoneal injection of Ad5sPD1PVR markedly recruited lymphocytes and activated antitumor immune responses. Ad5sPD1PVR exerted a profound antitumor effect on ascitic HCC. Furthermore, we found that Ad5sPD1PVR-H expressing sPD1PVR of human origin exhibited potent antitumor effects in a HCC humanized mouse model. We also found that CD8⁺ T cells mediated the antitumor effects and long-term tumor-specific immune surveillance induced by Ad5sPD1PVR. Finally, when combined with fludarabine, the antitumor efficacy of Ad5sPD1PVR was found to be further improved in the ascitic HCC model. Conclusions From our data we conclude that the newly designed recombinant Ad5sPD1PVR virus significantly enhances CD8⁺ T cell-mediated antitumor efficacy with long-term tumor-specific immune surveillance in hepatocellular carcinoma, and that fludarabine is a promising therapeutic partner for Ad5sPD1PVR.  

5.3305           Gene therapy for a murine model of eosinophilic esophagitis

Camilleri, A.E., Nag, S., Russo, A.R., Stiles, K.M., Crystal, R.G. and Pagovich, O.E. Allergy, 76, 2740-2752 (2021)   Background Eosinophils are specialized granulocytic effector cells that store and release highly active mediators used in immune defense. Eosinophils are also implicated in the pathogenesis of allergic disorders, including eosinophilic esophagitis (EoE), a chronic disorder characterized by infiltration of eosinophils into the esophagus and release of mediators that damage tissue, resulting in gastrointestinal morbidity, food impaction, and dysphagia. Treatment with elimination diets and/or topical corticosteroid therapy slow disease progression, but are complicated by adverse effects, limited compliance, and loss of response to therapy. We hypothesized that a single administration of an adeno-associated virus (AAV) coding for an anti-eosinophil monoclonal antibody that induces eosinophil clearance (anti-Siglec-F) would treat on a persistent basis a murine model of EoE. Methods A mouse model of peanut-induced EoE that mimics the human disease was established by sensitization and challenge with peanut extract. After challenge, these mice exhibited an EoE phenotype demonstrated by elevated levels of blood eosinophils, infiltration of eosinophils in the esophagus with associated esophageal remodeling and food impaction. Results The mice were treated with a single intravenous administration (10¹¹ genome copies) of AAVrh.10mAnti-Eos, a serotype rh.10 AAV vector coding for an anti-Siglec-F monoclonal antibody. Vector administration resulted in persistent, high levels of anti-Siglec-F antibody expression. Administration of AAVrh.10mAnti-Eos to the mouse model of EoE reduced blood (P < 0.02) and esophageal eosinophil numbers (P < 0.002) protected from esophageal tissue remodeling and minimized food impaction. Conclusion These results suggest that a single treatment with AAVrh.10mAnti-Eos has the potential to provide persistent therapeutic benefit to patients with EoE.  

5.3306           Enoyl coenzyme A hydratase 1 alleviates nonalcoholic steatohepatitis in mice by suppressing hepatic ferroptosis

Liu, B., Yi, W., Mao, X., Yang, L. and Rao, C. Am. J. Physiol. Endocrinol. Metab., 320, E925-E937 (2021)   Nonalcoholic steatohepatitis (NASH) is a common metabolic disorder that is a major contributor to health care expenditures worldwide. Enoyl coenzyme A hydratase 1 (ECH1) is initially recognized as a key component in mitochondrial fatty acid β-oxidation, and subsequent studies have demonstrated that it regulates multiple pathophysiological processes. However, the relationship between ECH1 and NASH has remained largely unknown. Herein, we investigated the role of ECH1 in NASH progression. Adeno-associated virus-mediated genetic engineering was used to investigate the role of ECH1. Alterations in hepatic steatosis, inflammation, fibrogenesis, oxidative stress, apoptosis, and liver injury were monitored using liver or serum samples from mice. ECH1 expression was significantly higher in human NASH biopsy specimens and in methionine choline-deficient (MCD) diet-fed mice. ECH1 overexpression significantly alleviated hepatic steatosis, inflammation, fibrogenesis, apoptosis, and oxidative stress in livers of mice. In addition, ECH1 overexpression also reduced alanine aminotransferase and proinflammatory cytokine levels in serum and triglyceride levels in livers. Consistently, ECH1 knockdown suppressed this beneficial phenotype. Mechanistically, ECH1-knockdown mice treated with ferrostatin-1 (Fer-1) showed an alleviated NASH phenotype compared with the untreated knockdown mice. Meanwhile, we detected changes in Erk signaling pathway when ECH1 was overexpressed or knocked down, which may partially explain the potential mechanism of ECH1 regulation of ferroptosis.      

5.3307           Frustration and Direct-Coupling Analyses to Predict Formation and Function of Adeno-Associated Virus

Thadani, N.N., Zhou, Q., Gamas, K.R., Butler, S., Bueno, C., Schafer, N.P., Morcos, F., Wolynes, P.G. and Suh, J. Biophys J., 120, 489-503 82021)   Adeno-associated virus (AAV) is a promising gene therapy vector because of its efficient gene delivery and relatively mild immunogenicity. To improve delivery target specificity, researchers use combinatorial and rational library design strategies to generate novel AAV capsid variants. These approaches frequently propose high proportions of nonforming or noninfective capsid protein sequences that reduce the effective depth of synthesized vector DNA libraries, thereby raising the discovery cost of novel vectors. We evaluated two computational techniques for their ability to estimate the impact of residue mutations on AAV capsid protein-protein interactions and thus predict changes in vector fitness, reasoning that these approaches might inform the design of functionally enriched AAV libraries and accelerate therapeutic candidate identification. The Frustratometer computes an energy function derived from the energy landscape theory of protein folding. Direct-coupling analysis (DCA) is a statistical framework that captures residue coevolution within proteins. We applied the Frustratometer to select candidate protein residues predicted to favor assembled or disassembled capsid states, then predicted mutation effects at these sites using the Frustratometer and DCA. Capsid mutants were experimentally assessed for changes in virus formation, stability, and transduction ability. The Frustratometer-based metric showed a counterintuitive correlation with viral stability, whereas a DCA-derived metric was highly correlated with virus transduction ability in the small population of residues studied. Our results suggest that coevolutionary models may be able to elucidate complex capsid residue-residue interaction networks essential for viral function, but further study is needed to understand the relationship between protein energy simulations and viral capsid metastability.  

5.3308           CRISPR/Cas9 Mediated Deletion of the Angiotensinogen Gene Reduces Hypertension: A Potential for Cure?

Sun, H., Hodgkinson, C.P., Pratt, R.E. and Dzau, V.J. Hypertension, 77(6), 1990-2000 (2021)   Graphical AbstractHypertension is a major contributor to the global burden of disease. Unfortunately, hypertension is controlled in less than one-fifth of patients worldwide due to either failure to treat or lack of compliance to medication. An ideal therapy would be administered one time only and yield lifelong blood pressure control. We investigated our hypothesis that CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9)-mediated disruption of a key gene in the renin-angiotensin system, AGT (angiotensinogen), specifically in the liver, would result in sustained and possibly lifelong reduction in blood pressure. We demonstrated in vitro that the CRISPR/Cas9 system led to a significant reduction in AGT expression in hepatocytes. Delivery of the CRISPR/Cas9 system into the liver via the hepatocyte-targeting adeno-associated virus 8 reduced both AGT expression (40% decrease) and circulating AGT levels (30% decrease). In the SHR (spontaneously hypertensive rat) model of hypertension, CRISPR/Cas9-mediated loss of AGT expression reduced blood pressure in adult animals with established hypertension and prevented the spontaneous development of hypertension in young SHR. Moreover, reductions in blood pressure were prolonged and sustained up to 1 year of follow-up. In addition, the partial disruption of the hepatic AGT gene was sufficient to control hypertension but did not affect the homeostatic response to cardiovascular stress such as sodium depletion and furosemide. In summary, we have demonstrated that targeting the CRISPR/Cas9 system to hepatic AGT results in sustained reduction of blood pressure and is a potential therapy to achieve sustained and possibly lifelong control of human hypertension.  

5.3309           Gene therapy using Aβ variants for amyloid reduction

Park, K-W., Wood, C.A., Li, J., Taylor, B.C., Oh, S., Young, N.L. and Jankowsky, J.L. Molecular Therapy, 29(7), 2294-2307 (2021)   Numerous aggregation inhibitors have been developed with the goal of blocking or reversing toxic amyloid formation in vivo. Previous studies have used short peptide inhibitors targeting different amyloid β (Aβ) amyloidogenic regions to prevent aggregation. Despite the specificity that can be achieved by peptide inhibitors, translation of these strategies has been thwarted by two key obstacles: rapid proteolytic degradation in the bloodstream and poor transfer across the blood-brain barrier. To circumvent these problems, we have created a minigene to express full-length Aβ variants in the mouse brain. We identify two variants, F20P and F19D/L34P, that display four key properties required for therapeutic use: neither peptide aggregates on its own, both inhibit aggregation of wild-type Aβ in vitro, promote disassembly of pre-formed fibrils, and diminish toxicity of Aβ oligomers. We used intraventricular injection of adeno-associated virus (AAV) to express each variant in APP/PS1 transgenic mice. Lifelong expression of F20P, but not F19D/L34P, diminished Aβ levels, plaque burden, and plaque-associated neuroinflammation. Our findings suggest that AAV delivery of Aβ variants may offer a novel therapeutic strategy for Alzheimer’s disease. More broadly our work offers a framework for identifying and delivering peptide inhibitors tailored to other protein-misfolding diseases.  

5.3310           Gene therapy reforms photoreceptor structure and restores vision in NPHP5-associated Leber congenital amaurosis

Aguirre, G.D:, Cideciyan, A.V., Dufour, V:L., Ripolles-garcia, A., Sudharsan, R., Swider, M., Nikonov, R., Iwabe, S., Boye, S.L., Hauswirth, W.W., Jacobson, S.G. and Beltran, W.A. Molecular Therapy, 29(8), 2456-2468 (2021)   The inherited childhood blindness caused by mutations in NPHP5, a form of Leber congenital amaurosis, results in abnormal development, dysfunction, and degeneration of photoreceptors. A naturally occurring NPHP5 mutation in dogs leads to a phenotype that very nearly duplicates the human retinopathy in terms of the photoreceptors involved, spatial distribution of degeneration, and the natural history of vision loss. We show that adeno-associated virus (AAV)-mediated NPHP5 gene augmentation of mutant canine retinas at the time of active degeneration and peak cell death stably restores photoreceptor structure, function, and vision with either the canine or human NPHP5 transgenes. Mutant cone photoreceptors, which failed to form outer segments during development, reform this structure after treatment. Degenerating rod photoreceptor outer segments are stabilized and develop normal structure. This process begins within 8 weeks after treatment and remains stable throughout the 6-month posttreatment period. In both photoreceptor cell classes mislocalization of rod and cone opsins is minimized or reversed. Retinal function and functional vision are restored. Efficacy of gene therapy in this large animal ciliopathy model of Leber congenital amaurosis provides a path for translation to human treatment.  

5.3311           Ligand-activated RXFP1 gene therapy ameliorates pressure overload-induced cardiac dysfunction

Sasipong, N., Schlegel, P., Wingert, J., Lederer, C., Meinhardt, E., Ziefer, A., Schmidt, C., Rapti, K., Thöni, C., Frey, N., Most, P., katus, H.A. and Raake, P.W.J. Molecular Therapy, 29(8), 2499-2513 (2021)   Recurrent episodes of decompensated heart failure (HF) represent an emerging cause of hospitalizations in developed countries with an urgent need for effective therapies. Recently, the pregnancy-related hormone relaxin (RLN) was found to mediate cardio-protective effects and act as a positive inotrope in the cardiovascular system. RLN binds to the RLN family peptide receptor 1 (RXFP1), which is predominantly expressed in atrial cardiomyocytes. We therefore hypothesized that ventricular RXFP1 expression might exert potential therapeutic effects in an in vivo model of cardiac dysfunction. Thus, mice were exposed to pressure overload by transverse aortic constriction and treated with AAV9 to ectopically express RXFP1. To activate RXFP1 signaling, RLN was supplemented subcutaneously. Ventricular RXFP1 expression was well tolerated. Additional RLN administration not only abrogated HF progression but restored left ventricular systolic function. In accordance, upregulation of fetal genes and pathological remodeling markers were significantly reduced. In vitro, RLN stimulation of RXFP1-expressing cardiomyocytes induced downstream signaling, resulting in protein kinase A (PKA)-specific phosphorylation of phospholamban (PLB), which was distinguishable from β-adrenergic activation. PLB phosphorylation corresponded to increased calcium amplitude and contractility. In conclusion, our results demonstrate that ligand-activated cardiac RXFP1 gene therapy represents a therapeutic approach to attenuate HF with the potential to adjust therapy by exogenous RLN supplementation.  

5.3312           A comprehensive study of a 29-capsid AAV library in a non-human primate central nervous system

Kondratov, O., Kondratova, L., Mandel, R.J., Coleman, K., Savage, M.A., Gray-Edwards, H.L., Ness, T.J., Rodriguez-Lebron, E., Bell, R.D., Rabinowitz, J., Gamlin, P.D. and Zolotukhin, S. Molecular Therapy, 29(9), 2806-2820 (2021)   Non-human primates (NHPs) are a preferred animal model for optimizing adeno-associated virus (AAV)-mediated CNS gene delivery protocols before clinical trials. In spite of its inherent appeal, it is challenging to compare different serotypes, delivery routes, and disease indications in a well-powered, comprehensive, multigroup NHP experiment. Here, a multiplex barcode recombinant AAV (rAAV) vector-tracing strategy has been applied to a systemic analysis of 29 distinct, wild-type (WT), AAV natural isolates and engineered capsids in the CNS of eight macaques. The report describes distribution of each capsid in 15 areas of the macaques’ CNS after intraparenchymal (putamen) injection, or cerebrospinal fluid (CSF)-mediated administration routes (intracisternal, intrathecal, or intracerebroventricular). To trace the vector biodistribution (viral DNA) and targeted tissues transduction (viral mRNA) of each capsid in each of the analyzed CNS areas, quantitative next-generation sequencing analysis, assisted by the digital-droplet PCR technology, was used. The report describes the most efficient AAV capsid variants targeting specific CNS areas after each route of administration using the direct side-by-side comparison of WT AAV isolates and a new generation of rationally designed capsids. The newly developed bioinformatics and visualization algorithms, applicable to the comparative analysis of several mammalian brain models, have been developed and made available in the public domain.  

5.3313           Best of most possible worlds: Hybrid gene therapy vectors based on parvoviruses and heterologous viruses

Fakhiri, J. and Grimm, D. Molecular Therapy, 29(12), 3359-3382 (2021)   Parvoviruses and especially the adeno-associated virus (AAV) species provide an exciting and versatile platform for the rational design or molecular evolution of human gene-therapy vectors, documented by literature from over half a century, hundreds of clinical trials, and the recent commercialization of multiple AAV gene therapeutics. For the last three decades, the power of these vectors has been further potentiated through various types of hybrid vectors created by intra- or inter-genus juxtaposition of viral DNA and protein cis elements or by synergistic complementation of parvoviral features with those of heterologous, prokaryotic, or eukaryotic viruses. Here, we provide an overview of the history and promise of this rapidly expanding field of hybrid parvoviral gene-therapy vectors, starting with early generations of chimeric particles composed of a recombinant AAV genome encapsidated in shells of synthetic AAVs or of adeno-, herpes-, baculo-, or protoparvoviruses. We then dedicate our attention to two newer, highly promising types of hybrid vectors created via (1) pseudotyping of AAV genomes with bocaviral serotypes and capsid mutants or (2) packaging of AAV DNA into, or tethering of entire vector particles to, bacteriophages. Finally, we conclude with an outlook summarizing critical requirements and improvements toward clinical translation of these original concepts.  

5.3314           Long-term functional correction of cystathionine β-synthase deficiency in mice by adeno-associated viral gene therapy

Lee, H-O., Salami, C.O., Sondhi, D., Kaminsky, S.M., Crystal, R.G. and kruger, W.D.

1. Inherit. Metab. Dis., 44, 1382-1392 (2021)

  Cystathionine β-synthase (CBS) deficiency is a recessive inborn error of sulfur metabolism characterized by elevated blood levels of total homocysteine (tHcy). Patients diagnosed with CBS deficiency are currently treated by a combination of vitamin supplementation and restriction of foods containing the homocysteine precursor methionine, but the effectiveness of this therapy is limited due to poor compliance. A mouse model for CBS deficiency (Tg-I278T Cbs^−/−) was used to evaluate a potential gene therapy approach to treat CBS deficiency utilizing an AAVrh.10-based vector containing the human CBS cDNA downstream of the constitutive, strong CAG promoter (AAVrh.10hCBS). Mice were administered a single dose of virus and followed for up to 1 year. The data demonstrated a dose-dependent increase in liver CBS activity and a dose-dependent decrease in serum tHcy. Liver CBS enzyme activity at 1 year was similar to Cbs^+/− control mice. Mice given the highest dose (5.6 × 10¹¹ genomes/mouse) had mean serum tHcy decrease of 97% 1 week after injection and an 81% reduction 1 year after injection. Treated mice had either full- or substantial correction of alopecia, bone loss, and fat mass phenotypes associated with Cbs deficiency in mice. Our findings show that AAVrh.10-based gene therapy is highly effective in treating CBS deficiency in mice and supports additional pre-clinical testing for eventual use human trials.  

5.3315           Alleviation of neuropathic pain by over-expressing a soluble colony-stimulating factor 1 receptor to suppress microgliosis and macrophage accumulation

Gushchina, S., Yip, P.K., parry, G.A., Sivakumar, H., Li, J., Liu, M. and Bo, X. Glia, 69, 2963-2980 (2021)   Microglial proliferation and activation and macrophage accumulation are implicated in neuropathic pain development. In this study, we aim to suppress microgliosis and macrophage accumulation by over-expressing a non-functional soluble colony stimulating factor-1 receptor (sCSF1R) using an adeno-associated virus 9 vector (AAV9). AAV9/sCSF1R and the control vector AAV9/GFP were intrathecally administered into the lumbar spine of adult C57BL/6 mice. Two weeks later, these mice underwent partial sciatic nerve ligation to induce neuropathic pain. GFP and sCSF1R were highly expressed in lumbar dorsal root ganglia (DRG) and spinal cord of AAV9-injected mice. A significant increase in microglia densities in the dorsal and ventral horns of lumbar spinal cords and macrophage densities in DRG and sciatic nerves were observed in the mice with either ligation alone or pre-treated with AAV9/GFP. In nerve-ligated mice pre-treated with AAV9/sCSF1R the microglia densities in the dorsal and ventral horns and macrophage densities in DRG and sciatic nerves were significantly lower compared to nerve-ligated mice pre-treated with AAV9/GFP. Behavioral tests showed that nerve-ligated mice pre-treated with AAV9/sCSF1R had a significantly higher paw withdrawal threshold, indicating the alleviation of neuropathic pain. The results implicate that viral vector-mediated expression of sCSF1R may represent a novel strategy in the alleviation of neuropathic pain.  

5.3316           Serum Antibodies to N-Glycolylneuraminic Acid Are Elevated in Duchenne Muscular Dystrophy and Correlate with Increased Disease Pathology in Cmah−/−mdx Mice

Martin, P.T., Kawanishi, K., Ashbrook, A., Golden, B., Samraj, A., Crowe, K.E., Zygmunt, D.A., Okerblom, J., Yu, H., Maki, A., Diaz, S., Chen, X., jjanssen, P.M.L. and Varki, A. Am. J. Pathol., 191(8), 1474-1486 (2021)   Humans cannot synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) because of an inactivating deletion in the cytidine-5'-monophospho-(CMP)–N-acetylneuraminic acid hydroxylase (CMAH) gene responsible for its synthesis. Human Neu5Gc deficiency can lead to development of anti-Neu5Gc serum antibodies, the levels of which can be affected by Neu5Gc-containing diets and by disease. Metabolic incorporation of dietary Neu5Gc into human tissues in the face of circulating antibodies against Neu5Gc-bearing glycans is thought to exacerbate inflammation-driven diseases like cancer and atherosclerosis. Probing of sera with sialoglycan arrays indicated that patients with Duchenne muscular dystrophy (DMD) had a threefold increase in overall anti-Neu5Gc antibody titer compared with age-matched controls. These antibodies recognized a broad spectrum of Neu5Gc-containing glycans. Human-like inactivation of the Cmah gene in mice is known to modulate severity in a variety of mouse models of human disease, including the X chromosome–linked muscular dystrophy (mdx) model for DMD. Cmah^−/−mdx mice can be induced to develop anti–Neu5Gc-glycan antibodies as humans do. The presence of anti-Neu5Gc antibodies, in concert with induced Neu5Gc expression, correlated with increased severity of disease pathology in Cmah^−/−mdx mice, including increased muscle fibrosis, expression of inflammatory markers in the heart, and decreased survival. These studies suggest that patients with DMD who harbor anti-Neu5Gc serum antibodies might exacerbate disease severity when they ingest Neu5Gc-rich foods, like red meats.  

5.3317           Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses

Chan, Y.K., Wang, S.K., Chu, C.J., Copland, D.A., Letizia, A.J. et al Sci. Transl. Med., 13, eabd3438 (2021)   Nucleic acids are used in many therapeutic modalities, including gene therapy, but their ability to trigger host immune responses in vivo can lead to decreased safety and efficacy. In the case of adeno-associated viral (AAV) vectors, studies have shown that the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA. Here, we engineered AAV vectors to be intrinsically less immunogenic by incorporating short DNA oligonucleotides that antagonize TLR9 activation directly into the vector genome. The engineered vectors elicited markedly reduced innate immune and T cell responses and enhanced gene expression in clinically relevant mouse and pig models across different tissues, including liver, muscle, and retina. Subretinal administration of higher-dose AAV in pigs resulted in photoreceptor pathology with microglia and T cell infiltration. These adverse findings were avoided in the contralateral eyes of the same animals that were injected with the engineered vectors. However, intravitreal injection of higher-dose AAV in macaques, a more immunogenic route of administration, showed that the engineered vector delayed but did not prevent clinical uveitis, suggesting that other immune factors in addition to TLR9 may contribute to intraocular inflammation in this model. Our results demonstrate that linking specific immunomodulatory noncoding sequences to much longer therapeutic nucleic acids can “cloak” the vector from inducing unwanted immune responses in multiple, but not all, models. This “coupled immunomodulation” strategy may widen the therapeutic window for AAV therapies as well as other DNA-based gene transfer methods.            

5.3318           In vivo optogenetic stimulation of the primate retina activates the visual cortex after long-term transduction

Chaffol, A., Provansal, M., Joffrois, C., Blaize, K., Labernede, G., Gulet, R., Burban, E., Brazhnikova, E., Duebel, J., Pouget, P., Sahel, J.A., Picaud, S., Arcizet, F. and Gauvain, G. Molecular Therapy-Methods Clin. Develop., 24, 1-10 (2022)   Over the last 15 years, optogenetics has changed fundamental research in neuroscience and is now reaching toward therapeutic applications. Vision restoration strategies using optogenetics are now at the forefront of these new clinical opportunities. But applications to human patients suffering from retinal diseases leading to blindness raise important concerns on the long-term functional expression of optogenes and the efficient signal transmission to higher visual centers. Here, we demonstrate in non-human primates continued expression and functionality at the retina level ∼20 months after delivery of our construct. We also performed in vivo recordings of visually evoked potentials in the primary visual cortex of anesthetized animals. Using synaptic blockers, we isolated the in vivo cortical activation resulting from the direct optogenetic stimulation of primate retina. In conclusion, our work indicates long-term transgene expression and transmission of the signal generated in the macaque retina to the visual cortex, two important features for future clinical applications.  

5.3319           Novel human liver-tropic AAV variants define transferable domains that markedly enhance the human tropism of AAV7 and AAV8

Cabanes-Creus, M., Navarro, R.G., Zhu, E., Baltazar, G., Liao, S.H.Y., Drouyer, M., et al Molecular Therapy-Methods Clin. Develop., 24, 88-101 (2022)   Recent clinical successes have intensified interest in using adeno-associated virus (AAV) vectors for therapeutic gene delivery. The liver is a key clinical target, given its critical physiological functions and involvement in a wide range of genetic diseases. Here, we report the bioengineering of a set of next-generation AAV vectors, named AAV-SYDs (where “SYD” stands for Sydney, Australia), with increased human hepato-tropism in a liver xenograft mouse model repopulated with primary human hepatocytes. We followed a two-step process that staggered directed evolution and domain-swapping approaches. Using DNA-family shuffling, we first mapped key AAV capsid regions responsible for efficient human hepatocyte transduction in vivo. Focusing on these regions, we next applied domain-swapping strategies to identify and study key capsid residues that enhance primary human hepatocyte uptake and transgene expression. Our findings underscore the potential of AAV-SYDs as liver gene therapy vectors and provide insights into the mechanism responsible for their enhanced transduction profile.  

5.3320           Differential T cell immune responses to deamidated adeno-associated virus vector

Bing, S.J., Justesen, S., Wu, W.W., Sajib, A.M., Warrington, S., baer, A., Thorgrimsen, S., Shen, R-F. and Mazor, R. Molecular Therapy, 24, 255-267 (2022)   Despite the high safety profile demonstrated in clinical trials, the immunogenicity of adeno-associated virus (AAV)-mediated gene therapy remains a major hurdle. Specifically, T-cell-mediated immune responses to AAV vectors are related to loss of efficacy and potential liver toxicities. As post-translational modifications in T cell epitopes have the potential to affect immune reactions, the cellular immune responses to peptides derived from spontaneously deamidated AAV were investigated. Here, we report that highly deamidated sites in AAV9 contain CD4 T cell epitopes with a Th1 cytokine pattern in multiple human donors with diverse human leukocyte antigen (HLA) backgrounds. Furthermore, some peripheral blood mononuclear cell (PBMC) samples demonstrated differential T cell activation to deamidated or non-deamidated epitopes. Also, in vitro and in silico HLA binding assays showed differential binding to the deamidated or non-deamidated peptides in some HLA alleles. This study provides critical attributes to vector-immune-mediated responses, as AAV deamidation can impact the immunogenicity, safety, and efficacy of AAV-mediated gene therapy in some patients.  

5.3321           Structural characterization of an envelope-associated adeno-associated virus type 2 capsid

Hull, J.A., Mietzch, M., Chipman, P., Strugatsky, D. and McKenna, R. Virology, 565, 22-28 (2022)   Adeno-associated virus (AAV) are classified as non-enveloped ssDNA viruses. However, AAV capsids embedded within exosomes have been observed, and it has been suggested that the AAV membrane associated accessory protein (MAAP) may play a role in envelope-associated AAV (EA-AAV) capsid formation. Here, we observed and selected sufficient homogeneous EA-AAV capsids of AAV2, produced using the Sf9 baculoviral expression system, to determine the cryo-electron microscopy (cryo-EM) structure at 3.14 Å resolution. The reconstructed map confirmed that the EA-AAV capsid, showed no significant structural variation compared to the non-envelope capsid. In addition, the Sf9 expression system used implies the notion that MAAP may enhance exosome AAV encapsulation. Furthermore, we speculate that these EA-AAV capsids may have therapeutic benefits over the currently used non-envelope AAV capsids, with advantages in immune evasion and/or improved infectivity.  

5.3322           Discovery of novel fish papillomaviruses: From the Antarctic to the commercial fish market

Kraberger, S., Austin, C., farkas, K., Desvignes, T., Postlethwait, J.H., Fontenele, R.S., Schmidlin, K., Bradley, R.W., Warzybok, P., Van Doorslaeer, K., Davison, W., Buck, C.B. and Varsani, A. Virology, 565, 65-72 (2022)   Fish papillomaviruses form a newly discovered group broadly recognized as the Secondpapillomavirinae subfamily. This study expands the documented genomes of the fish papillomaviruses from six to 16, including one from the Antarctic emerald notothen, seven from commercial market fishes, one from data mining of sea bream sequence data, and one from a western gull cloacal swab that is likely diet derived. The genomes of secondpapillomaviruses are ∼6 kilobasepairs (kb), which is substantially smaller than the ∼8 kb of terrestrial vertebrate papillomaviruses. Each genome encodes a clear homolog of the four canonical papillomavirus genes, E1, E2, L1, and L2. In addition, we identified open reading frames (ORFs) with short linear peptide motifs reminiscent of E6/E7 oncoproteins. Fish papillomaviruses are extremely diverse and phylogenetically distant from other papillomaviruses suggesting a model in which terrestrial vertebrate-infecting papillomaviruses arose after an evolutionary bottleneck event, possibly during the water-to-land transition.  

5.3323           Chronic neuronal excitation leads to dual metaplasticity in the signaling for structural long-term potentiation

Ueda, H.H., Nagasawa, Y., Sato, A., Onda, M. and Murakoshi, H. Cell Reports, 38, 110153 (2022)   Synaptic plasticity is long-lasting changes in synaptic currents and structure. When neurons are exposed to signals that induce aberrant neuronal excitation, they increase the threshold for the induction of long-term potentiation (LTP), known as metaplasticity. However, the metaplastic regulation of structural LTP (sLTP) remains unclear. We investigate glutamate uncaging/photoactivatable (pa)CaMKII-dependent sLTP induction in hippocampal CA1 neurons after chronic neuronal excitation by GABA_A receptor antagonists. We find that the neuronal excitation decreases the glutamate uncaging-evoked Ca²⁺ influx mediated by GluN2B-containing NMDA receptors and suppresses sLTP induction. In addition, single-spine optogenetic stimulation using paCaMKII indicates the suppression of CaMKII signaling. While the inhibition of Ca²⁺ influx is protein synthesis independent, the paCaMKII-induced sLTP suppression depends on it. Our findings demonstrate that chronic neuronal excitation suppresses sLTP in two independent ways (i.e., dual inhibition of Ca²⁺ influx and CaMKII signaling). This dual inhibition mechanism may contribute to robust neuronal protection in excitable environments.  

5.3324           Host glycocalyx captures HIV proximal to the cell surface via oligomannose-GlcNAc glycan-glycan interactions to support viral entry

Spillings, B.L., Day, C.J., Garcia-Miambres, A., Stow, J.L., Jennings, M.P. and Mak, J. Cell Reports, 38, 110296 (2022)   Here, we present ultrastructural analyses showing that incoming HIV are captured near the lymphocyte surface in a virion-glycan-dependent manner. Biophysical analyses show that removal of either virion- or cell-associated N-glycans impairs virus-cell binding, and a similar glycan-dependent relationship is observed between purified HIV envelope (Env) and primary T cells. Trimming of N-glycans from either HIV or Env does not inhibit protein-protein interactions. Glycan arrays reveal HIV preferentially binds to N-acetylglucosamine and mannose. Interfering with these glycan-based interactions reduces HIV infectivity. These glycan interactions are distinct from previously reported glycan-lectin and non-specific electrostatic charge-based interactions. Specific glycan-glycan-mediated attachment occurs prior to virus entry and enhances efficiency of infection. Binding and fluorescent imaging data support glycan-glycan interactions as being responsible, at least in part, for initiating contact between HIV and the host cell, prior to viral Env-cellular CD4 engagement.  

5.3325           Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins

Banskota, S., Raguram, A., Suh, S., Musunuru, K., Palczewski, K. and Liu, D.R. Cell, 185, 250-265 (2022)   Methods to deliver gene editing agents in vivo as ribonucleoproteins could offer safety advantages over nucleic acid delivery approaches. We report the development and application of engineered DNA-free virus-like particles (eVLPs) that efficiently package and deliver base editor or Cas9 ribonucleoproteins. By engineering VLPs to overcome cargo packaging, release, and localization bottlenecks, we developed fourth-generation eVLPs that mediate efficient base editing in several primary mouse and human cell types. Using different glycoproteins in eVLPs alters their cellular tropism. Single injections of eVLPs into mice support therapeutic levels of base editing in multiple tissues, reducing serum Pcsk9 levels 78% following 63% liver editing, and partially restoring visual function in a mouse model of genetic blindness. In vitro and in vivo off-target editing from eVLPs was virtually undetected, an improvement over AAV or plasmid delivery. These results establish eVLPs as promising vehicles for therapeutic macromolecule delivery that combine key advantages of both viral and nonviral delivery.  

5.3326           2-Arachidonoylglycerol mobilization following brief synaptic stimulation in the dorsal lateral striatum requires glutamatergic and cholinergic neurotransmission

Liput, D.J., Puhl, H.L., Dong, A., He, K. and Li, Y. Neuropharmacol., 205, 108916 (2022)   Several forms of endocannabinoid (eCB) signaling have been described in the dorsal lateral striatum (DLS), however most experimental protocols used to generate eCBs do not recapitulate the firing patterns of striatal-projecting pyramidal neurons in the cortex or firing patterns of striatal medium spiny neurons. Therefore, it is unclear if current models of eCB signaling in the DLS provide a reliable description of mechanisms engaged under physiological conditions. To address this uncertainty, we investigated mechanisms of eCB mobilization following brief synaptic stimulation that mimics in vivo patterns of neural activity in the DLS. To monitor eCB mobilization, the novel genetically encoded fluorescent eCB biosensor, GRAB_eCB2.0, was expressed presynaptically in corticostriatal afferents of C57BL6J mice and evoked eCB transients were measured in the DLS using a brain slice photometry technique. We found that brief bouts of synaptic stimulation induce long lasting eCB transients that were generated predominantly by 2-arachidonoylglycerol (2-AG) mobilization. Efficient 2-AG mobilization required coactivation of AMPA and NMDA ionotropic glutamate receptors and muscarinic M1 receptors. Dopamine D2 receptors expressed on cholinergic interneurons inhibited 2-AG mobilization by inhibiting acetylcholine release. Collectively, these data uncover unrecognized mechanisms underlying 2-AG mobilization in the DLS.  

5.3327           The activity of glyoxylase 1 is regulated by glucose-responsive phosphorylation on Tyr136

Cortizo, F.G., Pfaff, D., Wirth, A., Schlotterer, A., Medert, R., Morgenstern, J., Weber, T., Hammes, H-P., Fleming, T., Nawroth, P.P., Freichel, M. and Teleman, A.A. Mol. Metabolism, 55, 101406 (2022)   Objective Methylglyoxal (MG) is a highly reactive α-oxoaldehyde that glycates proteins. MG has been linked to the development of diabetic complications: MG is the major precursor of advanced glycation end products (AGEs), a risk marker for diabetic complications in humans. Furthermore, flies and fish with elevated MG develop insulin resistance, obesity, and hyperglycemia. MG is detoxified in large part through the glyoxalase system, whose rate-limiting enzyme is glyoxalase I (Glo1). Hence, we aimed to study how Glo1 activity is regulated. Methods We studied the regulation and effect of post-translational modifications of Glo1 in tissue culture and in mouse models of diabetes. Results We show that Glo1 activity is promoted by phosphorylation on Tyrosine 136 via multiple kinases. We find that Glo1 Y136 phosphorylation responds in a bimodal fashion to glucose levels, increasing in cell culture from 0 mM to 5 mM (physiological) glucose, and then decreasing at higher glucose concentrations, both in cell culture and in mouse models of hyperglycemia. Conclusions These data, together with published findings that elevated MG leads to hyperglycemia, suggest the existence of a deleterious positive feedback loop whereby hyperglycemia leads to reduced Glo1 activity, contributing to elevated MG levels, which in turn promote hyperglycemia. Hence, perturbations elevating either glucose or MG have the potential to start an auto-amplifying feedback loop contributing to diabetic complications.  

5.3328           Defining biological and biophysical properties of SARS-CoV-2 genetic material in wastewater

Robinson, C.A., Hsieh, H-Y., Hsu, S-Y., Wang, Y., Salcedo, B.T. et al Science of the Total Environment, 807, 150786 (2022)   SARS-CoV-2 genetic material has been detected in raw wastewater around the world throughout the COVID-19 pandemic and has served as a useful tool for monitoring community levels of SARS-CoV-2 infections. SARS-CoV-2 genetic material is highly detectable in a patient's feces and the household wastewater for several days before and after a positive COVID-19 qPCR test from throat or sputum samples. Here, we characterize genetic material collected from raw wastewater samples and determine recovery efficiency during a concentration process. We find that pasteurization of raw wastewater samples did not reduce SARS-CoV-2 signal if RNA is extracted immediately after pasteurization. On the contrary, we find that signal decreased by approximately half when RNA was extracted 24–36 h post-pasteurization and ~90% when freeze-thawed prior to concentration. As a matrix control, we use an engineered enveloped RNA virus. Surprisingly, after concentration, the recovery of SARS-CoV-2 signal is consistently higher than the recovery of the control virus leading us to question the nature of the SARS-CoV-2 genetic material detected in wastewater. We see no significant difference in signal after different 24-hour temperature changes; however, treatment with detergent decreases signal ~100-fold. Furthermore, the density of the samples is comparable to enveloped retrovirus particles, yet, interestingly, when raw wastewater samples were used to inoculate cells, no cytopathic effects were seen indicating that wastewater samples do not contain infectious SARS-CoV-2. Together, this suggests that wastewater contains fully intact enveloped particles.  

5.3329           Breaking the sound barrier: Towards next-generation AAV vectors for gene therapy of hearing disorders

Fakhiri, J., Landegger, L.D. and Grimm, D. Hearing Res., 413, 108092 (2022)   Owing to the advances in transgenic animal technology and the advent of the next-generation sequencing era, over 120 genes causing hereditary hearing loss have been identified by now. In parallel, the field of human gene therapy continues to make exciting and rapid progress, culminating in the recent approval of several ex vivo and in vivo applications. Despite these encouraging developments and the growing interest in causative treatments for hearing disorders, gene therapeutic interventions in the inner ear remain in their infancy and await clinical translation. This review focuses on the adeno-associated virus (AAV), which nowadays represents one of the safest and most promising vectors in gene therapy. We first provide an overview of AAV biology and outline the principles of therapeutic gene transfer with recombinant AAV vectors, before pointing out major challenges and solutions for clinical translation including vector manufacturing and species translatability. Finally, we highlight seminal technologies for engineering and selection of next-generation "designer" AAV capsids, and illustrate their power and potential with recent examples of their application for inner ear gene transfer in animals.  

5.3330           Intravitreal gene therapy restores the autophagy-lysosomal pathway and attenuates retinal degeneration in cathepsin D-deficient mice

Liu, J., Bassal, M., Schlichting, S., Braren, I., Di Spiezio, A., Saftig, P. and Bartsch, U. Neurobiology of Disease, 164, 105628 (2022)   Loss of vision due to progressive retinal degeneration is a hallmark of neuronal ceroid lipofuscinoses (NCL), a group of fatal neurodegenerative lysosomal storage diseases. Enzyme substitution therapies represent promising treatment options for NCLs caused by dysfunctions of soluble lysosomal enzymes. Here, we compared the efficacy of a cell-based enzyme substitution strategy and a gene therapy approach to attenuate the retinal pathology in cathepsin D- (CTSD) deficient mice, an animal model of CLN10 disease. Levels of enzymatically active CTSD in mutant retinas were significantly higher after an adeno-associated virus vector-mediated CTSD transfer to retinal glial cells and retinal pigment epithelial cells than after intravitreal transplantations of a CTSD overexpressing clonal neural stem cell line. In line with this finding, the gene therapy treatment restored the disrupted autophagy-lysosomal pathway more effectively than the cell-based approach, as indicated by a complete clearance of storage, significant attenuation of lysosomal hypertrophy, and normalized levels of the autophagy marker sequestosome 1/p62 and microtubule-associated protein 1 light chain 3-II. While the cell-based treatment did not prevent the rapidly progressing loss of various retinal cell types, the gene therapy approach markedly attenuated retinal degeneration as demonstrated by a pronounced rescue of photoreceptor cells and rod bipolar cells.  

5.3331           Endogenous cathelicidin is required for protection against ZIKV-caused testis damage via inactivating virons

Liu, Z., Wu, J., Qin, Z., Dong, C., Yang, H., Sun, J., Xu, W. and Wei, L. Antiviral Res., 198, 105248 (2022)   Cathelicidins have been shown to effectively inhibit flavivirus replication in vitro. However, the effects of mouse and human endogenous cathelicidins on flavivirus infection in vivo are rarely known. We herein found that mouse endogenous cathelicidin CRAMP was significantly up-regulated upon Zika virus (ZIKV) infection. CRAMP deficiency markedly exacerbated ZIKV replication in testis, and aggravated ZIKV-induced testicular damage and spermatic damage in mice, indicating that endogenous cathelicidin is required for protection against ZIKV-caused male infertility in mice. In vitro antiviral assay showed that both mouse cathelidin CRAMP and human cathelicidin LL-37 obviously reduced ZIKV-caused cytopathic effect and inhibited ZIKV replication in Vero cells. Antiviral mechanism revealed that they both directly inactivated ZIKV virons by binding to ZIKV virons and inducing the leakage of ZIKV genomic RNA, consequently inactivated ZIKV virons. In vivo antiviral assay indicated that both of them effectively inhibited ZIKV replication in C57BL/6J and IFNα/β receptor-deficient (Ifnar1^−/−) mice when CRAMP or LL-37 was intravenously injected in parallel with or at 1 h after intravenous injection of ZIKV, implying that CRAMP and LL-37 effectively inactivated ZIKV particles and exhibited therapeutic potential against ZIKV infection in vivo. Our findings reveal that endogenous cathelicidin CRAMP and LL-37 act as inactivators of ZIKV, and effectively protect against ZIKV replication and ZIKV-induced male infertility, highlighting their potential for therapy of ZIKV infection.  

5.3332           High Level Forebrain Expression of Active Tau Kinase p38γ Exacerbates Cognitive Dysfunction in Aged APP-transgenic Alzheimer’s Mice

Asih, P.R., Stefanoska, K., Prikas, P. and Ittner, A. Neuroscience, 484, 53-65 (2022)   Persistent improvement of cognitive deficits in Alzheimer’s disease (AD), a common form of dementia, is an unattained therapeutic objective. Gene therapy holds promise for treatment of familial and sporadic forms of AD. p38γ, a member of the p38 mitogen-activated protein (MAP) kinase family, inhibits amyloid-β toxicity through regulation of tau phosphorylation. We recently showed that a gene delivery approach increasing p38γ resulted in markedly better learning and memory performance in mouse models of AD at advanced stages of amyloid-β- and tau-mediated cognitive impairment. Notably, low-to-moderate expression of p38γ had beneficial outcomes on cognition. The impact of high levels of p38γ on neuronal function remain unclear. Therefore, we addressed the outcomes of high levels of active p38γ on brain function, by direct injection of p38γ-encoding adeno-associated virus (AAV) into the forebrain of aged mice of an APP transgenic AD mouse model. While motor function in p38γ-expressing APP transgenic mice 2 months post-injection was comparable to control treated APP mice, their activity was markedly reduced in the open field test and included frequent bouts of immobility. Moreover, their learning and memory function was markedly impaired compared to control-treated aged APP mice. These results suggest that high neuronal levels of active p38γ emphasize a stress kinase role of p38γ, perturbing circuit function in motivation, navigation, and spatial learning. Overall, this work shows excessive neuronal p38γ levels can aggravate circuit dysfunction and advises adjustable expression systems will be required for sustainable AD gene therapy based on p38γ activity.  

5.3333           Multi-scale light microscopy/electron microscopy neuronal imaging from brain to synapse with a tissue clearing method, ScaleSF

Furuta, T., Yamauchi, K., Okamoto, C., Isa, K., Isa, T. and Hioki, H. iScience, 25, 103601 (2022)   The mammalian brain is organized over sizes that span several orders of magnitude, from synapses to the entire brain. Thus, a technique to visualize neural circuits across multiple spatial scales (multi-scale neuronal imaging) is vital for deciphering brain-wide connectivity. Here, we developed this technique by coupling successive light microscopy/electron microscopy (LM/EM) imaging with a glutaraldehyde-resistant tissue clearing method, ScaleSF. Our multi-scale neuronal imaging incorporates (1) brain-wide macroscopic observation, (2) mesoscopic circuit mapping, (3) microscopic subcellular imaging, and (4) EM imaging of nanoscopic structures, allowing seamless integration of structural information from the brain to synapses. We applied this technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in simultaneous interrogation of their circuit structure and synaptic connectivity in a targeted way. Our multi-scale neuronal imaging will significantly advance the understanding of brain-wide connectivity by expanding the scales of objects.  

5.3334           Inducers of the NF-κB pathways impair hepatitis delta virus replication and strongly decrease progeny infectivity in vitro

Michelet, M., Alfaiate, D., Chardes, B., Pons, C., Faure-Dupay, S. et al JHEP Reports, 4(3), 100415 (2022)   Background & Aims HDV superinfection of chronically HBV-infected patients is the most aggressive form of chronic viral hepatitis, with an accelerated progression towards fibrosis/cirrhosis and increased risk of liver failure, hepatocellular carcinoma, and death. While HDV infection is not susceptible to available direct anti-HBV drugs, suboptimal responses are obtained with interferon-α-based therapies, and the number of investigational drugs remains limited. We therefore analyzed the effect of several innate immune stimulators on HDV replication in infected hepatocytes. Methods We used in vitro models of HDV and HBV infection based on primary human hepatocytes (PHHs) and the non-transformed HepaRG cell line that are relevant to explore new innate immune therapies. Results We describe here, for the first time, anti-HDV effects of Pam3CSK4 and BS1, agonists of Toll-like receptor (TLR)-1/2, and the lymphotoxin-β receptor (LTβR), respectively. Both types of agonists induced dose-dependent reductions of total intracellular HDV genome and antigenome RNA and of HDV protein levels, without toxicity in cells monoinfected with HDV or co/superinfected with HBV. Moreover, both molecules negatively affected HDV progeny release and strongly decreased their specific infectivity. The latter effect is particularly important since HDV is thought to persist in humans through constant propagation. Conclusions Immune-modulators inducing NF-κB pathways in hepatocytes can inhibit HDV replication and should be further evaluated as a possible therapeutic approach in chronically HBV/HDV-infected patients.  

5.3335           Molecular and functional architecture of striatal dopamine release sites

Banerjee, A., Imig, C., Balakrishnan, K., Wojcik, S.M., Brose, N. and Kaeser, P.S. Neuron, 110, 248-265 (2022)   Despite the importance of dopamine for striatal circuit function, mechanistic understanding of dopamine transmission remains incomplete. We recently showed that dopamine secretion relies on the presynaptic scaffolding protein RIM, indicating that it occurs at active zone-like sites similar to classical synaptic vesicle exocytosis. Here, we establish using a systematic gene knockout approach that Munc13 and Liprin-α, active zone proteins for vesicle priming and release site organization, are important for dopamine secretion. Furthermore, RIM zinc finger and C₂B domains, which bind to Munc13 and Liprin-α, respectively, are needed to restore dopamine release after RIM ablation. In contrast, and different from typical synapses, the active zone scaffolds RIM-BP and ELKS, and RIM domains that bind to them, are expendable. Hence, dopamine release necessitates priming and release site scaffolding by RIM, Munc13, and Liprin-α, but other active zone proteins are dispensable. Our work establishes that efficient release site architecture mediates fast dopamine exocytosis.  

5.3336           Plant expression systems as an economical alternative for the production of iELISA coating antigen AHSV VP7

Fearon, S.H., Dennis, S.J., Hitzeroth, I.I., Rybicki, E.P. and Meyers, A.E: New Biotechnology, 68, 48-56 (2022)   African horse sickness (AHS) is a debilitating and highly infectious arthropod-borne disease affecting all species of Equidae. The causative agent of AHS is the non-enveloped dsRNA African horse sickness virus (AHSV), belonging in the genus Orbivirus, family Reoviridae. The identification and surveillance of AHSV by simple and reliable diagnostic tools is essential for managing AHS outbreaks. Indirect ELISAs utilising soluble AHSV antigen or recombinant VP7, an immunodominant and serogroup-specific major core structural protein, are commonly used for serological diagnostic assays. Plant production systems are a significant alternative for recombinant protein production, as they are safe, easily scalable, production rates are rapid and upstream processes are more cost-effective than more traditional expression systems. This pilot study reports the successful production of AHSV-5 VP7 quasi-crystals in Nicotiana benthamiana by Agrobacterium tumefaciens-mediated transient expression using the self-replicating pRIC3.0 plant expression vector. After purification by means of density gradient ultracentrifugation, yields of pure VP7 of 2.66 µg/g fresh leaf mass (FLM) were achieved. Purified plant-produced AHSV-5 VP7 detected AHSV-specific antibodies in horse sera in an indirect ELISA and was able to distinguish between AHSV-positive and negative sera. Additionally, plant-produced AHSV-5 VP7 detected AHSV-specific antibodies to the same degree as E. coli-produced VP7. These results justify further investigation into the diagnostic capability of plant-produced AHSV VP7 quasi-crystals. To the best of our knowledge, this is the first report of AHSV VP7 quasi-crystal production in N. benthamiana and the first time that plant-produced VP7’s potential as a diagnostic has been assessed.  

5.3337           A warm-start digital CRISPR/Cas-based method for the quantitative detection of nucleic acids

Wu, X., Chan, C., Springs, S.L., Lee, Y.H., Lu, T.K. and Yu, H. Anal. Chim. Acta, 1196, 339494 (2022)   Nucleic acids-based molecular diagnostic tools incorporating the CRISPR/Cas system are being developed as rapid and sensitive methods for pathogen detection. However, most CRISPR/Cas-based diagnostics lack quantitative detection ability. Here, we report Warm-Start RApid DIgital Crispr Approach (WS-RADICA) for the rapid, sensitive, and quantitative detection of nucleic acids. WS-RADICA detected as little as 1 copy/μl SARS-CoV-2 RNA in 40 min (qualitative detection) or 60 min (quantitative detection). WS-RADICA can be easily adapted to various digital devices: two digital chips were evaluated for both DNA and RNA quantification, with linear dynamic ranges of 0.8–12777 copies/μL for DNA and 1.2–18391 copies/μL for RNA (both R² values > 0.99). Moreover, WS-RADICA had lower detection limit and higher inhibitor tolerance than a bulk RT-LAMP-Cas12b reaction and similar performance to RT-qPCR and RT-dPCR. To prove its performance on nucleic acids derived from live virus, WS-RADICA was also validated to detect and quantify human adenovirus and herpes simplex virus. Given its speed, sensitivity, quantification capability, and inhibitor tolerance, WS-RADICA shows great promise for a variety of applications requiring nucleic acid quantification.  

5.3338           Bile Acid–Mediated Activation of Brown Fat Protects From Alcohol-Induced Steatosis and Liver Injury in Mice

Fan, M., Wang, Y., Jin, L., Fang, Z., Peng, J. et al Cell. Mol. Gastroenterol. Hepatol., 13(3), 809-826 (2022)   Background & Aims Alcohol-associated liver disease (AALD) is one of the most common causes of liver injury and failure. Limited knowledge of the mechanisms underlying AALD impedes the development of efficacious therapies. Bile acid (BA) signaling was shown to participate in the progression of AALD. However, the mechanisms remain poorly understood. Methods C57BL/6J wild-type (WT), Takeda G-protein–coupled bile acid receptor 5 (TGR5) knockout (KO) and brown adipose tissue (BAT)-specific TGR5 knockdown mice were subjected to ethanol feeding–induced AALD. Liver samples from alcoholic hepatitis patients were used to examine the BA circulation signaling. Human Embryonic Kidney Cells 293 were used for the TGR5 reporter assay. 23(S)-methyl-lithocholic acid was used as a molecular tool to confirm the regulatory functions of BAT in the AALD mouse model. Results Ethanol feeding increased the expression of the thermogenesis genes downstream of TGR5 in BAT of WT, but not TGR5 KO, mice. TGR5 deficiency significantly blocked BAT activity and energy expenditure in mice after ethanol feeding. Alcohol increased serum BA levels in mice and human beings through altering BA transportation, and the altered BAs activated TGR5 signaling to regulate metabolism. Compared with ethanol-fed WT mice, ethanol-fed TGR5 KO mice showed less free fatty acid (FFA) β-oxidation in BAT, leading to higher levels of FFA in the circulation, increased liver uptake of FFAs, and exacerbated AALD. BAT-specific TGR5 knockdown mice showed similar results with TGR5 KO mice in AALD. Agonist treatment significantly activated TGR5 signaling in BAT, increased thermogenesis, reduced serum FFA level, and ameliorated hepatic steatosis and injury in AALD mice, while these effects were lost in TGR5 KO mice. Conclusions BA signaling plays a protective role in AALD by enhancing BAT thermogenesis. Targeting TGR5 in BAT may be a promising approach for the treatment of AALD.  

5.3339           Hepatic CDP-diacylglycerol synthase 2 deficiency causes mitochondrial dysfunction and promotes rapid progression of NASH and fibrosis

Xu, J., Chen, S., Wang, W., Lam, S.M., Xu, Y. et al Science Bulletin, 67, 299-314 (2022)   Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of pathologies, ranging from steatosis to nonalcoholic steatohepatitis (NASH). The factors promoting the progression of steatosis to NASH are still unclear. Recent studies suggest that mitochondrial lipid composition is critical in NASH development. Here, we showed that CDP-DAG synthase 2 (Cds2) was downregulated in genetic or diet-induced NAFLD mouse models. Liver-specific deficiency of Cds2 provoked hepatic steatosis, inflammation and fibrosis in five-week-old mice. CDS2 is enriched in mitochondria-associated membranes (MAMs), and hepatic Cds2 deficiency impaired mitochondrial function and decreased mitochondrial PE levels. Overexpression of phosphatidylserine decarboxylase (PISD) alleviated the NASH-like phenotype in Cds2^f/f;AlbCre mice and abnormal mitochondrial morphology and function caused by CDS2 deficiency in hepatocytes. Additionally, dietary supplementation with an agonist of peroxisome proliferator-activated receptor alpha (PPARα) attenuated mitochondrial defects and ameliorated the NASH-like phenotype in Cds2^f/f;AlbCre mice. Finally, Cds2 overexpression protected against high-fat diet-induced hepatic steatosis and obesity. Thus, Cds2 modulates mitochondrial function and NASH development.  

5.3340           Kinetic monitoring of neuronal stress response to proteostasis dysfunction

Santiago-Lopez, A.J., Berglund, K., Gross, R.E. and Gutekunst, C-A.N. Mol. Cell. Neurosci., 118, 103682 (2022)   Proteostasis dysfunction and activation of the unfolded protein response (UPR) are characteristic of all major neurodegenerative diseases. Nevertheless, although the UPR and proteostasis dysfunction has been studied in great detail in model organisms like yeast and mammalian cell lines, it has not yet been examined in neurons. In this study, we applied a viral vector-mediated expression of a reporter protein based on a UPR transcription factor, ATF4, and time-lapse fluorescent microscopy to elucidate how mouse primary neurons respond to pharmacological and genetic perturbations to neuronal proteostasis. In in vitro models of endoplasmic reticulum (ER) stress and proteasome inhibition, we used the ATF4 reporter to reveal the time course of the neuronal stress response relative to neurite degeneration and asynchronous cell death. We showed how potential neurodegenerative disease co-factors, ER stress and mutant α-synuclein overexpression, impacted neuronal stress response and overall cellular health. This work therefore introduces a viral vector-based reporter that yields a quantifiable readout suitable for non-cell destructive kinetic monitoring of proteostasis dysfunction in neurons by harnessing ATF4 signaling as part of the UPR activation.  

5.3341           Challenges in downstream purification of gene therapy viral vectors

Singh, N. and Heldt, C.L. Current Opinion in Chem., Engineering, 35, 100780 (2022)   The diversity and application of viral products has continued to explode. These modalities can be vaccines, cancer therapies, and gene therapies. However, their diversity is also the challenge in the downstream processing. Viral particles can be very labile, thus requiring intimate knowledge of biology to create environments where they are stable. However, despite these challenges, we have created many processes that produce large amounts of viral products. Different purification methods are utilized throughout the process. New modalities of chromatography are overcoming many of the challenges of diffusion-limited beads. Of special concern for gene therapy vectors is the need to separate the empty capsids from the full capsids, which contain the therapeutic gene of interest. With the discovery of novel therapuetic modalities that could revolutionize care by finding cures, the downstream processing of viral therapies needs to find solutions to make these therapies and cures affordable.  

5.3342           Optimized protocols for efficient gene editing in mouse hepatocytes in vivo using CRISPR-Cas9 technology

Chen, Y. and Ding, Q. STAR Protocols, 3, 101062 (2022)   We provide a protocol for gene editing in mouse hepatocytes in vivo using the CRISPR-Cas9 technology via AAV delivery. This protocol describes the construction of AAV plasmids, AAV packaging, injection, and the detection of in vivo knockout efficiency. Using this protocol, we can get up to 10¹⁴ AAV and knock out genes in hepatocytes efficiently within 15 days. Moreover, we describe an optimized protocol to simultaneously target two genes via AAV delivery of CRISPR-Cas9 materials in the liver.  

5.3343           Small Heat Shock Protein 22 Improves Cognition and Learning in the Tauopathic Brain

Ospina, S.R., Blazier, D.M., Criado-marrero, M., Gould, L.A., Gebru, N.T. et al Int. J. Mol. Sci., 23:851 (2022)   The microtubule-associated protein tau pathologically accumulates and aggregates in Alzheimer’s disease (AD) and other tauopathies, leading to cognitive dysfunction and neuronal loss. Molecular chaperones, like small heat-shock proteins (sHsps), can help deter the accumulation of misfolded proteins, such as tau. Here, we tested the hypothesis that the overexpression of wild-type Hsp22 (wtHsp22) and its phosphomimetic (S24,57D) Hsp22 mutant (mtHsp22) could slow tau accumulation and preserve memory in a murine model of tauopathy, rTg4510. Our results show that Hsp22 protected against deficits in synaptic plasticity and cognition in the tauopathic brain. However, we did not detect a significant change in tau phosphorylation or levels in these mice. This led us to hypothesize that the functional benefit was realized through the restoration of dysfunctional pathways in hippocampi of tau transgenic mice since no significant benefit was measured in non-transgenic mice expressing wtHsp22 or mtHsp22. To identify these pathways, we performed mass spectrometry of tissue lysates from the injection site. Overall, our data reveal that Hsp22 overexpression in neurons promotes synaptic plasticity by regulating canonical pathways and upstream regulators that have been characterized as potential AD markers and synaptogenesis regulators, like EIF4E and NFKBIA.  

5.3344           Schwannoma Gene Therapy via Adeno-Associated Viral Vector Delivery of Apoptosis-Associated Speck-like Protein Containing CARD (ASC): Preclinical Efficacy and Safety

Ahmed, S.G., Maguire, C.A., Cao, S.A: and Brenner, C.J. Int. J. Mol. Sci., 23:819 (2022)   Schwannomas are tumors derived from Schwann-lineage cells, cells that protect and support myelinated nerves in the peripheral nervous system. They are typically slow-growing, encapsulated and benign. These tumors develop along peripheral, spinal and cranial nerves causing pain, sensory-motor dysfunction and death. Primary treatment for schwannoma is operative resection which can be associated with significant morbidity. Pharmacotherapy is largely restricted to bevacizumab, which has minimal or no efficacy for many patients and can be associated with treatment-limiting adverse effects. Given the suffering and morbidity associated with schwannoma and the paucity of therapeutic options, there is an urgent need for safe and effective therapies for schwannomas. We previously demonstrated that adeno-associated virus serotype 1 (AAV1) vector mediated delivery of the inflammasome adaptor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) under the control of the P0 promoter, produced a prolonged reduction in tumor volume and tumor-associated pain in human xenograft and mouse syngeneic schwannoma models. Here, we present data essential for the translation of our AAV1-P0-ASC schwannoma gene therapy to clinical trials. We determine the minimum effective dose of AAV1-P0-hASC required to induce an anti-tumor effect in the xenograft human-schwannoma model. We also show that the presence of preexisting AAV1 immunity does not alter the antitumor efficacy of AAV-P0-mASC in a syngeneic mouse schwannoma model. Furthermore, the maximum deliverable intratumoral dose of AAV1-P0-ASC was not associated with neuronal toxicity in immunocompetent mice. Taken together, these safety and efficacy data support the translation of the AAV1-P0-ASC schwannoma gene therapy strategy to clinical trials.  

5.3345           Inhibitory Effect of IL-1β on HBV and HDV Replication and HBs Antigen-Dependent Modulation of Its Secretion by Macrophages

Delphia, M., Faure-Dupuy, S., Osorce, N., Rivoire, M., Salvetti, A., Durantel, D. and Lucifora, J. Viruses, 14:65 (2022)   Co-infection with the hepatitis B virus and hepatitis delta virus (HDV) leads to the most aggressive form of viral hepatitis. Using in vitro infection models, we confirmed that IL-1β, a crucial innate immune molecule for pathogen control, was very potent against HBV from different genotypes. Additionally, we demonstrated for the first time a strong and rapid antiviral effect induced by very low doses of IL-1β against HDV. In parallel, using co-culture assays, we demonstrated that monocytes exposed to HBV, and in particular to HBsAg, during differentiation into pro-inflammatory macrophages secreted less IL-1β. Altogether, our data emphasize the importance of developing combined antiviral strategies that would, for instance, reduce the secretion of HBsAg and stimulate the immune system to produce endogenous IL-1β efficient against both HBV and HDV.  

5.3346           Induction of Hepatitis E Virus Anti-ORF3 Antibodies from Systemic Administration of a Muscle-Specific Adeno-Associated Virus (AAV) Vector

Maurer, L., El Andari, J., Rapti, K., Spreyer, l., Steinmann, E., Grimm, D. and Thi, V.L.D. Viruses, 14:266 (2022)   The hepatitis E virus (HEV) is a major global health problem, leading to large outbreaks in the developing world and chronic infections in the developed world. HEV is a non-enveloped virus, which circulates in the blood in a quasi-enveloped form. The quasi-envelope protects HEV particles from neutralising anti-capsid antibodies in the serum; however, most vaccine approaches are designed to induce an immune response against the HEV capsid. In this study, we explored systemic in vivo administration of a novel synthetic and myotropic Adeno-associated virus vector (AAVMYO3) to express the small HEV phosphoprotein ORF3 (found on quasi-enveloped HEV) in the musculature of mice, resulting in the robust and dose-dependent formation of anti-ORF3 antibodies. Neutralisation assays using the serum of ORF3 AAV-transduced mice showed a modest inhibitory effect on the infection of quasi-enveloped HEV in vivo, comparable to previously characterised anti-ORF3 antibodies used as a control. The novel AAVMYO3 capsid used in this study can serve as a versatile platform for the continued development of vector-based vaccines against HEV and other infectious agents, which could complement traditional vaccines akin to the current positive experience with SARS-CoV-2.  

5.3347           Time-Course of Alterations in the Endocannabinoid System after Viral-Mediated Overexpression of α-Synuclein in the Rat Brain

Kelly, R., Begelmans, A-P-. Josephine, C., Brouillet, E., McKernan, D.P. and Dowd, E. Molecules, 27:507 (2022)   Since the discovery of α-synuclein as the major component in Lewy bodies, research into this protein in the context of Parkinson’s disease pathology has been exponential. Cannabinoids are being investigated as potential therapies for Parkinson’s disease from numerous aspects, but still little is known about the links between the cannabinoid system and the pathogenic α-synuclein protein; understanding these links will be necessary if cannabinoid therapies are to reach the clinic in the future. Therefore, the aim of this study was to investigate the time-course of alterations in components of the endocannabinoid system after viral-mediated α-synuclein overexpression in the rat brain. Rats were given unilateral intranigral injections of AAV-GFP or AAV-α-synuclein and sacrificed 4, 8 and 12 weeks later for qRT-PCR and liquid chromatography–mass spectrometry analyses of the endocannabinoid system, in addition to histological visualization of α-synuclein expression along the nigrostriatal pathway. As anticipated, intranigral delivery of AAV-α-synuclein induced widespread overexpression of human α-synuclein in the nigrostriatal pathway, both at the mRNA level and the protein level. However, despite this profound α-synuclein overexpression, we detected no differences in CB₁ or CB₂ receptor expression in the nigrostriatal pathway; however, interestingly, there was a reduction in the expression of neuroinflammatory markers. Furthermore, there was a reduction in the levels of the endocannabinoid 2-AG and the related lipid immune mediator OEA at week 12 post-surgery, indicating that α-synuclein overexpression triggers dysregulation of the endocannabinoid system. Although this research does show that the endocannabinoid system is impacted by α-synuclein, further research is necessary to more comprehensively understand the link between the cannabinoid system and the α-synuclein aspect of Parkinson’s disease pathology in order for cannabinoid-based therapies to be feasible for the treatment of this disease in the coming years.  

5.3348           Hepatocellular Carcinoma Is a Natural Target for Adeno-Associated Virus (AAV) 2 Vectors

Meumann, N., Schmithals, C., Elenschneider, L., Hansen, T., Baalakrishnan, A. et al Cancers, 14:427 (2022)   Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current—or develop novel—strategies for treating HCC.  

5.3349           Selection of an Optimal Recombinant Egyptian H9N2 Avian Influenza Vaccine Strain for Poultry with High Antigenicity and Safety

An, S-H., Son, S-E., Song, J-H., Hong, S-M., Lee, C-Y., Lee, N-H., Jeong, Y-J., Choi, J-G., Lee, Y-J., Kang, H-M.,and Kwon, H-J. Vaccines, 10:162 (2022)   For the development of an optimized Egyptian H9N2 vaccine candidate virus for poultry, various recombinant Egyptian H9N2 viruses generated by a PR8-based reverse genetics system were compared in terms of their productivity and biosafety since Egyptian H9N2 avian influenza viruses already possess mammalian pathogenicity-related mutations in the hemagglutinin (HA), neuraminidase (NA), and PB2 genes. The Egyptian HA and NA genes were more compatible with PR8 than with H9N2 AIV (01310) internal genes, and the 01310-derived recombinant H9N2 strains acquired the L226Q reverse mutation in HA after passages in eggs. Additionally, the introduction of a strong promoter at the 3′-ends of PB2 and PB1 genes induced an additional mutation of P221S. When recombinant Egyptian H9N2 viruses with intact or reverse mutated HA (L226Q and P221S) and NA (prototypic 2SBS) were compared, the virus with HA and NA mutations had high productivity in ECES but was lower in antigenicity when used as an inactivated vaccine due to its high binding affinity into non-specific inhibitors in eggs. Finally, we substituted the PB2 gene of PR8 with 01310 to remove the replication ability in mammalian hosts and successfully generated the best recombinant vaccine candidate in terms of immunogenicity, antigenicity, and biosafety.  

5.3350           Scn1a gene reactivation after symptom onset rescues pathological phenotypes in a mouse model of Dravet syndrome

Valassina, N., Brusco, S., Salamone, A., Serra, L., Luoni, M., Gianelli, S., Bido, S., Massimino, L., Ungaro, F., Mazzara, P.G., D’Adamo, P., Lignani, G., Broccoli, V. and Colasante, G. Nature Comm., 13161 (2022)   Dravet syndrome is a severe epileptic encephalopathy caused primarily by haploinsufficiency of the SCN1A gene. Repetitive seizures can lead to endurable and untreatable neurological deficits. Whether this severe pathology is reversible after symptom onset remains unknown. To address this question, we generated a Scn1a conditional knock-in mouse model (Scn1a ^Stop/+) in which Scn1a expression can be re-activated on-demand during the mouse lifetime. Scn1a gene disruption leads to the development of seizures, often associated with sudden unexpected death in epilepsy (SUDEP) and behavioral alterations including hyperactivity, social interaction deficits and cognitive impairment starting from the second/third week of age. However, we showed that Scn1a gene re-activation when symptoms were already manifested (P30) led to a complete rescue of both spontaneous and thermic inducible seizures, marked amelioration of behavioral abnormalities and normalization of hippocampal fast-spiking interneuron firing. We also identified dramatic gene expression alterations, including those associated with astrogliosis in Dravet syndrome mice, that, accordingly, were rescued by Scn1a gene expression normalization at P30. Interestingly, regaining of Na_v1.1 physiological level rescued seizures also in adult Dravet syndrome mice (P90) after months of repetitive attacks. Overall, these findings represent a solid proof-of-concept highlighting that disease phenotype reversibility can be achieved when Scn1a gene activity is efficiently reconstituted in brain cells.  

5.3351           MicroRNA-365 regulates human cardiac action potential duration

Esfandyari, D., Idrissou, B.M.G., Hennis, K., Avramopoulos, P., Dueck, A. et al Nature Comm., 13:220 (2022)   Abnormalities of ventricular action potential cause malignant cardiac arrhythmias and sudden cardiac death. Here, we aim to identify microRNAs that regulate the human cardiac action potential and ask whether their manipulation allows for therapeutic modulation of action potential abnormalities. Quantitative analysis of the microRNA targetomes in human cardiac myocytes identifies miR-365 as a primary microRNA to regulate repolarizing ion channels. Action potential recordings in patient-specific induced pluripotent stem cell-derived cardiac myocytes show that elevation of miR-365 significantly prolongs action potential duration in myocytes derived from a Short-QT syndrome patient, whereas specific inhibition of miR-365 normalizes pathologically prolonged action potential in Long-QT syndrome myocytes. Transcriptome analyses in these cells at bulk and single-cell level corroborate the key cardiac repolarizing channels as direct targets of miR-365, together with functionally synergistic regulation of additional action potential-regulating genes by this microRNA. Whole-cell patch-clamp experiments confirm miR-365-dependent regulation of repolarizing ionic current I_ks. Finally, refractory period measurements in human myocardial slices substantiate the regulatory effect of miR-365 on action potential in adult human myocardial tissue. Our results delineate miR-365 to regulate human cardiac action potential duration by targeting key factors of cardiac repolarization.  

5.3352           Engineered bacterial voltage-gated sodium channel platform for cardiac gene therapy

Nguyen, H.X., Wu, T., Needs, D., Zhang, H., Perelli, R.M., DeLuca, s., Yang, R., Tian, M., Landstrom, A.P., Henriquez, C. and Bursac, N. Nature Comm., 13:620 (2022)   Therapies for cardiac arrhythmias could greatly benefit from approaches to enhance electrical excitability and action potential conduction in the heart by stably overexpressing mammalian voltage-gated sodium channels. However, the large size of these channels precludes their incorporation into therapeutic viral vectors. Here, we report a platform utilizing small-size, codon-optimized engineered prokaryotic sodium channels (BacNa_v) driven by muscle-specific promoters that significantly enhance excitability and conduction in rat and human cardiomyocytes in vitro and adult cardiac tissues from multiple species in silico. We also show that the expression of BacNa_v significantly reduces occurrence of conduction block and reentrant arrhythmias in fibrotic cardiac cultures. Moreover, functional BacNa_v channels are stably expressed in healthy mouse hearts six weeks following intravenous injection of self-complementary adeno-associated virus (scAAV) without causing any adverse effects on cardiac electrophysiology. The large diversity of prokaryotic sodium channels and experimental-computational platform reported in this study should facilitate the development and evaluation of BacNa_v-based gene therapies for cardiac conduction disorders.  

5.3353           Therapeutic potential of highly functional codon-optimized microutrophin for muscle-specific expression

Strarikova, A.V., Skopenkova, V.V., Polikarpova, A.V., Reshetov, D.A., Vassilieva, S.G., Velyaev, O.A., Shmidt, A.A., Savchenko, I.M., Soldatov, V.O., Egorova, T.V. and Bardina, M.V. Scientific Reports, 12:848 (2022)   High expectations have been set on gene therapy with an AAV-delivered shortened version of dystrophin (µDys) for Duchenne muscular dystrophy (DMD), with several drug candidates currently undergoing clinical trials. Safety concerns with this therapeutic approach include the immune response to introduced dystrophin antigens observed in some DMD patients. Recent reports highlighted microutrophin (µUtrn) as a less immunogenic functional dystrophin substitute for gene therapy. In the current study, we created a human codon-optimized µUtrn which was subjected to side-by-side characterization with previously reported mouse and human µUtrn sequences after rAAV9 intramuscular injections in mdx mice. Long-term studies with systemic delivery of rAAV9-µUtrn demonstrated robust transgene expression in muscles, with localization to the sarcolemma, functional improvement of muscle performance, decreased creatine kinase levels, and lower immunogenicity as compared to µDys. An extensive toxicity study in wild-type rats did not reveal adverse changes associated with high-dose rAAV9 administration and human codon-optimized µUtrn overexpression. Furthermore, we verified that muscle-specific promoters MHCK7 and SPc5-12 drive a sufficient level of rAAV9-µUtrn expression to ameliorate the dystrophic phenotype in mdx mice. Our results provide ground for taking human codon-optimized µUtrn combined with muscle-specific promoters into clinical development as safe and efficient gene therapy for DMD.  

5.3354           A behavioural correlate of the synaptic eligibility trace in the nucleus accumbens

Yamaguchi, K., Maeda, Y., Sawadda, T., Iino, Y., Tajiri, M., Nakazato, R., Ishii, S., Kasai, h. and Yagishita, S. Scientific Reports, 12:1921 (2022)   Reward reinforces the association between a preceding sensorimotor event and its outcome. Reinforcement learning (RL) theory and recent brain slice studies explain the delayed reward action such that synaptic activities triggered by sensorimotor events leave a synaptic eligibility trace for 1 s. The trace produces a sensitive period for reward-related dopamine to induce synaptic plasticity in the nucleus accumbens (NAc). However, the contribution of the synaptic eligibility trace to behaviour remains unclear. Here we examined a reward-sensitive period to brief pure tones with an accurate measurement of an effective timing of water reward in head-fixed Pavlovian conditioning, which depended on the plasticity-related signaling in the NAc. We found that the reward-sensitive period was within 1 s after the pure tone presentation and optogenetically-induced presynaptic activities at the NAc, showing that the short reward-sensitive period was in conformity with the synaptic eligibility trace in the NAc. These findings support the application of the synaptic eligibility trace to construct biologically plausible RL models.  

5.3355           Towards the direct detection of viral materials at the surface of protective face masks via infrared spectroscopy

Schorer, V., Haas, j., Stach, R., Kookoric, V., Gross, R., Muench, J., Hummel, T., Sobek, H., Mennig, j. and Mizaikoff, B. Scientific Reports, 12:2309 (2022)   The ongoing COVID-19 pandemic represents a considerable risk for the general public and especially for health care workers. To avoid an overloading of the health care system and to control transmission chains, the development of rapid and cost-effective techniques allowing for the reliable diagnosis of individuals with acute respiratory infections are crucial. Uniquely, the present study focuses on the development of a direct face mask sampling approach, as worn (i.e., used) disposable face masks contain exogenous environmental constituents, as well as endogenously exhaled breath aerosols. Optical techniques—and specifically infrared (IR) molecular spectroscopic techniques—are promising tools for direct virus detection at the surface of such masks. In the present study, a rapid and non-destructive approach for monitoring exposure scenarios via medical face masks using attenuated total reflection infrared spectroscopy is presented. Complementarily, IR external reflection spectroscopy was evaluated in comparison for rapid mask analysis. The utility of a face mask-based sampling approach was demonstrated by differentiating water, proteins, and virus-like particles sampled onto the mask. Data analysis using multivariate statistical algorithms enabled unambiguously classifying spectral signatures of individual components and biospecies. This approach has the potential to be extended towards the rapid detection of SARS-CoV-2—as shown herein for the example of virus-like particles which are morphologically equivalent to authentic virus—without any additional sample preparation or elaborate testing equipment at laboratory facilities. Therefore, this strategy may be implemented as a routine large-scale monitoring routine, e.g., at health care institutions, nursing homes, etc. ensuring the health and safety of medical personnel.  

5.3356           Development and Application of a Liquid Chromatography-Mass Spectrometry Method for Residual Iodixanol Quantification in AAV-Based Gene Therapy Product Development

Pu, Y., Katz, R., Chen, Y., Kostrusky, V., Clarner, P., Lp, S-C., Sosic, Z and Yeung, B. Human Gene Therapy, 33(1-2), 103-108 (2022)   Adeno-associated viruses (AAVs) are nonenveloped viruses that have become popular gene transfer vectors to deliver DNA to target cells in clinical gene therapy. Iodixanol-based density gradient is one of the widely used purification methods for serotype-independent AAVs. However, residual iodixanol in AAV could be a safety concern, and further purification to remove this process-related impurity is typically needed. An analytical assay with high sensitivity is essential for the detection of residual iodixanol to ensure the safety of AAV products. We developed a liquid chromatography-mass spectrometry method with the limit of quantification of 0.01 μg/mL for residual iodixanol measurement in AAVs. The method also demonstrated linearity over four orders of magnitude that allows quantifying a high iodixanol concentration in in-process samples with excellent recovery and accuracy. In addition, we further explored a highly efficient purification method for removal of the residual iodixanol, to minimize the safety concern from iodixanol as a process impurity.  

5.3357           Efficient and Precise Processing of the Optimized Primary Artificial MicroRNA in a Huntingtin-Lowering Adeno-Associated Viral Gene Therapy In Vitro and in Mice and Nonhuman Primates

Wang, W., Zhou, P., Wang, X., Chen, F., Christensen, E., Thompson, J., Ren, X., Kells, A., Stanek, L., Carter, T., Hou, J. and Sah, D.W.Y. Human Gene Therapy, 33(1-2), 37-60 (2022)   Huntington's disease is a fatal neurodegenerative disorder caused by an inherited mutation in the huntingtin (HTT) gene comprising an expanded cytosine-adenine-guanine (CAG) trinucleotide repeat sequence that results in a pathogenic huntingtin protein. Adeno-associated viral (AAV) gene therapy containing a primary artificial microRNA (pri-amiRNA) specifically targeting HTT messenger RNA (mRNA) has the potential to provide long-lasting therapeutic benefit, through durable reduction of mutant HTT expression after a single administration. The efficiency and precision of processing of the pri-amiRNA precursor to the mature guide (G) strand by transduced cells are critical for specific and potent HTT mRNA lowering. The selection of the optimized pri-amiRNA comprised a series of in vitro studies followed by in vivo studies in small and then large mammals. Our studies demonstrate the predictivity of certain cell culture systems and rodent models for nonhuman primates with respect to some, but not all key features of pri-amiRNA processing. In addition, our results show that the processing of pri-amiRNAs to the mature guide strand can differ greatly across different scaffolds and sequences while providing the same levels of target lowering. Importantly, our data demonstrate that there is a combinatorial effect of guide and passenger (P) strand sequences, together with the scaffold, on pri-amiRNA processing, with different guide and passenger strand sequences within the same scaffold dramatically altering pri-amiRNA processing. Taken together, our results highlight the importance of optimizing not only target lowering but also the efficiency and precision of pri-amiRNA processing in vitro, in rodents and in large mammals to identify the most potent and selective AAV gene therapy that harnesses the endogenous microRNA (miRNA) biogenesis pathway for target lowering without perturbing the endogenous cellular miRNA profile. The optimized pri-amiRNA was selected with this focus on efficiency and precision of pri-amiRNA processing in addition to its pharmacological activity on HTT mRNA lowering and general tolerability in vivo.  

5.3358           Constitutively active SARM1 variants that induce neuropathy are enriched in ALS patients

Bloom, A.J., Mao, X., Strickland, A., Sasaki, Y., Milbrandt, j. and DiAntonio, A. Mol. Neuurogeneration., 17:1 (2022)   Background In response to injury, neurons activate a program of organized axon self-destruction initiated by the NAD⁺ hydrolase, SARM1. In healthy neurons SARM1 is autoinhibited, but single amino acid changes can abolish autoinhibition leading to constitutively active SARM1 enzymes that promote degeneration when expressed in cultured neurons. Methods To investigate whether naturally occurring human variants might disrupt SARM1 autoinhibition and potentially contribute to risk for neurodegenerative disease, we assayed the enzymatic activity of all 42 rare SARM1 alleles identified among 8507 amyotrophic lateral sclerosis (ALS) patients and 9671 controls. We then intrathecally injected mice with virus expressing SARM1 constructs to test the capacity of an ALS-associated constitutively active SARM1 variant to promote neurodegeneration in vivo. Results Twelve out of 42 SARM1 missense variants or small in-frame deletions assayed exhibit constitutive NADase activity, including more than half of those that are unique to the ALS patients or that occur in multiple patients. There is a > 5-fold enrichment of constitutively active variants among patients compared to controls. Expression of constitutively active ALS-associated SARM1 alleles in cultured dorsal root ganglion (DRG) neurons is pro-degenerative and cytotoxic. Intrathecal injection of an AAV expressing the common SARM1 reference allele is innocuous to mice, but a construct harboring SARM1^V184G, the constitutively active variant found most frequently among the ALS patients, causes axon loss, motor dysfunction, and sustained neuroinflammation. Conclusions These results implicate rare hypermorphic SARM1 alleles as candidate genetic risk factors for ALS and other neurodegenerative conditions.  

5.3359           Brain-wide TVA compensation allows rabies virus to retrograde target cell-type-specific projection neurons

Han, Z., Luo, N., Kou, J., Xu, L.L.Z., Wei, S., Wu, Y., Wang, J., Ye, C., Lin, K. and Xu, F. Mol. Brain., 15:13 (2022)   Retrograde tracers based on viral vectors are powerful tools for the imaging and manipulation of upstream neural networks projecting to a specific brain region, and they play important roles in structural and functional studies of neural circuits. However, currently reported retrograde viral tracers have many limitations, such as brain area selectivity or the inability to retrograde label genetically defined brain-wide projection neurons. To overcome these limitations, a new retrograde tracing method, AAV-PHP.eB assisted retrograde tracing systems (PARTS) based on rabies virus, was established through brain-wide TVA-dependent targeting using an AAV-PHP.eB that efficiently crosses the blood–brain barrier in C57BL/6 J mice, and complementation of EnvA-pseudotyped defective rabies virus that specifically recognizes the TVA receptor. Furthermore, combined with Cre transgenic mice, cell-type-specific PARTS (cPARTS) was developed, which can retrograde label genetically defined brain-wide projection neurons. Our research provides new tools and technical support for the analysis of neural circuits.  

5.3360           Chapter 1 - High-efficiency of genetic modification using CRISPR/Cpf1 system for engineered CAR-T cell therapy

Ding, R., Chao, C-C. and Gao, Q. Methods in Cell Biol., 167, 1-14 (2022)   Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated promising efficacy in several kinds of blood cancers, including diffuse large B-cell lymphoma and acute and chronic lymphoblastic leukemia, etc. It is essential to effectively generate more potent and safer CAR-T cells through gene editing technologies for immune cell therapy. Conventional methods based on lentivirus, retrovirus and transposon, randomly integrate CAR sequence into T cell genome, which could lead to safety issues. Therefore, precise knock-in of CAR cassette into specific gene locus like TRAC and PDCD1 can lower the risks caused by random integration, as well as enhance the stability and function of the modified CAR-T cells. Current approaches of CRISPR/Cas9-based gene-editing have limitations in knock-in efficiency of the chimeric antigen receptor, while Cpf1, a CRISPR–Cas/RNA-guided nuclease, shows higher homology-directed repair (HDR) rate compared to Cas9 due to its unique biochemical characteristics. Here, we introduce a method combining electroporation and adeno-associated virus (AAV) infection to deliver CRISPR/Cpf1 components and a HDR template into T cells, thus precisely integrate CAR sequence at a specific gene locus with high efficiency.  

5.3361           Systemic and local immune responses to intraocular AAV vector administration in non-human primates

Ail, D., Ren, D., Brazhnikova, E., Nouvel-Jaillard, C., Bertin, S., Mirashrafi, S.B., Fisson, S. and Dalkara, D. Molecular Therapy-Methods Clin. Develop., 24, 306-316 (2022)   Positive clinical outcomes in adeno-associated virus (AAV)-mediated retinal gene therapy have often been attributed to the low immunogenicity of AAVs and immune privilege of the eye. However, several recent studies have shown potential for inflammatory responses. The current understanding of the factors contributing to inflammation, such as the pre-existence of serum antibodies against AAVs and their contribution to increases in antibody levels post-injection, is incomplete. The parameters that regulate the generation of new antibodies in response to the AAV capsid or transgene after intraocular injections are also insufficiently described. This study is a retrospective analysis of the pre-existing serum antibodies in correlation with changes in antibody levels after intraocular injections of AAV in non-human primates (NHPs) of the species Macaca fascicularis. In NHP serums, we analyzed the binding antibody (BAB) levels and a subset of these called neutralizing antibodies (NABs) that impede AAV transduction. We observed significantly higher pre-existing serum BABs against AAV8 compared with other serotypes and a dose-dependent increase in BABs and NABs in the serums collected post-injection, irrespective of the serotype or the mode of injection. Lastly, we were able to demonstrate a correlation between the serum BAB levels with clinical grading of inflammation and levels of transgene expression.  

5.3362           Metabolic control of adult neural stem cell self-renewal by the mitochondrial protease YME1L

Wani, G.A., Sprenger, H-G., Ndoci, K., Motori, E., langer, T. and Bergami, M. Cell Reports, 38, 110370 (2022)   The transition between quiescence and activation in neural stem and progenitor cells (NSPCs) is coupled with reversible changes in energy metabolism with key implications for lifelong NSPC self-renewal and neurogenesis. How this metabolic plasticity is ensured between NSPC activity states is unclear. We find that a state-specific rewiring of the mitochondrial proteome by the i-AAA peptidase YME1L is required to preserve NSPC self-renewal. YME1L controls the abundance of numerous mitochondrial substrates in quiescent NSPCs, and its deletion activates a differentiation program characterized by broad metabolic changes causing the irreversible shift away from a fatty-acid-oxidation-dependent state. Conditional Yme1l deletion in adult NSPCs in vivo results in defective self-renewal and premature differentiation, ultimately leading to NSPC pool depletion. Our results disclose an important role for YME1L in coordinating the switch between metabolic states of NSPCs and suggest that NSPC fate is regulated by compartmentalized changes in protein network dynamics.  

5.3363           An AAV vaccine targeting the RBD of the SARS-CoV-2 S protein induces effective neutralizing antibody titers in mice and canines

Liu, F., Feng, C., Xu, S., Wu, Q., Tang, J., Chen, Y., Xu, R. et al Vaccine, 40, 1208-1212 (2022)   The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in catastrophic damage worldwide. Accordingly, the development of powerful, safe, easily accessible vaccines with long-term effectiveness is understood as an urgently needed countermeasure against this ongoing pandemic. Guided by this strong promise of using AAVs, we here designed, optimized, and developed an AAV-based vaccines (including AAV-RBD(max), AAV-RBD(wt), AAV-2xRBD, and AAV-3xRBD) that elicit strong immune responses against the RBD domain of the SARS-CoV-2 S protein. These immunogenic responses have proven long-lived, with near peak levels for at least six months in mice. Notably, the sera immunized with AAV-3xRBD vaccine contains powerful neutralizing antibodies against the SARS-CoV-2 pseudovirus. Further evidence proven that potent specific antibodies could also be elicited in canines after vaccination with AAV-3xRBD vaccine.  

5.3364           Targeted gene silencing in the nervous system with CRISPR-Cas13

Powell, J.E., Lim, C.K.W., Krishnan, R., McCallister, T.X., Saporito-Magrina, C., Zeballos, M.A., McPheron, G.D. and Gaj, T. Sci. Adv., 8, eabk2485 (2022)   Cas13 nucleases are a class of programmable RNA-targeting CRISPR effector proteins that are capable of silencing target gene expression in mammalian cells. Here, we demonstrate that RfxCas13d, a Cas13 ortholog with favorable characteristics to other family members, can be delivered to the mouse spinal cord and brain to silence neurodegeneration-associated genes. Intrathecally delivering an adeno-associated virus vector encoding an RfxCas13d variant programmed to target superoxide dismutase 1 (SOD1), a protein whose mutation can cause amyotrophic lateral sclerosis, reduced SOD1 mRNA and protein in the spinal cord by >50% and improved outcomes in a mouse model of the disorder. We further show that intrastriatally delivering an RfxCas13d variant programmed to target huntingtin (HTT), a protein whose mutation is causative for Huntington’s disease, led to a ~50% reduction in HTT protein in the mouse brain. Our results establish RfxCas13d as a versatile platform for knocking down gene expression in the nervous system.  

5.3365           Cryo-ET of Env on intact HIV virions reveals structural variation and positioning on the Gag lattice

Prasad, V.M., Leaman, D.P., Lovendahl, K.N., Hodge, E.A., Zwick, M.B. and Lee, K.K. Cell, 185, 641-653 (2022)   HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here, we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1-Å sub-tomogram-averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers, and a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.  

5.3366           Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine

Kishi, K.E., Kim, Y.S., Fukuda, M., Inoue, K., Deisseroth, K. and Kato, H.E. Cell, 185, 672-689 (2022)   ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.  

5.3367           High-Titer Hepatitis C Virus Production in a Scalable Single-Use High Cell Density Bioreactor

Offersgaard, A., Hernandez, C.R.D., Pihl, A.F., Venkatesan, N.P., Krarup, H., Lin, X., Reichl, U., Bukh, J., Genzel, Y. and Gottwein, J.M. Vaccines, 10:249 (2022)   Hepatitis C virus (HCV) infections pose a major public health burden due to high chronicity rates and associated morbidity and mortality. A vaccine protecting against chronic infection is not available but would be important for global control of HCV infections. In this study, cell culture-based HCV production was established in a packed-bed bioreactor (CelCradle™) aiming to further the development of an inactivated whole virus vaccine and to facilitate virological and immunological studies requiring large quantities of virus particles. HCV was produced in human hepatoma-derived Huh7.5 cells maintained in serum-free medium on days of virus harvesting. Highest virus yields were obtained when the culture was maintained with two medium exchanges per day. However, increasing the total number of cells in the culture vessel negatively impacted infectivity titers. Peak infectivity titers of up to 7.2 log₁₀ focus forming units (FFU)/mL, accumulated virus yields of up to 5.9 × 10¹⁰ FFU, and a cell specific virus yield of up to 41 FFU/cell were obtained from one CelCradle™. CelCradle™-derived and T flask-derived virus had similar characteristics regarding neutralization sensitivity and buoyant density. This packed-bed tide-motion system is available with larger vessels and may thus be a promising platform for large-scale HCV production.  

5.3368           Antiviral Targeting of Varicella Zoster Virus Replication and Neuronal Reactivation Using CRISPR/Cas9 Cleavage of the Duplicated Open Reading Frames 62/71

Wu, B.W., Yee, M.B., Goldstein, R.S. and Kinchington, P.R. Viruses, 14:378 (2022)   Varicella Zoster Virus (VZV) causes Herpes Zoster (HZ), a common debilitating and complicated disease affecting up to a third of unvaccinated populations. Novel antiviral treatments for VZV reactivation and HZ are still in need. Here, we evaluated the potential of targeting the replicating and reactivating VZV genome using Clustered Regularly Interspaced Short Palindromic Repeat-Cas9 nucleases (CRISPR/Cas9) delivered by adeno-associated virus (AAV) vectors. After AAV serotype and guide RNA (gRNA) optimization, we report that a single treatment with AAV2-expressing Staphylococcus aureus CRISPR/Cas9 (saCas9) with gRNA to the duplicated and essential VZV genes ORF62/71 (AAV2-62gRsaCas9) greatly reduced VZV progeny yield and cell-to-cell spread in representative epithelial cells and in lytically infected human embryonic stem cell (hESC)-derived neurons. In contrast, AAV2-62gRsaCas9 did not reduce the replication of a recombinant virus mutated in the ORF62 targeted sequence, establishing that antiviral effects were a consequence of VZV-genome targeting. Delivery to latently infected and reactivation-induced neuron cultures also greatly reduced infectious-virus production. These results demonstrate the potential of AAV-delivered genome editors to limit VZV productive replication in epithelial cells, infected human neurons, and upon reactivation. The approach could be developed into a strategy for the treatment of VZV disease and virus spread in HZ.  

5.3369           NPC1-regulated dynamic of clathrin-coated pits is essential for viral entry

Li, G., Su, B., Fu, P., Bai, Y., Ding, G., Li, D., Wang, J., Yang, G. and Chu, B. Science China Life Sciences, 65(2), 341-361 (2022)   Viruses utilize cellular lipids and manipulate host lipid metabolism to ensure their replication and spread. Therefore, the identification of lipids and metabolic pathways that are suitable targets for antiviral development is crucial. Using a library of compounds targeting host lipid metabolic factors and testing them for their ability to block pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) infection, we found that U18666A, a specific inhibitor of Niemann-Pick C1 (NPC1), is highly potent in suppressing the entry of diverse viruses including pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). NPC1 deficiency markedly attenuates viral growth by decreasing cholesterol abundance in the plasma membrane, thereby inhibiting the dynamics of clathrin-coated pits (CCPs), which are indispensable for clathrin-mediated endocytosis. Significantly, exogenous cholesterol can complement the dynamics of CCPs, leading to efficient viral entry and infectivity. Administration of U18666A improves the survival and pathology of PRV- and influenza A virus-infected mice. Thus, our studies demonstrate a unique mechanism by which NPC1 inhibition achieves broad antiviral activity, indicating a potential new therapeutic strategy against SARS-CoV-2, as well as other emerging viruses.  

5.3370           Superior infectivity of the fiber chimeric oncolytic adenoviruses Ad5/35 and Ad5/3 over Ad5-delta-24-RGD in primary glioma cultures

Stepanenko, A.A., Sosnovtseva, A.O., Valikhov, M.P., Chernysheva, A.A., Cherepanov, S.A., Yusubalieva, G.M., Ruzsics, Z., Lipatova, A.V. and Chekhonin, V.P. Noleculr Therapy-Oncolytics, 24, 230-248 (2022)   Ad5-delta-24-RGD is currently the most clinically advanced recombinant adenovirus (rAd) for glioma therapy. We constructed a panel of fiber-modified rAds (Ad5RGD, Ad5/3, Ad5/35, Ad5/3RGD, and Ad5/35RGD, all harboring the delta-24 modification) and compared their infectivity, replication, reproduction, and cytolytic efficacy in human and rodent glioma cell lines and short-term cultures from primary gliomas. In human cells, both Ad5/35-delta-24 and Ad5/3-delta-24 displayed superior infectivity and cytolytic efficacy over Ad5-delta-24-RGD, while Ad5/3-delta-24-RGD and Ad5/35-delta-24-RGD did not show further improvements in efficacy. The expression of the adenoviral receptors/coreceptors CAR, DSG2, and CD46 and the integrins αVβ3/αVβ5 did not predict the relative cytolytic efficacy of the fiber-modified rAds. The cytotoxicity of the fiber-modified rAds in human primary normal cultures of different origins and in primary glioma cultures was comparable, indicating that the delta-24 modification did not confer tumor cell selectivity. We also revealed that CT-2A and GL261 glioma cells might be used as murine cell models for the fiber chimeric rAds in vitro and in vivo. In GL261 tumor-bearing mice, Ad5/35-delta-24, armed with the immune costimulator OX40L as the E2A/DBP-p2A-mOX40L fusion, produced long-term survivors, which were able to reject tumor cells upon rechallenge. Our data underscore the potential of local Ad5/35-delta-24-based immunovirotherapy for glioblastoma treatment.  

5.3371           Meningeal lymphatic endothelial cells fulfill scavenger endothelial cell function and cooperate with microglia in waste removal from the brain

Huisman, Y., Uphoff, K., Berger, M., Dobrindt, U., Schelhaas, M., Zobel, T., Bussmann, J., van Impel, A. and Schulte-merker, S. Glia, 70- 35-49 (2022)   Brain lymphatic endothelial cells (BLECs) constitute a group of loosely connected endothelial cells that reside within the meningeal layer of the zebrafish brain without forming a vascular tubular system. BLECs have been shown to readily endocytose extracellular cargo molecules from the brain parenchyma, however, their functional relevance in relation to microglia remains enigmatic. We here compare their functional uptake efficiency for several macromolecules and bacterial components with microglia in a qualitative and quantitative manner in 5-day-old zebrafish embryos. We find BLECs to be significantly more effective in the uptake of proteins, polysaccharides and virus particles as compared to microglia, while larger particles like bacteria are only ingested by microglia but not by BLECs, implying a clear distribution of tasks between the two cell types in the brain area. In addition, we compare BLECs to the recently discovered scavenger endothelial cells (SECs) of the cardinal vein and find them to accept an identical set of substrate molecules. Our data identifies BLECs as the first brain-associated SEC population in vertebrates, and demonstrates that BLECs cooperate with microglia to remove particle waste from the brain.  

5.3372           A human cell atlas of the pressure-induced hypertrophic heart

Nicin, L., Schroeter, S.M., Glaser, S.G., Schulze-Brüning, R., Pham, M-D. et al Nature Cardiovasc. Res., 1, 174-185 (2022)   Pathological cardiac hypertrophy is a leading cause of heart failure, but knowledge of the full repertoire of cardiac cells and their gene expression profiles in the human hypertrophic heart is missing. Here, by using large-scale single-nucleus transcriptomics, we present the transcriptional response of human cardiomyocytes to pressure overload caused by aortic valve stenosis and describe major alterations in cardiac cellular crosstalk. Hypertrophied cardiomyocytes had reduced input from endothelial cells and fibroblasts. Genes encoding Eph receptor tyrosine kinases, particularly EPHB1, were significantly downregulated in cardiomyocytes of the hypertrophied heart. Consequently, EPHB1 activation by its ligand ephrin (EFN)B2, which is mainly expressed by endothelial cells, was reduced. EFNB2 inhibited cardiomyocyte hypertrophy in vitro, while silencing its expression in endothelial cells induced hypertrophy in co-cultured cardiomyocytes. Our human cell atlas of the hypertrophied heart highlights the importance of intercellular crosstalk in disease pathogenesis and provides a valuable resource.  

5.3373           The AAV9 Variant Capsid AAV-F Mediates Widespread Transgene Expression in Nonhuman Primate Spinal Cord After Intrathecal Administration

Baharry, A., Gong, Y., Kim, J.C., Hanlon, K.S., Nammour, J., Hieber, K., Eichler, F., Cheng, M., Stemmer-Rachamimov, A., Stankovic, K.M., Welling, D.B., Ng, C. and Maguire, C.A. Human Gne Therapy, 33(1-2), 61-75 (2022)   Intrathecal delivery of AAV9 into the subarachnoid space has been shown to transduce spinal cord and brain and be less affected by preexisting antibodies, which are lower in cerebral spinal fluid. Still, efficiency of transduction needs to be improved. Recently, we identified a new capsid from a library selection in mice, called AAV-F, that allowed robust transduction of the spinal cord gray matter after lumbar injection. In this study, we test transduction of spinal cord by AAV-F (n = 3) compared to AAV9 (n = 2), using a reporter gene, in cynomolgus monkeys after lumbar intrathecal injection. Using an automated image analysis (IA) approach to sensitively quantitate reporter gene expression in spinal cord, we found that AAV-F capsid mediated slightly higher transgene expression (both in percentages of cells and intensity of immunostaining) in motor neurons and interneurons, in the lumbar and thoracic regions, compared to AAV9. Interestingly, although AAV-F mediated higher transgene expression in spinal cord, the number of genomes in spinal cord and periphery were on average lower for AAV-F than AAV9, which suggest that lower numbers of genomes were able to mediate higher transgene expression in spinal cord with this capsid. In contrast, dorsal root ganglion transduction efficiency was lower for AAV-F compared to AAV9 on average. Interestingly, we also observed transduction of Schwann cells in sciatic nerve in two nonhuman primates injected with AAV-F, but none with AAV9. Overall, our data demonstrate the utility of automated IA for quantitation of AAV transduction in the spinal cord and the favorable on-target:off-target transduction profile suggests that the AAV-F capsid be considered for gene therapy applications focused on treating the spinal cord after intrathecal delivery.  

5.3374           CRISPR-Cas9 Gene Editing Protects from the A53T-SNCA Overexpression-Induced Pathology of Parkinson's Disease In Vivo

Yoon, H.H., Ye, S., Lim, S., Jo, A., Lee, H., Hong, F. et al The CRISP J., 5(1), 95-108 (2022)   Mutations in specific genes, including synuclein alpha (SNCA) that encodes the α-synuclein protein, are known to be risk factors for sporadic Parkinson's disease (PD), as well as critical factors for familial PD. In particular, A53T-mutated SNCA (A53T-SNCA) is a well-studied familial pathologic mutation in PD. However, techniques for deletion of the mutated SNCA gene in vivo have not been developed. Here, we used the CRISPR-Cas9 system to delete A53T-SNCA in vitro as well as in vivo. Adeno-associated virus carrying SaCas9-KKH with a single-guide RNA targeting A53T-SNCA significantly reduced A53T-SNCA expression levels in vitro. Furthermore, we tested its therapeutic potential in vivo in a viral A53T-SNCA-overexpressing rat model of PD. Gene deletion of A53T-SNCA significantly rescued the overexpression of α-synuclein, reactive microgliosis, dopaminergic neurodegeneration, and parkinsonian motor symptoms. Our findings propose CRISPR-Cas9 system as a potential prevention strategy for A53T-SNCA-specific PD.  

5.3375           c-Myb protects cochlear hair cells from cisplatin-induced damage via the PI3K/Akt signaling pathway

Bu, C., Xu, L., Han, Y., Wang, M., Wang, X., Liu, W., Chai, R and Wang, H. Cell Death Discovery, 8:78 (2022)   The transcription factor c-Myb is vital for cell survival, proliferation, differentiation, and apoptosis. We have previously reported that c-Myb knockdown exacerbates neomycin-induced damage to cochlea cells. However, the function and regulation of c-Myb in the mammalian inner ear remains unclear. Here, we first found that the expression of c-Myb in cochlear HCs was downregulated after cisplatin damage in vivo. Next, to investigate the role of c-Myb in HCs treated with cisplatin, the recombinant virus AAV-ie-CAG-Myb-HA (AAV-c-Myb) that overexpresses c-Myb was constructed and transfected into HCs. The protein expression of c-Myb was effectively up-regulated in cultured cochlear HCs after the virus transfection, which increased cochlear HC viability, decreased HC apoptosis and reduced intracellular reactive oxygen species (ROS) levels after cisplatin injury in vitro. The overexpression of c-Myb in HCs after AAV-c-Myb transfection in vivo also promoted HC survival, improved the hearing function of mice and reduced HC apoptosis after cisplatin injury. Furthermore, c-Myb-HC conditional knockout mice (Prestin; c-Myb-cKO) in which c-Myb expression is downregulated only in cochlear OHCs were generated and the cisplatin-induced HCs loss, apoptosis and hearing deficit were all exacerbated in Prestin; c-Myb-cKO mice treated with cisplatin in vivo. Finally, mechanistic studies showed that upregulation of the PI3K/Akt signaling pathway by c-Myb contributed to the increased HC survival after cisplatin exposure in vitro. The findings from this work suggest that c-Myb might serve as a new target for the prevention of cisplatin-induced HC damage and hearing loss.  

5.3376           Hippocampal NR6A1 impairs CREB-BDNF signaling and leads to the development of depression-like behaviors in mice

Tan, P., Xue, T., Wang, Y., Hu, Z., Su, J., Yang, R., Ji, J., Ye, M., Chen, Z., Huang, C. and Lu, X. Neuropharmacol., 209, 108990 (2022)   Chronic stress exposure is a risk factor that can induce the development of depression-like behaviors by impairing the hippocampal cyclic adenosine monophosphate-response element binding protein (CREB)-brain-derived neurotrophic factor (BDNF) signaling, but its underlying mechanisms remain largely unknown. We identified an orphan receptor that can suppress the activity of CREB, nuclear receptor sub-family 6, group A, member 1 (NR6A1), in mouse brain neurons. Given the critical role of the impaired CREB-BDNF signaling in depression, we speculate that the neuronal NR6A1 may mediate the pathogenesis of depression. Results showed that chronic unpredictable stress (CUS) markedly increased the expression levels of hippocampal NR6A1 protein, which reduced hippocampal CREB phosphorylation and BDNF protein expression. Overexpression of hippocampal NR6A1 in stress-naïve mice simulated chronic stress, inducing depression-like behaviors in the tail suspension test, forced swimming test, and sucrose preference test, and impairing the hippocampal CREB-BDNF signaling cascade. Genetic knockdown of hippocampal NR6A1 did not affect mouse behaviors but prevented the CUS-induced depression-like behaviors in mice and impairment in hippocampal CREB-BDNF signaling. Furthermore, genetic knockdown of hippocampal CREB or BDNF abrogated the preventive effect of hippocampal NR6A1 down-regulation on CUS-induced depression-like behaviors in mice. Collectively, these results for the first time identified a nuclear expression of NR6A1 in mouse brain neurons, and showed that the abnormally increased NR6A1 protein in the hippocampus in mice treated with or without chronic stress can impair the CREB-BDNF signaling cascade and lead to the development of depression-like behaviors. Hippocampal NR6A1 could be a novel target for the development of antidepressants.  

5.3377           The carboxyl-terminal region of SDCCAG8 comprises a functional module essential for cilia formation as well as organ development and homeostasis

Tsutsumi, R., Chaya, T., Tsujii, T. and Furukawa, T.

1. Biol. Chem., 298(3), 101686 (2022)

  In humans, ciliary dysfunction causes ciliopathies, which present as multiple organ defects, including developmental and sensory abnormalities. Sdccag8 is a centrosomal/basal body protein essential for proper cilia formation. Gene mutations in SDCCAG8 have been found in patients with ciliopathies manifesting a broad spectrum of symptoms, including hypogonadism. Among these mutations, several that are predicted to truncate the SDCCAG8 carboxyl (C) terminus are also associated with such symptoms; however, the underlying mechanisms are poorly understood. In the present study, we identified the Sdccag8 C-terminal region (Sdccag8-C) as a module that interacts with the ciliopathy proteins, Ick/Cilk1 and Mak, which were shown to be essential for the regulation of ciliary protein trafficking and cilia length in mammals in our previous studies. We found that Sdccag8-C is essential for Sdccag8 localization to centrosomes and cilia formation in cultured cells. We then generated a mouse mutant in which Sdccag8-C was truncated (Sdccag8^ΔC/ΔC mice) using a CRISPR-mediated stop codon knock-in strategy. In Sdccag8^ΔC/ΔC mice, we observed abnormalities in cilia formation and ciliopathy-like organ phenotypes, including cleft palate, polydactyly, retinal degeneration, and cystic kidney, which partially overlapped with those previously observed in Ick- and Mak-deficient mice. Furthermore, Sdccag8^ΔC/ΔC mice exhibited a defect in spermatogenesis, which was a previously uncharacterized phenotype of Sdccag8 dysfunction. Together, these results shed light on the molecular and pathological mechanisms underlying ciliopathies observed in patients with SDCCAG8 mutations and may advance our understanding of protein–protein interaction networks involved in cilia development.  

5.3378           Benefits of therapy by dynamin-2-mutant-specific silencing are maintained with time in a mouse model of dominant centronuclear myopathy

Trocket, D., Prudhon, B., Mekzine, L., lemaitre, M., Beuvin, M., Julien, L., Benkhelifa-Ziyyat, S., Bui, M.T., Romero, N. and Bitoun, M. Molecular Therapy-Nuucleic Acids, 27, 1179-1190 (2022)   Dominant dynamin 2 (DNM2) mutations are responsible for the autosomal dominant centronuclear myopathy (AD-CNM), a rare progressive neuromuscular disorder ranging from severe neonatal to mild adult forms. We previously demonstrated that mutant-specific RNA interference is an efficient therapeutic strategy to rescue the muscle phenotype at the onset of the symptoms in the AD-CNM knockin-Dnm2^R465W/+ mouse model. Our objective was to evaluate the long-term benefit of the treatment along with the disease time course. We demonstrate here that the complete rescue of the muscle phenotype is maintained for at least 1 year after a single injection of adeno-associated virus expressing the mutant-specific short hairpin RNA (shRNA). This was achieved by a maintained reduction of the mutant Dnm2 transcript. Moreover, this long-term study uncovers a pathological accumulation of DNM2 protein occurring with age in the mouse model and prevented by the treatment. Conversely, a physiological DNM2 protein decrease with age was observed in muscles from wild-type mice. Therefore, this study highlights a new potential pathophysiological mechanism linked to mutant protein accumulation and underlines the importance of DNM2 protein expression level for proper muscle function. Overall, these results strengthen the allele-specific silencing approach as a robust, safe, and efficient therapy for AD-CNM.  

5.3379           Modulation of miR-181 influences dopaminergic neuronal degeneration in a mouse model of Parkinson’s disease

Stein, C.S., McLendon, J.M., Witmer, N.H. and Boudreau, R.L. Molecular Therapy-Nucleic Acids, 28, 1-15 (2022)   Parkinson's disease (PD) is caused by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Although PD pathogenesis is not fully understood, studies implicate perturbations in gene regulation, mitochondrial function, and neuronal activity. MicroRNAs (miRs) are small gene regulatory RNAs that inhibit diverse subsets of target mRNAs, and several studies have noted miR expression alterations in PD brains. For example, miR-181a is abundant in the brain and is increased in PD patient brain samples; however, the disease relevance of this remains unclear. Here, we show that miR-181 target mRNAs are broadly downregulated in aging and PD brains. To address whether the miR-181 family plays a role in PD pathogenesis, we generated adeno-associated viruses (AAVs) to overexpress and inhibit the miR-181 isoforms. After co-injection with AAV overexpressing alpha-synuclein (aSyn) into mouse SN (PD model), we found that moderate miR-181a/b overexpression exacerbated aSyn-induced DA neuronal loss, whereas miR-181 inhibition was neuroprotective relative to controls (GFP alone and/or scrambled RNA). Also, prolonged miR-181 overexpression in SN alone elicited measurable neurotoxicity that is coincident with an increased immune response. mRNA-seq analyses revealed that miR-181a/b inhibits genes involved in synaptic transmission, neurite outgrowth, and mitochondrial respiration, along with several genes having known protective roles and genetic links in PD.  

5.3380           Epigenetic Silencing of Recombinant Adeno-associated Virus Genomes by NP220 and the HUSH Complex

Das, A., Vijayan, M., Walton, E.M., Stafford, V.G., Fiflis, D.N. and Asokan, A.

1. Virol., 96(4), E2039-21 (2022)

  The single-stranded DNA genome of adeno-associated viruses (AAV) undergoes second-strand synthesis and transcription in the host cell nucleus. While wild-type AAV genomes are naturally silenced upon integration into the host genome, recombinant AAV (rAAV) genomes typically provide robust expression of transgenes persisting as extrachromosomal DNA or episomes. Episomal DNA associating with host histones is subject to epigenetic modifications, although the mechanisms underlying such are not well understood. Here, we provide evidence that the double-stranded DNA binding protein NP220, in association with the human silencing hub (HUSH) complex, mediates transcriptional silencing of single-stranded as well as self-complementary rAAV genomes. In cells lacking NP220 or other components of the HUSH complex, AAV genome transcript levels are increased and correlate with a marked reduction in repressive H3K9 histone methylation marks. We also provide evidence that the AAV capsid (serotype) can profoundly influence NP220-mediated silencing of packaged genomes, indicating potential role(s) for capsid-genome or capsid-host factor interactions in regulating epigenetic silencing of rAAV genomes.  

5.3381           Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes

Rudy, M:J., Coughlan, C., Hixon, A.M., Clarke, P. and Tyler, K.L. Microbiology Spectrum, 10(1), e2452-21 (2022)   Enterovirus D68 (EV-D68) is an emerging pathogen which causes respiratory disease and is associated with an acute flaccid myelitis that predominately affects children. EV-D68 can infect motor neurons, causing cell death and a loss of motor control leading to flaccid paralysis. However, it remains unknown how viral particles gain entry into the central nervous system (CNS). Here, we show that three distinct densities of EV-D68 particle can be isolated from infected muscle and neural cell lines (RD and SH-SY5Y) using high-speed density centrifugation to separate cell supernatant. The lowest-density peak is composed of viral particles, which have adhered to the exterior surface of a small extracellular vesicle called an exosome. Analysis of prototypic (historic) and contemporary EV-D68 strains suggests that binding to exosomes is a ubiquitous characteristic of EV-D68. We further show that interaction with exosomes increases viral infectivity in a neural cell line. Analysis of the two higher-density peaks, which are not associated with exosomes, revealed that a significant amount of viral titer in the modern (2014) EV-D68 strains is found at 1.20 g/cm³, whereas this density has a very low viral titer in the prototypic Fermon strain.  

5.3382           SARS-CoV-2 Variants Increase Kinetic Stability of Open Spike Conformations as an Evolutionary Strategy

Yang, Z., Han, Y., Ding, S., Shi, W., Zhou, T., Finzi, A., Kwong, P.D., Mothes, W. and Lu, M. mBio, 13(1), e3227-21 (2022)   Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) harbor mutations in the spike (S) glycoprotein that confer more efficient transmission and dampen the efficacy of COVID-19 vaccines and antibody therapies. S mediates virus entry and is the primary target for antibody responses, with structural studies of soluble S variants revealing an increased propensity toward conformations accessible to the human angiotensin-converting enzyme 2 (hACE2) receptor. However, real-time observations of conformational dynamics that govern the structural equilibriums of the S variants have been lacking. Here, we report single-molecule Förster resonance energy transfer (smFRET) studies of critical mutations observed in VOCs, including D614G and E484K, in the context of virus particles. Investigated variants predominately occupied more open hACE2-accessible conformations, agreeing with previous structures of soluble trimers. Additionally, these S variants exhibited slower transitions in hACE2-accessible/bound states. Our finding of increased S kinetic stability in the open conformation provides a new perspective on SARS-CoV-2 adaptation to the human population.  

5.3383           Low-dose metformin targets the lysosomal AMPK pathway through PEN2

Ma, T., Tian, X., Zhang, B., Li, M., Wang, Y., yang, C. et al Nature, 603, 159-165 (2022)   Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1^,2^,3^,4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4^,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.  

5.3384           RGS3L allows for an M2 muscarinic receptor-mediated RhoA-dependent inotropy in cardiomyocytes

Levay, M.K., Krobert, K.A., Vogt, A., Ahmad, A., Jungmann, A., Neuber, c., pach, S., Hansen, A., Müller, O.J., Lutz, s. and Wieland, T. Basic Res. in Cardiol., 117:8 (2022)   The role and outcome of the muscarinic M₂ acetylcholine receptor (M₂R) signaling in healthy and diseased cardiomyocytes is still a matter of debate. Here, we report that the long isoform of the regulator of G protein signaling 3 (RGS3L) functions as a switch in the muscarinic signaling, most likely of the M₂R, in primary cardiomyocytes. High levels of RGS3L, as found in heart failure, redirect the G_i-mediated Rac1 activation into a G_i-mediated RhoA/ROCK activation. Functionally, this switch resulted in a reduced production of reactive oxygen species (− 50%) in cardiomyocytes and an inotropic response (+ 18%) in transduced engineered heart tissues. Importantly, we could show that an adeno-associated virus 9-mediated overexpression of RGS3L in rats in vivo, increased the contractility of ventricular strips by maximally about twofold. Mechanistically, we demonstrate that this switch is mediated by a complex formation of RGS3L with the GTPase-activating protein p190RhoGAP, which balances the activity of RhoA and Rac1 by altering its substrate preference in cardiomyocytes. Enhancement of this complex formation could open new possibilities in the regulation of the contractility of the diseased heart.  

5.3385           CELF4 regulates spine formation and depression-like behaviors of mice

Shen, Y., Zhang, C., Xiao, K., Liu, D. and Xie, G. Biochem. Biophys. Res. Comm., 605, 39-44 (2022)   Chronic social stress is closed related to major depressive disorder, torturing millions of people and may destroy their lives. The prefrontal cortex is one of the core brain areas involved in pathological development and behavior changes in depression. CELF4 is a neuronal RNA-binding protein and plays an essential role in RNA processing. It is closely related to some neurological disorders, including seizures and neuroticism. Most recently, GWAS analysis indicates it is one of the significant genes associated with depression. Nonetheless, we are still unknown whether and how CELF4 gets involved in depression. Here, we reported that the protein and mRNA expression levels of CELF4 in the PFC were decreased in the CSDS depression model, as well as the spine number. Furthermore, we disturbed CELF4 expression in the PFC by using the AAV-shCELF4 virus. Unexpectedly, the spine number showed a decrease in PFC because of the impaired CELF4 expression, and the AAV-shCELF4 mice displayed depression-like behaviors. Our results suggest that CELF4 is critical for spine number and acts a critical role in depression-like behaviors of mice.  

5.3386           Effects of the age of vaccination on the humoral responses to a human papillomavirus vaccine

Nicoli, F., Mantelli, B., Gllerani, E., Telatin, V., Squarzon, L., Masiero, S., Gaavioli, R., Palu, G., Barrzon, L and Caputo, A. Npj Vaccines, 7:37 (2022)   Adult vaccination programs are receiving increasing attention however, little is known regarding the impact of age on the maintenance of the immune response. We investigated this issue in the context of a human papillomavirus (HPV) vaccination program collecting real-world data on the durability of humoral immunity in 315 female subjects stratified according to vaccination age (adolescents and adults) and sampled at early or late time points after the last vaccine dose. HPV-specific IgGs, but not memory B cells, were induced and maintained at higher levels in subjects vaccinated during adolescence. Nonetheless, antibody functions waned over time to a similar degree in adolescents and adults. To shed light on this phenomena, we analyzed quantitative and qualitative properties of lymphocytes. Similar biochemical features were observed between B-cell subsets from individuals belonging to the two age groups. Long term humoral responses toward vaccines administered at an earlier age were comparably maintained between adolescents and adults. The percentages of naïve B and CD4⁺ T cells were significantly higher in adolescents, and the latter directly correlated with IgG titers against 3 out of 4 HPV types. Our results indicate that age-specific HPV vaccine responsiveness is mostly due to quantitative differences of immune cell precursors rather than qualitative defects in B cells. In addition, our results indicate that adults also have a good humoral immunogenic profile, suggesting that their inclusion in catch-up programmes is desirable.  

5.3387           A systematic comparison of optogenetic approaches to visual restoration

Gilhooley, M.J., Lindner, M., Palomaa, T., Hughes, S. and Peirson, S.N. Molecular Therapy-Methods Clin. Develop., 25, 111-123 (2022)   During inherited retinal degenerations (IRDs), vision is lost due to photoreceptor cell death; however, a range of optogenetic tools have been shown to restore light responses in animal models. Restored response characteristics vary between tools and the neuronal cell population to which they are delivered: the interplay between these is complex, but targeting upstream neurons (such as retinal bipolar cells) may provide functional benefit by retaining intraretinal signal processing. In this study, our aim was to compare two optogenetic tools: mammalian melanopsin (hOPN4) and microbial red-shifted channelrhodopsin (ReaChR) expressed within two subpopulations of surviving cells in a degenerate retina. Intravitreal adeno-associated viral vectors and mouse models utilising the Cre/lox system restricted expression to populations dominated by bipolar cells or retinal ganglion cells and was compared with non-targeted delivery using the chicken beta actin (CBA) promoter. In summary, we found bipolar-targeted optogenetic tools produced faster kinetics and flatter intensity-response relationships compared with non-targeted or retinal-ganglion-cell-targeted hOPN4. Hence, optogenetic tools of both mammalian and microbial origins show advantages when targeted to bipolar cells. This demonstrates the advantage of bipolar-cell-targeted optogenetics for vision restoration in IRDs. We therefore developed a bipolar-cell-specific gene delivery system employing a compressed promoter with the potential for clinical translation.  

5.3388           In vivo base editing rescues cone photoreceptors in a mouse model of early-onset inherited retinal degeneration

Choi, E.H., Suh, S., Foik, A.T., Leinonen, H., Newby, G.A., Gao, X.D., Banskota, S. et al Nature Comm., 13:1830 (2022)   Leber congenital amaurosis (LCA) is the most common cause of inherited retinal degeneration in children. LCA patients with RPE65 mutations show accelerated cone photoreceptor dysfunction and death, resulting in early visual impairment. It is therefore crucial to develop a robust therapy that not only compensates for lost RPE65 function but also protects photoreceptors from further degeneration. Here, we show that in vivo correction of an Rpe65 mutation by adenine base editor (ABE) prolongs the survival of cones in an LCA mouse model. In vitro screening of ABEs and sgRNAs enables the identification of a variant that enhances in vivo correction efficiency. Subretinal delivery of ABE and sgRNA corrects up to 40% of Rpe65 transcripts, restores cone-mediated visual function, and preserves cones in LCA mice. Single-cell RNA-seq reveals upregulation of genes associated with cone phototransduction and survival. Our findings demonstrate base editing as a potential gene therapy that confers long-lasting retinal protection.  

5.3389           Directed evolution of adeno-associated virus 5 capsid enables specific liver tropism

Wang, Y., Yang, C., Hu, H., Chen, C., Yan, M., Ling, L., Wang, K.C. et al Molecular Therapy-Nucleic Acids, 28, 293-306 (2022)   Impressive achievements in clinical trials to treat hemophilia establish a milestone in the development of gene therapy. It highlights the significance of AAV-mediated gene delivery to liver. AAV5 is a unique serotype featured by low neutralizing antibody prevalence. Nevertheless, its liver infectivity is relatively weak. Consequently, it is vital to exploit novel AAV5 capsid mutants with robust liver tropism. To this aim, we performed AAV5-NNK library and barcode screening in mice, from which we identified one capsid variant, called AAVzk2. AAVzk2 displayed a similar yield but divergent post-translational modification sites compared with wild-type serotypes. Mice intravenously injected with AAVzk2 demonstrated a stronger liver transduction than AAV5, roughly comparable with AAV8 and AAV9, with undetectable transduction of other tissues or organs such as heart, lung, spleen, kidney, brain, and skeletal muscle, indicating a liver-specific tropism. Further studies showed a superior human hepatocellular transduction of AAVzk2 to AAV5, AAV8 and AAV9, whereas the seroreactivity of AAVzk2 was as low as AAV5. Overall, we provide a novel AAV serotype that facilitates a robust and specific liver gene delivery to a large population, especially those unable to be treated by AAV8 and AAV9.  

5.3390           A presynaptic phosphosignaling hub for lasting homeostatic plasticity

Müller, J.A., Betzin, J., Santos-Tejedor, J., Graham, M.E., Dietrich, D. and Schoch, S. Cell Reports, 39, 110696 (2022)   Stable function of networks requires that synapses adapt their strength to levels of neuronal activity, and failure to do so results in cognitive disorders. How such homeostatic regulation may be implemented in mammalian synapses remains poorly understood. Here we show that the phosphorylation status of several positions of the active-zone (AZ) protein RIM1 are relevant for synaptic glutamate release. Position RIMS1045 is necessary and sufficient for expression of silencing-induced homeostatic plasticity and is kept phosphorylated by serine arginine protein kinase 2 (SRPK2). SRPK2-induced upscaling of synaptic release leads to additional RIM1 nanoclusters and docked vesicles at the AZ and is not observed in the absence of RIM1 and occluded by RIM^S1045E. Our data suggest that SRPK2 and RIM1 represent a presynaptic phosphosignaling hub that is involved in the homeostatic balance of synaptic coupling of neuronal networks.  

5.3391           Gene repression through epigenetic modulation by PPARA enhances hepatocellular proliferation

Aibara, D., Takahashi, S., Yagai, T., Levi, M., Matsusue, K. and Gonzalez, F.J. iScience, 25, 104196 (2022)   Peroxisome proliferator-activated receptor α (PPARA) is a key mediator of lipid metabolism and inflammation. Activation of PPARA in rodents causes hepatocyte proliferation, but the underlying mechanism is poorly understood. This study focused on genes repressed by PPARA and analyzed the mechanism by which PPARA promotes hepatocyte proliferation in mice. Activation of PPARA by agonist treatment was autoregulated, and induced expression of the epigenetic regulator UHRF1 via activation of the newly described PPARA target gene E2f8, which, in turn, regulates Uhrf1. UHRF1 strongly repressed the expression of CDH1 via methylation of the Cdh1 promoter marked with H3K9me3. Repression of CDH1 by PPARA activation was reversed by PPARA deficiency or knockdown of E2F8 or UHRF1. Furthermore, a forced expression of CDH1 inhibited expression of the Wnt signaling target genes such as Myc after PPARA activation, and suppressed hepatocyte hyperproliferation. These results demonstrate that the PPARA-E2F8-UHRF1-CDH1 axis causes epigenetic regulation of hepatocyte proliferation.  

5.3392           Humanized mice reveal a macrophage-enriched gene signature defining human lung tissue protection during SARS-CoV-2 infection

Kenney, D.J., O’Connell, A.K., Turcinovic, J., Crossland, N.A., Ploss, A. and Douam, F. Cell Reports, 39, 110714 (2022)   The human immunological mechanisms defining the clinical outcome of SARS-CoV-2 infection remain elusive. This knowledge gap is mostly driven by the lack of appropriate experimental platforms recapitulating human immune responses in a controlled human lung environment. Here, we report a mouse model (i.e., HNFL mice) co-engrafted with human fetal lung xenografts (fLX) and a myeloid-enhanced human immune system to identify cellular and molecular correlates of lung protection during SARS-CoV-2 infection. Unlike mice solely engrafted with human fLX, HNFL mice are protected against infection, severe inflammation, and histopathological phenotypes. Lung tissue protection from infection and severe histopathology associates with macrophage infiltration and differentiation and the upregulation of a macrophage-enriched signature composed of 11 specific genes mainly associated with the type I interferon signaling pathway. Our work highlights the HNFL model as a transformative platform to investigate, in controlled experimental settings, human myeloid immune mechanisms governing lung tissue protection during SARS-CoV-2 infection.  

5.3393           Dynamic Ca2+ sensitivity stimulates the evolved SARS-CoV-2 spike strain-mediated membrane fusion for enhanced entry

Singh, P., Mukherji, S., Basak, S., Hoffmann, M. and Das, D.K. Cell Reports, 39, 110694 (2022)   Mutations in the spike protein generated a highly infectious and transmissible D614G variant, which is present in newly evolved fast-spreading variants. The D614G, Alpha, Beta, and Delta spike variants of SARS-CoV-2 appear to expedite membrane fusion process for entry, but the mechanism of spike-mediated fusion is unknown. Here, we reconstituted an in vitro pseudovirus-liposome fusion reaction and report that SARS-CoV-2 wild-type spike is a dynamic Ca²⁺ sensor, and D614G mutation enhances dynamic calcium sensitivity of spike protein for facilitating membrane fusion. This dynamic calcium sensitivity for fusion is found to be higher in Alpha and Beta variants and highest in Delta spike variant. We find that efficient fusion is dependent on Ca²⁺ concentration at low pH, and the fusion activity of spike dropped as the Ca²⁺ level rose beyond physiological levels. Thus, evolved spike variants may control the high fusion probability for entry by increasing Ca²⁺ sensing ability.  

5.3394           Hepatitis E virus is highly resistant to alcohol-based disinfectants

Behrendt, P., Friesland, M., Wissmann, J-E., Kinast, V., Stahl, Y., Praditya, D. et al

1. Hepatol., 76, 1062-1069 (2022)

  Background & Aims Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide and is mainly transmitted via the fecal-oral route or through consumption of contaminated food products. Due to the lack of efficient cell culture systems for the propagation of HEV, limited data regarding its sensitivity to chemical disinfectants are available. Consequently, preventive and evidence-based hygienic guidelines on HEV disinfection are lacking. Methods We used a robust HEV genotype 3 cell culture model which enables quantification of viral infection of quasi-enveloped and naked HEV particles. For HEV genotype 1 infections, we used the primary isolate Sar55 in a fecal suspension. Standardized quantitative suspension tests using end point dilution and large-volume plating were performed for the determination of virucidal activity of alcohols (1-propanol, 2-propanol, ethanol), WHO disinfectant formulations and 5 different commercial hand disinfectants against HEV. Iodixanol gradients were conducted to elucidate the influence of ethanol on quasi-enveloped viral particles. Results Naked and quasi-enveloped HEV was resistant to alcohols as well as alcohol-based formulations recommended by the WHO. Of the tested commercial hand disinfectants only 1 product displayed virucidal activity against HEV. This activity could be linked to phosphoric acid as an essential ingredient. Finally, we observed that ethanol and possibly non-active alcohol-based disinfectants disrupt the quasi-envelope structure of HEV particles, while leaving the highly transmissible and infectious naked virions intact. Conclusions Different alcohols and alcohol-based hand disinfectants were insufficient to eliminate HEV infectivity with the exception of 1 commercial ethanol-based product that included phosphoric acid. These findings have major implications for the development of measures to reduce viral transmission in clinical practice.  

5.3395           Gene therapy for guanidinoacetate methyltransferase deficiency restores cerebral and myocardial creatine while resolving behavioral abnormalities

Khoja, S., Lambert, J., Nitzahn, M., Eliav, A., Zhang, Y.C., Tamboline, M. et al Molecular Therapy-Methods Clin. Develop., 25, 278-296 (2022)   Creatine deficiency disorders are inborn errors of creatine metabolism, an energy homeostasis molecule. One of these, guanidinoacetate N-methyltransferase (GAMT) deficiency, has clinical characteristics that include features of autism, self-mutilation, intellectual disability, and seizures, with approximately 40% having a disorder of movement; failure to thrive can also be a component. Along with low creatine levels, guanidinoacetic acid (GAA) toxicity has been implicated in the pathophysiology of the disorder. Present-day therapy with oral creatine to control GAA lacks efficacy; seizures can persist. Dietary management and pharmacological ornithine treatment are challenging. Using an AAV-based gene therapy approach to express human codon-optimized GAMT in hepatocytes, in situ hybridization, and immunostaining, we demonstrated pan-hepatic GAMT expression. Serial collection of blood demonstrated a marked early and sustained reduction of GAA with normalization of plasma creatine; urinary GAA levels also markedly declined. The terminal time point demonstrated marked improvement in cerebral and myocardial creatine levels. In conjunction with the biochemical findings, treated mice gained weight to nearly match their wild-type littermates, while behavioral studies demonstrated resolution of abnormalities; PET-CT imaging demonstrated improvement in brain metabolism. In conclusion, a gene therapy approach can result in long-term normalization of GAA with increased creatine in guanidinoacetate N-methyltransferase deficiency and at the same time resolves the behavioral phenotype in a murine model of the disorder. These findings have important implications for the development of a new therapy for this abnormality of creatine metabolism.  

5.3396           Monosynaptic rabies virus tracing from projection-targeted single neurons

Masaki, Y., Yamaguchi, M., Takeuchi, R.F. and Osakada, F. Neuroscience Res., 178, 20-32 (2022)   A single neuron integrates inputs from thousands of presynaptic neurons to generate outputs. Circuit tracing using G-deleted rabies virus (RVΔG) vectors permits the brain-wide labeling of presynaptic inputs to targeted single neurons. However, the experimental procedures are complex, and the success rate of circuit labeling is low because of the lack of validation to increase the accuracy and efficiency of monosynaptic RVΔG tracing from targeted single neurons. We established an efficient RVΔG tracing method from projection target-defined single neurons using TVA950, a transmembrane isoform of TVA receptors, for initial viral infection. Presynaptic neurons were transsynaptically labeled from 80 % of the TVA950-expressing single starter neurons that survived after infection with EnvA-pseudotyped RVΔG in the adult mouse brain. We labeled single neuronal networks in the primary visual cortex (V1) and higher visual areas, namely the posteromedial area (PM) and anteromedial area (AM), as well as the single neuronal networks of PM-projecting V1 single neurons. Monosynaptic RVΔG tracing from projection-targeted single neurons revealed the input–output organization of single neuronal networks. Single-neuron network analysis based on RVΔG tracing will help dissect the heterogeneity of neural circuits and link circuit motifs and large-scale networks across scales, thereby clarifying information processing and circuit computation in the brain.  

5.3397           Dependency of EGFR activation in vanadium-based sensitization to oncolytic virotherapy

Wong, B., Bergeron, A., Alluqmani, N., Maznyi, G., Chen, A., Arulanandam, R. and Diallo, J-S. Molecular Therapy-Oncplytics, 25, 146-159 (2022)   Oncolytic virotherapy is a clinically validated approach to treat cancers such as melanoma; however, tumor resistance to virus makes its efficacy variable. Compounds such as sodium orthovanadate (vanadate) can overcome viral resistance and synergize with RNA-based oncolytic viruses. In this study, we explored the basis of vanadate mode of action and identified key cellular components in vanadate’s oncolytic virus-enhancing mechanism using a high-throughput kinase inhibitor screen. We found that several kinase inhibitors affecting signaling downstream of the epidermal growth factor receptor (EGFR) pathway abrogated the oncolytic virus-enhancing effects of vanadate. EGFR pathway inhibitors such as gefitinib negated vanadate-associated changes in the phosphorylation and localization of STAT1/2 as well as NF-κB signaling. Moreover, gefitinib treatment could abrogate the viral sensitizing response of vanadium compounds in vivo. Together, we demonstrate that EGFR signaling plays an integral role in vanadium viral sensitization and that pharmacological EGFR blockade can counteract vanadium/oncolytic virus combination therapy.  

5.3398           Redirecting anti-Vaccinia virus T cell immunity for cancer treatment by AAV-mediated delivery of the VV B8R gene

Cao, D., Song, Q., Li, J., Dunmall, L.S.C., Jiang, Y., Qin, B., Wang, J., Guo, H., Cheng, Z., Wang, Z., Lemoine, N.R., Lu, S., and Wang, Y. Molelcular Therapy-Oncolytycs, 25, 264-275 (2022)   Immunotherapies, such as immune checkpoint inhibitors (ICIs) and chimeric antigen receptor-T (CAR-T) cells, are only efficient in a small proportion of tumor patients. One of the major reasons for this is the lack of immune cell infiltration and activation in the tumor microenvironment (TME). Recent research reported that abundant bystander CD8⁺ T cells targeting viral antigens exist in tumor infiltrates and that virus-specific memory T cells could be recalled to kill tumor cells. Therefore, virus-specific memory T cells may be effective candidates for tumor immunotherapy. In this study, we established subcutaneous tumor mice models that were pre-immunized with Vaccinia virus (VV) and confirmed that tumor cells with ectopic expression of the viral B8R protein could be recognized and killed by memory T cells. To create a therapeutic delivery system, we designed a recombinant adeno-associated virus (rAAV) with a modified tumor-specific promoter and used it to deliver VV B8R to tumor cells. We observed that rAAV gene therapy can retard tumor growth in VV pre-immunized mice. In summary, our study demonstrates that rAAV containing a tumor-specific promoter to restrict VV B8R gene expression to tumor cells is a potential therapeutic agent for cancer treatment in VV pre-immunized or VV-treated mice bearing tumors.  

5.3399           MSD-based assays facilitate a rapid and quantitative serostatus profiling for the presence of anti-AAV antibodies

Haar, J., Blazevic, D., Strobel, B., Kreuz, S. and Michelfelder, S. Molecular Therapy-Moethods Clin. Develop., 25, 360-369 (2022)   Adeno-associated virus (AAV) vector applications are often limited by capsid-directed humoral immune responses, mainly through neutralizing antibodies (NAbs), which are present throughout the human population due to natural AAV infections. Currently, antibody levels are often quantified via ELISA-based protocols or by cellular NAb assays and less frequently by in vivo NAb assays in mice. These methods need optimization for each serotype and are often not applicable to AAV variants with poor in vitro transduction. To tackle these limitations, we have established Meso Scale Discovery (MSD)-based assays for the quantification of binding antibodies (BAbs) and NAbs against the three most commonly used AAV serotypes, AAV2, AAV8, and AAV9. Both assays detect anti-AAV-IgG_1–3 with high sensitivity and consistency as shown in a screen of sera from 40 healthy human donors. Subsequently, BAb and NAb titers were determined for identification of seronegative animals in a non-human primate (NHP) cohort. Moreover, the MSD-based BAb assay protocol was extended to a panel of 14 different AAV serotypes. In summary, our platform allows a rapid and quantitative assessment of the immunological properties of any natural or engineered AAV variant irrespective of transduction efficiency and enables high-throughput screens.  

5.3400           Improved engraftment and therapeutic efficacy by human genome-edited hematopoietic stem cells with Busulfan-based myeloablation

Poletto, E., Colella, P., Vera, L.N.P., Khan, S., Tomatsu, S., Baldo, G. and Gomez-Ospina, N. Molecular Therapy-Methods Clin. Develop., 25, 392-409 (2022)   Autologous hematopoietic stem cell transplantation using genome-edited cells can become a definitive therapy for hematological and non-hematological disorders with neurological involvement. Proof-of-concept studies using human genome-edited hematopoietic stem cells have been hindered by the low efficiency of engraftment of the edited cells in the bone marrow and their modest efficacy in the CNS. To address these challenges, we tested a myeloablative conditioning regimen based on Busulfan in an immunocompromised model of mucopolysaccharidosis type 1. Compared with sub-lethal irradiation, Busulfan conditioning enhanced the engraftment of edited CD34+ cells in the bone marrow, as well the long-term homing and survival of bone-marrow-derived cells in viscera, and in the CNS, resulting in higher transgene expression and biochemical correction in these organs. Edited cell selection using a clinically compatible marker resulted in a population with low engraftment potential. We conclude that conditioning can impact the engraftment of edited hematopoietic stem cells. Furthermore, Busulfan-conditioned recipients have a higher expression of therapeutic proteins in target organs, particularly in the CNS, constituting a better conditioning approach for non-hematological diseases with neurological involvement.  

5.3401           Quantitative single-cell transcriptome-based ranking of engineered AAVs in human retinal explants

Xi, Z., Öztürk, B.E., Johnson, M.E., Turunc, S., Stauffer, W.R. and Byrne, L.C. Molecular Therapy-Methods Clin. Develop., 25, 476-489 (2022)   Gene therapy is a rapidly developing field, and adeno-associated viruses (AAVs) are a leading viral-vector candidate for therapeutic gene delivery. Newly engineered AAVs with improved abilities are now entering the clinic. It has proven challenging, however, to predict the translational potential of gene therapies developed in animal models due to cross-species differences. Human retinal explants are the only available model of fully developed human retinal tissue and are thus important for the validation of candidate AAV vectors. In this study, we evaluated 18 wild-type and engineered AAV capsids in human retinal explants using a recently developed single-cell RNA sequencing (RNA-seq) AAV engineering pipeline (scAAVengr). Human retinal explants retained the same major cell types as fresh retina, with similar expression of cell-specific markers except for a photoreceptor population with altered expression of photoreceptor-specific genes. The efficiency and tropism of AAVs in human explants were quantified with single-cell resolution. The top-performing serotypes, K91, K912, and 7m8, were further validated in non-human primate and human retinal explants. Together, this study provides detailed information about the transcriptome profiles of retinal explants and quantifies the infectivity of leading AAV serotypes in human retina, accelerating the translation of retinal gene therapies to the clinic.  

5.3402           Single AAV-mediated CRISPR-Nme2Cas9 efficiently reduces mutant hTTR expression in a transgenic mouse model of transthyretin amyloidosis

Wen, J., Cao, T., Wu, J., Chen, Y., Zhi, S., Huang, Y., Zhen, P., Wu, G., Aagaard, L., Zhong, J., Liang, P. and Huang, J. Molecular Therapy, 30(1), 164-174 (2022)   Transthyretin (TTR) amyloidosis is a hereditary life-threatening disease characterized by deposition of amyloid fibrils. The main causes of TTR amyloidosis are mutations in the TTR gene that lead to the production of misfolded TTR protein. Reducing the production of toxic protein in the liver is a validated strategy to treat TTR amyloidosis. In this study, we established a humanized mouse model that expresses mutant human TTR (hTTR; V30M) protein in the liver to model TTR amyloidosis. Then, we compared the efficiency of reducing the expression of mutant hTTR by dual adeno-associated virus 8 (AAV8)-mediated split SpCas9 with that by single AAV8-mediated Nme2Cas9 in this model. With two gRNAs targeting different exons, dual AAV-mediated split SpCas9 system achieved efficiencies of 37% and 34% reduction of hTTR mRNA and reporter GFP expression, respectively, in the liver. Surprisingly, single AAV-mediated Nme2Cas9 treatment resulted in 65% and 71% reduction of hTTR mRNA and reporter GFP, respectively. No significant editing was identified in predicted off-target sites in the mouse and human genomes after Nme2Cas9 targeting. Thus, we provide proof of principle for using single AAV-mediated CRISPR-Nme2Cas9 to effectively reduce mutant hTTR expression in vivo, which may translate into gene therapy for TTR amyloidosis.  

5.3403           Dual-AAV delivering split prime editor system for in vivo genome editing

Zhi, S., Chen, Y., Wu, G., Wen, J., Wu, J., Liu, Q., Li, Y., Kang, R., Hu, S., Wang, J., Liang, P: and Huang, J. Molecular Theerapy, 30(1), 283-294 (2022)   Prime editor (PE), a new genome editing tool, can generate all 12 possible base-to-base conversions, insertion, and deletion of short fragment DNA. PE has the potential to correct the majority of known human genetic disease-related mutations. Adeno-associated viruses (AAVs), the safe vector widely used in clinics, are not capable of delivering PE (∼6.3 kb) in a single vector because of the limited loading capacity (∼4.8 kb). To accommodate the loading capacity of AAVs, we constructed four split-PE (split-PE994, split-PE1005, split-PE1024, and split-PE1032) using Rma intein (Rhodothermus marinus). With the use of a GFP-mutated reporter system, PE reconstituting activities were screened, and two efficient split-PEs (split-PE1005 and split-PE1024) were identified. We then demonstrated that split-PEs delivered by dual-AAV1, especially split-PE1024, could mediate base transversion and insertion at four endogenous sites in human cells. To test the performance of split-PE in vivo, split-PE1024 was then delivered into the adult mouse retina by dual-AAV8. We demonstrated successful editing of Dnmt1 in adult mouse retina. Our study provides a new method to deliver PE to adult tissue, paving the way for in vivo gene-editing therapy using PE.  

5.3404           Non-invasive administration of AAV to target lung parenchymal cells and develop SARS-CoV-2-susceptible mice

Yang, M-S., Park, M-J., Lee, J., Oh, B., Kang, K.W., Kim, Y., Lee, S-M. et al Molecular Therapy, 30(5), 1994-2004 (2022)   Adeno-associated virus (AAV)-mediated gene delivery holds great promise for gene therapy. However, the non-invasive delivery of AAV for lung tissues has not been adequately established. Here, we revealed that the intratracheal administration of an appropriate amount of AAV2/8 predominantly targets lung tissue. AAV-mediated gene delivery that we used in this study induced the expression of the desired protein in lung parenchymal cells, including alveolar type II cells. We harnessed the technique to develop severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-susceptible mice. Three kinds of immune function-relevant gene knockout (KO) mice were transduced with AAV encoding human angiotensin-converting enzyme 2 (hACE2) and then injected with SARS-CoV-2. Among these mice, type I interferon receptor (IFNAR) KO mice showed increased viral titer in the lungs compared to that in the other KO mice. Moreover, nucleocapsid protein of SARS-CoV-2 and multiple lesions in the trachea and lung were observed in AAV-hACE2-transduced, SARS-CoV-2-infected IFNAR KO mice, indicating the involvement of type I interferon signaling in the protection of SARS-CoV-2. In this study, we demonstrate the ease and rapidness of the intratracheal administration of AAV for targeting lung tissue in mice, and this can be used to study diverse pulmonary diseases.  

5.3405           Ex vivo and in vivo suppression of SARS-CoV-2 with combinatorial AAV/RNAi expression vectors

Becker, J., Stanifer, M.L., Leist, S.R., Stolp, B., Maiakovska, O., West, A., Wiedtke, E. et al Moleculr Therapy, 30(5), 2005-2023 (2022)   Despite rapid development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant modalities to curb the pandemic by directly attacking the virus on a genetic level remain highly desirable and are urgently needed. Here we comprehensively illustrate the capacity of adeno-associated virus (AAV) vectors co-expressing a cocktail of three short hairpin RNAs (shRNAs; RNAi triggers) directed against the SARS-CoV-2 RdRp and N genes as versatile and effective antiviral agents. In cultured monkey cells and human gut organoids, our most potent vector, SAVIOR (SARS virus repressor), suppressed SARS-CoV-2 infection to background levels. Strikingly, in control experiments using single shRNAs, multiple SARS-CoV-2 escape mutants quickly emerged from infected cells within 24–48 h. Importantly, such adverse viral adaptation was fully prevented with the triple-shRNA AAV vector even during long-term cultivation. In addition, AAV-SAVIOR efficiently purged SARS-CoV-2 in a new model of chronically infected human intestinal cells. Finally, intranasal AAV-SAVIOR delivery using an AAV9 capsid moderately diminished viral loads and/or alleviated disease symptoms in hACE2-transgenic or wild-type mice infected with human or mouse SARS-CoV-2 strains, respectively. Our combinatorial and customizable AAV/RNAi vector complements ongoing global efforts to control the coronavirus disease 2019 (COVID-19) pandemic and holds great potential for clinical translation as an original and flexible preventive or therapeutic antiviral measure.  

5.3406           Excessive release of inorganic polyphosphate by ALS/FTD astrocytes causes non-cell-autonomous toxicity to motoneurons

Arredonda, C., Cefaliello, C., Dyrda, A., Montecino, M., Brown, R.H. and van Zundert, B. Neuron, 110, 1656-1670 (2022)   Non-cell-autonomous mechanisms contribute to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), in which astrocytes release unidentified factors that are toxic to motoneurons (MNs). We report here that mouse and patient iPSC-derived astrocytes with diverse ALS/FTD-linked mutations (SOD1, TARDBP, and C9ORF72) display elevated levels of intracellular inorganic polyphosphate (polyP), a ubiquitous, negatively charged biopolymer. PolyP levels are also increased in astrocyte-conditioned media (ACM) from ALS/FTD astrocytes. ACM-mediated MN death is prevented by degrading or neutralizing polyP in ALS/FTD astrocytes or ACM. Studies further reveal that postmortem familial and sporadic ALS spinal cord sections display enriched polyP staining signals and that ALS cerebrospinal fluid (CSF) exhibits increased polyP concentrations. Our in vitro results establish excessive astrocyte-derived polyP as a critical factor in non-cell-autonomous MN degeneration and a potential therapeutic target for ALS/FTD. The CSF data indicate that polyP might serve as a new biomarker for ALS/FTD.  

5.3407           Cholesterol determines the cytosolic entry and seeded aggregation of tau

Tuck, B.J., Miller, L.V.C., katsinelos, T., Metzakopian, E., James, L.C. and McEwan, W. Cell Reports, 39, 110776 (2022)   Assemblies of tau can transit between neurons, seeding aggregation in a prion-like manner. To accomplish this, tau must cross cell-limiting membranes, a process that is poorly understood. Here, we establish assays for the study of tau entry into the cytosol as a phenomenon distinct from uptake, in real time, and at physiological concentrations. The entry pathway of tau is cell type specific and, in neurons, highly sensitive to cholesterol. Depletion of the cholesterol transporter Niemann-Pick type C1 or extraction of membrane cholesterol renders neurons highly permissive to tau entry and potentiates seeding even at low levels of exogenous tau assemblies. Conversely, cholesterol supplementation reduces entry and almost completely blocks seeded aggregation. Our findings establish entry as a rate-limiting step to seeded aggregation and demonstrate that dysregulated cholesterol, a feature of several neurodegenerative diseases, potentiates tau aggregation by promoting entry of tau assemblies into the cell interior.  

5.3408           Lateralized deficits after unilateral AAV-vector based overexpression of alpha-synuclein in the midbrain of rats on drug-free behavioral tests

Gubinelli, F., Cazzola, G., Negrini, M., Kulacz, I., Mehrdadian, A., Tomasello, G., Venuti, C., Sarauskyte, L., Jacobs, F., Manfredsson, F.P., Davidsson, M. and Heuer, A. Behavioural Brain Res., 429, 113887 (2022)   Background Preclinical rodent models of Parkinson’s aim to recapitulate some of the hallmarks of the disease as it presents in humans, including the progressive neuronal loss of dopaminergic neurons in the midbrain as well as the development of a behavioral phenotype. AAV vector-based models of alpha-synuclein overexpression are a promising tool to achieve such animal models with high face and predictive validity. Objective We have developed a preclinical rodent model of Parkinson’s disease using an AAV-vector based overexpression of human alpha-synuclein. In the present work we characterize this model on a behavioral and histopathological level. Methods We use a AAV9 vector for transgene delivery to overexpress human alpha-synuclein under a CBA promoter. We compare the behavioral and histopathological changes to a AAV vector control group where the transgene was omitted and to that of a 6-OHDA lesion control. We assessed the behavioral performance of these three groups on a series of tests (Cylinder, Stepping, Corridor) at baseline and up to 22 weeks post-injection at which point we performed electrochemical recordings of dopamine kinetics. Results The overexpression of human alpha-synuclein led to the progressive manifestation of behavioral deficits on all three behavioral tests. This was accompanied with impaired dopamine release and reuptake kinetics as demonstrated by electrochemical detection methods. Histopathological quantifications corroborated the findings that we induced a moderate cell loss with remaining cells displaying pathological markers which are abundant in the brains of human PD patients. Conclusions In the present work we developed a characterized a rat model of PD that closely mimics human disease development and pathology. Such model will be of great use for investigation of disease mechanisms and early therapeutic interventions.  

5.3409           Modulation of Zika virus replication via glycosphingolipids

Konan, K.V., Ogbamikael, S.A:, Yager, E., Yamaji, T., Cerone, J., Monaco-Bronwn, M., Barroso, M. and Hanada, K. Virology, 572, 17-27 (2022)   The enveloped positive-sense RNA viruses including Zika virus (ZIKV) need host lipids to successfully replicate. The nature of the lipids and the replication step(s) where lipids are utilized often vary amongst viruses. In this study, we demonstrate that ZIKV particle envelope is significantly enriched in distinct sphingolipid species. To determine the role of sphingolipids in ZIKV replication, we leveraged a panel of sphingolipid-deficient cell lines. Notably, knockout of glucosylceramide and lactosylceramide synthase encoding genes (GCS^KO; B4G5^KO) resulted in a marked decrease in ZIKV titers. GCS^KO or pharmacological inhibition of GCS also led to a significant decrease in ZIKV genome replication. Further analysis indicated that GCS^KO reduced intracellular virus titers but had minimal impact on ZIKV binding. Restoration of B4G5 expression in B4G5^KO cells or supplementing PDMP-treated cells with glucosylceramide led to a significant rescue of ZIKV replication. Altogether, our findings suggest that ZIKV needs glycosphingolipids to facilitate virus replication.  

5.3410           Mouthrinses against SARS-CoV-2 – High antiviral effectivity by membrane disruption in vitro translates to mild effects in a randomized placebo-controlled clinical trial

Meister, T.L., Gottsauner, J-M., Schmidt, B., Heinen, N., Todt, D., Audebert, F., Buder, F., Lang, H., Gessner, A., Steinmann, E., Vielsmeier, V., Pfaender, S. and Cieplik, F. Virus Res., 316, 198791 (2022)   The emergence of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) represents an unprecedented threat for the human population, necessitating rapid and effective intervention measures. Given the main infection route by airborne transmission, significant attention has been bestowed upon the use of antiseptic mouthrinses as a way to possibly reduce infectious viral titers. However, clinical evaluations are still sparse. Thus, we evaluated a wide variety of antiseptic agents that can be used as mouthrinses for their antiviral effects in vitro and their respective mode of action. One of the most promising antiseptic agents (benzalkoniumchloride, BAC) was used in a randomized placebo-controlled clinical trial with subsequent analysis of viral loads by RT-qPCR and virus rescue in cell culture. Mechanistic analysis revealed that treatment with BAC and other antiseptic agents efficiently inactivated SARS-CoV-2 in vitro by primarily disrupting the viral envelope, without affecting viral RNA integrity. However, the clinical application only resulted in a mild reduction of viral loads in the oral cavity. These results indicate that gargling with mouthrinses comprising single antiseptic agents may play a minor role towards a potential reduction of transmission rates and thus, these findings are of utmost importance when considering alternative COVID-19 prevention strategies.      

5.3411           Molecular Determinants of Human T-cell Leukemia Virus Type 1 Gag Targeting to the Plasma Membrane for Assembly

Herrmann, D., Hanson, H.M., Zhou, L.W., Addabbo, R., Willkomm, N.A., Angert, I., Mueller, J.D., Mansky, L.M. and Saad, J.S.

1. Mol. Biol., 434(12), 167609 (2022)

  Assembly of human T-cell leukemia virus type 1 (HTLV-1) particles is initiated by the trafficking of virally encoded Gag polyproteins to the inner leaflet of the plasma membrane (PM). Gag–PM interactions are mediated by the matrix (MA) domain, which contains a myristoyl group (myr) and a basic patch formed by lysine and arginine residues. For many retroviruses, Gag–PM interactions are mediated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]; however, previous studies suggested that HTLV-1 Gag–PM interactions and therefore virus assembly are less dependent on PI(4,5)P₂. We have recently shown that PI(4,5)P₂ binds directly to HTLV-1 unmyristoylated MA [myr(–)MA] and that myr(–)MA binding to membranes is significantly enhanced by inclusion of phosphatidylserine (PS) and PI(4,5)P₂. Herein, we employed structural, biophysical, biochemical, mutagenesis, and cell-based assays to identify residues involved in MA–membrane interactions. Our data revealed that the lysine-rich motif (Lys47, Lys48, and Lys51) constitutes the primary PI(4,5)P₂–binding site. Furthermore, we show that arginine residues 3, 7, 14 and 17 located in the unstructured N-terminus are essential for MA binding to membranes containing PS and/or PI(4,5)P₂. Substitution of lysine and arginine residues severely attenuated virus-like particle production, but only the lysine residues could be clearly correlated with reduced PM binding. These results support a mechanism by which HTLV-1 Gag targeting to the PM is mediated by a trio engagement of the myr group, Arg-rich and Lys-rich motifs. These findings advance our understanding of a key step in retroviral particle assembly.  

5.3412           PXR activation impairs hepatic glucose metabolism partly via inhibiting the HNF4α–GLUT2 pathway

Liu, P., Jiang, L., Kong, W., Xie, Q., Li, P., Liu, X., Zhang, J., Liu, M., Wang, Z., Zhu, L., Yang, H., Zhou, Y., Zou, J., Liu, X. and Liu, L. Acta Pharmaceutica Sinica B, 12(5), 2391-2405 (2022)   Drug-induced hyperglycemia/diabetes is a global issue. Some drugs induce hyperglycemia by activating the pregnane X receptor (PXR), but the mechanism is unclear. Here, we report that PXR activation induces hyperglycemia by impairing hepatic glucose metabolism due to inhibition of the hepatocyte nuclear factor 4-alpha (HNF4α)‒glucose transporter 2 (GLUT2) pathway. The PXR agonists atorvastatin and rifampicin significantly downregulated GLUT2 and HNF4α expression, and impaired glucose uptake and utilization in HepG2 cells. Overexpression of PXR downregulated GLUT2 and HNF4α expression, while silencing PXR upregulated HNF4α and GLUT2 expression. Silencing HNF4α decreased GLUT2 expression, while overexpressing HNF4α increased GLUT2 expression and glucose uptake. Silencing PXR or overexpressing HNF4α reversed the atorvastatin-induced decrease in GLUT2 expression and glucose uptake. In human primary hepatocytes, atorvastatin downregulated GLUT2 and HNF4α mRNA expression, which could be attenuated by silencing PXR. Silencing HNF4α downregulated GLUT2 mRNA expression. These findings were reproduced with mouse primary hepatocytes. Hnf4α plasmid increased Slc2a2 promoter activity. Hnf4α silencing or pregnenolone-16α-carbonitrile (PCN) suppressed the Slc2a2 promoter activity by decreasing HNF4α recruitment to the Slc2a2 promoter. Liver-specific Hnf4α deletion and PCN impaired glucose tolerance and hepatic glucose uptake, and decreased the expression of hepatic HNF4α and GLUT2. In conclusion, PXR activation impaired hepatic glucose metabolism partly by inhibiting the HNF4α‒GLUT2 pathway. These results highlight the molecular mechanisms by which PXR activators induce hyperglycemia/diabetes.  

5.3413           Optimizing rAAV6 transduction of primary T cells for the generation of anti-CD19 AAV-CAR-T cells

Wang, D., Zhou, Q., Qiu, X., Liu, X. and Zhang, C. Biomed. Pharmacother., 150, 113027 (2022)   Recombinant Adeno-associated virus(rAAV) is currently the most widely used gene delivery vector and has been successfully used in various disease models, benefiting from its low immunogenicity, almost no toxicity, and no reported pathogenicity in humans. However, its low transduction efficiency for primary cells, especially for T lymphocytes, limits its further application in the field of cell therapy. In this study, we optimized the protocol for rAAV6 transduction of primary T cells, significantly improved the expression efficiency of the rAAV6 delivered CAR gene, and successfully generated rAAV6-based CAR-T cells (AAV-CAR-T). The gene expression intensity (mean fluorescence intensity, MFI) of rAAV6 transduced T cells treated with the tyrosine kinase inhibitor, Genistein, was increased 1–3-fold. Moreover, our results showed that rAAV6 efficiently transduced T cells stimulated with OKT3 and the gene expression could be enhanced 3-fold with an OKT3 concentration of 50 ng/mL in the medium. The gene expression intensity of T cells treated with OKT3 together with genistein could be augmented by 7-fold. Based on the above-optimized method, CAR-T cells prepared with rAAV6 showed evident anti-tumor ability both in vitro and in vivo. Our findings established an efficient method for the AAV transduction of T cells and would provide an alternative way for the preparation of CAR-T cells.  

5.3414           CMYA5 establishes cardiac dyad architecture and positioning

Lu, F., Ma, Q., Xie, W., Liou, C.L., Zhang, D., Sweat, M.E., Jardin, B.D., Naya, F.J., Guo, Y., Cheng, H. and Pu, W.T. Naturee Comm., 13:2185 (2022)   Cardiac excitation-contraction coupling requires dyads, the nanoscopic microdomains formed adjacent to Z-lines by apposition of transverse tubules and junctional sarcoplasmic reticulum. Disruption of dyad architecture and function are common features of diseased cardiomyocytes. However, little is known about the mechanisms that modulate dyad organization during cardiac development, homeostasis, and disease. Here, we use proximity proteomics in intact, living hearts to identify proteins enriched near dyads. Among these proteins is CMYA5, an under-studied striated muscle protein that co-localizes with Z-lines, junctional sarcoplasmic reticulum proteins, and transverse tubules in mature cardiomyocytes. During cardiac development, CMYA5 positioning adjacent to Z-lines precedes junctional sarcoplasmic reticulum positioning or transverse tubule formation. CMYA5 ablation disrupts dyad architecture, dyad positioning at Z-lines, and junctional sarcoplasmic reticulum Ca²⁺ release, leading to cardiac dysfunction and inability to tolerate pressure overload. These data provide mechanistic insights into cardiomyopathy pathogenesis by demonstrating that CMYA5 anchors junctional sarcoplasmic reticulum to Z-lines, establishes dyad architecture, and regulates dyad Ca²⁺ release.  

5.3415           Teneurins assemble into presynaptic nanoclusters that promote synapse formation via postsynaptic non-teneurin ligands

Zhang, X., Lin, P-Y., Liakath-Ali, K. and Südhof, T.C: Nature Comm., 13:2297 (2022)   Extensive studies concluded that homophilic interactions between pre- and postsynaptic teneurins, evolutionarily conserved cell-adhesion molecules, encode the specificity of synaptic connections. However, no direct evidence is available to demonstrate that teneurins are actually required on both pre- and postsynaptic neurons for establishing synaptic connections, nor is it known whether teneurins are localized to synapses. Using super-resolution microscopy, we demonstrate that Teneurin-3 assembles into presynaptic nanoclusters of approximately 80 nm in most excitatory synapses of the hippocampus. Presynaptic deletions of Teneurin-3 and Teneurin-4 in the medial entorhinal cortex revealed that they are required for assembly of entorhinal cortex-CA1, entorhinal cortex-subiculum, and entorhinal cortex-dentate gyrus synapses. Postsynaptic deletions of teneurins in the CA1 region, however, had no effect on synaptic connections from any presynaptic input. Our data suggest that different from the current prevailing view, teneurins promote the establishment of synaptic connections exclusively as presynaptic cell-adhesion molecules, most likely via their nanomolar-affinity binding to postsynaptic latrophilins.  

5.3416           An in vivo gene amplification system for high level expression in Saccharomyces cerevisiae

Peng, B ., Esquirol, L., Lu, Z., Shen, Q., Cheah, L.C., Howard, C.B., Scott, C., Trau, M., Dumsday, G. and Vickers, C.E. Nature Comm., 13:2895 (2022)   Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L⁻¹ in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.  

5.3417           Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure

De Genst, E., Foo, K.S., Xiao, Y., Rohner, E., de Vries, E., Sohlmer, J. et al Nature Comm., 13:3018 (2022)   The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca²⁺ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.  

5.3418           Identification of adeno-associated virus variants for gene transfer into human neural cell types by parallel capsid screening

Flitsch, L.J., Börner, K., Stüllein, C., Ziegler, S., Sonntag-Buck, V. et al Scientific Reports, 12:8356 (2022)   Human brain cells generated by in vitro cell programming provide exciting prospects for disease modeling, drug discovery and cell therapy. These applications frequently require efficient and clinically compliant tools for genetic modification of the cells. Recombinant adeno-associated viruses (AAVs) fulfill these prerequisites for a number of reasons, including the availability of a myriad of AAV capsid variants with distinct cell type specificity (also called tropism). Here, we harnessed a customizable parallel screening approach to assess a panel of natural or synthetic AAV capsid variants for their efficacy in lineage-related human neural cell types. We identified common lead candidates suited for the transduction of directly converted, early-stage induced neural stem cells (iNSCs), induced pluripotent stem cell (iPSC)-derived later-stage, radial glia-like neural progenitors, as well as differentiated astrocytic and mixed neuroglial cultures. We then selected a subset of these candidates for functional validation in iNSCs and iPSC-derived astrocytes, using shRNA-induced downregulation of the citrate transporter SLC25A1 and overexpression of the transcription factor NGN2 for proofs-of-concept. Our study provides a comparative overview of the susceptibility of different human cell programming-derived brain cell types to AAV transduction and a critical discussion of the assets and limitations of this specific AAV capsid screening approach.  

5.3419           AAV-ie-K558R mediated cochlear gene therapy and hair cell regeneration

Tao, Y., Liu, X., Yang, Y., Chu, C., Tan, F., Yu, Z., Ke, J., Li, X. et al Signal Transduction and Targeted Ther., 7:109 (2022)   The cochlea consists of multiple types of cells, including hair cells, supporting cells and spiral ganglion neurons, and is responsible for converting mechanical forces into electric signals that enable hearing. Genetic and environmental factors can result in dysfunctions of cochlear and auditory systems. In recent years, gene therapy has emerged as a promising treatment in animal deafness models. One major challenge of the gene therapy for deafness is to effectively deliver genes to specific cells of cochleae. Here, we screened and identified an AAV-ie mutant, AAV-ie-K558R, that transduces hair cells and supporting cells in the cochleae of neonatal mice with high efficiency. AAV-ie-K558R is a safe vector with no obvious deficits in the hearing system. We found that AAV-ie-K558R can partially restore the hearing loss in Prestin KO mice and, importantly, deliver Atoh1 into cochlear supporting cells to generate hair cell-like cells. Our results demonstrate the clinical potential of AAV-ie-K558R for treating the hearing loss caused by hair cell death.  

5.3420           Elimination of receptor binding by influenza hemagglutinin improves vaccine-induced immunity

Hendin, H.E., Lavoie, P-O., Gravett, J.M., Pillet, S., Saxena, P., Landry, N., D’Aoust, M-A. and Ward, B.J. Npj Vaccines, 7:42 (2022)   The binding of influenza hemagglutinin (HA) to sialic acid (SA) receptors plays a well-defined role in shaping infection but the impact of such binding on vaccine responses has not yet been explored. We generated a virus-like particle (VLP) vaccine bearing the HA of H1N1 A/California/07/09 that is unable to bind to its α(2,6)-linked SA receptor (H1_Y98F-VLP) and compared its immunogenicity and efficacy to a wild-type H1-VLP (H1_WT-VLP) in mice. The H1_Y98F-VLP elicited significantly stronger and more durable antibody responses (hemagglutination inhibition and microneutralization titers) and greater avidity maturation, likely attributable to improved germinal center formation. H1_Y98F-VLP also resulted in a robust population of IL-2⁺TNFα⁺IFNγ⁻ CD4⁺ T cells that correlated with antibody responses. Compared to H1_WT-VLP vaccination, mice immunized with H1_Y98F-VLP had 2.3-log lower lung viral loads and significantly lower pulmonary inflammatory cytokine levels 5 days post-challenge. These findings suggest that abrogation of HA-SA interactions may be a promising strategy to improve the quality and durability of influenza vaccine-induced humoral responses.  

5.3421           A plant immune protein enables broad antitumor response by rescuing microRNA deficiency

Qi, Y., Ding, l., Zhang, S., Li, Y., Wu, H. and Du, P. Cell, 185, 1888-1904 (2022)   Cancer cells are featured with uncontrollable activation of cell cycle, and microRNA deficiency drives tumorigenesis. The RNA-dependent RNA polymerase (RDR) is essential for small-RNA-mediated immune response in plants but is absent in vertebrates. Here, we show that ectopic expression of plant RDR1 can generally inhibit cancer cell proliferation. In many human primary tumors, abnormal microRNA isoforms with 1-nt-shorter 3′ ends are widely accumulated. RDR1 with nucleotidyltransferase activity can recognize and modify the problematic AGO2-free microRNA duplexes with mononucleotides to restore their 2 nt overhang structure, which eventually rescues AGO2-loading efficiency and elevates global miRNA expression to inhibit cancer cell-cycle specifically. The broad antitumor effects of RDR1, which can be delivered by an adeno-associated virus, are visualized in multiple xenograft tumor models in vivo. Altogether, we reveal the widespread accumulation of aberrant microRNA isoforms in tumors and develop a plant RDR1-mediated antitumor stratagem by editing and repairing defective microRNAs.  

5.3422           The Process of Filopodia Induction during HPV Infection

Biondo, A. and Meneses, P.I. Viruses, 14:1150 (2022)   Human Papillomavirus 16 (HPV16) infects mucosal and epithelial cells and has been identified as a high-risk HPV type that is an etiologic agent of human cancers. The initial infectious process, i.e., the binding of the virus particle and its entry into the host cell, has been studied extensively, although it is not fully understood. There is still a gap in understanding the steps by which the virus is able to cross the plasma membrane after receptor binding. In this study, we demonstrate that after HPV16 comes into contact with a plasma membrane receptor, there are cytoskeletal changes resulting in an increase of filopodia numbers. This increase in filopodia numbers was transient and was maintained during the first two hours after virus addition. Our data show that there is a statistically significant increase in infection when filopodia numbers are increased by the addition of drug and virus simultaneously, and a decrease in virus infection when filopodia formation is inhibited. We describe that HPV16 binding results in the activation of Cdc42 GTPase that in turn results in an increase in filopodia. siRNA directed at Cdc42 GTPase resulted in a statistically significant reduction of infection and a corresponding lack of filopodia induction.  

5.3423           Preclinical Efficacy of a Capsid Virus-like Particle-Based Vaccine Targeting IL-1β for Treatment of Allergic Contact Dermatitis

Gogsøyr, L., Funch, A.B., Okholm, A.K., Theander, T.G., de Jongh, W.A., Bonefeld, C.W. and Sander, A.F. Vaccines, 10:828 (2022)   Hypersensitivity to a contact allergen is one of the most abundant forms of inflammatory skin disease. Today, more than 20% of the general population are sensitized to one or more contact allergens, making this disease an important healthcare issue, as re-exposure to the allergen can initiate the clinical disease termed allergic contact dermatitis (ACD). The current standard treatment using corticosteroids is effective, but it has side effects when used for longer periods. Therefore, there is a need for new alternative therapies for severe ACD. In this study, we used the versatile Tag/Catcher AP205 capsid virus-like particle (cVLP) vaccine platform to develop an IL-1β-targeted vaccine and to assess the immunogenicity and in vivo efficacy of the vaccine in a translational mouse model of ACD. We show that vaccination with cVLPs displaying full-length murine IL-1β elicits high titers of neutralizing antibodies, leading to a significant reduction in local IL-1β levels as well as clinical symptoms induced by treatment with 1-Fluoro-2,4-dinitrobenzene (DNFB). Moreover, we show that a single amino acid mutation in muIL-1β reduces the biological activity while maintaining the ability to induce neutralizing antibodies. Collectively, the data suggest that a cVLP-based vaccine displaying full-length IL-1β represents a promising vaccine candidate for use as an alternative treatment modality against severe ACD.  

5.3424           Administration of Glutaredoxin-1 Attenuates Liver Fibrosis Caused by Aging and Non-Alcoholic Steatohepatitis

Tsukahara, Y., Ferran, B., Minetti, E.T., Chong, B.S.H., Gower, A.C., Bachschnid, M.M. and Matsui, R. Antioxidants, 11:867 (2022)   Liver fibrosis is a sign of non-alcoholic fatty liver disease progression towards steatohepatitis (NASH) and cirrhosis and is accelerated by aging. Glutaredoxin-1 (Glrx) controls redox signaling by reversing protein S-glutathionylation, induced by oxidative stress, and its deletion causes fatty liver in mice. Although Glrx regulates various pathways, including metabolism and apoptosis, the impact of Glrx on liver fibrosis has not been studied. Therefore, we evaluated the role of Glrx in liver fibrosis induced by aging or by a high-fat, high-fructose diet. We found that: (1) upregulation of Glrx expression level inhibits age-induced hepatic apoptosis and liver fibrosis. In vitro studies indicate that Glrx regulates Fas-induced apoptosis in hepatocytes; (2) diet-induced NASH leads to reduced expression of Glrx and higher levels of S-glutathionylated proteins in the liver. In the NASH model, hepatocyte-specific adeno-associated virus-mediated Glrx overexpression (AAV-Hep-Glrx) suppresses fibrosis and apoptosis and improves liver function; (3) AAV-Hep-Glrx significantly inhibits transcription of Zbtb16 and negatively regulates immune pathways in the NASH liver. In conclusion, the upregulation of Glrx is a potential therapeutic for the reversal of NASH progression by attenuating inflammatory and fibrotic processes.  

5.3425           Immunogenicity of an AAV-Based COVID-19 Vaccine in Murine Models of Obesity and Aging

Maciorowsky, D., Diop, C., Bhatt, U., Estelien, R., Li, D., Chauhan, R., Vandenberghe, L.H. and Zabaleta, N. Viruses, 14:820 (2022)   The SARS-CoV-2 pandemic has had a disastrous impact on global health. Although some vaccine candidates have been effective in combating SARS-CoV-2, logistical, economical, and sociological aspects still limit vaccine access globally. Recently, we reported on two room-temperature stable AAV-based COVID-19 vaccines that induced potent and protective immunogenicity following a single injection in murine and primate models. Obesity and old age are associated with increased mortality in COVID-19, as well as reduced immunogenicity and efficacy of vaccines. Here, we investigated the effectiveness of the AAVCOVID vaccine candidates in murine models of obesity and aging. Results demonstrate that obesity did not significantly alter the immunogenicity of either vaccine candidate. In aged mice, vaccine immunogenicity was impaired. These results suggest that AAV-based vaccines may have limitations in older populations and may be equally applicable in obese and non-obese populations.

6     Carbon Nanotubes

 

6.1           Enrichment of Single-Walled Carbon Nanotubes by Diameter in Density Gradients

Arnold, M.S., Stupp, S.I. and Hersam, M.C. Nano Lett., 5(4), 713-718 (2005)   The bulk enrichment and separation of single-walled carbon nanotubes (SWNTs) by diameter has been achieved through ultracentrifugation of DNA-wrapped SWNTs in aqueous density gradients. The separation is identified by the visual formation of colored bands of SWNTs in the density range of 1.11−1.17 g cm^-3. The optical absorbance spectra of the separated SWNTs indicate that SWNTs of decreasing diameter are increasingly more buoyant. This nondestructive and scalable separation strategy is expected to impact the fields of molecular electronics, optoelectronics, and sensing where SWNTs of a monodisperse band gap are essential.  

6.2           Sorting carbon nanotubes by electronic structure using density differentiation

Arnold, M.S., Green, A.A., Hulvat, J.F., Stupp, S.I. and Hersam, M.C. Nature Nanotech., 1, 60-65 (2006)   The heterogeneity of as-synthesized single-walled carbon nanotubes (SWNTs) precludes their widespread application in electronics, optics and sensing. We report on the sorting of carbon nanotubes by diameter, bandgap and electronic type using structure-discriminating surfactants to engineer subtle differences in their buoyant densities. Using the scalable technique of density-gradient ultracentrifugation, we have isolated narrow distributions of SWNTs in which >97% are within a 0.02-nm-diameter range. Furthermore, using competing mixtures of surfactants, we have produced bulk quantities of SWNTs of predominantly a single electronic type. These materials were used to fabricate thin-film electrical devices of networked SWNTs characterized by either metallic or semiconducting behaviour.  

6.3           Ultracentrifugation of single-walled nanotubes

Green, A.A. and Hersam, M.C. Materials Today, 10(12), 59-60 (2007)   Single-walled carbon nanotubes (SWNTs) are high aspect ratio cylinders of carbon (~1 nm in diameter) whose walls are one atomic layer thick and have an atomic arrangement analogous to graphite. The SWNT atomic structure is defined by a twodimensional chiral vector whose components are specified by a pair of positive integers: (n,m). This chirality of the SWNT dictates its properties. Unfortunately, current methods for producing SWNTs lack control over chirality, leading to significant polydispersity in the properties of as-synthesized SWNTs. Consequently, the widespread use of SWNTs in electronics, photonics, and sensors has been limited by their inhomogeneity.  

6.4           Quantum Yield Heterogeneities of Aqueous Single-Wall Carbon Nanotube Suspensions

Crochet, J., Clemens, M. and Hertel, T.

1. Am. Chem. Soc., 129, 8058-8059 (2007)

  Aqueous suspensions of single-wall carbon nanotubes (SWNTs) prepared by traditional methods from the supernatant of ultracentrifuged micellar SWNT mixtures are shown to be strongly heterogeneous with respect to the quantum yield of their constituents. The heterogeneities are isolated using preparative ultracentrifugation in iodixanol density gradients and are optically characterized by photoluminescence and linear absorption spectroscopy. We find that the most buoyant fractions with a density of 1.055 ± 0.005 g·cm^-3 have the highest photoluminescence quantum yield η of 1.1%, a factor of 5 higher than that of the supernatant. Denser fractions with lower η make up for about ²/₃ of these samples and contain mostly small ropes in which the quantum yield is reduced by nonradiative decay through coupling to metallic tubes.  

6.5           Diameter sorting of carbon nanotubes by gradient centrifugation: role of endohedral water 

Hennrich, F., Arnold, K., Lebedkin, S., Quintilla, A., Wenzel, W. and Kappes, M.M. Phys. Stat. Sol. (b), 244(11) , 3896-3900 (2007)   We demonstrate that water filling has a significant, tube-diameter dependent effect on the effective mass density of individual single-walled carbon nanotubes suspended in aqueous suspension. On the basis of extensive molecular dynamics simulations of water-filled carbon nanotubes we have estimated the number density and properties of water molecules inside different types of nanotubes. As the tube diameter becomes comparable with the size of a water molecule, the number density of water molecules jumps discontinously. For tube diameters just above this threshold the water molecules organize into a single file. Shell-like arrangements are found for even larger radii. Buoyant densities are predicted to change accordingly  

6.6           Optical properties of structurally sorted single-wall carbon nanotube ensembles

Crochet, J., Clemens, M. and Hertel, T. Phys. Stat. Sol. (b), 244(11), 3964-3968 (2007)   Using density gradient ultracentrifugation and isopycnic fractionation we structurally sort traditionally prepared single-wall carbon nanotube suspensions, and show that fractions with photoluminescence (PL) quantum yields of over 1% can be isolated. Moreover, we find that traditionally prepared suspensions appear to be composed of a significant fraction of small nanotube aggregates which reduce the quantum yield of the ensembles by a factor of 5 with respect to the most photoluminescent fractions. In order to better understand the nature of the nonradiative decay processes that lead to lower ensemble PL quantum yields, nanotube ropes of diameter enriched tubes are made and compared with the aforementioned ensemble measurements. Changes in the optical properties are discussed in terms of energy transfer to metallic tubes, carbonaceous particles, and smaller band gap tubes.  

6.7           Combination of atomic force microscopy and photoluminescence microscopy for the investigation of individual carbon nanotubes on sapphire surfaces

Jester, S-S., Kiowski, O., Lebedkin, S., Henrich, F., Fischer, R., Stürzl, N., Hawecker, J. and Kappes, M.M. Phys. Stat. Sol. (b), 244(11), 3973-3977 (2007)   Atomic force microscopy (AFM) and photoluminescence (PL) spectroscopy were applied to characterize single walled carbon nanotubes (SWNTs) deposited on sapphire. The electronic properties and structure of luminescent semiconducting SWNTs can be probed by PL spectroscopy. The diameter, length, spatial position, and angular orientation of deposited SWNTs can be accurately determined by intermittent contact AFM. We describe an approach to combine and compare the spectroscopic (PL) and topographical (AFM) information for the same individual nanotubes as well as nanotube aggregates.  

6.8           Dynamics of Surfactant-Suspended Single-Walled Carbon Nanotubes in a Centrifugal Field

Nair, N., Kim, W-J., Braatz, R.D. and Strano, M.S. Langmuir, 24, 1790-1795 (2008)   A hydrodynamic model is used to describe the motion of surfactant-suspended single-walled carbon nanotubes in a density gradient, while being subjected to a centrifugal field. The number of surfactant molecules adsorbed on each nanotube determines its effective density and, hence, its position in the gradient after centrifugation has been completed. Analysis of the spatial concentration distributions of CoMoCAT nanotubes suspended with 2 w/v% sodium cholate yielded 2.09, 2.14, and 2.08 surfactant molecules adsorbed per nanometer along the length of the (6,5), (7,5), and (8,7) nanotubes, respectively. The estimates are commensurate with experimental values reported in the literature and can be used to predict the fate of sodium cholate-suspended nanotubes in the separation process. Since the density of the surfactant-nanotube assembly is highly sensitive to the number of adsorbed molecules, a perturbation would cause it to be enriched at a different location in the gradient. The level of sensitivity is also reflected in the 95% confidence levels that are reported in this work.  

6.9           Highly Stabilized Conductivity of Metallic Single Wall Carbon Nanotube Thin Films

Miyata, Y., Yanaga, K., Maniwa, Y. And Kataura, H.

1. Phys. Chem. C, 112, 3591-3596 (2008)

  The extremely stable conductivity against molecular physisorptions was revealed in thin films of high-purity metallic single wall carbon nanotubes (SWCNTs). The stability of the metallic SWCNTs results from their constant electronic density of state near the Fermi level. In the case of a mixture of metallic and semiconducting SWCNTs, the existence of semiconducting SWCNTs in the films makes the sheet resistance more sensitive to the adsorption of molecules mainly due to carrier doping. In the practical use for the transparent electrode, stability of the resistance is important and pure metallic SWCNTs have great advantages for such kind of applications.  

6.10           Covalent Functionalization of Single-Walled Carbon Nanotubes Alters Their Densities Allowing Electronic and Other Types of Separation

Kim, W-J., Nair, N., Lee, C.Y. and Strano, M.S.

1. Phys. Chem. C, 112, 7326-7331 (2008)

  We show that covalently attached functional groups can alter the densities of individual single-walled carbon nanotubes (SWNTs) in a predictable and highly controllable manner. A volume-additivity model based on molecular group contributions can be used to estimate the density difference between 4-hydroxyphenyl-functionalized and nonfunctionalized HiPco SWNTs as approximately 98.3 kg/m³, compared with 97.9 kg/m³ measured by density-gradient centrifugation. Conversely, the estimated density difference between the (6,5) (0.75 nm diameter) and (9,8) (1.17 nm diameter) SWNTs is smaller at 23.4 kg/m³. We conclude that covalent functionalization can provide an effective handle to separate particular SWNTs from a typical diameter distribution. We show that SWNT mixtures in which metallic SWNTs have been selectively reacted produce two distinct density fractions corresponding to functionalized metallic and pure semiconducting SWNTs. The results were confirmed by Raman spectroscopy, where the high-density fractions exhibit an increased disorder mode with a corresponding decrease in intensity for the low-density fraction. This method also allows for the first independent measure of (n,m) SWNTs having different chemical conversions with functional groups, which will allow for a more rigorous analysis of SWNT chemistry than is possible with uncalibrated spectroscopies such as Raman or photoluminescence.  

6.11           Optical Evaluation of the Metal-to-Semiconductor Ratio of Single-Wall Carbon Nanotubes

Miyata, Y., Yanagi, K., Maniwa, Y. And Kataura, H.

1. Phys. Chem. C., 112, 13187-13191 (2008)

  The metal-to-semiconductor ratio for single-wall carbon nanotubes (SWCNTs) produced by laser ablation was investigated using optical absorption spectroscopy. The SWCNTs were separated into metals and semiconductors using the density gradient centrifugation method. The optical absorption spectra of both SWCNTs were measured from the near-infrared to ultraviolet. A simple superposition analysis revealed that the pristine SWCNTs consist of 27 3% metallic SWCNTs and 73 3% semiconducting SWCNTs without using a theoretical correction. From the detailed oscillator fitting, it was found the absorption coefficient of the first excitonic transition of metallic SWCNTs M₁₁ was almost the same as that of semiconducting SWCNTs S₁₁ for a diameter distribution of 1.1~1.3 nm. This result provides a simple optical evaluation method to estimate the metal-to-semiconductor ratio of various SWCNT samples produced by different methods.  

6.12           Optical Properties of Ultrashort Semiconducting Single-Walled Carbon Nanotube Capsules Down to Sub-10 nm

Sun, X. et al J.Am. Chem. Soc., 130, 6551-6555 (2008)   Single-walled carbon nanotubes (SWNTs) are typically long ( 100 nm) and have been well established as novel quasi one-dimensional systems with interesting electrical, mechanical, and optical properties. Here, quasi zero-dimensional SWNTs with finite lengths down to the molecular scale (7.5 nm in average) were obtained by length separation using a density gradient ultracentrifugation method. Different sedimentation rates of nanotubes with different lengths in a density gradient were taken advantage of to sort SWNTs according to length. Optical experiments on the SWNT fractions revealed that the UV−vis−NIR absorption and photoluminescence peaks of the ultrashort SWNTs blue-shift up to ~30 meV compared to long nanotubes, owing to quantum confinement effects along the length of ultrashort SWNTs. These nanotube capsules essentially correspond to SWNT quantum dots.  

6.13           Transparent Conductive Single-Walled Carbon Nanotube Networks with Precisely Tunable Ratios of Semiconducting and Metallic Nanotubes

Blackburn,. J.L. et al ASCNano, 2(6), 1266-1274 (2008)   We present a comprehensive study of the optical and electrical properties of transparent conductive films made from precisely tuned ratios of metallic and semiconducting single-wall carbon nanotubes. The conductivity and transparency of the SWNT films are controlled by an interplay between localized and delocalized carriers, as determined by the SWNT electronic structure, tube−tube junctions, and intentional and unintentional redox dopants. The results suggest that the main resistance in the SWNT thin films is the resistance associated with tube−tube junctions. Redox dopants are found to increase the delocalized carrier density and transmission probability through intertube junctions more effectively for semiconductor-enriched films than for metal-enriched films. As a result, redox-doped semiconductor-enriched films are more conductive than either intrinsic or redox-doped metal-enriched films.  

6.14           Selective Enrichment of (6,5) and (8,3) Single-Walled Carbon Nanotubes via Cosurfactant Extraction from Narrow (n,m) Distribution Samples

Wei, L. et al

1. Phys. Chem. B, 112, 2771-2774 (2008)

  Highly selective enrichment of (6,5) and (8,3) SWCNTs (above 85% of the semiconducting tubes) was achieved through multistep extraction by sodium dodecyl sulfate (SDS) and sodium cholate (SC) cosurfactant solution from narrowly (n,m) distributed SWCNTs produced by the catalyst Co-MCM-41. A systematic change in the chirality selectivity was observed when the weight ratio between SDS and SC varied in cosurfactant solutions, with maximum enrichment selectivity for (6,5) tubes yielded at 1:4. Furthermore, surfactants were washed away easily to produce "clean" SWCNTs. This observation sheds light on the possibility of obtaining SWCNTs with the desired (n,m) structure via an easily scalable approach. No selectivity was detected when using sodium dodecyl benzene sulfonate (SDBS)/SC cosurfactants, hence suggesting the need for further exploration of various cosurfactant combinations for more effective extraction of different (n,m) species.  

6.15           Nano-graphene oxide for cellular imaging and drug delivery

Sun, X.S. et al Nano Res., 1, 203-212 (2008)   Two-dimensional graphene offers interesting electronic, thermal, and mechanical properties that are currently being explored for advanced electronics, membranes, and composites. Here we synthesize and explore the biological applications of nano-graphene oxide (NGO), i.e., single-layer graphene oxide sheets down to a few nanometers in lateral width. We develop functionalization chemistry in order to impart solubility and compatibility of NGO in biological environments. We obtain size separated pegylated NGO sheets that are soluble in buffers and serum without agglomeration. The NGO sheets are found to be photoluminescent in the visible and infrared regions. The intrinsic photoluminescence (PL) of NGO is used for live cell imaging in the near-infrared (NIR) with little background. We found that simple physisorption via π-stacking can be used for loading doxorubicin, a widely used cancer drug onto NGO functionalized with antibody for selective killing of cancer cells in vitro. Owing to its small size, intrinsic optical properties, large specific surface area, low cost, and useful non-covalent interactions with aromatic drug molecules, NGO is a promising new material for biological and medical applications.  

6.16           In Situ Raman Spectroelectrochemistry of Single-Walled Carbon Nanotubes: Investigation of Materials Enriched with (6,5) Tubes

Kavan, L. et al

1. Phys. Chem., 112, 14179-14187 (2008)

  Single-walled carbon nanotubes (CoMoCat) have been enriched with (6,5) tubes via density-gradient ultracentrifugation. Thin solid films of quasi-isolated nanotubes were fabricated from a solution of sorted nanotubes by vacuum filtration and extraction with water. Optical spectroscopy in the vis−NIR region and Raman spectroscopy were used to characterize these materials. The experimental studies were supported by a theoretical analysis of the electronic and vibrational structure of selected (n,m) tubes by using density functional theory. Besides the most abundant tubes (6,5), the experimental and theoretical data for tubes (6,4), (7,3), (7,5), (8,3), and (9,1) are also discussed. A detailed investigation by in situ Raman spectroelectrochemistry was focused on the effects of electrochemical p-/n-doping. The experimental analysis of the intensities and frequencies of the radial breathing mode and the tangential displacement modes were correlated with the theoretically calculated optical transition energies and Raman frequencies. It was demonstrated that electrochemical charging is a useful tool for the study of doping effects on the electronic structure of carbon nanotubes.  

6.17           Influence of Nanotube Length on the Optical and Conductivity Properties of Thin Single-Wall Carbon Nanotube Networks

Simien, D. et al ACS Nano, 2(9), 1879-1884 (2008)   We study the optical and electrical properties of transparent conducting films made from length-sorted single-wall carbon nanotubes (SWCNT). Thin films of length-sorted SWCNTs, formed through filtration from a dispersing solvent onto a filter substrate (“buckypaper”), exhibit sharp changes in their optical properties and conductivity (σ) with increasing SWCNT surface concentration. At a given surface concentration, tubes longer than 200 nm are found to form networks that are more transparent and conducting. We show that changes of σ with SWCNT concentration can be quantitatively described by the generalized effective medium (GEM) theory. The scaling universal exponents describing the “percolation” transition from an insulating to a conducting state with increasing concentration are consistent with the two-dimensional (2D) percolation model. Shorter tubes and mixed length tubes form 3D networks. Furthermore, we demonstrate that the conductivity percolation threshold (x_c) varies with the aspect ratio L as, x_c ~ 1/L, a result that is also in accordance with the percolation theory. These findings provide a framework for engineering the optical and electrical properties of SWCNT networks for technological applications where flexibility, transparency, and conductivity are required.  

6.18           Colored Semitransparent Conductive Coatings Consisting of Monodisperse Metallic Single-Walled Carbon Nanotubes

Green, A.A. and Hersam, M.C. Nano Lett., 8(5), 1417-1422 (2008)   Single-walled carbon nanotubes (SWNTs) are promising materials for transparent conduction as a result of their exceptional electrical, optical, mechanical, and chemical properties. However, since current synthetic methods yield polydisperse mixtures of SWNTs, the performance of SWNT transparent conductive films has previously been hindered by semiconducting species. Here, we describe the performance of transparent conductors produced using predominantly metallic SWNTs. Compared with unsorted material, films enriched in metallic SWNTs can enhance conductivity by factors of over 5.6 in the visible and 10 in the infrared. Moreover, by using monodisperse metallic SWNTs sorted with angstrom-level resolution in diameter, semitransparent conductive coatings with tunable optical transmittance can be produced.  

6.19           Chiral-Angle Distribution for Separated Single-Walled Carbon Nanotubes

Sato, Y. et al Nano Lett., 8(10), 3151-3154 (2008)   Chiral indices (n,m) of metallic and semiconducting single-walled carbon nanotubes (SWNTs) selectively separated via the density-gradient ultracentrifugation process were individually assigned by using an aberration-corrected transmission electron microscope (TEM) operated at 80 kV. Our statistical analysis revealed that armchair (n,n) and chiral (n,n-3) SWNTs with large chiral angles (>20°) are dominant metallic nanotubes in the separated samples, whereas such a noticeable preference of particular indices was not observed for semiconducting nanotubes. Some significant discrepancies were found between the TEM and spectroscopic results on the major chiral indices and the metal/semiconductor ratios in these SWNTs.  

6.20           Centrifugal Length Separation of Carbon Nanotubes

Fagan, J.A., Becker, M.L., Chun, J., Nie, P., Bauer, B.J., Simpson, J.R., Hight-Walker, A. and Hobbie, E.K. Langmuir, 24(24), 13880-13889 (2008)   Separation of single-wall carbon nanotubes (SWCNTs) by length via centrifugation in a high density medium, and the characterization of both the separated fractions and the centrifugation process are presented. Significant quantities of the separated SWCNTs ranging in average length from <50 nm to ∼2 μm were produced, with the distribution width being coupled to the rate of the separation. Less rapid separation is shown to produce narrower distributions; these length fractions, produced using sodium deoxycholate dispersed SWCNTs, were characterized by UV−visible−near-infrared absorption and fluorescence spectroscopy, dynamic light scattering, Raman scattering, and atomic force microscopy. Several parameters of the separation were additionally explored: SWCNT concentration, added salt concentration, liquid density, rotor speed, surfactant concentration, and the processing temperature. The centrifugation technique is shown to support 10 mg per day scale processing and is applicable to all of the major SWCNT production methods. The cost per unit of the centrifugation-based separation is also demonstrated to be significantly less than size exclusion chromatography-based separations.  

6.21           Assessment of (n,m) Selectively Enriched Small Diameter Single-Walled Carbon Nanotubes by Density Differentiation from Cobalt-Incorporated MCM-41 for Macroelectronics

Wei, L., Lee, C.W., Li, L-J., Sudibya, G., Wang, B., Chen, L.Q., Chen, P., Yang, Y., Chan-Park, M.B. and Chen, Y. Chem. Mater., 20, 7417-7424 (2008)   Uniformly semiconducting or metallic single-walled carbon nanotube (SWNT) networks are ideal materials for flexible and large-area electronics (macroelectronics). With the goal of developing optimal enrichment and evaluation solutions toward economical production of monodisperse SWNTs for macroelectronics, we selectively enriched SWNTs, which have small diameters (<0.9 nm) and a narrow (n,m) distribution, synthesized on cobalt-incorporated MCM-41 catalysts. The (7,5) enriched SWNTs were obtained from sodium cholate (SC) dispersion, whereas (6,5) were from cosurfactant mixtures of sodium dodecyl sulfate (SDS):SC at 1: 4. Density gradient ultracentrifugation was applied to further refine the separation. Subsequently, SWNT thin-film field effect transistors (FETs) were fabricated using enriched SWNTs. We characterized the chiralities by photoluminescence excitation spectroscopy, optical absorption spectroscopy, Raman spectroscopy, and electrical transport measurements. Among these techniques, results demonstrate that the electrical transport measurement (through I_on/I_off ratio) of thin-film FETs is the most sensitive technique to evaluate the purity of semiconducting SWNTs. Enriched SWNTs via only SC produced more devices with higher on-/off-current ratios (up to 1 × 10⁶) compared to SWNTs obtained from SDS/SC cosurfactants. These results are different from previous studies using laser-ablation-grown SWNTs (1.1−1.4 nm), encouraging more comprehensive models to explain diameter dependent chirality selection using surfactants.  

6.22           Thin Film Nanotube Transistors Based on Self-Assembled, Aligned, Semiconducting Carbon Nanotube Arrays

Engel, M., Small, J.P., Steiner, M., Freitag, M., Green, A.A., Hersam, M.C. and Avouris, P. ACS Nano, 2(12), 2445-2452 (2008) Thin film transistors (TFTs) are now poised to revolutionize the display, sensor, and flexible electronics markets. However, there is a limited choice of channel materials compatible with low-temperature processing. This has inhibited the fabrication of high electrical performance TFTs. Single-walled carbon nanotubes (CNTs) have very high mobilities and can be solution-processed, making thin film CNT-based TFTs a natural direction for exploration. The two main challenges facing CNT-TFTs are the difficulty of placing and aligning CNTs over large areas and low on/off current ratios due to admixture of metallic nanotubes. Here, we report the self-assembly and self-alignment of CNTs from solution into micron-wide strips that form regular arrays of dense and highly aligned CNT films covering the entire chip, which is ideally suitable for device fabrication. The films are formed from pre-separated, 99% purely semiconducting CNTs and, as a result, the CNT-TFTs exhibit simultaneously high drive currents and large on/off current ratios. Moreover, they deliver strong photocurrents and are also both photo- and electroluminescent.  

6.23           Exciton Energy Transfer in Pairs of Single-Walled Carbon Nanotubes

Qian, H., Georgi, C., Anderson, N., Green, A.A., Hersam, M.C., Novotny, L. and Hartschuh, A. Nano Lett., 8(5), 1363-1367 (2008)   We studied the exciton energy transfer in pairs of semiconducting nanotubes using high-resolution optical microscopy and spectroscopy on the nanoscale. Photoluminescence from large band gap nanotubes within bundles is observed with spatially varying intensities due to distance-dependent internanotube transfer. The range of efficient energy transfer is found to be limited to a few nanometers because of competing fast nonradiative relaxation responsible for low photoluminescence quantum yield.  

6.24           Visualizing the Local Optical Response of Semiconducting Carbon Nanotubes to DNA-Wrapping

Qian, H., Araujo, P.T., Georgi, C., Gokus, T., Hartmann, N., Green, A.A., Jorio, A., Hersam, M.C., Novotny, L. and Hartschuh, A. Nano Lett., 8(9), 2706-2711 (2008)   We studied the local optical response of semiconducting single-walled carbon nanotubes to wrapping by DNA segments using high resolution tip-enhanced near-field microscopy. Photoluminescence (PL) near-field images of single nanotubes reveal large DNA-wrapping-induced red shifts of the exciton energy that are two times higher than indicated by spatially averaging confocal microscopy. Near-field PL spectra taken along nanotubes feature two distinct PL bands resulting from DNA-wrapped and unwrapped nanotube segments. The transition between the two energy levels occurs on a length scale smaller than our spatial resolution of about 15 nm.  

6.25           Reversibility, Dopant Desorption, and Tunneling in the Temperature-Dependent Conductivity of Type-Separated, Conductive Carbon Nanotube Networks

Barnes, T.M., Blackburn, J.L., va de Lagemaat, J., Coutts, T.J. and Heben, M.J. ACS Nano, 2(9), 1968-1976 (2008)   We present a comprehensive study of the effects of doping and temperature on the conductivity of single-walled carbon nanotube (SWNT) networks. We investigated nearly type-pure networks as well as networks comprising precisely tuned mixtures of metallic and semiconducting tubes. Networks were studied in their as-produced state and after treatments with nitric acid, thionyl chloride, and hydrazine to explore the effects of both intentional and adventitious doping. For intentionally and adventitiously doped networks, the sheet resistance (R_s) exhibits an irreversible increase with temperature above 350 K. Dopant desorption is shown to be the main cause of this increase and the observed hysteresis in the temperature-dependent resistivity. Both thermal and chemical dedoping produced networks free of hysteresis. Temperature-programmed desorption data showed that dopants are most strongly bound to the metallic tubes and that networks consisting of metallic tubes exhibit the best thermal stability. At temperatures below the dopant desorption threshold, conductivity in the networks is primarily controlled by thermally assisted tunneling through barriers at the intertube or interbundle junctions.  

6.26           Hydrodynamic Characterization of Surfactant Encapsulated Carbon Nanotubes Using an Analytical Ultracentrifuge

Arnold, M.S., Suntivich, J., Stupp, S.I. and Hersam, M.C: ACS Nano, 2(11), 2291-2300 (2008)   The hydrodynamic properties of surfactant encapsulated single-walled carbon nanotubes (SWNTs) have been characterized by optically measuring their spatial and temporal redistribution in situ in an analytical ultracentrifuge. The measured redistribution profiles are fit to the Lamm equation, thus determining the sedimentation, diffusion, and hydrodynamic frictional coefficients of the surfactant encapsulated SWNTs. For sodium cholate encapsulated SWNTs, we demonstrate that the technique of analytical ultracentrifugation can be utilized to determine the linear packing density of surfactant molecules along the length of the SWNTs, 3.6 ± 0.8 nm⁻¹, and the anhydrous molar volume of the surfactant molecules on the SWNT surfaces, 270 ± 20 cm³ mol⁻¹. Additionally, analytical ultracentrifugation is used to measure and compare the sedimentation rates of bundled and isolated carbon nanotubes. This study should serve as a guide for designing centrifuge-based processing procedures for preparing samples of SWNTs for a wide variety of applications and studies. Additionally, the results obtained here should aid in understanding the hydrodynamic properties of SWNTs and the interactions between SWNTs and surfactants in aqueous solution.  

6.27           Ultrafast Exciton Dephasing in Semiconducting Single-Walled Carbon Nanotubes

Ma, Y-Z., Graham, M.W. and Fleming, G.R. Phys. Rev.Lett., 101, 217402-1-217402-4 (2008)   Femtosecond two-pulse degenerate four-wave mixing spectroscopy was applied to study the exciton dephasing in a broad range of excitation intensities and lattice temperatures. We find that both exciton-exciton and exciton-phonon scattering have profound effects on the dephasing process. The dominant phonon mode involved in the dephasing is identified as the out-of-plane, transverse optical mode with a frequency of 847 cm^-1. The extracted homogeneous linewidths at all measured temperatures are in excellent agreement with the results of a single-tube photoluminescence experiment.  

6.28           Length Fractionation of Carbon Nanotubes Using Centrifugation 

Fagan, J.A., Becker, M.L., Chun, J. and Hobbie, E.K. Advanced Materials, 20, 1609-1613 (2008)   Scalable separation of single-walled carbon nanotubes  (SWCNTs) by length and chirality is critical to the adaptation of these materials for applications. Ultracentrifugation of SWCNTs within a density gradient produces chiral separation of the NTs, and it is shown here that ultracentrifugation can also be used to produce length fractionated SWCNTs by exploiting their transient motion in response to applied centripetal acceleration.

6.29           Optical properties of metallic and semiconducting single-wall carbon nanotubes

Miyata, Y., Yanagi, K., Maniwa, Y., and Kataura, H. Phys. Stat. Sol. (b), 245(10), 2233-2238 (2008)   Optical properties of high purity metallic and semiconducting single-wall carbon nanotubes (SWCNTs) were investigated from far infrared to ultraviolet by optical absorption spectroscopy. It was found that the metallic SWCNT thin film has larger infrared absorption intensity than the semiconducting one. Furthermore, little changes in the optical absorption spectrum and the DC sheet resistance against molecular physisorptions were revealed in the metallic SWCNTs thin film. The stability of the metallic SWCNTs results from their constant electronic density of state near the Fermi level. On the other hand, the existence of semiconducting SWCNTs in the films makes their infrared absorption intensity and the sheet resistance more sensitive to the adsorption of molecules mainly due to carrier doping.  

6.30           In-situ Vis/NIR spectroelectrochemistry of single-walled carbon nanotubes enriched with (6,5) tubes

Frank, O., Kavan, L., Green, A.A., Hersam, M.C. and Dunsch, L. Phys. Stat. Sol. (b), 245(10), 2239-2242 (2008)   An in-situ optical Vis/NIR spectroelectrochemistry study was performed on single-walled carbon nanotubes (CoMoCat) enriched with (6,5) tubes via density-gradient ultracentrifugation. Thin semitransparent solid films were prepared from the solution of sorted nanotubes by vacuum filtration and purification with water. Despite the appearance of some bundling of the nanotubes during this procedure, peaks corresponding to optical transitions of tubes with distinct chiralities may be observed in the spectra. The effects of electrochemical redox doping on the electronic state of the nanotubes were investigated in an electrode potential window between –2 and +1.7 V vs. Ag-pseudoreference electrode. The bleaching of ΔE11S and ΔE22S optical transitions is traced for particular tubes, which allows a partial quantification of changes in the electronic density of states (DOS) caused by their filling with electrons or holes.  

6.31           Photoinduced Luminescence Blinking and Bleaching in Individual Single-Walled Carbon Nanotubes

Georgi, C., Hartmann, N., Gokus, T., Green, A.A., Hersam, M.C. and Hartschuh, A. Chemphyschem., 9, 1460-1464 (2008)   The temporal evolution of photoluminescence in individual single-walled carbon nanotubes (SWNT) under strong laser irradiation is studied and pronounced blinking and bleaching is observed, caused by photoinduced oxidation that subsequently quenches mobile excitons. The nanotubes are isolated with sodium cholate and spun onto either a glass or mica surface. Their bleaching behavior is investigated for variable laser intensities in air and argon atmosphere. The decay rate for luminescence bleaching generally increases with higher laser intensity, however saturating on mica substrates, which is attributed to limited availability of oxygen in the vicinity of the nanotubes. Step-like events in the luminescence time traces corresponding  

6.32           Processing and properties of highly enriched double-wall carbon nanotubes

Green, A.A. and Hersam, M.C. Nature Nanotech., 4, 64-70 (2009)   Carbon nanotubes consist of one or more concentric graphene cylinders and are under investigation for a variety of applications that make use of their excellent thermal, mechanical, electronic and optical properties. Double-wall nanotubes are ideal systems for studying the interwall interactions influencing the properties of nanotubes with two or more walls. However, current techniques to synthesize double-wall nanotubes produce unwanted single- and multiwall nanotubes. Here, we show how density gradient ultracentrifugation can be used to separate double-wall nanotubes from mixtures of single- and multiwall nanotubes through differences in their buoyant density. This technique results in samples that are highly enriched in either single- or double-wall nanotubes of similar outer wall diameter, with the double-wall nanotubes being, on average, 44% longer than the single-wall nanotubes. The longer average length of the double-wall nanotubes provides distinct advantages when they are used in transparent conductors.  

6.33           Self-Assembly of Ordered Nanowires in Biological Suspensions of Single-Wall Carbon Nanotubes

Hobbie, E.K., Fagan, J.A., Becker, M.L., Hudson, S.D., Fakhri, N. and Pasquali, M. Nano, 3(1), 189-196 (2009)   We investigate the self-assembly of ordered nanowires from length-purified single-wall carbon nanotubes (SWCNTs) in aqueous suspensions of the biological surfactant sodium deoxycholate. Macroscopically straight and nearly periodic linear arrangements of aligned individual SWCNTs are found to self-assemble in two-dimensional geometries from nanotube suspensions that are otherwise stable in the bulk, which we attribute to a dominance of surface effects under strong confinement. Directed self-assembly is explored through surface patterning, opening up new potential routes to nanotube manipulation for optical diagnostics and applications that require ordered arrangements of mutually aligned SWCNTs. The stability of these structures to thermal fluctuations and changes in solution chemistry are surveyed with near-infrared fluorescence microscopy.  

6.34           Electrolyte Tuning of Surfactant Interfacial Behavior for Enhanced Density-Based Separations of Single-Walled Carbon Nanotubes

Niyogi, S., Densmore, C.G. and Doorn, S.K.

1. Am. Chem. Soc., 131, 1144-1153 (2009)

  We study the interfacial behavior between the straight-chain alkyl surfactant sodium dodecyl sulfate (SDS) and single-walled carbon nanotubes (SWNTs) as a function of added electrolytes, including NaCl. We observe an increase in photoluminescence intensity and narrowing of spectral line widths with electrolyte addition, indicating a change in SDS aggregation number that leads to a pronounced volume change in the nanotube/SDS composite structure. By tuning the interfacial dynamics through NaCl addition and temperature change, we demonstrate that this volume change can be used to yield diameter-dependent separation of metallic and semiconducting SWNTs, without the use of any additional cosurfactant, through density gradient ultracentrifugation. The diameter-dependent fractionation follows the intrinsic relation expected for the density of unfunctionalized nanotubes, indicating a simple amplification of these inherent density differences as the mechanism for salt enhanced separations. Isolation of enriched metallic and semiconducting fractions further illustrates that the surface aggregation characteristics of SDS on metallic SWNTs are different from that on the semiconducting chiralities. These experiments illustrate the governing behavior of surface phenomena and interfacial forces on the diameter-dependent fractionation of SWNTs and point to new routes for enhancing existing separations strategies.  

6.35           Size-Dependent Cellular Uptake and Expulsion of Single-Walled Carbon Nanotubes: Single Particle Tracking and a Generic Uptake Model for Nanoparticles

Jin, H., Heller, D.A., Sharma, R. and Strano, M.S. Nano, 3(1), 149-158 (2009)   The cellular uptake and expulsion rates of length-fractionated single-walled carbon nanotubes (SWNT) from 130 to 660 nm in NIH-3T3 cells were measured via single particle tracking of their intrinsic photoluminescence. We develop a quantitative model to correlate endocytosis rate with nanoparticle geometry that accurately describes this data set and also literature results for Au nanoparticles. The model asserts that nanoparticles cluster on the cell membrane to form a size sufficient to generate a large enough enthalpic contribution via receptor ligand interactions to overcome the elastic energy and entropic barriers associated with vesicle formation. Interestingly, the endocytosis rate constant of SWNT (10⁻³ min⁻¹) is found to be nearly 1000 times that of Au nanoparticles (10⁻⁶ min⁻¹) but the recycling (exocytosis) rate constants are similar in magnitude (10⁻⁴ to 10⁻³ min⁻¹) for poly(d,l-lactide-co-glycolide), SWNT, and Au nanoparticles across distinct cell lines. The total uptake of both SWNT and Au nanoparticles is maximal at a common radius of 25 nm when scaled using an effective capture dimension for membrane diffusion. The ability to understand and predict the cellular uptake of nanoparticles quantitatively should find utility in designing nanosystems with controlled toxicity, efficacy, and functionality.  

6.36           Colors of carbon nanotubes

Yanagi, K., Miyata, Y., Tanaka, T., Fujii, S., Nishide, D. and Kataura, H. Diamond & Related Materials, 18, 935-939 (2009)   Single-wall carbon nanotubes (SWCNTs) are graphitic materials whose colors are usually considered to be black. Actually, graphite-like black color is observed for as-synthesized SWCNT samples which are in the mixture state of the metallic and the semiconducting SWCNTs. However, after the separation of the metallic and the semiconducting types from the mixture, SWCNTs can exhibit various colors depending on their electronic types and diameters. These colorful colors are caused by the boundary conditions for the circumferential directions of the cylindrical structures. Here we discuss the underlying physical backgrounds of the colors of SWCNTs and the techniques to obtain such colorful SWCNTs.

6.37           Microscale Polymer−Nanotube Composites

Hobbie, E.K., Fagan, J.A., Obrzut, J. and Hudson, S.D. Appl. Materials & Interfaces, 1(7), 1561-1566 (2009)   Polymer colloids with an interfacial coating of purified single-wall carbon nanotubes (SWCNTs) are synthesized from length- and type-sorted SWCNTs. Aqueous nanotube suspensions sorted through density-gradient ultracentrifugation are used to emulsify spherical polymer colloids of microscale dimensions that are characterized through a combination of optical microscopy, transmission electron microscopy, and impedance spectroscopy. The SWCNT−polymer composite particles exhibit electrical conductivities comparable to or better than those of bulk SWCNT−polymer composites at nanotube loadings of more than 1 order of magnitude lower. The composite particles retain the unique electronic and optical characteristics of the parent SWCNT solution with potential applications as microelectronic and microoptical components.    

6.38           Quantifying the Semiconducting Fraction in Single-Walled Carbon Nanotube Samples through Comparative Atomic Force and Photoluminescence Microscopies

Naumov, A.V., Kuznetsov, O.A., Harutyunyan, A.R., Green, A.A., Hersam, M.C., Resasco, D.E., Nikolaev, P.N. and Weisman, R.B. Nano Lett., 9(9), 3203-3208 (2009)   A new method was used to measure the fraction of semiconducting nanotubes in various as-grown or processed single-walled carbon nanotube (SWCNT) samples. SWCNT number densities were compared in images from near-IR photoluminescence (semiconducting species) and AFM (all species) to compute the semiconducting fraction. The results show large variations among growth methods and effective sorting by density gradient ultracentrifugation. This counting-based method provides important information about SWCNT sample compositions that can guide controlled growth methods and help calibrate bulk characterization techniques.  

6.39           Do Inner Shells of Double-Walled Carbon Nanotubes Fluoresce?

Tsyboulski, D.A., Hou, Y., Fakhri, N., Ghosh, S., Zhang, R., Bachilo, S.M., PAsquali, M., Chen, L., Liu, J. and Weisman, R.B. Nano Lett., 9(9), 3282-3289 (2009)   The reported fluorescence from inner shells of double-walled carbon nanotubes (DWCNTs) is an intriguing and potentially useful property. A combination of bulk and single-molecule methods was used to study the spectroscopy, chemical quenching, mechanical rigidity, abundance, density, and TEM images of the near-IR emitters in DWCNT samples. DWCNT inner shell fluorescence is found to be weaker than SWCNT fluorescence by a factor of at least 10 000. Observable near-IR emission from DWCNT samples is attributed to SWCNT impurities.  

6.40           Isolation of single-walled carbon nanotube enantiomers by density differentiation

Green, A.A., Duch, M.C. and hersam, M.C. Nano Res., 2, 69-77 (2009)   Current methods of synthesizing single-walled carbon nanotubes (SWNTs) result in racemic mixtures that have impeded the study of left- and right-handed SWNTs. Here we present a method of isolating different SWNT enantiomers using density gradient ultracentrifugation. Enantiomer separation is enabled by the chiral surfactant sodium cholate, which discriminates between left- and right-handed SWNTs and thus induces subtle differences in their buoyant densities. This sorting strategy can be employed for simultaneous enrichment by handedness and roll-up vector of SWNTs having diameters ranging from 0.7 to 1.5 nm. In addition, circular dichroism of enantiomer refined samples enables identification of high-energy optical transitions in SWNTs.  

6.41           Carbon nanotubes in biology and medicine: In vitro and in vivo detection, imaging and drug delivery

Liu, Z., Tabakman, S., Welsher, K. and Dai, H. Nano Res., 2, 85-120 (2009)   Carbon nanotubes exhibit many unique intrinsic physical and chemical properties and have been intensively explored for biological and biomedical applications in the past few years. In this comprehensive review, we summarize the main results from our and other groups in this field and clarify that surface functionalization is critical to the behavior of carbon nanotubes in biological systems. Ultrasensitive detection of biological species with carbon nanotubes can be realized after surface passivation to inhibit the non-specific binding of biomolecules on the hydrophobic nanotube surface. Electrical nanosensors based on nanotubes provide a label-free approach to biological detection. Surface-enhanced Raman spectroscopy of carbon nanotubes opens up a method of protein microarray with detection sensitivity down to 1 fmol/L. In vitro and in vivo toxicity studies reveal that highly water soluble and serum stable nanotubes are biocompatible, nontoxic, and potentially useful for biomedical applications. In vivo biodistributions vary with the functionalization and possibly also size of nanotubes, with a tendency to accumulate in the reticuloendothelial system (RES), including the liver and spleen, after intravenous administration. If well functionalized, nanotubes may be excreted mainly through the biliary pathway in feces. Carbon nanotube-based drug delivery has shown promise in various In vitro and in vivo experiments including delivery of small interfering RNA (siRNA), paclitaxel and doxorubicin. Moreover, single-walled carbon nanotubes with various interesting intrinsic optical properties have been used as novel photoluminescence, Raman, and photoacoustic contrast agents for imaging of cells and animals. Further multidisciplinary explorations in this field may bring new opportunities in the realm of biomedicine.  

6.42           Selective suspension in aqueous sodium dodecyl sulfate according to electronic structure type allows simple separation of metallic from semiconducting single-walled carbon nanotubes

Moshammer, K., Hennrich, F. and Kappes, M.M. Nano Res., 2, 599-606 (2009)   Both density gradient centrifugation and gel electrophoresis have been reported to allow high throughput separation of metallic from semiconducting single-walled carbon nanotubes (SWNTs) when using aqueous sodium dodecyl sulphate (SDS) suspensions. We show here that both methods rely on an initial dispersion-by-sonication step, which is already selective with respect to electronic structure type. The corresponding aqueous SDS “starting” suspensions obtained after sonication and purification by simple centrifugation (70,000 g, 1 h) contain semiconducting SWNTs primarily in the form of small bundles whereas metallic SWNTs are predominantly suspended as individual tubes. Density gradient centrifugation then separates the bundles from the individual tubes on the basis of differences in their overall buoyant densities. Gel electrophoresis separates the longer bundles from the shorter individual tubes on the basis of their different mobilities. We also demonstrate that such starting suspensions can be fractionated according to electronic structure type by even simpler techniques such as size exclusion chromatography or gel filtration, thus opening the way for simple scale-up.  

6.43           Anchoring of Rare-Earth-Based Single-Molecule Magnets on Single-Walled Carbon Nanotubes

Kyatskaya, S., Mascaros, J.R.G., Bogani, L., Hennrich, F., Kappes, M., Wernsdorfer, W. and Ruben, M.

1. Am. Chem. Soc., 131, 15143-15151 (2009)

  A new heteroleptic bis(phthalocyaninato) terbium(III) complex 1, bearing a pyrenyl group, exhibits temperature and frequency dependence of ac magnetic susceptibility, typical of single-molecule magnets. The complex was successfully attached to single-walled carbon nanotubes (SWNTs) using π−π interactions, yielding a 1−SWNT conjugate. The supramolecular grafting of 1 to SWNTs was proven qualitatively and quantitatively by high-resolution transmission electron microscopy, emission spectroscopy, and atomic force spectroscopy. Giving a clear magnetic fingerprint, the anisotropy energy barrier and the magnetic relaxation time of the 1−SWNT conjugate are both increased in comparison with the pure crystalline compound 1, likely due to the suppression of intermolecular interactions. The obtained results propose the 1−SWNT conjugate as a promising constituent unit in magnetic single-molecule measurements using molecular spintronics devices.  

6.44           Modulation of Single-Walled Carbon Nanotube Photoluminescence by Hydrogel Swelling

Barone, P.W., Yoon, H., Ortiz-Garcia, R., Zhang, J., Ahn, J-H., Kim, J-H. and Strano, M.S. ACS Nano, 3(12), 3869-3877 (2009)   We demonstrate the use of hydrogel swelling as a mechanism to reversibly induce solvatochromic shifting in single-walled carbon nanotube (SWNT) near-infrared emission within a biocompatible hydrogel. The optical sensor reports the degree of the swelled state and glucose concentration when apo-glucose oxidase is used to cross-link the hydrogel. Photoluminescence emission maxima from dispersed nanotubes in a poly(vinyl alcohol) hydrogel shift as cross-linking is increased, with a maximum of −48 meV for the (6,5) nanotube. The Raman tangential mode also red shifts up to 17 cm⁻¹, indicative of nanotube lattice strain equivalent to an effective hydrostatic pressure of 3 GPa. While the electronic band gaps of SWNTs are known to either increase or decrease with uniaxial strain or lattice deformation depending on chiral vector, we show that the mechanism of detection is counterintuitively non-strain-dependent. Instead, the data are well-described by a model that accounts for changes in dielectric screening of the 1-D exciton, as the osmotic pressure forces conformational distortions in the PVA by rotating more polar groups to the nanotube surface. The model describes observed changes with hydration state and cross-linking density variation from 0 to 14%. Cross-linking with apo-glucose oxidase renders the hydrogel glucose responsive, and we demonstrate rapid and reversible detection of glucose from these systems after repeated cycling of 10 mM glucose. We also demonstrate detection and imaging in the near-infrared of implanted hydrogel sensors in a mouse tissue model, showing excellent signal-to-noise of 8.6 and contrast with integration times of 60 s.  

6.45           Near Monochiral Single-Walled Carbon Nanotube Dispersions in Organic Solvents

Stürzl, N., Hennrich, F., Lebedkin, S. and Kappes, M.M.

1. Phys. Chem. C., 113, 14628-14632 (2009)

  We describe simple procedures to obtain near monochiral samples of several single-walled carbon nanotube (SWNT) species starting from SWNT raw material. (7,5), (7,6), (10,5), and (9,7) nanotubes were obtained with respective enrichments of up to 90% (as estimated from photoluminescence and absorption spectra) by their selective dispersion in toluene with various fluorene-based polymers and subsequent centrifugation. Further highly enriched samples, for instance of (6,5) nanotubes, were prepared by implementing density gradient centrifugation of dispersions of SWNTs in organic solvents with 2,4,6-tribromotoluene as the density gradient additive.  

6.46           Solution Phase Production of Graphene with Controlled Thickness via Density Differentiation

Green, A.A. and Hersam, M.C. Nano Lett., 9(12), 4031-4036 (2009)   Graphene flakes with controlled thicknesses are isolated in solution using density gradient ultracentrifugation. These stable graphene dispersions are produced using the bile salt sodium cholate, which promotes graphite exfoliation and results in graphene−surfactant complexes having buoyant densities that vary with graphene thickness. The sorted graphene flakes are characterized using atomic force microscopy and Raman spectroscopy. Graphene dispersions produced using density differentiation offer superior performance in transparent conductors than those produced using conventional sedimentation-based centrifugation techniques.  

6.47           Transmission Electron Microscopy and UV-vis-IR Spectroscopy Analysis of the Diameter Sorting of Carbon Nanotubes by Gradient Density Ultracentrifugation

Fleurier, R., Lauret, J-S., Lopez, U. and Loiseau, A. Adv. Funct. Mater., 19(4), 2219-2223 (2009)   Diameter separation of single-walled carbon nanotubes is achieved via the density gradient ultracentrifugation process. Statistical analysis of the separated samples is performed using high-resolution transmission electron microscopy (HRTEM). The evolution of the diameter distribution with respect to the gradient density is extracted by analyzing hundreds of HRTEM images, and the results are found to be consistent with those estimated by UV-vis-IR spectroscopy. The efficiency of the separation process can be quantitatively characterized by the standard deviation of the diameter distribution, which is determined from the TEM analyses. This particular study indicated that for electric arc nanotubes dispersed in sodium cholate, diameter sorting is more efficient in the upper part of the gradient.  

6.48           80 GHz field-effect transistors produced using high purity semiconducting single-walled carbon nanotubes

Nougaret, L., Happy, H., Dambrine, g., Derycke, V., Bourgoin, J.P., Green, A.A. and Hersam, M.C. Appl. Phys. Lett., 94, 243505-1-243505-3 (2009)   This paper presents the high frequency performance of single-walled carbon nanotube (SWNT) field-effect transistors, with channel consisting of dense networks of high purity semiconducting SWNTs. Using SWNT samples containing 99% pure semiconducting SWNTs, we achieved operating frequencies above ,80 GHz. This record frequency does not require aligned SWNTs, thus demonstrating the remarkable potential of random networks of sorted SWNTs for high frequency electronics.  

6.49           Fractioning HiPco and CoMoCAT SWCNTs via density gradient ultracentrifugation by the aid of a novel perylene bisimide derivative surfactant

Backes, C., Hauke, F., Schmidt, C.D. and Hirsch, A. Chem. Commun., 19, 2643-2645 (2009)  

6.50           Separation of Nanoparticles in a Density Gradient: FeCo@C and Gold Nanocrystals

Sun, X., Tabakman, S.M., Seo, W-S., Zhang, L., Shang, G., Sherlock, S., Bai, L and Dai, H. Angewandte Chemie Int. Ed., 48, 939-942 (2009)   HiPco and CoMoCAT single-walled carbon nanotubes (SWCNT) were fractionated with the aid of a novel perylene bisimide surfactant by combined co-surfactant and replacement density gradient ultracentrifugation (DGU).  

6.51           Efficient Separation of (6,5) Single-Walled Carbon Nanotubes Using a “Nanometal Sinker”

Kato, Y., Niidome, Y. and Nakashima, N. Angewandte Chemie Int. Ed., 48, 5435-5438 (2009)   Sort it out! Density-gradient ultracentrifugation is used to separate single-walled carbon nanotubes (SWNTs) of a single chirality. A “nanometal sinker” (AuCl₄⁻ ions) adsorbs onto specific SWNTs and allows the separation of adsorbed SWNTs and nonfunctionlized SWNTs (see picture). (6,5) SWNTs have been successfully separated from as-prepared SWNTs in a process termed “SWNT chirality fishing”.  

6.52           Metallic single-wall carbon nanotubes separated by density gradient ultracentrifugation

Chernov, A.I. and Obraztsova, E.O. Phys. Status Solidi B, 246(11-12), 2477-2481 (2009)   A density gradient ultracentrifugation (DGU) technique recently applied for separation of single-wall carbon nanotubes (SWNTs) over diameter also appeared to be efficient for the nanotube separation according to their electronic structure. In this work we used DGU for extraction of a highly metallic (>97 wt%) nanotube fraction from a raw soot of arc-discharge SWNTs. The process parameters (the surfactant type and concentration, the treatment time, the approach to the gradient formation) have been optimized. A variation of surfactant concentration allowed obtaining the highly metallic nanotube fractions with different dominant diameter distribution resulting in the “blue” or “green” top fractions. Sodium taurodeoxycholate (TDOC) was efficiently used for DGU separation of raw, non-purified SWNTs. The fractions have been studied with the UV–Vis–NIR absorption and Raman spectroscopy.  

6.53           Sorting and transmission electron microscopy analysis of single or double wall carbon nanotubes

Fleurier, R., Lauret, J-S., Flahaut, E. and Loiseau, A. Phys. Status Solidi B, 246(11-12), 2675-2678 (2009)   On the basis of the recent progress on the sorting of carbon nanotubes' structure with respect to their diameter or number of walls, we investigate by transmission electron microscopy the sorting efficiency, with a comparison with optical absorption spectroscopy measurements. We study density gradient ultracentrifugation sorted single walled or double walled carbon nanotubes, showing obviously the ability to separate carbon nanotubes of different diameters or/and number of walls. This microscopic approach affords accurate information about the sorted samples such as the real mean diameter, the relative concentration of double walled carbon nanotubes over single walled carbon nanotubes, standard deviation, and the real diameter distribution of carbon nanotubes, even beyond any possible accurate analysis from optical absorption spectroscopy. Therefore, we demonstrate that the diameter analysis of the sorted samples by TEM can indeed afford some information about the relevant optical properties of carbon nanotubes.  

6.54           Tuning of Sorted Double-Walled Carbon Nanotubes by Electrochemical Charging

Kalbac, M., Green, A.A., hersam, M.C. and Kavan, L. ACS Nano, 4(1), 459-469 (2010)   Double-walled carbon nanotubes sorted by density gradient ultracentrifugation were examined by Raman spectroscopy and by in situ Raman spectroelectrochemistry. The sorted samples had a narrow distribution of diameters of both inner and outer tubes, which enabled a comparison of the behavior of inner metallic tubes and inner semiconducting nanotubes as a function of the applied electrochemical potential. The metallic inner tubes were efficiently doped even though they were protected from electrolyte ions by the outer wall, whereas the doping of semiconducting inner tubes was observed only at high magnitudes of the electrode potential. These results indicate that the doping response of inner tubes is predominantly controlled by inner tube electronic properties. On the other hand, the effect of electronic structure of the outer tube on the behavior of inner tube is weak. Furthermore, the efficiency of the charge transfer from outer to inner wall depends on the doping level. A low doping level corresponds to a high efficiency of the charge-transfer, while a high doping level shows low charge-transfer efficiency.  

6.55           Sequence-Specifically Addressable Hairpin DNA−Single-Walled Carbon Nanotube Complexes for Nanoconstruction

Müller, K., Malik, S. and Richert, C. ACS Nano, 4(2), 649-656 (2010)   Single-walled carbon nanotubes (SWCNTs) are attractive building blocks for molecular electronics and novel materials. Generating functional architectures with SWCNTs requires methodologies for dispersing, purifying, and binding these highly insoluble quasi one-dimensional molecules. We have previously shown that unstructured DNA strands bind to carbon nanotubes so tightly that it is difficult to address them with complementary strands. Here we show that hairpin oligonucleotides give SWCNT suspensions more concentrated than those obtainable with previously optimized DNA sequences. Further, hairpin-forming oligonucleotides and (6,5)-SWCNTs form complexes that are addressable with complementary, triplex-forming oligonucleotides. As proof of principle, we show that DNA−SWCNT complexes can be bound sequence-specifically with oligonucleotides featuring fluorophores or quantum dots. The new method brings SWCNTs of exquisite purity into the realm of DNA-based nanostructuring.  

6.56           Controllable Expansion of Single-Walled Carbon Nanotube Dispersions Using Density Gradient Ultracentrifugation

Zhao, P., Einarsson, E., Xiang, R., Murakami, Y., and Maruyama, S.

1. Phys. Chem. C, 114, 4831-4834 (2010)

  We present a protocol to selectively isolate single-walled carbon nanotubes (SWNTs) with different chiralities in a full-colored “rainbow” expansion using density gradient ultracentrifugation (DGU). Starting with SWNTs synthesized by the alcohol catalytic chemical vapor deposition (ACCVD) method, we used sodium deoxycholate (DOC) and sodium dodecyl sulfate (SDS) as cosurfactant encapsulating agents to form a DOC-restricted SDS wrapping morphology around the SWNTs. This enhances the density differences between nanotubes of different diameters, which leads to efficient chirality redistribution when combined with an appropriate density gradient profile. UV−vis-NIR absorbance spectra and photoluminescence excitation (PLE) maps show the optical properties of each fraction, and 97% pure isolation of (6,5) SWNTs achieved from the rainbow is also reported.  

6.57           Enrichment of Armchair Carbon Nanotubes via Density Gradient Ultracentrifugation: Raman Spectroscopy Evidence

Haroz, E.H., Rice, W., Lu, B.Y., Ghosh, S., hauge, R.H., Weisman, R.B., Doorn, S.K. and Kono, J. ACS Nano, 4(4), 1955-1962 (2010)   We have used resonant Raman scattering spectroscopy to fully analyze the relative abundances of different (n,m) species in single-walled carbon nanotube samples that are metallically enriched by density gradient ultracentrifugation. Strikingly, the data clearly show that our density gradient ultracentrifugation process enriches the metallic fractions in armchair and near-armchair species. We observe that armchair carbon nanotubes constitute more than 50% of each (2n + m) family.  

6.58           Influence of Aromatic Environments on the Physical Properties of β-Carotene

Yanagi, K., Miyata, Y., Liu, Z., Suenaga, K., Okada, S. and Kataura, H. J: Phys. Chem. C, 114(6), 2524-2530 (2010)   The influence of the surrounding aromatic environment on the properties of β-carotene (Car) was investigated for a simple system comprising single-walled carbon nanotubes (SWCNTs) encapsulating Car molecules. Both metallic- and semiconducting-type SWCNTs encapsulating Car were prepared, and their physical properties were investigated using optical measurements and first-principles calculations. The optical absorption peaks of encapsulated Car in metallic and semiconducting SWCNTs were slightly different, which is thought to be caused by the difference in polarizability of the two types of SWCNTs. The Raman frequency of the C═C stretching mode of Car in the metallic SWCNTs was 3 cm⁻¹ down-shifted from that in the semiconducting SWCNTs. This down-shift could not be explained by the difference of dielectric environments of the metallic and the semiconducting SWCNTs. One possible origin for the shift is a difference in the amount of charge on the encapsulated Car, which was supported by theoretical calculations. From the results of this study, it can be concluded that the electronic structure of the nanotube walls influences the properties of encapsulated molecules.  

6.59           Engineering Therapeutic Nanocarriers with Optimal Adhesion for Targeting

Haun, J.B., Robbins, G.P. and Hammer, D.A.

1. Adhesion, 86, 131-159 (2010)

  There is considerable interest in developing therapeutic delivery carriers that can be targeted via receptor-ligand interactions to sites within the blood stream. The adhesion of carriers is determined by the combined effects of transport phenomena, hydrodynamic force, and the dynamics of multivalent receptor/ligand bonding. Optimizing the adhesion of carriers requires developing relationships between these factors and carrier properties such as size and receptor coating density. Recently, we developed canonical relationships for the binding of antibody-conjugated 200 nm particles to surfaces coated with a vascular adhesion molecule, intercellular adhesion molecule-1. Here we extend our previous studies of adhesion to particles of different size, including 40 nm and 1  m particles. Particle binding is assessed under fluid flow in a parallel plate flow chamber while varying particle receptor density, substrate ligand density, and flow rate. Using a stochastic simulation and transport-reaction model we then extract multivalent kinetic rate constants for particle attachment and detachment from the binding data. We demonstrate that particles go though a maximum in binding with particle size. For small particles, increasing size increases receptor-ligand encounter rates; for larger particles, fluid shear force begins to dominate, leading to higher forces and decreased adhesion. Our methods provide a means for optimizing particle size and receptor density for the selective binding of particles to vascular endothelium under flow.  

6.60           Advanced sorting of single-walled carbon nanotubes by nonlinear density-gradient ultracentrifugation

Ghosh, S., Bachilo, S.M. and Weisman, R.B. Nature Nanotech., 5, 443-450 (2010)   Existing methods for growing single-walled carbon nanotubes produce samples with a range of structures and electronic properties, but many potential applications require pure nanotube samples. Density-gradient ultracentrifugation has recently emerged as a technique for sorting as-grown mixtures of single-walled nanotubes into their distinct (n,m) structural forms, but to date this approach has been limited to samples containing only a small number of nanotube structures, and has often required repeated density-gradient ultracentrifugation processing. Here, we report that the use of tailored nonlinear density gradients can significantly improve density-gradient ultracentrifugation separations. We show that highly polydisperse samples of single-walled nanotubes grown by the HiPco method are readily sorted in a single step to give fractions enriched in any of ten different (n,m) species. Furthermore, minor variants of the method allow separation of the mirror-image isomers (enantiomers) of seven (n,m) species. Optimization of this approach was aided by the development of instrumentation that spectroscopically maps nanotube contents inside undisturbed centrifuge tubes.    

6.61           Separation and Characterization of Double-Wall Carbon Nanotube Subpopulations

Huh, J.Y., Walker, A.R.H., Ro, H.W., Obrzut, J., Mansfield, E., Geiss, R. andf Fagan, J.A

1. Phys. Chem. C., 114, 11343-11351 (2010)

  Surfactant-encapsulated double-wall carbon nanotubes (DWCNTs) synthesized by the high-pressure carbon monoxide decomposition (HiPco) process were separated by length and electronic characteristics using density gradient ultracentrifugation (DGU). To ensure our study focuses only on the behavior of DWCNTs, dispersed DWCNTs were first isolated following the method of Green et al. [Green, A. A.; Hersam, M. C. Nat. Nanotechnol. 2008, 264, 1], utilizing the differences in buoyant density of DWCNTs from that of impurity single-wall carbon nanotubes (SWCNTs) and multiwall carbon nanotubes contained in the parent soot. By increasing the density difference between the nanotubes and the density gradient medium, we exploited the length-dependent translation of the nanotubes in response to applied centrifugation to isolate narrow length distribution DWCNT fractions. The length-dependent intrinsic optical response of DWCNTs is consistent compared with the previously reported values for SWCNTs. The controlled addition of cosurfactants is shown to allow resolution of DWCNTs by electronic structure, as demonstrated through optical absorbance, Raman spectra, and electrical conductivity measurements. Measurements of conducting films prepared from separated fractions exhibit significant property differences in the enriched materials.  

6.62           Sorting Single-Walled Carbon Nanotubes by Electronic Type Using Nonionic, Biocompatible Block Copolymers

Antaris, A.L., Seo, J-W., T., Green, A.A. and Hersam, M.C. ACSNano, 4(8), 4725-4732 (2010)   As-synthesized single-walled carbon nanotubes (SWNTs) typically possess a range of diameters and electronic properties. This polydispersity has hindered the development of many SWNT-based technologies and encouraged the development of postsynthetic methods for sorting SWNTs by their physical and electronic structure. Herein, we demonstrate that nonionic, biocompatible block copolymers can be used to isolate semiconducting and metallic SWNTs using density gradient ultracentrifugation. Separations conducted with different Pluronic block copolymers reveal that Pluronics with shorter hydrophobic chain lengths lead to higher purity semiconducting SWNTs, resulting in semiconducting purity levels in excess of 99% obtained for Pluronic F68. In contrast, X-shaped Tetronic block copolymers display an affinity for metallic SWNTs, yielding metallic purity levels of 74% for Tetronic 1107. These results suggest that high fidelity and high yield density gradient separations can be achieved using nonionic block copolymers with rationally designed homopolymer segments, thus generating biocompatible monodisperse SWNTs for a range of applications.  

6.63           Printed, Sub-3V Digital Circuits on Plastic from Aqueous Carbon Nanotube Inks

Ha, M., Xia, Y., Green, A.A., Zhang, W., Renn, M.J., Kim, C.H., Hersam, M.C. and Frisbie, C.D. ACSNano, 4(8), 4388-4395 (2010)   Printing electronic components on plastic foils with functional liquid inks is an attractive approach for achieving flexible and low-cost circuitry for applications such as bendable displays and large-area sensors. The challenges for printed electronics, however, include characteristically slow switching frequencies and associated high supply voltages, which together impede widespread application. Combining printable high-capacitance dielectrics with printable high-mobility semiconductors could potentially solve these problems. Here we demonstrate fast, flexible digital circuits based on semiconducting carbon nanotube (CNT) networks and high-capacitance ion gel gate dielectrics, which were patterned by jet printing of liquid inks. Ion gel-gated CNT thin-film transistors (TFTs) with 50 μm channel lengths display ambipolar transport with electron and hole mobilities >20 cm²/V·s; these devices form the basis of printed inverters, NAND gates, and ring oscillators on both polyimide and SiO₂ substrates. Five-stage ring oscillators achieve frequencies >2 kHz at supply voltages of 2.5 V, corresponding to stage delay times of 50 μs. This performance represents a substantial improvement for printed circuitry fabricated from functional liquid inks.  

6.64           Solution-Processable Carbon Nanotubes for Semiconducting Thin-Film Transistor Devices

Lee, C.W., Han, X., Chen, F., Wei, J., Chen, Y., Chan-Park, M.B. and Li, L-J. Adv. Mater., 22, 1278-1282 (2010)   CoMoCat single-walled carbon nanotubes (SWNTs) treated with diazonium salts can be used to fabricate solution-processable field-effect transistors (FETs) with a full semiconductor device yield. By increasing the network thickness, the effective mobility of the devices can be raised to ∼10 cm2 V−1 s−1 while keeping the on–off ratio higher than 5000. The removal of impurities is essential to achieve high-on–off-ratio devices. This approach is promising for preparation of SWNT inks for printing high-performance devices in flexible electronics.  

6.65           Tailored Distribution of Single-Wall Carbon Nanotubes from Arc Plasma Synthesis Using Magnetic Fields

Volotskova, O., Fagan, J.A., Huh, J.Y., Phelan, F.R., Shashurin, A. and Keidar, M. ACS Nano, 4(9), 5187-51192 (2010)   We report a method for tuning the distribution of single-wall carbon nanotubes (SWCNTs) produced by the anodic arc production method via the application of nonuniform magnetic fields to the gap region during synthesis. Raman, ultraviolet−visible−near-infrared absorbance and near-infrared fluorescence spectroscopies were used to characterize samples together with scanning electron microscopy. Application of the nonuniform magnetic field 0.2−2 kG results in a broadening of the diameter range of SWCNTs produced toward decreased diameters, with substantial fractions of produced SWCNTs being of small diameter, less than 1.3 nm, at the highest field. The ability to tune production of the arc production method may allow for improvement in achievable SWCNT properties.  

6.66           Photoelectrochemical complexes for solar energy conversion that chemically and autonomously regenerate

Ham, M-H., Choi, J.H., Boghossian, A.A., Jeng, E.S., Graff, R.A., Heller, D.A., Chang, A.C., Mattis, A., Bayburt, T.H., Grinkova, Y.V., Zeiger, A.S., Van Nliet, K.J., Hobbie, E.K., Sligar, S.G., Wraight, C.A. and Strano, M.S. Nature Chem., 2, 929-936 (2010)   Naturally occurring photosynthetic systems use elaborate pathways of self-repair to limit the impact of photo-damage. Here, we demonstrate a complex consisting of two recombinant proteins, phospholipids and a carbon nanotube that mimics this process. The components self-assemble into a configuration in which an array of lipid bilayers aggregate on the surface of the carbon nanotube, creating a platform for the attachment of light-converting proteins. The system can disassemble upon the addition of a surfactant and reassemble upon its removal over an indefinite number of cycles. The assembly is thermodynamically metastable and can only transition reversibly if the rate of surfactant removal exceeds a threshold value. Only in the assembled state do the complexes exhibit photoelectrochemical activity. We demonstrate a regeneration cycle that uses surfactant to switch between assembled and disassembled states, resulting in an increased photoconversion efficiency of more than 300% over 168 hours and an indefinite extension of the system lifetime.  

6.67           Density Gradient Ultracentrifugation of Nanotubes: Interplay of Bundling and Surfactants Encapsulation

Bonaccorso, F., Hasan, T., Tan, P.H., Sciascia, C., Privitera, G., Di Marco, G., Gucciardi, P.G. and Ferrari, A.C.

1. Phys. Chem C, 114(41), 17267-17285 (2010)

  Density gradient ultracentrifugation (DGU) has emerged as a promising tool to prepare chirality enriched nanotube samples. Here, we assess the performance of different surfactants for DGU. Bile salts (e.g., sodium cholate (SC), sodium deoxycholate (SDC), and sodium taurodeoxycholate (TDC)) are more effective in individualizing Single Wall Carbon Nanotubes (SWNTs) compared to linear chain surfactants (e.g., sodium dodecylbenzene sulfonate (SDBS) and sodium dodecylsulfate (SDS)) and better suited for DGU. Using SC, a narrower diameter distribution (0.69−0.81 nm) is achieved through a single DGU step on CoMoCAT tubes, when compared to SDC and TDC (0.69−0.89 nm). No selectivity is obtained using SDBS, due to its ineffectiveness in debundling. We assign the reduced selectivity of dihydroxy bile salts (SDC and TDC) in comparison with trihydroxy SC to the formation of secondary micelles. This is determined by the number and position of hydroxyl (−OH) groups on the α-side of the steroid backbone. We also enrich CoMoCAT SWNTs in the 0.84−0.92 nm range using the Pluronic F98 triblock copolymer. Mixtures of bile salts (SC) and linear chain surfactants (SDS) are used to enrich metallic and semiconducting laser-ablation grown SWNTs. We demonstrate enrichment of a single chirality, (6,5), combining diameter and metallic versus semiconducting separation on CoMoCAT samples.  

6.68           Optical Properties of Single-Walled Carbon Nanotubes Separated in a Density Gradient: Length, Bundling, and Aromatic Stacking Effects

Tabakman, S.M., Welscher, K., Hong, G. and Dai, H.

1. Phys. Chem. C, 114(46), 19569-19575 (2010)

  Single-walled carbon nanotubes (SWNTs) are promising materials for in vitro and in vivo biological applications due to their high surface area and inherent near-infrared photoluminescence and Raman scattering properties. Here, we use density gradient centrifugation to separate SWNTs by length and degree of bundling. Following separation, we observe a peak in photoluminescence quantum yield (PL QY) and Raman scattering intensity where the SWNT length is maximized and bundling is minimized. Individualized SWNTs are found to exhibit a high PL QY and high resonance-enhanced Raman scattering intensity. Fractions containing long, individual SWNTs exhibit the highest PL QY and Raman scattering intensities compared with fractions containing single, short SWNTs or SWNT bundles. Intensity gains of approximately 1.7- and 4-fold, respectively, are obtained compared with the starting material. Spectroscopic analysis reveals that SWNT fractions at higher displacement contain increasing proportions of SWNT bundles, which causes reduced optical transition energies and broadening of absorption features in the UV−vis-NIR spectra and reduced PL QYs and Raman scattering intensities. Finally, we adsorb small aromatic species on “bright,” individualized SWNT sidewalls and compare the resulting absorption, PL, and Raman scattering effects to that of SWNT bundles. We observe similar effects in both cases, suggesting that aromatic stacking affects the optical properties of SWNTs in an analogous way to SWNT bundles, likely due to electronic structure perturbations, charge transfer, and dielectric screening effects, resulting in reduction of the excitonic optical transition energies and exciton lifetimes.  

6.69           Solution-Phase Extraction of Ultrathin Inner Shells from Double-Wall Carbon Nanotubes

Miyata, Y., Suzuki, M., Fujihara, M., Asada, Y., Kitaura, R. and Shinohara, H. ACS Nano, 4(10), 5807-5812 (2010)   We present an efficient method to extract inner shells of double-wall carbon nanotubes (DWCNTs) in liquid phase. The extraction of inner from outer shells is achieved by cutting the DWCNTs with vigorous sonication in water containing surfactants. The extracted shells are perfectly isolated single-wall carbon nanotubes (SWCNTs) and can be separated using density gradient ultracentrifugation. Statistical analysis using high-resolution transmission electron microscopy reveals that the enrichment of SWCNTs with narrow diameter (0.62−1.0 nm) up to 100% is achieved from highly pure DWCNTs. Furthermore, the (5,4) SWCNTs, which have the diameter of 0.62 nm, are concentrated. Our findings provide a novel way to obtain very narrow, highly isolated SWCNTs with ultraclean surface that have not been obtained in conventional synthesis methods.  

6.70           Prolonging Charge Separation in P3HT−SWNT Composites Using Highly Enriched Semiconducting Nanotubes

Holt, J.M., Ferguson, A.J., Kopidakis, N., Larsen, B.A., Bult, J., Rumbles, G. and Blackburn, J.L. Nano Lett., 10(11), 4627-4633 (2010)   Single-walled carbon nanotubes (SWNTs) have potential as electron acceptors in organic photovoltaics (OPVs), but the currently low-power conversion efficiencies of devices remain largely unexplained. We demonstrate effective redispersion of isolated, highly enriched semiconducting and metallic SWNTs into poly(3-hexylthiophene) (P3HT). We use these enriched blends to provide the first experimental evidence of the negative impact of metallic nanotubes. Time-resolved microwave conductivity reveals that the long-lived carrier population can be significantly increased by incorporating highly enriched semiconducting SWNTs into semiconducting polymer composites.  

6.71           Saturation of Surfactant Structure at the Single-Walled Carbon Nanotube Surface

Duque, J.G., Densmore, C.G. and Doorn, S.K.

1. Am. Chem. Soc., 132(45), 16165-16175 (2010)

  Density gradient ultracentrifugation (DGU) and fluorescence spectroscopy are used to probe the limiting behaviors of the dynamic response of surfactant structure at the single-walled carbon nanotube (SWNT) surface to reorganizing forces, including changes in surfactant concentration and electrolyte screening. DGU results indicate that, as surfactant (sodium dodecyl sulfate, SDS) concentration is increased, SDS adsorbed on metallic SWNTs becomes limited in its ability to reorganize before SDS adsorbed on semiconducting species. A diameter-dependent enhancement is observed in photoluminescence intensities from semiconducting SWNTS upon initial titration with NaCl. This response to electrostatic screening diminishes as SDS concentration is increased. The results are understood as a saturation of the surfactant structural response, defined as both a loss in ability to increase SDS loading at the SWNT surface and a loss in ability to reorient surface structure in response to a reorganizing force. Saturation of response is found to be reversible and also occurs as a result of restricting SDS mobility. These results confirm several aspects of recent molecular dynamics simulations of SDS behavior on SWNTs and have important implications for tunability of density-based separation approaches using cosurfactant systems that include SDS.    

6.72           Evaluation of sorted semi-conducting carbon nanotube films for gas sensing applications

Battie, Y., Ducloux, O., Thobois, P., Coffinier, Y. and Loiseau, A. Comptes Rendus Physique, 11(5-6), 397-404 (2010)   This work focuses on the elaboration of sorted semi-conducting single walled carbon nanotube films (SC-SWCNT films), and the evaluation of a gas microsensor based on these films. First, we show that semi-conducting carbon nanotubes could be sorted from solutions containing both metallic and semi-conducting nanotubes by ultracentrifugation in a density gradient. SC-SWCNT films were then obtained by filtration through a nitrocellulose membrane, then transferred on TLM (transmission line method) electrodes to separately measure the resistivity and the contact resistance. We finally show characterization results obtained using NO₂ and NH₃ as a target gas.  

6.73           Photoluminescence of single-wall carbon nanotube films

Chernov, A.I. and Obraztsova, E.D. Phys. Status Solidi B., 247(11-12), 2805-2809 (2010)   In this work, we report the photoluminescence (PL) studies of the films produced by different methods from pristine single-wall carbon nanotubes (SWNTs). The suspensions of CoMoCAT and HiPCO SWNTs were used to make two different types of films: the polymer films with embedded SWNTs and the SWNT films deposited onto quartz substrates through filtration. A density gradient ultracentrifugation (DGU) has been used for enrichment of the initial carbon nanotube suspensions with semiconducting SWNTs and extraction of various diameter nanotubes to reveal a role of the nanotube interaction in the films. Optical diagnostic techniques including Raman spectroscopy, UV–VIS–NIR absorption spectroscopy, and PL have been used for characterization and comparison of the initial suspensions and films formed from them. A PL mapping has revealed an arrangement of carbon nanotubes inside the films. A cross-linkage between the individual SWNTs appeared to be higher inside the films deposited via filtration than in the polymer films with dispersed SWNTs.  

6.74           Determination of the Surfactant Density on SWCNTs by Analytical Ultracentrifugation

Backes, C., Karabudak, E., Schmidt, C.D., Hauke, F., Hirsch, A. and Wohlleben, W. Chem. Eur. J., 16, 13176-13184 (2010)   We report on the extensive characterization of single-walled carbon nanotubes (SWCNTs) dispersed in a variety of surfactants, such as sodium dodecyl benzene sulfonate (SDBS), sodium cholate (SC), and three synthesized perylene-based surfactants, by using differential sedimentation in H₂O and D₂O. Multidimensional evaluation of the absorption profiles over radius, wavelength, and time allows the determination of the anhydrous specific volumes of the SWCNT–surfactant complexes as well as the concentration of the surfactant reservoir in free micelles with very slow sedimentation coefficients (<1 Svedberg). Among the perylene bisimide surfactants, the smallest derivative is densely adsorbed on the nanotube backbone with an anhydrous specific volume significantly above that of SC or SDBS. Bulky Newkome dendritic groups on one or both ends of the perylene moiety gradually reduce the adsorption density, in accord with the absolute adsorption between 0.66 and 1.7 mmol surfactant per gram SWCNTs. Furthermore, hydrodynamic analysis reveals that SDBS favors the “tails-on” configuration. The distribution of sedimentation coefficients of SWCNTs prepared by high-pressure carbon monoxide decomposition (HiPco) is broader and shifted to faster sedimentation than those prepared by using cobalt–molybdenum catalysis (CoMoCAT), which reflects the polydispersity in diameter and length.  

6.75           Diffusion Limited Photoluminescence Quantum Yields in 1-D Semiconductors: Single-Wall Carbon Nanotubes

Hertel, T., Himmelein, S., Ackermann, T., Stich, D. and Crochet, J. ACSNano, 4(12), 7161-7168 (2010)   Photoluminescence quantum yields and nonradiative decay of the excitonic S₁ state in length fractionated (6,5) single-wall carbon nanotubes (SWNTs) are studied by continuous wave and time-resolved fluorescence spectroscopy. The experimental data are modeled by diffusion limited contact quenching of excitons at stationary quenching sites including tube ends. A combined analysis of the time-resolved photoluminescence decay and the length dependence of photoluminescence quantum yields (PL QYs) from SWNTs in sodium cholate suspensions allows to determine the exciton diffusion coefficient D = 10.7 ± 0.4 cm²s⁻¹ and lifetime τ_PL for long tubes of 20 ± 1 ps. PL quantum yields Φ_PL are found to scale with the inverse diffusion coefficient and the square of the mean quenching site distance, here l_d = 120 ± 25 nm. The results suggest that low PL QYs of SWNTs are due to the combination of high-diffusive exciton mobility with the presence of only a few quenching sites.  

6.76           Electron Paramagnetic Resonance Investigation of Purified Catalyst-free Single-Walled Carbon Nanotubes

Zaka, M., Ito, Y., Wang, H., Yan, W., Robertson, A., Wu, Y.A., Rümmeli, M.H., Staunton, D., Hashimoto, T., Morton, J.J.L., Ardavan, A., Briggs, G.A.D. and Warner, J.H. ACSNano, 4(12), 7708-7716 (2010)   Electron paramagnetic resonance of single-walled carbon nanotubes (SWCNTs) has been bedevilled by the presence of paramagnetic impurities. To address this, SWCNTs produced by laser ablation with a nonmagnetic PtRhRe catalyst were purified through a multiple step centrifugation process in order to remove amorphous carbon and catalyst impurities. Centrifugation of a SWCNT solution resulted in sedimentation of carbon nanotube bundles containing clusters of catalyst particles, while isolated nanotubes with reduced catalyst particle content remained in the supernatant. Further ultracentrifugation resulted in highly purified SWCNT samples with a narrow diameter distribution and almost no detectable catalyst particles. Electron paramagnetic resonance (EPR) signals were detected only for samples which contained catalyst particles, with the ultracentrifuged SWCNTs showing no EPR signal at X-band (9.4 GHz) and fields < 0.4 T.  

6.77           Dispersion of single walled carbon nanotubes using poly(3-dodecylthiophene-2,5-diyl)

Stuparu, A., Stroh, C., Hennrich, F. and Kappes, M.M. Phys. Status Sollidi B, 247(11-12), 2653-2655 (2010)   We describe a procedure to suspend single walled carbon nanotubes with poly(3-dodecylthiophene-2,5-diyl) and to purify them using density gradient centrifugation with 2,4,6-tribromotoluene or dodecyloxy-2,4,6-triiodobenzene as density gradient medium. In contrast to fluorene-based polymers, (n,m) selective solubilization is not observed.  

6.78           Improved sorting of carbon nanotubes according to electronic type by density gradient ultracentrifugation

Posseckardt, J., Battie, Y., Fleurier, R., Lauret, J-S., Loiseau, A., Jost, O. and Mertig, M. Phys. Status Sollidi B, 247(11-12), 2687-2690 (2010)   A variety of fabrication methods for single-walled carbon nanotubes (SWCNTs) has been developed, but none of them has been proven to generate tubes of only one chirality. Hence, a sample of as-produced SWCNTs always consists of a mixture of metallic and semiconducting tubes. By this, the application of SWCNTs in electronics, optics and sensing is limited today. Recently, the sorting of carbon nanotubes using density gradient ultracentrifugation (DGU) has been demonstrated. We report on an improved sorting according to electronic type by applying a two-step procedure: In the first step, we enriched semiconducting SWCNTs via DGU. In the second step, the sorted fractions have been reinserted and a second DGU was performed, winnowing metallic SWCNTs. Employing this procedure, we were able to reduce the content of metallic SWCNTs from 38 to 11%. Furthermore, we assembled carbon nanotube field-effect transistors with a high ON/OFF ratio from the sorted fraction in a single step by dielectrophoresis.  

6.79           Sorting single-wall carbon nanotubes combining gel chromatography and density-gradient ultracentrifugation

Nishide, D., Liu, H., Tanaka, T. and Kataura, H. Phys. Status Sollidi B, 247(11-12), 2746-2749 (2010)   High level structure sorting and metal/semiconductor separation of single-wall carbon nanotubes (SWCNTs) was demonstrated by combining two separation techniques, gel chromatography and density gradient ultra-centrifugation (DGU). Pristine SWCNTs were firstly separated into metallic and semiconducting parts by a gel column chromatography. Then a structure sorting of semiconducting SWCNTs was done by DGU method. Finally, highly (6,5) enriched semiconducting SWCNTs were obtained. Structure sorted SWCNTs obtained by this method were free from metallic SWCNT impurities, which cannot be achieved by simple DGU method.    

6.80           Revealing new electronic behaviours in the Raman spectra of chirality-enriched carbon nanotube ensembles

Duque, J.G., Chen, H., Swan, A.K., Haroz, E.H., Kono, J., Tu, X., Zheng, M. and Doorn, S.K. Phys. Status Sollidi B, 247(11-12), 2768-2773 (2010)   We present Raman spectroscopy of single-walled carbon nanotubes (SWNTs) that are enriched in metallic species by density gradient ultracentrifugation (DGU) and enriched in single semiconducting chiralities through DNA-based separations. Radial breathing mode (RBM) spectra demonstrate that DGU samples are highly enriched in armchair chiralities. The enrichment allows acquisition of pure G-band spectra of the armchair SWNTs and reveals that the LO mode is absent in these structures. Raman excitation profiles for the G-band in nearly pure (10,2) samples reveals a strong asymmetry in the intensities of the resonance coupling to incident and scattered photons. The experimental data may be fit using a four-level molecular model for Raman scattering and the strong asymmetry can be understood as a consequence of the presence of non-Condon effects. The result requires a reassessment of the assumption that the incident and scattered resonances are equivalent. The consequences of such non-Condon effects on other SWNT electronic and optical processes will be an important topic for future study.  

6.81           High-Efficiency Separation of Single-Wall Carbon Nanotubes by Self-Generated Density Gradient Ultracentrifugation

Feng, Y., Miyata, Y., Matsuishi, K. and Kataura, H.

1. Phys. Chem. C, 115(5), 1752-1756 (2011)

  We report an efficient density gradient ultracentrifugation (DGU) method for separation of metallic and semiconducting single-walled carbon nanotubes (SWCNTs) using a self-generated density gradient. A uniform-density SWCNT dispersion (33% v/v in iodixanol) was used to completely fill a centrifuge tube. After ultracentrifugation, separation of the metallic and semiconducting SWCNTs was achieved with a purity comparable to that achieved by DGU using a steplike density gradient. Because the SWCNTs were dispersed within the entire tube during this procedure, a large amount of SWCNTs can be separated at one time.  

6.82           Improved Monodispersity of Plasmonic Nanoantennas via Centrifugal Processing

Tyler, T.P., Henry, A-I., Van Duyne, R.P and Hersam, M.C.

1. Phys. Chem. Lett., 2(3), 218-222 (2011)

  Noble metal nanoparticle clusters underlie a variety of plasmonic devices and measurements including surface-enhanced Raman spectroscopy (SERS). Because of the strong dependence of plasmonic properties on nanoparticle cluster aggregation state, the elimination of non-SERS-active structures and the refinement of the nanoparticle cluster population are critical to realizing uniform and reproducible structures for plasmonic nanoantenna applications such as SERS-based sensors. In this Letter, we report a centrifugal sorting technique for gold core/silica shell nanoparticles that host SERS reporter molecules at the gold/silica interface. The relatively massive nanoparticle clusters are sorted by sedimentation coefficient via centrifugation in a high-viscosity density gradient medium, iodixanol, which yields solutions that contain a preponderance of one aggregation state and a diminished monomer population, as determined by transmission electron microscopy, extinction spectroscopy, and SERS. A quantitative analysis of the nanoparticle sedimentation coefficients is presented, thus allowing this approach to be predictably generalized to other nanoparticle systems.  

6.83           Nanoseparations: Strategies for size and/or shape-selective purification of nanoparticles

Kowalczyk, B., Lagzi, I. and Grzybowski, B.A. Current Opinion in Colloid & Interface Science, 16(2), 135-148 (2011)   This paper reviews techniques currently available for size- and shape-selective purification of nanoscopic objects. The methods discussed range from variants of familiar chromatographic, centrifugation, or filtration techniques, to purification schemes deriving from nanoscale-specific phenomena, including shape-selective reactivity, or propensity to form organized superstructures.  

6.84           Properties and Application of Double-Walled Carbon Nanotubes Sorted by Outer-Wall Electronic Type

Green, A.A. and Hersam, M.C. ACSNano, 5(2), 1459-1467 (2011)   Double-walled carbon nanotubes (DWNTs) can adopt four distinct permutations arising from the electronic type (metallic or semiconducting) of their inner and outer walls. This polydispersity limits the utility of DWNTs in applications such as thin film electronics. We demonstrate that density gradient ultracentrifugation can be employed to address this source of heterogeneity by producing DWNTs with well-defined outer-wall electronic types. Optical absorption measurements of sorted DWNTs reveal outer-wall purities of 96% and 98% for sorted semiconducting and metallic samples, respectively. Electrical characterization of semiconducting and metallic outer-wall DWNTs in thin film transistors directly confirms the efficacy of these separations, with semiconducting DWNT devices yielding on/off ratios 2 orders of magnitude higher than comparable metallic DWNT devices.  

6.85           Structural Stability of Transparent Conducting Films Assembled from Length Purified Single-Wall Carbon Nanotubes

Harris, J.M., Iyer, G.R.S., Simien, D.O., Fagan, J.A., Huh, J.Y., Chung, J.Y., Hudson, S.D., Obrzut, J., Douglas, J.F., Stafford, C.M. and Hobbie, E.K.

1. Phys. Chem. C., 115(10), 3973-3981 (2011)

  Single-wall carbon nanotube (SWCNT) films show significant promise for transparent electronics applications that demand mechanical flexibility, but durability remains an outstanding issue. In this work, thin membranes of length purified single-wall carbon nanotubes (SWCNTs) are uniaxially and isotropically compressed by depositing them on prestrained polymer substrates. Upon release of the strain, the topography, microstructure, and conductivity of the films are characterized using a combination of optical/fluorescence microscopy, light scattering, force microscopy, electron microscopy, and impedance spectroscopy. Above a critical surface mass density, films assembled from nanotubes of well-defined length exhibit a strongly nonlinear mechanical response. The measured strain dependence reveals a dramatic softening that occurs through an alignment of the SWCNTs normal to the direction of prestrain, which at small strains is also apparent as an anisotropic increase in sheet resistance along the same direction. At higher strains, the membrane conductivities increase due to a compression-induced restoration of conductive pathways. Our measurements reveal the fundamental mode of elasto-plastic deformation in these films and suggest how it might be suppressed.  

6.86           Analyzing Absorption Backgrounds in Single-Walled Carbon Nanotube Spectra

Naumov, A.V., Ghosh, S., Tsyboulski, D.A., Bachilo, S.M. and Weisman, R.B. ACSNano, 5(3), 1639-1648 (2011)   The sources of broad backgrounds in visible−near-IR absorption spectra of single-walled carbon nanotube (SWCNT) dispersions are studied through a series of controlled experiments. Chemical functionalization of nanotube sidewalls generates background absorption while broadening and red-shifting the resonant transitions. Extensive ultrasonic agitation induces a similar background component that may reflect unintended chemical changes to the SWCNTs. No major differences are found between spectral backgrounds in sample fractions with average lengths between 120 and 650 nm. Broad background absorption from amorphous carbon is observed and quantified. Overlapping resonant absorption bands lead to elevated backgrounds from spectral congestion in samples containing many SWCNT structural species. A spectral modeling method is described for separating the background contributions from spectral congestion and other sources. Nanotube aggregation increases congestion backgrounds by broadening the resonant peaks. Essentially no background is seen in sorted pristine samples enriched in a single semiconducting (n,m) species. By contrast, samples enriched in mixed metallic SWCNTs show broad intrinsic absorption backgrounds far from the resonant transitions. The shape of this metallic background component and its absorptivity coefficient are quantitatively assessed. The results obtained here suggest procedures for preparing SWCNT dispersions with minimal extrinsic background absorptions and for quantifying the remaining intrinsic components. These findings should allow improved characterization of SWCNT samples by absorption spectroscopy.  

6.87           A Scalable, CMOS-Compatible Assembly of Ambipolar Semiconducting Single-Walled Carbon Nanotube Devices

Ganzhorn, M., Vijayaraghavan, A., Green, A.A., Dehm, S., Voigt, A., Rapp, M., hersam, M.C. and Krupke, R. Adv. Mater., 23, 1734-1738 (2011)   Semiconducting single-walled carbon nanotubes are integrated into high-density arrays using dielectrophoresis, which is a CMOS-compatible, bottom-up assembly technique. The devices are statistically analyzed by voltage-contrast scanning electron microscopy and electron transport measurements. Annealing and the choice of parylene substrate are shown to improve device performance.  

6.88           High-Performance Hydrogen Production and Oxidation Electrodes with Hydrogenase Supported on Metallic Single-Wall CarbonNanotube Networks

Svedruzic, D., Blackburn, J:L., Trenent, R.C., Rocha, J-D.R., Vinzant, T.B., heben, M.J. and King, P.W.

1. Am.Chem. Soc., 133, 4299-4306 (2011)

  We studied the electrocatalytic activity of an [FeFe]-hydrogenase from Clostridium acetobutylicum (CaH2ase) immobilized on single-wall carbon nanotube (SWNT) networks. SWNT networks were prepared on carbon cloth by ultrasonic spraying of suspensions with predetermined ratios of metallic and semiconducting nanotubes. Current densities for both proton reduction and hydrogen oxidation electrocatalytic activities were at least 1 order of magnitude higher when hydrogenase was immobilized onto SWNT networks with high metallic tube (m-SWNT) content in comparison to hydrogenase supported on networks with low metallic tube content or when SWNTs were absent. We conclude that the increase in electrocatalytic activities in the presence of SWNTs was mainly due to the m-SWNT fraction and can be attributed to (i) substantial increases in the active electrode surface area, and (ii) improved electronic coupling between CaH2ase redox-active sites and the electrode surface.  

6.89           Gas sensors based on thick films of semi-conducting single walled carbon nanotubes

Battie, Y., Ducloux, O., Thobois, P., Dorval, N., Lauret, J.S., Attal-Tretout, B. and Loiseau, A. Carbon, 49, 3544-3552 (2011)   A comparative study was made of sorted semi-conducting single walled carbon nanotube (SWCNT) films and unsorted SWCNT films for gas sensing applications. The transmission line method is used to monitor separately the SWCNTs film resistance and the contact resistance between electrodes and the SWCNTs, thus revealing that the sensing mechanism mainly relies on a modification of the tube conductivity during gas exposure. The fabricated sensors demonstrate a detection limit of 20 ppb NO₂ and 600 ppb NH₃ mainly attributed to experimental setup limitations. Moreover, semi-conducting nanotubes happened to be 2.5 times more sensitive to NH₃ than unsorted ones, thus proving that selectivity can be improved by sorting the SWCNTs. The temperature dependence of the sensor sensitivity was studied, and a good agreement was found between experimental results and the Langmuir adsorption model.  

6.90           Chirality-Dependent Changes in the Density of Single-Walled Carbon Nanotubes Oxidized by Tetrachloroaurate

Kato, Y., Niidome, Y. and Nakashima, N. Mol. Cryst. Liq. Cryst., 539, 184-189 (2011)   Density change of single-walled carbon nanotubes (SWCNTs) with tetrachloroaurate enables chirality sorting of the SWCNTs. In this study, we examined in detail about the density change behavior of individually solubilized (n,m)-SWCNTs caused by tetrachloroaurate using density gradients centrifugation and vis-near IR absorption spectroscopy. The (7,5)-, (8,4)- and (8,3)- SWCNTs showed a linear relationship between the density and the degree of the oxidation of the corresponding SWCNTs. Although the (6,5)-SWCNTs was hardly oxidized by tetrachloroaurate, their density increased by the addition of tetrachloroaurate.  

6.91           Electrodynamic and Excitonic Intertube Interactions in Semiconducting Carbon Nanotube Aggregates

Crochet, J.J., Sau, J.D., Duque, J.G., Doorn, S.K. and Cohen, M. ACS Nano, 5(4), 2611-2618 (2011)   The optical properties of selectively aggregated, nearly single chirality single-wall carbon nanotubes were investigated by both continuous-wave and time-resolved spectroscopies. With reduced sample heterogeneities, we have resolved aggregation-dependent reductions of the excitation energy of the S₁ exciton and enhanced electron−hole pair absorption. Photoluminescence spectra revealed a spectral splitting of S₁ and simultaneous reductions of the emission efficiencies and nonradiative decay rates. The observed strong deviations from isolated tube behavior are accounted for by enhanced screening of the intratube Coulomb interactions, intertube exciton tunneling, and diffusion-driven exciton quenching. We also provide evidence that density gradient ultracentrifugation can be used to structurally sort single-wall carbon nanotubes by aggregate size as evident by a monotonic dependence of the aforementioned optical properties on buoyant density.  

6.92           Separation of Empty and Water-Filled Single-Wall Carbon Nanotubes

Fagan, J.A., Huh, J.Y., Simpson, J.R., Blackburn, J.L., Holt, J.M., Larsen, B.A. and Hight Walker, A.R. ACS Nano, 5(5), 3943-3953 (2011)   The separation of empty and water-filled laser ablation and electric arc synthesized nanotubes is reported. Centrifugation of these large-diameter nanotubes dispersed with sodium deoxycholate using specific conditions produces isolated bands of empty and water-filled nanotubes without significant diameter selection. This separation is shown to be consistent across multiple nanotube populations dispersed from different source soots. Detailed spectroscopic characterization of the resulting empty and filled fractions reveals that water filling leads to systematic changes to the optical and vibrational properties. Furthermore, sequential separation of the resolved fractions using cosurfactants and density gradient ultracentrifugation reveals that water filling strongly influences the optimal conditions for metallic and semiconducting separation.  

6.93           Electron Correlation Effects on the Femtosecond Dephasing Dynamics of E₂₂ Excitons in (6,5) Carbon Nanotubes

Schneck, J.R., Walsh, A.G., Green, A.A., Hersam, M.C., Ziegler, L.D. and Swan, A.K.

1. Phys. Chem. A, 15(16), 3917-3923 (2011)

  Highly nonlinear pump fluence dependence was observed in the ultrafast one-color pump−probe responses excited by 38 fs pulses resonant with the E₂₂ transition in a room-temperature solution of (6,5) carbon nanotubes. The differential probe transmission (ΔT/T) at the peak of the pump−probe response (τ = 20 fs) was measured for pump fluences from 10¹³ to 10¹⁷ photons/pulse cm². The onset of saturation is observed at 2 × 10¹⁵ photons/pulse cm² ( 8 × 10⁵ excitons/cm). At pump fluences >4 × 10¹⁶ photons/pulse cm² ( 1.6 × 10⁶ excitons/cm), ΔT/T decreases as the pump fluence increases. Analogous signal saturation behavior was observed for all measured probe delays. Despite the high exciton density at saturation, no change in the E₂₂ population decay rate was observed at short times (<300 fs). The pump probe signal was modeled by a third-order perturbation theory treatment that includes the effects of inhomogeneous broadening. The observed ΔT/T signal is well-fit by a pump-fluence-dependent dephasing rate linearly dependent on the number of excitons created by the pump pulse. Therefore, the observed nonlinear pump intensity dependence is attributed to the effects of quasi-elastic exciton−exciton interactions on the dephasing rates of single carbon nanotubes. The low fluence total dephasing time is 36 fs, corresponding to a homogeneous width of 36 meV (290 cm⁻¹), and the derived E₂₂ inhomogeneous width is 68 meV (545 cm⁻¹). These results are contrasted with photon-echo-derived parameters for the E₁₁ transition.  

6.94           Hydrogen Spillover in Pt-Single-Walled Carbon Nanotube Composites: Formation of Stable C−H Bonds

Bhowmick, R., Rajasekaran, S., Friebel, D., Beasley, C., Jiao, L., Ogaswara, H., Dai, H., Clemens, b. and Nilsson, A.

1. Am. Chem. Soc., 133(14), 5580-5586 (2011)

  Using in situ electrical conductivity and ex situ X-ray photoelectron spectroscopy (XPS) measurements, we have examined how the hydrogen uptake of single-walled carbon nanotubes (SWNTs) is influenced by the addition of Pt nanoparticles. The conductivity of platinum-sputtered single-walled carbon nanotubes (Pt-SWNTs) during molecular hydrogen exposure decreased more rapidly than that of the corresponding pure SWNTs, which supports a hydrogenation mechanism facilitated by “spillover” of dissociated hydrogen from the Pt nanoparticles. C 1s XPS spectra indicate that the Pt-SWNTs store hydrogen by means of chemisorption, that is, covalent C−H bond formation: molecular hydrogen charging at elevated pressure (8.27 bar) and room temperature yielded Pt-SWNTs with up to 16 ± 1.5 at. % sp³-hybridized carbon atoms, which corresponds to a hydrogen-storage capacity of 1.2 wt % (excluding the weight of Pt nanoparticles). Pt-SWNTs prepared by the Langmuir−Blodgett (LB) technique exhibited the highest Pt/SWNT ratio and also the best hydrogen uptake.  

6.95           The Potential of Perylene Bisimide Derivatives for the Solubilization of Carbon Nanotubes and Graphene

Backes, C., Hauke, F. and Hirsch, A. Adv. Mater., 23, 2588-2601 (2011)   Carbon nanotubes and graphene are outstanding materials of the 21^st century with a broad spectrum of applications. However, major challenges are faced such as the intrinsically low solubility of both sp² carbon allotropes. To overcome this hurdle the potential of noncovalent functionalization is summarized with a special focus on the establishment of the perylene bisimide unit as aromatic anchor to the graphitic surface. Rational surfactant design is unmasked as the key to solubilization of the carbon allotropes, while at the same time tailoring their surface properties, or even electronic properties in a fully reversible fashion.  

6.96           Tunable separation of single-walled carbon nanotubes by dual-surfactant density gradient ultracentrifugation

Zhao, P., Einarsson, E., Lagoudas, G., Shiomi, J., Chiashi, S. and Maruyama, S. Nano Res., 4(7), 623-634 (2011)   We present a systematic study of the effects of surfactants in the separation of single-walled carbon nanotubes (SWNTs) by density gradient ultracentrifugation (DGU). Through analysis of the buoyant densities, layer positions, and optical absorbance spectra of SWNT separation using the bile salt sodium deoxycholate (DOC) and the anionic salt sodium dodecyl sulfate (SDS), we clarify the roles and interactions of these two surfactants in yielding different DGU outcomes. The separation mechanism described here can also help in designing new DGU experiments by qualitatively predicting outcomes of different starting recipes, improving the efficacy of DGU and simplifying post-DGU fractionation.  

6.97           Preparation of Surface-Enhanced Raman Scattering-Active Au/Al2O3 Colloids by Sonoelectrochemical Methods

Mai, F-D., Yu, C-C., Liu, Y-C., Yang, K-H., and Junag, M-Y.

1. Phys. Chem. C, 115(28), 13660-13666 (2011)

  As shown in the literature, surface-enhanced Raman scattering (SERS)-active Au or Ag nanoparticle films in agglomerated states on substrates were generally prepared by salting-out the colloidal solutions with NaClO₄ or NaCl. In this work, we report a new pathway to prepare SERS-active Au/Al₂O₃ colloids by sonoelectrochemical methods. The prepared colloids demonstrate strong SERS effects on substrates without an additional salting-out procedure. The particle size of prepared Au nanoparticles (NPs) coated on Al₂O₃ NPs is ca. 20 nm in diameter. Experimental results indicate that the synthesized SERS-active Au/Al₂O₃ colloids demonstrate a large Raman scattering enhancement for Rhodamine 6G with a detection limit of 2 × 10^–10 M, which is lower than most of those shown in the literature based on Au NPs.  

6.98           Molar Extinction Coefficient of Single-Wall Carbon Nanotubes

Schöppler, F., Mann, C., Hain, T.C., Neubauer, F.M., Privitera, G., Bonaccorso, F., Chu, D., Ferrari, A.C. and hertel, T.

1. Phys. Chem. C, 115(30), 14682-14686 (2011)

  The molar extinction coefficient of single-wall carbon nanotubes (SWNTs) is determined using fluorescence tagging, as well as atomic force microscopy (AFM) imaging, which facilitate the correlation of nanotube concentrations with absorption spectra. Tagging of SWNTs is achieved using fluorescence-labeled single-strand DNA oligomers as the dispersion additive, while AFM imaging is used to determine the mass of SWNTs in the retentate of vacuum-filtered colloidal SWNT suspensions. The resulting absorption cross section for the first exciton transition of (6,5) nanotubes of 1.7 × 10^–17 cm² per C-atom corresponds to an extinction coefficient of (4400 ± 1000) M^–1·cm^–1, which is equivalent to an oscillator strength of 0.010 per carbon atom.  

6.99           Synthesis of single-walled carbon nanotubes by an arc-discharge method using selenium as a promoter

Huang, L., Wu, B., Chen, J., Xue, Y., Liu, Y, Kajiura, H. and Li, Y. Carbon, 49, 4792-4800 (2011)   Single-walled carbon nanotubes (SWCNTs) with high purity and very narrow diameter distribution have been synthesized using the dc arc-discharge method with Y–Ni alloy as catalyst and selenium (Se) as promoter. The SWCNTs show a very narrow diameter distribution mainly at about 1.5 nm, and can further be readily purified up to >99% purity with traditional purification including HNO₃ reflux and air oxidation. The key factor of the wetting effect of Se in the SWCNTs growth improvement process is proposed and discussed. Moreover, a new less-destructive purification method including electrolysis, air-oxidation and centrifugation has been introduced, and SWCNTs with semiconducting content up to 94% have been produced through density gradient ultracentrifugation method.  

6.100           Selective Bundling of Zigzag Single-Walled Carbon Nanotubes

Blum, C., Stürzl, N., Hennrich, F., Lebedkin, S., Heeg, S., Dumlich, H., Reich, S. and Kappes, M.M. ACS Nano, 5(4), 2847-2854 (2011)   A simple, high throughput fractionation procedure for aqueous/SDS (sodium dodecyl sulfate) suspensions of single-walled carbon nanotubes (SWNTs) is presented, which yields thin bundles of semiconducting-SWNTs with small chiral angles. To demonstrate this we show the photoluminescence signatures of nanotube suspensions that contain almost exclusively zigzag and near-zigzag tubes. Starting suspensions and resulting fractions were characterized using optical absorption, resonance Raman and photoluminescence spectroscopies as well as scanning force microscopy. Taken together with literature observations, our findings suggest that near zigzag edge tubes of similar diameters in a bundle are harder to separate from each other than for other chiral index combinations. We discuss the implications of these observations for SWNT growth and dispersion.  

6.101           Density Gradient Ultracentrifugation on Carbon Nanotubes According to Structural Integrity as a Foundation for an Absolute Purity Evaluation

Backes, C., Bosch, S., Mundloch, U., Hauke, F. and Hirsch, A. ChemPhysChem., 12(14), 2576-2580 (2011)   Ultrapure: The absolute purity of defect-free and structurally perfect single-walled carbon nanotubes in a bulk sample can be determined with density gradient ultracentrifugation (see picture). The experimental protocol offers a quick and reliable tool and is applicable to a broad variety of nanotube materials to evaluation production and purification procedures.  

6.102           Dynamics and Transient Absorption Spectral Signatures of the Single-Wall Carbon Nanotube Electronically Excited Triplet State

Park, J., Deria, P. and Therien, M.J.

1. Am. Chem. Soc., 133(43), 17156-17159 (2011)

  We utilize femtosecond-to-microsecond time domain pump–probe transient absorption spectroscopy to interrogate for the first time the electronically excited triplet state of individualized single-wall carbon nanotubes (SWNTs). These studies exploit (6,5) chirality-enriched SWNT samples and poly[2,6-{1,5-bis(3-propoxysulfonic acid sodium salt)}naphthylene]ethynylene (PNES), which helically wraps the nanotube surface with periodic and constant morphology (pitch length = 10 ± 2 nm), providing a self-assembled superstructure that maintains structural homogeneity in multiple solvents. Spectroscopic interrogation of such PNES-SWNT samples in aqueous and DMSO solvents using E₂₂ excitation and a white-light continuum probe enables E₁₁ and E₂₂ spectral evolution to be monitored concomitantly. Such experiments not only reveal classic SWNT singlet exciton relaxation dynamics and transient absorption signatures but also demonstrate spectral evolution consistent with formation of a triplet exciton state. Transient dynamical studies evince that (6,5) SWNTs exhibit rapid S₁→T₁ intersystem crossing (ISC) (τ_ISC 20 ps), a sharp T₁→T_n transient absorption signal (λ_max(T₁→T_n) = 1150 nm; full width at half-maximum ≈ 350 cm^–1), and a substantial T₁ excited-state lifetime (τ_es ≈ 15 μs). Consistent with expectations for a triplet exciton state, T₁-state spectral signatures and T₁-state formation and decay dynamics for PNES-SWNTs in aqueous and DMSO solvents, as well as those determined for benchmark sodium cholate suspensions of (6,5) SWNTs, are similar; likewise, studies that probe the ³[(6,5) SWNT]* state in air-saturated solutions demonstrate ³O₂ quenching dynamics reminiscent of those determined for conjugated aromatic hydrocarbon excited triplet states.  

6.103           Photoluminescence from Inner Walls in Double-Walled Carbon Nanotubes: Some Do, Some Do Not

Yang, S.Y., parks, A.N., Saba, S.A., Ferguson, P.L. and Liu, J. Nano Lett., 11(10), 4405-4410 (2011)   Double-walled carbon nanotubes (DWNTs) have recently been recognized as important members in the carbon nanotube family because they are expected to have certain unique properties. For example, DWNTs are expected to replace single-walled carbon nanotubes (SWNTs) in biomarker applications and optoelectronics if the observed luminescence from DWNTs can be verified. However, due to unavoidable byproducts, such as SWNTs, optical properties of DWNTs still remain controversial. There is an ongoing debate concerning the ability of DWNTs to exhibit photoluminescence (PL). In this report, we aim to clearly resolve this debate through the study of carefully separated DWNTs. DWNTs were successfully separated from SWNTs using density gradient ultracentrifugation. Here we clearly show that light is emitted from the inner wall of DWNTs; however, the intensity of the emission is significantly quenched. Interestingly, it was found that a very narrow range of diameters of the inner walls of DWNTs is required for PL to be observable. All other diameters led to complete PL quenching in DWNTs. In short, we have shown that both sides of the debate are correct under certain situations. The real answer to the question is that some DWNTs do emit light but most DWNTs do not.  

6.104           Electronically Monodisperse Single-Walled Carbon Nanotube Thin Films as Transparent Conducting Anodes in Organic Photovoltaic Devices

Tyler, T.P., Brock, R.E., karmel, H.J., marks, T.J. and hersam, M.C. Adv. Energy Mater., 1(5), 785-791 (2011)   Single-walled carbon nanotubes (SWNTs) sorted by electronic type are employed as organic photovoltaic device anodes. Metal-enriched SWNT films yield device efficiencies that are fifty times greater than their semiconducting counterparts. Through sheet resistance, UV-vis-NIR optical absorbance, and X-ray photoelectron spectroscopy measurements, the OPV charge blocking layer PEDOT:PSS is found to reverse the original chemical doping of the SWNT films. The relative insensitivity of metallic SWNTs to chemical doping thus explains the improved performance of metal-enriched SWNT films as OPV anodes.  

6.105           Chemometric determination of the length distribution of single walled carbon nanotubes through optical spectroscopy

Si, R., Wang, K., Chen, T. and Chen, Y. Analytica Chimica Acta, 708, 28-36 (2011)   Current synthesis methods for producing single walled carbon nanotubes (SWCNTs) do not ensure uniformity of the structure and properties, in particular the length, which is an important quality indicator of SWCNTs. As a result, sorting SWCNTs by length is an important post-synthesis processing step. For this purpose, convenient analysis methods are needed to characterize the length distribution rapidly and accurately. In this study, density gradient ultracentrifugation was applied to prepare length-sorted SWCNT suspensions containing individualized surfactant-wrapped SWCNTs. The length of sorted SWCNTs was first determined by atomic force microscope (AFM), and their absorbance was measured in ultraviolet–visible near-infrared (UV–vis-NIR) spectroscopy. Chemometric methods are used to calibrate the spectra against the AFM-measured length distribution. The calibration model enables convenient analysis of the length distribution of SWCNTs through UV–vis-NIR spectroscopy. Various chemometric techniques are investigated, including pre-processing methods and non-linear calibration models. Extended inverted signal correction, extended multiplicative signal correction and Gaussian process regression are found to provide good prediction of the length distribution of SWCNTs with satisfactory agreement with the AFM measurements. In summary, spectroscopy in conjunction with advanced chemometric techniques is a powerful analytical tool for carbon nanotube research.  

6.106           Discovery of Surfactants for Metal/Semiconductor Separation of Single-Wall Carbon Nanotubes via High-Throughput Screening

Tanaka, T., Urabe, Y., Nishide, D. and Kataura, H.

1. Am. Chem. Soc., 133(44), 17610-17613 (2011)

  We report novel surfactants that can be used for the separation of metallic (M) and semiconducting (S) single-wall carbon nanotubes (SWCNTs). Among the M/S separation methods using surfactants in an aqueous solution, sodium dodecyl sulfate plays a key role in density gradient ultracentrifugation (DGU) and agarose gel separations. In this study, we screened 100 surfactants for M/S separation using a high-throughput screening system. We identified five surfactants, which could be used for both DGU and agarose gel separations, suggesting that the basic principle of these separations is common. These surfactants have relatively low dispersibilities, which is likely due to their common structural features, i.e., straight alkyl tails and charged head groups, and appeared to enable M- and S-SWCNTs to be distinguished and separated. These surfactants should stimulate research in this field and extend the application of electrically homogeneous SWCNTs not only for electronics but also for biology and medicine.  

6.107           A Mechanistic Study of the Selective Retention of SDS-Suspended Single-Wall Carbon Nanotubes on Agarose Gels

Silvera-Batista, C.A., Scott, D.C., McLeod, S.M. and Ziegler, K.J.

1. Phys. Chem. C, 115, 9361-9369 (2011)

    Elution chromatography through columns packed with agarose beads has been used to separate metallic from semiconducting single-wall carbon nanotubes (SWCNTs). Prior studies have attributed the separation to either selective adsorption or size-exclusion (due to selective aggregation) of semiconducting SWCNTs. Initial SWCNT suspensions with different aggregation states were prepared to test these competing theories. Retention characteristics of the SWCNT suspensions were not affected by changes to aggregation state, except when the centrifugation time was short and aggregation excessive. On the other hand, selective adsorption of nanotubes on the agarose matrix is confirmed by modifying the surfactant structure around the SWCNTs without changing the aggregation state of the suspension. In addition, salt-modifiers and solvent-modifiers allow systematic changes to the surfactant aggregation number, orientation, and sidewall coverage. The retention characteristics from these modified SWCNT suspensions suggest that surfactant orientation rather than the exposed regions on the surface of the nanotubes is the dominant factor in the adsorption process.  

6.108           Selective Polycarboxylation of Semiconducting Single-Walled Carbon Nanotubes by Reductive Sidewall Functionalization

Gebhardt, B., Hof, F., Backes, C., Müller, M., Plocke, t., Maultzsch, J., Thomsen, C., Hauke, F. and Hirsch, A.

1. Am. Chem. Soc., 133(48), 19459-19473 (2011)

  The efficient and controllable synthesis, the detailed characterization, and the chemical postfunctionalization of polycarboxylated single-walled carbon nanotubes SWCNT(COOH)_n are reported. This innovative covalent sidewall functionalization method is characterized by (a) the preservation of the integrity of the entire σ-framework of SWCNTs; (b) the possibility of achieving very high degrees of addition; (c) control of the functionalization degrees by the variation of the reaction conditions (reaction time, ultrasonic treatment, pressure); (d) the identification of conditions for the selective functionalization of semiconducting carbon nanotubes, leaving unfunctionalized metallic tubes behind; (e) the proof that the introduced carboxylic acid functionalities can serve as versatile anchor points for the coupling to functional molecules; and (f) the application of a subsequent thermal degradation step of the functionalized semiconducting tubes leaving behind intact metallic SWCNTs. Functional derivatives have been characterized in detail by means of Raman, UV–vis/nIR, IR, and fluorescence spectroscopy as well as by thermogravimetric analysis combined with mass spectrometry, atomic force microscopy, and zeta-potential measurements.  

6.109           Intensity-Dependent Exciton Dynamics of (6,5) Single-Walled Carbon Nanotubes: Momentum Selection Rules, Diffusion, and Nonlinear Interactions

Harrah, D.M., Schneck, J.R., Green, A.A., Hersam, M.C., Ziegler, L.D.a dn Swan, A.K. ACS Nano, 5(12), 9898-9906 (2011)   The exciton dynamics for an ensemble of individual, suspended (6,5), single-walled carbon nanotubes revealed by single color E₂₂ resonant pump–probe spectroscopy for a wide range of pump fluences are reported. The optically excited initial exciton population ranges from approximately 5 to 120 excitons per 725 nm nanotube. At the higher fluences of this range, the pump–probe signals are no longer linearly dependent on the pump intensity. A single, predictive model is described that fits all data for two decades of pump fluences and three decades of delay times. The model introduces population loss from the optically active zero momentum E₂₂ state to the rest of the E₂₂ subband, which is dark due to momentum selection rules. In the single exciton limit, the E₁₁ dynamics are well described by a stretched exponential, which is a direct consequence of diffusion quenching from an ensemble of nanotubes of different lengths. The observed change in population relaxation dynamics as a function of increasing pump intensity is attributed to exciton–exciton Auger de-excitation in the E₁₁ subband and, to a lesser extent, in the E₂₂ subband. From the fit to the model, an average defect density 1/ρ = 150 nm and diffusion constants D₁₁ = 4 cm²/s and D₂₂ = 0.2 cm²/s are determined.  

6.110           Minimizing Oxidation and Stable Nanoscale Dispersion Improves the Biocompatibility of Graphene in the Lung

Duch, M.C., Budinger, G.R.S., Liang, Y.T., Soberanes, S., Urich, D., Chiarella, S.E., Campochiaro, L.A., Gonzales, A., Chandel, N.S., Hersam, M.C. and Mutlu, G.M. Nano Lett., 11(12), 5201-5207 (2011)   To facilitate the proposed use of graphene and its derivative graphene oxide (GO) in widespread applications, we explored strategies that improve the biocompatibility of graphene nanomaterials in the lung. In particular, solutions of aggregated graphene, Pluronic dispersed graphene, and GO were administered directly into the lungs of mice. The introduction of GO resulted in severe and persistent lung injury. Furthermore, in cells GO increased the rate of mitochondrial respiration and the generation of reactive oxygen species, activating inflammatory and apoptotic pathways. In contrast, this toxicity was significantly reduced in the case of pristine graphene after liquid phase exfoliation and was further minimized when the unoxidized graphene was well-dispersed with the block copolymer Pluronic. Our results demonstrate that the covalent oxidation of graphene is a major contributor to its pulmonary toxicity and suggest that dispersion of pristine graphene in Pluronic provides a pathway for the safe handling and potential biomedical application of two-dimensional carbon nanomaterials.  

6.111           Design of a Polymer–Carbon Nanohybrid Junction by Interface Modeling for Efficient Printed Transistors

Kim, D.H., Shin, H-J., Lee, H.S., Lee, J., Lee, B-L., Lee, W.H., Lee, J-H., Cho, K., Kim, W-J., Lee, S.Y., Choi, J-Y. and Kim, J.M. ACS Nano, 6(1), 662-670 (2012)   Molecularly hybridized materials composed of polymer semiconductors (PSCs) and single-walled carbon nanotubes (SWNTs) may provide a new way to exploit an advantageous combination of semiconductors, which yields electrical properties that are not available in a single-component system. We demonstrate for the first time high-performance inkjet-printed hybrid thin film transistors with an electrically engineered heterostructure by using specially designed PSCs and semiconducting SWNTs (sc-SWNTs) whose system achieved a high mobility of 0.23 cm² V^–1 s^–1, no V_on shift, and a low off-current. PSCs were designed by calculation of the density of states of the backbone structure, which was related to charge transfer. The sc-SWNTs were prepared by a single cascade of the density-induced separation method. We also revealed that the binding energy between PSCs and sc-SWNTs was strongly affected by the side-chain length of PSCs, leading to the formation of a homogeneous nanohybrid film. The understanding of electrostatic interactions in the heterostructure and experimental results suggests criteria for the design of nanohybrid heterostructures.  

6.112           Electronic Durability of Flexible Transparent Films from Type-Specific Single-Wall Carbon Nanotubes

Harris, J.M., Iyver, G.R.S., Bernhardt, A.K., Huh, J.Y., Hudson, S.D., Fagan, J.A. and Hobbie, E.E. ACS Nano, 6(1), 881-887 (2012)   The coupling between mechanical flexibility and electronic performance is evaluated for thin films of metallic and semiconducting single-wall carbon nanotubes (SWCNTs) deposited on compliant supports. Percolated networks of type-purified SWCNTs are assembled as thin conducting coatings on elastic polymer substrates, and the sheet resistance is measured as a function of compression and cyclic strain through impedance spectroscopy. The wrinkling topography, microstructure and transparency of the films are independently characterized using optical microscopy, electron microscopy, and optical absorption spectroscopy. Thin films made from metallic SWCNTs show better durability as flexible transparent conductive coatings, which we attribute to a combination of superior mechanical performance and higher interfacial conductivity.  

6.113           Highly concentrated carbon nanotube admixture for nano-fiber reinforced cementitious materials

Metaxa, Z.S., Seo, J-W.T., Konsta-Gdoutos, M.S., Hersam, M.C. and Shah, S.P. Cement & Concrete Composites, 34, 612-617 (2012)   The use of effectively dispersed multiwalled carbon nanotube (MWCNT)/aqueous/surfactant suspensions in cement based materials have been shown to substantially improve their mechanical properties. The produced MWCNT suspensions have a high aqueous content, which corresponds to the mixing water. In the present work, a method for preparing highly concentrated MWCNT suspensions is presented, thus reducing the volume of the resulting admixture that is required in cement based materials. A centrifugal process, that uses two different ultracentrifuge rotors, was employed to reduce the quantity of water in the suspensions. Optical absorbance spectroscopy shows that the ultracentrifugation process increases the concentration of the MWCNT suspensions by a factor of 5. Using the highly concentrated MWCNT suspensions following dilution results in nanocomposites with mechanical properties that are comparable to the performance of samples prepared using the non-concentrated suspensions. These results verify that the ultracentrifugation concentration method successfully preserves the solubility of the MWCNT suspensions without affecting the reinforcing properties of the admixture. In this manner, the ultracentrifugation concentration method may constitute an effective preparation step for large-scale implementation of MWCNT admixtures.  

6.114           Density gradient ultracentrifugation and stability of SWNT–peptide conjugates

Hartleb, H., Kröker, K. and Hertel, T. Chem. Phys. Lett., 535, 131-135 (2012)   Density gradient ultracentrifugation was used for preparation of biocompatible single-wall carbon nanotube (SWNT)–peptide conjugates. Aggregate size selected SWNT suspensions were analyzed by absorption and photoluminescence spectroscopy to determine stability and structure of conjugates. The results suggest that conjugates with densities over consist of SWNT aggregates. More buoyant fractions with contain mostly individual SWNTs. Dilution experiments indicate that equilibration between SWNT-bound and free peptide in solution is approached within a few minutes making excess peptide in solution necessary to prevent peptide–SWNT conjugate aggregation. This has significant implications for the use of SWNT–peptide conjugates in biochemical applications.  

6.115           Evidence for Patchy Lipid Layers on Gold Nanoparticle Surfaces

Yang, J.A. and  Murphy, C.J. Langmuir, 28(12), 5404-5416 (2012)   Gold nanoparticles bearing multiple surface ligands are becoming favored candidates as multifunctional targeting, imaging, and therapeutic vehicles for biomedicine. The question of spatial location of different ligands on nanoparticle surfaces, especially with those of diameters less than 100 nm, is an important one that is difficult to quantitatively address. Here we functionalize the surface of 20, 50, and 90 nm gold nanoparticles with two different lipids, both single and mixed, using two different surface chemical procedures. Mass spectrometry supports the presence of both lipids in the mixed-lipid systems on nanoparticles, while electron microscopy evidence shows domain sizes for one lipid apparently a quarter to a half the projected diameter for 50 and 90 nm particles; but for 20 nm particles, there is no evidence for the existence of patches of the two lipids. Larger gold nanoparticles (90 nm) can be decorated with an array of 12 nm gold nanoparticles by use of a third lipid and antibody–antigen connectors; the display of the 12 nm particles about the 90 nm particles can be controlled to some extent by the initial surface chemistry and is quantified via a new angle analysis procedure.  

6.116           Confirmation of K-Momentum Dark Exciton Vibronic Sidebands Using 13C-labeled, Highly Enriched (6,5) Single-walled Carbon Nanotubes

Blackburn, J.L., Holt, J.M., Irurzun, V.M., Resasco, D.E. and Rumbles, G. Nano Lett., 12(3), 1398-1403 (2012)   A detailed knowledge of the manifold of both bright and dark excitons in single-walled carbon nanotubes (SWCNTs) is critical to understanding radiative and nonradiative recombination processes. Exciton–phonon coupling opens up additional absorption and emission channels, some of which may “brighten” the sidebands of optically forbidden (dark) excitonic transitions in optical spectra. In this report, we compare ¹²C and ¹³C-labeled SWCNTs that are highly enriched in the (6,5) species to identify both absorptive and emissive vibronic transitions. We find two vibronic sidebands near the bright ¹E₁₁ singlet exciton, one absorptive sideband 200 meV above, and one emissive sideband 140 meV below, the bright singlet exciton. Both sidebands demonstrate a 50 cm^–1 isotope-induced shift, which is commensurate with exciton–phonon coupling involving phonons of A₁^′ symmetry (D band, ω 1330 cm^–1). Independent analysis of each sideband indicates that both sidebands arise from the same dark exciton level, which lies at an energy approximately 25 meV above the bright singlet exciton. Our observations support the recent prediction of, and mounting experimental evidence for, the dark K-momentum singlet exciton lying 25 meV (for the (6,5) SWCNT) above the bright Γ-momentum singlet. This study represents the first use of ¹³C-labeled SWCNTs highly enriched in a single nanotube species to unequivocally confirm these sidebands as vibronic sidebands of the dark K-momentum singlet exciton.  

6.117           Unique Origin of Colors of Armchair Carbon Nanotubes

Haroz, E.H., Duque, J.G:, Lu, B.Y., Nikolaev, P., Arepalli, S., Hauge, R.H., Doorn, S.K. and Kono, J.

1. Am. Chem. Soc., 134(10), 4461-4464 (2012)

  The colors of suspended metallic colloidal particles are determined by their size-dependent plasma resonance, while those of semiconducting colloidal particles are determined by their size-dependent band gap. Here, we present a novel case for armchair carbon nanotubes, suspended in aqueous medium, for which the color depends on their size-dependent excitonic resonance, even though the individual particles are metallic. We observe distinct colors of a series of armchair-enriched nanotube suspensions, highlighting the unique coloration mechanism of these one-dimensional metals.  

6.118           Unraveling the 13C NMR Chemical Shifts in Single-Walled Carbon Nanotubes: Dependence on Diameter and Electronic Structure

Engtrakul, C., Irurzun, V.M., Gjersing, E.L., Holt, J.M., Larsen, B.A., Resasdco, D.E. and Blackburn, J.L.

1. Am. Chem. Soc., 134(10), 4850-4856 (2012)

  The atomic specificity afforded by nuclear magnetic resonance (NMR) spectroscopy could enable detailed mechanistic information about single-walled carbon nanotube (SWCNT) functionalization as well as the noncovalent molecular interactions that dictate ground-state charge transfer and separation by electronic structure and diameter. However, to date, the polydispersity present in as-synthesized SWCNT populations has obscured the dependence of the SWCNT ¹³C chemical shift on intrinsic parameters such as diameter and electronic structure, meaning that no information is gleaned for specific SWCNTs with unique chiral indices. In this article, we utilize a combination of ¹³C labeling and density gradient ultracentrifugation (DGU) to produce an array of ¹³C-labeled SWCNT populations with varying diameter, electronic structure, and chiral angle. We find that the SWCNT isotropic ¹³C chemical shift decreases systematically with increasing diameter for semiconducting SWCNTs, in agreement with recent theoretical predictions that have heretofore gone unaddressed. Furthermore, we find that the ¹³C chemical shifts for small diameter metallic and semiconducting SWCNTs differ significantly, and that the full-width of the isotropic peak for metallic SWCNTs is much larger than that of semiconducting nanotubes, irrespective of diameter.  

6.119           Measuring Single-Wall Carbon Nanotubes with Solid-State Nanopores

Hall, A.R., Keegstra, J.M., Duch, M.C., Hersam, M.C. and Dekker, C. Methods in Mol. Biol., 870, 227-239 (2012)   Solid-state nanopores have been used widely to study biological polymers. Here, we expand the technique to analyze single-wall carbon nanotubes. By wrapping them in an amphiphilic layer, individual tubes can be translocated electrically through a nanopore, resulting in temporary interruptions in the trans-pore current reminiscent of measurements on DNA, RNA, and proteins. The technique may fi nd use in discriminating nanotubes by size and thus electrical structure, facilitating their inclusion in electrical devices.  

6.120           Towards Rationally Designed Graphene-Based Materials and Devices

Liang, Y.T. and Hersam, M.C. Macromol. Chem. Physics, 213(10-11), 1091-1100 (2012)   With its exceptional charge transport properties, graphene has emerged as a potential replacement material in the electronics industry. For these applications, much effort has been devoted towards the synthesis of large defect-free graphene sheets. However, recent developments have enabled the efficient production of micrometer- and nanometer-sized graphene sheets in the solution phase. These suspensions have stimulated the development of novel materials and devices that more fully exploit the tunability and large specific surface area of pristine graphene. This review highlights advances in the understanding of the defect structure and properties of as-produced graphene as well as strategies for its chemical selection and modification that facilitates its use in functional materials and devices.    

6.121           Narrow Diameter Distributions of Metallic Arc Discharge Single-Walled Carbon Nanotubes via Dual-Iteration Density Gradient Ultracentrifugation

Tyler, T.P., Shastry, T.A., Leever, B.J: and Hersam, M.C. Adv. Mater., 24(35), 4765-4768 (2012)   Dual-iteration density gradient ultracentrifugation isolates nearly single diameters of monodisperse metallic arc discharge SWCNTs. Subsequently fabricated conductive thin films possess distinct colors due to well-defined transmittance windows flanked by sharp optical transitions. Measurements of uniform sheet resistances and work functions confirm the largely invariant electronic properties between metallic arc discharge SWCNT films of differing diameters.  

6.122           Probing and Tailoring pH-Dependent Interactions between Block Copolymers and Single-Walled Carbon Nanotubes for Density Gradient Sorting

Antaris, A.L., Jung-Wu. T. Seo., Brock, R.E., Herriman, J.E., Born, M.J., Green, A.A. and Hersam, M.C:

1. Phys. Chem. C, 116(37), 20103-20108 (2012)

  The method by which surfactants selectively interact with particular electronic types of single-walled carbon nanotubes (SWCNTs) and thereby enable the isolation of metallic and semiconducting species is not well understood. While density gradient ultracentrifugation (DGU) has demonstrated its potential as a powerful nanomaterial separation technique, this study utilizes DGU as an analytic tool to probe the interactions between amphiphilic block copolymers, surfactants capable of electronic type extraction, and the SWCNT surface. By modulating the pH during DGU, we find that the linear shaped Pluronic copolymers can extract either metallic or semiconducting SWCNTs at purities in excess of 99%. Furthermore, the first electronic type sorting mechanism is given by which oxygen absorption and subsequent protonation of the SWCNT surface acts to template copolymer adhesion. Detailed characterization reveals the underlying mechanism for pH-shifted DGU and is thus likely to enable future development of more efficient and facile SWCNT electronic type sorting methods.  

6.123           Fundamental Performance Limits of Carbon Nanotube Thin-Film Transistors Achieved Using Hybrid Molecular Dielectrics

Sangwan, V.K., Ortiz, R.P., alaboson, J.M.P., Emery, J.D., Bedzyk, M.J., Lauhon, L.J., Marks, T.J. and Hersam, M.C. ACS Nano, 6(8), 7480-7488 (2012)   In the past decade, semiconducting carbon nanotube thin films have been recognized as contending materials for wide-ranging applications in electronics, energy, and sensing. In particular, improvements in large-area flexible electronics have been achieved through independent advances in postgrowth processing to resolve metallic versus semiconducting carbon nanotube heterogeneity, in improved gate dielectrics, and in self-assembly processes. Moreover, controlled tuning of specific device components has afforded fundamental probes of the trade-offs between materials properties and device performance metrics. Nevertheless, carbon nanotube transistor performance suitable for real-world applications awaits understanding-based progress in the integration of independently pioneered device components. We achieve this here by integrating high-purity semiconducting carbon nanotube films with a custom-designed hybrid inorganic–organic gate dielectric. This synergistic combination of materials circumvents conventional design trade-offs, resulting in concurrent advances in several transistor performance metrics such as transconductance (6.5 μS/μm), intrinsic field-effect mobility (147 cm²/(V s)), subthreshold swing (150 mV/decade), and on/off ratio (5 × 10⁵), while also achieving hysteresis-free operation in ambient conditions.  

6.124           Manipulating Electron Transfer between Single-Walled Carbon Nanotubes and Diazonium Salts for High Purity Separation by Electronic Type

Do, Y-J., Lee, J-H., CHOI, H., Han, J-H., Chung, C-H., Jeong, M-G., Strano, M.S. and Kim, W-J. Chem. Mater, 24(21), 4146-4151 (2012)   Diazonium salts preferentially react with metallic single-walled carbon nanotubes (SWNT) over semiconducting SWNT, enabling the separation of SWNT by electronic type. Therefore, the reaction selectivity of diazonium salts for metallic SWNT is crucial for high purity separation of both metallic and semiconducting SWNT. Herein, we developed an efficient method of increasing the reaction selectivity by manipulating the redox potential of diazonium salts. The electron affinity of diazonium salts is effectively lowered when the para-substituent of the diazonium salts is an electron-donating group, (i.e., 4-hydroxy and 4-propargyloxy) rather than an electron-withdrawing group (i.e., 4-nitro, 4-carboxy, and 4-cholro). The reduction potential of 4-hydroxyphenyl and 4-propargyloxyphenyl diazonium salt was greater than the oxidation potential of semiconducting SWNT; therefore, the electron transfer reaction between these two reagents was effectively suppressed, leading to a highly selective reaction for metallic SWNT. We confirmed that this highly selective reaction scheme can be used to separate SWNT, and high purity semiconducting SWNT can be obtained via density-induced separation.  

6.125           Mild Bromination-Assisted Density-Gradient Ultracentrifugation to Sort Single-Walled Carbon Nanotubes by Metallicity

Wang, W., Mahasin, A.S., Gao, P.Q., Lim, K.H. and Chan-Park, M.B.

1. Phys. Chem. C, 116(43), 23027-23035 (2012)

  Brominated single-walled carbon nanotubes, with bromine covalently attached to the nanotube surface, have been synthesized by a mild reaction using n-bromosuccinimide (NBS). The latter preferentially attacks metallic single walled carbon nanotubes (SWNTs) over semiconducting ones, and the attached Br leads to a significant density differential between reacted and pristine nanotubes. The differential reactivity between semiconducting and metallic SWNTs enhances the density contrast between them, which may be more effectively spatially separated via density gradient ultracentrifugation than unchemically modified SWNTs. The results of optical absorbance, photoluminescence emission, and resonant Raman scattering show that bromination-assisted density gradient ultracentrifugation (hereafter labeled as Br-DGU) preferentially separated semiconducting nanotubes within a certain diameter range (0.829–0.966 nm, specifically (7,6), (8,4), (9,4), and (10,3)). We have applied the semiconducting species enriched SWNTs to prepare solution-processed FET devices with random nanotube network active channels. The devices exhibit stable p-type semiconductor behavior in air with very promising characteristics. The on–off current ratio reaches up to 1730 within a narrow gate voltage (−2 to 2 V) and an estimated hole mobility of 13 cm² V^–1 s^–1.  

6.126           Characterization and Quantitative Analysis of Single-Walled Carbon Nanotubes in the Aquatic Environment Using Near-Infrared Fluorescence Spectroscopy

Schierz, A., Parks, A.N., Washburn, K.M., Chandler, G.T. and Ferguson, P.L. Environ. Sci. Technol., 46(22), 12262-12271 (2012)   Near infrared fluorescence (NIRF) spectroscopy is capable of sensitive and selective detection of semiconductive, single-walled carbon nanotubes (SWNT) using the unique electronic bandgap properties of these carbon allotropes. We reported here the first detection and quantitation of SWNT in sediment and biota at environmentally relevant concentrations using NIRF spectroscopy. In addition, we utilized this technique to qualitatively characterize SWNT samples before and after ecotoxicity, bioavailability and fate studies in the aquatic environment. Sample preparation prior to NIRF analysis consisted of surfactant-assisted high power ultrasonication. The bile salt sodium deoxycholate (SDC) enabled efficient extraction and disaggregation of SWNT prior to NIRF analysis. The method was validated using standard-addition experiments in two types of estuarine sediments, yielding recoveries between 66 ± 7% and 103 ± 10% depending on SWNT type and coating used, demonstrating the ability to isolate SWNT from complex sediment matrices. Instrument detection limits were determined to be 15 ng mL^–1 SWNT in 2% SDC solution and method detection limits (including a concentration step) were 62 ng g^–1 for estuarine sediment, and 1.0 μg L^–1 for water. Our work has shown that NIRF spectroscopy is highly sensitive and selective for SWNT and that this technique can be applied to track the environmental and biological fate of this important class of carbon nanomaterial in the aquatic environment.  

6.127           Ultrashort Single-Walled Carbon Nanotubes: Density Gradient Separation, Optical Property, and Mathematical Modeling Study

Kuang, Y., Liu, J. and Sun, X.

1. Phys. Chem. C, 116(46), 24770-24776 (2012)

  The density gradient ultracentrifuge separation (DGUS) method for obtaining ultrashort single-walled carbon nanotubes (SWNTs) was systematically investigated. A twice separation was used for further narrowing the length distribution. Investigations on Raman and absorbance spectra evidenced the concomitant chirality separation of the NTs. The length-dependent blue shift recorded on the absorbance spectra confirmed band gap widening on finite semiconductive SWNTs, which showed a linear relationship to the inverse of the length of the NTs. Laser ablation NTs were used to demonstrate DGUS as a general method for separation of different type of SWNTs. A possible vertical sedimentation separation mechanism is proposed, and a mathematical model was set up to give a quantitive description on separation results, which was further demonstrated by time-dependence experiments.  

6.128           Surface-enhanced Raman scattering-active Au/TiO2 films prepared by electrochemical and photochemical methods

Yang, K-H. and Chang, C-M. Materials Research Bulletin, 48, 372-377 (2013)   In this work, we report a new strategy for the preparation of surface-enhanced Raman scattering (SERS)-active Au/TiO₂(P25) nanocomposites (NCs), using electrochemical and photochemical methods. First, Au substrates were subjected to electrochemical oxidation–reduction cycles (ORCs) in a deoxygenated aqueous solution containing 0.1 M HCl and 1 mM TiO₂. After the ORC treatment AuCl₄⁻-adsorbed TiO₂ complexes were produced in the solution. These complex-containing substrates were then irradiated with UV light at 310 nm to synthesize Au/TiO₂ NCs with strong SERS activities for probe molecules of rhodamine 6G (R6G) and conductive polymers of polypyrrole (PPy). Experimental results indicated that the wavelength of UV light and the presence of TiO₂ before and after the ORC procedure during the preparation process both affected the resulting SERS activities.  

6.129           Large-Area, Electronically Monodisperse, Aligned Single-Walled Carbon Nanotube Thin Films Fabricated by Evaporation-Driven Self-Assembly

Shastry, T.A., Seo, J-W.T., Lopez, J.J., Arnold, H.N., Kelter, J.Z., Sangwan, V.K., Lauhon, L.J., Marks, T.J. and Hersam, M.C. Small, 9(1), 45-51 (2013)   By varying the evaporation conditions and the nanotube and surfactant concentrations, large-area, aligned single-walled carbon nanotube (SWCNT) thin films are fabricated from electronically monodisperse SWCNT solutions by evaporation-driven self-assembly with precise control over the thin film growth geometry. Tunability is possible from 0.5 μm stripes to continuous thin films. The resulting SWCNT thin films possess highly anisotropic electrical and optical properties that are well suited for transparent conductor applications.  

6.130           High-Field Transport and Thermal Reliability of Sorted Carbon Nanotube Network Devices

Behnam, A., Sangwan, V.K., Zhong, X., Lian, F., Estrada, D., Jariwala, d., Hoag, A.J., Lauhon, L.J., Marks, T.J., Hersam, M.C. and Pop, E. ACS Nano, 7(1), 482-490 (2013)   We examine the high-field operation, power dissipation, and thermal reliability of sorted carbon nanotube network (CNN) devices, with <1% to >99% semiconducting nanotubes. We combine systematic electrical measurements with infrared (IR) thermal imaging and detailed Monte Carlo simulations to study high-field transport up to CNN failure by unzipping-like breakdown. We find that metallic CNNs carry peak current densities up to an order of magnitude greater than semiconducting CNNs at comparable nanotube densities. Metallic CNNs also appear to have a factor of 2 lower intrinsic thermal resistance, suggesting a lower thermal resistance at metallic nanotube junctions. The performance limits and reliability of CNNs depend on their makeup, and could be improved by carefully engineered heat dissipation through the substrate, contacts, and nanotube junctions. These results are essential for optimization of CNN devices on transparent or flexible substrates which typically have very low thermal conductivity.  

6.131           Single-Walled Carbon Nanotube Surface Control of Complement Recognition and Activation

Andersen, A.J., Robinson, J.T., Dai, H., Hunter, A.C., Andresen, T. and Moghimi, S.M. ACS Nano, 7(2), 1108-1119 (2013)   Carbon nanotubes (CNTs) are receiving considerable attention in site-specific drug and nucleic acid delivery, photodynamic therapy, and photoacoustic molecular imaging. Despite these advances, nanotubes may activate the complement system (an integral part of innate immunity), which can induce clinically significant anaphylaxis. We demonstrate that single-walled CNTs coated with human serum albumin activate the complement system through C1q-mediated classical and the alternative pathways. Surface coating with methoxypoly(ethylene glycol)-based amphiphiles, which confers solubility and prolongs circulation profiles of CNTs, activates the complement system differently, depending on the amphiphile structure. CNTs with linear poly(ethylene glycol) amphiphiles trigger the lectin pathway of the complement through both l-ficolin and mannan-binding lectin recognition. The lectin pathway activation, however, did not trigger the amplification loop of the alternative pathway. An amphiphile with branched poly(ethylene glycol) architecture also activated the lectin pathway but only through l-ficolin recognition. Importantly, this mode of activation neither generated anaphylatoxins nor induced triggering of the effector arm of the complement system. These observations provide a major step toward nanomaterial surface modification with polymers that have the properties to significantly improve innate immunocompatibility by limiting the formation of complement C3 and C5 convertases.  

6.132           High-Resolution Length Fractionation of Surfactant-Dispersed Carbon Nanotubes

Khripin, C.Y., Tu, X., Heddleston, J.M., Silvera-Batista, C., Hight Walker, A.R., Fagan, J. and Zheng, M. Anal. Chem., 85(3), 1382-1388 (2013)   Length fractionation of colloidal single-wall carbon nanotube (SWCNT) dispersions is required for many studies. Size-exclusion chromatography (SEC) has been developed as a reliable method for high-resolution length fractionation of DNA-dispersed SWCNTs but has not been applied to surfactant-dispersed SWCNTs due to their lower dispersion stability and tendency to adsorb onto SEC stationary phases. Here, we report that SEC length fractionation can be achieved for bile salt dispersed SWCNTs by using porous silica-based beads as the stationary phase and bile salt solution as the mobile phase. We demonstrate that the SEC length sorting method can be combined with existing ultracentrifugation SWCNT sorting methods to produce “orthogonally sorted” samples, including length sorted semiconducting SWCNTs, which are important for electronics applications as well as length sorted empty-core SWCNTs. Importantly, we show that unlike simple length fractionation by SEC or any other method, orthogonal sorting produces samples of consistent quality for different length fractions, with similar UV–vis-nearIR absorption and Raman spectral features.  

6.133           Scalable and Effective Enrichment of Semiconducting Single-Walled Carbon Nanotubes by a Dual Selective Naphthalene-Based Azo Dispersant

Sundramoorthy, A.K., Mesgari, S., Wang, J., Kumar, R., Sk, M.A,m Yeap, S.H., Zhang, Q., Sze, S.K., Lim, K.-H. and Chan-Park, M.B.

1. Am. Chem. Soc., 135(15), 5569-5581 (2013)

  Semiconducting single-walled carbon nanotubes (s-SWNTs) have emerged as a promising class of electronic materials, but the metallic (m)-SWNTs present in all as-synthesized nanotube samples must be removed for many applications. A high selectivity and high yield separation method has remained elusive. A separation process based on selective chemistry appears to be an attractive route since it is usually relatively simple, but more effective chemicals are needed. Here we demonstrate the first example of a new class of dual selective compounds based on polycyclic aromatic azo compounds, specifically Direct Blue 71 (I), for high-purity separation of s-SWNTs at high yield. Highly enriched ( 93% purity) s-SWNTs are produced through the simple process of standing arc-discharge SWNTs with I followed by centrifugation. The s-SWNTs total yield is up to 41%, the highest yet reported for a solution-based separation technique that demonstrates applicability in actual transistors. 91% of transistor devices fabricated with these s-SWNTs exhibited on/off ratios of 10³ to 10⁵ with the best devices showing mobility as high as 21.8 cm²/V s with on/off ratio of 10⁴. Raman and X-ray photoelectron spectroscopic shifts and ultraviolet–visible–near-infrared (UV–vis–NIR) show that I preferentially complexes with s-SWNTs and preferentially suspends them. Preferential reaction of naphthyl radicals (generated from I with ultrasonication) with m-SWNTs is confirmed by changes in the D-band in the Raman spectroscopy, matrix-assisted desorption–ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and molecular simulation results. The high selectivity of I stems from its unique dual action as both a selective dispersion agent and the generator of radicals which preferentially attack unwanted metallic species.  

6.134           Probing Carbon Nanotube–Surfactant Interactions with Two-Dimensional DOSY NMR

Shastry, T.A., Morris-Cohen, A.J., Weiss, E.A. and hersam, M.C.

1. Am. Chem. Soc., 135(18), 6750-6753 (2013)

  Two-dimensional diffusion ordered spectroscopy (2D DOSY) NMR was used to probe the micellar structure of sodium dodecyl sulfate (SDS) and sodium cholate (SC) in aqueous solutions with and without semiconducting and metallic single-walled carbon nanotubes (SWCNTs). The solutions contain SDS and SC at weight ratios of 1:4 and 3:2, the ratios commonly used to isolate semiconducting and metallic SWCNTs through density gradient ultracentrifugation (DGU). These results show that the coverage of surfactant on the semiconducting and metallic SWCNTs is nearly identical in the 1:4 surfactant mixture, and a lower degree of bundling is responsible for the greater buoyancy of semiconducting SWCNTs. In the 3:2 surfactant mixture, the metallic SWCNTs are only encapsulated in SC while the semiconducting SWCNTs remain encapsulated in a poorly packed two-surfactant micelle, leading to a large buoyant density difference between the electronic species. This work provides insight into future directions to increase the purity of semiconducting and metallic SWCNTs sorted through DGU and demonstrates the utility of 2D DOSY NMR in probing SWCNT–surfactant complexes.  

6.135           Spontaneous Partition of Carbon Nanotubes in Polymer-Modified Aqueous Phases

Khripin, C.Y., Fagan, J.A. and Zheng, M.

1. Am. Chem. Soc., 135(18), 6822-6825 (2013)

  The distribution of nanoparticles in different aqueous environments is a fundamental problem underlying a number of processes, ranging from biomedical applications of nanoparticles to their effects on the environment, health, and safety. Here, we study distribution of carbon nanotubes (CNTs) in two immiscible aqueous phases formed by the addition of polyethylene glycol (PEG) and dextran. This well-defined model system exhibits a strikingly robust phenomenon: CNTs spontaneously partition between the PEG- and the dextran-rich phases according to nanotube’s diameter and metallicity. Thermodynamic analysis suggests that this chirality-dependent partition is determined by nanotube’s intrinsic hydrophobicity and reveals two distinct regimes in hydrophobicity-chirality relation: a small diameter (<1 nm) regime, where curvature effect makes larger diameter tubes more hydrophobic than small diameter ones, and a large diameter (>1.2 nm) regime, where nanotube’s polarizability renders semiconducting tubes more hydrophobic than metallic ones. These findings reveal a general rule governing CNT behaviors in aqueous phase and provide an extremely simple way to achieve spatial separation of CNTs by their electronic structures.  

6.136           SERS Tags: Novel Optical Nanoprobes for Bioanalysis

Wang, Y., Yan, B. and Chen, L. Chemical Reviews, 113(3), 1391-1428 (2013)   No abstract available.  

6.137           Analyzing Surfactant Structures on Length and Chirality Resolved (6,5) Single-Wall Carbon Nanotubes by Analytical Ultracentrifugation

Fagan, J.A., Zheng, M., Rastogi, V., Simpson, J.R., Khripin, C.Y., Batista, C.A.S. and Walker, A.R.H. ASC Nano, 7(4), 3373-3387 (2013)   The structure and density of the bound interfacial surfactant layer and associated hydration shell were investigated using analytical ultracentrifugation for length and chirality purified (6,5) single-wall carbon nanotubes (SWCNTs) in three different bile salt surfactant solutions. The differences in the chemical structures of the surfactants significantly affect the size and density of the bound surfactant layers. As probed by exchange of a common parent nanotube population into sodium deoxycholate, sodium cholate, or sodium taurodeoxycholate solutions, the anhydrous density of the nanotubes was least for the sodium taurodeoxycholate surfactant, and the absolute sedimentation velocities greatest for the sodium cholate and sodium taurodeoxycholate surfactants. These results suggest that the thickest interfacial layer is formed by the deoxycholate, and that the taurodeoxycholate packs more densely than either sodium cholate or deoxycholate. These structural differences correlate well to an observed 25% increase in fluorescence intensity relative to the cholate surfactant for deoxycholate and taurodeoxycholate dispersed SWCNTs displaying equivalent absorbance spectra. Separate sedimentation velocity experiments including the density modifying agent iodixanol were used to establish the buoyant density of the (6,5) SWCNT in each of the bile salt surfactants; from the difference in the buoyant and anhydrous densities, the largest hydrated diameter is observed for sodium deoxycholate. Understanding the effects of dispersant choice and the methodology for measurement of the interfacial density and hydrated diameter is critical for rationally advancing separation strategies and applications of nanotubes.  

6.138           Ultra-Low Doses of Chirality Sorted (6,5) Carbon Nanotubes for Simultaneous Tumor Imaging and Photothermal Therapy

Antaris, A.L., Robinson, J.T., Yaghi, O.K., Hong, g., Diao, S., Luong, R. and Dai, H. ACS Nano, 7(4), 3644-3652 (2013)   Single-walled carbon nanotubes (SWCNTs) exhibit intrinsic fluorescence and strong optical absorption in the near-infrared (NIR) biological window (0.7–1.4 μm), rendering them ideal for in vivo imaging and photothermal therapy. Advances in SWCNT sorting have led to improved nanoelectronics and are promising for nanomedicine. To date, SWCNTs used in vivo consist of heterogeneous mixtures of nanotubes and only a small subset of chirality nanotubes fluoresces or heats under a NIR laser. Here, we demonstrate that separated (6,5) SWCNTs exchanged into a biocompatible surfactant, C₁₈-PMH-mPEG, are more than 6-fold brighter in photoluminescence on the per mass basis, afford clear tumor imaging, and reach requisite photothermal tumor ablation temperatures with a >10-fold lower injected dose than as-synthesized SWCNT mixtures while exhibiting relatively low (6,5) accumulation in the reticuloendothelial system. The intravenous injection of 4 μg of (6,5) SWCNTs per mouse (0.254 mg/kg) for dual imaging/photothermal therapy is, by far, the lowest reported dose for nanoparticle-based in vivo therapeutics.  

6.139           Ultrafast Charge Photogeneration in Semiconducting Carbon Nanotubes

Soavi, G., Scotognella, F., Brida, D., Hefner, ST., Späth, f., Antognazza, M.R., Hertel, T., Lanzani, G. and Cerullo, G.

1. Phys. Chem. C., 117(20), 10849-10855 (2013)

  We show that excitons are not the unique outcome of photoexcitation in single-walled carbon nanotubes (SWNTs). Our experiments of transient photoinduced absorption suggest that charge carriers are formed with quantum yield of a few percent and that such species strongly affect the long-lived transient spectrum. Photogenerated charge carriers induce strong local electric fields that shift by the Stark effect the second subband exciton absorption in SWNTs, resulting in a characteristic derivative shape of the transient absorption spectra.  

6.140           Aerosol Jet Printed, Low Voltage, Electrolyte Gated Carbon Nanotube Ring Oscillators with Sub-5 μs Stage Delays

Ha, M., Seo, J-W.T., Prabhumirashi, P.L., Zhang, W., Geier, M.L., Renn, M.J., Kim, C.H., Hersam, M.C. and Frisbie, C.D. Nano Lett., 13(3), 954-960 (2013)   A central challenge for printed electronics is to achieve high operating frequencies (short transistor switching times) at low supply biases compatible with thin film batteries. In this report, we demonstrate partially printed five-stage ring oscillators with >20 kHz operating frequencies and stage delays <5 μs at supply voltages below 3 V. The fastest ring oscillator achieved 1.2 μs delay time at 2 V supply. The inverter stages in these ring oscillators were based on ambipolar thin film transistors (TFTs) employing semiconducting, single-walled carbon nanotube (CNT) networks and a high capacitance ( 1 μF/cm²) ion gel electrolyte as the gate dielectric. All materials except the source and drain electrodes were aerosol jet printed. The TFTs exhibited high electron and hole mobilities ( 20 cm²/(V s)) and ON/OFF current ratios (up to 10⁵). Inverter switching times t were systematically characterized as a function of transistor channel length and ionic conductivity of the gel dielectric, demonstrating that both the semiconductor and the ion gel play a role in switching speed. Quantitative scaling analysis suggests that with suitable optimization low voltage, printed ion gel gated CNT inverters could operate at frequencies on the order of 1 MHz.  

6.141           Separation of graphene oxide by density gradient centrifugation and study on their morphology-dependent electrochemical properties

Li, S., Zhu, F., Meng, F., Li, H., Wang, L., Zhao, J., Yue, Q., Liu, J. and Jia, J.

1. Electroanal. Chem., 703, 135-145 (2013)

  Graphene oxide (GO) made from graphite oxidation by Hummer’s method is a mixture of GO sheets with different size and thickness. Previous work has shown that these GO sheets have different electrophoretic properties and can be separated via capillary electrophoresis according to their surface charge (J. Zhao et al., Anal. Chem. 83 (2011) 9100–9106). Herein, the GO sheets were separated by density gradient centrifugation to achieve preparation of bulk GO materials. GO sheets were separated by their thicknesses and the as-obtained sheets were characterized for their spectroscopic, electronic and electrochemical properties. After treated by hydrazine, GO was derivatized with N element and become active toward oxygen reduction reaction. The thin layer GO sheets incorporated with more hydrazine and the atomic ratio of N/C was higher than thick layer GO sheets. This work suggested that the physicochemical properties of GO sheets were N of layer related and it is necessary to separate GO sheets to prepare suitable materials for device fabrication or catalysts use.  

6.142           Nanoparticles as macromolecules

Miller, J.B. and Hobbie, E.K.

1. Polymer Science Part B, 51, 1195-1208 (2013)

  A review of recent trends in the dispersion, purification, and assembly of colloidal nanoparticles highlights a number of growing analogies with ideas borrowed from polymer science. Beyond the similar scales of size, several key concepts lying at the foundation of polymer physics—such as polydispersity, fractionation, phase ordering, and viscoelasticity—are taking on new and unique significance in the contemporary realm of nanotechnology. Leveraging “soft matter” at the nanoscale to simplify materials processing and improve material performance is becoming a reality, with potentially profound implications for a number of emerging technologies.  

6.143           Sorting Nanoparticles by Centrifugal Fields in Clean Media

Bonaccorso, F., Zerbetto, M., Ferrari, A.C. and Amendola, V.

1. Phys. Chem. C, 117(25), 13217-13229 (2013)

  The on-demand availability of nanomaterials with selected size and well-defined chemical/physical properties is of fundamental importance for their widespread application. We report two clean, rapid, and non-destructive approaches for nanoparticle (NP) size selection in centrifugal fields. The first exploits rate zonal separation in a high viscosity gradient. The second exploits selective sedimentation of NPs with different sizes. These methods are here applied to metallic nanoparticles (MNPs) with different compositions and surface chemistry, dispersed either in water or organic solvents. The approach is general and can also be exploited for the separation of NPs of any material. We selectively sort both Au and AgNPs with sizes in the 10–30 nm range, achieving chemical-free MNPs with low polydispersivity. We do not use solutes, thus avoiding contamination, and only require low centrifugal fields, easily achievable in benchtop systems.  

6.144           Raman Spectroscopic Investigation of Individual Single-Walled Carbon Nanotubes Helically Wrapped by Ionic, Semiconducting Polymers

Bonhommeau, S., Deria, P., Glesner, M.G., Talaga, D., Najjar, S., Belin, C., Auneau, L., trainini, S., Therien, M.J. and Rodriguez, V.

1. Phys. Chem. C, 117(28), 14840-14849 (2013)

  Raman-active vibrational modes of (6,5) chirality-enriched single-walled carbon nanotubes (SWNTs), helically wrapped by semiconducting poly[2,6-{1,5-bis(3-propoxysulfonic acid sodium salt)}naphthylene]ethynylene (PNES), are described in great detail. At an irradiation wavelength of 568.2 nm, the extent to which the environment impacts the nanotube vibrational signature can be probed; in particular, the absence of a G band shift for PNES–[(6,5) SWNT] samples relative to benchmark surfactant-coated nanotubes indicates the lack of any significant charge transfer between the PNES strand and the SWNT skeleton, but electronic spectra provide compelling evidence for polymer-to-SWNT energy transfer. At an irradiation wavelength of 457.9 nm, vibrational modes associated with PNES chains that wrap (6,5) SWNTs are conspicuously enhanced. Under 514.5 nm irradiation, PNES–[(6,5) SWNTs] are not excited in resonance but G and G′ bands associated with these nanohybrids are strongly enhanced, reflecting the excitation of a multiphonon-mediated vibronic transition of the (6,5) SWNT backbone. At a 488.0 nm irradiation wavelength, Raman spectral signatures of both the PNES polymer and the vibronically excited (6,5) SWNT skeleton through one-phonon-assisted processes are pronounced, demonstrating that a specific SWNT chirality and the corresponding semiconducting polymer helically wrapped about its surface can be probed using an excitation wavelength that does not resonantly excite the SWNT structure.  

6.145           Solution-processable exfoliated zeolite nanosheets purified by density gradient centrifugation

Agrawal, K.V., Topuz, B., Jiang, Z., Nguenkam, K., Elyassi, B., Francis, L.F. and Tsapatsis, M. AICHE J., 59(9), 3458-3467 (2013)   Highly crystalline exfoliated MFI-nanosheets can pave the way for large-scale deployment of sub-500-nm zeolite membranes due to their processing and packing advantages. Exfoliated MFI-nanosheets prepared by melt compounding contain a large amount of polymer and unexfoliated particles which are detrimental to the fabrication of ultrathin zeolite membranes. Complete removal of polystyrene from the nanosheet suspension in toluene is demonstrated by centrifugation of the suspension across chlorobenzene as confirmed by thermogravimetric analysis (TGA) data and transmission electron microscopy (TEM) images. Rate-zonal centrifugation in a nonlinear density gradient fractionated exfoliated MFI-nanosheets from unexfoliated particles. The purified nanosheets were highly crystalline as indicated by high-resolution TEM (HRTEM) and electron diffraction (ED). Coating of purified MFI-nanosheets on a smooth -alumina support, fabricated by filtration of -alumina suspension, led to a compact, b-oriented, 80-nm-thick film. A mild hydrothermal treatment of the film led to a 200-nm-thick membrane, which demonstrated molecular sieving properties.  

6.146           Diameter Refinement of Semiconducting Arc Discharge Single-Walled Carbon Nanotubes via Density Gradient Ultracentrifugation

Seo, J-W.T., Yoder, N.L., Shastry, T.A., Humes, J.J., Johns, J.E., Green, A.A. and hersam, M.C.

1. Phys. Chem. Lett., 4(17), 2805-2810 (2013)

  Arc discharge single-walled carbon nanotubes (SWCNTs) possess superlative optical and electronic properties that are of high interest for technologically important applications including fiber optic communications, biomedical imaging, and field-effect transistors. However, as-grown arc discharge SWCNTs possess a mixture of metallic and semiconducting species in addition to a wide diameter distribution (1.2 to 1.7 nm) that limit their performance in devices. While previous postsynthetic sorting efforts have achieved separation by electronic type and diameter refinement for metallic arc discharge SWCNTs, tight diameter distributions of semiconducting arc discharge SWCNTs have not yet been realized. Herein, we present two advances in density gradient ultracentrifugation that enable the isolation of high purity (>99%) semiconducting arc discharge SWCNTs with narrow diameter distributions centered at 1.6 and 1.4 nm. The resulting diameter-refined populations of semiconducting arc discharge SWCNTs possess monodisperse characteristics that are well-suited for high-performance optical and electronic technologies.  

6.147           Centrifugal Shape Sorting and Optical Response of Polyhedral Gold Nanoparticles

Shin, Y.J., Ringe, E., Personick, M.L., Cardinal, M.F., Mirkin, C.A., Marks, L.D., Van Duyne, R.P. and Hersam, M.C. Adv. Mater., 25(29), 4023-4027 (2013)   A centrifugal route for separating small {110}-faceted gold nanostructures, namely rhombic dodecahedra (RD) and triangular bipyramids (BPs), which form simultaneously during synthesis and cannot be separated by means of conventional filtration methods, is presented. The centrifuged solution shows two distinct bands: i) RD and ii) BPs, as verified in the corresponding scanning electron microscopy images. The sorted BPs show a refractive index dependence 2.5 times that of the as-synthesized, unsorted mixture.  

6.148           Growth of carbon nanotubes via twisted graphene nanoribbons

Lim, H.E., Miyata, Y., Kitaura, R., Nishimura, Y., Nishimoto, Y., Irle, S.A., Warner, J.H., Kataura, H. and Shinohara, H. Nature Communications, 4:2485 (2013)   Carbon nanotubes have long been described as rolled-up graphene sheets. It is only fairly recently observed that longitudinal cleavage of carbon nanotubes, using chemical, catalytical and electrical approaches, unzips them into thin graphene strips of various widths, the so-called graphene nanoribbons. In contrast, rolling up these flimsy ribbons into tubes in a real experiment has not been possible. Theoretical studies conducted by Kit et al. recently demonstrated the tube formation through twisting of graphene nanoribbon, an idea very different from the rolling-up postulation. Here we report the first experimental evidence of a thermally induced self-intertwining of graphene nanoribbons for the preferential synthesis of (7, 2) and (8, 1) tubes within parent-tube templates. Through the tailoring of ribbon’s width and edge, the present finding adds a radically new aspect to the understanding of carbon nanotube formation, shedding much light on not only the future chirality tuning, but also contemporary nanomaterials engineering.  

6.149           Subnanowatt Carbon Nanotube Complementary Logic Enabled by Threshold Voltage Control

Geier, M.L., Prabhumirashi, P., McMorrow, J.J., Xu, W., Seo, J-W.T., Everaerts, K., Kim, C.H., Marks, T.J. and Hersam, M.C. Nano Lett., 13(10), 4810-4814 82013)   In this Letter, we demonstrate thin-film single-walled carbon nanotube (SWCNT) complementary metal-oxide-semiconductor (CMOS) logic devices with subnanowatt static power consumption and full rail-to-rail voltage transfer characteristics as is required for logic gate cascading. These results are enabled by a local metal gate structure that achieves enhancement-mode p-type and n-type SWCNT thin-film transistors (TFTs) with widely separated and symmetric threshold voltages. These complementary SWCNT TFTs are integrated to demonstrate CMOS inverter, NAND, and NOR logic gates at supply voltages as low as 0.8 V with ideal rail-to-rail operation, subnanowatt static power consumption, high gain, and excellent noise immunity. This work provides a direct pathway for solution processable, large area, power efficient SWCNT advanced logic circuits and systems.  

6.150           Plasmonic Nature of the Terahertz Conductivity Peak in Single-Wall Carbon Nanotubes

Zhang, Q., Haroz, E.H., Jin, Z., Ren, L., Wang, X., Arvidson, R.S., Lüttge, A. and Kono, J. Nano Lett., 13(12), 5991-5996 (2013)   Plasmon resonance is expected to occur in metallic and doped semiconducting carbon nanotubes in the terahertz frequency range, but its convincing identification has so far been elusive. The origin of the terahertz conductivity peak commonly observed for carbon nanotube ensembles remains controversial. Here we present results of optical, terahertz, and direct current (DC) transport measurements on highly enriched metallic and semiconducting nanotube films. A broad and strong terahertz conductivity peak appears in both types of films, whose behaviors are consistent with the plasmon resonance explanation, firmly ruling out other alternative explanations such as absorption due to curvature-induced gaps.  

6.151           Purification, separation and extraction of inner tubes from double-walled carbon nanotubes by tailoring density gradient ultracentrifugation using optical probes

Rohringer, P., Shi, L., Liu, X., Yanagi, K. and Pichler, T. Carbon, 74, 282-290 (2014)   We studied the effect of varying sonication and centrifugation parameters on double-walled carbon nanotubes (DWCNT) by measuring optical absorption and photoluminescence (PL) of the samples. We found that by using a low sonication intensity before applying density gradient ultracentrifugation (DGU), only inner tube species with a diameter ⩽⩽0.8 nm can be identified in absorption measurements. This is in stark contrast to the result after sonicating at higher intensities, where also bigger inner tubes can be found. Furthermore, by comparing PL properties of samples centrifugated either with or without a gradient medium, we found that applying DGU greatly enhances the PL intensity, whereas centrifugation at even higher speeds but without a gradient medium results in lower intensities. This can be explained by extraction of inner tubes from their host outer tubes in a two-stage process: the different shearing forces from the sonication treatments result in some DWCNT to be opened, whereas others stay uncut. A subsequent application of DGU leads to the extraction of the inner tubes or not if the host nanotube stayed uncut or no gradient medium was used. This work shows a pathway to avoid this phenomenon to unravel the intrinsic PL from inner tubes of DWCNT.  

6.152           Influence of Electronic Type Purity on the Lithiation of Single-Walled Carbon Nanotubes

Jaber-Ansari, L., Iddir, H., Curtiss, L.A. and Hersam, M.C. ACS Nano, 8(3), 2399-2409 (2014)   Single-walled carbon nanotubes (SWCNTs) have emerged as one of the leading additives for high-capacity nanocomposite lithium ion battery electrodes due to their ability to improve electrode conductivity, current collection efficiency, and charge/discharge rate for high power applications. However, since as-grown SWCNTs possess a distribution of physical and electronic structures, it is of high interest to determine which subpopulations of SWCNTs possess the highest lithiation capacity and to develop processing methods that can enhance the lithiation capacity of underperforming SWCNT species. Toward this end, SWCNT electronic type purity is controlled via density gradient ultracentrifugation, enabling a systematic study of the lithiation of SWCNTs as a function of metal versus semiconducting content. Experimentally, vacuum-filtered freestanding films of metallic SWCNTs are found to accommodate lithium with an order of magnitude higher capacity than their semiconducting counterparts, which is consistent with ab initio molecular dynamics and density functional theory calculations in the limit of isolated SWCNTs. In contrast, SWCNT film densification leads to the enhancement of the lithiation capacity of semiconducting SWCNTs to levels comparable to metallic SWCNTs, which is corroborated by theoretical calculations that show increased lithiation of semiconducting SWCNTs in the limit of small SWCNT–SWCNT spacing. Overall, these results will inform ongoing efforts to utilize SWCNTs as conductive additives in nanocomposite lithium ion battery electrodes.    

6.153           Etching of Surfactant from Solution-Processed, Type-Separated Carbon Nanotubes and Impact on Device Behavior

Kane, A.A., Ford, A.C., Nissen, A., Krafcik, K.L. and Leonard, F. ACS Nano, 8(3), 2477-2485 (2014)   Semiconducting single-walled carbon nanotubes (SWCNTs) have great potential for use in electronic and optoelectronic devices. However, methods for synthesizing SWCNTs produce a mixture of metallic and semiconducting materials, which require additional processing to separate by electronic type. Purification and enrichment of the semiconducting fraction is readily achieved by using the centrifugation of aqueous suspensions of SWCNTs with the help of surfactants, but this leaves residual surfactant on the SWCNT surface that can impact their electronic and optical properties. Here, we present a detailed study of the sodium taurodeoxycholate (STDC) surfactant removal process during vacuum annealing, showing that it occurs through fragmentation of the surfactant, and that complete removal requires exceedingly high temperatures, which indicates strong binding to the SWCNTs. We then present an approach based on air oxidation and mild annealing to completely remove the surfactant while maintaining the SWCNT properties. Using this approach, we compare single SWCNT electronic devices with and without STDC and show that, despite the very strong surfactant binding, it does not affect device performance substantially.  

6.154           Rod Hydrodynamics and Length Distributions of Single-Wall Carbon Nanotubes Using Analytical Ultracentrifugation

Batista, C.A.S., Zheng, M., Khripin, C.Y., Tu, X. and Fagan, J.A. Langmuir, 30(17), 4895-4904 (2014)   Because of their repetitive chemical structure, extreme rigidity, and the separability of populations with varying aspect ratio, SWCNTs are excellent candidates for use as model rodlike colloids. In this contribution, the sedimentation velocities of length and density sorted single-wall carbon nanotubes (SWCNTs) are compared to predictions from rod hydrodynamic theories of increasing complexity over a range of aspect ratios from <50 to >400. Independently measuring all contributions to the sedimentation velocity besides the shape factor, excellent agreement is found between the experimental findings and theoretical predictions for numerically calculated hydrodynamic radius values and for multiterm analytical expansion approximations; values for the hydrodynamic radii in these cases are additionally found to be consistent with the apparent hydrated particle radius determined independently by buoyancy measurements. Lastly, we utilize this equivalency to calculate the apparent distribution of nanotube lengths in each population from their sedimentation coefficient distribution without adjustable parameters, achieving excellent agreement with distributions from atomic force microscopy. The method developed herein provides an alternative for the ensemble measurement of SWCNT length distributions and others rodlike particles.  

6.155           Structure-Dependent Mitochondrial Dysfunction and Hypoxia Induced with Single-Walled Carbon Nanotubes

Wang, L-R., Xue, X., Hu, X-M., Wei, M-Y., Zhang, C-Q., Ge, G-L. and Liang, X-J. Small, 10(14), 2859-2869 (2014)   Cytotoxicity of nanomaterials on living systems is known to be affected by their size, shape, surface chemistry, and other physicochemical properties. Exposure to a well-characterized subpopulation of specific nanomaterials is therefore desired to reveal more detailed mechanisms. This study develops scalable density gradient ultracentrifugation sorting of highly dispersed single-walled carbon nanotubes (SWNTs) into four distinct bands based on diameter, aggregation, and structural integrity, with greatly improved efficiency, yield, and reproducibility. With guarantee of high yield and stability of four SWNT fractions, it is possible for the first time, to investigate the structure-dependent bioeffects of four SWNT fractions. it is possible Among these, singly-dispersed integral SWNTs show no significant effects on the mitochondrial functions and hypoxia. The aggregated integral SWNTs show more significant effects on the mitochondrial dysfunction and hypoxia compared to the aggregated SWNTs with poor structure integrity. Then, it is found that the aggregated integral SWNTs induced the irregular mitochondria respiratory and pro-apoptotic proteins activation, while aggregated SWNTs with poor structure integrity greatly enhanced reactive oxygen species (ROS) levels. This work supports the view that control of the distinct structure characteristics of SWNTs helps establish clearer structure-bioeffect correlation and health risk assessment. It is also hoped that these results can help in the design of nanomaterials with higher efficiency and accuracy in subcellular translocation.  

6.156           High-Yield, Single-Step Separation of Metallic and Semiconducting SWCNTs Using Block Copolymers at Low Temperatures

Homenick, C.M., Rousina-Webb, A., Cheng, F., Jakubinek, M.B., Malenfant, P.R.L. and Simard, b.

J. Phys. Chem. C, 118(29), 16156-16164 (2014)

  Electronic type separation of SWCNT material is necessary to facilitate the development of carbon nanotube electronics. A convenient, high-yield, single-step separation of metallic and semiconducting SWCNTs has been developed using block copolymers and density gradient ultracentrifugation. In particular by varying the centrifugation temperature and dissolved oxygen content under acidic conditions, extraction efficiencies of up to 65% were achieved with both metallic and semiconducting SWCNT electronic purity exceeding 99% as determined by absorption spectroscopy. It was demonstrated that lowering the temperature during the DGU separation, which is expected to increase the difference in densities between metallic and semiconducting nanotube complexes, results in higher purity and yield. Semiconducting and metallic bands are separated simply with a disposable pipet such that specialized fractioning equipment is not required for effective isolation of enriched SWCNTs.  

6.157           Top-Down Patterning and Self-Assembly for Regular Arrays of Semiconducting Single-Walled Carbon Nanotubes

Wu, J., Antaris, A., Gong, M. and Dai, H. Adv. Mater., 26(35), 6151-6156 (2014)   Highly pure semiconducting single-walled carbon nanotubes (SWNTs), sorted by density-gradient ultracentrifugation, undergo self-assembly using depletion attraction forces into rafts along lithographically defined patterns of narrow pitch (100 or 200 nm). The arrays demonstrate high pattern fidelity and channel filling, along with large-scale homogeneity. Field-effect transistors made from these arrays exhibit high performance at on/off ratios > 1000.  

6.158           Fringing-field dielectrophoretic assembly of ultrahigh-density semiconducting nanotube arrays with a self-limited pitch

Cao, Q., Han, S-j. and Tulevski, G. Nature Communications, 5:5071 (2014)   One key challenge of realizing practical high-performance electronic devices based on single-walled ​carbon nanotubes is to produce electronically pure nanotube arrays with both a minuscule and uniform inter-tube pitch for sufficient device-packing density and homogeneity. Here we develop a method in which the alternating voltage-fringing electric field formed between surface microelectrodes and the substrate is utilized to assemble semiconducting nanotubes into well-aligned, ultrahigh-density and submonolayered arrays, with a consistent pitch as small as 21±6 nm determined by a self-limiting mechanism, based on the unique field focusing and screening effects of the fringing field. Field-effect transistors based on such nanotube arrays exhibit record high device transconductance (>50 μS μm⁻¹) and decent on current per nanotube (~1 μA per tube) together with high on/off ratios at a drain bias of −1 V.  

6.159           Thickness sorting of two-dimensional transition metal dichalcogenides via copolymer-assisted density gradient ultracentrifugation

Kang, J., Seo, J-W.T., Alducin, D., Ponce, A., Yacaman, M.J. and Hersam, M.C. Nature Communications, 5:5478 (2014)   Two-dimensional transition metal dichalcogenides have emerged as leading successors to graphene due to their diverse properties, which depend sensitively on sample thickness. Although solution-based exfoliation methods hold promise for scalable production of these materials, existing techniques introduce irreversible structural defects and/or lack sufficient control over the sample thickness. In contrast, previous work on ​carbon nanotubes and graphene has shown that isopycnic density gradient ultracentrifugation can produce structurally and electronically monodisperse nanomaterial populations. However, this approach cannot be directly applied to transition metal dichalcogenides due to their high intrinsic buoyant densities when encapsulated with ionic small molecule surfactants. Here, we overcome this limitation and thus demonstrate thickness sorting of pristine ​molybdenum disulfide (​MoS₂) by employing a block copolymer dispersant composed of a central hydrophobic unit flanked by hydrophilic chains that effectively reduces the overall buoyant density in aqueous solution. The resulting solution-processed monolayer ​MoS₂ samples exhibit strong photoluminescence without further chemical treatment.  

6.160           High Precision Fractionator for Use with Density Gradient Ultracentrifugation

Kadria-Vili, Y., Canning, g., Bachilo, S.M. and Weisman, R.B. Anal. Chem., 86(22), 11018-11023 (2014)   The recent application of density gradient ultracentrifugation (DGU) for structural sorting of single-walled carbon nanotube samples has created a need for highly selective extraction of closely spaced layers formed in the centrifuged tube. We describe a novel computer-controlled device designed for this purpose. Through the use of fine needles, systematic needle motions, and slow flow rates, multiple sample layers can be aspirated under program control with minimal cross contamination between layers. The fractionator’s performance is illustrated with DGU-sorted samples of single-walled carbon nanotubes.  

6.161           Diameter dependence of the optoelectronic properties of single walled carbon nanotubes determined by ellipsometry

Battie, Y., Broch, L., En Naciri, A., Lauret, J-S., Guezo, M. and Loiseau, A. Carbon, 83, 32-39 (2015)   We report ellipsometric measurement on single walled carbon nanotube (SWCNT) films performed in a large spectral range from 0.07 to 4.97 eV. The complex dielectric functions of SWCNTs are correlated to their diameter distribution extracted from transmission electron microscopy. Here we show that the transition energies between Van Hove singularities are directly related to the strong one dimensional confinement. In the infrared spectral range, the real part of the dielectric function becomes negative. The electronic properties of SWCNTs are extracted from ellipsometry by using a Drude model. The mobility and the mean free path of charge carriers are limited by the high number of SWCNT contacts. In accordance with tight binding simulation, the conductivity and the charge carrier concentration increase with the SWCNT diameter. Finally, we demonstrate that the π-plasmon energy depends on the charge carrier concentration.  

6.162           Separation of colloidal two dimensional materials by density gradient ultracentrifugation

Kuang, Y., Song, S., Huang, J. and Sun, X.

1. Solid State Chem., 224, 120-126 (2015)

  Two-dimensional (2D) materials have been made through various approaches but obtaining monodispersed simply by synthesis optimization gained little success, which highlighted the need for introducing nanoseparation methods. Density gradient ultracentrifugation method has emerged as a versatile and scalable method for sorting colloidal 2D nanomaterials. Isopycnic separation was applied on thickness-dependent separation of graphene nanosheets. And rate-zonal separation, as a more versatile separation method, demonstrated its capability in sorting nanosheets of chemically modified single layered graphene, layered double hydroxide, and even metallic Ag. Establishing such density gradient ultracentrifugation method not only achieves monodispersed nanosheets and provides new opportunities for investigation on size dependent properties of 2D materials, but also makes the surface modification possible by introducing “reaction zones” during sedimentation of the colloids.  

6.163           Determination of the Lateral Dimension of Graphene Oxide Nanosheets Using Analytical Ultracentrifugation

Walter, J., Nacken, T.J., Damm, C., Thajudeen, T., Eigler, S. and Peukert, W. Small, 11(7), 814-825 (2015)   In this paper, a method to determine the lateral dimensions of 2D nanosheets directly in suspension by analytical ultracentrifugation (AUC) is shown. The basis for this study is a well-characterized and stable dispersion of graphene oxide (GO) monolayers in water. A methodology is developed to correlate the sedimentation coefficient distribution measured by AUC with the lateral size distribution of the 2D GO nanosheets obtained from atomic force microscopy (AFM). A very high accuracy can be obtained by virtue of counting several thousand sheets, thereby minimizing any coating effects or statistical uncertainties. The AFM statistics are further used to fit the lateral size distribution obtained from the AUC to determine the unknown hydrodynamic sheet thickness or density. It is found that AUC can derive nanosheet diameter distributions with a relative error of the mean sheet diameter of just 0.25% as compared to the AFM analysis for 90 mass% of the particles in the distribution. The standard deviation of the size-dependent error for the total distribution is found to be 3.25%. Based on these considerations, an expression is given to calculate the cut size of 2D nanosheets in preparative centrifugation experiments.  

6.164           High energetic excitons in carbon nanotubes directly probe charge-carriers

Soavi, G., Scotognella, F., Viola, D., Hefner, T., Hertel, T., Cerullo, G. and Lanzani, G. Scientific Reports, 5:9681 (2015)   Theory predicts peculiar features for excited-state dynamics in one dimension (1D) that are difficult to be observed experimentally. Single-walled carbon nanotubes (SWNTs) are an excellent approximation to 1D quantum confinement, due to their very high aspect ratio and low density of defects. Here we use ultrafast optical spectroscopy to probe photogenerated charge-carriers in (6,5) semiconducting SWNTs. We identify the transient energy shift of the highly polarizable S₃₃ transition as a sensitive fingerprint of charge-carriers in SWNTs. By measuring the coherent phonon amplitude profile we obtain a precise estimate of the Stark-shift and discuss the binding energy of the S₃₃ excitonic transition. From this, we infer that charge-carriers are formed instantaneously (<50 fs) even upon pumping the first exciton, S₁₁. The decay of the photogenerated charge-carrier population is well described by a model for geminate recombination in 1D.  

6.165           Double-Walled Carbon Nanotube Processing

Moore, K.E., Tune, D.D. and Flavel, B.S. Advanced Materials, 27(20), 3105-3137 (2015)   Single-walled carbon nanotubes (SWCNTs) have been the focus of intense research, and the body of literature continues to grow exponentially, despite more than two decades having passed since the first reports. As well as extensive studies of the fundamental properties, this has seen SWCNTs used in a plethora of applications as far ranging as microelectronics, energy storage, solar cells, and sensors, to cancer treatment, drug delivery, and neuronal interfaces. On the other hand, the properties and applications of double-walled carbon nanotubes (DWCNTs) have remained relatively under-explored. This is despite DWCNTs not only sharing many of the same unique characteristics of their single-walled counterparts, but also possessing an additional suite of potentially advantageous properties arising due to the presence of the second wall and the often complex inter-wall interactions that arise. For example, it is envisaged that the outer wall can be selectively functionalized whilst still leaving the inner wall in its pristine state and available for signal transduction. A similar situation arises in DWCNT field effect transistors (FETs), where the outer wall can provide a convenient degree of chemical shielding of the inner wall from the external environment, allowing the excellent transconductance properties of the pristine nanotubes to be more fully exploited. Additionally, DWCNTs should also offer unique opportunities to further the fundamental understanding of the inter-wall interactions within and between carbon nanotubes. However, the realization of these goals has so far been limited by the same challenge experienced by the SWCNT field until recent years, namely, the inherent heterogeneity of raw, as-produced DWCNT material. As such, there is now an emerging field of research regarding DWCNT processing that focuses on the preparation of material of defined length, diameter and electronic type, and which is rapidly building upon the experience gained by the broader SWCNT community. This review describes the background of the field, summarizing some relevant theory and the available synthesis and purification routes; then provides a thorough synopsis of the current state-of-the-art in DWCNT sorting methodologies, outlines contemporary challenges in the field, and discusses the outlook for various potential applications of the resulting material.  

6.166           Bench-top aqueous two-phase extraction of isolated individual single-walled carbon nanotubes

Subbaiyan, N.K., Parra-Vasquez, A.N.G., Cambre, S., Cordoba, M.A.S., Yalcin, S.E:, Hamilton, C.E., Mack, N.H., Blackburn, J.L., Doom, S.K. and Duque, J.G. Nano Res., 8(5), 1755-1769 (2015)   Isolation and purification of single-walled carbon nanotubes (SWCNTs) are prerequisites for their implementation in various applications. In this work, we present a fast (∼5 min), low-cost, and easily scalable bench-top approach to the extraction of high-quality isolated SWCNTs from bundles and impurities in an aqueous dispersion. The extraction procedure, based on aqueous two-phase (ATP) separation, is widely applicable to any SWCNT source (tested on samples up to 1.7 nm in diameter) and independent of defect density, purity, diameter, and length. The extracted dispersions demonstrate that the removal of large aggregates, small bundles, and impurities is comparable to that by density gradient ultracentrifugation, but without the need for high-end instrumentation. Raman and fluorescence-excitation spectroscopy, single-nanotube fluorescence imaging, atomic force and transmission electron microscopy, and thermogravimetric analysis all confirm the high purity of the isolated SWCNTs. By predispersing the SWCNTs without sonication (only gentle stirring), full-length, pristine SWCNTs can be isolated (tested up to 20 μm). Hence, this simple ATP method will find immediate application in the generation of SWCNT materials for all levels of nanotube research and applications, from fundamental studies to high-performance devices.  

6.167           Gold Nanoparticles Stabilized with MPEG-Grafted Poly(l-lysine): in Vitro and in Vivo Evaluation of a Potential Theranostic Agent

Bogdanov, A.A., Gupta, S., Koshkina, N., Corr, S.J., Zhang, S., Curley, S.A. and Han, G. Bioconjugate Chem., 26(1), 39-50 (2015)   As the number of diagnostic and therapeutic applications utilizing gold nanoparticles (AuNPs) increases, so does the need for AuNPs that are stable in vivo, biocompatible, and suitable for bioconjugation. We investigated a strategy for AuNP stabilization that uses methoxypolyethylene glycol-graft-poly(l-lysine) copolymer (MPEG-gPLL) bearing free amino groups as a stabilizing molecule. MPEG-gPLL injected into water solutions of HAuCl₄ with or without trisodium citrate resulted in spherical (Z_av = 36 nm), monodisperse (PDI = 0.27), weakly positively charged nanoparticles (AuNP3) with electron-dense cores (diameter: 10.4 ± 2.5 nm) and surface amino groups that were amenable to covalent modification. The AuNP3 were stable against aggregation in the presence of phosphate and serum proteins and remained dispersed after their uptake into endosomes. MPEG-gPLL-stabilized AuNP3 exhibited high uptake and very low toxicity in human endothelial cells, but showed a high dose-dependent toxicity in epithelioid cancer cells. Highly stable radioactive labeling of AuNP3 with ^99mTc allowed imaging of AuNP3 biodistribution and revealed dose-dependent long circulation in the blood. The minor fraction of AuGNP3 was found in major organs and at sites of experimentally induced inflammation. Gold analysis showed evidence of a partial degradation of the MPEG-gPLL layer in AuNP3 particles accumulated in major organs. Radiofrequency-mediated heating of AuNP3 solutions showed that AuNP3 exhibited heating behavior consistent with 10 nm core nanoparticles. We conclude that PEG-pPLL coating of AuNPs confers “stealth” properties that enable these particles to exist in vivo in a nonaggregating, biocompatible state making them suitable for potential use in biomedical applications such as noninvasive radiofrequency cancer therapy.  

6.168           Solubilization of Single-Walled Carbon Nanotubes Using a Peptide Aptamer in Water below the Critical Micelle Concentration

Li, Z., Kameda, T., Isoshima, T., Kobatake, E., Tanaka, T., Ito, y. and Kawamoto, M. Langmuir, 31(11), 3482-3488 (2015)   The solubilizing ability of single-walled carbon nanotubes (SWCNTs) in water with several dispersants was investigated. Among the dispersants, including low-molecular-weight surfactants, peptides, DNA, and a water-soluble polymer, the peptide aptamer, A2 (IFRLSWGTYFS), exhibited the highest dispersion capability below the critical micelle concentration at a concentration of 0.02 w/v%. The dispersion of supernatant aqueous solution of SWCNTs containing aptamer A2 was essentially unchanged for several months after high-speed ultracentrifugation and gave rise to an efficient and stable dispersion of the SWCNTs in water. From the results of isothermal titration calorimetry and molecular dynamics simulations, the effective binding capability of A2 was due to π–π interaction between aromatic groups in the peptide aptamer and the side walls of SWCNTs. Interestingly, the peptide aptamer showed the possibility of diameter separation of semiconducting SWCNTs using a uniform density gradient ultracentrifuge. These phenomena are encouraging results toward an effective approach to the dispersion and separation of SWCNTs.  

6.169           Isolation of >1 nm Diameter Single-Wall Carbon Nanotube Species Using Aqueous Two-Phase Extraction

Fagan, J.A., haroz, E.H., Ihly, R., Gui, H., Blackburn, J.L., Simpson, J.R., Lam, S., Walker, A.R.H., Doorn, S.K. and Zheng, M. ACS Nano, 9(5), 5377-5390 (2015)   In this contribution we demonstrate the effective separation of single-wall carbon nanotube (SWCNT) species with diameters larger than 1 nm through multistage aqueous two-phase extraction (ATPE), including isolation at the near-monochiral species level up to at least the diameter range of SWCNTs synthesized by electric arc synthesis (1.3–1.6 nm). We also demonstrate that refined species are readily obtained from both the metallic and semiconducting subpopulations of SWCNTs and that this methodology is effective for multiple SWCNT raw materials. Using these data, we report an empirical function for the necessary surfactant concentrations in the ATPE method for separating different SWCNTs into either the lower or upper phase as a function of SWCNT diameter. This empirical correlation enables predictive separation design and identifies a subset of SWCNTs that behave unusually as compared to other species. These results not only dramatically increase the range of SWCNT diameters to which species selective separation can be achieved but also demonstrate that aqueous two-phase separations can be designed across experimentally accessible ranges of surfactant concentrations to controllably separate SWCNT populations of very small (∼0.62 nm) to very large diameters (>1.7 nm). Together, the results reported here indicate that total separation of all SWCNT species is likely feasible by the ATPE method, especially given future development of multistage automated extraction techniques.  

6.170           Semiconducting enrichment of arc discharge single-walled carbon nanotubes by density gradient ultracentrifugation

Scharfenberg, L. and Mertig, M. Phys. Status Solidi A, 6, 1395-1398 (2015)   Due to their intrinsic properties, single-walled carbon nanotubes (SWCNTs) are promising candidates for source-drain channels in field-effect transistors (FETs). However, their application in transistors requires semiconducting tubes, and thus, sorting of SWCNTs according to those. The basis for an efficient sorting is the dispersion of the material that usually includes but is not limited to applying tip sonication in the presence of appropriate amphiphilic molecules. We present a high semiconducting enrichment of surfactant-wrapped arc discharge SWCNTs via sorting according to electronic type by applying density gradient ultracentrifugation (DGU). We utilized a common combination of anionic surfactants, but optimized the sonication time during the dispersion step of the SWCNTs and the duration of performing DGU. Furthermore, we used UV–Vis spectroscopy to determine the differences in the content of metallic (m) and semiconducting (sc) SWCNTs of different samples. By the refinement of the conditions, we have achieved an enrichment of sc-SWCNTs up to 98% in two sorting steps.  

6.171           Bright Fraction of Single-Walled Carbon Nanotubes through Correlated Fluorescence and Topography Measurements

Nogaj, L.J., Smyder, J.A., Leach, K.E., Tu, X., Zheng, M. and Krauss, T.D.

1. Phys. Chem. Lett., 6(14), 2816-2821 (2015)

  Correlated measurements of fluorescence and topography were performed for individual single-walled carbon nanotubes (SWNTs) on quartz using epifluorescence confocal microscopy and atomic force microscopy (AFM). Surprisingly, only ∼11% of all SWNTs in DNA-wrapped samples were found to be highly emissive on quartz, suggesting that the ensemble fluorescence quantum yield is low because only a small population of SWNTs fluoresces strongly. Qualitatively similar conclusions were obtained from control studies using a sodium cholate surfactant system. To accommodate AFM measurements, excess surfactant was removed from the substrate. Though individual SWNTs on nonrinsed and rinsed surfaces displayed differences in fluorescence intensities and line widths, arising from the influence of the local environment on individual SWNT optical measurements, photoluminescence data from both samples displayed consistent trends.  

6.172           Sorting Semiconducting Single-Walled Carbon Nanotubes by Water-Soluble Polyfluorene Assisted Electrophoresis and Its Application in Field-effect Transistors

Zhu, H. and Wang, W. Chin. J. Chem., 33, 756-764 (2015)   We report a considerably promising method based on agarose gel electrophoresis (AGE) to separate single-walled carbon nanotubes by adding a water-soluble polyfluorene (w-PFO) as surfactant into the agarose gel. In this effective method, the AGE/w-PFO gel network will trap more semiconducting single-walled carbon nanotubes (SWNTs) with the assistance of w-PFO, for the strong interaction between w-PFO and semiconducting species. The optical absorbance, photoluminescence emission and resonant Raman scattering characterization were used to verify the separation effect. The purity of separated semiconducting species is as high as (98±1)%. The demonstrated field effect transistors give the on/off ratio and mobility about 27000 and 10.2 cm²·V⁻¹·s⁻¹, respectively.  

6.173           High-work-function metal/carbon nanotube/low-work-function metal hybrid junction photovoltaic device

Chen, C., Jin, T., Wei, L., Li, Y., Liu, X., Wang, Y., Zhang, L., Liao, C., Hu, N., Song, C. and Zhang, Y. NPG Asia Materials, 7, e220 (2015)   Photovoltaic devices based on nanotechnology have attracted much attention because of their great potential for application in electronic and energy fields. Here, a photovoltaic device based on a high-work-function metal/single-walled carbon nanotube (SWNT)/low-work-function metal hybrid junction was investigated. In the device, asymmetric metal electrodes (palladium and aluminum) were fabricated on opposite ends of a single semiconducting SWNT, which was used as the photosensitive material. This structure allowed a strong built-in electric field to be generated in the SWNT to efficiently separate photogenerated electron-hole pairs and achieve good photovoltaic effect. In the dark, the device behaved as a gate-dependent Schottky diode and exhibited the electrical characteristics of a rectifier. The SWNT diameter (band gap) was found to have a significant effect on the device characteristics. For the device fabricated with a 1.4-nm-diameter SWNT, a high rectification ratio (I_forward/I_reverse) of >10³ could be achieved in the dark. Under monochromatic illumination, this device had an open-circuit voltage of 0.15 V and a high quantum efficiency of ~75%.  

6.174           Solution-Processed Dielectrics Based on Thickness-Sorted Two-Dimensional Hexagonal Boron Nitride Nanosheets

Zhu, J., Kang, J., Kang, J.,Jjariwala, D., Wood, J.D., Seo, J-W.T., Chen, K-S., marks, T.J. and hersam, M.C. Nano Lett., 15(10), 7029-7036 (2015)   Gate dielectrics directly affect the mobility, hysteresis, power consumption, and other critical device metrics in high-performance nanoelectronics. With atomically flat and dangling bond-free surfaces, hexagonal boron nitride (h-BN) has emerged as an ideal dielectric for graphene and related two-dimensional semiconductors. While high-quality, atomically thin h-BN has been realized via micromechanical cleavage and chemical vapor deposition, existing liquid exfoliation methods lack sufficient control over h-BN thickness and large-area film quality, thus limiting its use in solution-processed electronics. Here, we employ isopycnic density gradient ultracentrifugation for the preparation of monodisperse, thickness-sorted h-BN inks, which are subsequently layer-by-layer assembled into ultrathin dielectrics with low leakage currents of 3 × 10^–9 A/cm² at 2 MV/cm and high capacitances of 245 nF/cm². The resulting solution-processed h-BN dielectric films enable the fabrication of graphene field-effect transistors with negligible hysteresis and high mobilities up to 7100 cm² V^–1 s^–1 at room temperature. These h-BN inks can also be used as coatings on conventional dielectrics to minimize the effects of underlying traps, resulting in improvements in overall device performance. Overall, this approach for producing and assembling h-BN dielectric inks holds significant promise for translating the superlative performance of two-dimensional heterostructure devices to large-area, solution-processed nanoelectronics.  

6.175           Optical detection of individual ultra-short carbon nanotubes enables their length characterization down to 10 nm

Gao, Z., Oudjedi, L., Faes, R., Morote, F., Jaillet, C., Poulin, P., Lounis, B. and Cognet, L. Scientific Reports, 5:17093 (2015)   Ultrashort single-walled carbon nanotubes, i.e. with length below ~30 nm, display length-dependent physical, chemical and biological properties that are attractive for the development of novel nanodevices and nanomaterials. Whether fundamental or applicative, such developments require that ultrashort nanotube lengths can be routinely and reliably characterized with high statistical data for high-quality sample production. However, no methods currently fulfill these requirements. Here, we demonstrate that photothermal microscopy achieves fast and reliable optical single nanotube analysis down to ~10 nm lengths. Compared to atomic force microscopy, this method provides ultrashort nanotubes length distribution with high statistics, and neither requires specific sample preparation nor tip-dependent image analysis.  

6.176           Single Chirality (6,4) Single-Walled Carbon Nanotubes for Fluorescence Imaging with Silicon Detectors

Antaris, A.L., Yaghi, O.K., Hong, G., Diao, S., Zhang, B., Yang, J., Chew, L. and Dai, H. Small, 11(47), 6325-6330 (2015)   Postsynthetic single-walled carbon nanotube (SWCNT) sorting methods such as density gradient ultracentrifugation, gel chromatography, and electrophoresis have all been inspired by established biochemistry separation techniques designed to separate subcellular components. Biochemistry separation techniques have been refined to the degree that parameters such as pH, salt concentration, and temperature are necessary for a successful separation, yet these conditions are only now being applied to SWCNT separation methodologies. Slight changes in pH produce radically different behaviors of SWCNTs inside a density gradient, allowing for the facile separation of ultrahigh purity (6,4) SWCNTs from as-synthesized carbon nanotubes. The (6,4) SWCNTs are novel fluorophores emitting below ≈900 nm and can be easily detected with conventional silicon-based charge-coupled device detectors without the need for specialized InGaAs cameras. The (6,4) SWCNTs are used to demonstrate their potential as a clinically relevant NIR-I fluorescence stain for the immunohistochemical staining of cells and cancer tissue sections displaying high endothelial growth factor receptor levels  

6.177           Comprehensive spectroscopic characterization of high purity metallicity-sorted single-walled carbon nanotubes

Kharlamova, M.V., Kramberger, C., Sauer, M., Yanagi, K. and Pichler, T. Phys. Status Sollidi B, 252(11), 2512-2518 (2015)   We have performed a thorough characterization of 1.4 nm-diameter single-walled carbon nanotubes (SWCNTs) separated according to their metallicity by optical absorption (OAS), X-ray photoelectron (XPS), ultraviolet photoelectron (UPS), and multifrequency Raman spectroscopy. The OAS, XPS, and UPS data prove a high purity ( 99%) of SWCNTs. The C 1s core level XPS spectra of the separated nanotubes show different spectral widths, asymmetry, and peak positions. The UPS spectra of the samples demonstrate clearly defined van Hove singularities. Significant differences are observed in the Raman spectra of the separated nanotubes. Our findings suggest that for further advances in SWCNT metallicity sorting, Raman spectroscopy has to be applied to assess purity levels beyond the detection limits of OAS, XPS, and UPS.  

6.178           Solution-processed carbon nanotube thin-film complementary static random access memory

Geier, M.L., McMorrow, J.J., Xu, W., Zhu, J., Kim, C.H., Marks, T.J. and Hersam, M.C. Nature Nanotechnol. Lett., 10, 944-948 (2015)   Over the past two decades, extensive research on single-walled carbon nanotubes (SWCNTs) has elucidated their many extraordinary properties^{1, 2, 3}, making them one of the most promising candidates for solution-processable, high-performance integrated circuits^{4, 5}. In particular, advances in the enrichment of high-purity semiconducting SWCNTs^{6, 7, 8} have enabled recent circuit demonstrations including synchronous digital logic⁹, flexible electronics^{10, 11, 12, 13, 14} and high-frequency applications¹⁵. However, due to the stringent requirements of the transistors used in complementary metal–oxide–semiconductor (CMOS) logic as well as the absence of sufficiently stable and spatially homogeneous SWCNT thin-film transistors^{16, 17, 18}, the development of large-scale SWCNT CMOS integrated circuits has been limited in both complexity and functionality^{19, 20, 21}. Here, we demonstrate the stable and uniform electronic performance of complementary p-type and n-type SWCNT thin-film transistors by controlling adsorbed atmospheric dopants and incorporating robust encapsulation layers. Based on these complementary SWCNT thin-film transistors, we simulate, design and fabricate arrays of low-power static random access memory circuits, achieving large-scale integration for the first time based on solution-processed semiconductors.  

6.179           Recent Progress in Obtaining Semiconducting Single-Walled Carbon Nanotubes for Transistor Applications

Islam, A.E., Rogers, J.A. and Alam, M.A. Adv. Mater., 27(48), 7908-7937 (2015)   High purity semiconducting single-walled carbon nanotubes (s-SWCNTs) with a narrow diameter distribution are required for high-performance transistors. Achieving this goal is extremely challenging because the as-grown material contains mixtures of s-SWCNTs and metallic- (m-) SWCNTs with wide diameter distributions, typically inadequate for integrated circuits. Since 2000, numerous ex situ methods have been proposed to improve the purity of the s-SWCNTs. The majority of these techniques fail to maintain the quality and integrity of the s-SWCNTs with a few notable exceptions. Here, the progress in realizing high purity s-SWCNTs in as-grown and post-processed materials is highlighted. A comparison of transistor parameters (such as on/off ratio and field-effect mobility) obtained from test structures establishes the effectiveness of various methods and suggests opportunities for future improvements.  

6.180           Misorientation-angle-dependent electrical transport across molybdenum disulfide grain boundaries

Ly, T.H., Perello, D.J., Zhao, J., Deng, Q., Kim, H., Han, G.H., Chae, S.H., Jeong, H.Y. and Lee, Y.H. Nature Communications, 7:10426 (2016)   Grain boundaries in monolayer transition metal dichalcogenides have unique atomic defect structures and band dispersion relations that depend on the inter-domain misorientation angle. Here, we explore misorientation angle-dependent electrical transport at grain boundaries in monolayer MoS₂ by correlating the atomic defect structures of measured devices analysed with transmission electron microscopy and first-principles calculations. Transmission electron microscopy indicates that grain boundaries are primarily composed of 5–7 dislocation cores with periodicity and additional complex defects formed at high angles, obeying the classical low-angle theory for angles <22°. The inter-domain mobility is minimized for angles <9° and increases nonlinearly by two orders of magnitude before saturating at ~16 cm² V⁻¹ s⁻¹ around misorientation angle≈20°. This trend is explained via grain-boundary electrostatic barriers estimated from density functional calculations and experimental tunnelling barrier heights, which are ≈0.5 eV at low angles and ≈0.15 eV at high angles (≥20°).  

6.181           Performance Enhancement of Polymer-Free Carbon Nanotube Solar Cells via Transfer Matrix Modeling

Pfohl, M., Glaser, K., Ludwig, J., Tune, D.F.D., Dehm, S., Kayser, C., Colsmann, A., Krupke, R. and Flavel, B.S. Advanced Energy Materials, 6, 1501345 (2016)   Polymer-free (6,5) single-walled carbon nanotubes (SWCNTs) prepared using the gel permeation approach are integrated into SWCNT:C₆₀ solar cells. Evaporation-driven self-assembly is used to form large-area SWCNT thin films from the surfactant-stabilized aqueous suspensions. The thicknesses of various layers within the solar cell are optimized by theoretical modeling using transfer matrix calculations, where the distribution of the electric field within the stack is matched to light absorption by the SWCNTs through either their primary (S₁₁) or secondary (S₂₂) absorption peaks, or a combination thereof. The validity of the model is verified experimentally through a detailed parameter study and then used to develop SWCNT:C₆₀ solar cells with high open-circuit voltage (0.44 V) as well as a cutting-edge internal quantum efficiency of up to 86% through the nanotube S₁₁ transition, over an active area of 0.105 cm².  

6.182           Resonance Raman Optical Activity Spectra of Single-Walled Carbon Nanotube Enantiomers

Magg, M., Kadria-Vili, Y., Oulevey, P., Weismann, R.B. and Bürgi, T.

1. Phys. Chem. Lett., 7, 221-225 (2016)

  We present experimental Raman optical activity (ROA) spectra of enantio-enriched single-walled carbon nanotubes (SWCNTs). Enantiomeric samples of (6,5) SWCNTs were prepared using nonlinear density gradient ultracentrifugation (DGU). Upon excitation at 2.33 eV, remarkably strong G-band signals are obtained due to strong resonance enhancement with the E₂₂^S transition of (6,5) SWCNTs. Enhancement allows measuring the vibrational optical activity (VOA) at unusually low concentrations. The obtained results are in good agreement with the single-excited-state theory (SES). To our knowledge, these are the first experimental VOA spectra of SWCNTs.  

6.183           Synthesis and Testing of Modular Dual-Modality Nanoparticles for Magnetic Resonance and Multispectral Photoacoustic Imaging

Bogdanov, Jr, A.A., Dixon, A.J., Gupta, S., Zhang, L., Zheng, s., Shazeeb, M.S., Zhang, S. and Klibanov, A.L. Bioconjugate Chem., 27, 383-390 (2016)   Magnetic resonance (MR) and photoacoustic (PA) imaging are currently being investigated as complementing strategies for applications requiring sensitive detection of cells in vivo. While combined MR/PAI detection of cells requires biocompatible cell labeling probes, water-based synthesis of dual-modality MR/PAI probes presents significant technical challenges. Here we describe facile synthesis and characterization of hybrid modular dextran-stabilized gold/iron oxide (Au-IO) multimetallic nanoparticles (NP) enabling multimodal imaging of cells. The stable association between the IO and gold NP was achieved by priming the surface of dextran-coated IO with silver NP resulting from silver(I) reduction by aldehyde groups, which are naturally present within the dextran coating of IO at the level of 19–23 groups/particle. The Au-IO NP formed in the presence of silver-primed Au-IO were stabilized by using partially thiolated MPEG5-gPLL graft copolymer carrying residual amino groups. This stabilizer served as a carrier of near-infrared fluorophores (e.g., IRDye 800RS) for multispectral PA imaging. Dual modality imaging experiments performed in capillary phantoms of purified Au-IO-800RS NPs showed that these NPs were detectible using 3T MRI at a concentration of 25 μM iron. PA imaging achieved approximately 2.5-times higher detection sensitivity due to strong PA signal emissions at 530 and 770 nm, corresponding to gold plasmons and IRDye integrated into the coating of the hybrid NPs, respectively, with no “bleaching” of PA signal. MDA-MB-231 cells prelabeled with Au-IO-800RS retained plasma membrane integrity and were detectable by using both MR and dual-wavelength PA at 49 ± 3 cells/imaging voxel. We believe that modular assembly of multimetallic NPs shows promise for imaging analysis of engineered cells and tissues with high resolution and sensitivity.  

6.184           Large scale, selective dispersion of long single-walled carbon nanotubes with high photoluminescence quantum yield by shear force mixing

Graf, A., Zakharko, Y., Schiessl, S.P., Backs, C., Pfohl, M., Flavel, B.S. and  Zaumseil, J. Carbon, 105, 593-599 (2016)   Selective dispersion of semiconducting single-walled carbon nanotube (SWCNTs) with conjugated polymers typically involves harsh sonication methods that damage and shorten the nanotubes. Here, we use simple high speed shear force mixing (SFM) to disperse nearly monochiral (6,5) SWCNTs with poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(6,6′-{2,2′-bipyridine})] (PFO-BPy) in toluene with high yield and in large volumes. This highly scalable process disperses SWCNTs of exceptional quality with an average tube length of 1.82 μm and an ensemble photoluminescence quantum yield (PLQY) of 2.3%. For the first time for SWCNTs, we describe and apply absolute PLQY measurements, without the need for any reference emitter. We directly compare values for average SWCNT length, PLQY, linewidth and Stokes shift to other dispersion methods, including bath and tip sonication, as well as other sorting methods such as gel chromatography. We find that SFM results in dispersions of longer SWCNT with higher average PLQY than any other technique, thus making it an ideal method for sorting large amounts of long, high quality and purely semiconducting SWCNTs.  

6.185           Characterizing the Effect of Salt and Surfactant Concentration on the Counterion Atmosphere around Surfactant Stabilized SWCNTs Using Analytical Ultracentrifugation

Lam, S., Zheng, M. and Fagan, J.A. Langmuir, 32(16), 3926-3936 (2016)   Accurate characterization of dispersed-phase nanoparticle properties such as density, size, solvation, and charge is necessary for their utilization in applications such as medicine, energy, and materials. Herein, analytical ultracentrifugation (AUC) is used to quantify bile salt surfactant adsorption on length sorted (7,6) single-wall carbon nanotubes (SWCNTs) as a function of bulk surfactant concentration and in the presence of varying quantities of a monovalent salt—sodium chloride. These measurements provide high precision adsorbed surfactant density values in the literature for only the second SWCNT structure to date and report the quantity of adsorbed surfactant across a broad range of bulk surfactant concentrations utilized in SWCNT dispersion processing. Second, the measurements presented herein unambiguously demonstrate, via AUC, a direct relation between the size of the counterion cloud around a surfactant-stabilized SWCNT and solution ionic strength. The results show that changes in the size of the counterion cloud around surfactant-stabilized SWCNT are attributable to electrostatic phenomenon and not to changes in the quantity of adsorbed surfactant with salt addition. These results provide important reference values for projecting SWCNT dispersion behavior as a function of solution conditions and extend the range of nanoparticle properties measurable via AUC.  

6.186           Competitive Impact of Nanotube Assembly and Contact Electrodes on the Performance of CNT-based FETs

Toader, M., Hermann, S., Scharfenberg, L., Hartmann, M., Mertig, M., Schulz, S.E. and Gessner, T.

1. Phys. Chem. C, 120(18), 10020-10026 (2016)

  We report the fabrication and characterization of highly dense field-effect-transistor (FET) arrays based on single-walled carbon nanotubes (SWCNTs). The nanotubes were sorted according to the electronic type by using density gradient ultracentrifugation (DGU). By employing dielectrophoresis (DEP), SWCNTs with enriched semiconducting (sc) content were systematically integrated as active elements into FETs. The performance of air-operating FETs was addressed via an extended statistic study involving both electrical and structural analyses. The competitive impact of nanotube purity and assembly as well as the metal electrode composition and a thermal treatment on the final device performance was shown. Regardless of the used sc-content, the device-to-device consistency was improved via employing annealing up to 250 °C for 1 h in a vacuum. The observed clockwise hysteresis, known so far only in connection with CNT–FETs built on ferroelectric substrates as well as electrolyte gated CNT–FETs, was found to reverse upon annealing. Moreover, a simple approach in producing air-stable ambipolar transistors is pointed out only via change of the adhesion layer for the Pd electrodes. The annealing study, repeated on such systems, supports the previous results and provides complementary information via a reliable monitoring of the off-state. Indications for a doping-like effect, which partially compromises the device performance in terms of threshold voltage shifts and increased off-state currents, are revealed and discussed.  

6.187           Aggregated single-walled carbon nanotubes attenuate the behavioural and neurochemical effects of methamphetamine in mice

Xue, X. et al Nature Nanotechnology, 11(7), 613-620 (2016)   Methamphetamine (METH) abuse is a serious social and health problem worldwide. At present, there are no effective medications to treat METH addiction¹. Here, we report that aggregated single-walled carbon nanotubes (aSWNTs) significantly inhibited METH self-administration, METH-induced conditioned place preference and METH- or cue-induced relapse to drug-seeking behaviour in mice. The use of aSWNTs alone did not significantly alter the mesolimbic dopamine system, whereas pretreatment with aSWNTs attenuated METH-induced increases in extracellular dopamine in the ventral striatum. Electrochemical assays suggest that aSWNTs facilitated dopamine oxidation. In addition, aSWNTs attenuated METH-induced increases in tyrosine hydroxylase or synaptic protein expression. These findings suggest that aSWNTs may have therapeutic effects for treatment of METH addiction by oxidation of METH-enhanced extracellular dopamine in the striatum.  

6.188           13 nm Exciton Size in (6,5) Single-Wall Carbon Nanotubes

Mann, C. and Hertel, T. Phys. Chem. Lett., 7(12), 2276-2280 (2016)   Electron–hole correlation lengths, also termed exciton size, for (6,5) single-wall carbon nanotubes (SWNTs) are determined using femtosecond time-resolved pump–probe spectroscopy. The phase space filling model is used to obtain the sizes of the first subband exciton in samples of isolated and of bundled SWNTs. The experiments indicate that the exciton size of (13 ± 3) nm is a factor of 6 higher than previous experimental estimates and theoretical predictions for vacuum suspended SWNTs. This surprising result may be attributed at least in part to the effect of the dielectric environment on exciton sizes and supports recent theoretical findings predicting that screening in SWNTs may enhance rather than reduce electron–hole interactions for separations larger than the tube diameter. Thereby, the work also points to the unique nature of screening and electronic correlations in one-dimensional semiconductors.  

6.189           Toxicological Profiling of Highly Purified Metallic and Semiconducting Single-Walled Carbon Nanotubes in the Rodent Lung and E. coli

Wang, X., Mansukhani, N.D., Guiney, L.M., Lee, J-H., Li, R., Sun, B., Liao, Y-P., Chang, C.H., Ji, Z., Xia, T., Hersam, M.C. and Nel, A.E. ACS Nano, 10(6), 6008-6019 (2016)   The electronic properties of single-walled carbon nanotubes (SWCNTs) are potentially useful for electronics, optics, and sensing applications. Depending on the chirality and diameter, individual SWCNTs can be classified as semiconducting (S-SWCNT) or metallic (M-SWCNT). From a biological perspective, the hazard profiling of purified metallic versus semiconducting SWCNTs has been pursued only in bacteria, with the conclusion that aggregated M-SWCNTs are more damaging to bacterial membranes than S-SWCNTs. However, no comparative studies have been performed in a mammalian system, where most toxicity studies have been undertaken using relatively crude SWCNTs that include a M:S mix at 1:2 ratio. In order to compare the toxicological impact of SWCNTs sorted to enrich them for each of the chirality on pulmonary cells and the intact lung, we used density gradient ultracentrifugation and extensive rinsing to prepare S- and M-SWCNTs that are >98% purified. In vitro screening showed that both tube variants trigger similar amounts of interleukin 1β (IL-1β) and transforming growth factor (TGF-β1) production in THP-1 and BEAS-2B cells, without cytotoxicity. Oropharyngeal aspiration confirmed that both SWCNT variants induce comparable fibrotic effects in the lung and abundance of IL-1β and TGF-β1 release in the bronchoalveolar lavage fluid. There was also no change in the morphology, membrane integrity, and viability of E. coli, in contradistinction to the previously published effects of aggregated tubes on the bacterial membrane. Collectively, these data indicate that the electronic properties and chirality do not independently impact SWCNT toxicological impact in the lung, which is of significance to the safety assessment and incremental use of purified tubes by industry.  

6.190           Bioaccumulation of Multiwall Carbon Nanotubes in Tetrahymena thermophila by Direct Feeding or Trophic Transfer

Mortimer, M., Petersen, E.J., Buchholz, B.A., Orias, E. and Holden, P.A. Environ. Sci. Technol., 50(16), 8876-8885 (2016)   Consumer goods contain multiwall carbon nanotubes (MWCNTs) that could be released during product life cycles into the environment, where their effects are uncertain. Here, we assessed MWCNT bioaccumulation in the protozoan Tetrahymena thermophila via trophic transfer from bacterial prey (Pseudomonas aeruginosa) versus direct uptake from growth media. The experiments were conducted using ¹⁴C-labeled MWCNT (¹⁴C-MWCNT) doses at or below 1 mg/L, which proved subtoxic since there were no adverse effects on the growth of the test organisms. A novel contribution of this study was the demonstration of the ability to quantify MWCNT bioaccumulation at low (sub μg/kg) concentrations accomplished by employing accelerator mass spectrometry (AMS). After the treatments with MWCNTs at nominal concentrations of 0.01 mg/L and 1 mg/L, P. aeruginosa adsorbed considerable amounts of MWCNTs: (0.18 ± 0.04) μg/mg and (21.9 ± 4.2) μg/mg bacterial dry mass, respectively. At the administered MWCNT dose of 0.3 mg/L, T. thermophila accumulated up to (0.86 ± 0.3) μg/mg and (3.4 ± 1.1) μg/mg dry mass by trophic transfer and direct uptake, respectively. Although MWCNTs did not biomagnify in the microbial food chain, MWCNTs bioaccumulated in the protozoan populations regardless of the feeding regime, which could make MWCNTs bioavailable for organisms at higher trophic levels.  

6.191           Stable aqueous dispersions of optically and electronically active phosphorene

Kang, J., Wells, S., Wood, J.D., Lee, J-H., Liu, X., Ryder, C.R., Zhu, J., Guest, J.R., Husko, C.A. and Hersam, M.C. PNAS, 113(42) 11688-11693 (2016)   Understanding and exploiting the remarkable optical and electronic properties of phosphorene require mass production methods that avoid chemical degradation. Although solution-based strategies have been developed for scalable exfoliation of black phosphorus, these techniques have thus far used anhydrous organic solvents in an effort to minimize exposure to known oxidants, but at the cost of limited exfoliation yield and flake size distribution. Here, we present an alternative phosphorene production method based on surfactant-assisted exfoliation and postprocessing of black phosphorus in deoxygenated water. From comprehensive microscopic and spectroscopic analysis, this approach is shown to yield phosphorene dispersions that are stable, highly concentrated, and comparable to micromechanically exfoliated phosphorene in structure and chemistry. Due to the high exfoliation efficiency of this process, the resulting phosphorene flakes are thinner than anhydrous organic solvent dispersions, thus allowing the observation of layer-dependent photoluminescence down to the monolayer limit. Furthermore, to demonstrate preservation of electronic properties following solution processing, the aqueous-exfoliated phosphorene flakes are used in field-effect transistors with high drive currents and current modulation ratios. Overall, this method enables the isolation and mass production of few-layer phosphorene, which will accelerate ongoing efforts to realize a diverse range of phosphorene-based applications.  

6.192           Photoluminescence Dynamics of Aryl sp3 Defect States in Single-Walled Carbon Nanotubes

Hartmann, N.F., Velizhanin, K.A., Haroz, E.H., Kim, M., Ma, X., Wang, Y., Htoon, H. and Doorn, S.K. ACS Nano, 10(9), 8355-8365 (2016)   Photoluminescent defect states introduced by sp³ functionalization of semiconducting carbon nanotubes are rapidly emerging as important routes for boosting emission quantum yields and introducing new functionality. Knowledge of the relaxation dynamics of these states is required for understanding how functionalizing agents (molecular dopants) may be designed to access specific behaviors. We measure photoluminescence (PL) decay dynamics of sp³ defect states introduced by aryl functionalization of the carbon nanotube surface. Results are given for five different nanotube chiralities, each doped with a range of aryl functionality. We find that the PL decays of these sp³ defect states are biexponential, with both components relaxing on time scales of ∼100 ps. Exciton trapping at defects is found to increases PL lifetimes by a factor of 5–10, in comparison to those for the free exciton. A significant chirality dependence is observed in the decay times, ranging from 77 ps for (7,5) nanotubes to >600 ps for (5,4) structures. The strong correlation of time constants with emission energy indicates relaxation occurs via multiphonon decay processes, with close agreement to theoretical expectations. Variation of the aryl dopant further modulates decay times by 10–15%. The aryl defects also affect PL lifetimes of the free E₁₁ exciton. Shortening of the E₁₁ bright state lifetime as defect density increases provides further confirmation that defects act as exciton traps. A similar shortening of the E₁₁ dark exciton lifetime is found as defect density increases, providing strong experimental evidence that dark excitons are also trapped at such defect sites.  

6.193           Electron Excess Doping and Effective Schottky Barrier Reduction on the MoS2/h-BN Heterostructure

Joo, M-K., Moon, B.H., Ji, H., Han, G.H., Kim, H., Lee, G., Lim, S.C., Suh, D., and Lee, Y.H. Nano Lett., 16, 6383-6389 (2016)   Layered hexagonal boron nitride (h-BN) thin film is a dielectric that surpasses carrier mobility by reducing charge scattering with silicon oxide in diverse electronics formed with graphene and transition metal dichalcogenides. However, the h-BN effect on electron doping concentration and Schottky barrier is little known. Here, we report that use of h-BN thin film as a substrate for monolayer MoS₂ can induce ∼6.5 × 10¹¹ cm^–2 electron doping at room temperature which was determined using theoretical flat band model and interface trap density. The saturated excess electron concentration of MoS₂ on h-BN was found to be ∼5 × 10¹³ cm^–2 at high temperature and was significantly reduced at low temperature. Further, the inserted h-BN enables us to reduce the Coulombic charge scattering in MoS₂/h-BN and lower the effective Schottky barrier height by a factor of 3, which gives rise to four times enhanced the field-effect carrier mobility and an emergence of metal–insulator transition at a much lower charge density of ∼1.0 × 10¹² cm^–2 (T = 25 K). The reduced effective Schottky barrier height in MoS₂/h-BN is attributed to the decreased effective work function of MoS₂ arisen from h-BN induced n-doping and the reduced effective metal work function due to dipole moments originated from fixed charges in SiO₂.  

6.194           Substrate-Mediated Cooperative Adsorption of Sodium Cholate on (6,5) Single-Wall Carbon Nanotubes

Bergler, F.F., Stahl, S., Goy, A., Schöppler, F. and Hertel, T. Langmuir, 32, 9598-9603 (2016)   The interaction of sodium cholate (NaC) with (6,5) single-wall carbon nanotubes (SWNTs) is investigated using photoluminescence spectroscopy. Dilution of SWNT-NaC suspensions is accompanied by changes in the exciton PL quantum yield and peak emission energy. An abrupt change of the exciton emission peak energy at NaC concentrations between 10 and 14 mM indicates strongly cooperative formation of a micellar phase on (6,5) SWNT surfaces with a Hill coefficient of n_H = 65 ± 6. This is in contrast to the formation of free NaC micelles with aggregation numbers of only about 4 and suggests that the cooperativity of NaC micelle formation on nanotube surfaces is strongly substrate-enhanced. The temperature dependence of this previously unobserved transition is used for a determination of Δ_micG^⊖/(1 + β) = −(11.4 ± 0.2) kJ·mol^–1 which, for typical Na⁺ counterion binding with β ≈ 0.2, yields a free SWNT-NaC micellization enthalpy Δ_micG^⊖ of −13.7 kJ·mol^–1.  

6.195           A simple quantitative estimate of the number of functional groups on the surfaces of single-walled carbon nanotubes

Ngoc, Q.B.V., Choi, M-S. and Kim, W-J. RSC Adv., 6, 6451-6458 (2016)   We develop a quantitative analytical method to estimate the number of functional groups on single-walled carbon nanotube (SWNT) surfaces by simply measuring the Raman features of functionalized SWNTs. We use diazonium chemistry to attach functional groups to the SWNT surfaces and calculate the number of functional groups on the SWNTs by measuring their densities and using the analytical method developed in this study. We successfully establish a linear relationship between the independently estimated number of functional groups per carbon atom and the area ratio of the disorder mode (D peak, 1289 cm⁻¹) to the tangential mode (G peak, 1582 cm⁻¹) from the Raman measurements. From this study, we find that the injected reagents do not completely react with SWNT after 0.0263 functional groups per carbon atom are already attached to SWNT surfaces, e.g., 2.354 functional groups per nm (6, 5) of SWNT, therefore above this range, there is no linear relationship between the number of functional groups on SWNTs estimated from injected reagent concentration and the D to G area ratio (A_D/G) of the Raman features. However, the number of functional groups per carbon atom (n_f) estimated using the analytical method developed in this study linearly depends on the area ratio of D to G peaks of the Raman spectrum even above this range (n_f = 0.02A_D/G), therefore this simple and quick estimation scheme can provide an analytical basis for a variety of SWNT applications. We take into account the decrease of the number of surfactants by the coverage of functional groups on SWNTs in this scheme, and also verified that this relation is valid irrespective of SWNT diameter distributions.  

6.196           Plasmonic nanostructures for surface enhanced spectroscopic methods

Jahn, M., Patze, S., Hidi, I.J., Knipper, R., Radu, A.I., Mühlig, A., Yüksel, S., Peksa, V., Weber, K., Mayerhöfer, T., Cialla-May, D. and Popp, J. Analyst, 141, 756-793 (2016)   A comprehensive review of theoretical approaches to simulate plasmonic-active metallic nano-arrangements is given. Further, various fabrication methods based on bottom-up, self-organization and top-down techniques are introduced. Here, analytical approaches are discussed to investigate the optical properties of isotropic and non-magnetic spherical or spheroidal particles. Furthermore, numerical methods are introduced to research complex shaped structures. A huge variety of fabrication methods are reviewed, e.g. bottom-up preparation strategies for plasmonic nanostructures to generate metal colloids and core–shell particles as well as complex-shaped structures, self-organization as well as template-based methods and finally, top-down processes, e.g. electron beam lithography and its variants as well as nanoimprinting. The review article is aimed at beginners in the field of surface enhanced spectroscopy (SES) techniques and readers who have a general interest in theoretical modelling of plasmonic substrates for SES applications as well as in the fabrication of the desired structures based on methods of the current state of the art.  

6.197           (n,m)-Specific Absorption Cross Sections of Single-Walled Carbon Nanotubes Measured by Variance Spectroscopy

Sanchez, S.R., bachilo, S.M., kadria-Vili, Y., Lin, C-W. and Weisman, R.B. Nano Lett., 16(11), 6903-6909 (2016)   A new method based on variance spectroscopy has enabled the determination of absolute absorption cross sections for the first electronic transition of 12 (n,m) structural species of semiconducting single-walled carbon nanotubes (SWCNTs). Spectrally resolved measurements of fluorescence variance in dilute bulk samples provided particle number concentrations of specific SWCNT species. These values were converted to carbon concentrations and correlated with resonant components in the absorbance spectrum to deduce (n,m)-specific absorption cross sections (absorptivities) for nanotubes ranging in diameter from 0.69 to 1.03 nm. The measured cross sections per atom tend to vary inversely with nanotube diameter and are slightly greater for structures of mod 1 type than for mod 2. Directly measured and extrapolated values are now available to support quantitative analysis of SWCNT samples through absorption spectroscopy.  

6.198           Layer-by-Layer Sorting of Rhenium Disulfide via High-Density Isopycnic Density Gradient Ultracentrifugation

Kang, J., Sangwan, V.K., Wood, J.D., Liu, X., balla, I., Lam, D. and hersam, M.C. Nano Lett., 16(11), 7216-7223 (2016)   Isopycnic density gradient ultracentrifugation (iDGU) has been widely applied to sort nanomaterials by their physical and electronic structure. However, the commonly used density-gradient medium iodixanol has a finite maximum buoyant density that prevents the use of iDGU for high-density nanomaterials. Here, we overcome this limit by adding cesium chloride (CsCl) to iodixanol, thus increasing its maximum buoyant density to the point where the high-density two-dimensional nanomaterial rhenium disulfide (ReS₂) can be sorted in a layer-by-layer manner with iDGU. The resulting aqueous ReS₂ dispersions show photoluminescence at ∼1.5 eV, which is consistent with its direct bandgap semiconductor electronic structure. Furthermore, photocurrent measurements on thin films formed from solution-processed ReS₂ show a spectral response that is consistent with optical absorbance and photoluminescence data. In addition to providing a pathway for effective solution processing of ReS₂, this work establishes a general methodology for sorting high-density nanomaterials via iDGU.      

6.199           Intramolecular p-i-n junction photovoltaic device based on selectively doped carbon nanotubes

Chen, C., Song, C., Yang, J., Chen, D., Zhu, W., Liao, C., Dong, X., Liu, X., Wei, L., Hu, N., He, R. and Zhang, Y. Nano Energy, 32, 280-286 (2017)   Photovoltaic devices show promising applications in detection and energy fields as well as in next-generation optoelectronic circuits. The use of the ideal photosensitive material and device design are critical for achieving a high-performance photovoltaic device. Here, an intramolecular p-i-n junction photovoltaic device based on selectively doped carbon nanotubes is investigated. In this kind of device, the opposite ends of an individual single-walled carbon nanotube (SWCNT) channel are doped selectively by triethyloxonium hexachloroantimonate (OA) and polyethylene imine (PEI) to obtain stable p- and n- type SWCNT segments respectively, while the middle segment of the SWCNT is kept intrinsic, causing the formation of an intra-tube p-i-n junction for the efficient separation of photogenerated electron-hole pairs. The optical-absorption and electrical testing demonstrate that the OA and PEI can dope the SWCNTs into the stable p- and n- types, respectively. In the dark, the prepared p-i-n junction device behaves as a diode with a high rectification ratio >10³ that can be tuned by the gate voltage. Under a 1550-nm monochromatic illumination, the device exhibits a good photovoltaic effect with a large open-circuit voltage of 0.41 V and an external power conversion efficiency of ~4.2%. The quantum efficiency of the device is estimated to be as high as ~73%.  

6.200           Assessing Inhomogeneity in Sorted Samples of Single-Walled Carbon Nanotubes through Fluorescence and Variance Spectroscopy

Kadria-Vili, Y., Sanchez, S.R., bachilo, WS.M. and Weisman, R.B. ECS J. Solid State Sci. Technol., 6(6), M3097-M3102 (2017)   Detailed spectroscopic analysis has been used to study the homogeneity of single-walled carbon nanotube fractions carefully prepared by nonlinear density gradient ultracentrifugation sorting. Two distinct colored bands containing (6,5) enantiomers were subdivided into several extracted fractions that were separately diluted with sodium cholate surfactant and characterized by fluorescence, absorption, and variance spectroscopy. Values were measured for emission and absorption peak positions, Stokes shifts, emission peak widths, and emissive quantum yields. In addition, variance data were used to find relative emission per nanotube and to plot covariance slices representing homogeneous emission spectra. It was found that emission from SWCNTs within the upper enantiomer band shifts to shorter wavelengths with increasing depth in the centrifuge tube. In the lower enantiomer band such spectral shifts were not observed, but the emissive quantum yields decreased with depth. Variance analysis revealed spectral differences among SWCNTs within the same fraction of the same band. It is concluded that current methods for density gradient ultracentrifugation sorting produce samples that retain measurable structural and spectral inhomogeneities.  

6.201           Separation of double-wall carbon nanotubes by electronic type and diameter

Streit, J.K., Lam, S., Piao, L.Y., Walker, A.R.H., Fagan, J.A. and Zheng, M. Nanoscale, 9, 2531-2540 (2017)   We introduce a new procedure for the efficient isolation and subsequent separation of double-wall carbon nanotubes (DWCNTs). A simplified, rate zonal ultracentrifugation (RZU) process is first applied to obtain samples of highly-enriched DWCNTs from a raw carbon nanotube material that has both single- and double-wall carbon nanotubes. Using this purified DWCNT suspension, we demonstrate for the first time that DWCNTs can be further processed using aqueous two-phase extraction (ATPE) for sequential separation by electronic structure and diameter. Additionally, we introduce analytical ultracentrifugation (AUC) as a new method for DWCNT characterization to assess DWCNT purity in separated samples. Results from AUC analysis are utilized to compare two DWCNT separation schemes. We find that RZU processing followed by sequential bandgap and diameter sorting via ATPE provides samples of highest DWCNT enrichment, whereas single-step redox sorting of the same raw material through ATPE yields SWCNT/DWCNT mixtures of similar diameter and electronic character. The presented methods offer significant advancement in DWCNT processing and separation while also providing a promising alternative for DWCNT sample analysis.    

6.202           K-Channel: A Multifunctional Architecture for Dynamically Reconfigurable Sample Processing in Droplet Microfluidics

Doonan, S.R. and Bailey, R.C: Anal. Chem., 89(7), 4091-4099 (2017)   By rapidly creating libraries of thousands of unique, miniaturized reactors, droplet microfluidics provides a powerful method for automating high-throughput chemical analysis. In order to engineer in-droplet assays, microfluidic devices must add reagents into droplets, remove fluid from droplets, and perform other necessary operations, each typically provided by a unique, specialized geometry. Unfortunately, modifying device performance or changing operations usually requires re-engineering the device among these specialized geometries, a time-consuming and costly process when optimizing in-droplet assays. To address this challenge in implementing droplet chemistry, we have developed the “K-channel,” which couples a cross-channel flow to the segmented droplet flow to enable a range of operations on passing droplets. K-channels perform reagent injection (0–100% of droplet volume), fluid extraction (0–50% of droplet volume), and droplet splitting (1:1–1:5 daughter droplet ratio). Instead of modifying device dimensions or channel configuration, adjusting external conditions, such as applied pressure and electric field, selects the K-channel process and tunes its magnitude. Finally, interfacing a device-embedded magnet allows selective capture of 96% of droplet-encapsulated superparamagnetic beads during 1:1 droplet splitting events at ∼400 Hz. Addition of a second K-channel for injection (after the droplet splitting K-channel) enables integrated washing of magnetic beads within rapidly moving droplets. Ultimately, the K-channel provides an exciting opportunity to perform many useful droplet operations across a range of magnitudes without requiring architectural modifications. Therefore, we envision the K-channel as a versatile, easy to use microfluidic component enabling diverse, in-droplet (bio) chemical manipulations.  

6.203           Colloidal 2D nanosheets of MoS2 and other transition metal dichalcogenides through liquid-phase exfoliation

Grayfer, E.D., Kozlova, M.N. and Fedorov, V.E. Adv. Colloid Interface Sci., 245, 40-61 (2017)   This review focuses on the exfoliation of transition metal dichalcogenides MQ₂ (TMD, M = Mo, W, etc., Q = S, Se, Te) in liquid media, leading to the formation of 2D nanosheets dispersed in colloids. Nowadays, colloidal dispersions of MoS₂, MoSe₂, WS₂ and other related materials are considered for a wide range of applications, including electronic and optoelectronic devices, energy storage and conversion, sensors for gases, catalysts and catalyst supports, biomedicine, etc. We address various methods developed so far for transferring these materials from bulk to nanoscale thickness, and discuss their stabilization and factors influencing it. Long-time known exfoliation through Li intercalation has received renewed attention in recent years, and is recognized as a method yielding highest dispersed concentrations of single-layer MoS₂ and related materials. Latest trends in the intercalation/exfoliation approach include electrochemical lithium intercalation, experimenting with various intercalating agents, multi-step intercalation, etc. On the other hand, direct sonication in solvents is a much simpler technique that allows one to avoid dangerous reagents, long reaction times and purifying steps. The influence of the solvent characteristics on the colloid formation was closely investigated in numerous recent studies. Moreover, it is being recognized that, besides solvent properties, sonication parameters and solvent transformations may affect the process in a crucial way. The latest data on the interaction of MoS₂ with solvents evidence that not only solution thermodynamics should be employed to understand the formation and stabilization of such colloids, but also general and organic chemistry. It appears that due to the sonolysis of the solvents and cutting of the MoS₂ layers in various directions, the reactive edges of the colloidal nanosheets may bear various functionalities, which participate in their stabilization in the colloidal state. In most cases, direct exfoliation of MQ₂ into colloidal nanosheets is conducted in organic solvents, while a small amount of works report low-concentrated colloids in pure water. To improve the dispersion abilities of transition metal dichalcogenides in water, various stabilizers are often introduced into the reaction media, and their interactions with nanosheets play an important role in the stabilization of the dispersions. Surfactants, polymers and biomolecules usually interact with transition metal dichalcogenide nanosheets through non-covalent mechanisms, similarly to the cases of graphene and carbon nanotubes. Finally, we survey covalent chemical modification of colloidal MQ₂ nanosheets, a special and different approach, consisting in the functionalization of MQ₂ surfaces with help of thiol chemistry, interaction with electrophiles, or formation of inorganic coordination complexes. The intentional design of surface chemistry of the nanosheets is a very promising way to control their solubility, compatibility with other moieties and incorporation into hybrid structures. Although the scope of the present review is limited to transition metal dichalcogenides, the dispersion in colloids of other chalcogenides (such as NbS₃, VS₄, Mo₂S₃, etc.) in many ways follows similar trends. We conclude the review by discussing current challenges in the area of exfoliation of MoS₂ and its related materials.  

6.204           Solution-Based Processing of Monodisperse Two-Dimensional Nanomaterials

Kang, J., Sangwan, V.K., Wood, J.D. and Hersam, M.C. Acc. Chem. Res., 50(4), 943-951 (2017)   Exfoliation of single-layer graphene from bulk graphite and the subsequent discovery of exotic physics and emergent phenomena in the atomically thin limit has motivated the isolation of other two-dimensional (2D) layered nanomaterials. Early work on isolated 2D nanomaterial flakes has revealed a broad range of unique physical and chemical properties with potential utility in diverse applications. For example, the electronic and optical properties of 2D nanomaterials depend strongly on atomic-scale variations in thickness, enabling enhanced performance in optoelectronic technologies such as light emitters, photodetectors, and photovoltaics. Much of the initial research on 2D nanomaterials has relied on micromechanical exfoliation, which yields high-quality 2D nanomaterial flakes that are suitable for fundamental studies but possesses limited scalability for real-world applications. In an effort to overcome this limitation, solution-processing methods for isolating large quantities of 2D nanomaterials have emerged. Importantly, solution processing results in 2D nanomaterial dispersions that are amenable to roll-to-roll fabrication methods that underlie lost-cost manufacturing of thin-film transistors, transparent conductors, energy storage devices, and solar cells. Despite these advantages, solution-based exfoliation methods typically lack control over the lateral size and thickness of the resulting 2D nanomaterial flakes, resulting in polydisperse dispersions with heterogeneous properties. Therefore, post-exfoliation separation techniques are needed to achieve 2D nanomaterial dispersions with monodispersity in lateral size, thickness, and properties. In this Account, we survey the latest developments in solution-based separation methods that aim to produce monodisperse dispersions and thin films of emerging 2D nanomaterials such as graphene, boron nitride, transition metal dichalcogenides, and black phosphorus. First, we motivate the need for precise thickness control in 2D nanomaterials by reviewing thickness-dependent physical properties. Then we present a succinct survey of solution-based exfoliation methods that yield 2D nanomaterial dispersions in organic solvents and aqueous media. The Account subsequently focuses on separation methods, including a critical analysis of their relative strengths and weaknesses for 2D nanomaterials with different buoyant densities, van der Waals interactions, and chemical reactivities. Specifically, we evaluate sedimentation-based density gradient ultracentrifugation (sDGU) and isopycnic DGU (iDGU) for post-exfoliation 2D nanomaterial dispersion separation. The comparative advantages of sedimentation and isopycnic methods are presented in both aqueous and nonaqueous media for 2D nanomaterials with varying degrees of chemical reactivity. Finally, we survey methods for forming homogeneous thin films from 2D nanomaterial dispersions and emerging technologies that are likely to benefit from these structures. Overall, this Account provides not only an overview of the present state-of-the-art but also a forward-looking vision for the field of solution-processed monodisperse 2D nanomaterials.  

6.205           Conjugates between photosystem I and a carbon nanotube for a photoresponse device

Nii, D., Miyachi, M., Shimada, Y., Nozawa, Y., Ito, M., Homma, Y., Ikehira, S., Yamanoi, Y., Nishihara, H. and Tomo, T. Photosynth. Res., 133(1-3), 155-162 (2017)   Photosystem I (PS I) is a large pigment–protein complex embedded in the thylakoid membranes that performs light-driven electron transfer across the thylakoid membrane. Carbon nanotubes exhibit excellent electrical conductivities and excellent strength and stiffness. In this study, we generated PSI–carbon nanotube conjugates dispersed in a solution aimed at application in artificial photosynthesis. PS I complexes in which a carbon nanotube binding peptide was introduced into the middle of the PsaE subunit were conjugated on a single-walled carbon nanotube, orienting the electron acceptor side to the nanotube. Spectral and photoluminescence analysis showed that the PS I is bound to a single-walled carbon nanotube, which was confirmed by transmission electron microscopy. Photocurrent observation proved that the photoexcited electron originated from PSI and transferred to the carbon nanotube with light irradiation, which also confirmed its orientated conjugation. The PS I–carbon nanotube conjugate will be a useful nano-optoelectronic device for the development of artificial systems.      

6.206           Edge Delamination of Monolayer Transition Metal Dichalcogenides

Ly, T.H., Yun, S.J., Thi, Q.H. and Zhao, J. ACS Nano, 11(7), 7534-7541 (2017)   Delamination of thin films from the supportive substrates is a critical issue within the thin film industry. The emergent two-dimensional, atomic layered materials, including transition metal dichalcogenides, are highly flexible; thus buckles and wrinkles can be easily generated and play vital roles in the corresponding physical properties. Here we introduce one kind of patterned buckling behavior caused by the delamination from a substrate initiated at the edges of the chemical vapor deposition synthesized monolayer transition metal dichalcogenides, led by thermal expansion mismatch. The atomic force microscopy and optical characterizations clearly showed the puckered structures associated with the strain, whereas the transmission electron microscopy revealed the special sawtooth-shaped edges, which break the geometrical symmetry for the buckling behavior of hexagonal samples. The condition of the edge delamination is in accordance with the fracture behavior of thin film interfaces. This edge delamination and buckling process is universal for most ultrathin two-dimensional materials, which requires more attention in various future applications.  

6.207           3D-printed smartphone-based point of care tool for fluorescence- and magnetophoresis-based cytometry

Knowlton, S., Joshi, A., Syrrist, P., Coskun, A.F. and Tasoglu, S. Lab on a Chip, 17, 2839-2851 (2017)   In developing countries, there are often limited resources available to provide important medical diagnostics, which severely limits our ability to diagnose conditions and administer proper treatment, leading to high mortality rates for treatable conditions. Here, we propose a multiplex tool capable of density-based cell sorting via magnetic focusing in parallel with fluorescence imaging to provide highly specific clinical assays. While many cell sorting techniques and fluorescence microscopes generally are costly and require extensive user training, limiting accessibility and usability in developing countries, this device is compact, low-cost, and portable. The device can separate cells on the basis of density, which can be used to identify cell type and cell activity, and image the cells in either brightfield, darkfield, or fluorescent imaging modes using the built-in smartphone camera. The combination of these two powerful and versatile techniques – magnetic focusing and fluorescence imaging – will make this platform broadly applicable to a range of biomedical assays. Clinical applications include cell cytometry and immunocytochemistry-based assays in limited-resource settings, which can ultimately help to improve worldwide accessibility to medical diagnostics.  

6.208           Skewness Analysis in Variance Spectroscopy Measures Nanoparticle Individualization

Sanchez, S.R., Bachilo, S.M., Kadria-Vili, Y. and Weisman, R.B.

1. Phys. Chem. Lett., 8, 2924-2929 (2017)

  An important enabling step in nanoparticle studies is the sorting of heterogeneous mixtures to prepare structurally homogeneous samples. It is also necessary to detect and monitor aggregation of the individual nanoparticles. Although variance spectroscopy provides a simple optical method for finding low concentrations of heteroaggregates in samples such as single-walled carbon nanotube dispersions, it cannot detect the homoaggregates that are relevant for well-sorted samples. Here we demonstrate that variance spectral data can be further analyzed to find third moments of intensity distributions (skewness), which reveal the presence of emissive homoaggregates. Using experimental measurements on variously processed nanotube dispersions, we deduce a simple numerical standard for recognizing aggregation in the highly sorted samples that are increasingly available to nanoscience researchers.  

6.209           A Low Energy Route to DNA-Wrapped Carbon Nanotubes via Replacement of Bile Salt Surfactants

Streit, J.K., Fagan, J.A. and Zheng, M. Anal. Chem., 89, 10496-1+503 (2017)   DNA-wrapped carbon nanotubes are a class of bionano hybrid molecules that have enabled carbon nanotube sorting, controlled assembly, and biosensing and bioimaging applications. The current method of synthesizing these hybrids via direct sonication of DNA/nanotube mixtures is time-consuming and not suitable for high-throughput synthesis and combinatorial sequence screening. Additionally, the direct sonication method does not make use of nanotubes presorted by extensively developed surfactant-based methods, is not effective for large diameter (>1 nm) tubes, and cannot maintain secondary and tertiary structural and functional domains present in certain DNA sequences. Here, we report a simple, quick, and robust process to produce DNA-wrapped carbon nanotube hybrids with nanotubes of broad diameter range and DNA of arbitrary sequence. This is accomplished by exchanging strong binding bile salt surfactant coating with DNA in methanol/water mixed solvent and subsequent precipitation with isopropyl alcohol. The exchange process can be completed within 10 min and converts over 90% nanotubes into the DNA wrapped form. Applying the exchange process to nanotubes presorted by surfactant-based methods, we show that the resulting DNA-wrapped carbon nanotubes can be further sorted to produce nanotubes with defined handedness, helicity, and endohedral filling. The exchange method greatly expands the structural and functional variety of DNA-wrapped carbon nanotubes and opens possibilities for DNA-directed assembly of structurally sorted nanotubes and high-throughput screening of properties that are controlled by the wrapping DNA sequences.  

6.210           Determination of the Absolute Enantiomeric Excess of the Carbon Nanotube Ensemble by Symmetry Breaking Using the Optical Titration Method

Sim, J., Kim, S., Jang, M., Park, M., Oh, H. and Ju, S-Y. Langmuir, 33, 11000-11109 (2017)   Symmetry breaking of single-walled carbon nanotubes (SWNTs) has profound effects on their optoelectronic properties that are essential for fundamental study and applications. Here, we show that isomeric SWNTs that exhibit identical photoluminescence (PL) undergo symmetry breaking by flavin mononucleotide (FMN) and exhibit dual PLs and different binding affinities (K_a). Increasing the FMN concentration leads to systematic PL shifts of SWNTs according to structural modality and handedness due to symmetry breaking. Density gradient ultracentrifugation using a FMN–SWNT dispersion displays PL shifts and different densities according to SWNT handedness. Using the optical titration method to determine the PL-based K_a of SWNTs against an achiral surfactant as a titrant, left- and right-handed SWNTs display two-step PL inflection corresponding to respective K_a values with FMN, which leads to the determination of the enantiomeric excess (ee) of the SWNT ensemble that was confirmed by circular dichroism measurement. Decreasing the FMN concentration for the SWNT dispersion leads to enantiomeric selection of SWNTs. The titration-based ee determination of the widely used sodium cholate-based SWNT dispersion was also demonstrated by using FMN as a cosurfactant.  

6.211           Separation of Nickelocene-Filled Single-Walled Carbon Nanotubes by Conductivity Type and Diameter

Kharlamova, M.V., Kramberger, C., Yanagi, K., Sauer, M., Saito, T. and Pichler, T. Phys. Status Solidi B., 254, 1700178 (2017)   We have performed the separation of nickelocene-filled single-walled carbon nanotubes (SWCNTs) with a mean diameter of 1.7 nm by conductivity type and diameter using density gradient ultracentrifugation. The separated nanotube fractions were characterized by optical absorption spectroscopy (OAS), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. The OAS investigation of the separated samples showed the absence of optical absorption bands of semiconducting tubes in metallic sample and vice versa, which proved a high purity of the nanotube fractions. The XPS investigation of the separated samples showed different positions, spectral widths and asymmetry of the C 1s core level spectra. The multifrequency Raman spectroscopy investigation of the separated samples allowed performing an analysis of the diameter range of nanotubes. The demonstrated successful separation of the filled SWCNTs by conductivity type and diameter enables demanding applications of ultra pure SWCNTs with tailored properties.  

6.212           Inner tube growth and electronic properties of metallicity-sorted nickelocene-filled semiconducting single-walled carbon nanotubes

Kharlamova, M.V., Kramberger, C., Sauer, M., Yanagi, K., Saito, T. and Pichler, T. Applied Physiscs A, 124:247 (2018)   In the present work, we have obtained metallicity-sorted nickelocene-filled semiconducting single-walled carbon nanotubes (SWCNTs) by density gradient separation of metallicity mixed filled nanotubes. Double-walled carbon nanotubes (DWCNTs) were obtained by annealing of filled SWCNTs in vacuum. The diameter distribution of inner tubes was analyzed by multifrequency Raman spectroscopy. The chemical transformation of nickelocene upon annealing was studied by X-ray photoelectron spectroscopy (XPS) at the Ni 2p core level. The thermally-induced transformation of nickelocene to nickel carbides and metallic nickel was revealed. The electronic properties of the filled SWCNTs and DWCNTs were investigated by XPS at the C 1s core level. By tracing the C 1s binding energy, it was shown that the annealing of nickelocene-filled SWCNTs at low temperatures (360–600 °C) led to electron doping of SWCNTs, whereas annealing at high temperatures and formation of DWCNTs (680–1200 °C) resulted in hole doping of nanotubes.  

6.213           Extended-conjugation π-electron systems in carbon nanotubes

Miyaura, K., Miyata, Y., Thendie, B., Yanagi, K., Kitaura, R., Yamamoto, Y., Arai, S., Kataura, H. and Shinohara, H. Scientific Reports, 8:8098 (2018)   Extending π-electron systems are among the most important topics in physics, chemistry and materials science because they can result in functional materials with applications in electronics and optics. Conventional processes for π-electron extension, however, can generate products exhibiting chemical instability, poor solubility or disordered structures. Herein, we report a novel strategy for the synthesis of π-conjugated polymers within the interiors of carbon nanotubes (CNTs). In this process, thiophene-based oligomers are encapsulated within CNTs as precursors and are subsequently polymerized by thermal annealing. This polymerization increases the effective conjugation length of the thiophenes, as confirmed by transmission electron microscopy and absorption peak red shifts. This work also demonstrates that these polythiophenes can serve as effective markers for individual CNTs during Raman imaging with single-wavelength laser excitation due to their strong absorbance. In addition, stable carrier injection into the encapsulated polythiophenes is found to be possible via electrochemical doping. Such doping has the potential to produce π-electron-based one-dimensional conductive wires and highly stable electrochromic devices.  

6.214           Density gradient ultracentrifugation for colloidal nanostructures separation and investigation

Li, P., Kumar, A., Ma, J., Kuang, Y., Luo, L. and Sun, X. Science Bulletin, 63, 645-662 (2018)   In this article, we review the advancement in nanoseparation and concomitant purification of nanoparticles (NPs) by using density gradient ultracentrifugation technique (DGUC) and demonstrated by taking several typical examples. Study emphasizes the conceptual advances in classification, mechanism of DGUC and synthesis-structure-property relationships of NPs to provide the significant clue for the further synthesis optimization. Separation, concentration, and purification of NPs by DGUC can be achieved at the same time by introducing the water/oil interfaces into the separation chamber. We can develop an efficient method “lab in a tube” by introducing a reaction zone or an assembly zone in the gradient to find the surface reaction and assembly mechanism of NPs since the reaction time can be precisely controlled and the chemical environment change can be extremely fast. Finally, to achieve the best separation parameters for the colloidal systems, we gave the mathematical descriptions and computational optimized models as a new direction for making practicable and predictable DGUC separation method. Thus, it can be helpful for an efficient separation as well as for the synthesis optimization, assembly and surface reactions as a potential cornerstone for the future development in the nanotechnology and this review can be served as a plethora of advanced notes on the DGUC separation method.  

6.215           Synaptic Computation Enabled by Joule Heating of Single-Layered Semiconductors for Sound Localization

Sun, L., Zhang, Y., Hwang, G., Jiang, J., Kim, D., Eshete, Y.A., Zhao, R. and Yang, H. Nano Lett., 18(5), 3229-3234 (2018)   Synaptic computation, which is vital for information processing and decision making in neural networks, has remained technically challenging to be demonstrated without using numerous transistors and capacitors, though significant efforts have been made to emulate the biological synaptic transmission such as short-term and long-term plasticity and memory. Here, we report synaptic computation based on Joule heating and versatile doping induced metal–insulator transition in a scalable monolayer-molybdenum disulfide (MoS₂) device with a biologically comparable energy consumption (∼10 fJ). A circuit with our tunable excitatory and inhibitory synaptic devices demonstrates a key function for realizing the most precise temporal computation in the human brain, sound localization: detecting an interaural time difference by suppressing sound intensity- or frequency-dependent synaptic connectivity. This Letter opens a way to implement synaptic computing in neuromorphic applications, overcoming the limitation of scalability and power consumption in conventional CMOS-based neuromorphic devices.  

6.216           Chapter 19 - Chromatography as an Efficient Technique for the Separation of Diversified Nanoparticles

Hameed, B.S., Bhatt, C.S., Nagaraj, B. and Suresh, N.A. Nanomaterials in Chromatography (Elsevier), 503-518 (2018)   Chromatography is emerging as an efficient technique for the separation of engineered nanoparticles (NPs) and has been gaining tremendous attention due to its ease, facile, and cost-effective nature. This chapter puts together the various chromatography techniques so far implemented to separate or fractionate various NPs in detail. The influence of morphological features of the NPs along with overall surface properties that can aid their separation using chromatography will be highlighted. Finally, this chapter also provides an overview of the various analytical and advanced physical characterization techniques often used to evaluate the successful separation of nanoparticles.  

6.217           Fermi level engineering of metallicity-sorted metallic single-walled carbon nanotubes by encapsulation of few-atom-thick crystals of silver chloride

Kharmanova, M.V., Kramberger, C., Domanov, O., Mittelberger, A., Yanagi, K., Pichler, T. and Eder, D.

1. Mater. Sci., 53(18), 13018-13029 (2018)

  In the present work, the channels of metallicity-sorted metallic single-walled carbon nanotubes (SWCNTs) have been filled with silver chloride. The data of high-resolution scanning transmission electron microscopy proved the filling of the nanotube channels and formation of few-atom-thick crystals of silver chloride. The electronic properties of the filled SWCNTs were investigated by Raman spectroscopy, X-ray photoelectron spectroscopy, and ultraviolet photoelectron spectroscopy. Our results indicate the p-doping of nanotubes by silver chloride accompanied by the charge transfer from the nanotubes to the encapsulated compound and the downshift of the Fermi level by 0.36 eV. The calculated number of transferred electrons per nanotube carbon atom and the charge transfer density per nanotube length amounted to 0.0024 e⁻ per carbon and 0.0406 e⁻/Å, respectively. It was found that the band gap opens up in the band structure of the filled SWCNTs resulting in their transition from metallic into a semiconducting state. This work reveals a direct influence of the incorporated silver chloride on the electronic properties of metallicity-sorted metallic SWCNTs and demonstrates the potential of precise Fermi level engineering of SWCNTs by filling their channels and achieving high doping levels, thus providing a platform for designing next-generation nanoelectronic devices.  

6.218           Direct growth of doping controlled monolayer WSe2 by selenium-phosphorus substitution

Kang, W.T., Lee, I.M., Yun, S.J., Song, Y.I., Kim, K., Kim, D-H., Shin, Y.S., Lee, K., Heo, J., Kim, Y-M., Lee, Y.H. and Yu, W.J. Nanoscale, 10, 11397-11402 (2018)   Although many studies have been carried out on the doping of transition metal dichalcogenides (TMDCs), introducing controllable amounts of dopants into a TMD lattice is still insufficient. Here we demonstrate doping controlled TMDC growth by the replacement of selenium with phosphorus during the synthesis of the monolayer WSe₂. The phosphorus doping density was precisely controlled by fine adjustment of the amount of P₂O₅ dopant powder along the pre-annealing time. Raman spectroscopy, photoluminescence (PL), X-ray photoelectron spectroscopy (XPS), and high-angle annular bright field scanning tunneling electron microscopy (HAADF STEM) provide evidence that P doping occurs within the WSe₂ crystal with P occupying the substitutional Se sites. With regard to its electrical characteristics, the hole majority current of P-doped WSe₂ is 100-times higher than that of the intrinsic WSe₂. The measured doping concentration ranged from ∼8.16 × 10¹⁰ to ∼1.20 × 10¹² depending on the amount of P₂O₅ dopant powder by pre-annealing.  

6.219           Functional inks and printing of two-dimensional materials

Hu, G., Kang, J., Ng, L.W.T., Zhu, X., Howe, R.C.T., Jones, C.G., Hersam, M.C. and Hasan, T. Chem. Soc. Rev., 47, 3265.3300 (2018)   Graphene and related two-dimensional materials provide an ideal platform for next generation disruptive technologies and applications. Exploiting these solution-processed two-dimensional materials in printing can accelerate this development by allowing additive patterning on both rigid and conformable substrates for flexible device design and large-scale, high-speed, cost-effective manufacturing. In this review, we summarise the current progress on ink formulation of two-dimensional materials and the printable applications enabled by them. We also present our perspectives on their research and technological future prospects.  

6.220           Ultrashort Carbon Nanotubes That Fluoresce Brightly in the Near-Infrared

Danne, N., Kim, M., Godin, A.G., Kwon, H., Gao, Z., Wu, X., Hartmann, N.F., Doorn, S.K., Lounis, B., Wang, Y. and Cognet, L. ACSNano, 12(6), 6059-6065 (2018)   The intrinsic near-infrared photoluminescence observed in long single-walled carbon nanotubes is known to be quenched in ultrashort nanotubes due to their tiny size as compared to the exciton diffusion length in these materials (>100 nm). Here, we show that intense photoluminescence can be created in ultrashort nanotubes (∼40 nm length) upon incorporation of exciton-trapping sp³ defect sites. Using super-resolution photoluminescence imaging at <25 nm resolution, we directly show the preferential localization of excitons at the nanotube ends, which separate by less than 40 nm and behave as independent emitters. This unexpected observation opens the possibility to synthesize fluorescent ultrashort nanotubes—a goal that has been long thought impossible—for bioimaging applications, where bright near-infrared photoluminescence and small size are highly desirable, and for quantum information science, where high quality and well-controlled near-infrared single photon emitters are needed.  

6.221           Extraction of Linear Carbon Chains Unravels the Role of the Carbon Nanotube Host

Shi, L., Yanagi, K., Cao, K., Kaiser, U., Ayala, P. and Pichler, T. ACSNano, 12(8), 8477-8484 (2018)   Linear carbon chains (LCCs) have been shown to grow inside double-walled carbon nanotubes (DWCNTs), but isolating them from this hosting material represents one of the most challenging tasks toward applications. Herein we report the extraction and separation of LCCs inside single-walled carbon nanotubes (LCCs@SWCNTs) extracted from a double-walled host LCCs@DWCNTs by applying a combined tip-ultrasonic and density gradient ultracentrifugation (DGU) process. High-resolution transmission electron microscopy, optical absorption, and Raman spectroscopy show that not only short LCCs but clearly long LCCs (LLCCs) can be extracted and separated from the host. Moreover, the LLCCs can even be condensed by DGU. The Raman spectral frequency of LCCs remains almost unchanged regardless of the presence of the outer tube of the DWCNTs. This suggests that the major importance of the outer tubes is making the whole synthesis viable. We have also been able to observe the interaction between the LCCs and the inner tubes of DWCNTs, playing a major role in modifying the optical properties of LCCs. Our extraction method suggests the possibility toward the complete isolation of LCCs from CNTs.  

6.222           Coating two-dimensional MoS2 with polymer creates a corrosive non-uniform interface

Thi, Q.H., Kim, H., Zhao, J. and Ly, T.H. NPJ/2D Materials and Applications, 2:34 (2018)   Two-dimensional (2D) materials and soft materials are both susceptible to mechanical instabilities, such as buckling, wrinkling, folding and creasing, especially when located on surfaces. Here, we report that weak van der Waals interactions cause the interface between 2D molybdenum disulphide (MoS₂) and a soft poly(methyl methacrylate) coating to demonstrate mechanical instability and delamination. The resulting non-uniform and buckled interface greatly hampers the ability of the coating to protect the MoS₂ substrate. Also, the corrosion rate of 2D MoS₂ and quench rate of intrinsic luminescence in 2D MoS2 were significantly accelerated by the soft coating. Owing to the formation of corrosive cavities at the interface, the geometry and size of the flakes became the dominating factor, and a critical size of 2D flakes for such interfacial instability was determined based on elasticity theory. Such hazardous corrosion in a 2D material caused by a soft coating raises concern for their use in electronic packaging, and for the processing of van der Waals-layered materials for future applications.  

6.223           Stretchable Transparent Conductive Films from Long Carbon Nanotube Metals

Wang, P., Peng, Z., Li, M. and Wang, Y. Small, 14(14), 1802625 (2018)   Flexible transparent conductors are an enabling component for large‐area flexible displays, wearable electronics, and implantable medical sensors that can wrap around and move with the body. However, conventional conductive materials decay quickly under tensile strain, posing a significant hurdle for functional flexible devices. Here, we show that high electrical conductivity, mechanical stretchability, and optical transparency can be simultaneously attained by compositing long metallic double‐walled carbon nanotubes with a polydimethylsiloxane substrate. When stretched to 100% tensile strain, thin films incorporating these long nanotubes (≈3.2 µm on average) achieve a record high conductivity of 3316 S cm⁻¹ at 100% tensile strain and 85% optical transmittance, which is 194 times higher than that of short nanotube controls (≈0.8 µm on average). Moreover, the high conductivity can withstand more than 1000 repeated stretch‐release cycles (switching between 100% and 0% strain) with a retention approaching 96%, whereas the short nanotube controls exhibit only 10%. Mechanistic studies reveal that long tubes can bridge the microscale gaps generated during stretching, thereby maintaining high electrical conductivity. When mounted on human joints, this elastic transparent conductor can accommodate large motions to provide stable, high current output. These results point to transparent conductors capable of attaining high electrical conductivity and optical transmittance under mechanical strain to allow large shape changes that may take place in the operation and use of flexible electronics.  

6.224           Enantiomers of Single-Wall Carbon Nanotubes Show Distinct Coating Displacement Kinetics

Zheng, Y., Bachilo, S.M. and Weisman, R.B.

1. Phys. Chem. Lett., 9(13), 3793-3797 (2018)

  It is known that specific oligomers of single-stranded DNA (ssDNA) can show remarkable selectivity when coating different structural species of single-wall carbon nanotubes (SWCNTs). We report that (ATT)₄ ssDNA coatings strongly distinguish between the two optical isomers of (7,5) SWCNTs. This causes resolvable shifts in their fluorescence spectra and differences of 2 orders of magnitude in the room temperature rates of coating displacement, as monitored through changes in nanotube fluorescence wavelength and intensity on exposure to sodium deoxycholate. During coating displacement, the enantiomer with high affinity for the ssDNA oligomer is deduced to form an intermediate hybrid that is not observed for the low affinity enantiomer. These results reveal that enantiomeric differences in SWCNTs complexed with ssDNA are more diverse and dramatic than previously recognized.  

6.225           Synthesis, purification, properties and characterization of sorted single-walled carbon nanotubes

Bati, A.S.R., Yu, L., Batrmunkh, M. and Shapter, J.G. Nanoscale, 10, 22087-22139 (2018)   Single-walled carbon nanotubes (SWCNTs) have attracted significant attention due to their outstanding mechanical, chemical and optoelectronic properties, which makes them promising candidates for use in a wide range of applications. However, as-produced SWCNTs have a wide distribution of various chiral species with different properties (i.e. electronic structures). In order to take full advantage of SWCNT properties, highly purified and well-separated SWCNTs are of great importance. Recent advances have focused on developing new strategies to effectively separate nanotubes into single-chirality and/or semiconducting/metallic species and integrating them into different applications. This review highlights recent progress in this cutting-edge research area alongside the enormous development of their identification and structural characterization techniques. A comprehensive review of advances in both controlled synthesis and post-synthesis separation methods of SWCNTs are presented. The relationship between the unique structure of SWCNTs and their intrinsic properties is also discussed. Finally, important future directions for the development of sorting and purification protocols for SWCNTs are provided.  

6.226           Size‐dependent antibacterial activity for laser‐generated silver nanoparticles

Korshed, P., Li, L., Liu, Z., Mironov, A. and Wang, T.

1. Interdisciplinary Nanomed., 4(1), 24-33 (2019)

  Silver nanoparticles (Ag NPs) have been used widely for antibacterial applications; however, the effects of their sizes on antibacterial activities and toxicities to human cells, particularly for the laser‐generated Ag NPs, are not fully understood. In this study, sucrose gradient centrifugation was used to separate laser‐generated Ag NPs into different fractions by size. Transmission electron microscopy was used to analyze the size distribution of the Ag NPs, and well diffusion method was used to evaluate the antibacterial activity of the Ag NP fractions against the Escherichia coli. Results showed that the antibacterial effects of laser‐generated Ag NPs inversely correlated to the particle size. Among Ag NP fractions with average sizes ranging 19–47 nm, the 19‐nm Ag NPs presented the highest bactericidal effect. The smaller sized laser Ag NPs also significantly induced the generation of reactive oxygen species when applied to E. coli, compared with that of the larger sized laser Ag NPs. Cytotoxicity analysis revealed that the different sized laser‐generated Ag NPs were not significantly toxic to the human fibroblasts and lung epithelial cells in a 72‐h in vitro cell culture period. Understanding the size‐dependent functional properties of the laser‐generated Ag NPs helps informing the designs for future applications of the laser‐generated Ag NPs.  

6.227           Passively Q-switched laser with specific chiral SWCNTs selected by N-CS as saturable absorber at 1 µm

Zhang, X., Chu, H., Li, Y., Zhang, Y., Zhao, S. and Li, D. Optics Comm., 462, 125315 (2020)   The single-wall carbon nanotubes (SWCNTs) with the specific chirality (6, 5) and (7, 5) were sorted out by the density gradient ultracentrifugation (DGU) method. 2D photoluminescence excitation–emission map and the Raman spectrum confirmed the successful chirality-selection. The nonlinear optical performance of the chirality-selected SWCNTs was investigated by an open-aperture Z-scan setup. By employing the prepared SWCNTs saturable absorber (SWCNTs-SA), the minimum pulse duration of 115 ns with a pulse repetition rate of 550 kHz was obtained in the passively Q-switched (PQS) laser operation.  

6.228           Near-infrared fluorescence as a method for determining single-wall carbon nanotube extraction conditions in aqueous two polymer phase extraction

Sims, C.M. and Fagan, J.A: Carbon, 165, 196-203 (2002)   Near-infrared fluorescence emission from single-wall carbon nanotubes (SWCNTs) is demonstrated as a measurement technique enabling the determination of aqueous surfactant concentration conditions yielding upper or lower polymer phase extraction in aqueous two-polymer phase extraction (ATPE) separations. From measurements on single-phase dispersions of multiple SWCNT specie mixtures, the fluorescence intensity of a given SWCNT (n,m) species is demonstrated to correlate directly to partitioning in an ATPE separation, as determined by the type of surfactant molecule comprising a preponderance of the adsorbed interfacial layer on that species. This methodology enables rapid quantitative determination of partitioning conditions for individual (n,m) species of SWCNTs without ambiguity of mass transfer limitations or interfacial adsorption present when conducting the ATPE separation itself, while also allowing for ready investigation of complicating factors such as temperature effects or SWCNT enantiomers.  

6.229           Exfoliation of 2D Materials for Energy and Environmental Applications

Le, T-H., Oh, Y., Kim, H. and Yoon, H. Chem. Eur. J., 26, 6360-6401 (2020)   The fascinating properties of single‐layer graphene isolated by mechanical exfoliation have inspired extensive research efforts toward two‐dimensional (2D) materials. Layered compounds serve as precursors for atomically thin 2D materials (briefly, 2D nanomaterials) owing to their strong intraplane chemical bonding but weak interplane van der Waals interactions. There are newly emerging 2D materials beyond graphene, and it is becoming increasingly important to develop cost‐effective, scalable methods for producing 2D nanomaterials with controlled microstructures and properties. The variety of developed synthetic techniques can be categorized into two classes: bottom‐up and top‐down approaches. Of top‐down approaches, the exfoliation of bulk 2D materials into single or few layers is the most common. This review highlights chemical and physical exfoliation methods that allow for the production of 2D nanomaterials in large quantities. In addition, remarkable examples of utilizing exfoliated 2D nanomaterials in energy and environmental applications are introduced.  

6.230           Vacancies on 2D transition metal dichalcogenides elicit ferroptotic cell death

Xu, S., Zheng, H., Ma, R., Wu, D., Pan, Y., Yin, C., Gao, M., Wang, W., Li, W., Liu, S., Chai, Z. and Li, R. Nature Communications, 11:3484 (2020)   Sustainable developments of nanotechnology necessitate the exploration of structure-activity relationships (SARs) at nano-bio interfaces. While ferroptosis may contribute in the developments of some severe diseases (e.g., Parkinson’s disease, stroke and tumors), the cellular pathways and nano-SARs are rarely explored in diseases elicited by nano-sized ferroptosis inducers. Here we find that WS₂ and MoS₂ nanosheets induce an iron-dependent cell death, ferroptosis in epithelial (BEAS-2B) and macrophage (THP-1) cells, evidenced by the suppression of glutathione peroxidase 4 (GPX4), oxygen radical generation and lipid peroxidation. Notably, nano-SAR analysis of 20 transition metal dichalcogenides (TMDs) disclosures the decisive role of surface vacancy in ferroptosis. We therefore develop methanol and sulfide passivation as safe design approaches for TMD nanosheets. These findings are validated in animal lungs by oropharyngeal aspiration of TMD nanosheets. Overall, our study highlights the key cellular events as well as nano-SARs in TMD-induced ferroptosis, which may facilitate the safe design of nanoproducts.  

6.231           Functional Inks for Printable Energy Storage Applications based on 2 D Materials

Wang, L., Chen, S., Shu, T. and Hu, X. ChemSusChem., 13, 1330-1353 (2020)   Ubiquitous portable electronics and the ever‐growing internet‐of‐things have necessitated the emergence of high‐end miniaturized devices as well as associated sophisticated printing technologies. With excellent solution processability and tunable electronic properties, 2 D materials stand as a promising candidate for functional inks that are readily printable for energy‐storage devices. In this Review, we outline the significance, status, and challenges that we are facing of the developments of 2 D materials‐based functional inks. Then, general ink formulation and basic knowledge of printing techniques together with their rheological requirements and enabled applications in energy storage are introduced, providing guidelines for developing inks that match well with the present printing techniques. Last, but not least, we also propose the perspectives on the development of 2 D materials‐based inks for energy‐storage applications.  

6.232           Solution-Processed Mixed-Dimensional Hybrid Perovskite/Carbon Nanotube Electronics

Ma, C., Clark, S., Liu, Z., Liang, L., Firdaus, Y., Tao, R., Han, A., Liu, X., Li, L-J., Anthopoulos, T.D., Hersam, M.C. and Wu, T. ACS Nano, 14, 3969-3979 (2020)   Benefiting from their extraordinary physical properties, methylammonium lead halide perovskites (PVKs) have attracted significant attention in optoelectronics. However, the PVK-based devices suffer from low carrier mobility and high operation voltage. Here, we utilize sorted semiconducting single-walled carbon nanotubes (95% s-SWCNTs) to enhance the performance of thin-film transistors (TFTs) based on the mixed-cation perovskite (MA_1–xFA_x)Pb(I_1–xBr_x)₃, enabling mixed-dimensional solution-processed electronics with high mobility (32.25 cm²/(V s)) and low voltage (∼3 V) operation. The resulting mixed-dimensional PVK/SWCNT TFTs possess ON/OFF ratios on the order of 10⁷, enabling the fabrication of high-gain inverters.  

6.233           Disentangling oxygen and water vapor effects on optoelectronic properties of monolayer tungsten disulfide

Zhang, H., Dunklin, J.R., Reid, O.G., Yun, S.J., Nanayakkara, S.U., Lee, Y.H., Blackburn, J.L. and Miller, E.M. Nanoscale, 12, 8344-8354 (2020)   By understanding how the environmental composition impacts the optoelectronic properties of transition metal dichalcogenide monolayers, we demonstrate that simple photoluminescence (PL) measurements of tungsten disulfide (WS₂) monolayers can differentiate relative humidity environments. In this paper, we examine the PL and photoconductivity of chemical vapor deposition grown WS₂ monolayers under three carefully controlled environments: inert gas (N₂), dry air (O₂ in N₂), and humid nitrogen (H₂O vapor in N₂). The WS₂ PL is measured as a function of 532 nm laser power and exposure time and can be decomposed into the exciton, trion, and lower energy state(s) contributions. Under continuous illumination in either O₂ or H₂O vapor environment, we find dramatic (and reversible) increases in PL intensity relative to the PL in an inert environment. The PL bathochromically shifts in an O₂ environment and is dominated by increased trion emission and diminished exciton emission. In contrast, the WS₂ PL increase in a H₂O environment results from an overall increase in emission from all spectral components where the exciton contribution dominates. The drastic increases in PL are anticorrelated with corresponding decreases in photoconductivity, as measured by time-resolved microwave conductivity. The results suggest that both O₂ and H₂O react photochemically with the WS₂ monolayer surface, modifying the optoelectronic properties, but do so via distinct pathways. Thus, we use these optoelectronic differences to differentiate the amount of humidity in the air, which we show with 0%, 40%, and 80% relative humidity environments. This deeper understanding of how ambient conditions impact WS₂ monolayers enables novel humidity sensors as well as a better understanding of the correlation between TMDC surface chemistry, light emission, and photoconductivity. Moreover, these WS₂ measurements highlight the importance of considering the impact of the local environment on reported results.  

6.234           Growth Mechanism of Alternating Defect Domains in Hexagonal WS2 via Inhomogeneous W-Precursor Accumulation

An, G.H., Yun, S.J., Lee, Y.H. and Lee, H.S. Small, 16, 2003326 (2020)   While a hexagonal WS₂ monolayer, grown by chemical vapor deposition, exhibits distinctive patterns in photoluminescence mapping, segmented with alternating S-vacancy (SV) and W-vacancy (WV) domains in a single crystal, the formation mechanism for native alternating defect domains remains unresolved to date. Here, the formation mechanism of alternating defect domains in hexagonal WS₂ via the precursor accumulation model is experimentally elucidated. A triangular WS₂ seed is initially formed, followed by a hexagonal flake. Alternating W-rich (SV) and W-deficient (WV) domains are constructed in hexagonal WS₂ flake, which is confirmed by confocal photoluminescence mapping and secondary ion mass spectroscopy. This is explained by the accumulation or scarcity of W-precursors at the edge of the WS₂ flake. The W-precursors accumulate near the edges of the initial triangular WS₂ seed over time, while they are deficient near the corners of the triangular WS₂, eventually forming WV domains in the truncated hexagonal domains. The heterogeneous accumulation becomes more prominent in the presence of H₂ gas through desorption of the W-precursors.  

6.235           12 - Production of graphene and other two-dimensional nanosheets by liquid phase exfoliation

Backes, C. Graphene (Second edition), 251-314(2021)   In 2008, it was first demonstrated that layered crystals such as graphite can be exfoliated down to monolayers in the liquid phase by a relatively simple and scalable methodology termed liquid phase exfoliation. Importantly, this strategy is widely applicably to many structures with only minor modifications to the process. By now, > 10 classes of layered materials have been made accessible as 2-dimensional nanosheets in a solution-processable for by this technique. Various exfoliation methods and stabilizers have been investigated to date. In this chapter, advancements in exfoliation and size selection are summarized and the current picture of the exfoliation mechanism outlined. Remaining challenges, open questions, and perspectives are also discussed.  

6.236           Selective filling of n-hexane in a tight nanopore

Qu, H., Rayabharam, A., Wu, X., Wang, P., Li, Y., Fagan, J., Aluru, N.R. and Wang, Y. Nature Comm., 12:310 (2021)   Molecular sieving may occur when two molecules compete for a nanopore. In nearly all known examples, the nanopore is larger than the molecule that selectively enters the pore. Here, we experimentally demonstrate the ability of single-wall carbon nanotubes with a van der Waals pore size of 0.42 nm to separate n-hexane from cyclohexane—despite the fact that both molecules have kinetic diameters larger than the rigid nanopore. This unexpected finding challenges our current understanding of nanopore selectivity and how molecules may enter a tight channel. Ab initio molecular dynamics simulations reveal that n-hexane molecules stretch by nearly 11.2% inside the nanotube pore. Although at a relatively low probability (28.5% overall), the stretched state of n-hexane does exist in the bulk solution, allowing the molecule to enter the tight pore even at room temperature. These insights open up opportunities to engineer nanopore selectivity based on the molecular degrees of freedom.  

6.237           Low background estimation of metallic-to-semiconducting carbon nanotube ratio by using infrared spectroscopy

Oi, K., Komoto, J., Kawai, T. and Nonoguchi, Y. Synthetic metals, 282, 116958 (2021)   The ratio of metallic and semiconducting carbon nanotubes (CNTs) populations is a crucial factor for elucidating their structure-property relationship. Visible-to-near infrared absorption spectroscopy with dispersion is mostly used for the estimation of the metal-to-semiconductor ratio. Due to spectral cofactors including background absorption, however, it is still uneasy to obtain correct population ratios. Herein the detection of pure semiconducting fractions is performed by using the Fourier transform infrared (FTIR) spectroscopy. The working principle is based on the fact that highly pure semiconducting CNTs show no far-infrared (FIR) plasmon resonance while unsorted films containing metallic CNTs have a strong FIR band. This off-on characteristics enables the evaluation of metallic-to-semiconducting CNTs population. Several limitations in this technique are discussed for avoiding significant artifacts. This FTIR-based, solid-state endpoint detection will lead to the straightforward investigation of purity-to-transport properties relationship in solid-state CNT thin films.  

6.238           Receptor-targeting nanomaterials alleviate binge drinking-induced neurodegeneration as artificial neurotrophins

Yang, J., Wang, L., Huang, L., Che, X., Zhang, Z. et al Exploration, 1, 61-74 (2021)   The distinguished properties of nanomaterials promote us to explore whether their intrinsic activities would be beneficial to disease treatment. Furthermore, understanding the molecular mechanism is thereby crucial for biomedical applications. Here, we investigate the therapeutic effects of single-walled carbon nanotubes (SWNTs) in a rat model of binge alcohol-induced neurodegeneration. With selection from four types of SWNT structures, bundled SWNTs (bSWNTs) facilitated the recovery of learning and memory via enhancing neuroprotection and neuroregeneration. We screened the potential target for bSWNTs, and found that bSWNTs have the abilities to directly interact with neurotrophic receptors, especially tropomyosin-related kinase B (TrkB). Moreover, similar to the actions of endogenous neurotrophins, bSWNTs could trigger the dimerization and phosphorylation of TrkB, while these conformational changes resulted in activating their downstream signals involved in neuroprotection and neuroregeneration. With relatively clear mechanisms, these “artificial neurotrophins” provide a proof-of-concept example as an efficiently therapeutic strategy for the treatment of neurodegenerative diseases.  

6.239           Local Optical Properties in CVD-Grown Monolayer WS2 Flakes

Magnozzi, M., Pflug, T., Ferrera, M., Pace, S., Ramo, L., Olbrich, M., Canepa, P., Agrican, H., Horn, A., Forti, S., Cavalleri, O., Coletti, C., Bisio, F. and Canepa, M.

1. Phys. Chem., 125, 16059-16065 (2021)

  Excitons dominate the light absorption and re-emission spectra of monolayer transition-metal dichalcogenides (TMD). Microscopic investigations of the excitonic response in TMD almost invariably extract information from the radiative recombination step, which only constitutes one part of the picture. Here, by exploiting imaging spectroscopic ellipsometry (ISE), we investigate the spatial dependence of the dielectric function of chemical vapor deposition (CVD)-grown WS₂ flakes with a microscopic lateral resolution, thus providing information about the spatially varying, exciton-induced light absorption in the monolayer WS₂. Comparing the ISE results with imaging photoluminescence spectroscopy data, the presence of several correlated features was observed, along with the unexpected existence of a few uncorrelated characteristics. The latter demonstrates that the exciton-induced absorption and emission features are not always proportional at the microscopic scale. Microstructural modulations across the flakes, having a different influence on the absorption and re-emission of light, are deemed responsible for the effect.  

6.240           Synthesis of Large-Scale Monolayer 1T′-MoTe2 and Its Stabilization via Scalable hBN Encapsulation

Pace, S., Martini, L., Convertino, D., Keum, D.H., Forti, S., Pezzini, S., Fabbri, F., Miseikis, V. and Coletti, C. ASC Nano, 15, 4213-4225 (2021)   Out of the different structural phases of molybdenum ditelluride (MoTe₂), the distorted octahedral 1T′ possesses great interest for fundamental physics and is a promising candidate for the implementation of innovative devices such as topological transistors. Indeed, 1T′-MoTe₂ is a semimetal with superconductivity, which has been predicted to be a Weyl semimetal and a quantum spin Hall insulator in bulk and monolayer form, respectively. Large instability of monolayer 1T′-MoTe₂ in environmental conditions, however, has made its investigation extremely challenging so far. In this work, we demonstrate homogeneous growth of large single-crystal (up to 500 μm) monolayer 1T′-MoTe₂via chemical vapor deposition (CVD) and its stabilization in air with a scalable encapsulation approach. The encapsulant is obtained by electrochemically delaminating CVD hexagonal boron nitride (hBN) from copper foil, and it is applied on the freshly grown 1T′-MoTe₂via a top-down dry lamination step. The structural and electrical properties of encapsulated 1T′-MoTe₂ have been monitored over several months to assess the degree of degradation of the material. We find that when encapsulated with hBN, the lifetime of monolayer 1T′-MoTe₂ successfully increases from a few minutes to more than a month. Furthermore, the encapsulated monolayer can be subjected to transfer, device processing, and heating and cooling cycles without degradation of its properties. The potential of this scalable heterostack is confirmed by the observation of signatures of low-temperature phase transition in monolayer 1T′-MoTe₂ by both Raman spectroscopy and electrical measurements. The growth and encapsulation methods reported in this work can be employed for further fundamental studies of this enticing material as well as facilitate the technological development of monolayer 1T′-MoTe₂.  

6.241           Selective Pattern Growth of Atomically Thin MoSe2 Films via a Surface-Mediated Liquid-Phase Promoter

Kang, W.T., Phan, T.L., Ahn, K.J., Lee, J., Kim, Y.R., Won, U.Y., Kim, J.E.K, Lee, Y.H. and Yu, W.J. ACS Appl. Mater. Interfaces, 13, 18056-18064 (2021)   Two-dimensional transition metal dichalcogenides (TMDs) offer numerous advantages over silicon-based application in terms of atomically thin geometry, excellent opto-electrical properties, layer-number dependence, band gap variability, and lack of dangling bonds. The production of high-quality and large-scale TMD films is required with consideration of practical technology. However, the performance of scalable devices is affected by problems such as contamination and patterning arising from device processing; this is followed by an etching step, which normally damages the TMD film. Herein, we report the direct growth of MoSe2 films on selective pattern areas via a surface-mediated liquid-phase promoter using a solution-based approach. Our growth process utilizes the promoter on the selective pattern area by enhancing wettability, resulting in a highly uniform MoSe2 film. Moreover, our approach can produce other TMD films such as WSe2 films as well as control various pattern shapes, sizes, and large-scale areas, thus improving their applicability in various devices in the future. Our patterned MoSe2 field-effect transistor device exhibits a p-type dominant conduction behavior with a high on/off current ratio of ∼106. Thus, our study provides general guidance for direct selective pattern growth via a solution-based approach and the future design of integrated devices for a large-scale application.  

6.242           One-Step Synthesis of NbSe2/Nb-Doped-WSe2 Metal/Doped-Semiconductor van der Waals Heterostructures for Doping Controlled Ohmic Contact

Vu, V.T., Vu, T.T.H., Phan, T.L., Kang, W.T., Kim, Y.R., Tran, M.D., Nguyen, H.T.T., Lee, H-L. and Yu, W.J. ACS Nano, 15, 13031-13040 (2021)   van der Waals heterostructures (vdWHs) of metallic (m-) and semiconducting (s-) transition-metal dichalcogenides (TMDs) exhibit an ideal metal/semiconductor (M/S) contact in a field-effect transistor. However, in the current two-step chemical vapor deposition process, the synthesis of m-TMD on pregrown s-TMD contaminates the van der Waals (vdW) interface and hinders the doping of s-TMD. Here, NbSe₂/Nb-doped-WSe₂ metal-doped-semiconductor (M/d-S) vdWHs are created via a one-step synthesis approach using a niobium molar ratio-controlled solution-phase precursor. The one-step growth approach synthesizes Nb-doped WSe₂ with a controllable doping concentration and metal/doped-semiconductor vdWHs. The hole carrier concentration can be precisely controlled by controlling the Nb/(W + Nb) molar ratio in the precursor solution from ∼3 × 10¹¹/cm² at Nb-0% to ∼1.38 × 10¹²/cm² at Nb-60%; correspondingly, the contact resistance R_C value decreases from 10 888.78 at Nb-0% to 70.60 kΩ.μm at Nb-60%. The Schottky barrier height measurement in the Arrhenius plots of ln(I_sat/T²) versus q/K_BT demonstrated an ohmic contact in the NbSe₂/W_xNb_1–xSe₂ vdWHs. Combining p-doping in WSe₂ and M/d-S vdWHs, the mobility (27.24 cm² V^–1 s^–1) and on/off ratio (2.2 × 10⁷) are increased 1238 and 4400 times, respectively, compared to that using the Cr/pure-WSe₂ contact (0.022 cm² V^–1 s^–1 and 5 × 10³, respectively). Together, the R_C value using the NbSe₂ contact shows 2.46 kΩ.μm, which is ∼29 times lower than that of using a metal contact. This method is expected to guide the synthesis of various M/d-S vdWHs and applications in future high-performance integrated circuits.  

6.243           Endohedral Filling Effects in Sorted and Polymer-Wrapped Single-Wall Carbon Nanotubes

Li, H., Gordeev, G., Toroz, D., Di Tommaso, D., Reich, S. and Flavel, B.S.

1. Phys. Chem., 125, 7476-7487 (2021)

  Widespread use of the polymer extraction method for single-wall carbon nanotubes (SWCNTs) and the ability of endohedral functionalization to alter their material properties make it important to examine the effect of filling on the separation result. Rate-zonal centrifugation is used to obtain empty, water-filled and perfluorooctane-filled large (∼1.45 nm), and single-chirality small (∼0.78 nm) diameter SWCNTs. These are transferred from water to toluene by dispersion with poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(6,6′-(2,2′-bipyridine))] (PFO-BPy), and absorption and Raman spectroscopy are used to follow the extent of endohedral filling at each step. Measurements are made before and after centrifugation (supernatant) but also in the pellet. PFO-BPy is found to stabilize endohedral-functionalized SWCNTs in the supernatant, but a clear preference for less-filled or empty species is shown. Molecular dynamics simulations are used to model the absorption spectrum of encapsulated water molecules in the PFO-BPy/SWCNT/toluene system and explain why partial water filling is difficult to directly measure. These findings are then used to interpret separation results obtained for raw soot (without intentional filling) and with a view to the low yield of polymer-based SWCNT extraction.  

6.244           Analysis of Single-Walled Carbon Nanotubes in Estuarine Sediments by Density Gradient Ultracentrifugation Coupled to Near-Infrared Fluorescence Spectroscopy Reveals Disassociation of Residual Metal Catalyst Nanoparticles

Montano, M.D., Liu, K., Sabo-Attwood, T. and Ferguson, P.L. Environ. Sci. Technol., 55, 1015-1023 (2021)   The continued growth of the nanotechnology industry and the incorporation of nanomaterials into consumer applications will inevitably lead to their release into environmental systems. Single-walled carbon nanotubes (SWCNTs) in particular have exhibited many attractive optical, mechanical, and electrical properties that lend themselves to new and exciting applications. Assessing their environmental impact upon release into the environment is contingent upon quantifying and characterizing SWCNTs in environmental matrixes. In this study, SWCNTs were isolated from estuarine sediments using density gradient ultracentrifugation (DGU), followed by online flow-through analysis of the density fractions via near-infrared spectroscopy. This approach yielded significant improvements in the quantitative detection limit, from 62 to 1.5 μg g–1. In addition, fractions of the density gradient were also obtained for further analysis by bulk inductively coupled plasma mass spectrometry (ICP–MS) and single-particle ICP–MS. Using fluorescent, semiconductive SWCNTs, the primary fluorescent nanotube fraction was found to be separated from the sediment matrix during DGU; however, the residual metal catalyst particles that had been assumed to be physically bound to the SWCNTs were found to form a separate band in the density gradient apart from the fluorescent SWCNTs. This result was repeated for a number of SWCNT types regardless of the metal catalyst and synthesis method, with a 0.1 g cm–3 density difference between most fractions. The apparent disconnect between the fluorescent fraction of SWCNTs and their metal-containing constituents potentially complicates CNT risk assessment as analysis techniques focusing solely on either CNT fluorescence or metal fingerprints may misrepresent exposure concentrations and their toxicological implications.  

6.245           Ideal PN photodiode using doping controlled WSe2–MoSe2 lateral heterostructure

Kim, J.E., Kang, W.T., Vu, V.T., Kim, Y.R., Shin, Y.S., Lee, I., Won, U.Y., Lee, B.H., Kim, K., Phan, T.L., Lee, Y.H. and Yu, W.J.

1. Mater. Chem. C, 9, 3504-3512 (2021)

  As the tight contact interface of the lateral PN junction enables high responsivity, specific detectivity, and fast response speed, atomic-scale two-dimensional (2D) lateral PN heterostructures are emerging as viable alternatives to silicon-based photodiodes. The optical properties of the current 2D heterostructures depend entirely on the intrinsic properties of 2D materials, which can be greatly improved by forming an ideal PN diode via the doping control of 2D heterostructures. In this study, we propose a high-performance photodiode using a doping-controlled WSe₂–MoSe₂ PN heterojunction. During the synthesis, the low chemical reactivity of Nb₂O₅ with WO₃ as compared to MoO₃ enables sequential growth and prevents niobium (Nb) doping during MoSe₂ growth at low temperatures. Conversely, in the WSe₂ growth at high temperatures, tungsten (W) to Nb is selectively substituted, resulting in the lateral heterostructure of Nb-doped WSe₂–MoSe₂. The Nb atoms in WSe₂ change the WSe₂ type from ambipolar to p-type dominant. Together with intrinsically n-type MoSe₂, Nb-doped WSe₂ forms a lateral PN heterostructure with a near-unity ideality factor (1.3) and a high forward/reverse current ratio of 10⁴. Our ideal 2D PN photodiode effectively suppresses the dark current in the reverse bias region (∼100 fA at an overall V_DS of 0 V to approximately −10 V) and enhances the photocurrent by the high built-in potential at the PN depletion layer (V_OC = 0.52 V). Thus, our device exhibits a high I_light/I_dark ratio (10⁵) and a corresponding ultra-high detectivity (5.78 × 10¹⁵ Jones), which are approximately 100 times higher than those of reported lateral 2D PN heterostructure photodiodes. These outstanding performances show that the doping-controlled transition metal dichalcogenide PN heterostructures are promising candidates for next-generation optoelectronics.  

6.246           Bandgap modulation in the two-dimensional core-shell-structured monolayers of WS2

Kang, S., Eshete, Y.A., Lee, S., Lee, S., Cho, S. and Yang, H. iScience, 25, 103563 (2022)   Tungsten disulfide (WS₂) has tunable bandgaps, which are required for diverse optoelectronic device applications. Here, we report the bandgap modulation in WS₂ monolayers with two-dimensional core-shell structures formed by unique growth mode in chemical vapor deposition (CVD). The core-shell structures in our CVD-grown WS₂ monolayers exhibit contrasts in optical images, Raman, and photoluminescence spectroscopy. The strain and doping effects in the WS₂, introduced by two different growth processes, generate PL peaks at 1.83 eV (at the core domain) and 1.98 eV (at the shell domain), which is distinct from conventional WS₂ with a primary PL peak at 2.02 eV. Our density functional theory (DFT) calculations explain the modulation of the optical bandgap in our core-shell-structured WS₂ monolayers by the strain, accompanying a direct-to-indirect bandgap transition. Thus, the core-shell-structured WS₂ monolayers provide a practical method to fabricate lateral heterostructures with different optical bandgaps, which are required for optoelectronic applications.